Rev-erb (cid:1) Regulates the Expression of Genes Involved in Lipid Absorption in Skeletal Muscle Cells EVIDENCE FOR CROSS-TALK BETWEEN ORPHAN NUCLEAR RECEPTORS AND MYOKINES*

Rev-erb (cid:1) is an orphan nuclear receptor that selectively blocks trans-activation mediated by the retinoic acid-related orphan receptor- (cid:2) (ROR (cid:2) ). ROR (cid:2) has been implicated in the regulation of high density lipoprotein cholesterol, lipid homeostasis, and inflammation. Rev-erb (cid:1) and ROR (cid:2) are expressed in similar tissues, including skeletal muscle; however, the pathophysiological function of Rev-erb (cid:1) has remained obscure. We hypoth-esize from the similar expression patterns, target genes, and overlapping cognate sequences of these nuclear receptors that Rev-erb (cid:1) regulates lipid metabolism in skeletal muscle. This lean tissue accounts for > 30% of total body weight and 50% of energy expenditure. Moreover, this metabolically demanding tissue is a primary site of glucose disposal, fatty acid oxidation, and cholesterol efflux. Consequently, muscle has a significant role in insulin sensitivity, obesity, and the blood-lipid profile. We utilize ectopic expression in skeletal muscle cells to understand the regulatory role of Rev-erb (cid:1) in this major mass peripheral tissue. Exogenous expression of a dominant negative version of mouse Rev-erb (cid:1) decreases the expression of many genes involved in fatty acid/lipid absorption (including Cd36 , and Fabp-3 and - coenzyme A dehydrogenase. A key enzyme involved in fatty acid oxidation pathway. Pdk-2 and - 4 Pyruvate dehydrogenase kinases: inhibiting the pyruvate dehydrogenase complex, thereby controlling glucose oxidation and maintaining pyruvate for gluconeogenesis. Scd-1 and - 2 Stearoyl CoA desaturase-1, and -2. Enzymes associated with adiposity, i.e . storage and esterification of cholesterol, and responsible for the cis saturation of stearoyl and palmitoyl-CoA converting them to oleate and palmitoleate, which are the monounsaturated fatty acids of triglycerides. Srebp-1c Sterol regulatory element-binding protein-1c, the hierarchical transcriptional activator of lipogenesis. Ucp-2 and - 3 Uncoupling proteins. Mitochondrial proteins that uncouple metabolic fuel oxidation from ATP-synthesis, regulating energy expenditure.

Members of the nuclear receptor (NR) 1 superfamily bind to specific DNA elements and function as transcriptional regulators (1,2). In addition to the ligand-activated NRs, many mem-bers within this superfamily have no known ligand, and are referred to as "orphan NRs" (3). The orphan receptor Rev-erb␤ (NR1D2, also known as Rev-erb␣-related receptor, RVR) belongs to the family of "Reverbs" that also contain Rev-erb␣ (4,5). The primary structure of these two receptors together with retinoic acid-related orphan receptor-␣ (ROR␣) and the Drosophila orphan receptor, E75A, is very similar especially in the DNA-binding domain and the putative ligand-binding domain (6).
Two Rev-erb␤ genes have been identified; Rev-erb␤1 and Rev-erb␤2, which are alternatively spliced products of the Rev-erb␤ gene (7). The mRNA expression data shows that Rev-erb␤ is abundantly expressed in most tissues, although higher levels of expression are observed in skeletal muscle, brain, kidney, and liver (Refs. 4

and 8 and references therein).
Rev-erb␣, Rev-erb␤, and ROR bind as monomers to the nuclear receptor half-site motif, PuGGTCA flanked 5Ј by an ATrich sequence ((A/T) 6 PuGGTCA). Although these receptors are closely related, and bind to the same motif, they function in an opposing manner. Whereas ROR activates gene transcription, Rev-erb␣ and Rev-erb␤ mediate transcriptional repression, and can repress trans-activation mediated by ROR (6, 9 -12). The inter-relationship between these nuclear receptors is underscored by the evidence that demonstrates ROR␣ trans-activates the Rev-erb␣ promoter (13).
