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Cell Biology International (2006) 30, 947–951 (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


Abstract

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.


1 Introduction

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, 100units/ml of penicillin, and 100μg/ml of streptomycin. To mimic foam cell formation, macrophages were incubated with 70μg/ml of OA (Sigma) for 16h. For the study of lipolysis, lipid-laden macrophage foam cells were stimulated with 30μM magnolol (Wako chemicals, Japan), 1mM l-NAME, 0.1mM SNP, or 5μg/ml LPS for 6h, and the medium was collected and subjected to free fatty acid analysis.

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 10min, and then stained with Nile red (Sigma) at a final concentration of 10μg/ml for 5min (Greenspan et al., 1985). Following brief washes with PBS, cells were mounted and observed under a Zeiss epifluorescent microscope. The surface areas of lipid droplets were calculated from 200 Nile red-stained cells in each group using Image-Pro Plus software.

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 30μM magnolol or 5μg/ml LPS for 6h, and the medium was collected and centrifuged at 1500×g for 5min. The supernatant containing extracellular free fatty acid was then determined using a commercial kit (Roche, Mannheim, Germany) following the instructions of the manufacturer.

2.4 Determination of NO production

Lipid droplet-containing cells were treated with various reagents for a definite time, with cells incubated with 5μg/ml LPS for 24h as the positive control. The supernatants were used directly to measure NO production by mixing with Griess reagent (Assay designs, MI, USA) as described previously (Chen et al., 1999), before reading the absorbance at 540nm in a microplate reader.

2.5 Statistical analysis

The experimental results are expressed as the mean±S.E., and comparisons were performed using Student's t-test. A P value less than 0.05 was considered statistically significant.

3 Results

Since NO has been implicated in regulating lipolysis, we first explored the possible involvement of NO in lipolysis. As shown in Fig. 1, magnolol (30μM) stimulated a remarkable lipolysis at 6h (Fig. 1A), but NO production remained unchanged at 3h (Fig. 1B) when compared with the controls. Time course examination revealed that treatment of cells with magnolol for only 3h stimulated significant lipolysis when NO production was not changed (data not shown). Moreover, LPS at a concentration of 5μg/ml prominently elevated NO levels (Fig. 1B) without stimulating lipolysis (Fig. 1A). Further support of the NO-independent lipolysis pathway was derived from the results that dose-dependent inhibition of NO production by l-NAME did not stimulate lipolysis (Fig. 2). All these results demonstrated that changes in NO levels are irrelevant in modulating lipolysis in OA-laden macrophages.


Fig. 1

Determination of (A) lipolysis (6h) and (B) NO production (3h) in control RAW 264.7 macrophages (C) and macrophages treated with LPS or magnolol (Mag). Results are a representative of three experiments. **P<0.001 vs. control.


Fig. 2

Effect of NO on lipolysis in lipid-laden macrophages. (A) Dose-dependent inhibition of NO production by l-NAME for 24h in macrophages loaded with 70μg/ml OA. N, normal control. *P<0.05 compared with OA group (second column). (B) Nile red staining of RAW 264.7 macrophages treated with 1mM l-NAME for 24h. The bright spots (arrow), rather than the amorphous background, represent Nile red-stained lipid droplets accumulated within the cytoplasm. l-NAME at this dose did not stimulate lipolysis, since the lipid droplets in cells treated with l-NAME were not notably different from those without treatment. Bar=25μm.



In fact, closer examination showed that treatment of RAW 264.7 macrophages with 5μg/ml LPS for 24h significantly increased accumulation of lipid droplets (Fig. 3B), as compared with cells loaded with OA alone (Fig. 3A). It seemed at this time that production of NO was related to increased lipid droplet formation over this time interval. To test this hypothesis, OA-laden macrophages were co-treated with 5μg/ml LPS and 1mM l-NAME and no significant decrease in lipid droplets was seen (Fig. 3C). In addition, cells incubated with 0.1mM SNP, an NO donor, did not enhance lipid droplet accumulation (Fig. 3D). Further analysis revealed that, although the surface area of lipid droplets increased about 6-fold under LPS stimulation, this effect was not obviously altered by l-NAME (Fig. 4A), which significantly inhibited NO production to control levels (Fig. 4B). The idea that an increase in lipid droplets is mediated via an NO-independent mechanism is further supported by the finding that SNP induced a similar level of NO production to that produced by LPS (Fig. 4B), without enhancing lipogenesis (Fig. 4A). It is therefore clear that NO is not involved in lipid accumulation in RAW 264.7 macrophages loaded with OA.


Fig. 3

Nile red staining of RAW 264.7 macrophages. Cells were loaded with (A) OA alone or incubated with LPS (B), LPS and l-NAME (C) or SNP (D) in the presence of 70μg/ml OA for 24h. Bar=25μm.


Fig. 4

Analysis of NO on the accumulation of lipid droplets in RAW 264.7 macrophages. (A) Effect of LPS, l-NAME and SNP on the surface area of lipid droplets. The values are means±S.E. from 200 cells. There is no significant difference of surface area between LPS+OA and LPS+OA+l-NAME groups (P>0.05). (B) Measurement of NO production caused by LPS, l-NAME and SNP. Results are a representative of three experiments. *P<0.05 and **P<0.001 compared with OA group. #P<0.001 compared with OA+LPS group. In both experiments, cells were first treated with various agents for 24h, followed by measurement of surface area of lipid droplets and NO production.



4 Discussion

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 6h, when complete lipolysis in lipid-laden RAW 264.7 macrophages occurred. In fact, magnolol stimulated prominent lipolysis at 3h without affecting NO. These results indicate that lipolysis in lipid-laden macrophages cannot be accounted for by changes in NO levels. Further support of this idea comes from the observation that both inhibition and elevation of NO production induced by l-NAME and LPS, respectively, failed to induce lipolysis.

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 l-NAME failed to suppress lipogenesis stimulated by LPS. Finally, PMA stimulated increased accumulation of lipid droplets in RAW 264.7 macrophages loaded with OA (Chen et al., 2001), while NO production remained unchanged (data not shown). All these results provide obvious evidence that in vitro formation of foam cells by incubation with OA, but not oxidized LDL, is mediated via a pathway other than NO, and that LPS stimulates lipogenesis in an NO-independent manner. The reason for the differential roles of NO in modulating lipolysis between adipocytes and lipid-laden macrophages is not known. Whether this difference is attributed to the properties of the lipid droplets requires further investigation.

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.

Acknowledgements

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 2006

doi:10.1016/j.cellbi.2006.06.014


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
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