Marcelina Párrizas

Insulin-like growth factor-1 inhibition of apoptosis is associated with increased expression of the bcl-xL gene product.

Differentiated PC12 cells, which become dependent on the presence of growth factors in the media and die by apoptosis after several hours of serum deprivation, were used to test the ability of IGF-1 to regulate the endogenous levels of the death-suppressing protein Bcl-xL, IGF-1 was capable of preventing apoptosis of serum-deprived differentiated PC12 cells at physiological concentrations, with optimal results seen at 10(-8) M. Incubation with the hormone resulted in a significant increase of Bcl-x mRNA after 3-6 h incubation and a doubling of Bcl-xL protein levels by 24 h incubation. Our results show that the protective effect of IGF-1 in PC12 cells is associated with an up-regulation of Bcl-xL mRNA and protein levels.

Specific Inhibition of Insulin-Like Growth Factor-1 and Insulin Receptor Tyrosine Kinase Activity and Biological Function by Tyrphostins

A series of the synthetic protein tyrosine kinase inhibitors known as tyrphostins were studied for their effect on insulin-like growth factor-1 and insulin-stimulated cellular proliferation on NIH-3T3 fibroblasts overexpressing either receptor, as well as for their ability to inhibit ligand-stimulated receptor autophosphorylation and tyrosine kinase activity toward exogenous substrates. Several of the tyrphostins tested demonstrated a dramatic effect by inhibiting hormone-stimulated cell proliferation, with IC50s in the submicromolar range, while being unable to block serum-stimulated cell proliferation. The tyrphostins also inhibited receptor autophosphorylation and tyrosine kinase activity, with a higher IC50, in the micromolar range. Most of the tyrphostins tested presented no clear preference for either receptor, although two of them (AG1024 and AG1034) showed significantly lower IC50s for IGF-1 than for insulin receptors. These results suggest that, in spite of the high homology of the kinase regions...

Histone Demethylase LSD1 Regulates Adipogenesis

Epigenetic mechanisms, in particular the enzymatic modification of histones, are a crucial element of cell differentiation, a regulated process that allows a precursor cell basically to turn into a different cell type while maintaining the same genetic equipment. We have previously described that the promoters of adipogenic genes display significant levels of dimethylation at the Lys4 of histone H3 (H3K4) in preadipocytes, where these genes are still silenced, thus maintaining the chromatin of the precursor cell in a receptive state. Here, we show that the expression of several histone demethylases and methyltransferases increases during adipogenesis, suggesting an important role for these proteins in this process. Knockdown of the H3K4/K9 demethylase LSD1 results in markedly decreased differentiation of 3T3-L1 preadipocytes. This outcome is associated with decreased H3K4 dimethylation and increased H3K9 dimethylation at the promoter of transcription factor cebpa, whose expression must be induced >200-fold upon stimulation of differentiation. Thus, our data suggest that LSD1 acts to maintain a permissive state of the chromatin in this promoter by opposing the action of a H3K9 methyltransferase. Knockdown of H3K9 methyltransferase SETDB1 produced the opposite results, by decreasing H3K9 dimethylation and increasing H3K4 dimethylation levels at the cebpa promoter and favoring differentiation. These findings indicate that the histone methylation status of adipogenic genes as well as the expression and function of the proteins involved in its maintenance play a crucial role in adipogenesis.

Histone H3 lysine 4 dimethylation signals the transcriptional competence of the adiponectin promoter in preadipocytes.

Adipogenesis is regulated by a coordinated cascade of sequence-specific transcription factors and coregulators with chromatin-modifying activities that are between them responsible for the establishment of the gene expression pattern of mature adipocytes. Here we examine the histone H3 post-translational modifications occurring at the promoters of key adipogenic genes during adipocyte differentiation. We show that the promoters of apM1, glut4, gpd1, and leptin are enriched in dimethylated histone H3 Lys4 (H3-K4) in 3T3-L1 fibroblasts, where none of these genes are yet expressed. A detailed study of the apM1 locus shows that H3-K4 dimethylation is restricted to the promoter region in undifferentiated cells and associates with RNA polymerase II (pol II) loading. The beginning of apM1 transcription at the early stages of adipogenesis coincides with promoter H3 hyperacetylation and H3-K4 trimethylation. At the coding region, H3 acetylation and dimethylation, as well as pol II binding, are found in cells at later stages of differentiation, when apM1 transcription reaches its maximal peak. This same pattern of histone modifications is detected in mouse primary preadipocytes and adipocytes but not in a related fibroblast cell line that is not committed to an adipocyte fate. Inhibition of H3-K4 methylation by treatment of 3T3-L1 cells with methylthioadenosine results in decreased apM1 gene expression as well as decreased adipogenesis. Taken together, our data indicate that H3-K4 dimethylation and pol II binding to the promoter of key adipogenic genes are distinguishing marks of cells that have undergone determination to a preadipocyte stage.

