Rebecca L. Fitzsimmons

The Orphan Nuclear Receptor, RORα, Regulates Gene Expression That Controls Lipid Metabolism STAGGERER (SG/SG) MICE ARE RESISTANT TO DIET-INDUCED OBESITY

Homozygous staggerer mice (sg/sg) display decreased and dysfunctional retinoic acid receptor-related orphan receptor alpha (RORalpha) expression. We observed decreases in serum (and liver) triglycerides and total and high density lipoprotein serum cholesterol in sg/sg mice. Moreover, the sg/sg mice were characterized by reduced adiposity (associated with decreased fat pad mass and adipocyte size). Candidate-based expression profiling demonstrated that the dyslipidemia in sg/sg mice is associated with decreased hepatic expression of SREBP-1c, and the reverse cholesterol transporters, ABCA1 and ABCG1. This is consistent with the reduced serum lipids. The molecular mechanism did not involve aberrant expression of LXR and/or ChREBP. However, ChIP and transfection analyses revealed that RORalpha is recruited to and regulates the activity of the SREBP-1c promoter. Furthermore, the lean phenotype in sg/sg mice is also characterized by significantly increased expression of PGC-1alpha, PGC-1beta, and lipin1 mRNA in liver and white and brown adipose tissue from sg/sg mice. In addition, we observed a significant 4-fold increase in beta(2)-adrenergic receptor mRNA in brown adipose tissue. Finally, dysfunctional RORalpha expression protects against diet-induced obesity. Following a 10-week high fat diet, wild-type but not sg/sg mice exhibited a approximately 20% weight gain, increased hepatic triglycerides, and notable white and brown adipose tissue accumulation. In summary, these changes in gene expression (that modulate lipid homeostasis) in metabolic tissues are involved in decreased adiposity and resistance to diet-induced obesity in the sg/sg mice, despite hyperphagia. In conclusion, we suggest this orphan nuclear receptor is a key modulator of fat accumulation and that selective ROR modulators may have utility in the treatment of obesity.Homozygous staggerer mice (sg/sg) display decreased and dysfunctional retinoic acid receptor-related orphan receptor α (RORα) expression. We observed decreases in serum (and liver) triglycerides and total and high density lipoprotein serum cholesterol in sg/sg mice. Moreover, the sg/sg mice were characterized by reduced adiposity (associated with decreased fat pad mass and adipocyte size). Candidate-based expression profiling demonstrated that the dyslipidemia in sg/sg mice is associated with decreased hepatic expression of SREBP-1c, and the reverse cholesterol transporters, ABCA1 and ABCG1. This is consistent with the reduced serum lipids. The molecular mechanism did not involve aberrant expression of LXR and/or ChREBP. However, ChIP and transfection analyses revealed that RORα is recruited to and regulates the activity of the SREBP-1c promoter. Furthermore, the lean phenotype in sg/sg mice is also characterized by significantly increased expression of PGC-1α, PGC-1β, and lipin1 mRNA in liver and white and brown adipose tissue from sg/sg mice. In addition, we observed a significant 4-fold increase in β2-adrenergic receptor mRNA in brown adipose tissue. Finally, dysfunctional RORα expression protects against diet-induced obesity. Following a 10-week high fat diet, wild-type but not sg/sg mice exhibited a ∼20% weight gain, increased hepatic triglycerides, and notable white and brown adipose tissue accumulation. In summary, these changes in gene expression (that modulate lipid homeostasis) in metabolic tissues are involved in decreased adiposity and resistance to diet-induced obesity in the sg/sg mice, despite hyperphagia. In conclusion, we suggest this orphan nuclear receptor is a key modulator of fat accumulation and that selective ROR modulators may have utility in the treatment of obesity.

CD36/Fatty Acid Translocase in Rats: Distribution, Isolation from Hepatocytes, and Comparison with the Scavenger Receptor SR-B1

