Inhibition of adipose differentiation by 9α, 11β-prostaglandin F2α

Prostaglandin F2α (PGF2α) is a potent adipose differentiation inhibitor for the adipogenic cell line 1246 and for adipocyte precursors in primary culture with an ED50 of 3×10⁻⁸ M. In this paper, we examined the effect of several prostaglandins which have structural similarities with PGF2α on the differentiation of 1246 cells and of adipocyte precursors in primary culture. The results show that only 9α,11β-PGF2α is as potent as PGF2α to inhibit differentiation of adipocyte precursors in primary culture and of the adipogenic cell line 1246. In the presence of 9α,11β-PGF2α, the cells remained fibroblast-like, typical of undifferentiated adipocyte precursors. Triglyceride accumulation and increase of specific activity for glycerol-3-phosphate dehydrogenase were inhibited. In addition, mRNA expression of early markers of differentiation such as lipoprotein lipase (LPL) and fatty acid binding protein (FAB) was decreased. The isomer 9β,11α-PGF2α and other PGF2α derivatives were inactive. These results provide new information on the biological activity of 9α,11β-PGF2α as an inhibitor of adipose differentiation and about the structural characteristics of prostaglandins required for maintenance of a high adipose differentiation inhibitory effect.     

Adipogenic activity produced by hepatocyte-derived cell lines and by normal hepatocytes in primary culture.

Culture media conditioned by several hepatocyte derived cell lines were analyzed for their ability to stimulate adipose differentiation of the adipogenic cell line 1246. The results presented here show that culture media from HepG2 and Hep3B cell lines contain a high level of the activity, whereas media from hepatoma and hepato adenocarcinoma cell lines Huh-7, PLC/PRF/5, and SK-Hep-1 do not contain adipogenic activity. Conditioned medium from HepG2 cells also stimulated differentiation of 3T3-L1 cells and of rat epididymal adipocyte precursors in primary culture. Partial biochemical characterization of the adipogenic activity carried out using HepG2 conditioned medium indicates that the hepatocyte derived adipogenic factor has an apparent molecular weight between 445 and 232 kDa, is destroyed by treatment at 100 degrees C, with protease, with 2-mercaptoethanol and in acidic conditions. The activity is stable at alkaline pH. Culture media conditioned by normal rat hepatocytes in primary culture also contained adipogenic activity. In contrast, medium conditioned by primary culture of nonhepatocyte cell also isolated from liver was deprived of this activity. The data presented in this paper suggest that hepatocytes could be a physiological site of production of adipogenic activity.     

Insulin but not IGF-I is required for the maintenance of the adipose phenotype in the adipogenic cell line 1246

Summary The mouse adipogenic cell line 1246 which possesses both insulin and insulin-like growth factor I (IGF-I) receptors was used to investigate the role of IGF-I and insulin on the proliferation of adipocyte precursors and their differentiation into mature adipocytes. Results indicate that both insulin and IGF-I stimulate the proliferation of the 1246 adipocyte precursors with IGF-I being slightly more potent than insulin. Dose-response studies indicated that both polypeptides acted at physiological concentrations corresponding to binding to their own receptors. In contrast, comparison of insulin and IGF-I capacity to stimulate terminal adipose differentiation indicated that only insulin was active when added at physiological concentrations. IGF-I could not stimulate adipocyte differentiation except at supraphysiological concentrations (100 ng/ml and above) permitting its binding to the insulin receptors on 1246 cells. Time course study of expression of early and late markers of adipose differentiation indicated that the induction of markers such as adipose differentiation-related protein (ADRP), lipoprotein lipase (LPL) and fatty acid binding protein (FAB) took place even in the absence of insulin. However, the level of early and late differentiation markers decreased to a level below the one found in undifferentiated cells when cells had been maintained in the absence of insulin after differentiation had been initiated. These data indicate that although insulin is not necessary for the early onset of the adipose differentiation program, it is stringently required for the maintenance of the adipocyte phenotype and cannot be substituted by IGF-I.     

