MITOCHONDRIAL GLUTAMINE AND GLUTAMATE METABOLISM IN HUMAN PLACENTA AND ITS POSSIBLE LINK WITH PROGESTERONE BIOSYNTHESIS

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Trophoblast Research 7:77-86,1993 MITOCHONDRIAL GLUTAMINE AND GLUTAMATE METABOLISM IN HUMAN PLACENTA AND ITS POSSIBLE LINK WITH PROGESTERONE BIOSYNTHESIS Jerzy Klimek, Wieslaw Makarewicz, Julian

1 Trophoblast Research 7:77-86,1993 MITOCHONDRIAL GLUTAMINE AND GLUTAMATE METABOLISM IN HUMAN PLACENTA AND ITS POSSIBLE LINK WITH PROGESTERONE BIOSYNTHESIS Jerzy Klimek, Wieslaw Makarewicz, Julian Swierczynski, Grazyna Bossy-Bukato, and Leon Zelewski Department of Biochemistry Medical School ul. Debinki Gdansk, Poland INTRODUCTION Glutamine seems to be the preferable substrate for energy production in highly proliferative tissues and glutamate is the only amino acid which shows a net uptake from the umbilical circulation into the placenta (Lemons et al., 1976). Placental mitochondria possess the ability to metabolize glutamine and glutamate to ammonia and lactate (Makarewicz and Swierczynski, 1982). Experimental data indicate that this pathway involves deamidation of glutamine to glutamate and deamination of glutamate to oxoglutarate. The carbon skeleton of glutamine can be converted via the tricarboxylic acid cycle to malate and further to pyruvate and lactate (glutaminolysis). Previous experiments revealed that human placental mitochondria contain the specific enzymes involved: glutaminase (EC ) and NAD(P)-dependent malic enzyme (EC ). Their kinetic properties have been investigated and the data on the effect of substrate concentration, ph, inorganic phosphate, and divalent cations were reported (Makarewicz and Swierczynski, 1988; Swierczynski et al., 1982; Swierczynski et al., 1987; Zolnierowicz et al., 1988). Mitochondria isolated from human term placenta contain an oxidative chain associated with cytochrome P-450 which is responsible for the cleavage of cholesterol side chain during the biosynthesis of progesterone (Ryan et al., 1966). This hydroxylase system shows a specific requirement for NADPH (Meigs and Ryan, 1968). Based on experiments with isolated mitochondria, three mechanisms have been proposed for the generation of intramitochondrial NADPH in human placenta: i) via energy-dependent transhydrogenase at the expense of NADH (Klimek et al.,!979); ii) by the action of mitochondrial NADP-dependent isocitrate dehydrogenase (EC ) (Klimek et al., 1976; Boguslawski et al., 1976; iii) through the mitochondrial NAD(P)-dependent malic enzyme (Swierczynski et al., 1985). It has been suggested that the stimulation of progesterone biosynthesis in human term placental mitochondria by malate results not only from the generation of NADPH by NAD(P)-dependent malic enzyme, but also from further metabolism of pyruvate to isocitrate which in turn is readily oxidized by NADP-dependent isocitrate dehydrogenase University of Rochester Press

78 Klimek et al. So far the role of amino acids metabolism (especially glutamine) in regulation of progesterone biosynthesis has not been established.

