Rosiglitazone inhibits acyl-CoA synthetase activity and fatty acid partitioning to diacylglycerol and triacylglycerol via a peroxisome proliferator-activated receptor-gamma-independent mechanism in human arterial smooth muscle cells and macrophages

Abstract

Rosiglitazone is an insulin-sensitizing agent that has recently been shown to exert beneficial effects on atherosclerosis. In addition to peroxisome proliferator-activated receptor (PPAR)-gamma, rosiglitazone can affect other targets, such as directly inhibiting recombinant long-chain acyl-CoA synthetase (ACSL)-4 activity. Because it is unknown if ACSL4 is expressed in vascular cells involved in atherosclerosis, we investigated the ability of rosiglitazone to inhibit ACSL activity and fatty acid partitioning in human and murine arterial smooth muscle cells (SMCs) and macrophages. Human and murine SMCs and human macrophages expressed Acsl4, and rosiglitazone inhibited Acsl activity in these cells. Furthermore, rosiglitazone acutely inhibited partitioning of fatty acids into phospholipids in human SMCs and inhibited fatty acid partitioning into diacylglycerol and triacylglycerol in human SMCs and macrophages through a PPAR-gamma-independent mechanism. Conversely, murine macrophages did not express ACSL4, and rosiglitazone did not inhibit ACSL activity in these cells, nor did it affect acute fatty acid partitioning into cellular lipids. Thus, rosiglitazone inhibits ACSL activity and fatty acid partitioning in human and murine SMCs and in human macrophages through a PPAR-gamma-independent mechanism likely to be mediated by ACSL4 inhibition. Therefore, rosiglitazone might alter the biological effects of fatty acids in these cells and in atherosclerosis.

FIG. 1

Expression of ACSL isoforms in human and murine SMCs and macrophages. Semiquantitative RT-PCR analysis of ACSL isoforms (A) was performed on total RNA. A total of 28 cycles of PCRs were performed, using the primers and annealing temperatures shown in Table 1. The products were resolved on 2% agarose gels and visualized via ethidium bromide staining. ACSL1 (B) and ACSL4 (C) protein expression was evaluated by Western blot analysis. Cell lysates (60 μg/lane) were separated using 7.5% SDS-PAGE and probed with anti-rat Acsl1 or Acsl4 antibodies (1:10,000). The blots were re-probed with an anti–β-actin antibody (Sigma) at a 1:10,000 dilution (C, lower panel). Recombinant rat Acsl4 (rAcsl4) was used as a positive control in C. Molecular weight markers are indicated by arrows. Analyses were repeated at least three times with similar results. HSS, high-speed supernatant; Mφ, macrophages.

FIG. 2

Rosiglitazone inhibits ACSL activity in SMCs and human macrophages, but not in murine macrophages. ACSL activity was analyzed in rosiglitazone or triacsin C–treated cell lysates. Lysates (200 μg total protein) from human SMCs (A and B), human macrophages (C and D), murine SMCs (E and F), and murine macrophages (G and H) were incubated in the presence of [³H]-OA, ATP, CoA, and rosiglitazone or triacsin C for 20 min at 37°C. Generated [³H]-OA-CoA was separated, and the radioactivity was determined via a scintillation counter. The results are the representative means of picomoles oleoyl-CoA formed/minute ± SE, performed in triplicates. The experiments were repeated three times with similar results. Mφ, macrophages.

FIG. 3

Analysis of OA partitioning in human SMCs. SMCs were pretreated for 30 min with 10 μmol/l rosiglitazone (A)or1 μmol/l triacsin C (B) before being labeled with [¹⁴C]-OA (1 μCi; 1 μmol/l) for the indicated times. The lipids were then extracted and separated by thin-layer chromatography. The plates were exposed to a PhosphorImager. CE, cholesteryl esters; PL, phospholipids. Representative experiments are shown, n = 3–5.

FIG. 4

Rosiglitazone transiently inhibits OA partitioning into phospholipids, whereas triacsin C exerts a sustained inhibition in human SMCs. Human SMCs were pretreated for 30 min with 10 μmol/l rosiglitazone (□)or1 μmol/l triacsin C (●) before being labeled with [¹⁴C]-OA (1 μCi; 1 μmol/l) for the indicated times. The lipids were then extracted and separated by thin-layer chromatography, and the radioactivity in the phospholipid spot was quantified. Values are means ± SE; n = 3–5; ***P < 0.001, **P < 0.01, *P < 0.05 compared with untreated cells (two-way ANOVA).

FIG. 5

Rosiglitazone inhibits fatty acid partitioning into DAG and TAG in human SMCs and macrophages, but not in murine macrophages. SMCs and macrophages were pretreated for 30 min with 10 μmol/l rosiglitazone, 1 μmol/l triacsin C (A and B), 5 μmol/l triacsin C (C), or 25 μmol/l 15-dPGJ₂ (A) before being labeled with [¹⁴C]-OA or [³H]-AA (1 μCi; 1 μmol/l) for 4 h. The lipids were then extracted and separated via thin-layer chromatography. Values are means ± SE, n = 3–7; ***P < 0.001, **P < 0.01, *P < 0.05 compared with nontreated controls (one-way ANOVA). The 15-dPGJ₂ experiments were repeated twice and at a 24-h time point (n = 4) with similar results.

FIG. 6

Inhibition of PPAR-γ does not block the inhibitory effect of rosiglitazone on fatty acid partitioning into DAG and TAG. Human SMCs were pretreated for 30 min with the indicated concentrations of the PPAR-γ inhibitor T0070907 (A) or were infected 48 h before analysis with 40 plaque-forming units/cell of a dominant-negative PPAR-γ mutant (Adx-D/N-PPARγ) or control (Adx-LacZ) (B). The SMCs were then incubated in the absence or presence of 10 μmol/l rosiglitazone and then in the presence of [¹⁴C]-OA (1 μCi; 1 μmol/l) for 4 h. The lipids were extracted and separated via thin-layer chromatography. Values are means ± SE; n = 3; ***P < 0.001, **P < 0.01, *P < 0.05 compared with nontreated controls (one-way ANOVA). PL, phospholipids.