Effect of mitomycin C on specific gene transcript levels in cultured mammalian cells

The effect of mitomycin C on the accumulation of specific mRNAs was studied in asynchronously growing Swiss 3T3 cells, as well as in synchronously growing serum stimulated ts13 cells (a temperature-sensitive mutant from the BHK cell line). It was observed that the steady-state level of p53 RNA experienced some increase in 3T3 cells treated for 24 h with the drug. In addition, mitomycin when applied to serum stimulated ts13 cells increased the level of p2F1 RNA. Mitomycin diminished the level of core histone H3 RNA, a finding consistent with the inhibitory action of this compound on DNA replication.     

Glutathione metabolism in cultured Sertoli cells and spermatogenic cells from hamsters

Isolated spermatocytes and spermatids from hamsters contained a large amount of glutathione (GSH) (approximately 40 and 30 nmol GSH/mg protein, respectively), but showed a spontaneous decrease of GSH content during prolonged incubation (t1/2 approximately 35 h). Incubation of the germ cells in the presence of the glutathione biosynthesis inhibitor buthionine sulphoximine (BSO) provided evidence that the cells can perform glutathione synthesis. This synthesis, however, was not sufficient to maintain the GSH content of the isolated cells, or to restore the cellular GSH pool after depletion caused by exposure of the cells to the glutathione S-transferase substrate, diethyl maleate (DEM). Cultured Sertoli cells, containing approximately 10 nmol GSH/mg protein, had a more active BSO-sensitive GSH synthesis system. The Sertoli cells, but also tubule fragments containing Sertoli cells and germ cells, were able to restore their GSH pool after DEM-induced depletion. DEM treatment of the tubule fragments resulted in a 90% decrease of the GSH content of the spermatocytes and spermatids present within the fragments. The GSH levels of the tubule fragments and the enclosed germ cells were restored during a subsequent incubation in the absence of DEM. As indicated above, such a recovery was not observed for isolated spermatocytes and spermatids. The results illustrate the importance of Sertoli cell-germ cell interaction, and point to a role of Sertoli cells in glutathione synthesis by the germ cells.     

In vitro metabolism of testosterone by cultured Sertoli cells and the effect of FSH.

Cultures of Sertoli cells isolated from testes of 18-and 36-day-old Long Evans rats were used to investigate their capacity to metabolize testosterone and the effect of FSH on such metabolism. Three different approaches were used: 1) investigation of the metabolism of radiolabeled testosterone under saturating substrate conditions; 2) study of the metabolism of radiolabeled testosterone utilizing trace amounts of high specific activity substrates; 3) the utilization of radioimmunoassay for measurement of estradiol-17 beta. The following steroids were isolated and identified by recrystallization to constant specific acitvity from the control and FSH-treated cultures; testosterone (unconverted substrate), androstenedione, dihydrotestosterone, 3 alpha-hydroxy-5 alpha-androstan-17-one and 5 alpha-androstane-3 alpha, 17 beta-diol. Radioimmunoassay data suggests that the Sertoli cells produce an estradiol-17 beta-like compound from unlabeled testosterone and that this production is stimulated by FSH. However, the radioactive metabolite from all our studies that behaved chromatographically like estradiol--17 beta failed to crystallize to constant specific activity, while in each experiment, authentic radiolabeled estradiol-17 beta added as recovery tracer did. The data demonstrate that : 1) cultures of Sertoli cells from immature rats have 5 alpha-reductase, 3 alpha- and 17 beta-hydroxysteroid oxidoreductase activities; 2) these enzymes may be affected by FSH; 3) based on radiolabeled metabolic techniques, Sertoli cells were unable to biotransform testosterone to estradiol-17 beta even in the presence of FSH.     

Effect of insulin on low-density-lipoprotein metabolism in human lymphocytes in vitro.

