The biosynthesis and turnover of different molecular species of rat testicular choline phosphoglycerides and triacylglycerols following intratesticular injection with [1(3)-14C]glycerol

21

Biochimica @ Elsevier

et Biophysics

Scientific

Acta,

Publishing

380 (1975) 21-30 Company, Amsterdam

- Printed

in The Netherlands

BBA 56529

THE BIOSYNTHESIS AND TURNOVER OF DIFFERENT MOLECULAR SPECIES OF RAT TESTICULAR CHOLINE PHOSPHOGLYCERIDES AND TRIACYLGLYCEROLS FOLLOWING INTRATESTICULAR INJECTION WITH [ l( 3)-’ “C] GLYCEROL

HOWARD

SPRECHER

Department Columbus,

of Physiological Chemistry, Ohio 43210 (U.S.A.)

(Received

and MARIE

P. DUFFY Ohio State

University,

333

W. Tenth

Avenue,

June 20th, 1974)

Summary Rats were sacrificed 1, 4, 8, 24, 48 and 96 h after an intratesticular injec[l(3)-’ 4 C] glycerol. The choline phosphoglycerides and triacylglycer01s were isolated and the choline phosphoglycerides were converted to diacylglycerols by treatment with phospholipase C. These diacylglycerols and the triacylglycerols were fractionated into molecular species using argentation thin-layer chromatography. At early time periods the percent of radioactivity incorporated into the oleate- and linoleate-containing diacylglycerols exceeded the molar contribution of these two fractions. The percent of radioactivity in the linoleate fraction declined rapidly at later time periods while the percent of radioactivity in the oleate fraction also declined but not as rapidly. In contrast the percent of radioactivity in the arachidonate and docosa-4,7,10,13,16-pentaenoate-containing diacylglycerols at early time periods was lower than the molar amounts of these diacylglycerols. At later time periods the percent of radioactivity in these two fractions increased. These findings suggests that oleateand linoleate-containing choline phosphoglycerides are made largely by total synthesis while a deacylation-acylation pathway plays a major role in the biosynthesis of the highly unsaturated choline phosphoglycerides. The calculated apparent half-lifes for the oleate-, linoleate-, arachidonate- and docosa4,7,10,13,16-pentaenoate-containing diacylglycerols were respectively 35, 18, 41 and 46 h. The percent. incorporation of [l(3)-’ 4 C] glycerol into the various triacylglycerol fractions did not differ markedly from the molar contribution of the respective molecular fractions suggesting that the various molecular species of rat testicular triacylglycerols are all synthesized and turn over at about the same rate. tion

with

22

Introduction The type and amount of any fatty acid found within a tissue lipid is determined by the factors which regulate the production of a given fatty acid as well as those controls which regulabe the incorporation of a specific fatty acid into a given lipid. Linoleate and arachidonate are found in large amounts in rat liver lipids. It is now apparent from numerous studies that linoleate is incorporated into liver choline phosphoglycerides primarily by a pathway involving total synthesis while arachidonate is incorporated into liver choline phosphoglycerides primarily by a deacylation-acylation reaction sequence [l-6] . Compositional studies have shown that rat testicular phospholipids contain significant amounts of linoleate and arachidonate as well as large amounts of other polyenoic acids containing 22 carbon atoms [7-lo]. Testicular triacylglycerols also differ from hepatic triacylglycerols in that they contain large amounts of polyenoic acids [S,9]. The study reported here was undertaken to study the in vivo incorporation of glycerol into the various molecular species of testicular triacylglycerols and choline phosphoglycerides and to ascertain whether glycerol was preferentially incorporated into any given molecular species and also to see whether different molecular species have different turnover rates. Methods Injection of [l(3)-’ ‘CT/ glycerol and lipid isolation Sprague-Dawley rats weighing between 200 and 250 g were lightly anesthesized with ether and 2.5 /..K!iof [l(3)-’ 4C]glycero1 (spec. act. 15.4 Ci/mole) in 25 1-11of isotonic saline was injected into each testes. The rats were sacrificed in groups of three at periods of 1, 4, 8, 24, 48 and 96 h after injection of the radioactive glycerol. The testes from three rats at a given time period were combined and extracted with 100 ml of chloroform-methanol 2 : 1 (v/v) and then with methanol. The combined extracts were evaporated to dryness and the residue was dissolved in 100 ml of chloroform-methanol 2 : 1 (v/v) and 20 ml of water was added and the phases were allowed to separate at 4°C. The next morning the bottom organic phase was removed and washed three times with the upper phase as described by Folch et al. [ll] to remove residual amounts of radioactive glycerol. The solvent was removed from the lipids under reduced pressure using a rotary evaporator. The lipids were redissolved in chloreform. Purification of choline phosphoglycerides and triacylglyceroLs The total lipids in chloroform were applied to a 20-g column of Unisil (Clarkson Chemical Company, Williamsport, Penn.) which was packed in chloroform in a 2 X 38 cm water-jacketed chromatographic column. The column was eluted successively with 200 ml of chloroform, 400 ml of redistilled acetone, 500 ml of chloroform-methanol 4 : 1 (v/v) and 400 ml of methanol. Thin-layer chromatography showed that triacylglycerols were eluted with chloroform while the choline phosphoglycerides were recovered free of ethanolamine phosphoglycerides in the methanol eluate.

