Bioscience Reports, Vol. 10, No. I, 1990

Comparison of Linoleate, Palmitate and Acetate Metabolism in Rat Ventral Prostate N. del H o y o , 1 J. A . Pulido, 1 M. T. Carretero ~ and M. A . P6rez-Albarsanz ~'2 Received 12 June, 1989, revised version 30 October, 1989.

Rat ventral prostate incorporated (1-1aC)acetate, (1-14C)palmitate and (1-14C)linoleate into different phospholipids in a time-dependent process. The rate of incorporation into total phospholipids was higher with linoleate (10.0 nmol/g) than with either palmitate (5.8 nmol/g) or acetate (4.7 nmol/g). Predominant labelling with all the radioactive substrates assayed was found in choline glycerophospholipids (PC). The radioactive profiles for linoleate in the other ventral prostate phospholipids differed from those obtained with palmitate and acetate. Specifically linoleate was incorporated into inositol glycerophospholipids plus lysoethanolamine glycerophospholipids (PI + LPE) and not into sphingomyelin (SM), while palmitate and acetate incorporated into SM but not into PI+ LPE. Acetate showed the highest oxidation to CO2 whereas no differences were observed in the radioactivity incorporated into CO2 from a saturated (palmitate) or an essential unsaturated fatty acid (linoleate). These studies also show zinc-dependence by the acetate to CO2 oxidation. KEY WORDS: phospholipids; free fatty acids; rat ventral prostate. ABBREVIATIONS: PL, total phospholipids; PC, choline glycerophospholipids; PE, ethanolamine glycerophospholipids; PI + LPE, inositol glycerophospholipids plus lysoethanolamine glycerophospholipids; PS, serine glycerophospholipids; SM, sphingomyelin.

INTRODUCTION L o n g chain fatty acids h a v e i m p o r t a n t functions in the rat ventral p r o s t a t e not only as structural c o m p o n e n t s (1, 2, 3) but also as m a j o r substrates for oxidation. C a r b o h y d r a t e oxidations, and fatty acid fl-oxidation are the m a i n sources of acetyl C o A for citrate synthesis in the p r o s t a t e (4, 5). In this regard rat ventral prostate, like the h u m a n p r o s t a t e , a c c u m u l a t e s a n d secretes e x t r a o r d i n a r y high levels of citrate (6). Palmitic acid (16: 0) and linoleic acid (18: 2(n - 6)) are, respectively the m o s t a b u n d a n t saturated and p o l y u n s a t u r a t e d fatty acids in the rat ventral p r o s t a t e (1, 2, 3). T h e essential n a t u r e of linoleate in m a m m a l s and the signs that result f r o m its deficiency in the diet, including sterility in b o t h the m a l e and f e m a l e , 1Departamento de Bioqulmica y Biologia Molecular, Universidad de Alcalfi, 28871 Alcalfi de Henares, Madrid, Spain. 2 To whom correspondence should be addressed. 105 0144-8463/90/0200-0105506.00/0~) 1990 Plenum Publishing Corporation

del Hoyo, Pulido, Carretero and P6rez-Aibarsanz have long been recognized (7, 8, 9). Animals can convert dietary linoleate into other members of the n - 6 series and arachidonic acid ( 2 0 : 4 ( n - 6)) is usually the most predominant polyunsaturated fatty acid in this series. This fatty acid is then preferentially esterified into phospholipids, primarily PC (10, 11) and ethanolamine glycerophospholipids (PE) (2) so that the role in the prostate of arachidonic acid as a precursor for production of eicosanoids, including prostaglandins, thromboxanes and leukotrienes, has received attention (12, 13). Previous data from our laboratory (1,3,14,15,16) indicate that (1-14C)acetate incorporation into triacylglycerols, total phospholipids (PL) and cholesterol is a development-dependent process and the relationships between androgen production and the changes of (1-14C)acetate incorporation into the above lipid classes have been reported in this light (14, 17). However, until now no information has been available on the utilization of fatty acids in rat ventral prostate for biosynthesis. In this paper we describe studies on the incorporation of palmitate and linoleate, into different phospholipid classes and compare our results with those for acetate incorporation. The oxidation of these labelled precursors into CO2 is also shown in in vitro experiments.

