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1. 1. We have studied fatty acid oxidation and ketone bodies production in liver mitochondria from rats exposed to cold for 10 days and in rats adapted to cold for 40 days. We have examined State 3 respiration and ketone bodies production with palmitoylcarnitine as substrate in isolated mitochondria, together with mitochondrial protein mass and hepatocyte oxygen consumption. 2. 2. Mitochondrial protein mass increases both in cold exposed and in cold adapted rats. 3. 3. State 3 respiration and hepatocyte respiration increase only in cold exposed rats. 4. 4. Ketone bodies production increases in cold adapted rats. 5. 5. Taken together, our results suggest that the increased hepatic mitochondrial protein mass and the increased fatty acid oxidation subserve two different roles: an increase in ATP production in cold exposed rats and an increase in ketone bodies production in cold adapted rats.

Tumor resistance to oxidative stress prevents the efficacy of cancer therapy based upon a free radical-mediated mechanism. K-ras transformed NIH 3T3 cells (E32-4-2) showed, under oxidative stress, reactive oxygen species (ROS) levels 10-fold lower and lipid peroxide levels 56% lower, compared to their nontransformed counterpart. Since p21(ras) activity depends upon farnesylation, we tested the effect of the inhibitors of farnesylation lovastatin and (alpha-hydroxyfarnesyl) phosphonic acid on susceptibility to oxidative stress in these cells. Preincubation of cells for 24 h with 10 mu M lovastatin resulted in a 10-fold increase of ROS levels and a 50% increase of lipid peroxide levels measured under pro-oxidant conditions. Similarly, preincubation of cells with 100 mu M (alpha-hydroxyfarnesyl) phosphonic acid fur 24 h enhanced stress-induced levels of either ROS (7.5-fold) or lipid peroxides (33%). The effect of lovastatin and (alpha-hydroxyfarnesyl) phosphonic acid is specifically due to their ability to inhibit p21(ras) activity. In fact, inhibition of p21(ras) by transfecting E32-4-2 cells with the transdominant negative mutant of H-ras (L61,S186) led, analogously to lovastatin or (alpha-hydroxyfarnesyl) phosphonic acid treatment, to a strong increase of stress-induced ROS levels. These results suggest that farnesylation inhibitors could be used as an adjuvant therapy to improve the tumoricidal effect of cancer treatment based upon free-radical production in ms-dependent tumors. (C) 1996 Academic Press

We measured mitochondrial protein mass as well as State 4 and 3 respiratory rates using different substrates in isolated liver mitochondria from 30-day cold-exposed rats. In addition, we measured the respiration under different conditions of stimulation in isolated hepatocytes from long-term cold-exposed rats. The results show that long-term cold exposure elicits a significant increase in hepatic mass and mitochondrial protein mass. No variation was found in oxygen consumption of isolated mitochondria and hepatocytes. On the whole, the results indicate that long-term exposure elicits an increase in hepatic mitochondrial protein mass but not in hepatic oxygen consumption.

The effects of thyroid state on liver mitochondrial protein mass was investigated in rats at 24 and 4 °C, as was oxidative phosphorylation using substrates which represent the final catabolic products of the metabolic fuels. In rats at 24 °C, a significant increase in mitochondrial protein mass (about +40%) was observed only in hyperthyroid animals, while a significant increase due to cold exposure was found in hypothyroid (+ 45%) and euthyroid (+ 35%) rats. In rats at 24 °C, hypothyroidism significantly decreased the oxidation of glutamate and palmitoyl carnitine but not of pyruvate, while hyperthyroidism only increased the oxidation of palmitoyl carnitine. On the other hand, exposure to cold significantly increased the oxidation of glutamate and pyruvate only in the presence of tri-iodothyronine. Our results underline not only the fact that a simple and single hypothesis for thyroid effects cannot be adopted, but also that any study concerning oxidative metabolism should be carried out using different substrates and involving different pathways of oxidation. Journal of Endocrinology (1991) 131, 67–73

We have examined the relationship between the changes in resting metabolic rate (RMR) and those in hepatic metabolism induced by hyperthyroidism and fasting for 24 h. We found that hyperthyroidism induced a significant increase in RMR, while fasting for 24 h reduced RMR in euthyroid but not in hyperthyroid rats. We have also measured oxygen consumption in isolated hepatocytes from euthyroid and hyperthyroid rats, fed or fasted for 24 h. Hyperthyroidism induced an increase in oxygen consumption in rat liver cells; fasting for 24 h increased respiratory rates in isolated liver cells from euthyroid but not from hyperthyroid rats. The findings showed that hyperthyroidism and fasting for 24 h have opposite effects on RMR but similar effects on hepatic metabolism. The results also indicated that the increase in RMR found in hyperthyroid rats is partly due to an increase in hepatic metabolism, while no correlation exists between variations in resting and hepatic metabolism induced by 24-h fasting. Journal of Endocrinology (1992) 135, 45–51

