Influence of Insulin and Contractile Activity on Muscle Size and Protein Balance

ALFRED L. GOLDBERG

he growth of skeletal muscle in young organisms and the maintenance of muscle mass in the adult both require an adequate supply of insulin and an adequate amount of contractile activity.1"5 Thus, muscle wasting is a characteristic feature of the diabetic or fasted state and is also a prominent consequence of muscle disease as seen in bedridden individuals. The growth or atrophy of skeletal muscle depends on the balance between the rates of synthesis and rates of breakdown of intracellular proteins. Experiments with animals or isolated muscles have shown that insulin and contractile activity can independently influence these processes and thereby affect the protein content of this tissue. In studies performed more than 10 yr ago,3-4 I showed that contractile work affects muscle size directly and not through effects on the levels of insulin or growth hormone. These investigations were undertaken to define more precisely the process of work-induced hypertrophy and to determine what role, if any, pituitary hormones, insulin, and caloric intake play in such growth. For such studies, it was essential to develop a simple, experimental system to induce muscle hypertrophy in a reproducible fashion.. In the rat, three muscles normally act together to extend the ankle. The large gastrocnerhius muscle and the smaller soleus fuse to form the Achilles' tendon; they extend the ankle in conjunction with the plantaris muscle which has its own tendon. It is possible to increase the work load of the soleus and plantaris on one limb by cutting the connections of the gastrocnemius to the Achilles' tendon. The contralateral limb received a sham operation and served as a control. Following such an operation, the muscles of the tenotomized limb, which were forced to support the bocjy weight on that side, grew at a markedly accelerated pace for 5 days.2-3 By this time the soleus was 30-50% and the plantaris about 20% heavier than the contralateral muscles (Table 1). Muscle wet weight changed in parallel
From the Department of Physiology, Harvard Medical School, Boston, Massachusetts 02115.

with dry weight and total protein content and was associated with an enlarged diameter of the muscle fibers, the classic morphologic criterion of work-induced hypertrophy.2-3 Presumably this increase in mass helps the tissue compensate for the increased physiologic demand. In fact, this rapid growth led to a greater total ability of the muscle to develop tension.2 With this technique, experiments were undertaken to determine whether growth hormone plays an important role in work-induced growth. It has long been known that hypophysectomy of young animals prevents normal body growth, including that of muscle, unless growth hormone is readministered. However, when tenotomy of the gastrocnemius was performed on one limb of hypophysectomized rats, the soleus and plantaris on that side underwent compensatory hypertrophy in a similar manner to that seen in normal rats.3 In fact, the gain in weight, relative to the contralateral muscle, and the rate of growth were indistinguishable in the two groups. Thus, hypertrophy of skeletal muscle does not require pituitary growth hormone, and in these nongrowing rats compensatory growth could be dissociated from the normal developmental process. In a similar fashion, it was possible to establish that work-induced hypertrophy is also independent of insulin.4 Normal growth requires insulin, and in the diabetic organism protein synthesis and amino acid accumulation in muscle are severely reduced.5 In our studies, rats were made severely diabetic with alloxan; body and muscle growth ceased unless the animals were treated with insulin. However, even when insulin was not injected in the diabetic animals, tenotomy of the gastrocnemius still induced hypertrophy of the soleus and the plantaris, as in normal rats4 (Table 1). In related studies we also investigated the effects of complete starvation on work-induced hypertrophy.2 Food deprivation prevents normal growth and leads to generalized muscle wasting which in rats is evident within 1 day. In the fasted animals, the control soleus and plantaris showed a clear loss of weight. However, increased contractile work of the soleus and plantaris of the operated limb not only prevented their atrophy but actually caused net growth of these muscles in the fasting animal2 (Table-2).
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ALFRED L. GOLDBERG

Together these experiments indicated that it is possible to differentiate two main processes through which muscle mass may increase: (a) developmental growth which requires pituitary hormones, insulin, and adequate diet; and (b) work-induced hypertrophy, in which growth can occur even in the absence of these factors. Furthermore, there seems to be some sort of physiologic hierarchy among the various factors that can signal muscle growth or atrophy. Contractile activity appears to be the fundamental determinant of muscle mass and can even take precedence over endocrine signals for muscle protein depletion, such as the lack of insulin in starving or diabetic organisms2-3-" or large (catabolic) doses of glucocorticoids.7 Whatever its cellular basis, this predominant influence of contractile activity probably insures that those muscles being continually used are spared under environmental conditions (e.g., fasting) that necessitate the mobilization of amino acids from protein reserves in muscle. In accord with this idea, we have found that fasting6 or treatment with large amounts of cortisol7 can cause a marked atrophy of the tonic (pale) muscles, such as the gastrocnemius, without causing significant loss of weight of the continually active (dark) soleus. However, if the soleus is made inactive, it becomes much more sensitive to these catabolic signals.7
INFLUENCE OF CONTRACTILE ACTIVITY AND INSULIN ON AMINO ACID UPTAKE BY MUSCLE

