Diana Vargas

Regulation of adipogenesis by nucelar receptor PPARγ is modulated by the histone demethylase JMJD2C

A potential strategy to combat obesity and its associated complications involves modifying gene expression in adipose cells to reduce lipid accumulation. The nuclear receptor Peroxisome Proliferator-activated receptor gamma (PPARγ) is the master regulator of adipose cell differentiation and its functional activation is currently used as a therapeutic approach for Diabetes Mellitus type 2. However, total activation of PPARγ induces undesirable secondary effects that might be set with a partial activation. A group of proteins that produce histone demethylation has been shown to modify the transcriptional activity of nuclear receptors. Here we describe the repressive action of the jumonji domain containing 2C/lysine demethylase 4 C (JMJD2C/KDM4C) on PPARγ transcriptional activation. JMJD2C significantly reduced the rosiglitazone stimulated PPARγ activation. This effect was mainly observed in experiments performed using the Tudor domains that may interact with histone deacetylase class 1 (HDAC) and this interaction probably reduces the mediated activation of PPARγ. Trichostatin A, a HDAC inhibitor, reduces the repressive effect of JMJD2C. When JMJD2C was over-expressed in 3T3-L1 cells, a reduction of differentiation was observed with the Tudor domain. In summary, we herein describe JMJD2C-mediated reduction of PPARgamma transcriptional activation as well as preadipocyte differentiation. This novel action of JMJD2C might have an important role in new therapeutic approaches to treat obesity and its complications.

Biology of Beige Adipocyte and Possible Therapy for Type 2 Diabetes and Obesity

All mammals own two main forms of fat. The classical white adipose tissue builds up energy in the form of triglycerides and is useful for preventing fatigue during periods of low caloric intake and the brown adipose tissue instead of inducing fat accumulation can produce energy as heat. Since adult humans possess significant amounts of active brown fat depots and their mass inversely correlates with adiposity, brown fat might play an important role in human obesity and energy homeostasis. New evidence suggests two types of thermogenic adipocytes with distinct developmental and anatomical features: classical brown adipocytes and beige adipocytes. Beige adipocyte has recently attracted special interest because of its ability to dissipate energy and the possible ability to differentiate itself from white adipocytes. Importantly, adult human brown adipocyte appears to be mainly composed of beige-like adipocytes, making this cell type an attractive therapeutic target for obesity and obesity-related diseases. Because many epigenetic changes can affect beige adipocyte differentiation, the knowledge of the circumstances that affect the development of beige adipocyte cells may be important for therapeutic strategies. In this review we discuss some recent observations arising from the great physiological capacity of these cells and their possible role as ways to treat obesity and diabetes mellitus type 2.

EID1-induces brown-like adipocyte traits in white 3T3-L1 pre-adipocytes.

PPARgamma and pRB play an important role in the development of adipose cells, and functional modification of these proteins may lead to beneficial changes in adipose cell physiology. In the present work, we show that over-expression of EID1 (E1A-like inhibitor of differentiation), an inhibitor of muscle cell differentiation, reduces PPARgamma ligand-dependent transactivation and decreases triglyceride stores in pre-adipocytes (3T3-L1 cells). Additionally, we found that EID1 binds to pRB at the onset of adipocyte differentiation and may act to reduce pRB levels. Over-expression of EID1 in 3T3-L1 cells leads to increased expression of UCP1 and PGC-1alpha, both of which are involved in caloric dissipation and thermogenesis, in brown adipose tissue. These results indicate that EID1 is able to reduce fat accumulation in adipose cells and induce expression of brown fat genes in pre-adipocytes (3T3-L1 cells) normally destined to become white fat cells. The functional reduction of PPARgamma and pRB mediated by EID1 in adipose cells may play an important role in insulin resistance and the metabolic syndrome.

