Studies with insulin and insulin-like growth factor-I show that the increased labeling of phosphatidylinositol-3,4-bisphosphate is not sufficient to elicit the diverse actions of epidermal growth factor on MA-10 Leydig tumor cells

In a recent publication we showed that addition of mouse epidermal growth factor (mEGF) to MA-10 Leydig tumor cells rapidly leads to an increase in the incorporation of [3H]inositol-derived radioactivity into an unusual lipid that was identified as phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2). Other ligands that are known to bind to MA-10 cells, such as hCG and arginine vasopressin, however, did not elicit this effect. Inasmuch as mEGF modulates the differentiated functions of MA-10 cells in a number of ways, our findings raised the possibility that PI-3,4-P2 may be an intracellular mediator of these actions of mEGF. In an attempt to answer this question, we set out to determine if other ligands increase the labeling of PI-3,4-P2 in MA-10 cells prelabeled with [3H]inositol, and if such ligands mimic the diverse biological actions of mEGF on these cells. The experiments presented herein show that insulin, insulin-like growth factor-I, and transforming growth factor-alpha increase the labeling of PI-3,4-P2 in MA-10 cells, but only transforming growth factor-alpha mimics the actions of mEGF on the differentiated functions of MA-10 cells. We conclude that an increase in the labeling of PI-3,4-P2 is not sufficient to elicit these actions of mEGF.     

Anti-phosphotyrosine immunoprecipitation of phosphatidylinositol 3' kinase activity in different cell types after exposure to epidermal growth factor.

In previous studies from this laboratory, it was shown that mouse epidermal growth factor (mEGF) or insulin increased the labeling of phosphaditylinositol-3,4-bisphosphate (PI-3,4-P2) in MA-10 cells prelabeled with different radioactive precursors (Pignataro, O.P., and Ascoli, M. (1990) J. Biol. Chem. 265, 1718-1723 and Mol. Endocrinol. (1990) 4, 758-765). In order to further characterize this phenomenon we sought to determine if we could use anti-phosphotyrosine antibodies to immunoprecipitate a phosphatidylinositol (PI) kinase activity from MA-10 cells treated with mEGF or insulin. Our data indicate that this is indeed the case, and that the PI kinase precipitated is a PI-3' kinase. A second cell type, A431 cells, in which we were unable to detect an increase in PI-3,4-P2 labeling when stimulated with mEGF or insulin, was also studied. It was found that, as in MA-10 cells, A431 cells also contain an immunoprecipitable PI-3' kinase activity that is increased in response to mEGF or insulin.     

The inositol phosphate/diacylglycerol pathway in MA-10 Leydig tumor cells. Activation by arginine vasopressin and lack of effect of epidermal growth factor and human choriogonadotropin

It is now well established that mouse epidermal growth factor (mEGF) modulates the hormonal responsiveness of MA-10 Leydig tumor cells but does not affect cell multiplication. The studies presented herein are the first in a series of experiments designed to characterize the intracellular signaling systems activated by mEGF and their possible roles in mediating the diverse biological actions of this growth factor in MA-10 cells. We show that (i) MA-10 cells express a hormone-sensitive inositol phosphate/diacylglycerol pathway that can be stimulated with arginine vasopressin (AVP), (ii) mEGF does not activate this pathway, and (iii) activation of this pathway with arginine vasopressin does not mimic the biological actions of mEGF. Other data presented show that lutropin/choriogonadotropin, the principal endocrine regulators of Leydig cell function, also do not stimulate the inositol phosphate/diacylglycerol pathway in MA-10 cells.     

Regulation of gonadotropin receptors and gonadotropin responses in a clonal strain of Leydig tumor cells by epidermal growth factor

The MA-10 line is a clonal strain of Leydig tumor cells that has receptors for human choriogonadotropin (hCG) and mouse epidermal growth factor (mEGF). These cells respond to hCG, cholera toxin, and 8-Br-adenosine 3':5'-monophosphate with increased steroid production. It is reported herein that exposure of the MA-10 cells to mEGF results in a substantial (80 to 90%) reduction in the number of hCG receptors per cell. The loss of hCG receptors is accompanied by a corresponding reduction in the ability of hCG to stimulate steroidogenesis. The steroidogenic responses to cholera toxin and 8-Br-adenosine 3':5'-monophosphate, however, are not affected. Other results presented show that mEGF is not a mitogen for these cells.     

