BMPs are members of the transforming growth factor-β superfamily and control multiple key steps of embryonic development and differentiation6
. BMPs appear to have different roles in adipogenesis7
. Although certain BMPs, in particular BMP-2 and BMP-4, enhance white adipogenesis when assisted by a hormonal induction cocktail8,9
, the role of BMPs on the differentiation and function of BAT or the balance between WAT and BAT is unknown. To this end, we studied the role of BMPs in the differentiation of brown preadipocytes10
and 3T3-L1 white preadipocytes in the absence of other hormonal or chemical inducers. Treatment of these cells with BMP-2, -4, -6 and -7 markedly increased lipid accumulation of brown preadipocytes in culture even in the absence of normally required induction cocktail or thiazolidinediones (). BMP-5 exhibited a weaker effect as compared to other BMPs, and BMP-3 had virtually no effect on brown fat differentiation. By contrast, under the same conditions, 3T3-L1 white preadipocytes exhibited little or no differentiation when treated with these BMPs.
BMP-7 induces brown, but not white, preadipocyte differentiation and the essential role of p38 MAPK in BMP-7-induced thermogenesis
In brown preadipocytes, while BMP-2, -4, -6, and -7 induced lipid accumulation to similar extents, BMP-7 was unique in that it markedly induced UCP-1 mRNA expression () to a level comparable to that achieved by standard induction protocols (Supplementary Table S1
). In addition, expression of several other brown fat selective genes was significantly induced by BMP-7 (Supplementary Fig. S1
). By contrast, BMP-4, an adipogenic factor for white fat9
, suppressed expression of UCP-1 in these brown preadipocytes, despite its effect on lipid accumulation. Western blot analysis confirmed the specific effect of BMP-7 on induction of UCP-1 protein expression in brown, but not white, preadipocytes (Supplementary Fig. S2
). Importantly, expression of PGC-1α and UCP-1 was markedly induced by cAMP to 6- and 18-fold, respectively, in BMP-7-treated cells (), indicating the differentiated lipid-containing cells induced by BMP-7 are bona fide
brown adipocytes with a complete capacity to initiate the thermogenic program. BMPs are known to stimulate osteogenic differentiation by inducing osteogenic transcription factor Runx2 expression11
. In brown preadipocytes, BMP-2, -6, and -7 significantly inhibited Runx2 expression, while BMP-4 had no effect (), suggesting that these BMPs function in brown fat precursors to promote adipogenesis and inhibit osteogenic differentiation. The specific effect of BMP-7 on brown preadipocyte differentiation was also verified in primary culture isolated from stromo-vascular fraction of interscapular BAT (Supplementary Fig S3
Cellular responses to BMPs have been shown to be mediated by the formation of a heteo-oligomeric complex of the type 1 and type 2 BMP receptors (BMPRs). Two major signaling pathways, the Smad pathway and p38 MAP kinase (MAPK) pathway, confer most of the biological function of BMPs12
. We found there is only subtle difference in expression levels of different BMPR isoforms between brown and white preadipocytes (Supplementary Fig. S4
). Interestingly, while BMP-7 increased phosphorylation of Smad1/5/8 in both brown and white preadipocytes, robust activation of p38 MAPK and its downstream transcription factor ATF-2 following BMP-7 stimulation was observed only in brown preadipocytes, while being blunted or almost completely absent in the 3T3-L1 white preadipocytes ().
To further investigate the role of p38 MAPK in BMP-7-induced brown adipogenesis. Three pharmacological inhibitors of p38 MAPK were added individually to medium 7 hours prior to and throughout BMP-7 treatment. After 10 days in culture, while none of these inhibitors had an effect on BMP-7-induced lipid accumulation (Supplementary Fig. S5
), all of these drugs effectively blocked expression of UCP-1 protein induced by BMP-7 (). p38 MAPK is known to regulate thermogenesis via nuclear coactivator PGC-113,14
. Indeed, we found that BMP-7-induced UCP-1 expression was markedly diminished in brown preadipocytes deficient in both PGC-1α and PGC-1β15
(Supplementary Fig. S6
). Together, these data reveal an essential role for p38 MAPK and PGC-1 coactivators for BMP-7-induced thermogenic program in brown adipocytes, while they are dispensable for its effect on lipid accumulation.
