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Comparison of hormonal effects on glucose metabolism in Tenebrio molitor larval fat body, muscle and brain tissues
Camp. Biochem.
Pergamon
Physiol. Vol. lOBA, No. I, pp. 97-105, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0300-9629/94 $7.00 + 0.00
Comparison of hormonal effects on glucose metabolism in Tenebrio molitor larval fat body, muscle and brain tissues Abdelhamid Mtioui, Ahmed Bahjou, Lucienne Gourdoux Robert Moreau
and
Laboratoire de Physiologie des Insectes, URA CNRS 1138 Neuroendocrinologie, Bordeaux I, Avenue des Facultks, F-33405 Talence-Cedex, France
Universitk
Pathways of glucose metabolism in mealworm Tenebrio molitor larval fat body, mandibular muscle and brain tissues were studied, in uitro, by means of a micro-radiorespirometric method. The respiratory gaseous mixture supplied to tissues was composed of 30% O2 and 70% N1. Glucose and trehalose were used as energy substrates. The effects of the addition of different metabolic hormones were evaluated. Variations in the relative utilization of the two main glucose metabolic pathways were evaluated by using either [l-‘4C]Glucose or [6-‘4C]Glucose as substrates. The total expired CO2 from the different tissues was measured in the presence of hormones. No significant variations were observed, except for the brain where total CO2 significantly increased following addition of corpora cardiaca (CC) extracts. The insect insulin-like peptide (ILP) stimulated pentose cycle utilization for glucose degradation in both fat body and muscle, but not in brain tissue. The Tenebrio CC hormonal extracts rapidly diverted glucose from the pentose cycle in fat body which exhibits a highly active pentose pathway in controls; this effect lasted for a long time. Muscle and brain tissue, which did not spontaneously exhibit activity of the pentose cycle, were insensitive to CC extracts. Cumulative yields of 14C02 derived from (l-‘4C]glucose and [6-‘4C]glucose were recorded. In brain tissue, neither of the hormones used had any effect. In contrast, in muscle and particularly in the fat body, cumulative yields of 14C02 derived from [6-‘4C]glucosewere modified significantly by both hormones. Key words: In vitro; Glucose catabolism; Insulin-like peptide; Corpora Cardiaca; Tenebrio molitor; Fat body; Muscle; Brain. Comp. Biochem. Physiol. 108A, 97-105, 1994.
Introduction The precise comparison of hormonal effects on different isolated insect tissues is not an
easy task, especially when the insects belong to a relatively small species. Micromethod analyses in these cases are particularly useful to resolve such quantitative problems. To this aim, we have recently developed a new experimental system, allowing the in vitro study of small pieces of insect tissues, isolated in incubation medium
to: Robert Moreau, Laboratoire de Physiologie des Insectes, URA CNRS 1138 Neuroendocrinologie, Universitt Bordeaux I, Avenue des Fact&es, F-33405 Talence-Cedex, France. Fax: 56 84 87 50. Received 18 June 1993; accepted 6 August 1993. Correspondence
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(Mtioui et al., 1993). Using this more sensitive apparatus, in vitro carbohydrate metabolism can be measured both in the presence and absence of anabolic or catabolic hormonal substances and will thus enable us to complete our knowledge regarding the regulation of glucose metabolism in insects. The in vivo study of the relative contributions of the glycolysis-TCA cycle and the of pentose phosphate cycle to carbohydrate catabolism in insects, has been investigated using different experimental methods since the initial study of Silva et al. (1958). Several authors since this period have accomplished technical advances (Chefurka, 1966; Chefurka et al., 1970; Horie et al., 1968; Dixon and Shuel, 1969; Ela et al., 1970; Moreau, 1973; Moreau et al., 1977, 1980; Gourdoux, 1979, 1980; Gourdoux and Dutrieu, 1974; Gourdoux et al., 1985; Romaschin and Taylor, 1981; Ben Khay et al., 1987). However, beyond these studies few further technical advances have been achieved to date for the in vitro metabolic study of small sample tissue, because of certain technical difficulties related to apparatus sensitivity. Thus, the in vitro approach has routinely been carried out in mammals using either indirect methods or more directly but without high sensitivity (Castex et al., 1987). The aim of this present study was to provide a comparison of the hormonal regulation of the relative utilization of the two major glucose metabolic pathways, in three different isolated larval Tenebrio molitor tissues. We have compared the in vitro effects of an endogenous mealworm anabolic hormone, extracted from the mesenteron, insulin-like peptide (ILP), with those induced by a natural catabolic hormone produced in the corpora cardiaca (CC) on mealworm tissues. Materials and Methods Experimental animals
Insect mealworms (Tenebrio molitor ) were reared in our laboratory with a diet of wheat flour and potatoes at 25”C, under permanent darkness and at 70% relative humidity. The insects were taken out of the colony at the end of the last larval instar. Their mean body weight was 170 f 10 mg.
Mtioui et al.
Preparation and incubation of tissues
After cold anaesthesia, Tenebrio larval fat bodies, free of Malpighian tubes, mandibular muscles and brain were removed by micro-dissection in cold (2°C) saline solution: 150 mM NaCl, 1.87 mM KCl, 0.81 mM CaCl,, 2.3 mM NaHC03 and 0.55 mM NaH,PO, (extraction time durations were about 3-5 min). Immediately afterwards, tissues were rinsed in saline, then adjusted to a constant fresh weight for each tissue type on a microscale (Sauter, Paris, France). Each tissue type was washed and placed in incubation medium, in a reaction flask for microradiorespiromet~c expe~mentation with 500 ~1 of sterile incubation medium. This medium, according to Candy et al. (1976), was composed of 150 mM NaCl, 10 mM KCl, 4 mM CaCl,, 2 mM MgC12, and 80 mM Hepes pH 7. Preparation of hormonal substances
We used an intestinal insulin-like peptide (ILP), extracted from larval mealworm midgut and purified as previously described (Moreau et al., 1981). After rapid microdissection, batches of 20 midguts were sonicated in saline buffer pH 7.4 and ultracentrifuged at 105g at 4°C for 30 min. The infra-supernatant was submitted to a Sephadex G- 100 (Pharmacia, France) column and eluted with the same buffer. The active fractions were detected by insulin radio-immunoassays (RIA), dialysed against distilled water, concentrated, then reapplied to a second Ultrogel (IBF France) column and eluted with the same buffer. After a second series of insulin RIA, the active samples were dialysed, pooled and lyophilised. Samples were diluted in the same sterile culture medium as used for the experiments, to obtain 2-60 pg insulin equivalents (pg Eq I) in 10 1.11and which was added by injection with a microsyringe, into the experimental incubation medium. Corpora cardiaca (CC) extracts were prepared from young adult mealworm brains as previously described (Gourdoux, 1980). After rapid microdissection, 20 pairs of whole CC were sonicated in saline Ephrussi and ultracentrifuged at 10sg at 4°C for 15 min. The supernatant was stored (at - 30°C) of less than 30 davs. ~, for a neriod , d
