The effect of cortisone on protein breakdown and synthesis in rat skeletal muscle

Molecular and Cellular Endocrinology, 6 (1977) 159-169 0 ElsevierjNorth-Holland Scientific Publishers Ltd.

THE EFFECT OF CORTISONE ON PROTEIN BREAKDOWN AND SYNTHESIS IN RAT SKELETAL MUSCLE Shin’ichi

SHOJI and Ronald J.T. PENNINGTON

Regional Neurological Centre, Newcastle General Hospital, Newcastle upon Tyne, NE4 6BE, U.K. Received

22 March 1976; accepted

12 May 1976

The effect of cortisone administration on the rates of muscle protein breakdown and synthesis has been studied in the rat extensor digitorum longus muscle. Cortisone acetate (100 mg/kg body weight/day) was administered intraperitoneally for 1-3 days. Muscle weight and protein content were significantly reduced by cortisone administration. Rates of protein breakdown were measured by tyrosine release from the isolated musdle into the intracellular pool and medium during a 2-h incubation with cycloheximide to block protein synthesis. Rates of protein synthesis were assayed by [ 14C] tyrosine incorporation into protein of the isolated muscle during a 2-h incubation. Cortisone administration inhibited significantly the rate of protein synthesis after l-3 days treatment and also reduced significantly the rate of protein breakdown per muscle after 3 days treatment. The synthesis of myofibrillar and soluble proteins was affected to the same extent. These results strongly suggest that the effect of cortisone administration on muscle protein is mainly through its inhibition of protein synthesis rather than through an acceleration of protein breakdown. Keywords:

tyrosine incorporation steroid myopathy.

; cortisone

administration;

muscle

protein

turnover;

Steroid myopathy, involving the loss of muscle protein, is one of the major side effects of glucocorticoids, which are now widely used in clinical practice. A myopathic condition is also a feature of Cushing’s syndrome, a condition characterized by increased secretion of cortisol. Since Long et al. (1940) showed that administration of adrenal cortical extract to rats caused a loss of nitrogen in the urine, the net protein catabolic effect of glucocorticoids has been well documented. Increases in the levels of free amino acids in muscle and in plasma of animals receiving glucocorticoids have also been reported (Friedberg and Greenberg, 1947; Kaplan and Shimizu, 1963; Ryan and Carver, 1963; Betheil et al., 1965). Muscle proteins, like those of most tissues, are in a state of continuous turnover. The measured average half-life of muscle proteins using isotopic techniques is only a few days when errors arising from the re-utilization of labelled amino acids are minimized (Millward, 1970). Hence the rapid net loss of muscle protein by glucocorticoids could be a result of a depression of protein synthesis or an acceleration of protein catabolism, or both. 159

