AMINO ACID METABOLISM IN THE KIDNEYS OF GENETIC AND NUTRITIONALLY OBESE RATS

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1 Vol. 42, No. 2, June 1997 BIOCHEMISTRY end MOLECULAR BIOLOGY INTERNATIONAL Pages AMINO ACID METABOLISM IN THE KIDNEYS OF GENETIC AND NUTRITIONALLY OBESE RATS M.Carmen Herrero I, Xavier Remesar 2, Cinta Blad61, Lluis Arola 1 1Departament de Bioquimica i Biotecnologia, Universitat Rovira i Virgil/, Tarragona and 2Departament de Bioquimica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain Received January 10, 1997 Received after revision March 11, 1997 SUMMARY The ability of the kidney to take up and/or release amino acids has been determined in two models of obesity in Zucker rats, one genetic and the other nutritional (diet-obese). There was a noticeable increase in gluconeogenic amino acids in the arterial blood of diet-obese animals whereas the genetically obese rats showed small variations in the levels of these amino acids. There were significant decreases in renal Gly and Ser, only in the genetically obese rats. Genetically obese animals showed an increase in Glutamine synthetase activity. The uptake and/or release of amino acids showed important variations between the groups. The diet-obese group exhibited greater variation, since this group took up Glu, Ala, Gy, Phe and Citrulline and released Gin, Ser, Arg and Tyr. Genetically obese rats took up Gin, His and Taurine and released Ser. These different patterns may be related to variations in the whole body metabolic rate, since the diet-obese group was more active than the genetically obese group. Key words: Zucker rats / Obesity / Cafeteria diet / Kidney / Amino acid uptake/release INTRODUCTION Whole body amino acid metabolism varies in different models of obesity. Thus, rats that are obese as a consequence of a hypercaloric diet tend to preserve nitrogen (1) with a decrease in the production and excretion of urea (1,2,3) and an increase in lean muscle mass (4). Conversely, genetically obese rats are characterized by their lack of ability to manage their nitrogen economy, and they have less muscle mass (5) and fewer protein levels in muscle tissue. These metabolic conditions affect the normal pattern of kidney amino acid metabolism, since this tissue plays a principal role in control the whole body ammonia Address for correspondence: Dr. Cinta Blad6; Departament de Bioquimica i Biotecnologia; Facultat de Quimica; Universitat Rovira i Virgil/; P(;a.lmperial Tarraco 1; Tarragona, SPAIN. mcbs@astor.urv.es /97/ /0 Copyright by Academic Press Australia. All rights o/repjvduction in any,/orm reserved.

2 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL dissimilation. Thus,-the utilization of the amino and amide groups of glutamine in the ammoniagenic process implies a net consumption of this amino acid (6); conversely there is a net production of other amino acids such as citrulline or serine. Moreover, the relative maintenance of amino acid levels in rat kidney can be affected by physiological variations such as starvation (7) as a consequence of changes in the activity of some of the amino acid metabolising enzymes. The increase in nitrogen conservation described in cafeteria-fed rats is associated with a decrease in urea production and excretion (1) and with an increase in the nitrogen accretion ratio (2) and with a lack of information relative to possible variations in the rate of excretion of individual amino acids. In the genetically obese rats (fa/fa) there is a progressive development of renal disease manifested by glomerulosclerosis (8) which is responsible for the leak of different substances (glucose, albumin, etc) into the urine (9). The aim of this study was to determine the ability of the kidney to take up or release individual amino acids using two different rat models of obesity, one of genetic origin and the other developed by nutritional management. MATERIALS AND METHODS 60-day old male Zucker rats (Harlan Olac, UK) were used, either lean (Fa/?) or obese (fa/fa). They were bred at the Animal Service of the University under controlled conditions of light (12 hours on/12 hours off), humidity (70-80%) and temperature (20-21~ The rats were housed in polypropylene-bottomed cages with wood shavings as absorbent materials. After delivery, Fa/? dams were randomly divided into two groups. One group was fed ad libitum with a standard laboratory chow diet (A03, Panlab, Barcelona, Spain) and tap water. The second group (Diet-Ob) was fed with a rich hypercaloric diet (cafeteria diet) with daily fresh offering of biscuits spread with liver pat~, bacon, banana, chow pellets (as indicated above) tap water and whole milk supplemented with 300 g/i sucrose plus 10 g/i of mineral and vitamin supplemented (Gevral, Cyanamid Ib6rica, Spain). All materials were previously weighed and presented in excess (c % of the expected consumption). This diet is a simplified version of an earlier diet developed here (10). Male rats were selected at weaning, on day 21, and reared with these diets up to 60 days. Obese fa/fa were fed ad libitum with standard laboratory chow diet and tap water. The three groups of rats, Fa/? lean, Diet-Ob and fa/fa obese were divided in two subgroups, one used to measure blood flow and the other used to estimate the arteriovenous differences in amino acids across the kidney. Arterio-venous differences were determined in anaesthetized rats in the same way as described above and blood was sampled firstly from the left renal vein and then from the abdominal aorta with heparinized syringes. Blood samples were deproteinized with cold acetone (11) and after centrifuging the supernatant fractions were stored at -20~ for amino acid analysis by HPLC (Gilson Asted System,) using the OPA (orto-phthaldialdehyde) and FMOC (9-fluorenyl methoxy carbonyl chloride) derivatives. Individual amino acids were separated in a Spherisorb ODS-2 column and a ternary solvent gradient of sodium phosphate/propionic acid/acetonitrile at room temperature. Homocysteic acid, homoserine, 262

