A.Th.M. van den Broek

Regulation of glycogenolysis in the locust fat body during flight

Glycogen reserves in the fat body of Locusta migratoria decrease dramatically during the first two hours of flight. In fat body of rested locusts only 10% of glycogen phosphorylase occurs in the active form. The enzyme is activated significantly during flight, when up to one-third of the total phosphorylase becomes active. Phosphorylase activation can also be accomplished by injection of corpus cardiacum extracts, to give a maximum of circa 75% active enzyme. Locust fat body is shown to contain protein kinase activity, which can be activated both by cyclic AMP and cyclic GMP. Apparent Ka-values are 0.13μM for cyclic AMP and 0.16 μM for cyclic GMP. Results are discussed in relation to regulation of substrate utilization during flight.

Regulation of glycogen phosphorylase activity in fat body of Locusta migratoria and Periplaneta americana

Saline extracts of the corpus cardiacum (CC) of Locusta migratoria activate glycogen phosphorylase in locust fat body. The response of phosphorylase to CC extracts and to synthetic adipokinetic hormone (AKH) suggests that the factor responsible for the activating effect of the CC on phosphorylase is AKH, supplemented to a minor degree with Compound II. Octopamine does not influence fat body phosphorylase activity in locusts, however, it elicits a rapid short-term hyperlipemia. In cockroaches, Periplaneta americana, injection of octopamine results in a strong activation of fat body phosphorylase within 1 min. Cockroach CC extract exerts a more prolonged effect on phosphorylase activity than does octopamine.

Hypertrehalosaemic and hyperlipaemic responses to adipokinetic hormone in fifth larval instar locusts, Locusta migratoria

In fifth instar larvae of Locusta migratoria the haemolymph lipid concentration is elevated after injection of adipokinetic hormone (AKH). This hyperlipaemic response in larvae remains substantially lower than in adults; over 75% of the mobilized lipid consists of diacylglycerol. In addition, unlike adult locusts, fifth instar larvae also exhibit a consistent, though moderate, hypertrehalosaemic response to AKH. The increases of both lipid and carbohydrate concentrations in larvae are dose-dependent, showing a significant linear regression on log dose in the range 0.2–20 pmol AKH. Glycogen phosphorylase in the fat body of fifth instar larvae as well as young adults is activated on injection of AKH, the percentage active phosphorylase increasing linearly with log dose in the range 0.04–20 pmol AKH. For a given response, a somewhat higher dose of AKH is needed in larvae than in young adults. Fat body glycogen phosphorylase is strongly activated during the period of the larval-adult ecdysis, when active phosphorylase accounts for almost half of the total enzyme, which is approximately ten times more than it is two days before, and two days after the ecdysis. The corpora cardiaca of fifth larval instar locusts already possess the potencies to elevate carbohydrate and lipid concentrations in larval haemolymph, and to activate fat body glycogen phosphorylase.

Development of catabolic pathways in insect flight muscles. A comparative study

Activities of enzymes representative of glycolytic and β-oxidative pathways and citric acid and glycerophosphate cycles were measured in the developing flight muscles of three species: Calliphora erythrocephala, Locusta migratoria, and Philosamia cynthia. The activities were measured in vitro under optimal conditions. The enzyme pattern of young flight muscles is quite different from the adult pattern. In the second half of the developmental period final differentiation towards the adult metabolic pattern takes place, in Calliphora leading to exclusively carbohydrate-oxidizing capacities, in Locusta to properties enabling both aerobic glycolytic and β-oxidative processes, whereas Philosamia becomes oriented to fatty acid oxidation. This differentiation starts after a temporary rise of lactate dehydrogenase activity, a phenomenon that seems to be connected with invagination of tracheoblasts into the muscle fibres. This tracheolization might be necessary for differentiation towards the species specific metabolic properties of the adult flight muscle. Theoretical aspects of the enzyme activities, as they were measured in the in vitro assays, are discussed and related to the physiological qualities of the flight muscles of the three species investigated.

Adipokinetic hormone is dependent on extracellular Ca2+ for its stimulatory action on the glycogenolytic pathway in locust fat body in vitro

Abstract Inclusion of glucose or trehalose in the medium during the incubation of locust fat body in vitro leads to a reduction of the relative amount of active (AMP-independent) glycogen phosphorylase. The presence of adipokinetic hormone (AKH I) results in a rapid activation of phosphorylase, reaching a maximum within 5 min. This AKH effect is highly dependent on added Ca 2+ , and requires ⩾ 1 mM Ca 2+ for maximal enzyme activation. Ca 2+ alone has no effect on phosphorylase activity, but it does activate the enzyme when the ionophore A23187 is also included in the medium. In a cell-free system from locust fat body the activation of endogenous phosphorylase by phosphorylase kinase is stimulated by Ca 2+ . Activity of the latter enzyme can be increased further by high doses of calmodulin. Both in the presence and in the absence of external calmodulin, the calmodulin antagonist trifluoperazine has an inhibitory effect on phosphorylase kinase. Results are discussed in relation to the possible mechanisms underlying hormonal control of glycogenolysis.

