Loss of skeletal muscle estrogen-related receptors leads to severe exercise intolerance.

OBJECTIVE: Skeletal muscle oxidative capacity is central to physical activity, exercise capacity and whole-body metabolism. The three estrogen-related receptors (ERRs) are regulators of oxidative metabolism in many cell types, yet their roles in skeletal muscle remain unclear. The main aim of this study was to compare the relative contributions of ERRs to oxidative capacity in glycolytic and oxidative muscle, and to determine defects associated with loss of skeletal muscle ERR function. METHODS: We assessed ERR expression, generated mice lacking one or two ERRs specifically in skeletal muscle and compared the effects of ERR loss on the transcriptomes of EDL (predominantly glycolytic) and soleus (oxidative) muscles. We also determined the consequences of the loss of ERRs for exercise capacity and energy metabolism in mice with the most severe loss of ERR activity. RESULTS: ERRs were induced in human skeletal muscle in response to an exercise bout. Mice lacking both ERRα and ERRγ (ERRα/γ dmKO) had the broadest and most dramatic disruption in skeletal muscle gene expression. The most affected pathway was "mitochondrial function", in particular Oxphos and TCA cycle genes, and transcriptional defects were more pronounced in the glycolytic EDL than the oxidative soleus. Mice lacking ERRß and ERRγ, the two isoforms expressed highly in oxidative muscles, also exhibited defects in lipid and branch chain amino acid metabolism genes, specifically in the soleus. The pronounced disruption of oxidative metabolism in ERRα/γ dmKO mice led to pale muscles, decreased oxidative capacity, histochemical patterns reminiscent of minicore myopathies, and severe exercise intolerance, with the dmKO mice unable to switch to lipid utilization upon running. ERRα/γ dmKO mice showed no defects in whole-body glucose and energy homeostasis. CONCLUSIONS: Our findings define gene expression programs in skeletal muscle that depend on different combinations of ERRs, and establish a central role for ERRs in skeletal muscle oxidative metabolism and exercise capacity. Our data reveal a high degree of functional redundancy among muscle ERR isoforms for the protection of oxidative capacity, and show that ERR isoform-specific phenotypes are driven in part, but not exclusively, by their relative levels in different muscles.

Perm1 regulates CaMKII activation and shapes skeletal muscle responses to endurance exercise training.

OBJECTIVE: Endurance exercise training remodels skeletal muscle, leading to increased mitochondrial content and oxidative capacity. How exercise entrains skeletal muscle signaling pathways to induce adaptive responses remains unclear. In past studies, we identified Perm1 (PGC-1 and ERR induced regulator, muscle 1) as an exercise-induced gene and showed that Perm1 overexpression elicits similar muscle adaptations as endurance exercise training. The mechanism of action and the role of Perm1 in exercise-induced responses are not known. In this study, we aimed to determine the pathway by which Perm1 acts as well as the importance of Perm1 for acute and long-term responses to exercise. METHODS: We performed immunoprecipitation and mass spectrometry to identify Perm1 associated proteins, and validated Perm1 interactions with the Ca2+/calmodulin-dependent protein kinase II (CaMKII). We also knocked down Perm1 expression in gastrocnemius muscles of mice via AAV-mediated delivery of shRNA and assessed the impact of reduced Perm1 expression on both acute molecular responses to a single treadmill exercise bout and long-term adaptive responses to four weeks of voluntary wheel running training. Finally, we asked whether Perm1 levels are modulated by diet or diseases affecting skeletal muscle function. RESULTS: We show that Perm1 associates with skeletal muscle CaMKII and promotes CaMKII activation. In response to an acute exercise bout, muscles with a knock down of Perm1 showed defects in the activation of CaMKII and p38 MAPK and blunted induction of regulators of oxidative metabolism. Following four weeks of voluntary training, Perm1 knockdown muscles had attenuated mitochondrial biogenesis. Finally, we found that Perm1 expression is reduced in diet-induced obese mice and in muscular dystrophy patients and mouse models. CONCLUSIONS: Our findings identify Perm1 as a muscle-specific regulator of exercise-induced signaling and Perm1 levels as tuners of the skeletal muscle response to exercise. The decreased Perm1 levels in states of obesity or muscle disease suggest that Perm1 may link pathological states to inefficient exercise responses.

