Btochimtca et Biopltrsica lit.t((. 1081 (1991) 151 15g
151
' 1991 Elsevier Science Publishers B,V. {Biomedical Division) 0005-2760/ql/503.5[) A D O N I S 000527609100070K
BBALIP 53"370
Sexual dimorphism in the preferential secretion of unsaturated lysophosphatidylcholine by rat hepatocytes but no secretion by sheep hepatocytes Annette Graham ~'*. Victor A. Zammit 2. William W. C ~ : s t i e "and David N. Brindley Department of Bzm'hemz.:'t O" and Lipid and l.tp,~protem Research Gr~mp. Fac'ul(v *(l"Ah'dzcme, ]~'tlll't'~tlt" r~f .4 ll~'rta (Canada) aad 2 ttaanah Rt'~earth ins/(fete..Ivr (/_.K
(Received 23 July 19~()) Key words: ArachidonJte: Es~',cntialfatty acid; Phonpha[id?,lch~hne: VLDL: Sex related difference
(i) Rat and ovine hepatocytes were incubated in monolayer culture with various fatty acids to determine their effects on the composition of the lysophosphatidylcholine that was secreted. (2) No lysophosphatidylcholine was detected in the medium from the ovine hepatocytes even though these cells were hormonally responsive and they ~creted phosphatidylcholine and triacylglycerol in very-low-densi~ lipoprotein. (3) Lysophosphatidylcholine was readily detected in the incubation medium of rat hepatocytes. The predominant forD' acids in this lipid were unsaturated. Stearate and arachidonate contributed 15 and 34%, and 24 and 26% of the total tatly acids when hepatocytes from male and female rats were used, respectively. The relative proportions of stearate and arachidonate in the phosphatidylcholine .secreted from the hepato,zytes were 20 and 14%, and 28 and 21% for the males and female~ respectively. The equivalent values for stearate and arachidonate for phosphatidylcholine in the hepatocytes were 18 and 17% and 33 and 22% for male and female rats. These results provide furlher indications of sex differences it, hepatic phospholipid metabolism and extend this to the secretion of phosphatidylcholine and lyosphosphatidylcholine. (4) The addition of ! mM stearate to the incubation na~lium did not significantly decrease the proportion of arachidonate in the lysophosphatidylcholine obtained from the hepatocytes of the male rats. However, the relative proportion of arachidonate was decreased in incubations that contained I mM oleate or linoleate. (5) The results provide evidence that the preferential .secretion of unsaturated lysophosphatidyleholine by the liver may provide a system for transporting unsaturated lath.' acids and choline to other organs in non.ruminant animals. However, this mechanism may not operate for ruminants. Introduction Per(used rat liver I1] and monolayer cultures of rat [2-6] can synthesize lysophosphatidylcholine which appears in the perfusion, or incubation medium. A large proportion of the fatty acids associated with this lysophosphatidylcholine are unsaturated [1,5] and. furthermore, the secretion of this lipid is
hepatocytes
Abbreviations: Fatty acids are indicated by their numbers of carbon atoms and double bonds. The family to which some acids belong has been indicated b~, ( n - x ) where x denotes the number of carbon atoms from the last double bond to the terminal melhyl group. * Present address: Welicome Research Laboratories. Biochemistry Department. Langley Court, Beckenham. Kent. BR3 3BS, U.K.
Correspondence: D,N. Brindley, University of Alberta, Lipid and Lipoprotein Research Group, Heritage Medical Research Centre. Edmonton, T6G 2S2, Canada.
promoted by the inclusion of unsaturated fatty acid,; in the incubation m e d i u m [3]. This secretion of lysophosphatidylcholine from the liver could account for a large proportion of this lipid which is found associated with albumin in the blood of m a n y mammals. Lysophosphatidylcholine is often the second most prevalent phospholipid in the plasma [7] and its concentration remains relatively high [8] in p a 6 e n t s with a deficiency of lecithin:cholesterol acyltransferase, LCAT. T h e unsaturated lysophosphatidylcholine produced by the liver could provide a novel transport system for choline and polyunsaturated fatty acids to other organs [3]. The present work was undertaken with three objectives, The first was to examine the effects of modifying the fatty acid composition of the incubation m e d i u m on the proportion of the fatty acids esterified in the secreted lysophosphatidylcholine. The second objective was to establish if there was any difference between the fatty acid c o m p o s i t i o n of the l y s o p h o s p h a t i d y l c h o l i n e
152 secreted by mate and female hepatocytes. The third was to determine whether cultures of sheep hepatocytes could also secrete lysophosphatidylcholine as a specific means of transporting unsaturated fatty acids from the liver to other organs. This was particularly significant since the sheep being a ruminant animal has much lower proportions of polyunsaturated fatty acids in it:, phospholipids. Malerials and Methods
Animals Rats were obtained from A. Tuck & Son (Battlebridge, Essex, U.K.) and they were fed a standard laboratory diet which was purchased from Oxoid (Basingstoke, Hants, U.K.). The sheep were Finn x Dorset Horn Cross-bred wethers, 9-12 months old, which were fed hay and concentrates ad libitum. The typical composition of 1 kg of concer,trate was 100 g of cooked flake maize, 507.5 g rolled barley, 100 g rolled oats. 100 g wheat feed pellets, 130 g soya, 50 g grouod nut, 3.3 g dicalcium phosphate, 3.2 g NaC1, 2.5 g vitamins/trace minerals, 2.5 g lime stone and 1 g calcined magnesite.
Materials The sources of most materials have been described previously [3,4]. Lipids and radioisotopes were purchased from Sigma Chemical and Amersham International, r,::zpectively. Bisbenzamide H33258 was from Fluka. Ph:3sphatidyl[Me-3H]choline (0.3 Ci/mmol) was kindly donated by Dr. Dennis Vance.
Preparation and incubation of hepato(Ttes Hepatocytes were isolated from male and female Wistar rats essentially as described by Cascales et al. [9], except that heparin was added to the first perfusion buffer rather than being injected and the perfusion with collagenase was performed through the hepatic portal vein with the inferior vena cava being cut. For sheep hepatocytes only the caudate lobe of the liver was perfused using a technique adopted from Donaldson et al. [10]. The sheep was anesthetized with 20 ml of Sagatal by intrajugular injection. The caudate lobe was rapidly excised, cannulated and perfused under gravity with 500 ml of modified Krebs-Henseleit medium containing 50 mg of heparin at about 80 m l / m i n and at 25-35°C. The flow of medium was maintained during transportation of the lobe from the slaughter house to the laboratory, which lasted about 4 min. The lobe was then attached to a perfusion apparatus and the flow switched to a recirculating perfusion containing cc.!lagenase at 0.04% at 37°C. The incubation then proceeded for 40-45 rain until leakage from the lobe surface occured. The lobe was then transferred to a petri dish containing about 10 ml of perfusion
medium. The capsule was cut and the cells gently washed and filtered, essentially as for the rat hepatocytes [9] except that cells were centrifuged at 50 g for 2-3 rain. Sheep hepatocytes normally demonstrated and 85-95% exclusion of trypan blue. Between 1.5 • l0 t' and 2 • 10 ~ rat hepatocytes or 3.106 and 3.5 • 10 ~ sheep hepatocytes were pipetted into collagen-coated tissue culture dishes containing a final volume of 3 ml of modified Leibovitz L-15 medium containing new-born calf serum [91. The plates were incubated for 37°C in a humidified air atmosphere for I to 2 h. The medium was replaced removing non-viable and unattached cells. A monolayer of hepatocytes was produced, 95% of which excluded trypan blue. After incubation for 6 to 8 h, the cells had flattened and spread from their initial round shape and the medium was replaced by medium that contained 0.2% fatty acid-poor bovine serum albumin instead of serum. Both types of hepatocyte were incubated for 14 h and then for an extra 2 h in fresh medium. The hepatocytes were then incubated for the specified time in modified Leibovitz L-15 medium [9] containing 0.5 mM albumin, I mM glycerol, 100 luM choline and fatty acid additions as indicated. Fatty acid solutions (80 t~M) were prepared by heating to 7 0 ° C in water containing a 50% molar excess of KOH and they were then pipetted slowly into the albumin containing medium with stirring [3].
(,'ell viability and expression of rates of lipid synthesis and secretion The assessment of the metabolic viability of the rat hepatocytes has been described previously [9,11]. The sheep hepatocytes produced 4.87 + 1.5/~mol of glucose per mg of DNA in 20 rain. This secretion was increased between 2-2.2-fold by the addition of 1 ~tM glucagon, 71 #M dibutyryl cAMP, 5 #M phenylephrine or 1.1 IuM isoproterenol. The rates of lipid synthesis for rat hepatocytes have been calculated relative to the units of lactate dehydrogenase present in the cells. This compensates for small variations among dishes of cells better than does the determination of protein or DNA [9,111 since some non-viable cells ma3 remain attached to the plate and contribute to the latter two values. Loss of viability is accompanied by leakage of lactate dehydrogenase into the incubation medium, The content of lactate dehydrogenase per dish of cells was about 1 t~mol of lactate oxidized/min at 25 o C. One dish of cells also contained about I mg of protein and so that results can be readily expressed relative to protein if this is required for comparison with other work. The present incubations also contained about 34 mg of albumin/ml and, therefore, the measurement of the relatively small concentration of hepatocyte protein might have been inaccurate for the routine determination cell number. Results for
153 ovine hepatocytes have been expressed relative to DNA rather than lactate dehydrogenase, since this activity was about 12-times lower than in rat hepatocytes ~hich made the assay unreliable.