The metabolism of low-density lipoproteins (LDL) in vitro in the presence of insulin was studied in freshly isolated human peripheral-blood lymphocytes. Insulin appeared to decrease the binding affinity of 125I-LDL to its cell-surface receptor, without any change in apparent Vmax or in the number of LDL receptors. As a consequence, the absolute amounts of 125I-LDL internalized and degraded were lower in the presence of insulin than in its abscence, although the fraction of internalized 125I-LDL degraded in either instance was quite similar. 3-Hydroxy-3-methylglutaryl-CoA reductase activity, and hence cholesterol synthesis, were stimulated by insulin. This effect of insulin was independent of the inhibitory effect of LDL on cholesterol synthesis. At the same time, acid cholesterol esterase and acyl-CoA: cholesterol O-acetyltransferase activities were lower in cells incubated with insulin than in controls. The net effect of these metabolic alterations seems to be that cells accumulate greater quantities of free and esterified cholesterol when treated with insulin.     

Effect of hormones on cyclic AMP levels in cultured human cells.

Cultured cells derived from human adipose tissue grew more slowly and had significantly higher basal levels of cyclic AMP than cultured fibroblasts. Cyclic AMP levels in cultured adipose tissue cells were unaffected by epinephrine and were elevated 15-fold by prostaglandin E₁ while fibroblast cyclic AMP levels were elevated 27-fold by epinephrine and 95-fold by prostaglandin E₁. These results support the postulate that the cultured adipose tissue cell is a distinct cell type which may represent an adipocyte or preadipocyte in culture.     

Uptake and metabolism of retinol in cultured Sertoli cells: evidence for a kinetic model

When cultured Sertoli cells derived from 20-day-old weanling rats were supplied [3H]retinol bound to serum retinol binding protein-transthyretin complex, [3H]retinol was rapidly incorporated and [3H]retinyl esters were synthesized. Within 28 h after administration, 83% of the labeled retinoids were accounted for as retinyl esters (64% as retinyl palmitate). Sertoli cells derived from vitamin A deficient rats and supplied [3H]retinol in culture under identical conditions likewise incorporated [3H]retinol and synthesized retinyl esters. In contrast to normal Sertoli cells, vitamin A deficient Sertoli cells eventually metabolized virtually all of the cellular [3H]retinol to retinyl esters. The primary metabolic fate of retinol administered to Sertoli cell cultures was the synthesis of retinyl esters under all conditions tested. However, administration of [3H]retinol bound to serum retinol binding protein gave metabolic profiles having a higher proportion of retinyl esters and lower proportions of unresolved polar material than administration of [3H]retinol alone. The kinetics of retinol uptake and intracellular retinyl ester synthesis in cultured Sertoli cells was complex. An initial, rapid phase of [3H]retinol incorporation lasting 30 min was followed by a slower rate of incorporation and a concomitant decrease in the intracellular concentration of [3H]retinol. During the time course the specific activity of [3H]retinyl palmitate eventually exceeded that of intracellular [3H]retinol. These observations suggest that two intracellular pools of retinol may exist in Sertoli cells.     

Immunosuppressive molecules produced by Sertoli cells cultured in vitro: biological effects on lymphocytes.

In the present study we have analyzed the proteins secreted in vitro by murine Sertoli cells to identify immunosuppressive factors. Our data show that Sertoli cells secrete molecules capable to inhibit proliferation of lymphocytes activated in vitro. Cytophluorimetric analysis indicates that treated cells are arrested in the G1 phase of cell cycle. The inhibitory activity is specific for both B or T lymphocytes but not for other non-lymphoid cells and is associated to proteins, heat and freeze stable, with Mr of more than 30 kDa. Lymphocytes treated with Sertoli immunosuppressive proteins drastically reduce the secretion of interleukin-2.     

Effects of troglitazone on cellular differentiation, insulin signaling, and glucose metabolism in cultured human skeletal muscle cells

To determine the immediate effect of thiazolidinediones on human skeletal muscle, differentiated human myotubes were acutely (1 day) and myoblasts chronically (during the differentiation process) treated with troglitazone (TGZ). Chronic TGZ treatment resulted in loss of the typical multinucleated phenotype. The increase of muscle markers typically observed during differentiation was suppressed, while adipocyte markers increased markedly. Chronic TGZ treatment increased insulin-stimulated phosphatidylinositol (PI) 3-kinase activity and membranous protein kinase B/Akt (PKB/Akt) Ser-473 phosphorylation more than 4-fold. Phosphorylation of p42/44 mitogen-activated protein kinase (42/44 MAPK/ERK) was unaltered. Basal glucose uptake as well as both basal and insulin-stimulated glycogen synthesis increased approximately 1.6- and approximately 2.5-fold after chronic TGZ treatment, respectively. A 2-fold stimulation of PI 3-kinase but no other significant TGZ effect was found after acute TGZ treatment. In conclusion, chronic TGZ treatment inhibited myogenic differentiation of that human muscle while inducing adipocyte-specific gene expression. The effects of chronic TGZ treatment on basal glucose transport may in part be secondary to this transdifferentiation. The enhancing effect on PI 3-kinase and PKB/Akt involved in both differentiation and glycogen synthesis appears to be pivotal in the cellular action of TGZ.     

