Dietary protein load affects the energy and nitrogen balance requiring liver glutamate dehydrogenase to maintain physical activity.

Provision of amino acids to the liver is instrumental for gluconeogenesis while it requires safe disposal of the amino group. The mitochondrial enzyme glutamate dehydrogenase (GDH) is central for hepatic ammonia detoxification by deaminating excessive amino acids towards ureagenesis and preventing hyperammonemia. The present study investigated the early adaptive responses to changes in dietary protein intake in control mice and liver-specific GDH knockout mice (Hep-Glud1-/-). Mice were fed chow diets with a wide coverage of protein contents; i.e. suboptimal 10%, standard 20%, over optimal 30%, and high 45% protein diets; switched every 4 days. Metabolic adaptations of the mice were assessed in calorimetric chambers before tissue collection and analyses. Hep-Glud1-/- mice exhibited impaired alanine induced gluconeogenesis and constitutive hyperammonemia. The expression and activity of GDH in liver lysates were not significantly changed by the different diets. However, applying an in situ redox-sensitive assay on cryopreserved tissue sections revealed higher hepatic GDH activity in mice fed the high-protein diets. On the same section series, immunohistochemistry provided corresponding mapping of the GDH expression. Cosinor analysis from calorimetric chambers showed that the circadian rhythm of food intake and energy expenditure was altered in Hep-Glud1-/- mice. In control mice, energy expenditure shifted from carbohydrate to amino acid oxidation when diet was switched to high protein content. This shift was impaired in Hep-Glud1-/- mice and consequently the spontaneous physical activity was markedly reduced in GDH knockout mice. These data highlight the central role of liver GDH in the energy balance adaptation to dietary proteins.

Glucolipotoxicity promotes the capacity of the glycerolipid/NEFA cycle supporting the secretory response of pancreatic beta cells.

AIMS/HYPOTHESIS: Chronic exposure of pancreatic beta cells to high glucose and fatty acids has been proposed to induce glucolipotoxicity. However, contradictory results suggest adaptations of the beta cells, which might be instrumental for partial preservation of the secretory response. In this context, we delineated the expression pattern of genes related to lipid pathways along with fat storage/mobilisation during glucose-stimulated insulin secretion. METHODS: Insulin-secreting cells were cultured for 3 days at different glucose concentrations (5.5, 11.1, 25 mmol/l) without or with BSA-complexed 0.4 mmol/l palmitate and oleate. Then, transcriptomic analyses of lipid pathways were performed in human islets by RNA-Seq and in INS-1E cells and rat islets by quantitative RT-PCR. Storage of fat was assessed in INS-1E cells by electron microscopy and Bodipy staining, which was also used for measuring lipid mobilisation rate. The secretory response was monitored during acute 15 mmol/l glucose stimulation using online luminescence assay for INS-1E cells and by radioimmunoassay for rat islets. RESULTS: In human islets, chronic exposure to palmitate and oleate modified expression of a panel of genes involved in lipid handling. Culture at 25 mmol/l glucose upregulated genes encoding for enzymes of the glycerolipid/NEFA cycle and downregulated receptors implicated in fatty acid signalling. Similar results were obtained in INS-1E cells, indicating enhanced capacity of the glycerolipid/NEFA cycle under glucotoxic conditions. Exposure to unsaturated C18:1 fatty acid favoured intracellular lipid accumulation in a glucose-dependent way, an effect also observed with saturated C16:0 fatty acid when combined with the panlipase inhibitor Orlistat. After the glucolipotoxic culture, intracellular fat mobilisation was required for acute glucose-stimulated secretion, particularly in oleate-treated cells under glucotoxic culture conditions. The lipid mobilisation rate was governed chiefly by the levels of stored fat as a direct consequence of the culture conditions rather than energetic demands, except in palmitate-loaded cells. CONCLUSIONS/INTERPRETATION: Glucolipotoxic conditions promote the capacity of the glycerolipid/NEFA cycle thereby preserving part of the secretory response. The cycle of fat storage/mobilisation emerges as a mechanism helping the beta cell to cope with glucotoxic conditions.

