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Metabolism of ╬Ф0- ╬Ф5- and ╬Ф7-Sterols by Larvae of Heliothis zea.

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Archives of Insect Biochemistry and Physiology 1:281-296 (1984)
Metabolism of A
'-, A5-,and A7-Sterols by
Larvae of Heliothis zea
Karla S . R i t t e r
Department of Biological Sciences, Drexel University, Philadelphia
Heliothis zea was reared on artificial diets containing A5-sterols (cholesterol,
campesterol, or sitosterol), A'-sterols (lathosterol, epifungisterol, or spinasterol), or A'-sterols (cholestanol, epicoprostanol, carnpestanol, or sitostanol)
in order to determine how different dietary sterols affect the type of sterols
present in the tissues of the late-sixth-instar larva. Although all of the dietary
sterols (except epicoprostanol) supported the growth of the larvae, not all of
the sterols were metabolized to the same end products. I n each case, at least
80% of the sterols in the tissues of the larvae retained the same nucleus as
that of the dietary sterol, indicating that H. zea carries out very little
metabolism of ring B of A5-, A7-, and A'-sterols. The larvae dealkylated the
A'-, A'-, and A'-alkylsterols to 24desalkylsterols, but a greater percentage of
the A5-alkylsterols were metabolized in this manner. The sterols present as
free sterols i n the larva were also present as esterifed sterols which accounted
for 2-4% of the total sterols. Therefore, the sterol composition of the tissues
of H. zea can be altered by varying the dietary sterols.
Key words: Heliothis zea, cholesterol, campesterol, sitosterol, lathosterol, epifungisterol,
spinasterol, cholestanol, epicoprostanol, campestanol, sitostanol
INTRODUCTION
Heliothis zea is similar to other insects in that exogenous sterols are essential
for its growth and maturation. Although cholesterol* is a satisfactory dietary
*The systematic names for the sterols used in this study are as follows: cholesterol, cholest5-en-3P-01; campesterol, 24a-methylcholest-5-en-3~-ol; sitosterol, 24a-ethylcholest-5-en-3P-ol;
lathosterol, 501-cholest-7-en-30-01; epifungisterol, 24a-methyl-5a-cholest-7-en-3~-01; spinasterol, 24a-ethyl-5a-cholesta-7,22-trans-dien-3~-ol; cholestanol, 5c~-cholestan-3/3-01; epicoprostanol, 5P-cholestan-3a-ol; campestanol, 24a-methyl-5a-cholestan-3P-ol; sitostanol, 24a-ethyl5a-cholestan-3P-ol.
Acknowledgments: I thank Mr Frederick A. Burton for his assistance in purifying the sitostanol and Mrs Josephine R. Landrey for performing the mass spectroscopy and obtaining
some of the 'H-NMR spectra on the 360-MHz instrument at the National Institutes of Healthsupported Middle Atlantic regional facility for 'H-NMR at the University of Pennsylvania. This
study was supported by NSF grant PCM-8206541.
Received February 27,1984; accepted April 9,1984.
Address reprint requests to Karla S. Ritter, Department of Biological Sciences, Drexel University, Philadelphia, PA 19104.
0 1984 Alan R. Liss, Inc.
282
Ritter
sterol for this lepidopteran [l], various A5-sterols, as well as A'- and A7sterols, can also support the development of the larva to the adult stage,
although the growth rate of the insect may be affected [2].
Since the degree of alkylation of the side chain of a dietary sterol, and the
position of double bonds in the molecule, can affect the growth of H . m,
this suggests that the structure of the sterol may affect the efficacy with
which it is utilized by this animal. For example, the structure of the molecule
may affect its rate of absorption andlor its ability to function as a precursor
for ecdysteroids and as a structural component of membranes.
It is known that the structural requirements of other insects for sterols
may vary depending upon the species [3,4]. For instance, cholesterol fulfills
the cellular requirements for sterol in many insects, when it is obtained
directly in the diet or derived from other sterols such as phytosterols; however, other species such as Drosophilu pachea [S], Epiluchnn vurivestis [6], and
A f f a cephalofes isthrnicoZu [7] may use unusual sterols as their principal tissue
sterols (ie, A7-, A'-, and A5!'-sterols, respectively); not only do Oncopeltus
fasciatus [8], Dysdercus fusciatus [9], Trogodema grunariurn [lo], and Apis mellifem [ll]dealkylate little if any C28and C29dietary sterol to 24-desalkylsterol,
but also the first two species have been shown to contain the C28 ecdysteroid,makisterone A [3,9].
