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 Synaptotagmin-7 phosphorylation mediates GLP-1–dependent potentiation of insulin secretion from β-cells

2015 ◽  
Vol 112 (32) ◽  
pp. 9996-10001 ◽  
Author(s):  
Bingbing Wu ◽  
Shunhui Wei ◽  
Natalia Petersen ◽  
Yusuf Ali ◽  
Xiaorui Wang ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Glucose Homeostasis ◽  
Drug Target ◽  
Β Cells ◽  
Receptor Agonists ◽  
Intracellular Ca ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
Kinase A

Glucose stimulates insulin secretion from β-cells by increasing intracellular Ca2+. Ca2+ then binds to synaptotagmin-7 as a major Ca2+ sensor for exocytosis, triggering secretory granule fusion and insulin secretion. In type-2 diabetes, insulin secretion is impaired; this impairment is ameliorated by glucagon-like peptide-1 (GLP-1) or by GLP-1 receptor agonists, which improve glucose homeostasis. However, the mechanism by which GLP-1 receptor agonists boost insulin secretion remains unclear. Here, we report that GLP-1 stimulates protein kinase A (PKA)-dependent phosphorylation of synaptotagmin-7 at serine-103, which enhances glucose- and Ca2+-stimulated insulin secretion and accounts for the improvement of glucose homeostasis by GLP-1. A phospho-mimetic synaptotagmin-7 mutant enhances Ca2+-triggered exocytosis, whereas a phospho-inactive synaptotagmin-7 mutant disrupts GLP-1 potentiation of insulin secretion. Our findings thus suggest that synaptotagmin-7 is directly activated by GLP-1 signaling and may serve as a drug target for boosting insulin secretion. Moreover, our data reveal, to our knowledge, the first physiological modulation of Ca2+-triggered exocytosis by direct phosphorylation of a synaptotagmin.

scholarly journals cAMP-mediated signaling normalizes glucose-stimulated insulin secretion in uncoupling protein-2 overexpressing β-cells

2006 ◽  
Vol 190 (3) ◽  
pp. 669-680 ◽  
Author(s):  
T S McQuaid ◽  
M C Saleh ◽  
J W Joseph ◽  
A Gyulkhandanyan ◽  
J E Manning-Fox ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Uncoupling Protein ◽  
Uncoupling Protein 2 ◽  
Β Cells ◽  
Camp Content ◽  
Rat Islets ◽  
Mediated Effects ◽  
Intracellular Ca ◽  
Glucose Stimulated Insulin Secretion ◽  
Glucose Responsiveness

We investigated whether an increase in cAMP could normalize glucose-stimulated insulin secretion (GSIS) in uncoupling protein-2 (UCP2) overexpressing (ucp2-OE) β-cells. Indices of β-cell (β-TC-6f7 cells and rodent islets) function were measured after induction of ucp2, in the presence or absence of cAMP-stimulating agents, analogs, or inhibitors. Islets of ob/ob mice had improved glucose-responsiveness in the presence of forskolin. Rat islets overexpressing ucp2 had significantly lower GSIS than controls. Acutely, the protein kinase A (PKA) and epac pathway stimulant forskolin normalized insulin secretion in ucp2-OE rat islets and β-TC-6f7 β-cells, an effect blocked by specific PKA inhibitors but not mimicked by epac agonists. However, there was no effect of ucp2-OE on cAMP concentrations or PKA activity. In ucp2-OE islets, forskolin inhibited ATP-dependent potassium (KATP) channel currents and 86Rb+ efflux, indicative of KATP block. Likewise, forskolin application increased intracellular Ca2+, which could account for its stimulatory effects on insulin secretion. Chronic exposure to forskolin increased ucp2 mRNA and exaggerated basal secretion but not GSIS. In mice deficient in UCP2, there was no augmentation of either cAMP content or cAMP-dependent insulin secretion. Thus, elevating cellular cAMP can reverse the deficiency in GSIS invoked by ucp2-OE, at least partly through PKA-mediated effects on the KATP channel.

scholarly journals Reprogramming Mouse Cells With a Pancreatic Duct Phenotype to Insulin-Producing β-Like Cells

Endocrinology ◽  
2015 ◽  
Vol 156 (6) ◽  
pp. 2029-2038 ◽  
Author(s):  
Takatsugu Yamada ◽  
Claudia Cavelti-Weder ◽  
Francisco Caballero ◽  
Philippe A. Lysy ◽  
Lili Guo ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Pancreatic Duct ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Β Cells ◽  
Β Cell ◽  
Cell Markers ◽  
Adenoviral Delivery ◽  
Glucagon Like Peptide 1 ◽  
Green Fluorescent

