Ornithine Aminotransferase Expression Research Articles

Role of lung ornithine aminotransferase in idiopathic pulmonary fibrosis: regulation of mitochondrial ROS generation and TGF-β1 activity

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant lung remodeling and the excessive accumulation of extracellular matrix (ECM) proteins. In a previous study, we found that the levels of ornithine aminotransferase (OAT), a principal enzyme in the proline metabolism pathway, were increased in the lungs of patients with IPF. However, the precise role played by OAT in the pathogenesis of IPF is not yet clear. The mechanism by which OAT affects fibrogenesis was assessed in vitro using OAT-overexpressing and OAT-knockdown lung fibroblasts. The therapeutic effects of OAT inhibition were assessed in the lungs of bleomycin-treated mice. OAT expression was increased in fibrotic areas, principally in interstitial fibroblasts, of lungs affected by IPF. OAT levels in the bronchoalveolar lavage fluid of IPF patients were inversely correlated with lung function. The survival rate was significantly lower in the group with an OAT level >75.659 ng/mL than in the group with an OAT level ≤75.659 ng/mL (HR, 29.53; p = 0.0008). OAT overexpression and knockdown increased and decreased ECM component production by lung fibroblasts, respectively. OAT knockdown also inhibited transforming growth factor-β1 (TGF)-β1 activity and TGF-β1 pathway signaling. OAT overexpression increased the generation of mitochondrial reactive oxygen species (ROS) by activating proline dehydrogenase. The OAT inhibitor L-canaline significantly attenuated bleomycin-induced lung injury and fibrosis. In conclusion, increased OAT levels in lungs affected by IPF contribute to the progression of fibrosis by promoting excessive mitochondrial ROS production, which in turn activates TGF-β1 signaling. OAT may be a useful target for treating patients with fibrotic lung diseases, including IPF.

Ornithine aminotransferase supports polyamine synthesis in pancreatic cancer.

There is a need to develop effective therapies for pancreatic ductal adenocarcinoma (PDA), a highly lethal malignancy with increasing incidence1 and poor prognosis2. Although targeting tumour metabolism has been the focus of intense investigation for more than a decade, tumour metabolic plasticity and high risk of toxicity have limited this anticancer strategy3,4. Here we use genetic and pharmacological approaches in human and mouse in vitro and in vivo models to show that PDA has a distinct dependence on de novo ornithine synthesis from glutamine. We find thatthis process, which is mediated through ornithine aminotransferase (OAT), supports polyamine synthesis and is required for tumour growth. This directional OAT activity is usually largely restricted to infancy and contrasts with the reliance of most adult normal tissues and other cancer types on arginine-derived ornithine for polyamine synthesis5,6. This dependency associates with arginine depletion in the PDA tumour microenvironment and is driven by mutant KRAS. ActivatedKRAS induces the expression of OAT and polyamine synthesis enzymes, leading to alterations in the transcriptome and open chromatin landscape in PDA tumour cells. The distinct dependence of PDA, but not normal tissue, on OAT-mediated de novo ornithine synthesis provides an attractive therapeutic window for treating patients with pancreatic cancer with minimal toxicity.

Ornithine metabolism and the osmotic stress response in mytilid congeners

ABSTRACT Previous transcriptomic studies have suggested that ornithine decarboxylase (ODC) expression in Mytilids (Mytilus spp.) may be a critical component of the osmotic stress response. Ornithine is catabolized into polyamines by ODC or into glutamate or proline by ornithine aminotransferase (OAT). To better understand how ornithine is metabolized under low salinity conditions, we examined species-specific variation in OAT and ODC expression for three species that vary in their salinity tolerance (M. trossulus, M. edulis, and M. galloprovincialis). We found a consistent decrease in ODC expression during hypoosmotic exposure in all three species but pronounced species-specific increases in OAT expression. During hyperosmotic stress, the patterns of expression of these genes reversed, suggesting that proline or glutamate synthesis is important during low salinity exposure, while polyamine synthesis may be more important during hyperosmotic exposure. These responses were most pronounced in M. galloprovincialis, with an 18-fold increase in OAT during hypoosmotic exposure.