3 Vol. 42, No. 2, ] 997 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL norvaline and thioproline were used as internal standards. The uptake of each amino acid by the leg muscle was calculated as: Uptake = ((Pr x Ca) - (~r x Cr) where ~r represent the renal vein flow, and Ca and Cr the concentration of each amino acid in the aorta and renal vein respectively. Blood flow determinations were performed minutes after the beginning of the light cycle when the rats were anaesthetized intraperitoneally with sodium pentobarbital (60 mg/kg body weight). Renal blood flow was measured as previously described (12). Briefly, a cannula (PE-10 polyethylene tubing (Clay-Adams, Perssipany, N J, USA)) was inserted into the left ventricle through the left carotid artery; then 0.1 ml (50 kbq) of 46Sc microspheres (New England Nuclear, Bad Homburg, Germany; mean diameter 15 Bm) were injected uniformly for 15 s through this cannula. Five seconds before the injection and for 60 s thereafter, blood was collected from the renal artery with a heparinized syringe driven by an infusion pump. This blood was weighed and used as a reference for blood radioactivity. The rats were killed by the injection of 1 ml of air through the carotid cannula. After dissection, the left kidney was blotted and weighed and its radioactivity was determined with a solid scintillation counter. Relative blood flow was calculated as: ~r = (~B x RK)/RRB, where ~r is the kidney blood flow (ml - min -1 9 g-l); (PRB is the reference blood sample (ml. min -1 - g-l); RK is the radioactivy found in the kidney (cpm 9 g-l) and RRB is the radioactivty of the reference blood (cpm 9 g-l). There were no differences in the values of blood flow between both kidneys. Metabolite and enzyme activities were determined in left kidney samples, which was excised, immediately frozen in liquid nitrogen and powdered. Then homogeneous samples were used for nitrogen (elemental analysis), protein (13) and amino acid determinations. Fractions of the samples were deproteinized with cold acetone:water (1:1.2) (11) and individual amino acids were determined in the supernatant fractions using the method described above (HPLC). Total free amino acids were determined in the kidney homogenate by the ninhydrin method (t4). Alanine transaminase (EC ) (15) and glutamine synthetase (EC ) (16) were also determined in kidney samples. 5-7 animals per group were used and all results are expressed as mean+s.e.m. Significance of the changes induced by obesity were tested by one factor ANOVA and the Scheffe test. RESULTS The weights of the animals on day 60 were: g for lean group, g for Diet-ob group and g for genetic obese group. The mean daily energy intake from 30 to 60 days was : kj for the lean group; kj for the diet-obese (p < 0.05 vs lean group) and kj for the genetic obese group (p < 0.05)(values obtained from reference 10). Amino acid levels in the arterial blood are shown in Table 1. Some gluconeogenic amino acids (Ala, Ser, Gly, Thr) were higher in the diet-obese animals, and contrasted with the decreases shown by Gin and Asp. Genetically obese animals showed small variation, the most significant being the decrease in the Gly and the increase in the Thr and Leu levels. 263

