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.

Multilayered Control of Alternative Splicing Regulatory Networks by Transcription Factors.

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.

ARGLU1 is a transcriptional coactivator and splicing regulator important for stress hormone signaling and development.

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.

3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) prevents high fat diet-induced insulin resistance via maintenance of hepatic lipid homeostasis.

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.

Cellular cholesterol accumulation modulates high fat high sucrose (HFHS) diet-induced ER stress and hepatic inflammasome activation in the development of non-alcoholic steatohepatitis.

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.

Glucocorticoids and Metabolic Control.

In response to stress, the central nervous system initiates a signaling cascade, which leads to the production of glucocorticoids (GCs). GCs act through the glucocorticoid receptor (GR) to coordinate the appropriate cellular response with the primary goal of mobilizing the storage forms of carbon precursors to generate a continuous glucose supply for the brain. Although GCs are critical for maintaining energy homeostasis, excessive GC stimulation leads to a number of undesirable side effects, including hyperglycemia, insulin resistance, fatty liver, obesity, and muscle wasting leading to severe metabolic dysfunction. Summarized below are the diverse metabolic roles of glucocorticoids in energy homeostasis and dysregulation, focusing specifically on glucose, lipid, and protein metabolism.

Vitamin D receptor activation down-regulates the small heterodimer partner and increases CYP7A1 to lower cholesterol.

BACKGROUND & AIMS: Little is known about the effects of the vitamin D receptor (VDR) on hepatic activity of human cholesterol 7α-hydroxylase (CYP7A1) and cholesterol metabolism. We studied these processes in mice in vivo and mouse and human hepatocytes. METHODS: Farnesoid X receptor (Fxr)(-/-), small heterodimer partner (Shp)(-/-), and C57BL/6 (wild-type control) mice were fed normal or Western diets for 3 weeks and were then given intraperitoneal injections of vehicle (corn oil) or 1α,25-dihydroxyvitamin D3 (1,25[OH]2D3; 4 doses, 2.5 µg/kg, every other day). Plasma and tissue samples were collected and levels of Vdr, Shp, Cyp7a1, Cyp24a1, and rodent fibroblast growth factor (Fgf) 15 expression, as well as levels of cholesterol, were measured. We studied the regulation of Shp by Vdr using reporter and mobility shift assays in transfected human embryonic kidney 293 cells, quantitative polymerase chain reaction with mouse tissues and mouse and human hepatocytes, and chromatin immunoprecipitation assays with mouse liver. RESULTS: We first confirmed the presence of Vdr mRNA and protein expression in livers of mice. In mice fed normal diets and given injections of 1,25(OH)2D3, liver and plasma concentrations of 1,25(OH)2D3 increased and decreased in unison. Changes in hepatic Cyp7a1 messenger RNA (mRNA) correlated with those of Cyp24a1 (a Vdr target gene) and inversely with Shp mRNA, but not ileal Fgf15 mRNA. Similarly, incubation with 1,25(OH)2D3 increased levels of Cyp24a1/CYP24A1 and Cyp7a1/CYP7A1 mRNA in mouse and human hepatocytes, and reduced levels of Shp mRNA in mouse hepatocytes. In Fxr(-/-) and wild-type mice with hypercholesterolemia, injection of 1,25(OH)2D3 consistently reduced levels of plasma and liver cholesterol and Shp mRNA, and increased hepatic Cyp7a1 mRNA and protein; these changes were not observed in Shp(-/-) mice given 1,25(OH)2D3 and fed Western diets. Truncation of the human small heterodimer partner (SHP) promoter and deletion analyses revealed VDR-dependent inhibition of SHP, and mobility shift assays showed direct binding of VDR to enhancer regions of SHP. In addition, chromatin immunoprecipitation analysis of livers from mice showed that injection of 1,25(OH)2D3 increased recruitment of Vdr and rodent retinoid X receptor to the Shp promoter. CONCLUSIONS: Activation of the VDR represses hepatic SHP to increase levels of mouse and human CYP7A1 and reduce cholesterol.

A novel 3-hydroxysteroid dehydrogenase that regulates reproductive development and longevity.

Endogenous small molecule metabolites that regulate animal longevity are emerging as a novel means to influence health and life span. In C. elegans, bile acid-like steroids called the dafachronic acids (DAs) regulate developmental timing and longevity through the conserved nuclear hormone receptor DAF-12, a homolog of mammalian sterol-regulated receptors LXR and FXR. Using metabolic genetics, mass spectrometry, and biochemical approaches, we identify new activities in DA biosynthesis and characterize an evolutionarily conserved short chain dehydrogenase, DHS-16, as a novel 3-hydroxysteroid dehydrogenase. Through regulation of DA production, DHS-16 controls DAF-12 activity governing longevity in response to signals from the gonad. Our elucidation of C. elegans bile acid biosynthetic pathways reveals the possibility of novel ligands as well as striking biochemical conservation to other animals, which could illuminate new targets for manipulating longevity in metazoans.

Liver X receptors preserve renal glomerular integrity under normoglycaemia and in diabetes in mice.

AIMS/HYPOTHESIS: Liver X receptors (LXRs) α and ß 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. METHODS: Diabetes was induced with streptozotocin (STZ) and maintained for 14 weeks in Lxrα/ß (+/+) (Lxrα, also known as Nr1h3; Lxrß, also known as Nr1h2) and Lxrα/ß (-/-) 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. RESULTS: Even in the absence of diabetes, Lxrα/ß (-/-) mice exhibited a tenfold increase in the albumin:creatinine ratio and a 40-fold increase in glomerular lipid accumulation compared with Lxrα/ß (+/+) mice. When challenged with diabetes, Lxrα/ß (-/-) 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. CONCLUSIONS/INTERPRETATION: 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.

