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Activation of fat body by 20-hydroxyecdysone for the selective incorporation of storage protein in Sarcophaga peregrina larvae
Insect Biochem., Vol. 12, No. 2, pp. 185 191, 1982. Printed in Great Britain.
0020-1790/82/020185-07503.00/0 © 1982 Per,qamon Press Ltd.
A C T I V A T I O N O F FAT B O D Y BY 2 0 - H Y D R O X Y E C D Y S O N E F O R THE SELECTIVE I N C O R P O R A T I O N O F S T O R A G E P R O T E I N IN S A R C O P H A G A P E R E G R I N A LARVAE KOHJI UENO and SHUNJI NATOR! Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan (Received 22 July 1981)
Abstract--When haemolymph of third instar larvae of Sarcophaga pere(jrina was radioiodinated in ritro, most of the radioactivity was incorporated into 75 k storage protein which constitutes about 70'5,, of the total haemolymph protein at this stage. Using radioiodinated haemolymph, the fate of storage protein during metamorphosis was followed. It was found that this protein was selectively incorporated into the fat body in the early pupal stage and that 20-hydroxyecdysone was essential in this process to activate the fat body to incorporate storage protein. This was demonstrated in vitro by incubating larval fat body in the presence of 20-hydroxyecdysone. It was suggested that storage protein was accumulated in large granules in fat body cells that appeared in the cells responding to 20-hydroxyecdysone. Key Word Index: Storage protein, fat body, 20-hydroxyecdysone, Sarcophaya pereyrina
INTRODUCTION
MATERIALS AND METHODS
THE PIONEERING studies using the blowfly Calliphora erythrocephala have shown that larval fat body contains abundant protein particles, named storage protein, of about 10 nm diameter (MuNN and GREVILLE, 1969; 1971). These particles contain a hexameric protein named calliphorin with a molecular weight of 87,000. This protein was first synthesized in the larval fat body and then secreted into the haemolymph, where it constitutes more than 75-80% of the total haemolymph protein at the end of the third instar (MuNN et al., 1969; KINNEAR et al., 1971). However, in the early pupal stage, the fat body takes up this protein again and accumulates it in specific granules in fat body cells (MARTIN et al., 1971). On histolysis of fat body during metamorphosis, these granules may be hydrolysed in situ or are released into the haemolymph and are transported to the developing adult tissues (KINNEAR and THOMPSON, 1975). Although these processes seem to be the same in many insects and to be essential for metamorphosis, the precise mechanisms of protein uptake and granule formation are unknown. It is assumed that the moulting hormone activates the fat body enabling it to incorporate storage protein, but no clear evidence has yet been obtained that the hormone is essential for this process (THOMASON and MITCHELL, 1972; BUTTERWORTH et al., 1979). We studied the mechanism of selective uptake of storage protein by the fat body on pupation in Sarcophaga pereorina larvae because of the ease by which their physiological age can be controlled by dry-wet treatment. Results showed that 20-hydroxyecdysone is essential for selective uptake of the 75 k storage protein by the fat body.
Animals The flesh-fly, Sarcophaya peregrina, was reared by the method of OHTAKI(1966). Third instar larvae were kept in plastic containers under wet conditions when pupariation does not occur because 20-hydroxyecdysone is not secreted. When transferred to dry conditions, they start to pupariate after 16hr at 27C. Larvae could be used for experiments for up to five days after leaving their food. Injections Larvae were immobilized by placing them on ice and then 5/A of insect saline (130 mM NaCI, 5 mM KCI and 1 mM CaCI2) containing radioiodinated haemolymph protein or 0.5 mg/ml of 20-hydroxyecdysone (Rohto Seiyaku Co., Japan) was injected into the posterior part of the body cavity with a microsyringe under a binocular microscope. Radioiodination of haemolymph protein This was done by the method of MIYACHI et al. (1972). The reaction mixture contained in a total volume of 50 #l, 0.1 M acetate buffer, pH 5.7, 0.5 mCi Iodine-125 (Amersham/Searle, 15 mCi//~g), 0.06 units of immobilized lactoperoxidase, 3/ag/ml of H202 and 100~g of haemolymph protein, which corresponds to about 1 #l of haemolymph prepared from third instar larvae. After incubation for 10min at 25°C, the reaction mixture was applied to a column of Sephadex G-10 (1 cm, i.d. x 30cm) which had been equilibrated with insect saline containing 2mM PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), pH 7.0. The excluded radioactivity was combined, bovine serum albumin (BSA) was added to a final concentration of 1 mg/ml, and the mixture was stored at -80°C. The specific activity of radioiodinated protein was usually 2 x 106 cpm//~g. Under these conditions, most of the radioactivity was selectively incorporated into 75 k storage protein, as described in the text.
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KOHJ[ UENO AND SHUNJI NATORI
186
SDS-polytwrylamide .qel electrophoresis Electrophoresis on polyacrylamide SDS slab gels were carried out by the method of LAEMMLI(1970). The stacking gel (3% acrylamide) was about 2 cm long, and the separating gel (10!~i, acrylamide)about 7cm long. After electrophoresis the gels were stained by the method of FAIRBANKS et al. (1971). The gels were then dried and autoradiographed using Fuji Rx-s film.
Autoradioyraphy of sections offat body embedded in parqlfin Fat body was fixed in 10% (v/v) formalin and embedded in paraffin. Sections of 5 um thickness were cut and fixed on slide glasses. The paraffin was removed by washing with xylene and the slide glasses were coated with autoradiographic emulsion (Konishiroku NR-M2). The exposure time ranged from two to three weeks depending upon the preparation. The slides were stained with Giemsa stain after development.
