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PubMed2004

Fifty years with bile acids and steroids in health and disease.

Cholesterol and its metabolites, e.g., steroid hormones and bile acids, constitute a class of compounds of great biological importance. Their chemistry, biochemistry, and regulation in the body have been intensely studied for more than two centuries. The author has studied aspects of the biochemistry and clinical chemistry of steroids and bile acids for more than 50 years, and this paper, which is an extended version of the Schroepfer Medal Award lecture, reviews and discusses part of this work. Development and application of analytical methods based on chromatography and mass spectrometry (MS) have been a central part of many projects, aiming at detailed characterization and quantification of metabolic profiles of steroids and bile acids under different conditions. In present terminology, much of the work may be termed steroidomics and cholanoidomics. Topics discussed are bile acids in human bile and feces, bile acid production, bacterial dehydroxylation of bile acids and steroids during the enterohepatic circulation, profiles of steroid sulfates in plasma of humans and other primates, development of neutral and ion-exchanging lipophilic derivatives of Sephadex for sample preparation and group separation of steroid and bile acid conjugates, profiles of steroids and bile acids in human urine under different conditions, hydroxylation of bile acids in liver disease, effects of alcohol-induced redox changes on steroid synthesis and metabolism, alcohol-induced changes of bile acid biosynthesis, compartmentation of bile acid synthesis studied with 3H-labeled ethanol, formation and metabolism of sulfated metabolites of progesterone in human pregnancy, abnormal patterns of these in patients with intrahepatic cholestasis of pregnancy corrected by ursodeoxycholic acid, inherited and acquired defects of bile acid biosynthesis and their treatment, conjugation of bile acids and steroids with N-acetylglucosamine, sulfate-glucuronide double conjugates of hydroxycholesterols, extrahepatic 7alpha-hydroxylation and 3-dehydrogenation of hydroxycholesterols, and extrahepatic formation of C27 bile acids. The final part discusses analysis of free and sulfated steroids in brain tissue by capillary liquid chromatography-electrospray MS and suggests a need for reevaluation of the function of steroid sulfates in rat brain.

Animals↗
PubMed1983

Analysis of the coordinate expression of 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase activities in Chinese hamster ovary fibroblasts.

Decreased activities of both 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase are observed in the presence of sterol in the Chinese hamster ovary (CHO) fibroblast. In three different genotypes of CHO cell mutants resistant to 25-hydroxycholesterol both enzyme activities exhibit a decreased response to 25-hydroxycholesterol compared to wild-type cells. Permanently repressed levels of both HMG CoA synthase and HMG CoA reductase activities are observed in another CHO mutant, phenotypically a mevalonate auxotroph. Mevinolin, a competitive inhibitor of HMG CoA reductase, has no effect on HMG CoA synthase activity measured in vitro. Incubation of CHO cells with sublethal concentrations of mevinolin produces an inhibition of the conversion of [14C]acetate to cholesterol and results in elevated levels of both HMG CoA synthase and HMG CoA reductase activities. Studies of CHO cells in sterol-free medium supplemented with cycloheximide indicate that continuous protein synthesis is not required for the maximal expression of HMG CoA synthase activity and provide an explanation for the lack of temporal similarity between HMG CoA synthase and reductase activities after derepression. These results support the hypothesis of a common mode of regulation for HMG CoA synthase and HMG CoA reductase activities in CHO fibroblasts.

Animals↗
PubMed1975

The substrate for cholesterol 7alpha-hydroxylase.

The rates of incorporation of (14C) cholesterol into cholesteryl esters and 5-cholestene-3beta,7alpha-diol (7alpha-hydroxycholesterol) by rat liver microsomes, measured under conditions in which esterification and 7alpha-hydroxylation are varied independently, indicated that cholesterol is the substrate for cholesterol 7alpha-hydroxylase. The specific activities of cholesteryl esters and 7alpha-hydroxycholeste ol in incubations of microsomes labelled with (14C)cholesterol in vitro or in vivo suggest that 7alpha-hydroxycholesterol and esterified cholesterol are not derived from the same pool of free cholesterol.

Adenosine Triphosphate↗
PubMed1980

Interaction between an organic hydroperoxide and an unsaturated phospholipid and alpha-tocopherol in model membranes.

