Effects of leptin,acetylcholine and vasoactive intestinal polypeptide on insulin secretion in isolated <Emphasis Type="Italic">ob/ob</Emphasis> mouse pancreatic islets

Abstract. Obesity is often accompanied by hyperleptinemia, hyperinsulinemia, and an increased parasympathetic tone. Obese-hyperglycemic mice (Umeå ob/ob) have functional leptin receptors and a raised parasympathetic tone. We studied insulin release in islets isolated from 9-monthold severely obese ob/ob mice. Leptin (0.5–18 nM) did not affect insulin release together with 2.8–20 mM glucose. Leptin (18 µM) had no effect in the presence of low glucose (2.8–5.5 mM), but increased insulin secretion in islets challenged with 11.1 or 16.7 mM glucose. Leptin at 18 µM increased insulin secretion stimulated by the parasympathetic neurotransmitters acetylcholine (ACh; 10 µM) or vasoactive intestinal peptide (VIP; 10 nM), and by 5 mM theophylline or 2.5 µM forskolin. Overnight culture increased the effect of 18 µM leptin, but no effects were observed with 18 nM leptin. Pretreatment of islets with phorbol 12-myristate 13-acetate (PMA) did not suggest any involvement of protein kinase C. In summary, a high concentration of leptin stimulates insulin release in the presence of stimulatory concentrations of glucose alone and with parasympathetic neurotransmitters. Hyperleptinemia and increased parasympathetic stimulation may in part cause the hyperinsulinemia observed in obesity. This may aggravate insulin resistance and the abnormal metabolism in diabetes mellitus.     

The Pancreatic β-Cell Recognition of Insulin Secretagogues: Does Cyclic AMP Mediate the Effect of Glucose?

Insulin release and the content of cAMP were studied in microdissected pancreatic islets of noninbred ob/ob (obese) mice. In the absence of 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, 20 mM glucose had no effect on cAMP save a very small initial rise detectable by a freeze-stop perifusion technique only. However, combined with this methylxanthine, 20 mM glucose produced significant increases of cAMP both in perifused islets and in islets conventionally incubated in closed vials. Glucose shared this capacity to raise the cAMP level with D-glyceraldehyde and 1,3-dihydroxyacetone. Isobutylmethylxanthine (0.05-1.0 mM) or 5 mug/ml of cholera toxin, an activator of adenylate cyclase, also increased the islet cAMP level; the effects of the methylxanthine, whether or not combined with cholera toxin, were potentiated by glucose. Isobutylmethylxanthine (0.05-1.0 mM) or 5 mug/ml of cholera toxin potentiated insulin release in response to 20 mM glucose. However, only 0.5-1.0 mM isobutylmethylxanthine stimulated insulin release in the presence of 3 mM glucose, whereas 0.05-0.1 mM isobutylmethylxanthine or 5 mug/ml of cholera toxin had no effect on secretion at the low glucose concentration. These discrepancies between cAMP-promoting and insulin-releasing activities suggest that glucose does not initiate insulin release by activating the beta-cell adenylate cyclase. By being metabolized in the beta-cells, glucose may both create a release-initiating signal not identical with cAMP and enhance cAMP formation, leading to potentiation of the effect of the initiator signal.     

Function of microdissected pancreatic islets cultured in a chemically defined medium. I. Insulin content and release

Summary Microdissected pancreatic islets from non-inbredob/ob-mice, were cultured for 6 or 7 days in serum-free tissue culture medium 199. The insulin content of the islets decreased 60% during culture in 17 mM or 28 mM glucose and about 70% in the presence of 3.3 mM or 5.6 mM glucose. At the end of a culture period in high glucose, the sum of the insulin in the islet plus that in the culture medium was almost twice as high as the insulin content of fresh islets, indicating an active insulin biosynthesis. The maximal insulin response to glucose after culture in 17 mM or 28 mM glucose was about 40% of that in fresh islets; after culture in 3.3 mM glucose it was 10%. Half-maximal stimulation was observed at a glucose concentration of 5 mM for islets cultured with high glucose as compared to 9 mM for fresh islets. Like glucose, glibenclamide was a more effective insulin stimulator after culture with a high glucose concentration than with a low one. However, leucine-induced insulin release was not affected by the glucose concentration in the preceding culture medium. Whereas potentiation of glucose-stimulated release by arginine or dibutyryl-cAMP was independent of glucose concentration during the culture, theophylline released three times more insulin when the islets had been cultured with high glucose.     

