Regulation of the Transport and Metabolism of Amino ...

THEJOURN&or BIOLOQICAL CR~MI~TRY Vol. 245.No. 15,Issueof August10,pp. 3872-3881, 1970 Ptinted

Regulation of Amino EFFECT

in

U.S.A.

of the Transport Acids in Isolated

OF INSULIN

AND

A POSSIBLE

ROLE

and Metabolism Fat Cells FOR ADENOSINE

3’, 5’-MONOPHOSPHATE* (Received for publication,

February

16, 1970)

T. MINEMURA,$ W. W. LACY, ANDOSCARB. CROFFORD~

From the Departments of Medicine and Physiology, Vanderbilt University School of Medicine, Nashville, Tennessee37203

SUMMARY

* This research was supported by United States Public Health Service Grant HE08195. Portions of the data have appeared in abbreviated form (1, 2). $ Present address, Shinshu University School of Medicine, Matsumoto, Japan. $ Investigator of the Howard Hughes Medical Institute.

The principal effects of insulin on glucose metabolism and lipolysis in adipose tissue can be explained by assigning insulin a site of action on the external membrane of the fat cell. Thus, the predominant effects of insulin on glucose metabolism result from acceleration of the carrier-mediated transport of glucose (3-ll), while the antilipolytic action of insulin probably results both from its effect on glucose transport (12-14) and from regulation of the intracellular concentration of adenosine 3’,5’-monophosphate (E-23). With respect to protein metabolism in adipose tissue, insulin is known to increase the l*C-labeled protein content of fat cells incubated with YXabeled amino acids (2442). Under certain conditions, the effect of insulin is reported to be at the level of amino acid transport (36, 42), while under other conditions insulin seems to exert its effect on the synthesis of proteins from intracellular amino acids (37, 41). Only a few reports have dealt with the possibility that insulin alters the rate of protein breakdown (31, 32). In muscle, insulin is reported to stimulate both amino acid transport and the metabolism of intracellular amino acids (43). In liver, there is evidence that insulin inhibits protein breakdown (44). These subjects have been recently reviewed elsewhere

(45). In the present report we have used the isolated fat cell system to evaluate the effects of insulin on amino acid transport, protein synthesis, and protein breakdown. We have attempted to dissociate these events as well as possible by using ‘“C-labeled nonmetabolizable amino acids and inhibitors of protein synthesis. We have also examined the possibility that the effect of insulin

3872

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We investigated the effects of insulin on ammo acid transport, protein synthesis, and protein breakdown in fat cells isolatedfrom rat epididymal adiposetissue. Insulin stimulated the accumulation of ‘F-labeled proteins in fat cells incubated with l*C-labeled L-leucine, Lalanine, glycine, and r.-serine. With L-leucine-l-14C as a model amino acid, we observed that the effects of insulin did not require the addition of glucose or other carbohydrate to the incubation medium, and were produced by insulin concentrations comparable to those required to accelerate glucose transport. The accumulation of 14C-labeledproteins was inhibited by adrenocorticotropic hormone (ACTH) and theophylline at concentrations known to increase adenosine 3’,5’-monophosphate (cyclic AMP) in isolated fat cells. The inhibitory effects of ACTH were reversed by insulin, while the inhibitory effects of theophylline and ACTH plus theophylline were only partially reversed. High concentrations of N6, 2l-O-dibutyryl cyclic AMP duplicated the effects of ACTH and theophylline. Insulin had only slight effects on the inhibition produced by dibutyryl cyclic AMP. Ammo acid transport was assessedby measuring the intracellular accumulation of 14C-labeledLu-aminoisobutyric acid (AIB), 1-aminocyclopentane-1-carboxylic acid, and certain natural amino acids. The labeled ammo acids achieved intracellular to extracellular concentration ratios which were similar to the concentration ratios of the natural ammo acids measured in fresh adipose tissue. AIB transport was Na+dependent and temperature-sensitive and inhibited by a 20: 1 molar excess of L-leucine, but was glucose-independent and not affected by ACTH, theophylline, or N6, 2l-O-dibutyryl cyclic AMP. Insulin did not changethe initial rate of AIB uptake, the initial rate of AIB ei%.rx, or the equilibrium concentration ratios of AIB and cycloleucine. When protein synthesis was blocked by lo-’ M puromycin, insulin had a slight inhibitory effect on protein breakdown.

This “antiproteolytic” effect of insulin could not fully account for the stimulatory effect of insulin on the accumulation of 14C-labeledproteins. We concluded that the predominant effect of insulin was exerted on some unidentsed step in protein synthesis rather than on the steps of amino acid transport or protein breakdown. Furthermore, we postulated that cyclic AMP might act as a negative effector for the action of insulin on protein synthesis.

Issue of August

T. Minemura,

10, 1970

W. W. Lacy, and 0. B. Croford

is mediated indirectly through regulation of the intracellular concentration of cyclic AMP.1 EXPERIMENTAL

PROCEDURE

Mat&A

ExperimentalAnimals Male rats of the Sprague-Dawleystrain (The Holtzman Company) were maintained with free accessto Purina laboratory chow and water. Fed rats, weighing 140 to 160 g, were killed by decapitation and the epididymal adiposetissuewasexcised. Incubation

Techniques

Analytical

Procedures

14C-Labeled Protein Content of Fat Cells Incubated with “C-Labeled Amino Acids--This was determined by the method of

Mans and Novelli (47). Briefly, an aliquot of the incubation mixture was acid-precipitated on filter paper discs,washed,and extracted to remove unincorporated radioactivity, and then counted in a liquid scintillation spectrometer. A modification of this method has been applied previously to isolatedfat cell protein by Miller and Beigelman(48). Further validation of the procedureas applied to isolatedfat cellsis given in Table I and Fig. 2. Intracellular-Extracellular Concentration Ratios of I%‘-Labekd Amino Acid AnaloguesThese were determinedby a mod&ation of the procedurepreviously used for measuringthe intracellular concentration of radioactive, nonmetabolizedsugaranaloguesin isolatedfat cells (49). Thus, the cellswere incubated with 3H-labeledinulin and a I%-labeledamino acid, for example, AIB. The cellswere separatedfrom the incubation mediumby vacuum filtration (Millipore filter, SM 5 p), the apparent distribution volume (space)for each of the isotopeswasmeasured, and the intracellular AIB spacewascalculated asthe difference between the AIB-14C spaceand the inulinzH space. The AIB concentration ratio was then calculated by the formulas: Intracellular-extracellular AIB concentrationratio =

intracellular

AIB

concentration

extracellular AIB concentration

where

AIB concentration All incubations were performed in Krebs-Ringer-bicarbonate Intracellular buffer with 3.5% bovine serumalbumin, equilibrated with 95% = intracellular AIB space X extracellular AIB concentration 02-5% CO,, pH 7.4. Isolated fat cells were prepared with the intracellular water volume epididymal adiposetissueby digestionof the tissuestroma with crude bacterial collagenaseas describedby Rodbell (46). The This simplifiesto isolated fat cells were then incubated under the conditions de- Intracellular-extracellular AIB concentration ratio scribedin the legendsof the figuresand tables.

