Synopsis The Slc30a8 gene encodes the islet-specific zinc transporter ZnT-8, which provides zinc for insulin-hexamer formation. Polymorphic variants in amino acid 325 of human ZnT-8 are associated with altered susceptibility to type 2 diabetes and ZnT-8 autoantibody epitope specificity changes in type 1 diabetes. To assess the physiological importance of ZnT-8, mice carrying a Slc30a8 exon 3 deletion were analyzed histologically and phenotyped for energy metabolism and pancreatic hormone secretion. No gross anatomical or behavioral changes or differences in body weight were observed between wild type and ZnT-8 −/− mice and ZnT-8 −/− mouse islets were indistinguishable from wild type in terms of their numbers, size and cellular composition. However, total zinc content was markedly reduced in ZnT-8 −/− mouse islets, as evaluated both by Timm’s histochemical staining of pancreatic sections and direct measurements in isolated islets. Blood glucose levels were unchanged in 16 week old, 6 hr fasted animals of either gender, however, plasma insulin concentrations were reduced in both female (~31%) and male (~47%) ZnT-8 −/− mice. Intraperitoneal glucose tolerance tests demonstrated no impairment in glucose clearance in male ZnT-8 −/− mice but glucose-stimulated insulin secretion from isolated islets was reduced ~33% relative to wild type littermates. In summary, Slc30a8 gene deletion is accompanied by a modest impairment in insulin secretion without major alterations in glucose metabolism.

Elevated fasting blood glucose (FBG) is associated with increased risk for the development of type 2 diabetes and cardiovascular-associated mortality. Genome-wide association studies (GWAS) have linked polymorphisms in G6PC2 with variations in FBG and body fat, although not insulin sensitivity or glucose tolerance. G6PC2 encodes an islet-specific, endoplasmic reticulum–resident glucose-6-phosphatase catalytic subunit. A combination of in situ perfused pancreas, in vitro isolated islet, and in vivo analyses were used to explore the function of G6pc2 in mice. G6pc2 deletion had little effect on insulin sensitivity and glucose tolerance, whereas body fat was reduced in female G6pc2 knockout (KO) mice on both a chow and high-fat diet, observations that are all consistent with human GWAS data. G6pc2 deletion resulted in a leftward shift in the dose-response curve for glucose-stimulated insulin secretion (GSIS). As a consequence, under fasting conditions in which plasma insulin levels were identical, blood glucose levels were reduced in G6pc2 KO mice, again consistent with human GWAS data. Glucose-6-phosphatase activity was reduced, whereas basal cytoplasmic calcium levels were elevated in islets isolated from G6pc2 KO mice. These data suggest that G6pc2 represents a novel, negative regulator of basal GSIS that acts by hydrolyzing glucose-6-phosphate, thereby reducing glycolytic flux.

Islet-specific glucose-6-phosphatase (G6Pase) catalytic subunit-related protein (IGRP) is a homolog of the catalytic subunit of G6Pase, the enzyme that catalyzes the terminal step of the gluconeogenic pathway. Its catalytic activity, however, has not been defined. Since IGRP gene expression is restricted to islets, this suggests a possible role in the regulation of islet metabolism and, hence, insulin secretion induced by metabolites. We report here a comparative analysis of the human, mouse, and rat IGRP genes. These studies aimed to identify conserved sequences that may be critical for IGRP function and that specify its restricted tissue distribution. The single copy human IGRP gene has five exons of similar length and coding sequence to the mouse IGRP gene and is located on human chromosome 2q28 -32 adjacent to the myosin heavy chain 1B gene. In contrast, the rat IGRP gene does not appear to encode a protein as a result of a series of deletions and insertions in the coding sequence. Moreover, rat IGRP mRNA, unlike mouse and human IGRP mRNA, is not expressed in islets or islet-derived cell lines, an observation that was traced by fusion gene analysis to a mutation of the TATA box motif in the mouse/human IGRP promoters to TGTA in the rat sequence. The results provide a framework for the further analysis of the molecular basis for the tissuerestricted expression of the IGRP gene and the identification of key amino acid sequences that determine its biological activity.

Glucose-6-phosphatase catalyzes the final step in the gluconeogenic and glycogenolytic pathways. Glucocorticoids stimulate glucose-6-phosphatase catalytic subunit (G6Pase) gene transcription and studies performed in H4IIE hepatoma cells demonstrate the presence of a glucocorticoid response unit (GRU) in the proximal G6Pase promoter. In vitro deoxyribonuclease I footprinting analyses show that the glucocorticoid receptor binds to three glucocorticoid response elements (GREs) in the -231 to -129 promoter region and transfection results indicate all three contribute to glucocorticoid induction of G6Pase gene transcription. Furthermore, binding sites for hepatocyte nuclear factor-1 and -4, CRE binding factors, and FKHR (FOXO1a) are required for the full glucocorticoid response. Chromatin immunoprecipitation assays show that dexamethasone treatment stimulates glucocorticoid receptor and FKHR binding to the endogenous G6Pase promoter. Surprisingly, although glucocorticoids stimulate G6Pase gene transcription, deoxyribonuclease I footprinting and transfection analyses demonstrate the presence of a negative GRE and an associated negative accessory factor element in the -271 to -225 promoter region, which inhibit the glucocorticoid response. This appears to be the first report of a promoter that contains both positive and negative GREs, which function within the same cellular environment. We hypothesize that targeted signaling to the negative accessory element within the GRU may provide tight regulation of the glucocorticoid stimulation.

Aims/hypothesis Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP, now known as glucose-6-phosphatase, catalytic, 2 [G6PC2]) has recently been identified as a major autoantigen in mouse and human type 1 diabetes. Strategies designed to suppress expression of the gene encoding G6PC2 might therefore be useful in delaying or preventing the onset of this disease. However, since the function of G6PC2 is unclear, the concern with such an approach is that a change in G6PC2 expression might itself have deleterious consequences. Methods To address this concern and assess the physiological function of G6PC2, we generated G6pc2-null mice and performed a phenotypic analysis focusing principally on energy metabolism. Results No differences in body weight were observed and no gross anatomical or behavioural changes were evident. In 16-week-old animals, following a 6-h fast, a small but significant decrease in blood glucose was observed in both male (−14%) and female (−11%) G6pc2 −/− mice, while female G6pc2 −/− mice also exhibited a 12% decrease in plasma triacylglycerol. Plasma cholesterol, glycerol, insulin and glucagon concentrations were unchanged. Conclusions/interpretation These results argue against the possibility of G6PC2 playing a major role in pancreatic islet stimulus secretion coupling or energy homeostasis under physiological conditions imposed by conventional animal housing. This indicates that manipulating the expression of G6PC2 for therapeutic ends may be feasible.