Tea catechins enhance the mRNA expression of uncoupling protein 1 in rat brown adipose tissue

The aim of the present study was to determine whether the antiobesity effects of tea catechins (TCs) are associated with the expression of uncoupling protein 1 (UCP1) in brown adipose tissue (BAT). Male Sprague–Dawley rats were fed a high-fat (HF; 35% fat) diet for 5 weeks, then divided into four groups and fed an HF, HF with 0.5% TC (HFTC), normal-fat (NF; 5% fat) or NF with 0.5% TC (NFTC) diet for 8 weeks. At the end of the experimental period, perirenal and epididymal white adipose tissues (WATs) and interscapular BAT were isolated. The NFTC group had significantly lower perirenal WAT weights than the NF group (NF: 12.7±0.53 g; NFTC: 10.2±0.43 g; P<.01), but the HF and HFTC groups did not differ significantly. TC intake had no effects on epididymal WAT weights. The NFTC and HFTC groups had significantly lower BAT weights than the NF and HF groups, respectively. The NFTC group had significantly higher UCP1 mRNA levels in BAT than the NF group (NF: 0.35±0.02; NFTC: 0.60±0.11; P<.05), but the HF and HFTC groups did not differ significantly. Thus, TC intake in the context of the NF diet reduced perirenal WAT weight and up-regulated UCP1 mRNA expression in BAT. These results suggest that the suppressive effect of TC on body fat accumulation is associated with UCP1 expression in BAT.     

Effects of running training on in vitro brown adipose tissue thermogenesis in rats

Brown adipose tissue (BAT) is a major site of nonshivering thermogenesis (NST) during cold acclimation for most mammals. Repetitive nonthermal stress such as immobilization has been shown to enhance the capacity of NST as cold acclimation. In the present study, the effects of running training, another type of nonthermal stress, were investigated on in vitro thermogenesis and the cellularity of interscapular BAT in rats. The rats were subjected to treadmill running for 30 min daily at 30 m/min under 8° inclination for 4–5 weeks. In vitro thermogenesis was then measured in minced tissue blocks incubated in a Krebs-Ringer phosphate buffer containing glucose and albumin at 37° C, using a Clark type oxygen electrode. The trained rats showed less body weight gain during the experiment. The weights of BAT and epididymal white adipose tissue were smaller in the trained rats. Noradrenaline- and glucagon-stimulated oxygen consumption were also significantly smaller in the trained rats. The tissue DNA level was greater in the trained rats, but the DNA content per tissue pad did not significantly differ. The results indicate that running training reduces BAT thermogenesis, possibly as an adaptation to conserve energy substrates for physical work.     

Immunoelectron microscopical identification of the uncoupling protein in brown adipose tissue mitochondria

The distribution of the uncoupling protein (UCP) in brown adipocyte mitochondria of the hibernant Muscardinus avellanarius was obtained by ultrastructural immunocytochemistry. In both cryosections and sections of Lowicryl-embedded material UCP was localized in the mitochondrial cristae of brown adipocytes, but not in liver mitochondria. It should now be possible to easily identify the morphology of cells committed to BAT differentiation in the tissue as well as in cell culture.     

Effect of unilateral surgical denervation of brown adipose tissue on uncoupling protein mRNA level and cytochrom-c-oxidase activity in the Djungarian hamster

