Genome-wide coexpression of steroid receptors in the mouse brain: Identifying signaling pathways and functionally coordinated regions.

Steroid receptors are pleiotropic transcription factors that coordinate adaptation to different physiological states. An important target organ is the brain, but even though their effects are well studied in specific regions, brain-wide steroid receptor targets and mediators remain largely unknown due to the complexity of the brain. Here, we tested the idea that novel aspects of steroid action can be identified through spatial correlation of steroid receptors with genome-wide mRNA expression across different regions in the mouse brain. First, we observed significant coexpression of six nuclear receptors (NRs) [androgen receptor (Ar), estrogen receptor alpha (Esr1), estrogen receptor beta (Esr2), glucocorticoid receptor (Gr), mineralocorticoid receptor (Mr), and progesterone receptor (Pgr)] with sets of steroid target genes that were identified in single brain regions. These coexpression relationships were also present in distinct other brain regions, suggestive of as yet unidentified coordinate regulation of brain regions by, for example, glucocorticoids and estrogens. Second, coexpression of a set of 62 known NR coregulators and the six steroid receptors in 12 nonoverlapping mouse brain regions revealed selective downstream pathways, such as Pak6 as a mediator for the effects of Ar and Gr on dopaminergic transmission. Third, Magel2 and Irs4 were identified and validated as strongly responsive targets to the estrogen diethylstilbestrol in the mouse hypothalamus. The brain- and genome-wide correlations of mRNA expression levels of six steroid receptors that we provide constitute a rich resource for further predictions and understanding of brain modulation by steroid hormones.

Mechanistic Insights in NeuroD Potentiation of Mineralocorticoid Receptor Signaling.

Mineralocorticoid receptor (MR)-mediated signaling in the brain has been suggested as a protective factor in the development of psychopathology, in particular mood disorders. We recently identified genomic loci at which either MR or the closely related glucocorticoid receptor (GR) binds selectively, and found members of the NeuroD transcription factor family to be specifically associated with MR-bound DNA in the rat hippocampus. We show here using forebrain-specific MR knockout mice that GR binding to MR/GR joint target loci is not affected in any major way in the absence of MR. Neurod2 binding was also independent of MR binding. Moreover, functional comparison with MyoD family members indicates that it is the chromatin remodeling aspect of NeuroD, rather than its direct stimulation of transcription, that is responsible for potentiation of MR-mediated transcription. These findings suggest that NeuroD acts in a permissive way to enhance MR-mediated transcription, and they argue against competition for DNA binding as a mechanism of MR- over GR-specific binding.

Quercetin Lowers Plasma Triglycerides Accompanied by White Adipose Tissue Browning in Diet-Induced Obese Mice.

Obesity and dyslipidemia are major risk factors for the development of cardiovascular diseases (CVD). Quercetin, a natural flavonoid, lowers plasma triglycerides (TG) in human intervention studies, and its intake is associated with lower CVD risk. The aim of this study was to elucidate the mechanism by which quercetin lowers plasma TG levels in diet-induced obesity. C57Bl/6J mice received a high-fat diet (45% of calories derived from fat) with or without quercetin (0.1% w/w) for 12 weeks. Quercetin decreased plasma TG levels from nine weeks onwards (−19%, p < 0.05), without affecting food intake, body composition, or energy expenditure. Mechanistically, quercetin did not reduce intestinal fatty acid (FA) absorption. Rather, quercetin induced a slight reduction in liver Apob expression (−13%, p < 0.05), which suggests decreased very-low density lipoprotein-TG production. Interestingly, quercetin also markedly increased the uptake of [³H]oleate, which was derived from glycerol tri[³H]oleate-labeled lipoprotein-like particles by subcutaneous white adipose tissue (sWAT, +60%, p < 0.05). Furthermore, quercetin also markedly increased mRNA expression of Ucp1 (+229%, p < 0.05) and Elovl3 (+138%, p < 0.05), specifically in sWAT. Accordingly, only quercetin-treated animals showed uncoupling protein-1 protein-positive cells in sWAT, which is fully compatible with increased browning. Taken together, the TG-lowering effect of quercetin may, at least in part, be due to increased TG-derived FA uptake by sWAT as a consequence of browning.

Validation of a simple and fast method to quantify in vitro mineralization with fluorescent probes used in molecular imaging of bone.

