Inhibitory control of growth hormone secretion by somatostatin in rat pituitary GC cells: sst(2) but not sst(1) receptors are coupled to inhibition of single-cell intracellular free calcium concentrations

Rat pituitary tumor cells (GC cells) exhibit spontaneous oscillations of intracellular free calcium concentration ([Ca(2+)](i)) that allow continuous release of growth hormone (GH). Of the somatostatin (SRIH) receptor subtypes (sst receptors) mediating SRIH action, sst(1) and sst(2) receptors are highly expressed by GC cell membranes. In the present study, the effects of sst(1) or sst(2) receptor activation on single-cell [Ca(2+)](i) were investigated in GC cells by confocal fluorescence microscopy. In addition, the effects of sst(1) or sst(2) receptor activation on GH secretion were also studied. Our results demonstrate that SRIH decreases [Ca(2+)](i) baseline and almost completely blocks Ca(2+) transients through activation of sst(2) but not of sst(1) receptors. In contrast, SRIH effectively inhibits GH secretion through activation of both sst(1) and sst(2) receptors. Blocking Ca(2+) transients is less efficient than SRIH to inhibit GH release. The cyclic octapeptide, CYN-154806, antagonizes sst(2) receptors at [Ca(2+)](i) since it abolishes the sst(2) receptor-mediated inhibition of [Ca(2+)](i) without affecting single-cell Ca(2+) signals. On the other hand, CYN-154806 alone potently inhibits GH secretion through the involvement of pertussis toxin-sensitive G proteins. In conclusion, the present results demonstrate that SRIH inhibition of GH release in GC cells involves mechanisms either dependent or independent on SRIH modulation of [Ca(2+)](i). The implications of CYN-154806 inhibition of GH secretion are discussed.     

Cell specificity of the cytoplasmic Ca2+ response to tolbutamide is impaired in beta-cells from hyperglycemic mice

We recently reported that the timing and magnitude of the nutrient-induced Ca(2+) response are specific and reproducible for each isolated beta-cell. We have now used tolbutamide and arginine to test if the cell specificity exists also for the response to non-nutrient stimulation of beta-cells and if so, whether it is disturbed in beta-cells from hyperglycemic ob/ob and db/db mice. Zn(2+) outflow measurements were used to study the correlation between Ca(2+) response and insulin secretion in individual beta-cells. Tolbutamide and arginine induced cell-specific Ca(2+) responses in lean mouse beta-cells both with regard to lag times for [Ca(2+)](i) rise and peak [Ca(2+)](i) heights. beta-Cells within intact islets also showed cell-specific timing of their Ca(2+) responses to tolbutamide. However, in tolbutamide- and arginine-stimulated single beta-cells from ob/ob and db/db mice only the magnitude of Ca(2+) response was cell-specific, not the timing. The lag time of tolbutamide-induced insulin secretion was cell-specific in lean mouse beta-cells but not in ob/ob mouse cells. Therefore, cell specificity seems to be a robust mechanism, and probably important for an adequate beta-cell function. The loss of temporal cell specificity for the response to tolbutamide in single beta-cells from hyperglycemic mice may be a sign of K(ATP)- or voltage-dependent calcium channel dysfunction.     

Regulation of growth hormone release in common carp pituitary cells by pituitary adenylate cyclase-activating polypeptide: signal transduction involves cAMP- and calcium-dependent mechanisms

