Stephen R. Farmer

Induction of peroxisome proliferator-activated receptor gamma during the conversion of 3T3 fibroblasts into adipocytes is mediated by C/EBPbeta, C/EBPdelta, and glucocorticoids.

The differentiation of 3T3 preadipocytes into adipocytes is accompanied by a transient induction of C/EBPbeta and C/EBPdelta expression in response to treatment of the cells with methylisobutylxanthine (MIX) and dexamethasone (DEX), respectively. In this report, we demonstrate that peroxisome proliferator-activated receptor gamma (PPARgamma) expression in 3T3-L1 preadipocytes is induced by MIX and DEX, suggesting that C/EBPbeta and C/EBPdelta may be involved in this process. Using a tetracycline-responsive expression system, we have recently shown that the conditional ectopic expression of C/EBPbeta in NIH 3T3 fibroblasts (beta2 cells) in the presence of DEX activates the synthesis of peroxisome PPARgamma mRNA. Subsequent exposure of these cells to PPAR activators stimulates their conversion into adipocytes; however, neither the expression of C/EBPbeta nor exposure to DEX alone is capable of inducing PPARgamma expression in the beta2 cell line. We find that unlike the case for 3T3 preadipocytes, C/EBPdelta is not induced by DEX in these 3T3 fibroblasts and therefore is not relaying the effect of this glucocorticoid to the PPARgamma gene. To define the role of glucocorticoids in regulating PPARgamma expression and the possible involvement of C/EBPdelta, we have established an additional set of NIH 3T3 cell lines expressing either C/EBPdelta alone (delta23 cells) or C/EBPdelta and C/EBPbeta together (beta/delta39 cells), using the tetracycline-responsive system. Culture of these cells in tetracycline-deficient medium containing DEX, MIX, insulin, and fetal bovine serum shows that the beta/delta39 cells express PPARgamma and aP2 mRNAs at levels that are almost equivalent to those observed in fully differentiated 3T3-L1 adipocytes. These levels are approximately threefold higher than their levels of expression in the beta2 cells. Despite the fact that these beta/delta39 cells produce abundant amounts of C/EBPbeta and C/EBPdelta (in the absence of tetracycline), they still require glucocorticoids to attain maximum expression of PPARgamma mRNA. Furthermore, the induction of PPARgamma mRNA by exposure of these cells to DEX occurs in the absence of ongoing protein synthesis. The delta23 cells, on the other hand, are not capable of activating PPARgamma gene expression when exposed to the same adipogenic inducers. Finally, attenuation of ectopic C/EBPbeta production at various stages during the differentiation process results in a concomitant inhibition of PPARgamma and the adipogenic program. These data strongly suggest that the induction of PPARgamma gene expression in multipotential mesenchymal stem cells (NIH 3T3 fibroblasts) is dependent on elevated levels of C/EBPbeta throughout the differentiation process, as well as an initial exposure to glucocorticoids. C/EBPdelta may function by synergizing with C/EBPbeta to enhance the level of PPARgamma expression.

Adiponectin Secretion Is Regulated by SIRT1 and the Endoplasmic Reticulum Oxidoreductase Ero1-Lα

ABSTRACT Adiponectin is secreted from adipose tissue in response to metabolic effectors in order to sensitize the liver and muscle to insulin. Reduced circulating levels of adiponectin that usually accompany obesity contribute to the associated insulin resistance. The molecular mechanisms controlling the production of adiponectin are essentially unknown. In this report, we demonstrate that the endoplasmic reticulum (ER) oxidoreductase Ero1-Lα and effectors modulating peroxisome proliferator-activated receptor γ (PPARγ) and SIRT1 activities regulate secretion of adiponectin from 3T3-L1 adipocytes. Specifically, adiponectin secretion and Ero1-Lα expression are induced during the early phase of adipogenesis but are then down-regulated during the terminal phase, coincident with an increased expression of SIRT1. Suppression of SIRT1 or activation of PPARγ enhances Ero1-Lα expression and stimulates secretion of high-molecular-weight complexes of adiponectin in mature adipocytes. Suppression of Ero1-Lα through expression of a corresponding small interfering RNA reduces adiponectin secretion during the differentiation of 3T3-L1 preadipocytes. Moreover, ectopic expression of Ero1-Lα in Ero1-Lα-deficient 3T3 fibroblasts stimulates the secretion of adiponectin following their conversion into adipocytes and prevents the suppression of adiponectin secretion in response to activation of SIRT1 by exposure to resveratrol. These findings provide a framework to understand the mechanisms by which adipocytes regulate secretion of adiponectin in response to various metabolic states.

