Absence of cardiac lipid accumulation in transgenic mice with heart-specific HSL overexpression.

Hormone-sensitive lipase (HSL) hydrolyzes triglyceride (TG) in adipose tissue. HSL is also expressed in heart. To explore the actions of cardiac HSL, heart-specific, tetracycline (Tc)-controlled HSL-overexpressing mice were generated. Tc-responsive element-HSL transgenic (Tg) mice were generated and crossed with myosin heavy chain (MHC)alpha-tTA Tg mice, which express the Tc-responsive transactivator (tTA) in the heart. The double-Tg mice (MHC-HSL) were maintained with doxycycline (Dox) to suppress Tg HSL. Upon removal of Dox, cardiac HSL activity and protein increased 12- and 8-fold, respectively, and the expression was heart specific. Although cardiac TG content increased twofold in control mice after an overnight fast, it did not increase in HSL-induced mice. Electron microscopy showed numerous lipid droplets in the myocardium of fasted control mice, whereas fasted HSL-induced mice showed virtually no droplets. Microarray analysis showed altered expression of cardiac genes for fatty acid oxidation, transcription factors, signaling molecules, cytoskeletal proteins, and histocompatibility antigens in HSL-induced mice. Thus cardiac HSL plays a role in controlling accumulation of triglyceride droplets and can affect the expression of a number of cardiac genes.

Expression of scavenger receptor class B type 1 (SR-BI) promotes microvillar channel formation and selective cholesteryl ester transport in a heterologous reconstituted system.

In the "selective" cholesteryl ester (CE) uptake process, surface-associated lipoproteins [high density lipoprotein (HDL) and low density lipoprotein] are trapped in the space formed between closely apposed surface microvilli (microvillar channels) in hormone-stimulated steroidogenic cells. This is the same location where an HDL receptor (SR-BI) is found. In the current study, we sought to understand the relationship between SR-BI and selective CE uptake in a heterologous insect cell system. Sf9 (Spodoptera frugiperda) cells overexpressing recombinant SR-BI were examined for (i) SR-BI protein by Western blot analysis and light or electron immunomicroscopy, and (ii) selective lipoprotein CE uptake by the use of radiolabeled or fluorescent (BODIPY-CE)-labeled HDL. Noninfected or infected control Sf9 cells do not express SR-BI, show microvillar channels, or internalize CEs. An unexpected finding was the induction of a complex channel system in Sf9 cells expressing SR-BI. SR-BI-expressing cells showed many cell surface double-membraned channels, immunogold SR-BI, apolipoprotein (HDL) labeling of the channels, and high levels of selective HDL-CE uptake. Thus, double-membraned channels can be induced by expression of recombinant SR-BI in a heterologous system, and these specialized structures facilitate both the binding of HDL and selective HDL-CE uptake.

Expression and microvillar localization of scavenger receptor class B, type I (SR-BI) and selective cholesteryl ester uptake in Leydig cells from rat testis.

This study investigates the relationship between the high density lipoprotein (HDL) receptor (scavenger receptors, SR-BI and SR-BII), selective lipoprotein-cholesteryl ester uptake, and testosterone production in Leydig cells of control, hypocholesterolemic and gonadotrophic hormone (hCG) treated rats. Leydig cells from mature control rats show poor efficiency in incorporation of labeled HDL-cholesteryl esters into testosterone, poor selective uptake of lipoprotein lipids overall, and a dramatic reduction of circulating levels of lipoproteins has no apparent effect on testosterone production or expression of intracellular enzymes synthesizing cholesterol. Leydig cells from control rats show minimal levels of SR-BI and SR-BII. However, similarly aged rats treated with hCG for several days undergo changes consistent with hormone-desensitization. Despite the resulting low levels of testosterone production, SR-BI levels are dramatically increased, Leydig cells now efficiently internalize HDL-supplied cholesteryl esters by the selective cholesterol uptake process, and various other cholesterol-sensitive genes of the cells are up-regulated. Only SR-BII expression remains negligible and unchanged throughout this period. It is of interest that Leydig cell SR-BI of hCG-treated rats is localized in surface microvilli, but is present also in an elaborate and complex channel system within the cytoplasm of the cells. In summary, Leydig cells differ from other rat steroidogenic cells in not depending on exogenous lipoprotein-cholesterol during periods of normal steroid hormone production. However, trophic hormone desensitization is accompanied by increased Leydig cell SR-BI expression and increased selective HDL-cholesteryl ester uptake, presumably in preparation for renewed testosterone production.

