scholarly journals Phosphorylation of SNAP-25 on serine-187 is induced by secretagogues in insulin-secreting cells, but is not correlated with insulin secretion

2002 ◽  
Vol 368 (1) ◽  
pp. 223-232 ◽  
Author(s):  
Carmen GONELLE-GISPERT ◽  
Maria COSTA ◽  
Masami TAKAHASHI ◽  
Karin SADOUL ◽  
Philippe HALBAN
Keyword(s):  
Insulin Secretion ◽  
Rat Islets ◽  
Kinase C ◽  
Protein Receptor ◽  
Pkc Isoforms ◽  
Target Membrane ◽  
Insulin Secreting Cells ◽  
Pheochromocytoma Pc12 ◽  
Hit Cells ◽  
Rat Insulinoma

The tSNARE (the target-membrane soluble NSF-attachment protein receptor, where NSF is N-ethylmaleimide-sensitive fusion protein) synaptosomal-associated protein of 25kDa (SNAP-25) is implicated in regulated insulin secretion. In pheochromocytoma PC12 cells, SNAP-25 is phosphorylated at Ser187, which lies in a region that is important for its function. The aims of the present study were to determine whether SNAP-25 is phosphorylated at Ser187 in insulin-secreting cells and, if so, whether this is important for regulated insulin secretion. The major findings are: (i) SNAP-25 is rapidly and reversibly phosphorylated on Ser187 in both rat insulinoma INS-1 cells and rat islets in response to the phorbol ester, PMA; (ii) less than 35% of SNAP-25 in INS-1 cells is phosphorylated in response to PMA, and phosphorylation is limited to plasma-membrane-associated SNAP-25; (iii) both SNAP-25 isoforms (a and b) are phosphorylated, with 1.8-fold greater phosphorylation for SNAP-25b in response to PMA; (iv) in rat islets, Ser187 phosphorylation is stimulated by glucose or carbachol, albeit to a lesser extent than by PMA, but not by cAMP; (v) insulin secretion from botulinum neurotoxin E-treated hamster insulinoma tumour (HIT) cells, transfected with toxin-resistant Ser187→Ala or Ser187→Asp mutant SNAP-25, was similar to that of wild-type HIT cells. Furthermore, in rat islets no correlation was found between the extent of SNAP-25 phosphorylation at Ser187 in response to secretagogues and stimulation of insulin release; (vi) use of protein kinase C (PKC) inhibitors suggests that glucose stimulates SNAP-25 phosphorylation via conventional and non-conventional PKC isoforms. In summary, although SNAP-25 phosphorylation at Ser187 occurs in insulin-secreting cells and is mediated by PKC, it does not appear to play a major role in regulated insulin secretion.

scholarly journals SNAP-25a and -25b isoforms are both expressed in insulin-secreting cells and can function in insulin secretion

1999 ◽  
Vol 339 (1) ◽  
pp. 159-165 ◽  
Author(s):  
Carmen GONELLE-GISPERT ◽  
Philippe A. HALBAN ◽  
Heiner NIEMANN ◽  
Michael PALMER ◽  
Stefan CATSICAS ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
B Cells ◽  
Resistant Mutant ◽  
Secreting Cell ◽  
Protein Receptor ◽  
Target Membrane ◽  
Insulin Secreting Cells ◽  
Hit Cells

