Effects of mevinolin,an inhibitor of cholesterol synthesis,on the morphology and function of differentiating and differentiated rat adrenocortical cells in primary culture

Summary Mevinolin, an inhibitor of cholesterol synthesis, was used to study the effect of endogenous cholesterol synthesis on the morphology and function of differentiating and differentiated fetal rat adrenocortical cells grown in primary culture. Upon adrenocorticotrophic hormone (ACTH) stimulation under conditions in which endogenous cholesterol synthesis was inhibited but exogenous (lipoprotein) cholesterol was available, the cells differentiated normally from glomerulosa-like to fasciculata-like cells; the steroid hormone secretion was maximally induced. Under conditions in which cholesterol synthesis was maximally inhibited by mevinolin and the cells had no access to exogenous cholesterol, the cells did not differentiate into fasciculata-like cells; the ACTH-induced steroid response was highly suppressed under these conditions. The addition of either human low-density lipoprotein (LDL) or high-density lipoprotein (HDL₃) to the culture medium restored the ACTH-induced differentiation and steroid secretion. Thus, in the absence of exogenous cholesterol, endogenous cholesterol synthesis was a prerequisite for differentiation. In cultures grown in the presence of exogenous cholesterol and ACTH with mevinolin-inhibited cholesterol synthesis and high steroid output, an increase in cytoplasmic lipids was evident, suggesting upregulation of LDL and HDL receptors. The results also demonstrated that induction of phenotypic differentiation from glomerulosalike into fasciculata-like cells can proceed in the presence of a cholesterol synthesis inhibitor like mevinolin; this differentiation in the absence of endogenous cholesterol synthesis is accompanied by the appearance of cytoplasmic cholesterol ester droplets, typical of fasciculata cells.     

Endogenous cholesterol synthesis is sufficient for ACTH-induced differentiation of rat adrenocortical cells in primary culture

Summary To define the role of endogenously synthesized cholesterol in the differentiation of adrenocortical cells in primary culture, fetal rat adrenal cells were cultured in the presence of exogenous cholesterol (serum-supplemented medium) or in the absence of it (serum-free medium or lipoprotein-free medium). Ultrastructurally the cells had features of glomerulosa cells: mitochondria were oval or rod shaped with lamellar inner membranes. The amount of smooth endoplasmic reticulum was small, and lipid droplets were few. When the cells were cultured in serum-free medium some intracytoplasmic vacuoles were seen. The undifferentiated zona glomerulosa-like cells secreted low amounts of corticosterone and 18-OH-deoxycorticosterone (18-OH-DOC) in all three media (serum-supplemented medium, serum-free medium, and lipoprotein-free medium). Stimulation of the adrenocortical cells with ACTH induced the ultrastructural features of differentiated zona fasciculata-like cells. Mitochondrial inner membranes were well developed in lipoprotein-free medium, but not in serum-free medium. The amount of intracellular lipids was increased in both media devoid of cholesterol. In the ACTH stimulated cultures the presence of exogenous cholesterol resulted in increased secretions of corticosterone and 18-OH-DOC. In the absence of an exogenous source of cholesterol, the amounts of steroids secreted were only half of that secreted in the presence of serum-supplemented medium. Endogenously synthesized cholesterol is sufficient for the morphologic differentiation of fetal rat adrenocortical cells under ACTH stimulation. However, without exogenously provided cholesterol, the steroid production accounts only for half of the maximal output achieved using serum-supplemented medium. This work was supported by Finnish Culture Foundation.     

Human crypt intestinal epithelial cells are capable of lipid production, apolipoprotein synthesis, and lipoprotein assembly

