Mouse 3T3-L1 Cells Research Articles

Lamins in development, tissue maintenance and stress

Lamins are nuclear intermediate filament proteins. They provide mechanical stability, organize chromatin and regulate transcription, replication, nuclear assembly and nuclear positioning. Recent studies provide new insights into the role of lamins in development, differentiation and tissue response to mechanical, reactive oxygen species and thermal stresses. These studies also propose the existence of separate filament networks for A- and B-type lamins and identify new roles for the different networks. Furthermore, they show changes in lamin composition in different cell types, propose explanations for the more than 14 distinct human diseases caused by lamin A and lamin C mutations and propose a role for lamin B1 in these diseases.

Dynamic hydroxymethylation of deoxyribonucleic acid marks differentiation-associated enhancers

Enhancers are developmentally controlled transcriptional regulatory regions whose activities are modulated through histone modifications or histone variant deposition. In this study, we show by genome-wide mapping that the newly discovered deoxyribonucleic acid (DNA) modification 5-hydroxymethylcytosine (5hmC) is dynamically associated with transcription factor binding to distal regulatory sites during neural differentiation of mouse P19 cells and during adipocyte differentiation of mouse 3T3-L1 cells. Functional annotation reveals that regions gaining 5hmC are associated with genes expressed either in neural tissues when P19 cells undergo neural differentiation or in adipose tissue when 3T3-L1 cells undergo adipocyte differentiation. Furthermore, distal regions gaining 5hmC together with H3K4me2 and H3K27ac in P19 cells behave as differentiation-dependent transcriptional enhancers. Identified regions are enriched in motifs for transcription factors regulating specific cell fates such as Meis1 in P19 cells and PPARγ in 3T3-L1 cells. Accordingly, a fraction of hydroxymethylated Meis1 sites were associated with a dynamic engagement of the 5-methylcytosine hydroxylase Tet1. In addition, kinetic studies of cytosine hydroxymethylation of selected enhancers indicated that DNA hydroxymethylation is an early event of enhancer activation. Hence, acquisition of 5hmC in cell-specific distal regulatory regions may represent a major event of enhancer progression toward an active state and participate in selective activation of tissue-specific genes.

Effect of valproic acid on mitochondrial epigenetics

Valproic acid (valproate), an anticonvulsant and a mood stabilizer, is a potent histone deacetylase inhibitor and a widely utilized pharmacological tool for neuroepigenetic research including DNA methylation. However, only nuclear but not mitochondrial DNA (mtDNA) has been investigated for the effects of valproate on the formation of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Using mouse 3T3-L1 cells, we investigated the effects of short (1 day) and prolonged (3 days) valproate treatment on global mtDNA 5mC content, global and mtDNA sequence-specific 5hmC content, mRNA levels for ten–eleven-translocation (TET) enzymes involved in 5hmC formation, and the mitochondrial content of TET proteins. Only 5hmC but not 5mC content in mtDNA was affected (decreased) by valproate, and only after the prolonged treatment. This action of valproate was mimicked by MS-275, a class I histone deacetylase inhibitor. The prolonged but not the short valproate treatment decreased the expression of Tet1 mRNA and reduced the mitochondrial content of the TET1 protein. Hence, a likely scenario for a valproate-induced 5hmC decrease in mtDNA may involve nuclear histone deacetylase inhibition (mitochondria do not contain histones) causing the initial increase of Tet1 transcription, which is followed by a delayed compensatory decrease of Tet1 expression and a reduced presence of TET1 protein in mitochondria. Further research is needed to elucidate the functional implications of epigenetic modifications of mtDNA. The observed effects of valproate on mitochondrial epigenetics may have implications for a better understanding of both therapeutic and unwanted effects of this drug and possibly other histone deacetylase inhibitors.

