F1F0-ATPase, early target of the radical initiator 2,2'-azobis-(2-amidinopropane) dihydrochloride in rat liver mitochondria in vitro.

This study was designed to determine which enzyme activities were first impaired in mitochondria exposed to 2,2'-azobis-(2-amidinopropane) dihydrochloride (AAPH), a known radical initiator. EPR spin-trapping revealed generation of reactive oxygen species although malondialdehyde formation remained very low. With increasing AAPH concentrations, State-3 respiration was progressively depressed with unaltered ADP/O ratios. A top-down approach demonstrated that alterations were located at the phosphorylation level. As shown by inhibitor titrations, ATP/ADP translocase activity was unaffected in the range of AAPH concentrations used. In contrast, AAPH appeared to exert a deleterious effect at the level of F1F0-ATPase, comparable with dicyclohexylcarbodi-imide, which alters Fo proton channel. A comparison of ATP hydrolase activity in uncoupled and broken mitochondria reinforced this finding. In spite of its pro-oxidant properties, AAPH was shown to act as a dose-dependent inhibitor of cyclosporin-sensitive permeability transition initiated by Ca2+, probably as a consequence of its effect on F1F0-ATPase. Resveratrol, a potent antiperoxidant, completely failed to prevent the decrease in State-3 respiration caused by AAPH. The data suggest that AAPH, when used under mild conditions, acted as a radical initiator and was capable of damaging F1F0-ATPase, thereby slowing respiratory chain activity and reducing mitochondrial antioxidant defences.

In vivo beta-adrenergic induction of the unmasking of the uncoupling protein in rat brown fat.

1. In 28 degrees C adapted rats (WA) both cold stress and norepinephrine (NE) led to a 4-fold increase of uncoupling protein dependent proton conductance which was abolished by propranolol (PRO). 2. In 4-day warm re-exposed rats (after 10 days at 5 degrees C) (WR) the same uncoupling by cold stress was observed but the NE effect was lower. Uncoupling by cold stress was not abolished by PRO. 3. In WR rats, uncoupling was not due to the involvement of an alpha-adrenergic pathway. 4. Both beta-agonist isoproterenol and beta 3-agonists BRL 35135A and ICI D7114 led to high levels of unmasking. 5. Interscapular brown adipose tissue surgical denervation, which abolished cold stress unmasking both in WA and, WR rats, indicates a mediation by direct sympathetic innervation. 6. Depending on the thermal history of the rat, the possibility that unmasking by cold stress could be mediated by different types of beta-receptors is discussed.

Effect of acute magnesium deficiency on the masking and unmasking of the proton channel of the uncoupling protein in rat brown fat.

The short term regulation of heat production in brown adipose tissue mitochondria (BAT) of acutely Mg-deficient rats was demonstrated by comparing several parameters of mitochondrial energization. Mg deficiency in vivo had absolutely no effect on the BAT uncoupling protein concentration (UCP) which was only modified by thermal conditions. The same high concentration was observed 10 d cold exposed control and Mg-deficient rats. Four days of warm re-exposure at thermal neutrality led to a moderate 26 per cent decrease with both diets which was not modified by cold stress for 1 h. Proton conductance. CmH+, and proton motive force, delta p, were calculated from membrane potential and respiration rate measurements. The same high level CmH+ was observed in cold exposed rats with both diets. Compared to warm re-exposed control rats, CmH+ was threefold higher in the corresponding Mg-deficient group which indicated a much lower masking of the proton channel of UCP with the Mg-deficient diet. This difference was not dependent on the presence of magnesium in vitro. The basal CmH+, independent of UCP, was not altered by magnesium deficiency. These results emphasize that acute regulation of thermogenic BAT activity through the masking and unmasking process is altered when magnesium supply is limited in vivo.

Effect of sympathetic de-activation on thermogenic function and membrane lipid composition in mitochondria of brown adipose tissue.

Male Long-Evans rats (9 weeks of age) were exposed to cold (5 degrees C) for 10 days. Then, sympathetic de-activation of brown adipose tissue (BAT) was performed either by BAT surgical denervation (Sy) or by warm re-exposure at 28 degrees C (WE) for 4 days. The incidence of the two treatments on thermogenic activity of BAT mitochondrial membranes and their lipid composition was investigated. Sy and WE induced a large decrease in GDP binding on the uncoupling protein (UCP) (43% and 82%, respectively). Several parameters of mitochondrial energization were investigated. Sy and WE substantially decreased UCP-dependent proton conductance (CmH+) over the whole range of protonmotive force. CmH+ showed greater variation than GDP binding. The low basal UCP-independent CmH+ was the same in all groups. Comparison of GDP binding and CmH+ with UCP content which is not modified revealed a masking of both the nucleotide binding site and the proton channel. Sy and WE induced the same increase of phosphatidylcholine to phosphatidylethanolamine ratio (16%) but had opposite effects on fatty acid unsaturation. The results were discussed with reference to functional significance of these variations in BAT mitochondrial thermogenic activity and lipid composition.

