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Hypertrehalosemic hormone-regulated gene expression for cytochrome P4504C1 in the fat body of the cockroach Blaberus discoidalis.

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Archives of Insect Biochemistry and Physiology 28:79-90 (1 995)
Hypertrehalosemic Hormone-Regulated Gene
Expression for Cytochrome P4504C1 in the
Fat Body of the Cockroach, Blaberus
Kuang-Hui Lu, James Y. Bradfield, and Larry L. Keeley
Laboratories for liiuertebrafe Neuroendocrirte Research, Departnlettt of Entomology, Texas A&M
Utziurrsity, Texas Agriciilttiral Experiment Sfafioiz, College Station, Texas
Hypertrehalosemic hormone (HTH) up-regulates expression of a gene for a
cytochrome P450 of family 4 (CYf4CI) in the fat body of adult male B. discoi& / i s cockroaches. Studies were undertaken to determine the characteristics of
HTH-dependent CYP4CI expression. A dot-blot assay was developed for routine measurement of the relative levels of CYP4C1-mRNA in fat body RNA
extracts. A single injection of 10 pmol H T H produced a maximum CYf4CI
response within 8 h. This dose corresponds with the dose needed for a maximum in vivo hypertrehalosemic response to HTH (Keeley et a[., 1991). Multiple treatments with HTH at 8 or 24 h intervals were no more effective than a
single treatment for producing CYf4C7 expression. These results indicate that
CYf4C1 expression i s sensitive to physiological doses of H T H and responds
rapidly. CYP4C1 expression was suppressed by treatment with a-amanitin, an
inhibitor of RNA polymerase II, but was unaffected by cycloheximide, an inhibitor of protein synthesis. HTH appears to influence CYP4C7 transcription
without involvement of intervening regulatory genes. These results suggest that
regulation of fat body CYf4CI expression is a major physiological action of
HTH. @ 1995 Wiley-Liss, Inc.
Key words: adipokinetic hormone, cockroach, cytochrome P450, fat body, gene
Acknowledgments: This research was supported by National Science Foundation grant
Received December 3, 1993; accepted June 24, 1994.
Address reprint requests to Larry
College Station, TX 77843-2475.
L. Keeley, Department of Entomology, Texas A&M University,
Kuang-Hui Lu is now at Department of Entomology, National Chung-Hsing University, Taichung,
Taiwan, ROC.
0 1995 Wiley-Liss, Inc.
Lu et al.
Cytochrome P450 enzymes are a superfamily of monooxygenases for which
more than 200 CYP genes are described (Nelson et al., 1993). P450 enzymes
have two main functions in animals. First, P450 enzymes degrade toxic chemicals such as drugs, insecticides, environmental pollutants, and natural plant
products. Second, they metabolize natural physiological substrates such as
steroids, fatty acids, and arachidonic acid (Juchau, 1990). The expression of
many CYP genes is induced by xenobiotics or hormones (Gonzalez, 1989;
Lund et al., 1991).
The hypertrehalosemic hormone (HTH) was isolated and sequenced from
the neotropical cockroach Blaberus discoidalis (Hayes et al., 1986) and belongs to the family of insect and crustacean adipokinetic hormone/red
pigment-concentrating hormones (AKH/RPCH) (Gade, 1990).HTH regulates
fat body metabolism in B. discoidalis to increase glycogenolysis for trehalose
biosynthesis and for elevation of hemolymph carbohydrate (Lee and Keeley,
1994a). In addition, HTH stimulates fat body heme synthesis and enhances
the rate of juvenile hormone-dependent protein synthesis in the fat body of
vitellogenic females (Keeley et al., 1991). In studies on the cellular action of
HTH, it was observed that HTH increased [3Hluridineincorporation into fat
body RNA and a-amanitin suppressed HTH-related heme synthesis (Lee and
Keeley, 199413).Furthermore, in vitro translation of fat body RNA from HTHtreated animals revealed increased synthesis of three polypeptides when compared to RNA from Ringer-treated controls. These results suggested that HTH
stimulated specific gene expression in addition to its direct actions on trehalose synthesis.
