The heavy subunit of ferritin stimulates NLRP3 inflammasomes in hepatic stellate cells through ICAM-1 to drive hepatic inflammation.

Serum ferritin concentrations increase during hepatic inflammation and correlate with the severity of chronic liver disease. Here, we report a molecular mechanism whereby the heavy subunit of ferritin (FTH) contributes to hepatic inflammation. We found that FTH induced activation of the NLRP3 inflammasome and secretion of the proinflammatory cytokine interleukin-1ß (IL-1ß) in primary rat hepatic stellate cells (HSCs) through intercellular adhesion molecule-1 (ICAM-1). FTH-ICAM-1 stimulated the expression of Il1b, NLRP3 inflammasome activation, and the processing and secretion of IL-1ß in a manner that depended on plasma membrane remodeling, clathrin-mediated endocytosis, and lysosomal destabilization. FTH-ICAM-1 signaling at early endosomes stimulated Il1b expression, implying that this endosomal signaling primed inflammasome activation in HSCs. In contrast, lysosomal destabilization was required for FTH-induced IL-1ß secretion, suggesting that lysosomal damage activated inflammasomes. FTH induced IL-1ß production in liver slices from wild-type mice but not in those from Icam1-/- or Nlrp3-/- mice. Thus, FTH signals through its receptor ICAM-1 on HSCs to activate the NLRP3 inflammasome. We speculate that this pathway contributes to hepatic inflammation, a key process that stimulates hepatic fibrogenesis associated with chronic liver disease.

The Role of Sonic Hedgehog in Human Holoprosencephaly and Short-Rib Polydactyly Syndromes.

The Hedgehog (HH) signalling pathway is one of the major pathways controlling cell differentiation and proliferation during human development. This pathway is complex, with HH function influenced by inhibitors, promotors, interactions with other signalling pathways, and non-genetic and cellular factors. Many aspects of this pathway are not yet clarified. The main features of Sonic Hedgehog (SHH) signalling are discussed in relation to its function in human development. The possible role of SHH will be considered using examples of holoprosencephaly and short-rib polydactyly (SRP) syndromes. In these syndromes, there is wide variability in phenotype even with the same genetic mutation, so that other factors must influence the outcome. SHH mutations were the first identified genetic causes of holoprosencephaly, but many other genes and environmental factors can cause malformations in the holoprosencephaly spectrum. Many patients with SRP have genetic defects affecting primary cilia, structures found on most mammalian cells which are thought to be necessary for canonical HH signal transduction. Although SHH signalling is affected in both these genetic conditions, there is little overlap in phenotype. Possible explanations will be canvassed, using data from published human and animal studies. Implications for the understanding of SHH signalling in humans will be discussed.

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

The Nuclear Receptor Nor-1 Is a Pleiotropic Regulator of Exercise-Induced Adaptations.

Exercise induces various physical and metabolic changes in skeletal muscle that adaptively reprograms this tissue to current physiological and environmental demands. Underlying these changes are broad modifications to gene expression. We postulate that the nuclear hormone receptor, Nor-1, is activated after exercise, and this transcription factor modifies gene expression to drive the molecular and cellular adaptations associated with contractile reorganization.

Lung and liver growth and retinoic acid status in human fetuses with congenital diaphragmatic hernia.

