Rat brown adipose tissue thermogenic features are altered during mid-pregnancy.

Brown adipose tissue (BAT) thermogenesis is inhibited during late-pregnancy and lactation in the rat. However, scarce information concerning BAT functionality during mid-pregnancy is available. The aim of this work was to investigate uncoupling proteins and leptin expression during placentation in rat BAT as well as other key parameters in the thermogenic function of the tissue. BAT mitochondrial content was found to be reduced 50% in 11 and 13 day pregnant rats as compared to nonpregnant controls, although uncoupling protein 1 (UCP1) content was not modified. Furthermore, UCP3 mRNA levels were found to be highly increased during this period. beta3-adrenergic receptor (beta3-AR) decreased expression resulted in a higher alpha2/beta3 ratio. Finally, leptin mRNA levels in BAT were found to be 3-fold up-regulated in pregnant animals. In conclusion, we show the existence of profound changes in thermogenic features in BAT during gestational days 11 and 13, pointing to the importance of this tissue during mid-pregnancy.     

Sex differences in brown adipose tissue thermogenic features during caloric restriction.

Caloric restriction (CR) studies have shown that females rats conserve energy more efficiently, showing a higher resistance to weight loss and higher protection of vital organs mass than male rats. Gender-dependent inactivation of thermogenesis in brown adipose tissue (BAT) has been proposed as one of these possible energy conserving mechanisms. To study the changes underlying this inactivation in rats, a three month study with 40% CR was undertaken to unravel the effects on BAT recruitment. Under ad libitum conditions female rats had greater BAT recruitment and greater oxygen consumption than their male counterparts. Total and mitochondrial protein, as well as triglyceride and DNA content were more reduced in restricted female rats than in restricted males. Similarly, the levels of key BAT functional proteins (UCP1, LPL, HSL, TFAM) were more reduced in restricted females, whereas no changes were found in mitochondrial DNA levels (mtDNA) and OXPHOS activities in males and females. Furthermore, alpha (2A)/beta (3) adrenergic receptor ratio remained constant in male rats whereas in female rats CR increased 60%. In conclusion, our results suggest that female rats, whose BAT thermogenic activity is higher in ad libitum conditions, is depressed during CR. This inactivation involves the mitochondrial differentiation process and lipolytic system and could be due, at least in part, to the unfavourable adrenergic receptor balance for thermogenic activation.     

Mfn2 is critical for brown adipose tissue thermogenic function

Mitochondrial fusion and fission events, collectively known as mitochondrial dynamics, act as quality control mechanisms to ensure mitochondrial function and fine‐tune cellular bioenergetics. Defective mitofusin 2 (Mfn2) expression and enhanced mitochondrial fission in skeletal muscle are hallmarks of insulin‐resistant states. Interestingly, Mfn2 is highly expressed in brown adipose tissue (BAT), yet its role remains unexplored. Using adipose‐specific Mfn2 knockout (Mfn2‐adKO) mice, we demonstrate that Mfn2, but not Mfn1, deficiency in BAT leads to a profound BAT dysfunction, associated with impaired respiratory capacity and a blunted response to adrenergic stimuli. Importantly, Mfn2 directly interacts with perilipin 1, facilitating the interaction between the mitochondria and the lipid droplet in response to adrenergic stimulation. Surprisingly, Mfn2‐adKO mice were protected from high‐fat diet‐induced insulin resistance and hepatic steatosis. Altogether, these results demonstrate that Mfn2 is a mediator of mitochondria to lipid droplet interactions, influencing lipolytic processes and whole‐body energy homeostasis.     

Development of thermogenic adipose tissue.

Besides having a metabolic and insulatory-supporting function, adipose tissue in endotherms also performs a thermogenic function. Thermogenic adipocytes contain specific UC-mitochondria with uncoupling protein (UCP) and produce heat. Thermogenic adipose tissue has two forms: brown adipose tissue (BAT) and convertible adipose tissue (CAT). Brown adipocytes have UC-mitochondria and express UCP throughout the entire life of small rodents, chiropterans, and insectivores. However, in other endotherms and in humans CAT participates as thermogenic tissue only during early postnatal period. Both BAT and CAT start to develop in utero, although in some animals (hamsters, marsupials) or in some particular areas (thoraco-periaortal and medio-perirenal areas in rats) development of thermogenic adipose tissue starts after birth. Postnatal development of BAT in small endotherms is characterized by quantitative changes (the amount of UC-mitochondria, UCP, and lipids). Postnatal development of CAT causes qualitative changes during which UC-mitochondria in convertible adipocytes are replaced by common, nonthermogenic C-mitochondria; vascularization of adipocytes drops to a low level and, with lipid accumulation, convertible adipocytes appear as lipid-store cells. Postnatal development of CAT can be modulated or reversed by the environmental temperature. The duration of postnatal changes varies between species; i.e., cats, rabbits and sheep, change their thermogenic form of CAT into the lipid-store form within the first postnatal month, while in humans the same process takes up to 15-20 years. In maturity all these large endotherms have CAT in lipid-store form. In light of these results, the question of participation of thermogenic adipose tissue in the regulation of human obesity needs to be answered.     

