High glucose potentiates palmitate-induced NO-mediated cytotoxicity through generation of superoxide in clonal beta-cell HIT-T15

Prolonged exposure to free fatty acids induces beta-cell cytotoxicity. We investigated whether this fatty-acid-induced cytotoxicity is affected by high glucose levels. In clonal beta-cell HIT-T15, palmitate-induced cytotoxicity was potentiated depending on elevated glucose concentrations due to increased apoptosis without cytotoxic effects of high glucose per se. This palmitate cytotoxicity was blocked by NO synthase inhibitors, and palmitate actually increased cellular NO production. The potentiation of palmitate cytotoxicity under high glucose was reversed by decreasing superoxide production, suggesting that superoxide overproduction under high glucose enhances NO-mediated cytotoxicity in beta-cells, which may explain the mechanism of synergistic deterioration of pancreatic beta-cells by free fatty acids and high glucose.     

Extracellular acidification parallels insulin secretion in INS-1 and HIT-T15 beta-cell lines

As an alternative to manual assays that track insulin secretion, we tested a silicon-based biosensor that allows automated monitoring of extracellular acidification. Glucose stimulation of INS-1 and HIT-T15 cells resulted in a rapid increase in extracellular acidification in a biphasic and concentration-dependent fashion much like insulin secretion (EC(50) INS-1=5 mM and HIT-T15=1 mM). This response was attenuated by verapamil (10 microM) and stimulated by administration of glybenclamide (100 nM) or KCl-induced (40 mM) depolarization. These experiments suggest that automated monitoring of extracellular pH may be a useful assay and support the relevance of linking metabolic activity to insulin secretion.     

Evidence for the involvement of cGMP and protein kinase G in nitric oxide-induced apoptosis in the pancreatic B-cell line, HIT-T15

Intracellular production of nitric oxide (NO) is thought to mediate the pancreatic B-cell-directed cytotoxicity of cytokines in insulin-dependent diabetes mellitus, and recent evidence has indicated that this may involve induction of apoptosis. A primary effect of NO is to activate soluble guanylyl cyclase leading to increased cGMP levels and this effect has been demonstrated in pancreatic B-cells, although no intracellular function has been defined for islet cGMP. Here we demonstrate that the NO donor, GSNO, induces apoptosis in the pancreatic B-cell line HIT-T15 in a dose- and time-dependent manner. This response was significantly attenuated by micromolar concentrations of a specific inhibitor of soluble guanylyl cyclase, ODQ, and both 8-bromo cGMP (100 μM) and dibutyryl cGMP (300 μM) were able to fully relieve this inhibition. In addition, incubation of HIT-T15 cells with each cGMP analogue directly promoted cell death in the absence of ODQ. KT5823, a potent and highly selective inhibitor of cGMP-dependent protein kinase (PKG), abolished the induction of cell death in HIT cells in response to either GSNO or cGMP analogues. This effect was dose-dependent over the concentration range of 10–250 nM. Overall, these data provide evidence that the activation of apoptosis in HIT-T15 cells by NO donors is secondary to a rise in cGMP and suggest that the pathway controlling cell death involves activation of PKG.     

Nutrient-secretion coupling in the pancreatic islet beta-cell: recent advances

Insulin secretion from pancreatic islet beta-cells is a tightly regulated process, under the close control of blood glucose concentrations, and several hormones and neurotransmitters. Defects in glucose-triggered insulin secretion are ultimately responsible for the development of type II diabetes, a condition in which the total beta-cell mass is essentially unaltered, but beta-cells become progressively "glucose blind" and unable to meet the enhanced demand for insulin resulting for peripheral insulin resistance. At present, the mechanisms by which glucose (and other nutrients including certain amino acids) trigger insulin secretion in healthy individuals are understood only in part. It is clear, however, that the metabolism of nutrients, and the generation of intracellular signalling molecules including the products of mitochondrial metabolism, probably play a central role. Closure of ATP-sensitive K+(K(ATP)) channels in the plasma membrane, cell depolarisation, and influx of intracellular Ca2+, then prompt the "first phase" on insulin release. However, recent data indicate that glucose also enhances insulin secretion through mechanisms which do not involve a change in K(ATP) channel activity, and seem likely to underlie the second, sustained phase of glucose-stimulated insulin secretion. In this review, I will discuss recent advances in our understanding of each of these signalling processes.     

Channel regulation of glucose sensing in the pancreatic beta-cell

Mammalian beta-cells are acutely and chronically regulated by sensing surrounding glucose levels that determine the rate at which insulin is secreted, to maintain euglycemia. Experimental research in vitro and in vivo has shown that, when these cells are exposed to adverse conditions like long periods of hypoglycemia or hyperglycemia, their capability to sense glucose is decreased. Understanding the normal physiology and identifying the main players along this route becomes paramount. In this review, we have taken on the task of looking at the role that ion channels play in the regulation of this process, delineating the different families, and describing the signaling that parallels the glucose sensing process that results in insulin release.     

