Androgenic control of acetate incorporation into phospholipids and triacylglycerols in rat ventral prostate

Comp. Biochem. Physiol. Vol. 78B, No. 1, pp. 299-302, 1984

0305-0491/84 $3.00+0.00 © 1984 Pergamon Press Ltd

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A N D R O G E N I C CONTROL OF ACETATE INCORPORATION INTO PHOSPHOLIPIDS A N D TRIACYLGLYCEROLS IN RAT VENTRAL PROSTATE* N. DEL HOYO, L. A. TORRE and M. A. PI~REZ-ALBARSANZ'~ Departamento de Bioquimica, Facultad de Ciencias, Universidad de Alcal~ de Henares, Madrid, Spain (Received 5 October 1983)

A~traet--1. To obtain information about androgenic hormone actions on the synthesis of lipids from [1J4C]acetate in rat ventral prostate, in vitro and in vivo experimentshave been carried out. The time course of incorporation of labelled acetate was studied. Also different solubilization media of androgenic hormones was investigated. We have selected Tween-80. 2. Results for in vitro experiments, show that increasing concentrations of testosterone and dihydrotestosterone correspond to an increase in the incorporation of labelled acetate into phospholipids and a decrease in the incorporation into triacylglycerols. 3. In vivo experiments of incorporation of [IJ4C]acetate have also been carried out and show a similar pattern to the situation found in our in vitro experiments.

INTRODUCTION In most mammalian species, the development of the prostate gland from embryo to adult is under the control of sex steroids. Androgens have been related with the growth and development of normal and abnormal prostates. A correlation between the rate of formation of dihydrotestosterone (17/3-hydroxy-5ct-androstan-3one), the main metabolite of testosterone (17/3hydroxy-4-androsten-3-one) in the prostate, and prostatic growth has been reported in 11 animal species (Gloyna and Wilson, 1969). The organogenesis of prostatic tissue in the embryonic period is initiated by the action of androgens (Wilson and Lasnitzki, 1971; Wilson, 1973; Siiteri and Wilson, 1974; Lasnitzki and Mizuno, 1977). During puberty, increase in sex hormone levels incites the growth of the gland together with a cellular reorganization and an increase of the secretory function (Siiteri and Wilson, 1970; Moger, 1977). In adult life, under androgen influence, the gland weight, its cellular arrangement and its exocrine function persist (Siiteri and Wilson, 1970). Associated with the aging of the rat, is a decrease of the gland secretionary activity and an accumulation of dihydrotestosterone. Also the androgen receptor content is reduced and this can lead to hypertrophy or benign hyperplasia (Huggins, 1945 and 1947; Shain et al., 1975). In this way, three developmental periods (neonatal, prepubertal and pubertal) may be crucial for acquisition of functional activity, of the male accessory sexual tissues, in the adult male rat (Higgins et al., 1981). Castration, resulting in a decline in serum testosterone levels, is immediately associated with a decline

in prostate weight and function. Administration of testosterone to castrated rats was shown to result in an increase of these parameters (Heyns et aL, 1977; Singhal and Schaffner, 1980; Huttunen et al., 1981). Indeed, it is well known that the condition and function of the prostate gland depends on testicular function, especially in the production of testosterone. We have shown (Prrez-Albarsanz et al., 1982) the efficiency of the incorporation of acetate into triacylglycerols in ventral prostate of immature, 35-day-old, rats. However, the incorporation into phospholipids was mainly observed in the case of adult rats. It is interesting to note the existence of a crossing zone between the incorporation curves of triacylglycerols and phospholipids. This crossing zone coincides with the puberty stage of the rats in the breeding conditions of our animal house. The data to justify the existence of this crossing zone are various. Testosterone levels in plasma and prostate reach a maximum at the puberty stage of the rats (Smith et al., 1977; Moger, 1977; Corp~chot et al., 1981). Testosterone stimulates lipid synthesis (Doeg, 1968) particularly that of cholesterol (Singhal et al., 1978, 1979, 1980). It is realistic to consider the crossing zone as a response to the production of testosterone during puberty. In this work we examine the possibility of a relationship between the androgenic production and the changes in the [1-t4C]acetate incorporation into triacylglycerols and phospholipids during puberty. MATERIALS AND METHODS

