Inhibition of the MAP3 kinase Tpl2 protects rodent and human ß-cells from apoptosis and dysfunction induced by cytokines and enhances anti-inflammatory actions of exendin-4.

Proinflammatory cytokines exert cytotoxic effects on ß-cells, and are involved in the pathogenesis of type I and type II diabetes and in the drastic loss of ß-cells following islet transplantation. Cytokines induce apoptosis and alter the function of differentiated ß-cells. Although the MAP3 kinase tumor progression locus 2 (Tpl2) is known to integrate signals from inflammatory stimuli in macrophages, fibroblasts and adipocytes, its role in ß-cells is unknown. We demonstrate that Tpl2 is expressed in INS-1E ß-cells, mouse and human islets, is activated and upregulated by cytokines and mediates ERK1/2, JNK and p38 activation. Tpl2 inhibition protects ß-cells, mouse and human islets from cytokine-induced apoptosis and preserves glucose-induced insulin secretion in mouse and human islets exposed to cytokines. Moreover, Tpl2 inhibition does not affect survival or positive effects of glucose (i.e., ERK1/2 phosphorylation and basal insulin secretion). The protection against cytokine-induced ß-cell apoptosis is strengthened when Tpl2 inhibition is combined with the glucagon-like peptide-1 (GLP-1) analog exendin-4 in INS-1E cells. Furthermore, when combined with exendin-4, Tpl2 inhibition prevents cytokine-induced death and dysfunction of human islets. This study proposes that Tpl2 inhibitors, used either alone or combined with a GLP-1 analog, represent potential novel and effective therapeutic strategies to protect diabetic ß-cells.

Quercetin induces insulin secretion by direct activation of L-type calcium channels in pancreatic beta cells.

BACKGROUND AND PURPOSE: Quercetin is a natural polyphenolic flavonoid that displays anti-diabetic properties in vivo. Its mechanism of action on insulin-secreting beta cells is poorly documented. In this work, we have analysed the effects of quercetin both on insulin secretion and on the intracellular calcium concentration ([Ca(2+)]i) in beta cells, in the absence of any co-stimulating factor. EXPERIMENTAL APPROACH: Experiments were performed on both INS-1 cell line and rat isolated pancreatic islets. Insulin release was quantified by the homogeneous time-resolved fluorescence method. Variations in [Ca(2+)]i were measured using the ratiometric fluorescent Ca(2+) indicator Fura-2. Ca(2+) channel currents were recorded with the whole-cell patch-clamp technique. KEY RESULTS: Quercetin concentration-dependently increased insulin secretion and elevated [Ca(2+)]i. These effects were not modified by the SERCA inhibitor thapsigargin (1 µmol·L(-1)), but were nearly abolished by the L-type Ca(2+) channel antagonist nifedipine (1 µmol·L(-1)). Similar to the L-type Ca(2+) channel agonist Bay K 8644, quercetin enhanced the L-type Ca(2+) current by shifting its voltage-dependent activation towards negative potentials, leading to the increase in [Ca(2+)]i and insulin secretion. The effects of quercetin were not inhibited in the presence of a maximally active concentration of Bay K 8644 (1 µmol·L(-1)), with the two drugs having cumulative effects on [Ca(2+)]i. CONCLUSIONS AND IMPLICATIONS: Taken together, our results show that quercetin stimulates insulin secretion by increasing Ca(2+) influx through an interaction with L-type Ca(2+) channels at a site different from that of Bay K 8644. These data contribute to a better understanding of quercetin's mechanism of action on insulin secretion.

Subplasmalemmal Ca(2+) measurements in mouse pancreatic beta cells support the existence of an amplifying effect of glucose on insulin secretion.

