Standard Isolation of Primary Adipose Cells from Mouse Epididymal Fat Pads Induces Inflammatory Mediators and Down-regulates Adipocyte Genes
2003; Elsevier BV; Volume: 278; Issue: 48 Linguagem: Inglês
10.1074/jbc.m305257200
ISSN1083-351X
AutoresHong Ruan, Mary Jane Zarnowski, Samuel W. Cushman, Harvey F. Lodish,
Tópico(s)Fatty Acid Research and Health
ResumoIsolation and subsequent in vitro culture of primary adipose cells are associated with down-regulation of GLUT4 mRNA and simultaneous induction of GLUT1 gene expression. Progressive loss of insulin-responsive GLUT4 contributes to the decrease in insulin-mediated glucose uptake in these cells when cultured in vitro. The mechanisms underlying these alterations are unknown. Here, we report that the standard procedure for isolating primary adipose cells from mouse adipose tissue triggers induction of many genes encoding inflammatory mediators including TNF-α, interleukin (IL)-1α, IL-6, multiple chemokines, cell adhesion molecules, acute-phase proteins, type I IL-1 receptor, and multiple transcription factors implicated in the cellular inflammatory response. Secretion of TNF-α protein was also significantly induced during the 2-h collagenase digestion of adipose tissue. Isolated primary adipose cells exhibit dramatic changes in expression of multiple mRNAs that are characteristic of TNF-α-treated 3T3-L1 adipocytes including down-regulation of many genes important for insulin action and triglyceride synthesis. Addition of TNF-α to primary adipose cells in culture did not change the kinetics or the extent of the repression of adipose cell-abundant genes. Moreover, TNF-α-neutralizing antibody failed to block the changes in gene transcription in isolated primary adipose cells. Also, the standard isolation procedure induced the expression of NF-κB family members and their target genes in primary adipose cells prepared from TNF-α–/– mice to the same extent as in cells isolated from wild-type mice and resulted in almost identical changes in global gene expression when these cells were cultured in vitro. Thus, these data suggest that the standard isolation procedure-triggered reprogramming of gene expression in primary adipose cells that results in decreased insulin sensitivity does not require TNF-α, at least in this in vitro model system, but may be dependent on other inflammatory cytokines produced by these cells. Isolation and subsequent in vitro culture of primary adipose cells are associated with down-regulation of GLUT4 mRNA and simultaneous induction of GLUT1 gene expression. Progressive loss of insulin-responsive GLUT4 contributes to the decrease in insulin-mediated glucose uptake in these cells when cultured in vitro. The mechanisms underlying these alterations are unknown. Here, we report that the standard procedure for isolating primary adipose cells from mouse adipose tissue triggers induction of many genes encoding inflammatory mediators including TNF-α, interleukin (IL)-1α, IL-6, multiple chemokines, cell adhesion molecules, acute-phase proteins, type I IL-1 receptor, and multiple transcription factors implicated in the cellular inflammatory response. Secretion of TNF-α protein was also significantly induced during the 2-h collagenase digestion of adipose tissue. Isolated primary adipose cells exhibit dramatic changes in expression of multiple mRNAs that are characteristic of TNF-α-treated 3T3-L1 adipocytes including down-regulation of many genes important for insulin action and triglyceride synthesis. Addition of TNF-α to primary adipose cells in culture did not change the kinetics or the extent of the repression of adipose cell-abundant genes. Moreover, TNF-α-neutralizing antibody failed to block the changes in gene transcription in isolated primary adipose cells. Also, the standard isolation procedure induced the expression of NF-κB family members and their target genes in primary adipose cells prepared from TNF-α–/– mice to the same extent as in cells isolated from wild-type mice and resulted in almost identical changes in global gene expression when these cells were cultured in vitro. Thus, these data suggest that the standard isolation procedure-triggered reprogramming of gene expression in primary adipose cells that results in decreased insulin sensitivity does not require TNF-α, at least in this in vitro model system, but may be dependent on other inflammatory cytokines produced by these cells. Insulin resistance is a fundamental defect that precedes the development of the cluster of abnormalities associated with type 2 diabetes (1Boden G. Endocrinol. Metab. Clin. North Am. 2001; 30: 801-815Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 2DeFronzo R.A. Bonadonna R.C. Ferrannini E. Diabetes Care. 1992; 15: 318-368Crossref PubMed Scopus (1903) Google Scholar, 3Goldstein B.J. Am. J. Cardiol. 