Estrogen-Related Receptors Mediate the Adaptive Response of Brown Adipose Tissue to Adrenergic Stimulation.

Adrenergic stimulation of brown adipose tissue (BAT) induces acute and long-term responses. The acute adrenergic response activates thermogenesis by uncoupling oxidative phosphorylation and enabling increased substrate oxidation. Long-term, adrenergic signaling remodels BAT, inducing adaptive transcriptional changes that expand thermogenic capacity. Here, we show that the estrogen-related receptors alpha and gamma (ERRα, ERRγ) are collectively critical effectors of adrenergically stimulated transcriptional reprogramming of BAT. Mice lacking adipose ERRs (ERRαγAd-/-) have reduced oxidative and thermogenic capacity and rapidly become hypothermic when exposed to cold. ERRαγAd-/- mice treated long term with a ß3-adrenergic agonist fail to expand oxidative or thermogenic capacity and do not increase energy expenditure in response to norepinephrine (NE). Furthermore, ERRαγAd-/- mice fed a high-fat diet do not lose weight or show improved glucose tolerance when dosed with ß3-adrenergic agonists. The molecular basis of these defects is the finding that ERRs mediate the bulk of the transcriptional response to adrenergic stimulation.

Complementary Roles of Estrogen-Related Receptors in Brown Adipocyte Thermogenic Function.

Brown adipose tissue (BAT) thermogenesis relies on a high abundance of mitochondria and the unique expression of the mitochondrial Uncoupling Protein 1 (UCP1), which uncouples substrate oxidation from ATP synthesis. Adrenergic stimulation of brown adipocytes activates UCP1-mediated thermogenesis; it also induces the expression of Ucp1 and other genes important for thermogenesis, thereby endowing adipocytes with higher oxidative and uncoupling capacities. Adipocyte mitochondrial biogenesis and oxidative capacity are controlled by multiple transcription factors, including the estrogen-related receptor (ERR)α. Whole-body ERRα knockout mice show decreased BAT mitochondrial content and oxidative function but normal induction of Ucp1 in response to cold. In addition to ERRα, brown adipocytes express ERRß and ERRγ, 2 nuclear receptors that are highly similar to ERRα and whose function in adipocytes is largely unknown. To gain insights into the roles of all 3 ERRs, we assessed mitochondrial function and adrenergic responses in primary brown adipocytes lacking combinations of ERRs. We show that adipocytes lacking just ERRα, the most abundant ERR, show only mild mitochondrial defects. Adipocytes lacking ERRß and ERRγ also show just mild defects. In contrast, adipocytes lacking all 3 ERRs have severe reductions in mitochondrial content and oxidative capacity. Moreover, adipocytes lacking all 3 ERRs have defects in the transcriptional and metabolic response to adrenergic stimulation, suggesting a wider role of ERRs in BAT function than previously appreciated. Our study shows that ERRs have a great capacity to compensate for each other in protecting mitochondrial function and the metabolic response to adrenergic signaling, processes vital to BAT function.

Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle.

Skeletal muscle mitochondrial content and oxidative capacity are important determinants of muscle function and whole-body health. Mitochondrial content and function are enhanced by endurance exercise and impaired in states or diseases where muscle function is compromised, such as myopathies, muscular dystrophies, neuromuscular diseases, and age-related muscle atrophy. Hence, elucidating the mechanisms that control muscle mitochondrial content and oxidative function can provide new insights into states and diseases that affect muscle health. In past studies, we identified Perm1 (PPARGC1- and ESRR-induced regulator, muscle 1) as a gene induced by endurance exercise in skeletal muscle, and regulating mitochondrial oxidative function in cultured myotubes. The capacity of Perm1 to regulate muscle mitochondrial content and function in vivo is not yet known. In this study, we use adeno-associated viral (AAV) vectors to increase Perm1 expression in skeletal muscles of 4-wk-old mice. Compared to control vector, AAV1-Perm1 leads to significant increases in mitochondrial content and oxidative capacity (by 40-80%). Moreover, AAV1-Perm1-transduced muscles show increased capillary density and resistance to fatigue (by 33 and 31%, respectively), without prominent changes in fiber-type composition. These findings suggest that Perm1 selectively regulates mitochondrial biogenesis and oxidative function, and implicate Perm1 in muscle adaptations that also occur in response to endurance exercise.