Inhibition of gonadotropin-responsive adenylate cyclase in MA-10 Leydig tumor cells by epidermal growth factor

We have previously shown that mouse epidermal growth factor (mEGF) attenuates the increase in intracellular cAMP provoked by human choriogonadotropin (hCG) in MA-10 Leydig tumor cells (Ascoli, M., Euffa, J., and Segaloff, D. L. (1987) J. Biol. Chem. 262, 9196-9203). The studies presented herein were designed to investigate the mechanism(s) responsible for this phenomenon. We show that mEGF attenuates the increase in cAMP accumulation provoked by hCG primarily, if not entirely, by inhibiting adenylate cyclase activity. This phenomenon has some specificity for the agonist used, but it is not cell-specific. Thus, mEGF inhibited hCG-activated adenylate cyclase in MA-10 cells and in rat luteal cells but had no effect on the forskolin-activated enzyme in MA-10 cells or the isoproterenol-activated enzyme in rat luteal cells.     

On the mechanisms involved in the regulation of the cell-surface receptors for human choriogonadotropin and mouse epidermal growth factor in cultured Leydig tumor cells

The MA-10 cells are a clonal strain of mouse Leydig tumor cells that have receptors for human choriogonadotropin (hCG) and mouse epidermal growth factor (mEGF). Exposure of the cells to hCG results in a reduction in the number of surface hCG receptors, and little or no change in the number of surface mEGF receptors. On the other hand, exposure of the cells to mEGF results in a reduction in the number of both surface mEGF receptors and surface hCG receptors. In order to study these phenomena, we assumed that the number of surface receptors is determined by the rate at which receptors appear at the surface and by the rate of receptor internalization. When these rates were measured, we found that hCG and mEGF reduce their respective surface receptors by increasing the rate of receptor internalization, and that mEGF reduces the surface hCG receptors by decreasing the rate of appearance of the receptor.     

Epidermal growth factor activates steroid biosynthesis in cultured Leydig tumor cells without affecting the levels of cAMP and potentiates the activation of steroid biosynthesis by choriogonadotropin and cAMP

The studies presented herein were designed to investigate the effects of mouse epidermal growth factor (mEGF) on steroid biosynthesis in a clonal strain of cultured murine Leydig tumor cells (designated MA-10). We show that in short-term incubations (up to 8 h), mEGF activates steroid biosynthesis without affecting cAMP levels. The maximal activation of steroid biosynthesis by mEGF (about 10-fold) is, however, much lower than the maximal activation detected with human choriogonadotropin (hCG) or cAMP analogues (about 1000-fold). We also show that mEGF has two (opposing) effects on the activation of steroidogenesis by hCG. Initially, it transiently attenuates the increase in intracellular cAMP and steroid biosynthesis provoked by submaximal concentrations of hCG. At later times, however, it potentiates the stimulatory effects of submaximal concentrations of hCG on steroid biosynthesis in a synergistic fashion. Last, we show that mEGF and submaximal concentrations of cAMP analogues also activate steroidogenesis in a synergistic fashion and that the degree of synergism attained with cAMP analogues plus mEGF is much higher than that attained with hCG plus mEGF. Taken together, our results show that mEGF (i) activates steroidogenesis without affecting cAMP levels and (ii) modulates the activation of steroidogenesis by the cAMP second messenger system.     

An in vitro cell model system to study the action of retinoids on Leydig cell steroidogenesis

In this study we examined the effects of retinol and retinoic acid on steroid production in MA-10 mouse Leydig tumor cells. Results showed that both retinol and retinoic acid greatly increased progesterone production in this cloned cell line. The stimulatory effect of retinoids is not inhibited by cycloheximide suggesting that de novo protein synthesis is not required. The presence of the retinoid binding proteins CRBP and CRABP could not be detected in MA-10 Leydig cell cytosol indicating that the stimulatory action of retinoids on progesterone production is not mediated through these cellular binding proteins. Both previous and present findings suggest that retinoids play an important role in the regulation of Leydig cell steroidogenesis and that MA-10 Leydig tumor cells may represent an ideal in vitro cell system to study the mechanism of action of retinoids in Leydig cell steroidogenesis.     

