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Esterification of cholesterol and cholestanol in the whole body tissues and frass of Heliothis zea.

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Archives of Insect Biochemistry and Physiology 7:237-247 (1988)
Esterification of Cholesterol and Cholestanol
in the Whole Body, Tissues, and Frass of
Heliothis zed
Arun Kuthiala and Karla S. Ritter
Department of Bioscience and Biotecknology, Drexel University, Philadelphia, Pennsylvania
The quantity of free and esterified sterols in the whole body, intestine,
hernolymph, fat body, and frass of 6th-instar larvae of H. zea, fed cholesterol
or cholestanol, was measured in order to determine if there was a difference
in the utilization of these two molecules. The principal sterol in the tissues
of the larvae was cholestanol or cholesterol, when they were fed diet
containing these two molecules, respectively; there was little, if any,
metabolism of dietary cholestanol t o cholesterol. There was little or no
difference in the amount of total sterol in the whole body, tissues, or frass of
larvae fed the two different diets, indicating that the absence of a A5-bond in
cholestanol does not prevent the uptake or distribution of this sterol to
various tissues. However, the relative percentage of steryl ester was
significantly higher in prepupae reared on a diet containing cholestanol
instead of cholesterol (6-7-, 4-,13-,4, and 2-fold increase, for the whole body,
intestine, hernolymph, fat body, and frass, respectively). The average
percentage of total sterol that was esterified in the tissues was greater in the
fat body (10.8 k 15.4 and 44.2 f 12.3%, respectively, for larvae fed cholesterol
and cholestanol) than in the hernolymph (0.5 f 0.1 and 6.3 f 0.8%) and
intestine (1.2 f 0.1 and 4.7 k 1.1%).The percentage of sterol that was
esterified in the frass of larvae was large (26.9 k 3.7 and 48.2 2 0.5%,
respectively, for larvae fed cholesterol and cholestanol). Therefore, the fact
that larvae of H. zea fed cholestanol, instead of cholesterol, contain this
saturated molecule as their principal tissue sterol and preferentially esterify
it may explain, at least in part, why their rate of growth on cholestanol is
slower than on cholesterol.
Key words: [4-'4C]cholestanol, [4-'4C]cholesterol, steryl ester, corn earworm
Acknowledgments: We thank Mrs. J.R. Landrey for her assistance in preparing the [414C]cholestanoland Dr. J.T. Billheimer for reading the manuscript. This study was supported
in part by National Science Foundation grant DCB-8502578.
Received January6,1988; accepted February 25,1988.
Dr. Kuthiala is now at the Department of Entomology, University of Missouri, Columbia, M O
65211.
Address reprint requests to Dr. Karla S. Ritter, Department of Bioscience and Biotechnology,
Drexel University, Philadelphia, PA 19104.
0 1988 Alan R. Liss, Inc.
238
Kuthiala and Ritter
INTRODUCTION
All of the members of Insecta that have been studied, including Heliothis
zeu, require exogenous sterols for their development and reproduction [l]. H.
zeu can utilize a variety of sterols as tissue sterols [2,3]; however, the rate of
growth of the larva varies with the degree of alkylation and unsaturation of
the molecule [4,5]. For example, when H. zeu is fed an artificial diet containing
cholestanol instead of cholesterol, the larva grows more slowly [5].
Cholesterol is the principal tissue sterol in larvae fed cholesterol, whereas
cholestanol is the principal one in larvae fed cholestanol [2]. This suggests
that although both cholesterol and cholestanol are absorbed and transported
to various sites for utilization (e.g., as components of membranes) andlor
storage (e.g., as steryl esters), cholestanol is used less efficiently. Interestingly, previous studies in this laboratory have shown that, in vitro, there is a
2- to 3-fold increase in the esterification of cholestanol, by ACAT*, in the
microsomes of the intestine, fat body, and ovary of H . zeu, when compared
with that of cholesterol [6]. If cholestanol is preferentially esterified in vivo
and stored, instead of being used for membrane synthesis, this fact might
help to explain the difference in the rate of growth of larvae on these two
sterols. The purpose of this study was to compare the quantities of free and
esterified sterols in the whole body, intestine, hemolymph, fat body, and
frass of 6th-instar larvae of H. zeu fed cholesterol versus cholestanol in order
to determine if there is a difference in the utilization of these two sterols in
the various tissues.
