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A protein extract from Drosophila melanogaster with insulin-like activity
Camp. Biochem. Physiol., 1975, VoL 51A,pp. 483 to 485. Pergamon Press. Printed in Great Britain
A PROTEIN EXTRACT FROM DROSOPHILA MELANOGASTER WITH INSULIN-LIKE ACTIVITY* PATRICIO MENESESAND MARIA DE LOS ANGELES ORTiZ MSBS Program, Catholic University of Puerto Rico, Ponce, Puerto Rico (Received 17 January 1974)
Abstract--A crude protein extract from whole Drosophila melanogaster has been shown to have an insulin-likeactivity in mice; the extract obtained from 1 g (fresh weight) of flies has an activity corresponding to 0.04 units of beef insulin. INTRODUCTION
Animals
ONE OF the strongest arguments in favour of the metabolic uniformity of living organisms is the fact that glucose is degraded in the majority of organisms by the same enzymic sequences. However, this "unity of biochemistry" has been challenged by the comparative approach, often based on rather subtle differences (Agosin & Aravena, 1959). The main polysaccharide in insects is trehalose rather than glycogen (Wyatt, 1961), but insect and mammalian tissues follow a similar pattern of glucose metabolism from glucose to pyrnvic acid (Bueding, 1962). Trehalose is degraded into glucose during insect starvation, and an equilibrium has been observed between this disaccharide and the glucose pool (Chefurka, 1965). This phenomenon has been explained by a homeostatic mechanism (Saito, 1963), possibly by the action of unknown hormones. On the other hand, in all vertebrates, from fish to man, glucose requires the presence of insulin for its absorption through the cellular membrane (Hellman, 1969). Thus, insulin must be present and active in extracts from the cellular environment (Candela & Castrillon, 1959). In the case of invertebrates, Arthropoda have a morphological distribution similar to vertebrates with a circulating fluid in its internal media that transports different substances to and from the cells. It is then possible to reason that Arthropoda must have a protein molecule that acts as insulin does in mammals. We wish to report the presence of insulin-like activity in protein extracts of Drosophila melanogaster. The results obtained indicate that the protein fraction obtained from 1 g of fly has an activity equivalent to 0-04 units of beef insulin.
Eight-week-old female mice of the Balb C strain, obtained from the Puerto Rico Nuclear Center, San Juan, Puerto Rico, were utilized. Protein extract
The procedure used by Grodsky & Tarver (1956) for the extraction of plasma insulin was essentially followed. One g fresh weight of Drosophila was suspended in 2 ml of 95% ethanol-H~O-HC1 (15 : 5 : 3, v/v) and the insects were homogenized in a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 1200 g for 15 min at 4°C. The pH of the supematant fluid was adjusted to 8.7 and 4 vol. of 95% ethanol-ether solution (3 : 5, v/v) were then added. The material was left standing at 4°C for 12 hr and the precipitate was collected by centrifugation as above. The precipitate was washed twice with ethanol and once with ether and finally dried under vacuum at 4°C. For use the precipitate was dissolved in 4.0 ml of dis.tilledwater.
MATERIALS AND METHODS Insects
Treatment of animals
Groups of three mice each were injected intraperitoneally with 0"5 ml of the fly extract and the same amount was given after 30 min. Insulin (kindly supplied by Eli Lilly, Indianapolis, U.S.A.) was injected intraperitoneally at a dosage of 0.01 unit per animal. Control mice received 1"0 ml of 0"9~oNaCI. A sample of blood was obtained from the tail, prior to treatment, and at timed intervals as indicated in Results and Discussion. The animals were starved during the entire duration of each experiment. Glucose determination
Blood glucose was determined by glucose destrotix strips and a reflectance meter (Ames Co., Elkhart, Indiana; Orzeck et al., 1971). The results were analyzed statistically (van Dalen & Meyer, 1971).
Non-sexed 1-week-old insects of a wild-type strain of Drosophila melanogaster were used throughout.
RESULTS AND DISCUSSION
* Supported by Grant RR-08067 from the General Research Support Branch, Division of Research Resources, National Institutes of Health.