A,lalysis of lipids Lipids were extracted from cells and medium from 25-30 dishes for each experimental condition. The medium was collected and centrifuged at 3000 rpm for 15-20 min in a bench centrifuge to remove detached cells and debris. The monolayers were washed three times with 2.5 ml portions of ice-cold serum-free incubation medium and then scraped from the dishes in 1 ml of ice-cold 0.25 M sucrose containing 0.5 mM dithiothreitol and 10 mM Hepes adjusted to pH 7.4 with KOH. The cell suspensions were sonicated for 6 s in i s bursts at 22 kHz with an amplitude of 8 a m peak to peak. The sonicates and media were stored at - 2 0 ° C under N_,. Samples of medium and cell homogenates were extracted using solvents that contained 0.0005-O.001% 2,6,di-tert-butyl-p-cresol as an antioxidant. The phases were separated as described by Bligh and Dyer [12] until after the addition of chloroform when 0.2 M K H z P O 4 and 2 M KCI was used instead of water. In a typical experiment 80 ml of medium and 20 ml of cell homogenates were extracted. T o the bottom phase of the extractions was added 50 tzg each of t.-c~-heptadecanoyl lysophosphatidylcholine and t-a-pentadecanoyl phosphatidyleholine in i ml of prot',an-2-ol as internal standards. 15:0 and 17:0 fatty acids do not naturally occur in liver phospholipids of the rat [131 and only to the extent o f about 3% in sheep liver phospholipids 1t4]. Lipid extracts were dried in a rotary evaporator and residual water was removed by adding a small volume of propan-2-ol and re-evaporating. The lipid was then dissolved in a small volume of chloroform and stored at - 2 0 ° C until required for HPLC. The stationary phase of the H P L C system was Spherisorb T M silica (5 ~tm particles~ 25 cm × 0.5 cm column, Hichrom, Reading, Berkshire, U.K.). A model 8770 isocratic H P L C pump (Spectra-Physics, St, AIhans, U.K.) equipped with a Knauer differential refractometer (Dr. H. Knauer, Oberusel. Taumus. F.R.G.) was used. The lipid extract was filtered through a 40 # m solvent-resistant filter, dried, taken up in 50 t.tl of CHCi~ and injected onto the H P L C column. The mobile phase which was degassed with helium, consisted of acetonitrile/ m e t h a n o l / water containing 1.5 /tM lithium acetate ( 5 0 : 4 5 : 6.5, v / v ) at 1 m l / m i n . [15]. The ct,lumn was calibrated using a 20 ~1 mixture of 11 m g / m l tff phosphatidylcholine/ml and 14 m g / m l of lysophosphatidylcholine. Samples containing phosphatidylcholine and lysophosphatidylcholine were collected manually as the peaks corresponding to the standards eluted from the column. T h e fractions were evaporated to dryness in
a stream of N_,. The lipid was covered with hexane containing 0.02% butylated hydroxytoluene and stored at - 20" C. The purified samples were evaporated to dryness under a s~ream of N, and phospholipids were methylated by sodium methoxide-catalyzed transesterification [16]. Fatty acid analysis was performed on a 25 m fused silica column with Silar 5CP as the stationary phase using a Carlo Erba gas chromatograph and H z as the carrier gas. The temperature was set at 155 ° C for 3 min and was programmed to increase at 4 C ° / m i n to a final temperature of 195°C where it remained constant for 17 rain. Peaks were identified with authentic standards and were quantified by electronic integration. The mol% of each of the fatty acids present was then calculated and related to the known amount of internal standard.
Analysis o/htctate dehydrogenase and DNA These were analyzed by the methods of Saggerson and Greenbaum [I7] and Switzer and Summer [!S;], respectively.
Results and Discussi,-m
Lysopho.~phatidyh.holJne is secreted into the medium by ~'iahle hepatocTtes In determining the physiological significance for the secretion of lysophosphatidylcholine, it was important that this lipid is produced by viable cells and that it is not the result of non-specific degradation of phosphatidylcholine in the medium. The albumin used in the medium might have been contaminated by phospholipase, or LCAT and it was therefore heated to 6 0 ° C for 30 rain [191. This did not cause any significant decrease in iyst;phosphatidylcholine production by rat hepatocytes (results not shown). Lysophosphatidylcholine might also be produced in the medium by the action of an extracellular phospholipase produced by the hcpatocytes, Hepatic lipase can act on phospholipids and exhibit phospholipase A n activity 120]. It can be secreted by rat hepatocytes I21] and by Hep G2 ceils [22]. However, in some experiments in the present work, rat hepatocytes were prelabelled with ['all]glycerol and [14Clcholinc in the presence of I mM oleate at 37°C, They were cooled to 4 ° C and washed with sufficient heparin (I0 units/ml) to remove hepatic lipase that was associated with the cell surface I23]. The subsequent prt~duction t,f lysophosphatidylcholine on incubating the cells at 37 ° C was not decreased (results not shown). thus making it unlikely that lysophosphatidylcholine was produced by surface bound hepatic lipase. Similar results and conclusions were reported by Baisted et al. [51. LCAT is also secreted from the hepatocytes [24], and it could produce lysophosphatidylcholine, especially that containing saturated fatty acid. in the medium (Table
154 TABLE I
t"al(r actd c,Jmpmttion of the I.~:rt,pho.~phant(rh'holine isolated frmn the medium of cultured rat hepatocytes Ra~ hepatocytes were preincubated as described in the Materials and Methods Section. The medium was then changed and the medium was supplemented with l mM fatty acid where indicated. The cultured medium and cell.,, were collected aRer a further incubation of 8 h and lysopho,~phatidylcholine and ph~sphafidylch(fline were isolated and their fatty acid compositions analyzed. The mtd% of the r,~;.IV acids in lysophosphatidylcholine is given fi~r the number of independent experiments indicated. Results are means+ ranges h~r two experiments, or means_+. S.D. h~r three experiments. The significance of the difference between incubations in the absence of exogenous fatty acids and those in Iheir presence was calculated by using a paired t-lest and this is indicated by: * P < 0.025, * * P .c 0.01 and ' P < 0.005, The incorporation of exogcnou~ la('-Iabelted fatty acids into lysophosphatidylcholine in three independent experiments for male rats was 0.93_+ 0.23. 3.24+ 2.36 and 2.14 + 1,33 nmol per unit of lactate dehydrogenase, respectively, for lg:0, 18:I and 18:2. n.d.. not detected. Fatty acid
Fatty acid added to medium: male
female
none 16:0
16:1 18:0 18:I ( n - 9 } 18:1 ( n - 7 1 18:2 20:4 22:61n-3)
l 8:0
| 8:1
18:2
none
18:0
18:1
18;2
10.9__+1.1 n.d. 23.1_+1.9 11.9-+1.1 1.9_+0.1 11.6_+1.0 34.5_+3.7 6.2+_0.3
8.94- 3.1 n.d. 8.9_+ 1.4 42.I_+10.6 ' 2.6+ 1.6 O.O~+ 2.8 21.8_+ 9.4* 5.34. 3.6
8.34- 3.3 n.d. tl.9_+ 1.9 17.2_+17.6 n.d. 33.7_+13.0' t8.9_+ 8 . 9 * * 4.3_+ 0.6
5.4_+ 1.3 n.d. 24.2_+ 2.1 22.9+13.0 n.d. 5.1_+ 1.g 26,44. 9.1 13.3+_ 3.1
11.74-3.6 n.d. 39.5-+6.1 19.3+_7.4 n.d. 5.0+_1.7 16.8+5.7 7.1_+3.1
8.2____.5.9 n.d. 19.4+_4.1 30.2-+5.3 n.d. 6.1±2.1 29.1_+4.8 9.04-2.4
16.64- 5.5 n.d. 20.9_+ 4.6 18.8+-10.1 n.d. 23.3+_10,4 14.5+ l.O 5.2+ 1.2
2.95-+(i.4
1.9+0.3
4.8-+ 1.4
4.2-+ 1.3
2.3+ 0.3
0.95:0.09
2.75:0.2
1.65:0.07
3
2
3
3
2
2
2
2
10.8 n.d. 14.5 16.6 2.6 14,7 34.0 5.8
Unsaturated Saturated N umber of experiments
4-3.6 +_4.0 _+2.1 ±0.7 _+2,5 4-2.5 _+0,4
1). W e t h e r e f o r e t o o k m e d i u m f r o m h e p a t o c y t e s t h a t h a d b e e n i n c u b a t e d f o r 5 h a n d a d d e d 3.7 # C i / m l of
lysophosphatidylcholine by the conditioned compared to non-conditioned medium d'.,ring 20 h was less than
phosphatidyl[-~H]choline followed by son±cation. The fal, c o f ',his p h o s p h a t i d y l c h o l i n e w a s t h e n f o l l o w e d d u r i n g a f u r l h e r i n c u b a t i o n f o r u p Io 2 0 h in t h e a b s e n c e o f
1% o f t h e o r i g i n a l p h o s p h a t i d y l [ 3 H ] c h o l i n e added. This r e s u l t a g r e e s w i t h t h a t o f B a i s t e d e t al. [5] w h o t o o k m e d i u m f r o m t h e h e p a t o c y t e s a n d i n c u b a t e d it f o r a f u r t h e r 5 h i n t h e a b s e n c e o f cells. T h e r e w e r e n o
heptocytes.
The
conversion
of phosphatidylcholine
to
TABLE II
l.'at(r acid comp,,sttion of the phr~,~ffhatit(t'h'holine isolated from the medium of cultured rat hepatotytes The experimental conditions and the method of expressing the results are described in the legend to Table I. The incorporation ol' exogenous 14('-labelled fatty acids into secreled phosphatidylcholine in three independent experiments for mate rats was 0.76 +0.21, 0.764-0.08 and 0,81.15:0.58 nm~fl per unit of laclate dehydrog:,enase, respectively, for 18:0, 18:1 and 18:2 Fatty acid
Fatty acid added to medium: male
female
none 16:1) 16:t 18:0 1 8 : l ( n 9) l~:l(n-7) 18:2 2(/:4 22:6(n-3) Unsaturated Saturated Number of experiments
41.4 n.d. 19.5 7.0 2.4 14.8 13.8 n,d.
18:0 ±10.2 4.. ± _+ +_ ±
4.8 2.9 0.7 1.6 3.2
34.8 n.d. 24.7 6.9 1.2 16.4 13.4 n,d.
18:1 _+3.2 +1.7 _+0.5 _+1.0 "+(I.3 +_4.2
38.2 2.9 14.9 15.2 "-.8 7.8 12.3 5.9
+6.9 +1.4 +0.4 -+3.0 +_0.5 _+6.6 _+4,9 ±3,7
18:2
none
18:0
18:1
! g:2
28.6± 0,6 * n.d. 21.3+11.8 14.1+11.3 2.1_+ 2.0 18.6_+ 3,8 12.4-+ 6.5 2.9__+ 0,9
22.2±9.5 n.d. 28.2_+4.5 7.6±1.6 n.d. 14.7_+4.3 20.7_+5.8 5.6-+0.9
23.g± 4.9 n.d. 32.0+12.1 24.8±!3.1 •.d. 9.4+_ 2.0 8.4_+ 4.6 n.d.
