Expression Of Adipocyte Marker Genes Research Articles

Actions of β-apo-carotenoids in differentiating cells: Differential effects in P19 cells and 3T3-L1 adipocytes

β-Apo-carotenoids, including β-apo-13-carotenone and β-apo-14′-carotenal, are potent retinoic acid receptor (RAR) antagonists in transactivation assays. We asked how these influence RAR-dependent processes in living cells. Initially, we explored the effects of β-apo-13-carotenone and β-apo-14′-carotenal on P19 cells, a mouse embryonal carcinoma cell line that differentiates into neurons when treated with all-trans-retinoic acid. Treatment of P19 cells with either compound failed to block all-trans-retinoic acid induced differentiation. Liquid chromatography tandem mass spectrometry studies, however, established that neither of these β-apo-carotenoids accumulates in P19 cells. All-trans-retinoic acid accumulated to high levels in P19 cells. This suggests that the uptake and metabolism of β-apo-carotenoids by some cells does not involve the same processes used for retinoids and that these may be cell type specific. We also investigated the effects of two β-apo-carotenoids on 3T3-L1 adipocyte marker gene expression during adipocyte differentiation. Treatment of 3T3-L1 adipocytes with either β-apo-13-carotenone or β-apo-10′-carotenoic acid, which lacks RAR antagonist activity, stimulated adipocyte marker gene expression. Neither blocked the inhibitory effects of a relatively large dose of exogenous all-trans-retinoic acid on adipocyte differentiation. Our data suggest that in addition to acting as transcriptional antagonists, some β-apo-carotenoids act through other mechanisms to influence 3T3-L1 adipocyte differentiation.

Reduction of PP2A Cα stimulates adipogenesis by regulating the Wnt/GSK-3β/β-catenin pathway and PPARγ expression

Serine/threonine protein phosphatase 2A (PP2A) regulates several physiological processes such as the cell cycle, cell growth, apoptosis, and signal transduction. In this study, we examined the expression and role of PP2A Cα in adipocyte differentiation. PP2A Cα expression and PP2A activity decreased during adipocyte differentiation in C3H10T1/2 and 3T3-L1 cells and the expression of adipocyte marker genes such as PPARγ and adiponectin increased. To further clarify the role of PP2A Cα in adipocyte differentiation, we constructed PP2A knockdown cells by infecting C3H10T1/2 cells with a lentivirus expressing a shRNA specific for the PP2A Cα (shPP2A cells). Silencing of PP2A Cα in C3H10T1/2 cells dramatically stimulated adipocyte differentiation and lipid accumulation, which were accompanied by expression of adipocyte marker genes. Silencing of PP2A Cα suppressed Wnt10b expression and reduced the levels of the inactivated form of GSK-3β (phospho-GSK-3β), leading to the reduction of β-catenin levels in the nucleus and its transcriptional activity. Treatment with LiCl, a GSK-3β inhibitor, and inhibition of PPARγ expression suppressed the accelerated adipogenesis of shPP2A cells. Our data indicate that PP2A Cα plays an important role in the regulation of adipocyte differentiation by regulating the Wnt/GSK-3β/β-catenin pathway and PPARγ expression.

Heat stress during differentiation of porcine adipocytes is accompanied by increased expression of adipocyte marker genes and downregulation of inflammatory genes (1033.2)

