Alexandrine During
email: alexandrine.during@univ-lille.fr

Osteoporosis: a role for lipids

Keywords: lipids, bone marrow, mineralized tissue, long bones, human osteoporosis, ovariectomized murine model AA, arachidonic acid, BM, bone marrow, BMD, bone mass density, DHA, docosahexaenoic acid, DXA, dual X-ray absorptiometry, CE, cholesteryl esters, EPA, eicosapentaenoic acid, FA, fatty acids, H-MR, proton magnetic-resonance, GC, gas-chromatography, MT, mineralized bone tissue, OP, osteoporosis, OVX, ovariectomized rats, PC, phosphatidylcholine, PGE2, prostaglandin E2, PS, phosphatidylserine, SCD1, stearoyl-CoA desaturase, SHAM, sham-operated rats, SM, sphingomyelin, TG, triglycerides

An inverse relationship between bone marrow (BM) adiposity and bone mass has been described in different physiological and pathological conditions, including osteoporosis (OP).

In osteoporotic patients, lower bone mass density is indeed associated with higher BM fat content, suggesting a potential role for bone lipids in the OP pathogenesis. Nevertheless, some questions remain. Is that BM adiposity a cause or a consequence of the bone loss? What kinds of lipids are involved? Human data are somehow contradictories regarding bone lipid signature related to OP, and animal data are needed to support on one or another way the human observations. Bone lipid signature associated to OP needs to be clarified if we want to understand better their roles in OP.

In that context, by using an ovariectomy-induced OP murine model and looking at lipids in two bone compartments: BM and mineralized tissue (MT), our first challenge was to identify local lipid changes in relation to OP, in view to explore later the mechanisms by which those compounds could alter bone quality, particularly during the mineralization process. As the most striking data, long-term OP resulted in an accumulation of triglycerides, reduced levels of arachidonic and docosahexaenoic acids, an increase of stearoyl-CoA desaturase indices and a reduction of sphingomyelin in the MT, and potential consequences on bone properties and cell activities are discussed. The reader will appreciate that we are at an early stage of understanding the roles of lipids in the OP development and more investigations will be necessary.

Overview of human data 1.Osteoporosis (OP) facts & low bone mass

Osteoporosis (OP) is a silent and age-related disease that is often detected too late when a first fracture has occurred, which is concomitant with a 50% increased risk to get a second one. It affects people over 50 year-old and more women (21%) than men (6%). Today, OP management has a high societal cost (€37 billion in Europe for 2010), with 95% going for surgical and long-term cares against only 5% devoted to preventive actions [START_REF] Hernlund | Osteoporosis in the European Union: medical management, epidemiology and economic burden. A report prepared in collaboration with the International Osteoporosis Foundation (IOF) and the European Federation of Pharmaceutical Industry Associations (EFPIA)[END_REF]. Because of population ageing and secondary effects of current treatments available, there is a real need of new strategies in terms of OP prevention that goes along with a better understanding of how this pathology takes place.

In the human healthy adult skeleton, there is a balance between bone resorption and bone formation, as results of osteoclast and osteoblast activities, respectively, in order to replace old and/or stressed bone by new bone and to maintain a constant bone mass and quality. This process -named bone remodeling -occurs at multi-sites of the skeleton to repair bone from micro-cracks. By this way, 10% of our skeleton is renewed each year. After 45 years of age, bone resorption takes over bone formation in association with a declined bone turnover; effects that are accentuated in women after menopause and with OP pathogenesis. As such, OP is characterized by a decrease of bone mass and an alteration of bone microarchitecture, leading to a reduction of bone strength and, consequently, in an increased risk of fracture. The clinical diagnosis of OP is made by measuring bone mass density (BMD) via non-invasive techniques, mainly dual X-ray absorptiometry (DXA) and quantitative computed tomography.

That allows to segregate individuals into three groups (normal, osteopenic and OP groups) in function of their BMD values (e.g., when DXA T-score ≥ -1.0 (normal subjects), between -1.0 to -2.5 (osteopenic subjects), and ≤ -2.5 (OP subjects)). These BMD measurements are mostly performed at the lumbar spine and hip sites.

OP: bone marrow fat expansion

In humans, marrow fat content can be assessed by non-invasive magnetic-resonance imaging approaches, such as proton magnetic-resonance (H-MR) spectroscopy. From H-MR spectra, marrow fat content is determined as a percentage relative to water content: 100*(Ifat/(Ifat + Iwater)), where Ifat corresponds to the saturated lipid peak at 1.3 ppm (smaller unsaturated and residual lipid peaks at 5.3 and 2.0 ppm being often ignored) [START_REF] Schwartz | Marrow fat and bone: review of clinical findings[END_REF] (Figure 1). By using this approach, it was reported that vertebral marrow fat content followed the increasing order: normal < osteopenic < OP groups, in both a postmenopausal women population [START_REF] Yeung | Osteoporosis is associated with increased marrow fat content and decreased marrow fat unsaturation: a proton MR spectroscopy study[END_REF] and an over 65 year-old men population [START_REF] Griffith | Vertebral bone mineral density, marrow perfusion, and fat content in healthy men and men with osteoporosis: dynamic contrast-enhanced MR imaging and MR spectroscopy[END_REF]. Note that changes in marrow fat content are age-and sex-related. In each gender group, vertebral marrow fat content increased with age, however, it was higher (6-10%) in men than women of comparable age between 20 and 60 years [START_REF] Kugel | Age-and sex-specific differences in the 1Hspectrum of vertebral bone marrow[END_REF] and, after 60 year-old, there was a switch, probably in association to the postmenopausal status of women taking over men [START_REF] Griffith | Bone marrow fat content in the elderly: a reversal of sex difference seen in younger subjects[END_REF].

Bone marrow (BM) adiposity can also be assessed in situ by a histomorphometry approach, by evaluating the different volumes occupied by adipose tissue, hematopoietic tissue and trabecular bone compartments from an analyzed bone biopsy section. By this approach on iliac crest biopsies issued from a mix-gender patient population, there was an increase of adipose tissue volume concomitant to a decrease of bone trabecular volume, first, with age in the two gender group and, secondly, in the OP female group compared to the age-matched control group [START_REF] Verma | Adipocytic proportion of bone marrow is inversely related to bone formation in osteoporosis[END_REF], in agreement with the observations obtained by non-invasive approaches. In sum, OP-related bone loss is associated with an increased BM adiposity.