In this paper, we have examined the relationship between the changes in the resting metabolic rate (RMR) and the changes in hepatic metabolism induced by hypothyroidism and 24 h fasting. The results show that hypothyroidism induces a significant decrease in RMR, while 24 h fasting reduces RMR in euthyroid but not in hypothyroid rats. We have also measured oxygen consumption in isolated hepatocytes from euthyroid and hypothyroid rats, fed or fasted for 24 h. The results show that hypothyroidism is characterized by reduced hepatic oxygen consumption. On the other hand, 24 h fasting induces an increase in oxygen consumption in both euthyroid and hypothyroid rat liver cells, although the respiratory rates of hypothyroid rats were lower than those of euthyroid rats. The above findings, as a whole, show that hypothyroidism and 24 h fasting have similar effects on RMR but opposite effects on hepatic metabolism. In addition, a normal thyroid state does not appear to be necessary for the observed changes in hepatic metabolism due to 24 h fasting.

The effects of glucose and O2 deprivation (OGD) on the survival of cortical and cerebellar neurons were examined to characterize the biochemical mechanisms involved in OGD and OGD followed by reoxygenation. To this aim, neurons were kept for different time periods in a hypoxic chamber with a controlled atmosphere of 95% N(2) and 5% CO2 in a glucose-free medium. After OGD, reoxygenation was achieved by exposing the cells to normal O2 and glucose levels. Neither MTT, an index of mitochondrial oxidative phosphorylation, nor malondialdehyde (MDA) production, a parameter measuring lipid peroxidation, were affected by 1 hr of OGD in cortical neurons. When OGD was followed by 24 hr of reoxygenation, MTT levels were reduced by 40% and MDA was significantly increased, whereas cellular ATP content did not change. Cerebellar granule cells, on the other hand, did not show any reduction of mitochondrial activity after exposure to 1 hr OGD or to 1 hr OGD plus 24 hr of reoxygenation. When OGD was prolonged for 2 hr, a significant reduction of the mitochondrial activity and of cellular ATP content occurred, coupled to a significant MDA increase in cerebellar granule cells, whereas in cortical neurons a reduction of MTT levels after 2 hr OGD was not accompanied by a decrease of cellular ATP content nor by an increase of MDA production. Moreover, 24 hr of reoxygenation further reinforced lipid peroxidation, LDH release, propidium iodide positive neurons and the reduction of ATP content in cerebellar granule cells. The results of the present study collectively show that cortical and cerebellar neurons display different levels of vulnerability to reoxygenation followed by OGD. Furthermore, the impairment of mitochondrial activity and the consequent overproduction of free radicals in neurons were observed for the first time occurring not only during the reoxygenation phase, but already beginning during the OGD phase.

In this work the protonmotive force (delta p), as well as the subcellular distribution of malate, ATP, and ADP were determined in perfused liver from rats fed a low fat or high fat diet, using density gradient fractionation in non aqueous solvents. Rats fed a high fat diet, despite an enhanced hepatic oxygen consumption, exhibit similar delta p to that found in rats fed a low fat diet, but when we consider the two components of delta p, we find a significant decrease in mitochondrial/cytosolic pH difference (delta pH(m)) and a significant increase in mitochondrial membrane potential (delta psi(m)) in rats fed a high fat diet compared to rats fed a low fat diet, which tend to compensate each other. In rats fed a high fat diet the concentration ratio of malate and ATP/ADP does not reflect the changes in delta pH(m) and delta psi(m), which represent the respective driving force for their transport. The findings are in line with an increase in substrate supply to the respiratory chain which is, however, accompanied by a higher energy turnover in livers from HFD rats. By this way the liver could contribute to the lack of weight gain from the high caloric intake in HFD rats.

The aim of this work was to assess the participation of the sympathetic nervous system in the thermogenic response to food in control and hyperphagic rats. Rats were fed either a control (CD) or energy dense (ED) diet. After 15 days, CD rats received a small (7 kJ) meal composed of either control or energy dense diet, while ED rats received a small meal composed of energy dense diet. The experiment was then repeated, with the exception that rats received a larger portion (35 kJ) of the test meal. The postprandial increase in oxygen consumption was measured for 30 min after the small meal and 90-180 min after the completion of the large meal. The measurements were made in saline-injected and propranolol-injected rats. ED rats exhibited hyperphagia as well as an increase of 32% in resting metabolic rate after a 16 h fast. The sympathetically-mediated postprandial increase in oxygen consumption was greater after an energy dense meal than after a control meal in CD rats, and was higher in ED rats than in CD rats fed an energy dense meal. It was concluded that the sympathetically-mediated increase in the thermogenic response to food, as well as the increase in fasting metabolic rate can help prevent obesity development in hyperphagic rats.

