Comp. Biochem. Physiol. Vol. 102B, No. 4, pp. 897-904, 1992
0305-0491/92 $5.00 + 0.00 © 1992 Pergamon Press Ltd
Printed in Great Britain
THE BIOSYNTHESIS OF P O L Y U N S A T U R A T E D FATTY ACIDS BY RAT SERTOLI CELLS H. OULI-IAJ,S. HUYNH* and A. NOUVELOT Laboratoire de Biochimie, URA CNRS 609, Universit6 de Caen, 14032 Caen Cedex, France (Received '27 November 1991)
Abstract--1. The biosynthesis of polyunsaturated fatty acids (PUFA) of the n-6 and n-3 series was investigated in cultured Sertoli cells. 18 : 2n-6, 18 : 3n-6, 20: 2n-6, 18 : 3n-3 and 20: 3n-3 were added individually at a concentration of 20 pmol to culture media. 2. Maximum incorporation of 20- and 22-carbon PUFA into membrane lipids was observed after 72 hr of incubation with all the exogenous substrates used. 3. As reported in other cell systems, the A6 desaturation was the first rate-limiting step; the major factor regulating this activity was the concentration of linoleic acid or ct-linolenic acid in the medium. 4. Our data show that the A5-desaturation represents a second regulatory step in PUFA biosynthesis. 5. The sum of n-6 and n-3 PUFA of the 22 carbon chain length constantly represented between 11 and 12% of total fatty acids, regardless of the exogenous substrate used. 6. Our kinetic studies of the incorporation of PUFA of the n-6 and n-3 series did not permit detection of a A8 desaturase activity.
INTRODUCTION
MATERIAL AND METHODS
It is generally accepted that Sertoli cells supply the nutritive substances required for the development of testicular germinal cells. Coniglio and Sharp (1989) were the first to show that cultured Sertoli cells are capable of synthesizing arachidonic acid from linoleic acid. We recently demonstrated (Huynh et al., 1991) that these cells are capable of incorporating and utilizing both linoleic and ~-linolenic acids and thus of synthesizing 20- and 22-carbon polyunsaturated fatty acids ( P U F A ) of the n-3 as well as of the n-6 families. In the other cell systems studied to date (Lemarchal, 1989; Brenner, 1989), the most c o m m o n metabolic pathway for P U F A biosynthesis is the one beginning with a A6 desaturation and followed alternately by elongation, A5 desaturation, another elongation and A4 desaturation. An active A8 desaturase has been reported in the rat testis, and although the cell type involved was not determined (Albert and Coniglio, 1977), this finding has raised the possibility of the existence of a P U F A metabolic pathway involving A8 desaturation in the Sertoli cell. The present work was designed to investigate the metabolic pathways involved in the biosynthesis of n-6 and n-3 P U F A in the Sertoli cell. Linoleate (18 : 2n-6), ~-linolenate (18 : 3n-3), y-linolenate (18 : 3n-6), eicosadienoate (20:2n-6) and eicosatrienoate (20:3n-3) were added individually to the culture medium, and kinetic studies were performed to establish whether or not the A8 desaturase is active in Sertoli cells.
*Author to whom correspondence should be addressed. 897
Reagents
Linoleic (18:2n-6), ~,-linolenic (18:3n-6), eicosa 11-14 dienoic (20: 2n-6), ~ -linolenic ( 18 : 3n-3) and eicosa 11- 14-17 trienoic (20:3n-3) acids, and fatty acid-free bovine serum albumin were purchased from Sigma Chemical Co. (France). Organic solvents were obtained from Merck (Germany). All other chemicals were of the highest chemical purity available commercially. Cell culture
Purified Sertoli cell-enriched aggregates were isolated from testes of 18-day-old rats by sequential enzymatic digestions, at 32°C, essentially as described by Tung et al. (1984). Briefly, decapsulated testes were treated by trypsin/DNAse in Hank's buffer for 25 mn. After addition of soybean trypsin inhibitor, interstitial cells were discarded, and seminiferous tubules were washed three times, submitted to digestion by a mixture of collagenase and testicular hyaluronidase (25 mn), and recovered by unit-gravity sedimentation. They were digested for another 25 mn with hyaluronidase alone. The resulting aggregates were centrifuged (400g, 10mn), washed twice with Hank's buffer containing 1% bovine serum albumin, then twice with 0.1 mM EDTA in Ca 2÷- and Mg2+-free Hank's buffer, and again twice with 1% bovine serum albumin/Hank's buffer. Cell aggregates were suspended in DMEM-HAM F12 (1 : 1; v/v) supplemented with 0.15 mmol HEPES, 2% Ultroser SF and antibiotics (50 UI/ml penicillin, 50 pg/ml streptomycin and 0.25 pg/ml fungizone). About 1500 aggregates/cm2 were seeded onto 75cm 2 plastic flasks to obtain confluent cultures. Cells were maintained in a humidified atmosphere containing 3% CO2 at 32°C. Culture medium was renewed 24 hr later and replaced by DMEM-HAM F12 without Ultroser SF. On Day 3, germ cells were removed by brief hypotonic treatment according to the method of Galdieri et al. (1981).
898
H. OULHAJ et al.
Addition of fatty acids to culture medium
RESULTS
Fatty acids (20/~M) were added to the culture medium on Day 5. They were introduced as fatty acid salts bound to fatty acid-free bovine serum albumin at a molar ratio of 12:1 according to the procedure of Hendry and Possmayer (1974) and Hamilton and Cistola (1986). Incubation of cultured cells in the presence o f linoleic acid was performed in eight flasks; four additional flasks, with cells in the presence o f the culture medium alone, served as controls. At 24 hr intervals, the cells from two experimental and one control flasks were harvested. Culture medium was changed at 48 hr in the flasks to be harvested at 72 and 96 hr. The same procedure was followed for each of the fatty acids studied. In the same conditions, other kinetic studies of incorporation o f 18:2n-6 or 20:2n-6, using short times (3, 5, 7, 9, 12 hr) were carried out. Cultured cells from each individual flask were first washed twice with 1 m M HNaCOa, pH 7.4, and harvested with a rubber spatula. Membrane fractions were prepared according to the method o f Lories et al. (1986) and Kartner et al. (1979). The cell suspension was rapidly cooled, supplemented with I m M EDTA and homogenized with a Potter; nuclei, large cellular particles and unbroken cells were removed by centrifugation at 4°C (25,000g for 30 mn). The results presented were obtained from four different cultures.
Fatty acid analysis Lipids were extracted with chloroform/methanol (2:1, v/v) according to the method of Folch et al. (1957), and fatty acid methyl esters were prepared as described by Hagenfeldt (1966). Before methylation, nonadecanoic acid was added to the mixture as internal standard. Fatty acids were analyzed as methyl esters on a Varian 3 300 gas chromatograph equipped with a flame ionization detector, using a CP Sil 88 capillary column (50 m x 0.2 mm i.d.). The temperature was programmed at 60°C for I min, from 60 to 150°C at 30°C/min, and from 150 to 210°C at 40°C/min. Fatty acids were identified by comparison with standards and GLC peak areas were measured with a Merck D 2000 integrator.
Addition o f -linolenate )
essential
fatty
acids
(linoleate
Linoleate (20/~mol) added to the culture medium (Table 1). A f t e r the a d d i t i o n o f 18:2n-6 to the culture m e d i u m , m a x i m u m i n c o r p o r a t i o n o f 20- a n d 22c a r b o n n-6 P U F A into m e m b r a n e lipids w a s o b t a i n e d after 72 h r o f i n c u b a t i o n . D u r i n g the first 24 hr, a n increase in all n-6 P U F A was o b s e r v e d , w i t h t h e e x c e p t i o n o f 22: 5n-6, w h i c h o n the c o n t r a r y , t e n d e d to decline. T h e p r o p o r t i o n o f s a t u r a t e d fatty acids r e m a i n e d virtually c o n s t a n t , w h e r e a s the p e r c e n t a g e o f 16- a n d 18-carbon m o n o - u n s a t u r a t e d acids s h o w e d a p r o gressive decrease, d r o p p i n g by a b o u t 4 9 % over t h e 96 h r o f i n c u b a t i o n . E x o g e n o u s 18:2n-6 d i d n o t a p p e a r to induce a n y n o t a b l e c h a n g e in t h e overall p r o p o r t i o n s o f 2 2 - c a r b o n n-3 P U F A (Table 1). T h e m a s s i v e i n c o r p o r a t i o n o f 1 8 : 2 n - 6 a n d the low p r o p o r t i o n o f 1 8 : 3 n - 6 o b s e r v e d in the m e m b r a n e lipids as early as the first h o u r s o f i n c u b a t i o n reflect a r a p i d i n h i b i t i o n o f the A6 d e s a t u r a s e . T h e c o n t i n ued increase in 2 0 : 2 n - 6 o v e r time revealed a c h a i n e l o n g a t i o n o f 18:2n-6 f r o m t h e b e g i n n i n g o f incub a t i o n , raising t h e possibility t h a t this 2 0 : 2 n - 6 c o u l d be c o n v e r t e d to 20: 3n-6 by a A8 d e s a t u r a t i o n . H o w ever, the existence o f a n active A8 d e s a t u r a s e d o e s n o t exclude t h e classic p a t h w a y involving t h e A6 d e s a t u r a s e f o l l o w e d by a n elongase, since 18:3n-6 s y n t h e s i z e d by A6 d e s a t u r a s e activity c a n b e r a p i d l y c o n v e r t e d into 20: 3n-6. -Linolenate (20/~mol) added to the culture medium (Table 2). A f t e r t h e a d d i t i o n o f 18:3n-3 to the culture m e d i u m , m a x i m u m i n c o r p o r a t i o n o f 20- a n d 22c a r b o n n-3 P U F A into m e m b r a n e lipids w a s o b t a i n e d at 48 h r o f i n c u b a t i o n . T h e a c c u m u l a t i o n o f 18:3n-3 a n d t h e low p r o p o r t i o n o f 18:4n-3 o b s e r v e d in the
Table 1. Distribution (in %) of fatty acids in lipids of Sertoli cell membranes as a function of incubation time in the presence of linoleate 20/~M added to the culture medium Incubation times (hr) Fatty acids 16:0 16:1n-9 16:In-7 18:0 18:1n-9 18:ln-7 18:2n-6 18:3n-6 18:4n-3 20: In-9 20: 2n-6 20:3n-6 20:4n-6 20: 5n-3 22: 0 22:4n-6 22:5n-6 22:5n-3 22:6n-3 T(n-3) T(n-6)
or
0 26.98 + 2.91 2.61 + 0.35 2.22 + 0.31 8.46 -t-0.79 21.65_ 2.11 10.57 + 1.10 1.79 __.1.18 0.00 0.01 0.51 1.47 + 0.18 0.76 + 0.11 9.10 + 0.85 0.06 0.76 2.72 + 0.29 5.48 + 0.52 1.92 3.94 + 0.40
24 23.70 + 2.24 1.25 + 0.15 1.52 __.0.14 8.31 + 0.80 16.83_ 1.55 6.81 + 0.62 14.06 + 1.45 0.11 0.11 0.44 2.67 _ 0.28 1.86 _+0.17 10.25 _+ 1.10 0.00 0.47 3.29 + 0.33 5.39 + 0.42 1.58 3.37 + 0.36
48 24.12 __.2.41 1.26 + 0.13 1.64 _ 0.17 8.81 _ 0.69 13.61 + 1.35 5.61 _ 0.48 16.50 + 1.61 0.13 0.13 0.26 3.44 + 0.34 2.10 _ 0.23 11.07 + 1.10 0.00 0.45 3.01 _ 0.26 4.10 + 0.39 1.65 3.20 + 0.27
72 23.45 +_2.21 1.14 _+0.12 1.13 +_0.13 8.71 _+0.67 11.61 -t- 1.01 5.07 + 0.48 15.30 __.1.39 0.35 0.20 0.23 3.97 + 0.31 2.18 + 0.19 11.97 _+ 1.20 0.00 0.68 3.44 + 0.29 4.22 + 0.31 1.61 3.09 + 0.29
96 24.29 + 3.01 1.01 + 0.18 1.45 + 0.15 9.00 + 0.81 11.42 __. 1.01 5.01 _ 0.49 13.23 +_ 1.29 0.30 0.21 0.25 3.77 __.0.36 1.90 + 0.18 12.45 + 1.24 0.00 0.46 3.68 + 0.25 3.95 + 0.23 1.52 2.77 + 0.26
5.94 21.30
5.06 37.67
4.98 40.35
4.90 41.45
4.51 39.38
T(n-6): totality of the n-6 PUFA; T(n-3): totality of the n-3 PUFA.
