FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2110668771> ?p ?o ?g. }

• W2110668771 endingPage "43747" @default.
• W2110668771 startingPage "43740" @default.
• W2110668771 abstract "An elevated plasma level of homocysteine is a risk factor for the development of cardiovascular disease. The purpose of this study was to investigate the effect of glucagon on homocysteine metabolism in the rat. Male Sprague-Dawley rats were treated with 4 mg/kg/day (3 injections per day) glucagon for 2 days while control rats received vehicle injections. Glucagon treatment resulted in a 30% decrease in total plasma homocysteine and increased hepatic activities of glycine N-methyltransferase, cystathionine β-synthase, and cystathionine γ-lyase. Enzyme activities of the remethylation pathway were unaffected. The 90% elevation in activity of cystathionine β-synthase was accompanied by a 2-fold increase in its mRNA level. Hepatocytes prepared from glucagon-injected rats exported less homocysteine, when incubated with methionine, than did hepatocytes of saline-treated rats. Flux through cystathionine β-synthase was increased 5-fold in hepatocytes isolated from glucagon-treated rats as determined by production of14CO2 and α-[1-14C]ketobutyrate froml-[1-14C]methionine. Methionine transport was elevated 2-fold in hepatocytes isolated from glucagon-treated rats resulting in increased hepatic methionine levels. Hepatic concentrations of S-adenosylmethionine and S-adenosylhomocysteine, allosteric activators of cystathionine β-synthase, were also increased following glucagon treatment. These results indicate that glucagon can regulate plasma homocysteine through its effects on the hepatic transsulfuration pathway. An elevated plasma level of homocysteine is a risk factor for the development of cardiovascular disease. The purpose of this study was to investigate the effect of glucagon on homocysteine metabolism in the rat. Male Sprague-Dawley rats were treated with 4 mg/kg/day (3 injections per day) glucagon for 2 days while control rats received vehicle injections. Glucagon treatment resulted in a 30% decrease in total plasma homocysteine and increased hepatic activities of glycine N-methyltransferase, cystathionine β-synthase, and cystathionine γ-lyase. Enzyme activities of the remethylation pathway were unaffected. The 90% elevation in activity of cystathionine β-synthase was accompanied by a 2-fold increase in its mRNA level. Hepatocytes prepared from glucagon-injected rats exported less homocysteine, when incubated with methionine, than did hepatocytes of saline-treated rats. Flux through cystathionine β-synthase was increased 5-fold in hepatocytes isolated from glucagon-treated rats as determined by production of14CO2 and α-[1-14C]ketobutyrate froml-[1-14C]methionine. Methionine transport was elevated 2-fold in hepatocytes isolated from glucagon-treated rats resulting in increased hepatic methionine levels. Hepatic concentrations of S-adenosylmethionine and S-adenosylhomocysteine, allosteric activators of cystathionine β-synthase, were also increased following glucagon treatment. These results indicate that glucagon can regulate plasma homocysteine through its effects on the hepatic transsulfuration pathway. S-adenosylmethionine S-adenosylhomocysteine high performance liquid chromatography bovine serum albumin An elevated plasma concentration of homocysteine, a sulfur-containing amino acid derived from methionine, has been recognized as an independent risk factor for the development of vascular disease (1Kang S.S. Wong P.W.K. Malinow M.R. Annu. Rev. Nutr. 1992; 12: 279-298Crossref PubMed Scopus (702) Google Scholar). Methionine is adenylated by methionine adenosyltransferase to form S-adenosylmethionine, an important biological methyl donor. Numerous methyltransferases catalyze the transfer of a methyl group from S-adenosylmethionine to a methyl acceptor, producing a methylated product and S-adenosylhomocysteine, which is subsequently hydrolyzed to form adenosine and homocysteine. Homocysteine has several possible fates: 1) remethylation to form methionine via either the cobalamin-dependent methionine synthase (using N 5-methyltetrahydrofolate as a methyl donor) or betaine:homocysteine methyltransferase (using betaine as a methyl donor); 2) catabolism by the transsulfuration pathway, ultimately forming cysteine; 3) export to the