Biomedical subjects

B E Corkey

Publications and source records attributed to B E Corkey.

At least 55 records · Page 3Linked to original sources

PubMed1996

Temporal sequence of metabolic and ionic events in glucose-stimulated clonal pancreatic beta-cells (HIT).

Stimulation of insulin release by glucose requires increased metabolism of glucose and a rise in cytosolic free Ca2+ in the pancreatic beta-cell. It is accompanied by increases in respiratory rate, pyridine and flavin nucleotide reduction state, intracellular pH and the ATP/ADP ratio. To test alternative proposals of the regulatory relationships among free Ca2+, mitochondrial metabolism and cellular energy state, we determined the temporal sequence of these metabolic and ionic changes following addition of glucose to clonal pancreatic beta-cells (HIT). Combined measurements of the native fluorescence of reduced pyridine nucleotides and oxidized flavin, intracellular pH, and free Ca2+ were performed together with simultaneous measurement of O2 tension or removal of samples for assay of the ATP/ADP ratio. The initial changes were detected in three phases. First, decreases occurred in the ATP/ADP ratio (<3 s) and increases in pyridine (2 +/- 1 s) and flavin (2 +/- 1 s) nucleotide reduction. Next, increases in the O2 consumption rate (20 +/- 5 s), the ATP/ADP ratio (29 +/- 12 s) and internal pH (48 +/- 5 s) were observed. Finally, cytosolic free Ca2+ rose (114 +/- 10 s). Maximal changes in the ATP/ADP ratio, O2 consumption and pyridine and flavin nucleotide fluorescence preceded the beginning of the Ca2+ change. These relationships are consistent with a model in which phosphorylation of glucose is the initial event which generates the signals that lead to an increase in respiration, a rise in the ATP/ADP ratio and finally influx of Ca2+. Our results indicate that Ca2+ does not function as the initiator of increased mitochondrial respiration.

Adenosine Diphosphate↗

PubMed1996

Temporal patterns of changes in ATP/ADP ratio, glucose 6-phosphate and cytoplasmic free Ca2+ in glucose-stimulated pancreatic beta-cells.

Closure of ATP-sensitive K+ (K(ATP)) channels is part of the stimulus-secretion coupling mechanism in the pancreatic beta-cell, leading to membrane depolarization and influx of Ca2+ through voltage-sensitive L-type Ca2+ channels. The elevated ATP/ADP ratio seen in the presence of high levels of glucose has been postulated to mediate the glucose-induced closure of the K(ATP) channels and rise in cytoplasmic free Ca2+ concentration ([Ca2+]i), or alternatively to be a consequence of activation of mitochondrial dehydrogenases by the increase in [Ca2+]i. To distinguish between these two possibilities, the time course of the change in the ATP/ADP ratio was determined in comparison with that of [Ca2+]i. We here show that a severalfold rise in the ATP/ADP ratio occurs rapidly on stimulation of suspensions of mouse pancreatic beta-cells with glucose. The change in the ATP/ADP ratio is an early event that begins within 20-40 s and precedes the rise in [Ca2+]i. The temporal relationship indicates that the adenine nucleotide changes cannot be a consequence of the [Ca2+]i changes and may indeed be the connecting link between glucose metabolism and [Ca2+]i changes. When the cells were sequentially treated with high glucose concentration, clonidine and finally high extracellular Ca2+ concentration to induce synchronized oscillations in [Ca2+]i in the cell suspension, corresponding oscillations in the ATP/ADP ratio were observed. Glucose 6-phosphate levels oscillated out of phase with the ATP/ADP ratio. These results support the hypothesis that the Ca2+ oscillations previously observed in glucose-stimulated single islets or beta-cells may reflect oscillations in the ATP/ADP ratio that accompany oscillatory glycolysis.

Adenosine Diphosphate↗

PubMed1996

Malonyl coenzyme A and adiposity in the Dahl salt-sensitive rat: effects of pioglitazone.