2002; 90: 3G-10GAbstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 4Olefsky J.M. Ciaraldi T.P. Kolterman O.G. Am. J. Med. 1985; 79: 12-22Abstract Full Text PDF PubMed Scopus (75) Google Scholar). Initially, the reduced insulin sensitivity is compensated by an over-production of insulin from β-cells. When insulin resistance progresses and β-cells are no longer able to produce sufficient insulin, overt type 2 diabetes develops (5LeRoith D. Am. J. Med. 2002; 113: 3S-11SAbstract Full Text Full Text PDF PubMed Google Scholar). Thus, improving the overall in vivo insulin sensitivity appears to be a key factor in the treatment of type 2 diabetes. Previous studies on the beneficial effects of the thiazolidinedione class of insulin-sensitizing compounds in the treatment of type 2 diabetes underscore this notion (6Olefsky J.M. Saltiel A.R. Trends Endocrinol. Metab. 2000; 11: 362-368Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 7Saltiel A.R. Olefsky J.M. Diabetes. 1996; 45: 1661-1669Crossref PubMed Scopus (0) Google Scholar, 8Maggs D.G. Buchanan T.A. Burant C.F. Cline G. Gumbiner B. Hsueh W.A. Inzucchi S. Kelley D. Nolan J. Olefsky J.M. Polonsky K.S. Silver D. Valiquett T.R. Shulman G.I. Ann. Intern. Med. 1998; 128: 176-185Crossref PubMed Scopus (289) Google Scholar). Because obesity with or without overt hyperglycemia is associated with insulin resistance (9Ferrannini E. Metabolism. 1995; 44: 15-17Abstract Full Text PDF PubMed Scopus (89) Google Scholar, 10Walker M. Metabolism. 1995; 44: 18-20Abstract Full Text PDF PubMed Scopus (56) Google Scholar), attention has focused on abnormalities in adipose tissue that could lead to decreased systemic insulin sensitivity. Many adipose cell-secreted factors have been implicated in the pathogenesis of insulin resistance in vivo (11Trayhurn P. Beattie J.H. Proc. Nutr. Soc. 2001; 60: 329-339Crossref PubMed Scopus (939) Google Scholar). Among them, tumor necrosis factor-α (TNF-α) 1The abbreviations used are: TNF-αtumor necrosis factor-αILinterleukinRTreverse transcriptaseNF-κBnuclear factor κBIRSinsulin receptor substratePPAR-γperoxisome proliferator activator receptor-γCEBP-αCCAAT/enhancer binding protein-αTRAFTNF-α receptor-associated factor. is of particular interest, because it is highly induced in adipose tissues of obese animals and human subjects and induces insulin resistance both in cell culture and in vivo (12Moller D.E. Trends Endocrinol. Metab. 2000; 11: 212-217Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar). However, it remains largely unknown which factors and pathways trigger the expression of TNF-α in disease settings such as obesity and diabetes. In addition, other potential adipose tissue-derived autocrine/paracrine and endocrine factors, and the signaling pathways that these factors might utilize to induce insulin resistance in vivo, still remain elusive. tumor necrosis factor-α interleukin reverse transcriptase nuclear factor κB insulin receptor substrate peroxisome proliferator activator receptor-γ CCAAT/enhancer binding protein-α TNF-α receptor-associated factor. One attractive cell model of insulin resistance is primary adipose cells. When these cells are cultured in vitro, they gradually lose the expression of insulin-responsive GLUT4 glucose transporters but increase the synthesis and cell-surface expression of GLUT1 (13Gerrits P.M. Olson A.L. Pessin J.E. J. Biol. Chem. 1993; 268: 640-644Abstract Full Text PDF PubMed Google Scholar). These changes in gene expression contribute to the development of insulin resistance in primary adipose cells in prolonged in vitro culture and mimic the insulin-resistant phenotype in adipose tissue in vivo. Yet the signals, as well as the molecular pathways these signals might use to initiate the changes in adipose cell gene expression, are unknown. Elucidating such mechanisms will provide new insight into our current understanding of the mechanisms of insulin resistance in vivo. We have demonstrated previously (14Ruan H. Hacohen N. Golub T.R. Van Parijs L. Lodish H.F. Diabetes. 