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Regulation of fat body glycogen phosphorylase activity during refeeding in Manduca sexta larvae.

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Archives of Insect Biochemistryand Physiology 19:225-235 (1992)
Regulation of Fat Body Glycogen Phosphorylase
Activity During Refeeding in Manduca
sexfa larvae
Karl J. Siegert and William Mordue
Department of Zoology, University of Aberdeen, Aberdeen, Scotland, United Kingdom
In 12-h-starved larvae of the tobacco hornworm, Manduca sexta, fat body glycogen phosphorylase was quickly inactivated when insects were refed with
normal diet and agar which contained 3% sucrose. Only the first 2 min of
refeeding were necessary to induce enzyme inactivation. During this short
period, larvae did not ingest enough sucrose to increase the hemolymph glucose concentration. This may indicate that the gut released a hormone(s) which
directly or indirectly led to the inactivation of fat body glycogen phosphorylase.
Inactivation of the enzyme could also be induced by injection of glucose
(30 mg) into the hernolymph of starving M. sexta larvae suggesting that there
may be separate control from a neuroendocrine site such as the brain or the
corpora cardiaca. Trehalose was less effective.
Bovine insulin (2 and 4 pg/starved larva) did not induce phosphorylase inactivation over 20 min or decrease hernolymph carbohydrate or lipid concentrations within 60 min. It is, therefore, necessary to screen insect tissues for
substances which could bring about inactivation of fat body glycogen phosphorylase. Q 1992 WiIey-~iss,Inc.
Key words: starvation, intermediary metabolism, hemolymph carbohydrates, AKH, insulinlike factor
Carbohydrate metabolism in starved Manduca sexta larvae is regulated at least
in part by an AKH+from the CC [1,2]. This peptide hormone induces activaAcknowledgments: The authors wish to thank Dr. Stuart E. Reynolds (Universityof Bath, School
for Biological Sciences) for the regular supply of Manduca sexta eggs. This study was supported by a grant from the Aberdeen University Research Committee.
Received September 3,1991; accepted December 3,1991.
Address reprint requests to Dr. Karl I. Siegert, Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB9 2TN, Scotland, UK.
*Abbreviations used: AKH = adipokinetic hormone; CC = corpus cardiacum; RP-HPLC =
reverse-phase high-performance liquid chromatography; TEA = triethanolamine; CAMP =
cyclic adenosine monophosphate.
0 1992 Wiley-Liss, Inc.
Siegert and Mordue
tion of fat body glycogen phosphorylase within 3 h of starvation, afterwards
enzyme inactivation occurs slowly over a period of ca. 24 h. This inactivation
may result from cessation of AKH release and/or after the release of a different
hormone(s). It is also important to understand how carbohydrate metabolism
is regulated when previously starved insects are refed. In vertebrates under
such conditions insulin is released from the pancreatic islets into the blood to
change the molar insulin/glucagon ratio in order to stimulate the uptake of
metabolites into tissues. Glycogen phosphorylase must be inactivated to limit
the release of glucose metabolites from glycogen and to allow glycogen synthesis to cope with the increasing concentration of blood carbohydrate. No
data are available as to the regulation of fat body glycogen phosphorylase or
synthase activities in insects during refeeding. There is evidence for the presence
of insulin or insulin-like material in M. sexta [3], other data suggest that bovine
insulin has hypolipemic activity in migratory locusts, Locust0 migrutoriu [4].
In the present study M. sextu larvae were starved for 12 h and then the effect
of refeeding on carbohydrate metabolism observed. The inactivation of fat body
glycogen phosphorylase was found to be very rapid and is likely to be controlled by a hormone with a function similar to vertebrate insulin.
M. sextu eggs were supplied weekly by Dr. Stuart E. Reynolds (Universityof
Bath). In Aberdeen larvae were reared on a standard wheat germ-based artificial diet [5]at 25-27°C and under a light regimen of 12 h light:12 h dark (lightsoff was at 1900).
Dissection of Insects and Sample Preparation
For all experiments, M. sextu larvae (fifth instar, initial body weight 5.5-7.5
g) were selected between 1800 h and 1900 h. After having been starved for 12
h insects were treated as outlined in the text, fat body samples placed into 1
ml of medium (50 mM TEA, 5 mM Na,EDTA, 20 mM NaF, 1 mM N-phenylthiourea, pH 7), and homogenized for 20-30 s with a MSE sonicator (Crawley,
England). Hemolymph samples were collected into centrifuge tubes containing a crystal of N-phenylthiourea and kept on wet ice until further use.
