Adipogenic Process Research Articles

Antiadipogenic effects of subthermal electric stimulation at 448kHz on differentiating human mesenchymal stem cells.

The 448 kHz capacitive-resistive electric transfer (CRET) is an electrothermal therapy currently applied in anticellulite and antiobesity treatments. The aim of the present study was to determine whether exposure to the CRET electric signal at subthermal doses affected early adipogenic processes in adipose-derived stem cells (ADSC) from human donors. ADSC were incubated for 2 or 9 days in the presence of adipogenic medium, and exposed or sham-exposed to 5 min pulses of 448 kHz electric signal at 50 µA/mm2 during the last 48 h of the incubation. Colorimetric, immunofluorescence, western blotting and reverse transcription-quantitative polymerase chain reaction assays were performed to assess adipogenic differentiation of the ADSC. Electric stimulation significantly decreased cytoplasmic lipid content, after both 2 and 9 days of differentiation. The antiadipogenic response in the 9 day samples was accompanied by activation of mitogen-activated protein kinase kinase 1/2, decreased expression and partial inactivation of peroxisome proliferator-activated receptor (PPAR) γ, which was translocated from the nucleus to the cytoplasm, together with a significant decrease in the expression levels of the PPARG1 gene, perilipin, angiopoietin-like protein 4 and fatty acid synthase. These results demonstrated that subthermal stimulation with CRET interferes with the early adipogenic differentiation in ADSC, indicating that the electric stimulus itself can modulate processes controlling the synthesis and mobilization of fat, even in the absence of the concomitant thermal and mechanical components of the thermoelectric therapy CRET.

Timcodar (VX-853) Is a Non-FKBP12 Binding Macrolide Derivative That Inhibits PPARγ and Suppresses Adipogenesis.

Nutrient overload and genetic factors have led to a worldwide epidemic of obesity that is the underlying cause of diabetes, atherosclerosis, and cardiovascular disease. In this study, we used macrolide drugs such as FK506, rapamycin, and macrolide derived, timcodar (VX-853), to determine their effects on lipid accumulation during adipogenesis. Rapamycin and FK506 bind to FK506-binding proteins (FKBPs), such as FKBP12, which causes suppression of the immune system and inhibition of mTOR. Rapamycin has been previously reported to inhibit the adipogenic process and lipid accumulation. However, rapamycin treatment in rodents caused immune suppression and glucose resistance, even though the mice lost weight. Here we show that timcodar (1 μM), a non-FKBP12-binding drug, significantly (p < 0.001) inhibited lipid accumulation during adipogenesis. A comparison of the same concentration of timcodar (1 μM) and rapamycin (1 μM) showed that both are inhibitors of lipid accumulation during adipogenesis. Importantly, timcodar potently (p < 0.01) suppressed transcriptional regulators of adipogenesis, PPARγ and C/EBPα, resulting in the inhibition of genes involved in lipid accumulation. These studies set the stage for timcodar as a possible antiobesity therapy, which is rapidly emerging as a pandemic.

A Member of the Nuclear Receptor Superfamily, Designated as NR2F2, Supports the Self-Renewal Capacity and Pluripotency of Human Bone Marrow-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells are characterized with self-renewal capacity and pluripotency. NR2F2 is a nuclear receptor that has been detected in the mesenchymal compartment of developing organs. However, whether NR2F2 plays a role in the stemness maintenance of mesenchymal stem cells has not been explored yet. In this study, we investigated the function of NR2F2 in bone marrow-derived mesenchymal stem cells via shRNA-mediated knock-down of NR2F2. The suppression of NR2F2 impaired the colony-forming efficacy of mesenchymal stem cells. The inhibition of colony-forming capacity may be attributed to the acceleration of senescence through upregulation of P21 and P16. The downregulation of NR2F2 also suppressed both osteogenic and adipogenic differentiation processes. In conclusion, NR2F2 plays an important role in the stemness maintenance of bone marrow-derived mesenchymal stem cells.

