Insulin and protein synthesis in muscle

Insulin and Protein Synthesis in Muscle’

SA:\IUEL GOLDSTEIN? AND WILLIAAI J. ItEDD‘1-3

Baker Research f,aboralor~y, -\-ew England Deaconess Hospital and Peter Bent Brigham Hospital, Departments of Medicine and Biochemistry, Harvard Medical School, Boston, Jfmsachuaetts 02115

Received April 6, 1970; accepted June 16, 1970

Insulin stimulates in vilro the incorporation of labeled amino acid into muscle

protein. A st ttdy of t,his phenomenon has been carried out in which various incubation

conditions have been varied. The insldin stimulatorv effect, is lost when sodium ion is removed from the me-

dium, when the amino acid concentration of the medium is increased to twice t)hat occurring physiologically, or when the experimental design is modified to minimize the effects of transport or turnover on intracellular specific radioactivity. These studies show that insulin exerts its effect almost entirely on amino acid transport,.

The incorporation of labeled amino acid into protein should not be equated with protein svnthesis wit,hout consideration of factors which affect the specific radioactivity of the immediate precursor.

Many articles during t,he past decade have presented evidence for hormonal stimulation of protein synthesis. The experimental design has usually involved t,he incubation of tissue in buffer containing a radioactively labeled amino acid in the presence and absence of l-he hormone. The assumption is then made, that incorporation of the labeled amino acid into prot’ein reflects the rate of protein synthesis. Our studies have been ad- dressed specifically t,o the question of insulin and protein synthesis in muscle. The evidence t’o be presented suggest.s that the effect’ of insulin on st,imulatjing the incorporation of labeled amino acid into protein cannot, bc equat>ed \\-ith protein synthesis without knolving the specific radioactivity of the immediate precursor.

’ This investigation was supported by a grant from MI-1 AM 09262.

p Postdoctoral Fellow of the Diabetic Training Grant NIH AM 05077. Present address: Depart,- ment of Medicine, M&laster University, IIamil-

ton, Ontario, Canada. a Present address: I)epartment of Medicine and

Uinchemistry, University of Alabama Medical

s(~ltool.

MATERIALS AN1 METHODS

Preparation of muscle. Charles River COBS

male rats, 160-200 g were sacrificed by decapita-

tion and exsanguination after a 16.hr fast. Bi- lateral caudofemoralis muscles were rapidly ex-

posed and excised intact at the origins and inser- tions. The suitability of this muscle preparation

for in vitro studies has previously been described (l-4). After longitudinal stripping illt,o halves,

right and left were divided so that each animal

contributed to test and control samples. One caudofemoralis muscle weighed 100-150 mg. After weighing, the tissues were kept on normal salinc-

soaked and iced filter paper till addition to flasks. Incubation proccdzrre. Krebs-Ringer phosphate

buffer, pH 7.2, modified by the omission of Ca2+

and Mg2+, was used ill all experiments except in

the low sodium or Na+-free buffers, where K+ re-

placed iYa+ in isoequivaleu t amounts. Our experiments have shown that omitting Ca?+ and hlg”+

does not materially affect the results, confirming an earlier report (5). III srlch experiments the

phosphate compouent remained constant. The buffer contained, except when otherwise indicated, a mixture of 18 unlabeled amino acids approxi-

mating intracellular concentrations.4 These values

4 Concelltration of nmitlo acids ill medium (ml%): Ala 1.69, Arg .M, Asp 31, C,-s 31, Cl11 2.x2.

Gly 6.83, His 1.23, Ilrrl .21, Leu 31, Lys .97, RIrl,

181

152 GOLDSTEIN AND REDDY

were derived from a report on thigh muscle of fasted male rats (6). All incubations except those for determinat,ion of %-amino acid specific radio-

activity were carried out in 5.0 ml of medium in 25-ml Erlenmeyer flasks fitted with rubber serum

sleeve-type stoppers. Flasks were charged with 100% O2 for the first 10 min of each incubation.

When additions were made after a preincubation interval, the flasks were recharged with 100% 02

for the first 10 min of the subsequent incubation. Ins&in. Insulin dissolved in 0.01 N HCl was

added, 0.1 ml to each flask so that the final concen-

tration was 0.1 unit/ml. To the control flasks, 0.1 ml of 0.01 N HCI was added. Insulin and control

solutions were present from t,he time indicated. The addition of tissues was synchronized meticu-

lously so that each pair of samples made contact

with the medium at the same time. In experiments where tissues were present in the medium before

insulin addition, similar care was taken to add insulin and control solutions synchronously

within each pair. Additions to all flasks were com- pleted within 2-3 min.

