James K. Oeser

Deletion of the mouse Slc30a8 gene encoding zinc transporter-8 results in impaired insulin secretion.

The Slc30a8 gene encodes the islet-specific zinc transporter ZnT-8, which provides zinc for insulin-hexamer formation. Polymorphic variants in amino acid residue 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 analysed histologically and phenotyped for energy metabolism and pancreatic hormone secretion. No gross anatomical or behavioural 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 Timms histochemical staining of pancreatic sections and direct measurements in isolated islets. Blood glucose levels were unchanged in 16-week-old, 6 h fasted animals of either gender; however, plasma insulin concentrations were reduced in both female (approximately 31%) and male (approximately 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 approximately 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.

Cloning and Characterization of the Human and Rat Islet-specific Glucose-6-phosphatase Catalytic Subunit-related Protein (IGRP) Genes

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 ratIGRP 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 IGRPgene has five exons of similar length and coding sequence to the mouseIGRP gene and is located on human chromosome 2q28–32 adjacent to the myosin heavy chain 1B gene. In contrast, the ratIGRP 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 tissue-restricted expression of theIGRP gene and the identification of key amino acid sequences that determine its biological activity.

The Three Insulin Response Sequences in the Glucose-6-phosphatase Catalytic Subunit Gene Promoter Are Functionally Distinct

Glucose-6-phosphatase catalyzes the terminal step in the gluconeogenic and glycogenolytic pathways. In HepG2 cells, the maximum repression of basal glucose-6-phosphatase catalytic subunit (G6Pase) gene transcription by insulin requires two distinct promoter regions, designated A (located between −231 and −199) and B (located between −198 and −159), that together form an insulin response unit. Region A binds hepatocyte nuclear factor-1, which acts as an accessory factor to enhance the effect of insulin, mediated through region B, on G6Pase gene transcription. We have previously shown that region B binds the transcriptional activator FKHR (FOXO1a) in vitro. Chromatin immunoprecipitation assays demonstrate that FKHR also binds the G6Pase promoter in situ and that insulin inhibits this binding. Region B contains three insulin response sequences (IRSs), designated IRS 1, 2, and 3, that share the core sequence T(G/A)TTTT. However, detailed analyses reveal that these three G6Pase IRSs are functionally distinct. Thus, FKHR binds IRS 1 with high affinity and IRS 2 with low affinity but it does not bind IRS 3. Moreover, in the context of the G6Pase promoter, IRS 1 and 2, but not IRS 3, are required for the insulin response. Surprisingly, IRS 3, as well as IRS 1 and IRS 2, can each confer an inhibitory effect of insulin on the expression of a heterologous fusion gene, indicating that, in this context, a transcription factor other than FKHR, or its orthologs, can also mediate an insulin response through the T(G/A)TTTT motif.

Hepatocyte nuclear factor-4 alpha mediates the stimulatory effect of peroxisome proliferator-activated receptor gamma co-activator-1 alpha (PGC-1 alpha) on glucose-6-phosphatase catalytic subunit gene transcription in H4IIE cells.

It has recently been shown that adenoviral-mediated expression of peroxisome proliferator-activated receptor gamma co-activator-1 alpha (PGC-1 alpha) in hepatocytes stimulates glucose-6-phosphatase catalytic subunit (G6Pase) gene expression. A combination of fusion gene, gel retardation and chromatin immunoprecipitation assays revealed that, in H4IIE cells, PGC-1 alpha mediates this stimulation through an evolutionarily conserved region of the G6Pase promoter that binds hepatocyte nuclear factor-4 alpha.

Upstream stimulatory factor (USF) and neurogenic differentiation/beta-cell E box transactivator 2 (NeuroD/BETA2) contribute to islet-specific glucose-6-phosphatase catalytic-subunit-related protein (IGRP) gene expression.

Islet-specific glucose-6-phosphatase (G6Pase) catalytic-subunit-related protein (IGRP) is a homologue of the catalytic subunit of G6Pase, the enzyme that catalyses the final step of the gluconeogenic pathway. The analysis of IGRP-chloramphenicol acetyltransferase (CAT) fusion-gene expression through transient transfection of islet-derived beta TC-3 cells revealed that multiple promoter regions, located between -306 and -97, are required for maximal IGRP-CAT fusion-gene expression. These regions correlated with trans -acting factor-binding sites in the IGRP promoter that were identified in beta TC-3 cells in situ using the ligation-mediated PCR (LMPCR) footprinting technique. However, the LMPCR data also revealed additional trans -acting factor-binding sites located between -97 and +1 that overlap two E-box motifs, even though this region by itself conferred minimal fusion-gene expression. The data presented here show that these E-box motifs are important for IGRP promoter activity, but that their action is only manifest in the presence of distal promoter elements. Thus mutation of either E-box motif in the context of the -306 to +3 IGRP promoter region reduces fusion-gene expression. These two E-box motifs have distinct sequences and preferentially bind NeuroD/BETA2 (neurogenic differentiation/beta-cell E box transactivator 2) and upstream stimulatory factor (USF) in vitro, consistent with the binding of both factors to the IGRP promoter in situ, as determined using the chromatin-immunoprecipitation (ChIP) assay. Based on experiments using mutated IGRP promoter constructs, we propose a model to explain how the ubiquitously expressed USF could contribute to islet-specific IGRP gene expression.

