Biophysical and functional properties of purified glucose-6-phosphatase catalytic subunit 1.

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.

Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose.

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 nonsynonymous 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.

Glucose-6-phosphatase catalytic subunit 2 negatively regulates glucose oxidation and insulin secretion in pancreatic ß-cells.

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 ß-cell metabolism and redox regulation are unknown. Here we used CRISPR/Cas9 gene editing and metabolic flux analysis in ßTC3 cells, a murine pancreatic ß-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 ß-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.

Evidence that Evolution of the Diabetes Susceptibility Gene SLC30A8 that Encodes the Zinc Transporter ZnT8 Drives Variations in Pancreatic Islet Zinc Content in Multiple Species.

Pancreatic islet zinc levels vary widely between species. Very low islet zinc levels in Guinea pigs were thought to be driven by evolution of the INS gene that resulted in the generation of an isoform lacking a histidine at amino acid 10 in the B chain of insulin that is unable to bind zinc. However, we recently showed that the SLC30A8 gene, that encodes the zinc transporter ZnT8, is a pseudogene in Guinea pigs, providing an alternate mechanism to potentially explain the low zinc levels. We show here that the SLC30A8 gene is also inactivated in sheep, cows, chinchillas and naked mole rats but in all four species a histidine is retained at amino acid 10 in the B chain of insulin. Zinc levels are known to be very low in sheep and cow islets. These data suggest that evolution of SLC30A8 rather than INS drives variation in pancreatic islet zinc content in multiple species.

The Diabetes Susceptibility Gene SLC30A8 that Encodes the Zinc Transporter ZnT8 is a Pseudogene in Guinea Pigs Potentially Contributing to Low Guinea Pig Islet Zinc Content.

In most mammals pancreatic islet beta cells have very high zinc levels that promote the crystallization and storage of insulin. Guinea pigs are unusual amongst mammals in that their islets have very low zinc content. The selectionist theory of insulin evolution proposes that low environmental zinc led to the selection of a mutation in Guinea pig insulin that negated the requirement for zinc binding. In mice deletion of the Slc30a8 gene, that encodes the zinc transporter ZnT8, markedly reduces islet zinc content. We show here that SLC30A8 is a pseudogene in Guinea pigs. We hypothesize that inactivation of the SLC30A8 gene led to low islet zinc content that allowed for the evolution of insulin that no longer bound zinc.

Crystal structures reveal a new and novel FoxO1 binding site within the human glucose-6-phosphatase catalytic subunit 1 gene promoter.

Human glucose-6-phosphatase plays a vital role in blood glucose homeostasis and holds promise as a therapeutic target for diabetes. Expression of its catalytic subunit gene 1 (G6PC1) is tightly regulated by metabolic-response transcription factors such as FoxO1 and CREB. Although at least three potential FoxO1 binding sites (insulin response elements, IREs) and one CREB binding site (cAMP response element, CRE) within the proximal region of the G6PC1 promoter have been identified, the interplay between FoxO1 and CREB and between FoxO1 bound at multiple IREs has not been well characterized. Here we present the crystal structures of the FoxO1 DNA binding domain in complex with the G6PC1 promoter. These complexes reveal the presence of a new non-consensus FoxO1 binding site that overlaps the CRE, suggesting a mutual exclusion mechanism for FoxO1 and CREB binding at the G6PC1 promoter. Additional findings include (i) non-canonical FoxO1 recognition sites, (ii) incomplete FoxO1 occupancies at the available IRE sites, and (iii) FoxO1 dimeric interactions that may play a role in stabilizing DNA looping. These findings provide insight into the regulation of G6PC1 gene transcription by FoxO1, and demonstrate a high versatility of target gene recognition by FoxO1 that correlates with its diverse roles in biology.

Identification of structural motifs critical for human G6PC2 function informed by sequence analysis and an AlphaFold2-predicted model.

