Role of cell cycle regulators in adipose tissue and whole body energy homeostasis.

In the course of the last decades, metabolism research has demonstrated that adipose tissue is not an inactive tissue. Rather, adipocytes are key actors of whole body energy homeostasis. Numerous novel regulators of adipose tissue differentiation and function have been identified. With the constant increase of obesity and associated disorders, the interest in adipose tissue function alterations in the XXIst century has become of paramount importance. Recent data suggest that adipocyte differentiation, adipose tissue browning and mitochondrial function, lipogenesis and lipolysis are strongly modulated by the cell division machinery. This review will focus on the function of cell cycle regulators in adipocyte differentiation, adipose tissue function and whole body energy homeostasis; with particular attention in mouse studies.

A Pro12Ala substitution in PPARgamma2 associated with decreased receptor activity, lower body mass index and improved insulin sensitivity.

The peroxisome proliferator-activated receptor-gamma (PPARgamma) is a transcription factor that has a pivotal role in adipocyte differentiation and expression of adipocyte-specific genes. The PPARgamma1 and gamma2 isoforms result from alternative splicing and have ligand-dependent and -independent activation domains. PPARgamma2 has an additional 28 amino acids at its amino terminus that renders its ligand-independent activation domain 5-10-fold more effective than that of PPARgamma1. Insulin stimulates the ligand-independent activation of PPARgamma1 and gamma2 (ref. 5), however, obesity and nutritional factors only influence the expression of PPARgamma2 in human adipocytes. Here, we report that a relatively common Pro12Ala substitution in PPARgamma2 is associated with lower body mass index (BMI; P=0.027; 0.015) and improved insulin sensitivity among middle-aged and elderly Finns. A significant odds ratio (4.35, P=0.028) for the association of the Pro/Pro genotype with type 2 diabetes was observed among Japanese Americans. The PPARgamma2 Ala allele showed decreased binding affinity to the cognate promoter element and reduced ability to transactivate responsive promoters. These findings suggest that the PPARgamma2 Pro12Ala variant may contribute to the observed variability in BMI and insulin sensitivity in the general population.

Transcriptional control of adipogenesis.

Adipocyte differentiation is coordinatedly regulated by several transcription factors. C/EBP beta, C/EBP delta and ADD-1/SREBP-1 are active early during the differentiation process and induce the expression and/or activity of the peroxisome proliferator activated receptor-gamma (PPAR gamma), the pivotal coordinator of the adipocyte differentiation process. Activated PPAR gamma induces exit from the cell cycle and triggers the expression of adipocyte-specific genes, resulting in increased delivery of energy to the cells. C/EBP alpha, whose expression coincides with the later stages of differentiation, cooperates with PPAR gamma in inducing additional target genes and sustains a high level of PPAR gamma in the mature adipocyte as part of a feedforward loop. Altered activity and/or expression of these transcription factors might underlie the pathogenesis of disorders characterized by increased or decreased adipose tissue depots.

CDK4, a new metabolic sensor that antagonizes AMPK.

Cyclin-dependent kinase 4 (CDK4) is a positive regulator of cell cycle progression, however, there is growing evidence demonstrating that its function exceeds the control of cell division. Here we show that CDK4 is an important regulator of cellular substrate utilization through direct inhibition of the metabolic regulator AMPK (AMP-activated protein kinase).

The retinoblastoma-like protein p130 is involved in the determination of reserve cells in differentiating myoblasts.

During skeletal muscle differentiation, a subset of myoblasts remains quiescent and undifferentiated but retains the capacity to self-renew and give rise to differentiating myoblasts [1] [2] [3]: this sub-population of muscle cells was recently termed 'reserve cells' [3]. In order to characterise genes that can regulate the ratio between reserve cells and differentiating myoblasts, we examined members of the retinoblastoma tumor suppressor family - Rb, p107 and p130 - an important family of negative regulators of E2F transcription factors and cell cycle progression [4]. Although pRb and p107 positively regulate muscle cell differentiation [5] [6] [7], the role of p130 in muscle cells remains unknown. We show here that p130 (protein and mRNA), but neither pRb nor p107, preferentially accumulates during muscle differentiation in reserve cells. Also, p130 is the major Rb-family protein present in E2F complexes in this sub-population of cells. Although forced expression of either p130 or pRb in mouse C2 myoblasts efficiently blocked cell cycle progression, only p130 inhibited the differentiation program. Furthermore, muscle cells overexpressing p130 had reduced levels of the muscle-promoting factor MyoD. In addition, p130 repressed the transactivation capacity of MyoD, an effect abolished by co-transfection of pRb. Thus, we propose that p130, by blocking cell cycle progression and differentiation, could be part of a specific pathway that defines a pool of reserve cells during terminal differentiation.

