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Steroid hormone signaling pathways are critical for organismal development and act through binding to nuclear receptors driving transcriptional regulation. In this review, we summarize evidence for another – underrated – mechanism of action for steroid hormones: their ability to modulate the alternative splicing of pre-mRNA. Thirty years ago, pioneering studies used in vitro transfection of plasmids expressing alternative exons under the control of hormone responsive promoters in cell lines. These studies demonstrated that steroid hormone binding to their nuclear receptor impacted both gene transcription and alternative splicing outcomes. The advent of exon arrays and next generation sequencing has allowed researchers to observe the impact of steroid hormones at the whole transcriptome level. These studies demonstrate that steroid hormones regulate alternative splicing in a time-, gene-, and tissue-specific manner. We provide examples of the mechanisms by which steroid hormones regulate alternative splicing including 1) recruitment of dual function proteins that behave as coregulators and splicing factors, 2) transcriptional regulation of splicing factor levels, 3) the alternative splicing of splicing factors or transcription factors that feed-forward regulate steroid hormone signaling, and 4) regulation of elongation rate. Experiments performed in vivo and in cancer cell lines highlight that steroid hormone mediated alternative splicing occurs in both physiological and pathophysiologic states. Studying the impact of steroid hormones on alternative splicing is a fruitful avenue for research that should be exploited to discover new targets for therapeutic intervention.

Steroid 5β-reductase (AKR1D1) plays important role in hepatic bile acid synthesis and glucocorticoid clearance. Bile acids and glucocorticoids are potent metabolic regulators, but whether AKR1D1 controls metabolic phenotype in vivo is unknown. Akr1d1–/– mice were generated on a C57BL/6 background. Liquid chromatography/mass spectrometry, metabolomic and transcriptomic approaches were used to determine effects on glucocorticoid and bile acid homeostasis. Metabolic phenotypes including body weight and composition, lipid homeostasis, glucose tolerance and insulin tolerance were evaluated. Molecular changes were assessed by RNA-Seq and Western blotting. Male Akr1d1–/– mice were challenged with a high fat diet (60% kcal from fat) for 20 weeks. Akr1d1–/– mice had a sex-specific metabolic phenotype. At 30 weeks of age, male, but not female, Akr1d1–/– mice were more insulin tolerant and had reduced lipid accumulation in the liver and adipose tissue yet had hypertriglyceridemia and increased intramuscular triacylglycerol. This phenotype was associated with sexually dimorphic changes in bile acid metabolism and composition but without overt effects on circulating glucocorticoid levels or glucocorticoid-regulated gene expression in the liver. Male Akr1d1–/– mice were not protected against diet-induced obesity and insulin resistance. In conclusion, this study shows that AKR1D1 controls bile acid homeostasis in vivo and that altering its activity can affect insulin tolerance and lipid homeostasis in a sex-dependent manner.

Glucocorticoid hormones were discovered to have use as potent anti-inflammatory and immunosuppressive therapeutics in the 1940s and their continued use and development have successfully revolutionized the management of acute and chronic inflammatory diseases. However, long-term use of glucocorticoids is severely hampered by undesirable metabolic complications, including the development of type 2 diabetes mellitus. These effects occur due to glucocorticoid receptor activation within multiple tissues, which results in inter-organ crosstalk that increases hepatic glucose production and inhibits peripheral glucose uptake. Despite the high prevalence of glucocorticoid-induced hyperglycaemia associated with their routine clinical use, treatment protocols for optimal management of the metabolic adverse effects are lacking or underutilized. The type, dose and potency of the glucocorticoid administered dictates the choice of hypoglycaemic intervention (non-insulin or insulin therapy) that should be provided to patients. The longstanding quest to identify dissociated glucocorticoid receptor agonists to separate the hyperglycaemic complications of glucocorticoids from their therapeutically beneficial anti-inflammatory effects is ongoing, with selective glucocorticoid receptor modulators in clinical testing. Promising areas of preclinical research include new mechanisms to disrupt glucocorticoid signalling in a tissue-selective manner and the identification of novel targets that can selectively dissociate the effects of glucocorticoids. These research arms share the ultimate goal of achieving the anti-inflammatory actions of glucocorticoids without the metabolic consequences.

Regulation of RNA processing contributes profoundly to tissue development and physiology. Here, we report that serine-arginine-rich splicing factor 1 (SRSF1) is essential for hepatocyte function and survival. Although SRSF1 is mainly known for its many roles in mRNA metabolism, it is also crucial for maintaining genome stability. We show that acute liver damage in the setting of targeted SRSF1 deletion in mice is associated with the excessive formation of deleterious RNA–DNA hybrids (R-loops), which induce DNA damage. Combining hepatocyte-specific transcriptome, proteome, and RNA binding analyses, we demonstrate that widespread genotoxic stress following SRSF1 depletion results in global inhibition of mRNA transcription and protein synthesis, leading to impaired metabolism and trafficking of lipids. Lipid accumulation in SRSF1-deficient hepatocytes is followed by necroptotic cell death, inflammation, and fibrosis, resulting in NASH-like liver pathology. Importantly, SRSF1-depleted human liver cancer cells recapitulate this pathogenesis, illustrating a conserved and fundamental role for SRSF1 in preserving genome integrity and tissue homeostasis. Thus, our study uncovers how the accumulation of detrimental R-loops impedes hepatocellular gene expression, triggering metabolic derangements and liver damage.

Metabolic diseases are increasing at staggering rates globally. The peroxisome proliferator-activated receptors (PPARα/γ/δ) are fatty acid sensors that help mitigate imbalances between energy uptake and utilization. Herein, we report compounds derived from phenolic lipids present in cashew nut shell liquid (CNSL), an abundant waste byproduct, in an effort to create effective, accessible, and sustainable drugs. Derivatives of anacardic acid and cardanol were tested for PPAR activity in HEK293 cell co-transfection assays, primary hepatocytes, and 3T3-L1 adipocytes.

Plasma free fatty acids (FFAs) are elevated in obesity and can induce insulin resistance via endoplasmic reticulum (ER) stress. However, it is unknown whether hepatic insulin resistance caused by the elevation of plasma FFAs is alleviated by chemical chaperones. Rats received one of the following i.v. treatments for 48 h: saline, intralipid plus heparin (IH), IH plus the chemical chaperone 4-phenylbutyric acid (PBA), or PBA alone and a hyperinsulinemic-euglycemic clamp was performed during the last 2 h. PBA co-infusion normalized IH-induced peripheral insulin resistance, similar to our previous findings with an antioxidant and an IκBα kinase β (IKKβ) inhibitor. Different from our previous results with the antioxidant and IKKβ inhibitor, PBA also improved IH-induced hepatic insulin resistance in parallel with activation of Akt. Unexpectedly, IH did not induce markers of ER stress in the liver, but PBA prevented IH-induced elevation of phosphorylated eukaryotic initiation factor-2α protein in adipose tissue. PBA tended to decrease circulating fetuin-A and significantly increased circulating fibroblast growth factor 21 (FGF21) without affecting markers of activation of hepatic protein kinase C-δ or p38 mitogen-activated protein kinase that we have previously involved in hepatic insulin resistance in this model. In conclusion: (i) PBA prevented hepatic insulin resistance caused by prolonged plasma FFA elevation without affecting hepatic ER stress markers; (ii) the PBA effect is likely due to increased FGF21 and/or decreased fetuin-A, which directly signal to upregulate Akt activation.