CRISPR correction of the Finnish ornithine delta-aminotransferase mutation restores metabolic homeostasis in iPSC from patients with gyrate atrophy

Hyperornithinemia with gyrate atrophy of the choroid and retina (HOGA) is a severe recessive inherited disease, causing muscular degeneration and retinochoroidal atrophy that progresses to blindness. HOGA arises from mutations in the ornithine aminotransferase (OAT) gene, and nearly one-third of the known patients worldwide are homozygous for the Finnish founder mutation OAT c.1205 T > C p.(Leu402Pro). We have corrected this loss-of-function OAT mutation in patient-derived induced pluripotent stem cells (iPSCs) using CRISPR/Cas9. The correction restored OAT expression in stem cells and normalized the elevated ornithine levels in cell lysates and cell media. These results show an efficient recovery of OAT function in iPSC, encouraging the possibility of autologous cell therapy for the HOGA disease.

Developmental and hormonal regulation of Arabidopsis thaliana ornithine-delta-aminotransferase.

Ornithine aminotransferase (OAT) catalyzes transfer of the delta-amino group from L-ornithine to oxo-glutarate. In plants, this reaction biochemically connects urea cycle, proline cycle, and polyamine biosynthesis pathway. OAT activity is shown to be associated with biotic and abiotic stress responses and nitrogen metabolism, but its physiological role is still unclear. In our study, we decided to investigate transcriptional regulation of the OAT gene in Arabidopsis thaliana under normal conditions and in response to various growth regulators. In the present work, the reporter gene construct containing the Escherichia coli β-glucuronidase gene (gus) under control of the A. thaliana OAT gene promoter was introduced into the genome of A. thaliana ecotype Columbia plants using the floral dip method; GUS activity was assayed in different experimental conditions including hormone treatment, low and high nitrogen and salinity. The GUS activity was analyzed histochemically. Plants were incubated with staining solution containing X-Gluc. We show that under standard growth conditions, the promoter is active during germination and in developing floral organs. OAT promoter activity specifically activates in response to different forms of auxin (IAA, NAA, and 2,4D), cytokinin (6- BAP), ethylene precursor (ACC), high nitrogen and salinity. Analysis of the OAT expression by qRT-PCR confirmed the pattern observed using the GUS reporter system. The OAT gene showed a significantly elevated expression in fourday- old seedlings and in plant roots in response to auxins and cytokinins. The analysis of the OAT promoter structure reveals cis-acting regulatory DNA elements associated with auxin regulation and abiotic stresses. The results of the study indicate that the OAT gene is involved in developmental processes and is regulated by auxin and cytokinins.

An Increase in Liver Polyamine Concentration Contributes to the Tryptophan-Induced Acute Stimulation of Rat Hepatic Protein Synthesis.

Tryptophan has a unique role as a nutritional signaling molecule that regulates protein synthesis in mouse and rat liver. However, the mechanism underlying the stimulating actions of tryptophan on hepatic protein synthesis remains unclear. Proteomic and metabolomic analyses were performed to identify candidate proteins and metabolites likely to play a role in the stimulation of protein synthesis by tryptophan. Overnight-fasted rats were orally administered L-tryptophan and then sacrificed 1 or 3 h after administration. Four differentially expressed protein spots were detected in rat liver at 3 h after tryptophan administration, of which one was identified as an ornithine aminotransferase (OAT) precursor. OAT is the main catabolic enzyme for ornithine, and its expression was significantly decreased by tryptophan administration. The concentration of ornithine was increased in the liver at 3 h after tryptophan administration. Ornithine is a precursor for polyamine biosynthesis. Significantly increased concentrations of polyamines were found in the liver at 3 h after administration of tryptophan. Additionally, enhanced hepatic protein synthesis was demonstrated by oral administration of putrescine. We speculate that the increase in ornithine level through suppression of OAT expression by tryptophan administration may lead to accelerated polyamine synthesis, thereby promoting protein synthesis in the liver.