2 78 Klimek et al. So far the role of amino acids metabolism (especially glutamine) in regulation of progesterone biosynthesis has not been established. The possible regulatory link between mitochondrial glutamine and glutamate metabolism and the progesterone biosynthesis could lie in the supply of reducing equivalents (NADPH)produced by the NADP-dependent glutamate dehydrogenase (EC ), NAD(P)-dependent malic enzyme and NADP-dependent isocitrate dehydrogenase. The experiments reported in this paper have been designed to investigate if in placental mitochondria glutamine and glutamate metabolism can support progesterone biosynthesis. Materials MATERIALS AND METHODS NAD, NADP, L-glutamate, L-malate, 2-oxoglutarate, malonate, hydroxymalonate (tartronic acid), and oxamic acid were obtained from Sigma Chemical Co. (St. Louis, MO, USA). L-glutamine was from Koch-Light Laboratories (England). [4-14C] cholesterol (58 mci/mmol) and [3I-I] progesterone (12 Ci/mmol) were products of Radiochemical Centre Amersham (England). All other materials were of the highest analytical grade available from POCh, Gliwice (Poland). Preparation of Placental Mitochondria Human term placental mitochondria were prepared essentially as described previously (Swierczynski et al., 1976). Briefly, the tissue (approximately 100 g) was passed through the meat grinder and suspended in 200 ml medium containing: 0.25 M sucrose, 5 mm EDTA, and 10 mm Tris-HCl ph 7.4 The resulting mince was homogenized manually in a glass Potter-Elvehjem homogenizer with the telfon pestle. The homogenate was centrifuged at 500 x g for 5 minutes and the pellet discarded. The supernatant obtained was centrifuged again at 2300 x g for 1 minute and the pellet discarded. The supernatant was centrifuged at x g for 3 minutes. The pellet was suspended in about 70 ml of 0.25 M sucrose buffered with 10 mbt Tris-HCl ph 7.4 and resulting suspension was centrifuged at 500 x g for 5 minutes, the small pellet was discarded and the supernatant centrifuged at 7000 x g for 10 minutes. The pellet so obtained was washed once with about 20 ml of 0.25 M sucrose containing 2 mm potassium phosphate ph 7.8 and centrifuged at 7000 x g for 10 minutes. The final pellet was suspended in 0.25 M sucrose buffered with 2 mm potassium phosphate ph 7.8 to obtain 40 mg of mitochondral protein per ml. Glutaminase Assay All experiments were carried out on disrupted mitochondria. The disruption was achieved by freezing at -15~ and thawing of the mitochondrial suspension. The incubation was carried out at 30~ for 30 minutes in 1 ml medium containing different concentration of potassium phosphate buffer ph 8.2 (Figure 2) or 50 mm potassium phosphate buffer ph 6.0 to 10.0 (Figure 3), 20 mm L-glutamine and 4 mg of mitochondrial protein (0.1 ml mitochondrial suspension in 0.25 M sucrose + 2 mm potassium phosphate ph 7.8). Appropriate controls (containing 0.1 ml of 0~25 M sucrose + 2 mm potassium phosphate ph 7.8 instead of mitochondrial suspension) for nonenzymatic degradation of glutamine were performed and substrated. The reaction was started by the addition of mitochondria and stopped by addition of 0.5 ml 15%

Glutamine Metabolism and Progesterone Biosynthesis 79 trichloroacetic acid. After 5 minutes the excess of acid was neutralized by addition of 0.5 ml of 0.9 M KOH.

3 Glutamine Metabolism and Progesterone Biosynthesis 79 trichloroacetic acid. After 5 minutes the excess of acid was neutralized by addition of 0.5 ml of 0.9 M KOH. The precipitated protein was removed and 0.1 ml of the supernatant was used for colorimetric determination of ammonia according to Channey and Marbach (1962). Malic Enzyme Assay The malic enzyme activity was followed with a Specord UV-VIS spectrophotometer at 30~ by recording the appearance of NAD(P)H at 340 nm. Incubation was carried out in the medium (final volume 1 ml) containing 50 mm imidazole-hcl buffer ph 6.0 to 8.0, 0.5 mm oxamic acid, 0.5 mm NADP, 0.5 mm NAD, 10 mm L-malate, 1 mm MnC12, and 0.2 mg of partly purified NAD(P)-dependent malic enzyme. Protein Determination Protein concentration was determined by the biuret method in 0.25% sodium deoxycholate with bovine serum albumin as the standard (Layne, 1957). Progesterone Biosynthesis Estimation The incubation was carried out at 30~ for 30 minutes with 4 mg of mitochondrial protein in the reaction mixture (final volume 2.5 ml) containing 100 mm potassium phosphate buffer ph 7.2 or 8.2, 5 mm MgSO4, 0.5 mm NADP, 0.5 mm NAD, 0.15 I.tCi of [4-14C] cholesterol and either L-glutamine, L-glutamate, L-malate, or 2- oxoglutarate in concentrations indicated. For isolation of progesterone the incubation mixtures were transferred to conical tubes containing known amounts of [3I-I] progesterone. Non-radioactive progesterone (2 mg) was also added as unlabeled carrier. The contents of tubes were extracted twice with 10 ml of chloroform-diethyl either (1:5, v/v). The organic and aqueous phases were separated by centrifugation and the organic phase was evaporated to dryness. The dry residue was subjected to thin-layer chromatography on Silica Gel G (Merck) impregnated with Rhodamine 6(3. On developing the chromatogram in (i) methylene chloride-diethyl ether (5:2, v/v), two fractions were obtained: progesterone and cholesterol. Progesterone fractions were further purified by thin-lyaer chromatography in the following systems: (ii) benzene-ethyl acetate (3:2, v/v), and (iii) benzene-ethanol (9:1, v/v). Purification was continued to a constant 14C/3H ratio in the end product. The radioactivity of isolated progesterone was measured with an efficiency of 40% for 3H and 60% for 14C in 10 ml of scintillation fluid containing 4 g of 2,5- diphenyloxazole and 0.2 g of 1,4-di-2-(5-pheyloxazolyl) benzene per liter of toluene, using a Nuclear Chicago Isocap 300 liquid scintillation spectrometer. Total incorporation of 14C into [14(] progesterone was found on the basis of tritium found in the final product of the purification procedure. Pregnenolone, which is an intermediate in progesterone biosynthesis, did not accumulate to any significant extent after 30 minutes of incubation.

