Adrenomedullin inhibits insulin exocytosis via pertussis toxin-sensitive G protein-coupled mechanism

2006 ◽  
Vol 291 (1) ◽  
pp. E9-E14 ◽  
Author(s):  
Nobuo Sekine ◽  
Koji Takano ◽  
Nako Kimata-Hayashi ◽  
Takashi Kadowaki ◽  
Toshiro Fujita
Keyword(s):  
Insulin Secretion ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Colorimetric Assay ◽  
Calcium Ionophore ◽  
Cytosolic Calcium ◽  
Inhibitory Effect ◽  
Calcium Currents ◽  
Β Cells ◽  
Insulin Exocytosis

Direct effects of adrenomedullin on insulin secretion from pancreatic β-cells were investigated using a differentiated insulin-secreting cell line INS-1. Adrenomedullin (1–100 pM) inhibited insulin secretion at both basal (3 mM) and high (15 mM) glucose concentrations, although this inhibitory effect was not observed at higher concentrations of adrenomedullin. The inhibition of glucose-induced insulin secretion by adrenomedullin was restored with 12-h pretreatment with 1 μg/ml pertussis toxin (PTX), suggesting that this effect could be mediated by PTX-sensitive G proteins. Cellular glucose metabolism evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide colorimetric assay was not affected by adrenomedullin at concentrations that inhibited insulin secretion. Moreover, electrophysiological studies revealed that 10 pM adrenomedullin had no effect on membrane potential, voltage-gated calcium currents, or cytosolic calcium concentration induced by 15 mM glucose. Finally, insulin release induced by cAMP-raising agents, such as forskolin plus 3-isobutyl-1-methylxanthine or the calcium ionophore ionomycin, was significantly inhibited by 10 and 100 pM adrenomedullin. In conclusion, adrenomedullin at picomolar concentrations directly inhibited insulin secretion from β-cells. This effect is likely due to the inhibition of insulin exocytosis through the activation of PTX-sensitive G proteins.

scholarly journals Arachidonic acid release from rat Leydig cells: the involvement of G protein, phospholipase A2 and regulation of cAMP production

2002 ◽  
Vol 172 (1) ◽  
pp. 95-104 ◽  
Author(s):  
AM Ronco ◽  
PF Moraga ◽  
MN Llanos
Keyword(s):  
Arachidonic Acid ◽  
Fatty Acid ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Leydig Cells ◽  
Inhibitory Effect ◽  
Receptor Interaction ◽  
Dependent Manner ◽  
Camp Production

We have previously demonstrated that the release of arachidonic acid (AA) from human chorionic gonadotropin (hCG)-stimulated Leydig cells occurs in a dose- and time-dependent manner. In addition, the amount of AA released was dependent on the hormone-receptor interaction and the concentration of LH-hCG binding sites on the cell surface. The present study was conducted to evaluate the involvement of phospholipase A(2) (PLA(2)) and G proteins in AA release from hormonally stimulated rat Leydig cells, and the possible role of this fatty acid in cAMP production. Cells were first prelabelled with [(14)C]AA to incorporate the fatty acid into cell phospholipids, and then treated in different ways to evaluate AA release. hCG (25 mIU) increased the release of AA to 180+/-12% when compared with AA released from control cells, arbitrarily set as 100%. Mepacrine and parabromophenacyl bromide (pBpB), two PLA(2) inhibitors, decreased the hormone-stimulated AA release to 85+/-9 and 70+/-24% respectively. Conversely, melittin, a PLA(2) stimulator, increased the release of AA up to 200% over control. The inhibitory effect of mepacrine on the release of AA was evident in hCG-treated Leydig cells, but not in the melittin-treated cells. To determine if the release of AA was also mediated through a G protein, cells were first permeabilized and subsequently treated with pertussis toxin or GTPgammaS, a non-hydrolyzable analog of GTP. Results demonstrate that GTPgammaS was able to induce a similar level of the release of AA as hCG. In addition, pertussis toxin completely abolished the stimulatory effect of hCG on the release of AA, indicating that a member of the G(i) family was involved in the hCG-dependent release of AA. Cells treated with PLA(2) inhibitors did not modify cAMP production, but exogenously added AA significantly reduced cAMP production from hCG-treated Leydig cells, in a manner dependent on the concentration of AA and hCG. Results presented here suggest an involvement of PLA(2) and G proteins in the release of AA from hCG-stimulated Leydig cells, and under particular conditions, regulation of cAMP production by this fatty acid in these cells.

