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Zinc transporter 8 (ZnT8) is a major humoral target in human type 1 diabetes (T1D). Polymorphic variants of Slc30A8 , which encodes ZnT8, are also associated with protection from type 2 diabetes (T2D). The current study examined whether ZnT8 might play a role beyond simply being a target of autoimmunity in the pathophysiology of T1D. The phenotypes of NOD mice with complete or partial global loss of ZnT8 were determined using a combination of disease incidence, histological, transcriptomic, and metabolic analyses. Unexpectedly, while complete loss of ZnT8 accelerated spontaneous T1D, heterozygosity was partially protective. In vivo and in vitro studies of ZnT8 deficient NOD.SCID mice suggested that the accelerated disease was due to more rampant autoimmunity. Conversely, beta cells in heterozygous animals uniquely displayed increased mitochondrial fitness under mild proinflammatory conditions. In pancreatic beta cells and immune cell populations, Zn2+ plays a key role as a regulator of redox signaling and as an independent secondary messenger. Importantly, Zn2+ also plays a major role in maintaining mitochondrial homeostasis. Our results suggest that regulating mitochondrial fitness by altering intra-islet zinc homeostasis may provide a novel mechanism to modulate T1D pathophysiology. • NOD. ZnT8−/− animals get accelerated spontaneous type 1 diabetes. • NOD. ZnT8+/− animals are partially protected from spontaneous type 1 diabetes. • Beta cell mitochondrial fitness is potentiated in NOD. ZnT8+/− animals exposed to low doses of pro-inflammatory cytokines. [ABSTRACT FROM AUTHOR]

Glucose-6-phosphatase catalyzes the hydrolysis of glucose 6-phosphate (G6P) to glucose and inorganic phosphate. It is a multicomponent system located in the endoplasmic reticulum that comprises several integral membrane proteins, namely a catalytic subunit (G6PC) and transporters for G6P, inorganic phosphate, and glucose. The G6PC gene family contains three members, designated G6PC, G6PC2, and G6PC3. The tissuespecific expression patterns of these genes differ, and mutations in all three genes have been linked to distinct diseases in humans. This minireview discusses the disease association and transcriptional regulation of the G6PC genes as well as the biological functions of the encoded proteins. [ABSTRACT FROM AUTHOR]

G6PC2 is predominantly expressed in pancreatic islet beta cells. G6PC2 hydrolyzes glucose-6-phosphate to glucose and inorganic phosphate, thereby creating a futile substrate cycle that opposes the action of glucokinase. This substrate cycle determines the sensitivity of glucose-stimulated insulin secretion to glucose and hence regulates fasting blood glucose (FBG) but not fasting plasma insulin (FPI) levels. Our objective was to explore the physiological benefit this cycle confers. We investigated the response of wild type (WT) and G6pc2 knockout (KO) mice to changes in nutrition. Pancreatic G6pc2 expression was little changed by ketogenic diet feeding but was inhibited by 24 hr fasting and strongly induced by high fat feeding. When challenged with either a ketogenic diet or 24 hr fasting, blood glucose fell to 70 mg/dl or less in G6pc2 KO but not WT mice, suggesting that G6PC2 may have evolved, in part, to prevent hypoglycemia. Prolonged ketogenic diet feeding reduced the effect of G6pc2 deletion on FBG. The hyperglycemia associated with high fat feeding was partially blunted in G6pc2 KO mice, suggesting that under these conditions the presence of G6PC2 is detrimental. As expected, FPI changed but did not differ between WT and KO mice in response to fasting, ketogenic and high fat feeding. Since elevated FBG levels are associated with increased risk for cardiovascular-associated mortality (CAM), these studies suggest that, while G6PC2 inhibitors would be useful for lowering FBG and the risk of CAM, partial inhibition will be important to avoid the risk of hypoglycemia. • G6pc2 deletion lowers fasting blood glucose (FBG) in chow and high fat fed mice. • Elevated FBG increases the risk of cardiovascular-associated mortality (CAM). • G6pc2 deletion results in hypoglycemia in mice on a ketogenic diet. • G6pc2 deletion results in hypoglycemia in mice following prolonged fasting. • G6PC2 inhibitors may prevent CAM but increase risk of hypoglycemia. [ABSTRACT FROM AUTHOR]