Loss of function studies in cell culture and in animal models has demonstrated a role of ROR␣ in lipid metabolism. ROR␣deficient staggerer mice are predisposed to the development of atherosclerosis. The staggerer mice possess an aberrant bloodlipid profile with low circulating levels of major lipoproteins such as HDL cholesterol, apolipoprotein (apo) C-III, and plasma triglycerides. Furthermore, decreases in specific apolipoprotein compartments, namely Apoa-I, the major constituent of HDL, and Apoa-II that lead to hypo-␣-lipoproteinemia have been reported in staggerer mice (15). Moreover, our recent studies demonstrate that ROR␣ regulates the expression of genes involved in lipid homeostasis and energy balance in skeletal muscle cells (16).
Several reports have demonstrated the role of Rev-erb␣ in lipid metabolism and inflammation. In the context of lipid metabolism, Rev-erb␣, together with peroxisome proliferator activated receptor-␣ (PPAR-␣) has been shown to regulate the expression of ApoA1 in a species-specific manner (17). Apoa1 has an important role in the formation of nascent HDL particles and apolipoprotein-mediated cholesterol efflux in mice (18,19). Furthermore, Rev-erb␣ has also been shown to regulate the expression of the ApoC-III gene that plays a key role in maintaining serum triglyceride levels (20,21). The inter-relationship between Rev-erb␤ and lipid metabolism has not been investigated. Within inflammatory pathways, Rev-erb␣ and ROR act opposingly on the NFB pathway. ROR␣ has been shown to directly up-regulate the expression of IB␣, the major inhibitor of the NFB signaling pathway by binding to the IB␣ promoter (22). The functional role of Rev-erb␤ in NFB-mediated inflammation still remains obscure.
Rev-erb␤ is abundantly expressed in skeletal muscle and brown fat. Skeletal muscle is one of the most metabolically demanding major mass peripheral tissues that accounts for 50% of energy expenditure. Moreover, this lean tissue relies heavily on fatty acids as an energy source, accounts for 75% of glucose disposal, and is involved in cholesterol efflux. Consequently, muscle has a significant role in insulin sensitivity and the blood-lipid profile. However, the role of Rev-erb␤ in skeletal muscle lipid and energy homeostasis has not been well studied.
The C2C12 in vitro cell culture model system has been used to investigate the regulation of lipid metabolism and cholesterol homeostasis by nuclear receptors such as LXR (23), PPAR␣, -␤/␦, -␥ (24 -28), and ROR␣ (16). Selective and synthetic agonists to these nuclear receptors induce similar effects on mRNAs encoding Abca1/g1, ApoE, sterol regulatory element-binding protein (Srebp)-1c, Scd-1, Fabp3, fatty acid synthase, lipoprotein lipase, Glut5, Ucp-2, and Ucp-3 in differentiated C2C12-myotubes and Mus musculus skeletal muscle tissue (23)(24)(25). The physiological validation of the cell culture model with respect to lipid homeostasis in the mouse corroborates the utility of this model system. This cell culture model provides an ideal platform to identify the Rev-erb␤-dependent regulation of genes involved in metabolism.
We have examined the regulation of gene expression involved in lipid homeostasis by Rev-erb␤ "loss of function" in skeletal muscle cell culture model. Our investigation demonstrates that Rev-erb␤ controls the expression of genes involved in lipid absorption. Moreover, we observed the regulation of genes encoding critical myokines that influence energy expenditure and inflammation.
C2C12 Transfections-Each well of a 24-well plate of C2C12 cells (ϳ50% confluence) was transfected with DNA using the liposome-mediated transfection procedure as described previously (16). Cells were transfected using a DOTAP and Metafectene (Biontex Laboratories GmbH, Munich, Germany) liposome mixture in 1ϫ HBS (HEPES-buffered saline (42 mM HEPES, 275 mM NaCl, 10 mM KCl, 0.4 mM Na 2 HPO 4 , 11 mM dextrose (pH 7.1)). The DNA/DOTAP/Metafectene mixture was added to the cells in 0.6 ml of Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum for experiments with ROR␣ or 10% serum supreme. After 16 -24 h, the culture medium was changed and after a further 24 h the cells were harvested for the assay of luciferase activity. Luciferase activity was measured as described previously (16).