All the reagents used were analytical grade. Testosterone and dihydrotestosterone were purchased from Merck (Darmstadt, West Germany). Sesame oil and liquid scintil*The work was partly supported by a grant from Comisi6n lation reagents were obtained from Sigma Chemical Co. (St. Louis, MO, USA). [1-J4C]Sodiumacetate, specificactivAsesora de Investigaci6n Cientifica y Tecnica. tCorrespondence to be addressed to Dr. M. A. P6rez- ity 58 mCi/mmol, was obtained from the Radiochemical Albarsanz, Departamento de Bioquimica, Facultad de Centre (Amersham, Bucks., UK). Male young mature Wistar rats (60-75 days old weighing Ciencias, Universidad de Alcal~i de Henares, Madrid, 280-340 g) were used in this study. They were housed in Spain. 299

300

N. DEL HoYo et al.

temperature controlled rooms and maintained on a standard laboratory diet and water ad libitum. The animals were killed by exsanguination under light ether anaesthesia. The ventral prostates were removed, stripped of connective and adipose tissue, weighed immediately and kept in ice bath for later use.

7000_ cpm/mg prostate ~

In vivo experiments Castrations, when performed, were done under ether anaesthesia via scrotal incision. We castrated young mature rats and waited 7 days before the administration of the hormones. Androgens (dose used in results, vehicle sesame oil 0.2 ml) were administered subcutaneously (1 injection per day during 4 days) in the inguinal region. Control animals, receiving only sesame oil, were treated identically to the hormone injected animals. Injection of labelled sodium acetate into each lobe of the ventral prostate, extraction and separation of lipid and the methods used for the determination of radioactivity in the different classes of lipids have been previously described (P+rez-Albarsanz et al., 1982). Treatment o f the results

The data are presented as mean values + SEM of three of four determinations. Student's t-test was applied in the experiment of [lJ4C]acetate incorporation into lipid classes and significant differences (P < 0.05) between each group of assays and the appropriate control were obtained. RESULTS

Figure 1 shows the results obtained in a time course experiment of incorporation of [IJ4C]acetate into total lipids. Also, shown is the capacity of radioactivity incorporation into different classes of lipids, as a percentage of the labelled total lipid, at two incubation times. Total incorporation into lipids increased with the time of incubation up to 270 rain. Nevertheless, the relative percentage of incorporation into triacylglycerols and phospholipids did not significantly change at the two incubation times studied. It should be noted that the +4C-labelled acetate was mainly incorporated, by ventral prostates of young mature rats (60-75 days old weighing . . . . . . . . . . . . . . . . . . *EDTA--ethylenediaminetetracetic acid.

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In vitro experiments Approx. 38-42 mg samples of minced tissues were used to study the incorporation of radioactive precursors into lipid classes. Tissues were incubated with 2ml of Hank's salt solution (pregassed with 95°~, 02 and 5?{, CO2) at 37C on a constant speed shaker for varying lengths of time. Two #Ci [1-t4C]acetate dissolved in 4#1 phosphate buffer 0.05 M, were the radioactivity contents of the incubation mixtures. In experiments with androgenic hormones, the ventral prostate tissues were preincubated for 30min. After this period testosterone or dihydrotestosterone dissolved in Tween-80 (5 mg of Tween-80 per ml of incubation) were added to respective tubes (concentrations in results). These tubes were again incubated at 37C for 2 hr. At the end of this period the medium was decanted from each tube, and 2 ml of fresh Hank's solution was added with the appropriate supplement of androgens and the labelled acetate. After the addition of precursor, the tubes were further incubated at 37~C for 2hr. The reaction was stopped by the addition of 2 ml chloroform and the mixture made into a single phase by the addition of 4.2 ml methanol. Total lipids were extracted+ purified and separated following the methods previously described (P6rez-Albarsanz et al., 1982).