TABLE 1 Work-induced hypertrophy of the soleus muscle in diabetic and normal rats Weight of soleus Control limb Tenotomized limb Average % hypertrophy*

(mg wt/100 g body wt) Normal rats Diabetic rats

42 3 40 1

56 3 54 2

33 4 37 3

Muscle size was measured 6 days after tenotomy of the gastrocnemius. Values given are the means SEM for six diabetic animals and six controls. Diabetic animals were tenotomized following the cessation of insulin treatment. During the subsequent 6 days these animals did not increase in weight, although control animals grew by 34%. The finding that the control soleus muscles did not differ in size per gram of body weight indicates that the cessation of body growth in the diabetic rats included cessation of growth of the soleus. In both diabetic and normal rats the plantaris muscles of the. tenotomized limb were 15-23% heavier than the contralateral muscles. * Average percent increase in weight of hypertrophied muscle relative to contralateral muscle.

It follows from these findings that increased muscular work must also be able to elicit the various biochemical actions of insulin that are essential for normal muscle growth. For example, it is well known that insulin directly promotes amino acid uptake by skeletal muscle,8-9 and an early event in muscle hypertrophy is an increased rate of amino acid transport.10 On the other hand, this process decreases rapidly after muscle denervation or inactivation by spinal section.11 Furthermore, more recent studies with isolated muscles showed that the rate of amino acid transport is directly linked to contractile activity.2)9j12-13 To investigate the uptake of amino acids as an isolated process, we have utilized the nonmetabolized amino acid analog, a-aminoisobutyric acid (AIB). Muscle accumulates this compound exclusively by the "A carrier," an active transport system preferred by short-chain amino acids such as glycine, alanine, and serine.8-14 Insulin has been shown to stimulate this transport system in skeletal and cardiac muscle.8-9 Experiments by Jablecki,2-12 in our laboratory, and by others9-13 have demonstrated that electrical stimulation of muscle in vitro promotes AIB uptake in a similar fashion to insulin. In our investigations2-12 rat hemidiaphragms were suspended in a chamber containing Krebs-Ringer bicarbonate buffer. One side was electrically stimulated for varying periods of time at approximately physiologic rates, while the contralateral muscle was inactive and served as a control. After stimulation for as little as 30 min, the muscle accumulated 14C-AIB more rapidly than the inactive control (Figure 1). Greater uptake was evident within 30 min after stimulation, the shortest time that could be measured, and this effect required neither RNA nor protein synthesis.2-12 When the diaphragm was stimulated for longer periods, the increase in AIB uptake was greater. In addition, increased uptake was more.easily demonstrated with higher frequencies of stimulation (Table 3). Thus when the diaphragm was stimulated with only one shock per second,
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significant changes in transport rates were obtained only after 1 h. In contrast, stimulation at 10 pulses/s consistently increased amino acid uptake after only 6 min. In general, these experiments suggested that the total number of contractions was an important determinant of the subsequent rate of amino acid transport. Related experiments also indicated that some biochemical consequences of the contractile process and not a neural or an electrical event in the muscle membrane regulated the transport process. To choose among these alternatives, we stimulated both hemidiaphragms at identical rates using the same electrode.2-12 However, one muscle was positioned so that it contracted isometrically, while the other hemidiaphragm shortened against no load. The muscle that contracted isometrically consistently concentrated AIB more rapidly (exactly as would have been predicted by "Charles Atlas" and other advocates of iso-

TABLE 2 Hypertrophy of soleus following tenotomy of gastrocnemius in fasting and normal rats Animal weight (g) Fasting Initial Final Percent change 202 4 163 7 -19 4 Fed 197 6 226 5 16 2

Weight of soleus (mg) Control limb Tenotomized limb Percent hypertrophy 79 6 107 5 36 3 95 5 127 7 34 4

