Commitment of 3T3-F442A cells to adipocyte differentiation takes place during the first 24-36 h after adipogenic stimulation: TNF-alpha inhibits commitment

We studied the commitment of 3T3-F442A cells during stimulation with adipogenic serum or growth hormone. Confluent 3T3-F442A preadipocytes were incubated with adipogenic medium for increasing times; the number of adipose clusters, GPDH activity, and lipid accumulation were evaluated. Results show that cell commitment took place during the first 24-36 h after stimulation under adipogenic conditions. Then, cultures underwent a 2-fold increase in total cell number through selective multiplication of committed cells, followed by a dramatic decrease in colony-forming ability and 300- to 1000-fold raise in GPDH activity. Cell commitment was not modulated by insulin, but this hormone stimulated clonal expansion of committed cells and lipogenesis. Commitment was inhibited by TNF-alpha at concentrations as low as 5 ng/ml, and by retinoic acid. The results show that TNF-alpha inhibits adipose conversion at two different levels: at concentrations as low as 5 ng/ml, it blocks commitment, and at concentrations of 100 ng/ml or higher the cytokine seems to block mitotic expansion and other steps of differentiation after cell commitment. The identification of a specific time for cell commitment would allow the study of the early genes that might regulate cell reprogramming into adipocytes.     

Commitment of 3T3-F442A cells to adipocyte differentiation takes place during the first 24-36 h after adipogenic stimulation: TNF-α inhibits commitment

We studied the commitment of 3T3-F442A cells during stimulation with adipogenic serum or growth hormone. Confluent 3T3-F442A preadipocytes were incubated with adipogenic medium for increasing times; the number of adipose clusters, GPDH activity, and lipid accumulation were evaluated. Results show that cell commitment took place during the first 24-36 h after stimulation under adipogenic conditions. Then, cultures underwent a 2-fold increase in total cell number through selective multiplication of committed cells, followed by a dramatic decrease in colony-forming ability and 300- to 1000-fold raise in GPDH activity. Cell commitment was not modulated by insulin, but this hormone stimulated clonal expansion of committed cells and lipogenesis. Commitment was inhibited by TNF-α at concentrations as low as 5 ng/ml, and by retinoic acid. The results show that TNF-α inhibits adipose conversion at two different levels: at concentrations as low as 5 ng/ml, it blocks commitment, and at concentrations of 100 ng/ml or higher the cytokine seems to block mitotic expansion and other steps of differentiation after cell commitment. The identification of a specific time for cell commitment would allow the study of the early genes that might regulate cell reprogramming into adipocytes.     

Culture on Tissue‐Specific Coatings Derived from α‐Amylase‐Digested Decellularized Adipose Tissue Enhances the Proliferation and Adipogenic Differentiation of Human Adipose‐Derived Stromal Cells

While extracellular matrix (ECM)‐derived coatings have the potential to direct the response of cell populations in culture, there is a need to investigate the effects of ECM sourcing and processing on substrate bioactivity. To develop improved cell culture models for studying adipogenesis, the current study examines the proliferation and adipogenic differentiation of human adipose‐derived stem/stromal cells (ASCs) on a range of ECM‐derived coatings. Human decellularized adipose tissue (DAT) and commercially available bovine tendon collagen (COL) are digested with α‐amylase or pepsin to prepare the coatings. Physical characterization demonstrates that α‐amylase digestion generates softer, thicker, and more stable coatings, with a fibrous tissue‐like ultrastructure that is lost in the pepsin‐digested thin films. ASCs cultured on the α‐amylase‐digested ECM have a more spindle‐shaped morphology, and proliferation is significantly enhanced on the α‐amylase‐digested DAT coatings. Further, the α‐amylase‐digested DAT provides a more pro‐adipogenic microenvironment, based on higher levels of adipogenic gene expression, glycerol‐3‐phosphate dehydrogenase (GPDH) enzyme activity, and perilipin staining. Overall, this study supports α‐amylase digestion as a new approach for generating bioactive ECM‐derived coatings, and demonstrates tissue‐specific bioactivity using adipose‐derived ECM to enhance ASC proliferation and adipogenic differentiation.     

