A detailed understanding of the brown fat cell and its unique ability to dissipate chemical energy may offer new treatment avenues for obesity and associated diseases. In small mammals that retain distinct brown fat pads as adults, the heat produced by brown fat is an important contributor to overall energy balance (Cannon and Nedergaard, 2004
; Cederberg et al., 2001
; Ghorbani et al., 1997
; Ghorbani and Himms-Hagen, 1997
; Guerra et al., 1998
; Kopecky et al., 1995
; Kopecky et al., 1996
; Lowell et al., 1993
; Rothwell and Stock, 1979
; Tsukiyama-Kohara et al., 2001
). Although adult humans do not have distinct brown fat depots, they do appear to have small numbers of brown adipocytes in their white fat depots, and these brown cells proliferate under certain circumstances, such as chronic cold exposure (Garruti and Ricquier, 1992
; Huttunen et al., 1981
; Lean et al., 1986
; Oberkofler et al., 1997
). Thus, the molecular mechanisms that regulate brown fat cell determination are of significant and escalating biomedical interest.
A great deal is known about the metabolism and function of mature white and brown adipocytes; however, the developmental origins of these cell lineages have remained elusive. The majority of brown fat develops prenatally and is mature and fully functional at birth, when thermogenic requirements are particularly high (Nedergaard et al., 1986
). Most WAT, on the other hand, develops postnatally in response to relative nutritional excess. It has been presumed that white and brown fat cells are closely related to each other developmentally because they express many common enzymes, and both require PPARγ for their differentiation. Interestingly, recent fate-mapping experiments in mice show that interscapular brown fat but not white fat arises from a population of Engrailed-1 expressing cells in the dermomyotome, a structure that also gives rise to muscle and skin (Atit et al., 2006
). These data suggests that the two types of fat cells may have quite different origins.
PRDM16 is expressed very selectively in brown fat cells versus white fat cells and stimulates nearly all the key characteristics of authentic brown fat cells when expressed at or near physiological levels. This includes enhanced mitochondrial gene expression and mitochondrial density, increased expression of PGC-α, UCP1 and a very large increase in the uncoupled fraction of respiration. Importantly, the expression of UCP1 and PGC-1α induced by PRDM16 is further enhanced by cAMP, as it is in authentic brown fat cells. At a global scale, a large majority of genes that are selectively expressed in brown adipocytes are positively regulated by PRDM16 (Table S1
). Conversely, PRDM16 expression suppressed the mRNA levels of several genes that are selectively enriched in white fat such as resistin and serpin3ak. Notably, PRDM16 expression does not influence the expression of those genes that are common to both brown and white fat cells. Interestingly, several BAT genes including UCP1 and PGC-1α were induced by PRDM16 in the genetic absence of PPARγ (and therefore fat cell differentiation). Importantly, PRDM16 is shown here to activate a brown fat gene program in many different kinds of adipocytes as long as it is introduced before cell differentiation. These results suggest a model in which PRDM16 functions to establish “brown” identity including UCP1 expression and increased mitochondria that is, at least partly, separable from the adipogenic differentiation pathway common to white and brown fat cells (Figure S4
). Why PRDM16 is not effective when expressed after adipogenic differentiation in these experiments is not known at present.
A very effective shRNA directed against PRDM16 allowed us to ask about the requirement for this factor in the expression of brown fat-selective genes in established brown fat cell lines, and in primary brown fat cells. The reduction of PRDM16 levels has no effect on morphological differentiation of these cells, but causes an almost complete suppression of brown fat-selective genes, including UCP1 mRNA and protein, while leaving intact the expression of genes common to both white and brown fat cells such as PPARγ and aP2. Clearly, PRDM16 is required for the expression of the brown fat phenotype in isolated cells. Examination of this feature will be important in mice ablated for PRDM16 in vivo.
