Influence of nicotinic acid on insulin release in vitro

Summary Nicotinic acid stimulates insulin output of isolated mice islets in correlation with the drug concentration but regardless of glucose supply. Nicotinamide and also the pyridine nucleotides were ineffective on the insulin output. 6-amino-nicotinamide inhibits the glucose-stimulated insulin release. The results are discussed in connexion with the significance of the pentose phosphate shunt for the regulation of insulin secretion.

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Zusammenfassung Nicotinsäure stimuliert glucoseunabhängig, aber dosisabhängig, die Insulinsekretion isolierter Mäuseinselnin vitro. Nicotinamid, wie auch die Pyridinnucleotide haben keine Wirkung, auf die Hormonabgabe, während 6-Aminonicotinamid die glucose-stimulierte Insulinsekretion hemmt. Die erhobenen Befunde werden im Zusammenhang mit der Bedeutung des Pentosephosphatshunts für die Regulation der Insulinsekretion diskutiert.

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Resumen El ácido nicotínico estimula la liberación de insulina por parte de islas aisladas de ratón, en relación con la concentración del producto, pero independientemente de la aportación de glucosa. La nicotinamida y los nucleótidos piridínicos carecen de efecto sobre la liberacción del hormón. La 6-amino-nicotinamida inhibe la liberación de insulina estimulada por glucosa. Estos resultados se discuten en relación con la importancia delshunt de los pentosa-fosfatos para la regulación de la secreción insulínica.

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Résumé L'acide de nicotine stimule la libération d'insuline de la part d'îlots isolés de rat, par rapport à la concentration du médicament, mais indépendamment de l'apport de glucose. La nicotinamide et les nucléotides pyridiniques n'ont aucun effet sur la libération de l'hormone. La 6-amino-nicotinamide inhibe la libération d'insuline stimulée par le glucose. Ces résultats sont examinés par rapport à l'importance dushunt des pentose-phosphates pour la régularisation de la sécrétion insulinique.

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Riassunto L'acido nicotinico stimola la liberazione di insulina da parte di isole isolate di topo, in rapporto alla concentrazione del farmaco, ma indipendentemente dell'apporto di glucosio. La nicotinamide ed i nucleotidi piridinici sono privi di effetto sulla liberazione dell'ormone. La 6-amino-nicotinamide inibisce la liberazione insulinica stimolata dal glucosio. Questi risultati vengono discussi in relazione all'importanza delloshunt dei pentoso-fosfati per la regolazione della secrezione insulinica.

     

Immaturity of insulin secretion by pancreatic islets isolated from one human neonate

Human β‐cells are functionally mature by the age of 1 year. The timeline and mechanisms of this maturation are unknown owing to the exceptional availability of testable tissue. Here, we report the first in vitro study of insulin secretion by islets from a 5‐day‐old newborn. Glucose was inefficient alone, but induced insulin secretion, which was concentration‐dependent, showed a biphasic time‐course and was of similar magnitude as in infant islets when β‐cell cyclic adenosine monophosphate was raised by forskolin. Tolbutamide alone was effective in low glucose, but its effect was not augmented by high glucose. Metabolic amplification by glucose was thus inoperative, in contrast to amplification by cyclic adenosine monophosphate. Newborn islets showed high basal insulin secretion that could be inhibited by diazoxide or omission of CaCl₂. Postnatal acquisition of functional maturity by human β‐cells implicates control of basal secretion and production of metabolic signals able to activate both triggering and amplifying pathways of insulin secretion.     

Influence of isolated insulin antibodies on the insulin secretion of the islets of Langerhans in vitro

Summary Mouse islets of Langerhans, isolated by microdissection after treatment with collagenase, were incubated either with pure insulin antibodies (IAB), which were prepared by immune precipitation, or with exogenous insulin. Insulin release was enhanced with increased concentrations of IAB and was inhibited by exogenous insulin. The results suggest that it was not the insulin per se, but probably its biological effect on the -cells that influenced insulin secretion.     

