Inorganic nitrite stimulates pancreatic islet blood flow and insulin secretion

Reactive nitrogen and oxygen species have been proposed to be involved in control of insulin release from the pancreatic β cell. Recent evidence suggests that the supposedly inert anions nitrate and nitrite are metabolized in blood and tissues to form nitric oxide (NO) and other bioactive nitrogen oxides. Here we present evidence for a novel stimulatory role of nitrite in influencing pancreatic islet physiology via a dual mechanism, involving both indirect enhancement (through microcirculation redistribution) and direct insulinotropic effects on the β cell. In rats, intraperitoneal injection of sodium nitrite increased pancreatic islet blood flow by 50% and serum insulin concentrations by 30%, while whole pancreatic blood flow and glycemia remained unaffected. Nitrite also dose dependently enhanced insulin secretion from rat β cells in vitro under nonstimulatory glucose concentrations. This effect was not mimicked by nitrate and was abolished by the guanylyl cyclase (GC) inhibitor ODQ and the NO scavenger cPTIO. It was also mimicked by a cyclic GMP agonist (8-CPT-cGMP) and a classical NO donor (NONOate). Interestingly, a reactive oxygen species scavenger (vitamin E analog, Trolox) abolished the insulin secretion induced by nitrite. We conclude that nitrite exerts dual stimulatory effects on pancreatic islet function, including enhancement of islet blood flow and subsequent insulin secretion in vivo and direct stimulation of insulin release in vitro. The insulinotropic effect of nitrite is cGMP-dependent and involves formation of reactive nitrogen and oxygen species.     

The CD38-cyclic ADP-ribose signaling system in insulin secretion

Glucose induces an increase in the intracellular Ca2+ concentration in pancreatic -cells to secrete insulin. CD38 occurs in -cells and has both ADP-ribosyl cyclase, which catalyzes the formation of cyclic ADP-ribose (cADPR) from NAD+, and cADPR hydrolase, which converts cADPR to ADP-ribose. ATP, produced by glucose metabolism, competes with cADPR for the binding site, Lys-129, of CD38, resulting in the inhibition of the hydrolysis of cADPR and thereby causing cADPR accumulation in -cells. Cyclic ADP-ribose then binds to FK506-binding protein 12.6 in the ryanodine receptor Ca2+ channel (RyR), dissociating the binding protein from RyR to induce the release of Ca2+ from the endoplasmic reticulum. Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) phosphorylates RyR to sensitize and activate the Ca2+ channel. Ca2+, released from the RyR, further activates CaM kinase II and amplifies the process. Thus, cADPR acts as a second messenger for Ca2+ mobilization to secrete insulin. The novel mechanism of insulin secretion described above is different from the conventional hypothesis in which Ca2+ influx from extracellular sources plays a role in insulin secretion by glucose.     

Transient receptor potential vanilloid subfamily 1 expressed in pancreatic islet beta cells modulates insulin secretion in rats

Capsaicin-sensitive afferent neurons including transient receptor potential vanilloid subfamily 1, TRPV1, and neurohormonal peptides participate in the physiological regulation of pancreatic endocrine. However, the direct effect of capsaicin on insulin secretion remains unknown. Our present study showed that TRPV1 is expressed in islet beta cells as well as in neurons in rat pancreas, and also in rat beta cell lines, RIN and INS1. Capsaicin (10(-11)-10(-9) M) dose-dependently increased insulin secretion from RIN cells, and this effect was inhibited by either a TRPV1 inhibitor capsazepine or EDTA. Systemic capsaicin (10 mg/kg, s.c.) increased plasma insulin level 1 h after the treatment. We demonstrated for the first time that TRPV1 is functionally expressed in rat islet beta cells and plays a role in insulin secretion as a calcium channel. This study may account for the influences of capsaicin on the food intake and energy consumption as well as on the pathophysiological regulation of pancreatic endocrine.     

Insulin-stimulated insulin secretion in single pancreatic beta cells

Functional insulin receptors are known to occur in pancreatic beta cells; however, except for a positive feedback on insulin synthesis, their physiological effects are unknown. Amperometric measurements at single, primary pancreatic beta cells reveal that application of exogenous insulin in the presence or absence of nonstimulatory concentrations of glucose evokes exocytosis mediated by the beta cell insulin receptor. Insulin also elicits increases in intracellular Ca2+ concentration in beta cells but has minimal effects on membrane potential. Conditions where the insulin receptor is blocked or cell surface concentration of free insulin is reduced during exocytosis diminishes secretion induced by other secretagogues, providing evidence for direct autocrine action of insulin upon secretion from the same cell. These results indicate that the beta cell insulin receptor can mediate positive feedback for insulin secretion. The presence of a positive feedback mechanism for insulin secretion mediated by the insulin receptor provides a potential link between impaired insulin secretion and insulin resistance.     