Differential screening of a fat body cDNA library prepared from HTHtreated cockroaches identified an HTH-sensitive gene that was cloned, sequenced, and characterized as a cytochrome P450 gene (Bradfield et al.,
1991). This P450 gene was identified as a member in family 4, as the first
representative of a new subfamily C, and was designated CYP4CI (Nebert
et al., 1991). CYP4CI regulation by HTH represents the first example of a
specific neurohormonal regulation of gene expression in insects and provides an excellent model in which to study the mode of action of AKHHTH neurohormones on fat body metabolism. HTH exerts an almost
immediate stimulation of fat body trehalose biosynthesis through activation of enzymes related to glycogenolysis, but at the same time produces
a longer term stimulation of CYP4CZ expression. Are both actions part of
the same second messenger cascade or are two separate signal transduction mechanisms involved-one for immediate enzyme activation and a
second for gene regulation?
In this study, we have characterized the regulation of CYP4C1 expression by HTH. CYP4C1-mRNA levels were maximally elevated within 8 h
after exposure to a single physiological dose of HTH and were sensitive
to the inhibition of RNA polymerase I1 but not to the inhibition of protein
biosynthesis. These results suggest that stimulation of CYP4CZ expression
is a significant regulatory action by HTH for fat body metabolism in B.
HTH-Regulated P450 Gene Expression
Experimental Animals
B. discoidalis were reared in wood shavings in 40 liter plastic buckets, fed
dry dog food and water ad libitum, and kept in a 12-h 1ight:lZh dark circadian cycle at 27 & 2°C (Keeley, 1971). Newly emerged (day 0) adults were
collected daily, separated according to gender, and housed in plastic rat cages
under rearing conditions until used for experiments. The day before being
used in experiments, animals were decapitated to eliminate the corpora cardiaca, the source for endogenous HTH, and the wound was sealed with hot
beeswax:petroleum jelly (1:l).
Injection Regimen
Experiment animals received HTH in 10 pl of insect Ringer (Ephrussi and
Beadle, 1936) by injection with a microsyringe through a ventral abdominal
intersegmental fold. Controls received 10 p1 Ringer in the same manner.
Dot-Blot Analysis
Fat bodies were pooled from 3-4 experimental animals, and total RNA
was extracted (Puissant and Houdebine, 1990). One microgram of total
RNA from each treatment sample was denatured with formaldehyde
(White and Bancroft, 1982) and loaded into the wells of a dot-blot Minifold@
I (Schleicher & Schuell, Keene, NH) containing a positively charged ZetaProbe nylon membrane (BioRad, Richmond, CA). The blotted membranes
were rinsed with l x SET (20x = 3.0 M NaC1; 20 mM EDTA; 0.5 M Tris-C1,
pH 7.5), and RNA was cross-linked to the filter by illumination with ultraviolet (304 nm) for 5 min. For a hybridization probe, cloned CYP4CZcDNA was labeled using [ C X - ~ ~ P ] ~and
C T Pa random priming method
(DECAprimeTMDNA labeling kit; Ambion Inc., Austin, TX). Hybridization was in 4x SSPE (20x = 3.0 M NaC1; 0.2 M Na-phosphate buffer, pH
7.4; 20 mM EDTA) and 2% SDS at 60°C overnight; washes were with 0 . 1 ~
SSPE, 1%SDS at the same temperature. Each hybridized dot was excised
and its radioactivity quantitated by liquid scintillation spectrometry. Therefore, all values reflect the proportional amount of CYP4Cl-mRNA per microgram of total fat body RNA. The specificity of our CYP4C1-cDNA probe
was demonstrated previously by Northern blotting of fat body total RNA
(Bradfield et al., 1991).
A titration experiment was performed wherein samples of fat body total
RNA (0-1.0 pg in increments of 0.1 pg) were applied to nylon membrane and
hybridized with CYP4CZ-cDNA as described above. To compensate for possible quenching, heterologous RNA (poly(A)RNA from adult female Manduca
sextn fat body) was added such that each sample contained 1.0 pg total RNA.
