Yuanqing Gao

Clarita Layritz

Beata Legutko

Thomas O Eichmann

Elise Laperrousaz

Valentine S Moullé

Celine Cruciani-Guglielmacci

Christophe Magnan

Serge Luquet

Stephen C Woods

Robert H Eckel

Chun-Xia Yi

Cristina Garcia-Caceres

Matthias H Tschöp
email: tschoep@helmholtz-muenchen.de

Disruption of Lipid Uptake in Astroglia Exacerbates Diet Induced Obesity

Keywords: astrocyte, diet induced obesity, lipoprotein lipase

Neuronal circuits in the brain help to control feeding behavior and systemic metabolism in response to afferent nutrient and hormonal signals. Although astrocytes have historically been assumed to be less relevant for such neuroendocrine control, we asked whether lipid uptake via lipoprotein lipase (LPL) in astrocytes is required for the central regulation of energy homeostasis. Ex vivo studies with hypothalamic-derived astrocytes showed that LPL expression is up-regulated by oleic acid; whereas it is decreased in response to palmitic acid or triglyceride. Likewise, astrocytic LPL deletion reduced the accumulation of lipid drops in those glial cells. Consecutive in vivo studies showed that the postnatal ablation of LPL in glial fibrillary acidic protein (GFAP)-expressing astrocytes induced exaggerated body weight gain and glucose intolerance in mice exposed to a high-fat diet (HFD). Intriguingly, astrocytic LPL deficiency also triggered increased ceramide content in the hypothalamus, which may contribute to hypothalamic insulin resistance. We conclude that hypothalamic LPL functions in astrocytes to ensure appropriately balanced nutrient sensing, ceramide distribution, body weight regulation and glucose metabolism.

Introduction

Metabolic homeostasis is regulated by a complex central nervous system (CNS) network that senses and integrates nutrient and hormonal signals from the periphery to regulate feeding behavior, energy expenditure and glucose homeostasis. Considerable evidence indicates that lipid sensing by the brain is a normal component of such regulation [START_REF] Clement | Intracerebroventricular infusion of a triglyceride emulsion leads to both altered insulin secretion and hepatic glucose production in rats[END_REF][START_REF] Cruciani-Guglielmacci | Beta oxidation in the brain is required for the effects of non-esterified fatty acids on glucose-induced insulin secretion in rats[END_REF][START_REF] Lam | Hypothalamic sensing of circulating fatty acids is required for glucose homeostasis[END_REF][START_REF] Lopez | Hypothalamic fatty acid metabolism: a housekeeping pathway that regulates food intake[END_REF][START_REF] Obici | Central administration of oleic acid inhibits glucose production and food intake[END_REF][START_REF] Oomura | Effect of free fatty acid on the rat lateral hypothalamic neurons[END_REF], and the mechanism underlying brain lipid sensing's role in metabolism has attracted considerable interest in the metabolic field in recent years.

Glial cells constitute around 50% of the total cells in the whole brain [START_REF] Azevedo | Equal numbers of neuronal and nonneuronal cells make the human brain an isometrically scaled-up primate brain[END_REF], but until recently have historically received less attention in metabolic research. Astrocytes are the most abundant and diverse glial cells and they are the primary cell population in the brain that synthesizes and metabolizes lipids [START_REF] Edmond | Energy metabolism in developing brain cells[END_REF][START_REF] Foll | Regulation of hypothalamic neuronal sensing and food intake by ketone bodies and fatty acids[END_REF], positioning them as major players in the regulation of lipid sensing and metabolism in the CNS. Consistent with this, fatty acid oxidation in the brain, although at a very low rate compared to glycolysis, occurs primarily in astrocytes [START_REF] Edmond | Energy metabolism in developing brain cells[END_REF][START_REF] Edmond | Capacity for substrate utilization in oxidative metabolism by neurons, astrocytes, and oligodendrocytes from developing brain in primary culture[END_REF]. Further, chronic consumption of a HFD results in both hyperlipidemia and hypothalamic astrogliosis [START_REF] Horvath | Synaptic input organization of the melanocortin system predicts diet-induced hypothalamic reactive gliosis and obesity[END_REF]. However, until now, the functional significance of astrocyte lipid metabolism in CNS metabolic regulation has been unknown.

Lipoprotein lipase (LPL) -the key enzyme to hydrolyze triglyceride -, has been implicated in CNS metabolic regulation [START_REF] Wang | Deficiency of lipoprotein lipase in neurons modifies the regulation of energy balance and leads to obesity[END_REF]. LPL is the "gate-keeper" for lipids in peripheral tissues, modulating the distribution of fatty acids derived from TG-rich lipoproteins [START_REF] Fielding | Lipoprotein lipase and the disposition of dietary fatty acids[END_REF]. Importantly, LPL is regulated by nutrients and hormones in a tissue-specific manner [START_REF] Wang | Lipoprotein lipase: from gene to obesity[END_REF]. LPL expression and enzyme activity have been detected in the brain in both neurons and glial cells [START_REF] Wang | Deficiency of lipoprotein lipase in neurons modifies the regulation of energy balance and leads to obesity[END_REF][START_REF] Huey | Lipoprotein lipase is expressed in cultured Schwann cells and functions in lipid synthesis and utilization[END_REF][START_REF] Nishitsuji | Lipoprotein lipase is a novel amyloid beta (Abeta)-binding protein that promotes glycosaminoglycan-dependent cellular uptake of Abeta in astrocytes[END_REF][START_REF] Wang | Lipoprotein lipase in the brain and nervous system[END_REF]. However, the functional significance of LPL in astrocytes is unknown.

In the current studies, we investigated the function of LPL in astrocytes in brain-lipid metabolism and potential contributions of astrocytic LPL to diet-induced obesity (DIO). We found that in astrocytes, LPL is responsible for controlling cellular lipid storage. In a loss-of-function study, mice lacking LPL uniquely in astrocytes had glucose intolerance and accelerated body weight gain when maintained on a HFD. We also found increased ceramide content in the hypothalamus of astrocytic LPL-deficient mice, accompanied by increased microglial reactivity and endoplasmic reticulum (ER) stress. Collectively, our data imply that LPL in astrocytes mediates lipid partitioning in the brain, and that it is required for appropriate CNS nutrient sensing and the regulation of energy homeostasis.

Research Methods and Design

Animals

All studies were approved by and performed according to the guidelines of the Institutional Animal Care and Use Committee of the University of Cincinnati and the Institutional Animal Care and Use Committee of the Helmholtz Center Munich, Bavaria, Germany. All mice were group housed on a 12-h light dark cycle at 23°C, with free access to food and water. Mice were fed either a standard chow diet, or a HFD (D12331; Research Diets).

