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Transfer of phospholipids from fat body to lipophorin in Rhodnius prolixus.

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Archives of Insect Biochemistry and Physiology 19:133-144 (1992)
Transfer of Phospholipids From Fat Body to
lipophorin in Rhodnius prolixus
Georgia C o d a Atella, Katia Calp Gondim, and Hatisaburo Masuda
Departamento de Bioqufmica Mkdica, lnstituto de Citncias Biomiddicas, Universidade Federal do
Rio de Janeiro, RJ, Brasil
32P-Labeledfat bodies (32P-fatbodies) of Rhodnius prolixus females were incubated in the presence of nonradioactive purified lipophorin and the release
of radioactivity to the medium was analysed to answer the question of whether
lipophorin i s a reusable shuttle for phospholipids. The radioactivity found in
the medium was associated with lipophorin phospholipids. When the 32P-fat
bodies were incubated in the absence of lipophorin, only a small amount of
radioactivity was released and it was not associated with lipophorin, indicating that there was no release of preLlabeled 32P-lipophorinby the tissue. Analysis of the 32P-phospholipidstransferred from fat bodies to the lipophorin
particles by thin-layer chromatography revealed a predominance of phosphatidylethanolamine and phosphatidylcholine, with minor amounts of phosphatidylserine, phosphatidylinositol, and sphingomyelin. The transfer of
phospholipids to lipophorin was linear with time up to 45 min and the process was inhibited at low temperature and by the metabolic inhibitors azide
and fluoride. The transfer of phospholipids from the fat bodies to lipophorin
was saturable with respect to the concentration of lipophorin, which was
half-maximalat about 8 mg/ml. A directional movement of phospholipids from
the fat body to lipophorin was observed. The net gain of phospholipids in 2 h
of incubation with fat body was 8.54 nmol per insect, which corresponds to
6.69% of increase in the lipophorin phospholipid content. The rate of 32Pphospholipid transfer from fat body to lipophorin particles varied during the
days after a blood meal increasing up to day 10 and then decreasing in parallel with the process of oogenesis.
Key words: lipid carrying protein, lipid, lipoprotein
Acknowledgments: We wish to express our gratitude to Dr. Martha M. Sorenson for a critical
reading of the manuscript; to Rosane O.M.M. Costa, Jose de S. Lima, Junior and Jose F. de
Sousa Net0 for excellent technical assistance. This work was supported by grants from Conselho
Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Financiadora de Estudos e
Projetos (Finep), Fundaqao de Amparo 21 Pesquisa do Rio de Janeiro (Faperif and Conselho de
Ensino para Graduados (CEPC) of the Universidade Federal do Rio de Janeiro.
Received July18,1991 ; accepted November 4,1991.
Address reprint requests to Katia Calp Condim, Departamentode Bioquimica Medica, lnstituto
de [email protected], Universidade Federal do Rio de Janiero, RJ, Brasil, 21910.
0 1992 Wiley-Liss, Inc.
134
Atella et al.
INTRODUCTION
In insects lipids are transported by lipophorin, a major hemolymph lipoprotein which contains a large amount of lipids [l-31. Diacylglycerols, cholesterol, hydrocarbons, and phospholipids are the major lipids associated with
lipophorin [4-71. Lipophorin takes up diacylglycerols from the fat body and
transports them to flight muscles and oocytes [5,8-lo]. Lipophorin also transports hydrocarbons from the oenocytes, where they are synthesized, to the
cuticle and takes up cholesterol from the digestive tract [6,11].
The transport of diacylglycerols is the most studied lipid transport system
in insects. In insect species that use lipids to obtain energy for sustained flight,
diacylglycerolsare loaded onto high density lipophorin particles at the fat body.
As a consequence, the ratio of lipids to protein increases and as lipophorin
becomes less dense, it is termed low density lipophorin [12J.These changes
are mediated by adipokinetichormone [9,13-151. The diacylglycerols from LDLp*
are transferred to the flight muscles to be used as fuel for flight [16,17]. After
unloading of the diacylglycerolsat the muscles the lipophorin particles become
denser. This HDLp can be reloaded with more diacylglycerols at the fat body
and recycled [18].
