Functional atrophy of brown adipose tissue during lactation in mice. Effects of lactation and weaning on mitochondrial GDP binding and uncoupling protein.

The thermogenic activity and capacity of brown adipose tissue were determined in mice during lactation and after weaning. There was a marked fall during lactation in the mitochondrial content of the tissue, and in GDP binding and the specific mitochondrial concentration of uncoupling protein. The lactation-induced functional atrophy of brown adipose tissue was fully reversible after weaning; mitochondrial content and the mitochondrial concentration of uncoupling protein were both restored, although GDP binding was not normalized.     

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.     

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.     

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.     

Sequential changes in brown adipose tissue composition, cytochrome oxidase activity and GDP binding throughout pregnancy and lactation in the rat

The sequential appearance of changes in interscapular brown adipose tissue composition, cytochrome oxidase activity and GDP binding was studied throughout pregnancy and lactation in the rat. Brown adipose tissue was hypertrophied during pregnancy because of progressive lipid accumulation, whereas its mitochondrial component and GDP binding to brown fat mitochondria were unchanged. In early lactation (day 5) there was a decrease in the overall GDP binding to brown fat only because of the lower mitochondrial protein content. In late stages of lactation (days 10 and 15), the amount of tissue and its mitochondrial protein content were minimal and the GDP binding per mitochondrial protein decreased substantially. Scatchard analysis in day-15-lactating rats indicated a large decrease in GDP binding sites without any changes in affinity. It is concluded that the diminished thermogenic activity of brown fat in lactation is attained through changes at different structural levels of the tissue occurring in a characteristic sequential trend; first a reduction in its mitochondrial component, and only later, at mid-lactation, a decrease in the specific mitochondrial proton conductance pathway activity.     

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 that fasting can induce a selective loss of uncoupling protein from brown adipose tissue mitochondria of mice

The effects of fasting and refeeding on the concentration of uncoupling protein in brown adipose tissue mitochondria have been investigated in mice. Fasting mice for 48 h led to a large decrease in the total cytochrome oxidase activity of the interscapular brown fat pad. Mitochondrial GDP binding and the specific mitochondrial concentration of uncoupling protein also fell on fasting. After 24 h refeeding both GDP binding and the mitochondrial concentration of uncoupling protein were normalized, but there was no alteration in the total tissue cytochrome oxidase activity. Fasting appears to induce a selective loss of uncoupling protein from brown adipose tissue mitochondria, which is rapidly reversible on refeeding.     

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.     

Thermogenesis and the energetics of pregnancy and lactation

Energy balance studies suggest that the overall efficiency of energy utilization does not increase during pregnancy in rodents, other than as a consequence of "hyperphagia". Diet-induced thermogenesis is not stimulated in response to the increased energy intake of the pregnant animal, the extra intake being retained at the maximum efficiency. Biochemical studies on brown adipose tissue, the main site of adaptive thermogenesis in rodents, are consistent with the energy balance data, at least in rats and mice. However, in hamsters (golden and Djungarian) some atrophy of the tissue is evident during pregnancy. In contrast to pregnancy, the thermogenic activity (mitochondrial GDP binding) and capacity (uncoupling protein content) of brown adipose tissue are substantially reduced during lactation in rats and mice. These changes result from a fall in sympathetic activity in the tissue in lactation. Sympathetic activity and thermogenic capacity are, however, fully restored following weaning of the pups. The functional atrophy of brown adipose tissue during lactation is linked to a substantial saving in maternal energy expenditure, reducing the energy requirements for milk production. The lactating-post-lactating animal provides an excellent example of a physiologically programmed reversible atrophy of brown adipose tissue.     

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.     

Effect of caging singly or in groups of different sizes on the thermogenic activity of interscapular brown adipose tissue in mice

The effects on the thermogenic activity of brown adipose tissue of caging mice singly or in groups of different sizes has been investigated. At 23 degrees C the total cytochrome oxidase activity and the level of mitochondrial GDP binding were higher in mice caged singly than in mice caged in groups of three or six. At 4 degrees C GDP binding and cytochrome oxidase activity were lower in mice caged in groups of two, three or six than in mice caged singly. The mitochondrial concentration of uncoupling protein was not clearly affected by the number of mice in each cage.     

