Effects of intestinal insulin-like peptide on glucose catabolism in mealworm larval fat body in vitroDependence on extracellular Ca2+ for its stimulatory action.

Archives of Insect Biochemistry and Physiology 24:113-I 28 (1993)
Effects of Intestinal Insulin-Like Peptide on
Glucose Catabolism in Mealworm Larval Fat
Body In Vitro: Dependence on Extracellular
Ca2+forIts Stimulatory Action
Abdelhamid Mtioui, Lucienne Gourdoux, Bernard Fournier, and
Robert Moreau
Laboratoire de Neuroendocrinologie, URA CNRS, UFR de Biologie Universitk Bordeaux I,
Talence. France
In vitro hormonally induced variations of glucose catabolism in mealworm fat body
tissue were examined by a microradiorespirometric method. An insulin-like peptide (ILP) extracted from the midgut of last larval instar mealworm larvae significantly modified glucose catabolism and was dependent on energy metabolism and
on the Ca2+concentration in the culture medium. Using two different labelled
substrate molecules, the stimulatory effects of ILP (compared with those of rnammalian insulin) on the relative use of the pentose cycle as opposed to the
glycolytic-citric acid cycle by the mealworm fat body were measured in vitro.
Metabolic variations were evaluated using either [I -'4C]glucose or [6-''Clglucose
as substrates. Time course and dose-response curves of ILP and the hormonally
induced variations in total CQ and I4C02kinetics weredetermined. Modification
in the specific radioactivity kinetics of 14C02 derived from [ 1 - l4C] glucose and
[6J4C] glucose molecules under hormonal effects were observed. As demonstrated in
in vivo studies, ILP stimulated the relative utilization of the pentose cycle. However,
this effect was observed much more rapidly, but for a shorter time, with fat body in
vitro. Mammalian insulin produced similar, but not identical effects. Variations in
transrnembranous Ca2 cellular exchanges, induced by either EGTA, nifedipine, or
calcium ionophore ionomycin included in the culture medium, indicated that the
stirnulatory effects of ILP depends on this cation. 6 1993 WiIey-iiss, Inc.
+
Key words: Tenebrio, insect, metabolism, pentose cycle
Acknowledgments: We thank L. Coste for her technical assistance, M.H. Davant for typing the
manuscript, and W. Cazenave for insect breeding.
Received July 22, 1992; accepted March 18, 1993.
Address reprint requests to Robert Moreau, Laboratoire de Neuroendocrinologie, URA CNRS 1 138,
UFR de Biologie Universitb Bordeaux I, Avenue des FacultCs 33405, Talence Cedex, France.
0 1993 Wiley-Liss, Inc.
114
Mtioui e t al.
INTRODUCTION
Over the past few years, we have been investigating different aspects o the
endocrinological control over glucose catabolism in vivo. In particular, we have
been interested in hormonal control of the pentose cycle and glycolysis-TCA*
cycle to carbohydrate metabolism, in several insect species [14].
In this context, we have been interested in extending our previous in vivo
studies to in vitro studies using different tissues, e.g., the mealworm fat body.
By using a new, sensitive respirometric apparatus developed in our laboratory,
we are able to conduct microradiorespirometric studies using small pieces of
functional insect tissues maintained in sterile incubating media. In this manner,
it is possible to compare various experimental situations and to confront results
with the data derived from the in vivo studies. In all insect species we studied,
the relative participation of the pentose cycle to glucose catabolism has been
predominant [5,6].
In the whole mealworm body in vivo, carbohydrate metabolism is governed
by the CC, which control the relative participation of the pentose phosphate
pathway [7] and in some cases, glucose oxidation in the glycolysis-TCA cycle.
In the locust, the inhibitory effect of CC on energy metabolism is attributed to
A M I [8,9], whereas in contrast [lo], ILP increases the relative participation of
the pentose cycle in carbohydrate catabolism. In the present study we investigated the effects of ILP on glucose catabolism pathways in vitro in the mealworm larval fat body, especially in regard to the time course of ILP effects and
to the comparison of the effects of mammalian insulin. Furthermore, we examined the role of extracellular calcium ions in mediating the metabolism effects
of the hormonal signal.
