Tyrosine Aminotransferase Expression Research Articles

Identification of TAT as a Biomarker Involved in Cell Cycle and DNA Repair in Breast Cancer.

Breast cancer (BC) is the most frequently diagnosed cancer and the primary cause of cancer-related mortality in women. Treatment of triple-negative breast cancer (TNBC) remains particularly challenging due to its resistance to chemotherapy and poor prognosis. Extensive research efforts in BC screening and therapy have improved clinical outcomes for BC patients. Therefore, identifying reliable biomarkers for TNBC is of great clinical importance. Here, we found that tyrosine aminotransferase (TAT) expression was significantly reduced in BC and strongly correlated with the poor prognosis of BC patients, which distinguished BC patients from normal individuals, indicating that TAT is a valuable biomarker for early BC diagnosis. Mechanistically, we uncovered that methylation of the TAT promoter was significantly increased by DNA methyltransferase 3 (DNMT3A/3B). In addition, reduced TAT contributes to DNA replication and cell cycle activation by regulating homologous recombination repair and mismatch repair to ensure genomic stability, which may be one of the reasons for TNBC resistance to chemotherapy. Furthermore, we demonstrated that Diazinon increases TAT expression as an inhibitor of DNMT3A/3B and inhibits the growth of BC by blocking downstream pathways. Taken together, we revealed that TAT is silenced by DNMT3A/3B in BC, especially in TNBC, which promotes the proliferation of tumor cells by supporting DNA replication, activating cell cycle, and enhancing DNA damage repair. These results provide fresh insights and a theoretical foundation for the clinical diagnosis and treatment of BC.

TRIM21-mediated ubiquitylation of TAT suppresses liver metastasis in gallbladder cancer

Liver metastasis is common in patients with gallbladder cancer (GBC), imposing a significant challenge in clinical management and serving as a poor prognostic indicator. However, the mechanisms underlying liver metastasis remain largely unknown. Here, we report a crucial role of tyrosine aminotransferase (TAT) in liver metastasis of GBC. TAT is frequently up-regulated in GBC tissues. Increased TAT expression is associated with frequent liver metastasis and poor prognosis of GBC patients. Overexpression of TAT promotes GBC cell migration and invasion in vitro, as well as liver metastasis in vivo. TAT knockdown has the opposite effects. Intriguingly, TAT promotes liver metastasis of GBC by potentiating cardiolipin-dependent mitophagy. Mechanistically, TAT directly binds to cardiolipin and leads to cardiolipin externalization and subsequent mitophagy. Moreover, TRIM21 (Tripartite Motif Containing 21), an E3 ubiquitin ligase, interacts with TAT. The histine residues 336 and 338 at TRIM21 are essential for this binding. TRIM21 preferentially adds the lysine 63 (K63)-linked ubiquitin chains on TAT principally at K136. TRIM21-mediated TAT ubiquitination impairs its dimerization and mitochondrial location, subsequently inhibiting tumor invasion and migration of GBC cells. Therefore, our study identifies TAT as a novel driver of GBC liver metastasis, emphasizing its potential as a therapeutic target.

Abstract 4428: Integrated multi-omics analysis reveals systemic and localized metabolic disruptions in colorectal cancer

Abstract Despite technological advances in molecular medicine over the last 30 years, no single approach has proven to be sufficient to meet the diagnostic needs in cancer care. Here we use a multiomic approach leveraging proteomic, metabolomic, and transcriptomic technologies to study the metabolic shifts that occur during colorectal cancer (CRC) progression, Our integrated analysis identified systemic changes to beta oxidation pathways and localized changes to tyrosine metabolism within the tumor, creating a mechanistic, actionable description of the core CRC metabolic program. We conducted untargeted proteomics and metabolomics profiling on serum samples from CRC patients (n=10) and healthy controls (n=10). Our serum proteomics data is generated through sample enrichment on the Seer Proteograph system followed by LC/MS analysis with the Thermo Orbitrap Astral. Our metabolomics approach employs LC/MS assays for unbiased profiling of the serum metabolome, exposome, and lipidome. We supplemented this discovery work with both targeted serum and tumor-specific approaches including targeted proteomics to quantify select targets, inflammatory profiling with Alamar proteomics, public transcriptomics data from TCGA, and in vitro metabolic flux data. These large and diverse datasets were then integrated through joint pathway analysis and network integration. The global serum proteomics and metabolomics profiles generated show distinct separation between healthy and diseased individuals. Joint pathway analysis of these discovery datasets highlighted enrichment in beta oxidation pathways and tyrosine metabolism, among others. Interestingly, the Alamar inflammatory panel revealed that many of the analytes in the tyrosine metabolism pathway were well correlated with inflammatory status, while the beta oxidation signature had a lower correlation. To determine the relationship of these systemic findings from serum to tumor metabolism itself, we integrated tumor-specific transcriptomics data and found alterations to tyrosine metabolism with differences in tyrosine aminotransferase expression. The beta oxidation related genes, on the other hand, were not concordant with the serum findings. However, our in vitro metabolic flux studies have shown beta oxidation in CRC cells is upregulated to provide additional fuel for oxidative phosphorylation. This result suggests that beta oxidation may not be transcriptionally regulated in the tumor but rather a consequence of organismal metabolic rewiring. The results of this work, which is currently expanding into a larger and more diverse patient population, underscores the power of multiomic profiling for enhancing our understanding of metabolic dysregulation in CRC. Ultimately, a comprehensive model of the molecular alterations in CRC will yield a better understanding of tumor phenotypes and inform better diagnostics and therapies. Citation Format: Gary Patti, Ethan Stancliffe, Adam Richardson, Ashima Mehta, Monil Gandhi, Kevin Cho. Integrated multi-omics analysis reveals systemic and localized metabolic disruptions in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4428.

