Action of protein disulfide isomerase on proinsulin exit from endoplasmic reticulum of pancreatic β-cells

For insulin synthesis, the proinsulin precursor is translated at the endoplasmic reticulum (ER), folds to include its three native disulfide bonds, and is exported to secretory granules for processing and secretion. Protein disulfide isomerase (PDI) has long been assumed to assist proinsulin in this process. Herein we have examined the effect of PDI knockdown (PDI-KD) in β-cells. The data establish that upon PDI-KD, oxidation of proinsulin to form native disulfide bonds is unimpaired and in fact enhanced. This is accompanied by improved proinsulin exit from the ER and increased total insulin secretion, with no evidence of ER stress. We provide evidence for direct physical interaction between PDI and proinsulin in the ER of pancreatic β-cells, in a manner requiring the catalytic activity of PDI. In β-cells after PDI-KD, enhanced export is selective for proinsulin over other secretory proteins, but the same effect is observed for recombinant proinsulin trafficking upon PDI-KD in heterologous cells. We hypothesize that PDI exhibits unfoldase activity for proinsulin, increasing retention of proinsulin within the ER of pancreatic β-cells.     

Inositol hexakisphosphate kinase 1 is a metabolic sensor in pancreatic β-cells

Diphosphoinositol pentakisphosphate (IP₇) is critical for the exocytotic capacity of the pancreatic β-cell, but its regulation by the primary instigator of β-cell exocytosis, glucose, is unknown. The high K_m for ATP of the IP₇-generating enzymes, the inositol hexakisphosphate kinases (IP6K1 and 2) suggests that these enzymes might serve as metabolic sensors in insulin secreting β-cells and act as translators of disrupted metabolism in diabetes. We investigated this hypothesis and now show that glucose stimulation, which increases the ATP/ADP ratio, leads to an early rise in IP₇ concentration in β-cells. RNAi mediated knock down of the IP6K1 isoform inhibits both glucose-mediated increase in IP₇ and first phase insulin secretion, demonstrating that IP6K1 integrates glucose metabolism and insulin exocytosis. In diabetic mouse islets the deranged ATP/ADP levels under both basal and glucose-stimulated conditions are mirrored in both disrupted IP₇ generation and insulin release. Thus the unique metabolic sensing properties of IP6K1 guarantees appropriate concentrations of IP₇ and thereby both correct basal insulin secretion and intact first phase insulin release. In addition, our data suggest that a specific cell signaling defect, namely, inappropriate IP₇ generation may be an essential convergence point integrating multiple metabolic defects into the commonly observed phenotype in diabetes.     

Activation of protein kinase C-α is essential for stimulation of cell proliferation by ceramide 1-phosphate

We previously demonstrated that ceramide-1-phosphate (C1P) stimulates fibroblast and macrophage proliferation, but the mechanisms involved in this action have only been partially described. Here we demonstrate that C1P induces translocation of protein kinase C-alpha (PKC-α) from the soluble to the membrane fraction of bone marrow-derived macrophages. Translocation of this enzyme was accompanied by its phosphorylation on Ser 657 residue. Activation of PKC-α was independent of prior stimulation of phosphatidylinositol-dependent or phosphatidylcholine-dependent phospholipase C activities, but required activation of sphingomyelin synthesis. Inhibition of PKC-α activation also blocked C1P-stimulated macrophage proliferation indicating that this enzyme is essential for the mitogenic effect of C1P.     

NAD kinase regulates the size of the NADPH pool and insulin secretion in pancreatic β-cells