23

The neutral lipids were fractionated by preparative thin-layer chromatography on silica gel G plates with petroleum ether-ether-acetic acid (80 : 20 : 2, v/v). The lipids were ~su~ized by spraying with a 0.2% ethanolic solution of 2 ‘,7 ‘dichlorofluorescein. The triacylglycerol band was scraped from the plates and the silica powder was transferred to a sma31column containing a bed of Unisil and the triacylglycerols were recovered by eluting with ether-methanol (9 : 1, v/v). Choline phosphoglycerides were purified by preparative thin-layer chromatography using silica gel H plates prepared with 0.01 M Na, CO3 and developed with chloroform-methanol-acetic acid-water (100 : 50 : 14 : 6, v/v) [12] . The plates were sprayed with 2 ‘,7 ‘dichlorofluorescein and the choline phosphoglycerides were extracted with chloroform-methanol-acetic acidwater (50 : 39 : 1 : 10, v/v) [3]. Specific uctiuity measurements The ester content of suitable aliquots of the triacylglycerols was determined by the hydroxamate method of Stem and Shapiro [13] . Trimyristin was used as a standard. Other aliquots were transferred to scintillation vials and the radioactivity was measured with a Model 3380 Packard liquid scintillation counter using the water-dioxane cocktail described by Snyder [14]. Counts per minute were converted to disintegrations per minute with a quench curve. The specific activity of the triacylglycerols was then calculated. The phosphorous content of suitable aliquots of the choline phosphoglycerides was determined by the method of Rouser et al. [15]. Other appropriate aliquots were used for radioactive measurements and the specific activity of the choline phosphoglycerides was then calculated. Fractionation into molecular species Choline phosphoglycerides were converted to diacylglycerols by treatment with phospholipase C from Ciostridium weichi [16]. The phospholipase C was obtained from Sigma Chemical Company, St. Louis, Missouri. The progress of the reaction was monitored by thin-layer chromato~aphy using silica G plates and developing with chloroform-methanol-water (65 : 25 : 4, v/v). When the reaction was complete, as judged by the absence of choline phosphoglycerides, the diacylglycerols were recovered by extracting with ether. The diacylglycerols were fractionated on thin layers of silica gel G’ impregnated with 12.5% AgNOJ and developed with the solvent system chloroform~thanol f95 : 6, v/v) [17]. Triacylglycerols were fractionated in a similar way except that the solvent system was chloroform-isopropanol (98.5 : 1.5, v/v) [18]. The plates were visualized by spraying with 2’,7 ‘dichlorofluorescein. The various bands were scraped from the plates and the di- and triacylglycerol fractions were recovered by extracting with a 90% methanol solution which contained 1% NaCl [l ] . Two subsequent extractions were carried out using ethe~meth~ol(9 : 1, v/v). To measure radioactivity the combined extracts were transferred to a scintillation vial and the solvent was removed and radioactivity was measured as outlined above. Gas-liquid chrom~to~aph~ Lipids were transferred to esterification tubes and converted to methyl

