Solubilization, purification, and characterization of a truncated form of rat hepatic squalene synthetase.

Rat hepatic microsomal squalene synthetase (EC 2.5.1.21) was induced 25-fold by feeding rats with diet containing the hydroxymethylglutaryl-coenzyme A reductase inhibitor, fluvastatin, and cholestyramine, a bile acid sequestrant. A soluble squalene synthetase protein with an estimated mass of 32-35 kDa, as determined by gel filtration chromatography on Sephacryl S-200 column, was solubilized out of the microsomes by controlled proteolysis with trypsin. Approximately 25% of the activity was recovered in a soluble form. The enzyme was purified to homogeneity utilizing a series of column chromatography purification steps on DEAE-cellulose, hydroxylapatite, and phenyl-Sepharose sequentially. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Initial kinetic analysis indicated an S0.5 values for trans-farnesyl diphosphate of 1.0 microM and for NADPH of 40 microM. The Vmax with respect to trans-farnesyl diphosphate was calculated at 1.2 mumol/min/mg. NADH also serves as substrate for the reaction with S0.5 value of 800 microM. Western blot analysis utilizing rabbit antisera raised against the purified, trypsin-truncated enzyme showed a single band for the isolated solubilized enzyme at 32-33 kDa and a band for the intact microsomal enzyme at about 45-47 kDa.

Jumping into the 20th century before it is too late: is laboratory robotics still in its infancy?

Successful management of laboratory robotic automation programmes in the environment of research and drug discovery within the pharmaceutical industry may perhaps be best compared to a chef preparing the perfect hollandaise sauce. All the ingredients must be available at the same time and be of highest quality for the right price. However, if components are not added in the right quantities and in the proper order, no amount of whipping together by the product champion will create the best product. In the past, managerial scepticism surrounding useful implementation of cost-effective, high-throughput robotic systems often placed these 'modern toys' at low priorities for research development laboratories. Management now recognizes the unique contributions of robotics in the research environment. Although the scientific director must still play the role of product champion, new questions are being proposed and new commitments are being made to bring the potential of robotic automation to every laboratory where repetitive functions can benefit from new applications. Research laboratory directors have become both the key ingredient, as well as the rate-limiting determinant in the development of new applications. Having fulfilled the promise of robotic automation to release talented personnel, the challenge now is for the 'end users', the bench scientists, to be provided with opportunities to invest the time and effort required for future applications and new career functions.

Hemodynamic, antivasoconstrictor, and antiatherosclerotic effects of calcium antagonists in animal models of atherosclerosis.

Calcium antagonists retard the development of atherosclerosis in cholesterol-fed rabbits and modestly enhance regression after their return to a normal diet. Proliferative lesions following endothelial damage (from, for example, balloon catheter, electrical stimulation) are also diminished. Many mechanisms for these effects have been proposed and their relative importance is not yet clear. However, changes in blood lipid levels do not play an important role. Only a few investigations into how atherosclerosis affects the hemodynamic actions of calcium antagonists have been carried out. Thus, the effects of isradipine were compared in atherosclerotic and normal rabbits. Isradipine increased heart rate and cardiac output less in atherosclerotic rabbits than in normal ones while having no effect on the surface electrocardiogram (ECG). In contrast, the arteriolar vasodilator, dihydralazine, induced ST-segment depression with similar falls in blood pressure, partly explainable by reflex tachycardia and intramyocardial maldistribution of coronary blood flow. Flow to the brain increased with isradipine and decreased with dihydralazine. In atherosclerotic animals, the pressor effects of norepinephrine, phenylephrine, and angiotensin II (Ang II) were amplified. Isradipine partly corrected this enhanced responsiveness. Calcium antagonists thus elicit beneficial hemodynamic and antivasoconstrictor effects in atherosclerotic experimental animals, in addition to having a long-term prophylactic antiatherosclerotic action.

Antiatherogenic properties of calcium antagonists. State of the art.