Upregulation of selective cholesteryl ester uptake pathway in mice with deletion of low-density lipoprotein receptor function.

This study examines the effect of mutation of the low-density lipoprotein receptor (LDLR) on cholesterol metabolism, and especially lipoprotein-derived cholesteryl ester uptake, in murine ovarian granulosa cells. Although the tests were conducted on cells prepared by two different procedures, the results are similar. Deletion of LDLR function did not noticeably affect key enzymes of the steroidogenic pathway or affect progestin production and secretion in granulosa cells. No change was found in expression of LDL-related protein (LRP). These data suggested that cholesterol turnover in cells from the knockout animals is within normal limits and that the cells are not stressed to acquire more cholesterol. Both biochemical and morphological data indicate that unstimulated granulosa cells from LDLR-/- mice are nonetheless programmed to take in double the amount of lipoprotein-derived cholesteryl ester (via the selective cholesteryl ester uptake pathway) and to process (hydrolyze, re-esterify, or utilize) more than twofold the cholesteryl ester processed by cells from wildtype (LDLR+/+) animals. Bt2cAMP stimulation of the murine granulosa cells increases the mass of cholesteryl ester taken up by the selective pathway by an additional 38%. To determine to what extent this increase is related to high-density lipoprotein (HDL) scavenger receptor protein (SR-BI) or caveolin function, Western blots and immunohistochemical studies were performed under a variety of conditions. SR-BI levels are found to be low in unstimulated cells of both LDLR+/+ and LDLR-/- animals, but highly expressed (approximately 20-fold increase over basal levels) in stimulated (Bt2cAMP) cells of both animal models. Thus, the functional relationship between selective cholesteryl ester uptake and SR-BI receptor protein is not as tight as in previously reported studies, suggesting a requirement for other tissue factors. Caveolin expression did not change under any of the conditions tested and appears not to be functionally involved in this process.

The selective pathway and a high-density lipoprotein receptor (SR-BI) in ovarian granulosa cells of the mouse.

We recently reported that rat luteinized ovary tissue and primary cultures of rat ovarian granulosa cells reveal a remarkably tight functional correlation between expressed selective uptake of lipoprotein cholesteryl esters and the expression of an HDL receptor protein, scavenger receptor, class B, type I (SR-BI). In the current study, we examine these same processes in C57 mouse granulosa cells and report a different correlation. Unlike the rat cells, non-hormone stimulated mouse granulosa cells are able to effectively carry out their selective pathway functions and secrete HDL-derived progestins despite low levels of SR-BI and barely detectable levels of SR-BII (an isoform of SR-BI). Once stimulated with trophic hormones or Bt2cAMP, small (30-40%) increases are observed in selective pathway functions, but major (approximately 20-fold) increases are seen in SR-BI and SR-BII expression: thus, relatively little is gained in selective cholesteryl ester uptake by mouse granulosa cells even though SR-BI and SR-BII levels are greatly increased. The importance of the HDL receptor proteins to the selective pathway remains clear, however, since a significant portion of the selective process in both basal and stimulated granulosa cells is inhibitable by the use of blocking antibody. Another surface protein, caveolin, previously reported to co-localize with SR-BI in mouse cells shows no change in expression during periods when SR-BI/BII levels are undergoing major shifts.

Simultaneous induction of an HDL receptor protein (SR-BI) and the selective uptake of HDL-cholesteryl esters in a physiologically relevant steroidogenic cell model.

