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• W1983996840 abstract "We demonstrated previously in a liver perfusion system that agmatine increases oxygen consumption as well as the synthesis of N-acetylglutamate and urea by an undefined mechanism. In this study our aim was to identify the mechanism(s) by which agmatine up-regulates ureagenesis. We hypothesized that increased oxygen consumption and N-acetylglutamate and urea synthesis are coupled to agmatine-induced stimulation of mitochondrial fatty acid oxidation. We used 13C-labeled fatty acid as a tracer in either a liver perfusion system or isolated mitochondria to monitor fatty acid oxidation and the incorporation of 13C-labeled acetyl-CoA into ketone bodies, tricarboxylic acid cycle intermediates, amino acids, and N-acetylglutamate. With [U-13C16] palmitate in the perfusate, agmatine significantly increased the output of 13C-labeled β-hydroxybutyrate, acetoacetate, and CO2, indicating stimulated fatty acid oxidation. The stimulation of [U-13C16]palmitate oxidation was accompanied by greater production of urea and a higher 13C enrichment in glutamate, N-acetylglutamate, and aspartate. These observations suggest that agmatine leads to increased incorporation and flux of 13C-labeled acetyl-CoA in the tricarboxylic acid cycle and to increased utilization of 13C-labeled acetyl-CoA for synthesis of N-acetylglutamate. Experiments with isolated mitochondria and 13C-labeled octanoic acid also demonstrated that agmatine increased synthesis of 13C-labeled β-hydroxybutyrate, acetoacetate, and N-acetylglutamate. The current data document that agmatine stimulates mitochondrial β-oxidation and suggest a coupling between the stimulation of hepatic β-oxidation and up-regulation of ureagenesis. This action of agmatine may be mediated via a second messenger such as cAMP, and the effects on ureagenesis and fatty acid oxidation may occur simultaneously and/or independently. We demonstrated previously in a liver perfusion system that agmatine increases oxygen consumption as well as the synthesis of N-acetylglutamate and urea by an undefined mechanism. In this study our aim was to identify the mechanism(s) by which agmatine up-regulates ureagenesis. We hypothesized that increased oxygen consumption and N-acetylglutamate and urea synthesis are coupled to agmatine-induced stimulation of mitochondrial fatty acid oxidation. We used 13C-labeled fatty acid as a tracer in either a liver perfusion system or isolated mitochondria to monitor fatty acid oxidation and the incorporation of 13C-labeled acetyl-CoA into ketone bodies, tricarboxylic acid cycle intermediates, amino acids, and N-acetylglutamate. With [U-13C16] palmitate in the perfusate, agmatine significantly increased the output of 13C-labeled β-hydroxybutyrate, acetoacetate, and CO2, indicating stimulated fatty acid oxidation. The stimulation of [U-13C16]palmitate oxidation was accompanied by greater production of urea and a higher 13C enrichment in glutamate, N-acetylglutamate, and aspartate. These observations suggest that agmatine leads to increased incorporation and flux of 13C-labeled acetyl-CoA in the tricarboxylic acid cycle and to increased utilization of 13C-labeled acetyl-CoA for synthesis of N-acetylglutamate. Experiments with isolated mitochondria and 13C-labeled octanoic acid also demonstrated that agmatine increased synthesis of 13C-labeled β-hydroxybutyrate, acetoacetate, and N-acetylglutamate. The current data document that agmatine stimulates mitochondrial β-oxidation and suggest a coupling between the stimulation of hepatic β-oxidation and up-regulation of ureagenesis. This action of agmatine may be mediated via a second messenger such as cAMP, and the effects on ureagenesis and fatty acid oxidation may occur simultaneously and/or independently. Agmatine (Agm) 2The abbreviations used are: Agm, agmatine; AcAc, acetoacetate; FAO, fatty acid oxidation; GC-MS, gas chromatography-mass spectrometry; β-HB, β-hydroxybutyrate; MPE, mol % excess; NAG, N-acetylglutamate; OXA, oxaloacetate; PC, pyruvate carboxylase; HPLC, high pressure liquid chromatography. 