Isotopes. 14C-amino acids (all uniformly labeled) were obtained from New England Nuclear Corp.

and diluted with the l&amino acid mixture giving

final specific radioactivities seen in Table I.

Total protein. One volume of 200% trichloroacetic acid was added to each flask upon comple- tion of incubation. Tissues were then finely minced

and transferred to centrifuge tubes, centrifuged, and the supernatant fluid discarded. The residue

was treated with boiling 5% trichloroacetic acid

to solubilize nucleic acids. The supernatant fluid was discarded. The resultant protein pellet was

dissolved in 1.0 N NaOH, and an aliquot was worked up for liquid-scintillation counting as

described below. Protein determination (7) was performed directly OJI another aliquot of the 1.0 N

NaOH protein solution using bovine albumin

standards. Protein fractionation. The rationale and pro-

cedure for preparing 0.2 ionic strength extracts

have already been described (3). In brief, incubation was terminated by cooling the flasks to 0” in

ice water. 14C-leucine incorporation into protein is negligible at this temperature. Five milliliters

of 0.22 M KC1 (ionic strength 0.22) were added to each flask containing 5 ml incubating medium (ionic strength 0.18) followed by transfer to the

homogenizing flasks. Tissues were homogenized in l-2 min to fine particles at high speed on the Virtis-45 homogenizer. One drop of 2-octanol was added to each flask to reduce foaming. The con- tents of the homogenizing flasks were centrifuged

.14, Phe .16, Pro .57, Ser 1.07, Thr .83, Try .lO, Tyr .27, and Val .39.

at 16009 for 20 min, and the supernatant was decanted.

Liquid scintillation counting. An aliquot of the

supernatant fluid was precipitated with 2 vol of 20yo trichloroacetic acid, boiled wit.h 2 vol of 57,

trichloroacetic acid, washed consecutively with 2 vol of ethanol-ether (1: 1) and et,her, then dried

under negative pressure in a desiccator. Two milliliters hyamine (1 M in methanol, Packard Instru-

ment Company) were added for overnight hy-

drolysis in sealed tubes at 60’. To the hydrolyzate were added 15 ml of toluene-2,5-diphenyloxazole,

1,i bis -2(4-methyl-5-phenyloxazolyl) -benzene

fluor solution followed by transfer to vials and counting in a liquid scintillation counter at an

efficiency of 33yo. Quenching, which was always

less than 595, was corrected by the internal stand-

ard method. Another aliquot of the supernatant fluid was treated identically except for omission

of boiling with 5% trichloroacetic acid. The cleaned and dried pellet was dissolved in 0.1 N

NaOH and quantitated as above. Determination of specific radioactivity of intra-

cellular K’-amino acids. Twenty-four normal male

rats, fasted overnight, were sacrificed, caudofemoralis muscles were excised, stripped into

halves and distributed so that approximately

1400 mg of tissue were incubated in each of four flasks. The 40 ml of medium contained the amino acid mixture, and inulin at 10 mgiml. Nine 14C-

amino acids were also present as the uniformly

labeled derivatives at specific radioactivities given in Table I. The volume of medium was chosen to

be about 50 times that of the intracellular fluid so that no significant decrease in amino a,cid concen-

tration or specific radioactivity was observed during the incubation. After incubation, total tissue

water and inulin space were det.ermined (8), and aliquots of deproteinized medium and tissue water

were applied to a Beckman amino acid autoana-

lyzer in two successive runs. In the first run, elIhI- ent was collected in fractions directly from the autoanalyzer’s ion-exchange columns for determi-

nation of radioactivity of each of the nine l*C- amino acids by liquid-scintillation counting as

below. In the second IXJI, amino acid concentrations were determined in the usual manner by the

autoanalyzer. The inulin space was calculated and taken to represent extracellular space. Ext,racellular tissue 14C-amino acid radioact,ivity and

concentration were then subtracted from the total, and results were expressed as cpm!pg itltra- cellular ‘4C-arnillo acid.