BalancedAmino Acid Mtiture In the experimentsinvolving protein synthesis,the incubation medium contained a mixture of amino acids, proportioned according to the approximate amino acid concentration of rat plasma. A stock solution was prepared with amino acids (L form) in the following millimolar concentrations:hydroxyproline, 0.43; aspartic acid, 0.15; threonine, 3.34; serine, 3.29; proline, 1.79; citrulline, 0.68; glycine, 4.42; alanine,4.75; cY-aminobutyric acid, 0.15; cysteine, 0.32; methionine, 0.51; isoleucine, 1.36; leucine, 2.42; tyrosine, 0.99; phenylalanine, 0.99; ornithine, 1.38; lysine, 4.20; histidine, 0.64; tryptophan, 0.61; arginine, 1.10; glutamic acid, 3.00; valine, 2.72. The total amino acid concentration was 39.2 mErl. The stock solution was diluted 1:lOO in the preparation of the incubation medium so that the total amino acid concentration of the incubation medium was 0.39 MM and each amino acid was present at 0.01 the concentration listed above.

=

intracellular

intracellular

AIB

water

space

volume

Rather than measurethe intracellular water volume with each experiment, a value of 5.6 &‘lOO mg of cells was used for all calculations. This value was determined in previous experiments (49) and is our best estimate of the intracellular water volume of fat cellsas measuredby this technique. It shouldbe emphasized,however, that there is an unavoidably large experimental error in this estimate and that all of the absolutevalues for intracellular concentrationsand concentration ratios might have an error of +50% due to this estimatealone. This should be a systematicerror, however, and would not alter the interpretation of the results. Concentrationsof Amino Acids in Intracellular Water of Rat Epididymal Adipose Tissue and in Rat Plasma-These concentrations were determinedin the following way. Fed rats were killed by decapitation, blood was collected by drainage from the neck, and the epididymal adiposetissuewas excised. Fresh adiposetissuefrom three rats was weighed(about 1 g), pooled, 1The abbreviations used are: cyclic AMP, adenosine 3’,5’and homogenizedin 10 ml of 1% picric acid. The homogenate monophosphate; AIB, a-aminoisobutyric acid; cycloleucine, was centrifuged at 30,000 x g for 30 min at 4”, and the protein1-aminocyclopentane-1-carboxylicacid; ACTH, adrenocorticofree supernatant was aspirated from beneath the solidified fat tropic hormone; dibutyryl cyclic AMP, NC, 2l-0-dibutyryl adenolayer. The blood was collected in a heparinized tube and censine 3’) 5’-monophosphate.


All radioactive compounds were obtained from New England Nuclear and used without further purification. The compounds were: inulin-methoxy-SH, 0.164 mCi per mg; n-glucose-uniformly labeled-“C, 4.88 mCi per mmole; cY-aminoisobutyric-1-14C acid, 8.7 mCi per mmole; l-aminocyclopentane-1-carboxylic acidcarboxyV4C, 4.35 mCi per mmole; n-leucine-l-*4C, 25.6 mCi per mmole; r.-alanine-uniformly labeled-%, 136 mCi per mmole; L-serine-uniformly labeled-14C, 118 mCi per mmole; and glycineuniformly labeled-“C, 116 mCi per mmole. Nonradioactive compounds and their sources were: ol-aminoisobutyric acid and aminocyclopentane-l-carboxylic acid, Calbiochem; all natural amino acids, Mann; puromycin dihydrochloride, Nutritional Biochemicals; cycloheximide, Sigma; and crude collagenase from Clostridium histolyticum, Worthington. The insulin was pork insulin (PJ5682), given by Eli Lilly ResearchLaboratories.

3873

3874

Amino Acid Transport TABLE

and Metabolism

I

TABLE

Effect of insulin on W-labeled protein content of fat cells incubated with A-leucine-1 -WY, with or zvithout added substrate

Isolated fat cells were incubated at 37” in 2 ml of an incubation medium containing a balanced mixture of amino acids (see “Experimental Procedure”). The L-leucine concentration of this incubation medium was 0.029 mM. Each incubation vessel contained 45 mg of fat cells, 4.2 X lo6 cpm as L-leucine-l-W, and other additions as noted in the table. The nonincubated control cells were in contact with the incubation medium only long enough to achieve good mixing (30 to 60 set). The other cells were incubated for 2 hours. At the end of the incubation period the Wlabeled protein content of the cells was determined (see “Experimental Procedure”) and the results were expressed as lo3 cpm/lOO mg of fat cells. W-Labeled

protein

content

Insulin Pyruvate

GIUCOSEZ mdl

Mean0

.-

II

insulin on W-labeled protein content of fat cells incubated with a variety of W-labeled amino acids The experimental conditions were the same as given for Table I except that each incubation vessel contained 4.4 X 105 cpm of the particular amino acid listed. The concentrations of the test amino acids in the incubation medium were: L-leucine, 0.032 mM; L-alanine, 0.049 mM; glycine, 0.046 mM; and L-serine, 0.035 mM. E$ect