Cyclin A is a mediator of p120E4F-dependent cell cycle arrest in G1.

E4F is a ubiquitously expressed GLI-Krüppel-related transcription factor which has been identified for its capacity to regulate transcription of the adenovirus E4 gene in response to E1A. However, cellular genes regulated by E4F are still unknown. Some of these genes are likely to be involved in cell cycle progression since ectopic p120E4F expression induces cell cycle arrest in G1. Although p21WAF1 stabilization was proposed to mediate E4F-dependent cell cycle arrest, we found that p120E4F can induce a G1 block in p21(-/-) cells, suggesting that other proteins are essential for the p120E4F-dependent block in G1. We show here that cyclin A promoter activity can be repressed by p120E4F and that this repression correlates with p120E4F binding to the cyclic AMP-responsive element site of the cyclin A promoter. In addition, enforced expression of cyclin A releases p120E4F-arrested cells from the G1 block. These data identify the cyclin A gene as a cellular target for p120E4F and suggest a mechanism for p120E4F-dependent cell cycle regulation.

Regulation of peroxisome proliferator-activated receptor gamma expression by adipocyte differentiation and determination factor 1/sterol regulatory element binding protein 1: implications for adipocyte differentiation and metabolism.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor implicated in adipocyte differentiation and insulin sensitivity. We investigated whether PPARgamma expression is dependent on the activity of adipocyte differentiation and determination factor 1/sterol regulatory element binding protein 1 (ADD-1/SREBP-1), another transcription factor associated with both adipocyte differentiation and cholesterol homeostasis. Ectopic expression of ADD-1/SREBP-1 in 3T3-L1 and HepG2 cells induced endogenous PPARgamma mRNA levels. The related transcription factor SREBP-2 likewise induced PPARgamma expression. In addition, cholesterol depletion, a condition known to result in proteolytic activation of transcription factors of the SREBP family, induced PPARgamma expression and improved PPRE-driven transcription. The effect of the SREBPs on PPARgamma expression was mediated through the PPARgamma1 and -3 promoters. Both promoters contain a consensus E-box motif that mediates the regulation of the PPARgamma gene by ADD-1/SREBP-1 and SREBP-2. These results suggest that PPARgamma expression can be controlled by the SREBP family of transcription factors and demonstrate new interactions between transcription factors that can regulate different pathways of lipid metabolism.

Tissue distribution and quantification of the expression of mRNAs of peroxisome proliferator-activated receptors and liver X receptor-alpha in humans: no alteration in adipose tissue of obese and NIDDM patients.

Members of the peroxisome proliferator-activated receptor (PPAR) family might be involved in pathologies with altered lipid metabolism. They participate in the control of the expression of genes involved in lipid metabolism and adipocyte differentiation. In addition, thiazolidinediones improve insulin resistance in vivo by activating PPAR gamma. However, little is known regarding their tissue distribution and relative expression in humans. Using a quantitative and sensitive reverse transcription (RT)-competitive polymerase chain reaction (PCR) assay, we determined the distribution and relative mRNA expression of the four PPARs (alpha,beta, gamma1, and gamma2) and liver X receptor-alpha (LXR alpha) in the main tissues implicated in lipid metabolism. PPAR alpha and LXR alpha were mainly expressed in liver, while PPAR gamma1 predominated in adipose tissue and large intestine. We found that PPAR gamma2 mRNA was a minor isoform, even in adipose tissue, thus causing question of its role in humans. PPAR beta mRNA was present in all the tissues tested at low levels. In addition, PPAR gamma mRNA was barely detectable in skeletal muscle, suggesting that improvement of insulin resistance with thiazolidinediones may not result from a direct effect of these agents on PPAR gamma in muscle. Obesity and NIDDM were not associated with change in PPARs and LXR alpha expression in adipose tissue. The mRNA levels of PPAR gamma1, the predominant form in adipocytes, did not correlate with BMI, leptin mRNA levels, or fasting insulinemia in 29 subjects with various degrees of obesity. These results indicated that obesity is not associated with alteration in PPAR gene expression in abdominal subcutaneous adipose tissue in humans.