A role for C/EBPbeta in regulating peroxisome proliferator-activated receptor gamma activity during adipogenesis in 3T3-L1 preadipocytes.

The differentiation of 3T3-L1 preadipocytes is regulated in part by a cascade of transcriptional events involving activation of the CCAAT/enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptor γ (PPARγ) by dexamethasone (DEX), 3-isobutyl-1-methylxanthine (MIX), and insulin. In this study, we demonstrate that exposure of 3T3-L1 preadipocytes to DEX and insulin fails to induce adipogenesis as indicated by a lack of C/EBPα, PPARγ2, and adipose protein 2/fatty acid-binding protein expression; however, PPARγ1 is expressed. Treatment of these MIX-deficient cells with a PPARγ ligand, troglitazone, induces C/EBPα expression and rescues the block in adipogenesis. In this regard, we also show that induction of C/EBPα gene expression by troglitazone in C3H10T1/2 cells ectopically expressing PPARγ occurs in the absence of ongoing protein synthesis, suggesting a direct transactivation of the C/EBPα gene by PPARγ. Furthermore, ectopic expression of a dominant negative isoform of C/EBPβ (liver-enriched transcriptional inhibitory protein (LIP)) inhibits the induction of C/EBPα, PPARγ2, and adipose protein 2/fatty acid-binding protein by DEX, MIX, and insulin in 3T3-L1 cells without affecting the induction of PPARγ1 by DEX. Exposure of LIP-expressing preadipocytes to troglitazone along with DEX, MIX, and insulin induces differentiation into adipocytes. Additionally, we show that sustained expression of C/EBPα in these LIP-expressing adipocytes requires constant exposure to troglitazone. Taken together, these observations suggest that inhibition of C/EBPβ activity not only blocks C/EBPα and PPARγ2 expression, but it also renders the preadipocytes dependent on an exogenous PPARγ ligand for their differentiation into adipocytes. We propose, therefore, an additional role for C/EBPβ in regulating PPARγ activity during adipogenesis, and we suggest an alternative means of inducing preadipocyte differentiation that relies on the dexamethasone-associated induction of PPARγ1 expression.

Decreases in tubulin and actin gene expression prior to morphological differentiation of 3T3 Adipocytes

The differentiation of 3T3-F442A preadipocytes is characterized by numerous enzymatic events and by a programmed change in cell morphology from a fibroblastic form to a nearly spherical shape. Accompanying this morphological change are large and specific decreases in biosynthetic rates for beta and gamma actin, vimentin and alpha and beta tubulin, as detected by one- and two-dimensional gel electrophoresis. In cells undergoing adipose differentiation, actin synthesis decreases by 90%, while the decrease in tubulin synthesis is more than 95%. Translation in vitro of mRNA isolated from differentiating cultures indicates that the decreases in biosynthetic rate for cytoskeletal proteins result from altered levels of active mRNA. Using cloned cDNA probes for beta actin and alpha tubulin, we show that changes in mRNA activity correspond to a specific loss of these sequences during cellular differentiation. Quantitatively, this loss of tubulin and actin mRNA sequences accounts virtually completely for the changes in protein biosynthetic rates. Examination of the synthesis and accumulation of cytoskeletal proteins and of their temporal relation to morphological conversion indicates that the biosynthetic changes are very early events in the differentiation, and suggests strongly that they participate in the development of the adipocyte morphology. The early occurrence of decreased cytoskeletal-protein synthesis also suggests that subsequent biosynthetic events specific to adipocyte differentiation may be influenced by alterations in the cytoskeleton.

