Abnormally decreased NO and augmented CO production in islets of the leptin-deficient ob/ob mouse might contribute to explain hyperinsulinemia and islet survival in leptin-resistant type 2 obese diabetes

The role of the gaseous messengers NO and CO for β-cell function and survival is controversial. We examined this issue in the hyperglycemic-hyperinsulinemic ob/ob mouse, an animal model of type 2 obese diabetes, by studying islets from obese vs lean mice regarding glucose-stimulated insulin release in relation to islet NO and CO production and the influence of modulating peptide hormones. Glucose-stimulated increase in ncNOS-activity in incubated lean islets was converted to a decrease in ob/ob islets associated with markedly increased insulin release. Both types of islets displayed iNOS activity appearing after ~60 min in high-glucose. In ob/ob islets the insulinotropic peptides glucagon, GLP-1 and GIP suppressed NOS activities and amplified glucose-stimulated insulin release. The insulinostatic peptide leptin induced the opposite effects. Suppression of islet CO production inhibited, while stimulation amplified glucose-stimulated insulin release. Nonincubated isolated islets from young and adult obese mice displayed very low ncNOS and negligible iNOS activity. In contrast, production of CO, a NOS inhibitor, was impressively raised. Glucose injections induced strong activities of islet NOS isoforms in lean but not in obese mice and confocal microscopy revealed iNOS expression only in lean islets. Islets from ob/ob mice existing in a hyperglycemic in vivo milieu maintain elevated insulin secretion and protection from glucotoxicity through a general suppression of islet NOS activities achieved by leptin deficiency, high CO production and insulinotropic cyclic-AMP-generating hormones. Such a beneficial effect on islet function and survival might have its clinical counterpart in human leptin-resistant type 2 obese diabetes with hyperinsulinemia.     

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.     

Pancreatic beta-cells communicate via intermittent release of ATP

The role of external ATP for intercellular communication was studied in glucose-stimulated pancreatic beta-cells isolated from ob/ob mice. Digital image analyses with fura-2 revealed spontaneous transients of cytoplasmic Ca2+ appearing in synchrony in the absence of cell contacts. After removal of slow oscillations with methoxyverapamil, addition of ATP (0.1-100 microM) resulted in prompt firing of a transient, followed by suppression of the generation and synchronization of spontaneously occurring transients. It was possible to trigger transients during the suppressive phase by raising the concentration of ATP. The dual action of ATP was mimicked by ADP or 2-methylthio-ATP but not by AMP or UTP. The number of spontaneous transients and their synchronization were reduced in the presence of the dephosphorylating agent apyrase. Additional evidence that intermittent release of ATP participates in the generation of spontaneous Ca2+ transients was obtained from the suppression observed from use of antagonists of the purinoceptors [suramin (0.3-30 microM), pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid (PPADS; 10-30 microM) and 2-deoxy-N-methyladenosine (MRS 2179; 0.3-30 microM)] or from counteracting beta-cell release of ATP by inhibiting exocytosis with 100 nM epinephrine, 100 nM somatostatin, or lowering the temperature below 30 degrees C. The data indicate that ATP has time-dependent actions (prompt stimulation followed by inhibition) on the generation of Ca2+ transients mediated by P2Y receptors. It is proposed that beta-cells both receive a neural ATP signal with coordinating effects on their Ca2+ oscillations and propagate this message to adjacent cells via intermittent release of ATP combined with gap junction coupling.     

Cell-specific Ca(2+) responses in glucose-stimulated single and aggregated beta-cells

