Insulin secretion and protein phosphorylation in PKC-depleted islets of Langerhans.

Protein kinase C (PKC)-dependent phosphorylation of endogenous substrates was measured in electrically permeabilised rat islets of Langerhans. The PKC-activating phorbol ester, 4 beta-phorbol myristate acetate (PMA), caused a slow but prolonged increase in insulin secretion from permeabilised islets, which was accompanied by increased 32P incorporation into several islet proteins of apparent M.W. 30-50 kDa. Depletion of islet PKC by prolonged exposure to PMA abolished subsequent secretory and phosphorylating responses to the phorbol ester. However, PKC-depleted islets did not show diminished responses to glucose, suggesting that PKC-mediated phosphorylation of these proteins is not essential for nutrient-induced insulin secretion.     

Identification and metabolism of polyphosphoinositides in isolated islets of Langerhans.

The effect of pH variation on the exchangeability with deuterium of protons strongly coupled to Mo(V) in the active and desulpho forms of xanthine oxidase was studied by e.p.r. and rapid freezing, in extension of the work of Gutteridge, Tanner & Bray [Biochem. J. (1978) 175, 887-897]. Above neutrality, exchange rates increased with increasing pH. Detailed studies were made on the desulpho enzyme under a variety of conditions, and exchange rate constants at 22 degrees C ranged from 0.16s -1 at pH 6.6 to 1.6s -1 at pH 11.3. The mechanism of proton exchange in the enzyme is discussed. The interpretation by the above workers that the strongly coupled proton of the active enzyme is on sulphur and that of the desulpho enzyme is on oxygen remains valid (and is in agreement with other work), as do their proposals for the structures of the protonated and deprotonated species. However, pK values cannot be calculated from the exchange data. It is likely that the relatively low rates of exchange observed are due to the difference of structure between the protonated and the deprotonated forms. In the case of the desulpho enzyme, an exchange mechanism, which involves the proton exchanging both as such and along with oxygen in the form of a hydroxyl ion, is discussed.     

Enzymes of the cholinergic system in islets of Langerhans.

Quantitative radiometric assays were employed to measure activities of choline acetyltransferase and acetylcholinesterase in freeze-dried pieces of islets of Langerhans and exocrine tissue from rat pancreas. The activities of both enzymes were about an order of magnitude higher in islets than in exocrine tissue. This difference in activity was found in rats made diabetic with streptozotocin as well as in the controls. Although the enzyme activities in islets from diabetic rats averaged about 30-40% higher than those in islets from control rats, the differences were statistically only marginally significant. Since the islets of diabetic rats are probably much smaller than those of control rats, it is suggested that cholinergic elements associated with pancreatic islets are lost following induction of streptozotocin diabetes.     

Pentitols and insulin release by isolated rat islets of Langerhans

1. Insulin secretion was studied in isolated islets of Langerhans obtained by collagenase digestion of rat pancreas. In addition to responding to glucose and mannose as do whole pancreas and pancreas slices in vitro, isolated rat islets also secrete insulin in response to xylitol, ribitol and ribose, but not to sorbitol, mannitol, arabitol, xylose or arabinose. 2. Xylitol and ribitol readily reduce NAD(+) when added to a preparation of ultrasonically treated islets. 3. Adrenaline (1mum) inhibits the effects of glucose and xylitol on insulin release. Mannoheptulose and 2-deoxy-glucose, however, inhibit the response to glucose but not that to xylitol. 4. The intracellular concentration of glucose 6-phosphate is increased when islets are incubated with glucose but not with xylitol, suggesting that xylitol does not promote insulin release by conversion into glucose 6-phosphate. 5. Theophylline (5mm) potentiates the effect of 20mm-glucose on insulin release from isolated rat islets of Langerhans, but has no effect on xylitol-mediated release. These results indicate that xylitol does not stimulate insulin release by alterations in the intracellular concentrations of cyclic AMP. 6. A possible role for the metabolism of hexoses via the pentose phosphate pathway in the stimulation of insulin release is discussed.     

Insulin and glucagon secretion from isolated islets of Langerhans. The effects of calcium ionophores.

