Impaired Ca2+ Signaling in β-Cells Lacking Leptin Receptors by Cre-loxP Recombination

Obesity is a major risk factor for diabetes and is typically associated with hyperleptinemia and a state of leptin resistance. The impact of chronically elevated leptin levels on the function of insulin-secreting β-cells has not been elucidated. We previously generated mice lacking leptin signaling in β-cells by using the Cre-loxP strategy and showed that these animals develop increased body weight and adiposity, hyperinsulinemia, impaired glucose-stimulated insulin secretion and insulin resistance. Here, we performed several in vitro studies and observed that β-cells lacking leptin signaling in this model are capable of properly metabolizing glucose, but show impaired intracellular Ca²⁺ oscillations and lack of synchrony within the islets in response to glucose, display reduced response to tolbutamide and exhibit morphological abnormalities including increased autophagy. Defects in intracellular Ca²⁺ signaling were observed even in neonatal islets, ruling out the possible contribution of obesity to the β-cell irregularities observed in adults. In parallel, we also detected a disrupted intracellular Ca²⁺ pattern in response to glucose and tolbutamide in control islets from adult transgenic mice expressing Cre recombinase under the rat insulin promoter, despite these animals being glucose tolerant and secreting normal levels of insulin in response to glucose. This unexpected observation impeded us from discerning the consequences of impaired leptin signaling as opposed to long-term Cre expression in the function of insulin-secreting cells. These findings highlight the need to generate improved Cre-driver mouse models or new tools to induce Cre recombination in β-cells.     

Computational Modeling of Glucose Transport in Pancreatic β-Cells Identifies Metabolic Thresholds and Therapeutic Targets in Diabetes

Pancreatic β-cell dysfunction is a diagnostic criterion of Type 2 diabetes and includes defects in glucose transport and insulin secretion. In healthy individuals, β-cells maintain plasma glucose concentrations within a narrow range in concert with insulin action among multiple tissues. Postprandial elevations in blood glucose facilitate glucose uptake into β-cells by diffusion through glucose transporters residing at the plasma membrane. Glucose transport is essential for glycolysis and glucose-stimulated insulin secretion. In human Type 2 diabetes and in the mouse model of obesity-associated diabetes, a marked deficiency of β-cell glucose transporters and glucose uptake occurs with the loss of glucose-stimulated insulin secretion. Recent studies have shown that the preservation of glucose transport in β-cells maintains normal insulin secretion and blocks the development of obesity-associated diabetes. To further elucidate the underlying mechanisms, we have constructed a computational model of human β-cell glucose transport in health and in Type 2 diabetes, and present a systems analysis based on experimental results from human and animal studies. Our findings identify a metabolic threshold or “tipping point” whereby diminished glucose transport across the plasma membrane of β-cells limits intracellular glucose-6-phosphate production by glucokinase. This metabolic threshold is crossed in Type 2 diabetes and results in β-cell dysfunction including the loss of glucose stimulated insulin secretion. Our model further discriminates among molecular control points in this pathway wherein maximal therapeutic intervention is achieved.     

Preconditioning with Associated Blocking of Ca2+ Inflow Alleviates Hypoxia-Induced Damage to Pancreatic β-Cells

Objective

Beta cells of pancreatic islets are susceptible to functional deficits and damage by hypoxia. Here we aimed to characterize such effects and to test for and pharmacological means to alleviate a negative impact of hypoxia.

Methods and Design

Rat and human pancreatic islets were subjected to 5.5 h of hypoxia after which functional and viability parameters were measured subsequent to the hypoxic period and/or following a 22 h re-oxygenation period. Preconditioning with diazoxide or other agents was usually done during a 22 h period prior to hypoxia.

Results

Insulin contents decreased by 23% after 5.5 h of hypoxia and by 61% after a re-oxygenation period. Preconditioning with diazoxide time-dependently alleviated these hypoxia effects in rat and human islets. Hypoxia reduced proinsulin biosynthesis (³H-leucine incorporation into proinsulin) by 35%. Preconditioning counteracted this decrease by 91%. Preconditioning reduced hypoxia-induced necrosis by 40%, attenuated lowering of proteins of mitochondrial complexes I–IV and enhanced stimulation of HIF-1-alpha and phosphorylated AMPK proteins. Preconditioning by diazoxide was abolished by co-exposure to tolbutamide or elevated potassium (i.e. conditions which increase Ca²⁺ inflow). Preconditioning with nifedipine, a calcium channel blocker, partly reproduced effects of diazoxide. Both diazoxide and nifedipine moderately reduced basal glucose oxidation whereas glucose-induced oxygen consumption (tested with diazoxide) was unaffected. Preconditioning with diaxoxide enhanced insulin contents in transplants of rat islets to non-diabetic rats and lowered hyperglycemia vs. non-preconditioned islets in streptozotocin-diabetic rats. Preconditioning of human islet transplants lowered hyperglycemia in streptozotocin-diabetic nude mice.