The relationship of bound probe (cpm) to CYP4C1-mRNA was linear (r2 =
0.98) throughout the range. Therefore, expression of the data in cpm provides a close estimate of the relative abundance of the CYP4C1 message within
each experiment. However, because specific activity of the probe fluctuated
slightly during the course of these studies, cpm values are not directly comparable between figures unless otherwise noted.
Lu etal.
Inhibitors of mRNA and Protein Biosynthesis
CYP4C1-mRNA was measured after treatment with a-amanitin to inhibit
fat body mRNA synthesis. Amanitin and 100 pmol HTH were administered
sequentially to test animals in 10 p1 of Ringer. Control insects were injected
twice with 10 p1 Ringer. Fat bodies were isolated after 8 h, and RNA was
extracted for analysis.
CYP4C1-mRNA was measured after inhibition of fat body protein synthesis with cycloheximide (CHXM). Experimental animals were treated with 100
pg CHXM to suppress fat body protein synthesis, and 100 pmole HTH was
administered 1 h later. A second 100 yg dose of CHXM was administered 4 h
after HTH to insure continued suppression of fat body protein synthesis. Fat
bodies were isolated 8 h after the HTH treatment, RNA was isolated, and
CYP4C1 transcript levels were determined. The inhibitory action of the CHXM
treatments was confirmed by administering 10 yCi ["S]methionine in place
of HTH to CHXM-treated insects and determining the extent of 35S-labelincorporated into fat body proteins during the 8 h study period. Fat body proteins were precipitated, lipid extracted, and redissolved in 1 N NaOH for
liquid scintillation spectrometry and protein determination (Keeley et al.,
1988). Fat body protein synthesis was suppressed by 87% in animals treated
with CHXM compared to Ringer-treated controls.
No animals died during the 8 h study period after treatment with any of
the doses of a-amanitin or CHXM. Animals treated with a-amanitin remained
normally active; animals treated with CHXM became sluggish.
All chemicals were reagent grade. CHXM and a-amanitin were purchased
from Sigma Chemical Co. (St. Louis, MO); [a-32PldCTPwas obtained from
NEN-DuPont (Boston, MA). HTH was synthesized and standardized by the
Texas A&M University System, Biotechnology Support Laboratory.
Means or experimental and control groups were based on three independent samples. Each sample consisted of the RNA extracted from 3 4 pooled
fat bodies from individual animals. Two aliquots from each RNA extract were
blotted and probed and the cpm averaged for a single sample value.
Means for experiments with multiple variables were analyzed by ANOVA
followed by a Tukey-Kramer multiple comparisons post-test to show statistical differences between individual means. An unpaired Students t-test was
used when statistical analysis involved comparing means between only single
experimental and control groups. Statistical analyses were performed using InStat
2.0@(GraphPad Software, San Diego, CA) for Apple Macintosh computers.
Expression Profiles of CYP4CZ in Response to HTH
CYP4C1 transcript was measured in fat body of decapitated adult males at
intervals during 24 h following injection of 100 pmol HTH (Fig. 1).CYP4C1-
HTH-Regulated P450 Gene Expression
.. .
- - -.- - ' I * * * . I * * * * -
Fig. 1 , CYP4C1 transcript accumulation in response to HTH in fat body of decapitated, 1 day-old adult male B . discoidalis. Values represent mean f SEM for three samples; each
sample consisted of pooled fat bodies from four animals. Animals were decapitated on day 0
and injected on day 1 with 100 pmol HTH in 10 1.11 Ringer. Fat bodies were removed for RNA
extraction at the designated time intervals after HTH administration. Control insects were
injected with 10 pI Ringer.
mRNA levels did not change significantly in Ringer-treated control animals
during the 24 h observation period. By comparison, CYP4C1-mRNA increased
rapidly in HTH-treated animals and was twice that of control animals by 4 h.
A maximum 2.75-fold increase in transcript levels was attained at 8 h. This
peak level persisted through 12 h and then began to decline, and according
to linear extrapolations of the rate of decline, CYP4C1-mRNA would have
returned to basal levels by 40 h.