Effect of intracellular alkalinization on pancreatic islet calcium uptake and insulin secretion.

Microdissected beta-cell-rich pancreatic islets of ob/ob mice were used in studies of the relationship between intracellular pH (pHi) and 45Ca2+ uptake and insulin release. Stepwise increases in extracellular pH (pHo) from 6.80 to 8.00 resulted in a parallel, although less pronounced, elevation of pHi from 7.24 to 7.69. Experimental conditions that alkalinize the islet cell interior, i.e. addition of 5 mM-NH4+, sudden withdrawal of extracellular bicarbonate buffer or increase in pHo, induced insulin secretion in the absence of other types of secretory stimulation (1 mM-D-glucose). Intracellular acidification by lowering pHo below 7.40 or sudden addition of bicarbonate buffer did not induce insulin secretion. The removal of extracellular bicarbonate buffer, increase in pHo from 7.40 to 8.00, or the addition of 5 mM-L-5-hydroxytryptophan or 5 mM-NH4+, which all alkalinize the islet cells and induce insulin secretion, also increased the La3+-non-displaceable 45Ca2+ uptake in the presence of 1 mM-D-glucose. The results suggest that intracellular alkalinization in beta-cells can trigger insulin secretion. Taken together with the fact that D-glucose increases pHi in the islet cells, the results also point to the possibility that alkalinization may be a link in the stimulus-secretion coupling sequence in beta-cells.     

Abundant expression of 150-kDa oxygen-regulated protein in mouse pancreatic beta cells is correlated with insulin secretion

The 150-kDa oxygen-regulated protein (ORP150) is a member of glucose-regulated proteins (GRPs), which are induced by stressful conditions such as oxygen or glucose deprivation. Here we investigated the highly abundant expression of ORP150 in mouse pancreas and its relationship with insulin secretion. Immunohistochemical analysis revealed that ORP150 expression was restricted to islets, especially to beta cells. The beta cell-specific expression was also observed in a mouse insulinoma cell line, MIN6, which secretes insulin in response to increased glucose concentration. Furthermore, ORP150 in islets dramatically diminished by fasting, concomitant with reduction of the serum insulin level. These results strongly suggest the role for ORP150 in insulin secretion.     

Kv2.1 ablation alters glucose-induced islet electrical activity, enhancing insulin secretion

Voltage-gated potassium currents (Kv), primarily due to Kv2.1 channels, are activated by glucose-stimulated pancreatic beta cell depolarization, but the exact role (or roles) of this channel in regulating insulin secretion remains uncertain. Here we report that, compared with controls, Kv2.1 null mice have reduced fasting blood glucose levels and elevated serum insulin levels. Glucose tolerance is improved and insulin secretion is enhanced compared to control animals, with similar results in isolated islets in vitro. Isolated Kv2.1(-/-) beta cells have residual Kv currents, which are decreased by 83% at +50 mV compared with control cells. The glucose-induced action potential (AP) duration is increased while the firing frequency is diminished, similar to the effect of specific toxins on control cells but substantially different from the effect of the less specific blocker tetraethylammonium. These results reveal the specific role of Kv2.1 in modulating glucose-stimulated APs of beta cells, exposing additional important currents involved in regulating physiological insulin secretion.     

Protein prenylation in glucose-induced insulin secretion from the pancreatic islet beta cell: a perspective.

Insulin secretion from the pancreatic beta-cell is regulated principally by the ambient concentration of glucose. However, the molecular and cellular mechanisms underlying the stimulus-secretion coupling of glucose-stimulated insulin secretion (GSIS) remain only partially understood. Emerging evidence from multiple laboratories suggests key regulatory roles for GTP-binding proteins (G-proteins) in the cascade of events leading to GSIS. This class of signaling proteins undergo a series of requisite post-translational modifications (e.g., prenylation) at their C-terminal cysteines, which appear to be necessary for their targeting to respective membranous sites for optimal interaction with their respective effector proteins. This communication represents a perspective on potential regulatory roles for protein prenylation steps (i.e., protein farnesylation and protein geranylgeranylation) in GSIS from the islet beta cell. Possible consequences of protein prenylation and potential mechanisms underlying glucose-induced regulation of prenylation, specifically in the context of GSIS are also discussed.     