It is known that ascorbate has a lipid lowering effect, accompanied by a drop of apo B, in rats fed a diet enriched with 1.5% of cholesterol (Nath diet). In order to better clarify the role exerted by ascorbate in lipid metabolism, the effect of ascorbate administration on apolipoprotein pattern in rats fed the Nath diet was investigated. Wistar male rats fed for two months the Nath diet were treated i.p. with 60 mg/kg of body weight of ascorbate for 10 days. Blood collection before and after the treatment was performed by intracardiac puncture. Lipoproteins were prepared by preparative ultracentrifugation and their apoprotein content was obtained by densitometric scanning of the apoprotein electhrophoretic pattern. The decrease of total plasma cholesterol and triglycerides and of cholesterol, triglycerides and protein content of all plasma lipoproteins observed in ascorbate treated rats, is accompanied by a marked modification of the apolipoprotein pattern of all lipoprotein classes studied, with an increase of apo E content in VLDL-IDL and LDL fractions (135 and 44% respectively), and a decrease of C (37%), AI (70%) and B (37.5%) apoproteins in VLDL-IDL and of apo C (36%) in LDL. On the contrary, in HDL fraction ascorbate induces an increase of C apoproteins (26%) and a decrease of E and B apoproteins (47% and 71% respectively). The data reported clearly show that in hypercholesterolemic rats the lipid lowering effect of ascorbate administration, is accompanied by a marked modification of the apoprotein pattern of all lipoprotein classes studied.

1. The effect of cold exposure on the respiratory capacity of rat liver mitochondria has been studied using succinate as the substrate. 2. The mitochondria obtained in this study were well coupled, as shown by the RCR and ADP/O ratios. 3. In addition, durohydroquinone was used to eliminate the regulation of substrate supply. Likewise, we measured uncoupled respiration to evaluate the maximal electron flow through the respiratory chain. 4. We found that oxygen consumption using succinate or durohydroquinone + FCCP as substrates, as well as ATP production were not affected by cold exposure. 5. Our results also show that, when succinate is used, the maximal capacity of the respiratory chain is measured. 6. The data obtained do not support a role of the electron transport chain as a target of cold action.

We show that remarkable differences exist among 3 mitochondrial subpopulations with regard to oxidation-phosphorilation coupling. In the heavy fraction (sedimenting at 3,000 g) ATP synthesis is optimized while in the medium and light fractions (sedimenting at 10,000 and 27,000 g, respectively) heat production tends to be optimized as the mitochondrial size decreases. As the oxygen consumption of the L-fraction drastically increases (+150%) after fifteen days of cold exposure, such differences have remarkable implications for the mechanism of cellular thermogenesis and its regulation. © 1988 Academic Press, Inc.

In this paper we report that three different rat liver mitochondrial fractions, differing in density, exhibit differential effects when the animals are made hypo- or hyperthyroid. The investigations have been performed by correlating the protein content, the succinic dehydrogenase behaviour and the respiratory features of the three fractions in different thyroid states with morphometric-stereologic analysis the electron micrographie level. The results indicate that the thyroid hormone influences both the mass and the functionality of the heavy (H) and light (L) fraction. In hypothyroid rats the H fraction increases (+43%) while the L fraction decreases (− 32%) and their respiratory activity is drastically reduced. Adenosine triphosphate (ATP) synthesis in the H fraction is also inhibited. Triiodothyronine (T 3 ) administration to the above animals restores the values observed in control rats. At morphometric level we note in hypothyroid rats an increase in the number of mitochondria together with a concomitant increase in the average volume of a single mitochondrion. We are inclined to explain the above results through an action exerted by T 3 on a hypothetical mitochondrial cycle starting with the formation of light organelles from heavy ones.

We have studied changes in hepatic mitochondrial efficiency induced by 24-h fasting or acclimation at 29 degrees C, two conditions of reduced thermogenesis. Basal and palmitate-induced proton leak, which contribute to mitochondrial efficiency, are not affected after 24-h fasting, when serum free triiodothyronine decreases significantly and serum free fatty acids increase significantly. In rats at 29 degrees C, in which serum free triiodothyronine and fatty acids decrease significantly, basal proton leak increases significantly, while no variation is found in palmitate-induced proton leak. The present results indicate that mitochondrial efficiency in the liver is not related to a physiological decrease in whole body thermogenesis.