In the case of H . zed, Svoboda et a1 demonstrated that this insect can
dealkylate the C-24 alkyl groups of sitosterol [12] and stigmasterol [13], and
we described cholesterol as the principal free and esterified sterol in this
insect (when it was reared on an undefined diet containing wheat germ and
corn oil in addition to cholesterol [14]);however, the metabolic products from
other dietary sterols in this organism have not been studied. For example,
the metabolic fates of A'- and A7-sterols, with or without alkyl groups at C24, are unknown. If H. zeu metabolizes different dietary sterols to different
end products andlor if it uses them unchanged in membranes, then the
resulting alterations in its sterol composition might have important physiological and ecological consequences. Not only might a change in sterol
composition affect the physiological characteristics of membranes (eg, fluidity), but also it might affect the development of certain parasites (eg, parasitoid insects) which depend on the sterols of their host to complete their own
development [15].
Therefore, the purpose of this investigation was to study the sterol composition of sixth-instar larvae of H. zed, which had been reared on diets
supplemented with various A5-, A7-, and A'-sterols, in order to compare and
contrast the metabolic fates of these different dietary sterols in this insect.
MATERIALS AND METHODS
H . zea
Larvae of H. zeu were reared individually on artificial diets, which contained no plant material but were supplemented with different sterols (15
mgllOO ml diet) as described previously [1,2].
Metabolism of Sterols by H. zed
283
Dietary Sterols
The dietary sterols used in these experiments were: cholesterol (J.T. Baker
Chemical Co, Phillipsburg, NJ); campesterol, sitosterol, and cholestanol (Applied Science Laboratories, Inc, State College, PA); campestanol, lathosterol,
epifungisterol (7-campesterol), and spinasterol (Research Plus Steroid Laboratories, Denville, NJ); epicoprostanol and sitostanol (stigmastanol) (Sigma
Chemical Company, St Louis, MO). Each sterol was recrystallized, as necessary, from ethanol and examined analytically by GLC,* RPLC, UV, MS and
'H-NMR and was shown to be at least 98% pure, with the exception of the
sitostanol. This latter sterol was found to be a mixture of sitostanol (65%),
campestanol (30%), and cholestanol (5%). Therefore, for some experiments,
the sitostanol was isolated from this mixture using a Perkin-Elmer Series 1
liquid chromatograph, equipped with a variable wavelength UV detector and
a Perkin-Elmer preparative CI8 column, at 30"C, a mobile phase of 100%
acetonitrile, and a flow rate of 10 mllmin. Fifty milligrams of the mixture was
injected at a time in 2 ml of isopropanol and 20-ml fractions were collected.
Because the mixture was composed of stanols, no peaks were observed at
205 nm using this procedure; however, when the acetonitrile was evaporated
and each fraction resuspended in cyclohexane and analyzed using a PerkinElmer Sigma 3B gas chromatograph, equipped with a 3% QF-1 column at
230-235"C, the sterol in each fraction was identified. Those fractions that
contained >98% sitostanol were pooled, dried under nitrogen, and used in
the diets.
Isolation of Sterols and Steryl Esters
Late-sixth-instar larvae were collected after they had consumed different
dietary sterols, and to ensure that no unabsorbed dietary sterols were extracted from the larvae, each larva was pinned open, after a dorsal incision
had carefully been made through its integument, and the fore-, mid-, and
hindgut gently removed. The cadavers, minus the intestines, were then
frozen at -20°C until they were extracted.
Each group of cadavers was homogenized in acetone using a Sorvall
Omnimixer and the sterols obtained by continuous extraction with acetone
in a Soxhlet extractor for 24 hr. In experiments to determine what percentage
of the tissue sterol was esterified, the sterols and steryl esters were separated
by TLC on Silica-Gel G using a solvent system of benzene-ethyl acetate (9:l
vlv), the bands corresponding to the 4-desmethylsterol region, 4,4-dimethylsterol region, and steryl ester region collected and the compounds eluted
with anhydrous ether. In all cases, the TLC plates were protected from light
and the recovery of sterols made as rapidly as possible to protect against
their oxidation. The steryl esters were then saponified overnight at 60°C in
*Abbreviations: acyl coenzyme A: cholesterol acyltransferase = ACAT; gas-liquid chrornatography = CLC; mass spectroscopy = MS; proton nuclear magnetic resonance spectroscopy
= 'H-NMR; retention time relative to cholesterol = RRT; reversed-phase liquid chrornatography = RPLC; tetrarnethylsilane = Si(CH& = TMS; thin-layer chromatography = TLC;
ultraviolet spectroscopy = UV.
284
Ritter
5% KOH in 90% ETOH. Otherwise, the acetone extracts were first saponified, the neutral lipids extracted with ether, and the sterols isolated using
TLC as above.
In one set of experiments, to test the purity of the dietary ingredients, all
of the dietary components (175.4 g), with the exception of the H20 and
sterols, were also extracted and processed as above to test for the presence
of contaminating sterols. In addition, one ingredient, the agar (Difco, Detroit,
MI) (305.6 g), was treated similarly by itself to check for trace amounts of
sterols.