A rise in the cytoplasmic calcium concentration ([Ca(2+)](i)) is a key event for insulin exocytosis. We have recently found that the 'early [Ca(2+)](i) response' in single ob/ob mouse beta-cells is reproduced during consecutive glucose stimulations. It, therefore, appears that the response pattern is a characteristic of the individual beta-cell. We have now investigated if a cell-specific [Ca(2+)](i) response is a general phenomenon in rodent beta-cells, and if it can be observed when cells are functionally coupled. With the use of the fura-2 technique, we have studied the 'early [Ca(2+)](i) response' in single dispersed beta-cells, in beta-cell clusters of different size and in intact islets from the ob/ob mouse during repeated glucose stimulation (20mM). beta-Cells from lean mouse and rat, and intact islets from lean mouse were also investigated. Significant correlations between the first and second stimulation were found for the parameters lag-time for Ca(2+) rise (calculated as the time from start of stimulation of the cell until the first value above an extrapolated baseline), nadir of initial lowering (difference between the baseline and lowest [Ca(2+)](i) value), and peak height (difference between baseline and the highest [Ca(2+)](i) value of the first calcium peak) in single dispersed beta-cells, in 'single beta-cell within a small cluster', in clusters of medium and large size, and in single dispersed beta-cells from lean mouse and rat. The lag-times for Ca(2+) rise and peak heights were correlated within the pairs of stimulation also in intact ob/ob islets. In summary, despite a large heterogeneity of the 'early [Ca(2+)](i) response' among individual cells, the lag-time for [Ca(2+)](i) rise, the nadir of initial lowering and the height of the first peak response can be identified as cell-specific markers in beta-cells.     

Chronic blockade of NO synthase paradoxically increases islet NO production and modulates islet hormone release

Islet production of nitric oxide (NO) and CO in relation to islet hormone secretion was investigated in mice given the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) in their drinking water. In these mice, the total islet NO production was paradoxically increased, reflecting induction of inducible NOS (iNOS) in background of reduced activity and immunoreactivity of constitutive NOS (cNOS). Unexpectedly, normal mice fasted for 24 h also displayed iNOS activity, which was further increased in L-NAME-drinking mice. Glucose-stimulated insulin secretion in vitro and in vivo was increased in fasted but unaffected in fed mice after L-NAME drinking. Glucagon secretion was increased in vitro. Control islets incubated with different NOS inhibitors at 20 mM glucose displayed increased insulin release and decreased cNOS activity. These NOS inhibitors potentiated glucose-stimulated insulin release also from islets of L-NAME-drinking mice. In contrast, glucagon release was suppressed. In islets from L-NAME-drinking mice, cyclic nucleotides were upregulated, and forskolin-stimulated hormone release, CO production, and heme oxygenase (HO)-2 expression increased. In conclusion, chronic NOS blockade evoked iNOS-derived NO production in pancreatic islets and elicited compensatory mechanisms against the inhibitory action of NO on glucose-stimulated insulin release by inducing upregulation of the islet cAMP and HO-CO systems.     

Significance of Na/Ca exchange for Ca2+ buffering and electrical activity in mouse pancreatic beta-cells

We have combined the patch-clamp technique with microfluorimetry of the cytoplasmic Ca2+ concentration ([Ca2+]i) to characterize Na/Ca exchange in mouse beta-cells and to determine its importance for [Ca2+]i buffering and shaping of glucose-induced electrical activity. The exchanger contributes to Ca2+ removal at [Ca2+]i above 1 microM, where it accounts for >35% of the total removal rate. At lower [Ca2+]i, thapsigargin-sensitive Ca2+-ATPases constitute a major (70% at 0.8 microM [Ca2+]i) mechanism for Ca2+ removal. The beta-cell Na/Ca exchanger is electrogenic and has a stoichiometry of three Na+ for one Ca2+. The current arising from its operation reverses at approximately -20 mV (current inward at more negative voltages), has a conductance of 53 pS/pF (14 microM [Ca2+]i), and is abolished by removal of external Na+ or by intracellularly applied XIP (exchange inhibitory peptide). Inhibition of the exchanger results in shortening (50%) of the bursts of action potentials of glucose-stimulated beta-cells in intact islets and a slight (5 mV) hyperpolarization. Mathematical simulations suggest that the stimulatory action of glucose on beta-cell electrical activity may be accounted for in part by glucose-induced reduction of the cytoplasmic Na+ concentration with resultant activation of the exchanger.     

Insulin-secreting INS-1 cells generate a novel type of poorly synchronized Ca2+ transients

Pancreatic beta-cells have an intrinsic oscillatory Ca2+ activity supposed to be synchronized among the islets by cytoplasmic Ca2+ transients elicited by nonadrenergic, noncholinergic (NANC) neurons. To improve the understanding of this process, the cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in two insulin-releasing cell lines using dual wavelength microfluorometry and the indicator fura-2. INS-1 cells but not RINm5F cells were found to generate transients of [Ca2+]i in the presence of the Ca2+ channel blocker methoxyverapamil. These transients differed from those occurring in native beta-cells persisting in the presence of thapsigargin or during prolonged exposure to ATP. Moreover, the [Ca2+]i transients were poorly synchronized whether or not the INS-1 cells had physical contact. If appearing in native beta-cells, the type of [Ca2+]i transients now observed may interfere with the coordination of the beta-cell rhythmicity evoked by NANC neurons.     