MATERIALS A N D M E T H O D S Chemicals All solvents and reagents used were of analytical grade. (1-14C)Sodium acetate, (1-14C)sodium palmitate and (1-14C)sodium linoleate with specific activities between 56-57 Ci/mol were purchased from the Radiochemical Center Amershand, Bucks, U.K.). Animals Male Wistar rats aged 120-130 days, were housed in a temperaturecontrolled room and maintained on a laboratory diet and water ad libitum. The animals for the zinc studies were separated into two groups of six animals each. A previously described semisynthetic control diet (18) containing 13 ppm of zinc was fed to an experimental group for 30 days before sacrifice. The other group was fed the same semisynthetic diet but without zinc. The animals were killed by exanguination under light ether anaesthesia. The ventral prostates were excised and stripped of connective and adipose tissues. In Vitro Experiments Rat ventral prostate slices weighing about 40 mg were incubated with 2 ml of Hanks' salt solution, p H 6 . 7 (pregassed with 95% O2 and 5% CO2) and 21xCi (1-14C)sodium acetate or 1 ~Ci of radioactive long chain fatty acids. The latter were suspended by sonication at 4~ in bovine serum albumin (lipid free) in a

Phospholipid synthesis in rat prostate

107

molar ratio of 0.75 : 1, giving a final concentration of 9 ~tM. The incubations were carried out at 37~ at a constant shaking speed. The reaction was stopped by the addition of 2 ml of chloroform and the mixture was made into a single phase by the addition of 4.2ml of methanol whereafter the total lipid extraction was carried out as described below. Incubations with (1-14C)acetate, (1-14C)palmitate or (1-~4C)linoleate to determine the amount of radioactivity incorporated into ~4CO2 were carried out in a flask equipped with a center well containing a fluted filter paper and sealed with a rubber cap. After incubation, as above, 0.5 ml of 10 M sulphuric acid was added to the incubation mixture with a syringe and the 14CO2formed was trapped by injecting 0.15 ml of 6M K O H into the center well. The flask was kept in an ice-bath for 2 hours. Radioactivity determinations were performed in a Kontron BETAmatic liquid scintillation counter. Lipid Analysis Total lipids were extracted and purified according to the method of Bligh and Dyer (19). The different classes of lipids were separated by thin layer chromatography on silica gel G plates (Merck Darmstadt, F.R.G.) as previously described (20, 21). Briefly, total phospholipids were separated employing hexane/ ether/acetic acid (70:30:1, v/v). Different classes of phospholipids were separated using propionic acid/propanol/chloroform/water (2:2: 1, v/v). Standard phospholipids were used for comparison. Thin-layer bands were scraped off the plate and directly transferred to scintillation vials for counting. Statistical Treatment of Results Results are expressed as mean • SE. The statistical significance of differences between groups was determined by the Student's t-test. Differences were considered significant when p < 0.05.

RESULTS The palmitate and linoleate incorporation into total phospholipids at 2 hours of incubation time, is shown in Table 1. The rate of incorporation into total phospholipids was significantly higher for linoleate (10.0 nmol/g prostate) than for palmitate (5.8 nmol/g prostate). Differences were also found in the incorporation of these labelled fatty acids into the individual phospholipid subclasses. The highest incorporation of the two labelled fatty acids was observed into PC followed by incorporation into PE. Low levels of palmitate (1.8%) and linoleate (2%) incorporation were found in serine glycerophospholipids (PS). Palmitic acid was incorporated into SM however no incorporation of palmitate was found into PI + LPE. Interestingly, the linoleate incorporation into both SM and PI + LPE was the reverse of the palmitate incorporation into these phospholipid classes because linoleate was incorporated into PI + LPE but not into SM.

108

del Hoyo, Pulido, Carretero and PErez-Albarsanz Table 1. Incorporation of (1-14C)palmitic acid and (1-14C) linoleic acid into rat ventral prostate phospholipids 40 mg of rat ventral prostrate were incubated with 1 ~tCi labelled fatty acid bound to albumin (0.75:1 molar ratio) for 2h at 37~ Results are mean+sD of six experiments

Lipid

Palm•

Total phospholipids (nmol/g prostate) Phospholipid classes (% total incorp, into phospholipid) Choline glycerophospholipids Ethanolamine glycerophospholipids Serine glycerophospholipids Sphingomyelin Inositol glyserophospholipids+ lysoetha.olamine glycerophospholipids

acid

Linoleicacid

5.8•

10.0•

85.5• 6.5• 1.8• 4.0•

77.1• 11.1• 2.0• 7.6•

( -- ) = not detected or present at less than 1%. of (1-14C)acetate into rat ventral prostate phospholipids 40mg of rat ventral prostate were incubated with 21tCi (1-~4C)acetate for 2 hours at 37~ Results are means • SDof six experiments. Table

2.

