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Targeting cannabinoid 1 receptors (CB1R) with peripherally restricted antagonists (or inverse agonists) shows promise to improve metabolic disorders associated with obesity. In this context, we designed and synthetized JM-00266, a new CB1R blocker with limited blood–brain barrier (BBB) permeability. Pharmacokinetics were tested with SwissADME and in vivo in rodents after oral and intraperitoneal administration of JM-00266 in comparison with Rimonabant. In silico predictions indicated JM-00266 is a non-brain penetrant compound and this was confirmed by brain/plasma ratios and brain uptake index values. JM-00266 had no impact on food intake, anxiety-related behavior and body temperature suggesting an absence of central activity. cAMP assays performed in CB1R-transfected HEK293T/17 cells showed that the drug exhibited inverse agonist activity on CB1R. In addition, JM-00266 counteracted anandamide-induced gastroparesis indicating substantial peripheral activity. Acute administration of JM-00266 also improved glucose tolerance and insulin sensitivity in wild-type mice, but not in CB1R−/− mice. Furthermore, the accumulation of JM-00266 in adipose tissue was associated with an increase in lipolysis. In conclusion, JM-00266 or derivatives can be predicted as a new candidate for modulating peripheral endocannabinoid activity and improving obesity-related metabolic disorders.

Targeting cannabinoid 1 receptors (CB1R) with peripherally restricted antagonists (or inverse agonists) shows promise to improve metabolic disorders associated with obesity. In this context, we designed and synthetized JM-00266, a new CB1R blocker with limited blood-brain barrier (BBB) permeability. Pharmacokinetics were tested with SwissADME and in vivo in rodents after oral and intraperitoneal administration of JM-00266 in comparison with Rimonabant. In silico predictions indicated JM-00266 is a non-brain penetrant compound and this was confirmed by brain/plasma ratios and brain uptake index values. JM-00266 had no impact on food intake, anxiety-related behavior and body temperature suggesting an absence of central activity. cAMP assays performed in CB1R-transfected HEK293T/17 cells showed that the drug exhibited inverse agonist activity on CB1R. In addition, JM-00266 counteracted anandamide-induced gastroparesis indicating substantial peripheral activity. Acute administration of JM-00266 also improved glucose tolerance and insulin sensitivity in wild-type mice, but not in CB1R

OBJECTIVE Dyslipidemia observed in type 2 diabetes (T2D) is atherogenic. Important features of diabetic dyslipidemia are increased levels of triglyceride-rich lipoproteins and small dense LDL particles, which all have apolipoprotein B100 (apoB100) as a major apolipoprotein. This prompted us to study the effect of the GLP-1 agonist liraglutide on the metabolism of apoB100-containing lipoproteins. RESEARCH DESIGN AND METHODS We performed an in vivo kinetic study with stable isotopes (L-[1-13C]leucine) in 10 patients with T2D before and after 6 months of treatment with liraglutide (1.2 mg/day). We also evaluated in mice the effect of liraglutide on the expression of genes involved in apoB100-containing lipoprotein clearance. RESULTS In patients with T2D, liraglutide treatment significantly reduced plasma apoB100 (0.93 ± 0.13 vs. 1.09 ± 0.11 g/L, P = 0.011) and fasting triglycerides (1.76 ± 0.37 vs. 2.48 ± 0.69 mmol/L, P = 0.005). The kinetic study showed a significant increase in indirect catabolism of VLDL1-apoB100 (4.11 ± 1.91 vs. 2.96 ± 1.61 pools/day, P = 0.005), VLDL2-apoB100 (5.17 ± 2.53 vs. 2.84 ± 1.65 pools/day, P = 0.008), and IDL-apoB100 (5.27 ± 2.77 vs. 3.74 ± 1.85 pools/day, P = 0.017) and in catabolism of LDL-apoB100 (0.72 ± 0.22 vs. 0.56 ± 0.22 pools/day, P = 0.005). In mice, liraglutide increased lipoprotein lipase (LPL) gene expression and reduced proprotein convertase subtilisin/kexin type 9 (PCSK9), retinol-binding protein 4 (RBP4), and tumor necrosis factor-α (TNF-α) gene expression in adipose tissue and decreased PCSK9 mRNA and increased LDL receptor protein expression in liver. In vitro, liraglutide directly reduced the expression of PCSK9 in the liver. CONCLUSIONS Treatment with liraglutide induces a significant acceleration of the catabolism of triglyceride-rich lipoproteins (VLDL1, VLDL2, IDL) and LDL. Liraglutide modifies the expression of genes involved in apoB100-containing lipoprotein catabolism. These positive effects on lipoprotein metabolism may reduce cardiovascular risk in T2D.