Effect of extracellular pH on insulin secretion and glucose metabolism in neonatal and adult rat pancreatic islets

Changes in extracellular pH are known to affect glucose-stimulated insulin secretion. In the present study, glucose metabolism in pancreatic islets cultured at different pHs was investigated. Also, for islet transplantation purposes, insulin secretion and glucose metabolism were compared in neonatal and adult islets at different pHs to determine which islet preparation is more tolerant to acidity and alkalinity. The results revealed a dependency of insulin secretion on the external pH in both neonatal and adult islets. Reduction of insulin secretion was observed at both the acidic and alkaline sides of pH 7.3. Glucose stimulated increases of insulin secretion in all cases. Similar results were obtained for ATP and pyruvate contents. Intracellular insulin increased with the increase of pH value. In contrast, calcium content decreased with the increase of pH. The results demonstrate that neonatal islets are more acid tolerant than adult islets. Both basal and glucose-stimulated insulin secretions, as well as other parameters of neonatal islets were significantly higher than those of adult islets in response to low pH. The differences under alkaline conditions were not significant but give an indication that neonatal islets are more tolerant to alkalinity than are adult islets. Received: 10 February 2001 / Accepted in revised form: 29 June 2001     

In vitro effect of exogenous insulin on insulin secretion. Studies with glucose,leucine, arginine,aminophylline and tolbutamide

Summary Using rat pancreatic islets and the perfused rat pancreas, the effect of exogenous insulin on insulin secretion mediated by glucose, leucine, arginine, aminophylline and tolbutamide was studied. (1) In both systems the insulin releasing capacity of glucose was inhibited by exogenous insulin. In the perfused pancreas the inhibition concerned the first and the second phase of insulin release; (2) the EC50 (half-maximal inhibitory effect of insulin on glucose-induced insulin secretion) in islets was 1.2 nM (=160 μU/ml) and 2.8 nM (390 μU/ml) in perfused pancreas; (3) exogenous insulin also inhibited insulin release in response to leucine and arginine in the isolated islet system and in the perfused pancreas; (4) using aminophylline and tolbutamide in combination with glucose, the extent of the inhibitory effect of insulin was in the range of the inhibitory effect when glucose was used alone as stimulator in islets. Data suggest that the insulinogenic action of physiological stimulators including glucose, leucine and arginine is inhibited by exogenous insulin whereas this seems not to be the case when insulin release was stimulated by aminophylline and tolbutamide. Comparing the EC50s, isolated islets seem to be more sensitive to inhibition than the perfused pancreas when glucose was used as stimulator. As far as glucose is concerned the inhibitory effect seems to depend on the extent of its concentration and/or the extent to which the mechanism of insulin release is sensitive to stimulation. The EC50 of the inhibitory effect of exogenous insulin was in the range of dissociation constant of binding of insulin to insulin receptors of islets. Preliminary data on this study have been presented at the 22th Spring Meeting of theDeutsche Pharmakologische Gesellschaft, Mainz, F.R.G., March 10–13, 1981. This study was supported by grants of theDeutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, F.R.G.     

Impairment of the priming effect of glucose on insulin secretion from isolated islets of aging rats

The time-dependent potentiation (TDP) of insulin release or priming effect exerted by glucose was evaluated in the islets of Langerhans of mature and old rats. Islets isolated from 12-and 26-month-old male Sprague-Dawley rats and incubated for two consecutive 60-min periods in the presence of various stimulating agents were unable to enhance their insulin responsiveness significantly during the second incubation period and showed other abnormalities in their sensitivity to secretagogues compared with islets from 3-month-old animals. The priming action of glucose plus arginine or isobutylmethylxanthine (IBMX) was not observed in islets from 12-month-old rats, but surprisingly, islets from senescent rats showed a restoration of the beta-cell memory in the presence of IBMX. Interestingly, the islets isolated from 2-month-old animals previously exposed to an intravenous glucose load in vivo released approximately twice as much insulin as the islets taken from fed rats not subjected to the load. This potentiation exerted by the intravenous glucose administration was reduced but not abolished in the islets of glucose-intolerant, 12-month-old rats. In conclusion, the glucose TDP of insulin secretion is impaired in islets of mature and old rats, confirming an early loss of sensitivity of beta-cells to secretagogues during aging.     