Table 1 shows the blood glucose levels expressed as per g of body weight in the three experimental mouse groups. The control group (NaC1) shows a
483
484
PATRICIO MENESES AND M A R L ~ DE LOS ANGELES O R T i Z
Table I. Blood Glucose media in mice injected with 0.9% NaCI, insulin and fly protein extract Time (min)
Interval First Second Third Fourth Fifth Sixth Seventh Eighth Ninth Tenth Eleventh
NaCI
0 30 60 90 150 210 270 300 330 360 400
Insulin*
4.30+ 1.74 5"05 + 2-02 4"22 + 1 "57 4.18+ 1.49 3-27+ 1.14 2.79 + 0.83 2"91 + 1.06 3.06 + 1.13 3.02+ 1.11 3'09+ 1.43 3.04+0.91
5-09 + 1.60 2.10 + 0.58 1"05 + 0"34 0.60+0"33 0.77+0"53 1"15 + 0'93 1.72+ 0.89 1-98 + 0"72 1-96+ 0"74 2"16+ 1"30 2.35+0.77
Fly protein 4.70+ 1.28 2.45 + 0.61 1.40 + 0"84 1.01 +0"76 0.79+0'61 1"09 + 0"79 1-81 + 1-50 2.20 + 1.79 2"33 + 1.60 2'17+ 1"43 2.02+1.38
* 0.01 u/ml.
Table 2. Rate of survival of NaCI-, insulin- and fly protein-injected mice
Interval First Second Third Fourth Fifth Sixth Seventh Eighth Ninth Tenth Eleventh
NaCI Time (min) NI* %1 0 30 60 90 150 210 270 300 330 360 400
30 30 30 30 30 30 30 30 30 30 30
100 100 100 100 100 100 100 100 100 100 100
Insulin N2t 30 30 30 29 22 18 17 17 17 17 17
%2 100 100 100 96 73 60 56 56 56 56 56
Fly protein N3} 30 30 30 30 26 22 20 20 20 20 20
%3 100 100 100 100 86 73 66 66 66 66 66
* N1, number of NaCI 0.9%-injected mice. 1 N2, number of insulin-injected mice. $ N3, number of fly protein-injected mice survivors in each interval.
12 -
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.
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hr Fig. 1. Comparison between t-values of blood glucose concentration in control and insulin ( . . . . . . . ), control and fly-protein (- . . . . -) and insulin and fly-proteininjected mice ( ). The limit of significance at P~<0.01 was 2.35
3
5
hr Fig. 2. Comparison between t-values of survival of control and insulin (A) control and fly-protein (A) and insulin and fly-protein-injected mice ( e ) during 400 min of experiment. The limit of significance at P a 0 . 0 1 was 2.4.
Insulin-like activity from Drosophila protein lower glycemia level at 210min (2.79+0.83 mg) after an increase at 30 min (5.05 + 2-02 mg). Insulin injected mice show a steady decrease in glycemia levels up to 90 rain, while this decrease persists at 150 min in the fly extract-injected mice. These values are significantly lower than the control group. Afterwards, the glucose levels increase in all groups to reach a normal value at the end of the experiment. However, these values are still lower than the initial levels because the animals were starved during the entire duration of the experiment. Figure 1 shows the t-values obtained from the results of Table 1. The values are highly significant from the second to the seventh interval for both insulin- and fly proteininjected mice and controls. On the other hand, insulin- and fly protein-injected mice show only the third interval with a low value of significance, while all other values are not significant. In addition, the survival time among insulin- and fly protein-injected mice was essentially the same. A statistical analysis of the survival time was considered of importance, since several animals could not survive the shock occurring during the critical stages of hypoglycemia. This is shown in Table 2 and Fig. 2 giving the tvalues of percentage of survival between insulin and control, fly-protein and control, and insulin- and fly-protein-injected mice, for every interval. The close similarity between insulin- and fly proteininjected mice is very evident. F r o m the above results it was calculated that the insulin activity from I g of fly corresponded to 0.04 units. It is important to point out that this is the first time that an insect protein extract has been proved to have a hypoglycemic effect in mice. In this connection Hemmingsen (1924) and Wenig & Joachim (1936, quoted by Krahl, 1961) were unable to demonstrate any effect of insulin in insects. This result suggests that insects possess an insulin-like molecule, perhaps similar to mammalian insulin. As a result, certain aspects related to protein evolution and hormonal metabolic control in insect homeostasis have to be re-evaluated. It is possible that the cell membrane in insects has the same role as in mammals in glucose transport. And this situation