2The abbreviations used are: Agm, agmatine; AcAc, acetoacetate; FAO, fatty acid oxidation; GC-MS, gas chromatography-mass spectrometry; β-HB, β-hydroxybutyrate; MPE, mol % excess; NAG, N-acetylglutamate; OXA, oxaloacetate; PC, pyruvate carboxylase; HPLC, high pressure liquid chromatography. is widely distributed in mammalian tissue (1Rasch W. Regunathan S. Li G. Reis D.J. Life Sci. 1995; 56: 2319-2330Crossref PubMed Scopus (245) Google Scholar, 2Regunathan S. Reis D.J. J. Neurochem. 2000; 74: 2201-2208Crossref PubMed Scopus (94) Google Scholar) and may act as a hormone affecting multiple metabolic functions (3Satriano J. Kelly C.J. Blantz R.C. Kidney Int. 1999; 56: 1252-1253Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 4Blantz R.C. Satriano J. Gabbai F. Kelly C. Acta Physiol. Scand. 2000; 168: 21-25Crossref PubMed Google Scholar, 5Moinard C. Cynober L. De Bandt J.-P. Clin. Nutr. 2005; 24: 184-197Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 6Grillo M.A. Colmbatto S. Amino Acids (Vienna). 2004; 26: 3-8Crossref PubMed Scopus (49) Google Scholar, 7Gen L. Regunathan D. Barrow C.J. Esraghi J. Cooper R. Reis D.J. Science. 1994; 263: 12231-12234Google Scholar, 8Reis D.J. Regunathan S. Trends Pharmacol. Sci. 2000; 5: 187-193Abstract Full Text Full Text PDF Scopus (372) Google Scholar). We demonstrated previously that addition of Agm to a liver perfusion system significantly stimulated oxygen consumption and synthesis of 15N-labeled NAG and urea from 15N-labeled glutamine (9Nissim I. Horyn O. Daikhin Y. Nissim I. Lazarow A. Yudkoff M. Am. J. Physiol. 2002; 283: E1123-E1134Crossref PubMed Scopus (25) Google Scholar). However, the nature and/or the mechanism(s) underlying these actions of Agm are unknown. One possibility is that Agm stimulates fatty acid oxidation (FAO), thereby providing more reducing equivalents (NADH and FADH) to the respiratory chain and increasing availability of substrates and/or ATP for NAG and urea synthesis. This possibility is in line with the increased oxygen consumption associated with agmatine up-regulation of urea synthesis (9Nissim I. Horyn O. Daikhin Y. Nissim I. Lazarow A. Yudkoff M. Am. J. Physiol. 2002; 283: E1123-E1134Crossref PubMed Scopus (25) Google Scholar).As illustrated in Fig. 1, Agm may stimulate FAO, thereby increasing the availability of reducing substrates and acetyl-CoA. The latter may be converted to β-HB and AcAc (ketone bodies), utilized for NAG synthesis, and/or incorporated into the tricarboxylic acid cycle. The increased [acetyl-CoA] and/or [NADH] is expected to up-regulate the production of oxaloacetate (OXA) via the pyruvate carboxylase (PC) reaction (10Owen O.E. Kalhan S.C. Hanson R.W. J. Biol. Chem. 2002; 277: 30409-30412Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar, 11Garland P.B. Shepherd D. Nicholls D.G. Ontko J. Adv. Enzyme Regul. 