Sex-dependent effects of neonatal maternal deprivation on endocannabinoid levels in the adipose tissue: influence of diet

Maternal deprivation (MD) during neonatal life has diverse long-term effects, including modification of metabolism. We have previously reported that MD modifies the metabolic response to high-fat diet (HFD) intake, with this response being different between males and females, while previous studies indicate that in mice with HFD-induced obesity, endocannabinoid (EC) levels are markedly altered in various brown and white adipose tissue depots. Here, we analyzed the effects of MD (24 h at postnatal day 9), alone or in combination with a HFD from weaning until the end of the experiment in Wistar rats of both sexes. Brown and white perirenal and subcutaneous adipose tissues were collected and the levels of anandamide (AEA), 2-arachidonoylglycerol (2-AG), palmitoylethanolamide (PEA), and oleoylethanolamide (OEA) were determined. In males, MD increased the content of OEA in brown and 2-AG in subcutaneous adipose tissues, while in females the content of 2-AG was increased in perirenal fat. Moreover, in females, MD decreased AEA and OEA levels in perirenal and subcutaneous adipose tissues, respectively. HFD decreased the content of 2-AG in brown fat of both sexes and OEA in brown and subcutaneous adipose tissue of control females. In contrast, in subcutaneous fat, HFD increased AEA levels in MD males and OEA levels in control and MD males. The present results show for the first time that MD and HFD induce sex-dependent effects on the main ECs, AEA, and 2-AG, and of AEA-related mediators, OEA and PEA, in the rat brown and white (visceral and subcutaneous) adipose tissues.     

Extracellular β‐nicotinamide adenine dinucleotide (β‐NAD) promotes the endothelial cell barrier integrity via PKA‐ and EPAC1/Rac1‐dependent actin cytoskeleton rearrangement

Extracellular β‐NAD is known to elevate intracellular levels of calcium ions, inositol 1,4,5‐trisphate and cAMP. Recently, β‐NAD was identified as an agonist for P2Y1 and P2Y11 purinergic receptors. Since β‐NAD can be released extracellularly from endothelial cells (EC), we have proposed its involvement in the regulation of EC permeability. Here we show, for the first time, that endothelial integrity can be enhanced in EC endogenously expressing β‐NAD‐activated purinergic receptors upon β‐NAD stimulation. Our data demonstrate that extracellular β‐NAD increases the transendothelial electrical resistance (TER) of human pulmonary artery EC (HPAEC) monolayers in a concentration‐dependent manner indicating endothelial barrier enhancement. Importantly, β‐NAD significantly attenuated thrombin‐induced EC permeability as well as the barrier‐compromising effects of Gram‐negative and Gram‐positive bacterial toxins representing the barrier‐protective function of β‐NAD. Immunofluorescence microscopy reveals more pronounced staining of cell–cell junctional protein VE‐cadherin at the cellular periphery signifying increased tightness of the cell‐cell contacts after β‐NAD stimulation. Interestingly, inhibitory analysis (pharmacological antagonists and receptor sequence specific siRNAs) indicates the participation of both P2Y1 and P2Y11 receptors in β‐NAD‐induced TER increase. β‐NAD‐treatment attenuates the lipopolysaccharide (LPS)‐induced phosphorylation of myosin light chain (MLC) indicating its involvement in barrier protection. Our studies also show the involvement of cAMP‐dependent protein kinase A and EPAC1 pathways as well as small GTPase Rac1 in β‐NAD‐induced EC barrier enhancement. With these results, we conclude that β‐NAD regulates the pulmonary EC barrier integrity via small GTPase Rac1‐ and MLCP‐ dependent signaling pathways. J. Cell. Physiol. 223: 215–223, 2010. © 2010 Wiley‐Liss, Inc.     