Endocytosis, recycling, and degradation of the insulin receptor. Studies with monoclonal antireceptor antibodies that do not activate receptor kinase

The endocytosis, recycling, and degradation of the insulin receptor were studied in IM-9 cells and U-937 cells by employing two monoclonal antibodies directed at the alpha subunit of the human insulin receptor, antibodies MA-5 and MA-10. Antibody MA-5 is an insulin agonist and MA-10 is an insulin antagonist (Forsayeth, J., Caro, J.F., Sinha, M.K., Maddux, B.A., and Goldfine, I.D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 3448-3451). Both monoclonal antibodies, like insulin, induced the endocytosis of the insulin receptor within 15 min. Upon removal of extracellular ligand the internalized receptor recycled to the cell surface. At this time there was no degradation of the receptor as measured by a sensitive insulin receptor radioimmunoassay. After 20 h of incubation, insulin and MA-5, but not MA-10, induced significant receptor degradation as measured by both insulin receptor radioimmunoassay and metabolic labeling studies. These studies demonstrated, therefore, that: 1) internalization and recycling of the receptor can be induced by antireceptor monoclonal antibodies that are either insulin agonists or insulin antagonists; 2) enhanced receptor degradation can be induced by monoclonal antibodies that are insulin agonists; and 3) the process of receptor internalization does not necessarily lead to enhanced receptor degradation. Since prior studies have indicated that neither MA-5 nor MA-10 enhance insulin receptor kinase activity, the present studies also suggest that insulin receptor endocytosis and degradation induced by ligands different than insulin can occur without activation of this process.     

The inhibitory effects of lead on steroidogenesis in MA-10 mouse Leydig tumor cells

Lead is an environmental and occupational pollutant. It has been reported that lead affects the male reproductive system in humans and animals. However, the cellular mechanism of the adverse effect of lead on Leydig cell steroidogenesis remains unknown. To clarify whether lead has a direct effect on Leydig cells and how lead affects Leydig cells, MA-10 cells, a mouse Leydig tumor cell line, were exploited in this study. Lead acetate significantly inhibited hCG- and dbcAMP-stimulated progesterone production in MA-10 cells at 2 h. Steroid production stimulated by hCG or dbcAMP were reduced by lead. The mechanism of lead in reducing MA-10 cell steroidogenesis was further investigated. The expression of Steroidogenic Acute Regulatory (StAR) protein and the activities of P450 side-chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) enzymes were detected. Cells were treated with dbcAMP, 22R-hydroxycholesterol or pregnenolone alone or in combination with lead acetate ranging from 10(-8) to 10(-5) M for 2 h. The expression of StAR protein stimulated by dbcAMP was suppressed by lead at about 50%. Progesterone productions treated with 22R-hydroxycholesterol or pregnenolone were reduced 30-40% in lead-treated MA-10 cells. These data suggest that lead directly inhibited steroidogenesis by decreasing StAR protein expression and the activities of P450scc and 3beta-HSD enzymes with a dose-response trend in MA-10 cells. Moreover, cadmium, a calcium channel blocker, abolished inhibitory effect of lead on MA-10 cell steroid production. This indicates that lead might act on calcium channel to regulate MA-10 cell steroidogenesis.     

Attenuation of cAMP-mediated responses in MA-10 Leydig tumor cells by genetic manipulation of a cAMP-phosphodiesterase.

