Keywords: Vieillissement, adipocytes, dicer, mir-1949 Aging, adipocytes, dicer, mir-1949 ABBREVIATION ACC

On the one part, dicer, the RNase III endoribonuclease responsible for microRNAs maturation, has been reported to be decreased in adipocytes during ageing. With the use of tamoxifen inducible dicer lox/lox / adiponectin-CreER T2 mice, we found that adipocyte dicer deficiency promoted the onset of some of the age-related complications, such as reduced adipocyte sizes and dysfunctions in systemic metabolism. The abrogation of white adipocyte markers such as Pparγ, Glut4 or Hsl, indicated that dicer is indispensible for the maintainance of white adipocyte identity. In addition, the results that there were lipid accumulation and fibrosis in liver in tamoxifen treated dicer lox/lox / adiponectin-CreER T2+ mice, indicated that adipocyte dicer deficiency might contribute to liver aging.

Mechanistically, mitochondrial function seemed to be upregulated due to adipocyte dicer deficiency, indicated by increased protein levels of OXPHOS components and PGC1α. In line, mitochondria repressors FOXO1 and FOXO3 were phosphorylated and inactivated, whose downstream antioxidant targets Catalase and Sod were also decreased. Moreover, P16, a marker of senescence, exhibited a trend to be increased due to adipocyte dicer deficiency. Since mitochondrial ROS surplus can lead to DNA damage and senescence, we assured that adipocyte dicer deficiency might induce a combination of mitochondrial activation and reduction in detoxification reduction possibly mediated by the inactivation of FOXO1 and FOXO3a. Finally, the result that nutrient restriction positively regulated dicer level in adipocytes further supported that there is a conserved aging pathway in adipocytes involving dicer.

On the other part, through microarray screen and RT-qPCR validation, we reported that aging increased mir-1949 in adipocytes in wild type mice and possibly promoted its secretion from perigonadal adipose tissue in vitro. Correspondingly, in vitro results also suggested that senescence increased mir-1949 production and secretion from adipocytes.

Functionally, upregulation of mir-1949 in 3T3-F442A adipocytes negatively regulated mitochondrial complex II protein level and oxygen consumption capability, associated with lipid accumulation. Interestingly, sustained upregulation of mir-1949 during adipogenesis of 3T3-F442A preadipocytes, tended to increase white adipocyte markers, such as Leptin, Glut4

or Hsl (N=2). Finally, combined with the results that 24h fasting significantly increased mir-1949 in perigonadal adipose tissue as well as that upregulation of mir-1949 exhibited a trend to increase lipid accumulation in senescent 3T3-F442A adipocytes (N=1), we assured that aging increases mir-1949 expression in adipocytes which might try to rescue age-related dysfunctions in adipocytes, such as impaired lipid storage. Nevertheless, the precise actions of mir-1949 need to be validated in vivo by injection of AAV-aP2-mir-1949 mimic into aged mice.

In parallel of my PhD project, we faced the matter of modulating gene expressions in differentiated adipocytes from immortalized cell lines with routine techniques like siRNA transfection. Therefore, we aimed to develop a technique enabling us to conserve isolated adipocytes for experiments. With the help of cell culture inserts, we managed to sequence buoyant mature adipocytes from floating and exposing into air. Moreover, by reducing surface tension with supplementation of co-solvents, the survival rate of cultured fat cells was apparently increased. Then, RT-qPCR results demonstrated that mRNAs levels of adipocytes markers increased after nearly one week of culture, in consistent with the increased cell sizes. Besides, cultured adipocytes in our culture system were proved to be functional, being able to response to catecholamine/ insulin stimulation for lipolysis and glucose transport, respectively. Therefore, the new way that we invited is capable of remaining mature adipocytes for in vitro study without losing its characteristics of morphology and function.

Taken together, these two aspects of my PhD would lead us to understand more in the field of adipocytes and aging.

RESUME

D'une part, le dicer, l'endoribonucléase RNase III responsable de la maturation des microARN, aurait été réduit chez les adipocytes au cours du vieillissement. Avec l'utilisation de souris dicerlox / lox / adiponectine-CreERT2 inductibles par le tamoxifène, nous avons constaté que le déficit en adipocytes favorisait l'apparition de certaines complications liées à l'âge, telles que la réduction de la taille des adipocytes et le dysfonctionnement du métabolisme. L'abrogation des marqueurs adipocytaires blancs, tels que Pparγ, Glut4 ou Hsl, indique que le dicer est indispensable au maintien de l'identité des adipocytes blancs. De plus, les résultats montrant une accumulation de lipides et une fibrose hépatique chez des souris traitées par le damerlox / lox / adiponectine-CreERT2 + traitées au tamoxifène ont indiqué que la carence en adicocyte pourrait contribuer au vieillissement du foie.

Mécaniquement, la fonction mitochondriale semblait être régulée à la hausse en raison d'une déficience en dicer adipocytaire, indiquée par une augmentation des taux de protéines des composants d'OXPHOS et de PGC1α. En ligne, les répresseurs des mitochondries FOXO1 et FOXO3 ont été phosphorylés et inactivés, dont les cibles antioxydantes en aval, la Catalase et le Sod, ont également diminué. En outre, P16, un marqueur de la sénescence, a montré une tendance à augmenter en raison de la carence en dicer. Comme le surplus de ROS mitochondrial peut entraîner des dommages à l'ADN et la sénescence, nous avons assuré que la déficience en dicer adipocytaire pourrait induire une combinaison d'activation mitochondriale et une réduction de la réduction de la détoxification possiblement induite par l'inactivation de FOXO1 et FOXO3a. Enfin, le résultat selon lequel la restriction en éléments nutritifs a régulé positivement le taux de dicer dans les adipocytes a également confirmé qu'il existe une voie de vieillissement conservée dans les adipocytes impliquant le dicer.

Par ailleurs, grâce à la sélection de microréseaux et à la validation RT-qPCR, nous avons rapporté que le vieillissement augmentait le mir-1949 dans les adipocytes chez les souris de type sauvage et favorisait probablement sa sécrétion à partir du tissu adipeux perigonadal in vitro. De manière correspondante, des résultats in vitro ont également suggéré que la sénescence augmentait la production de mir-1949 et la sécrétion d'adipocytes. Sur le plan fonctionnel, la régulation positive du mir-1949 dans les adipocytes 3T3-F442A régulait négativement le niveau du complexe mitochondrial II et la capacité de consommation d'oxygène, associées à l'accumulation de lipides. Fait intéressant, la régulation à la hausse soutenue du mir-1949 au cours de l'adipogenèse des préadipocytes 3T3-F442A avait tendance à augmenter les marqueurs adipocytaires blancs, tels que la leptine, Glut4 ou Hsl (N = 2). Enfin, combiné aux résultats indiquant que le jeûne de 24 heures a significativement augmenté le mir-1949 dans les tissus adipeux péri-radicaux et que la régulation positive du mir-1949 tendait à augmenter l'accumulation de lipides dans les adipocytes séniles 3T3-F442A (N = 1) augmente l'expression de mir-1949 dans les adipocytes, ce qui pourrait tenter de sauver des dysfonctionnements liés à l'âge dans les adipocytes, tels qu'une altération du stockage des lipides. Néanmoins, les actions précises de mir-1949 doivent être validées in vivo par injection de mimétique d'AAV-aP2-mir-1949 chez des souris âgées.

Parallèlement à mon projet de thèse, nous avons dû moduler les expressions géniques dans des adipocytes différenciés à partir de lignées cellulaires immortalisées avec des techniques de routine telles que les transfections d'ARNsi. Nous avons donc cherché à développer une technique permettant de conserver des adipocytes isolés pour des expériences. À l'aide d'inserts de culture cellulaire, nous avons réussi à séquencer des adipocytes matures flottants et à les exposer à l'air. En outre, en réduisant la tension de surface avec la supplémentation en co-solvants, le taux de survie des cellules adipeuses en culture a apparemment augmenté. Ensuite, les résultats de la qPCR ont démontré que les taux d'ARNm des marqueurs des adipocytes augmentaient après presque une semaine de culture, ce qui correspondait à la taille accrue des cellules. En outre, les adipocytes cultivés dans notre système de culture se sont avérés fonctionnels, pouvant répondre respectivement à la stimulation catécholamine / insuline pour la lipolyse et le transport du glucose. Par conséquent, la nouvelle façon dont nous avons invité est capable de rester des adipocytes matures pour une étude in vitro sans perdre ses caractéristiques de morphologie et de fonction.

Ensemble, ces deux aspects de ma thèse nous amèneraient à mieux comprendre le domaine des adipocytes et du vieillissement.

LISTS OF FIGURES AND TABLES FIGURES

Introduction part

Results part Result 1: Role and regulation of microRNAs in adipose tissue during aging

Result 2: A novel method of culturing isolated adipocytes in vitro

TABLES Introduction part

Results part Result 1: Role and regulation of microRNAs in adipose tissue during aging

The adipose tissue

The adipose tissue is a master regulator of energy balance and nutritional homeostasis. Viewed as an inert energy storage site and a form of connective tissue not long time ago, surging evidence now have revealed that adipose tissue is a remarkably complex organ with important endocrinal functions which enable its crosstalk with diverse organs.

Moreover, with the very recent discovery of brown and beige adipocytes, interests have been fueled in the study of adipose biology (Figure 1). Adipose tissue is a dynamic organ which can range from 4% to over 40% of total body composition in adult humans and is dispersed throughout human body. Founctionally, adipose tissue could be categorized into white adipose tissue (WAT) and brown adipose tissue (BAT). BAT generates heat during cold exposure by adaptive thermogenesis to maintain normal body temperature and is more abundant in neonates and young children, and gradually declined in human adults [START_REF] Enerback | Human brown adipose tissue[END_REF] . Here in our topic, we are going to focus mainly on WAT.

Geographically, WAT is categorized into subcutaneous and visceral adipose tissue (sWAT and vWAT, respectively) (Figure 2a). Surprisingly, a pioneering clinical study in the 1950s firstly reported that location of fat depots has a great influence on the likelihood of an individual to develop many of the complications of obesity 4 (Figure 2b). The data indicate that there is heterogeneity between sWAT and vWAT. 1.1.

Subcutaneous adipose tissue (sWAT)

In human, sWAT forms just below the dermis, and includes periscapular and inguinal depots. Increased amounts of subcutaneous fat, especially in the gluteofemoral regions, are associated with improved insulin sensitivity and a lower risk of developing metabolic syndromes [START_REF] Snijder | Associations of hip and thigh circumferences independent of waist circumference with the incidence of type 2 diabetes: the Hoorn Study[END_REF] . In mice, transplantation of subucutaneous fat into the cavity of visceral fat significantly decreases body weight and total fat mass as well as improves systemic glucose tolerance, whereas transplantation of vWAT into the depot of sWAT has little effects [START_REF] Tran | Beneficial effects of subcutaneous fat transplantation on metabolism[END_REF] .

The metabolic benefits of sWAT might be associated with its unique property: sWAT is capable of 'browning' in responsive to cold exposure, β-agonists or other hormone-like stimuli [START_REF] Wu | Adaptive thermogenesis in adipocytes: is beige the new brown?[END_REF] , which includes the induction of uncoupling protein 1 (UCP1) expression, a gene giving rise to uncoupled respiration and heat production. The UCP positive cells would emerge in white fat depots under certain stimuli and are termed beige or brite cells.

Adipocyte-specific mutant of PR domain containing 16 (PRDM16) markedly inhibites beige adipocyte function in sWAT following cold exposure or β3-agonist treatment, acquiring numerous properties of vWAT including decreased thermogenic and increased inflammatory gene expression as well as macrophage infiltration. Moreover, transplantation of the sWAT derived from PRDM16 mutants into obese mice shows a loss of metabolic benefit 9 .

Recently, it has been reported that inhibitors of the Janus kinase/ signal transducers and activators of transcription 1/3 (JAK/ STAT1/3) signaling, like tofacitinib and R406 are strongly inducers of UCP1 exressionin sWAT but not in vWAT [START_REF] Moisan | White-to-brown metabolic conversion of human adipocytes by JAK inhibition[END_REF] , indicating that inhibitions of JAK/ STAT1/3 signaling might mediate the browning process in sWAT, whereas it is not employed in the futile energy cycle in vWAT.

Visveral adipose tissue (vWAT)

In human, there are 6 visceral fat depots: perirenal, gonadal, epicardial, retroperitoneal, omental and mesenteric depots. In contrast to sWAT, it is revealed that the size of abdominal adipocyte is a significant risk predictor for the development of type 2 diabetes, as revealed by a follow-up study of around 245 women from 1974 to 2001 [START_REF] Lonn | Adipocyte size predicts incidence of type 2 diabetes in women[END_REF] .

Several plausible mechanisms may be associated with the properties of vWAT.

Firstly, Aliki Kosteli et al, demonstrat that vWAT has a higher rate of basal lipolysis and therefore releases more free fatty acids (FFAs) compared to sWAT [START_REF] Kosteli | Weight loss and lipolysis promote a dynamic immune response in murine adipose tissue[END_REF] . Since vWAT is anatomically drained by the portal vein, the increased levels of portal FFAs might induce metabolic complications in liver, such as hepatic steatosis or hepatic insulin resistance in obesity [START_REF] Wueest | Mesenteric Fat Lipolysis Mediates Obesity-Associated Hepatic Steatosis and Insulin Resistance[END_REF] . What's more, FFAs are also strong inducers of adipose tissue's recruitment of macrophages, whose chronic accumulation would lead to elevated local inflammatory state [START_REF] Kosteli | Weight loss and lipolysis promote a dynamic immune response in murine adipose tissue[END_REF] , which is negatively associated with insulin resistance. In addition, with the use of whole-body fluorodeoxyglucose positron emission tomography (FDG-PET) scans, it is found that vWAT has higher rates of glucose uptake compared to sWAT in both lean and obese humans. The isolated stromal vascular fractions (SVFs) from vWAT exhibites significant increase in both basal and cytokine stimulated glucose consumptions associated with a higher expression of hexokinase (HK) compared to that from sWAT [START_REF] Christen | Increased glucose uptake in visceral versus subcutaneous adipose tissue revealed by PET imaging[END_REF] . These results indicate that vWAT has higher metabolic rates in comparision to sWAT.

Interestingly, although to a lesser extent, visceral fat is capable of responding to chronic β3-adrenergic receptor stimulation demonstrated by the appearance of lipid droplet mutilocularity and robust mitochondrial biogenesis, which might be explained by a prominent increase in futile creatine cycling in visceral fat under stimulation [START_REF] Bertholet | Mitochondrial Patch Clamp of Beige Adipocytes Reveals UCP1-Positive and UCP1-Negative Cells Both Exhibiting Futile Creatine Cycling[END_REF] . However, the physiological role of futile cycling of vWAT remains to be investigated.

Secondly, vWAT is also an endocrine organ that actively secretes lots of cytokines and bioactive mediators, which influence systemic inflammation, lipid metabolism and insulin sensitivity [START_REF] Macdougall | Visceral Adipose Tissue Immune Homeostasis Is Regulated by the Crosstalk between Adipocytes and Dendritic Cell Subsets[END_REF] . For example, B cells isolated from vWAT expressed higher levels of inflammatory immune activation markers and significantly higher nuclear factor-κB (NF-κB) activation and phospho-STAT3 [START_REF] Blomberg | Visceral adipose tissue contributes to the generation of pro-inflammatory B cell subsets in aging mice[END_REF] . Expression of proinflammatory adipokines in vWAT could be elevated by obesity, which might contribute to their circulating elevation during obesity [START_REF] Horbelt | The novel adipokine WISP1 associates with insulin resistance and impairs insulin action in human myotubes and mouse hepatocytes[END_REF] .

White Adipocytes

Adipose tissue is composed of mature adipocytes and various other cell types in stromal vascular factions (SVFs) that include leukocytes (e.g., macrophages and lymphocytes)

and adipose tissue stromal cells (ATSCs) that include preadipocytes and fibroblasts.

Adipocytes contribute mainly to metabolism and functions of adipose tissue [START_REF] Vilaboa | Age influence on stromal vascular fraction cell yield obtained from human lipoaspirates[END_REF] .

Architecture

White adipocytes contain a single large lipid droplet (LD) occupying approximately 90% of the cytoplasm, with the nucleus being squeezed to the cell periphery and the cytoplasm forming a very thin rim. Mitochondria are scarce and internal cristae are poorly developed (Figure 3). Due to these characteristics, these cells are also termed unilocular adipocytes. At the tissue level, spherical adipocytes are typically arranged in an imperfect hexagonal packing architecture resembling honeycomb, with smaller preadipocytes and interweaving capillaries and nerves filling the interstitial space [START_REF] Veilleux | Visceral Adipocyte Hypertrophy is Associated With Dyslipidemia Independent of Body Composition and Fat Distribution in Women[END_REF][START_REF] Wree | Adipocyte cell size, free fatty acids and apolipoproteins are associated with non-alcoholic liver injury progression in severely obese patients[END_REF] . Adipocytes themselves are interconnected via gap junctions and thereby share cytoplasm and respond in concert to electrical stimuli [START_REF] Burke | Adipocytes in both brown and white adipose tissue of adult mice are functionally connected via gap junctions: implications for Chagas disease[END_REF] . Additionally, adipocytes are intensively interacted with the extracellular matrix (ECM), which provides both the flexibility needed for cell migration and hypertrophy and the stability needed for the structural and functional integrity of the tissue as a whole (Figure 4). For example, adipocytes are interacted with an isotropic network of collagen and elastic fibers, extracellular fibronectin and laminin [START_REF] Young | Injectable hydrogel scaffold from decellularized human lipoaspirate[END_REF] , via attachment points for integrins anchored in the adipocyte membrane [START_REF] Bonnans | Remodelling the extracellular matrix in development and disease[END_REF] . The rounded LDs are surrounded by a single lipid layer (of endoplasmic reticulum membrane origin), which is in turn ensheathed by endoplasmic reticulum (ER). LDs are very ofter surrounded by one or more proteins of the PAT (perilipin, adipose differentiationrelated protein (ADRP), and TIP47 (tail interacting protein of 47 kDa family) [START_REF] Miura | Functional conservation for lipid storage droplet association among Perilipin, ADRP, and TIP47 (PAT)-related proteins in mammals, Drosophila, and Dictyostelium[END_REF] . The LDs of adipocytes are encased in a coating of perilipins, which have an important role in regulating triglyceride hydrolysis (breakdown of triglyceride into glycerol and free fatty acids), with perilipin A being the most abundant isoform in adipocytes. Perilipin null adipocytes exhibites elevated basal lipolysis but attenuated sitmulated lipolytic activity, indicating that perilipin is required for maximal lipolytic activity 28 .

Homeostasis of lipid droplets in adipocytes

Adipocytes are central in the regulation of triglyceride (TG) and FFAs homeostasis during energy expenditure and deprivation. The storage and removal of TG in adipocytes is essential to determine the sizes of adipocytes. In vivo, the sizes of adipocytes vary greatly according to the depots and the nutritional states ( Several major functions of adipocytes determine the size of LDs in adipocytes: fatty acid uptake, de novo lipogenesis (DNL), lipolysis and possible burning of fat by beiging, which we will discuss in details in the following parts.

Synthesis of lipid droplets (LDs)



Fatty acid uptake

Most of the lipids stored in adipocytes are transferred directly from diets. Several proteins are involved in fatty acid uptake in adipocytes: caveolin-1, fatty acid translocase (FAT/CD36), the 43-kDa plasma membrane fatty acid binding protein (FABPpm), and fatty acid transport protein (FATP), adipose acyl CoA synthetase (ACSL).

Caveolin-1

Caveolae are a subset of so-called membrane lipid raft domains that are often experimentally defined by their resistance to solubilization by nonionic detergents.

The surface of adipocytes is abundantly covered by caveolae (nearly 30%) [START_REF] Kanzaki | Caveolin-associated filamentous actin (Cav-actin) defines a novel F-actin structure in adipocytes[END_REF] . Caveolin-1 is the primary protein driving the formation of caveolae in adipocytes [START_REF] Briand | Caveolin-1 expression and cavin stability regulate caveolae dynamics in adipocyte lipid store fluctuation[END_REF] . Caveolin-1 is speculated to bind the fatty acids accumulated with the caveolar membrane, leading to membrane asymmetry and budding of caveolae from plasma membrane to form vesicles. These fatty acid loaded caveolae vesicles could deliver fatty acids to subcellular compartments for further metabolism (Figure 5) [START_REF] Thompson | Fatty acid flux in adipocytes: the in's and out's of fat cell lipid trafficking[END_REF] .

FABPpm

Expression of FABPpm in 3T3-L1 preadipocytes that normally do not express FABPpm induces a significant increase in fatty acid uptake. Anti-FABPpm antibodies selectively inhibit uptake of oleate in 3T3-L1 adipocyte monolayer without affecting 2-deoxyglucose or octanoate (medium-chain fatty acid) uptake [START_REF] Zhou | Mitochondrial aspartate aminotransferase expressed on the surface of 3T3-L1 adipocytes mediates saturable fatty acid uptake[END_REF] . Currently, the mechanism by which FABPpm used in LCFA uptake is unknown.

CD36

CD36 is an 88-kDa single chain membrane glycoprotein expressed in many tissues and cell types, including adipocytes, heart, skeletal muscle and macrophages. C/EBPα and C/EBPβ would increase CD36 expression in different types of cells, with C/EBPα exhibits a significantly higher potency than C/EBPβ. A C/EBP responsive element is identified in the CD36 promoter [START_REF] Qiao | Transcriptional regulation of fatty acid Translocase/CD36 expression by CCAAT/Enhancer-binding protein alpha[END_REF] . It has been speculated that CD36 may bind and internalize long chain fatty acid (LCFA) by endocytosis or may bind LCFA and work in concert with FABP to provide a high concentration gradient of LCFA across the plasma membrane facilitating its uptake by other fatty acid transporters (Figure 5) [START_REF] Thompson | Fatty acid flux in adipocytes: the in's and out's of fat cell lipid trafficking[END_REF] .

FATP1 and ACSL

FATP1 and ACSL are two different classes of membrane bound enzymes catalyzing the ATP-dependent esterification of long-chain and very long-chain fatty acids into acyl-CoA derivatives [START_REF] Hall | Characterization of the Acyl-CoA synthetase activity of purified murine fatty acid transport protein 1[END_REF][START_REF] Hall | Enzymatic properties of purified murine fatty acid transport protein 4 and analysis of acyl-CoA synthetase activities in tissues from FATP4 null mice[END_REF] . FATP1 and ACSL1 are enriched in adipose tissue.

FATP1 is induced during the differentiation of 3T3-L1 adipocytes, and increased LCFA uptake. Insulin could trigger FATP1 translocation from a perinuclear compartment to the plasma membrane, indicating that FATP1 is facilitated by insulin [START_REF] Stahl | Insulin causes fatty acid transport protein translocation and enhanced fatty acid uptake in adipocytes[END_REF] . Since it is reported that the conversion of incoming LCFA to TG occurs on/or around the plasma membrane in rat adipocytes [START_REF] Ost | Triacylglycerol is synthesized in a specific subclass of caveolae in primary adipocytes[END_REF] , FATP1 might facilitate LCFA uptake by tightly coupling it to metabolism: by converting it into acyl-CoAs which is preferentially shunted into TG synthesis (Figure 5) [START_REF] Thompson | Fatty acid flux in adipocytes: the in's and out's of fat cell lipid trafficking[END_REF] .

ACSL1 is found to be involved in the reacylation of LCFA released from the LD during basal and hormone-stimulated lipolysis. Acylation of LCFA leads to reincorporation into the TG thereby preventing its cellular efflux [START_REF] Lobo | Functional Analysis of Long-chain Acyl-CoA Synthetase 1 in 3T3-L1 Adipocytes[END_REF] . are abundantly expressed in fat cells [START_REF] Herman | A novel ChREBP isoform in adipose tissue regulates systemic glucose metabolism[END_REF] . Surprisingly, DNL contributes only a few percent of fatty acids to the TG that accumulates in the lipid droplets of adipocytes [START_REF] Strawford | Adipose tissue triglyceride turnover, de novo lipogenesis, and cell proliferation in humans measured with (H2O)-H-2[END_REF] . In addition, it was found that in vitro 3T3-L1 adipocytes preferentially use glycolysis to depose glucose into lactate even with the abundant oxygen availability, while only a small fraction of carbon from glucose would be converted into TG [START_REF] Sabater | Cultured 3T3L1 adipocytes dispose of excess medium glucose as lactate under abundant oxygen availability[END_REF] . However, DNL contributes to a number of adipose-derived putative bioactive lipids, such as palmitoleate [START_REF] Cao | Identification of a lipokine, a lipid hormone linking adipose tissue to systemic metabolism[END_REF] , the novel fatty acid esters of hydroxy-fatty acids [START_REF] Yore | Discovery of a class of endogenous mammalian lipids with antidiabetic and anti-inflammatory effects[END_REF] and PPARγ ligand alkyl ether-lipid [START_REF] Lodhi | Inhibiting adipose tissue lipogenesis reprograms thermogenesis and PPARgamma activation to decrease diet-induced obesity[END_REF] . In addition, studies have shown that up-regulation of DNL in adipose tissue mediated the improved insulin sensitivity and glycemic control of GLUT4 overexpression in adipocytes, whereas loss of ChREBP has the opposite effects [START_REF] Herman | A novel ChREBP isoform in adipose tissue regulates systemic glucose metabolism[END_REF][START_REF] Iizuka | Deficiency of carbohydrate response element-binding protein (ChREBP) reduces lipogenesis as well as glycolysis[END_REF] . Besides, FAS was dramatically upregualted in WAT by caloric restriction (CR) process which is associated with increased insulin sensitivity and longevity, indicating that DNL might be important metabolic adaptations of caloric restriction benefits [START_REF] Bruss | Calorie restriction increases fatty acid synthesis and whole body fat oxidation rates[END_REF] .

Conversely, impairment of DNL, which is under rictor/mTORC2 signaling in adipose tissue, was found to be necessary for the loss of insulin sensitivity induced by high fat diet in wild type mice [START_REF] Tang | Adipose tissue mTORC2 regulates ChREBP-driven de novo lipogenesis and hepatic glucose metabolism[END_REF] .

Glucose transport:

Insulin signaling plays an important role in lipid storage and the process of adipogenesis for adipocytes (Figure 6). The phosphatidylinositol-3-OH kinase (PI(3)K)dependent signaling is initiated by binding of insulin onto its tyrosine kinase receptor on the cell surface, leading to the recruitment and tyrosine phosphorylation of the insulin receptor substrate (IRS). The phosphorylated IRS then serves as docking sites for the SH2 domain of the p85 regulatory subunit of class I PI3K, resulting the activation of PI3K, which promotes the synthesis of phosphatidylinositol-3,4,5-trisphosphate PtdIns (3,4,5) P3 from PtdIns (4,5) P 2 at the plasma membrane. PtdIns (3,4,5) P3 then serves as a docking site for several PH domain-containing Ser/Thr kinases that are implicated in glucose uptake, such as phosphoinositide-dependent kinase 1 (PDK 1) and AKT (also known as PKB) [START_REF] Cong | Physiological role of Akt in insulin-stimulated translocation of GLUT4 in transfected rat adipose cells[END_REF] . AKT was then activated by PDK1 (phosphoinositide-dependent kinase 1) and mTORC2 through dual Ser/Thr phosphorylation. Downstream, AKT kinase affects the exocytic arm of the GLUT4 (glucose transporter 4) trafficking itinerary, probably by regulating the translocation, targeting and fusion of GLUT4-containing vesicles [START_REF] Nagano | Tomosyn is a novel Akt substrate mediating insulin-dependent GLUT4 exocytosis[END_REF] .

On the other hand, insulin could also initiate a PI3K-independent signaling by recruiting the adaptor protein APS, which has high affinity to the activated insulin receptor [START_REF] Liu | APS facilitates c-Cbl tyrosine phosphorylation and GLUT4 translocation in response to insulin in 3T3-L1 adipocytes[END_REF] .

Following insulin stimulation, APS recruits a complex that comprises the proto-oncogene c-CBL and c-CBL-associated protein (CAP), triggering tyrosine phosphorylation of CBL by insulin receptor [START_REF] Ribon | Insulin stimulates tyrosine phosphorylation of the protooncogene product of c-Cbl in 3T3-L1 adipocytes[END_REF] . Upon phosphorylation, CBL is then translocated to lipid rafts, recruiting the CrkII-C3G complex to lipid rafts, where C3G specifically activates the small GTP-binding protein TC10, which interacts with effector proteins that regulate GLUT4 vesicle exocytosis [START_REF] Chiang | Insulin-stimulated GLUT4 translocation requires the CAPdependent activation of TC10[END_REF] .