80 Klimek et al. RESULTS AND DISCUSSION Progesterone biosynthesis can be supported by glutamine and glutamine as oxidizable substrates (Figure 1).

4 80 Klimek et al. RESULTS AND DISCUSSION Progesterone biosynthesis can be supported by glutamine and glutamine as oxidizable substrates (Figure 1). The maximum progesterone biosynthesis in the presence of glutamate is observed after incubation for 30 minutes while using glutamine as substrate similar extent of progesterone biosynthesis occurs after 60 minutes. After incubation for 30 minutes the progesterone biosynthesis supported by glutamine is about 60% as compared with glutamate as a substrate. This difference can be explained assuming that glutamine is at first converted to glutamate and ammonia in the reaction catalyzed by glutaminase. Placental tissue has been shown to possess the activity of the enzyme which is localized in mitochondria (Makarewicz and Swierczynski, 1988). Because glutaminase activity is strongly dependent on phosphate concentration, progesterone biosynthesis is measured in the presence of glutamine and placental glutaminase activity as a function of inorganic phosphate concentration. These experiments are performed at ph 8.2 which is near optimum ph for glutaminase activity. The results of these experiments are presented in Figure 2. Both phenomena depend on inorganic phosphate concentration in a parallel manner. The activity of glutaminase and progesterone biosynthesis increases greatly in the range of phosphate concentration 5-50 mm. At higher concentrations of phosphate ( mm) no significant increase either of glutaminase activity or progesterone biosynthesis is observed. The progesterone biosynthesis supported by glutamate does not depend on inorganic phosphate concentration in the range mm (data not shown). This strongly suggests that the glutamine effect on progesterone biosynthesis is mediated by its prior hydrolysis to glutamate. 2,5 ol ~) m a_ 2,0 i_ 1,5 oo 10 t" ~ 0 ~- X E m._. 0,0 I I I I I ~ Time (rain) Figure 1. Time dependent effect of glutamine (A) and glutamate (1) on progesterone biosynthesis. Final concentration of L-glutamate and glutamine was 20 mm.

5 Glutamine Metabolism and Progesterone Biosynthesis ~ o c r _~ r- a. ~ E >, O 0.9.o O. ~ X C ~ O O 0.6 ~- - O X X 1-0,3 o E I1. "o Phosphote (mm) Figure 2. The effect of inorganic phosphate concentration at ph 8.2 on the activity of glutaminase (A) and on glutamine supported progesterone biosynthesis (11) in human placental mitochondria c "3 52 :E a. "~o~ 39 c.e b ~.= 26.~E O E 13 C 16 ~- (o 8 O~ 9 ~-- 'c 5 O ~ ~ E 0 J i i i i i I i Z5 8, ~5 10,0 ph Figure 3. The effect of ph on the activity of partly purified NAD (P) - linked malic enzyme (I) and on the activity of glutaminase (A) in human placental mitochondria.

