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- Hoy, M, et al.
(författare)
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Imidazoline NNC77-0074 stimulates insulin secretion and inhibits glucagon release by control of Ca2+-dependent exocytosis in pancreatic alpha- and beta-cells
- 2003
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Ingår i: European Journal of Pharmacology. - 1879-0712. ; 466:1-2, s. 213-221
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Tidskriftsartikel (refereegranskat)abstract
- We have investigated the effects of the novel imidazoline compound (+)-2-(2-(4,5-dihydro-1H-imidazol-2-yl)-thiopene-2-yl-ethyl)pyridine (NNC77-0074) on stimulus-secretion coupling in isolated pancreatic alpha- and beta-cells. NNC77-0074 stimulated glucose-dependent insulin secretion in intact mouse pancreatic islets. No effect was observed at less than or equal to 2.5 mM glucose and maximal stimulation occurred at 10-15 mM glucose. NNC77-0074 produced a concentration-dependent stimulation of insulin secretion. Half-maximal (EC50) stimulation was observed at 24 muM and at maximally stimulatory concentrations insulin release was doubled. The stimulatory action of NNC77-0074 on insulin secretion was not associated with membrane depolarisation or a change in the activity of ATP-sensitive K+ channels. Using capacitance measurements, we found that NNC77-0074 stimulated depolarisation-induced exocytosis 2.6-fold without affecting the whole-cell Ca2+ current when applied via the extracellular medium. The concentration dependence of the stimulatory action was determined by intracellular application of NNC77-0074 through the recording pipette. NNC77-0074 stimulated exocytosis half-maximal at 44 nM and at maximally stimulatory concentrations the rate of exocytosis was increased twofold. NNC77-0074 stimulated depolarised-induced insulin secretion from islets exposed to diazoxide and high external KCl (EC50 = 0.45 muM). The stimulatory action of NNC77-0074 was dependent on protein kinase C activity. NNC77-0074 potently inhibited glucagon secretion from rat islets (EC50 = I I nM). This was not associated with a change in spontaneous electrical activity and ATP-sensitive K channel activity but resulted from a reduction of the rate of Ca2+-dependent exocytosis in single rat alpha-cells (EC50=9 nM). Inhibition of exocytosis by NNC77-0074 was pertussis toxin-sensitive and mediated by activation of the protein phosphatase calcineurin. In rat somatotrophs, PC12 cells and mouse cortical neurons NNC77-0074 did not stimulate Ca2+-evoked exocytosis, whereas the other imidazoline compounds phentolamine and efaroxan produced 2.5-fold stimulation of exocytosis. Our data suggest that the imidazoline compound NNC77-0074 constitutes a novel class of antidiabetic compounds that stimulates glucose-dependent insulin release while inhibiting glucagon secretion. These actions are exclusively exerted by modulation of exocytosis of the insulin- and glucagon-containing granules. (C) 2003 Elsevier Science B.V. All rights reserved.
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- Hoy, M, et al.