The tSNARE (the target-membrane soluble NSF-attachment protein receptor, where NSF is N-ethylmaleimide-sensitive fusion protein) synaptosomal-associated protein of 25 kDa (SNAP-25) is expressed in pancreatic B-cells and its cleavage by botulinum neurotoxin E (BoNT/E) abolishes stimulated secretion of insulin. In the nervous system, two SNAP-25 isoforms (a and b) have been described that are produced by alternative splicing. Here it is shown, using reverse transcriptase PCR, that messages for both SNAP-25 isoforms are expressed in primary pancreatic B and non-B cells as well as in insulin-secreting cell lines. After transfection, both isoforms can be detected at the plasma membrane as well as in an intracellular perinuclear region in the insulin-secreting cell line, HIT. To test for the functional role of the two isoforms in insulin secretion, mutant forms of SNAP-25a and b resistant against cleavage by BoNT/E were generated. Such mutant SNAP-25, when expressed in HIT cells, is not inactivated by BoNT/E and its ability to restore insulin secretion can thus be investigated. To obtain the toxin-resistant mutant isoforms, the sequence around the BoNT/E cleavage site (R176QIDRIM182) was changed to P176QIKRIT182. This is the sequence of the equivalent region of human SNAP-23 (P187–T194), which has been shown to be resistant to BoNT/E. The mutant SNAP-25 was resistant to BoNT/E in vitro and in vivo and both mutant isoforms were able to reconstitute insulin secretion from toxin-treated HIT cells.

Arachidonic acid-induced insulin secretion from rat islets of Langerhans

1992 ◽  
Vol 8 (2) ◽  
pp. 95-101 ◽  
Author(s):  
A. M. Band ◽  
P. M. Jones ◽  
S. L. Howell
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Insulin Secretion ◽  
Incubation Temperature ◽  
Temperature Dependent ◽  
Rat Islets ◽  
Kinase C ◽  
The Media ◽  
Rat Islets Of Langerhans ◽  
Stimulation Of

ABSTRACT There is growing evidence that arachidonic acid (AA) and/or its metabolites may be involved in the control of insulin secretion. We have now investigated the effect of AA on insulin secretion from rat islets, and the possible involvement of protein kinase C (PKC) in this process. Exogenous AA stimulated insulin secretion from intact islets at a substimulatory concentration of glucose (2 mm), but did not further enhance glucose-induced (20 mm) insulin secretion. AA-induced insulin secretion was temperature dependent. The secretory responses seen at 37°C were totally abolished by reducing the incubation temperature to ≤34°C. AA-induced insulin secretion was not dependent upon extracellular Ca2+ and was potentiated by omission of Ca2+ or bovine serum albumin from the media. PKC in rat islets can thus be stimulated by AA, but the stimulation of PKC is not required for AA-induced insulin secretion.

scholarly journals Parathyroid hormone-related peptide stimulates DNA synthesis and insulin secretion in pancreatic islets

1999 ◽  
Vol 163 (3) ◽  
pp. 403-408 ◽  
Author(s):  
ML Villanueva-Penacarrillo ◽  
J Cancelas ◽  
F de Miguel ◽  
A Redondo ◽  
A Valin ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Parathyroid Hormone ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Pancreatic Islet ◽  
Type I ◽  
Islet Mass ◽  
Rat Islets ◽  
Kinase C ◽  
Pthrp Receptor

Parathyroid hormone (PTH)-related protein (PTHrP) is present in the pancreatic islet. Recent data in transgenic mice suggest that PTHrP might modulate islet mass and insulin secretion. In the present study, we assessed the effect of the N-terminal PTH-like region of PTHrP on DNA synthesis in isolated rat islets. PTHrP (1-34), between 1 pM and 10 nM, for 48 h stimulated []thymidine incorporation into rat islets. This effect was maximally induced, about 2.5-fold over control, by 10 pM of this peptide, decreasing thereafter. In contrast, PTHrP (38-64) amide or PTHrP (107-139) were ineffective in increasing DNA synthesis in islets. Using reverse transcription followed by PCR, we confirmed that rat islets express PTHrP and the type I PTH/PTHrP receptor. Addition of a neutralizing anti-PTHrP antibody to the incubation medium of proliferating islets decreased islet DNA synthesis by 30%. The effect of a submaximal dose (30 pM) of PTHrP (1-34) on DNA synthesis in rat islets was abolished by 25 nM bisindolylmaleimide I, a protein kinase C (PKC) inhibitor, but not by 25 microM adenosine 3',5'-cyclic monophosphorothioate, Rp-isomer, a protein kinase A inhibitor. Moreover, 100 nM phorbol-12-myristate-13-acetate for 48 h also increased DNA synthesis 2-fold over controls in islets. PTHrP (1-34), at 100 nM, in contrast to 50 microM forskolin or 10 mM NaF, failed to affect adenylate cyclase activity in islet membranes. PTHrP, at 30 pM, was also found to increase 2-fold insulin released into the islet-conditioned medium within 24-48 h. Our results suggest that PTHrP is a modulator of pancreatic islet growth and/or function by a PKC-mediated mechanism.