MATERIALS AND METHODS
H . zea
Larvae of H . zeu were reared, as described previously [4,5], on artificial
media containing cholesterol or cholestanol (20 mgllOO ml). The medium
containing cholesterol (J. T. Baker Chemical Co., Phillipsburg, NJ), recrystallized from ethanol, also included either [4-14C]cholesterol(Amersham, Arlington Heights, IL; 55 mCilmol), so that the final specific activity of the
cholesterol was 192 pcilmmol, or [7-3H(N)]cholesterol(New England Nuclear, Boston, MA; 37 Cilmmol), so that the final specific activity of the
cholesterol was 2.58 mCilmmo1. The medium containing cholestanol (Sigma
Chemical Co., St. Louis, MO) also included [4-14C]cholestanol, so that the
final specific activity of the cholestanol was 128 or 129 pCilmmol. The [414C]cholestanolwas prepared by the hydrogenation of [4-14C]cholesterol[7].
The cholesterol, cholestanol, and radiolabeled cholestanol were shown to be
297% pure by GLC, RP-HPLC, MS and 'H-NMR (these procedures have
been described previously 121). In RP-HPLC, the dietary cholestanol was
*Abbreviations: ac = k' for test sterollk' for cholesterol; ACAT = acyl Coenzyme A:cholesterol
acyl transferase; CLC = gas-liquid chromatography; 'H-NMR = proton nuclear magnetic
resonance spectroscopy; k' = V , - V a 0 ; MS = mass spectrometry; RP-HPLC = reversedphase high-performance liquid chromatography; TLC = thin-layer chromatography; Vo =
void volume; V, = retention volume of sterol.
Esterification of Sterols
239
suspended in isopropanol and 1-ml fractions were collected from a Zorbax
ODS (CIS) column (at 45°C with a mobile phase of 80% acetonitrile and 20%
isopropanol at 2 mllmin) and counted. The percentage of the radioactivity
associated with those fractions corresponding to cholesterol (a,of 1.00) and
cholestanol (acof 1.30) was 1-3% and 97-99%, respectively.
Some larvae were derived from a colony of H . zed that had been maintained on a diet containing pinto beans, wheat germ, and torula yeast [8] for
18 to 23 generations (population A). Other larvae were from another population, which had been maintained on a diet containing wheat germ and corn
oil [4] for 11 to 26 generations (population B). (Note: In both cases, the
amount of maternal sterol present in the larvae was probably quite small
because when neonates were fed diet that lacked sterol, they remained in
the first instar and did not molt until they were provided with exogenous
sterol [4].)
Collection of Prepupae, Tissues, and Frass
Whole prepupae of H . zeu (1day after they had ceased feeding) as well as
hemolymph, fat body, intestines, and frass were collected in order to determine their sterol content. Hemolymph was collected after puncturing an
abdominal proleg with a needle. Fat body was gently removed with forceps
from larvae (after they had been opened dorsally and the hemolymph had
been washed from the hemocoel with cold distilled water) and blotted dry
with filter paper. In some experiments, after the removal of the fat bodies,
the intestines were also isolated from the larvae. The lumen of each gut was
washed with distilled water, introduced via a syringe, and the tissue was
blotted dry with filter paper. Frass was collected from rearing vials and
lyophilized.
Quantitation of Sterol in Prepupae, Tissues, and Frass
Method 1. In some experiments, where the total quantity of radioactivity
in the sample was measured, the fresh tissues (less than 150 mg) from
individual prepupae were solubilized in 1 ml of Protosol@(New England
Nuclear, Boston, MA). Hemolymph was always added directly to Protosol@
in a tared vial, in order to prevent melanization of the blood, and then
weighed; the fat body and intestinal tissue were weighed and then incubated
in the Protosol@at 55°C until they were visibly digested. After 12-24 h at
ambient temperature, the alkaline solution was neutralized with 70 p1 of
acetic acid to prevent chemiluminescence, suspended in 20 ml of scintillation
cocktail (Ready SolveTM El', Beckman Instruments, Fullerton, CA), and
counted in an LS 7500 scintillation counter (Beckman Instruments). The total
amount of sterol per 100 mg of tissue was then determined after adjusting
for background, quench, and the amount of unlabeled sterol present.