Incorporation

Lipid Acetate incorporated (nmol/g prostate) Phospholipid classes (% total incorporation) Choline glycerophospholipids Ethanolamine glycerophospholipids Serine glycerophospholipids Sphingomyelin

Total Lipids

Total Lipids-P

15.5•

4.7i0.2

22.8• 3.4• 1.5• 1.8•

76.0il.7 11.4• 5.3• 5.9•

The relative importance of the phospholipids f o r m e d endogenously f r o m (1-14C)acetate in the rat ventral prostate are shown in Table 2. Acetate incorporation into total phospholipids at 2 hours of incubation time was 4.7 nmol/g and accounted for 2 9 - 3 1 % of the total labelled lipids. A c e t a t e was mainly incorporated into PC (78%) and PE (11.4%) which, together, accounted for 89% of the total acetate incorporation into phospholipids. In our experimental conditions we did not observe acetate incorporation into PI + LPE. Figure 1 shows the time course of acetate, palmitate and linoleate incorporation into different phospholipid classes in the ventral prostate. T h e results are expressed per unit mass of phospholipid as determined by us in a previous p a p e r (2). The highest incorporation of acetate, palmitate and linoleate was observed into PC. H o w e v e r , SM incorporation of acetate and palmitate was higher than that of linoleate, whereas the incorporation of linoleate into PI + L P E was greatly augmented with respect to the incorporation of acetate and palmitate. Figure 1 also shows that the incorporation of the three substrates was almost lineal during the first 3 hours in all the phospholipid classes.

Phospholipid synthesis in rat prostate

E~ r

30

acetate

palmitate

t3.. :_: 0

Nm

20

~2

::3 e -

"- 0

10

2

4

0 2 4 0 Time (hours)

2

4

Fig. 1. Time course of linoleate palmitate and acetate incorporation into PC (A), PE (O), PS (O), SM (C)) and P I + L P E (~r) from rat ventral prostate. Slices (40 mg) were incubated with 1 ~Ci labelled fatty acid bound to albumin (0.75 : 1 molar ratio) or 2 l*Ci of labelled acetate at 37~ Results are the means of 6 experiments. Error bars are omitted for clarity. F i g u r e 2 s h o w s t h e results o b t a i n e d in a t i m e c o u r s e e x p e r i m e n t for t h e r a d i o a c t i v i t y i n c o r p o r a t e d into CO2 f r o m ( 1 - t a C ) a c e t a t e , (1-14C)palmitate a n d ( 1 J 4 C ) l i n o l e a t e o x i d a t i o n . W e can s e e f r o m this figure t h a t the d e g r a d a t i o n o f p a l m i t a t e into CO2 was s i m i l a r to t h a t o f l i n o l e a t e a n d b o t h i n c r e a s e d d u r i n g t h e first t h r e e h o u r s o f i n c u b a t i o n in rats fed with a z i n c - c o n t a i n i n g diet. A c e t a t e s h o w e d t h e h i g h e s t o x i d a t i o n to CO2, i n c r e a s i n g during t h e first h o u r o f i n c u b a t i o n , a n d r e m a i n i n g f a i r l y c o n s t a n t a f t e r this p e r i o d o f t i m e in rats t h a t w e r e f e d a z i n c - d e f i c i e n t diet. O n i n c u b a t i o n in vitro v e n t r a l p r o s t a t e s f r o m rats f e d a zinc-deficient d i e t s h o w e d a significantly h i g h e r level o f a c e t a t e o x i d a t i o n t h a n v e n t r a l p r o s t a t e s f r o m r a t s f e d a d i e t c o n t a i n i n g zinc. N o significant differences in a c e t a t e o x i d a t i o n to CO2 in t h e v e n t r a l p r o s t a t e w e r e f o u n d b e t w e e n rats f e d a c o m m e r c i a l diet a n d t h o s e f e d a s e m i s y n t h e t i c z i n c - c o n t a i n i n g d i e t ( d a t a n o t shown).