Differential regulation of genes co-involved in aroma production and stress amelioration during salt acclimation in indica rice cultivars

The present study investigated the effects of salt acclimation on the expression of genes, viz., proline dehydrogenase (PDH), Δ1-pyrroline-5-carboxylate-synthetase (P5CS), P5C dehydrogenase (P5CDH), ornithine aminotransferase (OAT), betaine aldehyde dehydrogenase 1 (BADH1), BADH2, triose phosphate isomerase (TPI), ornithine decarboxylase (ODC), arginine decarboxylase (ADC), S-adenosyl-methionine decarboxylase (SAMDC), spermidine synthase (SPDS), spermine synthase (SPMS), diamine oxidase (DAO) and polyamine oxidase (PAO) in four indigenous aromatic varieties, viz., Tulaipanji (TP), Radhunipagal (RP), Kalonunia (KN) and Gobindobhog (GB) along with a non-aromatic cultivar, IR-64. The PDH, P5CS, P5CDH and OAT expression showed the regulation of the P5C pathway of aroma synthesis, with OAT appearing to regulate the crosstalk between the P5C and the polyamine (PA) pathway for the production of aroma. The role of PA pathway was further validated by monitoring the expression of ODC, ADC, SAMDC, SPDS, SPMS, DAO and PAO. The BADH2 expression reflected the fact that the enzyme encoded by the gene was directly and negatively associated with aroma content in rice. The TPI expression established that the enzyme encoded by this gene leading to the synthesis of methylglyoxal (MG) is also a key regulator for aroma synthesis. The current study established the varietal differences in the expression of the aforementioned genes in the selected rice cultivars. The salt-stressed aromatic varieties followed the P5C pathway to synthesize aroma. TP and RP exhibited the capability to generate aroma via PA-dependent pathway. Salt-stressed KN, on the contrary, accumulated higher BADH2 and lower TPI transcripts, which correlated with our previous report of KN accumulating low aroma during salinization due to efficient metabolite channelization towards stress amelioration.

Quantitative proteomic analysis of mitochondrial proteins differentially expressed between small cell lung cancer cells and normal human bronchial epithelial cells

BackgroundSmall cell lung cancer (SCLC) is highly aggressive and is associated with a dismal prognosis. However, there are no clinically recognized biomarkers for early diagnosis. In this study, we used quantitative proteomics to build differential mitochondrial protein profiles that may be used for early diagnosis and investigated the pathogenesis of lung cancer.MethodsWe cultured SCLC cells (NCI‐H446) and normal human bronchial epithelial cells (16‐HBE); mitochondria were extracted and purified using differential and Percoll density gradient centrifugation. Subsequently, we used Western blot analysis to validate mitochondrial purity and labeled proteins/peptides from NCI‐H446 and 16‐HBE cells using relative and absolute quantification of ectopic tags. We then analyzed mixed samples and identified proteins using two‐dimensional liquid chromatography‐tandem mass spectrometry. Additionally, we performed subsequent bioinformatic proteome analyses using the programs ExPASy, GOA, and STRING. Finally, the relationship between ornithine aminotransferase expression and clinicopathological features in lung cancer patients was evaluated using immunohistochemistry.ResultsOne hundred and fifty‐three mitochondrial proteins were differentially expressed between 16‐HBE and NCI‐H446 cells. The expression of 30 proteins between 16‐HBE and NCI‐H446 cells increased more than 1.3‐fold. The upregulation of ornithine aminotransferase was associated with pathological grade and clinical tumor node metastasis stage.ConclusionOur experiment represented a promising method for building differential mitochondrial protein profiles between NCI‐H446 and 16‐HBE cells. Such analysis may also help to identify novel biomarkers of lung cancer.

Proteomic Study on the New Potential Mechanism and Biomarkers of Diabetes.

Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia. So far, the pathogenesis of diabetes has not been fully elucidated. Identifying new potential molecule mechanisms and biomarkers in this process could contribute to the understanding of pathophysiology. Proteomic changes in the liver of type 2 diabetic mice (n = 6) and normal mice (n = 6) are studied. Triplicate experiments are carried out for each sample. A total of 15 differentially expressed proteins (DEPS ) are identified and Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicates that DEPS mainly involved two inflammatory pathways: glutathione metabolic pathway and the arachidonic acid metabolic pathway. The core of protein-protein interaction is the tumor necrosis factor inflammatory pathway, indicating the connection between inflammation and diabetes. Ten out of fifteen gene transcript levels are consistent with proteomics by quantitative RT-PCR validation. The transcriptional levels of OAT (ornithine aminotransferase) and fructose-1,6-bisphosphatase1 (FBP1) were significantly increased, whereas fatty acid binding protein 5 (FABP5) and ef-2 transcription levels decreased significantly. In addition, western blotting results showed that the expression of OAT and FBP protein increased significantly in the diabetes group, while elongation factor 2 decreased significantly and FABP do not have significant difference in the diabetes group. Taken together, the present exploratory liver proteomic analysis might be seen as an important starting point for studies targeting specific liver proteins aimed at the implementation of new biomarkers for the early detection of type 2 diabetes mellitus-related potential mechanisms, hoping to provide biomarkers and clinical therapeutic targets of type 2 diabetes mellitus.

The Increase in Liver Polyamine Concentration Contributes to the Tryptophan‐induced Stimulation of Hepatic Protein Synthesis

Tryptophan has a unique role as a nutritional signaling molecule that regulates protein synthesis in mice and rat liver. However, the mechanism of the stimulating actions of tryptophan on hepatic protein synthesis has not been clearly elucidated. Tryptophan has been suggested to stimulate protein synthesis at the translational level, because polysome aggregation was observed in a short time after oral administration of tryptophan. Therefore, we focused on the acute effects of tryptophan on protein synthesis. In the present study, proteomic and metabolomic analysis was performed to identify candidate proteins and metabolites, which are likely to play a role in stimulation of protein synthesis by tryptophan. Eighteen hours fasted Wistar rats were divided into 3 groups. One group of animals was sacrificed without any treatment as a control. Remaining two groups of animals were administered 135mg/100g body weight L‐tryptophan by oral gavage and then sacrificed after 1h or 3h after administration. With two‐dimensional electrophoresis and image analysis, four differentially expressed protein spots were detected at 3h after administration of tryptophan, and one of which was identified as key enzyme in the biosynthetic pathway of ornithine, ornithine aminotransferase (OAT) precursor. OAT is the main catabolic enzyme of ornithine and its expression was significantly decreased by tryptophan administration. The concentration of ornithine was increased in both serum and liver at 3h after administration of tryptophan. Ornithine is a precursor for polyamine biosynthesis. Ornithine decarboxylase (ODC) catalyzes the first step in the polyamine biosynthetic pathway forming putrescine, which is then converted into the polyamines spermidine and spermine. It has been reported that oral administration of tryptophan to overnight‐fasted rats exhibited increase in the hepatic ODC activity. Significantly increased concentrations of polyamines, as well as ornithine were found in the liver at 3h after administration of tryptophan. Based on these results, we speculate that the increase in ornithine level through suppression of OAT expression and stimulation of ODC activity by tryptophan administration may lead to accelerated polyamine synthesis and thereby promote protein synthesis in the liver.

Expression and bioinformatic analysis of ornithine aminotransferase in non-small cell lung cancer

背景与目的已有的研究表明，鸟氨酸氨基转移酶（ornithine aminotransferase, OAT）可能参与多种恶性肿瘤的发生和发展，本研究旨在检测非小细胞肺癌（non-small cell lung cancer, NSCLC）中OAT mRNA和蛋白质的表达，并对其进行生物信息学分析。方法通过RT-PCR的方法比较A549和16HBE细胞间OAT mRNA水平的差异；采用免疫组织化学SP法检测55例肺癌组织和17例癌旁肺组织中OAT蛋白表达；利用生物信息学的方法对OAT蛋白的生物信息学特征和相互作用蛋白进行分析，并对筛选出来的相互作用蛋白进行GO注释和信号通路分析。结果① A549中OAT mRNA相对含量较16HBE中低，两者差异约2.85倍。②NSCLC中OAT蛋白的表达明显高于癌旁肺组织（P < 0.05）；OAT蛋白在鳞癌和腺癌间的表达差异有统计学意义，而与患者性别、年龄、有无淋巴结转移、肿瘤直径及TNM分期无关。③生物信息学分析提示OAT蛋白定位于线粒体、为高度保守的亲水性蛋白，具有Aminotran-3结构域，可能存在多个丝/苏氨酸磷酸化位点，在信号转导、转录和分子转运等方面发挥一定的作用；在筛选出来的54种可能和OAT存在相互作用的蛋白质中，TNF和TRAF6这两种蛋白参与了NF-κB信号通路。结论OAT可能在NSCLC的发生和发展中发挥重要作用，有望成为肺癌的早期诊断标志物和治疗的新靶点。