Transformation of Ob17 cells promotes proliferation and differentiation of Ob17 preadipocytes via distinct extracellular intermediates

Conditioned serum-free medium of Ob17 cells transformed by the middle-T-only gene of polyoma virus (Ob17MT cells) is able to support growth and adipose conversion of the parental Ob17 cells. Conditioned media from 3T3-F442A cells (untransformed preadipocyte clonal line) and MTT4 cells (middle-T-transformed non-preadipocyte clonal line) are inactive. The serum-free conditioned medium of Ob17MT cells is also active on growth and adipose conversion of 3T3-F442A cells. The morphological differentiation of Ob17 cells is accompanied by the expression of early (lipoprotein lipase, LPL) and late (glycerol-3-phosphate dehydrogenase, GPDH) biochemical markers of adipose conversion. Bio-Gel P-60 chromatography and SDS-PAGE have allowed characterization of a mitogenic fraction of apparent MW approximately equal to 28 Kd distinct from an adipogenic fraction of apparent MW less than 10 Kd. This adipogenic fraction is only required for the acquisition of the GPDH activity and is therefore active on terminal differentiation.     

The effects of retinoic acid on reversing the adipocyte differentiation into an osteoblastic tendency in ST2 cells, a murine bone marrow-derived stromal cell line

Although the mouse bone marrow stromal cell line ST2 has been known to be differentiated into osteoblasts, the differentiation characteristics of the cell into adipocyte and the concerned relationship between its adipogenesis and osteogenesis remains unknown. The adipogenic induction medium which is made up of insulin, dexamethasone (DEX) and 3-isobutyl-1-methylxanthine(IBMX), stimulated the expression of n early adipogenic marker PPAR γ and a late marker GPDH in ST2 cells. The triglyceride accumulation and lipid stain level generated by the induction medium in ST2 cells was inhibited by RA with IC₅₀ at about 1 nM. The induction medium up-regulated expression of PPARγ and GPDH was also inhibited by RA whereas RA (30 nM) exterted no effect on the cell growth. Interestingly, treatment of the cells with induction medium in the presense of RA caused a 3- or 10-fold higher in ALP activity respectively as compared to those treated with RA or the induction medium alone. RT-PCR analysis showed that such a synergistic effect of RA and the induction medium paralleled the process of inhibition on adipogenesis. Additional experiments showed that IBMX played a key role in increasing the effect of RA and ALP activity. Our results suggested that the relationship between adipogenesis and osteogenesis in ST2 cells was reciprocally interrelated and the process of adipogenesis could be potentially reversed into an osteoblastogenic tendency. This is the first report demonstrating that RA transforms adipogenic potential into an osteoblastic tendency. This revised version was published online in July 2006 with corrections to the Cover Date.     

Rho激酶抑制剂Y-27632对C3H10T1/2细胞增殖与成脂分化的作用

探究Rho激酶抑制剂Y-27632对间充质干细胞（mesenchymal stem cells,MSCs）C3H10T1/2增殖和成脂分化的影响.实验分为对照组、成脂诱导组和Y-27632处理组（Y-27632+成脂诱导）. 利用MTT检测细胞增殖情况,油红O染色,异丙醇萃取法检测细胞成脂分化情况,半定量RT-PCR检测过氧化物酶体增殖物激活受体γ(peroxisome proliferator activiated receptor γ, PPARγ)和CCAAT增强子结合蛋白α (CCAAT enhancer binding protein α, C/EBPα)基因表达. 结果表明,Y-27632能够显著抑制C3H10T1/2细胞的增殖(P<0.05),并呈一定的浓度依赖性;高浓度Y-27632对C3H10T1/2细胞成脂分化具有显著抑制作用（P<0.05）;半定量RT-PCR结果显示,成脂诱导处理组PPARγ和C/EBPα表达量在第3 d、5 d和7 d显著低于成脂诱导组（P<0.05）. 综上所述,Y-27632能够抑制C3H10T1/2细胞增殖与成脂分化.     

Various stimulators of the cyclic AMP pathway fail to promote adipose conversion of porcine preadipocytes in primary culture