Global increase in ambient temperature as a result of climate change imposes heat stress on both humans and livestock. For pigs, an important food animal and a biomedical model, the implication of sustained heat stress on growth, physiology and metabolism of the animal is still unclear. Because of the role that adipose tissue plays in the regulation of overall metabolism and growth efficiency in the pig, this study was conducted with the objective of determining the impact of heat stress during adipocyte differentiation on expression of mature adipocyte and inflammatory marker genes. Preadipocytes isolated from the subcutaneous adipose depot were differentiated under normal (37oC) or heat stress (41.5oC) condition for 3, 6 or 9 days. Gene expression was determined by RT‐PCR. Fold expression of genes at 41.5oC was determined relative to 37oC after normalization with 18s. Heat stress induced 1.5 and 1.3 fold increase in the expression of PPARγ on days 3 and 6, with no change on day 9. Expression of CEBPα was induced by heat stress by 2.4, 2.6 and 2.2 fold on days 3, 6 and 9 respectively. Expression of adiponectin (3.5, 1.9 and 3.3 fold), leptin (2.0, 2.7 and 2.3 fold), HSP70 (2.9, 3.5 and 2.2 fold), HSP90 (2.7, 5.2 and 4.5 fold) was higher in heat stressed adipocytes on days 3, 6 and 9 respectively. However, heat stress led to a reduction in the expression of IL‐6 (0.3, 0.3 and 0.2 fold) and TNFα (0.3, 0.5 and 0.1 fold) on days 3, 6 and 9 respectively, relative to cells differentiated at 37 oC. Therefore, heat stress leads to significant alteration in adipocyte metabolism in a way that may be consequential for the health and growth performance of pigs.

Genistein promotes a gene expression profile characteristic of brown rather than white adipocytes and increases Sirt1 expression in mouse NIH3T3‐L1 cells (372.7)

Various possible benefits have been attributed to the dietary isoflavone genistein. In common with resveratrol, which may act via sirtuin 1 (Sirt1), these benefits include protection from diet‐induced obesity. We thus aimed to determine if genistein affected adipogenesis and/or Sirt1 expression using a cell culture model.NIH3T3‐L1 cells were differentiated into adipocytes (from 48 h post‐confluence, taken as day 0) in the presence or absence of genistein. Gene expression was measured by RT‐qPCR.A low concentration of genistein (10 µM) and/or shorter exposure (days 0‐12) promoted differentiation to white adipocytes, indicated by large fat droplets and increased expression of adipocyte marker genes (Acaca (acetyl Co‐A carboxylase‐α), Fasn (fatty acid synthase), Fabp4 (fatty acid binding protein 4), Lipe (hormone sensitive lipase), Retn (resistin), Rarres2 (chemerin)). However, at higher concentrations of genistein (50‐100 µM) and/or after longer exposure (days 0‐12) cells had smaller fat droplets and lower expression of these genes, coupled with increased expression of Sirt1 and of genes characteristic of brown adipocytes (Ucp1 (uncoupling protein 1) and Cebpb (CCAAT/enhancer binding protein‐β)).Dietary genistein may thus protect against obesity by encouraging the development of brown, rather than white, adipose tissue, possibly through a mechanism involving Sirt1.Grant Funding Source: Kurdistan Regional Government

α/β-hydrolase domain containing protein 15 (ABHD15)--an adipogenic protein protecting from apoptosis.

Our knowledge about adipocyte metabolism and development is steadily growing, yet many players are still undefined. Here, we show that α/β-hydrolase domain containing protein 15 (Abhd15) is a direct and functional target gene of peroxisome proliferator-activated receptor gamma (PPARγ), the master regulator of adipogenesis. In line, Abhd15 is mainly expressed in brown and white adipose tissue and strongly upregulated during adipogenesis in various murine and human cell lines. Stable knockdown of Abhd15 in 3T3-L1 cells evokes a striking differentiation defect, as evidenced by low lipid accumulation and decreased expression of adipocyte marker genes. In preconfluent cells, knockdown of Abhd15 leads to impaired proliferation, which is caused by apoptosis, as we see an increased SubG1 peak, caspase 3/7 activity, and BAX protein expression as well as a reduction in anti-apoptotic BCL-2 protein. Furthermore, apoptosis-inducing amounts of palmitic acid evoke a massive increase of Abhd15 expression, proposing an apoptosis-protecting role for ABHD15. On the other hand, in mature adipocytes physiological (i.e. non-apoptotic) concentrations of palmitic acid down-regulate Abhd15 expression. Accordingly, we found that the expression of Abhd15 in adipose tissue is reduced in physiological situations with high free fatty acid levels, like high-fat diet, fasting, and aging as well as in genetically obese mice. Collectively, our results position ABHD15 as an essential component in the development of adipocytes as well as in apoptosis, thereby connecting two substantial factors in the regulation of adipocyte number and size. Together with its intricate regulation by free fatty acids, ABHD15 might be an intriguing new target in obesity and diabetes research.

