Enhanced protection against cytokine- and fatty acid-induced apoptosis in pancreatic beta cells by combined treatment with glucagon-like peptide-1 receptor agonists and insulin analogues.

We recently showed that insulin analogues exhibit a beta-cell protective function. The aim of this study was to test if the anti-apoptotic activity of GLP-1 agonists and insulin analogues is mediated by different pathways and if combined treatment may provide augmented protection against beta-cell death. Incubation of INS-1 cells with cytokines or fatty acids increased the number of apoptotic cells and caspase 3 activity, which was reduced by pretreatment with GLP-1 and its receptor agonists exendin-4 and AVE0010 by 50-60%. Similar effects (about 40% reduction) were observed after pretreatment with several insulin analogues. Combined treatment revealed additive activity and resulted in prevention of both cytokine- and fatty acid-induced apoptosis by up to 80%. No acute Akt-phosphorylation in response to GLP-1 receptor agonists could be observed, however, it became detectable after 24-hour stimulation. Gene silencing of Akt2 increased cytokine-induced apoptosis 2-fold. Under these conditions the beta-cell protective activity of AVE0010 remained completely unaltered. We show here that the anti-apoptotic activity of GLP-1 and its receptor agonists AVE0010 and exendin-4 is enhanced by addition of insulin analogues and that the anti-apoptotic action of GLP-1 mimetics is mostly unrelated to Akt2 signaling. It is suggested that combination of GLP-1 receptor agonists and insulin analogues, specifically insulin glargine, may represent a new therapeutic option for preservation of beta-cell mass in type 2 diabetic patients.     

Enhanced beta-cell mass without increased proliferation following chronic mild glucose infusion

The physiological mechanisms underlying pancreatic beta-cell mass (BCM) homeostasis are complex and not fully resolved. Here we examined the factors contributing to the increased BCM following a mild glucose infusion (GI) whereby normoglycemia was maintained through 96 h. We used morphometric and immunochemical methods to investigate enhanced beta-cell growth and survival in Sprague-Dawley rats. BCM was elevated >2.5-fold over saline-infused control rats by 48 h and increased modestly thereafter. Unexpectedly, increases in beta-cell proliferation were not observed at any time point through 4 days. Instead, enhanced numbers of insulin(+) cell clusters and small islets (400-12,000 microm(2); approximately 23- to 124-microm diameter), mostly scattered among the acini, were observed in the GI rats by 48 h despite no difference in the numbers of medium to large islets. We previously showed that increased beta-cell growth in rodent models of insulin resistance and pancreatic regeneration involves increased activated Akt/PKB, a key beta-cell signaling intermediate, in both islets and endocrine cell clusters. GI in normal rats also leads to increased Akt activation in islet beta-cells, as well as in insulin(+) and insulin(-) cells in the common duct epithelium and endocrine clusters. This correlated with strong Pdx1 expression in these same cells. These results suggest that mechanisms other than proliferation underlie the rapid beta-cell growth response following a mild GI in the normal rat and involve Akt-regulated enhanced beta-cell survival potential and neogenesis from epithelial precursors.     

Insulin receptor substrate 1-induced inhibition of endoplasmic reticulum Ca2+ uptake in beta-cells. Autocrine regulation of intracellular ca2+ homeostasis and insulin secretion.

