G6PC Enzymology, Structure, Function and Role in the Regulation of Fasting Blood Glucose

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Project Summary: Glucose-6-phosphatase catalytic subunits (G6PCs) hydrolyze glucose-6-phosphate (G6P) to glucose and inorganic phosphate. G6PC2 is predominantly expressed in pancreatic islet b cells. Genetic and molecular studies have linked increased G6PC2 activity to elevated FBG. Higher FBG in the non-diabetic and pre-diabetic ranges strongly influence the risk of cardiovascular-associated mortality (CAM) and developing type 2 diabetes (T2D) whereas, in individuals with T2D, elevated FBG increases the risk for diabetic complications and further increases the risk of CAM. These observations strongly suggest that G6PC2 inhibitors may represent a potential novel therapy to reduce FBG. We hypothesize that the proposed genetic, metabolic, physiological and biochemical studies will define the functions of G6PC2 in b cells, demonstrate that G6PC2 represents a viable target for lowering FBG and generate data that fosters the development of G6PC2 inhibitors. Our data suggest a new paradigm in which a glucokinase/G6PC2 futile substrate cycle, rather than glucokinase alone, is the key determinant of glycolytic flux in b cells. Consequently, deletion of G6pc2 increases the sensitivity of glucose- stimulated insulin secretion (GSIS) to glucose, which results in reduced FBG. We have developed novel assays for measuring G6PC2 activity in vitro and in intact cells. In Aim 1 we propose using these assays to identify non-synonymous G6PC2 single nucleotide polymorphisms (SNPs) that impair G6PC2 activity and then explore their effects on human health using Vanderbilt’s BioVU biobank. We will also explore the concept, arising from preliminary data, that G6PC2 regulates metabolic fluxes and pulsatile insulin secretion in b cells through a mechanism independent of the known action of G6PC2 on glycolysis, that involves altered ER calcium oscillations. We hypothesize the results of Aim 1 will define the functions of G6PC2 in b cells and highlight the positive as well as potential negative effects of G6PC2 inhibition in humans. The conspicuous absence of structure- function studies on G6PC2 originated from the absence of a high-resolution molecular model, the low inherent activity of the enzyme and an inability to purify active G6PC2. The groundbreaking publication of the AlphaFold2 algorithm for protein structure prediction, and our development of heterologous expression and purification protocols for isolation of stable and catalytically active G6PC2, have overcome these hurdles. In Aim 2, we will use mutagenesis to probe the functional importance of amino acids within various putative domains in G6PC2 and then leverage a toolkit of biochemical/biophysical assays to define their effects on G6PC2 folding, stability and catalysis. We will also perform studies to characterize the specificity of a human G6PC2 inhibitor and explore its ability to regulate GSIS in human islets. We hypothesize the results of Aim 2 will establish a new paradigm for investigation of G6PC2 by providing a molecular blueprint for understanding the structural basis of catalysis as well as generating data that will provide insight into the mode of action of a known G6PC2 inhibitor, which will inform the rational design of optimized compounds.