6 82 Klimek et al. Glutamate is metabolized via Krebs cycle and can produce reducing equivalents (NADPH) for progesterone biosynthesis in the reactions catalyzed by NADPdependent glutamate dehydrogenase. The existence of the enzyme in placental mitochondria has been shown by Gaull et al. (1960). Meigs and Sheean (1977) investigated the effect of several substrates on progesterone biosynthesis in isolated placental mitochondria and observed that glutamate supports this process to the same extent as did several other dicarboxylic Krebs cycle metabolites. The stimulatory effect of glutamate could be also associated with NADPH supply from the reaction catalyzed by NAD(P)-dependent malic enzyme. In the study of possible involvement of glutaminase and NAD(P)-dependent malic enzyme in the regulation of progesterone biosynthesis one has to bear in mind the marked difference in the ph-dependence of these two enzymatic reactions. Figure 3 presents the combined date on ph-dependence of glutaminase in isolated placental mitochondria and partly purified NAD(P)-dependent malic enzyme (Makarewicz and Swierczynski, 1988; Swierczynski et al., 1987). The ph optimum for glutaminase is 8.5 and shows a rather wide plateau betwen 8.2 and 9.5. Maximum activity of NAD(P)- dependent malic enzyme is observed at ph 6.0. At ph 8.2, there is nearly maximum glutaminase activity while at this ph the activity of NAD(P)-dependent malic enzyme is very low. On the contrary at ph 7.2, the relative activity of NAD(P)- dependent malic enzyme is substantially higher than glutaminase activity. Table I Effect of ph on Progesterone Biosynthesis Stimulated by Malate, Oxoglutarate, Glutamate, and Glutamine in Human Placental Mitochondria Additions Progesterone biosynthesis dpm 14C per sample % of conversion Phosvhate buffer vh 7.2 None Malate 1200_ Oxoglutarate Glutamate Glutamine Phosphate buffer ph 8.2 None Malate Oxoglutarate Glutamate Glutamine Final concentraton of L-malate, 2-oxoglutarate, L-glutmate, and L-glutamine was 20 mm. The values are means + S.D. from five experiments.

Glutamine Metabolism and Progesterone Biosynthesis 83 The experiments in which the stimulatory effect of glutamine, glutamate, malate, and 2-oxoglutarate at two different ph values (7.2 and 8.

7 Glutamine Metabolism and Progesterone Biosynthesis 83 The experiments in which the stimulatory effect of glutamine, glutamate, malate, and 2-oxoglutarate at two different ph values (7.2 and 8.2) were compared in Table I. Glutamine stimulated progesterone biosynthesis at ph 8.2 to a much greater extent than at ph 7.2. Stimulation by malate and 2-oxoglutarate is greater at ph 7.2 than at ph 8.2. Glutamate stimulates substantially at ph 8.2 values but to the greater extent at ph 7.2 which could reflect the involvement of both NADP-dependent glutamate dehydrogenase and NAD(P)-dependent malic enzyme activities. This also confirms the previous observation that stimulation of progesterone biosynthesis by glutamine results from its conversion to glutamate. To assess to what extent, under the conditions employed, the NAD(P)- dependent malic enzyme supplies the reducing equivalents to placental progesterone biosynthesis the effect of malonate (inhibitor of succinate dehydrogenase) and hydroxymalonate (inhibitor of NAD(P)-dependent malic enzyme, Klimek et al., 1987) has been investigated. The results of these experiments are presented in Table II. At ph 7.2, both malonate and hydroxymalonate significantly decrease progesterone biosynthesis stimulated by glutamate which indicates the involvement at both NAD(P)-dependent malic enzyme and NADP-dependent glutamate dehydrogenase. However, at ph 8.2 these inhibitors are without effect which is in accord with very low activity of NAD(P)-dependent malic enzyme at this ph. These results strongly support the idea that the reduction of NADP and NADP-dependent glutamate dehydrogenase plays an essential role in the glutamine and glutamate supported progesterone biosynthesis. Table H Effect of Malonate and Hydroxymalonate on Progesterone Biosynthesis in the Presence of Glutamate and Glutamine Additions Phosohate buffer ph 7.2 Gluta-mate, 20 mm + Malonate, 10 mm + Hydroxymalonate, 4 mm Phosphate buffer oh ~.2 Glutamate, 20 mm + Malonate, 10 mm + Hydroxymalonate, 4 mm Glutamine, 20 mm + Malonate, 10 mm +Hydroxymalonate, 4 mm Progesterone biosynthesis dpm 14C per sample % of conversion The values are menas + S.D. from five experiments.

