Conrado R. Ricci

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.

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.

Effects of Thyroxine on Rat Brown Fat and Muscle Thermogenesis in the Cold

We studied whether the activation of rat brown adipose tissue (BAT) by cold exposure or by the administration of beta-3-noradrenergic agonist CGP-12177 could be prevented by the inhibition of thyroxine (T4) to triiodothyronine (T3) conversion. Hypothyroid rats were treated with replacement doses of T4, T4 plus iopanoic acid (IA) or T3. Groups of rats were placed at 4°C for 24 h or kept at room temperature. Cold exposure induced a significant increase in guanosine diphosphate (GDP) binding to BAT mitochondrial proteins in T4-treated rats, an effect not abolished by IA. No significant changes were seen in T3-treated rats. In rats maintained at room temperature and injected with CGP-12177, T4 induced a significant rise in GDP binding which was not blocked by IA. T3 also induced a significant increase in binding. The study of mitochondrial oxygen consumption in muscle from cold-exposed rats showed a marked decrease in consumption in T3-treated rats as compared to values in the warm. Normal oxygen consumption was restored with 2-fold doses of T3 replacement, whereas 5-fold doses increased consumption above normal. The data suggest that in states with low or absent T3, T4 can stimulate heat production and preserve normothermia.

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.

NEUROPEPTIDE Y SUPPRESSED THE T3-INDUCED RISE IN BROWN FAT MITOCHONDRIAL RESPIRATION IN HYPOTHYROID RATS

It has been postulated that a cold-induced decrease in hypothalamic neuro-peptide Y (NPY) might disinhibit the sympathetic outflow that activates brown adipose tissue (BAT) thermogenesis. The present work has assessed the interscapular BAT NPY and norepinephrine (NE) concentrations 7 days after surgical excision of the sympathetic nerves (Sx) of each BAT lobe in rats exposed to 4°C or 24°C during 24 h. In addition, the effects of NPY treatment on BAT oxygen (O2) consumption in normal and T3-treated hypothyroid rats was determined. Sprague-Dawley rats weighing 220–240 g were used. BAT was removed and homogenized in 0.1 M HCl. BAT NPY content at 24°C was 109 ± 19 pg/mg protein. Sx decreased this value significantly (P < 0.001) and cold increased it by two-fold (P < 0.001). Neither cold nor Sx altered hypothalamic NPY. BAT O2 consumption was depressed by NPY treatment (P < 0.001) and increased by NE (P < 0.001). In euthyroid Sx rats, NPY decreased O2 consumption (P < 0.001). NPY failed to alter O2 consumption in hypothyroid rats and sharply decreased it in T3-treated rats. The results show that NPY blocked the effects of T3 on BAT O2 consumption, and that cold-induced activation of the thermogenic process did not produce measurable changes in hypothalamic NPY.