Uptake of haemolymph protein by fat body in vitro Fat body was dissected out from larvae or pupae under a binocular microscope and rinsed well with insect saline. The reaction mixture contained in a total volume of 0.4 ml, 20 mM HEPES (N-(2-hydroxyethyl)piperazine-N'-2-ethansulfonic acid), pH 7.5, 150raM NaCI, 0.1 mg/ml of bovine serum albumin, 5-7 x 104 cpm/ml of radioiodinated haemolymph protein and fat body prepared from a larva or a pupa. After incubation at 25~C for various times, the fat body was washed well and its radioactivity was counted. RESULTS
Selective uptake of storaye protein by fat body on pupation It was found that haemolymph from third instar larvae of Sarcophaga peregrina contained numerous protein particles similar to calliphorin, as shown in Fig. la. These particles contained storage protein which amounted to more than 700,i, of the total haemolymph protein at this stage. The storage protein of this fly seemed to be heterogeneous and besides the major 75 k protein several protein bands were identifiable in the 75 k region on SDS-polyacrylamide gel when protein in these particles was analyzed, as shown in Fig. lb. These proteins disappeared gradually from the haemolymph after puparium formation. To examine the fate of storage protein, we radioiodinated haemolymph protein in vitro, injected it into third instar larvae and studied the uptake of labelled protein into various tissues. Under the conditions used, radioactive iodine was mainly incorporated into 75 k protein. Thus, it was possible to trace the fate of storage protein using radioiodinated haemolymph. Results showed that when larvae were kept u n d e r wet conditions where secretion of 20-hYdroxyecdysone was inhibited after injection of radioactive protein, there was no significant incorporation of radioactivity into the fat body. However, when the larvae were transferred to dry conditions, significant radioactivity became detectable in the fat body after about 12 hr, as shown in Fig. 2. The radioactivity in the fat body increased with time and 50}o of the injected radioactivity was incorporated into the fat body after 30hr. Since the radioactivity in other tissues was almost negligible at this stage, it seemed possible that the fat body was activated by 20-hydroxyecdysone to take up storage protein from the haemolymph. This was confirmed by the fact that
as soon as 20-hydroxyecdysone was administered, uptake of injected radioactive protein by the fat body became obvious even under the wet conditions (Fig. 2). The rate of uptake under these conditions was about the same as that observed under dry conditions. Thus, it is clear that 20-hydroxyecdysone activates fat body cells to incorporate storage protein. To examine the specificity of this reaction, we compared the uptake of radioiodinated lectin prepared from the haemolymph of this animal with that of storage protein. This lectin was found to be induced in the haemolymph on pupation or on injury of the larval body wall (KOMANO et al., 1980). As shown in Fig. 3, uptake of lectin by the fat body was significantly less than that of storage protein under the same conditions when about the same amounts of radioactive proteins were injected. Since the amount of storage protein in the haemolymph is several orders of magnitude higher than that of lectin, radioactive storage protein should have been diluted many-fold. Nevertheless, uptake of storage protein was many times more than that of lectin, indicating that the incorporation of storage protein into the fat body is highly selective.
The fate of protein taken up by the fat body As shown above, the fat body selectively incorporates 75 k proteins in response to 20-hydroxyecdysone. However, it was hot clear whether the proteins are degraded first on the cell surface and then incorporated or whether they are incorporated as intact molecules. This point was examined in the following experiments. Radioiodinated protein was injected into third instar larvae and the larvae were kept under dry conditions. The fat body was isolated from larvae after various times, homogenized and subjected to SDS-polyacrylamide gel electrophoresis followed by autoradiography. As is evident from Fig. 4a lanes 4-8, the intensity of the band corresponding to storage protein increased with time. In parallel with this, the radioactivity of this band also increased as shown in Fig. 4b. These results indicate that the 75 k polypeptides of the storage protein was incorporated and accumulated in fat body cells as intact molecules. The injected protein was also found to remain intact in the haemolymph for at least 30 hr, since the mobility of these proteins in SDS polyacrylamide gel did not change, as shown in lanes 2 and 3. Next, we examined the localization of radioiodinated storage protein in fat body cells by autoradiography. Larvae were kept under wet or dry conditions for 30hr after administration of radioiodinated protein and then their fat body was dissected out, fixed and embedded in paraffin. Sections were cut, subjected to autoradiography and then stained with Giemsa stain. As shown in Fig. 5a, grains seemed to be concentrated in large granules that were not seen in fat body prepared from larvae kept under wet conditions as shown in Fig. 5b. Thus, it is clear that the protein was actually incorporated into the cells, not merely ~/dsorbed on the cell surface. The profiles of granules in the two preparations are very different. Large granules seem to appear in response to 20-hydroxyecdysone. However, the distribution of grains on large granules was not even: some granules had many
187
(o)
(b)
Fig. 1. Electron micrograph and electrophoretic patterns of storage protein particles. (a) Haemolymph was negatively stained with ammonium molybdate. The bar represents 500 A. (b) Storage protein particles were isolated by sucrose density gradient centrifugation and subjected to SDS-polyacrylamide gel electrophoresis with various molecular weight markers purchased from Sigma Chemical Co. 1. phosphorylase b, 92,000; 2. bovine serum albumin, 68,000; 3. ovalbumin, 45,000; 4. e-chymotrypsinogen, 25,000.