C2:Rev-erb␤⌬E Stably Transfected Cell Line-The transfection of pSG5-Rev-erb␤⌬E into mouse myogenic C2C12 cells and the selection of stably transfected polyclonal cells using G418 has been described previously (29). The cells were cultured in growth medium with G418, and differentiated into post-mitotic multinucleated myotubes by 4 days of serum withdrawal.
RNA Extraction and cDNA Synthesis-Total RNA was extracted from C2C12 cells using Tri-Reagent (Sigma) according to the manufacturer's protocol. After treatment with 2 units of Turbo DNase (Ambion, Austin, TX) for 60 min, DNase was inactivated by heating to 75°C for 10 min. RNA for quantitative real time (Q-RT)-PCR was further purified using the RNeasy RNA extraction kit (Qiagen, Clifton Hill, Victoria, Australia) according to the manufacturer's instructions.
Q-RT-PCR-RNA was normalized using UV spectrometry and agarose gel electrophoresis. Complementary DNA was synthesized from 3 g of total RNA using Superscript III Reverse Transcriptase (Invitrogen Australia Pty. Ltd., Mulgrave, Victoria, Australia) and random hexamers according to the manufacturer's instructions. Target cDNA levels were analyzed by Q-RT-PCR in 25-l reactions containing either SYBR green (ABI, Warrington, UK) or Taqman PCR master mix (ABI, Branchburg, NJ), 200 nM each of forward and reverse primers or Assays-on-Demand Taqman primers (ABI, Foster City, CA), and cDNA (derived from 50 nanograms of RNA) by using an ABI Prism 7000 Sequence Detector system. PCR was conducted over 45 cycles at 95°C for 15 s and 60°C for a 1-min two-step thermal cycling preceded by an initial 95°C for 10 min for activation of Amplitaq Gold DNA polymerase.

Rev-erb␤ mRNA Is Expressed in Skeletal Muscle Cells and
Repressed during Myogenic Differentiation-Many studies in-dicate that the C2C12 cell line is an excellent cell culture model to investigate myogenesis, and the NR-mediated regulation of lipid homeostasis in skeletal muscle (23)(24)(25)(26). In this culture system, proliferating C2C12 myoblasts can be induced to biochemically and morphologically differentiate into post-mitotic multinucleated myotubes by serum withdrawal in culture over a 48 -96-h period. This transition from a non-muscle phenotype to a contractile phenotype is associated with activation/expression of a structurally diverse group of genes responsible for contraction and the extreme metabolic demands on this tissue.
Initially, total RNA was isolated from proliferating myoblasts and post-mitotic myotubes after 4 days of serum withdrawal, converted to cDNA for the analysis of mRNA expression by Q-RT-PCR. We utilized the GenBank TM sequences of mouse Rev-erb␤ to design specific primers for detection of mouse Rev-erb␤ by Q-RT-PCR.
We observed that Rev-erb␤ is expressed in proliferating myoblasts and the transcript was decreased 2-3-fold as the cells exit the cell cycle and form differentiated multinucleated myotubes (Fig. 1A). Concomitant with this decrease in Rev-erb␤ mRNA was the striking induction of expression of mRNA that encodes the slow (type I, Tnni1) and fast (type II, Tnni2) isoforms of the contractile protein, troponin I, and myogenin that encodes the hierarchical basic helix loop helix regulator ( Fig. 1, B-D). These data confirmed that these cells had terminally differentiated and had acquired a contractile phenotype. Acyl-CoA synthetase-4. Enhances the uptake of fatty acids by catalyzing their activation to acyl-CoA esters for subsequent use in catabolic fatty acid oxidation pathways.

Adrp/adipophilin
Adipocyte differentiation-related protein. Involved in lipid storage. AdipoR1 and R2 Adiponectin receptors 1 and 2. Cell surface membrane receptors for adiponectin that regulate glucose uptake and fatty acid oxidation. Ampk 5ЈAMP-activated protein kinase. A fuel-sensing enzyme that responds to cellular stress by regulating carbohydrate and fat metabolism.

Cav3
Caveolin 3. Muscle-specific caveolin involves in membrane protein anchoring, caveolae, t-tubules formation and lipid trafficking. Cpt-1 and -2 Carnitine palmitoyltransferases. Transfers the long-chain fatty acyl group from coenzyme A to carnitine, the initial reaction of mitochondrial import of long-chain fatty acids and their subsequent oxidation.