To understand the role of the insulin receptor pathway in beta-cell function, we have generated stable beta-cells (betaIRS1-A) that overexpress by 2-fold the insulin receptor substrate-1 (IRS-1) and compared them to vector-expressing controls. IRS-1 overexpression dramatically increased basal cytosolic Ca2+ levels from 81 to 278 nM, but it did not affect Ca2+ response to glucose. Overexpression of the insulin receptor also caused an increase in cytosolic Ca2+. Increased cytosolic Ca2+ was due to inhibition of Ca2+ uptake by the endoplasmic reticulum, because endoplasmic reticulum Ca2+ uptake and content were reduced in betaIRS1-A cells. Fractional insulin secretion was significantly increased 2-fold, and there was a decrease in betaIRS1-A insulin content and insulin biosynthesis. Steady-state insulin mRNA levels and glucose-stimulated ATP were unchanged. High IRS-1 levels also reduced beta-cell proliferation. These data demonstrate a direct link between the insulin receptor signaling pathway and the Ca2+-dependent pathways regulating insulin secretion of beta-cells. We postulate that during regulated insulin secretion, released insulin binds the beta-cell insulin receptor and activates IRS-1, thus further increasing cytosolic Ca2+ by reducing Ca2+ uptake. We suggest the existence of a novel pathway of autocrine regulation of intracellular Ca2+ homeostasis and insulin secretion in the beta-cell of the endocrine pancreas.     

Pancreatic beta-cell growth and survival--a role in obesity-linked type 2 diabetes?

Obesity-linked type 2 diabetes is a disease of insulin resistance combined with pancreatic beta-cell dysfunction. Although a role for beta-cell mass in the pathogenesis of obesity-linked type 2 diabetes has recently gained prominence, the idea is still being developed. It is proposed that in early obesity an increase in beta-cell mass and function might compensate for peripheral insulin resistance. However, as time and/or the severity of the obesity continue, there is decay in such adaptation and the beta-cell mass becomes inadequate. This, together with beta-cell dysfunction, leads to the onset of type 2 diabetes. It is becoming evident that elements in insulin and insulin growth factor (IGF)-1 signal-transduction pathways are key to regulating beta-cell growth. Current evidence indicates that interference of insulin signaling in obesity contributes to peripheral insulin resistance. This article examines whether a similar interference of IGF-1 signaling in the beta-cell could hinder upregulation of beta-cell mass and/or function, resulting in a failure to compensate for insulin resistance.     

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.     

Overexpression of plasma membrane-associated sialidase attenuates insulin signaling in transgenic mice

Plasma membrane-associated sialidase is a key enzyme for ganglioside hydrolysis, thereby playing crucial roles in regulation of cell surface functions. Here we demonstrate that mice overexpressing the human ortholog (NEU3) develop diabetic phenotype by 18-22 weeks associated with hyperinsulinemia, islet hyperplasia, and increased beta-cell mass. As compared with the wild type, insulin-stimulated phosphorylation of the insulin receptor (IR) and insulin receptor substrate I was significantly reduced, and activities of phosphatidylinositol 3-kinase and glycogen synthase were low in transgenic muscle. IR phosphorylation was already attenuated in the younger mice before manifestation of hyperglycemia. Transient transfection of NEU3 into 3T3-L1 adipocytes and L6 myocytes caused a significant decrease in IR signaling. In response to insulin, NEU3 was found to undergo tyrosine phosphorylation and subsequent association with the Grb2 protein, thus being activated and causing negative regulation of insulin signaling. In fact, accumulation of GM1 and GM2, the possible sialidase products in transgenic tissues, caused inhibition of IR phosphorylation in vitro, and blocking of association with Grb2 resulted in reversion of impaired insulin signaling in L6 cells. The data indicate that NEU3 indeed participates in the control of insulin signaling, probably via modulation of gangliosides and interaction with Grb2, and that the mice can serve as a valuable model for human insulin-resistant diabetes.     

Endoplasmic reticulum stress-induced apoptosis and auto-immunity in diabetes

Increasing evidence suggests that stress signaling pathways emanating from the endoplasmic reticulum (ER) are important to the pathogenesis of both type 1 and type 2 diabetes. Recent observations indicate that ER stress signaling participates in maintaining the ER homeostasis of pancreatic beta-cells. Either a high level of ER stress or defective ER stress signaling in beta-cells may cause an imbalance in ER homeostasis and lead to beta-cell apoptosis and autoimmune response. In addition, it has been suggested that ER stress attributes to insulin resistance in patients with type 2 diabetes. It is necessary to study the relationship between ER stress and diabetes in order to develop new therapeutic approaches to diabetes based on drugs that block the ER stress-mediated cell-death pathway and insulin resistance.     