1968; 6: 3-30Crossref PubMed Scopus (30) Google Scholar, 12Di Donato L. Des Rosiers C. Montgomery J.A. David F. Garneau M. Brunengraber H. J. Biol. Chem. 1993; 268: 4170-4180Abstract Full Text PDF PubMed Google Scholar, 13McGarry J.D. Am. J. Clin. Nutr. 1998; 67: S500-S504Crossref PubMed Scopus (110) Google Scholar), thus increasing the anaplerotic incorporation of pyruvate carbon into glucose and/or the tricarboxylic acid cycle (10Owen O.E. Kalhan S.C. Hanson R.W. J. Biol. Chem. 2002; 277: 30409-30412Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar). Simultaneously, increased [NADH] can promote reductive amination of α-ketoglutarate and production of glutamate, i.e. cataplerosis (10Owen O.E. Kalhan S.C. Hanson R.W. J. Biol. Chem. 2002; 277: 30409-30412Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar). Alternatively, agmatine may stimulate deamidation of glutamine, thereby elevating the glutamate concentration, as indicated previously (9Nissim I. Horyn O. Daikhin Y. Nissim I. Lazarow A. Yudkoff M. Am. J. Physiol. 2002; 283: E1123-E1134Crossref PubMed Scopus (25) Google Scholar). Glutamate plus acetyl-CoA provide more substrates for the synthesis of NAG, an obligatory activator of carbamoylphosphate synthetase I (CPS-I) (14Fahien L. Schoder J.M. Gehred G.A. Cohen P.P. J. Biol. Chem. 1964; 239: 1935-1942Abstract Full Text PDF PubMed Google Scholar). In addition, some mitochondrial OXA will be transaminated to aspartate, which then is transported into the cytosol to support the synthesis of argininosuccinate (15Nissim I. Horyn O. Luhovyy B. Lazarow A. Daikhin Y. Nissim I. Yudkoff M. Biochem. J. 2003; 376: 179-188Crossref PubMed Scopus (23) Google Scholar, 16Nissim I. Luhovyy B. Horyn O. Daikhin Y. Nissim I. Yudkoff M. J. Biol. Chem. 2005; 280: 17715-17724Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). In addition, an elevation of mitochondrial [glutamate] supports production of ornithine, and then citrulline, in the mitochondrial matrix (16Nissim I. Luhovyy B. Horyn O. Daikhin Y. Nissim I. Yudkoff M. J. Biol. Chem. 2005; 280: 17715-17724Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Hence, the proposed metabolic cascade may lead to the following: (i) increased reducing substrates such as NADH and FADH, thus furnishing more ATP (for ureagenesis) via oxidative phosphorylation; (ii) increased mitochondrial synthesis of NAG and citrulline; and (iii) increased availability of aspartate for cytosolic synthesis of argininosuccinate. This sequence of events leads to up-regulation of urea synthesis. This study also examined an alternative possibility, i.e. the action of Agm may be mediated through a second messenger such as cAMP. Agm and/or its second messenger may act simultaneously and independently on ureagenesis and FAO, with the increase of urea synthesis and FAO being mediated by independent events.Hepatic oxidation of fatty acids begins in cytosol and finishes in the mitochondrion (17Laffel L. Diabetes Metab. Res. Rev. 1999; 15: 412-426Crossref PubMed Google Scholar, 18Kerner J. Hoppel C. Biochim. Biophys. Acta. 2000; 1486: 1-17Crossref PubMed Scopus (583) Google Scholar). The transport of long chain fatty acids into mitochondria involves their conversion into acylcarnitine esters at the outer mitochondrial membrane (17Laffel L. Diabetes Metab. Res. Rev. 1999; 15: 412-426Crossref PubMed Google Scholar, 18Kerner J. Hoppel C. Biochim. Biophys. Acta. 2000; 1486: 1-17Crossref PubMed Scopus (583) Google Scholar). Acylcarnitine is transported into the mitochondrial matrix and metabolized via the β-oxidation pathway (17Laffel L. Diabetes Metab. Res. Rev. 1999; 15: 412-426Crossref PubMed Google Scholar, 18Kerner J. Hoppel C. Biochim. Biophys. Acta. 2000; 1486: 1-17Crossref PubMed Scopus (583) Google Scholar). Agm may act either at the site of long chain fatty acid activation in the mitochondrial outer membrane or on the β-oxidation chain shortening in the mitochondrial matrix. Therefore, in this study we investigated the action of Agm in a liver perfusion system with [U-13C16]palmitate as tracer and in isolated mitochondria incubated with 13C-labeled octanoic acid, a medium chain fatty