In Vitro Characterization and Engraftment of Adipocytes Derived from Human Induced Pluripotent Stem Cells and Embryonic Stem Cells

Human induced pluripotent stem (iPS) and embryonic stem (ES) cells can differentiate into a variety of cell types. We reported on adipogenic potential of human iPS and ES cells in vitro. In the present study, we investigate the survival and maintenance of adipocytes differentiated in vitro from human iPS and ES cells after transplantation. Following adipogenic induction in vitro, the differentiated cells exhibited functional properties of adipocytes such as lipid storage, lipolysis, and insulin responsiveness. Subsequently, Matrigel containing the differentiated human iPS and ES cells was transplanted into the subcutaneous tissue of nude mice. After 1-4 weeks, the cells with adipocyte-like features were observed in transplanted Matrigel by histological analysis. The human origin of the cells, their lipid accumulation, and gene expression of adipocyte markers in transplanted cells were then confirmed, suggesting the presence of adipocytes in transplanted Matrigel. When the relative areas of these cells were calculated by dividing the adipocyte areas by the total Matrigel areas, we found that they peaked at 2 weeks after transplantation, and that the adipocytes persisted at 4 weeks. The present study demonstrates that human iPS and ES cells can differentiate into adipocytes with functional properties and that adipocytes derived from human iPS and ES cells can survive and maintain the differentiated properties of adipocytes for at least 4 weeks after transplantation. Adipocytes derived from human iPS and ES cells thus have the potential to open new avenues for stem cell-based research into metabolic diseases and future therapeutic applications.

Markers are shared between adipogenic and osteogenic differentiated mesenchymal stem cells

The stem cell differentiation paradigm is based on the progression of cells through generations of daughter cells that eventually become restricted and committed to one lineage resulting in fully differentiated cells. Herein, we report on the differentiation of adult human mesenchymal stem cells (hMSCs) towards adipogenic and osteogenic lineages using established protocols. Lineage specific geneswere evaluated by quantitative real-time PCR relative to two reference genes. The expression of osteoblast-associated genes (alkaline phosphatase, osteopontin, and osteocalcin)was detected in hMSCs that underwent adipogenesis. When normalized, the expression of adipocyte marker genes (adiponectin, fatty acid binding protein P4, and leptin) increasedin a time-dependent manner during adipogenic induction. Adiponectin and leptin were also detected in osteoblast-induced cells. Lipid vacuoles that represent the adipocyte phenotype were only present in the adipogenic induction group. Conforming to the heterogeneous nature of hMSCs and the known plasticity between osteogenic and adipogenic lineages, these data indicatea marker overlap between MSC-derived adipocytes and osteoblasts. Weproposea careful consideration of experimental conditions such as investigated timepoints, selected housekeeping genesand the evidence indicating lack of differentiation into other lineageswhen evaluating hMSC differentiation.

The impact of geranylgeraniol on the differentiation of murine 3T3‐F442A preadipocytes

The statins, competitive inhibitors of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase, suppress the differentiation of adipocytes via mevalonate deprivation. We hypothesize that geranylgeraniol, a diterpene shown to accelerate the degradation of HMG CoA reductase, mimics the impact of lovastatin in adipocytes by suppressing adipocyte differentiation and adipogenic gene expression. Adipo‐Red assay and oil Red O staining showed that a 7‐d incubation with 2.5 – 20 μmol/L geranylgeraniol dose‐dependently reduced the intracellular triglyceride content of murine 3T3‐F442A adipocytes. Concomitantly, geranylgeraniol down‐regulated the expression of peroxisome proliferator‐activated receptor γ (PPARγ), a key regulator of adipocyte differentiation as analyzed by real‐time qPCR. The expression of adipocyte marker genes including adiponectin, leptin, fatty acid binding protein 4 and lipoprotein lipase was also suppressed by geranylgeraniol. No changes in cell viability were observed in 3T3‐F442A cells incubated with geranylgeraniol (0–400 μmol/L) for 24 and 48 h. Mevalonate‐derived metabolites have essential roles in promoting adipocyte differentiation and adipogenic gene expression. Dietary mevalonate suppressors may have potential as anti‐adipogenesis compounds.