S. Shoji, R.J. T. Pennington

160

Several workers (Manchester et al., 1959; Wool and Weinshelbaum, 1960a,b; Shimizu and Kaplan, 1964), using isolated diaphragms, have shown that there was a reduction in the incorporation of labelled amino acids (glycine, histidine, phenylalanine and methionine) into muscle protein in rats which were given cortisone or cortisol. Kostyo and Redmond (1966) reported that corticosterone added in vitro to diaphragms could produce a similar effect. On the other hand, De Loecker (1966) claimed that in vitro addition of relatively low levels of cortisol stimulated the incorporation of labelled leucine and lysine into the protein of adductor muscles of rat hind limbs; he suggested that increased protein breakdown might, therefore, be the main cause of glucocorticoid-induced muscle atrophy. Goldberg (1969) administered cortisone acetate for 7 days to rats in which the muscle proteins had been labelled with [3H]leucine. Although the hormone decreased the total amount of labelled proteins in the muscle (plantaris) it did not alter their specific activity. It was concluded, therefore, that cortisone decreased protein synthesis. Hanoune et al. (1972) found that cortisone administration to fasted rats led, 5 h later, to a 43% decrease in the incorporation of administered [ r4C] leucine into muscle proteins. Experiments with isolated muscle ribosomes have also provided evidence that glucocorticoids can disturb protein synthesis. Bullock et al. (1968, 1971) reported that ribosomes from muscle of rats injected with various glucocorticoids showed a decreased ability to incorporate amino acids into protein. Subsequently (Bullock et al., 1972) they obtained evidence that this decrease was associated with a decreased proportion of muscle polyribosomes. Young et al. (1968) also found a decrease in the proportion of muscle polyribosomes after cortisol administration; the effect was observed within 4 h after giving the steroid. Less attention has been paid to the possible effect of glucocorticoids upon muscle protein catabolism. An increase in peptidase activity of diaphragm muscle after giving cortisone to rats was reported by Schwartz et al. (1965) and more recently Mayer et al. (1974) found that the rate of autolysis of a rat muscle myofibril preparation was increased by 60% after treatment for 3 days with triamcinolone. The increased loss of labelled protein from muscle of cortisone-treated rats found by Goldberg (1969) was taken to show that cortisone increased protein catabolism. We have attempted to assess the relative importance of changes in protein synthesis and breakdown in glucocorticoid-induced atrophy by measuring the rates of each in whole extensor digitorum longus muscles incubated in vitro. The extensor digitorum longus is a ‘white’ muscle, having a predominance of Type II fibres. One of the histochemical characteristics of steroid myopathy is a preferential atrophy of Type II fibres. Furthermore, Goldberg and Goodman (1969) showed that white muscles of rats were affected more than the red soleus muscle by cortisone. MATERIALS

AND METHODS

Animals and treatment

All experiments

involved

the use of young female rats (Wistar, 40-60

g) ob-

tained from Bantin and Kingman Ltd., Hull, U.K. Animals were fed on a laboratory chow diet (Diet 41B, Oxoid Laboratory, U.K.) and were divided into pairs according to body weight. One rat of each pair received 100 mg of cortisone acetate per kg of initial body weight intraperitoneally each day for l-3 days, as a suspension (100 mg/6 ml) in 0.9% NaCl solution. The control animals recieved an equal volume of 0.9% NaCl solution. Twenty-four hours after the last injection, the animals were killed by cervical dislocation and the extensor digitorum longus muscles (EDL) rapidly dissected out. The net weight of the muscles was determined rapidly on a torsion balance. Mea~u~e~e~t of protein breakdown l%e rate of protein breakdown was measured in these muscles according to the method used for rat diaphragm by Fulks et al. (1975). In this procedure the rate of protein breakdown is determined by measuring the release of tyrosine into intracellular amino acid pools and the surrounding medium, in the presence of cycloheximide to block protein synthesis and thus prevent re-utilization of the tyrosine released by protein breakdown. Tyrosine is suitable for such studies, since it is neither synthesized nor degraded by muscle (Guroff and Udenfriend, 1960). The muscle was shaken gently for 2 h at 37°C in a medium containing KrebsRinger bicarbonate, pH 7.4, glucose (10 mM) and cycloheximide (0.5 mM), and saturated with 95% 02[.5% COZ. Following the incubation, the muscle was homogenized, the homogenate and medium treated with trichloroacetic acid (TCA) and the acid-soluble supernatants assayed for tyrosine by the fluorimet~c method of Waalkes and Udenfriend (1957), as described by Fulks et al. (1975). The muscle of the opposite leg was used to measure the initial tyrosine content. Measurement of protein synthesis This was determined according to Fulks et al. (1975) by measuring the incorporation of tyrosine into muscle protein during a 2-h incubation in 3 ml of KrebsRinger bicarbonate, containing glucose (10 mM), 0.05 PCi L-[U-14C]tyrosine (495 Ci/mol; Radiochemical Centre, Amersham, U.K.), L-tyrosine (0.1 mM) and L-valine, L-leucine and L-isoleucine in concentrations similar to those in rat plasma (Morgan et al., 1971). The three branched-chain amino acids together stimulate protein synthesis and reduce breakdown in isolated d~apllragm, while the rem~ning plasma amino acids do not affect either process signi~canuy (Fulks et al., 1975). For measurements of radioactivity in protein, the acid-precipitated material was washed twice with 5% TCA and once with ethanol-ether (1 : 1). The pellets were dissolved in 0.5 ml of ‘Soluene-350’ (Packard Instrument Ltd., Caversham, Berks., U.K.) and were added to 10 ml of the scintillation fluid (250 ml Triton X-l 14,3 g 2,5diphenyloxazole, 5 g benzoic acid, made up to 1 1 with xylene). The acid-soluble radioactivity in the muscle homogenate and in the medium were determined with 0.5 ml aliquots added to 10 ml of the scintillation fluid. Calculation of the tyrosine incorporated was made by dividing the incorporated