Glucose-induced increase in cytoplasmic pH in pancreatic beta-cells is mediated by Na+/H+ exchange, an effect not dependent on protein kinase C.

Glucose-induced changes in cytoplasmic pH (pHi) were investigated using pancreatic beta-cells isolated from obese hyperglycemic mice. Glucose, at concentrations above 3-5 mM, depolarized the beta-cell and increased pHi, cytoplasmic free Ca2+ ([Ca2+]i), and insulin release. This increase in pHi was dependent on the presence of extracellular Na+ and was inhibited by 5-(N-ethyl-N-isopropyl) amiloride, a blocker of Na+/H+ exchange. Stimulation of protein kinase C with phorbol ester also induced an alkalinization. However, when protein kinase C activity was down-regulated, glucose stimulation still induced alkalinization. At 20 mM glucose, 10 mM NH4Cl induced a marked rise in pHi, paralleled by repolarization, inhibition of electrical activity, and decreases in both [Ca2+]i and insulin release. Reduction in [Ca2+]i was prevented by 200 microM tolbutamide, but not by 10 mM tetraethylammonium. At 4 mM glucose, NH4Cl induced a transient increase in insulin release, without changing [Ca2+]i. Exposure of beta-cells to 10 mM sodium acetate caused a persistent decrease in pHi, an effect paralleled by a small transient increase in [Ca2+]i. Acidification per se did not change the beta-cell sensitivity to glucose, not excluding that the activity of the ATP-regulated K+ channels may be modulated by changes in pHi.     

Synchronization of pancreatic beta-cell rhythmicity after glucagon induction of Ca2+ transients

Pancreatic beta-cells are biological oscillators requiring a coupling force for the synchronization of the cytoplasmic Ca(2+) oscillations responsible for pulsatile insulin release. Testing the idea that transients, superimposed on the oscillations, are important for this synchronization, the concentration of cytoplasmic Ca(2+) ([Ca(2+)](i)) was measured with ratiometric fura-2 technique in single beta-cells and small aggregates prepared from islets isolated from ob/ob-mice. Image analyses revealed asynchronous [Ca(2+)](i) oscillations in adjacent beta-cells lacking physical contact. The addition of glucagon stimulated the firing of [Ca(2+)](i) transients, which appeared in synchrony in adjacent beta-cells. Moreover, the presence of glucagon promoted synchronization of the [Ca(2+)](i) oscillations in beta-cells separated by a distance <100 microm but not in those >200 microm apart. The results support the proposal that the repolarizing effect of [Ca(2+)](i) transients provides a coupling force for co-ordinating the pulses of insulin release generated by pancreatic beta-cells.     

Attenuation of insulin secretion by insulin-like growth factor binding protein-1 in pancreatic beta-cells

IGFBP-1 is involved in glucohomeostasis, but the direct action of IGFBP-1 on the beta-cell remains unclear. Incubation of dispersed mouse beta-cells with IGFBP-1 for 30min inhibited insulin secretion stimulated by glucose, glucagon-like peptide 1 (GLP-1) or tolbutamide without changes in basal release of insulin and in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and NAD(P)H evoked by glucose. In contrast, IGFBP-1 augmented glucose-stimulated insulin secretion in intact islets, associated with a reduced somatostatin secretion. These results suggest a suppressive action of IGFBP-1 on insulin secretion in isolated beta-cells through a mechanism distal to energy generating steps and not involving regulation of [Ca(2+)](i). In contrast, IGFBP-1 amplifies glucose-stimulated insulin secretion in intact islets, possibly by suppressing somatostatin secretion. These direct modulatory influences of IGFBP-1 on insulin secretion may imply an important regulatory role of IGFBP-1 in vivo and in the pathogenesis of type 2 diabetes, in which loss of insulin release is an early pathogenetic event.     

