Michèle Manteghetti

Experimental NIDDM: Development of a New Model in Adult Rats Administered Streptozotocin and Nicotinamide

We took advantage of the partial protection exerted by suitable dosages of nicotinamide against the β-cytotoxic effect of streptozotocin (STZ) to create a new experimental diabetic syndrome in adult rats that appears closer to NIDDM than other available animal models with regard to insulin responsiveness to glucose and sulfonylureas. Among the various dosages of nicotinamide tested in 3-month-old Wistar rats (100–350 mg/kg body wt), the dosage of 230 mg/kg, given intraperitoneally 15 min before STZ administration (65 mg/kg i.v.) yielded a maximum of animals with moderate and stable nonfasting hyperglycemia (155 ± 3 vs. 121 ± 3 mg/dl in controls; P < 0.05) and 40% preservation of pancreatic insulin stores. We also evaluated β-cell function both in vitro and in vivo 4–9 weeks after inducing diabetes. In the isolated perfused pancreas, insulin response to glucose elevation (5–11 mmol/l) was clearly present, although significantly reduced with respect to controls (P < 0.01). Moreover, the insulin response to tolbutamide (0.19 mmol/l) was similar to that observed in normal pancreases. Perfused pancreases from diabetic animals also exhibited a striking hypersensitivity to arginine infusion (7 mmol/l). In rats administered STZ plus nicotinamide, intravenous glucose tolerance tests revealed clear abnormalities in glucose tolerance and insulin responsiveness, which were interestingly reversed by tolbutamide administration (40 mg/kg i.v.). In conclusion, this novel NIDDM syndrome with reduced pancreatic insulin stores, which is similar to human NIDDM in that it has a significant response to glucose (although abnormal in kinetics) and preserved sensitivity to tolbutamide, may provide a particularly advantageous tool for pharmacological investigations of new insulinotropic agents.

4-Hydroxyisoleucine: experimental evidence of its insulinotropic and antidiabetic properties

We have recently shown in vitro that 4-hydroxyisoleucine (4-OH-Ile), an amino acid extracted from fenugreek seeds, potentiates insulin secretion in a glucose-dependent manner. The present study was designed to investigate whether 4-OH-Ile could exert in vivo insulinotropic and antidiabetic properties. For this purpose, intravenous or oral glucose tolerance tests (IVGTTs and OGTTs, respectively) were performed not only in normal animals but also in a type II diabetes rat model. During IVGTT in normal rats or OGTT in normal dogs, 4-OH-Ile (18 mg/kg) improved glucose tolerance. The lactonic form of 4-OH-Ile was ineffective in normal rats. In non-insulin-dependent diabetic (NIDD) rats, a single intravenous administration of 4-OH-Ile (50 mg/kg) partially restored glucose-induced insulin response without affecting glucose tolerance; a 6-day subchronic administration of 4-OH-Ile (50 mg/kg, daily) reduced basal hyperglycemia, decreased basal insulinemia, and slightly, but significantly, improved glucose tolerance. In vitro, 4-OH-Ile (200 μM) potentiated glucose (16.7 mM)-induced insulin release from NIDD rat-isolated islets. So, the antidiabetic effects of 4-OH-Ile on NIDD rats result, at least in part, from a direct pancreatic B cell stimulation.We have recently shown in vitro that 4-hydroxyisoleucine (4-OH-Ile), an amino acid extracted from fenugreek seeds, potentiates insulin secretion in a glucose-dependent manner. The present study was designed to investigate whether 4-OH-Ile could exert in vivo insulinotropic and antidiabetic properties. For this purpose, intravenous or oral glucose tolerance tests (IVGTTs and OGTTs, respectively) were performed not only in normal animals but also in a type II diabetes rat model. During IVGTT in normal rats or OGTT in normal dogs, 4-OH-Ile (18 mg/kg) improved glucose tolerance. The lactonic form of 4-OH-Ile was ineffective in normal rats. In non-insulin-dependent diabetic (NIDD) rats, a single intravenous administration of 4-OH-Ile (50 mg/kg) partially restored glucose-induced insulin response without affecting glucose tolerance; a 6-day subchronic administration of 4-OH-Ile (50 mg/kg, daily) reduced basal hyperglycemia, decreased basal insulinemia, and slightly, but significantly, improved glucose tolerance. In vitro, 4-OH-Ile (200 microM) potentiated glucose (16.7 mM)-induced insulin release from NIDD rat-isolated islets. So, the antidiabetic effects of 4-OH-Ile on NIDD rats result, at least in part, from a direct pancreatic B cell stimulation.

