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Incorporation of glucose-U-14C into lipids of the blowfly during flight
7. Insect Ph,vsiol., 1969, Vol. 15, pp. 1567 to 1571. Pergnmon Press. Printed in Great Britairt
INCORPORATION OF GLUCOSE-U-14C INTO LIPIDS OF THE BLOWFLY DURING FLIGHT CAROLYN F. MATHUR and WILLIAM
J. YURKIEWICZ
Department of Biology, Millersville State College, Millersville, Pennsylvania 17551 (Receiwed 6 March 1969) Abstract-Incorporation of the label from glucose-U-i4C into lipids of the blovvfly, Phaenicia sericata, was studied during flight and at rest. After 3 hr of continuous flight following injection of the glucose, only 0.5 per cent of the radioactivity was found in lipids, whereas 1.0 per cent was recovered from Ries at rest for 3 hr. Resting females incorporated more glucose into lipid than resting malees, whereas during flight the rate of incorporation was the same for both sexes. The label in triglyceride increased with time in both resting and flying flies. Abcfut twice as much label was found in the glycerol portion of glycerides and phospholipids as compared to the fatty acid portion. Some label was found in hydrocarbons. .Flight activity, although accompanied by an increase in metabolism, appears to decrease the rate of conversion of glucose into lipid. Since carbohydrate is the main source of flight energy in Diptera, a mechanism to conserve the level of carbohydrate during flight would seem to be desirable. INTRODUCTION
INSECTS1:hat utilize either lipid or carbohydrate as fuel for flight have been found capable of converting glucose to lipid, although usually at very low levels. Moths, Bombyx mori, at various stages of development, could incorporate up to 8.4 per cent of labelled glucose into lipid (HORIE et al., 1968). In vitro studies of locust fat body showed glucose conversion to lipid (CLEMENTS,1959), whereas mosquitoes were found to synthesize triglyceride when fed glucose exclusively (VAN HANDEL and LUM, 1961). Earlier, YURKIEWICZ(1965) found 0.1 per cent of the radioactivity from labelled sucrose in the lipids of Phaeniciu sericata after flight to exhaustion. In a later study (YURKIEWICZand MATHUR, 1969) the distribution of labelled free fatty acid within the lipid fractions of P. sericata was compared during flight and at rest. Slight conversion of free fatty acid to glucose was also reported at that time. This paper reports on conversion of glucose-U -14C into lipids of the blowfly during flight and at rest. Percentage distribution of label within the lipid fractions is presented as well as comparisons between the radioactivity recovered in the free fatty acids, glycerol, hydrocarbons, and sterols. MATERIALS
AND METHODS
The %es, Phaenicia sericata (Meig.), were reared in the laboratory at 25 k 2°C. Larvae were fed on Alpo beef chunks (Allen Products) and adults were maintained 90
1567
1568
CAROLYNF. MATHURANDWILLIAMJ. YURKIEWICZ
on sucrose cubes and water. Both male and female flies from 7 to 14 days after ecdysis were used in all experiments. The insects were flown while tethered to light, wire turnabouts (YURKIEWICZ and SMYTH, 1966a) at 25”C, and those at rest were tethered and held at the same temperature. Injections of glucose-U-14C (Nuclear Chicago, Chicago) were made laterally in the abdomen between the second and third segments with a sharpened microlitre Hamilton syringe. Each injection of 1~1 in volume consisted of enough labelled glucose in water to produce 500,000 disintegrations/min. After the prescribed period of flight or rest, lipids were extracted, fractionated, and counted as described previously (YURKIEWICZand MATHUR, 1969). Fatty acids were recovered by saponification of lipid in 10% ethanolic KOH, followed by acidification with 50%HCl and removal of the free fatty acids with hexane. Sterols were precipitated from the unsaponifiable fraction with 1% digitonin in ethanol and repeatedly washed with ethanol and diethyl ether. Hydrocarbons were isolated by thin-layer chromatography after precipitation of the sterols. Glycerol was recovered from the aqueous phase of the saponification medium by eluting from an Amberlite MB-3 ion-exchange column with water.