Fas
Fatty acid synthase. Involved in de novo fatty acid production.

Fat and Fabp3/Fabp4
Fatty acid translocase, and fatty acid-binding proteins. Facilitate uptake of long chain fatty acids (LFCAs) and low density lipoproteins (LDLs).

Lpl
Lipoprotein lipase. Hydrolysis of lipoprotein triglycerides into free fatty acids and responsible for the uptake of free fatty acids.

Mcad
Medium chain acyl coenzyme A dehydrogenase. A key enzyme involved in fatty acid oxidation pathway. Pdk-2 and -4 Pyruvate dehydrogenase kinases: inhibiting the pyruvate dehydrogenase complex, thereby controlling glucose oxidation and maintaining pyruvate for gluconeogenesis.

Scd-1 and -2
Stearoyl CoA desaturase-1, and -2. Enzymes associated with adiposity, i.e. storage and esterification of cholesterol, and responsible for the cis saturation of stearoyl and palmitoyl-CoA converting them to oleate and palmitoleate, which are the monounsaturated fatty acids of triglycerides.
In summary, these data demonstrate that the mRNA transcript encoding Rev-erb␤ was expressed during myogenesis. Second, during the acquisition of a contractile and metabolic phenotype (consistent with the increased utilization of lipids in skeletal muscle) we observed an ϳ2.5-fold repression of this orphan NR. However, we note that Rev-erb␤ is still expressed at a significant level in differentiated cells, in concordance with the expression of this orphan NR in adult muscle tissue.
Deletion of the Rev-erb␤ Ligand Binding Domain Compromises Its Ability to Repress ROR␣-mediated Trans-activation-To understand the metabolic role of Rev-erb␤ in skeletal muscle lipid and energy homeostasis, and to identify target genes of this orphan receptor in muscle cells, we examined the effect of perturbing Rev-erb␤ function. To disrupt Rev-erb␤-mediated trans-repression of gene expression, we utilized Rev-erb␤⌬E, that encodes amino acids 1-394 but lacks the entire E region that encodes the putative ligand binding domain of Rev-erb␤. Deletion of this region has been reported to have a dominant negative effect on Rev-erb␤mediated trans-repression of gene expression (29). Moreover, this region has been implicated in associating with the nuclear receptor co-repressors (30). In transient transfection assays in C2C12 skeletal muscle cells, deletion of the E region ablated the ability of Rev-erb␤ to trans-repress the human Rev-erbA␣ promoter ( Fig. 2A). Moreover, the capacity of Rev-erb␤ to repress ROR␣-mediated transcription is diminished when the E region is absent (Fig. 2, B and C). This demonstrates that deletion of the E-region that encodes the ligand binding domain of Rev-erb␤ compromises the capacity of this orphan NR to trans-repress the Rev-erb␣ promoter, and to attenuate ROR␣-mediated trans-activation.
Ectopic Dominant Negative (Rev-erb␤⌬E) Expression Induces Precocious Differentiation and ROR␣ mRNA Expression-The stable C2C12 cell line expressing Rev-erb␤⌬E (C2:Rev-erb␤⌬E) has been described previously (29). Prior to more extensive analysis of this cell line, we decided to ascertain the Rev-erb␤ status of this cell line. We designed a SYBR green-mediated Q-RT-PCR assay to selectively detect the ectopically expressed Rev-erb␤⌬E transcript. We observed that the C2:Rev-erb␤⌬E cell line specifically expresses the ectopically introduced mRNA in myoblasts and myotubes (Fig. 3, A and B). The expression of the exogenous transcript is constitutive (Fig. 3, A and B) in contrast to the endogenous transcript that is down-regulated during differentiation of proliferating myoblasts to postmitotic myotubes (Fig. 1A). Moreover, the steady state levels of myogenin mRNA were elevated in myotubes from the C2:Rev-erb␤⌬E cell line relative to wild-type C2C12 cells (Fig. 3C). This is consistent with the previously reported precocious biochemical and morphological differentiation of the C2:Rev-erb␤⌬E cell line relative to wild-type C2C12 cells (29). For example, Burke et al. (29) reported induction of differentiation upon serum withdrawal leads to accelerated activation and expression of the myogenin, and p21 Cip-1/Waf-1 mRNAs. Moreover, reduced expression of cyclin D1 was observed, which correlated with the increased expression of the cdk inhibitor, p21 (29). In addition, the levels of the non-muscle cytoskeletal ␤-actin are repressed in the C2:Rev-erb␤⌬E cell line, relative to wild-type C2C12 cells (Fig. 3D) in concordance with the elevated myogenin mRNA and precocious differentiative capacity of this cell line (29).