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the glucagon/secretin peptide family and its molecular structure is highly conserved among vertebrates. In this study, the role of PACAP in regulating growth hormone (GH) secretion in fish was examined in vitro using common carp pituitary cells under column perifusion. A dose-dependent increase in GH release was observed after exposing pituitary cells to increasing doses of ovine PACAP38 (oPACAP38) and PACAP27 (oPACAP27), but not vasoactive intestinal polypeptide (VIP). A lack of GH response to VIP stimulation is consistent with the pharmacological properties of PAC-1 receptors, suggesting that this receptor subtype may be involved in PACAP-induced GH secretion in carp species. Although the maximal GH responses induced by oPACAP38 and oPACAP27 were similar, the minimal effective dose and ED50 value for oPACAP38 were significantly lower than that for oPACAP27. These results may indicate that common carp PAC-1 receptors are more sensitive to stimulation by oPACAP38 than by oPACAP27. In parallel studies, oPACAP38 and oPACAP27 were also effective in increasing cAMP release, cellular cAMP content, total cAMP production, and intracellular Ca(2+) ([Ca(2+)](i)) levels in common carp pituitary cells. Besides, the rise in [Ca(2+)](i) induced by oPACAP38 was blocked by removing extracellular Ca(2+) ([Ca(2+)](e)) or by treatment with nifedipine, an inhibitor of voltage-sensitive Ca(2+) channels (VSCC). The dose dependence of PACAP-stimulated GH release in common carp pituitary cells was mimicked by activating adenylate cyclase using forskolin, inhibiting cAMP degradation using IBMX, increasing functional levels of intracellular cAMP using CPT-cAMP, or inducing [Ca(2+)](e) entry using the Ca(2+) ionophore A23187. In contrast, the GH-releasing effect of oPACAP38 was suppressed by treatment with the adenylate cyclase inhibitor MDL12330A, protein kinase A inhibitor H89, and VSCC blocker nifedipine, or by perifusion with a Ca(2+)-free culture medium. These results, as a whole, suggest that PACAP functions as a GH-releasing factor in common carp by activating pituitary receptors resembling mammalian PAC-1 receptors. Apparently, the GH-releasing action of PACAP is mediated through the adenylate cyclase/cAMP/protein kinase A pathway and [Ca(2+)](e) influx through VSCC.     

Intrinsic cardiac muscle function, calcium handling and beta -adrenergic responsiveness is impaired in rats with growth hormone deficiency.

To evaluate whether growth hormone (GH) is required for normal cardiac muscle function, we studied left ventricular papillary muscles of mutant GH-deficient rats. Developed tension normalized by cross-sectional area (DT), intracellular [Ca(2+)](i)(aequorin method) and beta-adrenergic responsiveness were assessed with or without 3 weeks GH replacement therapy and compared to normal controls. Steady-state force-Ca(2+)relationship was determined in tetanized ryanodine-treated muscles. beta-adrenergic responsiveness was tested during graded isoproterenol stimulation. [Ca(2+)](i)at baseline and the EC(50)of the force-Ca(2+)relationship were similar in all groups. In dwarf rats, DT at baseline was reduced by 43% compared to controls, due to a decreased maximal Ca(2+)-activated force. beta-adrenergic responsiveness of systolic Ca(2+)-release and mechanical function were depressed in dwarf rats. GH treatment caused at least partial improvement of the depressed parameters. These data support the hypothesis that GH is required for normal intrinsic function of cardiac muscle by maintaining Ca(2+)- and beta-adrenergic responsiveness.     

Nuclear translocation and retention of growth hormone

We have previously demonstrated that GH is subject to rapid receptor-dependent nuclear translocation. Here, we examine the importance of ligand activation of the GH-receptor (GHR)-associated Janus kinase (JAK) 2 and receptor dimerization for hormone internalization and nuclear translocation by use of cells stably transfected with cDNA for the GHR. Staurosporine and herbimycin A treatment of cells did not affect the ability of GH to internalize but resulted in increased nuclear accumulation of hormone. Similarly, receptor mutations, which prevent the association and activation of JAK2, did not affect the ability of the hormone to internalize or translocate to the nucleus but resulted in increased nuclear accumulation of GH. These results were observed both by nuclear isolation and confocal laser scanning microscopy. Staurosporine treatment of cells in which human GH (hGH) was targeted to the cytoplasm (removal of secretion sequence) or to the nucleus (addition of the nuclear localization sequence of SV40 large T antigen) resulted in preferential accumulation of hGH in the nucleus. We further investigated the requirement of receptor dimerization for GH nuclear translocation using the non-receptor-dimerizing hGH antagonist, hGH-G120R, conjugated to fluorescein isothiocyanate. Confocal laser scanning microscopy demonstrated efficient internalization of both hGH and hGH-G120R but lack of nuclear translocation of hGH-G120R. Thus, we conclude that activation of JAK2 kinase and the subsequent tyrosine phosphorylation is not required for nuclear translocation of GH but is pivotal for the removal of the hormone from the nucleus, and that GH translocates into the nucleus in a GHR dimerized-dependent fashion.     