Inhibition of glucose-stimulated insulin release by alpha 2-adrenoceptor activation is parallelled by both a repolarization and a reduction in cytoplasmic free Ca2+ concentration

Effects of the alpha 2-adrenergic agonist clonidine on insulin release, membrane potential, and cytoplasmic free Ca2+ concentration ([Ca2+]i) were investigated using pancreatic beta-cells isolated from obese hyperglycemic mice. Addition of 2 microM clonidine promptly inhibited glucose-stimulated insulin release, an effect accompanied by a lowering in both membrane potential and [Ca2+]i. Within minutes, the effect on Ca2+ was partly reversed, [Ca2+]i attaining a new level, although still significantly lower than in the absence of agonist. This late increase in [Ca2+]i was inhibited by 50 microM D-600, a blocker of voltage-activated Ca2+ channels. The inhibitory effects of clonidine on membrane potential, [Ca2+]i, and insulin release were abolished by 5 microM of the alpha 2-adrenergic antagonist yohimbine. Depolarization with high K+ increased [Ca2+]i also in the presence of clonidine, conditions accompanied by only a minute release of insulin. Secretion was, however, partly restored by subsequent addition of 20 mM glucose. Addition of 5 mM Ca2+ transiently reversed the effects of clonidine on both membrane potential and [Ca2+]i. Although the clonidine-induced repolarization should be enough for closing the voltage-activated Ca2+ channels with a resulting decrease in [Ca2+]i, a direct interaction of the agonist with these channels cannot be excluded. The fact that it was possible to increase [Ca2+]i with only a minor effect on insulin release suggests that the inhibitory effect of clonidine not only is due to a reduction in [Ca2+]i, but also involves interference with some more distal step in the insulin secretory machinery.     

Restitution of defective glucose-stimulated insulin release of sulfonylurea type 1 receptor knockout mice by acetylcholine

Inhibition of ATP-sensitive K+ (K(ATP)) channels by an increase in the ATP/ADP ratio and the resultant membrane depolarization are considered essential in the process leading to insulin release (IR) from pancreatic beta-cells stimulated by glucose. It is therefore surprising that mice lacking the sulfonylurea type 1 receptor (SUR1-/-) in beta-cells remain euglycemic even though the knockout is expected to cause hypoglycemia. To complicate matters, isolated islets of SUR1-/- mice secrete little insulin in response to high glucose, which extrapolates to hyperglycemia in the intact animal. It remains thus unexplained how euglycemia is maintained. In recognition of the essential role of neural and endocrine regulation of IR, we evaluated the effects of acetylcholine (ACh) and glucagon-like peptide-1 (GLP-1) on IR and free intracellular Ca2+ concentration ([Ca2+]i) of freshly isolated or cultured islets of SUR1-/- mice and B6D2F1 controls (SUR1+/+). IBMX, a phosphodiesterase inhibitor, was also used to explore cAMP-dependent signaling in IR. Most striking, and in contrast to controls, SUR1-/-) islets are hypersensitive to ACh and IBMX, as demonstrated by a marked increase of IR even in the absence of glucose. The hypersensitivity to ACh was reproduced in control islets by depolarization with the SUR1 inhibitor glyburide. Pretreatment of perifused SUR1-/- islets with ACh or IBMX restored glucose stimulation of IR, an effect expectedly insensitive to diazoxide. The calcium channel blocker verapamil reduced but did not abolish ACh-stimulated IR, supporting a role for intracellular Ca2+ stores in stimulus-secretion coupling. The effect of ACh on IR was greatly potentiated by GLP-1 (10 nM). ACh caused a dose-dependent increase in [Ca2+]i at 0.1-1 microM or biphasic changes (an initial sharp increase in [Ca2+]i followed by a sustained phase of low [Ca2+]i) at 1-100 microM. The latter effects were observed in substrate-free medium or in the presence of 16.7 mM glucose. We conclude that SUR1 deletion depolarizes the beta-cells and markedly elevates basal [Ca2+]i. Elevated [Ca2+]i in turn sensitizes the beta-cells to the secretory effects of ACh and IBMX. Priming by the combination of high [Ca2+]i, ACh, and GLP-1 restores the defective glucose responsiveness, precluding the development of diabetes but not effectively enough to cause hyperinsulinemic hypoglycemia.     