BACKGROUND: Abnormal retinoic acid (RA) signalling is considered a major cause of congenital diaphragmatic hernia (CDH). Pulmonary hypoplasia and pulmonary hypertension are the major causes of morbidity and mortality in infants born with CDH. Experimental studies in animals have found that RA signalling is involved in lung and liver development, but animal models of CDH do not directly correlate with CDH in human fetuses. This study investigated if RA status is also linked to lung and liver growth in human fetuses with CDH. STUDY DESIGN AND PATIENTS: Hepatic stellate cells (HSC) in autopsy human fetal liver tissue were identified using cRBP-1 immunohistochemistry and the numbers of HSC manually counted. In mammals, RA is principally stored in HSC complexed to cRBP-1 and therefore cRBP-1+ HSC numbers were used as an indicator of fetal RA status. The number of HSCs was correlated with liver and lung weights, calculated relative to either normal biometric values or fetal body weight. RESULTS: The number of cRBP-1+ HSCs correlated with lung weight contralateral to the side of the diaphragmatic hernia (r=0.82, p=0.025) and combined lung weight (r=0.78, p=0.039) but not with ipsilateral lung weight (r=0.43, p=0.33), in fetuses with right and left CDH and a case of giant omphalocoele. Liver growth was influenced by contact with diaphragm but not significantly correlated with cRBP-1 expression (r=0.52, p=0.056). CONCLUSION: Fetal RA stores, reflected in the number of cRBP-1+ HSCs, influence lung growth as well as diaphragm development in human fetuses with CDH. Contact with diaphragm influenced liver growth.

Taurocholate Induces Biliary Differentiation of Liver Progenitor Cells Causing Hepatic Stellate Cell Chemotaxis in the Ductular Reaction: Role in Pediatric Cystic Fibrosis Liver Disease.

Cystic fibrosis liver disease (CFLD) in children causes progressive fibrosis leading to biliary cirrhosis; however, its cause(s) and early pathogenesis are unclear. We hypothesized that a bile acid-induced ductular reaction (DR) drives fibrogenesis. The DR was evaluated by cytokeratin-7 immunohistochemistry in liver biopsies, staged for fibrosis, from 60 children with CFLD, and it demonstrated that the DR was significantly correlated with hepatic fibrosis stage and biliary taurocholate levels. To examine the mechanisms involved in DR induction, liver progenitor cells (LPCs) were treated with taurocholate, and key events in DR evolution were assessed: LPC proliferation, LPC biliary differentiation, and hepatic stellate cell (HSC) chemotaxis. Taurocholate induced a time-dependent increase in LPC proliferation and expression of genes associated with cholangiocyte differentiation (cytokeratin 19, connexin 43, integrin ß4, and γ-glutamyltranspeptidase), whereas the hepatocyte specification marker HNF4α was suppressed. Functional cholangiocyte differentiation was demonstrated via increased acetylated α-tubulin and SOX9 proteins, the number of primary cilia+ LPCs, and increased active γ-glutamyltranspeptidase enzyme secretion. Taurocholate induced LPCs to release MCP-1, MIP1α, and RANTES into conditioned medium causing HSC chemotaxis, which was inhibited by anti-MIP1α. Immunofluorescence confirmed chemokine expression localized to CK7+ DR and LPCs in CFLD liver biopsies. This study suggests that taurocholate is involved in initiating functional LPC biliary differentiation and the development of the DR, with subsequent induction of chemokines that drive HSC recruitment in CFLD.

The Nuclear Receptor, Nor-1, Induces the Physiological Responses Associated With Exercise.

Skeletal muscle remodels metabolic capacity, contractile and exercise phenotype in response to physiological demands. This adaptive remodeling response to physical activity can ameliorate/prevent diseases associated with poor diet and lifestyle. Our previous work demonstrated that skeletal muscle-specific transgenic expression of the neuron-derived orphan nuclear receptor, Nor-1 drives muscle reprogramming, improves exercise endurance, and oxidative metabolism. The current manuscript investigates the association between exercise, Nor-1 expression and the role of Nor-1 in adaptive remodeling. We demonstrate that Nor-1 expression is induced by exercise and is dependent on calcium/calcineurin signaling (in vitro and in vivo). Analysis of fatigue-resistant transgenic mice that express Nor-1 in skeletal muscle revealed increased hypertrophy and vascularization of muscle tissue. Moreover, we demonstrate that transgenic Nor-1 expression is associated with increased intracellular recycling, ie, autophagy, involving 1) increased expression of light chain 3A or LC3A-II, autophagy protein 5, and autophagy protein 12 in quadriceps femoris muscle extracts from Tg-Nor-1 (relative to Wild-type (WT) littermates); 2) decreased p62 expression indicative of increased autophagolysosome assembly; and 3) decreased mammalian target of rapamycin complex 1 activity. Transfection of LC3A-GFP-RFP chimeric plasmid demonstrated that autophagolysosome formation was significantly increased by Nor-1 expression. Furthermore, we demonstrated a single bout of exercise induced LC3A-II expression in skeletal muscle from C57BL/6 WT mice. This study, when combined with our previous studies, demonstrates that Nor-1 expression drives multiple physiological changes/pathways that are critical to the beneficial responses of muscle to exercise and provides insights into potential pharmacological manipulation of muscle reprogramming for the treatment of lifestyle induced chronic diseases.