The bilateral lobe of interscapular brown adipose tissue of the Djungarian hamster was unilaterally denervated in order to study the role of the sympathetic innervation for maintenance and cold-induced increase of non-shivering thermogenesis. Denervation decreased the noradrenaline content of brown adipose tissue to less than 9% of the intact contralateral pad. This low noradrenaline level was maintained for 1–14 days after denervation. First, to study the role of the sympathetic innervation of brown adipose tissue in the maintenance of the high thermogenic capacity characteristic of the cold acclimated state, brown adipose tissue was denervated in hamsters either kept at thermoneutrality or acclimated to 5°C ambient temperature for 4 weeks. Cold-acclimated hamsters had elevated levels of uncoupling protein messenger ribonucleic acid (8.1-fold) and cytochrom-c oxidase-activity (3-fold). Denervation of brown adipose tissue decreased uncoupling protein-messenger ribonucleic acid level and cytochrom-c-oxidase-activity as compared to the intact pad in thermoneutral and in cold-acclimated hamsters. However, in cold-acclimated hamsters uncoupling protein-messenger ribonucleic acid level and cytochrom-c-oxidase-activity in denervated brown adipose tissue both were maintained on an elevated 6-fold higher levels as compared to thermoneutral controls. Second, to study the role of the sympathetic innervation of brown adipose tissue in the cold-induced increase in thermogenic capacity, hamsters were denervated prior to cold acclimation and responses were measured after 3 and 14 days of cold exposure. Uncoupling protein-messenger ribonucleic acid level and cytochrom-c-oxidase-activity of intact brown adipose tissue increased after 14 days cold acclimation. Denervation did not completely prevent a cold-induced 1.5-fold increase of cytochrom-c-oxidase-activity and a 3.2-fold increase of the uncoupling protein-messenger ribonucleic acid level in denervated brown adipose tissue after 14 days of cold acclimation. In conclusion, high levels of uncoupling protein-messenger ribonucleic acid and cytochrom-c-oxidase activity in brown adipose tissue of cold-acclimated hamsters can partially be maintained without intact sympathetic innervation, suggesting a considerable contribution of trophic factors not requiring sympathetic innervation for maintenance. The cold-induced increase of cytochrom-c-oxidase activity and expression of uncoupling protein-messenger ribonucleic acid largely depends upon sympathetic innervation of brown adipose tissue.Abbreviations ANOVA analysis of variance - BAT brown adipose tissue - COX cytochrom-c-oxidase - HPLC high performance liquid chromatography - mRNA messenger ribonucleie acid - NA noradrenaline - T _a ambient temperature - UCP uncoupling protein     

京尼平对小鼠棕色和白色脂肪组织UCP1表达的影响

目的:棕色脂肪组织活化和白色脂肪组织棕化是改善减肥的良好策略。本研究利用冷刺激作为阳性对照,观察京尼平对小鼠脂肪组织活化与棕化的作用。方法:8周龄雄性C57BL/6J小鼠30只,随机分为正常对照组、京尼平组、冷刺激组, 每组10只。京尼平组小鼠腹腔注射给予京尼平处理(15 mg/(kg·d),连续9 d),对照组用生理盐水处理,冷刺激组小鼠在室温(22℃±2℃)下处理4 d后,置于4℃环境中进行冷刺激处理5 d(24 h/d)。检测各组小鼠每天摄食量、体重和体温变化,取肩胛下区、腹股沟区及附睾周围部分脂肪组织观察形态学的变化,测定棕色脂肪组织、皮下白色脂肪组织以及内脏白色脂肪组织解偶联蛋白1(UCP1)的表达。结果:与正常对照组相比,京尼平组小鼠白色脂肪湿重下降16%,冷刺激组下降28%,均有明显差异(P＜0.05);京尼平组和冷刺激组白色脂肪组织颜色变深,HE染色显示脂肪细胞内的脂滴变小,数量增加;京尼平组小鼠的皮下、内脏白色脂肪组织和棕色3种脂肪组织中的UCP1表达量均明显增加(P＜0.05)。结论:京尼平通过上调UCP1的表达促进棕色脂肪组织活化和白色脂肪组织棕化,此效应是京尼平降脂减轻体重的作用机制之一。     

Uncoupling protein in human brown adipose tissue mitochondria. Isolation and detection by specific antiserum

A protein of Mr 32 000 has been isolated from human infant brown adipose tissue mitochondria following the procedure used to purify the uncoupling protein from rat brown adipose tissue mitochondria. A specific antiserum has been raised against the human 32 kDa protein, and used to detect it by probing mitochondrial proteins separated by SDS-PAGE. The protein is present in large amounts in brown adipose tissue but is undetectable in human liver, heart or white adipose tissue. It has strong immunological cross-reactivity with rat brown adipose tissue uncoupling protein.     

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.     

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.     

Downregulation of AMP-activated protein kinase by Cidea-mediated ubiquitination and degradation in brown adipose tissue

We previously showed that Cidea(-/-) mice are resistant to diet-induced obesity through the upregulation of energy expenditure. The AMP-activated protein kinase (AMPK), consisting of catalytic alpha subunit and regulatory subunits beta and gamma, has a pivotal function in energy homoeostasis. We show here that AMPK protein levels and enzymatic activity were significantly increased in the brown adipose tissue of Cidea(-/-) mice. We also found that Cidea is colocalized with AMPK in the endoplasmic reticulum and forms a complex with AMPK in vivo through specific interaction with the beta subunit of AMPK, but not with the alpha or gamma subunit. When co-expressed with Cidea, the stability of AMPK-beta subunit was dramatically reduced due to increased ubiquitination-mediated degradation, which depends on a physical interaction between Cidea and AMPK. Furthermore, AMPK stability and enzymatic activity were increased in Cidea(-/-) adipocytes differentiated from mouse embryonic fibroblasts or preadipocytes. Our data strongly suggest that AMPK can be regulated by Cidea-mediated ubiquitin-dependent proteosome degradation, and provide a molecular explanation for the increased energy expenditure and lean phenotype in Cidea-null mice.     

