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 that this 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. Activated KRAS 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.

Lactobacillus maintains healthy gut mucosa by producing L-Ornithine.

Gut mucosal layers are crucial in maintaining the gut barrier function. Gut microbiota regulate homeostasis of gut mucosal layer via gut immune cells such as RORγt (+) IL-22(+) ILC3 cells, which can influence the proliferation of mucosal cells and the production of mucin. However, it is unclear how gut microbiota execute this regulation. Here we show that lactobacilli promote gut mucosal formation by producing L-Ornithine from arginine. L-Ornithine increases the level of aryl hydrocarbon receptor ligand L-kynurenine produced from tryptophan metabolism in gut epithelial cells, which in turn increases RORγt (+)IL-22(+) ILC3 cells. Human REG3A transgenic mice show an increased proportion of L-Ornithine producing lactobacilli in the gut contents, suggesting that gut epithelial REG3A favors the expansion of L-Ornithine producing lactobacilli. Our study implicates the importance of a crosstalk between arginine metabolism in Lactobacilli and tryptophan metabolism in gut epithelial cells in maintaining gut barrier.

The Arabidopsis N-Acetylornithine Deacetylase Controls Ornithine Biosynthesis via a Linear Pathway with Downstream Effects on Polyamine Levels.

Arabidopsis thaliana At4g17830 codes for a protein showing sequence similarity with the Escherichia coli N-acetylornithine deacetylase (EcArgE), an enzyme implicated in the linear ornithine (Orn) biosynthetic pathway. In plants, N-acetylornithine deacetylase (NAOD) activity has yet to be demonstrated; however, At4g17830-silenced and mutant (atnaod) plants display an impaired reproductive phenotype and altered foliar levels of Orn and polyamines (PAs). Here, we showed the direct connection between At4g17830 function and Orn biosynthesis, demonstrating biochemically that At4g17830 codes for a NAOD. These results are the first experimental proof that Orn can be produced in Arabidopsis via a linear pathway. In this study, to identify the role of AtNAOD in reproductive organs, we carried out a transcriptomic analysis on atnaod mutant and wild-type flowers. In the atnaod mutant, the most relevant effects were the reduced expression of cysteine-rich peptide-coding genes, known to regulate male-female cross-talk during reproduction, and variation in the expression of genes involved in nitrogen:carbon (N:C) status. The atnaod mutant also exhibited increased levels of sucrose and altered sensitivity to glucose. We hypothesize that AtNAOD participates in Orn and PA homeostasis, contributing to maintain an optimal N:C balance during reproductive development.

Deciphering the Substrate Specificity of SbnA, the Enzyme Catalyzing the First Step in Staphyloferrin B Biosynthesis.

Staphylococcus aureus assembles the siderophore, staphyloferrin B, from l-2,3-diaminopropionic acid (l-Dap), α-ketoglutarate, and citrate. Recently, SbnA and SbnB were shown to produce l-Dap and α-ketoglutarate from O-phospho-l-serine (OPS) and l-glutamate. SbnA is a pyridoxal 5'-phosphate (PLP)-dependent enzyme with homology to O-acetyl-l-serine sulfhydrylases; however, SbnA utilizes OPS instead of O-acetyl-l-serine (OAS), and l-glutamate serves as a nitrogen donor instead of a sulfide. In this work, we examined how SbnA dictates substrate specificity for OPS and l-glutamate using a combination of X-ray crystallography, enzyme kinetics, and site-directed mutagenesis. Analysis of SbnA crystals incubated with OPS revealed the structure of the PLP-α-aminoacrylate intermediate. Formation of the intermediate induced closure of the active site pocket by narrowing the channel leading to the active site and forming a second substrate binding pocket that likely binds l-glutamate. Three active site residues were identified: Arg132, Tyr152, Ser185 that were essential for OPS recognition and turnover. The Y152F/S185G SbnA double mutant was completely inactive, and its crystal structure revealed that the mutations induced a closed form of the enzyme in the absence of the α-aminoacrylate intermediate. Lastly, l-cysteine was shown to be a competitive inhibitor of SbnA by forming a nonproductive external aldimine with the PLP cofactor. These results suggest a regulatory link between siderophore and l-cysteine biosynthesis, revealing a potential mechanism to reduce iron uptake under oxidative stress.