Selenium stimulates pancreatic beta-cell gene expression and enhances islet function

The present study investigated the role of selenium in the regulation of pancreatic beta-cell function. Utilising the mouse beta-cell line Min6, we have shown that selenium specifically upregulates Ipf1 (insulin promoter factor 1) gene expression, activating the -2715 to -1960 section of the Ipf1 gene promoter. Selenium increased both Ipf1 and insulin mRNA levels in Min6 cells and stimulated increases in insulin content and insulin secretion in isolated primary rat islets of Langerhans. These data are the first to implicate selenium in the regulation of specific beta-cell target genes and suggest that selenium potentially promotes an overall improvement in islet function.     

Oxidative stress is a mediator of glucose toxicity in insulin-secreting pancreatic islet cell lines

Pancreatic beta cells secrete insulin in response to changes in the extracellular glucose. However, prolonged exposure to elevated glucose exerts toxic effects on beta cells and results in beta cell dysfunction and ultimately beta cell death (glucose toxicity). To investigate the mechanism of how increased extracellular glucose is toxic to beta cells, we used two model systems where glucose metabolism was increased in beta cell lines by enhancing glucokinase (GK) activity and exposing cells to physiologically relevant increases in extracellular glucose (3.3-20 mm). Exposure of cells with enhanced GK activity to 20 mm glucose accelerated glycolysis, but reduced cellular NAD(P)H and ATP, caused accumulation of intracellular reactive oxygen species (ROS) and oxidative damage to mitochondria and DNA, and promoted apoptotic cell death. These changes required both enhanced GK activity and exposure to elevated extracellular glucose. A ROS scavenger partially prevented the toxic effects of increased glucose metabolism. These results indicate that increased glucose metabolism in beta cells generates oxidative stress and impairs cell function and survival; this may be a mechanism of glucose toxicity in beta cells. The level of beta cell GK may also be critical in this process.     

Enhancement of mouse pancreatic regeneration and HIT-T15 cell proliferation with rat pancreatic extract

In this study, the effects of rat pancreatic extract (RPE) on regeneration of impaired mouse pancreas and proliferation of beta-cell line (HIT-T15) were investigated. RPE from the regenerating pancreas (2 days after 60% pancreatectomy) was treated to cure streptozotocin (STZ) induced diabetes in BALB/c mice. RPE-treated BALB/c mice for 21 consecutive days became euglycemic by day 30 and remained normoglycemic during a 150 day follow-up. Saline treated mice remained hyperglycemic sustained uncontrolled hyperglycemia. Islet neogenesis was observed in RPE-treated mice and confirmed by use of immunocytochemistry. Morphometric analysis of pancreas in reverted RPE-treated mice showed a new population of small islets compared with saline controls and an increased islet number. When HIT-T15 cells were treated with RPE, HIT-T15 cell proliferation and insulin secretion increased with increases in the RPE concentration. These results imply that RPE have the regeneration factors and help in the cure of diabetes.     

Cytosolic O-GlcNAc accumulation is not involved in beta-cell death in HIT-T15 or Min6

O-linked N-acetylglucosamine (O-GlcNAc) is attached to and detached from proteins by O-GlcNAc transferase (OGT) and O-GlcNAcase, respectively. It has been proposed that streptozotocin induces pancreatic beta-cell death by blocking O-GlcNAcase and increasing O-GlcNAc. To elucidate the relationship between cytosolic O-GlcNAc accumulation and beta-cell death, we treated beta-cell lines HIT-T15 and Min6 with glucosamine. Glucosamine markedly reduced cell viability in both cell lines only at 10 mM. The measurement of cytosolic O-GlcNAc under glucosamine treatment revealed that O-GlcNAc accumulation was observed even at 2 mM glucosamine and maximized at 5 mM, but did not occur very well at 10 mM. Furthermore, 100 microM PUGNAc, an inhibitor of O-GlcNAcase, increased cytosolic O-GlcNAc but did not induce cell death in these cells. Therefore, no correlation between accumulation of cytosolic O-GlcNAc and beta-cell death was suggested. Alternatively, inosine partially rescued cell death induced by glucosamine in Min6 cells, suggesting that energy depletion partly contributes to beta-cell death by glucosamine.     

Temperature modulates the Ca current of HIT-T15 and mouse pancreatic β-cells

The effects of raising temperature on the Ca²⁺, currents of insulin-secreting HIT and mouse pancreatic β-cells were studied. Currents were measured in 3 mM Ca²⁺ containing solutions using standard whole-cell techniques. Increasing temperature from 22°C to 35°C increased peak Ca²⁺, current amplitude, percent (fast) inactivation and decreased the time-to-peak of the current. Ca²⁺ currents in HIT and mouse β-cells responded in the same manner to an imposed physiological burstwave with test-pulses: (i) application of the burstwave inactivated the test-pulse Ca²⁺ current at both high and low temperatures; (ii) Ca²⁺, current inactivation leveled off during the plateau phase at 20–22°C whereas there was an apparent continual decay at 33–35°C; and (iii) recovery from inactivation occurred during the interburst period at both temperatures. Application of a physiological burstwave without test-pulses to mouse β-cells before and after addition of 0.2 mM Cd²⁺ resulted in a Ca²⁺ difference current. This current activated during the hyperpolarized interburst phase, activated, inactivated and deactivated rapidly and continually during the plateau phase, and recovered from inactivation during the interburst. Although raising temperature strongly modified HIT and mouse β-cell Ca²⁺ current, our work suggests that other channels, in addition to Ca²⁺ channels, are likely to be involved in the control of islet bursts, particularly at different temperatures. In addition, the effect of temperature on islet cell Ca²⁺, current may be partly responsible for the well-known temperature dependence of glucose-dependent secretion.     