Astrocyte-specific postnatal LPL-knockout mice (GFAP-LPL -/-) were generated by crossing the LPL lox/lox mice [START_REF] Augustus | Cardiac-specific knock-out of lipoprotein lipase alters plasma lipoprotein triglyceride metabolism and cardiac gene expression[END_REF] with hGFAP-CreER T2 , a transgenic mouse harboring the tamoxifeninducible Cre recombinase driven by a human glial fibrillary acidic protein promoter (hGFAP) [START_REF] Ganat | Early postnatal astroglial cells produce multilineage precursors and neural stem cells in vivo[END_REF]. At the age of 6 wk, tamoxifen was given by IP injection for 5 consecutive d at a dose of 100 ug/d. LPL loxP-homozygous and Cre-positive mice were used to generate knockout mice (GFAP-LPL -/-). Their littermates, which were LPL loxP-homozygous but Cre negative, served as WT controls (GFAP-LPL +/+ ).

For the flow cytometry study, co-localization of ceramide in GFAP-positive astrocytes and the in-situ hybridization study, GFAP-LPL +/+ and GFAP-LPL -/-mice were crossed with hGFAP-eGFP reporter mice [START_REF] Nolte | GFAP promoter-controlled EGFP-expressing transgenic mice: a tool to visualize astrocytes and astrogliosis in living brain tissue[END_REF] to label the Cre-targeted cells with an eGFP tag. To evaluate the efficiency of the post-natal deletion in different brain regions, GFAP-LPL +/+ mice were crossed with Rosa26 ACTB-tdTomato reporter mice and received tamoxifen injection in the same way as the experimental group. To evaluate the presence of ceramide in microglia, we used microglia-GFP reporter mice (Jax mice 005582).

Hypothalamic Primary Culture

For astrocytes, hypothalami were isolated from 2-d-old C57BL6J mice and triturated in MEM containing 1% penicillin-streptomycin, 10% fetal calf serum and 5.5 mM glucose. The cell pellet was centrifuged, re-suspended and seeded in a 175-cm 3 cell culture flask. After 8-9 d, the mixed glial culture reached 90% confluency. Then the flasks were placed in a 37°C shaking incubator at 240 rpm overnight to remove microglia. The cells were then seeded for the experiments.

For neurons, the hypothalami were isolated from 14-d embryos. After tripsinization, single cells were plated on 12-well plates and cultured in neurobasal medium supplemented with B-27 and GlutaMAX I. After 7 days in culture, the presence of glia cells were efficiently reduced through adding mitotic inhibitor Ara C (cytosine-1-β-D-arabinofuranoside), and hypothalamic neurons started to develop synaptic processes. Cells were then harvested to store at -80ºC until RNA extraction.

Adenovirus Cre-mediated deletion of LPL in hypothalamic astrocyte cultures

To induce the ex vivo ablation of LPL, primary hypothalamic astrocytes were obtained from LPL lox/lox mice and were seeded in 6-well cell culture plates as described above. Once they reached 90% confluency, astrocytes were transfected with adeno-Cre and adeno-control virus (Vector biolabs) plus 1% adenoboost (ATCGbio Life Technology) for 4 h to generate astrocyte cultures with LPL (Astro-LPL +/+ ) or without LPL (Astro-LPL -/-).

Seahorse analysis

Astrocytes were seeded in an XF24 plate (Seahorse Bioscience) with 80,000 cells per well. 24 h after adeno-cre or control virus transfection, cells were washed with PBS and incubated with XF assay medium containing 5.5 mM glucose for 1 h in a 37°C air incubator. The XF24 plate was then transferred to a temperature-controlled (37°C) extracellular flux analyzer (Seahorse Bioscience) and subjected to an equilibration period. For the analysis of extracellular acidification rates (ECARs) derived from glycolysis, the measurements were ended by the addition of 2-deoxy-glucose (2DG; 100 mM). 2DG-sensitive PPR estimates the ATP production from glycolysis with a 1:1 ratio. To normalize respirometry readings to cell number per well, cells were stained with crystal violet after the flux experiment. L-Lactate from the astrocyte culture medium was assayed by EnzyChromTM lactate assay kit (Bioassays System).

Lipase activity assay

Cells were collected in 20 mM Tris 150 mM NaCl wtih 15 ug/ml heparin. After sonication, cell lysates were kept at 37°C for 45 min, following by 10,000 g centrifuge at 4°C for 10 min.

Lipase activity was determined by a fluorometric lipoprotein lipase activity assay kit (STA-610-CB, cell biolabs). Protein level was determined by a BCA assay. Lipase activity was normalized to protein content.

Real-time PCR

For gene expression analysis, hypothalamic tissue was harvested and total RNA was isolated by an RNeasy lipid tissue kit (Qiagen). After reverse transcription by a QuantiTect Rev.

Transcription Kit (Qiagen), gene expression was analyzed by a real-time PCR with Taqman probes (Applied Biosystems) and SYBR primers. Hypoxanthine phosphoribosyltransferase 1 (HPRT) was used as a housekeeping gene. Sequences information can be found in Table .1 (Supplemental information).

PCR

For PCR to detect the LPL delta band after Cre-loxP recombination, sequences of primers were set as follows: Forward 5'-CGCCCTGGAACATCACTAAT-3'; Reverse: 5'-CTTCTCAATTGT GGGCAGGT-3'. The primers would develop a band of about 2000 bp on WT DNA sequence and a LPL delta band of 409 bp after floxed sequences cut by Cre.

Flow cytometry

The isolation protocol has been described in detail before [START_REF] Fischer | Prospective isolation of adult neural stem cells from the mouse subependymal zone[END_REF]. Briefly, the forebrain was dissected and dissociated by enzymatic and mechanical approaches. Single cells were separated by sucrose gradient. Sorting was done by a flow cytometry cell sorter: FACS Aria l with FACS Diva software (BD Biosciences) based on GFP signals. Purified GFAPexpressing astrocytes from GFAP-LPL +/+ and GFAP-LPL -/-mice were used for validating the LPL knock-down by real-time PCR as described above.

Glucose Tolerance Test

An intraperitoneal glucose tolerance test (ipGTT) was performed by injection of glucose (2 g/kg, 25% wt/vol. d-glucose (Sigma) in 0.9% wt/vol NaCl after a 5-h fast. Tail blood glucose levels (mg/dl) were measured with a TheraSense Freestyle glucometer (Abbott Diabetes Care) before (0 min) and at 15, 30, 60 and 120 min after injection.

Feeding experiments and metabolic phenotyping

Body weight were measured weekly through the study. Food intake was measured on a daily basis for two wk at the end of the study. Animals were double-housed and daily food intake of each cage was measured and averaged to per mice per day. Measurements of energy expenditure and physical activity were performed by a customized indirect calorimetric system (TSE Systems). Mice were adapted to the system for 24 h before data collection for the following 4 d.