Identification of Sterols
The sterols isolated from the larvae were characterized and quantitated by
GLC, RPLC, LJV, MS, andlor 'H-NMR. GLC was carried out at 225-235°C
using a 1%XE-60 column, 0.75% SE-30 column, andlor 3% QF-1 column;
rates of movement of compounds were expressed as retention times relative
to cholesterol. Analytical RPLC was carried out using a Perkin-Elmer Series
3B liquid chromatograph, equipped with a variable wavelength detector and
stop-flow capabilities, a Zorbax ODS (C,,) column at 45"C, a mobile phase
of acetonitrile-isopropanol (80:20 vlv) and a flow rate of 2 mllmin. The k' for
cholesterol ([v,-V,]/V,; V, = the retention volume of the sterol and V, =
the void volume) was used to calculate the at's (k' for test sterollk' for
cholesterol) of the peaks. Ultraviolet-absorption spectra were obtained by
scanning the individual peaks from RPLC between 190 and 300 nm. MS was
performed via direct probe on a model 4000 Finnigan instrument with electron impact ionization at 70 eV. 'H-NMR was performed at 360 MHz at
ambient temperature on a Bruker instrument, model WH360, in CDC13 with
Si(CH3)4as the usual internal standard. In these studies, only those peaks
that constituted >1-2% of the sample were considered to be significant (ie,
distinguishable from background).
All experiments were done in duplicate and when & values are reported
they denote the actual range of the results.
RESULTS
Purity of Dietary Ingredients
No 4-desmethyl- or 4,4dimethylsterols were detected in the corresponding bands from TLC of the acetone extracts of the dietary ingredients (minus
sterol and H20), or the agar, when they were examined by GLC, RPLC, and
MS analysis.
Metabolism of A5-Sterols
Cholesterol. The control larvae, which were fed cholesterol as their sole
dietary sterol, contained only one peak by GLC and this peak had an RRT
similar to that of cholesterol (Table 1).RPLC at 205 nm showed one major
peak which also had an ac similar to cholesterol (Table 2). One minor peak,
with an ac of 0.74, was also present in the usual sterol region (ie, a, >0.55
and <1.30). Although the position of this peak indicated that it could be a
1.30 f 0.03
* 0.01
1.30 f 0.04
1.00 f 0.01
Campesterol
Cholesterol
1.00
1.58 & 0.07
0.08
1.57
Sitosterol
Dietary sterol
RRT of dietary sterol
XE-60
QF-1
+
+
+
1.61 f 0.01
1.01 0.01
1.33 0.01
1.01 f 0.01
1.01 0.01
RRT
14 & 4
86 & 4
27 & 2
73 & 2
100
4+3
96 f 3
15 It 3
84 f 3
100
Percent of
esterified
sterol
QF-1
* 0.01
RRT
1.65
1.02
1.35
1.02
1.01
Sterols isolated from larvae
Percent of
free sterol
XE-60
TABLE 1. GLC Analvsis of A’-Sterols and Their Metabolites in H . zed Larvae
14
86
26
74
100
Percent of
free sterol
P
U
2
-.
ul
3
$
286
Ritter
TABLE 2. RPLC Analysis at 205 nm of A5-and A'-Sterols and Their Metabolites in
H. zeu Larvae
Dietary sterol
(Ye of
dietary
sterol
Sterols isolated from larvae
Percent of
(YC
free sterol
Sitosterol
1.24 5 0.04
Campesterol
1.12 f 0.03
Cholesterol
Spinasterol
1.00 0.01
1.09 f 0.03
Epifungisterol
1.12 f 0.01
1.26
1.01
1.11
1.01
1.00 0.01
1.09 f 0.02
1.01 f 0.02a
1.13
Lathosterol
1.04
1.03"
*
0.03
*
lola
11
89
26
74
100
39 f 6
60 f 6
68
32
100
"This was a broad peak which indicated that more than one peak was present.
sterol which was more polar than cholesterol (eg, a standard of 7-dehydrocholesterol had an ac of 0.77), the UV-absorption spectrum of this peak gave
a spectrum characteristic of end absorption (similar to the scan of the peak
with cq of loo), indicating an absence of conjugated double bonds. Since
GLC, MS (the M+ value was 386), and 'H-NMR (Table 3) indicated that
cholesterol was the only sterol present, this evidence suggested that the
minor peak might not be a sterol.
Campesterol. Larvae fed campesterol were capable of demethylating and
converting this sterol to cholesterol because about 73% of the sterol recovered
from the cadavers had an RRT similar to cholesterol by GLC analysis; the
RRT of the rest of the recovered sterol was similar to that of a standard
of campesterol (Table 1).These results were confirmed by RPLC analysis
(Table 2).