Role of voltage-dependent ionic currents in coupling glucose stimulation to insulin secretion in canine pancreatic islet B-Cells

Summary Glucose-induced electrical activity in canine pancreatic islet B cells is distinct from that in rodent islets, though both display Ca²⁺-dependent insulin secretion. Rodent islet B cells undergo regular bursts of Ca²⁺-dependent action potentials, while canine islet B cells generate isolated Na⁺-dependent action potentials which often give way to a plateau depolarization. Here we present evidence to reconcile the species difference in electrical activity with the similarity of Ca²⁺ dependence of secretion. (i) In canine B cells increasing glucose concentrations produce membrane depolarization and increasing frequency of Na_o-dependent action potentials until a background membrane potential (-40mV) is reached where Na⁺ currents are inactivated. (ii) Voltage-dependent Ca²⁺ currents are present which are activated over the voltage excursion of the action potential (–50 to +20 mV) and inactivate slowly, (over seconds) in the range of the plateau depolarization (–40 to –25 mV). Hence, they are available to contribute to both phases of depolarization. (iii) Tetrodotoxin (TTX) reduces by half an early transient phase of glucosestimulated insulin secretion but not a subsequent prolonged plateau phase. The transient phase of secretion often corresponds well in time to the period of initial high frequency action potential activity. These latter results suggest that in canine B cells voltagedependent Na⁺ and Ca²⁺ currents mediate biphasic glucose-induced insulin secretion. The early train of Na⁺-dependent action potentials, by transiently activating Ca²⁺ channels and allowing pulsatile Ca²⁺ entry, may promote an early transient phase of insulin secretion. The subsequent sustained plateau depolarization, by allowing sustained Ca²⁺ entry, may permit steady insulin release.     

Glucagon and insulin secretion by dispersed islet cells: possible paracrine relationships

The ability of dispersed islet cells in a perifusion system to secret glucagon and insulin in response to physiologic stimuli was investigated. Normal hamster islets were isolated by collagenase digestion and the cells dispersed by sequential digestion with collagenase and trypsin. Following a 50-min period of equilibrium in buffer with high glucose concentrations (5.0 mg/ml), glucagon secretion was stimulated by glucopenia and subsequently, inhibited by increasing the concentration of glucose. The responsiveness to glucose inhibition was significantly less in dispersed islet cells than in intact islets. However, the dispersed islet cells showed significantly greater response to arginine. Glucagon secretion by dispersed islet cells was stimulated to tolbutamide and epinephrine but somatostatin had no effect. Dispersed islet cell preparations did not augment insulin secretion in response to glucose but did secrete more insulin in response to arginine. Intact islets secreted insulin in response to glucose but not arginine. We conclude that A cells in cell suspension do not need direct contact or an intact intra-islet environment in order to respond to glucose, arginine, epinephrine, or tolbutamide but the extent of response may be influenced by paracrine effects. However, paracrine relationships may be important in determining the response of B cells to secretagogues.     

Glucose increases cytosolic Ca2+ activity in pancreatic islet cells

Isolated cells prepared from rat pancreatic islets were labelled with the tetraacetoxymethyl ester of the fluorescent Ca2+ indicator quin-2. An increase in the extracellular concentration of glucose provoked a rapid and sustained increase in the fluorescence of the labelled cells. This indicates that glucose increases cytosolic Ca2+ activity in pancreatic islet cells.     

Electrical activity and insulin release in pancreatic beta cells

The pancreatic beta cell, which is responsible for insulin secretion from the islets of Langerhans, exhibits a complex pattern of membrane-potential oscillations called bursting. These oscillations are induced by glucose and are strongly correlated with the release of insulin. Recently a number of the ion channels which are responsible for carrying membrane currents in the beta cell have been uncovered. This has led to several hypothesized mechanisms for bursting and a good deal of mathematical modeling. In this paper the progress in understanding bursting in the beta cell is reviewed, including the impact of the discovery of the ATP-inactivated, ADP-modulated channel on modeling and the effect of single-channel events on electrical activity.     

