DNA Methylation-Based Assessment of Cell Composition in Human Pancreas and Islets.

Assessment of pancreas cell type composition is crucial to the understanding of the genesis of diabetes. Current approaches use immunodetection of protein markers, for example, insulin as a marker of ß-cells. A major limitation of these methods is that protein content varies in physiological and pathological conditions, complicating the extrapolation to actual cell number. Here, we demonstrate the use of cell type-specific DNA methylation markers for determining the fraction of specific cell types in human islet and pancreas specimens. We identified genomic loci that are uniquely demethylated in specific pancreatic cell types and applied targeted PCR to assess the methylation status of these loci in tissue samples, enabling inference of cell type composition. In islet preparations, normalization of insulin secretion to ß-cell DNA revealed similar ß-cell function in pre-type 1 diabetes (T1D), T1D, and type 2 diabetes (T2D), which was significantly lower than in donors without diabetes. In histological pancreas specimens from recent-onset T1D, this assay showed ß-cell fraction within the normal range, suggesting a significant contribution of ß-cell dysfunction. In T2D pancreata, we observed increased α-cell fraction and normal ß-cell fraction. Methylation-based analysis provides an accurate molecular alternative to immune detection of cell types in the human pancreas, with utility in the interpretation of insulin secretion assays and the assessment of pancreas cell composition in health and disease.

Phenotypic Characterization of Congenital Hyperinsulinism Due to Novel Activating Glucokinase Mutations.

The importance of glucokinase (GK) in the regulation of insulin secretion has been highlighted by the phenotypes of individuals with activating and inactivating mutations in the glucokinase gene (GCK). Here we report 10 individuals with congenital hyperinsulinism (HI) caused by eight unique activating mutations of GCK. Six are novel and located near previously identified activating mutations sites. The first recognized episode of hypoglycemia in these patients occurred between birth and 24 years, and the severity of the phenotype was also variable. Mutant enzymes were expressed and purified for enzyme kinetics in vitro. Mutant enzymes had low glucose half-saturation concentration values and an increased enzyme activity index compared with wild-type GK. We performed functional evaluation of islets from the pancreata of three children with GCK-HI who required pancreatectomy. Basal insulin secretion in perifused GCK-HI islets was normal, and the response to glyburide was preserved. However, the threshold for glucose-stimulated insulin secretion in perifused glucokinase hyperinsulinism (GCK-HI) islets was decreased, and glucagon secretion was greatly suppressed. Our evaluation of novel GCK disease-associated mutations revealed that the detrimental effects of these mutations on glucose homeostasis can be attributed not only to a lowering of the glucose threshold of insulin secretion but also to a decreased counterregulatory glucagon secretory response. ARTICLE HIGHLIGHTS: Our evaluation of six novel and two previously published activating GCK mutations revealed that the detrimental effects of these mutations on glucose homeostasis can be attributed not only to a lowering of the glucose threshold of insulin secretion but also to a decreased counterregulatory glucagon secretory response. These studies provide insights into the pathophysiology of GCK-hyperinsulinism and the dual role of glucokinase in ß-cells and α-cells to regulate glucose homeostasis.

Acute frataxin knockdown in induced pluripotent stem cell-derived cardiomyocytes activates a type I interferon response.

Friedreich ataxia, the most common hereditary ataxia, is a neuro- and cardio-degenerative disorder caused, in most cases, by decreased expression of the mitochondrial protein frataxin. Cardiomyopathy is the leading cause of premature death. Frataxin functions in the biogenesis of iron-sulfur clusters, which are prosthetic groups that are found in proteins involved in many biological processes. To study the changes associated with decreased frataxin in human cardiomyocytes, we developed a novel isogenic model by acutely knocking down frataxin, post-differentiation, in cardiomyocytes derived from induced pluripotent stem cells (iPSCs). Transcriptome analysis of four biological replicates identified severe mitochondrial dysfunction and a type I interferon response as the pathways most affected by frataxin knockdown. We confirmed that, in iPSC-derived cardiomyocytes, loss of frataxin leads to mitochondrial dysfunction. The type I interferon response was activated in multiple cell types following acute frataxin knockdown and was caused, at least in part, by release of mitochondrial DNA into the cytosol, activating the cGAS-STING sensor pathway.