Adipocyte GLUT4 ablation resulted in systemic insulin resistance in liver and muscle [START_REF] Abel | Adipose-selective targeting of the GLUT4 gene impairs insulin action in muscle and liver[END_REF] , indicating that GLUT4 mediated glucose transport in adipocytes is indispensable for whole body glucose homeostasis.

Hydrolysis of lipid droplets

Lipolysis represents the process for FFAs to be liberated from TG in LDs. Total lipolysis is the sum of basal lipolysis and stimulated lipolysis. Basal lipolysis is the relase of FFAs from adipocytes occurring in the absence of negative energy balance, which correlates positively with adipocyte size and is increased by obesity [START_REF] Duncan | Regulation of lipolysis in adipocytes[END_REF] . Stimulated lipolysis refers to the hormonally regulated release of FFAs in response to nutritional demands. The lipolysis of TG in adipocytes is under tight control of various pathways in vivo (Figure 7).

Typically, the main regulating signaling pathway of stimulated lipolysis in adipocytes is β-adrenergic signaling. At least three major enzymes are responsible for the lipolytic process. The primary cleavage of TG to diacylglycerols is performed by the adipose tissue lipase (ATGL), which is predominantly expressed in adipose tissue. In ATGL -/-mice, absence of ATGL induced severe lipid accumulation in adipose tissues and heart, with dramatic inhibition of basal and isoproterenol stimulated lipolysis as well as premature death. On the contrary, the inability of fat catabolism due to ATGL deficiency led to increased glucose use, indicating that ATGL is rate limiting in the catabolism of cellular fat [START_REF] Haemmerle | Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase[END_REF] . Administration of Atglistain, a small molecule inhibitor of ATGL effectively improves high fat diet-induced obesity associated insulin resistance and liver steatosis in mice. Moreover, the drug predominantly targeted adipose tissue and the liver, and therefore did not induce cardiac lipid accumulation or cardiomyopathy even after long-term treatment [START_REF] Schweiger | Pharmacological inhibition of adipose triglyceride lipase corrects high-fat diet-induced insulin resistance and hepatosteatosis in mice[END_REF] . ATGL is highly regulated at both the transcriptional and post-transcriptional levels, including multiple phosphorylation events and translocation to the surface of LDs. It could be activated by its coactivator comparative gene identification-58 (CGI-58), which is normally bound in an inactive state by the LD protein perilipin 1 (PLIN1). And upon activation of β-adrenergic signaling, PKA induced phosphorylation of PLIN1 leading to the release of CGI-58, allowing its binding and activation of ATGL [START_REF] Granneman | Perilipin controls lipolysis by regulating the interactions of AB-hydrolase containing 5 (Abhd5) and adipose triglyceride lipase (Atgl)[END_REF] .

Subsequently, hormone-sensitive lipase (HSL) is dispensable for lipolysis. HSL is the major diglyceride lipase in adipocytes, with monoglyceride lipase (MGL) completes the process by generating glycerol and FFAs. Together, these three enzymes account for over 90% of the lipolytic activity in the adipocytes [START_REF] Young | Biochemistry and pathophysiology of intravascular and intracellular lipolysis[END_REF] .

Physiologically, insulin is the major suppressor of lipolysis, which functions through several different ways to block lipolysis: 1) It activates phosphodiesterase 3b (PDE3b) in a AKT dependent manner, which antagonize the intracellular levels of cAMP and thereby blocking protein kinase A (PKA) activation [START_REF] Czech | Insulin signalling mechanisms for triacylglycerol storage[END_REF] . 2) Recently, a non-canonical pathway has been proposed in which insulin blocks activation of PKA selectively on PLIN1 through a PI3Kmediated, AKT-independent pathway [START_REF] Choi | Insulin regulates adipocyte lipolysis via an Akt-independent signaling pathway[END_REF] . 3) Insulin could also repress lipolysis by transcriptionally silencing lipase genes via repressions of transcription factors forkhead box protein O1 (FOXO1) and interferon regulatory factor 4 (IRF4) [START_REF] Chakrabarti | FoxO1 controls insulin-dependent adipose triglyceride lipase (ATGL) expression and lipolysis in adipocytes[END_REF] (Figure 7).

The size of fat cells per se also have an impact on both the spontaneous and hormone-stimulated rates of lipolysis, with big adipocytes exhibited higher rates of basal and hormone-stimulated hydrolysis, probably owing to the fact that hypertrophic adipocytes are enriched in the expressions of ATGL, HSL and PLIN1. However, the receptor sensitivities for hormones like insulin, natriuretic peptides, dobutamine, terbutaline and clonidine, are comparable between small and large fat cells [START_REF] Laurencikiene | Regulation of lipolysis in small and large fat cells of the same subject[END_REF] .

Lipid driplets "burning"

Over the last three decades, it has been realized that mature adipocytes displayed high plasticity, especially those in the subcutaneous depots. In response to some external stimuli, including chronic cold exposure, exercise, long-term treatment with PPARγ agonists or β3-adrenergic receptor agonists, cancer cachexia, and tissue injury, a group of beige (also called brite) adipocytes are induced within WAT which are characterized by possessions of abundant cristae-dense mitochondria that express UCP1 and mutilocular LDs 66 . Fatty acids are rapidly consumed and deprived by mitochondria β-oxidationa and UCP-mediated heat production in beige adipocytes, resulting in a smaller appearance of beige adipocytes compared to normal white adipocytes [START_REF] Altshuler-Keylin | Beige Adipocyte Maintenance Is Regulated by Autophagy-Induced Mitochondrial Clearance[END_REF] . It also has been demonstrated that beige-like adipocytes also exist in human adults [START_REF] Shinoda | Genetic and functional characterization of clonally derived adult human brown adipocytes[END_REF] .

Although some of beige adipocytes might directly come from adipogenesis of distinctive populations of progenitors within WAT [START_REF] Wang | Ebf2 is a selective marker of brown and beige adipogenic precursor cells[END_REF][START_REF] Vishvanath | Pdgfr beta(+) Mural Preadipocytes Contribute to Adipocyte Hyperplasia Induced by High-Fat-Diet Feeding and Prolonged Cold Exposure in Adult Mice[END_REF] , it has been reported that stimulation of β3-adrenergic receptor (Adrb3) in mature adipocytes could directly lead the transformation into mitochondrial abundant beige adipocytes via activation of PRDM [START_REF] Jiang | Distinct cellular and molecular mechanisms for beta 3 adrenergic receptor-induced beige adipocyte formation[END_REF] .

Conversely, after withdrawal of external stimuli such as cold acclimation or β3adrenergic agonists, the produced beige adipocytes then possibly transform into normal white adipocytes, by upregulation of mitophagy (mitochondrial clearance via autophagy) activated by which is initiated by the activation of MiT/TFF (microphthalmia/ transcription factor E) transcription factor-mediated lysosome biogenesis 67 .

Endocrinal functions

Adipocytes have been well acknowledged as a bona fide endocrine cell type that regulates systemic metabolic homeostasis in accordance to nutrient flux by equally matching the metabolic demands of positive or negative energy balance, via secreting a variety of bioactive peptides, known as adipokines (Figure 8). Secretion of adipokines, including leptin, adiponectin, bone morphogenetic protein (BMP)-4, dipeptidyl peptidase 4 (DPP-4), etc, is altered by adipose dysfunction and may contribute to obesity-associated diseases. Therefore, adipokines are promising candidates for pharmacological treatment and as diagnostic tools.

Leptin

Leptin was discovered more than 20 years ago [START_REF] Zhang | Positional cloning of the mouse obese gene and its human homologue[END_REF] . Circulating levels of leptin are proportional to fat mass, and leptin receptors are abundantly expressed in various tissues, including on the adipocyte. Leptin negatively regulate adiposity. Indirectly, leptin negatively regulate satiety via inhibiting the orexigenic pathway comprising neuropeptide-Y (NPY) and agouti-related protein (AGRP)-containing neurons, while upreguation of anorexigenic pathway consisting of proopiomelanocortin (POMC) and cocaine-and amphetamineregulated transcript (CART)-containing neurons in hypothalamus [START_REF] Baver | Leptin Modulates the Intrinsic Excitability of AgRP/NPY Neurons in the Arcuate Nucleus of the Hypothalamus[END_REF] . Moreover, leptin would drive adiposity depletion by stimulation of lipolysis through activating sympathetic neurons innervating adipocytes in WAT in vivo [START_REF] Zeng | Sympathetic neuro-adipose connections mediate leptin-driven lipolysis[END_REF] . Also, in vitro, leptin was reported to decrease lipogenesis, increase TG hydrolysis and oxidation of fatty acids in isolated rat white adipocytes [START_REF] William | Leptin controls the fate of fatty acids in isolated rat white adipocytes[END_REF] .

Adiponectin

Adiponectin was firstly reported in 1995 [START_REF] Scherer | A novel serum protein similar to C1q, produced exclusively in adipocytes[END_REF] , which is almost exclusively synthesized by adipocytes. Adiponectin concentrations inversely correlate with fat mass and are downregulated in obesity and type 2 diabetes [START_REF] Scherer | Adipose tissue -From lipid storage compartment to endocrine organ[END_REF] .Overexpression of adiponectin in 3T3-L1 cells enhanced adipogenesis and lipid storage [START_REF] Fu | Adiponectin promotes adipocyte differentiation, insulin sensitivity, and lipid accumulation[END_REF] . Also, adiponectin has been shown to increase insulin sensitivity and maintains healthy adipose tissue expansion while rescuing ectopic lipid accumulation in mice [START_REF] Berg | The adipocyte-secreted protein Acrp30 enhances hepatic insulin action[END_REF] .

The beneficial effects of adiponectin on insulin sensitivity seem to be mediated in part by its ability to activate AMP-activated protein kinase (AMPK) in skeletal muscle and liver [START_REF] Awazawa | Adiponectin suppresses hepatic SREBP1c expression in an AdipoR1/LKB1/AMPK dependent pathway[END_REF] .For example, adiponectin decreases hepatic lipogenesis and increases β-oxidation through activation of AMPK, a cellular energy sensor, inhibits lipogenesis by phosphorylating the rate-limiting enzyme of DNL, acetyl CoA carboxylase-1 (ACC-1). This decreases in ACC-1 activity which leads to decrease in malonyl CoA production, thereby relieves inhibition of carnitine palmitoul transferase-1 (CPT-1) activity and enhances fatty acid transport into the mitochondria to undergo β-oxidation.

Adiponectin is also associated with cardio-protection. Administration of adenovirusmediated adiponectin reduces atheroscleric lesion size in apolipoprotein E knockout mice, accompanied by reduction in the expression of inflammatory cytokines [START_REF] Wang | Adiponectin improves NF-kappa B-mediated inflammation and abates atherosclerosis progression in apolipoprotein E-deficient mice[END_REF] . Moreover, adiponectin is also reported to promote angiogenesis and inhibits endothelial apopotosis [START_REF] Stern | Leptin, and Fatty Acids in the Maintenance of Metabolic Homeostasis through Adipose Tissue Crosstalk[END_REF] .

Fatty acid binding protein 4 (FABP4/ aP2) aP2 was elevated in circulation in obese humans and mice [START_REF] Aeberli | The increase of fatty acid-binding protein aP2 in overweight and obese children: interactions with dietary fat and impact on measures of subclinical inflammation[END_REF] . Furthermore, aP2 was demonstrated to be an adipokine released from adipocytes under obesogenic conditions, such as hypoxia, to augment insulin secretion from pancreatic β-cells in vivo, while insulin could in turn inhibit aP2 secretion [START_REF] Wu | Identification of fatty acid binding protein 4 as an adipokine that regulates insulin secretion during obesity[END_REF] .

DPP-4

Dipeptidyl peptidase 4 (DPP4) is a 110 kDa glycoprotein, which is ubiquitously expressed on different cell types as well as in circulation. By proteomic profiling of the adipocyte secretome, soluble DPP4 is identified as a novel adipokine, for which mature adipocytes constitute are a major source [START_REF] Lamers | Dipeptidyl Peptidase 4 Is a Novel Adipokine Potentially Linking Obesity to the Metabolic Syndrome[END_REF] . DPP-4 may not only reflect, but also contribute to adipose tissue dysfunction. Compared with healthy lean individuals, patients with obesity and insulin resistance have higher DPP-4 expression in vWAT, which may contribute to the higher circulating DPP-4 levels [START_REF] Sell | Adipose Dipeptidyl Peptidase-4 and Obesity Correlation with insulin resistance and depot-specific release from adipose tissue in vivo and in vitro[END_REF] . In obesity, hypoxia-mediated upregulation of DPP-4

shedding from adipocytes by metalloprotease-9 might contribute to induced DPP-4 activity [START_REF] Rohrborn | Shedding of dipeptidyl peptidase 4 is mediated by metalloproteases and up-regulated by hypoxia in human adipocytes and smooth muscle cells[END_REF] .

Also, in mice, production of DPP-4 in adipocytes is shown to be negatively regulated by high glucose concentration under physiological conditions, such an effect is abolished in mouse models of diabetes [START_REF] Das | Regulation of dipeptidyl peptidase 4 production in adipocytes by glucose. Diabetes, metabolic syndrome and obesity : targets and therapy[END_REF] .

Under physiological conditions, DPP-4 rapidly degrades the incretin hormones including glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1

(GLP-1) [START_REF] Drucker | Incretin Action in the Pancreas: Potential Promise, Possible Perils, and Pathological Pitfalls[END_REF] , which have been shown to enhance glucose-dependent insulin secretion and inhibit glucagon secretion, which together may reduce hepatic gluconeogesis [START_REF] Nauck | Incretin-based therapies for type 2 diabetes mellitus: properties, functions, and clinical implications[END_REF] . Moreover, under physiologic concentrations, DPP-4 directly induced insulin resistance in adipocytes and skeletal muscle [START_REF] Lamers | Dipeptidyl Peptidase 4 Is a Novel Adipokine Potentially Linking Obesity to the Metabolic Syndrome[END_REF] Nicotinamide phosphoribosyltransferase (NAMPT)

Very recently, NAMPT, a key NAD + biosynthetic enzyme, which has two different forms, intra-and extracellular (iNAMPT and eNAMPT) in mammals, was found predisposed to secretion from adipocytes via deacetylation by the mammalian NAD + -dependent deacetylase SIRT1. Adipose tissue-specific NAMPT knockout and knockin mice show reciprocal changes in circulating eNAMPT, affecting the hypothalamic NAD + production, SIRT activity and neural activation accordingly [START_REF] Yoon | SIRT1-Mediated eNAMPT Secretion from Adipose Tissue Regulates Hypothalamic NAD+ and Function in Mice[END_REF] . The story provides an elegant example in which adipocyte-derived molecules could be transferred and exert their function in other organs.

BMP-4

BMP-4 is secreted by differentiated preadipocytes and adipocytes, with higher expression in larger adipocytes [START_REF] Gustafson | The WNT Inhibitor Dickkopf 1 and Bone Morphogenetic Protein 4 Rescue Adipogenesis in Hypertrophic Obesity in Humans[END_REF] . It has been recently shown that differentiated human adipocytes can promote adipogenesis via endogenous BMP-4 activation, a process that may be counter-balanced by the expression of the BMP-4 (and -7) inhibitor gremlin-1 [START_REF] Gustafson | The WNT Inhibitor Dickkopf 1 and Bone Morphogenetic Protein 4 Rescue Adipogenesis in Hypertrophic Obesity in Humans[END_REF] . In hypertrophic obesity, adipose precursor cells are resistant to BMP-4, which may contribute to the limited expandability of adipose tissue and, subsequently, to obesity-related metabolic diseases [START_REF] Gustafson | The WNT Inhibitor Dickkopf 1 and Bone Morphogenetic Protein 4 Rescue Adipogenesis in Hypertrophic Obesity in Humans[END_REF] .

Interestingly, adipose tissue has been very recently demonstrated as a major source of circulating microRNAs, and adipocyte-derived microRNAs could regulate gene expressions in liver in mice [START_REF] Thomou | Adipose-derived circulating miRNAs regulate gene expression in other tissues[END_REF] , indicating that, aside the classical view that most of the adipokines are proteins, other factors like microRNAs might be considered as novel adipokines. 2.4.

Origins of white adipocytes

Although adipose tissue development is poorly understood, the phenomenon that, in lipodystrophies, regionalized depot atrophy coexists with preservation and often expansion of other depots suggests that adipose tissue is an organ of multiple lineages [START_REF] Villarroya | Lipodystrophy associated with highly active anti-retroviral therapy for HIV infection: the adipocyte as a target of anti-retroviralinduced mitochondrial toxicity[END_REF] . In mice, it has been demonstrated that sWAT and vWAT form in an ordered and timed manner throughout embryogenesis and within the first few weeks of birth. sWAT begins to develop during embryogenesis and the compartment of progenitors is established for all the depots of sWAT before the first few days of life. Whereas, vWAT principally form postnatally: the perigonadal lineage forms approximately between postnatal day 3 and the second week of life. The mesenteric WAT adipose compartment completes its establishment of lineage between the second and third weeks of life. The retroperitoneal WAT is formed in-between these pre-and postnatal stages, and has a morphogenesis, texture, and histology that also seem intermediate [START_REF] Jiang | Independent stem cell lineages regulate adipose organogenesis and adipose homeostasis[END_REF] . Transcriptomic analysis of preadipocytes isolated from the SVF of various fat depots have revealed consistent differences in developmental gene profiles between vWAT and sWAT, indicating the possibility that sWAT and vWAT are of distinct origins [START_REF] Macotela | Intrinsic Differences in Adipocyte Precursor Cells From Different White Fat Depots[END_REF] .

Three critical events are necessary for the establishment and maintenance of functional adipocytes (Figure 9):

1) Determination of preadipocyte, commitment of multipotent progenitors into the adipocyte lineage.

2) Adipocyte differentiation, in which committed preadipocytes undergo a morphological and biochemical transition into mature adipocytes in response to appropriate stumuli.

3) Adipocyte maintenance, in which the cellular identity and functional properties if the terminally differentiated cells are maintained.

Adipocyte progenitor cells (APCs)

Pluripotent stem cells, residing in the vascular stroma of adipose tissue, are able to be recruited into preadipocytes, which when appropriately induced, undergo mitotic clonal expansion and then differentiate into adipocytes (Figure 9). Adipocyte progenitor cells (APCs) reside in the SVF and could be separated from non-adipose precursors (e.g., endothelial progenitors, immune cell populations) using early adipocyte progenitor cells markers (Lin-: CD29+;CD34+;SCA1+:CD24+) with the technology of fluorescence-activated cell sorting (FACS), which could successfully proliferate and differentiate into WAT in A-Zip lipodystrophic transgenic mice [START_REF] Rodeheffer | Identification of White Adipocyte Progenitor Cells In Vivo[END_REF] .

To investigate the architecture of the stromal vascular (SV) compartment, Wei T., et al, developed an SV particulate (SVP) isolation procedure designed to partially maintain the native SV structure while removing adipocytes that obscure visualization of the precursor location. In the SVPs, the majority of peroxisome proliferator-activated receptor γ (PPARγ)/GFP cells were arrayed in tubelike structures, which immunohistochemically expressed platelet endothelial cell adhesion molecule (PECAM) and mural cell markers: SMA (endogenous smooth muscle actin), PDGFRβ (platelet-derived growth factor receptor β),

indicating that these adipocyte progenitors reside in the mural cell compartment of the adipose vasculature [START_REF] Tang | White fat progenitor cells reside in the adipose vasculature[END_REF] .

The large C2H2 zinc-finger transcription factor, Zfp423, is a factor associated with preadipocyte commitment. Zfp423 GFP reporter mice show that Zfp423 distinguished adipogenic from inflammatory-like mural cells. Zfp423 GFP expression was found not only in mature adipocytes but also in a distinct subset of perivascular cells expressing mural cell marker PDGFRβ, supporting the hypothesis that preadipocytes resemble pericyte-like cells. Signal transducer and activator of transcription 3 (STAT3) was reported to be activated by Janus kinase 2 (JAK2) within 2h of adipogenesis induciotn on 3T3-L1 adipocytes, translocating from cytoplasm to the nucleus and regulating the transcription of C/EBPβ by binding the distal region of its promoter 108 (Figure 9).

PRDM16 is more enriched in sWAT compared to that in vWAT in mice, associated with its role in the development and function of beige cells. Ablation of PRDM16 caused a switch of sWAT to vWAT like phenotype, and impairment of thermogenic gene expression in response to cold exposure, indicating that PRDM16 pathway is necessary for the differentiation of brite adipocytes [START_REF] Cohen | Ablation of PRDM16 and beige adipose causes metabolic dysfunction and a subcutaneous to visceral fat switch[END_REF] . In human, the elevated levels of BMP4 in WAT correlated with a lean phenotype. Forced expression of BMP4 in mice induced a brown fatlike alterations in WAT through upregulation of mitochondrial biogenesis in association with increased energy expenditure and insulin sensitivity, which is mediated by the PGC1α/ p38/MAPK/ATF2 pathway 109 , indicating a potential role of BMP4 in the biogenesis of brite adipocytes (Figure 9).

Wnts are a family of paracrine and autocrine factors that regulate cell growth and cell fate. Signaling is initiated when Wnt ligands bind to transmembrane receptors of the Frizzled family, which signals through Dishevelled to inhibit the kinase activity of a complex containing glycogen synthase kinase 3 (GSK3), Axin, β-catenin, etc. This complex targets βcatenin for rapid degradation through phosphorylation. Thus, once hypophosphorylated due to Wnt signaling, β-catenin is stabilized and translocate to the nucleus where it binds the TCF/LEF family of transcription factors to regulate the expression of Wnt target genes. When

Wnt signaling was activated in 3T3-L1 or 3T3-F442A, adipogenesis was significantly impaired, with no alterations in the expressions of C/EBPδ and C/EBPβ, but decreased levels of C/EBPα and PPARγ. Consistently, over-expression of C/EBPα and PPARγ could partly rescue the impaired adipogenesis due to Wnt activation 110 (Figure 9).

Pref-1 is a transmembrane protein encoded by the pref-1/dlk1 gene and belongs to a family of EGF-like repeat-containing proteins that include Notch/Delta/Serrate, which are involved in cell fate determination.Interestingly, Pref-1 is highly expressed in 3T3-L1

preadipocytes, but almost abolished in adipocytes 111 . The extracellular domain of Pref-1 undergoes two proteolytic cleavage events that generate 50 and 25 kDa soluble products. By fusing Pref-1 to human immunoglobulin-γ constant region (Pref-1/hFc), transgenic mice expressing the Pref-1/hFc transgene under the aP2 promoter, showed reduced expression of adipocyte markers and adipokines, whereas the preadipocyte markers Pref-1 was increased.

Moreover, mice expressing the Pref-1/hFc transgene exclusively in liver under the control of the albumin promoter also showed a decrease in adipose mass and markers, suggesting an endocrine role of Pref-1 on adipogenesis 112 (Figure 9).

The seven Sirtuins in mammals (Sirt1-Sirt7), which are involved in the regulation of several essential cellular processes, also have important roles in adipogenesis. Sirtuin 1 (Sirt 1) is the ortholog of the yeast protein silent information regulator 2 (Sir2) and a nuclear NAD + -dependent class III histone deacetylase class III, and is a key component activated by CR. Sirt 1 partly mediated the inhibitory effects of CR on adiposity by attenuating adipogenesis through suppressing PPARγ by docking with its cofactors NCoR (nuclear receptor corepressor) and SMART (silencing mediator of retinoid and thyroid hormone receptors). Conversely, Sirt7 which is known to specifically deacetylate H3K18, is reported to promote adipogenesis by binding and repressing Sirt1 activity 113 . The forkhead transcription factor FOXO1 is an important downstream effector of insulin/PI3K/AKT signaling, which lead to its phosphorylation and thereby nuclear exclusion. Constitutively active FOXO1 attenuated adipogenesis, while dominant-negative FOXO1 restored adipogenesis of fibroblasts from insulin receptor deficient mice 114 . Interestingly, mammalian Sirt2 is localized mainly in the cytoplasm and is reported to be down regulated during adipogenesis, and its over-expression inhibits differentiation via enhancing insulin-stimulated phosphorylation of FOXO1 115 .

Alterations of WAT by aging

Over the last decades, the growing number of elderlies (aged 65 years and over) has become a considerable challenge to health and social care service provision and funding.

Moreover, the numbers of the very old, those aged 85 years and over, are set to double over the next 20 years 116 . Aging is defined as a decline in survival and fecundity with advancing age, which is caused by damage to macromolecules and tissues.

What are the consequences of aging on WAT?

In human, age per se is a strong risk factor for fat accumulation 117 . Morphologically, computational tomography scans in men and women revealed that, as age increases, sWAT decreases while vWAT increases 118 . Rodents also demonstrate the same trend to develop age-associated central obesity 119 . Consequently, the dramatic alterations in fat distributions and functions occurring during life, are linked with the increasing risks of the developments of various aging-associated diseases, such as diabetes, cognitive dysfunction and atherosclerosis 120 . Multi-isotope imaging mass spectrometry (MIMS) is an imaging method that enables multiplexed measurement of stable isotope tracers with subcellullar resolution, and utility of stable isotope tracers of DNA synthesis and DNL to measure cell birth and lipid turnover. It was demonstrated that there's an age-dependent decline in the plasticity of adipose tissue whih is characterized by impaired adipogenesis and adipocyte lipid turnover in subcutaneous fat, which might be mediated by a decline in insulin like growth factor-1

(IGF-1) in humans 121 . In addition, by measuring the 2 nd World Warnuclear bomb test-derived [START_REF] Christen | Increased glucose uptake in visceral versus subcutaneous adipose tissue revealed by PET imaging[END_REF] C in lipids accumulated in adipocytes (in Hiroshima), it was reported that during the average ten-year-life span of human adipocytes, TG were renewed six times. Adipocyte lipid turnover is strongly related to conditions with disturbed lipid metabolism 122 .

Therefore, in the following part, we will detail effects of aging on adipogenesis and lipid turnover, respectively.

Senescence of adipocyte progenitors

Compromised adipogenesis during aging contributed to the impaired ability of adipose tissue to store lipids, leading to FFAs spillover and ectopic lipid accumulation in liver or muscle 123 . Senescence of adipocyte progenitors mediates partly the impaired adipogenesis during aging.

Senescence of adipocytes progenitors is increased in aged adipose tissue, demonstratd with upregulation of senescence-associated markers: SA-β-galactosidase and p16/p53 expressions 124 .

The senescence burden could further accelerate age-related dysfucntions in fat via both intrinsic and autocrine manners 125 . For instance, senescent adipocyte progenitors would impede the adipogenesis of non-senescent cells, via the activation of the JAK/STAT and over production of the activin A protein 126 .

Growth hormone (GH) plays a central role in regulating mammalian growth, metabolic homeostasis and adiposity. Both mice and human who have excessive GH production have increased mortality rates compared to age-matched counterparts 127 . Ames and Snell dwarf mutant mice (Prop1 df and Pit1 dw , respectively) are the first GH-deficient genetic mutants to display extension of lifespan. Interestingly, deficiency of GH in Ames and Snell mice attenuated the age-related central accumulation of vWAT. Besides, the levels of senescent fat progenitors measured by p16 expressions or SA-β-galactosidase, were negatively associated with the levels of GH. Thus, attenuation of senescent burden in vWAT might mediate the life-extending effects of GH deficiency 128 .

Clearance of senescent cells confers counteractions to age-related irregulations in adipocytes. For example, genetic clearance of p16 positive cells delayed or attenuated the reduction of the volumes of adipocytes 129 . In addition, depletion of p53 ameliorated senescence like changes in Ay obese mice, associated with improved insulin resistance 130 .

Altered lipid turnover

As mentioned before, fat cells function to store cytotoxic lipids, for example FFAs into neutral LDs 131 . However, this lipid storage capacity of adipocytes is impaired by aging. In 26month-old B6D2F1 mice, the average size of vWAT adipocytes was dramatically smaller compared with those in the counterparts of 6-month-old animals, indicating of a disability of lipid handling for adipocytes with aging 132 . The situation could be further demonstrated by the fact that middle-aged mice of around 14 months exhibited greater susceptibility to the toxicity of high fat diet, gaining more weight and fat content compared to their 3-month-old littermate 133 . Furthermore, three days of high fat diet feeding specifically induced memory deficits and significant brain inflammation in the aged rats, possible owing the the spillover of FFAs in the brain in aged rats 134 .

How could these dysregulations be explained?

Reduced sensitivity to nutrient stimuli

Age can drive insulin resistance in adipocytes. Both basal and insulin stimulated lipogenesis are reduced in isolated adipocytes from humans and rats by aging 135 . In humans, glucose-mediated glucose disposal and glucose transport is decreased by 30-35% in the elderly (64 ± 4 years) compared to the nonelderly (32 ± 4 years) 136 .

