Lipid Droplet Accumulation In Macrophages Research Articles

Abstract 3058: Cofilin 1 Mediates Fatty Acid-induced Foam Cell Formation And Inflammation In Mouse Bone Marrow-derived Macrophages

Background: Macrophages contribute to the development of atherosclerosis, in part through the accumulation of lipid droplets and subsequent transformation into highly inflammatory, lipid laden foam cells. Our previous work suggested that the RAGE/DIAPH1 axis plays a key role in regulating lipid metabolism, as deletion of Diaph1 in hepatocytes reduced nuclear translocation of SREBP1 through the actin cytoskeleton. Cofilin 1 (CFL1) is an actin polymerization molecule which has also been linked to the nuclear translocation of SRBP1, as well as triglyceride synthesis through activation of phospholipase D1. In the aforementioned study by our group, Diaph1 deletion was found to increase the Serine 3 phosphorylation (i.e., inactivation) of Cfl1 . Hypothesis: Accordingly, we hypothesized that CFL1 plays a role in lipid droplet accumulation in macrophages, potentially increasing the risk of atherosclerotic development. Methods: To investigate its role in macrophage lipid handling, we silenced Cfl1 in bone marrow derived macrophages (BMDMs) extracted from male C57BL/6J mice, using silencing RNAs and scramble controls. BMDMs were treated with a mixture of fatty acids palmitate and oleate (250 uM). The effects of Cfl1 knockdown on lipid accumulation were assessed using immunohistochemical staining (Bodipy). Quantitative (q)PCR was used to interrogate the mechanisms underlying these effects, and their impact on inflammation. Results: Cfl1- silenced (75% knockdown shown by qPCR) vs. scramble cells exhibited a significant decrease in the ratio of Bodipy to DAPI % area (p=0.0001). By qPCR, compared to scramble controls, BMDMs subjected to silencing of Cfl1 and incubated with fatty acids (palmitate-oleate) displayed significantly lower mRNA expression of Cd36 (p=0.0018) ; Ppargc1a (p=0.004) , Il6 (p<0.0001), and Tnfa (p=0.013). Conclusions: Our results indicate that silencing of Cfl1 leads to a decrease in lipid accumulation and markers of inflammation in BMDMs. Collectively, our data suggest that the effects of Cfl1 silencing on lipid handling result from decreased fatty acid uptake, which limits fatty acid oxidation on account of fatty acid availability in the cell. Studies are underway to probe the implications of these findings in atherosclerosis.

Electronegative LDL promotes triglyceride-enriched lipid droplet accumulation in macrophages by inducing the expression of genes related to lipid metabolism

Background and Aims: Electronegative LDL (LDL(-)) is a modified LDL present in blood that induces an inflammatory response in monocytes. However, the role of LDL(-) in macrophage function and particularly in foam cell formation has not been analyzed. In the current work, we sought to study the lipid accumulation promoted by LDL(-) in THP1-CD14 macrophages

Abstract 428: An Unexpected Role for ACC in Lipid Droplet Formation in Macrophages in Response to Acellular Adipocyte Fat

In adipose tissues of obese mice, macrophages accumulate in crown-like structures (CLS) formed around dying adipocytes. The CLS macrophages are thought to clear the dead adipocytes by exophagy, which involves exocytosis of lysosomes and digestion of apoptotic adipocytes. After digesting the plasma membrane and other cytosolic contents of the adipocyte, CLS macrophages come in contact with the large lipid droplet, however, it is unknown how macrophages response to such acellular adipocytes. We hypothesized that the acelular adipocytes in CLSs promote inflammation and metabolically activate the macrophages to promote clearance of the lipid. To mimic the in vivo scenario, we exposed cultured murine bone marrow-derived macrophages to lipid droplets isolated from the adipocytes of high-fat diet-induced obese C57BL/6 mice. In response to adipocyte lipid, macrophages accumulated lipid droplets, which were similar in size and number to lipid droplets of CLS macrophages in vivo . Acellular lipid exposure significantly increased TNF-α gene expression, which was suppressed by the CD36 inhibitor sulfo-N-succinimidyl oleate, supporting CD36-mediated inflammatory response. Moreover, lipid exposure significantly lowered metabolic activity of macrophages and impaired clearance of apoptotic bodies from 3T3-L1 adipocytes. Using specific inhibitors, unexpectedly we found that lipid droplet accumulation in macrophages was independent of CD36 activity or processes involved in exophagy such as exocytosis of lysosomes, extracellular lipase activity, lipolysis and phagocytosis. Interestingly, lipid droplet accumulation was dependent on acetyl-CoA carboxylase (ACC) as determined by use of ACC inhibitors soraphen A and TOFA, and siRNA knock-down of ACC in macrophages. Furthermore, confocal microscopy of whole mount adipose tissue revealed expression of ACC in CLS macrophages. Altogether, using a novel in vitro model we demonstrate that acelular adipocytes suppress metabolic activity, but induce inflammation and de novo lipogenesis-mediated lipid droplet formation in macrophages.

