Effects of substrates for aromatic L-amino acid decarboxylase on insulin secretion

It has been shown that substrates for aromatic L-amino acid decarboxylase potentiate glucose-induced insulin release. Microdissected islets of obese-hyperglycemic mice (Ume? ob/ob) have now been used in a study of the effects of decarboxylase substrates on insulin release induced by secretagogues other than glucose. L-5-hydroxytryptophan (L-5-HTP) at 4 mmol/l potentiated the effect of 1 mumol/l glibenclamide, 20 mmol/l D,L-glyceraldehyde or 20 mmol/l K+, but not that of 50 mumol/l chloromercuribenzene-p-sulphonic acid. The potentiating effect of 4 mmol/l L-5-HTP, 4 mmol/l D,L-m-tyrosine, or 4 mmol/l D,L-o-tyrosine on insulin release induced by 20 mmol/l L-leucine was inhibited by 0.1 mmol/l benserazide. Benserazide did not reduce the effect of 10 mmol/l L-glutamine on L-leucine-induced insulin release. L-dihydroxyphenylalanine inhibited glucose-induced insulin secretion at 0.1 mmol/l with a tendency towards a reduction also at lower concentrations. The findings support the hypothesis that increased activity of aromatic L-amino acid decarboxylase can stimulate islet B cell function.     

Evidence for metabolic regulation of pancreatic glucagon secretion by L-glutamine

To investigate effects by L-glutamine on pancreatic A-cell secretion and intermediary metabolism, isolated pancreatic islets from normal and streptozotocin treated guinea pigs (A-cell rich islets) were incubated in the presence of glucose (5.5 mM) +/- L-glutamine (10 mM). Glutamine significantly enhanced glucagon release from 297 +/- 54 to 528 +/- 53 pg/micrograms DNA/h in normal islets and from 553 +/- 31 to 806 +/- 50 pg/micrograms DNA/h in A-cell rich islets. All results were expressed on the basis of islet DNA concentration, being 66 +/- 4 ng DNA per normal islet and 32 +/- 2 ng DNA per A-cell rich islet. Simultaneously, glutamine suppressed glucose oxidation to 64 per cent in normal islets and to 47 per cent of basal oxidation in A-cell rich islets. Islet content of ATP was also reduced by glutamine to about 60 per cent in A-cell rich islets, but not significantly changed in normal islets. Glutamine oxidation, at 5.5 mM-glucose, was considerably higher in A-cell rich islets (911 +/- 65 pmol/micrograms DNA/h) than in normal islets (313 +/- 52 pmol/micrograms DNA/h). Addition of porcine insulin (25 mU/ml) counteracted these effects by glutamine, i.e. suppressed glucagon release but increased glucose oxidation and ATP content of the A-cell rich islets. The present findings demonstrate that glutamine stimulates glucagon release and is readily metabolized by the A-cells. Furthermore, the regulation of glucagon secretion by glutamine appears to be reciprocally related to factors affecting glucose metabolism and ATP-levels in the A-cell.     

Differences in long-term effects of L-glutamine and D-glucose on insulin release from rat pancreatic islets

We have compared the effects of long-term exposure to L-glutamine or D-glucose on nutrient-induced insulin release from pancreatic islets of the rat. After 3 days of culture islets were finally tested in 1 h incubations for insulin responses to 16.7 mM of glucose, glutamine, leucine or a combination of leucine and glutamine. After culture at 11 mM glucose + 2 mM glutamine (A), glucose, leucine and glutamine stimulated release to a similar extent from islets. After culture at 1.7 mM glucose + 10 mM glutamine (B), only leucine stimulated insulin release. After culture at 11 mM glucose + 10 mM glutamine (C), both leucine and glutamine increased the insulin response. After culture at 1.7 mM glucose and 2 mM glutamine (D), only glutamine slightly stimulated release. After culture in high glutamine (B or C), a combination of leucine and glutamine significantly inhibited release as compared to leucine alone. A switch in culture media from B to A for 1 h prior to final incubations revived insulin release in response to glucose but not to glutamine. The reverse switch (A to B) abolished both subsequent glucose-and glutamine-induced insulin release. A switch from D to B revived an insulin response to leucine. Exposure of B-cells to 11 mM glucose during 30 min in another experimental system (perfused pancreas) induced a significant insulin response to subsequent stimulation with glutamine; this response was, however, only 17% of that to glucose per se observed in the same experiments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of 5-hydroxytryptophan potentiation of glucose-induced insulin release