The recent availability of spontaneously proliferating, non-transformed human crypt intestinal epithelial cells (HIEC) affords an opportunity to investigate lipid metabolism in undifferentiated enterocytes. The major purpose of this study was to explore the capability of undifferentiated crypt cells to synthesize, assemble, and secrete lipids and apolipoproteins. HIEC were cultured in medium with 5% fetal bovine serum for 5 to 21 d. The cells were clearly able to incorporate [(14)C]oleic acid (dpm/mg protein) into triglycerides (128,279 +/- 16,988), phospholipids (30, 278 +/- 2,107), and cholesteryl esters (2,180 +/- 207). Although improvement in lipid secretion was noted with prolongation of cell culture periods, low efficiency of lipid export (10.3 +/- 2.2% of intracellular content) characterized the HIEC. All phospholipid classes were elaborated, with phosphatidylcholine accounting for 79. 3 +/- 1.3% of cellular phospholipids. Chylomicrons were the dominant (46.4%) lipoproteins secreted, followed by high, low, and very low density lipoproteins (HDL, LDL, and VLDL) comprising 22.5, 20.2, and 10.8% of the total, respectively. HIEC elaborated most of the major apolipoprotein (apo) classes (A-I, A-IV, B-100, C, and E), but were less efficient in producing apoB-48. In contrast to the production of apoA-I and C as early as 5 days after confluence, apoA-I and A-IV were maximally expressed at 11 d. Culture media accumulated much more apoB-100 than apoB-48 (B-48/B-100 ratio 0.21 +/- 0.03), reflecting limited apoB mRNA editing. HIEC demonstrated both endogenous cholesterol synthesis and LDL receptor expression. Cholesterol synthesis was sensitive to 25-hydroxycholesterol and mevinolin, but unresponsive to LDL treatment, suggesting independent regulation pathways. In contrast, LDL inhibited receptor activity. The present findings provide the first solid evidence that immature HIEC are capable of key fat absorptive functions of well-differentiated enterocytes. The intracellular mechanisms required for lipid and apolipoprotein synthesis as well as for lipoprotein assembly are already present in intestinal crypt cells. These cells also retain the capacity for sterol enzyme and receptor expression. However, certain limitations, especially apoB-48 production and lipoprotein secretion as well as unresponsiveness of cholesterol synthesis to LDL, may be ascribed to the lack of differentiation.     

Stereological and functional investigations on isolated adrenocortical cells: Zona fasciculata/reticularis cells of chronically ACTH-treated rats

Summary The morphology and function of isolated inner (zona fasciculata/reticularis) adrenocortical cells of rats pretreated with ACTH for 3, 6, 9 or 12 days were investigated. ACTH treatment induced a notable time-dependent enhancement in the steroidogenic capacity (corticosterone production) and growth of inner cells. The volumes of cells, mitochondrial compartment, membrane space [the cellular space occupied by smooth endoplasmic reticulum (SER) membranes] and lipid-droplet compartment, as well as the surface area of mitochondrial cristae and SER tubules, were increased in relation to the duration of ACTH pretreatment, and showed a highly significant positive linear correlation with both basal and stimulated corticosterone production. The acute exposure of isolated cells to ACTH provoked a striking lipid-droplet depletion, the extent of which was linearly and positively correlated with stimulated corticosterone secretion. The hypertrophy of the mitochondrial compartment and SER are interpreted as the morphological counterpart of the enhanced steroidogenic capacity of inner adrenocortical cells, inasmuch as the enzymes of steroid synthesis are located in these two organelles, and it is well known that chronic ACTH exposure stimulates the de novo synthesis of many of them in vivo. The rise in the number of lipid droplets, in which cholesterol is stored, is interpreted as being due to the fact that, under chronic ACTH treatment, the processes leading to cholesterol accumulation in adrenocortical cells (exogenous uptake and endogenous synthesis) exceed those of its utilization in basal steroid secretion. Cholesterol accumulated in lipid droplets as a reserve material may be rapidly utilized after acute ACTH exposure to meet the needs of the enhanced steroidogenic capacity of adrenocortical cells.     

Studies on lipoprotein and adrenal steroidogenesis: II. Utilization of low density lipoprotein- and high density lipoprotein-cholesterol for steroid production in functioning human adrenocortical adenoma cells in culture