485
involve the conversion of trehalose into glucose prior to its transport through the cell membrane. The clarification of some of these problems is now being undertaken. REFERENCES AGOSIN M. & ARAVENAL. (1959) Studies on the metabolism of Echinococcus granulosus--III. Glycolysis, with special references to hexokinaser and related glycolyticenzymes. Biochim. biophys. Acta 34, 90-102. BUEDINO E. (1962) Comparative aspects of carbohydrate metabolism. Fedn Proc. Fedn Am. Socs exp. Biol. 21, 1039-1046. R. CANDELAJ. L. & CASIRmLONA. (1963) Augmentation of glucose uptake and protein fractions of normal human plasma. Med. Exp. 8, 12-14, CrIErUar.x W. (1965) Some comparative aspects of metabolism of carbohydrates in insects. A. Rev. Ent. 10, 345-382. GRODSKY G. M. & TARVERH. (1956) Paper chromatography of insulin. Nature, Lond. 177, 223-225. HELLrea~N B. (1969) Islet morphology and glucose metabolism in relation to the specific function of the pancreatic beta cells. Diabetes 18, 509-516. HEM~NGSEN A. M. (1924) Blood sugar in some invertebrates. Skand. Arch. Physiol. 45, 204-210. KgAHL M. E. (1961) The Action of Insulin on Cells. Academic Press, New York. ORZECK E., MOONEY J. • OWEN J. (1971) Diabetes detection with a comparison of screening methods. Diabetes 20, 109-116. SArrO S. (1963) Trehalose in the body fluid of the silkworm Bombyx mori L. J. Insect Physiol. 9, 509-519. VANDALEND. & MEYERW. J. (1971) Manual de T~cnica de la Investigaci6n. Editorial Paidos, Buenos Aires. WENIG K. & JOACHIMJ. (1936) Influence of insulin on silkworm. Biochem. Z. 285, 98-100. WYATT G. R. (1961) The biochemistry of insect hemolymph. A. Rev. Ent. 6, 75-102. Key Word Index--Fly protein extract with insulin-like activity; Drosophila melanogaster; glucose metabolism; insulin; mice blood glucose; hypoglycemia; protein evolution; insect homeostasis; metabolic control; insect cell membrane.
A PROTEIN EXTRACT FROM DROSOPHILA MELANOGASTER WITH INSULIN-LIKE ACTIVITY* PATRICIO MENESESAND MARIA DE LOS ANGELES ORTiZ MSBS Program, Catholic University of Puerto Rico, Ponce, Puerto Rico (Received 17 January 1974)
Abstract--A crude protein extract from whole Drosophila melanogaster has been shown to have an insulin-likeactivity in mice; the extract obtained from 1 g (fresh weight) of flies has an activity corresponding to 0.04 units of beef insulin. INTRODUCTION
Animals
ONE OF the strongest arguments in favour of the metabolic uniformity of living organisms is the fact that glucose is degraded in the majority of organisms by the same enzymic sequences. However, this "unity of biochemistry" has been challenged by the comparative approach, often based on rather subtle differences (Agosin & Aravena, 1959). The main polysaccharide in insects is trehalose rather than glycogen (Wyatt, 1961), but insect and mammalian tissues follow a similar pattern of glucose metabolism from glucose to pyrnvic acid (Bueding, 1962). Trehalose is degraded into glucose during insect starvation, and an equilibrium has been observed between this disaccharide and the glucose pool (Chefurka, 1965). This phenomenon has been explained by a homeostatic mechanism (Saito, 1963), possibly by the action of unknown hormones. On the other hand, in all vertebrates, from fish to man, glucose requires the presence of insulin for its absorption through the cellular membrane (Hellman, 1969). Thus, insulin must be present and active in extracts from the cellular environment (Candela & Castrillon, 1959). In the case of invertebrates, Arthropoda have a morphological distribution similar to vertebrates with a circulating fluid in its internal media that transports different substances to and from the cells. It is then possible to reason that Arthropoda must have a protein molecule that acts as insulin does in mammals. We wish to report the presence of insulin-like activity in protein extracts of Drosophila melanogaster. The results obtained indicate that the protein fraction obtained from 1 g of fly has an activity equivalent to 0-04 units of beef insulin.
