Phospholipase D1 Mediates AMP-Activated Protein Kinase Signaling for Glucose Uptake Jong Hyun Kim1,2, Ji-Man Park1, Kyungmoo Yea1, Hyun Wook Kim1, Pann-Ghill Suh1, Sung Ho Ryu 1 * 1 Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, Kyungbuk, Republic of Korea, 2 Laboratory of Molecular Cardiology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, United States of America Abstract Background: Glucose homeostasis is maintained by a balance between hepatic glucose production and peripheral glucose utilization. In skeletal muscle cells, glucose utilization is primarily regulated by glucose uptake. Deprivation of cellular energy induces the activation of regulatory proteins and thus glucose uptake. AMP-activated protein kinase (AMPK) is known to play a significant role in the regulation of energy balances. However, the mechanisms related to the AMPK-mediated control of glucose uptake have yet to be elucidated. Methodology/Principal Findings: Here, we found that AMPK-induced phospholipase D1 (PLD1) activation is required for 14C-glucose uptake in muscle cells under glucose deprivation conditions. PLD1 activity rather than PLD2 activity is significantly enhanced by glucose deprivation. AMPK-wild type (WT) stimulates PLD activity, while AMPK-dominant negative (DN) inhibits it. AMPK regulates PLD1 activity through phosphorylation of the Ser-505 and this phosphorylation is increased by the presence of AMP. Furthermore, PLD1-S505Q, a phosphorylation-deficient mutant, shows no changes in activity in response to glucose deprivation and does not show a significant increase in 14C-glucose uptake when compared to PLD1- WT. Taken together, these results suggest that phosphorylation of PLD1 is important for the regulation of 14C-glucose uptake. In addition, extracellular signal-regulated kinase (ERK) is stimulated by AMPK-induced PLD1 activation through the formation of phosphatidic acid (PA), which is a product of PLD. An ERK pharmacological inhibitor, PD98059, and the PLD inhibitor, 1-BtOH, both attenuate 14C-glucose uptake in muscle cells. Finally, the extracellular stresses caused by glucose deprivation or aminoimidazole carboxamide ribonucleotide (AICAR; AMPK activator) regulate 14C-glucose uptake and cell surface glucose transport (GLUT) 4 through ERK stimulation by AMPK-mediated PLD1 activation. Conclusions/Significance: These results suggest that AMPK-mediated PLD1 activation is required for 14C-glucose uptake through ERK stimulation. We propose that the AMPK-mediated PLD1 pathway may provide crucial clues to understanding the mechanisms involved in glucose uptake. Citation: Kim JH, Park J-M, Yea K, Kim HW, Suh P-G, et al. (2010) Phospholipase D1 Mediates AMP-Activated Protein Kinase Signaling for Glucose Uptake. PLoS ONE 5(3): e9600. doi:10.1371/journal.pone.0009600 Editor: Charleen T. Chu, University of Pittsburgh, United States of America Received November 13, 2009; Accepted February 16, 2010; Published March 9, 2010 Copyright: 2010 Kim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a grant from the 21st Frontier Proteomics Research of the Ministry of Science and Technology in the Republic of Korea and National Institutes of Health (NIH) grant GM 68849 (W.C.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sungho@postech.ac.kr Introduction Circulating glucose levels reflect a balance between glucose production by the liver and glucose utilization in skeletal muscles [1], [2]. Energy deprivation occurs when cellular glucose levels are depleted by nutritional and environmental stressors such as glucose starvation, pressure overload, oxidative stress or hypoxia [3]–[8]. Once the balance breaks down, regulatory proteins such as AMPK are stimulated to restore it. AMPK, a serine/threonine protein kinase, is known to play an important role in the regulation of glucose uptake [9]. AMPK is often referred to as an energy sensor because it maintains the balance of AMP:ATP ratios and its activity increases with decreasing levels of cellular ATP. Thus, AMPK turns on ATP- producing catabolic pathways and turns off ATP-consuming processes under energy deprivation conditions [10]. AMPK is a heterotrimeric protein complex composed of one catalytic subunit (a) and two regulatory subunits (b and c) Its activity is also regulated by both AMP and the tumor suppressor, LKB1. Regulation by both AMP and LKB1 accompanies the interaction with AMPK and its phosphorylation [2]. LKB1, a serine-threonine kinase, is the most well characterized upstream kinase for AMPK activation. LKB1 phosphorylates a conserved Thr-172 within the T-loop of the AMPK catalytic subunit (a), leading to its activation. Activated AMPK induces phosphorylation at the Ser-79 site of acetyl-CoA carboxylase (ACC) as a direct downstream target. ACC regulates the conversion of acetyl-CoA to malonyl-CoA in the de novo lipid synthesis pathway [2]. Recently, several groups have reported that AMPK plays a major role in the regulation of metabolic stress-induced glucose uptake in both heart and skeletal muscles [11]–[13]. Activation of AMPK by AICAR has also been shown to increase glucose uptake via a phosphoinositide-3 kinase (PI-3K)-independent mechanism [14]. However, until now, the downstream target molecules of AMPK- mediated glucose uptake have been largely unknown. PLoS ONE | www.plosone.org 1 March 2010 | Volume 5 | Issue 3 | e9600