Endothelial progenitor cells (EPCs) promote the maintenance of the endothelium by secreting vasoreparative factors. A population of EPCs known as early outgrowth cells (EOCs) is being investigated as novel cell‐based therapies for the treatment of cardiovascular disease. We previously demonstrated that the absence of liver X receptors (LXRs) is detrimental to the formation and function of EOCs under hypercholesterolemic conditions. Here, we investigate whether LXR activation in EOCs is beneficial for the treatment of atherosclerosis. EOCs were differentiated from the bone marrow of wild‐type (WT) and LXR‐knockout (Lxrαβ−/−) mice in the presence of vehicle or LXR agonist (GW3965). WT EOCs treated with GW3965 throughout differentiation showed reduced mRNA expression of endothelial lineage markers (Cd144, Vegfr2) compared with WT vehicle and Lxrαβ−/− EOCs. GW3965‐treated EOCs produced secreted factors that reduced monocyte adhesion to activated endothelial cells in culture. When injected into atherosclerosis‐prone Ldlr−/− mice, GW3965‐treated EOCs, or their corresponding conditioned media (CM) were both able to reduce aortic sinus plaque burden compared with controls. Furthermore, when human EOCs (obtained from patients with established CAD) were treated with GW3965 and the CM applied to endothelial cells, monocyte adhesion was decreased, indicating that our results in mice could be translated to patients. Ex vivo LXR agonist treatment of EOCs therefore produces a secretome that decreases early atherosclerosis in Ldlr−/− mice, and additionally, CM from human EOCs significantly inhibits monocyte to endothelial adhesion. Thus, active factor(s) within the GW3965‐treated EOC secretome may have the potential to be useful for the treatment of atherosclerosis., Bone marrow cells differentiated with liver x receptors agonist (GW) produce a population of early outgrowth cells (EOCs) with an alternative secretome, capable of decreasing monocyte‐endothelial adhesion in culture, and reducing atherosclerosis in vivo. GW‐treated EOCs derived from peripheral blood mononuclear cells (MNCs) of patients with coronary artery disease also produced a secretome that reduced monocyte‐endothelial adhesion in culture.

The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARα) is emerging as an important target in the brain for the treatment or prevention of cognitive disorders. The identification of high-affinity ligands for brain PPARα may reveal the mechanisms underlying the synaptic effects of this receptor and facilitate drug development. Here, using an affinity purification-untargeted mass spectrometry (AP-UMS) approach, we identified an endogenous, selective PPARα ligand, 7(

The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARα) is emerging as an important target in the brain for the treatment or prevention of cognitive disorders. The identification of high-affinity ligands for brain PPARα may reveal the mechanisms underlying the synaptic effects of this receptor and facilitate drug development. Here, using an affinity purification–untargeted mass spectrometry (AP-UMS) approach, we identified an endogenous, selective PPARα ligand, 7( S )-hydroxy-docosahexaenoic acid [7( S )-HDHA]. Results from mass spectrometric detection of 7( S )-HDHA in mouse and rat brain tissues, time-resolved FRET analyses, and thermal shift assays collectively revealed that 7( S )-HDHA potently activated PPARα with an affinity greater than that of other ligands identified to date. We also found that 7( S )-HDHA activation of PPARα in cultured mouse cortical neurons stimulated neuronal growth and arborization, as well as the expression of genes associated with synaptic plasticity. The findings suggest that this DHA derivative supports and enhances neuronal synaptic capacity in the brain.

Atherosclerosis is a chronic inflammatory condition in which macrophages play a major role. Janus kinase 2 (JAK2) is a pivotal molecule in inflammatory and metabolic signaling, and Jak2V617F activating mutation has recently been implicated with enhancing clonal hematopoiesis and atherosclerosis. To determine the essential in vivo role of macrophage (M)-Jak2 in atherosclerosis, we generate atherosclerosis-prone ApoE-null mice deficient in M-Jak2. Contrary to our expectation, these mice exhibit increased plaque burden with no differences in macrophage proliferation, recruitment or bone marrow clonal expansion. Notably, M-Jak2-deficient bone marrow derived macrophages show a significant defect in cholesterol efflux. Pharmacologic JAK2 inhibition with ruxolitinib also leads to defects in cholesterol efflux and accelerates atherosclerosis. Liver X receptor agonist abolishes the efflux defect and attenuates the accelerated atherosclerosis that occurs with M-Jak2 deficiency. Macrophages of individuals with the Jak2V617F mutation show increased efflux which is normalized when treated with a JAK2 inhibitor. Together, M-Jak2-deficiency leads to accelerated atherosclerosis primarily through defects in cholesterol efflux from macrophages.

Despite the use of antiretroviral therapy for the treatment of HIV-1 infection, cognitive impairments, that is, HIV-1-associated neurocognitive disorders remain prevalent potentially due to persistent viral replication, production of viral proteins, associated brain inflammation or in certain instances, antiretroviral neurotoxicity. Cellular targets in the brain include microglia which in response to infection release inflammatory markers and viral proteins. Evidence suggests that PPARγ agonists exert anti-inflammatory properties in neurological disorders. However, these agonists namely, thiazolidinediones have limited use in the clinic due to reported adverse side effects. INT131 is a novel non-thiazolidinedione compound that belongs to a new class of drugs known as selective PPARγ modulators. INT131 is considered to have a safer profile; however, its neuroprotective role in vivo is not known.The goal of this study was to examine the effect of INT131 in the context of EcoHIV-induced inflammation in vitro, in primary cultures of mouse glial cells and in vivo, in a mouse model of EcoHIV-associated brain inflammation, as well as characterize its pharmacokinetic properties and brain penetration. In primary cultures of glial cells and in the in vivo mouse model, EcoHIV exposure resulted in a significant elevation of inflammatory markers such as TNFα, IL-1β, CCL3, and C3 which were attenuated with INT131 treatment. Pharmacokinetic analyses revealed that INT131 penetrates into the brain with a brain to blood partition ratio Kp value of 8.5%. Overall, this is the first report to demonstrate that INT131 could be a potential candidate for the treatment of HIV-1-associated brain inflammation.

Regulation of RNA processing contributes profoundly to tissue development and physiology. Here, we report that serine-arginine-rich splicing factor 1 (SRSF1) is essential for hepatocyte function and survival. Although SRSF1 is mainly known for its many roles in mRNA metabolism, it is also crucial for maintaining genome stability. We show that acute liver damage in the setting of targeted SRSF1 deletion in mice is primarily mediated by the excessive formation of deleterious RNA–DNA hybrids (R-loops), which induce DNA damage. Combining hepatocyte-specific transcriptome, proteome, and RNA binding analyses, we demonstrate that widespread genotoxic stress following SRSF1 depletion results in global inhibition of mRNA transcription and protein synthesis, leading to impaired metabolism and trafficking of lipids. Lipid accumulation in SRSF1-deficient hepatocytes is followed by necroptotic cell death, inflammation, and fibrosis, resulting in NASH-like liver pathology. Importantly, SRSF1-depleted human liver cancer cells recapitulate this pathogenesis illustrating a conserved and fundamental role for SRSF1 in preserving genome integrity and tissue homeostasis. Thus, our study uncovers how accumulation of detrimental R-loops impedes hepatocellular gene expression, triggering metabolic derangements and liver failure.

Successful delivery of anticancer drugs to intracellular targets requires different properties of the nanocarrier to overcome multiple transport barriers. However, few nanocarrier systems, to date, possess such properties, despite knowledge about the biological fate of inorganic and polymeric nanocarriers in relation to their fixed size, shape and surface properties. Herein, a polymer-lipid hybrid nanoparticle (PLN) system is described with size and shape transformability and its mechanisms of cellular uptake and intracellular trafficking are studied. Pharmaceutical lipids were screened for use in transformable PLN. Mechanisms of cellular uptake and the role of fatty acid-binding proteins in intracellular trafficking of PLN were investigated in breast cancer cells. Intra-tumoral penetration and retention of doxorubicin (DOX) were evaluated by confocal microscopy. The lead PLNs showed time-dependent size reduction and shape change from spherical to spiky shape. This transformability of PLNs and lipid trafficking pathways facilitated intracellular transport of DOX-loaded PLN (DOX-PLN) into mitochondria and nuclei. DOX-PLN significantly increased DOX penetration and retention over free DOX or non-transformable liposomal DOX particles at 4 h post-intravenous administration. Transformability of PLN and lipid-biology interplay can be exploited to design new nanocarriers for effective drug delivery to tumor cells and intracellular targets.

BackgroundSteroid 5β-reductase (AKR1D1) plays important roles in hepatic glucocorticoid clearance and bile acid synthesis. Glucocorticoids and bile acids are potent metabolic regulators, but whether AKR1D1 controls metabolic phenotype in vivo is unknown.MethodsAkr1d1-/-mice were generated on a C57BL/6 background. Liquid chromatography / mass spectrometry, metabolomic and transcriptomic approaches were used to determine effects on glucocorticoid and bile acid homeostasis. Metabolic phenotypes including body weight and composition, lipid homeostasis, glucose tolerance and insulin sensitivity were evaluated. Molecular changes were assessed by RNASeq and western blotting. Male Akr1d1-/-mice were challenged with a 60% high fat diet.ResultsAkr1d1-/-mice had a sex specific metabolic phenotype. At 30-weeks of age male, but not female, Akr1d1-/-mice were more insulin sensitive and had reduced lipid accumulation in the liver and adipose tissue, concomitant with hypertriglyceridemia and increased intramuscular triacylglycerol. This phenotype was underpinned by sexually dimorphic changes in bile acid metabolism and composition, but without overt effects on glucocorticoid action. Male Akr1d1-/-mice were not protected against diet induced obesity and insulin resistance.ConclusionThis study shows that AKR1D1 controls bile acid homeostasis in vivo and that altering its activity can affect insulin sensitivity and lipid homeostasis in a sex dependent manner.