Modifications of Human Subcutaneous ADMSC after PPARγ Activation and Cold Exposition

Mesenchymal stem cells are a diverse population of cells with a wide range of potential therapeutic applications. In particular, cells from adipose tissue have the distinction of being easily accessible and contain a lot of stem cells. ADMSCs can be induced to mature adipocyte and activate the energy expenditure upon treatment with total PPARγ agonists. Additionally these cells may respond to cold by activating the thermogenic program. In the present study, we determined the effect of partial agonism of PPARγ and temperature reduction on phenotype and metabolic activity of ADMSCs from human adipose subcutaneous tissue. We found that adipocytes differentiated with total and partial agonists of PPARγ and exposed to 31°C are able to respond to cold significantly increasing the expression of thermogenic proteins such as UCP1, PGC1α, and CITED1, a marker of beige phenotype. Additionally, we found that adipocyte cells subjected to cold had a reduction in triglycerides and increased adiponectin levels. These data confirm the promising role of ADMSCs as a treatment for metabolic disorders since it is possible to induce them to mature adipocytes and modulate their phenotype toward a cell with high-energy expenditure and metabolic beneficial effect.

Diverse coactivator recruitment through differential PPARγ nuclear receptor agonism

The PPARγ nuclear receptor regulates the expression of genes involved in lipid and carbohydrate metabolism, and it has protective effects in some patients with type 2 diabetes. Nevertheless, the therapeutic value of the PPARγ nuclear receptor protein is limited due to the secondary effects of some PPARγ ligands. Because the downstream effects of PPARγ are determined by the binding of specific cofactors that are mediated by ligand-induced conformational changes, we evaluated the differential effects of various ligands on the binding of certain cofactors associated with PPARγ. The ligands used were rosiglitazone for treating type 2 diabetes and telmisartan for treating arterial hypertension. Functional, phenotypic, and molecular studies were conducted on pre-adipocyte 3T3-L1 and functional studies in U2OS cells. The moderating influence of various cofactor families was evaluated using transient transfection assays. Our findings confirm that telmisartan has a partial modulating effect on PPARγ activity compared to rosiglitazone. The cofactors SRC1 and GRIP1 mediate the activity of telmisartan and rosiglitazone and partially determine the difference in their effects. Studying the modulating activity of these cofactors can provide interesting insights for developing new therapeutic approaches for certain metabolic diseases.

Regulation of human subcutaneous adipocyte differentiation by EID1

Increasing thermogenesis in white adipose tissues can be used to treat individuals at high risk for obesity and cardiovascular disease. The objective of this study was to determine the function of EP300-interacting inhibitor of differentiation (EID1), an inhibitor of muscle differentiation, in the induction of beige adipocytes from adipose mesenchymal stem cells (ADMSCs). Subcutaneous adipose tissue was obtained from healthy women undergoing abdominoplasty. ADMSCs were isolated in vitro, grown, and transfected with EID1 or EID1 siRNA, and differentiation was induced after 48 h by administering rosiglitazone. The effects of EID1 expression under the control of the aP2 promoter (aP2-EID1) were also evaluated in mature adipocytes that were differentiated from ADMSCs. Transfection of EID1 into ADMSCs reduced triglyceride accumulation while increasing levels of thermogenic proteins, such as PGC1α, TFAM, and mitochondrial uncoupling protein 1 (UCP1), all of which are markers of energy expenditure and mitochondrial activity. Furthermore, increased expression of the beige phenotype markers CITED1 and CD137 was observed. Transfection of aP2-EID1 transfection induced the conversion of mature white adipocytes to beige adipocytes, as evidenced by increased expression of PGC1α, UCP1, TFAM, and CITED1. These results indicate that EID1 can modulate ADMSCs, inducing a brown/beige lineage. EID1 may also activate beiging in white adipocytes obtained from subcutaneous human adipose tissue.

Functional Characterization of Preadipocytes Derived from Human Periaortic Adipose Tissue

Adipose tissue can affect the metabolic control of the cardiovascular system, and its anatomic location can affect the vascular function differently. In this study, biochemical and phenotypical characteristics of adipose tissue from periaortic fat were evaluated. Periaortic and subcutaneous adipose tissues were obtained from areas surrounding the ascending aorta and sternotomy incision, respectively. Adipose tissues were collected from patients undergoing myocardial revascularization or mitral valve replacement surgery. Morphological studies with hematoxylin/eosin and immunohistochemical assay were performed in situ to quantify adipokine expression. To analyze adipogenic capacity, adipokine expression, and the levels of thermogenic proteins, adipocyte precursor cells were isolated from periaortic and subcutaneous adipose tissues and induced to differentiation. The precursors of adipocytes from the periaortic tissue accumulated less triglycerides than those from the subcutaneous tissue after differentiation and were smaller than those from subcutaneous adipose tissue. The levels of proteins involved in thermogenesis and energy expenditure increased significantly in periaortic adipose tissue. Additionally, the expression levels of adipokines that affect carbohydrate metabolism, such as FGF21, increased significantly in mature adipocytes induced from periaortic adipose tissue. These results demonstrate that precursors of periaortic adipose tissue in humans may affect cardiovascular events and might serve as a target for preventing vascular diseases.