Method 2. In other experiments, where the quantity of radioactivity in the
free sterol versus the esterified sterol fraction was measured, whole prepupae
as well as tissues and frass from several larvae were pooled and stored at
-20°C until the sterols and steryl esters were extracted with methanolchloroform with a modified Bligh-Dyer technique [9] or with acetone in a
240
Kuthiala and Ritter
Soxhlet apparatus. The intestines, hemolymph, and fat body were homogenized with a tissue grinder of 2-ml capacity; whole prepupae were homogenized in a Sowall@Omni-Mixer (Dupont Co., Newtown, CT). These samples
were extracted with methanol-chloroform (2:l) for 20 min at 60°C; the extraction was repeated twice with fresh solvent (methanol:chloroform:water,
2:l:O.S).Frass was extracted for 24 h with acetone in a Soxhlet apparatus.
The free sterols in the extracts were separated from the steryl esters
through TLC on polysilicic-acid-gel impregnated glass fiber sheets (Gelman
Sciences Inc., Ann Arbor, MI) with a mobile phase of benzene and ethyl
acetate (9:l) andlor hexane, diethyl ether, and acetic acid (90:lO:l).The spots
on the plates were visualized with phosphomolybdic acid and those areas
with Rf values corresponding to the standards of cholesterol and cholesteryl
oleate carefully noted. The different fractions of the sample were placed
directly in scintillation cocktail and counted. After the counts were corrected
for background and quench, the quantity of 4-desmethylsterol vs. steryl ester
in each sample was calculated.
Method 3. In some experiments, RP-HPLC was used to determine the type
and quantity of radioactive sterols in the saponified steryl ester andlor 4desmethylsterol regions (separated by preparatory TLC on Silica Gel G with
a mobile phase of benzene and ethyl acetate [9:1] and then eluted from the
silica with diethyl ether). Samples were saponified overnight at 60°C in 5%
KOH in 90% ETOH. In order to confirm that any radioactivity in the saponified steryl ester region actually was associated with sterol, this fraction was
rechromatographed on glass fiber TLC plates, as described earlier, and the
quantity of radioactivity in the free sterol region, vs. other regions, was
determined. For RP-HPLC, 20 p1 of sample in isopropanol was used and 1ml fractions were collected (from a Zorbax ODS [C,] column at 45°C with a
mobile phase of 80% acetonitrile and 20% isopropanol at 2 mllmin) and
counted.
Method 4. In one experiment, GLC was used to determine the type and
amount of free sterol vs. esterified sterol in the prepupae. In this case, the
free sterols and steryl esters in the methanol-chloroform extract were separated by preparatory TLC (on Silica Gel G, with a mobile phase of hexane,
diethyl ether, and acetic acid [90:10:1]); the areas corresponding to the 4desmethylsterol and steryl ester regions collected and extracted with diethyl
ether; and the steryl ester band saponified. The sterols were then characterized and quantitated by GLC with a QF-1 column, as described previously [2].
RESULTS
Prepupae
Larvae of H . zeu were fed diet containing cholesterol or cholestanol, and
the quantity of free and esterified sterol associated with the prepupae was
determined. There was no difference in the total amount of sterol between
cholesterol- and cholestanol-fed larvae (26 f 9 and 26 pgl100 mg wet weight,
respectively) (Table 1).In both populations A and B, the relative percentage
of steryl ester in the larvae fed cholestanol (7.3 and 61.7%, respectively) was
7.40
ND
-
+g
+h
-
82.0
(37,734)
37.7
(3,721)
86.9
(4,640)
92.9
(10,370)
90.3
(5,545)
N D ~
7.9
(3,638)
60.7
(5,996)
6.8
(364)
0.7
(75)
1.7
(103)
ND
10.1
(4,630)
1.6
(154)
6.3
(334)
6.5
(722)
8.0
(491)
ND
% of Total dpm'
(number of dpm)
in region corresponding to
4-DesmethylSteryl
sterols
esters
Other
aThe specific activity of [414C]cholesterol was 192 pCilmmol.
m e specific activity of [414C]cholestanol was 129 pCilmmol.