Effects of high glucose on caveolin-1 and insulin signaling in 3T3-L1 adipocytes

ABSTRACTAdipocytes exposed to high glucose concentrations exhibit impaired metabolic function, including an increase of oxidative and proinflammatory factors that might favor the development of insulin resistance. Caveolin-1 (Cav-1) is a key mediator of the insulin transduction pathway whose expression is significantly enhanced during adipocyte differentiation. In this work, we studied the effects of high glucose concentration on the regulation of Cav-1 expression and activation and its relation to the insulin signaling pathway during the adipogenic process and in long-term differentiated adipocytes. Both, long-term high glucose exposure during adipogenesis and short-term glucose incubation of mature adipocytes, promoted triglyceride accumulation in 3T3-L1 cells. The short-term exposure of mature adipocytes to high glucose significantly reduced the sensitivity to insulin of Cav-1, insulin receptor (IR) and potein kinase B (AKT-2) phosphorylation, as well as insulin-induced deoxyglucose uptake. Adipocytes differentiated in the presence of high glucose lost Cav-1 and IR response to insulin-stimulated phosphorylation, but maintained the insulin sensitivity of AKT-2 phosphorylation and deoxyglucose uptake. Although long-term high glucose exposure increased DNA methylation in Cav-1 promoter, Cav-1 expression was not affected. Moreover, these cells showed an increase of Cav-1, IR and AKT-2 protein content, pointing to an adaptive response induced by the long-term high glucose exposure.

Dual Regulatory Role of Polyamines in Adipogenesis

Adipogenesis is a complex process, accompanied by a chain of interdependent events. Disruption of key events in this cascade may interfere with the correct formation of adipose tissue. Polyamines were demonstrated necessary for adipogenesis; however, the underlying mechanism by which they act has not been established. Here, we examined the effect of polyamine depletion on the differentiation of 3T3-L1 preadipocytes. Our results demonstrate that polyamines are required early in the adipogenic process. Polyamine depletion inhibited the second division of the mitotic clonal expansion (MCE), and inhibited the expression of PPARγ and C/EBPα, the master regulators of adipogenesis. However, it did not affect the expression of their transcriptional activator, C/EBPβ. Additionally, polyamine depletion resulted in elevation of mRNA and protein levels of the stress-induced C/EBP homologous protein (CHOP), whose dominant negative function is known to inhibit C/EBPβ DNA binding activity. Conditional knockdown of CHOP in polyamine-depleted preadipocytes restored PPARγ and C/EBPα expression, but failed to recover MCE and differentiation. Thus, our results suggest that the need for MCE in the adipogenic process is independent from the requirement for PPARγ and C/EBPα expression. We conclude that de novo synthesis of polyamines during adipogenesis is required for down-regulation of CHOP to allow C/EBPβ activation, and for promoting MCE.

A Bioactive Constituent of Ginger, 6-Shogaol, Prevents Adipogenesis and Stimulates Lipolysis in 3T3-L1 Adipocytes

Zingiber officinale Roscoe, one of the most widely used spices, has been reported to have anti-obesity and anti-diabetes effects. In the present study, we investigated the effects of 6-shogaol, a bioactive compound present in ginger, on the adipogenic process in 3T3-L1 preadipocytes. The anti-adipogenic effects of 6-shogaol was significantly higher than the more widely investigated 6-gingerol, another major ginger constituent. We observed that 6-shogaol inhibited the expression of two master regulators of adipogenesis, PPARγ and C/EBPα, and also stimulated lipolysis in mature 3T3-L1 adipocytes. Collectively, these results suggest that 6-shogaol, not 6-gingerol, is the major compound present in ginger responsible for its reported anti-adipogenic properties. Practical Applications Ginger is widely consumed all over the world, and has been associated with various health benefits. At least some of these benefits have been previously attributed to 6-gingerol. In the present study, we observed that 6-shogaol has more potent anti-adipogenic effects than 6-gingerol in 3T3-L1 cells. This is the first study to investigate the anti-obesity effect of 6-shogaol in vitro, and provides a new perspective on future development of ginger-based anti-obesity strategies.