Postnatal appearance of uncoupling protein and formation of thermogenic mitochondria in hamster brown adipose tissue

Brown adipose tissue of developing hamster was characterized by western blotting, enzyme activity measurements and immunoelectron microscopy. During the first postnatal week the tissue contained significant amounts of differentiating mitochondria and comparable quantities of active cytochrome oxidase and ATP synthase. The uncoupling protein appeared on the 7/8th day and its specific content increased 80-times between day 8 and day 17. In parallel, the specific content and activity of cytochrome oxidase increased 3-times but ATP synthase decreased 2-times. The total content of uncoupling protein and of cytochrome oxidase in interscapular brown adipose tissue increased 360- and 11-times, respectively. Analysis of isolated mitochondria showed that the observed differences result mainly from changes of the enzymic equipment of the mitochondrial membrane. During the same interval, propylthiouracil-insensitive "type II' thyroxine 5'-deiodinase activity in brown adipose tissue increased 10-times. It was concluded that the thermogenic function of the hamster brown adipose tissue develops after the first postnatal week due to highly differentiated synthesis of mitochondrial proteins leading to replacement of preexisting, uncoupling protein-lacking nonthermogenic mitochondria by thermogenic ones, similarly as shown in brown adipose tissue of the embryonic mouse and rat (Houst?k, J., et al. (1988) Biochim. Biophys. Acta 935, 19-25).     

Effects of neonatal sympathectomy on brown fat development and susceptibility to high fat diet induced obesity in mice.

Injections of 6-hydroxydopamine in mouse neonates caused extensive and long lasting damage to the sympathetic nervous system and impaired brown fat development. Brown adipose tissue (BAT) thermogenic capacity of sympathectomized mice (up to 120 days old) was reduced because of marked reductions in the tissue mitochondrial protein content and the mitochondrial concentration of uncoupling protein, as assessed by [3H]GDP binding and immunoassay. Neonatal sympathectomy did not affect BAT DNA content. Sympathectomized mice also had reduced epinephrine-stimulated rates of oxygen consumption. BAT of sympathectomized mice failed to respond by increases in [3H]GDP binding to isolated mitochondria and uncoupling protein concentration when animals were offered a palatable high-fat dietary supplement that increased calorie intake of both normal and sympathectomized mice. The high-fat diet caused increases in body weight, carcass fat, and gonadal white fat pad weights in sympathectomized animals that were similar to those of control mice. These results show that inactivation of BAT metabolism did not accentuate the development of obesity caused by a dietary supplement rich in fat and suggest that stimulation of BAT metabolism was not very effective in counteracting the obesity-inducing effect of this diet.     

Differential interaction of fatty acids and fatty acyl CoA esters with the purified/reconstituted brown adipose tissue mitochondrial uncoupling protein

Proteoliposomes containing highly purified uncoupling protein generated by a modified purification/reconstitution procedure carried out active GDP dependent proton conductance. It was further established that long chain acyl CoA esters as well as fatty acids stimulated proton influx by the uncoupling protein, and, moreover, that the acyl CoA esters were partially effective in overcoming the inhibition by GDP. GDP binding to the purified uncoupling protein was inhibited by acyl CoA esters but not fatty acids. Phenylglyoxal which prevents GDP binding to the uncoupling protein eliminated the acyl CoA but not the fatty acid effect on proton conductance. These results substantiate the fact that nucleotides and acyl CoA esters act at the same regulatory site on the uncoupling protein, whereas, fatty acids act at a separate site. The properties of the purified/reconstituted uncoupling protein confirm they are identical to those inherent in brown adipose tissue mitochondria.     

Effects of ciglitazone on insulin resistance and thermogenic responsiveness to acute cold in brown adipose tissue of genetically obese (ob/ob) mice

Genetically obese (ob/ob) mice develop a marked insulin resistance in brown adipose tissue soon after weaning, and this is paralleled by a fall in the acute activation of the mitochondrial proton conductance pathway in the tissue on cold exposure. Treatment of ob/ob mice with ciglitazone, a new oral hypoglycaemic, led to a restoration of insulin sensitivity in brown adipose tissue. The amelioration of insulin resistance was accompanied by a normalization of the acute, cold-induced increase in mitochondrial GDP binding. These results support the hypothesis that the development of insulin resistance in brown adipose tissue is an important factor in the impaired thermogenic responsiveness of obese mice.     

The concentration of uncoupling protein correlates with Mg2+ activated mitochondrial binding of GDP

Rats were housed at 4 degrees C for periods of up to 26 days. As little as 2 h of cold exposure caused an increase in the binding of [3H]GDP to mitochondria from brown adipose tissue. Incubation of mitochondria in vitro with 10 mM Mg2+ caused a marked increase in the subsequent binding of GDP to mitochondria from rats housed at 28 degrees C and a smaller increase in that from rats exposed to 4 degrees C for 2 h. Chronic exposure to cold led to an even greater increase in the amount of GDP bound to mitochondria incubated with Mg2+. The time course for the increase in the concentration of uncoupling protein was compared with that for GDP binding to mitochondria with and without Mg2+ treatment. The concentration of uncoupling protein appears to be correlated with the GDP-binding values for mitochondria treated with Mg2+ (r = 0.70) but not with the GDP binding to untreated mitochondria (r = 0.36). Therefore, the binding of GDP to untreated mitochondria may represent thermogenic activity at the time of death, whereas that after treatment with Mg2+ may more closely reflect total thermogenic capacity of the mitochondrion.     