The behavior of an organic hydroperoxide in the presence of lipid and/or alpha-tocopherol in model membranes has been studied using 14C-labeled cholesterol-5 alpha-hydroperoxide as the organic hydroperoxide. Cholesterol-F alpha-hydroperoxide in saturated phospholipid micelles is rapidly isomerized to cholesterol-7 alpha-hydroperoxide. Such an isomerization is inhibited by alpha-tocopherol, but not by an alpha-tocopheryl analogue with a substituted OH groups, present in the micelles, indicating the formation of hydrogen bonds between OH group in alpha-tocopherol and OOH group in the 5 alpha-hydroperoxide. The double bonds in unsaturated phospholipid can also serve to form a hydrogen bond with OOH in the 5 alpha-hydroperoxide moiety in the micelles. The resulting hydrogen-bonded complex could be decomposed by iron-induced lipid peroxidation, accompanied by isomerization of the 5 alpha-hydroperoxide and the further degradation to the 7-ketocholesterol and 7 alpha-hydroxycholesterol. When three components, such as unsaturated phospholipid, 5 alpha-hydroperoxide and alpha-tocopherol, are present in the same micelles, they form hydrogen bonded complexes. Such complexes could be decomposed by iron in the ferrous state, yielding mainly 5 alpha-hydroxycholesterol without significant change in the structure of alpha-tocopherol and peroxidative cleavage of unsaturated phospholipid.

Animals↗
PubMed1991

Unstable amplification of the chromosomal gene for 3-hydroxy-3-methylglutaryl coenzyme A reductase in compactin-resistant CR200 cells.

CR200 cells, a compactin-resistant clone of mouse FM3A cells, overaccumulate 3-hydroxy-3-methylglutaryl coenzyme A reductase. The elevated reductase activity is not regulated normally by low-density lipoprotein, mevalonate and 25-hydroxycholesterol. The amounts of reductase protein and mRNA were elevated in CR200 cells by 40- to 60-fold, as compared to those in parental FM3A cells and the rate of reductase transcription and the number of copies of reductase gene were increased in CR200 cells by 20- to 50-fold. In the parental cells, mevalonate and 25-hydroxycholesterol suppressed reductase transcription by greater than 90%, while that in the mutant cells was suppressed by only 20-50%, suggesting a regulatory alteration in the gene transcription in CR200 cells. When CR200 cells were grown for 10-20 weeks in the absence of compactin, levels of the gene amplification were reduced from approx. 50-fold to approx. 2-fold, along with a marked decrease in reductase activity and compactin-resistance of the cells. While the gene amplification was unstable, minute chromosomes were not seen in the cells and centrifugal fractionation and in situ analysis demonstrated that the amplified reductase gene was present on 2-3 chromosomes in the pseudotetraploidal CR200 cells having approx. 78 chromosomes. From these results it was concluded that the amplified reductase gene, which is responsible for overaccumulation of reductase, is located on chromosomes but is unstable in CR200 cells.

Animals↗
PubMed1991

Antiproliferative effects of oxygenated sterols: positive correlation with binding affinities for the antiestrogen-binding sites.

Oxygenated derivatives of cholesterol have long been known to exhibit antiproliferative properties but the mechanism of this effect remains incompletely understood. Following up on a recent observation in our laboratory that certain oxysterols bind with high affinity to the microsomal antiestrogen-binding site, we attempted to determine if a relationship existed between the antiproliferative effect of oxysterols and their binding affinities for the antiestrogen-binding site. Using the human breast cancer cell line, MCF7, and 14 different compounds (4 nonsteroidal antiestrogens, 6 oxysterols with appreciable binding affinities for the antiestrogen-binding site, and 4 other sterols with little or no affinity for this site), we showed that for compounds which bound to the antiestrogen-binding site, there was a clear positive correlation between their relative cytotoxic potencies and their affinities for this binding site. Two sterols, namely 25-hydroxycholesterol and 20 alpha-hydroxycholesterol, were exceptions to this general pattern, both clearly inhibited cell proliferation without having significant binding affinity for the antiestrogen-binding site. The same general order of antiproliferative potency of the oxysterols seen with MCF7 cells was also observed with the murine lymphoma cell line EL4. These findings suggest the possibility that the antiestrogen-binding site may be involved in mediating the antiproliferative effects of nonsteroidal antiestrogens and oxygenated sterols.

Animals↗
PubMed1992

Cultured fibroblasts from patients with Niemann-Pick disease type C and type D exhibit distinct defects in cholesterol esterification.