International audience; N-acylethanolamines (NAEs) are endogenous lipid-signaling molecules derived from fatty acids that regulate numerous biological functions, including in the brain. Interestingly, NAEs are elevated in the absence of fatty acid amide hydrolase (FAAH) and following CO2-induced ischemia/hypercapnia, suggesting a neuroprotective response. Tetracosahexaenoic acid (THA) is a product and precursor to DHA; however, the NAE product, tetracosahexaenoylethanolamide (THEA), has never been reported. Presently, THEA was chemically synthesized as an authentic standard to confirm THEA presence in biological tissues. Whole brains were collected and analyzed for unesterified THA, total THA, and THEA in wild-type and FAAH-KO mice that were euthanized by either head-focused microwave fixation, CO2 + microwave, or CO2 only. PPAR activity by transient transfection assay and ex vivo neuronal output in medium spiny neurons (MSNs) of the nucleus accumbens by patch clamp electrophysiology were determined following THEA exposure. THEA in the wild-type mice was nearly doubled (P < 0.05) following ischemia/hypercapnia (CO2 euthanization) and up to 12 times higher (P < 0.001) in the FAAH-KO compared with wild-type. THEA did not increase (P > 0.05) transcriptional activity of PPARs relative to control, but 100 nM of THEA increased (P < 0.001) neuronal output in MSNs of the nucleus accumbens. Here were identify a novel NAE, THEA, in the brain that is elevated upon ischemia/hypercapnia and by KO of the FAAH enzyme. While THEA did not activate PPAR, it augmented the excitability of MSNs in the nucleus accumbens. Overall, our results suggest that THEA is a novel NAE that is produced in the brain upon ischemia/hypercapnia and regulates neuronal excitation.

N-acylethanolamines (NAEs) are endogenous lipid-signaling molecules derived from fatty acids that regulate numerous biological functions, including in the brain. Interestingly, NAEs are elevated in the absence of fatty acid amide hydrolase (FAAH) and following CO(2)-induced ischemia/hypercapnia, suggesting a neuroprotective response. Tetracosahexaenoic acid (THA) is a product and precursor to DHA; however, the NAE product, tetracosahexaenoylethanolamide (THEA), has never been reported. Presently, THEA was chemically synthesized as an authentic standard to confirm THEA presence in biological tissues. Whole brains were collected and analyzed for unesterified THA, total THA, and THEA in wild-type and FAAH-KO mice that were euthanized by either head-focused microwave fixation, CO(2) + microwave, or CO(2) only. PPAR activity by transient transfection assay and ex vivo neuronal output in medium spiny neurons (MSNs) of the nucleus accumbens by patch clamp electrophysiology were determined following THEA exposure. THEA in the wild-type mice was nearly doubled (P < 0.05) following ischemia/hypercapnia (CO(2) euthanization) and up to 12 times higher (P < 0.001) in the FAAH-KO compared with wild-type. THEA did not increase (P > 0.05) transcriptional activity of PPARs relative to control, but 100 nM of THEA increased (P < 0.001) neuronal output in MSNs of the nucleus accumbens. Here were identify a novel NAE, THEA, in the brain that is elevated upon ischemia/hypercapnia and by KO of the FAAH enzyme. While THEA did not activate PPAR, it augmented the excitability of MSNs in the nucleus accumbens. Overall, our results suggest that THEA is a novel NAE that is produced in the brain upon ischemia/hypercapnia and regulates neuronal excitation.

Stress hormones bind and activate the glucocorticoid receptor (GR) in many tissues including the brain. We identified arginine and glutamate rich 1 (ARGLU1) in a screen for new modulators of glucocorticoid signaling in the CNS. Biochemical studies show that the glutamate rich C-terminus of ARGLU1 coactivates multiple nuclear receptors including the glucocorticoid receptor (GR) and the arginine rich N-terminus interacts with splicing factors and binds to RNA. RNA-seq of neural cells depleted of ARGLU1 revealed significant changes in the expression and alternative splicing of distinct genes involved in neurogenesis. Loss of ARGLU1 is embryonic lethal in mice, and knockdown in zebrafish causes neurodevelopmental and heart defects. Treatment with dexamethasone, a GR activator, also induces changes in the pattern of alternatively spliced genes, many of which were lost when ARGLU1 was absent. Importantly, the genes found to be alternatively spliced in response to glucocorticoid treatment were distinct from those under transcriptional control by GR, suggesting an additional mechanism of glucocorticoid action is present in neural cells. Our results thus show that ARGLU1 is a novel factor for embryonic development that modulates basal transcription and alternative splicing in neural cells with consequences for glucocorticoid signaling.

In vitro cell-based assays are common screening tools used for the identification of new VDR ligands. For 25-hydroxyvitamin D3 [25(OH)D3] and 1α-hydroxyvitamin D3 [1α(OH)D3], protein expressions of CYP2R1 and CYP27B1, respectively, that form the active 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] ligand were detected in human embryonic kidney (HEK293) cells expressing the GAL4-hVDR, the human brain microvessel endothelial (hCMEC/D3) and adenocarcinoma colonic (Caco-2) cells. The impact of bioactivation enzymes was shown upon the addition of ketoconazole (10 μM KTZ), a pan-CYP inhibitor, which reduced the apparent potency of 25(OH)D3 and increased the EC50 from 272 to 608 nM in HEK293 cells. EIA assays verified that 1,25(OH)2D3 was formed and contributed to VDR activity independently of its precursors. In hCMEC/D3 cells where enzyme protein levels were lowest, changes in MDR1/P-gp expression with KTZ were minimal. In Caco-2 cells, the induction of TRPV6 (calcium channel), CYP24A1, CYP3A4, OATP1A2 and MDR1 mRNA expression was 1,25(OH)2D3 > 1α(OH)D3 > 25(OH)D3, with the magnitude of change being blunted by KTZ. Upon inclusion of KTZ in the cell-based assays, high transcriptional activities were observed for synthetic VDR activators from Teijin Pharma. Cyclopentanone derivatives: TPD-003, TPD-005, TPD-006, TPD-008 and TPD-009 (EC50s 0.06 to 67 nM, unchanged with KTZ) were found more potent over straight chain and lactone derivatives (antagonists). Most TPD compounds activated OATP1A2, CYP24A1, CYP3A4, and MDR1 (28–67%) and TRPV6 transcriptionally in Caco-2 cells. The results identified that cell-based assays with added KTZ could accurately identify new VDR activators, although these may be hypercalcemic with strong TRPV6 inducing properties.