Other physiological situations, such as oogenesis, also require large amounts
of lipids. The developing oocytes accumulate lipids in the form of droplets;
Wiemerslage [19] and Chino et al. [20] proposed that diacylglycerols are transported by lipophorin to the oocytes. This was confirmed by Kawooya and Law
[lo] in Manducu sextu. Gondim et al. [21] showed that during oogenesis, in
Rhodnius prolixus phospholipids can be transferred from lipophorin particles
to the growing oocytes and that the lipophorin apoproteins are not accumulated in the oocytes, suggesting that lipophorin may function as a shuttle for
phospholipids as well as for diacylglycerols. The question of whether phospholipids are transportable has been debated but is still not clear. Although
Thomas and Gilbert [22] have shown the release of phospholipids from fat
body to the hemolymph in Hyulopkaru cecropia, Katagiri and Chino [23] could
not confirm their results. On the basis of lipophorin structural studies, Katagiri
[24] proposed that phospholipids are components of the lipophorin particle
but are not transferred to tissues. To answer the question of whether Iipophorin
particles can be considered a reusable shuttle for phospholipids, it is necessary to show that the particles can be reloaded with more phospholipids. In
this report we demonstrate that lipophorin particles can be loaded with phospholipids at the fat body, thus supporting the concept that lipophorin can in
fact be considered a reusable shuttle for phospholipids.
MATERIALS AND METHODS
Insects
Insects were taken from a colony of Rkodnius prolixus maintained at 28°C
and 70-80% relative humidity. The experimental insects were adult, mated
females fed on rabbit blood at 2-week intervals.
*Abbreviations used: HDLp = high density lipophorin; LDLp = low density lipophorin; LTP =
lipid transfer particle; PBS = phosphate-buffered saline; SDS = sodium dodecyl sulfate.
Transfer of Phospholipids in Rhodnius
135
32PiPurification
Carrier-free 32Pipurchased from Comissao Nacional de Energia Nuclear (Siio
Paulo, Brasil) was purified by extraction of the phosphomolybdate complex [25].
Lipophorin Purification
Two to 5 days after a blood meal, hemolymph was collected in the presence
of phenylthiourea (30-130 Fg/pl), 5 mM EDTA, and a mixture of protease inhibitors prepared in 0.15 M NaC1, with final concentrations of 0.05 mg/ml soybean trypsin inhibitor, leupeptin, and antipain, and 1mM benzamidine. The
collected hemolymph was centrifuged at room temperature for 5 min at 13,0009
and lipophorin was purified from the supernatant as described previously [26].
The supernatant was diluted to 5 ml with 5 mM EDTA and PBS (10 mM phosphate, 0.15 M NaCI, pH 7.4) and 1.25 g KBr was added. This material was
centrifuged at 159,0009 in a Beckman (Santa Clara, CA)Type 50 rotor at 4°C for
20 h and lipophorin was collected from the top of the KBr gradient. The purified
lipophorin was extensively dialysed against PBS and 5 mM EDIA and then against
PBS, and was stored under liquid nitrogen until use. The degree of purification
was monitored by SDS-PAGE and the protein concentration was estimated according to Lowry et al. [27], using bovine serum albumin as standard.
Polyacrylamide Gel Electrophoresis
Polyacrylamide slab gels were run both under denaturing conditions (with
SDS, [28]) and under nondenaturing conditions [29]. For radioactive samples,
the gels were stained, photographed, dried, and autoradiographed.
Preparation of 32P-LabeledFat Body (32P-Fat Body)
Adult females were fed on blood enriched with 32Pi (lo9 c p d m l of blood)
[26], using a special feeder described by Garcia et al. [30]. Two days after a
blood meal, the insects were carefully dissected and the 32P-fatbody was left
untouched and associated with the abdominal cuticle.
Transfer of Radioactivity From 32P-Fat Bodies to Lipophorin
The 32P-fatbodies were extensively washed in an excess of Rhodnius Ringer
[31J and then in 0.15 M NaC1, to remove contaminating radioactive hemolymph.
Twenty microliters of culture medium (no. 199, Sigma, St. Louis, MO) containing
nonradioactive purified lipophorin was added to the washed 32P-fatbodies. Unless otherwise stated, the incubationswere performed at 28°C. At the desired time,
culture medium was taken (10 PI), diluted to 100 pl with 0.15 M NaCl, and centrifuged at 13,OOOg for 10 min. The supernatants were separately applied to Sephadex G-50 centrifuge columns [32] previously equilibrated with PBS to separate the
proteins from the small phosphorylated molecules. The eluted material was
analysed by PAGE and autoradiography or by scintillation counting. Controls
were done by incubating 32P-fatbodies in culture medium without lipophorin.
