Juvenile hormone regulation of phospholipid synthesis in the endoplasmic reticulum of vitellogenic fat body cells from Leucophaea maderae

Insect Biochem. Vol. 14, No. 1, pp. 27 36, 1984 Printed in Great Britain. All rights reserved

0020-1790/84 $3.00 + 0.00 Copyright © 1984 Pergamon Press Ltd

JUVENILE HORMONE REGULATION OF PHOSPHOLIPID SYNTHESIS IN THE ENDOPLASMIC RETICULUM OF VITELLOGENIC FAT BODY CELLS FROM LEUCOPHAEA MADERAE GuY DELLA-CIOPPA and FRANZ ENGELMANN Department of Biology, University of California, Los Angeles, CA 90024, U.S.A.

(Received 6 May 1983)

Abstract--The endoplasmic reticulum (ER) from fat body ceils of Leucophaea rnaderae was isolated as a purified population of rough and smooth-surfaced microsomal vesicles which was highly enriched in NADH-cytochrome c reductase activity. Phosphatidylcholine (PC) was the major structural-phospholipid of the ER membrane and it accounted for 58)o of the total lipid phosphorus. Juvenile hormone (JH) treatment of allatectomized adult females caused a 5-fold increase in the rate of [~4C]cholineincorporation into microsomal PC, and this was temporally coupled to the induction of vitellogenin synthesis and secretion by the fat body. Turnover experiments suggested that the proliferation of ER membranes in response to JH was due to an increase in de novo PC synthesis rather than a decrease in its rate of catabolism. We also measured microsomal phospholipid synthesis by [32p]-o-phosphate incorporation and showed that endogenous JH secretion from the corpora allata supported an enhanced rate of ER biosynthesis in the fat body during vitellogenesis. We conclude that JH augments vitellogenin synthesis and secretion in the fat body by co-ordinately regulating the rate of ER membrane proliferation. Key Word Index: Juvenile hormone, ER phospholipid synthesis, fat body, vitellogenesis, Leucophaea rnaderae

INTRODUCTION

Vitellogenin is the major yolk-protein precursor synthesized by the fat body during periods of vitellogenic egg growth. In Leucophaea maderae, approx. 80% of the protein secreted by the fat body during the peak of vitellogenesis consists of newly-synthesized vitellogenin (Koeppe and Ofengand, 1976), and the fat body tissue from a single female can produce 5-6 mg of vitellogenin per day (Brookes, 1976). As with most exportable proteins characterized to date, vitellogenin is synthesized on polysomes bound to the rough endoplasmic reticulum (ER). Nascent vitellogenin molecules have been detected immunologically within isolated microsomal vesicles obtained from fat body homogenates (Engelmann and Barajas, 1975), and within the rough ER cisternae of intact fat body cells (Chen et al., 1976). During vitellogenesis the development of an extensive network of rough ER has been observed ultrastructurally in the fat body cells of Locusta migratoria (Minks, 1967; Lauverjat, 1977, 1980; Couble et al., 1979), Leptinotarsa decemlineata (De Loof and Lagasse, 1970), Gryllus bimaculatus (Favard-S~r~no, 1973), Calliphora erythrocephala (Thomsen and Thomsen, 1974), Nauphoeta cinerea (Wriest, 1975, 1978), Aedes aegypti (Behan and Hagedorn, 1978; Tadkowski and Jones, 1979), Macrotermes bellicosus, Macrotermes subhyalinus and Cubitermes Jungifaber (Han and Bordereau, 1982). A highly developed rough ER is characteristic of cells which synthesize large amounts of exportable protein (Palade, 1975).

In addition to the ultrastructural studies of whole fat body tissue, Engelmann and Barajas (1975) showed that microsome pellets harvested from the vitellogenic fat body of L. maderae are composed primarily of ribosome-studded vesicles. Microsome pellets from non-vitellogenic fat body, by comparison, contain mostly smooth-surfaced vesicles. Furthermore, fat body homogenates from vitellogenic females contain a population of microsomes which are denser than those found in non-vitellogenic fat body (Engelmann, 1974). The implication is that during vitellogenesis new rough ER is produced in the fat body either by de novo synthesis or by polysome attachment to pre-existing smooth membrane. The cytological development of the fat body is temporally coupled to vitellogenin gene expression, and both phenomena appear to be regulated by juvenile hormone (JH). Although ultrastructural studies of fat body cells show that JH is required for the development of an extensive network of rough ER (Lauverjat, 1977, 1980), no one has quantitatively measured the kinetics of new membrane synthesis, and there is no direct biochemical evidence showing that membrane synthesis and assembly is hormonally regulated. We previously reported that JH treatment increases the rate of phospholipid synthesis in both rough and smooth ER of adult female fat body of L. maderae (della-Cioppa and Engelmann, 1980). In the present paper, we document in detail the isolation and characterization of rough and smooth ER from vitellogenic fat body of L. maderae. We describe the 27

28

GuY DELLA-CIOPPA and FRANZ ENGELMANN

effects of exogenously-applied or endogenouslysecreted J H on the synthesis a n d t u r n o v e r o f the m a j o r structural-phospholipid, p h o s p h a t i d y l c h o l i n e (PC), o f the m e m b r a n e . MATERIALS AND METHODS

Animals

Stock colonies of L. maderae were maintained at 26°C and 75~o relative humidity on a 12 hr-I 2 hr light-dark cycle. Newly emerged adult cockroaches were collected from the colonies and housed in plastic cages (2-6 animals per cage) with Purina rat chow and water ad libitum. For synchronization of the first egg maturation cycle, 8-13-day old females were segregated within 24 hr of mating. Mating is indicated by the presence of a spermatophore in the bursa copulatrix. Surgical procedures