Immunoprecipitation of a pertussis toxin substrate of the Go family from rat islets of Langerhans

1992 ◽  
Vol 12 (2) ◽  
pp. 95-100 ◽  
Author(s):  
Nicholas S. Berrow ◽  
Roger D. Hurst ◽  
Susan L. F. Chan ◽  
Noel G. Morgan
Keyword(s):  
Molecular Weight ◽  
Insulin Secretion ◽  
Adenylate Cyclase ◽  
G Protein ◽  
G Proteins ◽  
Islets Of Langerhans ◽  
Pertussis Toxin ◽  
Inhibitory Receptors ◽  
Rat Islets ◽  
Rat Islets Of Langerhans

Rat islets express a pertussis toxin sensitive G-protein involved in receptor-mediated inhibition of insulin secretion. This has been assumed previously to represent “Gi” which couples inhibitory receptors to adenylate cyclase. Incubation of islet G-proteins with32P-NAD and pertussis toxin resulted in the labelling of a band of molecular weight 40,000. This band was very broad and did not allow resolution of individual components. Incubation of the radiolabelled proteins with an anti-Go antiserum resulted in specific immunoprecipitation of a32P-labelled band. These results demonstrate that the complement of pertussis toxin sensitive G-proteins in rat islets includes Go.

scholarly journals Protein Kinase CK2 Controls CaV2.1-Dependent Calcium Currents and Insulin Release in Pancreatic β-cells

2020 ◽  
Vol 21 (13) ◽  
pp. 4668
Author(s):  
Rebecca Scheuer ◽  
Stephan Ernst Philipp ◽  
Alexander Becker ◽  
Lisa Nalbach ◽  
Emmanuel Ampofo ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Calcium Currents ◽  
Insulin Biosynthesis ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Voltage Dependent ◽  
Regulatory Impact ◽  
Kinase Ck2

The regulation of insulin biosynthesis and secretion in pancreatic β-cells is essential for glucose homeostasis in humans. Previous findings point to the highly conserved, ubiquitously expressed serine/threonine kinase CK2 as having a negative regulatory impact on this regulation. In the cell culture model of rat pancreatic β-cells INS-1, insulin secretion is enhanced after CK2 inhibition. This enhancement is preceded by a rise in the cytosolic Ca2+ concentration. Here, we identified the serine residues S2362 and S2364 of the voltage-dependent calcium channel CaV2.1 as targets of CK2 phosphorylation. Furthermore, co-immunoprecipitation experiments revealed that CaV2.1 binds to CK2 in vitro and in vivo. CaV2.1 knockdown experiments showed that the increase in the intracellular Ca2+ concentration, followed by an enhanced insulin secretion upon CK2 inhibition, is due to a Ca2+ influx through CaV2.1 channels. In summary, our results point to a modulating role of CK2 in the CaV2.1-mediated exocytosis of insulin.

scholarly journals Intragranular targeting of syncollin, but not a syncollinGFP chimera, inhibits regulated insulin exocytosis in pancreatic β-cells

