A novel fatty acid mimetic with pan-PPAR partial agonist activity inhibits diet-induced obesity and metabolic dysfunction-associated steatotic liver disease.

OBJECTIVE: The prevalence of metabolic diseases is increasing globally at an alarming rate; thus, it is essential that effective, accessible, low-cost therapeutics are developed. Peroxisome proliferator-activated receptors (PPARs) are transcription factors that tightly regulate glucose homeostasis and lipid metabolism and are important drug targets for the treatment of type 2 diabetes and dyslipidemia. We previously identified LDT409, a fatty acid-like compound derived from cashew nut shell liquid, as a novel pan-active PPARα/γ/δ compound. Herein, we aimed to assess the efficacy of LDT409 in vivo and investigate the molecular mechanisms governing the actions of the fatty acid mimetic LDT409 in diet-induced obese mice. METHODS: C57Bl/6 mice (6-11-month-old) were fed a chow or high fat diet (HFD) for 4 weeks; mice thereafter received once daily intraperitoneal injections of vehicle, 10 mg/kg Rosiglitazone, 40 mg/kg WY14643, or 40 mg/kg LDT409 for 18 days while continuing the HFD. During treatments, body weight, food intake, glucose and insulin tolerance, energy expenditure, and intestinal lipid absorption were measured. On day 18 of treatment, tissues and plasma were collected for histological, molecular, and biochemical analysis. RESULTS: We found that treatment with LDT409 was effective at reversing HFD-induced obesity and associated metabolic abnormalities in mice. LDT409 lowered food intake and hyperlipidemia, while improving insulin tolerance. Despite being a substrate of both PPARα and PPARγ, LDT409 was crucial for promoting hepatic fatty acid oxidation and reducing hepatic steatosis in HFD-fed mice. We also highlighted a role for LDT409 in white and brown adipocytes in vitro and in vivo where it decreased fat accumulation, increased lipolysis, induced browning of WAT, and upregulated thermogenic gene Ucp1. Remarkably, LDT409 reversed HFD-induced weight gain back to chow-fed control levels. We determined that the LDT409-induced weight-loss was associated with a combination of increased energy expenditure (detectable before weight loss was apparent), decreased food intake, increased systemic fat utilization, and increased fecal lipid excretion in HFD-fed mice. CONCLUSIONS: Collectively, LDT409 represents a fatty acid mimetic that generates a uniquely favorable metabolic response for the treatment of multiple abnormalities including obesity, dyslipidemia, metabolic dysfunction-associated steatotic liver disease, and diabetes. LDT409 is derived from a highly abundant natural product-based starting material and its development could be pursued as a therapeutic solution to the global metabolic health crisis.

PGP-14 establishes a polar lipid permeability barrier within the C. elegans pharyngeal cuticle.

The cuticles of ecdysozoan animals are barriers to material loss and xenobiotic insult. Key to this barrier is lipid content, the establishment of which is poorly understood. Here, we show that the p-glycoprotein PGP-14 functions coincidently with the sphingomyelin synthase SMS-5 to establish a polar lipid barrier within the pharyngeal cuticle of the nematode C. elegans. We show that PGP-14 and SMS-5 are coincidentally expressed in the epithelium that surrounds the anterior pharyngeal cuticle where PGP-14 localizes to the apical membrane. pgp-14 and sms-5 also peak in expression at the time of new cuticle synthesis. Loss of PGP-14 and SMS-5 dramatically reduces pharyngeal cuticle staining by Nile Red, a key marker of polar lipids, and coincidently alters the nematode's response to a wide-range of xenobiotics. We infer that PGP-14 exports polar lipids into the developing pharyngeal cuticle in an SMS-5-dependent manner to safeguard the nematode from environmental insult.

Regulation of Alternative Splicing by Steroid Hormones.

Steroid hormone signaling pathways are critical for organismal development and act through binding to nuclear receptors (NRs) 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-messenger RNA. 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 hormones binding to their NRs affected both gene transcription and alternative splicing outcomes. The advent of exon arrays and next-generation sequencing has allowed researchers to observe the effect 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 both in physiological and pathophysiologic states. Studying the effect of steroid hormones on alternative splicing is a fruitful avenue for research that should be exploited to discover new targets for therapeutic intervention.