Evidence for a role of the ileum in the control of nitrogen homeostasis via the regulation of arginine metabolism

As arginine plays a key role in the regulation of liver ureagenesis, we hypothesised that a modulation of enzymes involved in arginine metabolism within the intestine contributes to the regulation of N homeostasis according to protein supply. Our aim was to study the influence of variations in protein or amino acid (AA) supply on intestinal arginase, glutaminase, ornithine aminotransferase (OAT), argininosuccinate lyase and argininosuccinate synthetase. We evaluated in vivo in rats the responses of these enzymes to short-term (ST, 16 h) and long-term (LT, 15 d) variations in dietary protein (10, 17 or 25 % protein diet). In addition, in order to test whether these responses could involve a direct action of AA on the gene expression and activity of these enzymes, Caco-2/TC7 cells were cultured for 3 d with increasing AA concentrations. In vivo, in the ST, both high- and low-protein diets increased arginase activity in the intestinal mucosa (ST25 %: 46 (sem 2) μmol/g per min and ST10 %: 46 (sem 2) μmol/g per min v. ST17 %: 36 (sem 3) μmol/g per min, P < 0.05). In the LT, OAT expression was increased in the LT10 % group (+277 %, P < 0.05) compared with the LT17 % group. Caco-2/TC7 cells showed inverse relationships between AA supply and arginase (P = 0.058) and OAT (P = 0.035) expressions. The present study demonstrates the regulation of intestinal arginase and OAT expressions in response to protein supply. Our in vitro experiments further indicate a direct AA-induced regulation of the mRNA abundance of these enzymes. In situations of limited protein supply, this regulation would increase intestinal arginine catabolism and, possibly via a decrease in arginine portal release, decrease hepatic AA oxidation, thus promoting N sparing.

