Therapeutic potential of a novel peripherally restricted CB1R inverse agonist on the progression of diabetic nephropathy.

Objective: This study assessed the efficacy of INV-202, a novel peripherally restricted cannabinoid type-1 receptor (CB1R) inverse agonist, in a streptozotocin-induced type-1 diabetes nephropathy mouse model. Methods: Diabetes was induced in 8-week-old C57BL6/J male mice via intraperitoneal injection of streptozotocin (45 mg/kg/day for 5 days); nondiabetic controls received citrate buffer. Diabetic mice were randomized to 3 groups based on blood glucose, polyuria, and albuminuria, and administered daily oral doses for 28-days of INV-202 at 0.3 or 3 mg/kg or vehicle. Results: INV-202 did not affect body weight but decreased kidney weight compared with the vehicle group. While polyuria was unaffected by INV-202 treatment, urinary urea (control 30.77 ± 14.93; vehicle 189.81 ± 31.49; INV-202 (0.3 mg/kg) 127.76 ± 20; INV-202 (3 mg/kg) 93.70 ± 24.97 mg/24h) and albumin (control 3.06 ± 0.38; vehicle 850.08 ± 170.50; INV-202 (0.3 mg/kg) 290.65 ± 88.70; INV-202 (3 mg/kg) 111.29 ± 33.47 µg/24h) excretion both decreased compared with vehicle-treated diabetic mice. Compared with the vehicle group, there was a significant improvement in the urinary albumin to creatinine ratio across INV-202 groups. Regardless of the dose, INV-202 significantly reduced angiotensin II excretion in diabetic mice. The treatment also decreased Agtr1a renal expression in a dose-dependent manner. Compared with nondiabetic controls, the glomerular filtration rate was increased in the vehicle group and significantly decreased by INV-202 at 3 mg/kg. While the vehicle group showed a significant loss in the mean number of podocytes per glomerulus, INV-202 treatment limited podocyte loss in a dose-dependent manner. Moreover, in both INV-202 groups, expression of genes coding for podocyte structural proteins nephrin (Nphs1), podocin (Nphs2), and podocalyxin (Pdxl) were restored to levels similar to nondiabetic controls. INV-202 partially limited the proximal tubular epithelial cell (PTEC) hyperplasia and normalized genetic markers for PTEC lesions. INV-202 also reduced expression of genes contributing to oxidative stress (Nox2, Nox4, and P47phox) and inflammation (Tnf). In addition, diabetes-induced renal fibrosis was significantly reduced by INV-202. Conclusions: INV-202 reduced glomerular injury, preserved podocyte structure and function, reduced injury to PTECs, and ultimately reduced renal fibrosis in a streptozotocin-induced diabetic nephropathy mouse model. These results suggest that INV-202 may represent a new therapeutic option in the treatment of diabetic kidney disease.

Liraglutide reduces plasma dihydroceramide levels in patients with type 2 diabetes.