Genetic activation of glucokinase in a minority of pancreatic beta cells causes hypoglycemia in mice.

AIMS: Glucokinase (GK) is expressed in the glucose-sensing cells of the islets of Langerhans and plays a critical role in glucose homeostasis. Here, we tested the hypothesis that genetic activation of GK in a small subset of ß-cells is sufficient to change the glucose set-point of the whole islet. MATERIAL AND METHODS: Mouse models of cell-type specific GK deficiency (GKKO) and genetic enzyme activation (GKKI) in a subset of ß-cells were obtained by crossing the αGSU (gonadotropin alpha subunit)-Cre transgene with the appropriate GK mutant alleles. Metabolic analyses consisted of glucose tolerance tests, perifusion of isolated islets and intracellular calcium measurements. KEY FINDINGS: The αGSU-Cre transgene produced genetically mosaic islets, as Cre was active in 15 ± 1.2 % of ß-cells. While mice deficient for GK in a subset of islet cells were normal, unexpectedly, GKKI mice were chronically hypoglycemic, glucose intolerant, and had a lower threshold for glucose stimulated insulin secretion. GKKI mice exhibited an average fasting blood glucose level of 3.5 mM. GKKI islets responded with intracellular calcium signals that spread through the whole islets at 1 mM and secreted insulin at 3 mM glucose. SIGNIFICANCE: Genetic activation of GK in a minority of ß-cells is sufficient to change the glucose threshold for insulin secretion in the entire islet and thereby glucose homeostasis in the whole animal. These data support the model in which ß-cells with higher GK activity function as 'hub' or 'trigger' cells and thus control insulin secretion by the ß-cell collective within the islet.

Role and mechanism of REG2 depletion in insulin secretion augmented by glutathione peroxidase-1 overproduction.

We previously reported a depletion of murine regenerating islet-derived protein 2 (REG2) in pancreatic islets of glutathione peroxidase-1 (Gpx1) overexpressing (OE) mice. The present study was to explore if and how the REG2 depletion contributed to an augmented glucose stimulated insulin secretion (GSIS) in OE islets. After we verified a consistent depletion (90%, p < 0.05) of REG2 mRNA, transcript, and protein in OE islets compared with wild-type (WT) controls, we treated cultured and perifused OE islets (70 islets/sample) with REG2 (1 µg/ml or ml · min) and observed 30-40% (p < 0.05) inhibitions of GSIS by REG2. Subsequently, we obtained evidences of co-immunoprecipitation, cell surface ligand binding, and co-immunofluorescence for a ligand-receptor binding between REG2 and transmembrane, L-type voltage-dependent Ca2+ channel (CaV1.2) in beta TC3 cells. Mutating the C-type lectin binding domain of REG2 or deglycosylating CaV1.2 removed the inhibition of REG2 on GSIS and(or) the putative binding between the two proteins. Treating cultured OE and perifused WT islets with REG2 (1 µg/ml or ml · min) decreased (p < 0.05) Ca2+ influx triggered by glucose or KCl. An intraperitoneal (ip) injection of REG2 (2 µg/g) to OE mice (6-month old, n = 10) decreased their plasma insulin concentration (46%, p < 0.05) and elevated their plasma glucose concentration (25%, p < 0.05) over a 60 min period after glucose challenge (ip, 1 g/kg). In conclusion, our study identifies REG2 as a novel regulator of Ca2+ influx and insulin secretion, and reveals a new cascade of GPX1/REG2/CaV1.2 to explain how REG2 depletion in OE islets could decrease its binding to CaV1.2, resulting in uninhibited Ca2+ influx and augmented GSIS. These findings create new links to bridge redox biology, tissue regeneration, and insulin secretion.

α Cell dysfunction in islets from nondiabetic, glutamic acid decarboxylase autoantibody-positive individuals.

