Rodent Brown Adipose Tissue Research Articles

The control of UCP1 is dissociated from that of PGC-1α or of mitochondriogenesis as revealed by a study using β-less mouse brown adipocytes in culture

In rodent brown adipose tissue, the β-adrenergic signaling is believed, by an action on PGC-1α, to control UCP1 expression and mitochondriogenesis. We addressed this hypothesis using β 1/β 2/β 3-adrenoceptor knockout (β-less) brown adipocytes in primary culture. In these cells: (a) proliferation and differentiation into multilocular cells were normal; (b) UCP1 mRNA expression was dramatically decreased (by 93%), whereas PGC-1α and mtTFA mRNA expressions were not; (c) UCP1, PGC-1α and COX IV protein expressions were decreased by 97%, 62% and 22%, respectively. Altogether the data show a dissociation between the control of UCP1, which is mostly β-adrenoceptor-dependent and that of PGC-1α and of mitochondriogenesis which are not.

Sibutramine-dependent brown fat activation in rats: an immunohistochemical study.

To evaluate morphological aspects and immunohistochemical markers of brown adipose tissue (BAT) activation following chronic treatment with sibutramine, a novel anti-obesity drug which increases thermogenesis and energy expenditure in mammals, and to establish whether chronic sibutramine treatment induces recruitment of BAT in white adipose tissue (WAT) depots. Adult rats were administered 7 mg/kg/day oral sibutramine for 4 weeks. Body weight was monitored daily. At the end of the 4 weeks rats were perfused with buffered paraformaldehyde solution; interscapular BAT and retroperitoneal and epididymal WAT were carefully dissected for weight and volume measurements and processed for light microscopic studies and immunohistochemistry on paraffin-embedded sections. Where possible, semiquantitative morphometric analyses were performed. Chronic sibutramine treatment determined a significant (about 8%) reduction in body weight. Compared with controls, sibutramine-treated rats showed: (1) interscapular brown adipocytes staining more intensely for uncoupling protein 1 (UCP1), the thermogenic mitochondrial protein; (2) a significantly larger number (about 45%) of brown adipocyte nuclei positive for peroxisome proliferator-activated receptor gamma, the transcription factor driving UCP1 expression; (3) surprisingly, a significant reduction (about 30%) in BAT parenchymal noradrenergic nerve staining; and (4) a significant weight and volume reduction of WAT depots, but no significant signs of transdifferentiation of white into brown adipocytes. This study confirms the ability of sibutramine to induce weight loss by selective and sustained activation of BAT in rodents without recruitment of brown fat in WAT depots. The parallel findings of a high level of brown adipocyte activation and low parenchymal noradrenergic innervation are discussed and a possible direct effect of sibutramine and/or its active metabolites on peripheral BAT sympathetic nerve terminals is hypothesized.

Role of leptin in peroxisome proliferator-activated receptor gamma coactivator-1 expression.

Peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1), a cold-induced protein expressed in brown adipose tissue (BAT), plays a role in adaptive thermogenesis by up-regulating uncoupling proteins (UCP). Here, we explore its relationship to the thermogenic actions of leptin, which also up-regulates UCPs. We find that PGC-1 messenger RNA (mRNA) is markedly reduced in BAT of obese leptin-deficient (ob/ob mice) and leptin-unresponsive (db/db mice and Zucker diabetic fatty fa/fa rats) rodents. Whereas, after cold exposure (6 C for 7 h), PGC-1 mRNA increases 2.6-fold in BAT of lean +/+ rats, it rises only 30% in fa/fa rats. Four days after induction of hyperleptinemia (>30 ng/ml) in Wistar rats, by adenovirus gene transfer, PGC-1 mRNA in BAT was 2.3-fold and UCP-1, 4-fold above controls. In isolated white adipocytes, PGC-1 mRNA increased 4.4-fold within 6 h of incubation with 20 ng/ml of leptin. We conclude that leptin action is required for normal basal and cold-stimulated PGC-1 expression in BAT in rodents and that hyperleptinemia rapidly up-regulates its expression, at least in part, by direct action.

Sex-associated differences in cold-induced UCP1 synthesis in rodent brown adipose tissue.

The effects of acute and chronic acclimation to cold on uncoupling protein 1 (UCP1) levels, as well as on GDP-binding to mitochondria, cytochrome c oxidase activity and mitochondrial protein concentration in brown adipose tissue (BAT) of intact male and female rats have been analyzed. Results reveal that females rats are more sensitive to cold because their threshold temperature for the thermogenic response is set at a higher value (around 22 degreesC) than that of males (around 18 degreesC), hence leading to differences in BAT UCP1 levels between the sexes at different environmental temperatures. In vitro experiments showed that steroid hormones, beta-estradiol, estrone and progesterone, can reduce norepinephrine-induced UCP1 synthesis in brown adipocytes differentiated in primary culture. Thus the different sex-associated response of cold-induced thermogenesis in rats does not appear to be explained by a direct action of sex steroids upon the adipocyte, implying that other factors in the thermogenic regulatory system must be involved.