Peroxisome-proliferator-activated receptor gamma suppresses Wnt/beta-catenin signalling during adipogenesis.

The Wnt/beta-catenin signalling pathway appears to operate to maintain the undifferentiated state of preadipocytes by inhibiting adipogenic gene expression. To define the mechanisms regulating suppression of Wnt/beta-catenin signalling, we analysed the beta-catenin expression in response to activation of transcription factors that regulate adipogenesis. The results show an extensive down-regulation of nuclear beta-catenin that occurs during the first few days of differentiation of 3T3-L1 preadipocytes and coincides with the induction of the adipogenic transcription factors, C/EBPbeta (CCAAT-enhancer-binding protein) and PPARgamma (peroxisome-proliferator-activated receptor). To assess the role of each of these factors in this process, we conditionally overexpressed C/EBPbeta in Swiss mouse fibroblasts using the TET-off system. Abundant expression of C/EBPbeta alone had minimal effect on beta-catenin expression, whereas expression of C/EBPbeta, in the presence of dexamethasone, induced PPARgamma expression and caused a measurable decrease in beta-catenin. In addition, exposure of cells expressing both C/EBPbeta and PPARgamma to a potent PPARgamma ligand resulted in an even greater decrease in beta-catenin by mechanisms that involve the proteasome. Our studies also suggest a reciprocal relationship between PPARgamma activity and beta-catenin expression, since ectopic production of Wnt-1 in preadipocytes blocked the induction of PPARgamma gene expression. Moreover, by suppressing beta-catenin expression, ectopic expression of PPARgamma in Wnt-1-expressing preadipocytes rescued the block in adipogenesis after their exposure to the PPARgamma ligand, troglitazone.

Cell-extracellular matrix interactions can regulate the switch between growth and differentiation in rat hepatocytes: reciprocal expression of C/EBP alpha and immediate-early growth response transcription factors.

Previous investigations have shown that culture of freshly isolated hepatocytes under conventional conditions, i.e., on dried rat tail collagen in the presence of growth factors, facilitates cell growth but also causes an extensive down-regulation of most liver-specific functions. This dedifferentiation process can be prevented if the cells are cultured on a reconstituted basement membrane gel matrix derived from the Englebreth-Holm-Swarm mouse sarcoma tumor (EHS gel). To gain insight into the mechanisms regulating this response to extracellular matrix, we are analyzing the activities of two families of transcription factors, C/EBP and AP-1, which control the transcription of hepatic and growth-responsive genes, respectively. We demonstrate that isolation of hepatocytes from the normal quiescent rat liver by collagenase perfusion activates the immediate-early growth response program, as indicated by increased expression of c-jun, junB, c-fos, and c-myc mRNAs. Adhesion of these activated cells to dried rat tail collagen augments the elevated levels of these mRNAs for the initial 1 to 2 h postplating; junB and c-myc mRNA levels then drop steeply, with junB returning to normal quiescence and the c-myc level remaining slightly elevated during the 3-day culture period. Levels of c-jun mRNA and AP-1 DNA binding activity, however, remain elevated from the outset, while C/EBP alpha mRNA expression is down-regulated, resulting in a decrease in the steady-state levels of the 42- and 30-kDa C/EBP alpha polypeptides and C/EBP alpha DNA binding activity. In contrast, C/EBP beta mRNA production remains at near-normal hepatic levels for 5 to 8 days of culture, although its DNA binding activity decreases severalfold during this time. Adhesion of hepatocytes to the EHS gel for the same period of time dramatically alters this program: it arrests growth and inhibits AP-1 DNA binding activity and the expression of c-jun, junB, and c-myc mRNAs, but, in addition, it restores C/EBP alpha mRNA and protein as well as C/EBP alpha and C/EBP beta DNA binding activities to the abundant levels present in freshly isolated hepatocytes. These changes are not due merely to growth inhibition, because suppression of hepatocyte proliferation on collagen by epidermal growth factor starvation or addition of transforming growth factor beta does not inhibit AP-1 activity or restore C/EBP alpha DNA binding activity to normal hepatic levels. These data suggest that expression of the normal hepatic phenotype requires that hepatocytes exist in a G0 state of growth arrest, facilitated here by adhesion of cells to the EHS gel, in order to express high levels of hepatic transcription factors such as C/EBP alpha.