19.4±8.9 n.d. 26.0+3.2 15.0±2.6 1.3-+0.3 8.5_+0.009 19.5-+2.1 9.4_+0.9
19.35:2.8 n.d. 23.5_+ 7.1 18.8+10.0 n.d. 13.7+ 1.0 16.9_+ 3.3 6.2± 0.3
(I.64+ 0.15
0.69+0.14
0.91+0.24
1.1± 0.5
1.0_+0.2
0.8_+ (I.2
1.2±0.3
1.3± 0.2
3
2
2
3
2
2
2
2
~55 significant changes in the concentrations of phosphatidylcholine, lysophosphatidylcholine and glycerophosphocholine. Sekas et al. [1] also demonstrated no significant increase in the lysophosphatidylcholine concentration of the medium used to perfuse rat livers when this was incubated in isolation, or when the perfusion was continued after removal of the liver. These results and the predominantly unsaturated nature of the lysophosphatidylcholine exclude a significant participation of LCAT in its production. The production of lysophosphatidytcholine can be dissociated from the secretion of phosphatidylcholine and V L D L through the effects of colchicine, dexamethasone plus insulin [2], albumin [4,5]. fatty acid,; [3], verapamil, E G T A and chlorpromazine [4 i. The rates of secretion from rat hepatocytes of lysophosphatidylcholine, phosphatidylcholine and triacylglycerol was approximately constant during a 24 h period with our hepatocyte system [2]. This and the fact that less than about 5% of the lactate dehydrogenase was lost 'r0m the cells during the incubation period indicates that the hepatocytes remained viable. Also the magnitude of lysophosphatidylcholine secretion throughout was 4 12-fold higher than that for phosphatidylcholine when the incubations were supplemented with oleate and albumin (Ref. 2~4; Tables I and il). Therefore, lysophosphatidylcholine is not formed by the hydrolysis of phosphatidylcholine in the medium, nor is it a product of dead or dying cells. It is a major choline metabolite in rat hepatocyte cultures and is probably a major contributor to the lysophosphatidylcholine found in blood of rats and man.
Effects of exogenous fatty acids on the composition of lysophosphatidylcholine secreted by hepatocytes from mule and female rats The lysophosphatidylcholine produced by hepatocytes from male rats in the absence of exogenous fatty acid was predominantly unsaturated with 20:4 constituting about 34 mol~. The unsaturated to saturated fatty acid ratio was 2.95 which was noticeably more unsaturated than the ratio of 1.38 to 1.96 reported by Baisted et al. [5] from hepatocytes cultured in fetal calf serum. Their equivalent values for cells incubated with de!ipidated fetal calf serum was 0.55 to 0.95. The hepatocytes used in the present work were incubated for 16 h prior to the experiments in medium containing 0.2f~ of fatty acid poor bovine serum albumin. Addition of 1 mM 18:0 to the medium gave an enrichment of about 9% in the content of 18:0. This was accompanied by decreases in the percentage of 18:1 and 18:2, but no significant change in the proportion of 20:4. The unsaturated to saturated ratio of the lysophosphatidylcholine decreased from 2.95 to 1,90. Conversely, addition of 1 mM 18:1 or 18:2 increased the unsaturated to saturated ratio. 18:1 markedly increased
by about 25~ the percentage content of this fatty acid in lysophosphatidylcholine. This was accompanied by a decrease in the content particularly of 20:,1. There also appeared to be decreases in the percentages of 16:0 and 18:0 but these did not reach the level of statistical significance. Similarly. the addition of 18:2 to the incubation increased its content by about 19% in lvsophosphatidylcholine and it decreased by about 15% that of 20:4. However. 18:1 and lg:2 also stimulated the secretion of lysophosphatidylcholine by about 2-fold [3] and so part of their effects were probably mediated by providing additional glycerolipid for lysophosphatidylcholine secretion rather than by competing with 20:4. Therefore, the secretion of 20:4 in lysophosphatidylcholine is well preserved, despite the addition of exogenous fatty acids to the incubation medium. The composition of lysophosphatidylcholine secreted by hepatocytes by female rats is also shown in Table 1 and they confirm the general effects seen with the hepatocytes from the males. However, the relative proportion of 18:0 appeared to be higher and that of 18:2 and 20:4 lower when compared to the males. T h e incubation of female hepatocytes with 18:0, 18:1 or 18:2 again resulted in the enrichment of the secreted lysophosphatidylcholine with the particular exogenous fatty acid, Although incubation with 18:2 increased its conccntration in lysophosphatidylcholine, it did not increase the unsaturated to saturated ratio in the two experiments performed. This appeared to be partly caused by a higher content of 16:0. The phosphatidylcholine secreted into the medium by hepatocytes from male rats in the absence of exogenous fatty acids gave an unsaturated to saturated ratio of about 0.64 (Table I1~. This composition differs significantly from that of the lysophosphatidylcholine that was secreted, for example, the proportions of 16:0 and 18:0 were increased ( P < 0.005 and P < 0.025, respectively), whereas 18:1 (n - 9) and 20:4 were lower ( P < 0,025 and P < 0.005, respectively~. Addition of 18:0 to the medium gave a small enrichment in its content in the secreted phosphatidylcholine in the twe experiments performed with no significant change in the unsaturated to saturated ratio. Incubation with 1 mM 18:1 (n - 9~ increased this ratio in both experiments. This was related to the increased proportion of 18:1, but this enrichment was not as marked that in the secreted lysophosphatidylcholi::e (Table I). Addition of 18:2 to the incubation decreased the proportion of 16:0 in the secreted phosphatidylcholine by about 12~ with an increase in the concentration of 18:I ( n - 9 ) c o m p a r e d to when no fatty acid was added (Table !I). There appeared to be a small increase in the content of 18:2, but this was not statistically significant. Addition of 18:2 to hepatocytes from male rats gave an unsaturated to saturated ratio of 1.1 for the secreted phosphatidylcholine which was less ( P < 0.05} than 4.2 for lysophos-
i56 TABLE IV
p h a t i d y l c h o l i n e secreted from the i n c u b a t i o n s c o n t a i n ing 18:2 (Table I). T h e p e r c e n t a g e c o m p o s i t i o n o f 16:0 in the p h o s p h a tidylcholine f r o m the male h e p a t o c y t e s a p p e a r e d to be higher than that for the h e p a t o c y t e s o f the two female rat:;. Conversely, the p r o p o r t i o n s of 18:0 a n d 20:4 app e a r e d to be higher for p h o s p h a t i d y l c h o l i n e secreted from the female hepatoeytes. T h e r e were n o significant differences in the perc e n t a g e c o m e n t o f the fatty acids between the secreted p h o s p h a t i d y l c h o l i n e a n d that f o u n d in the cells (Table !11) f r o m the male rats in the a b s e n c e o f e x o g e n o u s fatty acid. A d d i t i o n o f 18:0, 18:1 a n d 18:2 increased the relatively p r o p o r t i o n of the respective faUy acids in the p h o s p h a t i d y l c h o l i n e o f the cells o f the male rats as expected. In the case o f the a d d i t i o n o f 18:2 there were lower p r o p o r t i o n s o f 18:1 ( n - 9) a n d a greater p r o p o r tion o f 18:2 in the cells as c o m p o s e d to the p h o s p h a tidylcholine in the m e d i u m . T h e p h o s p h a t i d y t c h o l i n e in the h e p a t o c y t e s o f the female rats had a high p r o p o r t i o n o f 18:0 a n d a lower p r o p o r t i o n o f 16:0 in the p h o s p h a t i d y l c h o l i n e t h a n for the males (Table IIl). This w~s also reflected in the p h o s p h a t i d y l c h o l i n e that was secreted into the m e d i u m (Table II). S u p p l e m e n t a t i o n o f the m e d i u m with exogen o u s fatty acids again increased the p r o p o r t i o n o f that p a r t i c u l a r acid in the p h o s p h a t i d y l c h o l i n e as e x p e c t e d (Table ili).
Fatty acid compositi~m of the phosphatt~lvh'holine that was isolated from the medium and from ~he cells of cultured oeine hepato(3"tes
Ovine hepatocytes were prepared and preincubated as described in '~hc Materials and Methods section. The cells were then incubated in the presence or absence of 18:1 as indicated for 24 h and phosphalidylchotine was isolated from the cells and medium and the fal'y acid composition was determined. The tool% of the fatty acids in the legend.,, are sit~wn as meatJs +_,z D. for three independent experiments, Fatty acid
In medium none
16:0 16:1 I8:0 18:1~n-9) 18:1(n-7) 16.2 20:4
27.5 2.5 27.8 31.1 0.9 6.6 3.6
Unsaturated S:~turated
In cells 18:1
+7.7 _+1.0 ±5.7 ±3.3 +0.7 +0.9 +_1.2
23.8 2.0 26.8 36.6 0.8 6.4 3.3
none
18: I
_+3.9 23,1 -+5.6 22.5-+4.8 -+1.8 1.7 +0.6 1.6_+0.2 +5.7 30.1 _+7.1 25.7_+0.7 _+0.9 34.8 _+4.4 -,4,1.9+5.1 -+0.7 i.3 +1.9 2.4+0,9 +1.3 6.1 +_2.4 4.64-1.2 *_0.2 3.2 4-0.5 2.8_+0.7
0.81 _+0.16 0.974-0.07
0.88+0.08
!.1 -+0.09
with 1 m M 114C]18:1. T h e i n c o r p o r a t i o n o f 18:1 a n d c h o l i n e into secreted p h o s p h a t i d y l c h o l i n e u n d e r these c o n d i t i o n s was 175 + 57 a n d 10.9 + 1.9 n m o l / m g o f D N A , respectively. A b o u t 90% o f the o l e a t e i n c o r p o rated into triacylglycerol t h a t w a s secreted i n t o the m e d i u m was isolated with the V L D L f r a c t i o n as described p r e v i o u s l y 12], T h e secreted p h o s p h a t i d y l c h o l i n e had an u n s a t u r a t e d t o s a t u r a t e d fatty acid r a t i o o f 0.81 in the a b s e n c e o f e x o g e n o u s oleate a n d 0,97 in the p r e s e n c e ( T a b l e IV), T h i s w a s n o t very d i f f e r e n t f r o m that seen with rat h e p a t o c y t e s ( T a b l e !I), except t h a t the relative c o n t e n t o f p o l y u n s a t u r a t e d acids w a s m u c h