Uncoupling Protein-3 Is a Mediator of Thermogenesis Regulated by Thyroid Hormone, β3-Adrenergic Agonists, and Leptin

Mitochondrial uncoupling proteins (UCPs) are transporters that are important for thermogenesis. The net result of their activity is the exothermic movement of protons through the inner mitochondrial membrane, uncoupled from ATP synthesis. We have cloned a third member of the UCP family, UCP3. UCP3 is expressed at high levels in muscle and rodent brown adipose tissue. Overexpression in yeast reduced the mitochondrial membrane potential, showing that UCP3 is a functional uncoupling protein. UCP3 RNA levels are regulated by hormonal and dietary manipulations. In contrast, levels of UCP2, a widely expressed UCP family member, showed little hormonal regulation. In particular, muscle UCP3 levels were decreased 3-fold in hypothyroid rats and increased 6-fold in hyperthyroid rats. Thus UCP3 is a strong candidate to explain the effects of thyroid hormone on thermogenesis. White adipose UCP3 levels were greatly increased by treatment with the beta3-adrenergic agonist, CL214613, suggesting another pathway for increasing thermogenesis. UCP3 mRNA levels were also regulated by dexamethasone, leptin, and starvation, albeit differently in muscle and brown adipose tissue. Starvation caused increased muscle and decreased BAT UCP3, suggesting that muscle assumes a larger role in thermoregulation during starvation. The UCP3 gene is located close to that encoding UCP2, in a chromosomal region implicated in previous linkage studies as contributing to obesity.

The β 3-adrenergic receptor in the obesity and diabetes prone rhesus monkey is very similar to human and contains arginine at codon 64

The β 3-adrenergic receptor (ADRβ 3) is a seven-membrane spanning, G-protein linked receptor expressed in brown adipose tissue in rodents, and visceral adipose tissue in humans. Stimulation of the receptor by norepinephrine leads to lipolysis and thermogenesis. In rodent models of obesity and diabetes, administration of β 3-agonists results in weight loss and improved glucose tolerance. Studies indicate that the pharmacological properties of the ADRβ 3 differ markedly between rodents and humans, making generalizations of rodent studies to humans difficult. We hypothesized that the obesity and diabetes prone rhesus monkey ( Macaca mulatta) would provide an excellent animal model to study the role of the ADRβ 3 in the development of obesity and diabetes as well as for assessment of the therapeutic efficacy of β 3-agonists. We sequenced the entire coding region of the rhesus ADRβ 3 gene. Like humans, the rhesus ADRβ 3 has two exons. There is 89% amino acid (aa) identity between human and rhesus compared to 82% aa identity between human and mouse. A single base deletion results in divergence of the intracellular carboxy terminus accounting for 26 of the 45 aa changes and 10 additional aa. Of the 15 rhesus monkeys studied, all were homozygous for Arg 64. In humans, Arg 64 (rather than Trp) is associated with increased body mass index, insulin resistance, and an earlier onset of type II diabetes mellitus. We conclude that the rhesus ADRβ 3 is more similar to the human ADRβ 3 than to the rodent ADRβ 3 suggesting that this primate model may be more appropriate for physiologic and therapeutic studies of the ADRβ 3 axis, and that Arg 64 may influence susceptibility in this species to obesity, insulin resistance, and type II diabetes.

Effects of age on beta adrenergic subtype activation of adenylyl cyclase in brown adipose tissue.

Thermogenesis in brown adipose tissue (BAT) is believed to be mediated mainly by beta3 adrenergic receptors. We previously demonstrated that the specific beta3 adrenergic agonist CGP-12177 increases whole body oxygen consumption and BAT GDP binding to a greater extent in young than in senescent rats. In contrast, the forskolin-induced increases were maintained with age, suggesting that early events in beta3 adrenergic signal transduction are impaired with age. To investigate whether beta1 or beta3 adrenergic function is decreased with age, we assessed beta1 and beta3 adrenergic receptor mRNA levels and the ability of beta1 and beta3 adrenergic receptors to activate adenylyl cyclase in BAT membranes from 4- and 24-month-old F-344 rats. Both beta1 and beta3 adrenergic receptor mRNA levels decreased by 50% with age. Adenylyl cyclase stimulated by the nonspecific agonist, isoproterenol, and by the specific beta3 agonist, BRL 37344, also declined by 50% with age, whereas glucagon stimulation decreased by more than 70%. The isoproterenol-stimulated adenylyl cyclase activation curves were resolved by two-site regression analysis to determine the contribution of beta1 and beta3 adrenergic receptors. The Vmax for both beta1 and beta3 adrenergic receptors decreased by 50% with age. However, stimulation of adenylyl cyclase by NaF and forskolin was also diminished by the same amount as beta adrenergic stimulation, suggesting that the activation with age may be limited by the amount of adenylyl cyclase catalytic unit rather than by receptor number. These data suggest both beta1 and beta3 adrenergic receptors and adenylyl cyclase catalytic units are deficient with age in rodent BAT.

