Changes in protein turnover in skeletal muscle in response to fasting.

Changes in protein turnover in response to fasting JEANNE B. LI, JUDITH Department of Physiology, State University, Hershey,

E. HIGGINS, AND College of Medicine, 17033 Pennsylvania

LI, JEANNE B., JUDITH E. HIGGINS, AND LEONARD S. JEFFERSON. Changes in protein turnover in skeletal muscle in response to fasting. Am. J. Physiol. 236(3): E222-E228, 1979 or Am. J. Physiol.: Endocrinol. Metab. Gastrointest. Physiol. 5(3): E222-E228, 1979.-In response to a 72-h fast, rats weighing approximately 220 g initially exhibited a 28% reduction in total body weight and a 25% reduction in the weight of the gastrocnemius muscle. Reduced muscle weight was paralleled by a reduction in muscle protein, which, as determined in the perfused hemicorpus preparation, was the result of a 50% decrease in the rate of protein synthesis and an unchanged rate of protein degradation. During the first 24 h of fasting, a decrease in the level of plasma insulin was associated with a block in peptidechain initiation and a reduction in the efficiency of protein synthesis. Longer periods of fasting caused a decrease in the level of muscle RNA and a loss of protein synthetic capacity. Perfusion of muscle in the presence of insulin completely removed the block in peptide-chain initiation, restoring the efficiency of synthesis to normal, whereas the capacity of synthesis was unchanged. Although protein degradation in the 220-g rats was not altered by food deprivation, the degradative rate of 100-g rats fasted for 48 h was 30-50% greater than that observed in fed controls. The increased rate of degradation in these preparations was accompanied by an increase in the specific activity of a lysosomal protease, cathepsin D. Although addition of insulin lowered the rate of protein degradation in the perfused muscle preparation under all conditions tested, the increase in degradation in the fasted, 100-g rats could not be explained solely on the basis of acute insulin deficiency.

perfused hemicorpus; cathepsin D; insulin; units

protein synthesis; protein degradation; peptide-chain initiation; ribosomal sub-

during fasting by utilizing endogenous stores of glycogen, fatty acids, and protein. Because the major protein reserve of the body is found in skeletal muscle, a tissue that comprises 40-45% of the body weight, an essential adaptation to fasting is the mobilization of amino acids from muscle protein. The amino acids provided by the breakdown of muscle protein serve as precursors for gluconeogenesis in liver (6) and kidney (18), as substrates for oxidation in muscle (14), and in the synthesis of new proteins essential for adaptation to fasting. The mechanisms responsible for the net loss of muscle protein during fasting are not well understood. Evidence from in vivo (13) and in vitro (20, 29) studies indicates that protein synthesis is reduced in skeletal muscle in AN ANIMAL

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muscle

LEONARD S. JEFFERSON Pennsylvania

response to food deprivation. An increased rate of protein degradation could also contribute to the loss of muscle protein during fasting, but the evidence for this is not conclusive. Some studies (20, 26, 36) suggest that protein degradation increases in rats after 2-4 days of starvation, whereas others (37) suggest a decrease in the degradative process in obese human subjects during prolonged fasts of 3 wk duration. The role of hormones in the adaptation of skeletal muscle to fasting is also poorly defined. The decreased level of plasma insulin in the fasted state (4, 25) may be of primary importance because this hormone stimulates protein synthesis (11, 16)) inhibits protein degradation (11, 16), and reduces the net release of amino acids (11, 16, 30) in skeletal muscle. In the studies reported here an isolated perfused muscle preparation (15, 16) was used to investigate the response of rat skeletal muscle to fasting. Because this preparation is free of endocrine glands, it is less complex than the intact animal, and it is a more physiological preparation than isolated muscle. By using this preparation, it was possible to make direct and simultaneous measurements of both the rate of protein synthesis and the rate of protein degradation. These rates were related to actual changes in muscle weight. Alterations in synthetic rates were identified as changes in either the efficiency or capacity of synthesis. The role of insulin in the adaptation of muscle protein turnover to fasting was also evaluated. In addition, the activity of cathepsin D, a lysosomal protease, was correlated with rates of protein degradation. METHODS

