Mitochondrial dysfunction may play a key role in the progression of steatosis to nonalcoholic steatohepatitis (NASH); however, the molecular mechanism that controls the structure and function of mitochondria in NASH is not clearly understood. Here, we demonstrated that RORα is a regulator of expression of Bnip3 and PGC-1α, and thereby enhances mitochondrial quality. First, we observed that liver-specific RORα knockout mice (RORα-LKO) were more susceptible to high-fat diet-induced NASH compared with control, probably due to mitochondrial dysfunction. Concordantly, mitochondrial fission in response to nutrient stimuli was abolished with downregulation of Bnip3 and phospho-Drp1 in the hepatocytes of RORα-LKO. RORα enhanced oxygen consumption rate and expression of genes associated with mitochondrial quality control. Finally, we observed the positive correlation of the expression levels of Bnip3 and PGC-1α with those of RORα in patients with steatohepatitis. Together, we demonstrated that RORα mediates mitochondrial quality under nutrient-overloaded conditions and propose RORα as a potential therapeutic target in treatment of NASH.
Nonalcoholic fatty liver disease (NAFLD) ranges from simple steatosis to nonalcoholic steatohepatitis (NASH), which can eventually progress to irreversible cirrhosis and to hepatocarcinoma (HCC). About 10% to 20% of patients with hepatic steatosis develop NASH, a disease stage that is characterized by increased oxidative stress and lipotoxicity and leads to cellular injury and chronic inflammation1,2,3. However, the molecular mechanism underlying the development of NASH from steatosis remains unclear. The features that distinguish NASH from steatosis include defects in mitochondrial structure and function, as the mitochondria in the hepatocytes of patients with NASH are swollen and have an abnormal morphology with a loss of cristae, and the activities of mitochondrial respiratory complexes are impaired in these patients4,5. Thus, mitochondrial dysfunction is considered as a central cause of the development of steatosis to NASH, and the identification of the molecular mechanisms that control the structure and function of mitochondria will contribute to the understanding of the progression of NAFLD.
Mitochondria are the main organelles that are required for energy-yielding metabolism via the oxidative phosphorylation (OXPHOS) of glucose and lipids. Maintaining mitochondrial quality is critical for cellular survival, because mitochondria are frequently vulnerable to reactive oxygen species (ROS), a side product of electron transfer. Multiple quality control mechanisms contribute to the maintenance of mitochondrial activity and function, including biogenesis, fission, fusion, and mitochondria-selective autophagy (mitophagy)6. New mitochondria are generated by mitochondrial fission resulting from a fragmentation of the mitochondrial network, especially to reduce oxidative stress in nutrient-overloaded conditions. In contrast, mitochondrial fusion is stimulated by energy demand, to increase metabolic efficiency7. The core machineries of mitochondrial dynamics include the dynamin-1-like protein (Drp1), fission 1 homolog (Fis1), mitochondrial fission factor, BCL2/adenovirus E1B 19 kDa interacting protein 3 (Bnip3), mitofusin 1, 2 (Mfn1, 2), and optic atrophy 1 (Opa1)8. Mitochondrial biogenesis is regulated by a nuclear–mitochondrial network9. In this network, the peroxisome proliferator-activated receptor γ coactivator (PGC-1α) acts as a master regulator that modulates the activity and expression of nuclear respiratory factors, followed by the induction of the transcription factor A, mitochondrial (TFAM), which stimulates mitochondrial gene expression10. Nuclear receptors, such as the estrogen-related receptor (ERR) and peroxisome proliferator-activated receptor (PPAR), are also reported to play a role in connecting the nutrient influx status to mitochondrial gene expression11.
