Peripheral endocannabinoid system activity in patients treated with sibutramine

Objective: The endocannabinoid system (ECS) promotes weight gain and obesity‐associated metabolic changes. Weight loss interventions may influence obesity‐associated risk indirectly through modulation of the peripheral ECS. We investigated the effect of acute and chronic treatment with sibutramine on components of the peripheral ECS. Methods and Procedures: Twenty obese otherwise healthy patients received randomized, double‐blind, crossover treatment with placebo and 15 mg/day sibutramine for 5 days each, followed by 12 weeks open‐label sibutramine treatment. We determined circulating anandamide and 2‐arachidonoylglycerol and expression levels of endocannabinoid genes in subcutaneous abdominal adipose tissue biopsies. Results: Body weight was stable during the acute treatment period and decreased by 6.0 ± 0.8 kg in those patients completing 3 months of sibutramine treatment (P < 0.05). Circulating endocannabinoids and the expression of ECS genes did not change with acute or chronic sibutramine treatment. Discussion: The ECS is activated in obesity. We did not find any influence of 5% body weight loss induced by sibutramine on circulating levels of endocannabinoids and adipose‐tissue expression of endocannabinoid genes in obese subjects. These data confirm our previous findings on dietary weight loss and suggest that the dysregulation of the ECS may be a cause rather than a consequence of obesity.     

Circulating Endocannabinoids and the Polymorphism 385C>A in Fatty Acid Amide Hydrolase (FAAH) Gene May Identify the Obesity Phenotype Related to Cardiometabolic Risk: A Study Conducted in a Brazilian Population of Complex Interethnic Admixture

The dysregulation of the endocannabinoid system is associated with cardiometabolic complications of obesity. Allelic variants in coding genes for this system components may contribute to differences in the susceptibility to obesity and related health hazards. These data have mostly been shown in Caucasian populations and in severely obese individuals. We investigated a multiethnic Brazilian population to study the relationships among the polymorphism 385C>A in an endocannabinoid degrading enzyme gene (FAAH), endocannabinoid levels and markers of cardiometabolic risk. Fasting plasma levels of endocannabinoids and congeners (anandamide, 2-arachidonoylglycerol, N-oleoylethanolamide and N-palmitoylethanolamide) were measured by liquid chromatography-mass spectrometry in 200 apparently healthy individuals of both genders with body mass indices from 22.5 ± 1.8 to 35.9 ± 5.5 kg/m² (mean ± 1 SD) and ages between 18 and 60 years. All were evaluated for anthropometric parameters, blood pressure, metabolic variables, homeostatic model assessment of insulin resistance (HOMA-IR), adiponectin, leptin, C-reactive protein, and genotyping. The endocannabinoid levels increased as a function of obesity and insulin resistance. The homozygous genotype AA was associated with higher levels of anandamide and lower levels of adiponectin versus wild homozygous CC and heterozygotes combined. The levels of anandamide were independent and positively associated with the genotype AA position 385 of FAAH, C-reactive protein levels and body mass index. Our findings provide evidence for an endocannabinoid-related phenotype that may be identified by the combination of circulating anandamide levels with genotyping of the FAAH 385C>A; this phenotype is not exclusive to mono-ethnoracial populations nor to individuals with severe obesity.     

Effects of the new C1q/TNF-related protein (CTRP-3) "cartonectin" on the adipocytic secretion of adipokines