acid, that does not require activation by the carnitine transport system at the mitochondrial outer membrane (18Kerner J. Hoppel C. Biochim. Biophys. Acta. 2000; 1486: 1-17Crossref PubMed Scopus (583) Google Scholar). Experiments with isolated mitochondria or a liver perfusion system allow differentiation between a possible action of Agm on carnitine acyltransferase and the β-oxidation pathway. In addition, perfusion of the structurally intact liver with physiological concentrations of [U-13C16]palmitate and other metabolites would reveal a possible coupling between Agm action on β-oxidation and the up-regulation of urea synthesis, as illustrated in Fig. 1. Furthermore, a comparison between experiments with isolated mitochondria and a liver perfusion system should differentiate between Agm actions on mitochondrial versus peroxisomal β-oxidation (19Kasumov T. Adams J.E. Bian F. David F. Thomas K.R. Jobbins K.A. Minkler P.E. Hoppel C.L. Brunengraber H. Biochem. J. 2005; 389: 397-401Crossref PubMed Scopus (34) Google Scholar, 20Leighton F. Bergseth S. Rortveit T. Christiansen E.N. Bremer J. J. Biol. Chem. 1989; 264: 10347-10350Abstract Full Text PDF PubMed Google Scholar).The results demonstrate that, in isolated mitochondria and liver perfusions, Agm stimulated the production of 13C-labeled ketone bodies and CO2, indicating a stimulation of β-oxidation in the mitochondrial matrix. The stimulation of FAO was accompanied by increased [NAG] and urea output, suggesting a coupling between urea synthesis and the stimulation of fatty acid oxidation by agmatine.EXPERIMENTAL PROCEDURESMaterials and Animals—Male Sprague-Dawley rats (Charles River Breeding Laboratories) were fed a standard rat chow diet ad libitum. Chemicals were of analytical grade and obtained from Sigma. Enzymes and cofactors for the analysis of adenine nucleotides, β-HB, AcAc, NADH, NAD, urea, lactate, pyruvate, and ammonia were obtained from Sigma. [U-13C16]Palmitate and [1,2,3,4-13C4]octanoic acid, 15NH4Cl, or [15N]glutamate, 99 mol % excess (MPE), were from Isotec.Experiments with Liver Perfusions—Livers from overnight fasted male rats were perfused in the non-recirculating mode and antegrade flow at the rate of 3–3.5 ml·g–1·min–1 as described previously (9Nissim I. Horyn O. Daikhin Y. Nissim I. Lazarow A. Yudkoff M. Am. J. Physiol. 2002; 283: E1123-E1134Crossref PubMed Scopus (25) Google Scholar, 16Nissim I. Luhovyy B. Horyn O. Daikhin Y. Nissim I. Yudkoff M. J. Biol. Chem. 2005; 280: 17715-17724Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The basic perfusion medium was Krebs saline, pH 7.4, continuously gassed with 95% O2, 5%CO2 and containing lactate (2.1 mm) and pyruvate (0.3 mm) as metabolic fuels. pO2 (in influent and effluent media) was monitored throughout, and oxygen consumption was calculated. After 10 min of conditioning with a basic perfusion medium (Medium A), perfusate was replaced with one containing lactate, pyruvate (as in Medium A), plus 0.3 mm NH4Cl, and 1 mm glutamine with or without 0.1 mm agmatine (Medium B). After a 10-min perfusion with Medium B, the perfusate was replaced with one that contained 0.5 mm [U-13C16]palmitate (as potassium salt bound to bovine serum albumin in a 5:1 molar ratio), NH4Cl, glutamine, lactate, and pyruvate (as in Medium A and B), with or without 0.1 mm agmatine. The perfusion was continued for an additional 5, 10, 15, 20, 25, and 30 min. Samples were taken from the influent and effluent media for chemical and GC-MS analyses. At the indicated times, perfusion was stopped, and the liver was freeze-clamped with aluminum tongs pre-cooled in liquid N2. The frozen liver was ground into a fine powder, extracted into perchloric acid, and used for