Unmasking of GDP binding sites on hamster brown adipose tissue mitochondria and uncoupling protein.

1. A rapid unmasking of GDP binding sites on brown adipose tissue (BAT) mitochondria was observed when hamsters acclimatized to 28 degrees C were exposed to a temperature of 4 degrees C for 2 hr. 2. No rapid unmasking of GDP binding sites was observed when hamsters housed at 22 degrees C were briefly exposed to 4 degrees C. 3. The amount of GDP bound to BAT mitochondria from hamsters increased during 2 weeks of exposure to 4 degrees C, but did not change between 2 weeks and 30 days of cold exposure. 4. Incubation of mitochondria with 10 mM Mg2+ prior to the GDP binding assay increased the subsequent GDP binding to BAT mitochondria from hamsters housed at 28, 22 or 4 degrees C, albeit to different degrees. 5. The amount of GDP bound to uncoupling proteins isolated from untreated and Mg(2+)-treated mitochondria of hamsters and rats was measured. Scatchard analyses of the binding of GDP to purified uncoupling protein indicate that increases in the number of binding sites due to Mg2+ treatment of mitochondria do not change the affinity of the protein for GDP.     

Thermogenic capacity of the brown adipose tissue of developing rats; effects of rearing temperature

A chronological study was performed to investigate the postnatal development of the thermogenic capacity of the brown adipose tissue (BAT) comparing rats born and reared at 16 degrees C (cold) or 28 degrees C (control). Mitochondrial mass, cytochrome-c-oxidase activity (index of oxidative capacity) and GDP binding to mitochondria (uncoupling test) were investigated in rats from 1 to 33 days of age. Specific cytochrome-c-oxidase activity was the same in both groups during the first week, then increased in the cold group and decreased in controls; from the 9th day it was always twice as high in the former as in the latter. Specific binding of GDP to mitochondrial proteins remained almost constant in control rats during the first week contrasting with a rapid increase in that for cold rats. Afterwards it decreased in both groups until weaning but remained five times as high in cold rats as in control rats. As growth of BAT is faster and mitochondrial content greater in cold reared rats, the capacity of the tissue for thermogenesis appeared to be greatly temperature dependent soon after birth and during the entire suckling period. However the mechanisms of this stimulation remain to be elucidated.     

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.     

Evidence for unmasking of rat brown-adipose-tissue mitochondrial GDP-binding sites in response to acute cold exposure. Effects of washing with albumin on GDP binding.

Rats, previously acclimated to 29 degrees C, were moved into the cold (4 degrees C) for 2 h. Scatchard analysis of GDP binding to the brown-adipose-tissue mitochondria of these animals showed a 2.3-fold increase in the number of high-affinity sites and a 1.5-fold increase in the number of low-affinity sites compared with binding in animals maintained at 29 degrees C. Immunochemical determination showed no increase in the amount of mitochondrial uncoupling protein during this period. This strongly suggests an unmasking of existing GDP-binding sites before a detectable increase in synthesis of uncoupling protein can occur. Washing with albumin increased the number of GDP-binding sites of brown-adipose-tissue mitochondria from both warm-housed and cold-exposed animals to the same extent. This indicates that the effects of washing with albumin and cold exposure are independent and additive.     

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.     

Increased nonshivering thermogenesis, brown fat cytochrome-c oxidase activity, GDP binding, and uncoupling protein mRNA levels after short daily cold exposure of Phodopus sungorus