Fig. 4. SDS-polyacrylamide gel electrophoresis of fat body extracts. Radioiodinated haemolymph protein was injected and fat body was removed as described in the legend to Fig. 3. Fat body from each animal was homogenated in 150,ul of insect saline containing 0.01~o SDS and 5 pl was subjected to electrophoresis. Staining pattern (a) and its radioactivity (b). Lane 1, 5 pl of radioiodinated haemolymph protein (the thick band in (a) is that of BSA added to labelled protein); lanes 2 and 3, 0.5 pl each of haemolymph prepared 0 and 30 hr later, respectively; lanes 4, 5, 6, 7 and 8, fat body extracts prepared 3, 7, 15, 20 and 30hr after transfer to dry conditions.
188
Fig. 5. Autoradiographs of fat body with incorporated haemolymph protein. Radioiodinated haemolymph protein was injected into third instar larvae and the larvae were kept under dry or wet conditions for 30 hr. Then the fat body was dissected out and autoradiographic specimens were prepared. Granules were stained with Giemsa stain after development. Fat body from larvae kept under dry conditions (a) and wet conditions (b).
Storage protein uptake by fat body
189
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20 30 40 50 Time (h) Fig. 2. Uptake of radioiodinated haemolymph protein by fat body. 5/al of radioiodinated haemolymph protein solution was injected into each animal at zero time. Fat bodies from five larvae were removed at intervals and the radioactivity was measured. Points are expressed per larva. (O), larvae kept under dry conditions: (@), larvae kept under wet conditions: (l), larvae kept under wet conditions but injected with 5#1 of 20-hydroxyecdysone (0.5 mg/ml) at the time indicated by an arrow. grains and others none. Thus, it is possible that these granules are not homogeneous and that only some of them accumulate storage protein taken up by the cells.
Uptake of storage protein in vitro by fat body When radioiodinated storage protein was administered to third instar larvae, it started to be incorporated into the fat body in parallel with the secretion of 20-hydroxyecdysone. We examined whether fat body from third instar larvae could incorporate storage protein in vitro in the presence of 20-hydroxyecdysone. Fat body was carefully removed, washed well and immersed in buffered insect saline. The radioiodinated storage protein was added and uptake
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Fig. 3. Specificity of the uptake of protein by fat body. The same amount of radioiodinated haemolymph protein (O) or lectin (A) was injected into third instar larvae at zero time. and then larvae were transferred to dry conditions. Fat bodies from five larvae were removed at intervals and the radioactivity was measured. Points are expressed per larva. Closed symbols are negative control: haemolymph protein (e) or lectin (&) was injected but larvae were kept under wet conditions.
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Fig. 6. Uptake of haemolymph protein by fat body in vitro. Fat body was incubated in 0.4 ml of 20 mM HEPES, pH 7.5, 150mM NaC1, 0.1 mg/ml of BSA with 5 7 x 104 cpm/ml of radioiodinated haemolymph protein at 25"C, in the presence or absence of 5 x 10 -7 M 20-hydroxyecdysone. Incorporation of radioactive protein into fat body was followed with time. (©), fat body from larvae kept under dry conditions for 30hr; (&), fat body from third instar larvae, with 20-hydroxyecdysone: (A), fat body from third instar larvae, without 20-hydroxyecdysone: (@), fat body from third instar larvae kept under dry conditions for 30 hr, but incubated at 0 C. Points + S.E.M. are average for five animals. of radioactivity by the fat body was followed with time at 25"C. As shown in Fig. 6, there was no significant incorporation of storage protein into fat body prepared from larvae kept under wet conditions. However, this fat body showed significant incorporation when 5 x 10-VM 20-hydroxyecdysone was added to the incubation medium. The incorporation started as soon as 20-hydroxyecdysone was added and proceeded linearly for up to 120 min. Thus, it is clear that fat body is activated to take up storage protein in the presence of 20-hydroxyecdysone in vitro. The same experiment was performed using fat body prepared from larvae kept under dry conditions for 30hr. This fat body had been activated, and so it incorporated twice as much radioactivity as larval fat body in the absence of 20-hydroxyecdysone. No uptake of storage protein was detected when activated fat body was incubated at 0:C, indicating that this process is temperature dependent. The dose-response to 20-hydroxyecdysone of uptake of storage protein by larval fat body in vitro is shown in Fig. 7. Uptake of storage protein reached maximum when 5 x 10 -7 M of 20-hydroxyecdysone was added to the reaction mixture and no significant uptake was detected when the concentration of the hormone was below 1 0 - S M or above 10 -5 M. The optimum concentration of 20-hydroxyecdysone for uptake of storage protein roughly coincided with the physiological concentration of this hormone necessary for inducing metamorphosis of this fly reported by OHTAK1 et al. (1968), which is 6.9 x 1 0 - 7 M. Clearly, fat body prepared from larvae kept under dry conditions showed significant incorporation of
190
KOHJI UENO ANI) SHUNJI NATORI
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20- Hy droxyecdysone (M) Fig. 7. Dose-response to 20-hydroxyecdysone of the uptake of haemolymph protein in vitro. Incorporation was measured under the conditions described in the legend to Fig. 7, with incubation for 120min in the presence of increasing amounts of 20-hydroxyecdysone. (©), fat body from larvae kept under dry conditions for 30 hr: (@), fat body from third instar larvae. storage protein even in the absence of 20-hydroxyecdysone and the hormone had no significant effect when it was added to the medium at lower concentration. However, when the concentration of the hormone was above 10 -6 M, the uptake of storage protein decreased sharply, indicating that the hormone was inhibitory at higher concentration. DISCUSSION The storage protein of insects found in larval haemolymph was shown to be used as a source of amino acids and energy for synthesis of new protein during metamorphosis (THOMSON, 1975). However, many questions remained to be answered about the