br Materials and methods br

Materials and methods
Conflict of interest
Acknowledgments We thank Christine Heiner (Department of Surgery, University of Pittsburgh) for her critical reading of the manuscript. This work was supported by grants from the US National Institutes of Health (R01GM115366, R01CA160417, and R01CA211070), the Natural Science Foundation of Guangdong Province (2016A030308011), the American Cancer Society (Research Scholar Grant RSG-16-014-01-CDD), the National Natural Science Foundation of China (31671435, 81402247, 81400132, and 81772508), and Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (2017). This project partly utilized University of Pittsburgh Cancer Institute shared resources supported by award P30CA047904.
AMPK: structure, functions and activators Adenosine monophosphate-activated protein kinase (AMPK) is a highly conserved serine/threonine protein kinase that plays a central role in the regulation of energy metabolism. AMPK is a heterotrimeric protein consisting of three subunits: a catalytic α subunit and regulatory β and γ subunits. Different α (α1 and α2), β (β1 and β2) and γ (γ1- γ2- γ3) isoforms are expressed in different mammalian tissues (Hardie, 2015, Hardie, 2007, Lage et al., 2008), with the ubiquitous expression of the α1-β1-γ1 complex in many tissues (Kim et al., 2016). Under conditions of energy deprivation (ATP depletion and increase in AMP), AMPK acts as an “adenylate charge” regulatory kinase that inhibits anabolic pathways that consume ATP, such as lipid and protein synthesis, and stimulates catabolic pathways that produce ATP, such as fatty PHA 543613 hydrochloride oxidation and mitochondrial oxidative phosphorylation (Steinberg and Kemp, 2009). In ATP-depleted conditions, the increased concentration of AMP binds to the AMPK γ-subunit and activates kinase activity in the α-catalytic subunit (Hardie et al., 2003, Xiao et al., 2013). The allosteric activation of AMPK by AMP makes the enzyme a much better substrate for upstream AMPK kinases (AMPKKs) and worse substrate for protein phosphatases. The phosphorylation of threonine residue (Thr172) of the α-catalytic subunit is a more potent modulator of AMPK activity than allosteric activation alone, increasing it nearly 100-fold. Three AMPKKs can phosphorylate Thr172: the upstream tumor suppressor liver kinase B1 (LKB1) (Alessi et al., 2006, Hawley et al., 2003), the Ca2+/calmodulin-dependent protein kinase kinaseβ (CaMKKβ) (Hurley et al., 2005) and the transforming growth factor-β-activated kinase 1 (Tak1 kinase) (Momcilovic et al., 2006). AMP also regulates the rate of AMPK activation by directly inhibiting the protein phosphatases PP2A and PP2C, which are responsible for removing phosphate from the Thr172 site and maintaining AMPK in its inactive state (Carling et al., 2012, Sanders et al., 2007). During exercise and fasting, AMPK promotes glucose uptake through the phosphorylation of Akt and other enzymes necessary to the translocation of glucose transporter type 4 (GLUT4) and plays a complex role in increasing insulin sensitivity. AMPK also stimulates fatty acid oxidation by phosphorylating and inactivating acetyl CoA carboxylase (ACC), which converts acetyl-CoA into malonyl-CoA, which is the regulator of the switch between fatty acid synthesis and oxidation (Foster, 2012, Kahn et al., 2005). Therefore, the inactivation of ACC by AMPK results in increased fatty acid transport into mitochondria and subsequent oxidation, which maintains NADPH and GSH levels (antioxidant defense). Moreover, AMPK upregulates several antioxidant genes (superoxide dismutase, uncoupling protein 2) by activating the nuclear factor E2-related factor 2 (Nrf2), which is a master regulator of the antioxidant response (Jeon, 2016). AMPK also phosphorylates and decreases the transcription factor sterol regulatory element-binding protein-1c (SREBP-1c), thereby reducing the expression of lipogenic genes (Steinberg and Kemp, 2009).