diabetes 1979 goldberg 18 24

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Influence of Insulin andContractile Activity onMuscle Size and Protein BalanceALFRED L. GOLDBERG

T he growth of skeletal muscle in young organismsand the maintenance of muscle mass in the adultboth require an adequate supply of insulin and anadequate amount of contractile activity.15 Thus,muscle wasting is a characteristic feature of the diabeticor fasted state and is also a prominent consequence ofmuscle disease as seen in bedridden individuals. Thegrowth or atrophy of skeletal mu scle depe nds on the balancebetween the rates of synthesis and rates of breakdown ofintracellular proteins. Experiments with animals or isolatedmuscles have shown that insulin and contractile activitycan independently influence these processes and therebyaffect the protein content of this tissue.In studies performed more than 10 yr ago,3-4 I showedthat contractile work affects muscle size directly and notthrough effects on the levels of insulin or growth hormone.These investigations were undertaken to define moreprecisely the process of work-induced hypertrophy and todetermine what role, if any, pituitary hormones, insulin,and caloric intake play in such growth. For such studies, itwas essential to develop a simple, experimental system toinduce muscle h ypertrophy in a reprodu cible fashion.. Inthe rat, three muscles normally act together to extend theankle. The large gastrocnerhius muscle and the smallersoleus fuse to form the Achilles' tendon; they extend theankle in conjunction with the plantaris muscle whichhas its own tendon. It is possible to increase the workload of the soleus and plantaris on one limb by cutting theconnections of the gastrocnemius to the Achilles' tendon.The contralateral limb received a sham operation and servedas a control. Following such an operation, the muscles ofthe tenotomized limb, which were forced to support thebocjy weight on that side, grew at a markedly acceleratedpace for 5 days.2-3 By this t ime the soleus was 30-50%and the plantaris about 20% heavier than the contralateralmuscles (Table 1). Muscle wet weight changed in parallel

From the Department of Physiology, Harvard Medical School,Massachusetts 02115. Boston,

with dry weight and total protein content and was associate dwith an enlarged diameter of the muscle fibers, the classicmorphologic criterion of work-induced hypertrophy.2-3 Pre-sumably this increase in mass helps the tissue compensatefor the increased physiologic demand. In fact, this rapidgrowth led to a greater total ability of the muscle to de veloptension.2With this technique, experiments were undertaken todetermine whether growth hormone plays an important rolein work-induced growth. It has long been known that hypo-physectomy of young animals prevents normal body growth,including that of muscle, unless growth hormone is read-ministered. However, when tenotomy of the gastrocnemiuswas performed on one limb of hypophysectomized rats,

the soleus and plantaris on that side underwent compensa-tory hypertrophy in a similar manner to that seen in normalrats.3 In fact, the gain in weight, relative to the contralateralmuscle, and the rate of growth were indistinguishable inthe two groups. Thus, hypertrophy of skeletal muscle doesnot require pituitary growth hormone, and in these non-growing rats compensatory growth could be dissociatedfrom the normal developmental process.In a similar fashion, it was possible to establish thatwork-induced hypertrophy is also independent of insulin. 4Normal growth requires insulin, and in the diabetic organismprotein synthesis and amino acid accumulation in muscleare severely reduced.5 In our studies, rats were madeseverely diabetic with alloxan; body and muscle growth

ceased unless the animals were treated with insulin. How-ever, even when insulin was not injected in the diabeticanimals, tenotomy of the gastrocnemius still induced hyper-trophy of the soleus and the plantaris, as in normal rats 4(Table 1). In related studies we also investigated the effectsof complete starvation on work-induced hypertrophy.2Food deprivation prevents normal growth and leads togene ralized muscle wasting w hich in rats is evident within 1day. In the fasted animals, the control soleus and plantarisshowed a clear loss of weight. However, increasedcontractile work of the soleus and plantaris of the operatedlimb not only prevented their atrophy but actually causednet growth of these m uscles in the fasting anim al2(Table-2).18 DIABETES, VOL. 28, SUPPL. 1, JANUARY 1979

