Characterization of big stanniocalcin variants in mammalian adipocytes and adrenocortical cells

The hormone stanniocalcin (STC) is widely distributed, and in rodents the highest levels of expression are in the ovaries. In both cows and rodents, ovarian STC consists of three high-molecular-weight variants collectively known as big STC. In the ovary, big STC is made by theca cells and interstitial cells and is targeted to lipid storage droplets of nearby luteal cells to inhibit progesterone release. An endocrine pathway is operative during pregnancy and lactation. Whether or not big STC is made by tissues other than ovary has never been addressed. Therefore, the purpose of this study was to determine via a detailed characterization of adrenal glands and adipocytes whether big STC is present in other cells that store and release lipids. The results showed that STC was made in bovine and mouse adrenals, mainly in steroidogenic, adrenocortical cells. The majority of ligand and receptor were likewise confined to cortical zone cells. As in luteal cells, high levels of ligand and receptor were found in the adrenocortical cell lipid droplet fraction. However, adrenals made only the largest (135 kDa) of the three big STC variants. Nonetheless, adrenal STC had much greater receptor affinity than a mixture of the three big STC variants. Adipocytes contained all three big STC variants, and both ligand and receptor were heavily concentrated on the lipid droplets. Moreover, adipocyte lipid storage droplets had 50-fold more receptors than those in steroidogenic cells, indicating that big STC is heavily targeted to adipose cells. The findings collectively support the hypothesis that big STC is not unique to ovarian steroidogenic cells but is in fact common to cells with a role in lipid storage and release.     

Evidence for cross-talk between stanniocalcins

There are 2 forms of stanniocalcin (STC) produced by the STC-1 gene; a 50 kDa polypeptide known as STC50 and a recently discovered group of higher molecular weight variants that are collectively referred to as big STC. Both have different tissue patterns of expression and different intracellular targeting pathways. STC50 functions locally in tissues such as muscle, liver, and kidney and is targeted to mitochondria. Big STC, on the other hand, is made by the ovaries. It signals both locally on nearby corpus luteal cells and systemically. Interestingly, however, receptor binding assays employing STC50 as the tracer have shown that the smaller ligand can bind equally to tissue receptors targeted by either form of the hormone. This suggests there may be cross-talk between ligands. The present study provides credence to this notion by demonstrating how the 2 hormones can compete for tissue receptors normally targeted by 1 form of the hormone (big STC). The results also reveal how STC50 can completely block the inhibitory effects of big STC on luteal cell progesterone release when added simultaneously. The findings therefore add credence to the possibility that there may be circumstances during which the 2 ligands functionally antagonize each other's actions.     

Morphology and histochemistry of ovarian changes in the field rat, Millardia meltada

A study has been made of the morphological and histochemical changes of the ovary of the field rat, Millardia meltada during its oestrous cycle and pregnancy. The follicular growth and atresia, ovulation and formation of corpora lutea occur throughout the year except severe winter months (December and January). Fluctuations in the follicular development occur on different days of the oestrous cycle and pregnancy. The granulosa cells show a progressive increase in their size in successive stages of follicle growth. The granulosae of normal follicles show some sparsely scattered lipid bodies which consist of phospholipids. Theca interna cells during follicular growth develop diffuse lipoproteins and lipid droplets consisting of triglycerides, phospholipids and cholesterol and/or its esters. The luteal cells of corpora lutea are formed by the granulosa cells as the theca interna cells degenerate and disappear. The fibroblast-like cells of thecal origin, alongwith the blood vessels, invade the luteal cell mass. The luteal cells during metoestrus, dioestrus and first half of pregnancy show abundant diffuse lipoproteins and a few lipid droplets composed mainly of phospholipids and some triglycerides, which are indicative of active steroidogenesis. The details of degenerative histological and histochemical alterations of corpora lutea during oestrous cycle and pregnancy are also described and discussed. Morphological and histochemical changes of follicular atresia are described. The granulosa cells of atretic follicle degenerate and disappear leaving behind theca interna cells which form patches of interstitial gland cells during the reproductive activity of the present rat. Interstitial gland cells show diffusely distributed sudanophilic lipoproteins and lipid droplets consisting of triglycerides, cholesterol and/or its esters and some phospholipids, which are indicative of steroidogenesis. The functional significance of histological and histochemical changes, which occur in various components of the ovary during oestrous cycle and pregnancy, has been discussed.     