Animals. Male Sprague-Dawley rats were obtained from Charles River Breeding Laboratories (Wilmington, MA). They were provided Wayne Lab Blox and water ad libitum and maintained on a 12-h-light-12-h-dark cycle for 2 wk prior to the beginning of a study. At the beginning of each study, rats were weighed at 1l:OO A.M. and divided into experimental groups. Fed rats were maintained as described above. Fasted rats were placed in separate wire-bottom cages and given free access to water. After 24 or 72 h of food deprivation, fasted rats and fed controls were either killed for analysis of muscle composition or used in perfusion experiments. Hemicorpus perfusion. Details of the perfusion technique were described previously (15. 16). The perfusate

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consisted of Krebs-Henseleit bicarbonate buffer, 3% bovine serum albumin (Pentex fraction V, Miles Laboratories), 15 mM glucose, normal plasma levels of amino acids (16) unless otherwise indicated, and sufficient washed bovine erythrocytes to give a hematocrit of 25%. It was gassed with 95% oxygen-5% carbon dioxide and maintained at 37OC. Bovine insulin (lot 615-D63-5, a gift from Eli Lilly Co.), cycloheximide (Sigma Chemical Co.), and L-[U-14C]phenylalanine (New England Nuclear Corp.) were added to the perfusate as indicated in the table and figure legends. The first 50 ml of perfusate to pass through the preparation were discarded. Then 100 ml of perfusate were recirculated at a flow rate of 7 ml/min for 100-g rats and 14 ml/min for 220-g rats. Estimates of rates of protein synthesis and degradation. Protein turnover measurements were made between 1 and 3 h of perfusion. At each of these times, a sample of perfusate was taken, and one gastrocnemius muscle was removed (without ligating vessels of the leg) and frozen for subsequent analyses. Protein synthesis was measured by determining the nanomoles of phenylalanine incorporated into protein when the perfusate contained 0.4 mM L-phenylalanine (5 times normal plasma levels) and 0.05 pCi/ml [U-14C]phenylalanine. The number of nanomoles incorporated was calculated by dividing the disintegrations per minute in protein by the average (l-3 h) intracellular specific activity of free [14C]phenylalanine. As described previously (16), phenylalanine is a suitable marker for determining protein turnover because it is neither synthesized nor degraded by this preparation. Furthermore, these concentrations of phenylalanine did not alter rates of protein turnover (unpublished observations) and ensured that the specific activity of phenylalanyl-tRNA, the immediate precursor of protein synthesis, was the same as that of the extracellular and intracellular pools of free phenylalanine (23). Protein degradation was measured under the same conditions by determining the dilution of [ 14C]phenylalanine specific activity by [12C]phenylalanine released from protein (16, 27). The specific activity decreased ZO-40% between 1 and 3 h. In some experiments, the release of [12C]phenylalanine was determined in the presence of 100 PM cycloheximide. Addition of cycloheximide inhibited protein synthesis by more than 95%, thus minimizing the reutilization of released phenylalanine. Rates of synthesis and degradation are presented as nanomoles phenylalanine incorporated per hour per gram gastrocnemius muscle and nanomoles phenylalanine released per hour per gram hemicorpus, respectively. Perfusate and tissue analyses. Powdered muscle samples and whole perfusate were extracted with trichloroacetic acid for determination of phenylalanine content and radioactivity (16). Phenylalanine in the acid soluble supernatants was assayed fluorometrically (7) using tissue blank corrections determined separately (1). Acid soluble radioactivity was determined by liquid spectrometry using a Beckman LS-150 counter and NEN-947 scintillation fluid. Corrections for quenching were made with an external standard. Incorporation of [14C]phenylalanine into total protein was determined by isolating the protein (27)) solubilizing it in NaOH, and determining the amount of radioactivity as above.

