Cytokines Promote Wnt Signaling and Inflammation and Impair the Normal Differentiation and Lipid Accumulation in 3T3-L1 Preadipocytes

Abstract

Obesity with enlarged fat cells is associated with high local concentrations of interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) in the adipose tissue. We examined the effects of this inflammatory state on 3T3-L1 preadipocyte development and differentiation to mature adipose cells. Both IL-6 and TNFα impaired the normal differentiation pattern and lipid accumulation. However, IL-6 allowed a normal early induction of differentiation with inhibition of Wnt10b and Pref-1, whereas expression of CCAAT/enhancer-binding protein α, in contrast to peroxisome proliferator-activated receptor γ, was markedly reduced. TNFα also allowed a normal early induction of differentiation, whereas the terminal differentiation to adipose cells was completely prevented. However, both cytokines induced an inflammatory phenotype of the cells but with different profiles. Remarkably, both IL-6 and TNFα maintained and augmented the canonical Wnt signaling associated with low axin and high low density lipoprotein receptor-related protein (LRD), Dishevelled, and β-catenin levels. TNFα, but not IL-6, activated Wnt10b expression, whereas IL-6 increased the apparent phosphorylation of Dishevelled. Thus, both IL-6 and TNFα prevent the normal development of preadipocytes to fully differentiated adipose cells and, instead, promote an inflammatory phenotype of the adipocytes. These results provide an explanation as to why obesity and diabetes are associated with both local and systemic inflammation, insulin resistance, and ectopic lipid accumulation. Obesity with enlarged fat cells is associated with high local concentrations of interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) in the adipose tissue. We examined the effects of this inflammatory state on 3T3-L1 preadipocyte development and differentiation to mature adipose cells. Both IL-6 and TNFα impaired the normal differentiation pattern and lipid accumulation. However, IL-6 allowed a normal early induction of differentiation with inhibition of Wnt10b and Pref-1, whereas expression of CCAAT/enhancer-binding protein α, in contrast to peroxisome proliferator-activated receptor γ, was markedly reduced. TNFα also allowed a normal early induction of differentiation, whereas the terminal differentiation to adipose cells was completely prevented. However, both cytokines induced an inflammatory phenotype of the cells but with different profiles. Remarkably, both IL-6 and TNFα maintained and augmented the canonical Wnt signaling associated with low axin and high low density lipoprotein receptor-related protein (LRD), Dishevelled, and β-catenin levels. TNFα, but not IL-6, activated Wnt10b expression, whereas IL-6 increased the apparent phosphorylation of Dishevelled. Thus, both IL-6 and TNFα prevent the normal development of preadipocytes to fully differentiated adipose cells and, instead, promote an inflammatory phenotype of the adipocytes. These results provide an explanation as to why obesity and diabetes are associated with both local and systemic inflammation, insulin resistance, and ectopic lipid accumulation. Adipose tissue is the major organ for storing and releasing surplus energy. Inability of the adipose cells to take up and store lipids, as seen in lipoatrophic and lipodystrophic conditions, is associated with the accumulation of ectopic triglycerides in the liver and skeletal muscles, insulin resistance, and diabetes (1Perseghin G. Petersen K. Shulman G.I. Int. J. Obes. Relat. Metab. Disord. 2003; 27 (Suppl. 3): S6-S11Crossref PubMed Scopus (190) Google Scholar). Transplantation of fat to animal models of lipoatrophy reverses these conditions (2Kim J.K. Gavrilova O. Chen Y. Reitman M.L. Shulman G.I. J. Biol. Chem. 2000; 275: 8456-8460Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). Interestingly, obesity and insulin resistance are also associated with ectopic lipid accumulation suggesting an insufficient uptake and storage of lipids in the adipose cells in these conditions, as well. The adipose tissue also plays an important role as an endocrine organ secreting different hormones and cytokines that can augment or impair whole-body insulin sensitivity (3Fukuhara A. Matsuda M. Nishizawa M. Segawa K. Tanaka M. Kishimoto K. Matsuki Y. Murakami M. Ichisaka T. Murakami H. Watanabe E. Takagi T. Akiyoshi M. Ohtsubo T. Kihara S. Yamashita S. Makishima M. Funahashi T. Yamanaka S. Hiramatsu R. Matsuzawa Y. Shimomura I. Science. 