Leonard Best

Stimulus-secretion coupling of arginine-induced insulin release: comparison between the cationic amino acid and its methyl ester.

The role currently ascribed to the accumulation of l-arginine in the pancreatic islet B-cell as a determinant of its insulinotropic action was reevaluated by comparing the uptake and the metabolic, ionic, electric, and secretory effects of the cationic amino acid with those of its more positively charged methyl ester in rat pancreatic islets. The response to l-arginine methyl ester differed from that evoked by the unesterified amino acid by a lower uptake and oxidation, lack of inhibitory action on d-glucose metabolism, more severe inhibition of the catabolism of endogenous l-glutamine, inhibition of 45Ca net uptake, decrease in both 86Rb outflow from prelabeled islets perifused at normal extracellular Ca2+ concentration and 45Ca efflux from prelabeled islets perifused in the absence of extracellular Ca2+, and delayed and lesser insulinotropic action. These findings reinforce the view that the carrier-meadiated entry of l-arginine into the islet B-cells, with resulting depolarization of the plasma membrane, represents the essential mechanism for stimulation of insulin release by this cationic amino acid.The role currently ascribed to the accumulation of L-arginine in the pancreatic islet B-cell as a determinant of its insulinotropic action was reevaluated by comparing the uptake and the metabolic, ionic, electric, and secretory effects of the cationic amino acid with those of its more positively charged methyl ester in rat pancreatic islets. The response to L-arginine methyl ester differed from that evoked by the unesterified amino acid by a lower uptake and oxidation, lack of inhibitory action on D-glucose metabolism, more severe inhibition of the catabolism of endogenous L-glutamine, inhibition of 45Ca net uptake, decrease in both 86Rb outflow from prelabeled islets perifused at normal extracellular Ca2+ concentration and 45Ca efflux from prelabeled islets perifused in the absence of extracellular Ca2+, and delayed and lesser insulinotropic action. These findings reinforce the view that the carrier-mediated entry of L-arginine into the islet B-cells, with resulting depolarization of the plasma membrane, represents the essential mechanism for stimulation of insulin release by this cationic amino acid.

A VOLUME-ACTIVATED ANION CONDUCTANCE IN INSULIN-SECRETING CELLS

The whole-cell patch-clamp recording technique was used to measure volume-activated currents in K+-free solutions in RINm5F and HIT-T15 insulinoma cells and in dispersed rat islet cells. Cell swelling, induced by intracellular hypertonicity or extracellular hypotonicity, caused activation of an outwardly rectifying conductance which could be subsequently inactivated by hypertonic extracellular solutions. The conductance required adenosine 5′-triphosphate (ATP) in the pipette solution but was Ca2+ independent. Na+ and Cl− substitution studies suggested that the swelling-activated current is Cl− selective with a halide permeability sequence of Br > Cl > 1. The conductance was reversibly inhibited by the anion channel inhibitors 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) and by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). Further evidence for a volume-activated anion conductance was provided by studies of volume regulation in insulin-secreting cells. When RINm5F cells were exposed to a hypotonic medium, the initial cell swelling was followed by a regulatory volume decrease (RVD). This RVD response was also inhibited by DIDS and by NPPB. These data therefore provide evidence for a volume-activated anion conductance in insulin-secreting cells which could be involved in the RVD following osmotic stress. A possible role for the conductance in hypotonically induced insulin release is also discussed.

The stimulus-secretion coupling of glucose-induced insulin release: Fuel metabolism in islets deprived of exogenous nutrient

The fuel hypothesis for insulin release postulates that the secretory response to nutrient secretagogues reflects their capacity to augment catabolic fluxes in pancreatic islet cells. Hence, both the oxidation of exogenous nutrients and their effect upon the metabolism of endogenous nutrients should be taken into consideration to account for the stimulation of insulin release. In the present work, an attempt was made to quantify the respective contribution of carbohydrates, fatty acids, and amino acids in the respiration of islets deprived of exogenous nutrient. The metabolism of glycerol, fatty acids, and amino acids was found to account for the major part of the basal respiratory rate. Glucose modestly decreased the oxidation of endogenous fatty acids, lowered the production of NH4+, but did not impair the oxidative catabolism of 2-keto acids derived from endogenous amino acids. These findings suggest that the catabolism of noncarbohydrate nutrients largely contributes to the respiration of the islets, even when the latter are exposed to circulating glucose in its physiological concentration.

