Abstract 527: Loss of Hepatic Insulin Signaling Promotes Apolipoprotein Ai Retention in Liver and Alters HDL in Plasma in Mice

Background: Insulin signaling regulates a variety of features of hepatic lipid and lipoprotein metabolism. Liver-specific deletion of insulin receptor (LIRKO) reduces plasma HDL, an effect attributed to a reduction in deiodinase 2 expression and apolipoprotein (Apo) AI mRNA expression. We report a concomitant accumulation of ApoAI protein in the liver of LIRKO mice in a yet to be fully characterized compartment within hepatocytes. Methods: Mice harboring loxP sites within the insulin receptor gene (IR fl/fl ) were administered a control adenoassociated viral vector (AAV-Empty) or an AAV encoding Cre recombinase under the control of a liver-specific promoter (AAV_TBG-Cre) at 8 weeks of age to generate Control and LIRKO mice. Male and female mice were evaluated for the abundance of ApoAI in liver and total and FPLC-fractionated plasma. ApoAI secretion rates were evaluated in primary hepatocytes from both genotypes. Subcellular localization of ApoAI in liver of Control and LIRKO mice was evaluated by immunofluorescence microscopy and biochemical fractionation. Results: The loss of hepatic insulin receptor and signaling was confirmed by rtPCR, immunoblot analysis and glucose tolerance test. Whereas ApoAI was unaffected in LIRKO mice compared to controls, protein levels were reduced in plasma and elevated in liver. In contrast, no changes were observed for ApoB or ApoE in either liver or plasma. FPLC fractionation of plasma indicates a shift in HDL size and an accumulation of ApoM in the HDL fractions. Secretion rates of newly synthesized ApoAI were modestly reduced in primary hepatocytes isolated from LIRKO mice. Immunolocalization of ApoAI demonstrated accumulation in large, membrane-bound organelles located peripherally in cells. These organelles stained positively for LampI and other markers of the endosomal-lysosomal system. However, markers of the endosomal-lysosomal compartment in perinuclear regions of the cells were negative for ApoAI. Conclusions: Insulin signaling is a regulator of ApoAI secretion that alters the intracellular itinerary of ApoAI in the secretory and endocytic pathways and alters the plasma HDL proteome in mice.