Rorα deficiency and decreased adiposity are associated with induction of thermogenic gene expression in subcutaneous white adipose and brown adipose tissue.

The Rar-related orphan receptor-α (Rorα) is a nuclear receptor that regulates adiposity and is a potential regulator of energy homeostasis. We have demonstrated that the Rorα-deficient staggerer (sg/sg) mice display a lean and obesity-resistant phenotype. Adaptive Ucp1-dependent thermogenesis in beige/brite and brown adipose tissue serves as a mechanism to increase energy expenditure and resist obesity. DEXA and MRI analysis demonstrated significantly decreased total fat mass and fat/lean mass tissue ratio in male chow-fed sg/sg mice relative to wt mice. In addition, we observed increased Ucp1 expression in brown adipose and subcutaneous white adipose tissue but not in visceral adipose tissue from Rorα-deficient mice. Moreover, this was associated with significant increases in the expression of the mRNAs encoding the thermogenic genes (i.e., markers of brown and beige adipose) Pparα, Errα, Dio2, Acot11/Bfit, Cpt1ß, and Cidea in the subcutaneous adipose in the sg/sg relative to WT mice. These changes in thermogenic gene expression involved the significantly increased expression of the (cell-fate controlling) histone-lysine N-methyltransferase 1 (Ehmt1), which stabilizes the Prdm16 transcriptional complex. Moreover, primary brown adipocytes from sg/sg mice displayed a higher metabolic rate, and further analysis was consistent with increased uncoupling. Finally, core body temperature analysis and infrared thermography demonstrated that the sg/sg mice maintained greater thermal control and cold tolerance relative to the WT littermates. We suggest that enhanced Ucp1 and thermogenic gene expression/activity may be an important contributor to the lean, obesity-resistant phenotype in Rorα-deficient mice.

Transgenic muscle-specific Nor-1 expression regulates multiple pathways that effect adiposity, metabolism, and endurance.

The mRNA encoding Nor-1/NR4A3 is rapidly and strikingly induced by ß2-adrenergic signaling in glycolytic and oxidative skeletal muscle. In skeletal muscle cells, Nor-1 expression is important for the regulation of oxidative metabolism. Transgenic skeletal muscle-specific expression of activated Nor-1 resulted in the acquisition of an endurance phenotype, an increase in type IIA/X oxidative muscle fibers, and increased numbers of mitochondria. In the current study, we used dual-energy x-ray absorptiometry and magnetic resonance imaging analysis to demonstrate decreased adiposity in transgenic (Tg) Nor-1 mice relative to that in wild-type littermates. Furthermore, the Tg-Nor-1 mice were resistant to diet-induced weight gain and maintained fasting glucose at normoglycemic levels. Expression profiling and RT-quantitative PCR analysis revealed significant increases in genes involved in glycolysis, the tricarboxylic acid cycle, oxidative phosphorylation, fatty acid oxidation, and glycogen synthesis, in concordance with the lean phenotype. Moreover, expression profiling identified several Z-disc and sarcomeric binding proteins that modulate fiber type phenotype and endurance, eg, α-actinin-3. In addition, we demonstrated that the Tg-Nor-1 mouse line has significantly higher glycogen content in skeletal muscle relative to that in wild-type littermates. Finally, we identified a decreased NAD(+)/NADH ratio with a concordant increase in peroxisome proliferator-activated receptor γ coactivator-1α1 protein/mRNA expression. Increased NADH was associated with an induction of the genes involved in the malate-aspartate shuttle and a decrease in the glycerol 3-phosphate shuttle, which maximizes aerobic ATP production. In conclusion, skeletal muscle-specific Nor-1 expression regulates genes and pathways that regulate adiposity, muscle fiber type metabolic capacity, and endurance.