2005; 307: 426-430Crossref PubMed Scopus (1620) Google Scholar). Accumulation of body fat in adults is initially characterized by an increase in fat cell size followed by an increased cell number and, thus, recruitment of preadipocytes (4Kissebah A.H. Vydelingum N. Murray R. Evans D.J. Hartz A.J. Kalkhoff R.K. Adams P.W. J. Clin. Endocrinol. Metab. 1982; 54: 254-260Crossref PubMed Scopus (1556) Google Scholar, 5Krotkiewski M. Bjorntorp P. Sjostrom L. Smith U. J. Clin. Invest. 1983; 72: 1150-1162Crossref PubMed Scopus (1164) Google Scholar, 6Weyer C. Foley J.E. Bogardus C. Tataranni P.A. Pratley R.E. Diabetologia. 2000; 43: 1498-1506Crossref PubMed Scopus (558) Google Scholar). Very little is known about factors that regulate the commitment of pluripotent stem cells into the adipose lineage (7Camp H.S. Ren D. Leff T. Trends Mol. Med. 2002; 8: 442-447Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 8Otto T.C. Lane M.D. Cox M.M. Crit. Rev. Biochem. Mol. Biol. 2005; 40: 229-242Crossref PubMed Scopus (409) Google Scholar). Once committed, the preadipocytes undergo an adipogenic program, which requires a coordinate activation of several pathways initiated by the down-regulation of the inhibitory preadipocyte factor-1 (Pref-1) 2The abbreviations used are: Pref-1, preadipocyte factor-1; C/EBP, CCAAT/enhancer binding protein; PPAR, peroxisome proliferator-activated receptor; Dvl, Dishevelled; PPRE, PPAR response element; GSK, glycogen synthase kinase; MCP-1, monocyte chemoattractant protein-1; Fz, Frizzled; IL-6, interleukin-6, TNFα, tumor necrosis factor α; PCNA, proliferation cell nuclear antigen; MAPK, mitogen-activate protein kinase; ELISA, enzyme-linked immunosorbent assay; sFRP2, secreted Frizzled-related protein 2. 2The abbreviations used are: Pref-1, preadipocyte factor-1; C/EBP, CCAAT/enhancer binding protein; PPAR, peroxisome proliferator-activated receptor; Dvl, Dishevelled; PPRE, PPAR response element; GSK, glycogen synthase kinase; MCP-1, monocyte chemoattractant protein-1; Fz, Frizzled; IL-6, interleukin-6, TNFα, tumor necrosis factor α; PCNA, proliferation cell nuclear antigen; MAPK, mitogen-activate protein kinase; ELISA, enzyme-linked immunosorbent assay; sFRP2, secreted Frizzled-related protein 2. and Wnt proteins (9Bennett C.N. Ross S.E. Longo K.A. Bajnok L. Hemati N. Johnson K.W. Harrison S.D. MacDougald O.A. J. Biol. Chem. 2002; 277: 30998-31004Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar, 10Mei B. Zhao L. Chen L. Sul H.S. Biochem. J. 2002; 364: 137-144Crossref PubMed Scopus (109) Google Scholar, 11Moldes M. Zuo Y. Morrison R.F. Silva D. Park B.H. Liu J. Farmer S.R. Biochem. J. 2003; 376: 607-613Crossref PubMed Scopus (238) Google Scholar). Induction of the nuclear peroxisome proliferator-activated receptorγ2 (PPARγ2) also plays a crucial role in the early differentiation of the preadipocytes into lipid-accumulating cells (8Otto T.C. Lane M.D. Cox M.M. Crit. Rev. Biochem. Mol. Biol. 2005; 40: 229-242Crossref PubMed Scopus (409) Google Scholar). Other key transcription factors are the family of CCAAT/enhancer-binding proteins: C/EBPα, C/EBPβ, and C/EBPδ. Several loss-of-function studies with cells either lacking PPARγ or C/EBPα have shown an absolute requirement of PPARγ for the induction of differentiation, whereas cells lacking C/EBPα, but expressing PPARγ, can still be induced to accumulate lipids. However, these cells are not the typical insulin-responsive and hormone-secreting adipose cells, because C/EBPα is required for the normal induction of many key adipogenic genes and proteins such as GLUT4, adiponectin, and IRS-1 (12Rosen E.D. Hsu C.H. Wang X. Sakai S. Freeman M.W. Gonzalez F.J. Spiegelman B.M. Genes Dev. 2002; 16: 22-26Crossref PubMed Scopus (1041) Google Scholar, 13Rosen E.D. Spiegelman B.M. J. Biol. Chem. 2001; 276: 37731-37734Abstract Full Text Full Text PDF PubMed Scopus (1063) Google Scholar). During preadipocyte differentiation, a carefully coordinated chain of events occurs where one of the earliest transcription factors to be expressed is C/EBPβ. 