Allatectomy was performed on unfed adult females within 5 days of the moult. Operations were carried out under carbon dioxide anaesthesia. The paired corpora allata (CA) were exposed by cutting a slit into the dorsal neck membrane at the base of the head capsule. Both glands were then removed with watchmakers forceps, 1.5 mg of streptomycin sulphate (in 10 #1 saline) was applied to the incision, and the neck membrane was sealed with hot paraffin. The mortality rate was <10%. Tissue fractionation

Abdominal fat body was collected under physiological saline (188raM NaCI, 19mM KCI and l mM calcium chloride) and stored at - 2 0 ° C or used immediately. Approximately 200 mg of fat body could be obtained from a single animal. Fat body tissue radiolabelled in vivo was washed in five 2.0ml vol of ice-cold saline to remove exogenous label. The pooled fat body from 3 to 4 animals was homogenized in 4-6 ml of ice-cold 35 mM sucrose-TKM buffer (50mM Tris-HC1 [pH 7.6], 25mM potassium chloride, 35 mM potassium bicarbonate and 10 mM magnesium acetate) in a loose-fitting Potter-Elvehjem, or Dounce (radial clearance 64-140 :tm), all-glass homogenizer with a motordriven pestle (15 strokes, 76 rpm). After a 3 min centrifugation at 1000g at 4°C the infranatant was aspirated from between a solid white pellet (urate crystals, nuclei and cell debris) and an upper layer of lipid. This infranatant was then spun at 9000g for 10rain at 4°C (IEC-refrigerated centrifuge) to pellet the "mitochondrial" fraction. The post-mitochondrial supernatant (PMS) was centrifuged for 1 hr at 105,000g at 4°C (Beckman L2-65B, SW 50.1) to pellet the microsomes. For subfractionation of the microsomes into rough and smooth-surfaced populations, the 9000g PMS was layered over a 0.6M/1.08M/2.0 M sucrose-TKM step gradient. After centrifugation at 105,000g for 20hr at 4°C, the "'smooth membranes" (at the 0.6 M/1.08 M interface) and the "rough membranes" (at the 1.08/2.0 M sucrose interface) were aspirated, suspended in TKM buffer, and pelleted at 105,000g for 1 hr at 4°C. Alternatively, in some experiments the PMS was adjusted to 2.0 M sucrose and overlaid with 1.08 M and 0.6 M sucrose-TKM and spun at 90,000g for 24 hr at 4°C in an SW 27.1 rotor. Under these conditions, the "rough and smooth microsotnes" were floated up into the gradient where they banded as described above. Generation of smooth microsomes from the pre-existing rough population was apparently minimal under the isolation conditions employed since cycloheximide (200 #g/ml) or emetine (25 #g/ml), when added to the homogenization and gradient media, did not increase the yield of rough membranes.

Lipid extraction and thin-layer chromatography

The microsomal pellet from the fat body of 3 to 4 animals were suspended in 1.0 ml TKM buffer and precipitated with an equal volume of 10~/o (w/v) TCA (trichloroacetic acid). After two additional washings in TCA, the lipids were extracted in (2:1, v/v) chlorofrom-methanol according to Folch et al. (1957). Aliquots of the lipid-extract were taken for radioassay, phosphorus determination and thin-layer chromatography on silica gel-H (New England Nuclear), or pre-absorbent silica gel-GHL (Analtech), 5 x 20cm thinlayer plates. The silica gel was activated by heating at 100'C for 30 rain. Phospholipids were chromatographed with chloroformmethanol-water (70:30:4, by vol) as the developing solvent. Lipids were visualized with iodine vapour and identified by co-chromatography with authentic phosphatidylcholine and phosphatidylethanolamine (Sigma) or silicic acid purified egg-yolk phospholipids (Singleton et al., 1965). For radioassay and phosphorus determination, silicic acid from individual lipid areas was scraped into conical glass centrifuge tubes and extracted three times with 10 vol of methanol. Recovery of lipid phosphorus from the silica-gel was 100~', using this procedure. Immunological procedures Rabbit antiserum to haemolymph proteins from vitellogenic females of L. maderae was prepared as reported previously (Engelmann and Penney, 1966). Monospecific antibody to vitellogenin was obtained by absorption of the non sex-specific antibodies with male haemolymph (Engelmann, 1971). lmmunoglobins were isolated from the rabbit serum by precipitation at 40~ ammonium sulphate saturation (Harboe and Ingild, 1973). For immunoprecipitation of radiolabelled haemolymph proteins, the haemolymph was diluted 1 : 1 (v/v) with 0.4 M NaCI and incubated with excess antibody at 37'C for 1 hr and then at 4°C for 24 hr. Immunoprecipitates were pelleted by centrifugation at 1600g for 5 rain at 18-20'C. washed twice in 0.4 M NaCI, and then precipitated with 5~'~,(w/v) TCA. The TCA precipitates were pelleted and then redissolved in hot 1 M NaOH for radioassay. Biochemical assays

All of the enzyme assays were run at room temperature (18-20°C) and the reaction rates were optimized for protein and substrate concentration, pH, and linearity with respect to incubation time for each enzyme. Cytochrome c oxidase (EC 1.9.3.1) activity was assayed by following the decrease in absorbance of reduced cytochrome c at 550nm (Cooperstein and Lazarow, 1951). Cytochrome c (horse heart type III, Sigma) was fully reduced with 0.1 ml of 1.2 M sodium dithionite per 30 ml of substrate/buffer (60 # M cytochrome c in 100 mM phosphate buffer, pH 7.5). The incubation mixture contained 1.0 ml of the reduced substrate/buffer and 40-80 #g of protein. Readings were taken in a 1 cm cuvette at 30 sec intervals for up to 5 rain. Cytochrome e was then completely oxidized with potassium ferricyanide and the extinction coeffÉcient determined. NADH-cytochrome c reductase (EC 1.6.2.2) activity was measured by a modification of the procedure of Ernster et al. (1962). Cytochrome c was fully oxidized by adjusting the pH to 3.0 with HCI and then re-adjusting it to pH 7.5 with NaOH. The increase in absorbance of cytochrome c was followed at 550 nm in 100mM phosphate buffer (pH 7.5), 60 # M cytochrome c, 0.1 mM NADH and 0.33 mM KCN. For determination of NADPH-cytochrome c reductase (EC 1.6.2.4) activity, NADH was replaced with 0.1 mM NADP + and a NADPH generating system (glucose-6-phosphate [0.2/tmol/ml] and glucose-6 phosphate dehydrogenase [0.5 units/ml, Sigma]). Readings were taken every 30 sec for up to 5rain in l cm cuvettes containing 1.0ml of