Background: Cartonectin (collagenous repeat‐containing sequence of 26‐kDa protein; CORS‐26) was described as a new adipokine of the C1q/TNF molecular superfamily C1q/TNF‐related protein‐3 (CTRP‐3), secreted by the adipocytes of mice and humans. The receptor and function of cartonectin are unknown and the recombinant protein is not commercially available. Objective: To investigate the effects of recombinant cartonectin on the secretion of adipokines such as adiponectin, leptin, and resistin from adipocytes of human and murine origin. The effect of the BMI of the adipocyte donor was also investigated. Methods and Procedures: Human adipocytes from pooled lean and preobese healthy individuals and murine 3T3‐L1 adipocytes were used for stimulation experiments. Recombinant cartonectin was expressed in insect H5 cells. Adipokine secretion was measured using enzyme‐linked immunosorbent assay. In addition, western blot analysis and luciferase reporter gene assays were employed. Results: Cartonectin (1, 10, 50, and 250 ng/ml) in higher doses stimulates the secretion of adiponectin and resistin from murine adipocytes. This effect is not caused by an induction of peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) protein expression, as confirmed by western blot analysis. Also, luciferase reporter gene assay revealed that cartonectin failed to induce luciferase activity at the peroxisome proliferator‐activated receptor responsive element site containing the adiponectin/luciferase promoter fragment. Human adipocytes from lean individuals secrete higher amounts of adiponectin and leptin when compared with adipocytes of individuals with a preobesity BMI (25–30 kg/m²). Cartonectin failed to stimulate adiponectin or leptin secretion from human adipocytes, irrespective of the BMI value. Discussion: Cartonectin is a new adipokine that differentially regulates the secretion of classical adipokines, with marked differences between the human and the murine systems. These effects are species‐dependent, while basal adipokine secretion is influenced by the BMI.     

Eicosapentaenoic Acid and Rosiglitazone Increase Adiponectin in an Additive and PPARγ‐Dependent Manner in Human Adipocytes

Adiponectin, an anti‐inflammatory and insulin‐sensitizing protein secreted from adipose tissue, may be modulated by dietary fatty acids, although the mechanism is not fully known. Our objective was to investigate the effect of long‐chain n‐3 polyunsaturated fatty acids (PUFAs) on adiponectin in cultured human adipocytes, and to elucidate the role of peroxisome proliferator‐activated receptor‐γ (PPARγ) in this regulation. Isolated human adipocytes were cultured for 48 h with 100 µmol/l eicosapentaenoic acid (C20:5n‐3, EPA), docosahexaenoic acid (C22:6n‐3, DHA), palmitic acid (C16:0), 100 µmol/l EPA plus 100 µmol/l DHA, or bovine serum albumin (control). Additionally, adipocytes were treated for 48 h with a PPARγ antagonist (BADGE) or agonist (rosiglitazone) in isolation or in conjunction with either EPA or DHA. At 48 h, EPA and DHA increased (P < 0.05) adiponectin secretion by 88 and 47%, respectively, while EPA, but not DHA, also increased (136%, P < 0.001) cellular adiponectin protein. Interestingly, PPARγ antagonism completely abolished the DHA‐mediated increase in secreted adiponectin, but only partially attenuated the EPA‐mediated response. Thus, EPA's effects on adiponectin do not appear to be entirely PPARγ mediated. Rosiglitazone increased (P < 0.001) the secreted and cellular adiponectin protein (90 and 582%, respectively). Finally, the effects of EPA and rosiglitazone on adiponectin secretion were additive (+230% at 48 h combined, compared to 121 and 124% by EPA or rosiglitazone alone, respectively). Overall, our findings emphasize the therapeutic importance of long‐chain n‐3 PUFA alone, or in combination with a PPARγ agonist, as a stimulator of adiponectin, a key adipokine involved in obesity and related diseases.     

Endocannabinoids measurement in human saliva as potential biomarker of obesity

Background

The discovery of the endocannabinoid system and of its role in the regulation of energy balance has significantly advanced our understanding of the physiopathological mechanisms leading to obesity and type 2 diabetes. New knowledge on the role of this system in humans has been acquired by measuring blood endocannabinoids. Here we explored endocannabinoids and related N-acylethanolamines in saliva and verified their changes in relation to body weight status and in response to a meal or to body weight loss.