extracellular space. Two vitamin B6-dependent enzymes comprise the transsulfuration pathway: cystathionine β-synthase, which condenses homocysteine with serine to form cystathionine, and cystathionine γ-lyase, which cleaves cystathionine to cysteine, NH4+, and α-ketobutyrate. Altered flux through the remethylation or transsulfuration pathways as a result of genetic mutations or impaired vitamin status has been shown to affect plasma homocysteine levels (2Ubbink J.B. Vermaak W.J.H. van der Merwe A. Becker P.J. Am. J. Clin. Nutr. 1993; 57: 47-53Crossref PubMed Scopus (442) Google Scholar, 3Kluijtmans L.A. Boers G.H. Kraus J.P. van der Heuvel L.P. Cruysberg J.R. Trijbels F.J. Blom H.J. Am. J. Hum. Genet. 1999; 65: 59-67Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In recent years it has also become apparent that certain hormones can affect homocysteine metabolism. It has been shown that hypothyroid patients tend to have elevated plasma homocysteine and that these levels are normalized when thyroid levels are restored by thyroxine treatment (4Nedrebo B.G. Ericsson U.B. Nygard O. Refsum H. Ueland P.M. Aakvaag A. Aanderud S. Lien E.A. Metabolism. 1998; 47: 89-93Abstract Full Text PDF PubMed Scopus (135) Google Scholar, 5Hussein W.I. Green R. Jacobsen D.W. Faimen C. Ann. Int. Med. 1999; 131: 348-351Crossref PubMed Scopus (100) Google Scholar). Estrogen therapy for post-menopausal women has been shown to lower plasma homocysteine (6van Baal W.M. Smolders R.G. van der Mooren M.J. Teerlink T. Kenemans P. Obstet. Gynecol. 1999; 94: 485-491Crossref PubMed Scopus (96) Google Scholar). Altered homocysteine metabolism has been observed in diabetes mellitus. Diabetic patients (Types 1 and 2) with signs of kidney dysfunction (i.e. elevated creatinine levels) tend to have increased plasma homocysteine (7Hultberg B. Agardh E. Andersson A. Brattstrom L. Isaksson A. Israelsson B. Agardh C.D. Scand. J. Clin. Lab. Invest. 1991; 51: 277-282Crossref PubMed Scopus (144) Google Scholar). However, in the absence of renal dysfunction, patients with Type 1 diabetes exhibit decreased plasma homocysteine relative to normal subjects (8Robillon J.F. Canivet B. Candito M. Sadoul J.L. Jullien D. Morand P. Chambon P. Freychet P. Diabetes Metab. 1994; 20: 494-496PubMed Google Scholar). Studies in our laboratory have shown that plasma homocysteine is decreased in the streptozotocin-diabetic rat (a model for Type 1 diabetes mellitus). Insulin treatment increased plasma homocysteine in these diabetic animals (9Jacobs R.L. House J.D. Brosnan M.E. Brosnan J.T. Diabetes. 1998; 47: 1967-1970Crossref PubMed Scopus (170) Google Scholar). We have also shown that enzyme activities of the hepatic transsulfuration pathway are increased during uncontrolled diabetes. These changes in activity were reversed by insulin treatment (9Jacobs R.L. House J.D. Brosnan M.E. Brosnan J.T. Diabetes. 1998; 47: 1967-1970Crossref PubMed Scopus (170) Google Scholar). The regulatory effects of glucagon on amino acid metabolism are well known. It can, for example, activate the glycine cleavage system (10Mabrouk G.M. Jois M. Brosnan J.T. Biochem. J. 1998; 330: 759-763Crossref PubMed Scopus (18) Google Scholar), stimulate the y+ transporter (11Handlogten M.E. Kilberg M.S. J. Biol. Chem. 1984; 259: 3519-3525Abstract Full Text PDF PubMed Google Scholar), and induce the five urea-cycle enzymes (12Snodgrass P.J. Lin R.C. Muller W.A. Aoki T.T. J. Biol. Chem. 1978; 23: 2748-2753Abstract Full Text PDF Google Scholar). Patients with a glucagonoma have diminished plasma amino acid levels, which are related to increased clearance by the liver (13Barazzoni R. Zanetti M. Tiengo A. Tessari P. Diabetologia. 1999; 42: 326-329Crossref PubMed Scopus (18) Google Scholar). In light of the broad effects of glucagon on amino acid metabolism, it appears likely that homocysteine metabolism would be similarly regulated, given that it is a product of the metabolism of dietary essential methionine, and a precursor to cysteine. In addition, plasma glucagon is frequently elevated in Type 1 diabetes (14Unger R.H. Diabetes. 