GADD45γ regulates the thermogenic capacity of brown adipose tissue.

The coactivator peroxisome proliferator-activated receptor-gamma coactivator 1 α (PGC-1α) is widely considered a central transcriptional regulator of adaptive thermogenesis in brown adipose tissue (BAT). However, mice lacking PGC-1α specifically in adipose tissue have only mild thermogenic defects, suggesting the presence of additional regulators. Using the activity of estrogen-related receptors (ERRs), downstream effectors of PGC-1α, as read-out in a high-throughput genome-wide cDNA screen, we identify here growth arrest and DNA-damage-inducible protein 45 γ (GADD45γ) as a cold-induced activator of uncoupling protein 1 (UCP1) and oxidative capacity in BAT. Mice lacking Gadd45γ have defects in Ucp1 induction and the thermogenic response to cold. GADD45γ works by activating MAPK p38, which is a potent activator of ERRß and ERRγ transcriptional function. GADD45γ activates ERRγ independently of PGC-1 coactivators, yet synergizes with PGC-1α to induce the thermogenic program. Our findings elucidate a previously unidentified GADD45γ/p38/ERRγ pathway that regulates BAT thermogenesis and may enable new approaches for the stimulation of energy expenditure. Our study also implicates GADD45 proteins as general metabolic regulators.

Peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1)- and estrogen-related receptor (ERR)-induced regulator in muscle 1 (Perm1) is a tissue-specific regulator of oxidative capacity in skeletal muscle cells.

Mitochondrial oxidative metabolism and energy transduction pathways are critical for skeletal and cardiac muscle function. The expression of genes important for mitochondrial biogenesis and oxidative metabolism are under the control of members of the peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1) family of transcriptional coactivators and the estrogen-related receptor (ERR) subfamily of nuclear receptors. Perturbations in PGC-1 and/or ERR activities have been associated with alterations in capacity for endurance exercise, rates of muscle atrophy, and cardiac function. The mechanism(s) by which PGC-1 and ERR proteins regulate muscle-specific transcriptional programs is not fully understood. We show here that PGC-1α and ERRs induce the expression of a so far uncharacterized muscle-specific protein, PGC-1- and ERR-induced regulator in muscle 1 (Perm1), which regulates the expression of selective PGC-1/ERR target genes. Perm1 is required for the basal as well as PGC-1α-enhanced expression of genes with roles in glucose and lipid metabolism, energy transfer, and contractile function. Silencing of Perm1 in cultured myotubes compromises respiratory capacity and diminishes PGC-1α-induced mitochondrial biogenesis. Our findings support a role for Perm1 acting downstream of PGC-1α and ERRs to regulate muscle-specific pathways important for energy metabolism and contractile function. Elucidating the function of Perm1 may enable novel approaches for the treatment of disorders with compromised skeletal muscle bioenergetics, such as mitochondrial myopathies and age-related/disease-associated muscle atrophies.

Nuclear receptor/microRNA circuitry links muscle fiber type to energy metabolism.

The mechanisms involved in the coordinate regulation of the metabolic and structural programs controlling muscle fitness and endurance are unknown. Recently, the nuclear receptor PPARß/δ was shown to activate muscle endurance programs in transgenic mice. In contrast, muscle-specific transgenic overexpression of the related nuclear receptor, PPARα, results in reduced capacity for endurance exercise. We took advantage of the divergent actions of PPARß/δ and PPARα to explore the downstream regulatory circuitry that orchestrates the programs linking muscle fiber type with energy metabolism. Our results indicate that, in addition to the well-established role in transcriptional control of muscle metabolic genes, PPARß/δ and PPARα participate in programs that exert opposing actions upon the type I fiber program through a distinct muscle microRNA (miRNA) network, dependent on the actions of another nuclear receptor, estrogen-related receptor γ (ERRγ). Gain-of-function and loss-of-function strategies in mice, together with assessment of muscle biopsies from humans, demonstrated that type I muscle fiber proportion is increased via the stimulatory actions of ERRγ on the expression of miR-499 and miR-208b. This nuclear receptor/miRNA regulatory circuit shows promise for the identification of therapeutic targets aimed at maintaining muscle fitness in a variety of chronic disease states, such as obesity, skeletal myopathies, and heart failure.

