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.     

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.     

Adaptive changes in the concentration of the mitochondrial ‘uncoupling’ protein in brown adipose tissue of hamsters acclimated at different temperatures

The effect of acclimation at different temperatures on the activity of interscapular brown adipose tissue has been investigated in the hamster, a hibernator. Between 31° and 4°C the cytochrome oxidase activity of the tissue increased 4- to 5-fold, mitochondrial GDP binding per mg of mitochondrial protein doubled, and the amount of uncoupling protein rose from 1.7% to 5.4% of total mitochondrial protein. It is concluded that there are clear adaptive changes induced by temperature in brown adipose tissue of the hamster, but the changes are limited in comparison with those in the mouse.     

Brown fat activity in fasted and refed rats

Four days of fasting in the rat reduced brown-adipose-tissue (BAT) mass, mitochondrial protein, and tissue protein content. Specific binding of guanosine diposphate (GDP) to BAT mitochondria was depressed by 55% in 4d-fasted rats. Rats fasted for 3 d, and then refed a single carbohydrate meal (40 kJ), showed a significant increase in specific GDP-binding (27% above fasted) 24 h later, and a large increase in total binding. Specific activities of cytochrome oxidase and -glycerophosphate dehydrogenase in BAT mitochondria were not significantly affected by fasting or refeeding. These results suggest that BAT may be partly responsible for the fall in metabolic rate associated with fasting and the delayed increase after carbo-hydrate refeeding. These effects may be due to changes in the mitochondrial proton-conductance pathway in brown fat.     

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.     

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).     

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.     

A commentary on the interpretation of in vitro biochemical measures of brown adipose tissue thermogenesis

In this article we comment on the various in vitro biochemical measurements employed to assess the thermogenic activity and capacity of brown adipose tissue. The meaning and significance of changes in tissue weight, protein content, cell number, and mitochondrial mass are each summarized. In addition, various indices of the proton conductance pathway-mitochondrial swelling, proton conductance, uncoupling protein concentration, and GDP binding studies--are discussed. The issue of unmasking and masking of GDP binding sites is reviewed; recent reports have clearly demonstrated unmasking and masking, and it is concluded that GDP binding studies are an index of the activity of uncoupling protein, rather than a measure of its concentration. It is suggested that tissue mass, mitochondrial content, mitochondrial GDP binding, and uncoupling protein concentration represent core measurements for the biochemical assessment of the thermogenic activity and capacity of brown adipose tissue. Auxiliary measurements include Scatchard analysis of GDP binding data to distinguish changes in the number of binding sites from potential changes in Kd, and mitochondrial swelling studies, as an additional index of proton permeability. The distinction between thermogenic activity (GDP binding, proton permeability) and capacity (uncoupling protein content), both on a per unit of mitochondrial protein and per tissue basis, is emphasized.     

Noradrenaline turnover in brown adipose tissue and the heart of fed and fasted golden hamsters

The effect of fasting on sympathetic activity in tissues of the golden hamster has been investigated using measurements of noradrenaline turnover. Fasting for 60 h did not have a significant effect on noradrenaline turnover, both fractional and total, in brown adipose tissue or the heart. Fasting did, however, result in a functional atrophy of brown adipose tissue; tissue weight, protein content, and cytochrome oxidase activity were each reduced after a 60-h fast. These results suggest that the atrophy of brown adipose tissue induced by fasting in the golden hamster does not relate to a major decrease in sympathetic activity. The findings add further support for the view that the thermogenic capacity of brown adipose tissue is not primarily dependent on sympathetic activity in the golden hamster.     

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.     

Effects of fasting and refeeding on body mass,thermogenesis and serum leptin in Brandt's voles (Lasiopodomys brandtii)

(1)

To investigate the effect of fasting and refeeding on the body mass, thermogenesis and serum leptin in Brandt's voles, the changes in body and body fat mass, resting metabolic rate (RMR), mitochondrial cytochrome c oxidase (COX) activity in liver and brown adipose tissue (BAT), uncoupling protein 1 (UCP1) content of BAT, serum leptin level and post-fasting food intake were monitored and measured.     

Measurement by radioimmunoassay of the mitochondrial uncoupling protein from brown adipose tissue of obese (ob/ob) mice and Zucker (fa/fa) rats at different ages

The concentration of the 'uncoupling protein' in brown adipose tissue mitochondria has been measured in lean and obese (ob/ob) mice and Zucker (fa/fa) rats at different ages using a specific radioimmunoassay. During the suckling period the concentration of the protein was similar in normal and mutant animals of both types, despite the decrease in mitochondrial GDP binding observed in the obese. The concentration of uncoupling protein was, however, decreased in adult ob/ob mice and adult Zucker rats compared with their respective lean siblings, in parallel with the decrease in GDP binding. It is concluded that there is a 'masked', or inactive, form of uncoupling protein in young ob/ob mice and fa/fa rats.     