Brown adipose tissue from fetal rhesus monkey ( Macaca mulatta): Morphological and biochemical aspects

1. 1. Brown adipose tissue (BAT) from fetal rhesus monkeys microscopically resembled adult rodent BAT containing multiocular fat cells with numerous mitochondria. 2. 2. Mitochondrial carnitine palmitoyl transferase activity was lower than that in adult rodents and adenine nucleotide translocase activity was similar to that reported for rats. 3. 3. Rhesus monkey BAT mitochondria (BATM) possess an uncoupling protein that is characteristic of BAT as evidenced by the binding of [ 3H]GDP, the inhibition by GDP of the high Cl − permeability or rapid α-glycerol-3-phosphate oxidation. Electrophoretic analysis of BATM showed the presence of a 32,000 mol.wt protein which was enriched by procedures established for the isolation of BATM uncoupling protein.

Pharmacological and physiological properties of benzodiazepine binding sites in rodent brown adipose tissue

1. 1. [ 3H]Diazepam and [ 3H]Ro5-4864 were used as ligands to identify and characterize peripheral-type benzodiazepine binding sites in mouse and rat brown adipose tissue (BAT) membranes. 2. 2. [ 3H]Diazepam and [ 3H]Ro5-4864 binding sites in BAT are pharmacologically similar to peripheraltype benzodiazepine binding sites in other tissues. 3. 3. Stimulators of central-type benzodiazepine receptors had no effect on or inhibited ligand binding to BAT membranes. 4. 4. Brown adipose tissue benzodiazepine binding sites are highly localized to mitochondria-containing subcellular fractions. 5. 5. These binding sites decrease with age in BAT from Fischer 344 rats. 6. 6. Stimulation of BAT thermogenesis in mice with 1-norepinephrine led to a decrease in [ 3H]Ro5-4864 binding in the tissue.

Glycerokinase activity in human brown adipose tissue

Brown adipose tissue (BAT) is known to be responsible for heat production in newborn and adult hibernating mammals. In rats and mice, BAT has been demonstrated to possess a much higher glycerokinase activity than white adipose tissue (WAT). It has been speculated that this high activity may cause the futile cycle of triglyceride breakdown and resynthesis to be activated, thus contributing to heat production. However, at present very little information is available regarding the location, function, and quantitative importance of BAT in adult human subjects. Our objective in this study was to locate BAT in human subjects and to characterize it biochemically, especially with respect to the enzyme glycerokinase. We have looked for histologically identifiable BAT in 32 human subjects and found it in 12 subjects. Most of the BAT samples were obtained from perirenal adipose depots in children undergoing surgery. Some of the samples were almost totally comprised of BAT cells, whereas others were a mixture of BAT cells and WAT cells. The glycerokinase activity per gram of tissue was higher in BAT than in WAT in all the subjects where the above comparison was made. The activity per mg protein or per microgram DNA was higher in most BAT samples. In one pure BAT specimen, the basal lipolytic rate and the lipoprotein lipase activity were measured and they were both higher in BAT than in the WAT obtained from the same patient. These results show that human brown adipose tissue possesses an enzymatic profile very similar to that of rodent brown adipose tissue.

Effect of diet on the weight of brown adipose tissue in rodents

Interscapular brown adipose tissue (BAT) weighed more in rats given access to a solution of sucrose in addition to a nutritionally complete basal diet than in rats eating only a basal diet. This incremental effect of drinking sucrose solution occurs across a variety of dietary conditions. In the first experiment, rats were fed diets containing either 9%, 18%, 27% or 36% casein. Rats given access to a sucrose solution had significantly larger brown fat pads than controls when the diets contained 9 or 18% casein, but not when diets contained either 27% or 36% casein. The second experiment examined the weight of brown adipose tissue as a function of the type of protein and the percentage of fat in the diet. Animals given a sucrose solution had significantly more BAT than animals not given sucrose. Neither the type of protein (casein or soy protein) nor the percentage of fat (14.5% or 36.4%) in the diet influenced the weight of BAT. Animals given access to either a sucrose solution or a glucose solution had significantly heavier BAT than animals given access to a fructose solution, granulated sucrose or water.