2005 ◽  
Vol 185 (1) ◽  
pp. 57-67 ◽  
Author(s):  
L B Hays ◽  
B Wicksteed ◽  
Y Wang ◽  
J F McCuaig ◽  
L H Philipson ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Fluorescent Protein ◽  
Zymogen Granule ◽  
Β Cells ◽  
Rat Islets ◽  
Intracellular Ca ◽  
Specific Localization ◽  
Glucagon Like Peptide 1 ◽  
Green Fluorescent ◽  
Insulin Exocytosis

Several proteins play a role in the mechanism of insulin exocytosis. However, these ‘exocytotic proteins’ have yet to account for the regulated aspect of insulin exocytosis, and other factors are involved. In pancreatic exocrine cells, the intralumenal zymogen granule protein, syncollin, is required for efficient regulated exocytosis, but it is not known whether intragranular peptides similarly influence regulated insulin exocytosis. Here, this issue has been addressed using expression of syncollin and a syncollin-green fluorescent protein (syncollinGFP) chimera in rat islet β-cells as experimental tools. Syncollin is not normally expressed in β-cells but adenoviral-mediated expression of both syncollin and syncollinGFP indicated that these were specifically targeted to the lumen of β-granules. Syncollin expression in isolated rat islets had no effect on basal insulin secretion but significantly inhibited regulated insulin secretion stimulated by glucose (16.7 mM), glucagon-like peptide-1 (GLP-1) (10 nM) and glyburide (5μM). Consistent with specific localization of syncollin to β-granules, constitutive secretion was unchanged by syncollin expression in rat islets. Syncollin-mediated inhibition of insulin secretion was not due to inadequate insulin production. Moreover, secretagogue-induced increases in cytosolic intracellular Ca2+, which is a prerequisite for triggering insulin exocytosis, were unaffected in syncollin-expressing islets. Therefore, syncollin was most likely acting downstream of secondary signals at the level of insulin exocytosis. Thus, syncollin expression in β-cells has highlighted the importance of intralumenal β-granule peptide factors playing a role in the control of insulin exocytosis. In contrast to syncollin, syncollinGFP had no effect on insulin secretion, underlining its usefulness as a ‘fluorescent tag’ to track β-granule transport and exocytosis in real time.

scholarly journals N-Type Ca2+-Channels in Murine Pancreatic β-Cells Are Inhibited by an Exclusive Coupling with Somatostatin Receptor Subtype 1

Endocrinology ◽  
2009 ◽  
Vol 150 (2) ◽  
pp. 741-748 ◽  
Author(s):  
Paul A. Smith
Keyword(s):  
Insulin Secretion ◽  
Somatostatin Receptor ◽  
Inhibitory Effect ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Somatostatin Receptor Subtypes ◽  
Perforated Patch Clamp ◽  
Ca2 Channels

Somatostatin (SRIF) is a well-established inhibitor of insulin secretion, an effect in part mediated by a direct inhibition of voltage-operated Ca2+-channels. However, the identity of the somatostatin receptor subtypes (SSTRs) and voltage-operated Ca2+-channels involved in this process are unknown. Whole-cell perforated patch-clamp methods were applied to the murine pancreatic β-cell line, MIN6, to explore the molecular pharmacology of this problem. SRIF-14 inhibited voltage-gated Ca2+ currents (ICa2+) by 19 ± 3% (n=24) with a pEC50 = 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 ICa2+ produced by SRIF and CH-275 was insensitive to pertussis toxin but was reversed by a prepulse to +100 mV. The inhibition of ICa2+ by SRIF-14 was unaffected by 20 μm nifedipine, an inhibitor of L-type Ca2+ channels. Application of the specific N-type Ca2+ channel (Cav2.2) inhibitor ω-conotoxin GV1A at 100 nm mimicked, and as a consequence abolished, the inhibitory effect of SRIF-14 on ICa2+. SRIF selectively inhibits N-type Ca2+-channels in murine pancreatic β-cells via exclusive coupling with SSTR1. These findings help explain how SSTR1 activation can inhibit insulin secretion in pancreatic β-cells and suggest a possible new therapeutic lead for treatment of hyperinsulinemia. In pancreatic β-cells, somatostatin selectively inhibits N-type, but not other, Ca2+-channels via a direct and exclusive coupling with somatostatin receptor subtype 1.