Fibroblast growth factor receptor-1 signaling in pancreatic islet beta-cells is modulated by the extracellular matrix

Maintenance of pancreatic beta-cell mass depends on extracellular stimuli that promote survival and proliferation. In the islet, these stimuli come from the beta-cell microenvironment and include extracellular matrix deposited by associated vascular endothelial cells. Fibroblast growth factor receptor-1 (FGFR1) has recently been implicated as a signaling pathway that is important for normal beta-cell function. We would like to understand how extracellular matrix and FGFR1 signaling interact to promote beta-cell survival and proliferation. To examine beta-cell-specific receptor responses, we created lentiviral vectors with rat insulin promoter-driven expression of Venus fluorescent protein-tagged full-length (R1betav) and kinase-deficient (KDR1betav) FGFR1. Significant FGF-1-dependent activation of ERK1/2 was observed in betaTC3 cells, dispersed beta-cells, and beta-cells in intact islets. This response was enhanced by R1betav expression and reduced by KDR1betav expression. Plating-dispersed beta-cells on collagen type IV resulted in enhanced expression of endogenous FGFR1 that was associated with sustained activation of ERK1/2. Conversely, plating cells on laminin reduced expression of FGFR1, and this reduction was associated with transient activation of ERK1/2. Addition of neutralizing antibodies to inhibit beta-cell attachment to laminin via alpha(6)-integrin increased high-affinity FGF-1-binding at the plasma membrane and resulted in sustained ERK1/2 activity similar to cells plated on collagen type IV. These data show that the FGF-stimulated beta-cell response is negatively affected by alpha(6)-integrin binding to laminin and suggest regulation associated with vascular endothelial cell remodeling.     

The FRK/RAK-SHB signaling cascade: a versatile signal-transduction pathway that regulates cell survival,differentiation and proliferation

Recent experiments have unravelled novel signal transduction pathways that involve the SRC homology 2 (SH2) domain adapter protein SHB. SHB is ubiquitously expressed and contains proline rich motifs, a phosphotyrosine binding (PTB) domain, tyrosine phosphorylation sites and an SH2 domain and serves a role in generating signaling complexes in response to tyrosine kinase activation. SHB mediates certain responses in platelet-derived growth factor (PDGF) receptor-, fibroblast growth factor (FGF) receptor-, neural growth factor (NGF) receptor TRKA-, T cell receptor-, interleukin-2 (IL-2) receptor- and focal adhesion kinase- (FAK) signaling. Upstream of SHB in some cells lies the SRC-like FYN-Related Kinase FRK/RAK (also named BSK/IYK or GTK). FRK/RAK and SHB exert similar effects when overexpressed in rat phaeochromocytoma (PC12) and beta-cells, where they both induce PC12 cell differentiation and beta-cell proliferation. Furthermore, beta-cell apoptosis is augmented by these proteins under conditions that cause beta-cell degeneration. The FRK/RAK-SHB responses involve FAK and insulin receptor substrates (IRS) -1 and -2. Besides regulating apoptosis, proliferation and differentiation, SHB is also a component of the T cell receptor (TCR) signaling response. In Jurkat T cells, SHB links several signaling components with the TCR and is thus required for IL-2 production. In endothelial cells, SHB both promotes apoptosis under conditions that are anti-angiogenic, but is also required for proper mitogenicity, spreading and tubular morphogenesis. In embryonic stem cells, dominant-negative SHB (R522K) prevents early cavitation of embryoid bodies and reduces differentiation to cells expressing albumin, amylase, insulin and glucagon, suggesting a role of SHB in development. In summary, SHB is a versatile signal transduction molecule that produces diverse biological responses in different cell types under various conditions. SHB operates downstream of GTK in cells that express this kinase.     

Notch signaling: a mediator of beta-cell de-differentiation in diabetes?