These studies were designed to assess the effects of pioglitazone, a new oral antidiabetic agent that acts by improving insulin sensitivity, on blood pressure, plasma and tissue lipids, and insulin resistance in the Dahl salt-sensitive (Dahl-S) rat. Reaven et al had reported that male Dahl-S rats are moderately hyperinsulinemic and insulin-resistant. This was of particular interest since these rats are not obese but are hypertriglyceridemic, and on a high-salt diet they become hypertensive. In the current study, male Sprague-Dawley control and Dahl-S rats were compared when fed standard chow of high-fat, high-sucrose (HFHS) diets with or without pioglitazone (20 mg/kg body weight/d) for 3 weeks. On the standard chow diet, Dahl-S rats were hypertriglyceridemic and had high tissue levels of malonyl coenzyme A ([CoA] Dahl-S 5.0 v control 3.3 nmol/g in muscle, and Dahl-S 15.6 v control 10.7 nmol/g in liver); however, they were not hyperinsulinemic. Pioglitazone therapy decreased both malonyl CoA and plasma triglycerides toward control values, but had no effect on plasma insulin levels. On the HFHS diet, both groups became glucose-intolerant and hyperinsulinemic; however, the hyperinsulinemia was greater and more sustained in Dahl-S rats. In addition, the HFHS diet appeared to increase the mass of retroperitoneal fat in the Dahl-S but not in the control group. Treatment with pioglitazone decreased retroperitoneal fat, but as reported previously, it increased the mass of the epididymal fat pad. The results suggest that the hypertriglyceridemia of the Dahl-S rat is associated with an increase in the concentration of malonyl CoA in both liver and muscle. They also show that pioglitazone reverses both of these abnormalities independently of its effect on plasma insulin. Whether these high levels of malonyl CoA predispose the Dahl-S rat to hyperinsulinemia and possibly obesity when placed on a HFHS diet remains to be determined.

Adipose Tissue↗

PubMed1996

Glucose-induced oscillatory insulin secretion in perifused rat pancreatic islets and clonal beta-cells (HIT).

Normal insulin secretion is oscillatory in vivo and from groups of perifused islets. Stimulation of rat islets with different glucose concentrations gave insulin oscillations of similar period (5-8 min) but increasing amplitude. It has been assumed that oscillatory secretion is due to oscillations in intracellular free Ca2+, as seen in single islets and single pancreatic beta-cells. However, when islets were perifused with diazoxide and high KCl to maintain high intracellular free Ca2+, insulin oscillations of similar amplitude and period still occurred on glucose stimulation, although superimposed on elevated basal secretion. Several likely possibilities for a diffusible synchronizing factor were tested, including pyruvate, lactate, ATP, and insulin itself; nevertheless, perifusion with high concentrations of these did not prevent insulin oscillations. Clonal pancreatic beta-cells (HIT) and dissociated islets also exhibited oscillatory insulin secretion, but with the 5- to 8-min period oscillations superimposed on 15- to 20-min period oscillations. These results indicate that the mechanisms for generating and synchronizing insulin oscillations reside in the beta-cell, although the structure of the islet may modulate the oscillation pattern.

Animals↗

PubMed1996

Evidence for an anaplerotic/malonyl-CoA pathway in pancreatic beta-cell nutrient signaling.