Excessive exposure to glucocorticoids (GCs), either from endogenous overproduction of cortisol, or exogenous pharmacological GC treatment, potentiates the development of diabetes and obesity in a fat depot-specific manner. Undesirable metabolic side effects resulting from the activation of the glucocorticoid receptor (GR) remain a key limitation to the long-term therapeutic use of GCs as immunosuppressants. GC treatment disrupts the thermogenic function of brown adipose tissue (BAT) and enhances futile cycling within white adipose tissue (WAT). Mice lacking the liver X receptors (LXRs) were previously shown to have smaller AT depots with enhanced BAT activity compared to wildtype (WT) mice. We previously demonstrated that LXRβ is required to mediate the side effects of GCs in the liver but not the beneficial anti-inflammatory effects of GCs. The discovery of this GC/LXRβ cross-talk led to the hypothesis that LXRβ antagonism may be therapeutically beneficial to prevent GC-induced dysfunction in AT. To test this idea, LXRα-/- mice were treated for 5 days with vehicle, 5 mg/kg dexamethasone (Dex, a synthetic GC agonist), and/or 40 mg/kg GSK2033 (GSK, non-selective LXR antagonist). As expected, Dex-treated mice showed significant accumulation of lipids in BAT and were unable to maintain their body temperature when exposed to cold, yet GSK was able to protect against these effects. Dex increased body fat mass and caused adipocyte enlargement in WAT which was not observed in mice co-treated with GSK. At the transcriptional level, GSK attenuated Dex-mediated downregulation of thermogenic genes in BAT, and upregulation of lipogenic genes in WAT. Similar beneficial changes were confirmed in WT mice. The protection afforded by GSK against Dex-induced fat accumulation was confirmed to be cell-autonomous from studies in adipose-specific LXRβ-/- mice (AdβKO). Dex-dependent increases in lipolysis in gonadal WAT and plasma free fatty acids (FFA) were also attenuated by GSK co-treatment. With GSK co-treatment, Dex-induced liver steatosis was diminished suggesting that LXRβ antagonism attenuated FFA shuttling to the liver. The lipolytic and lipotoxic effects of Dex in AT and liver were largely abrogated in AdβKO along with improved systemic insulin sensitivity. Overall, our data suggest that LXRβ antagonism prevents disruption of BAT and WAT (and indirectly liver) function caused by GC treatment in an in vivo model, highlighting the potential role of LXRβ antagonists in combating the negative effects of excessive GC exposure on the development of diabetes and obesity. The identification of this novel mechanism of interrupting GC adipose tissue action suggests therapeutic targeting of LXRβ with an antagonist could improve the health of patients currently taking GCs to control inflammation but suffer the detrimental side effects of drug treatment.

Recent human brain imaging studies implicate dysregulation of monoamine oxidase-A (MAO-A), in particular in the prefrontal cortex (PFC) and anterior cingulate cortex (ACC), in the development of major depressive disorder (MDD). This study investigates the influence of four alterations underlying important pathologies of MDD, namely, chronic elevation of glucocorticoid levels, glutathione depletion, changes in female gonadal sex hormones and serotonin concentration fluctuation, on MAO-A and MAO-B activities in rats. Young adult rats exposed chronically to the synthetic glucocorticoid dexamethasone at 0, 0.05, 0.5, and 2.0mg/kg/day (osmotic minipumps) for eight days showed significant dose-dependent increases in activities of MAO-A in PFC (+17%, p

Glucocorticoids (GCs) have unparalleled anti-inflammatory and immunosuppressive properties, which accounts for their widespread prescription and use. Unfortunately, a limitation to GC therapy is a wide range of negative side effects including Cushing's syndrome, a disease characterized by metabolic abnormalities including muscle wasting and osteoporosis. GC-induced osteoporosis occurs in 30% to 50% of patients on GC therapy and thus, represents an important area of study. Herein, we characterize the molecular and physiologic effects of GC-induced osteoporosis using the Cushing's mouse model, the corticotropin releasing hormone (CRH) transgenic mouse (CRH-Tg). The humeri, femurs, and tibias from wild-type (WT) and CRH-Tg male mice, aged 13 to 14 weeks old were subjected to multiple bone tests including, micro-computed tomography (μCT), static and dynamic histomorphometry, strength testing, and gene expression analyses. The CRH-Tg mice had a 38% decrease in cortical bone area, a 35% decrease in cortical thickness, a 16% decrease in trabecular thickness, a sixfold increase in bone adiposity, a 27% reduction in osteoid width, a 75% increase in bone-resorbing osteoclast number/bone surface, a 34% decrease in bone formation rate, and a 40% decrease in bone strength compared to WT mice. At the gene expression level, CRH-Tg bone showed significantly increased osteoclast markers and decreased osteoblast markers, whereas CRH-Tg muscle had increased muscle atrophy gene markers compared to WT mice. Overall, the CRH-Tg mouse model aged to 14 weeks recapitulated many features of osteoporosis in Cushing's syndrome and thus, represents a useful model to study GC-induced osteoporosis and interventions that target the effects of GCs on the skeleton. © 2017 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

ObjectiveEndothelial progenitor cells (EPCs) promote the maintenance of the endothelium by the secretion of vasoreparative factors. A population of EPCs known as early outgrowth cells (EOCs) are currently being investigated as novel cell-based therapies for the treatment of cardiovascular disease. We previously demonstrated that the absence of liver x receptors (LXRs) is detrimental to the formation and function of EOCs under hypercholesterolemic conditions. Here, we investigate whether LXR gain-of-function in EOCs is beneficial for the treatment of atherosclerosis.Approach and ResultsEOCs were differentiated from the bone marrow of wildtype (WT) and LXR-knockout (Lxrαβ-/-) mice in the presence of vehicle or LXR agonist (GW3965). WT EOCs treated with GW3965 throughout differentiation showed reduced expression of endothelial lineage markers (Cd144, Vegfr2) compared to WT vehicle andLxrαβ-/- cells. GW3965-treated EOCs produced secreted factors that reduced monocyte adhesion to activated endothelial cells in culture. When injected into atherosclerosis-proneLdlr-/- mice, GW3965-treated EOCs and concentrated conditioned media (CM) from GW3965-treated EOCs, reduced plaque burden within the aortic sinus. Furthermore, when CM from human EOCs (obtained from patients with established CAD) were treated with GW3965, monocyte to endothelial adhesion was decreased suggesting the translatability of the results.ConclusionsEx vivoLXR agonist treatment of EOCs produces a secretome that decreases early atherosclerosis inLdlr-/- mice. CM from human EOCs significantly inhibits monocyte to endothelial adhesion. Thus, active factor(s) within the GW3965-treated EOC secretome have the potential to be useful for the treatment of atherosclerosis.

The nematode Caenorhabditis elegans is a bacterivore filter feeder. Through the contraction of the worm’s pharynx, a bacterial suspension is sucked into the pharynx’s lumen. Excess liquid is then shunted out of the buccal cavity through ancillary channels made by surrounding marginal cells. We find that many worm-bioactive small molecules (a.k.a. wactives) accumulate inside of the marginal cells as crystals or globular spheres. Through screens for mutants that resist the lethality associated with one crystallizing wactive we identify a presumptive sphingomyelin-synthesis pathway that is necessary for crystal and sphere accumulation. We find that expression of sphingomyelin synthase 5 (SMS-5) in the marginal cells is not only sufficient for wactive accumulation but is also important for absorbing exogenous cholesterol, without which C. elegans cannot develop. We conclude that sphingomyelin-rich marginal cells act as a sink to scavenge important nutrients from filtered liquid that might otherwise be shunted back into the environment., The C. elegans nematode worm is a filter-feeder and requires dietary sources of cholesterol. Here, the authors show that the C. elegans pharynx works as a filter to scavenge hydrophobic small molecules from its surrounding liquid environment.

The widespread use of combination antiretroviral therapy (cART) has resulted in significantly reduced deaths from HIV-1 associated complications and opportunistic infections. However, it is estimated that up to 50% of HIV-1 infected individuals still develop HIV-1 associated neurocognitive disorders (HAND). With no treatment currently available for patients, there is a critical need to identify therapeutic approaches that can treat this disorder. Evidence suggests that targeting Peroxisome Proliferator-Activated Receptor-gamma (PPARγ) can be anti-inflammatory in neurological disorders. Here we show that treatment with PPARγ agonists (rosiglitazone or pioglitazone) in primary cultures of mouse glial cells reversed EcoHIV-induced inflammatory genes (TNFα, IL-1β, CCL2, CCL3, CXCL10) and indicator of oxidative stress (iNOS). Furthermore, in vivo, mice administered with EcoHIV through intracranial injection resulted in upregulation of inflammatory genes (TNFα, IL-1β, IFNγ, CCL2, CCL3, CXCL10) and oxidative stress marker (iNOS) in the brain which was reversed through intraperitoneal administration of PPARγ agonists (rosiglitazone or pioglitazone). Finally, we demonstrated that treatment with these compounds in vivo reduced EcoHIV p24 protein burden in the brain. Our results suggest that treatment with PPARγ agonists are anti-inflammatory and antiviral in an in vivo model of EcoHIV infection. These drugs hold promise as potential candidates for HAND treatment in the future.