Methodology/Principal Findings

Fasting plasma and salivary endocannabinoids and N-acylethanolamines were measured through liquid mass spectrometry in 12 normal weight and 12 obese, insulin-resistant subjects. Salivary endocannabinoids and N-acylethanolamines were evaluated in the same cohort before and after the consumption of a meal. Changes in salivary endocannabinoids and N-acylethanolamines after body weight loss were investigated in a second group of 12 obese subjects following a 12-weeks lifestyle intervention program. The levels of mRNAs coding for enzymes regulating the metabolism of endocannabinoids, N-acylethanolamines and of cannabinoid type 1 (CB₁) receptor, alongside endocannabinoids and N-acylethanolamines content, were assessed in human salivary glands.The endocannabinoids 2-arachidonoylglycerol (2-AG), N-arachidonoylethanolamide (anandamide, AEA), and the N-acylethanolamines (oleoylethanolamide, OEA and palmitoylethanolamide, PEA) were quantifiable in saliva and their levels were significantly higher in obese than in normal weight subjects. Fasting salivary AEA and OEA directly correlated with BMI, waist circumference and fasting insulin. Salivary endocannabinoids and N-acylethanolamines did not change in response to a meal. CB₁ receptors, ligands and enzymes were expressed in the salivary glands. Finally, a body weight loss of 5.3% obtained after a 12-weeks lifestyle program significantly decreased salivary AEA levels.

Conclusions/Significance

Endocannabinoids and N-acylethanolamines are quantifiable in saliva and their levels correlate with obesity but not with feeding status. Body weight loss significantly decreases salivary AEA, which might represent a useful biomarker in obesity.     

Leptin Levels Are Negatively Correlated with 2-Arachidonoylglycerol in the Cerebrospinal Fluid of Patients with Osteoarthritis

Background

There is compelling evidence in humans that peripheral endocannabinoid signaling is disrupted in obesity. However, little is known about the corresponding central signaling. Here, we have investigated the relationship between gender, leptin, body mass index (BMI) and levels of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in the serum and cerebrospinal fluid (CSF) of primarily overweight to obese patients with osteoarthritis.

Methodology/Principal Findings

Patients (20 females, 15 males, age range 44-78 years, BMI range 24-42) undergoing total knee arthroplasty for end-stage osteoarthritis were recruited for the study. Endocannabinoids were quantified by liquid chromatography – mass spectrometry. AEA and 2-AG levels in the serum and CSF did not correlate with either age or BMI. However, 2-AG levels in the CSF, but not serum, correlated negatively with CSF leptin levels (Spearman’s ρ -0.48, P=0.0076, n=30). No such correlations were observed for AEA and leptin.

Conclusions/Significance

In the patient sample investigated, there is a negative association between 2-AG and leptin levels in the CSF. This is consistent with pre-clinical studies in animals, demonstrating that leptin controls the levels of hypothalamic endocannabinoids that regulate feeding behavior.     

Mass‐Spectrometric Identification of Anandamide and 2‐Arachidonoylglycerol in Nematodes

The purpose of the study was to see if nematodes (Caenorhabditis elegans, Caenorhabditis briggsae, and Pelodera strongyloides) produce endocannabinoids; i.e., anandamide (AEA) and 2‐arachidonoylglycerol (2‐AG). In this study, AEA and 2‐AG were identified as endogenous products from nematodes by using electrospray‐ionization ion‐trap MS/MS (ESI‐IT‐MS) experiments operated in the positive‐ionization mode. Endocannabinoids were identified by product ion scan and concentrations were measured by triple quadrupole mass spectrometry in the multiple reaction monitoring mode (MRM). Both AEA and 2‐AG were identified in all of the nematode samples, even though these species lack known cannabinoid receptors. Neither AEA nor 2‐AG were detected in the fat‐3 mutant of C. elegans, which lacks the necessary enzyme to produce arachidonic acid, the fatty acid precursor of these endocannabinoids.     

