M. Roye

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.

Evidence for two different P2‐purinoceptors on β cell and pancreatic vascular bed

1 The effects of a 2‐substituted analogue of adenosine 5′‐triphosphate (ATP), 2‐methylthioadenosine triphosphate (2‐methylthio ATP) have been studied on insulin secretion and flow rate of the isolated pancreas of the rat, perfused in the presence of glucose (8.3 mm). 2 2‐Methylthio ATP (16.5–1650 nm) increased insulin secretion in a biphasic and concentration‐dependent manner; the kinetics were comparable to those previously obtained with ATP. A comparison of relative potency between ATP and 2‐methylthio ATP showed that 2‐methylthio ATP was 45 times more potent that ATP. 3 2‐Methylthio ATP also provoked a transient decrease of the flow rate in a concentration‐dependent manner but at concentrations (165–825 μm) about 1000 fold higher than those needed to increase insulin secretion. A comparison of relative potency between the natural derivative and 2‐methylthio ATP showed that 2‐methylthio ATP was only twice as potent as ATP. 4 These and other previous results (with phosphate‐modified analogues of ATP) provide evidence for two different types of P2‐purinoceptors on endocrine cell and vessel cells of the pancreas. A P2Y subtype, mediating an increase of insulin secretion, is present on the β cell of the pancreas. A P2X subtype, mediating vasoconstriction, is present on the vascular bed of the rat pancreas.

Evidence for an A2-subtype adenosine receptor on pancreatic glucagon secreting cells.

1 The effects of a 5′‐substituted analogue of adenosine, 5′‐N‐ethylcarboxamidoadenosine (NECA) have been studied on glucagon secretion in vitro, using the isolated pancreas of the rat perfused in the presence of glucose (2.8 mM). 2 NECA provoked a peak of glucagon secretion, the kinetics of which were comparable to those previously obtained with adenosine. The effect was concentration‐dependent and appeared at nanomolar concentrations. The EC50 was approximately 4 × 10−8 M. 3 A comparison of relative potency between adenosine and NECA showed that NECA was about 800 fold more potent than adenosine in inducing glucagon secretion. 4 Theophylline (50 μM) considerably decreased the peak of glucagon secretion induced by 1.65 μM NECA and totally suppressed the effect of 16.5 nM NECA. These results indicate the involvement of an adenosine receptor. 5 These and other previous results (low stereoselectivity of N6‐phenylisopropyladenosine) provide evidence for an adenosine receptor of the A2‐subtype being involved in glucagon secretion.

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.

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.

Vanadyl sulphate differently influences insulin response to glucose in isolated pancreas of normal rats after in vivo or in vitro exposure

The effect of the antidiabetic agent vanadyl sulphate (VOSO4) on the endocrine pancreas function of normal rats was studied using the isolated pancreas preparation. A short-term (8 days) i.p. treatment (15 mg/kg per day) resulted in attenuation of high glucose-stimulated insulin release, at day 9 but also at days 19, i.e., after full recovery of appetite and weight, while blood and pancreas vanadium concentrations were still elevated. Six months of oral VOSO4 treatment (0.75 mg/ml in drinking water) resulted in elevated vanadium concentrations while glucose-stimulated insulin release was attenuated as compared to pair-fed animals. Conversely, when directly perfused in pancreas, VOSO4 potentiated glucose-stimulated insulin release. These apparently opposite effects may be related to the ability of VOSO4 to exert both peripheral insulinomimetic effects-leading to chronic reduction in insulin demand-, and a direct pancreatic insulinotropic activity.

Adrenergic inhibition of insulin secretion involves pertussis toxin-sensitive and -insensitive mechanisms

We studied the involvement of Bordetella pertussis toxin (PTX)-sensitive G proteins in the inhibition by adrenaline of insulin secretion from the isolated rat pancreas. The -90% inhibition induced by adrenaline (0.05 microM) was partially abolished after in vivo PTX pretreatment. The residual inhibitory effect of adrenaline in PTX-pretreated rats was suppressed by the alpha 2-adrenoceptor antagonist, yohimbine, but was not modified by the alpha 1-adrenoceptor antagonist, prazosin. Thus, the alpha 2-inhibitory effect of adrenaline on B-cells is mediated by both PTX-sensitive and PTX-insensitive mechanisms.

Inhibition by chlordiazepoxide of adenosine‐stimulated glucagon secretion

Summary— The action of a water soluble benzodiazepine, chlordiazepoxide (CDZ) on the stimulatory effect of adenosine on glucagon secretion from the isolated pancreas of the rat perfused in presence of 2.8 mM glucose was studied. CDZ 10−7 and 10−6 M had no effect per se on glucagon secretion under our experimental conditions. In contrast, CDZ 10−6 M (but not 10−7 M) markedly reduced the peak of glucagon secretion provoked by adenosine, 2‐chloroadenosine (1.65 times 10−6 M) and by a stable analogue, 5′‐N‐ethylcarboxamidoadenosine or NECA (1.65 times 10−8 M). This peripheral interaction between CDZ and adenosine seemed to be specific, since CDZ did not modify the peak of glucagon secretion induced by (‐)isoproterenol (10−8 M). Our results demonstrate an inhibitory effect of CDZ on adenosine‐stimulated glucagon secretion.

Chlordiazepoxide potentiates R-phenylisopropyladenosine-induced inhibition of insulin secretion

Interactions between benzodiazepines and adenosine have been reported in the central nervous system (Phillis, 1984) as well as in peripheral tissues; accordingly, we have previously shown that the central-type benzodiazepine receptor agonist, chlordiazepoxide (CDZ), selectively decreased adenosine-induced glucagon secretion from pancreatic A cells (Chapal et al, 1990). Since there has been evidence for an inhibitory adenosine receptor on pancreatic insulin-secreting B cells (Hillaire-Buys et al, 1987), we have investigated whether an interaction could be found on these cells and whether CDZ could affect the inhibition of insulin secretion induced by the adenosine suuctural analogue, R-phenylisopropyladenosine (R-PIA).