BACKGROUNDMultiple islet autoantibodies (AAbs) predict the development of type 1 diabetes (T1D) and hyperglycemia within 10 years. By contrast, T1D develops in only approximately 15% of individuals who are positive for single AAbs (generally against glutamic acid decarboxylase [GADA]); hence, the single GADA+ state may represent an early stage of T1D.METHODSHere, we functionally, histologically, and molecularly phenotyped human islets from nondiabetic GADA+ and T1D donors.RESULTSSimilar to the few remaining ß cells in the T1D islets, GADA+ donor islets demonstrated a preserved insulin secretory response. By contrast, α cell glucagon secretion was dysregulated in both GADA+ and T1D islets, with impaired glucose suppression of glucagon secretion. Single-cell RNA-Seq of GADA+ α cells revealed distinct abnormalities in glycolysis and oxidative phosphorylation pathways and a marked downregulation of cAMP-dependent protein kinase inhibitor ß (PKIB), providing a molecular basis for the loss of glucose suppression and the increased effect of 3-isobutyl-1-methylxanthine (IBMX) observed in GADA+ donor islets.CONCLUSIONWe found that α cell dysfunction was present during the early stages of islet autoimmunity at a time when ß cell mass was still normal, raising important questions about the role of early α cell dysfunction in the progression of T1D.FUNDINGThis work was supported by grants from the NIH (3UC4DK112217-01S1, U01DK123594-02, UC4DK112217, UC4DK112232, U01DK123716, and P30 DK019525) and the Vanderbilt Diabetes Research and Training Center (DK20593).

Gq signaling in α cells is critical for maintaining euglycemia.

Glucagon, a hormone released from pancreatic α cells, plays a key role in maintaining euglycemia. New insights into the signaling pathways that control glucagon secretion may stimulate the development of novel therapeutic agents. In this study, we investigated the potential regulation of α cell function by G proteins of the Gq family. The use of a chemogenetic strategy allowed us to selectively activate Gq signaling in mouse α cells in vitro and in vivo. Acute stimulation of α cell Gq signaling led to elevated plasma glucagon levels, accompanied by increased insulin release and improved glucose tolerance. Moreover, chronic activation of this pathway greatly improved glucose tolerance in obese mice. We also identified an endogenous Gq-coupled receptor (vasopressin 1b receptor; V1bR) that was enriched in mouse and human α cells. Agonist-induced activation of the V1bR strongly stimulated glucagon release in a Gq-dependent fashion. In vivo studies indicated that V1bR-mediated glucagon release played a key role in the counterregulatory hyperglucagonemia under hypoglycemic and glucopenic conditions. These data indicate that α cell Gq signaling represents an important regulator of glucagon secretion, resulting in multiple beneficial metabolic effects. Thus, drugs that target α cell-enriched Gq-coupled receptors may prove useful to restore euglycemia in various pathophysiological conditions.

Beta-cell M3 muscarinic acetylcholine receptors as potential targets for novel antidiabetic drugs.

A key feature of type 2 diabetes (T2D) is that beta-cells of the pancreatic islets fail to release sufficient amounts of insulin to overcome peripheral insulin resistance. Glucose-stimulated insulin secretion (GSIS) is regulated by the activity of numerous neurotransmitters, hormones and paracrine factors that act by stimulating specific G protein-coupled receptors expressed by pancreatic beta-cells. Studies with both mouse and human islets suggest that acetylcholine (ACh) acts on beta-cell M3 muscarinic receptors (M3Rs) to promote GSIS. In mouse islets, beta-cell M3Rs are thought to be activated by ACh released from parasympathetic nerve endings. Interestingly, studies with human pancreatic islets suggest that ACh is synthesized, stored and released by alpha-cells, which, in human pancreatic islets, are intermingled with beta-cells. Independent of the source of pancreatic islet ACh, recent studies indicate that beta-cell M3Rs represent a potential target for drugs capable of promoting insulin release for therapeutic purposes. In this review, we will provide an overview about signaling pathways and molecules that regulate the activity of beta-cell M3Rs. We will also discuss a novel pharmacological strategy to stimulate the activity of these receptors to reduce the metabolic impairments associated with T2D.

Allosteric modulation of ß-cell M3 muscarinic acetylcholine receptors greatly improves glucose homeostasis in lean and obese mice.

