The Orphan Nuclear Receptor, NOR-1, a Target of -Adrenergic ...

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Endocrinology 149(6):2853–2865 Copyright © 2008 by The Endocrine Society doi: 10.1210/en.2007-1202

The Orphan Nuclear Receptor, NOR-1, a Target of ␤-Adrenergic Signaling, Regulates Gene Expression that Controls Oxidative Metabolism in Skeletal Muscle Michael A. Pearen, Stephen A. Myers, Suryaprakash Raichur, James G. Ryall, Gordon S. Lynch, and George E. O. Muscat Institute for Molecular Bioscience (M.A.P., S.A.M., S.R., G.E.O.M.), University of Queensland, Queensland 4072, Australia; and Basic and Clinical Myology Laboratory (J.G.R.), Department of Physiology, University of Melbourne, Victoria 3010, Australia ␤1–3-Adrenoreceptor (AR)-deficient mice are unable to regulate energy expenditure and develop diet-induced obesity on a high-fat diet. We determined previously that ␤2-AR agonist treatment activated expression of the mRNA encoding the orphan nuclear receptor, NOR-1, in muscle cells and plantaris muscle. Here we show that ␤2-AR agonist treatment significantly and transiently activated the expression of NOR-1 (and the other members of the NR4A subgroup) in slow-twitch oxidative soleus muscle and fast-twitch glycolytic tibialis anterior muscle. The activation induced by ␤-adrenergic signaling is consistent with the involvement of protein kinase A, MAPK, and phosphorylation of cAMP response element-binding protein. Stable cell lines transfected with a silent interfering RNA targeting NOR-1 displayed decreased palmitate oxidation and lactate accumulation. In concordance with these observations, ATP production in the NOR-1 silent interfering RNA (but not control)-transfected cells was resistant to (azide-me-

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HE NR4A SUBGROUP of orphan nuclear hormone receptors (NRs) includes Nur77 (NR4A1), Nurr1 (NR4A2), and NOR-1 (NR4A3). The expression and function of the NR4A subgroup is regulated by pleiotropic physiological stimuli including exercise (1); mechanical stress (2); fatty acids (3); glucose (4); low-density lipoprotein (5); growth factors (6 – 8); cytokines (9, 10); prostaglandins (11); and the anticancer compounds, 6-mercaptopurine (12) and diindoylmethanes (13, 14). The NR4A subgroup is part of the orphan members of the NR superfamily of DNA binding proteins. Structural studies suggested that these NRs encode atypical ligand binding domains occupied by bulky amino acid side chains, which precludes the binding of orthodox First Published Online March 6, 2008 Abbreviations: ␤-AR, ␤-Adrenoceptor; ATF, activating transcription factor; c, catalytic subunit; ChIP, chromatin immunoprecipitation; CREB, cAMP response element-binding protein; DMSO, dimethylsulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HIF, hypoxia inducible factor; MEK, MAP-ERK kinase; NR, nuclear hormone receptor; PDHC, pyruvate dehydrogenase complex; PDK, pyruvate dehydrogenase kinase; PDP, pyruvate dehydrogenase phosphatase; PGC, peroxisomal proliferator-activated receptor-␥ coactivator; PKA, protein kinase A; qRT-PCR, quantitative real-time PCR; r, regulatory subunit; siRNA, small interfering RNA; TCA, tricarboxylic acid; UCP, uncoupling protein. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

diated) inhibition of oxidative metabolism and expressed significantly higher levels of hypoxia inducible factor-1␣. In addition, we observed the repression of genes that promote fatty acid oxidation (peroxisomal proliferator-activated receptor-␥ coactivator-1␣/␤ and lipin-1␣) and trichloroacetic acid cyclemediated carbohydrate (pyruvate) oxidation [pyruvate dehydrogenase phosphatase 1 regulatory and catalytic subunits (pyruvate dehydrogenase phosphatases-1r and -c)]. Furthermore, we observed that ␤2-AR agonist administration in mouse skeletal muscle induced the expression of genes that activate fatty acid oxidation and modulate pyruvate use, including PGC-1␣, lipin-1␣, FOXO1, and PDK4. Finally, we demonstrate that NOR-1 is recruited to the lipin-1␣ and PDK-4 promoters, and this is consistent with NOR-1-mediated regulation of these genes. In conclusion, NOR-1 is necessary for oxidative metabolism in skeletal muscle. (Endocrinology 149: 2853–2865, 2008)

NR ligands (12, 15, 16). Both the anticancer compounds and prostaglandin A2 (11) activate the NR4A subgroup; however, the anticancer compounds do not appear to directly bind to the NR4A ligand binding domains (12, 14). The NR4A subgroup has also been implicated in atherogenesis (17, 18), steroidogenesis (19 –21), inflammation (10, 22), apoptosis (23–25), and carcinogenesis (22, 26). NRs are involved in the tissue/organ-specific regulation of metabolism. The NR4A subgroup are regulated in a circadian manner (27, 28), expressed in skeletal muscle and other metabolic tissues (adipose and liver) with onerous energy demands (29); this suggests a potential role for these receptors in metabolism and energy homeostasis. Skeletal muscle accounts for approximately 40% of the total body mass. Moreover, it is the major site of insulinstimulated glucose uptake. Energy requirements are primarily met by use of stored glycogen, glucose (and other carbohydrates), and fatty acids. Energy expenditure in skeletal muscle accounts for a very significant fraction of the body’s’ energy use and is an important tissue during metabolic responses to fasting and feeding. Therefore, this peripheral tissue plays a pivotal role in insulin sensitivity, the blood lipid profile, and energy balance. Consequently, this majormass tissue has a significant role in the progression of dyslipidemia, diabetes, and obesity. Several reports have demonstrated that ␤-adrenoceptor