We have previously shown that L-5-hydroxytryptophan (L-5-HTP) potentiates glucose-induced insulin release in islets isolated from ob/ob-mice. In the present study, the kinetics of this effect were further studied. The combined effect of glucose and L-5-HTP was dependent on the concentration of both compounds. The threshold concentration for the potentiating effect of 5-HTP was on the order of 0.01-0.1 mmol/l, and the maximal effect was reached at 1-4 mmol/l extracellular concentration. L-5-HTP enhanced glucose-induced insulin release without changing the apparent Km for glucose (9.5-10 mmol/l). The effect of L-5-HTP increased with time for about 40 to 60 min and was reversibly reduced when either L-5-HTP was omitted or the glucose concentration was lowered. These findings support the view that L-5-HTP is a strong potentiator of glucose-induced insulin release.     

Defective catabolism of D-glucose and L-glutamine in mouse pancreatic islets maintained in culture after streptozotocin exposure

We recently described a preferential reduction of the secretory response to nutrient secretagogues (glucose; leucine plus glutamine) in islets maintained in culture after in vitro exposure to streptozotocin (SZ). The present study is an attempt to further clarify the biochemical mechanisms behind this defective insulin response. Mouse pancreatic islets were collagenase isolated and, after 4-5 days in culture, exposed during 30 min at 37 C to 1.8 mM SZ or vehicle alone (controls). The islets were subsequently cultured for 7 days in medium RPMI 1640 plus 10% calf serum, before the enzymatic and metabolic studies were performed. The activities of the glycolytic enzymes, hexokinase, glucokinase, and glyceraldehyde 3-phosphate dehydrogenase, were similar in the control and SZ-exposed islets. The relative amount of cytosolic and mitochondria-bound hexokinase was also unaffected by SZ. However, there was a 30-40% decrease in the activity of NAD+- and NADP+-dependent glutamate dehydrogenase and glutamate-aspartate transaminase in the SZ-treated islets. This coincided with a 40% decrease in L-[U-14C]glutamine oxidation in the SZ-treated islets. The D-glucose catabolism was further examined in the presence of D-[5-3H] and D-[6-14C] glucose. There was no difference between control and SZ islets in terms of glucose utilization at either 1.7 or 16.7 mM glucose. The oxidation of D-[6-14C]glucose was nevertheless decreased by more than 50% in SZ islets incubated at 16.7 mM (but not 1.7 mM) glucose. Altogether, these converging observations suggest a perturbation of distal regulatory processes, apparently at the mitochondrial level, in the D-glucose and L-glutamine catabolism of SZ-exposed islets. Whether this reflects a primary action of SZ on the islet mitochondria, or an inhibitory effect of SZ on the synthesis of mitochondrial enzymes, as a result of nuclear DNA damage, remains to be elucidated.     

The stimulus-secretion coupling of glucose-induced insulin release XLVI. Physiological role of l-glutamine as a fuel for pancreatic islets

Exogenous L-glutamine is actively metabolized in rat pancreatic islets. The rate of L-glutamine deamidation largely exceeds the rate of glutamate conversion to γ-aminobutyrate and α-ketoglutarate. The latter conversion occurs in part by oxidative deamination, and in part by transamination reactions coupled with the conversion of 2-keto acids (pyruvate, oxaloacetate), themselves derived from the metabolism of glutamine, to their corresponding amino acids (alanine, aspartate). An important fraction of malate formed from α-ketoglutarate leaves the Krebs cycle and is converted to pyruvate, this process being apparently associated with the induction of a more reduced state in cytosolic redox couples. L-Glutamine abolishes the oxidation of endogenous fatty acids and stimulates lipogenesis. A sparing action of L-glutamine upon the utilization of endogenous nutrients is documented by the fact that the glutamine-induced increase in O₂ consumption is much lower than expected from the rate of ¹⁴CO₂ output from islets exposed to L-[U-¹⁴C]glutamine. L-Glutamine, although decreasing K⁺conductance, fails to stimulate insulin release both in the absence and presence of D-glucose. It is proposed that L-glutamine represents a major fuel for pancreatic islets under physiological conditions.     