Insulin release from a cloned hamster B-cell line (HIT-T15). The effects of glucose, amino acids, sulphonylureas and colchicine

Insulin release from statically incubated HIT-T15 cells was maximally stimulated by glucose, L-arginine and L-leucine. L-arginine stimulated insulin release in the absence of glucose. Glucose induced insulin release was potentiated by the addition of L-leucine, L-arginine and the two in combination. Both glibenclamide and chlorpropamide stimulated insulin release from HIT-T15 cells. Glibenclamide was the more potent and equivalent in insulinotrophic action to 7.5 mmol/l glucose. Only chlorpropamide significantly potentiated glucose induced insulin release. Perifused HIT-T15 cells produced a reproducible biphasic insulin response to glucose challenge which was characterised by a pronounced and sustained first phase and a reduced second phase. The stimulation of phase I by glibenclamide alone and the inhibition of phase II of glucose induced insulin release by colchicine suggested the presence of a readily available pool of insulin granules which was not rapidly restored by insulin biosynthesis and granule margination.     

A role for G(z) in pancreatic islet beta-cell biology

Glucose-stimulated insulin secretion and beta-cell growth are important facets of pancreatic islet beta-cell biology. As a result, factors that modulate these processes are of great interest for the potential treatment of Type 2 diabetes. Here, we present evidence that the heterotrimeric G protein G(z) and its effectors, including some previously thought to be confined in expression to neuronal cells, are present in pancreatic beta-cells, the largest cellular constituent of the islets of Langerhans. Furthermore, signaling pathways upon which G alpha(z) impacts are intact in beta-cells, and G alpha(z) activation inhibits both cAMP production and glucose-stimulated insulin secretion in the Ins-1(832/13) beta-cell-derived line. Inhibition of glucose-stimulated insulin secretion by prostaglandin E (PGE1) is pertussis-toxin insensitive, indicating that other G alpha(i) family members are not involved in this process in this beta-cell line. Indeed, overexpression of a selective deactivator of G alpha(z), the RGS domain of RGSZ1, blocks the inhibitory effect of PGE1 on glucose-stimulated insulin secretion. Finally, the inhibition of glucose-stimulated insulin secretion by PGE1 is substantially blunted by small interfering RNA-mediated knockdown of G alpha(z) expression. Taken together, these data strongly imply that the endogenous E prostanoid receptor in the Ins-1(832/13) beta-cell line couples to G(z) predominantly and perhaps even exclusively. These data provide the first evidence for G(z) signaling in pancreatic beta-cells, and identify an endogenous receptor-mediated signaling process in beta-cells that is dependent on G alpha(z) function.     

Influence of neuropeptide Y and pancreatic polypeptide on islet function and beta-cell survival

BackgroundIn the present study we assessed the impact of neuropeptide Y receptor (NPYR) modulators, neuropeptide Y (NPY) and pancreatic polypeptide (PP), on islet function and beta-cell survival.MethodsThe effects of NPY and PP on beta-cell function were examined in BRIN BD11 and 1.1B4 beta-cells, as well as isolated mouse islets. Involvement of both peptides in pancreatic islet adaptations to streptozotocin and hydrocortisone, as well as effects on beta-cell proliferation and apoptosis was also evaluated.ResultsNeither NPY nor PP affected in vivo glucose disposal or insulin secretion in mice. However, both peptides inhibited (p < 0.05 to p < 0.001) glucose stimulated insulin secretion from rat and human beta-cells. NPY exerted similar insulinostatic effects in isolated mouse islets. NPY and PP inhibited alanine-induced changes in BRIN BD11 cell membrane potential and (Ca^2 +)_i. Streptozotocin treatment decreased and hydrocortisone treatment increased beta-cell mass in mice. In addition, streptozotocin, but not hydrocortisone, increased PP cell area. Streptozotocin also shifted the normal co-localisation of NPY with PP, towards more pronounced co-expression with somatostatin in delta-cells. Both streptozotocin and hydrocortisone increased pancreatic exocrine expression of NPY. More detailed in vitro investigations revealed that NPY, but not PP, augmented (p < 0.01) BRIN BD11 beta-cell proliferation. In addition, both peptides exerted protective effects against streptozotocin-induced DNA damage in beta-cells.ConclusionThese data emphasise the involvement of PP, and particularly NPY, in the regulation of beta-cell mass and function.General significanceModulation of PP and NPY signalling is suitable for further evaluation and possible clinical development for the treatment of diabetes.