The transport and oxidation of glucose, the content of fructose 1,6-diphosphate, and the release of insulin were studied in microdissected pancreatic islets of ob/ob mice incubated in Krebs-Ringer bicarbonate medium. Under control conditions glucose oxidation and insulin release showed a similar dependence on glucose concentration with the steepest slope in the range 5-12mm. The omission of Ca(2+), or the substitution of choline ions for Na(+), or the addition of diazoxide had little if any effect on glucose transport. However, Ca(2+) or Na(+) deficiency as well as diazoxide (7-chloro-3-methyl-1,2,4-benzothiadiazine 1,1-dioxide) or ouabain partially inhibited glucose oxidation. These alterations of medium composition also increased the islet content of fructose 1,6-diphosphate, as did the addition of adrenaline. Phentolamine [2-N-(3-hydroxyphenyl)-p-toluidinomethyl-2-imidazoline] counteracted the effects of adrenaline and Ca(2+) deficiency on islet fructose 1,6-diphosphate. After equilibration in Na(+)-deficient medium, the islets exhibited an increase in basal insulin release whereas the secretory response to glucose was inhibited. The inhibitory effects of Na(+) deficiency on the secretory responses to different concentrations of glucose correlated with those on (14)CO(2) production. When islets were incubated with 17mm-glucose, the sudden replacement of Na(+) by choline ions resulted in a marked but transient stimulation of insulin release that was not accompanied by a demonstrable increase of glucose oxidation. Galactose and 3-O-methylglucose had no effect on glucose oxidation or on insulin release. The results are consistent with a metabolic model of the beta-cell recognition of glucose as insulin secretagogue and with the assumption that Ca(2+) or Na(+) deficiency, or the addition of adrenaline or diazoxide, inhibit insulin release at some step distal to stimulus recognition. In addition the results suggest that these conditions create a partial metabolic block of glycolysis in the beta-cells. Hence the interrelationship between the processes of stimulus recognition and insulin discharge may involve a positive feedback of secretion on glucose metabolism.

Insulin release and the content of cAMP were studied in microdissected pancreatic islets of noninbred ob/ob (obese) mice. In the absence of 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, 20 mM glucose had no effect on cAMP save a very small initial rise detectable by a freeze-stop perifusion technique only. However, combined with this methylxanthine, 20 mM glucose produced significant increases of cAMP both in perifused islets and in islets conventionally incubated in closed vials. Glucose shared this capacity to raise the cAMP level with D-glyceraldehyde and 1,3-dihydroxyacetone. Isobutylmethylxanthine (0.05-1.0 mM) or 5 ug/ml of cholera toxin, an activator of adenylate cyclase, also increased the islet cAMP level; the effects of the methylxanthine, whether or not combined with cholera toxin, were potentiated by glucose. Isobutylmethylxanthine (0.05-1.0 mM) or 5 4g/mIl of cholera toxin potentiated insulin release in response to 20 mM glucose. However, only 0.5-1.0 mM isobutylmethylxanthine stimulated insulin release in the presence of 3 mM glucose, whereas 0.05-0.1 mM isobutylmethylxanthine or 5 jig/ml of cholera toxin had no effect on secretion at the low glucose concentration. These discrepancies between cAMP-promoting and insulin-releasing activities suggest that glucose does not initiate insulin release by activating the f-cell adenylate cyclase. By being metabolized in the B-cells, glucose may both create a release-initiating signal not identical with cAMP and enhance cAMP formation, leading to potentiation of the effect of the initiator signal.Several compounds can raise the concentration of cAMP and stimulate protein kinase in pancreatic islets (1). These compounds include glucagon, which activates islet adenylate cyclase (2-4), and methylxanthines, which inhibit cyclic nucleotide phosphodiesterase activity in the islets (5-7). Glucagon and methylxanthines also potentiate the insulin-releasing action of glucose (8, 9), effects that may be due to the cAMP-mediated activation of protein kinase (1).In contrast to the above potentiators of insulin release, glucose in most studies was found not to activate the adenylate cyclase (2-4), not to inhibit the phosphodiesterase (5-7), not to increase the cAMP concentration (9, 10), and not to stimulate the protein kinase (1) in pancreatic islets. Although in a dissenting study Charles et al. (11) reported that glucose raised the cAMP level in rat islets, this effect was smaller than that of theophylline; theophylline, however, was less effective than glucose in stimulating insulin release. (14,15). The effects of D-glyceraldehyde and 1,3-dihydroxyacetone were also studied to test the hypothesis that glucose-induced changes of cAMP are due to interaction of the unmetabolized glucose molecule with a specific receptor. Previous studies suggested that D-glyceraldehyde and 1,3-dihydroxyacetone feed into the glycolytic pathway of the f,-cells and so mimic the capacity of glucose to initiate insulin release (16). MATERIALS AND METHODSAdult noninbred ...