Origins of the reciprocal relationship between bone mass and marrow adiposity

An inverse relationship between BM adiposity and bone mass has been established in different physiological and pathological conditions, including during bone acquisition, aging, diabetes, starvation, anorexia nervosa as well as OP [START_REF] Schwartz | Marrow fat and bone: review of clinical findings[END_REF][START_REF] Rosen | Mechanisms of disease: is osteoporosis the obesity of bone?[END_REF][START_REF] Reid | Fat and bone[END_REF][START_REF] Paccou | Marrow adiposity and bone: Review of clinical implications[END_REF] (Figure 2). One of the reasons is that marrow adipocytes (fat-storing cells) and osteoblasts (bone-forming cells) have a common origin: the mesenchymal stem cells, which are contributing to the osteoblastogenesis/ adipogenesis pathways fate [START_REF] Houghton | Immortalization of human marrow stromal cells by retroviral transduction with a temperature sensitive oncogene: identification of bipotential precursor cells capable of directed differentiation to either an osteoblast or adipocyte phenotype[END_REF]. Moreover, it is now more and more documented that marrow adipocytes and all bone cell types (i.e., osteoblasts, osteoclasts (bone-resorbing cells), and osteocytes (mechano-sensor cells)) participate in a local communication network by secreting factors, which can modulate marrow stem cell lineage decision as well as adipocyte and bone cell differentiation/ maturation [START_REF] Nuttall | Controlling the balance between osteoblastogenesis and adipogenesis and the consequent therapeutic implications[END_REF][START_REF] Negishi-Koga | Bone cell communication factors and semaphorins[END_REF][START_REF] Muruganandan | The impact of bone marrow adipocytes on osteoblast and osteoclast differentiation[END_REF]. In that regard, lipids were purported to play key roles in the bone physiology, by being actors in the mineralization process and regulators of important signaling pathways for bone health (review [START_REF] During | Understanding the local actions of lipids in bone physiology[END_REF]). As such, several fatty acids (FA) were reported to affect bone cell survival and functions in vitro as summarized in Table 1, supporting the idea that bone marrow adipocytes in vivo could directly influence neighboring bone cells via the release of FA, possibly a triggering factor for OP as will be discussed later. The bone tissue-level microenvironment is thus determinant and perturbations of its molecular composition, e.g., its lipid content, might contribute to changes in bone microarchitecture via a disequilibrium of the two cellular adipocyte/ osteoblast and osteoblast/ osteoclast balances that condition bone remodeling [START_REF] Fazzalari | Bone remodeling: A review of the bone microenvironment perspective for fragility fracture (osteoporosis) of the hip[END_REF].

Therefore, it is important to look locally at the lipid composition changes in relation to bone quality.

OP: low marrow unsaturation index or not?

If marrow adiposity is a now well-recognized fact in human OP, several questions remain unanswered. Among them, what kinds of lipids are increased within OP bones? Regarding the bone lipid signature, the only few human data available in the literature are somehow contradictories. The marrow unsaturation fat index can be determined from H-MR spectra as a relative percentage of the unsaturated lipid signal (IUL at 5.3 ppm) compared to the sum of all lipid signals (IUL at 5.3ppm + IRL at 2.0 ppm + ISL at 1.3 ppm) (Figure 1). By that way, in a postmenopausal women population, the vertebral marrow unsaturation index followed the decreasing order: normal > osteopenic > OP groups [START_REF] Yeung | Osteoporosis is associated with increased marrow fat content and decreased marrow fat unsaturation: a proton MR spectroscopy study[END_REF], suggesting a potential enrichment in saturated lipids during OP development. Moreover, in a postmenopausal women population without OP (DXA T-score > -1.5), low vertebral unsaturation index were reported in women with fragility fractures; an effect accentuated with a diabetic status [START_REF] Patsch | Bone marrow fat composition as a novel imaging biomarker in postmenopausal women with prevalent fragility fractures[END_REF], implying that vertebral marrow fat composition could be a new imaging tool for fracture risk assessment prior OP diagnosis [START_REF] Patsch | Bone marrow fat composition as a novel imaging biomarker in postmenopausal women with prevalent fragility fractures[END_REF].

In contrast, by using the gold standard gas-chromatography (GC) tool, no changes in marrow fatty acid profiles -and, at fortiori unsaturated fat levels -were observed between normal, osteopenic and OP groups, neither in a Chinese population over 65 year-old (with several bone sites and both gender combined) [START_REF] Griffith | A study of bone marrow and subcutaneous fatty acid composition in subjects of varying bone mineral density[END_REF], nor in BM supernatant fluids of iliac crests from postmenopausal women [START_REF] Miranda | Characterization of Fatty Acid Composition in Bone Marrow Fluid From Postmenopausal Women: Modification After Hip Fracture[END_REF]. Interestingly, in that later study [START_REF] Miranda | Characterization of Fatty Acid Composition in Bone Marrow Fluid From Postmenopausal Women: Modification After Hip Fracture[END_REF], when the OP group was sub-divided into women "without" and "with" a recent hip fracture, higher unsaturated fatty acid levels were observed in BM supernatant fluids from the sub-group "with fracture". In sum, human data show discrepancies about a potential marrow lipid pattern associated to OP pathogenesis; indeed, OP could be correlated to a low marrow unsaturation index or not, or even opposite. To the best of our knowledge, until recently, there was none animal study to support on one or another way the above human observations. It became clear that more investigations were needed in that direction to understand better the involvement of the marrow fat expansion in the OP pathophysiology.

