Molecular Changes Following Induction of Hepatocellular Carcinoma by Diethylnitrosamine and Thioacetamide, and Subsequent Treatment with Dioscorea membranacea Extract.

Hepatocellular carcinoma (HCC) is a primary liver cancer commonly found in adults. Previously, we showed the anticancer effects of Thai herbal plant extract, Dioscorea membranacea Pierre (DM), in HCC-bearing rats. In the present study, we further examined the proposed mechanism of DM, including apoptosis and antioxidant activity. Moreover, we used RNA sequencing (RNA-seq) to analyze molecular pathways in the rat model in which HCC was induced by diethylnitrosamine (DEN) and thioacetamide (TAA). The HCC-bearing rats were then treated with 40 mg/kg of DM for 8 weeks, after which experimental and control rats were sacrificed and liver tissues were collected. The RNA-seq data of DEN/TAA-treated rats exhibited upregulation of 16 hallmark pathways, including epithelial mesenchymal transition, inflammatory responses, and angiogenesis (p<0.01). DM extract expanded the Bax protein-positive pericentral zone in the tumor areas and decreased hepatic malondialdehyde levels, implying a decrease in lipid peroxidation in liver. However, DM treatment did not ameliorate the molecular pathways induced in DEN/TAA-treated livers. Our findings indicate that DM extract has antioxidant activity and exerts its pro-apoptotic effect on rat HCCs in vivo at the (post-)translational level.

Microcirculatory Function during Endotoxemia-A Functional Citrulline-Arginine-NO Pathway and NOS3 Complex Is Essential to Maintain the Microcirculation.

Competition for the amino acid arginine by endothelial nitric-oxide synthase (NOS3) and (pro-)inflammatory NO-synthase (NOS2) during endotoxemia appears essential in the derangement of the microcirculatory flow. This study investigated the role of NOS2 and NOS3 combined with/without citrulline supplementation on the NO-production and microcirculation during endotoxemia. Wildtype (C57BL6/N background; control; n = 36), Nos2-deficient, (n = 40), Nos3-deficient (n = 39) and Nos2/Nos3-deficient mice (n = 42) received a continuous intravenous LPS infusion alone (200 µg total, 18 h) or combined with L-citrulline (37.5 mg, last 6 h). The intestinal microcirculatory flow was measured by side-stream dark field (SDF)-imaging. The jejunal intracellular NO production was quantified by in vivo NO-spin trapping combined with electron spin-resonance (ESR) spectrometry. Amino-acid concentrations were measured by high-performance liquid chromatography (HPLC). LPS infusion decreased plasma arginine concentration in control and Nos3-/- compared to Nos2-/- mice. Jejunal NO production and the microcirculation were significantly decreased in control and Nos2-/- mice after LPS infusion. No beneficial effects of L-citrulline supplementation on microcirculatory flow were found in Nos3-/- or Nos2-/-/Nos3-/- mice. This study confirms that L-citrulline supplementation enhances de novo arginine synthesis and NO production in mice during endotoxemia with a functional NOS3-enzyme (control and Nos2-/- mice), as this beneficial effect was absent in Nos3-/- or Nos2-/-/Nos3-/- mice.

Glutamine Anabolism Plays a Critical Role in Pancreatic Cancer by Coupling Carbon and Nitrogen Metabolism.

Glutamine is thought to play an important role in cancer cells by being deaminated via glutaminolysis to α-ketoglutarate (aKG) to fuel the tricarboxylic acid (TCA) cycle. Supporting this notion, aKG supplementation can restore growth/survival of glutamine-deprived cells. However, pancreatic cancers are often poorly vascularized and limited in glutamine supply, in alignment with recent concerns on the significance of glutaminolysis in pancreatic cancer. Here, we show that aKG-mediated rescue of glutamine-deprived pancreatic ductal carcinoma (PDAC) cells requires glutamate ammonia ligase (GLUL), the enzyme responsible for de novo glutamine synthesis. GLUL-deficient PDAC cells are capable of the TCA cycle but defective in aKG-coupled glutamine biosynthesis and subsequent nitrogen anabolic processes. Importantly, GLUL expression is elevated in pancreatic cancer patient samples and in mouse PDAC models. GLUL ablation suppresses the development of KrasG12D-driven murine PDAC. Therefore, GLUL-mediated glutamine biosynthesis couples the TCA cycle with nitrogen anabolism and plays a critical role in PDAC.

The odd-carbon medium-chain fatty triglyceride triheptanoin does not reduce hepatic steatosis.