The Niemann-Pick group of diseases can be broadly classified into two types based on clinical and biochemical characteristics. Type I is characterized by a primary deficiency of lysosomal sphingomyelinase while Type II may have a defect in the regulation of intracellular cholesterol metabolism. We have studied cholesterol esterification in cultured fibroblasts from patients with two phenotypes of Type II disease: an Acadian population of southwestern Nova Scotia (Canada) with a form of the disease known as Niemann-Pick type D (NPD) and a group of panethnic origin with Niemann-Pick type C (NPC). Addition of whole serum to normal fibroblasts grown initially in lipoprotein-deficient serum caused a rapid (within 6 h) increase in cholesterol esterification, reaching maximum values at around 24 h, while NPC fibroblasts showed little increase (less than 10% of normal). In contrast, cholesterol esterification in NPD fibroblasts increased slowly during the first 6-12 h and reached 50% of normal values by 24 h. 25-Hydroxycholesterol, a non-lipoprotein stimulator of cholesterol esterification, caused a similar stimulation of cholesterol esterification in NPC, NPD and normal cells. This was inhibited by addition of serum in mutant but not in normal cells. Within 24 h of serum addition, free cholesterol accumulated in all cell types with NPC greater than NPD greater than normal. These observations indicate that (a) regulation of cholesterol esterification in response to serum lipoproteins (but not 25-hydroxycholesterol) is abnormal in both NPC and NPD fibroblasts, and (b) the biochemical phenotypes of fibroblasts from NPC and NPD patients are distinct.

Cells, Cultured↗
PubMed1995

Evaluation of cultured hamster hepatocytes as an experimental model for the study of very low density lipoprotein secretion.

The secretion of triacylglycerol, cholesterol and cholesteryl ester in very low density lipoprotein (VLDL) by cultured hamster hepatocytes was studied, and the results compared with those obtained previously using cultured rat hepatocytes and the human hepatoma cell line HepG2. The hamster cells secreted apolipoprotein B and VLDL triacylglycerol, cholesterol and cholesteryl ester linearly during 24 h in culture, and this time period was used in all experiments. Addition of oleate (1 mM) to the culture medium resulted in increased secretion of triacylglycerol, but cholesterol ester output were unchanged. Triacylglycerol secretion was also increased in the presence of lipogenic substrates (10 mM lactate + 1 mM pyruvate) plus dexamethasone (1 microM), but not with either of these agents alone. Inhibition of cholesterol synthesis in the hamster cells by incubation with mevinolin (2 micrograms/ml) did not change VLDL lipid secretion, but stimulation using mevalonate lactone resulted in decreased triacylglycerol output. Manipulation of the rate of cholesterol esterification in the hepatocytes by inhibiting or stimulating the activity of acyl coenzyme A cholesterol:acyl transferase using the inhibitor Dup128 (25 microM) and 25-hydroxycholesterol (50 microM), respectively, had no effect on the secretion of VLDL lipid. In the presence of 1 mM oleate plus 25-hydroxycholesterol, however, a rise in the output of triacylglycerol and cholesteryl ester was observed. Hepatocytes prepared from hamsters fed 2% cholestyramine secreted significantly less triacylglycerol than those from animals given the control diet, but cholesterol and cholesteryl ester output were unchanged, despite a decrease of about 40% in the total cholesterol content of the cells. These results show that the secretion of lipid in VLDL in hamster hepatocytes differs from that in rat and human liver in its response to dietary cholestyramine, and from rat hepatocytes and HepG2 cells in its response to changes in the rate of lipogenesis and cholesterol synthesis and esterification. Overall, hamster hepatocytes appear to be less susceptible to modification the rate of hepatic VLDL secretion, and should provide a useful additional tool for the investigation of this process.

Animals↗
PubMed1991

Nanosecond fluorescence of tryptophans in cytochrome P-450scc (CYP11A1): effect of substrate binding.

Fluorescence of eight tryptophan residues in cytochrome P-450scc with bound endogenous cholesterol could be fitted with a two component model: a single exponential and a "top-hat" distribution of lifetimes as the second component. The short-lived component (tau 1 about 700 ps) does not change significantly upon binding of substrate (22R-hydroxycholesterol). The parameters of the long-lived component (central lifetime tau m about 3.4 ns) change upon binding of carbon monoxide and substrate. 22R-hydroxycholesterol binding broadens the distribution of the long-lived component; that is the heterogeneity of the Trp environment is increased when this substrate displaces the endogenous cholesterol.

Adrenal Glands↗
PubMed1984

Effects of cholesterol autoxidation derivatives on hexose transport in cultured aortic smooth muscle cells.

