Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP/G6PC2) is a major autoantigen in both mouse and human type 1 diabetes. IGRP is selectively expressed in islet β cells and polymorphisms in the IGRP gene have recently been associated with variations in fasting blood glucose levels and cardiovascular-associated mortality in humans. Chromatin immunoprecipitation (ChIP) assays have shown that the IGRP promoter binds the islet-enriched transcription factors Pax-6 and BETA2. We show here, again using ChIP assays, that the IGRP promoter also binds the islet-enriched transcription factors MafA and Foxa2. Single binding sites for these factors were identified in the proximal IGRP promoter, mutation of which resulted in decreased IGRP fusion gene expression in βTC-3, Hamster insulinoma tumor (HIT), and Min6 cells. ChiP assays have shown that the islet-enriched transcription factor Pdx-1 also binds the IGRP promoter, but mutational analysis of four Pdx-1 binding sites in the proximal IGRP promoter revealed surprisingly little effect of Pdx-1 binding on IGRP fusion gene expression in βTC-3 cells. In contrast, in both HIT and Min6 cells mutation of these four Pdx-1 binding sites resulted in a ∼50% reduction in fusion gene expression. These data suggest that the same group of islet-enriched transcription factors, namely Pdx-1, Pax-6, MafA, BETA2, and Foxa2, directly or indirectly regulate expression of the two major autoantigens in type 1 diabetes.

In the endoplasmic reticulum (ER) lumen, glucose-6-phosphatase catalytic subunit 1 and 2 (G6PC1; G6PC2) hydrolyze glucose-6-phosphate (G6P) to glucose and inorganic phosphate whereas hexose-6-phosphate dehydrogenase (H6PD) hydrolyzes G6P to 6-phosphogluconate (6PG) in a reaction that generates NADPH. 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1) utilizes this NADPH to convert inactive cortisone to cortisol. HSD11B1 inhibitors improve insulin sensitivity whereas G6PC inhibitors are predicted to lower fasting blood glucose (FBG). This study investigated whether G6PC1 and G6PC2 influence G6P flux through H6PD and vice versa. Using a novel transcriptional assay that utilizes separate fusion genes to quantitate glucocorticoid and glucose signaling, we show that overexpression of H6PD and HSD11B1 in the islet-derived 832/13 cell line activated glucocorticoid-stimulated fusion gene expression. Overexpression of HSD11B1 blunted glucose-stimulated fusion gene expression independently of altered G6P flux. While overexpression of G6PC1 and G6PC2 blunted glucose-stimulated fusion gene expression, it had minimal effect on glucocorticoid-stimulated fusion gene expression. In the liver-derived HepG2 cell line, overexpression of H6PD and HSD11B1 activated glucocorticoid-stimulated fusion gene expression but overexpression of G6PC1 and G6PC2 had no effect. In rodents, HSD11B1 converts 11-dehydrocorticosterone (11-DHC) to corticosterone. Studies in wild-type and G6pc2 knockout mice treated with 11-DHC for 5 weeks reveal metabolic changes unaffected by the absence of G6PC2. These data suggest that HSD11B1 activity is not significantly affected by the presence or absence of G6PC1 or G6PC2. As such, G6PC1 and G6PC2 inhibitors are predicted to have beneficial effects by reducing FBG without causing a deleterious increase in glucocorticoid signaling.

G6PC2 encodes a glucose-6-phosphatase catalytic subunit that opposes the action of glucokinase in pancreatic islets, thereby modulating the sensitivity of insulin and glucagon secretion to glucose. In mice, G6pc2 is expressed at ~20-fold higher levels in β-cells than in α-cells, whereas in humans G6PC2 is expressed at only ~5-fold higher levels in β-cells. We therefore hypothesize that G6PC2 likely influences glucagon secretion to a greater degree in humans. With a view to generating a humanized mouse that recapitulates augmented G6PC2 expression levels in α-cells, we sought to identify the genomic regions that confer differential mouse G6pc2 expression in α-cells versus β-cells as well as the evolutionary changes that have altered this ratio in humans. Studies in islet-derived cell lines suggest that the elevated G6pc2 expression in mouse β-cells versus α-cells is mainly due to a difference in the relative activity of the proximal G6pc2 promoter in these cell types. Similarly, the smaller difference in G6PC2 expression between α-cells and β-cells in humans is potentially explained by a change in relative proximal G6PC2 promoter activity. However, we show that both glucocorticoid levels and multiple differences in the relative activity of eight transcriptional enhancers between mice and humans likely contribute to differential G6PC2 expression. Finally, we show that a mouse-specific non-coding RNA, Gm13613, whose expression is controlled by G6pc2 enhancer I, does not regulate G6pc2 expression, indicating that altered expression of Gm13613 in a humanized mouse that contains both the human promoter and enhancers should not affect G6PC2 function.