Human pancreatic β cells have exceptionally high zinc content. In β cells the highest zinc concentration is in insulin secretory granules, from which it is cosecreted with the hormone. Uptake of zinc into secretory granules is mainly mediated by zinc transporter 8 (ZnT8), the product of the SLC30A8 [solute carrier family 30 (zinc transporter), member 8] gene. The minor alleles of several single-nucleotide polymorphisms (SNPs) in SLC30A8 are associated with decreased risk of type 2 diabetes (T2D), but the precise mechanisms underlying the protective effects remain uncertain. In this article we review current knowledge of the role of ZnT8 in maintaining zinc homeostasis in β cells, its role in glucose metabolism based on knockout mouse studies, and current theories regarding the link between ZnT8 function and T2D. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

G6PC2 encodes a glucose-6-phosphatase (G6Pase) catalytic subunit that modulates the sensitivity of insulin secretion to glucose and thereby regulates fasting blood glucose (FBG). A common single-nucleotide polymorphism (SNP) in G6PC2, rs560887 is an important determinant of human FBG variability. This SNP has a subtle effect on G6PC2 RNA splicing, which raises the question as to whether nonsynonymous SNPs with a major impact on G6PC2 stability or enzyme activity might have a broader disease/metabolic impact. Previous attempts to characterize such SNPs were limited by the very low inherent G6Pase activity and expression of G6PC2 protein in islet-derived cell lines. In this study, we describe the use of a plasmid vector that confers high G6PC2 protein expression in islet cells, allowing for a functional analysis of 22 non-synonymous G6PC2 SNPs, 19 of which alter amino acids that are conserved in mouse G6PC2 and the human and mouse variants of the related G6PC1 isoform. We show that 16 of these SNPs markedly impair G6PC2 protein expression (>50% decrease). These SNPs have variable effects on the stability of human and mouse G6PC1, despite the high sequence homology between these isoforms. Four of the remaining six SNPs impaired G6PC2 enzyme activity. Electronic health record-derived phenotype analyses showed an association between high-impact SNPs and FBG, but not other diseases/metabolites. While homozygous G6pc2 deletion in mice increases the risk of hypoglycemia, these human data reveal no evidence that the beneficial use of partial G6PC2 inhibitors to lower FBG would be associated with unintended negative consequences. [ABSTRACT FROM AUTHOR]

Glucose-6-phosphatase catalytic subunit 1 (G6PC1) plays a critical role in hepatic glucose production during fasting by mediating the terminal step of the gluconeogenesis and glycogenolysis pathways. In concert with accessory transport proteins, this membrane-integrated enzyme catalyzes glucose production from glucose-6-phosphate (G6P) to support blood glucose homeostasis. Consistent with its metabolic function, dysregulation of G6PC1 gene expression contributes to diabetes, and mutations that impair phosphohydrolase activity form the clinical basis of glycogen storage disease type 1a. Despite its relevance to health and disease, a comprehensive view of G6PC1 structure and mechanism has been limited by the absence of expression and purification strategies that isolate the enzyme in a functional form. In this report, we apply a suite of biophysical and biochemical tools to fingerprint the in vitro attributes of catalytically active G6PC1 solubilized in lauryl maltose neopentyl glycol (LMNG) detergent micelles. When purified from Sf9 insect cell membranes, the glycosylated mouse ortholog (mG6PC1) recapitulated functional properties observed previously in intact hepatic microsomes and displayed the highest specific activity reported to date. Additionally, our results establish a direct correlation between the catalytic and structural stability of mG6PC1, which is underscored by the enhanced thermostability conferred by phosphatidylcholine and the cholesterol analog cholesteryl hemisuccinate. In contrast, the N96A variant, which blocks N-linked glycosylation, reduced thermostability. The methodologies described here overcome long-standing obstacles in the field and lay the necessary groundwork for a detailed analysis of the mechanistic structural biology of G6PC1 and its role in complex metabolic disorders. [ABSTRACT FROM AUTHOR]

Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is selectively expressed in islet β cells and is a major autoantigen in a mouse model of type I diabetes. The analysis of IGRP-chloramphenicol acetyltransferase (CAT) fusion gene expression through transient transfection of islet-derived βTC-3 cells revealed that a promoter region, located between -273 and -254, is essential for high IGRP-CAT fusion gene expression. The sequence of this promoter region does not match that for any known islet-enriched transcription factor. However, data derived from gel retardation assays, a modified ligation-mediated polymerase chain reaction in situ footprinting technique and a SDS-polyacrylamide separation/renaturation procedure led to the hypothesis that this protein might be Pax-6, a conclusion that was confirmed by gel supershift assays. Additional experiments revealed a second non-consensus Pax-6 binding site in the -306/-274 IGRP promoter region. Pax-6 binding to these elements is unusual in that it appears to require both its homeo and paired domains. Interestingly, loss of Pax-6 binding to the -273/-246 element is compensated by Pax-6 binding to the -306/-274 element and vice versa. Gel retardation assays revealed that another islet-enriched transcription factor, namely Pdx-1, binds four non-consensus elements in the IGRP promoter. However, mutation of these elements has little effect on IGRP fusion gene expression. Although chromatin immunoprecipitation assays show that both Pax-6 and Pdx-1 bind to the IGRP promoter within intact cells, in contrast to the critical role of these factors in β cell-specific insulin gene expression, IGRP gene transcription appears to require Pax-6 but not Pdx-1. [ABSTRACT FROM AUTHOR]

In liver, glucose-6-phosphatase catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate, the final step in the gluconeogenic and glycogenolytic pathways. Mutations in the glucose-6-phosphatase catalytic subunit (G6Pase) give rise to glycogen storage disease (GSD) type la, which is characterized in part by hypoglycemia, growth retardation, hypertriglyceridemia, hypercholesterolemia, and hepatic glycogen accumulation. Recently, a novel G6Pase isoform was identified, designated UGRP/G6Pase-β. The activity of UGRP relative to G6Pase in vitro is disputed, raising the question as to whether G6P is a physiologically important substrate for this protein. To address this issue we have characterized the phenotype of UGRP knock-out mice. G6P hydrolytic activity was decreased by ~50% in homogenates of UGRP-/- mouse brain relative to wild type tissue, consistent with the ability of UGRP to hydrolyze G6P. In addition, female, but not male, UGRP-/- mice exhibit growth retardation as do G6Pase-/- mice and patients with GSD type 1a. However, in contrast to G6Pase-/- mice and patients with GSD type 1a, UGRP-/- mice exhibit no change in hepatic glycogen content, blood glucose, or triglyceride levels. Although UGRP-/- mice are not hypoglycemic, female UGRP-/- mice have elevated (~60%) plasma glucagon and reduced (~20%) plasma cholesterol. We hypothesize that the hyperglucagonemia prevents hypoglycemia and that the hypocholesterolemia is secondary to the hyperglucagonemia. As such, the phenotype of UGRP-/- mice is mild, indicating that G6Pase is the major glucose-6-phosphatase of physiological importance for glucose homeostasis in vivo. [ABSTRACT FROM AUTHOR]

Reports that three insulin response sequences (IRS) in the glucose-6-phosphatase catalytic subunit gene promoter are functionally distinct. Maximum repression of basal glucose-6-phosphatase catalytic subunit gene transcription by insulin; Accessory factor to enhance the effect of insulin; Chromatin immuno-precipitation assays; Inhibitory effect of insulin on the expression of a heterologous fusion gene.