E-cadherin interactions regulate beta-cell proliferation in islet-like structures.

Islet function is dependent on cells within the islet interacting with each other. E-cadherin (ECAD) mediates Ca(2+)-dependent homophilic cell adhesion between b-cells within islets and has been identified as a tumour suppressor. We generated clones of the MIN6 beta-cell line that stably over- (S) and under-express (alphaS) ECAD. Modified expression of ECAD was confirmed by quantitative RT-PCR, immunoblotting and immunocytochemistry. Preproinsulin mRNA, insulin content and basal rates of insulin secretion were higher in S cells compared to aS and control (V) cells. However, stimulated insulin secretory responses were unaffected by ECAD expression levels. ECAD expression did affect proliferation, with enhanced ECAD expression being associated with reduced proliferation and vice versa. Formation of islet-like structures was associated with a significant reduction in proliferation of V and S cells but not alphaS cells. These data suggest that ECAD expression levels do not modulate insulin secretory function but are consistent with a role for ECAD in the regulation of beta-cell proliferation.     

Homotypic cell contact enhances insulin but not glucagon secretion

Intra-islet interactions influence beta-cell function, and disruption of islet architecture results in a reduction in glucose-induced insulin secretion, whereas re-aggregation improves secretory responsiveness. Our studies on MIN6 cells have shown that by configuring beta-cells as three-dimensional islet-like structures there is a marked improvement in glucose-induced insulin secretion compared to that of their monolayer equivalents. In the present study, we have used the mouse glucagon-secreting alphaTC1 cell line to see whether homotypic interactions are important in the regulation of glucagon secretion from alpha-cells. We found no significant difference in the secretory responses of alphaTC1 cells maintained as monolayers or as cell clusters. We also found that different cell adhesion molecules are involved in cell interactions between alpha- and beta-cells; MIN6 cells express ECAD, whereas alphaTC1 cells express NCAM. ECAD is necessary for cell cluster formation by MIN6 cells but not by alphaTC1 cells, whereas NCAM is not needed for the formation of cell clusters in either cell line.     

Immunocytochemical localization of alpha-protein kinase C in rat pancreatic beta-cells during glucose-induced insulin secretion

To investigate the role of protein kinase C (PKC) in the regulation of insulin secretion, we visualized changes in the intracellular localization of alpha-PKC in fixed beta-cells from both isolated rat pancreatic islets and the pancreas of awake unstressed rats during glucose-induced insulin secretion. Isolated, perifused rat islets were fixed in 4% paraformaldehyde, detergent permeabilized, and labeled with a mAb specific for alpha-PKC. The labeling was visualized by confocal immunofluorescent microscopy. In isolated rat pancreatic islets perifused with 2.75 mM glucose, alpha-PKC immunostaining was primarily cytoplasmic in distribution throughout the beta-cells. In islets stimulated with 20 mM glucose, there was a significant redistribution of alpha-PKC to the cell periphery. This glucose-induced redistribution was abolished when either mannoheptulose, an inhibitor of glucose metabolism, or nitrendipine, an inhibitor of calcium influx, were added to the perifusate. We also examined changes in the intracellular distribution of alpha-PKC in the beta-cells of awake, unstressed rats that were given an intravenous infusion of glucose. Immunocytochemical analysis of pancreatic sections from these rats demonstrated a glucose-induced translocation of alpha-PKC to the cell periphery of the beta-cells. These results demonstrate that the metabolism of glucose can induce the redistribution of alpha-PKC to the cell periphery of beta-cells, both in isolated islets and in the intact animal, and suggest that alpha-PKC plays a role in mediating glucose-induced insulin secretion.     

Tetracaine stimulates insulin secretion from the pancreatic beta-cell by release of intracellular calcium