2002; 51: 1319-1336Crossref PubMed Scopus (428) Google Scholar) that NF-κB activation in 3T3-L1 adipocytes is obligatory for TNF-α-mediated repression of most adipocyte-abundant genes, as well as induction of many immune response, proinflammatory, and preadipocyte genes (14Ruan H. Hacohen N. Golub T.R. Van Parijs L. Lodish H.F. Diabetes. 2002; 51: 1319-1336Crossref PubMed Scopus (428) Google Scholar). However, whether NF-κB and its upstream activating signals play an essential role in the reprogramming of gene expression in primary adipose cells is not known. To begin to understand the mechanisms underlying the coordinate changes in gene expression in isolated primary adipose cells, we sought to identify the immediate early changes in gene expression and to determine whether TNF-α is involved in initiating these changes. Here, we report that the standard procedure for isolating primary adipose cells from mouse adipose tissue induces genes encoding a variety of inflammatory mediators including TNF-α, IL-1α, IL-6, multiple chemokines, and several transcription factors implicated in the induction of acute-phase cytokines. Secretion of TNF-α protein is also significantly induced during the 2-h collagenase digestion. These inflammatory responses are accompanied by dramatic changes in primary adipose cell gene expression that are characteristic of TNF-α-treated 3T3-L1 adipocytes. Hence, we used adipose cells isolated from TNF-α–/– mice to provide a direct and definitive test for the role of TNF-α in the reprogramming of gene expression in isolated primary adipose cells. Herein we report these results. Isolation and Culture of Primary Adipose Cells—8- to 9-week-old male C57BL/6J and FVB mice were used to obtain primary adipose cells essentially as described previously (15Malide D. Ramm G. Cushman S.W. Slot J.W. J. Cell Sci. 2000; 113 (Pt 23): 4203-4210Crossref PubMed Google Scholar). Briefly, the epididymal fat pads were removed, minced, and digested using collagenase at 37 °C for 2 h. The primary adipose cells were then washed extensively and incubated at 37 °C in a KRBH buffer (Krebs-Ringer-bicarbonate-HEPES buffer, pH = 7.4) or Dulbecco's modified Eagle's medium containing 5% bovine serum albumin. Primary adipose cells and conditioned medium were taken at various times as indicated in the figure legends and were flash-frozen in liquid nitrogen and stored in –80 °C until use. Antibody—TNF-α neutralizing antibody was purchased from R&D Systems (catalog number AF-410-NA; Minneapolis, MN). The manufacturer selected this line of TNF-α neutralizing antibody by functional screening and assessed the specificity of the antibody by showing that it does not cross-react with other cytokines tested so far. When used at 0.02–0.05 μg/ml, this antibody caused 50% inhibition of the biological effects of TNF-α activity at a concentration of 0.25 ng/ml, as demonstrated by the mouse L-929 cytotoxicity assay (according to R&D product sheet for AF-410-NA). Enzyme-linked Immunosorbent Assay—Conditioned media were collected from various primary adipose cell cultures. The protein levels of TNF-α and IL-6 were determined using Quantikine M kits from R&D Systems. Real-time Quantitative RT-PCR—Total RNA was isolated from fresh epididymal fat pads, primary fat cells immediately after collagenase digestion, and collagen-digested primary adipose cells cultured in vitro for up to 24 h. mRNA was quantified by Taqman chemistry-based method using a ABI 7000 sequence detection system (Applied Biosystems, Foster City, CA). All primers and probes were purchased from Applied Biosystems. Reagents for 18 S ribosomal RNA or tubulin were used as control to normalize RNA sample loading. Each standard curve was linear between 0.02 and 200 ng of total RNA, a 104-fold range that covers the concentrations of any particular mRNA transcript in all of the samples. The efficiencies of amplification were identical or similar between genes of interest and controls. Oligonucleotide Microarray Data Collection and Analysis—Total RNA was prepared from various samples as indicated above, converted to biotin-labeled cRNA targets, hybridized to MG74AV2 oligonucleotide microarrays (Affymetrix, Santa Clara, CA), and scanned on Affymetrix scanners essentially as described previously (14Ruan H. Hacohen N. Golub T.R. Van Parijs L. Lodish H.F. Diabetes. 