Conclusions

1) Prior blocking of Ca²⁺ inflow associates with lesser hypoxia-induced damage, 2) preconditioning affects basal mitochondrial metabolism and accelerates activation of hypoxia-reactive and potentially protective factors, 3) results indicate that preconditioning by K⁺-ATP-channel openers has therapeutic potential for islet transplantations.     

Concerted Trafficking Regulation of Kv2.1 and KATP Channels by Leptin in Pancreatic β-Cells

In pancreatic β-cells, voltage-gated potassium 2.1 (Kv2.1) channels are the dominant delayed rectifier potassium channels responsible for action potential repolarization. Here, we report that leptin, a hormone secreted by adipocytes known to inhibit insulin secretion, causes a transient increase in surface expression of Kv2.1 channels in rodent and human β-cells. The effect of leptin on Kv2.1 surface expression is mediated by the AMP-activated protein kinase (AMPK). Activation of AMPK mimics whereas inhibition of AMPK occludes the effect of leptin. Inhibition of Ca²⁺/calmodulin-dependent protein kinase kinase β, a known upstream kinase of AMPK, also blocks the effect of leptin. In addition, the cAMP-dependent protein kinase (PKA) is involved in Kv2.1 channel trafficking regulation. Inhibition of PKA prevents leptin or AMPK activators from increasing Kv2.1 channel density, whereas stimulation of PKA is sufficient to promote Kv2.1 channel surface expression. The increased Kv2.1 surface expression by leptin is dependent on actin depolymerization, and pharmacologically induced actin depolymerization is sufficient to enhance Kv2.1 surface expression. The signaling and cellular mechanisms underlying Kv2.1 channel trafficking regulation by leptin mirror those reported recently for ATP-sensitive potassium (K_ATP) channels, which are critical for coupling glucose stimulation with membrane depolarization. We show that the leptin-induced increase in surface K_ATP channels results in more hyperpolarized membrane potentials than control cells at stimulating glucose concentrations, and the increase in Kv2.1 channels leads to a more rapid repolarization of membrane potential in cells firing action potentials. This study supports a model in which leptin exerts concerted trafficking regulation of K_ATP and Kv2.1 channels to coordinately inhibit insulin secretion.     

Store-operated Ca2+ Entry Mediated by Orai1 and TRPC1 Participates to Insulin Secretion in Rat β-Cells

Store-operated Ca²⁺ channels (SOCs) are voltage-independent Ca²⁺ channels activated upon depletion of the endoplasmic reticulum Ca²⁺ stores. Early studies suggest the contribution of such channels to Ca²⁺ homeostasis in insulin-secreting pancreatic β-cells. However, their composition and contribution to glucose-stimulated insulin secretion (GSIS) remains unclear. In this study, endoplasmic reticulum Ca²⁺ depletion triggered by acetylcholine (ACh) or thapsigargin stimulated the formation of a ternary complex composed of Orai1, TRPC1, and STIM1, the key proteins involved in the formation of SOCs. Ca²⁺ imaging further revealed that Orai1 and TRPC1 are required to form functional SOCs and that these channels are activated by STIM1 in response to thapsigargin or ACh. Pharmacological SOCs inhibition or dominant negative blockade of Orai1 or TRPC1 using the specific pore mutants Orai1-E106D and TRPC1-F562A impaired GSIS in rat β-cells and fully blocked the potentiating effect of ACh on secretion. In contrast, pharmacological or dominant negative blockade of TRPC3 had no effect on extracellular Ca²⁺ entry and GSIS. Finally, we observed that prolonged exposure to supraphysiological glucose concentration impaired SOCs function without altering the expression levels of STIM1, Orai1, and TRPC1. We conclude that Orai1 and TRPC1, which form SOCs regulated by STIM1, play a key role in the effect of ACh on GSIS, a process that may be impaired in type 2 diabetes.     

