Insulin and 20-hydroxyecdysone action in Bombyx mori: Glycogen content and expression pattern of insulin and ecdysone receptors in fat body.

Insulin and ecdysone signaling play a critical role on the growth and development of insects including Bombyx mori. Our previous study showed that Bombyx larvae reached critical weight for metamorphosis between day 3.5 and 4 of the fifth larval instar. The present study showed that the effect of insulin on the accumulation of glycogen in fat body of Bombyx larvae depends on the critical growth period. When larvae are in active growth period (before reaching critical weight), insulin caused increased accumulation of glycogen, while its treatment in larvae at terminal growth period (after critical period) resulted in an increased mobilization of glycogen. During terminal growth period, insulin and 20-hydroxyecdysone (20E) showed an antagonistic effect on the accumulation of fat body glycogen in fed, food deprived and decapitated larvae as well as in isolated abdomens. Insulin treatment decreased the glycogen content, whereas, 20E increased it. Food deprivation and decapitation caused an increase in the transcript levels of insulin receptor (InR) and this increase in InR expression might be attributed to a decrease in synthesis/secretion of insulin-like peptides, as insulin treatment in these larvae showed a down-regulation in InR expression. However, insulin showed an up-regulation in InR in isolated abdomens and it suggests that in food deprived and decapitated larvae, the exogenous insulin may interact with some head and/or thoracic factors in modulating the expression of InR. Moreover, in fed larvae, insulin-mediated increase in InR expression indicates that its regulation by insulin-like peptides also depends on the nutritional status of the larvae. The treatment of 20E in fed larvae showed an antagonistic effect on the transcript levels since a down-regulation in InR expression was observed. 20E treatment also led to a decreased expression of InR in food deprived and decapitated larvae as well as in isolated abdomens. Insulin and 20E also modulated the expression level of ecdysone receptors (EcRB1 and USP1). 20E treatment showed an up-regulation in expression of ecdysone receptors, but only in fed larvae, whereas insulin treatment showed a down-regulation in the expression of EcRB1 and USP1 in all the experimental larvae studied. Further, the data indicates that an up-regulation of ecdysone receptors is associated with an increase in fat body glycogen content, whereas an up-regulation of insulin receptor expression causes glycogen mobilization. The study, therefore, suggests that the insulin and ecdysone signaling are linked to each other and that both insulin and ecdysone are involved in regulating the carbohydrate reserves in B. mori.

The feeding status regulates the action of insulin and 20-hydroxyecdysone on haemolymph trehalose level in Bombyx larvae.

Insulin in mammals is known for its effect on carbohydrate metabolism and maintenance of blood sugar levels. In the present study, we explored the effect of exogenous insulin and 20-hydroxyecdysone (20E) on carbohydrate metabolism in Bombyx mori under the fed and food-deprived conditions. The study showed that insulin and 20E regulate the trehalose (major circulating sugar) level in B. mori, and larval feeding status plays a decisive role in influencing the action of these two hormones. At feeding, both insulin and 20E showed its hypertrehalosemic action but at food deprivation, these hormones acted as hypotrehalosemic factors. Although both insulin and 20E showed the same effect on the haemolymph trehalose level either at feeding or food deprivation, the metabolic regulation was different for these two hormones. Insulin treatment to fed larvae increased the haemolymph trehalose level without altering the effectiveness of trehalose utilization but possibly by inducing the activity of glycogen phosphorylase enzyme and releasing glucose-1-P for the increased synthesis of trehalose. The treatment of 20E to fed larvae also increased the trehalose level, but concurrently it also increased both the enzyme activity of trehalase and glycogen phosphorylase. Insulin treatment to food-deprived larvae decreased the circulating trehalose level by increasing the trehalose breakdown as the mRNA expression level of trehalase-2 and enzyme activity of trehalase increased in these larvae. The treatment of 20E to food-deprived larvae decreased the haemolymph trehalose possibly by decreasing its synthesis, as glycogen phosphorylase enzyme activity decreased in these larvae, thus restricting the availability of glucose-1-P for trehalose synthesis. The study, thus suggests that both insulin and 20E regulate carbohydrate metabolism in B. mori.

Larval feeding status regulates the transcript levels of genes encoding PTTH and allatoregulatory peptides in silkworm Bombyx mori.

In Bombyx mori, two dorsolateral neurosecretory cells (NSCs) in each of the two brain lobes have been identified as prothoracicotropic hormone (PTTH) producing cells. This neuropeptide in insects stimulates the prothoracic gland for the synthesis and release of ecdysone, responsible for the molting events. Allatotropin (AT) and allatostatin (AST) are allatoregulatory neuropeptides that regulate juvenile hormone biosynthesis. Here, by using RT-qPCR, we showed that in B. mori, nutritional stress modulates the mRNA expression of AT and AST-C (allatostain type C) in the central nervous system consisting of the brain lobes and all the associated ganglia. Using whole-mount in situ hybridization, we showed that the feeding status of Bombyx larvae also influences the expression of PTTH in the NSCs of the brain. Food deprivation significantly decreased the mRNA expression levels of PTTH in larvae at active or terminal growth period. Further, we showed that insulin modulates the expression level of PTTH. However, its action was dependent on the feeding status of the larvae. At feeding, the insulin decreased the PTTH expression level, while at food deprivation, the insulin increased the PTTH expression level. The data thus indicates that larval feeding status plays an important role in altering the mRNA expression levels of allatoregulatory peptide genes and PTTH.