MATERIALS AND METHODS
Insects
Last instar mealworm larvae (Tenebrio rnolitor L.), originally obtained from
Boisbelet (France), were reared in crowded conditions in darkness at 25°C and
70% relative humidity. Insects were fed a mixture of wheat flour, bran, and
potatoes. For the experiments, insects of 150-170 mg of fresh weight were used.
Insects were immobilized by exposure to cold (OOC).
Fat Body Preparation and Incubation Medium
Fat body, freed of Malpighian tubes, from each insect was rapidly removed
by microdissection in sterile saline-Ephrussi at 0°C: 145 mM NaC1, 1.87mM KCI,
0.81 mM CaC12,2.3 mM NaHC03, and 0.55 mM NaH2P04. Following removal,
fat bodies were immediately washed rinsed in saline, then adjusted to 100 mg
fresh weight (30 mg dry weight) on a microscale (Sauter Paris, France). They
were placed in reaction flask for microradiorespirometricexperiments with 500
*Abbreviations used: AKH-I = adipokinetic hormone I; CC = corporacardiaca; C, = [l-'4CJglucose;
C 6 = (l-'4Clglucose; ECTA = ethyleneglycol-bis (p-arnynoethylether) N , N, N',"-tetraacetic acid;
CGPDH = glucose 6-phosphatedeshydrogenase; ILP = intestinal insulin-like peptide; pg Eq.1 = pg
equivalent insulin; SR = specificradioactivity; TCA = tricarboxylicacid.
In Vitro Study of Intestinal Insulin-Like Peptide 115
p1 of sterile incubation medium, according to Candy et al. [ll]. The medium was
composed of 150 mM NaC1,lO mM KC1,4 mM CaC12,2 mM MgC12, and 80 mM
Hepes, pH 7. For control experiments, sterile medium (10 11.1) was injected into
the medium containing the fat bodies. For experimental assays, hormonal
substances, labeled precursors, or drugs were diluted in the medium and
injected either simultaneously or separately. The final volume of the incubation
medium was always 500 11.1. During the experiments it was possible to inject
labeled compounds, hormonal substances, drugs, or control volumes by means
of a microsyringe (10 pl) through a specially adaptated septum, According to
Candy et al. [ l l ] and to our own control experiments, the Ca2+ concentration
required for the maximal hormonal activation of incubating fat body was
determined to be 4 mM.
Intestinal Insulin-Like Peptide Isolation
Intestinal ILP was prepared from larval mealworm midguts as previously
described for the honey bee [12] and adaptated to the locust [9,10] and to the
mealworm larval midgut 1131. The organs were homogenized using an ultrasonic probe (Annemasse, France), sonicated and ultracentrifuged at 100,000 g
at 4°C for 30 min. The supernatant was applied to a Sephadex G-100 column
(length = 70 cm; diameter = 2 cm * ILP fractions (500 pl) were identified using
an insulin radioimmunoassay. I 2 k insulin was purchased from Amersham
(France) and guinea pig insulin antibody from Serono (France). The active
fractions were pooled, concentrated, and then applied to a AcA 54 Ultrogel (IBF,
France) column (length = 40 cm; diameter 1.2 cm). The ILP fractions were
identified using the insulin radioimmunoassay. After 12 h, dialysis against
distilled water at 2"C, control experiments conducted with protease (trypsin)
digestion of the active fractions showed the loss of their immunological and
biological properties. Elsewhere, denaturing polyacrylamide gel electrophoresis led to only one active fraction [14]. Freeze-dried ILP samples were diluted
in the similar sterile incubation medium used for the experimental tissues to
obtain 2-240 pg Eq.1 in 10 pl. This was injected into the incubation medium.
Chemicals
Bovine insulin was purchased from Sigma (Saint Quentin Fallavier, France).
Samples were diluted in the same culture medium as used for the experimental
tissues, to obtain 25-150 pg insulin in 500 p1.
