Orexin A affects HepG2 human hepatocellular carcinoma cells glucose metabolism via HIF-1α-dependent and -independent mechanism.

Xing Wan,Yuyan Zhao,Xiaoqi Sun,Dongxiao Fan,Yuanyuan Liu,Lei Guo,Pankaj K Singh

doi:10.1371/journal.pone.0184213

Xing Wan, Yuyan Zhao + Show 5 more

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https://doi.org/10.1371/journal.pone.0184213

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Abstract

Orexins are hypothalamic neuropeptides that regulate feeding, reward, wakefulness and energy homeostasis. The present study sought to characterize the involvement of orexin A in glucose metabolism in HepG2 human hepatocellular carcinoma cells, and investigated the role of hypoxia-inducible factor-1α (HIF-1α) in the response. HepG2 cells were exposed to different concentrations of orexin A (10−9 to 10−7 M) in vitro, without or with the orexin receptor 1 (OX1R) inhibitor (SB334867), HIF-1α inhibitor (YC-1) or a combination of both inhibitors. Subsequently, OX1R, HIF-1α expression and localization, glucose uptake, glucose transporter 1 (GLUT1) expression and ATP content were measured. We further investigated the intracellular fate of glucose by measuring the gene expression of pyruvate dehydrogenase kinase 1 (PDK1), lactate dehydrogenase (LDHA) and pyruvate dehydrogenase B (PDHB), as well as metabolite levels including lactate generation and mitochondrial pyruvate dehydrogenase (PDH) activity. The activity of phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway was also assessed. Our results showed that the expression of OX1R was predominantly located in the nucleus in HepG2 cells. Orexin A oxygen-independently promoted the mRNA and protein expression of HIF-1α as well as its nuclear accumulation in HepG2 cells and the elevated HIF-1α protein was associated, at least partly, with the activation of the PI3K/Akt/mTOR pathway. Orexin A stimulated GLUT1 expression, glucose uptake as well as ATP generation in HepG2 cells via OX1R acting through the HIF-1α pathway. Moreover, orexin A inhibited LDHA, PDK1 expression and lactate production, stimulated PDHB expression and PDH enzyme activity independent of HIF-1α. Our results indicated that orexin signaling facilitated the glucose flux into mitochondrial oxidative metabolism rather than glycolysis in HepG2 cells. These findings provide new insight into the regulation of glucose metabolism by orexin A in hepatocellular carcinoma cells.

Highlights

Orexin A and orexin B are a pair of hypothalamic neuropeptides that play critical roles in the regulation of feeding, wakefulness, reward system, energy homeostasis as well as other physiological process [1,2,3,4]
Administration of the LY294002 (10−5M, Akt inhibitor), temsirolimus (10−5 M, mammalian target of rapamycin (mTOR) inhibitor), or a combination of both inhibitors suppressed Akt/ mTOR (Fig 3A and 3B, respectively), and abrogated the elevated hypoxia-inducible factor-1α (HIF-1α) protein induced by orexin A (Fig 3C). These results indicated that the phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway participated in orexin A-mediated HIF-1α protein accumulation in HepG2 cells
Orexin A promoted the expression and nuclear accumulation of HIF-1α in HepG2 cells under normoxia and the enhanced HIF-1α protein was associated with the activation of PI3K/Akt/mTOR signaling pathway

Summary

Introduction

Orexin A and orexin B are a pair of hypothalamic neuropeptides that play critical roles in the regulation of feeding, wakefulness, reward system, energy homeostasis as well as other physiological process [1,2,3,4]. The actions of orexin peptides are mediated via interaction with two G protein-coupled receptors (GPCRs), orexin receptor types 1 and 2 (OX1R and OX2R, respectively). OX1R has a higher selectivity for orexin A, while OX2R binds both orexin peptides with similar affinity [5]. Orexin singling deficiency caused narcolepsy in human and animal models, which was accompanied by metabolic abnormalities, such as elevated incidence of obesity and type 2 diabetes [8, 9]. Recent study demonstrated that transgenic orexin overexpression efficiently protected mice from insulin resistance induced by high fat diet [10], whereas the deletion of orexin caused disarrangement of hepatic insulin signaling and abnormal gluconeogenic activity during ageing [11, 12], suggesting an important role of orexin in glucose metabolism

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