'25 pl from 1 ml of extract (for population A only).
dNot determined.
eDetermined by GLC (by method 4).
ff
Values represent the S.D.
Qhe specific activity of [7-3H]cholesterolwas 2.58 mCilmmo1.
hThe specific activity of [4-'4C]cholestanol was 128 pCilmmol.
(8)
ND
1.52
6.21
5
1.43
Prepupae
-
+
5
1.24
+
-
+
3
1.19
Prepupae
(A)
-
-
+
3
(g)
Number
of
larvae
Type of sterol
in diet
Choles- Cholesterola
tanolb
Sample
Weight
of
sample
ND
ND
31e
Avg: 26 f sf
26
16
32
Total sterol
(rg1100 mg
of Sample)
38.3
91.2
98.7 f 0.6
92.7
98.6
98.2
99.3
61.7
8.8
1.4
1.3 f 0.6
7.3
1.8
0.7
% of Total sterol
Free
Esterified
TABLE 1. Amount of Sterol and Steryl Ester (Determined by Method 2) in Larvae of Two Populations (A and B) of H . zea Fed Diet
Containing Cholesterol or Cholestanol
242
Kuthiala and Ritter
approximately 6- to 7-fold that of larvae fed cholesterol (1.3 f 0.6 and 8.8%,
respectively) (Table 1).
Cholesterol (a, of 1.00; RRT of 1.00) was the only sterol in the larvae fed
the diet containing cholesterol, when methods 3 and 4 were used to characterize the molecules. It is not known whether a small radioactive peak in the
RP-HPLC sterol region (i.e., 1.4% of the recovered dpm), with an a, of 0.69,
also corresponds to a sterol. In contrast, 95 to 99% of the sterol in larvae fed
the diet containing cholestanol was cholestanol (a, of 1.30; RRT of 1.08); the
remaining sterol eluted with cholesterol (a,of 1.00; RRT of 1.00).
Intestine
The average amount of total sterol in the intestines of the larvae of population A fed cholesterol was slightly less (52 & 4 and 29 f 7 pgll00 mg wet
weight for methods 1and 2, respectively) than in intestines from larvae fed
cholestanol (68 f 4 and 62 & 2 pg1100 mg for methods 1and 2, respectively)
(Fig. 1,Table 2). The average relative percentage of steryl ester in the intestine
of larvae fed cholestanol (4.7 f 1.1%)
was approximately 4 times that of the
steryl ester in the intestine from larvae fed cholesterol (1.2 f 0.1%) (Table 2).
Hemolymph
The average amount of total sterol in the hemolymph of larvae of population A fed cholesterol was similar (39 f 2 and 33 +_ 1pgll00 mg wet weight
for methods 1 and 2, respectively) to that of hemolymph from larvae fed
cholestanol (36 f 2 and 24 f 3 pgll00 mg for methods 1and 2, respectively)
(Fig. 1, Table 3). The average relative percentage of steryl ester in the hemolymph of larvae fed cholestanol(6.3 f O.80/,)was approximately 13 times that
of hemolymph from larvae fed cholesterol (0.5 i 0.1%) (Table 3).
Fat Body
The average amount of total sterol in the fat body of larvae of population
A fed cholesterol was similar (57 f 5 and 43 & 22 pg1100 mg wet weight for
methods 1and 2, respectively) to that of fat body from larvae fed cholestanol
(85 36 and 78 i 2 pgll00 mg for methods 1 and 2, respectively) (Fig. 1,
Table 4). The average relative percentage of steryl ester in the fat body of
larvae fed cholestanol (44.2 f 12.3%) was approximately 4 times that of fat
body from larvae fed cholesterol (10.8 & 15.4%) (Table 4). When method 3
was used to characterize the molecules, 94% of the sterol in larvae fed the
diet containing cholestanol was cholestanol (a, of 1.30); the remaining sterol
eluted with cholesterol (a, of 1.00).