Glucagon Induces Suppression of ATP-sensitive K+ Channel Activity Through a Ca2+/Calmodulin-dependent Pathway in Mouse Pancreatic β-Cells

Glucagon is known to increase intracellular cAMP levels and enhance glucose-induced electrical activity and insulin secretion in pancreatic β-cell perfused with Krebs-Ringer bicarbonate solution. The present experiments were aimed at evaluation of the hypothesis that changes in β-cells ATP-sensitive K⁺ (K_(ATP)) channel activity are involved in the glucagon-induced enhancement of electrical activity. Channel activity was recorded using the cell-attached configuration of the patch-clamp technique. Addition of glucagon (2.9 × 10⁻⁷ m) in the presence of 11.1 mm glucose caused closure of K_(ATP) channels followed by an increase in the frequency of biphasic current transients (action currents) due to action potential generation in the cell. Three calmodulin-antagonists (W-7, chlorpromazine, and trifluoperazine) restored with similar efficacy K_(ATP) channel activity in cells being exposed to glucagon. At 2.8 mm glucose, glucagon did not affect K_(ATP) channel activity until Ca²⁺ was released from Nitr-5 by flash photolysis, at which point channel activity was transiently suppressed. Similar effects were seen when db-cAMP was used instead of glucagon.These results support the view that glucagon and other cAMP-generating agonists enhance glucose-induced β-cell electrical activity through a Ca²⁺/calmodulin dependent-closure of K_(ATP) channels. Received: 26 May 1998/Revised: 18 September 1998     

Insulin Secretion and Ca2+ Dynamics in β-Cells Are Regulated by PERK (EIF2AK3) in Concert with Calcineurin

Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) (EIF2AK3) is essential for normal development and function of the insulin-secreting β-cell. Although genetic ablation of PERK in β-cells results in permanent neonatal diabetes in humans and mice, the underlying mechanisms remain unclear. Here, we used a newly developed and highly specific inhibitor of PERK to determine the immediate effects of acute ablation of PERK activity. We found that inhibition of PERK in human and rodent β-cells causes a rapid inhibition of secretagogue-stimulated subcellular Ca²⁺ signaling and insulin secretion. These dysfunctions stem from alterations in store-operated Ca²⁺ entry and sarcoplasmic endoplasmic reticulum Ca²⁺-ATPase activity. We also found that PERK regulates calcineurin, and pharmacological inhibition of calcineurin results in similar defects on stimulus-secretion coupling. Our findings suggest that interplay between calcineurin and PERK regulates β-cell Ca²⁺ signaling and insulin secretion, and that loss of this interaction may have profound implications in insulin secretion defects associated with diabetes.     

Inhibition of Autophagic Turnover in β-Cells by Fatty Acids and Glucose Leads to Apoptotic Cell Death

Autophagy, a cellular recycling process responsible for turnover of cytoplasmic contents, is critical for maintenance of health. Defects in this process have been linked to diabetes. Diabetes-associated glucotoxicity/lipotoxicity contribute to impaired β-cell function and have been implicated as contributing factors to this disease. We tested the hypothesis that these two conditions affect β-cell function by modulating autophagy. We report that exposure of β-cell lines and human pancreatic islets to high levels of glucose and lipids blocks autophagic flux and leads to apoptotic cell death. EM analysis showed accumulation of autophagy intermediates (autophagosomes), with abundant engulfed cargo in palmitic acid (PA)- or glucose-treated cells, indicating suppressed autophagic turnover. EM studies also showed accumulation of damaged mitochondria, endoplasmic reticulum distention, and vacuolar changes in PA-treated cells. Pulse-chase experiments indicated decreased protein turnover in β-cells treated with PA/glucose. Expression of mTORC1, an inhibitor of autophagy, was elevated in β-cells treated with PA/glucose. mTORC1 inhibition, by treatment with rapamycin, reversed changes in autophagic flux, and cell death induced by glucose/PA. Our results indicate that nutrient toxicity-induced cell death occurs via impaired autophagy and is mediated by activation of mTORC1 in β-cells, contributing to β-cell failure in the presence of metabolic stress.     

Control by Ca2+ of mitochondrial structure and function in pancreatic β-cells

Mitochondria play a central role in glucose metabolism and the stimulation of insulin secretion from pancreatic β-cells. In this review, we discuss firstly the regulation and roles of mitochondrial Ca²⁺ transport in glucose-regulated insulin secretion, and the molecular machinery involved. Next, we discuss the evidence that mitochondrial dysfunction in β-cells is associated with type 2 diabetes, from a genetic, functional and structural point of view, and then the possibility that these changes may in part be mediated by dysregulation of cytosolic Ca²⁺. Finally, we review the importance of preserved mitochondrial structure and dynamics for mitochondrial gene expression and their possible relevance to the pathogenesis of type 2 diabetes.     

