Interleukin-1β mediates macrophage-induced impairment of insulin signaling in human primary adipocytes

Abstract

Adipose tissue expansion during obesity is associated with increased macrophage infiltration. Macrophage-derived factors significantly alter adipocyte function, inducing inflammatory responses and decreasing insulin sensitivity. Identification of the major factors that mediate detrimental effects of macrophages on adipocytes may offer potential therapeutic targets. IL-1β, a proinflammatory cytokine, is suggested to be involved in the development of insulin resistance. This study investigated the role of IL-1β in macrophage-adipocyte cross-talk, which affects insulin signaling in human adipocytes. Using macrophage-conditioned (MC) medium and human primary adipocytes, we examined the effect of IL-1β antagonism on the insulin signaling pathway. Gene expression profile and protein abundance of insulin signaling molecules were determined, as was the production of proinflammatory cytokine/chemokines. We also examined whether IL-1β mediates MC medium-induced alteration in adipocyte lipid storage. MC medium and IL-1β significantly reduced gene expression and protein abundance of insulin signaling molecules, including insulin receptor substrate-1, phosphoinositide 3-kinase p85α, and glucose transporter 4 and phosphorylation of Akt. In contrast, the expression and release of the proinflammatory markers, including IL-6, IL-8, monocyte chemotactic protein-1, and chemokine (C-C motif) ligand 5 by adipocytes were markedly increased. These changes were significantly reduced by blocking IL-1β activity, its receptor binding, or its production by macrophages. MC medium-inhibited expression of the adipogenic factors and -stimulated lipolysis was also blunted with IL-1β neutralization. We conclude that IL-1β mediates, at least in part, the effect of macrophages on insulin signaling and proinflammatory response in human adipocytes. Blocking IL-1β could be beneficial for preventing obesity-associated insulin resistance and inflammation in human adipose tissue.

Keywords: IL-1β; cytokines; human adipocytes; insulin signaling; macrophages.

Fig. 1.

Macrophage-conditioned (MC) medium reduces protein expression of insulin signaling molecules in human adipocytes. Adipocytes (at day 12 postdifferentiation) were treated with RPMI 1640 medium (control) or THP-1 MC medium (25%) for 24 h. Cell lysates were analyzed by Western blotting and densitometry, using antibodies to IRS-1 and PI3K p85α (A, B, C) and GLUT4 (D, E). For measuring basal and insulin-stimulated Akt phosphorylation, adipocytes were incubated with RPMI 1640 or MC medium for 24 h before being stimulated with insulin (167 nmol/l) for 5 min; Akt phosphorylation at Ser⁴⁷³ (pAkt) was analyzed by Western blotting and densitometry. Total Akt and GAPDH were used as loading controls (F, G). Representative blots are shown; data are means ± SE (n = 3 per group). *P < 0.05, **P < 0.01, ***P < 0.001 vs. controls. Results were confirmed by 3 independent experiments.

Fig. 2.

Effects of IL-1β on expression profile of genes involved in glucose metabolism, insulin signaling, and inflammation in human adipocytes. Differentiated adipocytes were cultured in the presence of IL-1β (2 ng/ml) or vehicle (control) for 24 h. Expression levels of genes involved in glucose metabolism, insulin signaling, insulin sensitivity, lipid metabolism (A), and inflammation (B) were measured using a PCR array. Data are expressed as fold changes (n = 3 per group). *P < 0.05, **P < 0.01, ***P < 0.001 vs. controls.

Fig. 3.

IL-1β impairs the insulin signaling pathway in human adipocytes. Differentiated adipocytes were cultured in the presence of IL-1β (2 ng/ml) or vehicle (control) for 24 h. Protein abundance of IRS-1 (A, B), PI3K p85α (A, C), and GLUT4 (A, D) were determined in cell lysates by Western blotting and densitometry. Separate groups of adipocytes were treated with IL-1β (2 ng/ml) or vehicle (control) for 24 h followed by stimulation with insulin (167 nmol/l) for 5 min; Akt phosphorylation at Ser⁴⁷³ (pAkt) was analyzed by Western blotting and densitometry. Total Akt and GAPDH were used as loading controls (E, F). Data are expressed as means ± SE (n = 3 or 6 per group); **P < 0.01, ***P < 0.001 vs. indicated groups. Results were confirmed by 3 independent experiments.

Fig. 4.

Inhibition of IL-1β activity reduces the effect of macrophages on expression profile of genes related to glucose metabolism, insulin signaling, and inflammation in human adipocytes. To block the activity of IL-1β, MC medium was preincubated with an IL-1β neutralizing antibody (2 μg/ml) for 1 h at 37°C. Differentiated adipocytes were then treated with RPMI 1640 (control), MC medium, or MC medium neutralized by IL-1β antibody for 24 h. Expression levels of genes involved in glucose metabolism, insulin signaling, lipid metabolism, insulin sensitivity (A), and inflammation (B) were determined using a PCR array. Data are expressed as fold changes (n = 3 per group). *P < 0.05, **P < 0.01, ***P < 0.001 vs. controls; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. MC medium treatment.

Fig. 5.