Mitochondrial Carriers Regulating Insulin Secretion Profiled in Human Islets upon Metabolic Stress.

Chronic exposure of ß-cells to nutrient-rich metabolic stress impairs mitochondrial metabolism and its coupling to insulin secretion. We exposed isolated human islets to different metabolic stresses for 3 days: 0.4 mM oleate or 0.4 mM palmitate at physiological 5.5 mM glucose (lipotoxicity), high 25 mM glucose (glucotoxicity), and high 25 mM glucose combined with 0.4 mM oleate and/or palmitate (glucolipotoxicity). Then, we profiled the mitochondrial carriers and associated genes with RNA-Seq. Diabetogenic conditions, and in particular glucotoxicity, increased expression of several mitochondrial solute carriers in human islets, such as the malate carrier DIC, the α-ketoglutarate-malate exchanger OGC, and the glutamate carrier GC1. Glucotoxicity also induced a general upregulation of the electron transport chain machinery, while palmitate largely counteracted this effect. Expression of different components of the TOM/TIM mitochondrial protein import system was increased by glucotoxicity, whereas glucolipotoxicity strongly upregulated its receptor subunit TOM70. Expression of the mitochondrial calcium uniporter MCU was essentially preserved by metabolic stresses. However, glucotoxicity altered expression of regulatory elements of calcium influx as well as the Na+/Ca2+ exchanger NCLX, which mediates calcium efflux. Overall, the expression profile of mitochondrial carriers and associated genes was modified by the different metabolic stresses exhibiting nutrient-specific signatures.

AMPK Profiling in Rodent and Human Pancreatic Beta-Cells under Nutrient-Rich Metabolic Stress.

Chronic exposure of pancreatic ß-cells to elevated nutrient levels impairs their function and potentially induces apoptosis. Like in other cell types, AMPK is activated in ß-cells under conditions of nutrient deprivation, while little is known on AMPK responses to metabolic stresses. Here, we first reviewed recent studies on the role of AMPK activation in ß-cells. Then, we investigated the expression profile of AMPK pathways in ß-cells following metabolic stresses. INS-1E ß-cells and human islets were exposed for 3 days to glucose (5.5-25 mM), palmitate or oleate (0.4 mM), and fructose (5.5 mM). Following these treatments, we analyzed transcript levels of INS-1E ß-cells by qRT-PCR and of human islets by RNA-Seq; with a special focus on AMPK-associated genes, such as the AMPK catalytic subunits α1 (Prkaa1) and α2 (Prkaa2). AMPKα and pAMPKα were also evaluated at the protein level by immunoblotting. Chronic exposure to the different metabolic stresses, known to alter glucose-stimulated insulin secretion, did not change AMPK expression, either in insulinoma cells or in human islets. Expression profile of the six AMPK subunits was marginally modified by the different diabetogenic conditions. However, the expression of some upstream kinases and downstream AMPK targets, including K-ATP channel subunits, exhibited stress-specific signatures. Interestingly, at the protein level, chronic fructose treatment favored fasting-like phenotype in human islets, as witnessed by AMPK activation. Collectively, previously published and present data indicate that, in the ß-cell, AMPK activation might be implicated in the pre-diabetic state, potentially as a protective mechanism.

Palmitate and oleate modify membrane fluidity and kinase activities of INS-1E ß-cells alongside altered metabolism-secretion coupling.