Modulation of gene expression by nutritional state and hormones in Bombyx larvae in relation to its growth period.

Insect growth and development are mainly regulated via synchronization of many extrinsic and intrinsic factors such as nutrition and hormones. Previously we have demonstrated that larval growth period influences the effect of insulin on the accumulation of glycogen in the fat body of Bombyx larvae. In the present study we demonstrate that Bombyx larvae at the terminal growth period (TGP, after critical weight) had a significantly greater increase in the expression level of Akt in the fat body than at the active growth period (AGP, before critical weight). The larvae at TGP also showed an increase in the expression level of ecdysone receptors (EcRB1 and USP1) and ecdysone-induced early genes (E75A and broad). The treatment of bovine insulin and methoprene to larvae at AGP induced the transcript levels of Akt, irrespective of the nutritional status of the larvae. However, in larvae at TGP, insulin repressed the transcript level of Akt. On contrary, 20-hydroxyecdysone (20E) induced the expression level of Akt in TGP larvae, but at feeding only. Insulin and 20E thus showed an antagonistic action on the Akt expression level in TGP larvae under feeding. The studies thus showed that larval growth period influences the expression level of Akt and ecdysone receptors in Bombyx. Further, the growth period and nutrition modulate the effect of exogenous hormones on Akt expression.

Developmental expression and hormonal regulation of different isoforms of the transcription factor E75 in the tobacco hornworm Manduca sexta.

E75A and E75B, isoforms of the E75 orphan nuclear receptor, are sequentially up-regulated in the abdominal epidermis of the tobacco hornworm Manduca sexta by 20-hydroxyecdysone (20E) during larval and pupal molts, with E75A also increasing at pupal commitment (Zhou et al., Dev. Biol. 193, 127-138, 1998). We have now cloned E75C and show that little is expressed in the epidermis during larval life with trace amounts seen just before ecdysis. Instead, E75C is found in high amounts during the development of the adult wings as the ecdysteroid titer is rising, and this increase was prevented by juvenile hormone (JH) that prevented adult development. By contrast, E75D is expressed transiently during the larval and pupal molts as the ecdysteroid titer begins to decline and again just before ecdysis, but in the developing adult wings is expressed on the rise of 20E. Removal of the source of JH had little effect on either E75C or E75D mRNA expression during the larval and pupal molts. At the time of pupal commitment, in vitro experiments show that 20E up-regulates E75D and JH prevents this increase. Neither E75A nor E75D mRNA was up-regulated by JH alone. Thus, E75C is primarily involved in adult differentiation whereas E75D has roles both during the molt and pupal commitment.

Action of estradiol-17beta on the synthetic activity of the silk gland in Bombyx mori L.

The effects of estradiol-17beta (E(2)) were studied on several metabolic parameters in the silk gland of Bombyx mori L. race Nistari. Topical application of different doses (0.05-4.0&mgr;g/g body weight) of E(2) on the first and second day of the fifth instar larvae showed a dose dependent effect when studied on the fifth day. A significant increase in silk gland weight and fibroin content was observed between the doses 0.05 and 0.1, and 0.1 and 1.0&mgr;g/g of E(2). A similar pattern of dose-dependent rise in DNA and RNA content of posterior silk gland (PSG) was observed with the doses of E(2) when the contents were expressed per pair of PSG. Higher doses of E(2) (2.0 or 4.0&mgr;g/g) demonstrated relatively less increase, unchanged level or a decrease in the above parameters in comparison to the control values. The glutamate-pyruvate transaminase of PSG showed a significant increase from 0.1 to 2.0&mgr;g/g of E(2) doses in comparison to the control value. Simultaneous injection of ICI-182780 (1.0&mgr;g/g), a very pure and specific antiestrogenic compound, with E(2) (1.0&mgr;g/g) caused a significant counteraction of E(2)-induced increase in silk gland activity, which was reflected in DNA and RNA content of PSG, wet weight and fibroin content of silk gland, and on glutamate-pyruvate transaminase activity. Cycloheximide (0.5&mgr;g/g), a protein synthesis blocker, caused a significant inhibition of the E(2) (1.0&mgr;g/g)-induced silk gland activity when treated along with estradiol. From this study it appears that estradiol has a specific effect on silk gland function and that it may act in a nuclear mediated way.