Comprehensive Analysis of Whole-Transcriptome Profiles in Response to Acute Hypersaline Challenge in Chinese Razor Clam Sinonovacula constricta.

The Chinese razor clam (Sinonovacula constricta) is an important for Chinese aquaculture marine bivalve that naturally occurs across intertidal and estuarine areas subjected to significant changes in salinity level. However, the information on the molecular mechanisms related to high salinity stress in the species remain limited. In this study, nine gill samples of S. constricta treated with 20, 30, and 40 ppt salinity for 24 h were used for whole-transcriptome RNA sequencing, and a regulatory network of competing endogenous RNAs (ceRNAs) was constructed to better understand the mechanisms responsible for adaptation of the species to high salinity. A total of 83,262 lncRNAs, 52,422 mRNAs, 2890 circRNAs, and 498 miRNAs were identified, and 4175 of them displayed differential expression pattern among the three groups examined. The KEGG analyses of differentially expressed RNAs evidenced that amino acid synthesis and membrane transport were the dominant factors involved in the adaptation of the Chinese razor clam to acute salinity increase, while lipid metabolism and signaling played only a supporting role. In addition, lncRNA/circRNA-miRNA-mRNA regulatory networks (ceRNA network) showed clearly regulatory relationships among different RNAs. Moreover, the expression of four candidate genes, including tyrosine aminotransferase (TAT), hyaluronidase 4 (HYAL4), cysteine sulfinic acid decarboxylase (CSAD), and ∆1-pyrroline-5-carboxylate synthase (P5CS) at different challenge time were detected by qRT-PCR. The expression trend of TAT and HYAL4 was consistent with that of the ceRNA network, supporting the reliability of established network. The expression of TAT, CSAD, and P5CS were upregulated in response to increased salinity. This might be associated with increased amino acid synthesis rate, which seems to play an essential role in adaptation of the species to high salinity stress. In contrast, the expression level of HYAL4 gene decreased in response to elevated salinity level, which is associated with reduction Hyaluronan hydrolysis to help maintain water in the cell. Our findings provide a very rich reference for understanding the important role of ncRNAs in the salinity adaptation of shellfish. Moreover, the acquired information may be useful for optimization of the artificial breeding of the Chinese razor clam under aquaculture conditions.

Fe2 O3 nanoparticles induced biochemical responses and expression of genes involved in rosmarinic acid biosynthesis pathway in Moldavian balm under salinity stress.

The effect of iron oxide nanoparticle (NP) at four concentrations (0, 30, 60 and 90 ppm) and salinity at three levels (0, 50 and 100 mM) were investigated on rosmarinic acid (RA) production in 5-week-old Moldavian balm (Dracocephalum moldavica L.) plants. Salinity and spraying iron oxide NPs significantly affected tyrosine (Tyr), phenylalanine (Phe) and proline (Pro) amino acids content, Phenylalanine Ammonia-Lyase (PAL), Tyrosine Aminotransferase (TAT) and Rosmarinic Acid Synthase (RAS) genes expression levels, RA content, Polyphenol Oxidase (PPO), PAL and Superoxide Dismutase (SOD) activities, malondialdehyde (MDA) content and DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity. PAL, TAT and RAS genes expression rate and content of RA were enhanced in Moldavian balm plants exposed by NaCl + NPs. The results of high performance liquid chromatography (HPLC) revealed that simultaneous application of 50 mM NaCl and 90 ppm NPs increases the RA content in leaf by 81.15% as compared to control plants. The Tyr and Phe contents decreased in Moldavian balm plants exposed to salt stress. Application of NPs had a positive effect on the content of these amino acids. Proline content increased under salinity stress and application of iron NPs induced a significant increase in the Pro content of leaf. The results revealed that PAL, PPO and SOD enzymes activities increased under salinity conditions. The highest activity of PPO and SOD was observed in 100 mM NaCl + 60 ppm NPs treatment. Simultaneous application of 100 mM NaCl + 90 ppm NPs increased the MDA content and DPPH radical scavenging activity compared to control plants. It can be concluded that the application of appropriate levels of NPs moderates the effect of salinity stress in D. moldavica L. and results in an increased amount of RA compared to control plants.