18β-Glycyrrhetinic acid inhibits adipogenic differentiation and stimulates lipolysis

18β-Glycyrrhetinic acid (18β-GA) obtained from the herb liquorice has various pharmacological properties including anti-inflammatory and anti-bacterial activities. However, potential biological anti-obesity activities are unclear. In this study, novel biological activities of 18β-GA in the adipogenesis of 3T3-L1 preadipocytes and in lipolysis of differentiated adipocytes were identified. Mouse 3T3-L1 cells were used as an in vitro model of adipogenesis and lipolysis, using a mixture of insulin/dexamethasone/3-isobutyl-1-methylxanthine (IBMX) to induce differentiation. The amount of lipid droplet accumulation was determined by an AdipoRed assay. The expression of several adipogenic transcription factors and enzymes was investigated using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. 18β-GA dose-dependently (1–40μM) significantly decreased lipid accumulation in maturing preadipocytes. In 3T3-L1 preadipocytes, 10μM of 18β-GA down-regulated the transcriptional levels of the peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein α and adiponectin, which are markers of adipogenic differentiation via Akt phosphorylation. Also, in differentiated adipocytes, 18β-GA increased the level of glycerol release and up-regulated the mRNA of hormone-sensitive lipase, adipose TG lipase and perilipin, as well as the phosphorylation of hormone-sensitive lipase at Serine 563. The results indicate that 18β-GA alters fat mass by directly affecting adipogenesis in maturing preadipocytes and lipolysis in matured adipocytes. Thus, 18β-GA may be useful for the treatment of obesity.

이소플라본 genistein이 전지방세포 성장 및 지방세포형성과정에 미치는 영향

이소플라본 genistein이 마우스 전지방 3T3-L1세포주의 성장과 지방세포형성과정에 미치는 영향을 연구하였다. 그 결과, 마우스 전지방 3T3-L1 세포주의 성장이 처리한 genistein 농도 의존적으로 저해되었다. 또한, Oil Red O 염색에 의하여 50 <TEX>${\mu}M$</TEX> genistein 처리에 의해 지방세포형성과정이 억제됨을 확인하였다. 이러한 genistein에 의한 지방세포성장 억제효과의 분자생물학적 기전을 연구하기 위하여, oligo DNA 마이크로어레이 실험을 수행하였다. 발현이 증가된 유전자 중 항비만, 항염증 활성과 관련이 있는 것으로 알려져 있는 sirtuin-1 유전자를 선택하여 발현변화를 확인하였다. 처리한 네종류의 파이토케미칼(resveratrol, capsaicin, daidzein, genistein)에 의해 sirtuin-1의 발현이 증가됨을 확인하였고, 이 중 genistein에 의한 sirtuin-1 유전자의 발현이 가장 높았다. 또한, sirtuin-1-siRNA에 의한 sirtuin-1 유전자의 발현 감소에 의해 지방세포형성 억제과정이 복구됨을 확인하였다. 이러한 연구결과는, genistein에 의한 전지방세포의 성장 저해와 지방세포형성의 저해과정을 이해하는데 도움을 줄 것으로 기대된다. The effects of genistein on cell proliferation and adipogenesis were examined in mouse 3T3-L1 preadipocyte cells. Genistein decreased viability of 3T3-L1 pre-adipocytes in a dose-dependent manner. Oil Red O staining of these cells also indicated that adipogenesis was inhibited by 50 <TEX>${\mu}M$</TEX> genistein treatment. We investigated the molecular mechanisms involved in the decrease in cell viability in genistein-treated 3T3-L1 cells by conducting an oligo DNA microarray analysis. We selected the sirtuin-1 gene, one of the upregulated genes, for further experimentation because sirtuin-1 belongs to the sirtuin family, which is associated with anti-obesity and anti-inflammation activities. We found that four phytochemicals (resveratrol, capsaicin, daidzein, and genistein) could increase sirtuin-1 expression. Genistein was the strongest inducer of sirtuin-1 among the tested phytochemicals. The inhibition of adipogenesis by genistein was recovered by surtuin-1 siRNA transfection. Overall, these results may further our understanding of the molecular mechanisms underlying the inhibition of proliferation and adipogenesis by genistein in mouse 3T3-L1 cells.