of

-

W-Labeled Additions

protein

content

IWAill

--

Mean*

Range I

10’ c#m/100 mg fat cells

milliunils/ml

L-Leucine

100

71.4 134

GAlanine

0 100

27.8 41.1

26.5-29 .O 40.3-41.9

Glycine

0 100

43.7 64.6

42.5-45.3 62.0-69.4

L-Serine

0 100

41.7 57.1

41.342.4 56.5-58.1

-

Additions

Vol. 245, No. 15

in Fat Cells

Range

0

68.8-75.0 133-138

I milliunits/ml

0

0

0

0

0

0

0

0

1 1 0 0

0 0 1 1

100 0 100 0 100 0 100

2.0* 1.96 128 216 115 232 125 221

fat cells 1.7-2.3 1.7-2.1 119-132 205-225 102-130 223-239 121-129 216-228

a Mean of three observations. * Nonincubated control. trifuged at 4”, and 2 ml of the plasma were deproteinized with 10 ml of 1% picric acid. The total a-amino nitrogen concentrations of the picric acid filtrates were estimated by the ninhydrin-carbon dioxide method (50). Individual free amino acid concentrations were measured by gradient elution chromatography with a Technicon amino acid analyzer (51, 52). The intracellular concentrations were then estimated by assuming that (a) the extracellular concentration was equal to the plasma concentration, (b) the extracellular volume was 9% of the fresh weight of the tissue, and (c) the intracellular water volume was 4% of the fresh weight of the tissue. The estimates of the water volumes were based on previous measurements made on the epididymal adipose tissue from fed rats of comparable weight (8). These estimates are thought to be the most accurate available but, nevertheless, could have introduced a systematic error in the intracellular amino acid concentrations of up to l 50%. Concentrations of Indiwidual Amino Acids in Incubation Mixture-These concentrations were determined by gradient elution chromatography of the picric acid filtrates of the entire contents (incubation medium plus fat cells) of the incubation vessels. Since the intracellular water volume was small (approximately 0.008 ml in a typical experiment) relative to the extracellular water volume (approximately 1.82 ml), the intracellular amino acids, even though highly concentrated, probably comprised less than 1 y0 of the total free amino acid content of the incubation vessels. Experiments were performed to test the purity of the 14C-labeled AIB preparation which we used and to determine the extent to which it is metabolized by rat adipose cell. Thus (a) the AIB-14C obtained from the manufacturers was investigated

-

0 Mean of three observations. with ascending paper chromatography, developed in butanolacetic acid-water (24:4: 10, v/v/v). Greater than 99% of the radioactivity applied to the origin migrated as a single symmetrical peak with an RF value equal to that obtained by the manufacturers. (b) Isolated fat cells were incubated for 2 hours with AIB-14C, under conditions which simulated those used in the transport experiments presented subsequently. Less than 0.05% of the radioactivity in the incubation vial was recovered as the sum of CO2 total lipids, and total protein. (c) The intracellular 14C-labeled material, when chromatographed in the same manner as described above, migrated in a symmetrical peak which was indistinguishable from AIBJ4C added to the incubation medium. (d) The partition coefficient of AIBJ4C in a corn oil-water mixture (l:l, v/v) was approximately 0.0019. (e) The addition of nonradioactive AIB to the incubation medium was not injurious to the cells as judged by the observation that the linear rate of oxidation of glucoseJ4C and the capacity of insulin to stimulate this rate was not altered by the addition of AIB (0.5 mM) to the incubation medium. Based on the foregoing results, we assumed that AIB-14C could be used as a transport model in fat cells, subject to the same limitations as those which apply to other tissues. RESULTS

E$ect

AND

COMMENTS

and Other Agentson 14C-Labeled Protein Content of Fat CellsIncubated with 14C-Labeled Amino Acids

of Insulin

The magnitude of the effect of insulin on the W-labeled content of fat cells incubated with L-leucine-l-l% is shown in Table I. In many such experiments, the W-labeled protein content of the insulin-treated cells ranged from 1.5 to 2.5 times that of the control. The low count rate in the proteins isolated from the nonincubated control cells shows the completeness with which the acid precipitation and washing procedure removes ‘4C-labeled L-leucine. The data in Table I also show that t,he


0

108 c$9?&/100 ng

Issue of August lo3

10, 1970

T. Minemura,

W. W. Lacy, and 0. B. Crofford IO’ cpm

cpm P’

’

’

““1”

’

-

‘1”“’

b

’

103cpm

r”“‘r z

E E

rn 60

0L,I 10-l'

* CS(,,' 10-10 ' j""'110-g MOLAR

CONCENTRATION

OF

3875

-~~*~' 10. -6

INSULIN

effect of insulin does not require the addition of glucose or other carbohydrates to the incubation medium. The literature contains conflicting reports about this point (24-28, 48). Since the cells in all of our experiments had been isolated and repeatedly washed in a glucose-free buffer, it is highly improbable that the incubation medium contained glucose. In some experiments, although not in this particular example, the addition of glucose or pyruvate did cause a further increase of 10 to 20% over that produced by insulin alone. The effect of insulin was observed with W-labeled amino acids other than L-leucine. The results obtained with L-leucine, calanine, glycine, and L-serine are compared in Table II. The stimulatory effect of insulin was dependent on the concentration of insulin in the incubation medium. Fig. 1 shows a comparison between the effect of graded doses of insulin on the oxidation of glucose-uniformly labeled-W to 14CO2, and on the W-labeled protein content of fat cells incubated with n-leucine1 -W. The two concentration-response curves were essentially parallel with the half-maximum stimulation of both processes occurring with insulin concentrations of about 7 X 10-l’ M.’ These results are in general agreement with those reported previously by Miller and Beigelman (38, 39). Fig. 2 shows the results of an experiment in which the cells were incubated with 0.1 mM puromycin for varying periods of time. When puromycin was present throughout both the first and second incubation periods (only the second incubation 2 Equivalent to 10 runits per ml, based on a molecular of 6000 for insulin and a potency of 25 units per mg.

weight

CONTROL PUROMVCIN

CELLS TREATED

CELLS

60 SECOND

INCUBATION

MIN.