The effects of NOD activation on adipocyte differentiation

Obesity is associated with chronic inflammation. Toll-like receptors (TLR) and NOD-like receptors (NLR) are two families of pattern recognition receptors that play important roles in immune response and inflammation in adipocytes. It has been reported that TLR4 and TLR2 activation induce proinflammatory changes that impair adipocyte differentiation. However, the effects of activation of NOD1 and NOD2, the two prominent members of NLR, on adipocyte differentiation have not been studied. 3T3-L1 and human adipose-derived stem cells were tested for adipocyte differentiation in the presence or absence of NOD ligand. Adipocyte differentiation was evaluated by the adipocyte markers gene expression and Oil Red O staining for lipid accumulation. Activation of NOD1, but not NOD2, by a synthetic ligand dose-dependently suppressed 3T3-L1 adipocyte differentiation as revealed by Oil Red O stained cell morphology, lipid accumulation, and attenuated gene expression of adipocyte markers (PPARγ, C/EBPα, SCD, FABP4, Adiponectin). Activation of NOD1, but not NOD2, induced NF-κB activation, which correlated with their abilities to suppress ligand-induced PPARγ transaction. Moreover, the suppressive effect by NOD1 activation was reversed by IκB super-repressor which blocks NF-κB activation. The suppression by NOD1 ligand C12-iEDAP on adipocyte differentiation was reversed by small RNA interference targeting NOD1, demonstrating the specificity of NOD1 activation. In contrast, activation of NOD1 and NOD2 both significantly suppressed adipocyte differentiation of human adipose-derived adult stem cells, demonstrating the species specific effects of NOD activation. In contrast to enhanced leptin mRNA by LPS and TNFα, NOD1 activation suppressed leptin mRNA in adipocytes, suggesting the differential effects of NOD1 activation in adipocytes. Overall, our results suggest that NOD1 represents a novel target for adipose inflammation in obesity.

Selenium Significantly Inhibits Adipocyte Hypertrophy and Abdominal Fat Accumulation in OLETF Rats via Induction of Fatty Acid β-Oxidation

A combination of selenium (Se) with other trace element is associated with partially modulate fatty acid distribution as well as reduction of the body weight and feed efficiency. To investigate whether or not Se treatment has an impact on lipid metabolism, we examined the levels of lipid metabolism-related factors, including abdominal fat, adiponectin, cholesterol, very long chain dehydrogenase (VLCAD), and medium chain acyl-CoA dehydrogenase (MCAD) in 20-week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats following sodium selenite treatment for 2weeks. Herein, we observed that (a) Se treatment induced insulin-like effects by lowering the serum glucose level in rats; (b) Se-treated rats showed significance values decreases in abdominal fat mass, adipocyte size, and adiponectin, which are associated with lipid metabolism; (c) Se treatment led to reduced levels of cholesterol, triglycerides, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) cholesterol; (d) fat tissue in Se-treated rats displayed significantly lower expression of adipocyte marker genes along with increased expression of VLCAD and MCAD; and (e) fatty liver formation and β-oxidation gene expression were both significantly reduced in liver tissue of Se-treated rats. Therefore, our results suggest that Se may induce inhibition of adipocyte hypertrophy and abdominal fat accumulation along with suppression of fatty liver formation by the differential regulation of the gene expression for fatty acid β-oxidation in the OLETF model.

