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M Prentki

Publications and source records attributed to M Prentki.

At least 55 records · Page 3Linked to original sources
PubMed1996

Are the beta-cell signaling molecules malonyl-CoA and cystolic long-chain acyl-CoA implicated in multiple tissue defects of obesity and NIDDM?

Widely held theories of the pathogenesis of obesity-associated NIDDM have implicated apparently incompatible events as seminal: 1) insulin resistance in muscle, 2) abnormal secretion of insulin, and 3) increases in intra-abdominal fat. Altered circulating or tissue lipids are characteristic features of obesity and NIDDM. The etiology of these defects is not known. In this perspective, we propose that the same metabolic events, elevated malonyl-CoA and long-chain acyl-CoA (LC-CoA), in various tissues mediate, in part, the pleiotropic alterations characteristic of obesity and NIDDM. We review the evidence in support of the emerging concept that malonyl-CoA and LC-CoA act as metabolic coupling factors in beta-cell signal transduction, linking fuel metabolism to insulin secretion. We suggest that acetyl-CoA carboxylase, which synthesizes malonyl-CoA, a "signal of plenty," and carnitine palmitoyl transferase 1, which is regulated by it, may perform as fuel sensors in the beta-cell, integrating the concentrations of all circulating fuel stimuli in the beta-cell as well as in muscle, liver, and adipose tissue. The target effectors of LC-CoA may include protein kinase C sub-types, complex lipid formation, genes encoding metabolic enzymes or transduction factors, and protein acylation. We support the concept that only under conditions in which both glucose and lipids are plentiful will the metabolic abnormality, which may be termed glucolipoxia, become apparent. If our hypothesis is correct that common signaling abnormalities in the metabolism of malonyl-CoA and LC-CoA contribute to altered insulin release and sensitivity, it offers a novel explanation for the presence of variable combinations of these defects in individuals with differing genetic backgrounds and for the fact that it has been difficult to determine whether one or the other is the primary event.

Acyl Coenzyme A↗
PubMed1995

Glucose, other secretagogues, and nerve growth factor stimulate mitogen-activated protein kinase in the insulin-secreting beta-cell line, INS-1.

The signaling pathways whereby glucose and hormonal secretagogues regulate insulin-secretory function, gene transcription, and proliferation of pancreatic beta-cells are not well defined. We show that in the glucose-responsive beta-cell line INS-1, major secretagogue-stimulated signaling pathways converge to activate 44-kDa mitogen-activated protein (MAP) kinase. Thus, glucose-induced insulin secretion was found to be associated with a small stimulatory effect on 44-kDa MAP kinase, which was synergistically enhanced by increased levels of intracellular cAMP and by the hormonal secretagogues glucagon-like peptide-1 and pituitary adenylate cyclase-activating polypeptide. Activation of 44-kDa MAP kinase by glucose was dependent on Ca2+ influx and may in part be mediated by MEK-1, a MAP kinase kinase. Stimulation of Ca2+ influx by KCl was in itself sufficient to activate 44-kDa MAP kinase and MEK-1. Phorbol ester, an activator of protein kinase C, stimulated 44-kDa MAP kinase by both Ca(2+)-dependent and -independent pathways. Nerve growth factor, independently of changes in cytosolic Ca2+, efficiently stimulated 44-kDa MAP kinase without causing insulin release, indicating that activation of this kinase is not sufficient for secretion. In the presence of glucose, however, nerve growth factor potentiated insulin secretion. In INS-1 cells, activation of 44-kDa MAP kinase was partially correlated with the induction of early response genes junB, nur77, and zif268 but not with stimulation of DNA synthesis. Our findings suggest a role of 44-kDa MAP kinase in mediating some of the pleiotropic actions of secretagogues on the pancreatic beta-cell.

Calcium↗
PubMed1994

Calcium regulation of immediate-early response genes.

A rise in intracellular Ca2+ concentration induces the transcription of a number of eukaryotic genes through transcription factors interacting with calcium response elements. Immediate-early response genes encode proteins that couple extracellular signals to phenotypic alterations by modulating the transcription rates of target genes. Since the activation of early response genes occurs within minutes, this class of genes has served as a paradigm for the understanding of the molecular mechanisms by which external signals are conveyed to the nucleus to induce changes in genetic programs. In this review, we outline the recent information which has been gained specifically on how the Ca2+ messenger system modulates early response gene expression. We also discuss some lines of research with the intent of linking closer Ca2+ homeostasis and gene expression studies which in the past have followed their own separate routes.