metabolism of this protein. We are interested in the phenomenon that storage protein once secreted into haemolymph is taken up again by the fat body on pupation. The storage proteins that have been studied most extensively are calliphorin and storage protein of silkworm (ToJo et al., 1978). However, the relation between 20-hydroxyecdysone and the selective uptake of these proteins by fat body has not yet been clarified. In this paper we provided clear evidence that 20-hydroxyecdysone is essential for incorporation of storage protein by the fat body. Sarcophaga peregrina larvae are convenient for use in studies on the effect of 20-hydroxyecdysone on the uptake of storage protein by the fat body, since their secretion of the hormone can be controlled easily. In these larvae, uptake of storage protein was detected only under conditions when the ring gland secreted 20-hydroxyecdysone. The requirement for 20-hydroxyecdysone was confirmed in ritro by incubating storage protein with fat body in the presence of 20-hydroxyecdysone. This is the first demonstration that 20-hydroxyecdysone directly activates the fat body to incorporate storage protein. The uptake of protein by the fat body is not
random, but seems to be selective because storage protein was incorporated to a much greater extent than a lectin that is also a haemolymph protein induced on pupation or when the body wall of the larva is injured. This difference in the incorporation of protein may be simply due to a difference in the structures of the proteins. However, it is possible that incorporation of storage protein by fat body cells occurs through specific receptors on their surface like the incorporation of low density lipoprotein by fibroblasts in higher organisms (GOLDSTEIN and BROWN, 1977). These receptors may be masked in the larval stage and they may be activated by 20-hydroxyecdysone on pupation. Although 20-hydroxyecdysone activated the larval fat body to incorporate storage protein in vitro, its incorporation activity is about half that of fat body prepared from larvae kept under dry conditions. Thus another factor or factors besides 20-hydroxyecdysone may be necessary for full activation of the fat body. It is noteworthy that too high a concentration of the hormone inhibits incorporation of storage protein. Probably 20-hydroxyecdysone has pleiotropic effects on fat body cells and the balance of biological activities of the cells is disordered by excess hormone. Comparative studies on the biochemical characters of cytoplasmic membranes prepared from active and inactive fat body cells should provide useful information about the mechanism of selective uptake of haemolymph proteins. It is known that some lysosomal enzymes are selectively taken up by fibroblasts from the serum, although the mechanism of this uptake is unknown (NEUFELD et al., 1975). Selective uptake of storage protein by fat body may be a good model to use in studies on the general mechanism of protein uptake by eukaryotic cells. We observed the storage protein was incorporated into large granules that appeared in fat body cells on pupariation. During metamorphosis, these granules are thought to carry storage protein to developing adult tissues where it is used as a source of amino acids. Granule formation may also be controlled by 20-hydroxyecdysone. since no large granules are found in larval fat body. Apparent heterogeneity of the granules was observed in terms of incorporation of storage protein. Probably many kinds of granules with different functions are formed at this stage.
REFERENCES BUTTERWORTHF. M., TYSELL B. and WACLAWSK11. (1972) The effect of 20-hydroxyecdysone and protein on granule formation in the in vitro cultured fat body of Drosophila. d. Insect. Physiol. 18, 1885 1889. FA1RBANKSG., STECKT. L. and WALLACHD. L. H. (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10, 2606-2617. GOLDSTEINJ. L. and BROWN M. S. (1977) The low-density lipoproteins pathway and its relation to atherosclerosis. Ann. Rev. Biochem. 46, 897-930. KINNEAR J. F., MARTIN M. D. and THOMSONJ. A. (1971) Developmental changes in the late larva of Callipora stygia. Aust. J. Biol. Sci. 24, 275 289. KINNEARJ. F. and THOMSONJ. A. (1975) Nature. origin and fate of major haemolymph proteins in Calliphora. In.sect Biochem. 5, 531 552.
Storage protein uptake by fat body KOMANO H., M1ZUNO D. and NATORI S. (1980) Purification of lectin induced in the haemolymph of Sarcophaga peregrina larvae on injury. J. Biol. Chem. 255, 2919-2924. LAEMMLI U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. MARTIN M. D., KINNEAR J. F. and THOMSON J. A. (1971) Developmental changes in the late larva of Calliphora stygia. IV. Uptake of plasma protein by the fat body. AuNt. J. Biol. Sci. 24, 291 299. MIYACHI Y., VAITUKAITISJ. L., NIESHLAG E. and LIPSETT M. B. 0972) Enzymatic radioiodination of gonadotropins. J. clin. Endocr. Metab. 34, 23-28. MUNN E. A., FEINSTEIN A. and GREVILLE G. D. (1971) The isolation and properties of the proteins calliphorin. Biochem. J. 124, 367-374. MUNN E. A. and GREVILLE G. D. (1969) The soluble proteins of developing Calliphora erythrocephala, particularly calliphorin, and similar proteins in other insects. J. Insect Physiol. 15, 1935 1950. MUNN E. A., PRICE G. M. and GREVILLE G. D. (1969) The synthesis in citro of the protein calliphorin by fat body
191
from the larva of the blowfly, Calliphora erythrocephala. J. Insect Physiol. 15, 1601-1605. NEUFELD E. F., LIM T. W. and SHAPIRO L. T. (1975) Inherited disorders of lysosomal metabolism. Ann. Rev. Biochem. 44, 357-376. OHTAKI T. (1966) On the delayed pupation of the fleshfly, Sarcophaga peregrina Robineau-Desvoidy. Japan J. Med. Sci. Biol. 19, 97-104. OHTAKI T., MILKMAN R. D. and WILLIAMS C. M. (1968) Dynamics of ecdysone secretion and action in the fleshfly Sarcophaga peregrina. Biol. Bull. Woods Hole 135, 322-334. THOMASON W. A. and MITCHELL H. K. 0972) Hormonal control of protein granule accumulation in fat bodies of Drosophila melanoftaster larvae. J. Insect Physiol. 18, 1885-1889. THOMSON J. A. (1975) Major patterns of gene activity during development in holometabolous insects. Ade. Insect. Physiol. II, 321-398. ToJo S., BETCHAKU T., ZICCARDI V. J. and WYATT G. R. (1978) Fat body protein granules and storage proteins in silkmoth, Hyalophora cecropia. J. Cell. Biol. 78, 823-838.