Glucose metabolism in mealworm
Samples were diluted in the same sterile culture medium as used for the experimental tissues, to obtain 0.025-0.5 pair CC equivalents (pCC Eq) in 10 ~1 and were added into the culture medium using the same procedure as for ILP. >%ficro-radiorespirometric method
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pose, for its own energy metabolism, of non-labelled glucose (5.5 mM) and trehalose (27 mM) which were diluted in a final volume of 500 ~1 incubation medium. Chemicals
[ 1-‘4C]Glucose (C, ) (specific radioactivity 50.5 mCi/mmol) and [6-‘4C]Glucose (C,) We used a method previously described 51.2 mCi/mmol) were (Gourdoux, 1980; Gourdoux et al., 1985; (specific radioactivity purchased from the Commissariat $ Moreau et al., 1980; Ben Khay et al., 1987), Atomique (Saclay, France). but which has been specifically modified in 1’Energie the present study by the introduction of Labelled glucose was diluted in distilled water to give a final specific activity of a new micro-radiorespirometric apparatus Nonwith higher sensitivity, enabling in vitro 0.4 PCi (9.250 MBq)/4 ~1 medium. labelled glucose and trehalose were puranalysis (Mtioui et al., 1993). The minimal chased from Prolabo (Paris, France). rate volume of CO1 detectable was 0.01 + 0.001 plI/min and the minimal 14C02 radioactivity was 0.5 + 0.1 cpm. The gaseous mixture supplied consisted of a Data expression The specific radioactivities of 14C02 pure N2 (70%) and pure O2 (30%) mixture, ) and free of CO*. Expired total CO, and 14C0, (SR) derived from [l-‘4C]glucose (C, [6-‘4C]glucose (C,), were calculated as the were continually recorded at 25°C for 45 min, by placing mealworm tissues of ratio of counts per min (cpm) per unit volume of CO*, measured in the expired constant adjusted fresh weight (as indicated gaseous mixture every 10 min after the inabove) into a 2 ml shaking (1 cycle/s) reaction flask, in the presence of 500 ~1 troduction of labelled glucose into the reaction flask. The development of SR curves, (final volume) of incubation medium. The as a function of time, was taken as an index respiratory gas flow rate was 50 ml/min. of a stronger or lower level of participation The experimental flask was connected to an of 14C substrate in COZ formation. The infrared COZ analyser (Binos II Leybold, of Lyon, France) which gave CO, rates of C,/C, ratio of the specific radioactivities 14C0, released, was calculated 15 min after production directly in parts per million injection of labelled compounds for fat (ppm), and a 90% argon and 10% methane body and brain, since it is at this time that radiation counter (Berthold, France) for we observed a minimal C,/C, ratio in fat 14C02 analysis. An IBM computer recorded total CO1 and 14C02, continuously and body control experiments. For muscle, the ratio was calculated after 5 min incubation. cumulatively, by means of a specially adapted program (Imetronic, Bordeaux, The C,/C, ratio has been frequently used as France). a reliable qualitative index of the activity of For experimental assays, 4 ~1 of labelled the pentose cycle [see in vivo previous studprecursors and 10 ,~l of hormonal subies (Ela et al., 1970; Gourdoux, 1980; stances were simultaneously injected into Gourdoux et al., 1985; Moreau et al., 1980; the medium. For control experiments, 10 ,U1 Ben Khay et al., 1987)]. of the sterile medium solution replaced The 45 min cumulative yields of 14C02 hormonal preparations. The final volume of from labelled glucose were expressed as the the incubation medium was always 500 ~1. percentage of the total radioactivity introDuring the experiments it was possible to duced into the medium. Thus we could introduce labelled compounds, hormonal calculate the fraction of [l-‘4C]glucose and substances, or control volumes by means of [6-‘4C]glucose oxidized to COZ. It is known a microsyringe injection (10 ~1) through a that the oxidation rate of carbon 6 of special adaptated septum. Each batch of glucose is an index of the relative utilisation in vitro isolated tissue (two fat bodies, i.e. of the cycle glycolysis-TCA cycle, by 100 mg fresh weight, eight muscles, i.e. measuring the 14C02 production from [615 mg, and 10 brains, i.e. 2 mg) could dis“C]glucose.
Abdelhamid Mtioui ef
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Results Hormonal influences on total expired CO* (Fig. 1)
Fat body exhibited the lowest CO, production per unit dry weight (controls: 0.7 pl/mg/hr, Fig. la) as compared to or brain tissues (3.23 and muscle 4.02 pl/mg/hr, respectively, Fig. 1b and lc). No significant variations of total CO, (Fig. 1) were observed when the three Tenebrio tissues were incubated with ILP. Following incubation with CC, a nonsignificant increase of total CO* was obtained for fat body and muscle (Fig. la and lb). This increase, however, became significant in brain tissue (Fig. Ic). Hormonal influences on the kinetics of 14C02 spec@ radioactivity (Fig. 2)
When using C, as substrate, ILP increased the specific radioactivity (SR) of 14C02 in both fat body and muscular tissues, but these effects were not identical (Fig. 2a and b). The stimulation was more intensive (400% versus 30%) appeared more rapidly (5 versus 15 min) in muscle than in fat body but disappeared rapidly in muscle. In contrast, ILP failed to modify the brain metabolism of C, (Fig. 2~). CC extracts modified the SR of 14C0, from fat body (Fig. 2a) where glucose was diverted from the pentose cycle (- 71.5 and -64.1% after 25 and 35 min, respectively).
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al.
In the other tissues, CC extracts had no influence (Fig. 2b and c). When using C6 as substrate, ILP had no effect. CC extracts were active only in muscle (Fig. 2b). The decrease of the SR 14C02 was maximal after 45 min ;L 20%). C6/CI ratios (Fig. 2) In control tissues, we observed the lowest C6/C1 ratio 15 min after the addition of labelled compounds into the medium, for mealworm fat body (0.37; Fig. Za), while for mandibular muscle and brain the C,/C, ratio was very close to unity, particularly for muscle where it was calculated 5 min after the introduction of labelled glucose (Fig. 2b and c). In all experiments using hormones, labelled glucose and hormonal preparations were simultaneously introduced into the incubation medium. In these conditions the C6/C, ratios were modified in fat body and in muscle (Fig. 2a and 2b) but not modified in brain tissue (Fig. 2~). In muscle, ILP induced a rapid decrease of the C&/C, ratio calculated at 5 min, (0.4 versus l), while CC was inactive (Fig. 2b). In fat body, both hormonal substances were active (Fig. 2a), but with opposite effects. ILP significantly decreased the C,/C, ratio (0.22 versus 0.37) while CC induced a strong increase (0.5 versus 0.37).
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Fig. 1. Total CO2 produced by fat body (a), muscle (b), and brain (c), in controls (Ct), after ILP treatment (ILP) and CC extracts treatment (CC). Data are expressed as PI/ mg dry weight/hr; each result represents the mean of six measures + SEM. *P < 0.05.