The cyclic adenosine-monophosphate (cAMP) pathway is generally recognized as one of the essential pathways for the adipose conversion of rodent preadipocytes in vitro. However, divergent effects of cAMP on adipocyte differentiation have also been reported. Since there is very little data on non-rodent preadipose cells, the aim of the present work was to analyze the effects of classic activators of the cAMP pathway on the proliferation and differentiation of porcine preadipocytes grown either in serum-free or in serum-containing medium. In both media, the addition of 10 microM forskolin from day 1 after cell plating to day 3 or 7 did not affect cell proliferation. Such stimulations also failed to enhance preadipocyte differentiation, as assessed by the measurement of lipoprotein lipase (LPL) and glycerol 3-phosphate dehydrogenase (GPDH) activities, two markers of adipose conversion. Similar results were obtained when various concentrations of forskolin (0.1 nM-100 microM) were added for 2 days either during the growth phase (days 1-3) or after confluence (days 5-7). Addition of methylisobutylxanthine (MIX) or 8-bromo-cAMP was also found inefficient to stimulate porcine preadipocytes differentiation clearly. By contrast, post-confluence treatment of the murine 3T3-L1 cell line with either forskolin or MIX markedly enhanced lipid accumulation and led to a dramatic increase in GPDH activity (up to 120 times). This indicates that similar culture conditions are adipogenic for the murine 3T3-L1 preadipocytes but not for porcine preadipose cells. In summary, this work clearly highlights the finding that porcine preadipocytes do not respond to classic activators of the cAMP pathway like rodent cells do. This calls in question again the general model proposed for the action of this pathway in adipose conversion and suggests that the mechanisms regulating adipocyte differentiation may differ among species.     

大鼠前脂细胞和3T3-F442A细胞对猫血清因子的分子诱导作用的差异性反应

根据形态学变化,甘油-3-磷酸脱氢酶(GPDH)活力的升高和三酸甘油酯(TG)积累的增加与胚牛血清(EBS)相比,猫血清能显著地使元代培养中大鼠前脂细胞发生分化作用,GPDH酶活力因加猫血清者为373+/-45单位/mg蛋白质,而加入FBS者为118+/-23单位/mg蛋白质;N＝21,P<0.001,相对应的TG分别为16.1+/-2.7μmol/mg DNA与5.5+/-1.2μmol/mg DNA,N＝12,P<0.0005.分化细胞引发的脂肪细胞转化作用,GPDHFBA与胰岛素组为1247+/-82单位/mg蛋白质,而猫血清+FBS+胰岛素组为1145+/-80单位/mg蛋白质.猫血清还对大鼠前脂细胞具有促进有丝分裂的作用,尤其在接种后的第4天至第5天最为明显.此种促分化作用成分在56℃经45分钟后仍稳定,但经100℃30分钟处理即遭破坏.它是非透析性的,对胰蛋白酶、链霉菌蛋白酶和羧肽酶A只有抗性,但胰凝乳蛋白酶却可使其部分地失活.它对DTT和高碘酸盐不敏感,在pH2和pH12条件下不稳定,其等电点为5左右,它能与Con A琼脂糖相结合,看来是一种糖蛋白.凝胶过滤层析表明它的分子量为57KDa.结论猫血清含有一种能促使元代培养中的大鼠前脂细胞转化成脂肪细胞,但却没有使3T3细胞系细胞发生这种作用,表明这两种细胞存在着固有的差异.     

Development of a chemically defined serum-free medium for differentiation of rat adipose precursor cells

Stromal-vascular cells from the epididymal fat pad of 4-week-old rats, when cultured in a medium containing insulin or insulin-like growth factor, IGF-I, triiodothyronine and transferrin, were able to undergo adipose conversion. Over ninety percent of the cells accumulated lipid droplets and this proportion was reduced in serum-supplemented medium. The adipose conversion was assessed by the development of lipoprotein lipase (LPL) and glycerol-3-phosphate dehydrogenase (GPDH) activities, [14C]glucose incorporation into polar and neutral lipids, triacylglycerol accumulation and lipolysis in response to isoproterenol. Similar results were obtained with stromal-vascular cells from rat subcutaneous and retroperitoneal adipose tissues. Stromal-vascular cells required no adipogenic factors in addition to the components of the serum-free medium. Insulin was required within a physiological range of concentrations for the emergence of LPL and at higher concentrations for that of GPDH. When present at concentrations ranging from 2 to 50 nM, IGF-I was able to replace insulin for the expression of both LPL and GPDH. The development of a serum-free, chemically defined medium for the differentiation of diploid adipose precursor cells opens up the possibility of characterizing inhibitors or activators of the adipose conversion process.     

Adipocyte differentiation factor (ADF): a protein secreted by mature fat cells that induces preadipocyte differentiation in culture

A factor that is released into the culture medium of mature adipocytes and promotes the differentiation (adipogenic conversion) of preadipocytes has been partially characterized. The factor acts in a dose-dependent manner on preadipocytes to produce up to a four-fold increase in triacylglycerol (triglyceride) content and a nine-fold increase in glycerol-3-phosphate dehydrogenase (GPDH) activity, a marker of the late phase of differentiation of preadipocytes. The material appears to be a protein, since it has a molecular weight (Superose-12 gel exclusion chromatography) of about 53 kDa, an isoelectric point (pl) of 4.7-4.9, and is inactivated by the proteases papain and chymotrypsin and extremes of pH (2 and 12). Considerations of molecular weight, isoelectric point, stability to specific proteases, and especially to the action of chemical agents [the adipogenic activity is not affected by either an oxidizing (KIO4) or a reducing agent (DTT)], lead to the conclusion that the differentiation factor is distinct from known cytokines. The authors suggest that the protein be designated adipocyte differentiation factor (ADF). ADF in vivo may act as a cytokine paracrine agent to regulate the differentiation of preadipocytes.     

