Leptin receptor overlapping transcript (LepROT) gene participates in insulin pathway through FoxO

doi:10.1016/j.gene.2016.04.036

Gene

Volume 587, Issue 1, 1 August 2016, Pages 64-69

https://doi.org/10.1016/j.gene.2016.04.036 Get rights and content

Highlights

•
LepROT is constitutively expressed during larval development.
•
Knockdown of LepROT causes delay of the development of Helicoverpa armigera.
•
Knockdown of LepROT results in the upregulation of FoxO and downregulation of PI3K, causes subcellular translocation of FoxO from cytoplasm to nuclei.
•
Overexpression of LepROT in the HaEpi cell line inhibits FoxO expression.

Abstract

Leptin receptor overlapping transcript (LepROT) is co-transcribed with the leptin receptor (LepR). However, the function and mechanism of LepROT in insulin pathway is unclear. In this study, we report the function of LepROT in maintaining consistent FoxO transcription. LepROT is constitutively expressed during larval development. 20-Hydroxyecdysone, methoprene, and insulin have no effect on the transcription of LepROT. However, the knockdown of LepROT by dsRNA injection in larvae causes delay of the development of Helicoverpa armigera. Knockdown of LepROT results in the upregulation of FoxO and downregulation of PI3K. The knockdown of LepROT also results in the subcellular translocation of FoxO from cytoplasm to nuclei. By contrast, overexpression of LepROT in the HaEpi cell line inhibits FoxO expression. Results suggest that LepROT participates in insulin signaling.

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).

References (31)

A. Barthel et al.
FoxO proteins in insulin action and metabolism
Trends Endocrinol. Metab.
(2005)
R. Baserga
The IGF-I receptor in cancer research
Exp. Cell Res.
(1999)
C. Brand et al.
Protein kinase Cdelta participates in insulin-induced activation of PKB via PDK1
Biochem. Biophys. Res. Commun.
(2006)
X.J. Du et al.
Molecular cloning and characterization of a lipopolysaccharide and beta-1,3-glucan binding protein from fleshy prawn (Fenneropenaeus chinensis)
Mol. Immunol.
(2007)
J.A. Fabrick et al.
cDNA cloning, purification, properties, and function of a beta-1,3-glucan recognition protein from a pyralid moth, Plodia interpunctella
Insect Biochem. Mol. Biol.
(2003)
J.S. Flier
Obesity wars: molecular progress confronts an expanding epidemic
Cell
(2004)
D.C. Goberdhan et al.
The functions of insulin signaling: size isn't everything, even in Drosophila
Differentiation
(2003)
B.D. Manning et al.
AKT/PKB signaling: navigating downstream
Cell
(2007)
K.D. Niswender et al.
Insulin and its evolving partnership with leptin in the hypothalamic control of energy homeostasis
Trends Endocrinol. Metab.
(2004)
S. Oldham et al.
Insulin/IGF and target of rapamycin signaling: a TOR de force in growth control
Trends Cell Biol.
(2003)

L.A. Tartaglia

The leptin receptor

J. Biol. Chem.

(1997)

B. Bailleul et al.

The leptin receptor promoter controls expression of a second distinct protein

Nucleic Acids Res.

(1997)

S.H. Bates et al.

The role of leptin– > STAT3 signaling in neuroendocrine function: an integrative perspective

J. Mol. Med.

(2004)

N. Belgareh-Touzé et al.

Yeast Vps55p, a functional homolog of human obesity receptor gene-related protein, is involved in late endosome to vacuole trafficking

Mol. Biol. Cell

(2002)

R. Burcelin et al.

Acute intravenous leptin infusion increases glucose turnover but not skeletal muscle glucose uptake in ob/ob mice

Diabetes

(1999)

Cited by (3)