AIMS/HYPOTHESIS: Glucose-induced insulin secretion is attributed to a rise of beta cell cytosolic free [Ca(2+)] ([Ca(2+)](c)) (triggering pathway) and amplification of the action of Ca(2+). This concept of amplification rests on observations that glucose can increase Ca(2+)-induced insulin secretion without further elevating an imposed already high [Ca(2+)](c). However, it remains possible that this amplification results from an increase in [Ca(2+)] just under the plasma membrane ([Ca(2+)](SM)), which escaped detection by previous measurements of global [Ca(2+)](c). This was the hypothesis that we tested here by measuring [Ca(2+)](SM). METHODS: The genetically encoded Ca(2+) indicators D3-cpv (untargeted) and LynD3-cpv (targeted to plasma membrane) were expressed in clusters of mouse beta cells. LynD3-cpv was also expressed in beta cells within intact islets. [Ca(2+)](SM) changes were monitored using total internal reflection fluorescence microscopy. Insulin secretion was measured in parallel. RESULTS: Beta cells expressing D3cpv or LynD3cpv displayed normal [Ca(2+)] changes and insulin secretion in response to glucose. Distinct [Ca(2+)](SM) fluctuations were detected during repetitive variations of KCl between 30 and 32-35 mmol/l, attesting to the adequate sensitivity of our system. When the amplifying pathway was evaluated (high KCl + diazoxide), increasing glucose from 3 to 15 mmol/l consistently lowered [Ca(2+)](SM) while stimulating insulin secretion approximately two fold. Blocking Ca(2+) uptake by the endoplasmic reticulum largely attenuated the [Ca(2+)](SM) decrease produced by high glucose but did not unmask localised [Ca(2+)](SM) increases. CONCLUSIONS/INTERPRETATION: Glucose can increase Ca(2+)-induced insulin secretion without causing further elevation of beta cell [Ca(2+)](SM). The phenomenon is therefore a true amplification of the triggering action of Ca(2+).

Shortcomings of current models of glucose-induced insulin secretion.

Glucose-induced insulin secretion by pancreatic beta-cells is generally schematized by a 'consensus model' that involves the following sequence of events: acceleration of glucose metabolism, closure of ATP-sensitive potassium channels (K(ATP) channels) in the plasma membrane, depolarization, influx of Ca(2+) through voltage-dependent calcium channels and a rise in cytosolic-free Ca(2+) concentration that induces exocytosis of insulin-containing granules. This model adequately depicts the essential triggering pathway but is incomplete. In this article, we first make a case for a model of dual regulation in which a metabolic amplifying pathway is also activated by glucose and augments the secretory response to the triggering Ca(2+) signal under physiological conditions. We next discuss experimental evidence, largely but not exclusively obtained from beta-cells lacking K(ATP) channels, which indicates that these channels are not the only possible transducers of glucose effects on the triggering Ca(2+)signal. We finally address the identity of the widely neglected background inward current (Cl(-) efflux vs. Na(+) or Ca(2+) influx through voltage-independent channels) that is necessary to cause beta-cell depolarization when glucose closes K(ATP) channels. More attention should be paid to the possibility that some components of this background current are influenced by glucose metabolism and have their place in a model of glucose-induced insulin secretion.

Insulin crystallization depends on zinc transporter ZnT8 expression, but is not required for normal glucose homeostasis in mice.

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.

Rapid three-dimensional imaging of individual insulin release events by Nipkow disc confocal microscopy.

Minute-to-minute control of the release of insulin by pancreatic beta-cells in response to glucose or other stimuli requires the precise delivery of large dense-core vesicles to the plasma membrane and regulated exocytosis. At present, the precise spatial organization at the cell surface and the nature of these events ('transient' versus 'full fusion') are debated. In order to monitor secretory events simultaneously over most of the surface of clusters of single MIN6 beta-cells, we have expressed recombinant neuropeptide Y-Venus (an enhanced and vesicle-targeted form of yellow fluorescent protein) as an insulin surrogate. Individual exocytotic events were monitored using Nipkow spinning disc confocal microscopy, with acquisition of a three-dimensional complete image (eight to twelve confocal slices) in <1 s, in response to cell depolarization. Corroborating earlier studies using TIRF (total internal reflection fluorescence) microscopy, this approach indicates that events occur with roughly equal probability over the entire cell surface, with only minimal clustering in individual areas, and provides no evidence for multiple events at the same site. Nipkow disc confocal imaging may thus provide a useful tool to determine whether event types occur at different sites at the cell surface and to explore the role of endocytic proteins including dynamin-1 and -2 in terminating individual exocytotic events.

