Effect of melatonin treatment on oxygen consumption by rat liver mitochondria.

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 microg/ml) or vehicle during 45 days. For experiment 2, rats received melatonin in the drinking water (50 microg/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-beta-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.

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/1) in 10 or 44 mmol/1 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.

Favourable, significant effect of the dose-dependent treatment with RU 38486 (RU) on the alterations of the hepatic mitochondrial function of diabetic rats.

In the present work, the effect "in vivo' of increasing doses of RU 38486 upon the hepatic mitochondrial function of diabetic rats has been studied. At the same time, the action of adrenalectomy and corticosterone restitution on this function were comparatively demonstrated. The parameters measured were oxygen consumption with the substrates: 3-hydroxybutyrate (HB), succinate (Suc) and malate-glutamate (Mal-glut) in intact liver mitochondria and the activities of 3-hydroxybutyrate dehydrogenase (HBD) and cytochrome c oxidase (Cyt.c oxid.) enzymes in broken liver mitochondria. The groups of animals studied were normal controls (N) and the following groups of diabetic rats: rats without any treatment (D), adrenalectomized rats (D+ADX), rats that were adrenalectomized and treated with corticosterone (D+ADX+C) and four groups treated with increasing oral doses of RU (in mg/kg body wt.), that is, 12.5 (D+RU1), 25.0 (D+RU2), 37.5 (D+RU3) and 50.0 (D+RU4). The results showed a tendency of increasing values of mitochondrial oxygen consumption in diabetic animals treated with RU. The favourable effect of increasing doses of RU on O2 consumption of diabetic rat liver mitochondria with each of the substrates showed a significant association as indicated by the values obtained for the correlation coefficients r (0.95, 0.97 and 0.99 according to the substrate HB, Succ or Mal-glut, respectively). Likewise, the correlation between the treatment with increasing doses of RU and the recovery of enzyme activities showed a significant dose-effect association with r 0.94 for HBD and r = 0.95 for Cyt.c oxid. Adrenalectomy showed a similar effect to treatment with the maximum dose of RU while corticosterone restitution gave measured values similar to those of the D group. In conclusion, the favourable, significant variation of the hepatic mitochondrial function of diabetic rats was demonstrated by the dose-dependent treatment with RU as seen by the correlation statistical study performed. At the same time, the pernicious effect that glucocorticoids exert upon such function in experimental diabetes was confirmed.

Relative roles of the thyroid hormones and noradrenaline on the thermogenic activity of brown adipose tissue in the rat.

We have assessed the relative contribution of the thyroid hormones and noradrenaline (NA) on the calorigenic function of brown adipose tissue (BAT) as indicated by GDP binding and O2 consumption of BAT mitochondria. Male Wistar rats of 200 g body weight were made hypothyroid with 131I. Groups of animals were injected s.c., in divided doses, daily for 10 days, with thyroxine (2 micrograms/100 g body weight) or tri-iodothyronine (T3; 0.3 microgram/100 g body weight). Animals were used 7 days after bilateral or unilateral sympathetic nerve excision of BAT (Sx). Sham-operated rats were used as controls. In normal rats kept at 22 degrees C, GDP binding reached 94 +/- 24 pmol/mg protein; untreated hypothyroid rats had normal binding values whereas the T3-treated group showed an increased binding. Sx induced a sharp fall in the three groups (P < 0.01). After 24-h exposure to 4 degrees C GDP binding increased in normal rats to about 410% (P < 0.01) whereas binding failed to increase in response to cold in the untreated hypothyroid and the T3-treated groups. Sx reduced GDP binding in the three groups significantly (P < 0.01). The consumption of O2 by BAT mitochondria showed similar variations in response to Sx and to cold exposure as did GDP binding. The data indicated that, at room temperature, BAT calorigenesis can function without the thyroid hormones, though not without the catecholamines. The findings in rats exposed to cold showed that the lack of NA was significantly more effective than the lack of thyroid hormones in preventing the BAT hyperactive response. This does not negate an active role for T3 in BAT calorigenesis.

Effects of thyroid hormones on mitochondrial oxygen consumption in brown adipose tissue and heart from cold-exposed hypothyroid rats.