Sitosterol. Large quantities of dietary sitosterol were also metabolized by
the sixth instar. Approximately 86% of the sterol recovered from the larvae
appeared to be cholesterol by GLC and RPLC analysis; the rest of the
recovered sterol appeared to be unmetabolized dietary sitosterol (Tables 1,2).
Metabolism of A'-Sterols
Cholestanol. The larvae fed cholestanol contained this sterol as their principal, but not sole, tissue sterol. Although only one peak was observed when
the recovered sterols were analyzed by GLC on the XE-60 column, two peaks
were clearly resolved on the QF-1 column (Table 4). The major peak (about
84% of the sterol) had an RRT similar to a standard of cholestanol and the
rest of the sterol had an RRT similar to cholesterol (Tables 1,4).When 20 p g
of this sample was analyzed by analytical RPLC at 205 nm, only one peak
was observed. This peak had an ac similar to that of a 20-pg injection of
cholesterol (ie, 1-00) but an area of only about 10% of the standard. This
indicated that most of the sample represented stanols because saturated
sterols are not detected by UV-spectroscopy. 'H-NMR confirmed the presence of cholestanol as the principal tissue sterol and cholesterol as the minor
one (Table 3). Although the presence of cholesterol in the sample was not
t
d
d
d
S
S
Multiplicityb
-
-
0.68
1.01
0.91
0.86
0.87
A
-
0.68
1.01
0.91
0.86
0.87
B
Cholesterol
-
0.53
0.79
0.91
0.86
0.87
A
-
-
0.68
1.01
0.92
0.87
0.87
-
-
-
0.65
0.80
0.90
0.86
0.87
0.54
0.80
0.92
0.87
0.87
Lathosterol
B
Minor
Major
sterol
sterol
-
0.68
1.01
0.89d
0.86d
0.86d
-
-
0.65
0.80
0.89
0.86
0.86
Type of dietary sterol‘
Cholestanol
B
Minor
Major
A
sterol
sterol
-
0.54
0.80
0.92
0.78
0.86
0.81
Epifungisterol
A
aSi(CH3)4which was in CDC13.
b~ = Singlet, d = doublet (J = 6 Hz), t = triplet (J = 7 Hz).
‘“A” represents spectrum of dietary sterol and “B” represents spectrum of sterol@)isolated from larvae.
dHidden under corresponding peaks in other sterol.
C-28
C-29
C-18
C-19
c-21
C-26, 27
Chemical
shift in
ppm (from
TMF) of
‘H at:
TABLE 3. ‘H-NMRSpectra of Dietary Sterols and the Sterols Isolated from H . zea Larvae
0.81
-
0.56
0.80
1.03
0.80
0.85
A
-
0.80
0.55
0.79
1.02
0.81
0.84
-
-
0.53
0.80
0.91
0.86
0.86
Spinasterol
B
Minor
Major
sterol
sterol
4
-.
0
:
3
1.00
1.28
1.56
1.07 2 0.02"
1.39 k 0.02b
1.66 f 0.02'
"5%of sample.
b30%of sample.
'65% of sample.
+
1.00
1.28
1.55
Cholestanol,
+ campestanol,
sitostanol
1.00 f 0.01
1.08 2 0.03
* 0.01
1.00
Cholestanol
1.30 2 0.02
1.00 f 0.01
1.39 2 0.02
1.30 f 0.02
1.57
1.02
Campestanol
* 0.02
RRT
1.56 f 0.02
1.65
RRT of dietary sterol
XE-60
QF-1
Sitostanol
Dietary
sterol
TABLE 4. GLC Analysis of A'-Sterols and Their Metabolites in H. zea L w a e
40
22
38
100
53 f 3
46 3
63
37
-
-
-
44
57
-
-
Percent of
esterified
sterol
**
RRT
1.65
1.09
1.00
1.38 f 0.02
1.09 0.02
1.01 0.01
1.08 0.02
1.00 f 0.01
1.00
1.09
1.38
1.65
Sterols isolated from larvae
Percent of
free sterol
XE-60
QF-1
65
34
1
51 k 4
42 f 5
7+2
8421
16 f 1
<1
38
21
41
Percent of
free sterol
Metabolism of Sterols by H. zed
289
detected by MS, the M+ at 388, and the similarity of the fragmentation
pattern to that of a standard of cholestanol, confirmed that cholestanol was
the major sterol recovered.