10E12Z CLA alters adipocyte differentiation and adipocyte cytokine expression and induces macrophage proliferation

The trans-10, cis-12 (10e12z) conjugated linoleic acid (CLA) isomer of CLA is responsible for loss of lipid storage or adipose tissue in vitro or in vivo. This isomer also induces inflammatory signaling in both mouse and human adipocytes in vitro. However, when these events occur and whether they are significant enough to affect other cell types are unclear. In these experiments, the 3T3-L1 cell line has been used to examine the interaction between inflammatory signaling and decreased differentiation or lipid storage induced by 10e12z CLA. In assays measuring both lipid accumulation and gene expression, differentiating 3T3-L1 cells exhibit concurrent induction of inflammatory signaling, as measured by cyclooxygenase-2 expression, and a decrease in adipocyte marker gene expression. Furthermore, in fully differentiated adipocytes, as identified in microarray assays and confirmed with real-time polymerase chain reaction, 10e12z CLA also significantly affected expression of both matrix metalloprotein-3 (MMP-3), collagen VI α 3 ColVI alpha 3 (VIα3) and the cytokine epiregulin, demonstrating that the effects of 10e12z broadly impact adipocyte function. In agreement with other experimental systems, 10e12z CLA inhibited RAW 264.7 cell proliferation; however, in response to adipocyte-conditioned media, 10e12z-CLA-treated adipocytes induced proliferation of this cell line, suggesting that the effect of 10e12z CLA is context dependent. These results are largely consistent with the known activation of the inflammatory mediator nuclear factor-κB in adipocytes in vitro and in vivo by 10e12z CLA treatment and demonstrate that adipose is an important target tissue of this isomer that impacts other cell types.

Curcumin inhibits adipocyte differentiation through modulation of mitotic clonal expansion

Adipocyte differentiation is a key process in determining the number of mature adipocytes in the development of obesity. Here, we examined the function of curcumin, a dietary polyphenol found in turmeric, and its underlying mechanisms in adipocyte differentiation. Our study reveals that curcumin exerts an anti-adipogenic function both in 3T3-L1 murine cells and in human primary preadipocytes as determined by intracellular lipid accumulation assay, quantitative analysis of adipocyte marker gene expression and a noninvasive multimodal Coherent Anti-Stokes Raman Scattering (CARS) microscopic analysis of intracellular curcumin. The inhibitory action of curcumin was largely limited to the early stage of adipocyte differentiation, where curcumin was found to inhibit mitotic clonal expansion (MCE) process as evidenced by impaired proliferation, cell-cycle entry into S phase and the S to G2/M phase transition of confluent cells, and levels of cell cycle-regulating proteins with no significant effect on cell viability and cytotoxicity. This, in turn, resulted in inhibition of mRNA levels of early adipogenic transcription factors, particularly Krüppel-like factor 5 (KLF5), CCAAT/enhancer binding proteinα (C/EBPα) and peroxisome proliferator-activated receptorγ (PPARγ), in the early stage of adipocyte differentiation. Supplementation with rosiglitazone, a PPARγ ligand, during the early stage of adipocyte differentiation partially rescued curcumin-inhibited adipocyte differentiation. Collectively, our results show that curcumin is an anti-adipogenic dietary bioactive component largely involved in the modulation of the MCE process during the early stage of adipocyte differentiation.

Absence of Estrogen Receptor-Related-α Increases Osteoblastic Differentiation and Cancellous Bone Mineral Density

The nuclear orphan receptor human estrogen receptor-related receptor (ERR)-alpha is implicated in bone metabolism. We studied the effect of ERRalpha silencing in human mesenchymal stem cells (hMSCs) during osteoblastogenesis. We found that ERRalpha silencing led to an increase of bone sialoprotein and a decrease of osteopontin mRNA levels, suggesting enhanced osteoblastic differentiation. This was confirmed by an increased ability of hMSCs to deposit calcium. Concomitantly, knockdown of ERRalpha inhibited adipogenesis, resulting in a decrease in adipocyte number and adipocyte marker gene expression. In line with a negative role of ERRalpha in bone metabolism, we found that adult female and male ERRalpha-deficient mice displayed a moderate increase in femoral cancellous bone volume and density. Osteoblast surface was increased and marrow fat volume decreased in these animals. Furthermore, ERRalpha-deficient osteoblasts displayed increased differentiation properties in vitro in line with our observations in hMSCs. In summary, we identified a role for ERRalpha in bone mass regulation by affecting osteoblastic differentiation.

