In mammals there are two types of adipocytes with opposing functions. Brown adipocytes are characterized by a high number of mitochondria and are specialized for heat production (thermogenesis), expressing thermogenic genes such as UCP1 (uncoupling protein 1). White adipocytes, on the other hand, store energy. Although many key regulators in the differentiation of white adipocytes have been established, our current knowledge on the same proteins in brown adipogenesis is lagging behind. One example is Pref-1 (pre-adipocyte factor-1), which maintains white pre-adipocytes in an undifferentiated state, but is only poorly characterized in the brown pre-adipocyte lineage. In this issue of the Biochemical Journal, Armengol et al. now shed new light on the role and regulation of Pref-1 in brown pre-adipocytes. First, Pref-1 specifically inhibits the thermogenic gene programme in brown pre-adipocytes. Secondly, they identified the transcription factor C/EBPδ (CCAAT/enhancer-binding protein δ) as a direct positive regulator of Pref-1 expression, whereas this protein does not fulfil this role in white adipogenesis. Taken together, these findings indicate that specific manipulation of brown adipocyte differentiation and/or function without interfering with their white adipocyte counterparts may be possible, which may open up new therapeutic ways to combat obesity-associated health problems.

In mammals there are two types of functionally distinct adipose tissue: BAT (brown adipose tissue) and WAT (white adipose tissue) [1]. Brown adipocytes contain several small (multilocular) lipid droplets, and are characterized by a high number of mitochondria and high expression levels of UCP1 (uncoupling protein 1). BAT has a prominent role in non-shivering adaptive thermogenesis, owing to its capacity to dissipate energy as heat through uncoupled electron transport (‘burning off fat’). WAT, on the other hand, is specialized for the storage of excess energy, which can be released in times of need. In white adipocytes, energy is stored in the form of triacylglycerols (triglycerides) in one major (unilocular) lipid droplet. Brown and white adipocytes are both derived from mesenchymal stem cells, and both differentiation pathways require the transcription factor PPARγ (peroxisome-proliferator-activated receptor γ) [2,3]. Brown and white adipocytes, however, do not share the same precursor cell. Upon prolonged cold exposure or β-adrenergic receptor activation, cells may appear within WAT that display features typical for brown adipocytes, including increased numbers of mitochondria, increased UCP1 expression and a multilocular morphology [2]. These cells are referred to as ‘beige’ or ‘brite’ (brown-white). Given the opposing roles of BAT and WAT in energy handling, the possibility to modulate the differentiation fate of pre-adipocytes opens up new therapeutic ways for the treatment of obesity-related health problems, including Type 2 diabetes, fatty liver disease and cardiovascular disease [4].

Although several markers common to mature white and brown adipocytes have been identified (e.g. PPARγ), the only widely accepted common marker for the undifferentiated state is Pref-1 (pre-adipocyte factor-1). This gene, originally identified in 1993 by Smas and Sul [5] and also referred to as Dlk1 (Delta-like protein 1), is highly expressed in brown and white pre-adipocytes and markedly down-regulated upon differentiation. Pref-1 inhibits the differentiation of 3T3-L1 cells into mature white adipocytes [5], and overexpression of Pref-1 in mice results in decreased BAT and WAT mass [6,7]. Conversely, Pref1−/− mice display increased WAT and BAT depot size, with a higher degree of differentiation and higher adipocyte marker gene expression [8]. In the last decade, the mechanism(s) through which Pref-1 inhibits WAT differentiation has been elucidated (reviewed in [9]). The soluble active form of Pref-1 initiates a signal transduction cascade resulting in inhibition of the expression of two important early regulators of white adipocyte differentiation: the transcription factors C/EBP (CCAAT/enhancer-binding protein) β and δ. Pref-1-mediated reduction of C/EBPβ and δ expression therefore provides a plausible explanation for the inhibitory effect of Pref-1 on white adipocyte differentiation observed in vitro and in vivo. It is currently, however, largely unknown whether a similar Pref-1 signal transduction pathway is operational to inhibit the differentiation of brown pre-adipocytes. Given the central role of Pref-1 in maintaining pre-adipocytes in an undifferentiated state, expression of this gene needs to be tightly regulated. Information on the transcriptional mechanisms regulating Pref-1 expression in brown (pre-) adipocytes is, however, very limited.

In this issue of the Biochemical Journal, Armengol et al. [10] present novel insights into the regulation of Pref-1 in brown pre-adipocytes by using different mouse models and in vitro cell culture experiments.

In mice, BAT development mainly occurs before birth and its differentiation and activity continues to progress after birth, reaching its maximal activity in neonates and young pups [11]. This notion prompted the authors [10] to investigate the changes in Pref-1 expression during BAT development. Significant Pref-1 expression was observed in the early fetal period with a decrease prior to birth and a progressive decline after birth. They also evaluated the expression pattern of UCP1, a BAT marker, during development, which showed an opposite pattern: expression was first detected in late fetal life followed by a gradual increase, peaking in early neonatal life. The authors [10] therefore hypothesized that Pref1 plays a repressive role in brown adipocyte differentiation, similar to its role in white adipocytes. To test this idea, they made use of Pref1-KO (knockout) mice. These animals showed hyperactivation of thermogenic pathways in BAT of neonates and young pups, as indicated by reduced lipid accumulation and induced expression of the thermogenesis marker genes Pgc1a (encoding PPARγ co-activator-α) and Ucp1. However, Pref1-KO mice did not show any changes in mitochondrial maturation and expression of lipid-handling genes [e.g. Fabp4 (fatty-acid-binding protein 4)], suggesting that the inhibitory action of Pref-1 on brown pre-adipocytes is specifically aimed at the thermogenic genes.

