Laura M. Linares

Vascular reactivity in diabetic rats: effect of melatonin.

The aim of the present study was to evaluate the in vitro contractile response of rat aorta in mild and severe type I diabetes and the effect of melatonin on it. Aortic rings were obtained from male Wistar rats injected with streptozotocin 8–12 wks earlier. Rats were divided into three groups: non‐diabetic rats (NDR), mildly diabetic rats (MDR) and severely diabetic rats (SDR). Dose–response curves for acetylcholine‐induced, endothelium‐related relaxation of aortic rings (after previous exposure to phenylephrine) and for serotonin‐induced vasoconstriction were conducted in the presence or absence of 10–5 mol/L melatonin. This protocol was repeated with rings preincubated in a high glucose solution (44 mmol/L). The contractile response to phenylephrine decreased in SDR, an effect counteracted by preincubation with high glucose. Melatonin decreased phenylephrine‐induced vasoconstriction in MDR and counteracted the effect of high glucose in SDR. Acetylcholine‐evoked relaxation decreased significantly after exposure to a high glucose in SDR, this effect being counteracted by melatonin. Serotonin‐induced vasoconstriction decreased in SDR and augmented in MDR, but only after exposure to high glucose. Melatonin reduced the maximal tension of aortic contraction after serotonin in MDR, both under basal conditions and after preincubation in a high glucose solution. The results support the existence of differences in vasomotor responses as a function of the diabetes state and of an improvement of contractile performance in diabetic rats after exposure to melatonin at a pharmacological concentration (in terms of circulating melatonin levels but not necessarily for some other fluids or tissues).

Effect of melatonin treatment on oxygen consumption by rat liver mitochondria

Summary.The objective of this study was to examine the in vivo effect of melatonin on rat mitochondrial liver respiration. Two experiments were performed: For experiment 1, adult male rats received melatonin in the drinking water (16 or 50 µg/ml) or vehicle during 45 days. For experiment 2, rats received melatonin in the drinking water (50 µg/ml) for 45 days, or the same amount for 30 days followed by a 15 day-withdrawal period. At sacrifice, a liver mitochondrial fraction was prepared and oxygen consumption was measured polarographically in the presence of excess concentration of DL-3-β-hydroxybutyrate or L-succinate. Melatonin treatment decreased Krebs’ cycle substrate-induced respiration significantly at both examined doses. The stimulation of mitochondrial respiration caused by excess concentration of substrate recovered after melatonin withdrawal. Basal state 4 respiration was not modified by melatonin. Melatonin, by curtailing overstimulation of cellular respiration caused by excess Krebs’ cycle substrates, can protect the mitochondria from oxidative damage.

Melatonin restores endothelium-dependent relaxation in aortic rings of pancreatectomized rats.

Abstract:  In rats turned hyperglycemic by a subtotal pancreatectomy, a decreased relaxation response of aortic rings to acetylcholine (ACh) was found; this effect was amplified by preincubation in a high glucose medium (44 mmol/L). The relaxation response to ACh did not occur in endothelium‐denuded rings or after the aortic rings were exposed to l‐nitro‐arginine methyl ester [l‐NAME, a nitric oxide (NO) synthase inhibitor]. Incubation with the NO donor sodium nitroprusside (SNP) restored the impaired relaxation response seen in endothelium‐denuded or l‐NAME‐treated aortic rings. Pancreatectomy decreased the vasorelaxation of aortic rings caused by SNP. Only in pancreatectomized rats, incubation in a high glucose medium impaired the relaxation effect of SNP. To assess whether melatonin preincubation reversed the impaired relaxation response to ACh (intact endothelium aortic rings) or to SNP (endothelium‐denuded or l‐NAME‐treated rings) in hyperglycemic rats, cumulative dose–response curves were performed in the presence of 10−5 mol/L melatonin. Melatonin preincubation did not modify ACh‐induced relaxation of aortic rings in a normal glucose concentration but was highly effective in preventing the impairment of relaxation caused by a high glucose solution. Melatonin was also effective in restoring the impaired SNP‐induced vasorelaxation seen in endothelium‐denuded or l‐NAME‐treated aortic rings from hyperglycemic rats. The results further support the improvement by melatonin of the endothelial‐mediated relaxation in blood vessels of diabetic rats.