In their natural environment, burrowing rodents experience rather fluctuating ambient temperatures and are acutely cold exposed only for short periods outside their burrows. The effect of short daily cold exposure on basal metabolic rate, nonshivering thermogenesis, brown fat thermogenesis, and uncoupling protein mRNA was studied in the Djungarian hamster, Phodopus sungorus. They were kept at 23 degrees C and exposed to 5 degrees C daily either for one 4-h period or twice for 2 h (in 12-h intervals). At the same time control hamsters were kept continuously either at thermoneutrality (23 degrees C) or at 5 degrees C. Two 2-h cold exposures daily were sufficient to increase basal metabolic rate and nonshivering thermogenesis to the same level as continuous cold exposure, whereas one 4-h cold period per day did not result in a significant increase of both parameters. Brown fat thermogenesis (as measured by cytochrome-c oxidase activity and GDP binding to the mitochondrial uncoupling protein) increased to the same extent by both treatments with short daily cold exposure. However, this increase was less than in the chronically cold-exposed hamsters. A similar result was found for uncoupling protein mRNA: both short-term cold-exposed hamsters increased uncoupling protein mRNA levels to a similar extent, but less than after chronic cold treatment. It is concluded that short daily cold exposures are sufficient to cause adaptive increases of the capacity of metabolic heat production as well as brown fat thermogenic properties.     

Number of mice per cage influences uncoupling protein content of brown adipose tissue.

The effect of housing density of mice on the thermogenic state and capacity of their brown adipose tissue was studied. Mice were housed one, two, or six per cage at 28 degrees C for 15 days. Increased housing density suppressed the thermogenic capacity of brown adipose tissue (decreased the total amount of uncoupling protein) and decreased the thermogenic state of brown adipose tissue mitochondria (decreased GDP binding). A density of six mice per cage had a greater effect than a density of two mice per cage. The size of brown adipose tissue (wet weight and protein content), the content of mitochondria in it (cytochrome oxidase content), and the total activity of thyroxine 5'-deiodinase were not altered by housing density. We conclude that even at a temperature close to thermoneutrality (29-33 degrees C for the mouse), the occurrence of social thermoregulation (huddling) reduces the requirement for brown adipose tissue thermogenesis and results in a reduction in its thermogenic capacity. It is clearly of importance that the design of studies of mouse brown adipose tissue take into account not only the temperature at which the mice are housed, but also the number of mice housed per cage.     

Brown fat thermogenesis in cold-acclimated rats is not abolished by the suppression of thyroid function

The effects of long-term cold exposure on brown adipose tissue (BAT) thermogenesis in hypothyroid rats have been examined. Thyroid ablation was performed in normal rats after 2 mo of exposure to 4 degrees C, when BAT hypertrophy and thermogenic activity were maximal. After ablation, hypothyroid and normal controls remained in the cold for 2 additional months. At the end of the 4-mo cold exposure, all untreated hypothyroid rats were alive, had normal body temperature, and had gained an average 12.8% more weight than normal controls. Long-term cold exposure of hypothyroid rats markedly increased BAT weight, mitochondrial proteins, uncoupling protein (UCP)-1, mRNA for UCP-1, and oxygen consumption to levels similar to those seen in cold-exposed normal rats. The results indicate that thyroid hormones are required for increased thermogenic capacity to occur as an adaptation to long-term cold exposure. However, cold adaptation can be maintained in the absence of thyroid hormone.     

Evidence that the acute unmasking of GDP-binding sites in brown adipose tissue mitochondria is not dependent on mitochondrial swelling

The role of mitochondrial swelling in the unmasking of GDP-binding sites on brown adipose tissue mitochondria has been examined in mice. Acute cold exposure (6 degrees C for 1 h) led to increases in GDP binding without changes in the concentration of uncoupling protein, indicating that an unmasking of binding sites had occurred. Measurements of mitochondrial matrix volume suggested that an acute unmasking of GDP-binding sites took place without swelling of the mitochondria. In addition, the induction of a rapid preswelling of the mitochondria by incubation in KCl in the presence of valinomycin did not affect the cold-induced unmasking of GDP-binding sites. It is concluded that the acute unmasking of GDP-binding sites on uncoupling protein in brown adipose tissue is not due simply to mitochondrial swelling.     