Animal investigations

So far, only three studies have investigated in situ lipid changes in relation to the OP status independently to any nutritional intervention, by using a postmenopausal OP murine model and looking at lipid composition in long bones (femurs and tibiae) [START_REF] Poulsen | Identification of inflammatory and proresolving lipid mediators in bone marrow and their lipidomic profiles with ovariectomy and omega-3 intake[END_REF][START_REF] Zhao | Integrative Bone Metabolomics-Lipidomics Strategy for Pathological Mechanism of Postmenopausal Osteoporosis Mouse Model[END_REF][START_REF] During | Long Term Ovariectomy-Induced Osteoporosis is Associated with High Stearoyl-CoA Desaturase Indexes in Rat Femur[END_REF]. However, these three studies had different strategies making uneasy to compare data. The first one was a targeted approach looking at lipoxygenase-generated lipid metabolites in bone marrow (BM) of femurs/ tibiae [START_REF] Poulsen | Identification of inflammatory and proresolving lipid mediators in bone marrow and their lipidomic profiles with ovariectomy and omega-3 intake[END_REF]. Then, the second one was an integrative untargeted omics approach aiming to identify as many metabolites as feasible in the femoral tissue and providing complex data difficult to comprehend (104 changes, including 93 lipid metabolites for which only 43% were annotated) [START_REF] Zhao | Integrative Bone Metabolomics-Lipidomics Strategy for Pathological Mechanism of Postmenopausal Osteoporosis Mouse Model[END_REF]. Finally, the third one, our global biochemical approach, aimed to characterize and quantify the major neutral lipid and phospholipid classes and fatty acid profiles in the two femoral compartments (BM and mineral tissue) as detailed below [START_REF] During | Long Term Ovariectomy-Induced Osteoporosis is Associated with High Stearoyl-CoA Desaturase Indexes in Rat Femur[END_REF]. Thus, in this section, it will be mostly discussed our approach/ data and compared them to the two other studies when possible. Although it is clear that more studies are needed, the current data suggest several lipid pathways that may be involved in the pathophysiology of OP as it summarized in Figure 3.

The postmenopausal OP murine model

The ovariectomy-induced OP (OVX) murine model is a widely used and suitable preclinical model for postmenopausal OP research. Indeed, estrogen-deprivation induced by ovary removal results in an accelerated bone loss -by 50% at 5 weeks post-surgery in rats [START_REF] Wronski | Skeletal alterations in ovariectomized rats[END_REF] -in association to an increased bone fracture risk, like in postmenopausal women. In murine, the bone remodelling steady-state (balance between bone resorption and bone formation) is a sitespecific, age-dependent process and the skeletal maturity is reached at 6-7 mo.-old for rats [START_REF] Quinn | Comparing rat's to human's age: how old is my rat in people years?[END_REF] and 4 mo.-old for mice [START_REF] Beamer | Genetic variability in adult bone density among inbred strains of mice[END_REF]. Thus, for human OP comparison purpose, it is important to take in consideration the skeletal maturity of animals as done in the two OVX rat studies, for surgical procedures were performed at 6 or 7 mo. of age and OP effects studied at 4 mo. or 9 mo. post-surgery, respectively [START_REF] Poulsen | Identification of inflammatory and proresolving lipid mediators in bone marrow and their lipidomic profiles with ovariectomy and omega-3 intake[END_REF][START_REF] During | Long Term Ovariectomy-Induced Osteoporosis is Associated with High Stearoyl-CoA Desaturase Indexes in Rat Femur[END_REF]. In the OVX mice study [START_REF] Zhao | Integrative Bone Metabolomics-Lipidomics Strategy for Pathological Mechanism of Postmenopausal Osteoporosis Mouse Model[END_REF], animals had surgery at 2 mo. of age and OP effect was examined 2 mo. later, meaning that skeletal maturity of animals was reached at the end point of the study and, thus, bone growth could interfere with the OP status. Note that "OP" effects were evaluated against a control sham-operated (SHAM) animal group.

An in situ approach to study bone lipids

Lipids are present in two major compartments of the bone: the bone marrow (BM; soft tissue) and the mineralized tissue (MT; the hard part). However, bone lipids are usually regarded from the BM side in reason of their better accessibility and higher content where they can reach up to 90% (by marrow weight) and derived in majority from marrow adipocytes and in from hematopoietic cells [START_REF] During | Understanding the local actions of lipids in bone physiology[END_REF]. Nevertheless, the MT contains also lipids, even though at much lower levels (less than 5%, by weight). They can be considered as loosely-bound/ easy extractable or tightly-associated lipids, depending if they are issued from: a) bone cells (mostly osteocytes representing 90-95% of total bone cells) and blood circulation irrigating the MT, or b) complexes with macromolecules and mineral in the mineralized extracellular matrix [START_REF] During | Understanding the local actions of lipids in bone physiology[END_REF].

We recently proposed an in situ approach to characterize lipids in both BM and MT compartments, by taking into account several analytical issues such as the initial sample size, potential contamination of the MT by BM lipids as well as the poor accessibility/ extractability of lipids present in MT [START_REF] During | Lipid determination in bone marrow and mineralized bone tissue: From sample preparation to improved high-performance thin-layer and liquid chromatographic approaches[END_REF]. By applying this original approach to healthy rat femurs, we reported that each bone compartment had its own lipid signature. For instance, the MT was a more metabolically active tissue in terms of long chain fatty acids (i.e., fatty acid profile enriched in arachidonic (AA, 20:4n-6), eicosapentaenoic (EPA, 20:5n-3) and docosahexaenoic (DHA, 22:6n-3) acids), compared to BM [START_REF] During | Lipid determination in bone marrow and mineralized bone tissue: From sample preparation to improved high-performance thin-layer and liquid chromatographic approaches[END_REF], pointing out the rational to examine lipids in the two bone compartments, in particular for a BM adiposity-associated bone disease like OP. For comparison, in the two other studies, lipids were examined either in the BM fraction of femurs plus tibiae combined [START_REF] Poulsen | Identification of inflammatory and proresolving lipid mediators in bone marrow and their lipidomic profiles with ovariectomy and omega-3 intake[END_REF] or in the femoral tissue freed from BM, but without any further washing step, implying a potential contamination of the bone tissue by BM lipids [START_REF] Zhao | Integrative Bone Metabolomics-Lipidomics Strategy for Pathological Mechanism of Postmenopausal Osteoporosis Mouse Model[END_REF].