RESULTS The incorporation of label from glucose-U- 14C into lipids of the blowfly during flight was not affected by sex differences, but resting females incorporated about twice as much label into lipids as males. The data from both sexes were pooled, however, because the total amount recovered averaged only O-5 per cent of the injected label in flown flies and about 1-O per cent in rested flies, and because no significant differences between the sexes were found in the individual lipid fractions. As shown in Tables 1 and 2, most of the label was found in the phospholipid and hydrocarbon plus sterol ester fractions. During flight (Table 1) there appears to be very little change in the percentage distribution of radioactivity within the fractions except for triglyceride which increases with time. This increase in triglyceride was also found in rested flies and was accompanied by a decrease in phospholipid and an increase in the hydrocarbon plus sterol ester fraction. A number of flies were injected and killed immediately to check for contamination of the lipid samples with labelled glucose. Essentially no radioactivity was recovered in lipids. Flies flown 1 to 2 hr were analysed to determine the relative distribution of the label within the glycerol and fatty acid portions of glycerides and phospholipids. Almost twice as much label was recovered in glycerol as in fatty acid. The sterols precipitated by digitonin showed considerably less radioactivity than the sterol fraction scraped directly from the thin-layer plate. This suggests that much of the radioactivity in the sterol fraction from the plate is contained in components other than digitonin-precipitated sterols. The hydrocarbon fraction was isolated and was shown to contain considerable radioactivity although individual components within the fraction were not identified.
26031:617* 3563 k 1038
24782588
No.
7 6
6
1 2
3
Sterol 5.7 4.3 3.7
Monodiglyceride 6.4 5.0 6.0
Phospholipid 29.6 29.0 32.8
6
:
No.
4125k851” 4922k1217 7241+ 1878
Recovered label (countsjmin)
1.6 1.2 22
Free fatty acid 7.4 8.0 14.2
Triglyceride
Monodigfyceride 4.4 3.7 4.1
Phospholipid 72.5 34.9 32.2
2.6 3-S
2.9
Sterol
1.3 0.9 0*7
Free fatty acid
Recovered label in lipid fractions (%)
4‘5 8.9 12.6
Triglyceride
13.9 48.9 46.3
Hydrocarbon + sterol ester
49.2 52.0 40.7
Hydrocarbon + stem1 ester
FLIGHT
OF THE LABEL FROMGLUCOSE-U-% INTO LIPIDSOF THE BLOWFLYAT REST
Each injection contained 5 x lo5 disinte~rat~ons/min. * Standard error.
1 3 12
Rest time (hr)
TABLE %--INCORPORATION
Each injection contained 5 x lo6 disintegrations/min. * Standard error.
Recovered label (counts/min)
Flight time (hr)
UNTO LIPIDS OF THE BLOWFLYDURING
Recovered ia’bei in iipid fractions (7;)
TABLE I-INCORPORATION OF THELABELFROMCLUCOSE-LV4C
P ij g
2 2
P
B &
$ 0 c +Q
$ g
?
