Search

Communication between the plasma membrane and mitochondria is essential for initiating the Ca(2+) and metabolic signals required for secretion in β cells. Although voltage-dependent Na(+) channels are abundantly expressed in β cells and activated by glucose, their role in communicating with mitochondria is unresolved. Here, we combined fluorescent Na(+), Ca(2+), and ATP imaging, electrophysiological analysis with tetrodotoxin (TTX)-dependent block of the Na(+) channel, and molecular manipulation of mitochondrial Ca(2+) transporters to study the communication between Na(+) channels and mitochondria. We show that TTX inhibits glucose-dependent depolarization and blocks cytosolic Na(+) and Ca(2+) responses and their propagation into mitochondria. TTX-sensitive mitochondrial Ca(2+) influx was largely blocked by knockdown of the mitochondrial Ca(2+) uniporter (MCU) expression. Knockdown of the mitochondrial Na(+)/Ca(2+) exchanger (NCLX) and Na(+) dose response analysis demonstrated that NCLX mediates the mitochondrial Na(+) influx and is tuned to sense the TTX-sensitive cytosolic Na(+) responses. Finally, TTX blocked glucose-dependent mitochondrial Ca(2+) rise, mitochondrial metabolic activity, and ATP production. Our results show that communication of the Na(+) channels with mitochondria shape both global Ca(2+) and metabolism signals linked to insulin secretion in β cells.- Nita, I. I., Hershfinkel, M., Kantor, C., Rutter, G. A., Lewis, E. C., Sekler, I. Pancreatic β-cell Na(+) channels control global Ca(2+) signaling and oxidative metabolism by inducing Na(+) and Ca(2+) responses that are propagated into mitochondria.

We and others have previously identified a group of genes, dubbed “disallowed,” whose expression is markedly lower in pancreatic islets than in other mammalian cell types. Forced mis-expression of several members of this family leads to defective insulin secretion, demonstrating the likely importance of disallowance for normal beta cell function. Up to now, transcriptomic comparisons have been based solely on data from whole islets. This raises the possibilities that (a) there may be important differences in the degree of disallowance of family members between beta and other either neuroendocrine cells; (b) beta (or alpha) cell disallowed genes may have gone undetected. To address this issue, we survey here recent massive parallel sequencing (RNA-Seq) datasets from purified mouse and human islet cells. Our analysis reveals that the most strongly disallowed genes are similar in beta and alpha cells, with 11β-hydroxysteroid dehydrogenase (Hsd11b1) mRNA being essentially undetectable in both cell types. The analysis also reveals that several genes involved in cellular proliferation, including Yap1 and Igfbp4, and previously assumed to be disallowed in both beta and alpha cells, are selectively repressed only in the beta cell. The latter finding supports the view that beta cell growth is selectively restricted in adults, providing a mechanism to avoid excessive insulin production and the risk of hypoglycaemia. Approaches which increase the expression or activity of selected disallowed genes in the beta cell may provide the basis for novel regenerative therapies in type 2 diabetes.

The glucagon-like peptide-1 receptor (GLP-1R), a key pharmacological target in type 2 diabetes (T2D) and obesity, undergoes rapid endocytosis after stimulation by endogenous and therapeutic agonists. We have previously highlighted the relevance of this process in fine-tuning GLP-1R responses in pancreatic beta cells to control insulin secretion. In the present study, we demonstrate an important role for the translocation of active GLP-1Rs into liquid-ordered plasma membrane nanodomains, which act as hotspots for optimal coordination of intracellular signaling and clathrin-mediated endocytosis. This process is dynamically regulated by agonist binding through palmitoylation of the GLP-1R at its carboxyl-terminal tail. Biased GLP-1R agonists and small molecule allosteric modulation both influence GLP-1R palmitoylation, clustering, nanodomain signaling, and internalization. Downstream effects on insulin secretion from pancreatic beta cells indicate that these processes are relevant to GLP-1R physiological actions and might be therapeutically targetable., Nanodomain segregation and clustering of the glucagon-like peptide-1 receptor, a key target for type 2 diabetes therapy, is regulated by agonist binding, leading to compartmentalization of downstream signaling and clathrin-dependent internalization and impacting pancreatic beta cell responses.

Progressive age-dependent replacement of canonical H3.1/2 with H3.3 in post-mitotic cells is a crucial mechanism for maintaining genome integrity at heterochromatic sites. The histone chaperone DAXX cooperates with the chromatin remodeller ATRX for the deposition of the non-canonical histone H3.3 at heterochromatin, while it prevents the expression of endogenous retroviral elements in another complex involving the SETDB1 methyltransferases. Loss of function mutations affecting ATRX/DAXX and MEN-1 have been recently identified as a subgroup of pancreatic neuroendocrine tumours (PanNETs) leading to poor prognosis and more aggressive phenotype compared to the ATRX/DAXX and MEN-1 wild-type counterparts. We developed a mouse model for conditional Daxx inactivation in mouse islets to understand the impact of DAXX loss on pancreas homeostasis and PanNETs development. We found that loss of Daxx in the endocrine pancreas, using the Insulin-1Cre driver, leads to aberrant glucose metabolism with the development of hyperglycemia and consequently insulin resistance in mice. We have also shown that loss of DAXX is linked to increased expression of α-cell lineage genes upon glucose stimulation of isolated islets in vitro, resembling the recently reported phenotype of PanNETs harboring ATRX/DAXX or MEN1 mutations. Nonetheless, loss of Daxx was not sufficient to lead to PanNET development. We are currently investigating the chromatin and transcriptional perturbations caused by Daxx loss and how these could be linked to the aberration of cell lineage specification and tissue dysfunction in the pancreas. More generally, this work will provide new insight into how epigenetic alterations contribute to misregulation of developmental programmes and cancer development in the pancreas. Citation Format: Teresa Sposito, Marie-Sophie Nguyen Tu, Guy A. Rutter, Marco Novelli, Chrissie Thirlwell, Paolo Salomoni. The PanNET-related histone H3.3 chaperone Daxx regulates lineage specification and tissue homeostasis in the pancreas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5294.

The higher organization of β-cells into spheroid structures termed islets of Langerhans is critical for the proper regulation of insulin secretion. Thus, rodent β-cells form a functional syncytium that integrates and propagates information encoded by secretagogues, producing a “gain-of-function” in hormone release through the generation of coordinated cell-cell activity. By contrast, human islets possess divergent topology, and this may have repercussions for the cell-cell communication pathways that mediate the population dynamics underlying the intraislet regulation of insulin secretion. This is pertinent for type 2 diabetes mellitus pathogenesis, and its study in rodent models, because environmental and genetic factors may converge on these processes in a species-specific manner to precipitate the defective insulin secretion associated with glucose intolerance. The aim of the present minireview is therefore to discuss the structural and functional underpinnings that influence insulin secretion from human islets, and the possibility that dyscoordination between individual β-cells may play an important role in some forms of type 2 diabetes mellitus.

Genome-wide association studies (GWAS) have revealed multiple loci associated with increased type 2 diabetes (T2D) risk. Genes in the affected loci are likely therefore to play a role in disease aetiology, and may ultimately provide targets for intervention. The majority of identified polymorphisms alter insulin production, and often lie between or within several known genes. Determining which gene(s) contribute(s) to disease risk is likely to require complementary human, animal and cellular studies. Here I compare existing models for two of the most-studied GWAS-identified T2D risk genes, Tcf7l2 and ZnT8 ( SLC30A8 ) and discuss prospects for examining other variants.