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Glucose metabolism in mealworm
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Fig. 2. Specific activities of r4C02 from fl -‘4C]glucose (C, ) and [6-r4C]glucose (C,), and C,/C, ratio in fat body (a), muscle (b), and brain (c). Specific activities o 14C02 are expressed as cpm/unit volume CO,; Controls: +; ILP: ...@..; CC extracts: - -. The C,/C, ratios are calculated 15 min after simultaneous addition of hormonal prepara f ton and labelled glucose for fat body (a) and brain (c) in controls (Ct), after ILP treatment (ILP) and CC extracts treatment (CC); for muscle, it is calculated after 5min (b). It represents the ratio between specific activities of 14C02 from [I-‘4C]glucose (C,) and (6-‘4C]glucose (C,). Each result represents the mean of six measures f SEM. *P < 0.05, **P < 0.01.
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[1-14C] action, cumulative yields of 14C0, from Cl and C, are quite identical, whereas the ILP and CC efects on C, metabolism. differences between cumulative yields of “C0, from C, and cumulative yields of In controls, after 45 min of incubation, 14C0, from C6 were greatest after ILP the percentage of total radioactivity action (Fig. 3a). recovered as 14C02 was 1.33% for two
Cumulative yields of 14C02 from glucose and [6-‘4C]glucose (Fig. 3)
fat bodies (Fig. 3a), 1.1% for eight muscles (Fig. 3b) and 0.5% for 10 brains (Fig. 3~). After ILP action, the cumulative yields of 14C02 (Fig. 3) increased, but not significantly so, for fat body and muscle (Fig. 3a and b). CC extracts reduced this cumulative yield only for fat body (Fig. 3a). In brain, neither hormonal preparation had any effect. ILP and CC eflects on C6 metabolism. In controls, after 45 min of incubation, the percentage of total radioactivity recovered as 14C02 was 0.7% for two fat bodies (Fig. 3a), 1% for eight muscles (Fig. 3b) and 0.5% for 10 brains (Fig. 3~). After ILP action, cumulative yields of 14C02increased but not significantly (Fig. 3) for the three different Tenebrio tissues. CC extracts increased the percentage of total radioactivity recovered as ‘“CO,& fat body (Fig. 3a). Thus, in this tissue, after CC
(a)
Discussion The fact that different tissues may not have the same metabolic capacity or hormonal reactivity is not a new idea, and many previous reports have dealt with this possibility in insects as in mammals (Downer, 1981; Downer and Laufer, 1983; Beaulieu, 1978). However, our present comparison of hormonal effects on the two major metabolic pathways of glucose in different insect tissues confirm and extend these data. In insects, hormonal effects on fat body metabolism are to be well-expected since it is sensitive to a wide range of hormonal substances and is also the main target for AKI-iijs) and hyperglycemic hormones (Steele, 1961). Fat body is also a major target for ILP: in a previous paper, we showed that in mealworm fat body, in vitro,
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Fig. 3. Cumulative yields of 14C02 produced from either [I-‘4C]glucose (0) or [6-‘4C]glucose (B) by fat body (a), muscle (b) and brain (c), in controls (Ct), after ILP treatment (ILP) and CC extracts treatment (CC). Data are expressed as a percentage of total radioactivity added in the incubation medium calculated after 45 min. Each result represents the mean of six measures + SEM.
Glucose metabolism in mealworm
ILP induces lipogenesis (Bahjou et al., 1990). Furthermore, we have already demonstrated the in vivo hypoglycemic effects of honeybee ILP on honeybee hemolymph (Bounias et al., 1986), and the in vivo effects of locust ILP on locust hemolymph (Moreau et al., 1982). We have also demonstrated that these ILP molecules affect glucose metabolism in insects in a manner comparable to that of mammalian insulin (Bounias et al., 1986; Ben Khay, 1986; Ben Khay et al., 1987). In live locusts, ILP can modify the relative contributions of the pentose cycle and glycolysis TCA cycle to the production of the 14C02. The relative participation of the pentose pathway in glucose degradation is significantly increased (+30%) 30 min after hormonal stimulation, (Ben Khay et al., 1987). In this present in vitro study on mealworm fat body, the addition of insulin-like peptide extracted from the midgut of mealworm larvae modifies glucose catabolism. In this tissue, as in the intact insect, the basal participation of pentose cycle is large (C/C, = 0.37) and ILP increases this relative participation of pentose cycle to glucose catabolism (C,/C, = 0.22). In contrast, in muscle, catabolism of glucose is mainly carried out by glycolysis-TCA cycle (C,/C, = 1). When ILP is added, the formation of 14C02 from C, is strongly increased ( + 2 15%); this phenomenon is very rapid (5 min), but lasted for only 15 min. The participation of the pentose cycle is also strongly increased (C,/C, = 0.4). In the brain, the participation of pentose pathway is moderate (C,/C, = 0.8) and addition of ILP has no effect on the catabolism of glucose in this tissue. Thus, muscle reacts in a manner similar to fat body towards ILP (but more rapidly and strongly), while brain tissue seems to be insensitive to this anabolic hormone. Several in viva and in vitro studies on the hormonal action of CC extracts have been developed since the first studies of Steele (1961). CC extracts from many insect species have been shown to contain a number of neuropeptides which control carbohydrate and lipid metabolisms. In previous in vivo studies we showed that, in the adult male locust (Gourdoux et al.,
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1985) or in the mealworm (Gourdoux, 1980) that CC extracts were able to modify the relative contributions of the pentose cycle and the glycolysis-TCA cycle to glucose catabolism. In the present in vitro study of the mealworm fat body, the inhibitory effects of CC on C1 degradation were large (-63.2%) and induced rapidly (15 min after CC addition into the medium). The increase of C,/C, ratio under the action of CC is significant and its maximum is reached after 15 min. These results demonstrate that CC action on the pentose cycle in mealworm fat body in vitro is larger and more rapid but lasts only for a relatively short period (35 min), as compared to in vivo studies on the whole insect. Indeed, in vivo, the inhibitory effect of CC extract on C, degradation is moderate, but the C6/CI ratio is significantly increased; it appears 2 hr after injections of CC extracts (Gourdoux, 1980). When CC extracts are added simultaneously with C, into the mealworm fat body incubation medium, the cumulative yields of 14C02 are significantly stimulated but after a relatively long incubation period (75-90 min, results not shown here). The comparison with previous in vivo studies shows that, in this case the stimulation of the 14C02 production from Cg, is visible only after an incubation time of 3 hr (Gourdoux, 1980). CC extracts are able to stimulate the degradation of glucose in glycolysis-TCA in mealworm larvae, and the effect is more rapid and larger in vitro than in vivo. In muscle in vitro which does not exhibit pentose cycle activity when incubations are performed in the absence of hormones, CC extracts have no effect on this pathway (C,/C, = 1 either in controls or in treated subjects). However, they decrease glucose catabolism via glycolysis-TCA after a delay of 25 min. The weak effect of CC extracts on muscle is similar to the lack of stimulatory effects of glucagon in mammalian muscles : these muscles possess specific receptors to glucagon, but adenylate cyclase not stimulated by this hormone beaulieu, 1978). In brain in vitro, CC extracts do not modify the pathways of glucose catabolism. We obtained the same result with ILP. The brain of this insect thus seems to be insen-
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Abdelhamid Mtioui et al.