BACKGROUND: Emerging evidence supports that dihydroceramides (DhCer) and ceramides (Cer) contribute to the pathophysiology of insulin resistance and liver steatosis, and that their circulating concentrations are independently associated with cardiovascular outcomes. Circulating DhCer levels are increased in patients with type 2 diabetes (T2D). On the other hand, the GLP-1 receptor agonist liraglutide reduces major adverse cardiac events, insulin resistance and liver steatosis in T2D patients. The main purpose of the present study was therefore to investigate whether liraglutide decreases circulating levels of DhCer and Cer in T2D patients, which could be a mechanism involved in its cardiometabolic benefits. The secondary purpose was to assess the relationship between liraglutide-induced changes in DhCer/Cer levels and insulin resistance and liver steatosis. METHODS: Plasma concentrations of 11 DhCer and 15 Cer species were measured by a highly-sensitive mass spectrometry system in 35 controls and 86 T2D patients before and after 6 months of liraglutide (1.2 mg/day). Insulin resistance was estimated by the triglyceride-glucose (TyG) index. Liver fat content (LFC) was assessed in 53 patients by proton magnetic resonance spectroscopy. RESULTS: Plasma levels of total DhCer, 7 DhCer and 7 Cer species were increased in T2D patients compared to controls. Liraglutide decreased total DhCer by 15.1% (p = 0.005), affecting 16:0 (p = 0.037), 18:0 (p < 0.0001), 18:1 (p = 0.0005), 20:0 (p = 0.0003), 23:0 (p = 0.005) and 24:1 (p = 0.04) species. Total plasma Cer did not significantly change after liraglutide (p = 0.18), but 5 Cer species decreased significantly, i.e. 18:0 and 18:1 (both p < 0.0001), 19:0 and 24:1 (both p < 0.01) and 26:1 (p = 0.04). In multivariate analysis, the reduction in DhCer after liraglutide was independently associated with the reduction in LFC (p = 0.0005) and in TyG index (p = 0.05). CONCLUSIONS: Liraglutide reduces plasma levels of numerous DhCer and Cer species in T2D patients, which may contribute to the cardiovascular benefit observed in the LEADER trial. The independent association between the decrease in plasma DhCer level with the reduction in LFC and TyG index adds new insights regarding the relationship between DhCer, liver steatosis and insulin resistance. Trial registration ClinicalTrials.gov identifier: NCT02721888.

Downregulation of Elovl5 promotes breast cancer metastasis through a lipid-droplet accumulation-mediated induction of TGF-ß receptors.

Metastatic breast cancer cannot be cured, and alteration of fatty acid metabolism contributes to tumor progression and metastasis. Here, we were interested in the elongation of very long-chain fatty acids protein 5 (Elovl5) in breast cancer. We observed that breast cancer tumors had a lower expression of Elovl5 than normal breast tissues. Furthermore, low expression of Elovl5 is associated with a worse prognosis in ER+ breast cancer patients. In accordance with this finding, decrease of Elovl5 expression was more pronounced in ER+ breast tumors from patients with metastases in lymph nodes. Although downregulation of Elovl5 expression limited breast cancer cell proliferation and cancer progression, suppression of Elovl5 promoted EMT, cell invasion and lung metastases in murine breast cancer models. The loss of Elovl5 expression induced upregulation of TGF-ß receptors mediated by a lipid-droplet accumulation-dependent Smad2 acetylation. As expected, inhibition of TGF-ß receptors restored proliferation and dampened invasion in low Elovl5 expressing cancer cells. Interestingly, the abolition of lipid-droplet formation by inhibition of diacylglycerol acyltransferase activity reversed induction of TGF-ß receptors, cell invasion, and lung metastasis triggered by Elovl5 knockdown. Altogether, we showed that Elovl5 is involved in metastasis through lipid droplets-regulated TGF-ß receptor expression and is a predictive biomarker of metastatic ER+ breast cancer.

Chemical Synthesis, Pharmacokinetic Properties and Biological Effects of JM-00266, a Putative Non-Brain Penetrant Cannabinoid Receptor 1 Inverse Agonist.

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.

Endocannabinoids Produced by White Adipose Tissue Modulate Lipolysis in Lean but Not in Obese Rodent and Human.