A metabolic model of fuel sensing has been proposed in which malonyl-CoA and long-chain acyl-CoA esters may act as coupling factors in nutrient-induced insulin release (Prentki M, Vischer S, Glennon MC, Regazzi R, Deeney J, Corkey BE: Malonyl-CoA and long chain acyl-CoA esters as metabolic coupling factors in nutrient-induced insulin secretion. J Biol Chem 267:5802-5810, 1992). To gain further insight into the control of malonyl-CoA content in islet tissue, we have studied the short- and long-term regulation of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) in the beta-cell. These enzymes catalyze the formation of malonyl-CoA and its usage for de novo fatty acid biogenesis. ACC mRNA, protein, and enzymatic activity are present at appreciable levels in rat pancreatic islets and clonal beta-cells (HIT cells). Glucose addition to HIT cells results in a marked increase in ACC activity that precedes the initiation of insulin release. Fasting does not modify the ACC content of islets, whereas it markedly downregulates that of lipogenic tissues. This indicates differential regulation of the ACC gene in lipogenic tissues and the islets of Langerhans. FAS is very poorly expressed in islet tissue, yet ACC is abundant. This demonstrates that the primary function of malonyl-CoA in the beta-cells is to regulate fatty acid oxidation, not to serve as a substrate for fatty acid biosynthesis. The anaplerotic enzyme pyruvate carboxylase, which allows the replenishment of citric acid cycle intermediates needed for malonyl-CoA production via citrate, is abundant in islet tissue. Glucose causes an elevation in beta (HIT)-cell citrate that precedes secretion, and only those nutrients that can elevate citrate induce effective insulin release. The results provide new evidence in support of the model and explain why malonyl-CoA rises markedly and rapidly in islets upon glucose stimulation: 1) glucose elevates citrate, the precursor of malonyl-CoA; 2) glucose enhances ACC enzymatic activity; and 3) malonyl-CoA is not diverted to lipids. The data suggest that ACC is a key enzyme in metabolic signal transduction of the beta-cell and provide evidence for the concept that an anaplerotic/malonyl-CoA pathway is implicated in insulin secretion.

Acetyl-CoA Carboxylase↗

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

Phosphofructokinase isozymes in pancreatic islets and clonal beta-cells (INS-1).

Normal insulin secretion is oscillatory in vivo, and the oscillations are impaired in type II diabetes. We and others have shown oscillations in insulin secretion from isolated perifused islets stimulated with glucose, and in this study we show oscillations in insulin secretion from the glucose-sensitive clonal beta-cell line INS-1. We have proposed that the oscillatory insulin secretion may be caused by spontaneous oscillations of glycolysis and the ATP:ADP ratio in the beta-cell, analogous to those seen in glycolyzing muscle extracts. The mechanism of the latter involves autocatalytic activation of the key regulatory enzyme, phosphofructokinase (PFK), by its product fructose 1,6-bisphosphate (F16BP). However, of the three PFK subunit isoforms (M-[muscle], L-[liver], and C-type, predominant in fibroblasts), only M-type is activated by micromolar F16BP at near-physiological conditions. We therefore studied PFK isoforms in the beta-cell. Western analysis of PFK subunits in isolated rat islets and INS-1 cells showed the presence of M-type, as well as C-type and perhaps lesser amounts of L-type. Kinetic studies of PFK activity in INS-1 cell extracts showed strong activation by micromolar concentrations of F16BP at near-physiological concentrations of ATP (several millimolar) and AMP and fructose 6-phosphate (micromolar), indicative of the M-type isoform. Activation by submicromolar concentrations of fructose 2,6-bisphosphate (F26BP) and potent inhibition by citrate were also observed. The F16BP-stimulatable activity was about one-half of the F26BP-stimulatable activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗

PubMed1994

Long chain acyl coenzyme A and signaling in neutrophils. An inhibitor of acyl coenzyme A synthetase, triacsin C, inhibits superoxide anion generation and degranulation by human neutrophils.