To further analyse the role of Pref-1 in BAT development, the authors used mouse models with impaired BAT development {C/EBPα-null and C/EBPα [TG+ (transgenic), C/EBPα−/−] mice}. These animals displayed increased Pref1 expression levels in BAT in early life, suggesting that C/EBPα acts as a transcriptional repressor of the Pref1 gene. To gain more mechanistic insights into the regulation of Pref1 expression by C/EBPs in BAT differentiation, the authors switched to cellular models of brown adipocyte differentiation (primary brown pre-adipocytes and HIB-1B cells). Although their in vivo experiments indicated that C/EBPα may function as a direct regulator of the Pref1 gene, the down-regulation of Pref1 expression clearly preceded the induction of C/EBPα, making it unlikely that C/EBPα directly regulates the Pref1 gene. The related protein C/EBPδ, which is up-regulated upon C/EBPα depletion, turned out to be a more likely candidate to fulfil this role, as the expression of this factor correlated directly with Pref-1 expression: C/EBPδ expression was highest in non-differentiated cells and decreased markedly with brown adipocyte differentiation. A direct role for C/EBPδ as a positive regulator of Pref-1 expression was established through three different approaches. First, the authors analysed the transcriptional regulation of the Pref1 gene in HIB-1B cells transiently transfected with a Pref1 gene promoter–luciferase reporter construct and co-transfection of expression vectors for several C/EBP family members. C/EBPδ was the strongest inducer of the Pref1 gene promoter, targeting a C/EBP-responsive element in the −412/−47 region of the promotor. Secondly, ChIP (chromatin immunoprecipitation) assays were performed, showing binding of C/EBPδ to the proximal Pref1 promoter in fetal BAT samples, but no significant binding in 21-day-old mice when Pref-1 expression is low. Thirdly, C/EBPδ protein levels were reduced by siRNA (small interfering RNA)-mediated knockdown, resulting in a specific and significant reduction of Pref1 mRNA. Taken together, these findings firmly establish C/EBPδ as a direct positive regulator of Pref1 transcription in brown pre-adipocytes.

Finally, as GCs (glucocorticoids) are known to repress the expression of thermogenic genes in brown adipocytes [12], the authors [10] investigated whether this effect may occur through the C/EBPδ–Pref-1 pathway described above. Exposure of brown adipocyte precursors to GCs caused a significant induction in C/EBPδ expression and completely prevented the decay in Pref1 mRNA expression during differentiation. Interestingly, this effect was associated with impaired induction of the thermogenesis marker genes Ucp1 and Pgc1a, whereas brown adipocyte differentiation and morphology was completely preserved.

Taken together, Armengol et al. [10] have provided important new insights into the regulation of the Pref1 gene in BAT differentiation. Their findings indicate that Pref-1 expression in brown pre-adipocytes critically depends on the transcription factor C/EBPδ, which in turn can be regulated by GCs. An important conclusion from their studies is that, although Pref-1 is a marker of both brown and white pre-adipocytes, the regulation of Pref-1 expression is clearly different between the two cell types. Although the authors show that C/EBPδ regulates Pref-1 in brown pre-adipocytes, this transcription factor is unlikely to fulfil a similar role in white adipocytes on the basis of its expression pattern (Figure 1). Moreover, Pref-1 expression is down-regulated by GCs in white adipocyte differentiation, whereas the same steroids prevent Pref-1 down-regulation in brown adipocyte differentiation. Taken together, these findings underscore the fact that, although brown and white adipogenesis share several key players (e.g. Pref-1, C/EBPδ and PPARγ), the function and regulation of these proteins in the differentiation process is often fundamentally different. Potentially even more exciting, the studies by Armengol et al. [10] indicate that factors such as Pref-1 regulate specific gene programmes, in this case the thermogenic genes. Various molecular mechanisms may underly these phenomenona, including post-translational modifications [13,14], which may be cell-type-specific, and BAT/WAT-specific expression of transcriptional co-regulators [15]. Although additional research is clearly required to investigate these possibilities, the cell-type-specific differences in regulation and action of the key players suggest that specific manipulation of brown adipocyte differentiation and/or function without interfering with their white counterparts may be possible. The next challenge will be to translate these findings to the human situation. Recently, Ahfeldt et al. [16] showed that reprogramming of human pluripotent stem cells by the introduction of PPARγ alone gave rise to functional white adipocytes, whereas overexpression of PPARγ together with PRDM16 (PR domain containing 16) and C/EBPβ resulted in brown adipocyte differentiation. These approaches will help us to transfer observations made in mouse models and cell lines to human cells, as a first step to assess the potential of modulating BAT differentiation and function to combat obesity-associated health problems.

Schematic representation of Pref-1 and C/EBPδ expression patterns in brown and white adipocyte differentiation

Abbreviations

     
  • BAT

    brown adipose tissue

  •  
  • C/EBP

    CCAAT/enhancer-binding protein

  •  
  • GC

    glucocorticoid

  •  
  • KO

    knockout

  •  
  • PPARγ

    peroxisome-proliferator-activated receptor γ

  •  
  • Pgc1a

    PPARγ co-activator-α gene

  •  
  • Pref-1

    pre-adipocyte factor-1

  •  
  • UCP1

    uncoupling protein 1

  •  
  • WAT

    white adipose tissue

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