We examined the utilization of human low density lipoprotein (LDL)- and high density lipoprotein (HDL)-cholesterol for steroid production in primary monolayer culture cells from adenomas of primary aldosteronism and Cushing's syndrome and an adrenal of nodular hyperplasia of Cushing's syndrome. We compared the data obtained with findings in the case of cultured normal human adrenocortical cells. In the presence of 10(-7) M adrenocorticotropin (ACTH), the addition of either LDL or HDL to the culture medium at a cholesterol concentration of 100 micrograms/ml led to a significant increase in the daily secretion rates of cortisol, dehydroepiandrosterone sulfate (DHEA-S) and aldosterone in the adenoma and nodular hyperplasia cells, as in the normal cells. Although LDL greatly increased the secretion of steroid hormones, no significant difference in steroid secretion following the treatments with LDL and HDL were observed in these cultured cells. The contribution of endogenous cholesterol to steroid production was also high, thereby indicating that the neoplastic transformation did not have untoward effects. Cells from adenomas of primary aldosteronism secreted not only aldosterone, but also cortisol and DHEA-S. The daily secretion rates of these steroids were markedly increased when ACTH was added to the medium. With prolonged exposure to ACTH, however, the rate of aldosterone secretion showed a gradual decrease with the incubation time. This decrease might be due to the impaired conversion of corticosterone to 18-hydroxycorticosterone. In case of adenomas in patients with Cushing's syndrome, the secretion of steroid hormones varied in quantity and quality, depending on the type of plasma cortisol response to the rapid ACTH test in vivo, thereby suggesting that the adrenocortical adenoma of Cushing's syndrome might be divided into two subtypes. These results indicate that human functioning adrenocortical adenoma cells utilize plasma lipoproteins as a source of cholesterol for steroidogenesis during the prolonged stimulation of steroid secretion.     

Compartmentalization of cholesterol metabolism and cellular growth in cultured intestinal crypt cells

Growth of rat intestinal crypt derived cells IEC-6 ceased when the key enzyme of cholesterol synthesis, hydroxymethylglutaryl-CoA reductase, was blocked by the competitive inhibitor mevinolin. This effect was reversed by the addition of mevalonolactone. LDL suppressed reductase activity as well as cholesterol synthesis from [14C]octanoate and stimulated acyl-CoA cholesterol acyltransferase, but failed to support cell growth despite rapid receptor mediated degradation even in the presence of low mevalonolactone concentrations. Inhibition of cholesterol esterification by Sandoz-Compound 58-035 enhanced cell growth in the presence of mevinolin, but did not promote proliferation in the additional presence of low-density lipoproteins. HDL3 but not HDL2 or tetranitromethane-modified HDL3 totally reversed the mevinolin induced inhibition of cell growth. This rescue by HDL3 was overcome by an increased dose of mevinolin. HDL3 derepressed reductase, stimulated cholesterol synthesis and reduced cholesterol esterification, but did not reverse the cholesterol synthesis inhibition by mevinolin. It is concluded that IEC-6 cells preferentially use endogenously synthesized cholesterol for membrane formation rather than low-density lipoprotein cholesterol. High-density lipoproteins appear to normalize cell growth in the presence of mevinolin by inhibition of cholesterol esterification and probably by inducing the formation of non sterol products of mevalonate.     

Stirred culture of peripheral and cord blood hematopoietic cells offers advantages over traditional static systems for clinically relevant applications

The ability to culture hematopoietic cells in readily characterizable and scalable stirred systems, combined with the capability to utilize serum-free medium, will aid the development of clinically attractive bioreactor systems for transplantation therapies. We thus examined the proliferation and differentiation characteristics of peripheral blood (PB) mononuclear cells (MNC), cord blood (CB) MNC, and PB CD34(+) cells in spinner flasks and (control) T-flask cultures in both serum-containing and serum-free media. Hematopoietic cultures initiated from all sources examined (PB MNC, CB MNC, and PB CD34(+) cells) grew well in spinner vessels with either serum-containing or serum-free medium. Culture proliferation in spinner flasks was dependent on both agitator design and RPM as well as on the establishment of critical inoculum densities (ID) in both serum-containing (2 x 10(5) MNC/mL) and serum-free (3 x 10(5) MNC/mL) media. Spinner flask culture of PB MNC in serum-containing medium provided superior expansion of total cells and colony-forming cells (CFC) at high ID (1.2 x 10(6) cells/mL) as compared to T-flask controls. Serum-free spinner culture was comparable, if not superior, to that observed in serum-containing medium. This is the first report of stirred culture of PB or CB MNC, as well as the first report of stirred CD34(+) cell culture. Additionally, this is the first account of serum-free stirred culture of hematopoietic cells from any source.     