MicroRNA-33 Regulates Macrophage Autophagy in Atherosclerosis.

Defective autophagy in macrophages leads to pathological processes that contribute to atherosclerosis, including impaired cholesterol metabolism and defective efferocytosis. Autophagy promotes the degradation of cytoplasmic components in lysosomes and plays a key role in the catabolism of stored lipids to maintain cellular homeostasis. microRNA-33 (miR-33) is a post-transcriptional regulator of genes involved in cholesterol homeostasis, yet the complete mechanisms by which miR-33 controls lipid metabolism are unknown. We investigated whether miR-33 targeting of autophagy contributes to its regulation of cholesterol homeostasis and atherogenesis. Using coherent anti-Stokes Raman scattering microscopy, we show that miR-33 drives lipid droplet accumulation in macrophages, suggesting decreased lipolysis. Inhibition of neutral and lysosomal hydrolysis pathways revealed that miR-33 reduced cholesterol mobilization by a lysosomal-dependent mechanism, implicating repression of autophagy. Indeed, we show that miR-33 targets key autophagy regulators and effectors in macrophages to reduce lipid droplet catabolism, an essential process to generate free cholesterol for efflux. Notably, miR-33 regulation of autophagy lies upstream of its known effects on ABCA1 (ATP-binding cassette transporter A1)-dependent cholesterol efflux, as miR-33 inhibitors fail to increase efflux upon genetic or chemical inhibition of autophagy. Furthermore, we find that miR-33 inhibits apoptotic cell clearance via an autophagy-dependent mechanism. Macrophages treated with anti-miR-33 show increased efferocytosis, lysosomal biogenesis, and degradation of apoptotic material. Finally, we show that treating atherosclerotic Ldlr-/- mice with anti-miR-33 restores defective autophagy in macrophage foam cells and plaques and promotes apoptotic cell clearance to reduce plaque necrosis. Collectively, these data provide insight into the mechanisms by which miR-33 regulates cellular cholesterol homeostasis and atherosclerosis.

The Molecular Target of Rubimaillin in the Inhibition of Lipid Droplet Accumulation in Macrophages

The naphthohydroquinone rubimaillin, which has an angular-type three cyclic skeleton and was isolated from the Chinese medical plant Rubia cordifola, was found to inhibit lipid droplet accumulation in mouse macrophages and to selectively inhibit cholesteryl ester synthesis (IC(50): 18 microM). The metabolism of cholesterol from lysosomes to lipid droplets was inhibited by the compound with a similar IC(50) (45 microM). Moreover, rubimaillin inhibited acyl-CoA:cholesterol acyltransferase (ACAT1) activity in ACAT1-expressing cells (IC(50): 80 microM). Thus, these data strongly suggest that rubimaillin inhibits macrophage ACAT activity in order to decrease cholesteryl ester (CE) synthesis, leading to a reduction in the number of lipid droplets. Furthermore, rubimaillin was found to inhibit the ACAT2 isozyme in ACAT2-expressing cells (IC(50): 22 microM). We concluded that rubimaillin is a dual inhibitor of ACAT1 and ACAT2, but is more selective for the ACAT2 isozyme.

Inhibition of Lipid Droplet Accumulation in Mouse Macrophages by Stemphone Derivatives

From a study on the biological activity of fungal stemphones and their derivatives, five derivatives having an O-alkyl moiety at C-11 of stemphone C were found to inhibit lipid droplet accumulation in macrophages without any cytotoxic effect. Among the derivatives, those having O-isopropyl and O-isobutyl were the most potent inhibitors by blocking the synthesis of both cholesteryl ester (CE) and triacylglycerol (TG), the main constituents of lipid droplets in macrophages.

Molecular Target of Piperine in the Inhibition of Lipid Droplet Accumulation in Macrophages

An alkaloid piperine isolated from the Piper Nigrum was found to inhibit lipid droplet accumulation in mouse macrophages, and especially inhibited cholesteryl ester (CE) synthesis (IC50: 25 microM). The metabolism of cholesterol from lysosome to lipid droplet was inhibited with a similar IC50 (18 microM), indicating that the site of inhibition is one of the steps between the lysosomes and the endoplasmic reticulum. Therefore, effects of piperine on acyl-CoA:cholesterol acyltransferase (ACAT) activity in the microsomes prepared from mouse macrophage and liver were studied, to show that the compounds inhibited the activity in both cases (IC50: 9.1, 7.0 microM, respectively). Furthermore, piperine was found to inhibit both ACAT1 and ACAT2 isozymes to a similar extent (IC50: 16, 18 microM, respectively) in cell-based assays using ACAT1- or ACAT2-expressing cells. Thus, it was suggested that piperine inhibited macrophage ACAT to decrease CE synthesis, leading to a reduction of lipid droplets.

