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.

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 > 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.

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 inununoreactive 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 μM), 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.

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.

In the rat, the four-day estrous cycle requires a continuous program of cellular proliferation and differentiation. In each cycle, follicles initiate growth with the oocyte increasing in size and the initiation of mitotic division of the granulosa cell layer. Follicular development is controlled by the presence and number of receptors for gonadotropin and steroid hormones (FSH, LH, estrogen and testosterone). FSH induces the appearance of more FSH receptors and LH/hCG and prolactin receptors on the granulosa cells (l). FSH also induces the appearance of aromatose enzymes which are required for aromatization of androgens to estrogens by granulosa cells. As the follicle matures FSH stimulates the synthesis and secretion of progestins (progesterone and 20-∝-dihydroprogesterone). The granulosa cells maintain progestin synthesis during the ovulating and luteal phases of the cycle. Cholesterol is the precursor of progestin production via an FSH-induced mitochondrial side chain cleavage pathway which forms pregnenolone, which is then converted to progesterone and 20-∝-dihydroprogestin.

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.

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 μg protein/ml (240% increase) with an ED50 value of 25 μg/ml rHDL. At low concentrations (10–30 μg protein/ml), all lipoproteins tested had similar stimulatory effects on T production but at the highest dose tested (300 μg 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 the 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.

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.

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.

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.

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.

Conference Abstract| March 01 1981 Effect of Insulin and Glucose on Very-Low-Density Lipoprotein Triglyceride Secretion by Cultured Adult Rat Hepatocytes P. N. Durrington; P. N. Durrington 1Departments of Medicine, University of California, San Diego, U.S.A. and University of Manchester, U.K. Search for other works by this author on: This Site PubMed Google Scholar R. S. Newton; R. S. Newton 1Departments of Medicine, University of California, San Diego, U.S.A. and University of Manchester, U.K. Search for other works by this author on: This Site PubMed Google Scholar D. B. Weinstein; D. B. Weinstein 1Departments of Medicine, University of California, San Diego, U.S.A. and University of Manchester, U.K. Search for other works by this author on: This Site PubMed Google Scholar D. Steinberg D. Steinberg 1Departments of Medicine, University of California, San Diego, U.S.A. and University of Manchester, U.K. Search for other works by this author on: This Site PubMed Google Scholar Clin Sci (Lond) (1981) 60 (3): 1P. https://doi.org/10.1042/cs060001Pa Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation P. N. Durrington, R. S. Newton, D. B. Weinstein, D. Steinberg; Effect of Insulin and Glucose on Very-Low-Density Lipoprotein Triglyceride Secretion by Cultured Adult Rat Hepatocytes. Clin Sci (Lond) 1 March 1981; 60 (3): 1P. doi: https://doi.org/10.1042/cs060001Pa Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search nav search search input Search input auto suggest search filter All ContentAll JournalsClinical Science Search Advanced Search This content is only available as a PDF. © 1981 The Biochemical Society and the Medical Research Society1981 Article PDF first page preview Close Modal You do not currently have access to this content.

The addition of bacterial lipopolysaccharide (LPS) from Salmonella minnesota R595 to rabbit plasma results in a marked reduction of the hydrated buoyant density of the parent R595 LPS, from 1.38 to less than 1.2 g/cm3. Using immunopurified anti-R595 LPS antibody covalently linked to Sepharose 4B, we were able to separate the altered R595 LPS (d less than 1.2 g/cm3) from the remainder of the plasma proteins by elution of the bound material with 2.5 M KSCN. The KSCN eluate was shown to have a d less than 1.2 g/cm3 and to contain both R595 LPS as well as protein and lipid characteristic of high density lipoprotein (HDL). The major protein in the KSCN eluate is a single polypeptide chain with an apparent molecular weight of 26,000 in sodium dodecyl sulfate and an amino acid composition essentially identical to that of apoprotein AI, the major protein of rabbit HDL. The lipid composition of the KSCN eluate is similar to that of HDL, although marked differences in the cholesterol ester/cholesterol ration and the phosphatidyl choline/phosphatidyl ethanolamine ratio were observed when the KSCN eluate and rabbit HDL were compared. The formation of this R595 LPS-protein-lipid complex in plasma accounts for the marked reduction in buoyant density found when LPS is added to plasma.