NADPH is an important component of the antioxidant defense system and a proposed mediator in glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells. An increase in the NADPH/NADP(+) ratio has been reported to occur within minutes following the rise in glucose concentration in β-cells. However, 30 min following the increase in glucose, the total NADPH pool also increases through a mechanism not yet characterized. NAD kinase (NADK) catalyzes the de novo formation of NADP(+) by phosphorylation of NAD(+). NAD kinases have been shown to be essential for redox regulation, oxidative stress defense, and survival in bacteria and yeast. However, studies on NADK in eukaryotic cells are scarce, and the function of this enzyme has not been described in β-cells. We employed INS-1 832/13 cells, an insulin-secreting rat β-cell line, and isolated rodent islets to investigate the role of NADK in β-cell metabolic pathways. Adenoviral-mediated overexpression of NADK resulted in a two- to threefold increase in the total NADPH pool and NADPH/NADP(+) ratio, suggesting that NADP(+) formed by the NADK-catalyzed reaction is rapidly reduced to NADPH via cytosolic reductases. This increase in the NADPH pool was accompanied by an increase in GSIS in NADK-overexpressing cells. Furthermore, NADK overexpression protected β-cells against oxidative damage by the redox cycling agent menadione and reversed menadione-mediated inhibition of GSIS. Knockdown of NADK via shRNA exerted the opposite effect on all these parameters. These data suggest that NADK kinase regulates intracellular redox and affects insulin secretion and oxidative defense in the β-cell.     

Electrophysiology of pancreatic β-cells in intact mouse islets of Langerhans

When exposed to intermediate glucose concentrations (6–16 mol/l), pancreatic β-cells in intact islets generate bursts of action potentials (superimposed on depolarised plateaux) separated by repolarised electrically silent intervals. First described more than 40 years ago, these oscillations have continued to intrigue β-cell electrophysiologists. To date, most studies of β-cell ion channels have been performed on isolated cells maintained in tissue culture (that do not burst). Here we will review the electrophysiological properties of β-cells in intact, freshly isolated, mouse pancreatic islets. We will consider the role of ATP-regulated K⁺-channels (K_ATP-channels), small-conductance Ca²⁺-activated K⁺-channels and voltage-gated Ca²⁺-channels in the generation of the bursts. Our data indicate that K_ATP-channels not only constitute the glucose-regulated resting conductance in the β-cell but also provide a variable K⁺-conductance that influence the duration of the bursts of action potentials and the silent intervals. We show that inactivation of the voltage-gated Ca²⁺-current is negligible at voltages corresponding to the plateau potential and consequently unlikely to play a major role in the termination of the burst. Finally, we propose a model for glucose-induced β-cell electrical activity based on observations made in intact pancreatic islets.     

Iodoacetamide-induced sensitization of the pancreatic β-cells to glucose stimulation

At a glucose concentration of 3mm or less, iodoacetamide had no effect on the release of insulin from microdissected pancreatic islets of ob/ob-mice. At higher glucose concentrations, iodoacetamide exerted both an initial stimulatory and a subsequent inhibitory action. When islets were perifused with 1mm-iodoacetamide and 17mm-glucose the inhibitory action predominated after about 15min of transient stimulation. With decreasing concentrations of iodoacetamide the stimulatory phase was gradually prolonged, and with 0.003-0.1mm-iodoacetamide stimulation only was observed for 75min. Prolonged stimulation was also noted after a short pulse of iodoacetamide. Similar responses to 0.1mm-iodoacetamide were observed with islets from normal mice. With islets from ob/ob-mice the effect of 0.1mm-iodoacetamide was reproduced with 0.1mm-iodoacetate, whereas 0.1mm-acetamide had no apparent effect. Iodoacetamide increased the V(max.) of glucose-stimulated insulin release without altering the apparent K(m) for glucose. Leucine, glibenclamide or theophylline could not replace glucose in this synergistic action with iodoacetamide. Iodoacetamide rather inhibited the insulin-releasing action of theophylline. Iodoacetamide-induced potentiation of the glucose-stimulated insulin release was rapidly and reversibly inhibited by mannoheptulose, adrenaline, or calcium deficiency. The potentiating effect on insulin release was not paralleled by effects on glucose oxidation or on islet fructose 1,6-diphosphate. However, the inhibitory action of iodoacetamide might be explained by inhibition of glycolysis as evidenced by an inhibition of glucose oxidation and a rise of fructose 1,6-diphosphate. The results support our previous hypothesis that thiol reagents can stimulate insulin release by acting on relatively superficial thiol groups in the beta-cell plasma membrane. Glycolysis seems to be necessary in order for iodoacetamide to stimulate in this way.     

