Adenosine triphosphate (ATP) and insulin secretion: effect of different concentrations and effect of temperature]

In the presence of a glucose concentration of 1.5 g/1, ATP provokes a biphasic stimulation of insulin secretion from the isolated and perfused rat pancreas. For ATP concentrations ranging from 0.5 mg/1 to 200 mg/1, the increase in insulin secretion presents a linear relation with the logarithm of the concentration. Lowering the temperature from 37.5 degrees C to 28 degrees C provokes a decrease in insulin secretion induced by glucose (1.5 g/1). In response to the stimulation by ATP, the increase in insulin secretion measured during the first phase is weaker at 28 degrees C than at 37.5 degrees C when estimated in ng/min; however, when evaluated in percentage in relation to the baseline value, this increase is more important at the lower temperature.     

Influence of temperature on insulin secretion induced by different concentrations of glucose]

Lowering of the temperature from 37.5 degrees C to 28 degrees C provokes a decrease in the response of the beta cell to the stimulation by glucose (1.5 g/1, 3 g/1 and 5 g/1). The insulin secretion obtained at 28 degrees C, compared to that obtained at 37.5 degrees C, is weaker for strongly stimulating concentrations (3 g/1 and 5 g/1) than for a slightly stimulating concentration (1.5 g/1).     

Effects of temperature on basal and insulin-stimulated glucose transport activities in fat cells. Further support for the translocation hypothesis of insulin action

Effects of temperature on glucose transport in fat cells were studied. In this system, the basal (no insulin) glucose transport activity was higher at approximately 25-30 degrees C than at 37 degrees C, as previously reported (Vega, F. V., and Kono, T. (1979) Arch. Biochem. Biophys. 192, 120-127). The stimulatory effect of low temperature (or the insulin-like effect) was reversible and apparently required metabolic energy for both its forward and reverse reactions. By lowering the ATP level with 2,4-dinitrophenol, one could separately determine the insulin-like stimulatory effect of low temperature and its inhibitory effect on the transport process itself. The maximum level of stimulation by low temperature was greater at 10 degrees C than at 25-30 degrees C, but the rate of stimulation was considerably slower at 10 degrees C than at 25-30 degrees C. When cells were exposed to low temperature, the glucose transport activity in the plasma membrane-rich fraction was increased, while that in the Golgi-rich fraction was decreased. The Arrhenius plot of the basal glucose transport activity determined in the presence of dinitrophenol was apparently linear from 10 to 37 degrees C and parallel to that of the plus insulin activity measured either in the presence or absence of dinitrophenyl. Insulin itself slowly stimulated the glucose transport activity at 10 degrees C. These results are consistent with the view that (a) low temperature, like insulin, induces translocation of the glucose transport activity from an intracellular storage site to the plasma membrane, (b) insulin stimulates glucose transport activity without changing its activation energy, and (c) subcellular membranes do not entirely stop their movement at a low temperature, e.g, at 10 degrees C.     

Effect of temperature on glucagon secretion in the presence of different concentrations of glucose]

Lowering the temperature from 37.5 degrees C to 28 degrees C does not alter the glucagon secretion by the isolated perfused rat pancreas in response to different glucose concentrations (0 g/l 1.5 g/l, 3 g/l and 5 g/l).     

Oxygen and temperature dependence of stimulated insulin secretion in isolated rat islets of Langerhans

The effects of lowered O2 tension on insulin secretion and changes in cellular energy parameters were investigated in isolated rat pancreatic islets perifused with buffers equilibrated with 21, 9, 5, and 1% oxygen and containing 5 mM glucose. Decreasing the external [O2] reduced the amount of insulin released in response to 16 mM glucose, 20 mM alpha-ketoisocaproic acid, and 40 mM KCl. Secretion elicited by high glucose or KCl had declined significantly at 9% oxygen, whereas that caused by alpha-ketoisocaproic acid became inhibited below 5% O2. Lowering the oxygen tension also decreased the ability of islets to respond with a rise in [ATP]/[ADP] upon stimulation with metabolic secretagogues. This reduction in the evoked increase in the nucleotide ratios paralleled the inhibition of stimulated insulin secretion. Addition of 2 mM amytal markedly decreased the islet energy level and eliminated the secretory response to 16 mM glucose. The results suggest that enhancement of B-cell energy production and a consequent rise in [ATP] (or [ATP]/[ADP]) are a necessary event for the hormone release elicited by high glucose and alpha-ketoisocaproic acid. A decrease in temperature inhibited insulin secretion with all three secretagogues tested. The energies of activation were similar for high glucose and KCl-induced secretion, about 20 kcal/mol, but were higher for alpha-ketoisocaproic acid, about 35 kcal/mol. At 28 degrees C, the [ATP]/[ADP] was larger than that at 38 degrees C (8 versus 5) and was not increased further upon addition of 16 mM glucose. It is suggested that a decrease in the rate of energy production at lowered temperatures may contribute to the inhibition of insulin release caused by metabolic secretagogues.     

