G6PC1 and G6PC2 influence G6P flux but not HSD11B1 activity.

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. 11ß-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.

Effects of G6pc2 deletion on body weight and cholesterol in mice.

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.

Combined Deletion of Slc30a7 and Slc30a8 Unmasks a Critical Role for ZnT8 in Glucose-Stimulated Insulin Secretion.

Polymorphisms in the SLC30A8 gene, which encodes the ZnT8 zinc transporter, are associated with altered susceptibility to type 2 diabetes (T2D), and SLC30A8 haploinsufficiency is protective against the development of T2D in obese humans. SLC30A8 is predominantly expressed in pancreatic islet ß-cells, but surprisingly, multiple knockout mouse studies have shown little effect of Slc30a8 deletion on glucose tolerance or glucose-stimulated insulin secretion (GSIS). Multiple other Slc30a isoforms are expressed at low levels in pancreatic islets. We hypothesized that functional compensation by the Slc30a7 isoform, which encodes ZnT7, limits the impact of Slc30a8 deletion on islet function. We therefore analyzed the effect of Slc30a7 deletion alone or in combination with Slc30a8 on in vivo glucose metabolism and GSIS in isolated islets. Deletion of Slc30a7 alone had complex effects in vivo, impairing glucose tolerance and reducing the glucose-stimulated increase in plasma insulin levels, hepatic glycogen levels, and pancreatic insulin content. Slc30a7 deletion also affected islet morphology and increased the ratio of islet α- to ß-cells. However, deletion of Slc30a7 alone had no effect on GSIS in isolated islets, whereas combined deletion of Slc30a7 and Slc30a8 abolished GSIS. These data demonstrate that the function of ZnT8 in islets can be unmasked by removal of ZnT7 and imply that ZnT8 may affect T2D susceptibility through actions in other tissues where it is expressed at low levels rather than through effects on pancreatic islet function.

G6PC2 Modulates the Effects of Dexamethasone on Fasting Blood Glucose and Glucose Tolerance.

The glucose-6-phosphatase catalytic subunit 2 (G6PC2) gene encodes an islet-specific glucose-6-phosphatase catalytic subunit. G6PC2 forms a substrate cycle with glucokinase that determines the glucose sensitivity of insulin secretion. Consequently, deletion of G6pc2 lowers fasting blood glucose (FBG) without affecting fasting plasma insulin. Although chronic elevation of FBG is detrimental to health, glucocorticoids induce G6PC2 expression, suggesting that G6PC2 evolved to transiently modulate FBG under conditions of glucocorticoid-related stress. We show, using competition and mutagenesis experiments, that the synthetic glucocorticoid dexamethasone (Dex) induces G6PC2 promoter activity through a mechanism involving displacement of the islet-enriched transcription factor MafA by the glucocorticoid receptor. The induction of G6PC2 promoter activity by Dex is modulated by a single nucleotide polymorphism, previously linked to altered FBG in humans, that affects FOXA2 binding. A 5-day repeated injection paradigm was used to examine the chronic effect of Dex on FBG and glucose tolerance in wild-type (WT) and G6pc2 knockout mice. Acute Dex treatment only induces G6pc2 expression in 129SvEv but not C57BL/6J mice, but this chronic treatment induced G6pc2 expression in both. In 6-hour fasted C57BL/6J WT mice, Dex treatment lowered FBG and improved glucose tolerance, with G6pc2 deletion exacerbating the decrease in FBG and enhancing the improvement in glucose tolerance. In contrast, in 24-hour fasted C57BL/6J WT mice, Dex treatment raised FBG but still improved glucose tolerance, with G6pc2 deletion limiting the increase in FBG and enhancing the improvement in glucose tolerance. These observations demonstrate that G6pc2 modulates the complex effects of Dex on both FBG and glucose tolerance.

Functional Analysis of Mouse G6pc1 Mutations Using a Novel In Situ Assay for Glucose-6-Phosphatase Activity and the Effect of Mutations in Conserved Human G6PC1/G6PC2 Amino Acids on G6PC2 Protein Expression.

Elevated fasting blood glucose (FBG) has been associated with increased risk for development of type 2 diabetes. Single nucleotide polymorphisms (SNPs) in G6PC2 are the most important common determinants of variations in FBG in humans. Studies using G6pc2 knockout mice suggest that G6pc2 regulates the glucose sensitivity of insulin secretion. G6PC2 and the related G6PC1 and G6PC3 genes encode glucose-6-phosphatase catalytic subunits. This study describes a functional analysis of 22 non-synonymous G6PC2 SNPs, that alter amino acids that are conserved in human G6PC1, mouse G6pc1 and mouse G6pc2, with the goal of identifying variants that potentially affect G6PC2 activity/expression. Published data suggest strong conservation of catalytically important amino acids between all four proteins and the related G6PC3 isoform. Because human G6PC2 has very low glucose-6-phosphatase activity we used an indirect approach, examining the effect of these SNPs on mouse G6pc1 activity. Using a novel in situ functional assay for glucose-6-phosphatase activity we demonstrate that the amino acid changes associated with the human G6PC2 rs144254880 (Arg79Gln), rs149663725 (Gly114Arg) and rs2232326 (Ser324Pro) SNPs reduce mouse G6pc1 enzyme activity without affecting protein expression. The Arg79Gln variant alters an amino acid mutation of which, in G6PC1, has previously been shown to cause glycogen storage disease type 1a. We also demonstrate that the rs368382511 (Gly8Glu), rs138726309 (His177Tyr), rs2232323 (Tyr207Ser) rs374055555 (Arg293Trp), rs2232326 (Ser324Pro), rs137857125 (Pro313Leu) and rs2232327 (Pro340Leu) SNPs confer decreased G6PC2 protein expression. In summary, these studies identify multiple G6PC2 variants that have the potential to be associated with altered FBG in humans.

G6PC2 Modulates Fasting Blood Glucose In Male Mice in Response to Stress.

The glucose-6-phosphatase catalytic 2 (G6PC2) gene is expressed specifically in pancreatic islet beta cells. Genome-wide association studies have shown that single nucleotide polymorphisms in the G6PC2 gene are associated with variations in fasting blood glucose (FBG) but not fasting plasma insulin. Molecular analyses examining the functional effects of these single nucleotide polymorphisms demonstrate that elevated G6PC2 expression is associated with elevated FBG. Studies in mice complement these genome-wide association data and show that deletion of the G6pc2 gene lowers FBG without affecting fasting plasma insulin. This suggests that, together with glucokinase, G6PC2 forms a substrate cycle that determines the glucose sensitivity of insulin secretion. Because genome-wide association studies and mouse studies demonstrate that elevated G6PC2 expression raises FBG and because chronically elevated FBG is detrimental to human health, increasing the risk of type 2 diabetes, it is unclear why G6PC2 evolved. We show here that the synthetic glucocorticoid dexamethasone strongly induces human G6PC2 promoter activity and endogenous G6PC2 expression in isolated human islets. Acute treatment with dexamethasone selectively induces endogenous G6pc2 expression in 129SvEv but not C57BL/6J mouse pancreas and isolated islets. The difference is due to a single nucleotide polymorphism in the C57BL/6J G6pc2 promoter that abolishes glucocorticoid receptor binding. In 6-hour fasted, nonstressed 129SvEv mice, deletion of G6pc2 lowers FBG. In response to the stress of repeated physical restraint, which is associated with elevated plasma glucocorticoid levels, G6pc2 gene expression is induced and the difference in FBG between wild-type and knockout mice is enhanced. These data suggest that G6PC2 may have evolved to modulate FBG in response to stress.