Ribosomal subunits in psoas muscle homogenates were separated by sucrose density centrifugation (28), and the RNA content of fractions containing 60 S and 40 S particles was quantitated by alkaline hydrolysis (10). Psoas muscle was used in these studies to allow ribosomal subunit levels to be measured in the same hemicorpus preparation as protein synthesis. Psoas muscle is similar to gastrocnemius in its rate of protein synthesis (16) and its degree of ribosomal aggregation under different conditions in vivo and during perfusion (unpublished observations). RNA and DNA phosphorous (RNA-P and DNA-P) content of muscle were determined by the method of Manchester and Harris (21). One microgram RNA-P was equivalent to 10 lug RNA. Protein content was measured by the biuret method (19) using bovine serum albumin as a standard. Lysosomal cathepsin D activity was assayed in homogenates of psoas muscle prepared as described previously (9,16). Homogenates were divided and 10% Triton X-100 was added to one aliquot to give a final concentration of 0.2%. After 10 min in an ice bath, the homogenates were centrifuged at 15,000 rpm in a Sorvall RC-2B for 10 min. Cathepsin D activity in supernatants was assayed with 1% [ 14C]acetylated hemoglobin as substrate. Incubations were carried out for 30 min at 37OC in 0.1 M citrate buffer, pH 3.0 (9). Total activity represented the activity in the supernatant treated with Triton, whereas nonsedimentable activity represented the activity in the untreated supernatant. The specific activity of [ 14C]acetylated hemoglobin was used to calculate the micrograms of hemoglobin broken down to acid soluble products. Results are presented as the mean t 1 SE of the mean. Significance was tested using the two-tailed Student’s t test. A P value less than 0.05 was considered significant. RESULTS

Effects of fasting on body weight, muscle weight, muscle composition, and plasma insulin levels. The overall effects of food deprivation were evaluated by monitoring body, carcass, and gastrocnemius muscle weights during fasting (Table 1). Fed rats exhibited a 9% increase in body weight during the 72-h experimental period, whereas rats fasted for 24 h lost 14% of their initial body weight and those fasted for 72 h lost 28%. The weight of the eviscerated carcass, which was not influenced by variations in the food content of the gastrointestinal tract, decreased by 12 and 26%, respectively, after 24 and 72 h of fasting. The weight of the gastrocnemius, a muscle with mixed fiber types, decreased in parallel to the reduction in carcass weight. The DNA, protein, and RNA components of the gastrocnemius were measured to determine their relative proportions in muscle from fed and fasted rats (Table 1). The DNA content per total gastrocnemius muscle remained unchanged throughout the period of food deprivation, demonstrating that muscle atrophy was due to a decrease in cell components and not to a reduction in the number of cells. Protein and RNA concentrations decreased at rates proportional to or greater than the reduction in muscle weight.


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1. Effect of fasting on body and muscle weights, muscle composition, and plasma insulin concentration ---______