The exact mechanisms regarding to age-related insulin resistance is not well understand, but might be related to the alteraed immune-environments in adipose tissue in aging. For example, an increase in the portion of regulatory T (Treg) cells is observed in vWAT of 11-month-old mice compared to 1-month-old mice. Both genetic and pharmacological depletion of Treg cells in fat help to improve metabolic state and insulin stimulated glucose uptake 137 . Besides, aging is associated with a chronic state of low-grade inflammation in WAT. This is likely due to the fact that vWAT is a major source of cytokines, responsible mostly for the up-regulation of circulating levels of inflammatory and procoagulant factors such as, thrombospondin-1, plasminogen activator inhibitors-1 and -2, (PAI-1/-2), in comparison to liver or kidney. Furthermore, levels of these genes exhibited an age-associated increase in vWAT 138 .

Impaired breakdrown and oxidation of LDs -Mitochondrial oxidation

In humans, proteomic analysis of adipocyte mitochondria reveals global downregulation of the oxidative phosphorylation system (OXPHOS) components by aging process 142 , indicating dysfunction of mitochondria. Dysfunction of mitochondria, through various aspects, contributes to reduced efficiency of lipid turnover in visceral adipocytes.

Concomitant with this notion, oxygen influx stimulated by a combination of succinate and octanoyl carnitine during mitochondrial respiration is around 30% lower in older visceral WAT in relative to young vWAT 132 .

In the early stage of aging, mitochondrial abnormalities could be responsible for adipocyte enlargement in middle-aged mice. It has been reported that middle-age promote hypoxia inducible factor 1α (HIF 1α) in adipocytes which, as a transcriptional factor, repressed cytochrome C oxidase 5b (COX5b) expression by binding to its proximal promoter.

Reduced COX5b then inhibits complex IV assembly and impaires fatty acid oxidation then resulting in adipocyte enlargement 143 . In addition, reduction in β-oxidation could also lead to increased rates of FFA efflux out of cells in order to avoid toxic FFA buildup in the cytoplasm 144 . Moreover, adipocyte β-oxidation is also important in maintaining body temperature, since adipocyte specific CPT2 knockout leads to cold intolerance in mice 145 .

This might be one of the reasons to the cold intolerance by aging.

Contribution of WAT to aging

Adipose Noticeably, not only the quantity but also the quality of fat mass matters. The epigenetic marker H3K4me3 is remodeled during aging and cellular senescence in mammals 157 . In paradox, the depletion of H3K4me3 methyltransferase (the COMPASS chromatin complex) in Caenorhabditis elegans (C. elegans) promotes lifespan as well as fat accumulation accounted by specific enrichment of mono-unsaturated fatty acids (MUFAs).

Moreover, dietary MUFAs are sufficient to extend lifespan 158 . These data suggest that not only the amount of fat but also the characteristics of the fat influence longevity. Indeed, the fatty acid composition of TG in visceral WAT is altered during aging in rats, with a marked decrease in shorter long-chain saturated fatty acids, whereas the levels of linoleic and oleic acids increase 159 . In addition, there are increases in the levels of total ether- Since the FOXO is downstream and inactivated by the insulin/insulin-like growth factor (IGF) signaling in diverse organisms, these data suggest that attenuation of certain signaling pathways in fat might be associated with life-span extension. In addition, in D.

melanogaster, limited activation of dFOXO in the fat body increases lifespan, while repressing the synthesis of insulin-like peptide dilp-2 in neurons which then inhibites endogenous insulin-dependent signaling in peripheral fat body 163 . These findings suggest that insulin/IGF pathway in the adipose tissue could contribute to the control of aging through both autonomous and non-autonomous manners.

This relationship has also been demonstrated in the mammals. Mouse animal models with reduced growth hormone (GH) and/or IGF-1 signaling have been shown to have an extended lifespan, the action of which is partly associated with a delayed increase in the ratio of visceral to subcutaneous fat, and most likely a reduced fat cell progenitor turnover 164 .

Likewise, reduced dietary methionine intake prolongs lifespan in male Fish 344 rats, with reduced serum levels of lipids and IGF-1 as well as reduction in vWAT and increased energy expenditure 165 . Cre-Lox specific adipocyte depletion of insulin receptor protects mice from age-associated obesity and glucose response impairment, accounted mostly by an increase in small adipocyte populations less than 75µm 166,167 . Moreover, inducible knockout of insulin receptor in mice of median life-span extends the life span of these animals by 3.5 months and is associated with dramatic reduction in adiposity without alterations in food intake 167 .

Interestingly, mice deficient for vasohibin-1, a protein originally isolated as an endotheliumderived angiogenesis inhibitor and a stress tolerance promoter in endothelial cells, have improved longevity. Although the exact molecular mechanisms remain elusive, the attenuated levels of IR and IRS-1 in WAT levels might contribute to the life extending effects of vasohibin-1 deficiency 168 .

There are also other molecules/mechanisms in WAT that contribute to life span. Sirt1

(Sirtuin 1) is a member of the NAD + -dependent deacetylase family that catalyze the removal of acetyl groups from protein substrates. Mammalian Sirt1 acts as an energy and stress sensor modulating metabolic responses to nutrient availability. Selective over-expression of wild type human Sirt1 (AWSM) in adipose tissue in mice significantly prevents agingassociated insulin resistance and improves systemic energy homeostasis as well as enhanced spontaneous locomotory activity, which could be attributed to the promotion of lipid metabolism in adipose tissue and muscle 169 . Increased mitochondrial oxidation in adipocytes also inhibits aging and diet associated-obesity in TFAM lx/lx / adiponectin-Cre mice. Indeed, TFAM (mitochondrial transcription factor A) depletion in adipocytes results in an increased electron transport chain flux and a greater fat glucose oxidation due to complex I and IV deficiency 170 . Conversely, in WAT, in vivo adipocyte specific knockout of acyl-CoA synthetase, which mediates conversion of FAs to acyl-CoAs, the limited step for FA metabolism, results in increased adipocyte volumes and cold intolerance in mice via an impaired β-oxidaition in WAT, which is a hallmark of aging 171 .

Additionally, female mice deficient for diacylglycerol acyltransferase 1 (DGAT1), an enzyme that catalyzes the synthesis of TG, are protected from age-related fat accumulation, tissue TG accumulation as well as inflammation in WAT, which is accompanied with both increased mean and maximal lifespan. Moreover, these mice exhibit several longevityassociated features, such as decreased levels of circulating IGF1 172 .

Finally, in 2014, it was reported that FAT10 knockout mice had an extended lifespan in both female and male mice, associated with prevention of the age-associated obesity as well as metabolic dysfunctions 173 . The HLA-F adjacent transcript 10 (FAT10) is a member of the ubiquitin-like gene family that alters protein function/stability through covalent ligation.

Novel insights from microRNAs

While previous studies mainly focus on the nutrient sensing pathways in fat to lifespan, it has recently been reported that the abundance of microRNAs in adipose tissue is an important determinant of longevity. The biogenetic enzyme for microRNAs: dicer is decreased in sWAT during aging. On the contrary, overexpression of this enzyme in the intestine of C. elegans, increases the lifespan as well as resistance to heat shock and oxidative stress 174 . MicroRNAs from fat can play important roles in both cell autonomous and non-autonomous manners. For instance, mir-8 knockout drosophila exhibits a smaller body size compared to control drosophila, which could be rescued by its conditional overexpression in fat, which then leads to derepression of USH, an inhibitor of PI3K p85a

subunit 175 . This result then raises the question why PI3K inhibition in fat can lead to systemic effects, highlighting the endocrinal role of adipose tissue. For example, improved circulating profile of adipokines, e.g., reduced levels of leptin, TNF , IL-6 or C-reactive protein might mediate the cardio-protective effects of intermittent fasting and caloric restriction in human 176 .

However, the precise molecular pathways that microRNAs in adipose tissue employ to achieve those benefits, remain to be largely unclear.

PART II: MicroRNAs in white adipose tissue Biogenesis of miRNAs starts with transcribing genes into large primary transcript (pri-miRNAs), which is 5' capped and 3' polyadenylated in structure. The transcription is typically mediated by RNA polymerase II 181,182 . The pri-miRNAs are then cleaved by a microprocessor complex, consisting of the type III RNase Drosha and RNA-binding protein DGCR8, into an around 85 nucleotides stem-loop structure named precursor miRNA (pre-miRNA). Following transportation by Ran/GTP/Exportin 5 complex from nucleus to cytoplasm, the pre-miRNAs are processed by the RNase III enzyme Dicer to an around 20-22-nucleotide miRNA/miRNA* duplex (note: * indicates the passenger strand, while the other complementary strand is referred as the mature or guide strand).

After the duplex is unwound, mature miRNAs are incorporated into a protein complex termed RNA-induced silencing complex (RISC) and guide RISC to target mRNAs (Figure 10) 177,178 . In many cases, miRNA-target interactions are mediated by the seed region, a 6-to 8-nucleotide-long fragment at the 5'-end of the miRNA that forms Watson-Crick pairs with the cognate target 183 . Inside RISC, once binding to their target miRNAs, miRNAs would result in translational repression if imperfect complementarity, or target mRNA degradation if perfect complementarirty 184 .

The biogenesis of miRNAs is under tight control at multiple levels, including the levels of miRNA transcription, processing by Drosha and Dicer, transportation, RISC binding and miRNA decay. For example, the KH-type splicing regulatory protein (KSRP), serves as a component of both Drosha and Dicer complexes to regulate the biogenesis of a subset of miRNAs in mammalian cells [START_REF] Abel | Adipose-selective targeting of the GLUT4 gene impairs insulin action in muscle and liver[END_REF] . 1.2.

Regulation of miRNAs expression in adipose tissue

Expression of miRNAs in adipose tissue could be regulated via several mechanisms.

With the next generation sequencing technology, it has been reported that for a majority of miRNAs in adipose tissue, genetic regulation of expression was independent of the expression of the mRNA from which this miRNA is transcribed. Moreover, the degree of genetic variation of miRNA expression was substantially less than that of the mRNA 186 .

However, expression of some miRNAs is highly correlated with the mRNA from which the miRNAs are transcripted from. For instance, mir-378 resides in the sense strand of the first intron of PGC-1β, which is a transcriptional co-activator that regulates mitochondrial biogenesis. Similar to PGC-1β, mir-378 was also identified as key regulatory component underlying the biogenesis of mitochondria in adipose tissue 187 .

On the other hand, expression of some miRNAs in adipose tissue could be regulated directly through alterations in their promoters. regarding these alterations remain to be investigated.

2.

MiRNAs in the formation of adipose tissue

MiRNAs in differentiation of adipocytes progenitors

Adipogenesis is under tight regulations of various miRNAs in both mice and humans.Adipocyte dicer depletion resulted in extreme low adiposity due to depleted WAT 193 .

Since the fat mass is mainly determined by the number and the volume of adipocytes, it indicates that the loss of certain miRNAs due to dicer deficiency could have a role in adipogenesis or lipid homeostasis in adipocytes which lead to a loss fat mass. Subsequently, numerous evidence sare surging concerning various miRNAs affecting adipogenesis, via targeting classic components involved in adipogenic program. Here, we will review reported individual miRNA that exhibits either pro-or anti-effects on adipogenesis which are listed in Table 2.



The transcriptomes of miRNAs in sWAT and vWAT differ from each other 219 .

MirRNAs enriched in sWAT are mainly related to adipogenesis and lipid metabolism.

For example, mir-155-5p, mir-143-5p, mir-27b-3p or mir-193b-3p and mir-365, all of which are summarized in (Table 2) above, are amongst those miRNAs enriched in sWAT.

In contrast, the vast majority of miRNAs enriched in vWAT are associated with inflammation. For example, mir-145 is attenuated in the omental adipose tissue of obese Aside from beiging, subcutaneous fat could also acquire a visceral-like feature under certain conditions. For instance, 24-h fasting induces "visceralization" of sWAT in young mice, possibly accounted by a reduction in PRDM16 due to the upregulation of mir-149-3p in the fat depot 230 . Therefore, it remains interesting to investigate whether mir-149-3p is a vWAT enriched miRNA.

Involvement in adipose tissue functions

MiRNAs expression in human adipose tissue is fat-depot specific and related to parameters of obesity, fat distribution, glucose metabolism and adipose tissue morphology 231 .



MiRNAs in lipolysis

In demand of nutrients deprivation, such as fasting or exercise, TG in adipocytes are hydrolyzed to release fatty acids to support other organs [START_REF] Duncan | Regulation of lipolysis in adipocytes[END_REF] .

KSRP is a multifunctional RNA-binding protein involved in the posttranscriptional regulation of gene expressions, and is also important for pri-miRNA processing 232 . KSRP deletion is associated with increased lipolysis in adipose tissue, accompanied with the impaired processing of pri-miR-145 into mature mir-145, which directly targets pro-lipolytic genes: CGI58 and FOXO1 233 . Mir-277 depletion results in severe impairment in lipid storage in female mosquitoes, associated with cytoplasmic retention of FOXO, arguing a role of mir-277 depletion in the promotion of lipolysis via FOXO 234 . Moreover, the expression of mir-124a is inversely correlated with the level of ATGL and its coactivator CGI-58 in various murine tissues such as adipose tissue and liver. In addition, ectopic expression of mir-124a leads to lipolysis inhibition which could be rescued by expressing mutant ATGL lacking its 3'UTR 235 .

In parallel, insulin signaling pathway is crucial for the various processes like glucose uptake and lipogenesis in adipocytes 236 . Abnormality of insulin signaling in obesity or over nutrition has been reported to be associated with irregularities of miRNAs levels 237 .



MiRNAs in insulin-stimulated glucose uptake in adipose tissue Some miRNAs are positively associated with insulin-stimulated glucose uptake.

For example, mir-26b is reduced in vWAT in obese rodents, human obesity and insulin resistant adipocytes, and is negatively correlated with BMI or HOMA-IR (homeostatic model assessment for insulin resistance). In vitro studies with differentiated human adipocytes suggest that mir-26b could promote insulin-stimulated glucose uptake via increasing GLUT4 translocation onto the plasma membrane via inhibition of the phosphatase and tensin homologue (PTEN), which is an antagonist of PI3K signaling 238 .

Some miRNAs are negatively associated with insulin-stimulated glucose uptake.

Mir

Release from adipose tissue

Besides being present and identified in tissues, miRNAs are also stable in the circulations with a large fraction of them found in exosomes 254 (50-200 nm vesicles that are released from mutivesicular bodies) 255 . Since adipocytes are now well appreciated as a type of cell with endocrine functions and could secrete hundreds of bio-molecules, it is temptating to investigate whether adipocytes are able to secrete miRNAs and these secreted miRNAs can crosstalk with other cells or organs. In this context, it has been clearly demonstrated that adipose tissue is able to release extracellular vesicles (EVs). Indeed, adipose tissue derived extracellular vesicles (ADEV) include exosomes that are released by multivesicular bodies fusing with the cell membrane and mcirovesicles (100-1000 nm vesicles originated by shedding of the plasma membrane) [256][257][258] . Surprisingly, adipose tissuespecific knockout of the miRNA-processing enzyme dicer (ADicer KO), results in a substantial decrease in most of the circulating exosomal miRNAs, which could be reversed by transplantation of adipose tissues from wild type mice, indicating that adipose tissue might even be a major source of systemic exosomal miRNAs [START_REF] Thomou | Adipose-derived circulating miRNAs regulate gene expression in other tissues[END_REF] .

 Adipocytes-derived miRNAs as biomarkers Exosomal features of miRNAs could also be proposed as novel biomarkers. Treatment with cAMP substantially results in an approximately 5-fold increase in exosomes release from human brown adipocytes, containing more mir-92a insides, indicating that in response to different stimuli, fat could sort distinct individual or spectrum of miRNAs into exosomes for secretion 259 . Furthermore, with the study from a large cohort of humans, it has been demonstrated that exosomal mir-92a inversely correlates with human BAT activity measured by 18F-FDG PET/CT, suggesting that mir-92a represents a potential serum biomarker for BAT activity 259 .



Adipocytes-derived miRNAs are functional

Then, aside from their presences in exosomes, are these miRNAs functional? Current literature supports this hypothesis.

Under physiological state, adipose tissue extracts are shown to be able to promote adipogenesis, suggesting that adipose tissue could produce bioactive factors to regulate adipogenesis via paracrine signals 260,261 . In vitro studies have found that adipose tissuederived exosomes-like vesicles could be internalized into adipose tissue-derived stem cells (ADSCs), and promote the adipogenesis of the cells via the pro-adipogenic miR-450a-5p which inhibits WSIP2 (WUS-interacting protein 2) expression 262 .

In an endocrine view, FGF21 (fibroblast growth factor-21), produced by liver and other tissues, is elevated in blood and liver in ADicer KO mice, but could be reduced by injection of wild type exosomes. Moreover, in vitro, exosomes isolated from ADicer KO exhibit a less inhibitory effect on FGF21-3'UTR luciferase activity compared to those isolated from wild-type mice [START_REF] Thomou | Adipose-derived circulating miRNAs regulate gene expression in other tissues[END_REF] . These data indicate that adipocytes-derived miRNAs regulate FGF21 expression in liver. Pathologically, in response to high fat diet, adipocyte-derived mir-155 can be uptaken by macropahges, leading to M1 macrophage polarization 263 . Another study also elegantly demonstrates that miRNAs can mediate crosstalk between adipocytes and peripherical cells. One of the side effects of rosiglitazone (RSG) treatment is cardiac hypertrophy 264,265 , which is also a same case in mice lacking PPARγ specifically in cardiomyocytes 264,265 . This phenomenon indicates that PPARγ activation in noncardiac tissues might contribute to the cardiac hypertrophy. Interestingly, Xi Feng et al reported that PPARγ activation in adipocytes by RSG increases the content of mir-200a in adipocytederived exosomes, which then causes mTOR activation and hypertrophy in cardiomyocytes 266 . Therefore, adipocyte-derived mir-200a might mediate the hypertrophic effects of RSG in heart.

Although it has been reported that exosomes might be secreted in a ceramidedependent manner 267 , the exact mechanisms underlying the regulation of exosomes release and miRNAs sorting into exosomes remain largely unknown and worthy of further investigation.

PART III: MiRNAs in aging 1.

Dysregulations of miRNAs in aging

Molecular changes during the aging process are crucial to better understand the mechanisms of age-related diseases, such as cancer, cardiovascular, neurogenegerative and immunological diseases as well as diabetes, etc. Specific molecules such as telomerase, p16 INK4A , mTOR, NF-κB1 and SIRT1 have been found to be involved in the aging process, and have been used as predictors of longevity [268][269][270][271] . Recently, mounting evidences have suggested that miRNAs are also crucial determinants of the longevity of the organisms. For instance, the preservation of biogenesis of miRNAs is shown to be vital for the life-extending effects of metaformin. By activation of AMPK pathway, metformin alters the subcellular localization of the RBPAU-rich element-binding factor 1 (AUF1), disrupting its interaction with Dicer1 and protects it from being degraded by AUF1 272 . Therefore, study of miRNAs' involvement in aging, might provide new insights and pharmaceutical targets for healthy aging.

Currently, plenty of miRNAs have identified to be involved in aging as well as agerelated diseases. Currently, considerable attention has been devoted to age-associated changes in individual miRNA, which later is demonstrated to be critical in the regulation of mammalian lifespan. For example, depletion of mir-150 increased the survival rate from 7.7% to 28.1% in 30-month-old male mice. The suppression of renal insulin/IGF/AKT activity and upregulation of hepatic ACC activity might partially be responsible for the improved lifespan of mir-150 deficiency 275 . Conversely, over-expression of lin-4 increased the lifespan of C.elegans, with the insulin-like signaling pathway proposed to be the underlying targets 276 . Similarly, in drosophila, loss of mir-14 resulted in a reduced lifespan and semi-lethality, possibly owing to its effects of increasing the levels of TG and diacylglycerol as well as to the increase of its possible targets of apoptotic effectors 277 . Gradual increases in the levels of mir-669c and mir-709 were observed in aged liver in mice, with their potential targets involving in detoxification activity and regeneration capacity function declined in old liver 293 . The Ames dwarf mouse is one of the long-lived mammalian modelwith a point mutation in the PROP1 gene, which can liveup to 70% longer than wild-type counterparts. In the liver of Ames dwarf mouse, mir-27a was markedly upregulated compared to wild type counterparts, which was associated with a reciprocal down-regulation of its putative targets involved in polyamine biosynthesis (ODC1) and spermidine synthase (SRM). These data indicate that mir-27a was a key post-transcriptional regulator of intermediate metabolism in liver which might contribute to the long-lived phenotype in the Ames dwarf mouse 294 .

NGS analysis of miRNAs in the whole blood of mice of different ages helped to reveal several age-altered ones, amongst which, six were predicted to be involved in non-fatty liver disease: mir-16-5p, mir-17-5p, mir-21a-5p, mir-30c-5p, mir-103-3p and mir-130a-3p.

Moreover, transfection of the six miRNAs into 18-month-old mice actually reduced the accumulation of age-related genes in liver, including mir-11a, p16 and mTOR, providing preliminary data that reversing age-associated miRNAs could be applied for treatments of age phenotype in the future 295 .

Age-related immune defects

Hematopoietic stem cells (HSC) homeostasis is disrupted under physiological stresses such as aging and periodic bacterial encounters, associated with declines in HSC number and quality. Chronic inflammation is demonstrated to be the major cause of the age-related decline in HSC functions. The absence of mir-146a triggered a chronic inflammatory state through pathways involving TRAF6/NF-ƘB/IL-6, which chronically could lead to severe pathologies, such as HSC exhaustion, bone marrow failure or myeloproliferative disease.

These data indicate that the single mir-146a is crucial for guarding the quality and longevity of HSC in mice 296 .

Macrophages are critical effector cells of the innate immune system. Aging has been demonstrated to drive a functional drift in macrophages in mice, including epigenomic alterations, impaired cholesterol metabolism and inability to inhibit pathological angiogenesis, which is proposed to contribute to the pathogenesis of various aging disorders, such as age-related macular degeneration (AMD). The group of Rajendra S. Apte has revealed that the initiation of mir-150 by aging in macrophages of diverse origins, such as spleen, bone marrow and peritoneal localization, induced a reprogramming of the macrophage transcriptome, with altered phospholipid and ceramide profiles. Moreover, mir-150 targeted stearoyl-CoA desaturase-2 (SCD2), dysregulating lipid metabolism and promoting pathologic angiogenesis. Finally, the upregulation of mir-150 in human peripheral blood mononuclear cells was associated with AMD 297 .

MiRNAs to senescence

Cellular senescence

How our cells are affected by aging? The answer might be that as our body turns aged, our cells turn senescent (Figure 11). Cellular senescence was formally describled more than 40 years ago as a limited proliferation ability of normal human cells in culture 298 . The irreversibility of the senescence arrest is tightly associated with the senescent cell's special chromatin architecture, the so-called senescence-associated heterochromatin foci (SAHF). It constitutes a hallmark of senescent cells that contain several common markers of transcriptionally repressed heterochromatin and are hypothesized to silence genes important for cell proliferation, in particular those regulated by the E2F/RB1 repressor complex 299 . Despite an irreversible cell cycle, senescent cells actively secrete a spectrum of proinflammatory cytokines, chemokines, matrix metalloproteinases and growth factors, which is termed the senescence-associated secretory phenotype (SASP) 300 . SASP might be one of the manners that senescence employs to promote aging. Interestingly, miRNAs have emerged as key regulators in the cellular senescence associated gene programs. Disruption of global miRNAs biogenesis by silencing of DGCR8, Dicer or Drosha could lead to a senescence-like state in human and mouse primary fibroblasts demonstrated by flatted morphology, increased SA-β-gal activity or formation of SAHF 305,306 . So, how miRNAs could affect senescence? In the following part, we are going to focus on the effects of miRNAs on the two major pathways involved in senescence. In the p53 pathway, signals including the ARF (also known as p14 in human and p19 in mice), production of the CDKN2A locus, increase p53 levels by sequestering MDM2, which facilitates the degradation and inactivation of p53.

In the RB pathway, stress signals indude INK4A, the product of the CDKN2A locus. INK4A inhibits CDKs that phosphorylate, and therefore inactivate, RB during the G1 phase of the cell cycle. Reviewed in 1 .

complex perturbed the timely execution of senescence 307 , suggesting that some miRNAs could potentially affect senescence via modifying the promoters of p16/p53 and thereby Specifically, mir-31, a novel target of histone deacetylase inhibitor (HDACi) in breast cancer cells, induces cellular senescence via repression of the polycomb group (PcG) protein BMI1. Mir-31 overexpression leads to γH2AX foci formation largely due to induction of mitochondrial ROS 322 . Although in kidney the molecular basis for aging is not well understood, it was reported that mir-335 and mir-34a were increased in aged rat kindney, corresponding with down regulation of their targets superoxide dismutase 2 (SOD2) and thioredoxin reductase 2 (Txnrd2), respectively, which are critical antioxidants located in mitochondria. In vitro experiments demonstrated that overexpression of these two miRNAs leaded to senescence of young mesangial cells via suppression of SOD2 and Txnrd2 associated with a concomitant increase in ROS. Conversely, inhibition of these two actors in aged mesangial cells inhibited senescence via upregulation of SOD2 and Txnrd2 with decreased ROS 323 .

MiRNAs in conserved aging

Various conserved pathways have been unvealed to be essential for aging process.

For example, age-related oxidative stress is associated with functional decline of cellular repairing mechanisms and contributes to the onset of age-asscoiated pathologies, such as cardiovascular diseases. The accumulation of ROS could induce expression of TGF-β, activating fibroblasts and collagen deposition, which is the initial step of fibrotic diseases 324,325 . In drosophila, Jun-N-terminal Kinase (JNK) pathway confers tolerance to oxidative stress and extends life span, which is mediated by the activation and nuclear translocation of FOXO, repressing insulin/IGF-signaling pathway 326 . In drosophila melanogasterm, a heteroallelic, hypomorphic genotype of mutant InR, which is homologous to mammalian insulin receptor, could yield up to 85% of extension in longevity in females and reduced late age-specific mortality in males 327 . Ribosomal S6 protein kinase 1 (S6K1) is a downstream component of the nutrient-responsive mTOR (mammalian target of rapamycin) signaling pathway. Deletion of S6K1 leads to lifespan extension and resistance to age-related pathologies such as bone, immune, motor dysfunction and loss of insulin sensitivity 328 .

Evidence indicates that age-related miRNAs are intimately associated with ageconserved pathways. For example, bioinformatic analysis through gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) suggest the target genes of deregulated miRNAs in aged left ventricular (mir-132, mir-222 and mir-182, etc.) are enriched in conserved pathways such as mTOR signaling pathway, FOXO signaling pathway and adipocytokine signaling pathway 329 . Therefore these data suggest that miRNAs might impact aging process through affecting conserved aging pathways.

Currently, caloric restriction (CR) is one of the most robust interventions shown to delay aging in diverse species. Pharmaceutically, the polyphenol reveratrol, an activator of sirtuins, has been proposed to promote healthy aging and extend lifespan 330 . Therefore, in the following part, we mainly focus on relations between miRNAs with CR as well as sirtuin families to undertand the participation of miRNAs in aging. Other miRNAs, such as mir-71 and mir-228 are also critical for the response to dietary restriction in C. elegans, which are essentially and positively regulated by pHA-4 (a FOXA homolog) and SKN-1 (a NRF-related transcription factor) 333 (Figure 13).

Secondly, in rodents, it was also reported that the inhibition of miRNAs biogenesis by dicer depletion in adipocytes mitigated the metabolic benefit of CR, which was due to impaired insulin signaling caused by mTORc1 hyperactivation. In addition, there was a shifted metabolism from oxidative metabolism to amino-acid utilization accounted by altered mitochondrial morphology 334 . In liver, expression of mir-125a-5p was down regulated while its targets STAT3, CASP2 and STARD13, were increased with age. 30% of CR would significantly reverse the expression of mir-125a-5p and its three targets, suggesting that mir-125a-5p contributes to the CR-mediated delay of aging liver 335 (Figure 13). In brain, the decline in cognitive robustness with aging could be delayed by CR via upregulation of anti-apoptotic regulatory proteins that may prevent neuronal death. Interestingly, CR decreased the age-linked expressions of mir-30e and mir-34a, as well as reversed the decreased ratio of BCL2 to BAX in the brain. In vitro 3' UTR reporter assay demonstrated that BCl2 is a direct target of mir-30e and mir-34a. Therefore, these data indicates that down regulation of mir-34a and mir-30e could mediate neuroprotective roles of CR in the brain through targeting apoptosis 336 (Figure 10). Although levels of certain miRNAs per se might not be modulated substantially by aging, their alterations in response to CR could mediate its pro-survival effects. For instance, down-regulation of mir-34a, -34e,-181a-1* delayed brain aging via upregulation of BCL-2 pro-survival pathway under CR 336 .