1501 O3

100 Fig. 2. Time dependence of the formation of 14CO2 from linoleate (E]), palmitate (0) and acetate (solid symbols) in rat ventral prostate. Incubation conditions are as in Fig. 1. The results are expressed as nmol. of labelled substrate degradated per g of rat ventral prostate. (O), Acetate of rats feed with a control or commercial diet. (11), Acetate in rats feed with a zinc deficient diet.

-~ g 5c E

IZ

0

1

2

"flme (hours)

3

110

del Hoyo, Pulido, Carretero and P6rez-Albarsanz DISCUSSION

Results from the investigations reported in this paper indicate that palmitate, linoleate and acetate are all incorporated into rat ventral prostate phospholipids. Linoleate esterification to prostate phospholipids was faster than palmitate esterification. Some information has also been obtained in other tissues on the availability of these fatty acids as substrates for lipid esterification. Linoleate uptake by phospholipids was higher compared with that of palmitate in the incubations of gastroanemius muscle slices from the rat (22). In human platelets (23), similar results were also observed. However, in rats linoleate was not esterified to phospholipids faster than was palmitate in slices of red and white muscle (24). Our results showing high linoleate incorporation in rat prostate, cannot be explained by reference to the fatty acid composition of the total phospholipids because the amount of palmitic acid in the phospholipids is 5 or 6 times higher than the amount of linoleic acid (1). All substrates palmitate, linoleate and acetate show the highest incorporation into PC per unit mass of PC. These data agree with the high PC content reported by Pulido et al. (2) in rat ventral prostate. Our present results support the conclusion that the incorporation of linoleate into PC per unit mass of PC was greater than for palmitate. However in previous work we reported (2) that the concentration of palmitate in PC is about 10 times higher than was that of linoleate. A possible explanation of this discrepancy involves enzymatic factors since, in rat liver, it has been reported (25, 26) that microsome activity in 1-acylglycerophosphorylcholine acyltransferase has a higher selectivity for the polyunsaturated fatty acids than for the saturated ones. Studies on the incorporation of essential fatty acids and their products into complex lipids has shown that the fatty acid 2 0 : 4 ( n - 6 ) , the major metabolic product of the fatty acid 1 8 : 2 ( n - 6 ) , was preferentially esterified to PC (10, 11). In addition to this finding, the lower incorporation of palmitate into PC compared with linoleate incorporation may also be due to isotopic dilution with palmitate from de n o v o biosynthesis. The above data, together with the high PC content in the rat ventral prostate (2), may account for the major incorporation of linoleic acid into rat ventral prostate phospholipids. The specific activities of linoleate and palmitate in the other phospholipid classes studied by us corresponded to the previously reported mass fatty acid composition in these phospholipid classes (2). The radiolabelled fatty acid concentrations used in this study (9 ~tM) are similar to those reported in the literature (27). However, other authors have used higher (100-500~tM) (28) or lower (11-3600nM) (29) radiolabelled substrate concentrations. In this way, Chander et al. (30) demonstrated that 100 IxM of exogenous palmitate or linoleate increased the synthesis of PC by 50% during 3 hour incubations in rat granular pneumocytes. We have found that the incorporation pattern for acetate, in phospholipid classes, is similar to that for palmitate surely due to the fact that palmitate is the main product of de n o v o synthesis from acetyl CoA. Our results demonstrate a higher acetate oxidation to CO2 in rats fed a zinc-free diet compared with rats fed a diet containing zinc. These results are in

Phospholipid synthesis in rat prostate

111

agreement with Costello and Franklin (31) who have found that zinc inhibits citrate oxidation in in vitro incubations with rat ventral prostate. These authors explained this fact by an aconitase inhibition at high zi,a~l[~centrations. After 2 hours incubation ventral prostate oxidized much more acetate to CO2 as compared with palmitate and linoleate. This result can explain the lower rates of acetate incorporation into the different phospholipid classes compared with both palmitate and linoleate. Linoleate and palmitate showed the same oxidation to CO2 profile. These data suggest that the presence of double bonds in linoleic acid does not affect its utilization for energy in rat ventral prostate. ACKNOWLEDGEMENTS

This work was supported by the Fondo de Investigaciones Sanitarias de la Seguridad Social (grant 89/0314). REFERENCES 1. P6rez-Albarsanz, M. A., del Hoyo, N., Alcaide, A. and Reclo, M. N. (1982) Comp. Biochem. Physiol. 72B: 673-675. 2. Pulido, J. A., del Hoyo, N. and P6rez-Albarsanz, M. A. (1986) Biochim. Blophys. Acta 879: 51-55.