The role of Ca2+ in the secretion of insulin and glucagon was investigated by studying the effects of Ca2+ ionophores on hormone secretion from isolated perifused islets of Langerhans. Ionophore X537A (100 muM), which binds alkaline earth cations and also complexes some univalent cations, caused a rapid transient increase in insulin and glucagon secretion which was not dependent on the presence of Ca2+ in the perifusion medium. Ionophore A23187 (100 muM), which specifically binds bivalent cations at neutral pH values, similarly increased insulin secretion in complete and Ca2+-free medium, but only stimulated glucagon release in the presence of extracellular Ca2+. Since the stimulatory effects of both ionophores were associated with an increased Ca2+ flux in the islets, these experiments support the hypothesis that Ca2+ may trigger the release of insulin and suggest that it is also involved in the secretion of glucagon. The basal rate of both insulin and glucagon release was significantly increased when Ca2+ was omitted from the perifusion medium, but it is proposed that this finding may be due to adverse effects on cell-membrane function under these conditions.     

Insulin receptor activation inhibits insulin secretion from human islets of Langerhans

There is no consensus on the role of insulin secreted from pancreatic β-cells in regulating its own secretion, either in rodent islets or in human islets. We have now investigated whether there is an autocrine signalling role for insulin in human islets by determining insulin receptor expression and assessing the effects of insulin receptor activation using a non-peptidyl insulin mimetic termed L-783,281. Human insulin receptor mRNA was detected by PCR amplification of human islet cDNA, and translation of the message in human islets was confirmed by Western blotting. Perifusion experiments revealed that both glucose-stimulated and basal insulin secretion were significantly inhibited following human islet insulin receptor activation with L-783,281, and that signalling through phosphatidylinositol 3-kinase (PI 3-kinase) was responsible, at least in part, for this inhibitory effect. These studies indicate that human islets express insulin receptors and that they are functionally coupled to a PI 3-kinase-dependent inhibition of insulin secretion.     

Insulin release and eflux of [32P]Phosphate from islets of rat Langerhans treated with iodoacetic acid and the anomers of D-glucose.

To clarify the insulin-releasing mechanism, we studied insulin release and the efflux of [32P]phosphate by glucose at 0.1 mM/min of gradient level or at 16.7 mM, and other metabolism in islets of rat Langerhans. When treated with 1 mM iodoacetic acid (IAA) plus the anomers of D-glucose at 2.8 mM for 6 min at 37 degrees C, islets elicited insulin at half the control rate under the step-wise stimulation by glucose and at the same rate as the control under the slow-rise stimulation by glucose. Using islets treated with IAA plus the alpha anomer at 16.7 mM, the step-wise stimulation secreted insulin at half a rate of the control and the slow-rise stimulation at the rate lower than the control, which was not significantly different from the control rate. Treatment with IAA plus the beta anomer at 16.7 mM inhibited insulin release under both types of stimulations by glucose. The step-wise stimulation caused the same rapid efflux of [32P]phosphate from IAA-treated islets as from the control islets, except for islets treated with IAA plus the beta anomer at 16.7 mM. The rate of glucose utilization in islets was inhibited by all IAA-treatments to the same extent, being merely half the control rate. Treatments with IAA plus the anomers at 16.7 mM significantly reduced the formation of [3H]-cAMP and the activity of protein phosphokinase in islets, while in the presence of the anomers at 2.8 mM IAA produced no significant effect. Neither IAA-treatments altered the uptake of 45Ca and the ATP content in islets. The uptake of [14C]IAA was significantly enhanced by the presence of the beta anomer at 16.7 mM to two times the control level. On the basis of these results, we suggested that the B cell might contain both glucoreceptors and rate-sensors of glucose controlling insulin release and the former might be less sensitive to IAA as compared with the latter.     

Isolation and partial characterization of a tubulin-like protein from human and swine synaptosomal membranes.

Synaptic membranes from human and swine brains were solubilized with 8 M urea and the proteins were reduced and alkylated. A protein was isolated from both sources and had identical amino acid compositions and molecular weights as determined by electrophoresis on polyacrylamide-sodium dodecylsulfate gels and by ion-exchange chromatography and gel filtration on Bioglas 1000. The apparent molecular weight of the protein was 53 000 on the acrylamide-sodium dodecylsulfate gels. Neither neutral sugars nor sialic acid was a significant component of the protein. When the proteins were digested with trypsin and the resultant peptides subjected to chromatography (n-butanol/acetic acid/water) and electrophoresis (pH 3.7) the peptide maps were identical. The protein comprises 1-2 percent of the total synaptosomal protein. With regard to amino acid composition, molecular weight, peptide map characteristics, behavior on DEAE-cellulose columns, electrophoretic mobility and sugar content, the synaptic protein is quite similar to the monomer of swine tubulin.     

