Search

Great progress has been made in identifying positive regulators that activate adipocyte thermogenesis, but negative regulatory signaling of thermogenesis remains poorly understood. Here, we found that cardiotrophin-like cytokine factor 1 (CLCF1) signaling led to loss of brown fat identity, which impaired thermogenic capacity. CLCF1 levels decreased during thermogenic stimulation but were considerably increased in obesity. Adipocyte-specific CLCF1 transgenic (CLCF1-ATG) mice showed impaired energy expenditure and severe cold intolerance. Elevated CLCF1 triggered whitening of brown adipose tissue by suppressing mitochondrial biogenesis. Mechanistically, CLCF1 bound and activated ciliary neurotrophic factor receptor (CNTFR) and augmented signal transducer and activator of transcription 3 (STAT3) signaling. STAT3 transcriptionally inhibited both peroxisome proliferator-activated receptor-γ coactivator (PGC) 1α and 1β, which thereafter restrained mitochondrial biogenesis in adipocytes. Inhibition of CNTFR or STAT3 could diminish the inhibitory effects of CLCF1 on mitochondrial biogenesis and thermogenesis. As a result, CLCF1-TG mice were predisposed to develop metabolic dysfunction even without external metabolic stress. Our findings revealed a brake signal on nonshivering thermogenesis and suggested that targeting this pathway could be used to restore brown fat activity and systemic metabolic homeostasis in obesity.

Brown and beige adipocytes harbor the thermogenic capacity to adapt to environmental thermal or nutritional changes. Histone methylation is an essential epigenetic modification involved in the modulation of nonshivering thermogenesis in adipocytes. Here, we describe a molecular network leading by KMT5c, a H4K20 methyltransferase, that regulates adipocyte thermogenesis and systemic energy expenditure. The expression of Kmt5c is dramatically induced by a β3-adrenergic signaling cascade in both brown and beige fat cells. Depleting Kmt5c in adipocytes in vivo leads to a decreased expression of thermogenic genes in both brown and subcutaneous (s.c.) fat tissues. These mice are prone to high-fat-diet-induced obesity and develop glucose intolerance. Enhanced transformation related protein 53 ( Trp53 ) expression in Kmt5c knockout (KO) mice, that is due to the decreased repressive mark H4K20me3 on its proximal promoter, is responsible for the metabolic phenotypes. Together, these findings reveal the physiological role for KMT5c-mediated H4K20 methylation in the maintenance and activation of the thermogenic program in adipocytes., Competing Interests: The authors declare no competing interest.

Expression of bone morphogenetic protein 4 (BMP4) in adipocytes of white adipose tissue (WAT) produces "white adipocytes" with characteristics of brown fat and leads to a reduction of adiposity and its metabolic complications. Although BMP4 is known to induce commitment of pluripotent stem cells to the adipocyte lineage by producing cells that possess the characteristics of preadipocytes, its effects on the mature white adipocyte phenotype and function were unknown. Forced expression of a BMP4 transgene in white adipocytes of mice gives rise to reduced WAT mass and white adipocyte size along with an increased number of a white adipocyte cell types with brown adipocyte characteristics comparable to those of beige or brite adipocytes. These changes correlate closely with increased energy expenditure, improved insulin sensitivity, and protection against diet-induced obesity and diabetes. Conversely, BMP4-deficient mice exhibit enlarged white adipocyte morphology and impaired insulin sensitivity. We identify peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α) as the target of BMP signaling required for these brown fat-like changes in WAT. This effect of BMP4 on WAT appears to extend to human adipose tissue, because the level of expression of BMP4 in WAT correlates inversely with body mass index. These findings provide a genetic and metabolic basis for BMP4's role in altering insulin sensitivity by affecting WAT development.