(författare)
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Tolbutamide stimulates exocytosis of glucagon by inhibition of a mitochondrial-like ATP-sensitive K+ (KATP) conductance in rat pancreatic A-cells
- 2000
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Ingår i: Journal of Physiology. - 1469-7793 .- 0022-3751. ; 527:1, s. 109-120
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Tidskriftsartikel (refereegranskat)abstract
- 1. Capacitance measurements were used to examine the effects of the sulphonylurea tolbutamide on Ca2+-dependent exocytosis in isolated glucagon-secreting rat pancreatic A-cells. 2. When applied extracellularly, tolbutamide stimulated depolarization-evoked exocytosis 4.2-fold without affecting the whole-cell Ca2+ current. The concentration dependence of the stimulatory action was determined by intracellular application through the recording pipette. Tolbutamide produced a concentration-dependent increase in cell capacitance. Half-maximal stimulation was observed at 33 microM and the maximum stimulation corresponded to a 3.4-fold enhancement of exocytosis. 3. The stimulatory action of tolbutamide was dependent on protein kinase C activity. The action of tolbutamide was mimicked by the general K+ channel blockers TEA (10 mM) and quinine (10 microM). A similar stimulation was elicited by 5-hydroxydecanoate (5-HD; 10 microM), an inhibitor of mitochondrial ATP-sensitive K+ (KATP) channels. 4. Tolbutamide-stimulated, but not TEA-induced, exocytosis was antagonized by the K+ channel openers diazoxide, pinacidil and cromakalim. 5. Dissipating the transgranular K+ gradient with nigericin and valinomycin inhibited tolbutamide- and Ca2+-evoked exocytosis. Furthermore, tolbutamide- and Ca2+-induced exocytosis were abolished by the H+ ionophore FCCP or by arresting the vacuolar (V-type) H+-ATPase with bafilomycin A1 or DCCD. Finally, ammonium chloride stimulated exocytosis to a similar extent to that obtained with tolbutamide. 6. We propose that during granular maturation, a granular V-type H+-ATPase pumps H+ into the secretory granule leading to the generation of a pH gradient across the granular membrane and the development of a positive voltage inside the granules. The pumping of H+ is facilitated by the concomitant exit of K+ through granular K+ channels with pharmacological properties similar to those of mitochondrial KATP channels. Release of granules that have been primed is then facilitated by the addition of K+ channel blockers. The resulting increase in membrane potential promotes exocytosis by unknown mechanisms, possibly involving granular alkalinization.
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- Roeske-Nielsen, A., et al.
(författare)
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Beta-galactosylceramide increases and sulfatide decreases cytokine and chemokine production in whole blood cells
- 2004
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Ingår i: Immunol Lett. - : Elsevier BV. - 0165-2478. ; 91:2-3, s. 205-11
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Tidskriftsartikel (refereegranskat)abstract
- The glycosphingolipid sulfatide and its immediate precursor beta-galactosylceramide (GalCer) are present in the pancreatic beta-cell in equimolar concentrations and may play a role in islet pathology. Previous studies of mononuclear cells have shown that sulfatide tends to decrease and GalCer tends to increase the production of proinflammatory cytokines. In this study we investigated the influence of various isoforms of sulfatide on the production of cyto- and chemokines and tested whether the opposing effects of GalCer and sulfatide could counter one another in competition assays. PHA-, LPS-, or unstimulated whole blood cultures were incubated with 30 microg/ml of native sulfatide (isolated from pig brains), C:16:0 and C:24:0 analogues of sulfatide, or native GalCer preparations. After 24 h, the supernatant levels of proinflammatory cytokines and chemokines were quantitated by ELISA. The general trend was for the sulfatides to lower the production of the cytokines, and for GalCer to increase it. In competition assays, native sulfatide dampened the stimulatory effects of GalCer but did not abolish cytokine release; GalCer, on the other hand, nullified the effect of native sulfatide at a ratio of four sulfatide molecules to one GalCer molecule. C:16:0 sulfatide appeared to have a stronger effect than C:24:0 sulfatide. The C:16:0 analogue decreased IL-1beta, IL-6, TNF-alpha, MIP-1alpha and IL-8 to 3-56% of control values (P < 0.05-0.01), while GalCer increased their production 2- to 10-fold (P < 0.01). In conclusion, sulfatide decreases the in vitro production of proinflammatory cytokines, whereas GalCer has the opposite effect.
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- Andersson, Kerstin, et al.