Insulin secretion in the hibernating edible dormouse (Glis glis): in vivo and in vitro studies

Plasma glucose and insulin have been studied during lethargy and spontaneous arousal of hibernating edible dormouse. During lethargy blood glucose was low while plasma insulin remained at the same level as in other seasons. Plasma glucose and insulin did not fluctuate along the phase of lethargy. During spontaneous arousal plasma insulin rose strongly from the 17 degrees C stage, reaching the higher values at 26 degrees C while blood glucose was only 85 mg/100 ml, then decreased at 37 degrees C. The effect of glucose and temperature on insulin secretion was studied using perfused pancreas preparation from hibernating edible dormice. During the rewarming of the edible dormouse pancreas the insulin release did not occur in response to the absolute extracellular glucose level but occurred in response to a B cell membrane phenomenon which was dependent on the changing rate of glucose level. The effect of glucose and temperature on insulin secretion from perfused pancreas was compared between edible dormouse and homeotherm permanent, the rat. The B cell response to glucose of the dormouse pancreas increased up to 15 degrees C whereas that of the rat only from 25 degrees C. The dormouse insulin secretion reached a peak value at the 30 degrees C of temperature, whereas that of the rat progressively increased until 37 degrees C. These results showed that some biochemical adjustment or process of acclimatization took place in the B cells of the hibernators.     

Effect of hyperthermia on blood levels of corticosterone and certain metabolites in rats during thiobutabarbital anaesthesia

No changes were found in the serum levels of corticosterone, pyruvate and lactate in rats during general anaesthesia with thiobutabarbital (Brevinarcon) subjected to short-lasting hyperthermia in a high-temperature chamber (air temperature 50 degrees C, relative humidity 50%) in relation to a control group of rats during similar general anaesthesia at room temperature. However, in the serum of rats during hyperthermia (rectal temperature 40-41 degrees C) the glucose level was about 52% lower and FFA were about 39% lower than in rats kept under normothermic conditions (rectal temperature 36.5-37.5 degrees C) which may point to an increased requirement of tissues for energy-yielding substrates at higher body temperatures and/or increased insulin secretion.     

Temperature optimum of insulin-stimulated 2-deoxy-D-glucose uptake in rat adipocytes. Correlation of cellular transport with membrane spin-label and fluorescence-label data.

The effects of temperature alterations between 22 degrees C and 48 degrees C on basal and insulin-stimulated 2-deoxy-D-[1-14C]glucose uptake were examined in isolated rat adipocytes. A distinct optimum was found near physiological temperature for uptake in the presence of maximally effective insulin concentrations where insulin stimulation and hexose uptake were both conducted at each given assay temperature. Basal uptake was only subtly affected. Control and maximally insulin-stimulated cells incubated at 35 degrees C subsequently exhibited minimal temperature-sensitivity of uptake measured between 30 and 43 degrees C. The data are mostly consistent with the concept that insulin-sensitive glucose transporters are, after stimulation by insulin, functionally similar to basal transporters. Adipocyte plasma membranes were labelled with various spin- and fluorescence-label probes in lipid structural studies. The temperature-dependence of the order parameter S calculated from membranes labelled with 5-nitroxide stearate indicated the presence of a lipid phase change at approx. 33 degrees C. Membranes labelled with the fluorescence label 1,6-diphenylhexa-1,3,5-triene, or the cholesterol-like spin label nitroxide cholestane, reveal sharp transitions at lower temperatures. We suggest that a thermotropic lipid phase separation occurs in the adipocyte membrane that may be correlated with the temperature-dependence of hexose transport and insulin action in the intact cells.     