The role of intracellular calcium stores in stimulus-secretion coupling in the pancreatic beta-cell is largely unknown. We report here that tetracaine stimulates insulin secretion from collagenase-isolated mouse islets of Langerhans in the absence of glucose or extracellular calcium. We also found that the anesthetic evokes a dose-dependent rise of the intracellular free-calcium concentration ([Ca2+]i) in cultured rat and mouse beta-cells. The tetracaine-specific [Ca2+]i rise also occurs in the absence of glucose, or in beta-cells depolarized by exposure to a Ca(2+)-deficient medium (< 1 microM) or elevated [K+]o. Furthermore, tetracaine (> or = 300 microM) depolarized the beta-cell membrane in mouse pancreatic islets, but inhibited Ca2+ entry through voltage-gated Ca2+ channels in HIT cells, an insulin-secreting cell line. From these data we conclude that tetracaine-enhancement of insulin release occurs by mechanisms that are independent of Ca2+ entry across the cell membrane. The tetracaine-induced [Ca2+]i rise in cultured rat beta-cells and insulin secretion from mouse islets is insensitive to dantrolene (20 microM), a drug that inhibits Ca2+ release evoked by cholinergic agonists in the pancreatic beta-cell, and thapsigargin (3 microM), a blocker of the endoplasmic reticulum (ER) Ca2+ pump. We conclude that the Ca2+ required for tetracaine-potentiated insulin secretion is released from intracellular Ca2+ stores other than the ER. Furthermore, tetracaine-induced Ca2+ release was unaffected by the mitochondrial electron transfer inhibitors NaN3 and rotenone. Taken together, these data show that a calcium source other than the ER and mitochondria can affect beta-cell insulin secretion.     

Antidiabetic sulfonylurea stimulates insulin secretion independently of plasma membrane KATP channels

Understanding mechanisms by which glibenclamide stimulates insulin release is important, particularly given recent promising treatment by glibenclamide of permanent neonatal diabetic subjects. Antidiabetic sulfonylureas are thought to stimulate insulin secretion solely by inhibiting their high-affinity ATP-sensitive potassium (K(ATP)) channel receptors at the plasma membrane of beta-cells. This normally occurs during glucose stimulation, where ATP inhibition of plasmalemmal K(ATP) channels leads to voltage activation of L-type calcium channels for rapidly switching on and off calcium influx, governing the duration of insulin secretion. However, growing evidence indicates that sulfonylureas, including glibenclamide, have additional K(ATP) channel receptors within beta-cells at insulin granules. We tested nonpermeabilized beta-cells in mouse islets for glibenclamide-stimulated insulin secretion mediated by granule-localized K(ATP) channels by using conditions that bypass glibenclamide action on plasmalemmal K(ATP) channels. High-potassium stimulation evoked a sustained rise in beta-cell calcium level but a transient rise in insulin secretion. With continued high-potassium depolarization, addition of glibenclamide dramatically enhanced insulin secretion without affecting calcium. These findings support the hypothesis that glibenclamide, or an increased ATP/ADP ratio, stimulates insulin secretion in part by binding at granule-localized K(ATP) channels that functionally contribute to sustained second-phase insulin secretion.     

Glucose stimulates Ca2+ influx and insulin secretion in 2-week-old beta-cells lacking ATP-sensitive K+ channels

In adult beta-cells glucose-induced insulin secretion involves two mechanisms (a) a K(ATP) channel-dependent Ca(2+) influx and rise of cytosolic [Ca(2+)](c) and (b) a K(ATP) channel-independent amplification of secretion without further increase of [Ca(2+)](c). Mice lacking the high affinity sulfonylurea receptor (Sur1KO), and thus K(ATP) channels, have been developed as a model of congenital hyperinsulinism. Here, we compared [Ca(2+)](c) and insulin secretion in overnight cultured islets from 2-week-old normal and Sur1KO mice. Control islets proved functionally mature: the magnitude and biphasic kinetics of [Ca(2+)](c) and insulin secretion changes induced by glucose, and operation of the amplifying pathway, were similar to adult islets. Sur1KO islets perifused with 1 mm glucose showed elevation of both basal [Ca(2+)](c) and insulin secretion. Stimulation with 15 mm glucose produced a transient drop of [Ca(2+)](c) followed by an overshoot and a sustained elevation, accompanied by a monophasic, 6-fold increase in insulin secretion. Glucose also increased insulin secretion when [Ca(2+)](c) was clamped by KCl. When Sur1KO islets were cultured in 5 instead of 10 mm glucose, [Ca(2+)](c) and insulin secretion were unexpectedly low in 1 mm glucose and increased following a biphasic time course upon stimulation by 15 mm glucose. This K(ATP) channel-independent first phase [Ca(2+)](c) rise was attributed to a Na(+)-, Cl(-)-, and Na(+)-pump-independent depolarization of beta-cells, leading to Ca(2+) influx through voltage-dependent calcium channels. Glucose indeed depolarized Sur1KO islets under these conditions. It is suggested that unidentified potassium channels are sensitive to glucose and subserve the acute and long-term metabolic control of [Ca(2+)](c) in beta-cells without functional K(ATP) channels.     