The present work measured brown adipose tissue and heart mitochondrial oxygen consumption in hypothyroid rats treated with replacement doses of T3, T4 or T4 plus iopanoic acid and kept at 4 degrees C for 24 h. Heart oxygen consumption in normal, untreated hypothyroid and T4-treated hypothyroid rats was unaffected by cold exposure. In rats treated with T4 plus iopanoic acid, rates of oxygen consumption were normal in those maintained at 4 degrees C as well as in those kept at room temperature, despite serum T3 concentration being significantly decreased. The cold-exposed T3-treated hypothyroid rats showed a marked decrease in oxygen consumption (p less than 0.02) and alpha-glycerophosphate dehydrogenase activity, a T3-dependent enzyme. Mitochondrial oxygen consumption in brown fat from normal (p less than 0.01), T4 (p less than 0.02) and T4 plus iopanoic acid-treated (p less than 0.01) rats rose more than twofold in response to cold. In the T3-treated group, oxygen consumption at room temperature was higher (p less than 0.02) than in any other group at similar temperatures. However, the T3-treated group showed no changes in oxygen consumption in response to cold, perhaps because this group reached the maximal response at room temperature. The untreated and the T3-treated hypothyroid rats (both groups devoid of T4) did not survive at 4 degrees C unless T4 or several-fold replacement amounts of T3 were administered. The data demonstrate the crucial role of T4 in thermogenesis during cold exposure.

Improving effects obtained by the ovariectomy or treatment with tamoxifen of female diabetic rats over the function and enzyme activities of liver mitochondria.

In the present work we studied, in female chronic diabetic rats the effect of either the parenteral administration of tamoxifen (TAM) (500 micrograms.kg-1.day-1) for 15 days or the ovariectomy upon the respiration and oscillatory behaviour of intact mitochondria and the activities of 3-hydroxybutyrate dehydrogenase (HBD) and cytochrome c oxidase (Cox) of disrupted liver mitochondria. The treatment with TAM as well as the ovariectomy of diabetic animals significantly increased the respiratory control (RC) and the state 3 (S3) of respiration of intact liver mitochondria with the three substrates assayed (3-hydroxybutyrate, malate-glutamate and succinate). Both treatments also lowered significantly the damped factors of the oscillatory variation of liver intact mitochondria of diabetic rats. Moreover, the two above-mentioned treatments restored the activities of HBD and Cox of liver disrupted mitochondria to normal values. The effect of estrogens at level of its receptors in the modulation of liver mitochondrial function and liver HBD and Cox activities in chronic diabetes is discussed.

[Effects of cold on myocardial mitochondria respiration of rats with hypothyroidism treated with triiodothyronine]. / Efectos del frío sobre la respiración mitocondrial del miocardio de ratas hipotiroideas tratadas con triiodotironina.

The present work studied the effect of cold on oxygen consumption (OC) and alpha-glycerophosphate dehydrogenase activity (alpha-GPD) in heart mitochondria of hypothyroid rats (hypo) treated with T3, T4 or T4 plus Iopanoic Acid (IOP). 200 g male Wistar rats were made hypothyroid by 131I administration. Animals were injected s.c., in divided doses, for 10 days, with one of the following substances: T3, 300 ng/100 g BW/day; T4, 2 micrograms/100 g BW/day or T4 plus IOP, 5 mg/100 g BW/day, for 72 h preceding the experiment. One half of each group was housed in a cold room at 4 degrees C and the other at 22 degrees C, for 25 h, and thereafter decapitated. Heart mitochondria were isolated by routine methods. The OC was measured polarographically using L-malate, L-glutamate and malonate as substrates. Intramitochondrial alpha-GPD activity was measured by a microcolorimetric assay. The results from 16 or 20 rats/group (4 or 5 pools of 4 hearts each) were: In the rats kept at 22 degrees C the OC (in ng at. oxyg./min/mg prot.; State 3) in the hypo+T4 group was 69 +/- 10; in the rats treated with T4+IOP, 75 +/- 11 and in the hypo+T3, 102 +/- 5. When the animals were exposed to 4 degrees C no change was observed in the hypo+T4 and hypo+T4IOP groups. On the other hand, OC was significantly lower in the T3-treated animals (p less than 0.001, versus their controls at 22 degrees C). This group of rats did not survive when exposed to cold.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of withdrawal of glucocorticoids on improving the function and enzymatic activities of liver mitochondria in female diabetic rats.