Alizarin Red S staining is the standard method to indicate and quantify matrix mineralization during differentiation of osteoblast cultures. KS483 cells are multipotent mouse mesenchymal progenitor cells that can differentiate into chondrocytes, adipocytes and osteoblasts and are a well-characterized model for the study of bone formation. Matrix mineralization is the last step of differentiation of bone cells and is therefore a very important outcome measure in bone research. Fluorescently labelled calcium chelating agents, e.g. BoneTag and OsteoSense, are currently used for in vivo imaging of bone. The aim of the present study was to validate these probes for fast and simple detection and quantification of in vitro matrix mineralization by KS483 cells and thus enabling high-throughput screening experiments. KS483 cells were cultured under osteogenic conditions in the presence of compounds that either stimulate or inhibit osteoblast differentiation and thereby matrix mineralization. After 21 days of differentiation, fluorescence of stained cultures was quantified with a near-infrared imager and compared to Alizarin Red S quantification. Fluorescence of both probes closely correlated to Alizarin Red S staining in both inhibiting and stimulating conditions. In addition, both compounds displayed specificity for mineralized nodules. We therefore conclude that this method of quantification of bone mineralization using fluorescent compounds is a good alternative for the Alizarin Red S staining.

Dual wavelength tumor targeting for detection of hypopharyngeal cancer using near-infrared optical imaging in an animal model.

Optical imaging is a promising technique to visualize cancer tissue during surgery. In this study, we explored the use of combinations of near-infrared (NIR) fluorescence agents that emit fluorescence signal at different wavelengths and each target specific tumor characteristics. Two combinations of agents (ProSense680 combined with 2DG CW800 and MMPSense680 combined with EGF CW800) were used to detect hypopharyngeal cancer in an animal model. ProSense680 and MMPSense680 detect increased activity of cathepsins and matrix metalloproteinases, respectively. These enzymes are mainly found in the invasive tumor border due to degradation of the extracellular matrix. 2DG CW800 detects tumor cells with high glucose metabolism and EGF CW800 is internalized by the epidermal growth factor receptor of tumor cells. Whole-body imaging revealed clear demarcation of tumor tissue using all four agents. The tumor-to-background ratio (standard deviation, p-value) was 3.69 (0.72, p < 0.001) for ProSense680; 4.26 (1.33, p < 0.001) for MMPSense680; 5.81 (3.59, p = 0.02) for 2DG CW800 and 4.84 (1.56, p < 0.001) for EGF CW800. Fluorescence signal corresponded with histopathology and immunohistochemistry, demonstrating signal of ProSense680 and MMPSense680 in the invasive tumor border, and signal of 2DG CW800 and EGF CW800 in the tumor tissue. In conclusion, we demonstrated the feasibility of dual wavelength tumor detection using different targeting strategies simultaneously in an animal model. Combined targeting at different wavelengths allowed simultaneous imaging of different tumor characteristics. NIR fluorescence optical imaging has the potential to be translated into the clinic in order to improve the complete removal of tumors by real-time image-guided surgery.

Targeting integrins and enhanced permeability and retention (EPR) effect for optical imaging of oral cancer.

BACKGROUND AND OBJECTIVES: Near-infrared (NIR) fluorescence optical imaging is a promising technique to assess the tumor margins during cancer surgery. This technique requires targeting by specific fluorescence agents to differentiate tumor from normal surrounding tissue. We assessed the feasibility of cancer detection using NIR fluorescence agents that target either αvß3 integrins or the enhanced permeability and retention (EPR) effect in an orthotopic mouse model of oral cancer. METHODS: Binding of the integrin-targeted agent to tumor cells was assessed in vitro. Oral cancer was induced in 6 BALB/c nu/nu mice by submucosal inoculation of human OSC19-luc cells into the tongue. Tumor growth was followed with bioluminescence imaging. A combination of agents targeting integrins or EPR effect was injected followed by fluorescence imaging in vivo and ex vivo after resection of the tongues. RESULTS: Oral cancer was clearly demarcated in vitro; in vivo; and on histological analysis with sufficient tumor-to-background ratios of the contrast agents. CONCLUSION: This study demonstrates the feasibility of optical imaging of oral squamous cell carcinoma based on targeting of αvß3 integrins and the EPR effect. Once these NIR fluorescence agents become available for clinical testing, optical image-guided surgery could reduce residual disease after oral cancer surgery.

A physiological glucocorticoid rhythm is an important regulator of brown adipose tissue function.