the absence of insulin. These results indicated that measurements of protein turnover in the perfused hemicorpus preparation closely approximated actual rates of turnover in vivo in both the fed and fasted state. Fasted Changes in protein synthesis in perfused skeletal musFed 24 h 72 h cle in response to fasting. The net changes in free phenylalanine noted above could have resulted from alteraInitial body wt, g 22Ok 4 2202 7" 219t 4" 239 t, qbTc 19Ok 6" 158t 3" Final body wt, g tions in the rate of protein synthesis or degradation. 169t_ 3 149 z!z4" 125 -+ 3" Carcass wt, g Changes in synthesis rates in response to fasting are illustrated in Table 3. When insulin was omitted from 1.04 t 0.03 0.91 zk 0.03 0.78 t, 0.03d Muscle weight, g the perfusion medium, synthesis rates in muscle of rats DNA-P, pg/muscle 45t 2 45t, 2 45t 3 fasted for 24 and 72 h were reduced to 60 and 44% of the Protein, mg/muscle 202t 7 18Ok 6d 155 Ifr 7" RNA-P, pg/muscle 12Ok 6 972 5d 68t 5d fed control rate, respectively. Addition of insulin to the perfusion medium increased the rate of synthesis by 20% Plasma insulin, pU/ml 36 Z!I5 16 zk 3" 15t, 3" in the fed control, 88% in the 24-h fasted control, and Values are means 2 SE of 5-12 determinations. Rats weighing about 100% in the 72-h fasted control. The insulin stimulated 220 g were divided into 3 groups. One group was maintained on food for rates were the same in preparations from fed and 24-h 72 h, a second group was fasted for 24 h, and a third group was fasted fasted rats, but were lower after 72 h of fasting. When for 72 h. Rats were killed and the whole-body and eviscerated-carcass weights were obtained. The gastrocnemius muscles were dissected out, rates of protein synthesis were expressed on the basis of weighed, and analyzed for protein, RNA, and DNA content. Insulin the amount of RNA in the muscle, rates determined in was determined by radioimmunoassay (Pharmacia Fine Chemithe absence of insulin still showed decreases in synthesis a Body weight immediately before removal of food from fasted cals) . in response to fasting (Table 3). However, in the presence b rats were fed for 72 h; ’ P < 0.01 vs. initial weight or rats; dP < 0.025 vs. fed; "P c 0.005 vs. of insulin, rates of synthesis per unit of RNA were not weight of pair-fed controls; fed. significantly different. The stimulation of protein synthesis per unit of RNA by insulin indicated an increase in the efficiency of the The plasma concentration of insulin, a hormone known to be important in the regulation of protein levels in synthetic process. It was previously shown that the lack muscle (4), decreased precipitously after a 24-h fast and of insulin leads to the formation of a block in peptideremained at a low level (42% of the fed control) after 72 chain initiation in perfused skeletal muscle from fed rats (16). The block in initiation results in a lowered rate of h of fasting. protein synthesis, a disaggregation of polysomes, and an Correlation of changes in muscle weight in vivo with net protein turnover in perfused hemicorpus. Changes in accumulation of ribosomal subunits. To determine whether a block in initiation developed in muscles of muscle protein turnover in response to fasting and the fasted rats in vivo or during perfusion, the level of riborole of insulin in mediating these changes were further assessedin the perfused hemicorpus, a preparation con- somal subunits was measured (Fig. 1). After a 72-h fast, sisting mostly of skeletal muscle (15). Net changes in protein turnover were measured by the net changes in TABLE 2. Correlation of net protein turnover free phenylalanine content of the perfusate and the hem- in perfused hemicorpus with fractional icorpus preparation. A net release of phenylalanine oc- changes in carcass weight in vivo ~---~__--____---_____-___ curred when hemicorpus preparations were perfused in Calculated FracObserved the absence of insulin, indicating that the rate of muscle Net Change in Free Phenyltional Change in Fraction alanine Carcass Weight protein degradation exceeded the rate of protein syntheChange in Carca+TvoW t in sis (Table 2). Previous studies (16) demonstrated that -Insulin +Insulin -Insulin +Insulin muscle protein turnover in hemicorpus preparations from nmol/h per g A%,/day AS/day fed rats becomes insulin dependent after about 1 h of Fed +47-+4 -23k 7" -3.7 +1.8 +2.8 perfusion. The data in Table 2 confirm the previous observation and show that net phenylalanine release in Fasted 24 h +59 t, 9 -3k3.t -4.6 +0.2 -4.5 the absence of insulin was increased in response to fast+84 & 6' 34 t 6**t -6.6 -2.6 -6.6 ing. When preparations were perfused in the presence of Fasted 72 h insulin, there was a net uptake of phenylalanine in fed Values are means & SE of 4-12 determinations. Positive (+) values preparations, no change in 24-h fasted preparations, and represent release; negative (-) values represent uptake. Rats weighing 220 g were fed or deprived of food for 24 or 72 h. Hemicorpus a net release in 72-h fasted preparations. Taking the about preparations were then perfused for 180 min in the presence or absence observed release (or uptake) of phenylalanine to repre- of insulin (25 mu/ml) as indicated. Phenylalanine content of gastrosent the net change in protein turnover and using ‘the cnemius muscle and perfusate was measured at I and 3 h. The eviscerpreparation were considered to be known values for the phenylalanine content of muscle ated carcass and the hemicorpus of muscle. The fractional change in weight protein and the amount of protein per gram of muscle, it equivalent in the proportion was calculated from net phenylalanine released from the perfused was possible to calculate the fractional changes in carcass hemicorpus weight per day (AS/day). These values were in agree(-Anmol phe/h per g) x 24 h x 100 ment with the observed in vivo fractional rates of growth A%/day = (175 nmol phe/mg protein) x (175 mg protein/g) or atrophy when the perfusion conditions resembled the in vivo situation, i.e., fed preparations perfused in the * P < 0.005 vs. -insulin; t P c 0.001 vs. fed (same perfusion connresence of insulin and fasted preparations perfused in dition) . TABLE


