In contrast to the earlier contention, adult humans have been shown recently to possess active brown adipose tissue with a potential of being of metabolic significance. Up to now, brown fat precursor cells have not been available for human studies. We have shown previously that human multipotent adipose-derived stem (hMADS) cells exhibit a normal karyotype and high self-renewal ability; they are known to differentiate into cells that exhibit the key properties of human white adipocytes, that is, uncoupling protein two expression, insulin-stimulated glucose uptake, lipolysis in response to b-agonists and atrial natriuretic peptide, and release of adiponectin and leptin. Herein, we show that, upon chronic exposure to a specific PPARc but not to a PPARb/d or a PPARa agonist, hMADS cell-derived white adipocytes are able to switch to a brown phenotype by expressing both uncoupling protein one (UCP1) and CIDEA mRNA. This switch is accompanied by an increase in oxygen consumption and uncoupling. The expression of UCP1 protein is associated to stimulation of respiration by b-AR agonists, including b3-AR agonist. Thus, hMADS cells represent an invaluable cell model to screen for drugs stimulating the formation and/or the uncoupling capacity of human brown adipocytes that could help to dissipate excess caloric intake of individuals.

Brown adipose tissue uncoupling protein-1 (UCP1) plays a major role in the control of energy balance in rodents. It has long been thought, however, that there is no physiologically relevant UCP1 expression in adult humans. In this study we show, using an original approach consisting of sorting cells from various tissues and differentiating them in an adipogenic medium, that a stationary population of skeletal muscle cells expressing the CD34 surface protein can differentiate in vitro into genuine brown adipocytes with a high level of UCP1 expression and uncoupled respiration. These cells can be expanded in culture, and their UCP1 mRNA expression is strongly increased by cell-permeating cAMP derivatives and a peroxisome-proliferator-activated receptor-␥ (PPAR␥) agonist. Furthermore, UCP1 mRNA was detected in the skeletal muscle of adult humans, and its expression was increased in vivo by PPAR␥ agonist treatment. All the studies concerning UCP1 expression in adult humans have until now been focused on the white adipose tissue. Here we show for the first time the existence in human skeletal muscle and the prospective isolation of progenitor cells with a high potential for UCP1 expression. The discovery of this reservoir generates a new hope of treating obesity by acting on energy dissipation.

No abstract

Uncoupling protein-3 gene expression in skeletal muscle is up-regulated during postnatal development of mice. A high-carbohydrate diet at weaning induces a decrease in uncoupling protein-3 mRNA levels that does not occur when mice were weaned onto a high-fat diet. Uncoupling protein-3 mRNA levels do not increase in response to fasting in young pups. Only after day 15 of life, when fasting increases serum nonesterified fatty acids, uncoupling protein-3 mRNA is upregulated by starvation. Over-nutrition or under-nutrition during lactation increases or decreases, respectively, uncoupling protein-3 mRNA expression in skeletal muscle. Regulation of uncoupling protein-3 gene expression in skeletal muscle during development is mediated by ontogenic and nutritional factors determining changes in circulating non-esterified fatty acids.z 1999 Federation of European Biochemical Societies.

No abstract

Brown fat differentiation in mice is fully achieved in fetuses at term and entails the acquisition of not only adipogenic but also thermogenic and oxidative mitochondrial capacities. The present study of the mice homozygous for a deletion in the gene for CCAAT/enhancer-binding protein ␣ (C/EBP␣-null mice) demonstrates that C/EBP␣ is essential for all of these processes. Developing brown fat from C/EBP␣-null mice showed a lack of uncoupling protein-1 expression, impaired adipogenesis, and reduced size and number of mitochondria per cell when compared with wild-type mice. Furthermore, immature mitochondrial morphology was found in brown fat, but not in liver or heart, from C/EBP␣-null mice. Concordantly, expression of both nuclear and mitochondrial genome-encoded genes for mitochondrial proteins was reduced in C/EBP␣-null brown fat, although expression of mitochondrial rRNA and mitochondrial DNA content were unaltered. Expression of nuclear respiratory factor-2, thyroid hormone nuclear receptors, and peroxisome proliferator-activated receptor ␥ coactivator-1, was delayed in C/EBP␣-null brown fat. Iodothyronine 5-deiodinase activity and thyroid hormone content were also reduced in brown fat from C/EBP␣-null mice, indicating for the first time a crucial role for C/EBP␣ in controlling thyroid status in developing brown fat, which may contribute to impaired mitochondrial biogenesis and cell differentiation. When survival of C/EBP␣-null mice was achieved by transgenically expressing C/EBP␣ only in the liver, a substantial recovery in brown fat differentiation was found by day 7 of postnatal age, which is associated with a compensatory overexpression of C/EBP␦ and C/EBP␤.