Identification and expression profiling of rosmarinic acid biosynthetic genes from Satureja khuzistanica under carbon nanotubes and methyl jasmonate elicitation

Satureja khuzistanica is a medicinal herb endemic to Iran which can serve as a source of rosmarinic acid (RA). In the present study, the effect of different concentrations of methyl jasmonate (MeJA) and multi-walled carbon nanotubes (MWCNTs) on the rosmarinic acid accumulation and the expression of genes involved in its biosynthetic pathway was evaluated in nodal segment cultures of S. khuzistanica. The concentration of RA varied in plant extracts derived from the field, plants obtained under in vitro solid media, calli, suspension cultures and nodal segment cultures in liquid medium, with the highest amount recorded in nodal segment cultures. Phenylalanine ammonia-lyase (PAL), tyrosine aminotransferase (TAT), 4-hydroxyphenylpyruvate reductase (HPPR) and RA synthase (RAS) cDNA clones as key biosynthetic genes of RA production were identified and expression patterns were assayed in response to MeJA and MWCNTs, exogenously applied at a range of 0, 50, 100, 250 mg L−1. The expression levels of HPPR, PAL and TAT were up-regulated at 100 mg L−1 MWCNTs, whereas down-regulated levels were observed at 250 mg L−1. RAS was up-regulated in all MWCNTs treatments. HPPR, PAL and TAT expression increased sharply at 250 mg L−1 MeJA. The highest levels of RAS transcripts were observed at 100 mg L−1 MeJA. The highest HPPR, PAL and TAT expression levels were observed at 250 mg L−1 MeJA. In accordance, HPLC analysis showed a high amount of RA under MeJA and MWCNT elicitors. Our results provide helpful information on the expression profiles of biosynthetic genes in connection with RA accumulation.

Characteristics, tissue-specific expression, and hormonal regulation of expression of tyrosine aminotransferase in the avian female reproductive tract

Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine to p-hydroxyphenylpyruvate. Accumulation of tyrosine in the body due to a genetic mutation in the TAT gene causes tyrosomia type II in humans. The TAT gene is regarded as a model for studying steroid-inducible factors regulating a variety of biological functions of TAT. However, little is known of the effects of estrogen on the expression of the TAT gene in chickens. Therefore, in the present study, we identified expression of the avian TAT gene in various organs. The results showed the TAT was detected predominantly in the liver and reproductive organs including testis, oviduct, and ovary. Specifically, TAT mRNA was expressed abundantly in the glandular and luminal epithelia of the oviducts in response to endogenous and exogenous estrogens which also induce dramatic morphological changes in the oviduct of chickens. In addition, target microRNAs of TAT (miR-1460, miR-1626-3p, miR-1690-5p, and miR-7442-3p) were found to modulate expression of the TAT gene. Especially, miR-1690-5p influenced TAT gene transcription by binding directly to its 3′-UTR region. Moreover, the expression of TAT was abundant in glandular epithelia of cancerous but not normal ovaries from laying hens. Taken together, our findings suggest that TAT plays an important role in the cytodifferentiation of oviducts in response to estrogen and in the progression of ovarian cancer in chickens.