Refractoriness to growth hormone is associated with increased intracellular calcium in rat adipocytes.

In adipocytes that have been deprived of growth hormone (GH) for at least 3 hr, GH elicits a transient insulin-like response that is followed by a period of refractoriness to further insulin-like stimulation. Exposure of adipocytes to GH in the first hour of a 3-hr incubation prevents the appearance of insulin-like sensitivity. Intracellular Ca2+ concentration [( Ca2+]i) was measured in individual adipocytes that were loaded with fura-2 hexakis(acetoxymethyl) ester after preincubation in the presence (refractory) or absence (sensitive) of recombinant human GH at 100 ng/ml. Using a dual nitrogen laser imaging microscope with computer-assisted image processing to measure fluorescence changes, we observed that resting [Ca2+]i was 220 +/- 10 nM in refractory adipocytes and 110 +/- 6 nM in sensitive adipocytes (P less than 0.001). GH had no acute effect on [Ca2+]i in sensitive adipocytes but caused a sustained 3-fold increase in [Ca2+]i in refractory cells within 3 min (P less than 0.001). Insulin did not change [Ca2+]i in either sensitive or refractory adipocytes. In refractory cells treated with insulin and GH simultaneously, insulin completely blocked the rise in [Ca2+]i due to GH. Oxytocin elicited a prompt increase in [Ca2+]i followed by a quick return to resting levels in both sensitive and refractory cells. These findings indicate that basal [Ca2+]i is increased in refractory cells and that GH produces a sustained rise in [Ca2+]i only in refractory adipocytes. We suggest that the sustained increase in [Ca2+]i produced by GH in refractory cells prevents the expression of the insulin-like response.     

Goldfish brain somatostatin-28 differentially affects dopamine- and pituitary adenylate cyclase-activating polypeptide-induced GH release and Ca(2+) and cAMP signals

Dopamine (DA) and pituitary adenylate cyclase-activating polypeptide (PACAP) stimulate goldfish growth hormone (GH) release via cAMP- and Ca(2+)-dependent pathways while DA also utilizes NO. In this study, identified goldfish somatotropes responded to sequential applications of PACAP and the DA D1 agonist SKF38393 with increased intracellular Ca(2+) levels ([Ca(2+)](i)), indicating that PACAP and DA D1 receptors were present on the same cell. A native goldfish brain somatostatin (gbSS-28) reduced SKF38393-stimulated cAMP production and PACAP- and NO donor-elicited GH and [Ca(2+)](i) increases, but not PACAP-induced cAMP production nor the GH and [Ca(2+)](i) responses to forskolin, 8-bromo-cAMP and SKF38393. gbSS-28 might inhibit PACAP-induced GH release by interfering with PACAP's ability to increase [Ca(2+)](i) in a non-cAMP-dependent manner. However, DA D1 receptor activation bypassed gbSS-28 inhibitory effects on cAMP production and NO actions via unknown mechanisms to maintain a normal [Ca(2+)](i) response leading to unhampered GH release.     

Novel, thapsigargin-insensitive intracellular Ca(2+) stores control growth hormone release from goldfish pituitary cells

The relative contribution of intracellular Ca(2+) stores to basal and agonist-stimulated hormone release in pituitary cells is still not well understood, especially in non-mammalian vertebrates. Using ratiometric Ca(2+) imaging of single identified goldfish somatotropes, along with time-resolved measurements of growth hormone (GH) secretion, we investigated the Ca(2+)-dependent signal transduction of two endogenous regulators of GH release from the goldfish pituitary. Two gonadotropin-releasing hormones (sGnRH and cGnRH-II) initiated GH release in nominally Ca(2+) free conditions. GnRH-evoked GH release was additive to KCl-stimulated GH responses. Ca(2+) signals and GH release elicited by both GnRHs were abolished by pretreatment with TMB-8, which blocks the release of Ca(2+) from intracellular stores. GnRH-stimulated GH secretion is mediated by caffeine-sensitive intracellular Ca(2+) stores that are functionally independent from those sensitive to thapsigargin and other inhibitors of SERCA-type Ca(2+)/ATPases. The caffeine/TMB-8-sensitive Ca(2+) stores are also involved in spontaneous Ca(2+) signalling and the maintenance of prolonged GH release.     