Ligand-initiated activation of neutrophils triggers O2- generation, degranulation, phospholipid remodeling, and release of fatty acids such as arachidonate, oleate, and palmitate. Long chain acyl-CoA synthetase converts free fatty acids to acyl-CoA esters; a role for acyl-CoA esters as positive modulators of neutrophil functions is proposed. Physiologically relevant concentrations (1-10 microM) of acyl-CoA esters such as palmitoyl-CoA, enhanced O2- generation triggered by fMet-Leu-Phe or guanosine 5'-O-(thiotriphosphate) (GTP gamma S) but did not act as a trigger per se. Triacsin C, an inhibitor of acyl-CoA synthetase, inhibited fMet-Leu-Phe-elicited O2- generation and degranulation in a concentration-dependent manner. Triacsin C inhibited O2- generation elicited by fMet-Leu-Phe and GTP gamma S in electroporated neutrophils, indicating that acyl-CoA acted downstream from the receptor. Palmitoyl-CoA reversed the Triacsin C-induced inhibition of O2- generation. fMet-Leu-Phe elicited a prompt increase in total long chain acyl-CoA esters. Arachidonoyl-CoA and oleoyl-CoA were elevated 5 s after addition of fMet-Leu-Phe, while palmitoyl-CoA was not elevated until 60 s. Triacsin C inhibited fMet-Leu-Phe-initiated increases in arachidonoyl-CoA, oleoyl-CoA, and palmitoyl-CoA. These results suggest a role for acyl-CoA esters in regulating activation of O2- generation and degranulation at the G protein or subsequent step(s).

Acyl Coenzyme A↗

PubMed1994

Rab3A effector domain peptides induce insulin exocytosis via a specific interaction with a cytosolic protein doublet.

A key protein involved in the regulated exocytotic mechanism in neuroendocrine cells is the GTP-binding protein, Rab3A. Rab3A is thought to mediate exocytosis by an interaction of its effector domain with a putative effector protein. We demonstrate here that Rab3A effector domain peptides specifically stimulated insulin exocytosis in electroporated insulin-secreting cells (K0.5 activation, 6-8 microM) in a Ca(2+)-independent manner, although in the presence of Ca2+ insulin exocytosis was further potentiated. By using a 125I-radiolabeled photoactivated cross-linking Rab3A effector domain peptide, we identified a cytosolic protein doublet (REEP-1 and REEP-2), which specifically interacted with the Rab3A effector domain. Competitive inhibition studies revealed this protein-protein interaction to be at a concentration equivalent to that required for Rab3A effector domain peptides to trigger insulin exocytosis (Ki, 6-8 microM). Furthermore, under basal secretory conditions REEP-1 and -2 were membrane-associated, but upon stimulation of exocytosis they were released into a cytosolic fraction. Our results suggest that REEP-1 and -2 are part of the regulated exocytotic machinery, and their dissociation upon stimulation of hormone release (likely from a protein complex) may be essential to the mechanism that triggers regulated exocytosis in pancreatic beta-cells.

Amino Acid Sequence↗

PubMed1994

Changes in internal pH caused by movement of fatty acids into and out of clonal pancreatic beta-cells (HIT).

Cells require a constant influx of free fatty acids for lipid resynthesis and metabolic energy. Fatty acids also act as second messengers and modulate channel activities. In the pancreatic beta-cell, fatty acids have both acute and chronic effects on insulin secretion. We show that the addition of fatty acid to pancreatic beta-cells in vitro produces a persistent decrease in intracellular pH, which begins immediately after the addition of fatty acid and has an exponential time course with t1/2 approximately 60 s. The pH drop can be largely reversed by the addition of albumin. The observed pH effect can be explained by passive diffusion ("flip-flop") of un-ionized fatty acid across the plasma membrane. Acidification by a fatty acid dimer and alkalinization by an alkylamine also favor the flip-flop mechanism of transport rather than a protein-mediated mechanism. Our method provides for the first time a real-time measurement of fatty acid import into cells. The significant pH change may be important in mediating some of the regulatory effects of fatty acid, such as inhibition of glycolysis.

Biological Transport↗

PubMed1994

Glucose-stimulated increase in cytoplasmic pH precedes increase in free Ca2+ in pancreatic beta-cells. A possible role for pyruvate.