Nuclear targeting of stanniocalcin to mammary gland alveolar cells during pregnancy and lactation

In most mammalian tissues, the stanniocalcin-1 gene (STC-1) produces a 50-kDa polypeptide hormone known as STC50. Within the ovaries, however, the STC-1 gene generates three higher-molecular-mass variants known as big STC. Big STC is targeted locally to corpus luteal cells to block progesterone release. During pregnancy and lactation, however, ovarian big STC production increases markedly, and the hormone is released into the serum. During lactation, this increase in hormone production is dependent on a suckling stimulus, suggesting that ovarian big STC may have regulatory effects on the lactating mammary gland. In this report, we have addressed this possibility. Our results revealed that virgin mammary tissue contained large numbers of membrane- and mitochondrial-associated STC receptors. However, as pregnancy progressed into lactation, there was a decline in receptor densities on both organelles and a corresponding rise in nuclear receptor density, most of which were on milk-producing, alveolar cells. This was accompanied by nuclear sequestration of the ligand. Sequestered STC resolved as one approximately 135-kDa band in the native state and therefore had the appearance of a big STC variant. However, chemical reduction collapsed this one band into six closely spaced, lower-molecular-mass species (28-41 kDa). Mammary gland STC production also underwent a dramatic shift during pregnancy and lactation. High levels of STC gene expression were observed in mammary tissue from virgin and pregnant rats. However, gene expression then fell to nearly undetectable levels during lactation, coinciding with the rise in nuclear targeting. These findings have thus shown that the mammary glands are indeed targeted by STC, even in the virgin state. They have further shown that there are marked changes in this targeting pathway during pregnancy and lactation, accompanied by a switch in ligand source (endogenous to exogenous). They also represent the first example of nuclear targeting by STC.     

Characterization of mammalian stanniocalcin receptors. Mitochondrial targeting of ligand and receptor for regulation of cellular metabolism

The polypeptide hormone stanniocalcin (STC) is widely expressed in mammalian tissues. STC acts locally in kidney and gut to modulate calcium and phosphate excretion, and its overexpression in mice results in high serum phosphate, dwarfism, and increased metabolic rate. STC has also been linked to cancer, pregnancy, lactation, angiogenesis, organogenesis, cerebral ischemia, and hypertonic stress. In this report we have characterized the STC receptor and the functional targeting of ligand and receptor to mitochondria. For receptor binding analysis, a stanniocalcin-alkaline phosphatase fusion protein was engineered. Subsequent binding assays using the fusion protein indicated that kidney and liver contained the highest number of binding sites with affinities of 0.8 and 0.25 nm, respectively. Intriguingly, purified mitochondria from both tissues yielded similar high affinity binding sites. Fractionation analysis revealed that the majority of binding sites were localized to the inner mitochondrial membrane. In further studies, we characterized the time course of STC-alkaline phosphatase fusion protein sequestration by intact mitochondria. In situ ligand binding also revealed discrete, displaceable binding to plasma membranes and mitochondria of nephron cells and liver hepatocytes. The existence of mitochondrial receptors prompted a similar search for the ligand. Immunogold electron microscopy revealed that STC was preferentially concentrated in the mitochondria of all nephron segments targeted by STC. Subcellular fractionation revealed that >90% of cellular STC immunoreactivity was mitochondrial, confined to the inner matrix, and similar in size to recombinant STC (50 kDa). In functional studies, recombinant STC had concentration-dependent stimulatory effects on electron transfer by sub-mitochondrial particles. Collectively the evidence implies a role for STC in cell metabolism.     

Microtubules, organelle transport, and steroidogenesis in cultured adrenocortical tumor cells. 1. An ultrastructural analysis of cells in which basal and ACTH-induced steroidogenesis was inhibited by taxol