Specific stimulation of phospholipase activities with phosphatidylethanolamine in transformed cells

Phospholipase A1, A2 and C activities with phosphatidylethanolamine were enhanced in C6 cells relative to primary astrocytic cultures. Enhancement was a function of cell density. Phospholipase activities with phosphatidylcholine were unchanged as a function of cell density, while phospholipase C activity with phosphatidylinositol was reduced. All acid phospholipase activities measured were low or essentially absent in the three transformed cell lines examined. These results suggest that arachidonate release upon confluency is mainly from phosphatidylethanolamine.     

Molecular cloning of a differentiation-related mRNA in the adipogenic cell line 1246.

The 1246 cell line is a C3H mouse teratoma-derived adipogenic cell line that can proliferate and differentiate in defined medium. We have constructed a recombinant phage library containing complementary DNAs (cDNAs) prepared from mRNA of differentiated 1246 cells. This library was screened using a differential hybridization technique. We have isolated five different cDNA clones corresponding to mRNAs that are induced during adipogenesis of 1246 cells and one cDNA clone corresponding to mRNA that is decreased during adipogenesis. Among the mRNAs expressed during adipose differentiation, some are not expressed in undifferentiated cells, whereas some are expressed at very low levels under these conditions. Moreover, the level of induction during differentiation and the temporal expression of the mRNAs corresponding to these cDNAs varied. Our results indicate that one of the cDNA clones isolated, called 154, which selects a 2.2-kilobase mRNA, was induced 100-fold at a very early time during the onset of the differentiation program in 1246 cells and also in adipocyte precursors in primary culture. Direct sequencing of 154 cDNA insert revealed no homology with sequences in GenBank and PIR protein databases. The expression of 154 mRNA was stimulated by accelerators of differentiation such as dexamethasone and isobutylmethylxanthine and inhibited by tumor necrosis factor alpha, transforming growth factor beta, and epidermal growth factor, which are known inhibitors of 1246 cell differentiation. In addition, 154 mRNA level in adipocytes was down-regulated by tumor necrosis factor alpha, but not by transforming growth factor beta or epidermal growth factor. These results suggest that the increase in 154 mRNA expression is related to the onset of adipose differentiation. Further analysis of this clone should allow characterization of a novel protein induced early during the process of differentiation.     

Differentiation of newborn rat adipocyte precursors in defined serum-free medium

Summary Newborn rat adipocyte precursors, isolated from inguinal fat pads of 2 day-old NBR rats proliferate and undergo adipose differentiation in defined medium in the absence of serum when cultivated on polylysine coated dishes in DME-F12 medium supplemented with fibronectin, insulin, transferrin and FGF. After 7 days in culture in these conditions, 90% of the cells have undergone differentiation as measured by the increase of G3PDH specific activity and by the accumulation of triglycerides in their cytoplasm. In contrast, the cells cultivated in the presence of 10% fetal bovine serum, have a limited ability to differentiate. These results indicate that newborn rat adipocyte precursors from inguinal fat pads do not require the presence of an undefined adipogenic factor in order to differentiate in culture. In contrast, proliferation and differentiation are dependent on the presence of insulin in the culture medium. Moreover, the data presented in this paper show that the rat adipocyte precursor culture represents a rapid and reproducible system for investigating the processes of adipose tissue development and for studying the negative and positive regulators of the adipose differentiation in a controlled environment. This work was supported by grants from the Juvenile Diabetes Foundation, File #185221 and from the National Institutes of Health 1 PO1 CA37589. Editor’s Statement This paper extends to primary cultures the serum-free methods previously applied to studies of adipocyte differentiation in established lines. The observation that serum can block differentiation in this system suggests the existence of previously unrecognized circulating plasma or platelet factors affecting adipocyte differentiation, and the model developed provides an assay for the identification of these factors.     

Identification of insulin receptors on the insulin-independent variant 1246-3A cell line