Proinsulin synthesis in islets of Langerhans from spiny mice (Acomys cahirinus). comparison with rats and mice

Summary Proinsulin synthesis in response to different concentrations of glucose has been studied in islets of Langerhans of acomys, rat and mouse. The response to increased glucose was small in acomys when compared with rat or mouse. The time course of the effect of glucose on proinsulin synthesis was also studied in sequential 15 min periods up to 60 min. With 5.5 mM glucose the rate of³H-leucine incorporation for acomys islets was greater over the periods 15–30, 30–45 and 45–60 min after the addition of glucose to the islets than at 0–15 min. By contrast rat islets exhibited a greater glucose-induced stimulation between 15–30 and 30–45 min relative to the 0–15 min period and a further increase of the incorporation rate between 45 and 60 min. With 27.5 mM glucose acceleration of incorporation relative to the first 15 min period occurred at 15–30 min; it was then stable up to 60 min for islets both from acomys and from rat. Incorporation into total protein was much greater in acomys than in rat or mouse: thus in 2.75 mM glucose, acomys islets incorporated 5 and 19 times more³H-leucine into total protein than did islets from rat or mouse respectively. In 27.5 mM glucose the incorporation into total protein of the islets from acomys was 4 and 13 times greater. Islet content of insulin was similar for all three species. No significant changes in ATP content were observed in response to changes in glucose concentration from 2.75 to 27.5 mM. The results demonstrate a decreased responsiveness of proinsulin synthesis to glucose in acomys and are discussed in terms of the known decreased sensitivity of insulin release in this animal.This work was supported by Grant No. 3106073 from the Fonds National Suisse pour la Recherche Scientifique     

In vitro studies on insulin secretion in the genetically obese mouse

Summary Pancreatic insulin content and insulin secretion, from the pancreas of obese mice fed ad lib (ob/ob), obese mice maintained on a restricted diet (ob/ob RD) and lean mice has been studied using incubated pieces of pancreas in vitro and in a perifusion system. The ob/ob mice pancreas contained approximately twice as much insulin as the lean mice pancreas, whereas the ob/ob RD mice had a normal insulin content. Increasing the glucose concentration had a marked and prolonged stimulating effect on insulin secretion in the pancreas of the ob/ob and ob/ob RD mouse, but not in the lean mouse. Leucine stimulated insulin secretion in all three groups of animals both in the absence and presence of glucose in a biphasic manner; in the presence of glucose the insulin secreted from the pancreas of the ob/ob and ob/ob RD mice was abnormally high. Arginine stimulated insulin secretion from the lean mouse pancreas in the absence of glucose, whereas in the obese mouse pancreas stimulation was observed only in the presence of glucose, and the effect increased with increasing glucose concentration. The large amounts of insulin which can be secreted by the pancreas of the ob/ob and ob/ob RD mice under stimulation suggest that an abnormal response of the pancreas to biological stimuli of insulin secretion could be a primary defect in these animals.     

Insulin release in aging: dynamic response of isolated islets of Langerhans of the rat to D-glucose and D-glyceraldehyde