Stimulation of phosphoinositide breakdown in rat pancreatic islets by glucose and carbamylcholine

In the presence of Li+, glucose, 2-ketoisocaproate and carbamylcholine induced the rapid formation of 3H-inositol phosphates in rat pancreatic islets prelabelled with 3H-inositol. The production of labelled inositol phosphates continued up to 20 min of incubation. Glibenclamide and ionophore A23187 had no significant effect on labelled inositol phosphate production. The effects of carbamylcholine and to a lesser extent, glucose were found to persist in the absence of added Ca2+, but both were strongly inhibited by excess EGTA. In general, the rise in 3H-inositol phosphate production was associated with a fall in lipid bound radioactivity, although the latter was found to occur more slowly, and was of a smaller magnitude than labelled inositol phosphate formation. The results suggest that nutrient secretagogues and cholinergic agonists stimulate hydrolysis of phosphoinositides in pancreatic islets by a phospholipase C mechanism. This effect is Ca2+-dependent, but probably not triggered by increased Ca2+ uptake into the islet.

Glucose and a-ketoisocaproate induce transient inward currents in rat pancreatic beta cells

Summary The perforated patch technique was used to study changes in membrane potential and whole-cell currents in single isolated rat pancreatic beta-cells during stimulation with glucose or α-ketoisocaproate. Increasing the glucose concentration from 4 to 20 mmol/l, or addition of 15 mmol/l α-ketoisocaproate, caused depolarization and, in most cases, initiation of action potentials. Under voltage-clamp conditions close to a potassium equilibrium potential (EK) (–60 to –70 mV) these effects were accompanied by the appearance of transient inward currents. These transient currents resembled those elicited during cell swelling in response to a 10 % hypotonic bath solution, a manoeuvre which also caused beta-cell depolarization and electrical activity. Tolbutamide (0.2 mmol/l), in the absence of glucose depolarized beta-cells but did not induce transient inward currents. Nutrient-induced electrical activity and inward currents were abolished by the anion channel inhibitors 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid and 5-nitro-2-(3-phenylpropylamino) benzoic acid, compounds which also inhibited glucose-induced insulin release. It is concluded that nutrient secretagogues induce transient inward currents in isolated rat beta-cells, possibly by activating a volume-sensitive anion conductance. These inward currents could enhance the intensity of electrical, and hence secretory, activity in the beta-cell during nutrient stimulation. [Diabetologia (1997) 40: 1–6]

Activation of protein kinase C by a tumor-promoting phorbol ester in pancreatic islets

Rat pancreatic islet homogenates display protein kinase C activity. This phospholipid‐dependent and calcium‐sensitive enzyme is activated by diacylglycerol or the tumor‐promoting phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). In the presence of TPA, the K a for Ca2+ is close to 5 μM. TPA does not affect phosphoinositide turnover but stimulates [32P]‐ and [3H]choline‐labelling of phosphatidylcholine in intact islets. Exogenous phospholipase C stimulates insulin release, in a sustained and glucose‐independent fashion. The secretory response to phospholipase C persists in media deprived of CaCl2. It is proposed that protein kinase C participates in the coupling of stimulus recognition to insulin release evoked by TPA, phospholipase C and, possibly, those secretatogues causing phosphoinositide breakdown in pancreatic islets.

Phospholipids and islet function

ConclusionsThe work reviewed above provides evidence that enhanced phospholipid turnover in islets may be a determinant of the secretory response to nutrient secretagogues and certain neurotransmitter and hormonal stimuli. The available data are compatible with the hypothesis that stimulated phospholipid turnover may be involved in the control of calcium mobilisation in islets, although additional possibilities clearly exist, such as the facilitation of membrane fusion during exocytosis and the liberation of arachidonic acid for subsequent metabolism via the cyclo-oxygenase and lipoxygenase pathways. Phospholipid and protein methylation may also be involved at some stage in stimulus-secretion coupling in pancreatic islets. Continued investigation of these topics is likely to contribute to a better understanding of the control of secretory activity in pancreatic islets, which in turn may throw light on pathophysiological aspects of islet function and perhaps suggest novel therapeutic approaches.