Thirdly, in primates, regression analysis revealed correlations between body weight, adiposity, and insulin sensitivity for about 10 of the CR-regulated known plasma miRNAs in rhesus monkeys. Sequence alignment and target identification for these 10 miRNAs identified a role in signaling downstream of the insulin receptor. Among them, the highly abundant mir-125a-5p in plasma correlated positively with adiposity and negatively with insulin sensitivity and was negatively regulated by CR 337 (Figure 13).

In sum, the knowledge of the category of "aging-related miRNAs" as well as the precise mechanisms underlying the role of miRNAs in aging is still at the preliminary stages.

miRNAs affecting sirtuin families

Mammalian sirtuins comprise a conserved protein family of seven members of NAD +dependent deacetylases or, in case of SIRT4, ADP-ribosyltransferases. SIRT3, SIRT4 and SIRT5 are exclusively found in the mitochondrium. Increased expression of Sirtuins lowers the risk of age-related diseases. Various miRNAs have been demonstrated to affect aging via sirtuin family proteins.

 miRNAs with SIRT1

In peripheral blood mononuclear cells (PBMC), age-related decrease of SIRT1 was found negatively correlated with the expression of mir-34a and mir-9, with both of the two functionally interacted with the 3' UTR of SIRT1, indicating that mir-9/34a might impact the longevity through SIRT1 338 . The relation between mir-34a and SIRT1 was further confirmed in the case of kallistain, an endogenous protein protecting against vascular injury by inhibiting oxidative stress and inflammation in hypertensive rats and enhancing the mobility and function of endothelial progenitor cells (EPCs). In vitro data supported that kallistain could block the TNFα-induced senescence in EPCs via inhibiting the synthesis of mir-34a and mir-21, which negatively targeting SIRT1 and eNOS. Furthermore, mutation of mir-34 abrogated the life-extending effects of kallistain in wild-type C.elegans under oxidative or heat stress in a SIRT1-dependent manner 339 . Moreover, age-related hearing loss, also known as presbycusis, is thought to be the irreversible loss of cochlear hair cells in the inner ear.

Upregulation of mir-34a was again demonstrated to contribute to the apopotosis of HEI-OCI cell and hair cell loss in mice through targeting SIRT1 which resulted in an increase in p53 acetylation, suggesting the mir-34a/SIRT1/p53 signaling might serve as a potential target for age-related hearing loss treatment 340 (Figure 14A).

 miRNAs with SIRT4

Conversely, Sirt4 level is upregulated in senescence, inversely correlated with mir-15b in photoaged human skin. Inhibition of mir-15b promotes mitochondrial ROS generation and reduces mitochondrial membrane potential as well as impacts the expression of components of SASP in a SIRT4-dependent mannar. These data indicate the mir-15b/SIRT4 axis mediated the stress-induced senescence associated with mitochondrial dysfunction and SASP regulation 341 (Figure 14B).

 miRNAs with SIRT6.

SIRT6 specifically deacetylases lysine 9 on histone H3 (H3K9Ac) or lysine 56 on histone H3 (H3K56Ac). In addition, SIRT6 is able to form a macromolecular complex with the DNA double-strand break repair factor DNA-dependent protein kinase (DNA-PK) to promote DNA double-strand break repair 342 . Physically, it interacts with poly (adenosine diphosphateribose) polymerase 1 (PARP1) and mono-ADP-ribosylates PARP1 on lysine residue 521, thereby stimulating PARP1 poly-ADP-ribosylase activity and enhancing double-strand break repair under oxidative stress 343 . SIRT6 knockout mouse cells exhibit genomic instability and hypersensitivity to DNA damage. Moreover, SIRT6 -/-mice have been shown to develop a severe degenerative phenotype similar to that of premature aging. On the contrary, overexpression of SIRT6 in mice could increase the life span of male mice 344 . SIRT6 level was reduced in human dermal fibroblasts derived from older subjects compared to that from young subjects, coincided with an augmentation in the level of mir-766. In vitro experiments

show that mir-766 post-transcriptionally regulated the expression of SIRT6, which could negatively regulate mir-766 transcription via a feedback regulatory loop through modulating acetylation levels at its promoter (Figure 14C). These data indicate that miRNAs have implications in the post-transcriptional regulation of SIRT6, which is vital for longevity 345 . PART IV: A novel method of culturing isolated adipocytes

Current in vitro models of adipocytes

The main functional unit inside adipose tissue are adipocytes, characterized by a unilocular lipid droplet and a peripherally located nucleus. Knowledge of adipocytes is fundamental to enhance our understanding of adipose tissue physiology/pathology. As early as in 1964, Rodbell M. firstly isolated mature adipocytes from adipose tissue with collagenase digestion 346 . However, turning to literature, it is surprising that currently there is no effective system to maintain mature adipocytes for in vitro culture, which greatly limits our understanding of the fat cells. Then, why adipocytes could not been cultured in vitro routinely? Because of engaging in lipid storage, adipocytes are highly buoyant and will float on top of cell culture media, which will then result in malnutrition and osmotic stress. So, currently what are the models for us to study adipocytes in vitro?

1.1.

Two-dimensional (2D) adipogenesis of preadipocytes

Currently, in vitro culture of adipocytes is perfomed on generic 2D polystyrene.

Cellular model systems including pluripotent fibroblasts such as 10T1/2, BALB/c-3T3 and RCJ3.1, as well as fibroblast-like preadipocytes that are committed to differentiate into adipocytes such as 3T3-L1, 3T3-F422A, 1246, and 30A5, are widely used to investigate adipogenesis and functions of adipocytes. Among them, 3T3-F442A and 3T3-L1 are the two most extensively characterized and used preadipocyte cell lines. Both of these cell lines are derived from disaggregated 17-to 19-day old Swiss 3T3 mouse embryos 347 . However, dislike mature adipocytes which contain a single lipid droplet, in vitro differentiated adipocytes are characterized by multiple lipid droplets and accompanied undifferentiated preadipcoytes (Figure 13). Moreover, it has been reported that they could only produce 1-2 % of leptin of that of mature adipocytes 348 . Indeed, these cell lines are immortalized aneuploid cells whose genetic regulation may be distorted 349 . Beside the use of preadipocytes from immortalized cell lines, primary culture of the isolated stromal vascular fractions (SVFs) from adipose tissue is also widely used to study adipocytes in vitro (Figure 14). Adipogenesis of SVFs is induced by hormone cocktails stimulation including insulin, 3-isobutyl-1-methylxantine (IBMX), dexamethasone, etc 124 .

Similarly, SVFs would differentiate into multiple lipid droplets. that the 3D culture of mature adipocytes from the preadipocytes with scaffolds, allow the differentiated adipocytes acquired spheroid shapes and form muticellular aggregates.

Moreover, compared to 2D culture, the sizes as well as the viablility of mature adipocytes from 3D culture significantly are increased 357 (Figure 15). Unfortunately, porous materials often exceed the acceptable scaffold rigidity and preadipocytes embedded directly in hydrogel may remain functionally impaired. In another way, to mimic extracellular environment of adipocytes in vivo, which is composed of preadipocytes, adipocytes, interstitial cells and a microvascular system entwined with collagen-rich ECM, the conjugation of a genetically engineered elastin-like polypeptide (ELP) and a synthetic polymer, polyethyleneimine (PEI) (ELP-PEI) was coated on classical polystyrene plates, allowing the formation of spheroids of 3T3-L1 adipocyte aggregates with 72 hour after seeding 358 (Figure 16). These spheroids are cellular aggregates allowing cell interactions through junctional complexes.

First attempt to culture isolated adipocytes

Then, are there attempts having been made to culture isolated adipocytes directly?

The answer is sure.

In 1986, Hajime Suihara et al firstly reported the so-called "ceiling culture" of fat cells.

The authors took advantage of the buoyancy of these cells, allowing them to float and attach on the upper surface of a flask, which was previously fully-filled with medium and was airfree. After firm attachment, the flask was then placed upside down to allow regular observation. However, the author observed dedifferentiation of the cultured adipocyte on the 4 th day, which would continue so that the previous unilocular cells were finally replaced by multilocular fat cells or fibroblast-like fat cells, which in turn proliferated extensively, indicating that cultured adipocyte gradually lose their identity 359 (Figure 17).

Then, in 2000, HH Zhang et al tried to modify the "ceiling culture" by culturing adipocytes underneath a floating glass surface in wells of 6-well plate, so that their methods could be employed for fluorescence microscopy. However, there are still uncertainties since adipocytes would escape from the periphery of the glass slide, and it is quite unpractical because culture medium could not be changed within 10 to 14 days in order to ensure their attachment onto the covering glass. Similarily, the unilocular identity of fat cells could not be maintained and adipocytes were fragmented, which they mistakenly taken as budding of a daughter cells 360 . Besides 2D culture of isolated adipocytes, Hajime Sugihara et al, also tried to culture fat cells in 3D with collagen gel matrix, in which they embedded fat cells in 2mm in depth collagen bed to sequester the fat cell from floating. Unfortunately, the dedifferentiation process would still take place within one week of culture 361 .

Due to the dedifferentiation of these cultured adipocytes, the method of "ceiling culture" or 3D culture with collagen could therefore not be prevailed. Then why attenched adipocytes would undergo dramatic dedifferentiation? Unfortunately, there's no clear answer to this phenomenon. However, it might be related to the factor that white adipocytes nowadays have been demonstrated with plenty of plasticity. Despite their committed status, mature adipocytes maintain a siginificant expression of genes involved in stem cell pathways. Very interestingly, it has been reported that cytoplasmic gap can form in adipocytes at the early stage of "ceiling culture" and large LDs would be released directly outside adipocytes, which is termed liposecretion 362 .

Questions:

Although recent studies suggest that adipocyte dicer is decreased in aged mice and is involved in the determination of longevity in C. elegans and mice, the underlying mechanisms remain largely unknown 363 . Moreover, previous studies concerning the effects of adipocyte dicer are mostly obtained with aP2-dicer or adiponectin-dicer mice neglecting the influence of developmental defects in these models. In addition, more and more studies suggest that beside the classical drosha/dicer pathway, there are non-canonical pathways producing miRNAs in vivo, suggesting that the protein dicer per se might have a role in the control of longevity. In this way, the protein dicer per se in adipocytes might be proposed as a pharmaceutical target in the promotion of healthy aging. Based on these backgrounds, two aspects of age-associated dicer loss could be considered:

1) What are the consequences of adipocyte dicer deficiency in aging process?

2) Are there age-related microRNAs in adipocytes that might positively or negatively participate in age-related process?

Therefore, during my PhD, we develop different strategies to:

I.
confirm that adipocyte dicer (miRNAs) affects the physiology of adipose tissue or pheripheral organs. To that aim, we used the inducible dicer knockout mice model in which the floxed dicer allele is specifically turn down with the adiponectin-Cre which is under the control of ERT2 receptors and could be activated by tamoxifen treatment.

II.

better understand the mechanism concerning dicer alterations in adipocytes.

III.

screen by microarray the potentially interesting age-related adipocyte miRNA and validate by qPCR.

IV. assess the role of a selected age-related adipocyte miRNA in aging of adipose tissue.

B. Adipocyte dicer deficiency triggered a waste of perigonadal fat

To investigate the role of adipocyte dicer (miRNAs) in the physiology of adipose tissue per se, we characterized the body weight of the mice, the body fat proportion by EchoMRI as well as the sizes of adipocytes in subcutaneous and perigonadal adipose tissue by H&E staining at the end of the protocol.

Firstly, adipocyte dicer deficiency did not significantly alter the body weight of mice in this protocol (Figure 2A). However, adipocyte dicer deficiency significantly reduced the body fat proportion (Figure 2B). In agreement, the ratio of perigonadal fat to body weight was significantly reduced by over 50% in comparison to control group (Figure 2C), whereas the ratio of subcutaneous fat to body weight was unaltered (Figure 2D). Then, the result of H&E staining revealed that the average cell size of perigonadal adipocytes was reduced by near 50% by dicer deficiency, while deficiency of dicer had a trend to reduce the sizes of subcutaneous adipocytes (Figure 2E, 2F and 2G). Therefore, these data indicated that adipocyte dicer deficiency resulted in a loss of perigonadal fat mass accounted partly by reduced volumes of adipocytes.

Molecular studies were then performed to to confirm these data as well as to investigate the underlying mechanisms. RT-qPCR results demonstrated that dicer deficiency led to dramatic reductions in Leptin, Fsp27, two genes known to be positively associated with adipocyte sizes (Figure 2H). Moreover, metabolic functional genes such as Pparγ 1/2 , Glut4, Hsl, Atgl and Pepck were also markedly decreased following dicer deficiency, indicating a possibility of attenuated adipocyte functions such as glucose transport and lipolysis (Figure 2H and2I). The lipid droplet (LD) is stabilized by a surrounding monolayer of phospholipids with adherent proteins, in particular perilipin 1 (PLIN1), which could act dually to both suppress basal lipolysis and augment catecholamine stimulated lipolysis 362,363 . The result of immunofluorescent staining exhibited that PLIN1 was markedly reduced with dicer loss in perigonadal adipocytes (Figure 2G).

Therefore, these results indicated that dicer abundance is indispensible for the maintenance of adipocytes identities in perigonadal adipose tissue. Therefore, in the following part, we mainly focused on the study of this adipose tissue.

C. Possible mechanisms underlying the effects of adipocyte dicer deficiency

As adipocyte size is mainly determined by its lipid contents, so we went on to investigate whether adipocyte lipid metabolism was affected by dicer deficiceny, and we then turned our attention to mitochondria.

Surprisingly, PGC1α, one of the transcriptional factors important in the transcription of genes involved in mitochondrial biogenesis and functions, was significantly upregulated in perigonadal adipocytes due to dicer deficiency at both the level of mRNA and protein (Figure 3A and 3B). In addition, OXPHOS complex II/IV were significantly upregulated in perigonadal adipocytes due to dicer deficiency, and there was a strong trend for complex III to be increased (P=0.606) (Figure 3C). Although mitochondria recently have been suggested to be able to consume extra nutrients in subcutaneous adipose tissue 365 , it is also a major source of reactive oxidative species (ROS) 366 . Therefore, to better evaluate the state of mitochondria, antioxidant systems were checked. Interestingly, mRNA levels of antioxidants such as Catalase and Sodium oxide dismutase (Sod) were significantly reduced in perigonadal adipose tissue due to dicer deficiency (Figure 3D). Therefore, adipocyte dicer deficiency might induce a combination of mitochondrial activation and reduction in detoxification.

According to literature, the transcriptional factor FOXO families are able to promote the transcription of antioxidant genes, like Catalase and Sod 367 , but also negatively regulate the transcription of genes important for mitochondrial functions 368 . The phosphorylation of FOXO families would lead to their relocation from nucleus to cytoplasma, thereby inhibiting their transcriptional activities 369 . Interestingly, the result of immunoblot showed that the phosphorylation of both FOXO1 and FOXO3 was increased in perigonadal adipose tissue due to dicer deficiency (Figure 3F), suggesting that adipocyte dicer deficiency might lead to inactivation of FOXO1 and FOXO3. Additionally, reduction in autophagy, one of downstream effector of FOXOs 370 , was also possibly inhibited by adipocyte dicer deficiency demonstrated with Bnip3 reduction (Figure 3G), further in favor the possibility that adipocyte dicer deficiency reduced FOXOs activitions. Therefore, with these preliminary data, we assurmed that adipocyte dicer deficiency resulted in a combination of mitochondria activation and detoxification reduction possibly mediated by FOXO1 and FOXO3a inactivation.

D. Altered systemic energy homeostasis with adipocyte dicer deficiency

To better understand whether adipocyte dicer deficiency would lead to systemic effects, we characterized the plasma levels of various metabolites and hormones (Table 1).

Although reaching no significance, there was a trend for plasma lipids such as TG, NEFAs and cholesterol to be decreased due to dicer deficiency in adipocytes. Of note, the plasma level of glycerol, a byproduct of lipolysis, was significantly increased because of dicer deficiency in adipocytes, indicating either increased basal lipolysis or inability of adipocytes to recycle glycerol for the synthesis of TG. The results correlated with the reduced Pepck and PLIN1

expressions in adipose tissue from iADicer KO mice compared to that in control mice (Figure 2H and 2G). Surprisingly, plasma insulin exhibited a strong trend to increase to around 2.5 folds (3.82 ± 0.90 vs. 1.53 ± 0.54), indicating iADicer KO mice might have a higher risk of developing systemic insulin resistance (glycemia is not affected). The clinical variables for liver function: plasma ALT (alanine aminotransferase) and AST (aspartate aminotransferase) levels were comparable between the two groups of mice. In sum, adipocyte dicer deficiency led to alterations in systemic metabolism.

E. Abnormalities of liver associated with adipocyte dicer deficiency

To be more convincing that adipocyte dicer affected systemic metabolism, we went on to characterize liver, which is an important organ influencing systemic metabolism.

Interestingly, the result of H&E counter staining exhibited that white dots appeared in phase contract microscope illustrating lipid overfilling in livers from iADicer KO mice treated with tamoxifen, suggesting that adipocyte dicer deficiency possibly result in lipid overload in liver (Figure 4A). To confirm this result and better understand the source of lipids in liver from iADicer KO mice, RT-qPCR was performed to detect the genes involved in lipid metabolism. Fat-specific protein 27 (FSP27), a lipid droplet-associated protein that promotes lipid droplet growth and TG storage was induced from iADicer KO mice compared to that from from control mice (Figure 3B), supporting ectopic lipid development in liver with adipocyte dicer deficiency. Moreover, genes involved in DNL pathways such as PAPRγ 1/2 , FAS, and CD36 also significantly increased in liver from iADicer KO mice compared to that from control mice (Figure 3B). In addition, immunoblot results of PAPRγ 1/2 and FAS further confirmed that DNL pathway was activated in liver consecutively to adipocyte dicer deficiency (Figure 3C). These results indicated that adipocyte dicer deficiency resulted in ectopic lipids in liver associated with upregulation of DNL pathway.

Since the accumulation of lipids is proposed to be crucial in the activation of hepatic stellate cells (HSCs) and the development of fibrosis in liver 371 , we went on to investigate whether the ectopic lipids in liver resulted from adipocyte dicer deficiency was associated with fibrosis in the liver. Then, the result of semi-quantification of sirius red stained area suggested that fibrosis was indeed increased in liver from iADicer KO mice compared to control mice (Figure 3D). Furthermore, the results of gene markers involved in extracellular matrix (ECM) depositions had a trend to be increased by adipocyte dicer deficiency (Figure 3E). Moreover, genes involved in inflammatory pathways such as MCP1 and TGF β receptor 1 (TGFβ r1) were significantly increased in mice from the group of iADicer KO mice (Figure 3E).

Since MCP1 is an important chemokine that triggers infiltrations of immune cells, in particular macrophages, it suggested that increased infiltrations of macrophages might lead to the activation of TGF β pathway and accumulation of ECM and thereby fibrosis in liver by dicer deficiency in adipocytes.

F. Regulation of dicer level in adipocytes

According to our previous data that adipocyte dicer deficiency reduced the lipid storage capability of adipocytes as well as induced abnormalities in systemic metabolism, like ectopic lipid overload and fibrosis in liver. Therefore, adipocyte dicer might have a positive role in the prevention of age-related complications, and adipocyte dicer might be proposed as a target to delay the onset of age-related complications. So, to know how this protein is regulated in adipocytes will be helpful to develop pharmaceutical compounds targeting it. Since adipocytes are sensitive to nutrient availability, we then wondered whether nutrient availability would affect dicer in adipocytes.

Interestingly, 24h fasting significantly increased dicer mRNA level in perigonadal adipose tissue in wildtype mice (Figure 5A). Then, in vitro, overnight deprivation of glucose in the culture media markedly increased dicer protein level in 3T3-F442A adipocytes (Figure 5B). Additionally, fetal bovine serum (FBS) contents in the culture media negatively regulated dicer protein level in 3T3-F442A adipocytes (Figure 5C). Therefore, negative energy state can positively regulate dicer level in adipose tissue and adipocytes.

Since nutrient deprivation is known to activate the lipolysis process in adipocytes by β-adrenergic signaling pathway [START_REF] Duncan | Regulation of lipolysis in adipocytes[END_REF] . Therefore, we went on to see if dicer upregulation under nutrient deprivation is downstream of activation of β-adrenergic signaling pathway, by stimulation of adipocytes with 10 -6 isoproterenol overnight. However, immunoblot result indicated that isoproterenol did not exhibit a similar upregulation effect on dicer as starvation (Figure 5D). Therefore, activation of β-adrenergic signaling pathway might not affect dicer level in adipocytes.

To continue to explore the underlying mechanisms how nutrient availability regulates dicer in adipocytes, we went on to see whether classical cellular nutrient sensor AMPactivated protein kinase (AMPK) is involved. Under nutrient deprivation, AMPK can be activated by the increased ratio of AMP/ATP, ultimately turning up ATP-generating catabolic processes 372 . In line with the data that fasting or glucose/serum deprivation upregulated dicer, treatment of 1mM 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), the activator of AMPK, possibly induced dicer expression in 3T3-F442A adipocytes (N=2) (Figure 5E). In contrast, mild inhibition of AMPK with 20µM Compound C (CC) seemed to reduce the basal level of dicer while not affecting the effect of serum deprivation on dicer level (N=2) (Figure 5F). But strong inhibition of AMPK with 50µM CC exhibited no obvious effect on dicer expression in adipocyts (N=2) (Figure 5F).

In sum, AMPK might positively regulate dicer expression in adipocytes. However, there might be other unknown factors participating in the regulation of adipocyte dicer expression.

G. Selection of age-related microRNAs in adipocytes

In an effort to isolate potential aging-associated miRNA targets in adipocytes, previous microRNA-based microarray experiments in mouse visceral adipose tissue have been performed in the laboratory (by By Karine Tréguer and Jason S. Iacovoni). Wild type mice of 3-, 12-and 24-month-old were used. Through selection, eight potential candidates had been isolated including mir-1949, mir-672, mir-125b*, mir-805, mir-494, mir-143 and mir-133a. Therefore to continue, RT-qPCR was perfomed to further validate the 8 candidates from microarray.

Validation of miRNAs candidates from microarray

Surprisingly, among the candidates, only mir-1949 was found significantly upregulated in vWAT from 20-month-old mice compared young (6-month-old) control mice (Figure 6A and 6B). Therefore, to be sure of the result, another set of experiment was performed. Similarly, mir-1949 was found dramatically upregulated in both whole vWAT and sWAT collected from 24-month-old mice and compared to 3-month-old mice (Figure 6C and 6D). Moreover, the same result was obtained in isolated adipocytes from vWATand sWAT, (Figure 6C and 6D). Finally, its levels were relatively less modified in SVF isolated from vWAT and sWAT (Figure 6C and 6D). Therefore, we concluded that mir-1949 might be proposed as an age-related miRNA in adipocytes.

Since miRNAs are also stable in the circulation and could positively be secreted by cells, we went on to investigate whether there was also an increased secretion of mir-1949 from adipocytes by aging. To do so, vWAT was isolated and cut into small pieces and then cultured in vitro. Conditioned medium (CM) was collected at the time points of 0.5h and 2h.

Mir-1949 levels in CM were detected by qPCR. Interestingly, mir-1949 was elevated in CM collected from vWAT of 24-month-old mice compared to 3-month-old mice, indicating that aging might possibly increase the secretion of mir-1949 from adipose tissue (Figure 6E). To further confirm that aging promotes the upregulation and possibly the secretion of mir-1949 from adipocytes, we went on to investigate whether adipocyte senescence would also affect Data represent the mean ± SEM, *P<0.05, **P<0.01.

Possible actions of age-related mir-1949 on adipocytes

To investigate the possible actions of this age-related miRNA on adipocytes, in vitro experiments with miRNA-mimic transfection was performed in 3T3-F442A adipocytes. RT-qPCR result indicated that with transfection of 10nM mir-1949 mimic, its level was dramatically upregulated (Figure 7A). Classical functions of adipocytes, like glucose transport and lipolysis were not significantly affected by upregulation of mir-1949 (data not shown).

However, in silico analysis with public database like Target Scan 7.0, suggested that some of genes involved in mitochondria functions were proposed to be potential targets of mir-1949.

Therefore, we went back to in vitro expriments to investigate whether this miRNA would affect mitochondria in adipocytes. Interestingly, RT-qPCR results suggested that most of the mRNA levels were only slightly modified by the elevation of mir-1949, with Pgc1α was significantly decreased (Figure 7B). Then, the result of immunoblots of mitochondrial OXPHOS revealed that mir-1949 upregulation significantly decreased the level of complex II (Figure 7C), In agreement, functional analysis showed that the ability of oxygen consumption In sum, with this preliminary in vitro results, we hypothized that mir-1949 might be able to modulate the lipid storage capacity of adipocytes via negatively affecting mitochondrial complex II acitivies and positively affecting adipogenesis process.

Nevertheless, this hypothesis abosultely needs to be further validated by in vivo experiments. With the use of the inducible adipocyte dicer knockout transgenic mice, we demonstrated that adipocyte dicer deficieny promoted the onset of some age-related complications. For example, adipocyte dicer deficiency significantly reduced perigonadal adipocytes' lipid storage capability, one of the age-associated dysfunctions in aging 380 .

Moreover, the result that nutrient restriction, a process promoting healthy aging, positively regulates dicer level in adipocytes, further supports that there is a concered aging pathway in adipocytes involving dicer. Mechanistically, molecules like FOXOs, mitochondrial ROS and detoxification system might be involved in the actions of dicer in adipocytes in aging (Figure 8B).

A. Adipocyte dicer abundance has a positive role in the prevention of age-assoicated complications.

The dramatic reductions in classical white adipocytes markers, like Leptin and Glut 4, HSL, as well as the reduction in LD coating proteins like Fsp27 and PLIN1, suggesting that dicer is indispensible for the maintanence of white adipocyte identity as well as the homeostasis of LD inside white adipocytes.

Surprisingly, PGC1α, one of the transcriptional factors important for the mitochondrial biogenesis and functions was increased due to dicer deficiency in perigonadal adipocytes. In addition, mitochondrial OXPHOS electron transport chain protein complexes were significantly increased or had a trend to be increased. These data indicated that there was an activation of mitochondria due to dicer loss in perigonadal adipocytes.

Activation of mitochondria has recently been proposed to have metabolic benefits in subucutaneous adipose tissue 381 . However, the role of mitochondria in perigonadal adipocytes is relative less well understand. Since mitochondrial respiration is a major source of ROS in cells, therefore, to evaluate the state of mitochondria more comprehensively, it remains necessary to detect the ROS levels after dicer reduction in adipocytes. Surprisingly, there was a down regulation of antioxidants indicated by reduced mRNA levels of Catalase and Sod, indicating that there might be insufficiency in detoxification system. Moreover, the results that there was a trend for senescence markers like p16 and pP53 (Figure 8A) to be increased due to adipocyte dicer deficiency, further supported the possibility that the combination of mitochondrial activation and detoxification reduction leads to DNA damage in adipcoytes by dicer deficiency.

According to literature, the transcriptional factor FOXOs has been reported to be able to promote the transcription of antioxidant genes like Sod and Catalase 367 , whereas inhibiting the transcription of genes involved in mitochondrial biogenesis and functions 368 .

The transcriptional activity of FOXOs can be regulated by acetylation and phosphorylation 382 .

For example, phosphorylatin of FOXOs will lead its nuclear exclusion into cytoplasma, thereby inhibiting their transcriptional activies 367 . Interestingly, the result that adipocyte Therefore, to illustrate well the molecular mechanisms of adipocyte dicer in aging, On one hand, to further confirm the involvement of detoxidication system, several questions need to be addressed more clearly.

1. Firstly, adipocyte dicer level negatively associates with cellular ROS levels?

The answer to this question is probably crucial to determine whether adipcoyte dicer will induce oxidative stress and thereby DNA damage. Therefore, to resolve this question, in vivo, we propose to measure cellular ROS levels in adipocytes in control and iADicer KO mice. In vitro, we propose to measure cellular ROS levels in adipocytes in normal 3T3-F442A adipocytes or in 3T3-FF442A adipocytes overexpressising dominant-negative dicer.

2. Secondly, adipocyte dicer loss reduces the enzymatic activities of SOD and CATALASE?

We've already demonstrated that the mRNA levels of antioxidant proteins: Sod and Catalase were dramatically reduced due to adipocyte dicer deficiency.

Therefore, to be more convincing that adipocyte dicer deficiency induced detoxidication reduction, the enzymatic activities of these antioxidants need to be well addressed in adipocytes in control and iADicer KO mice.