0020-1790/82/020185-07503.00/0 © 1982 Per,qamon Press Ltd.
A C T I V A T I O N O F FAT B O D Y BY 2 0 - H Y D R O X Y E C D Y S O N E F O R THE SELECTIVE I N C O R P O R A T I O N O F S T O R A G E P R O T E I N IN S A R C O P H A G A P E R E G R I N A LARVAE KOHJI UENO and SHUNJI NATOR! Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan (Received 22 July 1981)
Abstract--When haemolymph of third instar larvae of Sarcophaga pere(jrina was radioiodinated in ritro, most of the radioactivity was incorporated into 75 k storage protein which constitutes about 70'5,, of the total haemolymph protein at this stage. Using radioiodinated haemolymph, the fate of storage protein during metamorphosis was followed. It was found that this protein was selectively incorporated into the fat body in the early pupal stage and that 20-hydroxyecdysone was essential in this process to activate the fat body to incorporate storage protein. This was demonstrated in vitro by incubating larval fat body in the presence of 20-hydroxyecdysone. It was suggested that storage protein was accumulated in large granules in fat body cells that appeared in the cells responding to 20-hydroxyecdysone. Key Word Index: Storage protein, fat body, 20-hydroxyecdysone, Sarcophaya pereyrina
INTRODUCTION
MATERIALS AND METHODS
THE PIONEERING studies using the blowfly Calliphora erythrocephala have shown that larval fat body contains abundant protein particles, named storage protein, of about 10 nm diameter (MuNN and GREVILLE, 1969; 1971). These particles contain a hexameric protein named calliphorin with a molecular weight of 87,000. This protein was first synthesized in the larval fat body and then secreted into the haemolymph, where it constitutes more than 75-80% of the total haemolymph protein at the end of the third instar (MuNN et al., 1969; KINNEAR et al., 1971). However, in the early pupal stage, the fat body takes up this protein again and accumulates it in specific granules in fat body cells (MARTIN et al., 1971). On histolysis of fat body during metamorphosis, these granules may be hydrolysed in situ or are released into the haemolymph and are transported to the developing adult tissues (KINNEAR and THOMPSON, 1975). Although these processes seem to be the same in many insects and to be essential for metamorphosis, the precise mechanisms of protein uptake and granule formation are unknown. It is assumed that the moulting hormone activates the fat body enabling it to incorporate storage protein, but no clear evidence has yet been obtained that the hormone is essential for this process (THOMASON and MITCHELL, 1972; BUTTERWORTH et al., 1979). We studied the mechanism of selective uptake of storage protein by the fat body on pupation in Sarcophaga pereorina larvae because of the ease by which their physiological age can be controlled by dry-wet treatment. Results showed that 20-hydroxyecdysone is essential for selective uptake of the 75 k storage protein by the fat body.
Animals The flesh-fly, Sarcophaya peregrina, was reared by the method of OHTAKI(1966). Third instar larvae were kept in plastic containers under wet conditions when pupariation does not occur because 20-hydroxyecdysone is not secreted. When transferred to dry conditions, they start to pupariate after 16hr at 27C. Larvae could be used for experiments for up to five days after leaving their food. Injections Larvae were immobilized by placing them on ice and then 5/A of insect saline (130 mM NaCI, 5 mM KCI and 1 mM CaCI2) containing radioiodinated haemolymph protein or 0.5 mg/ml of 20-hydroxyecdysone (Rohto Seiyaku Co., Japan) was injected into the posterior part of the body cavity with a microsyringe under a binocular microscope. Radioiodination of haemolymph protein This was done by the method of MIYACHI et al. (1972). The reaction mixture contained in a total volume of 50 #l, 0.1 M acetate buffer, pH 5.7, 0.5 mCi Iodine-125 (Amersham/Searle, 15 mCi//~g), 0.06 units of immobilized lactoperoxidase, 3/ag/ml of H202 and 100~g of haemolymph protein, which corresponds to about 1 #l of haemolymph prepared from third instar larvae. After incubation for 10min at 25°C, the reaction mixture was applied to a column of Sephadex G-10 (1 cm, i.d. x 30cm) which had been equilibrated with insect saline containing 2mM PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), pH 7.0. The excluded radioactivity was combined, bovine serum albumin (BSA) was added to a final concentration of 1 mg/ml, and the mixture was stored at -80°C. The specific activity of radioiodinated protein was usually 2 x 106 cpm//~g. Under these conditions, most of the radioactivity was selectively incorporated into 75 k storage protein, as described in the text.
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KOHJ[ UENO AND SHUNJI NATORI
186
SDS-polytwrylamide .qel electrophoresis Electrophoresis on polyacrylamide SDS slab gels were carried out by the method of LAEMMLI(1970). The stacking gel (3% acrylamide) was about 2 cm long, and the separating gel (10!~i, acrylamide)about 7cm long. After electrophoresis the gels were stained by the method of FAIRBANKS et al. (1971). The gels were then dried and autoradiographed using Fuji Rx-s film.