24

esters by refluxing for 2 h with 5% anhydrous HCl in methanol. The methyl esters were recovered by extracting with petroleum ether. Gas-liquid chromatography was carried out on an F and M Model 810 gas chromatograph equipped with a flame detector. The stainless-steel columns, 10 feet long by 0.25 inch diameter, were packed with 15% ethyleneglycol succinate on 80-100 mesh Gas-Chrom P. The over temperature was 180°C and the flow rate of helium was 60 ml/min. The various methyl esters were identified by comparing retention times with authentic methyl esters purchased from Nu-Chek Prep, Elysian, Minnesota. The various components were measured by triangulation and the area percent was converted to mole percent by using conversion factors which were determined by showing that the flame detector was linear on a weight basis. When the fatty acid composition of the various diand triacylglycerol fractions was analyzed, a known amount of methyl heneicosanoate was added to each sample immediately before gas chromatographic analysis. The area due to this internal standard was then compared with the summed area of the other methyl esters. From this type of comparison we were able to calculate the molar contribution of each di- and triacylglycerol fraction. Results and Discussion Choline phosphoglycerides The fatty acid composition of the choline phosphoglycerides and the various diacylglycerol fractions along with the molar contribution of each diacylglycerol fraction are depicted in Table I. The fatty acid composition of the choline phosphoglyceride was determined independently using the choline

TABLE

I

FATTY

ACID

COMPOSITION

CONTRIBUTION DERIVED All

fatty

AND

FROM acid

THE

OF

FATTY

THE ACID

CHOLINE

compositions

TOTAL

CHOLINE

PHOSPHOGLYCERIDES

COMPOSITION

OF

THE

AND

VARIOUS

THE

MOLECULAR

MOLAR SPECIES

PHOSPHOGLYCERIDE

are expressed

as mole

percent. _

DiacylgIycerol

Band

fraction

designation

Fatty

Diacyl-

acid

glycerol 14

: 0 16

: 0 16

:

1 18

:1

: 0 18

18

:

2 20

:

3 20

:

4 22

:

5 molar contribution

_ 1

-

2

Monoene

2

51

2

3

Diene

1

33

4

4

Triene

55

5

Tetraene

49

5

3

6

Pentaene

1

40

2

4

2

1

9

41

1

46

5

17

5

2

13

12

Trace

46 + 1 Trace

4+1

17+1

6fl

1+113~112fl

7

5

40

14

34

7

40

11 45

3 44

23

-

Calculated recombined composition Original*

1

choline

phosphoglycerides

* Average

of six determination

at the six different

time

periods

k the S.D.

30

25

phosphoglycerides isolated at the six different time periods following the injection of glycerol. The results in Table I show that there is excellent agreement between these six determinations. The fatty acid composition of total choline phosphoglycerides agrees well with that reported by other investigators [7-101 for rat testes phosphoglycerides except that we found only trace amounts of docosa-4,7,10,13,16,19-hexaenoic acid. Carpenter [9] has-reported that this acid is found in relatively large amounts in rat testes lipids. The fatty acid composition of the various diacylglycerol fractions was measured by combining equal amounts of the diacylglycerols from the six different time periods and doing argentation thin-layer chromatography on this combined sample. The molar contribution of each diacylglycerol fraction was then utilized to calculate the fatty acid composition of the original choline phosphoglycerides. As shown in Table I there is excellent agreement between this calculated value and the average of the six independent measurements. Five different diacylglycerol fractions were obtained. Fraction 1 was the solvent front and Fraction 7 was the origin. Subsequently these two fractions were shown to contain small amounts of radioactivity but insufficient material for fatty acid analysis. As shown in Table I each of the five fractions was characterized by a markedly different fatty acid composition. The results in Table II compare the distribution of radioactivity in the various fractions with the molar contribution of each fraction. If all molecular species were synthesized to the same extent by the same metabolic pathways with no selective compartmentalized pools and if all molecular species turned over by the way of the same catabolic pathways in the same cellular compartment at the same rates then the precent of the total radioactivity in any fraction at any time period should always be equal to the molar contribution of that fraction. The results in Table II show that the incorporation of glycerol deviated from this pattern in two different ways. In Fraction 2 and even more so in Fraction 3 the percent of radioactivity was high at early time periods but declined at later time period. Conversely in Fractions 4 and 5 the percent of radioactivity was low at early time intervals but increased at later time periods. These findings suggest that different biosynthetic pathways may be utilized to TABLE