Chronic exposure to elevated levels of glucose and free fatty acids impairs beta-cell function, leading to insulin secretion defects and eventually beta-cell failure. Using a semi-high throughput approach applied to INS-1E beta-cells, we tested multiple conditions of chronic exposure to basal, intermediate and high glucose, combined with saturated versus mono- and polyunsaturated fatty acids in order to assess cell integrity, lipid metabolism, mitochondrial function, glucose-stimulated calcium rise and secretory kinetics. INS-1E beta-cells were cultured for 3 days at different glucose concentrations (5.5, 11.1, 25 mM) without or with BSA-complexed 0.4 mM saturated (C16:0 palmitate), monounsaturated (C18:1 oleate) or polyunsaturated (C18:2 linoleate, C18:3 linolenate) fatty acids, resulting in 0.1-0.5 µM unbound fatty acids. Accumulation of triglycerides in cells exposed to fatty acids was glucose-dependent, oleate inducing the strongest lipid storage and protecting against glucose-induced cytotoxicity. The combined chronic exposure to both high glucose and either palmitate or oleate altered mitochondrial function as well as glucose-induced calcium rise. This pattern did not directly translate at the secretory level since palmitate and oleate exhibited distinct effects on the first and the second phases of glucose-stimulated exocytosis. Both fatty acids changed the activity of kinases, such as the MODY-associated BLK. Additionally, chronic exposure to fatty acids modified membrane physicochemical properties by increasing membrane fluidity, oleate exhibiting larger effects compared to palmitate. Chronic fatty acids differentially and specifically exacerbated some of the glucotoxic effects, without promoting cytotoxicity on their own. Each of the tested fatty acids functionally modified INS-1E beta-cell, oleate inducing the strongest effects.

Chronic fructose renders pancreatic ß-cells hyper-responsive to glucose-stimulated insulin secretion through extracellular ATP signaling.

Fructose is widely used as a sweetener in processed food and is also associated with metabolic disorders, such as obesity. However, the underlying cellular mechanisms remain unclear, in particular, regarding the pancreatic ß-cell. Here, we investigated the effects of chronic exposure to fructose on the function of insulinoma cells and isolated mouse and human pancreatic islets. Although fructose per se did not acutely stimulate insulin exocytosis, our data show that chronic fructose rendered rodent and human ß-cells hyper-responsive to intermediate physiological glucose concentrations. Fructose exposure reduced intracellular ATP levels without affecting mitochondrial function, induced AMP-activated protein kinase activation, and favored ATP release from the ß-cells upon acute glucose stimulation. The resulting increase in extracellular ATP, mediated by pannexin1 (Panx1) channels, activated the calcium-mobilizer P2Y purinergic receptors. Immunodetection revealed the presence of both Panx1 channels and P2Y1 receptors in ß-cells. Addition of an ectonucleotidase inhibitor or P2Y1 agonists to naïve ß-cells potentiated insulin secretion stimulated by intermediate glucose, mimicking the fructose treatment. Conversely, the P2Y1 antagonist and Panx1 inhibitor reversed the effects of fructose, as confirmed using Panx1-null islets and by the clearance of extracellular ATP by apyrase. These results reveal an important function of ATP signaling in pancreatic ß-cells mediating fructose-induced hyper-responsiveness.

[Exposure of beta-cells to chronic fructose potentiates glucose-stimulated insulin secretion through ATP signaling]. / L'exposition des cellules bêta au fructose potentialise la sécrétion d'insuline via une signalisationpar l'ATP.

While the use of fructose as a sweetener and its consumption are associated with increased fat storage prompted by the action of insulin, fructose alone does not acutely stimulate insulin exocytosis from the pancreatic beta-cell, as opposed to the chief secretagogue glucose. We investigated the effects of chronic exposure to fructose on beta-cell function. Our results reveal that chronic fructose induces extracellular ATP signaling in the beta-cell, resulting in the potentiation of glucose-stimulated insulin secretion. This effect is mediated by the activation of the purinergic P2Y1 receptors and is associated with the release of cellular ATP through pannexin-1 channels. Consequently, the interplay between pannexin channels and purinergic receptors, through ATP signaling, represents a novel cellular target with potential therapeutic implications.