4-Hydroxyisoleucine: effects of synthetic and natural analogues on insulin secretion.

4-Hydroxyisoleucine, a peculiar amino acid extracted from fenugreek seeds and never found in mammalian tissues, exhibits interesting insulinotropic activity. To investigate the structural requirements for this stimulating effect, the insulinotropic activity of the major isomer (2S,3R,4S) of 4-hydroxyisoleucine, in the presence of 8. 3 mM glucose, was compared to that of (1) its minor isomer (2R,3R, 4S) (2) its lactone form, (3) classical structurally related amino acids, and (4) synthetic monomethylated analogues. In the isolated, ex vivo, perfused rat pancreas, only the major isomer of 4-hydroxyisoleucine (200 microM) potentiated insulin release. On incubated isolated rat islets, the threshold concentration for a significant increase (P<0.05) in insulin release was 200 microM for (2S,3R,4S) 4-hydroxyisoleucine, 500 microM for (2S,4R) and (2S,4S) gamma-hydroxynorvalines as well as (2S,3S) and (2S,3R) gamma-hydroxyvalines, and 1 mM or more for other congeners. In conclusion, the insulinotropic properties of 4-hydroxyisoleucine, in the micromolar range, are seen only in the presence of the linear major isoform; they also require carbon alpha in S-configuration, full methylation and carbon gamma-hydroxylation.

Effects of a fenugreek seed extract on feeding behaviour in the rat: Metabolic-endocrine correlates

Fenugreek seeds (Trigonella foenum graecum L.) are assumed to have restorative and nutritive properties. The present work was designed to investigate the effects of a fenugreek seed extract on feeding behaviour. Experiments were performed to determine food consumption and motivation to eat as well as metabolic-endocrine changes in chronically treated animals. Male Wistar rats were given the seed extract orally (10 and 100 mg/day per 300 g body weight), mixed together with food, and control animals were monitored in parallel. The results show that chronic oral administration of the fenugreek extract significantly increases food intake and the motivation to eat. The treatment, however, does not prevent the anorexia nor the decreased motivation to eat induced by d-fenfluramine (2 mg/kg, IP). An increase in plasma insulin and a decrease in total cholesterol and very low-density lipoprotein (VLDL)-low-density lipoprotein (LDL) total cholesterol were also observed. In conclusion, chronic administration of a fenugreek seed extract enhances food consumption and motivation to eat in rats and also induces hyperinsulinemia as well as hypocholesterolemia.

Alterations of insulin response to different β cell secretagogues and pancreatic vascular resistance induced by Nω-nitro-L-arginine methyl ester

1 We studied a possible interplay of pancreatic NO synthase activity on insulin secretion induced by different β cell secretagogues and also on pancreatic vascular bed resistance. 2 This study was performed in the isolated perfused pancreas of the rat. Blockade of NO synthase was achieved with Nω‐nitro‐L‐arginine methyl ester (l‐NAME); the specificity of the antagonist was checked by using its D‐enantiomer as well as by substitutive treatments with sodium nitroprusside (SNP) as a NO donor in studies of glucose‐induced insulin secretion. 3 Arginine (5 mM) induced a monophasic insulin response which was, in the presence of L‐NAME at equimolar concentration, very strongly potentiated and converted into a 13 times higher biphasic one. D‐NAME (5 mM) was only able to induce a 3 times higher response, but provoked a similar vasoconstrictor effect. 4 The small biphasic insulin secretion induced by L‐leucine (5 mM) was also strongly enhanced, by 8 times, in the presence of L‐NAME (5 mM) vs 2 times in the presence of D‐NAME (5 mM). 5 β cell responses to KC1 (5 mM) and tolbutamide (0.185 mM) were only slightly increased by L‐NAME (5 mM) to values not far from the sum of the effects of L‐NAME and of the two drugs alone. D‐NAME (5 mM) was totally ineffective on the actions of both secretagogues. 6 L‐NAME, infused 15 min before and during a rise in glucose concentration from 5 to 11 mM, was able in the low millimolar range (0.1‐0.5 mM) to blunt the classical biphasic pattern of β cell response to glucose and, at 5 mM, to convert it into a significantly greater monophasic one. In contrast, D‐NAME (5 mM) was unable to induce similar effects. 7 SNP alone at 3 μm was ineffective but at 30 μm substantially reduced the second phase of insulin response to glucose; however, at both concentrations the NO donor partly reversed alterations in insulin secretion caused by L‐NAME (5 mM) and restored a biphasic pattern of insulin response. At a high (300 μm) concentration, SNP drastically reduced the second phase of β cell response, but in the presence of L‐NAME, provoked a significantly greater biphasic response. 8 When L‐NAME was infused only for the 15 min before high glucose, an exaggerated first phase of β cell response was followed by an abortive second one. SNP, at a low concentration (30 nM), given simultaneously with L‐NAME, restored a biphasic pattern and prevented the vasoconstrictor effect induced by the inhibitor. 9 L‐NAME, when infused only during high glucose, markedly enhanced the second phase of insulin response which could be significantly reduced by SNP (3 μm). The NO donor induced a dilator effect significantly greater in L‐NAME‐treated pancreata than in non‐treated ones. 10 In conclusion our data bring evidence that NO synthase activity exerts an inhibitory control on pancreatic β cell response to various nutrient secretagogues and may, at least partly, be implicated in the generation of the biphasic pattern of insulin response to glucose.