Fungal Isobisvertinol, a New Inhibitor of Lipid Droplet Accumulation in Mouse Macrophages

[structure: see text] New isobisvertinol and known bisvertinol were isolated from the culture broth of Aspergillus sp. FKI-1746. Isobisvertinol with the two alkenyl side chains extending in the same direction inhibited lipid droplet accumulation in macrophages, whereas bisvertinol with those extending in the reverse direction had almost no effect on the accumulation.

Absolute Stereochemistry of Fungal Beauveriolide III and ACAT Inhibitory Activity of Four Stereoisomers

Fungal beauveriolide III (BeauIII, 1b), a cyclodepsipeptide inhibiting acyl-CoA:cholesterol acyltransferase (ACAT) and showing antiatherogenic activity in mouse models, consists of L-Phe, L-Ala, D-allo-Ile, and 3-hydroxy-4-methyloctanoic acid (HMA) moieties, but the stereochemistry of the HMA part has not until now been fully defined. To determine it, four HMA stereoisomers were synthesized and labeled with (S)-(+)-2-(anthracene-2,3-dicarboximido)-1-propyl trifluoromethane sulfonate (AP-OTf), a chiral fluorescent reagent. The derivatives were separated by HPLC and compared with the natural HMA derivative, which was thereby identified as (3S,4S)HMA in BeauIII. Furthermore, the four beauveriolide III isomers ((3S,4S)BeauIII (23a), (3R,4R)BeauIII (23b), (3R,4S)BeauIII (23c), and (3S,4R)BeauIII (23d)) were synthesized, and it was shown that all the spectral data for 23a were identical with those for natural 1b. Isomers 23a and 23d showed potent inhibitory activity of lipid droplet accumulation in macrophages, while the other two isomers caused weak inhibition. Thus, the 3S configuration of BeauIII is important for this activity. Furthermore, 23a and 23d showed rather specific inhibition against the ACAT1 isozyme.

The Nine Lives of ACAT Inhibitors

Atherosclerosis is the product of excessive lipid accumulation and inflammation in the artery wall. The lipid that tops every list of suspects is cholesterol, which is the primary lipid in low-density lipoproteins (LDL). Cholesterol exists either as a simple molecule or as cholesterol esters, in which the hydroxyl group is linked to a fatty acyl moiety. In cells, cholesterol esters are synthesized in an intracellular reaction catalyzed by acyl coenzyme A (CoA):cholesterol acyltransferase (ACAT) enzymes.1,2 Owing to the discoveries of cholesterol esters in arterial lesions in 19103 and of ACAT activity in the mid 1900s,4 inhibiting ACAT has been considered as a strategy for preventing or treating atherosclerosis. Over the past 25 years, interest in ACAT inhibitors has waxed and waned as new studies advance knowledge in the field. Prominently reported and disappointing results from a recent human trial of an ACAT inhibitor5 dampened enthusiasm for this potential therapy. However, a study by Bell et al in this issue of Arteriosclerosis, Thrombosis, and Vascular Biology 6 revives the idea of targeting ACAT enzymes and highlights a key unanswered question: Can ACAT2-specific inhibitors lower plasma cholesterol and treat or prevent atherosclerosis? See page 1814 After the discovery of the ACAT reaction, two rationales for inhibiting ACAT emerged. One, in retrospect, was perhaps overly simplistic: blocking cholesterol esterification in macrophages would diminish macrophage “foam cell” formation and thereby decrease atherosclerotic lesion development. The other was to decrease hepatic and intestinal cholesterol ester formation, resulting in decreases in plasma levels …

Molecular Target of Decursins in the Inhibition of Lipid Droplet Accumulation in Macrophages

During screening for inhibitors of lipid droplet accumulation in mouse peritoneal macrophages, two coumarins identified as decursin and decursinol angelate were isolated from the roots of Angelicae gigantis. The cellular molecular target of these inhibitors in macrophages was studied. Decursin and decursinol angelate inhibited cholesteryl ester (CE) synthesis with IC50 values of 9.7 and 10.1 microM, respectively, whereas they enhanced triacylglycerol (TG) synthesis. Lysosomal metabolism of cholesterol to CE was inhibited by the compounds, indicating that the site of inhibition is one of the steps between the exiting of cholesterol from the lysosomes and CE synthesis in the endoplasmic reticulum. Therefore, acyl-CoA:cholesterol acyltransferase (ACAT) activity in the microsomal fractions prepared from mouse macrophages was studied, and the results showed inhibition of this activity by decursin and decursinol angelate with IC50 values of 43 and 22 microM, respectively. Thus, it was concluded that the compounds inhibit macrophage ACAT activity to decrease CE synthesis, leading to a reduction of lipid droplets in macrophages.