Arginase 1 is a negative regulator of osteoclast differentiation.

Arginase 1 (Arg1) limits the availability of l-arginine for producing nitric oxide (NO) and ornithine, a substrate for polyamine synthesis. Anti-osteoclastogenic activities of NO and polyamines, and the involvement of Arg1 on the dendritic cell differentiation of dendritic cells have been reported, but the relevance of Arg1 to osteoclast differentiation has not been investigated. Here, we observed Arg1 down-regulation during the RANKL-induced differentiation of bone marrow-derived macrophages into osteoclasts. Arg1 overexpression significantly inhibited osteoclast differentiation with low NO production, while Arg1 knockdown enhanced osteoclast differentiation with high NO production. These results suggest that Arg1 and NO have reciprocal roles as negative and positive regulators, respectively, of osteoclast differentiation. We conclude that Arg1 is down-regulated during osteoclast differentiation and may negatively regulate osteoclast differentiation by regulating NO production.

Implication of gluconate kinase activity in L-ornithine biosynthesis in Corynebacterium glutamicum.

With the purpose of generating a microbial strain for L-ornithine production in Corynebacterium glutamicum, genes involved in the central carbon metabolism were inactivated so as to modulate the intracellular level of NADPH, and to evaluate their effects on L-ornithine production in C. glutamicum. Upon inactivation of the 6-phosphoglucoisomerase gene (pgi) in a C. glutamicum strain, the concomitant increase in intracellular NADPH concentrations from 2.55 to 5.75 mmol g⁻¹ (dry cell weight) was accompanied by reduced growth rate and L-ornithine production, suggesting that L-ornithine production is not solely limited by NADPH availability. In contrast, inactivation of the gluconate kinase gene (gntK) led to a 51.8 % increase in intracellular NADPH concentration, which resulted in a 49.9 % increase in L-ornithine production. These results indicate that excess NADPH is not necessarily rate-limiting, but is required for increased L-ornithine production in C. glutamicum.

An enzymatic pathway for the biosynthesis of the formylhydroxyornithine required for rhodochelin iron coordination.

Rhodochelin, a mixed catecholate-hydroxamate type siderophore isolated from Rhodococcus jostii RHA1, holds two L-δ-N-formyl-δ-N-hydroxyornithine (L-fhOrn) moieties essential for proper iron coordination. Previously, bioinformatic and genetic analysis proposed rmo and rft as the genes required for the tailoring of the L-ornithine (L-Orn) precursor [Bosello, M. (2011) J. Am. Chem. Soc.133, 4587-4595]. In order to investigate if both Rmo and Rft constitute a pathway for L-fhOrn biosynthesis, the enzymes were heterologously produced and assayed in vitro. In the presence of molecular oxygen, NADPH and FAD, Rmo monooxygenase was able to convert L-Orn into L-δ-N-hydroxyornithine (L-hOrn). As confirmed in a coupled reaction assay, this hydroxylated intermediate serves as a substrate for the subsequent N(10)-formyl-tetrahydrofolate-dependent (N(10)-fH(4)F) Rtf-catalyzed formylation reaction, establishing a route for the L-fhOrn biosynthesis, prior to its incorporation by the NRPS assembly line. It is of particular interest that a major improvement to this study has been reached with the use of an alternative approach to the chemoenzymatic FolD-dependent N(10)-fH(4)F conversion, also rescuing the previously inactive CchA, the Rft-homologue in coelichelin assembly line [Buchenau, B. (2004) Arch. Microbiol.182, 313-325; Pohlmann, V. (2008) Org. Biomol. Chem.6, 1843-1848].

Oxidant-mediated modification of the cellular thiols is sufficient for arginase activation in cultured cells.