Secretion o f g(vcerolipids f r o m ovine hepatocytes
O v i n e h e p t o c y t e s secreted triacylglyceroi into the m e d i u m at a rate o f 165 + 53 n m o l o f 18:1 i n c o r p o r a t e d / 2 4 h per m g o f D N A ( m e a n s _+ S.E.M. for fi,,e i n d e p e n d e n t e x p e r i m e n t s ) w h e n they were i n c u b a t e d
TABLE !II Fatty acid cmnpo.~tlion of the pho.vphatidyicholine isolated from cultured rat hepatocytes
The experimental conditions and the methods of expressing the results are described in the legend to Table 1. Tile incorporation of exogenous 14C-labelled fatty acids into phosphatidylcholine in the ~:ells for the male rals was 35.74- 7.5 (3), 36.64- 10.4 (2) and 21.9+6.7 (3) nmol per unit of lactate dehydrogenase, respectively, for 18:0, 18:1 and 18:2. Fatty acid
Fatty acid added 1o medium: male
female
none 16:0 16:1 18:0 18:1 ( n - 9 ) 18:1(n-7) 18:2 20:4 22:6(n-3) Unsaturated Saturated Number of experiments
18: I
18:2
+_1,2 _+0.03 +2.0 +0.4 +_0.9 +_1.1 +-1.t +_.0.4
25.6+0.a i.3__.0,2 12.3_+1,1 36.9+5.3 3.0+0.03 7.8+_2,1 11.2_.+2.5 1.84_.1.0
27.1 n.d. 21.0 4.1 4.0 31.4 10.2 2.0
0.96+__0.t3
0.63+.+0.085
t.6+0.04
4
2
2
33.0 1.6 18.1 8.9 6.1 12.2 16.6 3.5
18:0 +_4.9 +_1.2 __.2.7 ±2.7 +_2.1 +2.0 -+1~5 +_0.87
27.1 0.8 34.2 8.9 1.2 10.2 14.5 3.0
none
18:0
18: i
18:2
16.2 +_4.0 1.1 ±0,62 33.4 _+5.0 9.2 _+_2.9 2.7 _+0.02 8.5 -+0,004 21.6 -+1.7 6.55+0.63
15.9 4-2.5 0.8 +0.08 39.3 +1.4 13.9 +0.51 1.35-+0.005 8.2 +_1.5 t6.7 _+1.5 3,9 +0.52
9.7 0.34 32.0 23.8 1.3 7.4 21.0 4,1
17.8 0.8 27.1 7,6 1.5 27.7 Irl.0 3.9
1.11 +0.33
1.20__+0.17
0.81 +0.14
1.38
1.23
3
2
2
1
I
+2.7 _+5,5 +0,45 * _+086 _+6.0" +-3.02" +0.23
157 lower. The fatty acid composition of the secreted phosphatidylehoiine was essentially similar to that remaining in the cells (Table IV). Medium was also removed form the cells after 24 b and then incubated for a further 4 h. There was n,, degradation of phosphatidyb.holine or triacylglycerol indicating no significant lipase or phospholipase activity in the medium. There were no detectable levels of fysophosphatidylcholine in the medium after incubations of up to 24 h. It is possible that the experimental conditions were inappropriate for detecting lysophosphatidylcholine secretion from ovine hepatocytes, but these were viable. hormonally responsive and they secreted very low density 1;poprotein. Furthermore. the secretion of lysophosphatidylcholine could be readily demonstrated under similar conditions with rat hepatocytes. The phosphatidylcholine of the ovine hepatocytes contained small quantities of polyunsaturated acids with a relatively high content of 18:1. This lipid should have been a suitable substrate for a putative phospholipase A~ which might be involved in releasing lysophosphatidylcholine from rat hepatocytes. Lysophosphatidylcholine secretion by rat hepatocytes ,~as markedly stimulated by exogenous 18:1 [3]+ but no effect was observed with ovine hepatocytes. The lysophosphatidylcholine of sheep serum [28] contains relatively high proportions of saturated fatty acids which means that it could be produced by LCAT. Consequently, we have no evidence that the sheep can transport unsaturated lysophosphatidyleholines from the liver. Conclusions
The results from the rat hepatocytes provide further indications of sexual dimorphism in the fatty acid composition of hepatic phospholipids. Two of the male rats were littermates of the females and they were fed on the same batch of diet and therefore the differences should not have resulted from differences in dietary composition. The lower proportion of 20:4 in the lysophosphatidylcholine from the fema!e rats was obtained despite a relatively higher proportion of this acid in the phosphatidylcholine in hepatocytes obtained from female rather than males. It is, therefore, possible that separate pools of phosphatidylcholine are used for the production of iysophosphatidylcholine. Such selection has been observed in the secretion of phosphatidylcholine in VLDL
[291. The fatty acid composition of phosphatidylcholine for hepatocytes of male rats (Table !il) is well supported by preuious work [27,301 as is the increased proportion of 18:0 and 20:4 in phosphatidylcholine from the female rats [25-271. Our results demonstrate that sexual dimorphism in the fatty acid composition of hepatic phosphatidylcholine persists after culturing hepatocytes for 34 h. Furthermore, a sexual dimorphism
is also seen in the fatty acid compositions of the iyso~ phosphatidylcholine and phosphatidylcholine that were secreted. The latter observation may contribute to the differences in lipoprotein metabolism that are observed between the sexes. The higher proportion of 18:0 and 20:4 in phosphatidylcholine from the livers of female rats may result from a greater contribution from the methylation of phosphatidylethanolamine to total phosphatidylcholine synthesis [25+26.31- 33]. Livers of female rats also have a higher ability to convert 16:0 to 18:0 than those of males [34]. Sexual dimorphism in phospholipid composition appears to result from the effects of estrogens [26.33] which ~ a y be mediated through the pattern of growth horn,~ne secretion [27.35,36]. In mature male rats growth hormone is secreted in pulses, but in females the pulses are lower and the concentrations of growth hormone are higher [37.381. Such differences have been implicated in the sexual dimorphism of hepatic steroid metabolism [391 and in the isoforms of cytochrome P-450 [40.41], The mechanism for lyosphosphatidylcholine secretion is not firmly established but it probably arises mainly by the action of a phospholipase A~ on phosphatidylcholine in the cell because of the unsaturated ha+ ture of the lysophosphatidylcholine. However. the presence of 25-30% of 16:0 and 18:0 in lysophosphatidylcholine produced in the absence of exogenous fatty acid (Table I) is far higher than would be expected in the sn-2-position of glycerolipids [30.42]. Therefore. phospholipase A , probably participates in the production the saturated lysophosphatidylcholine that appears in the medium. It was not feasible to determine meaningfully the position of the acyl groups on the lysophc,sphatidylcholine, since isomerization would have taken place during the 8 h incubation that was required to obtain sufficient material to analyse even when using 20-30 dishes of cells for each condition. Further, isomerization would also have occurred on the silica gel used to separate the isomers [43]. An alternative ap+ proach would have been to have used N M R spectroscopy [44], but this would only have given the average composition of 1- and 2-1ysophosphatidylcholines and not whether the unsaturated fatty acid was on the 2-position as expected. The putative phospholipase activities that produce lysophosphatidylcholine may be Ca" +-dependent. since Ca'-+-antagonists such as chlorpromazine and the Ca 2 t-channel blocker, verapamil, inhibit lysophosphatidylcholine production at lower concentrations than those that inhibit the secretion of very-low-density lipoprotein. However. addition of EDTA to the incubation medium had relatively more effect on the latter process [4]. An alternative explanation is that verapamil and chlorpromazine might inhibit hepatic phospholipase A I directly since this has been demonstrated using tissue from heart [451.
158 T h e l y s o p h o s p h a t i d y l c h o l i n e that is f o r m e d by the hepa'.ocytes is s e q u e s t e r e d in the m e d i u m b y a t t a c h m e n t to a l b u m i n [41. It is likely tha~ this p r o c e s s w o u l d also o p e r a t e in vivo in n o n - r u m i n a n t a n i m a l s , t h e r e b y t r a n s p o r t i n g the l y : ~ o p h o s p h a t i d y l c h o l i n e to the b l o o d . T h i s w o u l d s u p p l y o t h e r o r g a n s with c h o l i n e a n d u n s a t u r a t e d fatty acids. H o w e v e r , we failed to d e m o n s t r a t e the release of i y s o p h o s p h a t i d y l c h o t i n e f r o m o v i n e h e p a t o cytes a n d this r o u t e of m e t a b o l i s m m a y n o t o p e r a t e at a s i g n i f i c a n t level in sheep. Acknowledgements W e wish to t h a n k the S E R C of G r e a t B r i t a i n for p r o v i d i n g A G with a C A S E research s t u d e n t s h i p , a n d the M R C ( G r e a t B r i t a i n ) a n d the A l b e r t a H e r i t a g e F o u n d a t i o n for M e d i c a l R e s e a r c h for f i n a n c i a l s u p p o r t , T h e skillful t e c h n i c a l a s s i s t a n c e of Mr. P a u l Hales is gratefully acknowledged.
References 1 Seka.,,, G., Patton, G.M., Lincoln, E,C, and Robins, S,J. (I985) J. Lab. Clin. Med. 105, 190-194. 2 Mangiapane, E.H. and Brindley, D.N. (1986) Biochem. J. 233, }51-}60.
3 Graham. A., Zarnmit, V.A. and Brindley, D.N, (1988} Biochem. J. 249, 727-733 4 Graham, A., Bennett, A.J., McLean, A.A., Zammil, V.A. and Brindley, D.N, (1988j Biochem, J. 253, 687-692. 5 Baisted, D J, Robinson. B.S. and Vance, D.E. (1988] Biochem. J. 253, 693-70I. 6 Robinson. B.S., Yao. Z., Baislcd, D.J. and Vance, D.E. (1989) Bi~x:hem. J. 260, 207-214. 7 Nelson, G.J. (1969) Lipids 2, 323-328. 8 N,~rum. K.R. and Gjone, E. (i967} Stand. J. Clin. Lab. Invesl. 20. 231 - 243. 9 C'ascales. C.. Mangi~pane. E.H, and Brindley, D.N. (1984) Binchem. J, 21g, 911-916. 10 Donaldson, A., Lomax. M.A. and Pogson, C.I. (1979) Proc. Nutr. soc. 38, 88A. }i Piltner. i.L-A., Fears, R. and Brmdley, D,N. (1985) Biochem. J. 225,455-462, 12 Bligh, [:-G. and Dyer, ~ J. (1959} Can. J, Biochenl. Physiol. 37, 911-917. 13 Christie, W.W, (1973) Lipid Analysis, pp. 1~28, Pergamon Press. Oxford. 14 Chris,i,z, W.W. (1981) in Lipid M¢tahol.ism in Ruminant Animal:,, (Christie, W.W., ed.), pp. ~)5-~193, Pergamon Press, Oxford. 15 Christie, W.W. t~nd I-louter, M.L, (1984) J. Chromatogr. 294, 489-494.
