Lipid Droplet Cholesterol Research Articles

Evidence of Differences in Cellular Regulation of Wolbachia-Mediated Viral Inhibition between Alphaviruses and Flaviviruses.

The intracellular bacterium Wolbachia is increasingly being utilised in control programs to limit the spread of arboviruses by Aedes mosquitoes. Achieving a better understanding of how Wolbachia strains can reduce viral replication/spread could be important for the long-term success of such programs. Previous studies have indicated that for some strains of Wolbachia, perturbations in lipid metabolism and cholesterol storage are vital in Wolbachia-mediated antiviral activity against the flaviviruses dengue and Zika; however, it has not yet been examined whether arboviruses in the alphavirus group are affected in the same way. Here, using the reporters for the alphavirus Semliki Forest virus (SFV) in Aedes albopictus cells, we found that Wolbachia strains wMel, wAu and wAlbB blocked viral replication/translation early in infection and that storage of cholesterol in lipid droplets is not key to this inhibition. Another alphavirus, o'nyong nyong virus (ONNV), was tested in both Aedes albopictus cells and in vivo in stable, transinfected Aedes aegypti mosquito lines. The strains wMel, wAu and wAlbB show strong antiviral activity against ONNV both in vitro and in vivo. Again, 2-hydroxypropyl-β-cyclodextrin (2HPCD) was not able to rescue ONNV replication in cell lines, suggesting that the release of stored cholesterol caused by wMel is not able to rescue blockage of ONNV. Taken together, this study shows that alphaviruses appear to be inhibited early in replication/translation and that there may be differences in how alphaviruses are inhibited by Wolbachia in comparison to flaviviruses.

Differences in proteome perturbations caused by the Wolbachia strain wAu suggest multiple mechanisms of Wolbachia-mediated antiviral activity

Some strains of the inherited bacterium Wolbachia have been shown to be effective at reducing the transmission of dengue virus (DENV) and other RNA viruses by Aedes aegypti in both laboratory and field settings and are being deployed for DENV control. The degree of virus inhibition varies between Wolbachia strains. Density and tissue tropism can contribute to these differences but there are also indications that this is not the only factor involved: for example, strains wAu and wAlbA are maintained at similar intracellular densities but only wAu produces strong DENV inhibition. We previously reported perturbations in lipid transport dynamics, including sequestration of cholesterol in lipid droplets, with strains wMel/wMelPop in Ae. aegypti. To further investigate the cellular basis underlying these differences, proteomic analysis of midguts was carried out on Ae. aegypti lines carrying strains wAu and wAlbA: with the hypothesis that differences in perturbations may underline Wolbachia-mediated antiviral activity. Surprisingly, wAu-carrying midguts not only showed distinct proteome perturbations when compared to non-Wolbachia carrying and wAlbA-carrying midguts but also wMel-carrying midguts. There are changes in RNA processing pathways and upregulation of a specific set of RNA-binding proteins in the wAu-carrying line, including genes with known antiviral activity. Lipid transport and metabolism proteome changes also differ between strains, and we show that strain wAu does not produce the same cholesterol sequestration phenotype as wMel. Moreover, in contrast to wMel, wAu antiviral activity was not rescued by cyclodextrin treatment. Together these results suggest that wAu could show unique features in its inhibition of arboviruses compared to previously characterized Wolbachia strains.

Abstract 5262: Avasimibe abolishes the breast cancer preventative efficacy of statin in a spontaneous mouse model of breast cancer