BACKGROUND & AIMS: Non-alcoholic fatty-liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome. Previously, we showed that a high-protein diet minimized diet-induced development of fatty liver and even reversed pre-existing steatosis. A high-protein diet leads to amino-acid catabolism, which in turn causes anaplerosis of the tricarboxylic-acid (TCA) cycle. Therefore, we hypothesized that anaplerosis of the TCA cycle could be responsible for the high-protein diet-induced improvement of NAFLD by channeling amino acids into the TCA cycle. Next we considered that an efficient anaplerotic agent, the odd-carbon medium-chain triglyceride triheptanoin (TH), might have similar beneficial effects. METHODS: C57BL/6J mice were fed low-fat (8en%) or high-fat (42en%) oleate-containing diets with or without 15en% TH for 3 weeks. RESULTS: TH treatment enhanced the hepatic capacity for fatty-acid oxidation by a selective increase in hepatic Ppara, Acox, and Cd36 expression, and a decline in plasma acetyl-carnitines. It also induced pyruvate cycling through an increased hepatic PCK1 protein concentration and it increased thermogenesis reflected by an increased Ucp2 mRNA content. TH, however, did not reduce hepatic lipid content. CONCLUSION: The comparison of the present effects of dietary triheptanoin with a previous study by our group on protein supplementation shows that the beneficial effects of the high-protein diet are not mimicked by TH. This argues against anaplerosis as the sole explanatory mechanism for the anti-steatotic effect of a high-protein diet.

Citrulline Supplementation Improves Organ Perfusion and Arginine Availability under Conditions with Enhanced Arginase Activity.

Enhanced arginase-induced arginine consumption is believed to play a key role in the pathogenesis of sickle cell disease-induced end organ failure. Enhancement of arginine availability with L-arginine supplementation exhibited less consistent results; however, L-citrulline, the precursor of L-arginine, may be a promising alternative. In this study, we determined the effects of L-citrulline compared to L-arginine supplementation on arginine-nitric oxide (NO) metabolism, arginine availability and microcirculation in a murine model with acutely-enhanced arginase activity. The effects were measured in six groups of mice (n = 8 each) injected intraperitoneally with sterile saline or arginase (1000 IE/mouse) with or without being separately injected with L-citrulline or L-arginine 1 h prior to assessment of the microcirculation with side stream dark-field (SDF)-imaging or in vivo NO-production with electron spin resonance (ESR) spectroscopy. Arginase injection caused a decrease in plasma and tissue arginine concentrations. L-arginine and L-citrulline supplementation both enhanced plasma and tissue arginine concentrations in arginase-injected mice. However, only the citrulline supplementation increased NO production and improved microcirculatory flow in arginase-injected mice. In conclusion, the present study provides for the first time in vivo experimental evidence that L-citrulline, and not L-arginine supplementation, improves the end organ microcirculation during conditions with acute arginase-induced arginine deficiency by increasing the NO concentration in tissues.

Integrated visualization of a multi-omics study of starvation in mouse intestine.

Our understanding of complex biological processes can be enhanced by combining different kinds of high-throughput experimental data, but the use of incompatible identifiers makes data integration a challenge. We aimed to improve methods for integrating and visualizing different types of omics data. To validate these methods, we applied them to two previous studies on starvation in mice, one using proteomics and the other using transcriptomics technology. We extended the PathVisio software with new plugins to link proteins, transcripts and pathways. A low overall correlation between proteome and transcriptome data was detected (Spearman rank correlation: 0.21). At the level of individual genes, correlation was highly variable. Many mRNA/protein pairs, such as fructose biphosphate aldolase B and ATP Synthase, show good correlation. For other pairs, such as ferritin and elongation factor 2, an interesting effect is observed, where mRNA and protein levels change in opposite directions, suggesting they are not primarily regulated at the transcriptional level. We used pathway diagrams to visualize the integrated datasets and found it encouraging that transcriptomics and proteomics data supported each other at the pathway level. Visualization of the integrated dataset on pathways led to new observations on gene-regulation in the response of the gut to starvation. Our methods are generic and can be applied to any multi-omics study. The PathVisio software can be obtained at http://www.pathvisio.org. Supplemental data are available at http://www.bigcat.unimaas.nl/data/jib-supplemental/ , including instructions on reproducing the pathway visualizations of this manuscript.

Citrulline a more suitable substrate than arginine to restore NO production and the microcirculation during endotoxemia.