Downregulation of ADIPOQ and PPARγ2 Gene Expression in Subcutaneous Adipose Tissue of Obese Adolescents With Hepatic Steatosis

Hepatic steatosis is associated with hypoadiponectinemia. The mechanism(s) resulting in lower serum adiponectin levels in obese adolescents with fatty liver is unknown. In two groups of equally obese adolescents, but discordant for hepatic fat content, we measured adiponectin, leptin, peroxisome proliferator–activated receptor γ 2 (PPARγ2) and tumor necrosis factor‐α (TNFα) gene expression in the abdominal subcutaneous adipose tissue (SAT). Twenty six adolescents with similar degrees of obesity underwent a subcutaneous periumbilical adipose tissue biopsy, in addition to metabolic (oral glucose tolerance test, and hyperinsulinemic—euglycemic clamp), and imaging studies (magnetic resonance imaging (MRI), DEXA). Using quantitative real‐time‐PCR; adiponectin, PPARγ2, TNFα, and leptin mRNA were measured. Based on a hepatic fat content (hepatic fat fraction, HFF) >5.5%, measured by fast MRI, the subjects were divided into low and high HFF group. In addition to the hypoadiponectinemia in the high HFF group, we found that the expression of adiponectin as well as PPARγ2 in the SAT was significantly decreased in this group. No differences were noted for TNFα and leptin plasma or mRNA levels between the groups. An inverse relationship was observed between adiponectin or PPARγ2 expression and hepatic fat content, whereas, adiponectin expression was positively related to PPARγ2 expression. Independent of overall obesity, a reduced expression of adiponectin and PPARγ2 in the abdominal SAT is associated with high liver fat content, as well as with insulin resistance in obese adolescents.     

C-reactive protein modifies the association of plasma leptin with coronary calcium in asymptomatic overweight individuals

Evidence suggests putative interactions of leptin and C‐reactive protein (CRP) in the pathogenesis of adiposity‐related atherosclerotic cardiovascular disease (CVD). Therefore, we investigated whether CRP levels modify the relationship of leptin levels with coronary artery calcium (CAC). We examined 1,460 asymptomatic individuals from two community‐based cross‐sectional studies coordinated at a single, university‐based research center. We focused on subjects who were overweight or obese (BMI ≥25) given greater biologic plausibility in this setting. In multivariable CAC models, we analyzed the interaction of log‐transformed plasma leptin levels with higher CRP levels as defined by three cut‐points: two clinically based (2 mg/l, 3 mg/l) and one dataset specific (sex‐specific upper quartile). The association of plasma leptin with CAC was modified by higher CRP regardless of cut‐point (interaction term P values all <0.01 in fully adjusted models). Leptin levels were associated with CAC in those with high, but not low CRP levels (e.g., tobit ratio for a 1 unit increase in ln(leptin) (95% CI): 2.18 (1.29–3.66) if CRP level ≥3 mg/l; N = 461 vs. 0.94 (0.67–1.31) if CRP levels <3 mg/l; N = 999) in fully adjusted models. No interaction with CRP was present in control analyses with adiponectin, BMI and waist circumference. In conclusion, in asymptomatic overweight and obese adults, increased leptin levels were independently associated with increased CAC in the presence of high, but not low CRP levels, supporting a leptin‐CRP interface in atherosclerosis risk.     

Resistin,Adiponectin, Ghrelin,Leptin, and Proinflammatory Cytokines: Relationships in Obesity