Diabetic dyslipidemia (DD), characterized by increased plasma triglycerides (TGs) and decreased high-density lipoprotein cholesterol (HDL) levels, is a major factor contributing to non-alcoholic steatohepatitis (NASH) and cardiovascular risk in type-2 diabetes. Activation of both the cannabinoid-1 receptor (CB1R) and inducible nitric oxide synthase (iNOS) are associated with NASH progression. Here, we tested whether dual-targeting inhibition of hepatic CB1R and iNOS improves DD in diet-induced obese (DIO) mice. DIO mice were treated for 14 days with (S)-MRI-1867, a peripherally-restricted hybrid inhibitor of CB1R and iNOS. (R)-MRI-1867, the CB1R-inactive stereoisomer which retains iNOS inhibitory activity and JD-5037, a peripherally-restricted CB1R antagonist were used to assess the relative contribution of the two targets to the effects of (S)-MRI-1867. (S)-MRI-1867 reduced hepatic steatosis, the rate of hepatic VLDL secretion, upregulated hepatic LDLR expression and reduced the circulating levels of the proprotein convertase subtilisin/kexin type 9 (PCSK9). The decrease in VLDL secretion could be attributed to CB1R blockade while the reduction of PCSK9 levels and the related increase in LDLR resulted from iNOS inhibition via a mTORC1-dependent mechanism. In conclusion, this approach based on the concomitant inhibition of CB1R and iNOS represents a promising therapeutic strategy for the treatment of dyslipidemia.

Diabetic dyslipidemia, characterized by increased plasma triglycerides and decreased HDL cholesterol levels, is a major factor contributing to nonalcoholic steatohepatitis and cardiovascular risk in type 2 diabetes. Activation of the cannabinoid-1 receptor (CB1R) and activation of inducible nitric oxide synthase (iNOS) are associated with nonalcoholic steatohepatitis progression. Here, we tested whether dual-targeting inhibition of hepatic CB1R and iNOS improves diabetic dyslipidemia in mice with diet-induced obesity (DIO mice). DIO mice were treated for 14 days with (S)-MRI-1867, a peripherally restricted hybrid inhibitor of CB1R and iNOS. (R)-MRI-1867, the CB1R-inactive stereoisomer that retains iNOS inhibitory activity, and JD-5037, a peripherally restricted CB1R antagonist, were used to assess the relative contribution of the two targets to the effects of (S)-MRI-1867. (S)-MRI-1867 reduced hepatic steatosis and the rate of hepatic VLDL secretion, upregulated hepatic LDLR expression, and reduced the circulating levels of proprotein convertase subtilisin/kexin type 9 (PCSK9). The decrease in VLDL secretion could be attributed to CB1R blockade, while the reduction of PCSK9 levels and the related increase in LDLR resulted from iNOS inhibition via an mTOR complex 1–dependent mechanism. In conclusion, this approach based on the concomitant inhibition of CB1R and iNOS represents a promising therapeutic strategy for the treatment of dyslipidemia.

Impaired mitochondrial fatty acid β-oxidation has been correlated with many metabolic syndromes, and the metabolic characteristics of the mammalian models of mitochondrial dysfunction have also been intensively studied. However, the effects of the impaired mitochondrial fatty acid β-oxidation on systemic metabolism in teleost have never been investigated. In the present study, we established a low-carnitine zebrafish model by feeding fish with mildronate as a specific carnitine synthesis inhibitor (0.05% body weight (BW) /d) for 7 weeks, and the systemically changed nutrient metabolism, including carnitine and triglyceride (TG) concentrations, fatty acid (FA) β-oxidation capability, and other molecular and biochemical assays of lipid, glucose and protein metabolism, were measured. The results indicated that mildronate markedly decreased hepatic carnitine concentrations but not in muscle carnitine concentrations. Liver TG concentrations increased by more than 50% in mildronate-treated fish. Mildronate decreased the efficiency of liver mitochondrial β-oxidation, increased the hepatic mRNA expression of genes related to FA β-oxidation and lipolysis, and decreased the expression of lipogenesis genes. Mildronate decreased whole body glycogen content, increased glucose metabolism rate, and upregulated the expression of glucose uptake and glycolysis genes. Mildronate also increased whole body protein content and hepatic mRNA expression of mechanistic target of rapamycin (mtor), and decreased the expression of a protein catabolism-related gene. Liver, rather than muscle, was the primary organ targeted by mildronate. In short, mildronate-induced hepatic inhibited carnitine synthesis in zebrafish caused decreased mitochondrial FA β-oxidation efficiency, greater lipid accumulation, and altered glucose and protein metabolism. This reveals the key roles of mitochondrial fatty acid β-oxidation in nutrient metabolism in fish, and this low-carnitine zebrafish model could also be used as a novel fish model for future metabolism studies.