29

Phospholipid synthesis in the ER substrate/buffer and 10080/~g of protein. Cytochrome c was then fully reduced with sodium dithionite and the extinction coefficient determined. Acid phosphatase (EC 3.1.3.2) activity was measured by following the production of p-nitrophenol from the substrate, p-nitrophenyl phosphate (ICN). The 1.0 ml reaction mixture contained 5 m M p-nitrophenyl phosphate, 0.1% Triton X-100, and 100 mM sodium acetate buffer (pH 5.0). The reaction was started with 50-150#g of protein and stopped after 15min by the addition of 5.0ml of 0.1 M NaOH. The 6.0 ml solution was read immediately against a time-zero blank at 420 nm using p-nitrophenol (ICN) as a standard. Microsomal RNA was solubilized from precipitated membranes in 5% (w/v) TCA (90°C, 15min) and quantitatively assayed either by the orcinol method (Schneider, 1957) or by direct absorbancy measurement at 254nm. Yeast RNA (Calbiochem) was used as a standard. Lipid phosphorus was determined by ascorbic acid reduction of o-phosphate as a phosphomolybdate complex (Chen et al., 1956). O-phosphate was liberated from the phospholipids by perchloric acid (70%) hydrolysis at 155°C for 2 hr. The absorbance was measured at 820 nm using potassium phosphate as a standard. Intracellular choline was quantitatively assayed according to Hayashi et al. (1962) with choline chloride used as a standard. Protein was measured according to Lowry et al. (1951) with bovine serum albumin (Calbiochem) used as a standard. Radioisotopes All radioisotopes were diluted in insect saline and injected in 5-20 #1 vol through the intersegrnental membranes between the abdominal tergites. Injections were made with a 50 or 100 #1 Hamilton syringe with insects under carbon dioxide anaesthesia. The following labelled compounds were used: [U14C]leucine (314mCi/mmol), [methylJ4C]choline chloride (49-58mCi/mmol), [l,2-14C]ethanolamine HC1 (95mCi/ mmol), [UJ4C]glycerol (20mCi/mmol), and [32P]-o-phosphate (carrier-free). All t4C-labelled compounds were purchased from New England Nuclear and 32p was obtained from ICN. The incorporation of 32p into microsomal phospholipids is expressed as a "relative specific radioactivity" to correct for differential uptake of labelled precursor into fat body cells of vitellogenic versus inactive animals. The specific radioactivity of the microsomal phospholipids from vitellogenic females was multiplied by the intracellular precursor-pool ratio, inactive 32P~/~mol P~: vitellogenic 32Pi/#mol Pi, to normalize the incorporation relative to the pool size of Pi of inactive females.

Radioactivity was measured in either a Nuclear Chicago or a Beckman LS 7000 liquid scintillation counter with ~4C counting efficiencies of 73 and 96%, respectively. The counting efficiency for 32p was nearly 100% in all cases. Samples were counted in glass scintillation vials with 10 ml of 0.4% (w/v) PPO and 0.005% (w/v) POPOP in toluene-methanol (70: 30, v/v). Juvenile hormone Juvenile hormone-III (Calbiochem) was dissolved in acetone and topically applied (100/~g JH-III in 2-4#1 acetone) to the intersegmental membranes of the abdomen. Controls received acetone only. RESULTS

The isolation o f f a t body E R D u r i n g fat b o d y h o m o g e n i z a t i o n the E R breaks up into small vesicles (microsomes) which can be sedim e n t e d at > 100,000g from the p o s t - m i t o c h o n d r i a l s u p e r n a t a n t . M i c r o s o m e pellets o b t a i n e d from fat b o d y h o m o g e n a t e s of L. rnaderae were previously s h o w n by electron microscopy to be c o m p o s e d alm o s t entirely o f r i b o s o m e - s t u d d e d a n d s m o o t h surfaced vesicles ( E n g e l m a n n a n d Barajas, 1975). In order to determine the degree of c o n t a m i n a t i o n of m i c r o s o m a l m e m b r a n e s with o t h e r organelles, the biochemical m a r k e r s for m i t o c h o n d r i a ( c y t o c h r o m e c oxidase), E R ( N A D H - c y t o c h r o m e c reductase) a n d lysosomes (acid p h o s p h a t a s e ) were measured. T a b l e 1 shows t h a t the 105,000g pellet (microsomes) contained a b o u t 43~o of the N A D H - c y t o c h r o m e c reductase activity a n d only small a m o u n t s o f the cytoc h r o m e c oxidase (2.5~o) a n d acid p h o s p h a t a s e (1.1~o) activity. A b o u t 5 3 ~ o f the E R sedimented at 9000 g together with the m i t o c h o n d r i a a n d intact lysosomes. Based on specific activity, N A D H - c y t o c h r o m e c reductase was enriched m o r e t h a n 7-fold (over the starting material) in the m i c r o s o m a l pellet, whereas c y t o c h r o m e c oxidase a n d acid p h o s p h a t a s e were reduced m o r e t h a n 5-fold in this fraction (Table 1). N A D P H - c y t o c h r o m e c reductase activity was also f o u n d in the m i c r o s o m e fraction [68.3 _ 11.3 n m o l / min-g fat body ( + S E M , n = 3 ) ] . In c o n t r a s t to N A D H - c y t o c h r o m e c reductase, the N A D P H - l i n k e d reductase lost m u c h of its activity within a few h o u r s of tissue h o m o g e n i z a t i o n , a n d the activity was lost

