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- Brown, H, et al.
(författare)
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Synaptotagmin III isoform is compartmentalized in pancreatic beta-cells and has a functional role in exocytosis
- 2000
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Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 49:3, s. 383-391
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Tidskriftsartikel (refereegranskat)abstract
- Synaptotagmin is involved in Ca2+-regulated secretion and has been suggested to serve as a general Ca2+ sensor on the membrane of secretory vesicles in neuronal cells. Insulin exocytosis from the pancreatic beta-cell is an example of a Ca2+-dependent secretory process. Previous studies of pancreatic beta-cells were unable to show presence of synaptotagmin I. We now present biochemical and immunohistochemical data showing that synaptotagmin III is present in pancreatic beta-cells as well as in the insulin-secreting cell line HIT-T15 and in rat insulinoma. By subcellular fractionation, we found synaptotagmin III in high-density fractions together with insulin and secretogranin I, indicating colocalization of synaptotagmin III and insulin in secretory granules. We could also show that blockade of synaptotagmin III by a specific antibody inhibited Ca2+-induced changes in beta-cell membrane capacitance, suggesting that synaptotagmin III is part of the functional protein complex regulating beta-cell exocytosis. The synaptotagmin III antibody did not affect the activity of the voltage-gated L-type Ca2+-channel. These findings are compatible with the view that synaptotagmin III, because of its distinct localization in the pancreatic beta-cell, functionally modulates insulin exocytosis. This indicates that synaptotagmin may have a general role in the regulation of exocytosis not only in neuronal cells but also in endocrine cells.
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- Hu, LP, et al.
(författare)
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Regulation of lipolytic activity by long-chain acyl-coenzyme A in islets and adipocytes
- 2005
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Ingår i: American Journal of Physiology: Endocrinology and Metabolism. - : American Physiological Society. - 1522-1555 .- 0193-1849. ; 289:6, s. 1085-1092
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Tidskriftsartikel (refereegranskat)abstract
- Intracellular lipolysis is a major pathway of lipid metabolism that has roles, not only in the provision of free fatty acids as energy substrate, but also in intracellular signal transduction. The latter is likely to be particularly important in the regulation of insulin secretion from islet beta-cells. The mechanisms by which lipolysis is regulated in different tissues is, therefore, of considerable interest. Here, the effects of long-chain acyl-CoA esters (LC-CoA) on lipase activity in islets and adipocytes were compared. Palmitoyl-CoA (Pal-CoA, 1-10 mu M) stimulated lipase activity in islets from both normal and hormone-sensitive lipase (HSL)-null mice and in phosphatase-treated islets, indicating that the stimulatory effect was neither on HSL nor phosphorylation dependent. In contrast, we reproduced the previously published observations showing inhibition of HSL activity by LC-CoA in adipocytes. The inhibitory effect of LC-CoA on adipocyte HSL was dependent on phosphorylation and enhanced by acyl-CoA-binding protein (ACBP). In contrast, the stimulatory effect on islet lipase activity was blocked by ACBP, presumably due to binding and sequestration of LC-CoA. These data suggest the following intertissue relationship between islets and adipocytes with respect to fatty acid metabolism, LC-CoA signaling, and lipolysis. Elevated LC-CoA in islets stimulates lipolysis to generate a signal to increase insulin secretion, whereas elevated LC-CoA in adipocytes inhibits lipolysis. Together, these opposite actions of LC-CoA lower circulating fat by inhibiting its release from adipocytes and promoting fat storage via insulin action.
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- Kamp, F, et al.
(författare)
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Sulfonylureas rapidly cross phospholipid bilayer membranes by a free-diffusion mechanism
- 2003
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Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 52:10, s. 2526-2531
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Tidskriftsartikel (refereegranskat)abstract
- Because sulfonylureas directly activate the exocytotic machinery, we were interested in the extent to which these compounds penetrate the β-cell plasma membrane and the underlying molecular mechanism(s). We now provide evidence that sulfonylureas cross phospholipid bilayer membranes rapidly and effectively by a free-diffusion mechanism. Two sulfonylurea compounds investigated by 1H nuclear magnetic resonance spectroscopy, glibenclamide and tolbutamide, were found to incorporate into phospholipid bilayers, with the ionizable sulfonamide exposed to the aqueous interface and its apparent dissociation constant (pKa) increased to ∼7.0. Diffusion of weak amphiphilic acids across membranes is associated with a measurable change in pH. Thus, by using a fluorescence-based pH assay, we could investigate the diffusion of sulfonylurea compounds across phospholipid bilayer membranes. A fluorescent pH indicator (pyranin or [2′,7′-bis (2-carboxyethyl)-5(6)-carboxyfluorescein] [BCECF]) was trapped in egg phosphatidylcholine vesicles. Addition of glibenclamide decreased internal pH (pHin), and addition of albumin reversed this drop by 50%. With the same amount of tolbutamide, the decrease in pHin was much smaller, primarily because of the lower partitioning of tolbutamide into phospholipid bilayers. Using similar protocols, we also demonstrated diffusion by the same mechanism across the β-cell plasma membrane. Thus, we now provide a molecular mechanism by which sulfonylureas can penetrate the plasma membrane and reach intracellular sites regulating exocytosis.
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- Nilsson, T, et al.
(författare)
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Temporal patterns of changes in ATP/ADP ratio, glucose 6-phosphate and cytoplasmic free Ca2+ in glucose-stimulated pancreatic beta-cells
- 1996
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Ingår i: The Biochemical journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 314314 ( Pt 1), s. 91-94
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Tidskriftsartikel (refereegranskat)abstract
- Closure of ATP-sensitive K+ (KATP) channels is part of the stimulus–secretion coupling mechanism in the pancreatic β-cell, leading to membrane depolarization and influx of Ca2+ through voltage-sensitive L-type Ca2+ channels. The elevated ATP/ADP ratio seen in the presence of high levels of glucose has been postulated to mediate the glucose-induced closure of the KATP channels and rise in cytoplasmic free Ca2+ concentration ([Ca2+]i), or alternatively to be a consequence of activation of mitochondrial dehydrogenases by the increase in [Ca2+]i. To distinguish between these two possibilities, the time course of the change in the ATP/ADP ratio was determined in comparison with that of [Ca2+]i. We here show that a severalfold rise in the ATP/ADP ratio occurs rapidly on stimulation of suspensions of mouse pancreatic β-cells with glucose. The change in the ATP/ADP ratio is an early event that begins within 20–40 s and precedes the rise in [Ca2+]i. The temporal relationship indicates that the adenine nucleotide changes cannot be a consequence of the [Ca2+]i changes and may indeed be the connecting link between glucose metabolism and [Ca2+]i changes. When the cells were sequentially treated with high glucose concentration, clonidine and finally high extracellular Ca2+ concentration to induce synchronized oscillations in [Ca2+]i in the cell suspension, corresponding oscillations in the ATP/ADP ratio were observed. Glucose 6-phosphate levels oscillated out of phase with the ATP/ADP ratio. These results support the hypothesis that the Ca2+ oscillations previously observed in glucose-stimulated single islets or β-cells may reflect oscillations in the ATP/ADP ratio that accompany oscillatory glycolysis.
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