1976; 25: 136-151Crossref PubMed Google Scholar). We therefore examined the role of glucagon in regulating homocysteine metabolism in the rat. We show that glucagon treatment lowered plasma homocysteine levels and those of related amino acids. Glucagon administration elevated the activity of both enzymes of the hepatic transsulfuration pathway. Increased hepatic cystathionine β-synthase mRNA was also observed. In addition, higher SAM1 and SAH concentrations provide positive allosteric modulation of cystathionine β-synthase, the committed step for the conversion of homocysteine to cysteine. The increased enzymes and positive effectors of the transsulfuration pathway could therefore provide a viable mechanism for the decrease in plasma homocysteine by stimulating flux through this pathway. Such an increased transsulfuration flux was directly demonstrated in isolated hepatocytes. Finally, we report novel data on the hepatic concentration of the relevant amino acids. In particular, we demonstrate that glucagon treatment, which decreases plasma levels of most amino acids, including methionine, actually results in an increase in hepatic levels of this amino acid. This, which would also contribute to the increased transsulfuration flux, was brought about by marked increase in methionine transport into hepatocytes. Injectable glucagon was obtained from Eli Lilly Canada Inc. (Toronto, Canada). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted. All procedures were approved by Memorial University's Institutional Animal Care Committee and were in accordance with guidelines of the Canadian Council on Animal Care. Male Sprague-Dawley rats (supplied from our University's breeding colony) weighing between 225 and 275 g were used in all studies. Animals were placed in individual cages and exposed to a 12-h light/dark cycle beginning with light at 8:00 a.m. The animals ate commercial rodent chow (20% crude protein; ProLab® RMH 3000 rat chow) ad libitum and had free access to water. Glucagon treatment followed the procedure of Snodgrass et al. (12Snodgrass P.J. Lin R.C. Muller W.A. Aoki T.T. J. Biol. Chem. 1978; 23: 2748-2753Abstract Full Text PDF Google Scholar). Glucagon (4 mg/kg/day, subcutaneously) was administered in three equal daily doses (at 8:00 a.m., 4:00 p.m., and 12:00 midnight) for 2 days while control rats received the vehicle (Eli Lilly Canada Inc). Two hours following the last injection, animals were anesthetized with 65 mg/kg intraperitoneal sodium pentobarbital. Following a midline abdominal incision, a blood sample was collected from the abdominal aorta. The liver was then rapidly removed, and a portion was freeze-clamped at −70 °C while the remaining tissue was placed in ice-cold 50 mm potassium phosphate buffer (pH 6.9). Heparinized tubes containing the blood samples were placed on ice until plasma was separated by centrifugation in a clinical centrifuge (15 min, 3700 × g). The plasma was then frozen (−20 °C) for later use. Fresh tissues were diluted 1:5 with phosphate buffer and then homogenized with a Polytron (Brinkman Instruments, Toronto, Canada) for 20 s at 50% output. Homogenates were centrifuged at 18,000 × g at 4 °C for 30 min, and the supernatant was retained. All enzyme assays were carried out on this 18,000 × g, post-mitochondrial supernatant. Total homocysteine and cysteine concentrations were determined in plasma and liver using reverse-phase HPLC and fluorescence detection of ammonium 7-fluoro 2-oxa-1,3-diazole-4-sulfonate thiol adducts, using the method of Vester and Rasmussen (15Vester B. Rasmussen K. Eur. J. Clin. Chem. Clin. Biochem. 1991; 29: 549-554PubMed Google Scholar). For amino acid determination, plasma and freeze-clamped liver were first deproteinized with 10% sulfosalicylic acid. Following centrifugation, the resulting supernatant was adjusted to pH 2.2 with lithium citrate buffer. The samples were then analyzed on a Beckman 121 MB amino acid analyzer using Benson D-X, 0.25 Cation Xchange Resin and a single-column, three-buffer lithium method as per Beckman 121MB-TB-017 application notes. Results were quantitated using a Hewlett Packard Computing Integrator Model 3395A. Plasma glucose concentrations were determined enzymatically (16Bergmeyer H.U. Bernt E. Schmidt F. Stork H. Methods of Enzymatic Analysis, Vol. 3. 2nd Ed. Verleg Chemie International, Deerfield Beach, FL1981Google Scholar). Plasma insulin and glucagon levels were measured by Linco Research Inc. (St. Charles, MO) using rat insulin and glucagon, respectively, as standards. We measured the activities of enzymes responsible for the remethylation of homocysteine to methionine: methionine synthase (17Kolbin D.D. Watson J.E. Deady J.E. Stokstad E.L.R. Eger II, E.I. Anesthesiology. 