Differential sensitivity to beta-cell secretagogues in cultured rat pancreatic islets exposed to human interleukin-1 beta

The early stages of insulin-dependent diabetes mellitus are characterized by a selective inability to secrete insulin in response to glucose, coupled to a better response to nonnutrient secretagogues. The deficient glucose response may be a result of the autoimmune process directed toward the beta-cells. Interleukin-1 (IL-1) has been suggested to be one possible mediator of immunological damage of the beta-cells. In the present study we characterized the sensitivity of beta-cells to different secretagogues after human recombinant IL-1 beta (rIL-1 beta) exposure. Furthermore, experiments were performed to clarify the biochemical mechanisms behind the defective insulin response observed in these islets. Rat pancreatic islets were isolated and kept in tissue culture (medium RPMI-1640 plus 10% calf serum) for 5 days. The islets were subsequently exposed to 60 pM human recombinant IL-1 beta during 48 h in the same culture conditions as above and examined immediately after IL-1 exposure. The rIL-1 beta-treated islets showed a marked reduction of glucose-stimulated insulin release. Stimulation with arginine plus different glucose concentrations, and leucine plus glutamine partially counteracted the rIL-1 beta-induced reduction of insulin release. The activities of the glycolytic enzymes hexokinase, glucokinase, and glyceraldehyde 3-phosphate dehydrogenase, were similar in control and IL-1-exposed islets. Treatment with IL-1 also did not impair the activities of NADH+- and NADPH+-dependent glutamate dehydrogenase, glutamate-aspartate transaminase, glutamate-alanine transaminase, citrate synthase, and NAD+-linked isocitrate dehydrogenase. The oxidation of D-[6-14C]glucose and L-[U-14C]leucine were decreased by 50% in IL-1-treated islets. Furthermore, there was a significant decrease in the ratios of [2-14C]pyruvate oxidation/[1-14C]pyruvate decarboxylation and L-[U-14C]leucine oxidation/L-[1-14C]leucine decarboxylation, indicating that IL-1 decreases the proportion of generated acetyl-coenzyme-A residues undergoing oxidation. However, in the presence of IL-1 there was a significant increase in L-[U-14C]glutamate oxidation. These combined observations suggest that exposure to IL-1 induces a preferential decrease in glucose-mediated insulin release and mitochondrial glucose metabolism. This mitochondrial dysfunction seems to reflect an impairment in proximal steps of the Krebs cycle. It is conceivable that the IL-1-induced suppression and shift in islet metabolism can be an explanation for the beta-cell insensitivity to glucose observed in the early phases of human and experimental insulin-dependent diabetes mellitus.     

The effects of vitamin A on insulin release and glucose oxidation in isolated rat islets.

We tested the effects of vitamin A, a membrane surface-active agent, on glucose (16.7 mM)-induced biphasic insulin release from collagenase-isolated rat islets. Also, efforts were made to correlate the effects of vitamin A with glucose oxidation. Vitamin A (10(-4) M) inhibited first- and second phase insulin release; 10(-5) M vitamin A inhibited second phase release only and to a lesser extent than that observed with 10(-4) M vitamin A; and 10(-6) M vitamin A had no effect. Vitamin A (10(-7) M) stimulated biphasic insulin release. Exposure to high glucose (27.8 mM) overcame the effects of 10(-4) M vitamin A on first phase release, but not on second phase release of insulin. Exposure to 10(-5) M hydrocortisone opposed the effects of 10(-4) M vitamin A on both phases of insulin release. Vitamin A (10(-4) and 10(-5) M) inhibited glucose oxidation by islets, as measured by the production of 14CO2 from [14C]glucose. The effects of vitamin A on insulin release were dissociated in part from those effects on glucose oxidation, in that hydrocortisone opposed the effect of vitamin A on insulin release but not on glucose oxidation. The effects of vitamin A on insulin secretion can best be explained by the interaction of vitamin A at multiple sites affecting the membrane and intracellular glucose oxidation.     

