Key points are not available for this paper at this time.
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. 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 whether TNF-α is in these Here, we report that the standard procedure for isolating primary adipose cells from mouse adipose tissue induces genes encoding a of inflammatory mediators including TNF-α, IL-6, multiple chemokines, and transcription factors implicated in the induction of acute-phase Secretion of TNF-α protein is also significantly induced during the 2-h collagenase These inflammatory are by dramatic changes in primary adipose cell gene expression that are characteristic of TNF-α-treated 3T3-L1 we used adipose cells isolated from TNF-α–/– mice to provide a and for the role of TNF-α in the reprogramming of gene expression in isolated primary adipose cells. we report these Isolation and of to and mice were used to primary adipose cells as previously D. G. J. 2000; PubMed Google Scholar). the were and collagenase at for 2 The primary adipose cells were and at in a or adipose cells and were at as in the and were in and in antibody was from The this of TNF-α antibody by and the of the antibody by that it does not with other cytokines When used at this antibody of the effects of TNF-α at a of as demonstrated by the mouse to for were from primary adipose cell The protein of TNF-α and were from was isolated from primary cells collagenase and primary adipose cells cultured in vitro for to mRNA was by a and were from for or were used as to standard was and of a that the of particular mRNA in of the The of were identical or genes of and and was prepared from as to to and on as previously (14Ruan H. Hacohen N. Golub T.R. Van Parijs L. Lodish H.F. Diabetes. 2002; 51: 1319-1336Crossref PubMed Scopus (428) Google Scholar, H. Golub T.R. Olefsky J.M. Lodish H.F. Diabetes. 2002; 51: PubMed Scopus Google Scholar). for were with with prepared from the by the of the signals of genes with to as a factor. We gene expression of isolated primary adipose cells with cells that have been cultured in vitro for to We used a Proc. Scopus Google Scholar, P. D. J. S. E. Golub T.R. Proc. S. PubMed Scopus Google Scholar) to and cluster genes that were induced or in primary adipose cells as a of in the mRNA of genes by were or for from TNF-α mRNA and TNF-α Secretion from Isolated a standard for a primary adipose cell we mouse with collagenase at for 2 by with Because the of including TNF-α be by and the may have on adipose cell and we the of TNF-α during and collagenase treatment by the of TNF-α in the of the digestion and subsequent cell collagenase to mouse adipose tissue to adipose the of TNF-α in the the of the 2-h collagenase the of TNF-α to when the isolated adipose cells were collagenase digestion and in at a of a but of TNF-α as by the of TNF-α in the the of the the of TNF-α in In this standard isolation procedure did not the of IL-6, as no protein was in the of the digestion the 2-h collagenase treatment To whether adipose tissue the we mRNA mRNA was in adipose tissue but was highly induced at the of the collagenase digestion The at when the isolated primary adipose cells were cultured in vitro for to with the expression kinetics of the protein of significantly in the 2 in vitro at the of the with a of is induced by nuclear which is in to a of such as tissue and inflammatory Because TNF-α is by adipose cells during collagenase digestion TNF-α-mediated NF-κB activation is a potential of gene expression in the cultured primary adipose cells. The kinetics of induction in adipose cells during collagenase treatment and with that of TNF-α, is in with this potential However, other adipose cell-secreted factors by collagenase digestion could also play a role in the induction of mRNA and protein procedure for isolating primary adipose cells to induction of multiple mRNAs encoding inflammatory collagenase collagenase and receptor, type receptor receptor adhesion cell adhesion early factors factor mRNAs type receptor in a new In many cell such as immune cells, TNF-α is not in cells but is induced in to a of such as The induction of TNF-α in these cells is of protein synthesis. To whether TNF-α gene transcription in adipose cells is induced by the standard isolation procedure including collagenase we used to the of TNF-α mRNA in adipose as well as in isolated adipose cells collagenase TNF-α mRNA is in from mice and is induced a 2-h collagenase The of TNF-α at this the collagenase the of TNF-α mRNA when cells were in for the TNF-α still adipose tissue Thus, our data that the standard for isolating primary adipose cells from adipose tissue triggers TNF-α gene transcription