61. |
- Monstein, H J, et al.
(författare)
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Cholecystokinin-A and cholecystokinin-B/gastrin receptor mRNA expression in the gastrointestinal tract and pancreas of the rat and man. A polymerase chain reaction study
- 1996
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Ingår i: Scandinavian Journal of Gastroenterology. - : Informa UK Limited. - 1502-7708 .- 0036-5521. ; 31:4, s. 383-390
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Gastrin and cholecystokinin (CCK) are thought to exert trophic effects on the gastrointestinal tract and pancreas. Two types of receptors have been cloned, CCK-A and CCK-B/ gastrin. We have examined the occurrence of CCK-A and CCK-B receptor mRNA in the brain, digestive tract, pancreas, and kidney of the rat and man by Northern blot and reverse transcribed polymerase chain reaction (RT-PCR). METHODS: Total RNA was isolated from rat tissues and reverse transcribed into cDNA. cDNA from brain, kidney, and pancreas of the rat and man and from human whole stomach were commercially available. Northern blot and a PCR technique based on Taq polymerase-antibody interaction and using CCK-A and CCK-B receptor-specific primers, followed by Southern blot analysis, were the methods used. RESULTS: By means of Northern blots, CCK-A receptor mRNA was detected in rat fundus mucosa and pancreas but not in the remaining GI tract or brain. By means of RT-PCR, CCK-A receptor mRNA was demonstrated in the brain and the mucosa of the fundus, antrum, duodenum, and colon, kidney, pancreas and pancreatic islets. CCK-B receptor mRNA was detected by Northern blot analysis in the brain and the fundus mucosa but not in the rest of the digestive tract and not in the pancreas, pancreatic islets, or kidney. By RT-PCR, expression of CCK-B receptor mRNA could also be detected in antrum mucosa. In man, CCK-A receptor mRNA was detected in the brain, stomach, pancreas, and kidney, whereas CCK-B receptor mRNA was found in the brain, stomach, and pancreas but not in the kidney. Cloning and DNA-sequence analysis of the PCR-amplified rat and human CCK-A and CCK-B receptor DNA fragments, which cover the protein-encoding regions of the intracellular loop C3, showed complete sequence homology as compared with published rat and human sequences. CONCLUSIONS: It appears unlikely that CCK will have effects in the ileum, at least not effects mediated by CCK-A receptors. It also appears unlikely that physiologic concentrations of gastrin in the circulation will promote growth (or exert other effects) in the pancreas, duodenum, ileum, and colon, since CCK-B receptor mRNA is not expressed or is poorly expressed in these tissues.
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62. |
- Mosén, Henrik, et al.
(författare)
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Defective glucose-stimulated insulin release in the diabetic Goto-Kakizaki (GK) rat coincides with reduced activity of the islet carbon monoxide signaling pathway
- 2005
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Ingår i: Endocrinology. - : The Endocrine Society. - 0013-7227 .- 1945-7170. ; 146:3, s. 1553-1558
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Tidskriftsartikel (refereegranskat)abstract
- The Goto-Kakizaki (GK) rat displays a markedly reduced insulin response to glucose, a defect that is thought to be coupled to an impaired glucose signaling in the beta-cell. We have examined whether carbon monoxide (CO), derived from beta-cell heme oxygenase (HO), might be involved in the secretory dysfunction. Immunocytochemical labeling of constitutive HO (HO-2) showed no overt difference in fluorescence pattern in islets from GK vs. Wistar controls. However, isolated islets from GK rats displayed a markedly impaired HO activity measured as CO production (-50%), and immunoblotting revealed an approximately 50% reduction of HO-2 protein expression compared with Wistar controls. Furthermore, there was a prominent expression of inducible HO (HO-1) in GK islets. Incubation of isolated islets showed that the glucose-stimulated CO production and the glucose-stimulated insulin response were considerably reduced in GK islets compared with Wistar islets. Addition of the HO activator hemin or gaseous CO to the incubation media brought about a similar amplification of glucose-stimulated insulin release in GK and Wistar islets, suggesting that distal steps in the HO-CO signaling pathway were not appreciably affected. We conclude that the defective insulin response to glucose in the GK rat can be explained, at least in part, by a marked impairment of the glucose-HO-CO signaling pathway as manifested by a prominent decrease in glucose stimulation of islet CO production and a reduced expression of HO-2. A possible role of HO-1 expression as a compensatory mechanism in the GK islets is presently unclear.
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63. |
- Mosén, Henrik, et al.
(författare)
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Impaired glucose-stimulated insulin secretion in the GK rat is associated with abnormalities in islet nitric oxide production.