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ALFRED L. GOLDBERGTogether these experiments indica ted that it is possible todifferentiate two main processes through which musclemass may increase: (a) developmental growth which re-quires pituitary hormones, insulin, and adequate diet; and (b)work-induced hypertrophy, in which growth can occur evenin the absence of these factors. Furthermore, there seemsto be some sort of physiologic hierarchy among the variousfactors that can signa l m uscle growth or atrophy. C ontractile

activity appears to be the fundamental determinant ofmuscle mass and can even take precedence over endo-crine signals for muscle protein depletion, such as the lackof insulin in starving or diabetic organisms2-3- or large(catabolic) doses of glucocorticoids.7 Whatever its cellularbasis, this predominant influence of contractile activityprobably insures that those muscles being continuallyused are spared under environmental conditions (e.g.,fasting) that necessitate the mobilization of amino acidsfrom protein reserves in mu scle. In acc ord with this ide a, wehave found that fasting 6or treatment with large amounts ofcortisol7 can cause a marked atrophy of the tonic (pale)muscles, such as the gastrocnemius, without causing sig-nificant loss of weight of the continually active (dark)soleus. However, if the soleus is made inactive, it becomesmuch more sensitive to these catabolic signals. 7INFLUENCE OF CONTRACTILE ACTIVITY AND INSULINON AMINO ACID UPTAKE BY MUSCLEIt follows from these findings that increased muscular workmust also be able to elicit the various biochemical ac-tions of insulin that are essential for normal muscle growth.For example, it is well known that insulin directly promotesamino acid uptake by skeletal muscle, 8-9and an early eventin muscle hypertrophy is an increased rate of amino acidtransport.10 On the other hand, this process decreasesrapidly after muscle denervation or inactivation by spinalsection.11 Furthermore, more recent studies with isolatedmuscles showed that the rate of amino acid transport isdirectly linked to contractile activity.2)9j12-13To investigate the uptake of amino acids as an isolatedprocess, we have utilized the nonmetabolized amino acidanalog, a-aminoisobutyric acid (AIB). Muscle accumulatesthis com poun d exclusively by the A carrier, an activetransport system preferred by short-chain amino acids suchas glycine , alanine, and serine.8-14Insulin has been shown tostimulate this transport system in skeletal and cardiacmuscle.8-9Experiments by Jablecki,2-12 in our laboratory, andby others9-13have demonstrated that electrica l stimulation ofmuscle in vitro promotes AIB uptake in a similar fashion toinsulin. In our investigations2-12 rat hemidiaphragms weresuspended in a chamber containing Krebs-Ringe rbicarbonate buffer. One side was electrically stimulated forvarying periods of time at approximately physiologic rates,while the contralateral muscle was inactive and served as acontrol. After stimulation for as little as 30 min, the muscleaccumulated 14C-AIB more rapidly than the inactive control(Figure 1). Greater uptake was evident within 30 min afterstimulation, the shortest time that could be measured, andthis 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, in-creased uptake was more.easily demonstrated with higherfrequencies of stimulation (Table 3). Thus when the dia-phragm was stimulated with only one shock per second,

TABLE 1Work-induced hypertrophy of the soleus muscle in diabeticand normal rats

Weight of soleusControl Tenotomized Average %limb limb hypertrophy*

(mg wt/100 g body wt)Normal ratsDiabetic rats 42 340 1 56 354 2 33 437 3

Muscle size was measured 6 days after tenotomy of thegastrocnemius. Values given are the means SEM for six diabeticanimals and six controls. Diabetic animals were tenotomizedfollowing the cessation of insulin treatment. During the subsequent6 days these animals did not increase in weight, although controlanimals grew by 34%. The finding that the control soleus musclesdid not differ in size per gram of body weight indicates thatthe cessation of body growth in the diabetic rats includedcessation of growth of the soleus. In both diabetic and normalrats the plantaris m uscles of the. tenotom ized lim b were 1 5- 23 %heavier than the contralateral muscles.* Average perce nt increase in weight of hype rtrophied musc lerelative to contralateral muscle.

significant changes in transport rates were obtained onlyafter h. In contrast, stimulation at 10 pulses/s consistentlyincreased amino acid uptake after only 6 min. In general,these experiments suggested that the total number of con-tractions was an important determinant of the subsequentrate of amino acid transport.Related experiments also indicated that some biochemi-cal consequences of the contractile process and not aneural or an electrical event in the muscle membrane regu-lated the transport process. To choose among thesealternatives, we stimulated both hemidiaphragms at identi-cal 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 con-centrated AIB more rapidly (exactly as would have beenpred icted by Charles Atlas and other advocates of iso-