scholarly journals Astragaloside IV inhibits palmitic acid-induced apoptosis through regulation of calcium homeostasis in mice podocytes

2021 ◽  
Author(s):  
Yingjun Zang ◽  
Shuang Liu ◽  
Aili Cao ◽  
Xiangyu Shan ◽  
Wenjuan Deng ◽  
...  
Keyword(s):  
Palmitic Acid ◽  
Mitochondrial Membrane ◽  
Protective Efficacy ◽  
Calcium Ionophore ◽  
Cytosolic Calcium ◽  
Inhibitory Effect ◽  
Astragaloside Iv ◽  
Cytochrome C Release ◽  
Induced Apoptosis ◽  
Cellular Ca

AbstractLoss of podocytes is a hallmark of diabetic nephropathy, and a growing body of evidence indicates that podocytes are susceptible to palmitic acid (PA). We have previously shown that AS-IV inhibited PA-induced podocyte apoptosis by activating sarcoendoplasmic reticulum Ca2+ ATPase (SERCA), which indicate calcium regulation may involve in the process. Immunofluorescence staining, Western blot and flow cytometry were used to measure the protective efficacy of AS-IV to ameliorate PA-induced ER stress and podocyte apoptosis. Meanwhile, AS-IV inhibited cytochrome c release, decreased mitochondrial membrane potential, accompany with the depletion of endoplasmic reticulum Ca2+ and elevation of cytosolic and mitochondrial Ca2+. Sequestration of cytosolic calcium with BAPTA-AM limited the response of podocyte apoptosis, while during the process the effect of AS-IV was also restrained. In contrast, elevation of cytosolic calcium with calcium ionophore ionomycin was depressed by AS-IV addition. Furthermore, inhibiting TRPC6 expression with SKF96365 or TRPC6 siRNA counteracted the beneficial effect of AS-IV. Our study provides further evidence to conclude the inhibitory effect of AS-IV to podocyte apoptosis is Ca2+-dependent. And the efficacy correlates with inhibiting TRPC6-mediated Ca2+ influx, and then cellular Ca2+ disturbance was coordinated.

Structural finding of R/S-3,4-dihydro-2,2-dimethyl-6-halo-4-(substituted phenylaminocarbonylamino)-2H-1-benzopyrans as selective pancreatic β-cells KATP-pβ channel openers

2007 ◽  
Vol 85 (12) ◽  
pp. 1053-1063 ◽  
Author(s):  
Sk. Mahasin Alam ◽  
Soma Samanta ◽  
Amit Kumar Halder ◽  
Soumya Basu ◽  
Tarun Jha
Keyword(s):  
Insulin Secretion ◽  
Negative Impact ◽  
Inhibitory Effect ◽  
Van Der Waals Interactions ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Qsar Study ◽  
Water Partition Coefficient ◽  
Qsar Models ◽  
Leave One Out

R/S-3,4-Dihydro-2,2-dimethyl-6-halo-4-(substituted phenylaminocarbonyl-amino)-2H-1-benzopyrans are pancreatic β-cells potassium (KATP-pβ) channel openers with inhibitory effect on insulin secretion. To find the more active and effective benzopyrans as selective potassium (KATP-pβ) channel openers towards the pancreatic tissues, quantitative structure–activity relationships (QSAR) study was performed using E-state and R-state indices along with Wang–Ford charges, n-octanol/water partition coefficient, molar refractivity, and indicator parameters. QSAR models were developed by statistical techniques, e.g., multiple linear regression (MLR), principle component regression analysis (PCRA), and partial least squares (PLS) analysis. The generated equations were validated by the leave-one-out cross-validation method. The models show the importance of ETSA indices of atom numbers 16, 17, 18, 19, 21 as well as 22. The positive coefficient of S16, S17, S18, S19, S21, and S22 indicate that with the increase of the value of E-state indices, desired activity decreases. RTSA index is also important for the biological activity, and the atom numbers 16, 17, 18, 19, 20 and 22 are involved in van der Waals interactions. RTSA index also possesses negative impact on the inhibition of residual insulin secretion. Wang–Ford charges of some particular atoms are also important for the inhibition. Increase of n-octanol/water partition coefficients of compounds inhibit insulin secretion, and the presence of chlorine atom at m- and p- positions of the phenyl ring B is necessary for the inhibition of residual insulin secretion.Key words: benzopyran derivatives, potassium channel openers, PCRA, PLS, QSAR.