Dickkopf-1 promotes the differentiation and adipocytokines secretion via canonical Wnt signaling pathway in primary cultured human preadipocytes

Dickkopf-1, a newly recognized antagonist for canonical Wnt signaling, is secreted in the early stage of human adipose-derived stem cells (ASCs) adipogenic differentiation. This study was aimed to investigate whether human recombinant DKK-1 (rhDKK-1) could affect the differentiation and metabolism as well as adipocytokines secretion in primary cultured human ASCs. Human ASCs were isolated from omental adipose tissue and induced to adipogenic differentiation in the absence or presence of Wnt signaling antagonist rhDKK-1 and agonist SB216763, respectively. mRNA and protein expression profiles of adipogenic factors during the differentiation process were analyzed using quantitative RT-PCR and Western blotting. Adipocytokines secretion levels in the culture medium were measured by ELISA method. Our results showed that DKK-1 was already expressed during the early stage of adipogenesis and reached the peak on the 9th day. Exogenous rhDKK-1 exposure accelerated the differentiation by up-regulating PPAR-γ and C/EBP-α, down-regulating Wnt3a, Wnt10b and β-catenin, without affecting non-canonical Wnt signaling marker (Wnt5a). In addition, rhDKK-1 treatment increased the secretion of leptin, RBP4, TNF-α and adiponectin during differentiation. rhDKK-1 treatment also significantly increased the intracellular accumulation of lipids and lipolysis. Thus, Wnt signal pathway agonist SB216763 down-regulated DKK-1 transcriptional and secretion levels during adipogenic process. Our results suggest that rhDKK-1 could promote ASCs differentiation and increase adipocytokines secretion via canonical Wnt signaling pathway.

The critical and specific transcriptional regulator of the microenvironmental niche for hematopoietic stem and progenitor cells.

It has been assumed that the special microenvironments known as niches in the marrow play an essential role in maintaining hematopoietic stem and progenitor cells (HSPCs), and the identity of the HSPC niche has been a subject of long-standing debate. Recent studies identified cells, which create microenvironments meeting the criteria for HSPC niches and the critical transcriptional regulators of their development and maintenance. Osterix as well as Ebf2 and Bmi1 are critical but not specific transcriptional regulators of HSPC niche development. The transcription factor Foxc1 is expressed preferentially in a population of adipo-osteogenic progenitors, termed CXCL12-abundant reticular (CAR) cells, which create HSPC niches and are largely equivalent to stem cell factor and Lepr-expressing cells, in developing and adult bone marrow. Foxc1 is essential for CAR cell development and maintenance of bone marrow niches for HSPCs upregulating CXCL12 and SCF expression and inhibition of adipogenic processes in CAR cell progenitors. Foxc1 is the first critical and specific transcriptional regulator that is required for development and maintenance of cells creating HSPC niches, including a specialized population of adipo-osteogenic progenitors in bone marrow.

Di and tripeptides from marine sources can target adipogenic process and contribute to decrease adipocyte number and functions

The effect of 11 marine-derived cryptides was investigated on proliferation, differentiation and maturation of human white pre-adipocytes (HWP). They were all formerly identified as potent Angiotensin-Converting-Enzyme inhibitors. Val-Trp (VW), Val-Tyr (VY), Lys-Tyr (KY), Lys-Trp (KW), Ile-Tyr (IY), Ala-Pro (AP), Val-Ile-Tyr (VIY), Leu-Lys-Pro (LKP), Gly-Pro-Leu (GPL), Ala-Lys-Lys (AKK) and Val-Ala-Pro (VAP) were previously found in fish products and co-products as well as other marine resources like wakame. Treatment with AP, VAP and AKK greatly affected viability of HWP during the proliferation period while KW and VW treatment reduced the number of viable cells during the differentiation stage. A GPL and IY incubation during the differentiation stage allowed the decrease of their final lipid content, of the GPDH activity and of the mRNA level of adipocyte markers (aP2, GLUT4, LPL and AGT). Moreover, a down regulation of both PPARγ and C/EBPα expression, two key regulators of adipogenesis, was observed. These findings indicate that small bioactive peptides from marine protein hydrolysates can target adipogenesis and thus could regulate energy metabolism disorders.