metabolite determination and 13C enrichment.Metabolic Studies with Isolated Mitochondria—Mitochondria were isolated from the liver of overnight fasted rats by differential centrifugation as described previously (21Vatamaniuk M.Z. Horyn O.V. Vatamaniuk O.K. Doliba N.M. Life Sci. 2003; 72: 1871-1882Crossref PubMed Scopus (35) Google Scholar). Respiratory control and oxygen consumption were determined in each mitochondrial preparation (16Nissim I. Luhovyy B. Horyn O. Daikhin Y. Nissim I. Yudkoff M. J. Biol. Chem. 2005; 280: 17715-17724Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 22Horyn O. Luhovyy B. Lazarow A. Daikhin Y. Nissim I. Yudkoff M. Nissim I. Biochem. J. 2005; 388: 419-425Crossref PubMed Scopus (40) Google Scholar). Metabolic studies were carried out with mitochondria having a state 3/state 2 respiratory ratio greater than 3.The mitochondrial suspension (3–4 mg of protein/ml) was incubated in Erlenmeyer flasks (2 ml final volume) at 30 °C in a shaking water bath for 10 min and with the addition of substrates as indicated below. The basic incubation medium consisted of the following (mm): Tris (50Zollner H. Adv. Exp. Med. Biol. 1982; 153: 197-205Crossref PubMed Scopus (7) Google Scholar), EDTA (2Regunathan S. Reis D.J. J. Neurochem. 2000; 74: 2201-2208Crossref PubMed Scopus (94) Google Scholar), KCl (5Moinard C. Cynober L. De Bandt J.-P. Clin. Nutr. 2005; 24: 184-197Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar), MgCl2 (5Moinard C. Cynober L. De Bandt J.-P. Clin. Nutr. 2005; 24: 184-197Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar), KHCO3 (15Nissim I. Horyn O. Luhovyy B. Lazarow A. Daikhin Y. Nissim I. Yudkoff M. Biochem. J. 2003; 376: 179-188Crossref PubMed Scopus (23) Google Scholar), KH2PO4 (5Moinard C. Cynober L. De Bandt J.-P. Clin. Nutr. 2005; 24: 184-197Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar), 15NH4Cl (2Regunathan S. Reis D.J. J. Neurochem. 2000; 74: 2201-2208Crossref PubMed Scopus (94) Google Scholar), α-ketoglutarate (5Moinard C. Cynober L. De Bandt J.-P. Clin. Nutr. 2005; 24: 184-197Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar), and ornithine (5Moinard C. Cynober L. De Bandt J.-P. Clin. Nutr. 2005; 24: 184-197Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar), pH 7.4.In the first series of experiments, mitochondria were incubated for 10 min at 30 °C with basic medium plus 2 mm [1,2,3,4-13C4]octanoic acid and an increasing concentration (0–1 mm) of Agm. The second series of experiments was carried out with broken mitochondria (22Horyn O. Luhovyy B. Lazarow A. Daikhin Y. Nissim I. Yudkoff M. Nissim I. Biochem. J. 2005; 388: 419-425Crossref PubMed Scopus (40) Google Scholar), basic medium, increasing concentrations of Agm, 5 mm ATP, 2 mm acetyl-CoA, 5 mm succinate, and 5 mm [15N]glutamate without octanoic acid.At the end of the incubation, an aliquot (100 μl) was taken for protein determination, and the incubation was stopped with 100–150 μl of HClO4 (60%). Metabolite measurements were done in neutralized extracts. Three to five independent experiments were carried out for each series.GC-MS and NMR Methodology; Determination of 13C-Labeled Metabolites—GC-MS measurements of 13C isotopic enrichment were performed on either a Hewlett-Packard 5970 MSD and/or 5971 Mass Selective Detector (MSD), coupled with a 5890 HP-GC, GC-MS Agilent System (6890 GC-5973 MSD) or Hewlett-Packard (HP-5970 MSD), using electron impact ionization with an ionizing voltage of –70 eV and an electron multiplier set to 2000 V.For measurement of the 13C enrichment in amino acids, organic acids (α-ketoglutarate or citrate), or ketone bodies, samples were prepared as described previously (9Nissim I. Horyn O. Daikhin Y. Nissim I. Lazarow A. Yudkoff M. Am. J. Physiol. 