This study addresses the question of whether the level of expression of SR-BI (an HDL receptor) is linked to the expression of selective lipoprotein-cholesteryl ester delivery in a steroidogenic cell model. Rat ovarian granulosa cells are physiologically normal cells which show no selective uptake of HDL-cholesteryl esters and no progestin production until luteinized by trophic hormones or adenylate cyclase stimulators, after which expression of the selective cholesterol pathway and production of steroid hormone is dramatically up-regulated. The current study demonstrates that at every cell stage studied, the protein content and level of expression of SR-BI mRNA are linked to changes that occur in HDL-cholesteryl ester uptake; i.e., SR-BI is not present in basal (non-luteinized) cells, develops slowly (from 6-9 h) after hormone treatment, increases robustly from 9-48 h after stimulation, and remains high after incubation with HDL. In contrast, another structural protein, caveolin, did not follow this pattern; caveolin expression showed an inverse relationship to selective cholesteryl ester uptake, and was most prominent in basal cells and least prominent in luteinized, HDL-incubated cells. Morphologically, SR-BI appears to be associated with cell surface sites showing high levels of cholesteryl ester uptake (after luteinization and/or incubation with HDL labeled with fluorescent cholesteryl esters), and at the electron microscope level, SR-BI is most clearly associated with microvillar regions on the cell surface which also bind HDL-labeled with colloidal gold. Thus, induction of the SR-BI receptor system and induction of the HDL-selective cholesterol uptake pathway in rat granulosa cells appear to be linked morphologically, biochemically, and functionally.

Expression and microvillar localization of scavenger receptor, class B, type I (a high density lipoprotein receptor) in luteinized and hormone-desensitized rat ovarian models.

Steroidogenic cells in rats and mice obtain most of their cholesterol for steroid production and cholesteryl ester (CE) storage via the selective uptake pathway in which high density lipoprotein CE (HDL-CE) is taken into the cell without the uptake and degradation of the HDL particle. A number of recent studies show that the scavenger receptor, class B, type I (SR-BI) can mediate HDL-CE selective uptake in cultured cells and suggest that this receptor may be responsible for HDL-CE selective uptake in steroidogenic cells in vivo. In the current study we examine the relationship between SR-BI expression and HDL-CE selective uptake in the gonadotropin-primed, luteinized rat ovary and in the ovary that is desensitized by multiple gonadotropin treatments. Results from this study demonstrate a tight association between expression of SR-BI and measurements of HDL-CE selective uptake regardless of the steroidogenic state of the ovary. Thus, in the luteinized ovary (which is actively producing progestins), HDL-CE selective uptake is high, as is the expression of SR-BI. In the desensitized ovary (where CE content is reduced by 90% and progestin production is virtually absent), HDL-CE selective uptake and SR-BI are induced 2- to 3-fold compared with those in the luteinized ovary. These data argue that SR-BI can be regulated by the cholesterol status of the luteal cell independently of gonadotropic stimulation. Immunostaining at the light microscopic level showed strong expression of SR-BI specifically on the surface of luteal cells in the luteinized and desensitized ovary. Immunolocalization at the electron microscopic level showed that SR-BI was associated with microvilli and microvillar channels of the luteal cell surface. This result supports the hypothesis that microvilli and microvillar channels represent a cell surface compartment that is specialized for the selective uptake of lipoprotein cholesterol into steroidogenic cells.

Human granulosa cells use high density lipoprotein cholesterol for steroidogenesis.

This study examines the ability of human high density lipoproteins (HDL3) to deliver cholesteryl esters to human granulosa cells and describes the selective cholesterol pathway by which this occurs. Luteinized cells obtained from subjects undergoing in vitro fertilization-embryo transfer procedures were incubated with native HDL3 (or radiolabeled or fluorescently labeled HDL cholesteryl esters) to determine whether cells from humans (in which HDL is not the primary circulating lipoprotein species) can nevertheless interiorize and appropriately process cholesteryl esters for steroidogenesis. The results indicate that hormone-stimulated granulosa cells actively and efficiently use human HDL-derived cholesterol for progesterone production. More than 95% of the mass of HDL cholesteryl esters entering cells does so through the nonlysosomal (selective) pathway, i.e. cholesteryl esters released from HDL are taken up directly by the cells without internalization of apoproteins. Once internalized, the cholesteryl esters are either hydrolyzed and directly used for steroidogenesis or stored in the cells as cholesteryl esters until needed. The utilization of the internalized cholesteryl esters is a hormone-regulated event; i.e. luteinized human granulosa cells internalize and store large quantities of HDL-donated cholesteryl esters when available, but further processing of the cholesteryl esters (hydrolysis, re-esterification, or use in steroidogenesis) does not occur unless the cells are further stimulated to increase progesterone secretion.