Resveratrol Promotes Gluconeogenesis by Inhibiting SESN2-mTORC2-AKT Pathway in Calf Hepatocytes
2023, Journal of Nutrition
The glucose requirement of dairy cows is mainly met by increasing the rate of hepatic gluconeogenesis. However, due to negative energy balance, the liver of periparturient cows is under oxidative stress induced by lipid over-mobilization, and hepatic gluconeogenesis is reduced. Studies have demonstrated that resveratrol, which is widely known for its antioxidant properties, can alter hepatic gluconeogenesis. However, it is not clear whether resveratrol could regulate hepatic gluconeogenesis by its antioxidant properties.
This study aims to investigate the precise effect of resveratrol in hepatic gluconeogenesis, the role of resveratrol on hydrogen peroxide (H₂O₂)-induced oxidative stress in hepatocytes and the potential mechanism using primary hepatocytes.
Primary hepatocytes were isolated from 5 healthy Holstein calves (1 d old, 30 to 40 kg, fasted) and treated with different concentrations of resveratrol (0, 5, 10, 25, or 50 μM) combined with or without H₂O₂ (0, 100, or 200 μM) induction for 12 h.
Resveratrol enhanced the expression of gluconeogenic genes of calf hepatocytes in a dose-dependent manner (P < 0.05). Conversely, H₂O₂ suppressed the expression of gluconeogenic genes and induced oxidative stress (P < 0.05), which was improved by resveratrol in calf hepatocytes (P < 0.001). Furthermore, the mechanistic target of rapamycin complex 2 (mTORC2)-AKT pathway was found to negatively regulate gluconeogenesis. An AKT inhibitor was used to assess the role of the mTORC2-AKT pathway in the effects of resveratrol. The results showed resveratrol promoted hepatic gluconeogenesis by inhibiting the mTORC2-AKT pathway. Moreover, sestrin 2 (SESN2) upregulated the activity of mTORC2. We further found that resveratrol decreased SESN2 levels (P < 0.001).
This study indicated that resveratrol enhances the gluconeogenic capacity of calf hepatocytes by improving H₂O₂-induced oxidative stress and modulating the activity of the SESN2-mTORC2-AKT pathway, implying that resveratrol may be a promising target for ameliorating liver oxidative stress in transition cows.
Endospanin1 affects oppositely body weight regulation and glucose homeostasis by differentially regulating central leptin signaling
2017, Molecular Metabolism
Citation Excerpt :
Our finding that Endo1 is involved in the PI3K pathway is in line with a very recent study defining the function of Endo1 in insects. The latter suggested that Endo1 silencing in Helicoverpa armigera larvae was correlated with a decrease in the transcript levels of PI3K in larvae and an increased level of the Forkhead transcription factor FoxO, known to be regulated by the PI3K signaling pathway [31]. However, unlike in insects, the absence of Endo1 in mammals does not impact on the protein levels of p85 (PI3K) or FoxO as investigated in the mouse hypothalamus, cortex, and brainstem of Endo1 KO mice compared to WT, and in human HeLa cells silenced for Endo1 (Supplem Figure S4).
The hypothalamic arcuate nucleus (ARC) is a major integration center for energy and glucose homeostasis that responds to leptin. Resistance to leptin in the ARC is an important component of the development of obesity and type 2 diabetes. Recently, we showed that Endospanin1 (Endo1) is a negative regulator of the leptin receptor (OBR) that interacts with OBR and retains the receptor inside the cell, leading to a decreased activation of the anorectic STAT3 pathway. Endo1 is up-regulated in the ARC of high fat diet (HFD)-fed mice, and its silencing in the ARC of lean and obese mice prevents and reverses the development of obesity.
Herein we investigated whether decreased Endo1 expression in the hypothalamic ARC, associated with reduced obesity, could also ameliorate glucose homeostasis accordingly.
We studied glucose homeostasis in lean or obese mice silenced for Endo1 in the ARC via stereotactic injection of shRNA-expressing lentiviral vectors.
We observed that despite being leaner, Endo1-silenced mice showed impaired glucose homeostasis on HFD. Mechanistically, we show that Endo1 interacts with p85, the regulatory subunit of PI3K, and mediates leptin-induced PI3K activation.
Our results thus define Endo1 as an important hypothalamic integrator of leptin signaling, and its silencing differentially regulates the OBR-dependent functions.
Resveratrol affects hepatic gluconeogenesis via histone deacetylase 4
2019, Diabetes, Metabolic Syndrome and Obesity

^☆: The nucleotide sequence LepROT has been submitted to the GenBank with accession number: KT963072

View full text

Research paperLeptin receptor overlapping transcript (LepROT) gene participates in insulin pathway through FoxO☆