Phenylenediamine derivatives induce GDF-15/MIC-1 and inhibit adipocyte differentiation of mouse 3T3-L1 Cells

Phenylenediamine derivatives can function as a hydrogen donor and reportedly exert various biological actions including cytoprotective effects against oxidative stress, possibly by acting as an antioxidant. Previous studies showed that feeding of such compounds to mice reduced their body weight, but the precise mechanism remains unknown at present. Here, we found that these compounds inhibited the in vitro differentiation of mouse preadipocytes, 3T3-L1 cells, into adipocytes, suggesting that, at least in part, reduced generation of adipocytes might contribute to the observed weight loss in mice. Next, we performed array analysis and found that the expression of GDF-15/MIC-1, which is a TGFβ superfamily cytokine, and Trib 3, an intracellular downstream effector of the cytokines, was up-regulated by these derivatives. Thus, we identified the compounds as inducers of GDF-15/MIC-1 and suggest that such induction may have led to inhibition of adipocyte differentiation, which could account for the weight-loss effect of these compounds.

TGF-β Mediates Suppression of Adipogenesis by Estradiol through Connective Tissue Growth Factor Induction

In the bone marrow cavity, adipocyte numbers increase, whereas osteoblast progenitor numbers decrease with aging. Because adipocytes and osteoblasts share a common progenitor, it is possible that this shift is due to an increase in adipocyte-lineage cells at the expense of osteoblast-lineage commitment. Estrogens inhibit adipocyte differentiation, and in both men and women, circulating estrogens correlate with bone loss with aging. In bone cells, estrogens stimulate expression of TGF-β and suppress mesenchymal cell adipogenesis. Using a tripotential mesenchymal cell line, we have examined whether estradiol suppression of adipocyte differentiation is due to stimulation of TGF-β and the mechanism by which TGF-β suppresses adipogenesis. We observed that estradiol-mediated suppression of adipogenic gene expression required at least 48 h treatment. TGF-β expression increased within 24 h of estradiol treatment, and TGF-β inhibition reversed estradiol influences on adipogenesis and adipocyte gene expression. Connective tissue growth factor (CTGF) mediates TGF-β suppression of adipogenesis in mouse 3T3-L1 cells. CTGF expression was induced within 24 h of TGF-β treatment, whereas estradiol-mediated induction required 48 h treatment. Moreover, estradiol-mediated induction of CTGF was abrogated by TGF-β inhibition. These data support that estradiol effects on adipogenesis involves TGF-β induction, which then induces CTGF to suppress adipogenesis.

Nuclear factor erythroid-derived factor 2-related factor 2 regulates transcription of CCAAT/enhancer-binding protein β during adipogenesis

Nuclear factor erythroid-derived factor 2-related factor 2 (Nrf2) is a cap-n-collar basic leucine zipper transcription factor that is involved in the cellular adaptive response to oxidative stress. Our previous study reported that targeted disruption of the Nrf2 gene in mice decreases adipose tissue mass and protects against obesity induced by a high-fat diet. Deficiency of Nrf2 in preadipocytes and mouse embryonic fibroblasts led to impaired adipogenesis. Consistent with these findings, the current study found that lack of Nrf2 in primary cultured mouse preadipocytes and 3T3-L1 cells hampered adipogenic differentiation induced by hormonal cocktails. Stable knockdown of Nrf2 in 3T3-L1 cells blocked the enhanced adipogenesis caused by deficiency of kelch-like ECH-associated protein 1 (Keap1), a Cul3-adapter protein that allows for Nrf2 to be ubiquinated and degraded by the 26S protesome complex. In addition, increased production of reactive oxygen species (ROS) and activation of Nrf2 occurred at the very early stage upon adipogenic hormonal challenge in 3T3-L1 cells, followed by an immediate induction of CCAAT/enhancer-binding protein β (C/EBPβ). Knockdown of Nrf2 led to reduced expression of C/EBPβ induced by adipogenic hormonal cocktails, chemical Nrf2 activators or Keap1 silencing. Cebpβ promoter-driven reporter assays and chromatin immunoprecipitation suggested that Nrf2 associates with a consensus antioxidant response element (ARE) binding site in the promoter of the Cebpβ gene during adipogenesis and upregulates its expression. These findings demonstrate a novel role of Nrf2 beyond xenobiotic detoxification and antioxidant response, and suggest that Nrf2 is one of the transcription factors that control the early events of adipogenesis by regulating expression of Cebpβ.