The retinoic acid receptor-related orphan receptor α (RORα) is an orphan nuclear receptor that is associated with the regulation of various genes in hepatic lipid metabolism and inflammation12. RORα binds to a specific DNA sequence, called ROR response element (RORE), consisting of the monomeric RGGTCA motif or Rev-DR2 sites of direct repeats13,14. Natural and synthetic ligands, such as cholesterol sulfates, SR1078, and JC1-40, reversibly bind to RORα and increase the transcriptional activity of target genes via the induction of the DNA binding of RORα and recruitment of coactivators such as p300 and PGC-1α15,16,17,18. In patients with NAFLD and animal NASH models, the hepatic expression levels of RORα are significantly decreased, suggesting that RORα may be associated with pathogenesis in NASH19,20. Recently, we demonstrated that RORα has an inhibitory effect on lipid accumulation and oxidative stress, thereby attenuating hepatic steatosis and NASH15,19. The protective function of RORα against NASH strongly suggests that RORα plays roles in mitochondrial quality control under the pathological condition of oversupply of nutrients.
The physiological functions of RORα have been studied mainly using the RORα-deficient staggerer mice (RORαsg/sg), which carry a C-terminal deleted form of RORα21,22. RORαsg/sg mice fed a high-fat diet (HFD) are resistant to the development of hepatic steatosis and exhibit decreased fasting blood glucose levels and increased insulin sensitivity23,24,25. However, this animal model has limitations regarding liver studies, because the systemic expression of the staggerer gene leads to the development of pathological phenotypes, such as immunodeficiencies, osteoporosis, cerebellar degeneration, atherosclerosis, and muscular atrophy21. Moreover, homozygous RORα−/− mice exhibit tremor and abnormal body balance and die between 24 and 28 days of life26. Thus, we generated mice with a liver-specific deletion of the RORα gene using albumin-cre, to study the hepatic roles of RORα in the progression of NASH. Here, we demonstrated that RORα is a potent regulator of mitochondrial quality control in the liver in response to metabolic input. In particular, Bnip3 and PGC-1α were the transcriptional targets of RORα in the regulation of mitochondrial fission and biogenesis.
Liver-specific KO of the RORα gene enhances susceptibility to HFD-induced steatohepatitis
To study the hepatic role of RORα during the progression of NAFLD, we generated a liver-specific RORα-null mouse, RORα-LKO (Supplementary Fig. S1a,c). The absence of RORα gene expression was demonstrated in the liver of RORα-LKO mice, whereas the hepatic expression of RORβ or RORγ remained at control levels (Supplementary Fig. S1b). We fed an HFD to RORα-LKO mice for 12 weeks, to monitor the effects of deletion of the RORα gene on symptoms of NASH. Hepatic steatosis was severe in RORα-LKO mice compared with flox/flox (f/f) mice. The livers of RORα-LKO mice weighed more than did those of f/f mice, and the accumulation of lipid droplets was evident in the hepatocytes of the former (Fig. 1a,b). The indicators of liver injury, i.e., serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and the marker of lipid peroxidation 4-hydroxynonenal (4-HNE) were increased largely in the livers of RORα-LKO mice (Supplementary Fig. S2c, Fig. 1c). Consistently, hepatic expression of a proinflammatory cytokine, tumor necrosis factor alpha (TNFα), were increased in RORα-LKO mice (Fig. 1d). Also, the expression level of F4/80, a marker of infiltrated macrophages, was increased in the livers of RORα-LKO mice (Supplementary Fig. S2d). In addition, collagen deposition was increased in the liver tissues of RORα-LKO mice and the expression of alpha-smooth muscle actin (α-SMA) and transforming growth factor β1 (TGFβ1), pro-fibrotic factors, were significantly increased in the livers of RORα-LKO mice (Fig. 1e,f). The mRNA levels of genes involved in lipogenesis such as liver X receptor alpha and fatty acid synthase, inflammation such as TNFα and NACHT, LRR and PYD domains-containing protein 3 (NLRP3), and fibrosis such as collagen Type I (Col1a1) and α-SMA were significantly increased in the livers of RORα-LKO mice (Fig. 1g). Together, these data showed that the hepatic expression of RORα is closely associated with the development of NASH.
