RORα and 25-Hydroxycholesterol Crosstalk Regulates Lipid Droplet Homeostasis in Macrophages.

Nuclear hormone receptors have important roles in the regulation of metabolic and inflammatory pathways. The retinoid-related orphan receptor alpha (Rorα)-deficient staggerer (sg/sg) mice display several phenotypes indicative of aberrant lipid metabolism, including dyslipidemia, and increased susceptibility to atherosclerosis. In this study we demonstrate that macrophages from sg/sg mice have increased ability to accumulate lipids and accordingly exhibit larger lipid droplets (LD). We have previously shown that BMMs from sg/sg mice have significantly decreased expression of cholesterol 25-hydroxylase (Ch25h) mRNA, the enzyme that produces the oxysterol, 25-hydroxycholesterol (25HC), and now confirm this at the protein level. 25HC functions as an inverse agonist for RORα. siRNA knockdown of Ch25h in macrophages up-regulates Vldlr mRNA expression and causes increased accumulation of LDs. Treatment with physiological concentrations of 25HC in sg/sg macrophages restored lipid accumulation back to normal levels. Thus, 25HC and RORα signify a new pathway involved in the regulation of lipid homeostasis in macrophages, potentially via increased uptake of lipid which is suggested by mRNA expression changes in Vldlr and other related genes

The nuclear receptor, Nor-1, induces the physiological responses associated with excercise

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

The Nuclear Receptor, RORγ, Regulates Pathways Necessary for Breast Cancer Metastasis

We have previously reported that RORγ expression was decreased in ER − ve breast cancer, and increased expression improves clinical outcomes. However, the underlying RORγ dependent mechanisms that repress breast carcinogenesis have not been elucidated. Here we report that RORγ negatively regulates the oncogenic TGF-β/EMT and mammary stem cell (MaSC) pathways, whereas RORγ positively regulates DNA-repair. We demonstrate that RORγ expression is: (i) decreased in basal-like subtype cancers, and (ii) inversely correlated with histological grade and drivers of carcinogenesis in breast cancer cohorts. Furthermore, integration of RNA-seq and ChIP-chip data reveals that RORγ regulates the expression of many genes involved in TGF-β/EMT-signaling, DNA-repair and MaSC pathways (including the non-coding RNA, LINC00511). In accordance, pharmacological studies demonstrate that an RORγ agonist suppresses breast cancer cell viability, migration, the EMT transition (microsphere outgrowth) and mammosphere-growth. In contrast, RNA-seq demonstrates an RORγ inverse agonist induces TGF-β/EMT-signaling. These findings suggest pharmacological modulation of RORγ activity may have utility in breast cancer

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 beta(2)-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, alpha-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 gamma coactivator-1 alpha 1 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

Ror-alpha deficiency and decreased adiposity are associated with induction of thermogenic gene expression in subcutaneous white and brown adipose tissue

The Rarrelated orphan receptor-alpha (Ror alpha) is a nuclear receptor that regulates adiposity and is a potential regulator of energy homeostasis. We have demonstrated that the Ror alpha-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 Ror alpha-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) Ppar alpha, Err alpha, Dio2, Acot11/Bfit, Cpt1 beta, 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 litter-mates. We suggest that enhanced Ucp1 and thermogenic gene expression/activity may be an important contributor to the lean, obesity-resistant phenotype in Ror alpha-deficient mice

Assessment of lipid uptake in WT and <i>sg/sg</i> BMMs.

<p>(a) TLC was performed on lipid extracted from WT and <i>sg/sg</i> BMMs (n = 3 littermate pairs). Lanes 2–4 and 5–7 correspond to untreated WT and <i>sg/sg</i> samples respectively while lanes 8–10 and 11–13 correspond to 4 h 400 μM oleic acid-treated WT and <i>sg/sg</i> samples respectively. Lanes 1 and 14 are ladder standards. Quantification was performed by normalizing the relative density of triglyceride bands in each lane to WT untreated controls (arbitrarily set as 1.0) and is expressed as the mean ± S.E.M. relative fold-difference (n = 3 littermate pairs). Statistical analyses were performed using two-way ANOVAs with Bonferroni post test applied where *P<0.05. (b) Representative images of WT and <i>sg/sg</i> BMMs with endocytosed fluorescent-acLDL were captured using the Olympus upright wide-field epifluorescence microscope with actin (phalloidin; white) and nucleus (DAPI; blue) labeling, and fluorescent-acLDL (red). Quantification of fluorescent-acLDL uptake is expressed as the mean ± S.E.M. fluorescence intensity (integrated density) normalized to number of cells analyzed (n = ~200 cells per mouse) from n = 4 biological replicates (littermate pairs). Relative fold-difference is calculated with the mean of WT values set as 1.0 and is available on the right Y-axis. Statistical analysis was performed using an unpaired Student’s t-test where **P<0.01. Scale bars = 20 μm. (c) Electron microscopy of LDs. Representative images of WT and <i>sg/sg</i> BMMs treated with 400 μM oleic acid overnight show clusters of LDs accumulated in the cytoplasm. And interspersed with endoplasmic reticulum. N = nucleus, scale bars = 1 μm. Quantification of LD size is expressed as a histogram of area/LD frequency distribution.</p