PERIOD

FIG. 2. Effect of puromycin on the ‘%-labeled protein content A single pool of isoof fat cells incubated with n-leucine-1-“C. lated fat cells was divided into four portions and each was incubated for 60 min at 37”. The cells were then washed three times and incubated for a second 60-min period. In the second incubation, each vial contained 100 mg of fat cells and 4 X 106 cpm as n-leucine-l-l% in a total volume of 2 ml of incubation medium, fortified with the balanced amino acid mixture. Each vial was sampled in triplicate at the times indicated, the WXabeled protein content of the cells was determined (see “Experimental Procedure”), and the results are expressed as 1Oa cpm/lOO mg of fat cells. The first portion (designated CONTROL CELLS and illustrated by the open bars) was not exposed to puromycin during either the first or the second incubation period. The second portion (hatched bars) was incubated with 0.1 mM puromycin during the last 30 min of the first incubation period. The third portion (stippled bars) was incubated with puromycin throughout the first 60-min incubation period. The fourth portion (black bars) was incubated with 0.1 mM puromycin throughout both the first and second incubation periods. In the figure, the height of each bar indicates the mean of the three observations with the range being comparable to that presented in Tables I and II.

period included L-leucine-1-i4C in the incubation medium), the low level of W activity in the isolated proteins indicates that protein synthesis was strongly inhibited. This emphasizes that the acid precipitation and washing procedure was effective in removing W-labeled compounds other than protein. When puromycin was not present in the medium during the second incubation period, the i4C activity of the isolated proteins accumulated linearly. This indicates that the processes under investigation were not irreversibly inactivated during the initial It was necessary to establish this point puromycin exposure. to interpret subsequent experiments. E$ects of Dibutyryl Cyclic AMP and Agents Known to Innrrease Intracellular Cyclic AMP Concentration on 14C-Labeled Protein Content of Fat Cells Incubated with T-Labeled Leucine-Since the antilipolytic action of insulin is mediated, at least in part, by modulation of the cyclic AMP concentration, experiments were performed to see whether the effects of insulin on the accumulation of W-labeled proteins were modified by agents influencing intracellular levels of cyclic AMP or mimicking its effects. The results shown in Table III indicate that the r4Clabeled protein content of fat cells incubated with L-leucine-1-14C was unaltered when adrenocorticotropin was added at a concentration of 0.2 milliunit per ml. When the dose of ACTH was increased to 20 milliunits per ml, the accumulation of 14C-labeled proteins by the unstimulated cells was reduced, but could be restored to the control level by the addition of insulin. When theophylline was present at a concentration of 1.5 mM, a dose


1. Effect of graded doses of insulin on the W-labeled protein content of fat cells incubated with n-leucine-l-“C and on the oxidation of glucoseJ4C to WO, by isolated fat cells. Aliquots of a single pool of isolated fat cells were incubated at 37’ with the insulin concentration indicated in this figure. Each incubation vessel contained 50 mg of fat cells in a total volume of 2.5 ml. The incubation medium in half of the vials contained a balanced mixture of amino acids (see “Experimental Procedure”) and 5.5 X lo6 cpm as n-leucine-l-l%, but no glucose. The Lleucine concentration of this incubation medium was 0.032 mm. At the end of 2 hours, the “C-labeled protein content of the cells was determined (see “Experimental Procedure”) and the results were expressed as lo3 cpm/lOO mg of fat cells. The incubation medium in the other half of the vials contained no amino acids, 1 mM glucose, and 2.8 X lo6 cpm as uniformly labeled glucoseJ4C. At the end of 2 hours, “COa production was measured (see “Experimental Procedure”) and the results were expressed as lo* cpm/lOO mg of fat cells. In the figure, each point represents the average of two determinations. FIG.

[7

3876

Ej’ect

Amino Acid Transport

of lipolytic

agents incubated

TABLE III on W’-labeled

with

protein

content

of fat

and Metabolism

Vol. 245, No. 15

in Fat Cells

cells

A-leucine-1-W

The experimental conditions were the same as given for Table I except that each incubation vial contained 2.2 X 106 cpm of L-leucine-1-W and other additions as noted in the table. -

W-Labeled

protein

content

Insulin

Additions --

n Eicroun~ts/r

None

10 mg fat cells

108 CM

50.3-54.7 86.2-91.8

0 200

53.9 88.4

50.7-56.0 85.8-92.5

0 200

35.9 83.8

34.5-38.2 80.6-88.0

0 200

36.0 65.1

34.0-38.8 64.1-65.9

ACTH, 0.2 mu/ml, + theophylline, 1.5 mM

0 200

29.5 33.8

28.0-31.0 33.4-34.3

Dibutyryl

cyclic AMP, 2 mM

0 200

34.6 45.9

32.6-36.9 45.2-46.7

Dibutyryl

cyclic AMP,

0 200

29.0 35.7

27.8-29.8 35.5-35.8

ACTH,

0.2 milliunits/ml

ACTH,

20 mu/ml

Theophylline,

1.5 mM

6 mM

-

-

a Mean of three observations. TABLE

Effect

of

palmitic

IV

acid on W-labeled protein incubated with L-leucine-1-W

content

of fat

cells

The experimental conditions were the same as given for Table I except that each incubation vial contained 70 mg of fat cells and 8.8 X lo6 cpm as L-leucine-l-l% in a total volume of 2.5 ml. The palmitic acid was added to the incubation via.ls as a palmitatealbumin complex. The total albumin concentration of the incubation medium was 3.5 g/100 ml in all incubation vials. W-Labeled Palmitic

acid

None None 0.25 0.5 1.0 2.0 2.0

microunits/ml

0 100 0 0 0 0 100

TIME

125 227 132 133 152 176 299

I

I

I

I

60

60

100

120

(MINI

FIG. 3. Effect of extracellular AIB concentration on the intracellular-extracellular concentration ratio of 14C-labeled AIB. A single pool of isolated fat cells was divided into four portions and each was incubated separately at 27”. Each incubation vial contained 300 mg of fat cells, 7 X lo6 cpm as “‘C-labeled AIB, and 7 X 106 cpm as inulin-methoxy-3H in a total volume of 6 ml. The AIB concentration of each incubation vessel is indicated in the figure. At the times indicated, 0.5 ml of the cell suspension was removed from each incubation vial and used for determination of the AIB concentration ratio (see “Experimental Procedure”). produced by ACTH and by theophylline can be reproduced by the addition of dibutyryl cyclic AMP to the incubation medium. Insulin has only a slight tendency to stimulate the accumulation of *4C-labeled proteins which has been depressed by dibutyryl cyclic AMP. The concentrations of dibutyryl cyclic AMP are high but are consistent with those required to produce maximum stimulation of lipolysis in intact fat cells (21). Since ACTH, theophylline, and dibutyryl cyclic AMP all stimulate lipolysis it seemed possible that fatty acids might inhibit the accumulation of W-labeled proteins when fat cells were incubated with I,-leucine-1-W. Such a mechanism has been proposed for diaphragm (53) and heart muscle (54). In isolated fat cells, however, increasing the palmitic acid concentration of the palmitic acid-albumin complex added to the incubation medium slightly increased the accumulation of “C-labeled proteins in fat cells and did not prevent the stimulatory effect of insulin (Table IV).