Animals↗
PubMed1994

Induction of c-fos in pituitary cells by thyrotrophin-releasing hormone and phorbol 12-myristate 13-acetate depends upon Ca2+ influx.

The role of cytosolic free Ca2+ ([Ca2+]i) in the induction of the immediate early gene c-fos by TRH or by phorbol 12-myristate 13-acetate (PMA) was studied in the clonal pituitary cell line GH4C1. It was found that c-fos mRNA levels were rapidly and transiently increased by TRH at physiological concentrations (1-100 nM). The effect of TRH was dependent on a rise in [Ca2+]i, and TRH stimulation of Ca2+ influx was essential for c-fos induction. Cell depolarization with K+, which produces a [Ca2+]i rise by soliciting Ca2+ influx via voltage-gated Ca2+ channels, was insufficient to induce c-fos. Blockade or downregulation of protein kinase C (PKC) strongly attenuated TRH stimulation of c-fos expression. Direct stimulation of PKC by PMA raised c-fos mRNA levels, but only under conditions permitting Ca2+ influx. We conclude that TRH induces c-fos mRNA by a mechanism dependent on PKC activation and on Ca2+ influx. The essential role of Ca2+ influx for PMA stimulation of c-fos mRNA suggests a novel pathway linking PKC stimulation to early gene expression.

Alkaloids↗
PubMed1993

Glucose regulates acetyl-CoA carboxylase gene expression in a pancreatic beta-cell line (INS-1).

Acetyl-CoA carboxylase (ACC) catalyzes the production of malonyl-CoA which may act as a metabolic coupling factor in nutrient-induced insulin release. We have studied the long term regulation of ACC by nutrients using the cell line INS-1. Glucose, from 5 to 20 mM, elicited a 15-fold increase in ACC mRNA. The effect was detected after 4 h and reached a maximum by 24 h. ACC protein accumulation followed that of ACC mRNA, and glucose did not modify the half-life of the ACC transcript. Glucose caused a dose-dependent rise in the glucose 6-phosphate content of INS-1 cells. 2-Deoxyglucose, which is phosphorylated by glucokinase but is not further metabolized, induced ACC mRNA. The effect of glucose was blocked by the glucokinase inhibitors mannoheptulose and glucosamine and was not mimicked by the 3-O-methyl or 6-deoxy analogues of glucose, which are not phosphorylated. Activation of the Ca2+, cAMP, and C-kinase pathways with high K+, forskolin, and phorbol 12-myristate 13 acetate, respectively, caused insulin release but not ACC mRNA induction. Basal insulin release, at 5 mM glucose, correlated with the ACC protein content of INS-1 cells preincubated for 24 h at various glucose concentrations. In conclusion, glucose is a potent inducer of the ACC gene, and glucose 6-phosphate may mediate its effect. Different signaling systems mediate the action of glucose on insulin release and ACC gene expression. The data strengthen the view that ACC plays a pivotal role in nutrient-induced insulin release.

Acetyl-CoA Carboxylase↗
PubMed1993

Intracellular Ca2+ and the regulation of early response gene expression in HL-60 myeloid leukemia cells.

To gain direct insight into the action of the second messenger Ca2+ on transcriptional regulation, we have developed an intact cell model in which the intracellular free Ca2+ concentration ([Ca2+]i) can be measured, set, and varied at any level within the physiological range and in which the expression of early response genes is assayed in parallel. Using promyelocytic HL-60 cells, we have observed an exquisite sensitivity to Ca2+ of c-fos, c-jun, and zif268 mRNA accumulation, since early and maximal inductions were observed at 200-300 nM [Ca2+]i. At early times (10-20 min), the [Ca2+]i dose dependence of c-fos transcription and mRNA accumulation displayed a bell shape since c-fos expression was barely modified at high (700-1,250 nM) [Ca2+]i. The threshold [Ca2+]i concentration for prolonged (60 min) c-fos mRNA accumulation was greater than 200 nM. This indicates that the quantitative effects of Ca2+ on a given gene can vary markedly as a function of both the [Ca2+]i concentration and the duration of stimulation. Strikingly, a [Ca2+]i perturbation of only 1 min was sufficient for full induction of c-fos and zif268 transcripts. This demonstrates that a transient perturbation of [Ca2+]i has long term effects on gene expression. The half-life of c-fos mRNA (16 min) was unaltered by Ca2+. Nuclear run-on analysis of the distribution of RNA polymerase II along the c-fos locus indicated that Ca2+ promotes a small increase in transcriptional initiation and a pronounced relief of a block to transcriptional elongation beyond intron 1. The extreme sensitivity to [Ca2+]i, in terms of both the length of time and the dose of [Ca2+]i required for maximal gene induction, demonstrates that Ca2+ is a major physiological regulator of early response gene expression. In addition, the results indicate that a c-fos intragenic element is the main target of Ca(2+)-regulated transcriptional activation.