Given the global epidemic in type 2 diabetes, novel antidiabetic drugs with increased efficacy and reduced side effects are urgently needed. Previous work has shown that M3 muscarinic acetylcholine (ACh) receptors (M3Rs) expressed by pancreatic ß cells play key roles in stimulating insulin secretion and maintaining physiological blood glucose levels. In the present study, we tested the hypothesis that a positive allosteric modulator (PAM) of M3R function can improve glucose homeostasis in mice by promoting insulin release. One major advantage of this approach is that allosteric agents respect the ACh-dependent spatiotemporal control of M3R activity. In this study, we first demonstrated that VU0119498, a drug known to act as a PAM at M3Rs, significantly augmented ACh-induced insulin release from cultured ß cells and mouse and human pancreatic islets. This stimulatory effect was absent in islets prepared from mice lacking M3Rs, indicative of the involvement of M3Rs. VU0119498 treatment of wild-type mice caused a significant increase in plasma insulin levels, accompanied by a striking improvement in glucose tolerance. These effects were mediated by ß-cell M3Rs, since they were absent in mutant mice selectively lacking M3Rs in ß cells. Moreover, acute VU0119498 treatment of obese, glucose-intolerant mice triggered enhanced insulin release and restored normal glucose tolerance. Interestingly, doses of VU0119498 that led to pronounced improvements in glucose homeostasis did not cause any significant side effects due to activation of M3Rs expressed by other peripheral cell types. Taken together, the data from this proof-of-concept study strongly suggest that M3R PAMs may become clinically useful as novel antidiabetic agents.

Metabolic stress activates an ERK/hnRNPK/DDX3X pathway in pancreatic ß cells.

OBJECTIVE: Pancreatic ß cell failure plays a central role in the development of type 2 diabetes (T2D). While the transcription factors shaping the ß cell gene expression program have received much attention, the post-transcriptional controls that are activated in ß cells during stress are largely unknown. We recently identified JUND as a pro-oxidant transcription factor that is post-transcriptionally upregulated in ß cells during metabolic stress. Here we seek to uncover the mechanisms underlying this maladaptive response to metabolic stress. METHODS: RNA-protein and protein-protein interactions were measured using RNA immunoprecipitation and co-immunoprecipitation, respectively, in Min6 cells and mouse islets. Phos-tag analyses were used to assess hnRNPK phosphorylation in primary mouse and human islets and Min6 cells. Translating ribosome affinity purification (TRAP) followed by RT-qPCR was used to identify changes in the ribosome occupancy of mRNAs in Min6 cells. Gene depletion studies used lentiviral delivery of CRISPR-Cas9 to Min6 cells. Apoptosis was measured in primary islets using a cell-permeable dye with a fluorescence readout of activated cleaved caspase-3 and-7. RESULTS: A de novo motif analysis was performed on a subset of genes previously found to be regulated at the level of ribosome binding during PDX1-deficiency, which identified a poly-cytosine (polyC) motif in the 3'UTR of the transcript encoding JUND. The polyC-binding protein hnRNPK bound to the mRNA encoding JUND, leading us to hypothesize that hnRNPK regulates JUND expression during glucolipotoxicity. Indeed, loss of hnRNPK blocked the post-transcriptional upregulation of JUND during metabolic stress. hnRNPK was phosphorylated in mouse and human islets during glucolipotoxicity and in islets of diabetic db/db mice. The MEK/ERK signaling pathway was both necessary and sufficient for the phosphorylation of hnRNPK, upregulation of JUND levels, and induction of pro-oxidant and pro-inflammatory genes. Further, we identified the RNA helicase DDX3X as a new binding partner for hnRNPK that is required for efficient translation of JUND mRNA. Loss of hnRNPK reduced DDX3X binding to translation machinery, suggesting that these factors cooperate to regulate translation in ß cells. CONCLUSIONS: Our results identify a novel ERK/hnRNPK/DDX3X pathway that influences ß cell survival and is activated under conditions associated with T2D.

JUND regulates pancreatic ß cell survival during metabolic stress.