Efect of 108 c~m/loo

40

content

Insulin Mean=

?nY

protein

20

Insulin

on Amino

Acid

Tran,qort

Range mg fat cells

121-128 226-229 124-137 131-136 143-157 168-181 293-304

0 Mean of three observations. which produces near maximum stimuk&on of lipolysis, the accumulation of 14C-labeled proteins by the unstimulated cells was again reduced, but only partially restored by the addition of insulin. When present together, the effects of ACTH (0.2 milliunit per ml) and theophylline (1.5 mM) were synergistic and the capacity of insulin to overcome this combined effect was minimal. The last four lines of Table III show that the effects

Since the results presented thus far could have resulted from a change in the rate of a number of steps, we attempted to dissociate amino acid transport from protein synthesis. a-Aminoisobutyric-1-W acid is transported but not significantly metabolized by most tissues (55-57) and has been used previously to assess amino acid transport in adipose tissue (37), isolated fat cells (42), and fat cell “ghosts” (11). The curves shown in Fig. 3 illustrate the time course of the intracellular-extracellular concentration ratio of AIB-1% in isolated fat cells. In addition, Fig. 3 shows that the intracellularextracellular concentration ratio at equilibrium is independent of the AIB concentration in the incubation medium. During the early stages of this time course, however, low concentrations of AIB showed an exaggerated intracellular-extracellular concentration ratio. Our interpretation of this last observation assumes competition for the same transport system between AIB and certain of the natural amino acids originating within the cells themselves (57). Thus, when the cells are first added to the


200

52.6 89.4

0

T. Minemura,

Issue of August 10, 1970

W. W. Lacy, and 0. B. Croford

TABLE V amino acids in intracellular water of rat adipose tissue, in rat plasma, and intracellular water to plasma concentration ratios of free amino acids The amino acid concentrations listed are the mean of three experiments. Each experiment was performed on the pooled adipose tissue and plasma from three rats (see “Experimental Procedure”). Concentrations epididymal

3877

I

----

+

insulin

of free

-

-

Concentrations Amino acid

Intracellular

--

water

Concentration ratio --

?nM

Glycine ................

11.52

Alanine ................ Valine ................. Methionine ............

0.87 0.47 0.02 0.30 0.73 0.64 0.45 0.21 1.62 0.40 0.89

Cystine................ Isoleucine ............. Leucine. ............... Tyrosine. .............. Phenylalanine. ......... Ornithine .............. Lysine. ................ Histidine .............. Arginine ............... Total ..................

63.6

0.016 0.316 0.314 0.767 0.075 0.058 0.408 0.465 0.210 0.032 0.027 0.098 0.170 0.095 0.071 0.089 0.394 0.058 0.169

242 12.0 15.2 14.3 18.5 188 23.8 24.8 3.9 14.7 1.0 3.1 4.3 6.7 6.3 2.4 4.1 6.9 5.3

3.84

-

-

16.5

incubation medium, there is minimal competition betweenAIB and other aminoacidsfor the entry processsinceno other amino acids are present in the incubation medium. On the inside of the cell, however, the AIB which enters must competewith certain natural amino acidsfor the exit process. This results in an exaggeratedintracellular-extracellularAIB concentrationratio which would: (a) diminish as the natural amino acids become distributed on both sidesof the cell membrane,and (b) be most apparent when low concentrationsof AIB were used. This interpretation wasvalidated by two additional experiments (data not shown). The first showedthat this “overshoot” was not observedwhen the incubation mediumcontained0.2 mM glycine. The second showed that the intracellular-extracellular AIB concentration ratio could be rapidly forced to reach its final concentrationratio if 0.2 mMglycine wasaddedto the incubation mediumat the peak of the overshoot (this would be at t = 5 min in Fig. 3). The results of certain other experiments are stated only in sentenceform, sincethey simply showthat certain observations previously

made in other cell types are also applicable

to isolated

fat cells. Thus: (a) the final intracellular-extracellular AIB concentration ratio and the rate at which it is establishedare inhibited at 17”, (a) the intracellular-extracellular AIB concentration ratio is reducedby 75% by replacing the Na+ in buffer by K+, (c) the initial rate of accumulation of AIB is strongly inhibited

10

by a 2O:l molar excess of L-leueine,

and (d) the final

intracellular-extracellular AIB concentration ratio and the rate

20

30 MINUTES

40

50

60

FIG. 4. Effect of insulin on the time course of the intracellularextracellular concentration ratios of W-labeled AIB and cvcloleucine. Isolated fat cells were incubated at 24” in 8 ml of an kcubation medium containing no gludose and no amino acids other than 0.1 mM AIB or 0.1mM cycloleucine. Each incubation vessel contained 9 X lo6 cpm as the W-labeled amino acid and 9 X lo8 cpm as inulin-methoxy-SH. Insulin, when present, was at a concentration of 1 milliunit per ml. At the times indicated, 0.5 ml of cell suspension was removed from each incubation vial and used to determine the concentration ratio (see “Experimental Procedure”).

at which it is establishedare not alteredby the addition of glucose to the incubation

medium.