Eight-week-old female mice of the Balb C strain, obtained from the Puerto Rico Nuclear Center, San Juan, Puerto Rico, were utilized. Protein extract
The procedure used by Grodsky & Tarver (1956) for the extraction of plasma insulin was essentially followed. One g fresh weight of Drosophila was suspended in 2 ml of 95% ethanol-H~O-HC1 (15 : 5 : 3, v/v) and the insects were homogenized in a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 1200 g for 15 min at 4°C. The pH of the supematant fluid was adjusted to 8.7 and 4 vol. of 95% ethanol-ether solution (3 : 5, v/v) were then added. The material was left standing at 4°C for 12 hr and the precipitate was collected by centrifugation as above. The precipitate was washed twice with ethanol and once with ether and finally dried under vacuum at 4°C. For use the precipitate was dissolved in 4.0 ml of dis.tilledwater.
MATERIALS AND METHODS Insects
Treatment of animals
Groups of three mice each were injected intraperitoneally with 0"5 ml of the fly extract and the same amount was given after 30 min. Insulin (kindly supplied by Eli Lilly, Indianapolis, U.S.A.) was injected intraperitoneally at a dosage of 0.01 unit per animal. Control mice received 1"0 ml of 0"9~oNaCI. A sample of blood was obtained from the tail, prior to treatment, and at timed intervals as indicated in Results and Discussion. The animals were starved during the entire duration of each experiment. Glucose determination
Blood glucose was determined by glucose destrotix strips and a reflectance meter (Ames Co., Elkhart, Indiana; Orzeck et al., 1971). The results were analyzed statistically (van Dalen & Meyer, 1971).
Non-sexed 1-week-old insects of a wild-type strain of Drosophila melanogaster were used throughout.
RESULTS AND DISCUSSION
* Supported by Grant RR-08067 from the General Research Support Branch, Division of Research Resources, National Institutes of Health.
Table 1 shows the blood glucose levels expressed as per g of body weight in the three experimental mouse groups. The control group (NaC1) shows a
483
484
PATRICIO MENESES AND M A R L ~ DE LOS ANGELES O R T i Z
Table I. Blood Glucose media in mice injected with 0.9% NaCI, insulin and fly protein extract Time (min)
Interval First Second Third Fourth Fifth Sixth Seventh Eighth Ninth Tenth Eleventh
NaCI
0 30 60 90 150 210 270 300 330 360 400
Insulin*
4.30+ 1.74 5"05 + 2-02 4"22 + 1 "57 4.18+ 1.49 3-27+ 1.14 2.79 + 0.83 2"91 + 1.06 3.06 + 1.13 3.02+ 1.11 3'09+ 1.43 3.04+0.91
5-09 + 1.60 2.10 + 0.58 1"05 + 0"34 0.60+0"33 0.77+0"53 1"15 + 0'93 1.72+ 0.89 1-98 + 0"72 1-96+ 0"74 2"16+ 1"30 2.35+0.77
Fly protein 4.70+ 1.28 2.45 + 0.61 1.40 + 0"84 1.01 +0"76 0.79+0'61 1"09 + 0"79 1-81 + 1-50 2.20 + 1.79 2"33 + 1.60 2'17+ 1"43 2.02+1.38
* 0.01 u/ml.
Table 2. Rate of survival of NaCI-, insulin- and fly protein-injected mice
Interval First Second Third Fourth Fifth Sixth Seventh Eighth Ninth Tenth Eleventh
NaCI Time (min) NI* %1 0 30 60 90 150 210 270 300 330 360 400
30 30 30 30 30 30 30 30 30 30 30
100 100 100 100 100 100 100 100 100 100 100
Insulin N2t 30 30 30 29 22 18 17 17 17 17 17
%2 100 100 100 96 73 60 56 56 56 56 56
Fly protein N3} 30 30 30 30 26 22 20 20 20 20 20
%3 100 100 100 100 86 73 66 66 66 66 66
* N1, number of NaCI 0.9%-injected mice. 1 N2, number of insulin-injected mice. $ N3, number of fly protein-injected mice survivors in each interval.
12 -
.K, /
\ B
"~T~
',r
.