Gender-related differences in morphology and thermogenic capacity of brown adipose tissue mitochondrial subpopulations

To investigate the possible existence of a gender dimorphism in the morphology and functionality of brown adipose tissue (BAT) mitochondrial subpopulations, we obtained three mitochondrial fractions - heavy, medium and light - by differential centrifugation. Electron microscopic analysis was carried out and mitochondrial protein content, cytochrome c oxidase and ATP synthase activities, mitochondrial DNA content and UCP1 protein levels were measured in each mitochondrial fraction. Female rats showed a greater mitochondrial size than males, with a different distribution pattern of the subpopulations. These differences were accompanied by higher oxidative and thermogenic capacities and a higher protein content in female rat BAT. This tissue also showed a greater tendency to respiratory chain uncoupling, as well as a close coordination between the oxidative, phosphorylative and thermogenic processes. These differences were found in the heavy subpopulation but not in the light one. Our results demonstrate that female rat BAT shows a highly differentiated mitochondrial pool, with the heavy mitochondrial subpopulation as the main responsible for the greater thermogenic activity of this tissue. In addition, it seems that there is a differential regulation of the mitochondrial growth cycle between genders in BAT, which leads to enhanced thermogenic capacity in female rat mitochondria.     

The thermogenic activity of rat brown adipose tissue and rabbit white muscle Ca2+-ATPase

The Ca2+-ATPase (SERCA) found in vesicles derived from the sarco/endoplasmic reticulum vesicles of rats brown adipose tissue and rabbit white muscle were identified by gel electrophoresis, Western blot, electron microscopy and immunolabeling with gold particles. In both tissues, the isoform found was SERCA 1. The Ca2+ affinity of the fat SERCA 1 was different from the muscle isoform. The degree of uncoupling is estimated measuring the ratio between Ca2+ transport and ATP cleaved. In brown fat vesicles the degree of uncoupling varied depending on the Ca2+ concentration of the medium. This was not observed in vesicles derived from muscle. At all Ca2+ concentrations tested, the uncoupling was not related to Ca2+ leakage from the membrane and was far more pronounced in fat than in muscle vesicle. When a Ca2+ gradient was formed across the vesicles membrane the heat released during ATP hydrolysis varied between 22 and 26 Kcal/mol in both fat and muscle vesicles but in the absence of a gradient the heat released was 17 Kcal/mol in fat and 12 Kcal/mol in muscle. The data reported indicate that the SERCA 1 of brown adipocytes is far more thermogenic than the white muscle SERCA 1, and suggest that, in addition to storing Ca2+ inside the endoplasmic reticulum, the SERCA 1 may represent a source of heat production contributing to the thermogenic function of brown adipose tissue.     

The relationship between the thermogenic response of brown adipose tissue and age during noradrenaline infusion in the young rabbit

This work was undertaken to determine if the thermogenic activity of brown fat decreased with age in young rabbits despite the morphological evidence indicating persistence of the brown adipocytes at 4 weeks of age. Data obtained by infusing five doses of noradrenaline and measuring oxygen consumption were used to construct cumulative dose-response curves for five age groups between 3 and 32 days of age. Blood flow to brown fat and other tissues was measured by the microsphere method at 1 and 3 weeks of age. The noradrenaline-induced increase in oxygen consumption when expressed as a percentage of resting oxygen consumption in millilitres per 100 g of body weight decreased (p less than 0.05) with age. However, the absolute noradrenaline-induced increase in metabolic rate (millilitres per minute) increased with age. Total blood flow to brown fat (millilitres per minute) during noradrenaline infusion was unchanged between 1 and 3 weeks of age, but when the blood flow was expressed in millilitres per minute per gram of tissue flow decreased significantly (p less than 0.05) probably because of infiltration of brown fat with white fat. These data suggest that the amount of brown fat and its thermogenic capacity remain relatively constant between 1 and 3 weeks of age, but as a thermogenic organ, brown fat becomes proportionally less effective with age because of the large increase in body mass.     