Cytokines are mediators of pancreatic beta-cell dysfunction and death in type 1 diabetes mellitus. Microarray analyses of insulin-producing cells exposed to interleukin-1beta+interferon-gamma showed decreased expression of genes related to beta-cell-differentiated functions and increased expression of members of the Notch signaling pathway. Re-expression of this developmental pathway may contribute for loss-of-function of beta-cells exposed to an autoimmune attack. In this study, we show that rat primary beta-cells exposed to cytokines up-regulate several Notch receptors and ligands, and the target gene Hes1. Transfection of insulin-producing INS-1E cells and primary rat beta-cells with a constitutively active form of the Notch receptor down-regulated Pdx1 and insulin expression in INS-1E cells but not in primary beta-cells. Thus, activation of the Notch pathway inhibits differentiated functions in dividing but not in terminally differentiated beta-cells.     

Exercise improves glucose homeostasis that has been impaired by a high-fat diet by potentiating pancreatic beta-cell function and mass through IRS2 in diabetic rats.

In this study, we investigated the effects of a high-fat diet and exercise on pancreatic beta-cell function and mass and its molecular mechanism in 90% pancreatectomized male rats. The pancreatectomized diabetic rats were given control diets (20% energy) or a high-fat (HF) diet (45% energy) for 12 wk. Half of each group was given regular exercise on an uphill treadmill at 20 m/min for 30 min 5 days/wk. HF diet lowered first-phase insulin secretion with glucose loading, whereas exercise training reversed this decrease. However, second-phase insulin secretion did not differ among the groups. Exercise increased pancreatic beta-cell mass. This resulted from stimulated beta-cell proliferation and reduced apoptosis, which is associated with potentiated insulin or IGF-I signaling through insulin receptor substrate-2 (IRS2) induction. Although the HF diet resulted in decreased proliferation and accelerated apoptosis by weakened insulin and IGF-I signaling from reduction of IRS2 protein, beta-cell mass was maintained in HF rats just as much as in control rats via increased individual beta-cell size and neogenesis from precursor cells. Consistent with the results of beta-cell proliferation, pancreas duodenal homeobox-1 expression increased in the islets of rats in the exercise groups, and it was reduced the most in rats fed the HF diet. In conclusion, exercise combined with a moderate fat diet is a good way to maximize beta-cell function and mass through IRS2 induction to alleviate the diabetic condition. This study suggests that dietary fat contents and exercise modulate beta-cell function and mass to overcome insulin resistance in two different pathways.     

IRS proteins and the common path to diabetes

Although a full understanding of insulin/insulin-like growth factor (IGF) action is evolving, the discovery of insulin receptor substrate (IRS) proteins and their role to link cell surface receptors to the intracellular signaling cascades provided an important step forward. Moreover, Insulin/IGF receptors use common signaling pathways to accomplish many tasks, the IRS proteins add a unique layer of specificity and control. Importantly, the IRS-2 branch of the insulin/IGF-signaling pathway is a common element in peripheral insulin response and pancreatic beta-cell growth and function. Failure of IRS-2 signaling might explain the eventual loss of compensatory hyperinsulinemia during prolonged periods of peripheral insulin resistance. Moreover, short-term inhibition of IRS protein functions by serine phosphorylation, or sustained inhibition by ubiquitin-targeted proteosome-mediated degradation suggests a common molecular mechanism for insulin resistance during acute injury or infection, or the sensitivity of beta-cells to autoimmune destruction. The broad role of IRS-1 and IRS-2 in cell growth and survival reveals a common regulatory pathway linking development, somatic growth, fertility, neuronal proliferation, and aging to the core mechanisms used by vertebrates for nutrient sensing.     

Beta-cell ion channels: keys to endodermal excitability.