Effect of amino-acids on secretion of insulin by isolated islets of the monkey

Summary Insulin secretion was monitored in monkey islets isolated by collagenase digestion and exposed to leucine and arginine with and without glucose. Leucine by itself (10 to 40 mmol/l) elicited concentration-dependent insulin secretion. At 40 mmol/l, leucine was approximately 60% as effective as glucose (16.7 mmol/l). The response to leucine was increased at low glucose concentrations. In high concentrations (20 and 40 mmol/l), arginine by itself was a poor stimulant. The effect of arginine was enhanced at low glucose concentrations (2.8 to 5.6 mmol/l). At high glucose concentrations neither amino-acid produced any significant further increase in insulin release.     

The effect of monooleoylglycerol on insulin secretion from isolated perifused rat islets

Summary The effect of monooleoylglycerol on cholecystokinin-and tolbutamide-induced insulin secretion was examined in isolated perifused rat islets. In the presence of 5.5 mmol/l glucose, addition of 10 nmol/l cholecystokinin or 50 mol/l tolbutamide had practically no effect on insulin secretion. Combined tolbutamide and cholecystokinin led to a biphasic insulin secretory response which was significantly enhanced by addition of 50 mol/l monooleoylglycerol, an inhibitor of diacylglycerol kinase. Monooleoylglycerol (50 mol/l) alone had a minimal stimulatory effect on insulin release in the presence of 5.5 mmol/l glucose. Perifusion of islets with 1 mol/l forskolin had no significant effect on basal insulin secretion in the presence of 5.5 mmol/l glucose, but markedly enhanced the responses to both cholecystokinin plus tolbutamide, and to the combination of cholecystokinin, tolbutamide and monooleoylglycerol. Lowering the glucose level to 2.75 mmol/l abolished the profound stimulatory effect to these agonist combinations on insulin release. Finally, monooleoylglycerol also enhanced the first and second phase insulin secretory responses induced by 20 mmol/l glucose. These results are discussed in relationship to the possible role of protein kinase C in mediating insulin secretion.     

Mechanisms of octanoic acid potentiation of insulin secretion in isolated islets

ABSTRACT

A potentiating effect of medium-chain triglycerides on glucose-stimulated insulin secretion (GSIS) has been observed since the 1960s. Subsequent observations identified octanoic acid (OA), the main component of medium-chain triglyceride, as the potentiator of GSIS, but the mechanism was unclear. We used wild-type (WT), short-chain 3-hydroxyacyl-CoA dehydrogenase knockout (Hadh^-/-), and sulfonylurea receptor 1 knockout (Sur1^-/-) mouse islets to define the mechanism of OA potentiation of insulin secretion. Application of OA alone induced a 2- to 3- fold increase of insulin secretion with an apparent threshold of 3 mM in WT mouse islets, suggesting that OA itself is a weak insulin secretagogue. However, OA at 1 mM strongly potentiated fuel-stimulated insulin secretion, especially GSIS. The potentiating effect on fuel-stimulated insulin secretion by OA did not require fatty acid β-oxidation because OA also potentiated amino acid-stimulated insulin secretion in islets isolated from Hadh^-/- mice, which cannot fully oxidize OA. Measurements using Sur1^-/- islets indicated that the potentiating effect of OA on fuel-stimulated insulin secretion is Ca²⁺ dependent and is often accompanied by β-cell membrane potential depolarization, and may also involve the Ca²⁺/calmodulin complex. Experiments using DCPIB, an ethacrynic acid derivative, to inhibit volume-sensitive anion channels (VSACs) in Sur1^-/- islets demonstrated that the potentiation effects of OA on insulin secretion are in part medicated by activation of VSAC. In addition, inhibition of IP3 receptor also abolishes the OA-induced intracellular Ca²⁺ increase in Sur1^-/- islets.     

Effect of enkephalins and morphine on insulin secretion from isolated rat islets

Summary The direct effects of an enkephalin analogue, (D-Ala²/MePhe⁴/Met/(O)-ol) enkephalin (DAMME), on insulin release from isolated islets of Langerhans of the rat have been investigated. DAMME had a dose-dependent effect on insulin secretion: low concentrations (10^–10 to 10^–8 mol/l) were stimulatory while high concentrations (10^–5mol/l) were inhibitory in the presence of 8 mmol/l glucose. Similar effects were found with met-enkephalin, and with the longer acting alanine substituted metenkephalin. Morphine sulphate (5 sx 10^–7 mol/l) also stimulated insulin release. The effects of enkephalin and morphine were blocked by the specific opiate antagonist naloxone hydrochloride (1.2 × 10^–6 mol/l). The insulin secretory response of perifused islets to enkephalins and morphine was rapid, corresponding to the first phase of glucose induced insulin release. These observations suggest that there may be opiate receptors in islets, and that opioid peptides could modulate insulin release.     