Calcium-dependent enzyme activation and vacuole formation in the apical granular region of pancreatic acinar cells

The pancreatic acinar cell produces powerful digestive enzymes packaged in zymogen granules in the apical pole. Ca(2+) signals elicited by acetylcholine or cholecystokinin (CCK) initiate enzyme secretion by exocytosis through the apical membrane. Intracellular enzyme activation is normally kept to a minimum, but in the often-fatal human disease acute pancreatitis, autodigestion occurs. How the enzymes become inappropriately activated is unknown. We monitored the cytosolic Ca(2+) concentration ([Ca(2+)](i)), intracellular trypsin activation, and its localization in isolated living cells with specific fluorescent probes and studied intracellular vacuole formation by electron microscopy as well as quantitative image analysis (light microscopy). A physiological CCK level (10 pM) eliciting regular Ca(2+) spiking did not evoke intracellular trypsin activation or vacuole formation. However, stimulation with 10 nM CCK, evoking a sustained rise in [Ca(2+)](i), induced pronounced trypsin activation and extensive vacuole formation, both localized in the apical pole. Both processes were abolished by preventing abnormal [Ca(2+)](i) elevation, either by preincubation with the specific Ca(2+) chelator 1, 2-bis(O-aminophenoxy)ethane-N,N-N',N'-tetraacetic acid (BAPTA) or by removal of external Ca(2+). CCK hyperstimulation evokes intracellular trypsin activation and vacuole formation in the apical granular pole. Both of these processes are mediated by an abnormal sustained rise in [Ca(2+)](i).     

Inhibition of protein synthesis sequentially impairs distinct steps of stimulus-secretion coupling in pancreatic beta cells

Proteins with a short half-life are potential sites of pancreatic ss cell dysfunction under pathophysiological conditions. In this study, mouse islets were used to establish which step in the regulation of insulin secretion is most sensitive to inhibition of protein synthesis by 10 microM cycloheximide (CHX). Although islet protein synthesis was inhibited approximately 95% after 1 h, the inhibition of insulin secretion was delayed and progressive. After long (18-20 h) CHX-treatment, the strong (80%) inhibition of glucose-, tolbutamide-, and K(+)-induced insulin secretion was not due to lower insulin stores, to any marked impairment of glucose metabolism or to altered function of K(+)-ATP channels (total K(+)-ATP currents were however decreased). It was partly caused by a decreased Ca(2+) influx (whole-cell Ca(2+) current) resulting in a smaller rise in cytosolic Ca(2+) ([Ca(2+)](i)). The situation was very different after short (2-5 h) CHX-treatment. Insulin secretion was 50-60% inhibited although islet glucose metabolism was unaffected and stimulus-induced [Ca(2+)](i) rise was not (2 h) or only marginally (5 h) decreased. The efficiency of Ca(2+) on secretion was thus impaired. The inhibition of insulin secretion by 15 h of CHX treatment was more slowly reversible (>4 h) than that of protein synthesis. This reversibility of secretion was largely attributable to recovery of a normal Ca(2+) efficiency. In conclusion, inhibition of protein synthesis in islets inhibits insulin secretion in two stages: a rapid decrease in the efficiency of Ca(2+) on exocytosis, followed by a decrease in the Ca(2+) signal mediated by a slower loss of functional Ca(2+) channels. Glucose metabolism and the regulation of K(+)-ATP channels are more resistant. Proteins with a short half-life appear to be important to ensure optimal Ca(2+) effects on exocytosis, and are the potential Achille's heel of stimulus-secretion coupling.     