TABLE 2Hypertrophy of soleus following tenotomy of gastrocnemiusin fasting and normal rats

InitialFinal

Percent change

Control limbTenotomized limbPercent hypertrophy

Animal weight (g)Fasting202 4163 7

Fed197 6226 5

-1 9 4 16 2Weight of soleus (mg)79 6107 536 3

95 5127 734 4

Animals were sacrificed 5 days after tenotomy of gastrocnemiusand 6 days after food deprivation. Each value is the mean SEMfor six animals and is for tissue wet weight. In both food-deprivedand normal animals, the plantaris also underwent compensatorygrowth.

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

1

5/s for h

30 60 90Minutes of incubation

120

FIGURE 1. Effects of muscle contractions on accumulation of14C-a-amino isobutyric acid (AIB) by isolated rat diaphragm fromhypophysectomized rats.11 One hemidiaphragm was stimulatedrepetitively at the rate of 5 per second for 1 h. AIB uptake was thencompared 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/extracellularconcentration ratio. Extracellular space was defined by the'H-insulin space.

metric work). It is interesting in this context that, followingtenotomy of the gastrocnemius, the increased work that in-duces the marked hypertrophy of the soleus involvescontinuous isometric contractions to support the organismagainst gravity. The biochemical reasons why isometricexercise appears more effective in promoting amino acidtransport and probably muscle growth are not at all clearnor are the cellular events that couple contractile work tothe transport process.Repetitive isometric contractions were thus able to mimicone of the important anabolic effects of insulin.8-9Muscularactivity specifically stimulates uptake of amino acids thatshare the A transport system. 2 Others have previouslyshown that insulin specifically accelerates this same up-

take system.8-14 It is attractive (see below) to concludethat insulin and contractile activity act through some com-mon intracellular signals. However, the effects of insulinand increased work are not identical. For example, insulinpromotes both the uptake of amino acids and their in-corporation into protein in these preparations. 15-16 However,in repeated attempts, we have been unable to accelerateprotein synthesis in muscle by electrical stimulation invitro.

2 Increased protein synthesis definitely occurs in vivoduring hypertrophy induced by increased muscular work.1-2The reasons for this difference between our in vitro andin vivo results are still not clear despite extensive experi-ments in our laboratory.

INFLUENCE OF MUSCULAR ACTIVITY ON PROTEINDEGRADATIONThe protein content of any tissue is determined by the netbalance between the overall rate of protein synthesis anddegradation. Consequently, muscle growth and atrophy mayoccur through changes in either or both of these processes.In recent years, appreciable progress has been made incharacterizing the physiologic factors regulating proteinturnover in skeletal and cardiac muscle,17 primarily throughin vitro studies of isolated rodent muscles.

Our own studies2-6-16-18 have utilized the rat soleus, ex-tensor digitorum longus, and diaphragm muscles incubatedunder defined conditions in vitro. When these muscles areincubated in unsupplemented Krebs-Ringer bicarbonatebuffer, they undergo net protein breakdown, i.e. proteincatabolism occurs several times more rapidly than proteinsynthesis. Consequently, these muscles undergo net re-lease of amino acids and thus resemble muscle in vivo dur-ing fasting. We have used these preparations to examinewhat physiologic factors influence overall nitrogen baiancein the muscles and thus the rate of release of amino acidsfrom proteins. The methods used for measu ring protein syn-thesis and degradation have been described elsewhere indetail.16 Protein synthesis was estimated from the rate of in-corporation of 14C-tyrosine into protein after correction forintracellular specific activity. Protein breakdown was esti-mated from the net release of tyrosine from muscle proteins.