Frass
There was slightly less total sterol in the frass of larvae of population A,
fed the diet containing cholesterol, than in the frass of larvae fed cholestanol
(32 + 2 versus 39 3 &lo0 mg dry weight, respectively, as determined by
method 2) (Table 5). The average relative percentage of steryl ester in the
frass of larvae fed cholestanol(48.2 i 0.5%) was approximately twice that of
Esterification of Sterols
Intestine
Hemolymph
243
Fat Body
T
I2Ol
100
Intestine
Hemolymph
Fat Body
Fig. 1. Quantity of sterol in various tissues from larvae of H. zea (population A) fed [4''C]cholesterol (A) or [414C]cholestanol (6).Total sterol (
(determined
I
) by measuring the
amount of radioactivity associated with the solubilized tissues through method 1). Free sterol
(0)
and steryl ester (M) (determined by measuring the amount' of radioactivity associated
with these fractions separated by TLC through method 2). (Bars represent S.D. of the mean
for the fat body and the actual range of data for the intestine and hernolymph.)
larvae fed cholesterol (26.9 & 3.7%) (Table 5). When the steryl esters from
the frass of larvae fed cholesterol or cholestanol were saponified and analyzed by TLC by method 3, 96.6% (1,602 dpm) and 90.3% (1,295 dpm),
respectively, of the radioactivity was associated with the 4-desmethylsterol
region; the remainder was associated with the other regions. Cholesterol (ac
of 1.00) was the only sterol present in the steryl esters of frass from larvae
fed the diet containing cholesterol, and cholestanol (acof 1.30) was the only
sterol present in the steryl esters of frass from larvae fed the diet containing
cholestanol, when method 3 was used to characterize the molecules.
244
Kuthiala and Ritter
TABLE 2. Amount of Sterol and Steryl Ester (Determined by Method 2) in the Intestine of Larvae of
H. zea (Population A) Fed Diet Containing Cholesterol or Cholestanol
Weight
of
intestine
(mg)
Type of sterol
in diet
Choles- Cholesterola
tanoIb
50
+
-
60
+
-
% of Total dpmC
(number of dpm)
in region corresponding to
4-Desmethyl- Steryl
sterols
esters Other
96.4
(3,975)
96.3
(1,454)
1.1
(49)
1.0
(15)
Total sterol
( p g l lmg
~~
of intestine)
2.4
(99)
2.7
(41)
% of Total sterol
Free
Esterified
36
98.8
1.2
22
98.9
1.1
Avg: 29 rt 7d 98.9 rt 0.1 1.2 f 0.1
25
48
+
-
88.8
(720)
92.1
(1,529)
+
-
5.4
(44)
3.4
(57)
5.8
(47)
4.5
60
94.2
5.8
64
96.4
3.6
95.3
1.1 4.7
(75)
Avg:
1.1
"The specific activity of [4-14C]cholesterol was 192 pciimmol.
bThe specific activity of [4-14C]cholestanolwas 129 pCi/mmol.
'In 50 or 100 p1 from 0.5 ml of extract.
df Values represent the actual range of the results.
TABLE 3. Amount of Sterol and Steryl Ester (Determined by Method 2) in the Hemolymph of Larvae
of H . zea (Population A) Fed Diet Containing Cholesterol or Cholestanol
Weight
of
Type of sterol
in diet
70of Total dpm'
(number of dpm)
in region corresponding to
Total sterol
hemolymph Choles- Choles- 4-Desmethyl- Steryl
(pg1100 mg
(mg)
terola tanolb
sterols
esters Other of hemolymph)
195
+
-
241
+
-
98.1
(6,827)
98.1
(8,973)
0.4
(26)
0.4
(41)
1.5
(103)
1.4
(132)
-
+
270
-
+
91.7
(2,692)
90.2
(2,703)
5.3
(157)
6.9
(207)
2.9
(86)
2.9
(86)
Avg:
Free
Esterified
32
99.6
0.4
34
99.5
0.5
Avg: 33 f Id
212
% of Total sterol
99.6 & 0.1 0.5 f 0.1
26
94.5
5.5
21
92.9
7.1
93.7 rt 0.8
"The specific activity of [4-14C]cholesterolwas 192 pcilmmol.
bThe specific activity of [4-14C]cholestanolwas 129 pCilmmo1.
