Obesity
Original Article OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY
Influence of Dietary Fat Intake on the Endocannabinoid System in Lean and Obese Subjects Stefan Engeli1, Anne-Christin Lehmann1, Jana Kaminski2, Verena Haas3, J€ urgen Janke4, Alexander A. Zoerner1, 3 1 1 Friedrich C. Luft , Dimitrios Tsikas and Jens Jordan
Objective: Endocannabinoid system (ECS) activation promotes obesity-associated metabolic disease. Increased dietary fat intake increases blood endocannabinoids and alters adipose and skeletal muscle ECS gene expression in human. Methods: Two weeks isocaloric low- (LFD) and high-fat diets (HFD) in obese (n 5 12) and normal-weight (n 5 17) subjects in a randomized cross-over study were compared. Blood endocannabinoids were measured in the fasting condition and after food intake using mass spectrometry. Adipose and skeletal muscle gene expression was determined using real-time RT-PCR. Results: Baseline fasting plasma endocannabinoids were similar with both diets. Anandamide decreased similarly with high- or low-fat test meals in both groups. Baseline arachidonoylglycerol plasma concentrations were similar between groups and diets, and unresponsive to eating. In subcutaneous adipose tissue, DAGL-a mRNA was upregulated and fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) mRNAs were down-regulated in obese subjects, but the diets had no influence. In contrast, the HFD produced pronounced reductions in skeletal muscle CB1-R and MAGL mRNA expression, whereas obesity did not affect muscular gene expression. Conclusions: Weight-neutral changes in dietary fat intake cannot explain excessive endocannabinoid availability in human obesity. Obesity and dietary fat intake affect ECS gene expression in a tissuespecific manner. Obesity (2014) 00, 00–00. doi:10.1002/oby.20728
Introduction Endocannabinoid system (ECS) dysregulation in peripheral tissues with excessive availability of the endocannabinoids anandamide and 2-arachidonoyl glycerol (2-AG) promotes obesity-associated metabolic disease (1). Indeed, weight loss and metabolic improvements observed with genetic deletion or pharmacological blockade of CB1receptors in animals cannot solely be explained by central nervous system influences on food intake (2-4). In clinical trials, the CB1receptor antagonist rimonabant increased circulating adiponectin and improved lipid and glucose metabolism. These changes exceeded the response expected from weight loss alone (5). The most persuasive observation linking peripheral ECS activation to obesityassociated metabolic disease is the fact that peripherally-restricted CB1-receptor blockade ameliorates body weight and improves metabolism (6,7). The mechanisms that increase ECS activity in
obesity are unknown. ECS gene variants have been discussed, but the findings are not conclusive (8-11). Other possible mechanisms include insulin resistance and hyperinsulinemia (12,13), as well as changes in endocannabinoid turnover due to changes in dietary fat intake (14,15). Fatty acid amide hydrolase (FAAH) is the main anandamide-degrading enzyme (16). FAAH activity was rapidly reduced in the liver of mice fed HFD, while anandamide increased (17). Adipose tissue FAAH mRNA expression is reduced in obese subjects (18,19), suggesting that they may be particularly susceptible to increased dietary fat intake in terms of ECS dysregulation. To address this issue, we conducted a randomized cross-over study comparing isocaloric LFD and HFD in obese and normal weight subjects. We hypothesized that weight-neutral high-fat dieting increases blood endocannabinoids and influences ECS gene expression in adipose tissue and muscle.
1
Institute of Clinical Pharmacology, Hannover Medical School, Hannover, Germany. Correspondence: Stefan Engeli ([email protected]) Department of Psychology, University of Potsdam, Potsdam, Germany 3 Franz-Volhard Clinical Research Center at the Experimental & Clinical Research Center, Charite University Medicine, Berlin, Germany 4 Molecular Epidemiology Group, Max Delbr€ uck Center for Molecular Medicine, Berlin, Germany
2
Disclosure: The authors declared no conflict of interest. Funding agency: The study was supported by the German Competence Network of Obesity (project 01Gl1122D to S.E.) through the Federal Ministry for Research and Education. Received: 22 January 2014; Accepted: 17 February 2014; Published online 20 February 2014. doi:10.1002/oby.20728
www.obesityjournal.org
Obesity | VOLUME 00 | NUMBER 00 | MONTH 2014
1
Obesity
Methods Study Participants Women and men aged 18–60 years were eligible. BMI had to be 25 kg/m2 (lean) or 30 kg/m2 (obese), and waist circumference 80 cm in women and 94 cm in men (lean) or 88 cm in women and 102 cm in men (obese). Exclusion criteria were any cardiovascular, hepatic, renal, or metabolic disease, >3 kg weight change in the last three months, bariatric surgery at any time, pregnancy, or nursing. The Ethics Committee of the Charite University Medicine in Berlin approved the study. All procedures were performed according to the Declaration of Helsinki (Tokyo 2004) after written informed consent was obtained. ICH-GCP guidelines were followed where appropriate.