899
Biosynthesis o f P U F A b y Sertoli cells Table 2. Distribution (in %) of fatty acids in lipids of Scrtoli cell membranes as a function of incubation time in the pre~nce of ~-Iinolenate (20/tM) added to the culture medium Incubation times (hr) Fatty acids 16:0 16:In-9 16:ln-7 18:0 18:1n-9 18: I n-7 18:2n-6 18: 3n-6 18:3n-3 18: 4n-3 20:0 20: In-9 20: 2n-6 20:3n-6 20:4n-6 20: 3n-3 20:4n-3 20:5n-3 22:4n-6 22:5n-6 22:5n-3 22:6n-3 T(n-3) T(n-3)- i 8: 3
Standards
24
48
72
32.16 ± 1.94 1.55 ± 0.15 2.08 ± 0.17 9.12 ± 0.99 19.89 ± 0.97 6.02 ± 0.46 1.35 ± 0.11 0.00 0.19 ± 0.09 0.00 0.21 0.91 0.17 0.99 ± 0.14 10.87 ± 1.01 0.11 _ 0.04 0.17 ± 0.07 0.14 ± 0.06 3.01 ± 0.18 6.17 _ 0.28 0.49 ± 0.07 4.21 ± 0.11
31.53 + 2.02 1.18 + 0.11 1.66 ± 0.13 8.99 ± 0.80 17.67 ± 0.99 5.63 ± 0.37 1.21 ± 0.12 0.00 2.87 ± 0.21 0.08 0.12 0.89 0.00 0.93 ± 0.11 10.09 ± 0.87 0.88 ± 0.04 0.32 ± 0.04 1.46 + 0.09 2.83 ± 0.21 5.80 5:0.22 0.69 ± 0.09 5.18 ± 0.13
30.28 5:0.99 0.79 ± 0.04 1.21 ± 0.10 8.81 ± 0.66 16.01 + 0.90 4.84 ± 0.38 1.19 ± 0.10 0.00 6.01 ± 0.54 0.22 0.00 0.71 0.09 0.81 ± 0,12 9.52 ± 0.54 1.84 ± 0,11 0.90 ± 0,12 2.14 ± 0,13 2.65 ± 0.21 5.43 ± 0.26 1.91 ± 0.15 5.94 ± 0.14
29.90 ± 1.22 0.58 ± 0.11 0.97 ± 0.11 8.62 ± 0.57 14.85 ± 1.01 4.27 ± 0.33 1.11 ± 0.09 0.00 9.01 ± 0.79 0.27 0.11 0.52 0.00 0.65 ± 0.13 9.09 ± 0.61 1.98 ± 0.24 0.86 + 0.15 2.21 _ 0.13 2.52 ± 0.17 5.13 ± 0.33 1.78 + 0.09 4.69 + 0.21
11.48 8.61
18.96 12.95
20.80 11.79
5.31 5.12
T(n-3): totality of the n-3 PUFA; T(n-3)-lS:3: totality of the n-3 P U F A without a-linolenate.
same concentration (20gmol), to different culture media.
membrane lipids reflected an inhibition of the A6 desaturase. Exogenous 18:3n-3 did not appear to induce change in the proportion of saturated fatty acids whereas the percentage of 16- and 18- carbon monounsaturated acids showed a progressive decrease. From the beginning of incubation, the proportion of 20: 3n-3 increased reflecting a chain elongation of 18:3n-3, raising the possibility that this 20:3n-3 could be converted to 20: 4n-3 by a A8 desaturation. In order to test the relative importance of the A6 and A8 metabolic pathways, the precursors of 20:3n-6 (18:3n-6 and 20:2n-6) and the precursor of 20:4n-3 (20:3n-3) were added individually, at the
Addition of precursors of 20:3n-6 and 20:4n-3 18:3n-6 (20#mol) added to the culture medium (Table 3). As with 18:2n-6, maximum incorporation of n-6 PUFA into membrane lipids was obtained after 72 hr of incubation. The proportion of 18:3n-6 did not exceed 1.5% of total fatty acids, even after 72 hr; in contrast, the proportion of 20:3n-6 increased from 0.7 to 16.5% over the same period, reflecting, on the one hand, an excellent incorporation of 18:3n-6 into the cells, and, on the other, a rapid elongation to 20:3n-6. This substantial
Table 3. Distribution (in %) of fatty acids in lipids of Sertoli cell membranes as a function of incubation time in the presence of 18:3n-6 (20 #M) added to the culture medium Incubation times (hr) Fatty acids 16:0 16:In-9 16: I n-7 18:0 18:In-9 18 : ln-7 18:2n-6 18:3n-6 18: 3n-3 18:4n-3 20:1n-9 20:2n-6 20:3n-6 20:4n-6 20: 5n- 3 22: 0 22:4n-6 22:5n-6 22: 5n-3 22:6n-3 T(n-3) T(n-6)
0
48
26.98 _ 2.54 2.61 _ 0.28 2.22 + 0.27 9.45 + 0.85 21.65 4- 2.10 9.57 ± 0.79 1.79 ± 0.20 0.00 0.01 0.01 0.51 1.47 ± 0.11 0.76 5:0.12 9.10 ± 0.84 0.06 0,76 2.72 ± 0.28 5.48 ± 0.52 1.92 3.94 ± 0.32
25.59 _ 2.71 1.55 5- 0.19 1.38 _ 0.14 9.06 _ 0.85 13.19 ± 1.29 3.37 + 0.31 1.71 5:0.18 1.16 ± 0.12 0.07 0.01 0.17 0.78 ± 0.09 14.33 _+ 1.41 14.62 ± 1.39 0.03 0.31 3.47 ± 0.31 4.42 ± 0.39 1.70 3.08 ± 0.35
24.97 _ 2.31 1.52 _ 0.17 1.22 ± 0.12 8.51 ± 0.80 ll.13 _ 1.10 3.19 + 0.29 1.49 5:0.16 1.49 _+0.15 0.00 0.01 0.16 0.51 ± 0.09 16.49 ± 1.51 15.83 ± 1.40 0.01 0.49 3.95 5:0.38 4,35 5- 0.45 1.63 3.06 ± 0.29
72
26.77 ± 3.01 1.56 ± 0.18 1.25 ± 0.13 9.72 ± 0.87 12.39 ± 1.21 3.45 ± 0.31 1.73 _+ 0.17 1.54 _+ 0.16 0.00 0.03 0.66 0.68 ± 0.15 13.40 5:1.32 14.13 ± 1.39 0.06 0.75 3.50 ± 0.37 3.43 5:0.31 1.88 2.96 5- 0.30
96
5.94 21.31
4.89 40.49
4.71 44.11
4.93 38.41
900
H. OULHAJ et al. Table 4. Distribution (in %) of fatty acids in lipids of Sertoli cell membranes as a function of incubation time in the presence of 20:2n-6 (20 ~M) added to the culture medium Incubation times (hr) Fatty acids
0
24
48
72
96
16:0 16:1n-9 16:ln-7 18:0 18:1n-9 18:1n-7 18:2n-6 18 : 3n-6 20: I n-9 20:2n-6 20:3n-6 20:4n-6 20:5n-3 22 : 0 22:4n-6 22:5n-6 22:5n-3 22: 6n-3
30.68 ± 2.24 1.66 ± 0.17 2.06 i 0.20 9.62 ± 0.87 19.85 ± 2.01 6.62 ± 0.65 1.43 ± 0.15 0.09 0.22 0.19 _+0.09 1.18 ± 0.12 10.16 ± 1.08 0.08 0.77 2.93 ± 0.27 6.03 ± 0.59 0.36 6.06
28.74 ± 2.77 0.93 ± 0.10 0.93 ± 0.09 9.21 ± 0.89 11.77 ± 1.10 1.87 ± 0.18 6.41 ± 0.61 0.10 0.13 10.31 ± 1.10 1.33 ± 0.20 12.59 ± 1.30 0.11 0.49 3.12 ± 0.29 6.02 ± 0.55 0.36 5.48
27.16 ± 2.65 0.45 ± 0.10 0.88 ± 0.11 8.25 ± 0.81 10.67 ± 1.09 1.68 ± 0.17 6.88 ± 0.65 0.05 0.08 8.37 ± 0.82 1.92 ± 0.19 15.77 ± 1.55 0.08 0.41 3.87 ± 0.36 6.06 ± 0.61 0.29 4.74
27.07 ± 2.65 0.37 ± 0.10 0.72 ± 0.12 8.24 ± 0.77 9.64 _+ 0.91 1.57 ± 0.16 8.15 ± 0.79 0.07 0.06 8.30 ± 0.79 2.34 ± 0.20 16.35 ± 1.61 0.14 0.35 3,96 ± 0.40 5,98 ± 0.61 0.25 3.95
26.63 ± 3.01 0.27 ± 0.08 0.62 ± 0.09 8.25 ± 0.80 9.25 ± 0.91 1.42 ± 0. l 5 8.89 ± 0.79 0.07 0.07 8.24 + 0.75 3.02 ± 0.23 16.23 ± 1.62 0.09 0.42 3.94 ± 0.35 5.16 ± 0.49 0.24 3.81
T(n-6) T(n-3)
22.02 6.50
39.87 5.66
42.92 5.10
45.16 4.34
45.54 4.14
18:2n-6 maintained the proportion of linoleic acid between 6 and 9%, permitting a more efficient activity of the A6 desaturase compared to that observed with exogenous linoleic acid (Tables 1 and 3). The proportion of 20:4n-6 obtained with exogenous 20:2n-6 was comparable to that obtained with 18:3n-6, that is, when the first regulatory step (A6 desaturation) was skipped. With both these substrates, the proportion of arachidonic acid was close to 16% at 72 hr. In contrast, the proportion of its precursor (20:3n-6) was 16.5% with 18:3n-6 and 2.3% with 20:2n-6. These observations appear to indicate that the A5 desaturation represents a second rate-limiting step in P U F A biosynthesis in the Sertoli cell. 20:3n-3 (20gM) added to the culture medium (Table 5). Addition of 20:3n-3 induced principally increases in the proportions of 20: 3n-3 and 18 : 3n-3 in lipids of Sertoli cells membranes. The utilization
elongase activity thus explains our preceding results relative to 18:2n-6. The 18:3n-6 obtained by A6 desaturation cannot accumulate since it is immediately converted into 20:3n-6. The overall proportion of 22-carbon PUFA of the n-3 and n-6 series consistently represented between 11 and 12% of total fatty acids, regardless of the exogenous precursor used (Tables 1, 2 and 3). 20:2n-6 (20/~mol) added to the culture medium (Table 4). The addition of 20:2n-6 induced only slight increases in the proportion of 20: 3n-6, and the values observed remained comparable to those obtained after the addition of 18:2n-6. A A8 desaturation thus appears to be unlikely. However, the increase in the proportion of 18:2n-6 to about 6% of total membrane PUFA within the first 24 hr indicates that the utilization of 20:2n-6 involves a retroconversion to 18:2n-6 which then becomes substrate for the A6 desaturase. The retroconversion of 20:2n-6 to