Animals were sacrificed 5 days after tenotomy of gastrocnemius and 6 days after food deprivation. Each value is the mean SEM for six animals and is for tissue wet weight. In both food-deprived and normal animals, the plantaris also underwent compensatory growth. 19

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INFLUENCE OF INSULIN AND CONTRACTILE ACTIVITY ON MUSCLE SIZE AND PROTEIN BALANCE

5/s for 1 h

take system.8-14 It is attractive (see below) to conclude that insulin and contractile activity act through some common intracellular signals. However, the effects of insulin and increased work are not identical. For example, insulin promotes both the uptake of amino acids and their incorporation into protein in these preparations.15-16 However, in repeated attempts, we have been unable to accelerate protein synthesis in muscle by electrical stimulation in vitro.2 Increased protein synthesis definitely occurs in vivo during hypertrophy induced by increased muscular work.1-2 The reasons for this difference between our in vitro and in vivo results are still not clear despite extensive experiments in our laboratory.
INFLUENCE OF MUSCULAR ACTIVITY ON PROTEIN DEGRADATION

30

60 Minutes of incubation

90

120

FIGURE 1. Effects of muscle contractions on accumulation of 14 C-a-amino isobutyric acid (AIB) by isolated rat diaphragm from hypophysectomized rats.11 One hemidiaphragm was stimulated repetitively at the rate of 5 per second for 1 h. AIB uptake was then compared in the stimulated muscle and the contralateral control. The differences in rate of accumulation are highly significant (P < 0.001). Each point is the mean SEM of at least five observations. Distribution ratio was taken as the intracellular/extracellular concentration ratio. Extracellular space was defined by the 'H-insulin space.

metric work). It is interesting in this context that, following tenotomy of the gastrocnemius, the increased work that induces the marked hypertrophy of the soleus involves continuous isometric contractions to support the organism against gravity. The biochemical reasons why isometric exercise appears more effective in promoting amino acid transport and probably muscle growth are not at all clear nor are the cellular events that couple contractile work to the transport process. Repetitive isometric contractions were thus able to mimic one of the important anabolic effects of insulin.8-9 Muscular activity specifically stimulates uptake of amino acids that share the "A transport system."2 Others have previously shown that insulin specifically accelerates this same upTABLE 3 Effect of electrical stimulation on

The protein content of any tissue is determined by the net balance between the overall rate of protein synthesis and degradation. Consequently, muscle growth and atrophy may occur through changes in either or both of these processes. In recent years, appreciable progress has been made in characterizing the physiologic factors regulating protein turnover in skeletal and cardiac muscle,17 primarily through in vitro studies of isolated rodent muscles. Our own studies2-6-16-18 have utilized the rat soleus, extensor digitorum longus, and diaphragm muscles incubated under defined conditions in vitro. When these muscles are incubated in unsupplemented Krebs-Ringer bicarbonate buffer, they undergo net protein breakdown, i.e. protein catabolism occurs several times more rapidly than protein synthesis. Consequently, these muscles undergo net release of amino acids and thus resemble muscle in vivo during fasting. We have used these preparations to examine what physiologic factors influence overall nitrogen baiance in the muscles and thus the rate of release of amino acids from proteins. The methods used for measuring protein synthesis and degradation have been described elsewhere in detail.16 Protein synthesis was estimated from the rate of incorporation of 14C-tyrosine into protein after correction for intracellular specific activity. Protein breakdown was estimated from the net release of tyrosine from muscle proteins.

14

C-AIB uptake by rat diaphragm Difference between distribution ratios (stimulated muscle-control)

Duration of stimulation (min) 3 6 12 24 40 60 0.05 0.05

Frequency of stimulation (pulse/s) 2. 5 (NS)* (NS) (NS) ( P < 0.001) 0.01 0.10 0.33 0.39 0.68 0.82 0.10 0.10 0.14 0.08 5 (NS) (P < 0.03) (P < 0.02) (P<0:01) ( P < 0.001) 0.10 0.22 0.36 0.43 0.40 1.04 0.06 0.06 0.12 0.09 0.06 0.06 10 (P < (P < (P < (P < (P< 0.02) 0.03) 0.005) 0.005) 0.001)

0.11 0.17 0.28 0.19 0.87 0.11

One hemidiaphragm (from 60-80 g rat) was stimulated at the indicated rate for the indicated time while pinned in a stretched position (approximately 10% greater than resting length). The contralateral muscle was fixed in analogous fashion within the same chamber but was not stimulated. The electrode consisted of a silver wire placed under the middle of the muscle, and the bath was perfused with Krebs-Ringer bicarbonate buffer containing glucose. Following stimulation, the hemidiaphragms were removed from the apparatus and incubated in-medium containing 14C-AIB (0.1 mM) for 45 min and their total AIB content was then measured. The distribution ratio was taken as the ratio of the total muscle AIB content to that in the medium. (No correction for extracellular space was performed.) Each point represents the SEM of diaphragms from six rats. * NS, not significant.