Abstract Background: The cholesterol biosynthesis pathway plays a central role in the normal cellular development and carcinogenesis. Relevant to breast cancer prevention, the breast epithelium in women with atypical hyperplasia has been shown to have increased in cholesterol levels and oxidative products of cholesterol. Additionally, cholesterol pathway genes such as HMGCR and HMGCS1 are known to be upregulated during progression of breast cancer in patients. We have previously shown in SV40C3TAg mice that the cholesterol lowering drug-fluvastatin reduces breast tumor incidence and burden by 50% and have noted that the efficacy of statin is reduced due to the tight regulation of the cholesterol biosynthesis pathway through multiple restorative feedback loops, thus bypassing the effect of statin blockade. Hypothesis: We hypothesize that fluvastatin efficacy can be improved by co-targeting the restorative feedback pathways that are involved in resistance to statins. Methodology: RNA seq data from statin resistant MCF10.AT1-R cell clones was compared to the statin sensitive parental MCF10.AT1 cells in order to identify pathways and targets involved in statin resistance. Genes that were also found to be upregulated in the statin non-responders mice mammary tumors relative to responders mice were tested for dual targeting. Efficacy of avasimibe, an ACAT inhibitor, to sensitize MCF10.DCIS cells to statin therapy was studied by the colony formation assay. In vivo validation of the statin + avasimibe dual therapy was performed in SV40C3TAg mice that spontaneously develop triple negative breast cancer. Results: We found ACAT1 and ACAT2 to be overexpressed in statin resistance cell clones and non-responder mouse breast tumors. ACATs are involved in cholesterol esterification that is required for storing cholesterol in lipid droplets. Cholesterol esterification in conjunction with cholesterol synthesis and export constitute key mechanisms involved in maintaining cholesterol homeostasis. Thus, we tested if avasimibe treatment potentiates the fluvastatin efficacy to inhibit colonizing ability. Avasimibe and fluvastatin combination completely abolished the ability of MCF10.DCIS cells to form colonies. Next, we tested the efficacy of combination treatment to prevent breast tumors in SV40C3TAg mice. The treatments started at the age of 6 weeks, prior to the onset of cancer and continued until 22 weeks of age. We found fluvastatin and avasimibe combination to be completely ineffective and 90% mice developed tumors. In addition, tumor burden was not reduced with dual treatment. Conclusions: We postulate that avasimibe enhanced metabolism of fluvastatin in mouse system and thus completely abolishing the chemopreventive effects of statin. Genomically derived rationale drug combinations may result in unanticipated interactions and/or ancillary effects that limit efficacy in vivo/in patients. Citation Format: Anjana Bhardwaj, Zhenlin Ju, Alexander Koh, Rhea Bhala, Jing Wang, Isabelle Bedrosian. Avasimibe abolishes the breast cancer preventative efficacy of statin in a spontaneous mouse model of breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5262.

BORC-ARL8-HOPS ensemble is required for lysosomal cholesterol egress through NPC2.

Lysosomes receive extracellular and intracellular cholesterol and redistribute it throughout the cell. Cholesterol egress from lysosomes is critical for cholesterol homeostasis, and its failure underlies the pathogenesis of genetic disorders such as Niemann-Pick C (NPC) disease. Here we report that the BLOC one-related complex (BORC)-ARL8-homotypic fusion and protein sorting (HOPS) ensemble is required for egress of free cholesterol from lysosomes and for storage of esterified cholesterol in lipid droplets. Depletion of BORC, ARL8, or HOPS does not alter the localization of the lysosomal transmembrane cholesterol transporter NPC1 to degradative compartments but decreases the association of the luminal transporter NPC2 and increases NPC2 secretion. BORC-ARL8-HOPS depletion also increases lysosomal degradation of cation-independent (CI)-mannose 6-phosphate (M6P) receptor (MPR), which normally sorts NPC2 to the endosomal-lysosomal system and then is recycled to the trans-Golgi network. These defects likely result from impaired HOPS-dependent fusion of endosomal-lysosomal organelles and an uncharacterized function of HOPS in CI-MPR recycling. Our study demonstrates that the BORC-ARL8-HOPS ensemble is required for cholesterol egress from lysosomes by enabling CI-MPR-dependent trafficking of NPC2 to the endosomal-lysosomal system.

Enhanced acyl-CoA:cholesterol acyltransferase activity increases cholesterol levels on the lipid droplet surface and impairs adipocyte function

Cholesterol plays essential structural and signaling roles in mammalian cells, but too much cholesterol can cause cytotoxicity. Acyl-CoA:cholesterol acyltransferases 1 and 2 (ACAT1/2) convert cholesterol into its storage form, cholesteryl esters, regulating a key step in cellular cholesterol homeostasis. Adipose tissue can store >50% of whole-body cholesterol. Interestingly, however, almost no ACAT activity is present in adipose tissue, and most adipose cholesterol is stored in its free form. We therefore hypothesized that increased cholesterol esterification may have detrimental effects on adipose tissue function. Here, using several approaches, including protein overexpression, quantitative RT-PCR, immunofluorescence, and various biochemical assays, we found that ACAT1 expression is significantly increased in the adipose tissue of the ob/ob mice. We further demonstrated that ACAT1/2 overexpression partially inhibited the differentiation of 3T3-L1 preadipocytes. In mature adipocytes, increased ACAT activity reduced the size of lipid droplets (LDs) and inhibited lipolysis and insulin signaling. Paradoxically, the amount of free cholesterol increased on the surface of LDs in ACAT1/2-overexpressing adipocytes, accompanied by increased LD localization of caveolin-1. Moreover, cholesterol depletion in adipocytes by treating the cells with cholesterol-deficient media or β-cyclodextrins induced changes in cholesterol distribution that were similar to those caused by ACAT1/2 overexpression. Our results suggest that ACAT1/2 overexpression increases the level of free cholesterol on the LD surface, thereby impeding adipocyte function. These findings provide detailed insights into the role of free cholesterol in LD and adipocyte function and suggest that ACAT inhibitors have potential utility for managing disorders associated with extreme obesity.