Campestanol. The presence of a saturated ring B in dietary campestanol
did not prevent the demethylation of this sterol; however, less of this dieta
sterol was dealkylated by the sixth instar than was the corresponding A sterol, campesterol. (The MS s ectrum of the dietary campestanol revealed
that its M+ value was 402; the H-NMR spectrum of this stanol is shown in
Table 5.) By GLC analysis, approximately one half of the dietary campestanol
was recovered unmetabolized by the larvae (Table 4) whereas only about one
quarter of the dietary campesterol was unmetabolized (Table 1).That portion
of dietary campestanol which was metabolized was converted to 24-desmethylsterol. The principal 24-desmethylsterol recovered had an RRT similar to
cholestanol and was clearly resolved, on the QF-1 column, from another
minor peak; this latter peak had the same RRT as cholesterol and represented
only 7% of the total sterol (Table 4). No peak that corresponded to campesterol was detected. The results from RPLC, after analyzing the sterols recovered from larvae fed campestanol at 205 nm, were similar to those obtained
from larvae fed cholestanol-that is, only a small peak with ac similar to
cholesterol was observed, indicating that the rest of the sterol in the sample
consisted of stanols. These RPLC studies substantiated the evidence that the
principal sterols in the larvae fed campestanol consisted of stanols.
Sitostanol. Dietary sitostanol was partially dealkylated to 24-desalkylsterol
just as campestanol had been. (The MS spectrum of the dietary sitostanol
revealed that its M+ value was 416; the 'H-NMR spectrum of this stanol is
shown in Table 5.) By GLC analysis, 34% of the recovered sterol was
cholestanol and about 1%was cholesterol; the rest of the sterol in the tissues
of the larvae was identified as sitostanol by its RRT on the QF-1 column
(Table 4). Only a small peak for cholesterol was observed by RPLC analysis
at 205 nm, indicating that, again, most of the sterol in the larval tissues was
stanol.
Mixture of sitostanol, campestanol, and cholestanol. This dietary sterol,
shown by GLC to be a mixture of 65% sitostanol, 30% campestanol, and 5%
r
P
TABLE 5. 'H-NMR Spectra of the Dietary Ao-Sterols
Chemical
shift in ppm
(from TMSa
bf 1~ at)
C-18
C-19
c-21
C-26,27
C-28
C-29
MultipIicityb
S
S
d
d
d
t
Dietary A'-sterol
Cholestanol
Campestanol
Sitostanol
0.65
0.80
0.90
0.86
0.87
0.65
0.80
0.90
0.77
0.85
0.80
0.65
0.79
0.90
0.81
0.83
-
0.84
-
aSi(CH3)4which was in CDC13.
s = Singlet, d = doublet (J= 6 Hz), t = triplet (J
=
7 Hz).
-
290
Ritter
cholestanol (Table 4), was fed to the larvae to determine if any of these sterols
were preferentially utilized by H. zeu, since all three of these sterols had been
shown to be absorbed and partially metabolized by the larvae (see above).
GLC analysis indicated that about 41% of the sterol recovered from the larvae
fed this mixture was sitostanol and about 21% of the sterol recovered was
campestanol (Table 4); therefore, assuming no difference in the rates of
absorption, about 62% of the sitostanol fraction and 73% of the campestanol
fraction remained unmetabolized. Once again, cholestanol was the principal
24-desalkylsterol present; less than 1%of the recovered sterol was cholesterol
as determined by GLC on the QF-1 column (Table 4) and by RPLC analysis
at 205 nm.
Epicoprostanol. When 5&cholestan-3a-o1 (epicoprostanol) was used in
place of 5a-cholestan-3/3-01 (cholestanol) in the diet, no larvae molted,
although they ate the diet, produced frass, and lived for as long as 2 weeks.
Their rate of mortality was similar to that of larvae fed diet which was not
supplemented with sterol (Fig. 1).In contrast, the mortality of larvae fed diet
supplemented with cholestanol was very low and similar to that of larvae fed
5
10
15
20
TIME (DAYS)
Fig. 1. Mortality of larvae on diets which were supplemented with epicoprostanol (O), no
and cholesterol (A).
sterol (O),cholestanol (0)
Metabolism of Sterols by H. zed
291
diet supplemented with cholesterol (Fig. 1).These results indicated that a
3a-hydroxyl group (instead of 30) andlor a 5fLhydrogen (instead of 5a)
prevents the normal utilization of sterols in H.zea.
Metabolism of A7-Sterols
Lathosterol. The fate of this dietary sterol was similar to that of cholestanol
in that it was utilized as the major tissue sterol in the larvae. About 82% of
the recovered sterol from the larvae had an RRT similar to that of a standard
of lathosterol (Table 6). The remainder of the sterol had an RRT similar to
that of cholesterol. No peak for cholestanol was observed on the QF-1
column. The broadness of the single peak observed by RPLC analysis (Table
2) indicated that two sterols (eg, cholesterol and lathosterol) were present.
(A mixture of standards of cholesterol and lathosterol also gave a single
broad peak.) 'H-NMR confirmed that about 80% of the recovered sterol had
a spectrum similar to a standard of lathosterol and 20% had a spectrum
similar to cholesterol (Table 3). The mass spectrum with an M+ of 386
substantiated the evidence that only mono-unsaturated 24-desalkylsterols
were present.
