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. 2007 Jan;292(1):E166-74.
doi: 10.1152/ajpendo.00284.2006. Epub 2006 Aug 22.

Macrophages block insulin action in adipocytes by altering expression of signaling and glucose transport proteins

Carey N Lumeng  1 , Stephanie M Deyoung, Alan R Saltiel
Affiliations

Macrophages block insulin action in adipocytes by altering expression of signaling and glucose transport proteins

Carey N Lumeng et al. Am J Physiol Endocrinol Metab. 2007 Jan.

Abstract

Obesity leads to a proinflammatory state with immune responses that include infiltration of adipose tissue with macrophages. These macrophages are believed to alter insulin sensitivity in adipocytes, but the mechanisms that underlie this effect have not been characterized. We have explored the interaction between macrophages and adipocytes in the context of both indirect and direct coculture. Macrophage-secreted factors blocked insulin action in adipocytes via downregulation of GLUT4 and IRS-1, leading to a decrease in Akt phosphorylation and impaired insulin-stimulated GLUT4 translocation to the plasma membrane. GLUT1 was upregulated with a concomitant increase in basal glucose uptake. These changes recapitulate those seen in adipose tissue from insulin-resistant humans and animal models. TNF-alpha-neutralizing antibodies partially reversed the insulin resistance produced by macrophage-conditioned media. Peritoneal macrophages and macrophage-enriched stromal vascular cells from adipose tissue also attenuated responsiveness to insulin in a manner correlating with inflammatory cytokine secretion. Adipose tissue macrophages from obese mice have an F4/80(+)CD11b(+)CD68(+)CD14(-) phenotype and form long cellular extensions in culture. Peritoneal macrophages take on similar characteristics in direct coculture with adipocytes and induce proinflammatory cytokines, suggesting that macrophage activation state is influenced by contact with adipocytes. Thus both indirect/secreted and direct/cell contact-mediated factors derived from macrophages influence insulin sensitivity in adipocytes.