Lipid changes in OP bones

Unlike humans, in animal studies, invasive tools are commonly used. First, lipids can be extracted from bones, e.g., by using the above mentioned in situ approach, and then analysed either by classical chromatography HPLC and HP-TLC methods [START_REF] During | Long Term Ovariectomy-Induced Osteoporosis is Associated with High Stearoyl-CoA Desaturase Indexes in Rat Femur[END_REF] or by high-throughput liquid chromatography tandem mass spectrometry (LC-or UPLC-MS/MS) tools [START_REF] Poulsen | Identification of inflammatory and proresolving lipid mediators in bone marrow and their lipidomic profiles with ovariectomy and omega-3 intake[END_REF][START_REF] Zhao | Integrative Bone Metabolomics-Lipidomics Strategy for Pathological Mechanism of Postmenopausal Osteoporosis Mouse Model[END_REF].

Triglycerides (TG) accumulation in BM and MT of OP femurs

Like in post-menopausal OP women, a low bone mass was associated with a significant increase of fat content in BM of OVX rat femurs, when compared to control SHAM rats. That increased adiposity affected the MT compartment of OVX femurs as well [START_REF] During | Long Term Ovariectomy-Induced Osteoporosis is Associated with High Stearoyl-CoA Desaturase Indexes in Rat Femur[END_REF].

In the BM of OVX rat femurs, there was a marked increase in amounts of both energy-storage lipids (i.e., TG) and membrane-associated lipids (i.e., the phospholipids SM, PC and PS), suggesting a hypertrophy and/or increased number of marrow adipocytes to allow a greater TG storage capacity [START_REF] During | Long Term Ovariectomy-Induced Osteoporosis is Associated with High Stearoyl-CoA Desaturase Indexes in Rat Femur[END_REF]. In support, in healthy tibia of mice fed with a high fat diet for 6 weeks, there was an increased volume of BM adipose tissue -assessed by histology -in association to an increased size of adipocytes and a higher content of TG in BM [START_REF] Adler | High fat diet rapidly suppresses B lymphopoiesis by disrupting the supportive capacity of the bone marrow niche[END_REF].

In the MT of OVX rat femurs, the increased fat content concerned only neutral lipids (amount of phospholipids was unchanged), and mainly TG [START_REF] During | Long Term Ovariectomy-Induced Osteoporosis is Associated with High Stearoyl-CoA Desaturase Indexes in Rat Femur[END_REF]. In agreement, all of the 19 identified TG species (15 annotated/ 4 putative) were increased in the femoral tissue of OVX mice [START_REF] Zhao | Integrative Bone Metabolomics-Lipidomics Strategy for Pathological Mechanism of Postmenopausal Osteoporosis Mouse Model[END_REF].

That accumulation of TG in the mineral compartment of OP bones raises questions of whether MT is a passive tissue undergoing an infiltration of medullary TG or a metabolically active tissue in which osteocytes could be capable of TG synthesis. In that regard, in two alcoholinduced rabbit osteonecrosis and rat bone loss models for which histological examinations were performed at the femoral head and tibia diaphysis sites, respectively [START_REF] Wang | Alcohol-induced adipogenesis in bone and marrow: a possible mechanism for osteonecrosis[END_REF][START_REF] Maurel | Osteocyte apoptosis and lipid infiltration as mechanisms of alcohol-induced bone loss[END_REF], it was reported that alcohol induced an accumulation of TG droplets within osteocytes, suggesting an intracellular TG synthesis following fatty acid uptake rather than the pinocytosis of large TG vesicles by osteocytes [START_REF] Maurel | Osteocyte apoptosis and lipid infiltration as mechanisms of alcohol-induced bone loss[END_REF]. In these two studies [START_REF] Wang | Alcohol-induced adipogenesis in bone and marrow: a possible mechanism for osteonecrosis[END_REF][START_REF] Maurel | Osteocyte apoptosis and lipid infiltration as mechanisms of alcohol-induced bone loss[END_REF], the cytosolic accumulation of TG droplets was associated with a cell apoptosis and an increased number of empty lacunaspaces in which are lodged osteocytes -. Thus, it is possible that similar events, i.e., changes in osteocyte metabolic activities and survival, occur in OP bones.

Cholesteryl esters (CE) increase in the MT of OP femurs

Among neutral lipids, cholesteryl esters (CE) amount was also increased in the MT of OVX rat femurs [START_REF] During | Long Term Ovariectomy-Induced Osteoporosis is Associated with High Stearoyl-CoA Desaturase Indexes in Rat Femur[END_REF], raising similar questions than for TG. Are they issued from an infiltration by blood circulating CE -since BM does not contain CE -, or locally produced? Note that CE, as a tightly associated-to-mineral constituent, might play a role in the mineralization process [START_REF] During | Lipid determination in bone marrow and mineralized bone tissue: From sample preparation to improved high-performance thin-layer and liquid chromatographic approaches[END_REF], in regards to what happens during the atherosclerotic vascular plaque formation. Indeed, in that later process, CE accumulates preferentially in the mineral part, making it a potential initiator/ activator of the vascular calcification [START_REF] Reid | Lipids in biocalcification: contrasts and similarities between intimal and medial vascular calcification and bone by NMR[END_REF]. Note that both vascular calcification and bone mineralization are similar mechanisms, implying matrix vesicles production by vascular smooth cells and osteoblasts, respectively. Thus, an unusual accumulation of CE in OP bones might affect the bone mineralization events driven by osteoblasts, resulting in adverse effects on the original properties of the mineral. Note that three sterol metabolites, including one from the "cholesterol and derivatives" category, were enhanced in the femoral tissue of OVX mice [START_REF] Zhao | Integrative Bone Metabolomics-Lipidomics Strategy for Pathological Mechanism of Postmenopausal Osteoporosis Mouse Model[END_REF]. In sum, local cholesterol metabolism should require further attention in OP pathophysiology.

