RNA-Seq analysis of islets from Akita mice treated with the SGLT2 inhibitor Dapagliflozin and wild-type controls

In diabetes, hyperglycemia drives the progression of b-cell failure and multi-organ complications. Prolonged exposure to high glucose stimulates mTORC1, resulting in impaired glucose metabolism and exacerbation of ER stress and inflammation. However, the precise mechanism by which glucose regulates mTORC1 activity in diabetes remains unclear. We performed metabolic labeling with 13C6- glucose in diabetic animals treated with or without the SGLT2 inhibitor (SGLT2i) dapagliflozin or insulin, followed by targeted metabolomics and metabolic flux analysis. We found that tissue glucose concentrations strongly correlate with glucosamine, rather than with other glucose or amino acid metabolites. Plasma glucosamine levels are increased in patients with impaired fasting glucose (IFG) and type 2 diabetes (T2D) and are inversely correlated with b-cell function. Glucosamine is synthesized by amine group transfer from glutamine to glucose, which enhances O-GlcNAcylation (O-GlcN) of mTORC1- regulating proteins, including Raptor, via O-GlcNAc transferase (OGT), subsequently activating mTORC1. Genetic inhibition of b-cell mTORC1 by heterozygous Raptor KO and inhibition of the glucosamine-mTORC1 pathway by SGLT2i improved diabetes and b-cell function. We propose that the glucosamine-mTORC1 pathway is a key mediator of glucose toxicity in diabetes. Overall design: To investigate the molecular mechanisms underlying glucotoxicity in beta cells, we performed RNA-seq analysis on whole islets from wild-type (WT) and Akita mice, a model of endoplasmic reticulum (ER) stress-mediated diabetes. Akita mice were treated with or without a glucose-lowering sodium-glucose cotransporter 2 inhibitor (SGLT2i). SGLT2i-treated Akita mice received the drug in their drinking water for two weeks immediately after weaning. Islets were subsequently collected from all groups and processed for RNA-seq analysis. Each experimental group included three biological replicates, with each replicate consisting of pooled islets (50 islets pooled) from 2–3 mice.