The progression of Type 2 diabetes is accompanied by diminishing islet β-cell mass and function. It has been proposed that β-cells are lost not only through apoptosis, but also by dedifferentiating into progenitor-like cells. There is therefore much interest in the mechanisms which define and maintain β-cell identity. The advent of genome-wide analyses of chromatin modifications has highlighted the role of epigenetic factors in determining cell identity. There is also evidence from both human populations and animal models for an epigenetic component in susceptibility to Type 2 diabetes. The mechanisms responsible for defining the epigenetic landscape in individual cell types are poorly understood, but there is growing evidence of a role for lncRNAs (long non-coding RNAs) in this process. In the present paper, we discuss some of the mechanisms through which lncRNAs may contribute to β-cell identity and Type 2 diabetes risk.

Polycomb-mediated gene repression is essential for embryonic development, yet its precise role in lineage-specific programming is poorly understood. Here we inactivated Ring1b, encoding a polycomb-repressive complex 1 subunit, in pancreatic multipotent progenitors (Ring1bprogKO). This caused transcriptional derepression of a subset of direct Ring1b target genes in differentiated pancreatic islet cells. Unexpectedly, Ring1b inactivation in differentiated islet β cells (Ring1bβKO) did not cause derepression, even after multiple rounds of cell division, suggesting a role for Ring1b in the establishment but not the maintenance of repression. Consistent with this notion, derepression in Ring1bprogKO islets occurred preferentially in genes that were targeted de novo by Ring1b during pancreas development. The results support a model in which Ring1b bookmarks its target genes during embryonic development, and these genes are maintained in a repressed state through Ring1b-independent mechanisms in terminally differentiated cells. This work provides novel insights into how epigenetic mechanisms contribute to shaping the transcriptional identity of differentiated lineages. © 2013 by Cold Spring Harbor Laboratory Press., Ministerio de Ciencia e Innovacion (SAF2011-27086 to J.F., and BFU2010-18146 to M.V.), Fundacion Ramon Areces to M.V.), and the Biology of Liver and Pancreatic Development and Disease Marie Curie Training Program (to M.C. and J.F.).

Excessive glucocorticoid exposure has been shown to be deleterious for pancreatic β-cell function and insulin release. However, glucocorticoids at physiological levels are essential for many homeostatic processes, including glycemic control. We show that corticosterone and cortisol and their less active precursors 11-dehydrocorticosterone (11-DHC) and cortisone suppress voltage-dependent Ca2+ channel function and Ca2+ fluxes in rodent as well as in human β-cells. However, insulin secretion, maximal ATP/ADP responses to glucose, and β-cell identity were all unaffected. Further examination revealed the upregulation of parallel amplifying cAMP signals and an increase in the number of membrane-docked insulin secretory granules. Effects of 11-DHC could be prevented by lipotoxicity and were associated with paracrine regulation of glucocorticoid activity because global deletion of 11β-hydroxysteroid dehydrogenase type 1 normalized Ca2+ and cAMP responses. Thus, we have identified an enzymatically amplified feedback loop whereby glucocorticoids boost cAMP to maintain insulin secretion in the face of perturbed ionic signals. Failure of this protective mechanism may contribute to diabetes in states of glucocorticoid excess, such as Cushing syndrome, which are associated with frank dyslipidemia.

Changes in cellular free Zn

Adenosine 5' monophosphate-activated protein kinase (AMPK) is a regulator of cellular and systemic energy homeostasis. It mediates some of the effects of peripheral hormones such as leptin, ghrelin and adiponectin, and it is involved in the insulin-sensitizing role of the antidiabetic drug metformin. There is increasing evidence that AMPK has a central role in mediating the appetite-modulating and metabolic effects of many other hormones and substances, including the cannabinoids. Recent studies have illustrated the interaction between hormones and AMPK, and highlighted AMPK as a potential target for the development of tissue-specific AMPK modulators in the treatment of obesity and the metabolic syndrome.

Increased levels of the second messenger lipid diacylglycerol (DAG) induce downstream signaling events including the translocation of C1-domain-containing proteins toward the plasma membrane. Here, we introduce three light-sensitive DAGs, termed PhoDAGs, which feature a photoswitchable acyl chain. The PhoDAGs are inactive in the dark and promote the translocation of proteins that feature C1 domains toward the plasma membrane upon a flash of UV-A light. This effect is quickly reversed after the termination of photostimulation or by irradiation with blue light, permitting the generation of oscillation patterns. Both protein kinase C and Munc13 can thus be put under optical control. PhoDAGs control vesicle release in excitable cells, such as mouse pancreatic islets and hippocampal neurons, and modulate synaptic transmission in Caenorhabditis elegans. As such, the PhoDAGs afford an unprecedented degree of spatiotemporal control and are broadly applicable tools to study DAG signaling.

The 2-oxoglutarate dehydrogenase (OGDH) complex is an important control point in vertebrate mitochondrial oxidative metabolism, including in the citrate cycle and catabolism of alternative fuels including glutamine. It is subject to allosteric regulation by NADH and the ATP/ADP ratio, and by Ca2+ through binding to the E1 subunit. The latter involves a unique Ca2+-binding site which includes D114ADLD (site 1). Here, we describe three splice variants of E1 in which either the exon expressing this site is replaced with another exon (loss of site 1, LS1) or an additional exon is expressed leading to the insertion of 15 amino acids just downstream of site 1 (Insert), or both changes occur together (LS1/Insert). We show that all three variants are essentially Ca2+-insensitive. Comparison of massive parallel sequence (RNA-Seq) databases demonstrates predominant expression of the Ca2+-sensitive archetype form in heart and skeletal muscle, but substantial expression of the Ca2+-insensitive variants in brain, pancreatic islets and other tissues. Detailed proteomic and activity studies comparing OGDH complexes from rat heart and brain confirmed the substantial difference in expression between these tissues. The evolution of OGDH variants was explored using bioinformatics, and this indicated that Ca2+-sensitivity arose with the emergence of chordates. In all species examined, this was associated with the co-emergence of Ca2+-insensitive variants suggesting a retained requirement for the latter in some settings. Tissue-specific expression of OGDH splice variants may thus provide a mechanism that tunes the control of the enzyme to the specialized metabolic and signalling needs of individual cell types.

Encoding acyl-CoA thioesterase-7 (Acot7) is one of ∼60 genes expressed ubiquitously across tissues but relatively silenced, or disallowed, in pancreatic β-cells. The capacity of ACOT7 to hydrolyze long-chain acyl-CoA esters suggests potential roles in β-oxidation, lipid biosynthesis, signal transduction, or insulin exocytosis. We explored the physiological relevance of β-cell–specific Acot7 silencing by re-expressing ACOT7 in these cells. ACOT7 overexpression in clonal MIN6 and INS1(832/13) β-cells impaired insulin secretion in response to glucose plus fatty acids. Furthermore, in a panel of transgenic mouse lines, we demonstrate that overexpression of mitochondrial ACOT7 selectively in the adult β-cell reduces glucose tolerance dose dependently and impairs glucose-stimulated insulin secretion. By contrast, depolarization-induced secretion was unaffected, arguing against a direct action on the exocytotic machinery. Acyl-CoA levels, ATP/ADP increases, membrane depolarization, and Ca2+ fluxes were all markedly reduced in transgenic mouse islets, whereas glucose-induced oxygen consumption was unchanged. Although glucose-induced increases in ATP/ADP ratio were similarly lowered after ACOT7 overexpression in INS1(832/13) cells, changes in mitochondrial membrane potential were unaffected, consistent with an action of Acot7 to increase cellular ATP consumption. Because Acot7 mRNA levels are increased in human islets in type 2 diabetes, inhibition of the enzyme might provide a novel therapeutic strategy.