2002; 283: E1123-E1134Crossref PubMed Scopus (25) Google Scholar, 16Nissim I. Luhovyy B. Horyn O. Daikhin Y. Nissim I. Yudkoff M. J. Biol. Chem. 2005; 280: 17715-17724Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 22Horyn O. Luhovyy B. Lazarow A. Daikhin Y. Nissim I. Yudkoff M. Nissim I. Biochem. J. 2005; 388: 419-425Crossref PubMed Scopus (40) Google Scholar). Briefly, an aliquot of effluent, liver, or mitochondrial extract was purified by passage on an AG-1 (Cl–, 100–200 mesh; 0.5 × 2.5 cm) or AG-50 (H+, 100–200 mesh) column, and then converted into the t-butyldimethylsilyl derivatives. Isotopic enrichment in glutamate isotopomers was monitored using ions at m/z 432, 433, 434, 435, 436, and 437 for M, M + 1, M + 2, M + 3, M + 4 and M + 5 (containing 1–5 13C atoms). Isotopic enrichment in aspartate isotopomers was monitored using ions at m/z 418, 419, 420, 421, and 422 for M + 1, M + 2, M + 3 and M + 4 (containing 1–4 13C atoms). Isotopic enrichment inβ-HB was monitored using ions at m/z 275, 276, 277, 278, and 279 for M + 1, M +2, M + 3, and M + 4 (containing 1–4 13C atoms), respectively, and 13C enrichment in AcAc was monitored using ions at m/z 273, 274, 275, 276, and 277 for M + 1, M + 2, M + 3, and M + 4 (containing 1–4 13C atoms), respectively.The concentration and 13C enrichment in N-acetylglutamate in liver or mitochondrial extracts were determined using GC-MS and an isotope dilution approach (9Nissim I. Horyn O. Daikhin Y. Nissim I. Lazarow A. Yudkoff M. Am. J. Physiol. 2002; 283: E1123-E1134Crossref PubMed Scopus (25) Google Scholar, 16Nissim I. Luhovyy B. Horyn O. Daikhin Y. Nissim I. Yudkoff M. J. Biol. Chem. 2005; 280: 17715-17724Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The formation of 13C-labeled NAG isotopomers was monitored using ions at m/z 158, 159, 160, 161, 162, and 163, for M + 1, M + 2, M + 3, M + 4, and M + 5 (containing 1–5 13C atoms). In experiments with isolated mitochondria, the production of 15N-labeled citrulline from 15NH4Cl was determined as described (16Nissim I. Luhovyy B. Horyn O. Daikhin Y. Nissim I. Yudkoff M. J. Biol. Chem. 2005; 280: 17715-17724Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar).The production of 13CO2 during liver perfusion was monitored in the effluent as follows: 1 ml of effluent was added to a sealed tube free of CO2 and containing 1 ml of 1 mm NaOH; 0.5 ml of 40% phosphoric acid was then added. Tubes were left for about 30 min to liberate 13CO2. The latter was removed with a sealed syringe and transferred to auto-sampler tubes for analysis. Isotopic enrichment in 13CO2 was determined by an isotope ratio-mass spectrometer (Thermoquest Finnigan Delta Plus) using the m/z 45/44 ratio.To evaluate further the effect of Agm on hepatic catabolism of [U-13C16]palmitate, a portion (about 2 g wet weight) of the neutralized liver extracts was analyzed by 13C NMR methodology using a Bruker DMX 400 wide bore equipped with a Silicon Graphic O2 computer. The chemical shifts of analyte containing 13C atoms were measured relative to the resonance of trimethylsilylpropionic acid at –2.7 ppm. Data acquisition and calculation of % 13C enrichment in various glutamate carbons were done as described (23Wherli S.L. Reynolds R. Chen J. Yager C. Segal S. NMR Biomed. 2001; 14: 192-198Crossref PubMed Scopus (9) Google Scholar).Analytical Measurements—The concentration of amino acids was determined by HPLC, utilizing pre-column derivatization with o-phthalaldehyde (24Jones B.N. Gilligan J.P. J. Chromatogr. 1983; 266: 471-482Crossref PubMed Scopus (453) Google Scholar). The levels of ammonia and urea were measured (9Nissim I. Horyn O. Daikhin Y. Nissim I. Lazarow A. Yudkoff M. Am. J. Physiol. 