Calcium↗
PubMed1992

Acyl-CoA esters modulate intracellular Ca2+ handling by permeabilized clonal pancreatic beta-cells.

Cytosolic free Ca2+ rises in pancreatic beta-cells in response to glucose stimulation and is part of the coupling to insulin secretion. This study evaluates a possible role for cytosolic long chain acyl-CoA esters in modulating Ca2+ handling by clonal beta-cells (HIT). Intact cells incubated with 20 microM free palmitic acid exhibited a 40% decrease in basal cytosolic free Ca2+. In contrast, acyl-CoA esters, up to a chain length of 16, but not the corresponding fatty acids, significantly lowered the Ca2+ set point maintained by cells permeabilized with saponin. The maximum response to the various acyl-CoA esters increased with increasing chain length, with no differences in the half-maximally effective concentration of 0.5 microM. Long chain acyl-CoA esters caused a 40-50% increase in 45Ca2+ influx into a non-mitochondrial pool in the permeabilized HIT cells, consistent with a stimulatory effect on the endoplasmic reticulum Ca(2+)-ATPase activity, but did not affect inositol 1,4,5-trisphosphate-induced Ca(2+)-efflux. Thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase activity, blocked the decrease in the Ca2+ set point caused by acyl-CoA esters. The ability of acyl-CoA esters to lower the Ca2+ set point depended on the ATP/ADP ratio (or free ADP); the Ca2+ set point was lowered by 36 +/- 3.6% at an ATP/ADP ratio of 90 and by 14 +/- 1.9% at an ATP/ADP ratio of 7. Depletion of cellular protein kinase C did not prevent the acyl-CoA-induced lowering of the Ca2+ set point. These findings suggest that the increases in long chain acyl-CoA esters may play a role in restoring cytosolic free Ca2+ through activation of Ca(2+)-ATPases.

Acyl Coenzyme A↗
PubMed1992

Malonyl-CoA and long chain acyl-CoA esters as metabolic coupling factors in nutrient-induced insulin secretion.

Several approaches were used to test the hypothesis proposing a role for acyl-CoA esters in nutrient-induced insulin release (Prentki, M., and Matschinsky, F. M. (1987) Physiol. Rev. 67, 1185-1248; Corkey, B. E., Glennon, M. C., Chen, K. S., Deeney, J. T., Matschinsky, F. M., and Prentki, M. (1989) J. Biol. Chem. 264, 21608-21612). Exogenous saturated long chain fatty acids markedly potentiated glucose-induced insulin release and elevated long chain acyl-CoA esters in the clonal beta-cell line (HIT). The secretory action depended on the fatty acid chain length, occurred in the range 3-20 microM (free concentration of palmitate), and was reversible and inhibitable by the neuromodulator somatostatin. 2-Bromopalmitate, an inhibitor of carnitine palmitoyl transferase I, suppressed the oxidation of endogenous fatty acids and promoted release of insulin. Only the nutrients or the combination of nutrients that caused secretion elevated malonyl-CoA. The short-chain acyl-CoA profile of HIT cells stimulated by various nutrients was determined in the presence of the nonstimulatory fuel glutamine. Glucose and leucine each provoked similar changes in acyl-CoA compounds. Both secretagogues elevated malonyl-CoA 3-6-fold, whereas succinyl-CoA, free CoASH, acetyl-CoA, and the free CoASH to acetyl-CoA ratio remained unaltered. Furthermore, only when inhibition of fatty acid oxidation was associated with a rise in malonyl-CoA did the total (mitochondrial plus cytoplasmic) content of long chain acyl-CoA esters correlate inversely with insulin release promoted by various nutrients. The results are consistent with the concept that fuel stimuli cause a rise in malonyl-CoA which by inhibiting fatty acid oxidation increase cytosolic long chain acyl-CoA esters. These data provide further support for a model in which malonyl-CoA and long chain acyl-CoAs esters serve as metabolic coupling factors when pancreatic beta-cells are stimulated with glucose and other nutrient secretagogues.

