Glucose regulates secretion of exogenously expressed insulin from HepG2 cells in vitro and in a mouse model of diabetes mellitus in vivo

Abstract

Glucose-controlled insulin secretion is a key component of its regulation. Here, we examined whether liver cell secretion of insulin derived from an engineered construct can be regulated by glucose. Adenovirus constructs were designed to express proinsulin or mature insulin containing the conditional binding domain (CBD). This motif binds GRP78 (HSPA5), an endoplasmic reticulum (ER) protein that enables the chimeric hormone to enter into and stay within the ER until glucose regulates its release from the organelle. Infected HepG2 cells expressed proinsulin mRNA and the protein containing the CBD. Immunocytochemistry studies suggested that GRP78 and proinsulin appeared together in the ER of the cell. The amount of hormone released from infected cells varied directly with the ambient concentration of glucose in the media. Glucose-regulated release of the hormone from infected cells was rapid and sustained. Removal of glucose from the cells decreased release of the hormone. In streptozotocin-induced diabetic mice, when infected with adenovirus expressing mature insulin, glucose levels declined. Our data show that glucose regulates release of exogenously expressed insulin from the ER of liver cells. This approach may be useful in devising new ways to treat diabetes mellitus.

Modification of a Constitutive to Glucose-Responsive Liver-Specific Promoter Resulted in Increased Efficacy of Adeno-Associated Virus Serotype 8-Insulin Gene Therapy of Diabetic Mice

“…While the strategy of using a one-time administration of AAV8-R3GHLP-hINSco may still require multiple daily injections of rapid/short-acting insulin to reduce post-meal glucose excursions [37], it may simulate the endogenous constitutive insulin secretion more effectively than the long/ultra-acting insulin, thereby reducing glycemic variability [4,38]. Further research to design strategies to store the excess insulin in hepatocytes and secrete it upon induction by glucose challenge [39,40] may potentially help to overcome the limitations of the current glucose responsive promoters.…”