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White and brown adipocytes are believed to occupy different sites in the body. We studied the anatomical features and quantitative histology of the fat depots in obesity and type 2 diabetes-prone C57BL/6J mice acclimated to warm or cold temperatures. Most of the fat tissue was contained in depots with discrete anatomical features, and most depots contained both white and brown adipocytes. Quantitative analysis showed that cold acclimation induced an increase in brown adipocytes and an almost equal reduction in white adipocytes; however, there were no significant differences in total adipocyte count or any signs of apoptosis or mitosis, in line with the hypothesis of the direct transformation of white into brown adipocytes. The brown adipocyte increase was accompanied by enhanced density of noradrenergic parenchymal nerve fibers, with a significant correlation between the density of these fibers and the number of brown adipocytes. Comparison with data from obesity-resistant Sv129 mice disclosed a significantly different brown adipocyte content in C57BL/6J mice, suggesting that this feature could underpin the propensity of the latter strain to develop obesity. However, the greater C57BL/6J browning capacity can hopefully be harnessed to curb obesity and type 2 diabetes in patients with constitutively low amounts of brown adipose tissue.

This study reports the metabolic and morphological characteristics of bovine intermuscular adipose tissue (AT) throughout foetal growth. Our hypothesis was that the histological and molecular features of intermuscular AT would be different from those previously reported for foetal perirenal AT, based on its anatomical location near the muscle and the recent identification of two distinct adipocyte precursors in mouse AT depending on their locations. To address this question, intermuscular AT was sampled from Charolais and Blond d'Aquitaine foetuses at 180, 210 and 260 days post conception (dpc). The two bovine breeds were chosen because of the higher adiposity of Charolais than Blond d'Aquitaine cattle during the postnatal life. Regardless of the breed, adipocyte volume increased slightly (+38%, P < 0.01) with increasing foetal age. This was concomitant with a decrease (P < 0.05) in the activity of enzymes involved in de novo fatty acid (FA) synthesis (FA synthase and glucose-6-phosphate dehydrogenase) and FA esterification (glycerol-3-phosphate dehydrogenase) when expressed per million adipocytes, and with an increase (P ⩽ 0.01) in mRNA abundances for uncoupling protein 1, adiponectin and leptin (LEP) between 180 and 260 dpc. No difference was observed in the adipocyte volume between breeds, which was consistent with the lack of major between-breed differences in mRNA abundances or activities of enzymes involved in lipid metabolism. The mRNA abundance of lipoprotein lipase was maintained across ages, suggesting a storage of circulating FA rather than of FA synthesized de novo. Plasma LEP increased with foetal age, but only in the Charolais breed (+71%, P ⩽ 0.01), and was two- to threefold higher in Charolais than Blond d'Aquitaine foetuses. Regardless of the breed, bovine intermuscular AT contained predominantly unilocular adipocytes believed to be white adipocytes that were larger at 260 dpc than at 180 dpc. These data thus challenge current concepts of the largely brown nature of bovine foetal AT (based on histological and metabolic features of perirenal AT as previously reported a few days before or after birth).

Catecholamines play an important role in controlling white adipose tissue function and development. beta- and alpha 2-adrenergic receptors (ARs) couple positively and negatively, respectively, to adenylyl cyclase and are co-expressed in human adipocytes. Previous studies have demonstrated increased adipocyte alpha 2/beta-AR balance in obesity, and it has been proposed that increased alpha 2-ARs in adipose tissue with or without decreased beta-ARs may contribute mechanistically to the development of increased fat mass. To critically test this hypothesis, adipocyte alpha 2/beta-AR balance was genetically manipulated in mice. Human alpha 2A-ARs were transgenically expressed in the adipose tissue of mice that were either homozygous (-/-) or heterozygous (+/-) for a disrupted beta 3-AR allele. Mice expressing alpha 2-ARs in fat, in the absence of beta 3-ARs (beta 3-AR -/- background), developed high fat diet-induced obesity. Strikingly, this effect was due entirely to adipocyte hyperplasia and required the presence of alpha2-ARs, the absence of beta 3-ARs, and a high fat diet. Of note, obese alpha 2-transgenic beta 3 -/- mice failed to develop insulin resistance, which may reflect the fact that expanded fat mass was due to adipocyte hyperplasia and not adipocyte hypertrophy. In summary, we have demonstrated that increased alpha 2/beta-AR balance in adipocytes promotes obesity by stimulating adipocyte hyperplasia. This study also demonstrates one way in which two genes (alpha 2 and beta 3-AR) and diet interact to influence fat mass.

