René Gross

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.

Evidence for a glutamate receptor of the AMPA subtype which mediates insulin release from rat perfused pancreas

1 The effect of l‐glutamate has been studied on insulin secretion by the isolated perfused pancreas of the rat. The glutamate receptor subtype involved has been characterized. 2 In the presence of a slightly stimulating glucose concentration (8.3 mm), l‐glutamate (5 × 10−5−4 × 10−3 m) induced an immediate, transient and concentration‐dependent insulin response. On the other hand, in the presence of a non stimulating glucose concentration (2.8 mm), l‐glutamate (10−3 m) did not modify the basal insulin secretion. 3 The three non‐NMDA receptor agonists, kainate (10−4−10−3 m), α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA, 5 × 10−5−10−4 m) and quisqualate (5 × 10−6−5 × 10−5 m) all provoked a transient and concentration‐dependent insulin response from pancreas perfused with 8.3 mm glucose. Compared with glutamate, kainate exhibited a similar efficacy, whereas AMPA and quisqualate elicited only a 3 fold lower maximal insulin response. In contrast, NMDA (10−4−10−3 m) was ineffective. 4 An antagonist of non‐NMDA receptors, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 5 × 10−5 m) totally prevented the stimulatory effect of l‐glutamate (4 × 10−4 m) and kainate (2 × 10−4 m). In contrast, the NMDA receptor antagonist, (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine ((+) MK801) was without effect. 5 The insulin secretory effect of glutamate (4 × 10−4 m) was not affected by atropine (3 × 10−7 m) or tetrodotoxin (3 × 10−6 m). 6 Quisqualate at a high maximally effective concentration (4 × 10−4 m) inhibited glutamate (10−3 m) or kainate (4 × 10−4 m)‐induced insulin release. 7 This study shows that l‐glutamate stimulates insulin secretion in rat pancreas, by acting on an excitatory amino acid receptor of the AMPA subtype.

Glutamate stimulates glucagon secretion via an excitatory amino acid receptor of the AMPA subtype in rat pancreas.

The effect of L-glutamate was studied on glucagon secretion from rat isolated pancreas perfused with 2.8 mM glucose. L-Glutamate (3.10(-5)-10(-4)M) induced an immediate, transient and concentration-dependent glucagon release. The three non-N-methyl-D-aspartate (NMDA) receptor agonists, kainate (3.10(-5)-3.10(-3)M), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (3.10(-5)-10(-4)M) and quisqualate (3.10(-6)-10(-5)M), all elicited a peak-shaped glucagon response. Compared to glutamate, AMPA and quisqualate exhibited a similar efficacy, whereas kainate caused a 4-fold higher maximal glucagon response. In contrast, NMDA (10(-3)M) was ineffective. The selective antagonist of non-NMDA receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 5.10(-5)M), totally prevented the glucagon response to 10(-4) M glutamate (IC50 congruent to 0.8 +/- 0.3 10(-6)M) and 3.10(-4)M kainate. Furthermore, quisqualate at a maximal effective concentration (3.10(-4)M) inhibited the response to kainate (10(-3)M). This study showed that L-glutamate stimulates glucagon release in rat pancreas by activating a receptor of the AMPA subtype.

P2 purinergic signalling in the pancreatic β-cell: Control of insulin secretion and pharmacology

Extracellular adenosine triphosphate is able to modulate pancreatic beta-cell function, acting on P2 purinergic ionotropic (P2X) and metabotropic (P2Y) receptors. Physiologically, ATP entrains beta-cells into a common rhythm by coordinating Ca(2+) oscillations; it plays a central role in insulin secretion pulsatility. ATP also triggers a positive feedback signal amplifying glucose-induced insulin release, which argues for a potential pharmacological application. ATP has consistently been shown to increase cytoplasmic free calcium concentration, notably in human tissue. Acting on P2X receptors, of which different molecular subtypes are expressed in beta-cells, it leads to a transient insulin release that may involve a closure of K(ATP) channels or a rapidly decaying inward current. Activation of G-protein-coupled P2Y receptors triggers different signalling pathways and amplifies insulin release in a glucose-dependent way. It has recently been shown that pancreatic beta-cells express different molecular subtypes of receptors, which may explain the complex interaction of P2Y ligands on high- and low-affinity binding sites. Despite the complexity of this purinergic pharmacology, consistent pre-clinical data suggest the potential of P2Y receptor agonists as drug candidates for type 2 diabetes.