Cyclic AMP-dependent protein phosphorylation and insulin secretion in intact islets of Langerhans.

Effects on insulin release, cyclic AMP content and protein phosphorylation of agents modifying cyclic AMP levels have been tested in intact rat islets of Langerhans. Insulin release induced by glucose was potentiated by dibutyryl cyclic AMP, glucagon, cholera toxin and 3-isobutyl-1-methylxanthine (IBMX); the calmodulin antagonist trifluoperazine reversed these potentiatory effects. Inhibition by trifluoperazine of IBMX-potentiated release was, however, confined to concentrations of IBMX below 50 microM; higher concentrations, up to 1 mM, were resistant to inhibition by trifluoperazine. IBMX-potentiated insulin release was also inhibited by 2-deoxyadenosine, an inhibitor of adenylate cyclase. In the absence of glucose, IBMX at concentrations up to 1 mM did not stimulate insulin release and in the presence of 3.3 mM-glucose IBMX was effective only at a concentration of 1 mM; under the latter conditions trifluoperazine again did not inhibit insulin secretion. The maximum effect on insulin release was achieved with 25 microM-IBMX. Islet [cyclic AMP] was increased by IBMX, with the maximum rise occurring with 100 microM-IBMX. The increase in [cyclic AMP] elicited by IBMX was more rapid than that induced by cholera toxin. Trifluoperazine did not significantly affect islet cyclic AMP levels under any of the conditions tested. When islets were incubated with [32P]Pi, radioactivity was incorporated into islet ATP predominantly in the gamma-position. The rate of equilibration of label was dependent on medium Pi and glucose concentration and at optimal concentrations of these 100% equilibration of internal [32P]ATP with external [32P]Pi required a period of 3h. Radioactivity was incorporated into islet protein and, in response to an increase in islet [cyclic AMP], the major effect was on a protein of Mr 15 000 on sodium dodecyl sulphate/polyacrylamide gels. The extent of phosphorylation of the Mr-15 000 protein was correlated with the level of cyclic AMP: phosphorylation in response to IBMX was inhibited by 2-deoxyadenosine but not by trifluoperazine. Fractionation of islets suggested that the Mr-15 000 protein was of nuclear origin: the protein co-migrated with histone H3 on acetic acid/urea/Triton gels. In the islet cytosol a number of proteins were phosphorylated in response to elevation of islet [cyclic AMP]: the major species had Mr values of 18 000, 25 000, 34 000, 38 000 and 48 000. Culture of islets with IBMX increased the rate of [3H]-thymidine incorporation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Opioid peptide effects on insulin release and c-AMP in islets of Langerhans

The time course and specificity of the effect of opioid peptides on c-AMP production in the islets of Langerhans was examined. An enkephalin analogue, d-Ala2Me Phe4 Met(O)-ol enkephalin (DAMME, Sandoz) produced a significant stimulation of basal c-AMP levels, with a peak of stimulation at 5 minutes and a decline thereafter. These changes in intracellular c-AMP levels were of the same order of magnitude as those induced by other secretagogues, but did not coincide in time with the more rapid peak of enkephalin-induced insulin release. The rise in islet c-AMP and insulin secretion induced by DAMME and alpha-endorphin but not leu enkephalin was antagonised by naloxone. The effects of high and low concentrations of a variety of opioid peptides and naloxone on insulin release and islet c-AMP levels were determined, alpha-endorphin, dynorphin, leu enkephalin and met enkephalin all stimulated insulin secretion significantly, though not to the same extent. Higher concentrations of alpha-endorphin, dynorphin and met enkephalin inhibited insulin release relative to effects at low opiate concentrations. However, higher concentrations of leu enkephalin stimulated insulin release further. We conclude from these results that the mode of action of opioid peptides in stimulating insulin release is not via increased islet c-AMP exclusively. Furthermore, the results obtained with different classes of opioid suggest the presence of distinctive types of opiate receptor in islets of Langerhans.     

The stimulus-secretion coupling of glucose-induced insulin release. Insulin release due to glycogenolysis in glucose-deprived islets.