2002; 51: 1319-1336Crossref PubMed Scopus (428) Google Scholar, 16Ruan H. Miles P.D.G. Ladd C.M. Ross K. Golub T.R. Olefsky J.M. Lodish H.F. Diabetes. 2002; 51: 3176-3188Crossref PubMed Scopus (228) Google Scholar). Array measurements for all samples were normalized with arrays hybridized with cRNA targets prepared from the appropriate control samples by using the median of the hybridization signals of all genes with P calls (P-call, according to Affymetrix algorithm) as a scaling factor. We compared gene expression profiles of freshly isolated primary adipose cells with cells that have been cultured in vitro for up to 24 h. We used a self-organizing feature map method (SOM) (17Kohonen T. Proc. IEEE. 1991; 78: 1464-1480Crossref Scopus (6088) Google Scholar, 18Tamayo P. Slonim D. Mesirov J. Zhu Q. Kitareewan S. Dmitrovsky E. Lander E.S. Golub T.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2907-2912Crossref PubMed Scopus (2414) Google Scholar) to analyze and cluster genes that were induced or repressed in primary adipose cells as a function of time. Changes in the steady-state mRNA levels of several genes identified by oligonucleotide microarray were confirmed using real-time quantitative RT-PCR or semi-quantitative RT-PCR. Standard Procedure for Isolating Primary Adipose Cells from Adipose Tissue Induces TNF-α mRNA and Triggers TNF-α Secretion from Isolated Adipose Cells—Following a standard protocol for preparing a primary adipose cell culture, we digested freshly harvested mouse epididymal fat pads with crude collagenase at 37 °C for 2 h, followed by extensive washes using KRBH buffer supplemented with 5% bovine serum albumin. Because the ectodomains of certain plasma membrane proteins including TNF-α can be readily released by proteolysis, and the resulting bioactive molecules may potentially have significant impact on adipose cell biology and function, we monitored the production of TNF-α during and after collagenase treatment by determining the concentration of TNF-α in the supernatant of the digestion mixture and subsequent cell culture. After collagenase addition to mouse adipose tissue equivalent to ∼106 adipose cells/ml, the concentration of TNF-α in the conditioned medium increased dramatically within 1 h, reaching 0.93 ng/ml (Fig. 1A, top panel). At the end of the 2-h collagenase digestion, the level of TNF-α increased to 1.9 ng/ml. Notably, when the isolated adipose cells were washed extensively after collagenase digestion and incubated in growth medium at a concentration of ∼2 × 105 cells/ml, a steady but lower release of TNF-α continued as measured by the increasing amount of TNF-α in the conditioned medium (Fig. 1A, top panel). At the end of the incubation, the concentration of TNF-α in conditioned media reached 0.17 ng/ml. In contrast, this standard isolation procedure did not affect the secretion of IL-6, as no detectable IL-6 protein was present in the supernatant of the digestion mixture throughout the 2-h collagenase treatment (Fig. 1A, bottom panel). To determine whether adipose tissue expresses the IL-6 gene, we first assessed IL-6 mRNA levels using oligonucleotide microarrays. IL-6 mRNA was barely detectable in fresh adipose tissue but was highly induced at the end of the collagenase digestion (Table I). The IL-6 transcript remained at high levels when the isolated primary adipose cells were cultured in vitro for up to 24 h (see below). Consistent with the expression kinetics of IL-6 mRNA, the protein levels of IL-6 increased significantly in the conditioned medium 2 h after in vitro culture, peaking at the end of the 24-h incubation with a concentration of 4 ng/ml (2 × 105 cells/ml; Fig. 1A, bottom panel). IL-6 is normally induced by nuclear factor-κB (NF-κB), which is activated in response to a variety of extracellular stimuli such as tissue injury, oxidative stress, and inflammatory cytokines. Because TNF-α is secreted by adipose cells during collagenase digestion (Fig. 1A, top panel), TNF-α-mediated NF-κB activation is thus a potential inducer