Effect of tetracaine and lidocaine on insulin release in isolated mouse pancreatic islets

The effect of tetracaine and lidocaine on insulin secretion and glucose oxidation by islets of ob/ob-mice was measured. Tetracaine, at a concentration of 1 microM to 0.1 mM, did not markedly influence the basal (3 mM glucose) insulin secretion, whereas 0.5-3.5 mM induced a marked increase. At 7 mM glucose, there was a dose-dependent increase with 0.1-2.5 mM tetracaine. Insulin release induced by 20 mM glucose was potentiated by 0.1 mM and 0.5 mM tetracaine, but this effect disappeared at 1 mM tetracaine. The stimulatory effect of 0.5-1 mM tetracaine on basal insulin release was blocked by the secretory inhibitors, adrenaline (1 microM), clonidine (1 microM) and by Ca2+-deficiency, but the stimulation by 3.5 mM tetracaine was not reduced by 1 microM clonidine or Ca2+ deficiency. Atropine (10 microM) did not affect the stimulation by 0.5 mM tetracaine at 3 mM glucose or by 0.25 mM tetracaine at 20 mM glucose. Tetracaine, at 0.1 mM, potentiated the secretory stimulation of 20 mM L-leucine, 20 mM D-mannose, or 1 microM glibenclamide. Mannoheptulose, 10 mM, abolished the combined effects of 0.1 mM tetracaine and 10 mM glucose. Lidocaine, 1-5 mM, stimulated basal insulin release, but 1 microM-1 mM of the drug did not affect glucose-induced (20 mM glucose) insulin release and 5 mM lidocaine inhibited glucose stimulation. The oxidation of 10 mM D-[U-14C]glucose was slightly enhanced by 0.1 and 1 mM tetracaine. The results indicate that tetracaine and lidocaine, at certain concentrations, can induce insulin release and that tetracaine potentiates secretion induced by other secretagogues. It is concluded that these effects may be associated with beta-cell functions related to the adrenergic receptors but probably not to cholinergic receptors.     

Insulin secretion by rat lacrimal glands: effects of systemic and local variables

To understand the secretory mechanisms and physiological role of insulin in the tear film, the present study examined 1) the time course of insulin secretion in the tear film under glucose intravenous stimulation, 2) the glucose- and carbachol-induced insulin secretion from isolated lacrimal gland (LG), 3) the effect of insulin on glucose consumption by the cornea, and 4) the expression of insulin, pancreatic duodenal homeobox-1 (PDX-1), and glucose transport proteins (GLUTs) in LG tissue. The insulin level in the tear film of 8-wk-old male Wistar rats increased from 0.6 +/- 0.45 to 3.7 +/- 1.3 ng/ml in the initial minutes after glucose stimulation. In vitro assays demonstrated that higher glucose concentrations from 2.8 to 16.7 mM, 200 microM carbachol, or 40 mM KCl significantly increased insulin secretion from lacrimal glands compared with controls, but did not detect C-peptide as measured by RIA. Glucose consumption by corneal tissue, evaluated by radiolabeled D-[U-14C]glucose uptake, was 24.07 +/- 0.61 and was enhanced to 31.63 +/- 3.15 nmol x cornea(-1) x 2 h(-1) in the presence of 6 nM insulin (P = 0.033) and to 37.5 +/- 3.7 nmol x cornea(-1) x 2 h(-1) in the presence of 11.2 mM glucose (P = 0.015). Insulin and PDX-1 mRNA was detected in LG. Insulin was located in the apical areas of acinar cells by immunoperoxidase and the expression of GLUT-1, but not PDX-1, was confirmed by Western blot. These findings suggest that insulin secretion in the tear film is influenced by local stimuli such as nutrient and neural inputs and that this hormone plays a metabolic role in ocular surface tissues. These data also indicate that under normal conditions the insulin secreted by LG is stored, but it is not clear that is locally produced in the LG.     

Control of insulin secretion in the developing pancreatic rudiment.