Somatostatin actions on a protein kinase C-dependent growth hormone secretagogue cascade

In mammals, the ability of somatostatin (SS) to block growth hormone (GH) secretion is due, in part, to the inhibition of two key intracellular mediators, cAMP and Ca2+. We examined whether or not inhibition of Ca2+ signaling was mediating SS-induced inhibition basal, as well as gonadotropin-releasing hormone (GnRH; a protein kinase C (PKC)-dependent growth hormone secretagogue)-stimulated growth hormone (GH) release. Although SS reduced basal GH release from populations of pituitary cells, parallel reductions in [Ca2+]i were not observed within single, identified somatotropes. Similarly, application of GnRH and the PKC activator DiC8 elicited increases in [Ca2+]i and GH release, but abolition of the Ca2+ responses did not accompany SS inhibition of the GH responses. Surprisingly, while DiC8 potentiated SS inhibition of GH release, SS paradoxically increased DiC8-stimulated increases in [Ca2+]i. These data establish that abolition of Ca2+ signals is not a primary mechanism through which SS lowers basal, or inhibits GnRH-stimulated hormone release.     

Somatostatin stimulates GH secretion in two porcine somatotrope subpopulations through a cAMP-dependent pathway

Somatostatin (SRIF) inhibits GH release from rat somatotropes by reducing adenylate cyclase (AC) activity and the free cytosolic calcium concentration ([Ca(2+)](i)). In contrast, we have reported that SRIF can stimulate GH release in vitro from pig somatotropes. Specifically, 10(-7) and 10(-15) M SRIF stimulate GH release from a subpopulation of high density (HD) somatotropes isolated by Percoll gradient centrifugation, whereas in low density (LD) somatotropes only 10(-15) M SRIF induces such an effect. To ascertain the signaling pathways underlying this phenomenon, we assessed SRIF effects on second messengers in cultured LD and HD cells by measuring cAMP, IP turnover, and [Ca(2+)](i). Likewise, contribution of the corresponding signaling pathways to SRIF-induced GH release was evaluated by blocking AC, PLC, extracellular Ca(2+) influx, or intracellular Ca(2+) mobilization. Both 10(-7) and 10(-15) M SRIF increased cAMP, IP turnover, and [Ca(2+)](i) in HD cells. Conversely, in LD cells 10(-7) M SRIF reduced [Ca(2+)](i), but did not alter cAMP or IP, and 10(-15) M SRIF was without effect. Interestingly, SRIF-stimulated GH release was abolished in both subpopulations by AC blockade, but not by PLC inhibition. Furthermore, SRIF-induced GH release was not reduced by blockade of extracellular Ca(2+) influx through voltage-sensitive channels or by depletion of thapsigargin-sensitive intracellular Ca(2+) stores. Therefore, SRIF stimulates GH secretion from cultured porcine somatotrope subpopulations through an AC/cAMP pathway-dependent mechanism that is seemingly independent of net increases in IP turnover or [Ca(2+)](i). These novel actions challenge classic views of SRIF as a mere inhibitor for somatotropes and suggest that it may exert a more complex, dual function in the control of porcine GH release, wherein molecular heterogeneity of somatotropes would play a critical role.     

Extracellular ATP stimulates estradiol secretion in rat Sertoli cells in vitro: modulation by external sodium

In this study, we examined the effects of extracellular ATP (ATPe) on [Ca(2+)](i), [Na(+)](i), plasma membrane potential changes and estradiol secretion in rat Sertoli cells. ATPe caused a rapid rise of [Ca(2+)](i) with an initial spike followed by a long lasting plateau. The first rapid spike was dependent on the release of Ca(2+) from internal stores as it also occurred in Ca(2+)-free medium while the long lasting plateau phase was dependent on Ca(2+) influx from the external medium. ATPe stimulated a rapid plasma membrane depolarization that was dependent on an influx of Na(+) from the external medium as demonstrated by plasma membrane potential monitoring in Na(+)-free medium and by [Na(+)](i) measurement with the Na(+)-sensitive fluorescent dye SBFI. ATPe stimulated estradiol secretion in a dose dependent manner and was fully dependent on the presence of Na(+) in the external medium while the presence of Ca(2+) was not necessary. Among the different nucleotides tested, only ATP, ATP-5'-[gamma-thio]triphosphate, UTP, alpha,beta-methylene-ATP were effective in stimulating estradiol secretion. These results demonstrate that rat Sertoli cells possess P2-purinergic receptors belonging to the P2X and P2Y subfamily which activation induces [Ca(2+)](i) and [Na(+)](i) rise and Na(+)-dependent plasma membrane depolarization leading to estradiol secretion.     