EGTA (Sigma, France) was used as Ca2+ chelator (1.2 mM EGTA for 0.8
mM Ca2+).The voltage-gated L-type Ca2+ channel blocker nifedipine (10
pM), and calcium ionophore ionomycin were purchased from Calbiochem
(San Diego CA).
[l-l~]Glucose(SR = 50.5 mCi/mmol) and [6-14C]glucose(SR = 51.2 mCi/mol)
were purchased from the Commissariat ii l'Energie Atomique (Saclay, France).
Labeled glucose was diluted in distilled water to give 0.4 pCi (9,250 MBq) per 4 11.1
medium by experimental assay.
Microradiorespirometry
A previously described method [7,8] was modified in the present study by
the use of a new, more sensitive microradiorespirometricapparatus. The min-
116
Mtioui et al.
imal rate volume C02 sensitivity was 0.01 5 0.001 kl/min and the minimal 14C02
radioactivity was 0.1 2 0.02 cpm. The gaseous mixture supplied was constituted
of pure N2 (70%)and pure 0 2 (30%).Expired CQ and 14C02were measured at
25°C for durations varying from 45 rnin to 1 h, by placing two mealworm fat
body tissues (100 f 0.5 mg) into a shaken (1 cycle/s) reaction flask (2 ml),
containing 500 kl of incubation medium. The respiratory gas flow rate was 50
mlimin. The experimental flask was connected to an infrared COz analyser
(Binos I1 Leybold, Lyon, France), and a 90% argon and 10% methane radiation
counter Berthold (France) for I4CO2 analysis. An IBM computer recorded total
CO;! and 14C02, continuously and cumulatively, by means of a specially
adapted program Imetronic (Bordeaux, France).
Data Expression
The SR of 14C02 was calculated as cpm per unit volume of C@ contained in
the expired gas mixture after the introduction of the labeled compound into the
reaction flask for C1 and C6.
The c6/c 1 ratio of the SR of 'TO2 released was calculated at 15 rnin after
injection of labeled compounds. We chose this duration because it was at this
time that we obtained the minimal c6/c 1 ratio in control experiments. This
c6/c 1 ratio was often taken as an index of the activity of the pentose cycle (see
in vivo previous studies 171).
The cumulative yield of 14C02 from labeled glucose was expressed as a
percentage of the total cpm introduced in the reaction flask.
RESULTS
Time Course of the Effects of Intestinal Insulin-Like Peptide
ILP was introduced 30, 15, 10, and 5 min before, simultaneously with (0) or
5, 10 min after the introduction of the labeled compounds. The SR of 'YO2 and
the CdC 1 ratio were calculated 15 min after introduction of labeled glucose into
the culture medium, i.e., 45, 30, 25, 20, 15, 10, and 5 rnin after ILP introduction
in the culture medium.
The effects of ILP on the SR of 14C02 produced from the G degradation by
the mealworm fat body are presented in Table 1. The stimulatory effect of ILP
was maximal and significant as compared to controls (27%),when it was present
for at least 10 min. This increase was also significant after 15 min (26.4%) and
after 20 min (24.3%) of ILP introduction into the culture medium.
When C6 was used as the substrate, the SR of 'YO2 derived from its degradation by the mealworm fat body was not significantly modified after ILP
introduction in the culture medium compared to controls (Table 1).
Since Cs/C 1 ratios were decreased following short times of ILP stimulation,
especially at 15 min after (-24%; Table l), this time delay was chosen for all
following experimental measures regarding 14C02 SR evolution.