II

PERCENT DISTRIBUTION OF RADIOACTIVITY IN THE VARIOUS DIACYLGLYCEROL TIONS DERIVED FROM CHOLINE PHOSPH~GLYCERIDES ALONG WITH THE MOLAR BUTION OF THE VARIOUS DIACYLGLYCEROL FRACTIONS

Diacy1-

glycerol fraction

Band designation

Percent distribution of radioactivity Hours after injection:

1 2 3 4 5 6 7

Monoene Diene tiene Tetraene Pentaene -

1 1 27 36 3 12 17 4

8

24

48

96

1

1

24 26 4 15 25 5

23 24 4 17 26 5

1 21 18 4 24 26 5

2 19 11 4 25 34 5

I 18 8 I 22 28 8

4

FRACCONTRI-

Molar contribution in %

34 11 3 23 30

26

TABLE

III

SPECIFIC

ACTIVITIES

OF

Time*

Spec.

CHOLINE

act.

PHOSPHOGLYCERIDES

AND

TRIACYLGLYCEROLS

(dpm//.onole)

Choline

Triacul-

phospho-

gIycerols

glycerides

1

2547

3317

4

2539

2832

8

2609

2258

24

2040

1172

48

1251

872

96

651

419

* Time

in h after

the injection

of

[ l(3)-14C]

giycerol.

different degrees for the production of different molecular species and also that different molecular species have different turnover rates. This hypothesis is supported by comparing the specific activities of the choline phosphoglycerides isolated at the various time periods (Table III) with the various diacylglycerols derived from the choline phosphoglycerides isolated at the six different time periods (Fig. 1). The specific activities of the various diacylglycerol fractions were computed by multiplying the specific activity of the choline phosphoglyceride isolated at a given time period (Table III) by the percent of radioactivity in a given diacylglycerol fraction at the same time period. This value was then divided by the molar contribution of that fraction to give a specific activity expressed as dpm/pmole of a given molecular species. As shown in Fig. 1 the oleate fraction (Fraction 2) and the linoleate fraction (Fraction 3) initially have high specific activities but after only 1 h the specific activities declined rapidly. The maximum specific activity of the arachidonate fraction (Fraction 5) was not reached until sometime between 8 and 48 h after glycerol

FRACTION

2

-

FRFlCTlON3 FRACTION

5 -

FRACTION

6

-

I 48

24 HOURS

Fig.

1.

The

specific

phosphoglycerides

AFTER

activity following

72

96

lNJECTlON

of

the

injection

various with

molecular [1(3)-14C]glycerol.