Glucose-stimulated insulin release is diminished in islets of Langerhans from older rats compared to that in islets from young controls. The causes of this age-related decrease in hormone release and its relationship to the hyperglycemia seen in aging populations have not been fully elucidated. In attempts to define this secretory defect, we demonstrated in static studies that the insulin secretion to D-glyceraldehyde is not diminished in aging. To gain further insight into the effects of D-glyceraldehyde vs. D-glucose in aging and to understand the dynamics of insulin release from islets of older rats, dynamic insulin release from isolated islets of 2.5- and 13-month-old rats was studied by the technique of perifusion to 2.8 mM and 16.7 mM D-glucose or 2.8 mM D-glucose with 5, 10, or 14 mM D-glyceraldehyde. Insulin secretion at nonstimulatory glucose concentrations (2.8 mM) was similar in the two groups of islets. Insulin release was reduced by 36% from islets of older rats incubated in the presence of 16.7 mM D-glucose, and the first phase of insulin release was largely blunted compared with that in islets from young controls. In the presence of 5.0, 10.0, or 14.0 mM D-glyceraldehyde (plus 2.8 mM D-glucose), total insulin secretion was similar from islets of older and young rats, and normal biphasic release was restored to islets from older rats. Response to the secretagogues was delayed by 1 min in studies on islets from older rats. These findings demonstrate that while the aging process leads to a profound defect in glucose-stimulated insulin release from the pancreatic beta-cell, this defect is not present with every secretagogue, since the normal secretory response is restored in the presence of D-glyceraldehyde. The differences in the insulin secretory responses to D-glucose and D-glyceraldehyde in islets from older rats support the hypothesis that the major rate-limiting step in stimulus-secretion coupling in aging is before the metabolism of the trioses.     

Further studies on the relationship between insulin release and lanthanum-nondisplaceable 45Ca2+ uptake by pancreatic islets: effects of fructose and starvation.

Relationships between the release of insulin and the incorporation of 45Ca2+ into a lanthanum-nondisplaceable (intracellular) pool were studied in islets microdissected from the pancreatic glands of non-inbred ob/ob mice. In comparison with D-glucose, D-fructose was slowly oxidized and had only marginal effects on insulin release. However, fructose was as effective as glucose in stimulating the lanthanum-nondisplaceable 45Ca2+ uptake. The 45Ca2+ uptake was dose-dependent on the concentration of fructose in the range 0-20 mM; the same dose-dependence was obtained with glucose. Fasting the mice for 3 days caused a total block of the insulin secretory response to 20 mM glucose, but it produced an enhancement of the glucose-induced 45Ca2+ uptake. Both the inhibition of insulin release and the enhancement of 45Ca2+ uptake were counteracted by pretreating the isolated islets with 20-40 mM D-glucose; pretreatment with L-glucose or fructose could not counteract the effects of fasting. Although some functional relationship may exist between the lanthanum-nondisplaceable uptake of 45Ca2+ and the insulin secretory apparatus, it is concluded that the uptake of Ca2+ is not simply the result of stimulated insulin release.     

Multiple effects of growth hormone on insulin release from isolated pancreatic islets

The direct effects of growth hormone (GH) on the endocrine pancreas were studied in isolated islets of rats. To study GH-induced insulin release, islets were incubated in RPMI 1640 medium containing 1 or 10 micrograms/ml of bovine GH for 120 minutes. Islets incubated in the absence of GH served as controls. During the incubation, GH significantly increased the insulin concentration in the medium. To study the effect of GH on subsequent glucose-induced insulin release, islets were preincubated with GH; then the islets were transferred to perifusion chambers and after a 30-minute stabilization period with 2.8 mM glucose, the islets were stimulated by addition of 16.7 mM glucose for 60 minutes. These perifusions were performed in the absence of GH. Glucose-induced insulin release from control and GH-pretreated islets peaked at five minutes during the first phase (0-8 minutes) and plateaued at 25 minutes during the second phase (9-60 minutes). Preincubation with GH (1 microgram/ml) did not change baseline or first phase release, but significantly suppressed the second phase of insulin release. When islets were preincubated with 10 micrograms/ml of GH, both phases of glucose-induced insulin release were suppressed; the total amount of insulin released by GH-pretreated islets was suppressed by 36.6% during the subsequent glucose stimulation period. These data indicate that GH stimulates insulin release by itself (GH-induced insulin release) but inhibits subsequent glucose-induced insulin release in a dose-dependent manner.     

Characterization of the insulinotropic potency of polyunsaturated fatty acids.