Striated muscle activator of Rho signalling (STARS) is a PGC-1α/oestrogen-related receptor-α target gene and is upregulated in human skeletal muscle after endurance exercise.

The striated muscle activator of Rho signalling (STARS) is an actin-binding protein specifically expressed in cardiac, skeletal and smooth muscle. STARS has been suggested to provide an important link between the transduction of external stress signals to intracellular signalling pathways controlling genes involved in the maintenance of muscle function. The aims of this study were firstly, to establish if STARS, as well as members of its downstream signalling pathway, are upregulated following acute endurance cycling exercise; and secondly, to determine if STARS is a transcriptional target of peroxisome proliferator-activated receptor gamma co-activator 1-α (PGC-1α) and oestrogen-related receptor-α (ERRα). When measured 3 h post-exercise, STARS mRNA and protein levels as well as MRTF-A and serum response factor (SRF) nuclear protein content, were significantly increased by 140, 40, 40 and 40%, respectively. Known SRF target genes, carnitine palmitoyltransferase-1ß (CPT-1ß) and jun B proto-oncogene (JUNB), as well as the exercise-responsive genes PGC-1α mRNA and ERRα were increased by 2.3-, 1.8-, 4.5- and 2.7-fold, 3 h post-exercise. Infection of C2C12 myotubes with an adenovirus-expressing human PGC-1α resulted in a 3-fold increase in Stars mRNA, a response that was abolished following the suppression of endogenous ERRα. Over-expression of PGC-1α also increased Cpt-1ß, Cox4 and Vegf mRNA by 6.2-, 2.0- and 2.0-fold, respectively. Suppression of endogenous STARS reduced basal Cpt-1ß levels by 8.2-fold and inhibited the PGC-1α-induced increase in Cpt-1ß mRNA. Our results show for the first time that the STARS signalling pathway is upregulated in response to acute endurance exercise. Additionally, we show in C2C12 myotubes that the STARS gene is a PGC-1α/ERRα transcriptional target. Furthermore, our results suggest a novel role of STARS in the co-ordination of PGC-1α-induced upregulation of the fat oxidative gene, CPT-1ß.

Estrogen-related receptor gamma promotes mesenchymal-to-epithelial transition and suppresses breast tumor growth.

Estrogen-related receptors (ERR), ERR alpha (ERRα) and ERR gamma (ERRγ), are orphan nuclear receptors implicated in breast cancer that function similarly in the regulation of oxidative metabolism genes. Paradoxically, in clinical studies, high levels of ERRα are associated with poor outcomes whereas high levels of ERRγ are associated with a favorable course. Recent studies suggest that ERRα may indeed promote breast tumor growth. The roles of ERRγ in breast cancer progression and how ERRα and ERRγ may differentially affect cancer growth are unclear. In mammary carcinoma cells that do not express endogenous ERRγ, we found that ectopic expression of ERRγ enhanced oxidative metabolism in vitro and inhibited the growth of tumor xenografts in vivo. In contrast, ectopic expression of the ERRα coactivator PGC-1α enhanced oxidative metabolism but did not affect tumor growth. Notably, ERRγ activated expression of a genetic program characteristic of mesenchymal-to-epithelial transition (MET). This program was apparent by changes in cellular morphology, upregulation of epithelial cell markers, downregulation of mesenchymal markers, and decreased cellular invasiveness. We determined that this program was also associated with upregulation of E-cadherin, which is activated directly by ERRγ. In contrast, PGC-1α activated only a subset of genes characteristic of the MET program and, unlike ERRγ, did not upregulate E-cadherin. In conclusion, these results show that ERRγ induces E-cadherin, promotes MET, and suppresses breast cancer growth. Our findings suggest that ERRγ agonists may have applications in the treatment of breast cancer.