Because of the importance of brown adipose tissue (BAT) as a natural defense against hypothermia and obesity1
, and its demonstrated presence in adult humans4–7
, understanding the formation of this cell type in mechanistic detail may open new avenues to the development of novel classes of therapeutics for metabolic diseases such as obesity and type-2 diabetes. Several transcriptional regulators have been identified that positively or negatively control BAT development including Rb8
. Most recently, we have shown that PRDM16, a 140 kDa zinc finger protein, functions as a bi-directional switch in brown fat cell fate by stimulating the development of brown fat cells from white preadipocytes12,13
and from myf5
-positive myoblastic precursors3
and in vivo
. At a molecular level, PRDM16 works as a transcriptional coregulatory protein by coactivating PPAR-γ (peroxisome-proliferator-activated receptor-γ), which is considered the “master” gene of fat cell differentiation14,15
, and this is almost certainly an important event in the myoblast to brown adipocyte conversion3
. However, both isoforms of PPAR-γ are expressed at very low levels in both primary and immortalized myoblasts, while they are abundantly expressed in white and brown preadipocytes (Supplementary Fig. 1
). Hence, it is very likely that PRDM16 initiates the process of myoblast to brown fat conversion by complexing with other DNA-binding factors, well before the coactivation of PPAR-γ.
We therefore devised a strategy to address this, as illustrated in . In brief, we performed proteomic analyses of transcriptional complexes formed with wild type PRDM16 or different mutant alleles that were differentiation-competent or -incompetent. Transcription factors that co-purified preferentially with differentiation-competent PRDM16 proteins were identified; their expression in white and brown fat was then analyzed and compared to that of PRDM16. Subsequently, we examined their function in the process of myoblast-brown fat conversion through PRDM16.
Identification of C/EBP-β as a critical binding partner in the PRDM16 transcriptional complex
As shown in , wild type (WT) and a mutant protein lacking the PR (P
IZ1 homologous)-domain (ΔPR; a.a.91–223), sharing high homology with a SET domain16
, induced brown fat cell differentiation from myoblasts. In contrast, a mutant allele lacking zinc finger domain-1 (ΔZF-1; a.a. 224–447) completely lost its adipogenic function. The brown fat gene program was also induced by both WT and ΔPR, but not by ΔZF-1 (Supplementary Fig. 2
). To avoid comparing proteomic analyses of complexes from cells of very different phenotypes, we expressed all three PRDM16 forms in bona-fide
brown fat cells. PRDM16 complexes were then immunopurified to apparent homogeneity (), and subjected to high-resolution “shotgun” sequencing by liquid chromatography with tandem mass spectrometry (LC-MS/MS)18
. In total, 49 proteins were identified in differentiation-competent PRDM16 complexes, but only 8 of these (Bclaf1, Zfp655, p53, C/ebp-β, Zcchc8, Zkscan3, Zfp143
) are known or predicted transcription factors (Supplementary Table 1
). Since we have assumed that the expression of a key initiating
transcription factor would not be extinguished during the brown fat cell adipogenesis, and PRDM16 is highly enriched in BAT relative to white adipose tissue (WAT)13
, we asked whether any of these factors were similarly enriched in BAT. As shown in , the expression of only C/EBP-β was co-enriched with PRDM16 in BAT versus WAT. In addition, C/EBP-β protein was enriched in BAT, and further induced by cold exposure (Supplementary Fig. 3
). Importantly, both primary and immortalized myoblasts express C/EBP-β at similar levels to those seen in preadipocytes (Supplementary Fig. 4
), where this factor is thought to play a very important role in adipogenesis19,20
. Our analyses have therefore been focused on C/EBP-β and its function in complex with PRDM16.
Brown fat cells express three forms of C/EBP-β, two active forms, named LAP (liver-enriched transcriptional activator protein) and a dominant-negative form, LIP (liver-enriched transcriptional inhibitory protein)21
(, left). Interestingly, PRDM16 preferentially bound to LAP, but not to LIP (, right and Supplementary Fig. 5
). Independent co-expression assays in HEK293 cells confirmed the physical binding of PRDM16 and C/EBP-β. In addition, PRDM16 interacts with other C/EBP family members, C/EBP-α and -δ (Supplementary Fig. 6
). This interaction is likely to be direct through the two zinc finger domains, because the zinc finger domains of the purified glutathione S-transferase (GST)-fused PRDM16 bound to in vitro
translated C/EBP-β (Supplementary Fig. 7
). Lastly, we asked if PRDM16 could affect the transcriptional activity of C/EBP-β. Since C/EBP-β is known to induce PGC-1α gene expression22
, we performed a luciferase reporter assay using the -2kb PGC-1α
promoter where the C/EBP binding sites have been characterized22
. As shown in , the expression of PRDM16 and C/EBP-β synergistically stimulated PGC-1α
promoter activity. These data suggest that PRDM16 forms a transcriptional complex with active forms of C/EBP-β through direct interaction, and regulates its transcriptional activity.