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(␤-AR) agonists increase glucose transport, energy expenditure, lipolysis, and thermogenesis in skeletal muscle (30 –34). Although cAMP response element-binding protein (CREB) activation has been associated with ␤-AR signaling, the pathways, mechanisms, and mode of action that accounts for these effects have not been completely resolved. Interestingly, mice that are deficient in the three known ␤-ARs are predisposed to obesity on a high-fat diet (35). New insights into the mechanisms (and associated signaling pathways) have been provided by studies demonstrating the induction of the mRNA encoding two members of NR4A subgroup (Nur77 and NOR-1) by ␤-AR agonists in skeletal muscle cells (36, 37) and cultured brown adipocytes (38). Furthermore, the expression of mRNA encoding NOR-1 is hyper-activated after ␤2-AR agonist treatment in skeletal muscle tissue (37) and cold exposure in Nur77 null mice in brown adipose (38). However, the in vivo response in slow (type I) oxidative and fast (type II) glycolytic skeletal muscle of the other NR4A subgroup members to ␤-AR signaling has not been ascertained, and the functional role of NOR-1 in skeletal muscle metabolism has not been addressed. Preliminary analysis of an NOR-1 small interfering RNA (siRNA) transfected cell line indicated aberrant FABP4, UCP-2, and UCP-3 expression (37). However, the metabolic implications of regulatory cross talk between the NOR-1 and ␤-AR signaling pathways in skeletal muscle has not been addressed. Understanding NOR-1 function (and the ␤-AR dependent expression) in muscle may provide the foundation for therapeutic manipulation in the future. In the current study, we identified that expression of the mRNAs encoding the three members of the NR4A subgroup were strikingly activated in slow oxidative and fast glycolytic dominant skeletal muscle tissues 1– 4 h after treatment of mice with a ␤2-AR agonist. The increased gene expression induced by ␤-adrenergic signaling involved protein kinase A (PKA), MAPK (predominantly p38 mediated), and phosphorylation of CREB. NOR-1 siRNA expression induced changes in gene expression associated with reduced fatty acid oxidation and the anaerobic use of glucose/pyruvate. These changes correlated with increased lactate accumulation and anaerobic ATP production. In addition ␤-AR signaling (in vivo and in vitro) induced gene expression that modulates fatty acid oxidation in skeletal muscle. Materials and Methods Animals, ␤-AR agonist administration, and tissue collection All procedures were approved by the Animal Experimentation Ethics Committees of The University of Melbourne. All procedures conformed to the Guidelines for the Care and Use of Experimental Animals described by the National Health and Medical Research Council of Australia. Male C57BL/10 ScSn (C57BL/10, wild-type, 6 –7 wk old) were obtained from the Animal Resource Centre (Canning Vale, Western Australia, Australia) and were randomly assigned to either saline control or formoterol (a specific ␤2-AR agonist)-treated groups (n ⫽ 4 mice per time point per treatment). The mice were housed in the Biological Research Facility at The University of Melbourne and maintained on a 12-h light, 12-h dark cycle, with standard mouse chow and water provided ad libitum. Treated mice received a single ip injection of formoterol (100 ␮g/kg in 0.2 ml saline; Astra Zeneca, Cheshire, UK) and control mice (n ⫽ 4) received an equivalent volume of sterile saline. We demonstrated pre-

Pearen et al. • NOR-1 Is Necessary for Oxidative Metabolism

viously that this is the most efficacious dose for eliciting skeletal muscle hypertrophy in rodents (39). Mice were anesthetized at 1, 4, 8, and 24 h after treatment, and the soleus and tibialis anterior muscles (C57BL/10) were surgically excised. Tissue was also removed from anesthetized untreated mice (n ⫽ 4) that did not receive an ip injection (No treatment group). All samples were snap frozen in liquid nitrogen for RNA extraction, cDNA synthesis, and quantitative real-time PCR (qRT-PCR) analysis.

RNA extraction and cDNA synthesis RNA was extracted from C2C12 cells and skeletal muscle tissue using TRI-Reagent (Sigma-Aldrich, Castle Hill, Australia) according to the manufacturer’s protocol. Extracted RNA was treated with 2 U Turbo DNase (Ambion, Austin, TX) for 30 min. RNA was further purified using a mini-RNeasy kit (QIAGEN, Valencia, CA) according to the manufacturer’s instructions. cDNA was synthesized from 3 ␮g of total RNA for cell culture experiments and 1 ␮g for animal muscle experiments (normalized via UV spectroscopy) using Superscript III primed by random hexamers, according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA).

qRT-PCR Target cDNA levels were compared by qRT-PCR in 25-␮l reactions containing either 1⫻ SYBR green (Applied Biosystems, Foster City, CA) or Taqman PCR master mix (Roche Molecular Systems, Pleasanton, CA), 100 nm of each forward and reverse primers for SYBR green or 1⫻ Assay-on-Demand Taqman primers (Applied Biosystems) and the equivalent of 0.3 ␮l cDNA. Using an ABI Prism 7500 sequence detection system (Applied Biosystems), PCR was conducted over 45 cycles of 95 C for 15 sec and 60 C for 1 min, preceded by an initial step 95 C for 10 min. Results were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or 18S as stated and compared by relative expression. cDNA used in 18S samples was diluted 1:100 and relative expression was contracted for this dilution.

qRT-PCR primers Primers for qRT-PCR analysis of the mRNA populations using SYBR green have been described in detail (36, 37, 40, 41) except for primers used to detect pyruvate dehydrogenase kinase (PDK)-1 (forward, 5⬘GAA AAG GAA GTC CAT CTC ATC GA-3⬘; reverse, 5⬘-AAA GCC GCC TAG CGT TCT C-3⬘); PDK-3 (forward, 5⬘-AGC GCT ACT CCC GCT TCT C-3⬘; reverse, 5⬘-GCG CAG AAA CAT ATA GGA AGT TTT T-3⬘); pyruvate dehydrogenase phosphatase (PDP)-1 catalytic subunit (c) (forward, 5⬘-GAA AGG AGA GCA AAG ATG TCA TCA-3⬘; reverse, 5⬘-TGA TCA ACA GCC CCA AAT TCA-3⬘); PDP1 regulatory subunit (r) (forward, 5⬘-TGT TGG GCC TGG CTT AAC A-3⬘; reverse, 5⬘-CTC GAG GGC CAA TCA GAT-3⬘); and citrate synthase (forward, 5⬘-GGT GGG CCA GAT CAC TGT G-3⬘; reverse, 5⬘-AAC GGA TGC CCT CGT CAG-3⬘). All SYBR primers were designed using Primer Express (Applied Biosystems). Assay-on-Demand Taqman primer/probe sets (Applied Biosystems) were used to assay expression of Nurr1, peroxisomal proliferator-activated receptor-␥ coactivator (PGC)-1␤, hypoxia inducible factor (HIF)1␣, lipin-1␣, and FOXO1.

Cell culture C2C12 mouse myoblasts (American Type Culture Collection, Manassas, VA) were expanded in growth medium [DMEM (Invitrogen Australia, Mount Waverly, Australia) supplemented with 10% heat-inactivated Serum Supreme (Cambrex Life Sciences, Verviers, Belgium)] in 7.5% CO2. Confluent myoblasts were differentiated into multinucleated myotubes using DMEM supplemented with 1% heat-inactivated fetal calf serum (Invitrogen Australia) and 1% heat-inactivated Serum Supreme up to 5 d. Cells were harvested at indicated time points.

Cell culture drug treatments Confluent myoblasts were differentiated over 4 d before all drug treatments. Myotubes were treated with 100 nm isoprenaline hydrochloride (Calbiochem), 10 ␮m forskolin (Sigma-Aldrich), or vehicles


[ethanol and dimethylsulfoxide (DMSO), respectively]. For MAPK inhibitor experiments, myotubes were pretreated with 20 ␮m SB203580 (Sigma-Aldrich), 20 ␮m PD98059 (Sigma-Aldrich), or DMSO (vehicle) for 15 min.

Western blot analysis Whole cell lysates were prepared from cells disrupted in lysis buffer [50 mm Tris HCl, 75 mm NaCl, 25 mm NaF, 5 mm MgCl2, 5 mm EGTA, 100 ␮m sodium orthovanadate, 1 mm dithiothreitol, 1 mm sodium pyrophosphate, 1% Nonidet P-40 and complete protease inhibitors (Roche Diagnostics Australia, Castle Hill, Australia)] resolved on 12% SDSPAGE gels (42). Proteins were transferred to a polyvinyl difluoride membrane (Immobilon-P; Millipore, Bisley, UK) and blocked for 1 h with 5% fraction V BSA (Sigma-Aldrich) in Tris-buffered saline with 0.1% Tween 20. Blots were initially probed with antiphospho-CREB (ser133; Cell Signaling, Beverly, MA) at 1:1000 in blocking buffer. Blots were also stripped with Reblott (Millipore), blocked with 5% skim milk powder in Tris-buffered saline, and reprobed with anti-CREB (Cell Signaling) in blocking buffer. Secondary antirabbit-horseradish peroxidase (Pierce Biotechnology, Rockford, IL) was used at 1:4000. Horseradish peroxidase localization was detected with Amersham ECL Plus (GE Healthcare, Little Chalfont, UK) according to the manufacturer’s instructions and visualized by autoradiography.