Intracellular events in the "selective" transport of lipoprotein-derived cholesteryl esters.

The current study utilizes human, apoE-free high density lipoprotein reconstituted with a highly specific fluorescent-cholesteryl ester probe to define the initial steps and regulatory sites associated with the "selective" uptake and intracellular itinerary of lipoprotein-derived cholesteryl esters. Bt2cAMP-stimulated ovarian granulosa cells were used as the experimental model, and both morphological and biochemical fluorescence data were obtained. The data show that cholesteryl ester provided through the selective pathway is a process which begins with a temperature-independent transfer of cholesteryl ester to the cell's plasma membrane. Thereafter transfer of the lipid proceeds rapidly and accumulates prominently in a perinuclear region (presumed to be the Golgi/membrane sorting compartment) and in lipid storage droplets of the cells. The data suggest that lipid transfer proteins (or other small soluble proteins) are not required for the intracellular transport of the cholesteryl esters, nor is an intact Golgi complex or an intact cell cytoskeleton (although the transfer is less efficient in the presence of certain microtubule-disrupting agents). The intracellular transfer of the cholesteryl esters is also somewhat dependent on an energy source in that a glucose-deficient culture medium or a combination of metabolic inhibitors reduces the efficiency of the transfer. A protein-mediated event may be required for cholesteryl ester internalization from the plasma membrane, in that N-ethylmaleimide dramatically blocks the internalization phase of the selective uptake process. Taken together these data suggest that the selective pathway is a factor-dependent, energy-requiring cholesteryl ester transport system, in which lipoprotein-donated cholesteryl esters probably flow through vesicles or intracellular membrane sheets and their connections, rather than through the cell cytosol.

Regulation of cholesterol responsive genes in ovary cells: impact of cholesterol delivery systems.

The "selective" cholesterol uptake pathway represents a bulk pathway by which many steroidogenic cells internalize lipoprotein-delivered cholesteryl esters. In the current report, we question whether cholesteryl esters entering cells via this pathway are capable of governing standard cholesterol end product feedback repression mechanisms. Cultured rat ovary granulosa cells which utilize both the "selective" and "endocytic" pathways to internalize lipoprotein-derived cholesteryl esters were used as a model system. ApoE-free hHDL3 was used to deliver cholesteryl esters to the cells exclusively by the selective pathway; hLDL was used as a control ligand which when internalized by the endocytic pathway releases cholesteryl esters which subsequently regulate the expression of the B/E (LDL)-receptor, HMG CoA reductase, and acyl-CoA:cholesterol acyltransferase (ACAT). Whereas trophic hormone (Bt2cAMP) stimulation by itself increased the activity, mRNA, and protein levels of both B/E-receptor and HMG CoA reductase, pretreatment with either lipoprotein (adjusted for equal cholesterol ester content) down-regulated this expression. Linked with these lipoprotein-related changes was an increase in activity (though not gene expression) of ACAT. The level of change in mRNA levels, protein content, and activity for the examined regulatory proteins was essentially equivalent whether the lipoprotein provided to the cells was hLDL or hHDL3. Thus, similar signals appear to have been received by the cells despite differences in the uptake and processing of the ligand-derived cholesteryl esters, and these signals resulted in identical homeostatic responses by the cells.

Alteration of the adrenal antioxidant defense system during aging in rats.

The goal of this study was to determine to what extent aging affects the antioxidant defense system of the rat adrenal and to evaluate the impact of any change in this system on the recognized age-related decline in steroidogenic capacity of adrenocortical cells. The studies were conducted on young (2-5 mo) and aging (12-27 mo) Sprague-Dawley rats and involved procedures measuring steroidogenesis; oxidative damage to tissue; non enzymatic antioxidants such as vitamin C, E, and glutathione; and tissue antioxidant enzyme (Mn and CuZn superoxide dismutases, catalase, and glutathione peroxidase) activity and expression (mRNA, protein mass, and location). Some measurements were made also on rats maintained on vitamin E-deficient diets. The data show that adrenals from young animals are especially well protected against oxidative events; i.e., these adrenals show the least endogenous lipid peroxidation and the highest level of resistance to prooxidant-induced damage (of various tissues measured) and show exceedingly high levels of tissue antioxidants. Aging, on the other hand, results in oxidative changes in adrenal tissue that are generally linked in time to a reduction in efficiency of the normally protective antioxidant defense system and to the decline in corticosterone production. We speculate that these events are causally related, i.e., that the age-related reduction in oxidative mechanisms in adrenal tissues leads to oxidative damage of membrane or cytosolic factors important to cholesterol transport, and, as a consequence of this damage, cholesterol cannot reach appropriate mitochondrial cholesterol side chain cleavage sites, and corticosterone production fails.

