216-OR: CREG1 Promotes Insulin Receptor Recycling to Enhance Insulin Signaling in Hepatocytes

CREG1 is an endolysosomal protein that promotes endocytic trafficking and lysosomal biogenesis (Autophagy, 2021). It is highly expressed in the liver. Recently, we demonstrated that the hepatocyte-specific deletion of the Creg1 gene in mice resulted in insulin resistance, elevated glycemia, and hyperinsulinemia. To investigate the role of CREG1 in hepatic insulin signaling and understand the underlying mechanisms, we stably transfected human and mouse hepatocytes with human CREG1 cDNA and mouse Creg1 shRNAs, respectively. CREG1 overexpression enhanced insulin signaling, as evidenced by phosphorylation of insulin receptor (InsR) and its downstream effectors Akt and GSK-3beta. Conversely, CREG1 depletion inhibited insulin-stimulated phosphorylation of these proteins. Immunofluorescence microscopy revealed a significant decrease in InsR expression on the hepatocyte surface, while the total InsR level remained unchanged in hepatocyte-specific Creg1 knockout mice. These novel findings suggest that loss of CREG1 may affect InsR trafficking. To test this hypothesis, we conducted live cell imaging of an InsR-GFP fusion protein and surface protein biotinylation. Pulse-chase experiments revealed that CREG1 overexpression increased insulin receptor recycling to the cell surface, while CREG1 depletion had the opposite effect. Furthermore, adenovirus-mediated InsR transfer to CREG1 knockdown hepatocytes not only restored InsR surface expression but also rescued insulin signaling. These results suggest that CREG1 regulates hepatic insulin signaling through its effects on insulin receptor recycling.