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Once C/EBPα its binding in the is important for the terminal differentiation and maintained C/EBPα expression in the Lane M.D. Proc. Natl. U. S. A. 2000; PubMed Scopus Google Scholar). C/EBPα is also and its expression clonal mitotic the genes to the fully differentiated are and TNFα are major inflammatory cytokines that have to the development of insulin resistance and diabetes D. Diabetologia. 40: PubMed Scopus Google Scholar). IL-6 is secreted by both adipose cells and inflammatory cells, and high IL-6 are to the of diabetes in S. A. S. J. Clin. Endocrinol. Metab. PubMed Google Scholar). TNFα is secreted by inflammatory cells, because the is not and in the by the H. P. 2002; PubMed Scopus Google Scholar). studies have shown that the adipose tissue of IL-6 in is to the size of the fat cells and that the concentrations are the M. B. A. M. X. P.A. Smith U. Obes. 2004; PubMed Scopus Google Scholar). The recruitment of the to the adipose tissue in obesity an D. M. M. J. Clin. Invest. 2003; PubMed Scopus Google Scholar, H. G. D. J. A. Ross Chen H. J. Clin. Invest. 2003; PubMed Scopus Google Scholar). The high concentrations that IL-6 is an important of the preadipocytes M. B. A. M. X. P.A. Smith U. Obes. 2004; PubMed Scopus Google Scholar). examined the effects of IL-6 concentrations to be in the adipose tissue in obesity M. B. A. M. X. P.A. Smith U. Obes. 2004; PubMed Scopus Google as well as TNFα, on the preadipocyte differentiation the well characterized 3T3-L1 cells. of high and with the proliferation cell nuclear C/EBPα C/EBPβ protein PPARγ and and and and β-catenin preadipocytes in with and of the cells with and for followed by insulin for an and TNFα, to the differentiation and to cell the of differentiation as lipid cells in with and with O. the cells with density for was by the cells with and, of of was for and the density was in a was by of with the on and allowed to with with or the induction of differentiation and the cells for with to and the number of cells cells was PPRE, C/EBP, and capacity in nuclear of PPARγ to PPARγ C/EBPα and C/EBPβ to binding and binding to binding ELISA, as by the and cells, in with and in and The was by for and the and was a of cell used for either or and to The for with in and in the with for the with for and the with as M. Mol. Cell. Biol. PubMed Google Scholar). 3T3-L1 cells one in and for The cells in and and in a The was by for and with of nuclear proteins was by of with for on The for and the was to was the protein was the cells with expression was with the and and the used are on The was as of both and and of in a of The or of was as with of was used as in and used to S.E. was used to expression and transcription factor binding and of was IL-6, TNFα, Accumulation and a the of IL-6, was a inhibition in the early lipid accumulation and and a in the accumulation was also seen and as well as not many of the preadipocytes maintained a in the of IL-6 and these cells lipids, to a the of IL-6, the cells lipids, to the and a that in and which induction of of the PPARγ the cells up and accumulate lipids but the and These induced by IL-6 that the cells in a was by which was also differentiation The proliferation was also associated with an increased expression of the both in the and of these that the of IL-6 both and the lipid accumulation and, in the cell and size as well as the pattern the differentiation We also examined the of TNFα on the differentiation was IL-6 and lipid accumulation of the was seen in the of as low a as and Thus, early preadipocytes are to the inhibitory of cytokines on IL-6 and TNFα the examined the effects of IL-6 on the expression of genes to the differentiation and development of a normal genes to adipose cell differentiation and such as adiponectin, and whereas which is by PPARγ, was not a number of important genes induced differentiation and to insulin lipid and such as insulin receptor protein and lipoprotein markedly in the of IL-6 IL-6 increased the expression of IL-6 as well as and the of the the IL-6 and and this was also the protein and TNFα induces inflammatory induction of 3T3-L1 preadipocytes differentiated for with or IL-6 or was and with The to to