sitive to metabolic hormones, at least as far as concerns glucose catabolism. We may conclude that the relative interventions of the major pathways of glucose catabolism is different according to the tissue in the same insect. The metabolism of fat body seems to be responsible for the greater part of general metabolism of mealworm larvae. Indeed the relative importance of pentose cycle observed in viuo is essentially carried out by the fat body in which the phenomenon is amplified. This tissue represents 30% of wet weight and it is the site of synthesis for stores, essentially lipids, the need for NADPH, produced by pentose cycle, being large. The pathways of glucose catabolism are well regulated by metabolic hormones; catabolic hormones (contained in CC) reduce the importance of pentose cycle, anabolic hormone (ILP) increasing the contribution of this pathway. Both the other tissues studied need energy for their functioning, glucose being catabolized by the glycolysis-TCA cycle and supplying ATP. If the catabolic pathways are regulated by ILP in muscle, they are not modified by CC extracts, and brain seems to be insensitive to both metabolic hormones. This comparative study may be extended in future to other larval tissues which participate in metabolism and further during the metamorphosis of certain larval tissues, such as imaginal discs, digestive tract and Malpighian tubes. Acknowledgements-We wish to thank L. Coste for her technical assistance, M. H. Davant for typing the manuscript and W. Cazenave for breeding the insects.
References Bahjou A., Gourdoux L., Moreau R. and Dutrieu J. (1990) In vitro lipid metabolism of mealworm fat body. Arch. Insect Biochem. Physiol. 15, 21-32. Beaulieu E. E. (1978) Hormones. Aspects fondamentaux et physio-pathologiques. Hermann Editeurs des Sciences et des Arts. pp. l-549. Ben Khay A. (1986) Metabolisme glucidique et lipidique du criquet migrateur : effets compares des hormones des corpora cardiaca et d’un peptide extrait du mesenteron. These Doctorat d’universite Bordeaux I, (171 pp.). Ben Khay A., Gourdoux L., Moreau R. and Dutrieu J. (1987) Effects of insulin-like peptide on glucose catabolism in male adult Locusta migratoriu. Arch. Insect Biochem. Physiol. 4, 233-239.
Bounias M., Moreau R. and Gourdoux L. (1986) Effects of honey bee insulin immunoreacted peptide on the heamolymph lipid and carbohydrates: interaction of vertebrate somatostatine. Insect Biothem. 16, 721-731. Candy D. L., Hall L. J. and Spencer I. M. (1976) The metabolism of glycerol in Locust Schistocerca greguriu during flight. (Orth. Acrid.). J. Insect Physiof. 22, 583-536. Castex C., Tahri A., Hoo-Paris R. and Sutter B. Ch. J. (1987) Glucose oxidation by adipose tissue of the edible dormouse (Glis g/is) during hibernation and arousal: effect of insulin. Comp. Biochem. Physiol. MA, 33-36. Chefurka W. (1966) Estimation of pathways of carbohydrate metabolism in insect. Proc. Entomol. Sot. Ontario. 9, 17-26. Chefurka W., Horie Y. and Robinson J. R. (1970) Contribution of the pentose cycle in glucose metabolism by insects. Comp. Biochem. Physiol. 37, 143-152. Dixon S. E. and Shuel R. W. (1969) Respiration of queen and worker honeybee larvae on differentially labelled glucose 14C. Comp. Biochem. Physiol. 30, 105-l 12. Downer R. G. H. (1981) Energy Metabolism in Insects, pp. l-244. Plenum Press, New York. Downer R. G. H. and Laufer H. (1983) Invertebrate Endocrinology. Vol. 1. Alan R. Liss, Inc., New York. Ela R., Chefurka W. and Robinson J. R. (1970) In viuo glucose metabolism in the normal and poisoned cockroach, Periplaneta americana. J. Insect Physiol. 16, 2137-2156. Gourdoux L. (1979) Quelques aspects du mitabolisme glucidolipidique et de sa regulation par les Corpora Cardiaca chez le coleoptere Tenebrio molitor L. au tours de son developpement. These Doctorat d’etat, Bordeaux (217 pp). Gourdoux L. (1980) The catabolism of glucose in Tenebrio molitor: the effects of corpora cardiaca. J. Insect Physiol. 26, 729-738. Gourdoux L. and Dutrieu J. (1974) Le cycle des pentoses pendant le developpement du Coliopthe Tenebrio molitor. CR Sot. Biol. 168, 1289-1292. Gourdoux L., Moreau R., Ben Khay A. and Dutrieu J. (1985) In viuo glucose catabolism in male adult Locusta migratoriu: effects of corpora cardiaca. Comp. Biochem. Physiol. 81B. 485489. Horie Y., Nakasone S. and Ito T. (1968) The conversion of [“‘C] carbohydrates into CO> and lipid by the silkworm Bombyx mori. J. Insect Physiol. 14, 971-983. Moreau R. (1973) Recherches sur quelques aspects des phenomenes physiques, metaboliques et physiologiques qui accompagnent ou conditionnent l’expansion des ailes des Ltpidopteres. These Doctorat d’etat, Bordeaux (141 pp.). Moreau R., Gourdoux L. and Cava-Ruart M. (1980) Neuroendocrine control possibility of the glucose degradation pathways in non-diapausing Pieris brassicue L. Lepidoptera. Camp. Biochem. Physiol. 66B, 589~-592. Moreau R., Gourdoux L. and Dutrieu J. (1977) Utilisation comparee du cycle des pentoses en fonction des variations thermiques chez deux
Glucose metabolism in mealworm Lepidopteres Bombyx mori L. et Pieris brassicae L. Comp. Biochem. Physiol. 56, 175-179. Moreau R., Gourdoux L., Lequellec Y. and Dutrieu J. (1982) Endocrine control of hemolymph carbohydrates in Locusta migratoria: comparison between effects of two endogenous hormonal extracts and effects of insulin and glucagon. Comp. Biochem. Physiol. 74, 666-673. Moreau R., Raoelison C. and Sutter B. Ch. J. (1981) An intestinal insulin-like molecule in Apis mellijica (L). Comp. Biochem. Physiol. 69A, 79-83. Mtioui A., Gourdoux L., Fournier B. and Moreau R. I 1993) The effects of insulin-like peptide on glucose
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catabolism in mealworm larval fat body in tritro; dependance on extracellular Ca2+ for its stimulatory action. Arch. Insect Biochem. Physiol. 24, 113-128. Romaschin A. and Taylor N. F. (1981) The in vioo effects of 3-deoxy-3-fluoro D-glucose on respiration in Locusta migratoria. Can. J. Biochem. 59, 261-269. Silva G. M.. Doyle W. P. and Wang C. H. (1958) Glucose catabolism in the American cockroach. Nature 182, 102-103. Steele J. E. (1961) Occurence of a hyperglycaemic factor in the corpus cardiacum in an insect. Nature, London, 192, 680-68 I.
