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Cell Biology International (2006) 30, 947951 (Printed in Great Britain)
Nitric oxide-independent lipid metabolism in RAW 264.7 macrophages loaded with oleic acid
Wen‑Chen Huanga and Jin‑Shan Chenb*
aDivision of Plastic Surgery, Department of Surgery, Mackay Memorial Hospital, Taipei 110, Taiwan, ROC
bDepartment of Anatomy, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan, ROC
The role of nitric oxide (NO) in the regulation of lipogenesis and lipolysis in RAW 264.7 macrophages loaded with oleic acid (OA) was investigated in this paper. Magnolol stimulated full lipolysis without affecting NO levels. Both inhibition and elevation of NO production in OA-loaded macrophages did not induce lipolysis. Besides, lipopolysaccharide (LPS)-induced increased accumulation of lipid droplets was not reduced by down-regulation of NO levels. Moreover, incubation of macrophages with sodium nitroprusside (SNP), an NO donor, stimulated significant NO production without altering the lipid droplet accumulation. All these results clearly demonstrate that NO is not involved in the modulation of lipid metabolism in macrophages loaded with OA.
Keywords: Macrophage, Oleic acid, Nitric oxide, Lipid droplet, Lipogenesis, Lipolysis, Lipid metabolism.
*Corresponding author. Tel.: +886 2 27361661; fax: +886 2 27388852.
It is known that lipolysis in adipocytes depends on the elevation of cAMP and its activation of cAMP-dependent protein kinase (PKA), which in turn phosphorylates hormone-sensitive lipase (HSL) and stimulates its translocation to the lipid droplet surface for lipid catabolism (Egan et al., 1992). In addition to the cAMP-PKA-HSL system, recent studies have revealed the role of NO as another pathway in regulating lipolysis. Ribière and coworkers first reported NO synthase (NOS) in adipose tissues of rats (Ribière et al., 1996) and humans (Elizalde et al., 2000; Andersson et al., 1999). Further investigations showed that NO donors inhibited isoproterenol-stimulated lipolysis (Andersson et al., 1999; Gaudiot et al., 1998; Kawanami et al., 2002; Klatt et al., 2000).
Surprisingly, lipolysis stimulated by dibutyryl cAMP (Gaudiot et al., 2000) or leptin (Frühbeck and Gómez-Ambrosi, 2001) was decreased, rather than increased, by inhibition of NOS activity. Dobashi et al. (2003) further showed that enhancers of cAMP inhibited NO production elicited by LPS/tumor necrosis factor-α/interferon-γ co-treatment in white adipocytes, but increased in brown adipocytes. Despite these inconsistent findings, however, it is clear that NO, other than the cAMP-PKA-HSL system may play a significant role in modulating lipolysis in adipocytes through a cross-talk with cAMP. Nevertheless, the effects of NO in regulating lipid metabolism in other lipid-laden cells have not been studied.
Accumulation of lipid droplets in macrophages results in formation of foam cells, which represents one of the early steps in atherosclerosis (Ross, 1999). Thus, understanding the mechanism through which lipid droplets accumulate and disintegrate is critical for this pathological process. In vitro lipogenesis in macrophages has been shown to be accompanied by the induction of adipophilin (formerly called adipose differentiation-related protein), a lipid droplet-associated protein (Chen et al., 2001). Adipophilin mRNA expression was significantly induced in human macrophages stimulated with oxidized low-density lipoprotein and in atherosclerotic lesions (Wang et al., 1999). In addition, iNOS mRNA (Luoma and Yla-Herttuala, 1999) and protein (Baker et al., 1999) have been detected in macrophages in atherosclerotic lesions.
Whether NO release via iNOS in macrophage foam cells plays a role in modulating atherosclerosis or simply represents an inflammatory process stimulated by cytokines remains obscure (Napoli and Ignarro, 2001). In hepatocytes, NO donors inhibits fatty acid metabolism (Roediger et al., 2004; Garcia-Villafranca et al., 2003). More importantly, NO also impairs cholesterol synthesis (Roediger et al., 2004) and reduces hypercholesterolemia in rabbits fed with casein diet (Kurowska and Carroll, 1998). Although NO donors stimulate lipogenesis in macrophages (Senna et al., 1998), the effect of inhibiting NO release on lipid metabolism is not known. Moreover, whether NO participates in regulating lipogenesis in macrophages has not been studied.
In this paper, we first investigate the role of NO in modulating lipid metabolism in macrophages loaded with OA and discuss its action between adipocytes and lipid-laden macrophages. We provide here clear morphological and biochemical evidence that, unlike those in adipocytes, both lipolysis and lipogenesis in macrophages are mediated via an NO-independent pathway.