Heparin-binding growth factor type one and platelet-derived growth factor are required for the optimal expression of cell surface low density lipoprotein receptor binding activity in human adult arterial smooth muscle cells

Summary Purified heparin-binding growth factor-1 (HBGF-1) stimulated low density lipoprotein binding, internalization, and degradation in isolated human adult arterial smooth muscle cells. Exposure of quiescent cells to HBGF-1 in serum-free, defined medium increased both low density lipoprotein (LDL) receptor activity and de novo cholesterol biosynthesis. Both events preceded the onset of DNA synthesis by 6 to 9 h. HBGF-1 acted additively with platelet-derived growth factor (PDGF) to maximally stimulate cell surface LDL receptor binding activity and DNA synthesis in the smooth muscle cells. The presence of LDL was required for maximal mitogenic activity of HBGF-1 and PDGF. In the presence of LDL, growth factor-stimulated, proliferating human smooth muscle cells accumulated cholesterol ester and triglycerides. The results suggest that HBGF-1, PDGF, and LDL act together to promote the maximal proliferation of smooth muscle cells in culture. Chronic exposure to the three growth promoters may contribute to the smooth muscle cell hyperplasia and lipid accumulation observed in atherosclerotic lesions. This work was supported by the National Cancer Institute grants CA 37589 and HD 03275, National Council for Tobacco Research grant 1718, and a grant from RJR Nabisco, Inc.     

Synthetic low-density lipoprotein, a novel biomimetic lipid supplement for serum-free tissue culture

Lipid supplementation in serum-free tissue culture employs solubilization techniques to permit the addition of lipids, but these systems are potentially cytotoxic and do not present lipid in a natural form. In this research a simplified preparation method for synthetic low-density lipoprotein (sLDL) has been developed that involves microfluidization of a solvent lipid solution in a simple aqueous solution. This produces material with size and zeta potential characteristics similar to those of native LDL. sLDL supplementation in tissue culture media provides cholesterol concentrations higher than those achieved by 10% serum supplementation and existing chemically defined lipid supplements. sLDL stimulates NS0 and U937 cellular proliferation in completely serum-free media, the former in a lipid concentration dependent manner that is also related to both the receptor peptide structure employed and its concentration on the particle. The greatest NS0 cellular proliferation was obtained at the highest cholesterol concentration tested (0.5 mg/mL), which was 10 times higher than the cholesterol concentration achieved by standard 10% serum supplementation. U937 cellular proliferation was influenced by variation of sLDL's fatty acid constituents with a natural mixture producing maximal effect. Cell uptake studies in NS0 with fluorescently labeled sLDL indicated that assimilation is reduced by competition from native LDL. The planktonic nature of NS0 cell growth meant that cell binding and uptake experiments were difficult to conduct because of cellular aggregation. However, sLDL-induced U937 proliferation is ablated by the presence of an anti-LDL receptor antibody. The results indicate that sLDL uptake is via the LDL receptor and that sLDL can function as a lipid supplement for serum-free media capable of supplementation to cholesterol concentrations up to 0.5 mg/mL. Cellular uptake studies also suggest that sLDL will be useful for the targeting and delivery of materials to cells. sLDL therefore represents a new and promising synthetic biomimetic alternative to native LDL with multiple applications.     

Analysis of plasma protein and lipoprotein synthesis in long-term primary cultures of baboon hepatocytes maintained in serum-free medium