624 PROTEOMICS AND GENOMICS INVESTIGATIONS OF DIETHYLSTILBESTROL ACTION ON A PROSTATE CANCER CELL LINE 22RV1

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 2011624 PROTEOMICS AND GENOMICS INVESTIGATIONS OF DIETHYLSTILBESTROL ACTION ON A PROSTATE CANCER CELL LINE 22RV1 Pierre Bigot, Kevin Mouzat, Souhil Lebdai, Catherine Guette, Géraldine Cancel-tassin, Abdel-Rahmène Azzouzi, and Olivier Cussenot Pierre BigotPierre Bigot Angers, France More articles by this author , Kevin MouzatKevin Mouzat Paris, France More articles by this author , Souhil LebdaiSouhil Lebdai Angers, France More articles by this author , Catherine GuetteCatherine Guette Angers, France More articles by this author , Géraldine Cancel-tassinGéraldine Cancel-tassin Paris, France More articles by this author , Abdel-Rahmène AzzouziAbdel-Rahmène Azzouzi Angers, France More articles by this author , and Olivier CussenotOlivier Cussenot Paris, France More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1477AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Diethylstilbestrol (DES) is a synthetic estrogen used for prostate cancer (PCa) treatment. Its action is independent from the estrogen receptors and is preserved after a first line hormonotherapy treatment. By using 2-Dimensional-Differential in-Gel Electrophoresis (2D-DIGE), we presented the expression variations of 14 proteins in the prostate cancer cell line 22RV1 after DES exposure. Our objective was to study the action of DES on the prostate cancer cell line 22RV1 by using a complementary quantitative proteomic approach by isobaric tagging iTRAQ followed by an assessment of variations of the identified protein of interest by RT-PCR. METHODS The prostate cancer 22RV1 cells were exposed to standard and DES (10 and 20 μM) enriched medium. After extraction, trypsine digestion and iTRAQ labeling, peptides were fractionated using offgel expression levels. Each iTRAQ set was analyzed by matrix assisted laser desorption-time-of-flight (MALDI-TOF/TOF) mass spectrometry. Statistical analysis was performed by iQuantitator. RNA were also extracted and quantitative RT-PCR was performed. RESULTS After proteomic analysis, 895 constitutive proteins of the 22RV1 proteome were identified. Among these proteins, 66 had a modified expression due to DES exposure (23 over-expressed, 46 under-expressed). Most of them were implicated in apoptosis and redox processes and had a mitochondrial expression. Gene expression analysis confirmed that there estrogens receptors ER1 and ER2 were not expressed in our model. We assessed by RT-PCR the expression of ornithine aminotransferase (OAT), Heat shock protein beta-1 (HSPB1) and Insulin-like growth factor-binding protein 2 (IGFB2), which are implicated in natural history of PCa. Their expression was changed by DES exposure according to proteomic analysis. These analyses confirmed the over-expression of OAT (p=0.006) and HSBP1 (p=0.046). However, the variation of IGFB2 was contradictory (it was over-expressed according to iTRAQ analysis and under-expressed according to RT-PCR (p=0,011)), suggesting that DES exerts both transcriptional and post-transcriptional effects on 22RV1 cells. CONCLUSIONS The proteomic complementary approach by 2D-DIGE method and iTRAQ analysis allowed identifying 67 proteins with an expression variation depending on the presence of DES. Among them, several mitochondrial proteins were identified. Thus a hypothesis on the apoptosis consecutive to an oxidative stress induced by DES can be formulated. The over expression of OAT and HSBP1 has been confirmed by genomic and proteomic analysis. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e251 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Pierre Bigot Angers, France More articles by this author Kevin Mouzat Paris, France More articles by this author Souhil Lebdai Angers, France More articles by this author Catherine Guette Angers, France More articles by this author Géraldine Cancel-tassin Paris, France More articles by this author Abdel-Rahmène Azzouzi Angers, France More articles by this author Olivier Cussenot Paris, France More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

P34 The mechanism behind synergistic action of l-ornithine and phenylacetate to reduce ammonia in bile-duct ligation rats

AimThis study was designed to test the hypothesis that OP has additional actions on the key ammonia regulating enzymes glutamine synthetase (GS) and glutaminase (GA), which results in the observed...

Adaptative response of nitrogen metabolism in early endotoxemia: role of ornithine aminotransferase

Arginine (Arg) and glutamine (Gln) utilization is greatly increased during catabolic stress. While the supply of both amino acids has been advocated in this situation, arginine administration is possibly associated with deleterious effects. From a metabolic point of view, these two amino acids are reciprocal precursors via ornithine aminotransferase (OAT). We hypothesized that OAT plays a key role in the interconversion between Arg and Gln. To test this hypothesis, we evaluated the influence of OAT activity in a model of septic shock induced by intraperitoneal injection of lipopolysaccharide (LPS) in wild-type (WT) and transgenic mice overexpressing OAT (OAT) in the liver, kidney and intestine, i.e. the three main organs of OAT expression. Plasma and tissue amino acid concentrations and tissue OAT expression and activity were measured. Five hours after LPS injection, WT and OAT mice showed a similar response to LPS in terms of inflammatory cytokine production and protein catabolism, suggesting that the interconversion between Arg and Gln through this pathway remains limited. Endotoxemia led to a significant decrease in plasma Orn levels and an increase in liver Orn levels. Of note, Orn levels were always lower in OAT mice. While only plasma Arg and Gln remained unaffected by LPS treatment, hepatic Gln was significantly increased without any difference between the two genotypes. In this model of early endotoxemia, arginine and glutamine maintained their metabolic homeostasis. Our results show an inhibition of OAT activity and expression in the liver following LPS treatment. These data highlight the importance of OAT in ornithine metabolism, especially in the liver, and suggest a post-transcriptional regulation of OAT by LPS in the liver.