The Zn2+-transporting pathways in pancreatic beta-cells: a role for the L-type voltage-gated Ca2+ channel

In pancreatic beta-cells Zn(2+) is crucial for insulin biosynthesis and exocytosis. Despite this, little is known about mechanisms of Zn(2+) transport into beta-cells or the regulation and compartmentalization of Zn(2+) within this cell type. Evidence suggests that Zn(2+) in part enters neurons and myocytes through specific voltage-gated calcium channels (VGCC). Using a Zn(2+)-selective fluorescent dye with high affinity and quantum yield, FluoZin-3 AM and the plasma membrane potential dye DiBAC(4)(3) we applied fluorescent microscopy techniques for analysis of Zn(2+)-accumulating pathways in mouse islets, dispersed islet cells, and beta-cell lines (MIN6 and beta-TC6f7 cells). Because the stimulation of insulin secretion is associated with cell depolarization, Zn(2+) (5-10 mum) uptake was analyzed under basal (1 mm glucose) and stimulatory (10-20 mm glucose, tolbutamide, tetraethylammonium, and high K(+)) conditions. Under both basal and depolarized states, beta-cells were capable of Zn(2+) uptake, and switching from basal to depolarizing conditions resulted in a marked increase in the rate of Zn(2+) accumulation. Importantly, L-type VGCC (L-VGCC) blockers (verapamil, nitrendipine, and nifedipine) as well as nonspecific inhibitors of Ca(2+) channels, Gd(3+) and La(3+), inhibited Zn(2+) uptake in beta-cells under stimulatory conditions with little or no change in Zn(2+) accumulation under low glucose conditions. To determine the mechanism of VGCC-independent Zn(2+) uptake the expression of a number of ZIP family Zn(2+) transporter mRNAs in islets and beta-cells was investigated. In conclusion, we demonstrate for the first time that, in part, Zn(2+) transport into beta-cells takes place through the L-VGCC. Our investigation demonstrates direct Zn(2+) accumulation in insulin-secreting cells by two pathways and suggests that the rate of Zn(2+) transport across the plasma membrane is dependent upon the metabolic status of the cell.     

Glucose refractoriness of beta-cells from fed fa/fa rats is ameliorated by nonesterified fatty acids

The aim of this study was to characterize the glucose responsiveness of individual beta-cells from fa/fa rats under ad libitum feeding conditions. Enlarged intact islets from fed fa/fa rats had a compressed insulin response curve to glucose compared with smaller islets. Size-sorted islets from obese rats yielded beta-cells whose glucose responsiveness was assessed by reverse hemolytic plaque assay to determine whether glucose refractoriness was caused by a decreased number of responsive cells or output per cell. In addition, the effects of palmitic acid on glucose-stimulated insulin secretion were assessed because of evidence that nonesterified fatty acids have acute beneficial effects. Two- to threefold more beta-cells from >250 microm diameter (large) islets than <125 microm diameter (small) or lean islets responded to low glucose. Increasing the glucose (8.3-16.5 mM) induced a >10-fold increase in recruitment of active cells from small islets, compared with only a 2.6-fold increase in large islets. This refractoriness was partially reversed by preincubation of the cells in low glucose for 2 h. In addition, secretion per cell of the large islet beta-cell population was significantly reduced compared with lean beta-cells, so that the overall response capacity of large but not small islet beta-cells was significantly reduced at high glucose. Therefore, continued near-normal function of the beta-cells from small islets of fa/fa rats seems crucial for glucose responsiveness. Incubation of beta-cells from large islets with palmitic acid normalized the secretory capacity to glucose mainly by increasing recruitment and secondarily by increasing secretion per cell. In conclusion, these studies demonstrate refractoriness to glucose of beta-cells from large islets of fa/fa rats under ad libitum feeding conditions. When acutely exposed to nonesterified fatty acids, islets from fa/fa rats have a potentiated insulin response despite chronic elevation of plasma lipids in vivo.     