In order to assess the effect of increased cAMP degradation on the responsiveness on an endocrine cell, we have obtained stable transfectants of MA-10 Leydig tumor cells that overexpress a mammalian cAMP-phosphodiesterase. Two novel cell lines, designated MA-10(P+8) and MA-10(P+29), that express high levels of the transfected enzyme were characterized. Although the basal levels of cAMP in the mutant cell lines are comparable to those of the wild-type cells, the increase in cAMP accumulation elicited by human choriogonadotropin (hCG) is severely blunted. Further studies with MA-10(P+29) show that the ability of hCG to stimulate adenylyl cyclase activity is normal. The failure of MA-10(P+29) cells to accumulate cAMP in response to hCG can be correlated with a similar reduction in hCG-stimulated steroidogenesis. On the other hand, the maximal steroidogenic response of MA-10(P+29) cells to dibutyryl cAMP, a cAMP analogue that is fairly resistant to phosphodiesterase degradation, is normal. We also show that the ability of these cells to respond to hCG with increased cAMP accumulation and steroid synthesis can be restored with a specific phosphodiesterase inhibitor. These results demonstrate that overexpression of a cAMP-phosphodiesterase in MA-10 cells limits the levels of cAMP attained under hCG stimulation and supresses the steroidogenic response of these cells to hCG. Since gonadotropins increase the cAMP-phosphodiesterase activity in their target cells, these findings also provide evidence that this regulation plays a major role in the modulation of cell responsiveness. Last, these new cell lines should be valuable in the study of the actions of cAMP because they express a conditional and reversible cAMP-resistant phenotype.     

Cordyceps sinensis mycelium activates PKA and PKC signal pathways to stimulate steroidogenesis in MA-10 mouse Leydig tumor cells

Cordyceps sinensis (CS) mycelium stimulates steroidogenesis in MA-10 mouse Leydig tumor cells, but the mechanisms remain unclear. In this study, MA-10 cells were treated with different reagents in the presence or absence of CS (10 mg/ml) for 3 h to determine the mechanisms. Results illustrated that CS activated the Gsalpha protein subunit, but not Gialpha, to induce cell steroidogenesis. Moreover, PKA inhibitors inhibited 37% of CS-stimulated steroidogenesis, which demonstrated that CS might enhance the cAMP-PKA pathway to affect MA-10 cell steroidogenesis. Because of incomplete inhibition by PKA inhibitors, we also examined the PKC pathway. PKC inhibitor, phospholipase C inhibitor, and calmodulin antagonist blocked 35-52% of CS-stimulated steroidogenesis in MA-10 cells, strongly suggesting that CS had activated the PKC pathway. Co-treatment with PKA and PKC inhibitors abolished 61% of CS-stimulated steroid production, indicating that CS simultaneously activated PKA and PKC pathways. Moreover, CS induced the expression of steroidogenic acute regulatory (StAR) protein in dose- and time-dependent relationships, and PKA inhibitor, PKC inhibitor, or co-treatment with both inhibitors suppressed it. These data support that CS activates both PKA and PKC signal transduction pathways to stimulate MA-10 cell steroidogenesis.     

Role of the 6-20 disulfide bridge in the structure and activity of epidermal growth factor

Two synthetic analogues of murine epidermal growth factor, [Abu6, 20] mEGF4-48 (where Abu denotes amino-butyric acid) and [G1, M3, K21, H40] mEGF1-48, have been investigated by NMR spectroscopy. [Abu6, 20] mEGF4-48 was designed to determine the contribution of the 6-20 disulfide bridge to the structure and function of mEGF. The overall structure of this analogue was similar to that of native mEGF, indicating that the loss of the 6-20 disulfide bridge did not affect the global fold of the molecule. Significant structural differences were observed near the N-terminus, however, with the direction of the polypeptide chain between residues four and nine being altered such that these residues were now located on the opposite face of the main beta-sheet from their position in native mEGF. Thermal denaturation experiments also showed that the structure of [Abu6, 20] mEGF4-48 was less stable than that of mEGF. Removal of this disulfide bridge resulted in a significant loss of both mitogenic activity in Balb/c 3T3 cells and receptor binding on A431 cells compared with native mEGF and mEGF4-48, implying that the structural changes in [Abu6, 20] mEGF4-48, although limited to the N-terminus, were sufficient to interfere with receptor binding. The loss of binding affinity probably arose mainly from steric interactions of the dislocated N-terminal region with part of the receptor binding surface of EGF. [G1, M3, K21, H40] mEGF1-48 was also synthesized in order to compare the synthetic polypeptide with the corresponding product of recombinant expression. Its mitogenic activity in Balb/c 3T3 cells was similar to that of native mEGF and analysis of its 1H chemical shifts suggested that its structure was also very similar to native.     