162

S. Shoji, R.J.T. Pennington

[14C] tyrosine counts by the specific radioactivity of the intracellular pool. The specific radioactivity (cpm/nmol) of the intracellular tyrosine pool was calculated using the following formula: acid soluble cpm-cpm in inulin space nmoles acid-soluble tyr-nmol tyr in inulin space The inulin space was measured by incubating muscles in 3 ml of Krebs-Ringer bicarbonate containing 0.09 PCi [ 14COOH]inulin (2.51 &i/mg), Radiochemical Centre, Amersham, U.K.) and inulin (1.2 PM). At the end of a 2-h incubation, the [14C]inulin content per mg of muscle was found to be equal to the [14C]inulin in 0.178 + 0.005 fi (S.E.) of the medium in control muscles, 0.214 + 0.017 ~1 in test muscles. Measurement of relative rates of synthesis of myofibrillar and soluble proteins To measure relative rates of synthesis of myotibrillar and soluble proteins, 6 test (l-day treatment) and 6 control EDL muscles were individually incubated in the medium used for the measurement of protein synthesis. After a 2-h incubation, the muscles in each group were pooled, and centrifuged (600 g, 10 min) to sediment the myofibrils. The supernatant was centrifuged at 40,000 g for 30 min to obtain the soluble proteins and a pellet consisting largely of mitochondria and fragmented sarcoplasmic reticulum. The fractions were treated with TCA and washed as described above. Muscle protein was measured by the method of Lowry et al. (195 1) using bovine serum albumin as a standard, after dissolving the TCA precipitates in 0.1 N NaOH. All statistical analysis was done using the Student’s t test on the paired data. Muscle potassium was measured in the TCA-soluble fraction of the muscle, using a flame photometer. RESULTS These are shown in taChanges in body weight and muscle weight and protein ble 1. Test animals showed a significant decline of gain in body weight after l-3 days treatment. The wet weight of the EDL muscle was significantly decreased in the test animals compared to the controls. The protein content of the whole EDL muscle was significantly iess after 3 days of treatment, although the concentration of protein in the muscle was increased. There were no significant Food consumption eaten by the control and test rats.

differences

in the amount

of food

The potassium content of the incuPotassium content of incubated muscles bated muscles was found to be the same or slightly less than that of the non-incu-

Protein turnover in steroid myopathy

163

Table 1 Effect of cortisone on changes in body weight and muscle weight and protein. Growing female rats daily received cortisone acetate (100 mg/kg of initial body weight) (Test) or 0.9% NaCl solution (Control), intraperitoneally for 1 to 3 days. Animals were sacrificed 24 h after the last injection. All values are mean f S.E. of 6 rats. Statistical analysis of the differences between test and control was done using Student’s paired t test. Duration