White adipose tissue (WAT) possesses the endocannabinoid system (ECS) machinery and produces the two major endocannabinoids (ECs), arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG). Accumulating evidence indicates that WAT cannabinoid 1 receptors (CB1R) are involved in the regulation of fat storage, tissue remodeling and secretory functions but their role in controlling lipid mobilization is unclear. In the present study, we used different strategies to acutely increase ECS activity in WAT and tested the consequences on glycerol production as a marker of lipolysis. Treating lean mice or rat WAT explants with JLZ195, which inhibits ECs degrading enzymes, induced an increase in 2-AG tissue contents that was associated with a CB1R-dependent decrease in lipolysis. Direct treatment of rat WAT explants with AEA also inhibited glycerol production while mechanistic studies revealed it could result from the stimulation of Akt-signaling pathway. Interestingly, AEA treatment decreased lipolysis both in visceral and subcutaneous WAT collected on lean subjects suggesting that ECS also reduces fat store mobilization in Human. In obese mice, WAT content and secretion rate of ECs were higher than in control while glycerol production was reduced suggesting that over-produced ECs may inhibit lipolysis activating local CB1R. Strikingly, our data also reveal that acute CB1R blockade with Rimonabant did not modify lipolysis in vitro in obese mice and human explants nor in vivo in obese mice. Taken together, these data provide physiological evidence that activation of ECS in WAT, by limiting fat mobilization, may participate in the progressive tissue remodeling that could finally lead to organ dysfunction. The present findings also indicate that acute CB1R blockade is inefficient in regulating lipolysis in obese WAT and raise the possibility of an alteration of CB1R signaling in conditions of obesity.

Liraglutide Increases the Catabolism of Apolipoprotein B100-Containing Lipoproteins in Patients With Type 2 Diabetes and Reduces Proprotein Convertase Subtilisin/Kexin Type 9 Expression.

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.

Simultaneous Inhibition of Peripheral CB1R and iNOS Mitigates Obesity-Related Dyslipidemia Through Distinct Mechanisms.

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.

Orosensory Detection of Dietary Fatty Acids Is Altered in CB1R-/- Mice.

Obesity is one of the major public health issues, and its prevalence is steadily increasing all the world over. The endocannabinoid system (ECS) has been shown to be involved in the intake of palatable food via activation of cannabinoid 1 receptor (CB1R). However, the involvement of lingual CB1R in the orosensory perception of dietary fatty acids has never been investigated. In the present study, behavioral tests on CB1R-/- and wild type (WT) mice showed that the invalidation of Cb1r gene was associated with low preference for solutions containing rapeseed oil or a long-chain fatty acid (LCFA), such as linoleic acid (LA). Administration of rimonabant, a CB1R inverse agonist, in mice also brought about a low preference for dietary fat. No difference in CD36 and GPR120 protein expressions were observed in taste bud cells (TBC) from WT and CB1R-/- mice. However, LCFA induced a higher increase in [Ca2+]i in TBC from WT mice than that in TBC from CB1R-/- mice. TBC from CB1R-/- mice also exhibited decreased Proglucagon and Glp-1r mRNA and a low GLP-1 basal level. We report that CB1R is involved in fat taste perception via calcium signaling and GLP-1 secretion.

Liraglutide Reduces Postprandial Hyperlipidemia by Increasing ApoB48 (Apolipoprotein B48) Catabolism and by Reducing ApoB48 Production in Patients With Type 2 Diabetes Mellitus.

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 (D8-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.

Inhibited Carnitine Synthesis Causes Systemic Alteration of Nutrient Metabolism in Zebrafish.

Impaired mitochondrial fatty acid ß-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 ß-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) ß-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 while it had no effect in muscle. Liver TG concentrations increased by more than 50% in mildronate-treated fish. Mildronate decreased the efficiency of liver mitochondrial ß-oxidation, increased the hepatic mRNA expression of genes related to FA ß-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 ß-oxidation efficiency, greater lipid accumulation, and altered glucose and protein metabolism. This reveals the key roles of mitochondrial fatty acid ß-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.

Impairment of the Ability of HDL From Patients With Metabolic Syndrome but Without Diabetes Mellitus to Activate eNOS: Correction by S1P Enrichment.

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<0.01) and 15% poorer in sphingosine-1-phosphate (P<0.05; n=23 in each group). eNOS activity, assessed by the conversion of L-[3H]arginine to L-[3H]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<0.0001). In addition, the activating phosphorylation of eNOS at serine (Ser) 1177 and of Akt (protein kinase B) at Ser473 was 37% (P<0.001) and 39% (P<0.05) lower, respectively, with HDL from MetS patients. Sphingosine-1-phosphate enrichment of HDL from MetS patients restored their ability to stimulate eNOS activity (P<0.05), in relation with a significant increase in eNOS phosphorylation at Ser1177 (P<0.05) and in Akt phosphorylation at Ser473 (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.