In the experiments performed in the presence of metabolic inhibitors the
32P-fatbodies were preineubated for 1 h at 28°C in the culture medium containing the inhibitors but in the absence of lipophorin. After the preincubation, nonradioactive lipophorin was added and the incubation was continued
for 30 min. The samples were treated as described above.
136
Atella et al.
Determination of the Amount of Phospholipids Transferred From Fat Body
to Lipophorin
Nonradioactive fat bodies prepared in the same way described for radioactive fat bodies were incubated for 2 h in the presence of 17.0 mg/ml of nonradioactive lipophorin. After incubation of 8 fat bodies, the culture media were
pooled and centrifuged for 10 min at 13,OOOg and the lipophorin was repurified from supernatant in a KBr ultracentrifugation gradient as described
before. After dialysis against PBS, the repurified lipophorin was subjected to
lipid extraction [33] as described elsewhere [26], and the amount of phospholipids was determined by measuring the phosphate content according to a
modified procedure of Bartlett [34] as described by Kates [35].
Phospholipid Analysis
After incubation of 32P-fatbodies with purified lipophorin, the culture media
were pooled and centrifuged for 10 min at 13,OOOg and the supernatant was
applied to a KBr gradient as described above for the repurification of lipophorin
(now radioactive). The purified 32P-lipophorinwas separated from KBr by dialysis and was then subjected to lipid extraction [26]. The radioactivity of the
lipid moiety was determined separately by scintillation counting.
The 32P-phospholipidsextracted from the 32P-lipophorinwere analysed by
two-dimensional thin-layer chromatography, as described by Yavin and Zutra
[36] with slight modifications [26]. The plates were stained with iodine and
autoradiographed.
RESULTS
When 32P-fatbodies were incubated in culture medium in the presence of
nonradioactive purified lipophorin, a significant amount of radioactivity was
secreted to the medium, where it was associated with lipophorin (Fig. 1, lane
4). Without addition of lipophorin no radioactivity appeared in the medium,
showing that there was no release of 32P-lipophorinby the 32P-fatbody during the incubation period (Fig. 1, lane 2). To be sure that the 32P-phospholipids
were transferred to lipophorin from the fat body and not from the cuticle,
lipophorin was incubated in the abdominal cuticle after removal of the 32P-fat
bod No transfer of radioactivity to lipophorin was observed (Fig. 1, lane 3).
No 1;P-vitellogenin was detected in the medium under all conditions used,
probably because of the short incubation period.
To ascertain whether the radioactivity transferred from the 32P-fatbodies to
nonradioactive lipophorin was associated with phospholipids, the 32P-lipophorin obtained after incubation was purified and subjected to lipid extraction, as described in Materials and Methods. All the radioactivity was found
in the lipid fraction. The phospholipids were analysed by thin-layer chromatography followed by autoradiography (Fig. 2); phosphatidylethanolamineand
phosphatidylcholinewere the major phospholipids found. Phosphatidylserine,
phosphatidylinositol, and sphingomyelin were also observed.
The time-course of the transfer of "P-phospholipids from 32P-fatbodies to
lipophorin is shown in Figure 3. After a rapid initial phase, the phospholipids
were transferred to lipophorin at a constant rate for at least 45 min. In the
Transfer of Phospholipidsin Rhodnius
137
Fig. 1. PACE analysis under nondenaturing conditions (5-10% polyacryiamide gradient) of the
culture medium obtained after incubation of 32P-fatbodies with nonradioactive lipophorin. A:
Coomassie blue stained gel. B: Autoradiography of the gel. lane 1. Hemolymph from a female
2 days after feeding 32Piin a blood meal (control to show the positions of (LP) lipophorin and
(VG) vitellogenin). lane 2. Culture medium from incubation of 32P-fatbody without purified
lipophorin. Lane 3. Culture medium containing nonradioactive lipophorin incubated in abdominal cuticle after removal of 32P-fatbody. lane 4. Culture medium from incubation of 32P-fat
body with nonradioactive lipophorin. The incubations were for 15 min at 28°C and the lipophorin
concentration was 12 mg/ml. Other experimental conditions were as indicated in Materials
and Methods.
absence of lipophorin (lower curve, Fig. 3), a negligible amount of radioactivity appeared in the medium. The transfer of phospholipids to lipophorin was
abolished at 0°C and reduced in the presence of metabolic inhibitors such as
sodium azide or sodium fluoride (Fig. 4).