The possible proton translocating activity of the mitochondrial uncoupling protein of brown adipose tissue. Reconstitution studies in liposomes

Loose coupling of thermogenic mitochondria of brown adipose tissue is related to a high proton (or hydroxyl) conductance of the inner membrane and to the presence of a unique 32 kDa uncoupling protein. Reconstitution experiments of the purified protein in liposomes are reported which suggest that this component could form proton channels in the membrane.     

Immunochemical detection and quantitation of brown adipose tissue uncoupling protein

A polyclonal antisera against rat brown adipose tissue mitochondrial uncoupling protein was used to examine mitochondrial samples from liver and white and brown adipose tissue from several mammalian species. A sodium dodecyl sulfate--polyacrylamide gel electrophoretic separation of proteins combined with an immunochemical method allowed for visualization of antigen--antibody complexes on nitrocellulose blots. Hamster, cavy, monkey, and mouse brown adipose tissue mitochondrial samples cross-reacted with the antisera. Mitochondria prepared from white fat obtained from young swine and sheep contained two closely migrating, antigenically active proteins. Hepatic mitochondria samples did not contain antigenically active protein. Reflectance densitometry was used for quantitation of the uncoupling protein in various mitochondrial samples. In rats fed diets low in protein, there appears to be a dissociation between the concentration of uncoupling protein and the number of nucleotide binding sites as given by the [3H]GDP binding assay. These results are indicative of a physiological activation of the uncoupling protein.     

Studies on the activity of brown adipose tissue in suckling,pre-obese, obob mice

The properties and activity of brown adipose tissue have been investigated in suckling, pre-obese, $\text{ob}\text{ob}$ mice in order to determine whether decreased thermogenesis in the tissue precedes the development of obesity in this mutant. At 14 days of age there was no difference between the $\text{ob}\text{ob}$ and normal animals in the total amount of interscapular brown adipose tissue, and the DNA content, protein content, and cytochrome oxidase activity of the tissue were similar in the two groups of mice. Respiration rates of brown adipose tissue mitochondria in the presence of albumin were, however, greater in the normal than the $\text{ob}\text{ob}$ animals, although after the addition of GDP to recouple the mitochondria there was no difference between the two groups. The mitochondrial membrane potential, measured with [³H]methyltriphenylphosphonium, was less affected by exogenous GDP in $\text{ob}\text{ob}$ mice than in normal animals. GDP binding to brown adipose tissue mitochondria, an index of the proton conductance pathway, was much greater in normal than in $\text{ob}\text{ob}$ mice at both 10 and 14 days of age; the decreased GDP binding in the mutant animals was found to result from a reduction in the number of binding sites. It is concluded that brown adipose tissue mitochondria of pre-obese $\text{ob}\text{ob}$ mice are more tightly coupled than those of normal siblings, and that the activity of the ‘thermogenic’ proton conductance pathway is lower in the mutant animals. A decrease in thermogenesis in brown adipose tissue is therefore an early event in the development of the $\text{ob}\text{ob}$ mouse and precedes the appearance of obesity.     

Long photophase is not a sufficient stimulus to reduce thermogenic capacity in winter-acclimatized short-tailed field voles (Microtus agrestis) during long-term cold acclimation

The thermogenic capacity of brown adipose tissue in winter- and summer-acclimatized short-tailed field voles (Microtus agrestis) was investigated by examining changes in mass of brown adipose tissue, the ratio of white adipose tissue to brown adipose tissue, the concentration of the uncoupling protein (thermogenin) in whole depots (μg) and in mitochondrial mass (μg·mg^-1) and the activity of cytochrome c oxidase in the depots (mmol·min^-1). The concentration of thermogenin in winter-acclimatized voles (n=8), per brown adipose tissue depot and per mitochondrial mass, was significantly higher than in summer-acclimatized voles (n=6). There was no significant difference in the level of cytochrome c oxidase activity between these two groups. Four groups of winter-acclimatized voles (n=6 in each group) were exposed to 5°C for 10, 20, 50 and 100 days in a 14L:10D photoperiod. Body mass, brown adipose tissue mass, white adipose tissue mass and basal metabolic rate were significantly positively related to the length of time cold exposed up to 100 days. There was a significant inverse relationship between the ratio of white to brown adipose tissue mass and the duration of cold exposure. There was no significant relationship between thermogenin concentration, either per depot or in mitochondrial mass of brown adipose tissue, with the length of time cold exposed. The level of cytochrome c oxidase activity increased significantly from control levels to a maximum after 10 days in the cold but decreased from 10 days onwards. In winter-acclimatized M. agrestis, a 14L:10D photoperiod is not a sufficient stimulus to reduce thermogenic capacity during cold acclimation. Indeed, some changes in the indirect parameters reflecting thermogenesis, notably the increase in basal metabolic rate and the decrease in the ratio of white to brown adipose tissue mass, indicated that despite the long photophase the thermogenic capacity was slightly further enhanced during the cold acclimation.