Possible links between glucose-induced changes in the energy state of pancreatic B cells and insulin release. Unmasking by decreasing a stable pool of adenine nucleotides in mouse islets.

Abstract

IntroductionWhether adenine nucleotides in pancreatic B cells serve as second messengers during glucose stimulation of insulin secretion remains disputed. Our hypothesis was that the actual changes in ATP and ADP are obscured by the large pool of adenine nucleotides (ATP/ADP ratio close to 1) in insulin granules. Therefore, mouse islets were degranulated acutely with a cocktail of glucose, KCl, forskolin, and phorbol ester or during overnight culture in RPMI-1640 medium containing 10 mM glucose. When these islets were then incubated in 0 glucose + azide (to minimize cytoplasmic and mitochondrial adenine nucleotides), their content in ATP+ADP+AMP was decreased in proportion to the decrease in insulin stores. After incubation in 10 mM glucose (no azide), the ATP/ADP ratio increased from 2.4 to > 8 in cultured islets, and only from 2 to < 4 in fresh islets. These differences were not explained by changes in glucose oxidation. The glucose dependency (0-30 mM) of the changes in insulin secretion and in the ATP/ADP ratio were then compared in the same islets. In nondegranulated, fresh islets, the ATP/ADP ratio increased between 0 and 10 mM glucose and then stabilized although insulin release kept increasing. In degranulated islets, the ATP/ADP ratio also increased between 0 and 10 mM glucose, but a further increase still occurred between 10 and 20 mM glucose, in parallel with the stimulation of insulin release. In conclusion, decreasing the granular pool of ATP and ADP unmasks large changes in the ATP/ADP ratio and a glucose dependency which persists within the range of stimulatory concentrations. The ATP/ADP ratio might thus serve as a coupling factor between glucose metabolism and insulin release. (J. Clin. Invest. 1995. 96:1738-1745 Measurements of adenine nucleotides in incubated islets. The aliquot of medium used for insulin assay was taken while the incubation tubes remained at 37°C. The islets were then incubated for another 5 min before the incubation was stopped by addition of 0.6 ml of icecold trichloroacetic acid to a final concentration of 5%. The tubes were vortex-mixed, left on ice for 15 min and centrifuged for 5 min in a microfuge (Eppendorf Inc., Fremont, CA). A fraction (0.5 ml) of the supernatant was then mixed with 1.5 ml diethylether, and the ether phase containing trichloracetic acid was discarded. This step was repeated three times to ensure complete elimination of trichloracetic acid. The extracts were eventually diluted with 0. Batches of five freshly isolated islets were incubated for 120 min in 1 ml medium containing 10 mM glucose or a stimulatory cocktail composed of 25 mM glucose, 30 mM KCI, 250 ,uM diazoxide, 1 pM forskolin, and 50 nM PMA. At the end of this incubation, the medium was removed and replaced, for 60 min, by 1 ml medium containing no glucose and 10 mM azide. At the end of this second incubation, certain batches were used for measurement of insulin content and others for measurement of adenine nucleotides. Values are means±SEM. Experiment A was repeated three times with eight...

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