Sphingosine kinase is induced in mouse 3T3-L1 cells and promotes adipogenesis

Sphingosine 1-phosphate (S1P) is a lysophospholipid mediator that exerts numerous biological activities both as a receptor ligand and as an intracellular second messenger. In the present study, we explored roles of sphingosine kinase (SphK), an S1P-producing enzyme, in adipose tissue. We utilized mouse 3T3-L1 cells as an in vitro model of adipogenesis, using a mixture of insulin/dexamethasone/3-isobutyl-1-methylxanthine (IBMX) to induce differentiation. Real-time quantitative PCR (qRT-PCR) assays revealed that the expression levels of transcripts encoding both isoforms of SphK-1 and SphK-2 are up-regulated during adipogenesis (37.6- and 6.6-fold vs. basal, P < 0.05, respectively). Concomitantly, SphK-1/SphK-2 protein abundance and S1P contents of these cells increased at 3 days after hormonal stimulation. Loss-of-function approaches by pharmacological inhibition of SphK activity as well as by transfection with small interfering RNA (siRNA) against SphK-1 led to significant attenuation of lipid droplet accumulation and adipocyte marker gene expression. We detected marked elevation of SphK-1 mRNA in adipose tissue derived from 13-week-old ob/ob mice with obese phenotype than their lean littermates. These results suggest that increased expression of SphK, an S1P-producing enzyme, plays a significant role during adipogenesis, potentially providing a novel point of control in adipose tissue.

Hedgehog Signaling Alters Adipocyte Maturation of Human Mesenchymal Stem Cells

Human stem cells are powerful tools by which to investigate molecular mechanisms of cell growth and differentiation under normal and pathological conditions. Hedgehog signaling, the dysregulation of which causes several pathologies, such as congenital defects and cancer, is involved in several cell differentiation processes and interferes with adipocyte differentiation of rodent cells. The present study was aimed at investigating the effect of Hedgehog pathway modulation on adipocyte phenotype using different sources of human mesenchymal cells, such as bone marrow stromal cells and human multipotent adipose-derived stem cells. We bring evidence that Hedgehog signaling decreases during human adipocyte differentiation. Inhibition of this pathway is not sufficient to trigger adipogenesis, but activation of Hedgehog pathway alters adipocyte morphology as well as insulin sensitivity. Analysis of glycerol-3-phosphate dehydrogenase activity and expression of adipocyte marker genes indicate that activation of Hedgehog signaling by purmorphamine impairs adipogenesis. In sharp contrast to reports in rodent cells, the maturation process, but not the early steps of human mesenchymal stem cell differentiation, is affected by Hedgehog activation. Hedgehog interferes with adipocyte differentiation by targeting CCAAT enhancer-binding protein alpha and peroxisome proliferator-activated receptor (PPAR) gamma2 expression, whereas PPARgamma1 level remains unaffected. Although Hedgehog pathway stimulation does not modify the total number of adipocytes, adipogenesis appears dramatically impaired, with reduced lipid accumulation, a decrease in adipocyte-specific markers, and acquisition of an insulin-resistant phenotype. This study indicates that a decrease in Hedgehog signaling is necessary but not sufficient to trigger adipocyte differentiation and unveils a striking difference in the adipocyte differentiation process between rodent and human mesenchymal stem cells.

Organotin Compounds Promote Adipocyte Differentiation as Agonists of the Peroxisome Proliferator-Activated Receptor γ/Retinoid X Receptor Pathway

Nuclear receptors play important roles in the maintenance of the endocrine system, regulation of organ differentiation, and fetal development. Endocrine disruptors exert their adverse effects by disrupting the endocrine system via various mechanisms. To assess the effects of endocrine disruptors on nuclear receptors, we developed a high-throughput method for identifying activators of nuclear receptors. Using this system, we found that triphenyltin and tributyltin were activators of peroxisome proliferator-activated receptor (PPAR) gamma and retinoid X receptor. Because PPARgamma is a master regulator of adipocyte differentiation, we assessed the effect of organotin compounds on preadipocyte 3T3-L1 cells. We found that organotin compounds stimulated differentiation of 3T3-L1 cells as well as expression of adipocyte marker genes.