Pergamon
Physiol. Vol. lOBA, No. I, pp. 97-105, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0300-9629/94 $7.00 + 0.00
Comparison of hormonal effects on glucose metabolism in Tenebrio molitor larval fat body, muscle and brain tissues Abdelhamid Mtioui, Ahmed Bahjou, Lucienne Gourdoux Robert Moreau
and
Laboratoire de Physiologie des Insectes, URA CNRS 1138 Neuroendocrinologie, Bordeaux I, Avenue des Facultks, F-33405 Talence-Cedex, France
Universitk
Pathways of glucose metabolism in mealworm Tenebrio molitor larval fat body, mandibular muscle and brain tissues were studied, in uitro, by means of a micro-radiorespirometric method. The respiratory gaseous mixture supplied to tissues was composed of 30% O2 and 70% N1. Glucose and trehalose were used as energy substrates. The effects of the addition of different metabolic hormones were evaluated. Variations in the relative utilization of the two main glucose metabolic pathways were evaluated by using either [l-‘4C]Glucose or [6-‘4C]Glucose as substrates. The total expired CO2 from the different tissues was measured in the presence of hormones. No significant variations were observed, except for the brain where total CO2 significantly increased following addition of corpora cardiaca (CC) extracts. The insect insulin-like peptide (ILP) stimulated pentose cycle utilization for glucose degradation in both fat body and muscle, but not in brain tissue. The Tenebrio CC hormonal extracts rapidly diverted glucose from the pentose cycle in fat body which exhibits a highly active pentose pathway in controls; this effect lasted for a long time. Muscle and brain tissue, which did not spontaneously exhibit activity of the pentose cycle, were insensitive to CC extracts. Cumulative yields of 14C02 derived from (l-‘4C]glucose and [6-‘4C]glucose were recorded. In brain tissue, neither of the hormones used had any effect. In contrast, in muscle and particularly in the fat body, cumulative yields of 14C02 derived from [6-‘4C]glucosewere modified significantly by both hormones. Key words: In vitro; Glucose catabolism; Insulin-like peptide; Corpora Cardiaca; Tenebrio molitor; Fat body; Muscle; Brain. Comp. Biochem. Physiol. 108A, 97-105, 1994.
Introduction The precise comparison of hormonal effects on different isolated insect tissues is not an
easy task, especially when the insects belong to a relatively small species. Micromethod analyses in these cases are particularly useful to resolve such quantitative problems. To this aim, we have recently developed a new experimental system, allowing the in vitro study of small pieces of insect tissues, isolated in incubation medium
to: Robert Moreau, Laboratoire de Physiologie des Insectes, URA CNRS 1138 Neuroendocrinologie, Universitt Bordeaux I, Avenue des Fact&es, F-33405 Talence-Cedex, France. Fax: 56 84 87 50. Received 18 June 1993; accepted 6 August 1993. Correspondence
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(Mtioui et al., 1993). Using this more sensitive apparatus, in vitro carbohydrate metabolism can be measured both in the presence and absence of anabolic or catabolic hormonal substances and will thus enable us to complete our knowledge regarding the regulation of glucose metabolism in insects. The in vivo study of the relative contributions of the glycolysis-TCA cycle and the of pentose phosphate cycle to carbohydrate catabolism in insects, has been investigated using different experimental methods since the initial study of Silva et al. (1958). Several authors since this period have accomplished technical advances (Chefurka, 1966; Chefurka et al., 1970; Horie et al., 1968; Dixon and Shuel, 1969; Ela et al., 1970; Moreau, 1973; Moreau et al., 1977, 1980; Gourdoux, 1979, 1980; Gourdoux and Dutrieu, 1974; Gourdoux et al., 1985; Romaschin and Taylor, 1981; Ben Khay et al., 1987). However, beyond these studies few further technical advances have been achieved to date for the in vitro metabolic study of small sample tissue, because of certain technical difficulties related to apparatus sensitivity. Thus, the in vitro approach has routinely been carried out in mammals using either indirect methods or more directly but without high sensitivity (Castex et al., 1987). The aim of this present study was to provide a comparison of the hormonal regulation of the relative utilization of the two major glucose metabolic pathways, in three different isolated larval Tenebrio molitor tissues. We have compared the in vitro effects of an endogenous mealworm anabolic hormone, extracted from the mesenteron, insulin-like peptide (ILP), with those induced by a natural catabolic hormone produced in the corpora cardiaca (CC) on mealworm tissues. Materials and Methods Experimental animals
Insect mealworms (Tenebrio molitor ) were reared in our laboratory with a diet of wheat flour and potatoes at 25”C, under permanent darkness and at 70% relative humidity. The insects were taken out of the colony at the end of the last larval instar. Their mean body weight was 170 f 10 mg.
Mtioui et al.
Preparation and incubation of tissues
After cold anaesthesia, Tenebrio larval fat bodies, free of Malpighian tubes, mandibular muscles and brain were removed by micro-dissection in cold (2°C) saline solution: 150 mM NaCl, 1.87 mM KCl, 0.81 mM CaCl,, 2.3 mM NaHC03 and 0.55 mM NaH,PO, (extraction time durations were about 3-5 min). Immediately afterwards, tissues were rinsed in saline, then adjusted to a constant fresh weight for each tissue type on a microscale (Sauter, Paris, France). Each tissue type was washed and placed in incubation medium, in a reaction flask for microradiorespiromet~c expe~mentation with 500 ~1 of sterile incubation medium. This medium, according to Candy et al. (1976), was composed of 150 mM NaCl, 10 mM KCl, 4 mM CaCl,, 2 mM MgC12, and 80 mM Hepes pH 7. Preparation of hormonal substances
We used an intestinal insulin-like peptide (ILP), extracted from larval mealworm midgut and purified as previously described (Moreau et al., 1981). After rapid microdissection, batches of 20 midguts were sonicated in saline buffer pH 7.4 and ultracentrifuged at 105g at 4°C for 30 min. The infra-supernatant was submitted to a Sephadex G- 100 (Pharmacia, France) column and eluted with the same buffer. The active fractions were detected by insulin radio-immunoassays (RIA), dialysed against distilled water, concentrated, then reapplied to a second Ultrogel (IBF France) column and eluted with the same buffer. After a second series of insulin RIA, the active samples were dialysed, pooled and lyophilised. Samples were diluted in the same sterile culture medium as used for the experiments, to obtain 2-60 pg insulin equivalents (pg Eq I) in 10 1.11and which was added by injection with a microsyringe, into the experimental incubation medium. Corpora cardiaca (CC) extracts were prepared from young adult mealworm brains as previously described (Gourdoux, 1980). After rapid microdissection, 20 pairs of whole CC were sonicated in saline Ephrussi and ultracentrifuged at 10sg at 4°C for 15 min. The supernatant was stored (at - 30°C) of less than 30 davs. ~, for a neriod , d