Table h Marker enzyme activity in fat body homogenates (a) Distribution among subcellular fractions NADH-cytochrome c Fraction Cytochrome c oxidase reductase Acid phosphatase 9000g pellet 876 + 32 97.1% 3280 + 94 52.9% 577 + 69 33.0% 105,000g pellet 22 + 4 2.4% 2660 + 210 42.9% 19 +_3 1.1°.o 105,000g supernatant 4 4- 0.6 0.4% 264 4- 25 4.2?/0 1150 _+240 65.9% (b) Specific activity

NADH-cytochrome c reductase Acid phosphatase Fraction Cytochrome c oxidase 1.0t 198 4- 10 1.0"I" 46.9 _+2.3 1.0"t Homogenate* 64.6 4- 5.2 1.09 213 _+12 1.08 46.2 _+4.5 0.98 9000g pellet 70.2 __.1.9 0.15 1470+230 7.44 8.8+2.9 0.19 105,000g pellet 9.8 ± 0.15 0.004 9+ 1 0.05 41.8 _ 8.2 0.89 105,000g supernatant 0.23 +_0.09 *1000g (3 min) "debris" supernatant. ?Relative specific activity. Enzyme activities are expressed as (a) nmol/min-g fat body and (b) nmol/min-mg protein. The values represent the mean + SE based on three determinations. Fat bodies from 4-5 vitellogenic females (0.8-1.1 g fresh wt) were used for each determination.

30

G u Y DELLA-CIOPPA a n d FRANZ ENGELMANN

entirely after storage of the microsomes at 0°C for 24 hr. The instability of this enzyme may explain, in part, its low activity in the microsome fraction ( < 3~/o of the NADH-linked enzyme activity). The equilibrium density of rough and smooth microsomes is sufficiently different to allow separation by centrifugation in sucrose density-gradients. Centrifugation of microsomes (105,000g, 20hr) in 0.6 M/1.08 M/2.0 M sucrose step-gradients yielded rough (high density) and smooth (low density) vesicles with a 14-fold difference in their RNA/phospholipid (pg/pg) ratios (rough=0.42, smooth = 0.03). We observed that the R N A content of the rough microsomal fraction was due to the presence of large quantities of 18s and 26s ribosomal RNA (data not shown). The protein/phospholipid (pg/pg) ratio, in addition, was 37~ higher in the rough than in the smooth microsomes (rough = 2.02, smooth = 1.47).

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Synthesis of ER phospholipids Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were the major membrane phospholipids; they made up approx. 58 and 34~o, respectively, of the total lipid phosphorus of the membrane. The PC/PE mass ratio based on eight replicate determinations was 1.8 + 0.06 ( + SEM). Incorporation of [~4C]glycerol into microsomes showed that about 23~ of the radioactivity in the lipid was non-polar and that PC and PE comprised most of the identifiable phospholipid (Table 2). The ratio of labelled PC/PE was 2.0 and thus similar to the observed mass ratio of 1.8. Also, the distribution of [3H]glycerol among the lipids of both rough and smooth microsomes (data not shown) was nearly identical to that observed in the total microsome fraction. In insects, PC is synthesized from dietary choline by way of cytidine-pathway intermediates (cf. Bridges, 1972). The alternate route for PC formation, i.e. stepwise N-methylation of PE, appears to be absent in almost all insect species studied. In the present context, it was determined to what extent PE methylation might contribute to PC synthesis in the fat body of L. maderae. Vitellogenic females were labelled with [~4C]ethanolamine in vivo for 4, 24 and 48 hr. As seen in Fig. 1, nearly all of the [~4C]ethanolamine was contained in PE at any time. A minor amount of radioactivity co-migrated with PC but it did not increase significantly with longer pulse duration. In another experiment, it was observed that [methyl-3H]methionine did not supply labelled methyl-groups to newly-synthesized microsomal PC. These results show that the PETable 2. Distribution of [U, 14C]glycerol in microsomal lipids

Phosphatidylcholine Phosphatidylethanolamine Other phosphatides Non-polar lipids

dpm 340+47 174 ± 32 38 +_ 5 163 + 18

Per cent of total 48% 24~o 5% 23~

Vitellogenic females were labelled in vivo with 2.0,uCi of [~4C]glycerol for 4 hr. Fat bodies from four animals (about 0.8 g fresh wt) were used for each determination (mean ± SE, n = 4).

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Fig. 1. Incorporation of [14C]ethanolamine into fat body microsomal PE and PC. Vitellogenic females were labelled in vivo with 2.0/~Ci of [14Clethanolaminefor 4, 24 and 48 hr. Fat body tissue from three animals was pooled for each labelling time. The phospholipids were isolated by thin-layer chromatography. methylation pathway was of only minor importance in microsomal PC formation. In many experiments, therefore, we measured the incorporation of [t4C]choline into microsomal phospholipids and used this as an index of the rate of ER biosynthesis.