Perinatal programming of obesity later in life by the endocrine disruptor bisphenol A in a mouse model

The 3T3-L1 preadipocyte culture system has been used to examine numerous compounds that influence adipocyte differentiation or function. The perfluoroalkyl acids (PFAAs), used as surfactants in a variety of industrial applications, are of concern as environmental contaminants that are detected worldwide in human serum and animal tissues. This study was designed to evaluate the potential for PFAAs to affect adipocyte differentiation and lipid accumulation using mouse 3T3-L1 cells. Cells were treated with perfluorooctanoic acid (PFOA) (5–100 µM), perfluorononanoic acid (PFNA) (5–100 µM), perfluorooctane sulfonate (PFOS) (50–300 µM), perfluorohexane sulfonate (PFHxS) (40–250 µM), the peroxisome proliferator activated receptor (PPAR) PPARα agonist Wyeth-14,643 (WY-14,643), and the PPARγ agonist rosiglitazone. The PPARγ agonist was included as a positive control as this pathway is critical to adipocyte differentiation. The PPARα agonist was included as the PFAA compounds are known activators of this pathway. Cells were assessed morphometrically and biochemically for number, size, and lipid content. RNA was extracted for qPCR analysis of 13 genes selected for their importance in adipocyte differentiation and lipid metabolism. There was a significant concentration-related increase in cell number and decreased cell size after exposure to PFOA, PFHxS, PFOS, and PFNA. All four PFAA treatments produced a concentration-related decrease in the calculated average area occupied by lipid per cell. However, total triglyceride levels per well increased with a concentration-related trend for all compounds, likely due to the increased cell number. Expression of mRNA for the selected genes was affected by all exposures and the specific impacts depended on the particular compound and concentration. Acox1 and Gapdh were upregulated by all six compounds. The strongest overall effect was a nearly 10-fold induction of Scd1 by PFHxS. The sulfonated PFAAs produced numerous, strong changes in gene expression similar to the effects after treatment with the PPARγ agonist rosiglitazone. By comparison, the effects on gene expression were muted for the carboxylated PFAAs and for the PPARα agonist WY-14,643. In summary, all perfluorinated compounds increased cell number, decreased cell size, increased total triglyceride, and altered expression of genes associated with adipocyte differentiation and lipid metabolism.

Genome-wide profiling of H3K56 acetylation and transcription factor binding sites in human adipocytes.

The growing epidemic of obesity and metabolic diseases calls for a better understanding of adipocyte biology. The regulation of transcription in adipocytes is particularly important, as it is a target for several therapeutic approaches. Transcriptional outcomes are influenced by both histone modifications and transcription factor binding. Although the epigenetic states and binding sites of several important transcription factors have been profiled in the mouse 3T3-L1 cell line, such data are lacking in human adipocytes. In this study, we identified H3K56 acetylation sites in human adipocytes derived from mesenchymal stem cells. H3K56 is acetylated by CBP and p300, and deacetylated by SIRT1, all are proteins with important roles in diabetes and insulin signaling. We found that while almost half of the genome shows signs of H3K56 acetylation, the highest level of H3K56 acetylation is associated with transcription factors and proteins in the adipokine signaling and Type II Diabetes pathways. In order to discover the transcription factors that recruit acetyltransferases and deacetylases to sites of H3K56 acetylation, we analyzed DNA sequences near H3K56 acetylated regions and found that the E2F recognition sequence was enriched. Using chromatin immunoprecipitation followed by high-throughput sequencing, we confirmed that genes bound by E2F4, as well as those by HSF-1 and C/EBPα, have higher than expected levels of H3K56 acetylation, and that the transcription factor binding sites and acetylation sites are often adjacent but rarely overlap. We also discovered a significant difference between bound targets of C/EBPα in 3T3-L1 and human adipocytes, highlighting the need to construct species-specific epigenetic and transcription factor binding site maps. This is the first genome-wide profile of H3K56 acetylation, E2F4, C/EBPα and HSF-1 binding in human adipocytes, and will serve as an important resource for better understanding adipocyte transcriptional regulation.

