Abstract 9137: Isolated Liver Perfusion and Electron Microscopy Reveal Defect in Vldl Lipidation Associated With Inactivation of Tm6sf2

Abstract

Background: A missense variant (E167K) in Transmembrane 6 Superfamily Member 2 (TM6SF2) is associated with lower plasma lipid levels and protection from coronary artery disease. Inactivation of Tm6sf2 in mice results in increased hepatic triglyceride (TG) content associated with a reduced rate of secretion of very low-density lipoprotein (VLDL)-TG, but not VLDL-Apolipoprotein B (ApoB). To determine the mechanism responsible for the hypolipidemic effect of Tm6sf2 inactivation, we performed liver perfusion and electron microscopy studies in wildtype (WT) and Tm6sf2 -/- mice. Methods: Ad libitum chow-fed male mice (11-13 weeks old, n=4/group) Tm6sf2 -/- and age- and sex-matched WT mice (C57BL/6N background) were anaesthetized with ketamine/xylazine and treated with heparin (4U/g body weight). Livers were harvested and perfused for 60 min without recirculation with buffer contained free fatty acids (0.8 mM). VLDL was isolated from the perfusate by ultracentrifugation ( d =1.006 g/ml) and VLDL-lipids were assayed using enzymatic assays (Sigma-Aldrich) and liquid chromatography mass-spectrometry (LC-MS). Amounts of VLDL-ApoB and -ApoE were compared between strains by immunoblotting. Electron microscopy was performed on liver sections that had been stained with imidazole/osmium tetroxide to visualize lipoproteins. Results: The secretion rates of VLDL-ApoB and -ApoE did not differ between the Tm6sf2 -/- and WT mice. Hepatic secretion of VLDL lipids (TG, cholesterol, phosphatidylcholine) per ApoB was reduced by 53%, 63% and 67%, respectively, in Tm6sf2 -/- mice compared to WT controls. Electron microscopy showed that the VLDL particles in the secretory pathway of Tm6sf2 -/- mice were smaller than those in the WT animals. Conclusions: Taken together these findings are consistent with TM6SF2 reducing plasma TG and cholesterol levels through limiting addition of neutral lipids to nascent VLDL particles in the hepatocyte prior to secretion.