species

of

diacylglycerols

derived

from

choline

27

injection while the maximum specific activity of the pentaene fraction (Fraction 6) was reached sometime between 4 and 24 h after the injection of [l(3)’ 4 C] glycerol. If a given molecular species is made exclusively or primarily by total synthesis then the specific activity of that component should be high at early time periods. This was found for the linoleate fraction and to a lesser degree for the oleate fraction. If biosynthesis and catabolism of these two components proceeded through the same pathways at the same rates and there were no selective intracellular metabolic pools then the specific activities of these two components should always be identical. Since the specific activity of the linoleate fraction was initially higher than that of the oleate fraction it suggests that de novo synthesis was more important in the biosynthesis of the linoleate fraction. If the specific activities of the oleate- and linoleate-containing diacylglycerols are plotted in semilog form it can be calculated that the apparent half-life for the oleate- and linoleate-containing diacylglycerols were respectively 35 and 18 h. These differences thus not only suggest that different pathways are involved to different extents in the biosynthesis of these two components but also that they have different turnover rates. Since the arachidonate and pentaene fractions do not reach their maximum specific activities until later time periods (Fig. 1) it suggests that a deacylationacylation pathway plays a more important role in producing this molecular species. When the oleate or linoleate fraction turns over these molecular species may be completely degraded. The radioactive glycerol would then enter the metabolic pool. Alternatively these two molecular species may be deacylated to yield 1-acyl-sn-glycero-3-phosphorycholine. If this compound is reacylated with either arachidonate or docosa-4,7,10,13,16-pentaenoate the radioactive glycerol would be transferred to either the tetraene or pentaene fraction. This type of metabolism is consistent with the finding that the tetraene and pentaene fractions do not reach their maximum specific activities until later time periods. It is also consistent with the finding that the linoleatecontaining diacylglycerol has a high initial specific activity and turns over rapidly thus making available a potential supply of l-acyl-sn-glycero-3-phosphorylcholine which can be utilized for the synthesis of the tetraene and pentaene fractions. When the specific activities of the tetraene and pentaene fractions as shown in Fig. 1 are plotted in semilog form it can be calculated that the respective apparent half-lifes of these two components are 41 and 46 h. The similarity in the specific activities of these two components during all the time periods coupled with the apparent similar turnover rates suggests that these two fractions are both synthesized and catabolized by the same metabolic pathway at the same rates. Studies by numerous investigators generally support the concept that arachidonate is incorporated into liver phospholipids primarily by an acylation-deacylation reaction sequence while linoleate is incorporated primarily via total synthesis [l-6]. Failure to produce major amounts of arachidonatecontaining choline phosphoglycerides by de novo synthesis can probably be attributed to the slow rate of formation of the required arachidonate-containing diacylglycerol. Compositional studies by Possmayer et al. [19] have shown that the unsaturated fatty acids are located primarily at the 2-position in phos-

28

phatidic acid. This important finding supports enzymatic studies showing that the acylation of a-glycerophosphate exhibits specificity relative to both saturated and unsaturated fatty acids [ZO] . Short-time in vivo experiments have shown that the labeling pattern of molecular species of phosphatidic acid was almost identical with that found for diacylglycerols thus showing that phosphatidate phosphatase (EC 3.1.3.4) was not substrate specific [3]. Short-time in vivo studies have shown that once an arehidonate-containing diacylglycerol is produced the rate constant for conversion of this molecular species to a choline phos~hoglyceride is more rapid than for other molecular species of diacylglycerols [ 31. These combined findings suggest that once an arachidonate-containing diacylglycerol is produced it is readily incorporated into a choline phosphoglyceride. The studies which we described here suggest that the various molecular species or rat testicular choline phosphoglycerides are synthesized by the same pathways as are operative in liver. Short-time in vivo labeling studies as well as tissue slice experiments with liver have shown a similar labeling pattern as that obtained here in that glycerol is preferentially incorporated into the dienoic species followed by the monoenoic fraction and then the arachidollate-co~~taining species [l--3]. Our studies do not eliminate the possibility that the arachidonate- and pentaene-containing choline phosphoglycerides may have been produced by selective methylation of these molecular species in the ethanolamine phosphoglycerides. If glycerol was rapidly incorporated into arachidonateand docosa-4,7,10,13,16-pentaenoate-containing ethanolamine phosphoglycerides and if these molecular species were selectively methylated with S-adenosylmethionine it would he possible to obtain the same labeling pattern which we have observed. This seems unlikely since no evidence1 has been presented to indicate that this pathway is operative in rat test~es. In addition enzymatic studies have shown that rat testes readily incorporate unsaturated fatty acids into l-acyl-so-glycero-3-phosphorycholine 1211. Recent studies also suggest that arachidonate is preferentially incorporated into testicular lipids by an acyl exchange reaction and that arachidonate is more selectively retained by the ethanolamine plasmalogen than the diacyl analog (22,231. In addition our studies do not provide any information as to whether either intracellular mctabolic pools or the reutilization of radioactive glycerol play selective roles in the biosynthesis, catabolism or turnover of individual molecular species of choline phosphoglycerides.