Zinc (as Zn2+), increasingly recognized as an important trace element, has physiological role in mammalian cell function. Resting intracellular free Zn2+ level ([Zn2+]i) can be greatly increased by thiol-reactive oxidants or high glucose that contributes to oxidant-induced alterations in EC-coupling of cardiomyocytes. Since Zn2+ is important in cellular physiology, very little is known how [Zn2+]i is controlled and stored intracellularly in cardiomyocytes. Therefore, in the present study we aimed to investigate the role of ZIP7 on regulation of [Zn2+]i in cardiomycoytes. We first used newly-developed genetically-encoded FRET-based sensors to monitor cytosolic (CYS), mitochondrial (MitC) and endoplasmic reticulum (ER) [Zn2+]. Our data showed that [Zn2+] levels in these compartments were about 0.5, 5.0 and 0.2 nM, respectively in isolated left ventricular cardiomyocytes. Since expression but not location of ZIP7 has been shown in the heart tissue, inhere, we tested whether Zn2+ transport from ER into cytosol by a transporter ZIP7 thereby responsible for cytosolic/S(E)R Zn2+ homeostasis in cardiomyocytes under pathological condition. Our data have shown that both protein level and expression of ZIP7 increased in either cardiomyocytes isolated from streptozotocin-induced diabetic rat heart or high glucose (25mM; 24-hours) incubated H9c2 cells. Our immunofluorescence analysis also demonstrated that ZIP7 is localised in ER but not in Golgi apparatus in these cells. Furthermore, ZIP7 knocked-down in cardiomycoytes induced a significant reduction of cytosolic [Zn2+]. All experimental data showed that ER is a Zn2+ pool and ZIP7 has a crucial role in regulation of cytosolic Zn2+ in mammalian cardiomyocytes (Supported by TUBITAK SBAG-113S466, COST Action TD1304, EFSD Fellowships).

Zn(2+) ions are essential for the normal processing and storage of insulin and altered pancreatic insulin content is associated with all forms of diabetes mellitus. Work of the past decade has identified variants in the human SLC30A8 gene, encoding the zinc transporter ZnT8 which is expressed highly selectively on the secretory granule of pancreatic islet β and α cells, as affecting the risk of Type 2 Diabetes. Here, we review the regulation and roles of Zn(2+) ions in islet cells, the mechanisms through which SLC30A8 variants might affect glucose homeostasis and diabetes risk, and the novel technologies including recombinant targeted zinc probes and knockout mice which have been developed to explore these questions.

Allosteric regulation promises to open up new therapeutic avenues by increasing drug specificity at G‐protein‐coupled receptors (GPCRs). However, drug discovery efforts are at present hampered by an inability to precisely control the allosteric site. Herein, we describe the design, synthesis, and testing of PhotoETP, a light‐activated positive allosteric modulator of the glucagon‐like peptide‐1 receptor (GLP‐1R), a class B GPCR involved in the maintenance of glucose homeostasis in humans. PhotoETP potentiates Ca2+, cAMP, and insulin responses to glucagon‐like peptide‐1 and its metabolites following illumination of cells with blue light. PhotoETP thus provides a blueprint for the production of small‐molecule class B GPCR allosteric photoswitches, and may represent a useful tool for understanding positive cooperativity at the GLP‐1R.

Background The human SLC30A8 gene encodes the secretory granule-localised zinc transporter ZnT8 whose expression is chiefly restricted to the endocrine pancreas. Single nucleotide polymorphisms (SNPs) in the human SLC30A8 gene have been associated, through genome-wide studies, with altered type 2 diabetes risk. In addition to a role in the control of insulin release, recent studies involving targeted gene ablation from the pancreatic α cell (Solomou et al., J Biol Chem 290(35):21432-42) have also implicated ZnT8 in the control of glucagon release. Up to now, however, the possibility that increased levels of the transporter in these cells may impact glucagon secretion has not been explored. Methods Here, we use a recently-developed reverse tetracyline transactivator promoter-regulated ZnT8 transgene to drive the over-expression of human ZnT8 selectively in the α cell in adult mice. Glucose homeostasis and glucagon secretion were subsequently assessed both in vivo during hypoglycemic clamps and from isolated islets in vitro. Results Doxyclin-dependent human ZnT8 mRNA expression was apparent in both isolated islets and in fluorescence-activated cell sorting- (FACS) purified α cells. Examined at 12 weeks of age, intraperitoneal glucose (1 g/kg) tolerance was unchanged in transgenic mice versus wild-type littermates (n = 8-10 mice/genotype, p > 0.05) and sensitivity to intraperitoneal insulin (0.75U/kg) was similarly unaltered in transgenic animals. In contrast, under hyperinsulinemic-hypoglycemic clamp, a ~45 % (p 0.05). Over-expression of ZnT8 in glucagonoma-derived αTC1-9 cells increased granule free Zn2+ concentrations consistent with a role for Zn2+ in this compartment in the action of ZnT8 on glucagon secretion. Conclusions Increased ZnT8 expression, and a likely increase in intragranular free Zn2+ concentration, is deleterious in pancreatic α cells for stimulated glucagon release. These data provide further evidence that type 2 diabetes-associated polymorphisms in the SLC30A8/ZnT8 gene may act in part via alterations in glucagon release and suggest that ZnT8 activation may restrict glucagon release in some settings.

Background and Aim: It has been shown that Zn 2+ release during cardiac cycle results mostly increases in intracellular free Zn 2+ levels ([Zn 2+ ] i ),which is further triggering reactive oxygen species (ROS) and reactive nitrogen species (RNS) production. Very small cytosolic free Zn 2+ level in cardiomyocytes can increase with pathological stimuli, inducing further oxidations/phosphorylation in proteins as well as changes in protein expression levels of Zn 2+ –transporters. In here, we aimed to examine the possible correlation between the changes in the level of either [Zn 2+ ] cyt , [Zn 2+ ] ER or [Zn 2+ ] mit and production of ROS and RNS in cardiomyocytes under hyperglycemic condition. Methods and Results: By using confocal imaging, we monitored [Zn 2+ ] cyt , [Zn 2+ ] ER and [Zn 2+ ] mit changes, as well as production of ROS and RNS and changes in mitochondrial membrane potential (MMP) by using Zn 2+ sensitive eCALWY FRET probes and specific fluorescence dyes, DAF, DCDFA and JC-1, respectively, in cardiac ventricular cell line H9c2 cells. We also measured cytosolic, ER and mitochondrial free Zn 2+ levels in H9c2 cells with adenovirally infected eCALWY FRET probes. Results We measured high [Zn 2+ ] cyt and [Zn 2+ ] mit and low [Zn 2+ ] ER in high glucose incubated cells comparison to those of controls. The basal levels of ROS and RNS are significantly high in hyperglycemic cardiomyocytes compared to the controls. When either ROS or RNS is acutely increased, it turns to induce marked increase in total intracellular free [Zn 2+ ] i , while those can be reversed by a thiol reducing agent DTT or a Zn 2+ -chelator TPEN. Moreover, an important depolarization of MMP is observed with increases in [Zn 2+ ] i via an increase in either ROS or RNS production as well as decrease in ATP production. Furthermore, Western blot analysis showed that Zn 2+ –transporter ZIP7 is increased and hyperphosphorylated with a marked decrease in ZnT7, both are localized on ER, in hyperglycemic cardiomyocytes. Additionally, we measured markedly high phosphorylation in some members of macromolecular protein complex of cardiac ryanodine receptors such as PKA, CaMKII and PP2A in these cells, which are similar to those of obtained with high [Zn 2+ ]. Discussion Overall, our present data demonstrate that hyperglycemia causes increase of [Zn 2+ ] cyt and [Zn 2+ ] mit , with a marked decrease in [Zn 2+ ] ER via changes in ZnT7 and ZIP7 expression levels, which further causes depolarization of MMP and increases of ROS and RNS production in H9c2 cells under hyperglycemia. These changes altogether can contribute, in part, to hyperglycemia-induced cardiac dysfunction. Supported by TUBITAK-SBAG-113S466, COSTAction-TD1304 and EFSD-Fellowships