Glycogen phosphorylase in the fat body of Locusta migratoria

Abstract Incubation of fat body extracts of Locusta migratoria with ATP and Mg2+ results in the conversion of AMP dependent phosphorylase to AMP independent phosphorylase. Upon incubation with EDTA, phosphorylase became absolutely dependent on AMP. This conversion could be prevented with NaF. These results indicate activities of phosphorylase kinase and phosphorylase phosphatase. Fractionation of partially purified phosphorylase on DEAE-Sephacel revealed three peaks (I, II and III), the dependence on AMP for activity being total for I (phosphorylase b ), high for II, and low for III (phosphorylase a ). Flight-induced activation of phosphorylase resulted in a shift of the relative amounts of I, II and III from, respectively, 80, 14 and 6% of total activity at rest to 28, 18 and 54% after flight. Peak II phosphorylase can be converted to phosphorylase a in a phosphorylase kinase catalyzed reaction. This was demonstrated by incubating peak II in the presence of phosphorylase kinase, which reduced the AMP dependence of its activity to the same low level as known for phosphorylase a ; besides, chromatography of the incubated peak II on DEAE-Sephacel revealed a shift of activity from peak II to III. Under conditions that permitted phosphorylase phosphatase activity peak II phosphorylase was converted to phosphorylase b . The results suggest that peak II phosphorylase is a partially phosphorylated intermediate between phosphorylase b and a . This intermediate form may be referred to as phosphorylase ab .

Hormonal control of fat-body glycogen mobilization for locust flight

Fat-body phosphorylase in locusts injected with adipokinetic hormone (AKH I) is highly activated, as revealed by the relative proportions of the three forms present. Activation of phosphorylase during flight is strongly reduced when locusts are ligated at the neck, indicating that this activation is due to a factor from the head, which upon flight is released into the hemolymph. Flight-induced activation of phosphorylase is prevented when the release of AKH from the corpus cardiacum is blocked by the presence of high trehalose levels in the hemolymph, and also when the production of AKH is made impossible by prior removal of the corpus cardiacum glandular lobe. These results are discussed in relation to the possible involvement of AKH in the control of fat-body phosphorylase activity during flight.

Influence of juvenile hormone on growth and digestion in fifth instar larvae and adults of Locusta migratoria

Food utilization was measured in female fifth instar larvae and adults of the migratory locust by following the weight of food ingested, the weight of faeces produced, and the increase in body weight. These parameters were measured in normally developing locusts, in locusts that had been implanted with a pair of active corpora allata (CA) in the beginning of the fifth instar period, and in allatectomized locusts, operated on the first day after adult ecdysis. A high titre of corpus allatum hormone results in a considerably higher water content of the insects; allatectomy reduces this content. The dry weight of the locusts is not essentially influenced by excess or absence of juvenile hormone. Food consumption in corpora-allata-implanted locusts does not differ from that in normally developing insects. Within each developmental period the digestive capacity remains constant, but the absolute value of this capacity may differ between the two developmental periods. The approximate digestibility is somewhat higher after CA-implantation and lower after allatectomy. The efficiency of conversion of digested food into body substance is greater in normally developing larvae than in adults. CA-implantation lowers this efficiency in developing larvae. Allatectomy slightly raises the efficiency of conversion in adult locusts. In the second half period of larval development, CA-implantation raises the respiratory rate, as estimated by measuring oxygen consumption. During adult development no significant influence of CA-implantation on respiration was established. Relations between the amount of food digested, the efficiency of conversion of digested food into body substance, and the respiratory activity are discussed.

Isolation and partial characterization of three forms of glycogen phosphorylase from fat body of Locusta migratoria

Abstract Glycogen phosphorylases a, ab and b were isolated simultaneously from fat body of flown Locusta migratoria , and purified by pH-shock treatment, DEAE-Sephacel and 5′AMP-Sepharose chromatography. A 330- to 380-fold purification resulted in final specific activities of 27–31 U/mg protein. The molecular weight of the native enzymes was determined by gel filtration and was found to be 180,000–193,000. SDS gel electrophoresis gave a subunit molecular weight of 92,500, indicating that Locusta fat-body phosphorylases a, ab and b are dimers. Optimum pH for activity of the three enzyme forms lies in the range of 6.9–7.2. AMP concentrations needed for maximal stimulation of phosphorylases a, ab and b are, respectively, 0.01, 1 and 2 mM. IMP also stimulates activity of the a and ab forms, but this nucleotide does not activate phosphorylase b . ATP and ADP potently inhibit the three enzyme forms. Glucose is also a strong inhibitor, while trehalose and UDP-glucose have only weak negative effects on activity. In general, phosphorylase b activity is more subject to inhibition than phosphorylase a and ab activities are. Inhibitory effects on phosphorylase a are counteracted by AMP.

Development of imaginal competence to adipokinetic hormone in Locusta: Lipid and carbohydrate levels, and glycogen phosphorylase activity in azadirachtin-induced over-aged nymphs

Abstract 1. 1. Adipokinetic responses to injection of synthetic adipokinetic hormone I (sAKH) and relevant physiological parameters were studied in azadirachtin-induced 31 to 34-day-old Vth-instar over-aged male nymphs of Locusta migratoria which did not undergo the metamorphic moult to adult. 2. 2. The resting lipid and carbohydrate levels in the haemolymph and sAKH-induced activation of fat body glycogen phosporylase did not differ markedly between over-aged nymphs, normal nymphs and normal adults. 3. 3. Total fat body glycogen phosphorylase activity and the effect of sAKH on haemolymph carbohydrate level in over-aged nymphs were similar to those in normal adults and differed from those in normal nymphs. 4. 4. sAKH-induced elevation of haemolymph lipid level in the over-aged nymphs was higher than in normal nymphs but lower than in normal adults; thus, the over-agad nymphs attained only a partial adult competence in this respect.