3. Thirdly, antioxidants will delay the age-related complications caused by adipocyte dicer deficiency?

To well support the hypothesis that oxidative stress partly contribute to the accelerated onset of age-related complications by adipocyte dicer deficiency, it's worthy of investigation whether the supplementation of antioxidants (vitamin C, N-acetylcysteine, and Trolox) to iADicer KO mice, would help to resuce the accelerated age-associated complications in iADicer KO mice.

On the other hand, the involvement of FOXO1/FOXO3a in the role of adipocyte dicer in aging process needs to be further clarified.

1. Firstly, our present data suggests that dicer deficiency induces increased phosphorylation FOXO1/FOXO3a, indicating their nuclear exclusion into cytoplasma. Therefore, to better support this result, their cellular compartmental distributions need to be determined by immunohistochemistry or by protein extrations from nuclear and cytoplasma in adipocytes from control and iADicer KO mice.

2. Secondly, the transcriptional activities of FOXO1 and FOXO3a also should be determined in adipocytes from control and iADicer KO mice. Aging is associated with the severity and poor prognosis of various liver diseases including nonalcoholic fatty liver disease (NASH) and firbosis. For example, aged populations (those aged over 65 years), was reported to have a higher NASH prevalence rate than their younger counter parts (56 vs. 72%, P=0.02), and also displayed a higher rate of liver fibrosis 378 . Interestingly, by using iADicer KO mice, our results propose that adipocyte dicer deficiency might accelerate liver aging, through promoting lipid accumulation and fibrosis in liver.

There are 2 possible mechanisms might be involved.

Firstly, there's a crosstalk between adipocytes and liver in aging through adipocytesderived miRNAs.

1. To test this possiblility, we can make use of ZsGreen lx/lx / Adiponectin-CreER T2 mice, in which adipocyte-derived proteins will have GFP signal induced by tamxofien treatment (Figure 9). By co-immunostaining of GFP and exosomes markers like CD63 in liver, it can help to determine whether liver can uptake adipocytederived exosomes under physiological states.

2. In addition, through comparision of miRNA profile in exosomes extracted from circulations as well as miRNA profile in liver in control and iADicer KO mice by microarray, it might help to determine if adipocyte dicer deficiency would lead to specific miRNA reduction in both circulating exosomes as well as in liver, which is needed for the maintenance of liver integrity and function in aging.

Secondly, inefficiency of adipocytes' capability of lipid storage leads to ectopic lipids in pheripheral liver.

To test this possibility, the functions of adipose tissue like lipolysis, glucose transport and de novo lipogenesis need to be well studied in control and iADicer KO mice. In our above data, we've demonstrated that adipocyte dicer deficiency would promote the onset of some age-related complications, such as impaired lipid storage and systemic metabolic dysfunctions. However, in that part, we could not determine whether these are direct consequences of the dicer protein per se, or indirect consequence of some dicer produced miRNAs.

MATERIALS AND METHODS

Reagents and cell culture

The 3T3-F442A cell line (ATCC) was maintained in Dulbecco's modified Eagle's medium (DMEM) medium supplemented with 10% of fetal bovine serum (FBS, Gibco®).

Differentiation was induced by incubating confluent cells in differentiation medium (DMEM supplemented with 10% FBS plus 50nM insulin for up to 14 days. Cells were routinely cultured in 5% CO2 at 37°C. SiRNA for Dicer were purchased from Santa Cruz.

AMPK activator AICAR and L-Ascorbic acid were obtained from sigma.

Animal studies and Genotyping

Mice used in this study were males of C57/BL6J background.

Dicer lx/lx :adipoQ-Cre ERT2 mice were generated by crossing Dicer-floxed mice that possessloxP sites flanking exon 23 of the Dicer1 locus 386 , with adipoq-Cre/ERT2 mice, which express a tamoxifen-inducible CRE recombinase in adipocytes 387 .

For genotyping, DNA was extracted from the tips of mice' tails with the REDExtract-N-Amp Tissue PCR Kit (Sigma) and PCR was performed with the KAPA Mouse Genotyping Kit (KPAB BIOSYSTEMS).

Tamoxifen (sigma) was dissolved in coin oil at 40°C overnight. Mouse wasorally administrated with tamoxifen (200mg/kg) for 5 consecutive days for 3 times with around 2 months.

Immunofluorescence

PLIN1 were performed with commercial kits (PROGEN and ThermoFisher Scientific, respectively) with slight modifications. Briefly, cells were rinsed with PBS, fixed with methanol for 5 mins on ice, and then rinsed with PBS twice. Primary antibody for PLIN1 was diluted in 1 to 200 inside 1 % BSA inside PBS, and laid on the surface of slides for incubation at 4°C overnight. Fluorescent second goat anti-Rabbit antibody (InvivoGen), which was diluted in1 to 100 in 1 % BSA was incubated with cells for 30mins at RT in dark. Slides were rinsed with PBS and then mounted with fluorescent mounting medium (Dako). Pictures were taken with microscopy. Lipid droplets were stained with BODIPY TM 493/503 (ThermoFisher) as described 388 .

Histological Analysis

Adipose tissues and liver were immediately excised and fixed in 4% paraformaldehyde and embedded in paraffin. Sections of 5μm were stained with hematoxylin and eosin (H&E), and imaged with Nikon microscope. Adipocyte sizes were quantified with Adiposoft software as described 389 .

Sirud red staining

The liver sections of 5μm were stained with 1% Sirus Red F3B (Sigma) in saturated picric acid (Sigma). The quantification of the red-stained collagen area was performed with Image J software.

Quantification of plasma parameters

Blood samples were taken from the portal vein of mouse, with EDTA, plasma were separated by centrifugation at 10000 rpm for 10 min at 4°C and were stored at -80°C

Glucose transport

Cells were starved in serum-free DMEM for 6 h and then incubated in the absence or presence of 100nM insulin in Krebs-Ringer phosphate buffer (pH 7.4) supplemented with 0.2% bovine serum albumin, for 45minutes at 37°C. Transport was started by adding 4μCi /1 ml

Triglyceride content measurement

Cells were rinsed with PBS and lysed in PBS by brief sonication. TG was measured by determination of the concentration of glycerol released after lysing and enzymatically hydrolyzing the triglyceride molecules. Free glycerol reagent and Triglyceride reagent kit were obtained from sigma.

The striking rise of obesity-related metabolic disorders has focused attention on adipocytes as critical mediators of disease phenotypes. To better understand the role played by excess adipose in metabolic dysfunction, it is crucial to decipher the characteristics of adipocyte per se. However, during my PhD project, we found that it was very hard to successfully modulate gene expressions in vitro in differentiated adipocytes from immortalized cell lines, such as 3T3-F442A cells, with routine techniques like siRNA transfections. Therefore, a practical method for studying adipocytes in vitro is absolutely necessary for development of therapeutic targeting adipocyte.

Taking into considerations of the present situations of in vitro adipocytes models that are introduced in the introduction part, we are wondering why there is no established protocol for directly culturing isolated adipocytes. Two major questions might need to be uncovered as following:

-What is the main technical difficulty preventing the possibility of culturing adipocytes in vitro directly?

-Through what kind of interventions tha we could overcome these roadblocks?

Result 2: A novel method of culturing isolated adipocytes in vitro 1.

Working scheme for culturing isolated adipocytes in vitro

The unique characteristics of adipocyte come from their structure: one big lipid droplet (LD) taking place of nearly all the cytoplasm (around 90%), which will result in extreme buoyancy forces to fat cells in aqueous system. Naturally, adipocyte would float on top of the culture medium which has a higher density than lipids. Consequently adipocytes will shortly die beacause of surface tension between atmosphere and the liquid culture medium, as well as poor nutrition. Therefore, it is necessary to solve this question in order to successfully culture mature adipocytes in vitro. Possibly, the solution is either to prevent the floating of buoyant adipocyte mechanistically or to modify the density of culture medium making it lower than that of fat. Herein, we tried to sequester adipocyte with the application of cell culture inserts by immersing them underneath the membrane of inserts (Figure 1A).

However, since previous "ceiling culture", where adipocyte were allowed floating on top of a glass flask or a cover slide on top of wells of culture plates which were fully filled with medium, resulted in dedifferentiation, deform or death of cells 359,390 , we suggest that there are other factors affecting the lipid storage capability of adipocyte (LDs stability) or viability of adipocyte in vitro.

To reslove this question, it is crucial to understand the unique properties of LDs inside adipocyte. Structurally, LDs are composed of a neutral lipid core, consisting primarily of triglyceride and sterol esters, which are covered by a monolayer of phospholipids as well as other amphiphilic lipids, and proteins 391,392 . For example, perilipin is the most abundant amphipathic protein on the surfaces of adipocyte LDs 393 , which is the major substrate for cAMP-dependent protein kinase (PKA) in lipolytically stimulated adipocytes. Perilipin serves important functions in the regulation of basal and hormonally stimulated lipolysis. Unlike the two leaflets of a bilayer, which are physically coupled, this monolayer can be stretched infinitely, resulting in an increasing surface tension. LDs with high surface tension are unstable and tend to fuse 394 .

Between the LDs and the bilayer phopholipid cell membrane is the scarce cytosol, a complex mixture of cellular organelles and substances dissolved in water. Due to the extreme limited proportion of cytosol in adipocyte, its buffer effects of preventing the LDs from alterations in extracellular compartments, are dramatically weakened. For instance, it would be possible that immediate immerse adipocytes into pure H 2 O would lead to rapid osmosis of water into cytosol through cellular membrane, and then because of the extreme limited room of cytoplasm inside adipocyte, this swelling of cytosol might lead to the final destruction of cellular membrane and thereby cell death. On the other hand, it would be also possible that immerse adipocytes into a solution which is higher concentrated than the cytosol of adipocytes would result in dehydration of adipocyte cytosol and thereby reduced cell viability (Figure 1B). Therefore, it is crucial to control the osmotic pressure of extracellular medium in order to maintain the homeostasis of adipocyte cytosol.

Glycerol is a well-known hyperosmolar agent and has a low toxicity. In addition, glycerol protects red cells against the trauma of freezing and thawing 395 . Therefore, in an attempt to modify the osmosis of culture medium, we tested thether the supplement of glycerol into culture medium would help to inmporve the survival rates of adipocytes (Figure 1C).

Modifications of culture medium

To investigate the morphology of cultured adipocyte, microscopy observation was applied and it was shown that fat cells exhibited a ball like spherical 3D structure (Figure 2A and 2B), with a relatively smaller nucleus which is peripherally located (Figure 2A and 2C), and the LDs nearly taking up of all the cytoplasma of adipocytes (Figure 2D). In addition, PHK26 which is a dye that identify the membrane structures, indicated that the cultured adipocytes maintained the integrity of cell membranes (Figure 2A). These data indicate that isolated adipocyte cultured with our working scheme maintained the spherical integrity at least for a short period (within 3 days). However, at the same time, it was also found that the survival rates of these adipocyte were very low with dead cells exhibiting an appearance of shrinking cell membranes (data not shown), confirming that other factors aside buoyancy are affecting the vitality of the in vitro cultured adipocytes. Therefore, we went on to investigate whether modification of the osmolarity of culture medium by supplement of glycerol would help to improve the survival rates of adipocytes.

Interestingly, glycerol supplement dose-dependently increased the survival rates of fat cells demonstrated with TUNEL staining (Figure 2E). The reason why there was the appearance of shrinking of cell membranes cultured with low concentration of glycerol is not well understand. Here we proposed that one of the factors leading to the crushes of the spherical 3D adipocytes might come from the osmosis of hypotonic culture medium into cell cytosol, resulting in imbalance of the homeostasis of cell cytoplasm and plasma membrane integrity and finally cytotoxicity.

On the one hand, to set up a better culture condition, we went on to select a suitable fetal bovine serum (FBS) concentration. Interestingly, we observed that when cultured without serum, fat cells started to exhibit strange alterations, which was "browning of the color" while still retaining the morphological integrity (Figure 2F). TUNEL assay helped to demonstrate that fat cells started apoptosis without serum, indicating that serum deprivation could also lead to apoptosis of terminal differentiated adipocytes. On the other hand, fat cells cultured with 20% FBS, exhibited "crushed" structures, which was demonstrated with TUNEL assay as dead cells. The reason that very high concentrations of FBS led to death of fat cells might due to the increased osmotic pressure that lead to dehydration (drain) of the very limited aqueous cytosol of adipocyte and then death.

Adipocyte cultured with 10 to 15% FBS remain relatively normal in term of structure.

Therefore, the concentration of FBS is also vital for the viability of adipocytes in culture, with both low and high concentrations would lead to apoptosis.

Functional investigation of cultured adipocyte in vitro

To investigate whether cultured adipocyte are biologically functional, we analysed various functional analysis.

It is well known that insulin signal transduction triggers the phosphorylation of AKT at serine 473 through insulin substrate 1 (IRS-1) and phosphatidylinositol (PI) 3-kinase (PI3K), which is crucial for insulin stimulated glucose uptake into adipocyte. The result demonstrated that 100nM insulin stimulation increased the level of pAKT (s473) in cultured adipocytes (Figure 3A), indicating that sensitivity of insulin pathway is conserved.However, it was also noted that the basal level of AKT phosphorylation of AKT at serine 473 was high even after deprivation, which might due to the structure of culturing system, because it is impossible to change all the medium at one time (some medium have to be remained to cover the sequestered adipocytes), instead, dual dilutions of previous culture medium with serum and glucose with DMEM.

To investigate the glucose uptake ability of cultured adipocyte, we took advantage of a fluorescent D-glucose analog 2-[N- (7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino ]-2-deoxy-Dglucose (2-NBDG), which could directly measure glucose uptake in living cells 379 . The result shows that 100 or 1000nM insulin stimulation had a clear trend to increase the uptake of fluorescent 2-NBDG into cultured adipocyte (Figure 3B). Unfortunately, because of the hyper-sizes of adipocytes, flow cytometry could not be applied to detect the difference of fuorescence. However, the result obtained with [ 3 H] 2-deoxy-D-glucosetracer further demonstrated that insulin would significantly stimulate the glucose uptake into culture adipocyte by around 50% at the day 5 of culture (Figure 3C). Therefore, in vitro cultured adipocytes are capable of extacellular glucose intake.

On the other hand, lipolysis was performed to evaluate the responsiveness of β3adrenergic signaling pathway of cultured adipocytes (Figure 3D). The result demonstrated that isoproterenol at the concentrations of 0.1 or 1μM successfully increased the glycerol release into medium by around 40 to 50% compared to basal release even at the day 8 of culture (0.39 ± 0.025 vs. 0.55 ± 0.037 and 0.60 ± 0.056 mM). In fact, it would apparently be observed that the size of adipocytes even reduced with isoproterenol stimulation (data not shown), indicating the dynamic of adipocytes. Technically, since adipocytes were cultured

Characterization of adipocytes during chronic culture in vitro

The results of PLIN1 and BODIPY double staining demonstrated that there was a preserved expression of PLIN1 on the surfaces of LDs inside adipocytes as long as at D13 of in vitro culture (Figure 4A), suggesting that the LDs might be stable during the chronic in vitro culture. Therefore, we then decided to further characterize the influence of chronic in vitro culture on adipocytes by studying their cell sizes and gene expressions.

Interestingly, characterization of the alteratioins in adipocyte size at D1, D3, D6 and D9, revelt that the average size of adipocytes had a trend to increase and peak at D6, after which it dropped again (Figure 4B) (N=1). Since the size of adipocytes reflect mainly the size of LDs inside adipocytes, which is mostly determined by the balance between lipolysis and lipogenesis. Thus, the possible alterations in adipocyte size might suggest that the cultured fat cells remain dynamic in terms of lipolysis and lipogenesis. To help find more clues about the effects of in vitro culture on adipocytes, RT-qPCR was performed to study the genetic profile of cultured adipocytes at D1, D3, D6 and D9. The adipocyte lipid binding protein aP2

(fatty acid binding protein (FABP) 4) gradually increased and peaked at D6, after which it dropped back. It was also the same trend other genes like Adiponectin, Cebpα, Pparγ 2 , Pgc1β, CD36 and Fatp (fatty acid transport protein) (Figure 4C). These results might coincide with the morphological alterations of these fat cells (Figure 4B). However, genes like Leptin and Glut4 surprisingly and strangly kept decreasing at the onset of culturing (Figure 4C). The reasons why these were abnormal alterations of genes are not fully understood.

Nevertheless, we then went on to see whether this system is applicable to human adipocytes. Preliminary results show that cultured human adipocytes had a much yellow appearance and could maintain their spherical morphology as long as at D11 (Figure 4D). With the help of mechanical sequestering by cell culture inserts and supplement of glycerol into culture medium, we firstly reported that isolated adipocytes were able to remain spherical 3D structure for as long as nearly 2 weeks.

PHK26 dye staining demonstrated the integrity of plasma membrane of cultured adipocytes, with DAPI detecting a very small and peripherally located nucleus after chronic culture. Dual staining of BODIPY and PLIN1, demonstrated that the preserved expression of lipid coating protein on the surface of LDs inside cultured adipocytes as long as 2 weeks of in vitro culture, indicating of preserved stability of LDs inside adipocytes with our novel culture system.

The protection of glycerol supplement to adipocytes might due to the increased tonicity of hypertonic culture medium, which then modified the osmosis of water into the limited cytosol of adipocytes and thereby maintained the homeostasis of adipocytes cytoplasm and then the survival rates of the adipocytes. Next, the pro-apopotic effects of high concentrations of FBS (20 to 25%) on the other hand suggested that hypertonicty of medium by FBS might lead to dehydration of adipocyte cytosol and thereby cell death. All these data indicated that the balance of the tonictiy of culture medium is vital from culture mature adipocytes in vitro.

Moreover, functional analysis revealed that cultured fat cells were responsive to insulin stimulated Akt phosphorylation and glucose uptake, as well as responsive to catecholamine stimulation demonstrated by increased lipolysis. Genomic analysis revealed that there were abnormalities in terms of gene expressions with present culture conditions.

They might be related to the effects of collagen digestion and need to be further investigated. The reasons why these are abnormal alterations of genes were not fully understood. However, several factors might contribute to the above phenomenon:

1) Trauma from collagenase digestion.

The process of isolation of adipocytes from adipose tissue with collagenase A digestion was firstly set up by Rodbell M. in 1964 345 . Since then, this method was widely applied for the study of adipocyte biology with minor modifications.

However, precise actions of the digestion process per se on adipocytes are largely unclear. We tried to evaluate the effects of digestion on the viability of adipocytes by staining isolated adipocytes with trypan blue shortly after digestion process. The result showed that most of the isolated adipocytes were positively stained at only one point of cell surface by trypan blue (data not shown), a dye that is endocytosed only by cells with impaired plasma membranes, indicating that digestion process per se might lead to reduced viability of adipocytes and thereby affecting their genome integrity during the in vitro culture thereafter.

2) Toxicity from fatty acids from broken adipocytes.

One of the limits of the present culture system with cell insert is that it could not prevent certain adipocytes from escaping through the peripheral rim covered without cell insert, which later would float on top of medium and break due to surface tension between air and medium, releasing large amount of lipids onto the medium. Therefore, lipid toxicity on adipocytes from broken adipocytes could not be excluded. To resolve this possibility, supplementation of BSA (free fatty acid-free) into medium might be helpful to neutralize spilled lipids from dead adipocytes.

3) Some nutrients or hormones are lacking in the medium to help preserve the genomic integrity of fat cells.

In vivo, the physiology of adipocytes is elegantly maintained by various hormones like insulin, leptin, catecholamine, etc. Therefore, it is plausible that proper supplementation of a cocktail of hormones might be necessary to maintain the normal gene expressions of cultured adipocytes.

In sum, for the first time, we demonstrated the possibility to culture isolated adipocytes in vitro with preserved morphology and LDs coating protein expressions as long as 2 weeks by glycerol supplementation. Functional studies confirmed that the cultured adipocytes were responsive to insulin and catecholamine stimulation. However, intriguing gentic profiles at different stage of in vitro culture, indicated that this technique is still at a preliminary stage with uncertainties, thereby might not be recommended for formal experiments.

Nevertheless, we image that in the further with progress in our technique protocol, we could directly modulate gene expressions in culture adipocytes by runtine gene therapy techniques like siRNA or CRISPR, which will better mimic in vivo situations.

MATERIALS AND METHODS

Isolation and culture mature adipocytes

Cell culture insert transparent PET membrane 12well 1.0μm pore size (Falcon, corining) were previous covered with 50µg/ml type I collagen (Santa Cruz, sc-136157) dissolved in 0.02N acetic acid for 1h at room temperature. Inserts were rinsed well with PBS and air dried under laminar flow hood before usage. Perigonadal adipose tissue was dissected from C57BL/6J background mice in accordance with the guidelines of the local ethical committee.

Adipocytes were isolated after digestion in 1 mg/mL collagenase A (Roche, USA) for around 40minutes at 37°C in a heat bath with brief shaking at intervals. The digested tissue was filtered through a 100μm nylon mesh. The filteredadipocyte were washed four to five times with DMEM/F12 containing 20% fetal bovine serum (FBS, Gibco  ). The floating layer of adipocytes was isolated for culture. 300μl DMEM/F12 supplemented with 20 percent fetal bovine serum were seeded into each well of 12 well plates to cover the surface.Then, 30 μL of packed and floated fat cells were carefully seeded. After, 300ul of culture medium were carefully added into the upper side of the insert. Medium was changed every two days using 1ml syringe equipped with 23G needle.Cells were routinely cultured in 5%CO2, 37°C. Cells were observed with light microscopy.

PLIN1/ BODIPY staining

PLIN1/ BODIPY staining were performed with commercial kits (PROGEN and ThermoFisher Scientific, respectively) with slight modifications. Briefly, cells were rinsed with PBS, fixed with 100 percent methanol for 5 mins on ice, and then removed the methanol and rinsed cells with PBS twice, and keep the cells on ice for 15 mins. Primary antibody of perilipin was diluted in 1 to 200 inside 1 percent of BSA inside PBS. Carefully lay the primary antibody on the membrane of MILLICELL insert and incubated at 4 degree for overnight. Removed the primary antibody from the upper or down of the membrane, rinsed 3 times with PBS.

Fluorescent second goat anti-Rabbit antibody (InvivoGen), which was diluted at the ratio of 1 to 100 in 1 percent BSA in PBS was incubated with cells at RT for 30mins in dark. After, cells were rinsed with PBS for 3 times, and further incubated with Bodipy (10l/ml) and Hoechst (1 to 50000) solution for another 30 min. Pictures were taken with microscophy.

TUNEL assay

Apoptotic or necrotic cells were detected by the terminal deoxynucleotidyltransferase (TdT)mediated dUTP nick end labeling (TUNEL) method using TUNEL Assays kit from RδD Systems.

Western blot

Cells were lysed with Laemmli buffer containing 0.2 % SDS. Proteins were electrophoresed on 15 % SDS-polyacrylamine gel and transferred to nitrocellulose membrane (Bio-Rad).

Membranes were blocked and incubated with primary antibodies in 5 % BSA in PBST overnight at 4°C. Blots were the rinsed and incubated with secondary antibodies conjugated to horseradish peroxidase (HRP) (Sigma). Immunoreactivity was detected by Amersham ECL prime Western Blotting Detection Reagent (GE Healthcare) and visualized with the

Quantitative real-time PCR

Total RNA was isolated from cultured adipocytes, using MasterPure TM RNA purification kit (Epicentre, USA) according to manufacturer's instructions. The cDNA templates were prepared by reverse transcription of RNA (500ng) using High-Capacity cDNA Reverse

Transcription kit (ThermoFisher scientific, USA)and gene expressions were measured using a MESA blue qPCR TM Mastermix plus for SYBR  assay (Eurogentec, Belgium). The amplification data were analyzed using Viia7 (Applied Biosystems).

Lipolysis assay

10μl of packed adipocytes were seeded into each well of corning  HTS transwell  96 well permeable supports, pore size of which was 3.0 μm (sigma, cat. No.:CLS3385). At the 8 th day of culture, 100 μl lipolysis buffer (KRBS buffer plus 0.1% glucose and 3.5% fatty acid-free bovine serum albumin, sigma) containing 0 or 10 -6 isoproterenol was added into the 96-well plate for 4h at 37°C. The glycerol assay (Sigma) was used to measure glycerol concentrations in the medium, as manufacturer's instructions.

Glucose transport

Cells were starved in serum-free DMEM without glucose for 6 h. Cells were then transferred into 1.5 mL eppendorf for incubationwith or without insulinin Krebs-Ringer phosphate buffer (pH 7.4) supplemented with 3% fatty acid-free bovine serum albumin (Sigma) for 45minutes at 37°C.Transport was started by adding 4μCi /1 ml [ 3 H]2-deoxy-D-glucose (NEN, Boston, MA) for10 minutes precisely at 37°C and stopped by addition of cytochalasin B (Sigma). After aliquots of adipocytes were then transferred into microcentrifuge tubes with 200μl and then separated from incubation buffer by brief centrifugation and were used for liquid scintillation counting. Resting aliquots of adipocytes in incubation buffer were used for DNA detection with Quanti-iT TM PicoGreen TM dsDNA Assay Kit (ThermoFisher). Final results of DMP were then normalized to DNA levels.

2-NBDG uptake

Cultured adipocytes were recoverd into 1.5mL eppendof, rinsed briefly with PBS, and then incubated in DMEM containing 0 or 100nM insulin for 60 min at 37°C. Then, 250mM 2-NBDG was incubated with cells for another 30 min at 37°C. Fluorensencent uptake of 2-NBDG were imaged with microscope.

Statistical analysis

Statistical analysis was performed using GraphPad Prism7.0 software (GraphPad Software, USA). Adipocyte sizes were analyzed with image J software. P<0.05 was considered statistically significant.

Figure 1 :

 1 Figure 1: Growing interests in adipose biology over the last century. .................................................Figure 2: Adipose tissue distribution associates with the incidence of diabetes development. ..........Figure 3: Representation and transmission electron microscopy of the morphology of white adipocytes. ............................................................................................................................................ Figure 4: Extracellular support of adipocytes in vivo. ...........................................................................Figure 5: Schematic representation of FFA uptake mediated by CD36 in caveolin-1-FABP rich microdomain. ........................................................................................................................................Figure 6: Insulin signaling regulates GLUT4 exocytosis by engaging the trafficking machinery. ..........Figure 7: Tight control of lipolysis process by IIS, β-adrenergic signaling and NP receptor A signaling pathways. ..............................................................................................................................................Figure 8 : Adipocytes crosstalk with various organs through adipokines. ............................................ Figure 9 : Simplified scheme of white adipocyte origins. ...................................................................... Figure 10: The classical pathway of microRNA biogenesis....................................................................Figure 11: Hallmarks of senescent cells. ...............................................................................................Figure 12 : The p53 and RB tumor-suppresor pathways. ......................................................................Figure 13: Simple illustrations of microRNAs' participations in calorie restriction process in different species. ..................................................................................................................................................Figure 14: Examples of effects of microRNAs in sirtuins during aging or senescence. .........................Figure 15 : 3T3-F442A adipocytes differentiated from 3T3-F442A fibroblasts. ....................................Figure 16: Isolation of SVF from adipose tissue with collagenase digestion and centrigugation for in vitro adipogenesis. ................................................................................................................................ Figure 17: 3D adipogenesis of 3T3-L1 cells inelectrospun polycaprolactone scaffolds. .......................Figure 18: Formation of spheroids of 3T3-L1 adipocyte aggregates on surface tethered with ELP-PEI. ............................................................................................................................................................... Figure 19: Ceiling culture of isolated adipocytes. .................................................................................

Figure 2 :

 2 Figure 1: Growing interests in adipose biology over the last century. .................................................Figure 2: Adipose tissue distribution associates with the incidence of diabetes development. ..........Figure 3: Representation and transmission electron microscopy of the morphology of white adipocytes. ............................................................................................................................................ Figure 4: Extracellular support of adipocytes in vivo. ...........................................................................Figure 5: Schematic representation of FFA uptake mediated by CD36 in caveolin-1-FABP rich microdomain. ........................................................................................................................................Figure 6: Insulin signaling regulates GLUT4 exocytosis by engaging the trafficking machinery. ..........Figure 7: Tight control of lipolysis process by IIS, β-adrenergic signaling and NP receptor A signaling pathways. ..............................................................................................................................................Figure 8 : Adipocytes crosstalk with various organs through adipokines. ............................................ Figure 9 : Simplified scheme of white adipocyte origins. ...................................................................... Figure 10: The classical pathway of microRNA biogenesis....................................................................Figure 11: Hallmarks of senescent cells. ...............................................................................................Figure 12 : The p53 and RB tumor-suppresor pathways. ......................................................................Figure 13: Simple illustrations of microRNAs' participations in calorie restriction process in different species. ..................................................................................................................................................Figure 14: Examples of effects of microRNAs in sirtuins during aging or senescence. .........................Figure 15 : 3T3-F442A adipocytes differentiated from 3T3-F442A fibroblasts. ....................................Figure 16: Isolation of SVF from adipose tissue with collagenase digestion and centrigugation for in vitro adipogenesis. ................................................................................................................................ Figure 17: 3D adipogenesis of 3T3-L1 cells inelectrospun polycaprolactone scaffolds. .......................Figure 18: Formation of spheroids of 3T3-L1 adipocyte aggregates on surface tethered with ELP-PEI. ............................................................................................................................................................... Figure 19: Ceiling culture of isolated adipocytes. .................................................................................