Splicing factor SRSF1 deficiency in the liver triggers NASH-like pathology and cell death.

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.

The omega-3 hydroxy fatty acid 7(S)-HDHA is a high-affinity PPARα ligand that regulates brain neuronal morphology.

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.

Fresh insights into glucocorticoid-induced diabetes mellitus and new therapeutic directions.

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.

AKR1D1 knockout mice develop a sex-dependent metabolic phenotype.

Steroid 5ß-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.

Macrophage Jak2 deficiency accelerates atherosclerosis through defects in cholesterol efflux.

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.

Phenolic Lipids Derived from Cashew Nut Shell Liquid to Treat Metabolic Diseases.

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. In vivo studies using PPAR-expressing zebrafish embryos identified CNSL derivatives with varying tissue-specific activities. LDT409 (23) is an analogue of cardanol with partial agonist activity for PPARα and PPARγ. Pharmacokinetic profiling showed that 23 is orally bioavailable with a half-life of 4 h in mice. CNSL derivatives represent a sustainable source of selective PPAR modulators with balanced intermediate affinities (EC50 ∼ 100 nM to 10 µM) that provide distinct and favorable gene activation profiles for the treatment of diabetes and obesity.

Activation and gut-homing of peripheral T cells in HIV immunologic non-responders despite long term viral suppression.

OBJECTIVE: Serious 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. METHODS: Blinded 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. RESULTS: INR participants demonstrated a reduced CD4/CD8 ratio (p<0.001), 35% more CD8 activation (p = 0.02), 36% greater α4ß7+ CD4 T cells (p<0.01), 54% more HLA-DR+ CD4 T cells (p<0.001), and 20% higher plasma VCAM (p<0.01) compared to CRs. The INR phenotype was not associated with levels of Kyn/Trp, CRP, TNF, IFNγ, IL-8, IL-6, sCD14, D-Dimer, I-FABP, MCP-1, ICAM or CD8%HLA-DR+. CONCLUSIONS: Peripheral 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.

4-Phenylbutyric acid improves free fatty acid-induced hepatic insulin resistance in vivo.

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 ß (IKKß) inhibitor. Different from our previous results with the antioxidant and IKKß 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.

Exploring the transformability of polymer-lipid hybrid nanoparticles and nanomaterial-biology interplay to facilitate tumor penetration, cellular uptake and intracellular targeting of anticancer drugs.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Transformability of PLN and lipid-biology interplay can be exploited to design new nanocarriers for effective drug delivery to tumor cells and intracellular targets.

The secretome of liver X receptor agonist-treated early outgrowth cells decreases atherosclerosis in Ldlr-/- mice.

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 (Lxrαß-/-) 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 Lxrαß-/- 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.

Tetracosahexaenoylethanolamide, a novel N-acylethanolamide, is elevated in ischemia and increases neuronal output.

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.

Selective peroxisome proliferator-activated receptor-gamma modulator, INT131 exhibits anti-inflammatory effects in an EcoHIV mouse model.

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-1ß, 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.

Essential role of STAT-3 dependent NF-κB activation on IL-6-mediated downregulation of hepatic transporters.

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 h post-treatment. This temporal mismatch suggested that the downregulation of transporters following IL-6 at 6 h 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 min prior to receiving a single dose of IL-6 or saline. Mice were euthanized after 6 h 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.

The marginal cells of the Caenorhabditis elegans pharynx scavenge cholesterol and other hydrophobic small molecules.

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.

Peroxisome Proliferator-Activated Receptor-gamma agonists exhibit anti-inflammatory and antiviral effects in an EcoHIV mouse model.

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-1ß, 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-1ß, 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.

Quantification of Oxysterol Nuclear Receptor Ligands by LC/MS/MS.

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 LXRß). 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.

A miR-29a-driven negative feedback loop regulates peripheral glucocorticoid receptor signaling.

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., Blüher, M., Tuckermann, J., Uhlenhaut, H., Herzig, S., Scheideler, M. A miR-29a-driven negative feedback loop regulates peripheral glucocorticoid receptor signaling.