In adrenocortical cells, the first step in the enzymatic processing of cholesterol to steroid end products occurs in the mitochondria. ACTH increases mitochondrial cholesterol and steroidogenesis. In cultured mouse adrenocortical tumor cells, microtubule-based organelle motility may increase the proximity of mitochondria to the SER, lipid droplets and endoscome-derived lysosomes, thereby facilitating the transfer of cholesterol from these organelles to the mitochondrial outer membrane. ACTH may increase opportunities for the transfer by promoting organelle motility and by increasing the number of lysosomes. Taxol, a microtubule polymerizer, inhibits basal and ACTH-induced steroidogenesis in these cells, presumably at the step where mitochondria obtain cholesterol. We examined the ultrastructure of taxol-treated, unstimulated and ACTH-stimulated cells, seeking alterations which conceivably could interefer with the proposed organelle transport and encounters, and thus correlate with taxol's inhibition of steroidogenesis. Primary cultured cells were incubated in serum-containing medium for 4 hr with and without ACTH (10 mU/ml), with 10 micrograms/ml and 50 micrograms/ml of taxol, and with ACTH and taxol 10 or taxol 50 simultaneously. Culture media were analyzed for the presence of secreted steroids at the end of 1, 2, and 4 hr of incubation. At the end of the fourth hour, unstimulated cells and cells treated with ACTH, taxol 50, and both agents simultaneously, were fixed and processed for EM. Taxol inhibited basal and ACTH-induced steroidogenesis in a dose-dependent fashion. In both unstimulated and ACTH-stimulated cells, taxol 50 formed numerous microtubule bundles, but did not markedly change the distribution of mitochondria and lipid droplets. SER tubules, and clusters of Golgi fragments, endosomes, and lysosomes appeared to be translocated towards the cell periphery along some of the microtubules. Taxol permitted an ACTH-induced cell rounding and microfilament rearrangement considered to facilitate organelle motility. Our data indicate that taxol disrupts the formation of lysosomes by these adrenal cells, but it seemed unlikely that taxol's ultrastructural effects could prevent organelle transport proposed to cause meetings between mitochondria and the SER or lipid droplets, or prevent ACTH-caused increases in these encounters. Taxol may instead prevent the transfer of lipid droplet or SER-contained cholesterol to adjacent mitochondria, by a means not detectable in our electron micrographs.     

Ultrastructural evidence for estradiol synthesis in the ovary of persistent-estrous rats exposed to continuous illumination

The relation between sex hormone levels in blood and ultrastructural changes of ovarian follicles was examined in persistent-estrous rats exposed to continuous illumination (LL) for 100 days. Plasma LH showed a tonic level secretory pattern, and circulating estradiol and estrone concentrations were relatively high, while both levels of FSH and progesterone were low. Various stages of growing and degenerating follicles were observed in the ovary of the LL-exposed rats. The early stage of antral follicle did not seem to possess the ability of steroidogenesis. Theca cells around mature antral follicles contained prominent Golgi apparatuses, plenty of smooth endoplasmic reticulum (ER), abundant free ribosomes and many round-mitochondria. A few newly formed lipid droplets were seen in some of theca cells. Granulosa cells contained much distended rough ER, well-developed mitochondria, several lipid droplets and microfilaments. The theca cells of abnormal follicles with hyperplastic and infolded layers of granulosa cells contained many lipid droplets. However, the development of the smooth ER became hindered with increasing lipid droplets in the theca cell. On the other hand, granulosa cells of abnormal follicles contained greater numbers of lipid droplets than those of antral mature follicles, and were equipped with well-developed cytoplasmic organelles as were those of mature antral follicles. Theca interna cells of abnormal follicles may be more involved in the secretion of androgen, which has already been accumulated in the lipid droplets, than the cells involved in the active synthesis of the hormone, while the granulosa cells may convert its androgen to estrogen. The present findings suggest that both follicles of mature and abnormal types in the LL-exposed rat retain enough capacity of estradiol production and participate in the continued elevation of circulating estradiol, probably resulting in the stimulation of the theca cells by the tonic level of LH and of the granulosa cells by the levels of FSH, which are lower than the basal values during the normal 4-day estrous cycle.     

Microtubules, organelle transport, and steroidogenesis in cultured adrenocortical tumor cells. 2. Reversibility of taxol's inhibition of basal and ACTH-induced steroidogenesis is unaccompanied by reversibility of taxol-induced changes in cell ultrastructure