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.

Lecithin:cholesterol acyltransferase deficiency protects against cholesterol-induced hepatic endoplasmic reticulum stress in mice.

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.

Dafadine inhibits DAF-9 to promote dauer formation and longevity of Caenorhabditis elegans.

The DAF-9 cytochrome P450 is a key regulator of dauer formation, developmental timing and longevity in the nematode Caenorhabditis elegans. Here we describe the first identified chemical inhibitor of DAF-9 and the first reported small-molecule tool that robustly induces dauer formation in typical culture conditions. This molecule (called dafadine) also inhibits the mammalian ortholog of DAF-9(CYP27A1), suggesting that dafadine can be used to interrogate developmental control and longevity in other animals.

Ketamine supresses REM sleep and markedly increases EEG gamma oscillations in the Wistar Kyoto rat model of treatment-resistant depression.

Wistar-Kyoto (WKY) rats exhibit depression-like characteristics and decreased sensitivity to monoamine-based antidepressants, making them a suitable model of treatment-resistant depression (TRD). Ketamine has emerged recently as a rapidly acting antidepressant with high efficacy in TRD. Our aim was to determine whether subanaesthetic doses of ketamine can correct sleep and electroencephalogram (EEG) alterations in WKY rats and whether any ketamine-induced changes differentially affect WKY rats compared to Sprague-Dawley (SD) rats. Thus, we surgically implanted 8 SD and 8 WKY adult male rats with telemetry transmitters and recorded their EEG, electromyogram, and locomotor activity after vehicle or ketamine (3, 5 or 10 mg/kg, s.c.) treatment. We also monitored the plasma concentration of ketamine and its metabolites, norketamine and hydroxynorketamine in satellite animals. We found that WKY rats have an increased amount of rapid eye movement (REM) sleep, fragmented sleep-wake pattern, and increased EEG delta power during non-REM sleep compared to SD rats. Ketamine suppressed REM sleep and increased EEG gamma power during wakefulness in both strains, but the gamma increase was almost twice as large in WKY rats than in SD rats. Ketamine also increased beta oscillations, but only in WKY rats. These differences in sleep and EEG are unlikely to be caused by dissimilarities in ketamine metabolism as the plasma concentrations of ketamine and its metabolites were similar in both strains. Our data suggest an enhanced antidepressant-like response to ketamine in WKY rats, and further support the predictive validity of acute REM sleep suppression as a measure of antidepressant responsiveness.

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.

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.

Low Doses of Psilocybin and Ketamine Enhance Motivation and Attention in Poor Performing Rats: Evidence for an Antidepressant Property.

Long term benefits following short-term administration of high psychedelic doses of serotonergic and dissociative hallucinogens, typified by psilocybin and ketamine respectively, support their potential as treatments for psychiatric conditions such as major depressive disorder. The high psychedelic doses induce perceptual experiences which are associated with therapeutic benefit. There have also been anecdotal reports of these drugs being used at what are colloquially referred to as "micro" doses to improve mood and cognitive function, although currently there are recognized limitations to their clinical and preclinical investigation. In the present studies we have defined a low dose and plasma exposure range in rats for both ketamine (0.3-3 mg/kg [10-73 ng/ml]) and psilocybin/psilocin (0.05-0.1 mg/kg [7-12 ng/ml]), based on studies which identified these as sub-threshold for the induction of behavioral stereotypies. Tests of efficacy were focused on depression-related endophenotypes of anhedonia, amotivation and cognitive dysfunction using low performing male Long Evans rats trained in two food motivated tasks: a progressive ratio (PR) and serial 5-choice (5-CSRT) task. Both acute doses of ketamine (1-3 mg/kg IP) and psilocybin (0.05-0.1 mg/kg SC) pretreatment increased break point for food (PR task), and improved attentional accuracy and a measure of impulsive action (5-CSRT task). In each case, effect size was modest and largely restricted to test subjects characterized as "low performing". Furthermore, both drugs showed a similar pattern of effect across both tests. The present studies provide a framework for the future study of ketamine and psilocybin at low doses and plasma exposures, and help to establish the use of these lower concentrations of serotonergic and dissociative hallucinogens both as a valid scientific construct, and as having a therapeutic utility.

Impact of acute streptozotocin-induced diabetes on ABC transporter expression in rats.

Hepatic ABC efflux transporters control the cellular uptake (in basolateral membranes) and excretion (in apical membranes) of many substrates. Since type-1 diabetes mellitus (T1DM) is associated with altered hepatobiliary excretion of many endogenous and exogenous substances, we examined key hepatic ABC transporters and levels of the endogenous substrate glutathione in rats with acute streptozotocin-induced T1DM. Renal transporters and inflammatory markers were also examined. Abcb1, Abcc1-4, and Abcg2 were measured using qRT-PCR. Glutathione was measured in liver tissue, plasma, and urine. Inflammatory markers, including C-reactive protein (CRP), were measured in plasma via ELISA. In diabetic rats, Abcb1a, Abcc2, and Abcg2 (apical) were decreased, while Abcc4 (basolateral) was increased. Abcb1a and Abcc2 inversely correlated with plasma CRP. Diabetic and control rats exhibited similar hepatic glutathione, but levels in diabetic plasma were lower. When standardized to urinary output, diabetic rats excreted 6.7-fold more glutathione in urine than controls. Renal transporter levels were normal in diabetic rats. Results show apical transporters involved in hepatobiliary excretion are downregulated in T1DM, possibly through an inflammation-mediated process. Findings suggest that there may be a vectorial shift from hepatic to renal excretion for some substrates in T1DM.

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.

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.

Strategies and limitations associated with in vitro characterization of vitamin D receptor activators.

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.