2 Materials and methods
2.1 Cell culture and drug treatment
RAW 264.7 macrophages (ATCC TIB-71) were obtained from the American Type Culture Collection (Rockville, MD, USA) and cultured in DMEM (GibcoBRL, Long Island, NY, USA) supplemented with 10% fetal bovine serum, 100
2.2 Nile red staining and calculation of surface area of lipid droplets
For observation of lipid droplets, macrophages were first fixed in 10% formalin (in PBS) for 10
2.3 Measurement of fatty acid release
Lipolysis was assayed by the amount of fatty acid released by RAW 264.7 macrophages into the medium. Briefly, cells were first treated with 30
2.4 Determination of NO production
Lipid droplet-containing cells were treated with various reagents for a definite time, with cells incubated with 5
2.5 Statistical analysis
The experimental results are expressed as the mean
Since NO has been implicated in regulating lipolysis, we first explored the possible involvement of NO in lipolysis. As shown in Fig. 1, magnolol (30
Determination of (A) lipolysis (6
Effect of NO on lipolysis in lipid-laden macrophages. (A) Dose-dependent inhibition of NO production by
In fact, closer examination showed that treatment of RAW 264.7 macrophages with 5
Nile red staining of RAW 264.7 macrophages. Cells were loaded with (A) OA alone or incubated with LPS (B), LPS and
Analysis of NO on the accumulation of lipid droplets in RAW 264.7 macrophages. (A) Effect of LPS,
Unlike macrophages, lipolysis in adipocytes is apparently modulated by NO. Exogenous addition of NO donors S-nitroso-N-acetyl-penicillamine (SNAP) and 1-propamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazine) (PAPA-NONOate) inhibited isoproterenol-stimulated lipolysis in adipocytes (Gaudiot et al., 1998; Kawanami et al., 2002), whereas inhibition of endogenous NO production by iNOS inhibitors decreased stimulated lipolysis (Gaudiot et al., 2000). SNAP may reduce isoproterenol-stimulated lipolysis by blocking the pathway upstream of adenylate cyclase (Gaudiot et al., 1998). This suggestion was supported by the finding that pre-treatment of isoproterenol with NO abolished the lipolytic activity of this catecholamine by oxidative inactivation (Klatt et al., 2000).
In contrast, PAPA-NONOate inhibited stimulated lipolysis without affecting cAMP production (Gaudiot et al., 1998). Interestingly, increase in cAMP and inhibition of PKA reduced and stimulated NO production, respectively, in 3T3-L1 cells (Dobashi et al., 2003), indicating a close relationship between cAMP and NO. Moreover, the effect of NO on modulating lipolysis may be accounted for by its antioxidant ability to protect PKA activity (Gaudiot et al., 2000). It is thus possible that lipolysis in adipocytes is not mediated by two distinct signal transduction pathways; instead, regulation between NO and the cAMP-PKA system may be responsible for the lipolytic process.
In the present study, however, lipolysis in lipid-laden macrophages was mediated in an NO-independent manner. Although previous results demonstrated that magnolol inhibited NO production in LPS-activated macrophages (Son et al., 2000; Matsuda et al., 2001), it did not notably alter the basal NO level at 6
It is thus very clear that lipolysis in lipid-laden macrophages is mediated through a mechanism unrelated to NO. Since magnolol stimulated steroidogenesis in rat adrenal cells (Wang et al., 2000) and lipolysis in lipid-laden macrophages (Chen et al., 2005) through a pathway independent of the cAMP-PKA system, all these results indicate that lipolysis in adipocytes and lipid-laden macrophages is mediated via different signal transduction pathways. Further studies are required to investigate the underlying mechanism through which magnolol regulates lipid metabolism in macrophages.
NO has been implicated in regulation of lipogenesis by inhibiting fatty acid and cholesterol synthesis in hepatocytes (Roediger et al., 2004; Garcia-Villafranca et al., 2003) and rat macrophages (Senna et al., 1998). However, all these results were obtained by the addition of exogenous NO donors, which, in the normal physiological state, come largely from release of endothelium. Thus, the above data may be explained by the regulatory action of NO derived from endothelium, but not by endogenous production of NO from hepatocytes or macrophages. In contrast, the data presented in this paper demonstrate that production and inhibition of endogenous NO has nothing to do with lipid metabolism in RAW 264.7 macrophages loaded with OA. Although LPS-enhanced accumulation of lipid droplets was accompanied by marked production of NO, similar increases in NO levels by SNP did not result in increased formation of lipid droplets.
Moreover, inhibition of LPS-induced NO generation by
Interestingly, iNOS expression was up-regulated in macrophages in atherosclerotic lesions (Luoma and Yla-Herttuala, 1999; Baker et al., 1999). Nevertheless, the significance of this fact should be interpreted with caution, as atherosclerosis represents an inflammatory process, during which NO is generated in bulk by macrophages (Ross, 1999). The elevated expression of iNOS, therefore, may be merely a result of macrophages responding to inflammation, instead of being regulated during formation of foam cells. Whether oxidized LDL results in macrophage foam cell formation by modulating NO generation remains to be elucidated. Similarly, the increased generation of lipid droplets may be explained as one of the inflammatory responses of macrophages elicited by LPS, since LPS repressed the expression of PPARγ (Welch et al., 2003), which in turn inhibited the expression of proinflammatory genes (Ricote et al., 2004). Besides, PPARs have been shown to promote lipid accumulation resulting in the formation of macrophage foam cells (Vosper et al., 2001; Nagy et al., 1998). The possibility regarding LPS-regulated expression of PPARs in foam cell formation requires further investigation.
In summary, our present data demonstrate that NO is not involved in either lipogenesis or lipolysis in OA-loaded RAW 264.7 macrophages, unlike in adipocytes, since changes in NO levels does not affect lipid metabolism. It is also not involved in lipid droplet accumulation. The mechanisms through which macrophages undergo lipid metabolism require further investigation.
This study was sponsored by the Mackay Memorial Hospital (94MMH-TMU-09). We thank Dr. C.H. Wu for his helpful discussions during the preparation of this manuscript.
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Received 25 April 2006/2 June 2006; accepted 26 June 2006doi:10.1016/j.cellbi.2006.06.014