Sphingomyelin (SM) reduction in the MT of OP femurs

Knowing that local SM catabolism is essential for the mineralization process in healthy bones [START_REF] During | Understanding the local actions of lipids in bone physiology[END_REF], it was surprising to find a significant reduction of SM in the MT part of OVX rat femurs [START_REF] During | Long Term Ovariectomy-Induced Osteoporosis is Associated with High Stearoyl-CoA Desaturase Indexes in Rat Femur[END_REF]. Nevertheless, this finding is in agreement with the data reported in OVX mice femurs, indicating a significant reduction of three to five SM species concomitantly to an increase of six metabolites -one glycosphingolipid and five ceramide species -, but only one of them was annotated [START_REF] Zhao | Integrative Bone Metabolomics-Lipidomics Strategy for Pathological Mechanism of Postmenopausal Osteoporosis Mouse Model[END_REF]. Thus, OP could be associated with a local dysregulation of sphingolipid metabolism, such as an exacerbated catabolism of SM that would not allow mineral to be produced properly. In support, patients with rare forms of OP associated with a defective bone mineralization had mutations in the SGMS2 gene encoding for the SM synthase 2 (or nSMS2) [START_REF] Pekkinen | Osteoporosis and skeletal dysplasia caused by pathogenic variants in SGMS2[END_REF]; a membrane-bound enzyme -catalyzing SM hydrolysis to form phosphocholine and ceramides -highly expressed in bones and required for normal mineralization [START_REF] During | Understanding the local actions of lipids in bone physiology[END_REF]. Overall, more investigations will be needed in the future regarding the potential role of SM metabolism in the development of OP.

Individual fatty acids (FA) changes in OP femurs

The unsaturation index (% mono-plus poly-unsaturated FA) and, at fortiori the saturation index (% saturated FA), were not modified in OVX rat femurs, compared to control SHAM femurs [START_REF] During | Long Term Ovariectomy-Induced Osteoporosis is Associated with High Stearoyl-CoA Desaturase Indexes in Rat Femur[END_REF]. These data are in agreement with several human reports [START_REF] Griffith | A study of bone marrow and subcutaneous fatty acid composition in subjects of varying bone mineral density[END_REF][START_REF] Miranda | Characterization of Fatty Acid Composition in Bone Marrow Fluid From Postmenopausal Women: Modification After Hip Fracture[END_REF] and, in some extents, with the previous OVX rat study [START_REF] Poulsen | Identification of inflammatory and proresolving lipid mediators in bone marrow and their lipidomic profiles with ovariectomy and omega-3 intake[END_REF]. Nevertheless, several individual FA were affected by the long-term ovariectomy in rats, i.e., an enrichment in myristic (14:0), palmitoleic (16:1n-7) and linolenic (18:3) acids and a reduction in oleic (18:0), arachidonic (AA, 20:4n-6) and docosahexaenoic (DHA, 22:6n-3) acids with OP [START_REF] During | Long Term Ovariectomy-Induced Osteoporosis is Associated with High Stearoyl-CoA Desaturase Indexes in Rat Femur[END_REF]. These FA changes were consistently observed in both BM and MT compartments of OVX rat femurs [START_REF] During | Long Term Ovariectomy-Induced Osteoporosis is Associated with High Stearoyl-CoA Desaturase Indexes in Rat Femur[END_REF], suggesting a more global -rather than a tissue-specific -OP-associated mechanism, possibly via a bone infiltration by a pool of circulating FA according to the hypothesis of a local TG synthesis discussed above. In agreement, a significant reduction of AA and DHA percentages, but not for eicosapentaenoic acid (EPA, 22:5n-3), was also noticed in BM of OP long bones at 4 mo. post-ovariectomy (other FA were not mentioned) [START_REF] Poulsen | Identification of inflammatory and proresolving lipid mediators in bone marrow and their lipidomic profiles with ovariectomy and omega-3 intake[END_REF]. Note that, independently of our results, all of the 31 fatty acyl metabolites, including six unusual fatty acids (2 annotated/ 6 putative), were enhanced in OVX mice femurs [START_REF] Zhao | Integrative Bone Metabolomics-Lipidomics Strategy for Pathological Mechanism of Postmenopausal Osteoporosis Mouse Model[END_REF].

The local reduction of AA observed in OP rats [START_REF] Poulsen | Identification of inflammatory and proresolving lipid mediators in bone marrow and their lipidomic profiles with ovariectomy and omega-3 intake[END_REF][START_REF] During | Long Term Ovariectomy-Induced Osteoporosis is Associated with High Stearoyl-CoA Desaturase Indexes in Rat Femur[END_REF] was consistent with previous OP women data [START_REF] Miranda | Characterization of Fatty Acid Composition in Bone Marrow Fluid From Postmenopausal Women: Modification After Hip Fracture[END_REF] showing a decrease of AA in medullar fluids of the sub-group "with" a recent hip fracture, compared to the "without" fracture sub-group, suggesting that local AA disappearance might be a marker of bone fracture. Furthermore, AA is a precursor of prostaglandin E2 (PGE2, a cyclooxygenase (COX)-derived product), locally produced and known as a potent regulator in bone physiology by influencing both bone formation and bone resorption in a concentration-dependent manner [START_REF] During | Understanding the local actions of lipids in bone physiology[END_REF]. In rabbits, local PGE2 synthesis was enhanced in response to bone fracture [START_REF] Dekel | Release of prostaglandins from bone and muscle after tibial fracture. An experimental study in rabbits[END_REF] and its inhibition by indomethacin (a COXinhibitor) impaired bone fracture healing [START_REF] Keller | Effects of indomethacin and local prostaglandin E2 on fracture healing in rabbits[END_REF], suggesting a PGE2 requirement during bone trauma conditions. In humans, high dietary intakes of AA were associated with a reduced risk of hip fracture in elderly adults [START_REF] Farina | Dietary intakes of arachidonic acid and alpha-linolenic acid are associated with reduced risk of hip fracture in older adults[END_REF] and, inversely, low unsaturated FA n-6 intakes aggravated that risk in old women [START_REF] Virtanen | Dietary intake of polyunsaturated fatty acids and risk of hip fracture in men and women, Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA[END_REF]. Thus, in OP, the local AA disappearance may reflect a higher PGE2 production in response to an increased demand for bone repair, a point that needs to be checked. In support, ovariectomy resulted in a significant increase in PGE2 release ex vivo from cultured OVX rat parietal bones [START_REF] Feyen | Prostaglandin production by calvariae from sham operated and oophorectomized rats: effect of 17 beta-estradiol in vivo[END_REF] as well as an increase of an eicosanoid metabolite (the 13,14-dihydro-15-keto PGE2; a primary metabolite of PGE2) in situ in OVX mice femurs [START_REF] Zhao | Integrative Bone Metabolomics-Lipidomics Strategy for Pathological Mechanism of Postmenopausal Osteoporosis Mouse Model[END_REF]. In addition, AA, like DHA and EPA, is a precursor of lipoxygenase (LOX)derived lipid mediators, such as leukotrienes, lipoxins, and hydroxyl-eicosatetraenoic acids, having potent pro-inflammatory or anti-inflammatory/proresolving activities. Ovariectomy resulted in an altered profile of these compounds in murine BM, with a relative increase in proportion of LOX products derived from AA relative compared to those from DHA [START_REF] Poulsen | Identification of inflammatory and proresolving lipid mediators in bone marrow and their lipidomic profiles with ovariectomy and omega-3 intake[END_REF], thus probably impacting the inflammatory status within the BM compartment of OP bones. In that context, it would be interesting to see if these local LOX-derived mediator changes could be directly associated to some inflammatory cytokine changes in BM [START_REF] Poulsen | Identification of inflammatory and proresolving lipid mediators in bone marrow and their lipidomic profiles with ovariectomy and omega-3 intake[END_REF].