e
2
q % %
z 8
1570
CAROLYNF. MATHURANDWILLIAMJ. YURKIEWICZ DISCUSSION
Blowflies, which use carbohydrate for flight fuel, convert very little labelled glucose to lipid while at rest or during flight. However, there is a gradual increase of label in triglyceride with time. VAN HANDEL and LUM (1961), in comparing houseflies and mosquitoes, reported that the triglyceride level increases dramatically in female mosquitoes maintained exclusively on glucose, whereas in male mosquitoes and male and female houseflies it decreases. This does not show that there is no conversion of glucose to triglyceride in houseflies but does suggest that any contributions from glucose are small. Some moths, which utilize lipid for flight, show similarly low levels of conversion from glucose to lipid. SRIDHARAand BHAT (1965) found 0.6 per cent of the injected labelled glucose in the total lipids of B. mori larvae after 24 hr. Higher percentages of 2.4 to 8.4 per cent were reported by HORIE et al. (1968) for the same insect. They found most of the radioactivity in glycerol and fatty acids, with glycerol containing a higher percentage of label than fatty acid, no matter how the glucose was labelled. This agrees with our findings using glucose-U-14C. CHINO and GILBERT (1964) recovered 2.5 per cent of the labelled glucose in the glycerides of developing Hyatophora cecropia pupae. The free fatty acids contained low but significant labelling while hydrocarbons contained none. We found that blowflies incorporate some label into hydrocarbons, and this has been shown in some other insects using labelled acetate as a precursor (LAMBREMONT and BENNETT, 1966; SRIDHARAand BHAT, 1964). In a previous paper (YURKIEWICZ and MATHUR, 1969), we reported a gradual increase of label in triglyceride from palmitate-l-14C over time in both resting and flying flies. The data in this paper show a similar increase in triglyceride, but whether this is from label in fatty acids or glycerol cannot be said with certainty since radioassays were made of the total glycerol and fatty acid components from the combined lipid fractions and not from triglyceride alone. D’COSTA and BIRT (1966) f ound that the neutral lipid fraction in adult male flies, Lucilia cuprina, decreased slowly during the first 5 days of adult life whereas it increased in the female during the same time period. This may in part account for the higher level of incorporation from glucose found in resting females when compared with resting males. It appears that resting blowflies incorporate more radioactive glucose into lipid than flies in flight, in spite of the greatly increased metabolic rate and elevated internal temperature of the fly during flight (YURKIEWICZ and SMYTH, 196613). Earlier we found an increase in the rate of fatty acid esterification into triglycerides during flight using palmitate-l -14C (YURKIEWICZ and MATHUR, 1969). Therefore, flight activity, although accompanied by an increase in general metabolism and in interconversions within the lipid fractions, appears to decrease the rate of conversion of glucose into lipid. Any mechanism which would conserve the level of carbohydrate in flies during flight would be desirable since carbohydrate is believed to be the main source of flight energy in Diptera and, considering the higher metabolic rate, is being rapidly oxidized. A lower rate of conversion of glucose into lipid might be an expected consequence of this high rate of carbohydrate utilization during flight.
INCORPORATION OF GLUCOSE INTO LIPIDSIN BLOWFLYFLIGHT
1571
REFERENCES CHINOII. and GILBERTL. I. (1964) Studies on the interconversion of carbohydrate and fatty acid in Hyalophora cecropia. J. Insect Physiol. l&287-295. CLEMEN::SA. N. (1959) Studies on metabolism of locust fat body. r. exp. BioZ. 36, 665-675. D’COSTA M. A. and BIRT L. M. (1966) Changes in the lipid content during the metamorphosis of the blowfly, Lucilia. J. Insect Physiol. 12, 1377-1394. HORIE Y., NAKASONES., and ITO T. (1968) The conversion of i4C-carbohydrates into CO, and lipid by the silkworm, Bombyx mori. J. Insect Physiol. 14, 971-981. LAMBRR~~ONT E. N. and BENNETTA. F. (1966) Lipid biosynthesis in the boll weevil: Formation (3f the acetate precursor for lipid synthesis from glucose and related carbohydrates. Can.J. Biochem. 44,1597-1606. SRIDHARAS. and BHAT J. V. (1964) Incorporation of 1-14C-acetate into the lipids of the silkworm, Bombyx mori L. BiochemJ. 91,120-123. SRIDHARAS. and BHATJ. V. (1965) Interconversion of carbohydrate and fat in the silkworm Bombyx mori L. Life Sci. 4,979-982. VAN HANDELE. and LUM P. T. M. (1961) Sex as regulator of triglyceride metabolism in the mosquito. Science, N. Y. 134,1979-1980. YURKIE~IICZW. J. (1965) Flight exhaustion studies on the blowfly Phaenicia sericata using r4C labeled sucrose. Ann. ent. Sot. Am. 58, 766. YURKIE~ICZW. J. and MATHURC. F. (1969) Incorporation of palmitate-l-i% into lipids of the blowfly during flight. J. Insect Physiol. 15,439-444. YURKIEP~ICZ W. J. and SMYTH T., JR. (1966a) Effect of temperature on flight speed of the sheep blowfly. r. Insect Physiol. 12, 189-194. YUKIEWICZW. J. and SMYTHT., JR. (1966b) Effects of temperature on oxygen consumption and fuel utilization by the sheep blowfly. J. Insect PhysioE. 12, 403408.