Autoradioyraphy of sections offat body embedded in parqlfin Fat body was fixed in 10% (v/v) formalin and embedded in paraffin. Sections of 5 um thickness were cut and fixed on slide glasses. The paraffin was removed by washing with xylene and the slide glasses were coated with autoradiographic emulsion (Konishiroku NR-M2). The exposure time ranged from two to three weeks depending upon the preparation. The slides were stained with Giemsa stain after development.
Uptake of haemolymph protein by fat body in vitro Fat body was dissected out from larvae or pupae under a binocular microscope and rinsed well with insect saline. The reaction mixture contained in a total volume of 0.4 ml, 20 mM HEPES (N-(2-hydroxyethyl)piperazine-N'-2-ethansulfonic acid), pH 7.5, 150raM NaCI, 0.1 mg/ml of bovine serum albumin, 5-7 x 104 cpm/ml of radioiodinated haemolymph protein and fat body prepared from a larva or a pupa. After incubation at 25~C for various times, the fat body was washed well and its radioactivity was counted. RESULTS
Selective uptake of storaye protein by fat body on pupation It was found that haemolymph from third instar larvae of Sarcophaga peregrina contained numerous protein particles similar to calliphorin, as shown in Fig. la. These particles contained storage protein which amounted to more than 700,i, of the total haemolymph protein at this stage. The storage protein of this fly seemed to be heterogeneous and besides the major 75 k protein several protein bands were identifiable in the 75 k region on SDS-polyacrylamide gel when protein in these particles was analyzed, as shown in Fig. lb. These proteins disappeared gradually from the haemolymph after puparium formation. To examine the fate of storage protein, we radioiodinated haemolymph protein in vitro, injected it into third instar larvae and studied the uptake of labelled protein into various tissues. Under the conditions used, radioactive iodine was mainly incorporated into 75 k protein. Thus, it was possible to trace the fate of storage protein using radioiodinated haemolymph. Results showed that when larvae were kept u n d e r wet conditions where secretion of 20-hYdroxyecdysone was inhibited after injection of radioactive protein, there was no significant incorporation of radioactivity into the fat body. However, when the larvae were transferred to dry conditions, significant radioactivity became detectable in the fat body after about 12 hr, as shown in Fig. 2. The radioactivity in the fat body increased with time and 50}o of the injected radioactivity was incorporated into the fat body after 30hr. Since the radioactivity in other tissues was almost negligible at this stage, it seemed possible that the fat body was activated by 20-hydroxyecdysone to take up storage protein from the haemolymph. This was confirmed by the fact that
as soon as 20-hydroxyecdysone was administered, uptake of injected radioactive protein by the fat body became obvious even under the wet conditions (Fig. 2). The rate of uptake under these conditions was about the same as that observed under dry conditions. Thus, it is clear that 20-hydroxyecdysone activates fat body cells to incorporate storage protein. To examine the specificity of this reaction, we compared the uptake of radioiodinated lectin prepared from the haemolymph of this animal with that of storage protein. This lectin was found to be induced in the haemolymph on pupation or on injury of the larval body wall (KOMANO et al., 1980). As shown in Fig. 3, uptake of lectin by the fat body was significantly less than that of storage protein under the same conditions when about the same amounts of radioactive proteins were injected. Since the amount of storage protein in the haemolymph is several orders of magnitude higher than that of lectin, radioactive storage protein should have been diluted many-fold. Nevertheless, uptake of storage protein was many times more than that of lectin, indicating that the incorporation of storage protein into the fat body is highly selective.
The fate of protein taken up by the fat body As shown above, the fat body selectively incorporates 75 k proteins in response to 20-hydroxyecdysone. However, it was hot clear whether the proteins are degraded first on the cell surface and then incorporated or whether they are incorporated as intact molecules. This point was examined in the following experiments. Radioiodinated protein was injected into third instar larvae and the larvae were kept under dry conditions. The fat body was isolated from larvae after various times, homogenized and subjected to SDS-polyacrylamide gel electrophoresis followed by autoradiography. As is evident from Fig. 4a lanes 4-8, the intensity of the band corresponding to storage protein increased with time. In parallel with this, the radioactivity of this band also increased as shown in Fig. 4b. These results indicate that the 75 k polypeptides of the storage protein was incorporated and accumulated in fat body cells as intact molecules. The injected protein was also found to remain intact in the haemolymph for at least 30 hr, since the mobility of these proteins in SDS polyacrylamide gel did not change, as shown in lanes 2 and 3. Next, we examined the localization of radioiodinated storage protein in fat body cells by autoradiography. Larvae were kept under wet or dry conditions for 30hr after administration of radioiodinated protein and then their fat body was dissected out, fixed and embedded in paraffin. Sections were cut, subjected to autoradiography and then stained with Giemsa stain. As shown in Fig. 5a, grains seemed to be concentrated in large granules that were not seen in fat body prepared from larvae kept under wet conditions as shown in Fig. 5b. Thus, it is clear that the protein was actually incorporated into the cells, not merely ~/dsorbed on the cell surface. The profiles of granules in the two preparations are very different. Large granules seem to appear in response to 20-hydroxyecdysone. However, the distribution of grains on large granules was not even: some granules had many
187
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(b)
Fig. 1. Electron micrograph and electrophoretic patterns of storage protein particles. (a) Haemolymph was negatively stained with ammonium molybdate. The bar represents 500 A. (b) Storage protein particles were isolated by sucrose density gradient centrifugation and subjected to SDS-polyacrylamide gel electrophoresis with various molecular weight markers purchased from Sigma Chemical Co. 1. phosphorylase b, 92,000; 2. bovine serum albumin, 68,000; 3. ovalbumin, 45,000; 4. e-chymotrypsinogen, 25,000.