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200~

100( 60rain v 30

i 60

120

180

270rain

i 240

270 rain

Fig. 1. Time course of acetate in vitro incorporation into total lipids by rat ventral prostate. Incorporation of labelled acetate, as a percentage of the labelled total lipids, into phospholipids (first bars from the left), triacylglycerols (second bars) and free fatty acids (third bars) at 60 and 270 rain of incubation. 280-340 g), into phospholipids; whereas free fatty acids and triacylglycerols exhibited lower radioactive incorporation. These incorporation levels into the different lipid classes reached in [1J4C]acetate in vitro experiments (Fig. 1) are in agreement with the levels of incorporation of acetate in in vivo experiments (P6rez-Albarsanz et al., 1982) at the same development stage of the rat ventral prostate. In order to obtain information about androgenic hormone actions on in vitro systems, the method used for dispersion of these hormones in the incubation mixture must be established previously. Different solubilization media for steroid hormones have been used by previous investigators (Tveter and Aakvaag, 1970; Levy et al., 1974; Singhal and Schaffner, 1980; Hudson, 1981; Kline et al., 1981; Hata et al., 1982). In view of this, we have checked several media obtaining results shown in Fig. 2. Compared with control, organic solvents such as acetone invert the percentage of [1-t4C]acetate incorporated into triacylglycerols and phospholipids, while albumin, E D T A * , Tween-20 and Tween-80 do not alter this percentage significantly. Therefore, we selected Tween-80 among the latter. It offers the additional advantage of giving more stable suspensions. Figure 3 shows, for in vitro experiments, how the concentration of testosterone and dihydrotestosterone affects the percentage of radioactivity incorporated into triacylglycerols and phospholipids with respect to the total lipids involved. The results show that increasing concentrations of testosterone and dihydrotestosterone lead to a significant (P < 0.05) increase in the incorporation of labelled acetate into phospholipids and a decrease into triacylglycerols. Moreover, we obtained larger effects for testosterone than dihydrotestosterone.

Androgenic control of acetate incorporation into lipids

301

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30

20

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C Ac A[ EDTA Tw20 Tw80 2¢ Fig. 2. Effects of different dispersion media for steroid hormones on in vitro incorporation of [1-~4C]acetate into phospholipids (first bars from the left), triacylglycerols 0:4 61o ,;~ ~;6 (second bars) and free fatty acids (third bars). C, control; mlVt Ac, control plus acetone 300 mM; AI, control plus albumin 5 mg/ml; EDTA, control plus EDTA 0.05 mM; Tw20, con- Fig. 3. Influence of testosterone ( - - ) and dihydrotrol plus Tween-20 5 mg/ml; Tw80, control plus Tween-80 testosterone ( - - - - ) on the [1-z4C]acetateincorporation (~ 5 mg/ml. Incubation time 60 min. total labelled lipids) into phospholipids (0) and triacylgtycerols (©) by rat ventral prostate. Incubations were carried out as described in in vitro experiments with anOnce it had been established in vitro that the drogenic hormones (Materials and Methods). incorporation of acetate into lipid classes is affected by androgenic hormones, it would be interesting to see if a similar pattern occurs in vivo. For this purpose, castrated rats have been chosen in order to 6.2.1.1), since acetate activation to acetyl CoA occurs suppress the action of endogenic androgens. We readily in the rat prostate and it is not assumed to be castrated young mature rats and waited for 7 days under hormonal control (Williams-Ashman and before the administration of the hormones. After this Banks, 1954; H/irk6nen et al., 1982). More reasontime androgens were undetectable in plasma (Cor- ably, hormonal influence is over the formation pathprchot et aL, 1981). The injection of androgenic ways of phospholipids provoking an increase in the hormones, testosterone and dihydrotestosterone, to synthesis of these structural lipids. The last would castrated rats caused an increase in the percentage of agree with results in the literature, which established [1-14C]acetate incorporated into phospholipids and a a direct relationship between increments of the andecrease into triacylglycerols (Table 1). This result drogen levels in plasma and prostate and the prosfrom our in vivo experiments agrees closely with our tatic growth (Gloyna and Wilson, 1969; Moger, 1977; Higgins et al., 1981). On the other hand, phosin vitro results, but the variations are smaller in vivo. pholipids, as a fundamental component of the prostatic cellular membrane, are needed for prostatic DISCUSSION growth. Toghrol et al. (1980) has suggested that fatty acids, This work shows that the rat ventral prostate can utilize acetate for the synthesis of fatty acids, which through fatty acid oxidation, are a much better are incorporated posteriorily into different lipid source of acetyl CoA used in citrate synthesis. This classes. This incorporation depends on testosterone citrate is accumulated and secreted by the ventral and, its active metabolite, dihydrotestosterone. Both prostate of the rat. In addition, Barron and Huggins hormones provoked an increase in the incorporation (1946) showed the absence of a functional triof acetate into phospholipids and a decrease in the carboxylic acid cycle in the rat ventral prostate, incorporation into triacylglycerols. aconitase (EC 4.2.1.3) being ultimately responsible This hormonal control on the acetate incorpo- for this fact (Costello and Franklin, 1981). On the ration into lipid classes cannot be interpreted as an other hand, the citrate accumulation in this gland is action of androgens on acetyl CoA synthetase (EC under androgenic control (H/irk6nen et aL, 1982). Table 1. Influence of testosterone (T) and dihydrotestosterone (DHT) on the [l-t4C]acetate incorporation (~ total labelled lipids) into phospholipids (PL), triacylglycerols (TG) and free fatty acids (FFA). In vivo experiments were carried out with castrated young mature rats as described in Materials and Methods. Control: only sesame oil, (1) 0,07 mg/100 g body wt/day in 0.2 ml sesame oil. (2) 0.7mg/100g body wt/day in 0.2ml sesame oil PL