Cholesterol uptake by the 'selective' pathway of ovarian granulosa cells: early intracellular events.

Although the 'selective' pathway is a major cholesteryl ester (CE) uptake pathway used by steroidogenic cells, essentially nothing is known about the itinerary of the CE once it is extracted from lipoproteins at the cell surface. In the current report we have begun to trace 'selective' pathway internalized-CE using both native and reconstituted human (h) high density lipoproteins (hHDL3) with variously labeled and tagged CEs to provide information from either a biochemical or morphological perspective. It appears that the amount of hHDL3-CE that is internalized and processed through the 'selective' pathway is directly related to the amount of cholesterol used for steroidogenesis at any given time point. There is a time-related correlation between the level of the Bt2cAMP-stimulated cell steroidogenic response, the level of conversion of freshly obtained hHDL3-CE into progestins, increases in hHDL3-derived CE internalization, hHDL3-CE hydrolysis, re-esterification and/or storage. None of this processing takes place in non-stimulated (non-Bt2cAMP treated) cells which do not secrete progestins despite the availability of hHDL3 as a cholesterol source. The data suggest that the 'selective' pathway has a special role in steroidogenic cells-one of providing sufficient cholesterol to fuel the required production of steroid hormones.

Enhanced expression of granulosa cell low density lipoprotein receptor activity in response to in vitro culture conditions.

Previous studies have shown that the B/E (low density lipoprotein [LDL]) receptor pathway plays a minor role in cholesterol uptake in the intact rat ovary, but when granulosa cells are isolated and maintained in culture, the cells develop a fully functional B/E receptor system. In the current study we examined the development of the B/E receptor over time (96 h) in culture and compared its physiological function, expression of mRNA and protein levels, and morphological events to the upregulation induced in 24 h by hormone (human chorionic gonadotropin [hCG] or Bt2cAMP). With both protocols, increased progestin production occurs and is associated with elevated binding, uptake, and degradation of LDL in the medium although the impact of Bt2cAMP stimulation on all these measurements is several times that observed with time alone. Only the hormone-stimulated LDL receptor response was associated with an increase in receptor protein (Western blot) or mRNA levels (RNase protection assay). We conclude that unstimulated granulosa cells show posttranslational increases in B/E receptor activity with time in culture, but transcriptional changes in B/E receptor follow stimulation with trophic hormone or its second messenger, cAMP.

Effect of age on cholesterol uptake and utilization by rat adrenals: I. Internalization of lipoprotein-derived cholesteryl esters.

Previous studies from this laboratory have documented a progressive age-related decline in trophic hormone (or second messenger cAMP) stimulated corticosterone production in isolated adrenocortical cells. In the current study, we examined the possibility that the aging process exerts this effect by interfering with an early step in the delivery of lipoprotein-derived cholesteryl esters to the cell. As such, we monitored the ability of two different rat adrenocortical cell model systems (intact perfused adrenal glands and primary cultures of adrenocortical cells from 5- and 18- to 20-month-old rats) to internalize lipoprotein cholesteryl esters, and to convert the newly internalized cholesteryl esters to corticosterone production. The results indicate that lipoprotein (hHDL3 and rHDL) cholesteryl ester internalization (by both the endocytic and 'selective' pathways) is comparable in adrenocortical cells of the young and old rats. However, despite this, both the mass of corticosterone produced and the ratio of newly internalized (radiolabeled) cholesteryl ester incorporated into corticosterone is dramatically reduced in cells of the older animals. Thus, the lipoprotein uptake pathway appears to be intact in adrenals of older rats, but the intracellular processing of internalized cholesteryl ester is defective.

