Abstract
Elevated fasting blood glucose (FBG) is associated with increased risks of developing type 2 diabetes (T2D) and cardiovascular-associated mortality. G6PC2 is predominantly expressed in islets, encodes a glucose-6-phosphatase catalytic subunit that converts glucose-6-phosphate (G6P) to glucose, and has been linked with variations in FBG in genome-wide association studies. Deletion of G6pc2 in mice has been shown to lower FBG without affecting fasting plasma insulin levels in vivo. At 5 mM glucose, pancreatic islets from G6pc2 knockout (KO) mice exhibit no glucose cycling, increased glycolytic flux, and enhanced glucose-stimulated insulin secretion (GSIS). However, the broader effects of G6pc2 KO on β-cell metabolism and redox regulation are unknown. Here we used CRISPR/Cas9 gene editing and metabolic flux analysis in βTC3 cells, a murine pancreatic β-cell line, to examine the role of G6pc2 in regulating glycolytic and mitochondrial fluxes. We found that deletion of G6pc2 led to ∼60% increases in glycolytic and citric acid cycle (CAC) fluxes at both 5 and 11 mM glucose concentrations. Furthermore, intracellular insulin content and GSIS were enhanced by approximately two-fold, along with increased cytosolic redox potential and reductive carboxylation flux. Normalization of fluxes relative to net glucose uptake revealed upregulation in two NADPH-producing pathways in the CAC. These results demonstrate that G6pc2 regulates GSIS by modulating not only glycolysis but also, independently, citric acid cycle activity in β-cells. Overall, our findings implicate G6PC2 as a potential therapeutic target for enhancing insulin secretion and lowering FBG, which could benefit individuals with prediabetes, T2D, and obesity.
Highlights
Glucose-6-phosphatase, a multicomponent system located in the endoplasmic reticulum (ER), catalyzes the conversion of glucose-6-phosphate (G6P) to glucose [1]
Similar to data obtained in primary KO islets [4], we measured a significant elevation in cytoplasmic calcium levels within the KO cells at 5 and 11 mM glucose concentrations (Fig. 5D). These results show that deletion of G6pc2 leads to a broad acceleration in glycolytic and mitochondrial metabolism, primarily by rerouting more glucose and glutamine toward oxidative pathways that supply ATP and redox cofactors linked to glucose-stimulated insulin secretion (GSIS)
G6PC2 is predicted to control the fate of G6P, its effect on islet metabolism remains largely unknown
Summary
Glucose-6-phosphatase, a multicomponent system located in the endoplasmic reticulum (ER), catalyzes the conversion of glucose-6-phosphate (G6P) to glucose [1]. C, extracellular uptake and excretion rates measured in βTC3 cells incubated at 5 and 11 mM glucose concentrations. Contrary to primary islets, where longer incubation times (>24 h) led to normalization of GSIS in KO cells due to metabolic adaptation [8], we saw a consistent elevation of media insulin production by βTC3 KO cells compared with WT cells at both glucose concentrations examined (Fig. 3A).
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