In this study we have assessed the individual abilities of the essential fatty acids, linoleic and linolenic acids, to release insulin and compared their insulinotropic potencies with those of the more established nutrient insulin secretagogues, glucose and arginine. In each experiment, a total of six islets microdissected from three mice were preperifused at the rate of 1 ml/min with Krebs-Ringer bicarbonate buffer, pH 7.4, containing 2% bovine albumin and 5.5 mM glucose (basal) with a continuous supply of 95% O2-5% CO2 at 37 C for 1 h. After collecting basal samples, the effects of 27.7 mM glucose, 20 mM arginine, 10 mM linoleic acid (18:2, omega 6), and 5 mM linolenic acid (18:3, omega 3) were tested using a sandwich protocol that entails 20-min alternating periods of stimulation with a secretagogue and a washout with basal perifusion. These nutrient concentrations were selected from initial experiments performed to characterize their dose-response effects on insulin secretion. Effluent samples were collected throughout each experiment for measurement of insulin by RIA. In one series of experiments, islets were challenged three times with 27.7 mM glucose, 10 mM linoleic acid, and 5 mM linolenic acid. In another set of experiments, islets were perifused with 20 mM arginine, 27.7 mM glucose, and 10 mM linoleic acid. All of these nutrients stimulated insulin release in a dose-dependent manner. In comparing the insulinotropic potencies of these secretagogues, we assessed insulin secretion as the integrated areas under the curve during 20 min of perifusion with a given nutrient. Thus, the mean integrated area under the curve per 20 min above basal in the presence of 27.7 mM glucose was 6,516 +/- 1,435 pg, which was not significantly different from the value of 4,772 +/- 866 pg obtained during arginine perifusion. However, the area under the curve during 20 min above basal obtained in the presence of linoleate and linolenic acid (8,712 +/- 1,949 and 10,506 +/- 1,490 pg, respectively) were significantly different (P less than 0.05) from those calculated during arginine and glucose perifusions. There was no statistically significant difference between the effects of these two fatty acids at the concentrations tested. In conclusion, our data suggest that linoleic acid and linolenic acid may be, at least in this murine islet preparation, as effective in stimulating insulin release as glucose and arginine, hitherto used to assess the abilities of nutrients to stimulate insulin secretion. However, it remains to be seen whether the efficacy of these polyunsaturated fatty acids in insulin release by murine islets will be obtained in experiments performed on human islets.     

Association between 5-hydroxytryptamine release and insulin secretion.

The efflux of radioactivity after loading with trace amounts of tritiated 5-hydroxytryptamine ([3H]5-HT) or 5-hydroxytryptophan ([3H]5-HTP) was studied in perifused beta-cell-rich pancreatic islets from ob/ob mice. Analysis of the effluent revealed that more than 90% of the radioactivity was released as [3H]5-HT after loading with [3H]5-HTP. Increasing the concentration of glucose in the perifusion medium from 3 to 20 mmol/l enhanced the efflux when islets from fed mice were used and this effect was potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Whereas 20 mM-glucose alone did not stimulate the efflux of 5-HT from islets isolated from mice starved for 3 days, a stimulatory effect was observed in the presence of IBMX. Stimulation of the efflux of radioactivity by glucose was inhibited if calcium was omitted from or adrenaline added to the medium. The results are consistent with the concept of exocytotic release of 5-HT occurring in response to stimulation of insulin secretion, although basal non-exocytotic transport must also be occurring across the beta-cell membrane.     

Glucose-induced insulin secretion from islets of fasted rats: modulation by alternate fuel and neurohumoral agonists

Islets from fed and 24-h-fasted rats were studied immediately after collagenase isolation. (1) After a 24-h fast, the insulin secretory responses to 8 mM glucose measured during perifusion were reduced by more than 90% from islets of fasted donors. (2) Increasing glucose to 11 or 27.5 mM resulted in enhanced insulin secretion from islets of fasted animals. (3) Fasting did not reduce islet insulin content. (4) Responses to 8 or 27.5 mM glucose were not affected if fatty acid-free albumin was used during the perifusion. (5) Inclusion of alpha-ketoisocaproate (5 mM), monomethyl succinate (10 mM) or carbachol (10 microM) significantly amplified insulin release from fasted islets in the simultaneous presence of 8 mM glucose. (6) Phospholipase C activation by glucose, carbachol or their combination was not adversely affected by fasting. (7) The response to the protein kinase C activator, phorbol 12-myristate 13-acetate (500 nM), was reduced by about 60% after fasting. (8) Extending the fast to 48 h resulted in a severe decline in response to 11 mM glucose; however, the further addition of 10 microM carbachol still enhanced release from these islets. The results confirm that caloric restriction impairs islet sensitivity to glucose stimulation and that protein kinase C may be involved in the reduction of glucose-induced insulin release from these islets. The activation of phospholipase C by cholinergic stimulation may contribute to the maintenance of insulin secretion from calorically restricted animals. These results also demonstrate that free fatty acids are not essential for glucose to evoke secretion from isolated islets of fasted donors.     