To the best of our knowledge, we are first to report a local increase of palmitoleic acid (16:1) in OP rat femurs. This result is not so surprising, knowing that, first, 16:1 is a marker of yellow marrow and its precursor, palmitic acid (16:0), of red marrow [START_REF] Tavassoli | Fatty acid composition of adipose cells in red and yellow marrow: A possible determinant of haematopoietic potential[END_REF] and, secondly, there is a red-to-yellow marrow shift occurring with ageing. That shift might be more pronounced during the age-related OP process and thus results in higher 16:1 levels. As mentioned earlier, several FA, including 16:0, were purported to affect bone cells in vitro (Table 1). It is only recently that 16:1 was seen as beneficial for bone health, by affecting negatively osteoclasts [START_REF] Van Heerden | Palmitoleic Acid Inhibits RANKL-Induced Osteoclastogenesis and Bone Resorption by Suppressing NF-kappaB and MAPK Signalling Pathways[END_REF]; however, these anti-resorptive effects would need to be accompanied by pro-osteoblast actions, a point that has not been studied yet (Table 1). Nevertheless, we will see right after this increase of 16:1 participates in the elevation of one of the stearoyl-Coa desaturase indexes.

Increase of stearoyl-CoA desaturase indices in OP femurs

Moreover, there was a local elevation of the two stearoyl-CoA desaturase (SCD1) indices (16:1/16:0 and 18:1/18:0 ratios) in both compartments of OP rat femurs [START_REF] During | Long Term Ovariectomy-Induced Osteoporosis is Associated with High Stearoyl-CoA Desaturase Indexes in Rat Femur[END_REF]. These indexes reflect the ∆9 desaturase (SCD1) activity that converts saturated FA (mostly 16:0 and 18:0) into monounsaturated FA (16:1 and 18:1); a key enzyme in de novo lipogenesis that has been implicated in a variety of metabolic disorders such as obesity, diabetes, insulin resistance and atherosclerosis [START_REF] Flowers | Role of stearoyl-coenzyme A desaturase in regulating lipid metabolism[END_REF]. Whether or not these indices are indeed the result of a local increase of SCD1 activity in OP boned remains to be checked. Nevertheless, these observations should be related to two earlier human findings showing, first, higher 18:1/18:0 ratio in BM fluids from OP women with a recent hip fracture [START_REF] Miranda | Characterization of Fatty Acid Composition in Bone Marrow Fluid From Postmenopausal Women: Modification After Hip Fracture[END_REF] and, secondly, a positive association between serum the SCD1 index (assessed as the 16:1/16:0 ratio) and risk of fracture in men (enrolled at 50 years and followed-up up to 35 years) [START_REF] Melhus | A high activity index of stearoyl-CoA desaturase is associated with increased risk of fracture in men, Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA[END_REF]. Taken together, OP could be regarded as a metabolic disorder in which de novo lipogenesis pathway would play a key role, however more investigations are needed, first, to see if its products are generated locally or are part of a more global process and, secondly, to understand the consequence of their accumulation in bone.

Perspectives

The main lipid changes in both compartments (BM and mineralized tissue) of murine OP long bones in association with their potential effects are summarized in Figure 3. Long-term OP resulted in an accumulation of TG, a decrease of AA (20:4n-6) and DHA (22:6 n-3), and an increase of SCD1 indices (or 16:1/16:0 and 18:1/18:0 ratios) in the two bone compartments.

In addition, in the mineral part, there were an elevation of CE and an increase of SM catabolism, two compounds that may play a role in the mineralization. Thus, it is clear that more data are required, in particular to understand how local fatty acid perturbations could affect functions of 'fatty acid-including' lipids involved in the bone physiology, e.g., CE and phospholipids, regarding to bone cell activities as well as the mineralization process. Finally, it would be interesting to see if those purported lipid changes in osteoporotic femurs occur also at earlier stages of OP in view to better understand how this disease takes place. a Osteoblast-specific bone formation markers: TNAP (tissue non-specific alkaline phosphatase) is a key enzyme in the mineralization process and RunX2 (Runt related transcription factor 2) is a transcription factor that controls osteoblastic differentiation by targeting osteoblast-specific genes, including the one for TNAP.

b Osteoclast-specific bone resorption markers: TRAP (tartrate resistant acid phosphatase), MMP-9 (matrix metalloproteinase) and CTSK (cathepsin K) are involved in the bone matrix organic degradation and the transcription factor NF-κB (nuclear factor κB) targets osteoclastspecific genes, including the TRAP, MMP-9 and CTSK genes. c ↑, increasing effect; ↓, decreasing effect; → no effect.

Figure 1 :

 1 Figure1: Typical proton-magnetic resonance (H-MR) spectrum obtained on one vertebral body (L1 to L4) of women with OP by using a higher resolution (1.5 or 3 Tesla) scanner and positioning the spectroscopy measurement box in the central portion of the lumbar (limited area). On the spectrum, lipids are identifiable as unsaturated lipids (UL, olefinic double bond -CH=CH-protons at 5.3 ppm), residual lipids (RL, methylene -CH=CHCH2-protons at 2.0 ppm) and saturated lipids (SL, bulk methylene -(CH2)n-protons at 1.3 ppm). A spectrum acquired from OP women exhibits a relatively high SL peak compared to the water peak (H2O at 4.7 ppm), while it is opposite in non-OP women.