Disruption of Rorα1 and cholesterol 25-hydroxylase expression attenuates phagocytosis in male Rorαsg/sg mice.

We and others have previously demonstrated that congenital deficiency of the nuclear hormone receptor, Rorα1, in staggerer (sg/sg) mice results in resistance to diet-induced obesity and increased insulin sensitivity. Paradoxically, the sg/sg mice are susceptible to atherosclerosis and display impaired innate immunity, underscoring the regulatory links between metabolic disease, inflammation, and susceptibility to infection. Here, we present novel evidence that Rorα1 regulates innate immune function by demonstrating impaired phagocytosis in sg/sg mice. The early stages of Fc-γ receptor-mediated phagocytosis in lipopolysaccharide-activated sg/sg bone marrow-derived macrophages (BMMs) were significantly impaired compared with wild-type cells. Moreover, in sg/sg BMMs, the phagocytic cup membranes had reduced levels of cholesterol. Expression profiling revealed dysregulated expression of genes involved in inflammation and lipid metabolism in sg/sg BMMs. Notably, we identified decreased expression of the mRNA encoding cholesterol 25-hydroxylase (Ch25h), an enzyme that converts cholesterol to 25-hydroxycholesterol (25HC), an oxysterol with emerging roles in immunity. Treatment of sg/sg BMMs with 25HC rescued phagocytosis in a dose-dependent manner, whereas small interfering RNA knockdown of Ch25h mRNA expression in wild-type cells attenuated phagocytosis. Hence, we propose that 25HC is essential for optimizing membrane internalization during phagocytosis and that aberrant Ch25h expression in Rorα1-deficient sg/sg macrophages disrupts phagocytosis. Our studies reveal new roles for Rorα1, Ch25h, and 25HC in phagocytosis. Aberrant 25HC underpins the paradoxical association between insulin sensitivity and impaired innate immunity in Rorα1-deficient mice, heralding a wider and essential role for this oxysterol at the nexus of metabolism and immunity.

Orphan nuclear receptors and the regulation of nutrient metabolism: understanding obesity.

Nuclear hormone receptors (NRs) are a superfamily of eukaryotic ligand-dependent transcription factors that translate endocrine, metabolic, nutritional, developmental, and pathophysiological signals into gene regulation. Members of the NR superfamily (on the basis of sequence homology) that lack identified natural and/or synthetic ligands are/were classified as "orphan" NRs. These members of the NR superfamily are abundantly expressed in tissues associated with major metabolic activity, such as skeletal muscle, adipose, and liver. Subsequently, in vivo genetic studies on these orphan NRs and exploitation of novel natural and synthetic agonists has revealed that orphan NRs regulate 1) carbohydrate, lipid, and energy homeostasis in a tissue-specific manner, and 2) the pathophysiology of dyslipidemia, obesity, Type 2 diabetes, and cardiovascular disease. This review discusses key studies that have implicated the orphan NRs as organ-specific regulators of metabolism and mediators of adverse pathophysiological effects. The emerging discovery of novel endogenous orphan NR ligands and synthetic agonists has provided the foundation for therapeutic exploitation of the orphans in the treatment of metabolic disease.

Protein arginine methyltransferase 6-dependent gene expression and splicing: association with breast cancer outcomes.

