E. Hubel

Sortilin deficiency improves the metabolic phenotype and reduces hepatic steatosis of mice subjected to diet-induced obesity

BACKGROUND & AIMS Sortilin traffics newly synthesized molecules from the trans-Golgi apparatus along secretory pathways to endosomes, lysosomes or to the cell surface. Sortilin trafficking of acid sphingomyelinase (aSMase) may regulate ceramide levels, a major modulator of insulin signalling. We therefore tested whether sortilin deficiency reduces hepatic and adipose tissue aSMase activity, improving insulin sensitivity in diet-induced obesity (DIO). METHODS DIO in C57BL/6 (WT) and sortilin(-/-) mice was induced by high-fat diet feeding for 10 weeks. RESULTS Sortilin(-/-) mice gained less body weight and less visceral fat, despite similar food intake compared to WT type mice and had enhanced glucose uptake in insulin tolerance tests, which was further corroborated by enhanced hepatic pAkt expression. Sortilin deficiency led to attenuated hepatic steatosis, reduced expression of genes involved in lipogenesis, ceramide synthesis and inflammatory cytokine production and reduced activity of ceramide synthase 5/6 (CerS5/6). Sortilin(-/-) mice had reduced hepatic aSMase activity under both steady-state and DIO. Likewise, sortilin(-/-) hepatocytes displayed hypersensitivity to insulin, due to enhanced insulin receptor downstream signalling. In adipose tissue, sortilin(-/-) mice exhibited lower expression of inflammatory cytokines and lower expression and activity of CerS5/6. As in liver, adipose tissue displayed increased insulin signalling, accompanied by attenuated aSMase activity. CONCLUSIONS Sortilin deficiency induces a beneficial metabolic phenotype in liver and adipose tissue upon DIO, mediated in part by reduced aSMase activity.

Hepatic Amiodarone Lipotoxicity Is Ameliorated by Genetic and Pharmacological Inhibition of Endoplasmatic Reticulum Stress

Amiodarone is a commonly used antiarrhythmic drug and can cause liver steatosis. We investigated the role of endoplasmic reticulum (ER) stress/unfolded protein response in the pathogenesis of amiodarone-induced steatosis. Amiodarone-induced liver injury was obtained by 1 intraperitoneal injection to wild-type (WT) or C/EBP homologous protein knock-out mice (Ddit3-/-). Amiodarone directly reduced intracellular ATP and Ca2+ in hepatocytes invitro, inducing ER stress and lipid accumulation. In vivo, amiodarone-driven liver damage and lipid accumulation was accompanied by activation of ER stress/unfolded protein response, as demonstrated by up-regulation of genes encoding key ER stress mediators and by phosphorylation of eIF2α. In contrast to WT mice, Ddit3-/- mice were protected from amiodarone-induced ER stress and lipid accumulation. Importantly, amiodarone-induced lipid accumulation was not mediated by de novo hepatic lipogenesis, increased adipose tissue lipolysis or increased hepatic uptake of triglycerides or free fatty acids. Rather, amiodarone strongly increased hepatic mRNA expression of lipid droplet proteins, particularly Cidea and Cidec, in WT, but less so in Ddit3-/- mice, suggesting a link between ER stress and increased triglyceride storage. Moreover, while insulin attenuated amiodarone-induced phosphorylation of hormone sensitive lipase (HSL) in WT, it did not affect pHSL in Ddit3-/-, indicating increased lipolysis and therefore reduced lipid accumulation in these mice. Finally, ER stress attenuation using 2 different pharmacological chaperones reduced lipid accumulation, accompanied by reduced mRNA expression of Cidec. In conclusion, amiodarone-induced ER stress drives liver steatosis and may be considered for therapeutic targeting.