Dose-Reponse Curve of ILP
A dose-response curve for ILP effects was established (Fig. 1)by introducing
ILP in the mealworm fat body incubation medium, simultaneously with C1. As
a function of increasing concentrations of hormone on in vitro adipose tissue
3.81
4.01 2 0.42
NS
+ 17.9%
0.25
0.26
+4
0.27
0.30
+11.1
Controls
ILP injection
% Variation
0.23
0.15
* 0.05
0.24
0.24
0
1.02 i: 0.08
NS
0.93
NS
k
k
+ 16.7%
4.23
3.81
25
2
0.30
2
0.21
0.26
0.22
- 15.3
CJC, ratio
NS
1.10 i 0.12
0.98
[6-14C]Glucose
4.70 2 0.17
<0.05
+24.3%
3.78
[ l-'4C]Glucose
20
0.25
0.I9
- 24
1.01 f 0.29
NS
1.06 2 0.14
5.31 f 0.09
co.01
+ 26.4%
4.20 i 0.41
15
0.27
0.23
- 14.8
NS
1.20 i: 0.06
1.10 k 0.13
0.26
0.27
+3.8
1.35 c 0.36
NS
1.14 i 0.21
5 . 0 5 k 0.25
c0.05
+ 16.6%
5.22 t 0.40
<0.05
27%
+
4.33 c 0.23
?
5
0.33
4.11
10
*Data of specific radioactivities are expressed as cpm per unit volume COz. Each result represents the mean of n = 6 measurements
& S.E.; P = statistical significance between control insect tissues and ILP-treated insect tissues by Student's t-test. CdC, ratio was calculated 15
min after addition of labelIed compounds. NS = nonsignificant.
NS
NS
0.09
0.99
1.20 2 0.19
&
0.91 t 0.10
0.92 t 0.08
k 0.38
NS
+5.2%
3.62 c 0.12
30
3.40 2 0.14
45
P
Controls
(Candy saline addition)
ILP
70 Variation
P
Controls
(Candy saline addition)
ILP
Time (min) after ILP treatment
TABLE 1. Time Course of Effects of ILP (60 pg Eq.I) on Specific Radioactivity of I4CO2Derived From [l-'4C]Glucose and [6-'4C]Glucose,
Calculated 15 Min After Addition of Labeled Compound Into Incubation Medium*
118
Mtioui et al.
6
.4
4
I
0
1
40
'
1
'
1
.
1
1
1
80
120
pg Equivalents Insulin
'
1
L
240
Fig. 1. Dose-responsecurve of ILP. Thespecific radioactivityof 14C02after introductionof increasing
doses of ILP into mealworm fat body incubation medium is calculated 15 rnin after the injection of
[ I -'4C]glucose. Data are expressed as cpm per unit volume COz. Each result represents the mean of
n = 6measurements -+ S.E.
(40, 60, 80, 120, and 240 pg Eq.1) the SR of the I4CO2 increased in parallel, with
a maximum of 43.8%at 15 min after the simultaneous introduction of both ILP
and labeled compound in the medium.
Effects of Bovine Insulin
Bovine insulin (80 pg) was introduced into the mealworm fat body incubation
medium and the time course of its effects on the SR of "CO2 derived from C1
was calculated 15 min after introduction of the labeled compound into the
culture medium (Table 2). Bovine insulin increased the C1 catabolism (41.8%
more C1 after 30 min insulin action compared with controls). The effect appeared later than in ILP-stimulated fat bodies (10 min).
Bovine insulin had no significant effect on the amount of I4C02derived from
Cg. The c6/c1 ratio was decreased after 30 min bovine insulin action.
Cumulative Yields of l4C02From C6
When a 60 pg Eq.1 dose ILP was added simultaneously with I4C6 into the
mealworm fat body incubation medium, the total I4CO2measured 25 or 45 rnin
after addition of the labeled compound was significantly decreased (Table 3)
5.94 -+ 0.59
1.01 -t- 0.23
0.17
4.19 ? 0.32
1.01 r 0.12
0.24
NS
c0.05
P
* 0.34
0.23
0.25
4.39
1.09
Controls
(45 min)
4.67 2 0.58
1.26 2 0.18
0.27
Bovin insulin
addition
(45 min after)
NS
NS
P
*Data represent the mean of n = 6 measurements t S.E.; P = statistical significance between control insect fat body and bovine insulin
stirnulatory effects on fat body in culture medium by Student's t-test. NS = nonsignificant.