Increased arginase activity in the vasculature has been implicated in the regulation of nitric oxide (NO) homeostasis, leading to the development of vascular disease and the promotion of tumor cell growth. Recently, we showed that cysteine, in the presence of iron, promotes arginase activity by driving the Fenton reaction. In the present report, we showed that induction of oxidative stress in erythroleukemic cells with the thiol-specific oxidant, diamide, led to an increase in arginase activity by 42% (P = 0.02; vs. control). By using specific antibodies, it was demonstrated that this increase correlated with an increase in arginase-1 levels in the cells and with corresponding decreases in glutathione and protein thiol levels. Treatment of cells with aurothiomalate (ATM), a protein thiol-complexing agent, diminished the activity of arginase and arginase-1 levels by 19.5 and 35.2%, respectively (vs. control) and significantly decreased both glutathione and protein thiol levels, further implicating the thiol redox system in the cellular activation of arginase. Furthermore, diamide significantly altered the kinetics of arginase, resulting in the doubling of its V(max) (vs. control). Our presented data demonstrate, for the first time that the intracellular arginase activation is may be enhanced in part, via a cellular thiol-mediated mechanism.

Living with high putrescine: expression of ornithine and arginine biosynthetic pathway genes in high and low putrescine producing poplar cells.

Arginine (Arg) and ornithine (Orn), both derived from glutamate (Glu), are the primary substrates for polyamine (PA) biosynthesis, and also play important roles as substrates and intermediates of overall N metabolism in plants. Their cellular homeostasis is subject to multiple levels of regulation. Using reverse transcription quantitative PCR (RT-qPCR), we studied changes in the expression of all genes of the Orn/Arg biosynthetic pathway in response to up-regulation [via transgenic expression of mouse Orn decarboxylase (mODC)] of PA biosynthesis in poplar (Populus nigra × maximowiczii) cells grown in culture. Cloning and sequencing of poplar genes involved in the Orn/Arg biosynthetic pathway showed that they have high homology with similar genes in other plants. The expression of the genes of Orn, Arg and PA biosynthetic pathway fell into two hierarchical clusters; expression of one did not change in response to high putrescine, while members of the other cluster showed a shift in expression pattern during the 7-day culture cycle. Gene expression of branch point enzymes (N-acetyl-Glu synthase, Orn aminotransferase, Arg decarboxylase, and spermidine synthase) in the sub-pathways, constituted a separate cluster from those involved in intermediary reactions of the pathway (N-acetyl-Glu kinase, N-acetyl-Glu-5-P reductase, N-acetyl-Orn aminotransferase, N (2)-acetylOrn:N-acetyl-Glu acetyltransferase, N (2)-acetyl-Orn deacetylase, Orn transcarbamylase, argininosuccinate synthase, carbamoylphosphate synthetase, argininosuccinate lyase, S-adenosylmethionine decarboxylase, spermine synthase). We postulate that expression of all genes of the Glu-Orn-Arg pathway is constitutively coordinated and is not influenced by the increase in flux rate through this pathway in response to increased utilization of Orn by mODC; thus the pathway involves mostly biochemical regulation rather than changes in gene expression. We further suggest that Orn itself plays a major role in the regulation of this pathway.

Biosynthesis and defensive function of Nδ-acetylornithine, a jasmonate-induced Arabidopsis metabolite.

Since research on plant interactions with herbivores and pathogens is often constrained by the analysis of already known compounds, there is a need to identify new defense-related plant metabolites. The uncommon nonprotein amino acid N(δ)-acetylornithine was discovered in a targeted search for Arabidopsis thaliana metabolites that are strongly induced by the phytohormone methyl jasmonate (MeJA). Stable isotope labeling experiments show that, after MeJA elicitation, Arg, Pro, and Glu are converted to Orn, which is acetylated by NATA1 to produce N(δ)-acetylornithine. MeJA-induced N(δ)-acetylornithine accumulation occurs in all tested Arabidopsis accessions, other Arabidopsis species, Capsella rubella, and Boechera stricta, but not in less closely related Brassicaceae. Both insect feeding and Pseudomonas syringae infection increase NATA1 expression and N(δ)-acetylornithine accumulation. NATA1 transient expression in Nicotiana tabacum and the addition of N(δ)-acetylornithine to an artificial diet both decrease Myzus persicae (green peach aphid) reproduction, suggesting a direct toxic or deterrent effect. However, since broad metabolic changes that are induced by MeJA in wild-type Arabidopsis are attenuated in a nata1 mutant strain, there may also be indirect effects on herbivores and pathogens. In the case of P. syringae, growth on a nata1 mutant is reduced compared with wild-type Arabidopsis, but growth in vitro is unaffected by N(δ)-acetylornithine addition.