The role of UCP 1 in production of reactive oxygen species by mitochondria isolated from brown adipose tissue

We provide evidence that ablation or inhibition of, uncoupling protein 1 increases the rate of reactive oxygen containing species production by mitochondria from brown adipose tissue, no matter what electron transport chain substrate is used (succinate, glycerol-3-phosphate or pyruvate/malate). Consistent with these data are our observations that (a) the mitochondrial membrane potential is maximal when uncoupling protein 1 is ablated or inhibited and (b) oxygen consumption rates in mitochondria from uncoupling protein 1 knock-out mice, are significantly lower than those from wild-type mice, but equivalent to those from wild-type mice in the presence of GDP. In summary, we show that uncoupling protein 1 can affect reactive oxygen containing species production by isolated mitochondria from brown adipose tissue.     

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.     

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.     

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.     

Synthesis of mitochondrial uncoupling protein in brown adipocytes differentiated in cell culture

In order to characterize the biogenesis of unique thermogenic mitochondria of brown adipose tissue, differentiation of precursor cells isolated from mouse brown adipose tissue was studied in cell culture. Synthesis of mitochondrial uncoupling protein (UCP), F1-ATPase, and cytochrome oxidase was examined by L-[35S]methionine labeling and immunoblotting. For the first time, synthesis of physiological amounts of the UCP, a key and tissue-specific component of thermogenic mitochondria, was observed in cultures at about confluence (day 6), indicating that a complete differentiation of brown adipocytes was achieved in vitro. In postconfluent cells (day 8) the content of UCP decreased rapidly, in contrast to some other mitochondrial proteins (beta subunit of F1-ATPase, cytochrome oxidase). In these cells, it was possible, by using norepinephrine, to induce specifically the synthesis of the UCP but not of F1-ATPase or cytochrome oxidase. The maximal response was observed at 0.1 microM norepinephrine and the synthesis of UCP remained activated for at least 24 h. Detailed analysis revealed a major role of the beta-adrenergic receptors and elevated intracellular concentration of cAMP in stimulation of UCP synthesis. A quantitative recovery of the newly synthesized UCP in the mitochondrial fraction indicated completed biogenesis of functionally competent thermogenic mitochondria.     

Comparative subcellular fractionation of control and cold-adapted rat brown and white adipose tissue with special reference to peroxisomal and mitochondrial distributions

A new technique for single-step subcellular fractionation of adipose tissue homogenates by analytical sucrose density gradient centrifugation in a vertical pocket reorientating rotor is described. The density gradient distributions of mitochondrial and peroxisomal marker enzymes in brown and white adipose tissue of control and cold exposed rats are compared. The equilibrium density of brown fat mitochondria was found to be significantly increased compared with white fat mitochondria. GDP binding activity was localized solely to the mitochondria in both control and cold-adapted brown adipose tissue. Brown and white fat mitochondria fractions were isolated by differential centrifugation and the specific activities of various enzymes in the homogenate and mitochondrial preparations determined. The specific activity of creatine kinase in brown adipose tissue was found to be ten-fold higher than in white fat and subcellular fractionation studies showed the activity to have an exclusively cytosolic distribution in both tissues. GDP binding activity and some of the mitochondrial enzymes showed, in brown adipose, a striking increase in total activity in cold adapted rats compared to control animals. For some enzyme activities there was a small increase when expressed per mg tissue or per mg mitochondrial protein. When expressed per mg DNA i.e. per cell, there was a reduced specific activity of the mitochondrial and peroxisomal enzymes in both brown and white adipose tissue on cold adaptation.     

Selective loss of uncoupling protein from mitochondria of surgically denervated brown adipose tissue of cold-acclimated mice

The effects of unilateral surgical denervation on brown adipose tissue (BAT) composition were evaluated to assess the importance of the sympathetic innervation in the maintenance of a high concentration of the uncoupling protein thermogenin in cold-acclimated (CA) mice and to assess whether suppression of neural activity could account for BAT atrophy observed during fasting or when CA mice are returned to a thermoneutral environment (33 degrees C). Denervation-induced BAT atrophy was characterized by protein and thermogenin losses in absence of changes in the tissue cellularity (DNA content). There was a marked reduction in the concentration of thermogenin in mitochondria isolated from denervated BAT, but the concentration of the adenine nucleotide translocator was unchanged. Fasting or exposure of CA mice to 33 degrees C induced a rapid and extensive loss of tissue protein from both innervated and denervated BAT. In CA mice exposed to 33 degrees C, there was also reduction in tissue cellularity and loss of thermogenin from BAT mitochondria. Since surgical denervation suppressed BAT hyperplasia and the increase in the mitochondrial concentration of thermogenin observed during cold exposure, these results indicate that an intact innervation is required for both synthesis and maintenance of a high mitochondrial content of thermogenin in CA mice. In addition, the lesser changes in tissue composition caused by denervation compared with those caused by fasting or exposure of CA mice to 33 degrees C question the importance of the suppression of neural activity as the exclusive cause of rapid BAT atrophy in mice.