Bien que la consommation du fructose soit associée à un stockage accru de graisses par l'action de l'insuline, le fructose seul n'induit pas la sécrétion de l'insuline par la cellule bêta-pancréatique, contrairement au glucose. Nous avons étudié les effets d'une exposition chronique au fructose sur la fonction des cellules bêta. Nos résultats révèlent que le fructose potentialise la sécrétion de l'insuline stimulée par le glucose en activant la voie de signalisation de l'adénosine triphosphate (ATP) extracellulaire. Cet effet est médié par l'activation des récepteurs purinérgiques P2Y1 et est associé à la libération d'ATP cellulaire par les canaux pannexines-1. En conséquence, l'interaction entre les canaux pannexines et les récepteurs purinérgiques via l'ATP extracellulaire représente une nouvelle cible cellulaire, offrant de potentielles implications thérapeutiques.

Beta-cell mitochondrial carriers and the diabetogenic stress response.

Mitochondria play a central role in pancreatic beta-cells by coupling metabolism of the secretagogue glucose to distal events of regulated insulin exocytosis. This process requires transports of both metabolites and nucleotides in and out of the mitochondria. The molecular identification of mitochondrial carriers and their respective contribution to beta-cell function have been uncovered only recently. In type 2 diabetes, mitochondrial dysfunction is an early event and may precipitate beta-cell loss. Under diabetogenic conditions, characterized by glucotoxicity and lipotoxicity, the expression profile of mitochondrial carriers is selectively modified. This review describes the role of mitochondrial carriers in beta-cells and the selective changes in response to glucolipotoxicity. In particular, we discuss the importance of the transfer of metabolites (pyruvate, citrate, malate, and glutamate) and nucleotides (ATP, NADH, NADPH) for beta-cell function and dysfunction. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.

PAX4 Defines an Expandable ß-Cell Subpopulation in the Adult Pancreatic Islet.

PAX4 is a key regulator of pancreatic islet development whilst in adult acute overexpression protects ß-cells against stress-induced apoptosis and stimulates proliferation. Nonetheless, sustained PAX4 expression promotes ß-cell dedifferentiation and hyperglycemia, mimicking ß-cell failure in diabetic patients. Herein, we study mechanisms that allow stringent PAX4 regulation endowing favorable ß-cell adaptation in response to changing environment without loss of identity. To this end, PAX4 expression was monitored using a mouse bearing the enhanced green fluorescent protein (GFP) and cre recombinase construct under the control of the islet specific pax4 promoter. GFP was detected in 30% of islet cells predominantly composed of PAX4-enriched ß-cells that responded to glucose-induced insulin secretion. Lineage tracing demonstrated that all islet cells were derived from PAX4(+) progenitor cells but that GFP expression was confined to a subpopulation at birth which declined with age correlating with reduced replication. However, this GFP(+) subpopulation expanded during pregnancy, a state of active ß-cell replication. Accordingly, enhanced proliferation was exclusively detected in GFP(+) cells consistent with cell cycle genes being stimulated in PAX4-overexpressing islets. Under stress conditions, GFP(+) cells were more resistant to apoptosis than their GFP(-) counterparts. Our data suggest PAX4 defines an expandable ß-cell sub population within adult islets.

Diabetogenic milieus induce specific changes in mitochondrial transcriptome and differentiation of human pancreatic islets.

In pancreatic ß-cells, mitochondria play a central role in coupling glucose metabolism to insulin secretion. Chronic exposure of ß-cells to metabolic stresses impairs their function and potentially induces apoptosis. Little is known on mitochondrial adaptation to metabolic stresses, i.e. high glucose, fatty acids or oxidative stress; being all highlighted in the pathogenesis of type 2 diabetes. Here, human islets were exposed for 3 days to 25 mm glucose, 0.4 mm palmitate, 0.4 mm oleate and transiently to H2O2. Culture at physiological 5.6 mm glucose served as no-stress control. Expression of mitochondrion-associated genes was quantified, including the transcriptome of mitochondrial inner membrane carriers. Targets of interest were further evaluated at the protein level. Three days after acute oxidative stress, no significant alteration in ß-cell function or apoptosis was detected in human islets. Palmitate specifically increased expression of the pyruvate carriers MPC1 and MPC2, whereas the glutamate carrier GC1 and the aspartate/glutamate carrier AGC1 were down-regulated by palmitate and oleate, respectively. High glucose decreased mRNA levels of key transcription factors (HNF4A, IPF1, PPARA and TFAM) and energy-sensor SIRT1. High glucose also reduced expression of 11 mtDNA-encoded respiratory chain subunits. Interestingly, transcript levels of the carriers for aspartate/glutamate AGC2, malate DIC and malate/oxaloacetate/aspartate UCP2 were increased by high glucose, a profile suggesting important mitochondrial anaplerotic/cataplerotic activities and NADPH-generating shuttles. Chronic exposure to high glucose impaired glucose-stimulated insulin secretion, decreased insulin content, promoted caspase-3 cleavage and cell death, revealing glucotoxicity. Overall, expression profile of mitochondrion-associated genes was selectively modified by glucose, delineating a glucotoxic-specific signature.