Juvenile hormone stimulation of ER phospholipid and vitellogenin synthesis in the fat body The time-course of [~4C]choline incorporation into fat body was measured over a period of 5 days after a single JH dose (100/~g given on day zero); these females were pulse-labelled for 4 hr in vivo (Fig. 2). The maximum rate of choline incorporation into membrane phospholipid occurred between the third and fourth day after JH treatment. Of the three tissue-fractions analyzed, the microsomes (105,000g pellet) had the highest specific radioactivity. The incorporation of choline into microsomal phospholipid was stimulated 5-fold over the untreated (day zero) controls. Choline incorporation into the "mitochondrial" fraction (9000g pellet) was stimulated by JH in a similar fashion as that observed for the microsomes, presumably because some of the ER was pelleted with the mitochondria (see Discussion). Nuclei and cell debris (1000g pellet) were poorly labelled with [14C]choline. The [T4C]choline that was incorporated into the microsomes was specific for membrane phospholipid. All of the radioactivity in the 105,000g pellet was extractable with chloroform-methanol, and > 95'~ of the label co-migrated with PC on silica-gel thin-layer plates. Even with longer in vitro labelling times (up to 18 hr) the [14C]choline label was entirely within the chloroform-methanol extractable material (i.e. lipid). The available [~4C]choline within the fat body cells, as well as the endogenous unlabelled choline pool within the cytosol, was also determined (Fig. 2). JH

31

Phospholipid synthesis in the ER

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(e) Microsomes (o) 9 0 0 0 g pellet (Z~) IO00g .e pellet eJ \

The proliferation of ER membranes observed during vitellogenesis may be due to an enhanced rate of ER synthesis or a decreased rate of membrane degradation. To determine whether JH affects ER turnover in the fat body of normal vitellogenic females, microsomal PC was first steady-state labelled with [~4C]choline for three days. These vitellogenic females had well-developed ER that were being maintained by JH secretion from the corpora allata. The hormone was then withdrawn by removal of the glands, the label diluted with unlabelled choline, and the decay of membrane radioactivity followed in operated and sham operated controls (Fig. 4). Both decay curves are nearly superimposable thus indicating that JH had no major effect on the rate of membrane phospholipid turnover.

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Fig. 2. The effects of JH treatment on cytosolic choline pools and [14C]choline incorporation into fat body organelles. CA- females were treated on day zero with 100 #g of JH-III. The animals were then labelled with 2.0#Ci of [14C]cholinefor 4 hr at the times shown. Each point is based on pooled fat body samples from nine (days 0-2) or five (days 3-5) animals. Aliquots of the acid-soluble 105,000g supernatant of the fat body homogenates were taken for determination of cytosol [~4C]cholineand endogenous unlabelled choline.

The native vitellogenin molecule of L. maderae is a complex glyco-phospholipo-phosphoprotein (Engelmann and Friedel, 1974). Since vitellogenin accounts for 80~o of the pulse-labelled secretory protein contained within microsomal vesicles (unpublished), some of the [~4C]choline radioactivity in microsomal phospholipids may be due to intraluminal vitellogenin lipid. To test for this possibility, vitellogenic females were labelled with [~4C]choline in vivo for various durations, and vitellogenin was then isolated from the lumen of ruptured microsomal vesicles. Only a small amount of the total radioactivity (3~,~o) was found in labelled vitellogenin within the microsomes (Table 3); this indicates that vitellogenin was either poorly-labelled with [t4C]choline and/or that vitellogenin was retained only briefly in the ER after

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treatment had no effect on the choline content of the tissue or the rate of uptake of the injected [~4C]choline. The 5-fold increase in the rate of choline incorporation into microsomal phospholipid was, therefore, not due to an increase in the specific radioactivity of the available precursor in the cells of the hormone-treated animals. To determine the relationship between membrane production and exportable protein synthesis in the fat body, C A - females were treated with 100/~g of JH-III (as in Fig. 2) and labelled with 32p to measure the rate of vitellogenin synthesis (32p labels the phosphoserine residues of vitellogenin). Newlysynthesized 32p-labelled vitellogenin that was released into the haemolymph during a 4 hr pulse was then precipitated with anti-vitellogenin (Fig. 3). The timecourse of vitellogenin secretion was similar to that observed for ER stimulation; the rate of vitellogenin secretion by the fat body was maximal on the third day after hormone treatment. These results show that ER phospholipid synthesis in the fat body was stimulated co-ordinately with the induction and release of vitellogenin.

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Fig. 3. The effects of JH treatment on 32p-incorporation into haemolymph vitellogenin. CA- adult females (3-4 weeks old) were treated with 100 #g of JH-III on day zero and then labelled with 0.15 mCi of 32p for 3 hr on the days indicated. Haemolymph vitellogenin was obtained by antibody precipitation. Each point shows the mean _+SE for three animals.

32

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DELLA-CIOPPA a n d ERANZ ENGELMANN

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Fig. 4. Turnover of ER phospholipids in the presence or absence of JH. Vitellogenic females were steady-state labelled for 72hr with a single 1.0#Ci injection of [14C]choline, and then either allatectomized ( C A ) or sham operated (sham). Immediately after surgery, each animal was injected with 2.5 mg of unlabelled choline (except day zero controls) to dilute out the remaining labelled precursor (this represented a 900-fold dilution of the originally injected [14C]choline). The day zero value (mean + SE) is based on three separate experiments using pooled fat body from 4-5 animals. All of the other points are based on pooled fat bodies from 3-4 animals.

synthesis. After labelling the vitellogenin with 32p, only 1% of the counts of microsomal or haemolymph vitellogenin were extractable with c h l o r o f o r m methanol. F r o m this we conclude that the radiolabelled phospholipids obtained from the microsomes were derived primarily from the vesicle lipid-bilayer and not from the hormone-induced vitellogenin lipid from within the vesicle lumen. When JH-treated females were pulse-labelled for 4 h r in vivo with ['4C]acetate, > 9 8 % of the total haemolymph vitellogenin radioactivity was in the non-polar lipid fractions (data not shown). These lipids were tentatively identified by thin-layer chromatography as cholesterol ester and diacylglycerol. Vitello~enin also contained free cholesterol, triacylglycerol and a small a m o u n t of phospholipid; these lipids were either unlabelled, or only poorly labelled, with ['4C]acetate. Our findings thus show that the lipid moiety of vitellogenin consisted largely of non-polar lipid species.

