These results suggest that apoCIII downregulates adiponectin via a TLR2CNF-B signaling pathway. Open in a separate window Figure 5 ApoCIII decreases adiponectin in 3T3L1 adipocytes. it was shown that apoCIII activates proinflammatory signals through toll-like receptor (TLR) 2. TLR2-blocking Rabbit polyclonal to TGFB2 antibody abolished activation of nuclear factor B and extracellular signalCregulated 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 Pargyline hydrochloride of wild-type mice but not of TLR2-deficient mice in vivo. Conclusion ApoCIII may activate extracellular signalCregulated 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. amebocyte lysate chromogenic test were 0.03 EU/mL. Free fatty acid levels in apos determined enzymatically were 20 nmol/L, which is much lower than is reported to induce inflammation in endothelial cells. In some experiments, apoCIII was incubated with inert acrylic beads attached to lipase of (immobilized lipase), as previously described.22 After the beads were removed by centrifugation, the effects of apoCIII were assayed using 3T3L1 adipocytes. Cell Culture Mouse 3T3L1 preadipocytes were purchased from American Type Culture Collection, Manassas, Va. The differentiation of 3T3L1 preadipocytes to adipocytes was described elsewhere.23 Wild-type human embryonic kidney (HEK) 293 cells and HEK293 cells stably transfected with human toll-like receptor (TLR) 2 were purchased. Animals Six-week-old male C57BL/6 (wild-type) mice (Oriental Yeast, Tokyo, Japan) or TLR2-deficient mice consumed a standard diet. Food and water were provided ad libitum. Then, wild-type and TLR2-deficient mice were administered apoCIII, 400 g/body, or PBS, IP24 once per day. After 1 week, fasting plasma was collected for ELISA and metabolic parameters; and epididymal white adipose tissue (WAT) was dissected Pargyline hydrochloride for RT-PCR and immunoblotting. In some experiments, the stromal vascular fraction and the adipocyte fraction were separated from WAT, as previously described.25 All animal experiments were conducted in accordance with the guidelines of the Tokyo Medical and Dental University Committee on Animal Research, Tokyo. Lipoprotein Preparation Blood was drawn in tubes containing EDTA from 10 healthy volunteers after 12 hours of fasting. The subjects were not taking cardiovascular medications, antioxidants, or estrogen. VLDL (d 1.006) with apoIII (VLDL CIII+) or without apoCIII (VLDL CIII?) was isolated from plasma, as previously described.11 The protocol of this study complied with the guidelines for the conduct of research involving human subjects by the Committee on Human Research at Tokyo Medical and Dental University. Endotoxin levels in VLDL preparations were 0.03 EU/mL. RT-PCR and ELISA Total RNA was isolated from 3T3L1 adipocytes or mice WAT using a kit (RNeasy mini kit) and then converted Pargyline hydrochloride to cDNA using a reverse-transcript method with transcriptase (Superscript III RNase H Reverse Transcriptase). Real-time PCR was performed using primer sets Pargyline hydrochloride (supplemental Table I; available online at http://atvb.ahajournals.org) with a sequence detection system (ABI PRISM 7900HT). Relative mRNA transcript levels were calculated using the comparative computed tomographic method, which is based on the difference in cycle threshold values between the target mRNA and -actin, used as an internal control. Concentrations of monocyte chemoattractant protein (MCP) 1, IL-6, and adiponectin in the culture medium of 3T3L1 adipocytes or in the plasma of mice were determined by an ELISA kit. Immunoblotting After treatment with apoCIII or VLDL fractions, 3T3L1 adipocyte lysates or mice WAT lysates were prepared as previously described.12 Lysate was assayed with immunoblotting using antibodies to antiphosphorylated p65 NF-B antibody, antiphosphorylated extracellular signalCregulated kinase (ERK) antibody, antiphosphorylated p38 antibody, or antiCCD68 antibody. Immunofluorescence Microscopy For NF-B p65 staining, 3T3L1 adipocytes were fixed with 4% formaldehyde for 30 minutes, then treated with 0.05% Triton X-100 for 5 minutes. The cells were incubated with fluorescein isothiocyanateCconjugated NF-B p65 antibody (1:200) for 45 minutes. The cells were rinsed and mounted onto slides, then analyzed and imaged using a fluorescent microscope with a 400-fold magnification. Statistical Analysis Data are expressed as the meanSE. Statistical analysis was performed using a Pargyline hydrochloride Student test or ANOVA. em P /em 0.05 was considered statistically significant. Results ApoCIII Induces MCP-1 and IL-6 Expression in 3T3L1 Adipocytes We first examined the direct effect of apoCIII on proinflammatory adipokine expression in 3T3L1 adipocytes. ApoCIII increased MCP-1 and IL-6 mRNA levels of 3T3L1 adipocytes in a time- and concentration-dependent manner (Figure 1A and B). ApoCIII significantly increased the concentrations of MCP-1 and IL-6 in the culture media (Figure 1C). In contrast, apoCI, apoCII, or.
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