Summary The analysis of lipoprotein synthesis and secretion in primary hepatocytes has been restricted by the short-term viability and low proliferative response of hepatocytes in vitro. During this investigation a serum-free medium formulation was developed that supports long-term maintenance (>70 d) and active proliferation of primary baboon hepatocytes. Examination of proliferating cells by electron microscopy revealed a distinctive hepatocyte ultrastructure including intercellular bile canaliculi and numerous surface microvilli. High levels of secreted apolipoproteins A-I and E were detected in the tissue culture medium by gel electrophoresis and immunoblot analysis. Immunoprecipitation of proteins from [³⁵S]-methionine labeled tissue culture medium revealed the synthesis and secretion of numerous plasma proteins. Metabolic labeling of cells with [³⁵S]-methionine followed by single-spin density gradient flotation of the media demonstrated that apolipoproteins were being secreted in the form of lipoprotein particles with buoyant densities corresponding to the very low density lipoprotein and low density lipoprotein range, and to the high density lipoprotein range. The labeled apolipoproteins included B _h , E, and A-I. This system for primary hepatocyte culture should prove very useful in future investigations on the regulation of lipoprotein production by hepatocytes. This investigation was supported in part by a research grant from the Southwest Foundation Forum, by program project HL 28972 from the National Heart, Lung and Blood Institute, Bethesda, MD, and by grants to R. V. H. from the National Institutes of Health (HL 15062), the American Heart Association, and the Louis Block Fund.     

Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serum-free or serum-containing media.

In this study, the quantitative fluctuation of cytoplasmic lipid droplets (LD) and cryotolerance were investigated in bovine embryos derived from in vitro-matured (IVM) and in vitro-fertilized (IVF) oocytes developed in different culture systems using serum-free or serum-containing media. The serum-free cultures were grown using IVMD101 medium in conjunction with bovine cumulus/granulosa cell (BCGC) cocultures or IVD101 medium without BCGC cocultures, and the serum-containing cultures were grown in the presence of BCGC cocultures using HPM199 medium supplemented with 5% calf serum (HPM199 + CS). Large numbers of sudanophilic LD were present in the cytoplasm of bovine embryos from 2-cell to hatched blastocyst stages, and the number and size differed between the embryos cultured in serum-free and serum-supplemented media. In the embryos cultured in HPM199 + CS, large (2-6 microm in diameter) sudanophilic LD increased significantly from the morula to the blastocyst stages. Throughout the embryonic development, the embryos developed in serum-free cultures with and without BCGC cocultures had numerous sudanophilic LD, but most of these droplets were small (<2 microm in diameter) and large LD were less numerous than those embryos cultured in HPM199 + CS. Giant LD (>6 microm in diameter) were frequently observed in morulae and blastocysts (including early blastocysts) developed in HPM199 + CS. Electron microscopic observations demonstrated that large LD were abundant in the cytoplasm of trophoblast and embryonic (inner cell mass) cells of blastocysts cultured in HPM199 + CS. These large LD were identified as osmophilic LD, an indication that these lipid inclusions contained a significant proportion of unsaturated lipids. Many elongated mitochondria were found in embryos developed in IVMD101 and IVD101 at the morula and early blastocyst stages, whereas many of the mitochondria in the morulae developed in HPM199 + CS were of an immature form such as spherical or ovoid shape. The survival and hatching rates of embryos (morulae, early blastocysts, and blastocysts) produced in serum-free media (both IVMD101 and IVD101) after post-thaw culture were superior to those of embryos produced in serum-containing medium. These results showed that bovine embryos cultured in serum-containing medium abnormally accumulated cytoplasmic lipids into their cytoplasm and the excess accumulation of cytoplasmic LD in embryos may affect the cryotolerance of embryos.     

HDL Subspecies in Young Adult Twins: Heritability and Impact of Overweight

The association between abdominal obesity and atherogenic lipid profile emerges from complex interactions of genes and environment. We aimed to explore the heritability and effects of overweight on serum lipid profile (high‐density lipoprotein‐cholesterol (HDL‐C), HDL mean particle size, percentages of HDL_{2b, 2a, 3a, 3b, and 3c}, low‐density lipoprotein‐cholesterol (LDL‐C), LDL peak particle size and triglycerides (TGs)) in healthy, young adults. HDL‐C, LDL‐C, and TG were measured in 52 monozygotic (MZ) and 89 dizygotic (DZ) twin pairs, aged 23–32 years, chosen to represent a wide range of BMIs (17.6–42.9 kg/m²). Of them, 24 MZ and 26 DZ pairs were chosen at random for measurements of HDL mean and LDL peak particle sizes and percentages of HDL subspecies. The heritabilities of the lipid parameters adjusted for BMI were HDL‐C 73%, HDL mean particle size 56%, HDL subspecies 46–63%, LDL‐C 79%, LDL peak particle size 49%, and TG 64%. Genetic and environmental correlations between BMI and HDL‐C, LDL‐C, and TG were modest (0.3–0.4). Abdominal overweight (waist circumference ≥94 cm for males and ≥80 cm for females) associated with decreased HDL‐C, increased LDL‐C, and TG concentrations, smaller HDL mean particle size, lower HDL_2b, and higher HDL_3c percentages in both genders. Within MZ twins, controlling for genetic influences, within‐pair differences in HDL_3c percentage were associated with those in waist (r = 0.46, P = 0.032) and BMI (r = 0.51, P = 0.013). In conclusion, serum lipid parameters, including LDL peak and HDL mean particle sizes and HDL subspecies distribution are under strong genetic control. Overweight associated with significant lipid profile changes, particularly, small HDL_3c increased in overweight independent of genetic influences.     