Mitochondrial function is a target of hepatic RORα
Next, we carried out a transcriptomics study to identify the target genes of RORα associated with the development of NASH. Results from an RNA-seq analysis combined with the data from a public ChIP-seq analysis (GSE59486) revealed that a total of 2,639 genes were specifically altered by RORα (Supplementary Fig. S3a). A gene ontology (GO) analysis of the altered genes revealed that “oxidation-reduction” was the top-ranked GO biological process that was targeted by RORα. Interestingly, the GO term “electron transport chain” was the top GO biological process among the 134 genes in the cluster of oxidation-reduction (Supplementary Fig. S3b). Moreover, we found that the GO cellular component included mitochondrial components with statistical significance (Supplementary Fig. S3c). Surprisingly, the relative expression of most of the genes in the GO terms of “electron transport chain”, “carboxylic catabolic process”, and “ATP biosynthetic process” were significantly decreased in the livers of HFD-fed RORα-LKO mice, suggesting that mitochondrial function may be a target of RORα-mediated transcriptional regulation (Supplementary Fig. S3d).
Therefore, we examined whether mitochondrial function was defective in the livers of RORα-LKO mice. First, we found that the expression of mitochondrial OXPHOS proteins, such as NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8 (NDUFB8), succinate dehydrogenase [ubiquinone] iron-sulfur subunit (SDHB), cytochrome b-c1 complex subunit 2 (UQCRC2), mitochondrially encoded cytochrome c oxidase I (MTCO1), and ATP synthase, H + transporting, mitochondrial F1 complex, alpha 1 (ATP5A), was decreased in the livers of HFD-fed RORα-LKO mice (Fig. 2a). Similarly, the activity of the mitochondrial complex I was decreased in the liver tissues of RORα-LKO mice (Fig. 2b). Moreover, we observed that mitochondria in the hepatocytes of RORα-LKO animals were deformed and showed an enlarged or swollen phenotype after HFD feeding (Fig. 2c). Together, these data showed that mitochondrial function may be a target of hepatic RORα.
RORα is a novel modulator of mitochondrial fission in response to nutrient status
Based on the observation of enlarged mitochondria in the livers of HFD-fed RORα-LKO mice, we hypothesized that the deletion of RORα causes defects in the process of mitochondrial fission, especially under conditions of nutrient overload. First, we measured the oxygen consumption rate (OCR) in hepatocytes isolated from RORα-LKO mice. Although the basal OCR was not much different between RORα-LKO and f/f control mice, the maximal OCR was significantly lower in RORα-LKO hepatocytes (Fig. 3a). To investigate mitochondrial dynamics further, we employed ad-COX8a-GFP to label mitochondria. The culture of primary hepatocytes from control mice in medium with low glucose (5.5 mM), which is a condition of energy demand, led to a hyperfused and elongated mitochondrial network. After the culture was challenged by high glucose (25 mM) with palmitic acid (300 μM), mitochondria were rapidly fragmented (Fig. 3b). However, the morphology and dynamics of mitochondria in RORα-LKO hepatocytes were largely different from those of control animals, in that mitochondria were swollen and remained not much different after the nutrient challenge (Fig. 3b). Along the time lapse after the high-nutrient challenge, the protein levels of Bnip3 and phospho-Drp1 (pDrp1), which are fission proteins, were decreased in RORα-LKO hepatocytes, whereas those of Fis1 was not (Fig. 3c). The decreases in Bnip3 and Drp1 levels were also observed in the liver tissues of HFD-fed RORα-LKO mice (Supplementary Fig. S4). Together, these data showed that mitochondrial fission in response to nutrient stimuli was abolished in RORα-LKO hepatocytes and suggest that the downregulation of Bnip3 and phospho-Drp1 might cause defects in mitochondrial quality control in RORα-LKO hepatocytes.