Figure 2 :

 2 Figure 2: OP-related bone loss is inversely associated to an increased marrow fat content.

Figure 3 :

 3 Figure 3: Summary of the current data showing lipid changes in both compartments of murine OP long bones from a During et al. [22], b Zhao et al. [21], and c Poulsen et al. [20] studies. ↑, increasing effect; ↓, decreasing effect Abbreviations: AA, arachidonic acid; DHA, docosahexaenoic acid; CE, cholesteryl esters; PC, phosphatidylcholine; PGE2, prostaglandin E2; PS, phosphatidylserine; SCD1, stearoyl-CoA desaturase activity; SM, sphingomyelin; TG, triglycerides.

Table 1 .

 1 Summary of in vitro effects of exogenous fatty acids (FA) on bone cells (adapted from Table4of the review[START_REF] During | Understanding the local actions of lipids in bone physiology[END_REF])

	FA	Osteoblasts [15]

The OVX rat study -that we performed and which is discussed in the present review -was partly financed by the French Society of Rheumatology (SFR) and our research activity on bone lipids is now supported by the foundation MSDAvenir (LIPIDOs project).


Jay Ram Lamichhane
email: jay-ram.lamichhane@inrae.fr

Parsimonius use of pesticide-treated seeds: An IPM framework

Keywords: Seed germination, seedling emergence, crop establishment, seed treatment, soil biodiversity, sustainable agriculture

Pesticide-treated seeds are usually supplied in "default" packages that leave farmers little choice for a tailor-made management of soil-borne pests and pathogens. This has led to a socio-economic impasse questioning the sustainability of planting pesticidetreated seeds. Here I propose an IPM framework to overcome the current impasse.

The rationale behind the pesticide seed treatment

.

Pesticide seed treatment and treatment frequency index

The frequency of chemical pesticides applied via seed treatment is lower (i.e. only once per cropping season) compared with that applied via foliar applications. In most EU

countries, pesticide use is generally measured as the treatment frequency index (TFI), which is defined as the number of pesticide applications per hectare per calendar year, assuming the use of a standard dose for each authorized use of pesticides [START_REF] Lamichhane | Toward a reduced reliance on conventional pesticides in European agriculture[END_REF]. In France, as in most EU countries, PST was not considered in the calculation of TFI until a few years ago, but now the TFI calculation takes PST into acount. The data show that TFI related to seed treatment represents an important portion of pesticides introduced into the environment (Figure 1). Nevertheless, there is an acute lack of information on PST data which are mainly inaccesible in several parts of the world, including Europe and North America [START_REF] Hitaj | Sowing Uncertainty: What We Do and Don't Know about the Planting of Pesticide-Treated Seed[END_REF].

The current socio-economic impasse for farmers

A great majority of pesticide-seed treatment is performed by the seed suppliers, who market treated seeds in "default" packages. This means that farmers can not chose freely between untreated or treated seeds and do not have access to tailored pesticide use based on the specific field situations (e.g. fields with a history of post-planting problems). The marketing of pesticide-treated seeds in default packages constitutes an important problem for farmers as most often they are unaware of the specific active ingredients in these packages and the pests and pathogens that would be targeted [START_REF] Hitaj | Sowing Uncertainty: What We Do and Don't Know about the Planting of Pesticide-Treated Seed[END_REF]. This routine-based planting of PTSs for certain crops such as soybean has led farmers to a socio-economic impasse where farmers systematically bear seed treatment costs without important economic return [4,[START_REF] Rossman | Profitability and efficacy of soybean seed treatment in Michigan[END_REF]. This dilemma is further exacerbated by recent findings that the planting of PTSs does not, as thought previously, result in lower human-health and environmental impacts compared with foliar applications of pesticides, including potential non-target effects [START_REF] Nettles | Influence of pesticide seed treatments on rhizosphere fungal and bacterial communities and leaf fungal endophyte communities in maize and soybean[END_REF]7].

No, low or inconsistent effectiveness of pesticide treated seeds answered?

Even when targeted to the right pests and pathogens, the planting of PTSs may not be effective due to a high diversity of environmental conditions, cropping systems and the diversity of soil-borne pests and pathogens across the world. Sartori et al. [START_REF] Fadel Sartori | Soybean seed treatment: how do fungicides translocate in plants?[END_REF] investigated the translocation pattern of three fungicides --viz. pyraclostrobin, carbendazim, and metalaxyl --with different modes of action and most often applied as a seed treatment, in interaction with soil type in soybean, the most important leguminuous crop worldwide. The authors showed that, following germination of PTSs, only a small fraction of the pesticides (i.e. 15%), compared with the quantity used for seed treatment, was translocated to plant parts that need protection (i.e. stem, roots and leaves) while a large proportion of these pesticides remained in cotyledons, independent of the fungicide type. This study also confirmed two key findings of previous studies: i) the rate of soil organic matter of a given soil affects the mobility of certain fungicides in the soil [START_REF] Sharom | The adsorption, mobility, and persistence of metalaxyl in soil and aqueous systems[END_REF], and ii) the soil texture in general and the clay content in particular of a given soil affects absorbtion rate of such fungicides [START_REF] Liu | Uptake and distribution characteristics of the novel fungicide pyraoxystrobin in cucumber plants[END_REF].