of IL-6 gene expression in the cultured primary adipose cells. The delayed kinetics of IL-6 induction in adipose cells during collagenase treatment and isolation, compared with that of TNF-α, is in accordance with this potential relationship. However, other adipose cell-secreted factors triggered by collagenase digestion and/or oxidative stress could also play a role in the induction of IL-6 mRNA and protein secretion (see below).Table IStandard procedure for isolating primary adipose cells leads to induction of multiple mRNAs encoding inflammatory mediatorsGenBank™ accession numberGene nameBefore collagenase digestionAfter collagenase digestionFold inductionChemokines J04596Chemokine (C-X-C motif) ligand 178516766.2 X53798Chemokine (C-X-C motif) ligand 2N.D.655* U27267Chemokine (C-X-C motif) ligand 5N.D.50* M33266Chemokine (C-X-C motif) ligand 10N.D.208* L12030Chemokine (C-X-C motif) ligand 121433502.4 AV139913Chemokine (C-X-C motif) ligand 12N.D.201* M19681Chemokine (C-C motif) ligand 2239349914.6 J04491Chemokine (C-C motif) ligand 3N.D.133* X62502Chemokine (C-C motif) ligand 4N.D.124* X70058Chemokine (C-C motif) ligand 7766288.3 U77462Chemokine (C-C motif) ligand 11762002.6Cytokines M14639Interleukin 1-αN.D.50* X54542Interleukin 6N.D.663*Chemokine and cytokine receptors M20658Interleukin 1 receptor, type I382516.6 Z80112Chemokine (C-X-C motif) receptor 432672.1 AF022990Chemokine (C-C motif) receptor 5N.D.50*Cell Adhesion Molecules M72332Selectin, plateletN.D.519* M80778Selectin, endothelial cellN.D.127* M90551Intercellular adhesion moleculeN.D.576* X53177Integrin α-4N.D.60* U12884Vascular cell adhesion molecule 1331203.6Acute phase proteins U60438Serum amyloid A2N.D.344* X03505Serum amyloid A3N.D.1237* U77630Adrenomedullin4646010 M28845Early growth response 152829055.5 X67644Immediate early response 323621559.1 X83601Pentaxin-related geneN.D.2289*Transcription factors AF017128fos-like antigen 1N.D.218* M21065Interferon regulatory factor 1204243511.9 X61800C/EBP-δ39210652.7 M61007C/EBP-β51712782.5 U20735Jun-B oncogene36614093.8 X12761Jun oncogene1876883.7 AI837104STAT 32224462.0Control mRNAs X56123Talin3663621.0 M28729Tubulin-α1498557031.1 M28739Tubulin, β26576351.0 M12481β-Actin198112860.65 Z48745ATP-binding cassette 82692330.9 M19381Calmodulin271324340.9 M73329Phospholipase C-α5565020.9 M17516Lactate dehydrogenase A-4531748270.9 X66405Procollagen, type VI, α-1194718691.0 M18194Fibronectin4724741.0 AB0056231-Acyl-sn-glycerol-3-phosphate acyltransferase3133191.0 U35312Nuclear receptor co-repressor2812881.0 Open table in a new tab In many cell types such as immune cells, TNF-α is not expressed in resting cells but is rapidly induced in response to a number of extracellular stimuli such as pathogens. The induction of TNF-α in these cells is independent of de novo protein synthesis. To determine whether TNF-α gene transcription in adipose cells is induced by the standard isolation procedure including collagenase treatment, we used real-time quantitative RT-PCR to measure the steady state levels of TNF-α mRNA in fresh adipose tissue, as well as in isolated adipose cells after collagenase digestion. TNF-α mRNA is readily detectable in epididymal fat pads freshly harvested from lean mice and is induced over 420-fold following a 2-h collagenase treatment. The levels of TNF-α transcript remained at this high level even 4 h after completing the collagenase digestion. Notably, although the levels of TNF-α mRNA dropped 90% when cells were incubated in growth medium for 24 h, the TNF-α transcript still remained about 40-fold higher than fresh adipose tissue (Fig. 1B). Thus, our data indicate that the standard method for isolating primary adipose cells from adipose tissue triggers TNF-α gene transcription and protein secretion and that adipose cell-derived TNF-α may potentially affect the expression of at least a set of genes, including IL-6, in isolated primary adipose cells in culture. Standard Procedure for Isolating Primary Adipose Cells from Adipose Tissue Triggers Major and Rapid Changes in Gene Expression That Are Characteristic of TNF-α-treated 3T3-L1 Adipocytes—To determine whether adipose cell gene transcription in general is significantly altered during standard preparation of isolated cells, we examined gene expression profiles in isolated adipose