Effect of Multiple Treatments with HTH on CYP4C1 Expression
Previous studies used three daily injections of HTH to produce maximum
CYP4C1 expression (Bradfield et al., 1991). The present experiment was undertaken to determine if repeated HTH treatments resulted in greater accumulation of CYP4Cl-mRNA. Decapitated animals were injected daily with
100 pmol HTH for 1, 2, or 3 days. The results show that HTH stimulated
CYP4C1 transcript accumulation by two- to three-fold over Ringer-treated
control animals for all injection regimens (Fig. 2A). No significant differences
were observed in the levels of CYP4C1-mRNA at 24 h after the last HTH
treatment, regardless of the number of treatments.
The results shown in Figure 1 indicate that maximal CYP4C1 transcript
levels were attained 8 h after HTH administration. Therefore, injections of
100 pmol HTH were administered at 8 h intervals to determine if additional
exposures to hormone at the time of maximum response reinforced CYP4Cl
Lu et al.
One Treatment
Two Treatments
Three Treatments
CYP4C 1-mRNA (cprn)
One Treatment
Two Treatments
Three Treatments
CYP4C1-mRNA (cpm)
Fig. 2. Effects of multiple HTH treatments on CYP4Cl expression in decapitated, 1-day-old
adult male 6 discoidalis. Values represent mean f S E M for three samples; each sample consisted of pooled fat bodies from four animals. All animals were decapitated on day 0. A:
Animals were injected with 100 pmol HTH in 10 PI Ringer on days 1, 2, andlor 3 with
measurements of CYP4C1 -mRNA 24 h after the last injection. 6: Animals were injected with
100 pmol HTH in 10 pI Ringer at 8, 16, and/or 24 h with measurements of CYP4C1-mRNA
8 h after the last injection. Control animals received 10 pl Ringer at the same time intervals.
Both the daily and the 8 h membranes were hybridized together so that cpms presented in
both A and B are comparable,
transcript accumulation (Fig. 28). Although values for CYP4Cl-mRNA did
increase modestly with increasing injections, the increases were not statistically significant. Therefore, no additional accumulation of transcript was evident in response to multiple 8 h treatments with HTH as compared to a single
HTH-Regulated P450 Gene Expression
Effects of Transcriptional/TranslationalInhibitors on CYP4C1 Expression
Experiment animals were treated with a-amanitin to determine if inhibition of DNA-dependent RNA polymerase-I1 suppressed HTH-dependent
CYP4CZ expression. Animals were injected with four different doses of aamanitin along with 100 pmol HTH; CYP4C1-mRNA levels were measured
in the fat body 8 h later. HTH (100 pmol) increased CYP4C1-mRNA by twofold over control animals. All doses of a-amanitin at or above 1.25 pg suppressed CYP4C1-mRNA to control levels in HTH-treated animals (Fig. 3A).
CHXM was used to determine if protein synthesis was required for HTHdependent CYP4C1 expression. The CHXM treatment did not suppress
CYP4CZ expression in HTH-treated, decapitated animals (Fig. 3B); instead,
CHXM appeared to increase CYP4Cl-mRNA levels compared to Ringertreated control animals.
Dose Response of CYP4C1 Expression to HTH Treatments
HTH dose sensitivity was measured for CYP4CZ expression. Test doses of
HTH were administered to day 1, decapitated adult male B. discoidalis, and
the amount of fat body CYP4Cl-mRNA was determined 8 h later. CYP4C1mRNA increased in a dose-dependent manner between 0.65 and 11 pmol
HTH per animal (Fig. 4). The ED5(,dose was 3 pmol HTH.
The relationship between HTH and CYP4C1 expression in B . discoidalis constitutes the first insect examples for both neurohormone-regulated gene expression and endocrine-sensitive cytochrome P450 genes. CYP4CZ was
discovered based on the abundance of its transcript in the fat body of HTHtreated cockroaches relative to HTH-deprived animals (Bradfield et al., 1991).
However, the early experiments employed high doses of HTH administered
daily for several days and did not examine the sensitivity of CYP4C1 expression to HTH or the kinetics of transcript accumulation. The present research
defines more precisely the temporal relationship between HTH action and
CYP4C1 expression.