Obesity is accompanied by an increase in both adipocyte number and size. The increase in adipocyte number is the result of recruitment to the adipocyte lineage of pluripotent stem cells present in the vascular stroma of adipose tissue. These pluripotent cells have the potential to undergo commitment and then differentiate into adipocytes, as well as myocytes, osteocytes, and chondrocytes. In this article, we show that both bone morphogenetic protein (BMP)2 and BMP4 can induce commitment of C3H10T1/2 pluripotent stem cells into adipocytes. After treatment of C3H10T1/2 stem cells with these BMPs during proliferation followed by exposure to differentiation inducers at growth arrest, nearly all cells enter the adipose development pathway, express specific adipocyte markers, and acquire the adipocyte phenotype. Overexpression of constitutively active BMP receptor (CA)-BMPr1A or CA-BMPr1B induces commitment in the absence of BMP2/4, whereas overexpression of a dominant-negative receptor dominant-negative-BMPr1A suppresses commitment induced by BMP. Also, knockdown of the expression of Smad4 (coregulator in the BMP/Smad signaling pathway) with RNAi disrupts commitment by the BMPs. However, knockdown of expression of p38 MAPK (an intermediary in the BMP/MAPK signaling pathway) with RNAi had little effect on BMP-induced commitment. Together, these findings indicate that the BMP/Smad signaling pathway has a dominant role in adipocyte lineage determination. Proteomic analysis identified lysyl oxidase (LOX), a bona fide downstream target gene of the BMP signaling pathway. Expression of LOX is induced by BMP2/4 during adipocyte lineage commitment, and knockdown of its expression disrupts the commitment process.

Upon induction of differentiation, growth-arrested (G(1) phase) 3T3-L1 preadipocytes express CCAAT/enhancer binding protein-beta (C/EBPbeta), initiating a transcriptional cascade. C/EBPbeta immediately undergoes a priming phosphorylation (on Thr(188)) by MAPK/ERK. However, the acquisition of DNA binding and transactivation capacity of C/EBPbeta is delayed until further phosphorylation (on Ser(184) or Thr(179)) by GSK3beta occurs. Phosphorylation by glycogen synthase kinase-3beta (GSK3beta) induces S phase entry and thereby mitotic clonal expansion (MCE), a requirement for terminal differentiation. Because MAPK activity is down-regulated before S phase is completed, we sought to identify the kinase that maintains C/EBPbeta in the primed phosphorylated state throughout S phase and MCE. We show here that cdk2/cyclinA, whose expression is activated at the onset of S phase, functions in this capacity. Ex vivo and in vitro experiments show that cdk2/cyclinA catalyzes this delayed priming phosphorylation. Mass spectrometric analysis revealed that cdk2/cyclinA phosphorylates C/EBPbeta on Thr(188) and is required for phosphorylation (on Ser(184) or Thr(179)) of C/EBPbeta by GSK3beta and maintenance of DNA binding activity. Suppression of cdk2 activity by RNA interference or pharmacologic inhibitor disrupts subsequent events in the differentiation program. Thus, MAPK and cdk2/cyclinA act sequentially to maintain Thr(188) of C/EBPbeta in the primed phosphorylated state during MCE and thereby progression of terminal differentiation.

CCAAT enhancer binding protein beta (C/EBPbeta) plays an essential role in the cascade that triggers adipocyte differentiation. C/EBPbeta activates transcription of C/EBPalpha and peroxisome proliferator-activated receptor-gamma, transcriptional activators of genes that give rise to the adipocyte phenotype. Sequential phosphorylation of C/EBPbeta/liver activating protein (LAP) on Thr(188) by MAPK and on Ser(184) or Thr(179) by glycogen synthase kinase beta (GSK3beta) is required for acquisition of DNA binding activity and transcriptional activation. To investigate how phosphorylation and dimerization of C/EBPbeta/LAP alter these activities, wild-type (Wt) and mutant rC/EBPbetas were prepared and purified to assess DNA binding and transcription in cell-free systems. rC/EBPbeta/LAP, phosphorylated by MAPK and GSK3beta in vitro, produced a >100-fold increase in DNA binding activity. Mutation of the phosphorylation to Glu increased DNA binding activity. Using a cell-free transcription system with nuclear extract from 3T3-L1 preadipocytes and rC/EBPbeta/LAP, only doubly phosphorylated rC/EBPbeta/LAP (by MAPK and GSK3beta) activated transcription driven by Wt C/EBPalpha, 422/aP2, and SCD1 promoters. Oxidation-induced dimerization of doubly phosphorylated Wt rC/EBPbeta/LAP increased DNA binding, whereas unphosphorylated Wt rC/EBPbeta/LAP lacked DNA binding activity. Mutation of the C-terminal Cys(296) adjacent to the leucine zipper and Cys(143) just upstream of the DNA binding domain eliminated phosphorylation-, oxidation-, and dimerization-dependent DNA binding activity, whereas mutation of Cys(201) within the basic DNA binding domain had little effect on DNA binding. These findings indicate that dual phosphorylation of C/EBPbeta/LAP caused a conformational change that facilitates S-S bond formation and dimerization, rendering the basic region accessible to the C/EBP regulatory element.