Osteoporosis-associated alteration in the signalling status of BMP-2 in human MSCs under adipogenic conditions.

Postmenopausal osteoporosis is characterized by decreased bone quality and mineral density. Mesenchymal stem cells (MSCs) found in the bone marrow, are pluripotent cells able to differentiate into several phenotypes, including osteoblasts and adipocytes. In osteoporosis, MSCs' commitment and differentiation into osteoblast/adipocyte is unbalanced, favoring adipocyte formation. The osteo and adipogenic processes are modulated by the bone morphogenetic protein-2 (BMP-2). This cytokine regulates the expression of transcription factors PPARγ and Runx 2, but its action on cells under adipogenic conditions is poorly understood. In this work we studied BMP-2 signaling in MSCs obtained from bone marrow of control or osteoporotic volunteer postmenopausal women. MSCs were cultured under basal, adipogenic (AD) or AD plus BMP-2 conditions. The protein content of PPARγ, p-PPARγ, Runx2, bone morphogenetic receptor IA (BMPR IA), phosphorylated Smad-1/5/8 (p-Smad) and Smad 4 were determined by specific western blots. mRNA level for BMPRs was determined by PCR and cell localization of p-Smad-1/5/8 were detected by immunocytochemistry. Control MSCs showed a differential response to both AD and AD plus BMP-2 treatments: BMP-2 exerted an anti-adipogenic effect increasing both transcription factors analyzed. Moreover, p-Smads-1/5/8 were detected in nuclei after short term BMP-2 treatment. Osteoporotic MSCs showed no response to exogenous added BMP-2, as shown by p-PPARγ/PPARγ ratio and Runx2 levels, although BMPR-IA level was significantly higher in osteoporotic than in control MSCs. In addition, staining for p-Smad-1/5/8 in o-MSCs was observed around nuclei at all experimental conditions. Taken together results demonstrate failure of BMP-2 signaling in osteoporotic MSCs.

Lithium Chloride Modulates Adipogenesis and Osteogenesis of Human Bone Marrow-Derived Mesenchymal Stem Cells

Background/Aims: Lithium chloride (LiCl) has long been used as a psychiatric medication; however, its role in the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) remains largely unknown. The aim of this study is to explore the effect of LiCl on the differentiation of BMSCs. Methods: The roles of LiCl in osteogenic and adipogenic processes were observed using alizarin red staining and oil red O staining, respectively. The effects of LiCl on the Wnt and Hedgehog (Hh) pathways were investigated. Results: Our data showed that LiCl effectively promoted osteogenesis and inhibited adipogenesis by simultaneously affecting the Wnt and Hh pathways. Conclusion: These results suggest that LiCl influences the differentiation of BMSCs directly through the Wnt and Hh pathways and thus may be a candidate drug for the treatment of osteoporosis.

Metformin enhances the anti-adipogenic effects of atorvastatin via modulation of STAT3 and TGF-β/Smad3 signaling

Adipocyte accumulation is associated with the development of obesity and obesity-related diseases. Interactions of master transcription factors and signaling cascades are required for adipogenesis. Regulation of excessive adipogenic processes may be an attractive therapeutic for treatment of obesity and obesity-related diseases. In this study, we found that atorvastatin exerts an anti-adipogenic activity in 3T3-L1 pre-adipocytes, and that this activity is elevated in combination with metformin. Expression of the adipogenic master regulators PPARγ and C/EBPα, and their target gene aP2, was suppressed by atorvastatin. Furthermore, atorvastatin treatment resulted in increased activation of the key master regulator of cellular energy homeostasis, AMPK. These biological activities of atorvastatin were elevated in combination with metformin. These anti-adipogenic activities were associated with regulation of the STAT3 and TGF-β signaling cascades, resulting in the regulation of the expression of STAT3 target genes, such as KLF5, p53, and cyclin D1, and TGF-β signaling inhibitory genes, such as SMAD7. Our results suggest that combination therapy with atorvastatin and metformin may have therapeutic potential for the treatment of obesity and obesity-related diseases caused by excessive adipogenesis.