Cold-induced changes in brown adipose tissue thermogenic capacity of immunocompetent and immunodeficient hairless mice

Mild cold acclimation (22°C, 3 weeks) of hairless mice was shown to increase 5-fold the brown adipose tissue uncoupling protein content in immunodeficient BALB/c nu/nu mice, but by only 2.3-fold in immunocompetent BFU mice. The difference in activation of brown adipose tissue thermogenic capacity was due to a 2-fold increase in the content of brown adipose tissue in nu/nu mice only, which was paralleled by an increase in brown adipose tissue protein but not DNA content. Likewise, only in nu/nu mice the cold acclimation increased the reaction of natural killer cells in blood and peritoneal exudate with a shift from spleen to lymph nodes and increased the phagocytic index. The results indicate that the immune system may influence the defence against cold at the level of brown adipose tissue thermogenesis.Abbreviations AU arbitrary unit(s) - bw body weight - HEMA 2-hydromethyl-metacrylate copolymer - BAT brown adipose tissue - UCP uncoupling protein - ATPase mitochondrial F_oF₁-ATPsynthase - IL-1 interleukin 1 - TNF tumour necrosis factor - NK cells natural killer cells - T _a ambient temperature     

Cold-induced expression of the VEGF gene in brown adipose tissue is independent of thermogenic oxygen consumption

To examine whether cold-induced vascular endothelial growth factor (VEGF) gene expression in brown adipose tissue involved generation of hypoxic oxygen levels by thermogenic processes, we cold-exposed wild-type mice, as well as uncoupling protein-1 (UCP1)-ablated mice in which no thermogenesis in brown adipocytes can be induced. Cold exposure stimulated VEGF expression in both wild-type and UCP1-ablated mice. Unexpectedly, the effect was 3-fold higher in UCP1-ablated animals, whereas cultured brown adipocytes from both genotypes responded identically to norepinephrine stimulation. These results demonstrate that generation of low oxygen levels does not contribute to cold-induced VEGF expression in brown adipose tissue, but the results are consistent with an adrenergic regulation of expression.     

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 resveratrol and its derivative pterostilbene on brown adipose tissue thermogenic activation and on white adipose tissue browning process

Journal of Physiology and Biochemistry - The main function of brown adipose tissue (BAT) is thermogenesis, a process mediated by uncoupling protein 1 (UCP1), which is located in the inner...     

Differential response of rat brown and white adipose tissue to environmental or nutritional stress.

1. In vivo fatty acid synthesis by brown adipose tissue was enhanced in rats exposed to cold (5 degrees C) or altitude (4300 m) for 7 days but was unaltered in rats exposed to heat (35 degrees C) for an equivalent period. In vivo fatty acid synthesis by white adipose tissue was depressed by cold exposure while altitude and heat exposure had no effect. 2. In vitro, CO2 production and lipid synthesis were elevated in brown adipose tissue from rats fasted for 4 days. Refeeding (4 days) such rats reversed these effects, leading to depressed values relative to those of control rats. In contrast, these metabolic events in white adipose tissue were decreased by fasting and increased compared to controls during subsequent refeeding.     

Decreased brown adipose tissue thermogenic activity following a reduction in brain serotonin by intraventricular p-chlorophenylalanine

The effects of reducing brain serotonin (5-HT) levels by means of intracerebral-ventricular injections of the tryptophan antagonist p-chlorophenylalanine (PCPA) were investigated in male rats. Six days after the operation, PCPA-treated rats, either fedad libitum or pair-fed to the food intake of control rats, showed decreased thermogenic activity and capacity in their interscapular brown adipose tissue (BAT) and also increased fat storage in their white adipose tissue (WAT). These results indicate that serotonergic synapses might play a regulatory role in the sympathetic control of BAT thermogenesis and in the rate of WAT deposition (by an as yet unidentified mechanism), in addition to their well established role in controlling food intake.     

Differentiation of brown adipose tissue and biogenesis of thermogenic mitochondria in situ and in cell culture

Differentiation and biogenesis of mitochondria in brown adipose tissue (BAT) was studied in situ and in cell culture by Western blotting, enzyme activity measurements, [35S]methionine incorporation and immunofluorescence microscopy. In different rodent species the perinatal development of BAT thermogenic function resulted from the formation of thermogenic mitochondria which replaced the preexisting nonthermogenic mitochondria. Their biogenesis was characterized by the sudden appearance and rapid increase of the uncoupling protein (UCP), increase of cytochrome oxidase (COX) and decrease of H(+)-ATPase. In primary cell culture, differentiation of precursor cells from mouse BAT to typical multilocular adipocytes was accompanied by increasing content of COX and H(+)-ATPase. A selective synthesis of UCP was induced by activation of beta-adrenergic receptors or by elevated levels of cellular cAMP. UCP was quantitatively incorporated into mitochondria and within 24 h after stimulation reached near physiological concentration. Both in situ and in cell culture, the conditions enabling the expression of UCP gene were accompanied by activation of intracellular thyroxine 5'-deiodinase.