We used Q-RT-PCR with an ABI Taqman primer set that does not discriminate between ectopic Rev-erb␤⌬E and endogenous Rev-erb␤ mRNA expression to determine the total quantity of Rev-erb␤ mRNA in the stable cell line. We observed that the total pool of Rev-erb␤ mRNA is increased by ϳ20 -30% in myotubes from the C2:Rev-erb␤⌬E cell line relative to wild type C2C12s (data not shown). Considering the efficient expression of the exogenous Rev-erb␤⌬E mRNA transcript (Fig. 3,  A and B), this data suggests that the endogenous transcript expression is reduced, in concordance with the previous analysis of this cell line (29). The down-regulation of the endogenous transcript is not surprising. During myogenesis, mRNA pool sizes in muscle tissue are under strict control (31) and mechanisms in skeletal muscle exist that sense total output from exogenous and endogenous genes (32). Furthermore, exogenous expression of a number of different contractile protein transgenes in the mouse (e.g. myosin light chain 2, troponin I fast, skeletal and cardiac actin) results in the decline of the expression of the corresponding endogenous gene (32)(33)(34)(35)(36).
Ectopic Rev-erb␤⌬E mRNA expression does not affect the mRNA expression level of the other member of the Rev-erb family, Rev-erbA␣ (Fig. 3E). Interestingly, the closely related but opposingly acting orphan receptor, ROR␣, was dramati-cally increased ϳ6-fold (Fig. 3F) in the C2:Rev-erb␤⌬E cell line. However, the levels of ROR-␥ remain unchanged (see Table IV). These data indicate that there is cross-talk between these two related, but opposingly acting, orphan receptors in skeletal muscle cells.

Dominant Negative Rev-erb␤ Regulates the Expression of Genes Involved in Lipid Absorption and Energy Expenditure-
The expression of many metabolic markers is altered during skeletal muscle differentiation and acquisition of the musclespecific and contractile phenotype (Fig. 1). We therefore analyzed the expression of the genes involved in differentiation and skeletal muscle metabolism in the C2:Rev-erb␤⌬E cell line, relative to wild-type C2C12 cells (see Table I and II). Before analysis of the mRNAs encoding the metabolic enzymes we first characterized the key markers of differentiation in the C2:Rev-erb␤⌬E cell line to determine a threshold of significance with which to assess putative changes in a relevant context.
We observed that after mitogen withdrawal, the C2:Rev-erb␤⌬E cells formed post-mitotic multinucleated myotubes and thus had morphologically differentiated. The induction and activation of myogenin (Fig. 3C) demonstrated that the cells had also acquired a muscle-specific phenotype. Transcripts for the contractile proteins troponin I slow ((Tnni1) Fig. 4A), and troponin I fast ((Tnni2) (Fig. 4B)) were also induced after serum withdrawal demonstrating that the C2:Rev-erb␤⌬E cell line had acquired a contractile phenotype and was biochemically differentiated. Analysis of the relative expression of these biochemical markers in wild type C2C12 versus C2:Rev-erb␤⌬E myotubes after 4 days of serum withdrawal revealed that myo- genin (Fig. 3C) and fast type II (Tnni2) mRNA expression (Fig.  4, C and D) were increased ϳ1.5-2-fold. The TNNI1 mRNA encoding the slow type I isoform was actually slightly reduced. This indicated that any major changes in the expression of metabolic markers were not because of compromised morphological and/or biochemical differentiation. Moreover, these data provide a threshold of significance whereby altered expression of genes involved in skeletal muscle metabolism would only be considered to be of importance if it varied more than 2-fold.