The fatty acid composition of the triacylglycerols was measured independently at the six different time periods following the injection of [l(3)’ 4 C] glycerol. As shown in Table IV there is excellent agreement between these six separate determinations. The fatty acid composition of the various triacylglycerol fractions was determined by combining equal amounts of the triacylglyeerols isolated at each time period and doing argentation thin-layer chromatography on this combined sample. As shown in Table IV there is excellent agreement between the calculated recombined fatty acid composition with the measured corn position. Seven different triacylglycerol fractions were obtained (Table IV). Frac-

29 TABLE IV FATTY FATTY

ACID COMPOSITION OF TRIACYLGLYCEROLS AND THE MOLAR CONTRIBUTION AND ACID COMPOSITION OF THE VARIOUS TRIACYLGLYCEROL MOLECULAR SPECIES

All fatty acid compositions are expressed as mole percent. TriacylgIycerol fraction

Fatty acid 14 : 0 16 : 0 16

1 2 3 4 5 6 7 8 9 Calculated recombined composition Original* triacylgIycero1

:1

18 : 0 18

:1

18

5 4 2 3 3

87 56 30 2% 25

5 11 11 8

7 7 4 5 4

26 52 39 29

5 31

2 2

41 24

3 2

4 3

17 10

13 7

3

36

5

4

23 23fl

2.4+032&l

4+04*1

:

2 20

:

4 22

:

4 22

:

5

Triacylglycerol molar contribution

4 10 10 10 10

3

5 7

3 6

12 40

10

3

2

14

ll_?1

4-I.l

3f9

17fl

30 26

* Average of six determinations at the six different time periods t the S.D.

tion 1 was the solvent front and although this fraction was subsequently shown to contain small amounts of radioactivity there was insufficient material for fatty acid analysis. Fraction 7 corresponded to an area on the thin-layer plate which also contained low levels of radioactivity but no detectable lipid. It is apparent from the fatty acid composition of the remaining triacylglycerol fractions that the complex nature of the triacylglycerols makes it impossible to obtain fractions cont~ning acids which are relatively specific for only that molecular species. A given species may contain a well defined fatty acid composition but a given unsaturated fatty acid may be a major component in several fractions. In Table V the molar amounts of the different triacylglycerol fractions are TABLE V PERCENT DISTRIBUTION OF RADIOACTIVITY IN THE VARIOUS TRIACYLGLYCEROL FRACTIONS ALONG WITH THE MOLAR CONTRIBUTION OF THE VARIOUS TRIACYLGLYCEROL FRACTIONS Percent distribution of radioactivity

TriacYl-

MC&r contribution in C

glycerol fraction

Hours after injection:

1

4

8

24

48

96

0 9 13 7 Q 7 2 34 21

0

0

0 3 7 4 4 4 2 24 53

1 6 7 6 5 2 4 29 41

2 6 8 7 4 5 4 30 35

6 8 4 6 3 1 37 36

5 6 4 4 6 1 30 43

30

compared with the amount oi radioactivity found in each fraction at the six different time periods. It is apparent from these results that the amount of radioactivity in each fraction at any given time period does not correlate exactly with the molar amount of that molecular species. The incorporation pattern differs from that found in the diacylglycerol fractions in that there is neither a consistent increase or decrease in radioactivity during the duration of the experiment. These findings suggest, that in contrast to the various molecular species of the choline phosphoglycerides, that the various triacylglycerol molecular species are all synthesized and turn over at about the same rate. If the specific activities of the total triacylglycerols are compared with the total choline phosphoglycerides (Table III) it appears that the triacylglycerols are synthesized and turn over somewhat more rapidly than do the total choline phosphoglyceride. These findings are in general agreement with tissue slice experiments [24 ] and in vivo studies [25] which have shown that rat liver triacylglycerols are produced at the same rates with a random utilization of various diacylglycerol species.

This study of Elealth.

was supported

by grant AM-09758

from the National

Institute

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