The pleiotropic functions of peroxisome proliferator-activated receptor gamma.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors, initially described as molecular targets for synthetic compounds that induce peroxisome proliferation. PPARgamma is the best characterized of the PPARs. The heterodimer of PPARgamma with the retinoid X receptor (RXR) plays a crucial role in adipogenesis and insulin sensitization. The RXR/PPARgamma heterodimer furthermore has been reported to have important immunomodulatory activities and to affect cell proliferation/differentiation pathways in various malignancies. PPARgamma is activated by a number of naturally occurring fatty acid derivatives and by several synthetic compounds, including the thiazolidinediones and L-tyrosine-based insulin sensitizers. This review gives an overview of the pleiotropic functions of PPARgamma and discusses the wide-ranging medical implications that modulation of PPARgamma activity might have for various diseases, ranging from obesity and type 2 diabetes to cancer and inflammation.

PPARgamma3 mRNA: a distinct PPARgamma mRNA subtype transcribed from an independent promoter.

PPARgamma is a member of the peroxisome proliferator activated receptors (PPAR) subfamily of nuclear receptors. So far two PPARgamma isoforms, PPARgamma1 and PPARgamma2, were known in mammals. We describe the structure of a novel human PPARgamma subtype, PPARgamma3. The PPARgamma3 mRNA is transcribed from a novel promoter localized 5' of exon A2. PPARgamma3 mRNA expression was restricted to adipose tissue and large intestine. Similar to human PPARgamma1 and -2, PPARy3 is activated by thiazolidinediones and prostaglandin J derivatives and binds with high affinity to a PPRE.

A CDE/CHR-like element mediates repression of transcription of the mouse RB2 (p130) gene.

The bipartite repressor elements, termed cell cycle-dependent element (CDE)/cell cycle regulatory element (CCRE)-cell cycle homology region (CHR) control the growth-dependent transcription of the cyclin A, cdc25C, cdc2 genes. Here, we have identified a functional element displaying the signature of the CDE-CHR in the promoter of the mouse RB2 (p130) gene, encoding the retinoblastoma protein family (pRB)-related protein p130. This element locates close to the major transcription start site where it makes major groove contacts with proteins that can be detected in a cellular context using in vivo genomic footprinting techniques. Inactivation of either the CDE or CHR sequence strongly up-regulates the p130 promoter activity in exponentially growing cells, a situation where endogenous p130 gene expression is almost undetectable. Electrophoretic mobility shift assays suggest that two different protein complexes bind independently to the p130 CDE and CHR elements, and that the protein(s) bound to the CDE might be related to those bound on cyclin A and cdc2 promoters.

Peroxisome proliferator-activated receptor-gamma: from adipogenesis to carcinogenesis.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors, initially described as molecular targets for synthetic compounds inducing peroxisome proliferation. PPAR-gamma, the best characterized of the PPARs, plays a crucial role in adipogenesis and insulin sensitization. Furthermore, PPAR-gamma has been reported to affect cell proliferation/differentiation pathways in various malignancies. We discuss in the present review recent advances in the understanding of the function of PPAR-gamma in both cell proliferation and adipocyte differentiation.

Peroxisome proliferator-activated receptor gamma is induced during differentiation of colon epithelium cells.

Peroxisome proliferator-activated receptor gamma (PPARgamma), a fatty acid-activated nuclear receptor, is implicated in adipocyte differentiation and insulin sensitisation. In view of the association of dietary fat intake and bowel disease, the expression of PPARgamma in rodent and human intestine was studied. Expression of PPARgamma mRNA was examined by Northern blot hybridisation, RNase protection, and/or competitive RT-PCR assays, whereas PPARgamma protein levels were evaluated by immunoblotting and immunohistochemistry. PPARgamma mRNA and protein were abundantly expressed in colon relative to the small intestine both in rodents and in man. Interestingly, expression of PPARgamma was primarily localised in the more differentiated epithelial cells in the colon. The level of expression of PPARgamma in colon was similar to the levels seen in adipose tissue. Expression of PPARgamma increased from proximal to distal segments of the colon in man. In Caco-2 and HT-29 human adenocarcinoma cells, PPARgamma expression increased upon differentiation, consistent with PPARgamma being associated with a differentiated epithelial phenotype. High-level expression of PPARgamma was observed in the colon, but not in the small intestine, suggesting a potential role of this nuclear receptor in the colon.