Figure 3 :

 3 Figure 1: Growing interests in adipose biology over the last century. .................................................Figure 2: Adipose tissue distribution associates with the incidence of diabetes development. ..........Figure 3: Representation and transmission electron microscopy of the morphology of white adipocytes. ............................................................................................................................................ Figure 4: Extracellular support of adipocytes in vivo. ...........................................................................Figure 5: Schematic representation of FFA uptake mediated by CD36 in caveolin-1-FABP rich microdomain. ........................................................................................................................................Figure 6: Insulin signaling regulates GLUT4 exocytosis by engaging the trafficking machinery. ..........Figure 7: Tight control of lipolysis process by IIS, β-adrenergic signaling and NP receptor A signaling pathways. ..............................................................................................................................................Figure 8 : Adipocytes crosstalk with various organs through adipokines. ............................................ Figure 9 : Simplified scheme of white adipocyte origins. ...................................................................... Figure 10: The classical pathway of microRNA biogenesis....................................................................Figure 11: Hallmarks of senescent cells. ...............................................................................................Figure 12 : The p53 and RB tumor-suppresor pathways. ......................................................................Figure 13: Simple illustrations of microRNAs' participations in calorie restriction process in different species. ..................................................................................................................................................Figure 14: Examples of effects of microRNAs in sirtuins during aging or senescence. .........................Figure 15 : 3T3-F442A adipocytes differentiated from 3T3-F442A fibroblasts. ....................................Figure 16: Isolation of SVF from adipose tissue with collagenase digestion and centrigugation for in vitro adipogenesis. ................................................................................................................................ Figure 17: 3D adipogenesis of 3T3-L1 cells inelectrospun polycaprolactone scaffolds. .......................Figure 18: Formation of spheroids of 3T3-L1 adipocyte aggregates on surface tethered with ELP-PEI. ............................................................................................................................................................... Figure 19: Ceiling culture of isolated adipocytes. .................................................................................

Figure 5 :

 5 Figure 1: Growing interests in adipose biology over the last century. .................................................Figure 2: Adipose tissue distribution associates with the incidence of diabetes development. ..........Figure 3: Representation and transmission electron microscopy of the morphology of white adipocytes. ............................................................................................................................................ Figure 4: Extracellular support of adipocytes in vivo. ...........................................................................Figure 5: Schematic representation of FFA uptake mediated by CD36 in caveolin-1-FABP rich microdomain. ........................................................................................................................................Figure 6: Insulin signaling regulates GLUT4 exocytosis by engaging the trafficking machinery. ..........Figure 7: Tight control of lipolysis process by IIS, β-adrenergic signaling and NP receptor A signaling pathways. ..............................................................................................................................................Figure 8 : Adipocytes crosstalk with various organs through adipokines. ............................................ Figure 9 : Simplified scheme of white adipocyte origins. ...................................................................... Figure 10: The classical pathway of microRNA biogenesis....................................................................Figure 11: Hallmarks of senescent cells. ...............................................................................................Figure 12 : The p53 and RB tumor-suppresor pathways. ......................................................................Figure 13: Simple illustrations of microRNAs' participations in calorie restriction process in different species. ..................................................................................................................................................Figure 14: Examples of effects of microRNAs in sirtuins during aging or senescence. .........................Figure 15 : 3T3-F442A adipocytes differentiated from 3T3-F442A fibroblasts. ....................................Figure 16: Isolation of SVF from adipose tissue with collagenase digestion and centrigugation for in vitro adipogenesis. ................................................................................................................................ Figure 17: 3D adipogenesis of 3T3-L1 cells inelectrospun polycaprolactone scaffolds. .......................Figure 18: Formation of spheroids of 3T3-L1 adipocyte aggregates on surface tethered with ELP-PEI. ............................................................................................................................................................... Figure 19: Ceiling culture of isolated adipocytes. .................................................................................

Figure 6 :

 6 Figure 1: Growing interests in adipose biology over the last century. .................................................Figure 2: Adipose tissue distribution associates with the incidence of diabetes development. ..........Figure 3: Representation and transmission electron microscopy of the morphology of white adipocytes. ............................................................................................................................................ Figure 4: Extracellular support of adipocytes in vivo. ...........................................................................Figure 5: Schematic representation of FFA uptake mediated by CD36 in caveolin-1-FABP rich microdomain. ........................................................................................................................................Figure 6: Insulin signaling regulates GLUT4 exocytosis by engaging the trafficking machinery. ..........Figure 7: Tight control of lipolysis process by IIS, β-adrenergic signaling and NP receptor A signaling pathways. ..............................................................................................................................................Figure 8 : Adipocytes crosstalk with various organs through adipokines. ............................................ Figure 9 : Simplified scheme of white adipocyte origins. ...................................................................... Figure 10: The classical pathway of microRNA biogenesis....................................................................Figure 11: Hallmarks of senescent cells. ...............................................................................................Figure 12 : The p53 and RB tumor-suppresor pathways. ......................................................................Figure 13: Simple illustrations of microRNAs' participations in calorie restriction process in different species. ..................................................................................................................................................Figure 14: Examples of effects of microRNAs in sirtuins during aging or senescence. .........................Figure 15 : 3T3-F442A adipocytes differentiated from 3T3-F442A fibroblasts. ....................................Figure 16: Isolation of SVF from adipose tissue with collagenase digestion and centrigugation for in vitro adipogenesis. ................................................................................................................................ Figure 17: 3D adipogenesis of 3T3-L1 cells inelectrospun polycaprolactone scaffolds. .......................Figure 18: Formation of spheroids of 3T3-L1 adipocyte aggregates on surface tethered with ELP-PEI. ............................................................................................................................................................... Figure 19: Ceiling culture of isolated adipocytes. .................................................................................

Figure 7 :

 7 Figure 1: Growing interests in adipose biology over the last century. .................................................Figure 2: Adipose tissue distribution associates with the incidence of diabetes development. ..........Figure 3: Representation and transmission electron microscopy of the morphology of white adipocytes. ............................................................................................................................................ Figure 4: Extracellular support of adipocytes in vivo. ...........................................................................Figure 5: Schematic representation of FFA uptake mediated by CD36 in caveolin-1-FABP rich microdomain. ........................................................................................................................................Figure 6: Insulin signaling regulates GLUT4 exocytosis by engaging the trafficking machinery. ..........Figure 7: Tight control of lipolysis process by IIS, β-adrenergic signaling and NP receptor A signaling pathways. ..............................................................................................................................................Figure 8 : Adipocytes crosstalk with various organs through adipokines. ............................................ Figure 9 : Simplified scheme of white adipocyte origins. ...................................................................... Figure 10: The classical pathway of microRNA biogenesis....................................................................Figure 11: Hallmarks of senescent cells. ...............................................................................................Figure 12 : The p53 and RB tumor-suppresor pathways. ......................................................................Figure 13: Simple illustrations of microRNAs' participations in calorie restriction process in different species. ..................................................................................................................................................Figure 14: Examples of effects of microRNAs in sirtuins during aging or senescence. .........................Figure 15 : 3T3-F442A adipocytes differentiated from 3T3-F442A fibroblasts. ....................................Figure 16: Isolation of SVF from adipose tissue with collagenase digestion and centrigugation for in vitro adipogenesis. ................................................................................................................................ Figure 17: 3D adipogenesis of 3T3-L1 cells inelectrospun polycaprolactone scaffolds. .......................Figure 18: Formation of spheroids of 3T3-L1 adipocyte aggregates on surface tethered with ELP-PEI. ............................................................................................................................................................... Figure 19: Ceiling culture of isolated adipocytes. .................................................................................

Figure 8 :

 8 Figure 1: Growing interests in adipose biology over the last century. .................................................Figure 2: Adipose tissue distribution associates with the incidence of diabetes development. ..........Figure 3: Representation and transmission electron microscopy of the morphology of white adipocytes. ............................................................................................................................................ Figure 4: Extracellular support of adipocytes in vivo. ...........................................................................Figure 5: Schematic representation of FFA uptake mediated by CD36 in caveolin-1-FABP rich microdomain. ........................................................................................................................................Figure 6: Insulin signaling regulates GLUT4 exocytosis by engaging the trafficking machinery. ..........Figure 7: Tight control of lipolysis process by IIS, β-adrenergic signaling and NP receptor A signaling pathways. ..............................................................................................................................................Figure 8 : Adipocytes crosstalk with various organs through adipokines. ............................................ Figure 9 : Simplified scheme of white adipocyte origins. ...................................................................... Figure 10: The classical pathway of microRNA biogenesis....................................................................Figure 11: Hallmarks of senescent cells. ...............................................................................................Figure 12 : The p53 and RB tumor-suppresor pathways. ......................................................................Figure 13: Simple illustrations of microRNAs' participations in calorie restriction process in different species. ..................................................................................................................................................Figure 14: Examples of effects of microRNAs in sirtuins during aging or senescence. .........................Figure 15 : 3T3-F442A adipocytes differentiated from 3T3-F442A fibroblasts. ....................................Figure 16: Isolation of SVF from adipose tissue with collagenase digestion and centrigugation for in vitro adipogenesis. ................................................................................................................................ Figure 17: 3D adipogenesis of 3T3-L1 cells inelectrospun polycaprolactone scaffolds. .......................Figure 18: Formation of spheroids of 3T3-L1 adipocyte aggregates on surface tethered with ELP-PEI. ............................................................................................................................................................... Figure 19: Ceiling culture of isolated adipocytes. .................................................................................

Figure 9 :

 9 Figure 1: Growing interests in adipose biology over the last century. .................................................Figure 2: Adipose tissue distribution associates with the incidence of diabetes development. ..........Figure 3: Representation and transmission electron microscopy of the morphology of white adipocytes. ............................................................................................................................................ Figure 4: Extracellular support of adipocytes in vivo. ...........................................................................Figure 5: Schematic representation of FFA uptake mediated by CD36 in caveolin-1-FABP rich microdomain. ........................................................................................................................................Figure 6: Insulin signaling regulates GLUT4 exocytosis by engaging the trafficking machinery. ..........Figure 7: Tight control of lipolysis process by IIS, β-adrenergic signaling and NP receptor A signaling pathways. ..............................................................................................................................................Figure 8 : Adipocytes crosstalk with various organs through adipokines. ............................................ Figure 9 : Simplified scheme of white adipocyte origins. ...................................................................... Figure 10: The classical pathway of microRNA biogenesis....................................................................Figure 11: Hallmarks of senescent cells. ...............................................................................................Figure 12 : The p53 and RB tumor-suppresor pathways. ......................................................................Figure 13: Simple illustrations of microRNAs' participations in calorie restriction process in different species. ..................................................................................................................................................Figure 14: Examples of effects of microRNAs in sirtuins during aging or senescence. .........................Figure 15 : 3T3-F442A adipocytes differentiated from 3T3-F442A fibroblasts. ....................................Figure 16: Isolation of SVF from adipose tissue with collagenase digestion and centrigugation for in vitro adipogenesis. ................................................................................................................................ Figure 17: 3D adipogenesis of 3T3-L1 cells inelectrospun polycaprolactone scaffolds. .......................Figure 18: Formation of spheroids of 3T3-L1 adipocyte aggregates on surface tethered with ELP-PEI. ............................................................................................................................................................... Figure 19: Ceiling culture of isolated adipocytes. .................................................................................

Figure 10 :

 10 Figure 1: Growing interests in adipose biology over the last century. .................................................Figure 2: Adipose tissue distribution associates with the incidence of diabetes development. ..........Figure 3: Representation and transmission electron microscopy of the morphology of white adipocytes. ............................................................................................................................................ Figure 4: Extracellular support of adipocytes in vivo. ...........................................................................Figure 5: Schematic representation of FFA uptake mediated by CD36 in caveolin-1-FABP rich microdomain. ........................................................................................................................................Figure 6: Insulin signaling regulates GLUT4 exocytosis by engaging the trafficking machinery. ..........Figure 7: Tight control of lipolysis process by IIS, β-adrenergic signaling and NP receptor A signaling pathways. ..............................................................................................................................................Figure 8 : Adipocytes crosstalk with various organs through adipokines. ............................................ Figure 9 : Simplified scheme of white adipocyte origins. ...................................................................... Figure 10: The classical pathway of microRNA biogenesis....................................................................Figure 11: Hallmarks of senescent cells. ...............................................................................................Figure 12 : The p53 and RB tumor-suppresor pathways. ......................................................................Figure 13: Simple illustrations of microRNAs' participations in calorie restriction process in different species. ..................................................................................................................................................Figure 14: Examples of effects of microRNAs in sirtuins during aging or senescence. .........................Figure 15 : 3T3-F442A adipocytes differentiated from 3T3-F442A fibroblasts. ....................................Figure 16: Isolation of SVF from adipose tissue with collagenase digestion and centrigugation for in vitro adipogenesis. ................................................................................................................................ Figure 17: 3D adipogenesis of 3T3-L1 cells inelectrospun polycaprolactone scaffolds. .......................Figure 18: Formation of spheroids of 3T3-L1 adipocyte aggregates on surface tethered with ELP-PEI. ............................................................................................................................................................... Figure 19: Ceiling culture of isolated adipocytes. .................................................................................

Figure 11 :

 11 Figure 1: Growing interests in adipose biology over the last century. .................................................Figure 2: Adipose tissue distribution associates with the incidence of diabetes development. ..........Figure 3: Representation and transmission electron microscopy of the morphology of white adipocytes. ............................................................................................................................................ Figure 4: Extracellular support of adipocytes in vivo. ...........................................................................Figure 5: Schematic representation of FFA uptake mediated by CD36 in caveolin-1-FABP rich microdomain. ........................................................................................................................................Figure 6: Insulin signaling regulates GLUT4 exocytosis by engaging the trafficking machinery. ..........Figure 7: Tight control of lipolysis process by IIS, β-adrenergic signaling and NP receptor A signaling pathways. ..............................................................................................................................................Figure 8 : Adipocytes crosstalk with various organs through adipokines. ............................................ Figure 9 : Simplified scheme of white adipocyte origins. ...................................................................... Figure 10: The classical pathway of microRNA biogenesis....................................................................Figure 11: Hallmarks of senescent cells. ...............................................................................................Figure 12 : The p53 and RB tumor-suppresor pathways. ......................................................................Figure 13: Simple illustrations of microRNAs' participations in calorie restriction process in different species. ..................................................................................................................................................Figure 14: Examples of effects of microRNAs in sirtuins during aging or senescence. .........................Figure 15 : 3T3-F442A adipocytes differentiated from 3T3-F442A fibroblasts. ....................................Figure 16: Isolation of SVF from adipose tissue with collagenase digestion and centrigugation for in vitro adipogenesis. ................................................................................................................................ Figure 17: 3D adipogenesis of 3T3-L1 cells inelectrospun polycaprolactone scaffolds. .......................Figure 18: Formation of spheroids of 3T3-L1 adipocyte aggregates on surface tethered with ELP-PEI. ............................................................................................................................................................... Figure 19: Ceiling culture of isolated adipocytes. .................................................................................

Figure 12 :

 12 Figure 1: Growing interests in adipose biology over the last century. .................................................Figure 2: Adipose tissue distribution associates with the incidence of diabetes development. ..........Figure 3: Representation and transmission electron microscopy of the morphology of white adipocytes. ............................................................................................................................................ Figure 4: Extracellular support of adipocytes in vivo. ...........................................................................Figure 5: Schematic representation of FFA uptake mediated by CD36 in caveolin-1-FABP rich microdomain. ........................................................................................................................................Figure 6: Insulin signaling regulates GLUT4 exocytosis by engaging the trafficking machinery. ..........Figure 7: Tight control of lipolysis process by IIS, β-adrenergic signaling and NP receptor A signaling pathways. ..............................................................................................................................................Figure 8 : Adipocytes crosstalk with various organs through adipokines. ............................................ Figure 9 : Simplified scheme of white adipocyte origins. ...................................................................... Figure 10: The classical pathway of microRNA biogenesis....................................................................Figure 11: Hallmarks of senescent cells. ...............................................................................................Figure 12 : The p53 and RB tumor-suppresor pathways. ......................................................................Figure 13: Simple illustrations of microRNAs' participations in calorie restriction process in different species. ..................................................................................................................................................Figure 14: Examples of effects of microRNAs in sirtuins during aging or senescence. .........................Figure 15 : 3T3-F442A adipocytes differentiated from 3T3-F442A fibroblasts. ....................................Figure 16: Isolation of SVF from adipose tissue with collagenase digestion and centrigugation for in vitro adipogenesis. ................................................................................................................................ Figure 17: 3D adipogenesis of 3T3-L1 cells inelectrospun polycaprolactone scaffolds. .......................Figure 18: Formation of spheroids of 3T3-L1 adipocyte aggregates on surface tethered with ELP-PEI. ............................................................................................................................................................... Figure 19: Ceiling culture of isolated adipocytes. .................................................................................

Figure 13 :

 13 Figure 1: Growing interests in adipose biology over the last century. .................................................Figure 2: Adipose tissue distribution associates with the incidence of diabetes development. ..........Figure 3: Representation and transmission electron microscopy of the morphology of white adipocytes. ............................................................................................................................................ Figure 4: Extracellular support of adipocytes in vivo. ...........................................................................Figure 5: Schematic representation of FFA uptake mediated by CD36 in caveolin-1-FABP rich microdomain. ........................................................................................................................................Figure 6: Insulin signaling regulates GLUT4 exocytosis by engaging the trafficking machinery. ..........Figure 7: Tight control of lipolysis process by IIS, β-adrenergic signaling and NP receptor A signaling pathways. ..............................................................................................................................................Figure 8 : Adipocytes crosstalk with various organs through adipokines. ............................................ Figure 9 : Simplified scheme of white adipocyte origins. ...................................................................... Figure 10: The classical pathway of microRNA biogenesis....................................................................Figure 11: Hallmarks of senescent cells. ...............................................................................................Figure 12 : The p53 and RB tumor-suppresor pathways. ......................................................................Figure 13: Simple illustrations of microRNAs' participations in calorie restriction process in different species. ..................................................................................................................................................Figure 14: Examples of effects of microRNAs in sirtuins during aging or senescence. .........................Figure 15 : 3T3-F442A adipocytes differentiated from 3T3-F442A fibroblasts. ....................................Figure 16: Isolation of SVF from adipose tissue with collagenase digestion and centrigugation for in vitro adipogenesis. ................................................................................................................................ Figure 17: 3D adipogenesis of 3T3-L1 cells inelectrospun polycaprolactone scaffolds. .......................Figure 18: Formation of spheroids of 3T3-L1 adipocyte aggregates on surface tethered with ELP-PEI. ............................................................................................................................................................... Figure 19: Ceiling culture of isolated adipocytes. .................................................................................

Figure 14 :

 14 Figure 1: Growing interests in adipose biology over the last century. .................................................Figure 2: Adipose tissue distribution associates with the incidence of diabetes development. ..........Figure 3: Representation and transmission electron microscopy of the morphology of white adipocytes. ............................................................................................................................................ Figure 4: Extracellular support of adipocytes in vivo. ...........................................................................Figure 5: Schematic representation of FFA uptake mediated by CD36 in caveolin-1-FABP rich microdomain. ........................................................................................................................................Figure 6: Insulin signaling regulates GLUT4 exocytosis by engaging the trafficking machinery. ..........Figure 7: Tight control of lipolysis process by IIS, β-adrenergic signaling and NP receptor A signaling pathways. ..............................................................................................................................................Figure 8 : Adipocytes crosstalk with various organs through adipokines. ............................................ Figure 9 : Simplified scheme of white adipocyte origins. ...................................................................... Figure 10: The classical pathway of microRNA biogenesis....................................................................Figure 11: Hallmarks of senescent cells. ...............................................................................................Figure 12 : The p53 and RB tumor-suppresor pathways. ......................................................................Figure 13: Simple illustrations of microRNAs' participations in calorie restriction process in different species. ..................................................................................................................................................Figure 14: Examples of effects of microRNAs in sirtuins during aging or senescence. .........................Figure 15 : 3T3-F442A adipocytes differentiated from 3T3-F442A fibroblasts. ....................................Figure 16: Isolation of SVF from adipose tissue with collagenase digestion and centrigugation for in vitro adipogenesis. ................................................................................................................................ Figure 17: 3D adipogenesis of 3T3-L1 cells inelectrospun polycaprolactone scaffolds. .......................Figure 18: Formation of spheroids of 3T3-L1 adipocyte aggregates on surface tethered with ELP-PEI. ............................................................................................................................................................... Figure 19: Ceiling culture of isolated adipocytes. .................................................................................

Figure 15 :

 15 Figure 1: Growing interests in adipose biology over the last century. .................................................Figure 2: Adipose tissue distribution associates with the incidence of diabetes development. ..........Figure 3: Representation and transmission electron microscopy of the morphology of white adipocytes. ............................................................................................................................................ Figure 4: Extracellular support of adipocytes in vivo. ...........................................................................Figure 5: Schematic representation of FFA uptake mediated by CD36 in caveolin-1-FABP rich microdomain. ........................................................................................................................................Figure 6: Insulin signaling regulates GLUT4 exocytosis by engaging the trafficking machinery. ..........Figure 7: Tight control of lipolysis process by IIS, β-adrenergic signaling and NP receptor A signaling pathways. ..............................................................................................................................................Figure 8 : Adipocytes crosstalk with various organs through adipokines. ............................................ Figure 9 : Simplified scheme of white adipocyte origins. ...................................................................... Figure 10: The classical pathway of microRNA biogenesis....................................................................Figure 11: Hallmarks of senescent cells. ...............................................................................................Figure 12 : The p53 and RB tumor-suppresor pathways. ......................................................................Figure 13: Simple illustrations of microRNAs' participations in calorie restriction process in different species. ..................................................................................................................................................Figure 14: Examples of effects of microRNAs in sirtuins during aging or senescence. .........................Figure 15 : 3T3-F442A adipocytes differentiated from 3T3-F442A fibroblasts. ....................................Figure 16: Isolation of SVF from adipose tissue with collagenase digestion and centrigugation for in vitro adipogenesis. ................................................................................................................................ Figure 17: 3D adipogenesis of 3T3-L1 cells inelectrospun polycaprolactone scaffolds. .......................Figure 18: Formation of spheroids of 3T3-L1 adipocyte aggregates on surface tethered with ELP-PEI. ............................................................................................................................................................... Figure 19: Ceiling culture of isolated adipocytes. .................................................................................

Figure 16 :

 16 Figure 1: Growing interests in adipose biology over the last century. .................................................Figure 2: Adipose tissue distribution associates with the incidence of diabetes development. ..........Figure 3: Representation and transmission electron microscopy of the morphology of white adipocytes. ............................................................................................................................................ Figure 4: Extracellular support of adipocytes in vivo. ...........................................................................Figure 5: Schematic representation of FFA uptake mediated by CD36 in caveolin-1-FABP rich microdomain. ........................................................................................................................................Figure 6: Insulin signaling regulates GLUT4 exocytosis by engaging the trafficking machinery. ..........Figure 7: Tight control of lipolysis process by IIS, β-adrenergic signaling and NP receptor A signaling pathways. ..............................................................................................................................................Figure 8 : Adipocytes crosstalk with various organs through adipokines. ............................................ Figure 9 : Simplified scheme of white adipocyte origins. ...................................................................... Figure 10: The classical pathway of microRNA biogenesis....................................................................Figure 11: Hallmarks of senescent cells. ...............................................................................................Figure 12 : The p53 and RB tumor-suppresor pathways. ......................................................................Figure 13: Simple illustrations of microRNAs' participations in calorie restriction process in different species. ..................................................................................................................................................Figure 14: Examples of effects of microRNAs in sirtuins during aging or senescence. .........................Figure 15 : 3T3-F442A adipocytes differentiated from 3T3-F442A fibroblasts. ....................................Figure 16: Isolation of SVF from adipose tissue with collagenase digestion and centrigugation for in vitro adipogenesis. ................................................................................................................................ Figure 17: 3D adipogenesis of 3T3-L1 cells inelectrospun polycaprolactone scaffolds. .......................Figure 18: Formation of spheroids of 3T3-L1 adipocyte aggregates on surface tethered with ELP-PEI. ............................................................................................................................................................... Figure 19: Ceiling culture of isolated adipocytes. .................................................................................

Figure 18 :

 18 Figure 1: Growing interests in adipose biology over the last century. .................................................Figure 2: Adipose tissue distribution associates with the incidence of diabetes development. ..........Figure 3: Representation and transmission electron microscopy of the morphology of white adipocytes. ............................................................................................................................................ Figure 4: Extracellular support of adipocytes in vivo. ...........................................................................Figure 5: Schematic representation of FFA uptake mediated by CD36 in caveolin-1-FABP rich microdomain. ........................................................................................................................................Figure 6: Insulin signaling regulates GLUT4 exocytosis by engaging the trafficking machinery. ..........Figure 7: Tight control of lipolysis process by IIS, β-adrenergic signaling and NP receptor A signaling pathways. ..............................................................................................................................................Figure 8 : Adipocytes crosstalk with various organs through adipokines. ............................................ Figure 9 : Simplified scheme of white adipocyte origins. ...................................................................... Figure 10: The classical pathway of microRNA biogenesis....................................................................Figure 11: Hallmarks of senescent cells. ...............................................................................................Figure 12 : The p53 and RB tumor-suppresor pathways. ......................................................................Figure 13: Simple illustrations of microRNAs' participations in calorie restriction process in different species. ..................................................................................................................................................Figure 14: Examples of effects of microRNAs in sirtuins during aging or senescence. .........................Figure 15 : 3T3-F442A adipocytes differentiated from 3T3-F442A fibroblasts. ....................................Figure 16: Isolation of SVF from adipose tissue with collagenase digestion and centrigugation for in vitro adipogenesis. ................................................................................................................................ Figure 17: 3D adipogenesis of 3T3-L1 cells inelectrospun polycaprolactone scaffolds. .......................Figure 18: Formation of spheroids of 3T3-L1 adipocyte aggregates on surface tethered with ELP-PEI. ............................................................................................................................................................... Figure 19: Ceiling culture of isolated adipocytes. .................................................................................

Figure 1 :

 1 Figure 1: Inducible reduction of dicer in adipocytes by tamoxifen in dicer Lox/Lox / adiponectin-CreER T2+ mice. ......................................................................................................................................................Figure 2: Reduction in the average sizes of perigonadal adipocytes due to dicer deficiency. .............Figure 3: Possible involved mechanisms underlying the effects of adipocyte dicer deficiency. ..........Figure 4: Adipocyte dicer deficiency promoted lipid accumulation and fibrosis in liver. .....................Figure 5: Regulation of dicer level in adipose tissue and in in vitro adipocytes. ..................................Figure 6: Age-associated increase of mir-1949. ....................................................................................Figure 7: In vitro actions of mir-1949 on adipcoytes. ..........................................................................

Figure 2 :

 2 Figure 1: Inducible reduction of dicer in adipocytes by tamoxifen in dicer Lox/Lox / adiponectin-CreER T2+ mice. ......................................................................................................................................................Figure 2: Reduction in the average sizes of perigonadal adipocytes due to dicer deficiency. .............Figure 3: Possible involved mechanisms underlying the effects of adipocyte dicer deficiency. ..........Figure 4: Adipocyte dicer deficiency promoted lipid accumulation and fibrosis in liver. .....................Figure 5: Regulation of dicer level in adipose tissue and in in vitro adipocytes. ..................................Figure 6: Age-associated increase of mir-1949. ....................................................................................Figure 7: In vitro actions of mir-1949 on adipcoytes. ..........................................................................

Figure 3 :

 3 Figure 1: Inducible reduction of dicer in adipocytes by tamoxifen in dicer Lox/Lox / adiponectin-CreER T2+ mice. ......................................................................................................................................................Figure 2: Reduction in the average sizes of perigonadal adipocytes due to dicer deficiency. .............Figure 3: Possible involved mechanisms underlying the effects of adipocyte dicer deficiency. ..........Figure 4: Adipocyte dicer deficiency promoted lipid accumulation and fibrosis in liver. .....................Figure 5: Regulation of dicer level in adipose tissue and in in vitro adipocytes. ..................................Figure 6: Age-associated increase of mir-1949. ....................................................................................Figure 7: In vitro actions of mir-1949 on adipcoytes. ..........................................................................