OBJECTIVE: In type 2 diabetes (T2D), oxidative stress contributes to the dysfunction and loss of pancreatic ß cells. A highly conserved feature of the cellular response to stress is the regulation of mRNA translation; however, the genes regulated at the level of translation are often overlooked due to the convenience of RNA sequencing technologies. Our goal is to investigate translational regulation in ß cells as a means to uncover novel factors and pathways pertinent to cellular adaptation and survival during T2D-associated conditions. METHODS: Translating ribosome affinity purification (TRAP) followed by RNA-seq or RT-qPCR was used to identify changes in the ribosome occupancy of mRNAs in Min6 cells. Gene depletion studies used lentiviral delivery of shRNAs to primary mouse islets or CRISPR-Cas9 to Min6 cells. Oxidative stress and apoptosis were measured in primary islets using cell-permeable dyes with fluorescence readouts of oxidation and activated cleaved caspase-3 and-7, respectively. Gene expression was assessed by RNA-seq, RT-qPCR, and western blot. ChIP-qPCR was used to determine chromatin enrichment. RESULTS: TRAP-seq in a PDX1-deficiency model of ß cell dysfunction uncovered a cohort of genes regulated at the level of mRNA translation, including the transcription factor JUND. Using a panel of diabetes-associated stressors, JUND was found to be upregulated in mouse islets cultured with high concentrations of glucose and free fatty acid, but not after treatment with hydrogen peroxide or thapsigargin. This induction of JUND could be attributed to increased mRNA translation. JUND was also upregulated in islets from diabetic db/db mice and in human islets treated with high glucose and free fatty acid. Depletion of JUND in primary islets reduced oxidative stress and apoptosis in ß cells during metabolic stress. Transcriptome assessment identified a cohort of genes, including pro-oxidant and pro-inflammatory genes, regulated by JUND that are commonly dysregulated in models of ß cell dysfunction, consistent with a maladaptive role for JUND in islets. CONCLUSIONS: A translation-centric approach uncovered JUND as a stress-responsive factor in ß cells that contributes to redox imbalance and apoptosis during pathophysiologically relevant stress.

Intra-islet glucagon signaling is critical for maintaining glucose homeostasis.

Glucagon, a hormone released from pancreatic alpha-cells, plays a key role in maintaining proper glucose homeostasis and has been implicated in the pathophysiology of diabetes. In vitro studies suggest that intra-islet glucagon can modulate the function of pancreatic beta-cells. However, because of the lack of suitable experimental tools, the in vivo physiological role of this intra-islet cross-talk has remained elusive. To address this issue, we generated a novel mouse model that selectively expressed an inhibitory designer G protein-coupled receptor (Gi DREADD) in α-cells only. Drug-induced activation of this inhibitory designer receptor almost completely shut off glucagon secretion in vivo, resulting in significantly impaired insulin secretion, hyperglycemia, and glucose intolerance. Additional studies with mouse and human islets indicated that intra-islet glucagon stimulates insulin release primarily by activating ß-cell GLP-1 receptors. These new findings strongly suggest that intra-islet glucagon signaling is essential for maintaining proper glucose homeostasis in vivo. Our work may pave the way toward the development of novel classes of antidiabetic drugs that act by modulating intra-islet cross-talk between α- and ß-cells.

The Role of Sodium in Diabetic Cardiomyopathy.

Cardiovascular complications are the major cause of mortality and morbidity in diabetic patients. The changes in myocardial structure and function associated with diabetes are collectively called diabetic cardiomyopathy. Numerous molecular mechanisms have been proposed that could contribute to the development of diabetic cardiomyopathy and have been studied in various animal models of type 1 or type 2 diabetes. The current review focuses on the role of sodium (Na+) in diabetic cardiomyopathy and provides unique data on the linkage between Na+ flux and energy metabolism, studied with non-invasive 23Na, and 31P-NMR spectroscopy, polarography, and mass spectroscopy. 23Na NMR studies allow determination of the intracellular and extracellular Na+ pools by splitting the total Na+ peak into two resonances after the addition of a shift reagent to the perfusate. Using this technology, we found that intracellular Na+ is approximately two times higher in diabetic cardiomyocytes than in control possibly due to combined changes in the activity of Na+-K+ pump, Na+/H+ exchanger 1 (NHE1) and Na+-glucose cotransporter. We hypothesized that the increase in Na+ activates the mitochondrial membrane Na+/Ca2+ exchanger, which leads to a loss of intramitochondrial Ca2+, with a subsequent alteration in mitochondrial bioenergetics and function. Using isolated mitochondria, we showed that the addition of Na+ (1-10 mM) led to a dose-dependent decrease in oxidative phosphorylation and that this effect was reversed by providing extramitochondrial Ca2+ or by inhibiting the mitochondrial Na+/Ca2+ exchanger with diltiazem. Similar experiments with 31P-NMR in isolated superfused mitochondria embedded in agarose beads showed that Na+ (3-30 mM) led to significantly decreased ATP levels and that this effect was stronger in diabetic rats. These data suggest that in diabetic cardiomyocytes, increased Na+ leads to abnormalities in oxidative phosphorylation and a subsequent decrease in ATP levels. In support of these data, using 31P-NMR, we showed that the baseline ß-ATP and phosphocreatine (PCr) were lower in diabetic cardiomyocytes than in control, suggesting that diabetic cardiomyocytes have depressed bioenergetic function. Thus, both altered intracellular Na+ levels and bioenergetics and their interactions may significantly contribute to the pathology of diabetic cardiomyopathy.