- 2008
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Ingår i: Regulatory Peptides. - : Elsevier BV. - 1873-1686 .- 0167-0115. ; 151, s. 139-146
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Tidskriftsartikel (refereegranskat)abstract
- We investigated implications of nitric oxide (NO) derived from islet neuronal constitutive NO synthase (ncNOS) and inducible NOS (iNOS) on insulin secretory mechanisms in the mildly diabetic GK rat. Islets from GK rats and Wistar controls were analysed for ncNOS and iNOS by HPLC, immunoblotting and immunocytochemistry in relation to insulin secretion stimulated by glucose or l-arginine in vitro and in vivo. No obvious difference in ncNOS fluorescence in GK vs control islets was seen but freshly isolated GK islets displayed a marked iNOS expression and activity. After incubation at low glucose GK islets showed an abnormal increase in both iNOS and ncNOS activities. At high glucose the impaired glucose-stimulated insulin release was associated with an increased iNOS expression and activity and NOS inhibition dose-dependently amplified insulin secretion in both GK and control islets. This effect by NOS inhibition was also evident in depolarized islets at low glucose, where forskolin had a further amplifying effect in GK but not in control islets. NOS inhibition increased basal insulin release in perfused GK pancreata and amplified insulin release after glucose stimulation in both GK and control pancreata, almost abrogating the nadir separating first and second phase in controls. A defective insulin response to l-arginine was seen in GK rats in vitro and in vivo, being partially restored by NOS inhibition. The results suggest that increased islet NOS activities might contribute to the defective insulin response to glucose and l-arginine in the GK rat. Excessive iNOS expression and activity might be deleterious for the beta-cells over time.
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64. |
- Mosén, Henrik, et al.
(författare)
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Nitric oxide, islet acid glucan-1,4-alpha-glucosidase activity and nutrient-stimulated insulin secretion
- 2000
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Ingår i: Journal of Endocrinology. - : Bioscientifica. - 1479-6805 .- 0022-0795. ; 165:2, s. 293-300
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Tidskriftsartikel (refereegranskat)abstract
- The mechanism of nutrient-evoked insulin release is clearly complex. One part of that mechanism is postulated to be the activation of the glycogenolytic enzyme acid glucan-1,4-alpha-glucosidase. As nitric oxide (NO) has been found to be a potent inhibitor of glucose-stimulated insulin secretion, we have now investigated a possible influence of exogenous NO and inhibition of endogenous NO production on islet acid glucan-1,4-alpha-glucosidase activity in relation to insulin release stimulated by glucose and l-arginine. In isolated islets, NO derived from the intracellular NO donor hydroxylamine inhibited the activation of acid glucan-1, 4-alpha-glucosidase and its isoform acid alpha-glucosidase in parallel with inhibition of glucose-stimulated insulin release. In comparison, other lysosomal enzymes were largely unaffected. Similarly, the spontaneous NO donor sodium nitroprusside, as well as NO gas, when added to islet homogenates, suppressed the activities of these acid alpha-glucosidehydrolases and, to a lesser extent, the activities of other lysosomal enzymes. Finally, in the presence of the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester, insulin release from isolated islets stimulated by glucose or l-arginine was markedly potentiated in parallel with an accompanying increase in the activities of acid glucan-1,4-alpha-glucosidase and acid alpha-glucosidase. Other lysosomal enzymes and neutral alpha-glucosidase were not influenced. We propose that an important inhibitory effect of NO on the insulin secretory processes stimulated by glucose and l-arginine is exerted via inactivation of islet acid glucan-1,4-alpha-glucosidase, a putative key enzyme in nutrient-stimulated insulin release.
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65. |
- Mårtensson, Ulrika, et al.
(författare)
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Deletion of the G protein-coupled receptor 30 impairs glucose tolerance, reduces bone growth, increases blood pressure, and eliminates estradiol-stimulated insulin release in female mice.
- 2009
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Ingår i: Endocrinology. - : The Endocrine Society. - 1945-7170 .- 0013-7227. ; 150:2, s. 687-98
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Tidskriftsartikel (refereegranskat)abstract
- In vitro studies suggest that the G protein-coupled receptor (GPR) 30 is a functional estrogen receptor. However, the physiological role of GPR30 in vivo is unknown, and it remains to be determined whether GPR30 is an estrogen receptor also in vivo. To this end, we studied the effects of disrupting the GPR30 gene in female and male mice. Female GPR30((-/-)) mice had hyperglycemia and impaired glucose tolerance, reduced body growth, increased blood pressure, and reduced serum IGF-I levels. The reduced growth correlated with a proportional decrease in skeletal development. The elevated blood pressure was associated with an increased vascular resistance manifested as an increased media to lumen ratio of the resistance arteries. The hyperglycemia and impaired glucose tolerance in vivo were associated with decreased insulin expression and release in vivo and in vitro in isolated pancreatic islets. GPR30 is expressed in islets, and GPR30 deletion abolished estradiol-stimulated insulin release both in vivo in ovariectomized adult mice and in vitro in isolated islets. Our findings show that GPR30 is important for several metabolic functions in female mice, including estradiol-stimulated insulin release.
