Publications by authors named "Hedeskov C"

Continuing formation of inositol phosphates during stimulation of pancreatic beta-cells by hormones and neurotransmitters requires the continued synthesis of the polyphosphoinositides phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5 bisphosphate (PIP2) from phosphatidylinositol (PI). In the present study we have investigated how this pathway and the activity of phosphoinositide-specific phospholipase C (PI-PLC) are regulated by carbamoylcholine (CCh), Ca2+, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), GTP gamma S and NaF in 44-h [3H]inositol-labelled, dispersed and digitonin-permeabilized mouse pancreatic islet cells. CCh stimulated not only PI-PLC (G-protein-mediated) but also, by an as yet unknown mechanism, significantly enhanced PI 4-kinase activity, estimated as the PIP:PI ratio, by 100%, and further increased the flux from PI to PIP and PIP2, GTP gamma S and NaF mimicked the effects of CCh on PI-PLC but had no effect on the levels of PIP and PIP2, TPA raised the PIP:PI ratio by 75%.

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The role of glucose metabolism and phosphoinositide hydrolysis in glucose-induced sensitization/desensitization of insulin secretion was studied. A change in glucose concentration from 5.5 to 16.

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The effect of arachidonic acid on protein kinase C activity and insulin secretion in mouse islets was investigated. Arachidonic acid stimulated protein kinase C activity in islet cytosol and membrane fractions by substituting for phosphatidylserine. Stimulation by arachidonic acid was dependent on either Ca2+ or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, was potentiated by the combined addition of Ca(2+) + 12-O-tetradecanoylphorbol 13-acetate, and did not further increase protein kinase C activity in the presence of saturating concentrations of phosphatidylserine.

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Insulin secretion, insulin biosynthesis and islet glucose oxidation were studied in pancreatic islets isolated from fat-fed diabetic mice of both sexes. Insulin secretion from isolated islets was studied after consecutive stimulation with alpha-ketoisocaproic acid + glutamine, glucose, forskolin, and 12-O-tetradecanoylphorbol 13-acetate. Glucose-induced insulin secretion was impaired in islets from fat-fed mice.

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Postreceptor insulin resistance was studied in liver, muscle and adipose tissue from NMRI mice of both sexes made diabetic by long-term fat-feeding. Intravenous glucose tolerance tests showed a combination of impaired glucose tolerance and increased plasma insulin concentrations consistent with insulin resistance and reduced peripheral and hepatic uptake of glucose. In the morning, the fat-fed mice were normoinsulinaemic and hyperglycaemic.

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Phosphoinositide-specific phospholipase C (PI-PLC) activity in whole homogenates of mouse pancreatic islets decreased 60-85% when the homogenates were incubated at 37 degrees C for 1 h in the presence of down to micromolar concentrations of Ca2+. Ca(2+)-induced inactivation was augmented by calmodulin, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate in the presence of ATP-Mg, and by Mg2+. Inactivation was inhibited when ATP was removed and completely abolished by trifluoperazine and EGTA.

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Mice of both sexes were fed diets with 80 per cent animal or vegetable fat for 3 months. Half of the animals also received SuperEPA, which contains 61% omega-3 fatty acids. At the end of the feeding period the mice receiving animal fat had gained more weight than the controls and the mice receiving vegetable fat, and all fat diet groups, irrespective of sex or kind of diet, had become hyperglycaemic and had impaired intravenous glucose tolerance.

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In pancreatic islets the bulk of phosphoinositide-specific phospholipase C (PI-PLC) activity was cytosolic. The soluble enzyme was activated by submicromolar concentrations of Ca2+, independent of calmodulin. It was unaffected by glucose and a series of glycolytic intermediates, including glyceraldehyde 3-phosphate.

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The influence of down-regulation of protein kinase C on glucose-induced insulin secretion was studied. A 22-24 h exposure of mouse pancreatic islets to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 0.16 microM) in RPMI 1640 culture medium (8.

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The effect of interference with diacylglycerol metabolism was investigated in pancreatic mouse islets. In the presence of the diacylglycerol lipase inhibitor RHC 80,267, glucose-induced insulin secretion was reduced 50-60%; whereas carbacholin-induced insulin secretion was unaffected. Addition of the diacylglycerol kinase inhibitor R 59,022 did not change glucose-stimulated insulin secretion but abolished the inhibition seen in the presence of RHC 80,267.