Glucose metabolism in mealworm
Samples were diluted in the same sterile culture medium as used for the experimental tissues, to obtain 0.025-0.5 pair CC equivalents (pCC Eq) in 10 ~1 and were added into the culture medium using the same procedure as for ILP. >%ficro-radiorespirometric method
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pose, for its own energy metabolism, of non-labelled glucose (5.5 mM) and trehalose (27 mM) which were diluted in a final volume of 500 ~1 incubation medium. Chemicals
[ 1-‘4C]Glucose (C, ) (specific radioactivity 50.5 mCi/mmol) and [6-‘4C]Glucose (C,) We used a method previously described 51.2 mCi/mmol) were (Gourdoux, 1980; Gourdoux et al., 1985; (specific radioactivity purchased from the Commissariat $ Moreau et al., 1980; Ben Khay et al., 1987), Atomique (Saclay, France). but which has been specifically modified in 1’Energie the present study by the introduction of Labelled glucose was diluted in distilled water to give a final specific activity of a new micro-radiorespirometric apparatus Nonwith higher sensitivity, enabling in vitro 0.4 PCi (9.250 MBq)/4 ~1 medium. labelled glucose and trehalose were puranalysis (Mtioui et al., 1993). The minimal chased from Prolabo (Paris, France). rate volume of CO1 detectable was 0.01 + 0.001 plI/min and the minimal 14C02 radioactivity was 0.5 + 0.1 cpm. The gaseous mixture supplied consisted of a Data expression The specific radioactivities of 14C02 pure N2 (70%) and pure O2 (30%) mixture, ) and free of CO*. Expired total CO, and 14C0, (SR) derived from [l-‘4C]glucose (C, [6-‘4C]glucose (C,), were calculated as the were continually recorded at 25°C for 45 min, by placing mealworm tissues of ratio of counts per min (cpm) per unit volume of CO*, measured in the expired constant adjusted fresh weight (as indicated gaseous mixture every 10 min after the inabove) into a 2 ml shaking (1 cycle/s) reaction flask, in the presence of 500 ~1 troduction of labelled glucose into the reaction flask. The development of SR curves, (final volume) of incubation medium. The as a function of time, was taken as an index respiratory gas flow rate was 50 ml/min. of a stronger or lower level of participation The experimental flask was connected to an of 14C substrate in COZ formation. The infrared COZ analyser (Binos II Leybold, of Lyon, France) which gave CO, rates of C,/C, ratio of the specific radioactivities 14C0, released, was calculated 15 min after production directly in parts per million injection of labelled compounds for fat (ppm), and a 90% argon and 10% methane body and brain, since it is at this time that radiation counter (Berthold, France) for we observed a minimal C,/C, ratio in fat 14C02 analysis. An IBM computer recorded total CO1 and 14C02, continuously and body control experiments. For muscle, the ratio was calculated after 5 min incubation. cumulatively, by means of a specially adapted program (Imetronic, Bordeaux, The C,/C, ratio has been frequently used as France). a reliable qualitative index of the activity of For experimental assays, 4 ~1 of labelled the pentose cycle [see in vivo previous studprecursors and 10 ,~l of hormonal subies (Ela et al., 1970; Gourdoux, 1980; stances were simultaneously injected into Gourdoux et al., 1985; Moreau et al., 1980; the medium. For control experiments, 10 ,U1 Ben Khay et al., 1987)]. of the sterile medium solution replaced The 45 min cumulative yields of 14C02 hormonal preparations. The final volume of from labelled glucose were expressed as the the incubation medium was always 500 ~1. percentage of the total radioactivity introDuring the experiments it was possible to duced into the medium. Thus we could introduce labelled compounds, hormonal calculate the fraction of [l-‘4C]glucose and substances, or control volumes by means of [6-‘4C]glucose oxidized to COZ. It is known a microsyringe injection (10 ~1) through a that the oxidation rate of carbon 6 of special adaptated septum. Each batch of glucose is an index of the relative utilisation in vitro isolated tissue (two fat bodies, i.e. of the cycle glycolysis-TCA cycle, by 100 mg fresh weight, eight muscles, i.e. measuring the 14C02 production from [615 mg, and 10 brains, i.e. 2 mg) could dis“C]glucose.
Abdelhamid Mtioui ef
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Results Hormonal influences on total expired CO* (Fig. 1)
Fat body exhibited the lowest CO, production per unit dry weight (controls: 0.7 pl/mg/hr, Fig. la) as compared to or brain tissues (3.23 and muscle 4.02 pl/mg/hr, respectively, Fig. 1b and lc). No significant variations of total CO, (Fig. 1) were observed when the three Tenebrio tissues were incubated with ILP. Following incubation with CC, a nonsignificant increase of total CO* was obtained for fat body and muscle (Fig. la and lb). This increase, however, became significant in brain tissue (Fig. Ic). Hormonal influences on the kinetics of 14C02 spec@ radioactivity (Fig. 2)
When using C, as substrate, ILP increased the specific radioactivity (SR) of 14C02 in both fat body and muscular tissues, but these effects were not identical (Fig. 2a and b). The stimulation was more intensive (400% versus 30%) appeared more rapidly (5 versus 15 min) in muscle than in fat body but disappeared rapidly in muscle. In contrast, ILP failed to modify the brain metabolism of C, (Fig. 2~). CC extracts modified the SR of 14C0, from fat body (Fig. 2a) where glucose was diverted from the pentose cycle (- 71.5 and -64.1% after 25 and 35 min, respectively).
(4
al.
In the other tissues, CC extracts had no influence (Fig. 2b and c). When using C6 as substrate, ILP had no effect. CC extracts were active only in muscle (Fig. 2b). The decrease of the SR 14C02 was maximal after 45 min ;L 20%). C6/CI ratios (Fig. 2) In control tissues, we observed the lowest C6/C1 ratio 15 min after the addition of labelled compounds into the medium, for mealworm fat body (0.37; Fig. Za), while for mandibular muscle and brain the C,/C, ratio was very close to unity, particularly for muscle where it was calculated 5 min after the introduction of labelled glucose (Fig. 2b and c). In all experiments using hormones, labelled glucose and hormonal preparations were simultaneously introduced into the incubation medium. In these conditions the C6/C, ratios were modified in fat body and in muscle (Fig. 2a and 2b) but not modified in brain tissue (Fig. 2~). In muscle, ILP induced a rapid decrease of the C&/C, ratio calculated at 5 min, (0.4 versus l), while CC was inactive (Fig. 2b). In fat body, both hormonal substances were active (Fig. 2a), but with opposite effects. ILP significantly decreased the C,/C, ratio (0.22 versus 0.37) while CC induced a strong increase (0.5 versus 0.37).
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Fig. 1. Total CO2 produced by fat body (a), muscle (b), and brain (c), in controls (Ct), after ILP treatment (ILP) and CC extracts treatment (CC). Data are expressed as PI/ mg dry weight/hr; each result represents the mean of six measures + SEM. *P < 0.05.