C/EBPbeta (CCAAT/enhancer-binding protein beta) is a transcriptional regulator of the UCP1 (uncoupling protein-1) gene, the specific marker gene of brown adipocytes that is responsible for their thermogenic capacity. To investigate the role of C/EBPbeta in brown fat, we studied the C/EBPbeta-null mice. When placed in the cold, C/EBPbeta(-/-) mice did not maintain body temperature. This cold-sensitive phenotype occurred, although UCP1 and PGC-1alpha (peroxisome-proliferator-activated receptor gamma co-activator-1alpha) gene expression was unaltered in brown fat of C/EBPbeta(-/-) mice. The UCP1 gene promoter was repressed by the truncated inhibitory C/EBPbeta isoform LIP (liver-enriched transcriptional inhibitory protein, the truncated inhibitory C/EBPbeta isoform). Since C/EBPbeta-null mice lack both C/EBPbeta isoforms, active LAP (liver-enriched transcriptional activatory protein, the active C/EBPbeta isoform) and LIP, the absence of LIP may have a stronger effect than the absence of LAP upon UCP1 gene expression. Gene expression for UCP2 and UCP3 was not impaired in all tissues analysed. In primary brown adipocytes from C/EBPbeta(-/-) mice, induction of gene expression by noradrenaline was preserved. In contrast, the expression of genes related to lipid storage was impaired, as was the amount of triacylglycerol mobilized after acute cold exposure in brown fat from C/EBPbeta(-/-) mice. LPL (lipoprotein lipase) activity was also impaired in brown fat, but not in other tissues of C/EBPbeta(-/-) mice. LPL protein levels were also diminished, but this effect was independent of changes in LPL mRNA, suggesting that C/EBPbeta is involved in the post-transcriptional regulation of LPL gene expression in brown fat. In summary, defective thermoregulation owing to the lack of C/EBPbeta is associated with the reduced capacity to supply fatty acids as fuels to sustain brown fat thermogenesis.

Objective:We have recently shown the in vivo anti-obesity effects of sodium tungstate. In this study, we investigate the in vitro effects of sodium tungstate on adipocyte differentiation and function. Methods: 3T3-F442A cells were allowed to differentiate in the presence of sodium tungstate, and were analyzed for triglyceride (TG) accumulation, adipocyte differentiation and mitochondrial oxygen consumption. Results: Sodium tungstate treatment of adipose cells decreased TG accumulation and adipocyte differentiation. Expression of key genes for adipocyte function (aP2, ACC, fatty acid synthase (FAS) and lipoprotein lipase (LPL)) and differentiation (CCAAT enhancer-binding protein (C/EBP)a and peroxisome proliferator-activated receptor gamma (PPARg)) was reduced by sodium tungstate, whereas C/EBPb isoform LIP expression level was increased. TG accumulation and changes in C/EBPb expression were partially recovered by inactivating the erk1/2 pathway. Finally, tungstate treatment increased the oxygen consumption of adipose cells without changes in the expression of oxidative genes. Conclusions: Sodium tungstate inhibits adipocyte differentiation by promoting the translation of LIP, a master dominantnegative regulator of this process, and regulates the mitochondrial oxygen consumption of adipose cells. These effects contribute to the anti-obesity activity of sodium tungstate and confirm its potential as a powerful alternative for the treatment of obesity.