'In 50 p1 from 0.5 ml of extract.
d + Values represent the actual range of the results.
DISCUSSION
The ability of insects to utilize cholestanol as a dietary sterol varies with
the species. Cholestanol will support growth as well as cholesterol does in
some insects, whereas in others it is used less effectively or not at all [lo].
-
-
+
+
-
-
-
85
95
61
69
72
46.7
(1,289)
61.2
(1,896)
45.6
(1,322)
97.2
(1,986)
96.5
(2,994)
69.1
(5,205)
43.7
(1,204)
26.7
(828)
53.3
(1,544)
1.3
(26)
2.5
(79)
27.7
(2,089)
% of Total dpm'
(number of dpm)
in region correspondingto
Steryl
4DesmethYlsterols
esters
'The specific activity of [4-14C]cholesterolwas 192 pcilrnmol.
%e specific activity of [4-14C]cholestanolwas 129 pcilmmol.
'In 50 pl from 0.5 ml of extract.
d + Values represent the S.D.
+
+
+
-
+
Type of sterol
in diet
CholesCholesterol'
tanolb
64
Weight
of
fat body
(mg)
9.6
(265)
12.1
(375)
1.1
(32)
1.6
(32)
0.9
(28)
3.1
(237)
Other
Avg:
55.8
78+2
69.6
51.7
89.1
46.1
* 22d
71.4
97.4
98.7
12.3
* 15.4
44.2 k 12.3
53.9
30.4
48.3
10.8 k 15.4
28.6
2.6
1.3
Esterified
YO of Total sterol
Free
78
77
80
Avg: 43
69
33
28
of sample)
(PLg/100 mg
Total sterol
TABLE 4. Amount of Sterol and Steryl Ester (Determined by Method 2) in the Fat Body of Larvae of H. zea (Population A) Fed Diet
Containing Cholesterol or Cholestanol
246
Kuthiala and Ritter
TABLE 5. Amount of Sterol and Steryl Ester (Determined by Method 2) in the Frass of Larvae of H.
zeu (PopulationA) Fed Diet Containing Cholesterol or Cholestanol
Ezght
of
frass
Type of sterol
in diet
(g)
Cholesterola
0.5
+
-
1.0
+
-
% of Total dpmC
(number of dpm)
in region corresponding to
Choles- 4-Desmethyl- Steryl
esters
tanolb
sterols
65.6
(3,602)
62.8
(5,743)
19.9
(1,090)
27.5
(2,516)
Total sterol
(llg1100 mg
Other
of frass)
14.6
(799)
9.7
(887)
34
76.8
23.2
30
69.5
30.5
Avg: 32
0.5
-
+
1.0
-
+
43.2
(1,171)
47.1
(2,845)
40.9
(1,110)
43.0
(2,598)
15.9
(432)
9.9
(596)
Avg:
% of Total sterol
Free
Esterified
2d
73.2
3.7
26.9 k 3.7
36
51.3
48.7
42
52.3
47.7
51.8 f 0.5
48.2 & 0.5
"The specific activity of [4-14C]cholesterolwas 192 pCilmmol.
bThe specific activity of [4-14C]cholestanolwas 129 pcilmmol.
'25 pl from 1ml of extract.
d + Values represent the actual range of the results.
Previous studies have shown that the larva of H. zed develops more slowly
when fed a diet containing cholestanol instead of cholesterol [5] and contains
cholestanol as its principal tissue sterol instead of cholesterol [2]. In the
present study, because there was little or no difference in the amount of total
cholesterol vs. cholestanol in the whole body, intestine, hemolymph, fat
body, and frass of prepupae fed the two different diets, the absence of a A5bond in cholestanol does not prevent the uptake or distribution of this sterol
to various tissues.
The RP-HPLC studies indicated that there was little, if any, metabolism of
dietary cholestanol to cholesterol. Although 1-6% of the radiolabeled tissue
sterols had an ac corresponding to cholesterol, 1-3% of the radiolabeled
dietary cholestanol also had an a, of 1.00. Because no other sterols were
detected in the dietary cholestanol, when it was analyzed by GLC, MS and
'H-NMR, it is not clear whether the small amount of tissue sterol that eluted
in the cholesterol region was a contaminant or a metabolite of the dietary
stanol.
