Effect of exogenous cortisone on amino acid metabolism in rats

Effect of exogenous cortisone on amino acid metabolism in rats

far. J. Biochem. Vol. 22, No. 1, pp. 83-87, 1990 Primed in Great Britain. All rights reserved EFFECT 0020-711X/90 $3.00 C 0.00 Copyright cfl 1990 Pe...

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far. J. Biochem. Vol. 22, No. 1, pp. 83-87, 1990 Primed in Great Britain. All rights reserved


0020-711X/90 $3.00 C 0.00 Copyright cfl 1990 Pergamon Press plc





of Medicine, Shinshu University School of Medicine, Asahi 3-l-1, Matsumoto 390, Japan (Received 9



Abstract-1 The effect of exogenous cortisone on concentration of free amino acids in serum, skeletal muscle, kidney, small intestine and liver was studied. 2. The amino acid pool in serum, skeletal muscle and small intestine decreased significantly. 3. Glutamine synthesis increased significantly in skeletal muscle. 4. Levels of branched amino acids increased in serum and small intestine. 5. Levels of alanine increased in kidney and liver.


centages of each amino acid in total free amino acid were calculated. The mean and SE of the mean were determined for the control and test groups. Student’s t test was used to compare the mean values in the test and control groups and to analyze the significance between them. A level of P < 0.05 was accepted as statistically significant.

Myopathy with severe weakness is one of the most serious side effects of glucocorticoid administration. It is known that glucocorticoids inhibit muscle

protein synthesis (Reiss and Kipnis, 1959; Shimizu and Kaplan, 1964; Kostyo and Redmond, 1966; Goldberg, 1969; Kovalenko et al., 1971; Hanoue et al., 1972; Shoji and Pennington, 1977). It has been suggested that a loss of muscle protein and dysfunction of sarcoplasmic reticulum might be the main causes of such muscle weakness (Shoji et al., 1976). Glucocorticoids induced the activity of gIutamine synthetase in skeletal muscle cell culture (Smith et al., 1984; Max et al., 1987). To examine the precise relationship between the induction of glutamine synthetase and the inhibition of protein synthesis in the skeletal muscle, I studied the effects of cortisone administration on the concentration of free amino acids in the serum, skeletal muscle, kidney, small intestine and liver of test and control rats. This appears to be the first systematic examination of free amino acids in the serum and various viscera in rats following the administration of glucocorticoids.

RESULTS Initially

the mean weights

of the control

Table 1. Total free amino acid concentration MATERIALS

and test

rats were 113 It 4 and 112 rt 3 g, respectively. After 7 days treatment (cortisone or vehicle), final body weights for control and test rats were 129 _t 4 and 100 f 2 g, respectively. During the experiment, control rats gained an average of 16 g while test rats lost an average of 12 g body wt. Total concentrations of free amino acids in serum or organs are shown in Table 1. Concentrations of amino acids were significantly decreased by cortisone admi~stration in serum, skeletal musle and the small intestine but not in liver or kidney. The ratio of glutamine to glutamate in serum and organs is shown in Table 2. The ratio exceeded



Serum Skeletal muscle Small intestine Kidney Liver

Female Wistar rats (110-12Og body wt) were fed a laboratory chow diet ad Mhm. Six test rats received cortisone acetate (10 mg/lOO g body wt/day) injected subcutaneously for 7 days while 6 control female Wistar rats received injections of the same volume of vehicle (0.9% benzyl alcohol) for 7 days, 24 hr following the last injection, rats were killed by a blow to the head and cervical dislocation. For detailed study, blood, gastrocnemius muscle, kidney, small intestine and liver were removed from each animal. Tissues were homogenized in 9 vol of 0.25 M saccharose. After centrifugation at 1OOOgfor 5 min, and the supernatant was separated. Blood was centrifuged and the serum was removed. The supematant and serum were then deproteinized with sulfo~~icylic acid at the concentration of 4%. After deproteinization the supematant was used for free amino acid analysis. Measurement of amino acids was done using an amino acid autoanalyzer Hitachi Model 835 (Hitachi Manufacturing Co., Tokyo). The concentrations of each amino acid was expressed as nmoles per ml. Per-

4,99+0.11 39.0 + 0.8 109.9 + 3.5 94.8 + 2.0 78.6 i: I .7

in serum and organs Test 4.34 + 0.12’ 34.8 + 0.8, 91.9 +#3.4’ 94.5 i_ 2.1 71.64 1.6

Unit is q~oljml for serum and pmol/g wet weight for other organs. Mean i SEM. lP < 0.05 “S controi. No. of samples: six for each value.

Table 2. Glutamine to glutamate ratio in serum and organs Control Serum Skeletal muscle Small intestine Kidney Liver

0.39 4 0.04 3.2 + 0.2 0.16*0.02 0.08 + 0.01 0.43 f 0.05

Mean k SEM. lP < 0.05 “S control. No. of samples: six for each value.


