2195-P: Identification of the Regulatory Mechanism of mTORC1 Signaling Activity in Pancreatic Beta Cells in GCN2 Knockout Mice

Abstract

Background and Objective: A single-nucleotide polymorphism (SNP) analysis of Japanese type 2 diabetes (T2DM) patients has revealed a significant correlation between a SNP of GCN2 and T2DM. We have demonstrated that islets isolated from GCN2 knockout (GCN2-/-) mice fed a high-fat diet (HFD) exhibited decreased expression of ATF4, a downstream molecule of GCN2, and pancreatic β-cell failure via hyperactivation of mTORC1 signaling. However, the mechanism of activated mTORC1 signaling has remained unclear. Therefore, our objective is to identify the regulatory mechanism of mTORC1 signaling activity in pancreatic β-cells of GCN2-/- mice fed an HFD. Methods: We analyzed using islets isolated from GCN2-/- mice and wild type mice fed an HFD, and pancreatic β-cell lines. Results: In pancreatic islets isolated from GCN2-/- mice fed an HFD, the phosphorylation of TSC2 was enhanced. Therefore, we speculated that there would be a pathway which regulated mTORC1 signaling via TSC2. It has been reported that TSC2 phosphorylation is regulated by its association with 14-3-3. We started to identify the molecule X downstream of GCN2, which involved in the regulation of mTORC1 signaling activity by competitively inhibiting the binding of TSC2 and 14-3-3. We carried out a proteome analysis to explore the molecules whose binding with 14-3-3 varied depending on the presence or the absence of GCN2. As a result, five molecules were listed, and Asparaginase was identified as the candidate of the molecule X. We revealed that Asparaginase was abundantly expressed in pancreatic islets following heart in mice tissues. In glucose loaded MIN6 cells and amino acid deficient MIN6 cells, the expression of Asparaginase was increased. Furthermore, the increased expression of Asparaginase by amino acid deficiency was decreased by ATF4 knocking down. Conclusion: It was suggested that Asparaginase was involved in the regulation of mTORC1 signaling activity downstream of GCN2-ATF4 pathway. Disclosure M. Kudo: None. S. Asahara: Research Support; Spouse/Partner; AstraZeneca. A. Kanno: None. Y. Kido: Research Support; Self; AstraZeneca.