Sex- and Dose-Specific Effects of Maternal Bisphenol A Exposure on Pancreatic Islets of First- and Second-Generation Adult Mice Offspring.

BACKGROUND: Exposure to the environmental endocrine disruptor bisphenol A (BPA) is ubiquitous and associated with the increased risk of diabetes and obesity. However, the underlying mechanisms remain unknown. We recently demonstrated that perinatal BPA exposure is associated with higher body fat, impaired glucose tolerance, and reduced insulin secretion in first- (F1) and second-generation (F2) C57BL/6J male mice offspring. OBJECTIVE: We sought to determine the multigenerational effects of maternal bisphenol A exposure on mouse pancreatic islets. METHODS: Cellular and molecular mechanisms underlying these persistent changes were determined in F1 and F2 adult offspring of F0 mothers exposed to two relevant human exposure levels of BPA (10µg/kg/d-LowerB and 10mg/kg/d-UpperB). RESULTS: Both doses of BPA significantly impaired insulin secretion in male but not female F1 and F2 offspring. Surprisingly, LowerB and UpperB induced islet inflammation in male F1 offspring that persisted into the next generation. We also observed dose-specific effects of BPA on islets in males. UpperB exposure impaired mitochondrial function, whereas LowerB exposure significantly reduced ß-cell mass and increased ß-cell death that persisted in the F2 generation. Transcriptome analyses supported these physiologic findings and there were significant dose-specific changes in the expression of genes regulating inflammation and mitochondrial function. Previously we observed increased expression of the critically important ß-cell gene, Igf2 in whole F1 embryos. Surprisingly, increased Igf2 expression persisted in the islets of male F1 and F2 offspring and was associated with altered DNA methylation. CONCLUSION: These findings demonstrate that maternal BPA exposure has dose- and sex-specific effects on pancreatic islets of adult F1 and F2 mice offspring. The transmission of these changes across multiple generations may involve either mitochondrial dysfunction and/or epigenetic modifications. https://doi.org/10.1289/EHP1674.

Inhibition of cholinergic potentiation of insulin secretion from pancreatic islets by chronic elevation of glucose and fatty acids: Protection by casein kinase 2 inhibitor.