Test 0.53 4.5 0.24 0.07 0.63

+ 0.04 IO.2’ i 0.02 & 0.01 + 0.06



1.0 only in the skeletal muscle, and only this tissue in cortisone-treated rats differed from controls (P < 0.05). No significant differences were seen in those values measured in serum, small intestine, kidney or liver. The profile of free amino acids in serum, skeletal muscle, small intestine, kidney and liver are shown in Figs l-5 for the control and treated animals. Levels of such branched amino acids as valine, isoleucine and leucine increased in the serum (Fig. 1) and small intestine (Fig. 3). Glycine levels decreased in serum following glucocorticoid administration (Fig 1). In-


creased concentrations of alanine were found in the kidney (Fig. 4) and the liver (Fig. 5). DISCUSSION

Levels of branched amino acids increased in the serum (Fig. I), and small intestine (Fig. 3). Increased levels of valine or isoleucine in serum were previously reported (Rannels and Jefferson, 1980) suggesting the breakdown of protein occurs in peripheral organs. It has been reported that cortisone may induce glutamine synthetase in skeletal muscle (King et cd..

Fig. I. Profile of free amino acids in serum. The vertical axis represents the percentage of the amino acid mole concentration. Each amino acid has two columns; the left represents the mean control value while the right the mean test value. Each group consisted of six rats. The height of each column indicates the mean value. The bar above the column shows the SEM. An asterisk indicates a significant difference between the control and test groups (P < 0.05).



Fig. 2. Profile of free amino acids in skeletal muscle. Same as Fig. 1 legend

Steroid and amino acids

1983; Smith et al., 1984; Max et al., 1987; Tischler et al., 1988). In the present study, the glutamine-toglutamate ratio increased significantly in the skeletal muscle of the test rats (Table 2) suggesting that exogenous cortisone increased the activity of glutamate synthetase (Jaspers et al., 1986). Glutamine is synthesized de nouo from carbon chains of glutamate, valine, isoleucine, aspartate and aspargine and amino groups from many amino acids, and is released into the blood (Ruderman and Berger, 1974; Garber et al., 1976; Caldwell et al., 1978; Chang and Goldberg, 1978b; Goldberg and Chang, 1978; Muhlbacher et al., 1984). Glutamine is taken up by the intestine and the kidney (Matsutaka et al., 1973; Windmueller and Spaeth 1974, 1975; Souba et al., 1985; Welbourne, 1988) where it is converted to alanine and serine



(Matsutaka et al., 1973; Windmueller and Spaeth, 1974, 1975; Souba et al., 1983, 1985). As previously reported, glycine levels decreased in serum following glucocorticoid administration (Ryan and Carver 1963; Rannels and Jefferson, 1980). This suggests that glycine is taken up by the kidney and is used for synthesizing serine from glutamine. Increased concentrations of alanine were found in the kidney (Fig. 4) suggesting active alanine synthesis. Alanine is taken up by the liver (Souba et al., 1983) and it is used as a main substrate for gluconeogenesis and conversion into urea (Feilig et al., 1970; Feilig, 1973; Sapir et al., 1977; Goldberg and Chang, 1978). Our finding of increased tendency of alanine levels in the liver further supports this active hepatic uptake.


Fig. 3. Profile of free amino acids in small intestine. Same as Fig. 1 legend.

Fig. 4. Profile of free amino acids in kidney. Same as Fig. 1 legend.



Fig. 5. Profile of free amino acids in liver. Same as Fig. I legend

Glucose synthesized in the liver is used to maintain the blood glucose level. Blood glucose is taken up by the skeleta1 muscle. Pyruvate is generated from glycolysis using glucose; alanine is produced from the pyruvate and amino groups from many amino acids ~~uderman and Berger, 1974; Garber cut al.. 1976; Karl ef ui., 1976; Spydevold, 1976; Snell and Duff, 1977; Caldwell ef ul,, 1978; Chang and Goldberg, 1978a) and is released into the blood. Alanine and glutamine synthesis in the skeletal muscle utilize glutamate as a common substrate. In the presence of excess glucocorticoid, glutamine synthetase is induced and carbon and nitrogen are both diverted from afanine formation toward glutamine synthesis. Therefore, with glucocorticoid excess, mainly glutamine synthesis increased in skeletal muscle. Glutamine is converted mainly into alaninc in the small intestine and the kidney. Afanine is used for gluconeogenes~s and urea synthesis in the liver. The overall metabolic result of excess giucocorticoid is the breakdown of protein in the peripheral

Glucose -.--+


organs. especially in skeletal muscle, while the synthesis of glucose, giycogen and urea takes place in the liver as described above. This study suggests that the administration of cortisone acetate activates part uf the Row as the amino acids illustrated in Fig. 6, which shows the glucose-alanine cycle and the flow of other amino acids between Ever, skeletat muscle, small intestine and kidney. With exogenous cortisone administration, the amino acid pool of skeletal muscle decreased gradually much as water seeps from a leaky container, mainly as glutamine. Protein equilibrates dynamically with the ammo acid pool. The synthesis of protein is inhibited by the decrease of the amino acid poo! in skeletal muscle, which may be one of the main causes of the decreased skeletal muscle protein synthesis observed in steroid myopathy. Ark-nou/cd~L~~~enrs-This study was supported by Grant No: 62-2 from National center of N&ology anh Psvchiatrv 1NCNPj of the Minis&v of Health and Welfare. Jaian a&i a grant’from the Shimabara Science Promotion Foundation.


I i


SerinesKidney Fig. 6. Amino acid flow between organs. Illustrated is the gln~ose-alan~ne cycle between the liver and the skeletal muscle, glutamine release from the skeletal muscle and the conversion of glutamine to alanine in the small intestine and the kidney. The arrow shows the primary flow during excess glucocorticoid administration.

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