INCORPORATION OF GLUCOSE-U-14C INTO LIPIDS OF THE BLOWFLY DURING FLIGHT CAROLYN F. MATHUR and WILLIAM
J. YURKIEWICZ
Department of Biology, Millersville State College, Millersville, Pennsylvania 17551 (Receiwed 6 March 1969) Abstract-Incorporation of the label from glucose-U-i4C into lipids of the blovvfly, Phaenicia sericata, was studied during flight and at rest. After 3 hr of continuous flight following injection of the glucose, only 0.5 per cent of the radioactivity was found in lipids, whereas 1.0 per cent was recovered from Ries at rest for 3 hr. Resting females incorporated more glucose into lipid than resting malees, whereas during flight the rate of incorporation was the same for both sexes. The label in triglyceride increased with time in both resting and flying flies. Abcfut twice as much label was found in the glycerol portion of glycerides and phospholipids as compared to the fatty acid portion. Some label was found in hydrocarbons. .Flight activity, although accompanied by an increase in metabolism, appears to decrease the rate of conversion of glucose into lipid. Since carbohydrate is the main source of flight energy in Diptera, a mechanism to conserve the level of carbohydrate during flight would seem to be desirable. INTRODUCTION
INSECTS1:hat utilize either lipid or carbohydrate as fuel for flight have been found capable of converting glucose to lipid, although usually at very low levels. Moths, Bombyx mori, at various stages of development, could incorporate up to 8.4 per cent of labelled glucose into lipid (HORIE et al., 1968). In vitro studies of locust fat body showed glucose conversion to lipid (CLEMENTS,1959), whereas mosquitoes were found to synthesize triglyceride when fed glucose exclusively (VAN HANDEL and LUM, 1961). Earlier, YURKIEWICZ(1965) found 0.1 per cent of the radioactivity from labelled sucrose in the lipids of Phaeniciu sericata after flight to exhaustion. In a later study (YURKIEWICZand MATHUR, 1969) the distribution of labelled free fatty acid within the lipid fractions of P. sericata was compared during flight and at rest. Slight conversion of free fatty acid to glucose was also reported at that time. This paper reports on conversion of glucose-U -14C into lipids of the blowfly during flight and at rest. Percentage distribution of label within the lipid fractions is presented as well as comparisons between the radioactivity recovered in the free fatty acids, glycerol, hydrocarbons, and sterols. MATERIALS
AND METHODS
The %es, Phaenicia sericata (Meig.), were reared in the laboratory at 25 k 2°C. Larvae were fed on Alpo beef chunks (Allen Products) and adults were maintained 90
1567
1568
CAROLYNF. MATHURANDWILLIAMJ. YURKIEWICZ
on sucrose cubes and water. Both male and female flies from 7 to 14 days after ecdysis were used in all experiments. The insects were flown while tethered to light, wire turnabouts (YURKIEWICZ and SMYTH, 1966a) at 25”C, and those at rest were tethered and held at the same temperature. Injections of glucose-U-14C (Nuclear Chicago, Chicago) were made laterally in the abdomen between the second and third segments with a sharpened microlitre Hamilton syringe. Each injection of 1~1 in volume consisted of enough labelled glucose in water to produce 500,000 disintegrations/min. After the prescribed period of flight or rest, lipids were extracted, fractionated, and counted as described previously (YURKIEWICZand MATHUR, 1969). Fatty acids were recovered by saponification of lipid in 10% ethanolic KOH, followed by acidification with 50%HCl and removal of the free fatty acids with hexane. Sterols were precipitated from the unsaponifiable fraction with 1% digitonin in ethanol and repeatedly washed with ethanol and diethyl ether. Hydrocarbons were isolated by thin-layer chromatography after precipitation of the sterols. Glycerol was recovered from the aqueous phase of the saponification medium by eluting from an Amberlite MB-3 ion-exchange column with water.