expression in the cytokines both IL-6 and, in TNFα increase expression of and as IL-6 induces expression of and as are as the S.E. with We also examined the of TNFα in to the lipid also completely the normal differentiation However, the expression of and cytokines IL-6 and was increased and for IL-6, whereas and not induced not TNFα, in contrast to IL-6, not an increase but expression The Induction of by IL-6 such effects on the and differentiation of the to the of is required for preadipocytes to undergo adipose However, was induction of differentiation or not IL-6 was Wnt signaling and also a key role for the cells in an state M. Zuo Y. Morrison R.F. Silva D. Park B.H. Liu J. Farmer S.R. Biochem. J. 2003; 376: 607-613Crossref PubMed Scopus (238) Google Scholar). However, also Wnt10b shown to differentiation S.E. Hemati N. Longo K.A. C.N. MacDougald O.A. Science. 2000; PubMed Scopus Google induction of differentiation or not IL-6 was We also and but these not expressed conditions used not of the family and is with the into the clonal mitotic expansion induction of differentiation, the expression of these proteins is with an increase in and a in K. Y. R.E. Mol. Cell. Endocrinol. 2002; PubMed Scopus Google Scholar, M. P. 1999; PubMed Scopus Google Scholar). IL-6 not with the normal Thus, IL-6 not with these required for the induction of and are expressed induction of C/EBPβ is by binding to CHOP-10, a of the However, and C/EBPβ is Biochem. PubMed Scopus Google Scholar, Q.Q. Lane M.D. Proc. Natl. U. S. A. 2000; PubMed Scopus Google Scholar). 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The protein in nuclear with a both in the and of IL-6 The transcription factor also as a of the C/EBPα The binding the binding and the activation Lane M.D. Proc. Natl. U. S. A. 2000; PubMed Scopus Google Scholar). induction of differentiation, and the C/EBPα can be activated by and M.D. Q.Q. Biochem. 1999; PubMed Scopus (238) Google Scholar). for with a in the or of IL-6 Thus, IL-6 not the activation of the C/EBPα an impaired of the and the C/EBPα also one for which can as a and the binding of proteins to R. 1999; PubMed Scopus Google examined IL-6 with the binding of to the binding but such was not IL-6, TNFα, the Wnt in β-catenin induction of PPARγ and C/EBPα, is in the phosphorylation by and S. T. M. A. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Farmer S.R. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). of is also required for because of preadipocytes with of differentiation and PPARγ and C/EBPα induction S.E. Hemati N. MacDougald O.A. Mol. Cell. Biol. 1999; PubMed Google Scholar). The of IL-6 not the normal down-regulation of or Wnt10b and or increase Wnt and to a role in preadipocytes S.E. Hemati N. Longo K.A. C.N. MacDougald O.A. Science. 2000; PubMed Scopus Google Scholar). However, β-catenin in the of IL-6 both in and nuclear not the differentiation The of the PPARγ this of IL-6, whereas the of the Wnt signaling Y. R. J. 2003; PubMed Scopus Google secreted Frizzled-related protein TNFα was in the β-catenin expression, and, this was not by the of TNFα also increased the expression of a of cell factor Both IL-6 and TNFα maintained a high expression of the important Wnt signaling in the cells the differentiation Interestingly, the pattern of in the of IL-6 an increased phosphorylation of this which shown to L. H. Mol. Cell. Biol. 2004; 24: PubMed Scopus Google was also to with not We also examined the expression of axin and the the is activated Wnt and this was also the in the of IL-6 or TNFα low in the of IL-6 and in the of TNFα on the The axin protein expression was not due to in not that was by axin protein expression a pattern as β-catenin these that β-catenin by IL-6 and TNFα was both activation and axin is that important the effects of IL-6 and TNFα on Wnt TNFα, in contrast to IL-6, maintained high Wnt10b and expression in the preadipocytes and whereas was seen on or not TNFα, also in contrast to IL-6, completely the activation of both PPARγ2 and C/EBPα these that preadipocytes are to which can the cells in an or state by Wnt signaling and a normal PPARγ C/EBPα the cells are differentiated an inflammatory is a which these cells can Wnt signaling in response to because the β-catenin not increased cytokines or