BACKGROUND: Impaired microcirculation during endotoxemia correlates with a disturbed arginine-nitric oxide (NO) metabolism and is associated with deteriorating organ function. Improving the organ perfusion in endotoxemia, as often seen in patients with severe infection or systemic inflammatory response syndrome (SIRS) is, therefore, an important therapeutic target. We hypothesized that supplementation of the arginine precursor citrulline rather than arginine would specifically increase eNOS-induced intracellular NO production and thereby improve the microcirculation during endotoxemia. METHODOLOGY/PRINCIPAL FINDINGS: To study the effects of L-Citrulline and L-Arginine supplementation on jejunal microcirculation, intracellular arginine availability and NO production in a non-lethal prolonged endotoxemia model in mice. C57/Bl6 mice received an 18 hrs intravenous infusion of endotoxin (LPS, 0.4 µg • g bodyweight(-1) • h(-1)), combined with either L-Citrulline (6.25 mg • h-1), L-Arginine (6.25 mg • h(-1)), or L-Alanine (isonitrogenous control; 12.5 mg • h(-1)) during the last 6 hrs. The control group received an 18 hrs sterile saline infusion combined with L-Alanine or L-Citrulline during the last 6 hrs. The microcirculation was evaluated at the end of the infusion period using sidestream dark-field imaging of jejunal villi. Plasma and jejunal tissue amino-acid concentrations were measured by HPLC, NO tissue concentrations by electron-spin resonance spectroscopy and NOS protein concentrations using Western blot. CONCLUSION/SIGNIFICANCE: L-Citrulline supplementation during endotoxemia positively influenced the intestinal microvascular perfusion compared to L-Arginine-supplemented and control endotoxemic mice. L-Citrulline supplementation increased plasma and tissue concentrations of arginine and citrulline, and restored intracellular NO production in the intestine. L-Arginine supplementation did not increase the intracellular arginine availability. Jejunal tissues in the L-Citrulline-supplemented group showed, compared to the endotoxemic and L-Arginine-supplemented endotoxemic group, an increase in degree of phosphorylation of eNOS (Ser 1177) and a decrease in iNOS protein level. In conclusion, L-Citrulline supplementation during endotoxemia and not L-Arginine reduced intestinal microcirculatory dysfunction and increased intracellular NO production, likely via increased intracellular citrulline and arginine availability.

Effects of L-arginine pretreatment on nitric oxide metabolism and hepatosplanchnic perfusion during porcine endotoxemia.

BACKGROUND: Sepsis is accompanied by an increased need for and a decreased supply of arginine, reflecting a condition of arginine deficiency. OBJECTIVE: The objective was to evaluate the effects of l-arginine pretreatment on arginine-nitric oxide (NO) production and hepatosplanchnic perfusion during subsequent endotoxemia. DESIGN: In a randomized controlled trial, pigs (20-25 kg) received 3 µg . kg(-1) . min(-1) lipopolysaccharide (LPS; 5 endotoxin units/ng) intravenously and saline resuscitation. l-Arginine (n = 8; 5.3 µmol . kg(-1) . min(-1)) or saline (n = 8) was infused starting 12 h before LPS infusion and continued for 24 h after the endotoxin infusion ended. Whole-body appearance rates, portal-drained viscera (PDV), and liver fluxes of arginine, citrulline, NO, and arginine de novo synthesis were measured by using stable-isotope infusion of [(15)N(2)]arginine and [(13)C-(2)H(2)]citrulline. Hepatosplanchnic perfusion was assessed by using a primed continuous infusion of para-aminohippuric acid and jejunal intramucosal partial pressure of carbon dioxide and was related to systemic hemodynamics. RESULTS: Arginine supplementation before LPS increased whole-body NO production in the PDV but not in the liver. Furthermore, it increased blood flow in the portal vein but not in the aorta and hepatic artery. During endotoxin infusion, arginine pretreatment was associated with an increased whole-body arginine appearance and NO production in the gut. Additional effects included a preserved mean arterial pressure, the prevention of an increase in pulmonary arterial pressure, an attenuated metabolic acidosis, and an attenuated increase in the intramucosal partial pressure of carbon dioxide. CONCLUSION: Arginine treatment starting before endotoxemia appears to be beneficial because it improves hepatosplanchnic perfusion and oxygenation during prolonged endotoxemia, probably through an enhancement in NO synthesis, without causing deleterious systemic side effects.

Arginine deficiency causes runting in the suckling period by selectively activating the stress kinase GCN2.

Suckling "F/A2" mice, which overexpress arginase-I in their enterocytes, develop a syndrome (hypoargininemia, reduced hair and muscle growth, impaired B-cell maturation) that resembles IGF1 deficiency. The syndrome may result from an impaired function of the GH-IGF1 axis, activation of the stress-kinase GCN2, and/or blocking of the mTORC1-signaling pathway. Arginine deficiency inhibited GH secretion and decreased liver Igf1 mRNA and plasma IGF1 concentration, but did not change muscle IGF1 concentration. GH supplementation induced Igf1 mRNA synthesis, but did not restore growth, ruling out direct involvement of the GH-IGF1 axis. In C2C12 muscle cells, arginine withdrawal activated GCN2 signaling, without impacting mTORC1 signaling. In F/A2 mice, the reduction of plasma and tissue arginine concentrations to ∼25% of wild-type values activated GCN2 signaling, but mTORC1-mediated signaling remained unaffected. Gcn2-deficient F/A2 mice suffered from hypoglycemia and died shortly after birth. Because common targets of all stress kinases (eIF2α phosphorylation, Chop mRNA expression) were not increased in these mice, the effects of arginine deficiency were solely mediated by GCN2.