A microchemical technic was applied to elucidate the possible role of ATP in insulin secretion by measuring the levels of this metabolite in pancreatic islets from obese hyperglycemic mice. The )3 cell content of ATP was markedly reduced within the first minute after interruption of the blood supply. A steady-state level of about 5 mmoles of ATP was noted in islets incubated in the absence of glucose. The corresponding ATP level was twice as high when at least 1 mg./ml. of glucose was present in the incubation medium. While the contents of ATP and glycogen remained constant when the pancreatic islets were incubated with diazoxide, the amounts of both these metabolites were significantly reduced by concentrations of sulfonylurea compounds known to stimulate the insulin release. The sulfonylurea effect on the islet ATP content implies a change in the "phosphate potential" of the /9 cells, which might stimulate glycogenolysis and increase the glycolytic flux. The sulfonylurea stimulation of insulin release might thus be related to a product of glucose degradation beyond the level of glucose-6-phosphate. DIABETES 28:509-16, August, 1969.

Acetylcholine potentiated the glucose-induced insulin release from microdissected mouse islets of Langerhans but had no effect on basal insulin release. Significant potentiation was obtained with 0.1 micron acetylcholine in the presence of 10 micron eserine and with 1 micron or more acetylcholine in the absence of a choline esterase inhibitor. Carbamylcholine, too, potentiated insulin release. Potentiation was blocked by methylatropine, whereas methylatropine alone had no effect on insulin release. Acetylcholine or carbamylcholine (5-500 micron) had no obvious effect on cyclic GMP or cyclic AMP in the islets. In the presence of 11.1 mM D-glucose, the membrane potential of beta-cells oscillated slowly between a polarized silent state of -50 to -55 mV and a depolarized active state of -33 to -39 mV, at which a fast spike activity occurred. Acetylcholine made the potential stay at the plateau and induced a continuous spike activity pattern. Atropine inhibited the electrical effects of acetylcholine but not those of glucose alone. It is suggested that cholinergic potentiation of insulin release is mediated by changes of transmembrane ionic fluxes, probably without the intervention of cyclic GMP or cyclic AMP.

The effects of p-chloromercuribenzoic acid and chloromercuribenzene-p-sulphonic acid on pancreatic islets were studied in vitro. Obese-hyperglycaemic mice were used as the source of microdissected islets containing more than 90% beta-cells. p-Chloromercuribenzoic acid and chloromercuribenzene-p-sulphonic acid stimulated insulin release at concentrations of 0.01mm or above. This stimulation was significantly inhibited by the omission of Ca(2+) or the addition of adrenaline, diazoxide or 2,4-dinitrophenol. p-Chloromercuribenzoic acid or chloromercuribenzene-p-sulphonic acid did not interfere with the insulin-releasing ability of glucose. Micro-perifusion experiments revealed that the release of insulin in response to organic mercurial occurred almost instantaneously, was reversible, and was biphasic. The two mercurials inhibited glucose transport as well as glucose oxidation, and increased the mannitol and sucrose spaces of isolated islets. Compared with the effects on insulin release, those on glucose transport and membrane permeability were characterized by a longer latency and/or required higher concentrations of organic mercurial. Apart from a seemingly higher proportion of beta-cells exhibiting certain degenerative features, in islets exposed to 0.1mm-chloromercuribenzene-p-sulphonic acid for 60min, no significant differences with respect to beta-cell fine structure were noted between non-incubated islets and islets incubated with chloromercuribenzene-p-sulphonic acid or glucose or both. It is suggested that insulin release may be regulated by relatively superficial thiol groups in the beta-cell plasma membrane.