1981; 54: 318-324Crossref PubMed Scopus (67) Google Scholar), betaine:homocysteine methyltransferase (18Wang J.A. Dudman N.P.B. Lynch J. Wilcken D.E.L. Clin. Chim. Acta. 1991; 204: 239-249Crossref PubMed Scopus (15) Google Scholar), and methylenetetrahydrofolate reductase (19Engbersen A.M.T. Franken D.G. Boers G.H.J. Stevens E.M.B. Trijbels F.J.M. Blom H.J. Am. J. Hum. Genet. 1995; 56: 142-150PubMed Google Scholar). Four enzymes that catabolize methionine conversion to cysteine were also measured:S-adenosylmethionine synthase (20Mudd S.H. Finkelstein J.D. Irreverre F. Laster L. J. Biol. Chem. 1965; 240: 4382-4392Abstract Full Text PDF PubMed Google Scholar), glycine N-methyltransferase (21Wagner C. Decha-Umphai W. Corbin J. J. Biol. Chem. 1989; 264: 9638-9642Abstract Full Text PDF PubMed Google Scholar), cystathionine β-synthase (22Miller J.W. Nadeau M.R. Smith J. Smith D. Selhub J. Biochem. J. 1994; 298: 415-419Crossref PubMed Scopus (221) Google Scholar), and cystathionine γ-lyase (23Stipanuk M.H. J. Nutr. 1979; 109: 2126-2139Crossref PubMed Scopus (63) Google Scholar). For the determination of betaine:homocysteine methyltransferase and cystathionine β-synthase activity, methionine and cystathionine, respectively, were measured by reverse-phase HPLC (24Jones B.N. Gillingham T.P. J. Chromatogr. 1983; 266: 471-482Crossref PubMed Scopus (452) Google Scholar). Protein concentration was determined using the Biuret method (25Gornall A.G. Bardawill C.J. David M.M. J. Biol. Chem. 1949; 177: 751-766Abstract Full Text PDF PubMed Google Scholar), after solubilization with deoxycholate, using bovine serum albumin as a standard (26Jacobs E.E. Jacob J. Sanadi D.R. Bradley L.B. J. Biol. Chem. 1956; 223: 147-156Abstract Full Text PDF PubMed Google Scholar). All assays were demonstrated to be linear with respect to time and protein concentration. Enzyme activities are expressed as nanomoles of product per milligram of protein per minute. Hepatocytes were isolated by the method of Berry et al. (27Berry M.N. Edwards A.M. Barritt G.J. Burdon R.H. van Knippenburg P.H. Laboratory Techniques in Biochemistry and Molecular Biology. 21. Elsevier, Oxford1991: 44-57Google Scholar), and viability was assessed by 0.2% (w/v) Trypan Blue exclusion. Viability was at least 95% in all cases. Hepatocytes were preincubated for 20 min at 4–6 mg of dry weight of cells/ml (1 ml of final volume) in Krebs-Henseleit medium equilibrated with 95% O2/5% CO2 and containing 1.25% (w/v) BSA. Following preincubation, 1 mm methionine was added and cells were incubated for another 30 min. Cells were gassed with 95% O2/5% CO2 at the beginning of preincubation and at the addition of methionine. At the end of the incubation, the contents of the flasks were immediately centrifuged at 14,000 ×g for 2 min to sediment the cells. The supernatant was then frozen at −20 °C until analyzed. Homocysteine export was determined by subtracting a zero time point and is expressed as nanomoles of homocysteine per milligram of hepatocytes per 30 min. In a separate series of experiments, hepatocytes were incubated with 1 mml-[1-14C]methionine (American Radiolabeled Chemicals, Inc., St. Louis, MO) in the presence and absence of propargylglycine and α-cyanocinnimate. Following the addition of methionine, flasks were incubated for 30 min, after which they were equipped with rubber septa with suspended plastic center wells containing NCS tissue solubilizer. Incubations were terminated by injection of 0.3 ml of 30% (w/v) perchloric acid through the septa, and 14CO2 was collected for 1 h. Radioactivity was then measured by placing the center wells in scintillation vials containing Omnifluor scintillation fluid. To collect 14CO2 from intermediate α-ketoacids, a new center well containing NCS was then added to each flask and 0.3 ml of 30% (w/v) hydrogen peroxide was injected through the septa (28Stead L.M. Brosnan M.E. Brosnan J.T. Biochem. J. 2000; 350: 685-692Crossref PubMed Scopus (95) Google Scholar).14CO2 was collected for 1 h and measured as described above. Methionine transport into isolated hepatocytes was measured as described by Salter et al. (29Salter M. Knowles R.G. Pogson C.I. Biochem. J. 1986; 233: 499-506Crossref PubMed Scopus (49) Google Scholar). Following a 20-min preincubation period, [1-14C]methionine