1 receptor regulates cMyc [43], which is reported to be a transcription factor of PHB [44]. IBMX, a cyclic adenosine monophosphate (cAMP) phosphodiesterase inhibitor, prevents the inactivation of the intracellular cAMP, and therefore enhances expression of C/EBPb, a critical transcription factor at the early stage of adipogenic program [45]. Taken together, we postulate that the induction of PHBs is probably initiated via IGF1, cMyc and/or cAMP molecules. Interestingly, we further observed that the expression of PHBs in WAT from obese mice is not more than that from lean ones. In fact, the decrease of the genes, that are characteristic of mature adipocytes and transcription factors critical to the maintenance of terminally differentiated fat cells, are reported in WAT of obese mice. This implies that some degree of dedifferentiation has taken place in the adipose tissue of obese mice [30]. PHB1 and PHB2 are highly homologous proteins that are evolutionarily conserved and ubiquitously expressed [46,47]. A study in yeast has initially shown that PHB1 and PHB2 act as mitochondrial chaperones in the inner mitochondrial membrane [48]. The interdependence of both PHBs was subsequently reported in nematode [12] and some types of mammalian cells by several independent groups including ours [14,17,18,49]. To study the function of PHBs in 3T3-L1 cells, we employed a loss-of- function strategy and found that the loss of one simultaneously leads to the loss of the other at the protein level. Upon silencing of the PHB1 or PHB2, we observed a lower degree of fat accumulation in adipogenic 3T3-L1 cells. Indeed, a recent observation has shown that PHB deficiency markedly reduces intestine fat content early in adulthood of wild-type nematodes. Interestingly, in both nhr-49 and fat-7 mutant nematodes, which causes fat accumulation due to decreased synthesis of monoun- saturated fatty acids, deficiency of PHB not only reduces intestinal fat but also prevents shortage of lifespan [27]. Since either the PHB1- or PHB2-conventional knockout mice do not survive, adipocyte-specific PHB conditional knockout mice may be used in future adipogenic studies. Besides fat accumulation, we detected a downregulation of the adipogenic markers, C/EBPb at the early stage and the PPARc and aP2 at the late stage, upon silencing of PHBs in 3T3-L1 cells, which confirms the essential role of PHBs during adipogenesis. This also implies that PPARc, a key molecule in adipogenesis, may be located downstream of PHBs during adipocyte differentiation. Interestingly, upon forced expression of PHB1 in human ASC, our data demonstrated that the adipocyte differentiation was reduced rather than enhanced. This result is in line with a recent report that adipogenesis is inhibited in 3T3-L1 cells. However, in the absence of insulin, overexpression of PHB1 facilitates adipogenesis of 3T3-L1 cells after adipogenic initiation with adipocyte-induction cocktail. This may be one of the underlying mechanisms involved in enhanced adipogenesis under insulin-resistance condition [34]. It will be interesting to determine the effects of insulin-lacking adipocyte-induction cocktail on adipogenesis and mitochondrial biology in human ASC upon overexpression of PHB1. PHB plays an important role in the Ras-mediated activation of the Raf/MEK/ERK pathway [31,50], which is a highly conserved signaling module that regulates a multitude of essential cellular functions such as proliferation and differentiation [51]. In addition, the activation of MEK/ERK signaling promotes adipogenesis by enhancing PPARc and C/EBPa gene expression during the early phase of the differentiation of 3T3-L1 preadipocytes [32,33]. Our data, in agreement with the above observations, further demonstrate that PHBs are required for the phosphorylation of ERK as early as 15 minutes post adipogenic induction in 3T3-L1 cells. Prohibitins Are Required for Adipogenesis Mitochondrial biogenesis is essential in adipocyte differentia- tion. A 20- to 30-fold increase in the concentration of many mitochondrial proteins has been observed during adipogenesis in a proteomic analysis [4]. It is reported that inhibition of mitochon- drial citrate export causes a significant reduction in fat accumulation in 3T3-L1 cells [6]. In addition, DNA binding of PPARc induced by the adipogenic cocktail is completely prevented in preadipocytes treated with an inhibitor of mitochon- drial respiration [7]. In the present study, we detected a remarkable increment of mitochondrial content and PHBs in differentiated adipocytes when compared to preadipocytes. Furthermore, in the mitochondrial fraction, the concentration of PHB1 or PHB2 is much higher in adipocyte-differentiated 3T3-L1 cells, suggesting that the increase of mitochondrial PHB1 or PHB2 is beyond the increase of the mitochondrial mass during adipogenesis. The mitochondrial content is reduced in PHB- deficient 3T3-L1 cells, which may be one of the mechanisms to explain our previous findings that PHB is essential for stabilizing the mitochondrial integrity and membrane potential [19,20]. Interestingly, our data demonstrate that the protein levels of PHB1 and PHB2 in nuclear fractions are slightly increased in 3T3-L1 cells upon adipocyte differentiation. The mitochondrial-nuclear communication by PHBs shuttling under cell stress or cell differentiation has been recently reported [17,52,53]. However, the existence and influence of nuclear translocation of PHBs during 3T3-L1 cell adipogenesis remain unclear. The analysis of the native structure of PHB1 and PHB2 in yeast, nematodes and mammals has revealed that both proteins are present in a high molecular-weight complex in the inner membrane of mitochondria [10,12,13]. The mitochondrial network is composed of highly interconnected tubules formed by balanced fusion and fission events [54]. Our previous study has shown that PHB1 down-regulation resulted in a transition of mitochondrial morphology from a normal reticular network to vesicular punctiform in ovarian cancer cells [20]. Here, we observed the loss of mitochondrial cristae and the fragmentation of mitochondrial network in either PHB1 or PHB2 knockdown 3T3- L1 preadipocytes. These findings are in agreement with the reports on PHB2-deficient MEFs and PHB1- or PHB2-silencing in HeLa cells [17,18], which suggests that the fusion of mitochondrial membranes is impaired in the absence of PHBs. The abnormal mitochondrial morphology observed in the absence of PHBs may be explained by an altered processing of OPA1 [18], a large dynamin-like GTPase that is found in the mitochondrial intermembrane space and regulates both mitochondrial fusion and cristae morphogenesis [54]. The mechanism by which PHBs affect OPA1 processing remains to be determined. Mitochondria are described as power plants because they generate most of the cellular supply of ATP, which is used as a source of chemical energy. We have not seen significant changes in ATP levels in 3T3-L1 preadipocytes upon PHB1- or PHB2- silencing. These observations are in accordance with the reports in PHB2-deficient MEFs and in PHB1- or PHB2-deficient wild-type C. elegans [18,27]. Schleicher et al. has also reported that the degree of mitochondrial coupling of the respiratory chain in PHB1- knockdown endothelial cells was similar to the control cells [23]. In addition to their crucial role in energy homeostasis, mitochondria are the main site of ROS generation. Mitochondrial ROS have been proven to act as signaling molecules that impact many basic cellular functions such as cell differentiation. It has been demonstrated that mitochondrial ROS strongly inhibits adipocyte differentiation by specifically up-regulating C/EBPf, a dominant- negative inhibitor which forms heterodimers with other C/EBP members [24]. By inhibiting adipogenesis, mitochondrial ROS PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e34315