Analysis of the expression of the genes involved in skeletal muscle metabolism in these cell lines revealed that attenuation of Rev-erb␤ had a number of significant effects in C2C12 cells (Table II). In the context of lipid and fatty acid absorption we observed repression of the mRNAs encoding Fabp-3 and -4 (ϳ5 and ϳ24-fold) and Fat/Cd36 (ϳ5-fold) (Fig. 5, A-C). Analysis of genes involved in the regulation of energy expenditure and lipid utilization showed that the expression of Ucp3 mRNA (Fig. 5D) was significantly repressed (ϳ7.5-fold). In contrast, Ucp2 mRNA showed minimum changes in the cell line overexpressing Rev-erb␤⌬E, relative to the ϳ2-fold changes observed in the markers of differentiation (Table II). Moreover, the mRNA encoding Ampk␥3 (preferentially expressed in glycolytic fast twitch muscle fibers), a regulator of glucose uptake, and energy balance was unchanged by Rev-erb␤⌬E expression. The expression of Scd-1, a key enzyme involved in adiposity, is repressed ϳ4-fold in the cell line overexpressing Rev-erb␤⌬E (Fig. 5E). In contrast, the expression of mRNA encoding the Scd-2 was relatively unaffected. Surprisingly, Srebp-1c mRNA expression increased 2.8-fold (Fig. 5F, Table II).
The expression of the transcripts encoding Cpt1, Mcad, and Acs-4 that are involved in lipid catabolism and preferential fuel utilization were not affected in the Rev-erb␤⌬E expressing cell line (Table II). Additionally, we did not observe any changes in the expression of mRNAs encoding ABCA1, ABCA8/G1, apoE, Cav-3, and ADRP, which are involved in cholesterol homeostasis and lipid storage (Table II). Analysis of the expression of many other nuclear hormone receptors that have been demonstrated to regulate lipid and carbohydrate metabolism, including ERR␣, PPAR-␣, -␤/␦, and -␥, LXR␣ and -␤, Rev-erb␣, and Nur77 demonstrated that Rev-erb␤⌬E expression did not affect FIG. 4. The C2:Rev-erb␤⌬E cell line acquires contractile markers of skeletal muscle. A and B, expression of troponin I type I (slow; Tnni1) and II (fast; Tnni2) mRNA levels, respectively, in the C2:Rev-erb␤⌬E cell line. Total RNA was extracted from proliferating myoblasts (PMB) or myotubes were harvested after 4 days of serum withdrawal (MT4), reverse transcribed to cDNA, and analyzed by Q-RT-PCR. Genespecific primers were used to examine the expression of Tnni1 (A) and Tnni2 (B). C and D, total RNA was extracted from wild type (C2C12) and dominant negative (C2:Rev-erb␤⌬E) cell lines from myotubes after 4 days of serum withdrawal, reverse transcribed to cDNA, and analyzed by Q-RT-PCR. Gene-specific primers were used to examine the expression of Tnni1 (C) and Tnni2 (D). Normalized expression was calculated relative to the expression of 18S rRNA determined with the same cDNA. All reactions were performed in triplicate and results are shown as average Ϯ S.D. the expression of the mRNAs encoding these NRs (see Table IV).
Rev-erb␤ Modulates Myokine Expression-Muscle cytokines (myokines) include IL-6, IL-15, and myostatin control inflammation, energy expenditure, muscle growth, and fat deposition (see Table I). The Rev-erb␣ nuclear receptor has been implicated in the control of inflammation, and the modulation of the NFB-dependent pathway. Therefore, we decided to investigate the expression of several myokines and inflammatory markers/regulators in the wild-type and C2C12 cells expressing Rev-erb␤⌬E.
Initially, we examined the expression of IL-6, IL-15, myostatin, and IB␣ (the regulator of NFB) during differentiation of native C2C12 cells. We isolated total RNA from wild type C2C12 proliferating myoblasts, and post-mitotic multinucleated myotubes after 4 days of serum withdrawal, and analyzed the expression levels of mRNAs by Q-RT-PCR. First, we observed that these myokines, and modulators of the inflammatory process, were expressed in wild type C2C12 cells (Fig. 6). Second, we observed that IL-15 and myostatin were induced (ϳ31-and 25-fold, respectively), whereas IB␣ and IL-6 were repressed (ϳ2.5-and 5.5-fold, respectively) during myogenic differentiation (Fig. 6, A-D).