The temporal relationship of glucose-induced increases in cytoplasmic pH (pHi) and cytoplasmic free Ca2+ was studied in single mouse pancreatic beta-cells and suspensions of clonal beta-cells (HIT). In both preparations of cells the increase in pHi preceded the cytoplasmic free Ca2+ increase. Therefore the alkalinization cannot be a consequence of the Ca2+ influx. A potential metabolic mechanism for the increase in pHi, involving stimulation of pyruvate transport and oxidation, was demonstrated in a model system of liver mitochondria incubated with pyruvate, ATP, and hexokinase to which glucose was then added to initiate ATP use. The involvement of this mechanism in beta-cells is suggested by the observation that the alkalinization was prevented in most cells by incubation with 3-hydroxycyanocinnamate, a mitochondrial pyruvate transport inhibitor. On the other hand, the inhibited cells exhibited normal Ca2+ responses to glucose stimulation. This indicates that neither pyruvate metabolism nor the alkalinization is of critical importance for the Ca2+ signal, though pyruvate oxidation or its metabolites may be important in downstream regulation of secretion.

Animals↗

PubMed1994

Cytokines inhibit fatty acid oxidation in isolated rat hepatocytes: synergy among TNF, IL-6, and IL-1.

We have developed an in vitro rat hepatocyte model in which cytokines inhibit fatty acid oxidation. Cytokine administration resulted in decreased fatty acid oxidation, ketone body production and acetyl CoA/CoA ratios. The inhibitory effects of TNF on fatty acid oxidation were enhanced by either IL-1 or IL-6. TNF (20 U/ml) + IL-6 (30 ng/ml) produced maximal inhibition, whereas IL-1 enhanced inhibition at submaximal TNF concentrations. The key to our model is that substrate input into the tricarboxylic acid cycle in the form of either alanine or pyruvate was required for cytokine mediated inhibition of fatty acid oxidation. Alanine or pyruvate may serve as a source for increased production of malonyl CoA, a potent inhibitor of fatty acid oxidation. We hypothesize that cytokines cause an inappropriate switch from fatty acid oxidation to fatty acid synthesis which has serious consequences for energy levels in the liver and can lead to end organ failure.

Acetyl Coenzyme A↗

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↗

PubMed1992

Two sites for adenine-nucleotide regulation of ATP-sensitive potassium channels in mouse pancreatic beta-cells and HIT cells.

ATP-inhibited potassium channels (K(ATP)) were studied in excised, inside-out patches from cultured adult mouse pancreatic beta-cells and HIT cells. In the absence of ATP, ADP opened K(ATP) channels at concentrations as low as 10 microM and as high as 500 microM, with maximal activation between 10 and 100 microM ADP in mouse beta-cell membrane patches. At concentrations greater than 500 microM, ADP inhibited K(ATP) channels while 10 mM virtually abolished channel activity. HIT cell channels had a similar biphasic response to ADP except that more than 1 mM ADP was required for inhibition. The channel opening effect of ADP required magnesium while channel inhibition did not. Using creatine/creatine phosphate solutions with creatine phosphokinase to fix ATP and ADP concentrations, we found substantially different K(ATP)-channel activity with solutions having the same ATP/ADP ratio but different absolute total nucleotide levels. To account for ATP-ADP competition, we propose a new model of channel-nucleotide interactions with two kinds of ADP binding sites regulating the channel. One site specifically binds MgADP and increases channel opening. The other, the previously described ATP site, binds either ATP or ADP and decreases channel opening. This model very closely fits the ADP concentration-response curve and, when incorporated into a model of beta-cell membrane potential, increasing ADP in the 10 and 100 microM range is predicted to compete very effectively with millimolar levels of ATP to hyperpolarize beta-cells. The results suggest that (i) K(ATP)-channel activity is not well predicted by the "ATP/ADP ratio," and (ii) ADP is a plausible regulator of K(ATP) channels even if its free cytoplasmic concentration is in the 10-100 microM range as suggested by biochemical studies.