Several cholesterol autoxidation derivatives known to be cytotoxic to arterial smooth muscle cells both in vivo and in vitro were shown to inhibit hexose transport in these cells in culture. Cholestane-3 beta, 5 alpha, 6 beta-triol was the most potent inhibitory sterol. The rapid onset of inhibition (15 min) and the reversibility of the effect upon removal of the sterol from the tissue culture medium suggests that the effect may be due to incorporation of the sterol into the plasma membrane. 25-Hydroxycholesterol, a potent inhibitor of sterol biosynthesis in cultured arterial smooth muscle cells, did not affect hexose transport up to 8 hr of incubation. The cytotoxic effect of 25-hydroxycholesterol, therefore, may be a consequence of the reduced sterol biosynthesis caused by this sterol.

Animals↗
PubMed1988

Clonal nature of atherosclerotic plaques.

The clonal nature of atherosclerotic plaques has been examined in diet-induced atherosclerosis of interspecies hybrid hare females (Lepus timidus (female) X Lepus europaeus (male], which exhibit genetic mosaicism with respect to glucose-6-phosphate dehydrogenase (G6P-D). Four diet groups have been used: control pellet diet, hyperlipemic (HL) diet, and HL diet supplemented with two different concentrations of 25-hydroxycholesterol. Lesion and nonlesion tissue samples and primary cultures developed from similar samples were used to determine the G6P-D variant patterns. A close correlation was observed, using regression analysis, between the in vivo and the in vitro phenotype. As has been reported earlier by us, most lesions in this animal model were heterozygous and the few homozygous areas recorded were all in the diet groups supplemented with 25-hydroxycholesterol. However, in tissue culture, homozygosity appeared with greater frequency, still of the T phenotype. In order to rule out maternal dominance as a factor, preliminary experiments involving G6P-D analysis have been carried out using tissue samples and cell cultures derived from a reverse cross. These also show a strong bias toward the T phenotype. The studies presented here highlight the difficulties in interpretation using G6P-D analysis as the sole criterion to determine the clonal nature of the atherosclerotic plaques. It also seems likely that the kind of diet given to induce atherosclerosis might influence the nature of the clonal growth. Studies using transfection are essential to determine whether transforming sequences are present in atherosclerotic lesions.

Animals↗
PubMed1988

Inhibition of cholesterol side-chain cleavage by intermediates of an alternative steroid biosynthetic pathway.

Mitochondrial preparations from endocrine tissues were incubated with radioactive cholesterol and the effect of hydroxylated metabolites of 23,24-dinor-5-cholen-3 beta-ol (23,24-dinor-5-cholene-3 beta,20-diol and 23,24-dinor-5-cholene-3 beta,21-diol) on the production of pregnenolone was measured. These compounds are intermediates in an alternative, sesterterpene pathway for steroid hormone biosynthesis. It was found that these materials, like the analogous side-chain-hydroxylated derivatives of cholesterol (20 alpha-hydroxycholesterol and 22S-hydroxycholesterol), inhibit cholesterol side-chain cleavage. The possibility that there could be a control mechanism whereby metabolites of 23,24-dinor-5-cholen-3 beta-ol inhibit steroidogenesis occurring by the cholesterol pathway is discussed.

Adrenal Glands↗
PubMed1989

Benign recurrent intrahepatic cholestasis: altered bile acid metabolism.

Altered bile acid metabolism has been claimed to play a role in the etiology of benign recurrent intrahepatic cholestasis (BRIC). Therefore, we studied bile acid metabolism in detail in 10 patients with this syndrome. Pool sizes of both primary bile acids were estimated simultaneously, using deuterated cholic acid and chenodeoxycholic acid. The pool sizes of cholic acid and chenodeoxycholic acid, expressed in micromoles per kilogram body weight, were significantly contracted in BRIC patients during a cholestasis-free period: 8.0 +/- 4.2 and 11.7 +/- 4.7, respectively, versus 24.1 +/- 11.7 and 22.9 +/- 7.8 in controls. Fractional turnover rates (per day) for cholic acid and chenodeoxycholic acid were increased: 0.70 +/- 0.29 and 0.58 +/- 0.27, respectively, versus 0.29 +/- 0.12 and 0.23 +/- 0.10 in controls. Bile acid pool composition expressed as percentages in BRIC patients was cholic acid 34 +/- 17, chenodeoxycholic acid 38 +/- 9, deoxycholic acid 27 +/- 18, and lithocholic acid 1 +/- 1, with a glycine to taurine conjugation ratio of 6.7 +/- 4.9. Corresponding values for 32 controls were cholic acid 57 +/- 13, chenodeoxycholic acid 29 +/- 9, deoxycholic acid 14 +/- 9, and lithocholic acid less than 1, with a glycine to taurine conjugation ratio of 2.4 +/- 1.3. Fecal bile acid loss, in micromoles per kilogram body weight per day, was 11.2 +/- 9.0 in BRIC patients compared with 2.8 +/- 1.4 in controls. The serum 7 alpha-hydroxycholesterol level (nanomoles per liter) was significantly increased in BRIC patients: 326 +/- 179 versus 171 +/- 90 in controls. These results suggest that in BRIC patients spillover of bile acids into the colon occurs, which leads to increased fecal bile acid loss and a reduced bile acid pool size. Increased serum 7 alpha-hydroxycholesterol is probably indicative of an accelerated bile acid synthesis rate due to increased activity of cholesterol 7 alpha-hydroxylase, the enzyme catalyzing the first step in the major pathway of bile acid synthesis. The results of our study suggest that in BRIC patients a contracted bile acid pool increases the susceptibility of the liver for cholestatic agents.