Atherosclerosis is an arterial disease characterized by localized accumulation of collagen, elastin, lipids, and calcium at sites associated with macrophage infiltration and altered smooth muscle metabolism. Studies in several types of animal models, especially cholesterol-fed rabbits, have shown that calcium competitors, calcium chelators, anticalcifying agents, and calcium antagonists can reduce the accumulation of atherogenic lesion components and decrease the progression of lesions. Although there are some conflicting data in the animal model studies, it is now apparent that several classes of calcium antagonists inhibit the progression of early arterial lesions induced by cholesterol-feeding in animals. The dihydropyridine class of calcium antagonists may be more potent as anti-atherosclerotic agents than the other classes. Mechanisms involving regulation of endothelial cell, smooth muscle cell, and macrophage metabolism may be responsible for the effects of calcium antagonists on early lesion progression. Recent studies in cell culture-model systems suggest that calcium antagonists may significantly alter activities that regulate lipoprotein-derived cholesterol accumulation by arterial wall cells. Some of these activities are independent of calcium flux across voltage-operated calcium channels. Thus, calcium antagonists may reduce the progression of atherogenic lesions by a combination of decreasing calcium accumulation within arterial wall cells and by altering calcium channel-independent metabolic activities, which affect lesion development.

Protective action of calcium channel antagonists in atherogenesis and experimental vascular injury.

Experimental research using in vitro and in vivo models of vascular injury have delineated several common mechanisms that characterize the arterial damage in diseases such as atherosclerosis and hypertension. Changes in endothelial permeability, smooth muscle cell proliferation, and accumulation of connective tissue matrix are major common mechanisms. Chronic hyperlipidemia is a major determinant of the proliferative arterial lesions in atherogenic models. Calcium antagonists of very diverse structure and function have been shown to have antiatherogenic potential in several animal model systems of arterial injury. Calcium channel-blockers of several chemical classes have been demonstrated to alter endothelial function, intimal smooth muscle proliferation, and lipid accumulation in the arterial wall. Cell culture model systems have elucidated several potential mechanisms that may contribute to the antiatherogenic potential of the calcium channel-blockers. These activities may in part involve protection of arterial cells from calcium overload via inhibition of calcium flux across voltage-regulated ion channels. However, other activities of these drugs, such as inhibition of cholesterol esterification and matrix protein formation, appear to function independently of calcium flux. A hypothesis is presented that lipophilic calcium channel-blockers are accumulated in cell membranes and perturb metabolic function as a result of altering local membrane structure.

The antiatherogenic potential of calcium antagonists.

Atherosclerosis is an arterial disease characterized by focal accumulation of collagen, elastin, lipids, and calcium at sites associated with macrophage infiltration and altered smooth muscle metabolic function. Studies in several types of animal models, especially cholesterol-fed rabbits, have shown that calcium competitors, calcium chelators, anticalcifying agents, and calcium channel blockers can reduce the accumulation of atherogenic lesion components and thus apparently decrease the progression of lesions. Although there are some conflicting data in the animal model studies using calcium channel antagonists, as a result of differences in experimental designs, it is now apparent that several classes of calcium channel blockers inhibit the progression of early arterial lesions induced by cholesterol feeding. The dihydropyridine calcium channel blockers appear to be more potent antiatherosclerotic agents than other classes of calcium channel antagonists. Several mechanisms involving regulation of endothelial cell, smooth muscle cell, and macrophage metabolic functions may be responsible for the calcium channel blocker effects on early lesion progression. For example, recent studies in cell culture model systems suggest that calcium channel blockers may significantly alter activities that regulate lipoprotein-derived cholesterol accumulation by cells. Some of these activities are independent of calcium flux across voltage-operated calcium channels. Thus, calcium channel blockers may reduce the progression of atherogenic lesions by a combination of decreasing calcium accumulation within arterial wall cells and by altering calcium-independent metabolic activities.

Antiatherogenic properties of calcium antagonists.

A generalized accumulation of cholesterol, calcium and matrix materials (collagen, elastin and proteoglycans) occurs in an age-dependent manner in major arteries. Human atherogenesis is a disease of arteries characterized by a focal accumulation of fibrous matrix elements, lipids and calcium at lesion sites. Studies in cholesterol-fed animal models have indicated that calcium competitors and chelating agents can reduce calcium, lipid and matrix accumulation in arterial lesions and reduce the extent of lesion formation. These agents generally alter soft and hard tissue calcium pools or have deleterious side-effect profiles. Antiatherogenic studies with calcium antagonists (which have been shown to be safe in human clinical studies) have created confusion because of conflicting results. It is apparent, however, that high doses of calcium antagonists can significantly decrease atherogenic lesion development in cholesterol-fed rabbits. The antiatherogenic effects of calcium antagonists may be the result of changes in intracellular calcium pools within smooth muscle cells, which may lead to alterations in cellular metabolic activity or may be due to activities not related to calcium channel effects. Several mechanisms involving regulation of lipoprotein receptor synthesis, lipoprotein uptake or degradation, cholesterol ester hydrolytic activity and arterial matrix synthesis are discussed as potential sites of activity for calcium antagonists. A dihydropyridine channel antagonist, PN 200-110 (isradipine), has been shown to be a very potent antiatherogenic agent in the rabbit and also to be a potent inhibitor of smooth muscle cell matrix synthesis.