OBJECTIVES: Chronic hyperlipidemia and hyperglycemia are characteristic features of type 2 diabetes (T2DM) that are thought to cause or contribute to ß-cell dysfunction by "glucolipotoxicity." Previously we have shown that acute treatment of pancreatic islets with fatty acids (FA) decreases acetylcholine-potentiated insulin secretion. This acetylcholine response is mediated by M3 muscarinic receptors, which play a key role in regulating ß-cell function. Here we examine whether chronic FA exposure also inhibits acetylcholine-potentiated insulin secretion using mouse and human islets. METHODS: Islets were cultured for 3 or 4 days at different glucose concentration with 0.5 mM palmitic acid (PA) or a 2:1 mixture of PA and oleic acid (OA) at 1% albumin (PA/BSA molar ratio 3.3). Afterwards, the response to glucose and acetylcholine were studied in perifusion experiments. RESULTS: FA-induced impairment of insulin secretion and Ca2+ signaling depended strongly on the glucose concentrations of the culture medium. PA and OA in combination reduced acetylcholine potentiation of insulin secretion more than PA alone, both in mouse and human islets, with no evidence of a protective role of OA. In contrast, lipotoxicity was not observed with islets cultured for 3 days in medium containing less than 1 mM glucose and a mixture of glutamine and leucine (7 mM each). High glucose and FAs reduced endoplasmic reticulum (ER) Ca2+ storage capacity; however, preserving ER Ca2+ by blocking the IP3 receptor with xestospongin C did not protect islets from glucolipotoxic effects on insulin secretion. In contrast, an inhibitor of casein kinase 2 (CK2) protected the glucose dependent acetylcholine potentiation of insulin secretion in mouse and human islets against glucolipotoxicity. CONCLUSIONS: These results show that chronic FA treatment decreases acetylcholine potentiation of insulin secretion and that this effect is strictly glucose dependent and might involve CK2 phosphorylation of ß-cell M3 muscarinic receptors.

Ndfip1 restricts mTORC1 signalling and glycolysis in regulatory T cells to prevent autoinflammatory disease.

Foxp3+ T regulatory (Treg) cells suppress immune cell activation and establish normal immune homeostasis. How Treg cells maintain their identity is not completely understood. Here we show that Ndfip1, a coactivator of Nedd4-family E3 ubiquitin ligases, is required for Treg cell stability and function. Ndfip1 deletion in Treg cells results in autoinflammatory disease. Ndfip1-deficient Treg cells are highly proliferative and are more likely to lose Foxp3 expression to become IL-4-producing TH2 effector cells. Proteomic analyses indicate altered metabolic signature of Ndfip1-deficient Treg cells and metabolic profiling reveals elevated glycolysis and increased mTORC1 signalling. Ndfip1 restricts Treg cell metabolism and IL-4 production via distinct mechanisms, as IL-4 deficiency does not prevent hyperproliferation or elevated mTORC1 signalling in Ndfip1-deficient Treg cells. Thus, Ndfip1 preserves Treg lineage stability and immune homeostasis by preventing the expansion of highly proliferative and metabolically active Treg cells and by preventing pathological secretion of IL-4 from Treg cells.

Functional and Metabolomic Consequences of KATP Channel Inactivation in Human Islets.

Loss-of-function mutations of ß-cell KATP channels cause the most severe form of congenital hyperinsulinism (KATPHI). KATPHI is characterized by fasting and protein-induced hypoglycemia that is unresponsive to medical therapy. For a better understanding of the pathophysiology of KATPHI, we examined cytosolic calcium ([Ca2+] i ), insulin secretion, oxygen consumption, and [U-13C]glucose metabolism in islets isolated from the pancreases of children with KATPHI who required pancreatectomy. Basal [Ca2+] i and insulin secretion were higher in KATPHI islets compared with controls. Unlike controls, insulin secretion in KATPHI islets increased in response to amino acids but not to glucose. KATPHI islets have an increased basal rate of oxygen consumption and mitochondrial mass. [U-13C]glucose metabolism showed a twofold increase in alanine levels and sixfold increase in 13C enrichment of alanine in KATPHI islets, suggesting increased rates of glycolysis. KATPHI islets also exhibited increased serine/glycine and glutamine biosynthesis. In contrast, KATPHI islets had low γ-aminobutyric acid (GABA) levels and lacked 13C incorporation into GABA in response to glucose stimulation. The expression of key genes involved in these metabolic pathways was significantly different in KATPHI ß-cells compared with control, providing a mechanism for the observed changes. These findings demonstrate that the pathophysiology of KATPHI is complex, and they provide a framework for the identification of new potential therapeutic targets for this devastating condition.

ß-arrestin-2 is an essential regulator of pancreatic ß-cell function under physiological and pathophysiological conditions.