In the present work the effects of corticosterone restitution were examined in female rats with chronic streptozotocin (SZ)-induced diabetes upon intact liver mitochondrial function and the activities of 3-hydroxybutyrate dehydrogenase (HBD), succinate dehydrogenase (SD) and cytochrome c oxidase (Cox) of the ruptured organelle. The liver mitochondrial function was analyzed by the respiration and the osmotic oscillatory behaviour. Respiration was measured by polarographic method and both the state 3 of active respiration (S3) and the respiratory control (RC) were determined using the following substrates: 3-hydroxybutyrate, succinate and malate-glutamate. The oscillatory behaviour was measured using as parameters the damping factors (DF) which are the ratios of amplitudes of two consecutive peaks or troughs of the spectrophotometrical tracings of this phenomenon. A group of control normal rats (N) and the following three groups of diabetic rats were studied: controls (D), adrenalectomized (D + ADX) and adrenalectomized with corticosterone restitution (D + ADX + C). The results of mitochondrial respiration showed that the mean values of S3 and RC decreased with the three substrates in the group D + ADX + C compared with D + ADX group (p < 0.001). This group demonstrated a significant increase of S3 and RC values of the respiration compared with the D group. The oscillatory behaviour of liver mitochondria of D + ADX + C group demonstrated a significant increase in the DF of peaks and troughs compared with D + ADX group. The values of DF of the latter group were not significantly different from the N group. The behaviour of the enzymes activities of ruptured liver mitochondria were different for each enzyme in the different groups of treated rats. Thus, in the D + ADX + C group the mean value of the activity of HBD significantly decreased, that of the Cox increased (p < 0.02) and that of SD did not show any variation compared with the corresponding values of the D + ADX group. Likewise, the mean value of HBD activity in this latter group was similar to that of the N group and that of Cox activity was lesser (p < 0.01) than that of the D group. The conclusion is drawn that corticosterone has significant additional diabetogenic effects upon biochemical functions of liver mitochondria in the SZ-induced diabetic state which could occur through the hormone cellular receptors.

Efectos del frío sobre la respiración mitocondrial del miocardio de ratas hipotiroideas tratadas con triiodotironina / Effects of cold on myocardial mitochondria respiration of rats with hypothyroidism treated with triiodothyronine

El presente trabajo estudió el efecto del frío sobre el consumo del oxígeno (CO) y la actividad *-glicerofosfato deshidrogenasa (*-GPD) en mitocondria cardíaca de ratas hipotiroideas (hipo) tratadas con T3, T4 o T4 más Acido lopanoico (IOP). Se usaron ratas Wistar macho de 200g de peso hechas hipotiroideas con la administración de I. Los animales fueron inyectadoss s.c., en dosis divididas, por 10 días, con una de las siguientes sustancias: T3, 300 ng/100g peso/día; T4, 2 *g/100g peso/día o T4 más IOP, 5 mg/100g peso/día, por 72 hs. previas al experimento. Una mitad de cada grupo fue mantenida a 4§C y el resto a 22§C, por 24 hs., y logo decapitados. Se aislaron las mitocondrias de corazón por métodos de rutina. El CO se midió polarográficamente usando L-malato, L-glutamato y malonato como sustratos. La actividad mitocondrial *-GPD se medió por un método microcolorimétrico. Los resultados correspondientes a 16 o 20 ratas/grupo (4 o 5 "pooles" de 4 corazones cada uno) fueron los isguientes: En las ratas mantenidas a 22§C el CO (en ng at. de oxíg./min/mg prot.; Estado 3) de las Hipo+T4 fue de 69 ñ 10; en el grupo tratado con T4+IOP fue de 75 ñ 11 y ...