2002; 283: E1123-E1134Crossref PubMed Scopus (25) Google Scholar, 16Nissim I. Luhovyy B. Horyn O. Daikhin Y. Nissim I. Yudkoff M. J. Biol. Chem. 2005; 280: 17715-17724Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) as well as ATP (25Trautschold I. Lamprecht W. Schweitzer G. Bergmeyer H.U. Methods of Enzymatic Analysis. 7. Verlag Chemie, Weinheim, Germany1985: 346-357Google Scholar), ADP, and AMP as in Ref. 26Jaworek D. Gruber W. Bergmeyer H.U. Bergmeyer H.U. Methods of Enzymatic Analysis. 2. Academic Press, New York1974: 2127-2131Crossref Google Scholar. cAMP was measured as described (27Pradelles P. Graassi J. Chabandes D. Guiso N. Anal. Chem. 1989; 61: 447-452Crossref PubMed Scopus (153) Google Scholar) using a cAMP EIA kit (Cayman Chemical Co.). We also measured NAD and NADH (28Gibon Y. Larher F. Anal. Biochem. 1997; 251: 153-157Crossref PubMed Scopus (120) Google Scholar), AcAc and β-HB (29Kientsch-Engel R.I. Siess E.A. Bergmeyer H.U. Methods of Enzymatic Analysis. 6. Academic Press, New York1985: 60-69Google Scholar), lactate (30Czok R. Lamprecht W. Bergmeyer H.U. Methods of Enzymatic Analysis. 3. Academic Press, New York1974: 1446-1451Google Scholar), pyruvate (31Gutmann I. Wahlefeld A.W. Bergmeyer H.U. Methods of Enzymatic Analysis. 3. Academic Press, New York1974: 1464-1472Google Scholar), and glucose (32Bergmeyer H.U. Bernt E. Schmidt F. Stork H. Bergmeyer H.U. Methods of Enzymatic Analysis. 3. Academic Press, New York1974: 1196-1201Google Scholar).Calculations and Statistical Analyses—During liver perfusions, the rate of uptake or the output of metabolites was determined by the measurement of metabolite concentration in the influent and effluent (nmol/ml), normalized to the flow rate (ml/min) and liver wet weight (9Nissim I. Horyn O. Daikhin Y. Nissim I. Lazarow A. Yudkoff M. Am. J. Physiol. 2002; 283: E1123-E1134Crossref PubMed Scopus (25) Google Scholar, 15Nissim I. Horyn O. Luhovyy B. Lazarow A. Daikhin Y. Nissim I. Yudkoff M. Biochem. J. 2003; 376: 179-188Crossref PubMed Scopus (23) Google Scholar, 16Nissim I. Luhovyy B. Horyn O. Daikhin Y. Nissim I. Yudkoff M. J. Biol. Chem. 2005; 280: 17715-17724Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). 13C enrichment in a given mass isotopomer is expressed by molar percent enrichment (MPE), which is the mol fraction (%) of analyte containing 13C atoms above natural abundance. The MPE was calculated using the peak area from GC-MS ions corrected for natural abundance as described (33Des Rosiers C. Di Donato L. Comte B. Laplante A. Marcoux C. David F. Fernandez C.A. Brunengraber H. J. Biol. Chem. 1995; 270: 10027-10036Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 34Robert R.W. Robert R.W. Radioactive and Stable Isotope Tracers in Biomedicine. Wiley-Liss, Inc., New York1992: 49-85Google Scholar). The appearance of 13C-labeled glutamate, aspartate, or NAG isotopomers was calculated by the product of 13C enrichment (MPE) in a given isotopomer/100 times concentration (nmol/g wet wt) and is expressed as nanomoles of 13C-labeled metabolite/g wet wt. The rate of appearance of 13C-labeled isotopomers was calculated by fitting the time course appearance of 13C-labeled glutamate, aspartate, or NAG isotopomers to a one-phase exponential association or to a linear regression analysis using GraphPad Prism-4 software for linear and nonlinear curve fitting as indicated (16Nissim I. Luhovyy B. Horyn O. Daikhin Y. Nissim I. Yudkoff M. J. Biol. Chem. 2005; 280: 17715-17724Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The output of 13C-labeled ketone bodies was calculated by the product of MPE/100 times rate of output (nmol·g–1·min–1 wet wt) and is expressed