It should be noted, however, that all

of our experimentswereperformedon cellsisolatedfrom fed rats. Statement d may not be valid for cellsisolatedfrom fasted rats (42). Sincethe intracellular-extracellular concentrationratio for AIB was higher than anticipated, we determined,for rat epididymal adiposetissue,the intracellular-plasmaconcentrationratios of 19 natural aminoacids. Thesedata are presentedin Table V. The intracellular concentrationsof free amino acids were in general agreementwith those observedpreviously in heart muscle(43) and adiposetissue (30). The very high apparent concentration ratios for aspartic acid and glutamic acid probably indicate that their intracellular concentrationsare determinedmoreby intracellular events (i.e. transaminationreactions)than by transport from the extracellular to the intracellular compartment (43). Nevertheless,the intracellular-plasmaconcentration ratio of the total amino acids was approximately 17, which is of the same order as we had observedfor AIB in the incubatedcell preparation. In five additional experimentsthe total a-amino nitrogen concentrations(micromolesper ml) of rat adiposetissueand rat plasmawere (mean f standarderror) : intracellular water space, 108 f 10; and, plasma,5.9 f 0.4; with the meanof the concentration ratios being 18 =t 1. For isolated fat cells the total a-amino nitrogen was approximately 73 pmolesper ml of intracellular water. Sincethis value did not decreasewith up to 60 min of incubation, we concludedthat: (a) intracellular protein was hydrolyzed at a rate sufficient to replace the amino acids which left the intracellular water space,and (b) the capacity of incubated fat cellsto concentratenatural aminoacidswasmaintained for at least 60 min. We alsomeasuredthe intracellularextracellular concentration ratios of certain 14C-labeled natural amino acidsin fat cellswhosecapacity to accumulateYXabeled proteins was blocked with puromycin (10m4 M beginning60 min before the addition of the 14C-labeledamino acids). The observed concentration ratios were: n-leucine-1-‘4C,4.6; Lalanine-uniformly labeled-14C,18.4; glycine-uniformly labeled14C,10.6; n-serine-uniformlylabeledJ4C,13.2. This procedure wasnot altogethersatisfactory becauseof the variable accumula-


3.88 3.79 4.77 10.98 1.39 10.93 9.73

Aspartic acid. ......... Threonine. ............. Serine ................. Glutamine ............. Asparagine. ............ Glutamic acid..........

I I

PlaSma

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~Vol. 245, No. 15

“Km”

(AIB

(AIB

mM)

s 13mM

mM)

noted previously to behave atypically sinceit appearsto enter a “bound pool” andrequirelongerto equilibrate (58). The effect of insulin on the intracellular-extracellular concentration ratios of AIBJ”C and on cycloleucine-14C is shownin Fig. 4. We have observedno effect of insulin on the “steady state” concentration ratio. Attempts to measurethe rate at which the concentration ratio becomesestablishedhave likewise shown that this rate is not altered by the addition of insulin. This point is consideredin greater detail in the subsequent paragraph. Since the intracellular-extracellular concentration ratio is the net result of the influx and efflux processes, we have usedAIB-1% to examinethe effect of insulin on theseprocesses independently (59). Fig. 5 showsthat insulin doesnot alter the initial velocity of AIB uptake. Our best estimate of the apparent K, of this processis 0.7 mM. Fig. 6 showsthat insulin doesnot alter the initial velocity of AIB efflux from “‘AIB-loaded” cel!s. Our best estimateof the apparent Km of thii latter processis 13 mM. The ratio of the apparent Km values for these two processesis 13:0.7 or 18. As expected (59), this value is of the samemagnitude as the measuredsteady state concentration ratio (see Fig. 4). Since we were unable to detect an effect of insulin on the amino acid transport process,we did not investigate extensively the effects of dibutyryl cyclic AMP or agentsknown to alter

servations.

the intracellular concentration of cyclic AMP. In experiments analogousto that shown in Fig. 4, however, we observedno effects of dibutyryl cyclic AMP (2 mM), ACTH (20 milliunits per ml), or theophylline (1.5 mrsr>. Effect of Insulin

on Protein Breakdown

If insulin inhibited the breakdown of ‘%-labeled proteins, the rate of accumulationof “C-labeled proteins would be greater in the insulin-treated cells even if the rate of protein synthesis was not stimulated by insulin. In addition, if insulin inhibited the breakdown of proteins from a pool of low specificactivity, then the specificactivity of the L-leucine-l-14cin the incubation medium would be higher in the insulin-treated cells and the rate of -accumulationof 14C-labeledproteins would again be greater even if the rate of protein synthesis was not stimulated by insulin. The following experimentsweredoneto seewhether we could detect an effect of insulin on protein breakdown,and, if suchan effect wasdetectable,to try to determineits contribution to the over-all processof ‘“C-labeledprotein accumulation. In the experiment whoseresults are shownin Table VI, the cellswere incubated with Irleucine-1-14Cto label the intracellular proteins and then washedto remove the excessleucineand reincubated to measurethe effect of insulin on the decreasein


FIG. 6. Effect of insulin on the initial velocity of W-labeled AIB ef&x. A single pool of isolated fat cells was divided into four portions and incubated for 60 min at 37” with varying concentrations of WXabeled AIB and 3H-labeled inulin. The snecific activity of the AIB ranged from 2.5 PCi per amole in the vi& with the lowest AIB concentration to 0.2 pCi per pmole in the vials with the highest AIB concentration while that of inulin was 0.16 pCi per pg in all vials. At the end of the 60-min incubation period each of the four portions was distributed evenly into six incubation vials, and then cooled to 0” in an ice bath and washed once at 0’ with incubation medium containing no AIB or inulin. The second incubation period was at 37” in 1 ml of incubation medium containing no AIB or inulin. Insulin, when present, was at a concentration of 100 milliunits per ml. At the beginning and after 1 min of the second incubation period the cells were separated from the incubation medium by vacuum filtration through a meviouslv - weighed Millinore filter and the intracellular AIB from the meanof three observations. concentration was determined (see “Experimental Procedure”). The AIB lost, from the cells in 1 min was calculated from the tion of 14CJabeled lipids. Nevertheless,except for glycine, these difference between the zero and I-min concentrations and exconcentration ratios are in reasonableagreement with those pressed as millimicromoles per 100 mg of cells per min. In measuredin fresh adiposetissue (Table V). Glycine has been this figure, each point was derived from the mean of three ob-

FIG. 5. Effect of insulin on the initial velocity of W-labeled AIB uptake. Aliquots from a single pool of isolated fat cells were incubated for 60 min at 37’, centrifuged, and reincubated for 1 min in fresh incubation medium containing ‘*C-labeled AIB and *H-labeled inulin. Each incubation vessel contained 50 mg of fat cells in a total volume of 1 ml. Insulin, when present, was at, a concentration of 100 milliunits per ml. The specific activity of the AIB ranged from 0.4 &i per rmole in the vials with the lowest AIB concentration to 0.13 pCi per pmole in the vials with the highest AIB concentration while that of inulin was 0.16 &i per pg. At the end of the 1-min incubation period the cells were separated from the incubation medium by vacuum filtration through a previously weighed Millipore filter, the intracellular AIB concentration was determined (see “Experimental Procedure”), and the results are expressed as millimicromoles of AIB taken up per 100 mg of cells per min. In the figure, each point was derived

Issue of August

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T. Minemura,

W.

