The origin of brown adipocytes arising in white adipose tissue (WAT) after cold acclimatization is unclear. Here, we demonstrate that several UCP1-immunoreactive brown adipocytes occurring in WAT after cold acclimatization have a mixed morphology (paucilocular adipocytes). These cells also had a mixed mitochondrioma with classic "brown" and "white" mitochondria, suggesting intermediate steps in the process of direct transformation of white into brown adipocytes (transdifferentiation). Quantitative electron microscopy disclosed that cold exposure (6 degrees C for 10 days) did not induce an increase in WAT preadipocytes. beta(3)-adrenoceptor-knockout mice had a blunted brown adipocyte occurrence upon cold acclimatization. Administration of the beta(3)-adrenoceptor agonist CL316,243 induced the occurrence of brown adipocytes, with the typical morphological features found after cold acclimatization. In contrast, administration of the beta(1)-adrenoceptor agonist xamoterol increased only the number of preadipocytes. These findings indicate that transdifferentiation depends on beta(3)-adrenoceptor activation, whereas preadipocyte recruitment is mediated by beta(1)-adrenoceptor. RT-qPCR experiments disclosed that cold exposure induced enhanced expression of the thermogenic genes and of genes expressed selectively in brown adipose tissue (iBAT) and in both interscapular BAT and WAT. beta(3)-adrenoceptor suppression blunted their expression only in WAT. Furthermore, cold acclimatization induced an increased WAT expression of the gene coding for C/EBPalpha (an antimitotic protein), whereas Ccna1 expression (related to cell proliferation) was unchanged. Overall, our data strongly suggest that the cold-induced emergence of brown adipocytes in WAT predominantly reflects beta(3)-adrenoceptor-mediated transdifferentiation.

The Ebf (O/E) family of helix-loop-helix transcription factors plays a significant role in B lymphocyte and neuronal development. The three primary members of this family, Ebf1, 2, and 3, are all expressed in adipocytes, and Ebf1 promotes adipogenesis when overexpressed in NIH 3T3 fibroblasts. Here we report that these three proteins have adipogenic potential in multiple cellular models and that peroxisome proliferatoractivated receptor ␥ (PPAR␥) is required for this effect, at least in part due to direct activation of the PPAR␥1 promoter by Ebf1. Ebf1 also directly binds to and activates the C/EBP␣ promoter, which exerts positive feedback on C/EBP␦ expression. Despite this, C/EBP␣ is dispensable for the adipogenic action of Ebf proteins. Ebf1 itself is induced by C/EBP␤ and ␦, which bind and activate its promoter. Reduction of Ebf1 and Ebf2 proteins by specific short hairpin RNA blocks differentiation of 3T3-L1 cells, suggesting a critical role for these factors and the absence of functional redundancy between members of this family. Altogether, these data place Ebf1 within the known transcriptional cascade of adipogenesis and suggest critical roles for Ebf1 and Ebf2.The last decade has seen an enormous surge of interest in the biology of adipocytes, including the developmental processes by which these cells are formed. This has been fuelled by the convergence of a worldwide epidemic of obesity and diabetes (26, 37), with the recent realization that adipose tissue is an active secretory organ regulating a wide array of physiological processes.Adipogenesis represents a complex series of transcriptional events through which multipotent mesenchymal precursor cells become committed to the adipocyte lineage and ultimately express all of the genes typical of mature fat cells (31). These transcriptional events integrate a variety of extracellular signals that direct fat cell formation in time and space. Most of our knowledge concerning the transcriptional events mediating adipogenesis has come from cultured cell lines such as 3T3-L1 and 3T3-F442A (16, 17) that can be differentiated into fat cells by empirically determined hormonal cocktails. These in vitro systems appear to recapitulate most of the developmental events that take place in vivo and offer the advantage of synchronous differentiation and ease of manipulation. Studies of these cells, as well as cultured mouse embryonic fibroblasts (MEFs) derived from mice with various targeted gene ablations (and more recently, studies from intact animals) have revealed a transcriptional cascade in which the nuclear hormone receptor peroxisome proliferator-activated receptor ␥ (PPAR␥) plays a critical role. In cultured adipocytes and fat pads in vivo, PPAR␥ is both necessary and sufficient for adipogenesis (4, 33, 41). Other transcription factors have also been shown to play an important role in adipogenesis, including the CCAAT/enhancer binding proteins C/EBP␣, C/EBP␤, and C/EBP␦ and the basic helix-loop-helix protein SREBP1c (34). In 3T3-L1 cells exposed to a proadipog...