Overexpression of RORα enhances mitochondrial dynamics via the induction of Bnip3 and pDrp1
Finally, we confirmed the function of RORα in mitochondria quality control via the transient overexpression of RORα. Viral infusion of Ad-GFP-RORα1 into primary hepatocytes dramatically enhanced OCR at both the basal and maximal levels (Fig. 4a). The expression levels of Bnip3 and pDrp1 after nutrient overload were significantly higher in RORα1-overexpressing hepatocytes (Fig. 4b). Consistently, the mRNA expression of Bnip3, but not that of the PTEN-induced putative kinase 1, Mfn1, and Opa1, increased in the presence of overexpression of RORα1, indicating that Bnip3 is a potential target of RORα. In addition, the overexpression of RORα1 resulted in increases in the mRNA expression of genes associated with mitochondrial biogenesis, including PGC-1α, and oxidative phosphorylation (Fig. 4c). Indeed, data from the ChIP-seq analysis showed that RORα-binding signals were present on the regulatory regions of Bnip3 and PGC-1α (Fig. 4d). Additional ChIP assays confirmed that RORα bound to the reads-enriched regions in the Bnip3 and PGC-1α genes. Signals of transcription activation, such as the recruitment of p300 and the acetylation of H3K27, were clearly observed in the presence of RORα in some of these regions (Fig. 4e). Analysis of the reporter genes encoding the putative ROREs present in the regulatory regions of Bnip3 and PGC-1α showed that RORα induced transcriptional activities of the RORE/Bnip3-Luc and the RORE(I)/PGC-1α-Luc by 9-fold and 2-fold, respectively. However, the RORα1 ΔDBD, which lacked DNA binding domain, did not induce the reporters (Fig. 4f). These results indicate direct regulation of Bnip3 and PGC-1α by RORα.
Expression levels of hepatic Bnip3 and PGC-1α were low and correlated positively with those of RORα in patients with steatohepatitis
To assess the clinical relevance of our findings, we analyzed the expression levels of RORα, Bnip3, and PGC-1α in the livers of patients with steatohepatitis, including NASH, using publicly available databases (GSE33814 and GSE48452)27,28. As shown in previous reports, the expression level of RORα was diminished in patients with steatohepatitis19,20. Interestingly, the levels of Bnip3 and PGC-1α were also decreased in the livers of patients with steatohepatitis compared with those of healthy obese patients (Fig. 5a). The observation that the expression levels of Bnip3 and PGC-1α correlated positively with those of RORα suggests a potential application of our findings to diagnostic and therapeutic interventions for NASH (Fig. 5b).
Here, we report that the loss of the function of hepatic RORα resulted in the development of severe NASH in mice, which supports a protective role of RORα against the progression of NAFLD. The function of RORα in hepatic lipid metabolism has been controversial for some decades. RORαsg/sg mice fed an HFD are resistant to diet-induced hepatic steatosis and insulin resistance23,25. In contrast, we found that RORα activates AMPK, while it represses the transcription function of LXRα, thereby protecting from hepatic lipid accumulation15. Moreover, RORα upregulated antioxidative and anti-inflammatory genes, which ameliorated the symptoms of NASH in the methionine and choline deficient diet mouse model19. Although the reasons for this discrepancy are not fully understood, the secondary effects generated by the sg/sg phenotype may contribute, at least in part, to this discordance in hepatic lipid metabolism. For example, the secretion of hormones that are important for energy balance, such as leptin, norepinephrine, adrenocorticotropic hormone, and corticosterone, was abnormal in RORαsg/sg mice25,29,30. These mice were lean despite their hyperphagia, probably because of an enhancement of the energy metabolism in brown adipose tissue29. This imbalanced whole-body metabolism may cover or surpass the primary phenotype of RORα depletion in the liver; thus, the liver-specific RORα mice may provide a better animal model for studying the hepatic function of RORα. Recently, two research groups reported a protective role of RORα against hepatic steatosis by employing the liver-specific depletion of RORα or RORα/γ. They showed that loss of negative regulation of the peroxisome proliferators-activated receptor-γ or overactivation of the sterol regulatory element-binding proteins exacerbated diet-induced hepatic steatosis in these animals31,32. Here, we revealed a novel role of RORα in mitochondrial quality control that inhibits further progression of hepatic steatosis to NASH.