Adrenocorticotrophic hormone, cortisol and catecholamine concentrations during insulin hypoglycaemia in dogs anaesthetized with thiopentone

Glucose homeostasis is maintained by complex neuroendocrine control mechanisms. Increases in plasma concentrations of various glucose-raising hormones such as glucagon, catecholamines, adrenocorticotrophic hormone (ACTH), and cortisol are observed under certain conditions associated with stress (haemorrhage and hypoglycaemia). The purpose of this study was to determine the effect of thiopentone anaesthesia on the cathecholamine, ACTH and cortisol response to insulin hypoglycaemia in dogs. Blood sugar (BS), plasma cathecholamine, and ACTH, and serum cortisol concentrations were measured during the course of (1) an intravenous insulin test (ITT) and (2) an ACTH test in conscious and in anaesthetized fasted dogs. During the ITT, the anaesthetized dogs showed a moderate resistance, compared with conscious dogs, to the hypoglycaemic action induced by insulin (blood sugar concentration 30 min after insulin injection: 2.91 ± 0.25 vs 1.93 ± 0.12 mM · L−1; P < 0.01). In addition, decreased epinephrine (220 ± 27 vs 332 ± 32 pg · ml−1 ACTH (65 ± 6 vs 90 ± 5 pg · ml−1) and cortisol (4.48 ± 0.3 vs 6.25 ± 0.5 μg · ml−1) concentrations were detected 60 min after insulin injection (P < 0.01). The norepinephrine response to hypoglycaemia was not altered by anaesthesia (273 ± 33 vs 325 ± 25 pg · ml−1). Anaesthetized dogs showed a decreased cortisol response to ACTH at 45 min (5.68 ± 0.54 vs 8.87 ± 0.47 μg · ml−1) when compared with control dogs (P < 0.001). Haemodynamic variables during anaesthesia showed little changes (P < NS); while respiratory rate was altered (P < 0.01 between 60 and 105 min). Arterial pH was decreased (7.29 ± 0.03 vs 7.36 ± 0.04; P < 0.05) and PaCO2 was increased (6.8 ± 0.3 vs 5.2 ± 0.3; P < 0.01) at 30 min from induction of anaesthesia but little change was seen after the beginning of the ITT and ACTH tests. We conclude that thiopentone anaesthesia provokes a moderate resistance to the hypoglycaemic action of insulin. This does not appear to be related to increases in plasma concentrations of cathecholamines, cortisol or ACTH. Since the hyperglycaemic effects of cathecholamines and glucagon are synergistic it is possible that glucagon plays an important role in the altered blood sugar response to insulin administration.RésuméL’homéostase du glucose sanguin est maintenue par des mécanismes de contrôle neuroendocriniens complexes. On observe une augmentation des concentrations plasmatiques (ou sériques) de plusieurs hormones hyperglycémiantes telles que le glucagon, l’ACTH et le cortisol sous certaines conditions liées au stress chirurgical comme l’hémorragie et l’hypoglycémie. Le but principal de cette étude est de déterminer les effets de l’anesthésie au thiopental sur la réponse, à l’hypoglycémie insulinique, des catécholamines, de l’ACTH et du cortisol. On détermine la glycémie et les concentrations plasmatiques des catécholamines et de l’ACTH, et les niveaux sériques du cortisol pendant 1) le test de tolérance à l’insuline, 2) l’épreuve à l’ACTH chez des chiens éveillés et anesthésiés. Lorsqu’on compare les chiens anesthesias avec les chiens éveillés, on constate que l’injection iv d’insuline provoque sur des mesures aux 30 minutes une insulino-résistance (glycémie: 2,91 ± 0,25 vs 1,93 ± 0,12 mM · L−1; P < 0,01) et une baisse de concentration plasmatique mesurée aux 60 minutes de l’épinéphrine (220 ± 27 vs 332 ±32 pg · ml−1), de l’ACTH (65 ±6 vs 90 ± 5 pg · ml−1) et du cortisol (4,48 ± 0,3 vs 6,25 ± 0,5 μg · ml−1) (P < 0,01). La réponse de la norépinéphrine plasmatique n’est pas modifiée par l’anesthésie (273 ± 33 vs 325 ± 25 pg · ml−1). Quarantecinq minutes après l’injection iv d’ACTH, on constate chez les chiens anesthésiés une diminution de la réponse au cortisol (5,68 ± 0,54 vs 8,87 ± 0,47) comparativement aux contrôles (P < 0,001). Les paramètres hémodynamiques pendant l’anesthésie demeurent inchangés (P < NS). Par contre, la fréquence respiratoire change (P < 0,01 entre 60 et 105 min). Trente minutes après l’induction de l’anesthésie, le pH artériel diminue (7,29 ± 0,03 vs 7,36 ± 0,04: P < 0,05), la PaCO2 augmente (6,8 ± 0,3 vs 5,2 ± 0,3; P < 0,01). On note peu de changements après le début des test de tolérance au glucose et l’épreuve à l’ACTH. A partir de ces données on peut conclure que l’anesthésie au thiopental produit une insulino-résistance modérée. Cet effet ne résulte pas d’une augmentation plasmatique des catécholamines, de l’ACTH ou du cortisol. Au contraire, le niveau sanguin de ces hormones diminue. On énonce comme postulat l’intervention probable du glucagon dans les mécanismes de régulation aigus de la glycémie chez les chiens anesthésiés.