1246-3A cell line is an insulin-independent variant isolated from the adipogenic cell line 1246 which can proliferate in the absence of insulin, has lost the ability to differentiate, and secretes an insulin-related factor called IRF similar to pancreatic insulin and different from IGFs. In contrast, the parent adipogenic cell line 1246 is dependent on the presence of insulin to proliferate and differentiate in defined medium. In the present paper, we examined if the loss of response to insulin observed for 1246-3A cells was accompanied by alterations in the insulin receptor properties. Insulin binding and tyrosine kinase activity of insulin receptors isolated from 1246-3A cells and from the parent cell line 1246 were measured; 125I-insulin binding to intact cells was 75% lower for the 1246-3A cells than for the 1246 cells. This was due to a decrease in receptor number without major change in receptor affinity. However, when the cells were solubilized in 1% Triton X-100 and the insulin receptor was partially purified by chromatography on wheat germ agglutinin-agarose, a similar pattern of binding was observed for both cell lines. Down regulation of insulin receptors by insulin occurred in a dose-dependent fashion, which was similar for both cell lines. Phosphorylation experiments were performed by incubation of the partially purified insulin receptor with insulin and [gamma-32P]ATP. They indicated that insulin stimulated phosphorylation of the 95-kDa molecular weight beta subunit of the receptor, in a similar fashion for both cell types. These data suggest that the insulin-independent cell line 1246-3A does not possess a specific defect in the insulin receptor which alters both its binding and autophosphorylation properties and that the loss of response to insulin can be attributed to the fact the 1246-3A cells secrete IRF which bind to cell surface receptors and stimulate their proliferation.     

Expression profiling during adipocyte differentiation of 3T3-L1 fibroblasts

The 3T3-L1 cell line is a well-established and commonly used in vitro model to assess adipocyte differentiation. Over the course of several days confluent 3T3-L1 cells can be converted to adipocytes in the presence of an adipogenic cocktail. Changes in gene expression were measured by DNA microarrays at three time points (24 h, 4 days, and 1 week) during the course of differentiation from preadipocytes to mature adipocytes. Several functional categories of genes were affected by adipocyte conversion. In addition, seven genes were found to be commonly altered by 5-fold or more by adipocyte conversion at all three time points. Lipocalin 2, haptoglobin, serum amyloid A3, stearoyl-CoA desaturase, and 11beta-hydroxysteroid dehydrogenase 1 were induced while actin alpha2 and procollagen VIII alpha1 were suppressed by adipocyte differentiation. Further study of the regulation of these genes and pathways will lead to an increased understanding of the biochemical pathways involved in adipocyte differentiation and possibly to the identification of new therapeutic targets for treatment of obesity and other metabolic diseases.     

Changes of phospholipase A2 inhibitory activity in the K+-sensitive actin gelation factor during the differentiation of myeloid leukemia cells

The phospholipase A2 inhibitory activity of a 38 kDa K+-sensitive actin gelation factor in a murine leukemia cell line (M1) was examined. A specific antibody against 38 kDa protein was found to cross-react with 37 kDa protein (lipocortin) in rat peritoneal exudates. Although the native 38 kDa protein from M1 cells did not block phospholipase A2 activity, pretreatment with alkaline phosphatase produced a form that did inhibit this enzyme. However, a purified 38 kDa protein from differentiated M1 cells blocked phospholipase A2 activity without pretreatment with alkaline phosphatase. Phospholipase A2 inhibitory activity of the 38 kDa protein was not altered by addition of actin. These findings suggest that the phospholipase A2 inhibitory of our 38 kDa protein was induced during differentiation. We also proposed that our 38 kDa protein has the same epitope as lipocortin.     

Bone morphogenetic proteins inhibit adipocyte differentiation by bone marrow stromal cells

The bone morphogenetic proteins were originally identified based on their ability to induce ectopic bone formation in vivo and have since been identified as members of the transforming growth factor-β gene superfamily. It has been well established that the bone morphogenetic cytokines enhance osteogenic activity in bone marrow stromal cells in vitro. Recent reports have described how bone morphogenetic proteins inhibited myogenic differentiation of bone marrow stromal cells in vitro. In vivo, bone marrow stromal cells differentiate along the related adipogenic pathway with advancing age. The current work reports the inhibitory effects of the bone morphorphogenetic proteins on adipogenesis in a multipotent murine bone marrow stromal cell line, BMS2. When exposed to bone morphogenetic protein-2, the pre-adipocyte BMS2 cells exhibited the expected induction of the osteogenic-related enzyme, alkaline phosphatase. Following induction of the BMS2 cells with adipogenic agonists, adipocyte differentiation was assessed by morphologic, enzymatic, and mRNA markers. Flow cytometric analysis combined with staining by the lipophilic fluorescent dye, Nile red, was used to quantitate the extent of lipid accumulation within the BMS2 cells. By this morphologic criteria, the bone morphogenetic proteins inhibited adipogenesis at concentrations of 50 to 500 ng/ml. This correlated with decreased levels of adipocyte specific enzymes and mRNAs. The BMS2 pre-adipocytes constitutively expressed mRNA encoding bone morphogenetic protein-4 and this was inhibited by adipogenic agonists. Together, these findings demonstrate that bone morphogenetic proteins act as adipogenic antagonists. This supports the hypothesis that adipogenesis and osteogenesis in the bone marrow microenvironment are reciprocally regulated.     

