Activation of the extracellular calcium-sensing receptor initiates insulin secretion from human islets of Langerhans: involvement of protein kinases

The extracellular calcium-sensing receptor (CaR) is usually associated with systemic Ca(2+) homeostasis, but the CaR is also expressed in many other tissues, including pancreatic islets of Langerhans. In the present study, we have used human islets and an insulin-secreting cell line (MIN6) to investigate the effects of CaR activation using the calcimimetic R-568, a CaR agonist that activates the CaR at physiological concentrations of extracellular Ca(2+). CaR activation initiated a marked but transient insulin secretory response from both human islets and MIN6 cells at a sub-stimulatory concentration of glucose, and further enhanced glucose-induced insulin secretion. CaR-induced insulin secretion was reduced by inhibitors of phospholipase C or calcium-calmodulin-dependent kinases, but not by a protein kinase C inhibitor. CaR activation was also associated with an activation of p42/44 mitogen-activated protein kinases (MAPK), and CaR-induced insulin secretion was reduced by an inhibitor of p42/44 MAPK activation. We suggest that the beta-cell CaR is activated by divalent cations co-released with insulin, and that this may be an important mechanism of intra-islet communication between beta-cells.     

Long-term inhibition of protein tyrosine kinase impairs electrophysiologic activity and a rapid component of exocytosis in pancreatic beta-cells

Dysfunction of pancreatic beta-cells is a fundamental feature in the pathogenesis of type 2 diabetes. As insulin receptor signaling occurs via protein tyrosine kinase (PTK), we investigated the role of PTK activity in the etiology of beta-cell dysfunction by inhibiting PTK activity in primary cultured mouse pancreatic beta-cells and INS-1 cells with genistein treatment over 24 h. Electrophysiologic recordings showed genistein treatment significantly attenuated ATP-sensitive K(+) (K(ATP)) and voltage-dependent Ca(2+) currents, and depolarized the resting membrane potential in primary beta-cells. When stimulated by high glucose, genistein-treated beta-cells exhibited a time delay of both depolarization and Ca(2+) influx, and were unable to fire action potentials, as well as displaying a reduced level of Ca(2+) influx and a loss of Ca(2+) oscillations. Semiquantitative PCR analysis revealed decreased expression of K(ATP) and L-type Ca(2+) channel mRNA in genistein-treated islets. PTK inhibition also significantly reduced the rapid component of secretory vesicle exocytosis, as indicated by membrane capacitance measurements, and this is likely to be due to the reduced Ca(2+) current amplitude in these cells. These results illustrate that compromised PTK activity contributes to pancreatic beta-cell dysfunction and may be involved in the etiology of type 2 diabetes.     

Identification of signal transduction pathways that modulate dibutyryl cyclic adenosine monophosphate activation of stanniocalcin gene expression in neuroblastoma cells

Stanniocalcin (STC) is a new mammalian polypeptide hormone and appears to be a regulator of neuronal function. We have already shown that the induction of STC mRNA and protein expression by cAMP is integral to neuroblastoma cell differentiation, particularly neurite outgrowth. In this study, we examined the cAMP pathway in greater detail. Some common neuritogenic agents, euxanthone (PW1) and trans-retinoic acid (RA), were studied for possible interactions with the dibutyryl cAMP (dbcAMP)-mediated response. Our results showed that STC mRNA induction by dbcAMP was mediated by protein kinase A-cAMP response element binding protein (CREB) pathway, accompanied with phosphorylation of CREB and a reduction of p50, p65, and phosphorylated inhibitor kappaBalpha levels. Using a synthetic peptide nuclear factor-kappaB SN50, stimulation of dbcAMP-mediated STC expression was observed; indicating the nuclear translocation of nuclear factor kappaB might possibly repress STC expression. dbcAMP-induced STC mRNA expression was enhanced by PW1. In contrast, RA had highly suppressive effects. Cotreatment of cell with PW1 and cAMP provoked an increase in phosphorylated CREB (pCREB). Conversely, cotreatment with RA suppressed pCREB. The results highlighted the importance of phosphorylation of CREB in mediating STC gene expression. Taking a step further to dissect the possible regulatory pathways involved, with the aid of phorbol 12-myristate 13-acetate or ionomycin, additive effects on STC gene expression were observed. The induction was aided by further elevation of pCREB, which was completely abolished by G? 6976, a Ca2+-dependent protein kinase C (PKC) alpha and PKCbeta1 inhibitor. Our results indicated that cross-talk with PKC and/or Ca2+ signaling pathways might sensitize cAMP-mediated effects, on CREB phosphorylation and STC gene expression.     