Preserving β-cell function during the development of obesity and insulin resistance would limit the worldwide epidemic of type 2 diabetes. Endoplasmic reticulum (ER) calcium (Ca2+) depletion induced by saturated free fatty acids and cytokines causes β-cell ER stress and apoptosis, but the molecular mechanisms behind these phenomena are still poorly understood. Here, we demonstrate that palmitate-induced sorcin downregulation and subsequent increases in glucose-6-phosphatase catalytic subunit-2 (G6PC2) levels contribute to lipotoxicity. Sorcin is a calcium sensor protein involved in maintaining ER Ca2+ by inhibiting ryanodine receptor activity and playing a role in terminating Ca2+-induced Ca2+ release. G6PC2, a genome-wide association study gene associated with fasting blood glucose, is a negative regulator of glucose-stimulated insulin secretion (GSIS). High-fat feeding in mice and chronic exposure of human islets to palmitate decreases endogenous sorcin expression while levels of G6PC2 mRNA increase. Sorcin-null mice are glucose intolerant, with markedly impaired GSIS and increased expression of G6pc2. Under high-fat diet, mice overexpressing sorcin in the β-cell display improved glucose tolerance, fasting blood glucose, and GSIS, whereas G6PC2 levels are decreased and cytosolic and ER Ca2+ are increased in transgenic islets. Sorcin may thus provide a target for intervention in type 2 diabetes.

Outside of the biological arena the term 'repression' often has a negative connotation. However, in the pancreatic β-cell a small group of genes, which are abundantly expressed in most if not all other mammalian tissues, are highly selectively repressed, with likely functional consequences. The two 'founder' members of this group, lactate dehydrogenase A (Ldha) and monocarboxylate transporter-1 (MCT-1/Slc16a1), are inactivated by multiple mechanisms including histone modifications and microRNA-mediated silencing. Their inactivation ensures that pyruvate and lactate, derived from muscle during exercise, do not stimulate insulin release inappropriately. Correspondingly, activating mutations in the MCT-1 promoter underlie 'exercise-induced hyperinsulinism' (EIHI) in man, a condition mimicked by forced over-expression of MCT-1 in the β-cell in mice. Furthermore, LDHA expression in the β-cell is upregulated in both human type 2 diabetes and in rodent models of the disease. Recent work by us and by others has identified a further ∼60 genes which are selectively inactivated in the β-cell, a list which we refine here up to seven by detailed comparison of the two studies. These genes include key regulators of cell proliferation and stimulus-secretion coupling. The present, and our earlier results, thus highlight the probable importance of shutting down a subset of 'disallowed' genes for the differentiated function of β-cells, and implicate previously unsuspected signalling pathways in the control of β-cell expansion and insulin secretion. Targeting of deregulated 'disallowed' genes in these cells may thus, in the future, provide new therapeutic avenues for type 2 diabetes.

Background ZAC (Zinc finger protein that regulates apoptosis and cell-cycle arrest) is a candidate gene for transient neonatal diabetes mellitus (TNDM). This condition involves severe insulin deficiency at birth that reverses over weeks or months but may relapse with diabetes recurring in later life. ZAC overexpression in transgenic mice has previously been shown to result in complex changes in both beta-cell mass and possibly function. The present study therefore aimed to examine the role of ZAC in beta-cell function in vitro, independent of the confounder of a reduced beta-cell mass at birth. Methods Overexpression of ZAC was achieved through the tetracycline-regulatable system in the beta-cell line, INS-1. Results We found that ZAC overexpression exerted no significant effect on proliferation in this transformed cell line at any of the glucose concentrations examined. By contrast, glucose-stimulated insulin secretion was impaired through a mechanism downstream of cytosolic Ca2+ increases. Furthermore, glucose-stimulated proinsulin biosynthesis was inhibited despite an increase in insulin transcript level. Finally, we found that glucose downregulated ZAC expression in both INS-1 cells and primary mouse islets. Conclusions These results indicate that ZAC is a negative regulator of the acute stimulatory effects of glucose on beta-cells, and provide a possible explanation for both insulin deficiency in the neonate and the later relapse of diabetes in patients with transient neonatal diabetes mellitus cases. Copyright © 2012 John Wiley & Sons, Ltd.

Stimulation of mitochondrial oxidative metabolism by Ca2+ is now generally recognised as important for the control of cellular ATP homeostasis. Here, we review the mechanisms through which Ca2+ regulates mitochondrial ATP synthesis. We focus on cardiac myocytes and pancreatic β-cells, where tight control of this process is likely to play an important role in the response to rapid changes in workload and to nutrient stimulation, respectively. We also describe a novel approach for imaging the Ca2+-dependent regulation of ATP levels dynamically in single cells.

Individuals carrying type 2 diabetes risk alleles in TCF7L2 display decreased beta cell levels of T cell factor 7 like-2 (TCF7L2) immunoreactivity, and impaired insulin secretion and beta cell sensitivity to glucagon-like peptide 1 (GLP-1). Here, we sought to determine whether selective deletion of Tcf7l2 in mouse pancreas impairs insulin release and glucose homeostasis. Pancreas-specific Tcf7l2-null (pTcf7l2) mice were generated by crossing mice carrying conditional knockout alleles of Tcf7l2 (Tcf7l2-flox) with mice expressing Cre recombinase under the control of the Pdx1 promoter (Pdx1.Cre). Gene expression was assessed by real-time quantitative PCR and beta cell mass by optical projection tomography. Glucose tolerance, insulin secretion from isolated islets, and plasma insulin, glucagon and GLP-1 content were assessed by standard protocols. From 12 weeks of age, pTcf7l2 mice displayed decreased oral glucose tolerance vs control littermates; from 20 weeks they had glucose intolerance upon administration of glucose by the intraperitoneal route. pTcf7l2 islets displayed impaired insulin secretion in response to 17 (vs 3.0) mmol/l glucose (54.6 ± 4.6%, p

Exercise-induced hyperinsulinism (EIHI) is an autosomal dominant disorder characterized by inappropriate insulin secretion in response to vigorous physical exercise or pyruvate injection. Activating mutations in the monocarboxylate transporter-1 (MCT1, SLC16A1) promoter have been linked to EIHI. Expression of this pyruvate transporter is specifically repressed (disallowed) in pancreatic β-cells, despite nearly universal expression across other tissues. It has been impossible to determine, however, whether EIHI mutations cause MCT1 expression in patient β-cells. The hypothesis that MCT1 expression in β-cells is sufficient to cause EIHI by allowing entry of pyruvate and triggering insulin secretion thus remains unproven. Therefore, we generated a transgenic mouse capable of doxycycline-induced, β-cell–specific overexpression of MCT1 to test this model directly. MCT1 expression caused isolated islets to secrete insulin in response to pyruvate, without affecting glucose-stimulated insulin secretion. In vivo, transgene induction lowered fasting blood glucose, mimicking EIHI. Pyruvate challenge stimulated increased plasma insulin and smaller excursions in blood glucose in transgenic mice. Finally, in response to exercise, transgene induction prevented the normal inhibition of insulin secretion. Forced overexpression of MCT1 in β-cells thus replicates the key features of EIHI and highlights the importance of this transporter’s absence from these cells for the normal control of insulin secretion.