Prunella vulgaris L. hairy roots: Culture, growth, and elicitation by ethephon and salicylic acid

Hairy roots have gained popularity as an alternative source for secondary metabolites not only because of their biosynthetic and bioactive potentials, but also because of studies of experimental systems aiming to understand secondary metabolic pathways. While many secondary metabolites have been identified in Prunella vulgaris, a comprehensive understanding of the metabolic pathways has remained elusive. Here, Agrobacterium rhizogenes (ATCC15834)‐mediated hairy root induction of P. vulgaris resulted in 15–30 times more rosmarinic acid (RA) than in the intact plant. After the growth and RA biosynthesis of P. vulgaris, hairy roots were investigated over a 1‐month period, we studied elicitation by ethephon (Eth) and salicylic acid (SA) and found 200 μg L−1 Eth and 6.9 mg L−1 SA exhibited the optimum induction efficiency. These concentrations were further selected for a time‐course assay of hairy root cultures to assess exposure to these compounds over 8 days. During that period, RA reached maximum accumulations of 1.66‐fold 8 days after Eth elicitation and 1.48‐fold 2 days post‐SA addition. We also examined the transcripts of genes involved in the RA biosynthesis pathway, and found the expression of tyrosine aminotransferase (TAT) and phenylalanine ammonia‐lyase (PAL) were increased by Eth, and SA heightened the expression of TAT, 4‐hydroxyphenylpyruvate reductase (HPPR), PAL, 4‐coumaric acid CoA‐ligase 1 (4CL1), and cytochrome P450‐dependent monooxygenase (CYP). Altogether, our results indicate that the convenient hairy root culture system of P. vulgaris can provide an alternate source for RA production and would be conducive for the engineering of secondary metabolic pathways in P. vulgaris.

Salicylic acid-induced cytosolic acidification increases the accumulation of phenolic acids in Salvia miltiorrhiza cells

Salicylic acid (SA) is an elicitor widely used to promote the synthesis of secondary metabolites. In order to determine the relevance between SA-induced pH changes and secondary metabolite synthesis, Salvia miltiorrhiza suspension cells were exposed to either SA, fusicoccin (FC), sodium orthovanadate (OVA), methylamine (ME) and their combination (FC+SA and ME+SA) for investigating the effect of cytosolic pH change on phenolic acids production, such as caffeic acid (CA), rosmarinic acid (RA), and salvianolic acid B (Sal B). The variations of cytosolic pH were sensed by using the fluorescent probe BCECF-AM, and combination with laser scanning confocal microscopic technique. Meanwhile, the genes expression of phenylalanine ammonia-lyase (PAL), tyrosine aminotransferase (TAT), and rosmarinic acid synthase (RAS) were determined using real time PCR. The contents of CA, RA, and Sal B were measured by high performance liquid chromatography. The results showed that SA decreased the cytosolic pH by inhibiting the activity of plasma membrane H+-ATPase. OVA induced cytosolic acidification too, while both FC and ME inhibited the acidification induced by SA. SA also up-regulated the genes expression of TAT, PAL, and RAS, and as a result enhanced the accumulation of those phenolic acids.

Impairment of the cortisol stress response mediated by the hypothalamus–pituitary–interrenal (HPI) axis in zebrafish (Danio rerio) exposed to monocrotophos pesticide

In teleosts, an important component of the stress response is coordinated by the hypothalamic–pituitary–interrenal (HPI) axis. Environmental contaminants might disrupt the stress axis and consequently affect the stress response in fish. To investigate the effect of monocrotophos (MCP) pesticide on the stress response of fish and its potential mechanisms, adult zebrafish (Danio rerio) were exposed to 0, 1, 10, and 100μg/L of a 40% MCP-based pesticide for 21d, after which time fish were subjected to a 3-min air-exposure stressor. Concentrations of the whole-body cortisol were measured by radioimmunoassay and abundances of transcripts of proteins involved in the HPI axis were determined using quantitative real-time PCR. Results showed that 100μg/L of MCP pesticide decreased whole-body cortisol levels of female zebrafish in response to an acute stressor, but without any effect on the cortisol response in males. 100μg/L MCP pesticide reduced POMC and GR expression in the brain, MC2R and P45011β expression in the head kidney, but enhanced 20β-HSD2 expression in the head kidney, suggesting that MCP damaged the HPI axis involving acting at pituitary regulatory levels, inhibiting cortisol synthesis and stimulating cortisol catabolism, or disturbing the negative feedback regulation. Additionally, MCP depressed liver GR transcription but did not affect phosphoenolpyruvate carboxykinase and tyrosine aminotransferase expression in zebrafish, suggesting a role for this pesticide in reducing target tissue responsiveness to cortisol. Considered together, the reduced ability to elevate cortisol levels in response to an acute stress may be an endocrine dysfunction occurring in zebrafish subchronically exposed to MCP pesticide.

Hepatic SRC-1 activity orchestrates transcriptional circuitries of amino acid pathways with potential relevance for human metabolic pathogenesis.

