Macrophages and neutrophils are necessary for ER stress-induced ß cell loss.

Persistent endoplasmic reticulum (ER) stress induces islet inflammation and ß cell loss. How islet inflammation contributes to ß cell loss remains uncertain. We have reported previously that chronic overnutrition-induced ER stress in ß cells causes Ripk3-mediated islet inflammation, macrophage recruitment, and a reduction of ß cell numbers in a zebrafish model. We show here that ß cell loss results from the intricate communications among ß cells, macrophages, and neutrophils. Macrophage-derived Tnfa induces cxcl8a in ß cells. Cxcl8a, in turn, attracts neutrophils to macrophage-contacted "hotspots" where ß cell loss occurs. We also show potentiation of chemokine expression in stressed mammalian ß cells by macrophage-derived TNFA. In Akita and db/db mice, there is an increase in CXCL15-positive ß cells and intra-islet neutrophils. Blocking neutrophil recruitment in Akita mice preserves ß cell mass and slows diabetes progression. These results reveal an important role of neutrophils in persistent ER stress-induced ß cell loss.

Effects of Arachidonic Acid and Its Metabolites on Functional Beta-Cell Mass.

Arachidonic acid (AA) is a polyunsaturated 20-carbon fatty acid present in phospholipids in the plasma membrane. The three primary pathways by which AA is metabolized are mediated by cyclooxygenase (COX) enzymes, lipoxygenase (LOX) enzymes, and cytochrome P450 (CYP) enzymes. These three pathways produce eicosanoids, lipid signaling molecules that play roles in biological processes such as inflammation, pain, and immune function. Eicosanoids have been demonstrated to play a role in inflammatory, renal, and cardiovascular diseases as well type 1 and type 2 diabetes. Alterations in AA release or AA concentrations have been shown to affect insulin secretion from the pancreatic beta cell, leading to interest in the role of AA and its metabolites in the regulation of beta-cell function and maintenance of beta-cell mass. In this review, we discuss the metabolism of AA by COX, LOX, and CYP, the roles of these enzymes and their metabolites in beta-cell mass and function, and the possibility of targeting these pathways as novel therapies for treating diabetes.

Pharmacological modulation of prostaglandin E2 (PGE2 ) EP receptors improves cardiomyocyte function under hyperglycemic conditions.

Type 2 diabetes (T2D) affects >30 million Americans and nearly 70% of individuals with T2D will die from cardiovascular disease (CVD). Circulating levels of the inflammatory signaling lipid, prostaglandin E2 (PGE2 ), are elevated in the setting of obesity and T2D and are associated with decreased cardiac function. The EP3 and EP4 PGE2 receptors have opposing actions in several tissues, including the heart: overexpression of EP3 in cardiomyocytes impairs function, while EP4 overexpression improves function. Here we performed complementary studies in vitro with isolated cardiomyocytes and in vivo using db/db mice, a model of T2D, to analyze the effects of EP3 inhibition or EP4 activation on cardiac function. Using echocardiography, we found that 2 weeks of systemic treatment of db/db mice with 20 mg/kg of EP3 antagonist, beginning at 6 weeks of age, improves ejection fraction and fractional shortening (with no effect on heart rate). We further show that either EP3 blockade or EP4 activation enhances contractility and calcium cycling in isolated mouse cardiomyocytes cultured in both normal and high glucose. Thus, peak [Ca2+ ]I transient amplitude was increased, while time to peak [Ca2+ ]I and [Ca2+ ]I decay were decreased. These data suggest that modulation of EP3 and EP4 activity has beneficial effects on cardiomyocyte contractility and overall heart function.

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.

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.

Pharmacological blockade of the EP3 prostaglandin E2 receptor in the setting of type 2 diabetes enhances ß-cell proliferation and identity and relieves oxidative damage.

OBJECTIVE: Type 2 diabetes is characterized by hyperglycemia and inflammation. Prostaglandin E2, which signals through four G protein-coupled receptors (EP1-4), is a mediator of inflammation and is upregulated in diabetes. We have shown previously that EP3 receptor blockade promotes ß-cell proliferation and survival in isolated mouse and human islets ex vivo. Here, we analyzed whether systemic EP3 blockade could enhance ß-cell mass and identity in the setting of type 2 diabetes using mice with a spontaneous mutation in the leptin receptor (Leprdb). METHODS: Four- or six-week-old, db/+, and db/db male mice were treated with an EP3 antagonist daily for two weeks. Pancreata were analyzed for α-cell and ß-cell proliferation and ß-cell mass. Islets were isolated for transcriptomic analysis. Selected gene expression changes were validated by immunolabeling of the pancreatic tissue sections. RESULTS: EP3 blockade increased ß-cell mass in db/db mice through enhanced ß-cell proliferation. Importantly, there were no effects on α-cell proliferation. EP3 blockade reversed the changes in islet gene expression associated with the db/db phenotype and restored the islet architecture. Expression of the GLP-1 receptor was slightly increased by EP3 antagonist treatment in db/db mice. In addition, the transcription factor nuclear factor E2-related factor 2 (Nrf2) and downstream targets were increased in islets from db/db mice in response to treatment with an EP3 antagonist. The markers of oxidative stress were decreased. CONCLUSIONS: The current study suggests that EP3 blockade promotes ß-cell mass expansion in db/db mice. The beneficial effects of EP3 blockade may be mediated through Nrf2, which has recently emerged as a key mediator in the protection against cellular oxidative damage.

