Abstract
Alcoholism can result in fatty liver that can progress to steatohepatitis, cirrhosis, and liver cancer. Mice fed alcohol develop fatty liver through endocannabinoid activation of hepatic CB(1) cannabinoid receptors (CB(1)R), which increases lipogenesis and decreases fatty acid oxidation. Chronic alcohol feeding also up-regulates CB(1)R in hepatocytes in vivo, which could be replicated in vitro by co-culturing control hepatocytes with hepatic stellate cells (HSC) isolated from ethanol-fed mice, implicating HSC-derived mediator(s) in the regulation of hepatic CB(1)R (Jeong, W. I., Osei-Hyiaman, D., Park, O., Liu, J., Bátkai, S., Mukhopadhyay, P., Horiguchi, N., Harvey-White, J., Marsicano, G., Lutz, B., Gao, B., and Kunos, G. (2008) Cell Metab. 7, 227-235). HSC being a rich source of retinoic acid (RA), we tested whether RA and its receptors may regulate CB(1)R expression in cultured mouse hepatocytes. Incubation of hepatocytes with RA or RA receptor (RAR) agonists increased CB(1)R mRNA and protein, the most efficacious being the RARgamma agonist CD437 and the pan-RAR agonist TTNPB. The endocannabinoid 2-arachidonoylglycerol (2-AG) also increased hepatic CB(1)R expression, which was mediated indirectly via RA, because it was absent in hepatocytes from mice lacking retinaldehyde dehydrogenase 1, the enzyme catalyzing the generation of RA from retinaldehyde. The binding of RARgamma to the CB(1)R gene 5' upstream domain in hepatocytes treated with RAR agonists or 2-AG was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift and antibody supershift assays. Finally, TTNPB-induced CB(1)R expression was attenuated by small interfering RNA knockdown of RARgamma in hepatocytes. We conclude that RARgamma regulates CB(1)R expression and is thus involved in the control of hepatic fat metabolism by endocannabinoids.
Highlights
Alcoholism can result in fatty liver that can progress to steatohepatitis, cirrhosis, and liver cancer
The present findings indicate that retinoic acid acting through RA receptor (RAR)␥ is involved in the up-regulation of hepatic CB1R observed in both alcohol- and high fat diet-induced fatty liver and may be involved in mediating the autoinduction of CB1R expression by endocannabinoids
Agonist resulted in increased expression of CB1R mRNA and protein, and the effect of the RAR panagonist was lost in cells with small interfering RNA (siRNA) knockdown of RAR␥
Summary
Mice with hepatocyte-specific knock-out of CB1R (LCB1Ϫ/Ϫ mice) were generated as described [1]. Raldh1Ϫ/Ϫ mice on a mixed 129/C57Bl/6 background were generated as described [14]. For diet-induced obesity studies, a high fat diet with 60% of calories derived from fat (D12492, Research Diets) was fed to the mice for 14 –16 weeks as described earlier [3, 16]. RAR␥ protein was purchased from ProteinOne. Isolation and Culture of Pure Fractions of Hepatocytes— Hepatocytes were isolated by collagenase perfusion of liver and separated from nonparenchymal cells using Percoll (GE Healthcare) density gradient centrifugation [18]. Tissue Levels of Lipids—For measuring triglyceride and cholesterol levels in liver, mice were sacrificed, and their livers were removed and extracted. Real-time PCR Analyses—Total RNA was isolated from liver homogenate or from purified hepatocyte fractions using TRIzol reagents (Invitrogen) according to the manufacturer’s instructions. Equal amounts (10 or 25 g/lane) were fractionated on a Criterion 4 –12% BisTris gel (Bio-Rad)
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