Prostaglandin E2 signals white-to-brown adipogenic differentiation

The formation of new adipocytes from precursor cells is a crucial aspect of normal adipose tissue function. During the adipogenic process, adipocytes differentiated from mesenchymal stem cells give rise to two main types of fat: white adipose tissue (WAT) characterized by the presence of adipocytes containing large unilocular lipid droplets, and brown adipose tissue (BAT) composed by multilocular brown adipocytes packed with mitochondria. WAT is not only important for energy storage but also as an endocrine organ regulating whole body homeostasis by secreting adipokines and other mediators, which directly impact metabolic functions in obesity. By contrast, BAT is specialized in dissipating energy in form of heat and has salutary effects in combating obesity and associated disorders. Unfortunately, WAT is the predominant fat type, whereas BAT is scarce and located in discrete pockets in adult humans. Luckily, another type of brown adipocytes, called beige or brite (brown-in-white) adipocytes, with similar functions to those of “classical” brown adipocytes has recently been identified in WAT. In this review, a close look is given into the role of bioactive lipid mediators in the regulation of adipogenesis, with a special emphasis on the role of the microsomal prostaglandin E (PGE) synthase-1, a terminal enzyme in PGE2 biosynthesis, as a key regulator of white-to-brown adipogenesis in WAT.

Relationship between impaired adipogenesis of retroperitoneal adipose tissue and hypertrophic obesity: role of endogenous glucocorticoid excess

Although the pro-adipogenic effect of glucocorticoid (GC) on adipose tissue (AT) precursor cell differentiation is openly accepted, the effect of chronically high peripheral levels of GC on AT mass expansion is not fully understood. In the present study, we aim to assess the in vitro adipogenic capacity of AT precursor cells isolated from retroperitoneal (RP) AT pads of the hypercorticosteronaemic, adult neonatally treated monosodium L-glutamate (MSG) male rat. To ascertain this issue, we explored the in vitro adipogenic process of stromal-vascular fraction (SVF) cells isolated from RPAT pads of 60-day-old MSG rats. The data recorded indicated that RPAT-SVF cells from hypercorticosteronaemic MSG rats, although displaying an enhanced proliferation capacity, differentiated slower than normal cells. This dysfunction was associated with a reduction in key parameters indicative of precursor cell commitment, differentiation capacity and the percentage of fully differentiated adipocytes, with a retarded maturation process. The distorted adipogenic capacity was highly conditioned by RPAT-SVF cells displaying a low committed population and both excessive and reduced expression of anti- (Pref-1 and Wnt-10b) and pro-adipogenic (mineralocorticoid receptor) signals respectively. Notably, the normalization of peripheral corticosterone levels in MSG rats, as a result of bilateral adrenalectomy combined with GC replacement therapy, fully prevented reduced RPAT precursor cell commitment and overall impaired adipogenesis. Our study strongly supports that the impaired adipogenic process observed in the adult hypertrophic obese MSG male rat is a GC-dependent mechanism, thus explaining the unhealthy RPAT expansion observed in human hypertrophic obese phenotypes, such as in the Cushing's syndrome.