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66. |
- Obermüller, Stefanie, et al.
(författare)
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Defective secretion of islet hormones in chromogranin-B deficient mice
- 2010
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 5:1, s. e8936-
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Tidskriftsartikel (refereegranskat)abstract
- Granins are major constituents of dense-core secretory granules in neuroendocrine cells, but their function is still a matter of debate. Work in cell lines has suggested that the most abundant and ubiquitously expressed granins, chromogranin A and B (CgA and CgB), are involved in granulogenesis and protein sorting. Here we report the generation and characterization of mice lacking chromogranin B (CgB-ko), which were viable and fertile. Unlike neuroendocrine tissues, pancreatic islets of these animals lacked compensatory changes in other granins and were therefore analyzed in detail. Stimulated secretion of insulin, glucagon and somatostatin was reduced in CgB-ko islets, in parallel with somewhat impaired glucose clearance and reduced insulin release, but normal insulin sensitivity in vivo. CgB-ko islets lacked specifically the rapid initial phase of stimulated secretion, had elevated basal insulin release, and stored and released twice as much proinsulin as wildtype (wt) islets. Stimulated release of glucagon and somatostatin was reduced as well. Surprisingly, biogenesis, morphology and function of insulin granules were normal, and no differences were found with regard to beta-cell stimulus-secretion coupling. We conclude that CgB is not required for normal insulin granule biogenesis or maintenance in vivo, but is essential for adequate secretion of islet hormones. Consequentially CgB-ko animals display some, but not all, hallmarks of human type-2 diabetes. However, the molecular mechanisms underlying this defect remain to be determined.
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67. |
- Olofsson, Charlotta, et al.
(författare)
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Long-term exposure to glucose and lipids inhibits glucose-induced insulin secretion downstream of granule fusion with plasma membrane.
- 2007
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Ingår i: Diabetes. - : American Diabetes Association. - 1939-327X .- 0012-1797. ; 56:7, s. 1888-1897
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Tidskriftsartikel (refereegranskat)abstract
- Mouse beta-cells cultured at 15 mmol/l glucose for 72 h had reduced ATP-sensitive K+ (K-ATP) channel activity (-30%), increased voltage-gated Ca2+ currents, higher intracellular free Ca2+ concentration ([Ca-i(2+]) +160%), more exocytosis (monitored by capacitance measurements, +100%), and greater insulin content (+230%) than those cultured at 4.5 mmol/l glucose. However, they released 20% less insulin when challenged with 20 mmol/l glucose. Glucose-induced (20 mmol/l) insulin secretion was reduced by 60-90% in islets cocultured at 4.5 or 15 mmol/l glucose and either oleate or palmitate (0.5 mmol/l). Free fatty acid (FFA)induced inhibition of secretion was not associated with any major changes in [Ca2+](i) or islet ATP content. Palmitate stimulated exocytosis by twofold or more but reduced V-induced secretion by up to 60%. Basal (1 mmol/l glucose) K-ATP channel activity was 40% lower in islets cultured at 4.5 mmol/l glucose plus palmitate and 60% lower in islets cultured at 15 mmol/l glucose plus either of the FFAs. Insulin content decreased by 75% in islets exposed to FFAs in the presence of high (15 mmol/l), but not low (4.5 mmol/l), glucose concentrations, but the number of secre tory granules was unchanged. FFA-induced inhibition of insulin secretion was not associated with increased tran script levels of the apoptosis markers Bax (BclII-associated X protein) and caspase-3. We conclude that glucose and FFAs reduce insulin secretion by interference with the exit of insulin via the fusion pore.
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68. |
- Olofsson, Charlotta S, 1971, et al.
(författare)
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Impaired insulin exocytosis in neural cell adhesion molecule-/- mice due to defective reorganization of the submembrane F-actin network.