Inhibitors of phosphatidylinositol 3-kinase amplify insulin release from islets of lean but not obese mice

We examined the effects of phosphatidylinositol 3-kinase (PI3K) inhibition by wortmannin or LY294002 on glucose-induced secretion from mouse islets. Islets were collagenase isolated and perifused or subjected to Western blot analyses and probed for insulin receptor-signaling components. In agreement with previous studies, mouse islets, when compared with rat islets, were minimally responsive to 10 mM glucose stimulation. The inclusion of 50 nM wortmannin or 10 microM LY294002 significantly amplified 10 mM glucose-induced release from mouse islets. The effect of wortmannin was abolished by the calcium channel antagonist nitrendipine or by lowering the glucose level to 3 mM. Wortmannin had no effect on 10 mM alpha-ketoisocaproate-induced secretion. In contrast to its potentiating effect on islets from CD-1 mice, wortmannin had no effect on 10 mM glucose-induced release from ob/ob mouse islets. Western blot analyses revealed the presence of the insulin receptor, insulin receptor substrate proteins 1 and 2 and PI3K in CD-1 islets. These results support the concept that a PI3K-dependent signaling pathway exists in beta-cells and that it may function to restrain glucose-induced insulin secretion from beta-cells. They also suggest that, as insulin resistance develops in peripheral tissues, a potential result of impaired PI3K activation, the same biochemical anomaly in beta-cells promotes a linked increase in insulin secretion to maintain glucose homeostasis.     

Detailed protocol for evaluation of dynamic perifusion of human islets to assess β-cell function

The definitive measure of β-cell quality in an islet is the measurement of β-cell function, i.e., the ability of the islets to release insulin in a controlled manner in response to minute changes in ambient glucose levels. Continuous flow or dynamic perifusion of the solution containing glucose and secretagogues through the islets is the most accurate assessment of regulated insulin release in vitro. Here, we describe in detail a low cost, mini-perifusion system that can be adapted to any laboratory to assess islet function by examining dynamic insulin release in response to elevated glucose concentrations and addition of secretagogues. Human islets with purity >80% and viability >90% were perifused with low glucose (1 mM) and subsequently challenged with high glucose (16.8 mM ± KCl, 25 mM). A prototypical biphasic response to elevated glucose concentrations was observed with an average 8-fold (above basal) increase in insulin concentration at peak values. Similarly, perifusion with carbachol or exendin-4 (Byetta) with glucose (6 mM) resulted in 1.32- and 1.35-fold increase in insulin secretion above basal. Islets could be maintained in the perifusion apparatus and continued to respond to glucose for up to 3 h. At minimal financial cost and technical expertise, this apparatus can be set-up in any biological laboratory to evaluate regulated hormone release from many cell types in less than 6 h. This will allow other laboratories to measure insulin responses to their drug or modifier of interest in vitro, in a manner that better approximates islet function in vivo.     

Influence of the pituitary gland on insulin secretion in the genetically obese (ob/ob) mouse.