Objective— Treatment with liraglutide, a GLP-1 (glucagon-like peptide-1) agonist, has been shown to reduce postprandial lipidemia, an important feature of diabetic dyslipidemia. However, the underlying mechanisms for this effect remain unknown. This prompted us to study the effect of liraglutide on the metabolism of ApoB48 (apolipoprotein B48). Approach and Results— We performed an in vivo kinetic study with stable isotopes (D 8 -valine) in the fed state in 10 patients with type 2 diabetes mellitus before treatment and 6 months after the initiation of treatment with liraglutide (1.2 mg/d). We also evaluated, in mice, the effect of a 1-week liraglutide treatment on postload triglycerides and analysed in vitro on jejunum, the direct effect of liraglutide on the expression of genes involved in the biosynthesis of chylomicron. In diabetic patients, liraglutide treatment induced a dramatic reduction of ApoB48 pool (65±38 versus 162±87 mg; P =0.005) because of a significant decrease in ApoB48 production rate (3.02±1.33 versus 6.14±4.27 mg kg -1 d -1 ; P =0.009) and a significant increase in ApoB48 fractional catabolic rate (5.12±1.35 versus 3.69±0.75 pool d -1 ; P =0.005). One-week treatment with liraglutide significantly reduced postload plasma triglycerides in mice and liraglutide, in vitro, reduced the expression of ApoB48, DGAT1 (diacylglycerol O-acyltransferase 1), and MTP (microsomal transfer protein) genes. Conclusions— We show that treatment with liraglutide induces a significant reduction of the ApoB48 pool because of both a reduction of ApoB48 production and an increase in ApoB48 catabolism. In vitro, liraglutide reduces the expression of genes involved in chylomicron synthesis. These effects might benefit cardiovascular health. Clinical Trial Registration— URL: https://www.clinicaltrials.gov . Unique identifier: NCT02721888.

Evidence has accumulated that obesity-related metabolic dysregulation is associated with overactivation of the endocannabinoid system (ECS), which involves cannabinoid receptor 1 (CB1R), in peripheral tissues, including adipose tissue (AT). The functional consequences of CB1R activation on AT metabolism remain unclear. Since excess fat mobilization is considered an important primary event contributing to the onset of insulin resistance, we combined in vivo and in vitro experiments to investigate whether activation of ECS could alter the lipolytic rate. For this purpose, the appearance of plasma glycerol was measured in wild-type and CB1R−/− mice after acute anandamide administration or inhibition of endocannabinoid degradation by JZL195. Additional experiments were conducted on rat AT explants to evaluate the direct consequences of ECS activation on glycerol release and signaling pathways. Treatments stimulated glycerol release in mice fasted for 6 h and injected with glucose but not in 24-h fasted mice or in CB1R−/−, suggesting that the effect was dependent on plasma insulin levels and mediated by CB1R. We concomitantly observed that Akt cascade activity was decreased, indicating an alteration of the antilipolytic action of insulin. Similar results were obtained with tissue explants exposed to anandamide, thus identifying CB1R of AT as a major target. This study indicates the existence of a functional interaction between CB1R and lipolysis regulation in AT. Further investigation is needed to test if the elevation of ECS tone encountered in obesity is associated with excess fat mobilization contributing to ectopic fat deposition and related metabolic disorders.

Objective— High-density lipoprotein (HDL) from nondiabetic patients with metabolic syndrome (MetS) displays abnormalities in their lipidome, such as triglyceride enrichment and sphingosine-1-phosphate depletion. We hypothesized that these abnormalities could impair the ability of HDL to stimulate endothelial nitric oxide synthase (eNOS). Approach and Results— Compared with HDL from control subjects, HDL from normoglycemic patients with MetS was 39% richer in triglycerides ( P P 3 H]arginine to L-[ 3 H]citrulline, was 69% lower in human umbilical vein endothelial cells incubated with HDL from MetS patients than in cells incubated with HDL from controls ( P P P P P P =0.05). By contrast, triglyceride enrichment of HDL from control subjects did not modify eNOS activity ( P =0.90) and phosphorylation at Ser1177 ( P =0.87). Conclusions— We provide evidence that the activation of eNOS by HDL is decreased in MetS patients before the appearance of diabetes mellitus and that sphingosine-1-phosphate depletion of HDL is the main factor responsible for this defect. This has important consequences on the impairment of HDL functionality and antiatherogenic properties in these patients.

Hallmarks of neuronal differentiation are neurite sprouting, extension, and branching. We previously showed that increased expression of CTP:phosphocholine cytidylyltransferase beta2 (CTbeta2), an isoform of a key phosphatidylcholine (PC) biosynthetic enzyme, accompanies neurite outgrowth (Carter, J. M., Waite, K. A., Campenot, R. B., Vance, J. E., and Vance, D. E. (2003) J. Biol. Chem. 278, 44988-44994). CTbeta2 mRNA is highly expressed in the brain. We show that CTbeta2 is abundant in axons of rat sympathetic neurons and retinal ganglion cells. We used RNA silencing to decrease CTbeta2 expression in PC12 cells differentiated by nerve growth factor. In CTbeta2-silenced cells, numbers of primary and secondary neurites were markedly reduced, suggesting that CTbeta2 facilitates neurite outgrowth and branching. However, the length of individual neurites was significantly increased, and the total amount of neuronal membrane was unchanged. Neurite branching of PC12 cells is known to be inhibited by activation of Akt and promoted by the Akt inhibitor LY294002. Our experiments showed that LY294002 increases neurite sprouting and branching in control PC12 cells but not in CTbeta2-deficient cells. CTbeta2 was not phosphorylated in vitro by Akt. However, inhibition of Cdk5 by roscovitine blocked CTbeta2 phosphorylation and reduced neurite outgrowth and branching. These results highlight the importance of CTbeta2 in neurons for promoting neurite outgrowth and branching and represent the first identification of a lipid biosynthetic enzyme that facilitates these functions.