Endocannabinoids and High-Fat Diet Engeli et al.
kit and the RNase-free DNase set (Qiagen, Hilden, Germany). cDNA synthesis from 2 mg total RNA employed the high-capacity cDNA RT Kit (Applied Biosystems/Ambion, Darmstadt, Germany). Gene expression was determined with the 7500 Fast Real-Time PCR System using premixed primer and probe kits for ECS genes by the same manufacturer (CB1: #Hs00275634_m1; CB2: #Hs00275 635_m1; FAAH: #Hs01038660_m1; MGL: #Hs00200752_m1; NAPE-PLD: #Hs00419593_m1; DAGL: #Hs00391374_m1; 18S: #4319413E; Applied Biosystems). Data were analyzed by the DCtmethod with 18S rRNA as the internal control. Insulin, glucose, blood lipids, free fatty acids, and hs-CRP were determined in a certified clinical chemistry laboratory.
Statistical Analysis Dietary Intervention Following inclusion, resting energy expenditure was measured over 45 min using a ventilated hood (Deltatrac II, Datex Ohmeda, Duisburg, Germany). Based on 7-days food diaries, a nutritionist estimated daily caloric requirements. Subjects started a 2-week isocaloric run-in period with approximately 30% calories from fat. We then compared an isocaloric HFD (>40% calories from fat) with an isocaloric LFD (10% between the diets to include subjects in the analysis. Secondary endpoints included differences in fasting endocannabinoids and ECS gene expression between lean and obese subjects and between diets, and the time course of plasma endocannabinoids after test meals. Kolmogorov–Smirnov testing revealed normal distribution of all variables, thus all data are expressed as mean6 SEM. Differences between interventions and groups were compared by paired t-test or 2-way ANOVA as stated. A P-value
Original Article OBESITY BIOLOGY AND INTEGRATED PHYSIOLOGY
Influence of Dietary Fat Intake on the Endocannabinoid System in Lean and Obese Subjects Stefan Engeli1, Anne-Christin Lehmann1, Jana Kaminski2, Verena Haas3, J€ urgen Janke4, Alexander A. Zoerner1, 3 1 1 Friedrich C. Luft , Dimitrios Tsikas and Jens Jordan
Objective: Endocannabinoid system (ECS) activation promotes obesity-associated metabolic disease. Increased dietary fat intake increases blood endocannabinoids and alters adipose and skeletal muscle ECS gene expression in human. Methods: Two weeks isocaloric low- (LFD) and high-fat diets (HFD) in obese (n 5 12) and normal-weight (n 5 17) subjects in a randomized cross-over study were compared. Blood endocannabinoids were measured in the fasting condition and after food intake using mass spectrometry. Adipose and skeletal muscle gene expression was determined using real-time RT-PCR. Results: Baseline fasting plasma endocannabinoids were similar with both diets. Anandamide decreased similarly with high- or low-fat test meals in both groups. Baseline arachidonoylglycerol plasma concentrations were similar between groups and diets, and unresponsive to eating. In subcutaneous adipose tissue, DAGL-a mRNA was upregulated and fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) mRNAs were down-regulated in obese subjects, but the diets had no influence. In contrast, the HFD produced pronounced reductions in skeletal muscle CB1-R and MAGL mRNA expression, whereas obesity did not affect muscular gene expression. Conclusions: Weight-neutral changes in dietary fat intake cannot explain excessive endocannabinoid availability in human obesity. Obesity and dietary fat intake affect ECS gene expression in a tissuespecific manner. Obesity (2014) 00, 00–00. doi:10.1002/oby.20728
Introduction Endocannabinoid system (ECS) dysregulation in peripheral tissues with excessive availability of the endocannabinoids anandamide and 2-arachidonoyl glycerol (2-AG) promotes obesity-associated metabolic disease (1). Indeed, weight loss and metabolic improvements observed with genetic deletion or pharmacological blockade of CB1receptors in animals cannot solely be explained by central nervous system influences on food intake (2-4). In clinical trials, the CB1receptor antagonist rimonabant increased circulating adiponectin and improved lipid and glucose metabolism. These changes exceeded the response expected from weight loss alone (5). The most persuasive observation linking peripheral ECS activation to obesityassociated metabolic disease is the fact that peripherally-restricted CB1-receptor blockade ameliorates body weight and improves metabolism (6,7). The mechanisms that increase ECS activity in
obesity are unknown. ECS gene variants have been discussed, but the findings are not conclusive (8-11). Other possible mechanisms include insulin resistance and hyperinsulinemia (12,13), as well as changes in endocannabinoid turnover due to changes in dietary fat intake (14,15). Fatty acid amide hydrolase (FAAH) is the main anandamide-degrading enzyme (16). FAAH activity was rapidly reduced in the liver of mice fed HFD, while anandamide increased (17). Adipose tissue FAAH mRNA expression is reduced in obese subjects (18,19), suggesting that they may be particularly susceptible to increased dietary fat intake in terms of ECS dysregulation. To address this issue, we conducted a randomized cross-over study comparing isocaloric LFD and HFD in obese and normal weight subjects. We hypothesized that weight-neutral high-fat dieting increases blood endocannabinoids and influences ECS gene expression in adipose tissue and muscle.