Effect of melatonin on vascular responses in aortic rings of aging rats

In old animals a marked reduction in endothelium-dependent relaxation occurs. Since there is evidence that the endothelial dysfunction associated with aging may be partly related to the local formation of reactive oxygen species, the purpose of this study was to examine the effect of the natural antioxidant melatonin (10(-5)mol/l) on in vitro contractility of aged aortic rings under conditions of increased oxidative stress (40 m mol/l glucose concentration in medium). Experiments were carried out in 18-20 months old, Wistar male rats, using adult (6-7 months old) animals as controls. A higher plasma lipid peroxidation was found in aged rats as compared to the younger ones. In a first experiment, dose-response curves for acetylcholine-induced relaxation of aortic rings were conducted. Analyzed as a main factor in a factorial ANOVA, age decreased and melatonin augmented the relaxing response to acetylcholine. melatonins restoring effect on aortic ring relaxation was found in aged aortic rings only and was more pronounced in the presence of a high glucose medium. In a second experiment, the effect of melatonin on the contractility response to phenylephrine of intact or endothelium-denuded aortic rings obtained from aged or control rats was examined in normal or high glucose medium. A main factor analysis in the factorial ANOVA indicated that age and operation augmented, and melatonin decreased, aortic ring contractility response to phenylephrine. Melatonins restoring effect on aortic contractility was seen in aged aortic rings. The effect of age or a high glucose medium on phenylephrine-induced contractility was more pronounced in the absence of an intact endothelium. Aging did not affect the relaxant response of intact or endothelium-denuded rings to sodium nitroprusside. The results support the improvement by melatonin of vascular response in aging rats, presumably via its antioxidant activity.

Blood sugar, serum insulin and serum non- esterified fatty acid levels during thiopentone anaesthesia in dogs