Cellular cholesterol metabolism in mitogen-stimulated lymphocytes--requirement for de novo synthesis

The relationship between cholesterol synthesis and uptake in proliferating lymphocytes has been examined. [14C]Acetate incorporation into lymphocytes cultured under lipoprotein-deficient conditions increased initially in response to mitogen, decreased after 24 h, and increased rapidly between 72 and 96 h. Addition of LDL (10 micrograms/ml) to the culture during the 'trough' period caused [14C]acetate incorporation to return rapidly to baseline, while at peak periods LDL suppression of cholesterol synthesis was minimal. Lymphocytes cultured in the presence of the HMG-CoA reductase inhibitor, mevinolin, exhibited a time-dependent increase in their capacity to incorporate [14C]acetate into cholesterol, evident when mevinolin was removed by washing prior to assay. PHA enhanced 125I-labelled LDL receptor-mediated binding by lymphocytes cultured in lipoprotein-deficient medium over a 4 day period and mevinolin augmented the effect. [3H]Thymidine incorporation into mitogen-stimulated lipoprotein-deficient cultures was inhibited up to 75% by mevinolin (1 mumol/l). LDL (2.5-10 micrograms/ml) substantially reversed this inhibition in 72 h cultures, but only partially overcame inhibition in cells cultured for 96 h. Results suggest that endogenous cholesterol synthesis may be obligatory for lymphocyte proliferation after the initial round of cell division.     

Characterization of metabolic interrelationships and in silico phenotyping of lipoprotein particles using self-organizing maps

Plasma lipid concentrations cannot properly account for the complex interactions prevailing in lipoprotein (patho)physiology. Sequential ultracentrifugation (UCF) is the gold standard for physical lipoprotein isolations allowing for subsequent analyses of the molecular composition of the particles. Due to labor and cost issues, however, the UCF-based isolations are usually done only for VLDL, LDL, and HDL fractions; sometimes with the addition of intermediate density lipoprotein (IDL) particles and the fractionation of HDL into HDL₂ and HDL₃ (as done here; n = 302). We demonstrate via these data, with the lipoprotein lipid concentration and composition information combined, that the self-organizing map (SOM) analysis reveals a novel data-driven in silico phenotyping of lipoprotein metabolism beyond the experimentally available classifications. The SOM-based findings are biologically consistent with several well-known metabolic characteristics and also explain some apparent contradictions. The novelty is the inherent emergence of complex lipoprotein associations; e.g., the metabolic subgrouping of the associations between plasma LDL cholesterol concentrations and the structural subtypes of LDL particles. Importantly, lipoprotein concentrations cannot pinpoint lipoprotein phenotypes. It would generally be beneficial to computationally enhance the UCF-based lipoprotein data as illustrated here. Particularly, the compositional variations within the lipoprotein particles appear to be a fundamental issue with metabolic and clinical corollaries.     