G6PC2 encodes a glucose-6-phosphatase (G6Pase) catalytic subunit, primarily expressed in pancreatic islet ß cells, which modulates the sensitivity of insulin secretion to glucose and thereby regulates fasting blood glucose (FBG). Mutational analyses were conducted to validate an AlphaFold2 (AF2)-predicted structure of human G6PC2 in conjunction with a novel method to solubilize and purify human G6PC2 from a heterologous expression system. These analyses show that residues forming a predicted intramolecular disulfide bond are essential for G6PC2 expression and that residues forming part of a type 2 phosphatidic acid phosphatase (PAP2) motif are critical for enzyme activity. Additional mutagenesis shows that residues forming a predicted substrate cavity modulate enzyme activity and substrate specificity and residues forming a putative cholesterol recognition amino acid consensus (CRAC) motif influence protein expression or enzyme activity. This CRAC motif begins at residue 219, the site of a common G6PC2 non-synonymous single-nucleotide polymorphism (SNP), rs492594 (Val219Leu), though the functional impact of this SNP is disputed. In microsomal membrane preparations, the L219 variant has greater activity than the V219 variant, but this difference disappears when G6PC2 is purified in detergent micelles. We hypothesize that this was due to a differential association of the two variants with cholesterol. This concept was supported by the observation that the addition of cholesteryl hemi-succinate to the purified enzymes decreased the Vmax of the V219 and L219 variants ∼8-fold and ∼3 fold, respectively. We anticipate that these observations should support the rational development of G6PC2 inhibitors designed to lower FBG.

Multiple promoter and enhancer differences likely contribute to augmented G6PC2 expression in human versus mouse pancreatic islet alpha cells.

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 ß-cells than in α-cells, whereas in humans G6PC2 is expressed at only ~5-fold higher levels in ß-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 ß-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 ß-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 ß-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.

Integrative analysis of pathogenic variants in glucose-6-phosphatase based on an AlphaFold2 model.

Mediating the terminal reaction of gluconeogenesis and glycogenolysis, the integral membrane protein glucose-6-phosphate catalytic subunit 1 (G6PC1) regulates hepatic glucose production by catalyzing hydrolysis of glucose-6-phosphate (G6P) within the lumen of the endoplasmic reticulum. Consistent with its vital contribution to glucose homeostasis, inactivating mutations in G6PC1 causes glycogen storage disease (GSD) type 1a characterized by hepatomegaly and severe hypoglycemia. Despite its physiological importance, the structural basis of G6P binding to G6PC1 and the molecular disruptions induced by missense mutations within the active site that give rise to GSD type 1a are unknown. In this study, we determine the atomic interactions governing G6P binding as well as explore the perturbations imposed by disease-linked missense variants by subjecting an AlphaFold2 G6PC1 structural model to molecular dynamics simulations and in silico predictions of thermodynamic stability validated with robust in vitro and in situ biochemical assays. We identify a collection of side chains, including conserved residues from the signature phosphatidic acid phosphatase motif, that contribute to a hydrogen bonding and van der Waals network stabilizing G6P in the active site. The introduction of GSD type 1a mutations modified the thermodynamic landscape, altered side chain packing and substrate-binding interactions, and induced trapping of catalytic intermediates. Our results, which corroborate the high quality of the AF2 model as a guide for experimental design and to interpret outcomes, not only confirm the active-site structural organization but also identify previously unobserved mechanistic contributions of catalytic and noncatalytic side chains.

The role of glucose-6-phosphatase activity in glucose homeostasis and its potential for diabetes therapy.

Glucose-6-phosphatase catalytic subunit (G6PC)1 and G6PC2 are crucial for glucose metabolism, regulating processes like glycolysis, gluconeogenesis, and glycogenolysis. Despite their structural and functional similarities, G6PC1 and G6PC2 exhibit distinct tissue-specific expression patterns, G6P hydrolysis kinetics, and physiological functions. This review provides a comprehensive overview of their enzymology and distinct roles in glucose homeostasis. We examine how inactivating mutations in G6PC1 lead to glycogen storage disease, and how elevated G6PC1 and G6PC2 expression can affect the incidence of diabetic complications, risk for type 2 diabetes mellitus (T2DM) and various cancers. We also discuss the potential of inhibiting G6PC1 and G6PC2 to protect against complications from elevated blood glucose levels, and highlight drug development efforts targeting G6PC1 and G6PC2, and the therapeutic potential of inhibitors for disease prevention.

Biochemical and metabolic characterization of a G6PC2 inhibitor.