Extracorporeal photochemotherapy induces arginase 1 in patients with graft versus host disease.

The benefits of extracorporeal photochemotherapy (ECP; psoralen and UVA exposure of blood mononuclear cells) in graft-versus-host-disease (GVHD) are well-recognized, but the mechanisms of action remain elusive. As the metabolism of l-arginine in immune cells is known to play a role in immune tolerance, we investigated the effect of ECP on arginine metabolism, and the influence of extracellular l-arginine concentration on the response to ECP in cells from patients on therapy by ECP for a GVHD and healthy donors cultured before and after ECP in the presence of different concentrations of arginine (0, 50, 100, 200 and 1000 µmol/l). At baseline arginine was not metabolized through the same pathway in patients and donors. When cells were exposed to ECP, the production of ornithine but not NO° was enhanced, while mRNA of arginase 1 was up-regulated but not INOS. In GVHD patients, increasing arginine concentration resulted in down-regulation of IFNγ and TNFα mRNA expression, whereas IL10 was up-regulated especially at physiological plasma levels (between 0 and 100 µM). Overall, our study shows that ECP orients the metabolism of arginine toward the arginase pathway together with shifting the cytokine profile toward IL-10, providing new insights into the enigmatic mechanism of action of ECP.

Hyperoxia increases hepatic arginase expression and ornithine production in mice.

Hyperoxic exposure affects the levels and activities of some hepatic proteins. We tested the hypothesis that hyperoxic exposure would result in greater hepatic .NO concentrations. C3H/HeN mice were exposed to >95% O(2) for 72 or 96 h and compared to room air-breathing controls. In contrast to our working hypothesis, exposure to >95% O(2) for 96 h decreased hepatic nitrite/nitrate NO(X) concentrations (10.9 +/- 2.2 nmol/g liver versus 19.3 +/- 2.4 nmol/g liver in room air, P < 0.05). The hepatic levels of endothelial NO synthase (eNOS) and inducible NOS (iNOS) proteins were not different among the groups. The arginases, which convert L-arginine to urea and L-ornithine, may affect hepatic NOS activities by decreasing L-arginine bioavailability. Hepatic ornithine concentrations were greater in hyperoxic animals than in controls (318 +/- 18 nmol/g liver in room air, and 539 +/- 64, and 475 +/- 40 at 72 and 96 h of hyperoxia, respectively, P < 0.01). Hepatic arginase I protein levels were greater in hyperoxic animals than in controls. Hepatic carbamoyl phosphate synthetase (CPS) protein levels and activities were not different among groups. These results indicate that increases in hepatic levels of arginase I in mice exposed to hyperoxia may diminish .NO production, as reflected by lower liver levels of NO(X). The resultant greater hepatic ornithine concentrations may represent a mechanism to facilitate tissue repair, by favoring the production of polyamines and/or proline.

Enterocyte metabolism during early adaptation after extensive intestinal resection in a rat model.

OBJECTIVE: A better knowledge of intestinal adaptation after resection is required to improve the nutritional support that is given to patients. The aim of this study was to understand the metabolic changes underlying early adaptation after massive intestinal resection. METHODS: Rats were assigned to either 80% intestinal resection or transection. All animals received the same intragastric nutrition. On day 8, plasma glutamine turnover was measured. Substrate use was determined on isolated enterocytes that were incubated in the presence of D-[U-(14)C] glucose (2 mmol/L), L-[U-(14)C] glutamine (2 mmol/L), L-[U-(14)C] arginine (1 mmol/L), or L-[1-(14)C] ornithine (1 mmol/L). RESULTS: Plasma glutamine turnover was similar in both groups. The rate of enterocyte glutamine use was significantly increased in the resection group, although the maximal glutaminase activity was unchanged. Glutathione generation was enhanced 3-fold in remnant intestine as compared with transected intestine (P <.05). L-ornithine decarboxylation was increased markedly in resected animals (P <.05), without any detectable change of maximal ornithine decarboxylase activity. CONCLUSION: The early phase of intestinal adaptation after resection induces changes in enterocyte glutamine and ornithine metabolism that may be related, in part, to increased de novo polyamine synthesis. This observation suggests that a supplementation of artificial nutrition by nutrients that lead to the generation of trophic agents may be of potential interest.