was added to a final concentration of 0.5 mm (0.2 μCi). At 5, 35, 65, 95, and 125 s following the addition of methionine, 1-ml aliquots were transferred to 1.5-ml microcentrifuge tubes containing 0.25 ml of silicone oil mixture (2:1 (v/v) Dow Corning 550 silicone oil and dinonyl phthalate) layered on top of 0.1 ml of 6% (v/v) perchloric acid. The tubes were centrifuged at 14,000 × g for 15 s to sediment cells through the silicone oil and into the acid layer, leaving the extracellular fluid on top of the oil. Following centrifugation, the tubes were frozen in liquid nitrogen and then the tubes were cut at the end of the silicone oil layer. The bottom layer, containing the intracellular [14C]methionine, was placed in a scintillation vial containing 10 ml of Omnifluor scintillation mixture, and radioactivity was determined by a scintillation counter. The volume of extracellular space that was carried through the silicone oil was determined by measuring radioactivity in the bottom layer following cell incubation with [carboxyl-14C]inulin. This value was used to correct rates of methionine transport. Methionine transport rates were determined by subtracting 5-s rates, representing primarily amino acid binding to membranes, from the values obtained at subsequent time points. SAM and SAH were measured in the liver using the method of Wagner 2C. Wagner, personal communication.. with minor modifications. Freeze-clamped liver was quickly added to 5 volumes of 8% cold trichloroacetic acid. Samples were homogenized with a Polytron for 10 s and were placed on ice for 10 min. The samples were then centrifuged for 10 min at 13,000 × g. The supernatant was retained and analyzed by HPLC using a Vydac C18 column (model 2187P54) that was equilibrated with 96% 50 mmNaH2PO4, 10 mm heptane sulfonic acid (adjusted to pH 3.2 with concentrated sulfuric acid), and 4% acetonitrile. A 15-min gradient from 4% to 20% acetonitrile was used to separate SAM and SAH. Peaks were monitored by UV detection at 258 nm and quantified using a 3390A Hewlett Packard integrator. Total RNA was isolated from rat livers by the acid guanidinium thiocyanate-phenol-chloroform method as described by Chomczynski and Sacchi (30Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62909) Google Scholar). 15 μg of total RNA was separated on a 1% agarose gel containing formaldehyde and transferred to a Hybond-Nylon membrane (Amersham Pharmacia Biotech). cDNAs for rat cystathionine β-synthase, cystathionine γ-lyase, and β-actin (CLONTECH) were labeled with [α-32P]dCTP using a random-labeling kit (Ambion). The probes (specific activity ∼109 cpm/μg) were allowed to hybridize to the membrane for 16 h at 42 °C in Northern Max ultrahybridization buffer (Ambion). Following high stringency washing (0.1% SSC, 0.1% SDS) and radioautography, the bands were quantitated by densitometry. The cystathionine β-synthase and cystathionine γ-lyase mRNA abundance were normalized to the amount of RNA applied, using β-actin mRNA. Intracellular hepatic amino acid concentrations were calculated using published data from our laboratory on hepatic cellular spaces (31Qian D. Brosnan J.T. Biochem. J. 1998; 313: 479-486Crossref Scopus (25) Google Scholar). The measured hepatic concentration for an amino acid is the sum of the amino acid in the intracellular fluid (ICF) and the amino acid in the extracellular fluid (ECF). The following equation was used to determine the hepatic intracellular concentration of an amino acid (AA). Total liver AA(nmol/g)=[plasma AA(nmol/ml)×ECF(ml/g liver)+intracellular AA×ICF(ml/g liver)]Eq. 1 Intracellular water was previously found to be 0.45 ml/g liver, whereas the extracellular water was 0.25 ml/g liver. Data are presented as means ± S.D. unless otherwise noted. There were three to eight samples in each experimental group. Student’s unpaired t test was performed to compare means unless otherwise specified. A p value of <0.05 was taken to indicate a significant difference. Table I gives information on body weight, food intake, plasma glucose, plasma insulin, and plasma glucagon. Two-day glucagon treatment did not affect weight gain in male rats. Likewise, there was no change in food intake following glucagon treatment. Therefore, none of the metabolic changes observed can be attributed to alterations in vitamin or amino acid intakes. Plasma glucose was increased