Insulin resistance is improved in high‐fat fed mice by photobiomodulation therapy at 630 nm

Photobiomodulation therapy (PBMT) in the infrared spectrum exerts positive effects on glucose metabolism, but the use of PBMT at the red spectrum has not been assessed. Male Swiss albino mice were divided into low‐fat control and high‐fat diet (HFD) for 12 weeks and were treated with red (630 nm) PBMT or no treatment (Sham) during weeks 9 to 12. PBMT was delivered at 31.19 J/cm², 60 J total dose per day for 20 days. In HFD‐fed mice, PBMT improved glucose tolerance, insulin resistance and fasting hyperinsulinemia. PBMT also reduced adiposity and inflammatory infiltrate in adipose tissue. Phosphorylation of Akt in epididymal adipose tissue and rectus femoralis muscle was improved by PBMT. In epididymal fat PBMT reversed the reduced phosphorylation of AS160 and the reduced Glut4 content. In addition, PBMT reversed the alterations caused by HFD in rectus femoralis muscle on proteins involved in mitochondrial dynamics and β‐oxidation. In conclusion, PBMT at red spectrum improved insulin resistance and glucose metabolism in HFD‐fed mice.      

A new perspective on cannabinoid signalling: complementary localization of fatty acid amide hydrolase and the CB1 receptor in rat brain.

CB1-type cannabinoid receptors in the brain mediate effects of the drug cannabis. Anandamide and sn-2 arachidonylglycerol (2-AG) are putative endogenous ligands for CB1 receptors, but it is not known which cells in the brain produce these molecules. Recently, an enzyme which catalyses hydrolysis of anandamide and 2-AG, known as fatty acid amide hydrolase (FAAH), was identified in mammals. Here we have analysed the distribution of FAAH in rat brain and compared its cellular localization with CB1-type cannabinoid receptors using immunocytochemistry. High concentrations of FAAH activity were detected in the cerebellum, hippocampus and neocortex, regions of the rat brain which are enriched with cannabinoid receptors. Immunocytochemical analysis of these brain regions revealed a complementary pattern of FAAH and CB1 expression with CB1 immunoreactivity occurring in fibres surrounding FAAH-immunoreactive cell bodies and/or dendrites. In the cerebellum, FAAH was expressed in the cell bodies of Purkinje cells and CB1 was expressed in the axons of granule cells and basket cells, neurons which are presynaptic to Purkinje cells. The close correspondence in the distribution of FAAH and CB1 in rat brain and the complementary pattern of FAAH and CB1 expression at the cellular level provides important new evidence that FAAH may participate in cannabinoid signalling mechanisms of the brain.     

Cannabinoid-based drugs targeting CB1 and TRPV1, the sympathetic nervous system,and arthritis

Chronic inflammation in rheumatoid arthritis (RA) is accompanied by activation of the sympathetic nervous system, which can support the immune system to perpetuate inflammation. Several animal models of arthritis already demonstrated a profound influence of adrenergic signaling on the course of RA. Peripheral norepinephrine release from sympathetic terminals is controlled by cannabinoid receptor type 1 (CB₁), which is activated by two major endocannabinoids (ECs), arachidonylethanolamine (anandamide) and 2-arachidonylglycerol. These ECs also modulate function of transient receptor potential channels (TRPs) located on sensory nerve fibers, which are abundant in arthritic synovial tissue. TRPs not only induce the sensation of pain but also support inflammation via secretion of pro-inflammatory neuropeptides. In addition, many cell types in synovial tissue express CB₁ and TRPs. In this review, we focus on CB₁ and transient receptor potential vanilloid 1 (TRPV1)-mediated effects on RA since most anti-inflammatory mechanisms induced by cannabinoids are attributed to cannabinoid receptor type 2 (CB₂) activation. We demonstrate how CB₁ agonism or antagonism can modulate arthritic disease. The concept of functional antagonism with continuous CB₁ activation is discussed. Since fatty acid amide hydrolase (FAAH) is a major EC-degrading enzyme, the therapeutic possibility of FAAH inhibition is studied. Finally, the therapeutic potential of ECs is examined since they interact with cannabinoid receptors and TRPs but do not produce central side effects.     