Determination of intracellular spwijic ratlioac- tivity of proline. The incubation was carried out as

described with all additions to the medium, in- cluding insulin, present from zero time. In these experiments, inulin was agait presellt at a concell-

INSULIN AND PROTEIS SYNTHESIS lS3

tration of 10 mg/ml. After incubation, tissues were

drained and blotted, then boiled in water for 5 miu

to liberate amino acids (9). After centrifugation,

the supernat,ant fluid was deproteinixed with trichloroacetic acid, and aliquots were removed for

three determinations. Radioactivity was deter-

miucd directly in an ethanol-toluene-2,5-di-

pherryloxazole, 1,4-bis-2(4 -methyl -5 -pherryloxa-

zolyl) -benzene fluor mixture which maintains a monophasic state. Counting efficiency was 20’,; in

this system. Proline concentration was measived accorditrg to Troll and Lindsley (10) while iiiuliir

det,erminat,ion and subsequent calculatioiis were

doue as above. End-group analysis. A modification of the

N-terminal analysis described by Porter (11) was

carried orit, and over 98yc of the labeled arniiio acids were found to be protected from reactiug

wit.h IINFB, indicating that incorporation into interior peptide linkage had occurred. Hydrolysis

of other protein aliquots, followed by isolation of

iGle\icine iising descending paper chromatogra- phy with butanol:acetic acid:water (5:1:4) as de-

veloping solvents, showed that over 99(-i of the

iucorporated radioactivity had a mobility identical to leucine standards. When identical chromato-

graphic analysis was done in the r4C-proline experiments, the corresponding value was foulid to

exceed 95:-i. Statistical analysis was by the paired ( test.

RESULTS

It can be seen (Table I) that) after 15 min of incubation? none of the amino acids

TABLE I

}‘:qrILII~ILATION OF SPECIFIC RADIOACTIVITIES OF AMINO ACIDS WITH TIME”

Specific

LIC. amino

Specific radioactivity (cpm/wde X 106)

radibactirity ‘$G equilibration

(intracellulari extracellular 1

rrrirl - - -“.- Extracellular Intracellular fluid

fluid After After After After (medium) 1.5 min 60 min 15 min 60 min

Thr

Pro \-al

Met

Leu

Phe

LT’S His

82.B 23.9 113.6 03.5

lii.8 104.7 378.8 190.8

191.8 104.6 333.0 133.8

54.9 5.8 44.1 29.3

100.1 31.7

29 ~

i5.2 5G (iG

59 ~- 50 ~-

138.2 55 72 40 ~~ 11

38.8 GG 88 32

n IJach value represents the average of two determinations.

TIME (min)

FIG. 1. Effect of insulin on intracellular specific

radioactivity of r4C-proline as a fuuctiou of time.

Sn = intracellular specific radioactivity, Sa = extracellular specific radioactivity-. Details de-

scribed in Methods.

in the intracellular fluid of caudofemoralis muscle has attained the specific radioactivity of the extracellular fluid. Or+ histidine has equilibrated appreciably beyond 50 70. Indeed after 60 min, the t,hree amino acids shown have fallen distinctly short of equilibration. The rate of equilibration of specific radioactivity appears to be a property of each amino acid.

Figure 1 reveals that] insulin accelerates the rate of increase in the specific radioactivity of 14C-proline within int~racellular fluid. kisulin’s greatest effect both in abso- lute and percentage terms is at 10 min, the earliest time studied. The effect, is seen to diminish at 20 min, and after 60 min specific radioactivity in the control is not) signifi- cantly different from that obtained in the insulintreated samples.

In experiments reported in Fig. 2, the tissues were added at zero time to the medium containing 14C-leucine and, where appropriate, insulin and control solutions. It can be seen t,hat insulin has produced a significant stimulation of *4C-leucine incorporation into iota1 protein of the caudofemoralis muscle. In subsequent experiments, the specific radioactivity of protein in the 0.2 ionic strength extract, is reported.

The results of incubations carried or1 in buffers where Na+ concentration is first markedly reduced and, secondly, eliminated, being replaced by I<+ in isorquivalcnt

1x4 (;oLI)s’lY~:l~

amounts, is sIron-ii in I’ig. 3. Iicsults of :tn incubation \vit 11 a physiological S:t+ cow

ceikation are shown at the extreme Icft for comparison of basrlittc 14C-lwcittr> incorporation and iis stimulation b!. insulin. The marked reduction in incorporation of 14C- leucine at, a JJ:t+ concentration of 7 meq with :I further reduction at zero Sa+ is obvious. The loss of insulin’s effed on this parameter is seen :tt both lo\v and zc’ro S:t+ concent,rations.

300 CPM PER

MGM. PROTEIN 200

100

0 INSULIN CONTROL

FIG. 2. Effect of insulin on ‘4C-leucine incorporation into total protein of catldofemoralis.

Tissues were added at zero time to 5.0 ml medium

without carrier amino acids. Specific radioactivity of leucine was 223 fiCi/pmole. Insulin was present

from zero time in half of the flasks. Incubation time was 1 hr. The vertical bars represent the SE

of the mean. N = 8 for each column. Insulin effect

p < .Ol.