Table 5. Distribution (in %) of fatty acids in lipids of Sertoli cell membranes as a function of incubation time in the presence of 20:3n-3 (20,uM) added to the culture medium Incorporation times (hr) Fatty acids 16:0 16:1n-9 16:1n-7 18:0 18:In-9 18:1n-7 18:2n-6 18:3n-3 18:4n-3 20: 0 20:In-9 20: 2n-6 20:3n-6 20:4n-6 20:3n-3 20:4n-3 20:5n-3 22:4n-6 22:5n-6 22:5n-3 22:6n-3 T(n-3)
Standards
24
48
72
30.58 ___1.89 1.41 ±0.19 1.96 + 0.17 8.97 ± 0.90 19.06 ± 1.00 5.32 ± 0.56 1.56 ± 0.21 0.19 ± 0.11 0.00 0.32 0.90 0.28 1.00 ± 0.09 11.38 ± 0.55 0.11 ± 0.10 0.17 ± 0.09 0.11 ± 0.05 2.77 ± 0.21 5.55 + 0.54 0.37 ± 0.03 4.17 ± 0.33
29.59 ± 1.46 1.58 ± 0.21 1.62 ± 0.18 8.24 ± 0.77 17.04 ± 0.98 5.84 ± 0.37 1.56 ± 0.31 2.56 ± 0.12 0.10 0.28 0.86 0.16 1.46 ± 0.09 13.68 ± 0.41 3.21 ± 0.26 0.23 ± 0.04 0.19 ± 0.04 1.98 ± 0,20 4.92 ± 0,47 0.46 ± 0.05 4.64 ± 0,39
27.22 ±- 2.04 1.25 ± 0.23 1.56 ± 0.20 7.14 ± 0.79 16.37 ± 0.84 4.15 ± 0.43 1.47 ± 0.21 4.19 ± 0.23 0.06 0.33 0.49 0.22 0.97 ± 0.11 13.11 ± 0.99 4.46 ± 0.34 0.88 ± 0.10 1.93 ± 0.06 1.75 ± 0.21 4.46 ± 0.41 2.12 ± 0.30 6.13 ± 0.53
26.14 ± 2.00 1.11 +0.21 1.24 ± 0.13 7.02 ± 0.55 14.06 ± 0.85 2.35 ± 0.19 1.47 _+0.14 6.97 ± 0.27 0.06 0.322 0.40 0.23 0.82 ± 0.10 12.93 ± 1.01 6.96 ± 0.55 1.48 ± 0.09 2.93 ± 0.12 1.41 + 0.15 4.33 ± 0.33 2.73 + 0.17 5.14 + 0.43
5.11
11.40
19.71
26.27
Biosynthesis o f P U F A by Sertoli cells
901
Table 6. Distribution (in %) of fatty acids in lipids of Sertoli cell membranes as a function of incubation time (short) in the presence of linoleate (20 # M ) added to the culture medium Incubation times (hr) Fatty acids 16:0 16:1n-9 16:ln-7 18:0 18:1n-9 18:1n-7 18:2n-6 18: 3n-6 18 : 3n-3 20:ln-9 20:2n-6 20:3n-6 22: 0 20:4n-6 20: 5n-3 22:4n-6 22:5n-6 22:5n-3 22:6n-3 T(n-6) T(n-6)-I 8: 2
0
3
5
7
9
28.19 + 1.81 2.21 _ 0.25 1.94 + 0.20 8.57 + 0.81 18.58+ 1.53 11.06 + 0.99 1.65 + 0.22 0.00 0.00 0.46 0.97 + 0.18 0.85 + O.19 0.36 11.14 + 1.01 0.04 2.68 + 0.29 5.55 + 0.59 1.63 4.11
28.20 + 1.71 2.01 + 0.24 1.75 4- 0,21 8.51 + 0,84 17.61 + 1,41 10.11 + 0.95 3.61 + 0.31 0.00 0.00 0.45 1.10 + 0.21 1.09 + 0.22 0.36 11.09 + 1.01 0.00 2.75 + 0.31 5.64 + 0.60 1.62 4.01
28.11 + 1.52 1.99 4- 0.25 1.76 + 0.21 8.55 + 0.82 17.10+ 1.32 9.87 + 0.89 4.30 + 0.52 0.00 0.00 0.45 1.32 + 0.20 1.19 + 0.22 0.37 11.35 + 1.02 0.00 2.68 + 0.30 5.44 + 0.57 1.53 3.99
27.91 + 1.95 1.78 + 0.20 1.53 + 0.23 8.33 + 0.81 15.84+ 1.41 9.01 + 0.87 7.11 + 0.67 0.00 0.00 0.41 1.49 + 0.19 1.38 + 0.20 0.31 11.63 + 1.09 0.00 2.60 + 0.30 5.31 + 0.58 1.49 3.87
27.64 _+ 1.87 1.49 + 0.21 1.22 4- 0.24 8.12 + 0.75 14.92 + 1.36 8.04 4- 0.79 10.03 + 0.87 0.00 0.00 0.39 1.41 + 0.17 1.61 +_0.23 0.30 11.81 + 1.11 0.00 2.54 _ 0.29 5.11 + 0.56 1.50 3.87
27.14 + 1.53 1.27 + 0.27 1.11 + 0.26 8.01 + 0.79 13.11 + 1.28 7.01 -t- 0.80 13.47 + 1.11 0.00 0.00 0.36 1.51 +_0.21 1.83 + 0.19 0.27 12,11 +_ 1.19 0.00 2,66 + 0.29 5.08 + 0.60 1.38 3.84
26.28 21.98
29.52 22.41
32.51 22.48
36.66 23.19
22.84 21.19
25.28 21.67
of 20:3n-3 involves a rctroconversion to 18:3n-3 which then becomes substrate for the A6 desaturase. This retroconversion maintained the proportion of ct-linolenic acid between 2 and 7%, permitting a more efficient activity of the A6 desaturase compared to that observed with exogenous ~t-linolenic acid. The addition of 20:3n-3 induced only slight increases in the proportion of 20:4n-3 and the values observed were nearly the same as those obtained after addition of 18:3n-3. A A8 desaturation thus appears to be unlikely. However it is possible that there is A8 as well as A5 desaturase activity present in Sertoli cells. The major pathway using A6, A5 desaturases may be able to hide the second one as soon as retroconversion of 20: 2n-6 to 18:2n-6 or 20:3n-3 to 18:3n-3 supplies the A6 desaturase substrate. In order to test the relative importance of these two metabolic pathways we made brief kinetic
12
studies of 18:2n-6 incorporation or 20:2n-6 incorporation. Incorporations Linoleate (20#M) added to the culture medium (Table 6). After the addition of 18 : 2n-6 to the culture medium, an increase of all n-6 PUFA into membrane lipids was obtained from the three first hours. This change was particularly important for linoleate and reflects a rapid inhibition of the A6 desaturase. 20:2n-6 (20#M) added to the culture medium (Table 7). When Sertoli cells were incubated with 20: 2n-6 in the culture medium, an increase of all n-6 PUFA in Sertoli cells membrane lipids was observed. For every experiment, this increase was more important than those obtained with linoleate incubation. The increase in the proportion of 18:2n-6 indicates that the utilization of 20: 2n-6 involves a retroconversion to 18:2n-6 observable after 3 hr.
Table 7. Distribution (in %) of fatty acids in lipids of Sertoli cell membranes as a function of incubation time (short) in the presence of 20:2n-6 (20/a M) added to the culture medium Incubation times (br) Fatty acids 16:0 16:1n-9 16:ln-7 18:0 18:In-9 18:1n-7 18:2n-6 18: 3n-6 20: In-9 20:2n-6 20:3n-6 22:0 20:4n-6 20:5n-3 22:4n-6 22:5n-6 22: 5n-3 22: 6n-3 T(n-6)
0
3
5
7
9
30.11 + 1.91 1.92 + 0.25 1.68 + 0.23 9.15 + 0.87 17.93 + 1.60 9.81 + 0.79 1.48 + 0.35 0.00 0.68 0.35 + 0.20 1.03 + 0.35 1.28 9.92 + 0.78 0.15 2.55 + 0.29 5.13 + 0.52 1.68 5.14
30.04 +_ 1.85 1.55 + 0.23 1.54 + 0.24 9.07 _+ 0.85 15.60 + 1.35 8.10 + 0.76 2.69 + 0.44 0.00 0.57 3.12 _ 0.44 1.06 + 0.31 1.18 9.96 + 0.83 0.13 2.56 _ 0.30 5.14 _+ 0.57 1.62 5.01
30.23 _ 1.99 1.44 + 0.30 1.33 _+ 0.31 9.10 + 0.90 14.99 + 1.39 7.91 + 0.80 3.01 _+0.42 0.00 0.59 4.31 + 0.45 1.28 + 0.29 1.19 10.29 _+ 0.99 0.14 2.60 + 0.32 5.18 + 0.60 1.60 4.97
29.40 _+ 1.96 1.38 _+ 0.40 1.27 _+ 0.36 8.98 _+0.85 14.53 _+ 1.46 7.50 +_0.81 3.97 _+ 0.45 0.00 0.56 4.98 + 0.52 1.39 + 0.40 1.16 10.67 + 1.02 0.11 2.61 _ 0.31 5.28 + 0.53 1.58 4.94
28.94 + 1.78 I. 13 _+0.32 1.11 + 0.23 8.91 + 0.87 14.07 + 1.37 7.31 + 0.75 4.35 + 0.44 0.00 0.54 5.54 + 0.49 1.61 + 0.27 1.14 11.19 + 1.01 0.10 2.67 + 0.37 5.40 + 0.58 1,59 4,93
28.51 + 1.77 1.07 + 0.29 1.01 + 0.22 8.62 _+0.89 13.21 + 1.21 7.01 +_ 0.69 4.96 + 0.47 0.00 0.53 6.62 + 0.60 1.86 + 0.30 1.12 I 1.52 _+ 0.99 0.10 2.69 _+ 0.28 5.44 + 0.53 1.57 4.92
12
20.66
24.53
26.67
28.90
30,76
33.09
902
H. OULHAJet al.
The proportion of linoleate in membrane lipids was always smaller than those observed after linoleate addition to culture medium, permitting a more efficient activity of the A6 desaturase and a better biosynthesis of n-6 PUFA. No increase in 20:3n-6 in Sertoli cells membrane lipids was observed during the first hours, and a A8 desaturase activity was not established.