Fig. 4. SDS-polyacrylamide gel electrophoresis of fat body extracts. Radioiodinated haemolymph protein was injected and fat body was removed as described in the legend to Fig. 3. Fat body from each animal was homogenated in 150,ul of insect saline containing 0.01~o SDS and 5 pl was subjected to electrophoresis. Staining pattern (a) and its radioactivity (b). Lane 1, 5 pl of radioiodinated haemolymph protein (the thick band in (a) is that of BSA added to labelled protein); lanes 2 and 3, 0.5 pl each of haemolymph prepared 0 and 30 hr later, respectively; lanes 4, 5, 6, 7 and 8, fat body extracts prepared 3, 7, 15, 20 and 30hr after transfer to dry conditions.
188
Fig. 5. Autoradiographs of fat body with incorporated haemolymph protein. Radioiodinated haemolymph protein was injected into third instar larvae and the larvae were kept under dry or wet conditions for 30 hr. Then the fat body was dissected out and autoradiographic specimens were prepared. Granules were stained with Giemsa stain after development. Fat body from larvae kept under dry conditions (a) and wet conditions (b).
Storage protein uptake by fat body
189
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Uptake of storage protein in vitro by fat body When radioiodinated storage protein was administered to third instar larvae, it started to be incorporated into the fat body in parallel with the secretion of 20-hydroxyecdysone. We examined whether fat body from third instar larvae could incorporate storage protein in vitro in the presence of 20-hydroxyecdysone. Fat body was carefully removed, washed well and immersed in buffered insect saline. The radioiodinated storage protein was added and uptake
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Fig. 3. Specificity of the uptake of protein by fat body. The same amount of radioiodinated haemolymph protein (O) or lectin (A) was injected into third instar larvae at zero time. and then larvae were transferred to dry conditions. Fat bodies from five larvae were removed at intervals and the radioactivity was measured. Points are expressed per larva. Closed symbols are negative control: haemolymph protein (e) or lectin (&) was injected but larvae were kept under wet conditions.
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Fig. 6. Uptake of haemolymph protein by fat body in vitro. Fat body was incubated in 0.4 ml of 20 mM HEPES, pH 7.5, 150mM NaC1, 0.1 mg/ml of BSA with 5 7 x 104 cpm/ml of radioiodinated haemolymph protein at 25"C, in the presence or absence of 5 x 10 -7 M 20-hydroxyecdysone. Incorporation of radioactive protein into fat body was followed with time. (©), fat body from larvae kept under dry conditions for 30hr; (&), fat body from third instar larvae, with 20-hydroxyecdysone: (A), fat body from third instar larvae, without 20-hydroxyecdysone: (@), fat body from third instar larvae kept under dry conditions for 30 hr, but incubated at 0 C. Points + S.E.M. are average for five animals. of radioactivity by the fat body was followed with time at 25"C. As shown in Fig. 6, there was no significant incorporation of storage protein into fat body prepared from larvae kept under wet conditions. However, this fat body showed significant incorporation when 5 x 10-VM 20-hydroxyecdysone was added to the incubation medium. The incorporation started as soon as 20-hydroxyecdysone was added and proceeded linearly for up to 120 min. Thus, it is clear that fat body is activated to take up storage protein in the presence of 20-hydroxyecdysone in vitro. The same experiment was performed using fat body prepared from larvae kept under dry conditions for 30hr. This fat body had been activated, and so it incorporated twice as much radioactivity as larval fat body in the absence of 20-hydroxyecdysone. No uptake of storage protein was detected when activated fat body was incubated at 0:C, indicating that this process is temperature dependent. The dose-response to 20-hydroxyecdysone of uptake of storage protein by larval fat body in vitro is shown in Fig. 7. Uptake of storage protein reached maximum when 5 x 10 -7 M of 20-hydroxyecdysone was added to the reaction mixture and no significant uptake was detected when the concentration of the hormone was below 1 0 - S M or above 10 -5 M. The optimum concentration of 20-hydroxyecdysone for uptake of storage protein roughly coincided with the physiological concentration of this hormone necessary for inducing metamorphosis of this fly reported by OHTAK1 et al. (1968), which is 6.9 x 1 0 - 7 M. Clearly, fat body prepared from larvae kept under dry conditions showed significant incorporation of
190
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20- Hy droxyecdysone (M) Fig. 7. Dose-response to 20-hydroxyecdysone of the uptake of haemolymph protein in vitro. Incorporation was measured under the conditions described in the legend to Fig. 7, with incubation for 120min in the presence of increasing amounts of 20-hydroxyecdysone. (©), fat body from larvae kept under dry conditions for 30 hr: (@), fat body from third instar larvae. storage protein even in the absence of 20-hydroxyecdysone and the hormone had no significant effect when it was added to the medium at lower concentration. However, when the concentration of the hormone was above 10 -6 M, the uptake of storage protein decreased sharply, indicating that the hormone was inhibitory at higher concentration. DISCUSSION The storage protein of insects found in larval haemolymph was shown to be used as a source of amino acids and energy for synthesis of new protein during metamorphosis (THOMSON, 1975). However, many questions remained to be answered about the metabolism of this protein. We are interested in the phenomenon that storage protein once secreted into haemolymph is taken up again by the fat body on pupation. The storage proteins that have been studied most extensively are calliphorin and storage protein of silkworm (ToJo et al., 1978). However, the relation between 20-hydroxyecdysone and the selective uptake of these proteins by fat body has not yet been clarified. In this paper we provided clear evidence that 20-hydroxyecdysone is essential for incorporation of storage protein by the fat body. Sarcophaga peregrina larvae are convenient for use in studies on the effect of 20-hydroxyecdysone on the uptake of storage protein by the fat body, since their secretion of the hormone can be controlled easily. In these larvae, uptake of storage protein was detected only under conditions when the ring gland secreted 20-hydroxyecdysone. The requirement for 20-hydroxyecdysone was confirmed in ritro by incubating storage protein with fat body in the presence of 20-hydroxyecdysone. This is the first demonstration that 20-hydroxyecdysone directly activates the fat body to incorporate storage protein. The uptake of protein by the fat body is not