as nanomoles of 13C-labeled metabolite·g–1·min–1. The output of 13CO2 (nmol·g–1·min–1) was calculated by the product of 13CO2 enrichment MPE/100 times 25 mm, the concentration of NaHCO3 in the perfusate. Data obtained from mitochondrial incubations were analyzed with GraphPad Prism-4 software for linear and nonlinear curve fitting.Each series of experiments was repeated 3–4 times with different mitochondrial preparations or with individual liver perfusion systems as outlined above. Statistical analysis was carried out using In-STAT 1.14 software for the Macintosh. The Student's t test or analysis of variance test was employed to compare two groups or differences among groups as needed. A p value less than 0.05 was taken as indicating a statistically significant difference.RESULTSThe initial series of experiments was designed to determine the relationship between the products of FAO, i.e. β-HB, AcAc (ketone bodies), CO2, and urea output in the effluent. Fig. 2 illustrates the effect of Agm on the output of 13C-labeled ketone bodies and CO2 in the effluent during perfusion with 0.5 mm [U-13C16]palmitate, glutamine, and ammonia, with or without 0.1 mm Agm. This dose of Agm was found to exert the maximum effect on the up-regulation of urea synthesis (9Nissim I. Horyn O. Daikhin Y. Nissim I. Lazarow A. Yudkoff M. Am. J. Physiol. 2002; 283: E1123-E1134Crossref PubMed Scopus (25) Google Scholar). During perfusions with Agm and [U-13C16]palmitate, the output of both 13C-labeled ketone bodies and CO2 was significantly higher compared with perfusions without Agm. The elevated output of 13C-labeled ketone bodies (Fig. 2A) and CO2 (Fig. 2B) was accompanied by a significant increase in urea output (Table 1), indicating a possible coupling between the oxidation of [U-13C16]palmitate and ureagenesis. In perfusion without 0.5 mm [U-13C16]palmitate (Medium B, see under “Experimental Procedures”), the release of ketone bodies in the effluent was about 54 ± 9 n mol·g–1·min–1, and the β-HB/AcAc ratio was about 1. When 0.5 mm [U-13C16]palmitate was added to the perfusate, the release of ketone bodies was increased by about 6-fold (Table 1), and the β-HB/AcAc ratio was increased to 2.7–2.9. These values are similar to those reported previously during liver perfusions with octanoate (35McGarry J.D. Foster D.W. J. Biol. Chem. 1971; 246: 1149-1159Abstract Full Text PDF PubMed Google Scholar, 36Dennis S.C. DeBuysere M. Scholz R. Olson M.S. J. Biol. Chem. 1978; 253: 2229-2237Abstract Full Text PDF PubMed Google Scholar) or palmitate (37Nishiyama P. Ishii-Iwamoto E.L. Bracht A. Cell Biochem. Funct. 1997; 15: 223-228Crossref PubMed Scopus (4) Google Scholar).FIGURE 2The effect of agmatine on the output of 13C-labeled ketone bodies (A) or CO2 (B) during the course of perfusions with [U-13C16]palmitate. Preperfusions (10 min) were carried out with basic perfusate containing 2.1 mm lactate and 0.3 mm pyruvate. Liver was then perfused for an additional 10 min with perfusate supplemented with 1 mm glutamine and 0.3 mm NH4Cl, lactate, and pyruvate with or without 0.1 mm agmatine. Finally, liver was perfused for the times indicated with lactate, pyruvate, glutamine and ammonia (as indicated above) plus 0.5 mm [U-13C16]palmitate, with (•) or without (▴) 0.1 mm agmatine. The indicated time represents time of collection of effluent samples following the start of [U-13C16]palmitate infusion. Bars are mean ± S.D. of 3–4 independent liver perfusions.View Large Image Figure ViewerDownload Hi-res image" @default.
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• W1983996840 title "Agmatine Stimulates Hepatic Fatty Acid Oxidation" @default.
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