Serum-free medium conditions for steroidogenesis of bovine follicular thecal cells cultured on collagen gel matrix

Summary Thecal cells isolated from bovine ovarian follicles were cultured with a serum-free basal medium or a serum-free complete medium in the presence or absence of collagen gel matrix, and their cellular proliferation and steroidogenesis were compared with those of cells cultured with a serum-containing medium. The cells cultured with the serum-free basal medium produced larger amounts of progesterone, androstenedione, and estradiol than the cells cultured with the serum-containing medium, but no appreciable cell proliferation was observed in the serum-free medium. Response of thecal cells to 8 bromo-cAMP, a steroidogenic agent, varied according to the type of steroid production examined and the type of culture medium used. In a cultivation period of 4 d, progesterone production was stimulated about five-fold by 8 bromo-cAMP in the serum-free complete medium on collagen gel matrix and in the serum-free basal medium without collagen matrix, whereas androstenedione production was stimulated about three- to fourfold in the serum-free complete medium on collagen gel matrix and in the serum-free basal medium with or without collagen matrix. Estradiol production, however, was significantly suppressed by 8 bromo-cAMP in the serum-free complete medium on collagen gel matrix and also in the serum-containing medium. Thus, among the conditions examined, the most suitable primary culture media for steroidogenesis of thecal cells were the serum-free media, especially serum-free complete medium on collagen gel matrix.     

Promotion of human T lymphocyte activation and proliferation by fatty acids in low density and high density lipoproteins

Mitogen-induced lymphocyte DNA synthesis measured by [3H]thymidine incorporation and lymphocyte proliferation assessed by counting the number of cells were reduced by greater than 95% when cells were cultured at low density in the absence of serum. Supplementation with either transferrin or lipoprotein alone only partially restored lymphocyte responses. Addition of both transferrin and lipoproteins of each major subclass permitted mitogen-induced lymphocyte DNA synthesis and proliferation equal to that observed in serum-containing medium. The degree of enhancement was dependent on the concentration of the lipoprotein added and could not be explained by the nonspecific addition of protein to the defined medium. The mechanisms of growth promotion by various lipoprotein fractions did not appear to be explained by provision of cholesterol to the cells. Neither cholesterol nor cholesteryl ester from endogenous sources or supplied exogenously was able to enhance mitogen-induced lymphocyte responses. In contrast, fatty acids, phospholipid, and triglyceride alone supported lymphocyte responses. Furthermore, lipoproteins retained the capacity to enhance lymphocyte responses following extraction of neutral lipid. Both low density lipoprotein and high density lipoprotein, subclass 3, increased the number of cells initially activated by mitogenic stimulation and supported the subsequent continued growth of the activated cells. Low density lipoprotein was more efficient than high density lipoprotein, subclass 3, in this latter regard. These results indicate that lipoproteins can promote maximal growth of mitogen-activated lymphocytes in transferrin-containing medium by providing growth factors other than cholesterol necessary for initial activation and required for continued lymphocyte proliferation.     

Serum suppresses the expression of hormonally induced functions in cultured granulosa cells

Growth and function of primary cultures of granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were compared in serum-containing and serum-free media. In serum-free medium (1:1 mixture of DMEM:F-12) supplemented with insulin, hydrocortisone, transferrin and fibronectin (4F medium), the cells remained healthy and steroidogenically responsive for at least 60 days in culture. The growth profile of the granulosa cells in 4F medium was similar to that obtained in serum-containing medium. In both media cell proliferation did not exceed more than one cell doubling. DMEM:F-12 alone did not support the cell viability. Upon FSH stimulation, the cells produced 25 fold more progestin and estrogen per cell in 4F medium than in medium supplemented with 5% serum. This effect was not directly related to serum proteins which mediate cell adhesion since cells cultured in dishes precoated with serum remained steroidogenically responsive to FSH. Cholera toxin and Bt₂-cAMP readily stimulated progestin production in the presence of serum. The inhibitory effect of serum was not reversed by adding the four factors to serum-containing medium. The factors were essential for the FSH-induced steroidogenesis in serum-free medium. After four days of incubation in 4F medium, the cells showed a transient loss of their ability to produce progestin in response to FSH. In both 4F medium as well as in serum-containing medium, the cells regained their hormonal responsiveness after 35 days in culture. Since the loss of hormonal responsiveness occurred at the same time as growth was initiated in the cultures, it is suggested that the FSH-induced steroidogenesis is negatively controlled by growth-related processes.     