Inhibition of IL-1β activity abolishes the inhibitory effect of macrophages on protein expression of IRS-1, PI3K p85α, and GLUT4 by human adipocytes. Differentiated adipocytes were incubated with RPMI 1640 medium (control), MC medium, MC medium neutralized by IL-1β antibody (2 μg/ml), or mouse IgG (2 μg/ml, as negative control) for 24 h. Cell lysates were analyzed by Western blotting and densitometry using antibodies to IRS-1 (A, B), PI3K p85α (C, D), and GLUT4 (E, F). Separate groups of adipocytes were treated with various agents as above, followed by stimulation with or without insulin (167 nmol/l) for 5 min; Akt phosphorylation at Ser⁴⁷³ (pAkt) was analyzed by Western blotting and densitometry. Total Akt and GAPDH were used as loading controls (G, H). Representative blots are shown; data are means ± SE (n = 3per group). *P < 0.05, **P < 0.01, ***P < 0.001 vs. indicated groups. Results were confirmed by 3 independent experiments.

Fig. 6.

Inhibition of IL-1β activity reverses macrophage-induced cytokine release by human adipocytes. Differentiated adipocytes were incubated with RPMI 1640 (control), MC medium, MC medium neutralized by IL-1β antibody (2 μg/ml), mouse IgG (2 μg/ml, as negative control), or MC medium alone (without cells) for 24 h. The release of IL-6 (A), MCP-1 (B), IL-8 (C) and RANTES (D) by adipocytes was measured as protein concentrations in cell culture medium by ELISAs. Data are means ± SD (n = 6 per group). ***P < 0.001 vs. indicated groups. Results were confirmed by 3 independent experiments.

Fig. 7.

Blocking IL-1 receptor with IL-1 receptor antagonist reverses the effects of MC medium on protein expression of IRS-1, PI3K p85α, and GLUT4 and cytokine release by human adipocytes. Differentiated adipocytes were incubated with RPMI 1640 (control), MC medium, or MC medium with a recombinant IL-1 receptor antagonist (IL-1RA; 1 μg/ml) for 24 h. Cell lysates were analyzed by Western blotting and densitometry, using antibodies to IRS-1 (A, B), PI3K p85α (C, D), and GLUT4 (E, F). On the blot shown, there is an empty lane between MC and MC+IL-1RA groups (C). Data are expressed as means ± SE (n = 3 per group). *P < 0.05, **P < 0.01, ***P < 0.001 vs. indicated groups. Release of IL-6 (G), MCP-1 (H), and IL-8 (I) by adipocytes was measured as protein concentrations in cell culture medium by ELISAs. Data are means ± SD (n = 6 per group). ***P < 0.001 vs. indicated groups. Results were confirmed by 2 independent experiments.

Fig. 8.

Inhibition of IL-1β production by THP-1 macrophages reduces the effect of MC medium on protein expression of IRS-1, PI3K p85α, and GLUT4 and cytokine release by adipocytes. THP-1 macrophages were incubated with RPMI 1640 (control) or caspase-1 inhibitor (50 μM) for 48 h (with freshly changed medium at 24 h), and the culture medium was collected. Differentiated adipocytes were then treated with RPMI 1640 (control), MC medium, or MC medium in the presence of caspase-1 inhibitor for 24 h. Cell lysates were analyzed by Western blotting and densitometry, using antibodies to IRS-1, PI3K p85α, and GLUT4 (A–D). Data are expressed as means ± SE (n = 6 per group). Protein release of IL-6 (E), IL-8 (F), and RANTES (G) by adipocytes was measured by ELISAs. Data are expressed as means ± SD (n = 6 per group). *P < 0.05, **P < 0.01, ***P < 0.001 vs. indicated groups. Results were confirmed by 3 independent experiments.

Fig. 9.

MC medium generated from human PBMC-derived macrophages decreases protein expression of insulin signaling molecules and induces cytokine release by human adipocytes and the effect of blocking IL-1β. Differentiated human adipocytes were treated with RPMI 1640 (control), MC medium, MC medium neutralized by IL-1β antibody (15 μg/ml), MC medium neutralized by IL-1β antibody (7.5 μg/ml), and TNFα (7.5 μg/ml), mouse IgG (15 μg/ml), or IL-1RA (1 μg/ml) for 24 h. Cell lysates were analyzed by Western blotting and densitometry, using antibodies to IRS-1 (A, B) and GLUT4 (C, D). For measuring basal and insulin-stimulated Akt phosphorylation, adipocytes were incubated with RPMI 1640 or MC medium for 24 h before being stimulated with insulin (167 nmol/l) for 5 min; Akt phosphorylation at Ser⁴⁷³ (pAkt) was analyzed by Western blotting and densitometry; GAPDH was used as loading controls (E, F). Representative blots are shown. Data are means ± SE (n = 3 per group). *P < 0.05, **P < 0.01, ***P < 0.001 vs. controls; §P < 0.05, §§P < 0.01 vs. MC group. IL-6 release by adipocytes was measured by ELISA (G). Data are expressed as means ± SD (n = 6 per group). **P < 0.01, ***P < 0.001 vs. controls; §§§P < 0.001 vs. MC group; †††P < 0.001 vs. MC + IL-1β neutralizing antibody group. Results were confirmed by 3 independent experiments.

Fig. 10.

IL-1β mediates macrophage-induced alteration of glucose and lipid metabolism in human adipocytes. Adipocytes were treated with RPMI 1640 (control), THP-1 MC medium, MC medium neutralized by IL-1β antibody (2 μg/ml), MC medium with IgG (2 μg/ml), RPMI 1640 only (control), or RPMI 1640 with IL-1β (2 ng/ml) for 24 h. Glucose consumption was measured as the glucose concentration in culture medium by glucose oxidase method (A). Lipolysis was determined as glycerol release into culture medium (B, C). Data are means ± SE (n = 6 per group). **P < 0.01, ***P < 0.001 vs. indicated groups. Results were confirmed by 3 independent experiments. D: schematic diagram of IL-1β in mediating the effect of human macrophages on insulin signaling in human adipocytes.