..r
",,
./t
I
hr Fig. 1. Comparison between t-values of blood glucose concentration in control and insulin ( . . . . . . . ), control and fly-protein (- . . . . -) and insulin and fly-proteininjected mice ( ). The limit of significance at P~<0.01 was 2.35
3
5
hr Fig. 2. Comparison between t-values of survival of control and insulin (A) control and fly-protein (A) and insulin and fly-protein-injected mice ( e ) during 400 min of experiment. The limit of significance at P a 0 . 0 1 was 2.4.
Insulin-like activity from Drosophila protein lower glycemia level at 210min (2.79+0.83 mg) after an increase at 30 min (5.05 + 2-02 mg). Insulin injected mice show a steady decrease in glycemia levels up to 90 rain, while this decrease persists at 150 min in the fly extract-injected mice. These values are significantly lower than the control group. Afterwards, the glucose levels increase in all groups to reach a normal value at the end of the experiment. However, these values are still lower than the initial levels because the animals were starved during the entire duration of the experiment. Figure 1 shows the t-values obtained from the results of Table 1. The values are highly significant from the second to the seventh interval for both insulin- and fly proteininjected mice and controls. On the other hand, insulin- and fly protein-injected mice show only the third interval with a low value of significance, while all other values are not significant. In addition, the survival time among insulin- and fly protein-injected mice was essentially the same. A statistical analysis of the survival time was considered of importance, since several animals could not survive the shock occurring during the critical stages of hypoglycemia. This is shown in Table 2 and Fig. 2 giving the tvalues of percentage of survival between insulin and control, fly-protein and control, and insulin- and fly-protein-injected mice, for every interval. The close similarity between insulin- and fly proteininjected mice is very evident. F r o m the above results it was calculated that the insulin activity from I g of fly corresponded to 0.04 units. It is important to point out that this is the first time that an insect protein extract has been proved to have a hypoglycemic effect in mice. In this connection Hemmingsen (1924) and Wenig & Joachim (1936, quoted by Krahl, 1961) were unable to demonstrate any effect of insulin in insects. This result suggests that insects possess an insulin-like molecule, perhaps similar to mammalian insulin. As a result, certain aspects related to protein evolution and hormonal metabolic control in insect homeostasis have to be re-evaluated. It is possible that the cell membrane in insects has the same role as in mammals in glucose transport. And this situation
485
involve the conversion of trehalose into glucose prior to its transport through the cell membrane. The clarification of some of these problems is now being undertaken. REFERENCES AGOSIN M. & ARAVENAL. (1959) Studies on the metabolism of Echinococcus granulosus--III. Glycolysis, with special references to hexokinaser and related glycolyticenzymes. Biochim. biophys. Acta 34, 90-102. BUEDINO E. (1962) Comparative aspects of carbohydrate metabolism. Fedn Proc. Fedn Am. Socs exp. Biol. 21, 1039-1046. R. CANDELAJ. L. & CASIRmLONA. (1963) Augmentation of glucose uptake and protein fractions of normal human plasma. Med. Exp. 8, 12-14, CrIErUar.x W. (1965) Some comparative aspects of metabolism of carbohydrates in insects. A. Rev. Ent. 10, 345-382. GRODSKY G. M. & TARVERH. (1956) Paper chromatography of insulin. Nature, Lond. 177, 223-225. HELLrea~N B. (1969) Islet morphology and glucose metabolism in relation to the specific function of the pancreatic beta cells. Diabetes 18, 509-516. HEM~NGSEN A. M. (1924) Blood sugar in some invertebrates. Skand. Arch. Physiol. 45, 204-210. KgAHL M. E. (1961) The Action of Insulin on Cells. Academic Press, New York. ORZECK E., MOONEY J. • OWEN J. (1971) Diabetes detection with a comparison of screening methods. Diabetes 20, 109-116. SArrO S. (1963) Trehalose in the body fluid of the silkworm Bombyx mori L. J. Insect Physiol. 9, 509-519. VANDALEND. & MEYERW. J. (1971) Manual de T~cnica de la Investigaci6n. Editorial Paidos, Buenos Aires. WENIG K. & JOACHIMJ. (1936) Influence of insulin on silkworm. Biochem. Z. 285, 98-100. WYATT G. R. (1961) The biochemistry of insect hemolymph. A. Rev. Ent. 6, 75-102. Key Word Index--Fly protein extract with insulin-like activity; Drosophila melanogaster; glucose metabolism; insulin; mice blood glucose; hypoglycemia; protein evolution; insect homeostasis; metabolic control; insect cell membrane.