Temporal Proteomic Analysis of Pancreatic β-Cells in Response to Lipotoxicity and Glucolipotoxicity

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Highlights

• •Temporal proteome profiling of lipotoxicity and glucolipotoxicity in β-cells
• •Palmitate induced cholesterol metabolism earlier than fatty acid metabolism
• •Setd8 promotes palmitate + glucose-stimulated INS-1 cell proliferation
• •PA induced apoptosis partially via upregulation of Rhob in INS-1 cells

     

Mitochondrial Ca2+ Uptake 1 (MICU1) and Mitochondrial Ca2+ Uniporter (MCU) Contribute to Metabolism-Secretion Coupling in Clonal Pancreatic ��-Cells

In pancreatic β-cells, uptake of Ca²⁺ into mitochondria facilitates metabolism-secretion coupling by activation of various matrix enzymes, thus facilitating ATP generation by oxidative phosphorylation and, in turn, augmenting insulin release. We employed an siRNA-based approach to evaluate the individual contribution of four proteins that were recently described to be engaged in mitochondrial Ca²⁺ sequestration in clonal INS-1 832/13 pancreatic β-cells: the mitochondrial Ca²⁺ uptake 1 (MICU1), mitochondrial Ca²⁺ uniporter (MCU), uncoupling protein 2 (UCP2), and leucine zipper EF-hand-containing transmembrane protein 1 (LETM1). Using a FRET-based genetically encoded Ca²⁺ sensor targeted to mitochondria, we show that a transient knockdown of MICU1 or MCU diminished mitochondrial Ca²⁺ uptake upon both intracellular Ca²⁺ release and Ca²⁺ entry via L-type channels. In contrast, knockdown of UCP2 and LETM1 exclusively reduced mitochondrial Ca²⁺ uptake in response to either intracellular Ca²⁺ release or Ca²⁺ entry, respectively. Therefore, we further investigated the role of MICU1 and MCU in metabolism-secretion coupling. Diminution of MICU1 or MCU reduced mitochondrial Ca²⁺ uptake in response to d-glucose, whereas d-glucose-triggered cytosolic Ca²⁺ oscillations remained unaffected. Moreover, d-glucose-evoked increases in cytosolic ATP and d-glucose-stimulated insulin secretion were diminished in MICU1- or MCU-silenced cells. Our data highlight the crucial role of MICU1 and MCU in mitochondrial Ca²⁺ uptake in pancreatic β-cells and their involvement in the positive feedback required for sustained insulin secretion.     

Glucose inhibits 45Ca efflux from pancreatic β-cells also in the absence of Na+-Ca2+ countertransport

During perifusion with medium deprived of Ca²⁺, addition of glucose or omission of Na⁺ resulted in prompt and quantitatively similar inhibitions of ⁴⁵Ca efflux from β-cell rich pancreatic islets microdissected from ob / ob mice. Glucose had no additional inhibitory effect when Na⁺ was isoosmotically replaced by sucrose or choline⁺. When K⁺ was used as a substitute for Na⁺, the inhibitory effect of Na⁺ removal on ⁴⁵Ca efflux became additive to that of glucose. The observation that glucose can be equally effective in inhibiting ⁴⁵Ca efflux in the presence or absence of Na⁺ is difficult to reconcile with the postulate that the Na⁺-Ca²⁺ countertransport mechanism is a primary site of action for glucose.     

The GTPase RalA Regulates Different Steps of the Secretory Process in Pancreatic β-Cells

Background

RalA and RalB are multifuntional GTPases involved in a variety of cellular processes including proliferation, oncogenic transformation and membrane trafficking. Here we investigated the mechanisms leading to activation of Ral proteins in pancreatic β-cells and analyzed the impact on different steps of the insulin-secretory process.

Methodology/Principal Findings

We found that RalA is the predominant isoform expressed in pancreatic islets and insulin-secreting cell lines. Silencing of this GTPase in INS-1E cells by RNA interference led to a decrease in secretagogue-induced insulin release. Real-time measurements by fluorescence resonance energy transfer revealed that RalA activation in response to secretagogues occurs within 3–5 min and reaches a plateau after 10–15 min. The activation of the GTPase is triggered by increases in intracellular Ca²⁺ and cAMP and is prevented by the L-type voltage-gated Ca²⁺ channel blocker Nifedipine and by the protein kinase A inhibitor H89. Defective insulin release in cells lacking RalA is associated with a decrease in the secretory granules docked at the plasma membrane detected by Total Internal Reflection Fluorescence microscopy and with a strong impairment in Phospholipase D1 activation in response to secretagogues. RalA was found to be activated by RalGDS and to be severely hampered upon silencing of this GDP/GTP exchange factor. Accordingly, INS-1E cells lacking RalGDS displayed a reduction in hormone secretion induced by secretagogues and in the number of insulin-containing granules docked at the plasma membrane.