Occurrence and possible biological role of the endocannabinoid system in the sea squirt Ciona intestinalis

A cannabinoid receptor orthologue (CiCBR) has been described in the sea squirt Ciona intestinalis. Here we report that CiCBR mRNA expression is highest in cerebral ganglion, branchial pharynx, heart and testis of C. intestinalis, and that this organism also contains cannabinoid receptor ligands and some of the enzymes for ligand biosynthesis and inactivation. Using liquid chromatography-mass spectrometry, the endocannabinoid anandamide was found in all tissues analysed (0.063-5.423 pmol/mg of lipid extract), with the highest concentrations being found in brain and heart. The endocannabinoid 2-arachidonoylglycerol (2-AG) was fivefold more abundant than anandamide, and was most abundant in stomach and intestine and least abundant in heart and ovaries (2.677-50.607 pmol/mg of lipid extract). Using phylogenomic analysis, we identified orthologues of several endocannabinoid synthesizing and degrading enzymes. In particular, we identified and partly sequenced a fatty acid amide hydrolase (FAAH) orthologue, showing 44% identity with human FAAH and containing nearly all the amino acids necessary for a functional FAAH enzyme. Ciona intestinalis also contained specific binding sites for cannabinoid receptor ligands, and an amidase enzyme with pH-dependency and subcellular/tissue distribution similar to mammalian FAAHs. Finally, a typical C. intestinalis behavioural response, siphon reopening after closure induced by mechanical stimulation, was inhibited by the cannabinoid receptor agonist HU-210, and this effect was significantly attenuated by mammalian cannabinoid receptor antagonists.     

Mesenteric adipose tissue B lymphocytes promote local and hepatic inflammation in non‐alcoholic fatty liver disease mice

Mesenteric adipose tissue (MAT) inflammation is associated with non‐alcoholic fatty liver disease (NAFLD), and immune cells play pivotal roles in the inflammation of adipose tissue. Here, we investigated the roles of MAT B lymphocytes in NAFLD. Mice fed with high‐fat diet (HFD) and normal diet (ND) were killed in time gradients (4, 8 and 12 weeks). Compared with ND‐fed mice, intra‐hepatic CD45⁺CD19⁺ B lymphocytes increased after 4 weeks (P < 0.01) of HFD feeding, and lasted until the 12th week, infiltrated earlier than CD45⁺CD3⁺ T lymphocytes and CD45⁺F4/80⁺ macrophages. The mRNA expression of tumour necrosis factor (TNF)‐α, interleukin (IL)‐6 and monocyte chemotactic protein (MCP)‐1 decreased in MAT of B^null HFD‐fed mice compared to that in wild‐type HFD‐fed mice, along with lesser macrophages. Mesenteric adipose tissue B cells from HFD‐fed mice promoted macrophage differentiation to type‐Ι macrophages and expression of pro‐inflammatory cytokines in vitro. Macrophages pre‐treated with MAT B cells from HFD‐fed mice showed elevated mRNA expression of IL‐6 and TNF‐α and declined IL‐10 levels in adipocytes compared to ND MAT B cell pre‐treated macrophages. Besides, internal near‐infrared scanning and external transwell assay showed that HFD MAT B cells migrated to the liver more than ND MAT B cells. High‐fat diet MAT B cells induced higher MCP‐1 and lower IL‐10 expression in primary hepatocytes compared to ND MAT B cells in co‐culture experiment. These data indicate that B lymphocytes infiltrate early in MAT during the development of NAFLD, which may not only promote MAT inflammation by regulating macrophages but also migrate to the liver and induce hepatocytes inflammation.     

Pancreatic ectopic fat is characterized by adipocyte infiltration and altered lipid composition