Plasmids The plasmids pSilencer 3.1 neomycin NOR-1 (37), pSilencer 3.1 neomycin-negative (Ambion), pGL2-p (Promega, Madison, WI), VP16 (43), and VP16-NOR-1 (44) have been previously described. For pGL2-pPDK-4 (⫺351/⫺308), a known responsive region of the PDK4 promoter (45), was synthesized (Geneworks, Adelaide, Australia) with BglII restriction sites. Equal molar ratios of both the sense (5⬘-GAT CGA GAT GGC TCT GGA GTT GTA AAC AAG GAC AAG TCT GGG CGG GC-3⬘) and antisense (5⬘-GAT CGC CCG CCC AGA CTT GTC CTT GTT TAC AAC TCC AGA GCC ATC TC-3⬘) strands were incubated at 95 C for 7 min followed by cooling to reverse transcription for 1 h. A single copy of the double-stranded PDK4 element was then cloned into pGL2-p vector at BglII sites and sequenced.

Stable C2C12 transfection C2C12 myoblasts cultured in growth medium were transfected at approximately 40% confluence with 4 ␮g of pSilencer 3.1 neomycin NOR-1 (C2C12-siNOR-1) or pSilencer 3.1 neomycin negative (C2C12siNEG) by Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol in 25-cm2 flasks. Cells were selected for 12 d by treatment with 650 ␮g/ml geneticin (G418; Invitrogen Australia) in growth medium. The polyclonal pool of G418-resistant cells (comprised of ⬎ 50 colonies to avoid clonal bias) were maintained in growth medium with 300 ␮g/ml geneticin. Polyclonal populations were passaged into three replicates, differentiated into myotubes as described above, and RNA independently extracted from each replicate of cells.

Palmitate oxidation assay C2C12 cells were grown and differentiated in 24-well plates. After 4 d of differentiation, cells were serum starved for 4 h in pure high-glucose DMEM. Palmitate oxidation was determined by measuring tritiated water production using the method described elsewhere (46). Briefly, after serum withdrawal, 500 ␮l of equilibrated (warm and gassed) assay media [high glucose DMEM (Invitrogen Australia) containing 2% fatty acid-free BSA (Sigma-Aldrich), 0.25 mm potassium palmitate, and 1␮Ci/ml (20 nm) [9,10 (n)-3H]palmitic acid (GE Healthcare)] was placed on cells. After 2 h of assay media treatment, media were removed, acidified with hydrochloric acid, and unoxidized palmitate removed via chloroform/methanol extraction as described previously (47). The aqueous phase containing titrated water was assayed in OptiPhase Supermix (PerkinElmer Life Sciences, Waltham, MA) cocktail using the Wallac Trilux microbeta 1496 scintillation detector (PerkinElmer Life Sciences). Serial dilutions of [9,10 (n)-3H]palmitic acid mixed with water were used as activity standards. Protein was assayed via the method of Bradford


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(48) using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hemel Hempstead, UK).

L-lactate assay L-lactate in cell culture media was measured using an enzymatic assay kit (Megazyme, Wicklow, Ireland) according to the manufacturer’s protocol. Cell culture media was diluted 1:25 to be within the recommended assay range. Confluent myoblasts were differentiated over 4 d before all drug treatments.

ATP assay and azide treatment analysis Total cellular ATP was assayed via ATPlite reagent (PerkinElmer Life Sciences) according to the manufacturer’s protocol. C2C12 myotubes were differentiated in 96-well plates, washed with PBS, and frozen in 50 ␮l ATPlite lysis buffer at ⫺80 C for analysis. For the 5-min azide treatment, myotubes were concurrently treated with 1 mm sodium azide in PBS or PBS without azide. After treatment with azide, cells were concurrently washed three times with PBS and quickly frozen in ATPlite lysis buffer at ⫺80 C for analysis. Protein was assayed via the method of Bradford (48) using the Bio-Rad protein assay reagent (Bio-Rad Laboratories).

Chromatin immunoprecipitation (ChIP) C2C12 cells were differentiated for 5 d, and expression of NOR-1 was induced by treatment with the ␤2-AR agonist salbutamol (100 nm; Sigma-Aldrich) for 2 h before harvesting. Cells were subsequently washed twice in ice-cold PBS and cross-linked in 1% formaldehyde solution. ChIP was performed as described by Li et al. (49) using anti-NOR-1 (sc-22519; Santa Cruz Biotechnology, Santa Cruz, CA), IgG (Santa Cruz), and anti-GAPDH (Santa Cruz). The following lipin-1␣ and PDK4 qPCR ChIP primers were used: lipin-1␣ NR1 (forward, 5⬘-CTA GCT TGT GAC AGG CAC ATC AG-3⬘ and reverse, 5⬘-GGA GTA GGC AGC CAA AGT CTT CT-3⬘), lipin-1␣ NR3 (forward, 5⬘-AGA ACA CCT GGG CTT CAG AGA CT-3⬘ and reverse, 5⬘-GAA GAT CCC ACA AAC AGG TCA CT-3⬘), PDK4 NR1 (forward, 5⬘-GAG AAT GGC CTG GGA TCT CTA TC-3⬘ and reverse, 5⬘-ACC CGT GAT TCC CAG ACA AA-3⬘), PDK4 NR3 (forward, 5⬘-CAT GAC CGA TGC TAG GAC CAT A-3⬘ and reverse, 5⬘-TCT CAC ATA ACA GAT GAC ACA TAT AGC TTT-3⬘), and GAPDH promoter region (used as negative control; forward, 5⬘-GCT CAC TGG CAT GGC CTT CCG-3⬘ and reverse, 5⬘GTA GGC CAT GAG GTC CAC CAC-3⬘.

Transient transfection C2C12 myoblasts were transfected at 50 –70% confluence in 24-well plates. Transfections were carried out in growth medium using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Growth medium was replaced after 14 –16 h. After an additional 24 h, cells were harvested and assayed for luciferase activity using LucLite reagent (PerkinElmer Life Sciences) according to the manufacturer’s protocol. Luminescence was measured using a Wallac 1450 Microbeta Trilux (PerkinElmer Life Sciences).

Statistical analysis Statistical analyses were performed using GraphPad Prism software (GraphPad, San Diego, CA). All data were analyzed using the Student’s t test or one-way ANOVA with Bonferroni’s or Dunnett’s posttest, as indicated.