TABLE 3Effect of electrical stimulation on 14C-AIB uptake by rat diaphragm

Duration ofstimulation(min)3612244060

2.0.05 0.050.11 0.170.28 0.190.87 0.11

Difference

5(NS)*(NS)(NS)(P < 0.001)

between distribution ratios (stimulatedFrequency of stimulation (pulse/s)

0.01 0.100.33 0.100.39 0.100.68 0.140.82 0.08

5(NS)(P < 0.03)(P < 0.02)( P < 0 : 0 1 )(P < 0.001)

muscle-control)

100.10 0.060.22 0.060.36 0.120.43 0.090.40 0.061.04 0.06

(P < 0.02)(P < 0.03)(P < 0.005)(P < 0.005)(P< 0.001)One hemidiaphragm (from 60-80 g rat) was stimulated at the indicated rate for the indicated time while pinned in a stretchedposition (approximately 10% greater than resting length). The contralateral muscle was fixed in analogous fashion within the samechamber but was not stimulated. The electrode consisted of a silver wire placed under the middle of the muscle, and the bath wasperfused with Krebs-Ringer bicarbonate buffer containing glucose. Following stimulation, the hemidiaphragms were removed from theapparatus and incubated in-medium containing 14C-AIB (0.1 mM) for 45 min and their total AIB content was then measured. Thedistribution ratio was taken as the ratio of the total muscle AIB content to that in the medium. (No correction for extracellular spacewas performed.) Each point represents the SEM of diaphragms from six rats.* NS, not significant.

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ALFRED L. GOLDBERGTABLE 4Effect of stimulation and passive tension on protein degradation in rat soleus and diaphragm

Tyrosine (pmol/mg muscle)

Muscle Treatment In musclepools Released intomedium/hDifference in proteindegradation/h(exper imental-control)

I Soleus No stretch 0.195 0. 01 7 0.175 0 .0 10Stimulated and stretched 0.163 0.007 0.137 0.005II Soleus Stretched 0.152 0 .0 07 0.195 0. 00 8Stimulated and stretched 0.143 0.005 0.177 0 .0 09III Diaphragm No stretch 0.187 0.014 0.318 0.005Stretched only before incuba tion 0.194 0 .0 05 0.287 0.010IV Soleus No stretch 0.226 0.014 0.282 0.0333Stretched during incuba tion 0.192 0.008 0.238 0.01

-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)

Since this amino acid is neither synthesized nor degrad ed inmuscle, its production must reflect the net breakdown ofmuscle protein.16 By this approach, we and others havedemo nstrated that a variety of factors that influence musclegrowth in vivo, including insulin, thyroxine, food intake, andexercise, alter the rate of protein degradation in muscle.Of special interest was the finding that repeated contrac-tions decrease protein catabolism in incubated rat soleus ordiaphragm.2-12 If the soleus or diaphragm was stimulatedfor 1 h at 5 pulses/s, the muscles during the su bsequenthour showed less net release of amino acids than thecontrol muscle (Table 4). Decreased release of tyrosinefrom protein could be demonstrated both in the presence orabsence of cycloheximide, and thus must reflect a reductionin the rate of protein degrada tion that occurs indepen dentlyof any chan ge in protein synthesis. Similar effects were alsofound when the diaphragm was stimulated in medium lack-ing glucose and amino acids. Thus the ability of contractileactivity to retard protein degradation is not a consequenceof increased glucose or amino acid uptake. Presumablythese effects of contraction can account for the apparentdecrease in protein breakdown during work-inducedhypertrophy1 '2 and in denervated muscle for the accelera-tion of protein breakdown that is primarily responsiblefor the muscle atrophy.19

One unexpected finding in these studies was that theeffects of repeated contraction appeared smaller when thecontrol (unstimulated) muscle was maintaine d in a stretchedposition (Table 4). This observation suggested that passivetension by itself might also reduce protein breakdown. In

fact, as shown in Table 4, when the soleus or diaphragmwas stretched beyond its resting length by 10-20%, the rateof protein degradation was also slower than in the contra-lateral muscle incubated at resting length. This reductionin protein breakdown was evident for some time after theperiod of passive stretch. More recent studies by Goldspinkhave confirmed and extended these observations.20In addi-tion to their biological interest, these findings may be ofappreciable pract ical relevance in the f ield of physicaltherapy. These observations support the long-held belief byphysical therapists that passive tension on muscle can re-tard the process of muscle atrophy. Previously there hasbeen very litt le documentation for such claims. The f ind-ing of reduced degradation by passive stretch is also ofspecial interest because of a variety of recent reportssuggesting that passive stretch of muscle by itself mayinitiate muscular hypertrophy.2

It is unknown whether contractile work and passivetension act through the same mechanisms to reduceproteolysis. Unfortunately, we have no clear informationas to how either factor influences rates of protein break-down. Changes in the rates of protein degradation mayoccur by two general mechanisms:17 (a) The amount of ten-sion might affect the sensitivity of muscle proteins to intra-cellular proteases, (b) Tension development may regulatethe activity of the cell's p roteolytic machinery as several hor-mones are known to do. Thus gluc ago n, insu lin, and thyroxineappear to influence overall proteolysis in liver by alteringeither the activity17-21-22 or total amount23 of lysosomalproteases.

