The stimulus-secretion coupling of glucose-induced insulin release: XVI. A glucose-like and calcium-independent effect of cyclic AMP

Theophylline increases the synthesis of proinsulin and, to a lesser extent, that of non insulinic peptides in isolated islets of Langerhans. Similar to its stimulant action on ⁴⁵Ca²⁺ uptake by insular tissue, the preferential stimulant action of theophylline on proinsulin biosynthesis is most marked at low glucose concentration (4.2 mM). It apparently represents a Ca²⁺-independent process. Since theophylline does not augment glucose uptake by the isolated islets, the glucose-like enhancing action of theophylline on both ⁴⁵Ca²⁺ uptake and proinsulin synthesis could be due, in part at least, to a cyclic AMP-mediated stimulation of glycogenolysis.     

The stimulus-secretion coupling of glucose-induced insulin release. Insulin release due to glycogenolysis in glucose-deprived islets.

1. When pancreatic islets are preincubated for 20h in the presence of glucose (83.3mM) and thereafter transferred to a glucose-free medium, theophylline (1.4mM) provokes a dramatic stimulation of insulin release. This phenomenon does not occur when the islets are preincubated for either 20h at low glucose concentration (5.6mM) or only 30 min at the high glucose concentration (83.3mM). 2. The insulinotropic action of theophylline cannot be attributed to contamination of the islets with exogenous glucose and is not suppressed by mannoheptulose. 3. The secretory response to theophylline is an immediate phenomenon, but disappears after 60min of exposure to the drug. 4. The release of insulin evoked by theophylline is abolished in calcium-depleted media containing EGTA. Theophylline enhances the net uptake of 45Ca by the islets. 5. Glycogen accumulates in the islets during the preincubation period, as judged by both ultrastructural and biochemical criteria. Theophylline significantly increases the rate of glycogenolysis during the final incubation in the glucose-free medium. 6. The theophylline-induced increase in glycogenolysis coincides with a higher rate of both lactate output and oxidation of endogenous 14C-labelled substrates. 7. These data suggest that stimulation of glycolysis from endogenous stores of glycogen is sufficient to provoke insulin release even in glucose-deprived islets, as if the binding of extracellular glucose to hypothetical plasma-membrane glucoreceptors is not an essential feature of the stimulus-secretion coupling process.     

The stimulus-secretion coupling of glucose-induced insulin release. Effect of exogenous pyruvate on islet function.

1. In isolated pancreatic islets, pyruvate causes a shift to the left of the sigmoidal curve relating the rate of insulin release to the ambient glucose concentration. The magnitude of this effect is related to the concentration of pyruvate (5--90 mM) and, at a 30 mM concentration, is equivalent to that evoked by 2 mM-glucose. Pyruvate also enhances insulin release in the presence of fructose, leucine and 4-methyl-2-oxopentanoate. 2. In the presence of glucose 8 mM), the secretory response to pyruvate is an immediate process, displaying a biphasic pattern. 3. The insulinotropic action of pyruvate coincides with an inhibition of 45Ca efflux and a stimulation of 45Ca net uptake. The relationship between 45Ca uptake and insulin release displays its usual pattern in the presence of pyruvate. 4. Exogenous pyruvate rapidly accumulates in the islets in amounts close to those derived from the metabolism of glucose. The oxidation of [2-14C]pyruvate represents 64% of the rate of [1-14C]pyruvate decarboxylation and, at a 30 mM concentration, is comparable with that of 8 mM-[U-14C]glucose. 5. When corrected for the conversion of pyruvate into lactate, the oxidation of 30 mM-pyruvate corresponds to a net generation of about 314 pmol of reducing equivalents/120 min per islet. 6. Pyruvate does not affect the rate of glycolysis, but inhibits the oxidation of glucose. Glucose does not affect pyruvate oxidation. 7. Pyruvate (30 mM) does not affect the concentration of ATP, ADP and AMP in the islet cells. 8. Pyruvate (30 mM) increases the concentration of reduced nicotinamide nucleotides in the presence but not in the absence of glucose. A close correlation is seen between the concentration of reduced nicotinamide nucleotides and the net uptake of 45Ca. Menadione inhibits the effect of pyruvate on insulin release, without altering its rate of oxidation. 9. Pyruvate, like glucose, modestly stimulates lipogenesis. 10. Pyruvate, in contrast with glucose, markedly inhibits the oxidation of endogenous nutrients. The latter effect accounts for the apparent discrepancy between the rate of pyruvate oxidation and the magnitude of its insulinotropic action. 11. Dichloroacetate fails to affect glucose oxidation and glucose-stimulated insulin release. 12. It is concluded that the effect of pyruvate to stimulate insulin release depends on its ability to increase the concentration of reduced nicotinamide nucleotides in the islet cells.     