by 80% (as expected) following glucagon administration. Plasma glucagon was increased 35-fold versuscontrol rats, and there was no change in plasma insulin.Table IBody weight, food intake, plasma glucose, insulin, and glucagon concentrations in control and glucagon-treated ratsUnitsControlGlucagonInitial weightg240 ± 15245 ± 7Final weightg272 ± 10271 ± 7Food intakeg/day26.3 ± 4.527.5 ± 3.7Plasma glucagonpg/ml79.5 ± 12.12630 ± 10401-aSignificant difference versus control rats, p < 0.05.Plasma insulinng/ml8.9 ± 1.77.1 ± 2.6Plasma glucosemm8.0 ± 0.814.1 ± 1.31-aSignificant difference versus control rats, p < 0.05.Rats were administered glucagon (4 mg/kg/day) for 2 days while control rats received the vehicle. Food intake and body weight were measured daily. Blood samples were taken from the abdominal aorta and centrifuged for plasma separation. Means ± S.D. are shown for three to six measurements.1-a Significant difference versus control rats, p < 0.05. Open table in a new tab Rats were administered glucagon (4 mg/kg/day) for 2 days while control rats received the vehicle. Food intake and body weight were measured daily. Blood samples were taken from the abdominal aorta and centrifuged for plasma separation. Means ± S.D. are shown for three to six measurements. Total plasma homocysteine levels were decreased by 30% by glucagon treatment (Table II). This is the first report, to our knowledge, of the homocysteine-lowering effects of glucagon. Table II also shows the effects of the glucagon treatment on plasma and liver concentrations of the other amino acids involved in sulfur amino acid metabolism. All plasma amino acids shown were significantly decreased by glucagon treatment. Hepatic amino acid levels are also reported. Liver homocysteine and glycine levels were significantly decreased by glucagon treatment, whereas the concentration of taurine was increased. No change was observed in the hepatic levels of methionine, cysteine, and serine. However, calculation of intracellular amino acid concentrations revealed increased methionine and taurine in the glucagon-treated rat, whereas intracellular homocysteine was decreased.Table IIPlasma and hepatic amino acids involved in homocysteine metabolismControlGlucagonPlasmaLiverHepatic intracellular concentrationL:P ratioPlasmaLiverHepatic intracellular concentrationL:P ratioμmnmol/mgμmμmnmol/mgμmMethionine56.9 ± 2.956.0 ± 6.094.1 ± 10.11.714.8 ± 2.82-aSignificant difference versus control, p < 0.05.60.6 ± 6.0125 ± 132-aSignificant difference versus control, p < 0.05.8.5Homocysteine9.7 ± 1.19.0 ± 1.014.8 ± 1.71.56.4 ± 0.72-aSignificant difference versus control, p < 0.05.6.9 ± 1.02-aSignificant difference versus control, p < 0.05.11.9 ± 1.72-aSignificant difference versus control, p < 0.05.1.9Cysteine318 ± 25443 ± 110808 ± 2002.5223 ± 282-aSignificant difference versus control, p < 0.05.358 ± 158672 ± 2973.0Taurine177 ± 192850 ± 4356235 ± 95035101 ± 182-aSignificant difference versus control, p < 0.05.3600 ± 5572-aSignificant difference versus control, p < 0.05.7943 ± 12282-aSignificant difference versus control, p < 0.05.78Serine180 ± 17311 ± 51591 ± 972.985 ± 312-aSignificant difference versus control, p < 0.05.330 ± 48686 ± 1008.0Glycine239 ± 38920 ± 1411912 ± 2938.096 ± 342-aSignificant difference versus control, p < 0.05.830 ± 772-aSignificant difference versus control, p < 0.05.1804 ± 16619.0Plasma and liver amino acids were determined using a Beckman amino acid analyzer, except for total homocysteine and total cysteine, which were determined by HPLC. Intracellular amino acid concentrations were calculated as described under “Experimental Procedures.” Liver:plasma (L:P) ratio is calculated by dividing the hepatic intracellular amino acid concentration (μm) by the plasma amino acid concentration (μm). Means ± S.D. for five rats are shown.2-a Significant difference versus control, p < 0.05. Open table in a new tab Plasma and liver amino acids were determined using a Beckman amino acid analyzer, except for total homocysteine and total cysteine, which were determined by HPLC. Intracellular amino acid concentrations were calculated as described under “Experimental Procedures.” Liver:plasma (L:P) ratio is calculated by dividing the hepatic intracellular amino acid concentration (μm) by the plasma amino acid concentration (μm). Means ± S.D. for five rats are shown. The liver is the central organ in sulfur amino acid metabolism. It contains a full complement of enzymes involved in the methionine cycle and the transsulfuration pathway and is the site of 85% of all methylation reactions in the body (32Wyss M. Wallimann T. Mol. Cell. Biochem. 