TG

FFA

CONTROL

52.6Z2.5

31°5~2.0

0.5±0.l

DHT(1)

53.6±2.2

24°3~i.i

1.4~0,3

DHT(2)

57.6~3.1

25.5~1.4

0.7±0.2

T

(i)

61.1±4.1

22.1~1.2

1.7~0.3

T

(2)

63.0±3.1

18,3ZI.3

1.5Z0.3

302

N. DEL HoYo et al.

Hence we consider, a n androgenic c o n t r i b u t i o n to the mobilization o f triacylglycerols into fatty acids necessary in citrate biosynthesis, There are several methods, none of which are ideal, that influence in vivo the h o r m o n a l system that acts over the prostatic gland, to suppress the participation of the endogen androgens, One m e t h o d consists in the t r e a t m e n t of animals with an a n t a g o n i s t (oestrogens) for a limited time (Callaway et al., 1982). This m e t h o d has the inconvenience o f including a p e r t u r b a t i n g factor in the system: it can have a n u m b e r o f actions p e r se. O t h e r m e t h o d s are ablative, such as c a s t r a t i o n a n d h y p o p h y s e c t o m y . T h e disadv a n t a g e o f such m e t h o d s is the difficulty in rep r o d u c i n g conditions similar to the original if the p e r t u r b a t i o n is not to be indelinite, T h e experiments with castrated rats, in this work has confirmed the influence of testosterone and dihydrotestosterone o n the i n c o r p o r a t i o n of radioactive acetate into triacylglycerols a n d phospholipids. On the o t h e r h a n d , the effects were greater for testosterone than d i h y d r o t e s t o s t e r o n e b o t h in cit:o a n d in ritro. Since d i h y d r o t e s t o s t e r o n e is the principal metabotite of testosterone in prostate ( G l o y n a and Wilson, 1969: Tveter a n d A a k v a a g , 1970), an explan a t i o n o f the experimental data can be given as the difference in m o b i l i z a t i o n a n d t r a n s p o r t for b o t h types o f h o r m o n e s . We can conclude that there is a relationship between increments of the a n d r o g e n levels a n d the increase a n d decrease of acetate i n c o r p o r a t i o n into phospholipids and triacylglycerols respectively. This is similar in a way to the correlations found in rico d u r i n g the male rat puberty. Acknowledgement--We

thank Dr, Luque for wduablc

criticism. REFERENCF.S Barron E. S. G. and Huggins C. (19463 The metabolism of the prostate: transamination and citric acid. J. Urot. 55, 385-396. Callaway T. W., Bruchovsky N., Rennie P. S. and Comeau T. (19823 Mechanisms of action of androgens and antiandrogens: effects of antiandrogens on translocation of cytoplasmic androgen receptor and nuclear abundance of dihydrotestosterone. The Prostate 3, 599-610. Corpechot C., Baulieu E. E. and Robel P, (t9813 Testosterone, dihydrotestosterone and androstanediols in plasma, testes and prostates of rats during development. Acta Endoer. 96, 127-135. Costello k. C. and Franklin R B. ( 1981 ) Aconitase activity, citrate oxidation and zinc inhibition in rat ventral prostate, Enzyme 26, 281-287. Doeg K. A. (19683 Control of mitochondrial lipid biosynthesis by testosterone in male sex accessory gland tissue and liver of castrate rats. Endocrinology 82, 535-539. Gloyna R. E. and Wilson J. D. (19693 A comparative study of the conversion of testosterone to 17fi-hydroxy-5~ androstan-3-one (dihydrotestosterone) by prostate and epididymis. J. olin, En&~cr, Metah. 29, 970-977. H~irk6nen P. L, Kostian M. L. and Santti R. S. (I982) Indirect androgenic control of citrate accumulation in rat ventral prostate. Archs Andrology 8, 107- 116. Hata S,, Nishino T_ Ariga N. and Katsuki H. (19823 Effect of detergents on sterol synthesis in a cell-free system of yeast. J. Lipids Res. 23, 803-810.