A potential role of IL-6 in the chito-oligosaccharide-mediated inhibition of adipogenesis

Recent studies have suggested that chito-oligosaccharides can have anti-adipogenic properties. The objectives of the present study were to evaluate the anti-adipogenic potential of four different chito-oligosaccharides (molecular weight (MW) < 1000, 1000-3000, 3000-5000 and 5000-10,000 Da) and to identify molecular mechanisms underlying the chito-oligosaccharide-mediated inhibition of adipogenesis. Mouse 3T3-L1 cells were allowed to differentiate in the presence of chito-oligosaccharide. At day 8 post-induction of differentiation, lipid accumulation, free glycerol release and the quantitative expression of adipogenic marker genes were evaluated. Chito-oligosaccharides had concentration- and MW-dependent inhibitory effects on lipid accumulation (P < 0·001 and < 0·05, respectively), as well as a concentration-dependent effect (P < 0·001) on free glycerol release and the expression of adipogenic marker genes. The 5000-10,000 Da chito-oligosaccharide was selected for subsequent molecular studies. A panel of forty-four lipid metabolic pathway-specific genes was analysed by quantitative real-time PCR. Chito-oligosaccharide-mediated inhibition of adipogenesis was associated with the up-regulation of the IL-6 gene at all concentrations of chito-oligosaccharide examined and the PG-endoperoxide synthase 2 (PTGS2) gene at higher concentrations of chito-oligosaccharide. The effect of chito-oligosaccharide on gene expression was validated by measuring IL-6 protein concentrations in the media. Finally, an IL-6 promoter assay was developed to characterise the effect of chito-oligosaccharide on the transcriptional activity of the IL-6 promoter, which was increased in a concentration-dependent manner (P < 0·001). We conclude that IL-6 is a candidate signalling molecule in the chito-oligosaccharide-mediated inhibition of adipogenesis in 3T3-L1 cells.

Niacin promotes adipogenesis by reducing production of anti-adipogenic PGF 2α through suppression of C/EBPβ-activated COX-2 expression

Niacin is converted to NAD and NADP in tissues, whose products are involved in a number of cellular processes; and it is associated with the regulation of adipogenesis. In this study, we identified the molecular mechanism by which niacin promotes the adipogenesis in mouse 3T3-L1 cells. When the cells were cultured with niacin, the expression of adipogenic peroxisome proliferator-activated receptor γ, CCAAT enhancer binding protein (C/EBP)α, and their target genes was enhanced concomitant with an increase in triglyceride storage. Moreover, niacin suppressed the expression of cyclooxygenase-2 and decreased the production of prostaglandin (PG) F 2α in the early phase of adipogenesis, which PG suppresses the progression of adipogenesis via the PGF 2α receptor. Furthermore, niacin decreased the C/EBPβ level in the early phase of adipogenesis. These results indicate that niacin promoted adipogenesis by suppressing the production of the anti-adipogenic PGF 2α through down-regulation of C/EBPβ-activated cyclooxygenase-2 expression in adipocytes.

Forkhead transcription factor Foxa1 is a novel target gene of C/EBPβ and suppresses the early phase of adipogenesis