Before entering the adipogenic program, preadipocytes must be released from suppressive factors and become committed to terminal differentiation3
. Necdin acts as a negative modulator of brown preadipocyte differentiation, coordinating early adipogenic events, including suppression of Pref-1 and Wnt10a expression16
. Treatment of brown preadipocytes with BMP-7 significantly suppressed expression of necdin (). In addition, BMP-7 also markedly suppressed expression of other inhibitors of adipogenesis, including Pref-1 and Wnt10a (). As a consequence of release from suppression by BMP-7 treatment, these brown preadipocytes initiated the full transcriptional program of adipogenesis as shown by a significant increase in expression of PPARγ, C/EBPα and aP2 (). Importantly, BMP-7 robustly induced expression of PRDM16, a zinc-finger binding protein recently identified as an early regulator determining brown fat fate4
, by 6.3-fold at day 3 (). This led to increased expression of other molecular characteristics of brown fat, including PGC-1α, PGC-1β () and UCP-1 ().
Molecular mechanisms by which BMP-7 induces brown adipogenesis and mitochondrial biogenesis
Differentiation of brown fat is accompanied by mitochondrial biogenesis17
. In the brown preadipocyte cell line, BMP-7 treatment significantly increased the expression of genes involved in mitochondrial biogenesis and function ( and Supplementary Fig. S1
), including PGC-1α and PGC-1β as well as nuclear respiratory factor (NRF)-1, mitochondrial transcription factor A (Tfam) and cytochrome C. This coincided with a 5-fold increase in mitochondrial density in BMP-7-treated cells compared to control (). Thus, BMP-7 activated a full program of brown adipogenesis by suppression of early adipogenic inhibitors, induction of factors determining brown fat fate, increased expression of adipogenic transcription factors and mitochondrial biogenesis.
BMPs are important in control of stem cell commitment to various lineages18
. To determine if BMP-7 could also trigger commitment of the mesenchymal progenitor/stem cells into a brown adipocyte lineage, we treated the multipotent C3H10T1/2 cells with BMP-7 for 3 days prior to treatment with a standard adipogenic differentiation cocktail10
. Cells pretreated with BMP-7, but not vehicle, displayed a mature brown adipocyte phenotype with marked increases in lipid accumulation, and induction of the brown fat specific protein UCP-1 (). Expression of specific markers indicated that the C3H10T1/2 cells had become committed to the brown adipocyte lineage within 3 days of BMP-7 treatment (). By this time point, BMP-7 pre-treatment had increased expression of C/EBPδ19
, followed by increased expression of C/EBPβ, C/EBPα, PPARγ and aP2 at later stage of differentiation, consistent with previously described gene patterns in committed white and brown preadipocytes 20,21
. Interestingly, BMP-7 pretreatment also caused a transient induction of PGC-1α expression and a significant increase in expression of NRF-1 and Tfam, followed by a later increase of cytochrome C expression (), indicating increased mitochondrial biogenesis in these BMP-7 pretreated cells during the course of brown adipogenesis.
BMP-7 triggers commitment of mesenchymal progenitor cells to brown adipocyte lineage in vitro and in vivo
To verify the cell culture findings in vivo
, we implanted BMP-7-treated C3H10T1/2 cells subcutaneously into athymic nude mice in the sternal region. Six weeks after implantation, the BMP-7-treated cells developed into a fat pad containing a large number of multi-locular and UCP-1-positive brown adipocytes and a small portion of uni-locular white adipocytes, while no additional tissue was found in mice receiving cells treated with vehicle ( and Supplementary Fig. S7
). Additionally, BMP-7, in concert with other differentiating agents, induced brown adipogenesis in two more primitive fibroblastic cells with no adipogenic character (Supplementary Fig. S8 and S9
). Thus, BMP-7 triggers commitment of multipotent mesenchymal cells to the brown adipocyte lineage in both in vitro
and ex vivo
To determine the physiological necessity of BMP-7 for BAT development, we analyzed brown fat morphology and function in BMP-7 knockout mice. Since BMP-7 null mice die shortly after birth due to renal failure22,23
, we focused our study in newborns and embryos. In rodents at these stages brown fat is already developed, while white fat is still not grossly visible. Strikingly, at birth BMP-7 knockout mice displayed a marked 50–70 % decrease in interscapular BAT mass compared with wild-type littermates, while the size of other internal organs, such as the liver, as well as the size of whole animal, were not altered (). The decrease of BAT mass in BMP-7 knockout animals was also evident at embryonic stages. Cross-sections of 17.5 days postcoitum (dpc) embryos showed a dramatic decrease in brown fat mass, due to a marked decrease in the number of brown adipocytes ( and Supplementary Fig. S10
). Most importantly, expression of UCP-1 protein was markedly decreased or completely absent in brown fat from 18.5 dpc BMP-7 null embryos, while expression of insulin receptor, another protein involved in adipogenesis, remained unaltered (). This was accompanied with by a significant decrease in expression of a number of brown fat selective genes (Supplementary Figure S11
). These data establish an essential role of BMP-7 in brown fat development in vivo
and a near absolute requirement for BMP-7 in maintaining brown fat specific thermogenic program.