Changes in mitochondrial carriers exhibit stress-specific signatures in INS-1Eß-cells exposed to glucose versus fatty acids.

Chronic exposure of ß-cells to metabolic stresses impairs their function and potentially induces apoptosis. Mitochondria play a central role in coupling glucose metabolism to insulin secretion. However, little is known on mitochondrial responses to specific stresses; i.e. low versus high glucose, saturated versus unsaturated fatty acids, or oxidative stress. INS-1E cells were exposed for 3 days to 5.6 mM glucose, 25 mM glucose, 0.4 mM palmitate, and 0.4 mM oleate. Culture at standard 11.1 mM glucose served as no-stress control and transient oxidative stress (200 µM H2O2 for 10 min at day 0) served as positive stressful condition. Mito-array analyzed transcripts of 60 mitochondrion-associated genes with special focus on members of the Slc25 family. Transcripts of interest were evaluated at the protein level by immunoblotting. Bioinformatics analyzed the expression profiles to delineate comprehensive networks. Chronic exposure to the different metabolic stresses impaired glucose-stimulated insulin secretion; revealing glucotoxicity and lipo-dysfunction. Both saturated and unsaturated fatty acids increased expression of the carnitine/acylcarnitine carrier CAC, whereas the citrate carrier CIC and energy sensor SIRT1 were specifically upregulated by palmitate and oleate, respectively. High glucose upregulated CIC, the dicarboxylate carrier DIC and glutamate carrier GC1. Conversely, it reduced expression of energy sensors (AMPK, SIRT1, SIRT4), metabolic genes, transcription factor PDX1, and anti-apoptotic Bcl2. This was associated with caspase-3 cleavage and cell death. Expression levels of GC1 and SIRT4 exhibited positive and negative glucose dose-response, respectively. Expression profiles of energy sensors and mitochondrial carriers were selectively modified by the different conditions, exhibiting stress-specific signatures.

NADPH oxidase NOX2 defines a new antagonistic role for reactive oxygen species and cAMP/PKA in the regulation of insulin secretion.

In insulin-secreting cells, expression of NADPH oxidase (NOX), a potent source of ROS, has been reported, along with controversial findings regarding its function. Here, the role of NOXs was investigated: first by expression and cellular localization in mouse and human pancreatic islets, and then by functional studies in islets isolated from Nox isoform-specific knockout mice. Both human and mouse ß-cells express NOX, in particular NOX2. With use of Nox isoform-specific knockout mice, functional analysis revealed Nox2 as the predominant isoform. In human islets, NOX2 colocalized with both insulin granules and endosome/lysosome membranes. Nox2-deficient islets stimulated with 22.8 mmol/L glucose exhibited potentiation of insulin release compared with controls, an effect confirmed with in vitro knockdown of Nox2. The enhanced secretory function in Nox2-deficient islets was associated with both lower superoxide levels and elevated cAMP concentrations. In control islets, GLP-1 and other cAMP inducers suppressed glucose-induced ROS production similarly to Nox2 deficiency. Inhibiting cAMP-dependent protein kinase reduced the secretory response in Nox2-null islets, although not in control islets. This study ascribes a new role for NOX2 in pancreatic ß-cells as negative modulator of the secretory response, reducing cAMP/PKA signaling secondary to ROS generation. Results also show reciprocal inhibition between the cAMP/PKA pathway and ROS.