RESULTS The incorporation of label from glucose-U- 14C into lipids of the blowfly during flight was not affected by sex differences, but resting females incorporated about twice as much label into lipids as males. The data from both sexes were pooled, however, because the total amount recovered averaged only O-5 per cent of the injected label in flown flies and about 1-O per cent in rested flies, and because no significant differences between the sexes were found in the individual lipid fractions. As shown in Tables 1 and 2, most of the label was found in the phospholipid and hydrocarbon plus sterol ester fractions. During flight (Table 1) there appears to be very little change in the percentage distribution of radioactivity within the fractions except for triglyceride which increases with time. This increase in triglyceride was also found in rested flies and was accompanied by a decrease in phospholipid and an increase in the hydrocarbon plus sterol ester fraction. A number of flies were injected and killed immediately to check for contamination of the lipid samples with labelled glucose. Essentially no radioactivity was recovered in lipids. Flies flown 1 to 2 hr were analysed to determine the relative distribution of the label within the glycerol and fatty acid portions of glycerides and phospholipids. Almost twice as much label was recovered in glycerol as in fatty acid. The sterols precipitated by digitonin showed considerably less radioactivity than the sterol fraction scraped directly from the thin-layer plate. This suggests that much of the radioactivity in the sterol fraction from the plate is contained in components other than digitonin-precipitated sterols. The hydrocarbon fraction was isolated and was shown to contain considerable radioactivity although individual components within the fraction were not identified.
26031:617* 3563 k 1038
24782588
No.
7 6
6
1 2
3
Sterol 5.7 4.3 3.7
Monodiglyceride 6.4 5.0 6.0
Phospholipid 29.6 29.0 32.8
6
:
No.
4125k851” 4922k1217 7241+ 1878
Recovered label (countsjmin)
1.6 1.2 22
Free fatty acid 7.4 8.0 14.2
Triglyceride
Monodigfyceride 4.4 3.7 4.1
Phospholipid 72.5 34.9 32.2
2.6 3-S
2.9
Sterol
1.3 0.9 0*7
Free fatty acid
Recovered label in lipid fractions (%)
4‘5 8.9 12.6
Triglyceride
13.9 48.9 46.3
Hydrocarbon + sterol ester
49.2 52.0 40.7
Hydrocarbon + stem1 ester
FLIGHT
OF THE LABEL FROMGLUCOSE-U-% INTO LIPIDSOF THE BLOWFLYAT REST
Each injection contained 5 x lo5 disinte~rat~ons/min. * Standard error.
1 3 12
Rest time (hr)
TABLE %--INCORPORATION
Each injection contained 5 x lo6 disintegrations/min. * Standard error.
Recovered label (counts/min)
Flight time (hr)
UNTO LIPIDS OF THE BLOWFLYDURING
Recovered ia’bei in iipid fractions (7;)
TABLE I-INCORPORATION OF THELABELFROMCLUCOSE-LV4C
P ij g
2 2
P
B &
$ 0 c +Q
$ g
?
e
2
q % %
z 8
1570
CAROLYNF. MATHURANDWILLIAMJ. YURKIEWICZ DISCUSSION
Blowflies, which use carbohydrate for flight fuel, convert very little labelled glucose to lipid while at rest or during flight. However, there is a gradual increase of label in triglyceride with time. VAN HANDEL and LUM (1961), in comparing houseflies and mosquitoes, reported that the triglyceride level increases dramatically in female mosquitoes maintained exclusively on glucose, whereas in male mosquitoes and male and female houseflies it decreases. This does not show that there is no conversion of glucose to triglyceride in houseflies but does suggest that any contributions from glucose are small. Some moths, which utilize lipid for flight, show similarly low levels of conversion from glucose to lipid. SRIDHARAand BHAT (1965) found 0.6 per cent of the injected labelled glucose in the total lipids of B. mori larvae after 24 hr. Higher percentages of 2.4 to 8.4 per cent were reported by HORIE et al. (1968) for the same insect. They found most of the radioactivity in glycerol and fatty acids, with glycerol containing a higher percentage of label than fatty acid, no matter how the glucose was labelled. This agrees with our findings using