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ALFRED L. GOLDBERG

TABLE 4 Effect of stimulation and passive tension on protein degradation in rat soleus and diaphragm Tyrosine (pmol/mg muscle) In muscle pools 0.195 0.017 0.163 0.007 0.152 0.007 0.143 0.005 0.187 0.014 0.194 0.005 0.226 0.014 0.192 0.008 Released into medium/h 0.175 0.010 0.137 0.005 0.195 0.008 0.177 0.009 0.318 0.005 0.287 0.010 0.282 0.0333 0.238 0.01 Difference in protein degradation/h (experimental-control) -0.09 0.011 ( P < 0.002) -0.026 0.009 (P < 0.025) -0.024 0.007 (P < 0.005) -0.081 0.030 (P < 0.05)

Muscle I Soleus

Treatment No stretch Stimulated and stretched Stretched Stimulated and stretched No stretch Stretched only before incubation No stretch Stretched during incubation

II Soleus III Diaphragm IV Soleus

Since this amino acid is neither synthesized nor degraded in muscle, its production must reflect the net breakdown of muscle protein.16 By this approach, we and others have demonstrated that a variety of factors that influence muscle growth in vivo, including insulin, thyroxine, food intake, and exercise, alter the rate of protein degradation in muscle. Of special interest was the finding that repeated contractions decrease protein catabolism in incubated rat soleus or diaphragm.2-12 If the soleus or diaphragm was stimulated for 1 h at 5 pulses/s, the muscles during the subsequent hour showed less net release of amino acids than the control muscle (Table 4). Decreased release of tyrosine from protein could be demonstrated both in the presence or absence of cycloheximide, and thus must reflect a reduction in the rate of protein degradation that occurs independently of any change in protein synthesis. Similar effects were also found when the diaphragm was stimulated in medium lacking glucose and amino acids. Thus the ability of contractile activity to retard protein degradation is not a consequence of increased glucose or amino acid uptake. Presumably these effects of contraction can account for the apparent decrease in protein breakdown during work-induced hypertrophy1'2 and in denervated muscle for the acceleration of protein breakdown that is primarily responsible for the muscle atrophy.19 One unexpected finding in these studies was that the effects of repeated contraction appeared smaller when the control (unstimulated) muscle was maintained in a stretched position (Table 4). This observation suggested that passive tension by itself might also reduce protein breakdown. In

fact, as shown in Table 4, when the soleus or diaphragm was stretched beyond its resting length by 10-20%, the rate of protein degradation was also slower than in the contralateral muscle incubated at resting length. This reduction in protein breakdown was evident for some time after the period of passive stretch. More recent studies by Goldspink have confirmed and extended these observations.20 In addition to their biological interest, these findings may be of appreciable practical relevance in the field of physical therapy. These observations support the long-held belief by physical therapists that passive tension on muscle can retard the process of muscle atrophy. Previously there has been very little documentation for such claims. The finding of reduced degradation by passive stretch is also of special interest because of a variety of recent reports suggesting that passive stretch of muscle by itself may initiate muscular hypertrophy.2 It is unknown whether contractile work and passive tension act through the same mechanisms to reduce proteolysis. Unfortunately, we have no clear information as to how either factor influences rates of protein breakdown. Changes in the rates of protein degradation may occur by two general mechanisms:17 (a) The amount of tension might affect the sensitivity of muscle proteins to intracellular proteases, (b) Tension development may regulate the activity of the cell's proteolytic machinery as several hormones are known to do. Thus glucagon, insulin, and thyroxine appear to influence overall proteolysis in liver by altering either the activity17-21-22 or total amount23 of lysosomal proteases.

