Role Of The Aryl Hydrocarbon Receptor-CD36 Pathway In Hyperhomo Cysteinemia-Induced Hepatic Steatosis In Mice

Nonalcoholic fatty liver disease(NAFLD) is an increasingly recognized disease that caused by factors other than alcohol and characterized by liver lipid accumulation.It refers to a spectrum of liver damage, ranging from simple steatosis to steatohepatitis, advanced fibrosis, and cirrhosis. Obesity, type 2 diabetes mellitus, and hyperlipidemia are important risk factors of NAFLD, but the pathogenesis of NAFLD is still poorly understood. Homocysteine(Hcy) is a non-protein sulfur containing amino acid, formed from methionine metabolism. Clinically, Hcy level(> 15 μM) is defined as hyperhomocysteinemia(HHcy), an important and independent factor for several disorders including atherosclerosis, diabetes, immune and neurodegenerative diseases. Recent studies have shown that HHcy also plays a vital role in NAFLD.Plasma Hcy level is higher in NAFLD patients than controls. In mice, a high methionine diet(HMD) caused HHcy and hepatic steatosis. Although it has been reported that HHcy caused lipid disturbance in different HHcy models, the underlying mechanism remains largely unknown. In present study, we aimed to explore the molecular mechanism of HHcy-induced hepatic steatosis. The results would provide a potential novel target for treating HHcy-induced fatty liver.We fed 6-week-old male C57BL/6 mice an HMD for 4 or 8 weeks to establish HHcy models at different stages. Plasma Hcy level was significantly higher. Oil-red O staining of lipids in liver sections and hepatic lipid extraction analysis showed that HMD-fed mice developed obvious hepatic steatosis with a noticeable increase in liver triglycerides(TG) compared with normal controls. To understand the mechanism by which HHcy induced hepatic steatosis, we examined the expression of genes involved in lipid metabolism. The mRNA and protein levels of total cellular CD36 were markedly increased as early as 4 weeks with an HMD and were maintained to 8weeks as compared with controls. Our in vitro experiments demonstrated that intracellular lipids were accumulated and CD36 expression was increased in primary cultured murine hepatocytes treated with Hcy(100 ?M) for 24 hr. As an important membrane receptor, CD36 mediates long-chain fatty acid(FA) lipid uptake by hepatocytes. Lipid accumulation in hepatocytes and lipid uptake by hepatocytes were both increased with Hcy stimulation, which was reversed by CD36 siRNA. These results suggested that CD36 was an early response factor in HHcy-induced fatty liver.Given that CD36 is transcriptionally regulated by several transcriptional factors, weinvestigated the effect of these factors on Hcy-induced CD36 upregulation. Among of them, aryl hydrocarbon receptor(AHR) was activated with both 4- and 8-week HMD,as evidenced by elevated levels of its target genes(Cyp1a1, Cyp1a2 and CD36).Furthermore, AHR knockdown or AHR antagonist CH223191 both could inhibite the Hcy-induced upregulation of AHR target genes, including CD36; lipid deposition;and FA uptake in hepatocytes. Overexpression of AHR promoted hepatic steatosis by upregulating CD36 expression. Results from chromatin immunoprecipitation assay and transient transfection assay revealed that Hcy facilitated AHR recruitment to the CD36 promoter and ligand-dependently activated AHR.As a nuclear receptor, AHR is a ligand dependent transcriptional factor.Metabolites of arachidonic acid(AA) have long been considered potential endogenous ligands of the AHR. In addition, Hcy participates in regulating metabolic enzyme activity toward AA. We screened the changes in the AA metabolite profile by a metabolomic approach and found that levels of lipoxin A4(LXA4), a metabolite of lipoxygenase, was significantly increased in livers of 8-week HMD-fed mice. The analysis of genes involved in LXA4 biosynthesis and inactivation showed that HMD-fed directly reduced 15-hydroxy/oxo-eicosanoid 13 oxidoreductase 1(Ptgr1)expression,which is a LXA4 inactivation enzyme. These data indicated that HMD-fed may increase hepatic LXA4 content by encumbering LXA4 inactivation. To gain further insights into whether LXA4 was essential for Hcy-induced lipid accumulation in vitro, overexpression of Ptgr1 to inactivate LXA4 effectively inhibited the upregulation of AHR target genes, including CD36; suppressed Hcy-induced lipid accumulation and FA uptake. Importantly, LXA4 significantly increased the luciferase activity in hepatocytes transfected with AHRE or Gal4-AHR/Gal4.Moreover, LXA4 upregulated the expression of AHR target genes Cyp1a1, Cyp1a2 and CD36, which were abolished by AHR silencing. Hence, LXA4ligand-dependently activated the AHR-CD36 pathway and played an important role in Hcy-induced lipid accumulation in liver.To further show the crucial role of AHR on HHcy-induced hepatic steatosis in vivo, we treated mice with HMD with and without the AHR antagonist CH223191(10 mg/kg/day in drinking water) for 4 or 8 weeks. CH223191 treatment ameliorated HMD-induced hepatic lipid accumulation and TG content. Accordingly, the hepatic mRNA levels of Cyp1a1, Cyp1a2 and CD36; In addition, protein levels of total and membrane bound CD36 were effectively inhibited by CH223191 treatment comparedwith HMD-fed group. Therefore, inhibiting AHR protected mice against HMD-induced hepatic steatosis by downregulation of CD36. To establish the clinical relevance of our study, we performed a continuous-variable meta-analysis to ascertain the linkage between Hcy and NAFLD and found that plasma Hcy level was elevated in NAFLD group when compared with controls, suggesting HHcy is an important risk factor for NAFLD.In this study, we demonstrated that activation of AHR by HHcy caused hepatic steatosis in vivo and in vitro. LXA4 derived from AA participates in this HHcy-induced activation of AHR, and CD36 was identified a transcriptional target of AHR. Moreover, AHR antagonism protected mice against HHcy-induced liver lipid accumulation. Our results may have important implications for therapeutic targets of NAFLD.