Epifungisterol. GLC and RPLC indicated that H. zeu can demethylate this
sterol just as it can campesterol and campestanol; however, a large amount
(about 70%) of the recovered sterol represented unmetabolized epifungisterol
(Tables 2,6). Sixteen percent of the recovered sterol was the 24-desmethylsterol lathosterol, and the rest was cholesterol. These latter two sterols were
not separated by RPLC but were resolved by GLC (Tables 2,6). No cholestanol, campestanol, or campesterol was detected.
Spinasterol. This dietary sterol, which differs from all of the other sterols
in this study because of the presence of a A22-bond,was dealkylated just as
sitosterol and sitostanol were. By GLC analysis, the principal tissue sterol
had an RRT similar to lathosterol, and 34% of the recovered sterol had an
RRT which indicated that it was unmetabolized spinasterol (Table 6). Less
than 10% of the remaining sterol had the same RRT as cholesterol, and no
dihydrospinasterol was detected (the RRT of a standard of this molecule was
1.73 on the QF-1 column). Although the lathosterol and cholesterol peaks
were not separated by RPLC, the 24-desalkylsterol peak was resolved from
the spinasterol peak using this technique (Table 2). The UV-spectrum of each
of these peaks was characteristic of end absorption, indicating an absence of
metabolites with conjugated double bonds. The presence of lathosterol as
the principal metabolite of spinasterol in the tissues was confirmed by 'HNMR; although the spinasterol was also detected by this technique, the *HNMR spectrum did not indicate that cholesterol was present (Table 3).
Summary of the Metabolism Studies
The results of the metabolism of dietary sterols by H. z
summarized in Table 7.
e are
~
averaged and
Esterification of Dietary Sterols
The amount of sterol which was esterified in larvae fed different dietary
sterols was small and averaged between 2% and 4%. Those sterols detected
1.56 f 0.02
1.47 f 0.02
1.12 f 0.01
Epifungisterol
Lathosterol
SE-30
1.14 It 0.02
1.45 f 0.03
1.59 f 0.05
XE-60
OF-1
1.14 f 0.01
1.45 k 0.03
1.50 f 0.02
RRT of dietary sterol
Spinasterol
Dietary
sterol
1.57 f 0.02
1.11f 0.02
1.00 f 0.01
1.46
1.13
1.02
1.12
1.01
RRT
SE-30
37
63
<1
74
15
12
84
16
Percent
of free
sterol
TABLE 6. GLC Analysis of A7-Sterols and Their Metabolites in H . zea Larvae
1.57 f 0.05
1.16 f 0.01
1.02 f 0.01
1.44 f 0.02
1.12 f 0.01
1.01 0.01
1.15 f 0.02
1.00 f 0.01
23
68
9
70
17
13
79
22
4 f l
39 f 2
51 f 3
78 & 5
19 f 6
3+2
Sterols isolated from larvae
XE-60
Percent
of
Percent
esteriof free
fied
RRT
sterol
sterol
1.49 f 0.01
1.13 f 0.01
1.01 f 0.01
1.48
1.14
1.01
1.15
1.00
RRT
QF-1
+
34 f 7
4
5+4
70
15
15
78
22
62
Percent
of free
sterol
A5-24a-ethyl
A5-24a-methyl
A'
Dietary sterol
Sitosterol
Campesterol
Cholesterol
Spinasterol
Epifungisterol
Lathosterol
13.4
9.7
42.2
A7
-
-
34*7
*2
-
70
Epifungisterol
Spinasterol
15.9
40
34,l
42 f 5
84+1
38
51 4
22 1
-
M*1
3.9
15.5
62.7
-
+3
-
*
+
Lathosterol
63 i- 5
16 5 1
81 k 3
100
Cholestanol
Campestanol
Sitostanol
+
27 2
-
-
4.7
45.2
Cholesterol
86+4
73 f 2
-
Campesterol
Sitosterol
14 +_ 4
Percent of free sterols isolated from larvae
13.7
Weight of
larva
extracted
(g)
A7,"-24a-ethyl
A'-24a-methyl
Sitostanol
24a-ethyl
Campestanol
24a-methyl
Cholestanol
Sitostanol (65%)
(as above)
+ Campestanol(30%)
+ Cholestanol ( 5%)
Structural
differences from 5acholestan-3@-01
TABLE 7. Summary of the Free Tissue Sterols Isolated from H. zeu Larvae Fed Different Dietary Sterols
+
Cholesterol
5+4
13 i- 2
19 3
<1
1
7+2
16 i- 1
Cholesterol
W
bn
h)
3
h(
0
r:
U
s
4
x
8
9
e
U
3
a
a4
294
Ritter
as free sterols in larvae were also found in the esterified form; sitosterol and
spinasterol, though, appeared to be esterified in exceptionally small amounts
(Tables 1,4,6).