ß-arrestins are critical signalling molecules that regulate many fundamental physiological functions including the maintenance of euglycemia and peripheral insulin sensitivity. Here we show that inactivation of the ß-arrestin-2 gene, barr2, in ß-cells of adult mice greatly impairs insulin release and glucose tolerance in mice fed with a calorie-rich diet. Both glucose and KCl-induced insulin secretion and calcium responses were profoundly reduced in ß-arrestin-2 (barr2) deficient ß-cells. In human ß-cells, barr2 knockdown abolished glucose-induced insulin secretion. We also show that the presence of barr2 is essential for proper CAMKII function in ß-cells. Importantly, overexpression of barr2 in ß-cells greatly ameliorates the metabolic deficits displayed by mice consuming a high-fat diet. Thus, our data identify barr2 as an important regulator of ß-cell function, which may serve as a new target to improve ß-cell function.

CK2 acts as a potent negative regulator of receptor-mediated insulin release in vitro and in vivo.

G protein-coupled receptors (GPCRs) regulate virtually all physiological functions including the release of insulin from pancreatic ß-cells. ß-Cell M3 muscarinic receptors (M3Rs) are known to play an essential role in facilitating insulin release and maintaining proper whole-body glucose homeostasis. As is the case with other GPCRs, M3R activity is regulated by phosphorylation by various kinases, including GPCR kinases and casein kinase 2 (CK2). At present, it remains unknown which of these various kinases are physiologically relevant for the regulation of ß-cell activity. In the present study, we demonstrate that inhibition of CK2 in pancreatic ß-cells, knockdown of CK2α expression, or genetic deletion of CK2α in ß-cells of mutant mice selectively augmented M3R-stimulated insulin release in vitro and in vivo. In vitro studies showed that this effect was associated with an M3R-mediated increase in intracellular calcium levels. Treatment of mouse pancreatic islets with CX4945, a highly selective CK2 inhibitor, greatly reduced agonist-induced phosphorylation of ß-cell M3Rs, indicative of CK2-mediated M3R phosphorylation. We also showed that inhibition of CK2 greatly enhanced M3R-stimulated insulin secretion in human islets. Finally, CX4945 treatment protected mice against diet-induced hyperglycemia and glucose intolerance in an M3R-dependent fashion. Our data demonstrate, for the first time to our knowledge, the physiological relevance of CK2 phosphorylation of a GPCR and suggest the novel concept that kinases acting on ß-cell GPCRs may represent novel therapeutic targets.

AMP promotes oxygen consumption and ATP synthesis in heart mitochondria through the adenylate kinase reaction: an NMR spectroscopy and polarography study.

Adenylate kinase plays an important role in cellular energy homeostasis by catalysing the interconversion of adenine nucleotides. The goal of present study was to evaluate the contribution of the adenylate kinase reaction to oxidative ATP synthesis by direct measurements of ATP using (31) P NMR spectroscopy. Results show that AMP can stimulate ATP synthesis in the presence or absence of ADP. In particular, addition of 1 mM AMP to the 0.6 mM ADP superfusion system of isolated superfused mitochondria (contained and maintained in agarose beads) led to a 25% increase in ATP synthesis as measured by the increase in ßATP signal. More importantly, we show that AMP can support ATP synthesis in the absence of ADP, demonstrated as follows. Superfusion of mitochondria without ADP led to the disappearance of ATP γ, α and ß signals and the increase of Pi . Addition of AMP to the medium restored the production of ATP, as demonstrated by the reappearance of γ, α and ß ATP signals, in conjunction with a decrease in Pi , which is being used for ATP synthesis. Polarographic studies showed Mg(2+) dependence of this process, confirming the specificity of the adenylate kinase reaction. Furthermore, data obtained from this study demonstrate, for the first time, that different aspects of the adenylate kinase reaction can be evaluated with (31) P NMR spectroscopy. SIGNIFICANCE OF RESEARCH PARAGRAPH: The data generated in the present study indicate that (31) P NMR spectroscopy can effectively be used to study the adenylate kinase reaction under a variety of conditions. This is important because understanding of adenylate kinase function and/or malfunction is essential to understanding its role in health and disease. The data obtained with (31) P NMR were confirmed by polarographic studies, which further strengthens the robustness of the NMR findings. In summary, (31) P NMR spectroscopy provides a sensitive tool to study adenylate kinase activity in different physiological and pathophysiological conditions, including but not exclusive of, cancer, ischemic injury, hemolytic anemia and neurological problems such as sensorineural deafness.