Objective: Sustained exposure to lipids is deleterious for pancreatic islet function. This could be mediated through increased pancreatic fat following increased dietary fat and in obesity, which has implications for the onset of type 2 diabetes. The aims of this study were to determine changes in extent and composition of pancreatic, hepatic, and visceral fat in mice fed a high‐fat diet (HFD, 40% by weight) compared with a control diet (5% fat) of similar fatty acid composition, and to compare composition and extent of pancreatic fat in human type 2 diabetes. Methods and Procedures: Mice were fed HFD for 3 or 15 weeks. Human postmortem pancreas was examined from subjects with type 2 diabetes (n = 9) and controls (n = 7). Tissue lipid content and composition were determined by gas chromatography and pancreatic adipocyte infiltration quantified by morphometry. Results: Pancreatic triacylglycerol (TG) content was 20× greater (P < 0.05) in HFD mice and there were more pancreatic perilipin‐positive adipocytes compared with controls after 15 weeks. The proportions of 18:1n ?9 and 18:2n ?6 in pancreatic TG and the 20:4n ?6/18:2n ?6 ratio in phospholipids, were higher (both P < 0.05) after HFD compared with controls. Human pancreatic TG content was correlated with the proportion of pancreatic perilipin‐positive adipocytes (r = 0.64, P < 0.05) and associated with unsaturated fatty acid enrichment (P < 0.05). Discussion: Adipocyte infiltration in pancreatic exocrine tissue is associated with high‐fat feeding in mice and pancreatic TG content in humans. This alters the fatty acid milieu of the islet which could contribute to islet dysfunction.     

Caralluma fimbriata and metformin protection of rat pancreas from high fat diet induced oxidative stress

A high fat diet promotes oxidative stress, which contributes to the development of pancreatic fibrosis. We compared the protective effects of a hydroalcoholic extract of Caralluma fimbriata (CFE) to metformin (Met) in the pancreas of Wistar rats fed a high fat diet. The experimental animals were divided into five groups: control (C), treated with CFE (C + CFE), treated with high fat diet (HFD), high fat diet treated with CFE (HFD + CFE), and high fat diet treated with metformin (Met) (HFD + Met). CFE was administered orally to groups C + CFE and HFD + CFE rats for 90 days. Met was given to the HFD + Met group. After 90 days, oxidative stress markers in the pancreas including reduced glutathione (GSH), lipid oxidation (LO), protein oxidation (PO), and activities of antioxidant and polyol pathway enzymes, aldose reductase (AR) and sorbitol dehydrogenase (SDH) were assayed and tissue histology was examined. Establishment of oxidative stress in high fat diet fed rats was verified by elevated LO and PO, decreased GSH, decreased activities of antioxidants and increased activities of polyol pathway enzymes. Oxidative stress was prevented in HFD + CFE and HFD + Met groups. Group C + CFE exhibited improved antioxidant status compared to group C. CFE treatment prevented high fat diet induced acinar cell degeneration, necrosis, edema and hemorrhage. CFE could be used as adjuvant therapy for preventing or managing high fat diet induced pancreatic damage.     

Coevolution between cannabinoid receptors and endocannabinoid ligands

Genes for receptors and ligands must coevolve to maintain coordinated gene expression and binding affinities. Researchers have debated whether anandamide or 2-arachidonyl glycerol (2-AG) is a more "intrinsic" ligand of cannabinoid receptors. We addressed this debate with a coevolutionary analysis, by examining genes for CB1, CB2, and ten genes that encode ligand metabolic enzymes: abhydrolase domain containing 4 protein, cyclooxygenase 2, diacylglycerol lipase paralogs (DAGLalpha, DAGLbeta), fatty acid amide hydrolase paralogs (FAAH1, FAAH2), monoglyceride lipase, N-acylethanolamine acid amidase, NAPE-selective phospholipase D, and protein tyrosine phosphatase non-receptor type 22. Gene trees (cladograms) of CB1, CB2, and ligand enzymes were obtained by searching for orthologs (tBLASTn) in the genomes of nine phylogenetically diverse species, aligning ortholog sequences with ClustalX, and applying Bayesian analysis (MrBayes). Mirrored cladograms provided evidence of coevolution (i.e., parallel cladogenesis). Next we constructed phylograms of CB1, CB2, and the ten enzymes. Phylogram branch lengths were proportional to three sets of maximum likelihood metrics: all-nucleotide-substitutions and NS/SS ratios (using PAUP()), and Ka/Ks ratios (using FUGE). Spurious correlations in all-nucleotide-substitutions trees (due to phylogenetic bias) and in Ka/Ks ratio trees (due to simplistic modeling) were parsed. Branch lengths from equivalent branches in paired trees were correlated by linear regression. Regression analyses, mirrored cladograms, and phylogenetic profiles produced the same results: close associations between cannabinoid receptors and DAGL enzymes. Therefore we propose that cannabinoid receptors initially coevolved with a fatty acid ester ligand (akin to 2-AG) in ancestral metazoans, and affinity for fatty acid ethanolamide ligands (e.g., AEA) evolved thereafter.     