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3. Changes in protein synthesis in perfused skeletal muscle in response to fasting ~_-~. ____-___-- _____--_________ --p----p-

TABLE

Phenylalanine

Incorporation

Expt Control

+Insulin nmol/h

I

Fed Fasted Fasted

24 h 72 h

55 f; 3 (14) 33-+ 4 (7)" 24 I?I2 (21)*

24 h

0.398 t 0.021 0.278 z!z0.036* 0.244 t 0.016"

nmol/h

II

Fed Fasted Fasted

72 h

per g muscle

66 * 2 (15)-j62 & 4 (9)t 48 t 2 (12)"qper M RNA-P

0.481 t, 0.012t 0.522 z!z0.030.f 0.501 Ik 0.021-t

Values are means t SE for the number of determinations indicated in parentheses. Hemicorpus preparations were perfused as described in Table 2. The perfusion medium contained 0.4 m-M [U-‘4C]phenylalanine at a specific activity of 0.05 @i/ml. Insulin (25 mu/ml) was added as indicated. * P < 0.001 vs. fed (same perfusion condition); t P < 0.005 vs. control (same pretreatment of rat).

/------&

FED +INSULIN

60 MINUTES

120 OF

hormones or other factors not present in the perfusion system. Insulin maintained the in vivo level of subunits during perfusion of muscle from fed rats and, within 60 min of perfusion, returned the level that existed in the 72-h fasted animal to that seen in the fed controls (data not shown). Thus, during fasting two changes occurred that reduced the rate of protein synthesis. These changes are referred to as efficiency of protein synthesis and capacity for protein synthesis (see ref. 25). In the shorter, 24-h fast, the efficiency of synthesis (synthesis/RNA) was impaired by the lack of insulin. In the longer, 72-h fast, the capacity for synthesis, represented by the RNA concentration, was also decreased, further reducing the rate of synthesis.

Changes in protein degradation in perfused skeletal muscle in response to fasting. Rates of protein degradation were measured to determine whether an increase in this process contributed to the loss of muscle protein during fasting. As noted earlier, enhanced degradation as well as inhibited synthesis could account for the observed increase in net phenylalanine release. In hemicorpus preparations perfused in the absence of insulin, degradation rates were 143 t 4 and 142 t 6 nmol/h per g for fed and 72-h fasted rats, respectively. Addition of insulin reduced the rate of degradation to 115 t 7 nmol/h per g in preparations from both fed and 72-h fasted rats. Comparable results were obtained when protein degradation was assessedin the presence of cycloheximide. These results were in contrast to those of other workers who observed increased rates of protein breakdown in skeletal muscle during fasting (20, 26, 36). In an attempt to reconcile the present data with the earlier reports, small rats weighing loo-130 g were used to conform to the size animal used by others (20, 26, 36). The smaller rats were fasted for only 48 h. During this time they lost about 30% of their initial body weight, a loss equivalent to that seen after a 72-h fast in 220-g rats (Table 1). When hemicorpus preparations from the fasted small rats were perfused in the absence of insulin, an increase (130%) in the net release of phenylalanine was the result of a 53% decrease in the rate of protein synthesis and a 32% increase in protein degradation compared to the fed controls (Table 4). An increase in the rate of degradation

FASTED 72”R FED

0

E225

FASTING

180 PERFUSION

240

FIG. 1. Effects of fasting, perfusion, and insulin on levels of ribosomal subunits. Ribosomal subunits were isolated from psoas muscles of fed and fasted rats or hemicorpus preparations perfused with or without insulin as described in Table 2. Total RNA in combined 60 S and 40 S ribosomal subunits was determined and is expressed per mg of muscle RNA. Points represent mean of 6-18 determinations, and vertical bars represent 2 SE.