Arginine metabolism during macrophage autocrine activation and infection with mouse hepatitis virus 3.

In contrast to BALB/c mouse macrophages (Mphi), Mphi from the A/J mouse strain, upon activation by exogenous interferon gamma (IFNgamma), develop an anti-mouse hepatitis virus 3 (MHV3) state which correlates with resistance to virus infection. To investigate the autocrine activation of BALB/c and A/J Mphi, we activated them with interleukin-12 (IL-12) and/or IL-18, and quantified IFNgamma production, the anti-MHV3 state and arginine metabolism. Synergistic activation by IL-12/IL-18 induced the expression of the IFNgamma gene in Mphi from both mouse strains. In bone marrow (BM) or peritoneal (P) Mphi of specific pathogen-free (spf) mice of both strains, IFNgamma synthesis occurred only with a synergistic IL-12/IL-18 activation and showed increasing levels from 24 to 72 h of activation. In contrast, when non-spf mice were used in the assay, their PMphi synthesized higher IFNgamma levels upon activation with only IL-12 or only IL-18 or both. The BALB/c Mphi were always capable of synthesizing higher amounts of IFNgamma than the A/J Mphi. An anti-MHV3 state was observed only in A/J Mphi upon activation with IL-12/IL-18 or IFNgamma regardless of their origin from the peritoneum or bone marrow. Arginine metabolism in activated and/or virus infected BMMphi was investigated through nitric oxide (NO) and arginase induction as well as the consumption of arginine and synthesis of citrulline, ornithine and spermine. The results showed that both BALB/c and A/J BMMphi populations released NO only after activation with IL-12/IL-18 or IFNgamma. Arginase was not induced in BMMphi from both strains by IL-12/IL-18 or IFNgamma but only by IL-4/IL-10. Higher arginine consumption was observed in BMMphi from both strains upon activation with IL-4 or IFNgamma which further increased, in this case, when the cells were infected with MHV3. As a consequence of nitric oxide synthase synthesis and arginine consumption in IFNgamma activated BMMphi, we observed a higher synthesis of citrulline. High levels of ornithine were induced only upon IL-4 activation. Polyamine synthesis was higher in A/J BMMphi than in BALB/c ones, which correlated with the slightly lower levels of ornithine observed. Upon infection with MHV3, we observed a higher synthesis of spermine. IL-12/IL-18 or IFNgamma activation, mainly in MHV3 infected cells, led to a decreased synthesis of polyamines, notably spermine, only in A/J BMMphi. Difluoromethylornithine treatment, which leads to inhibition of polyamine synthesis, induced a decreased MHV3 multiplication in both BALB/c and A/J BMMphi. Altogether these data show the relevance of IFNgamma, from the autocrine or paracrine pathway, and arginine metabolism for the control of MHV3 replication in Mphi of a resistant mouse strain.

Glutamine metabolism in endothelial cells: ornithine synthesis from glutamine via pyrroline-5-carboxylate synthase.

L-Glutamine (the most abundant free amino acid in plasma and the body) is a potent inhibitor of endothelial NO synthesis. However, little is known about glutamine metabolism in endothelial cells (EC). As an initial step toward understanding the role of glutamine in endothelial physiology, the present study was conducted to quantify glutamine catabolism in microvascular, aortic and venous EC. For metabolic studies, EC were incubated for 1 h in Krebs bicarbonate buffer containing 5 mM glucose and 0.5-4 mM L-[U-(14)C]-glutamine. For enzymological studies, cell extracts and mitochondrial fractions were prepared to determine the activities of glutamine-degrading enzymes. Our results reveal extensive hydrolysis of glutamine to glutamate and ammonia in a concentration-dependent manner via phosphate-dependent glutaminase in all EC studied. In addition, both metabolic and enzymological evidence indicate a novel pathway for endothelial synthesis of ornithine from glutamine via pyrroline-5-carboxylate synthase. This new knowledge of glutamine metabolism may pave a new path for understanding the physiological role of glutamine in vascular function.

De novo synthesis of arginine and ornithine from citrulline in human colon carcinoma cells: metabolic fate of L-ornithine.

