Glucose stimulation of insulin release involves closure of ATPsensitive K+ channels (K+-ATP channels), depolarization, and Ca" influx in B cells. However, by using diazoxide to open K+-ATP channels, and 30 mM K to depolarize the membrane, we could demonstrate that another mechanism exists, by which glucose can control insulin release independently from changes in K+-ATP channel activity and in membrane potential (Gembal et al. 1992. J. Clin. Invest. 89:1288-1295). A similar approach was followed here to investigate, with mouse islets, the nature of this newly identified mechanism. The membrane potential-independent increase in insulin release produced by glucose required metabolism of the sugar and was mimicked by other metabolized secretagogues. It also required elevated levels of cytoplasmic CO', but was not due to further changes in CO'. It could not be ascribed to acceleration of phosphoinositide metabolism, or to activation of protein kinases A or C. Thus, glucose did not increase inositol phosphate levels and hardly affected cAMP levels. Moreover, increasing inositol phosphates by vasopressin or cAMP by forskolin, and activating protein kinase C by phorbol esters did not mimic the action of glucose on release, and down-regulation of protein kinase C did not prevent these effects. On the other hand, it correlated with an increase in the ATP/ADP ratio in islet cells. We suggest that the membrane potential-independent control of insulin release exerted by glucose involves changes in the energy state of B cells. (J. Clin. Invest. 1993.91:871-880.)

In pancreatic beta cells, the increase in the ATP/ADP ratio that follows a stimulation by glucose is thought to play an important role in the Ca2+-dependent increase in insulin secretion. Here we have investigated the possible interactions between Ca2+ and adenine nucleotides in mouse islets. Measurements of both parameters in the same single islet showed that the rise in the ATP/ADP ratio precedes any rise in the cytoplasmic free-Ca2+ concentration ([Ca2+]i) and is already present during the initial transient lowering of [Ca2+]i produced by the sugar. Blockade of Ca2+ influx with nimodipine did not prevent the concentration-dependent increase in the ATP/ADP ratio produced by glucose and even augmented the ratio at all glucose concentrations which normally stimulate Ca2+ influx. In contrast, stimulation of Ca2+ influx by 30 mM K+ or 100 microM tolbutamide lowered the ATP/ADP ratio. This lowering was of rapid onset and reversibility, sustained and prevented by nimodipine or omission of extracellular Ca2+. It was, however, not attenuated after blockade of secretion by activation of alpha2-adrenoceptors. The difference in islet ATP/ADP ratio during blockade and stimulation of Ca2+ influx was similar to that observed between threshold and submaximal glucose concentrations. The results suggest that the following feedback loop could control the oscillations of membrane potential and [Ca2+]i in beta cells. Glucose metabolism increases the ATP/ADP ratio in a Ca2+-independent manner, which leads to closure of ATP-sensitive K+ channels, depolarization and stimulation of Ca2+ influx. The resulting increase in [Ca2+]i causes a larger consumption than production of ATP, which induces reopening of ATP-sensitive K+ channels and arrest of Ca2+ influx. Upon lowering of [Ca2+]i the ATP/ADP ratio increases again and a new cycle may start.