Role of Histone Demethylases in Cardiomyocytes Induced to Hypertrophy

Epigenetic changes induced by histone demethylases play an important role in differentiation and pathological changes in cardiac cells. However, the role of the jumonji family of demethylases in the development of cardiac hypertrophy remains elusive. In this study, the presence of different histone demethylases in cardiac cells was evaluated after hypertrophy was induced with neurohormones. A cell line from rat cardiomyocytes was used as a biological model. The phenotypic profiles of the cells, as well as the expression of histone demethylases, were studied through immunofluorescence, transient transfection, western blot, and qRT-PCR analysis after inducing hypertrophy by angiotensin II and endothelin-1. An increase in fetal gene expression (ANP, BNP, and β-MHC) was observed in cardiomyocytes after treatment with angiotensin II and endothelin-1. A significant increase in JMJD2A expression, but not in UTX or JMJD2C expression, was observed. When JMJD2A was overexpressed in cardiomyocytes through transient transfection, the effect of neurohormones on fetal cardiac gene expression was increased. We conclude that JMJD2A plays a principal role in the regulation of fetal cardiac genes, which increase in expression during the pathological hypertrophic process.

Modulation of thyroid hormone receptor transactivation by the early region 1A (E1A)-like inhibitor of differentiation 1 (EID1)

Transcriptional activation (TA) mediated by the effect of thyroid hormones on target genes requires co-activator proteins such as the early region 1A (E1A) associated 300 kDa binding protein (p300) and the cAMP response element binding protein (CREB) binding protein (CBP), known as the p300/CBP complex, which acetylate histones 3 and 4 to allow transcriptional machinery access to the target gene promoter. Little is known on the role of p300 in thyroid hormone receptor (TR) mediated TA but the E1A-like inhibitor of differentiation 1 (EID1), an inhibitor of p300 histone acetyltransferase (HAT), is a functional homolog of E1A and may inhibit myogenic differentiation factor D (MyoD) transcriptional activity and reduces muscle cell differentiation. We evaluated the influence of EID1 on TR-mediated transcriptional activity using transfection and mammalian two-hybrid studies to show that EID1 may partially reduces TA activity of the TR receptor, probably due to p300 blockage since EID1 mutants cannot reduce TR-mediated TA. The EID1 does not affect the function of p160 co-activator proteins (160 kDa proteins of steroid receptor co-activators) and is functionally independent of co-repressor proteins or TR binding. Summarizing, EID1 reduces TR-mediated transcriptional activity by blocking p300 and may play an important role in thyroid receptor activity in muscle and other tissues.

Human ADMC-Derived Adipocyte Thermogenic Capacity Is Regulated by IL-4 Receptor

Type two innate immune system is anti-inflammatory and may play an important role as the means whereby “browning” is induced in subcutaneous adipocytes. It was shown that IL-4 may influence the fate of adipose cell precursors by promoting differentiation towards more thermogenic adipocytes in mice. Here, we investigated the influence of IL-4 and IL-4 receptor, a type two immune cytokine pathway, on the metabolic activity and thermogenic potential of human adipocytes differentiated from adipose-derived mesenchymal stem cells (ADMSCs) obtained from subcutaneous samples of healthy women undergoing abdominoplasty. Western blot analysis, qPCR, and biochemical analyses were performed 10 days after ADMSC differentiation into mature adipocytes was induced. IL-4 receptor was expressed in both precursor and differentiated adipocytes, and IL-4 treatment increased phosphorylation Y641 of signal transducer and activator of transcription 6 (STAT6) in both cell types. IL-4 treatment also increased expression of thermogenic proteins PGC-1α, UCP-1, and CITED1. In addition, IL-4 increased the secretion of adiponectin, leptin, and FGF21 and promoted lipolysis in differentiated adipocytes. In conclusion, IL-4 may directly modulate differentiation of human adipocytes towards a beige phenotype acting through IL-4 receptors on both adipose precursors and differentiated human adipocytes, metabolic effect that must be considered in some antiallergic drugs.