Results The mRNAs encoding the three members of the NR4A subgroup (Nur77, Nurr1, and NOR-1) are significantly induced by the ␤2-AR-specific agonist, formoterol, in redoxidative and white-glycolytic skeletal muscle

As discussed, we previously observed that 0.5– 8 h after ␤2-AR agonist treatment, the expression of the mRNA encoding the orphan nuclear receptor, NOR-1, is significantly

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activated in both mouse white-glycolytic muscle tissue (plantaris) and C2C12 skeletal muscle cells (37). We subsequently examined whether ␤-AR agonists induced the expression of NOR-1 (and the other members of the NR4A subgroup) in white-glycolytic (fast fiber dominant) tibialis anterior and red-oxidative (slow fiber dominant) soleus skeletal muscle tissue. Skeletal muscle tissues were isolated from groups of male mice, treated with either the specific ␤2-AR agonist formoterol or saline (vehicle control) and assayed (0, 1, 4, 8, and 24 h after treatment) for the expression of mRNAs encoding Nur77, Nurr1, and NOR-1, respectively. Tibialis anterior is a white-glycolytic tissue and fast fiber dominant, composed of type IIB (and IIX) fibers. The red-oxidative soleus muscle has a predominantly slow-twitch fiber phenotype and composed of type I and IIA fibers (and is rich in mitochondria). Using qRT-PCR, we observed an induction of (4 –15-fold) in the mRNA encoding Nur77 (normalized to 18S rRNA) in soleus (Fig. 1Ai) and tibialis anterior muscles (Fig. 1Bi) 1– 4 h after formoterol treatment but not after saline treatment. The induction of Nur77 relative to the vehicle controls was statistically significant at 1 and 4 h (P ⬍ 0.001) but not at 8 and 24 h after treatment. Similarly, we observed an induction of (16- to 85-fold) in the mRNA encoding Nurr1 (normalized to 18S rRNA) in soleus (Fig. 1Aii) and tibialis anterior muscles (Fig. 1Bii) after 1– 4 h of ␤2-AR agonist treatment but not after saline treatment. The induction of Nurr1 relative to the vehicle controls

FIG. 1. The ␤2-AR agonist formoterol increases NR4A mRNA expression in slow oxidative (soleus) and fast glycolytic (tibialis anterior) skeletal muscle. qRT-PCR was used to assay the expression of Nur77 (i), Nurr1 (ii), and NOR-1 (iii) mRNA soleus (A) and tibialis anterior (B). Muscles were removed at 1, 4, 8, and 24 h after a single ip injection of formoterol or saline vehicle (NT ⫽ no treatment). Results were normalized against 18S at each time point. C, Relative expression values from both tissues were converted to fold inductions relative to vehicle controls at each time point for Nur77 (i), Nurr1 (ii), and NOR-1 (iii). D, RNA was extracted from soleus and tibialis anterior muscles. Using qRT-PCR, the expression of TNNI1 (i), and TNNI2 (ii) was compared in both muscle tissues normalized to GAPDH. Data are expressed as mean ⫾ SEM (n ⫽ 4). Statistical significance was assessed using a one-way ANOVA with Bonferroni’s posttest where P ⬍ 0.05 (*), P ⬍ 0.01 (**), and P ⬍ 0.001 (***).


was statistically significant at both 1 and 4 h (P ⬍ 0.001) but not at 8 and 24 h after treatment. Similarly to the previously reported induction of NOR-1 in fast-twitch plantaris muscle after formoterol treatment (37), we observed an induction of (50- to 80-fold, respectively) in the mRNA encoding NOR-1 (normalized to 18S rRNA) in soleus (Fig. 1Aiii) and tibialis anterior (Fig. 1Biii) after 1– 8 h of ␤2-AR agonist treatment but not after saline treatment. The induction of NOR-1 relative to the saline controls was statistically significant at 1 (P ⬍ 0.05), 4 (P ⬍ 0.001), and 8 h (P ⬍ 0.05) after treatment. For convenience, ␤2-AR agonist-induced changes in NR4A gene expression are shown as fold changes (relative to vehicle) for both tissues (Figs. 1, Ci–Ciii). (type II) muscle tissue from C57BL/10 mice. The fiber type identity of tibialis anterior and soleus muscle was confirmed by the analysis of type I (slow) and II (fast) troponin I mRNA expression (Fig. 1 Di and Dii, respectively). In addition, we observed that treatment with the nonselective ␤-AR agonist isoprenaline significantly induced the expression of the mRNAs encoding the NR4A subgroup in the mixed fiber-type gastrocnemius muscles (composed of type IIA, IIB, and IIX fibers) from C57BL/6 mice (data not shown). We conclude that the three members of the NR4A subgroup, Nur77, Nurr1 and NOR-1, are significantly and transiently induced by acute ␤-adrenergic signaling in


slow- and fast-twitch oxidative and glycolytic skeletal muscle, respectively. Induction of the NR4A subgroup involves PKA, MAPK, and phosphorylation of CREB in skeletal muscle cells

It is well documented that ␤-adrenergic signaling is coupled to intracellular cAMP levels and signaling (50). Moreover, NR4A expression (and promoter activity) has been demonstrated to be dependent on the cAMP response element and CREB and correlate with activation of the PKA and MAPK pathways and phosphorylation of CREB in several cell culture systems (5, 24, 51–58). We have previously shown that ␤-adrenergic agonists induce NOR-1 mRNA expression and the activity of the NOR-1 promoter in skeletal muscle cells (37). We conducted several experiments to analyze the involvement of PKA, MAPK, and CREB phosphorylation in response to ␤-AR-mediated induction of the NR4A subgroup in skeletal muscle cells. We observed that the PKA activator forskolin strikingly and significantly (P ⬍ 0.01) induces the expression of the three members of the NR4A subgroup (see Fig. 2Ai-iii). The extent of activation is similar to that of the ␤-adrenergic agonist isoprenaline (see Fig. 2Ai-iii). Moreover, the MAPK kinase (MEK) 1/2 inhibitor, PD98059, significantly attenuated ␤-adrenergic-mediated Nur77 induction (Fig. 2Bii); however, it failed to inhibit the ␤-adrenergic-mediated induction of Nurr1 and NOR-1 (see Fig. 2Bii-iii). The p38 MAPK inhibitor, SB203580, blocked the isoprenaline-mediated induction of the mRNAs encoding the three members of the NR4A subgroup (see Fig. 2Bi-iii).

FIG. 2. Induction of the NR4A subgroup involves phosphorylation of CREB, the PKA, and MAPK pathways in skeletal muscle cells. A, Differentiated C2C12 myotubes were treated with 10 ␮M forskolin (Fsk; a PKA activator), 100 nM isoprenaline hydrochloride (ISO) or vehicle (ethanol and DMSO). Cells were harvested after 1 h and qRTPCR was used to assay the expression of Nur77 (i), Nurr1 (ii), and NOR-1 (iii) mRNA. Results were normalized against 18S. B, Differentiated myotubes were pretreated with 20 ␮M SB203580 (SB; a MEK1/2 inhibitor), 20 ␮M PD98059 (PD; a p38 inhibitor), or DMSO (vehicle) for 15 min. Pretreated cells were then treated with isoprenaline or vehicle (ethanol). Cells were harvested after 1 h of isopenaline/vehicle treatment and qRT-PCR was used to assay the expression of Nur77 (i), Nurr1 (ii), and NOR-1 (iii) mRNA. Results were normalized against 18S. Data are expressed as mean ⫾ SEM (n ⫽ 3). Statistical significance was assessed using a one-way ANOVA with Dunnett’s posttest where P ⬍ 0.05 (*) and P ⬍ 0.01 (**). C, In triplicate, cells were treated for 20, 40, 60, or 120 min with 100 nM isoprenaline (ISO) or vehicle (ethanol) or left untreated (UN). Levels of phosphorylated (Ser133) CREB, ATF-1, and total CREB were visualized by Western blotting. D, Schematic diagram showing how ␤-adrenergic signaling may cause induction of NR4A expression via PKA, p38 and phospho-CREB in skeletal muscle cells.