OBJECTIVE: Brown adipose tissue (BAT) displays a strong circadian rhythm in metabolic activity, but it is unclear how this rhythm is regulated. As circulating levels of corticosterone coincide with the rhythm of triglyceride-derived fatty acid (FA) uptake by BAT, we investigated whether corticosterone regulates BAT circadian rhythm. METHODS: Corticosterone levels were flattened by implanting mice with subcutaneous corticosterone-releasing pellets, resulting in constant circulating corticosterone levels. RESULTS: Flattened corticosterone rhythm caused a complete loss of circadian rhythm in triglyceride-derived fatty acid uptake by BAT. This effect was independent of glucocorticoid receptor expression in (brown) adipocytes and was not caused by deregulation of clock gene expression or overexposure to glucocorticoids, but rather seemed mediated by reduced sympathetic innervation of BAT. In a mouse model of hyperlipidemia and metabolic syndrome, long-term experimental flattening of corticosterone - and thus rhythm in BAT function - resulted in adiposity. CONCLUSIONS: This study highlights that a physiological rhythm in glucocorticoids is an important regulator of BAT function and essential for the maintenance of metabolic health.

PXR agonism decreases plasma HDL levels in ApoE3-Leiden.CETP mice.

Pregnane X receptor (PXR) agonism has been shown to affect multiple steps in both the synthesis and catabolism of HDL, but its integrated effect on HDL metabolism in vivo remains unclear. The aim of this study was to evaluate the net effect of PXR agonism on HDL metabolism in ApoE3-Leiden (E3L) and E3L.CETP mice, well-established models for human-like lipoprotein metabolism. Female mice were fed a diet with increasing amounts of the potent PXR agonist 5-pregnen-3beta-ol-20-one-16alpha-carbonitrile (PCN). In E3L and E3L.CETP mice, PCN increased liver lipids as well as plasma cholesterol and triglycerides. However, whereas PCN increased cholesterol contained in large HDL-1 particles in E3L mice, it dose-dependently decreased HDL-cholesterol in E3L.CETP mice, indicating that CETP expression dominates the effect of PCN on HDL metabolism. Analysis of the hepatic expression of genes involved in HDL metabolism showed that PCN decreased expression of genes involved in HDL synthesis (Abca1, Apoa1), maturation (Lcat, Pltp) and clearance (Sr-b1). The HDL-increasing effect of PCN, observed in E3L mice, is likely caused by a marked decrease in hepatic SR-BI protein expression, and completely reversed by CETP expression. We conclude that chronic PXR agonism dose-dependently reduces plasma HDL-cholesterol in the presence of CETP.

Deletion of haematopoietic Dectin-2 or CARD9 does not protect from atherosclerosis development under hyperglycaemic conditions.

BACKGROUND: C-type lectin receptors, including Dectin-2, are pattern recognition receptors on monocytes and macrophages that mainly recognize sugars and sugar-like structures present on fungi. Activation of C-type lectin receptors induces downstream CARD9 signalling, leading to the production of cytokines. We hypothesized that under hyperglycaemic conditions, as is the case in diabetes mellitus, glycosylated protein (sugar-like) structures activate C-type lectin receptors, leading to immune cell activation and increased atherosclerosis development. METHODS: Low-density lipoprotein receptor-deficient mice were lethally irradiated and transplanted with bone marrow from control wild-type, Dectin-2-/- or Card9-/- mice. After 6 weeks of recovery, mice received streptozotocin injections (50 mg/g BW; 5 days) to induce hyperglycaemia. After an additional 2 weeks, mice were fed a Western-type diet (0.1% cholesterol) for 10 weeks. RESULTS AND CONCLUSION: Deletion of haematopoietic Dectin-2 reduced the number of circulating Ly6Chi monocytes, increased pro-inflammatory cytokine production, but did not affect atherosclerosis development. Deletion of haematopoietic CARD9 tended to reduce macrophage and collagen content in atherosclerotic lesions, again without influencing the lesion size. Deletion of haematopoietic Dectin-2 did not influence atherosclerosis development under hyperglycaemic conditions, despite some minor effects on inflammation. Deletion of haematopoietic CARD9 induced minor alterations in plaque composition under hyperglycaemic conditions, without affecting lesion size.

Identification of mineralocorticoid receptor target genes in the mouse hippocampus.