Mechanisms and physiological significance of the cholinergic control of pancreatic beta-cell function

Acetylcholine (ACh), the major parasympathetic neurotransmitter, is released by intrapancreatic nerve endings during the preabsorptive and absorptive phases of feeding. In beta-cells, ACh binds to muscarinic M(3) receptors and exerts complex effects, which culminate in an increase of glucose (nutrient)-induced insulin secretion. Activation of PLC generates diacylglycerol. Activation of PLA(2) produces arachidonic acid and lysophosphatidylcholine. These phospholipid-derived messengers, particularly diacylglycerol, activate PKC, thereby increasing the efficiency of free cytosolic Ca(2+) concentration ([Ca(2+)](c)) on exocytosis of insulin granules. IP3, also produced by PLC, causes a rapid elevation of [Ca(2+)](c) by mobilizing Ca(2+) from the endoplasmic reticulum; the resulting fall in Ca(2+) in the organelle produces a small capacitative Ca(2+) entry. ACh also depolarizes the plasma membrane of beta-cells by a Na(+)- dependent mechanism. When the plasma membrane is already depolarized by secretagogues such as glucose, this additional depolarization induces a sustained increase in [Ca(2+)](c). Surprisingly, ACh can also inhibit voltage-dependent Ca(2+) channels and stimulate Ca(2+) efflux when [Ca(2+)](c) is elevated. However, under physiological conditions, the net effect of ACh on [Ca(2+)](c) is always positive. The insulinotropic effect of ACh results from two mechanisms: one involves a rise in [Ca(2+)](c) and the other involves a marked, PKC-mediated increase in the efficiency of Ca(2+) on exocytosis. The paper also discusses the mechanisms explaining the glucose dependence of the effects of ACh on insulin release.     

Mechanism of D(2) agonist-induced inhibition of GH secretion from human GH-secreting adenoma cells

The mechanism of dopamine D(2) agonist-induced inhibition of GH secretion from GH-secreting adenoma cells was investigated by measurement of intracellular calcium concentration ([Ca(2+)] (i)) and static incubation experiment. Bromocriptine decreased [Ca(2+)](i) in a concentration-dependent manner through D(2) receptor. The inhibition was abolished by pertussis toxin pretreatment. Bromocriptine did not decrease [Ca (2+)](i) after nitrendipine had decreased it. 8Br-cAMP increased [Ca(2+)](i) but application of bromocriptine decreased it, suggesting that bromocriptine-induced inhibition of [Ca(2+)](i) is not dependent on bromocriptine-induced inhibition of adenylyl cyclase. Static incubation experiment revealed that bromocriptine inhibited GH secretion in a concentration-dependent manner. The inhibition was through D(2) receptor and was abolished by pertussis toxin pretreatment. 8Br-cAMP increased GH secretion. Bromocriptine decreased GH secretion even after 8Br-cAMP pretreatment. However, the GH release from cells incubated with bromocriptine alone was significantly less than that from cells incubated with bromocriptine after 8Br-cAMP pretreatment, suggesting a modulatory action of cAMP system in bromocriptine response.     

Mechanism of estrogen-mediated neuroprotection: regulation of mitochondrial calcium and Bcl-2 expression