Abstract A06: Cholesteryl ester accumulation induced by PTEN loss and PI3K/AKT activation underlies human prostate cancer aggressiveness

Abstract Altered lipid metabolism is increasingly recognized as a signature of cancer cells. Enabled by label-free Raman spectromicroscopy, we performed quantitative analysis of lipogenesis at single-cell level in human patient cancerous tissues. Our imaging data revealed an unexpected, aberrant accumulation of esterified cholesterol in lipid droplets of high-grade prostate cancer and metastases. Biochemical study showed that such cholesteryl ester accumulation was a consequence of loss of tumor suppressor PTEN and subsequent activation of PI3K/AKT pathway in prostate cancer cells. Furthermore, we found that such accumulation arose from significantly enhanced uptake of exogenous lipoproteins and required cholesterol esterification. Depletion of cholesteryl ester storage significantly reduced cancer proliferation, impaired cancer invasion capability, and suppressed tumor growth in mouse xenograft models with negligible toxicity. These findings open opportunities for diagnosing and treating prostate cancer by targeting the altered cholesterol metabolism. Citation Format: Hyeon Jeong Lee, Shuhua Yue, Junjie Li, Seung-Young Lee, Tian Shao, Bing Song, Liang Cheng, Timothy A. Masterson, Xiaoqi Liu, Timothy L. Ratliff, Ji-Xin Cheng. Cholesteryl ester accumulation induced by PTEN loss and PI3K/AKT activation underlies human prostate cancer aggressiveness. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A06.

Cholesteryl Ester Accumulation Induced by PTEN Loss and PI3K/AKT Activation Underlies Human Prostate Cancer Aggressiveness

Altered lipid metabolism is increasingly recognized as a signature of cancer cells. Enabled by label-free Raman spectromicroscopy, we performed quantitative analysis of lipogenesis at single-cell level in human patient cancerous tissues. Our imaging data revealed an unexpected, aberrant accumulation of esterified cholesterol in lipid droplets of high-grade prostate cancer and metastases. Biochemical study showed that such cholesteryl ester accumulation was a consequence of loss of tumor suppressor PTEN and subsequent activation of PI3K/AKT pathway in prostate cancer cells. Furthermore, we found that such accumulation arose from significantly enhanced uptake of exogenous lipoproteins and required cholesterol esterification. Depletion of cholesteryl ester storage significantly reduced cancer proliferation, impaired cancer invasion capability, and suppressed tumor growth in mouse xenograft models with negligible toxicity. These findings open opportunities for diagnosing and treating prostate cancer by targeting the altered cholesterol metabolism.

New technique to quantify the lipid composition of lipid droplets in porcine oocytes and pre-implantation embryos using Nile Red fluorescent probe

The principal objective of this study was to develop a novel method based on confocal microscopy and a solvatochromic fluorescent dye, Nile red (NR) to quantify the main types of lipids in a single mammalian oocyte and embryo. We hypothesize that NR staining followed by the decomposition of NR-spectra identifies and quantifies the triglycerides, phospholipids, and cholesterol in a single oocyte and embryo. We analyzed the lipid droplets in porcine oocytes and pre-implantation embryos up to the hatched blastocyst stage developed in vivo and in cultured blastocysts. The emission spectrum of NR-stained mixture of different lipid types is a convolution of several component spectra. The principal component analysis (PCA) and a multivariate curve resolution-alternating least squares method (MCR-ALS) allowed to decompose the emission spectrum and quantify the relative amount of each lipid type present in mixture. We reported here that the level of the triglycerides, phospholipids and cholesterol in lipid droplets significantly decreases by 17.7%, 26.4% and 23.9%, respectively, from immature to mature porcine oocytes. The content of triglycerides and phospholipids remains unchanged in droplets of embryos from the zygote up to the morula stage. Then the triglyceride level decreases in the blastocyst by 15.1% and in the hatched blastocyst by 37.3%, whereas the amount of phospholipids decreases by 10.5% and 12.5% at the blastocyst and hatched blastocyst stages, respectively. In contrast, the content of cholesterol in droplets does not change during embryo cleavage. The lipid droplets in the blastocyst produced in vivo contain lower amounts of triglycerides (by 26.1%), phospholipids (by 14.2%) and cholesterol (by 34.8%) than those in the blastocyst cultured in NCSU-23 medium. In conclusion, our new technique is suitable to quantify the content of triglycerides, phospholipids and cholesterol in individual mammalian oocytes and embryos. Our findings indicate an important role for lipids during porcine oocyte maturation and early embryonic development, and suggest an altered lipid metabolism in cultured embryos.