1
Institute of Clinical Pharmacology, Hannover Medical School, Hannover, Germany. Correspondence: Stefan Engeli ([email protected]) Department of Psychology, University of Potsdam, Potsdam, Germany 3 Franz-Volhard Clinical Research Center at the Experimental & Clinical Research Center, Charite University Medicine, Berlin, Germany 4 Molecular Epidemiology Group, Max Delbr€ uck Center for Molecular Medicine, Berlin, Germany
2
Disclosure: The authors declared no conflict of interest. Funding agency: The study was supported by the German Competence Network of Obesity (project 01Gl1122D to S.E.) through the Federal Ministry for Research and Education. Received: 22 January 2014; Accepted: 17 February 2014; Published online 20 February 2014. doi:10.1002/oby.20728
www.obesityjournal.org
Obesity | VOLUME 00 | NUMBER 00 | MONTH 2014
1
Obesity
Methods Study Participants Women and men aged 18–60 years were eligible. BMI had to be 25 kg/m2 (lean) or 30 kg/m2 (obese), and waist circumference 80 cm in women and 94 cm in men (lean) or 88 cm in women and 102 cm in men (obese). Exclusion criteria were any cardiovascular, hepatic, renal, or metabolic disease, >3 kg weight change in the last three months, bariatric surgery at any time, pregnancy, or nursing. The Ethics Committee of the Charite University Medicine in Berlin approved the study. All procedures were performed according to the Declaration of Helsinki (Tokyo 2004) after written informed consent was obtained. ICH-GCP guidelines were followed where appropriate.
Endocannabinoids and High-Fat Diet Engeli et al.
kit and the RNase-free DNase set (Qiagen, Hilden, Germany). cDNA synthesis from 2 mg total RNA employed the high-capacity cDNA RT Kit (Applied Biosystems/Ambion, Darmstadt, Germany). Gene expression was determined with the 7500 Fast Real-Time PCR System using premixed primer and probe kits for ECS genes by the same manufacturer (CB1: #Hs00275634_m1; CB2: #Hs00275 635_m1; FAAH: #Hs01038660_m1; MGL: #Hs00200752_m1; NAPE-PLD: #Hs00419593_m1; DAGL: #Hs00391374_m1; 18S: #4319413E; Applied Biosystems). Data were analyzed by the DCtmethod with 18S rRNA as the internal control. Insulin, glucose, blood lipids, free fatty acids, and hs-CRP were determined in a certified clinical chemistry laboratory.
Statistical Analysis Dietary Intervention Following inclusion, resting energy expenditure was measured over 45 min using a ventilated hood (Deltatrac II, Datex Ohmeda, Duisburg, Germany). Based on 7-days food diaries, a nutritionist estimated daily caloric requirements. Subjects started a 2-week isocaloric run-in period with approximately 30% calories from fat. We then compared an isocaloric HFD (>40% calories from fat) with an isocaloric LFD (10% between the diets to include subjects in the analysis. Secondary endpoints included differences in fasting endocannabinoids and ECS gene expression between lean and obese subjects and between diets, and the time course of plasma endocannabinoids after test meals. Kolmogorov–Smirnov testing revealed normal distribution of all variables, thus all data are expressed as mean6 SEM. Differences between interventions and groups were compared by paired t-test or 2-way ANOVA as stated. A P-value