Mechanism for antioxidative effects of thiazolidinediones in pancreatic β-cells

Thiazolidinediones (TZDs) are synthetic ligands of peroxisome proliferator-activated receptor-γ (PPARγ), a member of the nuclear receptor superfamily. TZDs are known to increase insulin sensitivity and also to have an antioxidative effect. In this study, we tested whether TZDs protect pancreatic β-cells from oxidative stress, and we investigated the mechanism involved in this process. To generate oxidative stress in pancreatic β-cells (INS-1 and βTC3) or isolated islets, glucose oxidase was added to the media. The extracellular and intracellular reactive oxygen species (ROS) were measured to directly determine the antioxidant effect of TZDs. The phosphorylation of JNK/MAPK after oxidative stress was detected by Western blot analysis, and glucose-stimulated insulin secretion and cell viability were also measured. TZDs significantly reduced the ROS levels that were increased by glucose oxidase, and they effectively prevented β-cell dysfunction. The antioxidative effect of TZDs was abolished in the presence of a PPARγ antagonist, GW9662. Real-time PCR was used to investigate the expression levels of antioxidant genes. The expression of catalase, an antioxidant enzyme, was increased by TZDs in pancreatic β-cells, and the knockdown of catalase significantly inhibited the antioxidant effect of TZDs. These results suggest that TZDs effectively protect pancreatic β-cells from oxidative stress, and this effect is dependent largely on PPARγ. In addition, the expression of catalase is increased by TZDs, and catalase, at least in part, mediates the antioxidant effect of TZDs in pancreatic β-cells.     

RyR channels and glucose-regulated pancreatic β-cells

Ryanodine receptor channel model is introduced to a dynamical model of pancreatic beta-cells to discuss the effects of RyR channels and glucose concentration on membrane potential. The results show Ca(2+) concentration changes responding to enhance of glucose concentration is more quickly than that of activating RyR channels, and both methods can induce bursting action potential and increase free cytosolic Ca(2+) concentration. An interesting finding is that moderate stimulation to RyR channels will result in a kind of "complex bursting", which is more effective in enhancing average Ca(2+) concentration and insulin section.     

Haplotype-specific sequence encoding the protein kinase, interferon-inducible double-stranded RNA-dependent activator in the human leukocyte antigen class II region

The protein kinase, interferon-inducible double-stranded (ds)RNA-dependent activator (PRKRA) is a dsRNA-binding protein which activates a protein kinase participating in the antiviral activity of interferon. Our previous studies indicated that the nucleotide sequence encoding PRKRA, which appeared to be an intronless gene, was present in PAC HS265J14 containing the human leukocyte antigen (HLA) DR subregion. In this study, we further investigated and characterized the PRKRA gene on the human genome by means of Southern blotting and polymerase chain reaction with homozygous typing cell lines for HLA genes. Results indicated that the presence of PRKRA in the DR subregion was dependent on the DR53 group. Consistently, fluorescence in situ hybridization profiles with PRKRA as a probe showed that the hybridization signal on Chromosome (Chr) 6p21.3 was seen only in the samples carrying the DR haplotypes that belonged to the DR53 group. Interestingly, another hybridization signal, which was mapped on Chr 2q31.2-q32.1, was always detected in the samples examined, i.e., even in the samples negative for the DR53 group. The outcome of a sequence-database homology search further indicated that the PRKRA gene with introns appeared to be present in a recently opened draft-sequence, RP11-65L3 (GenBank accession number AC009948), which is located between D2S335 and D2S2257. Together, the data presented here indicate that the PRKRA gene in the DR subregion is a processed pseudogene (PRKRApsi), which could have been generated only on the DR53 common ancestor's genome, and that the master copy of PRKRApsi is most probably present on Chr 2q31.2-q32.1.     