and protein and that adipose TNF-α may the expression of at least a of genes, including IL-6, in isolated primary adipose cells in for from and in of TNF-α-treated 3T3-L1 whether adipose cell gene transcription in is significantly during standard of isolated cells, we gene expression in isolated adipose cells cultured in vitro for to We a of the data from to identify of genes with of expression kinetics during the The that this standard procedure for isolation of primary adipose cells and changes in gene expression, including down-regulation of essential adipose cell-abundant genes and of immune and genes. the isolated adipose cells of gene expression kinetics that are identical to in 3T3-L1 adipocytes with Here, we focused on a of genes play a role in we the expression of key that are essential for glucose uptake and in the mRNA of insulin receptor and are in insulin signals in adipose cells, were or by in culture to the of the the mRNA of many of insulin such as and were at least with the of the In addition, the mRNA of transcription factors, and were and in primary adipose cells a culture in vitro In to in the expression of many adipose cell-secreted factors was when primary adipose cells were cultured in vitro Among them, the expression of and were the mRNA of did not increase from the induced by the collagenase treatment In other TNF-α family members such as and many in TNF-α signaling including TNF-α receptor-associated and and TNF-α were highly induced when adipose cells were cultured in vitro adipocyte-abundant D.E. Biol. PubMed Google Scholar, H. J. Shulman G.I. 2001; PubMed Scopus Google Scholar) mRNA was in cultured primary adipose cells, the mRNA encoding a of may in to the loss of insulin in cultured primary adipose cells. We have 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 of genes. Among the genes in 2 and the mRNA of and were by TNF-α in 3T3-L1 adipose cells in a because the expression of these genes were by TNF-α in 3T3-L1 adipose cells a of NF-κB activation induction of by TNF-α NF-κB and H. TNF-α also the expression of and and induced and in 3T3-L1 adipocytes multiple of However, TNF-α cell in adipose cells and we could not whether the effects of TNF-α on the expression of these genes were dependent on NF-κB The signals for the genes IL-6, and were in our on TNF-α-treated 3T3-L1 and we did not the for this are to be these data that the standard procedure for isolating primary adipose cells from adipose tissue triggers reprogramming of adipose cell gene expression that is characteristic of TNF-α-treated 3T3-L1 TNF-α for the of in Isolated the isolated primary adipose cells dramatic changes in gene expression that were to the changes in 3T3-L1 and because of TNF-α were from primary adipose cells during collagenase we that primary adipose TNF-α triggers the changes in global gene expression during isolation of primary adipose cells. To this we whether the of the primary adipose cell to the standard isolation the collagenase be by the of We the expression of key genes in primary adipose cells with TNF-α for to in 2 and the expression kinetics of genes that are induced or in primary adipose cells during in vitro were not significantly by the of TNF-α, the induction of genes was by TNF-α not that the standard for adipose cell isolation may the same pathways by TNF-α to gene expression. the standard isolation procedure could trigger cellular pathways in to by TNF-α, and these pathways could at the of and in changes that are characteristic of Because primary adipose cells to a of TNF-α when they are cultured in vitro, we used a TNF-α antibody to whether the TNF-α by isolated adipose cells in culture is for the changes in gene expression. in and of mRNA that was and GLUT1 was induced in primary adipose cells a in vitro with 2 in and with our Addition of of TNF-α-neutralizing antibody did not block the changes of the expression of these genes with 2 in and to the of the TNF-α and as the of antibody we used in this is sufficient to the of TNF-α at a of which is the at the of the Thus, our data suggest that TNF-α produced during 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 it is that this to of TNF-α during collagenase treatment is sufficient to trigger changes in gene expression in primary adipose cells. Thus, we used primary adipose cells isolated from TNF-α–/– mice to whether TNF-α is for the reprogramming of gene expression in isolated primary adipose cells. We that TNF-α was in primary adipose cells from TNF-α–/– in primary adipose cells isolated from wild-type mice of TNF-α at the of the standard collagenase When these primary adipose cells were and cultured in