The influence of the pituitary gland of lean and genetically obese (ob/ob) mice on insulin secretion from microdissected pancreatic islets of lean and ob/ob mice has been studied by perifusing the pituitaries of these animals in series with the isolated islets and measuring insulin secretion at 5-min intervals over a period of 60 min. It has been shown that the pituitary perifusate of both lean and obese mice stimulate insulin secretion from lean mouse islets but not from obese mouse islets. The maximum stimulation occurs in the first 10 min and with the lean mouse pituitaries returns to the basal level in about 20 min, whereas with the obese mouse pituitaries insulin secretion is about double that from the control islets even after 40 min. A concentration of pure porcine ACTH equivalent to about three times the amount released from the pituitary gland under the experimental conditions used, caused only a small stimulation of insulin release. Possible interpretations of these findings and further lines of investigation are discussed.     

Nesfatin-1 exerts a direct, glucose-dependent insulinotropic action on mouse islet β- and MIN6 cells

Nesfatin-1 is a recently discovered multifunctional metabolic hormone abundantly expressed in the pancreatic islets. The main objective of this study is to characterize the direct effects of nesfatin-1 on insulin secretion in vitro using MIN6 cells and islets isolated from C57BL/6 mice. We also examined the expression of the nesfatin-1 precursor protein, nucleobindin 2 (NUCB2) mRNA, and nesfatin-1 immunoreactivity (ir) in the islets of normal mice and in the islets from mice with streptozotocin-induced type 1 diabetes and diet-induced obese (DIO) mice with type 2 diabetes. Nesfatin-1 stimulated glucose-induced insulin release in vitro from mouse islets and MIN6 cells in a dose-dependent manner. No such stimulation in insulin secretion was found when MIN6 cells/islets were incubated with nesfatin-1 in low glucose. In addition, a fourfold increase in nesfatin-1 release from MIN6 cells was observed following incubation in high glucose (16.7 mM) compared to low glucose (2 mM). Furthermore, we observed a significant reduction in both NUCB2 mRNA expression and nesfatin-1-ir in the pancreatic islets of mice with type 1 diabetes, while a significant increase was observed in the islets of DIO mice. Together, our findings indicate that nesfatin-1 is a novel insulinotropic peptide and that the endogenous pancreatic islet NUCB2/nesfatin is altered in diabetes and diet-induced obesity.     

Effects of 5-hydroxytryptamine on rubidium ion fluxes and insulin release in cultured pancreatic islets

We have incubated pancreatic islets isolated from noninbred ob/ob mice and NMRI mice for 3 days with or without 5-hydroxytryptamine (5-HT) in the medium and tested the effect of such long term treatment on subsequent insulin release and 86Rb+ accumulation and efflux. Two tenths millimolars of 5-HT abolished insulin release in response to 20 mM glucose. Two tenths millimolars of 5-HT also diminished the ability of islets to accumulate 86Rb+ and the effect of 10 mM glucose on 86Rb+ efflux. One one-hundredth millimolars of 5-HT had no effect on insulin release or 86Rb+ fluxes. Clearly, islets subjected to 5-HT for 3 days at concentrations that do not elicit demonstrable effects in short term incubations show a reduced secretory response. However, the physiological role of the high affinity uptake system for 5-HT in islet cells [Michaelis-Menten constant (Km) = 1.6 microM] remains unknown.     

Detailed protocol for evaluation of dynamic perifusion of human islets to assess β-cell function

The definitive measure of β-cell quality in an islet is the measurement of β-cell function, i.e., the ability of the islets to release insulin in a controlled manner in response to minute changes in ambient glucose levels. Continuous flow or dynamic perifusion of the solution containing glucose and secretagogues through the islets is the most accurate assessment of regulated insulin release in vitro. Here, we describe in detail a low cost, mini-perifusion system that can be adapted to any laboratory to assess islet function by examining dynamic insulin release in response to elevated glucose concentrations and addition of secretagogues. Human islets with purity > 80% and viability > 90% were perifused with low glucose (1 mM) and subsequently challenged with high glucose (16.8 mM ± KCl, 25 mM). A prototypical biphasic response to elevated glucose concentrations was observed with an average 8-fold (above basal) increase in insulin concentration at peak values. Similarly, perifusion with carbachol or exendin-4 (Byetta) with glucose (6 mM) resulted in 1.32- and 1.35-fold increase in insulin secretion above basal. Islets could be maintained in the perifusion apparatus and continued to respond to glucose for up to 3 h. At minimal financial cost and technical expertise, this apparatus can be set-up in any biological laboratory to evaluate regulated hormone release from many cell types in less than 6 h. This will allow other laboratories to measure insulin responses to their drug or modifier of interest in vitro, in a manner that better approximates islet function in vivo.     