Forkhead/winged helix transcription factors (Foxs) regulate differentiation, metabolism, and development. Although Foxa1 is expressed in adipocytes, the roles and regulation of Foxa1 in them remain unclear. Here, we found that under the control of C/EBPβ, Foxa1 suppressed lipid accumulation and concomitantly caused a decrease in adipogenic gene expression in adipocytes. Foxa1 was expressed in undifferentiated mouse 3T3-L1 cells and in the early phase of adipogenesis, with its highest expression at 3 h after the initiation of adipogenesis, which was followed by a subsequent decrease. SiRNA-mediated suppression of Foxa1 expression activated the expression of adipogenic genes such as PPARγ. Moreover, siRNAs for C/EBPβ, but not those for C/EBPδ, reduced Foxa1 mRNA and protein levels. The results of a promoter–reporter assay and chromatin immunoprecipitation assay demonstrated that C/EBPβ bound to the C/EBP binding element at − 529 of the mouse Foxa1 promoter. Furthermore, siRNA-mediated knockdown of C/EBPβ decreased the promoter activity of mouse Foxa1 gene. These results suggest that Foxa1 plays a suppressive role in the early phase of adipogenesis, acting under the control of C/EBPβ, and might be involved in the regulation of the rate of progression of the early phase of adipogenesis.

Fad104, a positive regulator of adipogenesis, negatively regulates osteoblast differentiation

Fad104 (factor for adipocyte differentiation 104) is a novel gene expressed temporarily in the early stages of adipocyte differentiation. Previously, we showed that fad104 promotes adipocyte differentiation in mouse 3T3-L1 cells and mouse embryonic fibroblasts (MEFs). Furthermore, we reported that implanted wild-type MEFs could develop into adipocytes, whereas fad104-deficient MEFs could not. Interestingly, bone-like tissues were only observed in the implants derived from fad104-deficient MEFs. This result implies that fad104 is involved in osteoblast differentiation. However, the functions of fad104 during osteogenesis are unknown. In this paper, we show that fad104 negatively regulates osteoblast differentiation. During the differentiation process, the level of fad104 expression decreased. Deletion of fad104 facilitated osteoblast differentiation in MEFs, and elevated the level of runx2, a master regulator of osteoblast differentiation. Disruption of fad104 suppressed BMP-2-mediated adipocyte differentiation in MEFs. In conclusion, we demonstrate that fad104 reciprocally regulates differentiation of adipocytes and osteoblast; functions as a positive regulator in adipocyte differentiation and as a negative regulator in osteoblast differentiation.

Mathematical modeling of preadipocyte fate determination

White adipose tissue is the major energy storage depot for neutral lipids and is also a key endocrine regulator of a host of homeostatic activities, including metabolism, feeding behaviors, cardiovascular functions and reproduction. Abnormal fat accretion in the setting of obesity can lead to insulin resistance and type 2 diabetes, and has been linked to some cancers and arteriosclerosis. Thus, a thorough appreciation of the intricate signaling events that must take place as quiescent adipocyte precursors are recruited into the proliferating cell population that then must ‘decide’ to differentiate into fully functional fat cells is critical to our understanding of diseases related to excess adipogenesis. We are beginning to gain insights into the molecular regulators of adipocyte differentiation. A significant advance would be to construct mathematical modeling tools that would assist cell biologists in predicting how environmental and/or intrinsic inputs could influence preadipocyte fate decision making. We have developed a model of how preadipocytes may use the dynamic interplay of two transcription factors, nuclear factor- κ B ( NF ‐ κ B ) and peroxisome proliferator-activated receptor- γ (PPAR- γ ) in early proliferation and differentiation events in vitro. Critical to the model is the feedback signaling between NF ‐ κ B and its inhibitor, I κ B . The model is based on differential equations that describe the interactions of stimuli for NF ‐ κ B activation and mitogen concentrations and allows one to make predictions about how mouse 3T3-L1 preadipocytes choose between proliferation, differentiation or quiescence. Those predictions are supported by experiments on mouse 3T3-L1 cells.