cells cultured in vitro for up to 24 h. We performed a self-organizing feature map (SOM) analysis of the data collected from oligonucleotide microarrays to identify clusters of genes with distinct patterns of expression kinetics during the 24-h period. The SOM analysis shows that this standard procedure for isolation of primary adipose cells evoked major and rapid changes in adipocyte gene expression, including down-regulation of essential adipose cell-abundant genes and up-regulation of immune response and pre-adipocyte genes. Strikingly, the isolated adipose cells exhibited distinct patterns of gene expression kinetics that are identical to those seen in 3T3-L1 adipocytes treated with TNF-α. Here, we focused on a subset of genes whose encoded proteins play a critical role in adipocyte function. First, we assessed the expression pattern of key adipocyte proteins that are essential for insulin-stimulated glucose uptake and metabolism. As shown in Fig. 2, the steady state mRNA levels of insulin receptor substrate-2 (IRS-2), phosphatidylinositol 3-kinase, AKT-2, and c-Cbl-associated protein, whose encoded proteins are involved in transducing insulin signals in adipose cells, were down-regulated 80% or more by 24 h in culture relative to the beginning of the incubation (Fig. 2, A–D, empty squares). Similarly, the mRNA levels of many effector molecules of insulin signaling, such as GLUT4, glycogen synthase, fatty acid synthase, phosphoenolpyruvate carboxykinase, glycerol-3-phosphate acyltransferase, and diacylglycerol acyltransferase were repressed at least 80% compared with the beginning of the incubation (Fig. 2, E–J, empty squares). In addition, the mRNA levels of two adipocyte master transcription factors, PPAR-γ and CEBP-α, were repressed 95 and 77%, respectively, in primary adipose cells after a 24-h culture in vitro (Fig. 2, K–L, empty squares). In addition to proteins involved in energy metabolism, the expression of many adipose cell-secreted factors was altered when primary adipose cells were cultured in vitro (Fig. 3, A–D, empty squares). Among them, the expression levels of leptin, resistin, and ACRP30 were all down-regulated whereas the mRNA level of IL-6 did not further increase from the already high level induced by the collagenase treatment (Table I). In contrast, other TNF-α family members such as Fas antigen and many proteins involved in TNF-α signaling including TNF-α receptor-associated proteins (TRAF-2, 3, and 4) and TNF-α receptor-2 were highly induced when adipose cells were cultured in vitro (Fig. 3, F–J, empty squares). Notably, whereas adipocyte-abundant AKT-2 (19Hill M.M. Clark S.F. Tucker D.F. Birnbaum M.J. James D.E. Macaulay S.L. Mol. Cell Biol. 1999; 19: 7771-7781Crossref PubMed Google Scholar, 20Cho H. Mu J. Kim J.K. Thorvaldsen J.L. Chu Q. Crenshaw III, E.B. Kaestner K.H. Bartolomei M.S. Shulman G.I. Birnbaum M.J. Science. 2001; 292: 1728-1731Crossref PubMed Scopus (1558) Google Scholar) mRNA was repressed in cultured primary adipose cells, the mRNA encoding AKT-1, a ubiquitously expressed isoform of AKT, increased steadily (Fig. 3E, empty squares). This AKT isoform switch may contribute, in part, to the loss of insulin response in cultured primary adipose cells. We have shown previously that in 3T3-L1 adipocytes NF-κB activation is obligatory for TNF-α-mediated repression of most of the adipocyte-abundant genes and induction of a subset of genes. Among the 22 genes shown in Figs. 2 and 3, the mRNA levels of phosphatidylinositol 3-kinase, AKT-2, c-Cbl-associated protein, GLUT4, glycogen synthase, phosphoenolpyruvate carboxykinase, glycerol-3-phosphate acyltransferase, diacylglycerol acyltransferase, PPAR-γ, and CEBP-α were all repressed by TNF-α in 3T3-L1 adipose cells in a NF-κB-dependent manner, because the expression levels of these genes were unaffected by TNF-α in 3T3-L1 adipose cells expressing a dominant inhibitor of NF-κB activation (IκBα-DN). Similarly, induction of Fas antigen by TNF-α requires NF-κB activation. 