3. 4. 5. 6.

Recio, M. N., del Hoyo, N. and P6rez-Albarsanz, M. A. (1988) Biochem. Int. 16: 1-13. Franklin, R. B., Costello, L. C. and Littleton, G. K. (1977) Enzyme 22, 45-51. H~irk6nen, P. L., Kostian, M. L. and Santii, R. S. (1982) Arch. Andrology 8, 107-116, 1982. Franklin, R. B., Kahng, M. W., Akuffo, V. and Costello, L. C. (1986) Horm. Metabol. Res.

7. 8. 9. 10. 11. 12.

Burr, G. O. and Burr, M. N. (1930) J. Biol. Chem. 86, 587-621. Holman, R. T. and Hofstetter, H. H. (1965) J. Am. Oil. Chem. Soc. 42:540-544. Hansen, H. S. (1986) Trends Biochem. Sct. 11, 263-365. Cook, H. W., Clarke, J. T. R. and Spence, M. W. (1983) J. Biol. Chem. 258, 7586-759l. Cook, H. W. and Spence, M. W. (1987) Biochim. Biophys. Acta 918, 217-229. Cavanaugh, A. H., Farnsworth, W. E., Greizerstain, H. B. and Wojtowicz, C. (1980) Ltfe Sci. 26, 29-34. Farnsworth, W. E. (1981) In: The Prostattc Cell: Structure and Function Part A, (Murphy, Sandberg and Karr eds.) A.R. Liss New York. pages 225-230. Hoyo del, N., Torre, L. A. and P6rez-Albarsanz, M. A. (1984) Comp. Biochem. PhysLol. 78B : 299-302. Carmena, M. J., P6rez-Albarsanz, M. A. and Recio, M. N. (1984) Comp. Btochem. Physiol. 79B : 633-636. Recio, M. N., del Hoyo, N., Carmena, M. J. and P6rez-Albarsanz, M. A. (1985) Int. J. Biochem.

18:177-181.

13. 14. 15. 16.

17:1129-1132.

17. Schaffner, C. P., (1981) In: The Prostatic Cell: Structure and Function Part A. (Murphy, Sandberg and Karr eds.) A.R. Liss New York. pages 279-324. 18. Lopez-Aparicio, P., Hoyo del, N. and Perez-Albarsanz, M. A. (1988) Pest. Blochem. Physiol. 31: 109-119. 19. Bligh, E. G. and Dyer, W. J. (1959) Can. J. Biochem. Phystol. 37:911-917. 20. Alonso, F., Garcia Gil, M. Sanchez-Crespo, M. and Mato, J. M. (1982) J. Biol. Chem. 257 : 3376-3378. 21. Gutlerrez Ocafia, M. T., Recio, M. N., Lopez-Aparicio, P. and P6rez-Albarsanz, M. A. (1989) Biochem. Biophys. Acta. (In press.) 22. Spector, A. A., Hoac, J. C., Warner, E. D. and Fry, G. L. (1970) J. Chn. Invest., 49: 1489-1496. 23. Suzuki, and Kimura, S. (1971) Proc. J. C. B. L., 134:143-146 (In Japanese). 24. Okano, E. and Shimojo, T. (1982) Biochem. Biophys. Acta 710:122-127.

112

del Hoyo, Pulido, Carretero and PErez-Albarsanz

25. Lands, W. E. M., Inoue, M., Suguiura, Y. and Okuyama, H. (1982) J. Biol. Chem. 257:14968-14972. 26. Iritani, N., Ikeda, Y. and Kajitani, H. (1984) Biochim. Biophys. Acta 793:416-422. 27. Tocher, R. D. and Sargent, J. R. (1986) Biochim. Biophys. Acta 876:592-600. 28. Glatz, J. F. C., Jacobs, A. E. M. and Veerkamp, J. H. (1983) Biochim. Biophys. Acta 794: 454-465. 29. Swendsen, C. L., Chilton, F. H., O'Flaherty, J. T., Surles, J. R., Piantadosi, C., Waite, M. and Wykle, R. L. (1987) Biochim. Biophy. Acta 919:79-89. 30. Chander, A. and Fisher, A. B. (1988) Biochim. Biophys. Acta 958:343-351. 31. Costello, L. C. and Franklin, R. B. (1981) Enzyme 26:281-287.