High fat diet‐induced biliary lipoapoptosis, senescence, hepatic steatosis and fibrosis are reduced in secretin receptor knockout (SR−/−) mice

Abstract

BackgroundNonalcoholic fatty liver disease (NAFLD) is derived from simple steatosis, and can develop to nonalcoholic steatohepatitis (NASH) if left untreated. Recently, a cholestatic variant of NAFLD in patients has been identified as having portal inflammation and ductular reaction. Free fatty acids (FFAs) are increased in the serum of NAFLD patients and can induce lipoapoptosis. We have shown that the secretin/SR axis stimulates liver fibrosis, and it has been indicated that secretin can influence FFA uptake in adipocytes. Based on these data we aimed to evaluate the effect of SR−/− on biliary lipoapoptosis, senescence, hepatic steatosis and fibrosis following high fat diet (HFD) feeding.MethodsWT and SR−/− mice were fed beginning at 4 weeks of age either control diet (CD) or HFD with a high fructose corn syrup equivalent for 20 weeks. Liver and serum were obtained, and cholangiocytes were isolated by laser capture microdissection. Hepatic steatosis and inflammation were evaluated by H&E and Oil Red O staining. Lipid metabolism was evaluated in total liver and cholangiocytes by qPCR for peroxisome proliferator activated receptor‐α (PPAR‐α), and the PPAR‐α‐dependent enzymatic genes carnitine palmitoyl‐transferase 1α (CPT1α) and long chain Acyl CoA synthetase (ACSL1). Lipoapoptosis was detected by TUNEL staining and qPCR for Bax in cholangiocytes. Senescence was examined by SA‐β‐gal staining in liver sections and qPCR for p18 and p21 in total liver and cholangiocytes. Liver fibrosis was measured by Sirius red staining and qPCR for TGF‐β1 and collagen‐1a in total liver. In vitro, control or shRNA‐SR transfected murine cholangiocytes were treated with 800 μM of FFAs (palmitate, stearate or oleate) added to FFA‐free media for 24 hr before evaluating lipoapoptosis and senescence.ResultsWT HFD mice had increased hepatic steatosis and inflammation, but this was significantly reduced in HFD SR−/− mice. Total liver and cholangiocytes from HFD WT mice had increased expression of PPAR‐α, CPT1α and ACSL1, but these lipogenesis markers were decreased in HFD SR−/− mice. Biliary lipoapoptosis, senescence and liver fibrosis were increased in HFD WT mice; however, these parameters were decreased in HFD SR−/− mice. In vitro, FFAs induced biliary lipoapoptosis and senescence gene expression in murine cholangiocytes, but this FFA‐induced injury was reduced in murine cholangiocytes transfected with shRNA‐SR.ConclusionKnockout of SR decreases PPAR‐α signaling in cholangiocytes, thereby decreasing lipogenesis. Furthermore, knockout of SR decreases HFD‐induced biliary lipoapoptosis, senescence, hepatic steatosis and fibrosis. Modulation of secretin/SR signaling may be a therapeutic option for patients suffering from NASH.