Determination of Hemolymph Carbohydrates
Hemolymph was centrifuged (5 min, 12,0008)and 100 p1 mixed with 500 pl
0.3 N perchloric acid. An aliquot of the supernatant (100 pl) was used for the
enzymaticdetermination of glucose [6]. Glucose concentrationswere expressed
as millimolar, Another 100 pl of supernatant was diluted with 1,400 pl distilled water for the measurement of total hernolymph carbohydrates using the
anthrone reagent [6] with glucose as standard, from this value the glucose
concentration was subtracted and the data converted to mM trehalose.
Determination of Hemolymph Lipid Concentrations
Twenty microliters of hemolymph were pipetted into 500 p1 concentrated
sulfuric acid for the determination of hemolymph lipid concentrations [7]. All
absorbance measurements were performed with a Philips PU 8625 UVNIS
spectrophotometer (Cambridge, England).
Refeeding in M. sexta larvae
Determination of Glycogen Phosphorylase Activities
Fat body homogenate was centrifuged (10 min, 12,OOOg) and the supernatant (25-50 p1) used for activity measurements in the direction of glycogen
breakdown as described elsewhere [8] with a Philips 8720 UV/VIS scanning
spectrophotometer. Values for active phosphorylase are given as the percentage of total phosphorylase activity present.
Preparation of Agar for the Refeeding Experiment
One hundred milliliters of filtered water (Milli-Q water purification system
equipped with an organic-free kit, pore-size 0.22 pm, Millipore; Watford,
England) was boiled, 3 g agar blended into the water, and the mixture poured
into a flat dish. In some cases 3 g of sucrose were mixed into the agar. Agar
was kept at room temperature and used within 24 h.
Material to be injected was dissolved in filter-sterilized saline (110 mM KCl,
4 mM NaCl, 15 mM MgClz, 4 mM CaQ, 5 mM KH2P04, pH 6.5 [9]). Carbohydrate (25 p1) and insulin (10 p1) solutions were administered with Hamilton
syringes equipped with 33 gauge needles.
Reverse-Phase High-Performance Liquid Chromatography
RP-HPLC was used to check our bovine insulin preparation. An aliquot (10
pg) was run on the HPLC equipment described by Siegert et al. [lo] using an
Aquapore RP-300 column (C-8,220mm x 4.6 mm; Brownlee, obtained through
Pierce & Warriner, Chester, England). The sample (in 5% acetonitrile in 0.1%
trifluoroacetic acid) was injected at 15% acetonitrile/O.1% trifluoroacetic acid
and a linear gradient with an increase of 1%acetonitrile minpl applied at a
flow rate of 1ml min-'.
Effects of Continuous Refeeding in M. sexta Larvae
Activities of fat body glycogen phosphorylase. After 12 h of starvation ca.
15% of total fat body glycogen phosphorylase was in the active form (Fig. 1).
The same value was found after 3 h (data not shown). Since it was more convenient to starve the insects 12 h overnight, this procedure was chosen. After
an additional hour of starvation the average value was 19%.Fed control insects
showed values of ca. 3%.
All starved insects which were offered either diet or agar started refeeding
within 5-8 min. How much the insects ingested was not quantified. It was
possible to tell that insects had consumed food since the contents of the intestine
changed in consistency and color. The intestine of starved larvae usually
contained a dark brown watery liquid with few solids. Insects which had fed
even only for 2-5 min contained a light brown watery mass with more solids.
Agar-fed insects had active phosphorylase values comparable to starved larvae
with averages between 15 and 20% throughout. The percentage of active phosphorylase decreased in diet-fed and agar/sucrose-fed insects over 20 min to values
found in fed insects and remained low until 60 min after the start of refeeding.
Siegert and Mordue
Fig. 1. Effect of continuous refeeding on fat body glycogen phosphorylase activities in
12-h-starvedM. sexta larvae, Starved insects were either placed on normal diet (A)
or on 3%
agar containing 3% sucrose (-A-) or on 3% agar without any additions (
and sacrificed at
A second group
the times indicated. Control insects continued to starve for 30 or 60 rnin (--o--).
of control insects were of the same developmental stage but had been continuously fed (--a--).
At least N = 4 per individual data point.
Hemolymph carbohydrate cancentrations. In starved larvae the trehalose concentration was ca. 40-45 mM. During refeeding with normal diet, agar, and
agar containing 3% sucrose the values changed only slightly and in no case
were these changes statistically significant; the concentrations in fed control
larvae were ca. 30-35 mM (data not shown).
During the 60 min of the experiment, the hernolymph glucose concentration in starved insects and in those refed with agar remained low, i.e., at ca.