Adult↗
PubMed1987

Transport of cholesterol autoxidation products in rabbit lipoproteins.

Radiolabeled pure [4-14C]cholesterol was kept at 60 degrees C under air to autoxidize for 5 weeks, after which approximately 12% cholesterol oxidation products were formed. The mixture, suspended in gelatin, was given to rabbits by gastric gavage. Rabbits were killed 4, 24, and 48 h after treatment. Cholesterol and its autoxidation products were separated by thin-layer chromatography into 5 fractions and radioactivities of each fraction were measured. Percentages of each fraction of cholesterol oxidation products and cholesterol in the original mixture before administration and in the rabbit sera after administration were similar, suggesting that the rates of absorption of cholesterol oxidation products are not significantly different from that of cholesterol. Lipoproteins were fractionated by ultracentrifugation into VLDL, LDL and HDL. Radioactivities of each fraction in lipoproteins separated by thin layer chromatography showed that fractions containing cholestane-3 beta,5 alpha,6 beta-triol, 7 alpha- and 7 beta-hydroxycholesterol and 7-ketocholesterol were more selectively transported in VLDL, whereas most of the 25-hydroxycholesterol was present in LDL. HDL contained only minute amounts of cholesterol oxidation products.

Absorption↗
PubMed1996

The effect of cholesterol oxidation products on human platelet aggregation.

The cholesterol oxidation products (oxysterols) cholest-3,5-diene-7-one, cholestan-5 alpha, 6 alpha-epoxy-3 beta-ol (cholesterol 5 alpha-epoxide), cholestan-5 beta, 6 beta-epoxy-3 beta-ol (cholesterol 5 beta-epoxide), cholest-5-ene-3 beta-ol-7-one (7-ketocholesterol), cholest-5-ene-3 beta, 7 alpha-diol (7 alpha-hydroxycholesterol), cholestan-3 beta, 5 alpha, 6 beta-triol (cholestane triol), and cholest-5-ene-3 beta, 26-diol (27-hydroxycholesterol) potentiated platelet aggregation and increased thromboxane A2 formation in platelets challenged with thrombin, ADP or collagen. These effects were observed at oxysterol concentrations in the range 5-100 microM. Cholesterol 5 beta-epoxide and 7-ketocholesterol increased the mobilization of 3H-arachidonic acid from prelabelled platelet phospholipids in response to thrombin and collagen.

Adenosine Diphosphate↗
PubMed1988

Stimulation of progesterone production by phorbol-12-myristate-13-acetate in MA-10 Leydig tumor cells.

The tumor-promoting phorbol ester, phorbol-12-myristate-13-acetate (PMA) markedly stimulated progesterone production in MA-10 Leydig tumor cells. A slight but significant increase (35%) in the activity of the cholesterol side-chain cleavage (CSCC) enzyme was observed in mitochondria isolated from the PMA-treated MA-10 Leydig cells when compared to mitochondria isolated from non-treated cells. However, this stimulation of CSCC activity appears to be of limited importance when compared to the 240-fold increase observed in progesterone production following PMA stimulation. In contrast, the inactive phorbol ester 4 alpha-phorbol-12,13-didecanoate (alpha-PD) had no effect on either progesterone production or CSCC activity. PMA had no effect on the conversion of 25-hydroxycholesterol and 22R-hydroxycholesterol into progesterone suggesting that one of the mechanism(s) of PMA action may involve the delivery of cholesterol to the mitochondria and/or the affinity of cholesterol with cytochrome P-450scc. Stimulation of steroidogenesis by PMA was also shown to be inhibited by cycloheximide. When PMA was added together with a submaximal dose of hCG, hCG-stimulated steroidogenesis was inhibited. However, at a maximal dose of human chorionic gonadotropin (hCG), PMA inhibited steroid synthesis at 1 and 2 h but had no significant effect at 3 h. Conversely, PMA had an additive effect on cAMP induced steroidogenesis. It was further demonstrated that PMA resulted in a decrease in the hCG-induced accumulation of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Inhibition of hormonal-induced cAMP and steroid production by inhibitors of pregnenolone metabolism in adrenal and Leydig cells.