Immunohistochemistry and Image Analysis

Immunohistochemistry was carried out as described before [START_REF] Gao | Hormones and diet, but not body weight, control hypothalamic microglial activity[END_REF]. Briefly, mice were perfused and fixed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4) at 4°C. After being equilibrated for 48 h with 30% sucrose in TBS, coronal sections (30 µm) were cut on a cryostat. Sections were washed and incubated with primary antibodies at 4°C overnight: rabbit anti-GFAP (DAKO), rabbit anti-iba1 (synaptic systems), mouse anti-ceramide (Enzo life science), rabbit anti-NeuN (cell signaling). On the second day, sections were rinsed and incubated with biotinylated secondary antibodies and avidin-biotin complex (Vector Laboratories). The reaction product was visualized by incubation in 1% diaminobenzidine with 0.01% hydrogen peroxide. For immunofluorescence, sections were incubated with corresponding alexa fluorescent secondary antibodies (Jackson ImmunoResearch).

Immunoreactivity was analyzed by ImageJ. Appropriate color threshold for DAB staining was set up manually for the first picture and applied to all the rest pictures. The brain area covered by the DAB staining signals (iba1, GFAP and AGRP) above the color threshold of each individual brain section in the fixed hypothalamic region was used to quantify the immunoreactivity. Two brain sections (at the similar anatomical level) of each individual animal were used to calculate the immunoreactivity average of each animal.

In-situ hybridization

For in-situ hybridization, 30-um coronal sections were treated with 2 ug/ml proteinase K 30 min at 37°C, 0.2% glycine buffer 30 s and 0.1% Triton X-100 for 10 min. Brain sections were hybridized in hybridization buffer (Sigma-Aldrich) containing 200 nM locked nucleic acid for UPLC-qTOF analysis. Chromatographic separation was performed using an AQUITY-UPLC system (Waters Corporation), equipped with a HSS T3 column (2.1x100 mm, 1.8µm;

Waters Corporation) as previously described [START_REF] Knittelfelder | A versatile ultra-high performance LC-MS method for lipid profiling[END_REF]. A SYNAPT™G1 qTOF HD mass spectrometer (Waters Corporation) equipped with an ESI source was used for detection. Data acquisition was done by the MassLynx 4.1 software (Waters Corporation). Ceramides were analyzed with the "Lipid Data Analyzer 1.6.2" software. Extraction efficacy and lipid recovery was normalized using ISTDs.

Statistical Analysis

All statistical analyses were performed using GraphPad Prism. Two groups were compared by using two-tailed unpaired Student's t test. Two-way ANOVA was performed to detect significant interactions between genotype and diet, and multiple comparisons were analyzed following Bonferroni´s post hoc tests. P values lower than 0.05 were considered significant.

All results are presented as means ± SD when n<10, as means ± SEM when n>10.

Results

Astrocytic LPL is regulated by nutrients in the hypothalamus

LPL activity has been detected in the hypothalamus, with changes occurring in response to a prolonged fast [START_REF] Eckel | Lipoprotein lipase is produced, regulated, and functional in rat brain[END_REF]. We first confirmed the presence of LPL in the hypothalamus, and particularly in primary astrocytes, as well as its modulation in response to different nutrient stimuli. Consistent with previous reports, LPL was expressed by both neurons and glial cells [START_REF] Wang | Deficiency of lipoprotein lipase in neurons modifies the regulation of energy balance and leads to obesity[END_REF][START_REF] Nimmerjahn | Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo[END_REF]. We compared the relative abundance of LPL in primary isolated hypothalamic neurons and astrocytes, and found higher enriched-LPL mRNA levels in astrocytes than in neurons (Fig. 1A). Next, we evaluated the impact of the exposure to lipid on LPL expression in vivo and in vitro. An increase in LPL mRNA levels was observed in the hypothalamus of 8-mo HFD-fed mice (Fig. 1B). When hypothalamic-derived astrocytes were exposed to triglyceride (TG) and palmitic acid, LPL expression was reduced, whereas the opposite effect was seen with oleic acid (Fig. 1C&D). These data suggest that astrocytic LPL is regulated by nutrients and responds differentially to saturated versus non-saturated fatty acids.

The loss of LPL reduces cellular lipid content and increases glycolytic capacity in a

hypothalamic astrocyte culture.

To analyze whether LPL in astrocytes regulates cellular lipid accumulation and glucose metabolism, we isolated hypothalamic astrocytes from LPL lox/lox mice and performed virusmediated excision of LPL by in vitro transfection with Cre-expressing adenoviruses (AAV-Cre), to finally obtain astrocytes with (Astro-LPL +/+ ) and without LPL (Astro-LPL -/-). A reduction of LPL expression (Fig. 2A) and lipase activity (Fig. 2B) were confirmed by qPCR and the lipase activity assay, respectively, after virus transfection. Loss of LPL activity leads to a decrease of lipid droplet content in astrocytes as assessed by Oil red O staining (Fig. 2C).

Lipogenesis-related genes including cluster of differentiation 36 (CD36) and fatty acid synthase (FASN) were upregulated in LPL-deficient astrocytes (Fig. 2A), indicating a compensatory response to the lipid-derived situation. We next determined whether LPL regulates glycolysis in astrocytes (Fig. 2D). Indeed, Astro-LPL -/-cells had increased cellular acidification and maximal glycolytic capacity (Fig. 2E&F). More ATPs were produced from enhanced glycolysis compared with what occurred in Astro-LPL +/+ (Fig. 2G). Accordingly, lactate secretion was increased in the medium from Astro-LPL -/-(Fig. 2H), consistent with the increased glycolysis. These data indicate that LPL participates in regulating lipid content and energy metabolism in astrocytes. Specifically, we found that the lack of LPL reduces astrocytic lipid storage and enhances astrocytic glycolysis.