The lutropin/choriogonadotropin receptor-induced phosphorylation of the extracellular signal-regulated kinases in leydig cells is mediated by a protein kinase a-dependent activation of ras

The pathways involved in activation of the ERK1/2 cascade in Leydig cells were examined in MA-10 cells expressing the recombinant human LH receptor (hLHR) and in primary cultures of rat Leydig cell precursors. In MA-10 cells expressing the recombinant hLHR, human choriogonadotropin-induced activation of ERK1/2 is effectively inhibited by overexpression of a cAMP phosphodiesterase (a manipulation that blunts the human choriogonadotropin-induced cAMP response), by addition of H89 (a selective inhibitor of protein kinase A), or by overexpression of the heat-stable protein kinase A inhibitor, but not by overexpression of an inactive mutant of this inhibitor. Stimulation of hLHR did not activate Rap1, but activated Ras in an H89-sensitive fashion. Addition of H89 to MA-10 cells that had been cotransfected with a guanosine triphosphatase-deficient mutant of Ras almost completely inhibited the hLHR-mediated activation of ERK1/2. We also show that 8-bromo-cAMP activates Ras and ERK1/2 in MA-10 cells and in primary cultures of rat Leydig cells precursors in an H89-sensitive fashion, whereas a cAMP analog 8-(4-chloro-phenylthio)-2'-O-methyl-cAMP (8CPT-2Me-cAMP) that is selective for cAMP-dependent guanine nucleotide exchange factor has no effect. Collectively, our results show that the hLHR-induced phosphorylation of ERK1/2 in Leydig cells is mediated by a protein kinase A-dependent activation of Ras.     

Insulin-stimulated phosphoinositide metabolism in isolated fat cells

Treatment of isolated fat cells with insulin produced increases of up to 4.8-fold in the incorporation of [3H]inositol into phosphatidylinositol. This effect of insulin was both time- and dose-dependent with half-maximal stimulation at 30 microunits/ml of insulin. Insulin increased the labeling of phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate but not phosphatidylinositol 4-monophosphate in cells which had been preincubated with [3H]inositol for 90 min. Incubation of the cells in a Ca2+-free buffer increased the basal level of phosphatidylinositol labeling and enhanced the effect of insulin. Glucagon and isoprenaline, both of which stimulate lipolysis, had no effect on phosphatidylinositol labeling but did potentiate insulin-stimulated incorporation of [3H]inositol into phosphatidylinositol. Phosphoinositide breakdown was measured by the accumulation of inositol phosphates. Insulin did not increase the level of the inositol phosphates at all concentrations of the hormone tested. By comparison, phenylephrine and vasopressin were able to stimulate phosphoinositide breakdown. Pretreatment of the cells with insulin enhanced the effect of phenylephrine on inositol phosphates' accumulation, suggesting that insulin may potentiate phenylephrine-mediated phosphoinositide turnover. From these data we conclude that insulin stimulates the de novo synthesis of phosphatidylinositol and phosphatidylinositol 4,5-biphosphate, but has no effect on phosphoinositide breakdown.     

Swiss 3T3 cells preferentially incorporate sn-2-arachidonoyl monoacylglycerol into sn-1-stearoyl-2-arachidonoyl phosphatidylinositol.