Serum drug fluctuations after administration of two sustained-release oral theophylline preparations in asthmatic children.

Serum theophylline levels produced by two bands of sustained-release theophylline tablets, TheoDur and Respbid, were compared in a double-blind crossover study in 13 asthmatic children. The children, aged 9 to 17 years, remained involved in their daily school activities throughout the study. Each patient took 250 mg every 12 hours of one brand of anhydrous theophylline for five days and the other preparation for the remaining five days of the ten-day study. Trough and peak theophylline levels at steady state were measured on the fifth and tenth days of the study. Results indicated no significant differences between peak and trough with either preparation and no difference between the two preparations in mean theophylline concentrations.

Lipoprotein synthesis and secretion by cultured rat hepatocytes. Parallel inhibition of secretion of VLDL, HDL and albumin by monensin.

The biosynthesis and secretion of very-low-density lipoproteins (VLDL) and high-density lipoproteins (HDL) by cultured normal rat hepatocytes was investigated with particular emphasis on its modification by monensin. This acidic ionophore coordinately inhibited the rates of secretion of the several VLDL apolipoproteins and the VLDL lipids, suggesting an effect late in the process of biosynthesis and secretion, probably at the stage of exiting from the Golgi apparatus. The secretion of immunoreactive albumin into the medium was comparably inhibited, implying that the pathway and mechanisms involved in albumin secretion may be closely similar to those for VLDL synthesis and secretion. Secretion of phospholipids and of apolipoproteins E and A-I in the HDL fraction increased progressively with time over 18 h in control incubations but was strongly inhibited by monensin. During extended incubation with monensin at high concentrations (10 microM), there was a net release to the medium of a number of hepatocyte proteins, including some that comigrated with apolipoprotein A-I and apolipoprotein C, making it appear that monensin increased the secretion of these apolipoproteins. However, using labeled amino acids, it was shown by autoradiography and by immunoprecipitation that secretion of newly-synthesized, radioactive apolipoprotein A-I and apolipoprotein C was actually inhibited by monensin. These results are compatible with the conclusion that HDL synthesis and secretion may occur by mechanisms closely related to those for synthesis and secretion of albumin and VLDL.

Androgen and FSH synergistically stimulate lipoprotein degradation and utilization by ovary granulosa cells.

Androgen can directly modulate the induction of steroidogenic enzymes by FSH (follicle stimulating hormone) in ovary granulosa cells. In studies of its mechanism of action, we examined the androgen effect on granulosa cell interaction with lipoproteins, the physiologic source of cholesterol. After granulosa cells were cultured for 48 hours with and without androgen and/or FSH, the cells were incubated for 24 hours with 125I-lipoproteins [human high density lipoprotein (HDL), rat HDL, or human low density lipoprotein (LDL)]. The media were then analyzed for lipoprotein protein coat degradation products (mainly 125I-monoiodotyrosine) and progestin [mainly 20 alpha-dihydroprogesterone (20 alpha-DHP)]. In the absence of FSH and androgen, 2 X 10(5) granulosa cells degraded basal levels of all three lipoproteins, but produced no measurable 20 alpha-DHP. The addition of 10(-7) M androstenedione (A), testosterone (T), or 5 alpha-dihydrotestosterone (DHT) had no effect on lipoprotein protein degradation or 20 alpha-DHP production. FSH alone stimulated lipoprotein protein degradation by 50 to 300% while the addition of androgen synergistically augmented the FSH-stimulated 20 alpha-DHP production as well as protein coat degradation of all three lipoproteins. DHT and T were both effective, indicating that androgens themselves, and not estrogen products, were responsible for the effect on lipoprotein protein degradation and 20 alpha-DHP production. The addition of a 10-fold excess cyproterone acetate (an anti-androgen) inhibited the effect of T, suggesting that the action of T was mediated by the granulosa cell androgen receptor. Androgen and FSH also synergistically stimulated the production of 3H-progestin when the granulosa cells were incubated with either 3H-cholesterol ester core labeled human HDL or similarly labeled human LDL. This report demonstrates that androgen, in combination with FSH, augments the steroidogenic pathway of the granulosa cell from the degradation of lipoprotein and utilization of the cholesterol ester core, to the production of progestin product.