White and brown adipocytes are usually located in distinct depots; however, in response to cold, brown adipocytes appear in white fat. This response is mediated by b-adrenoceptors but there is a controversy about the subtype(s) involved. In the present study, we exposed to cold b 3 -adrenoceptor knockout mice (b 3 KO) on a C57BL/6J genetic background and measured in white adipose tissue the density of multilocular cells and the expression of the brown adipocyte marker uncoupling protein-1 (UCP1). In brown fat of b 3 KO mice, UCP1 expression levels were normal at 24°C as well as after a 10-day cold exposure. Strikingly, under both conditions, in the white fat of b 3 KO mice the levels of UCP1 mRNA and protein as well as the density of multilocular cells were decreased. These results indicate that b 3 -adrenoceptors play a major role in the appearance of brown adipocytes in white fat and suggest that the brown adipocytes present in white fat differ from those in brown fat.Keywords: adipose tissue; cold exposure; differentiation; knockout; uncoupling protein 1.Brown adipose tissue (BAT) and white adipose tissue (WAT) both play an important role in the control of energy balance in mammals. Indeed, BAT is involved in the control of body temperature and body weight via nonshivering and diet-induced thermogenesis [1,2] whereas the primary function of WAT is to store and release energy. BAT function is particularly important for thermoregulation in small mammals in which the surface to volume ratio is unfavorable [1]. Moreover, activation of BAT prevents obesity induced by overfeeding in rodents [2].Cold exposure, via activation of the sympathetic nervous system, induces heat production in BAT [3,4] and lipolysis in WAT [5]. The effects of norepinephrine released at the nerve endings are mediated in brown and white adipocytes mainly by the b 3 -adrenoceptor subtype [6].Brown and white adipocytes are usually located in distinct depots [4] and can be distinguished morphologically [7]: brown adipocytes contain multiple (multilocular) lipid droplets and numerous mitochondria whereas white adipocytes display a main (unilocular) lipid droplet and few mitochondria. Nevertheless, the morphological aspect alone is not sufficient to distinguish between these two cell types [8]. In fact, the only criterion is the specific expression in mitochondria of brown adipocytes, of the uncoupling protein-1 (UCP1), which uncouples the oxidative phosphorylations from electron transport, thus dissipating energy as heat [9].An observation made initially by Young et al.[10] was the presence of multilocular mitochondria-rich brown adipocytes in depots previously defined as typical WAT. The emergence of these ectopic cells was found to be induced by cold acclimation in WAT of rats [11,12] and of mice [13,14]. The new cells were found to be sympathetically innervated [15] and to remain present as long as a sympathetic nervous system stimulation persisted [16]. Several reports also showed that the administration of selective b 3 -adrenoceptor agonis...

Catecholamines are viewed as major stimulants of diet-and cold-induced thermogenesis and of-adrenoceptor triple knockout (TKO) mice and compared them to wild type animals. TKO mice exhibited normophagic obesity and cold-intolerance. Their brown fat had impaired morphology and lacked responses to cold of uncoupling protein-1 expression. In contrast, TKO mice had higher circulating levels of free fatty acids and glycerol at basal and fasted states, suggesting enhanced lipolysis. Hence, L L-adrenergic signalling is essential for the resistance to obesity and cold, but not for the lipolytic response to fasting.