Original Contribution NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes Emilie E. Vomhof-DeKrey a , Matthew J. Picklo a,b,c,n a Grand Forks Human Nutrition Research Center, USDA-ARS, Grand Forks, ND 58203, USA b Department of Pharmacology, Physiology & Therapeutics, University of North Dakota, Grand Forks, ND, USA c Department of Chemistry, University of North Dakota, Grand Forks, ND, USA article info Article history: Received 7 May 2012 Accepted 29 May 2012 Available online 8 June 2012 Keywords: Adipogenesis Insulin signaling Antioxidant response element Electrophile response element Oxidative stress Obesity miRNA regulation abstract The nuclear factor E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) pathway responds to oxidative stress via control of several antioxidant defense gene expressions. Recent efforts demonstrate that Nrf2 modulates development of adiposity and adipogenesis. One of the major Nrf2-regulated proteins, NAD(P)H:quinone oxidoreductase 1 (NQO1), is implicated in the development of adipose tissue and obesity. However, little is known about in situ disposition of Nrf2, Keap1, and NQO1 during adipogenesis in isolated adipocytes. Based on literature data, we hypothesized that adipocyte differentiation would increase expression of the Nrf2/Keap1 pathway and NQO1. Using murine 3T3-L1 preadipocytes, we mapped an increase in NQO1 protein at limited clonal expansion and postmitotic growth arrest (Days 1–3) stages and a decrease in terminally differentiated (Day 8) adipocytes that lasted for several days afterward. Conversely, NQO1, Nrf2, and Keap1 mRNA expres- sions were all increased in differentiated adipocytes (Days 11–14), indicating a discrepancy between steady-state mRNA levels and resulting protein. Treatment of differentiated 3T3-L1 adipocytes with glycogen synthase kinase-3b (GSK-3b) inhibitor, LiCl, led to 1.9-fold increase in NQO1 protein. Sulforaphane enhanced NQO1 protein (10.5-fold) and blunted triglyceride and FABP4 accumulation. The decrement in triglyceride content was partially reversed when NQO1 activity was pharmacologi- cally inhibited. These data demonstrate a biphasic response of Nrf2 and NQO1 during adipocyte differentiation that is regulated by Keap1- and GSK-3b-dependent mechanisms, and that hypertrophy is negatively regulated by NQO1 activity. Published by Elsevier Inc. Introduction Obesity is associated with an increase in systemic oxidative stress [1,2]. Nuclear factor E2-related factor 2 (Nrf2) is a key transcription factor that responds to oxidative stress via binding to the antioxidant response elements (ARE) in the promoter of several antioxidant defense proteins, such as those involved in glutathione synthesis, NAD(P)H:quinine oxidoreductase 1 (NQO1), glutathione S-transferase (GST) isoenzymes, and heme oxygenase-1 (HO-1) [3,4]. Under basal conditions, the Kelch repeat region of Kelch-like ECH-associated protein 1 (Keap1) binds to the Neh2 domain of Nrf2, so that Nrf2 is sequestered to the cytoplasm and continually shuttled to the proteasome. Elevated reactive oxygen species, chemical inducers, or dietary factors such as sulforaphane cause modifications in cysteine residues of Keap1, allowing for disruption of Keap1 binding to Nrf2 [5–7]. This disruption permits Nrf2 to translocate to the nucleus to bind to the ARE-DNA sequence (TGAG/CNNNGC) within antioxidant gene promoters [4,8,9]. The Nrf2/Keap1 pathway is linked to the development of obesity, adipose deposition, and hepatic fat deposition in animal models [10,11]. Mechanistic studies show that the Nrf2/Keap1 pathway regulates lipid synthesis and adipocyte differentiation [12–15]. There is a discrepancy in the literature on whether a deficiency in Nrf2 impairs or enhances adipogenesis. Shin et al. Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/freeradbiomed Free Radical Biology and Medicine 0891-5849/$ - see front matter Published by Elsevier Inc. http://dx.doi.org/10.1016/j.freeradbiomed.2012.05.047 Abbreviations: Nrf2, nuclear factor E2-related factor 2; Keap1, Kelch-like ECH-associated protein 1; NQO1, NAD(P)H:quinine oxidoreductase 1; GSK-3b, glycogen synthase kinase-3b; GST, glutathione S-transferase, HO-1, heme oxygenase-1; ARE, antioxidant response element, MEFs, mouse embryonic fibroblasts; PPARg, peroxisome proliferator-activated receptor g; BMI, body mass index; NADPH, nicotinamide adenine dinucleotide phosphate; DMEM, Dulbeccos modified Eagle’s medium, BCS, bovine calf serum; DX, dexamethasone; IBMX, isobutylmethylxanthine; HBSS, Hank’s balanced salt solution; RT, room tempera- ture; FBS, fetal bovine serum; PBS, phosphate-buffered saline; DTT, dithiothreitol; FABP4, fatty acid binding protein 4; SFN, sulforaphane; MAC, MAC220; C/EBP, CCAAT/enhancer-binding protein; bHLH, basic helix-loop-helix; ROS, reactive oxygen species. n Corresponding author at: USDA-ARS Human Nutrition Research Center, 2420 2nd Ave North, Grand Forks, ND 58203, United States. Fax: þ1 701 795 8240. E-mail address: matthew.picklo@ars.usda.gov (M.J. Picklo). Free Radical Biology and Medicine 53 (2012) 690–700