Figure 4 :

 4 Figure 1: Inducible reduction of dicer in adipocytes by tamoxifen in dicer Lox/Lox / adiponectin-CreER T2+ mice. ......................................................................................................................................................Figure 2: Reduction in the average sizes of perigonadal adipocytes due to dicer deficiency. .............Figure 3: Possible involved mechanisms underlying the effects of adipocyte dicer deficiency. ..........Figure 4: Adipocyte dicer deficiency promoted lipid accumulation and fibrosis in liver. .....................Figure 5: Regulation of dicer level in adipose tissue and in in vitro adipocytes. ..................................Figure 6: Age-associated increase of mir-1949. ....................................................................................Figure 7: In vitro actions of mir-1949 on adipcoytes. ..........................................................................

Figure 5 :

 5 Figure 1: Inducible reduction of dicer in adipocytes by tamoxifen in dicer Lox/Lox / adiponectin-CreER T2+ mice. ......................................................................................................................................................Figure 2: Reduction in the average sizes of perigonadal adipocytes due to dicer deficiency. .............Figure 3: Possible involved mechanisms underlying the effects of adipocyte dicer deficiency. ..........Figure 4: Adipocyte dicer deficiency promoted lipid accumulation and fibrosis in liver. .....................Figure 5: Regulation of dicer level in adipose tissue and in in vitro adipocytes. ..................................Figure 6: Age-associated increase of mir-1949. ....................................................................................Figure 7: In vitro actions of mir-1949 on adipcoytes. ..........................................................................

Figure 6 :

 6 Figure 1: Inducible reduction of dicer in adipocytes by tamoxifen in dicer Lox/Lox / adiponectin-CreER T2+ mice. ......................................................................................................................................................Figure 2: Reduction in the average sizes of perigonadal adipocytes due to dicer deficiency. .............Figure 3: Possible involved mechanisms underlying the effects of adipocyte dicer deficiency. ..........Figure 4: Adipocyte dicer deficiency promoted lipid accumulation and fibrosis in liver. .....................Figure 5: Regulation of dicer level in adipose tissue and in in vitro adipocytes. ..................................Figure 6: Age-associated increase of mir-1949. ....................................................................................Figure 7: In vitro actions of mir-1949 on adipcoytes. ..........................................................................

Figure 7 :

 7 Figure 1: Inducible reduction of dicer in adipocytes by tamoxifen in dicer Lox/Lox / adiponectin-CreER T2+ mice. ......................................................................................................................................................Figure 2: Reduction in the average sizes of perigonadal adipocytes due to dicer deficiency. .............Figure 3: Possible involved mechanisms underlying the effects of adipocyte dicer deficiency. ..........Figure 4: Adipocyte dicer deficiency promoted lipid accumulation and fibrosis in liver. .....................Figure 5: Regulation of dicer level in adipose tissue and in in vitro adipocytes. ..................................Figure 6: Age-associated increase of mir-1949. ....................................................................................Figure 7: In vitro actions of mir-1949 on adipcoytes. ..........................................................................

Figure 8 :

 8 Figure 8: Proposed molecular mechanisms underlying adipocyte dicer in aging. ..............................Figure 9: GFP expression in adipocytes from ZsGreen lx/lx /adiponectin-CreER T2+ mice by 4hydrotamoxifen. .................................................................................................................................. Figure 10: Possible role of mir-1949 in the prevention of age-related dysfunctions. ........................

Figure 9 :

 9 Figure 8: Proposed molecular mechanisms underlying adipocyte dicer in aging. ..............................Figure 9: GFP expression in adipocytes from ZsGreen lx/lx /adiponectin-CreER T2+ mice by 4hydrotamoxifen. .................................................................................................................................. Figure 10: Possible role of mir-1949 in the prevention of age-related dysfunctions. ........................

Fig 1 :

 1 Fig 1: Illustration of the working scheme. ........................................................................................... Fig 2: Image of adipocytes in culture under different condtions. ....................................................... Fig 3: Functional investigation of adipocyte in vitro. .......................................................................... Fig 4: Characterizations of adipocytes during chronic culture in vitro. ...............................................

Figure 1 :

 1 Figure 1: Growing interests in adipose biology over the last century. Adopted from 2 .

Figure 2 :

 2 Figure 2: Adipose tissue distribution associates with the incidence of diabetes development. (a) Distributions of WAT in human. (b) Incidence of development of diabetes associated with the fat distributions. Modified from 5 .

Figure 3 :

 3 Figure 3: Representation and transmission electron microscopy of the morphology of white adipocytes. Abbreviations: L, lipid; M, mitochondria; N, nucleus. Modified from 20 .

Figure 4 :

 4 Figure 4: Extracellular support of adipocytes in vivo. Structural support is provided by type I (100 nm to 100 µm diameter) and type VI (50 nm diameter) collagen networks. The LD is caged in vimentin (10 nm, brown filament). The nucleus (blue) moves to the cell periphery and is deformed by the LD. Modified from 26 .

Figure 5 :

 5 Figure 5: Schematic representation of FFA uptake mediated by CD36 in caveolin-1-FABP rich microdomain. Adopted from 33 .

Figure 6 :

 6 Figure 6: Insulin signaling regulates GLUT4 exocytosis by engaging the trafficking machinery. Modified from 56 .

Figure 7 :

 7 Figure 7: Tight control of lipolysis process by IIS, β-adrenergic signaling and NP receptor A signaling pathways. Adopted from 62 .

Figure 8 :

 8 Figure 8 : Adipocytes crosstalk with various organs through adipokines. Adopted from 94 .

Figure 9 :

 9 Figure 9 : Simplified scheme of white adipocyte origins.

  levels of glycerol and FFAs release in visceral fat were significantly reduced in aged mice139 , indicating a decreased efficiency of hydrolysis of TG in adipocytes during aging. Aging is associated with reduced expression of lipolytic genes such as HSL and ATGL in adipose tissue 140 . There is also a concerted decline in fatty acid reesterification (PEPCK: phosphoenolpyruvate carboxykinase) enzyme expression. This leads to reduced adipocyte's ability to recycle FFAs from extracellular pool, in 24-month-old mice compared to young mice 140 . Age-related alterations in the functionality of macrophages also contribute to age-reduced lipolysis. Camell et al, demonstrated that aging up-regulate the Nod-like receptor 3 (NLRP3) inflammasome in adipose macrophages, which is a driving factor for the catabolism of noradrenaline by downregulating growth differentiation factor-3 and monoamine oxidase. Consequently, catecholamine-induced lipolysis is negatively influenced in aged vWAT and thereby leads to age-related central fat accumulation 141 .

  phosphatidylcholine, sphingomyelin, and free cholesterol in the lipid composition of adipocyte plasma membrane, which favor an enlarged cell size with aging 159 . Therefore, it remains interesting to investigate how the composition of adipocyte fatty acids alters during aging in various fat depots and what are the effects of different fatty acids on longevity. 4.2. Removal or mobilization of vWAT increases longevity Due to the notorious associations of vWAT with age-associated pathologies, it has been proposed that surgical removal or stimulation of lipid mobilization might exhibit positive actions on life extension-associated benefits. On one hand, Radhika M. et al demonstrat that removal of vWAT in 5-month-old mice increases the mean and maximum lifespan and reduces the incidence of severe renal diseases compared to sham control mice 160 . On the other hand, in C. elegans, fat mobilization activation through the endocrine axis, which consists of nuclear translocation of the forkhead transcription factor DAF-16 (the equivalent of mammalian FOXO3a) and subsequent activation of the k04a8.5 lipase, mediates the prolonged longevity of germline stem cell arrest. Constitutive expression of this lipase in fat storage tissue generates lean and long-lived animals 161 . In a conserved manner, over-expression of dFOXO in the adult fruitfly (D. melanogaster) fat body, which is fly equivalent of mammalian liver and adipose tissue, increases longevity and reduces fecundity of females 162 .

Figure 10 :

 10 Figure 10: The classical pathway of microRNA biogenesis. Adopted from 185 .

  For example, NF-κB stimulation in 3T3 adipocytes resulted in inhibition of PPAR γ transcription, with miR-130 participating in this process. Chromatin immunoprecipitation (ChIP) assay demonstrated that activation of NF-κB pathway by TNFα induced increased binding of p65 to the promoters of both mir-130a and mir-130b. Deletion of these two miRNAs could partly block the inhibition of PPARγ by TNFα 188 . Similary, NF-κB would directly bound to the promoter of mir-1275 and inhibited its transcription in human mature adipocytes 189 . Glucocorticoids (GCs) could transcriptionally regulate the expression of miR-19b via a GC receptor-mediated binding to its promoter and significantly enhance its expression 190 . Still, for most of miRNAs, how their expressions are regulated in adipose tissue remain to be investigated. For example, the expression of mir-21 in human adipocytes was elevated following inflammatory stimulators such as TNFα, IL-6, leptin or FFAs 191 . Mir-224-5p was elevated in 3T3 adipocytes upon TNFα treatment. Knock down of mir-2224 suppressed NF-κB activation and rendered adipocytes susceptible to apoptosis 192 . The exact mechanisms

  patients and is proposed to improve macrophage-mediated inflammation by targeting ADPribosylation factor 6 (ARF6)220 . In human adipose tissue, mir-99a expression is negatively correlated with inflammation, possibly regulating macrophage phenotype-switching by targeting TNFα 221 . Finally, let-7 family is strongly associated with fat mass and systemic glucose tolerance222 . Additionally, a group of vWAT-enriched miRNAs are found to be involved in various pathologies. For instance, mir-125a is down regulated in adipose tissue by fat diet in mice or in obese patients and in in vitro insulin-resistant 3T3-L1 adipocytes223 .Similarly, mir-103 is associated with insulin sensitivity in adpocytes 224 .MiRNAs mediate the metabolic flexibility of adipose tissue.As mentioned before, human subcutaneous fat is capable of substantial metabolic flexibility, such as acquiring features of brown fat in response to stimuli of cold exposure, βadrengic receptor or PPARγ agonists, associated with increased mitochondria biogenesis and UCP1 expression. MiRNAs are necessary for this identity switch.Indeed, mir-30a-5p, which is less expressed in subcutaneous fat compared to its level in BAT and inversely associates with the level of ubiquitin carrier protein 9 (UCP9) between these two fat depots, would result in browning program in human white adipocytes: mitochondrial biogenesis and brown transcription factors expression via targeting and inhibiting UCP9 225 . In vitro, forced expression of mir-30b/c significantly increases thermogenic gene expressions and mitochondrial respiration in primary adipocytes from sWAT, by targeting the 3'UTR of the receptor-interacting protein 140 (RIP140) 226 . Conversely, let-7i-5p is decreased in brite adipocytes in sWAT and injection of let-7i-5p mimic into sWAT in mice partially inhibits the β3-adrenergic activaton of the browning process 227 . Similarly, mir-125b-5p level is reduced in sWAT upon β3-adrenergic receptor stimulation, with injection of mir-125b-5p mimic directly into WAT inhibiting β3-adrenoceptor-mediated induction of UCP1 and mitochondriogenesis 228 . Further results demonstrate that the thermogenic gene program of PPARγ /PRDM16 in sWAT is under the control of miRNAs: mir-30a-5p affecting PPARγ/PRDM16 via targeting the E2 SUMO ligase UBC9 in sWAT, a factor repressing acquisition of brown fat features in human adipocytes 225 ; mir-133a (mir-133a1/a2) directly targeting the 3' UTR of PRDM16, with mir-133 depletion in mice increasing UCP1 expression and browning of sWAT 229 .

  -223 is increased in the adipose tissue of women with insulin resistance and is positively associated with HOMA-IR. Overexpression of mir-223 in human adipocytes directly reduces protein levels of GLUT4 and blocks insulin stimulated glucose uptake239 . Likewise, mir-93 is up-regulated in the adipose tissue of women with insulin resistance as well as polycystic ovary syndrome patients (approximately 70% of who have intrinsic insulin resistance), correlating reversely with GLUT4 level and positively with HOMA-IR 240 . The mimic of mir-146b is directly transfected into porcine primary adipocytes, leading to impaired glucose uptake due to its inhibitory effect on IRS1 and GLUT4 expressions 241 .MiRNAs are found to be vital mediators of the obesity associated insulin resistance in adipose tissue. For example, in the context of obesity or diabetes in mice, mir-103 and mir-107 are elevated, while their silencing through systemic injection of its inhibitor significantly reduces adipocyte sizes and restores systemic glucose tolerance, arguing for a role of these two miRNAs in insulin resistance. Mechanical study reveals that caveolin-1, the principal protein of caveolae, a structure on the plasma membrane of adipocytes associated with insulin receptor stability, is a direct target of mir-103/mir-107 224 .Mir-377 is increased in visceral fat in mice by high fat diet and impairs insulin stimulated AKT phosphorylation via suppression of SIRT1, which improves insulin sensitivity by repressing protein tyrosine phosphatase (PTP1B) 242 . Similarly, mir-221 could also target SIRT1 and contribute to insulin resistance in WAT by high fat diet induced obesity 243 . At the level of adipose tissue, adipose tissue macrophages (ATMs) infiltration in the context of obesity is a leading cause of the chronic inflammatory state in fat tissue, a strong contributor to insulin resistance. It has been reported that ATMs-derived mir-155 strongly contributes to the impaired insulin stimulated glucose transport in adipocytes, possibly related to its direct suppression of the target gene PPARγ 244 .  MiRNAs in lipogenesis Various miRNAs could also modulate the sizes of adipocytes by targeting pathways related to lipogenesis. As an example, mir-192* is negatively correlated with serum TG and positively with high density lipoproteins (HDL) in morbidly obese subjects. In vitro experiments show that over expression of this miRNA in adipocytes, leads to a reduction in lipid storage accounted by inhibited expression of lipogenic enzymes such as stearoyl coenzyme A desaturase-1 (SCD-1) and aldehyde dehydrogenase 3 family memeber A2 (ALDH3A2) 245 . Located in the first intron of PGC-1β, miR-378/378* are coorninately expressed during adipogenesis and expressed in the same tissues as PGC-1β. Overexpression of miR-378/378* in 3T3 adipocytes leads to lipid accumulation associated with increased expression of lipogenic genes, such as C/EBPα and C/EBPβ 246 . In fat cells, mir-143-3p can increase insulin-stimulated lipogenesis, possibly related to the upregulation of protein kinase AMP-activated catalytic subunit α2 (PRKAA), which encodes a catalytic subunit of AMPK 247 .  MiRNAs in other aspects of adipocytes MiRNAs also participate in other aspects of adipocytes, such as apoptosis, inflammation response and energy expenditure process, etc. For instance, natural conjugated linoleic acids (CLAs) show strong anti-obesity actions in humans and animals, which could reduce the TG content of mature adiocytes and induce apoptosis. The expression of mir-23a is inhibited in adipose tissue in mice administrated with CLAs and its inhibition mediates the apoptotic action of CLAs on adipocytes via its direct effect on apoptotic protease-activating factor 1 (APAF1) 248 . Very recently, mir-103/107 have been identified to promote ER stress-mediated apoptosis by targeting the Wnt3a/β-catenin/ATF6 (activating transcription factor-6) pathway in preadipocytes 249 . The obesity associated mir-146a is found to dampen the inflammatory response in human adipocytes demonstrated by reducing phosphorylation of p38 and c-Jun N-terminal kinase (JNK) via targeting TNF receptor-associated factor 6 (TRAF6) and IL-1 receptorassociated kinase 1 (IRAK1), which act downstream of the IL-1 receptor and toll-like receptors 250 . MiR-155-5p knockout prevents high fat diet induced adipocyte hypertrophy and weight gain, possibly owing to the increased energy expenditure 251 . Likewise, elevation of mir-34a in adipocytes from obese individuals 252 inhibits the browning of adipose tissue partly by suppressing fibroblast growth factor (FGF21)/ SIRT1. In constrast, inhibition of mir-34a expression reduces high fat diet induced adiposity, associated with improved oxidative stress in mice 253 .

1. 1 .

 1 MiRNAs involved in overall longevityEvidence from C. elegans revealed that miRNA biogenesis genes are highly networked with transcription factors and aging-associated miRNAs. For instance, mir-71 is upregulated during aging, repressing ALG-1/AGO expression post-transcriptionally. Reversely, mir-71 loss of function leads to increased ALG-1 expression followed by a widespread mRNA expression dysregulations. Thus, the progressive molecular decline often thought to be the result of accumulated damage over an organism's lifespan, may be partially explained by a miRNA-directed mechanism of age-associated decline273 . Additionally, Christopher et al investigated the liver transcriptome changes in mice model of dietary plus lifestyle interventions that modify aging-related phenotypes. By high-throughput deep sequencing, they found a significant larger proportion to have decreased expressions of miRNAs with longer lifespan. Furthermore, they experimentally validated mir-34a, mir-107 and mir-212-3p targeted the chromatin remodeler CHD1, knockdown of which mimic high-fat diet and aging induced gene expression profiles 274 .

  Nowadays, it has been demonstrated that senescent cells accumulate in various tissues and organs overtime demonstrated by accumulations of senescence markers such as senescence-associated β-galactosidase (SA-β-gal), p53 and cyclin-dependent kinase inhibitors p21 and p16. Senescence contributes to aging process. For example, Darren J.B. et al has shown that genetic or pharmacological removal of p 16Ink4a -positve cells (senescent cells) would induce extended lifespan associated with reduced pathological alterations in various organs such as adipose tissue, heart and kidney 301 .It has been demonstrated that various stimuli could induce cellular senescence. On the one hand, the telomeric DNA loss during each S phase would result in telomeres erosion, which then generates a persistent DNA damage reponse (DDR). DDR then could further reinforce the senescence-related growth arrest 302 . On the other hand, senescence could also occur without DDR development under various culture stressors such as serum deprivation or oxidative stress, etc 303 .

Figure 11 :

 11 Figure 11: Hallmarks of senescent cells. Hallmarks of senescent cells include an essentially irreversible growth arrest; expression of SA-Bgal and p16; robust secretion of SASP); nuclear foci contanining DDR proteins (DNA-SCARS/TIF) or heterochromatin (SAHF). Adapted from 304 .

2. 2 .

 2 miRNAs in p53/RB tumor-supressor pathways Proliferative arrest, one of hallmarks of senescent cells, is induced by both endogenous and external influences that ultimately converge on either or both of the p16 INK4a / RB and p14 ARF / p53 pathways. The binding of p16 to the cyclin-dependent kinase 4-6/ cyclin D complex inhibits the phosphorylation of pRb family proteins and causes a G1 cell cycle arrest (Figure 12). miRNAs upstream of p53/ RB pathways In senescence, let-7/agronaute2 (AGO2) signaling induced implementation of silentstate chromatin modification at target promoters. Inhibition of the let-7/AGO2 effector

Figure 12 :

 12 Figure 12 : The p53 and RB tumorsuppresor pathways.

  their expressions. Similarly, in human mammary epithelial cells, senescence-associated miRNAs (SA-miRs) such as mir-26b, mir-181a function in concert to repress expressions of polycomb group proteins, embryonic ectoderm development or enhancer of zeste homologue 2, thereby activating expression of p16 and senescence308 . Likewise, amongst the human cancer-associated mir-17-92 cluster, mir-17/mir-20a seed family are necessary and sufficient for conferring resistance to oncogene-induced senescence, by directly targeting p21 WAF1309 . Senescence in the type II alveolar epithelial cells (AECs) contributes to the development of idiopathic pulmonary fibrosis (IPE). The upregulation of SA-miRs including mir-34 family and mir-20a, mir-29c and let-7f might participate in the induction of senescence in AECs, with its ectopic overexpression inducing senescence markers: p16, p53 and SA-β-gal 310 . In addition, induction of mir-29 and mir-30 is necessary for the RB pathway driven-senescence through targeting and inhibition of the 3'UTR of B-Myb 311 , a transcription factor that negatively regulates senescence 312 . Moreover, mir-29 overexpression induced by Wnt-3a in muscle progenitor cells (MPC) during aging, induces senescence via targeting the 3' UTR of p85α, IGF and B-Myb, suppressing the translation of these mediators of myoblast proliferation. Finally, electroporation of mir-29 into muscles of young mice recapitulates aging-induced responses 313 . Amongst these SA-miRs in human keratinocytes (NHKs), ectopic overexpression of mir-137 or mir-668 induced senescence in rapidly proliferating NHKs, demonstrated with increased SA-β gal activity and expression of p16 and p53 314 . P16 could improve and stabilize the levels of p53 through negatively regulated p53 suppressor MDM2 oncoprotein, which was mainly mediated by mir-141 and mir-146-5p dependent negative regulation of MDM2 315 .  miRNAs downstream of p53/RB pathways On the other hand, some miRNAs are identified as down stream effectors of senescence marker genes. Recent evidence indicated that p16 is a modulator of gene expression. It has been demonstrated that p16 could contribute to senescence through modulating miRNAs expression. For instance, through the formation of the p16-CDK4-Sp1 heterocomplex which binds to the Sp1 consensus-binding motifs present in the promoters of mir-141 and mir-146b-5p, p16 enables the transcription of mir-141 and mir-146b-5p, which mediate p16's response to ultraviolet (UV) induced DNA damage 316 . 2.3. miRNAs in DNA damage pathways The most significant phenotype associated with mammalian cellular aging is telomere shortening which induces disruption of chromosome integrity by DNA damage and apoptosis 317 . Telomerase lengthens telomeres in DNA strands by adding DNA sequence repeats (TTAGGG in all vertebrates) to the 3' end of DNA strands in the telomere regions, which are found at the ends of eukaryotic chromosomes, thereby preventing senescent cells from postmitotic condition and apoptosis 318 . However, aging is linked with reduced telomerase activity which reduces the regenerative capacity of proliferative organs and increases incidences of diseases onset and development 319 . Interestingly, mir-195 was overexpressed in old mesenchymal stem cells (OMSCs) in mice, which targeted telomerase reverse transcriptase. Abrogation of mir-195 in OMSCs resulted in increased length of telomere in the nucleus, markedly improving their proliferative abilities. Moreover, transplantation of mir-195-knockout OMSCs reduced infarction size and improved heart function in mice 320 . MiRNAs are also involved in the regulation of cellular reactive oxygen species (ROS) level which is major contributor to DDR. Surprisingly, in human HEK293 cells, induction of DDR by exposure to H 2 O 2 resulted in phosphorylation of dicer (the miRNA producing enzyme) and subsequent nuclear recruitment of dicer to promote DNA repairment mediated by repair factors MDC1 and 53BP1 321 , indicating that some miRNAs might be involved in this process.

3. 1 .

 1 miRNAs affecting caloric restriction process Similar to caloric restriction (CR), intermittent fasting extends lifespan of C. elegans and also mammals. Interestingly, the expressions of miRNAs machinery genes, such as Drosha, Argonaute and GW 182, are increased by this process. Conversely, the beneficial effects of fasting would be totally abrogated with Drosha depletion. These data, therefore, imply that miRNAs abundance might be upregulated by CR and be positively associated with CR-linked longevity. Microarray data further propose DAF-16, an effector of insulin/IGFsignaling, might be a downstream target of cellular miRNAs abandance's pro-longevity action331 . So, what are the miRNAs involved in CR in diverse organisms? Firstly in C.elegans, it has been reported that mir-80 is a major regulator of the CR process. Mir-80 depletion conferred healthy aging, including maintained cardiac-like and skeletal muscle-like function at advanced age, and reduced accumulation of lipofuscin and extended lifespan, coincident with induction of physiological features of CR. Mechanistically, the expression of mir-80 is positively associated with the food availability. The acetyltransferase transcription co-factor CBP-1, interacting transcription factors daf-16/FOXO and heat shock factor-1 are essential for mir-80 deficiency's benefits 332 (Figure10).

Figure 13 :

 13 Figure 13: Simple illustrations of microRNAs' participations in calorie restriction process in different species.

Figure 14 :

 14 Figure 14: Examples of effects of microRNAs in sirtuins during aging or senescence. (A) Aging-associated increase in mir-34a promotes p53 expression and apopotosis through inhibition of SIRT1. (B) Loss of mir-15b accounts for upregulation of mitochondrial ROS production by increased SIRT4 in cellular senescence. (C) Loop regulations of mir-766 and SIRT6 during aging.

Figure 15 :

 15 Figure 15 : 3T3-F442A adipocytes differentiated from 3T3-F442A fibroblasts.

Figure 16 :

 16 Figure 16: Isolation of SVF from adipose tissue with collagenase digestion and centrigugation for in vitro adipogenesis.

Figure 17 :

 17 Figure 17: 3D adipogenesis of 3T3-L1 cells inelectrospun polycaprolactone scaffolds. (A) Once hydrated the scaffold can be easily handled with fine scissors. (B) SEM observation of the dehydrated scaffold. (C) Observations of growing cells and fluorescence images of the live/dead assay at day 28 (scale bar: 100μm). Data is from 357 .

Figure 18 :

 18 Figure 18: Formation of spheroids of 3T3-L1 adipocyte aggregates on surface tethered with ELP-PEI. 3T3-L1 cells were differentiated on surface tethered without (left) or with (right) ELP-PEI. Adopted from 358 .

Figure 19 :

 19 Figure 19: Ceiling culture of isolated adipocytes. (a) Experimental scheme ofceiling culture. (b-d) SEM observations of adipocytes gradually change the unilocular LD into multiple LD, scale bar, 10μM. Adopoted from 359 .

Figure 1 :

 1 Figure 1: Inducible reduction of dicer in adipocytes by tamoxifen in dicer Lox/Lox / adiponectin-CreER T2+ mice. (A) Genotyping results of dicer Lox/Lox / adiponectin-CreER T2 transgenic mice. (B) Experimental protocol: around

Figure 2 :

 2 Figure 2: Reduction in the average sizes of perigonadal adipocytes due to dicer deficiency. (A) Body weight between the two different genotypes at the end of the sacrifice. (B) Body fat proportion of the mice measured by EchoMRI at the end of the protocol. (C and D) Perigonadal and subcutaneous fat to body weight ratios. (E) H&E staining of the perigonadal and subcutaneous adipose tissue. (F and G) Average cell sizes of perigonadal and subcutaneous adipocytes. (G) Dramatic downregulations of white adipocyte gene markers associated with dicer decline in adipocytes by qPCR. (H) Reduced protein level of HSL in perigonadal fat revealed by immunoblotting. (I) Reduced protein level of PLIN1 in perigonadal adipocytes exhibited by IF staining. Data represent the mean ± SEM, *P<0.05, **P<0.01. N=4-8.

Figure 3 :

 3 Figure 3: Possible involved mechanisms underlying the effects of adipocyte dicer deficiency. (A) RT-qPCR of genes involved in mitochondrial functions in perigonadal adipocytes. (B) Protein level of PGC1α in perigonadal adipocytes. (C) Quantification of protein levels of OXPHOS components in perigonadal adipocytes. (D) Downregulations of antioxidant genes of Sod and Catalase due to dicer deficiency in perigonadal adipocytes. (F) Increased phophorylations of FOXO1 and FOXO3a in perigonadal adipose tissue due to reduced adipocyte dicer deficiency. (G) Autophagy gene Bnip3 was negatively affected in perigonadal adipose tissue due to dicer deficiency. Data represent the mean ± SEM, *P<0.05, **P<0.01. N=4-8.

Figure 4 :

 4 Figure 4: Adipocyte dicer deficiency promoted lipid accumulation and fibrosis in liver. (A) The result of H&E staining suggested that adipocyte dicer deficiency resulted in lipid accumulation in liver. (B) Increased mRNA levels of genes involved in DNL pathway in liver as a result of dicer deficiency in adipocytes. (C) Increased levels of PPARγ and FAS in liver due to adipocyte dicer deficiency by immunobot. (D) Sirius red staining of liver exhibited increased fibrosis as a result of adipocyte dicer deficiency. (E) mRNA levels of genes involved in fibrosis and inflammation in liver. Data represent the mean ± SEM, *P<0.05, **P<0.01. N=4-8.

Figure 5 :

 5 Figure 5: Regulation of dicer level in adipose tissue and in in vitro adipocytes. (A) 24h fasting significantly increased mRNA level of dicer in perigonadal adipose tissue in wildtype mice (N=5). (B) Overnight glucose deprivation significantly increased dicer protein level in 3T3-F442A adipocytes (N=3). (C) Fetal bovine serum contents negatively affected dicer protein levels in 3T3-F442A adipocytes (N=3). (D) Overnight stimulation of 10 -6 isoproterenol did not increase dicer protein expression as serum starvation (N=3). (E) Overnight treatment of 1mM AICAR might increase dicer protein level in 3T3-F442A adipocytes (N=2). (F) Overnight treatment of various concentrations of AMPK inhibitor compound C differentially affected dicer protein level in 3T3-F442A adipocytes (N=2). Data represent the mean ± SEM, *P<0.05.