TABLE 5Effects 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 proteinReleased from protein 0.279 0.0150.734 0.015 +0.101 0.041*-0.073 0.026f 0.048 0.025-0.068 0.018| +0.168 0 048f-0 .136 0.010fProtein synthesis (i.e. tyrosine inc orporation into protein and protein degrada tion (i.e. tyrosine releas e from protein) were meas uredas described previously.16 Protein degradation rates were determined in the presence of cyclohe ximide to prevent concomitant proteinsynthesis. Quarter diaphragms were incubated for 2 h in Krebs-Ringer bicarbonate buffer* P < 0.025.t P < 0 . 0 1 .

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INFLUENCE OF INSULIN AND CONTRACTILE ACTIVITY ON MUSCLE SIZE AND PROTEIN BALANCETABLE 6Effects of insulin and amino acids on the degradation ofdifferent classes of proteins in rat hepatocytes

Rate of degradationInsulin plusControl amino acids Percentinhibition

Average cell protein 5.5 3.2Short-lived proteins 15.6 16.2Abnormal proteins(containing amino acidanalogs ) 40.3 42.6

420

0Hepatocytes were prepared by perfusion of rat livers withcollagenase, and the dissociated cells were cultured on collagen-containing Petri dishes. Average cell proteins were labeled byexposing cells to 3H-leucine (1 Ci/ml) for 24 h. To selectively label short-lived proteins, the cultures were exposed to 3H-leucine for1 h. To induce the production of abnormal proteins, cells wereincubated 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-Ringerbicarbonate buffer or medium supplemented with insulin (0.5

U/ml) and plasma amino acids at four times their normal con-centration in rat plasma.

INSULIN AND THE REGULATION OF PROTEIN BREAKDOWNAlthough our understanding of the mechanisms by whichcontractile activity influences the rate of protein turnoveris still very limited, significant progress has been maderecently in defining further the effects of insulin on intra-cellular proteolysis in skeletal and cardiac muscle. Thisability of insulin to retard proteolysis in muscle is of ap-preciable physiologic importance in regulating the releaseof gluco neog enic precursors from this tissue.5-16-18In fastingand the postprandial state, the net breakdown of muscleproteins and the subsequent release of amino acids intothe circulation represent important steps that limit the rateof gluconeogenesis. In fact, insulin probably is the mostimportant factor regulating protein balance in skeletaland cardiac muscle.5>15~17 The rise in insulin after foodintake promotes the net uptake of amino acids and net pro-tein accumulation in muscle, while the fall in insulin uponfasting leads to a net release of amino acids from thistissue. Experiments with isolated muscles in vitro have shownthat this hormone not only stimulates protein synthesis 15but also inhibits protein degradation in skeletal muscle(Table 5) as well as in liver, heart, and fibroblasts. 17-21-22These two actions of insulin, along with its ability to stimu-late amino ac id uptake into muscle,8-9act in complementaryfashion in vivo to reduce the flow of gluconeogenic aminoacids to liver and kidney.Even in the absence of insulin, glucose can also inhibitprotein degradation,16 but unlike insulin, glucose does notaffect protein synthesis4 (Table 5). The effects of insulinand glucose in improving overall protein balance appearadditive. Glucose may retard proteolysis by providing anenergy source, since similar effects can be demonstratedupon addition of the ketone bodies, /3-hydroxybutyrateand acetoacetate. Like glucose, ketone bodies decreaseprotein breakdown but do not affect protein synthesissignifica ntly. It has, inciden tally, been sugge sted that duringprolonged starvation, the loss of body nitrogen decreases

in humans as a consequence of the marked accumula-tion of ketone bodies, although there is litt le direct evi-dence for such a conclusion. 17This discussion of protein degradation has thus fartreated the cell as a homogenous black box, whose con-stituents all turn over in a uniform fashion. This view, how-ever, is an incorrect simplification and is potentially mis-leading.Actually, cell proteins vary markedly in their rates of