CdCi
{1-'4C]Glucose
[6-"C]Glucose
Bovine insulin
addition
(30 min after)
Controls
(30 min)
TABLE 2. Effects of Bovine Insulin (80 pg) 30 or 45 Min After Addition Into Mealworm Fat Body Incubation Medium on the Specific
Radioactivity of 14C02Derived From [1-'4Cl- or [6-'4CJGlucose and on CJC, Ratio, Calculated 15 Min After Introduction Into Medium of
Labeled Glucose*
120
Mtioui et a[.
when compared to controls (-25.8% at 45 min). The same phenomenon appeared with a 80 pg bovine insulin dose, but was not significant.
ILP Ca2+Dependent Stimulatory Effects
Effects of different amounts of external Ca2+ ions in the medium: a dose-response curve for the SR of ' T O 2 derived from C1 was obtained with the
concentration of external Ca2+ ions (Fig. 2). Following incubations of fat bodies
with low or high Ca2+ concentrations (either < 4 mM or from 8 mM), ILP (100
pg Eq. I) failed to increase the SR of
derived from C1 (Table 4), whereas
the maximum effects of ILP were observed with 4 mM external Ca2+.
Effects of EGTA or nifedipine on the SR of I4CO2 kinetics derived from (21:
both EGTA and nifedipine injected into the incubation medium (at 15 min)
provoked a decreased in the SR when compared t o the controls. During the
action of the ILP (100 pg Eq. I), a marked reduction in the stimulatory effects of
ILP was observed (Table 5) in the presence of either EGTA or nifedipine. The
ILP,
inhibitory effect was maximal 10 min after addition of EGTA or EGTA
nifedipine or nifedipine ILP, and this effect continued during at least 30 min.
Effects of ionomycin on the SR of ' T O 2 kinetics derived from C1: a significant
increase of the 1 4 C 0 2 SR was obtained in 3 mM Ca2+ medium when the Ca2+
ionophore ionomycin was added. Maximum levels of SR were reached rapidly
(15 min) and the steady level of SR was maintained for a long time (45 min). In
4 mM Ca2+ medium, no significant modifications were observed.
When ionomycin was complemented simultaneously with ILP in 3mM Ca2+
medium, a significant increase was obtained 15 min after hormonal stimulation, whereas in 4 mM Ca2+ medium, the 14C@ SR slightly decreased but not
significantly (Table 6).
+
+
DISCUSSION
In a previous study, we showed the hypoglycemic effects of honeybee ILP on
honeybee hemolymph [IS] and the effects of locust ILP on locust hemolymph
TABLE 3. Comparison of ILP (60 pg Eq.1) and Bovine Insulin (80 pg) Effects on Cumulative
Yields of 14C02From [6-14C]Glurose25 or 45 Min After Additioa of Labeled Glucose Into
Mealworm Fat Bodv Incubation Medium*
Time (min) after labeled
compound addition
25
45
0.24 2 0.03
0.19 ? 0.02
NS
0.62 & 0.03
0.46 2 0.02
<0.01
0.26 2 0.04
0.26 t 0.03
NS
0.67 0.12
0.61 k 0.13
ILP
Controls
ILP
P
Bovine insulin
Controls
Insulin
P
*
NS
*Data are expressed as a percentage of total injected radioactivity. Each result represents the mean
of n = 6 measurements ? S.E.; P = statistical significance between control insect fat bodies and
experimental insect fat bodies by Student's t-test. ILP = insulin-like peptide. NS = not significant.
In Vitro Study of Intestinal Insulin-Like Peptide 121
0
2
4
6
8
mM Ca2'
Fig. 2. Dose-response curve of calcium. The specific radioactivity of 14C02 after introduction of
increasing doses of calcium into mealworm fat body incubation medium is calculated 15 min after
the injection of [l -'4C]glucose.DataareexpressedascpmperunitvolumeCO2. Each resultrepresents
the mean of n = 6 measurements k S.E.
[8j. We have also demonstrated that these ILP molecules affected the metabolism in insects comparable to those of mammalian insulin [13,15].
