Peripheral endocannabinoids regulate skeletal muscle development and maintenance

Endocannabinoids regulate skeletal muscle European Journal Translational Myology 2010; 1 (4): 167-179 Peripheral endocannabinoids regulate skeletal muscle development and maintenance Dongjiao Zhao (1), Amber Pond (1), Bruce Watkins (2), Dave Gerrard (3), Yefei Wen (4), Shihuan Kuang (4), Kevin Hannon (1) (1) Department of Basic Medical Sciences, Purdue University, West Lafayette, IN; (2) Department of Food Sciences, Purdue University, West Lafayette, IN; (3) Department of Animal Sciences, Virginia Tech University, Blacksburg, VA; (4) Department of Animal Sciences, Purdue University, West Lafayette, IN, USA. Abstract As a principal tissue responsible for insulin-mediated glucose uptake, skeletal muscle is important for whole-body health. The role of peripheral endocannabinoids as regulators of skeletal muscle metabolism has recently gained a lot of interest, as endocannabinoid system disorders could cause peripheral insulin resistance. We investigated the role of the peripheral endocannabinoid system in skeletal muscle development and maintenance. Cultures of C2C12 cells, primary satellite cells and mouse skeletal muscle single fibers were used as model systems for our studies. We found an increase in cannabinoid receptor type 1 (CB1) mRNA and endocannabinoid synthetic enzyme mRNA skeletal muscle cells during differentiation. We also found that activation of CB1 inhibited myoblast differentiation, expanded the number of satellite cells, and stimulated the fast-muscle oxidative phenotype. Our findings contribute to understanding of the role of the endocannabinoid system in skeletal muscle metabolism and muscle oxygen consumption, and also help to explain the effects of the peripheral endocannabinoid system on whole-body energy balance. Key Words: Endocannabinoids, skeletal muscle, development, differentiation, metabolism European Journal Translational Myology 2010; 1 (4): 167-179 synthesis [36,43] and 2-AG is degraded by monoacylglyceride lipase (MAGL) [16]. It is known that the endocannabinoid system is involved in metabolic regulation and glycemic control. For example, in the obese state the endocannabinoid system is overactivated. Further, studies of both genetically and diet-induced obese animal models studies determined elevated levels of endocannabinoids in the hypothalamus and peripheral tissues [9,12,18,26]. In obese or type 2 diabetic patients, circulating levels of AEA and 2-AG are increased and levels of 2-AG are elevated in visceral adipose tissue [12]. Further, CB1 gene knockout (Cnr−/−) mice were resistant to diet-induced obesity and remain lean [33]. Skeletal muscle plays an important role in metabolic regulation and glycemic control [6,24,35]. It is likely that skeletal muscle exerts these effects though the endocannabinoid system, however this has not been studied in sufficient detail. CB1 receptors have been detected in skeletal muscle [30] and studies suggest that blockade of CB1 may have direct effects on The endocannabinoid system (ECS) is a complex network that regulates a variety of physiological processes including appetite, energy homeostasis, body weight, drug addiction, pain-sensation, mood, and memory [2,7,11,15,26,28,29,39]. The endocannabinoid system comprises a group of neuromodulatory lipids, including anandamide (AEA) and 2arachidonoylglycerol (2-AG) [31,39], and their receptors, the cannabinoid receptors type 1 (CB1) and 2 (CB2). Endocannabinoids are derivatives of arachidonic acid conjugated with ethanolamine or glycerol [21,37]. Signals induced by the endocannabinoids are terminated rapidly by transporter-mediated uptake [3] and subsequent degradation [4,14]. Anandamide is released from a membrane lipid precursor, NAPE (N-arachidonoylphosphatidylethanolamine) [32] and is catalyzed by NAPE phospholipase D (NAPE-PLD) [32]. The degradation of AEA is performed by a specific enzyme, fatty acid amide hydrolase (FAAH) [10]. Diacylglycerol lipase α (DAGLα) is essential for 2-AG - 167 - Endocannabinoids regulate skeletal muscle European Journal Translational Myology 2010; 1 (4): 167-179 (Invitrogen, Carlsbad, CA, USA) in PBS at room temperature for 10 min and then rinsed in PBS for 10 min. For each treatment depicted, 6 random images were captured from each culture using a Leaf MicroLumina scanning digital camera. From these images, the following data were obtained: total cell number; number of myofibers (cells with greater than or equal to 3 nuclei); number of nuclei per myofiber (# of nuclei in a cell containing 3 or more nuclei). skeletal muscle by modulating energy homeostasis [8,33]. For example, glucose uptake and oxygen consumption were significantly increased in the isolated soleus of mice treated for 7 days with the CB1 antagonist SR141716 compared with control mice [33]. Further, expression of CB1 mRNA in soleus muscle from obese mice was increased compared with soleus muscle from lean mice [30]. All these findings suggest that CB1 plays an important role in skeletal muscle metabolism, especially in glucose uptake [30,33]. Currently, data on the effects of the ECS on skeletal muscle are much less than for other tissues. The purpose of this study was to investigate the involvement of the peripheral endocannabinoid system on skeletal muscle development and establishment of metabolic function. Real time PCR (RT-PCR) Total RNA was extracted from skeletal muscle using TriZol reagent according to the manufacturer’s protocols (Life Technologies, Bethesda, MD, USA). The concentration and purity of the RNA were determined by measurement of the optical densities at 260 and 280 nm and analyzed by gel electrophoresis. Contaminating DNA was removed from total RNA by two 10-min treatments with RQ1 (RNA Qualified) RNase-Free DNase (Promega, Madison, WI, USA). The RNA solutions were diluted to a working concentration of 1 µg/µl in DEPC treated water (0.1% DEPC to water, Invitrogen). cDNA was prepared from RNA samples as following: A 20 µl reaction mix was made of 1x 1st standard buffer (Life Technologies), 10 mM DTT, 1 mM dNTPs, and 5 µM random hexamers. To this, 200 ng of RNA were added and the mixtures were heated to 65°C for 10 minutes. The reactions were then cooled to 25°C for 5 minutes and 1 μl (200 units) superscript II Reverse Transcriptase (Invitrogen) was added. Identical reaction mixtures were made for each RNA sample without adding superscript II Reverse Transcriptase. These reactions served as noRT controls. The reaction was heated to 37°C for 90 min followed by heat deactivation at 90°C for 10 min. The reaction was then diluted to 100 µl with H2O and stored at -20°C for later use. RT-PCR was performed using a GeneAmp 5700 Sequence Detection System (PE Applied Biosystems, Carlsbad, CA, USA) and the SYBR Green PCR core reagents kit (PE Applied Biosystems). RT-PCR was performed using Integrated DNA Technologies, Inc. primers. Sequences of primers are shown in Table 1. Results are presented as a ratio of target gene mRNA/18S mRNA (...truncated)