Overactivation of the endocannabinoid system alters the antilipolytic action of insulin in mouse adipose tissue.

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.

Early Low-Fat Diet Enriched With Linolenic Acid Reduces Liver Endocannabinoid Tone and Improves Late Glycemic Control After a High-Fat Diet Challenge in Mice.

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.

Acute activation of cannabinoid receptors by anandamide reduces gastrointestinal motility and improves postprandial glycemia in mice.

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.

Dietary eicosapentaenoic acid supplementation accentuates hepatic triglyceride accumulation in mice with impaired fatty acid oxidation capacity.

Reduced mitochondrial fatty acid (FA) ß-oxidation can cause accumulation of triglyceride in liver, while intake of eicosapentaenoic acid (EPA) has been recommended as a promising novel therapy to decrease hepatic triglyceride content. However, reduced mitochondrial FA ß-oxidation also facilitates accumulation of EPA. To investigate the interplay between EPA administration, mitochondrial activity and hepatic triglyceride accumulation, we investigated the effects of EPA administration to carnitine-deficient mice with impaired mitochondrial FA ß-oxidation. C57BL/6J mice received a high-fat diet supplemented or not with 3% EPA in the presence or absence of 500 mg mildronate/kg/day for 10 days. Liver mitochondrial and peroxisomal oxidation, lipid classes and FA composition were determined. Histological staining was performed and mRNA level of genes related to lipid metabolism and inflammation in liver and adipose tissue was determined. Levels of pro-inflammatory eicosanoids and cytokines were measured in plasma. The results showed that mildronate treatment decreased hepatic carnitine concentration and mitochondrial FA ß-oxidation and induced severe triglyceride accumulation accompanied by elevated systemic inflammation. Surprisingly, inclusion of EPA in the diet exacerbated the mildronate-induced triglyceride accumulation. This was accompanied by a considerable increase of EPA accumulation while decreased total n-3/n-6 ratio in liver. However, inclusion of EPA in the diet attenuated the mildronate-induced mRNA expression of inflammatory genes in adipose tissue. Taken together, dietary supplementation with EPA exacerbated the triglyceride accumulation induced by impaired mitochondrial FA ß-oxidation. Thus, further thorough evaluation of the potential risk of EPA supplementation as a therapy for NAFLD associated with impaired mitochondrial FA oxidation is warranted.

Antagonism of peripheral hepatic cannabinoid receptor-1 improves liver lipid metabolism in mice: evidence from cultured explants.

UNLABELLED: It is well established that inactivation of the central endocannabinoid system (ECS) through antagonism of cannabinoid receptor 1 (CB1R) reduces food intake and improves several pathological features associated with obesity, such as dyslipidemia and liver steatosis. Nevertheless, recent data indicate that inactivation of peripheral CB1R could also be directly involved in the control of lipid metabolism independently of central CB1R. To further investigate this notion, we tested the direct effect of the specific CB1R antagonist, SR141716, on hepatic carbohydrate and lipid metabolism using cultured liver slices. CB1R messenger RNA expression was strongly decreased by SR141716, whereas it was increased by the CB1R agonist, arachidonic acid N-hydroxyethylamide (AEA), indicating the effectiveness of treatments in modulating ECS activity in liver explants both from lean or ob/ob mice. The measurement of O(2) consumption revealed that SR141716 increased carbohydrate or fatty acid utilization, according to the cellular hormonal environment. In line with this, SR141716 stimulated ß-oxidation activity, and the role of CB1R in regulating this pathway was particularly emphasized when ECS was hyperactivated by AEA and in ob/ob tissue. SR141716 also improved carbohydrate and lipid metabolism, blunting the AEA-induced increase in gene expression of proteins related to lipogenesis. In addition, we showed that SR141716 induced cholesterol de novo synthesis and high-density lipoprotein uptake, revealing a relationship between CB1R and cholesterol metabolism. CONCLUSION: These data suggest that blocking hepatic CB1R improves both carbohydrate and lipid metabolism and confirm that peripheral CB1R should be considered as a promising target to reduce cardiometabolic risk in obesity.