Immunological detection of cDNA clones encoding the uncoupling protein of brown adipose tissue: Evidence for an antigenic determinant within the C-terminal eleven amino acids

Poly(A)⁺ RNA was isolated from brown adipose tissue of cold acclimated rats and a fraction enriched for uncoupling protein mRNA was used to generate a cDNA library in pBR 322, Immunological screening of 1,500 colonies with an affinity-purified antiserum against the uncoupling protein yielded five positive clones, pUCP_ratl–5. Clone pUCP_rat2 encoded the C-terminal 54 amino acids of rat uncoupling protein and exhibited 90% amino acid homology with the hamster protein. Clones pUCP_rat3–5 encoded only the C-terminal 11 amino acids suggesting that an antigenic determinant lies within this sequence.     

Interaction between heat acclimation and exogenous insulin in brown adipose tissue of rats

Seventy-one male Wistar strain rats (7 weeks old) were kept at 5, 25, or 34° C, respectively, for 2 weeks with or without insulin administration. Insulin (Novo Lente MC) was given subcutaneously in a dose of 3.62 nmol/125 µl saline per 100 g body weight. An apparent effect of insulin treatment was noted only in heat-exposed rats, resulting in a remarkable gain in inter-scapular brown adipose tissue (BAT) mass of heat-acclimated, insulin-treated rats in terms of weight or weight per unit body weight. The BAT from heat-acclimated, insulin-treated rats had significantly higher levels of protein, DNA, RNA, and triglyceride than BAT from heat-acclimated, saline-treated rats. Therefore, it seems likely that the growth of BAT in heat-acclimated, insulin-treated rats was mostly due to the anabolic effects of insulin. The uncoupling protein mRNA was, however, present in BAT of heat-acclimated, insulin-treated rats at rather a depressed level, explaining a corresponding decrease in cold tolerance. On the other hand, the expression of insulin receptor mRNA was attenuated in BAT of rats from all the insulin-treated groups, possibly due to the down-regulation of insulin. Thus, there appeared to be some linkage among BAT, heat acclimation, and insulin.     

Quantitative evaluation of gap junctions in rat brown adipose tissue after cold acclimation

Summary The decrease in the metabolic capacity of rat brown adipose tissue during the late postnatal period can be reversed by cold acclimation of the animals. In order to find out whether a parallel decrease in capability for intercellular communication observed during this period is also reversed by cold acclimation, gap junction size and number per unit area of cell surface have been quantified in freeze-fracture replicas; cell diameters have been measured in semi-thin sections. It was found that the specific number of gap junctions remains unchanged during cold acclimation. However, the mean gap junction size increases by 75% and the ratio of gap junctional area per cell volume, an index for intercellular exchange capacity, is doubled. This result illustrates further the parallelism between metabolic capacity and cell communication in brown fat.     

Subcellular fractionation of brown adipose tissue

The present study proposes a technique, using Metrizamide, which permits the preparation of brown adipose tissue plasma membranes from the crude mitochondria as well as from the crude microsome fraction. These plasma membranes have high relative specific activities of their marker enzyme, 5′-nucleotidase (15 ± 3 and 14 ± 2 respectively) and, particularly those originating in the crude microsomes, are relatively free of mitochondria contamination. This study also shows the influence of the mode of cell disruption on microsome integrity. When cell disruption was achieved by grinding in liquid nitrogen the purified microsome NADPH cytochrome c reductase specific activity was found to be 3.5 times greater than that of microsomes obtained after homogenization of the tissue.     