Isolation and analysis of lipoproteins secreted by rat liver hepatocytes.

A procedure has been developed for the small-scale isolation and characterization of lipoproteins secreted by cultured rat liver hepatocytes. The lipoproteins in the culture medium were separated into VLDL, LDL, HDL and a fraction with d greater than 1.21 on single-spin density-gradients. The lipoproteins were removed from the gradients by adsorption onto Cab-O-Sil, a hydrated colloidal silica. The lipid components were extracted from the silica with CHCl3/CH3OH and the apoproteins solubilized in a buffer that contained 2% sodium dodecyl sulfate and 6 M urea. The proteins were analyzed on 3-20% acrylamide electrophoresis gels that contained 1% sodium dodecyl sulfate. The two major rat-plasma lipoproteins, VLDL and HDL, were well separated by the gradients. The Cab-O-Sil was shown to bind 90-95% of the HDL and VLDL in the fractions from the gradient. The recovery of the lipid components was essentially quantitative. The recovery of the apolipoproteins was only about 60% but with very good precision. Over a 20 h period, the lipid phosphorus associated with secreted lipoproteins increased linearly. The secretion of apolipoprotein A1 and apolipoprotein E associated with HDL and apolipoprotein B associated with VLDL also increased as a nearly linear function with time. The secretion of apolipoprotein E associated with VLDL was linear only up to approx. 6 h. The availability of this procedure should greatly facilitate further studies on the characterization of lipoproteins secreted by hepatocytes and mechanisms that regulate lipoprotein synthesis and secretion.

Activity of acyl-CoA: cholesterol acyltransferase and 3-hydroxy-3-methylglutaryl-CoA reductase in subfractions of hepatic microsomes enriched with cholesterol.

The influence of membrane cholesterol on the activities of acyl-CoA: cholesterol acyltransferase and 3-hydroxy-3-methylglutaryl-CoA reductase was examined in three microsomal subfractions (RNA-rich, RNA-poor, and smooth) that had been enriched with cholesterol by incubation with mixed lipoproteins from hypercholesterolemic rabbit serum. Acyl-CoA: cholesterol acyltransferase activity was significantly stimulated in the three subfractions, particularly in the RNA-rich microsomal component. 3-Hydroxy-3-methylglutaryl-CoA reductase, on the other hand, was suppressed (30%) in only one (RNA-poor) of the three microsomal subfractions, despite a 1.4-fold increase in the concentration of membrane cholesterol. An attempt was made to distinguish between an effect based exclusively on an increase in available cholesterol substrate and an activation of acyl-CoA: cholesterol acyltransferase in RNA-rich microsomes enriched with cholesterol. An experimental design was devised so that substrate cholesterol was provided in the form of heated smooth microsomes and acyl-CoA: cholesterol acyltransferase was provided as a separate preparation in the form of RNA-rich microsomes. Appropriate controls were carried out to test for transfer of cholesteryl ester between the two sets of particles. The results suggested that cholesterol enhanced acyl-CoA: cholesterol acyltransferase activity by serving both as a substrate and as a non-substrate modulator.

Dissociation of tissue uptake of cholesterol ester from that of apoprotein A-I of rat plasma high density lipoprotein: selective delivery of cholesterol ester to liver, adrenal, and gonad.

The metabolic fate of homologous high density lipoprotein (HDL) was studied in the rat, tracing the apoprotein A-I (apo A-I) and cholesterol ester moieties simultaneously. The apo A-I was labeled with covalently linked 125I-labeled tyramine cellobiose, which accumulates in the cells degrading the apoprotein; [3H]cholesterol ethers, which cannot be hydrolyzed or mobilized after uptake, were incorporated into the lipid core of reconstituted HDL to reflect the fate of the cholesterol esters. Several lines of evidence, including direct comparison with biologically labeled HDL, are presented to support the validity of this approach. The liver was the major organ of cholesterol ether uptake, accounting for 65% of the total; the adrenal gland and ovary were the most active organs per gram (wet) of weight. Uptake of cholesterol ether was 7-fold greater than that of apo A-I in adrenal, 4-fold greater in the ovary, and greater than 2-fold greater in the liver. The remaining tissues took up apo A-I and cholesterol ethers at more nearly equal rates. Transfer of HDL-associated cholesterol ethers and 125I-labeled apo A-I to other lipoprotein fractions was not observed; thus, the results reflect direct uptake from HDL itself. Whereas uptake of low density lipoprotein appears to involve endocytosis of intact particles, uptake of HDL in at least some rat tissues involves additional, more complex, transfer mechanisms.