W.

Lacy, and 0. B. Crofford

TABLE VI insulin on breakdown of 14C-labeled protein in fat cells A single pool of isolated fat cells (800 mg) was incubated for 60 min at 37” in 10 ml of an incubation medium containing a balanced mixture of amino acids and 2.4 X lo6 cpm as n-leucine-l-14C. The cells were then washed three times, subdivided, and incubated for 90 min with or without 0.1 mM puromycin, and with or without insulin. At the times indicated, the incubation vials were sampled in triplicate, the W-labeled protein content of the cells was determined (see “Experimental Procedure”), and the results were expressed as lo3 cpm/lOO mg of fat cells. E$ect

of

Puromycin

Incubation time

min 0 90 90

10-4 M

0 0 0

I I

W-Labeled Mean=

milliw~:s/ml 0 0

100

+ +

0 0

100

Range

103 cpm/100

?ng fat cells

21.0 18.1 19.9

20.7-21.4 17.7-18.4 18.8-21.1

20.3 17.5 19.0

20.0-20.4 17.4-17.8 18.7-19.2

a Mean of three observations. VII

TABLE Effect

of insulin

on free

amino

acid

production

by isolated

fat

cells

A single pool of isolated fat cells was incubated for 60 min at 37’ in an incubation medium containing a balanced mixture of amino acids and 0.1 mM puromycin. The cells were then removed from the incubation medium, washed, dispensed into 15 incubation vessels, and incubated either with or without insulin. Each incubation vessel contained 142 mg of fat cells in a total volume of 2 ml of fresh medium, but of the same composition as used in the first incubation. At the times indicated, 10 ml of 1% picric acid were added to the incubation vessels and the filtrates were used for amino acid analyses by gradient elution chromatography (see “Experimental Procedure”). The initial free amino acid content of three of the incubation vessels was determined 60 set after adding the cells to the incubation medium and the values for the free amino acid production were calculated from the difference between the free amino acid content of the incubation vessels at the times indicated and the initial content. Although the data on 19 different amino acids were calculated, only those for leucine and the total are presented. These are shown as the mean of three observations and the range. Amino acid production Amino acid

Insulin

2 hrs

1 hr Mean=

milliunit

Range ~mole/1oo

Meana

Range

m g fat cells

Leucine

0 100

0.032 0.025

0.0324.035 0.018-0.027

0.063 0.044

0.061-0.066 0.0394.048

Total free amino acids

0 100

0.32 0.21

0.23-0.38 0.14-9.28

0.58 0.36

0.54-0.69 0.29-0.47

a Mean of three observations.

the content of intracellular 14C-labeledproteins. In certain cases, resynthesis of labeled proteins was blocked with low4 M puromycin. After a 90-min incubation period there was only a slight decrease in the content of intracellular 14C-labeled pro-

teins and the effect of insulin was small. In other experiments, protein breakdown, as measured in Table VI, was not significantly altered by ACTH (0.2 and 20 milliunits per ml) with or without insulin (200 microunits per ml), theophylline (1.5 mM) with or without insulin (200 microunits per ml), or dibutyryl cyclic AMP (6 mM) with or without insulin (200 microunits per ml). Even though the effect of insulin on the breakdown of labeled protein was small (Table VI), the possibility still existed of a substantial insulin effect on the release of unlabeled amino acids into the incubation medium if only a small fraction of the total cellular protein was labeled. This possibility was examined by measuring the effect of insulin on the production of free amino acids when protein synthesis was blocked by puromycin. The production of free leucine and of total amino acids is shown in Table VII. It is apparent that insulin inhibits the production of free amino acids. The other individual amino acids, with the exception of alanine, behaved in a manner which was similar to the total. In contrast to all of the others, the alanine content of the incubation vessels declined with time and the magnitude of this decline was increased by insulin. Our data do not explain this observation. The degree to which the effect of insulin on protein breakdown might account for the effect of insulin on the accumulation of 14C-labeled proteins in cells incubated with ‘4C-labeled leucine (Table I) is considered under “Discussion.” DISCUSSION

Our initial experiments (Tables I and II, Figs. 1 and 2) confirmed earlier reports of other investigators that insulin stimulates the accumulation of 14C-labeled proteins in fat cells incubated with 14C-labeled amino acids (26-40). Beyond this, however, we have attempted a systematic investigation as to whether the predominant site of action of insulin on this composite process is at the level of amino acid transport, protein breakdown, or protein synthesis. Our data failed to show a direct effect of insulin on amino acid transport. This observation agrees with those made by Goodman, in incubated adipose tissue (37), and by Rodbell, in fat cell ghosts (11). Tauabi and Jeanrenaud have reported that ACTH, adrenaline, caffeine, and theophylline inhibit the net uptake of i4C-labeled AIB and that, especially under conditions associated with reduced ATP levels, insulin stimulates AIB uptake (42). They were not investigating initial rates of AIB flux but rather net AIB uptake after 30 to 40 min of incubation. They concluded that their observations were probably not direct effects of the hormones on the membrane transport system but effects on the energy level of the cell which in turn governs the intracellular accumulation of AIB. We do not dispute the energy requirement of a high intracellular-extracellular concentration ratio and see no fundamental disagreement between their observations and those in this report. Turning to the question of protein breakdown, we observed only a small decrease in the intracellular content of 14C-labeled proteins when labeled cells were incubated for 90 min in fresh medium containing puromycin (Table VI). This suggests that, the labeled proteins were mixed in a large protein pool of low specific activity and slow turnover, rather than being localized in a small pool of high specific activity and rapid turnover. Since the proteins were not fractionated and the specific activities of the various pools were not determined, this point has