Cyclic AMP signaling stimulates proteasome degradation of thioredoxin interacting protein (TxNIP) in pancreatic β-cells

Thioredoxin interacting protein (TxNIP) functions as an effector of glucotoxicity in pancreatic β-cells. Exendin-4 (Ex-4), a long-term effective GLP-1 receptor agonist, reduces TxNIP level in pancreatic β-cells. Mechanisms underlying this reduction, however, remain largely unknown. We show here that Ex-4, 8-bromo-cAMP, the cAMP promoting agent forskolin, as well as activators of protein kinase A (PKA) and exchange protein activated by cAMP (Epac), all attenuated the effect of high glucose (20 mM) on TxNIP level in the pancreatic β-cell line Ins-1. Forskolin and Ex-4 also reduced TxNIP level in cultured primary rat islets. This repressive effect is at least partially mediated via stimulating proteasome-dependent TxNIP degradation, since the proteasomal inhibitor MG132, but not the lysosomal inhibitor chloroquine, significantly blocked the repressive effect of forskolin. Furthermore, forskolin enhanced TxNIP ubiquitination. Both PKA inhibition and Epac inhibition partially blocked the repressive effect of forskolin on TxNIP level. In addition, forskolin and Ex-4 protected Ins-1 cells from high glucose-induced apoptotic activity, assessed by measuring caspase 3 activity. Finally, knockdown of TxNIP expression led to reduced caspase 3 expression levels and blunted response to forskolin treatment. We suggest that proteasome-dependent TxNIP degradation is a novel mechanism by which Ex-4-cAMP signaling protects pancreatic β cells.     

Voltage-dependent metabolic regulation of Kv2.1 channels in pancreatic β-cells

Voltage-gated potassium channels (Kv channels) play a crucial role in formation of action potentials in response to glucose stimulation in pancreatic β-ells. We previously reported that the Kv channel is regulated by glucose metabolism, particularly by MgATP. We examined whether the regulation of Kv channels is voltage-dependent and mechanistically related with phosphorylation of the channels. In rat pancreatic β-cells, suppression of glucose metabolism with low glucose concentrations of 2.8 mM or less or by metabolic inhibitors decreased the Kv2.1-channel activity at positive membrane potentials, while increased it at potentials negative to −10 mV, suggesting that modulation of Kv channels by glucose metabolism is voltage-dependent. Similarly, in HEK293 cells expressing the recombinant Kv2.1 channels, 0 mM but not 10 mM MgATP modulated the channel activity in a manner similar to that in β-cells. Both steady-state activation and inactivation kinetics of the channel were shifted toward the negative potential in association with the voltage-dependent modulation of the channels by cytosolic dialysis of alkaline phosphatase in β-cells. The modulation of Kv-channel current-voltage relations were also observed during and after glucose-stimulated electrical excitation. These results suggest that the cellular metabolism including MgATP production and/or channel phosphorylation/dephosphorylation underlie the physiological modulation of Kv2.1 channels during glucose-induced insulin secretion.     

Systems analysis of GLP-1 receptor signaling in pancreatic β-cells

Glucagon-like peptide-1 (GLP-1) elevates intracellular concentration of cAMP ([cAMP]) and facilitates glucose-dependent insulin secretion in pancreatic β-cells. There has been much evidence to suggest that multiple key players such as the GLP-1 receptor, G(s) protein, adenylate cyclase (AC), phosphodiesterase (PDE), and intracellular Ca(2+) concentration ([Ca(2+)]) are involved in the regulation of [cAMP]. However, because of complex interactions among these signaling factors, the kinetics of the reaction cascade as well as the activities of ACs and PDEs have not been determined in pancreatic β-cells. We have constructed a minimal mathematical model of GLP-1 receptor signal transduction based on experimental findings obtained mostly in β-cells and insulinoma cell lines. By fitting this theoretical reaction scheme to key experimental records of the GLP-1 response, the parameters determining individual reaction steps were estimated. The model reconstructed satisfactorily the dynamic changes in [cAMP] and predicted the activities of cAMP effectors, protein kinase A (PKA), and cAMP-regulated guanine nucleotide exchange factor [cAMP-GEF or exchange protein directly activated by cAMP (Epac)] during GLP-1 stimulation. The simulations also predicted the presence of two sequential desensitization steps of the GLP1 receptor that occur with fast and very slow reaction rates. The cross talk between glucose- and GLP-1-dependent signal cascades for cAMP synthesis was well reconstructed by integrating the direct regulation of AC and PDE by [Ca(2+)]. To examine robustness of the signaling system in controlling [cAMP], magnitudes of AC and PDE activities were compared in the presence or absence of GLP-1 and/or the PDE inhibitor IBMX.(1).     