Glucose metabolism by pancreatic ␤ and ␣ cells is essential for stimulation of insulin secretion and inhibition of glucagon secretion. Studies using rodent islets have suggested that the ATP/ADP ratio serves as second messenger in ␤ cells. This study compared the effects of glucose on glucose oxidation ([U-14 C]glucose) and adenine nucleotides (luminometric method) in purified rat ␣ and ␤ cells. The rate of glucose oxidation at 1 mM glucose was higher in ␤ than ␣ cells (4.5-fold, i.e. ϳ2-fold after normalization for cell size). It was more strongly stimulated by 10 mM glucose in ␤ cells (9-fold) than in ␣ cells (5-fold). At 1 mM glucose, ATP levels were similar in both cell types, which corresponds to an approximately 2-fold higher concentration in ␣ cells (ϳ6.5 mM) than in ␤ cells (ϳ3 mM). In ␤ cells, glucose dose-dependently increased ATP and decreased ADP levels, causing a rise in the ATP/ADP ratio from 2.4 to 11.6 at 1 and 10 mM, respectively. In ␣ cells, glucose did not affect ATP and ADP levels, and the ATP/ADP ratio remained stable around 7.5. In human islets, the ATP/ADP ratio progressively increased between 1 and 10 mM glucose. In duct cells, which often contaminate human islet preparations, an increase in the ATP/ADP ratio sometimes occurred between 1 and 3 mM glucose. In conclusion, the present observations establish that the regulation of glucagon secretion by glucose does not involve changes in ␣ cell adenine nucleotides and further support the role of the ATP/ADP ratio in the control of insulin secretion.Glucose homeostasis is largely regulated in the endocrine pancreas through opposite effects of glucose on insulin and glucagon secretion. Pancreatic ␤ cells are fuel sensors that adjust the rate of insulin secretion to the rate at which they metabolize glucose (reviewed in Refs. 1-3). Two major transduction pathways are involved. The first one uses ATP-sensitive K ϩ channels (K ] i ) then triggers exocytosis of insulin granules (reviewed in Refs. 4 -6). The second pathway, known as the K ϩ -ATP channel-independent pathway, increases the effectiveness of Ca 2ϩ on exocytosis by as yet incompletely elucidated mechanisms (7-9). Much less information is available on how glucose inhibits glucagon secretion from ␣ cells (10). Measurements of glucose metabolism in ␣ cell-rich islets (11) and purified ␣ cells (12) and studies using metabolic inhibitors in whole islets or pancreas (13-15) suggest that the inhibition of glucagon secretion is mediated by glucose metabolism in the ␣ cells.Although early studies reported that glucose increases ATP levels in rodent islets (16, 17), a rise in the ATP/ADP ratio was not a consistent finding (reviewed in Ref. 18). Recently, we demonstrated that glucose causes a large, concentration-dependent increase in the ATP/ADP ratio in mouse islets and that this effect might be involved in the regulation of insulin secretion through both pathways (19,20). However, the changes measured in whole islets might not exactly reflect those occurring in ␤ cells. It is also not known whet...

Changes in the ATP:ADP ratio in pancreatic B cells may participate in the regulation of insulin secretion by glucose. Here, we have investigated the possible role of guanine nucleotides. Mouse islets were incubated in a control medium (when K ؉ -ATP channels are the major site of regulation) or in a high K ؉ medium (when glucose modulates the effectiveness of cytosolic Ca 2؉ on exocytosis). Glucose induced a concentration-dependent (0 -20 mM) increase in GTP and a decrease in GDP in both types of medium, thus causing a progressive rise of the GTP:GDP ratio. ATP and ADP levels were 4 -5-fold higher but varied in a similar way as those of guanine nucleotides. Insulin secretion was inversely correlated with ADP and GDP levels and positively correlated with the ATP:ADP and GTP:GDP ratios between 6 and 20 mM glucose in control medium and between 0 and 20 mM glucose in high K ؉ medium. The increases in the GTP: GDP and ATP:ADP ratios induced by a rise of glucose were faster than the decreases induced by a fall in glucose, but the changes of both ratios were again parallel. In conclusion, glucose causes large, concentration-dependent changes in guanine as well as in adenine nucleotides in islet cells. This raises the possibility that both participate in the regulation of nutrient-induced insulin secretion.The regulation of pancreatic B cell function differs from that of other secretory cells in that the control of insulin secretion does not depend on the binding of the major physiological stimulator, glucose, to a receptor, but on its metabolism within B cells (reviewed in Refs. 1-5). It is now widely accepted that this metabolism generates several signals that close ATP-sensitive K ϩ channels (K ϩ -ATP channels) 1 in the plasma membrane. This closure (hereafter referred to as the "primary mechanism" of control) leads to membrane depolarization with subsequent opening of voltage-dependent Ca 2ϩ channels, Ca 2ϩ influx, rise in cytoplasmic free Ca 2ϩ concentration ([Ca 2ϩ ] i ), and eventual triggering of insulin secretion (4 -9). A "second mechanism" of control by glucose exists, which also depends on changes in metabolism (10 -12). It does not involve a further change in [Ca 2ϩ ] i but an increase in the effectiveness of Ca 2ϩ on its intracellular targets (12). Despite numerous studies, the nature of the signals that link the acceleration of metabolism to the closure of K ϩ -ATP channels and to the increase in Ca 2ϩ action has only partially been elucidated. Purine nucleotides clearly stand out as potential candidates.The popular hypothesis that variations in the cytosolic concentrations or ratio of adenine nucleotides are involved in the primary mechanism of control by glucose rests primarily on the fact that K ϩ channels, which control the B cell membrane potential, are regulated by intracellular ATP and ADP (6,8,9,13). Although this hypothesis has long been disputed (reviewed in Ref. 14), our demonstration of a correlation between insulin release and the ATP:ADP ratio in islets incubated in the presence of...