In human colon carcinoma cells (HT-29 cells), L-arginine is the common precursor of L-ornithine which generates polyamines strictly necessary for cellular growth, and nitric oxide which has a strong antiproliferative activity. We show here that proliferative HT-29 cells possess the capacity for de novo synthesis of L-arginine from L-citrulline, but not from L-ornithine. L-Ornithine is apparently not an L-arginine precursor due to the absence of any detectable ornithine carbamoyltransferase activity. In contrast, the newly synthesized L-arginine was competent for urea and thus L-ornithine production in a context of a high putrescine production in the ornithine decarboxylase pathway and a low degradation of this polyamine in the diamine oxidase pathway. However, cells grown in an arginine-free culture medium containing added L-citrulline were unable to reach confluency. Furthermore, the low amount of nitric oxide produced from L-arginine by these cells was apparently not involved in the control of cell growth since inhibition of nitric oxide synthase activity was without effect. On the other hand, the capacity of more differentiated and less proliferative HT-29 cells for de novo L-arginine synthesis from L-citrulline was increased. It is concluded that L-citrulline is a precursor of L-arginine and L-ornithine in proliferative HT-29 cells and that the metabolic fate of L-ornithine in these cells is mainly devoted to polyamine synthesis. The similarity between differentiated HT-29 cells and the enterocytes of newborn animals in terms of L-arginine metabolism is finally discussed.

Selective induction of metabolic activation programs in peritoneal macrophages by lipopolysaccharide substructures.

The structural elements of Salmonella typhimurium lipopolysaccharides (LPS) that are able to stimulate peritoneal macrophages to produce increased amounts of prostaglandin E2, ornithine, and citrulline, agents known to modulate immune responses, are described. Two different incomplete lipid A structures which lack the carbohydrate portion, the nonhydroxylated fatty acids lauric acid and myristic acid (lipid A precursor IB), and additional palmitic acid (lipid A precursor IA) stimulated increased prostaglandin E2 synthesis but were unable to augment ornithine and citrulline production at concentrations of up to 0.5 microgram/ml. Acyl-deficient smooth LPS containing lipid A precursors IA and IB substituted by the complete carbohydrate region were able to augment prostaglandin E2 and ornithine production but failed, even at a high concentration (0.5 microgram/ml), to stimulate citrulline production. Moreover, Re glycolipids and smooth intact LPS containing the lipid A region with 3-acyloxyacyl residues possessed all of the structural requirements to induce increased prostaglandin E2, ornithine, and citrulline synthesis. Finally, all of the LPS structures, including lipid A precursors IA and IB stimulated, in combination with gamma interferon, production of citrulline with similar efficiencies. These results demonstrate that LPS contains various substructures including regions of the carbohydrate and lipid A structure that can deliver signals for the activation of peritoneal macrophages. Signals for partial activation of macrophages to produce prostaglandins and ornithine can be delivered by acyl-deficient LPS structures. In contrast, full activation of macrophages to produce citrulline requires an additional signal that is delivered by 3-acyloxyacyl residues of the lipid A region or gamma interferon.

Effect of dietary arginine restriction upon ornithine and polyamine metabolism during two-stage epidermal carcinogenesis in the mouse.