Taxol inhibits the basal and ACTH-stimulated steroidogenesis of cultured mouse adrenocortical tumor cells, presumably by preventing the arrival of cholesterol in mitochondria. In these cells, taxol polymerizes and rearranges microtubules, disperses SER masses, disrupts the Golgi, and impedes the formation of cholesterol-containing lysosomes. However, taxol's alterations in ultrastructure appear likely to permit both a microtubule-based organelle transport proposed to bring mitochondria of unstimulated cells close to alternate sources of cholesterol--the SER and lipid droplets--and postulated ACTH-caused increases in these encounters. Conceivably, taxol may prevent the transfer of cholesterol from the SER and lipid droplets to mitochondria, once the meetings are achieved. To investigate this possibility, we determined the reversibility of taxol's ultrastructural effects and inhibition of steroidogenesis. Primary cultured adrenal tumor cells were incubated for 4 hr with and without ACTH (10 mU/ml). with taxol (50 micrograms/ml), and with ACTH and taxol 50 simultaneously. Some cultures from each set were washed with fresh medium and re-incubated for 1.5 hr. with and without ACTH. Media taken from cultures at the ends of pre- and post-washout incubations were analyzed for the presence of secreted steroids. Sample cultures were fixed for electron microscopy at the ends of both incubations. Data derived from pre-washout incubations confirmed previous reports of taxol's ultrastructural changes and inhibition of steroidogenesis. When cells recovered from taxol in the absence of ACTH, the inhibition of steroidogenesis was completely reversed. In the presence of ACTH, ex-taxol-treated cells demonstrated a "rounding up' and an increased steroid production that are characteristic responses to the hormone. However, in all cases, there was a persistence of taxol's alterations in organelle numbers and arrangements. Our findings establish that the ultrastructural effects of taxol which we recorded cannot prevent mitochondria of unstimulated and ACTH-stimulated adrenal tumor cells from gaining cholesterol. They strengthened the possibility that in pre-washout incubations, taxol allowed organelle motility to bring mitochondria adjacent to cholesterol-containing SER tubules and lipid droplets, but inhibited steroidogenesis by preventing the cholesterol transfer. Taxol might limit the availability of a protein required for the transfer, an effect not visible in our electron micrographs.     

Interaction of hormone-sensitive lipase with steroidogenic acute regulatory protein: facilitation of cholesterol transfer in adrenal

Hormone-sensitive lipase (HSL) is responsible for the neutral cholesteryl ester hydrolase activity in steroidogenic tissues. Through its action, HSL is involved in regulating intracellular cholesterol metabolism and making unesterified cholesterol available for steroid hormone production. Steroidogenic acute regulatory protein (StAR) facilitates the movement of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane and is a critical regulatory step in steroidogenesis. In the current studies we demonstrate a direct interaction of HSL with StAR using in vitro glutathione S-transferase pull-down experiments. The 37-kDa StAR is coimmunoprecipitated with HSL from adrenals of animals treated with ACTH. Deletional mutations show that HSL interacts with the N-terminal as well as a central region of StAR. Coexpression of HSL and StAR in Chinese hamster ovary cells results in higher cholesteryl ester hydrolytic activity of HSL. Transient overexpression of HSL in Y1 adrenocortical cells increases mitochondrial cholesterol content under conditions in which StAR is induced. It is proposed that the interaction of HSL with StAR in cytosol increases the hydrolytic activity of HSL and that together HSL and StAR facilitate cholesterol movement from lipid droplets to mitochondria for steroidogenesis.     

Expression of mRNA of lipoprotein receptor related protein 8, low density lipoprotein receptor, and very low density lipoprotein receptor in bovine ovarian cells during follicular development and corpus luteum formation and regression

Lipoproteins in the plasma are the major source of cholesterol obtained by the ovarian theca and granulosa cells for steroidogenesis. In this study, we have identified mRNA expression in bovine theca and granulosa cells of two lipoprotein receptors, low density lipoprotein receptor (LDLr) and very low density lipoprotein receptor (VLDLr) in granulosa cells from small antral follicles through preovulatory follicles and in theca cells from large and medium sized antral follicles. In the corpus luteum (CL) both these receptors were found in the developing and differentiating stages whereas only mRNA for VLDLr was detected in the regression stage. This study also described for the first time, the presence of lipoprotein receptor related protein (LRP8) in granulosa cells from small antral follicles through preovulatory follicles and in theca cells from large and medium sized antral follicles. This may indicate a role of LRP8 in cholesterol delivery to steriodogenic cells. LRP8 was not detected in any of the CL stages. The roles of the LDLr superfamily in lipid transport to ovarian cells and its participation in follicular and CL development and regression is discussed.     