The stimulus-secretion coupling of glucose-induced insulin release. Metabolic effects of menadione in isolated islets.

Pancreatic islets contain an enzyme system which catalyzes the donation of hydrogen from NAD(P)H to menadione (2-methyl-1,4-naphthoquinone). In high concentrations (20 to 50 micrometer), menadione, in addition to lowering the concentration of reduced pyridine nucleotides in the islets, also impairs glycolysis and glucose oxidation, decreases ATP concentration, and inhibits proinsulin biosynthesis. However, at a 10 micrometer concentration, menadione fails to affect the concentration of adenine nucleotides, the utilization of glucose, the production of lactate and pyruvate, the oxidation of [6-14C]glucose and the synthesis of proinsulin; whereas the metabolism of glucose through the pentose shunt is markedly increased. The sole inhibitory effect of menadione 10 micrometer upon metabolic parameters is to reduce the concentration of both NADH and NADPH, such an effect being noticed in islets exposed to glucose 11.1 mM but not in those incubated at a higher glucose level (27.8 mM). Since, in the presence of glucose 11.1 mM, menadione 10 micrometer also severely decreases glucose-stimulated45 calcium net uptake and subsequent insulin release, it is concluded that the availability of reduced pyridine nucleotides may play an essential role in the secretory sequence by coupling metabolic to cationic events. Thus, when insulinotropic nutrients are oxidized in the B-cell, the increased availability of reduced pyridine nucleotides could modify the affinity for cations of native ionophoretic systems, eventually leading to the accumulation of calcium up to a level sufficient to trigger insulin release.     

The stimulus-secretion coupling of glucose-induced insulin release: effects of valinomycin upon pancreatic islet function.

In isolated rat pancreatic islets, valinomycin (0.01 to 1.0 μm) caused a dose-related facilitation of ⁸⁶Rb⁺ outflow and a dose-related inhibition of the glucose-induced changes in both outflow and net uptake of ⁸⁶Rb⁺. At high concentrations (0.1–1.0 μm), the ionophore also inhibited the oxidation of glucose and endogenous nutrients, decreased the adenylate charge, and lowered the concentration of reduced pyridine nucleotides in the islet cells. However, as little at 1.0 to 10.0 nm valinomycin caused anomalies in the handling of ⁴⁵Ca²⁺ (suppression of the early inhibitory effect of glucose upon ⁴⁵Ca²⁺ efflux, and reduction in the amount of ⁴⁵Ca²⁺ recovered in the islets after an extensive washing procedure) and inhibition of insulin release. Moreover, when the effect of glucose upon K⁺ conductance was abolished by high concentrations of valinomycin (0.1–1.0 μm), the glucose-induced secondary rise in ⁴⁵Ca²⁺ efflux was still observed. These findings suggest that the effects of glucose upon ⁸⁶Rb⁺ and ⁴⁵Ca²⁺ handling, respectively, although normally concomitant with one another, can be dissociated, in part at least, from one another. It is concluded that the glucose-induced reduction in K⁺ outflow may be unnecessary for the sugar to cause a partial remodeling of Ca²⁺ fluxes in the islet cells.     

The stimulus-secretion coupling of glucose-induced insulin release. Environmental influences on L-glutamine oxidation in pancreatic islets.