Validation of postnatal ablation of LPL in GFAP-expressing cells in the brain

To evaluate the impact of the loss of LPL in astrocytes in vivo, we generated postnatal astrocyte-specific LPL-knockout mice by the Cre/lox system. We first validated the deletion of LPL in astrocytes after peripheral tamoxifen injection at 6 wk of age by multiple approaches. We confirmed the Cre-driven deletion of LPL in hGFAP positive cells by in-situ hybridization (Fig. 3A). Primers were designed based on genetic construction of LPL-floxed sequences to detect a specific band after Cre-induced recombination (Fig. 3B). Purified GFAP-expressing astrocytes were sorted from the brain of GFAP-LPL +/+ (LPL lox/lox mice injected with tamoxifen) or GFAP-LPL -/-mice (hGFAP-CreER T2 -LPL lox/lox injected with tamoxifen), which were crossed with hGFAP-eGFP reporter mice. qPCR of sorted brain GFP-expressing cells confirmed the knockdown of LPL (Fig. 3C). Using Rosa26 ACTB-tdTomato reporter mice which were crossed with GFAP-LPL -/-mice, we confirmed that Cremediated postnatal ablation occurred in the brain after tamoxifen injection, (Fig. S1A). The majority of the Cre-recombined GFAP-expressing astrocytes were located in the hypothalamus (46.3% ± 2.1%) in comparison with other brain regions such as thalamus, hippocampus and cortex (Fig. 3D). Even though GFAP is also expressed in peripheral tissues including stellate cells in liver and pancreas [START_REF] Ding | Glial fibrillary acidic protein promoter targets pancreatic stellate cells[END_REF][START_REF] Tennakoon | Analysis of glial fibrillary acidic protein (GFAP)-expressing ductular cells in a rat liver cirrhosis model induced by repeated injections of thioacetamide (TAA)[END_REF], no alterations in LPL expression were found in the liver between GFAP-LPL +/+ and GFAP-LPL -/-mice (Fig. 3E). Glucose-induced insulin secretion in isolated islets was also not affected between groups (Fig. 3F). These findings confirm that the loss of LPL occurs specifically in GFAP-expressing cells in the brain.

The loss of LPL in astrocytes causes ceramide accumulation, microglial activation and increased Agouti-related peptide (AGRP) in the hypothalamus and not in other brain areas

Previously, it has been reported that ceramide synthesis in the brain is associated with LPL activity [START_REF] Picard | Hippocampal lipoprotein lipase regulates energy balance in rodents[END_REF]. Ceramide is an important multi-functional intracellular signaling molecule. In the periphery, ceramide accumulation causes lipid toxicity and insulin resistance [START_REF] Chavez | A ceramide-centric view of insulin resistance[END_REF][START_REF] Holland | Lipid-induced insulin resistance mediated by the proinflammatory receptor TLR4 requires saturated fatty acid-induced ceramide biosynthesis in mice[END_REF]. In the mouse brain, after knocking out LPL from GFAP-positive astrocytes, total ceramide content was increased in the hypothalamus (Fig. 4A). Ceramide species having different acylchains were also analyzed (Fig. 4B). Mice lacking astrocytic LPL had increased C18:0, C18:1 and C22:0 levels in the hypothalamus. By ceramide staining, we also found that ceramide immunoreactivity (-ir) positive cells, which are mainly neurons (Fig. S2), were markedly increased in GFAP-LPL -/-mice in the hypothalamus (Fig. S1B&C), consistent with the lipidomics results.

In accordance with several studies that have reported that ceramide accumulation in the hypothalamus causes endoplasmic reticulum (ER) stress and interrupts energy balance [START_REF] Contreras | Central ceramide-induced hypothalamic lipotoxicity and ER stress regulate energy balance[END_REF],

we observed that the loss of LPL in astrocytes increased the expression of several ER stress markers in the hypothalamus. Specifically, we found that chaperone GRP78 (glucoseregulated protein 78 kDa), total Xbp1 (X-box binding protein 1) and sliced Xbp1 (sXbp1),

were each increased in the hypothalamus of GFAP-LPL -/-mice (Fig. 4C); whereas were unchanged in the hippocampus or cortex of those mice (Fig. S1F-G).

We also analyzed the effect of the loss of LPL in astrocytes by immunohistochemistry. GFAP immunoreactivity did not differ between GFAP-LPL +/+ and GFAP-LPL -/-mice (Fig. 4D&E).

On the other hand, the major immune responsive cells -microglia, had increased reactivity in GFAP-LPL -/-mice medial basal hypothalamus (Fig. 4F&G), but not in other brain regions (Fig. S1D&E), suggesting that microglia responded to the ceramide-induced lipid toxicity, and that this may contribute to the hypothalamic insulin resistance and metabolic disorders.

Such glial and ceramide alterations in the hypothalamus were associated with changes in specific neuronal populations involved in the regulation of energy balance: AGRP neurons and Proopiomelanocortin (POMC) neurons. While POMC cell number did not change (Fig. 4H&I), AGRP immunoreactivity was significantly elevated in the GFAP-LPL -/-mice (Fig. 4J&K).

Collectively, these data suggest that in the current model, lack of LPL in astrocytes causes ceramide accumulation in hypothalamus, and is associated with microglial activation, ER stress and increased AGRP.

Mice lacking LPL in astrocytes have exaggerated body weight gain and glucose

intolerance when maintained on HFD diet.

To assess whether the loss of LPL in astrocytes affects whole-body energy metabolism, mice were maintained on chow or HFD for 10 wk beginning at 10 wk of age. Only modest metabolic phenotypes were initially observed when the animals were fed the chow diet (see Fig. S3). Briefly, GFAP-LPL -/-mice exhibited a glucose intolerance phenotype and reduced activation of AKT (phospho-AKT; phosphorylation in Ser 473 of protein kinase B) in the hypothalamus 10 min after peripheral insulin stimulation. Although the mice had a slightly higher food intake at 24 wk of age compared with GFAP-LPL +/+ mice, no changes were detected in body weight or fat mass.

When these mice were fed with a HFD, GFAP-LPL -/-mice had accelerated body weight gain (Fig. 5A) relative to controls, and this was mainly due to increased fat mass (Fig. 5B&C).

Calorimetric measurements after 10 wk on the HFD revealed that GFAP-LPL -/-mice had increased food intake (Fig. 5D) and lower locomotor activity (Fig. 5F), and these likely contributed to the increased body weight gain. Higher energy expenditure (Fig. 5E) might be the consequence of the higher body weight of GFAP-LPL -/-mice. Plasma leptin level was elevated in GFAP-LPL -/-mice (Fig. 5G), which was consistent with the increased fat mass.

Plasma insulin level did not change (Fig. 5H). GFAP-LPL -/-mice had enhanced glucose intolerance (Fig. 5I&J). Since GFAP-LPL -/-mice on the chow diet already exhibited glucose intolerance without changes in body weight or fat mass, we determined that the genotype is determinant of the glucose intolerance phenotype. In GFAP-LPL -/-mice maintained with the HFD, the increased adiposity exaggerates the glucose intolerance. Thus, the loss of LPL in astrocytes induces a glucose intolerance and exaggerated body weight on a HFD, implying that LPL-related lipid metabolism in astrocytes has an important role in regulating metabolic homeostasis.

