Inhibiting CB1 receptors improves lipogenesis in an in vitro non-alcoholic fatty liver disease model

Shi et al. Lipids in Health and Disease 2014, 13:173 http://www.lipidworld.com/content/13/1/173 RESEARCH Open Access Inhibiting CB1 receptors improves lipogenesis in an in vitro non-alcoholic fatty liver disease model Dongmei Shi1, Xi zhan2, Xiaofeng Yu1, Minglei Jia1, Ying Zhang1, Jianfeng Yao1, Xiaona Hu1 and Zhijun Bao1* Abstract Background: The endocannabinoids system (ECs) mediated mainly by CB1 and CB2 receptors plays an important role in non-alcoholic fatty liver disease by regulating lipid metabolism. This study is to further investigate the expression of CB1 and CB2 in the fat accumulation liver cells and to identify possible underlying mechanism by detecting the key lipogenesis factors. Methods: Sodium oleate and sodium palmitate were added into the HepG2 cell line for forming fat accumulation liver cell. MTT assay was used to test the cell’s cytotoxicity. The accumulation rate of fat in HepG2 cell was analyzed by the fluorescent staining. The mRNA and protein expression levels of CB1, CB2, SREBP-1c, ChREBP, L-PK, ACC1, FAS, LXRs and RXR were detected by RT-PCR and Western blot before and after the use of the antagonist. Results: The receptors of CB1 were expressed in HepG2 cells with low levels while in HepG2 fatty liver cells with higher levels (p < 0.05). However, after the application of antagonist, the expressions were significantly decreased (p < 0.05). The expressions of SREBP-1c, ChREBP and LXRs were detectable in HepG2 cells and the expressions were increased in HepG2 fatty liver cells (p < 0.05). After using the antagonists, the expressions of SREBP-1c, ChREBP, LXRs, ACC1 and FAS were significantly decreased (p < 0.05). But L-PK and RXR changed little in two groups (p > 0.05). Conclusion: Results of the present study demonstrated that CB1 receptors had important pathophysiological effects on the formation of fatty liver. CB1 receptors could be regulated by SREBP-1c, ChREBP and LXRs. Therefore, targeting CB1 receptors for the treatment of NAFLD might have a potential application value. Keywords: Endocannabinoids (ECs), Lipogenesis, Nonalcoholic fatty liver disease (NAFLD), Receptor cannabinoid (CB1,CB2) Introduction Non-alcoholic fatty liver disease (NAFLD) is a spectrum ranging from pure fatty liver to the more severe steatohepatitis, a condition that may progress to cirrhosis and even hepatocellular carcinoma [1]. Lipogenesis plays a critical role in the progression of NAFLD [2]. Though the potential mechanisms of lipogenesis in NAFLD are discussed a lot in previous studies, the etiology remains elusive [3]. Recently, it is reported that the endocannabinoids system (ECs) plays an important role in NAFLD and even its complications such as cardiovascular diseases are through modulating lipid metabolism [4]. ECs is primarily comprised of three components: endocannabinoids, endocannabinoid * Correspondence: 1 Department of Gastroenterology, Huadong Hospital, Medical School of Shanghai Fudan University, Shanghai 200040, China Full list of author information is available at the end of the article receptors, and endocannabinoid-metabolizing enzymes. Endocannabinoids, including arachidonoyl ethanolamide (anandamide) and 2-arachidonoylglycerol (2-AG), are lipid mediators that interact with cannabinoid receptors to produce effects similar to those of delta 9-tetrahydrocannabinol (THC), which is the main psychoactive component of cannabis. It has been reported that ECs widely participates in central and peripheral lipid metabolism through activating G protein-coupled cannabinoid receptors type 1 and type 2 (CB1 and CB2) [5]. ECs can not only stimulate the appetite to increase energy intake through the central nervous system, but also promote lipogenesis of peripheral tissues such as the adipose tissue, liver and skeletal muscle, thus leading to obesity and fatty liver disease [6,7]. High fat diet, alcohol and endotoxin can stimulate the production of ECs [8]. Considering that CB1 receptors are distributed in the brain, adipose tissue, pancreas, gastrointestinal tract, © 2014 Shi et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Shi et al. Lipids in Health and Disease 2014, 13:173 http://www.lipidworld.com/content/13/1/173 Page 2 of 7 skeletal muscle, heart and reproductive system, while CB2 receptors are mainly expressed in the immune system [9-11], it’s interesting to explore how CB1 and CB2 receptors involved in endocannabinoids to induce obesity and fatty liver. More and more evidences show that high-fat dietinduced steatosis resulting from increased fatty acid synthesis is mediated via anandamide-induced CB1 receptor activation. Activation of CB1 receptors can increase hepatic gene expression of the lipogenic transcription factor SREBP-1 and its target enzymes, acetylCoA carboxylase-1 (ACC1) and FAS. These effects are blocked or prevented by CB1 antagonist. CB1 receptor activation also appears to regulate fatty acid oxidation by modulating the activity of hepatic carnitine palmitoyltransferase1(CPT-1), the rate-limiting enzyme in fatty acid β-oxidation [12]. In past decades, some major lipogenesis-controlling factors have been identified in fatty liver disease, such as sterol regulatory element binding protein (SREBP1c), carbohydrate responsive elements binding protein (ChREBP) and liver X receptors (LXRs) [13]. Induced by insulin, SREBP1c regulates some of the key enzymes in fatty acid synthesis such as acetyl-CoA carboxylase (ACC1), fatty acid synthase (FAS). But studies find out that SREBP1c can only controlled 50% of fatty acid synthesis in vivo [14]. ChREBP can act on lipogenic gene promoters and regulate glucose to go into lipid synthesis pathway through the key enzymes liver pyruvate kinase (L-PK) [15]. As the oxysterols-activated nuclear receptors, LXRs are involved in cholesterol metabolism and also can induce liver lipogenesis. They act with the retinoid X receptors (RXRs) forming heterodimers to induce the expression of ACC, FAS and Stearoyl coenzyme A desaturase 1(SCD1). Moreover, LXRs can directly modulate the transcription of SREBP1c and ChREBP [16]. This study attempted to explore the possible mechanism underlying lipogenesis in the fat accumulation liver cells through investigating the expression of CB1 and CB2 receptors as well as SREBP1c, ChREBP, LXRs and the downstream factors ACC1, FAS, L-PK and RXRs. concentrations. Sodium oleate and sodium palmitate (Sigma Aldrich, St. Louis, MO, USA) were added into the cultured cells for 24 hr at ratios of 3:0, 2:1, 1:1, 1:2 and 0:3, respectively. (...truncated)