Carbohydrate-responsive element-binding protein (ChREBP) is a regulator of pancreatic β-cell gene expression and an important mediator of glucotoxicity. Glucose increases the activity and nuclear localization of ChREBP by still ill-defined mechanisms. Here we reveal, using both MIN6 and primary mouse β-cells, a unique mechanism behind ChREBP nuclear translocation. At low glucose concentrations, ChREBP interacts with sorcin, a penta EF hand Ca2+ binding protein, and is sequestered in the cytosol. Sorcin overexpression inhibits ChREBP nuclear accumulation at high glucose and reduced the activity of L-type pyruvate kinase (L-PK) and TxNIP promoters, two well-characterized ChREBP target genes. Sorcin inactivation by RNA interference increases ChREBP nuclear localization and in vivo binding to the L-PK promoter at low glucose concentrations. Ca2+ influx was essential for this process since Ca2+ chelation with EGTA, or pharmacological inhibition with diazoxide and nifedipine, blocked the effects of glucose. Conversely, mobilization of intracellular Ca2+ with ATP caused the nuclear accumulation of ChREBP. Finally, sorcin silencing inhibited ATP-induced increases in intracellular Ca2+ and glucose-stimulated insulin secretion. We therefore conclude that sorcin retains ChREBP in the cytosol at low glucose concentrations and may act as a Ca2+ sensor for glucose-induced nuclear translocation and the activation of ChREBP-dependent genes.

An ability to convert between pancreatic islet cell types may provide a new approach to replace insulin-secreting β cells destroyed by autoimmune attack in Type 1 diabetes. Two papers, which have recently appeared in Cell, describe how this might be achieved.

Understanding how pancreatic β cells achieve and maintain functional identity as insulin-secreting cells is a major roadblock in our understanding of the aetiology of diabetes mellitus. Three new papers reveal the roles, in the adult β cell, of four transcription factors that are crucial for reprogramming a multipotent endocrine progenitor.

Current methods of monitoring insulin secretion lack the required spatial and temporal resolution to adequately map the dynamics of exocytosis of native insulin granules in intact cell populations in three dimensions. Exploiting the fact that insulin granules contain a high level of Zn 2+ , and that Zn 2+ is coreleased with insulin during secretion, we have developed a fluorescent, cell surface-targeted zinc indicator for monitoring induced exocytotic release (ZIMIR). ZIMIR displayed a robust fluorescence enhancement on Zn 2+ chelation and bound Zn 2+ with high selectivity against Ca 2+ and Mg 2+ . When added to cultured β cells or intact pancreatic islets at low micromolar concentrations, ZIMIR labeled cells rapidly, noninvasively, and stably, and it reliably reported changes in Zn 2+ concentration near the sites of granule fusion with high sensitivity that correlated well with membrane capacitance measurement. Fluorescence imaging of ZIMIR-labeled β cells followed the dynamics of exocytotic activity at subcellular resolution, even when using simple epifluorescence microscopy, and located the chief sites of insulin release to intercellular junctions. Moreover, ZIMIR imaging of intact rat islets revealed that Zn 2+ /insulin release occurred largely in small groups of adjacent β cells, with each forming a “secretory unit.” Concurrent imaging of ZIMIR and Fura-2 showed that the amplitude of cytosolic Ca 2+ elevation did not necessarily correlate with insulin secretion activity, suggesting that events downstream of Ca 2+ signaling underlie the cell-cell heterogeneity in insulin release. In addition to studying stimulation-secretion coupling in cells with Zn 2+ -containing granules, ZIMIR may find applications in β-cell engineering and screening for molecules regulating insulin secretion on high-throughput platforms.

Background: The glucose sensor PAS kinase (PASK) plays a fundamental role in pancreatic islet physiology. Results: A single amino acid substitution in the human PASK kinase domain stimulates enzyme activity and increases insulin secretion at low glucose. Conclusion: A rare, naturally occurring mutation in PASK modulates insulin release in man. Significance: We provide direct genetic evidence for a role for PASK in controlling insulin secretion in man., PAS kinase (PASK) is a glucose-regulated protein kinase involved in the control of pancreatic islet hormone release and insulin sensitivity. We aimed here to identify mutations in the PASK gene that may be associated with young-onset diabetes in humans. We screened 18 diabetic probands with unelucidated maturity-onset diabetes of the young (MODY). We identified two rare nonsynonymous mutations in the PASK gene (p.L1051V and p.G1117E), each of which was found in a single MODY family. Wild type or mutant PASKs were expressed in HEK 293 cells. Kinase activity of the affinity-purified proteins was assayed as autophosphorylation at amino acid Thr307 or against an Ugp1p-derived peptide. Whereas the PASK p.G1117E mutant displayed a ∼25% increase with respect to wild type PASK in the extent of autophosphorylation, and a ∼2-fold increase in kinase activity toward exogenous substrates, the activity of the p.L1051V mutant was unchanged. Amino acid Gly1117 is located in an α helical region opposing the active site of PASK and may elicit either: (a) a conformational change that increases catalytic efficiency or (b) a diminished inhibitory interaction with the PAS domain. Mouse islets were therefore infected with adenoviruses expressing wild type or mutant PASK and the regulation of insulin secretion was examined. PASK p.G1117E-infected islets displayed a 4-fold decrease in glucose-stimulated (16.7 versus 3 mm) insulin secretion, chiefly reflecting a 4.5-fold increase in insulin release at low glucose. In summary, we have characterized a rare mutation (p.G1117E) in the PASK gene from a young-onset diabetes family, which modulates glucose-stimulated insulin secretion.

Zn²⁺ is an important cofactor for insulin biosynthesis and storage in pancreatic β-cells. Correspondingly, polymorphisms in the SLC30A8 gene, encoding the secretory granule Zn²⁺ transporter ZnT8, are associated with type 2 diabetes risk. Using a genetically engineered (FRET)-based sensor (eCALWY-4), we show here that elevated glucose time-dependently increases free cytosolic Zn²⁺ ([Zn²⁺](cyt)) in mouse pancreatic β-cells. These changes become highly significant (853 ± 96 pm versus 452 ± 42 pm, p < 0.001) after 24 h and are associated with increased expression of the Zn²⁺ importer family members Slc39a6, Slc39a7, and Slc39a8, and decreased expression of metallothionein 1 and 2. Arguing that altered expression of the above genes is not due to altered [Zn²⁺](cyt), elevation of extracellular (and intracellular) [Zn²⁺] failed to mimic the effects of high glucose. By contrast, increases in intracellular cAMP prompted by 3-isobutyl-1-methylxanthine and forskolin partially mimicked the effects of glucose on metallothionein, although not ZiP, gene expression. Modulation of intracellular Ca²⁺ and insulin secretion with pharmacological agents (tolbutamide and diazoxide) suggested a possible role for changes in these parameters in the regulation of Slc39a6 and Slc39a7 but not Slc39a8, nor metallothionein expression. In summary, 1) glucose induces increases in [Zn²⁺](cyt), which are then likely to facilitate the processing and/or the storage of insulin and its cocrystallization with Zn²⁺, and 2) these increases are associated with elevated expression of zinc importers. Conversely, a chronic increase in [Zn²⁺](cyt) following sustained hyperglycemia may contribute to β-cell dysfunction and death in some forms of diabetes.

SUMMARY AMP-activated protein kinase (AMPK) has long been recognized as a master energy sensor. Activation of AMPK in response to metabolic stress preserves energy stores by switching on catabolic pathways, whilst its inhibition consumes the energy by switching on anabolic pathways. Over the past 10–15 years, much attention has been focused on the role of AMPK in mammalian metabolism, and particularly in diabetes. As a consequence, AMPK has emerged as much more than a simple energy regulator and is now recognized as a kinase involved in controlling numerous cellular processes, including cell growth, apoptosis, autophagy and polarity. Using different in vitro and in vivo tools, AMPK has also been found to play important roles in different glucose-sensing organs and to serve as a key regulator of glucose homeostasis in mammals. Perhaps most importantly, AMPK appears to be the major target for several antidiabetic drugs. Here, we review recent advances in the field and particularly those emerging from the ge...