vitro, the of TNF-α in the at the of the However, the of TNF-α was as in adipose cells from TNF-α–/– mice we the of IL-6, which is induced by a of signals including TNF-α, from wild-type and TNF-α–/– adipose cells. the of was induced to the same extent in TNF-α–/– adipose cells as that in wild-type adipose cells We the gene expression of adipose cells from TNF-α–/– and wild-type mice these cells been cultured for in vitro. identical of changes in gene expression were in both of adipose cells The of TNF-α did not isolation reprogramming of gene expression, including down-regulation of key genes and induction of genes encoding inflammatory NF-κB was in both TNF-α–/– and wild-type adipose cells, as by the induction of members of the NF-κB family including and and NF-κB target genes such as cell adhesion and I and procedure for isolating primary adipose cells triggers identical of changes in gene expression in cells prepared from wild-type or TNF-α adipose tissue protein proliferator activator protein of TNF-α receptor, type cell adhesion activator of to in a new Thus, our data suggest that TNF-α may contribute to the induction of changes in global gene expression during isolation of primary adipose cells, other isolation procedure-triggered pathways also play a the in NF-κB activation and the induction of the changes in adipose cell gene expression that in decreased insulin sensitivity. for to of mRNAs in Addition to an in factors that may NF-κB global gene expression in isolated primary adipose cells, we the mRNA of important chemokines, and members of the TNF-α that have been implicated in the inflammatory and NF-κB The results in I that the mRNAs encoding chemokines, IL-6, TNF-α, and the type I IL-1 receptor are during the 2-h collagenase in to the of of mRNAs signals to the mRNAs encoding TNF-α members were not in the prepared from primary adipose cells not genes encoding transcription factors that have been implicated in the induction of synthesis of cytokines were also induced in primary adipose cells during the 2-h collagenase Among them, and mRNA were induced and These contribute to the induction of TNF-α, IL-6, and other cytokines in and N. S. Proc. S. 1993; 90: PubMed Scopus Google Scholar, J. Clin. PubMed Scopus Google Scholar, J. Google Scholar, 1995; PubMed Scopus Google Scholar), and may in the induction of cytokines in adipose cells, as transcription factors including NF-κB family members and and of transcription and also induced in primary adipose cells the 2-h collagenase digestion are in the induction of the cytokines Thus, our data suggest that the standard collagenase procedure for isolating primary adipose cells triggers induction of a of inflammatory mediators that could have on NF-κB activation and on gene expression. data that of TNF-α and at collagenase is not to the adipose tissue to the isolation procedure-triggered effects on adipose cell gene expression may not collagenase but of adipose tissue from the We report changes in primary adipose cells a standard isolation procedure collagenase we for the that the standard procedure for isolating primary adipose cells is a of TNF-α gene transcription and protein as well as multiple other important inflammatory we that the isolation procedure changes in global gene expression in primary adipose cells that are characteristic of TNF-α-treated 3T3-L1 we that adipose TNF-α, which was highly induced during the standard isolation is for the reprogramming of gene expression in cultured primary adipose cells and that primary adipose cells in gene transcription in to a of inflammatory adipose cells have been used as a model to the of adipose cells for Diabetes. PubMed Scopus Google Scholar). is that isolated primary adipose cells gradually lose their to insulin when cultured in vitro, as by decreased GLUT4 and glucose uptake and These alterations in cultured primary adipose cells mimic the development of insulin resistance in vivo. Thus, it is of both and to identify the factors that initiate these alterations and in insulin resistance in isolated primary adipose cells. of the insulin receptor of have been implicated as a potential for the decreased insulin in cells. Here, we demonstrated that the mRNA of genes encoding these and other essential of the as well as the including were significantly in primary adipose cells this standard isolation the standard isolation procedure resulted in changes in global gene expression, including down-regulation of adipocyte-abundant genes and of immune response, and preadipocyte genes, that in cell and of insulin resistance such as TNF-α-treated 3T3-L1 adipocytes and Because TNF-α induces changes in gene