0.3 mM (Fig. 2); fed control larvae showed levels of about 1.5 mM. In insects
_ _ _ I _ _ _ _ _ _ _ _ _ _ _ _ _ I - - - - -
Fig. 2. Effect of continuous refeeding on hernolymph glucose concentrations in 12-h-starved
M . sexta larvae. See legend to Figure 1 for details of the experiment and an explanation of
symbols. At least N = 4 per individual data point.
Refeeding in M.sexta Larvae
which had been refed with normal diet for only 10 min, the hernolymph glucose concentration had increased significantly to 0.75 mM ( P < 0.01), and
reached feeding levels within 20 min. Thereafter, an overshoot was observed
(60 min; P < 0.025). In larvae which received 3% sucrose in agar, the glucose
concentration remained low during the first 20 min and then increased significantly to feeding levels (30 min), but was significantly lower than in feeding
larvae after 60 min; P < 0.025).
Since the glycogen phosphorylase results obtained in insects refed with n o r
ma1 diet and agar containing 3% sucrose were superimposable, but smaller
changes of the hemolymph glucose concentration were observed on refeeding with the latter diet, the following experiment was performed using agar
supplemented with 3% sucrose.
Effects of Refeeding M . sexta Larvae for Short Periods
In this experiment larvae were precisely timed from the start of refeeding. After 2, 5, and 10 min of refeeding the larvae were removed from
the diet and either killed immediately or starved until the time indicated
in Figure 3.
Activities of fat body glycogen phosphorylase. Inactivation of fat body glycogen
phosphorylase was initiated in all three sets of insects, even when the insects
only had a 2-min access to the diet. Within 10 min of the start of refeeding
phosphorylase had decreased to levels found in continuously fed larvae. After
5 rnin, the differences were already significant ( P < 0.025; for t = 0 and 5 min).
Carbohydrate concentrationsof hemolymph. The hemolymph trehalose concentrations were found to be in the same range as in the above experiment
and did not undergo significant changes (data not shown).
The hemolymph glucose concentrations in starved and fed control larvae
(Fig. 4)were comparable to those found in the above experiment (Fig. 2). When
the insects were only refed for 2 min, the glucose concentrationsdid not change
at all during the entire experiment, i.e., they remained as low as in starved
larvae. In insects refed for 5 and 10 min, glucose concentrations increased but
only after 10 min. After 20 min the values had increased significantly ( P <
0,025) and were still significantly higher after 30 min ( P < 0.005).
Thus, only 2 min of refeeding were required to induce complete inactivation of fat body glycogen phosphorylase and this was achieved without any
significant changes of hemolymph glucose concentrations.
Effects of Injection of Glucose and Trehalose on Glycogen Phosphorylase
Activities in Starved M . sexta Larvae
Since the ingestion of sucrose led to the inactivation of fat body glycogen
phosphorylase, it was of interest to investigate whether the injection of 30 rng
glucose or trehalose into 12-h-starved insects had the same effect. The amount
was chosen in order to approximately double the hemolymph concentration
of total carbohydrates. This amount administered as glucose led to enzyme
inactivation within 5 min and during the remainder of the experiment, phosphorylase activities remained low (Fig. 5 ) .
The injection of trehalose led to an intermediate effect. During the first 10
min this sugar had no effect, but then the activities were significantly lower
Siegert and Mordue
Fig. 3. Effect of refeeding starved M. sexta larvae for short periods of time on glycogen phosphorylase activities. Twelve-hour-starved larvae were placed on 3% agar in water containing
3% sucrose, timed from the resumption of feeding, and allowed to feed only for 2 (-c-),
or 10 min (--A4
and then starved again until the time indicated. Control insects were
starved for 30 min (--O--) or continuouslyfed (--m--).At least N = 4 per individual data point.
than in saline-injected starved controls (20 min: P < 0.005; 30 min: P < O.OOl),
but significantly higher than in glucose-injected animaIs (20 min: P < 0.025; 30
min: P < 0.005). The controls were injected with insect saline and over the
30-min period of the experiment the phosphorylase activity increased significantly from ca. 16 to 22% (P < 0.025).
Fig. 4. Effect of refeeding starved M. sexta for short periods of time on hemolymph glucose
concentrations. See legend to Figure 3 for details of the experiment and explanation of symbols. At least N = 4 per individual data point.