SUMMARY Axon degeneration is observed in neurodegenerative diseases and neuroinflammatory disorders, such as Alzheimer’s disease, Parkinson’s disease and multiple sclerosis. The molecular basis of this process remains largely unknown. Here, we show that mice deleted for the tumour suppressor LKB1 (also called STK11) in the spinal cord, some parts of the brain and in the endocrine pancreas (βLKB1KO mice) develop hind-limb dysfunction and axon degeneration at about 7 weeks. Demyelination and macrophage infiltration are observed in the white matter of these mice, predominantly in the bilateral and anterior funiculi of the thoracic segment of the spinal cord, suggesting damage to the ascending sensory signalling pathway owing to LKB1 deletion in the brain. Microtubule structures were also affected in the degenerated foci, with diminished neurofilament and tubulin expression. Deletion of both PRKAA1 genes, whose products AMPKα1 and AMPKα2 are also downstream targets of LKB1, with the same strategy was without effect. We thus define LKB1 as an intrinsic suppressor of axon degeneration and a possible target for strategies that can reverse this process.

OBJECTIVE Hypothalamic nutrient sensing regulates glucose production, but the neuronal circuits involved remain largely unknown. Recent studies underscore the importance of N-methyl-d-aspartate (NMDA) receptors in the dorsal vagal complex in glucose regulation. These studies raise the possibility that hypothalamic nutrient sensing activates a forebrain-hindbrain NMDA-dependent circuit to regulate glucose production. RESEARCH DESIGN AND METHODS We implanted bilateral catheters targeting the mediobasal hypothalamus (MBH) (forebrain) and dorsal vagal complex (DVC) (hindbrain) and performed intravenous catheterizations to the same rat for infusion and sampling purposes. This model enabled concurrent selective activation of MBH nutrient sensing by either MBH delivery of lactate or an adenovirus expressing the dominant negative form of AMPK (Ad-DN AMPK α2 [D157A]) and inhibition of DVC NMDA receptors by either DVC delivery of NMDA receptor blocker MK-801 or an adenovirus expressing the shRNA of NR1 subunit of NMDA receptors (Ad-shRNA NR1). Tracer-dilution methodology and the pancreatic euglycemic clamp technique were performed to assess changes in glucose kinetics in the same conscious, unrestrained rat in vivo. RESULTS MBH lactate or Ad-DN AMPK with DVC saline increased glucose infusion required to maintain euglycemia due to an inhibition of glucose production during the clamps. However, DVC MK-801 negated the ability of MBH lactate or Ad-DN AMPK to increase glucose infusion or lower glucose production. Molecular knockdown of DVC NR1 of NMDA receptor via Ad-shRNA NR1 injection also negated MBH Ad-DN AMPK to lower glucose production. CONCLUSIONS Molecular and pharmacological inhibition of DVC NMDA receptors negated hypothalamic nutrient sensing mechanisms activated by lactate metabolism or AMPK inhibition to lower glucose production. Thus, DVC NMDA receptor is required for hypothalamic nutrient sensing to lower glucose production and that hypothalamic nutrient sensing activates a forebrain-hindbrain circuit to lower glucose production.

OBJECTIVE The fuel sensor AMP-activated protein kinase (AMPK) in the hypothalamus regulates energy homeostasis by sensing nutritional and hormonal signals. However, the role of hypothalamic AMPK in glucose production regulation remains to be elucidated. We hypothesize that bidirectional changes in hypothalamic AMPK activity alter glucose production. RESEARCH DESIGN AND METHODS To introduce bidirectional changes in hypothalamic AMPK activity in vivo, we first knocked down hypothalamic AMPK activity in male Sprague-Dawley rats by either injecting an adenovirus expressing the dominant-negative form of AMPK (Ad-DN AMPKα2 [D157A]) or infusing AMPK inhibitor compound C directly into the mediobasal hypothalamus. Next, we independently activated hypothalamic AMPK by delivering either an adenovirus expressing the constitutive active form of AMPK (Ad-CA AMPKα1312 [T172D]) or the AMPK activator AICAR. The pancreatic (basal insulin)-euglycemic clamp technique in combination with the tracer-dilution methodology was used to assess the impact of alternations in hypothalamic AMPK activity on changes in glucose kinetics in vivo. RESULTS Injection of Ad-DN AMPK into the hypothalamus knocked down hypothalamic AMPK activity and led to a significant suppression of glucose production with no changes in peripheral glucose uptake during the clamps. In parallel, hypothalamic infusion of AMPK inhibitor compound C lowered glucose production as well. Conversely, molecular and pharmacological activation of hypothalamic AMPK negated the ability of hypothalamic nutrients to lower glucose production. CONCLUSIONS These data indicate that changes in hypothalamic AMPK activity are sufficient and necessary for hypothalamic nutrient-sensing mechanisms to alter glucose production in vivo.

The tumor suppressor liver kinase B1 (LKB1), also called STK11, is a protein kinase mutated in Peutz-Jeghers syndrome. LKB1 phosphorylates AMP-activated protein kinase (AMPK) and several related protein kinases. Whereas deletion of both catalytic isoforms of AMPK from the pancreatic β-cell and hypothalamic neurons using the rat insulin promoter (RIP2). Cre transgene (βAMPKdKO) diminishes insulin secretion in vivo, deletion of LKB1 in the β-cell with an inducible Pdx-1.CreER transgene enhances insulin secretion in mice. To determine whether the differences between these models reflect genuinely distinct roles for the two kinases in the β-cell or simply differences in the timing and site(s) of deletion, we have therefore created mice deleted for LKB1 with the RIP2.Cre transgene. In marked contrast to βAMPKdKO mice, βLKB1KO mice showed diminished food intake and weight gain, enhanced insulin secretion, unchanged insulin sensitivity, and improved glucose tolerance. In line with the phenotype of Pdx1- CreER mice, total β-cell mass and the size of individual islets and β-cells were increased and islet architecture was markedly altered in βLKB1KO islets. Signaling by mammalian target of rapamycin (mTOR) to eIF4-binding protein-1 and ribosomal S6 kinase was also enhanced. In contrast to Pdx1- CreER-mediated deletion, the expression of Glut2, glucose-induced changes in membrane potential and intracellular Ca2+ were sharply reduced in βLKB1KO mouse islets and the stimulation of insulin secretion was modestly inhibited. We conclude that LKB1 and AMPK play distinct roles in the control of insulin secretion and that the timing of LKB1 deletion, and/or its loss from extrapancreatic sites, influences the final impact on β-cell function.

Zinc is highly concentrated in pancreatic beta cells, is critical for normal insulin storage and may regulate glucagon secretion from alpha cells. Zinc transport family member 8 (ZnT8) is a zinc efflux transporter that is highly abundant in beta cells. Polymorphisms of ZnT8 (also known as SLC30A8) gene in man are associated with increased risk of type 2 diabetes. While global Znt8 knockout (Znt8KO) mice have been characterised, ZnT8 is also present in other islet cell types and extra-pancreatic tissues. Therefore, it is important to find ways of understanding the role of ZnT8 in beta and alpha cells without the difficulties caused by the confounding effects of ZnT8 in these other tissues. We generated mice with beta cell-specific (Znt8BKO) and alpha cell-specific (Znt8AKO) knockout of Znt8, and performed in vivo and in vitro characterisation of the phenotypes to determine the functional and anatomical impact of ZnT8 in these cells. Thus we assessed zinc accumulation, insulin granule morphology, insulin biosynthesis and secretion, and glucose homeostasis. Znt8BKO mice are glucose-intolerant, have reduced beta cell zinc accumulation and atypical insulin granules. They also display reduced first-phase glucose-stimulated insulin secretion, reduced insulin processing enzyme transcripts and increased proinsulin levels. In contrast, Znt8AKO mice show no evident abnormalities in plasma glucagon and glucose homeostasis. This is the first report of specific beta and alpha cell deletion of Znt8. Our data indicate that while, under the conditions studied, ZnT8 is absolutely essential for proper beta cell function, it is largely dispensable for alpha cell function.