International audience; Evidence suggests that alterations of glucose and lipid homeostasis induced by obesity are associated with the elevation of endocannabinoid tone. The biosynthesis of the two main endocannabinoids, N-arachidonoylethanolamine and 2-arachidonoyl-glycerol, which derive from arachidonic acid, is influenced by dietary fatty acids (FAs). We investigated whether exposure to n-3 FA at a young age may decrease tissue endocannabinoid levels and prevent metabolic disorders induced by a later high-fat diet (HFD) challenge. Three-week-old mice received a 5% lipid diet containing lard, lard plus safflower oil, or lard plus linseed oil for 10 weeks. Then, mice were challenged with a 30% lard diet for 10 additional weeks. A low n-6/n-3 FA ratio in the early diet induces a marked decrease in liver endocannabinoid levels. A similar reduction was observed in transgenic Fat-1 mice, which exhibit high tissue levels of n-3 FA compared with wild-type mice. Hepatic expression of key enzymes involved in carbohydrate and lipid metabolism was concomitantly changed. Interestingly, some gene modifications persisted after HFD challenge and were associated with improved glycemic control. These findings indicate that early dietary interventions based on n-3 FA may represent an alternative strategy to drugs for reducing endocannabinoid tone and improving metabolic parameters in the metabolic syndrome.

A 4-week diet containing 1% of the conjugated trans-10,cis-12 linoleic acid (CLA2) induces, in C57BL/6 mice, a lipodystrophy, a liver steatosis and a reduction of plasma triglycerides. We demonstrate that, first, hepatic lipid accumulation is not due to an in vivo alteration of lipoprotein production. Fates of CLA2 relative to ß-oxidation reactions are then studied in mitochondrial fractions isolated from liver of control animals and the data indicate not only that the CLA is poorly oxidised but also that it modifies the oxidation of usual fatty acids. We also demonstrate that oxidation capacities are increased by CLA2 treatment concomitantly to carnitine palmitoyltransferase I (CPT I) activity. Besides, the increases in acetyl-CoA carboxylase activity and malonyl-CoA concentration in the liver of CLA2 treated mice provide evidence of a high rate of lipogenesis. The increased CPT I sensibility to malonyl-CoA inhibition due to the treatment suggests that the whole fatty acid ß-oxidation is finally reduced in vivo. The mechanisms implicated in the setup of the hepatic steatosis and in the reduction of the lipaemia despite the increased VLDL secretion are discussed.

International audience; The endocannabinoid system (ECS) is associated with an alteration of glucose homeostasis dependent on cannabinoid receptor-1 (CB1R) activation. However, very little information is available concerning the consequences of ECS activation on intestinal glucose absorption. Mice were injected intraperitoneally with anandamide, an endocannabinoid binding both CB1R and CB2R. We measured plasma glucose and xylose appearance after oral loading, gastrointestinal motility, and glucose transepithelial transport using the everted sac method. Anandamide improved hyperglycemia after oral glucose charge whereas glucose clearance and insulin sensitivity were impaired, pointing out some gastrointestinal events. Plasma xylose appearance was delayed in association with a strong decrease in gastrointestinal transit, while anandamide did not alter transporter-mediated glucose absorption. Interestingly, transit was nearly normalized by coinjection of SR141716 and AM630 (CB1R and CB2R antagonist, respectively), and AM630 also reduced the delay of plasma glucose appearance induced by anandamide. When gastric emptying was bypassed by direct glucose administration in the duodenum, anandamide still reduced plasma glucose appearance in wild-type but not in CB1R(-/-) mice. In conclusion, our findings demonstrated that acute activation of intestinal ECS reduced postprandial glycemia independently on intestinal glucose transport but rather inhibiting gastric emptying and small intestine motility and strongly suggest the involvement of both CB1R and CB2R.