4 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL Table 1: Amino acid levels (pmol/l) in arterial blood of obese rats. Each value represents the mean + SEM of 5-7 animals. Statistical analysis by one factor ANOVA and Scheff6 test: * P < 0.05 when compared with lean animals ; 1 P < 0.05 when compared with obese animals. Lean Diet-obesity Genetic-obesity Aspartic acid "1" * Glutamic acid "1" 177_+14 Glutamine 376+_ Alanine "t Serine " 176_+8 Glycine 16~+~ "t" 32_+13* Threonine 163_ _24* * Histidine 60_ "i" Arginine 81_+3 102_ Methionine 18+_2 26_+2"1" Taurine 300_ _+6"I" Citrulline Phenylalanine * Tyrosine 50_+6 46_ * Valine Leucine _ _20"1" Isoleucine 33_+5 39_+5 34_+6 These patterns illustrate the differences between the two obese groups, especially in the levels of the gluconeogenic amino acids. The levels of free amino acids in the kidney are shown in Table 2. In diet-obese animals there are no significant changes when compared with the control group. Animals in the genetically obese group exhibited a marked decrease in the levels of Gly and Ser. The activities of Alanine Transaminase were the same in all groups. Conversely, Glutamine Synthetase activity is increased in the genetically obese animals (Table 3). Figure 1 shows the uptake or release of individual amino acids by the kidney. The control group released Asp, Gin, Ser, Thr and Val, in contrast with the wide variations of release shown by the other groups. Thus, diet-obese animals released Gin, Ser, Arg and Tyr 264

5 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL Table 2: Amino acid levels (nmol/g of kidney) in the kidneys of obese rats. Each value represents the mean + SEM of 5-7 animals. Statistical analysis by one factor ANOVA and Scheff~ test: * P < 0.05 when compared with lean animals ; 1" P < 0.05 when compared obese animals. Lean Diet-obesity Genetic-obesity Aspartic acid _272 Glutamic acid _ _+682 Glutamine Alanine _ !145 Serine "1" Glycine _ "t" Threonine _20 686_+49t" Histidine i Arginine _ Taurine 12740_ Methionine Citrulline Tyrosine Phenylalanine 66+_ Valine 570+_ _+60 Leucine _ ~_80 Isoleucine _ _25 Table 3: Effect of dietary and genetic obesity on selected amino acid enzymatic activities in the kidney. Each value represents the mean + SEM of 5-7 animals. ALT = Alanine aminotransferase, GSA = Glutaminase. Statistical analysis by one factor ANOVA and Scheff6 test: * P < 0.05 when compared with lean animals. Lean Diet-obesity Genetic-obesity ALT (nkat/g of kidney) 28_ _+6 29+_2 GSA _2" (nkat/.q of kidney) 265

6 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL 400- '! 200- i~ I ~-200- "O t :g t Y ~ *t Asp Glu Gin Ala Ser Thr Gly Cit Arg Tau Met His Tyr Val Phe lie Leu Figure 1: Amino acid uptake (nmol/min.g of kidney) by kidneys of lean and obese rats Each value represents the mean + SEM of 5-7 animals. Positive values represent uptake; negative values represent release. ": Lean rats, [] : Diet obese rats and r-i: genetic obese rats. Statistical analysis by one factor ANOVA and Scheffe test: * P < 0.05 when compared with lean animals; t P < 0.05 when compared with obese animals. and took up Glu, Ala, Gly, Citruline and Phe. The fa/fa group did not show significative uptake or release of amino acids, except for Glu, Taurine, His and Ser. DISCUSSION The physiological status of these experimental animals was nearer to that of a postprandial state than that of a prandial one. This is considered to be a consequence of the model chosen (3). This needs be taken into account in considering the notable metabolic activity shown by the small intestine, which takes up glutamine and releases alanine (17). In the diet-obese group, the discernible increase in gluconeogenic amino acids in the arterial blood may generate a gradient that facilitates the uptake by different tissues of many amino acids - mainly Ala and Arg. This may be propitiated by the notable decrease in amino acid uptake by the liver (17).The trend to increased levels of insulin (18,19) in diet-obese rats may favour an increase in the uptake of alanine by the extrahepatic tissues, since different 266