4. Effect of fasting on protein turnover in small rats ----___. _______- --------- ---..----..- -.----______-.______--______-___----

TABLE

the combined level of 60 S and 40 S subunits in unperfused muscle (0 min) was 2.5fold greater than that observed in the fed controls, indicating that polysomes had become disaggregated in response to fasting. Perfusion of muscle from 72-h fasted rats did not cause any further change in subunit levels. In muscle from fed rats, however, subunit levels increased after 60 min of perfusion, reflecting the development of the insulin-dependent block in initiation that occurs in the absence of the hormone (16). The degree of subunit accumulation in perfused muscle from fed rats did not reach the level seen in fasted animals, a result that agreed with the different synthesis rates observed for these conditions (Table 2). In the fasted rats, the block in initiation appears to be greater than that produced by insulin deficiency alone, suggesting the involvement in vivo of

Fed nmol Phe/h

per g tissue

Net release

47 t 6

108 zk 8*

Protein

85 I!I 6

40t 4*

151 + 7 128 t 4

190 t, 9* 176 t 9*

synthesis

Protein degradation -Cycloheximide +Cycloheximide

_

Fasted

Values are means t SE of 6-10 determinations. Small, lOO- to 130-g rats were fed or fasted for 48 h. Hemicorpus preparations were then perfused as described in Tables 2 and 3 for determinations of protein synthesis, net phenylalanine release, and protein degradation in the absence of cycloheximide; or they were perfused in the presence of 100 PM cycloheximide as another way of estimating protein degrada* P c 0.005 vs. fed. tion.


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was also observed in the presence of cycloheximide. Thus, in contrast to the results with larger rats, protein degradation in the small rats increased in response to fasting. Changes in cathepsin D activity in response to fasting. Total and nonsedimentable cathepsin D activities were measured in unperfused psoas and gastrocnemius muscles to determine whether they changed in association with the increased rate of protein degradation noted above (Table 5). When small rats (loo-130 g) were fasted for 48 h, the total cathepsin D activity (per mg muscle protein) increased in both muscles, although the psoas (+68%) was more responsive than the gastrocnemius (+25%). The increase in protease activity in gastrocnemius was accounted for by a selective loss of other muscle components because the total amount of cathepsin D per muscle did not change. The nonsedimentable cathepsin D activity in psoas muscle increased by a small but significant amount in response to fasting, whereas no change was noted in this parameter in the gastrocnemius.

DISCUSSION

Starvation leads to a decrease in total body weight and a concomitant reduction in the amount of protein in skeletal muscle, as well as other tissues of the body. Reduction of muscle mass during fasting is a selective process. The decrease in weight of different muscles varies depending on the contractile activity of the muscle (20), and the rate of breakdown of proteins within a given muscle differs, e.g., myofibrillar proteins are degraded more rapidly than sarcoplasmic proteins (24). Amino acids derived from the breakdown of muscle protein, alanine in particular, are important precursors for hepatic gluconeogenesis (6). Previous studies (12, 16, 30, 34) show that alanine and glutamine are released from muscle in greater amounts than any of the other amino acids and in amounts that far exceed their relative 5. Changes in cathepsin D activity in response to fasting ____-___- ..--..---- _--_-.----- --_--. _~- .- -_-.---~.-_.-- _- -_ - -----.--___--~_~----~---.-- _------------..-.---