Rat ovarian luteinizing hormone/human choriogonadotropin binding sites were labelled with 125I-choriogonadotropin in vivo, and the resulting 125I-choriogonadotropin-receptor complexes were solubilized by Triton X-100 and purified by use of antibodies to choriogonadotropin immobilized to agarose. The purified 125I-choriogonadotropin-receptor complex was treated with glutaraldehyde to crosslink radiolabelled hormone to the receptor. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the crosslinked product revealed a labelled Mr 130 000 major band in addition to the hormone and its alpha-subunit, indicating that a single receptor component was linked to the hormone. Unoccupied binding sites for luteinizing hormone were also solubilized by Triton X-100 from pseudopregnant rat ovaries, and attached to choriogonadotropin-agarose. The agarose gel was washed, and eluted with 0.1 M-sodium acetate, pH 4. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the pH 4 eluate revealed an Mr 90 000 major band which was abolished when ovaries presaturated with choriogonadotropin were used as starting material. These observations suggest that the hormone-binding component of the luteinizing hormone receptor is a polypeptide of Mr 90 000. This polypeptide was isolated and labelled with Na 125I. The labelled polypeptide showed a single band on sucrose density gradient centrifugation and on gel filtration on agarose.     

The stimulus-secretion coupling of glucose-induced insulin release. XLIII. Na-Ca countertransport mediated by pancreatic islet native ionophores

Native ionophores extracted from isolated pancreatic islets were able to transport Ca2+ from one aqueous medium into another across an organic immiscible phase. In the presence of a K+, Na+, Li+, or H+ gradient, Ca2+ was transported against its own concentration gradient from the medium of low monovalent-cation concentration to the opposite medium. The transport of Ca2+ was abolished by the organic calcium-antagonist suloctidil. These findings provide a model for the process of Na-Ca countertransport in islet cells and its inhibition in response to the conversion of nutrient secretagogues to their acidic metabolites.     

The stimulus-secretion coupling of glucose-induced insulin release. Fasting-induced adaptation of key glycolytic enzymes in isolated islets.

The rate of glucose and fructose 6-phosphate phosphorylation in islet homogenates is reduced by prior fasting of the donor rats. In fed rats, the velocity of glucose phosphorylation at increasing glucose concentrations (0.1 to 100 mM) is compatible with the presence of two enzyme activities. A preferential effect of fasting upon the high Km enzyme activity can be documented either at low ATP concentration which enhances the fractional contribution of the high Km enzyme activity, or in the presence of glucose 6-phosphate, which suppresses the low Km enzyme activity. Islet phosphofructokinase activity was characterized by inhibition by citrate or high ATP concentrations, and relief from ATP inhibition by AMP. Fasting reduces the activity of phosphofructokinase without altering its sensitivity to ATP and AMP. Cyclic AMP fails to overcome the effect of fasting upon phosphofructokinase. The activity of phosphoglucoisomerase is unaffected by fasting. The fasting-induced adaptation of key glycolytic enzymes could account, in part at least, for reduced metabolism of glucose in islets from fasted rats.     

The stimulus-secretion coupling of glucose-induced insulin release. Cationic and secretory effects of menadione in the endocrine pancreas.

1. Menadione (2-methyl-1,4-naphthoquinone) inhibits insulin release evoked in the rat endocrine pancreas by glucose or glyceraldehyde, but fails to affect the secretory response to Ca2+, Ba2+, theophylline or gliclazide. The inhibitory effect of menadione upon glucose-induced insulin release is a dose-related, rapid and reversible phenomenon, menadione and glucose acting apparently as competitive antagonists. Menadione affects both the early and late phase of the secretory response to glucose. Menadione also antagonizes in a dose-related fashion the ability of glucose to reduce 86Rb efflux, to provoke 86Rb accumulation, to cause biphasic changes in 45 Ca efflux and to stimulate 45 Ca net uptake in pancreatic islets. 2. It is concluded that menadione impairs the insulinotropic action of glucose and other nutrients by impeding the remodelling of cationic fluxes normally provoked by these secretagogues in islet cells. Menadione, however, does not affect the capacity of divalent cations to activate the effector system which controls the release of secretory granules. Menadione may therefore represent a valuable tool to elucidate the mechanism by which glucose normally modifies the movement of cations in the pancreatic B-cell.     