A key question is how does PRDM16 stimulate the development of a brown fat gene program? This protein is annotated in databases as a potential transcription factor because it possesses two zinc-fingers. However, while zinc finger proteins are often DNA binding factors, it is also clear that zinc finger domains can mediate protein-protein interactions (Leon and Roth, 2000
). We have confirmed that PRDM16 does indeed bind to DNA in a sequence-specific manner, but this is not required for its regulation of many BAT-selective genes. On the other hand, it is clear that PRDM16 activates the expression as well as the transcriptional function of both
PGC-1α and PGC-1β, apparently through direct physical binding. While functions of PRDM16 in other aspects of brown fat regulation are by no means ruled out, the PRDM16-stimulated activity of PGC-1α and PGC-1β may explain many actions of PRDM16 in brown fat determination (Figure S4
As shown previously, PGC-1α can activate many of the genes that comprise the thermogenic program of brown fat, such as UCP1 and Deiodinase d2 (Puigserver et al., 1998
). On the other hand, PRDM16 is shown to have certain actions here that are not consistent with a function solely
through modulation of PGC-1α expression and/or function. In fact, genetic studies have shown conclusively that mice lacking PGC-1α retain identifiable, though abnormal, brown fat tissue (Lin et al., 2004
). Similarly, isolated brown fat cells lacking PGC-1α still express several genes characteristic of brown fat (Uldry et al., 2006
). However, it is notable that shRNA-mediated suppression of PGC-1β in the cells lacking PGC-1α caused a further loss of the brown fat phenotype (Uldry et al., 2006
). Thus, it is possible that PRDM16 functions as a brown fat determination factor, at least in part, by robustly stimulating PGC-1α and PGC-1β simultaneously. PRDM16 may also increase PGC-1 coactivator function in other tissues where it is expressed, such as heart, brain and kidney, with important physiological consequences.
How PRDM16 achieves this activation of the PGC-1s remains to be determined. The P
RDI-BF1 and R
IZ homology (PR) domain present in a subclass of zinc finger proteins, including PRDM16, is highly homologous to the SET domain that is noted for its histone lysine methyltransferase activity and diverse functions in regulating chromatin structure (Huang et al., 1998
; Rea et al., 2000
). Other PR-domain containing factors such as RIZ1 (R
) and Meisitz have intrinsic histone methyltransferase activity, while PRISM (PR-domain in smooth muscle) binds and recruits a histone methyltransferase (Davis et al., 2006
; Hayashi et al., 2005
; Kim et al., 2003
). Interestingly, another methyltransferase protein, PRMT1, has been shown to bind to PGC-1α and activate it via arginine methylation (Teyssier et al., 2005
). Whether the PR-domain of PRDM16 has enzymatic function and whether this activity is required for stimulating brown fat gene expression remains to be established.
Components that can influence the brown fat phenotype, in addition to the PGC-1s, have been identified, such as FOXC2, Rb, p107 and RIP140 (Cederberg et al., 2001
; Christian et al., 2005
; Hansen et al., 2004
; Leonardsson et al., 2004
; Powelka et al., 2006
; Scime et al., 2005
). It will be important to investigate their genetic interactions with PRDM16. The absolute requirement for PRDM16 in the formation of brown adipocytes suggests that the mechanism of action of these other factors may involve PRDM16. Whether they act upstream or downstream of PRDM16 in the differentiation program of brown fat remains to be elucidated.
The replacement of BAT with WAT in humans, and in obese mice and rats, has shown that these tissues can interconvert to some extent in vivo
. Similarly, prolonged exposure to cold or β–adrenergic agonists induces the appearance of many brown fat cells within classic white fat depots (Himms-Hagen et al., 2000
). This so-called “transdifferentiation” of fat by cold-exposure or β–adrenergic agonists could be due to the acquisition of brown fat cell features in preformed white fat cells and/or by the differentiation of resident committed brown preadipocytes into mature brown fat cells. While this important issue is not entirely closed, the latter scenario is supported by the observation that preadipocytes contained in fat tissues are committed to the brown or white fate (Klaus, 1997
; Klaus et al., 1994
; Klaus et al., 1995
; Kozak and Kozak, 1994
). In light of these data, it is very intriguing that PRDM16 expression did not convert white mature adipocytes into brown type cells. However, the white fat cell precursors present in whole fat tissues could be converted to a brown fat-like phenotype very efficiently when PRDM16 was introduced before differentiation. Furthermore, the emergence of UCP1-positive brown fat cells in the white fat depots of transgenic mice suggests strongly that PRDM16 directs the differentiation of resident white fat progenitors into the BAT fate. The small clusters of BAT cells in wildtype WAT most likely arise from BAT progenitors that are present in small numbers within white depots. The stimulation of PRDM16 action by β-adrenergic signaling is not surprising, given the known importance of this cascade in BAT development (Cannon and Nedergaard, 2004
). The mechanism by which cAMP signaling modulates the activity of PRDM16 is an important outstanding question from this study.
Taken together, inducing PRDM16 expression in preadipocytes could constitute a strategy to raise whole body energy expenditure and prevent excess fat accumulation. This can be done, in theory, by using drugs that raise PRDM16 levels in fat cell precursors, or by engineering preadipocytes ex vivo, and then reinjecting them, analogous to the transplantation experiments done here. More experimentation will be necessary to determine how this brown fat determination factor might be used to fight obesity in the context of a whole animal.