Mitochondrial dysfunction in pancreatic ß cells.

In pancreatic ß cells, mitochondria play a central role in coupling glucose metabolism to insulin exocytosis, thereby ensuring strict control of glucose-stimulated insulin secretion. Defects in mitochondrial function impair this metabolic coupling, and ultimately promote apoptosis and ß cell death. Various factors have been identified that may contribute to mitochondrial dysfunction. In this review we address the emerging concept of complex links between these factors. We also discuss the role of the mitochondrial genome and mutations associated with diabetes, the effect of oxidative stress and reactive oxygen species, the sensitivity of mitochondria to lipotoxicity, and the adaptive dynamics of mitochondrial morphology. Better comprehension of the molecular mechanisms contributing to mitochondrial dysfunction will help drive the development of effective therapeutic approaches.

In vivo conditional Pax4 overexpression in mature islet ß-cells prevents stress-induced hyperglycemia in mice.

OBJECTIVE: To establish the role of the transcription factor Pax4 in pancreatic islet expansion and survival in response to physiological stress and its impact on glucose metabolism, we generated transgenic mice conditionally and selectively overexpressing Pax4 or a diabetes-linked mutant variant (Pax4R129W) in ß-cells. RESEARCH DESIGN AND METHODS: Glucose homeostasis and ß-cell death and proliferation were assessed in Pax4- or Pax4R129W-overexpressing transgenic animals challenged with or without streptozotocin. Isolated transgenic islets were also exposed to cytokines, and apoptosis was evaluated by DNA fragmentation or cytochrome C release. The expression profiles of proliferation and apoptotic genes and ß-cell markers were studied by immunohistochemistry and quantitative RT-PCR. RESULTS: Pax4 but not Pax4R129W protected animals against streptozotocin-induced hyperglycemia and isolated islets from cytokine-mediated ß-cell apoptosis. Cytochrome C release was abrogated in Pax4 islets treated with cytokines. Interleukin-1ß transcript levels were suppressed in Pax4 islets, whereas they were increased along with NOS2 in Pax4R129W islets. Bcl-2, Cdk4, and c-myc expression levels were increased in Pax4 islets while MafA, insulin, and GLUT2 transcript levels were suppressed in both animal models. Long-term Pax4 expression promoted proliferation of a Pdx1-positive cell subpopulation while impeding insulin secretion. Suppression of Pax4 rescued this defect with a concomitant increase in pancreatic insulin content. CONCLUSIONS: Pax4 protects adult islets from stress-induced apoptosis by suppressing selective nuclear factor-κB target genes while increasing Bcl-2 levels. Furthermore, it promotes dedifferentiation and proliferation of ß-cells through MafA repression, with a concomitant increase in Cdk4 and c-myc expression.

Resveratrol potentiates glucose-stimulated insulin secretion in INS-1E beta-cells and human islets through a SIRT1-dependent mechanism.