Intra- and inter-islet synchronization of metabolically driven insulin secretion

Insulin secretion from pancreatic beta-cells is pulsatile with a period of 5-10 min and is believed to be responsible for plasma insulin oscillations with similar frequency. To observe an overall oscillatory insulin profile it is necessary that the insulin secretion from individual beta-cells is synchronized within islets, and that the population of islets is also synchronized. We have recently developed a model in which pulsatile insulin secretion is produced as a result of calcium-driven electrical oscillations in combination with oscillations in glycolysis. We use this model to investigate possible mechanisms for intra-islet and inter-islet synchronization. We show that electrical coupling is sufficient to synchronize both electrical bursting activity and metabolic oscillations. We also demonstrate that islets can synchronize by mutually entraining each other by their effects on a simple model "liver," which responds to the level of insulin secretion by adjusting the blood glucose concentration in an appropriate way. Since all islets are exposed to the blood, the distributed islet-liver system can synchronize the individual islet insulin oscillations. Thus, we demonstrate how intra-islet and inter-islet synchronization of insulin oscillations may be achieved.     

Intact pancreatic islets and dispersed beta-cells both generate intracellular calcium oscillations but differ in their responsiveness to glucose

Pancreatic islets produce pulses of insulin and other hormones that maintain normal glucose homeostasis. These micro-organs possess exquisite glucose-sensing capabilities, allowing for precise changes in pulsatile insulin secretion in response to small changes in glucose. When communication among these cells is disrupted, precision glucose sensing falters. We measured intracellular calcium patterns in 6-mM-steps between 0 and 16 mM glucose, and also more finely in 2-mM-steps from 8 to 12 mM glucose, to compare glucose sensing systematically among intact islets and dispersed islet cells derived from the same mouse pancreas in vitro. The calcium activity of intact islets was uniformly low (quiescent) below 4 mM glucose and active above 8 mM glucose, whereas dispersed beta-cells displayed a broader activation range (2-to-10 mM). Intact islets exhibited calcium oscillations with 2-to-5-min periods, yet beta-cells exhibited longer 7–10 min periods. In every case, intact islets showed changes in activity with each 6-mM-glucose step, whereas dispersed islet cells displayed a continuum of calcium responses ranging from islet-like patterns to stable oscillations unaffected by changes in glucose concentration. These differences were also observed for 2-mM-glucose steps. Despite the diversity of dispersed beta-cell responses to glucose, the sum of all activity produced a glucose dose-response curve that was surprisingly similar to the curve for intact islets, arguing against the importance of “hub cells” for function. Beta-cells thus retain many of the features of islets, but some are more islet-like than others. Determining the molecular underpinnings of these variations could be valuable for future studies of stem-cell-derived beta-cell therapies.     

Cell swelling-induced signaling for insulin secretion bypasses steps involving G proteins and PLA2 and is N-ethylmaleimide insensitive.

BACKGROUND: This study was undertaken to examine putative mechanisms of calcium independent signal transduction pathway of cell swelling-induced insulin secretion. METHODS: The role of phospholipase A(2), G proteins, and soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) in insulin secretion induced by 30% hypotonic medium was studied using isolated rat pancreatic islets. RESULTS: In contrast to glucose stimulation, osmotically induced insulin secretion from pancreatic islets was not inhibited by 10 micromol/l bromoenol lactone, an iPLA(2) (Ca(2+) independent phospholipase) inhibitor. Similarly, preincubation of islets for 20 hours with 25 microg/ml mycophenolic acid to inhibit GTP synthesis fully abolished glucose-induced insulin secretion but was without effect on hypotonicity stimulated insulin release. Glucose-induced insulin secretion was prevented by preincubation with 20 nmol/l tetanus toxin (TeTx), a metalloprotease inactivating soluble SNARE. Cell swelling-induced insulin secretion was inhibited by TeTx in the presence of calcium ions but not in calcium depleted medium. The presence of N-ethylmaleimide (NEM, 5 mmol/l, another inhibitor of SNARE proteins) in the medium resulted in high basal insulin secretion and lacking response to glucose stimulation. In contrast, high basal insulin secretion from NEM treated islets further increased after hypotonic stimulation. CONCLUSION: G proteins and iPLA(2) - putative mediators of Ca(2+) independent signaling pathway participate in glucose but not in hypotonicity-induced insulin secretion. Hypotonicity-induced insulin secretion is sensitive to clostridial neurotoxin TeTx but is resistant to NEM.     

