Biomarkers for PPARalpha activation: A previous study identified the peroxisome proliferator-activated receptor alpha (PPARalpha) is involved in the control of lipid homeostasis. PPARalpha is activated during fasting where is controls expression of genes involved if fatty acid transport and metabolism. Metabolomics, using the ultra-performance chromatography-linked electrospray ionization quadrupole time-of-flight mass spectrometry (UPLCESI-QTOFMS) platform, was employed to discover biomarkers fof PPARalpha activation. The biomarkers 21-steroid carboxylic acids 11beta-hydroxy-3,20-dioxopregn-4-en-21-oic acid (HDOPA) and 11beta,20-dihydroxy-3-oxo-pregn-4-en-21-oic acid (DHOPA) were found to be very abundant in urine of mice treated with the PPARalpha activator Wy-14,643. The molecular mechanism and the metabolic pathway of HDOPA and DHOPA production were determined. The PPARalpha-specific time-dependent increases in HDOPA and 20alpha-DHOPA paralleled the development of adrenal cortex hyperplasia, hypercortisolism, and spleen atrophy, which was attenuated in adrenalectomized mice. Wy-14,643 activation of PPARalpha induced hepatic FGF21, which caused increased neuropeptide Y and agouti-related protein mRNAs in the hypothalamus, stimulation of the agouti-related protein/neuropeptide Y neurons, and activation of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in increased adrenal cortex hyperplasia and corticosterone production, revealing a link between PPARalpha and the HPA axis in controlling energy homeostasis and immune regulation. Corticosterone was demonstrated as the precursor of 21-carboxylic acids both in vivo and in vitro. Under PPARalpha activation, the classic reductive metabolic pathway of corticosterone was suppressed, whereas an alternative oxidative pathway was uncovered that leads to the sequential oxidation on carbon 21 resulting in HDOPA. The latter was then reduced to the end product 20alpha-DHOPA. Hepatic cytochromes P450, aldehyde dehydrogenase (ALDH3A2), and 21-hydroxysteroid dehydrogenase (AKR1C18) were found to be involved in this pathway. Activation of PPARalpha resulted in the induction of Aldh3a2 and Akr1c18, both of which were confirmed as target genes through introduction of promoter luciferase reporter constructs into mouse livers in vivo. This study revealed downstream metabolic biomarkers for PPARalpha and the corresponding upstream molecular mechanisms. Biomarkers for human PPARalpha activation: Metabolomics was also performed in humans administered the fibrate drug and PPARalpha activator fenofibrate in order to identify latent, endogenous biomarkers of PPARalpha activation associated with increased fatty acid beta-oxidation. Healthy human volunteers were given fenofibrate orally for 2 weeks and their urine was profiled by UPLCESI-QTOFMS. Biomarkers identified by the machine learning algorithm random forests included significant depletion by day 14 of both pantothenic acid ( greater than 5-fold) and acetylcarnitine ( greater than 20-fold), observations that are consistent with known targets of PPARalpha including pantothenate kinase and genes encoding proteins involved in the transport and synthesis of acylcarnitines. It was also concluded that serum cholesterol (-12.7%), triglycerides (-25.6%), uric acid (-34.7%), together with urinary propylcarnitine ( greater than 10-fold), isobutyrylcarnitine ( greater than 2.5-fold), (S)-(+)-2-methylbutyrylcarnitine (5-fold), and isovalerylcarnitine ( greater than 5-fold) were all reduced by day 14. Specificity of these biomarkers as indicators of PPARalpha activation was demonstrated using the Ppara-null mouse. Urinary pantothenic acid and acylcarnitines may prove useful indicators of PPARalpha-induced fatty acid beta-oxidation in humans. This study illustrates the utility of a pharmacometabolomic approach to understand drug effects on lipid metabolism in both human populations and in inbred mouse models. Biomarkers for Farnesoid X receptor (FXR) activation: FXR is a nuclear receptor that regulates the expression of genes involved in synthesis, metabolism, and transport of bile acids and thus plays a major role in maintaining bile acid homeostasis. Metabolomic responses monitored by UPLCESI-QTOFMS were investigated in urine of wild-type and Fxr-null mice fed cholic acid, an FXR ligand. Multivariate data analysis between wild-type and Fxr-null mice on a cholic acid diet revealed that the most increased ions were metabolites of p-cresol (4-methylphenol), corticosterone, and cholic acid in Fxr-null mice. The structural identities of the above metabolites were confirmed by chemical synthesis and by comparing retention time (RT) and/or tandem mass fragmentation patterns of the urinary metabolites with the authentic standards. Tauro-3alpha,6,7alpha,12alpha-tetrol (3alpha,6,7alpha,12alpha-tetrahydroxy-5beta-cholestan-26-oyltaurine), one of the most increased metabolites in Fxr-null mice on a CA diet, is a marker for efficient hydroxylation of toxic bile acids possibly through induction of Cyp3a11. A cholestatic model induced by lithocholic acid revealed that enhanced expression of Cyp3a11 is the major defense mechanism to detoxify cholestatic bile acids in Fxr-null mice. These results will be useful for identification of biomarkers for cholestasis and for determination of adaptive molecular mechanisms in cholestasis. Human pregnane X receptor (PXR) has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Rifaximin, a human PXR activator, is in clinical trials for treatment of IBD and has demonstrated efficacy in Crohn's disease and active ulcerative colitis. In the current study, the protective and therapeutic role of rifaximin in IBD and its respective mechanism were investigated. PXR-humanized (hPXR), wild-type, and Pxr-null mice were treated with rifaximin in the dextran sulfate sodium (DSS)-induced and trinitrobenzene sulfonic acid (TNBS)-induced IBD models to determine the protective function of human PXR activation in IBD. The therapeutic role of rifaximin was further evaluated in DSS-treated hPXR and Pxr-null mice. Results demonstrated that pre-administration of rifaximin ameliorated the clinical hallmarks of colitis in DSS-treated and TNBS-treated hPXR mice as determined by body weight loss and assessment of diarrhea, rectal bleeding, colon length, and histology. Additionally, higher survival rates and recovery from colitis symptoms were observed in hPXR mice, and not in Pxr-null mice when rifaximin was administered after the onset of symptoms. NF-kappaB target genes were markedly down-regulated in hPXR mice by rifaximin treatment. In vitro NF-kappaB reporter assays demonstrated inhibition of NF-kappaB activity following rifaximin treatment in colon-derived cell lines expressing hPXR. These findings demonstrated the preventive and therapeutic role of rifaximin on IBD through human PXR-mediated inhibition of the NF-kappaB signaling cascade, thus suggesting that human PXR may be an effective target for the treatment of IBD.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC005708-19
Application #
8157193
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
19
Fiscal Year
2010
Total Cost
$1,350,062
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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