Conclusions/Significance

Taken together, our data indicate that RalA activation elicited by the exchange factor RalGDS in response to a rise in intracellular Ca²⁺ and cAMP controls hormone release from pancreatic β-cell by coordinating the execution of different events in the secretory pathway.     

Frog α- and β-Ryanodine Receptors Provide Distinct Intracellular Ca2+ Signals in a Myogenic Cell Line

Background

In frog skeletal muscle, two ryanodine receptor (RyR) isoforms, α-RyR and β-RyR, are expressed in nearly equal amounts. However, the roles and significance of the two isoforms in excitation-contraction (E-C) coupling remains to be elucidated.

Methodology/Principal Findings

In this study, we expressed either or both α-RyR and β-RyR in 1B5 RyR-deficient myotubes using the herpes simplex virus 1 helper-free amplicon system. Immunological characterizations revealed that α-RyR and β-RyR are appropriately expressed and targeted at the junctions in 1B5 myotubes. In Ca²⁺ imaging studies, each isoform exhibited caffeine-induced Ca²⁺ transients, an indicative of Ca²⁺-induced Ca²⁺ release (CICR). However, the fashion of Ca²⁺ release events was fundamentally different: α-RyR mediated graded and sustained Ca²⁺ release observed uniformly throughout the cytoplasm, whereas β-RyR supported all-or-none type regenerative Ca²⁺ oscillations and waves. α-RyR but not β-RyR exhibited Ca²⁺ transients triggered by membrane depolarization with high [K⁺]_o that were nifedipine-sensitive, indicating that only α-RyR mediates depolarization-induced Ca²⁺ release. Myotubes co-expressing α-RyR and β-RyR demonstrated high [K⁺]_o-induced Ca²⁺ transients which were indistinguishable from those with myotubes expressing α-RyR alone. Furthermore, procaine did not affect the peak height of high [K⁺]_o-induced Ca²⁺ transients, suggesting minor amplification of Ca²⁺ release by β-RyR via CICR in 1B5 myotubes.

Conclusions/Significance

These findings suggest that α-RyR and β-RyR provide distinct intracellular Ca²⁺ signals in a myogenic cell line. These distinct properties may also occur in frog skeletal muscle and will be important for E-C coupling.     

Autocrine/Paracrine Function of Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) for Glucose Homeostasis in Pancreatic β-Cells and Adipocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger for mobilizing Ca²⁺ from intracellular stores in various cell types. Extracellular application of NAADP has been shown to elicit intracellular Ca²⁺ signals, indicating that it is readily transported into cells. However, little is known about the functional role of this NAADP uptake system. Here, we show that NAADP is effectively transported into selected cell types involved in glucose homeostasis, such as adipocytes and pancreatic β-cells, but not the acinar cells, in a high glucose-dependent manner. NAADP uptake was inhibitable by Ned-19, a NAADP mimic; dipyridamole, a nucleoside inhibitor; or NaN₃, a metabolic inhibitor or under Ca²⁺-free conditions. Furthermore, NAADP was found to be released from pancreatic islets upon stimulation by high glucose. Consistently, administration of NAADP to type 2 diabetic mice improved glucose tolerance. We propose that NAADP is functioning as an autocrine/paracrine hormone important in glucose homeostasis. NAADP is thus a potential antidiabetic agent with therapeutic relevance.     

Homotypic and Heterotypic Adhesion Induced by Odorant Receptors and the β2-Adrenergic Receptor

In the mouse olfactory system regulated expression of a large family of G Protein-Coupled Receptors (GPCRs), the Odorant Receptors (ORs), provides each sensory neuron with a single OR identity. In the wiring of the olfactory sensory neuron projections, a complex axon sorting process ensures the segregation of >1,000 subpopulations of axons of the same OR identity into homogeneously innervated glomeruli. ORs are critical determinants in axon sorting, and their presence on olfactory axons raises the intriguing possibility that they may participate in axonal wiring through direct or indirect trans-interactions mediating adhesion or repulsion between axons. In the present work, we used a biophysical assay to test the capacity of ORs to induce adhesion of cell doublets overexpressing these receptors. We also tested the β2 Adrenergic Receptor, a non-OR GPCR known to recapitulate the functions of ORs in olfactory axon sorting. We report here the first evidence for homo- and heterotypic adhesion between cells overexpressing the ORs MOR256-17 or M71, supporting the hypothesis that ORs may contribute to olfactory axon sorting by mediating differential adhesion between axons.