Metabolic intervention on lipid synthesis converging pathways abrogates prostate cancer growth.

One of the most conserved features of all cancers is a profound reprogramming of cellular metabolism, favoring biosynthetic processes and limiting catalytic processes. With the acquired knowledge of some of these important changes, we have designed a combination therapy in order to force cancer cells to use a particular metabolic pathway that ultimately results in the accumulation of toxic products. This innovative approach consists of blocking lipid synthesis, at the same time that we force the cell, through the inhibition of AMP-activated kinase, to accumulate toxic intermediates, such as malonyl-coenzyme A (malonyl-CoA) or nicotinamide adenine dinucleotide phosphate. This results in excess of oxidative stress and cancer cell death. Our new therapeutic strategy, based on the manipulation of metabolic pathways, will certainly set up the basis for new upcoming studies defining a new paradigm of cancer treatment.

Ciglitazone negatively regulates CXCL1 signaling through MITF to suppress melanoma growth.

We have previously demonstrated that the thiazolidinedione ciglitazone inhibited, independently of PPARγ activation, melanoma cell growth. Further investigations now show that ciglitazone effects are mediated through the regulation of secreted factors. Q-PCR screening of several genes involved in melanoma biology reveals that ciglitazone inhibits expression of the CXCL1 chemokine gene. CXCL1 is overexpressed in melanoma and contributes to tumorigenicity. We show that ciglitazone induces a diminution of CXCL1 level in different human melanoma cell lines. This effect is mediated by the downregulation of microphthalmia-associated transcription factor, MITF, the master gene in melanocyte differentiation and involved in melanoma development. Further, recombinant CXCL1 protein is sufficient to abrogate thiazolidinedione effects such as apoptosis induction, whereas extinction of the CXCL1 pathway mimics phenotypic changes observed in response to ciglitazone. Finally, inhibition of human melanoma tumor development in nude mice treated with ciglitazone is associated with a strong decrease in MITF and CXCL1 levels. Our results show that anti-melanoma effects of thiazolidinediones involve an inhibition of the MITF/CXCL1 axis and highlight the key role of this specific pathway in melanoma malignancy.

Metabolism and proliferation share common regulatory pathways in cancer cells.

Cancer development involves major alterations in cells' metabolism. Enhanced glycolysis and de novo fatty acids synthesis are indeed characteristic features of cancer. Cell proliferation and metabolism are tightly linked cellular processes. Others and we have previously shown a close relationship between metabolic responses and proliferative stimuli. In addition to trigger proliferative and survival signaling pathways, most oncoproteins also trigger metabolic changes to transform the cell. We present herein the view that participation of cell-cycle regulators and oncogenic proteins to cancer development extend beyond the control of cell proliferation, and discuss how these new functions may be implicated in metabolic alterations concomitant to the pathogenesis of human cancers.

Atypical transcriptional regulators and cofactors of PPARgamma.

Regulation of peroxisome proliferator-activated receptor gamma (PPARgamma) activity is the result of several events. The first control level is the regulation of the expression of PPARgamma. Examples of this regulation, during adipogenesis, is the transactivation of the PPARgamma promoter by transcription factors of the classical pathway, such as C/EBPs or ADD1/SREBP1, but also newly identified factors, such as E2Fs. When preadipocytes re-enter the cell cycle, PPARgamma expression is induced coincident with an increase in DNA synthesis, suggesting the involvement of the E2F family of cell cycle regulators. E2F1 induces PPARgamma transcription during clonal expansion, whereas E2F4 represses PPARgamma expression during terminal adipocyte differentiation. Hence, E2Fs represent the link between proliferative signaling pathways, triggering clonal expansion, and terminal adipocyte differentiation through regulation of PPARgamma expression. A second regulatory level of PPARgamma action is interaction with cofactors. We will focus our attention on the atypical PPARgamma modulators. We have described an interaction between PPARgamma and the retinoblastoma protein, RB, which is both dependent upon ligand binding by PPARgamma and upon the phosphorylation status of RB. The interaction between PPARgamma and RB decreases the transcriptional activity of PPARgamma through recruitment of the histone deacetylase HDAC3. Inhibition of HDAC activity consequently results in a strong activation of PPARgamma.