of treatment

(days) 2

1

3

Initial body wt. (g) Control Test

52.1 52.0

* 1.8 + 2.0

43.3 43.6

+ 1.2 f 1.0

42.0 42.2

f +

0.6 0.7

55.4 53.0

t 2.0 + 2.1 ***

50.9 44.9

f 1.2 f 0.7 ***

47.6 43.3

* f

0.6 0.7 ***

21.9 20.5

f 1.2 * 1.1 *

19.9 16.5

f 0.3 f 0.3 ***

20.9 16.9

+ 0.8 ?: 0.7 ***

Final body wt. (g) Control Test EDL wet wt. (mg) Control Test EDL wet wt./final

body wt. (o/oO) 0.393 0.387

Control Test Total protein

4.02 3.90

concentration

Control Test

0.392 0.368

i- 0.012 * 0.011

0.438 0.389

* *

0.012 0.013

f 0.29 f 0.26

3.59 3.10

f 0.17 f 0.14

3.71 3.25

c +

0.27 0.28 ***

***

(mg) in EDL

Control Test Protein

c 0.009 t 0.007

(mg/g wet wt.) in EDL

184 190

180 188

f 7 + 7

+7 *8

177 192

+ 7 ? 11** -

*P < 0.05; **p

< 0.01; ***p < 0.005.

bated muscles from the opposite ,uequiv/g wet weight. Rates of protein

side. All incubated

muscles had more than 70

breakdown Tyrosine release per whole muscle was not significantly altered after 1 or 2 days treatment with cortisone but was reduced after 3 days (table 2). When related to wet weight or muscle protein, there were no significant differences between the test and control groups.

164

S. Shoji, R.J. T. Pmnirzgion

Table 2 Effect of cortisone upon rate of protein breakdown by EDL muscie. All values are mean f S.E. of 6 rats. Group

Duration oi’ treatment (days)

Tyrosine released (nmol/2 h) Per whole muscle

Per mg wet wt.

Per mg protein

1

Control Test

6.71 2 0.29 6.34 -I:0.64

0.309 + 0.009 0.307 + 0.024

1.69 + 0.07 1.61 1: 0.08

2

Control Test

6.13 i 0.18 6.51 f 0.58

0.309 s 0.012 0.395 f 0.034

1.73 * 0.09 2.10 * 0.16

3

Control Test

6.06 F 0.23 5.21 + 0.09 *

0.292 r 0.015 0.313 + 0.018

1.67 t 0.13 1.66 ?z0.14 --

*P < 0.01.

Rates of protein synthesis The calculation of the amount of tyrosine incorporated into protein assumes that the specific radioactivity of the intracellular tyrosine is constant during the incubation. Li et al. (1973) showed that, in the case of diaphragm, the specific radioactivity reached a constant value after 20 min, hence the error for a 2-h incubation time would be relatively small. We measured the specific radioactivity of the intracellular tyrosine of the EDL after various times of incuhation and the results are shown in fig. 1. maximum specific radioactivity was reached after about 30 min using muscle from either normal or cortisone-treated

I 0

_I

30

60

90

120

MIN.

Fig. 1. Specific radioactivity of intracellular tyrosine in muscles incubated for varying periods in a medium containing [ 14C,3tyrosine. 0, muscle from cortisone-treated rats; o, muscle from control rats.

165

Protein turnover in steroid myopathy

Table 3 Effect of cortisone amount of tyrosine

upon rate of protein synthesis by EDL muscle. The calculations incorporated into the muscle protein were based upon the specific

tivity of the intracellular Duration

free tyrosine,

of treatment

Group

as described

of the radioac-

in the text. Values are mean + S.E.

[ ’ 4C] Tyrosine

incorporated

(nmol/2 h)

and (number of pairs) Per whole muscle

Per mg wet wt.

1 day (7)

Control Test

4.12 t 0.06 2.68 f 0.18 **

0.244 + 0.005 0.174 k 0.009 **

2 days (6)

Control Test

5.23 + 0.39 2.21 f 0.24 **

0.241 k 0.016 0.134 A 0.019 *

3 days (7)

Control Test

5.38 + 0.35 2.49 f 0.11 **

0.234 0.135

*P < 0.025;

**

k 0.010 + 0.007

**

P < 0.005.