L-谷氨酰胺对克隆的胰岛β细胞株INS-1E细胞和小鼠胰岛的急性刺激作用

目的探讨L-谷氨酰胺对INS-1E细胞和小鼠胰岛分泌胰岛素的作用。方法INS-1E细胞经传代培养2d后，在Krebs-Ringer缓冲液中37℃培养箱预培养30min，再用含有不同浓度葡萄糖和不同浓度L-谷氨酰胺的改良Krebs-Ringer缓冲液培养60min，然后留取上清液进行胰岛素测定。雌性NMRI小鼠，6～10周龄，苯巴比妥腹腔麻醉，应用胶原酶技术消化胰腺分离胰岛，置于RPMI1640培养皿中在37℃培养箱（5％CO2，95％空气）过夜培养。次日在Krebs-Ringer缓冲液中37℃水浴培养箱预培养30min，然后分别把单个胰岛小心放入100bd含有不同浓度葡萄糖和不同浓度L-谷氨酰胺的改良Krebs-Ringer缓冲液37℃水浴培养箱培养60min，然后留取50μ1上清液进行胰岛素测定。结果L-谷氨酰胺在0．1～5mmol／L范围不增加葡萄糖刺激的INS-1E细胞的胰岛素分泌，仅10～20mmol／L的L-谷氨酰胺促进葡萄糖诱导的胰岛素分泌。L-谷氨酰胺在0．1～10mmol／L范围不促进葡萄糖诱导的小鼠胰岛的胰岛素分泌，仅20mmol／L的L-谷氨酰胺促进葡萄糖诱导的胰岛素分泌。结论大剂量L-谷氨酰胺能增加葡萄糖诱导的INS-1E细胞和小鼠胰岛分泌胰岛素。     

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.     

Inhibitory effect of deuterium oxide on glucose oxidation by pancreatic islets

The inhibition of insulin release by deuterium oxide has been interpreted by its stabilizing action on the microtubular system of the β-cell. Because only indirect information exists about β-cell metabolism in the presence of D₂O, the effect of D₂O (99.7%) on glucose oxidation by pancreatic islets was studied.

Heavy water reduced ¹⁴CO₂ formation from [U-¹⁴C]glucose (16.7 mM) by ob/ob mouse and rat islets by 50 and 30%, resp. Omission of calcium from H₂O-containing media resulted in a 40% diminished glucose oxidation by ob/ob mouse islets. In the absence of calcium, D₂O led to a further decrease in ¹⁴CO₂ production (60%). Erythrocytes oxidized similar amounts of glucose in the absence and presence of D₂O.

Effects of D₂O on glucose transport and mitochondrial respiration are discussed as possible causes of the decreased ¹⁴CO₂ formation in islet tissue. It is concluded that the inhibition of insulin release by D₂O need not necessarily be ascribed to its stabilizing effect on microtubules, but may also be due — at least in part — to the reduction of islet glucose oxidation.     

Insulin secretion and the morphological and metabolic characteristics of pancreatic islets of hyperthyroid ob/ob mice.

Thyroxine treatment induced experimental hyperthyroidism in ob/ob mice, inhibited glucose-induced insulin secretion from the isolated perfused ob/ob mouse pancreas, and reduced total pancreas insulin content. In contrast, glucose-induced insulin release from incubated pancreatic islets and insulin content of pancreatic islets from ob/ob mice isolated by freehand microdissection were not reduced after thyroxine treatment when expressed per microgram dry islet. Histological examination of the ob/ob mouse pancreas revealed islets without degenerative lesions of islet cells. Granularity of beta cells was well preserved. The average number of pancreatic islets was unchanged. However, the beta cell area was significantly decreased in relation to the total pancreatic parenchyma after thyroxine treatment. This implies that insulin release and content per pancreatic islet was half of that of the controls. ATP content of islets was slightly reduced. Glucose oxidation and glucose utilization by islets from treated mice were slightly increased. Thyroxine treatment of the animals did not abolish the stimulation of 45Ca2+ uptake by glucose, but it did suppress the potentiating effect of fasting on the stimulatory effect of glucose on 45Ca2+ uptake. The metabolic characteristics of islets from experimentally hyperthyroid mice are those of all hyperthyroid tissues. The results provide no evidence for the view that the effects of thyroxine treatment may be due to disturbed metabolic function or energy deprivation of pancreatic islets. Inhibition of insulin secretion from the pancreas after thyroxine administration is apparently due to a reduction in pancreas insulin content and a diminished pancreatic islet volume. Reduced pancreatic islet volume represents most probably a reduction of individual islet cell volume.     