TABLE 5 Effects of insulin and glucose on protein synthesis and degradation in rat diaphragms Changes upon addition of: Control Insulin Glucose Insulin + glucose (nmol tyrosine/mg muscle)

Incorporated into protein Released from protein

0.279 0.015 0.734 0.015

+0.101 0.041* -0.073 0.026f

+0.048 0.025 -0.068 0.018|

+0.168 0.048f -0.136 0.010f

Protein synthesis (i.e. tyrosine incorporation into protein and protein degradation (i.e. tyrosine release from protein) were measured as described previously.16 Protein degradation rates were determined in the presence of cycloheximide to prevent concomitant protein synthesis. Quarter diaphragms were incubated for 2 h in Krebs-Ringer bicarbonate buffer. * P < 0.025. t P<0.01.

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INFLUENCE OF INSULIN AND CONTRACTILE ACTIVITY ON MUSCLE SIZE AND PROTEIN BALANCE

TABLE 6 Effects of insulin and amino acids on the degradation of different classes of proteins in rat hepatocytes Rate of degradation Insulin plus amino acids 3.2 16.2 Percent inhibition 42 0

Control Average cell protein Short-lived proteins Abnormal proteins (containing amino acid analogs) 5.5 15.6

40.3

42.6

Hepatocytes were prepared by perfusion of rat livers with collagenase, and the dissociated cells were cultured on collagencontaining Petri dishes. "Average cell proteins" were labeled by exposing cells to 3H-leucine (1 Ci/ml) for 24 h. To selectively label "short-lived proteins," the cultures were exposed to 3H-leucine for 1 h. To induce the production of abnormal proteins, cells were incubated in the medium containing the arginine analog, cavanine (2.5 mM); the phenylalanine analog, fluorophenylalanine (2.5 mM); and the tryptophan analog, azatryptophan (1.25 mM). After labeling, the cultures were incubated either in Krebs-Ringer bicarbonate buffer or medium supplemented with insulin (0.5 U/ml) and plasma amino acids at four times their normal concentration in rat plasma.

INSULIN AND THE REGULATION OF PROTEIN BREAKDOWN

Although our understanding of the mechanisms by which contractile activity influences the rate of protein turnover is still very limited, significant progress has been made recently in defining further the effects of insulin on intracellular proteolysis in skeletal and cardiac muscle. This ability of insulin to retard proteolysis in muscle is of appreciable physiologic importance in regulating the release of gluconeogenic precursors from this tissue.5-16-18 In fasting and the postprandial state, the net breakdown of muscle proteins and the subsequent release of amino acids into the circulation represent important steps that limit the rate of gluconeogenesis. In fact, insulin probably is the most important factor regulating protein balance in skeletal and cardiac muscle.5>15~17 The rise in insulin after food intake promotes the net uptake of amino acids and net protein accumulation in muscle, while the fall in insulin upon fasting leads to a net release of amino acids from this tissue. Experiments with isolated muscles in vitro have shown that this hormone not only stimulates protein synthesis15 but also inhibits protein degradation in skeletal muscle (Table 5) as well as in liver, heart, and fibroblasts.17-21-22 These two actions of insulin, along with its ability to stimulate amino acid uptake into muscle,8-9 act in complementary fashion in vivo to reduce the flow of gluconeogenic amino acids to liver and kidney. Even in the absence of insulin, glucose can also inhibit protein degradation,16 but unlike insulin, glucose does not affect protein synthesis4 (Table 5). The effects of insulin and glucose in improving overall protein balance appear additive. Glucose may retard proteolysis by providing an energy source, since similar effects can be demonstrated upon addition of the ketone bodies, /3-hydroxybutyrate and acetoacetate. Like glucose, ketone bodies decrease protein breakdown but do not affect protein synthesis significantly. It has, incidentally, been suggested that during prolonged starvation, the loss of body nitrogen decreases