1994; 134: 51-66Crossref Scopus (94) Google Scholar). In light of this, it is reasonable to assume that alterations in hepatic homocysteine metabolism would have a profound effect on circulating levels of this atherogenic amino acid. We therefore measured homocysteine output by isolated hepatocytes. Previously, our laboratory has showed that the half-maximal rate of homocysteine export occurs at a methionine concentration of 0.44 mm and is linear for at least 60 min (28Stead L.M. Brosnan M.E. Brosnan J.T. Biochem. J. 2000; 350: 685-692Crossref PubMed Scopus (95) Google Scholar). A methionine concentration of 1 mm was chosen for all experiments. Following incubation with methionine, hepatocytes isolated from the glucagon-treated rats exported less than half as much homocysteine as the control hepatocytes (Fig.1). Our earlier studies have shown that addition of serine (a substrate for cystathionine β-synthase), together with methionine, reduced homocysteine export (28Stead L.M. Brosnan M.E. Brosnan J.T. Biochem. J. 2000; 350: 685-692Crossref PubMed Scopus (95) Google Scholar). We therefore undertook experiments with both serine and methionine in the incubation medium. Serine incubation decreased homocysteine export from the control hepatocytes by 50%. However, serine did not reduce, any further, the homocysteine export of hepatocytes from glucagon-treated animals. There was no change in cysteine export found in any of the experimental groups (data not shown). Such a decrease in homocysteine export by hepatocytes coupled with a decreased intracellular homocysteine concentration suggests an appreciably altered metabolism. Therefore, we assayed the major enzymes involved in producing (transmethylation) and removing (transsulfuration and remethylation) homocysteine in the liver. Glucagon-treated rats exhibited increased hepatic activities of enzymes involved in the catabolism of methionine to cysteine (TableIII). The activities of glycine N-methyltransferase and cystathionine γ-lyase activity were elevated by 25% whereas cystathionine β-synthase activity was increased by 90%. These changes are still evident when activities are expressed per gram of liver or per 100 g of body weight (data not shown). Methionine adenosyltransferase activity was unaffected by glucagon treatment. These data suggest the importance of the hepatic transsulfuration pathway in glucagon's regulation of homocysteine metabolism. No changes were observed in methionine synthase, betaine:homocysteine methyltransferase, or methylenetetrahydrofolate reductase activity.Table IIIHepatic enzymes of methionine and homocysteine metabolism in glucagon-treated ratsControlGlucagonS-Adenosylmethionine synthase1.20 ± 0.051.25 ± 0.10Cystathionine β-synthase5.3 ± 0.99.3 ± 1.23-aSignificant difference versus control rats, p < 0.05.Cystathionine γ-lyase15.2 ± 2.019.6 ± 3.23-aSignificant difference versus control rats, p < 0.05.Glycine N-methyltransferase1.02 ± 0.041.25 ± 0.103-aSignificant difference versus control rats, p < 0.05.Methionine synthase0.11 ± 0.010.11 ± 0.02Betaine:homocysteine methyltransferase2.6 ± 0.72.2 ± 0.1Methylenetetrahydrofolate reductase0.16 ± 0.050.14 ± 0.02Liver samples were homogenized in potassium phosphate buffer and centrifuged at 17,000 × g for 30 min. All assays were performed on the resulting post-mitoch" @default.
• W2110668771 created "2016-06-24" @default.
• W2110668771 creator A5016378050 @default.
• W2110668771 creator A5038500014 @default.
• W2110668771 creator A5046069471 @default.
• W2110668771 creator A5079911358 @default.
• W2110668771 date "2001-11-01" @default.
• W2110668771 modified "2023-10-15" @default.
• W2110668771 title "Hyperglucagonemia in Rats Results in Decreased Plasma Homocysteine and Increased Flux through the Transsulfuration Pathway in Liver" @default.
• W2110668771 cites W1484712019 @default.
• W2110668771 cites W1506043065 @default.
• W2110668771 cites W1527275481 @default.
• W2110668771 cites W152764626 @default.
• W2110668771 cites W1548659851 @default.
• W2110668771 cites W1548746280 @default.
• W2110668771 cites W1560473424 @default.