TABLE

Cathepsin Expt

Animal

Muscle Total U/mg protein*

I

D Activity Nonsedimentable % of Total

Fed Fasted

Psoas Psoas

189 t 16 318 t 30-f

14.0 t 0.8 18.6 + 0.9t

Fed Fasted

Gastrocnemius Gastrocnemius

172 t 8 215 z!L 7$

9.2 -+ 0.4 9.2 Ik 0.7

Fed Fasted

Gastrocnemius Gastrocnemius

U/muscle$j

II

16,749 t 978 16,998 t 767

Values are means t SE of 4-7 determinations. Psoas or gastrocnemius muscles were removed from anesthetized, fed or fasted, 100-g rats and homogenized (see METHODS). Supernatants of homogenates pretreated with 0.2% Triton X-100 were assayed for total cathepsin D activity. Activity in supernatants of untreated homogenates represented nonsedimentable activity in the muscle after homogenization. Denatured hemoglobin was used as substrate. In experiment I the cathepsin D specific activity (U/mg protein) is reported. In experiment II the total cathepsin D activity per muscle is reported. * Hemoglobin (pg) degraded/h per mg muscle protein; t P < 0.02 vs. fed; $ P c 0.05 vs. fed: 6 hemoglobin (ug) degraded/h ner total muscle.

HIGGINS,

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proportions in muscle protein (17). Furthermore, uptake of alanine by the liver exceeds that of any other amino acid (8). This situation has led to the formulation of a “glucose-alanine” cycle (8) that stresses the importance of muscle-derived alanine in the control of gluconeogenesis in the liver. Release of amino acids from skeletal muscle protein may also provide substrates for other adaptive changes of starvation, for example, the increased oxidation of branched-chain amino acids in heart (3) and diaphragm (14) and the synthesis of new proteins essential for adaptation to fasting. The results presented here demonstrate that a reduced rate of protein synthesis is in part responsible for the loss of muscle protein and increased release of amino acids from muscle that occur in response to food deprivation. This finding is supported by previous observations in vivo (13) and in isolated diaphragm (11)) soleus (20)) and extensor digitorum longus muscle (20). At least two mechanisms appear to account for the decreased rate of protein synthesis in skeletal muscle during starvation. During the first 24 h of food deprivation, polysomes disaggregrate, giving rise to increased levels of ribosomal subunits. This change, in association with the observed decrease in the rate of synthesis, indicated formation of a block in peptide-chain initiation (16, 38). After longer periods of fasting, the RNA content of muscle that reflects the ribosome content and thus, capacity for synthesis (11, 26) also decreases, causing a further reduction in the rate of synthesis. The possible contribution of an increased rate of protein degradation to the loss of muscle protein during fasting appears to depend on the animal model tested. The results presented here suggest that fasting leads to an increased rate of breakdown of muscle protein in small (100-g) rats, but not in larger (220-g) animals. Measurements of the fractional rate of protein breakdown in vivo show that 4 days of starvation leads to a larger increase in degradation in 100-g rats than in 400-g animals (26). In these in vivo studies, degradation rates are actually decreased after 2 days of starvation even though they are increased after 4 days (26). In other studies, increases (20, 36) as well as decreases (37) in protein degradation in skeletal muscle in response to fasting have been observed. These variations in response to fasting may be due to the size and/or age of the rats employed in the different studies. Larger rats initially have a greater amount of body fat that can provide metabolic fuel and perhaps lessen or postpone the drain on muscle protein stores. Studies with mice suggest that protein degradation does not increase until the stores of triglycerides have been utilized (5). The possibility that free fatty acids or ketone bodies may inhibit protein degradation has been suggested from studies in human beings (35) and isolated perfused rat hearts (31). However, neither free fatty acids nor ketone bodies influence protein degradation in the perfused hemicorpus (16) or isolated diaphragm (11). Therefore, other factors may be responsible for the size- and/or age-associated variations in the response to protein turnover in skeletal muscle to fasting. Increased activity of lysosomal proteases like cathepsin D may account for the accelerated rate of protein degradation observed in skeletal muscle of fasted animals.