Resveratrol, a polyphenol compound, is known for its effects on energy homeostasis. With properties of energy sensors mediating effects of calorie restriction, sirtuins are targets of resveratrol. The mammalian sirtuin homolog SIRT1 is a protein deacetylase playing a role in glucose metabolism and islet function. Here, we investigated the effects of resveratrol and possible link with SIRT1 in ß-cells. Insulinoma INS-1E cells and human islets were cultured with resveratrol before analyzing their physiological responses. Treatment of INS-1E cells for 24 h with 25 µM resveratrol resulted in marked potentiation of glucose-stimulated insulin secretion. This effect was associated with elevated glycolytic flux, resulting in increased glucose oxidation, ATP generation, and mitochondrial oxygen consumption. Such changes correlated with up-regulation of key genes for ß-cell function, i.e. Glut2, glucokinase, Pdx-1, Hnf-1α, and Tfam. In human islets, chronic resveratrol treatment similarly increased both the glucose secretory response and expression of the same set of genes, eventually restoring the glucose response in islets obtained from one type 2 diabetic donor. Overexpression of Sirt1 in INS-1E cells potentiated resveratrol effects on insulin secretion. Conversely, inhibition of SIRT1 achieved either by expression of an inactive mutant or by using the EX-527 inhibitor, both abolished resveratrol effects on glucose responses. Treatment of INS-1E cells with EX-527 also prevented resveratrol-induced up-regulation of Glut2, glucokinase, Pdx-1, and Tfam. Resveratrol markedly enhanced the glucose response of INS-1E cells and human islets, even after removal of the compound from the medium. These effects were mediated by and fully dependent on active SIRT1, defining a new role for SIRT1 in the regulation of insulin secretion.

Role of mitochondria in beta-cell function and dysfunction.

Pancreatic beta-cells are poised to sense glucose and other nutrient secretagogues to regulate insulin exocytosis, thereby maintaining glucose homeostasis. This process requires translation of metabolic substrates into intracellular messengers recognized by the exocytotic machinery. Central to this metabolism-secretion coupling, mitochondria integrate and generate metabolic signals, thereby connecting glucose recognition to insulin exocytosis. In response to a glucose rise, nucleotides and metabolites are generated by mitochondria and participate, together with cytosolic calcium, to the stimulation of insulin release. This review describes the mitochondrion-dependent pathways of regulated insulin secretion. Mitochondrial defects, such as mutations and reactive oxygen species production, are discussed in the context of beta-cell failure that may participate to the etiology of diabetes.

Transient oxidative stress damages mitochondrial machinery inducing persistent beta-cell dysfunction.

Transient exposure of beta-cells to oxidative stress interrupts the transduction of signals normally coupling glucose metabolism to insulin secretion. We investigated putative persistence of effects induced by one transient oxidative stress (200 microm H(2)O(2), 10 min) on insulin secreting cells following recovery periods of days and weeks. Three days after oxidative stress INS-1E cells and rat islets exhibited persistent dysfunction. In particular, the secretory response to 15 mm glucose was reduced by 40% in INS-1E cells stressed 3 days before compared with naïve cells. Compared with non-stressed INS-1E cells, we observed reduced oxygen consumption (-43%) and impaired glucose-induced ATP generation (-46%). These parameters correlated with increased mitochondrial reactive oxygen species formation (+60%) accompanied with down-regulation of subunits of the respiratory chain and decreased expression of genes responsible for mitochondrial biogenesis (TFAM, -24%; PGC-1alpha, -67%). Three weeks after single oxidative stress, both mitochondrial respiration and secretory responses were recovered. Moreover, such recovered INS-1E cells exhibited partial resistance to a second transient oxidative stress and up-regulation of UCP2 (+78%) compared with naïve cells. In conclusion, one acute oxidative stress induces beta-cell dysfunction lasting over days, explained by persistent damages in mitochondrial components.

A focus on the role of Pax4 in mature pancreatic islet beta-cell expansion and survival in health and disease.

Blood glucose homeostasis is achieved by the regulation of insulin and glucagon secretion from the pancreatic islet beta- and alpha-cells. Diabetes mellitus, which comprises a heterogeneous group of hyperglycaemic disorders, results mainly from inadequate mass and function of islet beta-cells. Autoimmune destruction of beta-cells causes type 1 diabetes, while type 2 is characterized by impaired insulin secretion and is often associated with diminished insulin action on its target tissues. Interestingly, similar to type 1 diabetes, a gradual loss of beta-cell mass is observed in type 2 diabetes often requiring insulin therapy. Understanding the molecular mechanism that governs beta-cell mass plasticity may provide a means to develop strategies to countera,ct beta-cell death while increasing replication. Of particular interest is the islet-specific transcription factor paired box4 (Pax4) that was previously shown to be indispensable for the establishment of the beta-cell lineage during development. However, recent accumulating evidence now suggest that Pax4 is also crucial for mature beta-cell expansion and survival in response to physiological cues and that mutations or polymorphisms are associated with both type 1 and type 2 diabetes. In contrast, aberrant expression of Pax4 confers protection against apoptosis to insulinomas, whereas it promotes cell growth in lymphocytes. This review summarizes promising new published results supporting the important function of Pax4 in mature islet beta-cell physiology and its contribution to pathophysiology when deregulated.