CCAAT enhancer-binding protein (C/EBP)beta, C/EBPalpha, and peroxisome proliferator activated receptor (PPAR)gamma act in a cascade where C/EBPbeta activates expression of C/EBPalpha and PPARgamma, which then function as pleiotropic activators of genes that produce the adipocyte phenotype. When growth-arrested 3T3-L1 preadipocytes are induced to differentiate, C/EBPbeta is rapidly expressed but still lacks DNA-binding activity. After a long (14-hour) lag, glycogen synthase kinase 3beta enters the nucleus, which correlates with hyperphosphorylation of C/EBPbeta and acquisition of DNA-binding activity. Concurrently, 3T3-L1 preadipocytes synchronously enter S phase and undergo mitotic clonal expansion, a prerequisite for terminal differentiation. Ex vivo and in vitro experiments with C/EBPbeta show that phosphorylation of Thr-188 by mitogen-activating protein kinase "primes" C/EBPbeta for subsequent phosphorylation on Ser-184 and Thr-179 by glycogen synthase kinase 3beta, acquisition of DNA-binding function, and transactivation of the C/EBPalpha and PPARgamma genes. The delayed transactivation of the C/EBPalpha and PPARgamma genes by C/EBPbeta appears necessary to allow mitotic clonal expansion, which would otherwise be prevented, because C/EBPalpha and PPARgamma are antimitotic.

The increase of adipose tissue mass associated with obesity is due in part to an increase in the number of adipocytes. This hyperplasia results from recruitment of pluripotent stem cells present in the vascular stroma of adipose tissue. A model cell culture system has been developed that recapitulates this process both ex vivo and in vivo. After treatment of pluripotent C3H10T1/2 stem cells with bone morphogenic protein 4 (BMP4) during proliferation followed by differentiation inducers at growth arrest, the cells synchronously enter S phase and undergo mitotic clonal expansion, a hallmark of preadipocyte differentiation. Upon exiting the cell cycle, these cells express adipocyte markers and acquire adipocyte characteristics at high frequency. C3H10T1/2 cells treated with BMP4 in cell culture and implanted s.c. into athymic mice develop into tissue indistinguishable from adipose tissue in normal fat depots. We interpret the findings as evidence that BMP4 is capable of triggering commitment of pluripotent C3H10T1/2 stem cells to the adipocyte lineage.

Hormonal induction of growth-arrested 3T3-L1 preadipocytes rapidly activates expression of CCAAT/enhancer-binding protein (C/EBP) beta. Acquisition of DNA-binding activity by C/EBPbeta, however, is delayed until the cells synchronously enter the S phase of mitotic clonal expansion (MCE). After MCE, C/EBPbeta activates expression of C/EBPalpha and peroxisome proliferator-activated receptor gamma, which then transcriptionally activate genes that give rise to the adipocyte phenotype. A-C/EBP, which possesses a leucine zipper but lacks functional DNA-binding and transactivation domains, forms stable inactive heterodimers with C/EBPbeta in vitro. Infection of 3T3-L1 preadipocytes with an adenovirus A-C/EBP expression vector interferes with C/EBPbeta function after induction of differentiation. A-C/EBP inhibited events associated with hormone-induced entry of S-phase of the cell cycle, including the turnover of p27/Kip1, a key cyclin-dependent kinase inhibitor, expression of cyclin A and cyclin-dependent kinase 2, DNA replication, MCE, and, subsequently, adipogenesis. Although A-C/EBP blocked cell proliferation associated with MCE, it did not inhibit normal proliferation of 3T3-L1 preadipocytes. Immunofluorescent staining of C/EBPbeta revealed that A-C/EBP prevented the normal punctate nuclear staining of centromeres, an indicator of C/EBPbeta binding to C/EBP regulatory elements in centromeric satellite DNA. The inhibitory effects of A-C/EBP appear to be due primarily to interference with nuclear import of C/EBPbeta caused by obscuring its nuclear localization signal. These findings show that both MCE and adipogenesis are dependent on C/EBPbeta.