Effect of age on cholesterol uptake and utilization by rat adrenals: II. Lipoproteins from young and old rats.

The current study examines whether age-related changes in high density lipoproteins (HDL) influences how these particles are handled by adrenal cells. It appears that HDL from 18- to 20-month-old Sprague-Dawley rats show a seven- to eightfold increase in content of apolipoprotein E compared to HDL from 2- to 5-month-old rats. The 'aged' particles show increased binding to susceptible hepatic membranes, and show a doubling in whole particle endocytosis by cortical cells of the perfused adrenal gland and by isolated adrenal cells from all rats regardless of age. Despite this twofold increase in particle uptake, the increase in total cholesteryl ester uptake by either the perfused adrenal or incubated adrenal cells is minor, amounting to less than 10% of the total cholesteryl ester internalized. This discrepancy occurs since the high apo E content of the 'aged' HDL only affects cholesteryl ester uptake by the 'endocytic' pathway; uptake via the 'selective' pathway (where cholesteryl ester is separated from the rest of the particle at the cell surface and directly internalized) is not altered.

Effect of okadaic acid on utilization of lipoprotein-derived cholesteryl esters by rat steroidogenic cells.

This study examines various functional, biochemical, and structural changes in rat adrenocortical and ovarian granulosa cells that could account for the decline in lipoprotein-supported hormone production after cell treatment with the protein phosphatase inhibitor, okadaic acid. Although the steroidogenic pathway enzymes in these cells are not in themselves affected by okadaic acid, the intracellular transport of cholesterol to important cellular processing sites is defective. That is, okadaic acid does not interfere with the internalization of lipoprotein-derived cholesteryl esters, but the mitochondrial utilization of cholesterol obtained from intracellular cholesterol storage sites is 50% reduced as compared to control cells. Two-dimensional electrophoresis gels from okadaic acid-treated cells demonstrate a number of hyperphosphorylated proteins. Morphological examination of the affected cells reveal completely disrupted Golgi complexes with attendant structures, but otherwise the cells appear unchanged. The results suggest that some necessary sterol transport protein (or cofactor or associated membrane) is adversely phosphorylated by okadaic acid, and is rendered dysfunctional.

Age-related decline in the steroidogenic capacity of isolated rat Leydig cells: a defect in cholesterol mobilization and processing.

This study was designed to evaluate the effects of aging on steroidogenesis and intracellular cholesterol processing in rat Leydig cells. Maximum gonadotropin-induced testosterone secretion was significantly reduced in Leydig cells from 18 to 27-month-old rats compared to 2 to 5-month-old rats. The decreased production of testosterone in older groups persisted after incubation with cAMP analogs or other non-specific stimulatory agents. This age-related loss in testosterone response was not due to changes in gonadotropin receptor concentration, cAMP concentration, protein kinase A activation or the activity of key steroidogenic enzymes. The content of cellular cholesteryl esters doubled as rats aged from 5 to 18 months, and this high cholesteryl esters level remained constant through 27 months. The ability of hCG to mobilize (hydrolyze) stored cholesteryl ester for testosterone production was significantly reduced (65-75%) in cells from the older rats. This change could be accounted for by the decline in activity of neutral cholesteryl esterase in Leydig cells from 18-month-old rats. In contrast, the activity of a non-specific lysosomal acidic cholesteryl esterase did not change with age. The activity of HMG CoA reductase, the rate limiting enzyme in cholesterol biosynthesis decreased about 70% between 5 and 18 months and fell slightly further as the rats aged to 27 months. Also, [14C]acetate or [3H]H2O incorporation into cellular sterols showed a similar decline. Cyanoketone plus hCG stimulated pregnenolone production was reduced about 70-80% in old as compared to young cells. Leydig cells from young rats responded to hCG with increased accumulation of mitochondrial cholesterol in the presence and absence of steroidogenic inhibitors. On the other hand, old cells responded poorly to hCG and mitochondrial cholesterol levels were little affected by hCG plus cycloheximide or aminoglutethimide. Together, these data indicate that alterations in the intracellular processing and metabolism of cholesteryl esters occur in Leydig cells of aging rats, and we suggest they may be responsible for the observed age-related changes in testosterone production.