3oo

t

[Na*] = 145

CPM PER MGM. PROTEIN I

The effect of ittctwsing :miitio acitl entry \\-:I:: studird by doubling tltt‘ cottc.rtttr:~tiott of t,licb :tmitto :Icicls itt (hr. mr~tlittin (Fig. 4). Ko itisuliii rficct \v:~s &dctit, 1ltitler tllcse

cotidit ions. Tlw ittcorpotxt ion of 14(‘-lrucit~c into thr control wi~ples rq)or~~~l iii Fig. :5 \v:ts 130 cptn mg 1)roi An. Tl~f> ittc’orl)oL.:Ltiotl

200 1

CPM PER MGM. PROTEIN 100

0 INSULIN CONTROL

FIG. 4. IMect of do\tblillg amino arid concentration 011 illsulill stimulation of ‘G!er1cinr iucorpo-

ratio11 into 0.2 ionic strength protein extract. Tissues were :~tldctl at zero t,ime to 5.0 ml of me-

dium. 0.4 ,Ki 14C-lcrlcilx per ml was used here to keep specific radioactivit,y constnrlt, at 0.1; hCi/

&mole ill face of doubled collceutratiotls of III,-

labeled leucillc (ntld ot.hcr unlitlo acids). Insulin and collt,rol solutiotls were prcsetlt from zero time.

Incubation time was 1 hr. The vertical bars represent the SE of the mea11. L\- = 8 for each CY~I~I~II.

iY0 significant, elf’ect of itlsldill.

[Na’] = 7 [Nat] = 0

INSULIN

0 CONTROL

FIG. 3. ERect. of ionic composition on insulin stimrllation of ‘J(J~lrr~cine incorporalior~

into 0.2 ionic strength protein extract. Tissues were added at zero time to 5.0 ml of medium containing 0.2 PCi 1°C.leucine/ml, 0.6 &i/@nole, and the 1%amino acid mixtr1r.e in normal concentrations. Insulin was present from zero time. Inrubxtioll time was 1 hr. Thrl vertical bars represent the SE of t.he mean. LV = 8 for each column. Insrdin effect [N:t+i = 145, p <

.OOl; [Na+] = i, KS; [Na+] = 0: X3.

I:inal intracellular concentration tpmoles,‘~ wet weight1

Amino acid Incubated 30 min ~ Incubated 60 min

I,eucine

.‘U:tllill?

Aspartic :rcid

Glot amic acid

Glycine

Isoleucinrt Methioniirc l’he~~ylalar~i~~e

Prolillcl Serine

Threonine

Tyrosine \-alille

I

I 2 ,3

4.412

,377 2.213

10.585

,278 ,148

,250

,680 2.749

1.599

,382 ,515

2.395

.2i9 1.105

5 ,099

,155 ,024

,134

.3i9 1.456

,838

,495

1.017 ,191

2.623

10.721 ,312

,180 ,253

,721 2.292 1 ,443

,204 , ,393

,284 ~ ,587

,237

2.249

.298 1.112

5.973 ,151

Oi4 lli-4

.385 1.312

,770

,229

,207

a Ca\,dofemoralis muscles from 12 rats were

randomly distributed into four pools. Samples 2 altd 4 wrre incubated in Krrhs-Ringer phosphate

buffer caontaining a mixture of 18 amino acids at normal concentrations. Samples 1 and 3 were

incubated in the same bluffer hut, rontaining double the amino acid concentration. Incubation

times are shown. The specific radioactivity of

14C-lcucine in the medium was constant. After incubation, the tissues were rinsed, boiled in

distilled water, and deproteinized with trichloroacetic acid. After extraction with diethgl et,her

the supcrnatant fluid was examined on the Beck- man analyzer for amino acid concentrations.

into t,he cont,rol samples of Fig. 4, where the concentrations of amino acids in the medium were doubled, was essentially the same. The data in Table II show that the final coriceril rations of intracellular leucine and other amino acids double after being incubated in twice the normal extracellular concentration. However, intracellular specific radioactivities for leucine at each time were found to be essentially the same, 0.49, 0.46, 0.51, and 0.52 X lo6 cpm/pmole for samples 1, 2, 3, and 4, respect,ively.” Tlrcl

5 The precipitate obtained after boilily in water

was extracted with a potassium carbonate bklffet as described by Wolfenden, It ., Hiochemi.slr~/ 2,

~prn mg protein I’reincubalion ~~~ P

Insulin COiltD31

CA) 37" s2 zt x 87 + 12 ss (IS) 0" 30 It 4 2(i f 4 KS

n (I) Preincltbatcd in Krcbs-Ringer phosphate

buffer (A) at 37” or (H) at 0” for 30 min; (II) Washed with normal saline at 0” to drpl~t e crtr:i-

c~cllrllar fluid ‘C; (III) Final incrlbatioll in Krebs-

Itinger phosphate brlffer milllis amino acid mist IIre ai 37” for 60 min with and withorlt illsldil,.