Disturbances in amino acid metabolism are increasingly recognized as being associated with, and serving as prognostic markers for chronic human diseases, such as cancer or type 2 diabetes. In the current study, a quantitative metabolomics profiling strategy revealed global impairment in amino acid metabolism in mice deleted for the transcriptional coactivator steroid receptor coactivator (SRC)-1. Aberrations were hepatic in origin, because selective reexpression of SRC-1 in the liver of SRC-1 null mice largely restored amino acids concentrations to normal levels. Cistromic analysis of SRC-1 binding sites in hepatic tissues confirmed a prominent influence of this coregulator on transcriptional programs regulating amino acid metabolism. More specifically, SRC-1 markedly impacted tyrosine levels and was found to regulate the transcriptional activity of the tyrosine aminotransferase (TAT) gene, which encodes the rate-limiting enzyme of tyrosine catabolism. Consequently, SRC-1 null mice displayed low TAT expression and presented with hypertyrosinemia and corneal alterations, 2 clinical features observed in the human syndrome of TAT deficiency. A heterozygous missense variant of SRC-1 (p.P1272S) that is known to alter its coactivation potential, was found in patients harboring idiopathic tyrosinemia-like disorders and may therefore represent one risk factor for their clinical symptoms. Hence, we reinforce the concept that SRC-1 is a central factor in the fine orchestration of multiple pathways of intermediary metabolism, suggesting it as a potential therapeutic target that may be exploitable in human metabolic diseases and cancer.

Omeprazole and lansoprazole enantiomers induce CYP3A4 in human hepatocytes and cell lines via glucocorticoid receptor and pregnane X receptor axis.

Benzimidazole drugs lansoprazole and omeprazole are used for treatment of various gastrointestinal pathologies. Both compounds cause drug-drug interactions because they activate aryl hydrocarbon receptor and induce CYP1A genes. In the current paper, we examined the effects of lansoprazole and omeprazole enantiomers on the expression of key drug-metabolizing enzyme CYP3A4 in human hepatocytes and human cancer cell lines. Lansoprazole enantiomers, but not omeprazole, were equipotent inducers of CYP3A4 mRNA in HepG2 cells. All forms (S-, R-, rac-) of lansoprazole and omeprazole induced CYP3A4 mRNA and protein in human hepatocytes. The quantitative profiles of CYP3A4 induction by individual forms of lansoprazole and omeprazole exerted enantiospecific patterns. Lansoprazole dose-dependently activated pregnane X receptor PXR in gene reporter assays, and slightly modulated rifampicin-inducible PXR activity, with similar potency for each enantiomer. Omeprazole dose-dependently activated PXR and inhibited rifampicin-inducible PXR activity. The effects of S-omeprazole were much stronger as compared to those of R-omeprazole. All forms of lansoprazole, but not omeprazole, slightly activated glucocorticoid receptor and augmented dexamethasone-induced GR transcriptional activity. Omeprazole and lansoprazole influenced basal and ligand inducible expression of tyrosine aminotransferase, a GR-target gene, in HepG2 cells and human hepatocytes. Overall, we demonstrate here that omeprazole and lansoprazole enantiomers induce CYP3A4 in HepG2 cells and human hepatocytes. The induction comprises differential interactions of omeprazole and lansoprazole with transcriptional regulators PXR and GR, and some of the effects were enantiospecific. The data presented here might be of toxicological and clinical importance, since the effects occurred in therapeutically relevant concentrations.

In utero glucocorticoid exposure reduces fetal skeletal muscle mass in rats independent of effects on maternal nutrition

Maternal stress and undernutrition can occur together and expose the fetus to high glucocorticoid (GLC) levels during this vulnerable period. To determine the consequences of GLC exposure on fetal skeletal muscle independently of maternal food intake, groups of timed-pregnant Sprague-Dawley rats (n = 7/group) were studied: ad libitum food intake (control, CON); ad libitum food intake with 1 mg dexamethasone/l drinking water from embryonic day (ED)13 to ED21 (DEX); pair-fed (PF) to DEX from ED13 to ED21. On ED22, dams were injected with [(3)H]phenylalanine for measurements of fetal leg muscle and diaphragm fractional protein synthesis rates (FSR). Fetal muscles were analyzed for protein and RNA contents, [(3)H]phenylalanine incorporation, and MuRF1 and atrogin-1 (MAFbx) mRNA expression. Fetal liver tyrosine aminotransferase (TAT) expression was quantified to assess fetal exposure to GLCs. DEX treatment reduced maternal food intake by 13% (P < 0.001) and significantly reduced placental mass relative to CON and PF dams. Liver TAT expression was elevated only in DEX fetuses (P < 0.01). DEX muscle protein masses were 56% and 70% than those of CON (P < 0.01) and PF (P < 0.05) fetuses, respectively; PF muscles were 80% of CON (P < 0.01). Muscle FSR decreased by 35% in DEX fetuses (P < 0.001) but were not different between PF and CON. Only atrogin-1 expression was increased in DEX fetus muscles. We conclude that high maternal GLC levels and inadequate maternal food intake impair fetal skeletal muscle growth, most likely through different mechanisms. When combined, the effects of decreased maternal intake and maternal GLC intake on fetal muscle growth are additive.

