Interactive changes between macrophages and adipocytes

Abstract

Obesity is associated with a proinflammatory state, with macrophage infiltration into adipose tissue. We tested the hypothesis that communication between macrophages and adipocytes affects insulin resistance by disrupting insulin-stimulated glucose transport, adipocyte differentiation, and macrophage function. To test this hypothesis, we cocultured 3T3-L1 adipocytes with C2D macrophages or primary peritoneal mouse macrophages and examined the impacts of macrophages and adipocytes on each other. Adipocytes and preadipocytes did not affect C2D macrophage TNF-alpha, IL-6, or IL-1beta transcript concentrations relative to those obtained when C2D macrophages were incubated alone. However, preadipocytes and adipocytes increased PEC-C2D macrophage IL-6 transcript levels, while preadipocytes inhibited IL-1beta transcript levels compared to those obtained when PEC-C2D macrophages were incubated in medium alone. We found that adipocyte coculture increased macrophage consumption of tumor necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), and, in some cases, IL-6. C2D macrophages increasingly downregulated GLUT4 transcript levels in differentiated adipocytes. Recombinant TNF-alpha, IL-1beta, and IL-6 also downregulated GLUT4 transcript levels relative to those for the control. However, only IL-6 was inhibitory at concentrations detected in macrophage-adipocyte cocultures. IL-6 and TNF-alpha, but not IL-1beta, inhibited Akt phosphorylation within 15 min of insulin stimulation, but only IL-6 was inhibitory 30 min after stimulation. Lastly, we found that adipocyte differentiation was inhibited by macrophages or by recombinant TNF-alpha, IL-6, and IL-1beta, with IL-6 having the most impact. These data suggest that the interaction between macrophages and adipocytes is a complex process, and they support the hypothesis that the macrophage-adipocyte interaction affects insulin resistance by disrupting insulin-stimulated glucose transport, adipocyte differentiation, and macrophage function.

FIG. 1.

Secretion of TNF-α, IL-6, and IL-1β in macrophage and 3T3-L1 adipocyte cocultures. 3T3-L1 adipocytes and peritoneal (PC) macrophages were either incubated alone, coincubated directly (mix), or coincubated but separated in transwell plates for 4 days (from day 8 to day 12 in adipocyte medium). Cell-free supernatants were collected, and cytokine concentrations were determined by ELISA. (A) TNF-α; (B) IL-6; (C) IL-1β. The levels of each cytokine in supernatants from cocultures were compared to the sum of the amounts of that cytokine secreted by 3T3-L1 cells and macrophages when they were cultured alone. For this purpose, the bars for 3T3-L1 adipocytes and macrophages are stacked together. The data are presented as means ± standard errors of the means for 3 to 6 independent replicates per treatment. Different letters indicate a significant difference. A P value of < 0.05 was considered significant.

FIG. 2.

Percentage of CFDA-SE-positive C2D cells testing positive for a given marker. (A) CFDA-SE-labeled C2D macrophages grown in vitro were either cultured alone or cocultured with adipocytes or with preadipocytes. (B) CFDA-SE-labeled C2D macrophages were adoptively transferred to the peritoneal cavity for 24 h. After peritoneal cavity isolation, PEC-C2D macrophages were either cultured alone or cocultured with 3T3-L1 adipocytes or preadipocytes. Cell mixtures were dispersed with 0.025% EDTA and were resuspended in HBSS buffer. Cells were immunostained for flow cytometry as described in Materials and Methods. The data are presented as means ± standard errors of the means for 3 independent replicates per treatment. Different letters indicate a significant difference. A P value of <0.05 was considered significant.

FIG. 3.

Effects of recombinant TNF-α, IL-6, and IL-1β on GLUT4 protein expression. Fully differentiated adipocytes were incubated in medium containing recombinant mouse TNF-α (2 ng/ml), IL-6 (10 ng/ml), or IL-1β (20 ng/ml) for 2 days. Cells were fixed and immunostained for GLUT1 or GLUT4 expression using flow cytometry as described in Materials and Methods. The data are presented as means ± standard errors of the means for 3 to 6 independent replicates per treatment. Different letters indicate a significant difference. A P-value of <0.05 was considered significant.

FIG. 4.

TNF-α, IL-6, and IL-1β affect Akt phosphorylation differently. Fully differentiated adipocytes were incubated with or without (control) recombinant mouse TNF-α, IL-6, or IL-1β for 2 days. (A) Total Akt protein levels were detected after treatment with cytokines. Values were normalized to control values, set at 1. (B) Cells were serum starved for 2 h, stimulated with insulin (100 nM) for 20 min, fixed, and assessed for Akt and phosphorylated Akt (p-Akt) by flow cytometry as described in Materials and Methods. p-Akt was detected at 0, 5, 10, 15, or 30 min after insulin stimulation. (C) Fully differentiated adipocytes were incubated in DMEM with 10% fetal bovine serum containing different levels of TNF-α, IL-6, or IL-1β for 2 days. Cells were serum starved for 2 h, stimulated with insulin (100 nM) for 20 min, fixed, and assessed for Akt and p-Akt by flow cytometry. p-Akt was detected at 15 min after insulin stimulation. The data are presented as means ± standard errors of the means for 3 to 6 independent replicates per treatment. An asterisk identifies a significant difference between the control (no cytokine treatment) and the treatment at each time point. A P value of <0.05 was considered significant.

FIG. 5.

Effects of TNF-α, IL-6, and IL-1β on 3T3-L1 adipocyte differentiation. 3T3-L1 cells were differentiated for 4 days with thioglycolate-elicited peritoneal macrophages in direct coculture or in transwells. Cells were fixed and stained with Sudan IV dye as described in Materials and Methods. (A) Cells were imaged at ×200 magnification under a light microscope. “Mix” stands for direct coculture. (B) The lipid concentration was determined by measuring the absorbance at 550 nm. The number of PEC-C2D macrophages (Mφ) in each direct coculture is given at the bottom. Different letters indicate a significant difference. (C) 3T3-L1 cells were differentiated for 4 days and were then treated with TNF-α, IL-6, or IL-1β for an additional 4 days. High, medium, and low concentrations of the cytokines were as follows: 2, 1, and 0.5 ng/ml for TNF-α; 20, 10, and 1 ng/ml for IL-1β; and 10, 5, and 2 ng/ml for IL-6. At day 8, cells were fixed and stained with Sudan IV. Differentiation was determined by measuring the absorbance at 550 nm. The data are presented as means ± standard errors of the means for 3 to 6 independent replicates per treatment. An asterisk identifies a significant difference between the control and the treatment at each time point. A P value of <0.05 was considered significant.