The diabetes-linked transcription factor Pax4 is expressed in human pancreatic islets and is activated by mitogens and GLP-1.

We previously demonstrated that the transcription factor Pax4 is important for beta-cell replication and survival in rat islets. Herein, we investigate Pax4 expression in islets of non-diabetic and diabetic donors, its regulation by mitogens, glucose and the incretin GLP-1 and evaluate its effect on human islet proliferation. Pax4 expression was increased in islets derived from Type 2 diabetic donors correlating with hyperglycaemia. In vitro studies on non diabetic islets demonstrated that glucose, betacellulin, activin A, GLP-1 and insulin increased Pax4 mRNA levels. Glucose-induced Pax4 expression was abolished by the inhibitors LY294002, PD98050 or H89. Surprisingly, increases in Pax4 expression did not prompt a surge in human islet cell replication. Furthermore, expression of the proliferation marker gene Id2 remained unaltered. Adenoviral-mediated expression of human Pax4 resulted in a small increase in Bcl-xL expression while Id2 transcript levels and cell replication were unchanged in human islets. In contrast, overexpression of mouse Pax4 induced human islet cell proliferation. Treatment of islets with 5-Aza-2'-deoxycytidine induced Pax4 without stimulating Bcl-xL and Id2 expression. Human Pax4 DNA binding activity was found to be lower than that of the mouse homologue. Thus, human pax4 gene expression is epigenetically regulated and induced by physiological stimuli through the concerted action of multiple signalling pathways. However, it is unable to initiate the transcriptional replication program likely due to post-translational modifications of the protein. The latter highlights fundamental differences between human and rodent islet physiology and emphasizes the importance of validating results obtained with animal models in human tissues.

Transcriptional response of pancreatic beta cells to metabolic stimulation: large scale identification of immediate-early and secondary response genes.

BACKGROUND: Physiological long term adaptation of pancreatic beta cells is driven by stimuli such as glucose and incretin hormones acting via cAMP (e.g. GLP-1) and involves regulated gene expression. Several rapidly inducible immediate-early genes (IEGs) have been identified in beta cells. Many of these IEGs code for transcription factors and have the potential to control the transcription of downstream target genes likely involved in long term cellular adaptation. The identity of these target genes has not been determined, and the sequence of events occurring during beta cell adaptation is still unclear. RESULTS: We have developed a microarray-based strategy for the systematic search of targets. In Min6 insulin-secreting cells, we identified 592 targets and 1278 IEGs responding to a co-stimulation with glucose and cAMP. Both IEGs and targets were involved in a large panel of functions, including those important to beta cell physiology (metabolism, secretion). Nearly 200 IEGs were involved in signaling and transcriptional regulation. To find specific examples of the regulatory link between IEGs and targets, target promoter sequences were analyzed in silico. Statistically significant over-representation of AP-1 response elements notably suggested an important role for this transcription factor, which was experimentally verified. Indeed, cell stimulation altered expression of IEG-encoded components of the AP-1 complex, activating AP-1-dependent transcription. Loss and gain-of-function experiments furthermore allowed to validate a new AP-1 regulated gene (sulfiredoxin) among the targets. AP-1 and sulfiredoxin are sequentially induced also in primary cells from rat islets of Langerhans. CONCLUSION: By identifying IEGs and their downstream targets, this study brings a comprehensive description of the transcriptional response occurring after beta cell stimulation, as well as new mechanistic insights concerning the AP-1 transcription factor.