Phospholipid metabolism in red and white insect muscle

Insect Biochem., 1976, Vol. 6, pp. 381 to 384. Pergaraon Press. Printed in Great Britain.

PHOSPHOLIPID METABOLISM IN RED AND WHITE INSECT MUSCLE OLGA NOV~KOV.~, FRANTIgEKNOV.~K, and VgCLAV KUBIgTA Department of General Physiology, Charles University, Prague, Czechoslovakia (Received 25 February 1976)

Abstract--The red flight musculature of Schistocerca gregaria contains twice as much phospholipids than the white femoral musculature. In individual phospholipids the differenceis greatest in phosphatidylethanolamine and phosphatidylglycerol, lowest in sphingomyeline and phosphatidylinositol. The plasmalogen content is very low. After an injection of 32p orthophosphate the increase of specific activity during six days follows a similar course in both muscle types in phosphatidylethanolamine, sphingomyeline and phosphatidylserine but is more rapid in red than in white muscle in phosphatidylcholine (1.3 x ) and in phosphatidylinositol (5 x ). The incorporation into diphosphatidylglycerol is extremely slow. Flight induces an increase in the specific activity in phosphatidylinositol.

INTRODUCTION MUSCLES of flying insects are known to be differentiated into two distinct types: red flight muscles containing a high proportion of mitochondria and correspondingly high activity of respiratory enzymes, very low lactic dehydrogenase activity and low phosphagen content, and white leg muscles with low content of mitochondria, higher lactic dehydrogenase activity and phosphagen (ZEaE and M c S ~ , 1957; KtraI~TA, 1959). The muscles differ markedly even in their rate of the resting oxygen consumption (Ktrmg'rh, 1956). Recently it has been shown that the difference in the rate of the resting metabolism is reflected in similar' difference in rate of incorporation of inorganic phosphate into the energy-rich compounds of the tissue, viz phosphoarginine and ATP ( S r g t n ~ c i ~ and KtmI~TA, 1974); this, however, affects only the initial incorporation course as in insect muscle there seems to be a tendency for the specific activity to level off and remain at a steady level for a longer time (HE~OP and RAY, 1961; Ktrm~xA and FOUSTgA, 1963) similarly as in the vertebrate muscle (GRAFF et al., 1965a, b). The purpose of this work was to investigate the rate of incorporation of inorganic phosphate into phospholipids of red and white insect muscle. On one side, two skeletal muscles taken from the same animal are well comparable; on the other side the clear-cut difference between them offers the possibility to look for specific features in the metabolism of different phospholipids. MATERIALS AND METHODS All experiments were done on locusts, Schistocerca oreoaria. They were reared at 30°C, fed fresh grass and bran 381

(during winter wheat seedlings) and taken for experimentation 3 to 6 weeks after adult ecdysis. 30 ~C 32p orthophosphate without carrier in physiological saline pH 7.4 was injected in a volume of 50 gl into the abdomen of the animals. Hereafter they were kept individually in transparent vessels with enough distilled water but without food. Preparations of pterothoracic and femoral musculature (KUBI~,TA,1966) were weighed, frozen with liquid nitrogen, pulverised and extracted twice by chloroform-methanol 2:1, the third extraction being made with chloroformmethanol 7:1 saturated with ammonia (RouSeR and FLEISCHER, 1967). After each extraction the tissue was separated by centrifugation. Joined extracts were shaken with 20~ volume of 0.9~ sodium chloride solution in water to remove non-lipid impurities. The lower phase was separated and evaporated to dryness under reduced pressure. The residue was dissolved in chloroform-methanol 2:1 and analysed by thin layer chromatography. Glass plates with 0.25 mm layer of Kieselgel G (Merck) were predominantly used. The solution systems were chloroformmethanol-water (65:25:4) and butanol-acetic acid-water (60:20:20) (SL~KOTESand ROUSER, 1965). Later, for complete separation of phosphatidylinositol and phosphatidylserine, Kieselgel H (Merck) with magnesium silicate was used in several experiments. Chloroform--methanolammonia (65:25:5) and chloroform-acetone-methanolacetic acid-water (4:3:1:1:0,5) were used for development of the chromatograms (Rous~ et al., 1970). The spots of individual phospholipids were detected by iodine vapour, ninhydrine solution, and molybdate reagent (H/ffn~ and LUCrd-L~lqS,1956).The spots were scraped out carefully and the material used either for the determination of phosphate after Rous~ et al. (1970), or for the deterruination of radioactivity on the Mark I liquid scintillation spectrophotometer (Nuclear Chicago). For the determination of plasmalogens the alkali-labile phospholipids were first hydrolysed after DAWSON(1960), and the rest subjected to analysis by thin-layer chromatography.