The beneficial effects exerted by low amounts of conjugated linoleic acids (C222222237) suggest that CLA are maximally conserved and raise the question about their mitochondrial oxidizability. Cis-9,trans-11-C18:2 (CLA1) and trans-10,cis-12-C18:2 (CLA2) were compared to cis-9,cis-12-C18:2 (linoleic acid; LA) and cis-9-C16:1 (palmitoleic acid; PA), as substrates for total fatty acid (FA) oxidation and for the enzymatic steps required for the entry of FA into rat liver mitochondria. Oxygen consumption rate was lowest when CLA1 was used as a substrate with that on CLA2 being intermediate between it and the respiration on LA and PA. The order of the radiolabeled FA oxidation rate was PA >> LA > CLA2 > CLA1. Transesterification to acylcarnitines of the octadecadienoic acids were similar, while uptake across inner membranes of CLA1 and, to a lesser extent, of CLA2 was greater than that of LA or PA. Prior oxidation of CLA1 or CLA2 made re-isolated mitochondria much less capable of oxidising PA or LA under carnitine-dependent conditions, but without altering the carnitine-independent oxidation of octanoic acid. Therefore, the CLA studied appeared to be both poorly oxidizable and capable of interfering with the oxidation of usual FA at a step close to the beginning of the β-oxidative cycle.

OBJECTIVE The beneficial effects of the inactivation of endocannabinoid system (ECS) by administration of antagonists of the cannabinoid receptor (CB) 1 on several pathological features associated with obesity is well demonstrated, but the relative contribution of central versus peripheral mechanisms is unclear. We examined the impact of CB1 antagonism on liver and adipose tissue lipid metabolism in a mouse model of diet-induced obesity. RESEARCH DESIGN AND METHODS Mice were fed either with a standard diet or a high-sucrose high-fat (HSHF) diet for 19 weeks and then treated with the CB1-specific antagonist SR141716 (10 mg · kg−1 · day−1) for 6 weeks. RESULTS Treatment with SR141716 reduced fat mass, insulin levels, and liver triglycerides primarily increased by HSHF feeding. Serum adiponectin levels were restored after being reduced in HSHF mice. Gene expression of scavenger receptor class B type I and hepatic lipase was induced by CB1 blockade and associated with an increase in HDL-cholesteryl ether uptake. Concomitantly, the expression of CB1, which was strongly increased in the liver and adipose tissue of HSHF mice, was totally normalized by the treatment. Interestingly, in visceral but not subcutaneous fat, genes involved in transport, synthesis, oxidation, and release of fatty acids were upregulated by HSHF feeding, while this effect was counteracted by CB1 antagonism. CONCLUSIONS A reduction in the CB1-mediated ECS activity in visceral fat is associated with a normalization of adipocyte metabolism, which may be a determining factor in the reversion of liver steatosis induced by treatment with SR141716.

Feeding mice the trans-10, cis-12 (t10c12) conjugated linoleic acid (CLA) isomer is associated with lipodystrophy, insulin resistance, hyperinsulinemia, and liver steatosis. It has been hypothesized that CLA-induced liver steatosis is the result of increased hepatic lipogenesis stimulated by high insulin levels. We studied the effects of a 12-d t10c12CLA treatment (1 g/100 g diet) on liver carbohydrate and lipid metabolism in control and streptozotocin (STZ)-injected mice. STZ mice were characterized by insulin deficiency, hypertriglyceridemia, and depletion of liver triglyceride and glycogen. Remarkably, feeding t10c12CLA to diabetic mice (STZ-CLA) normalized these variables. Reconstitution of fat stores in the livers of STZ-CLA mice was associated with lower fatty acid (FA) oxidation rates and greater malonyl-CoA concentration than in STZ mice. FA translocase and VLDL receptor mRNA levels were greater in STZ-CLA than in STZ mice, suggesting that t10c12CLA increased liver lipid uptake. Phosphoenolpyruvate carboxykinase mRNA levels and AMP kinase phosphorylation were lower in STZ-CLA than in STZ mice, indicating that t10c12CLA may reduce glucogenic activity and promote glycogenesis in diabetic mice. Because glycemia and glucokinase expression were not modified by t10c12CLA treatment, we postulated that glycogen accumulation is likely not the result of an effect of t10c12CLA on plasma glucose utilization, but rather is due to the contribution of lactate, the concentration of which was higher in muscle of STZ-CLA mice. The results demonstrate that t10c12CLA stimulates liver lipid accumulation in the absence of insulin and, thus, suggest that t10c12CLA can improve liver carbohydrate and lipid metabolism in type I diabetic mice.

Rats with carnitine deficiency due to trimethylhydrazinium propionate (mildronate) administered at 80 mg/100 g body weight per day for 10 days developed liver steatosis only upon fasting. This study aimed to determine whether the transient steatosis resulted from triglyceride accumulation due to the amount of fatty acids preserved through impaired fatty acid oxidation and/or from up-regulation of lipid exchange between liver and adipose tissue. In liver, mildronate decreased the carnitine content by approximately 13-fold and, in fasted rats, lowered the palmitate oxidation rate by 50% in the perfused organ, increased 9-fold the triglyceride content, and doubled the hepatic very low density lipoprotein secretion rate. Concomitantly, triglyceridemia was 13-fold greater than in controls. Hepatic carnitine palmitoyltransferase I activity and palmitate oxidation capacities measured in vitro were increased after treatment. Gene expression of hepatic proteins involved in fatty acid oxidation, triglyceride formation, and lipid uptake were all increased and were associated with increased hepatic free fatty acid content in treated rats. In periepididymal adipose tissue, mildronate markedly increased lipoprotein lipase and hormone-sensitive lipase activities in fed and fasted rats, respectively. On refeeding, carnitine-depleted rats exhibited a rapid decrease in blood triglycerides and free fatty acids, then after approximately 2 h, a marked drop of liver triglycerides and a progressive decrease in liver free fatty acids. Data show that up-regulation of liver activities, peripheral lipolysis, and lipoprotein lipase activity were likely essential factors for excess fat deposit and release alternately occurring in liver and adipose tissue of carnitine-depleted rats during the fed/fasted transition.