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 development of insulin resistance in brown adipose tissue may impair the acute cold-induced activation of thermogenesis in genetically obese (ob/ob) mice

Genetically obese (ob/ob) mice develop insulin resistance in brown adipose tissue during the fifth week of life. Prior to this, at 26 days of age, oh/oh mice show a substantial increase in GDP binding to brownadipose-tissue mitochondria during acute cold exposure. When insulin resistance in brown fat develops, by 35 days of age, the increase in GDP binding in response to cold is markedly reduced. Studies with 2-deoxyglucose suggest that insulin resistance in brown adipose tissue could impair thermogenic responsiveness during acute cold exposure by limiting the ability of the tissue to take up glucose.     

Attenuated response to cold of brown adipose tissue of mice made obese with gold thioglucose

In a first study, mice made obese with gold thioglucose became hypothermic when exposed to 4 degrees C. In a second study, lean mice and mice made obese with gold thioglucose (dynamic phase) were acclimated to 14 degrees C for up to 2 weeks and their brown adipose tissue was studied. The cold-induced increase in thyroxine 5'-deiodinase activity was initially slightly smaller in obese mice, but by 24 h and 2 weeks in the cold the activity of thyroxine 5'-deiodinase was the same in lean and obese mice. Unexpectedly, the elevated activity of 5'-deiodinase returned to the low level seen in warm-acclimated mice in both lean and obese mice after 2 weeks of cold acclimation. In gold thioglucose obese mice, a progressive cold-induced increase in the binding of guanosine diphosphate to isolated mitochondria, an index of both acute thermogenic activation and a long-term increase in uncoupling protein concentration, paralleled that seen in normal lean mice and remained at a high level after 2 weeks in the cold, although still remaining slightly lower than normal. It is not clear how a high level of mitochondrial GDP binding is maintained in cold-acclimated mice at the same time as a low level of thyroxine 5'-deiodinase activity when both are believed to be controlled by the sympathetic nervous system. We conclude that the gold thioglucose obese mouse can activate its brown adipose tissue fairly normally when it is exposed to cold, but that some attenuation of this process may contribute to the impaired survival of this mouse at low temperatures.     

Metabolic Changes Induced by Cold Stress in Rat Liver Mitochondria

The mechanisms involved in the metabolic changes induced by cold stress in isolated rat liver mitochondria were studied. Respiration, ATP synthesis, and membrane potential as well as the contents of several metabolites were determined in liver mitochondria from cold-exposed rats. At different times of cold exposure, the force-flux relationships showed net variation in flux (enhanced respiration, diminished ATP synthesis) with no associated variation in force (H+ gradient); this suggested that decoupling rather than classical uncoupling was involved in the effects of cold stress. The flux control coefficient of the H+ leak on basal respiration was slightly increased by 380 h of cold exposure. Cold stress also induced a diminution in total membrane fatty acids, Zn2+, Fe3+, ATP, and ADP/O ratios; the content of cytochromes c + c1 and b oscillated. The contents of Ca2+, Na+, Pi, and cytochromes a + a3 were not affected, whereas matrix ADP, AMP, K+, and Mg2+ were markedly increased. Basal and oleic acid-stimulated respiration of mitochondria from cold-stressed rats was inhibited by GDP, carboxyatractyloside, or albumin. These agents did not affect basal respiration in control mitochondria. Western blot analysis showed enhanced expression of a protein of about 35 kDa, presumably the uncoupling protein 2, induced by long-term cold exposure. The overall data suggest that cold stress promoted decoupling of oxidative phosphorylation, and hence, changes in several matrix metabolites, by increasing free fatty acids and the UCP2 content.     

Increased GDP binding and thermogenic activity in brown adipose tissue mitochondria during arousal of the hibernating garden dormouse (Eliomys quercinus L.).

1. The thermogenic activity of brown adipose tissue in hibernating garden dormice during hypothermic torpor and at different states of arousal were studied. High levels of GDP binding were observed on isolated brown fat mitochondria, indicating that the thermogenic proton conductance pathway is very active in brown fat during arousal. 2. In order to investigate this phenomenon, the uncoupling protein was assessed by immunological assay and the mRNA for UCP was analysed. 3. Animals during arousal exhibited neither increase in UCPmRNA nor an increase in UCP. 4. Our results suggest that during the rewarming of garden dormice there is an acute unmasking of GDP binding sites on the protein.     