Figure 6 :

 6 Figure 6: Age-associated increase of mir-1949. (A and B) 8 candidates from microarray were further validated by qPCR in PG and SC from wild type mice of different ages (N=5-6 per group). (C and D) Further qPCR validation of mir-1949 expressions in whole PG, adipocytes and SVF from PG and in whole SC, adipocytes and SVF from SC, respectively (N=5-6). (E) Levels of mir-1949 in CM from ex vivo cultured PG isolated from mice of different ages was detected by qPCR (N=3-6 per group). (F and G) Senescence in 3T3 adipocytes induced with 0.2M glucose exposure for one week demonstrated with p16 elevation and SA-β staining (representivative of 3 independent manipulations). (H and I) elevated levels of mir-1949 in both senescent 3T3 adipocytes as wellCM from senescent 3T3 adipocytes (N=3).

  by complex II stimulated by its substrate succinate was significantly reduced by mir-1949 upregulation (Figure7D) in 3T3-F442A adipocytes. Therefore, mir-1949 might negatively regulate mitochondrial complex II level and function in adipocytes in vitro. Since mitochondria activation participates in the determination of lipid contents in adipocytes, we went on to investigate whether lipid contents was affected by mir-1949. Interestingly, BODIPY staining suggested that upregulation of mir-1949 promoted lipid accumulation in 3T3-F442A adipocytes (Figure7E). Moreover, upregulation of mir-1949 during adipogenesis by consecutively transfecting 3T3-F442A fibroblast every two days within 10 days , had a trend to increase classical white adipocyte gene markers such as Leptin and Glut 4 (n=2) (Figure7F).

Figure 7 :

 7 Figure 7: In vitro actions of mir-1949 on adipcoytes. (A) 3T3 differentiated adipocytes were transfected with 10nM of the mimic sequence of mir-1949 or a negative control sequence. The levels of mir-1949 in adipocytes were detected 72 hours post transfection (N=3). (B) Levels of mRNAs were measured in 3T3 adipocytes by qPCR 72 hours post transfection (N=3). (C) Protein levels of OXPHOS in 3T3 adipocytes were detected by immunoblots 72 hours post mir-1949 mimic transfection (N=3). (D) Succinate stimulated oxygen consumption by mitochondrial complex II was measured in 3T3 adipocytes 72 hours post transfection (N=3). (B) 3T3 adipocytes were stained by BODIPY 72 hours post transfection of mir-1949 mimic (representative of three independent manipulations). (F) 3T3 preadipocytes were consecutively transfected with 10nM mir-1949 mimic within differentiation. Then gene markers for adipocytes were detected by qPCR (N=2). Data represent the mean ± SEM, *P<0.05, ***P<0.001.

  dicer deficiency increased the phosphorylation of both FOXO1 and FOXO3a, indicating that adipocyte dicer deficiency might negatively affect the FOXOs activities. FOXOs are key regulators of cellular response to various stresses and have been identified as a concerved molecule in promoting healthy aging383 . One of the mechanisms that FOXOs contribute to longevity is through regulation of autophagy384 . For example, FOXO1 and FOXO3 active autophagy in various cell types to help to maintan protein homeostasis385 . Then, the result that dicer deficiency decreased authophagy gene maker Bnip3, further supported the idea that adipocyte dicer deficiency might lead to FOXOs inactivation. Since autophagy is also an important process removing organelles inside cells, it remains to be solved whether the inhibition of autophagy by dicer deficiency in adipocytes might have a role in mitochondrial degradation in vWAT.

Figure 8 :

 8 Figure 8: Proposed molecular mechanisms underlying adipocyte dicer in aging. (A) The protein level of pP53 in perigonadal adipose tissue in control and iADicer KO mice. (B) Simple scheme of possible mechanisms underlying adipocyte dicer in aging.

3 .

 3 Finally, whether overexpression of FOXO1 or FOXO3a specifically in adipocytes (achieved by injection of control AAV sequence or AAV-adiponetin-dicer into iADicer mice) will prevent the onset of age-related complications in iADicer KO mice? B. Our results suggested that adipocyte dicer deficiency promotes liver aging.

Figure 9 :

 9 Figure 9: GFP expression in adipocytes from ZsGreen lx/lx /adiponectin-CreER T2+ mice by 4-hydrotamoxifen.Perigonadal adipocytes were isolated from ZsGreen lx/lx /adiponectin-CreER T2+ mice and cultured in vitro. GFP expression was induced by 4-hydrotamoxifen treatment.

Figure 10 :

 10 Figure 10: Possible role of mir-1949 in the prevention of age-related dysfunctions. (A) Adipocyte senescence was induced by exposure to 0.2M glucose for one week. Then, senescent adipocytes were transfected with 10nM of the mimic sequence of mir-1949 or a negative control sequence (N=1). (B) Mir-1949 level in perigonadal adipose tissue in wildtype mice fasted for 24h (N=5). Data represent the mean ± SEM, *P<0.05.

[ 3 H

 3 ]2-deoxy-D-glucose (NEN, Boston, MA) for 10 minutes precisely and stopped by placing the cells on ice and rapidly washing them with ice cold 10mM glucose in PBS. Cells were lysed with 0.05N NaOH. Aliquots of the cell lysates were used for liquid scintillation counting and determination of protein contents by the Bradford method (Bio-Rod), respectively.Senecence-associated β-Galactosidase staining (SA-β-gal)Β-gal activity was detected at pH 6, a known characteristic of senescent cells not found in quiescent or immortal cells with commercial kit from Cell signaling TECHNOLOGY (Ref: 9860).

Fig 1 :

 1 Fig 1: Illustration of the working scheme. (A) Application of cell inserts to sequester buoyant adipocytes from floating for culturing in vitro. (B) Possible influence of the homeostasis of adipocyte cytosol by environmental tonicity. (C) The chemical structure of glycerol.

Fig 2 :

 2 Fig 2: Image of adipocytes in culture under different condtions. (A) PHK26 and DAPI staining demonstrated that cultured adipocytes maintained the integrity of structures with normal cell membrane and peripherally located nucleus at D10 of in vitro culture. (B, C and D) Microscopic observations show that cultured adipocytes exhibted ball-like 3D spherical structures, with DAPI identified an extreme small and peripherally located nucleus and bodipy staining demonstrated that a single LD took up nearly of all of the cytoplasm at D2 of in vitro culture. (E) Adipocytes were cultured with different concentrations of glycerol (2.5%, 5% and 10%) and were observed and imaged daily. Tunel assay were performed 2 weeks after to detect apoptosis. (F) Adipocytes were cultured with different concentrations of FBS (0, 10%, 15% and 20%) and were observed and imaged daily. Tunel assay were performed one week after to detect apoptosis. Representiative of 3 independent manipulations.

Fig 3 : 3 H

 33 Fig 3: Functional investigation of adipocyte in vitro. (A) Adipocytes at D5 of culture were previous starved in DMEM overnight followed by stimulation of 100nM insulin. Phosphorylation of Akt and total Akt were detected with immunoblot. (B) Adipocytes at D2 of culture were recovered into 1.5 ml tube, rinsed with DMEM, and then were stimulated with different concentration of insulin for 45 min, followed by incubation with 200mM 2-NBDG for 60 min. (C) Adipocytes at D5 of culture were previously starved in DMEM overnight followed by stimulation of 100 and 1000nM insulin. [ 3 H] 2-deoxy-D-glucose was used to evaluate glucose uptake. (D) Adipocytes at D8 of culture were previously starved in DMEM for 4h followed by stimulation of 0.1 and 1μM isoproterenol. Glycerol release in the medium was determined. Data represent the mean ± SEM, *P<0.05, **P<0.01.

Fig 4 :

 4 Fig 4: Characterizations of adipocytes during chronic culture in vitro. (A) Triple stainings of adipocytes with BODIPY, PLIN1 and DAPI at the D13 of in vitro culture. (B) Alterations of cell sizes during culture. (C) Alterations of gene expressions in adipocyte during culture. (D) Applications of the method to culture human adipocytes.

  ChemiDoc XRS+ system (Bio-Rad). Information about sources of antibodies was listed as following: GAPDH (Ref: mAb #2118, Cell Signaling TECHNOLOGY (CST)); Phospho-Akt (ser473) (Ref: 9271); AKT (Ref: 9272, CST).

Table 1 :

 1 The average sizes of adipocytesin developing and adult human ............................................

Table 2 :

 2 List of miRNAs involved in adipogenesis .................................................................................

Table 3 :

 3 MiRNAs involved in age-associated cardiovascular diseases .................................................

Table 1 :

 1 Plasma parameters (fed) .........................................................................................................

	eNOS	Endothelial NO systhesis
	EVs	Extracellular vesicles
	FABP4	Fatty acid binding protein 4
	FABPpm	Plasma membrane fatty acid binding protein
	FAT10	HLA-F adjacent transcript 10
	FAT/CD36	Fatty acid translocase
	FATP	Fatty acid transport protein
	FAS	Fatty acid synthesis
	FFAs	Free fatty acids
	FPLD	Familial partial lipodystrophic syndromes
	FOXO	Forkhead box O
	FGF21	Fibroblast growth factor 21

White adipose tissue and aging ________________________________________ 1. The adipose tissue ______________________________________________________

	1.1. Subcutaneous adipose tissue (sWAT) ______________________________________________
	1.2. Visveral adipose tissue (vWAT) ____________________________________________________
	2. White Adipocytes ______________________________________________________
	2.1. Architecture __________________________________________________________________________
	2.2. Homeostasis of lipid droplets in adipocytes _______________________________________
	2.2.1. Synthesis of lipid droplets (LDs) ___________________________________________________
	2.2.2. Hydrolysis of lipid droplets _________________________________________________________
	2.2.3. Lipid driplets "burning" _____________________________________________________________
	2.3. Endocrinal functions _________________________________________________________________
	2.4. Origins of white adipocytes _________________________________________________________
	2.4.1. Adipocyte progenitor cells (APCs) __________________________________________________
	2.4.2. Adipogenesis _________________________________________________________________________

3. Alterations of WAT by aging ______________________________________________

3.1. Senescence of adipocyte progenitors_______________________________________________ 3.2. Altered lipid turnover _______________________________________________________________ 4. Contribution of WAT to aging _____________________________________________ 4.1. Nutrition/age-associated obesity attenuates longevity___________________________ 4.2. Removal or mobilization of vWAT increases longevity ___________________________ 4.3. Novel insights from microRNAs ____________________________________________________

PART II: MicroRNAs in white adipose tissue _____________________________________ 1. Biogenesis and regulation of microRNAs ____________________________________

	PART III:
	1.1. Biogenesis of microRNAs ____________________________________________________________
	1.2. Regulation of miRNAs expression in adipose tissue ______________________________
	2. MiRNAs in the formation of adipose tissue __________________________________
	2.1. MiRNAs in differentiation of adipocytes progenitors _____________________________
	2.2. Determination of adipocytes identities ____________________________________________
	2.3. Involvement in adipose tissue functions ___________________________________________

MiRNAs in aging ___________________________________________________ 1. Dysregulations of miRNAs in aging ________________________________________

	1.1. MiRNAs involved in overall longevity ______________________________________________
	1.2.1. Age-associated cardiovascular diseases ___________________________________________
	1.2.2. Age-associated sarcopenia and osteoporosis ______________________________________
	1.2.3. Age-related liver dysfunctions ______________________________________________________
	1.2.4. Age-related immune defects ________________________________________________________
	2. MiRNAs to senescence __________________________________________________
	2.1. Cellular senescence __________________________________________________________________
	2.2. miRNAs in p53/RB tumor-supressor pathways ___________________________________
	2.3. miRNAs in DNA damage pathways _________________________________________________
	3. MiRNAs in conserved aging ______________________________________________
	3.1. miRNAs affecting caloric restriction process ______________________________________
	3.2. miRNAs affecting sirtuin families ___________________________________________________
	PART IV:

A novel method of culturing isolated adipocytes __________________________ 1. Current in vitro models of adipocytes_______________________________________

1.1. Two-dimensional (2D) adipogenesis of preadipocytes ___________________________ 1.2. Three-dimensional (3D) adipogenesis of preadipocytes _________________________ 2.

First attempt to culture isolated adipocytes _________________________________ Result 1: Role and regulation of microRNAs in adipose tissue during aging ____________ Result 2: A novel method of culturing isolated adipocytes in vitro __________________ Bibliography _____________________________________________________________ GENERAL INTRODUCTION Introduction PART I: White adipose tissue and aging 1.

Table

  [START_REF] Bluher | Role of insulin action and cell size on protein expression patterns in adipocytes[END_REF]

Table 1 : The average sizes of adipocytesin developing and adult human

 1

a 25-30 weeks gestation, b 1-3 months postpartum. Adopted from 30 .

  cancers and diabetes148 . Interestingly, a strong correlation between high BMI and the "epigenetic age" of liver in human exists, which might help to explain why obese people suffer from the early-onset of many age-related pathologies, such as liver cancer 149 . Indeed, the group of Steve Horvath has recently developped a biomarker of aging ("epigenetic clock") based on DNA methylation (DNAm) levels 150 , which is defined as a prediction method of chronological age based on the DNAm levels of 353 CpGs, also referred to as "DNAm age". The use of DNAm age could be seen as a proxy of epigenetic tissue age.Finally, obesity has been shown to induce DNA damage in adipose tissue via activation of p53 pathway 151 . Moreover, disorders derived from energy metabolism might also negatively influence age-related pathogenesis. For example, Alzeihmer's diseases patients are very often characterized by visceral fat accumulation, insulin resistance and abnormal secretion of leptin and adiponectin 152 .

tissue is at the nexus of mechanisms and pathways involved in longevity and genesis of age-related diseases by general mechanisms such as inflammation or metabolic dysfunction. It has been proven that genetic profiles of WAT may affect lifespan 146 . For instance, PPARγ signaling pathway is revealed as the most significantly enriched biological pathway within the aging sub-network. In agreement, two independent PPARγ knockout mice models (one lacks PPARγ ½ exclusively in WAT and the other lacks PPARγ2 in all tissues), exhibit severe lipodystrophy and remain insulin-resistant, both having a significant reduction in lifespan 147 . Under extreme conditions of adiposity depletion, e.g., lipodystrophy syndromes such as genetic Berardinelli-seipcongential generalized lipodystrophy (BSCL) or familial partial lipodystrophic syndromes (FPLD), patients are markedly short-lived, suggesting that malfunctions of adipose tissue affect life quality and longevity. 4.1. Nutrition/age-associated obesity attenuates longevity  The link between excessive fat development and aging Obesity is defined by a body mass index over 30 (BMI, an indicator of body fat calculated by dividing a person's weight in kilograms by their height in meter squared). Pooled analysis data of 20 prospective studies from the United States, Sweden and Australia, reveal that people with Class III obesity (BMI> 40 kg/m 2 ) have a dramatic reduction in life expectancy compared with people of normal weight, with most of the excess death due to heart diseases,  Age and adipose tissue Besides the nutrition-induced obesity, age-related central obesity, which is accounted mainly by fat accumulation in visceral depot, is also negatively associated with insulin sensistivity. Different studies showed that aging per se does not drive this phenomenon, but it's rather the augmented visceral fat during aging that is truly responsible for this agerelated insulin resistance 153 . Independently of other depots, vWAT plays an important role in modulating hepatic insulin action 154 . One of the most distinguished features of vWAT is that it is believed to be a source of harmful peptides such as resistin, PAI-1 and angiotensinogen, which might contribute to various pathological states in peripherical organs 155 . For instance, aging induces cardiac structural and functional changes associated with increased deposition of ECM proteins, such as osteopontin (OPN), resulting in progressive interstitial fibrosis. Visceral WAT strongly mediate this process 156 . Indeed, it has been demonstrated that plasma OPN is increased in mice during aging, with vWAT showing the strongest OPN production in contrast to myocardium that does not express this protein. Moreover, vWAT removal or OPN inhibition greatly reverses senescence in cardiac fibroblasts 156 .

  Ambros et al discovered the first miRNA, lin-4 in C.elegans, which was identified as a small non-coding RNA regulating the expression of the protein lin-14177 . Several years later, let-7, another famous miRNA was discovered in C. elegans, negatively regulating the expression of the gene lin-41 via RNA-RNA interactions with the 3'-UTR of lin-41178 . Subsequent reports further unveiled that miRNAs were abundant in both

	1.	Biogenesis and regulation of microRNAs
		1.1.	Biogenesis of microRNAs
		MicroRNAs (miRNAs) are a family of small non-coding RNAs that post-
	transcriptionally regulates mRNAs levels or stability by pairing to the 3'-untranslated regions
	(UTR) of their targets. In 1993,

invertebrates and vertebrates, with some are highly conserved, suggesting that miRNAmediated gene regulation is a general regulatory function across species

179,180

. Currently, there are up to 1872 annotated human miRNA precursor genes that are processed into around 2578 mature miRNA sequences (http://www.mirbase.org) with functions of lots of them are waiting to be clarified.

Table 2 : List of miRNAs involved in adipogenesis

 2

	miRNAs	Model	of	Targets/mechanisms		Pro/anti-	Reference
		adipogenesis				adipogenesis
	Mir-143-3p	ADSC		Repressing MAP2K5		Dual
	Mir-15a/b	Porcine		Repressing FOXO1		Pro
		preadipocytes
	Mir-17-92 cluster 3T3-L1		Down-regulating Rb2/p130	Pro
	Mir-30a/d	hMADS		Inhibition of the osteogenesis	Pro
				TR RUNX2
	Mir-30c	hMADS		Targeting PAI-1 and ALK2		Pro
	Mir-103	3T3-L1		Targeting	MEF2D	and	Pro
				activation of AKT/mTOR
	Mir-17-5p/mir-	hMADS		Suppressing osteogenesis via	Pro
	106a			BMP2
	Mir-146b	3T3-L1		Targeting the 3' UTR of SIRT1	Pro
				and induced acetylation of
				FOXO1
	Mir-342-3p	Human	adipose-	Releasing	C/EBPα	from	Pro
		derived MSCs	Binding with CtBP2
	Mir-355	Ob/ob, db/db, KKAy	Levels in WAT correlated with	Pro
		mice		body weight and adipocyte
				differentiation markers
	Mir-455	3T3-L1		Targeting UCP1		Pro
	Let-7	3T3-L1		Induction	during	Anti
				differentiation,
				Unclear
	Mir-27a	3T3-L1		Suppressing	PPARγ	Anti
				expression via 3' UTR
	Mir-27b	Zebrafish/ 3T3-L1	Negatively correlated with	Anti	207,208
				the levels of as PPARγ,
				C/EBPα and SREBP-1c.
	Mir-29a/b/c	3T3-L1		Targeting		DNA Anti

gluteofemoral and in subcutaneous abdominal fat in humans, with its level more abundant in adipocytes of the gluteofemoral region 218 . The favorably expressed mir-196a in gluteofemoral adipocyte might negatively regulate the homebox (HOX) family genes, which are involved in the transcriptional regulation of adipogenesis and less abundant in gluteofemoral fat

218

. These results indicate that the abundance of mature miRNAs is vital in the determination and maintenance of identities of different adipocytes, and linked to their intrinsic properties and functions.

Table 3 : MiRNAs involved in age-associated cardiovascular diseases

 3

	miRNAs	Organ		Direction Targets		Function	Reference
	Mir-29 family	Aortas/		up	ECM components:	Aortic	aneurysm
	(-29a,-29b,	WTmice			Collagens/		formation
	-29c)				elastin
	Mir-217	HUVECs		up	Sirt1/FoxO1		Pro-senescence
	Mir-34a	Heart/	wt	up	PNUTS(PPP1R10)	Induction	of
		mice/ ku80-/-				cardiomyocyte
		mice					apopotosis, telomere
							attribution and DDR.
	Mir-22	Cardiac		up	mimecan		Induction	of
		fibroblast/					senescence	and
		C57/B16N					chemotaxis
	Mir-17-3p	Mouse		down	PAWR/CEBP	and	Anti-senescence and
		cardiac			FAK signaling		apoptosis
		fibroblasts
	Mir-125a-5p	Endothelial	up	Transcriptional		Angiogenesis defects
		cells/	WT		enhancer factor-1/
		mice			eNOS/VEGF
	Mir-146a	Endothelial	down	Possible targets:	unvalidated
		cells/in vitro		NOX4
		human
	Mir-217	Human		up	Sirt1		Pro-senescenece
		HUVEC	in
		vitro

(TGFβ) signaling, indicating that age-associated mir-431 plays a key role in maintaining the myogenic capacity of sketetal muscle with age 290 .

Table 1 : Plasma parameters (fed)

 1

		Control	iADicer KO
	Glucose (mg/dl)	203.04 ± 11.59	192.1 ± 9.28
	TG (mg/dl)	1.16 ± 0.16	0.90± 0.26
	Glycerol (mM)	0.083 ±0.0089	0.114±0.0076*
	NEFAs (mmol/L)	0.60± 0.086	0.34± 0.084
	Cholesterol (mmol/L)	1.58 ±0.059	1.37±0.20
	ALT (U/L)	22.03 ±0.033	23.25± 1.74
	AST (U/L)	59.33 ±12.84	62.75± 3.48
	Insulin (µg/ml)	1.53 ± 0.54	3.82± 0.90
	Data represent the mean ± SEM, *P<0.05. N=4-8.

  The fact that caloric restriction promotes healthy aging in diverse species reveals that systemic metabolism is crucial in longevity373 . In following, several conserved nutrientsensing pathways, such as, insulin-like growth factor-1 signaling, mTOR, AMPK and sirtuins have been identified as concerved moleculars in aging374 . Aging witnesses dysfunctions in metabolisms. Specifically, during glucose tolerance test, old people could not handle the glucose stimulation as well as the young, needing to produce significant more insulin to get rid of the exogenous glucose375 , indicating that that the peripheral metabolic tissues have a reduced efficiency to utilize the glucose. So, what kind of tissues responsible for this phenomenon? Currently, plenty of researches have indicated that aged liver and age muscle make contributions to the insufficient insulin sensitivity in aging[376][377][378][379] . For example, senescence drives lipid accoumulation and thereby steostasis in liver impairing its metabolic activities 376 .However, relative less attention has been put on another important tissue in metabolism, which is white adipose tissue, one of the largest human organs 124 . Adipose tissue is crucial in the regulation of systemic energy homeostasis. Despite the classic view that adipose tissue as an inert lipid tanker, elevating novel molecules in adipose tissue are

	revealed to process vital functions in recent years. In 2012, the work of Ronald Kahn's group
	demonstrated that dicer was reduced in adipose tissue as well as in isolated adipocytes in
	aging in mice 174 , indicating that dicer/miRNAs might be novel molecules linking adipocytes
	and aging process.

  Therefore, in my second part, we aimed at the selection of age-

	mir-1949 from adipocytes. Moreover, in vitro data indicated that adipocytes senescence also
	increased mir-1949 production and secretion from adipocytes.
	Funtionally, however, we did not observe significant effects of this miroRNA in
	glucose transport and lipolysis in normal adipocytes in vitro. Interestingly, PGC1α was
	significantly reduced by mir-1949 upregulation in 3T3-F442A adipocytes, indicating that mir-
	1949 might affect mitochondria in adipocytes. Susequently, the results that mir-1949
	upregulation led to a reduction in mitochondrial complex II protein level and oxygen
	consumption ability, further suggesting that mir-1949 might negative regulate mitochondria
	in adipocytes.
	Interestingly, Bodipy staining suggested that upregulation of mir-1949 promoted lipid
	accumulation in adipocytes, coinside with that mir-1949 negatively regulates mitochondria
	and thereby a reduced lipid utility. Interestingly, at the same time, continuous upregulation
	of this miroRNA during adipogenesis of 3T3-F442A preadipcoytes, exhibited a trend to
	increase the expression of classical white adipocyte gene markers, indicating that mir-1949
	might promote lipid storage in adipocytes. Moreover, this effect was only observed at the
	end of differentiation but not during differentiation (data not shown), suggesting that
	mature adipocytes are more sensitive to mir-1949's effects.
	Then, interestingly, our preliminary results that upregulation of mir-1949 tended to
	increase lipid accumulation in senescent adipocytes as well as nutrient deprivation
	significantly increased its level in perigonadal adipose tissue in mice (Figure 10A and 10B),
	lead us to hyphothezse that this age-related microRNA might try to resuce age-related
	dysfunctions in adipocytes by modulating lipid storage capacity as well mitochondrial
	activities.
	Therefore, to further validate this hypothesis, in vivo experiments such as injection of
	AAV-aP2-mir-1949 into old mice (specific upregulation of mir-1949 in adipocytes), would be
	helpful to determine whether specific upregulation of this microRNA in aged adipocytes
	would help to rescue age-related complications in mice. In vitro, to better characterize the
	related miRNAs in adipocytes. actions of mir-1949 on senesencet adipocytes, such as, gene profiles, functions, etc.
	Interestingly, we firstly reported that aging increased mir-1949 expression in
	adipocytes in mice. Additionally and possiblity, aging might also promote the secretion of

EXPERIMENTAL RESULTS

Project I:

Role and regulation of microRNAs in adipose tissue during aging Result 1: Role and regulation of microRNAs in adipose tissue during aging

A. Validation of the inducible adipocyte dicer knockdown mice model

The AdipoCreER T2 -Dicer Lox/lox mice were obtained by crossing Adiponectin-CreER T2 mice and Dicer lox/lox mice. After breeding and backcrossing the strain over seven generations, we obtained a colony of AdipoCreER T2 -Dicer Lox/lox mice. After genotyping, around six-monthold male mice bearing homozyotes of dicer flox alleles plus adiponectin-CreER T2 heterozygotes were selected and paired (Figure 1A). As this construction contains an ERT2 cassette rendering it inducible, mice were subjected to three times of tamoxifen (200mg/kg) gavage within approximately two months as already described 364 (Figure 1B). Protein levels of dicer were demonstrated to be down regulated at the end of the protocol in Dicer Lox/lox / adiponectin-CreER T2+ (thereafter termed iADicer KO) mice than that in Dicer Lox/lox / adiponectin-CreER T2-(thereafter termed control) mice (Figure 1C and1D). RT-qPCR results further demonstrated that dicer levels were significantly reduced by around 30% to 40% with tamoxifen treatment in adipocytes isolated from perigonadal and subcutenous adipose tissue from the group of iADicer KO mice compared to that in control mice (Figure 1E and 1F, respectively). Correspondingly, mir-143, one of the adipocytes enriched microRNAs, was significantly decreased by around 40 to 50% in perigonadal adipocytes in iADicer KO mice compared to that from control mice (Figure 1G). this miRNA. To test this hypothesis, we set up an in vitro senescence model of adipocytes by long term exposure of 3T3 adipocytes to 0.2M glucose. Upregulations of senescence markers such as p16 expression (Figure 6F) and SA-β-galactosidase staining (Figure 6G) demonstrated the development of senescence in adipocytes treated by 0.2M glucose. Interestingly, mir-1949 was both upregulated in senescent adipocytes as well as in CM derived from these senescent adipocytes (Figure 6H and6I). Therefore, senescence of adipocytes also increases the production and secretion of mir-1949 in vitro.

In sum, we firstly reported that mir-1949 could be a potent age-associated miRNA in adipocytes.

Measurement of mitochondria oxygen consumption

Oxygen consumption was determined at 37°C using the Oxygraph-2k (O2k, OROBOROS instruments). Cells were permeabilized with 0.1% saponin (Sigma) in BIOPS ( 2

RNA Extraction and Quantitative RT-PCR (qPCR )

For the analysis of mRNAs, cells or tissues were lysed with QiAzol lysis reagent (QIAGEN), and RNA was extracted with RNeasy Mini Kit (QIAGEN). The cDNA templates were prepared by reverse transcription of RNA (500ng) using High-Capacity cDNA Reverse Transcription kit (ThermoFisher) and gene expressions were measured using a MESA blue qPCR TM Mastermix plus for SYBR  assay (Eurogentec).

For the analysis of miRNAs, total RNA was extracted with MasterPure TM Complete DNA δ RNA Purification kit 2.00 (EUROMEDEX) according to manufacturer's instructions. 10ng of total RNA were reverse transcribed using TaqMan TM MicroRNA Reverse Transcription Kit (Applied Biosystem) and qPCR and data evaluation were performed as described previously 228 . The amplification data were analyzed using Viia7 (Applied Biosystems).

Statistical Analysis

All calculations were performed by using Prism 5 (GraphPad Software, USA). Statistical significance between two groups was determined by the Student's t test. Statistical significance was set at p < 0.05.

Project II:

A novel method of culturing isolated adipocytes in vitro with 10% of glycerol in medium, it required thorough refresh of the medium with glycerol free DMEM before the performance of lipolysis, which might lead to trauma to the delight adipocytes. Therefore, whether technical defects would result in effects of the results remain unknown. Nevertheless, these above results indicated that cultured fat cells were functional.