Conjugated linoleic acids (CLA) have recently been recognized to reduce body fat and plasma lipids in some animals. This study demonstrated that the steatosis accompanying the fat loss induced by trans-10,cis-12-C18:2 (CLA2) and not cis-9,trans-11-C18:2 (CLA1) isomer in C57BL/6j mice was not due to an alteration of the liver lipoprotein production that was even increased. The 3-fold decrease in plasma triacylglycerol contents and the induction of mRNA expression of low-density lipoprotein receptors concomitantly observed in CLA2-fed mice suggested an increase in the lipoprotein clearance at the level of the liver itself. CLA1 feeding produced similar but attenuated effects on triglyceridaemia only.

Decreased body fat mass and liver steatosis have been reported in mice fed diets containing the conjugated linoleic acid trans-10,cis-12-C18:2 (CLA2), but not in those fed diets containing cis-9,trans-11-C18:2 (CLA1). Because the decrease in fatty acid (FA) oxidation may cause fat accumulation, we questioned whether the effects of both CLAs on enzyme activities and mRNA expression were related to liver FA oxidation. To address this question, 7-wk-old male C57BL/6J mice were fed for 4 wk a diet supplemented with 1% CLA1, CLA2, or cis-9-C18:1 (control) esterified as triacylglycerols. In CLA2-fed mice, the proportions of CLA2 in the total FA of liver lipids were substantially lower than those of CLA1 in mice fed CLA1. The mitochondrial protein content per total liver was about 56% greater in CLA2-fed mice than in CLA1-fed mice and controls. Mitochondrial carnitine palmitoyltransferase I (CPT I) and carnitine-dependent palmitate oxidation activities were also significantly greater in CLA2-fed mice than in the two other groups. The amounts of malonyl-CoA per gram of liver and the sensitivity of CPT I to malonyl-CoA inhibition were greater in both groups of CLA-fed mice than in the controls. L-CPT I mRNA expression doubled in CLA2-fed mice and was 3 and 2 times greater for M-CPT I in the CLA1 and CLA2 groups, respectively, compared with controls. Peroxisomal FA oxidation-related activities and acyl-CoA oxidase mRNA expression were increased in CLA1-fed mice, and to a larger extent in CLA2-fed mice, relative to controls. These data indicate that FA oxidation capacities were increased in mice fed CLA2, but were likely depressed in vivo through malonyl-CoA inhibition.