The embryonic rat pancreas, removed on the 14th day of gestation and cultivated in vitro, accumulates differentiated levels of exocrine enzymes and insulin. In the period corresponding to days 16–22 in vivo, 99% of the final insulin content accumulates. During this period we have studied the development of competence for insulin secretion, the regulation of this secretion by glucose and other secretatogues, and the rate of synthesis following a secretory challenge. Our results demonstrate that the capacity for insulin secretion develops in parallel with the accumulation of insulin in secretory granules since β granules appear at day 16. On day 16, after 48 hr of culture, both glucose and caffeine are required for detectable insulin secretion. At later stages, insulin release can be effectuated by glucose alone. In the fetal pancreas at day 20 of development, glucose is ten times more efficient than caffeine and fourfold more efficient than caffeine combined with either glucagon, cholera toxin or dibutyryl cyclic AMP. Glucagon, cholera toxin or cyclic AMP in the presence of caffeine increases equally (about tenfold) both the “basal” and the glucose-induced level of secretion. This suggests that glucose and caffeine act independently but synergistically. The integrity of the cells is maintained under the stimulation conditions, and there is a selective increase in insulin synthesis measured during 18 hr following stimulation of insulin release.     

Qualitative and quantitative comparison of glucose transport activity and glucose transporter concentration in plasma membranes from basal and insulin-stimulated rat adipose cells.

Conditions are described which allow the isolation of rat adipose-cell plasma membranes retaining a large part of the stimulatory effect of insulin in intact cells. In these membranes, the magnitude of glucose-transport stimulation in response to insulin was compared with the concentration of transporters as measured with the cytochalasin-B-binding assay or by immunoblotting with an antiserum against the human erythrocyte glucose transporter. Further, the substrate- and temperature-dependencies of the basal and insulin-stimulated states were compared. Under carefully controlled homogenization conditions, insulin-treated adipose cells yielded plasma membranes with a glucose transport activity 10-15-fold higher than that in membranes from basal cells. Insulin increased the transport Vmax. (from 1,400 +/- 300 to 15,300 +/- 3,400 pmol/s per mg of protein; means +/- S.E.M.; assayed at 22 degrees C) without any significant change in Km (from 17.8 +/- 4.4 to 18.9 +/- 1.4 nM). Arrhenius plots of plasma-membrane transport exhibited a break at 21 degrees C, with a higher activation energy over the lower temperature range. The activation energy over the higher temperature range was significantly lower in membranes from basal than from insulin-stimulated cells [27.7 +/- 5.0 kJ/mol (6.6 +/- 1.2 kcal/mol) and 45.3 +/- 2.1 kJ/mol (10.8 +/- 0.5 kcal/mol) respectively], giving rise to a larger relative response to insulin when transport was assayed at 37 degrees C as compared with 22 degrees C. The stimulation of transport activity at 22 degrees C was fully accounted for by an increase in the concentration of transporters measured by cytochalasin B binding, if a 5% contamination of plasma membranes with low-density microsomes was assumed. However, this 10-fold stimulation of transport activity contrasted with an only 2-fold increase in transporter immunoreactivity in membranes from insulin-stimulated cells. These data suggest that, in addition to stimulating the translocation of glucose transporters to the plasma membrane, insulin appears to induce a structural or conformational change in the transporter, manifested in an altered activation energy for plasma-membrane transport and possibly in an altered immunoreactivity as assessed by Western blotting.     

Effects of adenosine on the secretions of glucagon and insulin of isolated ad perfused pancreas of the rat

The effects of adenosine on insulin and glucagon secretions were studied using the isolated perfused rat pancreas. The secretion of glucagon was stimulated by adenosine at concentrations ranging from 1.65 to 165 mumol/l, in the presence of glucose 0.5 g/l; the stimulation was immediate, but transient and was dose-dependent. Insulin secretion was not changed by adenosine in the presence of glucose 0.5 g/l; in the presence of glucose 1.5 g/l, adenosine at 1.65 and 16.5 mumol/l did not significantly modify insulin secretion. But at 165 mumol/l adenosine induced a progressive increase in time after the 5th minute. The A cell appears then to be much more sensitive to adenosine than the B cell.     