degradation.17-24 In rat liver, for example, the half-lives ofenzymes range from as short as 20 min to several weeks.17-22In general those proteins with particularly short half-lives tend to be rate-limiting enzymes whose activitiesregulate the flux of metabolites through crucial pathways.17Rapid turnover of such proteins presumably aids in theregulation of cell metabolism. Animal and bacterial cellsalso degrade especially rapidly proteins with highly un-usual conformations.17-24 Thus protein degradation appearsto serve in part as a cellu lar sanitation system by selec-tively removing abnormal proteins that may arise throughmutations, biosynthetic errors, incorporation of amino acidanalogs, spontaneous denaturat ion, or postsynthet icchemical modifications. In fact, this process also helps to

prevent the intracellular accumulation of abnormal proteinsin various human diseases, such as the certain humanhemoglobin variants17 or even the glycosylated proteinsthat may arise spontaneously in cells of diabetics.25The rates of turnover of individual proteins not only varywide ly but they also differ in their responsiveness to insu lin.In general, insulin and nutrient supply appear to retardselectively the breakdown of those proteins with especiallylong half-lives. This selectivity is nicely illustrated in re-cent data on hepatocytes obtained by Drs. Neff and De-Martino in my laboratory (Table 6). In these cells, insulinand amino acids together cause a marked reduction inthe degra dation of more stable cell proteins but havelitt le or no effect on the removal of short-lived cell proteins.

Thus insulin and amino acids had no effect on the veryrapid degradation of abnormal proteins containing aminoacid analogs. This latter process thus appears to occur inall cells and is independent of nutrient supply or endocrinestatus. Similar selective effects have been found with serumor nutrient deprivation17 -26 28 which also appear only toaffect the degradation of the most stable cell proteins.These findings and related ones have led to the sugges-tion that discrete proteolytic systems exist within cellsand serve distinct physiologic functions. Recent biochemi-cal studies have supported this idea.17-2729 For example,we have demonstrated in reticulocytes29 a soluble nonlyso-somal degradation system that seems to be responsiblefor the selective hydrolysis of abnormal proteins. By con-

trast, there is appreciable evidence that the turnover ofthe most stable cell proteins involves the lysosomal ap-paratus whose activity appears to be under fine endocrinecontrol. For example, experiments by Glen Mortimore andcolleagues have shown that in the absence of insulin andamino acids, when proteolysis is accelerated, the livercytoplasm contains large numbers of autophagic vacu-o s 2i,22 yhes e structure s ap pea r to be large lysosom escontaining part ially degraded cellular materials and prob-ably represent the primary site of the accelerated pro-tein breakdown. In related studies, we have found that theability of the rat to accelerate protein breakdown in starva-

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ALFRED L. GOLDBERG

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FIGURE 2A. Relative degradative rates of liver proteins in normaland in diabetic rats: SDS-polyacrylamide gel electrophoresis. Adiabetic rat (170 g) that had been maintained on insulin received150fid of l4C-leucine intraperitoneally, after which its insulin waswithdrawn. Three days later it was killed and its tissues pooledwith those from a normal rat that had received 500 /*Ci of 3H-leucine4 h previously. "Severe diabetes" and "moderate diabetes" referto the degree of abnormality in serum glucose content and growthrate. These data were kindly provided by Dr. J. F.Dice,Jr., andco-workers.33

t ion depends upon an adequate supply of thyroid hor-mones.18 These hormones appear to control the lysosomalcontent of liver and skeletal muscles, the two tissueswhere protein breakdown changes most markedly duringfasting.23In other words, the rate of breakdown of individual pro-teins is dependent upon both inherent structural featuresof these molecules and the overall degradative activity ofthe cell , which is controlled by insulin and other hormones(e.g., thyroxine). However, these effects of insulin appearnegligible for that fraction of cell proteins with very shorthalf-lives, such as proteins with highly abnormal con-formations. In recent years, progress has been made inidentifying certain structural features of the proteins thatmay determine their degradative rates in vivo. Schimke andcolleagues have shown that, on the average, large proteinstend to be degraded more rapidly than smaller ones inmammalian tissues24-30 (see Figure 2). In addition, Diceand I, using the double-label technique of Arias et al.,31found that acidic proteins generally are degraded morerapidly than neutral or basic ones32 (Figure 2).It is noteworthy that these correlations, whose precise