Protein arginine methyltransferase-6 (PRMT6) regulates steroid-dependent transcription and alternative splicing and is implicated in endocrine system development and function, cell death, cell cycle, gene expression and cancer. Despite its role in these processes, little is known about its function and cellular targets in breast cancer. To identify novel gene targets regulated by PRMT6 in breast cancer cells, we used a combination of small interfering RNA and exon-specific microarray profiling in vitro coupled to in vivo validation in normal breast and primary human breast tumours. This approach, which allows the examination of genome-wide changes in individual exon usage and total transcript levels, demonstrated that PRMT6 knockdown significantly affected i) the transcription of 159 genes and ii) alternate splicing of 449 genes. The PRMT6-dependent transcriptional and alternative splicing targets identified in vitro were validated in human breast tumours. Using the list of genes differentially expressed between normal and PRMT6 knockdown cells, we generated a PRMT6-dependent gene expression signature that provides an indication of PRMT6 dysfunction in breast cancer cells. Interrogation of several well-studied breast cancer microarray expression datasets with the PRMT6 gene expression signature demonstrated that PRMT6 dysfunction is associated with better overall relapse-free and distant metastasis-free survival in the oestrogen receptor (ER (ESR1)) breast cancer subgroup. These results suggest that dysregulation of PRMT6-dependent transcription and alternative splicing may be involved in breast cancer pathophysiology and the molecular consequences identifying a unique and informative biomarker profile.

The nuclear receptor, Nor-1, markedly increases type II oxidative muscle fibers and resistance to fatigue.

Nuclear hormone receptors (NR) have been implicated as regulators of lipid and carbohydrate metabolism. The orphan NR4A subgroup has emerged as regulators of metabolic function. Targeted silencing of neuron-derived orphan receptor 1 (Nor-1)/NR4A3 in skeletal muscle cells suggested that this NR was necessary for oxidative metabolism in vitro. To investigate the in vivo role of Nor-1, we have developed a mouse model with preferential expression of activated Nor-1 in skeletal muscle. In skeletal muscle, this resulted in a marked increase in: 1) myoglobin expression, 2) mitochondrial DNA and density, 3) oxidative enzyme staining, and 4) genes/proteins encoding subunits of electron transport chain complexes. This was associated with significantly increased type IIA and IIX myosin heavy chain mRNA and proteins and decreased type IIB myosin heavy chain mRNA and protein. The contractile protein/fiber type remodeling driving the acquisition of the oxidative type II phenotype was associated with 1) the significantly increased expression of myocyte-specific enhancer factor 2C, and phospho-histone deacetylase 5, and 2) predominantly cytoplasmic HDAC5 staining in the Tg-Nor-1 mice. Moreover, the Nor-1 transgenic line displayed significant improvements in glucose tolerance, oxygen consumption, and running endurance (in the absence of increased insulin sensitivity), consistent with increased oxidative capacity of skeletal muscle. We conclude that skeletal muscle fiber type is not only regulated by exercise-sensitive calcineurin-induced signaling cascade but also by NR signaling pathways that operate at the nexus that coordinates muscle performance and metabolic capacity in this major mass tissue.

Minireview: Nuclear hormone receptor 4A signaling: implications for metabolic disease.

Numerous members of the nuclear hormone receptor (NR) superfamily have been demonstrated to regulate metabolic function in a cell- and tissue-specific manner. This review brings together recent studies that have associated members of the NR superfamily, the orphan NR4A subgroup, with the regulation of metabolic function and disease. The orphan NR4A subgroup includes Nur77 (NR4A1), Nurr1 (NR4A2), and Nor-1 (NR4A3). Expression of these receptors is induced in multiple tissues by a diverse range of stimuli, including stimuli associated with metabolic function, such as: ß-adrenoceptor agonists, cold, fatty acids, glucose, insulin, cholesterol, and thiazolidinediones. In vitro and in vivo gain- and loss-of-function studies in major metabolic tissues (including skeletal muscle, adipose, and liver cells and tissues) have associated the NR4A subgroup with specific aspects of lipid, carbohydrate, and energy homeostasis. Most excitingly, although these orphan receptors do not have known endogenous ligands, several small molecule agonists have recently been identified. The preliminary studies reviewed in this manuscript suggest that therapeutic exploitation of the NR4A subgroup may show utility against dyslipidemia, obesity, diabetes, and cardiovascular disease.

