Control of Steroid 21-oic Acid Synthesis by Peroxisome Proliferator-activated Receptor and Role of the Hypothalamic-Pituitary-Adrenal Axis

A previous study identified the peroxisome proliferator-activated receptor alpha (PPARalpha) activation 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). In the present study, the molecular mechanism and the metabolic pathway of their 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 underscores the power of mass spectrometry-based metabolomics combined with genomic and physiologic analyses in identifying downstream metabolic biomarkers and the corresponding upstream molecular mechanisms.

Peroxisome proliferator-activated receptor {gamma} coactivator 1{alpha} (PGC-1{alpha}) promotes skeletal muscle lipid refueling in vivo by activating de novo lipogenesis and the pentose phosphate pathway

Exercise induces a pleiotropic adaptive response in skeletal muscle, largely through peroxisome proliferator-activated receptor coactivator 1 (PGC-1 ). PGC-1 enhances lipid oxidation and thereby provides energy for sustained muscle contraction. Its potential implication in promoting muscle refueling remains unresolved, however. Here, we investigated a possible role of elevated PGC-1 levels in skeletal muscle lipogenesis in vivo and the molecular mechanisms that underlie PGC-1 -mediated de novo lipogenesis. To this end, we studied transgenic mice with physiological overexpression of PGC-1 and human muscle biopsies pre- and post-exercise. We demonstrate that PGC-1 enhances lipogenesis in skeletal muscle through liver X receptor -dependent activation of the fatty acid synthase (FAS) promoter and by increasing FAS activity. Using chromatin immunoprecipitation, we establish a direct interaction between PGC-1 and the liver X receptor-responsive element in the FAS promoter. Moreover, we show for the first time that increased glucose uptake and activation of the pentose phosphate pathway provide substrates for RNA synthesis and cofactors for de novo lipogenesis. Similarly, we observed increased lipogenesis and lipid levels in human muscle biopsies that were obtained post-exercise. Our findings suggest that PGC-1 coordinates lipogenesis, intramyocellular lipid accumulation, and substrate oxidation in exercised skeletal muscle in vivo.

Antagonistic effects of oxidized low density lipoprotein and alpha-tocopherol on CD36 scavenger receptor expression in monocytes: involvement of protein kinase B and peroxisome proliferator-activated receptor-gamma

Vitamin E deficiency increases expression of the CD36 scavenger receptor, suggesting specific molecular mechanisms and signaling pathways modulated by alpha-tocopherol. We show here that alpha-tocopherol down-regulated CD36 expression (mRNA and protein) in oxidized low density lipoprotein (oxLDL)-stimulated THP-1 monocytes, but not in unstimulated cells. Furthermore, alpha-tocopherol treatment of monocytes led to reduction of fluorescent oxLDL-3,3'-dioctadecyloxacarbocyanine perchlorate binding and uptake. Protein kinase C (PKC) appears not to be involved because neither activation of PKC by phorbol 12-myristate 13-acetate nor inhibition by PKC412 was affected by alpha-tocopherol. However, alpha-tocopherol could partially prevent CD36 induction after stimulation with a specific agonist of peroxisome proliferator-activated receptor-gamma (PPARgamma; troglitazone), indicating that this pathway is susceptible to alpha-tocopherol action. Phosphorylation of protein kinase B (PKB) at Ser473 was increased by oxLDL, and alpha-tocopherol could prevent this event. Expression of PKB stimulated the CD36 promoter as well as a PPARgamma element-driven reporter gene, whereas an inactive PKB mutant had no effect. Moreover, coexpression of PPARgamma and PKB led to additive induction of CD36 expression. Altogether, our results support the existence of PKB/PPARgamma signaling pathways that mediate CD36 expression in response to oxLDL. The activation of CD36 expression by PKB suggests that both lipid biosynthesis and fatty acid uptake are stimulated by PKB.