hepatoma cells (Iturralde et al., 1990), and pig renal cells (Mahfouz et al., 1989). In the Sertoli cell, the proportion of 18:3n-6 in membrane lipids both after its direct addition to the culture medium and after the desaturation of 18 : 2n-6 remained low. This is in agreement with results obtained with hepatocytes; the data of Voss and Sprecher (1988) and Christophersen et al. (1982) suggest that when 18:3n-6 is obtained by desaturation of 18:2n-6 or when it is added directly as exogenous substrate, the major fraction of this DISCUSSION 18:3n-6 is initially incorporated into a labile With all the exogenous fatty acid substrates used, phospholipid pool and is then rapidly released for except for ~-linolenate, maximum incorporation and metabolism to arachidonate. The rates of acylation utilization of n-6 and n-3 PUFA were observed after using 1-acyl-sn-glycero-3-phosphocholine as acceptor are 10-50 times greater than those involved in the 72 hr of incubation. As reported previously (Huynh et al., 1991), the desaturations and chain elongations (Bernert and incorporation of n-6 PUFA had little effect on the Sprecher, 1977; Ludwig and Sprecher, 1979). After its overall proportion of n-3 PUFA, which showed only release, 18:3n-6 is metabolized to 20:3n-6 and then 20:4n-6, which is incorporated into the membrane slight variations. The addition of individual n-6 or n-3 fatty acids to phospholipids. The accumulation of 20:3n-6 obtained with the culture medium did not alter the distribution of saturated fatty acids in the membrane lipids, but exogenous 18:3n-6 resulted from an inhibition of the did induce a marked decrease in the proportion of A5 desaturase, the second step in PUFA desaturpalmitoleic and oleic acids. These observations are ation. While little is known about the regulation of consistent with the findings of de Gomez Dumm who the A5 desaturase activity, recent findings have reported that A9 desaturase activity in rat liver suggested that it differs markedly from that of the A6 microsomes was regulated by dietary alterations desaturase. The early decrease of A5 desaturase en(de Gomez Dumm et al., 1982). The addition of zyme in the fat-deficient rat accompanies a decrease 18:2n-6, 18:3n-6 or 20:4n-6 to fat-free diets resulted of acids of n-6 series whereas the A6 desaturase is in an inhibition of the conversion of palmitic acid to generally increased (Brenner, 1989). Both enzymes follow different directions under the same dietary palmitoleic acid. Similar results were obtained in vitro with rat liver microsomes or hepatocytes (Sprecher, change, demonstrating that A5 desaturase induction 1972; Volpe and Vagelos, 1976) regarding the has its own regulatory mechanism dependent on the presence or absence of substrates or related fatty desaturation of stearic acid to oleic acid. Our kinetic studies of the incorporation of linoleic acids. Jeffcoat and James (1977) and de Alaniz et al. acid showed that A6 desaturase activity rapidly be- (1980) have shown that a high-fat diet rich in 18:2n-6 comes the rate-limiting factor in the metabolism of stimulates A5 desaturase activity. We reported n-6 PUFA. The 6(n-6) ratio [(n-6 PUFA-18:2n-6)/ (Huynh et al., 1991) that maximum A5 desaturase (18:2n-6)| dropped abruptly from 10.2 to 1.6 after activity in the Sertoli cell was observed after the 12 hr of incubation, and then remained at this low addition of about 0.7 #g/ml of 18:2n-6 to the culture level up to 96 hr. In the same way, incorporation medium and that with higher concentrations, A5 of ct-linolenate showed that A6 desaturase activity activity decreased. The proportion of 20:4n-6 aprepresents a first regulatory step of n-3 PUFA bio- pears to be one of the major factors in the regulation synthesis. The 66(n-3) ratio [(n-3 PUFA- 18 : 3n-3)/ of this A5 desaturase activity in the Sertoli cell. The (18:3n-3)] dropped abruptly from 27 to 3 after 24 hr chain elongation of 20: 4n-6 to 22: 4n-6 did not seem of incubation and then remained at this low level up to be very efficient. On the other hand, the addition of 18:3n-3 or 20:3n-3 did not show an important to 72 hr. The regulation of the A6 desaturase appears to be accumulation of 20:5n-3 but small increases of 20closely linked to the proportion of 18:2n-6 present and 22-carbon n-3 polyenes reflecting good activities in the membrane lipids. When the incorporation of of A5 desaturase and elongase. However, 22:6n-3 18:2n-6 was limited, either by the addition of produced by the same reaction, is an important lower concentrations to the culture medium (Huynh component of liver lipids. The low level of the et al., 1991) or by retroconversion of 20:2n-6 elongation of 20 : 4n-6 has been described in other cell as observed here, the ratio ~6(n-6) [(n-6 PUFA)- systems and appears to be specific to the n-6 series. (18:2n-6+20:2n-6)/18:2n-6] was far higher (4.5 According to Christophersen and Hagve (1985) the times), reflecting a more efficient utilization of linoleic difference lies in substrate's efficiency. At least, they acid. In our experiments, this phenomenon was not showed an efficient elongation of 20:5n-3 acid to so important after addition of n-3 fatty acids because 22:5n-3 acid in isolated rat hepatocytes in the presincorporation of n-3 PUFA was always smaller than ence of lactate and correspondingly an active A4 that of n-6 PUFA. The important role of the A6 desaturation to 22:6n-3 acid. Rosenthal and Hill desaturase as the first rate-limiting step in PUFA (1986) also compared the metabolism of 20:4n-6 and biosynthesis has been widely documented in other of 20:5n-3 in human vascular endothelial cells in subcellular or cellular systems, such as rat liver culture; the endothelial cells were found to elongate 23% of 20:4n-6 compared to 72% of 20:5n-3 after microsomes (Brenner, 1981; de Gomez Dumm et al., 1983), rat hepatocytes (Voss and Sprecher, 1988), rat 25hr of incubation. Hagve and Christophersen
Biosynthesis of PUFA by Sertoli cells (1984) reported that the chain elongation of 20- to 22-carbon fatty acids was far more efficient for the n-3 than for the n-6 series, resulting in elevated proportions of 22: 5n-3 and 22: 6n-3. Although there is little documentation on the conversion of 22:4n-6 to 22:5n-6, it is generally accepted that this activity is weak in the liver, but is fairly important in the testicles and adrenals (Hagve and Christophersen, 1984; Ayala et al., 1973). We noted that for all the tested n-6 fatty acids, the sum of 22:4n-6 + 22: 5n-6 remained constant. Any increase, however slight, in 22:4n-6 was accompanied by an equivalent decrease in 22:5n-6. The sensitivity of the A4 desaturase to an increase in 22:4n-6 maintained the proportion of 22: 5n-6 between 4 and 6% of total fatty acids in the Sertoli cell membrane lipids. The systematic retroconversion of 22:4n-6 to 20:4n-6 shows that arachidonate remains the major end product of the n-6 series. Other investigators (Klenk and Mohrhauer, 1960), and more recently, Coniglio and Sharp (1989) have suggested that in the testis, arachidonic acid may be synthesized via a metabolic pathway involving a A8 desaturation of 20:2n-6 to 20:3n-6. Our data have shown that exogenous 20:2n-6 induced a marked increase in 18 : 2n-6 within 3 hr, whereas a noticeable increase in 20: 3n-6 was observed only after 7-9 hr. It would thus appear that in the Sertoli cell the A8 desaturase is either absent or has only a very slight activity. Retroconversion of 20:2n-6 supplies the 18:2n-6 used as substrate by the A6 desaturase. The metabolic pathways involved in the biosynthesis of n-6 and n-3 P U F A from 18:2n-6 and 18:3n-3 in the Sertoli cell thus appear to be the same as those described in other cell systems. REFERENCES Albert D. H. and Coniglio J. G. (1977) Metabolism of eicosa-11,14 dienoic acid in rat testes. Evidence for A8 desaturase activity. Biochim. biophys. Acta 489, 390-396. Ayala S., Gaspar G., Brenner R. R., Peluffo R. O., Kunau W. (1973) Fate of linoleic, arachidonic and docosa 7,10,13,16-tetraenoic acids in rat testicles. J. Lipid Res. 14, 296-305. Bernert J. T. and Sprecher H. (1977) An analysis of partial reactions in the overall chain elongation of saturated and unsaturated fatty acids by rat liver microsomes. J. biol. Chem. 252, 6736-6744. Brenner R. R. (1981) Nutritional and hormonal factors influencing desaturation of essential fatty acids. Prog. Lipid Res. 20, 41-47. Brenner R. R. 0989) Factors influencing fatty acid chain elongation and desaturation. In The Role of Fats in Human Nutrition (Edited by Vergroesen A. J. and Crawford M.), 2nd edn, pp. 45-70. Academic Press, London. Christophersen B. O., Hagve T. A. and Norseth J. (1982) Studies on the regulation of arachidonic acid synthesis in isolated rat liver cells. Biochim. biophys. Acta 712, 305 -314. Christophersen B. O. and Hagve T. A. (1985) Eicosapentaenoic and arachidonic acid chain elongation and A4 desaturation in isolated liver cells. In Proceeding of the 2nd International Congress o f Essential Fatty Acids, Prostaglandins and Leukotrienes, p. 8. London. Coniglio J. G. and Sharp J. (1989) Biosynthesis of arachidonic acid from linoleate in primary cultures of Sertoli cells. Lipids 24, 84-85.
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De Alaniz, M. J. T., de Gomez Dumm I. N. T. and Brenner R. R. (1980) Effect of fasting on A5 desaturation activity in rat liver mierosomes and HTC cells. Mol. cell. Biochem. 33, 165-170. De Gomez Durum I. N. T., Peluffo R. O., De Alaniz M. J. T. and Brenner R. R. (1982) Role of specific fatty acids administered in the diet on rat liver microsomal A9 and A6 desaturation activity. Anal. Assoc. Quire. Arg. 70, 383-393. De Gomez Durum I. N. T., Alaniz M. J. T. and Brenner R. R. (1983) Effect of dietary fatty acids on A5 desaturation activity and biosynthesis of arachidonic acid in rat liver microsomes. Lipids 18, 781-788. Folch J., Lees M. and Sloane Stanley G. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem. 226, 497-509. Galdieri M., Ziparo E., Palombi F., Russo M. and Stefanini M. (1981) Pure Sertoli cell: a new model for the study of somatic germ cell interactions. J. Androl. 2, 249-254. Hagenfeldt L. (1966) A gas chromatographic method for the determination of individual free fatty acids in plasma. Clin. Chim. Acta 13, 266-268. Hagve T. A. and Christophersen B. O. (1984) Effect of dietary fats on arachidonic acid and eicosapentaenoic acid biosynthesis and conversion to C22 fatty acids in isolated rat liver cells. Biochim. biophys. Acta 796, 205-217. Hamilton J. A. and Cistola D. P. (1986) Transfer of oleic acid between albumin and phospholipid vesicles. Proc. nam. Acad. Sci. U.S.A. 83, 82-86. Hendry A. T. and Possmayer F. (1974) Pulmonary phospholipid biosynthesis. Properties of a stable microsomal glycerophosphate acetyltransferase preparation from rabbit lung. Biochim. biophys. Acta 369, 156-172. Huynh S., Oulhaj H., Bocquet J. and Nouvelot A. (1991) Metabolic utilization of linoleate and ~-linolenate in cultured Sertoli cells. Comp. Biochem. Physiol. 99B, 265-270. lturralde M., Gonzalez B. and Pineiro A. (1990) Linoleate and linolenate desaturation by rat hepatoma cells. Biochem. Int. 20, 37-43. Jeffcoat R. and James A. T. (1977) Interrelations between the dietary regulation of fatty acid synthesis and the fatty acyl-CoA desaturases. Lipids 12, 469-474. Kartner N., Alon W., Swift M., Buchwald M. and Riordan J. R. (1979) Isolation of plasma membranes from human skin fibroblasts. J. membr. Biol. 36, 191-211. Klenk E. and Mohrhauer H. (1960) Z. Physiol. Chem. 320, 218-232. Lemarchal P. (1989) La r6gulation hormonale et nutritionhelle des d6saturases. Cab. Nutr. Di~t. XXIV, 99-106. Lories V., David G., Cassiman J. J. and Van der Berghe M. (1986) Heparan sulfate proteoglycans of human lung fibroblasts. Eur. J. Biochem. 158, 351-360. Ludwig S. and Sprecher H. (1979) Substrate specificity on the malonyl-CoA-depcndent chain elongation of all cis polyunsaturated fatty acids by rat liver microsomes. Archs Biochem. Biophys. 197, 333-341. Mahfouz M. M., Smith T. L. and Kummerow F. A. (1989) Changes of linoleic acid metabolism and cellular phospholipid fatty acid composition in LLKC-PK cells cultured at low magnesium concentrations. Biochim. biophys. Acta 1006, 70-74. Rosenthal M. D. and Hill (1986) Elongation of arachidonic acid and eicosapentaenoic acids limit their availability for thrombin-stimulated release from the glycerolipids of vascular endothelial cells. Biochim. biophys. Acta 875, 382-391. Sprecher A. W. (1972) Studies on the regulation of polyunsaturated fatty acid biosynthesis in the rat. Fed. Proc. 31, 1451-1457. Stoffel W. (1961) Biosynthesis of polyenoic fatty acids. Biochem. biophys. Res. Comm. 6, 270-273.