Metabolic modulation induced by oestradiol and DHT in immature rat Sertoli cells cultured in vitro

Sertoli cells actively metabolize glucose that is converted into lactate, which is used by developing germ cells for their energy metabolism. Androgens and oestrogens have general metabolic roles that reach far beyond reproductive processes. Hence, the main purpose of this study was to examine the effect of sex hormones on metabolite secretion/consumption in primary cultures of rat Sertoli cells. Sertoli cell-enriched cultures were maintained in a defined medium for 50?h. Glucose and pyruvate consumption, and lactate and alanine secretion were determined, by 1H-NMR (proton NMR) spectra analysis, in the presence or absence of 100?nM E2 (17β-oestradiol) or 100?nM 5α-DHT (dihydrotestosterone). Cells cultured in the absence (control) or presence of E2 consumed the same amount of glucose (29±2?pmol/cell) at similar rates during the 50?h. After 25?h of treatment with DHT, glucose consumption and glucose consumption rate significantly increased. Control and E2-treated cells secreted similar amounts of lactate during the 50?h, while the amount of lactate secreted by DHT-treated cells was significantly lower. Such a decrease was concomitant with a significant decrease in LDH A [LDH (lactate dehydrogenase) chain A] and MCT4 [MCT (monocarboxylate transporter) isoform 4] mRNA levels after 50?h treatment in hormonally treated groups, being more pronounced in DHT-treated groups. Finally, alanine production was significantly increased in E2-treated cells after 25?h treatment, which indicated a lower redox/higher oxidative state for the cells in those conditions. Together, these results support the existence of a relation between sex hormones action and energy metabolism, providing an important assessment of androgens and oestrogens as metabolic modulators in rat Sertoli cells.     

Cobalamin metabolism in cultured human chorionic villus cells

Cobalamin (Cbl, vitamin B₁₂) metabolism was analyzed in cultures of human chorionic villus (CV) cells obtained at 9–10 weeks of gestation. CV cells were shown to synthesize transcobalamin II (TCII) and to possess a high affinity receptor for that molecule. The cells bound and internalized radioactive cyanocobalamin (CN[⁵⁷Co]Cbl) complexed to TCII. This internalized CN[⁵⁷Co]Cbl was found to be converted to both methylCbl and adenosylCbl, the two intracellular coenzyme forms of Cbl, and bound to the two known intracellular Cbl requiring enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase. Both enzyme systems were found to be functional in the intact cell by demonstrating the incorporation of the radioactive label from both [¹⁴C]CH₃-tetrahydrofolate and [¹⁴C]propionate into acid insoluble products. MS activity was also detected in lysed cell material. CV cells were shown not to be auxotrophic for methionine since they were able to utilize homocysteine in place of methionine for cell division. Since CV cells are capable of performing many of the complex events associated with Cbl metabolism, it may be possible to use these cells to diagnose genetic defects of Cbl metabolism. © 1993 Wiley-Liss, Inc.     