Synthesis and Biological Evaluation of a Beauveriolide Analogue Library

Synthesis of beauveriolide III (1b), which is an inhibitor of lipid droplet accumulation in macrophages, was achieved by solid-phase assembly of linear depsipeptide using a 2-chlorotrityl linker followed by solution-phase cyclization. On the basis of this strategy, a combinatorial library of beauveriolide analogues was carried out by radio frequency-encoded combinatorial chemistry. After automated purification using preparative reversed-phase HPLC, the library was tested for inhibitory activity of CE synthesis in macrophages to determine structure-activity relationships of beauveriolides. Among them, we found that diphenyl derivative 7{9,1} is 10 times more potent than 1b.

Spylidone, a Novel Inhibitor of Lipid Droplet Accumulation in Mouse Macrophages Produced by Phoma sp. FKI‐1840.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Discovery of halogenated eurypamide B analogues as inhibitors of lipid droplet accumulation in macrophages

Halogenated cyclic isodityrosine–tripeptides were synthesized as analogues of a marine natural product, eurypamide B. Although the original eurypamides showed no inhibitory activity, the new analogues were found to inhibit lipid droplet accumulation in macrophages with a low micromolar IC 50 value.

Spylidone, a Novel Inhibitor of Lipid Droplet Accumulation in Mouse Macrophages Produced by Phoma sp. FKI-1840

During our screening for microbial inhibitors of lipid droplet accumulation in macrophages, a new compound designated spylidone was isolated along with two structurally related known compounds, PF1052 and vermisporin, from the fermentation broth of Phoma sp. FKI-1840 by solvent extraction, silica gel column chromatography, ODS column chromatography and preparative HPLC. From the structure elucidation, spylidone has a spiro ring containing 2,4-pyrrolidinedione. Among the three compounds, only spylidone was found to inhibit lipid droplet accumulation in macrophages at 10 to approximately 50 microM without any cytotoxic effect.

Inhibition of Lipid Droplet Accumulation in Macrophages by Triterpenoids Produced from Torametes orientaris

Four triterpenoids 1-4 isolated from the fruit body of Torametes orientaris were found to inhibit lipid droplet accumulation in macrophages. From the biochemical analysis, compounds 2 and 3 inhibited selectively cholesteryl ester synthesis in macrophages, while compounds 1 and 4 showed inhibition of both cholesteryl ester and triacylglycerol syntheses.

Antiatherogenic activity of fungal beauveriolides, inhibitors of lipid droplet accumulation in macrophages.

Beauveriolides I and III, isolated from the culture broth of fungal Beauveria sp. FO-6979, showed potent inhibitory activity of lipid droplet accumulation in primary mouse peritoneal macrophages. The cellular molecular target of this inhibitory activity was studied in macrophages. Beauveriolides I and III strongly inhibited the cholesteryl ester (CE) synthesis with IC(50) values of 0.78 and 0.41 microM, respectively, without showing significant effects on the triacylglycerol and phospholipid synthesis. Furthermore, lysosomal cholesterol metabolism to CE in macrophages was inhibited by the compounds, indicating that the inhibition site lies within steps between cholesterol departure from the lysosome and CE synthesis in the endoplasmic reticulum. Therefore, acyl-CoA:cholesterol acyltransferase (ACAT) activity in the membrane fractions prepared from mouse macrophages was studied, resulting in a dose-dependent inhibition by beauveriolides I and III with IC(50) values of 6.0 and 5.5 microM, respectively. Thus, we showed that the beauveriolides inhibit macrophage ACAT activity specifically, resulting in blockage of the CE synthesis, leading to a reduction of lipid droplets in macrophages. ACAT activity in the membrane fractions prepared from mouse liver and Caco-2 cells was also inhibited, indicating that the beauveriolides block both ACAT-1 and -2. Moreover, beauveriolides I and III exert antiatherogenic activity in both low-density lipoprotein receptor- and apolipoprotein E-knockout mice without any side effects such as diarrhea or cytotoxicity to adrenal tissues as observed for many synthetic ACAT inhibitors. Beauveriolides I and III are the first microbial cyclodepsipeptides having an in vivo antiatherosclerotic effect and show promise as potential lead compounds for antiatherosclerotic agents.