Stimulation of lipolysis enhances the rate of cholesterol efflux to HDL in adipocytes

Adipose tissue constitutes a major location for cholesterol storage and, as such, it may play a role in the regulation of circulating cholesterol levels. A possible metabolic link between the lipolytic activity of adipocytes and their ability to release cholesterol to reconstituted human high density lipoprotein, HDL, was investigated in 3T3-L1 adipocytes. In the presence of HDL, composed of human apoA-I and phosphatidylcholine, adipocytes release cholesterol in a lipoprotein-dose and time dependent fashion. beta-adrenergic activation of the lipolysis promotes a 22% increase in the extent of cholesterol efflux to reconstituted discoidal HDL particles. Activation of lipolysis promotes a rapid decrease in the cholesterol content of the plasma membrane and a concomitant increase in lipid droplet cholesterol. This change is independent of the presence of HDL. Activation of the lipolysis does not affect the levels of ABCA1 and SR-BI. Therefore, the enhancement of cholesterol efflux is not due to the level of plasma membrane cholesterol, or to the levels of the cholesterol transporters ABCA1 and scavenger receptor SR-BI. Brefeldin A did not affect the rate of cholesterol efflux under basal lipolytic conditions, but it abolished the lipolysis-dependent enhancement of cholesterol efflux to HDL. This study suggests that activation of lipolysis is accompanied by an increase in BFA-sensitive vesicular transport that in turn enhances cholesterol efflux to HDL. The study supports a metabolic link between the lipolytic activity of adipocytes and the rate of cellular cholesterol efflux to HDL.

The Surface of Lipid Droplets Is a Phospholipid Monolayer with a Unique Fatty Acid Composition

We found that caveolin-2 is targeted to the surface of lipid droplets (Fujimoto, T., Kogo, H., Ishiguro, K., Tauchi, K., and Nomura, R. (2001) J. Cell Biol. 152, 1079-1085) and hypothesized that the lipid droplet surface is a kind of membrane. To elucidate the characteristics of the lipid droplet surface, we isolated lipid droplets from HepG2 cells and analyzed them by cryoelectron microscopy and by mass spectrometry. By use of cryoelectron microscopy at the stage temperature of 4.2 K, the lipid droplet surface was observed as a single line without any fixation or staining, indicating the presence of a single layer of phospholipids. This result appeared consistent with the hypothesis that the lipid droplet surface is derived from the cytoplasmic leaflet of the endoplasmic reticulum membrane and may be continuous to it. However, mass spectrometry revealed that the fatty acid composition of phosphatidylcholine and lysophosphatidylcholine in lipid droplets is different from that of the rough endoplasmic reticulum. The ample presence of free cholesterol in lipid droplets also suggests that their surface is differentiated from the bulk endoplasmic reticulum membrane. On the other hand, although caveolin-2beta and adipose differentiation-related protein, both localizing in lipid droplets, were enriched in the low density floating fraction, the fatty acid composition of the fraction was distinct from lipid droplets. Collectively, the result indicates that the lipid droplet surface is a hemi-membrane or a phospholipid monolayer containing cholesterol but is compositionally different from the endoplasmic reticulum membrane or the sphingolipid/cholesterol-rich microdomain.

Foam cell formation containing lipid droplets enriched with free cholesterol by hyperlipidemic serum