Inactivation of fatty acid amide hydrolase protects against ischemic reperfusion injury-induced renal fibrogenesis

Although cannabinoid receptors (CB) are recognized as targets for renal fibrosis, the roles of endogenous cannabinoid anandamide (AEA) and its primary hydrolytic enzyme, fatty acid amide hydrolase (FAAH), in renal fibrogenesis remain unclear. The present study used a mouse model of post-ischemia-reperfusion renal injury (PIR) to test the hypothesis that FAAH participates in the renal fibrogenesis. Our results demonstrated that PIR showed upregulated expression of FAAH in renal proximal tubules, accompanied with decreased AEA levels in kidneys. Faah knockout mice recovered the reduced AEA levels and ameliorated PIR-triggered increases in blood urea nitrogen, plasma creatinine as well as renal profibrogenic markers and injuries. Correspondingly, a selective FAAH inhibitor, PF-04457845, inhibited the transforming growth factor-beta 1 (TGF-β1)–induced profibrogenic markers in human proximal tubular cell line (HK-2 cells) and mouse primary cultured tubular cells. Knockdown of FAAH by siRNA in HK-2 cells had similar effects as PF-04457845. Tubular cells isolated from Faah^?/? mice further validated the protection against TGF-β1–induced damages. The CB 1 or CB2 receptor antagonist and exogenous FAAH metabolite arachidonic acid failed to reverse the protective effects of FAAH inactivation in HK-2 cells. However, a substrate-selective inhibitor of AEA-cyclooxygenase-2 (COX-2) pathway significantly suppressed the anti-profibrogenic actions of FAAH inhibition. Further, the AEA-COX-2 metabolite, prostamide E2 exerted anti-fibrogenesis effect. These findings suggest that FAAH activation and the consequent reduction of AEA contribute to the renal fibrogenesis, and that FAAH inhibition protects against fibrogenesis in renal cells independently of CB receptors via the AEA-COX-2 pathway by the recovery of reduced AEA.     

The Endocannabinoid/Endovanilloid N-Arachidonoyl Dopamine (NADA) and Synthetic Cannabinoid WIN55,212-2 Abate the Inflammatory Activation of Human Endothelial Cells

Although cannabinoids, such as Δ⁹-tetrahydrocannabinol, have been studied extensively for their psychoactive effects, it has become apparent that certain cannabinoids possess immunomodulatory activity. Endothelial cells (ECs) are centrally involved in the pathogenesis of organ injury in acute inflammatory disorders, such as sepsis, because they express cytokines and chemokines, which facilitate the trafficking of leukocytes to organs, and they modulate vascular barrier function. In this study, we find that primary human ECs from multiple organs express the cannabinoid receptors CB₁R, GPR18, and GPR55, as well as the ion channel transient receptor potential cation channel vanilloid type 1. In contrast to leukocytes, CB₂R is only minimally expressed in some EC populations. Furthermore, we show that ECs express all of the known endocannabinoid (eCB) metabolic enzymes. Examining a panel of cannabinoids, we demonstrate that the synthetic cannabinoid WIN55,212-2 and the eCB N-arachidonoyl dopamine (NADA), but neither anandamide nor 2-arachidonoylglycerol, reduce EC inflammatory responses induced by bacterial lipopeptide, LPS, and TNFα. We find that endothelial CB₁R/CB₂R are necessary for the effects of NADA, but not those of WIN55,212-2. Furthermore, transient receptor potential cation channel vanilloid type 1 appears to counter the anti-inflammatory properties of WIN55,212-2 and NADA, but conversely, in the absence of these cannabinoids, its inhibition exacerbates the inflammatory response in ECs activated with LPS. These data indicate that the eCB system can modulate inflammatory activation of the endothelium and may have important implications for a variety of acute inflammatory disorders that are characterized by EC activation.     