Developmental regulation of glycerol-3-phosphate dehydrogenase synthesis inDrosophila

Methods have been developed to measure the synthesis of glycerol-3-phosphate dehydrogenase (GPDH) during the development of Drosophila melanogaster. In emerged adult flies, GPDH is a principal component of protein synthesis, comprising between 1 and 2% of the protein synthetic effort. This high relative rate of protein synthesis continues throughout adult life during a period of stable enzyme concentration. Therefore, it is evident that GPDH undergoes continual turnover. Analysis of GPDH synthesis in the adult segments reveals that this enzyme is synthesized in head, thorax, and abdomen. In 5-day-old flies, the relative rates of GPDH synthesis in the thorax and abdomen are similar. However, the concentration of GPDH in the thorax greatly exceeds that found in the abdomen. Therefore, it appears that the turnover rate of GPDH in the abdomen must be greater than the turnover rate of GPDH in the GPDH-containing cells (flight muscle) of the thorax. GPDH represents between 0.5 and 0.9% of the protein synthetic effort of larvae. The principle GPDH-containing tissue of larvae is fat body. The turnover of GPDH in larvae is similar to that in adult abdomen. This may be related to the concurrent presence of GPDH isozyme-3 in both tissues. Our studies indicate that the cell type-specific control of GPDH occurs at several levels.     

The tyrosine kinase receptor HER2 (erbB-2): from oncogenesis to adipogenesis

Recent experimental evidences begin to support the notion that the proto-oncogene HER2 (erbB-2) might unexpectedly function to modulate the adipogenic conversion of preadipocytes. Two opposing scenarios have been proposed, however, to explain the influence of HER2 on adipocyte differentiation. In one hand, down-modulation of HER2 expression and pharmacological reduction of HER2 activity have been related to enhanced adipocyte differentiation. On the contrary, an increased abundance in HER2 has been described in differentiated adipocytes compared with preadipocytes. Considering that expression and activity of the lipogenic enzyme Fatty Acid Synthase (FASN) become up-regulated during adipogenic conversion, we recently hypothesized that a "HER2 --> FASN axis" -a "lipogenic benefit" that has been shown to enhance cancer cell proliferation, survival, chemoresistance and metastasis in biologically aggressive subgroups of breast carcinomas-might also naturally work during the differentiation of preadipocytes. To definitely clarify if the discrepancy between the opposing theories for a role of HER2 during adipocyte differentiation related to the experimental approach utilized to compare the abundance of HER2 in undifferentiated and differentiated adipocytes (i.e., cell lysates containing equivalent protein content versus cell lysates generated from similar cell numbers), we here took advantage of a high content microscopy approach. Using an automated confocal imaging platform, we monitored the expression status of the adipogenic marker FASN and its timing relationship with HER2 not only in individual 3T3-L1 cells but further in whole cultures of 3T3-L1 preadipocytes undergoing adipogenic conversion. Our findings not only confirm a non-oncogenic role for HER2 in the process of adipose differentiation but further suggest that HER2 might represent a previously unrecognized target to manage obesity via the lipogenic enzyme FASN.     

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.     

Preadipocyte stimulating factor in rat serum: evidence for a discrete 63 kDa protein that promotes cell differentiation of rat preadipocytes in primary cultures

