Research paperLeptin receptor overlapping transcript (LepROT) gene participates in insulin pathway through FoxO☆
Introduction
The leptin receptor overlapping transcript (LepROT) is co-transcribed with Leptin receptor (LepR) but without similarity to the LepR (Bailleul et al., 1997). LepR and LepROT co-expressed in the mouse brain (Mercer et al., 2000). In mouse, silencing of LepROT will increase leptin receptor signaling (Couturier et al., 2007). Mammals have a single LepROT homologue called LepROT like-1 (LepROTL1), which has 70% amino acid sequence similarity with LepROT. Vps55p, the homologue of LepROT in yeast, has functions in the transport of the proteins from Golgi to the vacuole (Belgareh-Touze et al., 2002). Nonetheless, the function of LepROT is not much reported.
Leptin is the major signal regulating food intake and energy homoeostasis (Burcelin et al., 1999). Leptin binds to LepR and activates Janus kinase 2 (JAK2) to phosphorylate the receptor itself, leading to the activation of other function molecules such as signal transducer and activator of transcription 3 (STAT-3) (Myers, 2004). The phosphorylated LepR makes STAT3 transmit the signal to the nucleus (Tartaglia, 1997). LepR also could phosphorylate insulin receptor substrate (IRS) and affect the IRS-phosphoinositide-3 kinase (PI3K) signaling pathway (Bates and Myers, 2004). Several studies have illustrated that leptin supplement could enhance glucose turnover in rodents (Friedman and Halaas, 1998) and could repair glucose metabolism in leptin-deficient mice (Pelleymounter et al., 1995). These data show that leptin and insulin may have overlapping effects in metabolism.
The insulin pathway is an evolutionarily conserved pathway (Goberdhan and Wilson, 2003) and regulates metabolism, cell growth, and proliferation (Hafen, 2004). Both in vertebrates and invertebrates, the insulin pathway shares the same signaling transduction. In Drosophila, the insulin receptor (Drosophila insulin receptor, dinr) will recognize insulin/insulin-like signal and activate the phosphorylation of PI3K (Oldham and Hafen, 2003). Phosphorylated PI3K increases phosphatidylinositol (3,4,5)-trisphosphate to recruit protein kinase B (PKB) to the plasma membrane (Oldham et al., 2002). PKB on the membrane is phosphorylated and activates several downstream targets (Manning and Cantley, 2007). In Drosophila, PI3K is necessary for insulin or insulin-like peptide signaling pathway to promote growth and proliferation (Goberdhan and Wilson, 2003).
FoxO, as the specific protein of Forkhead box family, has an important role in mediating the effects of insulin on diverse physiological functions, such as cell proliferation, apoptosis, and metabolism (Barthel et al., 2005). FoxO could be retained in the cytoplasm or could be translocated to the nucleus to control the insulin/insulin-like growth factor (IGF)-mediated growth. Under high insulin signal, PKB phosphorylates FoxO and makes it retain in cytoplasm; when insulin signal is low, FoxO will be dephosphorylated and translocated to the nucleus, thus activating the transcription of the target genes to inhibit insulin signaling pathway (Jünger et al., 2003, Kramer et al., 2003).
To understand the role of LepROT in the insect growth and development, we studied LepROT and found that the transcription of LepROT is constitutive after the challenge of 20-Hydroxyecdysone (20E), methoprene, and insulin. Knockdown of the gene LepROT will delay larvae development. FoxO was upregulated and PI3K was downregulated when the gene LepROT was knockdown by RNAi. In addition, RNAi of LepROT would make the FoxO remain in the nuclei on the cell line. LepROT overexpression blocks the expression of FoxO. These pieces of evidence indicated that LepROT plays roles in insulin pathway.
Section snippets
Insect
Helicoverpa armigera (cotton bollworms) were obtained from Wuhan Institute of Virology, Chinese Academy of Science, Wuhan, China. We fed the cotton bollworms with an artificial diet (mainly consisting of wheat and soybean) and the insects grown under 14:10 h light:dark cycles (Zhao et al., 2005).
Bioinformatics analysis
Homology analysis was achieved with BlastX (http://www.ncbi.nlm.nih.gov). Protein translation and prediction were completed with the software from ExPASy (http://www.au.expasy.org). The prediction of the
Identification of LepROT
H. armigera LepROT contains a 390 bp ORF (Sup.1). Through ExPASy software (http://www.au.expasy.org/prosite/), LepROT contains a vps55 domain (Leu 7-Asp 125) and four transmembrane segments, but no N-terminal signal peptide. LepROT has similarity with the insect LepROT from Aedes aegypti (56% identity) and Tribolium castaneum (72% identity) (Sup.2).
To analyze the relationships among the LepROTs in various organisms, a phylogenetic tree was constructed using the neighbor-joining methods. As shown
Discussion
In the present study, interference of LepROT led to growth slowdown and upregulated FoxO and downregulated PI3K. LepROT could control the localization of FoxO. The data suggested that the LepROT could mediate the insulin signaling pathway through regulating the gene expression and localization of FoxO.
Conflict of interest
The authors declare that they have no conflict of interest.
Research Involving Human Participants and/or Animals
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
Informed consent
Informed consent was obtained from all individual participants included in the study.
Acknowledgments
This work was supported by grants from PhD Start-up Foundation of Yuncheng University (grant no. YQ-2012012).
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The nucleotide sequence LepROT has been submitted to the GenBank with accession number: KT963072