T.-\I31,1~: r\-

Insulin Control II

232 f 18 “2X + 26 ss

/ (I) I’rtqr~ilihrat iota in Krebs-I’Lingrr phos- phatc bufl’er at 3i”; (II) Insrdin ;rnd cc~nlrol solr~-

tions added nfrcr 30 min; (III) In~l1b:lticln ~011. t inrled for addit ional GO rni11.

specific radio:tctivii\. of leucine in thr mc- dium \V:IS constant at 0.60 X lOti cpm/ pmole.

Further maneuvers were desigwd lo control the specific radioactivity \vithin the intracellular pool. In Table III, t,tre step- wise procedure employed in some such experiments is outlined. No insulin is present during the Wmin preincubation interval. This step is designed to accumulate 14C- leucine inside cell water nncl so separate amino acid entry from its subsequent incorporation into protein. The specific radioact,ivity of intracellular 14C-leucirrr: is about, 70(X of the extracellular after 1 hr at 37” (Table I). The tissues n-err then incubatetl in fresh buffer for 1 hr at 37” wit,h and n-itlr-

1090 (1963). The radioactivity solubilized by this treatrnellt presumably represents ‘Camino acid

bound to &X:8. The values obtained were 15,700, 9,400, 17,500 and 8,400 cpm/g of tissue for samples 1, 2, 3, alld A, respectively. (Note that this is total

radioactivity. not specific radioactivity.)

186 GOLDSTEIN ANI) I<EI)l)Y

500-

400-

300 -

CPM PER MGM. PROTEIN

200-

INSULIN

1

N.S.

HOURS OF INCUBATION

FIG. 5. Effect, of extracellular specific radioactivit,y on insulin stimulation of ‘Gleucine

incorporation. Tissues are added to flasks and incubated in Krebs-Ringer phosphate buffer in the presence of 0.2 pCi/ml for 1 hr specific radioactivity 0.6 /ICi/pmole. The l&amino acid

mixture is present in normal concentrations. Xo instdin is used in first hottr in either A (left) or B (right). In A after 1 hr, insulin and control solutions are added to appropriate flasks

and incubation continued for a further hour. In B after 1 hr, instdin and control solutions

are added to appropriate flasks, and 1 PCi i4C-letmine is added to all flasks and incubation continued for a further hour. A significant effect of instdin is seen in B.

out’ insulin. It, can be seen that. no insulin effect is evident, whether preaccumulation was effected at 37 or 0”. When the 14C- amino acid was added after 30 min of preincubation and washing a significant effect of insulin was obtained, control 57 (SE f 5), insulin 77 (SE f 9), N = S, p < .Ol, indicating that responsivity of the tissue was preserved after these manipulations.

In addition, anot,her design was employed, and the results are presented in Table IV. Here, rather than removing and washing t,he tissues after the preincubation interval, the tissues were kept. in the same medium and flasks throughout. During step I, intracellular fluid specific radioactivity is allowed to approach equilibrium in all tissues (re- ferred t,o as a preequilibration interval). Insulin and control solutions were then added direct,ly to the flasks and incubation continued for one more hour. So insulin effect was observed.

In the Fig. 5, are shown the results of an experiment in which the procedure in A was similar to that of Table III. That is, following a preequilibration interval of 1 hr

(in this case), insulin and control solutions were introduced into the flasks, followed bJ continued incubation for a further hour. As in the previous experiment, no significant insulin effect, R-US demonstrated. The re- maining samples sllowr~ in B, received the same insulin and control additions after 1 hr but simultaneously received an additional 1 PCi 14C-leucme, thereby acutelv doubling the extracellular fluid spe&c radid- activity. A clear-cut stimulation by insulin of 14C incorporation is shown. Similar results are found using “C-proline (Fig. 6).

DISCUSSION

Profound derangements in nitrogen bal- ance are seen in both experimentally in duced diabetes of animals and in uncon trolled spontjaneous diabetes of man. Their amenability to correction by insulin leaves little doubt of this hormone’s central role in the regulat,ion of pro1 ein synt,hesis. That, insulin stimulates the incorporation of labeled amino acids into the protein of a diverse array of tissues has long been known on the basis of both in vivo (12, 13) and in

INSULIN AND PROTEIN SYNTHESIS IS7

tiOURS OF INCUBATION

FIG. 6. Effect of extracellular specific radioactivity on insulin stimulation of ‘Gproline

incorporation. Conditions otherwise identical to Fig. 5.

vitro (14-19) experiments. These studies have employed the classical experimental design6 wherein Gssues are exposed from zero time t,o the labeled amino acid in t,he presence or absence of exogenous insulin. This design does not different)iate between an effect of insulin on membrane transport and an effect at, an intracellular &e.