Long-term leptin treatment of ob/ob mice improves glucose-induced insulin secretion.

OBJECTIVE: Previous studies have demonstrated that leptin inhibits glucose-stimulated insulin secretion from isolated islets, although a lack of leptin effect on insulin secretion has also been reported. The effect of long term in vivo leptin treatment of insulin secretion has, however, not been established. Therefore, in the present study, we have evaluated the effect of long term in vivo treatment of leptin on glucose-induced insulin secretion in ob/ob mice. METHODS: After 7 days' treatment of leptin (100 microg daily s.c.), insulin release was measured in isolated islets by batch incubation followed by radioimmunoassay. Glucose utilization and oxidation were measured by measuring the formation of (3)H(2)O and (14)CO(2) from [5-(3)H] and [U-(14)C] glucose, respectively. Glucose-6-phosphatase activity was measured by measuring the conversion of (14)C-glucose-6-P to (14)C-glucose. In addition, immunohistochemistry of pancreatic specimens was undertaken for study of expression of insulin, GLUT-2 and hormone-sensitive lipase (HSL). RESULTS: Leptin treatment significantly improved insulin secretion both at 5.5 mM (by 15%; P<0.05) and 16.7 mM (by 85%; P<0.001) glucose, compared to vehicle-treated controls. Furthermore, whereas leptin treatment did not affect islet insulin or DNA contents, a significant decrease in islet triglyceride content and glucose-6-phosphatase activity was observed. Moreover, the immunocytochemical data revealed an increased immunostaining for insulin, GLUT-2 and hormone-sensitive lipase (HSL) in islets from leptin-treated ob/ob mice. CONCLUSION: The results suggest that long-term leptin treatment of ob/ob mice improves glucose-stimulated insulin secretion in parallel with reduced glucose-6-phosphatase activity, increased HSL and decreased triglyceride levels in islets. These perturbations may explain the improvement of glucose-stimulated insulin secretion induced by leptin.     

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.     

Stimulation of pancreatic islet metabolism and insulin release by a nonmetabolizable amino acid.

The leucine analog beta-2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) activates glutamate dehydrogenase [L-glutamate:NAD+ oxidoreductase (deaminating), EC 1.4.1.2] in pancreatic islet homogenates. In intact islets, BCH increased the islet content or output of NH4+, 2-ketoglutarate, malate, pyruvate, and alanine. BCH caused a dose-related increase in 14CO2 output from islets prelabeled with L-[U-14C]glutamine. BCH increased the islet content of ATP and stimulated both 45Ca net uptake and insulin release. The capacity of seven distinct amino acids to activate glutamate dehydrogenase tightly correlated with their ability to augment 14CO2 output from islets prelabeled with [U-14C]-glutamine and to stimulate insulin release in the presence of L-glutamine. The activation of glutamate dehydrogenase by BCH may thus account for the insulin-releasing capacity of the leucine analog.     

Effect of a hypoglycaemic sulphonylurea (HB419) and a non-metabolizable amino acid (BCH) on the insulin release and endogenous substrate metabolism of rat pancreatic islets

The effects of the hypoglycaemia sulphonylurea glibenclamide (HB419) and the non-metabolizable leucine analogue beta-2-aminobicyclo(2.2.1)heptane-2-carboxylic acid (BCH) on insulin release and endogenous substrate metabolism were studied in isolated rat islets. Pre-labelling of the endogenous islet substrate was performed with [14C]glucose (20 mM) or [14C]glutamine (10 mM) during a 24 h tissue culture period before measurements of insulin release or 14CO2 production in short-term incubations. Both HB419 and BCH stimulated the insulin release of the cultured islets, although BCH only after culture of islets with glutamine. The rate of labelling of the islets with [14C]glucose reached an apparent plateau after 16 h in culture and the total islet accumulation of glucose carbon over the 24 h period averaged 12.9 +/- 3.0 nmol/25 islets. Less than 0.5% of the glucose residues was converted to glycogen whereas lipids represented about 2.5%. Fractionation of lipids showed 67% phospholipids, 18% triacylglycerols, 11% diacylglycerols and 6% non-esterified fatty acid. The islet accumulation of glutamine during 24 h corresponded to 11.5 +/- 1.5 nmol/25 islets. After pre-labelling of islets with [14C]glucose there was no effect on the 14CO2-evolution over a 30 min incubation period of either HB419 or BCH. There was also no effect of HB419 after pre-labelling with [14C]glutamine, whereas, in this latter situation, a significant stimulation was observed with BCH. It is concluded that the effects on the pancreatic B-cells by antidiabetic sulphonylureas are not mediated via nutrient receptors.     