Potentiation of insulin secretion by phorbol esters is mediated by PKC-α and nPKC isoforms

2002 ◽  
Vol 283 (5) ◽  
pp. E880-E888 ◽  
Author(s):  
Gordon C. Yaney ◽  
Jamison M. Fairbanks ◽  
Jude T. Deeney ◽  
Helen M. Korchak ◽  
Keith Tornheim ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Membrane Potential ◽  
Phorbol Esters ◽  
Myristate Acetate ◽  
Β Cells ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Enhanced Secretion ◽  
Intracellular Ca ◽  
Stimulation Of

Culturing clonal β-cells (HIT-T15) overnight in the presence of phorbol ester [phorbol myristate acetate (PMA)] enhanced insulin secretion while causing downregulation of some protein kinase C (PKC) isoforms and most PKC activity. We show here that this enhanced secretion required the retention of PMA in the cell. Hence, it could not be because of long-lived phosphorylation of cellular substrates by the isoforms that were downregulated, namely PKC-α, -βII, and -ε, but could be because of the continued activation of the two remaining diacylglycerol-sensitive isoforms δ and μ. The enhanced secretion did not involve changes in glucose metabolism, cell membrane potential, or intracellular Ca2+handling, suggesting a distal effect. PMA washout caused the loss of the enhanced response, but secretion was then stimulated by acute readdition of PMA or bombesin. The magnitude of this restimulation appeared dependent on the mass of PKC-α, which was rapidly resynthesized during PMA washout. Therefore, stimulation of insulin secretion by PMA, and presumably by endogenous diacylglycerol, involves the activation of PKC isoforms δ and/or μ, and also PKC-α.

scholarly journals The role of protein kinase C in cholinergic stimulation of insulin secretion from rat islets of Langerhans

1989 ◽  
Vol 264 (3) ◽  
pp. 753-758 ◽  
Author(s):  
S J Persaud ◽  
P M Jones ◽  
D Sugden ◽  
S L Howell
Keyword(s):  
Protein Kinase C ◽  
Insulin Secretion ◽  
Protein Kinase ◽  
Prolonged Exposure ◽  
Inositol Phosphates ◽  
Rat Islets ◽  
Phospholipid Hydrolysis ◽  
Kinase C ◽  
Short Term Exposure

The role of the Ca2+/phospholipid-dependent protein kinase C (PKC) in cholinergic potentiation of insulin release was investigated by measuring islet PKC activity and insulin secretion in response to carbachol (CCh), a cholinergic agonist. CCh caused a dose-dependent increase in insulin secretion from cultured rat islets at stimulatory glucose concentrations (greater than or equal to 7 mM), with maximal effects observed at 100 microM. Short-term exposure (5 min) of islets to 500 microM-CCh at 2 mM- or 20 mM-glucose resulted in redistribution of islet PKC activity from a predominantly cytosolic location to a membrane-associated form. Prolonged exposure (greater than 20 h) of islets to 200 nM-phorbol myristate acetate caused a virtual depletion of PKC activity associated with the islet cytosolic fraction. Under these conditions of PKC down-regulation, the potentiation of glucose-stimulated insulin secretion by CCh (500 microM) was significantly decreased, but not abolished. CCh stimulated the hydrolysis of inositol phospholipids in both normal and PKC-depleted islets, as assessed by the generation of radiolabelled inositol phosphates. These results suggest that the potentiation of glucose-induced insulin secretion by cholinergic agonists is partly mediated by activation of PKC as a consequence of phospholipid hydrolysis.

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