382

OLGA NOV~,KOV~.,FRANTI~EKNOV,~K,AND V,~CLAVKUBI~TA Table 1. The phospholipid content of red and white muscle of Schistocerca gregaria. Means from 14 determinations + S.E.M. (for PS and PI from 3 determinations)

RESULTS

Phospholipid content in red and white muscles Results of the analysis of red and white muscles of Schistocerca are given in Table 1. The total phospholipid content is twice as high in red than in white musculature; this no doubt reflects a higher content of membranous structures in red flight muscles. In the content of individual phosphotipids the differences are of the same direction but of different magnitude. The difference is greatest in diphosphatidylglycerol (DPG) and phosphatidylethanolamine (PE), lower in phosphatidylcholine (PC) and lowest in sphingomyeline (SM) and phosphatidylinositol (PI). The relation PE/PC is 1.02 in the red muscle and 0.66 in the white muscle, the respective values of the relation DPG/PC are 0.33 and 0.2 respectively. The content of plasmalogens in both tissue types is very low.

Content #mole P/g flesh weight Phospholipid

Red muscle White muscle

Phosphatidylcholine Phosphatidylethanolamine Diphosphatidylglycerol Phosphatidylserine + phosphatidylinositol Phosphatidylserine Phosphatidylinositol Sphingomyeline Phosphatidalcholine Phosphatidalethanolamine

8.87 + 0.35 9.15 +_ 0.30 2.95 + 0.22

Total lipid P

24.1 _+ 0.07

4.84+ 0.23 3.23 + 0.22 0.97_+ 0.11

0.98 + 0.19 0.74 + 0,08 0.69 + 0.14 0.39 + 0.10 0.56 + 0.05 0.51 + 0.09 0.79 + 0.14 0.51 +_ 0.07 0.06 not detected 0.30 0.18 12.3 + 0.4

The time course of the incorporation of 32p into phospholipids Specific activities of phospholipids in red and white muscle at three different intervals after a single dose of radioactive orthophosphate are given in Figs. 1-3. The curves for both types of tissue have many common features. The activity increases in all phospho, lipids during the whole period of observation. Of the three major phospholipids, phosphatidylcholine reaches highest values of specific activity, phosphatidylethanolamine incorporates considerably less rapidly and diphosphatidylglycerol by an order of magnitude more slowly. There are, however, characteristic differences between red and white muscle. Generally, the incorporation is higher in the red muscle. But sphingomyeline and phosphatidylethanolamine do not differ in their incorporation rate at all, in phosphatidylcholine there is a moderate difference and in the sum phosphatidylserine + phosphatidylinositol the incorporation is about three times as high in red muscle than in the white one. Additional

experiments in which these two phospholipids were separated (broken lines) show that specific activity of phosphatidylserine is low and not very different in both muscle types so that phosphatidylinositol is responsible for the difference seen in the sum; this phospholipid reveals greatest difference in the rate of incorporation between the two muscle types and its labelling in red muscle is highest of all phospholipids examined.

The influence of flight In a small series of three experiments the influence of flight of short duration on the specific activity of the flight muscle phospholipids was examined. If the locusts were allowed to fly for 15 min 2 hr after injection of 32p-orthophosphate, there was a distinct increase in the specific activity of phosphatidylinositol with respect to non-flying animals while the activity of both phosphatidylcholine and phosphatidylethano-

PC ~0-

(3o

E

E 5Q. o

4

24

144 hours

~4

,~4 hours

Fig. 1. The time course of the incorporation of 32p into phosphatidylcholine (PC) and diphosphatidylglycerol (DPG) of the red (left) and white (right) muscle of Schistocerca gregaria after a single injection of 50/~Ci 32P-orthophosphate. (Means from six experiments + S.E.M.)

Phospholipids in insect muscle

383

0..