Neonatal complete generalized glucocorticoid resistance and growth hormone deficiency caused by a novel homozygous mutation in Helix 12 of the ligand binding domain of the glucocorticoid receptor gene (NR3C1).

CONTEXT: Glucocorticoid resistance is a rare genetic condition characterized by reduced sensitivity to cortisol signaling and subsequent hyperactivation of the hypothalamic-pituitary-adrenal axis. OBJECTIVE: The objective was to confirm the diagnosis of glucocorticoid resistance in the patient, to determine the degree of suppression of cortisol and ACTH levels in response to dexamethasone, and to determine the underlying genetic abnormality and functional consequences of the mutation. PATIENT AND METHODS: The patient presented on the first day of life with profound hypoglycemia. Initial cortisol levels were appropriately elevated; however, the patient was found to have persistently elevated levels of both cortisol and ACTH. The baby developed a tanned appearance and severe hypertension and fatigued easily with feeding. Serial oral dexamethasone suppression tests were performed with doses escalating from 0.125 mg to 12 mg dexamethasone given at 2300 h. Sequencing of the glucocorticoid receptor gene was performed along with functional studies of the glucocorticoid receptor. GH secretion was assessed with an arginine glucagon stimulation test. RESULTS: Cortisol and ACTH levels did not suppress with doses of up to 12 mg dexamethasone. A 2-bp deletion was found at amino acid position 773 of the glucocorticoid receptor ligand binding domain. A complete lack of dexamethasone binding and in vitro biological effect was demonstrated. GH stimulation testing was consistent with GH deficiency. CONCLUSION: The homozygous mutation in the ligand-binding domain of the glucocorticoid receptor gene resulted in a functionally inactive glucocorticoid receptor and apparent complete glucocorticoid resistance with biochemical GH deficiency.

Expression profiling of skeletal muscle following acute and chronic beta2-adrenergic stimulation: implications for hypertrophy, metabolism and circadian rhythm.

BACKGROUND: Systemic administration of beta-adrenoceptor (beta-AR) agonists has been found to induce skeletal muscle hypertrophy and significant metabolic changes. In the context of energy homeostasis, the importance of beta-AR signaling has been highlighted by the inability of beta(1-3)-AR-deficient mice to regulate energy expenditure and susceptibility to diet induced obesity. However, the molecular pathways and gene expression changes that initiate and maintain these phenotypic modulations are poorly understood. Therefore, the aim of this study was to identify differential changes in gene expression in murine skeletal muscle associated with systemic (acute and chronic) administration of the beta(2)-AR agonist formoterol. RESULTS: Skeletal muscle gene expression (from murine tibialis anterior) was profiled at both 1 and 4 hours following systemic administration of the beta(2)-AR agonist formoterol, using Illumina 46K mouse BeadArrays. Illumina expression profiling revealed significant expression changes in genes associated with skeletal muscle hypertrophy, myoblast differentiation, metabolism, circadian rhythm, transcription, histones, and oxidative stress. Differentially expressed genes relevant to the regulation of muscle mass and metabolism (in the context of the hypertrophic phenotype) were further validated by quantitative RT-PCR to examine gene expression in response to both acute (1-24 h) and chronic administration (1-28 days) of formoterol at multiple timepoints. In terms of skeletal muscle hypertrophy, attenuation of myostatin signaling (including differential expression of myostatin, activin receptor IIB, phospho-Smad3 etc) was observed following acute and chronic administration of formoterol. Acute (but not chronic) administration of formoterol also significantly induced the expression of genes involved in oxidative metabolism, including hexokinase 2, sorbin and SH3 domain containing 1, and uncoupling protein 3. Interestingly, formoterol administration also appeared to influence some genes associated with the peripheral regulation of circadian rhythm (including nuclear factor interleukin 3 regulated, D site albumin promoter binding protein, and cryptochrome 2). CONCLUSION: This is the first study to utilize gene expression profiling to examine global gene expression in response to acute beta(2)-AR agonist treatment of skeletal muscle. In summary, systemic administration of a beta(2)-AR agonist had a profound effect on global gene expression in skeletal muscle. In terms of hypertrophy, beta(2)-AR agonist treatment altered the expression of several genes associated with myostatin signaling, a previously unreported effect of beta-AR signaling in skeletal muscle. This study also demonstrates a beta(2)-AR agonist regulation of circadian rhythm genes, indicating crosstalk between beta-AR signaling and circadian cycling in skeletal muscle. Gene expression alterations discovered in this study provides insight into many of the underlying changes in gene expression that mediate beta-AR induced skeletal muscle hypertrophy and altered metabolism.

