Leptin Enhances Hepatic Fibrosis and Inflammation in a Mouse Model of Cholestasis

Abstract

Leptin is an orexigenic peptide produced mainly in the adipose, with roles in food intake, body weight and energy metabolism. Leptin acts on specific neurons in the hypothalamus, suppressing food intake and stimulating energy expenditure. Leptin's roles in obesity, hypertension and cardiovascular function are well documented while its influence on liver injuries such as cholestasis is poorly understood. We investigated the effects of exogenously administered leptin and leptin-neutralizing antibody (Lep-Ab) on cholangiocyte proliferation, hepatic fibrosis and inflammation in the Mdr2-/- mouse model of cholestasis. Mdr2-/- and FVB/N (control) mice were treated with vehicle, recombinant leptin or Lep-Ab for 14 days. The expression of target genes in the liver was assessed by RT-qPCR. Serum AST, ALT and leptin were assayed. Markers of intrahepatic biliary duct mass (IBDM) and hepatic fibrosis were assessed by IHC. In vitro, mouse cholangiocytes (MsCh) and LX-2 cells were used to dissect the signaling pathway relating leptin to cholangiocyte proliferation and hepatic stellate cell (HSC) activation. P-Akt and cell proliferation were measured using commercial kits. Knockdown of LepR in MsCh was achieved using siRNA from OriGene. To test the influence of leptin-treated cholangiocytes on LX-2 cell proliferation, MsCh with wild type-LepR or silenced LepR were treated with vehicle or leptin for 24 h, then, LX-2 cells were incubated with MsCh-conditioned media for 24 h, followed by cell proliferation assay. Serum transaminases and leptin, as well as hepatic leptin and LepR mRNA's were increased in Mdr2-/- mice compared to FVB/N controls. Leptin-treated mice exhibited significant upregulation of genes associated with cholangiocyte proliferation (CK19, PCNA), the proliferation and activation of HSC (desmin, αSMA), fibrogenesis (TGFβ) and extracellular matrix remodeling (Col1A1, MMP2, TIMP1) in all experimental groups. Increases in IBDM and liver fibrosis were confirmed at protein level. In addition, leptin enhanced the hepatic expression of pro-inflammatory genes IL-1β, IL-6 and CCL2 in Mdr2-/- mice. Conversely, treatment of mice with Lep-Ab reduced serum ALT, AST, and markers of biliary hyperplasia, hepatic fibrosis and inflammation in Mdr2-/- mice when compared to FVB/N mice. Interestingly, there were gender-related differences, generally leptin having a greater effect in female than in male Mdr2-/- mice. In vitro experiments showed that leptin induced p-Akt in MsCh resulting in increased cell proliferation, while knockdown of LepR in MsCh caused a robust reduction in PCNA expression and decreased cell proliferation. LX-2 stellate cells also express LepR and displayed increased p-Akt levels when challenged with leptin, resulting in upregulation of genes involved in HSC activation (Col1A1, aSMA), fibrosis (TGFβ1, TGFβ2) and inflammation (CCL2, IL-6). LepR knockdown in MsCh prevented HSC proliferation via conditioned medium from Lep-treated MsCh. These data indicate that leptin through its receptor expressed in cholangiocytes and HSC, exacerbates biliary mass, fibrogenesis and inflammation in the liver during cholestasis injury.