SRSF10 Regulates Alternative Splicing and Is Required for Adipocyte Differentiation

During adipocyte differentiation, significant alternative splicing changes occur in association with the adipogenic process. However, little is known about roles played by splicing factors in this process. We observed that mice deficient for the splicing factor SRSF10 exhibit severely impaired development of subcutaneous white adipose tissue (WAT) as a result of defects in adipogenic differentiation. To identify splicing events responsible for this, transcriptome sequencing (RNA-seq) analysis was performed using embryonic fibroblast cells. Several SRSF10-affected splicing events that are implicated in adipogenesis have been identified. Notably, lipin1, known as an important regulator during adipogenesis, was further investigated. While lipin1β is mainly involved in lipogenesis, its alternatively spliced isoform lipin1α, generated through the skipping of exon 7, is primarily required for initial adipocyte differentiation. Skipping of exon 7 is controlled by an SRSF10-regulated cis element located in the constitutive exon 8. The activity of this element depends on the binding of SRSF10 and correlates with the relative abundance of lipin1α mRNA. A series of experiments demonstrated that SRSF10 controls the production of lipin1α and thus promotes adipocyte differentiation. Indeed, lipin1α expression could rescue SRSF10-mediated adipogenic defects. Taken together, our results identify SRSF10 as an essential regulator for adipocyte differentiation and also provide new insights into splicing control by SRSF10 in lipin1 pre-mRNA splicing.

Prostaglandin F2α Inhibits Adipogenesis Via an Autocrine‐Mediated Interleukin‐11/Glycoprotein 130/STAT1‐Dependent Signaling Cascade

Prostaglandin F2α (PGF2α) is a potent inhibitor of adipocyte differentiation in vitro, that has also recently been implicated in the regulation of the adipogenic process in vivo, by opposing adipose tissue accretion and the subsequent development of obesity and its attendant metabolic consequences. In previous studies, we have demonstrated that PGF2α inhibits adipocyte differentiation by means of a calcium-dependent signaling pathway that is critically dependent upon the activity of the calcineurin phosphatase. In the current study, we have now extended these findings to further elucidate the mechanism by which the PGF2α/calcineurin-pathway inhibits the adipogenic process. We now report that the IL-11 cytokine, a member of the gp130 cytokine co-receptor-related family, is a downstream transcriptional target of this pathway in 3T3-L1 preadipocytes and is actively secreted in differentiating cells in response to PGF2α stimulation. Using a combined shRNA and dominant-negative receptor mutant approach, we provide evidence that IL-11/gp130-signaling is required to mediate the inhibitory effects of PGF2α on adipogenesis. Moreover, by taking advantage of a well-characterized panel of chimeric gp130 mutant receptors, we demonstrate that gp130 signaling is sufficient to inhibit adipocyte differentiation and specifically requires the activation of the STAT1 transcription factor. Conversely, we find that depleting endogenous STAT1 levels rescues adipogenesis in the presence of both IL-11/gp130 signaling and PGF2α. Collectively, our findings support a model in which PGF2α inhibits adipocyte differentiation by means of an IL-11 mediated autocrine negative feedback loop, that acts via gp130 to block adipogenesis through the essential actions of the STAT1 transcription factor.

WNT Signaling Suppression in the Senescent Human Thymus

Human thymus is completely developed in late fetal stages and its function peaks in newborns. After the first year of life, the thymus undergoes a progressive atrophy that dramatically decreases de novo T-lymphocyte maturation. Hormonal signaling and changes in the microRNA expression network are identified as underlying causes of human thymus involution. However, specific pathways involved in the age-related loss of thymic function remain unknown. In this study, we analyzed differential gene-expression profile and microRNA expression in elderly (70 years old) and young (less than 10 months old and 11 years old) human thymic samples. Our data have shown that WNT pathway deregulation through the overexpression of different inhibitors by the nonadipocytic component of the human thymus stimulates the age-related involution. These results are of particular interest because interference of WNT signaling has been demonstrated in both animal models and in vitro studies, with the three major hallmarks of thymic involution: (i) epithelial structure disruption, (ii) adipogenic process, and (iii) thymocyte development arrest. Thus, our results suggest that secreted inhibitors of the WNT pathway could be explored as a novel therapeutical target in the reversal of the age-related thymic involution.