Objective: To evaluate interactions among leptin, adiponectin, resistin, ghrelin, and proinflammatory cytokines [tumor necrosis factor receptors (TNFRs), interleukin‐6 (IL‐6)] in nonmorbid and morbid obesity. Research Methods and Procedures: We measured these hormones by immunoenzyme or radiometric assays in 117 nonmorbid and 57 morbidly obese patients, and in a subgroup of 34 morbidly obese patients before and 6 months after gastric bypass surgery. Insulin resistance by homeostasis model assessment, lipid profile, and anthropometrical measurements were also performed in all patients. Results: Average plasma lipids in morbidly obese patients were elevated. IL‐6, leptin, adiponectin, and resistin were increased and ghrelin was decreased in morbidly obese compared with nonmorbidly obese subjects. After adjusting for age, gender, and BMI in nonmorbidly obese, adiponectin was positively associated with HDLc and gender and negatively with weight (β = ?0.38, p < 0.001). Leptin and resistin correlated positively with soluble tumor necrosis factor receptor (sTNFR) 1 (β = 0.24, p = 0.01 and β = 0.28, p = 0.007). In the morbidly obese patients, resistin and ghrelin were positively associated with sTNFR2 (β = 0.39, p = 0.008 and β = 0.39, p = 0.01). In the surgically treated morbidly obese group, body weight decreased significantly and was best predicted by resistin concentrations before surgery (β = 0.45, p = 0.024). Plasma lipids, insulin resistance, leptin, sTNFR1, and IL‐6 decreased and adiponectin and ghrelin increased significantly. Insulin resistance improved after weight loss and correlated with high adiponectin levels. Discussion: TNFα receptors were involved in the regulatory endocrine system of body adiposity independently of leptin and resistin axis in nonmorbidly obese patients. Our results suggest coordinated roles of adiponectin, resistin, and ghrelin in the modulation of the obesity proinflammatory environment and that resistin levels before surgery treatment are predictive of the extent of weight loss after bypass surgery.     

Improved insulin sensitivity and adiponectin level after exercise training in obese Korean youth

Objective: The objective of this study was to investigate the association among adiposity, insulin resistance, and inflammatory markers [high‐sensitivity C‐reactive protein (hs‐CRP), interleukin (IL)‐6, and tumor necrosis factor (TNF)‐α] and adiponectin and to study the effects of exercise training on adiposity, insulin resistance, and inflammatory markers among obese male Korean adolescents. Research Methods and Procedures: Twenty‐six obese and 14 lean age‐matched male adolescents were studied. We divided the obese subjects into two groups: obese exercise group (N = 14) and obese control group (N = 12). The obese exercise group underwent 6 weeks of jump rope exercise training (40 min/d, 5 d/wk). Adiposity, insulin resistance, lipid profile, hs‐CRP, IL‐6, TNF‐α, and adiponectin were measured before and after the completion of exercise training. Results: The current study demonstrated higher insulin resistance, total cholesterol, LDL‐C levels, triglyceride, and inflammatory markers and lower adiponectin and HDL‐C in obese Korean male adolescents. Six weeks of increased physical activity improved body composition, insulin sensitivity, and adiponectin levels in obese Korean male adolescents without changes in TNF‐α, IL‐6, and hs‐CRP. Discussion: Obese Korean male adolescents showed reduced adiponectin levels and increased inflammatory cytokines. Six weeks of jump rope exercise improved triglyceride and insulin sensitivity and increased adiponectin levels.     

Effect of peroxisome proliferator-activated receptor-alpha ligands in the interaction between adipocytes and macrophages in obese adipose tissue

Objective: This study was designed to examine the effect of peroxisome proliferator‐activated receptor‐α (PPAR‐α) ligands on the inflammatory changes induced by the interaction between adipocytes and macrophages in obese adipose tissue. Methods and Procedures: PPAR‐α ligands (Wy‐14,643 and fenofibrate) were added to 3T3‐L1 adipocytes, RAW264 macrophages, or co‐culture of 3T3‐L1 adipocytes and RAW264 macrophages in vitro, and monocyte chemoattractant protein‐1 (MCP‐1) and tumor necrosis factor‐α (TNF‐α) mRNA expression and secretion were examined. PPAR‐α ligands were administered to genetically obese ob/ob mice for 2 weeks. Moreover, the effect of PPAR‐α ligands was also evaluated in the adipose tissue explants and peritoneal macrophages obtained from PPAR‐α‐deficient mice. Results: In the co‐culture of 3T3‐L1 adipocytes and RAW264 macrophages, PPAR‐α ligands reduced MCP‐1 and TNF‐α mRNA expression and secretion in vitro relative to vehicle‐treated group. The anti‐inflammatory effect of Wy‐14,643 was observed in adipocytes treated with macrophage‐conditioned media or mouse recombinant TNF‐α and in macrophages treated with adipocyte‐conditioned media or palmitate. Systemic administration of PPAR‐α ligands inhibited the inflammatory changes in adipose tissue from ob/ob mice. Wy‐14,643 also exerted an anti‐inflammatory effect in the adipose tissue explants but not in peritoneal macrophages obtained from PPAR‐α‐deficient mice. Discussion: This study provides evidence for the anti‐inflammatory effect of PPAR‐α ligands in the interaction between adipocytes and macrophages in obese adipose tissue, thereby improving the dysregulation of adipocytokine production and obesity‐related metabolic syndrome.     