AMP-activated protein kinase (AMPK) is an evolutionarily conserved enzyme and a target of glucose-lowering agents, including metformin. However, the precise role or roles of the enzyme in controlling insulin secretion remain uncertain.The catalytic alpha1 and alpha2 subunits of AMPK were ablated selectively in mouse pancreatic beta cells and hypothalamic neurons by breeding Ampkalpha1 [also known as Prkaa1]-knockout mice, bearing floxed Ampkalpha2 [also known as Prkaa2] alleles (Ampkalpha1 ( -/- ),alpha2( fl/fl ),), with mice expressing Cre recombinase under the rat insulin promoter (RIP2). RIP2 was used to express constitutively activated AMPK selectively in beta cells in transgenic mice. Food intake, body weight and urinary catecholamines were measured using metabolic cages. Glucose and insulin tolerance were determined after intraperitoneal injection. Beta cell mass and morphology were analysed by optical projection tomography and confocal immunofluorescence microscopy, respectively. Granule docking, insulin secretion, membrane potential and intracellular free Ca(2+) were measured with standard techniques.Trigenic Ampkalpha1 ( -/- ),alpha2( fl/fl ) expressing Cre recombinase and lacking both AMPKalpha subunits in the beta cell, displayed normal body weight and increased insulin sensitivity, but were profoundly insulin-deficient. Secreted catecholamine levels were unchanged. Total beta cell mass was unaltered, while mean islet and beta cell volume were reduced. AMPK-deficient beta cells displayed normal glucose-induced changes in membrane potential and intracellular free Ca(2+), while granule docking and insulin secretion were enhanced. Conversely, betaAMPK transgenic mice were glucose-intolerant and displayed defective insulin secretion.Inhibition of AMPK activity within the beta cell is necessary, but not sufficient for stimulation of insulin secretion by glucose to occur. AMPK activation in extrapancreatic RIP2.Cre-expressing cells might also influence insulin secretion in vivo.

We have previously identified two genes, encoding lactate dehydrogenase (Ldha) and the monocarboxylate carrier, MCT1 (Slc16a1) whose expression is remarkably low in pancreatic β-cells and islets. We sought here to determine whether these may be part of a larger family of genes selectively repressed ("disallowed") in the pancreatic islet. Using new and publicly available microarray data, we undertook a bioinformatic analysis of gene expression in islets and a range of other murine tissues. We compared data sets from three sources of mouse pancreatic islets with a total of 30 datasets from nine tissues, to identify genes with at least five-fold down-regulation in islets. 39 genes were revealed as being specifically repressed in islets. These included Ldha and Slc16a1 as expected but also genes involved in several other metabolic pathways which could affect glucose stimulated insulin secretion. Of these, adenylate kinase 3 (AK3) is a mitochondrial enzyme which acts on GTP, and ornithine aminotransferase (OAT) lies on the pathway converting glutamate to ornithine. The removal of an enzyme which could dissipate mitochondrial GTP levels in beta cells provides support for the theory that mitochondrial GTP may be an important for regulating insulin secretion, whilst blocking an alternative metabolic fate for glutamate is consistent with a signalling role for glutamate. The identification of these genes should inform efforts to generate fully functional β-cells from stem cell sources, and may provide new targets in type 2 diabetes.

Research highlights ► ChREBP silencing enhances glucose-responsive gene expression in MIN6 β-cells. ► ChREBP modulation of Pdx-1 gene expression might be indirect. ► ChREBP over-expression decreases Pdx-1, MafA, Ins1, Ins2 and GcK mRNA levels in mouse pancreatic islets of Langerhans., Carbohydrate responsive element-binding protein (ChREBP) is a transcription factor whose expression and activity are increased in pancreatic β-cells maintained at elevated glucose concentrations. We show here that ChREBP inactivation in clonal pancreatic MIN6 β-cells results in an increase in Pdx-1 expression at low glucose and to a small, but significant, increase in Ins2, GcK and MafA gene expression at high glucose concentrations. Conversely, adenovirus-mediated over-expression of ChREBP in mouse pancreatic islets results in decreases in Pdx-1, MafA, Ins1, Ins2 and GcK mRNA levels. These data suggest that strategies to reduce ChREBP activity might protect against β-cell dysfunction in type 2 diabetes.

Research highlights ► Mouse PDX1 Ser-269 is phosphorylated in pancreatic islets of Langerhans and beta cells. ► High glucose concentration decreases the degree of phosphorylation on PDX1 Ser-269, as assessed by a phospho-specific anti-phospho-Ser269 antibody. ► HIPK2 is a potential kinase for PDX1 Ser-269. ► Phosphorylation at Ser-269 affects the subnuclear distribution of PDX1., Pancreatic and duodenal homeobox 1 (PDX1) regulates pancreatic development and mature β-cell function. We demonstrate by mass spectrometry that serine residue at position 269 in the C-terminal domain of PDX1 is phosphorylated in β-cells. Besides we show that the degree of phosphorylation, assessed with a phospho-Ser-269-specific antibody, is decreased by elevated glucose concentrations in both MIN6 β-cells and primary mouse pancreatic islets. Homeodomain interacting protein kinase 2 (HIPK2) phosphorylates PDX1 in vitro; phosphate incorporation substantially decreases in PDX1 S269A mutant. Silencing of HIPK2 led to a 51 ± 0.2% decrease in Ser-269 phosphorylation in MIN6 β-cells. Mutation of Ser-269 to phosphomimetic residue glutamic acid (S269E) or de-phosphomimetic residue alanine (S269A) exerted no effect on PDX1 half-life. Instead, PDX1 S269E mutant displayed abnormal changes in subnuclear localization in response to high glucose. Our results suggest that HIPK2-mediated phosphorylation of PDX1 at Ser-269 might be a regulatory mechanism connecting signals generated by changes in extracellular glucose concentration to downstream effectors via changes in subnuclear localization of PDX1, thereby influencing islet cell differentiation and function.

Mitochondrial Ca(2+) transport was initially considered important only in buffering of cytosolic Ca(2+) by acting as a "sink" under conditions of Ca(2+) overload. The main regulator of ATP production was considered to be the relative concentrations of high energy phosphates. However, work by Denton and McCormack in the 1970s and 1980s showed that free intramitochondrial Ca(2+) ([Ca(2+)](m)) activated dehydrogenase enzymes in mitochondria, leading to increased NADH and hence ATP production. This leads them to propose a scheme, subsequently termed a "parallel activation model" whereby increases in energy demand, such as hormonal stimulation or increased workload in muscle, produced an increase in cytosolic [Ca(2+)] that was relayed by the mitochondrial Ca(2+) transporters into the matrix to give an increase in [Ca(2+)](m). This then stimulated energy production to meet the increased energy demand. With the development of methods for measuring [Ca(2+)](m) in living cells that proved [Ca(2+)](m) changed over a dynamic physiological range rather than simply soaking up excess cytosolic [Ca(2+)], this model has now gained widespread acceptance. However, work by ourselves and others using targeted probes to measure changes in both [Ca(2+)] and [ATP] in different cell compartments has revealed variations in the interrelationships between these two in different tissues, suggesting that metabolic regulation by Ca(2+) is finely tuned to the demands and function of the individual organ.