The orphan nuclear receptor, NOR-1, a target of beta-adrenergic signaling, regulates gene expression that controls oxidative metabolism in skeletal muscle.

beta 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 beta2-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 beta2-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 beta-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-mediated) inhibition of oxidative metabolism and expressed significantly higher levels of hypoxia inducible factor-1alpha. In addition, we observed the repression of genes that promote fatty acid oxidation (peroxisomal proliferator-activated receptor-gamma coactivator-1alpha/beta and lipin-1alpha) and trichloroacetic acid cycle-mediated carbohydrate (pyruvate) oxidation [pyruvate dehydrogenase phosphatase 1 regulatory and catalytic subunits (pyruvate dehydrogenase phosphatases-1r and -c)]. Furthermore, we observed that beta2-AR agonist administration in mouse skeletal muscle induced the expression of genes that activate fatty acid oxidation and modulate pyruvate use, including PGC-1alpha, lipin-1alpha, FOXO1, and PDK4. Finally, we demonstrate that NOR-1 is recruited to the lipin-1alpha 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.

The orphan nuclear receptor, NOR-1, is a target of beta-adrenergic signaling in skeletal muscle.

beta-Adrenergic receptor (beta-AR) agonists induce Nur77 mRNA expression in the C2C12 skeletal muscle cell culture model and elicit skeletal muscle hypertrophy. We previously demonstrated that Nur77 (NR4A1) is involved in lipolysis and gene expression associated with the regulation of lipid homeostasis. Subsequently it was demonstrated by another group that beta-AR agonists and cold exposure-induced Nur77 expression in brown adipocytes and brown adipose tissue, respectively. Moreover, NOR-1 (NR4A3) was hyperinduced by cold exposure in the nur77(-/-) animal model. These studies underscored the importance of understanding the role of NOR-1 in skeletal muscle. In this context we observed 30-480 min of beta-AR agonist treatment significantly and transiently increased expression of the orphan nuclear receptor NOR-1 in both mouse skeletal muscle tissue (plantaris) and C2C12 skeletal muscle cells. Specific beta(2)- and beta(3)-AR agonists had similar effects as the pan-agonist and were blocked by the beta-AR antagonist propranolol. Moreover, in agreement with these observations, isoprenaline also significantly increased the activity of the NOR-1 promoter. Stable exogenous expression of a NOR-1 small interfering RNA (but not the negative control small interfering RNA) in skeletal muscle cells significantly repressed endogenous NOR-1 mRNA expression and led to changes in the expression of genes involved in the control of lipid use and muscle mass underscored by a dramatic increase in myostatin mRNA expression. Concordantly the myostatin promoter was repressed by NOR-1 expression. In conclusion, NOR-1 is highly responsive to beta-adrenergic signaling and regulates the expression of genes controlling fatty acid use and muscle mass.