P283 SORTILIN DEFICIENCY IMPROVES HEPATIC AND ADIPOSE TISSUE INSULIN RESISTANCE AND INFLAMMATION IN DIET-INDUCED OBESITY

Background and Aims: Sortilin is a trafficking molecule directing newly synthesized molecules from the trans-Golgi network to secretory pathways, endosomes, lysosomes or cell surface. Several of the molecules trafficked by sortilin, such as acidic sphingomyelinase (ASM) and sphingolipid activating proteins (SAPs), regulate the synthesis of ceramide, a major modulator of insulin signaling. We hypothesized that in sortilin mice, reduced hepatic ASM may improve insulin sensitivity and reduce steatosis in a diet-induced obesity (DIO) model. Methods: DIO and insulin resistance were induced by feeding high fat diet for 10 weeks to WT and sortilin mice. Results: Sortilin mice had significantly reduced body weight and visceral fat, despite similar food intake. Sortilin mice had better insulin tolerance test and displayed increased insulin signaling in both liver and adipose tissue as demonstrated by increased Akt phosphorylation. In accordance with the proposed role of sortilin in ASM trafficking, ASM activity in both liver and adipose tissue of sortilin mice was significantly reduced. Sortilin mice had almost no steatosis and a three-fold reduction in total hepatic triglyceride levels compared to WT mice. In addition, expression of proinflammatory cytokines was reduce in both livers and adipose tissue of sortilin. Looking at ceramide synthesis enzymes, we observed a significant reduction in ceramide synthase 6, which synthesizes long chain ceramides, in both liver and adipose tissue of sortilin. Conclusions: Sortilin deficiency induces a beneficial metabolic phenotype in DIO, with respect to both liver and adipose tissue, which may be mediated in part by reduced ASM activity and reduced ceramide levels.

Sa1683 Sortilin Deficiency Abolishes Ductular Reaction and Reduces Hepatocyte Apoptosis and Liver Fibrosis in Bile Duct Ligation-Induced Cholestasis

mice treated with cromolyn. Further, α-SMA, collagen I, fibronectin, TGF-β, MMPs, IL-6 and IL-10 were also significantly reduced in cromolyn-treated MDR2-/mice compared to MDR2-/control mice. Serum levels of HA and IL-6 were all increased in MDR2-/mice, but decreased after cromolyn treatment. In vitro, after co-culture with mast cells (containing stable levels of HA) there was increased cholangiocyte and HSC fibrotic marker expression as well as HA secretion. When cholangiocytes and HSCs were treated with mast cells lacking histamine (MC-HDC), these parameters decreased compared to basal-treatment.Conclusion: Our studies reveal that mast cell-derived histamine is contributor to HSC activation, liver inflammation and fibrosis. Modulation of mast cells may be an option for the treatment of chronic cholestatic disease and fibrotic injury.

963 SORTILIN-DEFICIENT HEPATOCYTES ARE LESS SUSCEPTIBLE TO BILE ACIDS-INDUCED INFLAMMATION AND APOPTOSIS

Background and Aims: Sortilin is a trafficking molecule directing newly synthesized molecules from the trans-Golgi network to secretory pathways or cell surface. One of the molecules trafficked by sortilin is acidic sphingomyelinase (ASMase), whose product ceramide participates in apoptosis signaling. We hypothesized due to less production of ceramide during liver injury, hepatocytes from sortilin−/− mice would be less sensitive to apoptosis. We have investigated liver injury, apoptosis and necrosis, inflammation and fibrosis in a bile duct ligation model in wild type (WT) and sortilin−/− mice. Methods: We have assessed liver damage and fibrosis 7 days after BDL using WT and sortilin−/− mice. The effect of bile acids on hepatocyte apoptosis and expression of inflammatory cytokines was determined in vitro in primary hepatocytes from WT and sortilin−/− mice. Results: Sortilin−/− mice displayed reduced liver damage compared to WT mice, as determined by lower serum AST, ALT, alkaline phosphatase and bilirubin. Sortilin−/− mice also had fewer areas of hepatocyte necrosis that result from bile infarcts, reduced hepatocyte apoptosis compared to WT mice, as well as reduced mRNA levels of collagen I. We determined the in vitro effect of bile acids on hepatocyte apoptosis and expression of proinflammatory cytokines and observed strong reduction of apoptosis and inflammatory cytokines in sortilin-deficient hepatocytes. Conclusion: Sortilin−/− mice have attenuated fibrosis and liver damage after BDL, due in part to reduced hepatocyte apoptosis and reduced inflammatory cytokines.