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

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.

The Orexin-A-Regulated Akt/mTOR Pathway Promotes Cell Proliferation Through Inhibiting Apoptosis in Pancreatic Cancer Cells

“…To determine the biological functions of orexin-A in pancreatic cancer, we activated or inactivated the stimulation of OX1Rby incubation with different concentrations (10 −5 , 10 −6 , 10 −7 , and 10 −8 M) of orexin-A with or without treatment of SB408124 (50 nM), an OX1 receptor antagonist to prevent the orexin-A effect on cell proliferation in PANC1 cells (data not shown). We found that treatment with10 −7 M orexin-A can significantly upregulate OX1R expression in PANC1 cells (Figure 3A ), which is consistent with that of previous reports ( 20 , 23 ).…”

“…Previous reports have indicated that orexin-A expression might be associated with malignancy in several tumors ( 20 , 23 , 24 ). Therefore, we examined the potential functions of orexin-A in human pancreatic cancer.…”

“…It is well-known that orexins and their receptors are widely distributed throughout the brain and peripheral tissues ( 16 , 17 ) and are implicated in food intake, energy homeostasis, the reward system, wakefulness, drug addiction, and neuroendocrine regulation ( 13 , 33 , 34 ). Recent studies have indicated that orexin-A was found to be highly expressed in prostate cancer ( 20 ) and was involved in glucose metabolism in hepatocellular carcinoma ( 23 , 24 ). The present study also indicated the high expression of orexin-A in pancreatic cancer (Figure 1 ).…”

“…Additionally, a recent study indicated that orexin-A can induce autophagy in HCT-116 human colon cancer cells through the ERK signaling pathway ( 22 ). Furthermore, several studies also indicated that stimulation of OX1R can mediate glucose metabolism through stimulating HIF-1α signaling and PI3K/Akt/mTOR pathways in human hepatocellular carcinoma cells ( 23 , 24 ), suggesting that orexin-A/OX1R might be a potential target for malignant cancer therapy. However, the possible roles of the orexin-A/OX1R signaling cascade are largely unknown in pancreatic cancer.…”

Orexin System and Avian Muscle Mitochondria

“…Human neuroblastoma cells treated with ORX-A had increased mitochondrial membrane potential [60]. Additionally, in studies using human hepatoma cells [61] and human embryonic kidney cells [62], treatment with ORX-A resulted in increased ATP production that shifted from glycolysis in the cytoplasm to oxidative phosphorylation in the mitochondria. Taken together, these studies indicate that orexin is able to enhance mitochondrial function, biogenesis, and ATP production in mammalian vertebrates.…”