We found that Bnip3 is one of the major downstream effectors of RORα-controlled mitochondrial quality maintenance (Fig. 4c,d). Bnip3 is a well-known mitochondrial fission factor that promotes the translocation of Drp1 to mitochondria and simultaneously decreases Opa1-induced mitochondrial fusion33. Consistently, the depletion of Bnip3 in mice resulted in the loss of mitochondrial membrane potential and structural integrity, probably because of impairment of mitochondrial fission and subsequent mitophagy34. Interestingly, the loss of Bnip3 led to typical features of steatohepatitis, such as reduced AMPK activity and fatty acid β-oxidation and increased lipid synthesis, which were observed in the livers of RORα-LKO mice34. In human patients with NASH, the hepatic levels of Bnip3 were decreased compared with those of healthy obese individuals and correlated positively with the expression levels of RORα (Fig. 5a,b). Together, these observations strongly support our proposed mechanism of Bnip3-mediated RORα action. Bnip3 is also involved in the mitophagic process by interacting with the microtubule-associated protein 1 light chain 3 (LC3)35. Mitophagy is a cellular process triggered by severe mitochondrial defects that eventually promotes the survival of cells under stress conditions6. It was reported that the mitophagic flux is impaired in the livers of both NAFLD patients and murine models of NAFLD, which could be caused by an elevation of endoplasmic reticulum (ER) stress36. The results of our RNA-seq and ChIP-seq analyses showed that the ER ranked at the top of the GO cellular components for RORα-regulated genes (Supplementary Fig. S3c). In addition, RORα induced the mRNA levels of Mfn2, which is enriched at the ER–mitochondria interface and promotes intercommunication (Fig. 4c)37. These observations suggest that RORα mediates the mitochondrial quality control, which is linked to the regulation of ER maintenance.
The observation that RORα increased the expression of markers for mitochondrial biogenesis, such as PGC-1α, Idh3a, and Acadm, suggests that RORα may enhance mitochondrial biogenesis (Fig. 4c). However, total mitochondrial number was not affected by knock-down of RORα (Supplementary Fig. S5). One of the possible causes of this discrepancy may be that quantity of mitochondria is regulated by the balance of two opposing processes, i.e., mitochondrial biogenesis and elimination of mitochondria. Thus, no effect on the mitochondrial number by RORα could be due to the RORα-induced biogenesis and simultaneous elimination of mitochondria by RORα-induced Bnip3, one of the key factors involved in the mitophagic process35.
As mitochondrial dysfunction is associated with various human diseases, including Alzheimer’s disease, multiple sclerosis (MS), and retinal diseases, the roles of RORα in the pathophysiology of these diseases should be addressed. Acquaah-Mensah et al. (2015) reported that RORα expression was abnormal in the hippocampus of patients with Alzheimer’s disease, and that RORα was highly connected in a network of differentially expressed genes, which included genes involved in mitochondrial dynamics such as Fis1 and Opa138. The pathobiology of MS, which is an autoimmune disorder of the central nervous system, is accompanied by mitochondrial dysfunction; the expression of nuclear-encoded mitochondrial genes and the activities of mitochondrial complexes were decreased in the MS motor cortex in patients with this disease39. Interestingly, polymorphism of intronic variations in the RORα gene was associated with susceptibility to MS40. Similarly, a significant link between single-nucleotide polymorphisms present in intron 1 of the RORα gene and a mitochondrial dysfunction-related disease, aged-related macular degeneration, was demonstrated using a systems biology-based approach41. Although there is no clear evidence currently, we suspect that RORα-mediated mitochondrial function might be associated with the development of these diseases. In addition, we found a potential that JC1-40, an agonistic ligand of RORα, improved mitochondrial function (Supplementary Fig. S6). Further studies of pharmacological interventions targeting RORα-mediated mitochondria quality control may provide therapeutic strategies against NASH as well as other diseases associated with defects in mitochondrial function.
The RORαf/f mutant embryo, which has loxP sites flanking exon 4 of the RORα gene, was obtained from the Institut Clinique de la Souris (Illkirch, France) and the mutant mouse was generated by in vitro fertilization (Korea Research Institute of Bioscience and Biotechnology). To produce the liver-specific RORα KO line (AlbCre-RORαf/f), RORαf/f animals were crossbred with AlbCre animals, which express Cre recombinase in hepatocytes under the control of the albumin promoter (Jackson Laboratories). Several backcrosses of the two mouse lines produced the liver-specific RORα KO mice on the C57BL/6 background. Offspring were genotyped to confirm the inclusion of loxP sites within RORα alleles and the presence of Cre recombinase via PCR using specific primers (Supplementary Fig. S1, Supplementary Table). All animals were maintained with 12 h light (7 am) and dark (7 pm) cycles.