The purpose of this study was to determine the effect of thiopentone anaesthesia on glucose metabolism. Blood sugar (BS), serum immunoreactive insulin (IRI) and serum non-esterified fatty acid (NEFA) concentrations were measured during the course of (1) an intravenous glucose tolerance test (IVGTT), and (2) an intravenous insulin test (ITT), in conscious and anaesthetized fasted dogs. The IVGTTs were repeated in dogs under alpha-or beta-adrenergic blockade, induced by phentolamine or propranolol. During the IVGTT, the anaesthetized dogs showed glucose intolerance (blood sugar levels were higher than in the control group) and little serum IRI response to hyperglycaemia was detected. An attenuated initial decrease and a slower rebound of NEFA concentration was observed in anaesthetized animals than in controls. Phentolamine administration (5 mg · kg−1 iv) partly restored the IRI response without affecting the BS levels; propanolol (1 mg · kg−1 iv) had no effect. Anaesthetized dogs showed a moderate resistance to insulin induced hypoglycaemic action and a lack of serum NEFA response during counter-regulation of hypoglycaemia, while in conscious controls an intense rebound was observed. Hyperinsulinaemia after iv insulin administration was longer in anaesthetized dogs than in controls. The insulin distribution space was 78% of body weight and insulin t1/2 in blood group compared with 54% and 16 min, in controls. We conclude that thiopentone provokes disturbances in glucose and serum NEFA metabolisms and abolishes the serum IRI response to hyperglycaemia. These effects are influenced by extrapancreatic factors regulating serum IRI levels and by an alpha-adrenergic mechanism, via the inhibition of insulin secretion.RésuméCette étude porte sur les effets de l’anesthésie au thiopental sur la glycémie, l’insulinémie et la concentration des acides gras non-estérifiés (AGNE) sur des chiens en réponse à un test d’hyperglycémie provoquée et à une injection intraveineuse d’insuline. Le groupe contrôle se compose de chiens éveillés. Chez les chiens anesthésiés, on constate après le test d’hyperglycémie provoquée, une intolérance au glucose, une absence de réponse insulinique 5 min après l’injection du glucose (29 ± 15 μU · ml−1 comparativement à 85 ± 8 μU · ml−1 pour les contrôles) et une baisse moins importante des AGNE. Un bloc β- adrénergique au propanolol n’a pas modifié ces résultats. Cependant, l’administration de phentolamine (blocage α- adrénergique) rétablit partiellement la réponse insulinique sans modifier la glycémie. Chez le chien anesthesié, l’injection intraveineuse d’insuline entraîne une résistance aux actions hypoglycémiantes de l’insuline et lipolytique des hormones antiinsuliniques. L’insuline injectée lors du test d’hyperglycémie provoquée avait une demi-vie plus longue (27,5 min contre 16 min) et un volume de distribution plus grand (78% contre 54%) chez les chiens anesthésiés que chez les contrôles. On peut conclure que la réponse insulinique à l’hyperglycémie diminue chez les chiens anesthésiés au thiopental. En toute probabilité, on peut attribuer ces résultats à une association de facteurs extrapancréatiques auxquels s’ajoute un facteur α-adrénergique inhibitoire de la sécrétion insulinique.

Comparative effects of melatonin and vitamin E in restoring aortic relaxation in pancreatectomized rats

In a previous study we reported the efficacy of melatonin to restore the decreased relaxation response to acetylcholine (ACh) or to sodium nitroprusside (SNP) in aortic rings of rats turned hyperglycemic by subtotal pancreatectomy. The effect was amplified by pre-incubation in a high (44 mmol/l) glucose solution, a situation that resulted in oxidative stress. We hereby compare the effect of another antioxidant, vitamin E, with that of melatonin on ACh response in intact aortic rings or on SNP response in endothelium-denuded aortic rings obtained from pancreatectomized or sham-operated rats. Dose-response curves to ACh or SNP were performed in the presence or absence of melatonin or vitamin E (10-5 mol/l) in 10 or 44 mmol/l glucose medium. Melatonin was more effective than vitamin E in restoring Ach- or SNP-induced relaxation of aortic rings in a high glucose medium. The differences between the two antioxidants may rely on the ability of melatonin to diffuse readily into intracellular compartments.ResumenEn un estudio previo se describe la eficacia de la melatonina para restablecer la respuesta disminuida a la acetilcolina (ACh) o al nitroprusiato de sodio (SNP) de anillos aórticos de rata con pancreatectomía subtotal. Este efecto fue mayor en el grupo de anillos preincubados en solución de Krebs con elevada concentración de glucosa (44 mmol/l), lo que favorece la producción de estrés oxidativo. En el presente trabajo se comparan los efectos de la vitamina E y la melatonina sobre la respuesta a la ACh y al SNP de anillos aórticos con endotelio intacto o denudado, obtenidos a partir de ratas con pancreatectomía subtotal o con operación simulada (controles). Se realizaron curvas dosis-respuesta a la ACh o al SNP en medios de incubación con glucosa normal o alta con melatonina o vitamina E (10−5 mol/l). La melatonina fue más efectiva que la vitamina E para restablecer la relajación provocada por ACh o SNP en anillos aórticos expuestos a un medio con elevada concentración de glucosa. La diferencia entre el efecto de ambas sustancias antioxidantes podría deberse a la capacidad de la melatonina para difundir hacia el compartimiento intracelular.