in humans as a consequence of the marked accumulation of ketone bodies, although there is little direct evidence for such a conclusion.17 This discussion of protein degradation has thus far treated the cell as a homogenous black box, whose constituents all turn over in a uniform fashion. This view, however, is an incorrect simplification and is potentially misleading. Actually, cell proteins vary markedly in their rates of degradation.17-24 In rat liver, for example, the half-lives of enzymes range from as short as 20 min to several weeks.17-22 In general those proteins with particularly short halflives tend to be rate-limiting enzymes whose activities regulate the flux of metabolites through crucial pathways.17 Rapid turnover of such proteins presumably aids in the regulation of cell metabolism. Animal and bacterial cells also degrade especially rapidly proteins with highly unusual conformations.17-24 Thus protein degradation appears to serve in part as a "cellular sanitation system" by selectively removing abnormal proteins that may arise through mutations, biosynthetic errors, incorporation of amino acid analogs, spontaneous denaturation, or postsynthetic chemical modifications. In fact, this process also helps to prevent the intracellular accumulation of abnormal proteins in various human diseases, such as the certain human hemoglobin variants17 or even the glycosylated proteins that may arise spontaneously in cells of diabetics.25 The rates of turnover of individual proteins not only vary widely but they also differ in their responsiveness to insulin. In general, insulin and nutrient supply appear to retard selectively the breakdown of those proteins with especially long half-lives. This selectivity is nicely illustrated in recent data on hepatocytes obtained by Drs. Neff and DeMartino in my laboratory (Table 6). In these cells, insulin and amino acids together cause a marked reduction in the degradation of "more stable" cell proteins but have little or no effect on the removal of short-lived cell proteins. Thus insulin and amino acids had no effect on the very rapid degradation of abnormal proteins containing amino acid analogs. This latter process thus appears to occur in all cells and is independent of nutrient supply or endocrine status. Similar selective effects have been found with serum or nutrient deprivation17-26"28 which also appear only to affect the degradation of the most stable cell proteins. These findings and related ones have led to the suggestion that discrete proteolytic systems exist within cells and serve distinct physiologic functions. Recent biochemical studies have supported this idea.17-27"29 For example, we have demonstrated in reticulocytes29 a soluble nonlysosomal degradation system that seems to be responsible for the selective hydrolysis of abnormal proteins. By contrast, there is appreciable evidence that the turnover of the most stable cell proteins involves the lysosomal apparatus whose activity appears to be under fine endocrine control. For example, experiments by Glen Mortimore and colleagues have shown that in the absence of insulin and amino acids, when proteolysis is accelerated, the liver cytoplasm contains large numbers of autophagic vacuo l e s 2i,22 yhese structures appear to be large lysosomes containing partially degraded cellular materials and probably represent the primary site of the accelerated protein breakdown. In related studies, we have found that the ability of the rat to accelerate protein breakdown in starva-

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ALFRED L. GOLDBERG

Liver 1.0 SEVERE DIABETES MODERATE DIABETES & NORMAL OO A

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FIGURE 2A. Relative degradative rates of liver proteins in normal and in diabetic rats: SDS-polyacrylamide gel electrophoresis. A diabetic rat (170 g) that had been maintained on insulin received 150 fid of l4C-leucine intraperitoneally, after which its insulin was withdrawn. Three days later it was killed and its tissues pooled with those from a normal rat that had received 500 /*Ci of 3H-leucine 4 h previously. "Severe diabetes" and "moderate diabetes" refer to the degree of abnormality in serum glucose content and growth rate. These data were kindly provided by Dr. J. F. Dice, Jr., and co-workers.33 FIGURE 2B. Relative degradative rates of liver proteins in normal and diabetic rats: isoelectric focusing. These were the same double-labeled proteins used in the experiment shown in Figure 2A. Proteins were separated according to their isoelectric points as described previously. These data were kindly provided by Dr. J. F. Dice, Jr., and co-workers."

tion depends upon an adequate supply of thyroid hormones.18 These hormones appear to control the lysosomal content of liver and skeletal muscles, the two tissues where protein breakdown changes most markedly during fasting.23 In other words, the rate of breakdown of individual proteins is dependent upon both inherent structural features of these molecules and the overall degradative activity of the cell, which is controlled by insulin and other hormones (e.g., thyroxine). However, these effects of insulin appear negligible for that fraction of cell proteins with very short half-lives, such as proteins with highly abnormal conformations. In recent years, progress has been made in identifying certain structural features of the proteins that may determine their degradative rates in vivo. Schimke and colleagues have shown that, on the average, large proteins tend to be degraded more rapidly than smaller ones in mammalian tissues24-30 (see Figure 2). In addition, Dice and I, using the double-label technique of Arias et al., 31 found that acidic proteins generally are degraded more rapidly than neutral or basic ones32 (Figure 2). It is noteworthy that these correlations, whose precise