Abstract Reprogramming technology has opened the possibility of converting one cell type into another by forced expression of transgenes. Transduction of adenoviral vectors encoding 3 pancreatic transcription factors, Pdx1, Ngn3, and MafA, into mouse pancreas results in direct reprogramming of exocrine cells to insulin-producing β-like cells. We hypothesized that cultured adult pancreatic duct cells could be reprogrammed to become insulin-producing β-cells by adenoviral-mediated expression of this same combination of factors. Exocrine were isolated from adult mouse insulin 1 promoter (MIP)-green fluorescent protein (GFP) transgenic mice to allow new insulin-expressing cells to be detected by GFP fluorescence. Cultured cells were transduced by an adenoviral vector carrying a polycistronic construct Ngn3/Pdx1/MafA/mCherry (Ad-M3C) or mCherry sequence alone as a control vector. In addition, the effects of glucagon-like peptide-1 (GLP-1) receptor agonist, exendin-4 (Ex-4) on the reprogramming process were examined. GFP+ cells appeared 2 days after Ad-M3C transduction; the reprogramming efficiency was 8.6 ± 2.6% by day 4 after transduction. Ad-M3C also resulted in increased expression of β-cell markers insulin 1 and 2, with enhancement by Ex-4. Expression of other β-cell markers, neuroD and GLP-1 receptor, were also significantly up-regulated. The amount of insulin release into the media and insulin content of the cells were significantly higher in the Ad-M3C-transduced cells; this too was enhanced by Ex-4. The transduced cells did not secrete insulin in response to increased glucose, indicating incomplete differentiation to β-cells. Thus, cultured murine adult pancreatic cells with a duct phenotype can be directly reprogrammed to insulin-producing β-like cells by adenoviral delivery of 3 pancreatic transcription factors.

scholarly journals Subpopulations of GFP-Marked Mouse Pancreatic β-Cells Differ in Size, Granularity, and Insulin Secretion

Endocrinology ◽  
2012 ◽  
Vol 153 (11) ◽  
pp. 5180-5187 ◽  
Author(s):  
Hitoshi Katsuta ◽  
Cristina Aguayo-Mazzucato ◽  
Rimiko Katsuta ◽  
Tomoyuki Akashi ◽  
Jennifer Hollister-Lock ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Fluorescent Protein ◽  
Plaque Assay ◽  
Gene Promoter ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Forward Scatter ◽  
Insulin Gene Expression ◽  
Green Fluorescent ◽  
Advance Knowledge

Abstract There is growing information about the heterogeneity of pancreatic β-cells and how it relates to insulin secretion. This study used the approach of flow cytometry to sort and analyze β-cells from transgenic mice expressing green fluorescent protein (GFP) under the control of the mouse insulin I gene promoter. Three populations of β-cells with differing GFP brightness could be identified, which were classified as GFP-low, GFP-medium, and GFP-bright. The GFP-medium population comprised about 70% of the total. The GFP-low population had less insulin secretion as determined by the reverse hemolytic plaque assay and reduced insulin gene expression. Additionally, all three subpopulations of β-cells were found in mice of varying ages (embryonic d 15.5 and postnatal wk 1–9). The three populations from the youngest had larger cells (forward scatter) and less granularity (side scatter) than those from the adults. This approach opens up new ways to advance knowledge about β-cell heterogeneity.

scholarly journals Localization of Cold Shock Proteins to Cytosolic Spaces Surrounding Nucleoids in Bacillus subtilis Depends on Active Transcription

2001 ◽  
Vol 183 (21) ◽  
pp. 6435-6443 ◽  
Author(s):  
Michael H. W. Weber ◽  
Arsen V. Volkov ◽  
Ingo Fricke ◽  
Mohamed A. Marahiel ◽  
Peter L. Graumann
Keyword(s):  
Bacillus Subtilis ◽  
Cold Shock ◽  
Fluorescent Protein ◽  
Cold Shock Protein ◽  
Initiation Of Translation ◽  
Specific Localization ◽  
Active Transcription ◽  
Green Fluorescent ◽  
Shock Proteins ◽  
Mutant Cells