Relationship between carbohydrate tolerance,insulin secretion,and insulin sensitivity of isolated fat cells from obese protodiabetics

Summary Six normal weight subjects without any heredity of diabetes (group 1), 3 obese subjects with normal (group 2) and 9 with pathological carbohydrate tolerance (group 3) were characterized by a 2-h glucose infusion test. Adipose tissue fragments were obtained from the abdominal wall by surgical biopsy under intracutaneous anesthesia. Adipocytes were isolated by collagenase digestion and incubated in buffer containing [1-¹⁴C] glucose and different concentrations of insulin. The metabolic effect of insulin was expressed as percent increase above control¹⁴CO₂ production. Maximal CO₂ raised to 207±25% and 154±9% in groups 1 and 2, respectively. These values were significantly higher than in obese subjects displaying a pathological carbohydrate tolerance (group 3; 119±6%). A negative correlation was found between blood glucose levels and biological activity of insulin on adipocytes. The results suggest that insulin sensitivity of target tissue seems to play an important role in the development of carbohydrate intolerance. These investigations have been carried out within the medical research project ‘Diabetes mellitus and disturbances of lipid metabolism’, Ministry of Health, GDR.     

Evidence for priming and inhibitory effects of glucose on insulin secretion from isolated islets of langerhans

Summary Biphasic insulin secretion from perifused rat islets of Langerhans was affected in three ways by the islet glucose environment prior to stimulation, (i) The secretory response to glucose was diminished if the basal concentration of glucose in the medium was reduced from 5.5 to 2.7 mmol/l for 2 h prior to stimulation. First phase secretion was affected more than the second, (ii) Secretion was potentiated if islets had been previously exposed to a stimulatory concentration of glucose of 22.2 mmol/l. Again first phase secretion was particularly affected and there was a positive correlation between the magnitude of the secretory response and the duration of the initial stimulus, (iii) In contrast, both phases of secretion were proportionately reduced if islets had been previously exposed to stimulatory concentrations of glucose of 8.3 mmol/l.     

The atypical antipsychotic clozapine impairs insulin secretion by inhibiting glucose metabolism and distal steps in rat pancreatic islets

Aims/hypothesis Diabetogenic effects of some atypical antipsychotic drugs have been reported, although the mechanisms are not fully understood. We investigated the long-term effects of culturing isolated rat pancreatic islets with atypical antipsychotic clozapine.Methods Glucose- and non-glucose-stimulated insulin secretion, glucose metabolism and intracellular Ca²⁺ concentration ([Ca²⁺]_i) were measured in islets cultured with or without clozapine.Results Although acute incubation or 3-day culture with clozapine did not affect glucose-stimulated insulin secretion, clozapine suppressed glucose-stimulated insulin secretion by 53.2% at 1.0 μmol/l (therapeutic concentration) after 7 days of culture. Islet glucose oxidation and [Ca²⁺]_i elevation by high glucose were not affected after 3 days of culture, but clozapine significantly inhibited islet glucose oxidation, ATP production, and [Ca²⁺]_i elevation by high glucose after 7 days of culture. Moreover, 7 days of culture with clozapine inhibited insulin secretion stimulated by: (1) membrane depolarisation induced by high K⁺; (2) protein kinase C activation; and (3) mastoparan at 16.7 mmol/l glucose under stringent Ca²⁺-free conditions. Elevation of [Ca²⁺]_i by high K⁺-induced membrane depolarisation was similar in control and clozapine-treated islets. Clozapine, a muscarinic blocker, acutely inhibited carbachol-induced insulin secretion, as did atropine, whereas after 7 days of culture atropine did not have the inhibitory effect shown by clozapine after 7 days. The impairment of glucose-stimulated insulin secretion recovered 3 days after the removal of clozapine treatment.Conclusions/interpretation The present study demonstrated that the atypical antipsychotic drug clozapine directly impaired insulin secretion via multiple sites including glucose metabolism and the distal step in insulin exocytosis in a long-term culture condition. These mechanisms may be involved in the form of diabetes mellitus associated with atypical antipsychotic drugs.     