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Glucose metabolism in mealworm
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Fig. 2. Specific activities of r4C02 from fl -‘4C]glucose (C, ) and [6-r4C]glucose (C,), and C,/C, ratio in fat body (a), muscle (b), and brain (c). Specific activities o 14C02 are expressed as cpm/unit volume CO,; Controls: +; ILP: ...@..; CC extracts: - -. The C,/C, ratios are calculated 15 min after simultaneous addition of hormonal prepara f ton and labelled glucose for fat body (a) and brain (c) in controls (Ct), after ILP treatment (ILP) and CC extracts treatment (CC); for muscle, it is calculated after 5min (b). It represents the ratio between specific activities of 14C02 from [I-‘4C]glucose (C,) and (6-‘4C]glucose (C,). Each result represents the mean of six measures f SEM. *P < 0.05, **P < 0.01.
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et al.
[1-14C] action, cumulative yields of 14C0, from Cl and C, are quite identical, whereas the ILP and CC efects on C, metabolism. differences between cumulative yields of “C0, from C, and cumulative yields of In controls, after 45 min of incubation, 14C0, from C6 were greatest after ILP the percentage of total radioactivity action (Fig. 3a). recovered as 14C02 was 1.33% for two
Cumulative yields of 14C02 from glucose and [6-‘4C]glucose (Fig. 3)
fat bodies (Fig. 3a), 1.1% for eight muscles (Fig. 3b) and 0.5% for 10 brains (Fig. 3~). After ILP action, the cumulative yields of 14C02 (Fig. 3) increased, but not significantly so, for fat body and muscle (Fig. 3a and b). CC extracts reduced this cumulative yield only for fat body (Fig. 3a). In brain, neither hormonal preparation had any effect. ILP and CC eflects on C6 metabolism. In controls, after 45 min of incubation, the percentage of total radioactivity recovered as 14C02 was 0.7% for two fat bodies (Fig. 3a), 1% for eight muscles (Fig. 3b) and 0.5% for 10 brains (Fig. 3~). After ILP action, cumulative yields of 14C02increased but not significantly (Fig. 3) for the three different Tenebrio tissues. CC extracts increased the percentage of total radioactivity recovered as ‘“CO,& fat body (Fig. 3a). Thus, in this tissue, after CC
(a)
Discussion The fact that different tissues may not have the same metabolic capacity or hormonal reactivity is not a new idea, and many previous reports have dealt with this possibility in insects as in mammals (Downer, 1981; Downer and Laufer, 1983; Beaulieu, 1978). However, our present comparison of hormonal effects on the two major metabolic pathways of glucose in different insect tissues confirm and extend these data. In insects, hormonal effects on fat body metabolism are to be well-expected since it is sensitive to a wide range of hormonal substances and is also the main target for AKI-iijs) and hyperglycemic hormones (Steele, 1961). Fat body is also a major target for ILP: in a previous paper, we showed that in mealworm fat body, in vitro,
(c)
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0 .S
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ct
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cc
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Fig. 3. Cumulative yields of 14C02 produced from either [I-‘4C]glucose (0) or [6-‘4C]glucose (B) by fat body (a), muscle (b) and brain (c), in controls (Ct), after ILP treatment (ILP) and CC extracts treatment (CC). Data are expressed as a percentage of total radioactivity added in the incubation medium calculated after 45 min. Each result represents the mean of six measures + SEM.
Glucose metabolism in mealworm
ILP induces lipogenesis (Bahjou et al., 1990). Furthermore, we have already demonstrated the in vivo hypoglycemic effects of honeybee ILP on honeybee hemolymph (Bounias et al., 1986), and the in vivo effects of locust ILP on locust hemolymph (Moreau et al., 1982). We have also demonstrated that these ILP molecules affect glucose metabolism in insects in a manner comparable to that of mammalian insulin (Bounias et al., 1986; Ben Khay, 1986; Ben Khay et al., 1987). In live locusts, ILP can modify the relative contributions of the pentose cycle and glycolysis TCA cycle to the production of the 14C02. The relative participation of the pentose pathway in glucose degradation is significantly increased (+30%) 30 min after hormonal stimulation, (Ben Khay et al., 1987). In this present in vitro study on mealworm fat body, the addition of insulin-like peptide extracted from the midgut of mealworm larvae modifies glucose catabolism. In this tissue, as in the intact insect, the basal participation of pentose cycle is large (C/C, = 0.37) and ILP increases this relative participation of pentose cycle to glucose catabolism (C,/C, = 0.22). In contrast, in muscle, catabolism of glucose is mainly carried out by glycolysis-TCA cycle (C,/C, = 1). When ILP is added, the formation of 14C02 from C, is strongly increased ( + 2 15%); this phenomenon is very rapid (5 min), but lasted for only 15 min. The participation of the pentose cycle is also strongly increased (C,/C, = 0.4). In the brain, the participation of pentose pathway is moderate (C,/C, = 0.8) and addition of ILP has no effect on the catabolism of glucose in this tissue. Thus, muscle reacts in a manner similar to fat body towards ILP (but more rapidly and strongly), while brain tissue seems to be insensitive to this anabolic hormone. Several in viva and in vitro studies on the hormonal action of CC extracts have been developed since the first studies of Steele (1961). CC extracts from many insect species have been shown to contain a number of neuropeptides which control carbohydrate and lipid metabolisms. In previous in vivo studies we showed that, in the adult male locust (Gourdoux et al.,
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1985) or in the mealworm (Gourdoux, 1980) that CC extracts were able to modify the relative contributions of the pentose cycle and the glycolysis-TCA cycle to glucose catabolism. In the present in vitro study of the mealworm fat body, the inhibitory effects of CC on C1 degradation were large (-63.2%) and induced rapidly (15 min after CC addition into the medium). The increase of C,/C, ratio under the action of CC is significant and its maximum is reached after 15 min. These results demonstrate that CC action on the pentose cycle in mealworm fat body in vitro is larger and more rapid but lasts only for a relatively short period (35 min), as compared to in vivo studies on the whole insect. Indeed, in vivo, the inhibitory effect of CC extract on C, degradation is moderate, but the C6/CI ratio is significantly increased; it appears 2 hr after injections of CC extracts (Gourdoux, 1980). When CC extracts are added simultaneously with C, into the mealworm fat body incubation medium, the cumulative yields of 14C02 are significantly stimulated but after a relatively long incubation period (75-90 min, results not shown here). The comparison with previous in vivo studies shows that, in this case the stimulation of the 14C02 production from Cg, is visible only after an incubation time of 3 hr (Gourdoux, 1980). CC extracts are able to stimulate the degradation of glucose in glycolysis-TCA in mealworm larvae, and the effect is more rapid and larger in vitro than in vivo. In muscle in vitro which does not exhibit pentose cycle activity when incubations are performed in the absence of hormones, CC extracts have no effect on this pathway (C,/C, = 1 either in controls or in treated subjects). However, they decrease glucose catabolism via glycolysis-TCA after a delay of 25 min. The weak effect of CC extracts on muscle is similar to the lack of stimulatory effects of glucagon in mammalian muscles : these muscles possess specific receptors to glucagon, but adenylate cyclase not stimulated by this hormone beaulieu, 1978). In brain in vitro, CC extracts do not modify the pathways of glucose catabolism. We obtained the same result with ILP. The brain of this insect thus seems to be insen-
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Abdelhamid Mtioui et al.