CYP4CZ expression occurs rapidly in response to HTH. A measurable increase in transcript levels was observed within 2 h after treatment with HTH,
and a maximum level was attained by 8 h (Fig. 1).There are no other reports
of neuropeptide-responsive gene expression in insects to which these times
for response can be compared, by 2 h appears to represent a rapid hormonedependent response. For example, juvenile hormone (JH) induces vitellogenin gene expression in Locusta rnigvatovia fat body, and vitellogenin mRNA is
not detected until 18 h after JH treatment (Chinzei et al., 1982; Wyatt, 1988).
The rapidity of the CYP4CZ response suggests that HTH likely regulates the
CYP4CZ gene in a relatively direct manner.
Multiple doses of HTH did not cause accumulation of CYP4C1-mRNA
above the optimal level detected at 8 h after a single treatment (Fig. 2). In our
initial discovery of CYP4CZ expression, we used a 3 day regimen of HTH
injections (Bradfield et al., 1991). It was hypothesized that multiple daily in-
Lu et al.
100 pmole HTH
HTH + 0.63 pg Amanitin
HTH + 1.25 pg Amanitin
HTH + 2.50 pg Amanitin
HTH + 5.00 c(gAmanitin
CYP4Cl-mRNA (cpm)
CYP4C1-mRNA (cpm)
Fig. 3 . Effects of a-amanitin (A) and cycloheximide (CHXM) (B) on HTH-related CYP4CI expression in the fat body of decapitated, 1 -day-old adult male R. discoidalis. Values represent
mean 2 SEM for three samples; each sample consisted ot pooled fat bodies from four animals.
jections might produce transcript accumulation exceeding that obtained with
a single dose; however, this was not the case, as transcript levels remained
essentially constant despite repeated administration of HTH. The data in Figure 2 were obtained using the same radiolabeled probe preparation so that
the cpm can be compared between the 24 h (Fig. 2A) and 8 h (Fig. 2B) injection regimens. The results appear to suggest that the amount of mRNA present
at 24 h was the same as the amount present at 8 h which was maximal. This
is a confounding result since by 24 h the transcript level should have declined by 50% based on the results shown in Figure 1. We have no explanation for this apparent discrepancy. It is possible that the values for one and
two 24 h treatments are in fact abnormally high, by chance, and should have
been closer to the value of the three 24 h treatments which was approxi-
HTH-Regulated P450 Gene Expression
pmol HTH
Fig. 4. HTH dose response for CYP4CI expression in the fat body of decapitated, 1-day-old adult
male 5. discoidalis. Values represent mean SEM for five samples; each sample consisted of pooled
fat bodies from three animals. Decapitated male 6. discoid;tlis were injected with the indicated dose
of HTH in 10 pI Ringer; fat bodies were removed after 8 h and pooled, and the RNA was extracted
and CYP4C1 -mRNA determined. Dose responses indicated by the dotted lines are as follows: minimum = 0.65 pmol HTH; maximum = 11 pmol HTH; EDSO= 3 pmol HTH.
mately 50% lower and closer to the expected value. Finally, fat body CYP4C1mRNA levels were increased two to three-fold over controls by 8 h after a
single HTH treatment, but these elevated transcript levels were not augmented
by repeated treatments with HTH at 8 h intervals (Fig. 2B). This is also a
confounding result since 8 h is maximal for transcript accumulation, and we
would anticipate that continued stimulation of CYP4CI expression by repeated
HTH injections at 8 h intervals would produce ongoing transcript accumulation. Both of these results suggest that there is an upper limit for HTH stimulation of pretranslational CYP4C1 gene expression beyond the constitutive
levels. Since we have little information on how HTH induces CYP4C1, it is
possible that a second messenger cascade is involved that may include feedback controls.
Inhibitors of RNA polymerase I1 and protein biosynthesis were used to
examine the mechanisms of CYP4C1 gene regulation by HTH. a-amanitin
completely blocked the HTH-dependent increase in fat body CYP4Cl-mRNA
(Fig. 3A), indicating that the increase in fat body CYP4C1-mRNA was due to
HTH stimulation of CYP4C1 transcription. On the other hand, the CHXM
treatment regimen strongly inhibited fat body protein synthesis but did not
inhibit the HTH-dependent increase in CYP4C1-mRNA (Fig. 3B). The latter
results suggest that HTH promoted CYP4CZ expression independent of the
translation of any regulatory products from other genes.