Human interleukin-1β induced stimulation of insulin release from rat pancreatic islets is accompanied by an increase in mitochondrial oxidative events

Summary Acute exposure of pancreatic islets to interleukinl-1 results in an increase in insulin release, while an extension of the exposure time induces a functional suppression and eventually, destruction of the B-cells. We have recently suggested that the interleukin-1 induced inhibition of islet function is mediated through an impairment in oxidative metabolism. The aim of the current study was to investigate if the acute, stimulatory effects of interleukin-1 on islet function could also be related to changes in the substrate metabolism. For this purpose, rat islets were exposed for 90–120 min to 30 pmol/l human recombinant interleukin-1 (biological activity of 2.5 U/ml) and their function and metabolism characterized during this period. The cytokine did not increase insulin release in the presence of 1.7 or 5.5 mmol/l glucose but in both the presence of 16.7 mmol/l glucose or 10 mmol/l leucine + 2 mmol/l glutamine there was a 50% increase in insulin release. Interleukin-1 exposure increased the oxidation of D-[U-¹⁴C]glucose at 5.5 mmol/l glucose by 25% and at 16.7 mmol/l glucose by 60%. Carbohydrate and amino acid metabolism were further examined in the presence of D-[5-³H] glucose, D-[6-¹⁴C]glucose, [1-¹⁴C]pyruvate, L-[U-¹⁴C]glutamine, L-[U-¹⁴C]leucine and L-[1-¹⁴C]leucine. There was no difference between control islets and interleukin-1 exposed islets in terms of D-[5-³H]glucose utilization or [1-¹⁴C]pyruvate decarboxylation, but the oxidation of D-[6-¹⁴C]glucose was increased by 64% in the interleukin-1 exposed islets. There was also an interleukin-1 induced 45–60% increase in the decarboxylation of L-[1-¹⁴C]leucine and oxidation of L-[U-¹⁴C]leucine and L-[U-¹⁴C]glutamine, all intramitochrondrial events. The stimulation of insulin release by interleukin-1 in the presence of 16.7 mmol/l glucose was abolished in islets incubated in Ca²⁺ depleted medium, but the rate of D-[6-¹⁴C] glucose oxidation remained elevated (47% increase at 16.7 mmol/l glucose). These data indicate an increase in substrate metabolism at the mitochondrial level during acute exposure of rat pancreatic islets to interleukin-1. The increase in oxidative events can explain the observed interleukin-1 induced increase in insulin release during glucose stimulation. Furthermore, these findings raise the possibility that mitochondria are primary targets of interleukin-1 action in the B-cells.     

Biochemical mechanisms involved in monomethyl succinate-induced insulin secretion.