PPARγ Expression After a High‐fat Meal Is Associated With Plasma Superoxide Dismutase Activity in Morbidly Obese Persons

Peroxisome proliferator‐activated receptor‐γ (PPARγ) may play a protective role in the regulation of vascular function, partly mediated by its effects on superoxide dismutase (SOD). The aim of this study was to determine the association between PPARγ expression in peripheral blood mononuclear cells (PBMCs) and SOD activity in morbidly obese persons with varying degrees of insulin resistance (IR). We studied in 10 morbidly obese persons (five with no IR and five with high IR) the effect of a high‐fat meal on the plasma activity of various antioxidant enzymes and the mRNA expression of PPARγ in PBMC. The high‐fat meal resulted in a significant decrease in plasma SOD activity, glutathione reductase (GSH‐Rd) activity, and mRNA expression of PPARγ only in the group of morbidly obese persons with high IR. PPARγ expression after the high‐fat meal correlated with the IR levels (r = ?0.803, P = 0.009) and the plasma SOD activity (r = 0.903, P = 0.001). Likewise, the reduction in PPARγ expression correlated with the increase in free fatty acids (FFA) (r = 0.733, P = 0.016). In conclusion, the decreased expression of PPARγ in PBMC in morbidly obese persons after a high‐fat meal was associated with the state of IR, the plasma SOD activity, and the changes in the concentration of FFA.     

Differential modulation of white adipose tissue endocannabinoid levels by n-3 fatty acids in obese mice and type 2 diabetic patients

n-3 polyunsaturated fatty acids (n-3 PUFA) might regulate metabolism by lowering endocannabinoid levels. We examined time-dependent changes in adipose tissue levels of endocannabinoids as well as in parameters of glucose homeostasis induced by n-3 PUFA in dietary-obese mice, and compared these results with the effect of n-3 PUFA intervention in type 2 diabetic (T2DM) subjects. Male C57BL/6J mice were fed for 8, 16 or 24?weeks a high-fat diet alone (cHF) or supplemented with n-3 PUFA (cHF?+?F). Overweight/obese, T2DM patients on metformin therapy were given for 24?weeks corn oil (Placebo; 5?g/day) or n-3 PUFA concentrate as above (Omega-3; 5?g/day). Endocannabinoids were measured by liquid chromatography-tandem mass-spectrometry. Compared to cHF-fed controls, the cHF?+?F mice consistently reduced 2-arachidonoylglycerol (up to ~2-fold at week 24) and anandamide (~2-fold) in adipose tissue, while the levels of endocannabinoid-related anti-inflammatory molecules N-eicosapentaenoyl ethanolamine (EPEA) and N-docosahexaenoyl ethanolamine (DHEA) increased more than ~10-fold and ~8-fold, respectively. At week 24, the cHF?+?F mice improved glucose tolerance and fasting blood glucose, the latter being positively correlated with adipose 2-arachidonoylglycerol levels only in obese cHF-fed controls, like fasting insulin and HOMA-IR. In the patients, n-3 PUFA failed to reduce 2-arachidonoylglycerol and anandamide levels in adipose tissue and serum, but they increased both adipose tissue and serum levels of EPEA and DHEA. In conclusion, the inability of n-3 PUFA to reduce adipose tissue and serum levels of classical endocannabinoids might contribute to a lack of beneficial effects of these lipids on glucose homeostasis in T2DM patients.