Brain mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) respond to the same glucocorticoid hormones but can have differential effects on cellular function. Several lines of evidence suggest that MR-specific target genes must exist and might underlie the distinct effects of the receptors. The present study aimed to identify MR-specific target genes in the hippocampus, a brain region where MR and GR are co-localised and play a role in the stress response. Using genome-wide binding of both receptor types, we previously identified MR-specific, MR-GR overlapping and GR-specific putative target genes. We now report altered gene expression levels of such genes in the hippocampus of forebrain MR knockout (fbMRKO) mice, killed at the time of their endogenous corticosterone peak. Of those genes associated with MR-specific binding, the most robust effect was a 50% reduction in Jun dimerization protein 2 (Jdp2) mRNA levels in fbMRKO mice. Down-regulation was also observed for the MR-specific Nitric oxide synthase 1 adaptor protein (Nos1ap) and Suv3 like RNA helicase (Supv3 l1). Interestingly, the classical glucocorticoid target gene FK506 binding protein 5 (Fkbp5), which is associated with MR and GR chromatin binding, was expressed at substantially lower levels in fbMRKO mice. Subsequently, hippocampal Jdp2 was confirmed to be up-regulated in a restraint stress model, posing Jdp2 as a bona fide MR target that is also responsive in an acute stress condition. Thus, we show that MR-selective DNA binding can reveal functional regulation of genes and further identify distinct MR-specific effector pathways.

Deletion of hematopoietic Dectin-2 or CARD9 does not protect against atherosclerotic plaque formation in hyperlipidemic mice.

Inflammatory reactions activated by pattern recognition receptors (PRRs) on the membrane of innate immune cells play an important role in atherosclerosis. Whether the PRRs of the C-type lectin receptor (CLR) family including Dectin-2 may be involved in the pathogenesis of atherosclerosis remains largely unknown. Recently, the CLR-adaptor molecule caspase recruitment domain family member 9 (CARD9) has been suggested to play a role in cardiovascular pathologies as it provides the link between CLR activation and transcription of inflammatory cytokines as well as immune cell recruitment. We therefore evaluated whether hematopoietic deletion of Dectin-2 or CARD9 reduces inflammation and atherosclerosis development. Low-density lipoprotein receptor (Ldlr)-knockout mice were transplanted with bone marrow from wild-type, Dectin-2- or Card9-knockout mice and fed a Western-type diet containing 0.1% (w/w) cholesterol. After 10 weeks, lipid and inflammatory parameters were measured and atherosclerosis development was determined. Deletion of hematopoietic Dectin-2 or CARD9 did not influence plasma triglyceride and cholesterol levels. Deletion of hematopoietic Dectin-2 did not affect atherosclerotic lesion area, immune cell composition, ex vivo cytokine secretion by peritoneal cells or bone marrow derived macrophages. Unexpectedly, deletion of hematopoietic CARD9 increased atherosclerotic lesion formation and lesion severity. Deletion of hematopoietic CARD9 did also not influence circulating immune cell composition and peripheral cytokine secretion. Besides a tendency to a reduced macrophage content within these lesions, plasma MCP-1 levels decreased upon WTD feeding. Deletion of hematopoietic Dectin-2 did not influence atherosclerosis development in hyperlipidemic mice. The absence of CARD9 unexpectedly increased atherosclerotic lesion size and severity, suggesting that the presence of CARD9 may protect against initiation of atherosclerosis development.

Chronic infusion of taurolithocholate into the brain increases fat oxidation in mice.

Bile acids can function in the postprandial state as circulating signaling molecules in the regulation of glucose and lipid metabolism via the transmembrane receptor TGR5 and nuclear receptor FXR. Both receptors are present in the central nervous system, but their function in the brain is unclear. Therefore, we investigated the effects of intracerebroventricular (i.c.v.) administration of taurolithocholate (tLCA), a strong TGR5 agonist, and GW4064, a synthetic FXR agonist, on energy metabolism. We determined the effects of chronic i.c.v. infusion of tLCA, GW4064, or vehicle on energy expenditure, body weight and composition as well as tissue specific fatty acid uptake in mice equipped with osmotic minipumps. We found that i.c.v. administration of tLCA (final concentration in cerebrospinal fluid: 1 µM) increased fat oxidation (tLCA group: 0.083 ±â€…0.006 vs control group: 0.036 ±â€…0.023 kcal/h, F = 5.46, P = 0.04) and decreased fat mass (after 9 days of tLCA infusion: 1.35 ±â€…0.13 vs controls: 1.96 ±â€…0.23 g, P = 0.03). These changes were associated with enhanced uptake of triglyceride-derived fatty acids by brown adipose tissue and with browning of subcutaneous white adipose tissue. I.c.v. administration of GW4064 (final concentration in cerebrospinal fluid: 10 µM) did not affect energy metabolism, body composition nor bile acid levels, negating a role of FXR in the central nervous system in metabolic control. In conclusion, bile acids such as tLCA may exert metabolic effects on fat metabolism via the brain.