To examine the functional role of the interaction between PRDM16 and C/EBP-β in the myoblast-brown fat conversion, retroviruses expressing a short hairpin (sh) scrambled control RNA (scr) or shRNAs targeting C/EBP-β (shβ-1 and -2) were transduced together with PRDM16 or an empty vector to C2C12 myoblasts (). As shown in , knock-down of C/EBP-β significantly blunted the induction of Pparγ2
expression by PRDM16 in undifferentiated C2C12 myoblasts. Consistent with this result, Oil-Red-O staining showed that depletion of C/EBP-β blunted the adipogenesis induced by PRDM16 (). Furthermore, induction of brown fat selective genes including Pgc-1α, Ucp1, Elovl3
were completely or partially blocked by knock-down of C/EBP-β, correlating with the knock-down efficacy (). In addition, we ectopically expressed LIP, a dominant-negative form of C/EBP-β and this also significantly blunted PRDM16-induced adipogenesis and brown fat-selective gene expression (Supplementary Fig. 8
C/EBP-β is required for the initiation of the myoblast to brown fat conversion through PRDM16
Next, we took a systematic approach to ask what fraction of the PRDM16-regulated genes require C/EBP-β at the initiating
step of the myoblast-brown fat conversion. To this end, RNAs from undifferentiated C2C12 myoblasts expressing PRDM16 or control together with scr or sh-C/EBP-β, maintained under conditions non-permissive for differentiation, were subjected to Affymetrix microarray analysis. As shown in , 316 genes were significantly elevated or reduced by PRDM16 (>2-fold, P
<0.05), which were clustered into 4 groups: 1) genes elevated by PRDM16 in a C/EBP-β dependent manner; 2) genes elevated by PRDM16 in a C/EBP-β independent manner; 3) genes repressed by PRDM16 in a C/EBP-β dependent manner; and 4) genes repressed by PRDM16 in a C/EBP-β independent manner. The expression of a subset of genes identified by microarray analyses was validated by RT-PCR (Supplementary Fig. 9
). Strikingly, the majority of genes activated by PRDM16 before differentiation (62/95, 65.3%) indeed required C/EBP-β, whereas most of the repressed genes (210/221, 95.0%) were not grossly altered by C/EBP-β depletion.
We further explored the genetic requirement for C/EBP-β in brown fat development by analyzing C/EBP-β deficient (KO) embryos. Defects in BAT of C/EBP-β null newborn or adult mice have been described, although the reported phenotype was inconsistent23,24
. Since a large number of these embryos died within first 24 h after birth23,25
, we have re-examined this issue in late gestation (stage E18.5). This should permit a clearer separation of developmental changes in the BAT, as opposed to those that might occur secondary to abnormalities in other tissues after birth. As shown in , haematoxylin and eosin (H&E) staining showed that brown fat cells in KO embryos contained significantly less lipid droplets compared with those in WT, suggesting a defects in brown fat development per se
(, top). Moreover, UCP1 expression was severely reduced in KO embryos (, bottom), consistent with the results of Tanaka et al23
. We also conducted a definitive molecular characterization of the BAT from WT and KO embryos. Remarkably, BAT from C/EBP-β KO mice nearly phenocopied that from PRDM16 KO mice at the gene expression level; i.e.
