Mitochondrial Dysfunction and Adipogenic Reduction by Prohibitin Silencing in 3T3-L1 Cells Dong Liu1,2*, Yiming Lin3, Ting Kang1,4, Bo Huang1,5, Wei Xu 6 , Minerva Garcia-Barrio1, Moshood Olatinwo6, Roland Matthews6, Y. Eugene Chen7, Winston E. Thompson 6 * 1 Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta, Georgia, United States of America, 2 Department of Physiology, Morehouse School of Medicine, Atlanta, Georgia, United States of America, 3 Department of Endocrinology, School of Medicine, Emory University, Atlanta, Georgia, United States of America, 4 Division of Cardiology, the First Affiliated Hospital, Nanchang University, Nanchang, China, 5 Department of Clinical Laboratory Sciences, the Second Affiliated Hospital, Nanchang University, Nanchang, China, 6 Department of Obstetrics and Gynecology, Morehouse School of Medicine, Atlanta, Georgia, United States of America, 7 Cardiovascular Center, University of Michigan, Ann Arbor, Michigan, United States of America Abstract Increase in mitochondrial biogenesis has been shown to accompany brown and white adipose cell differentiation. Prohibitins (PHBs), comprised of two evolutionarily conserved proteins, prohibitin-1 (PHB1) and prohibitin-2 (PHB2), are present in a high molecular-weight complex in the inner membrane of mitochondria. However, little is known about the effect of mitochondrial PHBs in adipogenesis. In the present study, we demonstrate that the levels of both PHB1 and PHB2 are significantly increased during adipogenesis of 3T3-L1 preadipocytes, especially in mitochondria. Knockdown of PHB1 or PHB2 by oligonucleotide siRNA significantly reduced the expression of adipogenic markers, the accumulation of lipids and the phosphorylation of extracellular signal-regulated kinases. In addition, fragmentation of mitochondrial reticulum, loss of mitochondrial cristae, reduction of mitochondrial content, impairment of mitochondrial complex I activity and excessive production of ROS were observed upon PHB-silencing in 3T3-L1 cells. Our results suggest that PHBs are critical mediators in promoting 3T3-L1 adipocyte differentiation and may be the potential targets for obesity therapies. Citation: Liu D, Lin Y, Kang T, Huang B, Xu W, et al. (2012) Mitochondrial Dysfunction and Adipogenic Reduction by Prohibitin Silencing in 3T3-L1 Cells. PLoS ONE 7(3): e34315. doi:10.1371/journal.pone.0034315 Editor: Paul McNeil, Medical College of Georgia, United States of America Received October 27, 2011; Accepted February 28, 2012; Published March 30, 2012 Copyright: 2012 Liu 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 study was supported by the National Institutes of Health grants G12RR003034 and G12RR003034-supplement to DL, U54HD41749 to WET (http:// grants.nih.gov/grants/oer.htm); and by the Department of Defense grant BC020050 to WET (http://cdmrp.army.mil/bcrp/default.shtml). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the munuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: dliu@msm.edu (DL); wthompson@msm.edu (WET) Introduction During the development of obesity, adipocytes become hypertrophic until a crucial cell size is reached, after which they further expand the fat mass by adipocyte hyperplasia [1,2]. Preadipocytes, even from elderly humans, retain the capacity to differentiate in vitro [3]. Mitochondrial biogenesis, found during mouse 3T3-L1 preadipocyte differentiation, is accompanied by the remodeling of the mitochondrion and is considered to be a necessary adjustment because the cells become increasingly active in metabolism [4]. Enhancement of mitochondrial biogenesis during adipogenesis may be the result of activation or enhanced expression of nuclear encoded mitochondrial genes that are under the control of adipogenic transcription factors [5]. The essential role of mitochondrial biogenesis during adipogenesis is further confirmed by the observation that induction of mitochondrial dysfunction inhibits adipogenesis in 3T3-L1 preadipocytes [6,7]. Mitochondria are extremely dynamic structures that fuse and divide continuously to adjust the shape and distribution of the mitochondrial network depending on cell type and energy demands, therefore playing critical roles in cell physiology. Prohibitin (PHB) proteins are highly expressed in cells that rely heavily on mitochondrial function [8]. PHBs comprise two evolutionarily conserved proteins, prohibitin-1 (PHB1) and prohibitin-2 (PHB2, a.k.a. REA). Both proteins associate in heterodimers in a high molecular-weight complex (,1.2 MDa) in the inner membrane of mitochondria [9,10,11,12,13]. About 12 to 16 PHB heterodimers associate to form a ring-like structure at the mitochondrial inner membrane [11]. PHB1 and PHB2 are physically interactive [9,14,15] and functionally interdependent in various organisms [12,14,16,17,18]. The absence of either PHB does not affect the expression of the other, but results in its posttranslational degradation. Our previous work revealed that PHB1 is essential for stabilizing the mitochondrial integrity and membrane potential in human ovarian cancer cells and rat ovarian granulosa cells [19,20]. Loss of PHBs brings about altered organization and reduced copy number of mtDNA, and unstabilized mitochondrial-encoded subunits of the respiratory chain [21]. Affected mtDNA within fragmented mitochondria may cause the disruption of OXPHOS and therefore promote the production of free radicals [22]. Indeed, lack of PHB1 results in increased levels of reactive oxygen species (ROS) in endothelial cells [23]. An increase in mitochondrial ROS generation is demonstrated to prevent preadipocyte differentiation through upregulation of C/EBPf, an adipogenic repressor [24]. Increased intracellular expression and decreased extracellular secretion of PHBs have been observed during adipogenesis [25,26]. A recent publication has shown that PHB deficiency in PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e34315