Endocrine Characterization of the Designer Steroid Methyl-1-Testosterone: Investigations on Tissue-Specific Anabolic-Androgenic Potency, Side Effects, and Metabolism

Various products containing rarely characterized anabolic steroids are nowadays marketed as dietary supplements. Herein, the designer steroid methyl-1-testosterone (M1T) (17β-hydroxy-17α-methyl-5α-androst-1-en-3-one) was identified, and its biological activity, potential adverse effects, and metabolism were investigated. The affinity of M1T toward the androgen receptor (AR) was tested in vitro using a yeast AR transactivation assay. Its tissue-specific androgenic and anabolic potency and potential adverse effects were studied in a Hershberger assay (sc or oral), and tissue weights and selected molecular markers were investigated. Determination of M1T and its metabolites was performed by gas chromatography mass spectrometry. In the yeast AR transactivation assay, M1T was characterized as potent androgen. In rats, M1T dose-dependently stimulated prostate and levator ani muscle weight after sc administration. Oral administration had no effect but stimulated proliferation in the prostate and modulated IGF-I and AR expression in the gastrocnemius muscle in a dose-dependent manner. Analysis of tyrosine aminotransferase expression provided evidence for a strong activity of M1T in the liver (much higher after oral administration). In rat urine, 17α-methyl-5α-androstane-3α,17β-diol, M1T, and a hydroxylated metabolite were identified. In humans, M1T was confirmed in urine in addition to its main metabolites 17α-methyl-5α-androst-1-ene-3α,17β-diol and 17α-methyl-5α-androstane-3α,17β-diol. Additionally, the corresponding 17-epimers as well as 17β-hydroxymethyl-17α-methyl-18-nor-5α-androsta-1,13-dien-3-one and its 17-epimer were detected, and their elimination kinetics was monitored. It was demonstrated that M1T is a potent androgenic and anabolic steroid after oral and sc administration. Obviously, this substance shows no selective AR modulator characteristics and might exhibit liver toxicity, especially after oral administration.

Selective amplification of glucocorticoid anti-inflammatory activity through synergistic multi-target action of a combination drug

IntroductionGlucocorticoids are a mainstay of anti-inflammatory therapy, but significant adverse effects ultimately limit their utility. Previous efforts to design glucocorticoid structures with an increased therapeutic window have focused on dissociating anti-inflammatory transcriptional repression from adverse effects primarily driven by transcriptional activation. An alternative to this medicinal chemistry approach is a systems biology based strategy that seeks to amplify selectively the anti-inflammatory activity of very low dose glucocorticoid in immune cells without modulating alternative cellular networks that mediate glucocorticoid toxicity.MethodsThe combination of prednisolone and the antithrombotic drug dipyridamole was profiled using in vitro and in vivo models of anti-inflammatory activity and glucocorticoid-induced adverse effects to demonstrate a dissociated activity profile.ResultsThe combination synergistically suppresses release of proinflammatory mediators, including tumour necrosis factor-α, IL-6, chemokine (C-C motif) ligand 5 (RANTES), matrix metalloproteinase-9, and others, from human peripheral blood mononuclear cells and mouse macrophages. In rat models of acute lipopolysaccharide-induced endotoxemia and delayed-type hypersensitivity, and in chronic models of collagen-induced and adjuvant-induced arthritis, the combination produced anti-inflammatory activity that required only a subtherapeutic dose of prednisolone. The immune-specific amplification of prednisolone anti-inflammatory activity by dipyridamole did not extend to glucocorticoid-mediated adverse effects, including corticosterone suppression or increased expression of tyrosine aminotransferase, in vivo after repeat dosing in rats. After 8 weeks of oral dosing in mice, treatment with the combination did not alter prednisolone-induced reduction in osteocalcin and mid-femur bone density, which are markers of steroid-induced osteoporosis. Additionally, amplification was not observed in the cellular network of corticotroph AtT-20/D16v-F2 cells in vitro, as measured by pro-opiomelanocortin expression and adrenocorticotropic hormone secretion.ConclusionsThese data suggest that the multi-target mechanism of low-dose prednisolone and dipyridamole creates a dissociated activity profile with an increased therapeutic window through cellular network selective amplification of glucocorticoid-mediated anti-inflammatory signaling.