Hormonal induction of growth-arrested 3T3-L1 preadipocytes triggers a signaling cascade that culminates in adipogenesis. CCAATenhancer-binding protein (CEBP)beta is expressed immediately but gains DNA-binding activity only after a long lag as the cells synchronously begin mitotic clonal expansion (MCE). After MCE, a process required for adipogenesis, CEBPbeta activates expression of CEBPalpha and peroxisome proliferator-activated receptor gamma, which then transcriptionally activate genes that produce the adipocyte phenotype. When mouse embryo fibroblasts (MEFs) are subjected to the same differentiation protocol, a subset of the MEFs undergoes a similar program of events. Similar to 3T3-L1 preadipocytes, the MEFs reenter the cell cycle (as indicated by the synchronous expression of cyclin A) and undergo MCE as evidenced by the incorporation of BrdUrd into DNA and the formation of mitotic foci of cells that undergo adipogenesis. CEBPbeta is expressed immediately after induction but exhibits delayed acquisition of DNA-binding activity followed by expression of adipocyte markers and the accumulation of cytoplasmic triglyceride. MEFs from CEBPbeta(-/-) mice, however, neither undergo MCE nor differentiate into adipocytes. Forced expression of CEBPbeta (LAP) but not dominant-negative CEBPbeta (LIP) in CEBPbeta(-/-) MEFs restores MCE, expression of adipocyte markers, and the capacity to form mitotic foci of cells that undergo adipogenesis. These findings demonstrate that expression of CEBPbeta is a prerequisite for MCE in the adipocyte-differentiation program.

When induced to differentiate, growth-arrested 3T3-L1 preadipocytes synchronously reenter the cell cycle and undergo mitotic clonal expansion (MCE) followed by expression of genes that produce the adipocyte phenotype. The preadipocytes traverse the G(1)S checkpoint synchronously as evidenced by the expressionactivation of cdk2-cyclin-EA, turnover of p27kip1, hyperphosphorylation of Rb, translocation of cyclin D(1) from nuclei to cytoplasm and GSK-3beta from cytoplasm to nuclei, and incorporation of [(3)H]thymidine into DNA. As the cells cross the G(1)S checkpoint, CEBPbeta acquires DNA-binding activity, initiating a cascade of transcriptional activation that culminates in the expression of adipocyte proteins. The mitogen-activated protein kinaseextracellular signal-regulated kinase kinase (MEK) inhibitor PD98059 delays, but does not block, MCE and differentiation, the extent of the delay causing a comparable delay in the expression of cell-cycle markers, MCE, and adipogenesis. The more potent and specific MEK inhibitor UO126 and the cyclin-dependent kinase inhibitor roscovitine, which inhibit the cell cycle at different points, block MCE, expression of cell cycle and adipocyte markers, as well as adipogenesis. These results show that MCE is a prerequisite for differentiation of 3T3-L1 preadipocytes into adipocytes.

Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic DNA virus that causes Kaposi sarcoma and AIDS-related primary effusion lymphoma (PEL). Here we show that KSHV lytic cycle replication in PEL cells induces G(1) cell cycle arrest, presumably to facilitate the progression of viral DNA replication. Expression of a KSHV-encoded early lytic protein referred to as RAP or K8 is induced within 12-24 h after the onset of lytic cycle induction in host PEL cells, and coincides with increased levels of both the endogenous C/EBPalpha and p21(CIP-1) proteins in the nucleus of the same cells. The KSHV RAP protein binds to C/EBPalpha in vitro and stimulates C/EBPalpha-induced expression from both the C/EBPalpha and p21 promoters in cotransfected cells. A recombinant adenovirus expressing the RAP protein induced the expression of both the C/EBPalpha and p21 proteins in primary human fibroblasts, and flow cytometric analysis revealed a dramatic inhibition of G(1) to S cell cycle progression in the same cells. All of these effects were abolished in cells that lack C/EBPalpha or by deletion of the basic/leucine zipper region in RAP that interacts with C/EBPalpha. Therefore, C/EBPalpha is essential for the p21-mediated inhibition of G(1) to S-phase progression by RAP in KSHV-infected host cells.

