AMPK is involved in mediation of erythropoietin influence on metabolic activity and reactive oxygen species production in white adipocytes

https://doi.org/10.1016/j.biocel.2014.06.008Get rights and content

Abstract

Erythropoietin, discovered for its indispensable role during erythropoiesis, has been used in therapy for selected red blood cell disorders in erythropoietin-deficient patients. The biological activities of erythropoietin have been found in animal models to extend to non-erythroid tissues due to the expression of erythropoietin receptor. We previously demonstrated that erythropoietin promotes metabolic activity and white adipocytes browning to increase mitochondrial function and energy expenditure via peroxisome proliferator-activated receptor alpha and Sirtuin1. Here we report that AMP-activated protein kinase was activated by erythropoietin possibly via Ca2+/calmodulin-dependent protein kinase kinase in adipocytes as well as in white adipose tissue from diet induced obese mice. Erythropoietin increased cellular nicotinamide adenine dinucleotide via increased AMP-activated protein kinase activity, possibly leading to Sirtuin1 activation. AMP-activated protein kinase knock down reduced erythropoietin mediated increase in cellular oxidative function including the increased oxygen consumption rate, fatty acid utilization and induction of key metabolic genes. Under hypoxia, adipocytes were found to generate more reactive oxygen species, and erythropoietin reduced the reactive oxygen species and increased antioxidant gene expression, suggesting that erythropoietin may provide protection from oxidative stress in adipocytes. Erythropoietin also reversed increased nicotinamide adenine dinucleotide by hypoxia via increased AMP-activated protein kinase. Additionally, AMP-activated protein kinase is found to be involved in erythropoietin stimulated increase in oxygen consumption rate, fatty acid oxidation and mitochondrial gene expression. AMP-activated protein kinase knock down impaired erythropoietin stimulated increases in antioxidant gene expression. Collectively, our findings identify the AMP-activated protein kinase involvement in erythropoietin signaling in regulating adipocyte cellular redox status and metabolic activity.

Introduction

Erythropoietin (EPO) binds to its cell surface receptor, EpoR to promote early erythroid progenitor cell survival, proliferation and differentiation. EPO is produced in the adult kidney and regulated by hypoxia inducible factor (HIF). However, EPO signaling is not restricted to the erythroid lineage and can be found in many non-hematopoietic cells including endothelial, muscle, adipocytes, cardiovascular and renal tissue (Noguchi et al., 2008, Teng et al., 2011). EPO stimulation of mitochondrial biogenesis in part by enhancement of peroxisome proliferator-activated receptor-gamma coactivator ((PGC)-1α) was suggested to mediate its cardioprotective activity (Carraway et al., 2010). EPO activity has also been reported for other non-hematopoietic tissues in animal models including brain protection against ischemia, enhanced neural progenitor production and anti-inflammatory effect (Sakanaka et al., 1998, Shingo et al., 2001, Tsai et al., 2006). Recently, a metabolic effect of EPO signaling in adipocytes was reported to provide protection against diet-induced obesity, increase glucose tolerance and mitochondrial function (Teng et al., 2011, Wang et al., 2013b). However, the detailed mechanism by which EPO regulates energy expenditure and metabolic activity still remains open for investigation. Although EPO was reported to promote metabolic activity of adipocytes via increasing Sirt1 and PGC-1α activity (Wang et al., 2013b), the involvement of other pathways in EPO/EpoR signaling in adipocytes remains largely unknown.