The new mAb UA009 recognizes an antigen expressed by microvascular endothelium, by lymphatic endothelium, and by some epithelia in a number of organs, including the small intestine, lactating mammary gland, kidney, lung, sebaceous glands, and circumvallate papillae of the tongue. This antigen is also expressed abundantly in the splenic red pulp and marginal zone and by monocytes, macrophages, and erythrocytes (but not by platelets). Among tissues that store or metabolize fatty acids, the antigen is expressed by adipocytes, cardiomyocytes, and red skeletal muscle. Importantly, it is expressed by steroidogenic cells in the adrenal gland, testis, and ovary, whereas in the liver it is expressed by hepatocytes in a pattern that is dependent on gender and genetic background. mAb UA009 immunoprecipitated a mol wt 85-kDa surface protein from detergent extracts of hepatocytes from Dark Agouti female rats. The N-terminal amino acid sequence of this protein was identical to fatty acid translocase (FAT), the rat cluster of differentiation 36 (CD36) ortholog. The mAb also reacted with COS-7 cells transfected with cDNA encoding FAT. cDNAs encoding a CD36/FAT-like polypeptide were prepared from both liver and heart RNA by RT-PCR. The nucleotide sequences obtained from these cDNAs (Dark Agouti rats) revealed identity and 99% similarity, respectively, with the published sequences of Cd36/Fat in rats of the Wistar and Sprague-Dawley strains. The absence of the UA009 antigen in CD36/FAT-deficient SHR/N rats confirmed the identity of the UA009 antigen and CD36/FAT. We suggest that CD36/FAT might function in the liver as a sex-regulated accessory molecule, either in reverse cholesterol transport and/or in fatty acid uptake.

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.

Homozygous staggerer (sg/sg) mice display improved insulin sensitivity and enhanced glucose uptake in skeletal muscle

Aims/hypothesisHomozygous staggerer (sg/sg) mice, which have decreased and dysfunctional Rorα (also known as Rora) expression in all tissues, display a lean and dyslipidaemic phenotype. They are also resistant to (high fat) diet-induced obesity. We explored whether retinoic acid receptor-related orphan receptor (ROR) α action in skeletal muscle was involved in the regulation of glucose metabolism.MethodsWe used a three-armed genomic approach, including expression profiling, ingenuity analysis and quantitative PCR validation to identify the signalling pathway(s) in skeletal muscle that are perturbed in sg/sg mice. Moreover, western analysis, functional insulin and glucose tolerance tests, and ex vivo glucose uptake assays were used to phenotypically characterise the impact of aberrant v-AKT murine thymoma viral oncogene homologue (AKT) signalling.ResultsHomozygous and heterozygous (sg/sg and sg/+) animals exhibited decreased fasting blood glucose levels, mildly improved glucose tolerance and increased insulin sensitivity. Illumina expression profiling and bioinformatic analysis indicated the involvement of RORα in metabolic disease and phosphatidylinositol 3-kinase–AKT signalling. Quantitative PCR and western analysis validated increased AKT2 (mRNA and protein) and phosphorylation in sg/sg mice in the basal state. This was associated with increased expression of Tbc1d1 and Glut4 (also known as Slc2a4) mRNA and protein. Finally, in agreement with the phenotype, we observed increased (absolute) levels of AKT and phosphorylated AKT (in the basal and insulin stimulated states), and of (ex vivo) glucose uptake in skeletal muscle from sg/sg mice relative to wild-type littermates.Conclusions/interpretationWe propose that Rorα plays an important role in regulation of the AKT2 signalling cascade, which controls glucose uptake in skeletal muscle.

Identification and validation of the pathways and functions regulated by the orphan nuclear receptor, ROR alpha1, in skeletal muscle

The retinoic acid receptor-related orphan receptor (ROR) alpha has been demonstrated to regulate lipid metabolism. We were interested in the RORα1 dependent physiological functions in skeletal muscle. This major mass organ accounts for ∼40% of the total body mass and significant levels of lipid catabolism, glucose disposal and energy expenditure. We utilized the strategy of targeted muscle-specific expression of a truncated (dominant negative) RORα1ΔDE in transgenic mice to investigate RORα1 signaling in this tissue. Expression profiling and pathway analysis indicated that RORα influenced genes involved in: (i) lipid and carbohydrate metabolism, cardiovascular and metabolic disease; (ii) LXR nuclear receptor signaling and (iii) Akt and AMPK signaling. This analysis was validated by quantitative PCR analysis using TaqMan low-density arrays, coupled to statistical analysis (with Empirical Bayes and Benjamini–Hochberg). Moreover, westerns and metabolic profiling were utilized to validate the genes, proteins and pathways (lipogenic, Akt, AMPK and fatty acid oxidation) involved in the regulation of metabolism by RORα1. The identified genes and pathways were in concordance with the demonstration of hyperglycemia, glucose intolerance, attenuated insulin-stimulated phosphorylation of Akt and impaired glucose uptake in the transgenic heterozygous Tg-RORα1ΔDE animals. In conclusion, we propose that RORα1 is involved in regulating the Akt2-AMPK signaling pathways in the context of lipid homeostasis in skeletal muscle.

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 β2-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.