The rate of 3ZP-phospholipidtransfer to lipophorin increased with increasing concentrations of lipophorin in the medium, with a tendency to saturation (Fig. 5). To show whether the plateau observed at the highest lipophorin
concentrations was due to a real saturation and not due to depletion of
32P-phospholipidsof fat bodies, a control was performed by incubating 32P-fat
bodies at the highest lipophorin concentration (40 mg/ml) for a longer period
of time. The transfer of phospholipids was twice that observed after 15 min of
incubation indicating that the plateau in Figure 5 reflects saturation and not
depletion. It seems that the transfer of phospholipids from fat body is, in fact,
mediated by a receptor for lipophorin. The concentration of lipophorin
138
Atella et al.
Fig. 2. Autoradiography of a two-dimensional thin-layer chromatogram of phospholipids
extracted from 32P-lipophorinobtained by incubation of nonradioactive lipophorin with 32P-fat
bodies. Nonradioactive lipophorin (6 mg/ml) incubated for 60 min with 32P-fatbodies was repurified and the phospholipids were extracted. Phosphatidylethanolarnine (PE); phosphatidylcholine (PC); phosphatidylserine (PS); sphingomyelin (SM);phosphatidylinositol (PI).The first
and the second dimensions are indicated in the figure. The experimental conditions were as
described in Materials and Methods.
required to produce the half maximal rate of phospholipid transfer was about
8 mg/ml.
The amounts of phospholipids associated with lipophorin before and after
the incubation with fat bodies were determined by their phosphate contents
and compared (Table 1) to show that the incorporation of radioactivity by
lipophorin was due to a net gain of phospholipids and not to an exchange.
The net gain of phospholipids was 8.54 nmolhnsect in 2 h incubation, which
corresponds to an increase of 6.69%in the lipophorin phospholipid content.
Analysis of the rate of 32P-phospholipidsecretion by 32P-fatbodies to lipophorin showed that during the days following the blood meal the rate increased
up to day 10 and then decreased (Fig. 6).
Transfer of Phospholipids in Rhodnius
10
0
20
30
139
40
TIME (min)
Fig. 3. Time-course of 32P-phospholipidtransfer from 32P-fatbodies to lipophorin in vitro.
"P-fat bodies were incubated without (0) or with ( 0 ) purified lipophorin (5 r n g h l ) and at different times an aliquot (10PI) of the medium was taken, the32P-lipophorinwas separated from
the small molecules and the amount of radioactivity associated with lipophorin was estimated
by scintillation counting. Other experimental conditions were as described in Materials and
Methods. The vertical bars represent the SE for 4 determinations.
A
C
D
E
Fig. 4. Effects of metabolic inhibitors and low temperature on 32P-phospholipidtransfer from
'*P-fat bodies to lipophorin. 32P-fatbodies were incubated for30 min at28"C in a culture medium
containing 8 mg/ml nonradioactivelipophorin and no added inhibitor (A); lipophorin plus 10 rnM
NaN3(6);lipophorin plus 10 mM NaF (C);no lipophorin (D);
or lipophorin with no added inhibitor
but at 0°C (E). After the incubation the radioactivity transferred to lipophorin was estimated as
described in Materials and Methods. The vertical bars represent the SE for 4 determinations.
140
Atella et al.
0
10
20
30
40
LIPOPHORIN CONCENTRATION (mg/ml)
Fig. 5. Effect of lipophorin concentration on the transfer of 32P-phospholipids.32P-Fatbodies
were incubated with different lipophorinconcentrations for 15 min ( 0 )or30 min (A).The radioactivity transferred to lipophorin was determined by scintillation counting, as described in Materials and Methods. The vertical bars represent the SE for 4 determinations.
DISCUSSION
We have shown previously that lipophorin transfers phospholipids to growing oocytes and that the lipophorin apoproteins are not accumulated by the
ovary [21]. Here we present evidence to support the concept that lipophorin
is a reusable shuttle for phospholipids by showing that lipophorin can be loaded
with phospholipids (by showing that lipophorin can be loaded with phospholipids) at the fat body. Prelabeled 32P-fatbodies incubated with nonradioactive
purified lipophorin released radioactivity to the lipoprotein (Fig. 1).The presence of two lipophorin bands was described earlier [26], but their biological
significance is still unknown. The radioactivity bound to lipophorin particles
TABLE 1. Transfer of Phospholipids From Fat Body to Lipophorint
Control incubated without fat body
After incubation with fat body
Phospholipids transferred
Phospholipid content
in lipophorin particles
(nmol)
Percentage
128.0 f 3.2*
136.5 k 3.3"
8.5 k 1.2
100
106.7
6.7
tThe amounts of phospholipids associated with lipophorin particles were measured as described
in Materials and Methods. The values represent the content of phospholipids associated with
340 kg of lipophorin protein, which was the total amount of lipophorin used to challenge the fat
body of one insect. The incubation period was 2 h. Data are means SE for 4 determinations.