Refeeding in M. sexfa larvae
Fig. 5. Effect of injection of glucose or trehalose (30 mg) on activities of fat body glycogen
phosphorylase in 12-h-starved M. sexta larvae. The amount chosen to approximately double
the concentration of total hemolymph carbohydrates [ I l l was injected at t = 0 min and the
trehaloseinsects were then starved until the time indicated. Glucose-injected larvae (--L);
saline-injected starved control specimens (--o--).At least N = 4 per indiinjected larvae (A);
vidual data point.
Effects of Bovine Insulin in Starved M. sextu Larvae
Since the inactivation of fat body glycogen phosphorylase can be achieved
very rapidly in starved M. sextu larvae by refeeding and the injection of glucose, a hormone(s) may be released from an as yet undisclosed site. Bovine
insulin was injected into 12-h-starved M. sextu larvae and its effects on fat
body glycogen phosphorylase activities and hemolymph metabolite concentrations measured (Table 1).The doses injected were chosen according to
Tager et al. [3] (2 and 4 pg/larva). Neither dose affected any of the measured
The expression of an insulin-like peptide in an insect has recently been concluded from sequences of cDNA clones from brain mRNA of L. rnigrutoria [12].
Insulin-immunoreactive material had been identified earlier in the neurosecretory system [3] and the hemolymph [13] of M . sexta. Partially purified
immunoreactive material reduced hemolymph carbohydrate concentrations
(hypotrehalosemic effect), but bovine insulin was ineffective [ 3 ] .In fed male
adult L. rnigmtoriu, however, insulin was found to reduce hemolymph lipid
levels, as did methanol or aqueous extracts of the storage lobe of the CC and
the brain [4] (both results could not be reproduced by the authors of the present investigation).In adult L. migutaria starvation leads to increased hemolymph
lipid levels, an effect which cannot be attributed to the action of AKH. Refeeding and the injection of sucrose into starved insects led to a rapid decrease in
lipid concentration [141. The decrease of hemolymph lipid levels upon refeeding and after injection of CC and brain extract may be caused by an insulin-
15.7 ? 3.0
60.0 f 5.3
0.15 ? 0.02
2.0 2 0.1
15.7 f 3.0
60.0 i 5.3
0.15 0.02
2.0 r 0.1
23.5 k 7.9
50.5 f 3.2
0.15 t 0.02
1.9 f 0.2
23.5 2 7.9
50.5 i 3.2
0.15 0.02
1.9 0.2
33.9 k 13.4
58.7 f 2.9
0.20 k 0.03
1.9 f 0.3
15.9 & 2.2
54.6 k 5.0
0.16 k 0.03
1.8 2 0.1
5 min
25.6 f 11.1
56.1 3.2
0.19 r 0.03
2.0 f 0.2
20.6 2 6.3
55.2 r 3.8
0.11 t 0.03
1.9 2 0.2
10 min
26.5 & 11.2
56.7 2 5.3
0.19 & 0.03
2.0 f 0.2
24.0 2 7.3
62.4 f 4.4
0.14 t 0.04
2.3 t 0.3
20 min
58.4 k 2.9
0.18 & 0.02
1.9 f 0.2
58.7 & 2.6
0.14 & 0.02
1.9 f 0.1
60 min
T h e doses (2 and 4 @insect equivalent to 8 and 16 x 1@ IU/g body weight) were chosen according to Tager et al. [3]. Insects were injected at t
= 0 min, starved, and hemolymph and fat body samples taken after the time indicated. Control specimens, injected with 10 11.1 saline, were sacrificed after 20 min. At least N = 4 per individual measurement. In this experiment the hemolymph trehalose concentrations were found to be significantly higher than in the above refeeding experiments. An explanation for this fact cannot be given. Since we have no direct way of testing
whether or not our insulin preparation was biologically active in an appropriate vertebrate assay, an aliquot (10 pg) of our stock solution was
chromatographed on a RP-HPLC column. We used a long-range gradient (see MateriaIs and Methods). A single peak eluted with a retention
time of 25.6 min indicating the integrity and purity of the preparation.
4 pg bovine insulin injected
(% of total)
Hemolymph trehalose
Hemolymph glucose
Hemolymph lipids
Hernolymph glucose
Hemolymph lipid
2 pg bovine insulin injected
Active phosphorylase (% of total)
Hemolymph trehalose
TABLE 1. Effects of Bovine Insulin (Sigma) on Some Parameters in 12-h-StarvedM . sextu Larvae*
Refeedingin M. sexta Larvae
like factor. Since this compound has yet to be isolated, its biological activities
and physiological functions have not been assessed. The present investigation provides the physiological framework for such a factor. It would be released
into the hemolymph to fine-tune energy metabolism during the switch from
starvation to the reavailability of nutrients when feeding occurs.