IL-6 markedly decreases the expression of numerous hepatic transporters. We previously demonstrated that IL-6-mediated downregulation of transporters occurs through STAT3, with partial involvement of PXR. However, while IL-6-mediated induction of STAT3 occurs rapidly, repression of transporter expression is not observed until 6 hours post-treatment. This temporal mismatch suggested that the downregulation of transporters following IL-6 at 6 hours might require additional signaling downstream of STAT3. Since NF-κB has been implicated in endotoxin-mediated downregulation of transporters, we hypothesized that NF-κB may be similarly involved in suppressing transporter expression following IL-6. Our objective was to investigate whether IL-6-mediated changes in transporter expression occur through STAT3-dependent NF-κB activation, and whether PXR is involved. PXR null (-/-) or wild type (+/+) mice were pre-dosed with the NF-κB inhibitor PHA408 or vehicle 30 minutes prior to receiving a single dose of IL-6 or saline. Mice were euthanized after 6 hours and transporter expression was analyzed using qRT-PCR. IL-6 imposed downregulation of Abcb1a, Abcb1b, Abcc3, Abcg2 and Cyp3a11 in both PXR (+/+) and PXR (-/-) mice, while downregulation of Abcb11, Abcc2, Slc10a1, and Slco2b1 was only significant in PXR (+/+) mice. PHA408 pretreatment fully inhibited NF-κB activation in PXR (+/+) but only partially inhibited NF-κB in PXR (-/-). Inhibition of NF-κB attenuated IL-6-mediated changes in transporters in PXR (+/+) mice. Transient transfection assays did not detect significant activation of human or mouse PXR by PHA408. Our findings suggest that IL-6 imposes significant downregulation of numerous ABC and SLC transporters in the liver via collaborative STAT3/NF-κB activation. Since drug transporters play an integral role in the pharmacokinetics of numerous clinically relevant drugs, understanding the signaling pathways involved in transporter regulation during inflammation will contribute to a better understanding of drug-disease interactions.

Oxidative derivatives of cholesterol such as 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 25-hydroxycholesterol, and (25S),26-hydroxycholesterol are endogenous ligands for the liver X receptors (LXRα and LXRβ). The LXRs are nuclear hormone receptors known as "intracellular cholesterol sensors" because of their ability to bind to and be activated by oxysterols at circulating concentrations. Oxysterols are expressed in a tissue-specific manner and are generally at least 10

The glucocorticoid receptor (GR) represents the crucial molecular mediator of key endocrine, glucocorticoid hormone-dependent regulatory circuits, including control of glucose, protein, and lipid homeostasis. Consequently, aberrant glucocorticoid signaling is linked to severe metabolic disorders, including insulin resistance, obesity, and hyperglycemia, all of which also appear upon chronic glucocorticoid therapy for the treatment of inflammatory conditions. Of note, long-term glucocorticoid exposure under these therapeutic conditions typically induces glucocorticoid resistance, requiring higher doses and consequently triggering more severe metabolic phenotypes. However, the molecular basis of acquired glucocorticoid resistance remains unknown. In a screen of differential microRNA expression during glucocorticoid-dependent adipogenic differentiation of human multipotent adipose stem cells, we identified microRNA 29a (miR-29a) as one of the most down-regulated transcripts. Overexpression of miR-29a impaired adipogenesis. We found that miR-29a represses GR in human adipogenesis by directly targeting its mRNA, and downstream analyses revealed that GR mediates most of miR-29a's anti-adipogenic effects. Conversely, miR-29a expression depends on GR activation, creating a novel miR-29-driven feedback loop. miR-29a and GR expression were inversely correlated both in murine adipose tissue and in adipose tissue samples obtained from human patients. In the latter, miR-29a levels were additionally strongly negatively correlated with body mass index and adipocyte size. Importantly, inhibition of miR-29 in mice partially rescued the down-regulation of GR during dexamethasone treatment. We discovered that, in addition to modulating GR function under physiologic conditions, pharmacologic glucocorticoid application in inflammatory disease also induced miR-29a expression, correlating with reduced GR levels. This effect was abolished in mice with impaired GR function. In summary, we uncovered a novel GR-miR-29a negative feedback loop conserved between mice and humans, in health and disease. For the first time, we elucidate a microRNA-related mechanism that might contribute to GR dysregulation and resistance in peripheral tissues.-Glantschnig, C., Koenen, M., Gil-Lozano, M., Karbiener, M., Pickrahn, I., Williams-Dautovich, J., Patel, R., Cummins, C. L., Giroud, M., Hartleben, G., Vogl, E., Bluher, M., Tuckermann, J., Uhlenhaut, H., Herzig, S., Scheideler, M. A miR-29a-driven negative feedback loop regulates peripheral glucocorticoid receptor signaling.

ObjectiveSerious non-AIDS disease events (SNAE) are experienced disproportionately by immunologic non-responders (INRs), HIV-infected individuals who do not restore CD4 T cells in blood despite effective viral suppression. We aimed to characterize the inflammatory biomarker profile of the INR phenotype.MethodsBlinded cross-sectional cohort study comparing markers of immune activation and gut homing between INR and non-INR individuals. HIV-positive participants had HIV RNA suppression on antiretroviral therapy and were categorized as either INR (N = 36) or Clinical Responders (“CR”; CD4>350/mm3; N = 47). 18 HIV-negative comparator individuals were included. Cellular markers were assessed by flow cytometry, with soluble markers assessed by ELISA and LC/MS-MS. Multivariable linear regression models estimated the association between INR phenotype and markers, adjusting for age, sex, duration of ART, and recent infection/vaccination.ResultsINR participants demonstrated a reduced CD4/CD8 ratio (pConclusionsPeripheral CD4 non-recovery during long-term treated HIV infection is characterized by elevated CD8 activation and CD4 gut homing. Gut-focused interventions may be warranted in the INR context, and CD8 activation may serve as a surrogate endpoint for clinical interventions.

Nuclear Receptors (NRs) are a family of ligand-activated transcription factors that control the expression of genes involved in a wide range of physiological processes. An atlas detailing the expression of all NRs at the mRNA level was completed in 2006 using quantitative PCR [Bookout et al. Cell 2006]. The comparative measurement of NRs at the protein level, however, has been hindered by the poor quality of commercially available antibodies, as well as the absence of a high throughput method for quantitation. To address this need, we are developing a mass spectrometry-based targeted proteomic assay to quantify the absolute amounts of NR protein in a panel of mouse tissues. NRs were overexpressed in HEK293 cells by transient transfection and protein was isolated. The cell lysates were digested with a combination of trypsin and Lys-C following the Multi-Enzyme Digestion Filter Aided Sample Preparation protocol. The peptides were desalted using an in-house made C18 tip, separated on an EASY-Spray C18 column (75 um x 50 cm, 3Å), and analyzed on a Thermo QExactive HF in Top20 data-dependent acquisition mode. Protein identifications were made using MaxQuant software, and the identifications were mined for members of the NR family. The NR peptides detected were searched against an in silico generated list of optimal NR peptides (filtered for uniqueness, length, absence of post translational modifications, and conservation between human and mouse). The matching peptides were validated by parallel reaction monitoring (PRM) and purchased as synthetic isotopes with a heavy terminal arginine or lysine. Peptide linearity, and lower limits of detection (LLOD) were estimated by spiking digests from a C57Bl/6 mouse liver lysate with increasing amounts of the labeled peptides and analyzing by PRM. Peptides that displayed non-linear behavior were excluded for quantitation. The LLOD were between 100 amol and 1.5 fmol on column. A test panel of tissues (cerebrum, hippocampus, cerebellum, liver, spleen, brown/white adipose, and kidney) showed that we could detect endogenous expression of NRs. To date, we have purchased and validated peptides for 44 of the 49 receptors. We used this assay to quantify the changes in NR protein expression in mouse livers in response to 16 hours of fasting. We found significant changes in the nuclear expression of CAR (3.1-fold increase), RXRβ (1.8-fold increase), SHP (3.9-fold decrease) and RARβ (2.0-fold decrease) in the fasted vs. fed state. Increased CAR activity with fasting was further supported by label-free quantitative proteomics on the same lysates which revealed 210 differentially expressed proteins (2-fold change, p