The curve depicting the rate of change of the specific radioactivit)y of free amino acid inside the cell (Fig. 1) is given by the equa- tion

where S, and Sa are intracellular and extracellular specific radionctivit,ies, v is velocity, R is intracellular concentration, and t is time (20). Under conditions where only the rate of uptake or the rate of exchange of the radioact,ively labeled amino acid is increased, a greater incorporation of label would be expected until equilibration had occurred. As shown in Table I, equilibration of specific radioact,ivity of the free amino acid is specific for each amino acid and requires at, least, 1 hr of incubation.

6 For purposes of convenience, the authors define classical experimental design in this way.

The amount, of label incorporated into prot,ein is determined by the specific radioactivity of the immediate precursor, 14C- amino acyl- sRNA, and the rate of protein synthesis (21). Under conditions where the rates of amino acid activation, amino ncyl- sRNA formation, and protein synt,hesis are constant but the rates of 14C-amino acid upt,ake or exchange are increased, an increased incorporation of label into protein should be observed. Ideally the specific radioactivity of the 14C-amino acyl-sRXA pool should be measured and,!or conditions for maint,aining it constant developed. Since technical difficulties precluded such studies, a series of indirect experiments were designed to test the hypothesis that the increased incorporation observed in the presence of insulin result,ed from an increased rate of uptake of label.

The insulin effect on stimulating incorporation of 14C-leucine into protein was abolished by incubating in Krebs-Ringer phosphate buffer with concentrations of 18 unlabeled amino acids increased tfo double those found in intracellular fluid. One would not expect, an insulin effect distal to the membrane, such as on the protein synthetic machinery, to be obliterated by a higher

188 GOLI)STEIN AND I~EI)I)Y

concentrat,ion of amino acids (22). Using the same design but, now replacing Na+ wi’ith Ii+ in the medium, the insulin effect, on stimulating 14C-leucine incorporation into 0.2 ionic strength extracts of tissue protein was again abolished (k’ig. 3). It, is known that amino acid transport in skeletal muscle is Na+- dependent. Blocking Ka+ transport with cardiac glycosides, complete removal of Ka-l- from the incubation medium, and stepivise replacement of Na+ with I<+, greatly reduces amino acid penetration into cell water of diaphragm and abolishes insulin’s effect on this parameter, in contrast to t,he case of sugars (23). Thus, the effect, of insulin \V:LS eliminated by both increased and decreased amino acid entry. P’urther- more, doubling the intracellular amino acid concentration does not seem to affect the rate of prot,ein synthesis. In tot,al, these observations suggest, that the increased incorporation of 14C-leucine observed in Fig. 2, and t’he left side of Fig. 3, results from insulin’s stimulation of transport.

In the experiments departing from the classical design, an at lempt’ was made to circumvent, the transport step. Upon pre- accumulating the labeled amino acid, followed by washing and transfer t,o fresh media, an insulin effect was not, demon- strable. A preequilibration of labeled amino acid bet’neen intm- and extracellular com- part,ments, followed hi- addition of insulin and furt,her incubation, virtually abolishes the insulin effect, seen in the classlcal design. Introduction of additional labeled amino acid concomitant with the insulin restores the insulin effect. In this latter group, additional label was introduced after the original label had approached equilibrium bet\\-een intra- and extracellular fluid compartments. The newly added label must non- equilibrate, and here a clearcut stimulation by insulin of 14C incorporation is shown. JIoreover, this effect was observed with t\vo amino acids, leucine and proline, handled by different transport mechanisms (24, 25). This provides evidence for the effect of insulin at the membrane, in rsser~e by putting a tag on t,he transport step. Parallel results were obt,ained in diaphragm (Goldstein and Reddy, unpublished data), and a third muscle preparation, isolated perfused rat lie& (26).

Wool et al. have proposed (27) that the primary act,ion of insulin is to form a “translat,ion fact,or” by initiating transla- tion of the stable template RKR for that factor. They have shown that, alloxan diabetes decreases the number of act,ive ribo- omes obtained from skeletal muscle of rats

(2S), and more recently that the decreased ability of diabetic ribosomes, to support polyuridylic acid-directed protein synthesis at, suboptimal Mg++ concentrat,ions, is a property of the 60s subunit, (29). These conclusions are based almost exclusively on studies of tissues from insulin-deficient animals compared to normals. Further, insulin’s effects on the ribosome are said to be independent of amino acid transport (30) using condit’ions, such that one or more amino acids may have been rate-limiting for prot’ein synthesis (31-34). In this situn- tion insulin may have restored optimum conditions by either increasing t,he transport, of effluent amino acids or retarding their exit from t.he intracellular space (35).

