Recent discoveries of metabolically active BAT in adult humans have highlighted BAT as a therapeutic target for treating obesity and its associated diseases. The brown adipocyte-like cells in WAT can be generated by cold exposure or β-adrenergic stimulation in rodents, but the molecular mechanisms underlying these phenomena have not been fully elucidated. In this work, we elucidated that miR-196a induces functional brown adipocytes in WAT in mice. miR-196a is upregulated in WAT-progenitor cells during brown adipogenesis induced by cold or β-adrenergic stimulations. miR-196a is required for the brown fat gene expression and is sufficient to induce metabolically functional brown adipocyte-like cells in mice. The target gene of miR-196a is Hoxc8, which is categorized as a white-fat gene with a previously undermined role in adipogenesis. Hoxc8 directly targets and represses C/EBPβ, a master switch of brown adipogenesis. Thus, the miR-196a-Hoxc8-C/EBPβ pathway underlies the brown adipogenesis in WAT () and might be a therapeutic target for the treatment of obesity and type 2 diabetes.
A schematic of miR-196a-regulated brown adipogenesis of WAT-progenitor cells.
Elucidation of the molecular mechanism regulating the brown adipogenesis in WAT is important from both a biological and clinical viewpoint. Recent studies uncovered the existence of WAT-progenitor cells that harbor a potential to differentiate to brown adipocytes 
. The molecular mechanism behind the inducible brown adipogenesis in WAT is relatively unknown, but recent studies elucidated the importance of cyclooxygenase-2
is an essential regulator of brown fat gene program 
, but whether C/EBPβ
has a significant role in the inducible brown adipogenesis was not fully understood. We found that miR-196a suppresses Hoxc8
, thereby derepressing C/EBPβ
, which leads to the activation of the brown fat gene program. Our findings imply the relevance of C/EBPβ
not only in the conventional brown adipogenesis but also in the inducible brown adipogenesis in WAT.
The cellular origin of the inducible brown adipocyte-like cells in WAT is an important question. Transdifferentiation is a significant mechanism that has been reported to contribute to brown adipocyte recruitment in WAT 
. Because the increase in Ucp1
mRNA is detectable within a few hours after cold stimulation 
, and in vitro SVF cell differentiation is a longer process, transdifferentiation might have a significant role in the rapid response to stimulation. The important questions include the relative contribution of transdifferentiation and the progenitor cell-mediated mechanism in brown adipocyte recruitment throughout the different phases upon exposure to a cold environment and physiological energy regulation.
miRNAs regulate the gene networks underlying various physiological and pathological phenomena and might be therapeutic targets 
. miR-196a has been implicated in the in vitro osteoblast differentiation of human fat progenitor cells, where miR-196a suppresses Hoxc8 
, but the in vivo relevance remains unknown. We elucidated that miR-196a is induced in the WAT-progenitor cells after the induction of brown adipogenesis, is required for the induction of brown fat gene expression, and is sufficient to induce the metabolically functional brown adipocyte-like cells in WAT.
Our observations indicate that miR-196a has only a modest, if any, effect on iBAT. The endogenous expression of Hoxc8 and miR-196a was much lower in iBAT than in ingWAT and epiWAT. The forced expression of miR-196a in mice did not yield appreciable effects in iBAT. Treatment of mice with β3-adrenergic receptor agonists usually leads to a much more moderate induction of Ucp1
expression in iBAT than in WAT depots. Although the primary cultures of brown adipocytes from iBAT are highly sensitive to β3-adrenergic activation 
, a moderate but significant induction of Ucp1
was reported in iBAT in response to β3-adrenoreceptor agonists in vivo 
. A relatively modest response from iBAT to the β3-adrenergic receptor agonist compared with subcutaneous and visceral WAT has also been reported in other studies 
. These results imply that distinct machinery regulates brown adipocyte recruitment in iBAT, which was previously suggested by Petrovic et al. 
A number of miRNAs function as a molecular switch 
, and further elucidating how the miRNAs influence the physiological output will enable better understanding and clinical use of miRNAs.
The significance of the distinct expression patterns of Hox genes between BAT and WAT has been unknown 
. We here demonstrate that Hoxc8 functions as an important determinant of white fat lineage and negatively regulates the induction of brown adipogenesis in WAT-progenitor cells by repressing C/EBPβ
, which is a master switch of brown adipogenesis 
. Mechanistically, Hoxc8 directly represses the C/EBPβ
expression through the 3′ regulatory sequence. Similar conserved non-coding regulatory elements have been reported for the Foxp3
, and previous studies suggested that the majority of transcription factors bind to sites other than the promoter 
. Hoxc8 recruits HDAC3, which is implicated in the regulation of metabolic genes 
. Since the HDAC proteins lack DNA-binding activity, they are recruited to target genes via association with transcriptional factors 
. Our findings imply the possible therapeutic efficacy of HDAC inhibitors for obesity through inducing brown adipogenesis, but further study is required to address the possibility.
The induction of brown adipogenesis in WAT has great therapeutic potential. Our findings suggest that the miR-196a-Hoxc8-C/EBPβ pathway may constitute a promising strategy for addressing the social and health problems caused by obesity and its associated diseases.