The present in vitro study on mealworm fat body strengthens and extends
these observations on the effects of ILP extracted from the midgut of mealworm
larvae on glucose catabolism. We have already shown that in in vivo locusts,
ILP can modify the relative contributions of the pentose cycle and glycolysisTCA cycle to the production of the 14C02 [lo]. The relative participation of the
pentose pathway in glucose degradation is significantly increased ( 30%) 30
rnin after ILP injection. We also observed a constant decrease in the C6/C I ratio
during the entire period of activity of ILP; the lowest C6/C 1 ratio was observed
when ILP was injected 15 min before the 14C-labeledglucose (i.e., 30 min after
ILP injection).
In the in vitro mealworm fat body study, the stimulatory effect of ILP is
significant (+42.3%)and it arises rapidly (10 min after addition of ILP in the
medium). The decrease in the C6/C 1 ratio under the action of ILP is significant
for the shortest times only (10, 15, 20, and 25 min), with a maximum decrease
(-24% at 15 min) when ILP and [I1*C]-labeledglucose are simultaneously added
+
2.93 ? 0.22
3.28 -c 0.38
NS
11.9
0
2.79 2 0.39
3.31 % 0.77
NS
18.6
2
3.55 k 0.35
4 . 9 % 0.47
NS
26.7
3
4.20 % 0.41
6.01 2 0.29
aJ.01
43.1
4
2 0.41
5.88 ? 0.37
c0.05
27.2
4.62
6
4.59 2 0.68
5.34 i- 0.61
NS
16.3
8
*Data are expressed as cpm per unit volume C 0 2 .Each result represents the mean of n = 6 measurements +- S.E.; P = statistical significance
between control mealworm fat body and ILP-treated mealworm fat body by Student's t-test. Incubations were performed in the presence of
100 pg Eq.1 ILPI500 ~1 medium. NS = nonsignificant; S.R. = specific radioactivity.
% Variation
P
S.R. of C1 controls
S.R. ofC1 + ILP
Extracellular free Ca" (mM)
TABLE 4. Dependence of ILP-Induced Stimulation of the S.R. of '*C02 Derived From [l-'4C]Glucose on Extracellular Caz+Mealworm Fat
Bodies Dissected and Preincubated in Physiological Saline, Incubated for 15 Min at Varying Concentrations of Ca2+*
In Vitro Study of Intestinal Insulin-Like Peptide 123
TABLE 5. Consequence on ILP-Induced Stimulation of the S.R. of 14C02Derived From
[l-'4C]Glucose (C1) of EGTA and Nifedipine Added Into 4 mM Incubation Medium*
Treatment
Effect (max)
4.59
+
3.23 f 0.42
+ ILP
+ ILP + EGTA
4.41 + 0.57
Controls
4.63 2 0.31
Nifedipine
3.47
+ ILP
7.61 & 0.77
+
ILP
+ Nifedipine
P
-29.6
<0.05
-37.2
<0.05
-25
<0.01
47.3
<0.01
* 0.31
Controls
EGTA
Variations (%)
7.02 & 0.59
* 0.21
* 0.68
6 measurements * S.E. P = statistical significance of differences
4.01
*Data represent the mean of n =
was determined using Student's t-test for paired comparisons. S.R. = specific radioactivity.
Mealworm fat bodies dissected and preincubated in physiological saline were incubated for 45
min with simultaneously added ILI' and labelled compound 15 min after the beginning of
incubation; 6 mM EGTA or 10 pM nifedipine was injected into the medium. Incubations were
performed in the presence of 100 pg Eq.1 ILP/500 p1 medium. Results are expressed at the time of
maximum effect (10 min of EGTA or nifedipine action).
to the incubated fat bodies (Le., 15 min after ILP addition) in the medium.
Moreover, when ILP was added after the labelled substrate, we observed
significant effects of ILP for a 10-min incubation with the decrease of the CdC 1
ratio remaining measurable (-14.8%). This result shows that the ILP action on
the mealworm fat body in vitro is rapid but lasts only for a relatively short
period (25 min), as compared to the in vivo studies performed in locusts.