Hormone induction of growth-arrested preadipocytes triggers mitotic clonal expansion followed by expression of CCAAT/enhancer-binding protein (C/EBP)alpha and differentiation into adipocytes. The order of these events is critical because C/EBPalpha is antimitotic and its expression prematurely would block the mitotic clonal expansion required for differentiation. C/EBPbeta, a transcriptional activator of the C/EBPalpha gene, is expressed early in the differentiation program, but lacks DNA-binding activity and fails to localize to centromeres until preadipocytes traverse the G(1)-S checkpoint of mitotic clonal expansion. Evidence is presented that dominant-negative CHOP-10 expressed by growth-arrested preadipocytes transiently sequesters C/EBPbeta by heterodimerization. As preadipocytes reach S phase, CHOP-10 is down-regulated, apparently releasing C/EBPbeta from inhibitory constraint and allowing transactivation of the C/EBPalpha gene. In support of these findings, up-regulation of CHOP-10 with the protease inhibitor N-acetyl-Leu-Leu-norleucinal prevents activation of C/EBPbeta, expression of C/EBPalpha, and adipogenesis.

During adipogenesis, CCAAT/enhancer binding protein alpha (C/EBPalpha) serves as a pleiotropic transcriptional activator of adipocyte genes. Previously, we identified dual repressive elements in the C/EBPalpha gene and a putative transacting factor (C/EBPalpha undifferentiated protein, or CUP) expressed by preadipocytes, but not adipocytes, that bind to these elements. In the present investigation, CUP was purified 17,000-fold from nuclear extracts of 3T3-L1 preadipocytes. Amino acid sequence and mass spectral analysis of tryptic peptides derived from purifed CUP (molecular mass approximately 50 kDa) revealed that the repressor is (or contains) an isoform of the transcription factor, AP-2alpha. Electrophoretic mobility shift and Western blot analysis on purified CUP and preadipocyte nuclear extracts confirmed the identity of CUP as AP-2alpha. Both AP-2alpha protein and CUP binding activity are expressed by preadipocytes and then decrease concomitantly during differentiation of 3T3-L1 preadipocytes into adipocytes. Consistent with a repressive role of AP-2alpha/CUP, an AP-2alpha1 expression vector, cotransfected with a C/EBPalpha promoter-reporter construct into 3T3-L1 adipocytes, inhibited reporter gene transcription. Taken together with previous results, these findings suggest that in preadipocytes the C/EBPalpha gene is repressed by AP-2alpha/CUP, which, upon induction of differentiation, is down-regulated, allowing expression of the gene.

During adipocyte differentiation, the expression of C/EBPalpha is activated, which in turn serves to transcriptionally activate numerous adipocyte genes. A previous search for cis elements that regulate transcription of the C/EBPalpha gene led to the identification of a potential repressive element within the proximal 5' flanking region of the gene. Nuclear extracts from 3T3-L1 preadipocytes, but not adipocytes, were found to contain a factor, CUP (C/EBPalpha undifferentiated protein), that binds to this site (the CUP-1 site). In the present investigation, we show that C/EBPalpha promoter-luciferase constructs containing both the proximal 5' flanking and the entire 5' untranslated regions of the gene exhibit an expression pattern during adipocyte differentiation comparable to that of the endogenous C/EBPalpha gene. Mutation of the CUP-1 site in these constructs had little effect on reporter gene expression; however, when this mutation was combined with deletion of the 5' untranslated region, reporter gene expression by preadipocytes was dramatically up-regulated. Consistent with this finding, a second CUP binding site (the CUP-2 site) was identified in the 5' untranslated region. Although mutation of either CUP element in constructs containing both the 5' flanking and 5' untranslated region had little effect on reporter gene transcription, mutation of both CUP elements markedly activated transcription. Thus, it appears that dual CUP regulatory elements repress transcription of the C/EBPalpha gene prior to induction of the adipocyte differentiation program.