$84 Klimek et al. ACETYL-CoA " ~ I~lic enzyme NADP H PY RU_VAT E MALATE ISOCITRATE \ A'NA ~ SUCCINATE \ OXOGLUTARAT E Glutamate ~.~.~ NADPH +H dehydrogenase I~.~-.$

8 84 Klimek et al. ACETYL-CoA " ~ I~lic enzyme NADP H PY RU_VAT E MALATE ISOCITRATE \ A'NA ~ SUCCINATE \ OXOGLUTARAT E Glutamate ~.~.~ NADPH +H dehydrogenase I~.~-.NADP GLUTAMATE I GLUTAMI N E Isocitrme dehydr ogen(zse NADP H + DI Scheme 1. Generation of reducing equivalents (NADPH) resulting from glutamine metabolism. It has been suggested (Swierczynski et al., 1985) that stimulation by malate of progesterone biosynthesis depends also on further metabolism of pyruvate (formed from malate) to isocitrate, which is subsequently oxidized by NADP-dependent isocitric dehydrogenase. Thus, four-carbon Krebs cycle intermediates derived from glutamine and glutamate could be converted within the mitochondria to isocitrate, providing NADPH for progesterone biosynthesis by the action of NADP-dependent isocitrate dehydrogenase. Mechanism by which metabolism of glutamine may generate NADPH responsible for the rise of progesterone biosynthesis is summarized in Scheme 1. The proposed metabolic scheme can account for all of the effects of glutamine on progesterone biosynthesis presented in this paper. Glutaminase, which was found in human placental several years ago (Hagerman, 1964) and also in ovine placenta (Pell et al., 1983), appears to be the main initiating step and glutamate dehydrogenase, malic enzyme, and isocitrate dehydrogenase the most important enzymes responsible for intramitochondrial NADPH generation during glutamine catabolism. Glutamine is the most abundant amino acid in human plasma and it has been shown that glutamate is extracted by placenta from fetal circulation (Schneider et al., 1979). The results of the current experiments provided evidence that glutamine and glutamate can support progesterone biosynthesis in placental mitochondria and suggest a role for placental glutaminase. This seems to be the most interesting observation indicating that in human placenta amino acid metabolism may be closely correlated to another basic physiological process namely steroid biosynthesis.

Glutamine Metabolism and Progesterone Biosynthesis 85 SUMMARY The effect of glutamine and glutamate on progesterone biosynthesis in human placental mitochondria has been investigated.

9 Glutamine Metabolism and Progesterone Biosynthesis 85 SUMMARY The effect of glutamine and glutamate on progesterone biosynthesis in human placental mitochondria has been investigated. Both amino acids stimulates this process, but glutamate to a greater extent. At ph 8.2 activity of placental mitochondrial glutaminase and progesterone biosynthesis in the presence of glutamine depend on inorganic phosphate concentration in the same manner. This suggests that glutaminase at first hydrolyses glutamine to glutamate and further metabolism of glutamate supports progesterone biosynthesis. Results of experiments in which progesterone biosynthesis has been measured at ph 7.2 and 8.2 indicate the possible involvement of NADP-dependent glutamate dehydrogenase, NAD(P)-dependent malic enzyme, and NADP-dependent isocitrate dehydrogenase in providing the reducing equivalents (NADPH) for progesterone biosynthesis from glutamine and glutamate. Data reported in this paper suggest that in human placental mitochondria metabolism of glutamine and glutamate may be closely correlated to progesterone biosynthesis. ACKNOWLEDGEMENTS This work has been supported by a grant from the Medical Academy in Gdansk within the projects W-95, ST40, and ST41. REFERENCES Boguslawski, W., Klimek, J., Tialowska, B., and Zelewski, L. (1976) Inhibition of Mn 2+ of citrate supported progesterone biosynthesis in mitochondrial fraction of human term placentae. J. Steroid Biochem. 7, Chaney, A.L. and Marbach, E.P. (1962) Modified reagents for determination of urea and ammonia. Clin. Chem. 8, Gaull, G., Hagerman, D.D., and Villee, C.A. (1960) Preparation and properties of glutamic dehydrogenase from human placenta. Biochim. Biophys. Acta 40, Hagerman, D.D. (1964) Enzymatic capabilities of the placenta. Fed. Proc. 23, Klimek, J., Boguslawski, W., Tialowska, B., and Zelewski, L. (1976) Regulation of progesterone biosynthesis in human placental mitochondria by Krebs cycle metabolites. Acta Biochim. Polon. 23, Klimek, J., Boguslawski, W., and Zelewski, L. (1979) The relationship between energy generation and cholesterol side-chain cleavage reaction in the mitochondria from human term placenta. Biochim. Biophys. Acta 587, Klimek, J., Swierczynski, J., and Zelewski, L. (1987) Inhibition by hydroxymalonate of malate dependent biosynthesis of progesterone in the mitochondrial fraction of human term placenta. J. Steroid Biochem. 26,