The relative percentage of steryl ester in prepupae was significantly higher
when they were reared on a diet containing cholestanol instead of cholesterol
(4-, 13-, 4-, and 2-fold increases for the intestine, hemolymph, fat body, and
frass, respectively). Although the relative percentage of steryl ester in the
whole body of the larvae was greater in population B than in population A,
there was still a 6-7-fold increase in the relative percentage of cholestanol
that was esterified, as compared with that of cholesterol, in these larvae.
Therefore, cholesterol appeared to be used in the free form preferentially
(e.g., for membrane synthesis), whereas cholestanol appeared to be esterified
(e.g., for storage). The results of these in vivo studies are in agreement with
Esterification of Sterols
247
those of previous in vitro studies, which demonstrated that there was an
increase (2- to 3-fold) in the esterification of cholestanol by ACAT in microsomes of the intestine and fat body of H. zeu, as compared with cholesterol
PI
*
The average percentage of the total sterol that was esterified in the tissues
was greater in the fat body (10.8 k 15.4 and 44.2
12.3% for larvae fed
cholesterol and cholestanol, respectively) than in the hemolymph (0.5 0.1
and 6.3 k 0.8%) and intestine (1.2 :& 0.1 and 4.7
1.lY0). This finding
suggests that in the 6th-instar larva, sterols are transported in the hemolymph of H. zed primarily in the free form, as they are in other insects [ll],
and that the fat body is a major storage site for steryl esters. It is not clear,
however, why such a high percentage of the total sterol in the frass of the
prepupae was esterified (26.9 and 48.2% for larvae fed cholesterol and cholestanol, respectively).
Because the larva of H. zea fed cholestanol instead of cholesterol contains
this molecule as its principal tissue sterol and preferentially esterifies it, this
may explain, at least in part, why its rate of growth on a diet containing
cholestanol is slower than on a diet containing cholesterol. However, other
factors that also might affect the growth and development of the larva, such
the rate of utilization of cholestanol relative to cholesterol for the synthesis
of membranes and ecdysteroids, remain to be investigated.
LITERATURE CITED
1. Svoboda JA, Thompson MJ: Steroids. In: Comprehensive Insect Physiology Biochemistry
and Pharmacology. Kerkut GA, Gilbert LI, eds. Pergamon Press, New York, Vol. 10, pp
137-175 (1985).
2. Ritter KS: Metabolism of A'-, A5-, and A7-sterols by larvae of Heliothis zea. Arch Insect
Biochem Physiol 1, 281 (1984).
3. Ritter KS: Utilization of A5j7- and As-sterols by larvae of Heliothis zeu. Arch Insect Biochem
Physiol3, 349 (1986).
4. Ritter KS, Nes WR: The effects of cholesterol on the development of Heliothis zea. J Insect
PhysiolZ7, 175 (1981).
5. Ritter KS, Nes WR: The effects of the structure of sterols on the development of Heliothis
zeu. J Insect Physiol27, 419 (1981).
6. Macauley SK, Billheimer JT, Ritter KS: Sterol substrate specificity of acyl coenzyme A:
Cholesterol acyltransferase from the corn earworm, Heliothis zea. J Lipid Res 27, 64 (1986).
7. Nace HR:An improved hydrogenation of cholesterol to cholestanol. J Am Chem SOC73,
2379 (1951).
8. Burton RL: Mass rearing the corn earworm in the laboratory. US Department of Agriculture Presentation Paper ARS 33-134,8 pp (1969).
9. Bligh EG, Dyer WJ: A rapid method of total lipid extraction and purification. Can J
Biochem Physiol37, 911 (1959).
10. Kircher HW, Gray MA: Cholestanol-cholesterol utilization by axenic Drosophila melanogasteu. J Insect Physiol24, 555 (1978).
11. Chino H, Kitazawa K: Diacylglycerol-carrying lipoprotein of hemolymph of the locust and
some other insects. J Lipid Res 22, 1042 (1981).
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