0 90 90

+

protein content

Insulin

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I

I


isolated from fasted rats, it does not occur if the cells are from not been established. If our interpretation is correct, however, then the “antiproteolytic” action of insulin would not be the fed rats or when dibutyryl cyclic AMP is used as the lipolytic major determinant of the insulin effect observed in Table I. agent.3 Although clearly unproven, it is physiologically attracIt also follows that, if the specific activity of the intracellular tive to think that cyclic AMP might be a negative effector for protein pool is low, the experimental approach used for Table VI the action of insulin on protein synthesis. Thus, in the fed is too insensitive to assess accurately how much the antiproteostate, insulin could favor anabolic processes by accelerating lytic effect of insulin would affect the specific activity of leucine glucose transport and by reducing the level of cyclic AMP. in the incubation medium. This, however, can be estimated The reduced level of cyclic AMP would then favor protein synfrom the data in Table VII. Thus, if the rates of nonlabeled thesis and deactivate the lipolytic system. In the fasted state, leucine production measured in the puromycin-treated cells with lower blood glucose and insulin levels, the glucose transport system would not be accelerated and the cyclic AMP level would (Table VII) are comparable to those which were undoubtedly occurring in our other experiments (e.g. Table I), then, after 2 rise. The higher cyclic AMP levels would inhibit protein synhours of incubation, the specific activity of leucine in the medium thesis and activate the lipolytic system. It will be interesting bathing the insulin-treated cells would have been approximately to examine this hypothesis in greater detail. If the theory is 10% higher than that of the cells incubated in the absence of valid, then the stimulatory effect of insulin on protein synthesis insulin, and about 10% of the effect of insulin in Table I could should be mimicked by agents which mimic the antilipolytic have been produced by an inhibition of protein breakdown. In action of insulin, e.g. prostaglandin El (63), insulin-sepharose fact, the rate of protein breakdown relative to the volume of (67)) and p-chloromercuribenzene sulfonic acid (68). Lavis and the incubation medium was such that, even if insulin had com- Williams have already noted that certain thiol compounds which are “insulin-like” in many respects (69) stimulate amino acid pletely blocked protein breakdown, only 50% of the stimulatory effect of insulin shown in Table I could be due to a change in metabolism in isolated fat cells (70). Thus far, the importance specific activity of the leucine in the incubation medium. This of cyclic AMP in the regulation of protein synthesis in other is not to imply that the rate of protein breakdown was small cell types has not been exhaustively investigated (71-74). and that the cells were in a state of net protein synthesis. Quite AcknowledgmentsThe authors are indebted to Mrs. Brenda the contrary, the production of free leucine in Table VII was approximately 0.032 pmole/lOO mg of fat cells during the 1st Flowers, Miss Melba McCullough, and Mr. E. H. Fryer for hour, whereas the average accumulation of leucine as protein skillful technical assistance. We are particularly indebted to in the labeling experiments was approximately 0.025 pmole/lOO student assistants Messrs. Bob Winton and Ben Kibler who mg of fat cells per hour, even when stimulated by insulin. Thus, discoveredthat the “effect of insulin on AIB transport,” which the cells were probably in a state of net protein breakdown and we had observed and reported in an abstract (l), was not an the major reason why the specific activity of the labeled leucine effect of insulin but was an inhibition of AIB transport by the was changed little by changes in the rate of protein breakdown glycine present in the pH 2 buffer originally usedfor dissolving was the relatively large volume of the incubation medium. Our the insulin crystals. We are also grateful to Dr. C. R. Park interpretation of these observations is that insulin has a de- for his valuable suggestions and critique of the manuscript. tectable inhibitory effect on protein breakdown, but that the REFERENCES predominant effect of insulin on the accumulation of 14C-labeled proteins in fat cells incubated with %-labeled amino acids re1. MINEMURA, T., CROFFORD, 0. B., LACY, W. W., AND NEWMAN, E. V., Fed. &oc., 28, 568 (1969). sults from a stimulation of some unidentified step in protein 0. B.. MINEMURA. T.. AND KONO. T.. Advan. En2. CROFFORD. synthesis. zyme Rebut., 8,‘219 (1970). ’ ’ It has been observed previously that certain lipolytic agents M., Recent Progr. Hormone Res., 3. LEVINE, R., AND GOLDSTEIN, inhibit protein synthesis (26). In the present report, we have 11, 343 (1955). demonstrated that changes in protein synthesis occur under D., HENDERSON, M. J., CADENAS, 4. PARK, C. R., REINWIZIN, E., AND MORGAN, H. E., Amer. J. Med., 26, 674 (1959). conditions which are now known to alter the intracellular conJ. L., J. Biol. Chem., 237, 5. FROESCH, E. R., AND GINSBERG, centration of cyclic AMP in isolated fat cells (60-64). It is 3317 (1962). entirely possible that changes in cyclic AMP and changes in D. L., Biochim. Biophys. Acta, 67, 305 (1963). 6. DIPIETRO, protein synthesis are only concurrent and that the inhibition of A., AND SOLS, A., Biochem. J., 86, 166 (1963). 7. 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Section 6: Adipose tissue, of glycerol or of fatty acids when isolated fat cells are incubated American Physiologi’eai So&&, Washington, 0. C., 1965; in the presence of 3.5% albumin. We did not measure ATP, p. 385. 14. STEINBERG, D., AND VAUGHAN, M., in A. E. RENOLD AND but there is some evidence that the ATP level does not limit G. F. CAHILL, JR. (Editors), Handbook of physiology, Secthe rate of protein synthesis under these conditions. Thus, tion 5: Adipose tissue, American Physiological Societ,p, stimulation of protein synthesis did not require added carboWashington, D. C., 1965, p. 344. hydrate substrate (Table I), and there is evidence that, although ATP depletion may occur when lipolysis is stimulated in cells a R. J. Ho, personal communication.

Issue of August

10, 1970

T. Minemura,

W. W. Lacy, and 0. B. Crofford

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46. 47. 48. 49. 50. 51.

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3881

Regulation of the Transport and Metabolism of Amino Acids in Isolated Fat Cells: EFFECT OF INSULIN AND A POSSIBLE ROLE FOR ADENOSINE 3',5'-MONOPHOSPHATE T. Minemura, W. W. Lacy and Oscar B. Crofford J. Biol. Chem. 1970, 245:3872-3881.

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