Addition of a 60 pg Eq.1 dose of ILP to c 6 in the mealworm fat body incubation
medium did not change the total l4C& measured at different periods after ILP
stimulation. However, the cumulative yields of 1 4 C 0 2 from Cj dropped when
compared to controls (-25.8% at 45 rnin). A same trend was observed using a
80 pg bovine insulin dose, but was not significant. These observations are in
agreement with previous data obtained in vivo using locusts [lo], in which the
decrease of the 14C02 SR derived from c6 was observed 90 min after ILP injection.
Moreover, the cumulative yields of 'TO2 from C6 were also signrficantly decreased
3 h after ILP injection in vivo. These results suggest a slight inhibition of the
glycolysis-TCA cycle by ILP, which is more rapid in the in vitro study. We failed
to observe similar effects after insulin injection; however, the inhibitory effects of
insulin on glycolysis TCA cycle in mammals are well known [lq.
From the present in vitro data, it appears that in a first step, ILP rapidly
increases (5 min) the relative importance of the pentose cycle in the mealworm
fat body and, in a second step, ILP seems to be able to slightly inhibit the glucose
catabolism by glycolysis and the TCA cycle. However, this phenomenon is
measurable only after a long incubation delay (minimum 45-60 min) in the
+ CII (h)
3.10 2 0.50
?
?
0.20
1NS
0.46
*
5.13 ? 0.23
) NS
4.88 i 0.30
3.80
4.03
5.57 i 0.31
5.03 ? 0.37
4.50 i 0.27
1%
?
) NS
0.50
5.30
?
0.70
1 NS
5.55 2 0.33
5.10 t 0.40
4.45
4.92 t 0.46
) NS
4.93 i 0.40
3.85 i 0.24
3.55 i 0.35
3.55 t 0.35
) *
35 min
15min
-C
1 NS
0.35
2
1 NS
0.60
5.30 ? 0.30
5.05
4.10 2 0.30
) NS
3.98 i 0.38
4.57
4.52 i 0.34
4.08 i 0.27
) *
3.15 i 0.44
45 min
‘Mealworm fat bodies dissected and preincubated in physiological saline were incubated for 45 min with simultaneously added ILP, labelled
compound, and CII. Incubations were performed in the presence of 100 pg Eq.1 ILP and 1 FM CII in 500 p1 medium. Data represent the mean
of n = 6 measurements ? S.E. CI1 = calcium ionophore ionomycin; NS = nonsignificant; S.R. = specific radioactivity.
“P = statistical significance of differences (between b vs. a, d vs. c, f vs. e, and h vs. g), determined using Student’s t-test for paired comparisons, P < 0.05.
4mM Ca2 + ILP
3.98 2 0.37
4mM Ca2 + ILP (g)
1 NS
2.80 t 0.20
4mM Ca2 + CII (f)
1NS
4.17 t 0.28
2.85 -c 0.13
+ CII (d)
4mM CaZ(e)
3mM Ca2 + ILP
3.22 ? 0.31
3.65 2 0.43
3mM Ca2 + CII (b)
3mM CaZ t ILP (c)
) NS
3.70 2 0.40
3mM Caz (a)
1 NS
10 min
Treatment
TABLE 6. Time Course of 1LP-Induced Stimulation of the S.R. of “COz Derived From ~l-’4ClGlucoseWhen CII Is Added in 3 mM or
4 mM Ca2’ Concentration Incubation Mediat
In Vitro Study of Intestinal Insulin-like Peptide 125
presence of ILP. Bovine insulin exhibits similar properties, but its stimulatory
effects on the pentose cycle of the mealworm fat body appear more slowly. This
relatively delayed action of insulin might be due to differences in hormone
binding to the specific ILP-receptors in the insect-cell membrane, since in
mammals it is known that insulin binds quickly to its receptors [17-191.
The major result of our study concerns the stimulatory effects of ILP on
utilization of the pentose cycle and agrees with previous findings obtained in
the laboratory [20] of the stimulatory effect of ILP on the specific activity of the
key enzyme (GGPDH) of this metabolic pathway. It is noteworthy that insulin
possesses the same function in mammals [16].