We investigated, in skeletal muscle mitochondria isolated from semistarved and refed rats, the relation between the protein expression of uncoupling protein 3 (UCP3) and mitochondrial oxidative capacity, assessed as state 4 and state 3 respiration rates in presence of substrates that are either non-lipids (glutamate, succinate) or lipids (palmitoyl CoA, palmitoylcarnitine). During semistarvation, when whole-body thermogenesis is diminished, state 3 respiration was lower than in fed controls by about 30% independently of substrate types, while state 4 respiration was lower by 20% only during succinate oxidation, but UCP3 was unaltered. After 5 days of refeeding, when thermogenesis is still diminished, neither state 4, state 3 nor UCP3 were lower than in controls. Refeeding on a high-fat diet, which exacerbates the suppression of thermogenesis, resulted in a two-fold elevation in UCP3 but no change in state 4 or state 3 respiration. These results during semistarvation and refeeding, in line with those previously reported for fasting, are not in support of the hypothesis that UCP3 is a mediator of adaptive thermogenesis pertaining to weight regulation, and underscore the need for caution in interpreting parallel changes in UCP3 and mitochondrial oxidative capacity as the reflection of mitochondrial uncoupling by UCP3.

To establish whether changes in skeletal muscle mitochondrial efficiency contribute to increased energy expenditure and decreased metabolic efficiency of overeating rats with increased thermogenesis, we measured basal proton leak, fatty acid-induced uncoupling and uncoupling protein 3 (UCP3) content in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria. Intermyofibrillar, but not subsarcolemmal, mitochondria from rats with increased thermogenesis exhibited a lower proton leak compared with controls. In both mitochondrial populations from rats with increased thermogenesis, fatty acid-induced uncoupling was increased significantly and a small recoupling effect of GDP was detected. In addition, intermyofibrillar and subsarcolemmal mitochondria from rats with increased thermogenesis showed higher UCP3 contents than controls. These results point out that metabolic efficiency in subsarcolemmal and intermyofibrillar mitochondria from rats with increased thermogenesis is differently regulated. In fact, in intermyofibrillar mitochondria both basal proton leak and fatty acid-induced uncoupling are altered, while in subsarcolemmal mitochondria only fatty acid-induced uncoupling increases. Both mitochondrial populations in skeletal muscle cells from rats with increased thermogenesis display an increased fatty acid-induced uncoupling and UCP3 content, which could contribute to avoiding obesity.

In this paper we have examined the effect of cold exposure on hepatic mitochondrial state 3 respiration and ATP synthesis, using succinate as the substrate, in euthyroid, hypothyroid and hyperthyroid rats. The results show that cold exposure does not elicit any variation in the above parameters in euthyroid and hyperthyroid rats, whereas when hypothyroid rats are exposed to cold, a significant increase (about +45%) occurs in state 3 respiration and ATP synthesis. We have also measured succinic dehydrogenase specific activity and uncoupled respiration during cold exposure in various thyroid states. The finding that cold exposure elicits no variation in the above parameters indicates that there is some control on ATP synthase and/or adenine nucleotide translocator. The above findings, as a whole, suggest that cold exposure acts on oxidative phosphorylation only if triiodothyronine is lacking, by controlling ATP synthase and/or adenine nucleotide translocator.

We have investigated whether changes in hepatic mitochondrial protein mass and respiration occur in hyperphagic rats. Hyperphagia was induced by a palatable diet. Rats fed such a diet showed a significantly greater energy intake (+40%) without a corresponding greater body weight gain when compared to control rats. They also showed a significantly higher resting metabolic rate (+44%) compared to control rats. The latter increase was abolished by propranolol administration and, as such, was considered to be an adaptative response. No difference in liver mitochondrial protein mass was found in hyperphagic rats. On the other hand, state 3 respiration using succinate as substrate increased, with no difference in ADP/O ratios and, hence, in the degree of coupling. This increase is mediated by the sympathetic nervous system, as it was abolished by propranolol. The above results indicate, an increase in ATP synthetic capacity and suggest one possible mechanism by which hepatic metabolism contributes to the increased energy expenditure of hyperphagic rats.

We studied the relationship between membrane potential and respiration rate in isolated liver mitochondria from rats fed an energy dense diet. We conceptually divided the system into blocks of reactions that produced or consumed mitochondrial membrane potential and then measured the kinetic response of these blocks of reactions to this potential using NAD-linked and FAD-linked substrates. We show that decreased respiration rate with an NAD-linked substrate is accounted for by decreased kinetic response of the substrate oxidation pathway to the potential. No variation in the kinetic response of the above blocks of reactions to the potential was found using an FAD-linked substrate. These results indicate that FAD-linked and NAD-linked pathways are differently affected in rats fed an energy dense diet.