Limited proteolysis reveals conformational changes in uncoupling protein-1 from brown adipose tissue mitochondria

Limited proteolytic digestion of uncoupling protein-1 (UCP1) from hamster brown adipose tissue mitochondria was studied. Under optimal conditions, trypsin and chymotrypsin cleave at Lys-292 and at Phe-102, yielding major products 31-kDa T1 and 22-kDa Ch1. Both T1 and Ch1 remained dimers, as in UCP1. Using fluorescent nucleotide derivative 2'-O-dansyl GTP, it is shown that T1 retains the nucleotide binding affinity (K(D)=1 microM for dansyl GTP) while Ch1 does not bind nucleotide. Previously kinetic binding and H(+) transport studies [Biochemistry 35 (1996) 7846] have shown that UCP1 forms tight complexes to varying degrees with nucleotides and their derivatives. Nucleotides strongly protect against tryptic digestion but less against chymotryptic digestion, because the chymotryptic product Ch1 does not bind nucleotide. The nucleotides and derivatives show the same potency profile in protecting against both trypsinolysis and chymotryptic digestion, suggesting that UCP1 undergoes a major conformational change upon nucleotide binding from an initial loose complex into a tight complex, in which the cleavage sites become masked from proteolysis.     

Effect ofE. coli endotoxin on temperature,oxygen consumption and brown adipose tissue thermogenesis in rats and mice

The effects ofE. coli endotoxin 0127 B8 on oxygen consumption, temperature, and on the activity of the proton conductance pathway in brown adipose tissue (BAT) were investigated in rats and mice. In rats an increase was observed in rectal and skin temperature, whole body oxygen consumption and GDP binding in BAT. In mice only the rise in rectal and skin temperature were significantly changed by endotoxin administration.These findings suggest that in some species BAT is involved in the production of endotoxin induced fever and increased energy expenditure.     

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.     

L-alanine transport in isolated cells of interscapular brown adipose tissue in rat

The pattern of L-alanine uptake in isolated cells of interscapular brown adipose tissue has been determined. The uptake can be divided into the diffusion component (Kd=0.55 min^–1) and a saturable Na⁺-dependent transport (K _M =0.87 mM andV _max=155 nmol/min/10⁶ cells). The saturable component can be subdivided into MeAIB-sensitive (K _M =1.63 mM andV _max=162 nmol/min/10⁶ cells) and MeAIB-insensitive (K _M =3.2 mM andV _max=39.5 nmol/min/10⁶ cells). This kinetic pattern could indicate the presence of transport system (s) that resemble the commonly described transport systems for alanine uptake in several tissues.Abbreviations MeAIB Methyl-aminoisobutyric acid - AIB Aminoisobutyric acid     

Intrauterine food restriction alters the expression of uncoupling proteins in brown adipose tissue of rat newborns

A previous study from our laboratory showed that maternal food restriction (MFR) delays thermoregulation in newborn rats. In neonates brown adipose tissue (BAT) is essential for thermogenesis due to the presence of uncoupling proteins (UCPs). The aim of this study was to evaluate the influence of MFR on the UCPs mRNA and protein expression in BAT and skeletal muscle (SM) of the newborn rat. Female Wistar EPM-1 control rats (CON) received chow ad libitum during pregnancy, whereas food-restricted dams (RES) received 50% of the amount ingested by CON. Fifteen hours after birth, the litters were weighed and sacrificed. Blood was collected for hormonal analysis. BAT and SM were used for determination of UCPs mRNA and protein expression, and Ca²⁺-ATPase sarcoplasmic reticulum (SERCA1). RES pups showed a significant reduction in body weight and fat content at birth. MFR caused a significant increase in the expression of UCP1 and UCP2 in BAT, without changes in UCP3 and SERCA1 expression in BAT and SM. No differences between groups were found for leptin, T4 and glucose levels. RES pups showed increased insulin and decreased T3 levels. The delay in development of thermoregulation previously described in RES animals appears not to result from impairment in thermogenesis, but from an increase in heat loss, since MFR caused low birth weight in pups, leading to greater surface/volume ratio. The higher expression of UCP1 and UCP2 in BAT suggests a compensatory mechanism to increased thermogenesis.     

Brown adipose tissue specific lack of uncoupling protein 3 is associated with impaired cold tolerance and reduced transcript levels of metabolic genes

Uncoupling protein 3 (Ucp3) is located within the mitochondrial inner membrane of brown adipose tissue and skeletal muscle. It is thought to be implicated in lipid metabolism and defense against reactive oxygen species. We previously reported on a mutation in our breeding colony of Djungarian hamsters (Phodopus sungorus) that leads to brown adipose tissue specific lack of Ucp3 expression. In this study we compared wildtype with mutant hamsters on a broad genetic background. Hamsters lacking Ucp3 in brown adipose tissue displayed a reduced cold tolerance due to impaired nonshivering thermogenesis. This phenotype is associated with a global decrease in expression of metabolic genes but not of uncoupling protein 1. These data implicate that Ucp3 is necessary to sustain high metabolic rates in brown adipose tissue.