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Tung P. S., Skinner M. K. and Fritz I. B. (1984) Fibronectin synthesis is a marker for peritubular cell contaminants in Sertoli cell-enriched cultures. Biol. Reprod. 30, 199-211. Volpe J. J. and Vagelos P. R. (1976) Mechanisms and regulation of biosynthesis of saturated fatty acids. Physiol. Rev. 56, 339-417.
Voss A. C. and Sprecher H. (1988) Regulation of the metabolism of linoleic acid to arachidonic acid in rat hepatocytes. Lipids 23, 660-665. Voss A. C. and Sprecher H. (1988) Metabolism of 6,9,12octadecatrienoic acid and 6,9,12,15-octadecatetraenoic acid by rat hepatocytes. Biochim. biophys. Acta 958, 153-162.
0305-0491/92 $5.00 + 0.00 © 1992 Pergamon Press Ltd
Printed in Great Britain
THE BIOSYNTHESIS OF P O L Y U N S A T U R A T E D FATTY ACIDS BY RAT SERTOLI CELLS H. OULI-IAJ,S. HUYNH* and A. NOUVELOT Laboratoire de Biochimie, URA CNRS 609, Universit6 de Caen, 14032 Caen Cedex, France (Received '27 November 1991)
Abstract--1. The biosynthesis of polyunsaturated fatty acids (PUFA) of the n-6 and n-3 series was investigated in cultured Sertoli cells. 18 : 2n-6, 18 : 3n-6, 20: 2n-6, 18 : 3n-3 and 20: 3n-3 were added individually at a concentration of 20 pmol to culture media. 2. Maximum incorporation of 20- and 22-carbon PUFA into membrane lipids was observed after 72 hr of incubation with all the exogenous substrates used. 3. As reported in other cell systems, the A6 desaturation was the first rate-limiting step; the major factor regulating this activity was the concentration of linoleic acid or ct-linolenic acid in the medium. 4. Our data show that the A5-desaturation represents a second regulatory step in PUFA biosynthesis. 5. The sum of n-6 and n-3 PUFA of the 22 carbon chain length constantly represented between 11 and 12% of total fatty acids, regardless of the exogenous substrate used. 6. Our kinetic studies of the incorporation of PUFA of the n-6 and n-3 series did not permit detection of a A8 desaturase activity.
INTRODUCTION
MATERIAL AND METHODS
It is generally accepted that Sertoli cells supply the nutritive substances required for the development of testicular germinal cells. Coniglio and Sharp (1989) were the first to show that cultured Sertoli cells are capable of synthesizing arachidonic acid from linoleic acid. We recently demonstrated (Huynh et al., 1991) that these cells are capable of incorporating and utilizing both linoleic and ~-linolenic acids and thus of synthesizing 20- and 22-carbon polyunsaturated fatty acids ( P U F A ) of the n-3 as well as of the n-6 families. In the other cell systems studied to date (Lemarchal, 1989; Brenner, 1989), the most c o m m o n metabolic pathway for P U F A biosynthesis is the one beginning with a A6 desaturation and followed alternately by elongation, A5 desaturation, another elongation and A4 desaturation. An active A8 desaturase has been reported in the rat testis, and although the cell type involved was not determined (Albert and Coniglio, 1977), this finding has raised the possibility of the existence of a P U F A metabolic pathway involving A8 desaturation in the Sertoli cell. The present work was designed to investigate the metabolic pathways involved in the biosynthesis of n-6 and n-3 P U F A in the Sertoli cell. Linoleate (18 : 2n-6), ~-linolenate (18 : 3n-3), y-linolenate (18 : 3n-6), eicosadienoate (20:2n-6) and eicosatrienoate (20:3n-3) were added individually to the culture medium, and kinetic studies were performed to establish whether or not the A8 desaturase is active in Sertoli cells.
*Author to whom correspondence should be addressed. 897
Reagents
Linoleic (18:2n-6), ~,-linolenic (18:3n-6), eicosa 11-14 dienoic (20: 2n-6), ~ -linolenic ( 18 : 3n-3) and eicosa 11- 14-17 trienoic (20:3n-3) acids, and fatty acid-free bovine serum albumin were purchased from Sigma Chemical Co. (France). Organic solvents were obtained from Merck (Germany). All other chemicals were of the highest chemical purity available commercially. Cell culture
Purified Sertoli cell-enriched aggregates were isolated from testes of 18-day-old rats by sequential enzymatic digestions, at 32°C, essentially as described by Tung et al. (1984). Briefly, decapsulated testes were treated by trypsin/DNAse in Hank's buffer for 25 mn. After addition of soybean trypsin inhibitor, interstitial cells were discarded, and seminiferous tubules were washed three times, submitted to digestion by a mixture of collagenase and testicular hyaluronidase (25 mn), and recovered by unit-gravity sedimentation. They were digested for another 25 mn with hyaluronidase alone. The resulting aggregates were centrifuged (400g, 10mn), washed twice with Hank's buffer containing 1% bovine serum albumin, then twice with 0.1 mM EDTA in Ca 2÷- and Mg2+-free Hank's buffer, and again twice with 1% bovine serum albumin/Hank's buffer. Cell aggregates were suspended in DMEM-HAM F12 (1 : 1; v/v) supplemented with 0.15 mmol HEPES, 2% Ultroser SF and antibiotics (50 UI/ml penicillin, 50 pg/ml streptomycin and 0.25 pg/ml fungizone). About 1500 aggregates/cm2 were seeded onto 75cm 2 plastic flasks to obtain confluent cultures. Cells were maintained in a humidified atmosphere containing 3% CO2 at 32°C. Culture medium was renewed 24 hr later and replaced by DMEM-HAM F12 without Ultroser SF. On Day 3, germ cells were removed by brief hypotonic treatment according to the method of Galdieri et al. (1981).
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Addition of fatty acids to culture medium
RESULTS
Fatty acids (20/~M) were added to the culture medium on Day 5. They were introduced as fatty acid salts bound to fatty acid-free bovine serum albumin at a molar ratio of 12:1 according to the procedure of Hendry and Possmayer (1974) and Hamilton and Cistola (1986). Incubation of cultured cells in the presence o f linoleic acid was performed in eight flasks; four additional flasks, with cells in the presence o f the culture medium alone, served as controls. At 24 hr intervals, the cells from two experimental and one control flasks were harvested. Culture medium was changed at 48 hr in the flasks to be harvested at 72 and 96 hr. The same procedure was followed for each of the fatty acids studied. In the same conditions, other kinetic studies of incorporation o f 18:2n-6 or 20:2n-6, using short times (3, 5, 7, 9, 12 hr) were carried out. Cultured cells from each individual flask were first washed twice with 1 m M HNaCOa, pH 7.4, and harvested with a rubber spatula. Membrane fractions were prepared according to the method o f Lories et al. (1986) and Kartner et al. (1979). The cell suspension was rapidly cooled, supplemented with I m M EDTA and homogenized with a Potter; nuclei, large cellular particles and unbroken cells were removed by centrifugation at 4°C (25,000g for 30 mn). The results presented were obtained from four different cultures.
Fatty acid analysis Lipids were extracted with chloroform/methanol (2:1, v/v) according to the method of Folch et al. (1957), and fatty acid methyl esters were prepared as described by Hagenfeldt (1966). Before methylation, nonadecanoic acid was added to the mixture as internal standard. Fatty acids were analyzed as methyl esters on a Varian 3 300 gas chromatograph equipped with a flame ionization detector, using a CP Sil 88 capillary column (50 m x 0.2 mm i.d.). The temperature was programmed at 60°C for I min, from 60 to 150°C at 30°C/min, and from 150 to 210°C at 40°C/min. Fatty acids were identified by comparison with standards and GLC peak areas were measured with a Merck D 2000 integrator.