16 Christie. W.W, (I9821 J. Lipid Res. 23, 1(]72-1075. 17 Saggerson, E.D. and dreenbaum. A.L. (196q) Bin,:hem. J, 115, 405 -417. 18 Switzer, B.B. ;rod Summer, G.K. ~lg71) Clin. Chim. Acta 32, 203- 226. 19 Glom.set, J.A. and Wright, J.L. (1965) Bioehim. Biophys, Attn. 89, 266- 276, 20 Kucera. G.L.. Sisson. P.J.. Thomas. M.J. and Waite, M. (1988) J. BioL Chem, 263. 1920-1928. 21 Parles, J.G.. Chan. P, and Goldberg, D.M. (1986) Biochem, Cell. B 64. t147--1152. 22 Kasim. S.E.. Khilnani, S. and Peterson, W.D. (1986} Clin. Res, 34, ASg6. 23 Cisar, L.A. and Bensadoun (1987) Biochim. Biophys. Aeta g27, 305-314. 24 Ridgway. N.D. and Dolphin. P.J. (1985) J. Lipid Res. 26, 13001313. 25 Lyman, R.L.. Tinoco, J.. Bouchard, P., Shehan, G., Ostwald, R. and Mdjanich, P. (1967) Biochim. Biophys. Aeta 137, 107-114. 26 Lyman, R.L. Hopkins. S.M,, Sheehan. G. and Tinoco, J. ~'968) Biochim. BioFhys. Acta 152, 197-204. 27 Oscarsson, J. and Ed._"~. S. (1988) B~ochim. Biophys. Aelal g59. 280-287. 28 Nelson. G.I. (1969) Comp. Biochem. Physiol. 30. 715-725. 29 Vance. J.E. and Vance. D.E. (1986) J. Biol, Chem. 261, 4486-4491. 30 Holub, B.J. and Kuksis, A. (19711 Can, J. Biochem. 49, 1347-1356. 31 Natori, Y. (1963) J. Biol. Chem. 238. 2075-2080. 32 Bjornslad, P. and Bremer, J. (1966) .I. Lipid Res. 7. 38-45. 33 Young, D.L. (1971) J. Lipid Re~,, 12, 590-595, 34 Christiansen, K., Gan, M.V. and Holman, R.T. (1969) Biochim, Biophys. A(,ta 187, 19-25. 35 Clejan, S. and Maddaiah, V. (1986) Lipids 2t, 677-683. 36 Oscarsson. J., Jansson. J.-O. and Ed6n, S. (19881 Acta Physiol. Scand. 133, 257-255. 37 tZd6n, S. (1979) Endocrinology 105, 555-560, 38 Jansson. J.-O., Eden, S. and lsaksson. O. (1985) Endocrine Rev. 6, 128-~50, 39 Mode, A., Guslaesson, j.-A., Jansson, J.-O., Ed,Sn, S. and Isaksson, O. (1982)Endocrinology. 111, 1692-1697, 40 MacG~,~ch, C.. Morgan. E.F. and Gustafsson, J.-h. (1985) Endocrinology. 117, 2085-2092, 41 Guzelian, P.S.. Li, D.. Schuetz, E.G.. Thomas, P., Levin. W, Mc~de, A. and Gustafssoq, J.-A, (1988) Proc Natl, Acad. Sti, USA 85, 9783-9787. 42 PJnd..~., Kuksis, A.. Myher, J.J. and Marai, L. (1985) Can. J. Biochem. Cell Biol. 63, 137-144. 43 Van den Bosch, H. and Van Deenen, L.L.M. 0965) Biochim. Biophys. Acta 106. 326-337. 44 Pt~ckthun, A. and Dennis, E.A, (1982) Biochemistry., 21, 17431750. 45 Hosteller, K.Y. and Jellison, E.J. (1989) Mol. Cell. Biochem. 88, 77-82.
151
' 1991 Elsevier Science Publishers B,V. {Biomedical Division) 0005-2760/ql/503.5[) A D O N I S 000527609100070K
BBALIP 53"370
Sexual dimorphism in the preferential secretion of unsaturated lysophosphatidylcholine by rat hepatocytes but no secretion by sheep hepatocytes Annette Graham ~'*. Victor A. Zammit 2. William W. C ~ : s t i e "and David N. Brindley Department of Bzm'hemz.:'t O" and Lipid and l.tp,~protem Research Gr~mp. Fac'ul(v *(l"Ah'dzcme, ]~'tlll't'~tlt" r~f .4 ll~'rta (Canada) aad 2 ttaanah Rt'~earth ins/(fete..Ivr (/_.K
(Received 23 July 19~()) Key words: ArachidonJte: Es~',cntialfatty acid; Phonpha[id?,lch~hne: VLDL: Sex related difference
(i) Rat and ovine hepatocytes were incubated in monolayer culture with various fatty acids to determine their effects on the composition of the lysophosphatidylcholine that was secreted. (2) No lysophosphatidylcholine was detected in the medium from the ovine hepatocytes even though these cells were hormonally responsive and they ~creted phosphatidylcholine and triacylglycerol in very-low-densi~ lipoprotein. (3) Lysophosphatidylcholine was readily detected in the incubation medium of rat hepatocytes. The predominant forD' acids in this lipid were unsaturated. Stearate and arachidonate contributed 15 and 34%, and 24 and 26% of the total tatly acids when hepatocytes from male and female rats were used, respectively. The relative proportions of stearate and arachidonate in the phosphatidylcholine .secreted from the hepato,zytes were 20 and 14%, and 28 and 21% for the males and female~ respectively. The equivalent values for stearate and arachidonate for phosphatidylcholine in the hepatocytes were 18 and 17% and 33 and 22% for male and female rats. These results provide furlher indications of sex differences it, hepatic phospholipid metabolism and extend this to the secretion of phosphatidylcholine and lyosphosphatidylcholine. (4) The addition of ! mM stearate to the incubation na~lium did not significantly decrease the proportion of arachidonate in the lysophosphatidylcholine obtained from the hepatocytes of the male rats. However, the relative proportion of arachidonate was decreased in incubations that contained I mM oleate or linoleate. (5) The results provide evidence that the preferential .secretion of unsaturated lysophosphatidyleholine by the liver may provide a system for transporting unsaturated lath.' acids and choline to other organs in non.ruminant animals. However, this mechanism may not operate for ruminants. Introduction Per(used rat liver I1] and monolayer cultures of rat [2-6] can synthesize lysophosphatidylcholine which appears in the perfusion, or incubation medium. A large proportion of the fatty acids associated with this lysophosphatidylcholine are unsaturated [1,5] and. furthermore, the secretion of this lipid is
hepatocytes
Abbreviations: Fatty acids are indicated by their numbers of carbon atoms and double bonds. The family to which some acids belong has been indicated b~, ( n - x ) where x denotes the number of carbon atoms from the last double bond to the terminal melhyl group. * Present address: Welicome Research Laboratories. Biochemistry Department. Langley Court, Beckenham. Kent. BR3 3BS, U.K.
Correspondence: D,N. Brindley, University of Alberta, Lipid and Lipoprotein Research Group, Heritage Medical Research Centre. Edmonton, T6G 2S2, Canada.
promoted by the inclusion of unsaturated fatty acid,; in the incubation m e d i u m [3]. This secretion of lysophosphatidylcholine from the liver could account for a large proportion of this lipid which is found associated with albumin in the blood of m a n y mammals. Lysophosphatidylcholine is often the second most prevalent phospholipid in the plasma [7] and its concentration remains relatively high [8] in p a 6 e n t s with a deficiency of lecithin:cholesterol acyltransferase, LCAT. T h e unsaturated lysophosphatidylcholine produced by the liver could provide a novel transport system for choline and polyunsaturated fatty acids to other organs [3]. The present work was undertaken with three objectives, The first was to examine the effects of modifying the fatty acid composition of the incubation m e d i u m on the proportion of the fatty acids esterified in the secreted lysophosphatidylcholine. The second objective was to establish if there was any difference between the fatty acid c o m p o s i t i o n of the l y s o p h o s p h a t i d y l c h o l i n e
152 secreted by mate and female hepatocytes. The third was to determine whether cultures of sheep hepatocytes could also secrete lysophosphatidylcholine as a specific means of transporting unsaturated fatty acids from the liver to other organs. This was particularly significant since the sheep being a ruminant animal has much lower proportions of polyunsaturated fatty acids in it:, phospholipids. Malerials and Methods
Animals Rats were obtained from A. Tuck & Son (Battlebridge, Essex, U.K.) and they were fed a standard laboratory diet which was purchased from Oxoid (Basingstoke, Hants, U.K.). The sheep were Finn x Dorset Horn Cross-bred wethers, 9-12 months old, which were fed hay and concentrates ad libitum. The typical composition of 1 kg of concer,trate was 100 g of cooked flake maize, 507.5 g rolled barley, 100 g rolled oats. 100 g wheat feed pellets, 130 g soya, 50 g grouod nut, 3.3 g dicalcium phosphate, 3.2 g NaC1, 2.5 g vitamins/trace minerals, 2.5 g lime stone and 1 g calcined magnesite.
Materials The sources of most materials have been described previously [3,4]. Lipids and radioisotopes were purchased from Sigma Chemical and Amersham International, r,::zpectively. Bisbenzamide H33258 was from Fluka. Ph:3sphatidyl[Me-3H]choline (0.3 Ci/mmol) was kindly donated by Dr. Dennis Vance.
Preparation and incubation of hepato(Ttes Hepatocytes were isolated from male and female Wistar rats essentially as described by Cascales et al. [9], except that heparin was added to the first perfusion buffer rather than being injected and the perfusion with collagenase was performed through the hepatic portal vein with the inferior vena cava being cut. For sheep hepatocytes only the caudate lobe of the liver was perfused using a technique adopted from Donaldson et al. [10]. The sheep was anesthetized with 20 ml of Sagatal by intrajugular injection. The caudate lobe was rapidly excised, cannulated and perfused under gravity with 500 ml of modified Krebs-Henseleit medium containing 50 mg of heparin at about 80 m l / m i n and at 25-35°C. The flow of medium was maintained during transportation of the lobe from the slaughter house to the laboratory, which lasted about 4 min. The lobe was then attached to a perfusion apparatus and the flow switched to a recirculating perfusion containing cc.!lagenase at 0.04% at 37°C. The incubation then proceeded for 40-45 rain until leakage from the lobe surface occured. The lobe was then transferred to a petri dish containing about 10 ml of perfusion
medium. The capsule was cut and the cells gently washed and filtered, essentially as for the rat hepatocytes [9] except that cells were centrifuged at 50 g for 2-3 rain. Sheep hepatocytes normally demonstrated and 85-95% exclusion of trypan blue. Between 1.5 • l0 t' and 2 • 10 ~ rat hepatocytes or 3.106 and 3.5 • 10 ~ sheep hepatocytes were pipetted into collagen-coated tissue culture dishes containing a final volume of 3 ml of modified Leibovitz L-15 medium containing new-born calf serum [91. The plates were incubated for 37°C in a humidified air atmosphere for I to 2 h. The medium was replaced removing non-viable and unattached cells. A monolayer of hepatocytes was produced, 95% of which excluded trypan blue. After incubation for 6 to 8 h, the cells had flattened and spread from their initial round shape and the medium was replaced by medium that contained 0.2% fatty acid-poor bovine serum albumin instead of serum. Both types of hepatocyte were incubated for 14 h and then for an extra 2 h in fresh medium. The hepatocytes were then incubated for the specified time in modified Leibovitz L-15 medium [9] containing 0.5 mM albumin, I mM glycerol, 100 luM choline and fatty acid additions as indicated. Fatty acid solutions (80 t~M) were prepared by heating to 7 0 ° C in water containing a 50% molar excess of KOH and they were then pipetted slowly into the albumin containing medium with stirring [3].