IFN-gamma and tumor necrosis factor-alpha. Cytotoxicity to murine islets of Langerhans

We have previously reported that the cytokines IFN-gamma and TNF-alpha each upregulate the expression of class I MHC proteins and, in combination, induce the expression of class II MHC proteins on pancreatic islet cells. IFN-gamma and TNF-alpha are therefore implicated in the immunologic destruction of beta-cells in insulin-dependent diabetes mellitus. The objective of the present study was to define the effects of IFN-gamma and TNF-alpha on the function and viability of murine pancreatic islet beta-cells in vitro. Exposure of islets for 3 days to 200 U/ml of either IFN-gamma or TNF-alpha did not affect glucose-stimulated insulin release, but at higher concentrations (2000 U/ml) of either cytokine there was significant inhibition of glucose-stimulated insulin release. In combination, IFN-gamma and TNF-alpha each at 200 U/ml caused significant inhibition of glucose-stimulated insulin release; at 2000 U/ml glucose-stimulated insulin release was abolished. In time-course experiments, glucose-stimulated insulin release from islets exposed to IFN-gamma and TNF-alpha each at 1000 U/ml was significantly increased at 4-h (twofold increase compared with control islets), decreased back to control levels at 18 h, significantly inhibited by 24 h (threefold decrease compared with control islets), and completely abolished by 48 h. The progressive impairment of beta-cell function mediated by IFN-gamma plus TNF-alpha was associated with morphologic derangement of the islets that were almost totally disintegrated by day 6 of exposure to the cytokines. At day 6, insulin content of the islets was significantly reduced by exposure to TNF-alpha but not IFN-gamma. The combination of IFN-gamma and TNF-alpha resulted in a further dose-dependent depletion in insulin content compared with TNF-alpha alone. The synergistic functional and cytotoxic effects of IFN-gamma and TNF-alpha are consistent with a direct role for these cytokines in the destruction of beta-cells in insulin-dependent diabetes.     

Overexpression of Bcl-x(L) in beta-cells prevents cell death but impairs mitochondrial signal for insulin secretion

To study effects of Bcl-x(L) in the pancreatic beta-cell, two transgenic lines were produced using different forms of the rat insulin promoter. Bcl-x(L) expression in beta-cells was increased 2- to 3-fold in founder (Fd) 1 and over 10-fold in Fd 2 compared with littermate controls. After exposure to thapsigargin (10 microM for 48 h), losses of cell viability in islets of Fd 1 and Fd 2 Bcl-x(L) transgenic mice were significantly lower than in islets of wild-type mice. Unexpectedly, severe glucose intolerance was observed in Fd 2 but not Fd 1 Bcl-x(L) mice. Pancreatic insulin content and islet morphology were not different from control in either transgenic line. However, Fd 2 Bcl-x(L) islets had impaired insulin secretory and intracellular free Ca(2+) ([Ca(2+)](i)) responses to glucose and KCl. Furthermore, insulin and [Ca(2+)](i) responses to pyruvate methyl ester (PME) were similarly reduced as glucose in Fd 2 Bcl-x(L) islets. Consistent with a mitochondrial defect, glucose oxidation, but not glycolysis, was significantly lower in Fd 2 Bcl-x(L) islets than in wild-type islets. Glucose-, PME-, and alpha-ketoisocaproate-induced hyperpolarization of mitochondrial membrane potential, NAD(P)H, and ATP production were also significantly reduced in Fd 2 Bcl-x(L) islets. Thus, although Bcl-x(L) promotes beta-cell survival, high levels of expression of Bcl-x(L) result in reduced glucose-induced insulin secretion and hyperglycemia due to a defect in mitochondrial nutrient metabolism and signaling for insulin secretion.     

Intracellular ATP mimics GTP-gamma-S in generating Ca2+ oscillations in pancreatic beta-cells

Intracellular free calcium ([Ca2+]i) was measured in individual pancreatic beta-cells from mice using dual emission microfluorometry and the indicator Indo-1 applied by a patch clamp pipette. GTP-gamma-S (100 microM) injected together with 0.3 or 3 mM ATP evoked repetitive [Ca2+]i transients with a frequency of about 1 per min in beta-cells kept at a membrane potential of -70 mV. The oscillatory pattern was unaffected by the Ca2+ channel blocker verapamil (50 microM). When omitting GTP-gamma-S from the pipette medium it became evident that 3 mM ATP alone can induce oscillations. The results provide additional evidence for an important role of ATP in the ionic control of insulin release, indicating that such regulation may also involve activation of G-proteins.     