It must, be considered that insulin acts primarily to increase the rate of amino acid utilization in t’lie intracellular pool, facili- tating the influx of extracellular amino acids and thus secondarily increasing the specific radioactivity. However, our data in con- junct’ion wit’li previous reports using the nonutilizable amino acid, cu-amino isobutyric acid, as well as natural amino acids (23, 36X9), provide good evidence for a primary effect, at. the transport level. In this regard, it’ has been proposed (40) that. transport may be mediat,ed in two different ways, by increasing t,he affinity of t,lie carrier syst.em for amino acids, and by initiating the sun- thesis of a specific protein or proteins which enhances transport. In any case, augment’ed transport, by increasing the availability of amino acids to a distal synthet,ic machinery could then enhance protein s\ntlwsis as shown under conditions of limited amino acid availabilit\. in liver slices (41), and several cell types grown in tissue culture (22). Indeed, A\lunro has shown that the supply of amino acids has :I profound effect, on ribosomal assembly into polysomes (42).

In the present. studies where amino acid concentrations arc not limiting, tile evidence indicates that neither basal nor “insu- liwstimulated” protein s)-nthesis increases

INSULIN ANI) I’ROTISI?; SYXTHKSIS lS9

wlim the intracellular concentrations are within the range of once to twice that occurring in normal rats fasted for 1G hr. It follon-s that increased incorporation of lu- beled amino acid is not equivnlentj t,o increased incorporation of unlnbeIet1 amino acid. Over :L decade ago Russell pointed out the pitfalls in estimating mtes of all met:l- bolic rwrt ions with isotopic tracers (20). This 11:~s been recently sl~own in studies of RNA (33, 44) and DY,4 (4.i, 46) synthesis, and is IIO\V repeated here for studies of protein s~III I~rsis.

ACKNOWLEL%MENTS

We thank I)r. M. Root of the IS. Lilly Co. for the illsulilt rued in these studies, ?v3. A. Brienzo, M. IIervart,h, and E. Boyle for expert technical

assistance, a11t1 Dr. 8. IIartman and Miss Henry

Jane Becherer, Department of Biochemistry, Harvard &Iedical School, for use of the amino acid

analyzer.

8.

9.

to.

11.

12.

I:(.

14.

15, 16. 17.