The effects of inhibitors of pregnenolone metabolism, WIN-24540 and spironolactone, on adrenocorticotropic hormone (ACTH)- and human chorionic gonadotropin (hCG)-induced cAMP and steroid production by bovine (BAC) and ovine (OAC) adrenal cells and pig Leydig cells (PLC) were investigated. The inhibitors reduced cAMP production by adrenal and Leydig cells by about 75% and 60%, respectively (P less than 0.001). Further, the inhibitors also reduced the cholera toxin- and forskolin-induced cAMP production by pig Leydig cells. In the presence of the inhibitors, corticosterone and testosterone production by BAC and PLC, respectively, following hormonal stimulation was reduced by more than 90%. However, pregnenolone production by BAC and PLC under these conditions represented only 12% and 42% of the corticosterone and testosterone production, respectively, in the absence of inhibitors. Moreover, the inhibitors also reduced the steroidogenic response of PLC to 8-Br-cAMP and the conversion of 22(R)-hydroxycholesterol to pregnenolone by BAC and PLC. The reduced production of pregnenolone in the presence of inhibitors was in part due to the weak inhibition of 17 alpha-hydroxylase by spironolactone. However, when OAC cells were incubated in the presence of WIN-24540 and SU-10603, a potent 17 alpha-hydroxylase inhibitor, the amount of pregnenolone produced in response to ACTH or 22(R)-hydroxycholesterol was only 10% and 19%, respectively, of the steroids (corticosterone plus cortisol) secreted in the absence of inhibitors. The results show that the inhibitors of pregnenolone metabolism reduced, in both adrenal and Leydig cells, the response of adenylate cyclase to several effectors and the activity of the cholesterol side-chain cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗
PubMed1992

Luteinizing hormone induction of the cholesterol side-chain cleavage enzyme in cultured immature rat Leydig cells: no role of insulin-like growth factor-I?

Long-term inductive effects of luteinizing hormone (LH) on cholesterol side-chain cleavage (CSCC) enzyme activity were studied, using cultured Leydig cells isolated from 21-day-old rats. Particular reference was given to the role of insulin-like growth factor-I (IGF-I) as an autocrine or paracrine modulator or as an essential extracellular mediator of LH action. The CSCC enzyme activity was measured using an excess of 22(R)-hydroxycholesterol as substrate to saturate the enzyme, and inhibitors of pregnenolone metabolism to concentrate all the products of the enzyme reaction in pregnenolone. The rate of sterol conversion into pregnenolone (CSCC enzyme activity) reflected the amount of cytochrome P-450scc (P-450scc), as was shown by Western blotting. In cells cultured without LH, the CSCC enzyme activity decreased to 10% on day 7 of the culture period. In the presence of various doses of LH ranging from 0.01 to 100 ng/ml, the CSCC enzyme activity also diminished during the first 3 days of culture, but during the following days, the amount of CSCC enzyme was stimulated by LH. In contrast to the absence of any LH effect on the activity of the CSCC enzyme during the first days of the culture, the endogenous steroid production (no added 22(R)-hydroxycholesterol) could be stimulated at least 10-fold by high doses of LH. When LH (1 ng/ml) was added to cells which had been cultured for 7 days without hormones, CSCC enzyme activity was elevated 8-fold after 4 days of exposure of LH. These effects of LH could be mimicked by dbcAMP (0.5 mM). No evidence could be provided that IGF-I plays any role in the LH induction of the CSCC enzyme; neither the addition of exogenous IGF-I or analogs that do not bind to IGF-I binding proteins (IGFBPs) nor the inactivation of endogenous IGF-I action (through binding to IGFBP and antibodies to IGF-I or via masking of IGF-I receptor by antibodies) could influence the LH induced CSCC enzyme activity. The present data raise the question under which conditions IGF-I is capable of modulating Leydig cell steroidogenesis.

Age Factors↗