Peroxisome proliferator-activated receptor-(gamma) receptor ligand partially prevents the development of endometrial explants in baboons: a prospective, randomized, placebo-controlled study

A prospective, randomized, placebo-controlled study was conducted in a baboon model to determine if a thiazolidinedione agonist of peroxisome proliferator-activated receptor-gamma, pioglitazone, can impede the development of endometriosis. Endometriosis was induced using laparoscopic, intrapelvic injection of eutopic menstrual endometrium, previously incubated with placebo or pioglitazone for 30 min, in 12 female baboons with a normal pelvis that had undergone at least one menstrual cycle since the time of captivity. At this point, the 12 baboons were randomized into two groups and treated from the day of induction. They received either PBS tablets (n = 6, placebo control, placebo tablets once a day by mouth) or pioglitazone (n = 6, test drug, 7.5 mg by mouth each day). A second and final laparoscopy was performed in the baboons to record the extent of endometriotic lesions between 24 and 42 d after induction (no difference in length of treatment between the two groups, P = 0.38). A videolaparoscopy was performed to document the number and surface area of endometriotic lesions. The surface area and volume of endometriotic lesions were significantly lower in pioglitazone treated baboons than the placebo group (surface area, 48.6 vs. 159.0 mm(2), respectively, P = 0.049; vol, 23.7 vs. 131.8 mm(3), respectively, P = 0.041). The surface area (3.5 vs. 17.8 mm(2), P = 0.017, pioglizatone vs. placebo) and overall number (1.5 vs. 9.5, P = 0.007, pioglizatone vs. placebo) of red lesions were lower in the pioglitazone group. A peroxisome proliferator-activated receptor-gamma ligand, pioglitazone, effectively reduced the initiation of endometriotic disease in the baboon endometriosis model. Using this animal model, we have shown that thiazolidinedione is a promising drug for preventive treatment of endometriosis.

The Ras inhibitors caveolin-1 and docking protein 1 activate peroxisome proliferator-activated receptor ? through spatial relocalization at helix 7 of its ligand-binding domain

Peroxisome proliferator-activated receptor ? (PPAR?) is a transcription factor that promotes differentiation and cell survival in the stomach. PPAR? upregulates and interacts with caveolin-1 (Cav1), a scaffold protein of Ras/mitogen-activated protein kinases (MAPKs). The cytoplasmic-to-nuclear localization of PPAR? is altered in gastric cancer (GC) patients, suggesting a so-far-unknown role for Cav1 in spatial regulation of PPAR? signaling. We show here that loss of Cav1 accelerated proliferation of normal stomach and GC cells in vitro and in vivo. Downregulation of Cav1 increased Ras/MAPK-dependent phosphorylation of serine 84 in PPAR? and enhanced nuclear translocation and ligand-independent transcription of PPAR? target genes. In contrast, Cav1 overexpression sequestered PPAR? in the cytosol through interaction of the Cav1 scaffolding domain (CSD) with a conserved hydrophobic motif in helix 7 of PPAR?'s ligand-binding domain. Cav1 cooperated with the endogenous Ras/MAPK inhibitor docking protein 1 (Dok1) to promote the ligand-dependent transcriptional activity of PPAR? and to inhibit cell proliferation. Ligand-activated PPAR? also reduced tumor growth and upregulated the Ras/MAPK inhibitors Cav1 and Dok1 in a murine model of GC. These results suggest a novel mechanism of PPAR? regulation by which Ras/MAPK inhibitors act as scaffold proteins that sequester and sensitize PPAR? to ligands, limiting proliferation of gastric epithelial cells.

Influence of pregnancy on the adipocytokine and peroxisome proliferator-activated receptor pathways in peripheral blood mononuclear cells from healthy donors and rheumatoid arthritis patients

To identify candidate genes that are regulated by human pregnancy and have the potential to modulate rheumatoid arthritis (RA) disease activity.

Ontogeny of mRNA abundance of nuclear receptors and nuclear receptor target genes in young cattle

After birth the development of appropriate detoxification mechanisms is important. Nuclear receptors (NR), such as constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor-alpha (PPARalpha), retinoid receptors (RAR, RXR), and NR target genes are involved in the detoxification of exogenous and endogenous substances. We quantified abundances of hepatic mRNA of NR and several NR target genes (cytochromes, CYP; cytochrome P450 reductase, CPR; UDP-glucuronosyl transferase, UDP) in calves at different ages. Gene expression was quantified by real-time RT-PCR. Abundance of mRNA of CAR and PXR increased from low levels at birth in pre-term calves (P0) and full-term calves (F0) to higher levels in 5-day-old calves (F5) and in 159-day-old veal calves (F159), whereas mRNA levels of PPARalpha did not exhibit significant ontogenetic changes. RARbeta mRNA levels were higher in F5 and F159 than in F0, whereas no age differences were observed for RARalpha levels. Levels of RXRalpha and RXRbeta mRNA were lower in F5 than in P0 and F0. Abundance of CYP2C8 and CYP3A4 increased from low levels in P0 and F0 to higher levels in F5 and to highest levels in F159. Abundance of CPR was transiently decreased in F0 and F5 calves. Levels of UGT1A1 mRNA increased from low levels in P0 and F0 to maximal level in F5 and F159. In conclusion, mRNA levels of NR and NR target genes exhibited ontogenetic changes that are likely of importance for handling of xeno- and endobiotics with increasing age.