Cold exposure differently influences mitochondrial energy efficiency in rat liver and skeletal muscle

This study deals with mitochondrial energy efficiency in liver and skeletal muscle mitochondria in 15 days cold exposed rats. Cold exposure strongly increases the sensitivity to uncoupling by added palmitate of skeletal muscle but not liver mitochondria, while mitochondrial energy coupling in the absence of fatty acids is only slightly affected by cold in liver and skeletal muscle. In addition, uncoupling protein 3 content does not follow changes in skeletal muscle mitochondrial coupling. It is therefore concluded that skeletal muscle could play a direct thermogenic role based on fatty acid-induced mild uncoupling of mitochondrial oxidative phosphorylation.     

Cold tolerance of UCP1-ablated mice: A skeletal muscle mitochondria switch toward lipid oxidation with marked UCP3 up-regulation not associated with increased basal,fatty acid- or ROS-induced uncoupling or enhanced GDP effects

Mice lacking the thermogenic mitochondrial membrane protein UCP1 (uncoupling protein 1) - and thus all heat production from brown adipose tissue - can still adapt to a cold environment (4 °C) if successively transferred to the cold. The mechanism behind this adaptation has not been clarified. To examine possible adaptive processes in the skeletal muscle, we isolated mitochondria from the hind limb muscles of cold-acclimated wild-type and UCP1(–/–) mice and examined their bioenergetic chracteristics. We observed a switch in metabolism, from carbohydrate towards lipid catabolism, and an increased total mitochondrial complement, with an increased total ATP production capacity. The UCP1(–/–) muscle mitochondria did not display a changed state-4 respiration rate (no uncoupling) and were less sensitive to the uncoupling effect of fatty acids than the wild-type mitochondria. The content of UCP3 was increased 3-4 fold, but despite this, endogenous superoxide could not invoke a higher proton leak, and the small inhibitory effect of GDP was unaltered, indicating that it was not mediated by UCP3. Double mutant mice (UCP1(–/–) plus superoxide dismutase 2-overexpression) were not more cold sensitive than UCP1(–/–), bringing into question an involvement of reactive oxygen species (ROS) in activation of any alternative thermogenic mechanism. We conclude that there is no evidence for an involvement of UCP3 in basal, fatty-acid- or superoxide-stimulated oxygen consumption or in GDP sensitivity. The adaptations observed did not imply any direct alternative process for nonshivering thermogenesis but the adaptations observed would be congruent with adaptation to chronically enhanced muscle activity caused by incessant shivering in these mice.     

Uncoupling protein and its mRNA in brown adipose tissue of newborn rabbits

Guanosine diphosphate binding to the uncoupling protein of isolated mitochondria of brown adipose tissue in newborn rabbits was measured as an index of thermogenic activity. The binding was 0.281 +/- 0.022 nmol GDP/mg mitochondrial protein at 1 day of age, 0.214 +/- 0.017 at 3 days, 0.428 +/- 0.038 at 5 days, and 0.208 +/- 0.016 at 7 days. The increase in binding between 3 and 7 days of age suggests that the brown fat has an increased thermogenic capacity at that age. In addition, the potential for synthesis of the uncoupling protein was investigated in 1- to 5-day-old newborn rabbits by probing the total cellular ribonucleic acid for the messenger that codes for uncoupling protein. The amount of uncoupling protein messenger was highest at 1 day of age and declined at least until 5 days of age. Because the amount of uncoupling protein messenger decreased as the GDP binding increased, the results suggest that either the initially translated uncoupling protein was unmasked at about 5 days of age or there was a delay in the incorporation of uncoupling protein into the mitochondrial inner membrane, or both.     

Acute effects of a beta-adrenoceptor agonist (BRL 26830A) on rat brown-adipose-tissue mitochondria. Increased GDP binding and GDP-sensitive proton conductance without changes in the concentration of uncoupling protein.