Three glucose-6-phosphatase catalytic subunits, that hydrolyze glucose-6-phosphate (G6P) to glucose and inorganic phosphate, have been identified, designated G6PC1-3, but only G6PC1 and G6PC2 have been implicated in the regulation of fasting blood glucose (FBG). Elevated FBG has been associated with multiple adverse clinical outcomes, including increased risk for type 2 diabetes and various cancers. Therefore, G6PC1 and G6PC2 inhibitors that lower FBG may be of prophylactic value for the prevention of multiple conditions. The studies described here characterize a G6PC2 inhibitor, designated VU0945627, previously identified as Compound 3. We show that VU0945627 preferentially inhibits human G6PC2 versus human G6PC1 but activates human G6PC3. VU0945627 is a mixed G6PC2 inhibitor, increasing the Km but reducing the Vmax for G6P hydrolysis. PyRx virtual docking to an AlphaFold2-derived G6PC2 structural model suggests VU0945627 binds two sites in human G6PC2. Mutation of residues in these sites reduces the inhibitory effect of VU0945627. VU0945627 does not inhibit mouse G6PC2 despite its 84% sequence identity with human G6PC2. Mutagenesis studies suggest this lack of inhibition of mouse G6PC2 is due, in part, to a change in residue 318 from histidine in human G6PC2 to proline in mouse G6PC2. Surprisingly, VU0945627 still inhibited glucose cycling in the mouse islet-derived ßTC-3 cell line. Studies using intact mouse liver microsomes and PyRx docking suggest that this observation can be explained by an ability of VU0945627 to also inhibit the G6P transporter SLC37A4. These data will inform future computational modeling studies designed to identify G6PC isoform-specific inhibitors.

Moving on from GWAS: functional studies on the G6PC2 gene implicated in the regulation of fasting blood glucose.

Genome-wide association studies (GWAS) have shown that single-nucleotide polymorphisms (SNPs) in G6PC2 are the most important common determinants of variations in fasting blood glucose (FBG) levels. Molecular studies examining the functional impact of these SNPs on G6PC2 gene transcription and splicing suggest that they affect FBG by directly modulating G6PC2 expression. This conclusion is supported by studies on G6pc2 knockout (KO) mice showing that G6pc2 represents a negative regulator of basal glucose-stimulated insulin secretion that acts by hydrolyzing glucose-6-phosphate, thereby reducing glycolytic flux and opposing the action of glucokinase. Suppression of G6PC2 activity might, therefore, represent a novel therapy for lowering FBG and the risk of cardiovascular-associated mortality. GWAS and G6pc2 KO mouse studies also suggest that G6PC2 affects other aspects of beta cell function. The evolutionary benefit conferred by G6PC2 remains unclear, but it is unlikely to be related to its ability to modulate FBG.

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.

Molecular mechanisms of catalytic inhibition for active site mutations in glucose-6-phosphatase catalytic subunit 1 linked to glycogen storage disease.

Mediating the terminal reaction of gluconeogenesis and glycogenolysis, the integral membrane protein G6PC1 regulates hepatic glucose production by catalyzing hydrolysis of glucose-6-phosphate (G6P) within the lumen of the endoplasmic reticulum. Consistent with its vital contribution to glucose homeostasis, inactivating mutations in G6PC1 cause glycogen storage disease (GSD) type 1a characterized by hepatomegaly and severe hypoglycemia. Despite its physiological importance, the structural basis of G6P binding to G6PC1 and the molecular disruptions induced by missense mutations within the active site that give rise to GSD type 1a are unknown. Exploiting a computational model of G6PC1 derived from the groundbreaking structure prediction algorithm AlphaFold2 (AF2), we combine molecular dynamics (MD) simulations and computational predictions of thermodynamic stability with a robust in vitro screening platform to define the atomic interactions governing G6P binding as well as explore the energetic perturbations imposed by disease-linked variants. We identify a collection of side chains, including conserved residues from the signature phosphatidic acid phosphatase motif, that contribute to a hydrogen bonding and van der Waals network stabilizing G6P in the active site. Introduction of GSD type 1a mutations into the G6PC1 sequence elicits changes in G6P binding energy, thermostability and structural properties, suggesting multiple pathways of catalytic impairment. Our results, which corroborate the high quality of the AF2 model as a guide for experimental design and to interpret outcomes, not only confirm active site structural organization but also suggest novel mechanistic contributions of catalytic and non-catalytic side chains.

Chronic Glucocorticoid Exposure Induced an S1PR2-RORγ Axis to Enhance Hepatic Gluconeogenesis in Male Mice.