Nr4a1 siRNA Expression Attenuates α-MSH Regulated Gene Expression in 3T3-L1 Adipocytes

Several recent investigations have underscored the growing role of melanocortin signaling in the peripheral regulation of lipid, glucose, and energy homeostasis. In addition, the melanocortins play a critical role in the central control of satiety. These observations, and the latest reports highlighting the emerging role of the nuclear hormone receptor (NR) 4A subgroup in metabolism, have prompted us to investigate the cross talk between [Nle(4), d-Phe(7)] (NDP)-α-MSH and Nr4a signaling in adipose. We have shown that NDP-MSH strikingly and preferentially induces the expression of the NR4A subgroup (but not any other members of the NR superfamily) in differentiated 3T3-L1 adipocytes. Utilization of quantitative PCR on custom-designed metabolic TaqMan low-density arrays identified the concomitant and marked induction of the mRNAs encoding Il-6, Cox2, Pdk4, and Pck-1 after NDP-MSH treatment. Similar experiments demonstrated that the mRNA expression profile induced by cAMP and NDP-MSH treatment displayed unique but also overlapping properties and suggested that melanocortin-mediated induction of gene expression involves cAMP-dependent and -independent signaling. Nr4a1/Nur77 small interfering RNA (siRNA) expression suppressed NDP-MSH-mediated induction of Nr4a1/Nur77 and Nr4a3/Nor-1 (but not Nr4a2/Nurr1). Moreover, expression of the siRNA-attenuated NDP-MSH mediated induction of the mRNAs encoding Il-6, Cox2/Ptgs2, and Pck-1 expression. In addition, Nur77 siRNA expression attenuated NDP-MSH-mediated glucose uptake. In vivo, ip administration of NDP-MSH to C57 BL/6J (male) mice significantly induced the expression of the mRNA encoding Nur77 and increased IL-6, Cox2, Pck1, and Pdk4 mRNA expression in (inguinal) adipose tissue. We conclude that Nur77 expression is necessary for MSH-mediated induction of gene expression in differentiated adipocytes. Furthermore, this study demonstrates cross talk between MSH and Nr4a signaling in adipocytes.

Retinoid-related orphan receptor alpha and the regulation of lipid homeostasis

Many nuclear hormone receptors (NRs) control lipid, glucose and energy homeostasis in an organ specific manner. Concordantly, dysfunctional NR signalling results in metabolic disease. The Retinoic acid receptor-related orphan receptor alpha (RORα), a member of the NR1F subgroup, is expressed in metabolic tissues. Previous studies identified the role of this NR in dyslipidemia, apo-lipoprotein metabolism and atherosclerosis. Recent data is underscoring the significant role of this orphan NR in the regulation of phase I/II metabolism (bile acids, xenobiotics, steroids etc.), adiposity, insulin signalling, and glucose tolerance. Moreover, oxygenated sterols, have been demonstrated to function as native ligands and inverse agonists. This review focuses on the rapidly emerging and evolving role of RORα in the control of lipid and glucose homeostasis in major mass metabolic tissues. Article from the special issue orphan receptors.

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, Cpt1β, 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.

Ski Overexpression in Skeletal Muscle Modulates Genetic Programs That Control Susceptibility to Diet-Induced Obesity and Insulin Signaling

Transgenic mice overexpressing chicken Ski (c‐Ski) have marked decrease in adipose mass with skeletal muscle hypertrophy. Recent evidence indicates a role for c‐Ski in lipogenesis and energy expenditure. In the present study, wild type (WT) and c‐Ski mice were challenged on a high‐fat (HF) diet to determine whether c‐Ski mice were resistant to diet‐induced obesity. During the HF feeding WT mice gained significantly more weight than chow‐fed animals, while c‐Ski mice were partially resistant to the effects of the HF diet on weight. Body composition analysis confirmed the decreased adipose mass in c‐Ski mice compared to WT mice. c‐Ski mice possess a similar metabolic rate and level of food consumption to WT littermates, despite lower activity levels and on chow diet show mild glucose intolerance relative to WT littermates. On HF diet, glucose tolerance surprisingly remained unchanged in c‐Ski mice, while it became worse in WT mice. Skeletal muscle of c‐Ski mice exhibit impaired insulin‐stimulated Akt phosphorylation and glucose uptake. In concordance, gene expression profiling of skeletal muscle of chow and HF‐fed mice indicated that Ski suppresses gene expression associated with insulin signaling and glucose uptake and alters gene pathways involved in myogenesis and adipogenesis. In conclusion, c‐Ski mice are partially resistant to diet‐induced obesity and display aberrant insulin signaling and glucose homeostasis which is associated with alterations in gene expression that inhibit lipogenesis and insulin signaling. These results suggest Ski plays a major role in skeletal muscle metabolism and adipogenesis and hence influences risk of obesity and diabetes.