Gluconeogenesis: re-evaluating the FOXO1-PGC-1alpha connection.

Arising from: P. Puigserver et al. 423, 550–555 (2003).Increased expression of the gene encoding the enzyme glucose-6-phosphatase (G6Pase) contributes to the increased production of glucose by the liver that occurs in individuals with diabetes. Puigserver et al. show that the transcription factor FOXO1 and the transcriptional co-activator PGC-1α act synergistically to stimulate the expression of genes in the gluconeogenesis pathway and propose that PGC-1α acts, in part, directly through FOXO1. Here we show that FOXO1 is neither required nor sufficient for the stimulation of G6Pase–luciferase fusion gene expression by PGC-1α. Our results indicate that the transcriptional interaction between FOXO1 and PGC-1α is indirect.

G6PC2 A Negative Regulator of Basal Glucose-Stimulated Insulin Secretion

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.

Genetic and Functional Assessment of the Role of the rs13431652-A and rs573225-A Alleles in the G6PC2 Promoter That Are Strongly Associated With Elevated Fasting Glucose Levels

OBJECTIVE Genome-wide association studies have identified a single nucleotide polymorphism (SNP), rs560887, located in a G6PC2 intron that is highly correlated with variations in fasting plasma glucose (FPG). G6PC2 encodes an islet-specific glucose-6-phosphatase catalytic subunit. This study examines the contribution of two G6PC2 promoter SNPs, rs13431652 and rs573225, to the association signal. RESEARCH DESIGN AND METHODS We genotyped 9,532 normal FPG participants (FPG <6.1 mmol/l) for three G6PC2 SNPs, rs13431652 (distal promoter), rs573225 (proximal promoter), rs560887 (3rd intron). We used regression analyses adjusted for age, sex, and BMI to assess the association with FPG and haplotype analyses to assess comparative SNP contributions. Fusion gene and gel retardation analyses characterized the effect of rs13431652 and rs573225 on G6PC2 promoter activity and transcription factor binding. RESULTS Genetic analyses provide evidence for a strong contribution of the promoter SNPs to FPG variability at the G6PC2 locus (rs13431652: β = 0.075, P = 3.6 × 10−35; rs573225 β = 0.073 P = 3.6 × 10−34), in addition to rs560887 (β = 0.071, P = 1.2 × 10−31). The rs13431652-A and rs573225-A alleles promote increased NF-Y and Foxa2 binding, respectively. The rs13431652-A allele is associated with increased FPG and elevated promoter activity, consistent with the function of G6PC2 in pancreatic islets. In contrast, the rs573225-A allele is associated with elevated FPG but reduced promoter activity. CONCLUSIONS Genetic and in situ functional data support a potential role for rs13431652, but not rs573225, as a causative SNP linking G6PC2 to variations in FPG, though a causative role for rs573225 in vivo cannot be ruled out.

Long-Range Enhancers Are Required to Maintain Expression of the Autoantigen Islet-Specific Glucose-6-Phosphatase Catalytic Subunit–Related Protein in Adult Mouse Islets In Vivo

OBJECTIVE—Islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP) is selectively expressed in islet β-cells and is a major autoantigen in both mouse and human type 1 diabetes. This study describes the use of a combination of transgenic and transfection approaches to characterize the gene regions that confer the islet-specific expression of IGRP. RESEARCH DESIGN AND METHODS—Transgenic mice were generated containing the IGRP promoter sequence from −306, −911, or −3911 to +3 ligated to a LacZ reporter gene. Transgene expression was monitored by 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside staining of pancreatic tissue. RESULTS—In all the transgenic mice, robust LacZ expression was detected in newborn mouse islets, but expression became mosaic as animals aged, suggesting that additional elements are required for the maintenance of IGRP gene expression. VISTA analyses identified two conserved regions in the distal IGRP promoter and one in the third intron. Transfection experiments demonstrated that all three regions confer enhanced luciferase reporter gene expression in βTC-3 cells when ligated to a minimal IGRP promoter. A transgene containing all three conserved regions was generated by using a bacterial recombination strategy to insert a LacZ cassette into exon 5 of the IGRP gene. Transgenic mice containing a 15-kbp fragment of the IGRP gene were then generated. This transgene conferred LacZ expression in newborn mouse islets; however, expression was still suppressed as animals aged. CONCLUSIONS—The data suggest that long-range enhancers 5′ or 3′ of the IGRP gene are required for the maintenance of IGRP gene expression in adult mice.

Deletion of the gene encoding the ubiquitously expressed glucose-6-phosphatase catalytic subunit-related protein (UGRP)/glucose-6-phosphatase catalytic subunit-β results in lowered plasma cholesterol and elevated glucagon

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 1a, 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.