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Figures

Fig. 1
Fig. 1
Macrophage-conditioned medium (CM) blocks insulin (Ins) action in 3T3-L1 adipocytes. A: insulin-stimulated 2-deoxyglucose (2-DG) uptake in 3T3-L1 adipocytes was examined after exposure to medium harvested from LPS-stimulated J774 macrophages. CM or unconditioned medium (Control) were added to differentiated 3T3-L1 adipocytes for 24 h. 2-DG uptake assessed without (open bars) or with (filled bars) 30 min of insulin (100 nM) stimulation. B: 2-DG uptake with indirect coculture. J774 macrophages were cultured in the upper cell culture insert with differentiated 3T3-L1 adipocytes in the lower chamber for 5 days. 2-DG uptake was assessed with and without insulin stimulation as in A. For A and B, data are expressed as means (SD) of triplicate determinants. Similar results were obtained from 3 independent experiments. *P < 0.05. C: effects of CM on adipocyte glucose transporter expression. Immunoblots (IB) of 3T3-L1 adipocytes lysates with or without treatment with CM for 24 h. Cells were stimulated with insulin (100 nM) for 10 min. Actin was used as loading control. D: effect of CM on insulin signaling in adipocytes. Following insulin stimulation, adipocyte lysates from cells treated with or without CM were probed for insulin receptor (IR), IRS-1, Akt/PKB, and phosphorylated Jun kinase (pJNK). Phosphotyrosine (pY) antibody was used on lysates to identify IR and IRS phosphorylation, and IRS phospho-Ser307-specific antibody was used to probe IRS serine phosphorylation. E: time course of exposure to CM. Adipocytes were treated with CM from unstimulated (J774 CM) and LPS-stimulated J774 cells (J774 + LPS CM) for 0, 1, 3, and 8 h prior to insulin treatment (100 nM for 10 min). Lysates were immunoprecipitated (IP) for IRS-1 and IR using specific antibodies and probed for pY or the immunoprecipitated protein. Actin from lysates was used as loading control.
Fig. 2
Fig. 2
CM and macrophage (MP) coculture impair insulin-stimulated GLUT4 plasma membrane (PM) translocation. A and B: insulin-stimulated GLUT4 translocation in adipocytes. 3T3-L1 adipocytes stably expressing Myc-GLUT4-eGFP fusion protein were serum starved and treated with medium without (A) or with (B) insulin (100 nM for 15 min). Cells were imaged for GFP expression, demonstrating translocation of Myc-GLUT4-GFP to the plasma membrane. C: CM blocks GLUT4 translocation. Myc-GLUT4-eGFP-expressing 3T3-L1 adipocytes were incubated for 24 h with CM. Insulin stimulation was performed as in A. D: coculture with macrophages blocks GLUT4 translocation. RAW264 macrophages (2 × 105 cells/well of 6-well dish) were cocultured with Myc-GLUT4-eGFP-expressing 3T3-L1 adipocytes for 24 h prior to insulin stimulation and imaging GLUT4 translocation. E: quantitation of GLUT4 plasma membrane translocation. Cells with translocation of Myc-GLUT4-eGFP to the plasma membrane were quantitated in 2 independent experiments (>200 cells/experiment) per condition. Data are presented as mean %GLUT4 translocation ± SE. *P < 0.05 vs. control; **P < 0.005 vs. control.
Fig. 3
Fig. 3
TNF-α blockade partially restores insulin sensitivity in adipocytes when exposed to CM. A: insulin-stimulated glucose uptake with CM in the presence or absence of TNF-α neutralizing antibodies (TNFNA). Adipocytes were treated for 16 h with control medium or CM from unstimulated (MP) or LPS-stimulated (MP + LPS) J774 cells. TNF-α neutralizing antibodies were added to the media from stimulated cells prior to addition to adipocytes (MP + LPS + TNFNA). Insulin-stimulated glucose uptake was then assessed. Data are expressed as means (SD) of triplicate determinants. Similar results were obtained from 2 independent experiments. *P < 0.05. B: anti-TNF-α antibodies restore IRS-1 protein levels in adipocytes exposed to CM. Immunoblots of 3T3-L1 adipocyte lysates from CM experiments were probed for IRS-1 expression and Akt serine phosphorylation at Ser473. Similar results were obtained from duplicate experiments, and a representative experiment is shown.
Fig. 4
Fig. 4
Indirect coculture with primary murine macrophages blocks insulin action in adipocytes. A: insulin-stimulated glucose uptake in 3T3-L1 adipocytes cocultured with peritoneal macrophages (PM) or stromal vascular fraction (SVF) cells from epididymal adipose tissue from C57Bl/6 mice. Cells (105) of PM or SVF were added to cell culture insert, which was cultured over differentiated 3T3-L1 cells in the lower chamber. After 48 h of coculture, glucose uptake was assessed with (filled bars) and without (open bars) insulin stimulation. Data are expressed as means (SD) of triplicate determinants. Similar results were obtained from 2 independent experiments. *P < 0.05. B: cytokine analysis of media from SVF and adipocyte coculture. Media from control 3T3-L1 cultures (open bars) and indirect cocultures of SVF with 3T3-L1 adipocytes (filled bars) were collected and examined for proinflammatory cytokines TNF-α, IL-6, MIP-2, and MCP-1; n = 5 samples/condition. *P < 0.05.
Fig. 5
Fig. 5
Adipose tissue macrophage (ATM) morphology and cell surface markers in obese mice. A: SVF cells were isolated from high-fat diet-fed mice and selected by adhesion on coverslips. Cultures were then stained with anti-F4/80 antibodies and visualized by immunofluorescence microscopy and phase contrast microscopy. B: SVF cells stained for macrophage marker CD11b (green) and counterstained with rhodamine-phalloidin (red) to visualize actin-enriched processes and filopodia that extend over fibroblasts (arrow). C: SVF cells stained for CD68 in permeablized cells and CD14 show that ATMs have a CD68+CD14− phenotype. Scale bar, 50 µm for all figures.
Fig. 6
Fig. 6
Direct coculture of macrophages with adipocytes alters macrophage morphology. A: phase contrast pictures of thioglycollate PMs in isolated culture. B: phase contrast pictures of differentiated 3T3-L1 adipocytes in isolated culture. C: direct coculture of PMs and adipocytes after 3 days demonstrates that macrophages take on an elongated phenotype with long cellular processes (arrows). Two representative views are shown. Increased accumulation of intracellular lipid (arrowhead) is also seen in macrophages. D: PMs were labeled with PKH26 (red) prior to plating on differentiated 3T3-L1 adipocytes. After 1 day of coculture with adipocytes (top), MPs appear small and oblong. After 3 days of coculture (bottom), PKH26+ macrophages appear more elongated and accumulate intracellular lipid (arrowhead).
Fig. 7
Fig. 7
Direct coculture of macrophages with adipocytes alters insulin sensitivity in a dose-dependent fashion. A: insulin-stimulated 2-DG uptake in direct coculture. Differentiated 3T3-L1 adipocytes were cultured in FBS (None) or after addition of 104 (10k) or 105 (100k) PM. 2-DG uptake was assessed with (filled bars) or without (open bars) 30 min of insulin stimulation. Although 10k PMs had minimal effects on glucose uptake, 100k PMs led to an increase in basal glucose uptake and a decrease in fold insulin stimulation. Data are expressed as means (SD) of triplicate determinants. Similar results were obtained from 2 independent experiments. *P < 0.05. B: cytokine analysis of coculture media. Media from the 3 conditions of coculture were evaluated for cytokine levels of TNF-α, IL-6, (top), and MCP-1 (bottom); n = 3 samples/condition. **P < 0.001; *P < 0.05.
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