Secretory phospholipase A2 is released from pancreatic beta-cells and stimulates insulin secretion via inhibition of ATP-dependent K+ channels

The release of sPLA(2) from single mouse pancreatic beta-cells was monitored using a fluorescent substrate of the enzyme incorporated in the outer leaflet of the plasma membrane. Stimulation of beta-cells with agents that increased cytosolic free Ca(2+) concentration ([Ca(2+)](i)) induced a rapid release of sPLA(2) to the extracellular medium. Exogenous sPLA(2) strongly stimulated insulin secretion in mouse pancreatic islets at both basal and elevated glucose concentrations. The stimulation of insulin secretion by sPLA(2) was mediated via inhibition of ATP-dependent K(+) channels and an increase in [Ca(2+)](i). Measurements of cell capacitance in single beta-cells revealed that sPLA(2) did not modify depolarisation-induced exocytosis. Our data suggest that a positive feedback regulation of insulin secretion by co-released sPLA(2) is operational in pancreatic beta-cells and point to this enzyme as an autocrine regulator of insulin secretion.     

Syntaxin 4 facilitates biphasic glucose-stimulated insulin secretion from pancreatic beta-cells

Numerous overexpression studies have recently implicated Syntaxin 4 as an effector of insulin secretion, although its requirement in insulin granule exocytosis is unknown. To address this, islets from Syntaxin 4 heterozygous (-/+) knockout mice were isolated and compared with islets from wild-type mice. Under static incubation conditions, Syntaxin 4 (-/+) islets showed a 60% reduction in glucose-stimulated insulin secretion compared with wild-type islets. Perifusion analyses revealed that Syntaxin 4 (-/+) islets secreted 50% less insulin during the first phase of glucose-stimulated insulin secretion and that this defect could be fully restored by the specific replenishment of recombinant Syntaxin 4. This essential role for Syntaxin 4 in secretion from the islet was localized to the beta-cells because small interfering RNA-mediated depletion of Syntaxin 4 in MIN6 beta-cells abolished glucose-stimulated insulin secretion. Moreover, immunofluorescent confocal microscopy revealed that Syntaxin 4 was principally localized to the beta-cells and not the alpha-cells of the mouse islet. Remarkably, islets isolated from transgenic mice that express 2.4-fold higher levels of Syntaxin 4 relative to wild-type mice secreted approximately 35% more insulin during both phases of insulin secretion, suggesting that increased Syntaxin 4 may be beneficial for enhancing biphasic insulin secretion in a regulated manner. Taken together, these data support the notion that Syntaxin 4-based SNARE complexes are essential for biphasic insulin granule fusion in pancreatic beta-cells.     

Both triggering and amplifying pathways contribute to fuel-induced insulin secretion in the absence of sulfonylurea receptor-1 in pancreatic beta-cells

In normal beta-cells glucose induces insulin secretion by activating both a triggering pathway (closure of K(ATP) channels, depolarization, and rise in cytosolic [Ca(2+)](i)) and an amplifying pathway (augmentation of Ca(2+) efficacy on exocytosis). It is unclear if and how nutrients can regulate insulin secretion by beta-cells lacking K(ATP) channels (Sur1 knockout mice). We compared glucose- and amino acid-induced insulin secretion and [Ca(2+)](i) changes in control and Sur1KO islets. In 1 mm glucose (non-stimulatory for controls), the triggering signal [Ca(2+)](i) was high (loss of regulation) and insulin secretion was stimulated in Sur1KO islets. This "basal" secretion was decreased or increased by imposed changes in [Ca(2+)](i) and was dependent on ATP production, indicating that both triggering and amplifying signals are involved. High glucose stimulated insulin secretion in Sur1KO islets, by an unsuspected, transient increase in [Ca(2+)](i) and a sustained activation of the amplifying pathway. Unlike controls, Sur1KO islets were insensitive to diazoxide and tolbutamide, which rules out effects of either drug at sites other than K(ATP) channels. Amino acids potently increased insulin secretion by Sur1KO islets through both a further electrogenic rise in [Ca(2+)](i) and a metabolism-dependent activation of the amplifying pathway. After sulfonylurea blockade of their K(ATP) channels, control islets qualitatively behaved like Sur1KO islets, but their insulin secretion rate was consistently lower for a similar or even higher [Ca(2+)](i). In conclusion, fuel secretagogues can control insulin secretion in beta-cells without K(ATP) channels, partly by an unsuspected influence on the triggering [Ca(2+)](i) signal and mainly by the modulation of a very effective amplifying pathway.     