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Migration distance (mm)FIGURE 2B. Relative degradative rates of liver proteins in normaland diabetic rats: isoelectric focusing. These were the samedouble-labeled proteins used in the experiment shown in Figure 2A.Proteinswere separated according to their isoelectric points asdescribed previously. These data w ere kindly provided by Dr. J. F.Dice,Jr., and co-workers."

biochemical explanation is still unclear, were originallydemonstrated for the breakdown of proteins in normallygrowing animals. Recently, Dice et al .33 found that thesecorrelations with size and change do not apply to the ac-celerated protein breakdown seen in liver and muscle indiabetes or starvation (Figure 2). In these animals, theselectivity for large and for acid ic molecu les is lost, and thiseffect is most marked in animals whose diabetic symptomsare most pronounced.This apparent loss of specificity in the degradativeprocess may be a reflection of the earlier finding thatthe acceleration of proteolysis in the absence of insulin

results from an increas ed breakdown of the more stable cellcomponents, which are necessarily the smaller, more basicproteins. Perhaps also the loss of selectivity in starvationor diabetes reflects the activation of a lysosomal process,which may act by engulfing cell proteins in a relativelynonspecif ic way.Whatever their basis, these results (Figure 2) may also beof appreciable clinical signif icance since they predictthat the accelerated protein breakdown in diabetes orstarvation should seriously alter cell composition. In otherwords, the proteins being lost in these ca tabo lic states shouldbe different from those degra ded when there is an adequate

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INFLUENCE OF INSULIN AND CONTRACTILE ACTIVITY ON MUSCLE SIZE AND PROTEIN BALANCEsupply of insulin. Since the lack of insulin decreases thesynthesis of various cell proteins to a similar extent, 15whilethe insulin def iciency promotes select ively the degrada-tion of the more stable cell components, there should bein diabetes a differential depletion of small and basicproteins. Such a change in cell composition could haveprofound physiologic consequences.SUMMARYAlthough the biochemical mechanism by which insulinand contractile activity affect protein turnover in muscleare still unclear, certain physiologic conclusions can bemade: (a) Increased work can induce muscle hypertrophyeven in the diabetic or starving animal. Thus, work-inducedgrowth differs from normal growth of muscle in not re-quiring insulin, (b) Like insulin, repeated contractions stimu-late the transport of amino acids into muscle. Contractileactivity or passive tension can also reduce the rate of pro-tein degradation in this tissue. These effects can be shownwith isolated muscles in vitro, but the mechanisms couplingcontractile activity to these anabolic processes are un-known, (c) Insulin also reduces overall protein breakdownin skeletal mu scle, heart, and liver, appare ntly by re gulatinglysosomal function, (d) Insulin reduces selectively the break-down of cell proteins with relatively long half-lives. Thishormone does not affect the rapid breakdown of abnormalproteins which probably does not occur within the lyso-some. (e) Normally, liver and muscle degrade large,acidic cell proteins quite rapidly, but this selectivity is lostin the tissues of diabetic or starved organisms, in whichproteolysis is accelerated.REFERENCES1Goldbe rg, A. L: Mechanisms of growth and atrophy of skeletalmuscle. In Muscle Biology, Vol. 1, Cassens, R. G., Ed. New York, MarcelDekker, 1972, pp. 89-118.2Goldbe rg, 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 m uscle innormal and hypophysectionized rats. Am. J. Physiol. 372:1193-98, 1967.4Goldbe rg, A. L: Role of insulin in work-induced growth of skeletalmuscle. Endocrinology 83:1071-73, 1968.5Cahill, G. F., Jr., Aoki, T. T., and Marliss, E. B.: Insulin and muscleprotein. In Handbook of Physiology. Endocrinology 7:563-77, 1972.6 Li, J. B., and Goldberg, A. L: Effects of food deprivation on proteinsynthesis and degradation in rat skeletal muscles. Am. J. Physiol. 237:441_48, 1976.7Goldberg , A. L, and Goodm an, H. M.: Relationship betweencortisone 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 ofL-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, trypsinand electrical stimulation on amino acid transport in muscle. J.Biol.Chem.249:5435, 1974.