STAT 5 activators can replace the requirement of FBS in the adipogenesis of 3T3-L1 cells

The 3T3-L1 cells differentiate into fat cells that have many properties of native adipocytes including: substantial lipid accumulation, insulin sensitivity, and the ability to secrete endocrine hormones. A substantial expense in using these cells is fetal bovine serum (FBS), a critical component of efficient adipogenesis. Our recent studies on STAT 5 proteins have revealed that these transcription factors are phosphorylated and translocate to the nucleus immediately after the initiation of differentiation. Studies by several other laboratories also suggest that STAT 5 proteins can have pro-adipogenic properties. Growth hormone (GH) and prolactin (PRL) are both potent activators of STAT 5A and STAT 5B proteins. Since, FBS has high concentrations of GH; we examined the ability of GH to replace FBS as a component of the differentiation cocktail for 3T3-L1 cells. Our studies revealed that FBS was not required for the adipogenesis of 3T3-L1 cells if GH or PRL was added to the differentiation cocktail. Adipogenesis was judged by Oil Red O staining and expression of adipocyte marker genes. Hence, we have developed a substantially less expensive method for differentiating 3T3-L1 cells without FBS, thiazolidinediones, or expensive cytokines.

The Novel Gene fad158, Having a Transmembrane Domain and Leucine-rich Repeat, Stimulates Adipocyte Differentiation

Adipocyte differentiation is known to be regulated by a complex array of genes known as master regulators. Using a subtraction method, we previously isolated 102 genes that are expressed in the early stage of adipocyte differentiation. One of these genes named fad158 (factor for adipocyte differentiation 158) seems to be a novel gene, since there is no significantly similar gene listed in databases. Both mouse and human fad158 encode 803 amino acids and contain 4 transmembrane regions and 8 leucine-rich repeat motifs. Expression of fad158 was induced at an early stage in differentiating 3T3-L1 cells and was observed in the skeletal muscle. When the expression was knocked down with an antisense method in 3T3-L1 cells, the accumulation of oil droplets was reduced. Moreover, on overexpression of fad158 in NIH-3T3 cells, which are fibroblasts and do not usually differentiate into adipocytes, stable transformants accumulated oil droplets and showed an elevated expression of adipocyte marker genes, indicating that these cells had differentiated into mature adipocytes. fad158 has the ability to regulate adipocyte differentiation positively, especially at an early stage.

Deregulated MAPK Activity Prevents Adipocyte Differentiation of Fibroblasts Lacking the Retinoblastoma Protein

A functional retinoblastoma protein (pRB) is required for adipose conversion of preadipocyte cell lines and primary mouse embryo fibroblasts (MEFs) in response to treatment with standard adipogenic inducers. Interestingly, lack of functional pRB in MEFs was recently linked to elevated Ras activity. Ras-dependent signaling plays a significant, although incompletely understood, role in adipocyte differentiation, because activated Ras has been reported to either promote or inhibit adipogenesis depending on the cellular context. In various cell types activation of Ras leads to activation of the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2 (ERK1/2), and protein kinase B (PKB)/Akt, which exert opposing effects on adipogenesis, with ERK1/2 inhibiting and PKB/Akt promoting terminal differentiation. Here we report that the levels of activated ERK1/2 and PKB/Akt are significantly increased in pRB-deficient MEFs both before and after the addition of adipogenic inducers. Consistently, we detected higher levels of activated Ras in MEFs lacking pRB. Suppression of ERK1/2 activation by the MEK inhibitor UO126 restored the ability of pRB-deficient MEFs to undergo adipocyte differentiation, as manifested by expression of adipocyte marker genes and lipid accumulation. Furthermore and reflecting the elevated levels of activated PKB/Akt in the pRB-deficient MEFs, differentiation proceeded in an insulin-independent manner. In conclusion, we suggest that pRB plays a pivotal role in adipogenesis by suppressing MAPK activity.