biochemical explanation is still unclear, were originally demonstrated for the breakdown of proteins in normally growing animals. Recently, Dice et al.33 found that these correlations with size and change do not apply to the accelerated protein breakdown seen in liver and muscle in diabetes or starvation (Figure 2). In these animals, the selectivity for large and for acidic molecules is lost, and this effect is most marked in animals whose diabetic symptoms are most pronounced. This apparent loss of specificity in the degradative process may be a reflection of the earlier finding that the acceleration of proteolysis in the absence of insulin results from an increased breakdown of the more stable cell components, which are necessarily the smaller, more basic proteins. Perhaps also the loss of selectivity in starvation or diabetes reflects the activation of a lysosomal process, which may act by engulfing cell proteins in a relatively nonspecific way. Whatever their basis, these results (Figure 2) may also be of appreciable clinical significance since they predict that the accelerated protein breakdown in diabetes or starvation should seriously alter cell composition. In other words, the proteins being lost in these catabolic states should be different from those degraded when there is an adequate

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INFLUENCE OF INSULIN AND CONTRACTILE ACTIVITY ON MUSCLE SIZE AND PROTEIN BALANCE

supply of insulin. Since the lack of insulin decreases the synthesis of various cell proteins to a similar extent,15 while the insulin deficiency promotes selectively the degradation of the more stable cell components, there should be in diabetes a differential depletion of small and basic proteins. Such a change in cell composition could have profound physiologic consequences. SUMMARY Although the biochemical mechanism by which insulin and contractile activity affect protein turnover in muscle are still unclear, certain physiologic conclusions can be made: (a) Increased work can induce muscle hypertrophy even in the diabetic or starving animal. Thus, work-induced growth differs from normal growth of muscle in not requiring insulin, (b) Like insulin, repeated contractions stimulate the transport of amino acids into muscle. Contractile activity or passive tension can also reduce the rate of protein degradation in this tissue. These effects can be shown with isolated muscles in vitro, but the mechanisms coupling contractile activity to these anabolic processes are unknown, (c) Insulin also reduces overall protein breakdown in skeletal muscle, heart, and liver, apparently by regulating lysosomal function, (d) Insulin reduces selectively the breakdown of cell proteins with relatively long half-lives. This hormone does not affect the rapid breakdown of abnormal proteins which probably does not occur within the lysosome. (e) Normally, liver and muscle degrade large, acidic cell proteins quite rapidly, but this selectivity is lost in the tissues of diabetic or starved organisms, in which proteolysis is accelerated.
REFERENCES

1 Goldberg, A. L: Mechanisms of growth and atrophy of skeletal muscle. In Muscle Biology, Vol. 1, Cassens, R. G., Ed. New York, Marcel Dekker, 1972, pp. 89-118. 2 Goldberg, A. L, Etlinger, J. D., Goldspink, D. F., and Jablecki, C: Mechanism of work-induced hypertrophy of skeletal muscle. Med. Sci. Sports 7:248-61, 1975. :| Goldberg, A. L.: Work-induced growth of skeletal muscle in normal and hypophysectionized rats. Am. J. Physiol. 372:1193-98, 1967. 4 Goldberg, A. L: Role of insulin in work-induced growth of skeletal muscle. Endocrinology 83:1071-73, 1968. 5 Cahill, G. F., Jr., Aoki, T. T., and Marliss, E. B.: Insulin and muscle protein. In Handbook of Physiology. Endocrinology 7:563-77, 1972. 6 Li, J. B., and Goldberg, A. L: Effects of food deprivation on protein synthesis and degradation in rat skeletal muscles. Am. J. Physiol. 237: 441_48, 1976. 7 Goldberg, A. L, and Goodman, H. M.: Relationship between cortisone and muscle work in determining muscle size. J. Physiol. 200: 667-75, 1969. 8 Riggs, T. R., and McKirahan, K. J.: Action of insulin on transport of L-alanine into rat diaphragm in vitro. J. Biol. Chem. 248:6450-55, 1973. 9 Narahara, H. T., and Holloszy, J. 0.: The actions of insulin, trypsin and electrical stimulation on amino acid transport in muscle. J. Biol. Chem. 249:5435, 1974.