Non-alcoholic steatohepatitis (NASH), is the form of non-alcoholic fatty liver disease posing risk to progress into serious long term complications. Human and pre-clinical models implicate cellular cholesterol dysregulation playing important role in its development. Mouse model studies suggest synergism between dietary cholesterol and fat in contributing to NASH but the mechanisms remain poorly understood. Our laboratory previously reported the primary importance of hepatic endoplasmic reticulum cholesterol (ER-Chol) in regulating hepatic ER stress by comparing the responses of wild type, Ldlr-/-xLcat+/+ and Ldlr-/-xLcat-/- mice, to a 2% high cholesterol diet (HCD). Here we further investigated the roles of ER-Chol and ER stress in HFHS diet-induced NASH using the same strains. With HFHS diet feeding, both WT and Ldlr-/-xLcat+/+ accumulate ER-Chol in association with ER stress and inflammasome activation but the Ldlr-/-xLcat-/- mice are protected. By contrast, all three strains accumulate cholesterol crystal, in correlation with ER-Chol, albeit less so in Ldlr-/-xLcat-/- mice. By comparison, HCD feeding per se (i) is sufficient to promote steatosis and activate inflammasomes, and (ii) results in dramatic accumulation of cholesterol crystal which is linked to inflammasome activation in Ldlr-/-xLcat-/- mice, independent of ER-Chol. Our data suggest that both dietary fat and cholesterol each independently promote steatosis, cholesterol crystal accumulation and inflammasome activation through distinct but complementary pathways. In vitro studies using palmitate-induced hepatic steatosis in HepG2 cells confirm the key roles by cellular cholesterol in the induction of steatosis and inflammasome activations. These novel findings provide opportunities for exploring a cellular cholesterol-focused strategy for treatment of NASH.

Oxidative derivatives of cholesterol such as 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 25-hydroxycholesterol, and (25S),26-hydroxycholesterol are endogenous ligands for the liver X receptors (LXRα and LXRβ). The LXRs are nuclear hormone receptors known as "intracellular cholesterol sensors" because of their ability to bind to and be activated by oxysterols at circulating concentrations. Oxysterols are expressed in a tissue-specific manner and are generally at least 104 to 106-fold less abundant than cholesterol. Thus, the extraction and measurement of oxysterols from plasma and tissues are facilitated by the removal of bulk sterols by solid phase extraction prior to quantitative analysis by mass spectrometry. In this chapter we describe step by step methods for extracting and quantitating oxysterols from biological samples using electrospray ionization LC/MS/MS.

Current peroxisome proliferator-activated receptor (PPAR)-targeted drugs, such as the PPARγ-directed diabetes drug rosiglitazone, are associated with undesirable side effects due to robust agonist activity in non-target tissues. To find new PPAR ligands with fewer toxic effects, we generated transgenic zebrafish that can be screened in high throughput for new tissue-selective PPAR partial agonists. A structural analog of coenzyme Q10 (idebenone) that elicits spatially restricted partial agonist activity for both PPARα and PPARγ was identified. Coenzyme Q10 was also found to bind and activate both PPARs in a similar fashion, suggesting an endogenous role in relaying the states of mitochondria, peroxisomes and cellular redox to the two receptors. Testing idebenone in a mouse model of type 2 diabetes revealed the ability to reverse fatty liver development. These findings indicate new mechanisms of action for both PPARα and PPARγ, and new potential treatment options for nonalcoholic fatty liver disease (NAFLD) and steatosis. This article has an associated First Person interview with the first author of the paper., Summary: A zebrafish screen identifies a novel PPARα/γ ligand, idebenone, with potential for treatment of fatty liver diseases, as seen by testing it in a mouse model of type 2 diabetes.

Atherosclerosis is a chronic condition associated with cardiovascular disease. While largely identified by the accumulation of lipid-laden foam cells within the aorta later on in life, atherosclerosis develops over several stages and decades. During atherogenesis, various cell types of the aorta acquire a pro-inflammatory phenotype that initiates the cascade of signaling events facilitating the formation of these foam cells. The liver X receptors (LXRs) are nuclear receptors that upon activation induce the expression of transporters responsible for promoting cholesterol efflux. In addition to promoting cholesterol removal from the arterial wall, LXRs have potent anti-inflammatory actions via the transcriptional repression of key pro-inflammatory cytokines. These beneficial functions sparked an interest in the potential to target LXRs and the development of agonists as anti-atherogenic agents. These early studies focused on mediating the contributions of macrophages to the underlying pathogenesis. However, further evidence has since demonstrated that LXRs reduce atherosclerosis through their actions in multiple cell types apart from those monocytes/macrophages that infiltrate the lesion. LXRs and their target genes have profound effects on multiple other cells types of the hematopoietic system. Furthermore, LXRs can also mediate dysfunction within vascular cell types of the aorta including endothelial and smooth muscle cells. Taken together, these studies demonstrate the whole-body benefits of LXR activation with respect to anti-atherogenesis, and that LXRs remain a viable target for the treatment of atherosclerosis, with a reach which extends beyond plaque macrophages.

Aim Omega-3 fatty acid ethyl ester supplements, available by prescription, are common in the treatment of dyslipidaemia in humans. Recent studies show that 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), a metabolite formed from fish oil supplementation, was able to prevent and reverse high fat diet (HFD)-induced fatty liver in mice. In the present study, we investigated the underlying molecular mechanisms responsible for CMPF's hepatic lipid-lowering effects. Materials and methods CD1 male mice were i.p. injected with CMPF (dosage, 6 mg/kg) for 7 days, followed by 5 weeks of a 60% HFD to induce a fatty liver phenotype. Metabolic parameters, liver morphology, lipid content, protein expression and microarray analysis were assessed. We also utilized primary hepatocytes, an in vitro model, to further investigate the direct effects of CMPF on hepatic lipid utilization and biosynthesis. Results CMPF-treated mice display enhanced hepatic lipid clearance while hepatic lipid storage is prevented, thereby protecting against liver lipid accumulation and development of HFD-induced hepatic insulin resistance. Mechanistically, as CMPF enters the liver, it acts as an allosteric acetyl-coA carboxylase (ACC) inhibitor, which directly induces both fatty acid oxidation and hepatic production of fibroblast growth factor 21 (FGF21). A feed-back loop is initiated by CMPF, which exists between ACC inhibition, fatty acid oxidation and production of FGF21. As a consequence, an adaptive decrease in Insig2/SREBP-1c/FAS protein expression results in priming of the liver to prevent a HFD-induced fatty liver phenotype. Conclusion CMPF is a potential driver of hepatic lipid metabolism, preventing diet-induced hepatic lipid deposition and insulin resistance in the long term.

Background The liver X receptors ( LXRs ; α/β) are nuclear receptors known to regulate cholesterol homeostasis and the production of select hematopoietic populations. The objective of this study was to determine the importance of LXR s and a high‐fat high‐cholesterol diet on global hematopoiesis, with special emphasis on endothelial progenitor cells ( EPC s), a vasoreparative cell type that is derived from bone marrow hematopoietic stem cells. Methods and Results Wild‐type and LXR double‐knockout ( Lxr αβ −/− ) mice were fed a Western diet ( WD ) to increase plasma cholesterol levels. In WD ‐fed Lxr αβ −/− mice, flow cytometry and complete blood cell counts revealed that hematopoietic stem cells, a myeloid progenitor, and mature circulating myeloid cells were increased; EPC numbers were significantly decreased. Hematopoietic stem cells from WD ‐fed Lxr αβ −/− mice showed increased cholesterol content, along with increased myeloid colony formation compared with chow‐fed mice. In contrast, EPC s from WD ‐fed Lxr αβ −/− mice also demonstrated increased cellular cholesterol content that was associated with greater expression of the endothelial lineage markers Cd144 and Vegfr2 , suggesting accelerated differentiation of the EPC s. Treatment of human umbilical vein endothelial cells with conditioned medium collected from these EPC s increased THP ‐1 monocyte adhesion. Increased monocyte adhesion to conditioned medium–treated endothelial cells was recapitulated with conditioned medium from Lxr αβ −/− EPC s treated with cholesterol ex vivo, suggesting cholesterol is the main component of the WD inducing EPC dysfunction. Conclusions LXR s are crucial for maintaining the balance of hematopoietic cells in a hypercholesterolemic environment and for mitigating the negative effects of cholesterol on EPC differentiation/secretome changes that promote monocyte‐endothelial adhesion.