Addition o f -linolenate )
essential
fatty
acids
(linoleate
Linoleate (20/~mol) added to the culture medium (Table 1). A f t e r the a d d i t i o n o f 18:2n-6 to the culture m e d i u m , m a x i m u m i n c o r p o r a t i o n o f 20- a n d 22c a r b o n n-6 P U F A into m e m b r a n e lipids w a s o b t a i n e d after 72 h r o f i n c u b a t i o n . D u r i n g the first 24 hr, a n increase in all n-6 P U F A was o b s e r v e d , w i t h t h e e x c e p t i o n o f 22: 5n-6, w h i c h o n the c o n t r a r y , t e n d e d to decline. T h e p r o p o r t i o n o f s a t u r a t e d fatty acids r e m a i n e d virtually c o n s t a n t , w h e r e a s the p e r c e n t a g e o f 16- a n d 18-carbon m o n o - u n s a t u r a t e d acids s h o w e d a p r o gressive decrease, d r o p p i n g by a b o u t 4 9 % over t h e 96 h r o f i n c u b a t i o n . E x o g e n o u s 18:2n-6 d i d n o t a p p e a r to induce a n y n o t a b l e c h a n g e in t h e overall p r o p o r t i o n s o f 2 2 - c a r b o n n-3 P U F A (Table 1). T h e m a s s i v e i n c o r p o r a t i o n o f 1 8 : 2 n - 6 a n d the low p r o p o r t i o n o f 1 8 : 3 n - 6 o b s e r v e d in the m e m b r a n e lipids as early as the first h o u r s o f i n c u b a t i o n reflect a r a p i d i n h i b i t i o n o f the A6 d e s a t u r a s e . T h e c o n t i n ued increase in 2 0 : 2 n - 6 o v e r time revealed a c h a i n e l o n g a t i o n o f 18:2n-6 f r o m t h e b e g i n n i n g o f incub a t i o n , raising t h e possibility t h a t this 2 0 : 2 n - 6 c o u l d be c o n v e r t e d to 20: 3n-6 by a A8 d e s a t u r a t i o n . H o w ever, the existence o f a n active A8 d e s a t u r a s e d o e s n o t exclude t h e classic p a t h w a y involving t h e A6 d e s a t u r a s e f o l l o w e d by a n elongase, since 18:3n-6 s y n t h e s i z e d by A6 d e s a t u r a s e activity c a n b e r a p i d l y c o n v e r t e d into 20: 3n-6. -Linolenate (20/~mol) added to the culture medium (Table 2). A f t e r t h e a d d i t i o n o f 18:3n-3 to the culture m e d i u m , m a x i m u m i n c o r p o r a t i o n o f 20- a n d 22c a r b o n n-3 P U F A into m e m b r a n e lipids w a s o b t a i n e d at 48 h r o f i n c u b a t i o n . T h e a c c u m u l a t i o n o f 18:3n-3 a n d t h e low p r o p o r t i o n o f 18:4n-3 o b s e r v e d in the
Table 1. Distribution (in %) of fatty acids in lipids of Sertoli cell membranes as a function of incubation time in the presence of linoleate 20/~M added to the culture medium Incubation times (hr) Fatty acids 16:0 16:1n-9 16:In-7 18:0 18:1n-9 18:ln-7 18:2n-6 18:3n-6 18:4n-3 20: In-9 20: 2n-6 20:3n-6 20:4n-6 20: 5n-3 22: 0 22:4n-6 22:5n-6 22:5n-3 22:6n-3 T(n-3) T(n-6)
or
0 26.98 + 2.91 2.61 + 0.35 2.22 + 0.31 8.46 -t-0.79 21.65_ 2.11 10.57 + 1.10 1.79 __.1.18 0.00 0.01 0.51 1.47 + 0.18 0.76 + 0.11 9.10 + 0.85 0.06 0.76 2.72 + 0.29 5.48 + 0.52 1.92 3.94 + 0.40
24 23.70 + 2.24 1.25 + 0.15 1.52 __.0.14 8.31 + 0.80 16.83_ 1.55 6.81 + 0.62 14.06 + 1.45 0.11 0.11 0.44 2.67 _ 0.28 1.86 _+0.17 10.25 _+ 1.10 0.00 0.47 3.29 + 0.33 5.39 + 0.42 1.58 3.37 + 0.36
48 24.12 __.2.41 1.26 + 0.13 1.64 _ 0.17 8.81 _ 0.69 13.61 + 1.35 5.61 _ 0.48 16.50 + 1.61 0.13 0.13 0.26 3.44 + 0.34 2.10 _ 0.23 11.07 + 1.10 0.00 0.45 3.01 _ 0.26 4.10 + 0.39 1.65 3.20 + 0.27
72 23.45 +_2.21 1.14 _+0.12 1.13 +_0.13 8.71 _+0.67 11.61 -t- 1.01 5.07 + 0.48 15.30 __.1.39 0.35 0.20 0.23 3.97 + 0.31 2.18 + 0.19 11.97 _+ 1.20 0.00 0.68 3.44 + 0.29 4.22 + 0.31 1.61 3.09 + 0.29
96 24.29 + 3.01 1.01 + 0.18 1.45 + 0.15 9.00 + 0.81 11.42 __. 1.01 5.01 _ 0.49 13.23 +_ 1.29 0.30 0.21 0.25 3.77 __.0.36 1.90 + 0.18 12.45 + 1.24 0.00 0.46 3.68 + 0.25 3.95 + 0.23 1.52 2.77 + 0.26
5.94 21.30
5.06 37.67
4.98 40.35
4.90 41.45
4.51 39.38
T(n-6): totality of the n-6 PUFA; T(n-3): totality of the n-3 PUFA.
899
Biosynthesis o f P U F A b y Sertoli cells Table 2. Distribution (in %) of fatty acids in lipids of Scrtoli cell membranes as a function of incubation time in the pre~nce of ~-Iinolenate (20/tM) added to the culture medium Incubation times (hr) Fatty acids 16:0 16:In-9 16:ln-7 18:0 18:1n-9 18: I n-7 18:2n-6 18: 3n-6 18:3n-3 18: 4n-3 20:0 20: In-9 20: 2n-6 20:3n-6 20:4n-6 20: 3n-3 20:4n-3 20:5n-3 22:4n-6 22:5n-6 22:5n-3 22:6n-3 T(n-3) T(n-3)- i 8: 3
Standards
24
48
72
32.16 ± 1.94 1.55 ± 0.15 2.08 ± 0.17 9.12 ± 0.99 19.89 ± 0.97 6.02 ± 0.46 1.35 ± 0.11 0.00 0.19 ± 0.09 0.00 0.21 0.91 0.17 0.99 ± 0.14 10.87 ± 1.01 0.11 _ 0.04 0.17 ± 0.07 0.14 ± 0.06 3.01 ± 0.18 6.17 _ 0.28 0.49 ± 0.07 4.21 ± 0.11
31.53 + 2.02 1.18 + 0.11 1.66 ± 0.13 8.99 ± 0.80 17.67 ± 0.99 5.63 ± 0.37 1.21 ± 0.12 0.00 2.87 ± 0.21 0.08 0.12 0.89 0.00 0.93 ± 0.11 10.09 ± 0.87 0.88 ± 0.04 0.32 ± 0.04 1.46 + 0.09 2.83 ± 0.21 5.80 5:0.22 0.69 ± 0.09 5.18 ± 0.13
30.28 5:0.99 0.79 ± 0.04 1.21 ± 0.10 8.81 ± 0.66 16.01 + 0.90 4.84 ± 0.38 1.19 ± 0.10 0.00 6.01 ± 0.54 0.22 0.00 0.71 0.09 0.81 ± 0,12 9.52 ± 0.54 1.84 ± 0,11 0.90 ± 0,12 2.14 ± 0,13 2.65 ± 0.21 5.43 ± 0.26 1.91 ± 0.15 5.94 ± 0.14
29.90 ± 1.22 0.58 ± 0.11 0.97 ± 0.11 8.62 ± 0.57 14.85 ± 1.01 4.27 ± 0.33 1.11 ± 0.09 0.00 9.01 ± 0.79 0.27 0.11 0.52 0.00 0.65 ± 0.13 9.09 ± 0.61 1.98 ± 0.24 0.86 + 0.15 2.21 _ 0.13 2.52 ± 0.17 5.13 ± 0.33 1.78 + 0.09 4.69 + 0.21
11.48 8.61
18.96 12.95
20.80 11.79
5.31 5.12
T(n-3): totality of the n-3 PUFA; T(n-3)-lS:3: totality of the n-3 P U F A without a-linolenate.
same concentration (20gmol), to different culture media.
membrane lipids reflected an inhibition of the A6 desaturase. Exogenous 18:3n-3 did not appear to induce change in the proportion of saturated fatty acids whereas the percentage of 16- and 18- carbon monounsaturated acids showed a progressive decrease. From the beginning of incubation, the proportion of 20: 3n-3 increased reflecting a chain elongation of 18:3n-3, raising the possibility that this 20:3n-3 could be converted to 20: 4n-3 by a A8 desaturation. In order to test the relative importance of the A6 and A8 metabolic pathways, the precursors of 20:3n-6 (18:3n-6 and 20:2n-6) and the precursor of 20:4n-3 (20:3n-3) were added individually, at the
Addition of precursors of 20:3n-6 and 20:4n-3 18:3n-6 (20#mol) added to the culture medium (Table 3). As with 18:2n-6, maximum incorporation of n-6 PUFA into membrane lipids was obtained after 72 hr of incubation. The proportion of 18:3n-6 did not exceed 1.5% of total fatty acids, even after 72 hr; in contrast, the proportion of 20:3n-6 increased from 0.7 to 16.5% over the same period, reflecting, on the one hand, an excellent incorporation of 18:3n-6 into the cells, and, on the other, a rapid elongation to 20:3n-6. This substantial
Table 3. Distribution (in %) of fatty acids in lipids of Sertoli cell membranes as a function of incubation time in the presence of 18:3n-6 (20 #M) added to the culture medium Incubation times (hr) Fatty acids 16:0 16:In-9 16: I n-7 18:0 18:In-9 18 : ln-7 18:2n-6 18:3n-6 18: 3n-3 18:4n-3 20:1n-9 20:2n-6 20:3n-6 20:4n-6 20: 5n- 3 22: 0 22:4n-6 22:5n-6 22: 5n-3 22:6n-3 T(n-3) T(n-6)
0
48
26.98 _ 2.54 2.61 _ 0.28 2.22 + 0.27 9.45 + 0.85 21.65 4- 2.10 9.57 ± 0.79 1.79 ± 0.20 0.00 0.01 0.01 0.51 1.47 ± 0.11 0.76 5:0.12 9.10 ± 0.84 0.06 0,76 2.72 ± 0.28 5.48 ± 0.52 1.92 3.94 ± 0.32
25.59 _ 2.71 1.55 5- 0.19 1.38 _ 0.14 9.06 _ 0.85 13.19 ± 1.29 3.37 + 0.31 1.71 5:0.18 1.16 ± 0.12 0.07 0.01 0.17 0.78 ± 0.09 14.33 _+ 1.41 14.62 ± 1.39 0.03 0.31 3.47 ± 0.31 4.42 ± 0.39 1.70 3.08 ± 0.35
24.97 _ 2.31 1.52 _ 0.17 1.22 ± 0.12 8.51 ± 0.80 ll.13 _ 1.10 3.19 + 0.29 1.49 5:0.16 1.49 _+0.15 0.00 0.01 0.16 0.51 ± 0.09 16.49 ± 1.51 15.83 ± 1.40 0.01 0.49 3.95 5:0.38 4,35 5- 0.45 1.63 3.06 ± 0.29
72
26.77 ± 3.01 1.56 ± 0.18 1.25 ± 0.13 9.72 ± 0.87 12.39 ± 1.21 3.45 ± 0.31 1.73 _+ 0.17 1.54 _+ 0.16 0.00 0.03 0.66 0.68 ± 0.15 13.40 5:1.32 14.13 ± 1.39 0.06 0.75 3.50 ± 0.37 3.43 5:0.31 1.88 2.96 5- 0.30
96
5.94 21.31
4.89 40.49
4.71 44.11
4.93 38.41
900