The sn-1-stearoyl-2-arachidonoyl phospholipids of animal cells appear to be formed by special mechanisms. To determine whether monoacylglycerol (MG) incorporation pathways are involved we incubated quiescent Swiss 3T3 cells with [3H]glycerol-labeled sn-2-arachidonoyl MG, then analyzed the radioactive cell lipids that accumulated. We also examined cell homogenates to identify enzyme activities that might promote the incorporation of sn-2-arachidonoyl MG into other cell lipids. The cell incubation experiments demonstrated rapid labeling of several lipids, including diacylglycerol, lysophosphatidic acid, phosphatidic acid, and phosphatidylinositol. They also demonstrated selective labeling of sn-1-stearoyl-2-arachidonoyl species of phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine. The cell homogenate experiments identified an sn-2-acyl MG acyltransferase activity, an MG kinase activity that phosphorylates sn-2-arachidonoyl MG in preference to sn-2-oleoyl MG, and a stearoyl-specific acyl transferase activity that converts sn-2-arachidonoyl lysophosphatidic acid into sn-1-stearoyl-2-arachidonoyl phosphatidic acid. The results also showed that this stearoyl transferase could act with other enzymes to convert sn-2-arachidonoyl lysophosphatidic acid into sn-1-stearoyl-2-arachidonoyl phosphatidylinositol. The combined results indicate that Swiss 3T3 cells incorporate sn-2-arachidonoyl MG into phospholipids by at least two different pathways, including one that specifically forms sn-1-stearoyl-2-arachidonoyl phosphatidylinositol.     

Mitochonic acid-5 attenuates TNF-α-mediated neuronal inflammation via activating Parkin-related mitophagy and augmenting the AMPK–Sirt3 pathways

Mitochondrial dysfunction has been found to be associated with neuronal inflammation; however, no effective drug is available to attenuate neuroinflammation via sustaining mitochondrial function. In the current study, experiments were performed to understand the beneficial effects of mitochonic acid 5 (MA-5) on tumor necrosis factor-α (TNF-α)-mediated neuronal injury and mitochondrial damage. Our data illustrated that MA-5 pretreatment reduced inflammation response induced by TNF-α in CATH.a cells. Molecular investigations demonstrated that MA-5 pretreatment repressed oxidative stress, inhibited endoplasmic reticulum stress, sustained cellular energy metabolism, and blocked cell apoptosis induced by TNF-α stress. Further, we found that MA-5 treatment elevated the expression of Sirtuin 3 (Sirt3) and this effect was dependent on the activation of AMP-activated protein kinase (AMPK) pathway. Blockade of AMPK abolished the promotive action of MA-5 on Sirt3 and thus mediated mitochondrial damage and cell death. Besides, we also found that MA-5 treatment augmented Parkin-related mitophagy and increased mitophagy promoted CATH.a cells survival via improving mitochondrial function. Knockdown of Parkin abolished the beneficial action of MA-5 on mitochondrial homeostasis and CATH.a cell survival. Altogether, our results confirm that MA-5 is an effective drug to attenuate neuroinflammation via sustaining mitochondrial damage and promoting CATH.a cell survival. The protective action of MA-5 on neuronal damage is associated with Parkin-related mitophagy and the activation of AMPK–Sirt3 pathways.     

An extracellular domain of the insulin receptor beta-subunit with regulatory function on protein-tyrosine kinase

Anti-insulin receptor monoclonal antibody MA-10 inhibits insulin receptor autophosphorylation of purified rat liver insulin receptors without affecting insulin binding (Cordera, R., Andraghetti, G., Gherzi, R., Adezati, L., Montemurro, A., Lauro, R., Goldfine, I. D., and De Pirro, R. (1987) Endocrinology 121, 2007-2010). The effect of MA-10 on insulin receptor autophosphorylation and on two insulin actions (thymidine incorporation into DNA and receptor down-regulation) was investigated in rat hepatoma Fao cells. MA-10 inhibits insulin-stimulated receptor autophosphorylation, thymidine incorporation into DNA, and insulin-induced receptor down-regulation without affecting insulin receptor binding. We show that MA-10 binds to a site of rat insulin receptors different from the insulin binding site in intact Fao cells. Insulin does not inhibit MA-10 binding, and MA-10 does not inhibit insulin binding to rat Fao cells. Moreover, MA-10 binding to down-regulated cells is reduced to the same extent as insulin binding. In rat insulin receptors the MA-10 binding site has been tentatively localized in the extracellular part of the insulin receptor beta-subunit based on the following evidence: (i) MA-10 binds to insulin receptor in intact rat cells; (ii) MA-10 immunoprecipitates isolated insulin receptor beta-subunits labeled with both [35S]methionine and 32P; (iii) MA-10 reacts with rat insulin receptor beta-subunits by the method of immunoblotting, similar to an antipeptide antibody directed against the carboxyl terminus of the insulin receptor beta-subunit. Moreover, MA-10 inhibits autophosphorylation and protein-tyrosine kinase activity of reduced and purified insulin receptor beta-subunits. The finding that MA-10 inhibits insulin-stimulated receptor autophosphorylation and reduces insulin-stimulated thymidine incorporation into DNA and receptor down-regulation suggests that the extracellular part of the insulin receptor beta-subunit plays a role in the regulation of insulin receptor protein-tyrosine kinase activity.     