Gestation stage-specific frequency of adipogenic cells in Syrian hamster cell cultures

High frequencies (up to 50%) of spontaneous adipocyte differentiation are observed in cultures of 9 day gestation Syrian hamster embryos (E9 cells) within six to eight population doublings after primary culture. This is in contrast to the absence of adipogenic cells in primary cultures derived from later gestation age Syrian hamster tissue. In addition, E9 primary cultures contain a transient subpopulation of presumptive mesenchymal stem or progenitor cells that lack density dependent inhibition of growth [contact-insensitive (CS-) cells]. Analysis of the temporal pattern of expression of the CS- and adipocyte phenotypes during the proliferative life span of E9 cells demonstrates that maximal expression of the CS- phenotype precedes maximal expression of adipocyte differentiation. In addition, lipid accumulation appears to occur primarily, if not exclusively, in the contact-sensitive (CS+) cells that are derived from CS- cells. These observations suggest that primary E9 cultures contain either adipoblasts or primordial mesenchymal cells that become determined to the adipocyte lineage early during the in vitro life span of the cultures, and that the CS- phenotype may be a marker for these earlier developmental cell stages.     

An essential role for Dicer in adipocyte differentiation

Dicer is a cellular enzyme required for the processing of pre‐miRNA molecules into mature miRNA, and Dicer and miRNA biogenesis have been found to play important roles in a variety of physiologic processes. Recently, reports of alterations in miRNA expression levels in cultured pre‐adipogenic cell lines during differentiation and findings of differences between the miRNA expression signatures of white and brown adipose have suggested that miRNA molecules might regulate adipocyte differentiation and the formation of adipose tissue. However, direct evidence that miRNAs regulate adipogenesis is lacking. To determine if Dicer and mature miRNA govern adipocyte differentiation, we utilized primary cells isolated from mice bearing Dicer‐conditional alleles to study adipogenesis in the presence or absence of miRNA biogenesis. Our results reveal that Dicer is required for adipogenic differentiation of mouse embryonic fibroblasts and primary cultures of pre‐adipocytes. Furthermore, the requirement for Dicer in adipocyte differentiation is not due to miRNA‐mediated alterations in cell proliferation, as deletion of the Ink4a locus and the prevention of premature cellular senescence normally induced in primary cells upon Dicer ablation fails to rescue adipogenic differentiation in fibroblasts and pre‐adipocytes. J. Cell. Biochem. 110: 812–816, 2010. © 2010 Wiley‐Liss, Inc.     

Histone demethylase KDM7A reciprocally regulates adipogenic and osteogenic differentiation via regulation of C/EBPα and canonical Wnt signalling

Recent emerging evidences revealed that epigenetic methylation of histone and DNA regulates the lineage commitment of mesenchymal progenitor cells. This study was undertaken to delineate the actions of histone lysine demethylase 7A (KDM7A) on osteogenic and adipogenic differentiation. Kdm7a expression was up‐regulated in primary marrow stromal cells and established stromal ST2 line after adipogenic and osteogenic treatment. Silencing of endogenous Kdm7a in the cells blocked adipogenic differentiation whereas promoted osteogenic differentiation. Conversely, overexpression of wild‐type Kdm7a in the progenitor cells enhanced adipogenic differentiation whereas inhibited osteogenic differentiation. However, the effect of KDM7A on cell differentiation was largely attenuated when the point mutation was made that abolishes enzymatic activity of KDM7A. Mechanism investigations revealed that silencing of Kdm7a down‐regulated the expression of the CCAAT/enhancer binding protein α (C/EBPα) and secreted frizzled‐related protein 1 (Sfrp1). Chromatin immunoprecipitation (ChIP) assay revealed that KDM7A directly binds to the promoters of C/EBPα and Sfrp1 and removes the histone methylation marks H3K9me2 and H3K27me2. Furthermore, silencing of Kdm7a activated canonical Wnt signalling. Thereafter, activation of canonical Wnt signalling through silencing of Sfrp1 in ST2 attenuated the stimulation of adipogenic differentiation and inhibition of osteogenic differentiation by KDM7A. Our study suggests that KDM7A balances adipogenic and osteogenic differentiation from progenitor cells through epigenetic control of C/EBPα and canonical Wnt signalling and implicates that control of KDM7A action has an epigenetic perspective of curtailing metabolic disorders like osteoporosis.     