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The mechanism of glucose-stimulated cyclic AMP accumulation in mouse pancreatic islets was studied. In the presence of 3-isobutyl-1-methylxanthine, both glucose and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, enhanced cyclic AMP formation 2.5-fold during 60 min of incubation.

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Secretin and glucagon potentiate glucose-induced insulin release. We have compared the effects of secretin and glucagon with that of four hybrid molecules of the two hormones on insulin release and formation of cyclic AMP (cAMP) in isolated mouse pancreatic islets. All six peptides potentiated the release of insulin at 10 mM D-glucose, and their effects were indistinguishable with respect to the dynamics of release, dose-response relationship, and glucose dependency.

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When the extracellular concentration of glucose was raised from 3 mM to 7 mM (the concentration interval in which beta-cell depolarization and the major decrease in K+ permeability occur), the cytosolic free [NADPH]/[NADP+] ratio in mouse pancreatic islets increased by 29.5%. When glucose was increased to 20 mM, a 117% increase was observed.

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The occurrence of polyamine-stimulated protein kinase (casein kinase II) in cytosol of mouse pancreatic islets was investigated. Islet protein phosphorylation was enhanced by spermidine, spermine, lysine-rich histone and polylysine; the major endogenous substrates in the cytosol were three proteins of Mr 50,000, 55,000 and 100,000. Cadaverine and putrescine were without effects.

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Column perifusion of mouse pancreatic islets was used to study the ability of amino acids and their methyl esters to influence insulin release and activate islet glutamate dehydrogenase activity. In the absence of L-glutamine, L-serine and the methyl ester of L-phenylalanine, but neither L-phenylalanine nor L-serine methyl ester, stimulate insulin secretion. In the presence of L-glutamine, however, the effect of L-serine was additive, while the methyl esters of L-serine and L-phenylalanine as well as native L-phenylalanine, potentiated the glucose-stimulated release of insulin.

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The presence of an enzyme that hydrolyzes ATP to AMP and PPi was demonstrated in a 27,000 X g particulate and supernatant fraction of mouse pancreatic islets. The enzyme was stimulated by addition of Ca2+, Zn2+, and Co2+. Addition of calmodulin or trifluoperazine had no effect.

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The occurrence and function of polyamines in protein kinase C activation and insulin secretion in mouse pancreatic islets were studied. Determination of polyamines in mouse islets revealed 0.9 +/- 0.

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The activity of cyclic AMP phosphodiesterase in mouse pancreatic islets was investigated. 85% of the total activity was found in a 27000 g supernatant fraction. The phosphodiesterase activity in the supernatant fraction, but not in the particulate fraction, was stimulated approximately 20% by Ca2+ (10(-5)M) and calmodulin (1 microM).

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Peptides representing the C-terminal end of secretin were synthetized and their effects tested along with secretin on column-perifused isolated mouse pancreatic islets. Insulin release induced by 10 mmol/l D-glucose was potentiated by secretin tested in a concentration range of 0.01-10 micrograms/ml; the maximal effect was obtained with 1 microgram/ml secretin.

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The mechanism of potentiation of insulin secretion by fructose was investigated. Twenty mM fructose + 3 mM glucose stimulated insulin secretion in a biphasic manner similar to what is found during stimulation with 20 mM glucose, whereas 20 mM fructose alone did not affect secretion. Fructose utilization was measured as formation of tritiated water from 5-3H-fructose.

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The occurrence of endogenous substrate proteins for Ca2+-dependent protein kinase, augmented by either phospholipid or calmodulin, and for cyclic AMP-dependent protein kinase was examined in homogenates and subcellular fractions of mouse pancreatic islets. Islet protein phosphorylation was enhanced by Ca2+-calmodulin; the major endogenous substrates in the homogenate were two proteins of Mr 53000 and 100000. The Mr-100000 phosphoprotein was localized to a 27000g-supernatant fraction, whereas the Mr-53000 phosphoprotein was present in a 27000g particulate fraction of mouse islets.

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The role of cytosolic components in the regulation of mouse pancreatic islet adenylate cyclase activity was studied. Addition of mouse islet cytosol (27000 g supernatant of mouse islet sonicate), devoid of adenylate cyclase activity itself, increased adenylate cyclase activity by 93 +/- 17% (n = 9) in the 27000 g total particulate fraction of mouse islets. Addition of GTP stimulated adenylate cyclase activity by 91 +/- 11% (n = 13) or to the same degree as cytosol.

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