Glucose-induced period-doubling cascade in the electrical activity of pancreatic β-cells

 In the presence of stimulatory concentrations of glucose, the membrane potential of pancreatic β-cells may experience a transition from periods of rapid spike-like oscillations alternating with a pseudo-steady state to spike-only oscillations. Insulin secretion from β-cells closely correlates the periods of spike-like oscillations. The purpose of this paper is to study the mathematical structure which underlines this transitional stage in a pancreatic β-cell model. It is demonstrated that the transition can be chaotic but becomes more and more regular with increase in glucose. In particular, the system undergoes a reversed period-doubling cascade leading to the spike-only oscillations as the glucose concentration crosses a threshold. The transition interval in glucose concentration is estimated to be extremely small in terms of the rate of change for the calcium dynamics in the β-cells. The methods are based on the theory of unimodal maps and the geometric and asymptotic theories of singular perturbations. Received: 25 October 1996/Revised version: 18 August 1997     

Insulinotropic effect of the non-steroidal compound STX in pancreatic β-cells

The non-steroidal compound STX modulates the hypothalamic control of core body temperature and energy homeostasis. The aim of this work was to study the potential effects of STX on pancreatic β-cell function. 1-10 nM STX produced an increase in glucose-induced insulin secretion in isolated islets from male mice, whereas it had no effect in islets from female mice. This insulinotropic effect of STX was abolished by the anti-estrogen ICI 182,780. STX increased intracellular calcium entry in both whole islets and isolated β-cells, and closed the K(ATP) channel, suggesting a direct effect on β-cells. When intraperitoneal glucose tolerance test was performed, a single dose of 100 μg/kg body weight STX improved glucose sensitivity in males, yet it had a slight effect on females. In agreement with the effect on isolated islets, 100 μg/kg dose of STX enhanced the plasma insulin increase in response to a glucose load, while it did not in females. Long-term treatment (100 μg/kg, 6 days) of male mice with STX did not alter body weight, fasting glucose, glucose sensitivity or islet insulin content. Ovariectomized females were insensitive to STX (100 μg/kg), after either an acute administration or a 6-day treatment. This long-term treatment was also ineffective in a mouse model of mild diabetes. Therefore, STX appears to have a gender-specific effect on blood glucose homeostasis, which is only manifested after an acute administration. The insulinotropic effect of STX in pancreatic β-cells is mediated by the closure of the K(ATP) channel and the increase in intracellular calcium concentration. The in vivo improvement in glucose tolerance appears to be mostly due to the enhancement of insulin secretion from β-cells.     

Activation of alpha adrenergic and muscarinic receptors modifies early glucose suppression of cytoplasmic Ca2+ in pancreatic β-cells

Elevation of glucose induces transient inhibition of insulin release by lowering cytoplasmic Ca²⁺ ([Ca²⁺]_i) below baseline in pancreatic β-cells. The period of [Ca²⁺]_i decrease (phase 0) coincides with increased glucagon release and is therefore the starting point for antisynchronous pulses of insulin and glucagon. We now examine if activation of adrenergic α_2A and muscarinic M₃ receptors affects the initial [Ca²⁺]_i response to increase of glucose from 3 to 20 mM in β-cells situated in mouse islets. In the absence of receptor stimulation the elevation of glucose lowered [Ca²⁺]_i during 90–120 s followed by rise due to opening of voltage-dependent Ca²⁺ channels. The period of [Ca²⁺]_i decrease was prolonged by activation of the α_2A adrenergic receptors (1 μM epinephrine or 100 nM clonidine) and shortened by stimulation of the muscarinic M₃ receptors (0.1 μM acetylcholine). The latter effect was mimicked by the Na/K pump inhibitor ouabain (10–100 μM). The results indicate that prolonged initial decrease (phase 0) is followed by slow [Ca²⁺]_i rise and shorter decrease followed by fast rise. It is concluded that the period of initial decrease of [Ca²⁺]_i regulates the subsequent β-cell response to glucose.