In M. sextu larvae fat body glycogen phosphorylase was inactivated very rapidly upon refeeding. The speed of the inactivation, the fact that hemolymph
glucose and trehalose levels did not change during early refeeding, and that
only short periods of refeeding were required to induce inactivation, point
strongly to a possible hormonal regulation of this effect. The low hemolymph
glucose concentrations early in refeeding indicate that this parameter is not
involved in the regulation of either enzyme activity or of hemolymph titer of
the presumptive hormone. Phosphorylase inactivation at this stage can only
be a preparative step for glycogen synthesis. There may be, however, a high
glucose turnover.
Since no significant increase in hemolymph carbohydrate levels occurred
during early stages of refeeding with agar containing 3%sucrose, the gut may
participate in the regulation of enzyme activity, which may be three-fold. 1)
The gut may release a hormone(s) which acts directly on the fat body and/or 2)
stimulate the release of another hormone(s). There is evidence for the presence of insulin-like material in the intestine of worker honeybees, Apis mellifica
[15], and L. rnigvutoriu [16]. 3) Nervous regulation of hormone release from a
site other than the gut is also possible, this would explain the speed of phosphorylase inactivation. Stretch-receptors do not seem to be involved in this
process since enzyme activities remained high when insects were fed with
agar. Since no changes in hemolymph carbohydrate concentrations were
observed, in addition to carbohydrate receptors (glucose, fructose, and/or
sucrose) on the mouthparts, such receptors may be present in the intestine.
The hemocoel may also contain such receptors since the injection of carbohydrates resulted in enzyme inactivation. These results are in agreement with
those reported by Siegert [17]; injection of glucose into starving M. sexta larvae and replacement of the normal diet with agar containing various sugars
prevented activation of fat body glycogen phosphorylase, while starved and
agar-fed controls showed enzyme activation.
The effect of injected glucose on glycogen phosphorylase activities may indicate that there is a direct action of glucose on the enzyme. This, however, cannot be the reason for the inactivation of phosphorylase during refeeding since
neither the glucose nor the trehalose concentration of the hemolymph increased
significantly when larvae were refed for 2 min. Additionally, no direct effect
of high glucose or trehalose concentrations on glycogen phosphorylase in larval fat body from M. sextu has been observed in an in vitro system (Siegert
and Mordue, in preparation).
In rats, the molar ratio of insulin/glucagon in the portal vein correlates well
with the hepatic CAMPlevels [18], such a ratio may also determine the overall
direction of metabolism in insects. AKH is released during starvation of M.
sexta larvae [l]and the molar insulin-like/AKH ratio would decrease, favoring
the release of metabolites from fat body stores into the hemolymph. Upon
refeeding the ratio would increase: the insulin-like material directs the storage
Siegert and Mordue
of metabolites in the fat body. Bovine insulin did not influence metabolism in
the present study. It also did not decrease hemolymph trehalose levels or
increase fat body glycogen levels in fifth instar M . sextu larvae [3]. At the moment
the insulin-likematerial from L. rnigrutoria and M. sextu is unavailable for physiological analysis. In order to isolate and characterize the full complement of
possible (peptide) hormone(s), it is required to screen the brain and the CC as
well as the intestine since it contains in L. rnigrutoriu insulin-like material which
is hypoglycemic [16] and hypolipemic [19].
The insulin-related peptide from the corpora cardiaca of Locustu rnigrutoriu
has been isolated and characterized by Hetru et al. [20], but as yet no biological
activity has been assigned to this molecule.
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carbohydrate metabolism in larvae of Munducu sexta. Peptides 22,1037 (1990).
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insect neurosecretory system. Biochem J 156,515 (1976).
4. Orchard I, Loughton BG: A hypolipaemic factor from the corpus cardiacum of locusts. Nature
286,494 (1980).
5. Bell RA, Joachim FG: Techniques for rearing laboratory colonies of tobacco hornworms and
pink bollworms. Ann Entomol SOCAm 69,365 (1976).
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257 (1986).
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cells of brains of Locusfu rnigruforia (Insecta, Orthoptera) encoding a novel member of the
superfamily of insulins. Eur J Biochem 287,249 (1990).
Refeeding in M. sexfa Larvae
13. Kramer KJ, Childs CN, Spiers RD, Jacobs RM: Purification of insulin-like peptides h-om insect
haemolymph and royal jelly. Insect Biochem 12,91(1982).
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phosphorylase, larvae, activity, fat, body, regulation, sexta, manduca, glycogen, refeeding
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