Acyl Coenzyme A↗
PubMed1991

Oscillations in cytosolic free Ca2+, oxygen consumption, and insulin secretion in glucose-stimulated rat pancreatic islets.

Insulin secretion in the intact organism, and by the perfused pancreas and groups of isolated perifused islets, is pulsatile. We have proposed a metabolic model of glucose-induced insulin secretion in which oscillations in the ATP/ADP ratio drive alterations in metabolic and electrical events that lead to insulin release. A key prediction of our model is that metabolically driven Ca2+ oscillations will also occur. Using the fluorescent Ca2+ probe, fura 2, digital image analysis, and sensitive O2 electrodes, we investigated cytosolic free Ca2+ responses and O2 consumption in perifused rat islets that had been maintained in culture for 1-4 days. We found that elevated ambient glucose increased the average cytosolic free Ca2+ level, the ATP/ADP ratio, and oxygen consumption, as previously found in freshly isolated islets. Oscillatory patterns were obtained for Ca2+, O2 consumption, and insulin secretion in the presence of 10 and 20 mM glucose. Very low amplitude oscillations in cytosolic free Ca2+ were observed at 3 mM nonstimulatory glucose levels. Evaluation of the Ca2+ responses of a large series of individual islets, monitored by digital image analysis and perifused at both 3 and 10 mM glucose, indicated that the rise in glucose concentration caused more than a doubling of the average cytosolic free Ca2+ value and a 4-fold increase in the amplitude of the oscillations with little change in period. The pattern of Ca2+ change within the islets was consistent with recruitment of responding cells. The coexistence of oscillations with similar periods in insulin secretion, oxygen consumption, and cytosolic free Ca2+ is consistent with the model of metabolically driven pulsatile insulin secretion.

Adenine Nucleotides↗
PubMed1990

Somatostatin inhibition of Ca2(+)-induced insulin secretion in permeabilized HIT-T15 cells.

Somatostatin inhibited Ca2(+)-induced insulin secretion in permeabilized HIT-T15 cells, albeit with decreased sensitivity relative to intact cells. The inhibitory action required the presence of GTP, whereas GDP could not substitute for GTP. Pertussis-toxin treatment before cell permeabilization abolished the inhibition of secretion. Thus somatostatin, by activating a G-protein, interferes with exocytosis distal to the generation of soluble intracellular messengers.

Animals↗
PubMed1990

EGF receptor down-regulation attenuates ligand-induced second messenger formation.

Epidermal growth factor (EGF)-induced increases in cytosolic Ca2+ and inositol polyphosphate production were compared in a human hepatocellular carcinoma-derived cell line, PLC/PRF/5, and in an EGF receptor-overexpressing subline, NPLC/PRF/5. Formation of these second messengers was correlated to EGF receptor display at the cell surface by monitoring ligand-induced EGF receptor down-regulation. Both cell lines exhibited a strikingly similar cytosolic Ca2+ increase upon exposure to EGF. The initial inositol phosphate responses were also similar in the two cell lines; inositol 1,4,5-trisphosphate increased within 10-15 s and returned to prestimulatory values after 2 min in both cell lines, while inositol tetrakisphosphate and inositol 1,3,4-trisphosphate were elevated after a 2-min exposure to EGF. At later times the responses were markedly different; NPLC/PRF/5 cells exhibited prolonged production of inositol 1,3,4-trisphosphate and inositol tetrakisphosphate (maximum at 1-3 h) but PLC/PRF/5 cells showed decreased levels of these isomers after 10 min and a return to basal values by 1 h. Exposure of PLC/PRF/5 cells to EGF caused a progressive decrease in the amount of EGF receptor at the cell surface whereas such treatment did not change the surface receptor levels in NPLC/PRF/5 cells. Kinetic analysis of EGF receptor down-regulation showed that receptor internalization was rapid enough to account for the transient nature of the inositol phosphate response in PLC/PRF/5 cells. Thus, the divergent patterns of signaling exhibited by the two cell lines may reflect differences in the efficiency of EGF-induced down-regulation of surface receptors.

Angiotensin II↗
PubMed1989

A role for malonyl-CoA in glucose-stimulated insulin secretion from clonal pancreatic beta-cells.