Thus the amount of tyrosine incorporated during 2 h would be slightly (about 10%) underestimated. However, the relative incorporation in the test and control muscles would be unaltered. Tyrosine incorporation into muscle protein was significantly inhibited in the test muscles after l-3 days treatment, when expressed in terms of wet weight (table 3); when calculated per whole muscle, the difference was significant at 2 and 3 days. As explained above, the calculation of the amount of tyrosine incorporated was rats.

Table 4 Rates of incorporation of tyrosine into muscle protein calculated on the assumption that extracellular tyrosine is directly incorporated. In most cases they were derived from the same experiments as the results in table 3. Values are mean c S.E. Duration of treatment and (number of pairs)

Group

[ 14C]Tyrosine Per whole

1

day (7)

2 days (5)

3 days (9)

*P < 0.025:

**p

<

0.005.

incorporated

muscle

(nmol/2

h)

Per mg wet wt.

Control Test

2.31 f 0.07 1.39 f 0.06 **

0.119 0.090

i: 0.003 * 0.002

**

Control Test

2.52 + 0.07 1.41 f 0.05 **

0.118 0.087

f 0.004 + 0.002

*

Control Test

3.13 f 0.09 1.56 + 0.06 **

0.131 0.083

+ 0.007 + 0.005

**

Table 5 Relative rates of protein synthesis in various muscle fractions, in control and cortisone-treated rats. Following incubation of the muscles with [ r4C] tyrosine, the muscle were fractionated as described ‘and the radioactivity measured in each fraction. In each experiment, 3 pairs of rats were used and the muscles combined for fractionation and measurement of radioactivity. Exp. No.

Group

Radioa~t~ity in fraction (%l 600g pellet

40,000 g pellet

40,000 g supernatant

I

Control Test

69.3 70.8

8.1 10.4

22.0 18.8

II

Control Test

75.8 67.9

6.0 11.9

18.2 20.2

based on the assumption that intracellular free tyrosine serves as the precursor for protein synthesis in muscle. Evidence that this is so was provided by Li et al. (1973). However, other researchers (Hider et al., 1969) have claimed that amino acids are incorporated into protein directly from the extracellular pool. In view of this, the results were also calculated using the specific radioactivity of the tyrosine in the incubation medium (table 4). A significant effect of cortisone at all time periods was again evident. Table 5 shows that cortisone inhibited protein synthesis to the same extent in the myoflbrillar and soluble proteins.

DISCUSSION The experiments reported in table 2 provide no support for the idea that glucocorticoids increase the catabolic rate of muscle protein. In fact, a significant reduction in total breakdown occurred after 3 days of cortisone administration, which may be a reflection of the decreased muscle weight. It seems likely, therefore, that gluco~orticoid-induced muscle wasting is primarily or wholly a result of interference with protein synthesis. In vivo protein turnover studies (Goldberg, 1969) which indicated an increased breakdown of muscle protein after cortisone treatment are subject to error due to re-utilization of the labelled amino acids derived from protein breakdown. Reutilization causes the rate of protein breakdown to be underestimated. It has been shown (Odessy and Goldberg, 1972; Ryan et al., 1974) that leucine, the labelled amino acid used by Goldberg, is readily oxidized by skeleta1 muscle and this oxidation is more rapid after treatment with ~uco~orticoid. Hence the reutilization of labelled leucine may be less in steroid-treated animals. The marked increase in the rate of autolytic breakdown of muscle myofibril