The priming effect of glucose on insulin secretion from isolated islets of Langerhans

Summary Biphasic insulin secretion from perifused rat islets of Langerhans was enhanced if islets had previously been stimulated with glucose 16.6 mmol/l. The priming effect of glucose was reduced if mannoheptulose (16.6 mmol/l), deuterium oxide (D₂O; 98% v/v) or adrenaline (10mol/l) was included in the medium during the initial stimulation period, or if Calcium was omitted. Glyceraldehyde (16.6 mmol/l) but not theophylline (5 mmol/l) could substitute for glucose during the initial stimulation and make islets more responsive to subsequent stimulation. The results suggest that the priming effect of glucose on insulin secretion may be related to 1) glucose metabolism and 2) Ca fluxes in the B cell and the consequent activation of the microtubular system. Neither the generation of intracellular cyclic AMP nor the release of insulin per se appears to be involved in the priming process.     

Somatostatin and insulin secretion in man II. The effect of theophylline

Summary This study was aimed at evaluating the effect of theophylline, a drug that increases the intra-cellular concentrations of cAMP by inhibiting phosphodiesterase activity, on somatostatin (SRIF)-mediated inhibition of insulin secretion in man. Acute insulin response (AIR) to i.v. glucose (mean change 3–10 min) was almost totally suppressed by SRIF (500 μg/h) and glucose utilization was reduced (p<0.0001). These SRIF-induced decreases failed to be eliminated by a concurrent infusion of theophylline (100 mg as a loading dose followed by a constant infusion of 5 mg/min). Theophylline alone resulted in a significant increase in both AIR (p<0.01) and glucose removal rates (p<0.05). Thus, our data disprove the involvement of the phosphodiesterase enzymes in the inhibitory action of SRIF on glucose-induced insulin secretion in man.     

Effect of human lymphoblastoid interferon on insulin synthesis and secretion in isolated human pancreatic islets

Summary Human islets of Langerhans were isolated from the pancreas removed from a 13-year-old female transplant donor. The islets were incubated in a culture medium for 24 h in the presence of human lymphoblastoid interferon (1000 units/ml). Insulin secretion, proinsulin biosynthesis, total protein biosynthesis and total insulin content were assessed at various concentrations of glucose in the presence of interferon. In interferon-treated islets glucose-stimulated insulin secretion was unaltered from that of control islets; however, glucose-stimulated proinsulin biosynthesis was specifically inhibited by interferon (48%, p<0.025). Total protein biosynthesis and total insulin content were not significantly affected by interferon.     

白藜芦醇对原代大鼠胰岛葡萄糖刺激的胰岛素分泌的影响

分离SD大鼠胰岛接种于24孔板中,用不同浓度的葡萄糖和白藜芦醇分别培养1 h或24 h,结果表明白藜芦醇孵育大鼠胰岛1 h可呈剂苗依赖地抑制大鼠高糖刺激的胰岛素分泌,1、10和100 μmol/L白藜芦醇可以分别使胰岛素的分泌降低10%、35%(P<0.05)和80%(P<0.01).显微离子成像技术爪10μmol/L的白藜芦醇可以使高糖引起的β细胞内Ca2+浓度的升高减少60%(P<0.05).白藜芦醇可使软脂酸孵育24 h大鼠胰岛的胰岛素分泌恢复到对照组的75%(P<0.01),提示白藜芦醇短期可通过调控细胞内的Ca+浓度,而抑制原代胰岛高精刺激的胰岛素分泌,长期可改善软脂酸引起的β细胞损伤.     

Feedback inhibition of insulin and glucagon secretion by insulin is altered in abdominal obesity with normal or impaired glucose tolerance