86 Klimek et al. Layne, E. (1957) Spectrophotometric and turbidimetric methods for measuring proteins. Methods Enzymol. 3, 447-454. Lemons, J.A., Adcock, E.W., Jones, M.D., Naughton, M.A., Meschia, G.

10 86 Klimek et al. Layne, E. (1957) Spectrophotometric and turbidimetric methods for measuring proteins. Methods Enzymol. 3, Lemons, J.A., Adcock, E.W., Jones, M.D., Naughton, M.A., Meschia, G., and Battaglia, F.C. (1976) Umbilical uptake of amino acids in the unstressed fetal lamb. J. Cfin. Invest. 58, Makarewicz, W. and Swierczynski, J. (1982) Ammonia formation from some amino acids by human term placental mitochondria. Biochem. Med. 28, Makarewicz, W. and Swierczynski, J. (1988) Phosphate-dependent glutaminase in the human term placental mitochondria. Biochem. Med. Metab. Biol. 39, Meigs, R.A. and Ryan, K.J. (1968) Cytochrome P-450 and steroid biosynthesis in the human placenta. Biochim. Biophys. Acta 165, Meigs, R.A. and Sheean, L.A. (1977) Mitochondria from human term placenta. III. The role of respiration and energy generation in progesterone biosynthesis. Biochim. Biophys. Acta 489, Pell, J.M., Jeacock, M.K., and Shepherd, D.A.L. (1983) Glutamate and glutamine metabolism on the ovine placenta. J. Agric. Sci. Camb. 101, Ryan, K.J., Meigs, R.A., and Petro, Z. (1966) The formation of progesterone bythe human placenta. Am. J. Obstet. Gynecol. 96, Schneider, H., Moehlen, K-H., Chollier, J.C., and Dancis, J. (1979) Transfer of glutamic acid across the human placenta perfused in vitro. Br. J. Obstet. Gynecol. 86, Swierczynski, J., Scislowski, P., and Aleksandrowicz, Z. (1976) High activity of (zglicerophosphate oxidation by human placental mitochondria. Biochim. Biophys. Acta 429, Swierczynski, J., Scislowski, P., Aleksandrowicz, Z., and Zelewski, L. (1982) NAD(P)- dependent malic enzyme activity in human term placental mitochondria. Biochem. Med. 28, Swierczynski, J., Klimek, J., and Zelewski, L. (1985) The role of malic enzyme in the malate dependent biosynthesis of progesterone in the mitochondrial fraction of human term placenta. J. Steroid Biochem. 22, Swierczynski, J., Aleksandrowicz, Z., and Zelewski, L. (1987) Stimulatory effect of ADP, ATP, NAD(P) on pyruvate production from malate by uncoupled human placental mitochondria. Biochem. Med. Metab. Biol. 38, Zolnierowicz, S., Swierczynski, J., and Zelewski, L. (1988) Purification and properties of the NAD(P)-dependent malic enzyme from human placenta mitochondria. Biochem. Med. Metab. Biol. 39,

4. Which step shows a split of one molecule into two smaller molecules? a. 2. d. 5

4. Which step shows a split of one molecule into two smaller molecules? a. 2. d. 5 1. Which of the following statements about NAD + is false? a. NAD + is reduced to NADH during both glycolysis and the citric acid cycle. b. NAD + has more chemical energy than NADH. c. NAD + is reduced