Norepinephrine‐ and Epinephrine‐deficient Mice Gain Weight Normally on a High‐fat Diet

Objective: Signaling through adrenergic receptors (ARs) by norepinephrine (NE) and epinephrine (Epi) regulates weight gain when mice are fed a high‐fat diet (HFD) by controlling diet‐induced thermogenesis. Thus, one would predict that mice unable to make NE/Epi because of inactivation of the dopamine β‐hydroxylase gene (Dbh‐null mice) would have a propensity to become obese. We characterized the response of Dbh‐null and control mice to a HFD. Research Methods and Procedures: Dbh‐null and control mice were fed an HFD or a regular diet (RD) for 2 months. Body weight, adiposity, muscle triglyceride levels, and adipocyte size were measured, as were circulating leptin, adiponectin, triglyceride, glucose, and insulin levels. A glucose tolerance test was also preformed. Results: Dbh‐null mice gain weight normally on an HFD and have the same adiposity. Their serum triglyceride and leptin levels are normal, but adipocytes are ～30% smaller than controls. Dbh‐null mice maintain low blood glucose levels and glucose tolerance when exposed to the HFD in contrast to controls. Discussion: Complete lack of NE/Epi does not predispose to obesity. Because mice lacking all three βARs become obese on an HFD, an imbalance of signaling through α‐ and βARs seems to be responsible for obesity. Surprisingly, Dbh‐null mice maintain glucose tolerance.     

A Prescribed Chinese Herbal Medicine Improves Glucose Profile and Ameliorates Oxidative Stress in Goto-Kakisaki Rats Fed with High Fat Diet

Oxidative stress (OS) plays a role in hyperglycemia induced islet β cell dysfunction, however, studies on classic anti-oxidants didn’t show positive results in treating diabetes. We previously demonstrated that the prescribed Chinese herbal medicine preparation “Qing Huo Yi Hao” (QHYH) improved endothelial function in type 2 diabetic patients. QHYH protected endothelial cells from high glucose-induced damages by scavenging superoxide anion and reducing production of reactive oxygen species. Its active component protected C2C12 myotubes against palmitate-induced oxidative damage and mitochondrial dysfunction. In the present study, we investigated whether QHYH protected islet β cell function exacerbated by high fat diet (HFD) in hyperglycemic GK rats. 4-week-old male rats were randomly divided into high HFD feeding group (n = 20) and chow diet feeding group (n = 10). Each gram of HFD contained 4.8 kcal of energy, 52% of which from fat. Rats on HFD were further divided into 2 groups given either QHYH (3 ml/Kg/d) or saline through gastric tube. After intervention, serum glucose concentrations were monitored; IPGTTs were performed without anesthesia on 5 fasting rats randomly chosen from each group on week 4 and 16. Serum malondialdehyde (MDA) concentrations and activities of serum antioxidant enzymes were measured on week 4 and 16. Islet β cell mass and OS marker staining was done by immunohistochemistry on week 16. QHYH prevented the exacerbation of hyperglycemia in HFD feeding GK rats for 12 weeks. On week 16, it improved the exacerbated glucose tolerance and prevented the further loss of islet β cell mass induced by HFD. QHYH markedly decreased serum MDA concentration, increased serum catalase (CAT) and SOD activities on week 4. However, no differences of serum glucose concentration or OS were observed on week 16. We concluded that QHYH decreased hyperglycemia exacerbated by HFD in GK rats by improving β cell function partly via its antioxidant effect.     