Characterization of apoB, E receptor function in the luteinized ovary

Recent findings from this laboratory have led to the suggestion that the hormone-producing cells of the rat luteinized ovary in situ may obtain a large share of low density lipoprotein (LDL) cholesterol without actually internalizing the intact lipoprotein particles. We have shown that the lipoproteins are trapped at the surface of the luteal cells in a rich network of "microvillar channels" and have theorized that these channel membranes, with their large surface area for interacting with lipoprotein particles, may function in the cholesterol transfer process. In the current study, we try to establish what proportion of the human (h)LDL-cholesterol transfer in the in situ perfused tissue occurs by a classical apoB, E receptor-mediated process versus a surface extraction process. We examine the tissue for the presence of apoB, E receptors, and characterize the structural/functional interaction of hLDL with the apoB, E receptor utilizing a variety of modified hLDL particles as probes. Then, using nonmetabolizable radiolabels for both the protein and cholesteryl ester moieties of these LDL probes, we attempt to quantify the extent to which apoB, E receptors in the ovary contribute to the uptake of hLDL-cholesterol during steroidogenesis. Our experiments show that although the luteinized ovary contains apoB, E receptor protein, hLDL interacts with the tissue atypically. That is, despite modifications of LDL amino acid residues to prevent interaction with the apoB, E receptor, the modified ligands continue to contribute cholesterol for luteal cell internalization and/or steroidogenesis. We conclude, therefore, that in this tissue much of the LDL-cholesterol is not delivered by the apoB, E receptor pathway.     

Ovarian interstitial tissue of the wood mouse, Apodemus sylvaticus

The ovary of the wood mouse contains an unusually large amount of interstitial tissue which appears to develop from undifferentiated cells of the ovarian stroma and also by transformation of theca or granulosa cells of atretic follicles. The cells are characterized by the presence of smooth endoplasmic reticulum, rounded mitochondria with tubular cristae, and abundant, large (1.5 micron diameter) lipid droplets containing cholesterol and its esters. 3 beta-Hydroxysteroid dehydrogenase activity occurs in the interstitial cells. Their ultrastructural characteristics suggest that the cells are not very active, but their abundance and the considerable amount of steroid hormone precursor they contain may compensate for low secretory activity and they may be an important and (from a developmental viewpoint) early source of steroid hormone.     

Rat ovarian apolipoprotein E: localization and gonadotropic control of messenger RNA.

Apolipoprotein E (apo E) is a 35-kDa protein found in association with various lipoproteins. It is synthesized by a wide variety of tissues, including the ovary. It can serve several functions, such as 1) transport of excess cholesterol from peripheral tissue to the liver; 2) directed movement of cholesterol from areas of high to low cholesterol concentration within tissue or organs; and 3) inhibition of the conversion of theca progesterone to androgen, thus acting as an autocrine or paracrine factor within the ovary. To better understand the physiological role of ovarian apo E, we employed the technique of in situ hybridization utilizing 35S-labeled apo E riboprobes to identify cells containing E mRNA. We studied ovaries of hypophysectomized immature rats administered various regimens of gonadotropins because of the uniform, predictable stimulation of follicular granulosa and theca development, ovulation, and corpus luteum formation. Apo E mRNA was localized predominantly in the theca, with an increase associated with theca hypertrophy. Apo E mRNA increased in granulosa cells with the development of preovulatory Graafian follicles, but decreased to baseline following ovulation and corpus luteum formation. These data are consistent with two roles for apo E in the ovary: 1) directing cholesterol to cells needing cholesterol as substrate for cell proliferation and steroidogenesis, and 2) acting as an autocrine regulatory factor to reduce theca androgen substrate for follicle estrogen production.     

Quantitative ultrastructural analysis of the follicular and luteal cells of the bovine ovary

Summary Ovaries were obtained from normal adult dairy cows at all days of the estrous cycle. The largest Graafian follicle and corpus luteum were excised, prepared for electron microscopic study, and their cell components quantitated using the linear scanning method and the counting of membrane crossings.The results indicated that in the theca interna cells during proestrus and estrus and in the large luteal cells during late metestrus and diestrus, enlarged mitochondria occupied an increased cytoplasmic percentage volume. During proestrus and estrus in the theca interna cells, the concentration of membranes of endoplasmic reticulum and Golgi apparatus also increased. The cytoplasmic percentage volumes of lipid bodies and of lysosomes increased in the small luteal cells; during luteal regression, they also increased in the large luteal cells. Similar rates of increase during follicular maturation, and decrease during luteal regression, occurred for measurements of succinic dehydrogenase and mitochondria.The quantitative observations were related to the production of steroid hormones by the ovary, and to the cyclic growth and regression of follicular and luteal cells. It was noted that an increased cytoplasmic percentage volume of mitochondria, an increased concentration of agranular cytoplasmic membranes, and low levels of lipid bodies and lysosomes, were generally present at times when ovarian steroid elaboration and cell growth were maximal.This investigation was supported by a General Research Support Grant to the College of Veterinary Medicine, University of Minnesota, and Research Grant No. GM-07009, of the United States Public Health Service. Approved for publication as Scientific Journal Series Paper No. 6343, Minnesota Agricultural Experiment Station. The work reported is taken from the senior author's Ph. D. thesis. Appreciation is expressed to Professor A.-M. CarPenter, Department of Anatomy, University of Minnesota, for her advice in matters concerning the quantitative techniques.     

