Patrick Lau

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.

Suppressor of cytokine signalling gene expression is elevated in breast carcinoma

Cytokines are important for breast cell function, both as trophic hormones and as mediators of host defense mechanisms against breast cancer. Recently, inducible feedback suppressors of cytokine signalling (SOCS/JAB/SSI) have been identified, which decrease cell sensitivity to cytokines. We examined the expression of SOCS genes in 17 breast carcinomas and 10 breast cancer lines, in comparison with normal tissue and breast lines. We report elevated expression of SOCS-1–3 and CIS immunoreactive proteins within in situ ductal carcinomas and infiltrating ductal carcinomas relative to normal breast tissue. Significantly increased expression of SOCS-1–3 and CIS transcripts was also shown by quantitative in situ hybridisation within both tumour tissue and reactive stroma. CIS transcript expression was elevated in all 10 cancer lines, but not in control lines. However, there was no consistent elevation of other SOCS transcripts. CIS protein was shown by immunoblot to be present in all cancer lines at increased levels, mainly as the 47 kDa ubiquitinylated form. A potential proliferative role for CIS overexpression is supported by reports that CIS activates ERK kinases, and by strong induction in transient reporter assays with an ERK-responsive promoter. The in vivo elevation of SOCS gene expression may be part of the host/tumour response or a response to autocrine/paracrine GH and prolactin. However, increased CIS expression in breast cancer lines appears to be a specific lesion, and could simultaneously shut down STAT 5 signalling by trophic hormones, confer resistance to host cytokines and increase proliferation through ERK kinases.

Rev-erbβ Regulates the Expression of Genes Involved in Lipid Absorption in Skeletal Muscle Cells EVIDENCE FOR CROSS-TALK BETWEEN ORPHAN NUCLEAR RECEPTORS AND MYOKINES

Rev-erbβ is an orphan nuclear receptor that selectively blocks trans-activation mediated by the retinoic acid-related orphan receptor-α (RORα). RORα has been implicated in the regulation of high density lipoprotein cholesterol, lipid homeostasis, and inflammation. Reverbβ and RORα are expressed in similar tissues, including skeletal muscle; however, the pathophysiological function of Rev-erbβ has remained obscure. We hypothesize from the similar expression patterns, target genes, and overlapping cognate sequences of these nuclear receptors that Rev-erbβ 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-erbβ in this major mass peripheral tissue. Exogenous expression of a dominant negative version of mouse Rev-erbβ decreases the expression of many genes involved in fatty acid/lipid absorption (including Cd36, and Fabp-3 and -4). Interestingly, we observed a robust induction (>15-fold) in mRNA expression of interleukin-6, an “exercise-induced myokine” that regulates energy expenditure and inflammation. Furthermore, we observed the dramatic repression (>20-fold) of myostatin mRNA, another myokine that is a negative regulator of muscle hypertrophy and hyperplasia that impacts on body fat accumulation. This study implicates Rev-erbβ in the control of lipid and energy homoeostasis in skeletal muscle. In conclusion, we speculate that selective modulators of Rev-erbβ may have therapeutic utility in the treatment of dyslipidemia and regulation of muscle growth.

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.

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.

Rev-erb beta regulates the Srebp-1c promoter and mRNA expression in skeletal muscle cells

The nuclear hormone receptor, Rev-erb beta operates as a transcriptional silencer. We previously demonstrated that exogenous expression of Rev-erb betaDeltaE in skeletal muscle cells increased Srebp-1c mRNA expression. We validated these in vitro observations by injection of an expression vector driving Rev-erb betaDeltaE expression into mouse tibialis muscle that resulted in increased Srebp-1c mRNA expression. Paradoxically, Rev-erb beta siRNA expression in skeletal muscle cells repressed Srebp-1c expression, and indicated that Rev-erb beta expression was necessary for Srebp-1c expression. ChIP analysis demonstrated that Rev-erb beta was recruited to the Srebp-1c promoter. Moreover, Rev-erb beta trans-activated the Srebp-1c promoter, in contrast, Rev-erb beta efficiently repressed the Rev-erb alpha promoter, a previously characterized target gene. Finally, treatment with the Rev-erb agonist (hemin) (i) increased the trans-activation of the Srebp-1c promoter by Rev-erb beta; and (ii) increased Rev-erb beta and Srebp-1c mRNA expression. These data suggest that Rev-erb beta has the potential to activate gene expression, and is a positive regulator of Srebp-1c, a regulator of lipogenesis.

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.

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.