Genes involved in amino acid metabolism are differentially regulated by saturated and polyunsaturated dietary fat

Previous studies have shown that Wy 14643, a synthetic PPAR‐alpha ligand, down‐regulates the expression of the histidine degrading enzyme, histidase (HAL) gene. In addition fatty acids are natural ligands of PPAR‐alpha with higher affinity for polyunsaturated fatty acids (PUFAs). Therefore, the aim of the present work was to study the effect of a diet rich in PUFAs (soybean oil) or saturated (coconut oil) dietary fat on the expression of genes involved in amino acid metabolism such as HAL, tyrosine aminotransferase (TAT), serine dehydratase (SDH), and the sodium‐dependant neutral amino acid transporter (SNAT2). We also measured the gene expression of PPAR‐alpha, and its coactivator PGC1. Our results showed that the gene expression of HAL, TAT, SDH and SNAT2 increased between 100–200% when the rats were fed with saturated fat when compared with rats fed with corn oil (control group). The gene expression of these genes showed a 20% to 50% decrease in the liver of the rats fed with soybean oil when compared with the control group. PPAR‐alpha and PGC‐1 gene expression decreased by approximately 30% in the rats fed with coconut oil, and about 60% in those fed with soybean oil, when compared with the rats fed with corn oil. Our results suggest that amino acid catabolism may be regulated by the kind of dietary fat via PPAR‐alpha.

Mechanism of Liver Tyrosine Aminotransferase Increase in Ethanol-Treated Mice and Its Effect on Serum Tyrosine Level

Liver tyrosine aminotransferase (TAT) activity is known to increase with ethanol treatment; however, the mechanism of this increase is unclear. Upon investigation we found that TAT activity and mRNA levels started to increase 2 h after ethanol administration and continued to increase until 6 h after ethanol administration. The increase in ethanol-induced TAT activity could not be explained by calorie loading after fasting, since ethanol loading increased TAT expression, while glucose loading decreased TAT expression. In addition, liver TAT activity was not related to serum tyrosine levels. TAT activity increased when an adenosine A2 agonist, 5'-N-ethylcarboxamide adenosine, was given. Since TAT activity is increased by cAMP, and ethanol increases cAMP production via an adenosine receptor-dependent mechanism, this increase in ethanol-induced TAT activity may occur via an adenosine receptor-dependent mechanism.

Curative effects of transplantation of hepatocytes differentiated from embryonic stem cells on treatment of fulminant hepatic failure: experiment with mice

To investigate the curative effects of transplantation of hepatocytes differentiated from embryonic stem cells (ESCs) on treatment of fulminant hepatic failure (FHF). Mouse ESCs transfected with green fluorescent protein were cultured. RT-PCR was used to detect the mRNA expression of alpha-fetoprotein (AFP), transthyretin (TTR), hepatic nuclear factor (HNF), albumin (ALB), glucose-6-phosphate (G6P), and tyrosine aminotransferase (TAT) at different time points. Immunocytochemistry (ICC) was used to detect the expression of AFP, ALB, cytokeratin 8 (CK8), and CK11 in the cells. The morphology and distribution of the cells were observed with microscope. Forty mice were randomly divided into 2 equal groups: ESC transplantation group, undergoing transplantation of ICC positive ESCs (hepatocytes) into the hepatic capsule, and control group, undergoing injection of normal saline in the hepatic capsule. Twenty-four hours later carbon tetrachloride was injected intra-peritoneally to induce FHF. The survival status of the mice was observed. Twenty-four hours later venous blood samples were collected to examine the levels of total bilirubin (TB), alanine aminotransferase (ALT), ALB, blood sugar (BS), and prothrombin time (PT). After the death of the mice, their livers were taken out to undergo microscopy and immunohistochemistry to observe the structure of liver tissue, growth of tumor, and expression of ALB. RT-PCR showed that the mRNA expression of AFP, TTR, HNF, ALB, G6P, and TAT emerged since days 3, 3, 5, 9, and 11 respectively and then gradually increased till day 19. ICC showed that the EB cells began to express AFP since day 7, to express CK8 and CK11 since day 9, and to express ALB since day 11. The ICC-positive cells were consistent with the mouse hepatocytes morphologically and were distributed only in the central and marginal areas of the EB cell community. Injected with carbon tetrachloride, the mice showed manifestations of FHF. However, the symptoms of central nervous system emerged later in the mice implanted with the hepatocytes in comparison with the control mice; 8 of the 20 mice in the transplantation group showed ascites in comparison with 14 in the control group. The mean survival time of the transplantation group was 62 hours, significantly longer than that of the control group (23 hours, P < 0.05). Compare with the normal mice, the FHF mice in both groups showed higher ALT and TB and lower ALB and BS (all P < 0.01), however, the levels of ALT and TB were lower, the level of BS was higher, and PT was shorter significantly in the transplantation group than in the control group (all P < 0.05). Pathology showed that no tumor formation was found in both groups and that the transplanted hepatocytes were incorporated well into the liver parenchymal structure and expressed ALB. Hepatocytes originating from ESCs exercise the functions of normal hepatocytes. Transplantation of hepatocytes differentiated from ESCs is able to improve the life quality and lengthen the survival time of FHF mice.