黄芪水提取物减轻高脂饮食引起的小鼠肥胖

目的:研究黄芪水提取物(Astragalus radix extract,ARE)对高脂饮食(High fat diet,HFD)引起的小鼠肥胖的作用及可能机制。方法:将30只C57 BL/6小鼠随机分为正常喂养组(ND组,n=10)、高脂喂养组(HFD组,n=10)和高脂喂养+黄芪水提取物处理组(ARE组,n=10)。记录三组小鼠体重及食物摄入。在喂养16周时,对小鼠附睾白色脂肪称重,并进行HE染色观察脂肪细胞大小;对小鼠肝脏进行进行HE染色观察肝脏脂肪变性情况。应用ELISA方法检测血清瘦素及脂联素水平。应用Western Blot检测脂肪组织过氧化物酶体增殖物激活受体γ(Peroxisome proliferator activated receptorγ,PPARγ)表达。结果:1与ND组相比,HFD组体重及热量摄入均显著增加,表明肥胖模型建立成功;ARE处理组的体重较HFD组显著下降,但其热量摄入与HFD组相当。2与ND组相比,HFD组白色脂肪组织重量增加、脂肪细胞增大、肝细胞出现显著脂肪变性;ARE处理组上述指标较HFD组明显改善。3与ND组相比,HFD组瘦素水平升高、脂联素水平下降;ARE处理组与HFD组相比,瘦素水平降低、脂联素水平升高。4与ND组相比,HFD组PPARγ表达显著增加,而ARE处理组较HFD组PPARγ表达下降。结论:黄芪水提取物可能通过抑制PPARγ减轻高质饮食引起的肥胖。     

Folic acid supplementation alters the DNA methylation profile and improves insulin resistance in high-fat-diet-fed mice

Folic acid (FA) supplementation may protect from obesity and insulin resistance, the effects and mechanism of FA on chronic high-fat-diet-induced obesity-related metabolic disorders are not well elucidated. We adopted a genome-wide approach to directly examine whether FA supplementation affects the DNA methylation profile of mouse adipose tissue and identify the functional consequences of these changes. Mice were fed a high-fat diet (HFD), normal diet (ND) or an HFD supplemented with folic acid (20 μg/ml in drinking water) for 10 weeks, epididymal fat was harvested, and genome-wide DNA methylation analyses were performed using methylated DNA immunoprecipitation sequencing (MeDIP-seq). Mice exposed to the HFD expanded their adipose mass, which was accompanied by a significant increase in circulating glucose and insulin levels. FA supplementation reduced the fat mass and serum glucose levels and improved insulin resistance in HFD-fed mice. MeDIP-seq revealed distribution of differentially methylated regions (DMRs) throughout the adipocyte genome, with more hypermethylated regions in HFD mice. Methylome profiling identified DMRs associated with 3787 annotated genes from HFD mice in response to FA supplementation. Pathway analyses showed novel DNA methylation changes in adipose genes associated with insulin secretion, pancreatic secretion and type 2 diabetes. The differential DNA methylation corresponded to changes in the adipose tissue gene expression of Adcy3 and Rapgef4 in mice exposed to a diet containing FA. FA supplementation improved insulin resistance, decreased the fat mass, and induced DNA methylation and gene expression changes in genes associated with obesity and insulin secretion in obese mice fed a HFD.     

CD36 is important for adipocyte recruitment and affects lipolysis

Objective: The scavenger receptor CD36 facilitates the cellular uptake of long‐chain fatty acids. As CD36‐deficiency attenuates the development of high fat diet (HFD)‐induced obesity, the role of CD36‐deficiency in preadipocyte recruitment and adipocyte function was set out to characterize. Design and Methods: Fat cell size and number were determined in gonadal, visceral, and subcutaneous adipose tissue of CD36^?/? and WT mice after 6 weeks on HFD. Basal lipolysis and insulin‐inhibited lipolysis were investigated in gonadal adipose tissue. Results: CD36^?/? mice showed a reduction in adipocyte size in all fat pads. Gonadal adipose tissue also showed a lower total number of adipocytes because of a lower number of very small adipocytes (diameter <50 μm). This was accompanied by an increased pool of preadipocytes, which suggests that CD36‐deficiency reduces the capacity of preadipocytes to become adipocytes. Regarding lipolysis, in adipose tissue from CD36^?/? mice, cAMP levels were increased and both basal and 8‐bromo‐cAMP stimulated lipolysis were higher. However, insulin‐mediated inhibition of lipolysis was more potent in CD36^?/? mice. Conclusions: These results indicate that during fat depot expansion, CD36‐deficiency negatively affects preadipocyte recruitment and that in mature adipocytes, CD36‐deficiency is associated with increased basal lipolysis and insulin responsiveness.