Selective Glucocorticoid Receptor Antagonist CORT125281 Activates Brown Adipose Tissue and Alters Lipid Distribution in Male Mice.

Glucocorticoids influence a wide range of metabolic processes in the human body, and excessive glucocorticoid exposure is known to contribute to the development of metabolic disease. We evaluated the utility of the novel glucocorticoid receptor (GR) antagonist CORT125281 for its potential to overcome adiposity, glucose intolerance, and dyslipidemia and compared this head-to-head with the classic GR antagonist RU486 (mifepristone). We show that, although RU486 displays cross-reactivity to the progesterone and androgen receptor, CORT125281 selectively inhibits GR transcriptional activity. In a mouse model for diet-induced obesity, rhythmicity of circulating corticosterone levels was disturbed. CORT125281 restored this disturbed rhythmicity, in contrast to RU486, which further inhibited endogenous corticosterone levels and suppressed adrenal weight. Both CORT125281 and RU486 reduced body weight gain and fat mass. In addition, CORT125281, but not RU486, lowered plasma levels of triglycerides, cholesterol, and free fatty acids and strongly stimulated triglyceride-derived fatty acid uptake by brown adipose tissue depots. In combination with reduced lipid content in brown adipocytes, this indicates that CORT125281 enhances metabolic activity of brown adipose tissue depots. CORT125281 was also found to increase liver lipid accumulation. Taken together, CORT125281 displayed a wide range of beneficial metabolic activities that are in part distinct from RU486, but clinical utility may be limited due to liver lipid accumulation. This warrants further evaluation of GR antagonists or selective modulators that are not accompanied by liver lipid accumulation while preserving their beneficial metabolic activities.

The GPR120 agonist TUG-891 promotes metabolic health by stimulating mitochondrial respiration in brown fat.

Brown adipose tissue (BAT) activation stimulates energy expenditure in human adults, which makes it an attractive target to combat obesity and related disorders. Recent studies demonstrated a role for G protein-coupled receptor 120 (GPR120) in BAT thermogenesis. Here, we investigated the therapeutic potential of GPR120 agonism and addressed GPR120-mediated signaling in BAT We found that activation of GPR120 by the selective agonist TUG-891 acutely increases fat oxidation and reduces body weight and fat mass in C57Bl/6J mice. These effects coincided with decreased brown adipocyte lipid content and increased nutrient uptake by BAT, confirming increased BAT activity. Consistent with these observations, GPR120 deficiency reduced expression of genes involved in nutrient handling in BAT Stimulation of brown adipocytes in vitro with TUG-891 acutely induced O2 consumption, through GPR120-dependent and GPR120-independent mechanisms. TUG-891 not only stimulated GPR120 signaling resulting in intracellular calcium release, mitochondrial depolarization, and mitochondrial fission, but also activated UCP1. Collectively, these data suggest that activation of brown adipocytes with the GPR120 agonist TUG-891 is a promising strategy to increase lipid combustion and reduce obesity.

Selective Glucocorticoid Receptor Modulation Prevents and Reverses Nonalcoholic Fatty Liver Disease in Male Mice.

Medication for nonalcoholic fatty liver disease (NAFLD) is an unmet need. Glucocorticoid (GC) stress hormones drive fat metabolism in the liver, but both full blockade and full stimulation of GC signaling aggravate NAFLD pathology. We investigated the efficacy of selective glucocorticoid receptor (GR) modulator CORT118335, which recapitulates only a subset of GC actions, in reducing liver lipid accumulation in mice. Male C57BL/6J mice received a low-fat diet or high-fat diet mixed with vehicle or CORT118335. Livers were analyzed histologically and for genome-wide mRNA expression. Functionally, hepatic long-chain fatty acid (LCFA) composition was determined by gas chromatography. We determined very-low-density lipoprotein (VLDL) production by treatment with a lipoprotein lipase inhibitor after which blood was collected to isolate radiolabeled VLDL particles and apoB proteins. CORT118335 strongly prevented and reversed hepatic lipid accumulation. Liver transcriptome analysis showed increased expression of GR target genes involved in VLDL production. Accordingly, CORT118335 led to increased lipidation of VLDL particles, mimicking physiological GC action. Independent pathway analysis revealed that CORT118335 lacked induction of GC-responsive genes involved in cholesterol synthesis and LCFA uptake, which was indeed reflected in unaltered hepatic LCFA uptake in vivo. Our data thus reveal that the robust hepatic lipid-lowering effect of CORT118335 is due to a unique combination of GR-dependent stimulation of lipid (VLDL) efflux from the liver, with a lack of stimulation of GR-dependent hepatic fatty acid uptake. Our findings firmly demonstrate the potential use of CORT118335 in the treatment of NAFLD and underscore the potential of selective GR modulation in metabolic disease.