Evidence for an inhibitory A1 subtype adenosine receptor on pancreatic insulin-secreting cells

The effects of L- and D-phenylisopropyladenosine (L- and D-PIA) were studied on glucose-induced insulin secretion from the isolated perfused rat pancreas. L-PIA at the low dose of 16.5 nM inhibited insulin secretion by 50%. In contrast, D-PIA at 16.5 and 82.5 nM was ineffective. D-PIA used at a 100-fold higher concentration (1.65 microM) than L-PIA induced a similar inhibition of insulin secretion. The inhibitory effect of L-PIA was abolished by 8-phenyltheophylline (1 microM), a potent P1 purinoceptor antagonist. The present experiments provide evidence for an adenosine receptor of the A1 subtype on the insulin-secreting pancreatic cell of rats.

Effects of imidazolines and derivatives on insulin secretion and vascular resistance in perfused rat pancreas

The effects of imidazolines and derivatives were studied on insulin secretion and vascular resistance in the isolated perfused rat pancreas. On insulin secretion, two imidazoline alpha 2-adrenoceptor antagonists, efaroxan (1-100 microM) and RX821002 (10 microM), had a stimulating response; however, idazoxan, like the non-imidazoline alpha 2-adrenoceptor antagonist yohimbine, was ineffective at 10 microM. The oxazoline rilmenidine with alpha 2-adrenergic activity at 10 microM), an imidazoline devoid of alpha 2-adrenergic activity, also had an insulin-releasing effect. On pancreatic vessels, all imidazolines tested (efaroxan, RX821002, antazoline and idazoxan), in contrast to yohimbine, induced vasoconstriction. Rilmenidine did not have a vasoconstrictor effect after blockade of alpha 2-adrenoceptors. Furthermore, the efaroxan-induced insulin release or vasoconstriction was not affected by the blockade of alpha 2- and alpha 1-adrenoceptors. This study shows that imidazolines and derivatives are able to stimulate insulin release and induce vasoconstriction in the rat pancreas. These effects cannot be ascribed to an interaction with alpha-adrenoceptors but may involve different types of imidazoline sites.

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.

Comparison of Effects of Neuropeptide Y and Norepinephrine on Insulin Secretion and Vascular Resistance in Perfused Rat Pancreas

Neuropeptide Y (NPY) and norepinephrine (NE) behave like cotransmitters in intrapancreatic adrenergic nerves. Therefore, in the isolated rat pancreas, we 1) studied and compared the effect of increasing concentrations of NE and NPY given alone on insulin secretion induced by 8.3 mM glucose and on pancreatic vascular flow rate and 2) investigated the effects of combinations of NPY and NE at low concentrations. NE induced a dose-dependent inhibition of insulin release between 1 and 50 nM (max 80%); β-cells appeared sensitive to NPY at 0.1 nM, but the maximal reduction of insulin release was comparatively weak (25–30%) at 10 nM. The study of the effect of combinations of NE and NPY at different concentrations suggests that the two neurotransmitters act in an additive way to inhibit insulin secretion. NPY (0.1–10 nM) induced a marked dose-dependent reduction of pancreatic outflow rate with a biphasic pattern between 1 and 10 nM. On the other hand, at low concentrations (1 and 2 nM), NE induced a progressive increase in pancreatic outflow rate; a clear but transient decrease could only be observed at 50 nM before a secondary increase. A combined treatment with two effective concentrations of NPY (0.1 nM) and NE (1 nM) resulted in a progressive reversal by NE of NPYs vasoconstrictive effect. We conclude that β-cell function and pancreatic vascular resistance were not affected in the same way by NPY and NE: NPY appeared to be a strong vasoconstrictor but only a slight inhibitor of insulin release, whereas NE strongly inhibited β-cells and was a comparatively poor vasoconstrictor, even able to increase vascular output at low concentration. When combined at the concentrations used, they showedinhibitory additive effects on insulin secretion but exerted opposite effects and were antagonistic on pancreatic flow rate.