Subsequently, we examined the effect of Rev-erb␤⌬E on the expression of the myokines, and inflammatory regulators in C2:Rev-erb␤⌬E cells after 4 days of serum withdrawal, relative to similarly differentiated native C2C12 cells by Q-RT-PCR (see Table III). Interestingly, we observed that ectopic expression of Rev-erb␤⌬E leads to an induction in the inhibitor of NFB-mediated gene expression, IB␣ (Fig. 6E). Consistent with increased expression of IB␣, mRNAs encoding the NFB and tumor necrosis factor target genes IL-15 (Fig. 6G) and Cox-2 (Table III) were not induced in cells expressing Rev-erb␤⌬E.
Curiously, we found that IL-6 mRNA is robustly increased (ϳ15-fold; Fig. 6F), and myostatin mRNA is dramatically reduced (ϳ24-fold; Fig. 6H) in the cells expressing Rev-erb␤⌬E, concordant with the precocious differentiation of the C2:Rev-erb␤⌬E cell line. In summary, this demonstrates that Rev-erb␤ regulates myokine expression, and as such is a critical modulator of muscle growth and inflammation in this tissue. DISCUSSION We have utilized the C2C12 in vitro cell culture model system and ectopic overexpression of dominant negative Rev-erb␤ to investigate the role of this orphan receptor in skeletal muscle lipid, and energy homeostasis. Here we report that perturbation of Rev-erb␤ function decreases the expression of genes involved in lipid absorption. Additionally, we observed dramatic induction and repression of two myokines, IL-6, and myostatin, respectively, which are involved in energy expenditure, inflammation, muscle hypertrophy and hyperplasia, and the accumulation of body fat.
These observations are consistent with genetic and molecular studies that demonstrate that the NR1D subfamily of nuclear receptors (Reverbs) has a direct role in lipid homeostasis. For example, the NR1D subfamily of orphan receptors regulate the expression of ApoC-III (20,21), a major component of triglyceride-rich remnant lipoprotein and associated with hypertriglyceridemia (37). In addition, Rev-erb␣ Ϫ/Ϫ mice have elevated levels of ApoC-III and very low density lipoprotein triglycerides. Moreover, Rev-erb␣ regulates ApoA1 gene expression in rodents treated with the hypolipidemic fibrate drugs. Finally, Rev-erb␣ is preferentially expressed in fast twitch/type IIB glycolytic, and intermediate type IIA oxidative fibers and null mutations lead to a transition to type I fibers (38). This suggests that this orphan NR subgroup regulates muscle fiber type, which can influence insulin sensitivity and the utilization of aerobic/anaerobic metabolism for the generation of ATP.
Specifically, we demonstrated that attenuation of Rev-erb␤mediated gene expression suppresses the expression of a subgroup of genes that include Fabp-3 and -4, Cd36, Ucp-3, and Scd-1 that are involved in lipid absorption and utilization. In this context, it is interesting to note that mice with a targeted disruption of SCD-1, a key target for Rev-erb␤, had lower levels of very low density lipoprotein, impaired triglyceride and cholesterol ester biosynthesis, and a lean phenotype (39). The Scd-1 Ϫ/Ϫ phenotype is very similar to the natural mutation called staggerer (ROR␣ sg/sg ) in an obese mouse strain, which leads to a functional knockout of ROR␣. The staggerer mice exhibit an aberrant blood-lipid profile with lower circulating plasma levels of HDL-C, apoC-III, and plasma triglycerides. It  should be noted that a dominant negative ROR␣ represses both Rev-erb␣ and -␤ mRNA expression in skeletal muscle (16). Moreover, lack of Fabp4, a key target for Rev-erb␤ in our model, protected the mice deficient in ApoE against atherosclerosis (40). Interestingly, we observed a suppression in the expression of genes involved in lipid absorption, and in Scd-1 that is involved in the formation of cholesterol esters. Moreover the activity of Scd-1 has also been linked to adiposity. Curiously, we observed an increase in Srebp-1c expression, the master regulator of fatty acid metabolism. This apparent contradiction is in concordance with several reports in the literature. For example, Raspe et al. (15), showed that staggerer mice have reduced plasma triglycerides, and Lau et al. (16) demonstrated that a dominant negative ROR␣ expression in muscle reduced Srebp-1c mRNA expression. The increase in SREBP-1c expression correlates with the Ͼ5-fold increase in ROR␣ mRNA expression in the C2:Rev-erb␤⌬E cell line. Second, in most tissues SREBP-1c and SCD-1 mRNA are coordinately expressed and regulated. However, it has been previously reported in skeletal muscle cells, and tissue treated with LXR agonists, that Srebp-1c and Scd-1 mRNA expression are uncoupled, and not co-ordinately regulated (23).