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We next analyzed the phosphorylation of CREB after ␤-adrenergic treatment. We report that 20 – 60 min treatment with isoprenaline induced the phosphorylation of CREB (at serine133) in C2C12 myotubes (see Fig. 2C). In addition, the antiphospho-CREB antibody cross-reacts with phospho-activating transcription factor (ATF)-1 [a CREB homolog (59)]. Moreover, the 20 – 60 min induction with isoprenaline induced phosphorylation of ATF-1. In summary, the isoprenaline-mediated induction of the NR4A subgroup appears to involve PKA, MAPK (predominantly p38 mediated), and phosphorylation of CREB (and the highly homologous ATF-1), and this is consistent with other reports in different cell systems (5, 24, 51–55, 58, 60). A possible signaling pathway for the induction of the NR4A subgroup in skeletal muscle cells is shown in Fig. 2D. Expression of NOR-1 siRNA decreases palmitate oxidation and increases lactate accumulation

Preliminary analysis of an NOR-1 siRNA-transfected cell line indicated aberrant uncoupling protein (UCP)-2 and -3 expression (37). However, the functional role of NOR-1 in the context of skeletal muscle lipid and carbohydrate metabolism has not been fully resolved. We observed that the oxidation of palmitate (measured by release of 3H2O into the cell culture media) was significantly (P ⬍ 0.01) decreased relative to total protein in the (stable) NOR-1 siRNA transfected mouse skeletal muscle cell line (C2C12-siNOR-1) relative to the negative control siRNA transfected stable mouse skeletal muscle cell line (C2C12siNEG) (Fig. 3A). Similarly, we observed the media from the

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FIG. 3. Expression of NOR-1 siRNA decreases palmitate oxidation and increases lactate accumulation. A, Oxidation of palmitate in differentiated stable cell lines expressing NOR-1 siRNA (C2C12siNOR-1) was compared with stable cell lines expressing negative control siRNA (C2C12-siNEG) over 2 h (normalized to total protein). Both cell lines have been previously described (37). B, Media L-lactate was assayed after 3 d of differentiation in C2C12-siNOR-1 and C2C12-siNEG cells. The data represent the mean ⫾ SEM (n ⫽ 3). Statistical significance was assessed using an unpaired Student’s t test where P ⬍ 0.05 (*), P ⬍ 0.01 (**), and P ⬍ 0.001 (***).

C2C12-siNOR-1 cells accumulated significantly (P ⬍ 0.001) higher levels of lactate relative to the C2C12-siNEG control cells (Fig. 3B). This was also evident by reduced media pH C2C12-siNOR-1 cell line if the media were not changed daily (data not shown). In summary, these data are consistent with reduced lipid oxidation and anaerobic use of pyruvate in skeletal muscle cells with attenuated NOR-1 mRNA expression. NOR-1 siRNA transfected cells are resistant to an inhibitor of aerobic metabolism and express significantly more HIF1␣ mRNA

The decreased palmitate oxidation and increased lactate accumulation suggested the NOR-1 siRNA-transfected cells lines were more dependent on anaerobic metabolism. We examined this hypothesis further by examining the effect of treating cells with azide, an inhibitor of aerobic metabolism (61). The levels of ATP in the C2C12-siNEG control cells were significantly (P ⬍ 0.001) reduced after 5 min of azide exposure (Fig. 3A). In contrast, the levels of ATP in the C2C12siNOR-1 cell line were refractory to azide exposure (Fig. 4A). This suggested that ATP production in the C2C12-siNOR-1 cells was primarily dependent on anaerobic mechanisms and consistent with the elevated levels of lactate produced by this cell line. Interestingly, the untreated levels of ATP were significantly (P ⬍ 0.001) lower in the C2C12-siNOR-1 cell line, compared with the C2C12-siNEG cell line. It has been demonstrated previously that increased lactate accumulation correlates with increased expression of HIF-1␣ (62, 63). Hence, we subsequently measured HIF-1␣ mRNA levels in our cell lines. We observed that the C2C12-siNOR-1


FIG. 4. Expression of NOR-1 siRNA prevents azide induced ATP loss over 5 min. A, ATP levels in both C2C12-siNEG and C2C12-siNOR-1 cells lines before and after 5 min of azide treatment relative to total protein. Values are expressed as mean ⫾ SEM (n ⫽ 6). Statistical significance was assessed using a one-way ANOVA with Bonferroni’s posttest where P ⬍ 0.05 (*), P ⬍ 0.01 (**), and P ⬍ 0.001 (***). B, Both C2C12-siNEG and C2C12-siNOR-1 cells were differentiated for 4 d, and RNA was isolated for qRT-PCR analysis of HIF1␣ expression (relative to GAPDH). Data represent the mean ⫾ SEM (n ⫽ 3). Statistical significance was assessed using an unpaired Student’s t test where P ⬍ 0.05(*), P ⬍ 0.01(**), P ⬍ 0.001(***), and no significant difference (NS).

control cells express significantly (P ⬍ 0.001) higher levels of HIF-1␣ mRNA relative to the C2C12-siNEG cells (Fig. 4B). In summary, the observations of reduced palmitate oxidation, increased lactate accumulation, azide-resistant ATP production, and increased HIF-1␣ (mRNA) in the NOR-1 siRNAtransfected lines are all concordant with a shift to anaerobic metabolism. This suggests NOR-1 expression is necessary for oxidative metabolism. NOR-1 siRNA-transfected cells attenuate/modulate the expression of genes that control lipid oxidation and pyruvate use

We subsequently examined the effect of attenuating NOR-1 mRNA expression on the expression of several genes that control fatty acid oxidation and oxidative (vs. anaerobic) pyruvate metabolism in skeletal muscle cells. We compared expression in NOR-1 siRNA-transfected cells (C2C12siNOR-1), compared with the negative control cells (C2C12siNEG). RNA was harvested from both cells lines after differentiation in culture for qRT-PCR analysis (both cells line were phenotypically similar). Both PGC-1␣ and -␤ control fatty acid oxidation and mitochondrial oxidative energy metabolism in type I (and IIA) slow twitch (64) and IIX (fast twitch) muscle fibers, respectively (65). We observed that the expression of the mRNAs encoding PGC-1␣ and -␤ were both significantly (P ⬍ 0.05 and P ⬍ 0.001,


respectively) attenuated in the NOR-1 siRNA-transfected cells, 1.5- and 3.7-fold, respectively (Fig. 5, A and B). Furthermore, we examined the expression of lipin-1␣. Recently it was demonstrated that lipin-1␣ expression is induced by PGC-1␣ and selectively activates fatty acid oxidation and mitochondrial fatty acid oxidation (66). We observed that the expression of the mRNA encoding lipin-1␣