DISCUSSION
This is the first qualitative and quantitative study of the tissue sterols of
H. zeu larvae fed diets containing different A'-, A5-, and A7-sterols.The results
indicate that H . zea carries out very little metabolism of ring B of those dietary
Po-, A5-, and A7-sterols destined to become incorporated into membranes
and esterified, because after the larvae consumed the different diets ring B of
their principal tissue sterols was the same as that of their dietary sterol. That
is, sitostanol and campestanol were dealkylated and converted to cholestanol; sitosterol and campesterol to cholesterol; and spinasterol and epifungisterol to lathosterol. Although H. zeu is capable of dealkylating A'-, A7-,
and A5-alkylsterols, since more of the latter type of sterol is metabolized to
24-desalkylsterol by the late sixth instar, the larva may dealkylate A5-sterols
more efficiently than those with a double bond at the 7 position or no double
bonds (the stanols). These studies indicate that the intestinal cells of H.zeu
can absorb A'-, A7-, and A5-sterolswith or without a 24-alkyl group; however,
whether the absorption rates are similar is not known. The fact that alkylated
sterols were found at all in the larvae, from which the intestines (and
therefore all unabsorbed dietary sterols) had been removed prior to analysis,
indicates that some alkylated sterols may be transported out of the intestine
without being metabolized first. It is not known whether these sterols may
be dealkylated later at some other location, such as in the fat body.
Since esters of each type of free sterol were also found in the tissues of H.
zeu, this indicates that the larva produces enzyme(s) for the esterification of
sterols with differences not only in the side chain but also in ring B of the
nucleus. Since we have found that ACAT activity is present not only in the
microsomes of the intestine but also in the microsomes of the fat body of H.
zeu [14], and since Tavani et a1 [16] have shown that ACAT from rat liver
microsomes may esterify a number of sterols (eg, cholestanol and lathosterol)
in addition to cholesterol, ACAT may at least in part be responsible for the
esterification of the different tissue sterols found in H. zed. Interestingly, the
24-ethylsterolsrsitosterol and spinasterol, were found in the smallest amounts
in the esterified form (1%and 4%, respectively, of the total esterified sterols)
which is similar to the results of Tavani et a1 [%I, who showed that the
percentage esterification of sitosterol and stigmasterol by ACAT from rat liver
was significantly less than with other sterols, such as lathosterol and
cholestanol.
There was evidence that the larvae convert A7- and A'-sterols to cholesterol, because (1)minor quantities of cholesterol were present in varying
amounts when the insects were fed these sterols, and (2) cholesterol was not
found to be a contaminant of the dietary ingredients. Although some of this
cholesterol may have been derived from maternal sterols, the contribution of
transovarially derived sterols to the sterol pool of the sixth instar is probably
quite small and doesn't explain why the amounts of cholesterol found in
Metabolism of Sterols by H. zea
295
these experiments varied from < 1% to 19%. (The weight of a neonate larvae
is about 0.1 mg, which is less than 0.03% of that of a later-sixth-instar larva
or a pupa; therefore, assuming that the percentage of sterol in the tissues
does not change, the maternal sterols represent <0.03% of the sterols in the
mature larva.) The absence of sitosterol (andlor stigmasterol) from the tissues
of larvae fed sitostanol and spinasterol, and the absence of campesterol in
the tissues of larvae fed campestanol and epifungisterol, indicate that if A'and A7-sterols are metabolized to A5-sterols, the introduction of the double
bond at the 5 position occurs after the dealkylation of the side chain. Interestingly, more desalkylsterol than alkylsterol appeared to be converted to
cholesterol. If larvae do introduce a A5 bond into A7-desalkylsterols, it is not
apparent whether the desaturation occurs before or after the reduction at the
7 position, because neither 7-deh drocholesterol nor cholestanol was detected as a metabolite of dietary A -sterols. These results indicate that final
proof of the metabolism of A7- and Ao-sterols to cholesterol in H. zea must
await further studies using dietary sterols which have been radiolabeled.
Although it is possible that at least minor amounts of cholesterol may be
essential to larvae so that they can carry out some critical metabolic function(s), the results corroborate the results of others, such as Clark and Bloch
and Thompson et a1 [18], and indicate that insects may function normally,
at least for one generation, with sterols other than A5-sterols as their principal
membrane sterols and that they can store these molecules as steryl esters.
The fact that the sterol composition of H. zeu can be changed, with the only
immediately obvious effect being a change in rate of growth, is unusual, but
understandable, considering that some insects (eg, D. pucheu [5] and E.
vurivestis [6]) may contain sterols other than cholesterol and yet function
normally.