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Fig. 5. Synthesis of rough and smooth ER in vitellogenic (stippled column) and inactive (white column) adult females. The vitellogenic females were 2-3 weeks old and had mated exactly 1 week prior to the experiment; inactive females (virgins) were 2 weeks old. The animals were labelled in vivo with 0.15mCi of 32p for 3hr. The values represent the mean +_ SE for three separate determinations based on pooled fat body samples from four animals.

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150

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Regulation of ER phospholipid synthesis by the corpora allata In L. maderae, the C A of the female become active shortly after mating and the animals then begin to produce mature eggs. To measure the effects of normal physiological titres of J H on the rate of E R Table 3. ['4C]choline incorporation into microsomal vitellogenin Microsomes Vitellogenin Labelling time (total) (hr) (dpm/mg microsomal protein) l 3090 0 6 10977 349 (3.2%) 12 13922 462 (3.3%) 18 31093 1247 (4.0%) Vitellogenic females were labelled in vivo with 2.0#Ci of [14C]choline, fat body microsomes were isolated and ruptured with 19/~Triton X-100, and labelled vitellogenin was obtained by antibody precipitation. Fat bodies from four animals (0.7-1.1 g fresh wt) were used for each labelling time.

¢.) l-J

I

5

5O

g

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I

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3

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Days p o s t o p e r a t i v e

Fig. 6. Effects of allatectomy on the incorporation of ['4C]leucine into haemolymph vitellogenin. Mid-vitellogenic females were labelled for 4 hr with 1.0 #Ci of [t4qleucine and haemolymph vitellogenin was then collected by antibody precipitation. The concentration of haemolymph vitellogenin was determined by rocket immunoelectrophoresis. Each point shows the mean + SE for the indicated number of animals.

33

Phospholipid synthesis in the ER production, the fat body from viteUogenic (7 days after mating) and control (virgin) females was labelled in vivo with 32p. The control animals had emerged two weeks prior to the experiment and showed as yet no evidence of CA activity, i.e. there was no visible egg growth or accessory sex gland activity. As seen in Fig. 5, vitellogenic females incorporated significantly more 32p into rough and smooth-microsomal phospholipids than did the inactive control animals. When the CA were removed from normal eggmaturing females, the rate of vitellogenin release by the fat body fell rapidly to nearly undetectable levels within 72hr (Fig. 6). To determine whether this reduction in vitellogenin secretion was correlated with a decline in the rate of ER biosynthesis in fat body, the synthesis of microsomal PC was measured over a 6-day period following allatectomy. The data shown in Fig. 7 indicate that 32p incorporation into microsomal PC dropped to about 50~o of the control value by day 3 after allatectomy and to 35~ by day 6. The rate of PC synthesis was not reduced in day-5 sham operated or day-6 unilaterally CA females. We also observed that the rate of synthesis of the second most abundant structural phospholipid of the membrane, PE, was reduced to 62~ of control values by day 6 following allatectomy (data not shown).

Quantities of ER obtained from the fat body As shown above, fat body cells synthesized both rough and smooth ER most rapidly under the influence of JH. Since JH had no measurable effect on the rate of turnover of ER phospholipids (see Fig. 4), newly-made membranes potentially accumulated within the fat body cells during vitellogenesis. Vitellogenic fat body yielded indeed more microsomal phospholipid, and more microsomal protein, than did non-vitellogenic fat body samples (Table 4). However, since the recovery of the ER (105,000g pellet) from fat body homogenates of vitellogenic females was < 50~o (based on NADH-cytochrome c reductase activity), these values most likely underrepresent the actual amount of ER that occurred in

vivo. Table 4 also shows the percentage of rough microsomes expressed in terms of the total microsomal phospholipid. These values may be taken as an index of the relative amount of rough ER that occurs in vivo, and thus show that vitellogenic fat body contained significantly more rough-microsomal membranes than did non-vitellogenic fat body.

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postoperative

Fig. 7. Effects of allatectomy on the incorporation of 32p into fat body microsomal PC. Mid-vitellogenicfemales were allatectomized (or sham operated) and then labelled with 0.15 mCi of 32p for 3 hr before sacrifice. Each point is based on pooled fat bodies from 3-5 animals. The bars on day-zero and day-3 values are standard errors based on three experiments. One group of sham operated animals (triangle) had one CA removed.

DISCUSSION The results presented here show that phospholipid synthesis in the fat body ER was substantially accelerated by JH. Since this conclusion is based primarily on the measurement of radioisotope incorporation into microsomal membranes, it is important to demonstrate that fat body microsomes are indeed derived from the ER. In this present study with L. maderae, the degree of purity of fat body microsomes is shown by the marker-enzyme distribution among subcellular fractions. Microsomal pellets (105,000g) were relatively free of mitochondrial and lysosomal membranes (these sedimented during the intial 9000g centrifugation). The specific activity of ER marker, NADH-cytochrome c reductase, in the microsome fraction was increased by more than 7-fold over that of the original homogenate. Based on the markerenzyme distribution shown here (Table 1), and the morphological criteria described previously (Engel-

Table 4. Microsomesobtained from fat body of adult females Total microsomes Rough microsomes /~g phospholipid g fat body 690 _+85(6)

mgprotein g fat body 4.04 _+0.54(9)

(~ total phospholipid) 54.8 + 2.6(13)

Vitellogenic: Non-vitellogenic: allatectomized 566 + 63(7) 2.93 + 0.34(4) 40.1 + 3.9(9) 72 hr post-emergence * * 26.9 _ 1.9(3) *Not determined. Each determinationwas made with fat bodies from 3-5 animals.The valuesshow the mean+ SE with the numberof determinationsin parentheses. The vitellogenicand allatectomizedfemaleswere 3-4 weeks old.