Mice deficient in interleukin-4 (IL-4) or IL-4 receptor alpha have higher resistance to sporozoite infection with Plasmodium berghei (ANKA) than do naive wild-type mice

BALB/c interleukin-4 (IL-4(-/-)) or IL-4 receptor-alpha (IL-4ralpha(-/-)) knockout (KO) mice were used to assess the roles of the IL-4 and IL-13 pathways during infections with the blood or liver stages of plasmodium in murine malaria. Intraperitoneal infection with the blood-stage erythrocytes of Plasmodium berghei (ANKA) resulted in 100% mortality within 24 days in BALB/c mice, as well as in the mutant mouse strains. However, when infected intravenously with the sporozoite liver stage, 60 to 80% of IL-4(-/-) and IL-4ralpha(-/-) mice survived, whereas all BALB/c mice succumbed with high parasitemia. Compared to infected BALB/c controls, the surviving KO mice showed increased NK cell numbers and expression of inducible nitric oxide synthase (iNOS) in the liver and were able to eliminate parasites early during infection. In vivo blockade of NO resulted in 100% mortality of sporozoite-infected KO mice. In vivo depletion of NK cells also resulted in 80 to 100% mortality, with a significant reduction in gamma interferon (IFN-gamma) production in the liver. These results suggest that IFN-gamma-producing NK cells are critical in host resistance against the sporozoite liver stage by inducing NO production, an effective killing effector molecule against Plasmodium. The absence of IL-4-mediated functions increases the protective innate immune mechanism identified above, which results in immunity against P. berghei infection in these mice, with no major role for IL-13.

Hypoxia aggravates non-alcoholic steatohepatitis in mice lacking hepatocellular PTEN

The metabolic disorders that predispose patients to NASH (non-alcoholic steatohepatitis) include insulin resistance and obesity. Repeated hypoxic events, such as occur in obstructive sleep apnoea syndrome, have been designated as a risk factor in the progression of liver disease in such patients, but the mechanism is unclear, in particular the role of hypoxia. Therefore we studied the influence of hypoxia on the development and progression of steatohepatitis in an experimental mouse model. Mice with a hepatocellular-specific deficiency in the Pten (phosphatase and tensin homologue deleted on chromosome 10) gene, a tumour suppressor, were exposed to a 10% O2 (hypoxic) or 21% O2 (control) atmosphere for 7 days. Haematocrit, AST (aspartate aminotransferase), glucose, triacylglycerols (triglycerides) and insulin tolerance were measured in blood. Histological lesions were quantified. Expression of genes involved in lipogenesis and mitochondrial beta-oxidation, as well as FOXO1 (forkhead box O1), hepcidin and CYP2E1 (cytochrome P450 2E1), were analysed by quantitative PCR. In the animals exposed to hypoxia, the haematocrit increased (60+/-3% compared with 50+/-2% in controls; P<0.01) and the ratio of liver weight/body weight increased (5.4+/-0.2% compared with 4.7+/-0.3% in the controls; P<0.01). Furthermore, in animals exposed to hypoxia, steatosis was more pronounced (P<0.01), and the NAS [NAFLD (non-alcoholic fatty liver disease) activity score] (8.3+/-2.4 compared with 2.3+/-10.7 in controls; P<0.01), serum AST, triacylglycerols and glucose were higher. Insulin sensitivity decreased in mice exposed to hypoxia relative to controls. The expression of the lipogenic genes SREBP-1c (sterol-regulatory-element-binding protein-1c), PPAR-gamma (peroxisome-proliferator-activated receptor-gamma), ACC1 (acetyl-CoA carboxylase 1) and ACC2 (acetyl-CoA carboxylase 2) increased significantly in mice exposed to hypoxia, whereas mitochondria beta-oxidation genes [PPAR-alpha (peroxisome-proliferator-activated receptor-alpha) and CPT-1 (carnitine palmitoyltransferase-1)] decreased significantly. In conclusion, the findings of the present study demonstrate that hypoxia alone aggravates and accelerates the progression of NASH by up-regulating the expression of lipogenic genes, by down-regulating genes involved in lipid metabolism and by decreasing insulin sensitivity.