Regulation of ornithine aminotransferase gene expression and activity by all-transretinoic acid in Caco-2 intestinal epithelial cells

Ornithine aminotransferase (OAT) is a crucial enzyme in the synthesis of citrulline and arginine from glutamine/glutamate and proline by enterocytes of the small intestine. However, a role for OAT in intestinal polyamine synthesis and cell growth is not known. All-transretinoic acid (RA), an active metabolite of vitamin A, regulates the activity of several metabolic enzymes related to OAT, including ornithine decarboxylase and arginase, which may influence the function of OAT through effects on substrate (ornithine) availability. The objective of the present study was to test the hypothesis that RA regulates OAT mRNA expression and enzymatic activity in intestinal epithelial cells. Caco-2 cells were cultured for 12–72 h in the presence of 0, 0.01 and 1 μM RA and then used for measurements of OAT mRNA levels and enzyme activity as well as ornithine and polyamines. Treatment with RA induced increases in OAT gene expression and enzymatic activity, which resulted in decreased intracellular concentrations of ornithine and polyamines (putrescine, spermidine and spermine) in a dose-dependent manner. These changes occurred concomitantly with a decrease in the total number of cells, and the increase in OAT activity was due to increased OAT mRNA expression. In cells treated with 1 μM RA, addition of 10 μM putrescine to culture medium restored both cellular levels of polyamines and cell numbers to the values for the control group (without addition of RA). We conclude that exposure of Caco-2 cells to RA induces OAT expression for increasing ornithine catabolism. This leads to a reduced availability of intracellular ornithine for polyamine synthesis, thereby decreasing cell proliferation. These novel findings indicate a functional role for OAT in regulating intestinal polyamine synthesis and growth.

Polyamine homeostasis in arginase knockout mice

The role of ornithine decarboxylase (ODC) in polyamine metabolism has long been established, but the exact source of ornithine has always been unclear. The arginase enzymes are capable of producing ornithine for the production of polyamines and may hold important regulatory functions in the maintenance of this pathway. Utilizing our unique set of arginase single and double knockout mice, we analyzed polyamine levels in the livers, brains, kidneys, and small intestines of the mice at 2 wk of age, the latest timepoint at which all of them are still alive, to determine whether tissue polyamine levels were altered in response to a disruption of arginase I (AI) and II (AII) enzymatic activity. Whereas putrescine was minimally increased in the liver and kidneys from the AII knockout mice, spermidine and spermine were maintained. ODC activity was not greatly altered in the knockout animals and did not correlate with the fluctuations in putrescine. mRNA levels of ornithine aminotransferase (OAT), antizyme 1 (AZ1), and spermidine/spermine-N(1)-acetyltransferase (SSAT) were also measured and only minor alterations were seen, most notably an increase in OAT expression seen in the liver of AI knockout and double knockout mice. It appears that putrescine catabolism may be affected in the liver when AI is disrupted and ornithine levels are highly reduced. These results suggest that endogenous arginase-derived ornithine may not directly contribute to polyamine homeostasis in mice. Alternate sources such as diet may provide sufficient polyamines for maintenance in mammalian tissues.