Functional Interaction between Peroxisome Proliferator-Activated Receptor γ and β-Catenin

ABSTRACT Studies have demonstrated cross talk between β-catenin and peroxisome proliferator-activated receptor γ (PPARγ) signaling pathways. Specifically, activation of PPARγ induces the proteasomal degradation of β-catenin in cells that express an adenomatous polyposis coli-containing destruction complex. In contrast, oncogenic β-catenin is resistant to such degradation and inhibits the expression of PPARγ target genes. In the present studies, we demonstrate a functional interaction between β-catenin and PPARγ that involves the T-cell factor (TCF)/lymphocyte enhancer factor (LEF) binding domain of β-catenin and a catenin binding domain (CBD) within PPARγ. Mutation of K312 and K435 in the TCF/LEF binding domain of an oncogenic β-catenin (S37A) significantly reduces its ability to interact with and inhibit the activity of PPARγ. Furthermore, these mutations render S37A β-catenin susceptible to proteasomal degradation in response to activation of PPARγ. Mutation of F372 within the CBD (helices 7 and 8) of PPARγ disrupts its binding to β-catenin and significantly reduces the ability of PPARγ to induce the proteasomal degradation of β-catenin. We suggest that in normal cells, PPARγ can function to suppress tumorigenesis and/or Wnt signaling by targeting phosphorylated β-catenin to the proteasome through a process involving its CBD. In contrast, oncogenic β-catenin resists proteasomal degradation by inhibiting PPARγ activity, which requires its TCF/LEF binding domain.

Reconstitution of insulin-sensitive glucose transport in fibroblasts requires expression of both PPARgamma and C/EBPalpha.

Adipocyte differentiation is regulated by at least two major transcription factors, CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ). Expression of PPARγ in fibroblasts converts them to fat-laden cells with an adipocyte-like morphology. Here, we investigate the ability of PPARγ to confer insulin-sensitive glucose transport to a variety of murine fibroblast cell lines. When cultured in the presence of a PPARγ ligand, Swiss-3T3 and BALB/c-3T3 cells ectopically expressing PPARγ accumulate lipid droplets, express C/EBPα, aP2, insulin-responsive aminopeptidase, and glucose transporter isoform 4 (GLUT4), and exhibit highly insulin-responsive 2-deoxyglucose uptake. In contrast, PPARγ-expressing NIH-3T3 cells, despite similar lipid accumulation, adipocyte morphology, and aP2 expression, do not express C/EBPα or GLUT4 and fail to acquire insulin sensitivity. In cells ectopically expressing PPARγ, the development of insulin-responsive glucose uptake correlates with C/EBPα expression. Furthermore, ectopic expression of C/EBPα in NIH-3T3 cells converts them to the adipocyte phenotype and restores insulin-sensitive glucose uptake. We propose that the pathway(s) leading to fat accumulation and morphological changes are distinct from that leading to insulin-dependent glucose transport. Our results suggest that although PPARγ is sufficient to trigger the adipogenic program, C/EBPα is required for establishment of insulin-sensitive glucose transport.