Dual effect of insulin on in vitro leptin secretion by adipose tissue

Although it is widely accepted that insulin stimulates leptin secretion, a dual action was observed using a validated in vitro system, i.e., an early (less than 48 h) inhibitory action, followed later (48-96 h) by a clear-cut stimulation. While the inhibitory phase was observed at every glucose concentration tested (from 1 to 25 mM), the stimulatory phase required the presence of physiological or supraphysiological glucose concentrations. In fact, leptin secretion was virtually eliminated in the presence of glucose uptake inhibitors. This dual effect of insulin was not due to modifications of the ob mRNA levels, suggesting that it depends entirely on posttranslational mechanisms. In conclusion, insulin appears to induce an early inhibition of leptin secretion by the adipose cell, followed later by a stimulatory effect secondary to the metabolic changes triggered by the insulin-induced increase in glucose uptake.     

The role of protein kinase C in cholinergic stimulation of insulin secretion from rat islets of Langerhans.

The role of the Ca2+/phospholipid-dependent protein kinase C (PKC) in cholinergic potentiation of insulin release was investigated by measuring islet PKC activity and insulin secretion in response to carbachol (CCh), a cholinergic agonist. CCh caused a dose-dependent increase in insulin secretion from cultured rat islets at stimulatory glucose concentrations (greater than or equal to 7 mM), with maximal effects observed at 100 microM. Short-term exposure (5 min) of islets to 500 microM-CCh at 2 mM- or 20 mM-glucose resulted in redistribution of islet PKC activity from a predominantly cytosolic location to a membrane-associated form. Prolonged exposure (greater than 20 h) of islets to 200 nM-phorbol myristate acetate caused a virtual depletion of PKC activity associated with the islet cytosolic fraction. Under these conditions of PKC down-regulation, the potentiation of glucose-stimulated insulin secretion by CCh (500 microM) was significantly decreased, but not abolished. CCh stimulated the hydrolysis of inositol phospholipids in both normal and PKC-depleted islets, as assessed by the generation of radiolabelled inositol phosphates. These results suggest that the potentiation of glucose-induced insulin secretion by cholinergic agonists is partly mediated by activation of PKC as a consequence of phospholipid hydrolysis.     

Interaction of caerulein, glucose, and amino acids on insulin secretion from the perfused rat pancreas

The effect of caerulein on insulin response to graded amounts of glucose from the isolated perfused rat pancreas was investigated in the presence or absence of an amino acids mixture. Caerulein at a concentration of 0.1 ng/ml which is a submaximal concentration for an effect on exocrine pancreatic secretion potentiated insulin responses to glucose concentrations less than 200 mg/dl, but produced no further increase when added to a glucose stimulus over a 200 mg/dl. However, in the presence of amino acids the insulin response to 200 mg/dl glucose was significantly potentiated by the stimulation of 0.1 ng/ml caerulein. The effectiveness of caerulein as an insulinotropic agent depended on the glucose concentration only when amino acids were present. These results indicate that caerulein, at a concentration which stimulate pancreatic exocrine secretion, has a synergistic effect on insulin response to glucose and amino acids and therefore raises the possibility that endogenously released CCK may contribute to the entero-insular axis.     

Difference in mechanism between glyceraldehyde- and glucose-induced insulin secretion from isolated rat pancreatic islets

The effects of D-glyceraldehyde and glucose on islet function were compared in order to investigate the difference between them in the mechanism by which they induce insulin secretion. The stimulation of insulin secretion from isolated rat islets by 10 mM glyceraldehyde was not completely inhibited by either 150 microM diazoxide (an opener of ATP-sensitive K(+) channels) or 5 microM nitrendipine (an L-type Ca(2+)-channel blocker), whereas the stimulation of insulin secretion by 20 mM glucose was completely inhibited by either drug. The insulin secretion induced by glyceraldehyde was less augmented by 100 microM carbachol (a cholinergic agonist) than that induced by glucose. The stimulation of myo-inositol phosphate production by 100 microM carbachol was more marked in islets incubated with the hexose than with the triose. The content of glyceraldehyde 3-phosphate, a glycolytic intermediate, in islets incubated with glyceraldehyde was far higher than that in islets incubated with glucose, whereas the ATP content in islets incubated with the triose was significantly lower than that in islets incubated with the hexose. These results suggest that glyceraldehyde not only mimics the effect of glucose on insulin secretion but also has the ability to cause the secretion of insulin without the influx of Ca(2+ )through voltage-dependent Ca(2+) channels. The reason for the lower potency of the triose than the hexose in stimulating insulin secretion is also discussed.     