Heyns W., Peelers B. and Mous J. lt977) lnttuencc ol androgens on the concentration of prostatic binding protein (PBP) and its mRNA in rat prostate. Biochem. hiophys. Res. Commun. 77, 1492-1499, Higgins S. J., Brooks D. E,, Fuller IL M., Jackson P. J. and Smith S. g. (19813 Functional devclopmcnt of sex accessory organs of the male rat. Biochem. J. 194, 895-905. Hudson R. W. (198I) Studies of the nuclear 57-reductasc of human hyperplastic prostatic tissue. J. steroid Biochem. 14, 579-584. Huggins C, (19453 The physiology of the prostate gland, Physiol. Rec. 25, 281 295. Huggins C. (19473 The etiology of benign prostatic hypertrophy, Butt. N.Y. Acad. Med. 23, 696-704. Huttunen E,, Romppanen T. and Helmmen H J. (19813 ,,\ histoquantitative study on the effects ot" castration ,m the rat ventral prostate lobe. J. Anat. 132, 357 37tL Kline L, D., [.el~bvre F. T. and kefebvre Y. A. (1981~ Uptake of androgens by intact and detergent-treated nuclei from the rat ventral prostate. ,L xleroid Biochcm. 14, 855-860. Lasnitzki I. and Mizuno T. (19773 Induction of the ral prostate gland by androgens in organ culture, t:'mbJcrmologv 74, 47 45. Levy C,, Marchut M., Baulieu Ii. E. and Robel P, (19743 Studies of the 3//-hydroxysteroid oxidoreductase activit 3 in rat ventral prostate. S1eroM 23, 291 300, Moger W. H. (i9773 Serum 5~-androstane-3~., 17fl-diol, androsterone and testosterone concentrations in the male rat, Influence of age and gonadotropin stimulation. Emtocrimdogy 100, 1027- 1032, Perez-Albarsanz M. A., del Hoyo N.. Atcaidc A. and Recio M. N. (1982) Lipogenesis during the development of the rat ventral prostate. Comp. Biochem. Phw, iol. 72B, 673675. Shain S, A. Boesel R. W. and Axclrod L. R. 11975) Aging in the rat prostate: reduction in detectable ventral prostale androgen receptor content. Archs Bioehem. Biophvs. 167, 247- 263, Siiteri P, K, and Wilson ,I. D. (t9703 Dihydrotestostcronc m prostatic hypertrophy. I. The I\~rmation and contenf of dihydrotestosterone in the hypertrophic prostate of man. .l. din, hu'est. 49, t737 1745. Siiteri P. K. and Wilson J. D. (19743 Testosterone formation and metabolism during male sexual differentiation in the human embryo. J. elin. £)utocr. Metab. 38, 113-125. Singhal A. K.. Bonner D. P. and Schaffner C. P, (19783 Kinetics of testosterone induced-cholesterol synthesis in rat ventral prostate. Proc. Sot'. exp. BioL Med. 159, 1 5. Singhal A. K., Brill D. R. and Schaffner C, P. (19791 Effect of elofibrate on cholesterol and DNA synthesis in rat ventral prostate. Proe. Soc. exp. BioL Med. 160, 405- 409. Singhal A. K, and Shaffner C. P. (19803 In vitro cifcct of testosterone and cAMP on cholesterol synthesis in rat ventral prostate. Proc. ,S'm'. evp. Biol. Med. 164, 45 50. Smith E. R.. Damassa D, A. and Davidson J. M. (I~,~77) Feedback regulation and mate puberty: testosteroneluteinizing hormone relationships in the developing rat, Endocrinology 101, 173-180. l'oghrol F., Franklin R. B. and Costello k. C. (t980) Citrate synthesis from fatty acids and amino acids in rat ventral prostate. Enzyme 25, 371 376, Tveter K..1. and Aakvaag A. (1970) Metabolism m vu,o of [*tt]androstenedione and pH]testosterone by androgen dependent tissues, Acta endocr. 65, 723-730. Williams-Ashman H, G. and Banks J. (1954) The synthesis and degradation of citric acid by ventral prostate tissue, J. l~iol. Chem. 20g, 337-344. Wilson J. D. (19733 Testosterone uptake by the urogenital tract of the rabbit embryo. Endocrinology 92, 1192--I 199. Wilson J. D. and Lasnitzki 1. (1971) Dihydrotcstosterone forrnation in fetal tissues of the rabbit and rat. Endocrinoto&v 89, 659- 668.