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FASTING

The results presented here show that an increase in protein degradation in muscles of smaller rats is associated with an increased specific activity as well as a decreased latency (sedimentability) of cathepsin D. The change in latency is consistent with the observed alterations in the sedimentation properties of lysosomes from skeletal muscle of fasted animals (2). Because the total amount of cathepsin D activity per muscle does not increase in response to fasting, it is doubtful that this protease is the rate-limiting enzyme in the degradative pathway. Other proteases that may initiate the breakdown of muscle protein are a protease that is tightly bound to the myofibrils and has an alkaline pH optimum (22) or a calcium-activated protease that has a neutral pH optimum (33). A number of hormones may be involved in the adaptation of an animal to fasting. Plasma levels of insulin decrease rapidly in response to food deprivation (4, 25) and the relative deficiency of this hormone appears to be primarily responsible for the changes in protein synthesis reported here. Acute insulin deficiency, induced either by perfusion of skeletal muscle from fed rats in the absence of the hormone or by a 24-h fast, leads to a disaggregation of polysomes and a decrease in the rate of protein synthesis due to a block in peptide-chain initiation. Replacement of insulin to the muscle in vitro increases the rate of protein synthesis and induces reaggregation of ribosomal subunits into polysomes (16). Insulin reversal of the initiation block can also be accomplished after a longer (72-h) period of fasting. In this case however, the loss of tissue RNA causes a decreased capacity for synthesis that is not influenced by acute exposure to insulin in vitro. When muscle is perfused in vitro under conditions in which peptide-chain initiation is not limiting (in the presence of insulin), the rate of synthesis is proportional to the RNA content. Although the role of insulin in the response of muscle protein synthesis to fasting is clearly established, its involvement in the effect of fasting on muscle protein degradation is not as well understood. As noted above, an acute insulin-dependent condition is produced by perfusing hemicorpus preparations from fed rats without insulin for more than 1 h. Therefore, an increase in degradation in association with the development of insulin deficiency in these preparations would be evidence in favor of an effect of insulin to inhibit muscle protein degradation. When this effect was tested experimentally, the rates of protein degradation between 1 and 3 h of perfusion were slightly, but not significantly, greater than those observed during the 1st h. The observation was the same regardless of whether the hemicorpus preparations were from 100-g or 220-g fed rats (unpublished data).

E227 Because reliable measurements of protein degradation during the 1st h of perfusion are difficult to obtain due to the relatively slow equilibration of [ 14C]phenylalanine between perfusate and muscle intracellular water (16), degradative activity in the present study was determined between 1 and 3 h. During this time period, protein degradation in preparations from 220-g rats was not influenced by fasting. In contrast, degradation in preparations from 100-g rats was increased 30-50% in response to a 48-h fast. Thus, in the smaller rats but not in the larger rats, fasting produced an increase in protein degradation that was demonstrable under in vitro conditions in which insulin deficiency existed in all preparations tested. It is possible that the reduction in plasma insulin levels in vivo is responsible for this effect; however, if this is so, then the explanation for the selectivity of the effect is not clear because insulin levels fall to the same extent during fasting in both 220-g and 100-g rats (compare Table 1 and ref. 25). Furthermore, because addition of insulin in vitro inhibits protein degradation to about the same extent in all preparations tested, the effect noted in the fasted, 100-g rats cannot be explained solely on the basis of acute insulin deficiency. Plasma levels of glucocorticoids, thyroid hormones, growth hormone, and catecholamines also change in response to food deprivation. These hormones are known to influence protein turnover and amino acid release in skeletal muscle; however, their role in the adaptation of muscle to fasting remains to be defined. In summary, the initial response to fasting involves a decreased protein synthesis rate per unit of muscle RNA, a reduced efficiency of synthesis. This reduced efficiency is followed by a fall in tissue RNA or a reduced capacity of the tissue to carry out protein synthesis. In small animals an increase in protein degradation accompanies these changes in synthesis. Together these changes account for the loss of muscle protein and the increased release of amino acids from skeletal muscle during fasting. A decrease in plasma insulin appears to be primarily responsible for the changes in synthesis, whereas other factors may be involved in the changes in degradation. The authors thank Mary E. Burkart and Elsie N. Culp for expert technical assistance and Jeanette Schwartz for help in the preparation of this manuscript. We also acknowledge the valuable discussions and criticisms of Dr. Kathryn Flaim. This study was supported by National Institutes of Arthritis, Metabolism, and Digestive Diseases Grant AM 15658 and grants from the Muscular Dystrophy Association and the American Diabetes Association. L. S. Jefferson is an Established Investigator of the American Diabetes Association. Received

27 March

1978; accepted

in final form

12 October

1978.

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