scholarly journals Calcium-activated K+ Channels of Mouse β-cells are Controlled by Both Store and Cytoplasmic Ca2+

2002 ◽  
Vol 120 (3) ◽  
pp. 307-322 ◽  
Author(s):  
P.B. Goforth ◽  
R. Bertram ◽  
F.A. Khan ◽  
M. Zhang ◽  
A. Sherman ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Action Potentials ◽  
Potassium Current ◽  
Cytosolic Calcium ◽  
Β Cells ◽  
Calcium Dependent ◽  
Extracellular Glucose Concentration ◽  
Extracellular Glucose ◽  
Experimental Findings

A novel calcium-dependent potassium current (Kslow) that slowly activates in response to a simulated islet burst was identified recently in mouse pancreatic β-cells (Göpel, S.O., T. Kanno, S. Barg, L. Eliasson, J. Galvanovskis, E. Renström, and P. Rorsman. 1999. J. Gen. Physiol. 114:759–769). Kslow activation may help terminate the cyclic bursts of Ca2+-dependent action potentials that drive Ca2+ influx and insulin secretion in β-cells. Here, we report that when [Ca2+]i handling was disrupted by blocking Ca2+ uptake into the ER with two separate agents reported to block the sarco/endoplasmic calcium ATPase (SERCA), thapsigargin (1–5 μM) or insulin (200 nM), Kslow was transiently potentiated and then inhibited. Kslow amplitude could also be inhibited by increasing extracellular glucose concentration from 5 to 10 mM. The biphasic modulation of Kslow by SERCA blockers could not be explained by a minimal mathematical model in which [Ca2+]i is divided between two compartments, the cytosol and the ER, and Kslow activation mirrors changes in cytosolic calcium induced by the burst protocol. However, the experimental findings were reproduced by a model in which Kslow activation is mediated by a localized pool of [Ca2+] in a subspace located between the ER and the plasma membrane. In this model, the subspace [Ca2+] follows changes in cytosolic [Ca2+] but with a gradient that reflects Ca2+ efflux from the ER. Slow modulation of this gradient as the ER empties and fills may enhance the role of Kslow and [Ca2+] handling in influencing β-cell electrical activity and insulin secretion.

scholarly journals Regulation of cytosolic calcium and insulin secretion by galanin and ATP receptors: interactions of pertussis-toxin-sensitive and -insensitive signalling pathways

1994 ◽  
Vol 303 (3) ◽  
pp. 885-891 ◽  
Author(s):  
J Lang ◽  
F Boulay ◽  
P Parker ◽  
P Gierschik ◽  
C B Wollheim
Keyword(s):  
Insulin Secretion ◽  
Pertussis Toxin ◽  
Calcium Concentration ◽  
Cytosolic Calcium ◽  
Signalling Pathways ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Insulin Secreting Cells ◽  
Agonist Concentration ◽  
Simultaneous Activation