GDP binding, proton conductance and the specific concentration of uncoupling protein were measured in brown-adipose-tissue mitochondria of rats treated acutely with the novel beta-agonist, BRL 26830A. At 1 h after dosing with BRL 26830A, mitochondrial GDP binding was increased more than 2-fold. The increase in binding resulted from an increase in the number of binding sites. An iterative analysis of Scatchard binding data suggested that there is only one high-affinity GDP-binding site (Kd 0.3 microM) in brown-adipose-tissue mitochondria. The acute increase in GDP binding produced by treatment with BRL 26830A occurred without any alteration in the specific mitochondrial concentration of uncoupling protein, as determined by radioimmunoassay. Treatment with the beta-agonist did, however, lead to a small increase in the GDP-sensitive component of mitochondrial proton conductance. These results indicate that GDP-binding sites on uncoupling protein can be rapidly unmasked after treatment with a brown-fat-specific beta-agonist, and that the increase in binding reflects an increase in the activity of the mitochondrial proton-conductance pathway.     

Cold-induced changes in the energy coupling and the UCP3 level in rodent skeletal muscles

The mechanism of thermoregulatory uncoupling of respiration and phosphorylation in skeletal muscles has been studied. It is found that 24 h cold exposure results in (i) a 3-fold increase in the amount of UCP3 protein in rat skeletal muscle mitochondria, and (ii) pronounced lowering of the membrane potential in isolated rat or mouse skeletal muscle mitochondria. The decrease in membrane potential is reversed by adding bovine serum albumin. Cold exposure is also found to sensitize the membrane potential to the uncoupling action of added fatty acid (laurate). After laurate addition, the recoupling effects of GDP and carboxyatractylate decrease whereas that of albumin increases in mitochondria from cold-treated rats or mice. Changes similar to those induced by cold can be initiated by the in vivo addition of thyroxine. Cold exposure does not affect energy coupling in liver mitochondria. The possible involvement of UCP3 isoforms in nucleotide-sensitive and -insensitive uncoupling is discussed.     

Factors affecting cold-induced hypertension in rats

A 3- to 4-week exposure of rats to a cold environment (5 +/- 2 degrees C) induces hypertension, including elevation of systolic, diastolic, and mean blood pressures and cardiac (left ventricular) hypertrophy. The studies described here were designed to investigate some factors affecting both the magnitude and the time course for development of cold-induced hypertension. The objective of the first study was to determine whether there was an ambient temperature at which the cold-induced elevation of blood pressure did not occur. The objective of the second experiment was to determine whether body weight at the time of exposure to cold affected the magnitude and time course for development of hypertension. To assess the first objective, male rats were housed in a chamber whose temperature was maintained at 5 +/- 2 degrees C while others were housed in an identical chamber at 9 +/- 2 degrees C. After 7 days of exposure to cold, the rats exposed to the colder temperature had a significant elevation of blood pressure (140 +/- 2 mm Hg) compared with the group maintained at 9 degrees C (122 +/- 3 mm Hg). The rats exposed to 9 degrees C had no significant elevation of systolic blood pressure at either 27 or 40 days after initiation of exposure to cold. At the latter time, the temperature in the second chamber was reduced to 5 +/- 2 degrees C. By the 25th day of exposure to this ambient temperature, the rats had a significant increase in systolic blood pressure above their levels at 9 degrees C. Thus, there appears to be a threshold ambient temperature for elevation of blood pressure during exposure to cold. That temperature appears to lie somewhere between 5 and 9 degrees C. The second objective was assessed by placing rats varying in weight from approximately 250 to 430 g in air at 5 degrees C. There was a highly significant direct relationship (r = 0.96) between body weight at the time of introduction to cold and the number of days required to increase systolic blood pressure by 10 mm Hg above pre-cold exposure level. The third objective was to make an initial assessment of potential differences among strains of rats with respect to development of cold-induced hypertension. To this end, rats of the Fischer 344 strain were used. Systolic blood pressures of these rats also increased during chronic exposure to cold.(ABSTRACT TRUNCATED AT 400 WORDS)