Effects of amphetamine on steroidogenesis in MA-10 mouse Leydig tumor cells

Amphetamine influences plasma and testicular testosterone levels. However, there is no evidence that amphetamine can directly influence Leydig cell functions. In the present study, a MA-10 mouse Leydig tumor cell line was used to determine whether and how amphetamine affected Leydig cell steroidogenesis. MA-10 cells were treated with different concentrations of amphetamine without or with human chorionic gonadotropin (hCG) and/or enzyme precursors over different time durations. Steroid production, enzyme activities and StAR protein expression were determined. Amphetamine alone had no any effect on MA-10 cell steroidogenesis. However, amphetamine (10(-11)M and 10(-10)M) significantly enhanced hCG-treated progesterone production at 3 hr in MA-10 cells (p < 0.05). Furthermore, amphetamine significantly induced more progesterone production upon treatment with 22R-hydroxycholesterol (p < 0.05), a precursor of P450 side-chain cleavage enzyme (P450scc). However, amphetamine did not induce more progesterone production when treated with pregnenolone (p > 0.05), a precursor of 3beta-hydroxysteroid dehydrogenase. In addition, the expressions of StAR protein and P450scc enzyme were not significantly different between hCG alone and hCG plus amphetamine treatment in MA-10 cells (p > 0.05). These results suggested that amphetamine enhanced hCG-induced progesterone production in MA-10 cells by increasing P450scc activity without influencing StAR protein and P450scc enzyme expression or 3beta-HSD enzyme activity.     

Ceramide increases steroid hormone production in MA-10 Leydig cells.

Ceramide is known to have major roles in the control of cell proliferation, differentiation, and apoptosis. Recent studies also have shown that ceramide affects steroid production by JEG-3 choriocarcinoma cells, acutely dispersed rat Leydig cells, and ovarian granulosa cells, but the mechanism by which this occurs is unknown. Because ceramide induces apoptosis in many different cell types, we hypothesized that ceramide might affect steroidogenesis and/or induce apoptosis in MA-10 murine Leydig cells. To test this, MA-10 cells were incubated with either the water soluble C2-ceramide, (N-acetyl-sphingosine, 0.01-10 cm); bacterial sphingomyelinase (1-100 mU/ml); or C2-dihydroceramide (N-acetyl-sphinganine, 0.1-10 microM). The data show that N-acetyl-sphingosine significantly increased basal (0.87 +/- 0.2 vs. 0.42 +/- 0.09 ng/mg cell protein, P < 0.01) and human chorionic gonadotropin (hCG) stimulated progesterone (P) synthesis (204 +/- 12 vs. 120 +/- 5 ng/mg cell protein, P < 0.001); as did sphingomyelinase (basal P = 0.83 +/- 0.1 ng/mg cell protein, P < 0.01; hCG stimulated P = 173 +/- 7 ng/mg cell protein, P < 0.001). C2-dihydroceramide also increased basal P synthesis but was less effective than ceramide on a molar basis. Neither sphingomyelinase (100 mU/ml) nor ceramide (10 microM) had any effect on cAMP production or human chorionic gonadotropin binding; and neither induced any signs of apoptosis (FragEL DNA fragmentation assay and electron microscopy). Cells incubated with anti-Fas (300 ng/ml) demonstrated DNA fragmentation, nuclear condensation, and frequent apoptotic bodies, but had no change in P synthesis. These data show that ceramide significantly increases MA-10 Leydig cell P synthesis but does not induce apoptosis. The mechanism by which ceramide increases steroid hormone synthesis remains unknown but does not appear to be linked to the induction of apoptosis in MA-10 cells.