A Diurnal Rhythm in Brown Adipose Tissue Causes Rapid Clearance and Combustion of Plasma Lipids at Wakening.

Many favorable metabolic effects have been attributed to thermogenic activity of brown adipose tissue (BAT). Yet, time of day has rarely been considered in this field of research. Here, we show that a diurnal rhythm in BAT activity regulates plasma lipid metabolism. We observed a high-amplitude rhythm in fatty acid uptake by BAT that synchronized with the light/dark cycle. Highest uptake was found at the onset of the active period, which coincided with high lipoprotein lipase expression and low angiopoietin-like 4 expression by BAT. Diurnal rhythmicity in BAT activity determined the rate at which lipids were cleared from the circulation, thereby imposing the daily rhythm in plasma lipid concentrations. In mice as well as humans, postprandial lipid excursions were nearly absent at waking. We anticipate that diurnal BAT activity is an important factor to consider when studying the therapeutic potential of promoting BAT activity.

Deuterium-reinforced polyunsaturated fatty acids protect against atherosclerosis by lowering lipid peroxidation and hypercholesterolemia.

BACKGROUND AND AIMS: Oxidative modification of lipoproteins is a crucial step in atherosclerosis development. Isotopic-reinforced polyunsaturated fatty acids (D-PUFAs) are more resistant to reactive oxygen species-initiated chain reaction of lipid peroxidation than regular hydrogenated (H-)PUFAs. We aimed at investigating the effect of D-PUFA treatment on lipid peroxidation, hypercholesterolemia and atherosclerosis development. METHODS: Transgenic APOE*3-Leiden.CETP mice, a well-established model for human-like lipoprotein metabolism, were pre-treated with D-PUFAs or control H-PUFAs-containing diet (1.2%, w/w) for 4 weeks. Thereafter, mice were fed a Western-type diet (containing 0.15% cholesterol, w/w) for another 12 weeks, while continuing the D-/H-PUFA treatment. RESULTS: D-PUFA treatment markedly decreased hepatic and plasma F2-isoprostanes (approx. -80%) and prostaglandin F2α (approx. -40%) as compared to H-PUFA treatment. Moreover, D-PUFAs reduced body weight gain during the study (-54%) by decreasing body fat mass gain (-87%) without altering lean mass. D-PUFAs consistently reduced plasma total cholesterol levels (approx. -25%), as reflected in reduced plasma non-HDL-cholesterol (-28%). Additional analyses of hepatic cholesterol metabolism indicated that D-PUFAs reduced the hepatic cholesterol content (-21%). Sterol markers of intestinal cholesterol absorption and cholesterol breakdown were decreased. Markers of cholesterol synthesis were increased. Finally, D-PUFAs reduced atherosclerotic lesion area formation throughout the aortic root of the heart (-26%). CONCLUSIONS: D-PUFAs reduce body weight gain, improve cholesterol handling and reduce atherosclerosis development by reducing lipid peroxidation and plasma cholesterol levels. D-PUFAs, therefore, represent a promising new strategy to broadly reduce rates of lipid peroxidation, and combat hypercholesterolemia and cardiovascular diseases.

Atorvastatin accelerates clearance of lipoprotein remnants generated by activated brown fat to further reduce hypercholesterolemia and atherosclerosis.