(författare)
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Patients with insulin-dependent diabetes but not those with non-insulin-dependent diabetes have anti-sulfatide antibodies as determined with a new ELISA assay
- 2002
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Ingår i: Autoimmunity. - : Informa UK Limited. - 0891-6934 .- 1607-842X. ; 35:7, s. 463-8
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: In sera from newly diagnosed insulin-dependent diabetes mellitus patients (IDDM type 1) autoantibodies occur against different antigen determinants often shared with neural tissues. The role of these autoantibodies in the disease process is not yet clarified but they can be used as a diagnostic tool in the detection of IDDM patients. METHODS: We have analysed the occurrence of sulfatide autoantibodies in serum from patients with type 1 diabetes (n = 20), individuals with pre-type 1 diabetes (n = 6), patients with type 2 diabetes (n = 32) and controls (n = 43). The method used for the determination of the autoantibodies was a newly developed microtitre-ELISA assay utilizing a complex of sulfatide-albumin as the ligand. RESULTS: The new assay procedure for serum sulfatide autoantibodies showed good reproducibility. The total (day-to-day) imprecision based on analyses of three different serum samples with positive titres varied between 11 and 14% during an assay period of 6 months. None of the controls (0/43) had positive titres of sulfatide antibodies. Of the patients with type 1 diabetes, 85% displayed positive titres of anti-sulfatide antibodies while none of the type 2 patients did so. All individuals with pre-type 1 diabetes had positive titres of sulfatide antibodies. CONCLUSIONS: We conclude that sulfatide autoantibodies in serum can be reproducibly assayed by the newly developed microtitre-ELISA procedure. Elevated titres of sulfatide autoantibodies are a constant finding in newly diagnosed type 1 patients.
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- Blomqvist, Maria K., 1975, et al.
(författare)
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Sulfatide is associated with insulin granules and located to microdomains of a cultured beta cell line
- 2002
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Ingår i: Glycoconj J. - 0282-0080. ; 19:6, s. 403-13
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Tidskriftsartikel (refereegranskat)abstract
- Previous studies using pancreas from various mammals and freshly isolated islets from rat pancreas have provided evidence supporting possible involvement of the glycosphingolipid sulfatide in insulin processing and secretion. In this study, sulfatide expression and metabolism in the beta cell line RINr1046-38 (RIN-38), commonly used as a model for beta cell functional studies, were investigated and compared with previous findings from freshly isolated islets. RIN-38 cells expressed similar amounts (2.7 +/- 1.1 nmol/mg protein, n = 19) of sulfatide as isolated rat islets and also followed the same metabolic pathway, mainly through recycling. Moreover, in agreement with findings in isolated islets, the major species of sulfatide isolated from RIN-38 cells contained C16:0 and C24:0 fatty acids. By applying subcellular isolations and electron microscopy and immunocytochemistry techniques, sulfatide was shown to be located to the secretory granules, the plasma membrane and enriched in detergent insoluble microdomains. In the electron microscopy studies, Sulph I staining was also associated with mitochondria and villi structures. In conclusion, RIN-38 cells might be an appropriate model, as a complement to isolated islets where the amount of material often limits the experiments, to further explore the role of sulfatide in insulin secretion and signal transduction of beta cells.
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| 8. |
- Blomqvist, M., et al.
(författare)
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Selective lack of the C16:0 fatty acid isoform of sulfatide in pancreas of type II diabetic animal models
- 2003
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Ingår i: Apmis. - 0903-4641. ; 111:9, s. 867-77
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Tidskriftsartikel (refereegranskat)abstract
- Sulfatide (3'-sulfogalactosyl-ceramide) is a glycosphingolipid mainly located in the nervous system, but has also been found in the islets of Langerhans. Previous studies have suggested that sulfatide is involved in insulin processing and secretion. In this study, sulfatide expression and metabolism in pancreas and isolated islets of the type II diabetes models, ob/ob- and db/db mouse, was investigated using TLC-ELISA, metabolic labelling and electron microscopy. As in non-diabetic Lewis rat and human pancreas, sulfatide was located in secretory granules of the beta cells. However, the type II diabetic animal models and their background strains had an altered sulfatide expression, involving the lack of the C16:0 sulfatide fatty acid isoform, compared to non-diabetic Lewis rat, BALB/c mouse and human pancreatic tissue, in which the two dominating pancreatic sulfatide isoforms C16:0 and C24:0 are expressed. Correspondingly, in isolated ob/ob islets, sulfatide synthesis excluded the production of C16:0 sulfatide. Insulin administration to ob/ob mouse, which lowers beta cell activity, resulted in significantly increased sulfatide expression in pancreas (p=0.0003), but still no expression of the C16:0 sulfatide isoform. In vitro, the C16:0 sulfatide was shown to be the isomer involved in the preservation of insulin crystals. Thus, it is hypothesized that the selection of sulfatide isomers in pancreas might be a genetic factor contributing to disease development in type II diabetic animal models.
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