Identification of a Domain within Peroxisome Proliferator-Activated Receptor γ Regulating Expression of a Group of Genes Containing Fibroblast Growth Factor 21 That Are Selectively Repressed by SIRT1 in Adipocytes

ABSTRACT Peroxisome proliferator-activated receptor γ (PPARγ) activity is regulated through association with ligands that include the thiazolidinedione class of antidiabetic drugs, as well as derivatives of polyunsaturated fatty acids. Induction of PPARγ target gene expression involves ligand-dependent reconfiguration of the ligand-binding domain (LBD), followed by recruitment of specific transcriptional coactivators. In this study, we have identified an amino acid (F372) within helix 7 of the LBD that is required for the response of PPARγ to endogenous ligands. Additionally, the data show that this amino acid is also required for expression of a novel subset of adipocyte genes (group 2), including fibroblast growth factor 21 (FGF21), and that the FGF21 gene is a direct target of PPARγ. Expression of the group 2 genes is selectively repressed by the NAD-dependent deacetylase SIRT1 in mature 3T3-L1 adipocytes, since knockdown of SIRT1 through the constitutive expression of a corresponding RNA interference enhances their expression without affecting the expression of classic adipogenic genes, such as adiponectin and FABP4/aP2. It appears that many of the group 2 genes repressed by SIRT1 in mature adipocytes correspond to the same set of genes that are selectively activated by treatment of fat cells with the PPARγ ligand, troglitazone. These data support a role for helix 7 of the LBD of PPARγ in regulating adipocyte function and suggest that inhibition of SIRT1 in adipocytes induces the same insulin-sensitizing action as PPARγ ligands.

Phosphorylation of C/EBPβ at a Consensus Extracellular Signal-Regulated Kinase/Glycogen Synthase Kinase 3 Site Is Required for the Induction of Adiponectin Gene Expression during the Differentiation of Mouse Fibroblasts into Adipocytes

ABSTRACT Stimulation of adipogenesis in mouse preadipocytes requires C/EBPβ as well as activation of the MEK/extracellular signal-regulated kinase (ERK) signaling pathway. In this study, we demonstrate that phosphorylation of C/EBPβ at a consensus ERK/glycogen synthase kinase 3 (GSK3) site regulates adiponectin gene expression during the C/EBPβ-facilitated differentiation of mouse fibroblasts into adipocytes. First, we show that exposure of 3T3-L1 preadipocytes to insulin, dexamethasone (DEX), and isobutylmethylxanthine (MIX) leads to the phosphorylation of C/EBPβ at threonine 188. Pretreating the cells with a MEK1-specific inhibitor (U0126) significantly attenuates this activity. Similarly, these effectors activate the phosphorylation of T188 within an ectopic C/EBPβ overexpressed in Swiss mouse fibroblasts, and this event involves both MEK1 and GSK3 activity. We further show that expression of C/EBPβ (p34kD LAP isoform) in Swiss mouse fibroblasts exposed to DEX, MIX, and insulin induces expression of peroxisome proliferator-activated receptor γ (PPARγ) and some adiponectin but that it does not activate expression of FABP4/aP2. In fact, complete conversion of these fibroblasts into lipid-laden adipocytes, which includes activation of FABP4 and adiponectin expression, requires their exposure to a potent PPARγ ligand such as troglitazone. Expression of a mutant C/EBPβ in which threonine 188 has been modified to alanine (C/EBPβ T188A) can induce PPARγ production in the mouse fibroblasts, but it is incapable of stimulating adiponectin expression in the absence or presence of troglitazone. Interestingly, replacement of T188 with aspartic acid creates a C/EBPβ molecule (C/EBPβ T188D) that possesses adipogenic activity similar to that of the wild-type molecule. The absence of adiponectin expression correlates with a reduced amount of C/EBPα in the adipocytes expressing the T188A mutant suggesting that C/EBPα is required for expression of adiponectin. In fact, ectopic expression of PPARγ in C/EBPα-deficient fibroblasts (NIH 3T3 cells) produces a modest amount of adiponectin, whereas expression of both PPARγ and C/EBPα in NIH 3T3 cells facilitates production of abundant quantities of adiponectin. These data demonstrate that phosphorylation of C/EBPβ at a consensus ERK/GSK3 site is required for both C/EBPα and adiponectin gene expression during the differentiation of mouse fibroblasts into adipocytes.