Proinflammatory cytokine production and insulin sensitivity regulated by overexpression of resistin in 3T3-L1 adipocytes

Abstract

Resistin is secreted from adipocytes, and high circulating levels have been associated with obesity and insulin resistance. To investigate whether resistin could exert autocrine effects in adipocytes, we expressed resistin gene in 3T3-L1 fibroblasts using a lentiviral vector, and selected several stably-transduced cell lines under blasticidin selection. We observed that 3T3-L1 adipocytes expressing resistin have a decreased gene expression for related transcriptional factors (CCAAT/enhancer binding protein alpha(C/EBPalpha), peroxisome proliferator-activated receptor gamma (PPARgamma), and adipocyte lipid binding protein (ALBP/aP2) which is one of target genes for the PPARgamma during adipocyte differentiation,. Overexpression of resistin increased the levels of three proinflammatory cytokines, tumor necrosis factor alpha (TNFalpha), interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), which play important roles for insulin resistance, glucose and lipid metabolisms during adipogenesis. Furthermore, overexpressing resistin in adipocytes inhibits glucose transport 4 (GLUT4) activity and its gene expression, reducing insulin's ability for glucose uptake by 30%. In conclusion, resistin overexpression in stably transduced 3T3-L1 cells resulted in: 1) Attenuation of programmed gene expression responsible for adipogenesis; 2) Increase in expression of proinflammatory cytokines; 3) Decrease in insulin responsiveness of the glucose transport system. These data suggest a new role for resistin as an autocrine/paracrine factor affecting inflammation and insulin sensitivity in adipose tissue.

Figure 1

Generation of recombinant resistin lentivirus and detection of recombinant gene expression. (A) A full length of mouse resistin coding sequence (0.34 kb) was cloned into a ViraPower-CMV vector (Invitrogen). This recombinant resistin gene construct included a V5 epitope tag and a CMV promoter. (B) A western blot analysis was performed to confirm the expression of the recombinant resistin genes in three different stably transduced cell lines (R18, R20 and R23) using anti-V5 antibodies. The lanes marked by the letter C were loaded with proteins from non-transduced control 3T3-L1 cells. The lane marked by the letter Z was loaded with proteins from cells transduced lentivirus containing recombinant LacZ gene including the V-5 tag and LacZ was detected using anti-V5 antibodies. (C) A western blot analysis was performed to examine the secretion of the recombinant exogenous resistin proteins (R18, R20, and R23) and the endogenous resistin proteins in the medium of 3T3-L1 cells. Exogenous and endogenous resistin proteins were detected using anti-resistin antibodies. The control lane marked by the letter C was loaded with proteins from non-transduced 3T3-L1 cells.

Figure 2

Effects of resistin on gene expression patterns during adipogenesis. Day 0 represents 3T3L1 fibroblasts reaching 100% confluence. Two days after full confluence (Day 2), cells were placed in DMEM containing 25 mM glucose, 0.5 mM isobutylmethylxanthine (IBMX), 1 μm dexamethasone (Dex), 10 μg/ml insulin, and 10% FBS for 3 days; then, on day 5 cells were placed in DMEM containing 25 mM glucose, 10 μg/ml insulin, and 10% FBS for 2 days. After day 7, cells were maintained in DMEM, 25 mM glucose, and 10% FBS. At the indicated time points, resistin and LacZ transduced preadipocytes and adipocytes were lysed and the mRNA levels encoding PPARγ, C/EBPα, and ALBP/aP2 were quantified by quantitative PCR. Results represent the mean ± SE from three separate experiments. Asterisk, P < 0.05; double asterisk, P < 0.01 for resistin versus control cells.

Figure 3

Effects of resistin on GLUT4 activity and expression. Adipocytes transduced with the LacZ gene (Control) or with the resistin gene were incubated for 12 days until fully-differentiated. (A) Resistin overexpressing adipocytes show an inhibition of insulin-stimulated glucose transport activity. Control cells (Bars 1 and 2) and cells over-expressing resistin (Bars 3 and 4), were incubated in the absence (Bars 1 and 3) or presence (Bars 2 and 4) of 100 nM insulin for 30 min at 37°C to induce maximal rates of glucose uptake. Double asterisk, P < 0.01 for (Resistin + Insulin) versus (Control + Insulin). (B) Conditioned medium from resistin transduced adipocytes inhibits the activity of insulin-stimulated glucose transport. 3T3-L1 adipocytes treated with conditioned control medium from LacZ transduced cells (control) (Bars 1 and 2) and conditioned medium from resistin transduced cells (Bars 3 and 4), were incubated in the absence (Bars 1 and 3) or presence (Bars 2 and 4) of 100 nM insulin for 30 min at 37°C to induce maximal rates of glucose uptake. Asterisk, P < 0.05 for (Resistin medium + Insulin) versus (Control medium + Insulin). (C) Resistin decreases the gene expression of GLUT4. Expression of glucose transporter GLUT4 gene was examined using a quantitative PCR assay in resistin transduced adipocytes and LacZ transduced adipocytes during their differentiation process as described in the Fig. 2. Results represent the mean ± SE from three separate experiments. Double asterisk, P < 0.01 for resistin versus control at Day 9.

Figure 4

Effects of resistin overexpression in adipocytes on proinflammatory and anti-inflammatory cytokines. Western blot analyses were performed to examine the levels of three proinflammatory cytokines, IL-6, TNFα, and MCP-1, and one anti-inflammatory cytokine IL-10 in resistin transduced 3T3-L1 cell lines (R18, R20 and R23) and in the LacZ transduced control cells (C) which had been fully differentiated into adipocytes (Day 12).