BACKGROUND AND AIMS: Activation of brown adipose tissue (BAT) reduces both hyperlipidemia and atherosclerosis by increasing the uptake of triglyceride-derived fatty acids by BAT, accompanied by formation and clearance of lipoprotein remnants. We tested the hypothesis that the hepatic uptake of lipoprotein remnants generated by BAT activation would be accelerated by concomitant statin treatment, thereby further reducing hypercholesterolemia and atherosclerosis. METHODS: APOE*3-Leiden.CETP mice were fed a Western-type diet and treated without or with the selective ß3-adrenergic receptor (AR) agonist CL316,243 that activates BAT, atorvastatin (statin) or both. RESULTS: ß3-AR agonism increased energy expenditure as a result of an increased fat oxidation by activated BAT, which was not further enhanced by statin addition. Accordingly, statin treatment neither influenced the increased uptake of triglyceride-derived fatty acids from triglyceride-rich lipoprotein-like particles by BAT nor further lowered plasma triglyceride levels induced by ß3-AR agonism. Statin treatment increased the hepatic uptake of the formed cholesterol-enriched remnants generated by ß3-AR agonism. Consequently, statin treatment further lowered plasma cholesterol levels. Importantly, statin, in addition to ß3-AR agonism, also further reduced the atherosclerotic lesion size as compared to ß3-AR agonism alone, without altering lesion severity and composition. CONCLUSIONS: Statin treatment accelerates the hepatic uptake of remnants generated by BAT activation, thereby increasing the lipid-lowering and anti-atherogenic effects of BAT activation in an additive fashion. We postulate that, in clinical practice, combining statin treatment with BAT activation is a promising new avenue to combat hyperlipidemia and cardiovascular disease.

A Mixed Glucocorticoid/Mineralocorticoid Selective Modulator With Dominant Antagonism in the Male Rat Brain.

Adrenal glucocorticoid hormones are potent modulators of brain function in the context of acute and chronic stress. Both mineralocorticoid (MRs) and glucocorticoid receptors (GRs) can mediate these effects. We studied the brain effects of a novel ligand, C118335, with high affinity for GRs and modest affinity for MRs. In vitro profiling of receptor-coregulator interactions suggested that the compound is a "selective modulator" type compound for GRs that can have both agonistic and antagonistic effects. Its molecular profile for MRs was highly similar to those of the full antagonists spironolactone and eplerenone. C118335 showed predominantly antagonistic effects on hippocampal mRNA regulation of known glucocorticoid target genes. Likewise, systemic administration of C118335 blocked the GR-mediated posttraining corticosterone-induced enhancement of memory consolidation in an inhibitory avoidance task. Posttraining administration of C118335, however, gave a strong and dose-dependent impairment of memory consolidation that, surprisingly, reflected involvement of MRs and not GRs. Finally, C118335 treatment acutely suppressed the hypothalamus-pituitary-adrenal axis as measured by plasma corticosterone levels. Mixed GR/MR ligands, such as C118335, can be used to unravel the mechanisms of glucocorticoid signaling. The compound is also a prototype of mixed GR/MR ligands that might alleviate the harmful effects of chronic overexposure to endogenous glucocorticoids.

Spatial and temporal expression of immunoglobulin superfamily member 1 in the rat.

Loss-of-function mutations in the immunoglobulin superfamily member 1 (IGSF1) gene cause an X-linked syndrome of central hypothyroidism, macroorchidism, variable prolactin and GH deficiency, delayed pubertal testosterone rise, and obesity. To understand the pathophysiology of this syndrome, knowledge on IGSF1's place in normal development is imperative. Therefore, we investigated spatial and temporal protein and mRNA expression of IGSF1 in rats using immunohistochemistry, real-time quantitative PCR (qPCR), and in situ hybridization. We observed high levels of IGSF1 expression in the brain, specifically the embryonic and adult choroid plexus and hypothalamus (principally in glial cells), and in the pituitary gland (PIT1-lineage of GH, TSH, and PRL-producing cells). IGSF1 is also expressed in the embryonic and adult zona glomerulosa of the adrenal gland, islets of Langerhans of the pancreas, and costameres of the heart and skeletal muscle. IGSF1 is highly expressed in fetal liver, but is absent shortly after birth. In the adult testis, IGSF1 is present in Sertoli cells (epithelial stages XIII-VI), and elongating spermatids (stages X-XII). Specificity of protein expression was corroborated with Igsf1 mRNA expression in all tissues. The expression patterns of IGSF1 in the pituitary gland and testis are consistent with the pituitary hormone deficiencies and macroorchidism observed in patients with IGSF1 deficiency. The expression in the brain, adrenal gland, pancreas, liver, and muscle suggest IGSF1's function in endocrine physiology might be more extensive than previously considered.