random, but seems to be selective because storage protein was incorporated to a much greater extent than a lectin that is also a haemolymph protein induced on pupation or when the body wall of the larva is injured. This difference in the incorporation of protein may be simply due to a difference in the structures of the proteins. However, it is possible that incorporation of storage protein by fat body cells occurs through specific receptors on their surface like the incorporation of low density lipoprotein by fibroblasts in higher organisms (GOLDSTEIN and BROWN, 1977). These receptors may be masked in the larval stage and they may be activated by 20-hydroxyecdysone on pupation. Although 20-hydroxyecdysone activated the larval fat body to incorporate storage protein in vitro, its incorporation activity is about half that of fat body prepared from larvae kept under dry conditions. Thus another factor or factors besides 20-hydroxyecdysone may be necessary for full activation of the fat body. It is noteworthy that too high a concentration of the hormone inhibits incorporation of storage protein. Probably 20-hydroxyecdysone has pleiotropic effects on fat body cells and the balance of biological activities of the cells is disordered by excess hormone. Comparative studies on the biochemical characters of cytoplasmic membranes prepared from active and inactive fat body cells should provide useful information about the mechanism of selective uptake of haemolymph proteins. It is known that some lysosomal enzymes are selectively taken up by fibroblasts from the serum, although the mechanism of this uptake is unknown (NEUFELD et al., 1975). Selective uptake of storage protein by fat body may be a good model to use in studies on the general mechanism of protein uptake by eukaryotic cells. We observed the storage protein was incorporated into large granules that appeared in fat body cells on pupariation. During metamorphosis, these granules are thought to carry storage protein to developing adult tissues where it is used as a source of amino acids. Granule formation may also be controlled by 20-hydroxyecdysone. since no large granules are found in larval fat body. Apparent heterogeneity of the granules was observed in terms of incorporation of storage protein. Probably many kinds of granules with different functions are formed at this stage.
REFERENCES BUTTERWORTHF. M., TYSELL B. and WACLAWSK11. (1972) The effect of 20-hydroxyecdysone and protein on granule formation in the in vitro cultured fat body of Drosophila. d. Insect. Physiol. 18, 1885 1889. FA1RBANKSG., STECKT. L. and WALLACHD. L. H. (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10, 2606-2617. GOLDSTEINJ. L. and BROWN M. S. (1977) The low-density lipoproteins pathway and its relation to atherosclerosis. Ann. Rev. Biochem. 46, 897-930. KINNEAR J. F., MARTIN M. D. and THOMSONJ. A. (1971) Developmental changes in the late larva of Callipora stygia. Aust. J. Biol. Sci. 24, 275 289. KINNEARJ. F. and THOMSONJ. A. (1975) Nature. origin and fate of major haemolymph proteins in Calliphora. In.sect Biochem. 5, 531 552.
Storage protein uptake by fat body KOMANO H., M1ZUNO D. and NATORI S. (1980) Purification of lectin induced in the haemolymph of Sarcophaga peregrina larvae on injury. J. Biol. Chem. 255, 2919-2924. LAEMMLI U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. MARTIN M. D., KINNEAR J. F. and THOMSON J. A. (1971) Developmental changes in the late larva of Calliphora stygia. IV. Uptake of plasma protein by the fat body. AuNt. J. Biol. Sci. 24, 291 299. MIYACHI Y., VAITUKAITISJ. L., NIESHLAG E. and LIPSETT M. B. 0972) Enzymatic radioiodination of gonadotropins. J. clin. Endocr. Metab. 34, 23-28. MUNN E. A., FEINSTEIN A. and GREVILLE G. D. (1971) The isolation and properties of the proteins calliphorin. Biochem. J. 124, 367-374. MUNN E. A. and GREVILLE G. D. (1969) The soluble proteins of developing Calliphora erythrocephala, particularly calliphorin, and similar proteins in other insects. J. Insect Physiol. 15, 1935 1950. MUNN E. A., PRICE G. M. and GREVILLE G. D. (1969) The synthesis in citro of the protein calliphorin by fat body
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from the larva of the blowfly, Calliphora erythrocephala. J. Insect Physiol. 15, 1601-1605. NEUFELD E. F., LIM T. W. and SHAPIRO L. T. (1975) Inherited disorders of lysosomal metabolism. Ann. Rev. Biochem. 44, 357-376. OHTAKI T. (1966) On the delayed pupation of the fleshfly, Sarcophaga peregrina Robineau-Desvoidy. Japan J. Med. Sci. Biol. 19, 97-104. OHTAKI T., MILKMAN R. D. and WILLIAMS C. M. (1968) Dynamics of ecdysone secretion and action in the fleshfly Sarcophaga peregrina. Biol. Bull. Woods Hole 135, 322-334. THOMASON W. A. and MITCHELL H. K. 0972) Hormonal control of protein granule accumulation in fat bodies of Drosophila melanoftaster larvae. J. Insect Physiol. 18, 1885-1889. THOMSON J. A. (1975) Major patterns of gene activity during development in holometabolous insects. Ade. Insect. Physiol. II, 321-398. ToJo S., BETCHAKU T., ZICCARDI V. J. and WYATT G. R. (1978) Fat body protein granules and storage proteins in silkmoth, Hyalophora cecropia. J. Cell. Biol. 78, 823-838.