AMP-activated protein kinase (AMPK), a serine/threonine kinase, is an evolutionarily conserved energy metabolic sensor and an important regulator of energy homeostasis. AMPK can be activated to block body weight gain and increase fatty acid oxidation by inducing transcription regulators involved in energy homeostasis such as peroxisome proliferator-activated receptor alpha (PPARα) and peroxisome proliferator-activated receptor gamma (PPARγ) co-activator 1α (PGC-1α) (Canto and Auwerx, 2009, Giri et al., 2006, Hardie, 2007, Lage et al., 2008, Rodgers et al., 2005, Towler and Hardie, 2007). AMPK activator, metformin and thiazolidinediones have shown important therapeutic benefits in the treatment of type 2 diabetes and metabolic syndrome (Fryer et al., 2002, Hardie, 2007, Zhou et al., 2001). AMPK is also implicated in the appearance of brown features and increased mitochondrial activity in white adipose tissue (WAT) via regulating or interacting with factors such as PR domain containing 16 (PRDM16), a master regulator of brown fat determination and uncoupled protein 1 (UCP1), a key regulator of brown fat thermogenesis (Ahmadian et al., 2011, Bostrom et al., 2012, Kajimura et al., 2008, Seale et al., 2008, Seale et al., 2011, Sun et al., 2011). In addition, AMPK also regulates Sirt1, another metabolic sensor and a Nicotinamide adenine dinucleotide (NAD+) dependent type III deacetylase sirtuin, that activates PGC-1α and various substrates including PPARγ (Canto and Auwerx, 2009, Canto et al., 2009). We previously demonstrated that EPO enhances AMPK activity in C2C12 myoblasts, which may mediate the EPO effect to promote slow muscle fiber specification (Wang et al., 2013a). EPO is also reported to trigger AMPK activity, leading to enhanced phosphorylation of β common receptor (βCR) and endothelial nitric oxide synthase (eNOS) to stimulate NO production and, ultimately, angiogenesis (Su et al., 2012). However, how EPO triggers AMPK activity is still largely unknown so far.

In this study, we demonstrate a novel action of endogenous EPO in WAT to facilitate energy expenditure by regulating AMPK via calcium/calmodulin-dependent protein kinase kinase (CaMKK). EPO alters cellular nicotinamide adenine dinucleotide (NADH) and NAD+ levels and modulates NAD+/NADH ratio through regulating AMPK activity, which may lead to increased Sirt1 activity. EPO mediated activation of AMPK also contributes to energy expenditure and reduction of hypoxia induced oxidative stress of adipocytes. These effects of EPO in adipocyte may account for the beneficial metabolic effects of EPO.

Section snippets

Animal studies

C57BL/6 mice (4 weeks; NCI-Frederick) were maintained under a 12-h light/dark cycle with free access to food and drinking water except as indicated for paired-fed mice. For pair-fed study in mice, the animal group having lesser intake of food is used as “control” and the amount of food ingested by this group is equal to the amount given to the “pair-fed” group. In this study, one group of EPO treated mice had free access to food while the other was pair-fed to the ad libitum intake of the EPO

EPO increases AMPK activity in adipocytes

AMPK activation is able to increase fatty acid oxidation and increase the NAD+/NADH ratio (Canto et al., 2009). To determine if AMPK activity is involved in adipocyte response to EPO, 3T3-L1 preadipocytes were allowed to differentiate for 9 days and then treated with EPO. We found EPO stimulation increased AMPKα activity reflected in the two-fold increase in the ratio of phosphorylated AMPKα to total AMPKα (p-AMPKα/AMPKα), while total AMPKα protein levels remained unchanged (Fig. 1A).

EPO

Discussion

In this study, we provide evidence to suggest that in white adipocytes, AMPK phosphorylation is activated in response to EPO, possibly through the CaMKK pathway, to modulate the redox status as reflected by changed NAD+/NADH ratio resulting in stimulation of downstream Sirt1 deacetylation activity and other metabolism related factors including PGC-1α (Fig. 6). AMPK is required for EPO effect including increasing metabolic activity and reducing oxidative stress in vitro in adipocytes and in vivo

Disclosure

The authors declare no competing financial interests.

Acknowledgements

This work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases (ZIA/DK025102) and the Funding from University of Macau (SRG2013-00044-FHS).

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    L.W. and LJ.D. contributed equally to this work.

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