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is also significantly repressed (2.4-fold) in the in the C2C12siNOR-1 cell line, relative to the negative control transfected cells (Fig. 5C). These results support the findings of reduced palmitate oxidation in the C2C12-siNOR-1 cell line. Citrate synthase, which controls entry of acetyl-CoA into the tricarboxylic acid cycle, was unchanged (Fig. 5D). The pyruvate dehydrogenase complex (PDHC) is the nexus that links the glycolytic production of pyruvate to anaerobic (lactate production) or oxidative (tricarboxylic acid cycle) metabolism. Hence, the PDHC is a critical module of glucose homeostasis because it regulates mitochondrial carbohydrate oxidation (and uncouples glycolytic flux from pyruvate oxidation). The activity of the PDHC is mediated by PDPs that induce dephosphorylation (promotes carbohydrate oxidation). Conversely, PDHC activity is attenuated by PDK-mediated phosphorylation (promotes conversion of pyruvate to lactate; Fig. 5E). This regulatory nexus is finetuned by the presence of several isoforms of PDK (1– 4) and PDP (1–2); however, PDK2, PDK4, and PDP1 [consisting of catalytic (c) and regulatory (r) subunits] are predominantly expressed in skeletal muscle. Hence, we measured the expression of the mRNAs encoding PDP1c and -r, and PDK1– 4 in the C2C12-siNOR-1 and C2C12-siNEG cell lines. Interestingly, we observed that the mRNAs encoding both subunits of PDP1 (c and r) are repressed, 1.8- and 2.1-fold, respectively, in the NOR-1 siRNA cells (Fig. 4, F and G, respectively) The expression of the mRNAs encoding PDK1– 4 are relatively unaffected (and the changes in expression very minor) in the NOR-1 siRNA relative to the negative control transfected cell line (data not shown). The expression profile of the PGC-1␣, lipin-1␣, PDP1c, and PDP1r is entirely consistent with decreased palmitate oxidation, increased lactate accumulation (anaerobic pyruvate metabolism), azide-resistant ATP production, and increased HIF-1␣ expression. The program of gene expression in the NOR-1 siRNA-transfected cells is concordant with a transition to anaerobic metabolism and reduced oxidative catabolism. Acute ␤2-AR signaling induces an in vivo program of gene expression that activates fatty acid oxidation

FIG. 5. Expression of NOR-1 siRNA results in differential changes in the expression of mRNAs encoding genes that regulate oxidative metabolism and the fate of pyruvate. Both C2C12-siNEG and C2C12siNOR-1 cells were differentiated for 4 d and RNA was isolated for qRT-PCR analysis of PGC-1␣ (A), PGC-1␤ (B, lipin-1␣ (C), and citrate synthase (CS; D) was compared in both differentiated C2C12siNOR-1 cell lines normalized to GAPDH. E, Metabolic diagram highlighting the control of pyruvate metabolism. Using qRT-PCR the expression of PDP1c (F) and PDP1r (G) in both cell lines normalized to GAPDH. Data represent the mean ⫾ SEM (n ⫽ 3). Statistical significance was assessed using an unpaired Student’s t test where P ⬍ 0.05 (*), P ⬍ 0.01 (**), P ⬍ 0.001 (***), and no significant difference (NS).

The analysis of the in vitro cell culture model suggested that attenuation of NOR-1 resulted in a shift to anaerobic metabolism. Hence, we hypothesized that activation of adrenergic signaling in vivo would activate gene expression involved in oxidative metabolism. We profiled the expression of genes that control the induction of aerobic metabolism. We observed no significant difference in the expression of the mRNA encoding NOR-1 in both muscles types (Fig. 1, A and Biii); however, we observed greater ␤2-AR agonist (formoterol)-induced increases of NOR-1 in the fast glycolytic tibialis anterior muscle (Fig. 1Ciii). Consequently, we focused on the ␤2-AR-mediated induction of gene expression in tibialis anterior muscle. To confirm whether ␤2-AR agonists modulate the expression of genes controlling oxidative (vs. anaerobic) metabolism in vivo, groups (n ⫽ 4) of mice treated with either the specific ␤2-AR agonist formoterol or saline control for 1, 4, 8,

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and 24 h were assayed for expression of mRNA in fast fiber dominant tibialis anterior muscle using qRT-PCR. The expression of the mRNAs encoding PGC-1␣ (Fig. 6A) and lipin-1␣ (Fig. 6B) were significantly induced in vivo by ␤2-AR agonist (formoterol) treatment (relative to saline) with optimal induction (relative to vehicle) observed 4 and 8 h after ip injection, respectively. Similarly, PGC-1␣ and lipin-1␣ (data not shown) were significantly induced in the in vitro C2C12 skeletal muscle cell culture model by isoprenaline treatment. In this context, we also observed a very significant increase of the forkhead transcription factor FOXO1 (also known as FKHR) mRNA expression (Fig. 6C) 4 h after ␤2-AR agonist


(formoterol) treatment, concomitant with the induction of PGC-1␣, a known coactivator of FOXO1 (67) that preferentially induces oxidative fatty acid metabolism and decreases oxidative tricarboxylic acid (TCA) cycle mediated glucose/ pyruvate use through induction of PDK4 (68). We explored this connection in our ␤2-AR agonist (formoterol)-treated skeletal muscle samples. We observed a significant induction of PDK4 (Fig. 6D) at 4 and 8 h after formoterol treatment. However, at 24 h, levels of PDK4 were significantly decreased (3.8-fold) relative to levels in salinetreated animals. At 24 h, these results are in accordance with the expression of PGC-1␣, lipin-1␣, and FOXO1 that have now returned to baseline levels. In summary, the parallel ␤-adrenergic-dependent induction of NOR-1 (and the other members of the NR4A subgroup), and the program of PGC-1-associated gene expression, correlates with the metabolic phenotype (and repression of PGC-1␣/␤, lipin-1␣ and PDP1c and -r) of the NOR-1 siRNA-expressing cells. This is consistent with the hypothesis that NOR-1 is involved in the regulation of oxidative metabolism. NOR-1 is recruited to the lipin-1␣ and PDK4 promoters

FIG. 6. The ␤2-AR agonist, formoterol, increases the mRNA expression of genes regulating oxidative metabolism in skeletal muscle. Tibialis anterior muscle was removed at 1, 4, 8, and 24 h after a single ip injection of formoterol or saline vehicle (NT ⫽ no treatment). qRTPCR was used to assay the expression PGC-1␣ (A), lipin-1␣ (B), FOXO1 (C), and PDK4 (D) in this tissue normalized against 18S at each time point. Data are expressed as the mean ⫾ SEM (n ⫽ 4 per time point). Statistical significance was assessed using a one-way ANOVA with Bonferroni’s posttest where P ⬍ 0.05 (*), P ⬍ 0.01 (**), and P ⬍ 0.001 (***).