Obesity is a pathology characterized by an excess of adipose tissue (AT) whose functions may be altered. Interestingly, strong evidence suggests that metabolic dysregulations linked to obesity is associated with an hyperactivation of the endocannabinoid system (ECS) in several organs including AT. This system, composed of cannabinoid receptors (CB1R and CB2R), their endogenous ligands (EndoCannabinoids - ECs) and their biosynthetic and degrading enzymes, is expressed both in the central nervous system and in various peripheral tissues.CB1R blockade with Rimonabant, the first antagonist marketed in 2006, has proven to be an effective therapeutic approach by reducing food intake, body mass and by significantly improving metabolic parameters. However, the associated severe centrally-mediated psychiatric disorders led to its market withdrawal 2 years later.Since then, the use of antagonists that do not cross the blood-brain barrier has demonstrated that inactivation of peripheral CB1R is sufficient to reduce the cardiometabolic risk in obese mice. Considering the central role played by AT in the etiology of obesity-associated pathologies, it appears important to clarify the existing relationship between ECS and adipocyte metabolism. In this context, these thesis objectives were to clarify the role of ECs on adipocyte lipolytic activity, to evaluate the secretory abilities of the different AT deposits and to study the impact of CB1R agonists and antagonists on adipogenesis. A last part is devoted to the characterization of the biological activity of new CB1R antagonists.First of all, studying the consequences of the ECS modulation on lipolytic activity allowed us to demonstrate that the activation of CB1R, by stimulating the PI3K/Akt signaling pathway, leads to a decrease in lipolysis. The results also suggest that AT-derived ECs could be the source of circulating ECs and be responsible for harmful exocrine effects.Studying ECs production in vitro, using visceral and subcutaneous AT explants from both obese mice and humans, confirmed the alteration of ECs secretory abilities. These preliminary results validated an original methodological approach that allows us to further explore the mechanisms of ECs production.In addition, the role of ECs on stem cells differentiation from the stroma-vascular fraction of healthy and pathological mice AT has been studied. Preliminary data are suggesting a link between adipocyte differentiation and CB1R activity.Finally, characterization studies of new CB1R antagonists have demonstrated interesting effects of the compounds JM-00266 and HR-0133 on body mass as well as on lipid and carbohydrate metabolism. Nonetheless, the optimization, development, and characterization of these new types of antagonists for therapeutic purposes is still incomplete and appears essential in the fight against obesity and obesity-associated disorders.Key words : Obesity, Endocannabinoid system, Adipose tissue, Lipid and carbohydrate metabolism, L’obésité est une pathologie dont la fréquence est en constante augmentation. Elle correspond à un excès de tissu adipeux (TA) dont les fonctions peuvent être altérées. Parmi les dérégulations métaboliques, il peut exister une hyperactivation du système endocannabinoïde (SEC). Ce système, composé de récepteurs aux cannabinoïdes (CB1R et CB2R), de leurs ligands endogènes (EndoCannabinoïdes – ECs) et des enzymes impliquées dans leur biosynthèse et leur dégradation, est présent dans le système nerveux central ainsi que dans divers tissus périphériques.Le blocage des CB1R par le Rimonabant, premier antagoniste commercialisé en 2006, s’est révélé être une approche thérapeutique efficace en réduisant la prise alimentaire, la masse corporelle et en améliorant significativement les paramètres métaboliques. Néanmoins, les troubles psychiatriques sévères associés, consécutifs aux effets centraux, ont valu à ce composé d’être retiré du marché 2 ans plus tard.Depuis, l’utilisation d’antagonistes ne franchissant pas la barrière hémato-encéphalique a permis de démontrer que l’inactivation des CB1R périphériques était suffisante pour diminuer le risque cardio-métabolique chez la souris obèse. Compte tenu du rôle central joué par le TA dans l’étiologie des pathologies associées à l’obésité, il apparait important de préciser la relation existante entre le SEC et le métabolisme adipocytaire. Dans ce contexte, ces travaux de thèse ont pour objectifs de préciser le rôle des ECs sur l’activité lipolytique adipocytaire, d’évaluer les capacités sécrétoires des différents dépôts de TA et d’étudier l’impact d’agonistes et d’antagonistes des CB1R sur l’adipogenèse. Une dernière partie, est consacrée à la caractérisation de l’activité biologique de nouveaux antagonistes des CB1R.Tout d’abord, l’étude des conséquences de la modulation du SEC sur l’activité lipolytique a permis de démontrer que l’activation des CB1R, en stimulant la voie de signalisation PI3K/Akt, conduit à une diminution de la lipolyse. Les résultats suggèrent également que les ECs produits par le TA, pourraient alimenter le pool d’ECs circulants et être à l’origine d’effets exocrines néfastes.L’étude de la production des ECs in vitro, par des explants de TA viscéral et sous-cutané chez la souris et chez l’Homme obèses, a confirmé la modification des capacités sécrétoires en ECs. Ces résultats préliminaires valident une approche méthodologique originale qui nous permet d’envisager une exploration plus poussée des mécanismes de la production des ECs.Par ailleurs, le rôle des ECs sur la différenciation de cellules souches issues de la fraction stroma-vasculaire de TA sain et pathologique de souris a été étudié. Des essais préliminaires ont permis de suggérer l’existence d’un lien entre différenciation adipocytaire et activité des CB1R.Enfin, les études de caractérisation de nouveaux antagonistes des CB1R ont démontré des effets intéressants des molécules JM-00266 et HR-0133 sur la masse corporelle et le métabolisme glucido-lipidique. Toutefois, l’optimisation, le développement et la caractérisation de ces nouveaux types d’antagonistes à des fins thérapeutiques apparait essentiel dans la lutte contre l’obésité et ses complications.Mots clés : Obésité, Système Endocannabinoïde, Tissu Adipeux, Métabolisme glucido-lipidique