Isorhamnetin-induced anti-adipogenesis is mediated by stabilization of β-catenin protein

Aims Previous studies have shown that isorhamnetin has anti-adipogenic effects in mouse 3T3-L1 cells. This study was conducted to elucidate the inhibitory mechanisms of isorhamnetin during adipogenic differentiation of human adipose tissue-derived stem cells (hAMSCs). Main methods The effect of isorhamnetin on adipogenic differentiation of hAMSCs was quantified by Oil Red O staining and a triglyceride assay. In addition, real-time PCR and Western blot were used to determine the expression of adipogenesis-related genes. Key findings Isorhamnetin inhibited the adipocyte differentiation of hAMSCs. Additionally, when the effects of Wnt antagonists that promote adipogenesis were evaluated, isorhamnetin was found to down-regulate the mRNA levels of sFRP1 and Dkk1, but had no effect on the mRNA levels of sFRP2, sFRP3, sFRP4 and Dkk3. Isorhamnetin also inhibited the expression of Wnt receptor and co-receptor genes. Furthermore, isorhamnetin increased the protein levels of β-catenin, an effector molecule of Wnt signaling, but had no effect on the mRNA levels of β-catenin. The phosphorylation level of GSK 3β was also increased by isorhamnetin. These results were confirmed by the fact that the expression of c-myc, cyclin D1 and PPARδ, which are target genes of β-catenin, was upregulated by isorhamnetin. Moreover, isorhamnetin reduced the mRNA expression levels of C/EBPα and PPARγ, which are known to be inhibited by c-myc or by cyclin D1 and PPARδ, respectively. Significance Our results indicate that isorhamnetin inhibits the adipogenic differentiation of hAMSCs and that its mechanisms are mediated by the stabilization of β-catenin.

Suppression of Adipocyte Differentiation by Aldo-keto Reductase 1B3 Acting as Prostaglandin F2α Synthase

Prostaglandin (PG) F(2alpha) suppresses adipocyte differentiation by inhibiting the function of peroxisome proliferator-activated receptor gamma. However, PGF(2alpha) synthase (PGFS) in adipocytes remains to be identified. Here, we studied the expression of members of the aldo-keto reductase (AKR) 1B family acting as PGFS during adipogenesis of mouse 3T3-L1 cells. AKR1B3 mRNA was expressed in preadipocytes, and its level increased about 4-fold at day 1 after initiation of adipocyte differentiation, and then quickly decreased the following day to a level lower than that in the preadipocytes. In contrast, the mRNA levels of Akr1b8 and 1b10 were clearly lower than that level of Akr1b3 in preadipocytes and remained unchanged during adipogenesis. The transient increase in Akr1b3 during adipogenesis was also observed by Western blot analysis. The mRNA for the FP receptor, which is selective for PGF(2alpha), was also expressed in preadipocytes. Its level increased about 2-fold within 1 h after the initiation of adipocyte differentiation and was maintained at almost the same level throughout adipocyte differentiation. The small interfering RNA for Akr1b3, but not for Akr1b8 or 1b10, suppressed PGF(2alpha) production and enhanced the expression of adipogenic genes such as peroxisome proliferator-activated receptor gamma, fatty acid-binding protein 4 (aP2), and stearoyl-CoA desaturase. Moreover, an FP receptor agonist, Fluprostenol, suppressed the expression of those adipogenic genes in 3T3-L1 cells; whereas an FP receptor antagonist, AL-8810, efficiently inhibited the suppression of adipogenesis caused by the endogenous PGF(2alpha). These results indicate that AKR1B3 acts as the PGFS in adipocytes and that AKR1B3-produced PGF(2alpha) suppressed adipocyte differentiation by acting through FP receptors.

Deficiency in the Nuclear Factor E2-related Factor-2 Transcription Factor Results in Impaired Adipogenesis and Protects against Diet-induced Obesity