The stimulus-secretion coupling of amino acid-induced insulin release

Summary Aminooxyacetate, an inhibitor of cytosolic transamination reactions, inhibited insulin release evoked by either 2-ketoisocaproate or L-leucine in rat pancreatic islets incubated in the presence of L-glutamine or L-asparagine. As a rule, aminooxyacetate also inhibited the oxidation of these nutrient secretagogues and impaired the respiratory response of the islets to the combinations of nutrients. However, the oxidative and secretory response to the combination of L-leucine and L-glutamine was less severely affected by aminooxyacetate than that evoked by the three other combinations of exogenous nutrients. These findings reinforce the view that the stimulus-secretion coupling of insulin release in response to L-leucine and 2-ketoisocaproate in association with either L-glutamine or L-asparagine tightly depends on the oxidation of these nutrient secretagogues, on their effect upon O₂ uptake and, within limits, on the intracellular site of generation of reducing equivalents in the pancreatic islet cells.This paper is the 16th in a series.     

The stimulus secretion coupling of glucose-induced insulin release.

In isolated rat pancreatic islets, exogenous l-lactate causes a dose-related enhancement of glucose-induced insulin release and shifts the sigmoidal curve relating insulin output to ambient glucose concentrations to the left. l-Lactate also enhances α-ketoisocaproate-induced insulin release and glucose-induced proinsulin biosynthesis. l-Lactate rapidly accumulates in the islet cells, is converted to pyruvate and CO₂, and raises the intracellular concentration of both ATP and NAD(P)H. On a molar basis, the insulinotropic capacity of nutrients ranges as follows d-glucose ? l-lactate > pyruvate = d/l-lactate > d-lactate and does not correlate with their respective oxidation rates. However, when allowance is made for the intracellular interconversion of these exogenous nutrients, for their reciprocal influence upon oxidation rates, and for their sparing action on the utilization of endogenous fuels, a close correlation is found between the aptitude of glucose, l-lactate, and pyruvate to generate reducing equivalents and to stimulate insulin release. It is proposed that the concentration of NAD(P)H in islet cells affects the ionophoretic fluxes of cations (K⁺, Ca²⁺) across membrane systems and, hence, regulates the net uptake of Ca²⁺ and subsequent release of insulin. The effect of l-lactate upon Ca²⁺ handling is sufficiently rapid to account for the immediate secretory response to this nutrient.     

The stimulus-secretion coupling of amino acid-induced insulin release. Insulinotropic action of L-alanine

Available information on the fate and insulinotropic action of L-alanine in isolated pancreatic islets is restricted to data collected in obese hyperglycemic mice. Recent data, however, collected mostly in tumoral islet cells of either the RINm5F line or BRIN-BD11 line, have drawn attention to the possible role of Na(+) co-transport in the insulinotropic action of L-alanine. In the present study conducted in islets prepared from normal adult rats, L-alanine was found (i) to inhibit pyruvate kinase in islet homogenates, (ii) not to affect the oxidation of endogenous fatty acids in islets prelabelled with [U-14C]palmitate, (iii) to stimulate 45Ca uptake in islets deprived of any other exogenous nutrient, and (iv) to augment insulin release evoked by either 2-ketoisocaproate or L-leucine, whilst failing to significantly affect glucose-induced insulin secretion. The oxidation of L-[U-14C]alanine was unaffected by D-glucose, but inhibited by L-leucine. Inversely, L-alanine decreased the oxidation of D-[U-14C]glucose, but failed to affect L-[U-14C]leucine oxidation. It is concluded that the occurrence of a positive insulinotropic action of L-alanine is restricted to selected experimental conditions, the secretory data being compatible with the view that stimulation of insulin secretion by the tested nutrient(s) reflects, as a rule, their capacity to augment ATP generation in the islet B cells. However, the possible role of Na(+) co-transport in the secretory response to L-alanine cannot be ignored.