a broad reduction of BAT-selective gene expression, and a broad induction of the skeletal muscle gene expression (). Together, these data indicate that the PRDM16-C/EBP-β transcriptional complex specifically plays a critical role in the initiation of myoblast-brown fat switch. This strongly suggests that PRDM16 acts in myf5
-positive myoblastic precursors, at least in part, by coactivation of C/EBP-β to induce the expression of PPARγ and PGC-1α. Subsequently, PRDM16 coactivates PPARγ and PGC-1α through direct binding events, which drives a complete brown fat differentiation program (Supplementary Fig. 10
This mechanistic model suggests a critical question: Are the two factors sufficient to reconstitute a brown fat program in naïve cells? To this end, PRDM16 and C/EBP-β were ectopically expressed in mouse embryonic fibroblasts (MEFs) or primary skin fibroblasts with no inherent adipose or brown fat character. As shown in , Pparγ2
mRNA expression was synergistically induced by PRDM16 and C/EBP-β in a dose-dependent manner in undifferentiated fibroblasts. After 6–8 days under adipogenic conditions, both MEFs and skin fibroblasts expressing these two factors uniformly differentiated into lipid-filled adipocytes, as shown by Oil-Red-O staining (). The single factors alone were not sufficient to robustly stimulate the differentiated state. Gene expression studies showed that PRDM16 and C/EBP-β powerfully induced mRNA levels of brown fat genes including Cox7a1
(16-fold) and Cidea
(170-fold) to levels comparable with or even higher than those seen in bona-fide
immortalized brown fat cells (). Importantly, as in authentic brown fat cells, mRNA level of thermogenic genes such as Pgc-1α
were further enhanced by cAMP treatment (). The mechanism underlying the augmentation of cAMP effects in the engineered brown fat cells remains unknown. To our surprise, mRNA level of those genes at the basal state were activated to levels seen in cAMP-stimulated brown fat cells. Lastly, the two factors were able to induce the brown fat gene program from primary mouse skin fibroblasts () and human skin fibroblasts isolated from newborn foreskin (Supplementary Fig. 11
Reconstitution of the brown fat gene program in fibroblasts through PRDM16 and C/EBP-β
An important characteristic of brown fat cells is their extraordinarily high rates of respiration, particularly uncoupled respiration in response to cAMP. As shown in , engineered brown fat cells induced by these two factors have significantly higher levels of total and uncoupled respiration than control cells, by 4.4 and 6.5 fold, respectively, at the basal state. It is notable that the engineered cells have greater basal respiration, both total and uncoupled, than bona-fide brown fat cells. However, while the bona-fide brown fat cells can further increase both total and uncoupled respiration (by 85% and 90%, respectively) in response to cAMP, engineered brown fat cells already seem to be at their maximal respiration. That these cells are responsive to cAMP is shown by the fact that expression of thermogenic genes such as such as Pgc-1α and Ucp1, are induced by cAMP treatment (). Hence, some other aspect of the respiratory apparatus, unknown at this point, seems to be limiting in the engineered brown fat cells.
The finding that the combination of PRDM16 and C/EBP-β is sufficient to reconstitute a near complete brown fat program offers an opportunity for controlling brown fat levels and function in vivo
. We conducted transplantation studies, as originally developed by Green and Kehinde26
, using undifferentiated MEFs expressing either: vector, PRDM16, C/EBP-β or combination of the two factors. As shown in hematoxylin and eosin staining (), the cells expressing vector nor either PRDM16 or C/EBP-β alone did not form visible fat tissues. In contrast, the cells expressing both PRDM16 and C/EBP-β formed very distinct fat pads in vivo
. Notably, at high magnification, the engineered fat tissue induced by the two factors contained “multilocular” fat cells, a morphological characteristic of brown fat in vivo
(). The population of mulilocular fat cells (area 1) is mixed with regions of “unilocular” fat cells (area 2). Importantly, immunohistochemical analyses showed that the engineered adipose tissue was UCP1 positive both in the multilucolar and unilocular fat cells ().
Generation of functional brown adipose tissue in vivo through expression of PRDM16 and C/EBP-β
To further characterize the activity of engineered brown fat tissue in vivo
, we utilized positron emission tomography (PET) with fluorodeoxyglucose (18
FDG), which has recently been used to detect active BAT in adult humans4–7
. This technique measures glucose uptake, with brown fat functioning in vivo
as an active “sink” for glucose. To this end, we engineered two adipose tissues with similar sizes in the same nude mice: a “brown” fat tissue induced by PRDM16 and C/EBP-β and a “white” fat tissue induced by PPARγ alone as a control (Supplementary Fig. 12a
). Induction of BAT-selective genes by PRDM16 and C/EBP-β were confirmed in the cultured cells by RT-PCR (Supplementary Fig. 12b
). As shown in , PET scanning detected a signal in mice from the engineered BAT. To enhance the sensitivity and specificity of the PET signal from the engineered fat tissues, the skin with these fat tissues attached were removed and scanned. The combination of computed tomography (CT) image (, left) and PET image (, right) clearly showed that the PET signal was detected from the engineered BAT, but not from the engineered WAT. These results indicate that the engineered brown fat cells function as a sink for active glucose disposal. Given the incredible capacity of BAT to dissipate stored chemical energy and thus counteract obesity, we are optimistic that the PRDM16 pathway can be used to drive brown fat development in vivo
in a therapeutic setting. Certainly natural or synthetic compounds that can induce PRDM16 in white fat precursors or in myoblastic cells could have great value in human metabolic disease. Alternatively, as shown here, autologous transplantation of engineered brown fat induced by PRDM16 and C/EBP-β in amounts that are both clinically acceptable and therapeutically useful may well be possible. Future experiments must define the optimal conditions to achieve maximal angiogenesis, innervation and resulting energy expenditure from autologous transplants.