Ultrastructure of the steroidogenic cells in the ovary of the Russian sturgeon before and after maturation under exposure to luliberin

Cells with the ultrastructure typical of steroid cells of vertebrates were found in the theca and stroma of the Russian sturgeon ovary. Mitochondria with the tubulo-vesicular internal structure, smooth and rough endoplasmic reticulum, lipid droplets or liposomes were observed in the cytoplasm of these cells. After the ovulation of fishes, the volumetric density and mean diameter of cell mitochondria and of the reticulum are found to increase; besides, mitochondria are seen vacuolized. The ultrastructural changes may indicate that the production of steroid sex hormones is more intensive in fish during ovulation. These suggestions are in accordance with biochemical data available in the literature.     

In vitro differentiation of bovine theca and granulosa cells into small and large luteal-like cells: morphological and functional characteristics

This study was undertaken to investigate whether bovine granulosa and theca interna cells could be luteinized in vitro into luteal-like cells. Granulosa and theca cells were cultured for 9 days in the presence of forskolin (10 microM), insulin (2 micrograms/ml), insulin-like growth factor I (100 ng/ml), or a combination of these agents. During the first day of culture, granulosa and theca cells secreted estradiol and androstenedione, respectively; progesterone rose only after 3-5 days in culture and reached a maximum on the ninth day of culture. Cells incubated in the presence of forskolin plus insulin exhibited morphological and functional characteristics of luteal cells isolated from the corpus luteum. It was found that cell diameter, basal and stimulated progesterone secretion, and pattern of cell replication for both cell types were comparable to those of luteal cells. Numerous lipid droplets and intensified mitochondrial adrenodoxin staining also indicated active steroidogenesis in luteinized cells. After 9 days in culture, stimulants were withdrawn, and the culture proceeded in basal medium for an additional 5 days; elevated progesterone levels were maintained by luteinized granulosa cells (LGC), whereas in contrast a dramatic drop in progesterone production was observed in luteinized theca cells (LTC). On Day 9, cells were challenged for 3 h with LH (10 ng/ml), forskolin (10 microM), or cholera toxin (100 ng/ml), resulting in a 4-fold increase in progesterone secretion by LTC; the same treatments failed to stimulate progesterone in LGC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of Triglyceride and Cholesterol Ester Synthesis Impairs Assembly of Infectious Hepatitis C Virus

In hepatitis C virus infection, replication of the viral genome and virion assembly are linked to cellular metabolic processes. In particular, lipid droplets, which store principally triacylglycerides (TAGs) and cholesterol esters (CEs), have been implicated in production of infectious virus. Here, we examine the effect on productive infection of triacsin C and YIC-C8-434, which inhibit synthesis of TAGs and CEs by targeting long-chain acyl-CoA synthetase and acyl-CoA:cholesterol acyltransferase, respectively. Our results present high resolution data on the acylglycerol and cholesterol ester species that were affected by the compounds. Moreover, triacsin C, which blocks both triglyceride and cholesterol ester synthesis, cleared most of the lipid droplets in cells. By contrast, YIC-C8-434, which only abrogates production of cholesterol esters, induced an increase in size of droplets. Although both compounds slightly reduced viral RNA synthesis, they significantly impaired assembly of infectious virions in infected cells. In the case of triacsin C, reduced stability of the viral core protein, which forms the virion nucleocapsid and is targeted to the surface of lipid droplets, correlated with lower virion assembly. In addition, the virus particles that were released from cells had reduced specific infectivity. YIC-C8-434 did not alter the association of core with lipid droplets but appeared to decrease production of infectious virus particles, suggesting a block in virion assembly. Thus, the compounds have antiviral properties, indicating that targeting synthesis of lipids stored in lipid droplets might be an option for therapeutic intervention in treating chronic hepatitis C virus infection.