Genome-wide association studies have shown that a polymorphic variant in SLC30A8, which encodes zinc transporter-8, is associated with altered susceptibility to type 2 diabetes (T2D). This association is consistent with the observation that glucose-stimulated insulin secretion is decreased in islets isolated from Slc30a8 knockout mice. In this study, immunohistochemical staining was first used to show that SLC30A8 is expressed specifically in pancreatic islets. Fusion gene studies were then used to examine the molecular basis for the islet-specific expression of SLC30A8. The analysis of SLC30A8-luciferase expression in βTC-3 cells revealed that the proximal promoter region, located between −6154 and −1, relative to the translation start site, was only active in stable but not transient transfections. VISTA analyses identified three regions in the SLC30A8 promoter and a region in SLC30A8 intron 2 that are conserved in the mouse Slc30a8 gene. Additional fusion gene experiments demonstrated that none of these Slc30a8 promoter regions exhibited enhancer activity when ligated to a heterologous promoter whereas the conserved region in SLC30A8 intron 2 conferred elevated reporter gene expression selectively in βTC-3 but not in αTC-6 cells. Finally, the functional effects of a single nucleotide polymorphism (SNP), rs62510556, in this conserved intron 2 enhancer were investigated. Gel retardation studies showed that rs62510556 affects the binding of an unknown transcription factor and fusion gene analyses showed that it modulates enhancer activity. However, genetic analyses suggest that this SNP is not a causal variant that contributes to the association between SLC30A8 and T2D, at least in Europeans.

Genome-wide association study (GWAS) data have linked the G6PC2 gene to variations in fasting blood glucose (FBG). G6PC2 encodes an islet-specific glucose-6-phosphatase catalytic subunit that forms a substrate cycle with the beta cell glucose sensor glucokinase. This cycle modulates the glucose sensitivity of insulin secretion and hence FBG. GWAS data have not linked G6PC2 to variations in body weight but we previously reported that female C57BL/6J G6pc2-knockout (KO) mice were lighter than wild-type littermates on both a chow and high-fat diet. The purpose of this study was to compare the effects of G6pc2 deletion on FBG and body weight in both chow-fed and high-fat-fed mice on two other genetic backgrounds. FBG was reduced in G6pc2 KO mice largely independent of gender, genetic background or diet. In contrast, the effect of G6pc2 deletion on body weight was markedly influenced by these variables. Deletion of G6pc2 conferred a marked protection against diet-induced obesity in male mixed genetic background mice, whereas in 129SvEv mice deletion of G6pc2 had no effect on body weight. G6pc2 deletion also reduced plasma cholesterol levels in a manner dependent on gender, genetic background and diet. An association between G6PC2 and plasma cholesterol was also observed in humans through electronic health record-derived phenotype analyses. These observations suggest that the action of G6PC2 on FBG is largely independent of the influences of environment, modifier genes or epigenetic events, whereas the action of G6PC2 on body weight and cholesterol are influenced by unknown variables.

The G6PC1, G6PC2 and G6PC3 genes encode distinct glucose-6-phosphatase catalytic subunit (G6PC) isoforms. In mice, germline deletion of G6pc2 lowers fasting blood glucose (FBG) without affecting fasting plasma insulin (FPI) while, in isolated islets, glucose-6-phosphatase activity and glucose cycling are abolished and glucose-stimulated insulin secretion (GSIS) is enhanced at submaximal but not high glucose. These observations are all consistent with a model in which G6PC2 regulates the sensitivity of GSIS to glucose by opposing the action of glucokinase. G6PC2 is highly expressed in human and mouse islet beta cells however, various studies have shown trace G6PC2 expression in multiple tissues raising the possibility that G6PC2 also affects FBG through non-islet cell actions. Using real-time PCR we show here that expression of G6pc1 and/or G6pc3 are much greater than G6pc2 in peripheral tissues, whereas G6pc2 expression is much higher than G6pc3 in both pancreas and islets with G6pc1 expression not detected. In adult mice, beta cell-specific deletion of G6pc2 was sufficient to reduce FBG without changing FPI. In addition, electronic health record-derived phenotype analyses showed no association between G6PC2 expression and phenotypes clearly unrelated to islet function in humans. Finally, we show that germline G6pc2 deletion enhances glycolysis in mouse islets and that glucose cycling can also be detected in human islets. These observations are all consistent with a mechanism by which G6PC2 action in islets is sufficient to regulate the sensitivity of GSIS to glucose and hence influence FBG without affecting FPI.