Six-week-old male RORαf/f or RORα-LKO mice were fed an HFD (D12492) or low-fat diet (D12450J) (Research Diets, New Brunswick, NJ) for 12 weeks. HFD-fed RORα-LKO mice gained more weight compared with control mice (Supplementary information, Figure S2A). After feeding, liver tissues were excised and cross sections of the left lobe of the liver were analyzed for protein and mRNA, or fixed in 10% neutral buffered formalin (Sigma-Aldrich) for immunohistochemistry. The activities of ALT and AST in the serum were measured using a Fuji DRI-CHEM 3500 s serum biochemistry analyzer (Fujifilm, Japan), and the amount of hepatic TG was measured using an EnzyChromTM Triglyceride Assay Kit (BioAssay Systems). For histological examinations, 3 μm sections of paraffin-embedded tissue were stained with hematoxylin and eosin (H&E). Frozen liver tissue sections were stained with Oil red O staining. All experiments were performed in a blinded and randomized fashion. The experimental protocols were approved by the Seoul National University Institutional Animal Care and Use Committee (permission number SNU-140424-2-5) and all experiments were conducted according to the committee’s guidelines.
Determination of OXPHOS protein expression and complex I activity
OXPHOS proteins in ETC complexes were analyzed by western blotting using a commercially available anti-total OXPHOS primary antibody cocktail (458099, Invitrogen, 1:20000). Proteins were extracted from liver tissues and quantified by bicinchoninic acid assay (Pierce). After quantification, we added sample buffer and let the samples stand for 30 min at 37 °C, for the detection of mitochondrially encoded cytochrome c oxidase I (MTCO1) proteins. An anti-Hsp60 antibody (ab45134, Abcam, 1:10000) was used as a control and the intensity of western blots was quantified by ImageJ. Complex I activity was measured using the Complex I Activity Assay Kit (AAMT001-1KIT, Novagen). Proteins were extracted from liver tissues by adding detergent, and each diluted sample (100 μg protein/200 μl) was added into a well coated with a monoclonal antibody against the NADH dehydrogenase complex. Complex I activity was determined based on the rate of NADH oxidation, which is linked to the reduction of a dye, leading to increased absorbance at 450 nm (according to the manufacturer’s protocol).
Liver tissues were excised and small blocks from the left lobe were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.0). The blocks were postfixed with osmium tetroxide, followed by En bloc staining with 0.5% uranyl acetate. After samples were dehydrated with 30%, 50%, 70%, 80%, 90%, and 100% ethanol, they were embedded in Spurr’s resin and polymerized. Ultrathin sections were cut using an EM UC7 ultramicrotome (Leica, Germany), and examined by JEM1010 transmission electron microscope (JEOL, Japan).
Assessment of oxygen consumption rate
Primary hepatocytes were isolated from 8–10-week-old male C57BL/6 mice via the perfusion of livers with collagenase type IV (Sigma-Aldrich), as described previously19. After perfusion, cells were suspended in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone) containing 10% fetal bovine serum (FBS). The oxygen consumption rate (OCR) was analyzed using the Seahorse XFp Extracellular Flux Analyzer. Mouse primary hepatocytes isolated from control and RORα-LKO mice were plated in assay plates at 104 cells/well with minimal DMEM (XF base medium) supplemented with 10 mM pyruvate, as described previously. In the case of assessment of the OCR of primary hepatocytes infected with either Ad-GFP or Ad-GFP-RORα, hepatocytes were plated in DMEM with 10% FBS 4 h prior to transduction. After 18 h, media were exchanged to unbuffered minimal DMEM supplemented with 10 mM pyruvate. To determine the basal and maximal respiration, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP, an inducer of maximal respiration; 0.25 μM) and antimycin A/rotenone (2 μM) were added to hepatocytes. The basal OCR was calculated by the OCR baseline levels before FCCP injection minus the average of three OCR levels after antimycin A/rotenone injection (non-mitochondrial respiration). Maximal OCR was produced by subtracting non-mitochondrial respiration from the OCR levels after FCCP injection42. The production and infusion of Ad-GFP and Ad-GFP-RORα1 were as previously described15.