The effect of starvation on insulin secretion and glucose metabolism in mouse pancreatic islets

1. Crude extracts of seeds of Pinus radiata catalysed acetate-, propionate-, n-butyrate- and n-valerate-dependent PP(i)-ATP exchange in the presence of MgCl(2), which was apparently due to a single enzyme. Propionate was the preferred substrate. Crude extracts did not catalyse medium-chain or long-chain fatty acid-dependent exchange. 2. Ungerminated dry seeds contained short-chain fatty acyl-CoA synthetase activity. The activity per seed was approximately constant for 11 days after imbibition and then declined. The enzyme was located only in the female gametophyte tissue. 3. The synthetase was purified 70-fold. 4. Some properties of the enzyme were studied by [(32)P]PP(i)-ATP exchange. K(m) values for acetate, propionate, n-butyrate and n-valerate were 4.7, 0.21, 0.33 and 2.1mm respectively. Competition experiments between acetate and propionate demonstrated that only one enzyme was involved and confirmed that the affinity of the enzyme for propionate was greater than that for acetate. CoA inhibited fatty acid-dependent PP(i)-ATP exchange. The enzyme catalysed fatty acid-dependent [(32)P]PP(i)-dATP exchange. 5. The enzyme also catalysed the fatty acyl-AMP-dependent synthesis of [(32)P]ATP from [(32)P]PP(i). Apparent K(m) (acetyl-AMP) and apparent K(m) (propionyl-AMP) were 57mum and 7.5mum respectively. The reaction was inhibited by AMP and CoA. 6. Purified enzyme catalysed the synthesis of acetyl-CoA and propionyl-CoA. Apparent K(m) (acetate) and apparent K(m) (propionate) were 16mm and 7.5mm respectively. The rate of formation of acetyl-CoA was enhanced by pyrophosphatase. 7. It was concluded that fatty acyl adenylates are intermediates in the formation of the corresponding fatty acyl-CoA.     

Effect of perchlorate on calcium uptake and insulin secretion in mouse pancreatic islets.

Microdissected beta-cell-rich pancreatic islets of non-inbred ob/ob mice were used in studies of how perchlorate (CIO4-) affects stimulus-secretion coupling in beta-cells. CIO4- at 16 mM potentiated D-glucose-induced insulin release, without inducing secretion at non-stimulatory glucose concentrations. The potentiation mainly applied to the first phase of stimulated insulin release. In the presence of 20 mM-glucose, the half-maximum effect of CIO4- was reached at 5.5 mM and maximum effect at 12 mM of the anion. The potentiation was reversible and inhibitable by D-mannoheptulose (20 mM) or Ca2+ deficiency. CIO4- at 1-8 mM did not affect glucose oxidation. The effects on secretion were paralleled by a potentiation of glucose-induced 45Ca2+ influx during 3 min. K+-induced insulin secretion and 45Ca2+ uptake were potentiated by 8-16 mM-CIO4-. The spontaneous inactivation of K+-induced (20.9 mM-K+) insulin release was delayed by 8 mM-CIO4-. The anion potentiated the 45Ca2+ uptake induced by glibenclamide, which is known to depolarize the beta-cell. Insulin release was not affected by 1-10 mM-trichloroacetate. It is suggested that CIO4- stimulates the beta-cell by affecting the gating of voltage-controlled Ca2+ channels.     

Phe-met-arg-phe-amide (FMRF-NH2) inhibits insulin and somatostatin secretion and anti-FMRF-NH2 sera detects pancreatic polypeptide cells in the rat islet

FMRF-NH2-like immunoreactivity was localized in the pancreatic polypeptide containing cells of the rat islet. FMRF-NH2 was investigated with regard to its effect on insulin, somatostatin and glucagon secretion from the isolated perfused rat pancreas. FMRF-NH2 (1 microM) significantly inhibited glucose stimulated (300 mg/dl) insulin release (p less than 0.005) and somatostatin release (p less than 0.01) from the isolated perfused pancreas. FMRF-NH2 (1 and 10 microM) was without effect on glucagon secretion, either in low glucose (50 mg/dl), high glucose (300 mg/dl), or during arginine stimulation (5 mM). These findings indicate that these FMRF-NH2 antisera recognize a substance in the pancreatic polypeptide cells of the islet which may be capable of modulating islet beta and D cell activity.