- 2009
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Ingår i: Endocrinology. - : The Endocrine Society. - 1945-7170 .- 0013-7227. ; 150:7, s. 3067-75
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Tidskriftsartikel (refereegranskat)abstract
- The neural cell adhesion molecule (NCAM) is required for cell type segregation during pancreatic islet organogenesis. We have investigated the functional consequences of ablating NCAM on pancreatic beta-cell function. In vivo, NCAM(-/-) mice exhibit impaired glucose tolerance and basal hyperinsulinemia. Insulin secretion from isolated NCAM(-/-) islets is enhanced at glucose concentrations below 15 mM but inhibited at higher concentrations. Glucagon secretion from pancreatic alpha-cells evoked by low glucose was also severely impaired in NCAM(-/-) islets. The diminution of insulin secretion is not attributable to defective glucose metabolism or glucose sensing (documented as glucose-induced changes in intracellular Ca(2+) and K(ATP)-channel activity). Resting K(ATP) conductance was lower in NCAM(-/-) beta-cells than wild-type cells, and this difference was abolished when F-actin was disrupted by cytochalasin D (1 muM). In wild-type beta-cells, the submembrane actin network disassembles within 10 min during glucose stimulation (30 mM), an effect not seen in NCAM(-/-) beta-cells. Cytochalasin D eliminated this difference and normalized insulin and glucagon secretion in NCAM(-/-) islets. Capacitance measurements of exocytosis indicate that replenishment of the readily releasable granule pool is suppressed in NCAM(-/-) alpha- and beta-cells. Our data suggest that remodeling of the submembrane actin network is critical to normal glucose regulation of both insulin and glucagon secretion.
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69. |
- Olsson, Anders H, et al.
(författare)
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Decreased expression of genes involved in oxidative phosphorylation in human pancreatic islets from patients with type 2 diabetes.
- 2011
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Ingår i: European Journal of Endocrinology. - 1479-683X. ; 165, s. 589-595
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: Gene expression alterations, especially in target tissues of insulin, have been associated with type 2 diabetes (T2D). Here, we examined if genes involved in oxidative phosphorylation (OXPHOS) show differential gene expression and DNA methylation in pancreatic islets from patients with T2D compared with non-diabetic donors. DESIGN AND METHODS: Gene expression was analyzed in human pancreatic islets from 55 non-diabetic donors and 9 T2D donors using microarray. RESULTS: While the expected number of OXPHOS genes with reduced gene expression is 7.21 we identified 21 down-regulated OXPHOS genes in pancreatic islets from patients with T2D using microarray analysis. This gives a ratio of observed over expected OXPHOS genes of 26.37 using a Χ(2)-test with p = 1.52•10-7. The microarray data was validated by qRT-PCR for four selected OXPHOS genes; NDUFA5, NDUFA10, COX11 and ATP6V1H. All four OXPHOS genes were significantly down-regulated in islets from patients with T2D compared with non-diabetic donors using qRT-PCR (p≤0.01). Furthermore, HbA1c levels correlated negatively with gene expression of NDUFA5, COX11 and ATP6V1H (p less than 0.05). Gene expression of NDUFA5, NDUFA10, COX11 and ATP6V1H correlated positively with glucose-stimulated insulin secretion (p less than 0.03). Finally, DNA methylation was analyzed upstream of the transcription start for NDUFA5, COX11 and ATP6V1H. However, none of the analyzed CpG sites in the three genes showed differences in DNA methylation in islets from donors with T2D compared with non-diabetic donors. CONCLUSION: Pancreatic islets from patients with T2D show decreased expression of a set of OXPHOS genes, which may lead to impaired insulin secretion.
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70. |
- Parandeh, Fariborz, et al.
(författare)
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Uridine diphosphate (UDP) stimulates insulin secretion by activation of P2Y(6) receptors.
- 2008
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 6;370:3, s. 499-503
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Tidskriftsartikel (refereegranskat)abstract
- We examined the transcriptional expression and functional effects of receptors for the extracellular pyrimidines uridine triphosphate (UTP) and uridine diphosphate (UDP), on insulin and glucagon secretion in isolated mouse pancreatic islets and purified beta-cells. Using real-time PCR, the UDP receptor P2Y(6) was found to be highly expressed in both whole islets and beta-cells purified by repeated counter-flow elutriation, whereas no mRNA expression for UTP receptors P2Y(4) and P2Y(2) could be detected. Functional in vitro experiments revealed that the P2Y(6) agonist UDPbetaS dose-dependently enhanced insulin and glucagon release during short-term incubation (1h), while P2Y(6) activation during a longer period (24h), selectively increased insulin release, especially at high glucose levels. The corresponding EC(50) value for UDPbetaS ranged from 3.2x10(-8)M to 1.6x10(-8)M for both glucose concentrations. The P2Y(6) antagonist MRS2578 inhibited the effects of UDPbetaS, supporting a P2Y(6) specific effect. In addition to negative RT-PCR results, the lack of response to UTPgammaS a selective P2Y(2/4) agonist further rule out the involvement of P2Y(2/4) receptors in the islet hormone release. Our results suggest a modulatory role for UDP via a functional active P2Y(6) receptor in the regulation of islet hormone release.
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