ATP and phosphate-modified adenine nucleotide analogues: effects on insulin secretion and calcium uptake

Our previous experiments on isolated rat pancreas gave evidence for a P2 purinergic receptor on the insulin-secreting B cell. This work was designed to investigate whether the stimulation of insulin release by phosphorylated adenosine derivatives could also be observed in rat isolated Langerhans islets and whether this stimulation was accompanied by changes in calcium uptake. The results indicate that two structural methylene analogues of ATP and ADP (alpha,beta-methylene ATP and alpha,beta-methylene ADP) display an insulin stimulatory effect comparable to that of ATP, confirming the membrane action of the latter. It was also found that calcium uptake increased concomitantly with insulin release under the effect of alpha,beta-methylene ADP; on the other hand this agent also increased the total exchangeable calcium content of islets at isotopic equilibrium. Verapamil, a blocker of voltage-sensitive calcium channels, counteracted the stimulation of insulin release and also blocked the increase in total exchangeable calcium content. These results demonstrate the involvement of calcium in the stimulus-secretion coupling of insulin release induced by an activator of P2 purinergic receptors and suggest the implication of voltage-sensitive calcium channels.

Mechanisms involved in the effect of nitric oxide synthase inhibition on L-arginine-induced insulin secretion

1 A constitutive nitric oxide synthase (NOSc) pathway negatively controls L‐arginine‐stimulated insulin release by pancreatic β cells. We investigated the effect of glucose on this mechanism and whether it could be accounted for by nitric oxide production. 2 NOSc was inhibited by N∞‐nitro‐L‐arginine methyl ester (l‐NAME), and sodium nitroprusside (SNP) was used as a palliative NO donor to test whether the effects of L‐NAME resulted from decreased NO production. 3 In the rat isolated perfused pancreas, L‐NAME (5 mM) strongly potentiated L‐arginine (5 mM)‐induced insulin secretion at 5 mM glucose, but L‐arginine and L‐NAME exerted only additive effects at 8.3 mM glucose. At 11 mM glucose, L‐NAME significantly inhibited L‐arginine‐induced insulin secretion. Similar data were obtained in rat isolated islets. 4 At high concentrations (3 and 300 μm), SNP increased the potentiation of arginine‐induced insulin output by L‐NAME, but not at lower concentrations (3 or 30 nM). 5 L‐Arginine (5 mM) and L‐ornithine (5 mM) in the presence of 5 mM glucose induced monophasic β cell responses which were both significantly reduced by SNP at 3 nM but not at 30 nM; in contrast, the L‐ornithine effect was significantly increased by SNP at 3 μm. 6 Simultaneous treatment with L‐ornithine and L‐arginine provoked a biphasic insulin response. 7 At 5 mM glucose, L‐NAME (5 mM) did not affect the L‐ornithine secretory effect, but the amino acid strongly potentiated the alteration by L‐NAME of L‐arginine‐induced insulin secretion. 8 L‐Citrulline (5 mM) significantly reduced the second phase of the insulin response to L‐NAME (5 mM) + L‐arginine (5 mM) and to L‐NAME + L‐arginine + SNP 3 μm. 9 The intermediate in NO biosynthesis, NG‐hydroxy‐L‐arginine (150–300 μm) strongly counteracted the potentiation by L‐NAME of the secretory effect of L‐arginine at 5 mM glucose. 10 We conclude that the potentiation of L‐arginine‐induced insulin secretion resulting from the blockade of NOSc activity in the presence of a basal glucose concentration (1) is strongly modulated by higher glucose concentrations, (2) is not due to decreased NO production but (3) is probably accounted for by decreased levels of NG‐hydroxy‐L‐arginine or L‐citrulline, resulting in the attenuation of an inhibitory effect on arginase activity.