Blocking protein synthesis with CHXM surprisingly increased CYP4ClmRNA significantly compared to Ringer-treated controls. A similar increase
Lu et a].
in female-specific P4503A1-mRNA was observed in the liver of rats after
CHXM treatment (Burger et al., 1990). The increase in CYP4C1-mRNA after
CHXM treatment suggests that noninduced fat body might synthesize proteins that suppress CYP4CZ expression or degrade CYP4C1-mRNA. If CHXM
inhibited the synthesis of such putative proteins, CYP4Cl transcription might
increase and/or transcripts might accumulate due to reduced degradation.
The dose-response relationship was determined for HTH and CYP4C1 expression. In a previous study, 100 pmols HTH were used to insure a maximum response (Bradfield et al., 1991). This was based on earlier in vivo
dose-response stimulation of fat body heme biosynthesis between 10 and 100
pmols HTH per animal (Keeley et al., 1991).The present studies showed that
0.65-11 pmols HTH was the dose-response range for CYP4CZ expression (Fig.
4). In vivo trehalose biosynthesis is dose-dependent between 1 and 10 pmoles
of HTH per animal for adult male B. discoidulis (Keeley et al., 1991). Therefore, within the limits of experimental variability, the HTH dose response for
CYP4CZ expression likely coincides with the physiological range for HTHdependent trehalose biosynthesis.
In general, we observed that HTH stimulates both trehalose and heme biosynthesis by two- to four-fold in B. discoidalis fat body (Keeley et al., 1991;
Lee and Keeley, 1994a). This agrees with the magnitude of the CYP4CZ response to HTH. The rapidity, sensitivity, and magnitude of the CYP4CI response relative to other HTH-sensitive physiological events suggest that HTH
stimulation of CYP4C1 expression is a direct action by the hormone and not
a delayed, secondary effect that occurs subsequent to other more primary
actions by the hormone. Therefore, CYP4C1 expression is likely a significant
physiological effect by HTH for the production of P4504C1, which is somehow related to the hypertrehalosemic action of the hormone.
As a peptide, HTH probably does not enter the cell and interact directly
with the CYP4CZ gene; rather the gene expression is probably implemented
through a presently undefined HTH-dependent second messenger transduction cascade. The nature of this cascade remains undetermined. Past studies
on HTH second messengers have demonstrated that HTH does not increase
fat body CAMPnor affect adenylyl cyclase activity (Lee and Keeley, 1994a).
Similarly, cGMP analogs have no effects on trehalose biosynthesis, but both
extra- and intracellular Ca2' are important for maximal HTH-dependent hypertrehalosemic activity (unpublished results). It is of interest to determine if
HTH stimulation of CYP4CZ expression is an extension of the second messenger cascade that stimulates trehalose biosynthesis or is a separate transduction cascade.
The enzymology of the P4504C1 protein is under investigation in our laboratory but is not yet elucidated. Based on mammalian research, CYP4 enzymes perform o-oxidation of medium-chain fatty acids such as lauric and
arachidonic acids (Bains et al., 1985; Hardwick et al., 1987). HTH is a member of the AKH/RPCH family, and a major action of AKHs is to mobilize fat
body fatty acids (Mayer and Candy, 1967) and to enhance fatty acid metabolism (Robinson and Goldsworthy, 1974, 1977). Therefore, it is possible that
HTH could affect lipid metabolism in addition to trehalose biosynthesis in
the cockroach fat body. CC extracts stimulate fatty acid oxidation as an alter-
HTH-Regulated P450 Gene Expression
native energy source in the fat body of Periplaueta arnericana while glycogenolytic products are being converted to trehalose (McDougall and Steele,
1988; Wiens and Gilbert, 1965). We speculate that a similar series of metabolic events may occur in B . discoidalis fat body during HTH-induced
hypertrehalosemia, and that o-oxidation by P4504C1 may participate in a
shift to fatty acid oxidation.
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