Another study by You et al. [START_REF] You | Understanding why Effective Fungicides against Individual Soilborne Pathogens are Ineffective with Soilborne Pathogen Complexes[END_REF], based on multi-year trials, analyzed the effectiveness of planting PTSs in controlling seed and seedling diseases of subterranean clover across contrasted soil and environmental conditions of Australia. The authors, tested nine combinations of pesticides (i.e. thiram, metalaxyl, iprodione, phosphonic acid, propamocarb, fluquinconazole, difenoconazole + metalaxyl, ipconazole + metalaxyl, sedaxane + difenoconazole + metalaxyl), against four prevalent soil-borne pathogens -viz. Pythium irregulare, Aphanomyces trifolii, Phytophthora clandestina and Rhizoctonia solani --causing seed and seedling diseases worldwide on a large number of crops. This study demonstrated that planting PTSs provided effective control of a seed or seedling disease only when a single soil-borne pathogen was associated with the disease, whereas this practice was ineffective when different soil-borne pathogens were associated with the disease complex that resulted by synergistic interactions of different soil-borne pathogens. Indeed, increasing evidence in the literature shows that a given plant disease is often caused by synergistic interactions among different soil-borne pests and pathogens that co-occur in a given plant or plant-parts under field conditions [START_REF] Harvey | The Pythium-Fusarium root disease complex -an emerging constraint to irrigated maize in southern New South Wales[END_REF][START_REF] Madriz-Ordeñana | Prevalence of Soil-borne Diseases in Kalanchoe blossfeldiana Reveals a Complex of Pathogenic and Opportunistic Fungi[END_REF][START_REF] Lamichhane | Synergisms between microbial pathogens in plant disease complexes: A growing trend[END_REF].

The studies by Sartori et al. [START_REF] Fadel Sartori | Soybean seed treatment: how do fungicides translocate in plants?[END_REF] and You et al [START_REF] You | Understanding why Effective Fungicides against Individual Soilborne Pathogens are Ineffective with Soilborne Pathogen Complexes[END_REF] provide an important insight into lack of, low or inconsistent effectiveness of PTSs in controlling soil-borne pathogens that affect seeds and seedlings both pre-germination, pre-and post-emergence across various soil and environmental conditions. These studies, further emphasize the need for future studies on other crops, such as cereals, for which seed treatment is even more common compared with leguminuous crops [START_REF] Hitaj | Sowing Uncertainty: What We Do and Don't Know about the Planting of Pesticide-Treated Seed[END_REF]. In particular, a better focus on how pesticides used for seed treatments affect soil water regime, soil properties, plant biology, and the diversity of pests and pathogens of a given soil can provide important insights into the potential effectiveness of PTSs across environmental gradients.

An integrated pest management framework for a parsimonious use of PTSs in agriculture

Integrated Pest Management (IPM) is a dynamic and flexible approach that takes into account the diversity and the complexity of agro-ecosystems with the aim to improve the sustainability of cropping systems [15]. IPM encourages the use of non-chemical and sustainable cropping practices while also allowing a parsimonious use of synthetic chemicals, when indispensable.

More specifically to the sustainability issues related to the use of PTSs, an IPM framework (Figure 2) that combines all best management practices can significantly increase economic profitability of this practice while reducing environmental risks and health hazards. Such a framework is not dogmatic and can be tailored to specific contexts giving the highest priority to non-chemical measures while allowing the use of PTSs as the last option. Because decisions about the type of seed to be planted are made much earlier in the growing season (i.e. months before planting), a strict implementation of IPM is needed while making these decisions. This IPM framework takes into account diverse types of crops grown across very different areas of the world; multiplicity of soil types, environmental conditions, and production practices; diversity in seed-and soil-borne pests and pathogens to improve crop emergence, stand development and yield performance. This is fundamental to achieve the ultimate goal of increasing economic profitability due to planting of PTSs while reducing their negative effects to human health and the environment. However, key for an effective implementation of IPM is to provide farmers with diverse choices of treated vs. differently treated seeds and the detailed information on the type of active ingredients used for seed treatments. This will allow farmers to make better use of PTSs and help solve the curent dilemma of planting PTSs as a routine-based practice even when it is not needed.

Concluding remarks

The planting of PTSs remains an important practice for farmers to ensure economic profitability across areas characterized by a high pressure of soil-borne pests and pathogens. Nevertheless, there is a need to re-evaluate the routine-based planting of PTSs that is unsustainable from economic, environmental and social points of view.

Therefore, farmers need market acess to a range of treated vs. untreated seeds, suitable for different crops and diverse field situations. Resolving the dilemma of marketing PTSs in "default" packages is the first step to get farmers out of the current socio-economic impasse of planting PTSs as a routine-based practice, and toward a proper adoption of IPM. I invite all stakeholders to re-think the current seed supply marketing policy, to re-assess the sustainability of planting PTSs as a routine-based practice, and propose an IPM framework for a judicious use of PTSs.

Figure legends : Figure 1 .

 :1 Figure legends:

Figure 2 .

 2 Figure 2. An Integrated Pest Management framework that combines all key crop management levers to improve seed germination, seedling emergence and thereby the quality of crop establishment. This framework prioritizes the non-chemical means of crop protection although the use of pesticide-treated seeds is allowed as the last option, based on a priori risk assessment of planting areas. The priority given to these measures decreases from left to right with the lever « inoculum management » and « chemical seed treatment » receiving the highest and lowest priority, respectively. More focus on crop diversification and, in general, on non-chemical measures, enhances soil microbial diversity that plays an important role in soil health. In this way, an increased level of beneficial soil microbial community helps to suppress soil-borne pests and pathogens and to reduce negative impact on seed and seedlings through biotic factors. Inoculation management refers both to the soil-and seed-borne inoculum and it is based on three key cropping practices : (a) crop diversification that includes crop rotation, inter-and cover-cropping; (b) tillage or no-tillage that comprise conventional, minimum or notillage, depending on specific situations; and (c) use of healthy seeds through using steps such as, testing seed sanitary quality before use, performing selective sorting of noncertified seeds, testing for good seed germination ability before planting, and applying biological seed treatments to manage seed-borne diseases when non-certified on-farm seeds have to be used. The horizontal arrow from left to right represents a set of IPM levers whereas the vertical arrows represent individual IPM levers. The order of the roman numerals indicate the increasing priority given to the individual IPM levers. The dotted arrows indicate that biological and chemical control should be applied for specific situations. The figure was partly created using BioRender (https://biorender.com/).

Acknowledgements

I thank the anonymous reviewer for providing very constructive feedback on the previous version of this paper. I apologize for the omission of any relevant articles that have not been cited due to space limit. This study was partially supported by the FAST project (Faisabilité et Evaluation de Systèmes de Cultures Economes en pesticides en l'Absence répétée de Semences Traitées) funded by the French Agency for Biodiversity, by credits from the royalty for diffuse pollution, attributed to the funding of the Ecophyto plan. I am grateful to Véronique Laudinot, Chamber of agriculture of the Vosges for her earlier feedback on this topic.