Since the sterol composition of H. zeu can be changed under laboratory
conditions by varying the type of sterol in the diet, similar changes in the
sterol composition of larvae might also occur naturally in the field because
some host plants, such as alfalfa [19], contain unusual sterols such as A7sterols as their dominant sterols, instead of A5-sterols (eg, corn [20]). Therefore, the sterol compositions of different populations of H. zeu may differ in
the field when they feed on plants (eg, corn vs alfalfa) with different sterol
compositions.
This change in the sterol composition of the larva may affect not only the
physiology of cellular membranes, but also the development of those parasites which derive their sterols from their host (eg, hymenopterous and
dipterous parasites). Consequently, the physiological as well as ecological
effects of changes in the sterol composition of H . zeu should be investigated.
Y
[ln
LITERATURE CITED
1. Ritter KS, Nes WR: The effects of cholesterol on the development of Heliothis z a . J Insect
Physiol27, 175 (1981).
2. Ritter KS, Nes WR: The effects of the structure of sterols on the development of Heliothis
zea. J Insect Physiol27, 419 (1981).
3. Svoboda JA, Thompson MJ, Robbins WE, Kaplanis JN: Insect steroid metabolism. Lipids
13, 742 (1978).
296
Ritter
4. Kircher HW:Sterols and insects. In: Cholesterol Systems in Insects and Animals. Dupont
J, ed. CRC Press, Boca Raton, FL, pp 1-50 (1982).
5. Goodnight KC, Kircher HW: Metabolism of lathosterol by Drosophila pacheu. Lipids 6 , 166
(1971).
6. Svoboda JA, Thompson MJ, Robbins WE: Unique pathways of sterol metabolism in the
Mexican bean beetle, a plant-feeding insect. Lipids 10, 524 (1975).
7. Ritter KS, Weiss BA, Norrbom AL, Nes WR: Identification of A517-24-methylene and
methylsterols in the brain and whole body of Atta cephalofes isthmicola. Comp Biochem
Physiol 71B, 345 (1982).
8. Svoboda JA, Dutky SR, Robbins WE, Kaplanis JN: Sterol composition and phytosterol
utilization and metabolism in the milkweed bug. Lipids 12, 318 (1977).
9. Gibson JM, Majumder MSI, Mendis AHW, Rees HH: Absence of phytosterol dealkylation
and identification of the major ecdysteroid as Makisterone A in Dysdercus fusciafus (Heteroptera, Pyrrhocoridae). Arch Insect Biochem Physiol1, 105 (1983).
10. Svoboda JA, Nair AMG, Agarwal N, Agarwal HC, Robbins WE: The sterols of the khapra
beetle, Trogoderma granarium Everts. Experientia 35, 1454 (1979).
11. Svoboda JA, Herbert EW Jr, Lusby WR, Thompson MJ: Comparison of sterols of pollens,
honeybee workers and prepupae from field sites. Arch Insect Biochem Physiol 1, 25 (1983).
12. Svoboda JA, Robbins WE: The inhibitive effects of azasterols on sterol metabolism and
growth and development in insects with special reference to the tobacco hornworm.
Lipids 6, 113 (1971).
13. Svoboda JA, Hutchins RFN, Thompson MJ, Robbins WE: 22-Trans-cholesta-5,22,24-trien3P-ol-an intermediate in the conversion of stigmasterol to cholesterol in the tobacco
hornworm, Manduca sexfa (Johannson). Steroids 14, 469 (1969).
14. Billheimer JT, Tavani DM, Ritter KS: Acyl coenzyme A: Cholesterol acyltransferase activity
in fat body and intestinal microsomes of Heliothis zea. Comp Biochem Physiol 76B, 127
(1983).
15. Ritter KS: Some unusual aspects of the sterol biochemistry of insects. In: Isopentenoids in
Plants: Biochemistry and Function. Nes WD, Fuller G, Tsai L, eds. Marcel Dekker, New
York, pp 389-400 (1984).
16. Tavani DM, Nes WR, Billheimer JT: The sterol substrate specificity of acyl CoA: Cholesterol acyltransferase from rat liver. J Lipid Res 23, 774 (1982).
17. Clark AJ, Bloch K: Function of Sterols in Demesfes vulpinus. J Biol Chem 234, 2583 (1959).
18. Thompson MJ, Louloudes SJ, Robbins WE, Waters JA, Steele JA, Mosettig E: The identity
of the major sterol from houseflies reared by the CSMA procedure. J Insect Physiol9, 615
(1963).
19. Itoh T, Tamura T, Matsumoto T: Sterols, methylsterols, and triterpene alcohols in three
Theaceae and some other vegetable oils. Lipids 9, 173 (1974).
20. Mohammed AJA, Hopkins TL: Dietary sterol utilization during development and reproduction of the corn borer, Diatraea grandiosella. Comp Biochem Physiol 71B, 637 (1982).
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