The pancreatic β-cell recognition of insulin secretagogues. Effects of calcium and sodium on glucose metabolism and insulin release

The transport and oxidation of glucose, the content of fructose 1,6-diphosphate, and the release of insulin were studied in microdissected pancreatic islets of ob/ob mice incubated in Krebs-Ringer bicarbonate medium. Under control conditions glucose oxidation and insulin release showed a similar dependence on glucose concentration with the steepest slope in the range 5-12mm. The omission of Ca(2+), or the substitution of choline ions for Na(+), or the addition of diazoxide had little if any effect on glucose transport. However, Ca(2+) or Na(+) deficiency as well as diazoxide (7-chloro-3-methyl-1,2,4-benzothiadiazine 1,1-dioxide) or ouabain partially inhibited glucose oxidation. These alterations of medium composition also increased the islet content of fructose 1,6-diphosphate, as did the addition of adrenaline. Phentolamine [2-N-(3-hydroxyphenyl)-p-toluidinomethyl-2-imidazoline] counteracted the effects of adrenaline and Ca(2+) deficiency on islet fructose 1,6-diphosphate. After equilibration in Na(+)-deficient medium, the islets exhibited an increase in basal insulin release whereas the secretory response to glucose was inhibited. The inhibitory effects of Na(+) deficiency on the secretory responses to different concentrations of glucose correlated with those on (14)CO(2) production. When islets were incubated with 17mm-glucose, the sudden replacement of Na(+) by choline ions resulted in a marked but transient stimulation of insulin release that was not accompanied by a demonstrable increase of glucose oxidation. Galactose and 3-O-methylglucose had no effect on glucose oxidation or on insulin release. The results are consistent with a metabolic model of the beta-cell recognition of glucose as insulin secretagogue and with the assumption that Ca(2+) or Na(+) deficiency, or the addition of adrenaline or diazoxide, inhibit insulin release at some step distal to stimulus recognition. In addition the results suggest that these conditions create a partial metabolic block of glycolysis in the beta-cells. Hence the interrelationship between the processes of stimulus recognition and insulin discharge may involve a positive feedback of secretion on glucose metabolism.     

Progesterone inhibits insulin secretion by a membrane delimited,non-genomic action

In rat islets, progesterone caused a prompt concentration-dependent inhibition of glucose-stimulated insulin release with an IC50 of 10 microM at 8.4mM glucose. The inhibition was specific since both testosterone and 17beta-estradiol had no such effect. The degree of inhibition was similar in islets from male and female rats. The inhibition was not blocked in PTX-treated islets thus ruling out the Gi/Go proteins as mediators of the inhibition. Progesterone inhibited both glucose- and BayK-8644-stimulated insulin secretion in HIT-T15 cells and the IC50 vs. 10 mM glucose was also 10 microM. There was no effect on intracellular cyclic AMP concentration in the presence 0.2 and 10 mM glucose. Progesterone decreased [Ca2+]i under all conditions tested. The decrease in [Ca2+]i was due to blockade of the L-type voltage-dependent Ca2+ channels. Under Ca(2+)-free conditions, progesterone did not inhibit the stimulation of insulin release due to the combination of glucose, phorbol ester and forskolin. Thus blockade of Ca2+ entry appears to be the sole mechanism by which progesterone inhibits insulin release. As progesterone covalently linked to albumin had a similar inhibitory effect as progesterone itself, it is concluded that the steroid acts at the outer surface of the beta-cell plasma membrane. These effects would be classified as either AI or AIIb in the Mannheim classification of nongenomically initiated steroid actions.     

Different insulin-secretory responses to calcium-channel blockers in islets of lean and obese (ob/ob) mice.

The purpose of these experiments was to determine whether the activity of the voltage-dependent Ca2+ channel was modulated in the same manner in islets of the ob/ob mouse as in islets of homozygous lean mice of the same strain. The effect of agents that are known to alter the concentrations and movements of intracellular Ca2+ were investigated in relation to glucose-stimulated insulin secretion and in relation to the effect of forskolin. In islets of obese mice, verapamil and nifedipine both inhibited glucose-induced insulin release, nifedipine being the more potent inhibitor. Forskolin-stimulated secretion was inhibited either not at all (verapamil) or much less (nifedipine) in islets of the ob/ob mouse compared with those of lean mice. At basal glucose concentrations, verapamil initiated insulin secretion in islets of the ob/ob mouse and acted synergistically with forskolin to evoke a secretory activity that was 3-fold greater than that evoked by 20 mM-glucose. Nifedipine also initiated secretion at basal glucose concentrations and acted synergistically with forskolin, but its effect was considerably smaller than that of verapamil. A comparison of the effect of forskolin in the presence of Ca2+-channel blockers and in the absence of Ca2+ suggests that, in the obese mouse, the operation of the voltage-dependent Ca2+ channel is impaired.     