It is well established that chronic glucocorticoid exposure causes hyperglycemia. While glucocorticoid receptor (GR) stimulates hepatic gluconeogenic gene transcription, additional mechanisms are activated by chronic glucocorticoid exposure to enhance gluconeogenesis. We found that chronic glucocorticoid treatment activated sphingosine-1-phosphate (S1P)-mediated signaling. Hepatic knockdown of hepatic S1P receptor 1 (S1PR1) had no effect on chronic glucocorticoid-induced glucose intolerance but elevated fasting plasma insulin levels. In contrast, hepatic S1PR3 knockdown exacerbated chronic glucocorticoid-induced glucose intolerance without affecting fasting plasma insulin levels. Finally, hepatic S1PR2 knockdown attenuated chronic glucocorticoid-induced glucose intolerance and reduced fasting plasma insulin levels. Here, we focused on dissecting the role of S1PR2 signaling in chronic glucocorticoid response on glucose homeostasis. We found that chronic glucocorticoid-induced hepatic gluconeogenesis, gluconeogenic gene expression, and GR recruitment to the glucocorticoid response elements (GREs) of gluconeogenic genes were all reduced in hepatic S1PR2 knockdown male mice. Hepatic S1PR2 knockdown also enhanced glucocorticoid suppression of RAR-related orphan receptor γ (RORγ) expression. Hepatic RORγ overexpression in hepatic S1PR2 knockdown mice restored glucocorticoid-induced glucose intolerance, gluconeogenic gene expression, and GR recruitment to their GREs. Conversely, RORγ antagonist and the reduction of hepatic RORγ expression attenuated such glucocorticoid effects. Thus, chronic glucocorticoid exposure induces an S1PR2-RORγ axis to cooperate with GR to enhance hepatic gluconeogenesis. Overall, this work provides novel mechanisms of and pharmaceutical targets against steroid-induced hyperglycemia.

An Enhancer Within Abcb11 Regulates G6pc2 in C57BL/6 Mouse Pancreatic Islets.

G6PC2 is predominantly expressed in pancreatic islet ß-cells where it encodes a glucose-6-phosphatase catalytic subunit that modulates the sensitivity of insulin secretion to glucose by opposing the action of glucokinase, thereby regulating fasting blood glucose (FBG). Prior studies have shown that the G6pc2 promoter alone is unable to confer sustained islet-specific gene expression in mice, suggesting the existence of distal enhancers that regulate G6pc2 expression. Using information from both mice and humans and knowledge that single nucleotide polymorphisms (SNPs) both within and near G6PC2 are associated with variations in FBG in humans, we identified several putative enhancers 3' of G6pc2. One region, herein referred to as enhancer I, resides in the 25th intron of Abcb11 and binds multiple islet-enriched transcription factors. CRISPR-mediated deletion of enhancer I in C57BL/6 mice had selective effects on the expression of genes near the G6pc2 locus. In isolated islets, G6pc2 and Spc25 expression were reduced ∼50%, and Gm13613 expression was abolished, whereas Cers6 and nostrin expression were unaffected. This partial reduction in G6pc2 expression enhanced islet insulin secretion at basal glucose concentrations but did not affect FBG or glucose tolerance in vivo, consistent with the absence of a phenotype in G6pc2 heterozygous C57BL/6 mice.

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

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-1alpha act synergistically to stimulate the expression of genes in the gluconeogenesis pathway and propose that PGC-1alpha 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-1alpha. Our results indicate that the transcriptional interaction between FOXO1 and PGC-1alpha is indirect.

The pancreatic islet ß-cell-enriched transcription factor Pdx-1 regulates Slc30a8 gene transcription through an intronic enhancer.

The SLC30A8 gene encodes the zinc transporter ZnT-8, which provides zinc for insulin-hexamer formation. Genome-wide association studies have shown that a polymorphic variant in SLC30A8 is associated with altered susceptibility to Type 2 diabetes and we recently reported that glucose-stimulated insulin secretion is decreased in islets isolated from Slc30a8-knockout mice. The present study examines the molecular basis for the islet-specific expression of Slc30a8. VISTA analyses identified two conserved regions in Slc30a8 introns 2 and 3, designated enhancers A and B respectively. Transfection experiments demonstrated that enhancer B confers elevated fusion gene expression in both ßTC-3 cells and αTC-6 cells. In contrast, enhancer A confers elevated fusion gene expression selectively in ßTC-3 and not αTC-6 cells. These data suggest that enhancer A is an islet ß-cell-specific enhancer and that the mechanisms controlling Slc30a8 expression in α- and ß-cells are overlapping, but distinct. Gel retardation and ChIP (chromatin immunoprecipitation) assays revealed that the islet-enriched transcription factor Pdx-1 binds enhancer A in vitro and in situ respectively. Mutation of two Pdx-1-binding sites in enhancer A markedly reduces fusion gene expression suggesting that this factor contributes to Slc30a8 expression in ß-cells, a conclusion consistent with developmental studies showing that restriction of Pdx-1 to pancreatic islet ß-cells correlates with the induction of Slc30a8 gene expression and ZnT-8 protein expression in vivo.

Zinc transporter 8 haploinsufficiency protects against beta cell dysfunction in type 1 diabetes by increasing mitochondrial respiration.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Glucose-6-phosphatase catalytic subunit gene family.

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 tissue-specific 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.