10 Goldberg, A. L, and Goodman, H. M.: Amino acid transport during work-induced growth of skeletal muscle. Am. J. Physiol. 276:1111-15, 1969. 11 Goldberg, A. L, and Goodman, H. M.: Effects of disuse and denervation on amino acid transport by skeletal muscle. Am. J. Physiol. 276:1116-19, 1969. 12 Goldberg, A. L, Jablecki, C. M., and Li, J. B.: Effects of use and disuse on amino acid transport and protein turnover in muscle. Ann. N.Y. Acad. Sci. 228:190-201, 1974. 13 Arvi11, A.: Relationship between the effects of contraction and insulin on the metabolism of the isolated levator ani muscle of the rat. Acta Endocrinol. 722:27-41, 1967. 14 Oxender, D. L, and Christensen, H. N.: Distinct mediating systems for the transport of neutral amino acids by the Ehrlich cell. J. Biol. Chem. 238: 3686-99, 1963. 15 Wool, I. L. G.: Effects of insulin on cellular protein synthesis. Handbook of Experimental Pharmacology, XXXII, 2:267-302, 1975. 16 Fulks, R., Li, J. B., and Goldberg, A. L: Effects of insulin, glucose and amino acids on protein turnover in rat diaphragm. J. Biol. Chem. 250:290-98, 1975. 17 Goldberg, A. L., and St. John, A. C: Intracellular protein degradation in mammalian and bacterial cells: Part II. Ann. Rev. Biochem. 45:747-803, 1976. 18 Goldberg, A. L, DeMartino, G. N., and Chang, T. W.: Release of gluconeogenic precursors from skeletal muscle in "Regulatory Mechanisms of Carbohydrate Metabolism," F.E.B.S. Lett. 42:347-58, 1978. 19 Goldberg, A. L:.Protein turnover in skeletal muscle. II. Effects of denervation and cortisone on protein catabolism in skeletal muscle. J. Biol. Chem. 244:3223-29, 1969. 20 Goldspink, D. F.: The influence of activity on muscle size and protein turnover. J. Physiol. 264 (7J:283-96, 1977. 21 Mortimore, G. E., and Neely, A. N.: Regulatory effects of insulin, glucogon, and amino acids on hepatic protein turnover in association with alterations of the lysosomal system: In Intracellular Protein Turnover. Shimke, R. T., and Katunuma, N., Eds. New York, Academic Press, 1975, pp. 265-79. 22 Ward, W. F., and Mortimore, G. E.: Compartmentation of intracellular amino acids in rat liver. J. Biol. Chem. 253:3581-87, 1978. 23 DeMartino, G. N., a n d G o l d b e r g , A. L : Thyroid hormones control lysosomal enzyme activities in liver a n d skeletal muscle. Proc. Natl. A c a d . Sci. U.S.A. 7 5 : 1 3 6 9 - 7 3 , 1978. 24 Goldberg, A. L , a n d Dice, J. F.: Intracellular protein degradation in mammalian and bacterial cells. Annu. Rev. Biochem. 43:835-69, 1974. 25 Bunn, H. F., Gabbay, K. H, and Gallop, P. M.: The glycosylation of hemoglobin: relevance to diabetes mellitus. Science 200:21-22, 1978. 26 Wibo, M., and Poole, B.: Protein digestion in cultured cells. I. The effect of fresh medium, fluoride, and iodoacetate on the digestion of cellular protein or rat fibroblasts. J. Biol. Chem. 248:6221-26, 1973. 27 Hopgood, M. F., Clark, M. G., and Ballard, F. J.: Inhibition of protein degradation in isolated rat hepatocytes. Biochem. J. 764:399-407, 1977. 28 Neff, N., and Goldberg, A. L: Effects of protease inhibitors on the degradation of different classes of proteins in rat liver. Submitted for publication. 29 Etlinger, J., and Goldberg, A. L: A soluble, ATP-dependent proteolytic system responsible for the degradation of abnormal proteins in reticulocytes. Proc. Natl. Acad. Sci. U.S.A. 74:54-58, 1977. 3(1 Dice, J. F., Dehlinger, P. J., and Schimke, R. T.: Studies on the correlation between size and relative degradation rate of soluble proteins. J. Biol. Chem. 248:4220-28, 1973. 31 Arias, I. M., Doyle, D., and Schimke, R. T.: Studies on the synthesis and degradation of proteins of the endoplasmic reticulum of rat liver. J. Biol. Chem. 244:3303-15, 1969. 32 Dice, J. F., and Goldberg, A. L: Relationship between in vivo degradative rates and isoelectric points of proteins. Proc. Natl. Acad. Sci. U.S.A. 72:3893-97, 1975. 33 Dice, J. F., Walker, C. D., Byrne, B., and Cardiel, A.: General characteristics of protein degradation in diabetes and starvation. Proc. Natl. Acad. Sci. U.S.A. 75:2093-97, 1978.

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