10Goldberg , A. L, and Goodman, H. M.: Amino acid transport duringwork-induced growth of skeletal muscle. Am. J.Physiol.276:1111-15, 1969.11Goldberg , A. L, and Goodm an, H. M.: Effects of disuse anddenervation on amino acid transport by skeletal muscle. Am. J. Physiol.276:1116-19, 1969.12Goldberg , A. L, Jableck i, C. M., and Li, J. B.: Effects of use anddisuse on amino acid transport and protein turnover in muscle. Ann. N.Y.Acad. Sci.228:190-201, 1974.13Arvi11, A.: Relationship between the effects of contraction andinsulin on the metabolism of the isolated levator ani muscle of the rat. ActaEndocrinol. 722:27-41, 1967.14Oxender, D. L, and Christe nsen, H. N.: Distinct m ediatin g systemsfor the transport of neutral amino acids by the Ehrlich cel l .J .Biol.Chem. 238:3686-99, 1963.15Wool, I. L. G.: Effects of insulin on ce llular protein sy nthesis.Handbook of Experimental Pharmacology, XXXII,2:267-302, 1975.16Fulks, R., Li, J. B., and Gold berg , A. L: Effects of insulin, gluc oseand amino acids on protein turnover in rat diaphragm. J. Biol. Chem.250:290-98, 1975.17Goldbe rg, A. L., and St. John, A. C : Intracellular proteindegradation in mammalian and bacterial cells: Part II. Ann. Rev. Biochem.45:747-803, 1976.18Goldberg , A. L , DeMartino, G. N., and Ch ang, T. W.: Release ofgluconeog enic precursors from skeletal muscle in Regulatory Mechanismsof Carbohydrate Metabolism , F.E.B.S. Lett. 42: 347 -58, 1978.19Goldberg , A. L:.P rotein turnover in skeletal muscle. II. Effectsof 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 pro-tein turnover. J. Physiol.2 64 (7J:283-96, 1977.21Mortimore, G. E., and Neely, A. N .: Regulatory e ffects of insulin,glucogon, and amino acids on hepatic protein turnover in associationwith 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 intracellularamino acids in rat liver. J.Biol. Chem. 253:3581-87, 1978.23DeMartino, G. N. , an d Go ldberg , A . L : Thyroid hormones contro llysosomal enzyme activi t ies in l iver a n d skeleta l muscle. Proc. Nat l .A c a d . Sci . U.S.A. 75 :1369-73 , 1978 .24Go ldberg , A . L , a n d D ice , J. F.: Intracel lu lar prote in degr ada-tion in mammalian and bacterial cells. Annu. Rev. Biochem. 43:835-69,1974. 25 Bunn, H. F., Gabbay, K. H, and Gallop, P. M.: The glycosylationof 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 ofcellular protein or rat fibroblasts. J. Biol. Chem. 248:6 221-2 6, 1973.27 Hopgood, M. F., Clark, M. G., and Ballard, F. J.: Inhibition of pro-tein degradation in isolated rat hepatocytes. Biochem. J. 764:399-407,1977. 28Neff, N., and Go ldbe rg, A. L: Effects of protease inhib itors onthe degradation of different classes of proteins in rat liver. Submittedfor publication.29Etlinger, J., and G oldberg, A. L: A soluble, ATP-dependentproteolytic system responsible for the degradation of abnormal proteinsin reticulocytes. Proc. Natl. Acad. Sci.U S A 74:54-58, 1977.3(1Dice , J. F., Dehlinger, P. J., and Schim ke, R. T.: Studies on thecorrelation between size and relative degradation rate of soluble pro-teins. J.Biol. Chem. 248:4220-28, 1973.31 Arias, I. M., Doyle, D., and Schimke, R. T.: Studies on the synthesisand degradation of proteins of the endoplasmic reticulum of rat liver. J.Biol. Chem. 244:3303-15, 1969.32Dice, J. F., and Goldb erg, A. L: Relationship between in vivodegradative rates and isoelectric points of proteins. Proc. Natl. Acad. Sci.U S A 72:3893-97, 1975.33Dice , J. F., Walker, C. D., Byrne, B., and Card iel, A.: Gene ralcharacteristics of protein degradation in diabetes and starvation. Proc.Natl. Acad. Sci.U S A 75:2093-97, 1978.

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