Estrogens are neuroprotective against glutamate excitotoxicity caused by an excessive rise in intracellular calcium ([Ca(2+)](i)). In this study, we demonstrate that 17 beta-estradiol (E(2)) treatment of hippocampal neurons attenuated the excitotoxic glutamate-induced rise in bulk-free [Ca(2+)](i) despite potentiating the influx of Ca(2+) induced by glutamate. E(2)-induced attenuation of bulk-free [Ca(2+)](i) depends on mitochondrial sequestration of Ca(2+), which is blocked in the presence of the combination of rotenone and oligomycin or in the presence of antimycin, which collapse the mitochondrial membrane potential, thereby preventing mitochondrial Ca(2+) transport. Release of mitochondrial Ca(2+) by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) after excitotoxic glutamate treatment resulted in a greater [Ca(2+)](i) in E(2)-treated cells, indicating an E(2)-induced increase in the mitochondrial calcium ([Ca(2+)](m)) load. The increased [Ca(2+)](m) load was accompanied by increased expression of Bcl-2, which can promote mitochondrial Ca(2+) load tolerance. These findings provide a mechanism of E(2)-induced neuronal survival by attenuation of excitotoxic glutamate [Ca(2+)](i) rise via increased mitochondrial sequestration of cytosolic Ca(2+) coupled with an increase in Bcl-2 expression to sustain mitochondrial Ca(2+) load tolerance and function.     

Nesfatin-1 enhances glucose-induced insulin secretion by promoting Ca(2+) influx through L-type channels in mouse islet β-cells

Nucleobindin-2 (NUCB2)-derived nesfatin-1 located in the brain has been implicated in the satiety and control of energy metabolism. Nesfatin-1 is also produced in the periphery and present in the plasma. It has recently been reported that NUCB2/nesfatin-1 is localized in pancreatic islet β-cells in mice and rats and released from islets. However, its function in islets remains largely unknown. This study examined direct effects of nesfatin-1 on insulin release from pancreatic islets and on cytosolic Ca(2+) concentration ([Ca(2+)](i)) in single β-cells from ICR mice. In the presence of 8.3 mmol/L glucose, nesfatin-1 at 10(-10)-10(-9) mol/L tended to increase and at 10(-8) mol/L increased insulin release from isolated islets, while at 2.8 mmol/L glucose nesfatin-1 had no effect. Furthermore, nesfatin-1 at 10(-10)-10(-8) mol/L increased [Ca(2+)](i) in single β-cells in the presence of 8.3 but not 2.8 mmol/L glucose. The nesfatin-1-induced [Ca(2+)](i) increase and insulin release were inhibited by removal of extracellular Ca(2+) and by addition of nitrendipine, a blocker of voltage-dependent L-type Ca(2+) channels. Unexpectedly, the [Ca(2+)](i) responses to nesfatin-1 were unaltered by inhibitors of protein kinase A (PKA) and phospholipase A(2) (PLA(2)). These results indicate that nesfain-1 potentiates glucose-induced insulin secretion by promoting Ca(2+) influx through L-type Ca(2+) channels independently of PKA and PLA(2) in mouse islet β-cells.     

Pathophysiology of impaired pulsatile insulin release

Plasma insulin displays 5-10 min oscillations. In Type 2 diabetes the regularity of the oscillations disappears, which may lead to insulin receptor down-regulation and glucose intolerance and explain why pulsatile delivery of the hormone has a greater hypoglycemic effect than continuous delivery. The rhythm is intrinsic to the islet. Variations in metabolism, cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), other hormones, neuronal signaling and possibly beta-cell insulin receptor expression have been implicated in the regulation of plasma insulin oscillations. Most of these factors are important for amplitude-regulation of the insulin pulses. Although evidence exists supporting a role of both metabolism and [Ca(2+)](i) as pacemakers of the pulses, metabolic oscillations probably have a primary role and [Ca(2+)](i) oscillations a permissive role. Results from islets from animal models of diabetes suggest that altered plasma insulin pattern could be due to lowering of pulse amplitude of insulin oscillations rather than alterations in their frequency. Supporting a role of metabolism, altered plasma insulin oscillations were found in MODY2, MIDD and glycogenosis Type VII, which are linked to alterations in glucokinase, mitochondrial tRNALeu(UUR) and phosphofructokinase. Plasma insulin oscillations require coordination of islet secretory activities in the pancreas. The intrapancreatic ganglia have been suggested as coordinators. The diabetes-associated neuropathy may contribute to the deranged pattern as indicated by glucose intolerance in chagasic patients. Continued investigation of the role and regulation of pulsatile insulin release will lead to better understanding of the pathophysiology of impaired pulsatile insulin release, which could lead to new approaches to restore normal plasma insulin oscillations in diabetes and related diseases.