Involvement of CTP:phosphocholine cytidylyltransferase-ß2 in axonal phosphatidylcholine synthesis and branching of neurons.

In the brain, phosphatidylcholine (PC) is synthesized by the CDP-choline pathway in which the rate-limiting step is catalyzed by two isoforms of CTP:phosphocholine cytidylyltransferase (CT): CTα and CTß2. In mice, CTß2 mRNA is more highly expressed in the brain than in other tissues, and several observations suggest that CTß2 plays an important role in the nervous system. We, therefore, investigated the importance of CTß2 for PC synthesis as well as for axon formation, growth and branching of primary sympathetic neurons. We show that in cultured primary neurons nerve growth factor increases the amount of CTß2, but not CTα, mRNA and protein. The brains of mice lacking CTß2 had normal PC content despite having 35% lower CT activity than wild-type brains. CTß2 mRNA and protein are abundant in distal axons of mouse sympathetic neurons whereas CTα mRNA and protein were not detected. Moreover, CTß2 deficiency in distal axons reduced the incorporation of [(3)H]choline into PC by 95% whereas PC synthesis in cell bodies/proximal axons was unaltered. These data suggest that CTß2 is the major CT isoform involved in PC synthesis in axons. Axons of CTß2-deficient sympathetic neurons contained 32% fewer branch points than did wild-type neurons although the number of axons/neuron and the rate of axon extension were the same as in wild-type neurons. We conclude that in distal axons of primary sympathetic neurons CTß2 is a major contributor to PC synthesis and promotes axon branching, whereas CTα appears to be the major CT isoform involved in PC synthesis in cell bodies/proximal axons.

Different effects of pioglitazone and rosiglitazone on lipid metabolism in mouse cultured liver explants.

BACKGROUND: Pioglitazone (PIO) and rosiglitazone (ROSI) are widely used as oral antidiabetic agents for treatment of type 2 diabetes. Although these medications exert similar effects on blood glucose, recent clinical studies indicated that PIO has a more pronounced beneficial effect on lipid parameters than ROSI. In order to get further insight into the lipid effects of both drugs, we tested whether PIO, compared to ROSI, could exert direct effects on lipid liver metabolism in relation with plasma lipids. METHODS: We performed in vitro studies using mice liver slices incubated 21 h either with ROSI (1 micromol/L) or PIO (7.5 micromol/L). RESULTS: We showed that both glitazones slightly reduced HMG-CoA reductase mRNA levels at the same degree but only PIO reduced intracellular cholesterol content, suggesting an alteration of cholesterol uptake rather than an inhibition of cholesterol biosynthesis. This concept was supported by the reduction of scavenger receptor class B type I expression, hepatic lipase activity and high-density lipoprotein cholesterol uptake in PIO-treated liver explants. Conversely, hepatic lipase mRNA levels were increased 3.5-fold. ROSI, but not PIO, induced acetyl-CoA carboxylase and fatty acid synthase gene expression and increased apoB secretion suggesting a stimulation of lipogenesis. Concurrently, peroxisome proliferator-activated receptor-gamma mRNA levels were induced by ROSI and not significantly changed by PIO. Besides, PIO appeared to be a more potent activator of AMP-Activated Protein Kinase than ROSI. CONCLUSIONS: PIO and ROSI exert specific direct effects on liver and extrapolating these data to humans could explain the significant improvements in plasma lipids observed in diabetic patients treated with PIO.

CB1 antagonism exerts specific molecular effects on visceral and subcutaneous fat and reverses liver steatosis in diet-induced obese mice.

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 x kg(-1) x 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.

Liver carbohydrate and lipid metabolism of insulin-deficient mice is altered by trans-10, cis-12 conjugated linoleic acid.

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.