Disorganization of cytoplasmic Ca(2+) oscillations and pulsatile insulin secretion in islets from ob/ obmice.

AIMS/HYPOTHESIS: In normal mouse islets, glucose induces synchronous cytoplasmic [Ca(2+)](i) oscillations in beta cells and pulses of insulin secretion. We investigated whether this fine regulation of islet function is preserved in hyperglycaemic and hyperinsulinaemic ob/ obmice. METHODS: Intact islets from ob/ ob mice and their lean littermates were used after overnight culture for measurement of [Ca(2+)](i) and insulin secretion. RESULTS: We observed three types of [Ca(2+)](i) responses during stimulation by 9 to 12 mmol/l of glucose: sustained increase, rapid oscillations and slow (or mixed) oscillations. They occurred in 8, 18 and 74% of lean islets and 9, 0 and 91% of ob/ ob islets, respectively. Subtle desynchronisation of [Ca(2+)](i) oscillations between regions occurred in 11% of lean islets. In ob/ ob islets, desynchronisation was frequent (66-82% depending on conditions) and prominent: oscillations were out of phase in different regions because of distinct periods and shapes. Only small ob/ ob islets were well synchronised, but sizes of synchronised lean and desynchronised ob/ ob islets were markedly overlapped. The occurrence of desynchronisation in clusters of 5 to 50 islet cells from ob/ obmice and not from lean mice further indicates that islet hypertrophy is not the only causal factor. In both types of islets, synchronous [Ca(2+)](i) oscillations were accompanied by oscillations of insulin secretion. In poorly synchronised ob/ ob islets, secretion was irregular but followed the pattern of the global [Ca(2+)](i) changes. CONCLUSIONS/INTERPRETATION: The regularity of glucose-induced [Ca(2+)](i) oscillations is disrupted in islets from ob/ ob mice and this desynchronisation perturbs the pulsatility of insulin secretion. A similar mechanism could contribute to the irregularity of insulin oscillations in Type II (non-insulin-dependent) diabetes mellitus.

Inhibition of protein synthesis sequentially impairs distinct steps of stimulus-secretion coupling in pancreatic beta cells.

Proteins with a short half-life are potential sites of pancreatic ss cell dysfunction under pathophysiological conditions. In this study, mouse islets were used to establish which step in the regulation of insulin secretion is most sensitive to inhibition of protein synthesis by 10 microM cycloheximide (CHX). Although islet protein synthesis was inhibited approximately 95% after 1 h, the inhibition of insulin secretion was delayed and progressive. After long (18-20 h) CHX-treatment, the strong (80%) inhibition of glucose-, tolbutamide-, and K(+)-induced insulin secretion was not due to lower insulin stores, to any marked impairment of glucose metabolism or to altered function of K(+)-ATP channels (total K(+)-ATP currents were however decreased). It was partly caused by a decreased Ca(2+) influx (whole-cell Ca(2+) current) resulting in a smaller rise in cytosolic Ca(2+) ([Ca(2+)](i)). The situation was very different after short (2-5 h) CHX-treatment. Insulin secretion was 50-60% inhibited although islet glucose metabolism was unaffected and stimulus-induced [Ca(2+)](i) rise was not (2 h) or only marginally (5 h) decreased. The efficiency of Ca(2+) on secretion was thus impaired. The inhibition of insulin secretion by 15 h of CHX treatment was more slowly reversible (>4 h) than that of protein synthesis. This reversibility of secretion was largely attributable to recovery of a normal Ca(2+) efficiency. In conclusion, inhibition of protein synthesis in islets inhibits insulin secretion in two stages: a rapid decrease in the efficiency of Ca(2+) on exocytosis, followed by a decrease in the Ca(2+) signal mediated by a slower loss of functional Ca(2+) channels. Glucose metabolism and the regulation of K(+)-ATP channels are more resistant. Proteins with a short half-life appear to be important to ensure optimal Ca(2+) effects on exocytosis, and are the potential Achille's heel of stimulus-secretion coupling.

The oscillatory behavior of pancreatic islets from mice with mitochondrial glycerol-3-phosphate dehydrogenase knockout.