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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Adenine nucleotides are thought to serve as second messengers in the control of beta-cell function by glucose, e.g. by regulating the activity of ATP-dependent K+ channels. However, their localization in different intracellular pools may mask the biologically relevant changes and complicate the interpretation of measurements in whole cells. The plasma membrane of mouse islet cells was selectively permeabilized by the alpha-toxin from Staphylococcus aureus to allow diffusion of cytoplasmic nucleotides. After permeabilization of cells from freshly isolated islets, approximately 68% of ATP, 45% of ADP, and 52% of AMP rapidly diffused out of the cells, whereas the insulin content hardly varied. The nondiffusible pool of nucleotides was stable for at least 90 min at 4 C, which suggests that it is contained in cellular organelles. The size of this nondiffusible pool decreased proportionally to insulin stores when these were lowered by stimulating secretion to different degrees during culture before permeabilization. From these results, it can be calculated that nondiffusible nucleotides are mainly contained in insulin secretory granules, with a small proportion in another, probably mitochondrial, compartment. Approximately 80% GTP and 30% GDP were present in the diffusible pool, and their relative proportions in the granular pool were only about 20% that of adenine nucleotides. Incubation of the cells in 20 instead of 2 mM glucose before permeabilization did not affect the nondiffusible pool, which indicates that the increase in the ATP/ADP ratio measured in intact cells occurred in the diffusible pool. Cytoplasmic nucleotide levels could be evaluated by subtracting the nondiffusible pool from the measurements in intact cells. It emerges that glucose induces large changes in the ATP/ADP ratio in the cytoplasmic pool, and that these changes are largely due to a fall in ADP.

The energy state of pancreatic B-cells may influence insulin release at several steps of stimulus-secretion coupling. By closing ATP-sensitive K+ channels (K(+)-ATP channels), a rise in the ATP/ADP ratio may regulate the membrane potential, and hence Ca2+ influx. It may also modulate the effectiveness of Ca2+ on its intracellular targets. To assess the existence of these two roles and determine their relative importance for insulin release, we tested the effects of azide, a mitochondrial poison, on mouse B-cell function under various conditions. During stimulation by glucose alone, when K(+)-ATP channels are controlled by cellular metabolism, azide caused parallel, concentration-dependent (0.5-5 mM), membrane repolarization, decrease in cytosolic Ca2+ concentration [Ca2+]i and inhibition of insulin release. When K(+)-ATP channels were closed pharmacologically (by tolbutamide in high glucose), azide did not repolarize the membrane or decrease [Ca2+]i, and was much less effective in inhibiting insulin release. A similar resistance to azide was observed when K(+)-ATP channels were opened by diazoxide, and high K+ was used to depolarize the membrane and raise [Ca2+]i. In contrast, azide similarly decreased ATP levels and increased ADP levels, thereby lowering the ATP/ADP ratio under all conditions. In conclusion, lowering the ATP/ADP ratio in B-cells can inhibit insulin release even when [Ca2+]i remains high. However, this distal step is much more resistant to a decrease in the energy state of B-cells than is the control of membrane potential by K(+)-ATP channels. Generation of the signal triggering insulin release, high [Ca2+]i, through metabolic control of membrane potential requires a higher global ATP/ADP ratio than does activation of the secretory process itself.