Inhibition of insulin secretion by betagranin, an N-terminal chromogranin A fragment

Betagranin, an N-terminal fragment of chromogranin A, results from a proteolytic processing, and is co-secreted with insulin. While other chromogranin A-derived peptides negatively modulate hormone secretion, the role of betagranin in pancreatic beta-cells is so far unknown. We have recently shown that pancreatic islet betagranin levels are down-regulated in obese, leptin-deficient mice. In the present study, we have investigated the distribution of betagranin in primary mouse islets and cells of the MIN6 line and have evaluated its effects on insulin secretion. We showed that betagranin co-localizes with insulin within secretory granules and strongly inhibited insulin secretion in response to both glucose and potassium, by blocking the influx of calcium. The data demonstrated a hitherto unknown inhibitory effect of betagranin on insulin secretion.     

Glucose regulates expression of the nerve growth factor (NGF) receptors TrkA and p75NTR in rat islets and INS-1E beta-cells

BACKGROUND: The function and survival of pancreatic beta-cells strongly depend on glucose concentration and on autocrine secretion of peptide growth factors. NGF and its specific receptors TrkA and p75NTR play a pivotal role in islet survival and glucose-dependent insulin secretion. We therefore investigated whether or not glucose concentration influences expression of TrkA and p75NTR in rat islets and in INS-1E beta-cells at the mRNA and protein level (INS-1E). METHODS: Gene expression of the NGF receptors TrkA and p75NTR but also of the metabolic gene liver-type pyruvate kinase (L-PK) and the neurotrophin receptors TrkB and TrkC was studied by semi-quantitative PCR and by real-time PCR in islets and INS-1E beta-cells. RESULTS: In rat islets, high glucose exposure (25 mmol/l) increased gene expression of TrkA, p75NTR and L-PK. Expression of TrkA, p75NTR and L-PK reflected insulin secretion at the respective glucose concentration. In rat INS-1E insulinoma cells, expression of L-PK and p75NTR was suppressed by low glucose as in the islets, while expression of TrkA was strongly increased by low glucose levels and thus was regulated differently than in islets. Expression of TrkB and TrkC was not regulated by glucose concentration at all. TrkA protein was regulated in the same fashion as its mRNA expression, while p75NTR protein was not significantly regulated within 24 h. CONCLUSION: Glucose interacts with gene expression of TrkA and p75NTR that are strongly involved in beta-cell growth and glucose-dependent insulin secretion. The fact that TrkA expression is regulated the opposite way in islets and in INS-1E beta-cells might reflect their specific grade of differentiation and tendency to proliferate.     

Glucose and insulin stimulate heparin-releasable lipoprotein lipase activity in mouse islets and INS-1 cells. A potential link between insulin resistance and beta-cell dysfunction

Lipoprotein lipase (LpL) provides tissues with triglyceride-derived fatty acids. Fatty acids affect beta-cell function, and LpL overexpression decreases insulin secretion in cell lines, but whether LpL is regulated in beta-cells is unknown. To test the hypothesis that glucose and insulin regulate LpL activity in beta-cells, we studied pancreatic islets and INS-1 cells. Acute exposure of beta-cells to physiological concentrations of glucose stimulated both total cellular LpL activity and heparin-releasable LpL activity. Glucose had no effect on total LpL protein mass but instead promoted the appearance of LpL protein in a heparin-releasable fraction, suggesting that glucose stimulates the translocation of LpL from intracellular to extracellular sites in beta-cells. The induction of heparin-releasable LpL activity was unaffected by treatment with diazoxide, an inhibitor of insulin exocytosis that does not alter glucose metabolism but was blocked by conditions that inhibit glucose metabolism. In vitro hyperinsulinemia had no effect on LpL activity in the presence of low concentrations of glucose but increased LpL activity in the presence of 20 mm glucose. Using dual-laser confocal microscopy, we detected intracellular LpL in vesicles distinct from those containing insulin. LpL was also detected at the cell surface and was displaced from this site by heparin in dispersed islets and INS-1 cells. These results show that glucose metabolism controls the trafficking of LpL activity in beta-cells independent of insulin secretion. They suggest that hyperglycemia and hyperinsulinemia associated with insulin resistance may contribute to progressive beta-cell dysfunction by increasing LpL-mediated delivery of lipid to islets.     