(,'ell viability and expression of rates of lipid synthesis and secretion The assessment of the metabolic viability of the rat hepatocytes has been described previously [9,11]. The sheep hepatocytes produced 4.87 + 1.5/~mol of glucose per mg of DNA in 20 rain. This secretion was increased between 2-2.2-fold by the addition of 1 ~tM glucagon, 71 #M dibutyryl cAMP, 5 #M phenylephrine or 1.1 IuM isoproterenol. The rates of lipid synthesis for rat hepatocytes have been calculated relative to the units of lactate dehydrogenase present in the cells. This compensates for small variations among dishes of cells better than does the determination of protein or DNA [9,111 since some non-viable cells ma3 remain attached to the plate and contribute to the latter two values. Loss of viability is accompanied by leakage of lactate dehydrogenase into the incubation medium, The content of lactate dehydrogenase per dish of cells was about 1 t~mol of lactate oxidized/min at 25 o C. One dish of cells also contained about I mg of protein and so that results can be readily expressed relative to protein if this is required for comparison with other work. The present incubations also contained about 34 mg of albumin/ml and, therefore, the measurement of the relatively small concentration of hepatocyte protein might have been inaccurate for the routine determination cell number. Results for
153 ovine hepatocytes have been expressed relative to DNA rather than lactate dehydrogenase, since this activity was about 12-times lower than in rat hepatocytes ~hich made the assay unreliable.
A,lalysis of lipids Lipids were extracted from cells and medium from 25-30 dishes for each experimental condition. The medium was collected and centrifuged at 3000 rpm for 15-20 min in a bench centrifuge to remove detached cells and debris. The monolayers were washed three times with 2.5 ml portions of ice-cold serum-free incubation medium and then scraped from the dishes in 1 ml of ice-cold 0.25 M sucrose containing 0.5 mM dithiothreitol and 10 mM Hepes adjusted to pH 7.4 with KOH. The cell suspensions were sonicated for 6 s in i s bursts at 22 kHz with an amplitude of 8 a m peak to peak. The sonicates and media were stored at - 2 0 ° C under N_,. Samples of medium and cell homogenates were extracted using solvents that contained 0.0005-O.001% 2,6,di-tert-butyl-p-cresol as an antioxidant. The phases were separated as described by Bligh and Dyer [12] until after the addition of chloroform when 0.2 M K H z P O 4 and 2 M KCI was used instead of water. In a typical experiment 80 ml of medium and 20 ml of cell homogenates were extracted. T o the bottom phase of the extractions was added 50 tzg each of t.-c~-heptadecanoyl lysophosphatidylcholine and t-a-pentadecanoyl phosphatidyleholine in i ml of prot',an-2-ol as internal standards. 15:0 and 17:0 fatty acids do not naturally occur in liver phospholipids of the rat [131 and only to the extent o f about 3% in sheep liver phospholipids 1t4]. Lipid extracts were dried in a rotary evaporator and residual water was removed by adding a small volume of propan-2-ol and re-evaporating. The lipid was then dissolved in a small volume of chloroform and stored at - 2 0 ° C until required for HPLC. The stationary phase of the H P L C system was Spherisorb T M silica (5 ~tm particles~ 25 cm × 0.5 cm column, Hichrom, Reading, Berkshire, U.K.). A model 8770 isocratic H P L C pump (Spectra-Physics, St, AIhans, U.K.) equipped with a Knauer differential refractometer (Dr. H. Knauer, Oberusel. Taumus. F.R.G.) was used. The lipid extract was filtered through a 40 # m solvent-resistant filter, dried, taken up in 50 t.tl of CHCi~ and injected onto the H P L C column. The mobile phase which was degassed with helium, consisted of acetonitrile/ m e t h a n o l / water containing 1.5 /tM lithium acetate ( 5 0 : 4 5 : 6.5, v / v ) at 1 m l / m i n . [15]. The ct,lumn was calibrated using a 20 ~1 mixture of 11 m g / m l tff phosphatidylcholine/ml and 14 m g / m l of lysophosphatidylcholine. Samples containing phosphatidylcholine and lysophosphatidylcholine were collected manually as the peaks corresponding to the standards eluted from the column. T h e fractions were evaporated to dryness in
a stream of N_,. The lipid was covered with hexane containing 0.02% butylated hydroxytoluene and stored at - 20" C. The purified samples were evaporated to dryness under a s~ream of N, and phospholipids were methylated by sodium methoxide-catalyzed transesterification [16]. Fatty acid analysis was performed on a 25 m fused silica column with Silar 5CP as the stationary phase using a Carlo Erba gas chromatograph and H z as the carrier gas. The temperature was set at 155 ° C for 3 min and was programmed to increase at 4 C ° / m i n to a final temperature of 195°C where it remained constant for 17 rain. Peaks were identified with authentic standards and were quantified by electronic integration. The mol% of each of the fatty acids present was then calculated and related to the known amount of internal standard.
Analysis o/htctate dehydrogenase and DNA These were analyzed by the methods of Saggerson and Greenbaum [I7] and Switzer and Summer [!S;], respectively.
Results and Discussi,-m
Lysopho.~phatidyh.holJne is secreted into the medium by ~'iahle hepatocTtes In determining the physiological significance for the secretion of lysophosphatidylcholine, it was important that this lipid is produced by viable cells and that it is not the result of non-specific degradation of phosphatidylcholine in the medium. The albumin used in the medium might have been contaminated by phospholipase, or LCAT and it was therefore heated to 6 0 ° C for 30 rain [191. This did not cause any significant decrease in iyst;phosphatidylcholine production by rat hepatocytes (results not shown). Lysophosphatidylcholine might also be produced in the medium by the action of an extracellular phospholipase produced by the hcpatocytes, Hepatic lipase can act on phospholipids and exhibit phospholipase A n activity 120]. It can be secreted by rat hepatocytes I21] and by Hep G2 ceils [22]. However, in some experiments in the present work, rat hepatocytes were prelabelled with ['all]glycerol and [14Clcholinc in the presence of I mM oleate at 37°C, They were cooled to 4 ° C and washed with sufficient heparin (I0 units/ml) to remove hepatic lipase that was associated with the cell surface I23]. The subsequent prt~duction t,f lysophosphatidylcholine on incubating the cells at 37 ° C was not decreased (results not shown). thus making it unlikely that lysophosphatidylcholine was produced by surface bound hepatic lipase. Similar results and conclusions were reported by Baisted et al. [51. LCAT is also secreted from the hepatocytes [24], and it could produce lysophosphatidylcholine, especially that containing saturated fatty acid. in the medium (Table
154 TABLE I
t"al(r actd c,Jmpmttion of the I.~:rt,pho.~phant(rh'holine isolated frmn the medium of cultured rat hepatocytes Ra~ hepatocytes were preincubated as described in the Materials and Methods Section. The medium was then changed and the medium was supplemented with l mM fatty acid where indicated. The cultured medium and cell.,, were collected aRer a further incubation of 8 h and lysopho,~phatidylcholine and ph~sphafidylch(fline were isolated and their fatty acid compositions analyzed. The mtd% of the r,~;.IV acids in lysophosphatidylcholine is given fi~r the number of independent experiments indicated. Results are means+ ranges h~r two experiments, or means_+. S.D. h~r three experiments. The significance of the difference between incubations in the absence of exogenous fatty acids and those in Iheir presence was calculated by using a paired t-lest and this is indicated by: * P < 0.025, * * P .c 0.01 and ' P < 0.005, The incorporation of exogcnou~ la('-Iabelted fatty acids into lysophosphatidylcholine in three independent experiments for male rats was 0.93_+ 0.23. 3.24+ 2.36 and 2.14 + 1,33 nmol per unit of lactate dehydrogenase, respectively, for lg:0, 18:I and 18:2. n.d.. not detected. Fatty acid
Fatty acid added to medium: male
female
none 16:0
16:1 18:0 18:I ( n - 9 } 18:1 ( n - 7 1 18:2 20:4 22:61n-3)
l 8:0
| 8:1
18:2
none
18:0
18:1
18;2
10.9__+1.1 n.d. 23.1_+1.9 11.9-+1.1 1.9_+0.1 11.6_+1.0 34.5_+3.7 6.2+_0.3
8.94- 3.1 n.d. 8.9_+ 1.4 42.I_+10.6 ' 2.6+ 1.6 O.O~+ 2.8 21.8_+ 9.4* 5.34. 3.6
8.34- 3.3 n.d. tl.9_+ 1.9 17.2_+17.6 n.d. 33.7_+13.0' t8.9_+ 8 . 9 * * 4.3_+ 0.6
5.4_+ 1.3 n.d. 24.2_+ 2.1 22.9+13.0 n.d. 5.1_+ 1.g 26,44. 9.1 13.3+_ 3.1
11.74-3.6 n.d. 39.5-+6.1 19.3+_7.4 n.d. 5.0+_1.7 16.8+5.7 7.1_+3.1
8.2____.5.9 n.d. 19.4+_4.1 30.2-+5.3 n.d. 6.1±2.1 29.1_+4.8 9.04-2.4
16.64- 5.5 n.d. 20.9_+ 4.6 18.8+-10.1 n.d. 23.3+_10,4 14.5+ l.O 5.2+ 1.2
2.95-+(i.4
1.9+0.3
4.8-+ 1.4
4.2-+ 1.3
2.3+ 0.3
0.95:0.09
2.75:0.2
1.65:0.07
3
2
3
3
2
2
2
2
10.8 n.d. 14.5 16.6 2.6 14,7 34.0 5.8
Unsaturated Saturated N umber of experiments
4-3.6 +_4.0 _+2.1 ±0.7 _+2,5 4-2.5 _+0,4
1). W e t h e r e f o r e t o o k m e d i u m f r o m h e p a t o c y t e s t h a t h a d b e e n i n c u b a t e d f o r 5 h a n d a d d e d 3.7 # C i / m l of
lysophosphatidylcholine by the conditioned compared to non-conditioned medium d'.,ring 20 h was less than
phosphatidyl[-~H]choline followed by son±cation. The fal, c o f ',his p h o s p h a t i d y l c h o l i n e w a s t h e n f o l l o w e d d u r i n g a f u r l h e r i n c u b a t i o n f o r u p Io 2 0 h in t h e a b s e n c e o f
1% o f t h e o r i g i n a l p h o s p h a t i d y l [ 3 H ] c h o l i n e added. This r e s u l t a g r e e s w i t h t h a t o f B a i s t e d e t al. [5] w h o t o o k m e d i u m f r o m t h e h e p a t o c y t e s a n d i n c u b a t e d it f o r a f u r t h e r 5 h i n t h e a b s e n c e o f cells. T h e r e w e r e n o
heptocytes.