Discussion

In the present experiments, we investigated the function of LPL in astrocytes and its role in systemic metabolic control. We first found that LPL expression is increased in the hypothalamus of long-term HFD-fed mice, suggesting a potential role of LPL in the dietinduced obesity pathology. We also found that LPL is more abundant in astrocytes than in neurons, consistent with the classic view that CNS astrocytes actively consume lipid. We found that LPL controls lipid storage in astrocytes in response to different nutrient inputs. A lack of LPL in astrocytes resulted in exaggerated body weight gain and glucose intolerance in animals fed a HFD, and these phenotypes were associated with increased ceramide content in the hypothalamus. These results indicate that astrocytes play a key role in TG sensing and the control of lipid homeostasis.

We found that the LPL level in astrocytes varies according to the amount and specific mix of extracellular lipids. LPL was down-regulated in the presence of TGs and palmitic acid, but up-regulated by oleic acid. These data suggest that LPL may protect astrocytes from saturated fatty acid overload, an essential regulatory function for astrocytes to function properly during HFD-induced hyperlipidemia.

Moreover, we also observed increased ceramide content in the hypothalamus of mice lacking astrocytic LPL, consistent with studies indicating that the inhibition of LPL activity triggers de novo ceramide synthesis [START_REF] Picard | Hippocampal lipoprotein lipase regulates energy balance in rodents[END_REF]. In our study, we found that the deletion of LPL from astrocytes resulted in ceramide accumulation in hypothalamic neurons. Given that astrocytes metabolically supply lipids to neurons [START_REF] Foll | Regulation of hypothalamic neuronal sensing and food intake by ketone bodies and fatty acids[END_REF][START_REF] Guzman | Is there an astrocyte-neuron ketone body shuttle?[END_REF], we cannot rule out that the LPL deficiency in astrocytes might also cause the reduction of neuronal FA availability, triggering de novo ceramide synthesis in neurons as we observed. In our mouse model, we also found changes in AGRP immunoreactivity in the hypothalamus that might be a consequence of reduced lipid storage in the microenvironment due to the LPL deletion in astrocytes. Indeed, Libby et al.,

reported that LPL is able to modulate AGRP level through the regulation of lipid storage in neurons [START_REF] Libby | Lipoprotein lipase is an important modulator of lipid uptake and storage in hypothalamic neurons[END_REF]. Besides the ceramide-induced lipid toxicity, we also observed elevation of ER stress markers and increased microglial immunoreactivity in the hypothalamus, both of which are associated with ceramide accumulation as reported in other studies [START_REF] Holland | Lipid-induced insulin resistance mediated by the proinflammatory receptor TLR4 requires saturated fatty acid-induced ceramide biosynthesis in mice[END_REF][START_REF] Contreras | Central ceramide-induced hypothalamic lipotoxicity and ER stress regulate energy balance[END_REF]. Thus, these data suggest that disrupted lipid uptake in astrocytes causes ceramide accumulation and consequentially contributes to the pathology of obesity.

Compared with previous studies using neuronal LPL knockout approach [START_REF] Wang | Deficiency of lipoprotein lipase in neurons modifies the regulation of energy balance and leads to obesity[END_REF], our astrocytic LPL-knockout mouse model had unchanged body weight on a chow diet. Given that LPL is highly expressed in the rat brain during the lactating period, and falls dramatically after weaning [START_REF] Tavangar | Developmental regulation of lipoprotein lipase in rats[END_REF], we induced the deletion of LPL in adult mice to avoid developmental complications. Moreover, this deletion was restricted to GFAP-expressing astrocytes, which induced mainly changes in the hypothalamus rather than other brain areas, to investigate the significance of LPL function in these glial cells in the adult stage.

Overall, our data highlight the role of astrocytic lipid uptake in energy homeostasis. Mice lacking LPL in astrocytes have disrupted lipid metabolism in astrocytes and ceramide accumulation in hypothalamic neurons, contributing to impaired glucose tolerance and accelerated body weight gain in animals fed a HFD. analysis demonstrated that GFAP-LPL -/-mice had higher energy expenditure (E) and lower physical activity (F). Plasma leptin levels increased in GFAP-LPL -/-mice (G); while plasma insulin levels were unchanged (H). GFAP-LPL -/-mice had impaired glucose tolerance (I) and increased area under the curve (AUC) compared with GFAP-LPL +/+ littermates (J). p <0.05 for *, p<0.01 for **, p<0.001 for. n= 6-12 mice for metabolic phenotyping and calorimetric study.

Figure 1 .

 1 Figure 1. Astrocytic LPL is regulated by nutrients in the hypothalamus. Expression

Figure 2 .

 2 Figure 2. The loss of LPL reduces cellular lipid content and increases glycolytic capacity

Figure 3 .

 3 Figure 3. Validation of postnatal ablation of LPL in GFAP-expressing cells in the mouse

Figure 4 .

 4 Figure 4. The loss of LPL in astrocytes causes ceramide accumulation, ER stress and

Figure 5 .

 5 Figure 5. Mice lacking LPL in astrocytes have exaggerated body weight gain and glucose intolerance when maintained on HFD. GFAP-LPL -/-mice fed on HFD had

Table 1 . qRT-PCR SYBR primer sequences or taqman probes for amplification of cDNA

 1

	Gene symbol	Sequence (5' -3')
	Hprt	Mm01545399_m1
	Hprt	GCAGTACAGCCCCAAAATGG
		AACAAAGTCTGGCCTGTATCCAA
	Lpl	Mm00434764_m1
	Fasn	MM00662319_m1
	Cd36	Mm01135198_m1
	sXbp1	CTGAGTCCGAATCAGGTGCAG
		GTCCATGGGAAGATGTTCTGG
	totalXbp1	CAGCACTCAGACTATGTGCA
		GTCCATGGGAAGATGTTCTGG
	Atf4	GGGTTCTGTCTTCCACTCCA
		AAGCAGCAGAGTCAGGCTTTC
	Chop	CCACCACACCTGAAAGCAGAA
		AGGTGAAAGGCAGGGACTCA
	Grp78	TTCAGCCAATTATCAGCAAACTCT
		TTTTCTGATGTATCCTCTTCACCAGT

Acknowledgement

The authors thank Carola Meyer and Elma Stapic (Helmholtz Diabetes Center) for the support of animal work organizing. The authors also appreciate the support from animal facility staff at University of Cincinnati (Metabolic Disease Institute) and Helmholtz Diabetes Center. This work was supported in part by funding to M.H.T from the Alexander von Humboldt Foundation, the Helmholtz Alliance ICEMED & the Helmholtz Initiative on Personalized Medicine iMed by Helmholtz Association, and the Helmholtz cross-program topic "Metabolic Dysfunction". Moreover, this work also was supported by funding of European Research Council ERC (AdG HypoFlam no. 695054).