Glucose stimulation of pancreatic beta cells induces oscillations of the membrane potential, cytosolic Ca(2+) ([Ca(2+)](i)), and insulin secretion. Each of these events depends on glucose metabolism. Both intrinsic oscillations of metabolism and repetitive activation of mitochondrial dehydrogenases by Ca(2+) have been suggested to be decisive for this oscillatory behavior. Among these dehydrogenases, mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH), the key enzyme of the glycerol phosphate NADH shuttle, is activated by cytosolic [Ca(2+)](i). In the present study, we compared different types of oscillations in beta cells from wild-type and mGPDH(-/-) mice. In clusters of 5-30 islet cells and in intact islets, 15 mM glucose induced an initial drop of [Ca(2+)](i), followed by an increase in three phases: a marked initial rise, a partial decrease with rapid oscillations and eventually large and slow oscillations. These changes, in particular the frequency of the oscillations and the magnitude of the [Ca(2+)] rise, were similar in wild-type and mGPDH(-/-) mice. Glucose-induced electrical activity (oscillations of the membrane potential with bursts of action potentials) was not altered in mGPDH(-/-) beta cells. In single islets from either type of mouse, insulin secretion strictly followed the changes in [Ca(2+)](i) during imposed oscillations induced by pulses of high K(+) or glucose and during the biphasic elevation induced by sustained stimulation with glucose. An imposed and controlled rise of [Ca(2+)](i) in beta cells similarly increased NAD(P)H fluorescence in control and mGDPH(-/-) islets. Inhibition of the malate-aspartate NADH shuttle with aminooxyacetate only had minor effects in control islets but abolished the electrical, [Ca(2+)](i) and secretory responses in mGPDH(-/-) islets. The results show that the two distinct NADH shuttles play an important but at least partially redundant role in glucose-induced insulin secretion. The oscillatory behavior of beta cells does not depend on the functioning of mGPDH and on metabolic oscillations that would be generated by cyclic activation of this enzyme by Ca(2+).

Oscillations of insulin secretion can be triggered by imposed oscillations of cytoplasmic Ca2+ or metabolism in normal mouse islets.

Glucose-induced insulin secretion depends on an acceleration of glucose metabolism, requires a rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i), and is modulated by activation of protein kinases in beta-cells. Normal mouse islets were used to determine whether oscillations of these three signals are able and necessary to trigger oscillations of insulin secretion. The approach was to minimize or abolish spontaneous oscillations and to compare the impact of forced oscillations of each signal on insulin secretion. In a control medium, repetitive increases in the glucose concentration triggered oscillations in metabolism [NAD(P)H fluorescence], [Ca2+]i (fura-PE3 method), and insulin secretion. In the presence of diazoxide, metabolic oscillations persisted, but [Ca2+]i and insulin oscillations were abolished. When the islets were depolarized with high K+ with or without diazoxide, [Ca2+]i was elevated, and insulin secretion was stimulated. Forced metabolic oscillations transiently decreased or did not affect [Ca2+]i and potentiated insulin secretion with oscillations of small amplitude. These oscillations of secretion followed metabolic oscillations only when [Ca2+]i did not change. When [Ca2+]i fluctuated, these changes prevailed over those of metabolism for timing secretion. Repetitive depolarizations with high K+ in the presence of stable glucose (10 mmol/l) induced synchronous pulses of [Ca2+]i and insulin secretion with only small oscillations of metabolism. Continuous stimulation of protein kinase A (PKA) and protein kinase C (PKC) did not dissociate the [Ca2+]i and insulin pulses from the high K+ pulses. However, the amplitude of the insulin pulses was consistently increased, whereas that of the [Ca2+]i pulses was either increased (PKA) or decreased (PKC). In conclusion, metabolic oscillations can induce oscillations of insulin secretion independently of but with a lesser effectiveness than [Ca2+]i oscillations. Although oscillations in metabolism may cyclically influence secretion through an ATP-sensitive K+ channel (K+-ATP channel)-independent pathway, their regulatory effects are characterized by a hysteresis that makes them unlikely drivers of fast oscillations, unless they also involve [Ca2+]i changes through the K+-ATP channel-dependent pathway.