Differential effects of purinergic and cholinergic activation on the hydrolysis of membrane polyphosphoinositides in rat pancreatic islets

This work was designed to investigate the effects of a P2 purinoreceptor agonist, alpha, beta-methylene ADP, on membrane polyphosphoinositide hydrolysis in relation to insulin release from rat isolated islets of Langerhans. The effects of this stable structural analogue of ADP (10(-4) M) were compared with those of a muscarinic cholinergic agonist, carbachol (10(-4) M). The interaction between alpha, beta-methylene ADP and carbachol was studied on polyphosphoinositide breakdown and insulin secretion. The experiments were performed in presence of a slightly stimulating glucose concentration (8.3 mM). Whereas carbachol-induced insulin release was accompanied by a concomitant increase in inositol phosphates accumulation, alpha, beta-methylene ADP at the same concentration produced a similar insulin secretion without eliciting an accumulation of inositol phosphates. The combined effect of both substances added simultaneously resulted in a significant increase in insulin release as compared with the secretion induced by either substance used separately. By contrast, the accumulation of inositol phosphates induced by both substances was not different from the accumulation induced by carbachol alone. These results seem to rule out the involvement of polyphosphoinositide hydrolysis in the coupling mechanism between P2 purinoreceptor activation and insulin response of the B cell. Moreover, purinergic stimulation appears not to interact with the effect of muscarinic stimulation on polyphosphoinositide breakdown.

A Different Action of Hypothermia on Insulin Release from the Isolated, Perfused Rat Pancreas, Depending on the Stimulating Agent

SUMMARY Two series of experiments were performed in parallel on the isolated perfused rat pancreas. The experimental conditions differed only as pertaining to temperature. In one series the organ and the perfusion liquid were maintained at 37.5°C and in the other at 28°C. The pancreases were perfused from the start of the experiments with a perfusion medium containing 8.3 mmol/l glucose. The effects of various stimulatory agents were studied (glucose 16.6 mmol/l, tolbutamide 0.4 mmol/l, acetylcholine 0.5 μmol/l, glucagon, 2.8 nmol/l, and L-isoprenaline 0.05 μmol/l). At 37.5°C the insulin secretion induced by high glucose or tolbutamide, acetylcholine, and glucagon was biphasic and not statistically different. In all cases the hypothermia (28°C) decreased insulin secretion. However, glucoseinduced and tolbutamide-induced insulin secretion was more decreased than the secretion induced by acetylcholine and glucagon. The study of the secretion ratios obtained at 28°C relative to 37.5°C showed that the ratios for the glucose and tolbutamide groups were significantly lower than those obtained for acetylcholine and glucagon gsroups for both the first and the second phase. The ratios were not significantly different between glucose and tolbutamide on the one hand and acetylcholine and glucagon on the other hand. In all groups the ratios 28°/37.5° for the second phase were lower than those obtained during the first phase. L-isoprenaline induced only a weak increase in insulin secretion and this was not long lasting; this increase was not statistically different at both temperatures.

Comparative effects of LN 5330 and diazoxide on insulin release and 86Rb+ permeability in perifused rat islets of Langerhans.

The benzothiadiazine derivative LN 5330 (chloro-7 trifluoromethyl-6 benzothiadiazine-1,2,4-dioxide-1,1) has been shown to inhibit insulin secretion and calcium uptake. The present study was carried out to investigate the effects of LN 5330 on insulin release and 86Rb- efflux from perifused rat pancreatic islets; a comparison was made with the structural analogue diazoxide. In the presence of 8.3 mM glucose, LN 5330 (100 microM) accelerated 86Rb+ efflux while reducing insulin output from the islets. LN 5330 induced a dose-dependent acceleration of 86Rb+ efflux and appeared to be a more potent activator of 86Rb+ efflux than diazoxide. The stimulatory effect of LN 5330 on 86Rb+ efflux persisted in the absence of extracellular calcium. In the absence of glucose, 86Rb+ permeability also increased, LN 5330 being again significantly more efficient than diazoxide at an equimolar concentration. These data indicate that the benzothiadiazine derivative LN 5330 inhibits insulin secretion by increasing the potassium permeability of the plasma membrane. It is suggested that, like diazoxide, this drug could activate the ATP-sensitive K+ channel.