The
conversion
of phosphatidylcholine
to
TABLE II
l.'at(r acid comp,,sttion of the phr~,~ffhatit(t'h'holine isolated from the medium of cultured rat hepatotytes The experimental conditions and the method of expressing the results are described in the legend to Table I. The incorporation ol' exogenous 14('-labelled fatty acids into secreled phosphatidylcholine in three independent experiments for mate rats was 0.76 +0.21, 0.764-0.08 and 0,81.15:0.58 nm~fl per unit of laclate dehydrog:,enase, respectively, for 18:0, 18:1 and 18:2 Fatty acid
Fatty acid added to medium: male
female
none 16:1) 16:t 18:0 1 8 : l ( n 9) l~:l(n-7) 18:2 2(/:4 22:6(n-3) Unsaturated Saturated Number of experiments
41.4 n.d. 19.5 7.0 2.4 14.8 13.8 n,d.
18:0 ±10.2 4.. ± _+ +_ ±
4.8 2.9 0.7 1.6 3.2
34.8 n.d. 24.7 6.9 1.2 16.4 13.4 n,d.
18:1 _+3.2 +1.7 _+0.5 _+1.0 "+(I.3 +_4.2
38.2 2.9 14.9 15.2 "-.8 7.8 12.3 5.9
+6.9 +1.4 +0.4 -+3.0 +_0.5 _+6.6 _+4,9 ±3,7
18:2
none
18:0
18:1
! g:2
28.6± 0,6 * n.d. 21.3+11.8 14.1+11.3 2.1_+ 2.0 18.6_+ 3,8 12.4-+ 6.5 2.9__+ 0,9
22.2±9.5 n.d. 28.2_+4.5 7.6±1.6 n.d. 14.7_+4.3 20.7_+5.8 5.6-+0.9
23.g± 4.9 n.d. 32.0+12.1 24.8±!3.1 •.d. 9.4+_ 2.0 8.4_+ 4.6 n.d.
19.4±8.9 n.d. 26.0+3.2 15.0±2.6 1.3-+0.3 8.5_+0.009 19.5-+2.1 9.4_+0.9
19.35:2.8 n.d. 23.5_+ 7.1 18.8+10.0 n.d. 13.7+ 1.0 16.9_+ 3.3 6.2± 0.3
(I.64+ 0.15
0.69+0.14
0.91+0.24
1.1± 0.5
1.0_+0.2
0.8_+ (I.2
1.2±0.3
1.3± 0.2
3
2
2
3
2
2
2
2
~55 significant changes in the concentrations of phosphatidylcholine, lysophosphatidylcholine and glycerophosphocholine. Sekas et al. [1] also demonstrated no significant increase in the lysophosphatidylcholine concentration of the medium used to perfuse rat livers when this was incubated in isolation, or when the perfusion was continued after removal of the liver. These results and the predominantly unsaturated nature of the lysophosphatidylcholine exclude a significant participation of LCAT in its production. The production of lysophosphatidytcholine can be dissociated from the secretion of phosphatidylcholine and V L D L through the effects of colchicine, dexamethasone plus insulin [2], albumin [4,5]. fatty acid,; [3], verapamil, E G T A and chlorpromazine [4 i. The rates of secretion from rat hepatocytes of lysophosphatidylcholine, phosphatidylcholine and triacylglycerol was approximately constant during a 24 h period with our hepatocyte system [2]. This and the fact that less than about 5% of the lactate dehydrogenase was lost 'r0m the cells during the incubation period indicates that the hepatocytes remained viable. Also the magnitude of lysophosphatidylcholine secretion throughout was 4 12-fold higher than that for phosphatidylcholine when the incubations were supplemented with oleate and albumin (Ref. 2~4; Tables I and il). Therefore, lysophosphatidylcholine is not formed by the hydrolysis of phosphatidylcholine in the medium, nor is it a product of dead or dying cells. It is a major choline metabolite in rat hepatocyte cultures and is probably a major contributor to the lysophosphatidylcholine found in blood of rats and man.
Effects of exogenous fatty acids on the composition of lysophosphatidylcholine secreted by hepatocytes from mule and female rats The lysophosphatidylcholine produced by hepatocytes from male rats in the absence of exogenous fatty acid was predominantly unsaturated with 20:4 constituting about 34 mol~. The unsaturated to saturated fatty acid ratio was 2.95 which was noticeably more unsaturated than the ratio of 1.38 to 1.96 reported by Baisted et al. [5] from hepatocytes cultured in fetal calf serum. Their equivalent values for cells incubated with de!ipidated fetal calf serum was 0.55 to 0.95. The hepatocytes used in the present work were incubated for 16 h prior to the experiments in medium containing 0.2f~ of fatty acid poor bovine serum albumin. Addition of 1 mM 18:0 to the medium gave an enrichment of about 9% in the content of 18:0. This was accompanied by decreases in the percentage of 18:1 and 18:2, but no significant change in the proportion of 20:4. The unsaturated to saturated ratio of the lysophosphatidylcholine decreased from 2.95 to 1,90. Conversely, addition of 1 mM 18:1 or 18:2 increased the unsaturated to saturated ratio. 18:1 markedly increased
by about 25~ the percentage content of this fatty acid in lysophosphatidylcholine. This was accompanied by a decrease in the content particularly of 20:,1. There also appeared to be decreases in the percentages of 16:0 and 18:0 but these did not reach the level of statistical significance. Similarly. the addition of 18:2 to the incubation increased its content by about 19% in lvsophosphatidylcholine and it decreased by about 15% that of 20:4. However. 18:1 and lg:2 also stimulated the secretion of lysophosphatidylcholine by about 2-fold [3] and so part of their effects were probably mediated by providing additional glycerolipid for lysophosphatidylcholine secretion rather than by competing with 20:4. Therefore, the secretion of 20:4 in lysophosphatidylcholine is well preserved, despite the addition of exogenous fatty acids to the incubation medium. The composition of lysophosphatidylcholine secreted by hepatocytes by female rats is also shown in Table 1 and they confirm the general effects seen with the hepatocytes from the males. However, the relative proportion of 18:0 appeared to be higher and that of 18:2 and 20:4 lower when compared to the males. T h e incubation of female hepatocytes with 18:0, 18:1 or 18:2 again resulted in the enrichment of the secreted lysophosphatidylcholine with the particular exogenous fatty acid, Although incubation with 18:2 increased its conccntration in lysophosphatidylcholine, it did not increase the unsaturated to saturated ratio in the two experiments performed. This appeared to be partly caused by a higher content of 16:0. The phosphatidylcholine secreted into the medium by hepatocytes from male rats in the absence of exogenous fatty acids gave an unsaturated to saturated ratio of about 0.64 (Table I1~. This composition differs significantly from that of the lysophosphatidylcholine that was secreted, for example, the proportions of 16:0 and 18:0 were increased ( P < 0.005 and P < 0.025, respectively), whereas 18:1 (n - 9) and 20:4 were lower ( P < 0,025 and P < 0.005, respectively~. Addition of 18:0 to the medium gave a small enrichment in its content in the secreted phosphatidylcholine in the twe experiments performed with no significant change in the unsaturated to saturated ratio. Incubation with 1 mM 18:1 (n - 9~ increased this ratio in both experiments. This was related to the increased proportion of 18:1, but this enrichment was not as marked that in the secreted lysophosphatidylcholi::e (Table I). Addition of 18:2 to the incubation decreased the proportion of 16:0 in the secreted phosphatidylcholine by about 12~ with an increase in the concentration of 18:I ( n - 9 ) c o m p a r e d to when no fatty acid was added (Table !I). There appeared to be a small increase in the content of 18:2, but this was not statistically significant. Addition of 18:2 to hepatocytes from male rats gave an unsaturated to saturated ratio of 1.1 for the secreted phosphatidylcholine which was less ( P < 0.05} than 4.2 for lysophos-
i56 TABLE IV
p h a t i d y l c h o l i n e secreted from the i n c u b a t i o n s c o n t a i n ing 18:2 (Table I). T h e p e r c e n t a g e c o m p o s i t i o n o f 16:0 in the p h o s p h a tidylcholine f r o m the male h e p a t o c y t e s a p p e a r e d to be higher than that for the h e p a t o c y t e s o f the two female rat:;. Conversely, the p r o p o r t i o n s of 18:0 a n d 20:4 app e a r e d to be higher for p h o s p h a t i d y l c h o l i n e secreted from the female hepatoeytes. T h e r e were n o significant differences in the perc e n t a g e c o m e n t o f the fatty acids between the secreted p h o s p h a t i d y l c h o l i n e a n d that f o u n d in the cells (Table !11) f r o m the male rats in the a b s e n c e o f e x o g e n o u s fatty acid. A d d i t i o n o f 18:0, 18:1 a n d 18:2 increased the relatively p r o p o r t i o n of the respective faUy acids in the p h o s p h a t i d y l c h o l i n e o f the cells o f the male rats as expected. In the case o f the a d d i t i o n o f 18:2 there were lower p r o p o r t i o n s o f 18:1 ( n - 9) a n d a greater p r o p o r tion o f 18:2 in the cells as c o m p o s e d to the p h o s p h a tidylcholine in the m e d i u m . T h e p h o s p h a t i d y t c h o l i n e in the h e p a t o c y t e s o f the female rats had a high p r o p o r t i o n o f 18:0 a n d a lower p r o p o r t i o n o f 16:0 in the p h o s p h a t i d y l c h o l i n e t h a n for the males (Table IIl). This w~s also reflected in the p h o s p h a t i d y l c h o l i n e that was secreted into the m e d i u m (Table II). S u p p l e m e n t a t i o n o f the m e d i u m with exogen o u s fatty acids again increased the p r o p o r t i o n o f that p a r t i c u l a r acid in the p h o s p h a t i d y l c h o l i n e as e x p e c t e d (Table ili).