ABSTRACT Using immunofluorescence microscopy and a fusion of a cold shock protein (CSP), CspB, to green fluorescent protein (GFP), we showed that in growing cells Bacillus subtilis CSPs specifically localize to cytosolic regions surrounding the nucleoid. The subcellular localization of CSPs is influenced by the structure of the nucleoid. Decondensed chromosomes in smc mutant cells reduced the sizes of the regions in which CSPs localized, while cold shock-induced chromosome compaction was accompanied by an expansion of the space in which CSPs were present. As a control, histone-like protein HBsu localized to the nucleoids, while β-galactosidase and GFP were detectable throughout the cell. After inhibition of translation, CspB-GFP was still present around the nucleoids in a manner similar to that in cold-shocked cells. However, in stationary-phase cells and after inhibition of transcription, CspB was distributed throughout the cell, indicating that specific localization of CspB depends on active transcription and is not due to simple exclusion from the nucleoid. Furthermore, we observed that nucleoids are more condensed and frequently abnormal incspB cspC and cspB cspDdouble-mutant cells. This suggests that the function of CSPs affects chromosome structure, probably through coupling of transcription to translation, which is thought to decondense nucleoids. In addition, we found that cspB cspD and cspB cspC double mutants are defective in sporulation, with a block at or before stage 0. Interestingly, CspB and CspC are depleted from the forespore compartment but not from the mother cell. In toto, our findings suggest that CSPs localize to zones of newly synthesized RNA, coupling transcription with initiation of translation.

Taste Receptor Cell Responses to the Bitter Stimulus Denatonium Involve Ca2+ Influx Via Store-Operated Channels

2002 ◽  
Vol 87 (6) ◽  
pp. 3152-3155 ◽  
Author(s):  
Tatsuya Ogura ◽  
Robert F. Margolskee ◽  
Sue C. Kinnamon
Keyword(s):  
Prolonged Exposure ◽  
Fluorescent Protein ◽  
Bitter Taste ◽  
Taste Receptor ◽  
Taste Receptor Cell ◽  
Cell Responses ◽  
Necturus Maculosus ◽  
Taste Cells ◽  
Intracellular Ca ◽  
Green Fluorescent

Previous studies in rat and mouse have shown that brief exposure to the bitter stimulus denatonium induces an increase in [Ca2+]i due to Ca2+ release from intracellular Ca2+ stores, rather than Ca2+influx. We report here that prolonged exposure to denatonium induces sustained increases in [Ca2+]i that are dependent on Ca2+ influx. Similar results were obtained from taste cells of the mudpuppy, Necturus maculosus, as well as green fluorescent protein (GFP) tagged gustducin-expressing taste cells of transgenic mice. In a subset of mudpuppy taste cells, prolonged exposure to denatonium induced oscillatory Ca2+responses. Depletion of Ca2+ stores by thapsigargin also induced Ca2+ influx, suggesting that Ca2+store-operated channels (SOCs) are present in both mudpuppy taste cells and gustducin-expressing taste cells of mouse. Further, treatment with thapsigargin prevented subsequent responses to denatonium, suggesting that the SOCs were the source of the Ca2+ influx. These data suggest that SOCs may contribute to bitter taste transduction and to regulation of Ca2+ homeostasis in taste cells.

scholarly journals A TRPM4 Inhibitor 9-Phenanthrol Inhibits Glucose- and Glucagon-Like Peptide 1-Induced Insulin Secretion from Rat Islets of Langerhans

2017 ◽  
Vol 2017 ◽  
pp. 1-5 ◽  
Author(s):  
Zuheng Ma ◽  
Anneli Björklund ◽  
Md. Shahidul Islam
Keyword(s):  
Insulin Secretion ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Monovalent Cation ◽  
Major Effect ◽  
Hyperinsulinemic Hypoglycemia ◽  
Rat Islets ◽  
Glucagon Like Peptide 1 ◽  
Transient Receptor ◽  
Rat Islets Of Langerhans

Pancreatic β-cells express several ion channels of the transient receptor potential family, which play important roles in mediating the stimulus-secretion coupling. One of these channels, the TRPM4 is a Ca2+-activated monovalent cation channel. This channel is inhibited by 9-phenanthrol, which also inhibits the TMEM16a Cl− channel, and activates the Ca2+-activated K+ channel, Kca3.1. The net effects of ion-channel modulation by 9-phenantherol on the insulin secretion remain unclear. We tested the effects of 9-phenanthrol on glucose- and GLP-1-induced insulin secretion from isolated rat islets in static incubations. When applied to the islets in the presence of 3.3 mM glucose, 9-phenanthrol caused a small increase in insulin secretion (~7% of the insulin secretion stimulated by 10 mM glucose). 10 μM 9-phenanthrol did not inhibit glucose- or GLP-1-induced insulin secretion. 20 μM and 30 μM 9-phenanthrol inhibited glucose-induced insulin secretion by ~80% and ~85%, respectively. Inhibition of the GLP-1-induced insulin secretion by 20 μM and 30 μM 9-phenanthrol was 65% and 94%, respectively. Our study shows that the major effect of 9-phenanthrol on the islets is a strong inhibition of insulin secretion, and we speculate that compounds related to 9-phenanthrol may be potentially useful in treating the pancreatogenous hyperinsulinemic hypoglycemia syndromes.