4

i 24

24

144

144 hours

hours

Fig. 2. The time course of the incorporation of 32p into phosphatidylethanolamine (PE) and sphingomyeline (SM) of the red (left) and white (right) muscle of Schistocerca gregaria after an injection of 50/zCi 32p-orthophosphate. (Means from six experiments + S.E.M. lamine did not change significantly. Thus the relation o f the specific activities PI/PE and PI/PC increased by 28, 54, and 42% and 44, 38 and 44, and 42% respectively. So a small 'phosphatidylinositol effect' is observable in insect muscle subjected to natural stimulation during flight. DISCUSSION When our values for the muscle of Schistocerca oregaria are compared with the data for vertebrate muscle some differences are apparent. In most vertebrate muscles phosphatidyleholine is present in higher amount than phosphatidylethanolamine (SIMON and Rouser, 1969; HOF and SLMON,1970). In phospholipid composition red muscles resemble the vertebrate heart muscle most closely; the similarity is no doubt due to a high content of mitochondria in both muscle types. Another important difference is the very low plasmalogen content in insect muscle, while in vertebrate muscle these compounds constitute a consider-

able proportion of choline and ethanolamine phospholipids (DAwm,ogr, 1964; DAWSON, 1962). Our results are in agreement with the findings of CRor,m and BRIDGES (1963) who found no plasmalogens in whole Musca domestica. These results suggest that plasmalogens are not essential for muscle function. Like in Vertebrates ( G ~ - r et al., 1965a, b), the specific activity of all phospholipids increases after a single injection of 32p-orthophosphate for several days (at least six days in Schistocerca gregaria). The most conspicuous difference between insect and vertebrate muscle is in the relation of the incorporation rate of diphosphatidylglycerol to other phospholipids. In insect muscle diphosphatidylglycerol incorporates extremely slowly. After four days the specific activity of this phospholipid reaches a level only about 20 times lower than phosphatidylcholine which according to the character of the curve approaches equilibrium with the precursors. The course of diphosphatidylglycerol labelling may be taken as linear within the four days and allows an extrapolation showing

PI + )5-

Ia

E

E o

PI+PS

5-

PI +

i

' hours

~

144 hours

Fig. 3. The time course of the incorporation of 32p into the sum phosphatidylserine + phosphatidylinositol (PS + P I ) - 6 experiments) and into phosphatidylserine (PS) and phosphatidylinositol (PI - three experiments) of the red (left) and white (right) muscle of Schistocerca gregaria after an injection of 50#Ci of 32p-orthophosphate. LH. 6 / 4 - ~ :

384

OLGA NOV~,KOV/~,FRANTI~EK NOVA,K, AND V,~CLAV KUBI~TA

that all phospholipid would turn over not earlier than after 80 days which is a period comparable with the life span of the animal. In rat muscle the relative rate of diphosphatidylglycerol turnover is much higher (GRAFF et al., 1965a, b). The cause may be that rats are growing continuously while in insects there is a sharp termination of growth after reaching maturity. Incorporation of 32p into energy-rich phosphates is less in white than in red muscle. The difference is apparent mainly on the first day after injection before steady level of radioactivity is reached. This seems not to have a great influence on the rate of incorporation into phospholipids since some of them, i.e. phosphatidylethanolamine and sphingomyeline, are labelled with the same speed in both types of muscle. Moreover their incorporation curves are practically identical during their whole course; in phosphatidylcholine, the specific activities are in the same relation (10:7) both after one and six days. Thus the differences seen in other phospholipids may be ascribed rather to the variations in their turnover rate than to the difference of the specific activity of the common precursors. It appears interesting that greatest differences in the incorporation rate are seen in phosphatidylinositol the metabolism of which has in many tissues been shown to be connected with their functional activity (HogaN, 1969; LAPETINA and MICHEL, 1973). Only in this phospholipid, the relation of the turnover rates in white and red muscle is comparable to the relation of the metabolic activity of the tissues. The increase in phosphatidylinositol labelling after flight confirms that even in insect flight muscle its turnover is more intimately associated with specific functions than that of other phospholipids, In conclusion, this comparison of the rate of labelling of individual phospholipids in two types of muscle tissue of the same animal shows that the muscles differ much more in the labelling pattern than in the overall turnover rate. This strongly suggest that metabolism of different phospholipids as measured by the relative rate of incorporation has different physiological significance.

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