34

G u y DELLA-C1OPPAand FRANZ ENGELMANN

mann and Barajas, 1975), microsome pellets obtained by differential centrifugation appear to consist largely of ER-derived membranes. A large percentage of the ER ( > 5 0 ~ ) , however, was pelleted during preliminary centrifugation to 9000g (Table 1). In some earlier experiments we studied the low-speed sedimentation kinetics of the ER marker enzyme, NADH-cytochrome c reductase, and observed that much of the enzyme activity pelleted at <4000g along with the bulk of the cytochrome c oxidase (unpublished). We speculate that the loss of ER to the "mitochondrial" fraction may be due, in part, to large sheets of lamellar ER which do not vesiculate during fat body homogenization. A "rapidly-sedimenting ER" fraction with these characteristics has also been demonstrated in homogenates of rat liver (Shore and Tata, 1977). Regardless of the substantial loss of ER during low-speed centrifugation, those portions of the membrane that sediment in the microsome fraction are relatively pure, and are considered to be representative of the ER as a whole. During tissue homogenization the ER breaks up into rough and smooth-surfaced microsomal vesicles presumably in proportion to the amount of rough and smooth ER that occurs in vivo. The differences in the RNA/phospholipid ratio, protein/phospholipid ratio, and the ribosomal RNA content in high and low-density microsomes indicate that these vesicles were indeed derived from rough and smooth ER. However, given some of the inherent limitations to cell fractionation, a microsome fraction may contain a mixture of vesicles that are derived from membranes other than ER, e.g. Golgi and plasma membranes (Beaufay et al., 1981). These contaminants, although minor, may tend to augment primarily the smooth-microsomal subfractions. Moreover, since marker-enzyme heterogeneity and/or redistribution may complicate the interpretation of findings based on enzyme distribution among subcellular fractions (Meldolesi et al., 1978), the origin of smoothmicrosomal vesicles is particularly subject to some uncertainty. For the present purposes, however, the rough microsomes (in contrast to the smootl:) represent a homogeneous population of vesicles which was derived essentially from the rough ER. In this study, we measured phospholipid synthesis in the ER by [methyl, ~4C]choline incorporation. With labelling times from 1-18 hr, all of the radioactivity of the microsome fraction was extractable with chloroform-methanol, and > 95%oof the counts were found in the PC fraction separated by thin-layer chromatography. The labelled PC was derived almost entirely from phospholipids of the vesicle bilayer, and only 4~o or less of the [~4C]choline radioactivity came from intraluminal vitellogenin. Even though vitellogenin is a PC-containing lipoprotein that makes up 80~o of the newly-synthesized microsomal protein, in these experiments, [~4C]choline labelled only the membrane phospholipids (primarily PC) and not vitellogenin (Table 3). In addition, our results with 32p pulse-labelling of the fat body showed that newlysynthesized phospholipids obtained from crude microsomal pellets were localized primarily in structural components of the membrane and not within intraluminal vitellogenin.

In vertebrates, PC can by synthesized in the ER by stepwise N-methylation of PE (Bremer et al., 1960). This pathway accounts for about 20'~/0 of the total PC formed in rat liver (Sundler and ~kesson, 1975). Since PE is the second most abundant phospholipid in the ER membrane of L. maderae, it is thus important to establish to what extent the PE methylation pathway contributes to microsomal PC formation; our studies with [~4C]choline incorporation measured only PC formed by the cytidine pathway. When [~4C]ethanolamine was used to label fat body microsomal phospholipids, nearly all of the radioactivity was incorporated into PE (Fig. 2). There was no evidence for a time-dependent conversion of labelled-PE to PC (which would be indicative of an active methylation pathway; ~kesson, 1980). Furthermore, no [methyl, 3H]methionine radioactivity was incorporated into microsomal PC in z~ivo in vitellogenic fat body. These results indicate that stepwise N-methylation of PE by S-adenosyl methionine is not an important pathway for PC formation in the ER of L. maderae. These findings agree with data obtained from other species which show that insects in general do not synthesize choline from ethanolamine (Bridges, 1972). Ultrastructural studies suggest that the extensive rough ER development in insect fat body occurs only during vitellogenesis, i.e. only when JH is available (Minks, 1967; De Loof and Lagasse, 1970: FavardS6r~no, 1973; Wriest, 1975, 1978; Lauverjat, 1977, 1980; Couble et al., 1979). In allatectomized females of L. migratoria, for example, these developments do not occur, and the fat body resembles inactive previtellogenic tissue (Chen et al., 1976). Implantation of active CA (Lauverjat, 1977, 1980) or topical application of the JH-analog methoprene (ZR-515) (Chen et al., 1976; Couble et al., 1979) causes the development of a cyto-architecture typical of that normally seen in vitellogenic fat body. In L. maderae, the presumed changes in the cytoarchitecture of the fat body were paralleled by a stimulation of phospholipid synthesis in the ER~ When females received a single dose of JH-IiI, choline incorporation during a 4 hr pulse in vivo was stimulated up to 5-fold (Fig. 2). The stimulation of [~4C]choline incorporation shown here cannot be interpreted as differential uptake of the labelled precursor in cells of treated and untreated animals because the specific radioactivity of intracellular choline was nearly identical in control and treated animals (Fig. 2). Since we show that the release of newlysynthesized vitellogenin from the fat body (Fig. 3) was co-ordinately stimulated with the increase in the rate of ER phospholipid synthesis, we speculate that ER membrane proliferation may be an important rate-limiting factor for vitellogenin secretion into the haemolymph. In some recent experiments~ we analyzed the relationship between ER synthesis and vitellogenin secretion during primary and secondary induction with JH. Our results show that in secondary induction an earlier time-course for ER proliferation in the tat body was correlated with a substantially accelerated response for vitellogenin secretion and vitellogenic egg growth (unpublished). The proliferation of ER can theoretically occur by increasing synthesis or slowing catabolism of mere-