Efectos del frío sobre la respiración mitocondrial del miocardio de ratas hipotiroideas tratadas con triiodotironina / Effects of cold on myocardial mitochondria respiration of rats with hypothyroidism treated with triiodothyronine

El presente trabajo estudió el efecto del frío sobre el consumo del oxígeno (CO) y la actividad *-glicerofosfato deshidrogenasa (*-GPD) en mitocondria cardíaca de ratas hipotiroideas (hipo) tratadas con T3, T4 o T4 más Acido lopanoico (IOP). Se usaron ratas Wistar macho de 200g de peso hechas hipotiroideas con la administración de I. Los animales fueron inyectadoss s.c., en dosis divididas, por 10 días, con una de las siguientes sustancias: T3, 300 ng/100g peso/día; T4, 2 *g/100g peso/día o T4 más IOP, 5 mg/100g peso/día, por 72 hs. previas al experimento. Una mitad de cada grupo fue mantenida a 4ºC y el resto a 22ºC, por 24 hs., y logo decapitados. Se aislaron las mitocondrias de corazón por métodos de rutina. El CO se midió polarográficamente usando L-malato, L-glutamato y malonato como sustratos. La actividad mitocondrial *-GPD se medió por un método microcolorimétrico. Los resultados correspondientes a 16 o 20 ratas/grupo (4 o 5 "pooles" de 4 corazones cada uno) fueron los isguientes: En las ratas mantenidas a 22ºC el CO (en ng at. de oxíg./min/mg prot.; Estado 3) de las Hipo+T4 fue de 69 ñ 10; en el grupo tratado con T4+IOP fue de 75 ñ 11 y ... (AU)

Efectos del frío sobre la respiración mitocondrial del miocardio de ratas hipotiroideas tratadas con triiodotironina. / [Effects of cold on myocardial mitochondria respiration of rats with hypothyroidism treated with triiodothyronine]

The present work studied the effect of cold on oxygen consumption (OC) and alpha-glycerophosphate dehydrogenase activity (alpha-GPD) in heart mitochondria of hypothyroid rats (hypo) treated with T3, T4 or T4 plus Iopanoic Acid (IOP). 200 g male Wistar rats were made hypothyroid by 131I administration. Animals were injected s.c., in divided doses, for 10 days, with one of the following substances: T3, 300 ng/100 g BW/day; T4, 2 micrograms/100 g BW/day or T4 plus IOP, 5 mg/100 g BW/day, for 72 h preceding the experiment. One half of each group was housed in a cold room at 4 degrees C and the other at 22 degrees C, for 25 h, and thereafter decapitated. Heart mitochondria were isolated by routine methods. The OC was measured polarographically using L-malate, L-glutamate and malonate as substrates. Intramitochondrial alpha-GPD activity was measured by a microcolorimetric assay. The results from 16 or 20 rats/group (4 or 5 pools of 4 hearts each) were: In the rats kept at 22 degrees C the OC (in ng at. oxyg./min/mg prot.; State 3) in the hypo+T4 group was 69 +/- 10; in the rats treated with T4+IOP, 75 +/- 11 and in the hypo+T3, 102 +/- 5. When the animals were exposed to 4 degrees C no change was observed in the hypo+T4 and hypo+T4IOP groups. On the other hand, OC was significantly lower in the T3-treated animals (p less than 0.001, versus their controls at 22 degrees C). This group of rats did not survive when exposed to cold.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of withdrawal of glucocorticoids on improving the function and enzymatic activities of liver mitochondria in female diabetic rats.

In the present work the effects of corticosterone restitution were examined in female rats with chronic streptozotocin (SZ)-induced diabetes upon intact liver mitochondrial function and the activities of 3-hydroxybutyrate dehydrogenase (HBD), succinate dehydrogenase (SD) and cytochrome c oxidase (Cox) of the ruptured organelle. The liver mitochondrial function was analyzed by the respiration and the osmotic oscillatory behaviour. Respiration was measured by polarographic method and both the state 3 of active respiration (S3) and the respiratory control (RC) were determined using the following substrates: 3-hydroxybutyrate, succinate and malate-glutamate. The oscillatory behaviour was measured using as parameters the damping factors (DF) which are the ratios of amplitudes of two consecutive peaks or troughs of the spectrophotometrical tracings of this phenomenon. A group of control normal rats (N) and the following three groups of diabetic rats were studied: controls (D), adrenalectomized (D + ADX) and adrenalectomized with corticosterone restitution (D + ADX + C). The results of mitochondrial respiration showed that the mean values of S3 and RC decreased with the three substrates in the group D + ADX + C compared with D + ADX group (p < 0.001). This group demonstrated a significant increase of S3 and RC values of the respiration compared with the D group. The oscillatory behaviour of liver mitochondria of D + ADX + C group demonstrated a significant increase in the DF of peaks and troughs compared with D + ADX group. The values of DF of the latter group were not significantly different from the N group. The behaviour of the enzymes activities of ruptured liver mitochondria were different for each enzyme in the different groups of treated rats. Thus, in the D + ADX + C group the mean value of the activity of HBD significantly decreased, that of the Cox increased (p < 0.02) and that of SD did not show any variation compared with the corresponding values of the D + ADX group. Likewise, the mean value of HBD activity in this latter group was similar to that of the N group and that of Cox activity was lesser (p < 0.01) than that of the D group. The conclusion is drawn that corticosterone has significant additional diabetogenic effects upon biochemical functions of liver mitochondria in the SZ-induced diabetic state which could occur through the hormone cellular receptors.