expression both in cell culture and in vivo, and because a of TNF-α is from primary adipose cells during the standard isolation we that primary adipose cell-secreted TNF-α is for the changes in global gene expression in adipose cells. However, of TNF-α antibody to the primary adipose cell culture collagenase digestion no on the changes in adipose cell gene the same changes were in TNF-α–/– Thus, our data provide and that TNF-α is not the or the primary of the changes in primary adipose cells. We to identify potential mediators that might initiate the changes in primary adipose cells the standard isolation We that a of inflammatory mediators were highly induced in primary adipose cells during the 2-h collagenase which mimic the that from tissue such as and Among the inflammatory mediators that are induced in the primary adipose cells at the of the 2-h collagenase is to multiple cellular pathways including 2 and and L. Scholar). The activation of NF-κB to induction of many cytokines and cell receptor proteins, as well as repression of adipocyte-abundant genes (14Ruan H. Hacohen N. Golub T.R. Van Parijs L. Lodish H.F. Diabetes. 2002; 51: 1319-1336Crossref PubMed Scopus (428) Google Scholar), and is sufficient to gene transcription in gene H. Lodish H.F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The and protein have also been implicated in the of gene transcription and the mRNA of multiple genes in L. M. 2001; PubMed Scopus Google Scholar, 1995; PubMed Scopus Google Scholar, J. Biol. 1996; PubMed Scopus Google Scholar). these cellular protein the and in NF-κB activation J. Full Text Full Text PDF PubMed Scopus Google Scholar). we have 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 is for TNF-α-mediated repression of key genes in 3T3-L1 adipocytes but that induction of many transcription factors and genes is not dependent on other potential factors, by the inflammatory cytokines that are induced during collagenase treatment of primary may play an important role in reprogramming gene expression in primary adipose cells, as the other the of many cytokines and including IL-6, and TNF-α be by IL-1 TNF-α in an or The could be and from the adipose tissue. In transcription factors implicated in the induction of the inflammatory cytokines such as and of transcription and are also in primary adipose cells the standard isolation a potential and multiple signaling The induction of multiple cytokines and inflammatory mediators with in primary adipose cells potential for the that TNF-α is not essential for induction of the coordinate changes in gene expression during in vitro culture of primary adipose cells the standard isolation The of which inflammatory is for the reprogramming of gene expression in primary adipose cells in animals protein or a of inflammatory of the role of inflammatory mediators in the of gene expression in primary adipose cells, to their potential in the repression of adipocyte-abundant genes, are from the and of the cytokines and their signaling Thus, in the current we could not which inflammatory is in the changes in adipose cell gene expression. our data at least that the inflammatory mediators produced by cell the adipose tissue the standard isolation procedure contribute to the activation of NF-κB and other transcription that in repression of the adipocyte-abundant genes in primary adipose cells cultured in vitro. The primary of the of to the changes in global gene expression in primary adipose cells is to the in adipose tissue during the standard isolation because our data that such changes in adipose cell gene transcription does not require collagenase treatment but may of adipose tissue from the In to a of such as and tissue and cells initiate acute-phase inflammatory in adipose immune cells a of inflammatory mediators including many and that trigger a of changes in to the the activation of multiple cellular mediators including NF-κB and other transcription In our data provide that adipose tissue is a of inflammatory mediators and that adipose cells are highly to a of inflammatory These are because they suggest that factors, including TNF-α, may in to the development of insulin resistance in a of settings cellular we that multiple inflammatory factors may trigger the activation of NF-κB and other transcription and induce the changes in global gene expression in isolated primary adipose cells, our does not TNF-α as an important in our data provide a for of the role of factors in the induction of insulin resistance in vivo and for the potential of the of NF-κB in the treatment of insulin resistance in the of obesity and type 2 diabetes.
Ruan et al. (Sat,) studied this question.