To gain insight into the relationship between acyl coenzyme A (CoA) esters and glucose-induced insulin release, acyl-CoA profiles were determined in clonal pancreatic beta-cells (HIT). A high sensitivity high performance liquid chromatography method was used to measure malonyl, succinyl, beta-hydroxy beta-methylglutaryl and acetyl-CoA esters and free CoASH. Malonyl-CoA content increased more than 3-fold following exposure of HIT cells to 10 mM glucose. The rise in malonyl-CoA, which preceded insulin secretion, was evident 2 min after exposure to glucose and was sustained for at least 30 min. The increase in malonyl-CoA was associated with inhibition of fatty acid oxidation, increased de novo lipid synthesis and a rise in diacylglycerol content. Succinyl-CoA levels, which may reflect anaplerotic influx into the citric acid cycle, were elevated in the presence of glucose. The concentration of acetyl-CoA and the ratio of free CoASH to acetyl-CoA was unchanged. The data are consistent with a metabolic model in which malonyl-CoA mediates the switch from fatty acid catabolism to lipid synthesis during glucose stimulation of beta-cells. We suggest that these changes in lipid metabolism, by leading to increased diacylglycerol synthesis or protein acylation could play a pivotal role in the regulation of the sustained phase of insulin secretion.

Acyl Coenzyme A↗
PubMed1989

Selective activation of Ca2+ influx by extracellular ATP in a pancreatic beta-cell line (HIT).

The action of exogenous ATP on cytoplasmic free Ca2+ ([Ca2+]i) was studied in insulin secreting cells using fura-2. Stimulation of clonal pancreatic beta-cells (HIT) with ATP (range 2-20 microM) evoked a sustained elevation in [Ca2+]i. ATP selectively promoted Ca2+ influx and not Ca2+ mobilization since (1) the effect required external Ca1+ and (2) was observed in cells in which internal stores were depleted with ionomycin (3) the rate of Mn2+ influx, measured as the quenching of the fura-2 signal, was accelerated by ATP. The action of ATP was unaffected by the voltage-sensitive Ca2+ channel blockers nifedipine and verapamil as well as by a depolarizing concentration of K+. The effect on [Ca2+]i was highly specific for ATP since AMP, ADP, adenosine 5'-[gamma-thio]triphosphate, adenosine 5'-[beta, gamma-methylene]triphosphate, GTP and adenosine were ineffective. In normal pancreatic islet cells, both exogenous ATP (range 0.2-2 microM) and ADP induced a transient Ca2+ elevation that did not require external Ca2+. The nucleotide specificity of the effect on [Ca2+]i suggests that ATP activates P2 gamma purinergic receptors in normal beta-cells. Thus, ATP evokes a Ca2+ signal in clonal HIT cells and normal islet cells by different transducing systems involving distinct purinoreceptors. A novel mechanism for increasing [Ca2+]i by extracellular ATP is reported in HIT cells, since the nucleotide specificity and the selective activation of Ca2+ influx without mobilization of internal Ca2+ stores cannot be explained by mechanisms already described in other cell systems.

Adenine Nucleotides↗
PubMed1988

Cell-specific patterns of oscillating free Ca2+ in carbamylcholine-stimulated insulinoma cells.

The effect of the muscarinic agonist carbamylcholine on cytoplasmic Ca2+ concentration ([Ca2+]i was examined at the single cell level in clonal pancreatic beta-cells (HIT). Cells were loaded with the indicator dye fura 2, and [Ca2+]i was measured by microfluorimetry. Carbamylcholine induced changes in Ca2+ that differed from cell to cell and provoked in some cells oscillatory Ca2+ fluctuations. During a transient, free Ca2+ rose to a peak within 1-3 s. The frequency of the oscillations increased with agonist concentration. Oscillations in [Ca2+]i occurred in the absence of external Ca2+. When cells were perifused for a sufficient period of time without carbamylcholine, near identical Ca2+ responses were elicited in each cell by successive applications of the agonist. Thus, individual cells displayed characteristic and reproducible Ca2+ responses with respect to amplitude, frequency, and shape of the transients as well as latency in onset of the initial Ca2+ rise. We propose that the biological response to a Ca2+ agonist in a given cell is not only determined by the frequency and amplitude of Ca2+ oscillations but is governed by the unique pattern of the Ca2+ signal of each cell, which may be termed "Ca2+ fingerprint."

Adenoma, Islet Cell↗
PubMed1988

Epidermal growth factor-induced increases in inositol trisphosphates, inositol tetrakisphosphates, and cytosolic Ca2+ in a human hepatocellular carcinoma-derived cell line.