Nuclear factor E2-related factor 2 (Nrf2) is a cap-n-collar basic leucine zipper (CNC-bZIP) transcription factor that is well established as a master regulator of phase II detoxification and antioxidant gene expression and is strongly expressed in tissues involved in xenobiotic metabolism including liver and kidney. Nrf2 is also abundantly expressed in adipose tissue; however, the exact function of Nrf2 in adipocyte biology is unclear. In the current study we show that targeted knock-out of Nrf2 in mice decreases adipose tissue mass, promotes formation of small adipocytes, and protects against weight gain and obesity otherwise induced by a high fat diet. In mouse embryonic fibroblasts, 3T3-L1 cells, and human subcutaneous preadipocytes, selective deficiency of Nrf2 impairs adipocyte differentiation. Deficiency of Nrf2 also leads to decreased expression of peroxisome proliferator-activated receptor gamma (PPARgamma), CCAAT enhancer-binding protein alpha (C/EBPalpha), and their downstream targets during adipocyte differentiation. Conversely, activation of Nrf2 in 3T3-L1 cells by stable knockdown of its negative regulator Keap1 enhances and accelerates hormone-induced adipocyte differentiation. Transfection of Nrf2 stimulates Ppargamma promoter activity, and stable knockdown of Keap1 enhances PPARgamma expression in 3T3-L1 cells. In addition, chromatin immunoprecipitation studies show that Nrf2 associates with consensus binding sites for Nrf2 in the Ppargamma promoter. These findings demonstrate a novel biologic role for Nrf2 beyond its participation in detoxification and antioxidant pathways and place Nrf2 within the limited network of transcription factors that control adipocyte differentiation by regulating expression of PPARgamma.

Gelsolin, an Actin Regulatory Protein, is Required for Differentiation of Mouse 3T3-L1 Cells into Adipocytes

To elucidate molecular mechanisms of adipocyte differentiation, we previously isolated TC10-like/ TC10betaLong (TCL/TC10betaL), which was transiently expressed in the early phase of adipogenesis of 3T3-L1 cells and seemed to be a positive regulator of adipogenesis. By using TCL/TC10betaL-overexpressing NIH-3T3 cells, we also isolated gelsolin as a gene whose expression was up-regulated by TCL/TC10betaL. However, the roles of gelsolin in adipocyte differentiation are unclear. In this paper we characterized the function of gelsolin in adipogenesis in 3T3-L1 cells. The level of gelsolin changed during adipocyte differentiation. Knockdown of the expression of gelsolin using RNAi inhibited adipocyte differentiation, and impaired the expression of peroxisome proliferator-activated receptor gamma (PPARgamma) and CCAAT/enhancer-binding protein (C/EBP) alpha. Interestingly, the knockdown also impaired mitotic clonal expansion (MCE), and increased cell size, though it reduced levels of C/EBPbeta and C/EBPdelta, markers for the early stage of adipogenesis, only slightly. Gelsolin plays a crucial role in the differentiation of 3T3-L1 cells into adipocytes.

Insulin-mediated Regulation of Decidual Protein Induced by Progesterone (DEPP) in Adipose Tissue and Liver

We analyzed the profile of the genes expressed in human adipose tissue and identified the fat-derived molecules, adiponectin and aquaporin 7, which modulate glucose and lipid metabolism. The same Bodymap analysis revealed abundant expression of the decidual protein induced by progesterone (DEPP) in the white adipose tissue. Northern blot analysis confirmed that human DEPP mRNA was highly expressed in white adipose tissue. Mouse DEPP mRNA was detected in heart, lung, skeletal muscle, and white adipose tissue under feeding state. In contrast, under fasting state, mouse DEPP mRNA was enhanced in lung, skeletal muscle, and white adipose tissue and it appeared also in the liver and kidney, suggesting up regulation of DEPP by fasting. Because fasting-induced DEPP expression was observed in insulin-sensitive organs, we investigated the regulation of DEPP in white adipose tissue and liver. During adipogenesis of mouse 3T3-L1 cells, DEPP mRNA increased in a differentiation-dependent manner similar to adiponectin and aquaporin 7. Treatment of cultured 3T3-L1 mature adipocytes, rat H4IIE, and human HepG2 hepatoma cells with insulin significantly decreased DEPP mRNA levels in dose- and time-dependent manners. IN VIVO experiments showed significant decrease of hepatic and adipose DEPP mRNA levels in refed mice, compared to fasted animals, and also showed significant increase in DEPP mRNA in streptozotocin-induced insulin-deficient diabetic mice. These results indicate that DEPP is a novel insulin-regulatory molecule expressed abundantly in insulin-sensitive tissues including white adipose tissue and liver.