G6PC2 confers protection against hypoglycemia upon ketogenic diet feeding and prolonged fasting.

OBJECTIVE: G6PC2 is predominantly expressed in pancreatic islet beta cells. G6PC2 hydrolyzes glucose-6-phosphate to glucose and inorganic phosphate, thereby creating a futile substrate cycle that opposes the action of glucokinase. This substrate cycle determines the sensitivity of glucose-stimulated insulin secretion to glucose and hence regulates fasting blood glucose (FBG) but not fasting plasma insulin (FPI) levels. Our objective was to explore the physiological benefit this cycle confers. METHODS: We investigated the response of wild type (WT) and G6pc2 knockout (KO) mice to changes in nutrition. RESULTS: Pancreatic G6pc2 expression was little changed by ketogenic diet feeding but was inhibited by 24 hr fasting and strongly induced by high fat feeding. When challenged with either a ketogenic diet or 24 hr fasting, blood glucose fell to 70 mg/dl or less in G6pc2 KO but not WT mice, suggesting that G6PC2 may have evolved, in part, to prevent hypoglycemia. Prolonged ketogenic diet feeding reduced the effect of G6pc2 deletion on FBG. The hyperglycemia associated with high fat feeding was partially blunted in G6pc2 KO mice, suggesting that under these conditions the presence of G6PC2 is detrimental. As expected, FPI changed but did not differ between WT and KO mice in response to fasting, ketogenic and high fat feeding. CONCLUSIONS: Since elevated FBG levels are associated with increased risk for cardiovascular-associated mortality (CAM), these studies suggest that, while G6PC2 inhibitors would be useful for lowering FBG and the risk of CAM, partial inhibition will be important to avoid the risk of hypoglycemia.

Potential positive and negative consequences of ZnT8 inhibition.

SLC30A8 encodes the zinc transporter ZnT8. SLC30A8 haploinsufficiency protects against type 2 diabetes (T2D), suggesting that ZnT8 inhibitors may prevent T2D. We show here that, while adult chow fed Slc30a8 haploinsufficient and knockout (KO) mice have normal glucose tolerance, they are protected against diet-induced obesity (DIO), resulting in improved glucose tolerance. We hypothesize that this protection against DIO may represent one mechanism whereby SLC30A8 haploinsufficiency protects against T2D in humans and that, while SLC30A8 is predominantly expressed in pancreatic islet beta cells, this may involve a role for ZnT8 in extra-pancreatic tissues. Consistent with this latter concept we show in humans, using electronic health record-derived phenotype analyses, that the 'C' allele of the non-synonymous rs13266634 SNP, which confers a gain of ZnT8 function, is associated not only with increased T2D risk and blood glucose, but also with increased risk for hemolytic anemia and decreased mean corpuscular hemoglobin (MCH). In Slc30a8 KO mice, MCH was unchanged but reticulocytes, platelets and lymphocytes were elevated. Both young and adult Slc30a8 KO mice exhibit a delayed rise in insulin after glucose injection, but only the former exhibit increased basal insulin clearance and impaired glucose tolerance. Young Slc30a8 KO mice also exhibit elevated pancreatic G6pc2 gene expression, potentially mediated by decreased islet zinc levels. These data indicate that the absence of ZnT8 results in a transient impairment in some aspects of metabolism during development. These observations in humans and mice suggest the potential for negative effects associated with T2D prevention using ZnT8 inhibitors.

Ins1-Cre and Ins1-CreER Gene Replacement Alleles Are Susceptible To Silencing By DNA Hypermethylation.

Targeted gene ablation studies of the endocrine pancreas have long suffered from suboptimal Cre deleter strains. In many cases, Cre lines purportedly specific for beta cells also displayed expression in other islet endocrine cells or in a subset of neurons in the brain. Several pancreas and endocrine Cre lines have experienced silencing or mosaicism over time. In addition, many Cre transgenic constructs were designed to include the hGH mini-gene, which by itself increases beta-cell replication and decreases beta-cell function. More recently, driver lines with Cre or CreER inserted into the Ins1 locus were generated, with the intent of producing ß cell-specific Cre lines with faithful recapitulation of insulin expression. These lines were bred in multiple labs to several different mouse lines harboring various lox alleles. In our hands, the ability of the Ins1-Cre and Ins1-CreER lines to delete target genes varied from that originally reported, with both alleles displaying low levels of expression, increased levels of methylation compared to the wild-type allele, and ultimately inefficient or absent target deletion. Thus, caution is warranted in the interpretation of results obtained with these genetic tools, and Cre expression and activity should be monitored regularly when using these lines.

Pancreatic islet beta cell-specific deletion of G6pc2 reduces fasting blood glucose.

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.

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.