Disruption of cell-matrix interactions by heparin enhances mesenchymal progenitor adipocyte differentiation

Differentiation of marrow-derived mesenchymal progenitors to either the osteoblast or adipocyte lineage is reciprocally regulated. Factors that promote osteoblastogenesis inhibit adipogenesis, while adipogenic factors are inhibitory to osteoblast differentiation. Heparin, a soluble glycosaminoglycan, inhibits bone formation in vivo and osteoblast cell differentiation and function in vitro, and has been shown to promote adipocyte differentiation. To elucidate the role that heparin plays in the adipogenic induction of murine mesenchymal progenitors, we studied immortalized marrow stromal cells (IM-MSC), the MSC cell line, ST2, and 3T3L1 pre-adipocytes. Heparin alone was not sufficient to induce adipogenesis, but enhanced the induction under a variety of adipogenic cocktails. This effect was both dose- and time-dependent. Heparin showed a positive effect at concentrations > 0. 1 μg/ml when applied before day 3 during the induction course. Heparin's effect on adipogenesis was independent of cell proliferation, cell density, and extracellular lipid. This effect is likely related to the unique structure of heparin because another polyanionic glycosaminoglycan, dextran sulfate, did not promote adipogenic differentiation. Heparin treatment altered morphology and adhesion characteristics of progenitor cells, resulting in cell rounding and aggregation. As well, heparin counteracted the known inhibitory effect of fibronectin on adipogenesis and decreased basal focal adhesion kinase and paxillin phosphorylation. We conclude that heparin-mediated disruption of cell-matrix adhesion enhances adipogenic potential.     

Differential effects of fibroblast growth factor and tumor promoters on the initiation and maintenance of adipocyte differentiation

Fibroblast growth factor (FGF) has been shown to inhibit the differentiation of myogenic and adipogenic cell lines without inducing a proliferative response. We have previously shown that agents capable of activating protein kinase C (PKC), such as FGF and the phorbol ester tetradecanoyl phorbol-13-acetate (TPA), inhibit the differentiation of the adipogenic cell line TA1, as measured by the rapid loss of adipocyte-specific RNAs. We report here that the treatment of fully differentiated TA1 adipocytes with FGF at 10 ng/ml induces the reversal of adipocyte differentiation, even in cells where PKC activity has been down-regulated by TPA pretreatment. In contrast, TPA or lower concentrations of FGF (1 ng/ml), both effective inducers of c-fos RNA in adipocytes, fail to reverse adipocyte differentiation. The adipocytes, however, will extinguish differentiation-specific functions in response to TPA by the addition of a calcium ionophore. Therefore, we propose that there are two FGF-sensitive pathways in TA1 cells: one responsible for the initiation of differentiation (TPA sensitive) and another required for maintenance of the adipose phenotype (TPA insensitive). These results suggest that activation of two distinct signaling pathways--one PKC and calcium dependent, the other FGF activated but PKC independent--are capable of inhibiting the biochemical events responsible for the maintenance of adipocyte differentiation.     

Multiple growth factor mRNAs are expressed in chicken adipocyte precursor cells.

We have examined the expression of growth factor genes in primary cultures of chicken adipocyte precursors. RNA was extracted from proliferating and differentiated cells, reversed transcribed and amplified by PCR using gene specific primers. The identity of the PCR products was confirmed by restriction mapping. We show, for the first time, constitutive expression of TGF-beta 2, TGF-beta 3, TGF-beta 4 and bFGF genes in chicken adipocyte precursors. We also detect GH-independent, but differentiation-dependent IGF-I gene expression. The synthesis and action of these growth factors supports the hypothesis that they act as autocrine and/or paracrine regulators of adipocyte precursor cell proliferation and differentiation.