Effects of withdrawal of glucocorticoids on improving the function and enzymatic activities of liver mitochondria in female diabetic rats.

In the present work the effects of corticosterone restitution were examined in female rats with chronic streptozotocin (SZ)-induced diabetes upon intact liver mitochondrial function and the activities of 3-hydroxybutyrate dehydrogenase (HBD), succinate dehydrogenase (SD) and cytochrome c oxidase (Cox) of the ruptured organelle. The liver mitochondrial function was analyzed by the respiration and the osmotic oscillatory behaviour. Respiration was measured by polarographic method and both the state 3 of active respiration (S3) and the respiratory control (RC) were determined using the following substrates: 3-hydroxybutyrate, succinate and malate-glutamate. The oscillatory behaviour was measured using as parameters the damping factors (DF) which are the ratios of amplitudes of two consecutive peaks or troughs of the spectrophotometrical tracings of this phenomenon. A group of control normal rats (N) and the following three groups of diabetic rats were studied: controls (D), adrenalectomized (D + ADX) and adrenalectomized with corticosterone restitution (D + ADX + C). The results of mitochondrial respiration showed that the mean values of S3 and RC decreased with the three substrates in the group D + ADX + C compared with D + ADX group (p < 0.001). This group demonstrated a significant increase of S3 and RC values of the respiration compared with the D group. The oscillatory behaviour of liver mitochondria of D + ADX + C group demonstrated a significant increase in the DF of peaks and troughs compared with D + ADX group. The values of DF of the latter group were not significantly different from the N group. The behaviour of the enzymes activities of ruptured liver mitochondria were different for each enzyme in the different groups of treated rats. Thus, in the D + ADX + C group the mean value of the activity of HBD significantly decreased, that of the Cox increased (p < 0.02) and that of SD did not show any variation compared with the corresponding values of the D + ADX group. Likewise, the mean value of HBD activity in this latter group was similar to that of the N group and that of Cox activity was lesser (p < 0.01) than that of the D group. The conclusion is drawn that corticosterone has significant additional diabetogenic effects upon biochemical functions of liver mitochondria in the SZ-induced diabetic state which could occur through the hormone cellular receptors.

Modulation of D-3-hydroxybutyrate dehydrogenase activity by estrogens.

Liver D-3-hydroxybutyrate dehydrogenase (OHBD) is subjected to estrogen modulation. Estrogen action was demonstrated by (a) the lesser activity of liver OHBD in female rats, as compared with their male counterparts; (b) the increase of OHBD activity after ovariectomy of sexually mature rats; (c) the decrease of OHBD activity after treatment of gonadectomized or normal rats with 17 beta-estradiol or with artificial estrogens; (d) the decrease of OHBD activity in female rats during sexual development; (e) the effects of tamoxifen on the enzyme activity. The kinetics of OHBD reaction using liver mitochondria from estrogen-treated rats showed a 50% decrease of Vmax, as compared with the control value, in contrast to the other parameters which did not vary. These results, taken together with the effect of estrogens on liver mitochondrial phospholipids, point to a decreased content of OHBD in liver mitochondria from estrogen-treated rats. In contrast to OHBD, succinate dehydrogenase and cytochrome oxidase activities, mitochondrial protein synthesis and L-malate + L-glutamate oxidation by coupled liver mitochondria either increased or were not affected by estrogens. Kidney and heart OHBD were affected by ovariectomy and estrogens like the liver enzyme, though to a lesser degree.