Role of adipose tissue cannabinoid receptor 1 (CB1R) on lipolysis and consequences on lipid and carbohydrate metabolism in miceObesity is accompanied with lipolysis deregulation responsible for excessive Free Fatty Acid (FFA) release, which are key actors in the implementation of peripheral insulin resistance. In parallel, it is now well established that obesity is associated with EndoCannabinoid System (ECS) activity dysfunction in several metabolic tissues. If it is well described that overactivation of this system in the Adipose Tissue (AT) is favourable to its expansion, the consequences on lipolysis remain to be elucidated.The main objective of this thesis work was to precise the role of ECS on lipolysis regulation. To achieve this, AEA, a cannabinoid receptor 1 (CB1R) agonist and JZL195, an inhibitor of the enzymes responsible for the degradation of endocannabinoids, were used, in vivo in mice and in vitro on an explant model, to increase the endocannabinoid tone in the AT and analyse the consequences on lipolytic activity. Then, ECS blockade effect on lipolysis was studied in obese mice by using a specific CB1R antagonist, Rimonabant.This work demonstrates that activation of ECS under high insulinemia condition (postprandial state) alters insulin signalling limiting its antilipolytic effect and increasing FFA release which can ultimately be deleterious for peripheral tissues. Conversely, when insulin signalling pathways are weakly activated, as it is the case during fasting, ECS activation comes with PI3K/Akt activation impeding fasting induced lipolysis and promoting lipid storage and AT expansion. This experimental situation resembles that encountered in obesity which is associated, both with low activity of PI3K/Akt pathway (insulin resistance) and ECS overactivation. CB1R blockade by Rimonabant in obese mice lead to a strong stimulation of lipolysis which seems to be dependent on adenylate cyclase activation. Considering that Rimonabant treatment was also reported to be associated with improved fatty acid catabolism, it can be advanced that FFA released in excess in these conditions, do not have deleterious effects.In conclusion, this work suggests that ECS activation is involved in the onset of AT insulin resistance which ultimately could indirectly affect peripheral tissues via lipolysis derived FFA lipotoxicity. During obesity, the association of insulin resistance and ECS tone elevation would promote lipid storage and AT expansion. Finally, AT CB1R specific blockade could constitute an interesting therapeutic target limiting metabolic deregulations linked to obesity.Key words: Obesity, Insulin Resistance, Endocannabinoid System, Adipose Tissue, Lipolysis, Lipid and carbohydrate metabolism, Rôle des récepteurs aux cannabinoïdes 1 (CB1R) du tissu adipeux sur la lipolyse et conséquences sur le métabolisme glucido-lipidique de la souris.L’obésité s’accompagne d’une dérégulation de la lipolyse responsable d’une libération excessive d’Acides Gras Libres (AGL) qui sont des acteurs majeurs dans la mise en place de la résistance périphérique à l’insuline. En parallèle, il est maintenant bien établi qu’au cours de l’obésité, une altération de l’activité du Système EndoCannabinoïde (SEC) dans plusieurs tissus métaboliques est observée. S’il est bien décrit que l’hyperactivation de ce système au sein du Tissu Adipeux (TA) favorise son expansion, les conséquences sur la lipolyse adipocytaire restent à déterminer.Ces travaux de thèse ont pour objectif principal de préciser le rôle du SEC dans la régulation de la lipolyse. Pour cela, l’AEA, un agoniste des récepteurs aux cannabinoïdes 1 (CB1R) et le JZL195, un inhibiteur des enzymes de dégradation des endocannabinoïdes, ont été utilisés in vivo chez la souris et in vitro sur un modèle d’explants pour augmenter le tonus endocannabinoïde dans le TA et en mesurer les conséquences sur l’activité lipolytique. Ensuite, les effets du blocage du SEC sur la lipolyse ont été étudiés chez la souris obèse par l’utilisation d’un antagoniste spécifique des CB1R, le Rimonabant.Ces travaux montrent que l’activation du SEC adipocytaire dans des conditions d’insulinémie élevée (période postprandiale) conduit à une altération du signal insulinique et à une perte de son effet antilipolytique favorisant le relargage d’AGL et pouvant, à terme, conduire à des effets délétères sur les tissus périphériques. A l’inverse, lorsque les voies de signalisation à l’insuline sont peu actives comme c’est le cas au cours du jeûne, la stimulation du SEC s’accompagne d’une activation de la voie PI3K/Akt freinant la lipolyse induite par le jeûne et favorisant ainsi le stockage des lipides et l’expansion du TA. Cette situation expérimentale est comparable à celle rencontrée au cours de l’obésité qui est associée à la fois, à une faible activité de la voie PI3K/Akt (résistance à l’insuline) et à une hyperactivation du SEC. Le blocage des CB1R par le Rimonabant chez la souris obèse, conduit à une stimulation importante de la lipolyse qui semble dépendre d’une activation de l’adénylate cyclase. Etant donné qu’un traitement par le Rimonabant s’accompagne également d’une augmentation du catabolisme des acides gras, il est raisonnable de penser que dans ces conditions, les AGL libérés n’exercent pas leurs effets délétères.En conclusion, ces travaux suggèrent que l’activation du SEC participe à l’installation d’une résistance à l’insuline dans le TA qui pourrait, à plus long terme, s’étendre aux tissus périphériques via les effets lipotoxiques des AGL issus de la lipolyse. Au cours de l’obésité, l’association d’une insulinorésistance et d’un tonus endocannabinoïde élevé favoriserait le stockage des lipides et l’expansion de la masse grasse. Au final, le blocage ciblé des CB1R du TA pourrait constituer une piste thérapeutique intéressante pour lutter contre les dérégulations métaboliques associées à l’obésité.Mots clés : Obésité, Insulinorésistance, Système Endocannabinoïde, Tissu Adipeux, Lipolyse, Métabolisme glucido-lipidique