Adenosine Diphosphate↗

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↗

PubMed1991

Protein kinase C isotypes and signaling in neutrophils. Differential substrate specificities of a translocatable calcium- and phospholipid-dependent beta-protein kinase C and a phospholipid-dependent protein kinase which is inhibited by long chain fatty acyl coenzyme A.

Neutrophils possess a classical Ca2+, phosphatidyl serine (PS) and diglyceride (DG)-dependent protein kinase C (beta-PKC) which was translocatable from cytosol to membrane in response to elevated Ca2+ in the physiologic range or to pretreatment with phorbol myristate acetate (PMA). The translocatable beta-PKC was purified from neutrophil membranes prepared in the presence of Ca2+, eluted with EGTA and subjected to hydroxyapatite chromatography. An 80-kDa protein possessing Ca/DG/PS-dependent histone phosphorylating activity was recognized by a monoclonal antibody to beta-PKC but not to alpha-PKC or gamma-PKC. A cytosolic kinase activity remaining after Ca(2+)-induced translocation of beta-PKC was dependent on PS and DG but did not require Ca2+. This novel Ca(2+)-independent, PS/DG-dependent kinase, termed nPKC, eluted from hydroxyapatite between alpha-PKC and beta-PKC, ran as a 76-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was reactive to a polyclonal consensus antibody but not to monoclonal antibodies to alpha-PKC, beta-PKC, or gamma-PKC. Long chain fatty acyl-CoA, but not the corresponding free fatty acids, inhibited nPKC in the 1-10 microM range. The chemotactic peptide fMet-Leu-Phe triggered prompt but transient increases in neutrophil long chain fatty acid acyl-CoA, suggesting that nPKC is regulated by fatty acyl-CoA as well as DG during neutrophil activation. Purified beta-PKC phosphorylated a number of cytosolic proteins in a Ca(2+)-dependent manner, including a major 47-kDa cytosolic protein, which may be implicated in superoxide anion generation. In contrast, nPKC did not phosphorylate the 47-kDa protein, but phosphorylated numerous cytosolic proteins in a Ca(2+)-independent manner, including a 66-kDa protein which was not phosphorylated by beta-PKC. Differences in location, substrate specificity, and cofactor dependence between nPKC and beta-PKC suggest these kinases may play selective roles in the activation sequence of the neutrophil.

Acyl Coenzyme A↗

PubMed1991

ATP-dependent control of steady-state cytosolic calcium in cultured gastric smooth muscle.

The regulation of intracellular calcium uptake and release in cultured gastric smooth muscle cells was studied in saponin-permeabilized cells derived from the rabbit antrum. Cells were studied in an ATP-regenerating medium in which the value of the ATP-to-ADP ratio was fixed by variation of the relative concentrations of creatine and creatine phosphate in the presence of a constant concentration of adenine nucleotides and creatine kinase. Free calcium in the medium was measured through the use of the fluorescent probe fura-2. As the ratio of ATP/ADP was increased (8.5, 55.0, and 155.0), the rate of calcium sequestration was increased, resulting in a decrease of steady-state free calcium (275.2, 178.4, and 98.1 nM, respectively). The addition of glucose (5 mM) and hexokinase (15 U/ml), which results in an increase of ADP due to the phosphorylation of glucose in the medium, caused an increase of free calcium concentration to a new set point of approximately 400 nM. Mitochondrial blockade with antimycin A before permeabilization had no effect on calcium sequestration or the resultant free calcium concentration, indicating that under physiological conditions calcium is sequestered predominantly into nonmitochondrial storage sites. Specific variation of ATP/ADP had no effect on the concentration dependence of inositol trisphosphate-induced calcium efflux, suggesting the functional independence of intracellular calcium influx and efflux pathways. These results indicate a significant role for cytoplasmic ATP/ADP in the control of intracellular calcium sequestration and the regulation of steady-state calcium concentration in cultured gastrointestinal smooth muscle cells.

Adenosine Diphosphate↗