Phospholipid synthesis in the ER brane phospholipids. For example, the accumulation of smooth ER in phenobarbital-stimulated rat liver is due, in part, to a decreased rate of phospholipid turnover (Holtzman and Gillette, 1968). We are unaware, however, of any case where a hormone has been shown to decrease the rate of ER degradation, and our data for L. maderae showed no evidence for different rates of phospholipid catabolism in the presence or absence of JH (Fig. 4). Thus, JH exerts its primary effect by stimulating de novo phospholipid synthesis. We also studied the effects of endogenous JH secretion from the CA on the rate of ER synthesis. In normal adult females with active CA (seven days after mating), synthesis of rough and smooth-ER phospholipids in the fat body was significantly higher than in 14-day old virgin females (Fig. 5), and the rate of phospholipid synthesis in these animals was essentially the same as that observed in CA females treated with JH-III (100 #g, 72 hr) (della-Cioppa and Engeimann, 1980). These effects of JH on phospholipid synthesis in the ER are consistent with our observation that vitellogenic females contained quantitatively more ER per gram of fat body (based on microsome yield) than did non-vitellogenic animals. It is difficult, however, to quantitate the amount of ER that occurs in vivo based on microsome yield because harvesting of this organelle by high-speed centrifugation neglects those portions of the membrane which were lost during the preceding low-speed spins. We have not measured the distribution of ER marker enzymes in subcellular fractions from both vitellogenic and inactive fat body and, therefore, do not know whether the ER is recovered equally in both groups. A quantitative morphometric analysis of the tissue by electron microscopy remains the most accurate method for measuring the amount of membrane that occurs in vivo. There are no ultrastructural studies of fat body in L. maderae, and the morphological evidence for ER proliferation in fat body of other insect species during vitellogenesis has not been analyzed quantitatively. None the less, it seems clear that an enhanced rate of ER phospholipid synthesis in the fat body of L. maderae is correlated with CA activity, and that vitellogenic females contain more ER (particularly rough ER) than do nonvitellogenic animals. The maintenance of a well developed ER seems to be coupled to endogenous levels of hormone, since a decline in JH titres (by CA inactivation or allatectomy) is correlated with a loss of the large arrays of stacked rough-ER from the fat body ceils by in situ autophagy (Lauverjat, 1977; Wriest, 1978). We observed that vitellogenin synthesis was reduced in L. maderae by >95~o within 72hr after allatectomy (Fig. 6). While this reduction may be related primarily to a decline in vitellogenin m R N A transcription, we suspect that the concomitant decrease in the rate of ER phospholipid synthesis (Fig. 7) may cause an inactivation of the pathway for protein secretion from the cells. JH is, therefore, required for the maintainance of a high rate of ER phospholipid synthesis in the fat body, and this is correlated with the capacity of the tissue to synthesize and secrete large amounts of vitellogenin. The proliferation of ER may be essential for the IB 14/] ~C

35

production of vitellogenin during egg maturation. The pre-vitellogenic fat body transforms from an adipose-like storage organ into a highly active secretory tissue, and this transformation takes place in the absence of any known cell proliferation. While transcriptional control of vitellogenin m R N A production appears to be the primary mechanism whereby JH regulates vitellogenin synthesis in the fat body (cf. Engelmann, 1979), the mechanism underlying the co-ordinated stimulation of phospholipid synthesis and ER-membrane assembly is unknown. The findings described here for L. maderae may be analogous with the observations in barley aleurone where giberellic acid induces amylase secretion while coordinately stimulating cytidine-pathway enzyme synthesis in the ER (Johnson and Kende, 1971). Much of the work in this study has focused on the temporal relationship between phospholipid synthesis in the ER and viteUogenin production. The co-ordination of these two events has important consequences with respect to the efficiency of vitellogenin synthesis. In addition to potentially providing more binding sites for more vitellogenin polysomes, the newly-made ER membranes may also provide the enzymes required for post-translational modification of vitellogenin, in view of the extensive glycosylation, lipidation and phosphorylation that the molecule undergoes prior to secretion. The available evidence in L. maderae indicates that JH functions pleiotropically in the adult-female fat body to co-ordinate the complex series of events that constitute vitellogenesis (Engelmann, 1979). While vitellogenin-mRNA-induction in the fat body may be the single most important event leading to vitellogenin synthesis, it is becoming clear that JH regulates a host of other processes which eventually lead to the development of fully mature eggs. An increased rate of ER phospholipid synthesis, which is paralleled by a change in the fat body ultrastructure, may simply augment the rate of vitellogenin production, or it may be an absolute requirement for both vitellogenin synthesis and secretion. If one could selectively inhibit phospholipid synthesis in the fat body it might be possible to study vitellogenin production in the absence of increased ER proliferation. Our observation that fat body of last-instar females synthesizes only very small amounts of vitellogenin in responses to JH (unpublished), but does so in the absence of increased ER phospholipid synthesis, may provide some useful information for a further line of investigation. Some recent experiments indicate that the fat body of adult males, which normally does not synthesize vitellogenin, can also synthesize very small amounts of vitellogenin in response to high doses of JH. Interestingly, this response is coupled to an increased rate of phospholipid synthesis comparable to that observed in the adult female (unpublished). The investigation of these latter phenomena will undoubtedly shed more light on the normal course of events which occur during vitellogenesis in the adultfemale fat body. Acknowledgements--The work reported here was supported

in part by a grant (HD 15530) from the National Institutes of Health. We thank Dr E. C. Mundall for critically reading the manuscript.

36

GUY DELLA-CIOPPA and FRANZ ENGELMANN REFERENCES

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