To ascertain whether NOR-1 was directly involved in the modulation of genes that regulate fatty acid and carbohydrate oxidation, we examined whether the NOR-1 is recruited to the lipin-1␣ and PDK-4 promoters in skeletal muscle cells. Putative NOR-1/NR4A NR half-sites were identified using MatInspector (Genomatix, Ann Arbor, MI). We identified (using ChIP studies) strong and selective recruitment of NOR-1 to two sites between nt positions ⫺333 to ⫺232 (NR1) and ⫺1762 to ⫺1662 (NR3), respectively, from the start of transcription on the lipin-1␣ promoter (Fig. 7, A and C), relative to IgG, anti-GAPDH antibody, and no antibody controls (Fig. 7C). The specificity of NOR-1 recruitment at the lipin-1␣ NR1 and NR3 sites was highlighted by the lack of recruitment to the GAPDH promoter (Fig. 7C). Both NR1 (ATAAGGTCA) and NR3 (AACAGGTCA) sites in the lipin-1␣ promoter show strong similarity to the nerve growth factor-induced gene B response element originally defined as AAAAGGTCA with up to one mismatch in the 5⬘ A nucleotides (69). Second, we also observed efficient recruitment of NOR-1 to a site between ⫺2785 and ⫺2765 (NR1) from the start of transcription on the PDK-4 promoter (Fig. 7, B and D), relative to the IgG, anti-GAPDH antibody, and no antibody controls (Fig. 7D). However, no recruitment was observed at a site between ⫺4253 and ⫺4153 (NR3) from the start of transcription on the PDK-4 promoter (Fig. 7D). Moreover, specificity of NOR-1 recruitment at the PDK-4 NR1 site was again highlighted by the lack of recruitment to the GAPDH promoter. The NR1 sequence motif (CAAAGGTGA) also shows strong homology to the nerve growth factor-induced gene B response element consensus (69). Third, VP16-NOR-1 trans-activated a region spanning ⫺351 to ⫺308 of the PDK-4 promoter that encoded a defined NR half-site (45, 68). This region was subcloned into the pGL2 promoter-LUC reporter [PDK-4 (⫺351/⫺308)]. This previously defined NR half-site (45, 68) at ⫺351 is AACAAG-



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GACA. PGC-1␣ did not further coactivate VP16-NOR-1-mediated trans-activation of the heterologous PDK-4 (⫺351/ ⫺308) LUC reporter gene (data not shown). In summary, the selective recruitment of NOR-1 to NR sites in these promoters is consistent with the involvement of NOR-1 in the regulation of lipin-1␣ and PDK-4 expression, important modulators of fatty acid and glucose oxidation in skeletal muscle. Discussion

A variety of studies demonstrated that acute (and chronic) exposure of skeletal muscle to ␤-AR agonists modulate oxidative metabolism, energy expenditure, and lipolysis (30 – 34, 70). Moreover, treatment of rodent skeletal muscle cells and human skeletal muscle with ␤-AR agonists stimulated glucose transport (71) and glucose oxidation, respectively (70). However, the molecular basis for these observations is poorly understood. In this context, we and others have previously established that ␤-AR agonists significantly induced expression of Nur77 (36) mRNA in mouse skeletal muscle cells and brown adipocytes (38). Furthermore, we demonstrated that siRNA-mediated suppression of Nur77 expression in an in vitro cell culture system results in the attenuated expression of mRNA (and proteins) involved in lipid, carbohydrate, and energy use and homeostasis, including UCP3, Glut4, CD36, and AMPKg3 (36). Furthermore, Pei et al. (72) implicated the NR4A receptors in the regulation of liver gluconeogenesis. Previously we demonstrated that the mRNA encoding NOR-1 is hyperactivated after ␤2-AR agonist treatment in an in vitro skeletal muscle cell culture system and in vivo in mouse plantaris muscle. Preliminary analysis of an NOR-1 siRNAtransfected cell line indicated aberrant UCP-2, and -3 expression (37). However, the in vivo response of the other NR4A subgroup members to ␤-AR signaling in slow (type I) oxidative and fast (type II) glycolytic skeletal muscle has not been ascertained. Moreover, the metabolic implications of regulatory cross talk between the NR4A subgroup and ␤-AR signaling pathways in skeletal muscle remains to be addressed. In the present study, we identified that the expression of mRNAs encoding all three members of the NR4A orphan nuclear receptor subgroup (Nur77, Nurr1, and NOR-1) were significantly and transiently activated in slow oxidative and fast glycolytic dominant skeletal muscle after 1– 4 h of treatment with the ␤2-AR-specific agonist formoterol in mice. This is conFIG. 7. NOR-1 is recruited to the lipin-1␣ and PDK4 promoters. Diagrammatic representation of predicted NR4A half-sites on the promoters of mouse lipin-1␣ (A) and PDK4 (B). Please note that NR sites 2– 4 for lipin-1␣ and 1– 4 for PDK4 are on the reverse strand. C, The recruitment of NOR-1 onto the lipin-1␣ promoter in C2C12 myotubes by ChIP assay (representative assay) after 2 h of isoprenaline treatment. D, The recruitment of NOR-1 onto PDK4 promoter in C2C12 myotubes by ChIP assay after 2 h of isoprenaline treatment. Triplicate real-time PCR analysis was performed and the results are expressed as the mean ⫾ SD. Results are representative of two independent experiments. E, C2C12 myoblasts were cotransfected with pGL2-p or pGL2-p-PDK-4 (⫺351/⫺308) (PDK4 promoter fragment) and VP16-NOR-1 expression vector or empty vector (VP16). Each value is expressed as relative light units (RLU) ⫾ SEM and derived from six replicates. pGL2-B is arbitrarily set to 1. The result is representative of six independent experiments.

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sistent with the very recent report that 90 min isoprenaline (a pan agonist for ␤1–3-ARs) treatment induces the expression of the NR4A subgroup in quadriceps and soleus (73). In this study we identified that the marked increase in the expression of NOR-1 and the NR4A subgroup induced by ␤-adrenergic signaling is consistent with the involvement of PKA, MAPK (predominantly p38 mediated), and phosphorylation of CREB (and ATF-1) in skeletal muscle cells. This is in concordance with many previous reports in other systems. PKA, p38-MAPK signaling, and CREB phosphorylation have been associated with the induction of the NR4A subgroup in vascular smooth muscle (5, 51–53), breast cancer cells (24), synovial tissue (54), embryonic fibroblast cells (55), corticotroph cells (56, 57), and T cells (58). Interestingly, in our study, MEK1/2 and p38 MAPK inhibitors suppressed the induction of Nur77 expression. In contrast, the ␤-adrenergicinduced expression of Nurr1 and NOR-1 is only sensitive to p38 MAPK inhibition (and not MEK1/2). This not only underscores subtle differences between the pathways that induce the different NR4A subgroup members but also highlights the central role of p38 MAPK in the induction of NR4A subgroup expression. In this context, it has previously been demonstrated that increased PGC-1␣ expression (and subsequent increased energy expenditure) in response to ␤-adrenergic signaling in brown adipose cells/tissue is mediated by cAMP and p38 MAPK (74), suggesting that p38 may possibly be a key metabolic regulator in different tissues. Finally, we observed that ␤-AR signaling in mouse skeletal muscle induced the expression of genes that are associated with preferential oxidation of fatty acids, i.e. PGC-1␣, lipin1␣, FOXO1, and PDK4. Attenuation of NOR-1 expression resulted in decreased palmitate oxidation and increased lactate accumulation. Moreover, ATP production in the NOR-1 siRNA-transfected cells was resistant to (azide mediated) inhibition of aerobic/oxidative metabolism and expressed significantly higher levels of HIF-1␣. Furthermore, we observed the repression of genes that promote fatty acid oxidation [such as PGC-1 (␣/␤) and lipin-1␣] as well as oxidative carbohydrate (pyruvate) use (including PDP1r and -c). In conclusion, this suggests acute ␤-AR signaling activates expression of the NR4A subgroup and preferentially induces oxidative metabolism. The present study clearly identifies that acute ␤-AR signaling results in a significant and transient induction (1– 4 h after treatment) of the mRNAs encoding the NR4A subgroup. Concomitantly and/or subsequent to activation of NR4A expression, we observed the significant (5- to 16-fold) induction PGC-1␣ (4 and 8 h after treatment) in vivo (tibialis anterior muscle) and a similar response in vitro. PGC-1␣ is a major regulator of oxidative capacity via induction of mitochondrial gene expression, mitochondrial biogenesis (75–77), and induction of fatty acid oxidation (78 – 80). In this context it is worth noting that PGC-1␣ is an important estrogenrelated receptor-␣ coactivator in the modulation of oxidative metabolism in skeletal muscle (80, 81) and mitochondrial biogenesis (77). Interestingly, estrogen-related receptor-␣ and PGC1␣ have been implicated in the transcriptional activation of mitofusin 2 (82). Knockdown of mitofusin 2 in both muscle and nonmuscle cells reduces oxygen consumption and oxidation of glucose, pyruvate, and palmitate (83,