"The difference between the 2 groups is significant for P < .05.
Transfer of Phospholipids in Rhodnius
141
days after feeding
Fig. 6. Rate of phospholipid transfer from 32P-fatbody to lipophorin during the days after a
blood meal. Insects were fed on day zero with blood enriched with 32Pi, At different days following the meal the "P-fat bodies were isolated in the abdominal cuticle and incubated with
3.5 mg/rnl nonradioactive lipophorin for 30 min at 28°C. Data are corrected for 32P decay. The
vertical bars represent the SE for 4 determinations.
was associated solely with the phospholipid moieties (Fig. 2). As it has been
observed for diacylglycerols,hydrocarbons, and cholesterol [5,6,9,11],lipophorin
can be loaded with phospholipids (Table 1). This observation brings about
the conclusion that phospholipids are not only part of the lipoprotein vehicle
itself, but they are also a transportable lipid, confirming the initial results of
Thomas and Gilbert [22], who showed the release of phospholipids from fat
bodies incubated in the presence of hemolymph in Hyalophora cecropia. It is
not clear why Katagiri and Chino [23] did not observe the transfer of phospholipids to lipophorin in vitro in Philosamia cynthia or Hyalophora cecropia.
The transfer of 32P-phospholipidsto lipophotin was linear with time (Fig.
3). The control, in the absence of lipophorin in the medium, showed that even
after 45 min of incubation the fat bodies did not release a significant amount
of radioactivity. This is a good indication that the tissues were still healthy
under our experimental conditions because no leakage was observed.
The process of phospholipid loading of lipophorin at the fat body was inhibited by low temperature and by metabolic inhibitors (Fig. 4).Thus, the loading process is dependent on active metabolism as it is for diacylglycerols and
hydrocarbons [5,11].
The transfer of diacylglycerols from lipophorin to the flight muscles in Locusta
migratoriu [37] and to the fat body in Munduca sextn [38] is mediated by
lipophorin receptors found in these tissues, and the transfer of phospholipids
from the fat body to lipophorin of Rhodnius is also a saturable process (Fig. 5).
These data suggest that receptors are necessary for lipophorin to load and
unload different lipids. The lipophorin titer in Rhodnius female hemolymph
was shown to be about 40 mg/ml[21] and the lipophorin concentration required
142
Atella et al.
to produce half-maximal transfer of 32P-phospholipidfrom 32P-fatbodies was
estimated to be about 8 mg/ml (Fig. 5). Considering the observed constant
and the high level of circulating lipophorin concentration in the hemolymph,
it can be concluded that the loading system at the fat body works at maximal
velocity.
The fat body capacity for loading phospholipids onto lipophorin particles
increases up to day 10 after a blood meal (Fig. 6 ) coinciding with the period
when most of the eggs are produced (unpublished observation) and then
decreases in parallel with the process of oogenesis. The variable loading rates
in the days following a blood meal cannot be attributed to changes in the radioactivity associated with fat body phospholipids which remained constant
throughout the experiment (data not shown).
In Manduca sextu and Locustu migrutoriu a very high density lipoprotein in
the hemolymph mediates the transfer of lipids between lipophorin of different densities in vitro. This LTP [39,40] has been shown to be necessary for the
transfer of diacylglycerols from the fat body to lipophorin in Manducu [41].
Some LTP seems to be associated with the fat body, even after washing it. In
this work the transfer of phospholipids to lipophorin was obtained in the presence of purified lipophorin without addition of LTP, but the existence of LTP
bound to the fat body is possible.
Probably, the site of synthesis of lipophorin in Xhodnius prolixus is the fat
body, as it is in Munducu sextu larvae (421 and in Locusfa rnigratoria [43]. In our
experimental conditions we did not observe lipophorin secretion by the 32P-fat
bodies possibly because of the very short incubation periods employed and
the low turnover rate of lipophorin apoproteins, which has been measured in
Locustu migrutoriu [44].
Differing from Katagiri's [24] conclusion that phospholipids are only structural components of the carrier system, this study demonstrates that they are
also a transportable lipid. These data taken together with the demonstration
that phospholipids are in fact transferred from lipophorin to the oocytes [21]
support strongly the idea that lipophorin acts as a reusable shuttle for phospholipids.
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