Diabetes 78x39mm (300 x 300 DPI) Diabetes 150x255mm (300 x 300 DPI) Diabetes 71x31mm (300 x 300 DPI)

Author contributions

Y.G. performed the study, researched the data, analyzed results and wrote the manuscript.


Hayate Haddadi-Guemghar

Asma Tlili

Julien Dairou

Jean-Louis Paul

Khodir Madani

Nathalie Janel
email: janel@univ-paris-diderot.fr

Pr N Janel

Effect of lyophilized prune extract on hyperhomocysteinemia in mice

Keywords: Homocysteine, Prune extract, Mice, S-adenosylhomocysteine hydrolase, NAD(P)H:quinone oxydo reductase-1

Altered homocysteine metabolism defined as hyperhomocysteinemia is implicated as pathogenic factor in several cardiovascular diseases and atherosclerosis. The purpose of this study was to investigate the efficacy of prune extract, a good source of phenolic antioxidants, on lowering plasma homocysteine level in male hyperhomocysteinemic mice from average weight of 28g. The administration of lyophilized prune extract was carried out by intraperitoneal injection one day preceding and one hour before sacrifice of mice.

Prune extract decreased significantly plasma homocysteine level, correlated with an increased activity of Sadenosylhomocysteine (SAH) hydrolase and NAD(P)H: quinone oxydoreductase-1 activities. Our results suggest a beneficial effect of prune extract on hyperhomocysteinemia with reduction of homocysteine level by its conversion on to SAH by S-adenosylhomocysteine hydrolase, which is activated by NAD + , a byproduct of NAD(P)H: quinone oxydo reductase-1.

Introduction

Homocysteine (Hcy), a metabolic by-product of methionine, can cause widespread hazards to human body if its level is increased in plasma, defined by hyperhomocysteinemia [START_REF] Zhang | Inhibitory effect of Epimedium extract on S-adenosyl-l-homocysteine hydrolase and biomethylation[END_REF]. It can be remethylated into methionine by means of vitamin B12-dependent methionine synthase and 5methyltetrahydrofolate as a methyl donor. Hcy can also be catabolized into cysteine by the transsulfuration pathway via cystathionine beta synthase (CBS) and cystathioninase, both enzymes being vitamin B6dependent. A third way to remove Hcy is conversion to S-adenosylhomocysteine (SAH). The last reaction is mediated by SAH-hydrolase (SAHH) and favors the SAH formation in case of increased Hcy concentrations [START_REF] Obeid | Homocysteine and lipids: S-Adenosyl methionine as a key intermediate[END_REF]. Hcy is the main factor implicated or linked to several metabolic diseases like atherosclerosis, thrombosis, diabetes, Alzheimer, cerebral, and cardiovascular diseases [START_REF] Jacobsen | Caffeinated coffee, decaffeinated coffee, and the phenolic phytochemical chlorogenic acid up-regulate NQO1 expression and prevent H 2 O 2-induced apoptosis in primary cortical neurons[END_REF] Lawrence de [START_REF] Lawrence De Koning | Hyperhomocysteinemia and its role in the development of atherosclerosis[END_REF][START_REF] Buysschaert | Hyperhomocysteinemia and diabetic macroangiopathy: guilty or innocent bystander?: A literature review of the current dilemma[END_REF][START_REF] Van Dam | Hyperhomocysteinemia and Alzheimer's disease: A systematic review[END_REF]. Some phenolic compounds and plant extracts such as catechin, quercetin, chlorogenic acid, coffee, wine phenolics have been demonstrated to be effective in decreasing plasma Hcy level [START_REF] Nygård | Coffee consumption and plasma total homocysteine: The Hordaland Homocysteine Study[END_REF]Noll et al., 2009a;[START_REF] Noll | Effect of red wine polyphenol dietary supplementation on two phase II enzymes in liver of hyperhomocysteinemic mice[END_REF][START_REF] Jacobsen | Caffeinated coffee, decaffeinated coffee, and the phenolic phytochemical chlorogenic acid up-regulate NQO1 expression and prevent H 2 O 2-induced apoptosis in primary cortical neurons[END_REF]. Prune, dried fruits of plum (Prunus domestica) contain higher level of phenolic compounds. In according to the study of Donovan and colleagues (1998) the level of phenolic compounds in prunes (1840 mg/kg) surpasses the levels reported for many other popular fruits like as Red Flame seedless table grapes (<250 mg /kg of grape), white table varieties (<50 mg/kg of grape), apples (1200 mg/kg), oranges (830 mg/kg), pears (265 mg/kg), and cherries (850mg/kg of fruit) [START_REF] Donovan | Phenolic composition and antioxidant activity of prunes and prune juice (Prunus domestica)[END_REF]. Phenolic compounds in prunes have been found to inhibit human low-density lipoprotein oxidation in vitro, and thus might serve as preventive agents against chronic diseases, such as heart disease and cancer [START_REF] Stacewicz-Sapuntzakis | Chemical composition and potential health effects of prunes: a functional food? Critical reviews in food science and nutrition[END_REF]. Hyperhomocysteinemia is well defined as risk factor for cardiovascular diseases, therefore, the present study investigated the effects of polyphenolic prune extract, which contains notably chlorogenic acid and its isomer neochlorogenic acid, on plasma Hcy level in hyperhomocysteinemic mice and its implication on SAHH activity, a third way of Hcy metabolism.

Materials and methods

Extraction of prune phenolic compounds

The extraction of phenolic compounds from prune was carried out by microwave assisted extraction under optimized conditions [START_REF] Haddadi-Guemghar | Optimisation of microwave-assisted extraction of prune (Prunus domestica) antioxidants by response surface methodology[END_REF]. The fruit samples were prepared for extraction by cutting into small pieces by a knife. Two grams of sample were placed in a round-bottom flask with 40 mL of

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The flask was placed in a microwave oven and connected to condenser. After irradiation (1mn at 500W), the flask was taken out and cooled in ice water bath (4°C). The solution was filtered throug h Wathman n°1 paper under vacuum. After concentrating using rotavapor at 40°C, the extract was lyophiliz ed.

Total phenols and hydrogen peroxide determination

Total phenols (TP) content was determined by the Folin-Ciocalteu method. 1 ml of Folin-Ciocalteu reagent (diluted 10 times by water) was mixed with 100 µL of the prune extract. After 5min, 1 mL of aqueous solution of sodium carbonate (6%) was added. The mixture was kept for 60 min at room temperature. Absorbance was measured at 755 nm. Ethanol solution of gallic acid was used as standard [START_REF] Velioglu | Antioxidant Activity and Total Phenolics in Selected Fruits, Vegetables, and Grain Products[END_REF].

Hydrogen peroxide content was determined with the hydrogen peroxide assay kit (Abcam, France) as recommended by the supplier's instructions.