Excitation wave propagation as a possible mechanism for signal transmission in pancreatic islets of Langerhans

In response to glucose application, beta-cells forming pancreatic islets of Langerhans start bursting oscillations of the membrane potential and intracellular calcium concentration, inducing insulin secretion by the cells. Until recently, it has been assumed that the bursting activity of beta-cells in a single islet of Langerhans is synchronized across the whole islet due to coupling between the cells. However, time delays of several seconds in the activity of distant cells are usually observed in the islets of Langerhans, indicating that electrical/calcium wave propagation through the islets can occur. This work presents both experimental and theoretical evidence for wave propagation in the islets of Langerhans. Experiments with Fura-2 fluorescence monitoring of spatiotemporal calcium dynamics in the islets have clearly shown such wave propagation. Furthermore, numerical simulations of the model describing a cluster of electrically coupled beta-cells have supported our view that the experimentally observed calcium waves are due to electric pulses propagating through the cluster. This point of view is also supported by independent experimental results. Based on the model equations, an approximate analytical expression for the wave velocity is introduced, indicating which parameters can alter the velocity. We point to the possible role of the observed waves as signals controlling the insulin secretion inside the islets of Langerhans, in particular, in the regions that cannot be reached by any external stimuli such as high glucose concentration outside the islets.     

Critical role of cAMP-GEFII--Rim2 complex in incretin-potentiated insulin secretion

Incretins such as glucagon-like peptide-1 and gastric inhibitory polypeptide/glucose-dependent insulinotropic peptide are known to potentiate insulin secretion mainly through a cAMP/protein kinase A (PKA) signaling pathway in pancreatic beta-cells, but the mechanism is not clear. We recently found that the cAMP-binding protein cAMP-GEFII (or Epac 2), interacting with Rim2, a target of the small G protein Rab3, mediates cAMP-dependent, PKA-independent exocytosis in a reconstituted system. In the present study, we investigated the role of the cAMP-GEFII--Rim2 pathway in incretin-potentiated insulin secretion in native pancreatic beta-cells. Treatment of pancreatic islets with antisense oligodeoxynucleotides (ODNs) against cAMP-GEFII alone or with the PKA inhibitor H-89 alone inhibited incretin-potentiated insulin secretion approximately 50%, while a combination of antisense ODNs and H-89 inhibited the secretion approximately 80-90%. The effect of cAMP-GEFII on insulin secretion is mediated by Rim2 and depends on intracellular calcium as well as on cAMP. Treatment of the islets with antisense ODNs attenuated both the first and second phases of insulin secretion potentiated by the cAMP analog 8-bromo-cAMP. These results indicate that the PKA-independent mechanism involving the cAMP-GEFII--Rim2 pathway is critical in the potentiation of insulin secretion by incretins.     

Glucose-dependent expansion of pancreatic beta-cells by the protein p8 in vitro and in vivo

p8 protein expression is known to be upregulated in the exocrine pancreas during acute pancreatitis. Own previous work revealed glucose-dependent p8 expression also in endocrine pancreatic beta-cells. Here we demonstrate that glucose-induced INS-1 beta-cell expansion is preceded by p8 protein expression. Moreover, isopropylthiogalactoside (IPTG)-induced p8 overexpression in INS-1 beta-cells (p8-INS-1) enhances cell proliferation and expansion in the presence of glucose only. Although beta-cell-related gene expression (PDX-1, proinsulin I, GLUT2, glucokinase, amylin) and function (insulin content and secretion) are slightly reduced during p8 overexpression, removal of IPTG reverses beta-cell function within 24 h to normal levels. In addition, insulin secretion of p8-INS-1 beta-cells in response to 0-25 mM glucose is not altered by preceding p8-induced beta-cell expansion. Adenovirally transduced p8 overexpression in primary human pancreatic islets increases proliferation, expansion, and cumulative insulin secretion in vitro. Transplantation of mock-transduced control islets under the kidney capsule of immunosuppressed streptozotocin-diabetic mice reduces blood glucose and increases human C-peptide serum concentrations to stable levels after 3 days. In contrast, transplantation of equal numbers of p8-transduced islets results in a continuous decrease of blood glucose and increase of human C-peptide beyond 3 days, indicating p8-induced expansion of transplanted human beta-cells in vivo. This is underlined by a doubling of insulin content in kidneys containing p8-transduced islet grafts explanted on day 9. These results establish p8 as a novel molecular mediator of glucose-induced pancreatic beta-cell expansion in vitro and in vivo and support the notion of existing beta-cell replication in the adult organism.