84), similar to the phenotype of skeletal muscle cells with attenuated NOR-1 expression noted in this study. In the context of ␤-AR signaling, it should be noted that the expression of both PGC1␣ and mitofusin 2 are induced by both ␤-AR agonist treatment and cold exposure in skeletal muscle and brown adipose tissue (75, 82, 85). We also observed that the expression of the mRNAs encoding lipin-1␣ and FOXO1 were induced (4 – 8 h after treatment) by the ␤2-AR (formoterol) in vivo. These observations are entirely consistent with the induction of PGC-1␣ and the in vivo effects of ␤-AR signaling on muscle (that leads to increased mitochondrial activity, energy expenditure, lipolysis, etc.). For example, PGC-1␣ has been shown to activate the expression of lipin-1␣ in the liver. The study went on to demonstrate that lipin-1␣ selectively stimulates the PGC-1␣dependent pathways involved in oxidative mitochondrial metabolism and fatty acid oxidation (66). This suggests this regulatory cascade is not limited to hepatic tissue but also involves the peripheral metabolic tissues. Furthermore, the in vivo increase of FOXO1 (4 h after ␤2-AR agonist treatment) parallels the induction of PGC1␣, a known coactivator of FOXO1 (67). Concomitant with the results above, we observed a very significant and consistent 3- to 5-fold increase in PDK4 mRNA expression (4 – 8 h after ␤-AR agonist treatment). Interestingly, ChIP analysis demonstrated selective recruitment of NOR-1 to specific sites in the lipin-1␣ and PDK-4 promoters. This suggests and is consistent with the direct involvement of NOR-1 in the regulation of these important genes that are induced by ␤-AR signaling in skeletal muscle. The pattern of gene expression associated with the selective activation of oxidative fatty acid catabolism is consistent with the effect of ␤-blockers (propranolol and metoprolol) in skeletal muscle, which have been demonstrated to impair/compromise mitochondrial respiration (86). We subsequently used a skeletal muscle cell line expressing an NOR-1 siRNA to further investigate the link between NR4A expression (and function) and the ␤-AR-dependent gene expression program involved in preferential induction of pathways controlling oxidative fatty acid metabolism. In the present study, we found that in NOR-1 siRNA-positive cells, the expression of PGC-1␣, PGC-1␤, and lipin-1␣ mRNA was repressed. This finding is consistent with the finding of reduced fatty acid (palmitate) oxidation. These results support previous findings: 1) the induction of these genes in response to ␤-AR agonists in vivo and in vitro and 2) reports of increased fuel oxidation due to acute exposure of skeletal muscle to ␤-AR agonists and mitochondrial hyperplasia in response to chronic ␤-AR signaling in skeletal muscle (87). Second, we observed significant repression of PDP1c and PDP1r mRNA, suggesting that the activity of the PDHC complex under these conditions may be reduced, preventing aerobic use of pyruvate by the TCA cycle. This hypothesis is supported by the finding of greater accumulation of lactate in the NOR-1 siRNA-expressing cells, consistent with the anaerobic metabolism of pyruvate. Lastly, we observed azide (a metabolic poison of oxidative metabolism)-resistant ATP production in the NOR-1 siRNAexpressing cell lines, paralleled by increased HIF-1␣ mRNA expression. The insensitivity of steady-state ATP levels in the


C2C12-siNOR-1 cells to azide is consistent with previous reports that have found mitochondrial uncoupling results in a shift toward oxidative metabolism (88) and that UCP2 modulates sensitivity to cyanide (89). This observation is consistent with the reduced UCP2 expression we previously observed in this cell line (37). The results from the NOR-1 siRNA-transfected cells suggest a shift toward increased anaerobic metabolism. Analysis of the in vitro cell culture model suggests that NOR-1 is necessary for oxidative fatty acid catabolism and aerobic pyruvate use. The gene expression footprint in the NOR-1 attenuated cell line correlates with suppressed fatty acid oxidation and TCA driven pyruvate use. ␤-Adrenergic signaling (the induced the expression of the NR4A subgroup) modulated a shift in the opposite direction of the NOR-1 siRNA cells toward increased fatty acid oxidation. Careful examination of NOR-1-inhibited cells and ␤-AR agonisttreated muscles revealed ␤-AR treatment resulted in preferential activation of gene expression involved in fatty acid oxidation. Whereas the attenuation of NOR-1 suppressed gene expression involved in oxidative fatty acid and aerobic pyruvate metabolism. Recently it has been reported that exogenous Nur77 expression in mouse muscle cells (and rat muscle) induces the expression of several genetic programs involved in glucose homeostasis (73); this was consistent with the observation that Nur77 siRNA expression in mouse skeletal muscle cells repressed GLUT4 mRNA and protein expression (36). This study demonstrated that denervation attenuates nur77 expression and several genes associated with glucose metabolism. In the context of these studies, exercise, which induces sympathetic signaling, increases the mRNA levels of the NR4A subgroup (1), PGC-1␣ (1, 85, 90), and FOXO1 (1, 91). These studies are consistent with the changes in gene expression we observed from treatment of mice with ␤2-AR agonists. In summary, our results show that that expression of the mRNAs encoding the three members of the NR4A subgroup were strikingly activated in both slow (type I) oxidative and fast (type II) glycolytic dominant skeletal muscle tissues 1– 4 h after treatment of mice with ␤2-AR-specific agonist. Furthermore, (in vivo and in vitro) characterization of gene expression, coupled to analysis of several metabolic parameters in a cell culture model demonstrated NOR-1 is necessary for oxidative metabolism and that ␤2-AR-specific activation induces a program of gene expression that regulates fatty acid oxidation. In conclusion, these studies are consistent with the hypothesis that NOR-1 (and the NR4A subgroup) is involved in the regulation of genes that control of metabolism in skeletal muscle. Further studies will reveal whether this class of NRs has utility in the context of metabolic disease. Acknowledgments The authors thank Rachel Burow and Shayama Wijedasa for technical assistance. Received August 30, 2007. Accepted February 28, 2008. Address all correspondence and requests for reprints to: George Muscat, Institute for Molecular Bioscience, The University of Queensland, Queensland 4072, Australia. E-mail: [email protected].


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This work was supported by a National Health and Medical Research Council of Australia (NHMRC) project grant. G.E.O.M. is a principal research fellow of the NHMRC. The Institute for Molecular Bioscience is an Australian Research Council Special Research Centre in Functional and Applied Genomics. Disclosure Statement: The authors of this manuscript have nothing to disclose.

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