Aromatic amino acids and pancreatic islet function: a comparison of L-tryptophan and L-5-hydroxytryptophan

L-Tryptophan (4 mM) did not affect insulin release at 3 mM glucose but strongly potentiated glucose-induced (10 mM) insulin release in microdissected ob/ob mouse islets. The effect was concentration dependent with half-maximum at about 5 mM. 10 mM L-glutamate also enhanced the effect of 10 mM D-glucose on insulin release but L-phenylalanine, L-tyrosine, L-alanine, glycine and L-glutamine did not. 0.1 mM benserazide and 0.1 mM alpha-monofluoromethyldopa did not inhibit the effect of L-tryptophan. 1 mM aminooxyacetate reduced the potentiating effect of L-tryptophan but not that of L-5-hydroxytryptophan. 10 mM indole pyruvate stimulated basal insulin release but inhibited the effect of glucose. 10 mM L-glutamine did not enhance the stimulatory effect of indole pyruvate. 10 mM L-5-hydroxytryptophan reduced the effect of 10 mM L-glutamine on glucose oxidation. L-5-Hydroxytryptophan did not influence 14CO2 production from islets preloaded with [14C]glutamine but reduced the oxidation rate when [14C]glutamine was present in the incubation medium. Both L-tryptophan and L-5-hydroxytryptophan potentiate insulin release. The underlying mechanisms probably differ but do not seem to involve transaminations. The effect of L-5-hydroxytryptophan may be coupled to the activity of aromatic L-amino acid decarboxylase.     

Calcium and pancreatic beta-cell function. 12. Modification of 45Ca fluxes by excess of K+

Glucose stimulation of insulin release is supposed to result from depolarization of the pancreatic beta-cells with subsequent influx of Ca2+. Isolated islets from non-inbred ob/ob-mice were employed for elucidating whether the glucose effects on the beta-cell handling of Ca2+ could be simulated by the depolarization evoked by excess of K+. Addition of 25 mM K+ was as effective as 20 mM glucose in stimulating the intracellular uptake of 45Ca. In both instances the additional amounts of incorporated 45Ca appeared in the mitochondria and the secretory granules. When analysing the washout pattern for 45Ca it was evident that the effects of raising K+ differed from those evoked by glucose. Whereas glucose inhibited 45Ca efflux during perifusion with Ca2+-deficient medium the addition of K+ resulted in a slight stimulation. Furthermore, the 45Ca incorporated in response to K+ was more readily mobilised.     

Comparison of glucose-induced changes in electrical activity, insulin release, lactate output and potassium permeability between normal and ob/ob mouse islets: effects of cooling

A comparison has been made between the glucose-induced changes in electrical activity, insulin release, lactate output and potassium permeability in normal and ob/ob obese (Norwich strain) mice. The electrical response of the islet membrane to high glucose (22.2 mmol/l) stimulation was different in the two types of mice, generating continuous spike activity in normal but producing bursts of activity in ob/ob mouse islets. The absolute amounts of insulin and lactate produced by ob/ob islets in response to both basal and high glucose concentrations were greater than the absolute amounts produced by normal islets, though the ratio of the amount produced in high glucose concentrations to the amount produced in basal glucose concentrations was not significantly different between normal and ob/ob islets for both parameters. Glucose-induced changes in potassium permeability were smaller in ob ob than in normal mice. Cooling from 37 to 27 degrees C, during steady-state glucose stimulation, reduced both lactate output and insulin release, the temperature coefficients being similar in both types of mice. The effect of temperature reduction on electrical activity was more marked in the islets of ob/ob mice than in those of normal mice; spike frequency was unaffected in normal but reduced in ob/ob mice, whereas spike amplitude was decreased in both. Cooling-induced inhibition of potassium permeability was greater in the islets of ob/ob mice than in those of normal mice.(ABSTRACT TRUNCATED AT 250 WORDS)