Sterol regulatory element-binding protein 1 mediates liver X receptor-beta-induced increases in insulin secretion and insulin messenger ribonucleic acid levels

Liver X receptors (LXRalpha and LXRbeta) regulate glucose and lipid metabolism. Pancreatic beta-cells and INS-1E insulinoma cells express only the LXRbeta isoform. Activation of LXRbeta with the synthetic agonist T0901317 increased glucose-induced insulin secretion and insulin content, whereas deletion of the receptor in LXRbeta knockout mice severely blunted insulin secretion. Analysis of gene expression in LXR agonist-treated INS-1E cells and islets from LXRbeta-deficient mice revealed that LXRbeta positively regulated expression of ATP-binding cassette transporter A1 (ABCA1), sterol regulatory element-binding protein 1 (SREBP-1), insulin, PDX-1, glucokinase, and glucose transporter 2 (Glut2). Down-regulation of SREBP-1 expression with the specific small interfering RNA blocked basal and LXRbeta-induced expression of pancreatic duodenal homeobox 1 (PDX-1), insulin, and Glut2 genes. SREBP-1 small interfering RNA also prevented an increase in insulin secretion and insulin content induced by T0901317. Moreover, 5-(tetradecyloxy)-2-furoic acid, an inhibitor of the SREBP-1 target gene acetyl-coenzyme A carboxylase, blocked T0901317-induced stimulation of insulin secretion. In conclusion, activation of LXRbeta in pancreatic beta-cells increases insulin secretion and insulin mRNA expression via SREBP-1-regulated pathway. These data support the role of LXRbeta, SREBP-1, and cataplerosis/anaplerosis pathways in the control of insulin secretion in pancreatic beta-cells.     

N-type Ca(2+) -channels in murine pancreatic beta-cells are inhibited by an exclusive coupling with somatostatin receptor subtype 1

Somatostatin (SRIF) is a well-established inhibitor of insulin secretion, an effect in part mediated by a direct inhibition of voltage-operated Ca(2+)-channels. However, the identity of the somatostatin receptor subtypes (SSTRs) and voltage-operated Ca(2+)-channels involved in this process are unknown. Whole-cell perforated patch-clamp methods were applied to the murine pancreatic beta-cell line, MIN6, to explore the molecular pharmacology of this problem. SRIF-14 inhibited voltage-gated Ca(2+) currents (ICa(2+)) by 19 +/- 3% (n=24) with a pEC(50) = 9.05 (95% confidence limits 9-9.1). This action was mimicked solely by 100 nm CH-275, a selective agonist at the somatostatin type 1 receptor (SSTR1), but not by 100 nm BIM-23027, L-362855, or NNC-269100; agonists selective for the other four SSTRs known to exist in MIN6. The inhibition of ICa(2+) produced by SRIF and CH-275 was insensitive to pertussis toxin but was reversed by a prepulse to +100 mV. The inhibition of ICa(2+) by SRIF-14 was unaffected by 20 microm nifedipine, an inhibitor of L-type Ca(2+) channels. Application of the specific N-type Ca(2+) channel (Ca(v)2.2) inhibitor omega-conotoxin GV1A at 100 nm mimicked, and as a consequence abolished, the inhibitory effect of SRIF-14 on ICa(2+). SRIF selectively inhibits N-type Ca(2+)-channels in murine pancreatic beta-cells via exclusive coupling with SSTR1. These findings help explain how SSTR1 activation can inhibit insulin secretion in pancreatic beta-cells and suggest a possible new therapeutic lead for treatment of hyperinsulinemia.     

