Transcriptional regulation of the insulin signaling pathway genes by starvation and 20-hydroxyecdysone in the Bombyx fat body.

Genetic studies in the fruitfly, Drosophila melanogaster, have uncovered a conserved insulin/insulin growth factor signaling (IIS) pathway that regulates nutrition-dependent growth rates of insects. From the silkworm, Bombyx mori, we have identified and characterized several key genes involved in the IIS pathway, including InR, IRS, PI3K110, PI3K60, PTEN, PDK, and Akt. Tissue distribution analysis showed that most of these genes were highly expressed in the fat body implying that the IIS pathway is functionally important within insect adipose tissue. Developmental profile studies revealed that the expression levels of InR, IRS, PI3K110, and PDK were elevated in the fat body during molting and pupation, periods when animals ceased feeding and hemolymph levels of 20-hydroxyecdysone (20E) were high. Starvation rapidly up-regulated the mRNA levels of these same genes in the fat body, while 20E slowly induced their transcription. We conclude that 20E slowly reduces food consumption and then indirectly induces a state of starvation resulting in the elevation of the mRNA levels of InR, IRS, PI3K110, and PDK in the Bombyx fat body during molting and pupation.

Liu Y ，Zhou S ，Ma L ，Tian L ，Wang S ，Sheng Z ，Jiang RJ ，Bendena WG ，Li S ... -  《-》

Developmental regulation of glycolysis by 20-hydroxyecdysone and juvenile hormone in fat body tissues of the silkworm, Bombyx mori.

Tian L ，Guo E ，Wang S ，Liu S ，Jiang RJ ，Cao Y ，Ling E ，Li S ... -  《-》

20-Hydroxyecdysone-induced transcriptional activity of FoxO upregulates brummer and acid lipase-1 and promotes lipolysis in Bombyx fat body.

In a previous study, we have shown that the molting hormone, 20-hydroxyecdysone (20E), reduces insect food consumption resulting in fat body lipolysis during the non-feeding molting and pupation stages, and assumed that the transcription factor FoxO is involved in this process. To verify this hypothesis, we cloned foxO from the silkworm, Bombyx mori. During molting and pupation, FoxO is highly expressed and predominantly localizes in the nuclei of fat body cells. 20E induced foxO mRNA expression and FoxO nuclear localization resulting in an increase of FoxO transcriptional activity. RNAi of foxO prior to the 4th larval molting downregulated two lipase genes--the insect adipose triacylglycerol lipase homologue, brummer, and an acid lipase, acid lipase-1, in the fat body. Overexpression of the constitutively-active form of foxO (foxO(CA)) upregulated brummer and acid lipase-1 in both the fat body and Bombyx Bm-12 cells. Putative FoxO-response elements (FREs) are present in the promoter regions of brummer and acid lipase-1, and mutation of the FREs attenuated their FoxO-induced luciferase activities. ChIP assay revealed that FoxO binds directly to those FREs. Moreover, foxO(CA) overexpression in vivo doubled lipid concentration in the hemolymph, increased total lipase activity, and slightly but significantly reduced lipid content in the fat body. Taken together, we conclude that 20E increases the transcriptional activity of FoxO which, in turn, upregulates brummer and acid lipase-1 and induces lipolysis in the Bombyx fat body during molting and pupation.

Hossain MS ，Liu Y ，Zhou S ，Li K ，Tian L ，Li S ... -  《-》

20-hydroxyecdysone upregulates apoptotic genes and induces apoptosis in the Bombyx fat body.

During insect metamorphosis, obsolete larval tissues are removed by programed cell death (PCD), mainly apoptosis and autophagy, which is directed by the molting hormone, 20-hydroxyecdysone (20E) and the 20E-triggered transcriptional cascade. Here, we investigated how 20E regulates apoptosis at the transcriptional level in the fat body of the silkworm, Bombyx mori. As detected by TdT-mediated dUTP Nick-End Labeling (TUNEL), apoptosis weakly occurred during the fourth larval molting, decreased to undetected levels during the early fifth instar, and gradually increased from day 4 of fifth instar to the wandering stage to the prepupal stage. Meanwhile, as determined by quantitative real-time PCR, eight genes involved in apoptosis, including Apaf-1, Nedd2 like1, Nedd2 like2, ICE1, ICE3, ICE5, Arp, and IAP, were highly expressed during molting and pupation, when the 20E titer is high. Injection of 20E into day 2 of fifth instar larvae significantly induced apoptosis and upregulated apoptotic genes after 6 h of treatment, and in vitro treatment of larval fat body tissues with 20E upregulated all the eight apoptotic genes. Moreover, RNAi knockdown of USP, a component of the 20E receptor complex EcR-USP, at the early-wandering stage reduced apoptosis and downregulated apoptotic genes after 24 h of treatment. Taken together, we infer that 20E upregulates apoptotic genes and thus induces apoptosis in the Bombyx fat body during larval molting and the larval-pupal transition.

Tian L ，Liu S ，Liu H ，Li S ... -  《-》

The AMPK-PP2A axis in insect fat body is activated by 20-hydroxyecdysone to antagonize insulin/IGF signaling and restrict growth rate.

Yuan D ，Zhou S ，Liu S ，Li K ，Zhao H ，Long S ，Liu H ，Xie Y ，Su Y ，Yu F ，Li S ... -  《-》