H. OULHAJ et al. Table 4. Distribution (in %) of fatty acids in lipids of Sertoli cell membranes as a function of incubation time in the presence of 20:2n-6 (20 ~M) added to the culture medium Incubation times (hr) Fatty acids
0
24
48
72
96
16:0 16:1n-9 16:ln-7 18:0 18:1n-9 18:1n-7 18:2n-6 18 : 3n-6 20: I n-9 20:2n-6 20:3n-6 20:4n-6 20:5n-3 22 : 0 22:4n-6 22:5n-6 22:5n-3 22: 6n-3
30.68 ± 2.24 1.66 ± 0.17 2.06 i 0.20 9.62 ± 0.87 19.85 ± 2.01 6.62 ± 0.65 1.43 ± 0.15 0.09 0.22 0.19 _+0.09 1.18 ± 0.12 10.16 ± 1.08 0.08 0.77 2.93 ± 0.27 6.03 ± 0.59 0.36 6.06
28.74 ± 2.77 0.93 ± 0.10 0.93 ± 0.09 9.21 ± 0.89 11.77 ± 1.10 1.87 ± 0.18 6.41 ± 0.61 0.10 0.13 10.31 ± 1.10 1.33 ± 0.20 12.59 ± 1.30 0.11 0.49 3.12 ± 0.29 6.02 ± 0.55 0.36 5.48
27.16 ± 2.65 0.45 ± 0.10 0.88 ± 0.11 8.25 ± 0.81 10.67 ± 1.09 1.68 ± 0.17 6.88 ± 0.65 0.05 0.08 8.37 ± 0.82 1.92 ± 0.19 15.77 ± 1.55 0.08 0.41 3.87 ± 0.36 6.06 ± 0.61 0.29 4.74
27.07 ± 2.65 0.37 ± 0.10 0.72 ± 0.12 8.24 ± 0.77 9.64 _+ 0.91 1.57 ± 0.16 8.15 ± 0.79 0.07 0.06 8.30 ± 0.79 2.34 ± 0.20 16.35 ± 1.61 0.14 0.35 3,96 ± 0.40 5,98 ± 0.61 0.25 3.95
26.63 ± 3.01 0.27 ± 0.08 0.62 ± 0.09 8.25 ± 0.80 9.25 ± 0.91 1.42 ± 0. l 5 8.89 ± 0.79 0.07 0.07 8.24 + 0.75 3.02 ± 0.23 16.23 ± 1.62 0.09 0.42 3.94 ± 0.35 5.16 ± 0.49 0.24 3.81
T(n-6) T(n-3)
22.02 6.50
39.87 5.66
42.92 5.10
45.16 4.34
45.54 4.14
18:2n-6 maintained the proportion of linoleic acid between 6 and 9%, permitting a more efficient activity of the A6 desaturase compared to that observed with exogenous linoleic acid (Tables 1 and 3). The proportion of 20:4n-6 obtained with exogenous 20:2n-6 was comparable to that obtained with 18:3n-6, that is, when the first regulatory step (A6 desaturation) was skipped. With both these substrates, the proportion of arachidonic acid was close to 16% at 72 hr. In contrast, the proportion of its precursor (20:3n-6) was 16.5% with 18:3n-6 and 2.3% with 20:2n-6. These observations appear to indicate that the A5 desaturation represents a second rate-limiting step in P U F A biosynthesis in the Sertoli cell. 20:3n-3 (20gM) added to the culture medium (Table 5). Addition of 20:3n-3 induced principally increases in the proportions of 20: 3n-3 and 18 : 3n-3 in lipids of Sertoli cells membranes. The utilization
elongase activity thus explains our preceding results relative to 18:2n-6. The 18:3n-6 obtained by A6 desaturation cannot accumulate since it is immediately converted into 20:3n-6. The overall proportion of 22-carbon PUFA of the n-3 and n-6 series consistently represented between 11 and 12% of total fatty acids, regardless of the exogenous precursor used (Tables 1, 2 and 3). 20:2n-6 (20/~mol) added to the culture medium (Table 4). The addition of 20:2n-6 induced only slight increases in the proportion of 20: 3n-6, and the values observed remained comparable to those obtained after the addition of 18:2n-6. A A8 desaturation thus appears to be unlikely. However, the increase in the proportion of 18:2n-6 to about 6% of total membrane PUFA within the first 24 hr indicates that the utilization of 20:2n-6 involves a retroconversion to 18:2n-6 which then becomes substrate for the A6 desaturase. The retroconversion of 20:2n-6 to
Table 5. Distribution (in %) of fatty acids in lipids of Sertoli cell membranes as a function of incubation time in the presence of 20:3n-3 (20,uM) added to the culture medium Incorporation times (hr) Fatty acids 16:0 16:1n-9 16:1n-7 18:0 18:In-9 18:1n-7 18:2n-6 18:3n-3 18:4n-3 20: 0 20:In-9 20: 2n-6 20:3n-6 20:4n-6 20:3n-3 20:4n-3 20:5n-3 22:4n-6 22:5n-6 22:5n-3 22:6n-3 T(n-3)
Standards
24
48
72
30.58 ___1.89 1.41 ±0.19 1.96 + 0.17 8.97 ± 0.90 19.06 ± 1.00 5.32 ± 0.56 1.56 ± 0.21 0.19 ± 0.11 0.00 0.32 0.90 0.28 1.00 ± 0.09 11.38 ± 0.55 0.11 ± 0.10 0.17 ± 0.09 0.11 ± 0.05 2.77 ± 0.21 5.55 + 0.54 0.37 ± 0.03 4.17 ± 0.33
29.59 ± 1.46 1.58 ± 0.21 1.62 ± 0.18 8.24 ± 0.77 17.04 ± 0.98 5.84 ± 0.37 1.56 ± 0.31 2.56 ± 0.12 0.10 0.28 0.86 0.16 1.46 ± 0.09 13.68 ± 0.41 3.21 ± 0.26 0.23 ± 0.04 0.19 ± 0.04 1.98 ± 0,20 4.92 ± 0,47 0.46 ± 0.05 4.64 ± 0,39
27.22 ±- 2.04 1.25 ± 0.23 1.56 ± 0.20 7.14 ± 0.79 16.37 ± 0.84 4.15 ± 0.43 1.47 ± 0.21 4.19 ± 0.23 0.06 0.33 0.49 0.22 0.97 ± 0.11 13.11 ± 0.99 4.46 ± 0.34 0.88 ± 0.10 1.93 ± 0.06 1.75 ± 0.21 4.46 ± 0.41 2.12 ± 0.30 6.13 ± 0.53
26.14 ± 2.00 1.11 +0.21 1.24 ± 0.13 7.02 ± 0.55 14.06 ± 0.85 2.35 ± 0.19 1.47 _+0.14 6.97 ± 0.27 0.06 0.322 0.40 0.23 0.82 ± 0.10 12.93 ± 1.01 6.96 ± 0.55 1.48 ± 0.09 2.93 ± 0.12 1.41 + 0.15 4.33 ± 0.33 2.73 + 0.17 5.14 + 0.43
5.11
11.40
19.71
26.27
Biosynthesis o f P U F A by Sertoli cells
901
Table 6. Distribution (in %) of fatty acids in lipids of Sertoli cell membranes as a function of incubation time (short) in the presence of linoleate (20 # M ) added to the culture medium Incubation times (hr) Fatty acids 16:0 16:1n-9 16:ln-7 18:0 18:1n-9 18:1n-7 18:2n-6 18: 3n-6 18 : 3n-3 20:ln-9 20:2n-6 20:3n-6 22: 0 20:4n-6 20: 5n-3 22:4n-6 22:5n-6 22:5n-3 22:6n-3 T(n-6) T(n-6)-I 8: 2
0
3
5
7
9
28.19 + 1.81 2.21 _ 0.25 1.94 + 0.20 8.57 + 0.81 18.58+ 1.53 11.06 + 0.99 1.65 + 0.22 0.00 0.00 0.46 0.97 + 0.18 0.85 + O.19 0.36 11.14 + 1.01 0.04 2.68 + 0.29 5.55 + 0.59 1.63 4.11
28.20 + 1.71 2.01 + 0.24 1.75 4- 0,21 8.51 + 0,84 17.61 + 1,41 10.11 + 0.95 3.61 + 0.31 0.00 0.00 0.45 1.10 + 0.21 1.09 + 0.22 0.36 11.09 + 1.01 0.00 2.75 + 0.31 5.64 + 0.60 1.62 4.01
28.11 + 1.52 1.99 4- 0.25 1.76 + 0.21 8.55 + 0.82 17.10+ 1.32 9.87 + 0.89 4.30 + 0.52 0.00 0.00 0.45 1.32 + 0.20 1.19 + 0.22 0.37 11.35 + 1.02 0.00 2.68 + 0.30 5.44 + 0.57 1.53 3.99
27.91 + 1.95 1.78 + 0.20 1.53 + 0.23 8.33 + 0.81 15.84+ 1.41 9.01 + 0.87 7.11 + 0.67 0.00 0.00 0.41 1.49 + 0.19 1.38 + 0.20 0.31 11.63 + 1.09 0.00 2.60 + 0.30 5.31 + 0.58 1.49 3.87
27.64 _+ 1.87 1.49 + 0.21 1.22 4- 0.24 8.12 + 0.75 14.92 + 1.36 8.04 4- 0.79 10.03 + 0.87 0.00 0.00 0.39 1.41 + 0.17 1.61 +_0.23 0.30 11.81 + 1.11 0.00 2.54 _ 0.29 5.11 + 0.56 1.50 3.87
27.14 + 1.53 1.27 + 0.27 1.11 + 0.26 8.01 + 0.79 13.11 + 1.28 7.01 -t- 0.80 13.47 + 1.11 0.00 0.00 0.36 1.51 +_0.21 1.83 + 0.19 0.27 12,11 +_ 1.19 0.00 2,66 + 0.29 5.08 + 0.60 1.38 3.84
26.28 21.98
29.52 22.41
32.51 22.48
36.66 23.19
22.84 21.19
25.28 21.67
of 20:3n-3 involves a rctroconversion to 18:3n-3 which then becomes substrate for the A6 desaturase. This retroconversion maintained the proportion of ct-linolenic acid between 2 and 7%, permitting a more efficient activity of the A6 desaturase compared to that observed with exogenous ~t-linolenic acid. The addition of 20:3n-3 induced only slight increases in the proportion of 20:4n-3 and the values observed were nearly the same as those obtained after addition of 18:3n-3. A A8 desaturation thus appears to be unlikely. However it is possible that there is A8 as well as A5 desaturase activity present in Sertoli cells. The major pathway using A6, A5 desaturases may be able to hide the second one as soon as retroconversion of 20: 2n-6 to 18:2n-6 or 20:3n-3 to 18:3n-3 supplies the A6 desaturase substrate. In order to test the relative importance of these two metabolic pathways we made brief kinetic
12
studies of 18:2n-6 incorporation or 20:2n-6 incorporation. Incorporations Linoleate (20#M) added to the culture medium (Table 6). After the addition of 18 : 2n-6 to the culture medium, an increase of all n-6 PUFA into membrane lipids was obtained from the three first hours. This change was particularly important for linoleate and reflects a rapid inhibition of the A6 desaturase. 20:2n-6 (20#M) added to the culture medium (Table 7). When Sertoli cells were incubated with 20: 2n-6 in the culture medium, an increase of all n-6 PUFA in Sertoli cells membrane lipids was observed. For every experiment, this increase was more important than those obtained with linoleate incubation. The increase in the proportion of 18:2n-6 indicates that the utilization of 20: 2n-6 involves a retroconversion to 18:2n-6 observable after 3 hr.