Regulation of insulin secretion by overexpression of Ca2+/calmodulin-dependent protein kinase II in insulinoma MIN6 cells

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) may play a key role in Ca2+-induced insulin secretion. We have previously reported that treatment of insulinoma MIN6 cells with secretagogues activated CaM kinase II and increased the phosphorylation of synapsin I, followed by insulin secretion. Here, we identified isoforms of CaM kinase II in MIN6 cells and rat islets. Immunoblot analysis suggested that the major isoforms of CaM kinase II were beta'e and delta2 at the protein level in MIN6 cells. Only the beta'e isoform was detected in rat islets by both RT-PCR and immunoblot analysis. We transiently overexpressed beta'e and delta2 isoforms in MIN6 cells and confirmed that treatment of cells with tolbutamide and glucose activated the isoforms. Immunoblot analysis with an antibody against synapsin I phosphorylated by CaM kinase II demonstrated that treatment with tolbutamide and glucose rapidly increased phosphorylation of synapsin I and that phosphorylation was potentiated by overexpression of the isoforms. The secretagogue-induced insulin secretion was potentiated by overexpression of the isoforms. Our results further support our conclusion that activation of CaM kinase II and the concomitant phosphorylation of synapsin I contribute to insulin secretion from pancreatic beta-cells.     

Dual effect of cell-cell contact disruption on cytosolic calcium and insulin secretion

Cell-to-cell interactions play an important role in insulin secretion. Compared with intact islets, dispersed pancreatic beta-cells show increased basal and decreased glucose-stimulated insulin secretion. In this study, we used mouse MIN6B1 cells to investigate the mechanisms that control insulin secretion when cells are in contact with each other or not. RNAi-mediated silencing of the adhesion molecule E-cadherin in confluent cells reduced glucose-stimulated secretion to the levels observed in isolated cells but had no impact on basal secretion. Dispersed cells presented high cytosolic Ca(2+) activity, depolymerized cytoskeleton and ERK1/2 activation in low glucose conditions. Both the increased basal secretion and the spontaneous Ca(2+) activity were corrected by transient removal of Ca(2+) or prolonged incubation of cells in low glucose, a procedure that restored the ability of dispersed cells to respond to glucose (11-fold stimulation). In conclusion, we show that dispersed pancreatic beta-cells can respond robustly to glucose once their elevated basal secretion has been corrected. The increased basal insulin secretion of dispersed cells is due to spontaneous Ca(2+) transients that activate downstream Ca(2+) effectors, whereas engagement of cell adhesion molecules including E-cadherin contributes to the greater secretory response to glucose seen in cells with normal intercellular contacts.     

Mechanisms of mitogenic and anti-apoptotic signaling by glucose-dependent insulinotropic polypeptide in beta(INS-1)-cells

Glucose-dependent insulinotropic polypeptide (GIP) acts as a glucose-dependent growth factor for beta-cells. Here we show that GIP and glucose also act synergistically as anti-apoptotic factors for beta-cells, using the well-differentiated beta-cell line, INS-1. Mitogenic and anti-apoptotic signaling of GIP were dependent upon pleiotropic activation of protein kinase A (PKA)/cAMP regulatory element binder (CREB), mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-kinase)/PKB signaling modules. The signaling modules activated by GIP were dependent on glucose metabolism and calcium influx and were tightly linked by multiple activating and inhibiting cross-talk. These interactions included: (i) a central role of tyrosine phosphorylation for stimulation of PKA/CREB, MAPK and PI3-kinase/PKB, (ii) inhibition of PKA/CREB by the MAPK pathway at the level of MAPK kinase-1 or downstream, (iii) activation of MAPK signaling by PI3-kinase and PKA at the level of extracellular-signal regulated kinase 1/2 or upstream, and (iv) activation of PKB by MAPK and PKA signaling at the level of PKB or upstream. Furthermore, we demonstrated inhibition of CREB signaling by Ca(2+)/calmodulin kinase I/IV. These results indicated that GIP acts as a mitogenic and anti-apoptotic factor for beta-cells by pleiotropic activation of tightly linked signaling pathways in beta-cells.     