7 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL amino acid transport system activities are enhanced by the action of insulin (20). However, it is of note that hyperinsulinemia is not clearly established in the hypercaloric (mainly hyperlipidic) diet (21). Moreover, the Gin released by muscle can be actively taken up by the small intestine as previously described (17). The kidney of control animals followed an anticipated and coneventional pattern, in relation to Ser and Arg release, described in post prandrial conditions (22,23). Serine may be used by the small intestine and argjnine by the liver (17). The amino acid metabolism in kidney is quite active as deduced from the high activity values of the enzymes involved in amino acid metabolism (7). On the other hand, the release of glutamine may represent part of the mechanism for the normal maintenance of blood ph and the normal ammoniagenesis pattern from Glu. The high Glu levels in renal tissue support this proposition, and furthermore the high concentration of Glu may inhibit the ammoniagenesis pathway from Gin (24); thus, an uptake of Glu may initiate and regulate the release of Gin. The uptake of glycine and taurine is consistent with their limited metabolic fate and their pattern of elimination via the urine (25). On the other hand, the release of Ser may be a consequence of the synthesis of this amino acid from glycerate and the incorporation of amino groups from alanine and/or glutamate (26). Active renal metabolism is increased in the diet-obese group since the rates of release and/or uptake of amino acids are enhanced. Thus, the increased uptake of Glu is supported by its increased concentration, that may act to inhibit the activity of phosphate dependent glutaminases (27) thus propitiating the release of Gin and the possible participation of Glu as an amino-group donor for Ser synthesis. Alanine utilization in the Ser synthesis may also contribute by its increased uptake. The lack of significant variations in the release and/or uptake of amino acids in genetically obese rats may be a consequence of slow metabolic activity. However, the increase in glutamine synthetase activity may result from the requeriment to maintain ph homeostasis, since in these animals the functionality of renal filtration is decreased, because the progressive development of glomerulosclerosis (8), and its affect on the normal whole body amino acid balance. However, this lack of functionality may be selective, since these 267

8 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL animals do not exhibit disturbances in the excretion of some mineral elements such as Na, Ca, K or Mg (28). The low Gly concentration in renal tissue may also play an essential role in the reduced maintenance of tubule cell structural integrity (29). Thus impaired renal activity may be the basis for the increased urine production in these rats (2), which may account for an increased release of amino acids without acute changes in amino acid balances through the kidney. We conclude that amino acid metabolic patterns diverge in different models of obesity, since the diet-ob group appear to maintain a higher amino acid catabolism than genetically obese rats. Furthermore, the diminished protein content of fa/fa muscle is not due to a lower availability of amino acids. Acknowledgements Thanks are given to Robin Rycroft for editorial assistance. This study was supported by grants PB (from the "Direccibn General de Investigaci6n Cientifica y Tecnica") and 93/0218 (from the Fondo de Investigaciones Sanitarias) of the Government of Spain, as well as a personal grant (MC.Herrero) from the CIRIT of the Government of Catalonia. REFERENCES: 1. Barber, T., ViSa, J.R., ViSa, J. and Cabo, J. (1985) Biochem J. 230, Esteve, M., Rafecas, I., Remesar, X. and Alemany, M. (1992) Int. J. Obesity 16, Herrero, M.C., Angles, N., Remesar, X., Arola, LI. and Blade, C. (1994) Int. J. Obesity 18, Rafecas, M., Esteve, M., Fern~ndez-L6pez, J.A., Remesar, X. and Alemany, M. (1994) Nutr. Res. 14, Lanza-Jacoby, S. and Kaplan, M.C. (1984) Int. J. Obesity 8, Preuss, H.G. (1980) Life Science 27, Remesar, X., Arola, LL., Palou, A., Soley, M. and Alemany, M. (1983) Arch. Internat. Physiol. Biochim. 91, Shimamura, T. (1982) Exp. Mol. Pathol. 36, Kashise, B.L., Cleary, MP., O'Donnell, MP. and Keane, W.F. (1985) J. Lab. Clin. Med. 106, Salvad6, J., Segu6s, T., Alemany, M. and Arola, LI. (1986) Br. J. Nutr. 55, Arola, LI., Herrera, E. and Alemany, M. (1975) Anal. Biochem. 82, Closa, D., G6mez-Sierra, J.M., Latres, E., Alemany, M. and Remesar, X. (1993) Exp. Physiol. 78, Lowry, O.H., Rosebrough, N.J., Farr, A.L. and, Randall, R.J. (1951) J. Biol. Chem. 173, Yemm, E.V. and Cocking, E.C. (1955) Analyst 80,

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