Chromatin and chromatin-modifying proteins in adipogenesis.

Long considered scarcely more than an uninteresting energy depot, adipose tissue has recently achieved star status. Far from being mere fat droplets, the adipocytes secrete a number of hormones and bioactive peptides, collectively known as adipokines, which participate in the regulation of a variety of functions, from haemostasis to angiogenesis to energy balance. Adipose tissue constitutes a bona-fide endocrine organ whose main dysfunctions, obesity and lipodystrophy, are related to the development of diabetes, hypertension, or dyslipidemia. The renewed interest in this tissue has prompted an escalation in the number of studies focusing on every aspect of the biology of the adipose cell, in the belief that a detailed knowledge of the mechanisms involved in the differentiation and function of adipocytes may contribute new therapeutical approaches to the treatment of such alarming medical problems. Adipogenesis is the result of an intertwined network of transcription factors and coregulators with chromatin-modifying activities that together, are responsible for the establishment of the gene expression pattern of mature adipocytes. Although the exquisitely regulated transcription factor cascade controlling adipogenesis has been extensively studied, the role of chromatin and chromatin-modifying proteins has become apparent only in recent times.

Circulating miR-192 and miR-193b Are Markers of Prediabetes and Are Modulated by an Exercise Intervention

CONTEXT Diabetes is frequently diagnosed late, when the development of complications is almost inevitable, decreasing the quality of life of patients. However, early detection of affected individuals would allow the implementation of timely and effective therapies. OBJECTIVE Here we set to describe the profile of circulating microRNAs (miRNAs) in prediabetic patients with the intention of identifying novel diagnostic and therapeutic tools. DESIGN We used real-time RT-PCR to measure the abundance of 176 miRNAs in serum of a cohort of 92 control and prediabetic individuals with either impaired fasting glucose or impaired glucose tolerance, as well as newly diagnosed diabetic patients. We validated the results in a second cohort of control and prediabetic subjects undergoing a therapeutic exercise intervention, as well as in a mouse model of glucose intolerance. RESULTS We identified two miRNAs, miR-192 and miR-193b, whose abundance is significantly increased in the prediabetic state but not in diabetic patients. Strikingly, these miRNAs are also increased in plasma of glucose-intolerant mice. Moreover, circulating levels of miR-192 and miR-193b return to baseline in both prediabetic humans and glucose-intolerant mice undergoing a therapeutic intervention consisting in chronic exercise, which succeeded in normalizing metabolic parameters. CONCLUSIONS Our data show that the pattern of circulating miRNAs is modified by defects in glucose metabolism in a similar manner in mice and humans. This circulating miRNA signature for prediabetes could be used as a new diagnostic tool, as well as to monitor response to intervention.

The insulin-like growth factor-I receptor and apoptosis. Implications for the aging progress.

Apoptosis or programmed cell death is a process that is a genetically controlled response for cells to commit suicide, and is orchestrated by mechanisms involving various proteins. The physiological role of apoptosis is to kill unwanted cells. During development of the nervous system for example, a large excess of redundant neurons die off during the period when synapses are being formed between the neurons and their target cells (I). A second important role for apoptosis is as a defense mechanism whereby prevention of the spread of viruses to neighboring cells is achieved (2). It is also critical for the normal functioning of the immune system, where certain cells need to be removed, otherwise they may attack normal cells. In preventing the onset or progression of tumors, apoptosis is the antithesis of cellular proliferation and can impair cellular proliferation (3). Finally, apoptosis is adistinctive feature of the aging process, in which it leads to loss of essential cells and the impairment of normal functioning of specialized organs. Apoptosis is a highly ordered process that may be characterized by nuclear changes including chromatin condensation, fragmentation, and margination as well as internucleosomal DNA cleavage, which gives rise to the characteristic DNA laddering (4). In addition, there is cytoskeletal disruption, shrinkage of the cell and membrane blebbing with membrane-bound apoptotic bodies. The process often results in an undesirable inflammatory response. Insulin-like growth factors (IGF-I and IGF-II), like several other growth factors, prevent apoptosis in a number of different cells and tissues (5-7). The mechanisms invot-

Epigenetic regulation of adipogenesis.