Effects of insulin and glucose on very low density lipoprotein triglyceride secretion by cultured rat hepatocytes.

The effect of insulin on hepatic triglyceride synthesis and secretion is controversial. Previously, we have described a cell culture system of adult rat hepatocytes that synthesize and secrete very low density lipoprotein (VLDL) triglycerides with small and irreproducible effects of insulin on triglyceride metabolism. To study the primary effects of insulin on hepatic triglyceride metabolism a method was developed utilizing fibronectin-coated culture dishes that allowed adhesion, spreading, and maintenance of hepatocytes for 2-3 d in the absence of serum and insulin. This culture system allowed mass measurements of both cellular and secreted VLDL triglycerides for long time periods after the addition of physiological concentrations of insulin to hormone-free culture medium. In the absence of insulin and after an initial 4 h in culture, the medium was replenished and triglyceride mass was measured at the end of 18-h incubations. VLDL triglyceride accumulated in the culture medium at a linear rate over this time-course with increasing accumulation as the medium glucose concentration was raised from 2.5 to 25 mM glucose (1.77+/-0.24 to 3.09+/-0.76 mug triglyceride/mg cell protein per h). There was no apparent significant lipolysis or hepatocellular reuptake of secreted VLDL triglycerides. In the absence of insulin cellular triglyceride levels were unchanged between 3 and 24 h in culture while insulin (50-500 muU/ml) significantly increased cellular triglyceride content at all glucose concentrations tested (0-25 mM). The addition of insulin to the culture medium progressively reduced the rate of VLDL triglyceride secretion accompanied by an increase in cellular triglyceride at insulin concentrations > 50 muU/ml. Most or all of the observed increase in cell triglyceride content could in all experiments be accounted for by the insulin-induced inhibition of VLDL secretion. Incorporation of [2-(3)H]glycerol into cellular and VLDL triglycerides as a function of insulin concentration was also measured. Glycerol incorporation data at 20-22 h after plating of the cells closely paralleled the insulin-induced changes in cellular and VLDL triglyceride as determined by mass analysis. The observed effects of insulin occurred at concentrations close to the physiological range and suggest that the direct hepatic effect is to suppress VLDL secretion although the net effect in vivo will clearly reflect many additional accompanying changes.

Viscosity of culture medium as a regulator of synthesis and secretion of very low density lipoproteins by cultured hepatocytes.

It has been postulated that hyperlipidemia in the nephrotic syndrome is due to overproduction of lipoproteins and that low colloid osmotic pressure (due to hypoalbuminemia) triggers this. Secretion of very low density lipoproteins (VLDL) by cultured rat hepatocytes has been shown to be inhibited by albumin, globulins, and dextrans, but the effect did not correlate with osmolarity. In the present studies we tested the hypothesis that viscosity rather than osmolarity might be the parameter determining the effectiveness of macromolecules in inhibiting VLDL synthesis and secretion by cultured rat hepatocytes. Synthesis and secretion of VLDL was measured in terms of incorporation of [3H] glycerol into medium triglycerides and also from changes in the mass of secreted VLDL triglycerides and apoproteins. The viscosity of the culture medium was increased by addition of dextran-500, gelatin or methylcellulose MX 880. Synthesis and secretion of VLDL was inhibited in direct proportion to increasing viscosity. At a viscosity of 2, which is about that of normal plasma, VLDL secretion was reduced by 20%. An inhibition of 60-70% in secretion and 30-40% in synthesis of VLDL lipid and protein components was observed at a relative viscosity of approximately 3.7. This viscosity was obtained by addition of any of the following: 3% dextran, 3% gelatin, 0.2% methylcellulose, or a combination of 0.1% methylcellulose plus 2% gelatin. Thus, similar viscosities resulted in similar degrees of inhibition despite differences of up to 16-fold in mass concentration and up to 20-fold in osmolarity.