REFERENCES

~~~.~NF~I~H, w. II., .Q?D Lnozr, J. B., J. Viol.

Chem. 239, 40-17 (1961).

BOCIK, 1:. itI., PI:TEKSOS, It. D., .\XD BC~TTT, C. H., Bmer. J. Physiol. 210, 1101 (1966).

~:OLDWEIN, s., .\XD k<DDY, w. J., &)&,,I.

Bioph!/s. 4clu 141, 310 (1967).

~:OLDSW:IN, s., .\ND l~l~DDY, w. J., Rioc~hirn.

Biophys. Acta 150, 733 (19F8).

X~N~HI.;S’I~I~.R, K. I,., Uiochem. .I. 98, 711 (196fi 1.

S.\SSISNIL.\‘PH, I<. N., .\su C;~~I~:SX~I~I-;IX, 1). ?\1., Ctcncrr Res. 14, 563 (1954).

LO\VRY, 0. TT . , ~~OSl~IM~UGH, N. .J., F WI<:

A. L., ANI) ~kND.\I.L, I:. J., .I. niol. Chcril.

193, 365 (1951).

IiIG.\SHI, L4., .\SD PETlW3, II., .,. f,cIh. (‘ht. died. 35, 175 (1950).

M.\N(xI:s~I,x, K. L., .\su Youxc, F. (;., ISo-

chew. J. 75, 487 (1960). TI~OLL. W., .\XD LIX)RLI;Y, J., .J. Bid. U~em.

215, 653 (19553.

I’oI~~~P:II, 1:. Ii., in ‘Xethods it1 Ellzymology” (S. P. Colowick and N. 0. Kaplan, eds.), p ‘L21. Academic Press, New York (1957).

P\Irl~au, I. A., .,Ltuer. J. Physiol. 124, 569

(1938). FOlLKKl1, L. IA., CH \IICOFF, 1. 1, ~<NTI~:Nhl.\N,

C., .\NU T.ULVCIL, II., J. ISid. &m 188, 37

(1951). ~IsI.:s, F, &I., tiI.\C>~ULLEI\., J., \ND 11 SI’1StiS,

A. B., J. Hiol. Chcnt. 198, 615 (195“).

KR.\tII,, M. E., Science 116, 524 (1952). KIL.\HL, M. E., J. Viol. Chem. 200, 99 (1953).

Hls~~t~~caa, hI. G., .%NI) REZNOLD, A. I<., Hio-

chin. Biophgs. Actu. 44, 165 (19FO).

18.

19.

20.

21.

2’2.

23.

24.

25.

26.

"7.

28.

29.

30. 31.

32.

33.

34.

35.

Xi.

37.

38.

39.

40.

41.

42.

43.

4-1.

45.

4G.

NIXHIZLIGS, T., Fed. Proc. 20, 67 (1961).

Baasso~~~~, E. I)., JIZ., AND REDDY, W. J.,

Biochirn. Rio&/s.ilcfa 76, 641 (1963).

li~sscr,r,, J. il., Perspect. Riol. Med. 1, 138 (1958).

I%o.\GL.\ND, hl. B., in “The Nucleic Acids”

(E. Chargaff, and J. N. Davidson, eds.),

p 349. Academic Press, New York (1960). E.zLE,H., PIEZ, K. A., AND LEYY,M., J. &‘ol.

Chem. 236, 2039 (1961).

KIPSLS, 1). &I., .\sI) P~I~RISII, J. E., Fed. Proc.

24, 1051 (1965). CHRISTICNSEN, II. N., in “hIammalian Protein

Metabolism” (I-1. N. Mum-o and J. B.

Allison, eds.), p. 105. Academic Press, New York (1964).

CHI~ISTENSEX, II. X., Atlvan. Enzymol. Rel.

A~rens Mol. Biol. 32, 1 (1969).

FALLEN, E., THEMBL.~Y, C. M., AND GORLIN,

R., Fed. Proc. 25, 441 (1966).

WOOL, I. G., STIREW.\LT, W. S., KURIHARA, K., LO\\-, R. B., B\ILEY, P., .\ND OTER, I).,

Recent Progr. Harm. Res. 24, 139 (1968).

WOOL, I. G., ASD KURIHARA, K., Proc. Nat.

had. Sci. U.S.A. 58, 2401 (19Gi). MARTIN, T. E., AND WOOL, I. G., Proc. Nat.

Acud. Sci. 7T.S.A. 60, 569 (1968). WOOL, I. G., Fed. PIVC. 24, 10GO (1965).

WOOL, I. G., .WU KRAHL, ,M. E., Xutwe

London 183, 1399 (1959).

MANCH~STI~R, K. L., AND KRAHL, M. E.,

J. Biol. Gem. 234, 2938 (1959). WOOL, I. G. .1ND KRAHL, &I. E., Biochinn. Uio-

ph!/s. ;lcta 82, 606 (19Gi).

STIRlrn..\LT, w. S., Awl) Woor,, I. (+., Science

154, 284 (1966).

POZKFSKV, T., FJCLIG, I’., TOISIN, J. D.,

~Ol‘~LI)NI’;IL, J. 8., AND CAHILL, (;. F., J. Cl&.

Inrest. 43, 2273 (1969).

KIPNIS, I). PII., .\su N~.~LL, M. W., HiochinL.

Riophjys. ;tcta 28, 226 (1958).

FRITZ, (+. I<., .\ND KNOHIL, B., Xature London

200, (it2 (19G3).

C!.~SI’LES, J. J., .\xI) Woor,, I. (i., HiochenL. J.

91, llc: (19c;lj.

SCH.\RWF, Il., .\NII WOOL, 1. G., Biochem. d.

97, 257 (1965).

I<Ls.~s, I,. J., AIXUSCHT, I., UD ROSENHERG,

L. I<., 1. ISioZ. Chenz. 243, 18% (1968).

lIAmIrc, u., .\NU ItoHImrs, s., Sabztre Lontlon

207, 862 (1965).

Mr~sno, IT. N., Fed. Proc. 27, 1231 (1968).

~\‘IXRLICH, I). P., Science 168, 1186 (1967).

I3UCHI,:R, r. 1,. It., .\SD ~WAFFIELD, Rx. N., Biochim. Biophqs. rlctn 174, -191 (1969).

I~UCHI:U, 3. L. Ii., AND ().UCM.LN, N. J., ,%o-

chilrc. Biophys. 4cln 186, 13 (1969).

Sims, L. A., .I. Cell I’kysiol. 74, 63 (1969).

Insulin and protein synthesis in muscle

Documents

Transcript of Insulin and protein synthesis in muscle