Oleic acid interacts with GPR40 to induce Ca2+ signaling in rat islet beta-cells: mediation by PLC and L-type Ca2+ channel and link to insulin release

It has long been thought that long-chain free fatty acids (FFAs) stimulate insulin secretion via mechanisms involving their metabolism in pancreatic beta-cells. Recently, it was reported that FFAs function as endogenous ligands for GPR40, a G protein-coupled receptor, to amplify glucose-stimulated insulin secretion in an insulinoma cell line and rat islets. However, signal transduction mechanisms for GPR40 in beta-cells are little known. The present study was aimed at elucidating GPR40-linked Ca(2+) signaling mechanisms in rat pancreatic beta-cells. We employed oleic acid (OA), an FFA that has a high affinity for the rat GPR40, and examined its effect on cytosolic Ca(2+) concentration ([Ca(2+)](i)) in single beta-cells by fura 2 fluorescence imaging. OA at 1-10 microM concentration-dependently increased [Ca(2+)](i) in the presence of 5.6, 8.3, and 11.2 mM, but not 2.8 mM, glucose. OA-induced [Ca(2+)](i) increases at 11.2 mM glucose were inhibited in beta-cells transfected with small interfering RNA targeted to rat GPR40 mRNA. OA-induced [Ca(2+)](i) increases were also inhibited by phospholipase C (PLC) inhibitors, U73122 and neomycin, Ca(2+)-free conditions, and an L-type Ca(2+) channel blocker, nitrendipine. Furthermore, OA increased insulin release from isolated islets at 8.3 mM glucose, and it was markedly attenuated by PLC and L-type Ca(2+) channel inhibitors. These results demonstrate that OA interacts with GPR40 to increase [Ca(2+)](i) via PLC- and L-type Ca(2+) channel-mediated pathway in rat islet beta-cells, which may be link to insulin release.     

Nitric oxide-cyclic GMP system potentiates glucose-induced rise in cytosolic Ca2+ concentration in rat pancreatic beta-cells.

Involvement of nitric oxide (NO) in the regulation of insulin secretion from pancreatic beta-cells was investigated by measuring cytosolic Ca2+ concentration ([Ca2+]i) in isolated rat pancreatic beta-cells. At 7.0 mM glucose, L-arginine (0.1 mM) elevated [Ca2+]i in about 50% of the beta-cells examined. The response was partially inhibited by an NO synthase inhibitor, N(G)-monomethyl-L-arginine (L-NMA; 0.1 mM), suggesting that part of the response was mediated by the production of NO from L-arginine. D-Arginine at higher concentrations (3 or 10 mM) also increased [Ca2+]i at 7.0 mM glucose; however, the response was not affected by L-NMA (0.1 mM). Similar [Ca2+]i elevation was produced by NO (10 nM) and sodium nitroprusside (SNP; 10 microM) at 7.0 mM glucose. The SNP-induced increase in [Ca2+]i was abolished by nicardipine (1 microM), suggesting that the [Ca2+]i response is mediated by Ca2+ influx through L-type voltage-operated Ca2+ channels. In the presence of oxyhemoglobin (1 microM), the [Ca2+]i elevation induced by NO (10 nM) was abolished. Neither degradation products of NO, NO2- nor NO3-, caused any changes in [Ca2+]i. 8-Bromo-cyclic GMP (8-Br-cGMP; 3 mM) and atrial natriuretic peptide (0.1 microM) elevated [Ca2+]i at 7.0 mM glucose. We conclude that NO, which is produced from L-arginine in pancreatic islets, facilitates glucose-induced [Ca2+]i increase via the elevation of cGMP in rat pancreatic beta-cells. NO-cGMP system may physiologically regulate insulin secretion from pancreatic beta-cells.