Real-time confocal microscopy
For the time course of experiments, hepatocytes were plated in DMEM with 10% FBS 4 h prior to refreshment of media or transduction. After 18 h, hepatocytes were cultured in Earle’s balanced salt solution with 5.5 mM glucose for 2 h. Next, cells were exposed to DMEM with 25 mM glucose and 0.3 mM palmitic acid conjugated with bovine serum albumin for the indicated time course. For real-time confocal microscopy, mitochondria were tagged by Ad-COX8a-GFP for visualization, as described previously43. Subsequently, live images were acquired at 37 °C using a confocal microscope with a Nikon Plan Apochromat 20×/0.75 objective (Nikon Eclipse Ti; Nikon, Japan). Ad-COX8a-GFP was kindly provided by Dr. Lee C-H (Harvard University, MA).
Western blotting, ChIP, and quantitative real-time PCR (qRT–PCR)
Western blotting was performed as described previously using specific antibodies against α-SMA (ab7817, Abcam, 1:5000), TGFβ1 (sc-130348, Santa Cruz Biotechnology, 1:1500), Bnip3 (ab109362, Abcam, 1:7000), phospho-DRP1 (Ser616) (#4494, Cell Signaling Technology, 1:2000), Drp1 (#8570, Cell Signaling Technology, 1:2000), Fis1 (ab71498, Abcam, 1:2000), RORα (sc-6062, Santa Cruz Biotechnology, 1:2000), and actin (sc-1616, Santa Cruz Biotechnology, 1:2000). The ChIP assay was performed using anti-RORα (sc-6062, Santa Cruz Biotechnology), anti-p300 (sc-585, Santa Cruz Biotechnology), and anti-histone 3 (acetyl K27) (ab4729, Abcam) antibodies or a control IgG antibody (Santa Cruz Biotechnology), and specific primers (Supplementary Table)19. Relative mRNA expression was determined by qRT–PCR using the ABI StepOnePlusTM Real-Time PCR system (Applied Biosystems, Foster City, CA) using specific primers (Supplementary Table). The mRNA expression of genes was calculated relative to controls using the 2−ΔΔCT method19.
Reporter gene analysis
The RORE/Bnip3-Luc and RORE(I)/PGC-1α-Luc were constructed by inserting three copies of putative ROREs into a pGL2-promoter vector (Promega) using specific oligomers (Supplementary Table). An eukaryotic expression vector encoding RORα1 ΔDBD was constructed by a PCR-mediated deletion method. Chang liver cells (CCL-13TM, ATCC, Rockville, MD) were transfected with a plasmid mixture containing reporter plasmid, eukaryotic expression vector, and pCMV-β-galactosidase using the Polyfect (Qiagen) according to the manufacturer’s protocol. Cells were lysed using luciferase lysis buffer (Promega, Madison, WI) and luciferase activity was measured using LB9508 luminometer (Berthold, Bad Wildbad, Germany) and normalized to β-galactosidase activity.
Database-based gene expression analysis
Database-based gene expression analysis was conducted using public datasets obtained from GEO site at the NCBI gene expression (http://www.ncbi.nlm.nih.gov/geo/). The data from GSE33814 and GSE48452 processed as median-normalized signal intensity value. These public datasets were obtained from liver tissues of human patients. Normal (n = 13) and steatohepatitis (n = 12) for GSE3381428; healthy obese (n = 27) and NASH (n = 18) for GSE4845227.
All analyses were performed using the GraphPad Prism software. Statistical analyses between two groups were conducted using the nonparametric Mann–Whitney U test (two-tailed, Figs 1, 2a, 3a right, 3b, 4a right, 4c, 5a) or unpaired Student’s t-test (two-tailed, Fig. 4f). Two-way ANOVA followed by the Bonferroni posttest was used to analyze the statistical significance of complex I activity (Fig. 2b), OCRs (Fig. 3a left, 4a left), and of ex vivo studies of nutrient switch (Figs 3c and 4b). Data are presented as the mean ± SEM. Statistical significance was set at P < 0.05.
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This work was supported by grants, the NRF-2014R1A2A1A10052265, NRF-2012M3A9B6055338, NRF-2014M3A9D5A01073556, and a grant from the Health Fellowship Foundation.