glucose-U-14C. CHINO and GILBERT (1964) recovered 2.5 per cent of the labelled glucose in the glycerides of developing Hyatophora cecropia pupae. The free fatty acids contained low but significant labelling while hydrocarbons contained none. We found that blowflies incorporate some label into hydrocarbons, and this has been shown in some other insects using labelled acetate as a precursor (LAMBREMONT and BENNETT, 1966; SRIDHARAand BHAT, 1964). In a previous paper (YURKIEWICZ and MATHUR, 1969), we reported a gradual increase of label in triglyceride from palmitate-l-14C over time in both resting and flying flies. The data in this paper show a similar increase in triglyceride, but whether this is from label in fatty acids or glycerol cannot be said with certainty since radioassays were made of the total glycerol and fatty acid components from the combined lipid fractions and not from triglyceride alone. D’COSTA and BIRT (1966) f ound that the neutral lipid fraction in adult male flies, Lucilia cuprina, decreased slowly during the first 5 days of adult life whereas it increased in the female during the same time period. This may in part account for the higher level of incorporation from glucose found in resting females when compared with resting males. It appears that resting blowflies incorporate more radioactive glucose into lipid than flies in flight, in spite of the greatly increased metabolic rate and elevated internal temperature of the fly during flight (YURKIEWICZ and SMYTH, 196613). Earlier we found an increase in the rate of fatty acid esterification into triglycerides during flight using palmitate-l -14C (YURKIEWICZ and MATHUR, 1969). Therefore, flight activity, although accompanied by an increase in general metabolism and in interconversions within the lipid fractions, appears to decrease the rate of conversion of glucose into lipid. Any mechanism which would conserve the level of carbohydrate in flies during flight would be desirable since carbohydrate is believed to be the main source of flight energy in Diptera and, considering the higher metabolic rate, is being rapidly oxidized. A lower rate of conversion of glucose into lipid might be an expected consequence of this high rate of carbohydrate utilization during flight.
INCORPORATION OF GLUCOSE INTO LIPIDSIN BLOWFLYFLIGHT
1571
REFERENCES CHINOII. and GILBERTL. I. (1964) Studies on the interconversion of carbohydrate and fatty acid in Hyalophora cecropia. J. Insect Physiol. l&287-295. CLEMEN::SA. N. (1959) Studies on metabolism of locust fat body. r. exp. BioZ. 36, 665-675. D’COSTA M. A. and BIRT L. M. (1966) Changes in the lipid content during the metamorphosis of the blowfly, Lucilia. J. Insect Physiol. 12, 1377-1394. HORIE Y., NAKASONES., and ITO T. (1968) The conversion of i4C-carbohydrates into CO, and lipid by the silkworm, Bombyx mori. J. Insect Physiol. 14, 971-981. LAMBRR~~ONT E. N. and BENNETTA. F. (1966) Lipid biosynthesis in the boll weevil: Formation (3f the acetate precursor for lipid synthesis from glucose and related carbohydrates. Can.J. Biochem. 44,1597-1606. SRIDHARAS. and BHAT J. V. (1964) Incorporation of 1-14C-acetate into the lipids of the silkworm, Bombyx mori L. BiochemJ. 91,120-123. SRIDHARAS. and BHATJ. V. (1965) Interconversion of carbohydrate and fat in the silkworm Bombyx mori L. Life Sci. 4,979-982. VAN HANDELE. and LUM P. T. M. (1961) Sex as regulator of triglyceride metabolism in the mosquito. Science, N. Y. 134,1979-1980. YURKIE~IICZW. J. (1965) Flight exhaustion studies on the blowfly Phaenicia sericata using r4C labeled sucrose. Ann. ent. Sot. Am. 58, 766. YURKIE~ICZW. J. and MATHURC. F. (1969) Incorporation of palmitate-l-i% into lipids of the blowfly during flight. J. Insect Physiol. 15,439-444. YURKIEP~ICZ W. J. and SMYTH T., JR. (1966a) Effect of temperature on flight speed of the sheep blowfly. r. Insect Physiol. 12, 189-194. YUKIEWICZW. J. and SMYTHT., JR. (1966b) Effects of temperature on oxygen consumption and fuel utilization by the sheep blowfly. J. Insect PhysioE. 12, 403408.