sitive to metabolic hormones, at least as far as concerns glucose catabolism. We may conclude that the relative interventions of the major pathways of glucose catabolism is different according to the tissue in the same insect. The metabolism of fat body seems to be responsible for the greater part of general metabolism of mealworm larvae. Indeed the relative importance of pentose cycle observed in viuo is essentially carried out by the fat body in which the phenomenon is amplified. This tissue represents 30% of wet weight and it is the site of synthesis for stores, essentially lipids, the need for NADPH, produced by pentose cycle, being large. The pathways of glucose catabolism are well regulated by metabolic hormones; catabolic hormones (contained in CC) reduce the importance of pentose cycle, anabolic hormone (ILP) increasing the contribution of this pathway. Both the other tissues studied need energy for their functioning, glucose being catabolized by the glycolysis-TCA cycle and supplying ATP. If the catabolic pathways are regulated by ILP in muscle, they are not modified by CC extracts, and brain seems to be insensitive to both metabolic hormones. This comparative study may be extended in future to other larval tissues which participate in metabolism and further during the metamorphosis of certain larval tissues, such as imaginal discs, digestive tract and Malpighian tubes. Acknowledgements-We wish to thank L. Coste for her technical assistance, M. H. Davant for typing the manuscript and W. Cazenave for breeding the insects.
References Bahjou A., Gourdoux L., Moreau R. and Dutrieu J. (1990) In vitro lipid metabolism of mealworm fat body. Arch. Insect Biochem. Physiol. 15, 21-32. Beaulieu E. E. (1978) Hormones. Aspects fondamentaux et physio-pathologiques. Hermann Editeurs des Sciences et des Arts. pp. l-549. Ben Khay A. (1986) Metabolisme glucidique et lipidique du criquet migrateur : effets compares des hormones des corpora cardiaca et d’un peptide extrait du mesenteron. These Doctorat d’universite Bordeaux I, (171 pp.). Ben Khay A., Gourdoux L., Moreau R. and Dutrieu J. (1987) Effects of insulin-like peptide on glucose catabolism in male adult Locusta migratoriu. Arch. Insect Biochem. Physiol. 4, 233-239.
Bounias M., Moreau R. and Gourdoux L. (1986) Effects of honey bee insulin immunoreacted peptide on the heamolymph lipid and carbohydrates: interaction of vertebrate somatostatine. Insect Biothem. 16, 721-731. Candy D. L., Hall L. J. and Spencer I. M. (1976) The metabolism of glycerol in Locust Schistocerca greguriu during flight. (Orth. Acrid.). J. Insect Physiof. 22, 583-536. Castex C., Tahri A., Hoo-Paris R. and Sutter B. Ch. J. (1987) Glucose oxidation by adipose tissue of the edible dormouse (Glis g/is) during hibernation and arousal: effect of insulin. Comp. Biochem. Physiol. MA, 33-36. Chefurka W. (1966) Estimation of pathways of carbohydrate metabolism in insect. Proc. Entomol. Sot. Ontario. 9, 17-26. Chefurka W., Horie Y. and Robinson J. R. (1970) Contribution of the pentose cycle in glucose metabolism by insects. Comp. Biochem. Physiol. 37, 143-152. Dixon S. E. and Shuel R. W. (1969) Respiration of queen and worker honeybee larvae on differentially labelled glucose 14C. Comp. Biochem. Physiol. 30, 105-l 12. Downer R. G. H. (1981) Energy Metabolism in Insects, pp. l-244. Plenum Press, New York. Downer R. G. H. and Laufer H. (1983) Invertebrate Endocrinology. Vol. 1. Alan R. Liss, Inc., New York. Ela R., Chefurka W. and Robinson J. R. (1970) In viuo glucose metabolism in the normal and poisoned cockroach, Periplaneta americana. J. Insect Physiol. 16, 2137-2156. Gourdoux L. (1979) Quelques aspects du mitabolisme glucidolipidique et de sa regulation par les Corpora Cardiaca chez le coleoptere Tenebrio molitor L. au tours de son developpement. These Doctorat d’etat, Bordeaux (217 pp). Gourdoux L. (1980) The catabolism of glucose in Tenebrio molitor: the effects of corpora cardiaca. J. Insect Physiol. 26, 729-738. Gourdoux L. and Dutrieu J. (1974) Le cycle des pentoses pendant le developpement du Coliopthe Tenebrio molitor. CR Sot. Biol. 168, 1289-1292. Gourdoux L., Moreau R., Ben Khay A. and Dutrieu J. (1985) In viuo glucose catabolism in male adult Locusta migratoriu: effects of corpora cardiaca. Comp. Biochem. Physiol. 81B. 485489. Horie Y., Nakasone S. and Ito T. (1968) The conversion of [“‘C] carbohydrates into CO> and lipid by the silkworm Bombyx mori. J. Insect Physiol. 14, 971-983. Moreau R. (1973) Recherches sur quelques aspects des phenomenes physiques, metaboliques et physiologiques qui accompagnent ou conditionnent l’expansion des ailes des Ltpidopteres. These Doctorat d’etat, Bordeaux (141 pp.). Moreau R., Gourdoux L. and Cava-Ruart M. (1980) Neuroendocrine control possibility of the glucose degradation pathways in non-diapausing Pieris brassicue L. Lepidoptera. Camp. Biochem. Physiol. 66B, 589~-592. Moreau R., Gourdoux L. and Dutrieu J. (1977) Utilisation comparee du cycle des pentoses en fonction des variations thermiques chez deux
Glucose metabolism in mealworm Lepidopteres Bombyx mori L. et Pieris brassicae L. Comp. Biochem. Physiol. 56, 175-179. Moreau R., Gourdoux L., Lequellec Y. and Dutrieu J. (1982) Endocrine control of hemolymph carbohydrates in Locusta migratoria: comparison between effects of two endogenous hormonal extracts and effects of insulin and glucagon. Comp. Biochem. Physiol. 74, 666-673. Moreau R., Raoelison C. and Sutter B. Ch. J. (1981) An intestinal insulin-like molecule in Apis mellijica (L). Comp. Biochem. Physiol. 69A, 79-83. Mtioui A., Gourdoux L., Fournier B. and Moreau R. I 1993) The effects of insulin-like peptide on glucose
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catabolism in mealworm larval fat body in tritro; dependance on extracellular Ca2+ for its stimulatory action. Arch. Insect Biochem. Physiol. 24, 113-128. Romaschin A. and Taylor N. F. (1981) The in vioo effects of 3-deoxy-3-fluoro D-glucose on respiration in Locusta migratoria. Can. J. Biochem. 59, 261-269. Silva G. M.. Doyle W. P. and Wang C. H. (1958) Glucose catabolism in the American cockroach. Nature 182, 102-103. Steele J. E. (1961) Occurence of a hyperglycaemic factor in the corpus cardiacum in an insect. Nature, London, 192, 680-68 I.