Arginine deficiency affects early B cell maturation and lymphoid organ development in transgenic mice.

Apart from its role in the synthesis of protein and nitric oxide (NO), and in ammonia detoxification, the amino acid arginine exerts an immunosupportive function. We have studied the role of arginine in immune defense mechanisms in the developing postnatal immune system. In suckling mice, arginine is produced in the small intestine. In F/A-2(+/+) transgenic mice, which overexpress arginase in their enterocytes, circulating and tissue arginine concentrations are reduced to 30-35% of controls. In these mice, the development and composition of the T cell compartment did not reveal abnormalities. However, in peripheral lymphoid organs and the small intestine, B cell cellularity and the number and size of Peyer's patches were drastically reduced, and serum IgM levels were significantly decreased. These phenotypes could be traced to an impaired transition from the pro- to pre-B cell stage in the bone marrow. Cytokine receptor levels in the bone marrow were normal. The development of the few peripheral B cells and their proliferative response after in vitro stimulation was normal. The disturbance in B cell maturation was dependent on decreased arginine levels, as this phenotype disappeared upon arginine supplementation and was not seen in NO synthase- or ornithine transcarbamoylase-deficient mice. We conclude that arginine deficiency impairs early B cell maturation.

L-arginine supplementation in hyperdynamic endotoxemic pigs: effect on nitric oxide synthesis by the different organs.

OBJECTIVES: Under septic conditions, the protective role of nitric oxide in the organs may become compromised at a time of increased demand as a result of decreased availability of L-arginine. It remains unknown whether supplementation with L-arginine, as a substrate, can modulate organ nitric oxide production. DESIGN: Controlled study with laboratory animals. SETTING: University research laboratory. SUBJECTS: Female crossbred pigs. INTERVENTION: Pigs were challenged with Escherichia coli endotoxin (intravenously) and received intravenous fluid resuscitation for 24 hrs to reproduce a model of long-lasting hyperdynamic endotoxemia. Pigs were infused with either L-arginine or L-alanine intravenously during endotoxin and via the intragastric route after cessation of endotoxin infusion. The effects of L-arginine supplementation on nitric oxide synthesis and the relationships with arginine metabolism were determined with a stable isotope infusion protocol. Also, organ nitrite plus nitrate fluxes were measured. Implantation of multiple catheters enabled in vivo measurements across the hindquarter muscle, the portal-drained viscera, the liver, and the kidneys. MEASUREMENTS AND RESULTS: The isotope conversion method showed that L-arginine intervention significantly increased nitric oxide production by the portal-drained viscera, liver, and kidneys, resulting in elevated whole-body nitric oxide synthesis under endotoxemic and postendotoxemic conditions. Organ nitrite plus nitrate fluxes only tended to increase because of high variance among data. CONCLUSIONS: In this endotoxemia model, supplemental use of L-arginine favored nitric oxide synthesis in various organs.

L-arginine supplementation in pigs decreases liver protein turnover and increases hindquarter protein turnover both during and after endotoxemia.

BACKGROUND: Accumulating evidence suggests that L-arginine, under conditions of septicemia, not only enhances immune function but also improves protein metabolism. OBJECTIVE: Because the effect of L-arginine administration on the protein metabolism of different organs is unknown, the aim of the study was to elucidate the effects of exogenous supplementation of L-arginine during endotoxemia on the in vivo protein metabolism of individual organs and at the whole-body level. DESIGN: Female pigs were cannulated with catheters in the aorta and the splenic, caval, portal, hepatic, and renal veins, enabling measurements across the hindquarter, portal-drained viscera, liver, and kidneys. Endotoxemia was induced by a 24-h continuous intravenous infusion of endotoxin (3 microg x kg body wt(-1) x h(-1)). At 8 h, an intravenous infusion of L-arginine was started (n = 8). Control pigs (n = 6) received L-alanine. At 24 h, blood was sampled. After cessation of the endotoxin infusion, L-arginine and L-alanine infusions were continued as a supplement in the enterally infused diet. At 48 h, blood samples were obtained during the postendotoxemic and nutritionally supported conditions. Stable isotopes were used to assess protein metabolism and phenylalanine hydroxylation. RESULTS: Both during and after the endotoxin challenge, L-arginine administration enhanced protein synthesis and degradation across the hindquarter and simultaneously reduced protein synthesis and degradation in the liver at equal rates. Protein turnover across the kidneys and portal-drained viscera remained unaffected. After endotoxemia, L-arginine infusion decreased whole-body protein turnover without affecting the net protein balance. CONCLUSION: L-Arginine administration affects protein turnover of the muscle area and the liver oppositely.