Endocannabinoids And Energy Homeostasis

National Institute On Alcohol Abuse And Alcoholism

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Abstract

It is well established that wild-type (wt) C57Bl6 mice exposed to a high-fat diet (HFD) become obese and steatotic, whereas mice with global knockout of Cnr1 (the gene encoding the cannabinoid 1 receptor, CB1R) remain lean and do not accumulate fat in the liver, suggesting a major role of CB1R in obesity and hepatic steatosis. Hepatocytes express low but functional levels of CB1R, but the possible role of hepatocyte CB1R (hCB1R) in the control of hepatic fat metabolism remains unclear. We earlier reported that mice with transgenic re-expression of hepatic Cnr1 on a global Cnr1 knockout background accumulate very little hepatic triglyceride (TG) on a HFD. This indicates that CB1R in hepatocytes has a minimal role in the development of diet-induced hepatic steatosis, which is driven by the uptake of adipocyte-derived fatty acids (FAs) into hepatocytes via the FA translocase CD36. However, the removal of excess TG from the liver is not a simple reversal of the process of TG accumulation, and the most likely target of CB1R blockade for reversing steatosis is CB1R expressed by hepatocytes. In a recently published study, we reported that wt mice and their hepatocyte-specific littermates (hCB1RKO mice) become similarly obese and steatotic on HFD. Treatment with the peripheral CB1R antagonist JD5037 similarly reversed the weight gain in both strains, but only reversed the steatosis in wt and not in hCB1RKO mice, suggesting the involvement of hCB1R in the latter process. Treatment with monlunabant, another peripheral CB1R antagonist developed in our laboratory and currently in clinical development, induced a similar response pattern. Hepatic gene expression analyses, using RNA-SEQ and Nanostring and confirmed by RT-PCR, yielded a single gene, Cd36, whose expression in wt mice was strongly induced by HFD and the induction was fully reversed by JD5037 treatment, whereas in hCB1RKO mice HFD caused only a minor, statistically insignificant increase in Cd36 gene expression which remained unaffected by JD treatment. CD36 protein expression, analyzed by Western blotting, displayed a similar pattern of changes. The FA translocase CD36 plays a key role not only in promoting the uptake and transport of FAs into hepatocytes but also in tonically inhibiting the activity of AMP kinase (AMPK) and its downstream effect of increasing FA oxidation (FAO), processes involved in the removal of hepatic TGs. We therefore analyzed the role of hCB1R/CD36/AMPK signaling in the removal of hepatic TG by JD5037. Consistently, we found that AMPK activation, as deduced by the phosphorylation of its threonine-178 residue, was reduced by HFD and the reduction reversed by JD5037 treatment, changes opposite to those of CD36 expression. Interestingly, in lean wt mice on standard diet, CB1R activity had effects opposite to those in obese mice, i.e. exposing lean mice to HFD or treating them with the CB1R agonist anandamide decreased hepatic CD36 gene and protein expression and increased AMPK phosphorylation and FAO, and both effects were reversed by JD5037 treatment. These effects were again much smaller or non-existent in hCB1RKO mice on standard diet. The opposite effects of hCB1R in obese and lean mice were replicated in AML12 mouse hepatocytes incubated with or without oleic acid (OA), respectively. OA, a putative endogenous ligand of the orphan receptor GPR3, induced a switch in hCB1R signaling from Gi/o-alpha-mediated reduction in cAMP to Gs-alpha-mediated increase in cAMP. This OA-induced metabolic switch was prevented in the presence of AF64394, a potent and selective inhibitor of GPR3. In intact mice, the OA-induced metabolic switch was facilitated by a decrease in the hepatic expression of Gi/o-alpha proteins and a parallel increase in the expression of Gs-alpha protein, which were similarly prevented by in vivo treatment with AF64394. Together, these findings confirm the role of hCB1R in the control of liver TG content by regulating the CD36/AMPK signaling pathway. They also reveal, for the first time, the OA-dependent opposite regulation of hepatic lipid metabolism by hCB1R, being lipolytic in the lean state vs. lipogenic in obese conditions. The lipolytic effects of CB1R activation under lean conditions may account for the 'paradoxical' observation reported by several groups that chronic cannabis use by non-obese individuals protects from rather than promotes the development of fatty liver disease. These findings have just been published (Metabolism 170 (2025) 156308)