Sex-differential expression of ornithine aminotransferase in the mouse kidney

The mouse kidney expresses the gene of ornithine aminotransferase (Oat). Previous works suggest that Oat is differentially expressed in female and male mouse kidney (Alonso E, Rubio V. Biochem J 259: 131-138, 1989; Levillain O, Diaz JJ, Blanchard O, Dechaud H. Endocrinology 146: 950-959, 2005; Manteuffel-Cymborowska M, Chmurzynska W, Peska M, Grzelakowska-Sztabert B. Int J Biochem Cell Biol 27: 287-295, 1995; Natesan S, Reddy SR. Comp Biochem Physiol B Biochem Mol Biol 130: 585-595, 2001; Yu H, Yoo PK, Aguirre CC, Tsoa RW, Kern RM, Grody WW, Cederbaum SD, Iyer RK. J Histochem Cytochem 51: 1151-1160, 2003). This study was designed to provide a detailed description of the sexual dimorphism of Oat expression in the mouse kidney and to test the influence of sex hormones on its regulation. Experiments were performed on male and female Swiss OF1 mice during their postnatal development, at adulthood, and in orchidectomized and ovariectomized mice. Kidneys, dissected renal zones, and mitochondria were used to analyze OAT mRNA and protein levels and measure OAT activity. The results revealed that before puberty, Oat expression was similar between female and male kidneys whereas from puberty until adulthood Oat expression increased in the female kidney, becoming approximately 2.5-fold higher than in the male kidney. This sex-differential expression of Oat was associated with a sex-specific distribution of Oat along the corticopapillary axis and within the nephron. OAT was three- to fourfold more expressed in the female than the male cortex. In males, Oat was highly expressed in the medulla, mainly in the thick ascending limbs. Renal Oat distribution in orchidectomized mice resembled that in the females. Ovariectomy did not influence Oat expression. Sex differences are explained by the physiological increase in plasma testosterone in males. Expression of medium-chain acyl-CoA synthetase protein confirmed this finding. We report sexual dimorphism of Oat expression in the mouse kidney and show that Oat is naturally downregulated in the presence of testosterone.

Control of nitrogen homeostasis: intestinal arginine metabolism regulation according to protein supply.

Portal arginine (Arg) flow plays a key role in liver ureagenesis. In the intestine, Arg can be converted into citrulline (Cit) via arginase and ornithine carbamyl-transferase (OCT). Cit can also be synthesized from glutamine via glutaminase and ornithine aminotransferase (OAT). The aim of this work was to study the regulation of these enzymes in the intestine in relation to time and level of protein supply. 48 rats were divided in 6 groups to receive a 10% (10P), 17% (17P) or 25% protein (25P) diet for either 16 hours short-term (ST) or 15 days long-term (LT). LT group urines were collected to determine N balance. At sacrifice, blood was collected for plasma amino acid measurement, and the intestinal mucosa was removed for enzyme expression and activity analysis (results are given as means ± SEM, ANOVA + Newman-Keuls test [∗ vs. 17P: p < 0.05]). N balance remained similar in all groups. Cit plasma levels at LT varied with diet (10P: 87 ± 2 ∗; 17P: 69 ± 4; 25P: 88 ± 5 ∗ in mM). There was an increase in ileum at ST in arginase activity under 25P and 10P diets (respectively 46 ± 2 and 46 ± 2 vs. 36 ± 3 (17P) in μmol/g/min) and at LT, in OAT expression in the 10P group (+ 277 %∗). With an hypoproteic diet, Cit synthesis appears to be ensured by either OAT activation at LT or arginase at ST. Surprisingly, both low and high protein diets increased Cit production. Research is needed to gain further insight into the regulation of this complex metabolic network.

Ornithine deficiency in the arginase double knockout mouse

Knockout mouse models have been created to study the consequences of deficiencies in arginase AI and AII, both individually and combined. The AI knockout animals die by 14 days of age from hyperammonemia, while the AII knockout has no obvious phenotype. The double knockout (AI −/−/AII −/−) exhibits the phenotype of the AI-deficient mice, with the additional absence of AII not exacerbating the observed phenotype of the AI knockout animals. Plasma amino acid measurements in the double knockout have shown arginine levels increased roughly 100-fold and ornithine decreased roughly 10-fold as compared to wildtype. Liver ornithine levels were reduced to 2% of normal in the double knockout with arginine very highly elevated. Arginine and ornithine were also altered in other tissues in the double knockout mice, such as kidney, brain, and small intestine. This is the first demonstration that the fatal hyperammonemia in the AI knockout mouse is almost certainly due to ornithine deficiency, the amino acid needed to drive the urea cycle. Others have shown that the expression of ornithine aminotransferase (OAT) rapidly decreases in the intestine at the same age when the AI-deficient animals die, indicating that this enzyme is critical to the maintenance of ornithine homeostasis, at least at this early stage of mouse development. Although most human AI-deficient patients have no symptomatic hyperammonemia at birth, it is possible that clinically significant ornithine deficiency is already present.