Table 7. Distribution (in %) of fatty acids in lipids of Sertoli cell membranes as a function of incubation time (short) in the presence of 20:2n-6 (20/a M) added to the culture medium Incubation times (br) Fatty acids 16:0 16:1n-9 16:ln-7 18:0 18:In-9 18:1n-7 18:2n-6 18: 3n-6 20: In-9 20:2n-6 20:3n-6 22:0 20:4n-6 20:5n-3 22:4n-6 22:5n-6 22: 5n-3 22: 6n-3 T(n-6)
0
3
5
7
9
30.11 + 1.91 1.92 + 0.25 1.68 + 0.23 9.15 + 0.87 17.93 + 1.60 9.81 + 0.79 1.48 + 0.35 0.00 0.68 0.35 + 0.20 1.03 + 0.35 1.28 9.92 + 0.78 0.15 2.55 + 0.29 5.13 + 0.52 1.68 5.14
30.04 +_ 1.85 1.55 + 0.23 1.54 + 0.24 9.07 _+ 0.85 15.60 + 1.35 8.10 + 0.76 2.69 + 0.44 0.00 0.57 3.12 _ 0.44 1.06 + 0.31 1.18 9.96 + 0.83 0.13 2.56 _ 0.30 5.14 _+ 0.57 1.62 5.01
30.23 _ 1.99 1.44 + 0.30 1.33 _+ 0.31 9.10 + 0.90 14.99 + 1.39 7.91 + 0.80 3.01 _+0.42 0.00 0.59 4.31 + 0.45 1.28 + 0.29 1.19 10.29 _+ 0.99 0.14 2.60 + 0.32 5.18 + 0.60 1.60 4.97
29.40 _+ 1.96 1.38 _+ 0.40 1.27 _+ 0.36 8.98 _+0.85 14.53 _+ 1.46 7.50 +_0.81 3.97 _+ 0.45 0.00 0.56 4.98 + 0.52 1.39 + 0.40 1.16 10.67 + 1.02 0.11 2.61 _ 0.31 5.28 + 0.53 1.58 4.94
28.94 + 1.78 I. 13 _+0.32 1.11 + 0.23 8.91 + 0.87 14.07 + 1.37 7.31 + 0.75 4.35 + 0.44 0.00 0.54 5.54 + 0.49 1.61 + 0.27 1.14 11.19 + 1.01 0.10 2.67 + 0.37 5.40 + 0.58 1,59 4,93
28.51 + 1.77 1.07 + 0.29 1.01 + 0.22 8.62 _+0.89 13.21 + 1.21 7.01 +_ 0.69 4.96 + 0.47 0.00 0.53 6.62 + 0.60 1.86 + 0.30 1.12 I 1.52 _+ 0.99 0.10 2.69 _+ 0.28 5.44 + 0.53 1.57 4.92
12
20.66
24.53
26.67
28.90
30,76
33.09
902
H. OULHAJet al.
The proportion of linoleate in membrane lipids was always smaller than those observed after linoleate addition to culture medium, permitting a more efficient activity of the A6 desaturase and a better biosynthesis of n-6 PUFA. No increase in 20:3n-6 in Sertoli cells membrane lipids was observed during the first hours, and a A8 desaturase activity was not established.
hepatoma cells (Iturralde et al., 1990), and pig renal cells (Mahfouz et al., 1989). In the Sertoli cell, the proportion of 18:3n-6 in membrane lipids both after its direct addition to the culture medium and after the desaturation of 18 : 2n-6 remained low. This is in agreement with results obtained with hepatocytes; the data of Voss and Sprecher (1988) and Christophersen et al. (1982) suggest that when 18:3n-6 is obtained by desaturation of 18:2n-6 or when it is added directly as exogenous substrate, the major fraction of this DISCUSSION 18:3n-6 is initially incorporated into a labile With all the exogenous fatty acid substrates used, phospholipid pool and is then rapidly released for except for ~-linolenate, maximum incorporation and metabolism to arachidonate. The rates of acylation utilization of n-6 and n-3 PUFA were observed after using 1-acyl-sn-glycero-3-phosphocholine as acceptor are 10-50 times greater than those involved in the 72 hr of incubation. As reported previously (Huynh et al., 1991), the desaturations and chain elongations (Bernert and incorporation of n-6 PUFA had little effect on the Sprecher, 1977; Ludwig and Sprecher, 1979). After its overall proportion of n-3 PUFA, which showed only release, 18:3n-6 is metabolized to 20:3n-6 and then 20:4n-6, which is incorporated into the membrane slight variations. The addition of individual n-6 or n-3 fatty acids to phospholipids. The accumulation of 20:3n-6 obtained with the culture medium did not alter the distribution of saturated fatty acids in the membrane lipids, but exogenous 18:3n-6 resulted from an inhibition of the did induce a marked decrease in the proportion of A5 desaturase, the second step in PUFA desaturpalmitoleic and oleic acids. These observations are ation. While little is known about the regulation of consistent with the findings of de Gomez Dumm who the A5 desaturase activity, recent findings have reported that A9 desaturase activity in rat liver suggested that it differs markedly from that of the A6 microsomes was regulated by dietary alterations desaturase. The early decrease of A5 desaturase en(de Gomez Dumm et al., 1982). The addition of zyme in the fat-deficient rat accompanies a decrease 18:2n-6, 18:3n-6 or 20:4n-6 to fat-free diets resulted of acids of n-6 series whereas the A6 desaturase is in an inhibition of the conversion of palmitic acid to generally increased (Brenner, 1989). Both enzymes follow different directions under the same dietary palmitoleic acid. Similar results were obtained in vitro with rat liver microsomes or hepatocytes (Sprecher, change, demonstrating that A5 desaturase induction 1972; Volpe and Vagelos, 1976) regarding the has its own regulatory mechanism dependent on the presence or absence of substrates or related fatty desaturation of stearic acid to oleic acid. Our kinetic studies of the incorporation of linoleic acids. Jeffcoat and James (1977) and de Alaniz et al. acid showed that A6 desaturase activity rapidly be- (1980) have shown that a high-fat diet rich in 18:2n-6 comes the rate-limiting factor in the metabolism of stimulates A5 desaturase activity. We reported n-6 PUFA. The 6(n-6) ratio [(n-6 PUFA-18:2n-6)/ (Huynh et al., 1991) that maximum A5 desaturase (18:2n-6)| dropped abruptly from 10.2 to 1.6 after activity in the Sertoli cell was observed after the 12 hr of incubation, and then remained at this low addition of about 0.7 #g/ml of 18:2n-6 to the culture level up to 96 hr. In the same way, incorporation medium and that with higher concentrations, A5 of ct-linolenate showed that A6 desaturase activity activity decreased. The proportion of 20:4n-6 aprepresents a first regulatory step of n-3 PUFA bio- pears to be one of the major factors in the regulation synthesis. The 66(n-3) ratio [(n-3 PUFA- 18 : 3n-3)/ of this A5 desaturase activity in the Sertoli cell. The (18:3n-3)] dropped abruptly from 27 to 3 after 24 hr chain elongation of 20: 4n-6 to 22: 4n-6 did not seem of incubation and then remained at this low level up to be very efficient. On the other hand, the addition of 18:3n-3 or 20:3n-3 did not show an important to 72 hr. The regulation of the A6 desaturase appears to be accumulation of 20:5n-3 but small increases of 20closely linked to the proportion of 18:2n-6 present and 22-carbon n-3 polyenes reflecting good activities in the membrane lipids. When the incorporation of of A5 desaturase and elongase. However, 22:6n-3 18:2n-6 was limited, either by the addition of produced by the same reaction, is an important lower concentrations to the culture medium (Huynh component of liver lipids. The low level of the et al., 1991) or by retroconversion of 20:2n-6 elongation of 20 : 4n-6 has been described in other cell as observed here, the ratio ~6(n-6) [(n-6 PUFA)- systems and appears to be specific to the n-6 series. (18:2n-6+20:2n-6)/18:2n-6] was far higher (4.5 According to Christophersen and Hagve (1985) the times), reflecting a more efficient utilization of linoleic difference lies in substrate's efficiency. At least, they acid. In our experiments, this phenomenon was not showed an efficient elongation of 20:5n-3 acid to so important after addition of n-3 fatty acids because 22:5n-3 acid in isolated rat hepatocytes in the presincorporation of n-3 PUFA was always smaller than ence of lactate and correspondingly an active A4 that of n-6 PUFA. The important role of the A6 desaturation to 22:6n-3 acid. Rosenthal and Hill desaturase as the first rate-limiting step in PUFA (1986) also compared the metabolism of 20:4n-6 and biosynthesis has been widely documented in other of 20:5n-3 in human vascular endothelial cells in subcellular or cellular systems, such as rat liver culture; the endothelial cells were found to elongate 23% of 20:4n-6 compared to 72% of 20:5n-3 after microsomes (Brenner, 1981; de Gomez Dumm et al., 1983), rat hepatocytes (Voss and Sprecher, 1988), rat 25hr of incubation. Hagve and Christophersen
Biosynthesis of PUFA by Sertoli cells (1984) reported that the chain elongation of 20- to 22-carbon fatty acids was far more efficient for the n-3 than for the n-6 series, resulting in elevated proportions of 22: 5n-3 and 22: 6n-3. Although there is little documentation on the conversion of 22:4n-6 to 22:5n-6, it is generally accepted that this activity is weak in the liver, but is fairly important in the testicles and adrenals (Hagve and Christophersen, 1984; Ayala et al., 1973). We noted that for all the tested n-6 fatty acids, the sum of 22:4n-6 + 22: 5n-6 remained constant. Any increase, however slight, in 22:4n-6 was accompanied by an equivalent decrease in 22:5n-6. The sensitivity of the A4 desaturase to an increase in 22:4n-6 maintained the proportion of 22: 5n-6 between 4 and 6% of total fatty acids in the Sertoli cell membrane lipids. The systematic retroconversion of 22:4n-6 to 20:4n-6 shows that arachidonate remains the major end product of the n-6 series. Other investigators (Klenk and Mohrhauer, 1960), and more recently, Coniglio and Sharp (1989) have suggested that in the testis, arachidonic acid may be synthesized via a metabolic pathway involving a A8 desaturation of 20:2n-6 to 20:3n-6. Our data have shown that exogenous 20:2n-6 induced a marked increase in 18 : 2n-6 within 3 hr, whereas a noticeable increase in 20: 3n-6 was observed only after 7-9 hr. It would thus appear that in the Sertoli cell the A8 desaturase is either absent or has only a very slight activity. Retroconversion of 20:2n-6 supplies the 18:2n-6 used as substrate by the A6 desaturase. The metabolic pathways involved in the biosynthesis of n-6 and n-3 P U F A from 18:2n-6 and 18:3n-3 in the Sertoli cell thus appear to be the same as those described in other cell systems. REFERENCES Albert D. H. and Coniglio J. G. (1977) Metabolism of eicosa-11,14 dienoic acid in rat testes. Evidence for A8 desaturase activity. Biochim. biophys. Acta 489, 390-396. Ayala S., Gaspar G., Brenner R. R., Peluffo R. O., Kunau W. (1973) Fate of linoleic, arachidonic and docosa 7,10,13,16-tetraenoic acids in rat testicles. J. Lipid Res. 14, 296-305. Bernert J. T. and Sprecher H. (1977) An analysis of partial reactions in the overall chain elongation of saturated and unsaturated fatty acids by rat liver microsomes. J. biol. Chem. 252, 6736-6744. Brenner R. R. (1981) Nutritional and hormonal factors influencing desaturation of essential fatty acids. Prog. Lipid Res. 20, 41-47. Brenner R. R. 0989) Factors influencing fatty acid chain elongation and desaturation. In The Role of Fats in Human Nutrition (Edited by Vergroesen A. J. and Crawford M.), 2nd edn, pp. 45-70. Academic Press, London. Christophersen B. O., Hagve T. A. and Norseth J. (1982) Studies on the regulation of arachidonic acid synthesis in isolated rat liver cells. Biochim. biophys. Acta 712, 305 -314. Christophersen B. O. and Hagve T. A. (1985) Eicosapentaenoic and arachidonic acid chain elongation and A4 desaturation in isolated liver cells. In Proceeding of the 2nd International Congress o f Essential Fatty Acids, Prostaglandins and Leukotrienes, p. 8. London. Coniglio J. G. and Sharp J. (1989) Biosynthesis of arachidonic acid from linoleate in primary cultures of Sertoli cells. Lipids 24, 84-85.
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