Mice, genotyping and experimental protocol

Mice were maintained in a controlled environment with unlimited access to food and water on a 12-h light/dark cycle. All procedures were carried out in accordance with internal guidelines of the French Agriculture Ministry for animal handing. Number of mice and suffering were minimized as possible. Mice heterozygous for targeted disruption of the Cbs gene (Cbs +/-) were generously donated by Dr. N. Maeda (Department of Pathology, University of North Carolina, Chapel Hill, NC, USA) [START_REF] Watanabe | Mice deficient in cystathionine beta-synthase: animal models for mild and severe homocyst (e) inemia[END_REF]. Cbs +/-mice, on a C57BL/6 background, were obtained by mating male Cbs +/-mice with female wild-type C57BL/6 (Cbs +/+) mice. DNA isolated from 4-week-aged mice tail biopsies was subjected to genotyping of the targeted CBS allele using polymerase chain reaction assay [START_REF] Watanabe | Mice deficient in cystathionine beta-synthase: animal models for mild and severe homocyst (e) inemia[END_REF]. The study was carried out on male 2-month-old Cbs +/-and Cbs +/+. The administration of prune extract was carried out by intraperitoneal injection of 100 µL of prune extract at concentration of 1mg/mL (1mg of lyophilized prune extract was resuspended in 1 mL of NaCl 0.9%) to Cbs +/-and Cbs +/+ mice. The mice control, both Cbs +/and Cbs +/+, were treated by intraperitoneal injection of 100 µL of NaCl 0.9%. All mice were treated one day preceding and one hour before sacrifice. Two experimental protocols (one with five mice per group and the second with three mice per group) have been done, depending on the variability of the analysed biomarker.

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Preparation of serum samples, tissue collection, and plasma tHcy assay

At the time of sacrifice, blood samples were collected into tubes containing an 1/10 volume of 3.8% sodium citrate, placed on ice immediately. Plasma was isolated by centrifugation at 2500g for 15 min at 4 °C. Liver was harvested, snap-frozen and stored at -80 °C un til use. Plasma tHcy was assayed by using the fluorimetric high-performance liquid chromatography method described by [START_REF] Fortin | Measurement of homocyst (e) ine in the prediction of arteriosclerosis[END_REF].

Determination of SAHH activity

SAHH activity assay was performed on 200 µg of total proteins obtained from liver samples following the protocol described previously [START_REF] Villanueva | Hepatic transmethylation reactions in micropigs with alcoholic liver disease[END_REF].

Determination of NAD(P)H: quinone oxydo reductase-1 activity

Determination of NAD(P)H: quinone oxydo reductase-1 (NQO1) activity was assayed on 100 µg of total proteins obtained from liver samples following the protocol described by Ernster [START_REF] Ernster | DT diaphorase[END_REF], and modified by Benson and colleagues [START_REF] Benson | Increase of NAD (P) H: quinone reductase by dietary antioxidants: possible role in protection against carcinogenesis and toxicity[END_REF].

Data analysis

Statistical analysis was done with one-way ANOVA followed by Student's unpaired t test using Statview software. The results are expressed as medians with interquartile ranges. Data were considered significant when p < 0.05. A p value of 0.06-0.10 was considered to indicate a strong statistical tendency due to the small sample size. Correlations between tHcy level, SAHH activity and NQO1 activity were determined by using Spearman's rank correlation as data were not normally distributed according to Shapiro -Wilk test.

Data were analyzed using R software (http://www.R-project.org) and considered significant when p < 0.05.

Results and discussion

Effects of prune extract on plasma tHcy level in wild type and hyperhomocysteinemic mice

Previous study demonstrated that administration of wine polyphenols such as catechin, epicatechin or wine extract modifies significantly Hcy metabolism, this effect depending on quantity of polyphenols administered (Noll et al., 2009a). The concentration of prune exptract injected here to mice, in terms of total polyphenols, was measured by Folin ciochalteu method. It was 3mg GAE (gallic acid equivalents)/mL per injection. The volume of prune extract injected was 100 µL, thereby the quantity of total phenols expressed as equivalent of gallic acid was 300 µg. The effects of prune extract administration on plasma tHcy level in wild type mice (Cbs +/+) and hyperhomocysteinemic mice (Cbs +/-) were shown in Fig. 1. The prune extract administration decreased significantly plasma tHcy level in both Cbs +/+ and Cbs +/-mice, in comparison with mice on control diet respectively. Although the phenolic content of prune extract is different to phenolic content of red wine extract, the result was similar to those obtained previously (Noll et al., 2009a). The major polyphenols in wine extract are gallic acid, catechin, epicatechin and p-coumaric acid [START_REF] Porgali | Determination of phenolic composition and antioxidant capacity of native red wines by high performance liquid chromatography and spectrophotometric methods[END_REF], whereas neochlorogenic acid and chlorogenic acid are the predominant polyphenols in prune extract [START_REF] Donovan | Phenolic composition and antioxidant activity of prunes and prune juice (Prunus domestica)[END_REF].

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Effect of prune extract on SAHH activity in wild type and hyperhomocysteinemic mice

The SAHH reaction, which forms Hcy from SAH, is reversible, with the equilibrium actually favoring the condensation of Hcy and adenosine to generate SAH [START_REF] De La Haba | The enzymatic synthesis of S-adenosyl-L-homocysteine from adenosine and homocysteine[END_REF]. A third way to remove Hcy is its conversion to SAH. The last reaction is mediated by SAHH and favors the SAH formation in case of increased Hcy concentrations. The effect of prune extract administration on SAHH activity in wild type and hyperhomocysteinemic mice was shown in Fig. 2A. Prune extract administration increased significantly SAHH activity in both Cbs +/+ and Cbs +/-mice. We found a 1.6 fold increase in SAHH activity in liver of Cbs +/+ mice treated with prune extract in comparison with mice on control diet, and a two-fold increase in SAHH activity in liver of Cbs +/-mice treated with prune extract in comparison with mice on control diet. Using Spearman correlation test, we found a significant negative correlation between plasma tHcy level and SAHH activity (Fig. 2B, r = -0.55, p < 0.02). Noll and colleagues (Noll et al., 2009a) also found a decrease of plasma tHcy level, concomitant with an increase of hepatic SAHH activity, after wine polyphenolic extract administration.