A monoclonal antibody, ASH1a/256C (256C), which binds to atherosclerotic lesions in Watanabe heritable hyperlipidemic rabbit (WHHL) aorta in vivo, recognizes complex structures of phosphatidylcholine mixed with neutral lipids. In the present study, a cell culture system is described in which foam cells express 256C-positive lipid droplets. J774.1 macrophages were incubated in the presence of a small volume of WHHL serum for 24 h to produce foam cells, which were then incubated without the WHHL serum for 3 days. Oil red O-positive lipid droplets appeared on day 1, and were present in the cells during the whole incubation period. The lipid droplets in the cells were positively immunostained with antibody 256C on day 4, although they were negative on day 1. Expression of the antigenic lipid droplets was also induced by the addition of acetylated LDL or sera from patients with hyperlipidemia. When foam cells were induced by the addition of WHHL serum, cellular content of cholesteryl ester was greatly increased but then decreased to near basal levels by day 4. Concomitantly, cellular free cholesterol increased during the culture period, indicating that the cholesteryl ester changes to free cholesterol by day 4. The lipid droplets in the foam cells on day 4 were positively stained with filipin, a fluorescent probe for free cholesterol, as well as with 256C antibody, indicating that free cholesterol is enriched in antigenic lipid droplets. These observations suggest that hydrolysis and rearrangement of cellular cholesterol take place in foam cells to form complex structures of phosphatidylcholine and free cholesterol in lipid droplets.—Mori, M., H. Itabe, Y. Higashi, Y. Fujimoto, M. Shiomi, M. Yoshizumi, Y. Ouchi, and T. Takano. Foam cell formation containing lipid droplets enriched with free cholesterol by hyperlipidemic serum.

Cholesteryl ester transfer protein (CETP) mRNA abundance in human adipose tissue: relationship to cell size and membrane cholesterol content.

Cholesteryl ester transfer protein (CETP) has a well-defined role in plasma neutral lipid transport. CETP synthesized by human adipose tissue may contribute to the plasma CETP pool. CETP mRNA abundance increases in subcutaneous adipose tissue in response to cholesterol feeding and we have hypothesized that CETP gene expression is regulated by a specific pool of cellular sterol. In the present study, we have quantified CETP mRNA levels in subcutaneous adipose tissue of 10 female subjects using a solution hybridization RNase protection assay. Particulate (membrane cholesterol) and lipid droplet cholesterol (core cholesterol) were determined by gas chromatography. CETP mRNA abundance in these adipose tissue specimens correlated significantly with membrane cholesterol expressed as a fraction of membrane protein (r = 0.67, P = 0.031). There was also a linear relationship between CETP mRNA abundance and membrane cholesterol to core triglyceride ratio (r = 0.77, P = 0.009) and a strong correlation between the percentage of cellular cholesterol in the membrane fraction (ratio of membrane to core cholesterol) and CETP mRNA abundance (r = 0.91, P = 0.0002). In contrast, there was a negative relationship between each of lipid droplet cholesterol and triglyceride and CETP mRNA levels. Human adipose tissue maintained in organ culture for several days was shown to secrete CETP into the culture medium. Incubation with cholesterol-rich chylomicron remnants elicited a dose-dependent increase in both membrane and core cholesterol and a concomitant increase in the level of CETP mRNA. These studies demonstrate that adipose tissue CETP mRNA abundance is a function of membrane cholesterol concentration rather than lipid droplet cholesterol and that CETP mRNA increases with adipocyte cholesterol enrichment via chylomicron remnants. CETP gene expression is highest in small lipid-poor adipocytes, suggesting that CETP synthesized and secreted by adipocytes may have a role in promoting cellular cholesterol accumulation.

Sterol carrier protein2. Delivery of cholesterol from adrenal lipid droplets to mitochondria for pregnenolone synthesis.

The ability of sterol carrier protein2 (SCP2) to mediate transfer of unesterified cholesterol from adrenal lipid inclusion droplets to mitochondria has been tested in an in vitro model system. Unlike mitochondrial utilization of cholesterol added in acetone or dimethyl sulfoxide, the unesterified cholesterol of lipid droplets did not provide a readily available source of substrate for mitochondrial pregnenolone production, without the addition of a transport mediator. Addition of SCP2, but not albumin, stimulated mitochondrial utilization of droplet cholesterol in a concentration-dependent manner. In the absence of mitochondria, SCP2 sequestered lipid droplet cholesterol, and in the presence of mitochondria, which were unable to convert cholesterol to pregnenolone, this cholesterol was quantitatively accumulated by mitochondria. Both processes were concentration-dependent and demonstrated a molar ratio of SCP2 and cholesterol for both binding and transport of 1. SCP2 also enhanced pregnenolone formation by mitochondria which were incubated in the absence of an extramitochondrial source of cholesterol. However, SCP2 had no effect on steroid release from a crude particulate fraction. These studies suggest that the effects of SCP2 are related to delivery of cholesterol from preformed stores to and into mitochondria for initiation of steroid hormone synthesis, and may represent an important modulator of sterol metabolism in adrenal cortical cells.