1. When pancreatic islets are preincubated for 20h in the presence of glucose (83.3mM) and thereafter transferred to a glucose-free medium, theophylline (1.4mM) provokes a dramatic stimulation of insulin release. This phenomenon does not occur when the islets are preincubated for either 20h at low glucose concentration (5.6mM) or only 30 min at the high glucose concentration (83.3mM). 2. The insulinotropic action of theophylline cannot be attributed to contamination of the islets with exogenous glucose and is not suppressed by mannoheptulose. 3. The secretory response to theophylline is an immediate phenomenon, but disappears after 60min of exposure to the drug. 4. The release of insulin evoked by theophylline is abolished in calcium-depleted media containing EGTA. Theophylline enhances the net uptake of 45Ca by the islets. 5. Glycogen accumulates in the islets during the preincubation period, as judged by both ultrastructural and biochemical criteria. Theophylline significantly increases the rate of glycogenolysis during the final incubation in the glucose-free medium. 6. The theophylline-induced increase in glycogenolysis coincides with a higher rate of both lactate output and oxidation of endogenous 14C-labelled substrates. 7. These data suggest that stimulation of glycolysis from endogenous stores of glycogen is sufficient to provoke insulin release even in glucose-deprived islets, as if the binding of extracellular glucose to hypothetical plasma-membrane glucoreceptors is not an essential feature of the stimulus-secretion coupling process.     

Insulin release from mouse islets. Effect of glucose and hormones on adenylate cyclase

The adenylate cyclase system of normal mouse islets was characterized. The pH optimum of the system was 7.6. The enzyme preparation contained particulate phosphodiesterase activity. This could be removed by treatment with 0.4% (v/v) Triton X-100 or inhibited by 8mm-theophylline in the presence of 2mm-cyclic AMP (adenosine 3':5'-cyclic monophosphate). ATP at 0.32mm produced one-half maximal enzyme activity. The enzyme was stimulated in the presence of F(-) and strongly inhibited by Ca(2+). The isolated enzyme retained hormonal sensitivity and was stimulated by glucagon, pancreozymin and secretin at physiological concentrations. Glucose at 17mm, 8mm and 2mm had no direct effect on the activity of the enzyme; neither did galactose at the same concentrations. Groups of islets incubated in 17mm- or 2mm-glucose for 5 or 15min and then homogenized and assayed for adenylate cyclase activity showed no differences in adenylate cyclase activity. The results suggest that the mechanism of glucose-mediated insulin release is not via the adenylate cyclase system. Hormones, however, could mediate insulin secretion via their effects on the adenylate cyclase system.     

Partial purification and characterization of microtubular protein from Trypanosoma brucei

The tubulin proteins of the parasitic hemoflagellate Trypanosoma brucei brucei were purified and characterized. Cytoskeletal microtubules of trypanosomes do not disrupt under conditions used to solubilize brain tubulins. Trypanosomal tubulins, solubilized by extensive sonication, were partially purified from the crude cell extracts by taxol-mediated polymerization. Taxolinduced microtubules were identified by electron microscopy and analyzed biochemically. They consist predominantly of two proteins of about 52,000 and 56,000 Da. Their mobilities on sodium dodecyl sulfate gels differ slightly from those of bovine brain tubulins. Immunological cross-reactivity with antibodies raised against bovine brain tubulins confirmed the nature of the trypanosomal proteins. Peptide mapping of bovine and trypanosomal alpha- and beta-tubulins was performed by enzymatic digestion with staphylococcal protease V8 and chemical cleavage with N-chlorosuccinimide. In both cases, the peptide patterns generated from the trypanosomal alpha- and beta-tubulins were closely related to each other. This suggests that the trypanosomal alpha- and beta-tubulins may have remained more conserved during evolution than the tubulins from higher eukaryotes. The trypanosomal alpha-tubulin is post-translationally modified in vivo by the reversible addition of a tyrosine residue at its COOH terminus. As in higher eukaryotes, this reaction is completely specific for the alpha-polypeptide chain. Our observation represents the first documentation of the occurrence of COOH-terminal tyrosinolation of alpha-tubulin in an eukaryotic microorganism.     

Phosphorylation reactions in islets of Langerhans.

The possible significance of phosphorylation reaction in islets of Langerhans in relation to the secretion of insulin is discussed. The secretagogues, glucose and its metabolites, cAMP and Ca++ and their influences on protein-kinase activity are given particular attention. The data obtained by the authors, as well as by other groups, are in agreement that cAMP is a potent stimulator of protein kinase activity. Glucose and its metabolites influenced protein kinase activity in one instance. Ca++ in supraphysiological amounts inhibited protein phosphorylation. The links between phosphorylation reactions and insulin secretion are, at the present time, conjectural.