In primary cultures of rat preadipocytes (PA) isolated from epididymal or perirenal depots, rat serum is more effective than other animal sera (fetal calf, newborn calf, human, horse, rabbit, cat, sheep, goat, dog, pig) in promoting adipogenic conversion, biochemical differentiation, and mitogenesis. Only mouse serum is comparable to rat serum. This activity is attributable to a specific growth factor (preadipocyte stimulating factor, PSF). An assay for PSF in rat serum was devised using PA from perirenal fat of 3-month-old Fischer 344 rats grown first to confluence in FCS for 8 days and then for the next 3 days in test serum, followed by measurement of triglyceride (TG) and glycerol-3-phosphate dehydrogenase (GPDH). Rat serum induces dose-dependent rapid cell division, which coincides with accumulation of TG and increase of GPDH; for routine quantitation, TG is assayed. The biochemical characteristics of PSF in serum are as follows: stable at 4 degrees C for up to 1 year; inactivated at 100 degrees C (80% loss, 30 min) but stable at 56 degrees C for 1 hr; stable at pH 2-12; non-dialyzable; completely resistant to pepsin, trypsin, and chymotrypsin but destroyed by pronase and subtilisn; stable to DTT and periodate; and m.w. between 68 kDa (Sephacryl-300) and 58 kDa (Sephacryl-300 in 5 M urea). PSF activity is greater in serum from Wistar than from Fischer 344 rats, while activity of serum from Zucker obese (fa/fa) rats is at least as great as that from Wistar rats and, like serum of rats made obese by feeding a high-fat, high-carbohydrate diet, is not suppressed. PSF activity is not due to insulin, insulin-like growth factor-1 (IGF-1), growth hormone, glucocorticoids, or combinations of these hormones. PSF activity was not seen with a number of growth factors including colony-stimulating factor (CSF-1), GM-CSF, interleukins 1, 2, and 3, neuroleukin, tumor necrosis factor, and others. PSF is distinct from the low molecular weight (4-8 kDa) differentiation factor present in rat serum, FCS, and human serum that promotes the adipogenic conversion and cellular differentiation of 3T3-L1, 3T3-F442A, and Ob17 cells. PSF appears to be a new differentiation factor for rat preadipocytes, has properties suggestive of a highly glycosylated protein, and may be highly species specific.     

Developmentally regulated alternate modes of expression of the Gpdh locus of Drosophila.

Immunoblot analyses have been performed on extracts prepared from Drosophila melanogaster. Those analyses have revealed two subunit forms of enzyme glycerol 3-phosphate dehydrogenase (GPDH) in larval tissues and in adult abdominal tissues. Thoracic tissue, which accounts for the bulk of the adult GPDH, has only one subunit form, the smaller. The two subunit forms differ by approximately 2400 daltons. In agreement with previous genetic and biochemical data indicating that this enzyme is encoded by a single structural gene, analyses of extracts prepared from a strain carrying a GPDH null mutation detect no GPDH polypeptides in larvae or adults. Similarly, analyses of extracts prepared from a strain carrying a mutation which produces a GPDH polypeptide that differs in size from wild-type reveal a change in the adult thoracic GPDH polypeptide as well as a change in both GPDH polypeptides found in larvae. Total Drosophila RNA prepared from larvae or newly eclosed adults has been translated in a mRNA-dependent cell-free system. GDPH was immunoprecipitated from the translation products and analyzed. Two subunit forms of GPDH were immunoprecipitated from translation products whose synthesis was directed by larval RNA and only one was detected in the polypeptides synthesized from adult RNA. The GPDH polypeptides synthesized in vitro are approximately the same size as the corresponding polypeptides found in vivo. The relative proportion of total GPDH represented by each subunit form synthesized in vitro is similar to those found in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyamine metabolism is involved in adipogenesis of 3T3-L1 cells

Polyamines spermidine and spermine are known to be required for mammalian cell proliferation and for embryonic development. Alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC) a limiting enzyme of polyamine biosynthesis, depleted the cellular polyamines and prevented triglyceride accumulation and differentiation in 3T3-L1 cells. In this study, to explore the function of polyamines in adipogenesis, we examined the effect of polyamine biosynthesis inhibitors on adipocyte differentiation and lipid accumulation of 3T3-L1 cells. The spermidine synthase inhibitor trans-4-methylcyclohexylamine (MCHA) increased spermine/spermidine ratios, whereas the spermine synthase inhibitor N-(3-aminopropyl)-cyclohexylamine (APCHA) decreased the ratios in the cells. MCHA was found to decrease lipid accumulation and GPDH activity during differentiation, while APCHA increased lipid accumulation and GPDH activity indicating the enhancement of differentiation. The polyamine-acetylating enzyme, spermidine/spermine N ¹-acetyltransferase (SSAT) activity was increased within a few hours after stimulus for differentiation, and was found to be elevated by APCHA. In mature adipocytes APCHA decreased lipid accumulation while MCHA had the opposite effect. An acetylpolyamine oxidase and spermine oxidase inhibitor MDL72527 or an antioxidant N-acetylcysteine prevented the promoting effect of APCHA on adipogenesis. These results suggest that not only spermine/spermidine ratios but also polyamine catabolic enzyme activity may contribute to adipogenesis.