Protein turnover in steroid myopathy

167

preparations from rats treated with triamcinolone (Mayer et al., 1974) is difficult to interpret. Evidence has been obtained (Park et al., 1973) that the enzyme in muscle homogenates which is responsible for such breakdown may be a constituent of mast cells in the muscle and may therefore be of doubtful importance in the catabolism of muscle protein in vivo. The decline in the rate of muscle protein synthesis after giving cortisone substantiates the previous reports (see Introduction) that glucocorticoids cause a decline in amino acid incorporation by isolated diaphragms. It may be pointed out, however, that except in the report of Shimizu and Kaplan (1964) the results were expressed simply as the radioactivity incorporated into muscle protein; the specific radioactivity of the intracellular amino acid was not measured. The experiments did not, therefore, take account of any possible differences in the rate of transport of the labelled amino acids into the muscle tibres. However, our own results (fig. 1) show that, for tyrosine at least, there is no difference in this respect in the steroid-treated animals. The mechanism through which glucocorticoids inhibit protein synthesis in muscle still remains to be elucidated. In muscle, protein turnover is influenced by nutritional status (Munro, 1964) but Goodlad and Munro (1959) reported that the catabolic action of cortisone on the body as a whole was independent of the protein and energy content of the diet. In our experiments food consumption was not decreased during the period of cortisone administration. The possibility that the primary site of action of glucocorticoids is on the rate of transfer of amino acids from the extracellular compartment into the cell interior has been suggested. Transport of several natural amino acids and of a-aminoisobutyric acid into the extracellular pool of the isolated rat diaphragm was reduced after administration of cortisone to the animals (Wool, 1960). Hanoune et al. (1972) found a similar effect on the uptake of a-aminoisobutyric acid in vivo. Kostyo, (1965) reported that corticosteroids added in vitro inhibited the transport of aaminoisobutyric acid into rat diaphragms. However, Kostyo and Redmond (1966) found that in muscles in which protein synthesis is blocked by puromycin, corticosterone had no effect on amino acid uptake, and they suggested that this finding supports the idea that corticosterone inhibits amino acid transport in muscle through its inhibitory action on protein synthesis. Hanoune et al. (1972) obtained evidence that the effect of cortisone on muscle protein synthesis may be independent of amino acid penetration. The results of Shimizu and Kaplan (1964) and our own findings, in which the specific radioactivity of the intracellular amino acid was measured, confirm that there is an independent effect upon protein synthesis. Finally, the observation (Bullock et al., 1968; Young et al., 1968) that the glucocorticoid effect upon protein synthesis can be demonstrated with isolated ribosomes leads to the same conclusion. Evidence has been presented by Munck et al. (1972) that, in rat thymus cells, glucocorticoids inhibit glucose transport before affecting protein synthesis. It was postulated that cortisol stimulates the production of a specific protein that inhibits

168

S. Shoji, R.J. T. Pennington

glucose transport. The ability of glucocorticoids to induce the synthesis of specific proteins (e.g. tyrosine aminotransferase) is well known in liver. The possibility that this occurs in muscle and that the inhibition of glucose transport is responsible for a general inhibition of protein synthesis must therefore be considered. However, Munck (1971) concluded that glucocorticoids do not have a similar effect on glucose transport in muscle. On the other hand, Hanoune et al. (1972) reported that glucose completely inhibited the effect of cortisol on the incorporation of radioactive leucine into muscle protein, when administered before the steroid. However, the rapid oxidation of leucine by muscle throws doubt on the significance of this finding. Clearly more studies are required to decide whether the inhibitory effect of glucocorticoids on muscle protein synthesis is primary or secondary and to determine which step(s) of protein synthesis are affected. With regard to the latter problem, Ferguson and Wool (1962) reported that cortisone administration was without effect on the incorporation of 14C from adenine into RNA, suggesting a lack of effect on transcription of DNA, but little else has emerged in this field of study. An incidental observation in our work (table 1) was that the concentration of protein in the muscle was somewhat higher in the steroid-treated animals. This is presumably the effect of a more rapid loss of cell water (Kochakian and Robertson, 19.5 1) than of protein.

ACKNOWLEDGEMENTS The authors acknowledge the excellent technical assistance of Miss Penelope Cox in these investigations. This work was made possible by the financial support of the Wellcome Trust. It was supported also by the Medical Research Council.

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Protein furnover in steroid myopathy

169

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