2H. Ruan and H. F. Lodish, unpublished data. TNF-α also repressed the expression of fatty acid synthase, resistin, and ACRP30 and induced TNF-R2 and TRAF-4 in 3T3-L1 adipocytes after multiple hours of incubation. However, TNF-α caused extensive cell death in adipose cells expressing IκBα-DN after2hof incubation, and so we could not determine whether the effects of TNF-α on the expression of these genes were dependent on NF-κB activation. The hybridization signals for the remaining six genes (IRS-2, leptin, IL-6, AKT-1, TRAF-2, and TRAF-3) were low in our previous experiments on TNF-α-treated 3T3-L1 adipocytes, and we did not further investigate the cause for this observation, although technical limitations are likely to be involved. Nevertheless, these data indicate that the standard procedure for isolating primary adipose cells from adipose tissue triggers rapid reprogramming of adipose cell gene expression that is characteristic of TNF-α-treated 3T3-L1 adipocytes. TNF-α Is Not Necessary for the Reprogramming of Gene Expression in Isolated Primary Adipose Cells—Because the isolated primary adipose cells exhibited dramatic changes in gene expression that were similar to the TNF-α-induced changes in 3T3-L1 adipocytes, and because significant amounts of TNF-α were secreted from primary adipose cells during collagenase digestion, we hypothesized that primary adipose cell-derived TNF-α triggers the changes in global gene expression during isolation of primary adipose cells. To test this hypothesis, we first determined whether the dynamics of the primary adipose cell response to the standard isolation procedures, especially the collagenase exposure, would be further altered by the addition of TNF-α. We assessed the expression patterns of key adipocyte genes in collagenase-digested primary adipose cells incubated with TNF-α (1 nmol/liter) for up to 24 h. As shown in Figs. 2 and 3 (filled triangles), the expression kinetics of genes that are normally induced or repressed in primary adipose cells during in vitro incubation were not significantly affected by the presence of exogenous TNF-α, although the induction of some genes was further enhanced by TNF-α (data not shown). This suggests that the standard method for adipose cell isolation may activate the same pathways utilized by TNF-α to affect adipocyte gene expression. Alternatively, the standard isolation procedure could trigger cellular pathways in addition to those activated by TNF-α, and these different pathways could converge at the level of transcriptional machinery and result in transcriptional changes that are characteristic of TNF-α. Because primary adipose cells continue to release a significant amount of TNF-α when they are cultured in vitro, we used a TNF-α neutralizing antibody to test whether the TNF-α secreted by isolated adipose cells in culture is responsible for the changes in gene expression. As shown in Fig. 4, A and B, semi-quantitative RT-PCR analysis of mRNA levels indicates that glyceraldehyde 3-phosphate dehydrogenase was down-regulated, and GLUT1 was induced in primary adipose cells following a 24-h incubation in vitro (compare lanes 1 with lanes 2 in Fig. 4, A and B), consistent with our microarray data. Addition of increasing amounts of TNF-α-neutralizing antibody (0.008–0.4 μg/ml) did not block the changes of the expression of these two representative genes (compare lanes 3–6 with lanes 2 in Fig. 4, A and B). According to the efficacy of the TNF-α neutralizing antibody, and as described under "Experimental Procedures," the highest amount of antibody we used in this study is sufficient to fully inhibit the biological activities of TNF-α at a concentration of 0.75 ng/ml, which is about 4-fold the concentration seen at the end of the 24-h incubation period (Fig. 1A, top panel). Thus, our data suggest that TNF-α produced during incubation of adipose cells did not significantly contribute to the induction of the changes in global gene transcription in adipose cells. Because TNF-α was highly induced during collagenase digestion (Fig. 1A, top panel), it is possible that this brief exposure to high levels of TNF-α during collagenase treatment is sufficient to trigger major changes in gene expression in primary adipose cells. Thus, we used primary adipose cells isolated from TNF-α–/– mice to ascertain whether TNF-α is responsible for initiating the reprogramming of gene expression in isolated primary adipose cells. We first confirmed that TNF-α was indeed absent in primary adipose cells derived from TNF-α–/– mice. As shown in Fig. 5A
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