Effect of prune extract on NQO1 activity in wild type mice and hyperhomocysteinemic mice

Previous study demonstrated that an increase in SAHH activity is not due to an increase in SAHH expression, but occurs concomitantly with an increase in NQO1 activity (Noll et al., 2009b). NQO1, also

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referred to as DT-diaphorase, is a flavoprotein that catalyzes the two-electron reduction of quinones and quinonoid compounds to hydroquinones, using either NADH or NADPH as the electron donor [START_REF] Chen | Structure-function studies of DT-diaphorase (NQO1) and NRH: quinone oxidoreductase (NQO2)[END_REF]. NQO1 co-produces NAD+, which is a cofactor of SAHH. The administration of prune extract to mice increases NQO1 activity in both Cbs +/+ and Cbs +/-mice (Fig. 3A). Moreover, NQO1 activity was negatively correlated with plasma tHcy level (Fig. 3B, r = -0.65, p < 0.01). Several previous studies demonstrated that the induction of NQO1 is attributed to the NF-E2-related factor-2 (Nrf2), which binds to an upstream regulatory sequence, the antioxidant response element (ARE) and activates NQO1 gene expression [START_REF] Krajka-Kuźniak | Phloretamide, an apple phenolic compound, activates the Nrf2/ARE pathway in human hepatocytes[END_REF]. Experimental studies in animal models and cell cultures have shown that several naturally occurring polyphenols or plant extract demonstrate the ability to activate the Nrf2/ARE pathway.

Valerio and colleagues [START_REF] Valerio | Induction of human NAD (P) H: quinone oxidoreductase (NQO1) gene expression by the flavonol quercetin[END_REF] reported the induction of NQO1 expression by quercetin in human breast cancer cells. They show that treatment of human breast cancer cells for 24 h with 15 mM quercetin results in a two fold increase in NQO1 protein levels and enzyme activity, and a three-to four-fold increase in NQO1 mRNA expression [START_REF] Valerio | Induction of human NAD (P) H: quinone oxidoreductase (NQO1) gene expression by the flavonol quercetin[END_REF]. Krajka-Kuźniak and colleagues [START_REF] Krajka-Kuźniak | Phloretamide, an apple phenolic compound, activates the Nrf2/ARE pathway in human hepatocytes[END_REF] demonstrated the activation of Nrf2/ARE pathway by phloretamide, an apple phenolic compound in human hepatocytes. They have shown the translocation of Nrf2 from cytosol to nucleus and an increasing in the NQO1 transcript and protein after treatment of hepatocyte cells with phloretamide. Forman and colleagues proposed that the activation of the Nrf2/ARE pathway is the major mechanism of action for nutritional antioxidants in vivo [START_REF] Forman | How do nutritional antioxidants really work: Nucleophilic tone and para-hormesis versus free radical scavenging in vivo<[END_REF]. In prune, neochlorogenic acid is the principal phenolic compound (71%) followed by its isomer chlorogenic acid (24%). Kim and colleagues have shown the induction of NQO1 expression by chlorogenic acid in neuronal cultural cells, which suggested that prune phenolic compounds enhance NQO1 activity by inducing the expression of NQO1 [START_REF] Jacobsen | Caffeinated coffee, decaffeinated coffee, and the phenolic phytochemical chlorogenic acid up-regulate NQO1 expression and prevent H 2 O 2-induced apoptosis in primary cortical neurons[END_REF]. One other way to activate Nrf2 by polyphenols is the generation of hydrogen peroxide [START_REF] Erlank | Polyphenols activate Nrf2 in astrocytes via H2O2, semiquinones, and quinones[END_REF]. More importantly, polyphenols can undergo autoxidation with oxygen consumption and hydrogen peroxide production [START_REF] Matsuura | Structural specificity of electric potentials in the coulometric-array analysis of catechins and theaflavins[END_REF]. We therefore analyzed hydrogen peroxide content in prune extract and found generation (figure 5), with a positive correlation with the volume of prune extract (p < 0.0455; r= 1).

Therefore, even if we demonstrated that the use of microwaves in the extraction of phenolics compounds from prune not only gives the higher value but also increased the antioxidant activity [START_REF] Haddadi-Guemghar | Optimisation of microwave-assisted extraction of prune (Prunus domestica) antioxidants by response surface methodology[END_REF], the activation of Nrf2 by prune extract may also be due to oxidized species generated by autoxidation of polyphenols. In this sense, hydrogen peroxide can act as a cell-survival signaling molecule [START_REF] Groeger | Hydrogen peroxide as a cell-survival signaling molecule[END_REF]. NQO1. SAHH requires NAD+ to maintain its quaternary structure and catalytic activity, making the enzyme sensitive to the redox status of the cell that is reflected by the NAD + /NADH ratio [START_REF] Tehlivets | S-adenosyl-Lhomocysteine hydrolase and methylation disorders: Yeast as a model system[END_REF].

Conclusion

This study demonstrates that prune extract is effective in reducing plasma tHcy level in both Cbs +/+ mice and Cbs +/-mice. The possible mechanism is associated with the conversion of Hcy to SAH by SAHH, which is activated by NAD + , a by-product of NQO1 reaction. We suggest that prune extract enhances the NQO1 activity, which could increase the NAD + /NADH ratio.

  Correlation between SAHH activity and NQO1 activityAccording to Spearman test, we showed a strong tendency to positive correlation between SAHH activity and NQO1 activity (figure4, r = 0.42, p< 0.1). NQO1 activity and SAHH activity have an indirect relation since the SAHH enzyme is activated by NAD + , which is produce in oxydo-reduction reactions catalyzed by

Fig. 1 .Fig. 2 .

 12 Figure legends

Fig. 3 .

 3 Fig. 3. Effects of prune extract on hepatic NQO1 activity in wild type (Cbs +/+) and hyperhomocysteinemic (Cbs +/-) mice fed the control diet supplemented (prune extract: PE) or not (NaCl). Data of hepatic SAHH activity (A) correspond to the medians with interquartile ranges. Data were normalized to the mean of (Cbs +/+) mice on control diet not supplemented (NaCl). n = number of mice. Statistical analysis was done with one-way ANOVA followed by Student's unpaired t-tests. (B) Hepatic NQO1 activity was negatively correlated with tHcy level (p < 0.01; r=-0.65).

Fig. 4 .

 4 Fig. 4. Positive correlation between hepatic SAHH and NQO1 activity. Hepatic NQO1 activity was positively correlated with SAHH activity (p < 0.01; r= 0.42).

Fig. 5 .

 5 Fig. 5. Effects of prune extract on hydrogen peroxide generation. Hydrogen peroxide content was determined according to the volume of prune extract (p < 0.0455; r= 1).

Acknowledgements

We thank Dr. N. Maeda (Department of Pathology, University of North Carolina, Chapel Hill, NC) for providing heterozygous Cbs-null mice. This work was supported by the Association Gaspard Félix (L'AGAFE). We acknowledge the FlexStation3 Facility of the Functional and Adaptative Biology (BFA) laboratory.

Conflict of interest

The authors declare there are no conflicts of interest.

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