Ca(2+)-induced Ca(2+) release in the pancreatic beta-cell: direct evidence of endoplasmic reticulum Ca(2+) release

The role of the Ca(2+)-induced Ca(2+) release channel (ryanodine receptor) in MIN6 pancreatic beta-cells was investigated. An endoplasmic reticulum (ER)-targeted "cameleon" was used to report lumenal free Ca(2+). Depolarization of MIN6 cells with KCl led to release of Ca(2+) from the ER. This ER Ca(2+) release was mimicked by treatment with the ryanodine receptor agonists caffeine and 4-chloro-m-cresol, reversed by voltage-gated Ca(2+) channel antagonists and blocked by treatment with antagonistic concentrations of ryanodine. The depolarization-induced rise in cytoplasmic Ca(2+) was also inhibited by ryanodine, which did not alter voltage-gated Ca(2+) channel activation. Both ER and cytoplasmic Ca(2+) changes induced by depolarization occurred in a dose-dependent manner. Glucose caused a delayed rise in cytoplasmic Ca(2+) but no detectable change in ER Ca(2+). Carbamyl choline caused ER Ca(2+) release, a response that was not altered by ryanodine. Taken together, these results provide strong evidence that Ca(2+)-induced Ca(2+) release augments cytoplasmic Ca(2+) signals in pancreatic beta-cells.     

Glucose enhances human macrophage LOX-1 expression: role for LOX-1 in glucose-induced macrophage foam cell formation

Lectin-like oxidized LDL receptor-1 (LOX-1) is a newly identified receptor for oxidized LDL that is expressed by vascular cells. LOX-1 is upregulated in aortas of diabetic rats and thus may contribute to the pathogenesis of human diabetic atherosclerosis. In this study, we examined the regulation of human monocyte-derived macrophage (MDM) LOX-1 expression by high glucose and the role of LOX-1 in glucose-induced foam cell formation. Incubation of human MDMs with glucose (5.6 to 30 mmol/L) enhanced, in a dose- and time-dependent manner, LOX-1 gene and protein expression. Induction of LOX-1 gene expression by high glucose was abolished by antioxidants, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), nuclear factor-kappaB (NF-kappaB), and activated protein-1 (AP-1) inhibitors. In human MDMs cultured with high glucose, increased expression of PKCbeta2 and enhanced phosphorylation of extracellular signal-regulated protein kinase 1/2 was observed. Activation of these kinases was inhibited by the antioxidant N-acetyl-L-cysteine (NAC) and by the PKCbeta inhibitor LY379196. High glucose also enhanced the binding of nuclear proteins extracted from human MDMs to the NF-kappaB and AP-1 regulatory elements of the LOX-1 gene promoter. This effect was abrogated by NAC and PKC/MAPK inhibitors. Finally, high glucose induced human macrophage-derived foam cell formation through a LOX-1-dependent pathway. Overall, these results demonstrate that high glucose concentrations enhance LOX-1 expression in human MDMs and that this effect is associated with foam cell formation. Pilot data showing that MDMs of patients with type 2 diabetes overexpress LOX-1 support the relevance of this work to human diabetic atherosclerosis.     

Insulin secretagogues,but not glucose,stimulate an increase in [Ca2+]i in the fetal human and porcine beta-cell

Fetal pancreatic beta-cells release insulin poorly in response to glucose; however, the cellular mechanism for this is unknown. By using fura-2 to measure changes in the cytoplasmic free Ca(2+) concentration in beta-cells, we examined human/porcine fetal islet-like cell clusters (ICCs) and human adult islets for the presence of functional K(+)(ATP) and voltage-activated Ca(2+) ion channels. The effects of glucose, glyceraldehyde, leucine, KCl, and the channel effectors glipizide and BAY K8644 were studied. In fetal human/porcine ICCs and adult islets, KCl, glipizide, and BAY K8644 increased [Ca(2+)](i). Both glucose and glyceraldehyde increased [Ca(2+)](i) in islets but had no effect on ICCs. Leucine increased [Ca(2+)](i) in islets and porcine but not human ICCs. We hypothesize that the beneficial effect of leucine in fetal porcine, but not human ICCs, is attributable to time-dependent maturation of the beta-cells, because porcine ICCs examined were at 87% of the gestational period, and human ICCs were at 42%. Our data demonstrate that both K(+)(ATP) and voltage-activated Ca(2+) channels, required for glucose-stimulated increase in [Ca(2+)](i), are functional early in gestation. This suggests that the cause of the immaturity of fetal human/porcine beta-cells is at a more proximal step of glucose-induced metabolism than the channels on the cell surface.     