Glucose-induced electrical activity in rat pancreatic β-cells : dependence on intracellular chloride concentration

A rise in glucose concentration depolarizes the β‐cell membrane potential leading to electrical activity and insulin release. It is generally believed that closure of KATP channels underlies the depolarizing action of glucose, though work from several laboratories has indicated the existence of an additional anionic mechanism. It has been proposed that glucose activates a volume‐regulated anion channel, generating an inward current due to Cl− efflux. This mechanism requires that intracellular [Cl−] is maintained above its electrochemical equilibrium. This hypothesis was tested in rat β‐cells by varying [Cl−] in the patch pipette solution using the Cl−‐permeable antibiotic amphotericin B to allow Cl− equilibration with the cell interior. Under such conditions, a depolarization and electrical activity could be evoked by 16 mm glucose with pipette solutions containing 80 or 150 mm Cl−. At 40 or 20 mm Cl−, a subthreshold depolarization was usually observed, whilst further reduction to 12 or 6 mm abolished depolarization, in some cases leading to a glucose‐induced hyperpolarization. With a pipette solution containing gramicidin, which forms Cl−‐impermeable pores, glucose induced a depolarization and electrical activity irrespective of [Cl−] in the pipette solution. Under the latter conditions, glucose‐induced electrical activity was prevented by bumetanide, an inhibitor of the Na+–K+–2Cl− co‐transporter. This inhibition could be overcome by the use of amphotericin B with a high [Cl−] pipette solution. These findings suggest that the maintenance of high intracellular [Cl−] in the β‐cell is an important determinant in glucose‐induced depolarization, and support the hypothesis that β‐cell stimulation by glucose involves activation of the volume‐regulated anion channel and generation of an inward Cl− current.

Stimulation of insulin secretion by glucose in the absence of diminished potassium (86Rb+ permeability

Two inhibitors of the nucleotide-sensitive K+ (KATP) channel, tolbutamide and quinine, were utilized in order to assess the role of this channel in glucose-stimulated insulin release from perifused rat islets. In the absence of these drugs, the addition of 15 mM glucose elicited a marked biphasic stimulation of insulin secretion concomitant with a reduction in the rate of 86Rb+ efflux. In the presence of either 500 microM tolbutamide or 100 microM quinine, a reduced rate of efflux of 86Rb+ was observed together with an elevated rate of insulin release. Under such conditions, the addition of 15 mM glucose retained the ability to stimulate insulin secretion though this was associated with a marked increase in 86Rb+ efflux. It is concluded that a net reduction in beta-cell K+ permeability is not an obligatory step in glucose-stimulated insulin release. Thus, glucose is likely to exert depolarizing actions on the beta-cell in addition to the closure of K+ channels.

Phosphatidylinositol and phosphatidic acid metabolism in rat pancreatic islets in response to neurotransmitter and hormonal stimuli

Carbamylcholine produced a concentration-dependent stimulation of labelling of phosphatidylinositol and phosphatidic acid in rat islets of Langerhans following preincubation with 32PO43(-). The time course of these effects suggested that the initial action of carbamylcholine was to stimulate phosphatidic acid production, presumably by causing hydrolysis of phosphatidylinositol. This conclusion was substantiated by experiments in which islet phospholipids were pre-labelled with [3H]arachidonic acid. Under these conditions, carbamylcholine caused a loss of radioactivity from phosphatidylinositol, together with an increase in labelling of phosphatidic acid. The effects of carbamylcholine on islet phospholipid labelling were not dependent upon the presence of added Ca2+, but were abolished by EDTA and by atropine. An apparent stimulation of phosphatidylinositol and phosphatidic acid metabolism was also induced by cholecystokinin-pancreozymin, whereas glucagon, arginine, glibenclamide and thyrotropin had no significant effect. The data suggest that enhanced activity of the so-called phosphatidylinositol cycle may be an important event in regulating secretory activity of islets in response to certain neurotransmitter and hormonal stimuli. Furthermore, the results are compatible with the hypothesis that increased phospholipid metabolism may play a role in the modulation of ionic fluxes during stimulation by such agents.