Purpose of reviewEpigenetic regulation plays an essential role in cell differentiation, by allowing the establishment and maintenance of the gene-expression pattern of the mature cell type. Because of its importance in chronic diseases, adipogenesis is one of the best-studied differentiation processes. The hormonal and transcriptional cascades governing the differentiation of the adipocytes are well known, but the role of epigenetic mechanisms is only starting to emerge. In this review, we intend to summarize the recently described epigenetic events that participate in adipogenesis and their connections with the main factors that constitute the classical transcriptional cascade. Recent findingsThe advent of high-throughput technologies has made possible the exhaustive analysis of the epigenetic phenomenons taking place during adipogenesis. The cooperative recruitment of CCAAT/enhancer-binding protein (C/EBP&bgr;) and other early proadipogenic transcription factors to transcription factor hotspots shortly after induction of adipogenesis is required to establish a transient epigenomic state that then informs the recruitment of the later adipogenic transcription factors peroxisome proliferator-activated receptor (PPAR&ggr;) and C/EBP&agr; to their target genes. SummaryEpigenetic marks and chromatin-modifying proteins contribute to adipogenesis and, through regulation of the phenotypic maintenance of the mature adipocytes, to the control of metabolism.

A chromatin perspective of adipogenesis

The transcriptional cascade governing adipogenesis has been thoroughly examined throughout the years. Transcription factors PPARγ and C/EBPα are universally recognized as the master regulators of adipocyte differentiation and together they direct the establishment of the gene expression pattern of mature adipose cells. However, this familiar landscape has been considerably broadened in recent years by the identification of novel factors that participate in the regulation of adipogenesis, either favoring or inhibiting it, through their effects on chromatin. Epigenetic signals and chromatin-modifying proteins contribute to adipogenesis and, through regulation of the phenotypic maintenance of the mature adipocytes, to the control of metabolism. In this review we intend to summarize the recently described epigenetic events that participate in adipogenesis and their connections with the main factors that constitute the classical transcriptional cascade.

Coordinate Functional Regulation between Microsomal Prostaglandin E Synthase-1 (mPGES-1) and Peroxisome Proliferator-activated Receptor γ (PPARγ) in the Conversion of White-to-brown Adipocytes

Background: Microsomal prostaglandin E (PGE) synthase-1 (mPGES-1) is an inducible enzyme with unknown properties in adipose homeostasis. Results: mPGES-1 is necessary for pre-adipocyte differentiation into beige/brite adipocytes through functional interaction with peroxisome proliferator-activated receptor γ (PPARγ). Conclusion: A coordinate interaction between mPGES-1 and PPARγ is required for white-to-brown fat conversion. Significance: Increases in the number of beige cells in fat exerts beneficial metabolic actions. Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated nuclear receptor and a master regulator of adipogenesis. Microsomal prostaglandin E (PGE) synthase-1 (mPGES-1) is an inducible enzyme that couples with cyclooxygenase-2 for the biosynthesis of PGE2. In this study we demonstrate the existence of a coordinate functional interaction between PPARγ and mPGES-1 in controlling the process of pre-adipocyte differentiation in white adipose tissue (WAT). Adipocyte-specific PPARγ knock-out mice carrying an aP2 promoter-driven Cre recombinase transgene showed a blunted response to the adipogenic effects of a high fat diet. Pre-adipocytes from these knock-out mice showed loss of PPARγ and were resistant to rosiglitazone-induced WAT differentiation. In parallel, WAT from these mice showed increased expression of uncoupling protein 1, a mitochondrial enzyme that dissipates chemical energy as heat. Adipose tissue from mice lacking PPARγ also showed mPGES-1 up-regulation and increased PGE2 levels. In turn, PGE2 suppressed PPARγ expression and blocked rosiglitazone-induced pre-adipocyte differentiation toward white adipocytes while directly elevating uncoupling protein 1 expression and pre-adipocyte differentiation into mature beige/brite adipocytes. Consistently, pharmacological mPGES-1 inhibition directed pre-adipocyte differentiation toward white adipocytes while suppressing differentiation into beige/brite adipocytes. This browning effect was reproduced in knockdown experiments using a siRNA directed against mPGES-1. The effects of PGE2 on pre-adipocyte differentiation were not seen in mice lacking PPARγ in adipose tissue and were not mirrored by other eicosanoids (i.e. leukotriene B4). Taken together, these findings identify PGE2 as a key regulator of white-to-brown adipogenesis and suggest the existence of a coordinate regulation of adipogenesis between PPARγ and mPGES-1.