Calcium has a permissive role in interleukin-1beta-induced c-jun N-terminal kinase activation in insulin-secreting cells

The c-jun N-terminal kinase (JNK) signaling pathway mediates IL-1beta-induced apoptosis in insulin-secreting cells, a mechanism relevant to the destruction of pancreatic beta-cells in type 1 and 2 diabetes. However, the mechanisms that contribute to IL-1beta activation of JNK in beta-cells are largely unknown. In this study, we investigated whether Ca(2+) plays a role for IL-1beta-induced JNK activation. In insulin-secreting rat INS-1 cells cultured in the presence of 11 mm glucose, combined pharmacological blockade of L- and T-type Ca(2+) channels suppressed IL-1beta-induced in vitro phosphorylation of the JNK substrate c-jun and reduced IL-1beta-stimulated activation of JNK1/2 as assessed by immunoblotting. Inhibition of IL-1beta-induced in vitro kinase activity toward c-jun after collective L- and T-type Ca(2+) channel blockade was confirmed in primary rat and ob/ob mouse islets and in mouse betaTC3 cells. Ca(2+) influx, specifically via L-type but not T-type channels, contributed to IL-1beta activation of JNK. Activation of p38 and ERK in response to IL-1beta was also dependent on L-type Ca(2+) influx. Membrane depolarization by KCl, exposure to high glucose, treatment with Ca(2+) ionophore A23187, or exposure to thapsigargin, an inhibitor of sarco(endo)plasmic reticulum Ca(2+) ATPase, all caused an amplification of IL-1beta-induced JNK activation in INS-1 cells. Finally, a chelator of intracellular free Ca(2+) [bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-acetoxymethyl], an inhibitor of calmodulin (W7), and inhibitors of Ca(2+)/calmodulin-dependent kinase (KN62 and KN93) partially reduced IL-1beta-stimulated c-jun phosphorylation in INS-1 or betaTC3 cells. Our data suggest that Ca(2+) plays a permissive role in IL-1beta activation of the JNK signaling pathway in insulin-secreting cells.     

Somatostatin inhibits oxidative respiration in pancreatic beta-cells

Somatostatin potently inhibits insulin secretion from pancreatic beta-cells. It does so via activation of ATP-sensitive K+-channels (KATP) and G protein-regulated inwardly rectifying K+-channels, which act to decrease voltage-gated Ca2+-influx, a process central to exocytosis. Because KATP channels, and indeed insulin secretion, is controlled by glucose oxidation, we investigated whether somatostatin inhibits insulin secretion by direct effects on glucose metabolism. Oxidative metabolism in beta-cells was monitored by measuring changes in the O2 consumption (DeltaO2) of isolated mouse islets and MIN6 cells, a murine-derived beta-cell line. In both models, glucose-stimulated DeltaO2, an effect closely associated with inhibition of KATP channel activity and induction of electrical activity (r > 0.98). At 100 nm, somatostatin abolished glucose-stimulated DeltaO2 in mouse islets (n = 5, P < 0.05) and inhibited it by 80 +/- 28% (n = 17, P < 0.01) in MIN6 cells. Removal of extracellular Ca2+, 5 mm Co2+, or 20 microm nifedipine, conditions that inhibit voltage-gated Ca2+ influx, did not mimic but either blocked or reduced the effect of the peptide on DeltaO2. The nutrient secretagogues, methylpyruvate (10 mm) and alpha-ketoisocaproate (20 mm), also stimulated DeltaO2, but this was unaffected by somatostatin. Somatostatin also reversed glucose-induced hyperpolarization of the mitochondrial membrane potential monitored using rhodamine-123. Application of somatostatin receptor selective agonists demonstrated that the peptide worked through activation of the type 5 somatostatin receptor. In conclusion, somatostatin inhibits glucose metabolism in murine beta-cells by an unidentified Ca2+-dependent mechanism. This represents a new signaling pathway by which somatostatin can inhibit cellular functions regulated by glucose metabolism.