Zinc co-crystallizes with insulin in dense core secretory granules, but its role in insulin biosynthesis, storage and secretion is unknown. In this study we assessed the role of the zinc transporter ZnT8 using ZnT8-knockout ( ZnT8 −/− ) mice. Absence of ZnT8 expression caused loss of zinc release upon stimulation of exocytosis, but normal rates of insulin biosynthesis, normal insulin content and preserved glucose-induced insulin release. Ultrastructurally, mature dense core insulin granules were rare in ZnT8 −/− beta cells and were replaced by immature, pale insulin “progranules,” which were larger than in ZnT8 +/+ islets. When mice were fed a control diet, glucose tolerance and insulin sensitivity were normal. However, after high-fat diet feeding, the ZnT8 −/− mice became glucose intolerant or diabetic, and islets became less responsive to glucose. Our data show that the ZnT8 transporter is essential for the formation of insulin crystals in beta cells, contributing to the packaging efficiency of stored insulin. Interaction between the ZnT8 −/− genotype and diet to induce diabetes is a model for further studies of the mechanism of disease of human ZNT8 gene mutations.

OBJECTIVE Polymorphisms in the human TCF7L2 gene are associated with reduced insulin secretion and an increased risk of type 2 diabetes. However, the mechanisms by which TCF7L2 affect insulin secretion are still unclear. We define the effects of TCF7L2 expression level on mature β-cell function and suggest a potential mechanism for its actions. RESEARCH DESIGN AND METHODS TCF7L2 expression in rodent islets and β-cell lines was altered using RNAi or adenoviral transduction. β-Cell gene profiles were measured by quantitative real-time PCR and the effects on intracellular signaling and exocytosis by live cell imaging, electron microscopy, and patch clamp electrophysiology. RESULTS Reducing TCF7L2 expression levels by RNAi decreased glucose- but not KCl-induced insulin secretion. The glucose-induced increments in both ATP/ADP ratio and cytosolic free Ca2+ concentration ([Ca2+]i) were increased compared with controls. Overexpression of TCF7L2 exerted minor inhibitory effects on glucose-regulated changes in [Ca2+]i and insulin release. Gene expression profiling in TCF7L2-silenced cells revealed increased levels of mRNA encoding syntaxin 1A but decreased Munc18–1 and ZnT8 mRNA. Whereas the number of morphologically docked vesicles was unchanged by TCF7L2 suppression, secretory granule movement increased and capacitance changes decreased, indicative of defective vesicle fusion. CONCLUSION—TCF7L2 is involved in maintaining expression of β-cell genes regulating secretory granule fusion. Defective insulin exocytosis may thus underlie increased diabetes incidence in carriers of the at-risk TCF7L2 alleles.

OBJECTIVE— ATP-sensitive K+ channels (KATP channels) link glucose metabolism to the electrical activity of the pancreatic β-cell to regulate insulin secretion. Mutations in either the Kir6.2 or sulfonylurea receptor (SUR) 1 subunit of the channel have previously been shown to cause neonatal diabetes. We describe here an activating mutation in the ABCC8 gene, encoding SUR1, that is associated with the development of type 2 diabetes only in adults. RESEARCH DESIGN AND METHODS— Recombinant KATP channel subunits were expressed using pIRES2-based vectors in human embryonic kidney (HEK) 293 or INS1(832/13) cells and the subcellular distribution of c-myc–tagged SUR1 channels analyzed by confocal microscopy. KATP channel activity was measured in inside-out patches and plasma membrane potential in perforated whole-cell patches. Cytoplasmic [Ca2+] was imaged using Fura-Red. RESULTS— A mutation in ABCC8/SUR1, leading to a Y356C substitution in the seventh membrane-spanning α-helix, was observed in a patient diagnosed with hyperglycemia at age 39 years and in two adult offspring with impaired insulin secretion. Single KATP channels incorporating SUR1-Y356C displayed lower sensitivity to MgATP (IC50 = 24 and 95 μmol/l for wild-type and mutant channels, respectively). Similar effects were observed in the absence of Mg2+, suggesting an allosteric effect via associated Kir6.2 subunits. Overexpression of SUR1-Y356C in INS1(832/13) cells impaired glucose-induced cell depolarization and increased in intracellular free Ca2+ concentration, albeit more weakly than neonatal diabetes–associated SUR1 mutants. CONCLUSIONS— An ABCC8/SUR1 mutation with relatively minor effects on KATP channel activity and β-cell glucose sensing causes diabetes in adulthood. These data suggest a close correlation between altered SUR1 properties and clinical phenotype.

Defective insulin secretion is a hallmark of all forms of diabetes. Whereas Type 1 diabetes has long been known to result from the immune-mediated destruction of β-cells, Type 2 diabetes appears to involve both loss of β-cell mass and glucose sensitivity in the face of extrapancreatic insulin resistance. We summarize here the proceedings of a Biochemical Society Focused Meeting, held at the St Thomas campus of King's College London in December 2007, which highlighted recent research advances targeting the β-cell.

Previous studies have reported both positive and negative effects of culture of islets at high glucose concentrations on regulated insulin secretion. Here, we have re-examined this question in mouse islets and determined the role of changes in lipid synthesis in the effects of glucose. Glucose-stimulated insulin secretion (GSIS) and gene expression were examined in islets from C57BL/6 mice or littermates deleted for sterol-regulatory element binding protein-1 (SREBP1) after 4 days of culture at high glucose concentrations. Culture of control islets at 30 versus 8 mmol/1 glucose led to enhanced secretion at both basal (3 mmol/1) and stimulatory (17 mmol/1) glucose concentrations and to enhanced triacylglycerol accumulation. These changes were associated with increases in the expression of genes involved in glucose sensing (glucose transporter 2, glucokinase, sulfonylurea receptor 1, inwardly rectifying K + channel 6.2), differentiation (pancreatic duodenal homeobox 1), and lipogenesis (Srebp1, fatty acid synthase, acetyl-coenzyme A carboxylase 1, stearoyl-coenzyme A desaturase 1). When cultured at either 8 or 30 mmol/1 glucose, SREBP1-deficient (SREBP1 -/- ) islets displayed reduced GSIS and triacylglycerol content compared with normal islets. Correspondingly, glucose induction of the above genes in control islets was no longer observed in SREBP1 -/- mouse islets. We conclude that enhanced lipid synthesis mediated by SREBP1c-dependent genes is required for the adaptive changes in islet gene expression and insulin secretion at high glucose concentrations.

OBJECTIVE—Apoptotic destruction of insulin-producing pancreatic β-cells is involved in the etiology of both type 1 and type 2 diabetes. AMP-activated protein kinase (AMPK) is a sensor of cellular energy charge whose sustained activation has recently been implicated in pancreatic β-cell apoptosis and in islet cell death posttransplantation. Here, we examine the importance of β-cell AMPK in cytokine-induced apoptosis and in the cytotoxic action of CD8+ T-cells. RESEARCH DESIGN AND METHODS— Clonal MIN6 β-cells or CD1 mouse pancreatic islets were infected with recombinant adenoviruses encoding enhanced green fluorescent protein (eGFP/null), constitutively active AMPK (AMPK-CA), or dominant-negative AMPK (AMPK-DN) and exposed or not to tumor necrosis factor-α, interleukin-1β, and interferon-γ. Apoptosis was detected by monitoring the cleavage of caspase-3 and DNA fragmentation. The cytotoxic effect of CD8+ purified T-cells was examined against pancreatic islets from NOD mice infected with either null or the AMPK-DN–expressing adenoviruses. RESULTS— Exposure to cytokines, or expression of AMPK-CA, induced apoptosis in clonal MIN6 β-cells and CD1 mouse pancreatic islets. By contrast, overexpression of AMPK-DN protected against the proapoptotic effect of these agents, in part by preventing decreases in cellular ATP, and lowered the cytotoxic effect of CD8+ T-cells toward NOD mouse islets. CONCLUSIONS— Inhibition of AMPK activity enhances islet survival in the face of assault by either cytokines or T-cells. AMPK may therefore represent an interesting therapeutic target to suppress immune-mediated β-cell destruction and may increase the efficacy of islet allografts in type 1 diabetes.