Effect of hormones on phospholipid metabolism in human cultured fibroblasts

The effect of hormones on phospholipid metabolism, pool size, 32P labeling and changes in fatty acid of human adult fibroblasts was determined. Simultaneously the change in membrane fluidity of single cells was recorded via fluorescence recovery after photobleaching under the influence of hormones. From all substances tested (isoproterenol, phenylephrine, adrenalin, histamine, angiotensin II, dansylcadaverine, propranolol) only isoproterenol and adrenalin slightly decreased total amount of phosphatidylcholine (PC). The amount of the other phospholipids analyzed remained unchanged. The 32P incorporation rate into phospholipids (PC, phosphatidylinositol (PI), phosphatidylethanolamine (PE)) was affected basicly different analyzing either PC, PI or PE. Histamine and propranolol provoked the highest incorporation of 32P (240% increase in PI labeling). Isoproterenol and adrenalin decreased PC labeling (45% and 18%) whereas isoproterenol decreased 32P incorporation into PI (18%), and adrenalin led to an increase (37%). PE labeling showed no or a slight increase in 32P incorporation applying the other agonists or antagonists. The fatty acid pattern of the respective phospholipids changed only to a minor extend. A decrease in hexadecanoic acid content of PI was found after administration of either isoproterenol, adrenalin or histamine. Parallel determination of membrane fluidity of single cells by fluorescence recovery after photobleaching showed an increase in the diffusion coefficient of a fluorescent lipid probe sticking in the membrane, following administration of isoproterenol and adrenalin, other substances tested exerted no effect. A relationship to changes in phospholipid metabolism became obvious. These results are discussed considering known mechanisms of receptor coupling and change in phospholipid metabolism and fluidity.     

Metabolism of palmitate in cultured rat Sertoli cells

Isolated rat Sertoli cells were incubated in the presence of [1-14C]palmitate at a cell concentration of 1.54 +/- 0.31 mg protein/flask (n = 7). The oxidation of palmitate was concentration dependent and maximal oxidation was obtained at 0.35 mM-palmitate. At a saturating concentration of palmitate the oxidation was linear for at least 6 h. About 65% of the total amount of palmitate oxidized during 5 h at 0.52 mM-palmitate (109 +/- 44 nmol/flask, n = 5) was recovered as CO2 and the rest as acid-soluble compounds. Almost all radioactive acid-soluble compounds which were secreted by the Sertoli cells were shown to be 3-hydroxybutyrate and acetoacetate. The palmitate recovery in cellular lipids and triacylglycerols was 9.4 +/- 5.1 nmol/flask (n = 5) and 3.5 +/- 2.8 nmol/flask (n = 5) respectively. Addition of glucose had no significant effect on palmitate oxidation but caused a 9-fold increase in esterification of palmitate into triacylglycerols. We conclude that cultured rat Sertoli cells can oxidize palmitate to CO2 and ketone bodies and that fatty acids appear to be a major energy substrate for these cells.     

Uptake and metabolism of polyprenols by animal cells cultured in vitro

Several mammalian, chicken, and mosquito cells grown in vitro take up tritiated dolichol supplied to the incubation medium. The extent of labelling varied markedly between different cell cultures. After 20 h incubation most of the dolichol taken up was unchanged and the major product of metabolism of dolichol was identified as its fatty acid esters. Green-monkey kidney cells were tested with 8 fully unsaturated and 6 alpha-saturated polyprenols ranging from C35 to C105. In general the uptake of alpha-saturated polyprenols (dolichol type, was higher. Considerable differences were found between the uptake of polyprenols of differing chain lengths. Less than 1% of the polyprenols taken up was converted into more polar product, mainly polyprenyl phosphates and polyprenyl phosphate sugars. The short-chain polyprenols, from C35 to C65, were metabolized more rapidly than the long-chain polyprenols, as judged from the amount of polar products and fatty acid esters of polyprenols.     

Effect of temperature on insulin binding and action in cultured human fibroblasts

Insulin stimulation of glycogen synthase activity and insulin binding were measured in fibroblast monolayers at 24, 32, and 37°C. Insulin stimulation of %$\text{I}$ glycogen activity increased with increasing temperature. Maximum response was greater at 37°C than at 32°C, and half maximal stimulation required at 2.0 nM insulin at 37°C vs. 10nM at 32°C. Insulin stimulation of glycogen synthase was greater and somewhat faster at 37°C than at 32°C. No insulin effect was observed at 24°C. ¹²⁵I-insulin binding to monolayers became maximal in 15 min at 37°C, 60 min at 32°C, and 120 min at 24°C. However, insulin binding decreased with increasing temperature, and this decline was due to decreased numbers of receptors. Insulin binding and stimulation of glycogen synthase were comparable at 32°C, with half maxima at 10 nM, indicating no evidence of “spare” receptors. The data indicate that temperature effects on insulin binding and action in fibroblasts are not directly related. The results also suggest that a rate limiting step(s) of insulin action is temperature sensitive, and that this step is not insulin binding.