Abstract
The recent insight that brown adipocytes and muscle cells share a common origin and in this respect are distinct from white adipocytes has spurred questions concerning the origin and molecular characteristics of the UCP1-expressing cells observed in classic white adipose tissue depots under certain physiological or pharmacological conditions. Examining precursors from the purest white adipose tissue depot (epididymal), we report here that chronic treatment with the peroxisome proliferator-activated receptor gamma agonist rosiglitazone promotes not only the expression of PGC-1alpha and mitochondriogenesis in these cells but also a norepinephrine-augmentable UCP1 gene expression in a significant subset of the cells, providing these cells with a genuine thermogenic capacity. However, although functional thermogenic genes are expressed, the cells are devoid of transcripts for the novel transcription factors now associated with classic brown adipocytes (Zic1, Lhx8, Meox2, and characteristically PRDM16) or for myocyte-associated genes (myogenin and myomirs (muscle-specific microRNAs)) and retain white fat characteristics such as Hoxc9 expression. Co-culture experiments verify that the UCP1-expressing cells are not proliferating classic brown adipocytes (adipomyocytes), and these cells therefore constitute a subset of adipocytes ("brite" adipocytes) with a developmental origin and molecular characteristics distinguishing them as a separate class of cells.
Highlights
Expressed the homeobox transcription factor Engrailed 1, developed into three types of tissue: dermis, muscle, and brown adipose tissue (BAT),2 implying a close developmental relationship between brown adipocytes and myocytes
“Brite” Adipocytes in White Adipocyte Cultures few “adipomyocytes” that are resident in the white adipose tissue (WAT) depots, but not normally sufficiently conspicuous to be detected, but that may proliferate and differentiate given the correct stimulus, or whether the uncoupling protein-1 (UCP1) is found in certain “non-adipomyocyte” cells that despite a different origin from “true” brown adipocytes, can be forced to initiate UCP1 gene expression
PPAR␥ Activation Enables White (Pre)adipocyte Cultures to Acquire Brown Adipocyte-like Characteristics—To examine whether the effects of in vivo PPAR␥ stimulation on the occurrence of UCP1-expressing adipocytes in WAT can be mimicked by similar treatment in vitro, we continuously treated primary cultures of white adipocytes with the potent PPAR␥ agonist rosiglitazone, starting immediately after plating
Summary
Male outbred NMRI mice, purchased from a local supplier (B&K, Stockholm, Sweden), or, where indicated, male UCP1KO mice (on C57Bl/6 background) and UCP1-wild-type mice (C57Bl/6), bred at the Stockholm University animal facility, were used for the preparation of primary cultures of brown and white adipocytes. The medium was not changed on the day the cells were harvested. The cells were washed twice with PBS and fixed with 3% paraformaldehyde in PBS for 20 min at room temperature. Cells were washed three times with PBS and exposed to 5% glycine in PBS to quench unspecific fluorescence. The cells were washed three times with PBS and blocked with 8% bovine serum albumin in PBS for 1 h at room temperature. Cells were washed three times with PBS and incubated with 1:3000 diluted anti-UCP1 antibody and 1:3000 diluted anti-VDAC antibody in 8% bovine serum albumin in PBS overnight at 4 °C. Cells were washed three times with PBS and incubated with anti-rabbit-Alexa Fluor 488-labeled and anti-mouse-Texas Red-labeled secondary antibodies (Molecular Probes), diluted 1:1000 in 8% bovine serum albumin in PBS for 1 h at room temperature. Oxygen consumption rates of cells were monitored with a Clark-type oxygen electrode (Yellow Springs Instrument) as described before (21)
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