Foxc1 is a critical regulator of haematopoietic stem/progenitor cell niche formation

Haematopoietic stem and progenitor cells are maintained by special microenvironments known as niches in bone marrow. Many studies have identified diverse candidate cells that constitute niches for haematopoietic stem cells in the marrow, including osteoblasts, endothelial cells, Schwann cells, α-smooth muscle actin-expressing macrophages and mesenchymal progenitors such as CXC chemokine ligand (CXCL)12-abundant reticular (CAR) cells, stem cell factor-expressing cells, nestin-expressing cells and platelet-derived growth factor receptor-α (PDGFR-α)(+)Sca-1(+)CD45(-)Ter119(-) (PαS) cells. However, the molecular basis of the formation of the niches remains unclear. Here we find that the transcription factor Foxc1 is preferentially expressed in the adipo-osteogenic progenitor CAR cells essential for haematopoietic stem and progenitor cell maintenance in vivo in the developing and adult bone marrow. When Foxc1 was deleted in all marrow mesenchymal cells or CAR cells, from embryogenesis onwards, osteoblasts appeared normal, but haematopoietic stem and progenitor cells were markedly reduced and marrow cavities were occupied by adipocytes (yellow adipose marrow) with reduced CAR cells. Inducible deletion of Foxc1 in adult mice depleted haematopoietic stem and progenitor cells and reduced CXCL12 and stem cell factor expression in CAR cells but did not induce a change to yellow marrow. These data suggest a role for Foxc1 in inhibiting adipogenic processes in CAR progenitors. Foxc1 might also promote CAR cell development, upregulating CXCL12 and stem cell factor expression. This study identifies Foxc1 as a specific transcriptional regulator essential for development and maintenance of the mesenchymal niches for haematopoietic stem and progenitor cells.

Peroxisome Proliferator-activated Receptor γ Regulates Genes Involved in Insulin/Insulin-like Growth Factor Signaling and Lipid Metabolism during Adipogenesis through Functionally Distinct Enhancer Classes

The nuclear receptor peroxisome proliferator-activated receptor (PPAR) is a transcription factor whose expression is induced during adipogenesis and that is required for the acquisition and control of mature adipocyte functions. Indeed, PPAR induces the expression of genes involved in lipid synthesis and storage through enhancers activated during adipocyte differentiation. Here, we show that PPAR also binds to enhancers already active in preadipocytes as evidenced by an active chromatin state including lower DNA methylation levels despite higher CpG content. These constitutive enhancers are linked to genes involved in the insulin/insulin-like growth factor signaling pathway that are transcriptionally induced during adipogenesis but to a lower extent than lipid metabolism genes, because of stronger basal expression levels in preadipocytes. This is consistent with the sequential involvement of hormonal sensitivity and lipid handling during adipocyte maturation and correlates with the chromatin structure dynamics at constitutive and activated enhancers. Interestingly, constitutive enhancers are evolutionary conserved and can be activated in other tissues, in contrast to enhancers controlling lipid handling genes whose activation is more restricted to adipocytes. Thus, PPAR utilizes both broadly active and cell type-specific enhancers to modulate the dynamic range of activation of genes involved in the adipogenic process.

Long-term live cell microscopy studies of lipid droplet fusion dynamics in adipocytes

During the adipogenic differentiation process of mesenchymal stem cells, lipid droplets (LDs) grow slowly by transferring lipids between each other. Recent findings hint at the possibility that a fusion pore is involved. In this study, we analyze lipid transfer data obtained in long-term label-free microscopy studies in the framework of a Hagen-Poiseuille model. The data obtained show a LD fusion process in which the lipid transfer directionality depends on the size difference between LDs, whereas the respective rates depend on the size difference and additionally on the diameter of the smaller LDs. For the data analysis, the viscosity of the transferred material has to be known. We demonstrate that a viscosity-dependent molecular rotor dye can be used to measure LD viscosities in live cells. On this basis, we calculate the diameter of a putative lipid transfer channel which appears to have a direct dependence on the diameter of the smaller of the two participating LDs.