Human urinary metabolomic profile of PPARalpha induced fatty acid beta-oxidation

Activation of the peroxisome proliferator-activated receptor alpha (PPARalpha) is associated with increased fatty acid catabolism and is commonly targeted for the treatment of hyperlipidemia. To identify latent, endogenous biomarkers of PPARalpha activation and hence increased fatty acid beta-oxidation, healthy human volunteers were given fenofibrate orally for 2 weeks and their urine was profiled by UPLC-QTOFMS. Biomarkers identified by the machine learning algorithm random forests included significant depletion by day 14 of both pantothenic acid (>5-fold) and acetylcarnitine (>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 (>10-fold), isobutyrylcarnitine (>2.5-fold), (S)-(+)-2-methylbutyrylcarnitine (5-fold), and isovalerylcarnitine (>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.

Serum metabolomics reveals irreversible inhibition of fatty acid beta-oxidation through the suppression of PPARalpha activation as a contributing mechanism of acetaminophen-induced hepatotoxicity

Metabolic bioactivation, glutathione depletion, and covalent binding are the early hallmark events after acetaminophen (APAP) overdose. However, the subsequent metabolic consequences contributing to APAP-induced hepatic necrosis and apoptosis have not been fully elucidated. In this study, serum metabolomes of control and APAP-treated wild-type and Cyp2e1-null mice were examined by liquid chromatography-mass spectrometry (LC-MS) and multivariate data analysis. A dose-response study showed that the accumulation of long-chain acylcarnitines in serum contributes to the separation of wild-type mice undergoing APAP-induced hepatotoxicity from other mouse groups in a multivariate model. This observation, in conjunction with the increase of triglycerides and free fatty acids in the serum of APAP-treated wild-type mice, suggested that APAP treatment can disrupt fatty acid beta-oxidation. A time-course study further indicated that both wild-type and Cyp2e1-null mice had their serum acylcarnitine levels markedly elevated within the early hours of APAP treatment. While remaining high in wild-type mice, serum acylcarnitine levels gradually returned to normal in Cyp2e1-null mice at the end of the 24 h treatment. Distinct from serum aminotransferase activity and hepatic glutathione levels, the pattern of serum acylcarnitine accumulation suggested that acylcarnitines can function as complementary biomarkers for monitoring the APAP-induced hepatotoxicity. An essential role for peroxisome proliferator-activated receptor alpha (PPARalpha) in the regulation of serum acylcarnitine levels was established by comparing the metabolomic responses of wild-type and Ppara-null mice to a fasting challenge. The upregulation of PPARalpha activity following APAP treatment was transient in wild-type mice but was much more prolonged in Cyp2e1-null mice. Overall, serum metabolomics of APAP-induced hepatotoxicity revealed that the CYP2E1-mediated metabolic activation and oxidative stress following APAP treatment can cause irreversible inhibition of fatty acid oxidation, potentially through suppression of PPARalpha-regulated pathways.

Fenofibrate metabolism in the cynomolgus monkey using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry-based metabolomics

Fenofibrate, widely used for the treatment of dyslipidemia, activates the nuclear receptor, peroxisome proliferator-activated receptor alpha. However, liver toxicity, including liver cancer, occurs in rodents treated with fibrate drugs. Marked species differences occur in response to fibrate drugs, especially between rodents and humans, the latter of which are resistant to fibrate-induced cancer. Fenofibrate metabolism, which also shows species differences, has not been fully determined in humans and surrogate primates. In the present study, the metabolism of fenofibrate was investigated in cynomolgus monkeys by ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS)-based metabolomics. Urine samples were collected before and after oral doses of fenofibrate. The samples were analyzed in both positive-ion and negative-ion modes by UPLC-QTOFMS, and after data deconvolution, the resulting data matrices were subjected to multivariate data analysis. Pattern recognition was performed on the retention time, mass/charge ratio, and other metabolite-related variables. Synthesized or purchased authentic compounds were used for metabolite identification and structure elucidation by liquid chromatographytandem mass spectrometry. Several metabolites were identified, including fenofibric acid, reduced fenofibric acid, fenofibric acid ester glucuronide, reduced fenofibric acid ester glucuronide, and compound X. Another two metabolites (compound B and compound AR), not previously reported in other species, were characterized in cynomolgus monkeys. More importantly, previously unknown metabolites, fenofibric acid taurine conjugate and reduced fenofibric acid taurine conjugate were identified, revealing a previously unrecognized conjugation pathway for fenofibrate.