Effect of okadaic acid on hepatocyte structure and function.

The aim of this study with rat hepatocytes was to describe the effect of okadaic acid (OKA) (a potent and specific inhibitor of protein phosphatases) on the biosynthesis, processing and/or secretion of various lipid and protein molecules. Gel radioautograms indicated that low concentrations of okadaic acid (100 nM) induced hyperphosphorylation of a number of hepatocyte phosphoserine/threonine residues in the Mr range of 35-220 kDa. The effects of okadaic acid on the morphology of the hepatocytes was time and dose-dependent; early changes included cell rounding, loss of typical Golgi staining of beta COP, and fragmentation of the Golgi compartment at the EM level. General hepatocyte cell functions such as protein synthesis, lactate dehydrogenase activity, and ATP levels were unchanged with 100 nM okadaic acid as were all hepatocyte functions carried out in the endoplasmic reticulum of these cells. As such, incubation with okadaic acid did not alter the biosynthesis of phosphatidylcholine (from labeled choline), or very low density lipoproteins (VLDL) from labeled fatty acids or glycerol. Likewise, the biosynthesis of various endoplasmic reticulum synthesized proteins (transferrin, albumin, apolipoprotein E, and HMG CoA Reductase) continued normally in the presence of okadaic acid. However, incubation with okadaic acid led to major changes in all hepatocyte functions normally carried out in the Golgi compartment; i.e., the incorporation of labeled ceramide into sphingomyelin was profoundly reduced, as was the Golgi-required packaging and secretion of various proteins synthesized in the endoplasmic reticulum. These findings point to the Golgi compartment as an specific target for okadaic acid and suggest that one or more okadaic acid-sensitive phosphoproteins may be involved in maintaining its normal structure and function.

Reconstitution of the lipoprotein cholesteryl ester transfer process using isolated rat ovary plasma membranes.

Steroidogenic cells are able to utilize lipoprotein-derived cholesteryl esters for steroidogenesis without internalizing intact lipoproteins. In the current report, we provide evidence that an early step in this process may be the selective extraction of cholesteryl esters at the cell (plasma membrane) surface. We have used a highly purified plasma membrane preparation from rat luteinized ovaries for incubation with rat- and human-derived high density (HDL) and low density (LDL) lipoproteins. The lipoproteins were modified with residualizing [125I]apoprotein or [3H]cholesteryl ester markers. Following trypsin treatment to remove intact surface-bound apoprotein particles, the membranes were analyzed for transferred radioactive labels. The results show that all the lipoproteins tested could serve as cholesteryl ester donors. Although far more [3H]cholesteryl ester than [125I]apoprotein radioactivity was transferred to plasma membranes in each case, and varied with the ligand used, the total (net) mass of cholesteryl ester transferred was comparable with the different lipoproteins. These data were confirmed using direct chemical methodology. Transfer was found to be specific for cholesteryl esters or ethers and did not involve other lipoprotein core lipids tested. Endomembranes from the same tissue could not substitute for plasma membranes as the primary cholesteryl ester acceptor. These results provide evidence that a reconstituted lipoprotein-plasma membrane system can simulate the cholesteryl ester extraction process described in situ and suggest uses for this methodology in future experiments designed to understand the transfer process.

Okadaic acid interferes with lipoprotein-supported corticosterone production in adrenal cells.

Rat adrenocortical cells in culture respond to stimulation by ACTH alone (15 fold over basal) and to ACTH + added lipoproteins (as an exogeneous source of cholesterol), with an additional 25-30 fold rise in steroidogenesis. With the addition of okadaic acid (OKA, 100 nM), a potent protein phosphatase inhibitor, the lipoprotein-induced rise in steroidogenesis is blocked. If 20 alpha-hydroxycholesterol is provided instead of lipoprotein-cholesterol, OKA has no effect suggesting that OKA affects only actively transported cholesterol. Since the OKA block is preceded by specific morphological changes in the cell (i.e., the loss of Golgi-associated microtubules followed by the disruption of the Golgi apparatus itself), it is hypothesized that some OKA-sensitive phosphoprotein associated with the microtubule/Golgi network of adrenocortical cells is critical for lipoprotein-derived cholesterol uptake and/or transport during steroidogenesis.