Apolipoprotein CIII induces monocyte chemoattractant protein-1 and interleukin 6 expression via Toll-like receptor 2 pathway in mouse adipocytes

Abstract

Objective: To examine the direct effect of apolipoprotein CIII (apoCIII) on adipokine expressions that are involved in obesity, insulin resistance, or metabolic syndrome.

Methods and results: ApoCIII in triglyceride-rich lipoproteins is elevated in patients with obesity, insulin resistance, or metabolic syndrome. Its level is also associated with proinflammatory adipokines. Fully differentiated mouse 3T3L1 adipocytes were incubated with apoCIII. ApoCIII activated nuclear factor κB of 3T3L1 adipocytes and induced the expression of monocyte chemoattractant protein (MCP) 1 and interleukin (IL) 6. ApoCIII also activated extracellular signal-regulated kinase and p38. Mitogen-activated protein kinase kinase (MEK)-1 inhibitor PD98059, but not p38 inhibitor SB203580, inhibited apoCIII-induced upregulation of MCP-1 and IL-6. Previously, it was shown that apoCIII activates proinflammatory signals through toll-like receptor (TLR) 2. TLR2-blocking antibody abolished activation of nuclear factor κB and extracellular signal-regulated kinase induced by apoCIII and inhibited apoCIII-induced upregulation of MCP-1 and IL-6. ApoCIII also reduced adiponectin expression of 3T3L1 adipocytes, which was recovered by TLR2-blocking antibody. ApoCIII induced the expression of MCP-1 and IL-6 in TLR2-overexpressed human embryonic kidney 293 cells but not wild-type human embryonic kidney 293 cells without TLR2. ApoCIII induced the expression of MCP-1 and IL-6 and decreased adiponectin expression in white adipose tissue of wild-type mice but not of TLR2-deficient mice in vivo.

Conclusions: ApoCIII may activate extracellular signal-regulated kinase and nuclear factor kB through TLR2 and induce proinflammatory adipokine expression in vitro and in vivo. Thus, apoCIII links dyslipidemia to inflammation in adipocytes, which, in turn, may contribute to atherosclerosis.

Figure 1

ApoCIII induces MCP-1 and IL-6 expression in 3T3L1 adipocytes. A, 3T3L1 adipocytes were incubated with apoCIII at 100 µg/mL for the indicated hours. *P<0.05 vs 0 hours. B, 3T3L1 adipocytes were incubated with apoCIII at the indicated concentrations for 8 hours. *P<0.05 vs 0 µg/mL. C, 3T3L1 adipocytes were incubated with apoCIII (100 µg/mL for 8 hours) or PBS (control). *P<0.05 vs control.

Figure 2

ApoCIII activates NF-kB in 3T3L1 adipocytes. A and B, 3T3L1 adipocytes were incubated with apoCIII at the indicated concentrations for 30 minutes. In some conditions, apoCIII was pretreated with apoCIII-blocking antibody (50 µg/mL for 30 minutes). Blots represent 4 independent experiments using apoCIII from 4 different donors that yielded similar results. Arrows indicate NF-κB p65 located in the nucleus (B). C and D, 3T3L1 adipocytes were pretreated with SN50 (20 µmol/mL for 30 minutes) and then incubated with apoCIII (100 µg/mL for 8 hours) or PBS (control). *P<0.05 vs control and #P<0.05 vs ApoCIII. IB indicates immunoblotting; p, phosphorylated.

Figure 3

ApoCIII activates ERK mitogen-activated protein kinase (MAPK) in 3T3L1 adipocytes. A, 3T3L1 adipocytes were incubated with apoCIII at the indicated concentrations for 30 minutes. Blots represent 3 independent experiments using apoCIII from 3 different donors that yielded similar results. B and C, 3T3L1 adipocytes were pretreated with PD98059 or SB203580 (50 nmol/L for 30 minutes) and then incubated with apoCIII (100 µg/mL for 8 hours) or PBS (control). *P<0.05 vs control and #P<0.05 vs ApoCIII. IB indicates immunoblotting; p, phosphorylated.

Figure 4

TLR2 mediates apoCIII-induced MCP-1 and IL-6 expression in 3T3L1 adipocytes. A through C, 3T3L1 adipocytes were pretreated with the indicated antibodies (50 µg/mL for 30 minutes) and then incubated with apoCIII (100 µg/mL for 30 minutes) or PBS (control). Blots represent 3 independent experiments using apoCIII from 3 different donors that yielded similar results. Arrows indicate the NF-κB p65 located in the nucleus (B). D and E, 3T3L1 adipocytes were pretreated with the indicated antibodies (50 µg/mL for 30 minutes) and then incubated with apoCIII (100 µg/mL for 8 hours) or PBS (control). *P<0.05 vs control and #P<0.05 vs ApoCIII. IB indicates immunoblotting; p, phosphorylated.

Figure 5

ApoCIII decreases adiponectin in 3T3L1 adipocytes. A and B, 3T3L1 adipocytes were incubated with apoCIII at the indicated concentrations for 6 hours. *P<0.05 vs 0 µg/mL. C and D, 3T3L1 adipocytes were pretreated with SN50 (20 µmol/mL for 30 minutes) or TLR2-blocking antibody (50 µg/mL for 30 minutes) and then incubated with apoCIII (100 µg/mL for 8 hours) or PBS (control). *P<0.05 vs control and #P<0.05 vs ApoCIII.

Figure 6

ApoCIII modulates adipokine expression in human TRL2-overexpressed HEK293 cells. A through C, Wild-type HEK293 cells (WT) or human TLR2-overexpressed HEK293 cells (TLR2) were incubated with apoCIII (100 µg/mL) for 8 hours (A and B) or 30 minutes (C) or PBS (control). In some conditions, TLR2-overexpressed HEK293 cells were pretreated with TLR2-blocking antibody (50 µg/mL for 30 minutes). *P<0.05 vs control and #P<0.05 vs ApoCIII. Blots represent 3 independent experiments using apoCIII from 3 different donors that yielded similar results. IB indicates immunoblotting; p, phosphorylated.