Time-course variations of effective proton conductance and GDP binding in brown adipose tissue mitochondria of rats during prolonged cold exposure.

1. Time-course variations of the thermogenic pathway in rat brown adipose tissue (BAT) mitochondria were examined. 2. Several parameters of mitochondrial energization, protonmotive force and its components pH gradient and membrane potential were investigated. The specific binding of GDP was compared with the effective proton conductance (CmH+) of the membrane. 3. Ten-days cold exposure led to maximal GDP binding and GDP-dependent CmH+. 4. The subsequent relative decrease in GDP binding observed during prolonged cold exposure (40 days) was functional and led to a lower GDP-dependent CmH+. CmH+ showed greater variation than GDP binding. 5. The CmH+ decrease was not due to a masking of active sites of the uncoupling protein. 6. Basal GDP-independent CmH+ was not modified. 7. Results are discussed with reference to the significance of biochemical measures and to the physiological regulation of BAT thermogenesis.

Variations in energization parameters and proton conductance induced by cold adaptation and essential fatty acid deficiency in mitochondria of brown adipose tissue in the rat.

Male weanling Long-Evans rats were fed on a low-fat semipurified diet (control diet, 2% sunflower oil; essential fatty acid (EFA) deficient diet, 2% hydrogenated coconut oil) for 9 weeks. In order to modulate need for non-shivering thermogenesis, groups of rats on each diet were exposed at 28 degrees C (thermoneutrality) and at 5 degrees C (cold acclimation) for the last 5 weeks. In brown adipose tissue (BAT) mitochondria, several parameters of mitochondrial energization, protonmotive force (delta p) and its components delta pH and membrane potential, delta psi, were investigated. Simultaneous measurement of oxygen consumption and delta psi (the main component of delta p) was performed by varying alpha-glycerophosphate concentration and the force/flux relationship of the mitochondria was established by comparison of proton conductance, CmH+, over the whole range of protonmotive force. delta p. In the absence of GDP, at 28 degrees C, EFA deficiency induced a marked increase in CmH+. Cold acclimation led to comparable enhanced CmH+ in control and EFA-deficient mitochondria. In the presence of GDP which binds and inhibits the BAT 32 kDa uncoupling protein, CmH+ was the same in 28 degrees C and 5 degrees C control mitochondria, but EFA deficiency led to an enhanced GDP independent CmH+ at 28 degrees C and to a lesser extent at 5 degrees C. These results are discussed with reference to substantial changes in mitochondrial lipid composition induced by the deficiency.

The effects of essential fatty acid deficiency on brown adipose tissue activity in rats maintained at thermal neutrality.

1. The consequences of essential fatty acid (EFA) deficiency on the resting metabolism, food efficiency and brown adipose tissue (BAT) thermogenic activity were examined in rats maintained at thermal neutrality (28 C). 2. Weanling male Long-Evans rats were fed a hypolipidic semi-purified diet (control diet: 2% sunflower oil; EFA-deficient diet: 2% hydrogenated coconut oil) for 9 weeks. 3. They were kept at 28 C for the last 5 weeks. Compared to controls, in EFA-deficient rats the growth shortfall reached 21% at killing. 4. As food intake was the same in EFA-deficient and control rats, food efficiency was thus decreased by 40%. 5. Resting metabolism expressed per surface unit was 15% increased. 6. Non-renal water loss was increased by 88%. 7. BAT weight was 28% decreased but total and mitochondrial proteins were not modified. 8. Heat production capacity, tested by GDP binding per BAT was 69% increased in BAT of deficient rats. 9. The stimulation of BAT was established by two other tests: GDP inhibition of mitochondrial O2 consumption and swelling of mitochondria. 10. It is suggested that the observed enhancement of resting metabolism in EFA-deficient rats is, in part, due to an activation of heat production in BAT.

Involvement of the sympathetic nervous system in lipolytic activity in brown adipose tissue of cold acclimated rats.

In cold acclimated rats, in vitro, NE led to a significant increase in release of FFA and glycerol in denervated IBAT. In vivo, study of arteriovenous differences showed that the denervated BAT loses its full capacity to utilize FFA and glycerol released by NE. After denervation an increase of blood flow in Sulzer's vein was observed. This effect appeared immediately after intervention whereas the effect on fat metabolism appeared later. In cold acclimated rats, the sympathetic nervous system appears to be an important regulator of fatty acid metabolism in BAT.