Numerous studies on the mechanism of action of insect metabolic hormones
in vitro were developed. They concern catabolic hormones contained in CC
extracts [Zl-23, 341, hypertrehalosemic peptides from the CC [24], octopamine
[25], neuroparsin [26-281, and AKH I and I1 [29-341. Many of these insect in vitro
studies have demonstrated that hormonal activation of metabolism is highly
dependent on the external concentration of Ca2+ ions in the medium [22, 35,
361.
In contrast, the mechanism of action of insect anabolic hormones via in vitro
studies has been much less studied and is primarily concerned with glucidic, or
lipid metabolism [15-201. In the present study, we investigated the effects of ILP
on glucidic catabolism in mealworm fat body in vitro and also the dependence
of ILP on Ca2+ ions. We demonstrated a rapid and significant action of ILP on
the glucose catabolism effect, where the major result is a rise in the amount of
C1 metabolized in the pentose cycle. These phenomena are in line with previous
results [ZO] and suggest a direct stimulating action of ILP on GGPDH, on one
hand, combined with a glycolytic inhibition of the glycolysis Krebs cycle,
whereas, on the other hand, they suggest an inhibition of the enzyme chain. This
latter hypothesis requires further experiments.
Our present experiments using varying concentrations of Ca2+,a Ca2
ionophore, EGTA, and nifedipine, provide evidence that the stimulatory effects
of ILP on glucose metabolism depend on the level of external Ca2' concentration. Increasing concentrations of external Ca2+ induced a dose-response curve
of the 14C02 SR levels, which partially mimicks the ILP effects. Without ILP, a
maximum elevation of the amount of C1 oxidized was obtained with 6 mM Ca2+
concentration, but in the presence of ILP, 4 mM became sufficient to induce it,
i.e., in a physiological Ca2+ range equivalent to that measured in the insect
hemolymph [ll].Elsewhere, it is known that certain effects of insect hormones
(phosphorylase activation by AKH [30], phosphoinositide turnover [28]), can
be mimicked by calcium ionophores (as ionomycin), suggesting an increase in
the concentration of cytosolic free Ca2+. In our experiments, ionomycin mimicked the ILP action when external Ca2+ in medium lowered under 4 mM. The
ability of ILP to increase rapidly (within 10 min of incubation) the pentose cycle
utilization in mealworm fat body is inhibited by the L-type Ca2+ channel
blocker nifedipine and also by the divalent cation chelator EGTA. This suggests
a voltage-dependent Ca2+ channel intervention. All the data indicate that ILP
in vitro stirnulatory effects on mealworm fat bodies are clearly dependent on
Ca2+ ion. A comparison may be established with mammals, where expression
+
126
Mtioui et at.
of biological effects of insulin requires physiological Ca2' ion concentration in
the extracellular fluid [17].
In a recent study, Draznin et al. [38] showed that insulin increases the
concentration in cytosolic free-Ca2+ions in isolated rat adipocytes. Herein, such
an action of ILP on our biological model was not demonstrated (e.g., by
measurements of cytosolic free Ca2+ movements). However, it appears a priori
unprobable since higher levels of external Ca2+ (28 mM) or addition of 1 FM
ionomycin in incubating media containing 4 mM external Ca2+ caused a
blockage or even a slight decrease in ILP stimulatory effects, respectively.
Several analyses described involvement of antagonist effects with low and high
concentrations of Ca2+ ions. Thus a Ca2+-relatedcytotoxicity has been demonstrated recently [37], and with high amounts of cytosolic free Ca2+ it was
described as a feed-back action on transduction mechanisms in target cells by
increasing phosphodiesterase activities or inhibiting phosphoinositide turnover [28].
Thus we may conclude that the role of external Ca2+ in the ILP-induced
metabolic activation of mealworm fat body is certainly important, but it must
be considered as one aspect of the hormonal effects of ILP. It is evident that other
possible different steps of ILP signal-transduction need to be investigated.
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