Esters of succinic acid stimulate insulin secretion from pancreatic beta-cells. Using collagenase-isolated rat islets, the transduction mechanisms involved were investigated. In freshly isolated perifused islets, monomethyl succinate (MMSucc), in the presence of basal (2.75 mM) glucose, stimulated insulin release in a biphasic pattern. This secretory response was dependent on extracellular calcium movement into the beta-cell, since the calcium channel blocker nitrendipine (5 microM) abolished it. The glucokinase inhibitor mannoheptulose (20 mM) had no effect on its secretory action, while the protein kinase-C inhibitor staurosporine (20 nM) reduced secretion to MMSucc. In addition, while ineffective alone, the diacylglycerol kinase inhibitor monooleoylglycerol (25 microM) potentiated MMSucc-induced insulin release. A similarly amplified response occurred in the presence of forskolin (0.25 microM), a compound that elevates islet cAMP levels. The sodium salt of succinic acid (20 mM) had no effect on insulin release in the presence or absence of forskolin. Prior treatment with MMSucc in the presence of 2.75 mM glucose sensitized islets to the usually weak insulin secretory effect of 7.5 mM glucose. Other groups of islets were incubated for 2 h with myo-[2-3H]inositol to label their phosphoinositide pools. These islets were subsequently stimulated, and the kinetics of [3H]inositol efflux and insulin secretion were measured. MMSucc induced a rapid and sustained dose-dependent increase in [3H]inositol efflux rates. In batch-incubated islets, MMSucc increased inositol phosphate levels. Finally, MMSucc (20 mM), in the presence of 8 mM glucose, did not influence the detritiation of [5-3H]glucose, but reduced the oxidation of [U-14C] glucose. These results support the following conclusions. First, MMSucc is a potent activator of islet phosphoinositide hydrolysis. Second, the activation of protein kinase-C appears to contribute to the acute insulin secretory effect of MMSucc. Third, MMSucc-induced increases in phosphoinositide hydrolysis contribute at least in part to its ability to acutely stimulate insulin release and prime the beta-cell to subsequent stimulation. Finally, mitochondrial events associated with the oxidative metabolism of MMSucc may underlie its insulinotropic action.     

The stimulus-secretion coupling of glucose-induced insulin resease XXVI. Are the secretory and fuel functions of glucose dissociable by iodoacetate?

Iodoacetate inhibits glyceraldehyde-3-phosphate dehydrogenase activity in pancreatic islets and causes a time- and dose-related inhibition of glucose oxidation and lactate output by the islets. High concentrations of the drug (0.3 mM or more) fail to affect Ba2+-induced insulin secretion but inhibit glucose-stimulated proinsulin biosynthesis, 45Ca net uptake and insulin release. A mixture of fumarate, glutamate, and pyruvate, the oxidation of which is only partially reduced by iodoacetate, fails to protect the B-cell against the inhibitory effect of the drug. These findings are compatible with the view that glycolysis plays an essential role in the process of glucose-induced insulin release. At low concentrations of iodoacetate (up to 0.2 mM), the reduction in glucose metabolism coincides with a partial inhibition of proinsulin biosynthesis. However, the expected reduction in 45Ca net uptake and subsequent insulin release is masked by a concomitant facilitating action of iodoacetate, possibly due to interference with native ionophoretic processes. It is concluded that iodoacetate is not an adequate tool to dissociate, if they are dissociable, the fuel and secretory functions of glucose.     

Effects of porcine pancreastatin on secretion and biosynthesis of insulin and glucose oxidation of isolated rat pancreatic islets

The effects of porcine pancreastatin were studied on insulin secretion induced by glucose and nonnutrient stimuli, insulin biosynthesis, and glucose oxidation of cultured rat islets. Pancreastatin (100 nM) significantly suppressed, by 32-52%, the insulin response to 27, 16.7, 11, and 5.5 mM but not to 50 mM glucose, whereas 10 nM pancreastatin inhibited insulin release significantly only at 11 and 5.5 mM glucose. Pancreastatin (10 and 100 nM) also suppressed release induced by 20 mM arginine (by 26 and 30%) as well as by 1 microgram/ml of glibenclamide (by 56 and 72%, respectively). Pancreastatin (10 and 100 nM) furthermore inhibited insulin release induced by 0.1 mM 3-isobutyl-1-methylxanthine (IBMX) (by 40 and 61%, respectively) and 1.0 mM IBMX (by 44 and 76%, respectively). Neither glucose oxidation nor overall insulin biosynthesis in islets was significantly affected by pancreastatin, although a slight but significant enhancement of biosynthesis was noted at 1.7 mM glucose in the presence of 100 nM pancreastatin. In conclusion, these data demonstrate that porcine pancreastatin suppresses glucose-induced insulin response from isolated rat islets in a competitive manner. This effect seems not to be exerted through a suppression of (pro)insulin biosynthesis or glucose metabolism in the islets, and thus the effect mediated by pancreastatin must be on a step distal to the coupling between islet glucose metabolism and insulin secretion. The relatively strong inhibition by the peptide of IBMX-induced insulin release suggests that it acts on the cAMP system of islet B cells.