A human hepatocellular carcinoma-derived cell line, PLC/PRF/5, was examined for its ability to respond to epidermal growth factor (EGF) exposure with increased phosphatidylinositol 4,5-bisphosphate hydrolysis. Upon addition of EGF (25 ng/ml), a rapid (10-15 s) but transient increase in Ins(1,4,5)P3 levels and large, prolonged (2 min) increases in Ins(1,3,4,5)P4 and Ins(1,3,4)P3 levels were detected. Increases in cytosolic Ca2+ were observed after a 10 to 20 s lag, reaching peak value at 1 min, and remaining elevated for 10 min. The initial burst of cytosolic Ca2+ occurred in the absence of extracellular Ca2+ and probably reflects mobilization of intracellular Ca2+ stores. In cells pretreated with EGTA, the sustained component of the Ca2+ response was not observed. Comparison of the inositol phosphate and Ca2+ responses of PLC/PRF/5 cells to responses reported in other cell types indicates that this cell line is a good model for EGF action in liver.

Calcium↗
PubMed1988

Regulation of steady-state free Ca2+ levels by the ATP/ADP ratio and orthophosphate in permeabilized RINm5F insulinoma cells.

Stimulation of insulin secretion in the pancreatic beta-cell by a fuel such as glucose requires the metabolism of the fuel and is accompanied by increases in oxygen consumption and intracellular free Ca2+. A very early signal for these events could be a decrease in the cytosolic ATP/ADP ratio due to fuel phosphorylation. To test this hypothesis the regulation of free Ca2+ was evaluated in permeabilized RINm5F insulinoma cells that sequester Ca2+ and maintain a low medium free Ca2+ concentration (set point), between 100 and 200 nM, in the presence of Mg2+ and ATP. ATP, creatine, creatine phosphate, and creatine phosphokinase were added to the media to achieve various constant ratios of ATP/ADP. Free Ca2 was monitored using fura-2. The results demonstrated that the steady-state free Ca2+ concentration varied inversely with the ATP/ADP ratio and orthophosphate (Pi) levels. In contrast, no correlation between free Ca2+ and the phosphorylation potential (ATP/ADP.Pi) was found. Regulation of the Ca2+ set point by the ATP/ADP ratio was observed at ratios between 5 and 50 and at Pi concentrations between 1 and 7 mM, irrespective of whether mitochondria were participating in Ca2+ sequestration or were inhibited. Increasing the ATP/ADP ratio stimulated Ca2+ uptake by the nonmitochondrial pool but did not modify Ca2+ efflux. Glucose 6-phosphate (1 mM) had no effect on the Ca2+ set point. The data suggest that variations in the cytosolic ATP/ADP ratio induced by fuel stimuli may regulate Ca2+ cycling across nonmitochondrial compartments and the plasma membrane by modulating the activity of Ca2+ -ATPases. A mechanism linking fuel metabolism and cytosolic ATP/ADP ratio to activation of the Ca2+ messenger system in pancreatic beta-cells is proposed.

Adenoma, Islet Cell↗
PubMed1988

Linked oscillations of free Ca2+ and the ATP/ADP ratio in permeabilized RINm5F insulinoma cells supplemented with a glycolyzing cell-free muscle extract.

We show in the accompanying paper that the steady-state level of free Ca2+ maintained by the organelles of permeabilized RINm5F insulinoma cells varies inversely with the ATP/ADP ratio when this ratio is set by addition of creatine phosphokinase and fixed ratios of creatine to creatine phosphate. We, therefore, asked whether acute cyclic alterations in the cytosolic ATP/ADP ratio in the range known to modulate O2 consumption might be involved in regulating the physiological activity of Ca2+ -ATPases and the cytosolic free Ca2+ level. To explore this hypothesis we combined two experimental systems: 1) permeabilized RINm5F insulinoma cells that can maintain a low medium Ca2+ concentration and 2) a cell-free extract of rat skeletal muscle that spontaneously exhibits oscillatory behavior of glycolysis and linked oscillations in the ATP/ADP ratio, when provided with glucose. The free Ca2+ level maintained by the permeabilized cells oscillated in phase with the glycolytic oscillations and correlated closely with the ATP/ADP ratio but not with glucose 6-phosphate, fructose 6-phosphate, orthophosphate, or pH. When glucokinase replaced hexokinase as the glucose phosphorylating enzyme, Ca2+ oscillations were induced by increasing the glucose concentration from 2 to 8 mM. The results demonstrate a link between metabolite changes and free Ca2+ levels in a reconstituted physiological system. They support a model in which oscillations in glycolysis and the ATP/ADP ratio may cause oscillations in cytosolic free Ca2+, beta-cell electrical activity, and insulin release.

Adenoma, Islet Cell↗