Increased glucose sensitivity of both triggering and amplifying pathways of insulin secretion in rat islets cultured for 1 wk in high glucose

Chronic hyperglycemia has been shown to induce either a lack of response or an increased sensitivity to glucose in pancreatic beta-cells. We reinvestigated this controversial issue in a single experimental model by culturing rat islets for 1 wk in 10 or 30 mmol/l glucose (G10, Controls; or G30, High-glucose islets) before testing the effect of stepwise glucose stimulation from G0.5 to G20 on key beta-cell stimulus-secretion coupling events. Compared with Controls, the glucose sensitivity of High-glucose islets was markedly increased, leading to maximal stimulation of oxidative metabolism and both triggering and amplifying pathways of insulin secretion in G6 rather than G20, hence to loss of glucose effect above G6. This enhanced glucose sensitivity occurred despite an approximately twofold increase in islet uncoupling protein 2 mRNA expression. Besides this increased glucose sensitivity, the maximal glucose stimulation of insulin secretion in High-glucose islets was reduced by approximately 50%, proportionally to the reduction of insulin content. In High-glucose islets, changes in (45)Ca(2+) influx induced by glucose and diazoxide were qualitatively similar but quantitatively smaller than in Control islets and, paradoxically, did not lead to detectable changes in the intracellular Ca(2+) concentration measured by microspectrofluorimetry (fura PE 3). In conclusion, after 1 wk of culture in G30, the loss of glucose stimulation of insulin secretion in the physiological range of glucose concentrations (G5-G10) results from the combination of an increased sensitivity to glucose of both triggering and amplifying pathways of insulin secretion and an approximately 50% reduction in the maximal glucose stimulation of insulin secretion.     

Effects of cholinergic agonists on diacylglycerol and intracellular calcium levels in pancreatic β-cells

We have studied the effects of cholinegic agonists on the rates of insulin release and the concentrations of diacylglycerol (DAG) and intracellular free Ca²⁺ ([Ca²⁺]_i) in the β-cell line MIN6. Insulin secretion was stimulated by glucose, by glibenclamide and by bombesin. In the presence of glucose, both acetylcholine (ACh) and carbachol (CCh) produced a sustained increase in the rate of insulin release which was blocked by EGTA or verapamil. The DAG content of MIN6 β-cells was not affected by glucose. Both CCh and ACh evoked an increase in DAG which was maximal after 5 min and returned to basal after 30 min; EGTA abolished the cholinergic-induced increased in DAG. ACh caused a transient rise in [Ca²⁺]_i which was abolished by omission of Ca²⁺ or by addition of devapamil. Thus, cholinergic stimulation of β-cell insulin release is associated with changes in both [Ca²⁺]_i and DAG. The latter change persists longer than the former and activation of protein kinase C and sensitization of the secretory process to Ca²⁺ may underlie the prolonged effects of cholinergic agonists on insulin release. However, a secretory response to CCh was still evident after both [Ca²⁺]_i and DAG had returned to control values suggesting that additional mechanisms may be involved.     

Effects of muscarinic receptor type 3 knockout on mouse islet secretory responses

The impact of muscarinic type 3 receptor knockout (M3KO) on the cholinergic regulation of insulin secretion and phospholipase C (PLC) activation was determined. Islets isolated from control, wild-type mice or heterozygotes responded with comparable insulin secretory responses to 15 mM glucose. This response was markedly amplified by the inclusion of 10 microM carbachol. While 15 mM glucose-induced release remained similar to wild-type and heterozygote responses in M3KO mice, the stimulatory impact of carbachol was abolished. Stimulation with 15 mM glucose plus 50 microM carbachol increased fractional efflux rates of myo-[2-3H]inositol from control wild-type and heterozygote islets but not from M3KO islets. Fed plasma insulin levels of M3KO mice were reduced 68% when compared to values obtained from combined wild-type and heterozygote animals. These studies support the conclusion that the M3 receptor in islets is coupled to PLC activation and insulin secretion and that cholinergic stimulation of the islets may play an important role in the regulation of plasma insulin levels.