Fatty acid compositi~m of the phosphatt~lvh'holine that was isolated from the medium and from ~he cells of cultured oeine hepato(3"tes
Ovine hepatocytes were prepared and preincubated as described in '~hc Materials and Methods section. The cells were then incubated in the presence or absence of 18:1 as indicated for 24 h and phosphalidylchotine was isolated from the cells and medium and the fal'y acid composition was determined. The tool% of the fatty acids in the legend.,, are sit~wn as meatJs +_,z D. for three independent experiments, Fatty acid
In medium none
16:0 16:1 I8:0 18:1~n-9) 18:1(n-7) 16.2 20:4
27.5 2.5 27.8 31.1 0.9 6.6 3.6
Unsaturated S:~turated
In cells 18:1
+7.7 _+1.0 ±5.7 ±3.3 +0.7 +0.9 +_1.2
23.8 2.0 26.8 36.6 0.8 6.4 3.3
none
18: I
_+3.9 23,1 -+5.6 22.5-+4.8 -+1.8 1.7 +0.6 1.6_+0.2 +5.7 30.1 _+7.1 25.7_+0.7 _+0.9 34.8 _+4.4 -,4,1.9+5.1 -+0.7 i.3 +1.9 2.4+0,9 +1.3 6.1 +_2.4 4.64-1.2 *_0.2 3.2 4-0.5 2.8_+0.7
0.81 _+0.16 0.974-0.07
0.88+0.08
!.1 -+0.09
with 1 m M 114C]18:1. T h e i n c o r p o r a t i o n o f 18:1 a n d c h o l i n e into secreted p h o s p h a t i d y l c h o l i n e u n d e r these c o n d i t i o n s was 175 + 57 a n d 10.9 + 1.9 n m o l / m g o f D N A , respectively. A b o u t 90% o f the o l e a t e i n c o r p o rated into triacylglycerol t h a t w a s secreted i n t o the m e d i u m was isolated with the V L D L f r a c t i o n as described p r e v i o u s l y 12], T h e secreted p h o s p h a t i d y l c h o l i n e had an u n s a t u r a t e d t o s a t u r a t e d fatty acid r a t i o o f 0.81 in the a b s e n c e o f e x o g e n o u s oleate a n d 0,97 in the p r e s e n c e ( T a b l e IV), T h i s w a s n o t very d i f f e r e n t f r o m that seen with rat h e p a t o c y t e s ( T a b l e !I), except t h a t the relative c o n t e n t o f p o l y u n s a t u r a t e d acids w a s m u c h
Secretion o f g(vcerolipids f r o m ovine hepatocytes
O v i n e h e p t o c y t e s secreted triacylglyceroi into the m e d i u m at a rate o f 165 + 53 n m o l o f 18:1 i n c o r p o r a t e d / 2 4 h per m g o f D N A ( m e a n s _+ S.E.M. for fi,,e i n d e p e n d e n t e x p e r i m e n t s ) w h e n they were i n c u b a t e d
TABLE !II Fatty acid cmnpo.~tlion of the pho.vphatidyicholine isolated from cultured rat hepatocytes
The experimental conditions and the methods of expressing the results are described in the legend to Table 1. Tile incorporation of exogenous 14C-labelled fatty acids into phosphatidylcholine in the ~:ells for the male rals was 35.74- 7.5 (3), 36.64- 10.4 (2) and 21.9+6.7 (3) nmol per unit of lactate dehydrogenase, respectively, for 18:0, 18:1 and 18:2. Fatty acid
Fatty acid added 1o medium: male
female
none 16:0 16:1 18:0 18:1 ( n - 9 ) 18:1(n-7) 18:2 20:4 22:6(n-3) Unsaturated Saturated Number of experiments
18: I
18:2
+_1,2 _+0.03 +2.0 +0.4 +_0.9 +_1.1 +-1.t +_.0.4
25.6+0.a i.3__.0,2 12.3_+1,1 36.9+5.3 3.0+0.03 7.8+_2,1 11.2_.+2.5 1.84_.1.0
27.1 n.d. 21.0 4.1 4.0 31.4 10.2 2.0
0.96+__0.t3
0.63+.+0.085
t.6+0.04
4
2
2
33.0 1.6 18.1 8.9 6.1 12.2 16.6 3.5
18:0 +_4.9 +_1.2 __.2.7 ±2.7 +_2.1 +2.0 -+1~5 +_0.87
27.1 0.8 34.2 8.9 1.2 10.2 14.5 3.0
none
18:0
18: i
18:2
16.2 +_4.0 1.1 ±0,62 33.4 _+5.0 9.2 _+_2.9 2.7 _+0.02 8.5 -+0,004 21.6 -+1.7 6.55+0.63
15.9 4-2.5 0.8 +0.08 39.3 +1.4 13.9 +0.51 1.35-+0.005 8.2 +_1.5 t6.7 _+1.5 3,9 +0.52
9.7 0.34 32.0 23.8 1.3 7.4 21.0 4,1
17.8 0.8 27.1 7,6 1.5 27.7 Irl.0 3.9
1.11 +0.33
1.20__+0.17
0.81 +0.14
1.38
1.23
3
2
2
1
I
+2.7 _+5,5 +0,45 * _+086 _+6.0" +-3.02" +0.23
157 lower. The fatty acid composition of the secreted phosphatidylehoiine was essentially similar to that remaining in the cells (Table IV). Medium was also removed form the cells after 24 b and then incubated for a further 4 h. There was n,, degradation of phosphatidyb.holine or triacylglycerol indicating no significant lipase or phospholipase activity in the medium. There were no detectable levels of fysophosphatidylcholine in the medium after incubations of up to 24 h. It is possible that the experimental conditions were inappropriate for detecting lysophosphatidylcholine secretion from ovine hepatocytes, but these were viable. hormonally responsive and they secreted very low density 1;poprotein. Furthermore. the secretion of lysophosphatidylcholine could be readily demonstrated under similar conditions with rat hepatocytes. The phosphatidylcholine of the ovine hepatocytes contained small quantities of polyunsaturated acids with a relatively high content of 18:1. This lipid should have been a suitable substrate for a putative phospholipase A~ which might be involved in releasing lysophosphatidylcholine from rat hepatocytes. Lysophosphatidylcholine secretion by rat hepatocytes ,~as markedly stimulated by exogenous 18:1 [3]+ but no effect was observed with ovine hepatocytes. The lysophosphatidylcholine of sheep serum [28] contains relatively high proportions of saturated fatty acids which means that it could be produced by LCAT. Consequently, we have no evidence that the sheep can transport unsaturated lysophosphatidyleholines from the liver. Conclusions
The results from the rat hepatocytes provide further indications of sexual dimorphism in the fatty acid composition of hepatic phospholipids. Two of the male rats were littermates of the females and they were fed on the same batch of diet and therefore the differences should not have resulted from differences in dietary composition. The lower proportion of 20:4 in the lysophosphatidylcholine from the fema!e rats was obtained despite a relatively higher proportion of this acid in the phosphatidylcholine in hepatocytes obtained from female rather than males. It is, therefore, possible that separate pools of phosphatidylcholine are used for the production of iysophosphatidylcholine. Such selection has been observed in the secretion of phosphatidylcholine in VLDL
[291. The fatty acid composition of phosphatidylcholine for hepatocytes of male rats (Table !il) is well supported by preuious work [27,301 as is the increased proportion of 18:0 and 20:4 in phosphatidylcholine from the female rats [25-271. Our results demonstrate that sexual dimorphism in the fatty acid composition of hepatic phosphatidylcholine persists after culturing hepatocytes for 34 h. Furthermore, a sexual dimorphism
is also seen in the fatty acid compositions of the iyso~ phosphatidylcholine and phosphatidylcholine that were secreted. The latter observation may contribute to the differences in lipoprotein metabolism that are observed between the sexes. The higher proportion of 18:0 and 20:4 in phosphatidylcholine from the livers of female rats may result from a greater contribution from the methylation of phosphatidylethanolamine to total phosphatidylcholine synthesis [25+26.31- 33]. Livers of female rats also have a higher ability to convert 16:0 to 18:0 than those of males [34]. Sexual dimorphism in phospholipid composition appears to result from the effects of estrogens [26.33] which ~ a y be mediated through the pattern of growth horn,~ne secretion [27.35,36]. In mature male rats growth hormone is secreted in pulses, but in females the pulses are lower and the concentrations of growth hormone are higher [37.381. Such differences have been implicated in the sexual dimorphism of hepatic steroid metabolism [391 and in the isoforms of cytochrome P-450 [40.41], The mechanism for lyosphosphatidylcholine secretion is not firmly established but it probably arises mainly by the action of a phospholipase A~ on phosphatidylcholine in the cell because of the unsaturated ha+ ture of the lysophosphatidylcholine. However. the presence of 25-30% of 16:0 and 18:0 in lysophosphatidylcholine produced in the absence of exogenous fatty acid (Table I) is far higher than would be expected in the sn-2-position of glycerolipids [30.42]. Therefore. phospholipase A , probably participates in the production the saturated lysophosphatidylcholine that appears in the medium. It was not feasible to determine meaningfully the position of the acyl groups on the lysophc,sphatidylcholine, since isomerization would have taken place during the 8 h incubation that was required to obtain sufficient material to analyse even when using 20-30 dishes of cells for each condition. Further, isomerization would also have occurred on the silica gel used to separate the isomers [43]. An alternative ap+ proach would have been to have used N M R spectroscopy [44], but this would only have given the average composition of 1- and 2-1ysophosphatidylcholines and not whether the unsaturated fatty acid was on the 2-position as expected. The putative phospholipase activities that produce lysophosphatidylcholine may be Ca" +-dependent. since Ca'-+-antagonists such as chlorpromazine and the Ca 2 t-channel blocker, verapamil, inhibit lysophosphatidylcholine production at lower concentrations than those that inhibit the secretion of very-low-density lipoprotein. However. addition of EDTA to the incubation medium had relatively more effect on the latter process [4]. An alternative explanation is that verapamil and chlorpromazine might inhibit hepatic phospholipase A I directly since this has been demonstrated using tissue from heart [451.
158 T h e l y s o p h o s p h a t i d y l c h o l i n e that is f o r m e d by the hepa'.ocytes is s e q u e s t e r e d in the m e d i u m b y a t t a c h m e n t to a l b u m i n [41. It is likely tha~ this p r o c e s s w o u l d also o p e r a t e in vivo in n o n - r u m i n a n t a n i m a l s , t h e r e b y t r a n s p o r t i n g the l y : ~ o p h o s p h a t i d y l c h o l i n e to the b l o o d . T h i s w o u l d s u p p l y o t h e r o r g a n s with c h o l i n e a n d u n s a t u r a t e d fatty acids. H o w e v e r , we failed to d e m o n s t r a t e the release of i y s o p h o s p h a t i d y l c h o t i n e f r o m o v i n e h e p a t o cytes a n d this r o u t e of m e t a b o l i s m m a y n o t o p e r a t e at a s i g n i f i c a n t level in sheep. Acknowledgements W e wish to t h a n k the S E R C of G r e a t B r i t a i n for p r o v i d i n g A G with a C A S E research s t u d e n t s h i p , a n d the M R C ( G r e a t B r i t a i n ) a n d the A l b e r t a H e r i t a g e F o u n d a t i o n for M e d i c a l R e s e a r c h for f i n a n c i a l s u p p o r t , T h e skillful t e c h n i c a l a s s i s t a n c e of Mr. P a u l Hales is gratefully acknowledged.
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