Chimeric molecule IL-6/soluble IL-6 receptor is a potent mitogen for fetal hepatocytes.

A novel recombinant molecule, termed IL-6c and consisting of a chimera of interleukin 6 (IL-6) and its soluble receptor is extremely potent in stimulating proliferation of hematopoietic progenitors. We investigated the effect of the IL-6c on the proliferation and differentiation of E14 fetal hepatocytes. IL-6c, in a dose-dependent manner, stimulated proliferation of E14 fetal rat hepatocytes. Adult hepatocyte mitogens together with IL-6c showed no further effect on proliferation. Hematopoietic stem cells mitogens SCF and flt3 ligand (FL) were also mitogenic for fetal hepatocytes, but did not further enhance the effect of IL-6c on cell proliferation. IL-6c decreased expression of fetal markers alpha-fetoprotein (AFP) and gamma-glutamyltranspeptidase, and induced expression of adult enzyme glucose-6-phosphatase (Gluc-6-P) in E14 hepatocytes. On the other hand, IL-6c strongly reduced, in a dose-dependant manner, expression of albumin and tyrosine aminotransferase (TAT). However, when the cells were grown for 3 days with IL-6c, and IL-6c was removed for the next 5 days, expression of albumin and TAT returned to levels found in control cultures. In conclusion, IL-6c stimulated proliferation and affected gene expression in fetal hepatocytes in culture.

5α-Reduced Glucocorticoids, Novel Endogenous Activators of the Glucocorticoid Receptor

Metabolism of glucocorticoids to A-ring-reduced dihydro- and tetrahydro-derivatives by means of hepatic 5alpha- and 5beta-reductases has long been regarded as a pathway of irreversible inactivation. However, 5alpha-reduced metabolites of other steroids, e.g. testosterone and aldosterone, have significant biological activity. We investigated whether 5alpha-reduced metabolites of corticosterone are glucocorticoid receptor (GR) agonists. Corticosterone, 5alpha-tetrahydrocorticosterone (5alphaTHB), and 5alpha-dihydrocorticosterone (5alphaDHB) were similarly effective in displacing tritiated dexamethasone from binding sites in hepatocytes, whereas 5beta-reduced metabolites were less effective in binding. 5alphaTHB had glucocorticoid receptor agonist effects in vitro and in vivo. After transient co-transfection of hGR and a murine mammary tumor virus-luciferase reporter into HeLa cells, 5alphaTHB was active to a comparable extent as corticosterone (28-fold versus 37-fold induction, respectively, at 1 microm) and additive to the effect of corticosterone. 5beta-Reduced metabolites did not activate GR. In H4IIE hepatoma cells, both 5alphaTHB and corticosterone induced mRNA expression of tyrosine aminotransferase and phosphoenolpyruvate carboxykinase (6.0-versus 10.1-fold and 3.5-versus 3.9-fold at 1 microM, respectively), an effect that was inhibited by RU486. To assess in vivo glucocorticoid activity, suppression of plasma ACTH was demonstrated in adrenalectomized rats after intraperitoneal administration of vehicle (ACTH trough 80.2 pm), corticosterone (5 mg/kg; 22 pm, p < 0.001) or 5alphaTHB (5 mg/kg; 51.3 pm, p < 0.005). Similar endogenous concentrations of corticosterone and 5alphaTHB were detected in rat liver homogenates by gas chromatography mass spectrometry. We conclude that 5alpha-reduced glucocorticoids bind to and activate GR. Transcription of glucocorticoid-regulated genes in tissues that express 5alpha-reductases will thus be influenced by intracellular levels of both corticosterone and its 5alpha-reduced metabolites.