In a previous study it was found that the expression of the exogenous fMet-Leu-Phe-receptor (NFPR) in the insulin-secreting cell line RINm5F mediates inhibition of hormone release and additionally raises cytosolic calcium concentration ([Ca2+]i) by activating phospholipase C (PLC) in a pertussis-toxin (PTX)-sensitive manner. We investigated whether an endogenous receptor could elicit similar effects and examined the interaction with PTX-insensitive signalling pathways. The hormone galanin inhibited insulin release at subnanomolar concentrations and increased [Ca2+]i, mainly by a PTX-sensitive mechanism with an EC50 (50 nM) comparable with that for hyperpolarization of membrane potential. The effect of galanin or fMet-Leu-Phe on [Ca2+]i was inhibited by pre-activation of the P2-receptor by ATP, which mobilizes calcium in a PTX-insensitive fashion. Simultaneous activation of the P2- and peptide receptors caused additive increases in [Ca2+]i saturating at a calcium concentration corresponding to the optimal ATP response. This suggests a specific convergence of PTX-sensitive and -insensitive pathways. In contrast, galanin and FMLP inhibited the insulin secretion induced by ATP (1-100 microM), but only when added prior to the nucleotide. In permeabilized cells, FMLP added after the calcium stimulus still inhibited secretion, indicating that the inefficacy observed in intact cells was not due to the rapid ATP-evoked rise in [Ca2+]i. Thus, (i) insulin-secreting cells possess an endogenous PTX-sensitive pathway mobilizing [Ca2+]i, (ii) inhibitory hormones preferentially activate different effectors depending on the agonist concentration and (iii) activation of NFPR or galanin receptor reveals an unusual dissociation between [Ca2+]i rises and insulin secretion, pointing towards an overriding inhibitory control of exocytosis.

Protein acylation in the inhibition of insulin secretion by norepinephrine, somatostatin, galanin, and PGE2

2003 ◽  
Vol 285 (2) ◽  
pp. E287-E294 ◽  
Author(s):  
Haiying Cheng ◽  
Susanne G. Straub ◽  
Geoffrey W. G. Sharp
Keyword(s):  
Insulin Secretion ◽  
G Proteins ◽  
Inhibitory Effect ◽  
Specific Protein ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Acyltransferase Activity ◽  
Distal Site ◽  
Protein Acylation

The major physiological inhibitors of insulin secretion, norepinephrine, somatostatin, galanin, and prostaglandin E2, act via specific receptors that activate pertussis toxin (PTX)-sensitive G proteins. Four inhibitory mechanisms are known: 1) activation of ATP-sensitive K channels and repolarization of the β-cell; 2) inhibition of L-type Ca2+ channels; 3) decreased activity of adenylyl cyclase; and 4) inhibition of exocytosis at a “distal” site in stimulus-secretion coupling. We have examined the underlying mechanisms of inhibition at this distal site. In rat pancreatic islets, 2-bromopalmitate, cerulenin, and polyunsaturated fatty acids, all of which suppress protein acyltransferase activity, blocked the distal inhibitory effects of norepinephrine in a concentration-dependent manner. In contrast, control compounds such as palmitate, 16-hydroxypalmitate, and etomoxir, which do not block protein acylation, had no effect. Furthermore, 2-bromopalmitate also blocked the distal inhibitory actions of somatostatin, galanin, and prostaglandin E2. Importantly, neither 2-bromopalmitate nor cerulenin affected the action of norepinephrine to decrease cAMP production. We also examined the effects of norepinephrine, 2-bromopalmitate, and cerulenin on palmitate metabolism. Palmitate oxidation and its incorporation into lipids seemed not to contribute to the effects of 2-bromopalmitate and cerulenin on norepinephrine action. These data suggest that protein acylation mediates the distal inhibitory effect on insulin secretion. We propose that the inhibitors of insulin secretion, acting via PTX-sensitive G proteins, activate a specific protein acyltransferase, causing the acylation of a protein or proteins critical to exocytosis. This particular acylation and subsequent disruption of the essential and precise interactions involved in core complex formation would block exocytosis.

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