Reprogramming glutamine metabolism in pancreatic beta cells

Project Summary Progression to type 2 diabetes results from the inability of insulin secreting pancreatic  cells to compensate for increased insulin demand, usually in the setting of obesity. We have previously found that the homeodomain transcription factor and human diabetes gene Pdx1 coordinates islet compensation in the setting of diet-induced obesity and associated insulin resistance. We identified Gpt2 as a target of a PDX1- ATF transcriptional complex whose stress induction and PDX1 enrichment of regulatory CARE sites is conserved in both human and murine islets. Importantly, silencing of the transaminase Gpt2 ex vivo protects primary mouse β cells from stress induced apoptosis. GPT2 is a rapid equilibrium transaminase that catalyzes a bidirectional reaction converting glutamate and pyruvate to α-ketoglutarate (-KG) and alanine. Recent reports implicate Gpt2 upregulation as a key step in the reprogramming of glutamine metabolism of cancer cells. The production of -KG feeds into the tricarboxylic acid (TCA) cycle, resulting in synthesis of ATP which leads to closure of KATP channels, membrane depolarization, calcium influx and exocytosis. TCA cycle intermediates also act enzymes as a cofactors for several chromatin-modifying enzymes, including the Tet family of involved in DNA demethylation. Our preliminary findings support the exciting hypothesis that the induction of GPT2 during fuel excess reprograms beta cell metabolism, thereby causing beta cell dysfunction and death. This hypothesis and the underlying mechanisms will be tested in the following Aims: Aim 1: To uncover the mechanism whereby the transaminase Gpt2 promotes  cell dysfunction and death. We will (A) complete the in vivo characterization of Gpt2 deficiency in pancreatic  cells, (B) elucidate how Gpt2 deficiency affects mitochondrial morphology, function and metabolism, (C) explore the epigenetic impact of Gpt2 silencing and (D) assess the role of Gpt2 in human  cell function and survival during metabolic stress. Aim 2: To elucidate how Gpt2 influences sensitivity of the  cell to GLP1. We will (A) determine whether the sensitivity of GIP and other insulin secretagogues are also affected by Gpt2 expression, (B) investigate the molecular mechanism whereby GPT2 impacts GLP1 sensitivity, and (C) determine whether its impact on cell survival and function is linked to its effect on GLP1 sensitivity via intraislet paracrine signaling. These results will address an important gap in our understanding of how glucoliptoxicity promotes  cell dysfunction and death. Further we will then be poised to devise strategies to target Gpt2 activity or expression as a therapeutic means to protect beta cells during metabolic stress and to enhance sensitivity to Glp1 receptor agonists, an application of the proposed studies that is of the highest translational relevance.