Defective catabolism of low-density lipoprotein by fibroblasts from patients with I-cell disease.

Skin fibroblast cultures from patients with I-cell disease (mucolipidosis II) are characterized by multiple lysosomal enzyme deficiencies The present studies deal with the consequences of these deficiencies with respect to the metabolism of plasma low-density lipoproteins. Degradation of the protein moiety was defective in I-cells compared with control cells, but the binding and internalization of low density lipoprotein were much less affected. Measurements of low-density lipoprotein degradation in homogenates demonstrated directly for the first time a deficiency of acid proteinase activity in I-cell fibroblasts. Comparison of results in 6-h incubations with those in 24-h incubations showed accumulation of intracellular low-density lipoprotein in I-cell fibroblasts and an accelerating rate of degradation, possibly attributable to intracellular accumulation of low-density lipoprotein substrate. The significance of these findings with respect to low-density lipoprotein metabolism in vivo is discussed.

Degradation of rat and human lipoproteins by cultured rat ovary granulosa cells.

We have investigated the degradation of 125I-labeled rat and human lipoproteins by rat ovary granulosa cells cultured in serum-free medium. The granulosa cells degrade rat [125I]iodo high density lipoprotein (HDL) to acid-soluble products, mainly monoiodotyrosine. The degradation of 125I-labeled rat HDL is a specific, saturable, high affinity (Km = 21 micrograms protein/ml) process. In studies of rat [125I]iodo-HDL degradation and progestin (progesterone plus 20 alpha-dihydroprogesterone) production by the same granulosa cell cultures, the cholesterol potentially made available to the cells by degradation can account for the majority of the substrate necessary for the increased progestin production. Granulosa cells degrade human [125I]iodo-HDL by a specific, saturable, high affinity (Km = 20 micrograms protein/ml) process. The degradation of human [125I]iodo-HDL can account for only 20% of the cholesterol substrate necessary for increased progestin production. The degradation of human [125I]iodo-low density lipoprotein (LDL) is saturable and a high affinity (Km = 8 micrograms protein/ml) process, but can be inhibited significantly by a 10-fold excess of unlabeled human HDL. In contrast to both rat [125I]iodo-HDL and human [125I]iodo-HDL, the degradation of human [125I]iodo-LDL can potentially provide twice the cholesterol necessary for increased progestin production. Pronase treatment of the granulosa cells inhibits human [125I]iodo-LDL degradation but stimulates rat [125I]iodo-HDL degradation, indicating that the mechanisms of degradation are separate. The data demonstrate that cultured rat ovary granulosa cells degrade rat HDL, human HDL, and human LDL, and this process has the potential for providing cholesterol for cellular steroid hormone synthesis.

Lipoproteins stimulate androgen production by cultured rat testis cells.

The effects of human high density lipoprotein (hHDL), human low density lipoprotein (hLDL), and rat high density lipoprotein (rHDL) on androgen production by cultured rat testis cells were investigated. Enzymatically dissociated testis cells from hypophysectomized adult rats were cultured in a serum-free medium. During the first 2 days of culture, the addition of human or rat lipoprotein alone stimulated testis cell testosterone (T) production by 100 to 250% in a dose-dependent manner. Likewise, treatment with hCG caused a 2.5-fold increase in T production. Furthermore, the effect of lipoproteins plus hCG was synergistic; the stimulation of T production by concomitant treatment with hCG and lipoproteins was greater than the sum of each added individually. Rat HDL augmented hCG stimulated T production in a concentration-dependent manner and maximum synthesis was achieved at 100 micrograms protein/ml (240% increase) with an ED50 value of 25 micrograms/ml rHDL. At low concentrations (10-30 micrograms protein/ml), all lipoproteins tested had similar stimulatory effects on T production but at the highest dose tested (300 micrograms protein/ml), hHDL was more effective than rHDL. The stimulatory effect of lipoprotein was shown to be time-dependent. Maximum stimulation of T production by lipoprotein was seen during the initial 48 h of culture, whereas lipoproteins were ineffective during the first 5 h and during days 8 to 10 of culture. The present data are consistent with the concept that lipoproteins provide cholesterol substrate while gonadotropins stimulate tha rate limiting enzyme(s) which convert cholesterol to T. This primary culture model of testis cells in serum-free medium is responsive to serum lipoproteins and offers a unique opportunity to study the direct effect of lipoproteins and gonadotropins on testicular steroidogenesis.