We investigated the feedback inhibition of insulin and glucagon secretion during euglycemic-hyperinsulinemic clamp at about 350 pmol/l in 16 patients with abdominal obesity [8 with normal glucose tolerance (oNGT), 8 with impaired glucose tolerance (oIGT)] and 8 normal-weight subjects matched for age, sex and blood pressure. In oNGT and oIGT, fasting plasma C-peptide levels were twice those in the controls (962±51 and 915±85 vs 439±28 pmol/l,P<0.001) and their suppression was lower than in the controls, both in absolute terms (155±19 and 185±17 vs 274±18 pmol/l,P<0.001) and as a percentage decline from basal levels (16±2% and 21±2% vs 63±2%,P<0.001). Fasting plasma glucagon levels were similar in the patients and in the controls, but were less suppressed during clamp in oNGT and oIGT, both in absolute terms (7.0±0.9 and 5.6±0.6 vs 13.2±1.2 pmol/l,P<0.001) and as a percentage change from basal levels (23±3% and 19±2% vs 44±4%,P<0.001). These results suggest that the insulin feedback on B and A cells is impaired in abdominal obesity, and that this defect is of similar degree in oNGT and oIGT. These alterations could be implicated in the pathogenesis of hyperinsulinemia in obesity.     

Nutrient control of insulin secretion in perifused adult pig islets

OBJECTIVES: Xenotransplantation of pig islets is a potential solution to the shortage of human islets, but our knowledge of how these islets secrete insulin in response to nutrients is still fragmentary. This was the question addressed in the present study. METHODS: After 24 h culture adult pig islets were perifused to characterize the dynamics of insulin secretion. Some responses were compared to those in human islets. RESULTS: Increasing glucose from 1 to 15 mM weakly (approximately 2x) stimulated insulin secretion, which was potentiated (approximately 12x) by the cAMP-producing agent, forskolin. The effect of glucose was concentration-dependent (threshold at 3-5 mM and maximum at approximately 10 mM). The pattern of secretion was biphasic with a small first phase and an ascending second phase, and a paradoxical increase when the glucose concentration was abruptly lowered. Diazoxide abolished glucose-induced insulin secretion and tolbutamide reversed the inhibition. Glucose also increased secretion when islets were depolarized with tolbutamide or KCl. Insulin secretion was increased by leucine+glutamine, arginine, alanine or a mixture of amino acids, but their effect was significant only in the presence of forskolin. Upon stimulation by glucose alone, human islets secreted approximately 10x more insulin than pig islets, and the kinetics was characterized by a large first phase, a flat second phase, and rapid reversibility. CONCLUSIONS: Compared with human islets, in vitro insulin secretion by adult pig islets is characterized by a different kinetics and a major quantitative deficiency that can be corrected by cAMP.     

Differential mechanisms of glucose and palmitate in augmentation of insulin secretion in mouse pancreatic islets

Aims/hypothesis. To assess the possible importance of saturated fatty acids in glucose amplification of K⁺ _ATP channel-independent insulin secretion. Methods. Insulin release from perifused pancreatic islets of NMRI mice was determined by radioimmunoassay. Results. In the presence of K⁺ (20 mmol/l) and diazoxide (250 μmol/l), which stimulates Ca²⁺ influx and opens K⁺ _ATP channels, palmitate (165 μmol/l total; 1.2 μmol/l free) increased insulin secretion at 3.3, 10 and 16.7 mmol/l glucose while glucose (10; 16.7 mmol/l) did not increase insulin secretion. In the presence of K⁺ (60 mmol/l) and diazoxide (250 μmol/l), glucose (10; 16.7 mmol/l) stimulation of K⁺ _ATP channel-independent insulin secretion increased, whereas the effectiveness of palmitate (165 μmol/l total; 1.2 μmol/l free) on insulin secretion at both 3.3, 10 or 16.7 mmol/l glucose was reduced. Palmitate thereby mimicked the stimulatory pattern of the protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (0.16 μmol/l), which also failed to increase insulin secretion at maximum depolarising concentrations of K⁺ (60 mmol/l). Furthermore, the protein kinase C inhibitor calphostin C (1 μmol/l), led to a complete suppression of the effects of both palmitate (165 μmol/l total; 1.2 μmol/l free) and myristate (165 μmol/l total; 2.4 μmol/l free) stimulation of glucose (16.7 mmol/l)-induced insulin secretion. Calphostin C (1 μmol/l), however, failed to affect insulin secretion induced by glucose (16.7 mmol/l). Conclusion/interpretation. These data suggest that glucose could increase insulin secretion independently of saturated fatty acids like palmitate and myristate, which amplify glucose-induced insulin secretion by activation of protein kinase C. [Diabetologia (2001) 44: 738–746] Received: 30 October 2001 and in revised form: 31 January 2001