induction of differentiation not IL-6, TNFα, is an important and is to a major role for both the induction of as well as insulin resistance in of IL-6, and with TNFα, induced effects on the terminal differentiation and cells with both lipid accumulation and expression of several genes of for normal insulin sensitivity and GLUT4, adiponectin, lipoprotein and the cells an inflammatory However, the of the cytokines was not because TNFα increased and IL-6 IL-6 increased these cytokines in induced The in differentiation and lipid accumulation of with an increased an of the expression as shown for TNFα C. C. 2004; PubMed Scopus Google body insulin resistance and the accumulation of ectopic lipids in the liver and skeletal muscles, because lipids be up and in the adipose cells. The early for induction of differentiation, as activation of the C/EBPβ and was not by either TNFα or IL-6 also allowed a normal induction of and whereas this was not seen with Several that the cells in a state in the of IL-6, and the increased expression of its and effects in 3T3-L1 cells, in several cells such as and cells T.C. S.E. M.L. 2004; PubMed Scopus Google Scholar). of the effects of IL-6 on and differentiation of the 3T3-L1 cells can be to the impaired expression of We examined IL-6 the normal induction of C/EBPα the known of activation but of However, the of C/EBPα be induced to a IL-6 that the normal binding of C/EBPα to binding and the activation of the Remarkably, that the canonical Wnt which the cells activated the differentiation in the of either IL-6 or for an activation of the canonical Wnt by both cytokines was by the maintained expression of Dishevelled, low and high β-catenin levels. The secreted Wnt to the and these with the which as a high for axin L. H. 2004; PubMed Scopus Google β-catenin in the which shown to be by preadipocytes and to prevent differentiation S.E. Hemati N. Longo K.A. C.N. MacDougald O.A. Science. 2000; PubMed Scopus Google Scholar, K.A. S. I. MacDougald O.A. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google not an expression in the of IL-6, whereas was by also shown to S.E. Hemati N. Longo K.A. C.N. MacDougald O.A. Science. 2000; PubMed Scopus Google but this was not expressed in 3T3-L1 cells. results that the major of TNFα is to Wnt the cells in an state with to the normal phenotype instead, the induction of inflammatory However, a to was not seen these conditions, as expression of and of PPARγ is important for Wnt signaling by the of and this is on S.E. Hemati N. MacDougald O.A. Mol. Cell. Biol. 1999; PubMed Google Scholar). However, the expression of PPARγ, as well as the binding of nuclear to PPRE, not in the and of Thus, is that IL-6 Wnt signaling by this and and the X. M. K. X. 2004; PubMed Scopus Google plays an important role in Wnt signaling by as a to axin to the L. H. 2004; PubMed Scopus Google and also the binding and axin by phosphorylation of K. X. Liu C. X. Y. X. Mol. 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Chem. 2004; Full Text Full Text PDF PubMed Scopus Google that a of β-catenin a of adipogenic genes and that whereas adiponectin, of differentiation, was markedly The of the Wnt signaling the activation of many pathways and of the PPARγ markedly C/EBPα protein expression and the activation of the adipogenic genes in the of with also that the β-catenin protein an increased C. A. L. A. B. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, one important for the of PPARγ to differentiation of preadipocytes is by the activation by the β-catenin levels. have shown that IL-6 and TNFα increase the expression of inflammatory whereas the normal differentiation and lipid accumulation are with a maintained Wnt of the where the cytokines with the normal differentiation is shown in an in these can why conditions obesity and diabetes are associated with increased local and systemic inflammation, insulin resistance, and ectopic lipid accumulation. Interestingly, have that insulin resistance is characterized by an expression in the adipose tissue of several genes to adipose cell differentiation such as GLUT4, adiponectin, and C/EBPα P.A. A. M. H. K. M. R. E. Funahashi T. Matsuzawa Y. O. X. Smith U. J. 2003; PubMed Scopus Google Scholar). this also is a of an increased Wnt signaling is with