Evidence for an essential role of BMP-7 in BAT development and regulation of whole body energy expenditure by loss-of-function and gain-of-function approaches
Finally to explore the potential role of BMP-7 in regulation of brown adipogenesis and energy homeostasis in vivo
, we injected adenoviruses expressing BMP and LacZ control via the tail vein into 4-week old C57BL/6 mice and measured metabolic rate by indirect calorimetry. Adenoviruses are trophic for the liver, where they can drive release of secretory proteins, like BMPs, into the blood stream (Supplementary Fig. S12a
). Importantly, mice that received adenovirus expressing BMP-7 showed significant increases in whole-body energy expenditure and basal body temperature, leading to a significant reduction in weight gain as compared with mice that received LacZ adenovirus (). The increase in energy expenditure in BMP-7-treated mice was not due to an increase in physical activity or food intake (Supplementary Fig. S12
). By contrast, BMP-3, which had no effect on differentiation of brown preadipocytes in vitro
(), did not produce any differences compared to control mice. Fifteen days after adenoviral injection, mice that received BMP-7 treatment displayed a significant increase in brown fat mass with no change in WAT mass (Supplementary Fig. S13
). In a cohort of older mice, BMP-7 treatment specifically induced expression of PRDM16 and UCP-1, two key factors determining brown fat fate and function, in BAT (), while the expression of genes involved in energy homeostasis in other tissues, including WAT, muscle and liver, remained unaltered (Supplementary Fig. S14
). Together, these data not only recapitulate the brown adipogenic effect of BMP-7 in vivo
, but also reveal an important anti-obesity potential of BMP-7 via increasing whole body thermogenesis.
BAT and WAT are morphologically and functionally different tissues, and their developmental patterns are quite distinct. One of the remaining questions in adipocyte biology is how and when the developmental fate of brown versus white adipocytes is regulated and specified. On the basis of the present data and published observations from other investigators8,9,24
, we propose a model for the role of BMPs in determination of brown versus white fat cell fate, as illustrated in . While BMP-2 and BMP-4 can promote differentiation of white adipocyte lineage, we demonstrate in the present study that BMP-7 drives brown fat cell fate in both mesenchymal progenitor cells and committed brown preadipocytes. This is achieved by suppression of early adipogenic inhibitors, such as necdin, Pref-1 and Wnts, and by induction of key molecules that specify brown fat fate, such as PRDM16 and PGC-1α, leading to a mature brown adipocyte phenotype with UCP-1 expression and abundant mitochondria. BMP-7 null embryos display brown fat hypoplasia and almost complete absence of UCP-1 protein, highlighting an essential role of BMP-7 in brown fat development. When applied in vivo
, BMP-7 is able to increase brown fat mass and thermogenic energy expenditure in mice.
Whereas BMP-2, -4, -6, and -7 are able to induce massive lipid accumulation in brown preadipocytes, we find that only BMP-7 has a specific effect on induction of the brown fat specific protein UCP-1. Differential effects of various BMPs on other single cell types have also been observed25
. For example, BMP-7, but not BMP-4 or BMP-6, is able to reverse TGF-β-induced epithelial-to-mesenchymal transition in distal tubular epithelial cells26
. Exactly how the specificity of different BMPs is determined remains an unsolved question in the field. Our data have indicated an important role of p38 MAPK and PGC-1 coactivator in BMP-7-induced thermogenesis. Originally identified as a bone inducer27
, BMP-7 is now being recognized as a multifunctional cytokine and has been implicated as a potential therapeutic agent for cardiovascular, metabolic and degenerative diseases28
. In this study, our data reveal a novel function of BMP-7 in the regulation of energy homeostasis by promoting brown, but not white, fat differentiation and function. Thus, we propose that treatment of humans with BMP-7 or its molecular mimetic may recruit and activate brown fat differentiation, leading to an increase in energy expenditure, and thereby providing a new avenue to combat obesity.