Our studies also demonstrate that Rev-erb␤ has a crucial role in regulating UCP3. Abundant expression of this gene correlates with preferential lipid utilization, and increased energy expenditure. The role of uncoupling proteins in regulating energy balance have been extensively investigated through transgenic mice and cell culture studies (41)(42)(43). Repression of UCP3 mRNA also correlates with the decreased expression of genes involved in lipid absorption and utilization, in concordance with the observations derived from this study.
Recent studies (22,44,45) have demonstrated a role of ROR␣ in the inflammation process. For example, ROR␣ has an anti-  inflammatory role by inhibiting tumor necrosis factor-␣-induced gene expression (22). The molecular basis of this modulation involves the direct induction of IB␣ transcription, thereby inhibiting the NFB signaling cascade. Conversely, Rev-erb␣ induces the NFB-mediated activation of the inflammatory cascade, for example, Cox-2 (44). Our studies show that attenuation of Rev-erb␤-mediated gene regulation leads to elevated ROR␣ and IB␣ expression in skeletal muscle cells. This correlates with no change in expression of the NFB/ tumor necrosis factor target genes, Cox-2 and IL- 15. Surprisingly, these studies have revealed a dramatic induction (ϳ15-fold) of interleukin-6, in the presence of increased IB␣ expression. However, IL-6 is an exercised induced myokine that induces lipolysis in adipose tissue, and suppresses tumor necrosis factor production. The increased expression of IL-6 is consistent with decreased myostatin expression. For example, it has been reported that IL-6 deficiency leads to late-onset obesity (46), whereas myostatin deficiency leads to the reduced accumulation of body fat (47). Therefore, the inverse correlation between IL-6 and myostatin expression in our Rev-erb␤ cell line is in concordance with the phenotypic effects of these myokines on metabolism (48 -50). The study has implicated Rev-erb␤ in the regulatory cascade controlling genes involved in lipid homeostasis. Furthermore, the significant impact on myokine expression that regulates inflammation, lipolysis, muscle growth, and the accumulation of body fat underscores the potential therapeutic utility of Rev-erb modulation by inverse agonists/antagonists. However, identification of small molecule regulators will be hindered by the lack of a significant pocket in the ligand binding domain in the Rev-erb/ NR1D subgroup of orphan nuclear receptors (51).
It is becoming increasingly apparent that skeletal muscle is a critical target tissue in the fight against metabolic disorders associated with diet, lifestyle, and metabolism. For example, LXR, PPAR␣, -␤/␦, and -␥ in skeletal muscle have been shown to be involved in enhancing the insulin-stimulated glucose disposal rate, decreasing triglycerides, increasing energy expenditure, and increasing lipid catabolism, cholesterol efflux, and plasma HDL-C levels (14,(23)(24)(25)(26)(27)(28).
Activation of these NRs in muscle has lead to increased insulin sensitivity, resistance to diet-induced obesity, and atherosclerosis. Hence, orphan nuclear receptors (for example, Rev-erb␤) that regulate lipid homeostasis and inflammation in skeletal muscle have enormous homeostatic ramifications. As discussed, skeletal muscle is a major mass peripheral tissue, and this lean tissue accounts for ϳ40% of the total body mass (36% for females, and 42% for males) and 30 -50% of the energy expenditure. These results indicate significant homeostatic interrelationships between the muscular and other body systems including the cardiovascular, endocrine, and lymphatic networks for the treatment of dyslipidemia, syndrome X, and inflammation. In conclusion, we suggest that in skeletal muscle cells, Rev-erb␤ programs a cascade of gene expression that controls the regulatory cross-talk between lipid metabolism and inflammation.