Whereas pancreatic islet cells are not neurons, they are endodermally-derived specialized excitable cells that display many properties of neurons. Multiple ion channels in the pancreatic beta-cell regulate electrical excitability. Our focus for the last several years has been on the delayed rectifier (Kv) K+ channels, in an effort to define the individual roles of specific Kv channel genes in the overall regulation of insulin secretion. The many Kv channel genes, represented by more than 40 mammalian isoforms (termed Kv1 to Kv8), give rise to overlapping functions, primarily regulating repolarization of the plasma membrane. Experiments involving inhibition of Kv channel function have shown the important role Kv channels play in regulating beta-cell calcium oscillations in response to glucose stimulation. From our recent studies, we have concluded that although detectable mRNA for Kv1 family members is present in islets, Kv1 family channels are unlikely to play a significant role in the beta-cell, and we are now focusing on the roles of Kv2 and Kv3 channels.     

Glucose-stimulated Cdc42 signaling is essential for the second phase of insulin secretion

The small Rho family GTPases Cdc42 and Rac1 have each been shown to function in insulin exocytosis and are presumed to function in actin remodeling and insulin granule mobilization. However, whether either GTPase is required for the mobilization phase of insulin release (second phase) and are linked in a common signaling pathway has remained unknown. Here we demonstrate that small interfering RNA-mediated depletion of Cdc42 from isolated islets results in the selective loss of second phase insulin release. Consistent with a role in this nutrient-dependent phase, Cdc42 activation was detected exclusively in response to D-glucose and was unresponsive to KCl or non-metabolizable glucose analogs in MIN6 beta-cells. Cdc42 activation occurred early in secretion (3 min), whereas Rac1 activation required approximately 15-20 min, suggesting Cdc42 as proximal and Rac1 as distal regulators of second-phase secretion. Importantly, Rac1 activation and function was linked in a common pathway downstream of Cdc42; Cdc42 depletion ablated glucose-induced Rac1 activation, and expression of constitutively active Rac1 in Cdc42-depleted cells functionally restored glucose-stimulated insulin secretion. Occurring at a time midway between Cdc42 and Rac1 activations was the phosphorylation of p21-activated-kinase 1 (Pak1), and this phosphorylation event required Cdc42. Moreover, small interfering RNA-mediated Pak1 depletion abolished Rac1 activation and glucose-stimulated insulin release, suggesting that Pak1 may mediate the link between Cdc42 and Rac1 in this pathway. Taken together, these data substantiate the existence of a novel signaling pathway in the islet beta-cell whereby Cdc42 functions as a key proximal transmitter of the glucose signal early in stimulus-secretion coupling to support the later stage of insulin release.     

Inhibition of Ca2+-independent phospholipase A2 results in insufficient insulin secretion and impaired glucose tolerance

Islet Ca2+-independent phospholipase A2 (iPLA2) is postulated to mediate insulin secretion by releasing arachidonic acid in response to insulin secretagogues. However, the significance of iPLA2 signaling in insulin secretion in vivo remains unexplored. Here we investigated the physiological role of iPLA2 in beta-cell lines, isolated islets, and mice. We showed that small interfering RNA-specific silencing of iPLA2 expression in INS-1 cells significantly reduced insulin-secretory responses of INS-1 cells to glucose. Immunohistochemical analysis revealed that mouse islet cells expressed significantly higher levels of iPLA2 than pancreatic exocrine acinar cells. Bromoenol lactone (BEL), a selective inhibitor of iPLA2, inhibited glucose-stimulated insulin secretion from isolated mouse islets; this inhibition was overcome by exogenous arachidonic acid. We also showed that iv BEL administration to mice resulted in sustained hyperglycemia and reduced insulin levels during glucose tolerance tests. Clamp experiments demonstrated that the impaired glucose tolerance was due to insufficient insulin secretion rather than decreased insulin sensitivity. Short-term administration of BEL to mice had no effect on fasting glucose levels and caused no apparent pathological changes of islets in pancreas sections. These results unambiguously demonstrate that iPLA2 signaling plays an important role in glucose-stimulated insulin secretion under physiological conditions.