The liver x receptors (LXRα/β) are nuclear receptors that are highly expressed in macrophages and endothelial progenitor cells (EPCs). EPCs are bone marrow (BM) derived cells that facilitate endothelial repair through the secretion of factors. The importance of LXRs in preventing atherosclerosis has been attributed to promoting cholesterol efflux from macrophages. However, a role for EPCs in the LXR response has not been explored. We hypothesized that LXR activation in EPCs contributes to their anti-atherogenic effects by preventing endothelial activation during atherogenesis. In the Lxr -/- mice, circulating EPCs were decreased by 27% compared to WT, while myeloid populations were increased. Isolated hematopoietic stem cells treated with LXR agonist (1μM GW3965) ex vivo showed a 36% increase in the number of EPC colonies formed, while myeloid colonies were decreased by 25%. GW3965 treatment during EPC differentiation resulted in a 76% decrease in endothelial lineage marker ( Cd144 and Vegfr2 ) expression. To investigate endothelial cell activation, activated human umbilical vein endothelial cells were incubated with the conditioned media (CM) from GW3965-treated WT EPCs, which reduced THP-1 monocyte adhesion by 36% compared to the CM from vehicle treatment. To determine the effects of the EPC-derived CM in vivo , Ldlr -/- mice were injected with concentrated CM bi-weekly over an 8-week period, which decreased aortic sinus lesion area by 52% compared to the media control injections. Taken together, the loss of LXRs perturbed hematopoietic balance in vivo , while the activation of LXRs were associated with enhanced ex vivo EPC formation. Furthermore, LXR activation altered EPC differentiation and secretome resulting in decreased in vitro endothelial-monocyte adhesion. When administered to atherosclerotic-prone mice, these secreted factors reduced lesion formation in the aortic sinus. These data suggest that LXRs reduce the development of atherosclerosis in part through the release of EPC-derived factors that can reduce endothelial activation.

Hormones such as fibroblast growth factor 21 (FGF21) and glucocorticoids (GCs) play crucial roles in coordinating the adaptive starvation response. Here we examine the interplay between these hormones. It was previously shown that FGF21 induces corticosterone levels in mice by acting on the brain. We now show that this induces the expression of genes required for GC synthesis in the adrenal gland. FGF21 also increases corticosterone secretion from the adrenal in response to ACTH. We further show that the relationship between FGF21 and GCs is bidirectional. GCs induce Fgf21 expression in the liver by acting on the GC receptor (GR). The GR binds in a ligand-dependent manner to a noncanonical GR response element located approximately 4.4 kb upstream of the Fgf21 transcription start site. The GR cooperates with the nuclear fatty acid receptor, peroxisome proliferator-activated receptor-α, to stimulate Fgf21 transcription. GR and peroxisome proliferator-activated receptor-α ligands have additive effects on Fgf21 expression both in vivo and in primary cultures of mouse hepatocytes. We conclude that FGF21 and GCs regulate each other's production in a feed-forward loop and suggest that this provides a mechanism for bypassing negative feedback on the hypothalamic-pituitary-adrenal axis to allow sustained gluconeogenesis during starvation.

Neonicotinoids have become the most widely used class of insecticides world-wide. Although numerous studies have documented neonicotinoid toxicity in bees and other insects, the effects of exposure during early development in mammals remain largely unexplored. We assessed the effects of the neonicotinoid imidacloprid (IMI) in adult male and female mice after in utero and early postnatal exposure. Pregnant mice were infused with IMI (0.5 mg/kg/day) from gestational day 4 to the end of nursing at postnatal day 21. The young adult offspring were studied in a series of biochemical and behavioral tests. To assess reproducibility, the behavioral analyses were conducted in three separate studies using multiple exposed litters. Exposure to IMI reduced fecundity, and in adult offspring, decreased body weight in male but not female pups. Offspring from IMI-treated mothers displayed lower triglycerides, elevated motor activity, enhanced social dominance, reduced depressive-like behavior, and a diminution in social aggression compared to vehicle treated controls. Low levels of IMI were detected in the brains and livers of the treated mothers, while trace levels were detected in some offspring. Our results demonstrate that transient exposure to a neonicotinoid over the early developmental period induces long-lasting changes in behavior and brain function in mice., This study was supported by funds from the Leslie Dan Faculty of Pharmacy, University of Toronto, and the Natural Sciences and Engineering Research Council of Canada.

Liver X receptors (LXRs) α and β are nuclear hormone receptors that are widely expressed in the kidney. They promote cholesterol efflux from cells and inhibit inflammatory responses by regulating gene transcription. Here, we hypothesised (1) that LXR deficiency would promote renal decline in a mouse model of diabetes by accelerating intraglomerular cholesterol accumulation and, conversely, (2) that LXR agonism would attenuate renal decline in diabetes. Diabetes was induced with streptozotocin (STZ) and maintained for 14 weeks in Lxrα/β +/+ (Lxrα, also known as Nr1h3; Lxrβ, also known as Nr1h2) and Lxrα/β −/− mice. In addition, STZ-injected DBA/2J mice were treated with vehicle or the LXR agonist N,N-dimethyl-hydroxycholenamide (DMHCA) (80 mg/kg daily) for 10 weeks. To determine the role of cholesterol in diabetic nephropathy (DN), mice were placed on a Western diet after hyperglycaemia developed. Even in the absence of diabetes, Lxrα/β −/− mice exhibited a tenfold increase in the albumin:creatinine ratio and a 40-fold increase in glomerular lipid accumulation compared with Lxrα/β +/+ mice. When challenged with diabetes, Lxrα/β −/− mice showed accelerated mesangial matrix expansion and glomerular lipid accumulation, with upregulation of inflammatory and oxidative stress markers. In the DN-sensitive STZ DBA/2J mouse model, DMHCA treatment significantly decreased albumin and nephrin excretion (by 50% each), glomerular lipids and plasma triacylglycerol (by 70%) and cholesterol (by 48%); it also decreased kidney inflammatory and oxidative stress markers compared with vehicle-treated mice. These data support the idea that LXR plays an important role in the normal and diabetic kidney, while showing that LXR, through its inhibitory effect on inflammation and cholesterol accumulation in glomeruli, could also be a novel therapeutic target for DN.

The membrane-associated drug transporter P-glycoprotein (P-gp) plays an essential role in drug efflux from the brain. Induction of this protein at the blood-brain barrier (BBB) could further affect the ability of a drug to enter the brain. At present, P-gp induction mediated by antiretroviral drugs at the BBB has not been fully investigated. Since P-gp expression is regulated by ligand-activated nuclear receptors, i.e., human pregnane X receptor (hPXR) and human constitutive androstane receptor (hCAR), these receptors could represent potential pathways involved in P-gp induction by antiretroviral drugs. The aims of this study were (i) to determine whether antiretroviral drugs currently used in HIV pharmacotherapy are ligands for hPXR or hCAR and (ii) to examine P-gp function and expression in human brain microvessel endothelial cells treated with antiretroviral drugs identified as ligands of hPXR and/or hCAR. Luciferase reporter gene assays were performed to examine the activation of hPXR and hCAR by antiretroviral drugs. The hCMEC/D3 cell line, which is known to display several morphological and biochemical properties of the BBB in humans, was used to examine P-gp induction following 72 h of exposure to these agents. Amprenavir, atazanavir, darunavir, efavirenz, ritonavir, and lopinavir were found to activate hPXR, whereas abacavir, efavirenz, and nevirapine were found to activate hCAR. P-gp expression and function were significantly induced in hCMEC/D3 cells treated with these drugs at clinical concentrations in plasma. Together, our data suggest that P-gp induction could occur at the BBB during chronic treatment with antiretroviral drugs identified as ligands of hPXR and/or hCAR.

Synthetic glucocorticoids (GCs), including dexamethasone (DEX), are powerful anti-inflammatory drugs. Long-term use of GCs, however, can result in metabolic side effects such as hyperglycemia, hepatosteatosis, and insulin resistance. The GC receptor (GR) and liver X receptors (LXRα and LXRβ) regulate overlapping genes involved in gluconeogenesis and inflammation. We have previously shown that Lxrβ-/- mice are resistant to the diabetogenic effects of DEX but still sensitive to its immunosuppressive actions. To determine whether this finding could be exploited for therapeutic intervention, we treated mice with GSK2033, a pan-LXR antagonist, alone or combined with DEX. GSK2033 suppressed GC-induced gluconeogenic gene expression without affecting immune-responsive GR target genes. The suppressive effect of GSK2033 on DEX-induced gluconeogenic genes was specific to LXRβ, was liver cell autonomous, and occurred in a target gene-specific manner. Compared with DEX treatment alone, the coadministration of GSK2033 with DEX decreased the recruitment of GR and its accessory factors MED1 and C/EBPβ to the phosphoenolpyruvate carboxykinase promoter. However, GSK2033 had no effect on DEX-mediated suppression of inflammatory genes expressed in the liver or in mouse primary macrophages stimulated with lipopolysaccharides. In conclusion, our study provides evidence that the gluconeogenic and immunosuppressive actions of GR activation can be mechanistically dissociated by pharmacological antagonism of LXRβ. Treatment with an LXRβ antagonist could allow the safer use of existing GC drugs in patients requiring chronic dosing of anti-inflammatory agents for the treatment of diseases such as rheumatoid arthritis and inflammatory bowel disease.