Polyamine synthesis is required in normal or neoplastic tissues if they are to continue to grow or divide. The highly inducible enzyme ornithine decarboxylase (ODC) catalyzes the conversion of ornithine to putrescine as the initial step in polyamine biosynthesis. The level of substrate pools of ornithine in cultured cells has been reported to markedly alter mitogen-induced ODC activity, putrescine accumulation, and DNA synthesis (V. Wu and C. V. Byus, Biochim. Biophys. Acta, 804: 89-99, 1984; V. Wu et al., Cancer Res., 41: 3384-3391, 1981). We attempted to limit the amount of ornithine available for polyamine biosynthesis in an animal by using a dietary approach. Since arginine serves as one of the intermediate biosynthetic precursors of ornithine, female CD-1 mice were placed on a special synthetic amino acid diet deficient in arginine. The ability of this arginine-free diet to alter epidermal ornithine and polyamine metabolism and tumorigenesis was assessed in the mouse two-stage model of skin carcinogenesis. The basal level of ornithine in the epidermis in control animals receiving the amino acid complete diet was very high compared to other tissues (155 nmol/mg protein). However, when the mice were fed the isocaloric arginine-free diet for a 2-week period, the levels of epidermal ornithine and arginine decreased by 40% (P less than 0.01). This reduction was blocked by the addition of 2% ornithine to the drinking water of the arginine-restricted animals. Acute administration of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to the epidermis caused a transient (4 and 8 h) reduction in ornithine and arginine but not lysine in the animals receiving the control, and ornithine-supplemented diets. The animals fed the special arginine-free diet exhibited a 40-50% reduction in tumor multiplicity or papillomas/mouse (P less than 0.05) and had a significantly lower tumor incidence or percentage of animals with tumour throughout a 19-week promotion period (P less than 0.02). However, the major effect of arginine restriction was consistent with an increase in tumor latency. The addition of ornithine completely reversed the reduction in the rate and extent of tumorigenesis in the arginine-free animals. The accumulation of putrescine (but not spermidine or spermine) in the epidermis following a single administration of TPA was significantly reduced in the animals receiving the arginine-free diet. The papillomas or tumors from the animals deprived of arginine had markedly reduced (less than 35%) levels of putrescine compared to the tumors from control animals, and appeared to be more sensitive to dietary arginine restriction than was the chronically promoted but untransformed epidermis.(ABSTRACT TRUNCATED AT 400 WORDS)

Production of citrulline and ornithine by interferon-gamma treated macrophages.

Activated macrophages exert strong arginase (ASE) activity that converts L-arginine into ornithine, the key precursor for putrescine and polyamine biosynthesis. Macrophages were previously also shown to generate nitric oxide that is derived from the guanido group of arginine by the oxidative deiminase (OAD) reaction. In view of the physiological importance of ornithine and putrescine, we now investigated whether interferon-gamma (IFN-gamma), a principal stimulator of the OAD activity, may lead to the accumulation of the deiminated derivative citrulline at the expense of ornithine production, or whether the carbon backbone could be reutilized for the production of arginine and ornithine. Our experiments show that murine peritoneal macrophages treated with IFN-gamma in combination with tumor necrosis factor (TNF) or bacterial lipopolysaccharide (LPS) generate substantial amounts of citrulline as identified by amino acid analyzer and by thin-layer chromatography. Also, labeled citrulline is generated from [14C]L-arginine but not from [14C]L-ornithine. This suggests that macrophages have little or no capacity to convert ornithine into arginine. In the absence of IFN-gamma, TNF and LPS stimulate the conversion of arginine into ornithine but not citrulline. However, when TNF or LPS stimulated macrophages are simultaneously treated with IFN-gamma, ornithine production is relatively inhibited by the strong OAD reaction that competes with the ASE reaction for its substrate L-arginine. IFN-gamma thus down-regulates the availability of ornithine and putrescine. The lipid A precursor IA also induces, in conjunction with IFN-gamma, the production of citrulline but fails to stimulate the generation of ornithine.

The lipopolysaccharide-induced stimulation of peritoneal macrophages involves at least two signal pathways. Partial stimulation by lipid A precursors.

Bacterial lipopolysaccharides (LPS) stimulate the ability of macrophages to convert arginine into ornithine. This effect is inhibited by the cycloxygenase inhibitor indomethacin and reconstituted by application of exogenous prostaglandin E2 (PGE2) or cholera toxin, i.e. two substances which are known to raise the intracellular concentration of cyclic AMP. Moreover, the LPS-mediated effect is augmented in a synergistic manner by PGE2, cholera toxin and the dibutyryl-derivative of cyclic AMP. Lipid A precursor IA, which lacks lauric, myristic and palmitic acids, and lipid A precursor IB, which is devoid of lauric and myristic acids of the complete lipid A structure, are capable of augmenting PGE2 synthesis but do not stimulate the conversion of arginine into ornithine. Taken together, our experiments suggest that the LPS-induced production of PGE2 and the PGE2-induced increase of the intracellular cAMP concentrations are essential elements of an autoregulatory loop that controls the LPS-mediated stimulation of the ornithine production by macrophages. The stimulation of the autoregulatory loop is necessary but not sufficient for this effect, indicating that an additional signal is required which is provided by the complete lipid A structure but not by the incomplete lipid A structures IA and IB. This additional signal can be provided by a lipid A structure containing the 3-acyloxyacyl residues with lauric and myristic acids.