Lipid profile of a French-Canadian population: 1. Association of plasma lipid and lipoprotein levels with age,relative body weight and education

Plasma lipid and lipoprotein levels were determined in a randomly selected population of 1169 French-Canadian men in the Quebec City area. The mean levels of total plasma cholesterol and triglycerides were 224.0 and 166.5 mg/dL respectively. The mean level of low-density lipoprotein cholesterol was higher and the mean level of high-density lipoprotein (HDL) cholesterol lower than those reported in a recent study in English-Canadian men. The mean HDL₂ and HDL₃ levels were lower than those reported in American men. Stratification of plasma triglyceride levels for all age groups showed that mean HDL₂ levels decreased rapidly with moderate rises in triglyceride levels. Less than 9% of the variation in lipid or lipoprotein levels was related to age or relative body weight. Education had no significant effect on the levels.     

Studies on lipoprotein and adrenal steroidogenesis: I. Roles of low density lipoprotein- and high density lipoprotein-cholesterol in steroid production in cultured human adrenocortical cells

The roles of human low density lipoprotein (LDL)- cholesterol and high density lipoprotein (HDL)- cholesterol on adrenal steroidogenesis were investigated using cultured human adult and fetal adrenocortical cells and the findings were then compared to those obtained with bovine adrenocortical cells. The secretion of cortisol in both human and bovine adrenocortical cells was dose-dependently increased by the administration of LDL- or HDL-cholesterol in the presence of adrenocorticotropin (ACTH). LDL-cholesterol was utilized to a greater extent than HDL-cholesterol in both human and bovine adrenal steroidogenesis in the presence of ACTH. Exogenous lipoprotein-derived cholesterol was less utilized in human adrenal steroidogenesis than in bovine adrenal steroidogenesis, compared to the endogenous cholesterol. An increase in the secretion of cortisol and dehydroepi androsterone sulfate (DHEA-S) continued for the 5-day culture period, in the presence of lipoprotein cholesterol and ACTH in both human adult and fetal adrenocortical cells. The secretion of aldosterone increased on the first day of the culture period, then gradually decreased for the 5-day culture period in human adult adrenocortical cells, but not in human fetal adrenocortical cells in the presence of lipoprotein cholesterol and ACTH. These findings demonstrate that exogenous cholesterol utilized in the biosynthesis of steroids is mainly from LDL-cholesterol in both human adult and fetal adrenals and bovine adrenal and the proportion of cholesterol synthesized de novo is significantly larger in the human adult adrenal than in the bovine adrenal.     

The role of bovine lipoproteins in the regulation of steroidogenesis and HMG-CoA reductase in bovine adrenocortical cells.

The sources of cholesterol for steroid hormone production were examined using bovine adrenocortical (BAC) cells in primary culture. The experiments were designed to determine the effects of lipoproteins on cortisol production and the level of BAC cell 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Most studies on BAC cell lipoprotein requirements have been conducted using human low-density lipoprotein (hHDL); none have used the homologous bovine lipoproteins. BAC cells treated with corticotropin (ACTH) in a medium devoid of lipoproteins increased and maintained cortisol production 7- to 20-fold above basal levels. Under such conditions ACTH also increased the rate of HMG-CoA reductase activity. Inhibition of HMG-CoA reductase with mevinolin inhibited cortisol production by 85%, indicating that the cells were using cholesterol synthesized de novo for steroid production. Cortisol production was increased almost 40-fold above basal levels if hLDL (100 micrograms/ml) was included in the incubation medium. Human LDL also suppressed the levels of HMG-CoA reductase in a concentration-dependent fashion. Human HDL was without effect on either BAC cell steroidogenesis of HMG-CoA reductase. Addition of bovine LDL (bLDL) to the incubation medium also caused an increase in cortisol production and inhibited cholesterol synthesis. By contrast to hHDL, bHDL (100 micrograms/ml) increased the ability of BAC cells to produce cortisol production. Bovine HDL (bHDL) also was able to decrease HMG-CoA reductase, but not to the extent caused by hLDL or bLDL. These data demonstrate that bovine adrenal cells can use bHDL as a source of cholesterol for steroid hormone production. These findings may be of particular importance when one considers that in vivo, the bHDL content of bovine serum greatly surpasses the level of bLDL.