• W2110668771 cites W1738677317 @default.
• W2110668771 cites W1838741822 @default.
• W2110668771 cites W1840655861 @default.
• W2110668771 cites W1932472567 @default.
• W2110668771 cites W1965319078 @default.
• W2110668771 cites W1981595029 @default.
• W2110668771 cites W1988041601 @default.
• W2110668771 cites W1994831592 @default.
• W2110668771 cites W2005513272 @default.
• W2110668771 cites W2010870164 @default.
• W2110668771 cites W2011393897 @default.
• W2110668771 cites W2018756843 @default.
• W2110668771 cites W2025771789 @default.
• W2110668771 cites W2031360597 @default.
• W2110668771 cites W2036846090 @default.
• W2110668771 cites W2054847530 @default.
• W2110668771 cites W2060355646 @default.
• W2110668771 cites W2067377490 @default.
• W2110668771 cites W2070933212 @default.
• W2110668771 cites W2072620003 @default.
• W2110668771 cites W2073809240 @default.
• W2110668771 cites W2077382007 @default.
• W2110668771 cites W2111620111 @default.
• W2110668771 cites W2113377359 @default.
• W2110668771 cites W2118015493 @default.
• W2110668771 cites W2137394317 @default.
• W2110668771 cites W2164721380 @default.
• W2110668771 cites W2199432246 @default.
• W2110668771 cites W2417812919 @default.
• W2110668771 cites W2427694386 @default.
• W2110668771 cites W285674794 @default.
• W2110668771 cites W32741228 @default.
• W2110668771 cites W4234370759 @default.
• W2110668771 cites W4239075380 @default.
• W2110668771 cites W4294216491 @default.
• W2110668771 cites W87493514 @default.
• W2110668771 doi "https://doi.org/10.1074/jbc.m107553200" @default.
• W2110668771 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11559709" @default.
• W2110668771 hasPublicationYear "2001" @default.
• W2110668771 type Work @default.
• W2110668771 sameAs 2110668771 @default.
• W2110668771 citedByCount "54" @default.
• W2110668771 countsByYear W21106687712012 @default.
• W2110668771 countsByYear W21106687712013 @default.
• W2110668771 countsByYear W21106687712014 @default.
• W2110668771 countsByYear W21106687712015 @default.
• W2110668771 countsByYear W21106687712016 @default.
• W2110668771 countsByYear W21106687712018 @default.
• W2110668771 countsByYear W21106687712019 @default.
• W2110668771 crossrefType "journal-article" @default.
• W2110668771 hasAuthorship W2110668771A5016378050 @default.
• W2110668771 hasAuthorship W2110668771A5038500014 @default.
• W2110668771 hasAuthorship W2110668771A5046069471 @default.
• W2110668771 hasAuthorship W2110668771A5079911358 @default.
• W2110668771 hasConcept C126322002 @default.
• W2110668771 hasConcept C134018914 @default.
• W2110668771 hasConcept C16525657 @default.
• W2110668771 hasConcept C178790620 @default.
• W2110668771 hasConcept C185592680 @default.
• W2110668771 hasConcept C2777090595 @default.
• W2110668771 hasConcept C2778563252 @default.
• W2110668771 hasConcept C2780912031 @default.
• W2110668771 hasConcept C2908748658 @default.
• W2110668771 hasConcept C2910648213 @default.
• W2110668771 hasConcept C515207424 @default.
• W2110668771 hasConcept C55493867 @default.
• W2110668771 hasConcept C68709404 @default.
• W2110668771 hasConcept C71315377 @default.
• W2110668771 hasConcept C71924100 @default.
• W2110668771 hasConcept C86803240 @default.
• W2110668771 hasConceptScore W2110668771C126322002 @default.
• W2110668771 hasConceptScore W2110668771C134018914 @default.
• W2110668771 hasConceptScore W2110668771C16525657 @default.
• W2110668771 hasConceptScore W2110668771C178790620 @default.
• W2110668771 hasConceptScore W2110668771C185592680 @default.
• W2110668771 hasConceptScore W2110668771C2777090595 @default.
• W2110668771 hasConceptScore W2110668771C2778563252 @default.
• W2110668771 hasConceptScore W2110668771C2780912031 @default.
• W2110668771 hasConceptScore W2110668771C2908748658 @default.
• W2110668771 hasConceptScore W2110668771C2910648213 @default.
• W2110668771 hasConceptScore W2110668771C515207424 @default.
• W2110668771 hasConceptScore W2110668771C55493867 @default.

• next