Time-resolved metabolomics analysis of β-cells implicates the pentose phosphate pathway in the control of insulin release

2013 ◽  
Vol 450 (3) ◽  
pp. 595-605 ◽  
Author(s):  
Peter Spégel ◽  
Vladimir V. Sharoyko ◽  
Isabel Goehring ◽  
Anders P. H. Danielsson ◽  
Siri Malmgren ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Pentose Phosphate Pathway ◽  
Metabolic Response ◽  
Cell Metabolism ◽  
Pentose Phosphate ◽  
Β Cells ◽  
Β Cell ◽  
Time Resolved ◽  
Atp Production ◽  
Rat Islets

Insulin secretion is coupled with changes in β-cell metabolism. To define this process, 195 putative metabolites, mitochondrial respiration, NADP+, NADPH and insulin secretion were measured within 15 min of stimulation of clonal INS-1 832/13 β-cells with glucose. Rapid responses in the major metabolic pathways of glucose occurred, involving several previously suggested metabolic coupling factors. The complexity of metabolite changes observed disagreed with the concept of one single metabolite controlling insulin secretion. The complex alterations in metabolite levels suggest that a coupling signal should reflect large parts of the β-cell metabolic response. This was fulfilled by the NADPH/NADP+ ratio, which was elevated (8-fold; P<0.01) at 6 min after glucose stimulation. The NADPH/NADP+ ratio paralleled an increase in ribose 5-phosphate (>2.5-fold; P<0.001). Inhibition of the pentose phosphate pathway by trans-dehydroepiandrosterone (DHEA) suppressed ribose 5-phosphate levels and production of reduced glutathione, as well as insulin secretion in INS-1 832/13 β-cells and rat islets without affecting ATP production. Metabolite profiling of rat islets confirmed the glucose-induced rise in ribose 5-phosphate, which was prevented by DHEA. These findings implicate the pentose phosphate pathway, and support a role for NADPH and glutathione, in β-cell stimulus-secretion coupling.

scholarly journals Electrophysiological Characterization of Pancreatic Islet Cells in the Mouse Insulin Promoter-Green Fluorescent Protein Mouse

Endocrinology ◽  
2005 ◽  
Vol 146 (11) ◽  
pp. 4766-4775 ◽  
Author(s):  
Yuk M. Leung ◽  
Ishtiaq Ahmed ◽  
Laura Sheu ◽  
Robert G. Tsushima ◽  
Nicholas E. Diamant ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Katp Channels ◽  
Β Cells ◽  
Β Cell ◽  
Mouse Islet ◽  
Channel Density ◽  
Na Currents ◽  
Insulin Promoter ◽  
Green Fluorescent ◽  
Ca2 Channels

We recently reported a transgenic [mouse insulin promoter (MIP)-green fluorescent protein (GFP)] mouse in which GFP expression is targeted to the pancreatic islet β-cells to enable convenient identification of β-cells as green cells. The GFP-expressing β-cells of the MIP-GFP mouse were functionally indistinguishable from β-cells of normal mice. Here we characterized the ionic channel properties and exocytosis of MIP-GFP mouse islet β- and α-cells. β-Cells displayed delayed rectifying K+ and high-voltage-activated Ca2+ channels and exhibited Na+ currents only at hyperpolarized holding potential. α-Cells were nongreen and had both A-type and delayed rectifier K+ channels, both low-voltage-activated and high-voltage-activated Ca2+ channels, and displayed Na+ currents readily at −70 mV holding potential. α-Cells had ATP-sensitive K+ channel (KATP) channel density as high as that in β-cells, and, surprisingly, α-cell KATP channels were more sensitive to ATP inhibition (IC50 = 0.16 ± 0.03 mm) than β-cell KATP channels (IC50 = 0.86 ± 0.10 mm). Whereas α-cells were rather uniform in size [2–4.5 picofarad (pF)], β-cells varied vastly in size (2–12 pF). Of note, small β-cells (&lt;4.5 pF) showed little exocytosis, whereas medium β-cells (5–8 pF) exhibited vigorous exocytosis, but large β-cells (&gt;8 pF) had weaker exocytosis. We found no correlation between β-cell size and their Ca2+ channel density, suggesting that Ca2+ influx may not be the cause of the heterogeneity in exocytotic responses. The MIP-GFP mouse therefore offers potential to further explore the functional heterogeneity in β-cells of different sizes. The MIP-GFP mouse islet is therefore a reliable model to efficiently examine α-cell and β-cell physiology and should greatly facilitate examination of their pathophysiology when the MIP-GFP mice are crossed with diabetic models.

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.

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