Multilocular, mitochondria-rich adipocytes appear in white adipose tissue (WAT) of rats treated with the beta3-adrenoceptor agonist, CL-316243 (CL). Objectives were to determine whether these multilocular adipocytes derived from cells that already existed in the WAT or from proliferation of precursor cells and whether new mitochondria contained in them were typical brown adipocyte mitochondria. Use of 5-bromodeoxyuridine to identify cells that had undergone mitosis during the CL treatment showed that most multilocular cells derived from cells already present in the WAT. Morphological techniques showed that at least a subpopulation of unilocular adipocytes underwent conversion to multilocular mitochondria-rich adipocytes. A small proportion of multilocular adipocytes ( approximately 8%) was positive for UCP1 by immunohistochemistry. Biochemical techniques showed that mitochondrial protein recovered from WAT increased 10-fold and protein isolated from brown adipose tissue (BAT) doubled in CL-treated rats. Stained gels showed a different protein composition of new mitochondria isolated from WAT from that of mitochondria isolated from BAT. Western blotting showed new mitochondria in WAT to contain both UCP1, but at a much lower concentration than in BAT mitochondria, and UCP3, at a higher concentration than that in BAT mitochondria. We hypothesize that multilocular adipocytes present at 7 days of CL treatment have two origins. First, most come from convertible unilocular adipocytes that become multilocular and make many mitochondria that contain UCP3. Second, some come from a cell that gives rise to more typical brown adipocytes that express UCP1.

Leptin is synthesized exclusively by adipocytes and acts on the hypothalamus to regulate energy balance. Previous messenger RNA expression studies demonstrated that leptin is expressed in white adipocytes and also in brown adipose tissue, however expression in brown fat is markedly lower than in white fat. This suggests the possibility that leptin expression in brown adipose tissue is due to the presence of white adipocytes that reside within brown adipose tissue, and that brown adipocytes actually do not express leptin. To address this point, we performed immunohistochemistry on paraffin sections and studied leptin protein expression in different depots of white and brown fat of lean and obese (db/db) mice. To establish the cell type expressing leptin, we also assessed the size and organization of lipid droplets, the ultrastructural features of mitochondria, and the presence or absence of uncoupling protein, a brown fat-specific marker. In white adipose tissue of lean and obese (db/db) mice, leptin protein was expressed in adipocytes of various sizes (range examined: 19.67-200 microns), including adipocytes at the multilocular stage of differentiation. Leptin staining was more intense in some depots (retroperitoneal), and appeared to decrease with fasting. In brown adipose tissue of lean animals, multilocular uncoupling protein (UCP)-positive brown adipocytes had typical brown mitochondria and were leptin-negative, both in fed and fasted conditions. At the periphery of the interscapular brown adipose tissue depot, unilocular, UCP-negative adipocytes (mean diameter: 41.55 microns) with white-type mitochondria were observed, and these cells were leptin-positive. In obese (db/db) animals, brown fat was composed mainly of small unilocular, UCP-positive. adipocytes (mean diameter: 40.08 microns), which were also leptin-positive. At the periphery of the organ, numerous large, unilocular, UCP-negative adipocytes (mean diameter: 73.65 microns) with white-like mitochondria were present. As expected, these cells were also leptin-positive. In summary, classical brown adipocytes differ from white adipocytes, not only by their morphology and UCP expression, but also by their apparent lack of detectable leptin expression. db/db brown adipocytes, however, were unilocular and leptin-positive. The molecular mechanisms mediating expression of leptin in white but not brown adipocytes of lean animals, and the significant expression of leptin in brown adipocytes of db/db mice will be the focus of future studies.