Hypercholesterolemia is a key risk factor for atherosclerosis and leads to the uptake of native and oxidized low-density lipoprotein (oxLDL) by macrophages (Mϕs) and foam cell formation. Inflammatory processes accompany Mϕ foam cell formation in the artery wall, yet the relationship between Mϕ lipid loading and their response to inflammatory stimuli remains elusive. We investigated proinflammatory gene expression in thioglycollate-elicited peritoneal Mϕs, bone marrow–derived Mϕs and dendritic cells, and RAW264.7 cells. Loading with oxLDL did not induce peritoneal Mϕ apoptosis or modulate basal-level expression of proinflammatory genes. Upon stimulation of TLR4, the rapid induction of IFN-β was inhibited in cells loaded with oxLDL, whereas the induction of other proinflammatory genes by TLR4 (LPS), TLR3 (polyriboinosinic-polyribocytidylic acid), TLR2 (Pam3CSK4), and TLR9 (CpG) remained comparable within the first 2 h. Subsequently, the expression of a subset of proinflammatory genes (e.g., IL-1β, IL-6, CCL5) was reduced in oxLDL-loaded cells at the level of transcription. This phenomenon was partially dependent on NF erythroid 2–related factor 2 (NRF2) but not on nuclear liver X receptors α and β (LXRα,β), peroxisome proliferator-activated receptor-γ (PPARγ), and activating transcription factor 3 (ATF3). LPS-induced NF-κB reporter activity and intracellular signaling by NF-κB and MAPK pathways were comparable in oxLDL-loaded Mϕs, yet the binding of p65/RelA (the prototypic NF-κB family member) was reduced at IL-6 and CCL5 promoters. This study revealed that oxLDL loading of Mϕs negatively regulates transcription at late stages of TLR-induced proinflammatory gene expression and implicates epigenetic mechanisms such as histone deacetylase activity.

Networks of coordinated alternative splicing (AS) events play critical roles in development and disease. However, a comprehensive knowledge of the factors that regulate these networks is lacking. We describe a high-throughput system for systematically linking trans-acting factors to endogenous RNA regulatory events. Using this system, we identify hundreds of factors associated with diverse regulatory layers that positively or negatively control AS events linked to cell fate. Remarkably, more than one-third of the regulators are transcription factors. Further analyses of the zinc finger protein Zfp871 and BTB/POZ domain transcription factor Nacc1, which regulate neural and stem cell AS programs, respectively, reveal roles in controlling the expression of specific splicing regulators. Surprisingly, these proteins also appear to regulate target AS programs via binding RNA. Our results thus uncover a large “missing cache” of splicing regulators among annotated transcription factors, some of which dually regulate AS through direct and indirect mechanisms.

Endothelial progenitor cells (EPCs), critical for mediating vascular repair, are dysfunctional in a hyperglycemic and/or hypercholesterolemic environment. Their dysfunction contributes to the progression of diabetic macro- and microvascular complications. Activation of “cholesterol-sensing” nuclear receptors, the liver X receptors (LXRα/LXRβ), protects against atherosclerosis by transcriptional regulation of genes important in promoting cholesterol efflux and inhibiting inflammation. We hypothesized that LXR activation with a synthetic ligand would correct diabetes-induced EPC dysfunction and improve diabetic retinopathy. Studies were performed in streptozotocin (STZ)-injected DBA/2J mice fed a high-fat Western diet (DBA/STZ/WD) and treated with the LXR agonist GW3965 and in LXRα−/−, LXRβ−/−, and LXRα/β−/− mice. Retinas were evaluated for number of acellular capillaries and glial fibrillary acidic protein (GFAP) immunoreactivity. Bone marrow EPCs were analyzed for migratory function and gene expression. Compared with vehicle-treated DBA/STZ/WD mice, GW3965 treated mice showed fewer acellular capillaries and reduced GFAP expression. These mice also exhibited enhanced EPC migration and restoration of inflammatory and oxidative stress genes toward nondiabetic levels. LXRα−/−, LXRβ−/−, and LXRα/β−/− mice developed acellular capillaries and EPC dysfunction similar to the DBA/STZ/WD mice. These studies support a key role for LXR in retinal and bone marrow progenitor dysfunction associated with type 1 diabetes. LXR agonists may represent promising pharmacologic targets for correcting retinopathy and EPC dysfunction.

We recently reported that lecithin:cholesterol acyltransferase (LCAT) knock-out mice, particularly in the LDL receptor knock-out background, are hypersensitive to insulin and resistant to high fat diet-induced insulin resistance (IR) and obesity. We demonstrated that chow-fed Ldlr-/-xLcat+/+ mice have elevated hepatic endoplasmic reticulum (ER) stress, which promotes IR, compared with wild-type controls, and this effect is normalized in Ldlr-/-xLcat-/- mice. In the present study, we tested the hypothesis that hepatic ER cholesterol metabolism differentially regulates ER stress using these models. We observed that the Ldlr-/-xLcat+/+ mice accumulate excess hepatic total and ER cholesterol primarily attributed to increased reuptake of biliary cholesterol as we observed reduced biliary cholesterol in conjunction with decreased hepatic Abcg5/g8 mRNA, increased Npc1l1 mRNA, and decreased Hmgr mRNA and nuclear SREBP2 protein. Intestinal NPC1L1 protein was induced. Expression of these genes was reversed in the Ldlr-/-xLcat-/- mice, accounting for the normalization of total and ER cholesterol and ER stress. Upon feeding a 2% high cholesterol diet (HCD), Ldlr-/-xLcat-/- mice accumulated a similar amount of total hepatic cholesterol compared with the Ldlr-/-xLcat+/+ mice, but the hepatic ER cholesterol levels remained low in conjunction with being protected from HCD-induced ER stress and IR. Hepatic ER stress correlates strongly with hepatic ER free cholesterol but poorly with hepatic tissue free cholesterol. The unexpectedly low ER cholesterol seen in HCD-fed Ldlr-/-xLcat-/- mice was attributable to a coordinated marked up-regulation of ACAT2 and suppressed SREBP2 processing. Thus, factors influencing the accumulation of ER cholesterol may be important for the development of hepatic insulin resistance.

SummaryPreselection of compounds that are more likely to induce a phenotype can increase the efficiency and reduce the costs for model organism screening. To identify such molecules, we screened ∼81,000 compounds in Saccharomyces cerevisiae and identified ∼7500 that inhibit cell growth. Screening these growth-inhibitory molecules across a diverse panel of model organisms resulted in an increased phenotypic hit-rate. These data were used to build a model to predict compounds that inhibit yeast growth. Empirical and in silico application of the model enriched the discovery of bioactive compounds in diverse model organisms. To demonstrate the potential of these molecules as lead chemical probes, we used chemogenomic profiling in yeast and identified specific inhibitors of lanosterol synthase and of stearoyl-CoA 9-desaturase. As community resources, the ∼7500 growth-inhibitory molecules have been made commercially available and the computational model and filter used are provided.

Summary Bile acids are cholesterol-derived signaling molecules that regulate mammalian metabolism through sterol-sensing nuclear receptor transcription factors. In C. elegans, bile acid-like steroids called dafachronic acids (DAs) control developmental timing and longevity by activating the nuclear receptor DAF-12. However, little is known about the biosynthesis of these molecules. Here, we show that the DAF-36/Rieske oxygenase works at the first committed step, converting cholesterol to 7-dehydrocholesterol. Its elucidation as a cholesterol 7-desaturase provides crucial biochemical evidence that such oxygenases are key steroidogenic enzymes. By controlling DA production, DAF-36 regulates DAF-12 activities for reproductive development and longevity and may illuminate related pathways in metazoans.