Neutral

Amino

Acid Transport Into Rat Skeletal Muscle: Regulation, and Effects of Insulin

Competition,

Adaptive

Armando R. Tovar, Jean K. Tews, Nimbe Torres, and Alfred E. Harper Amino acid (AA) transport systems A and L, which transfer preferentially

small neutral AA (SNAA) and large neutral AA (LNAA), respectively, were studied in the isolated soleus muscle with the specific models, Z-(methylamino)isobutyrate (MeAIB) and 2-aminobicyclo[2,2,1]heptane-2-carboxylate (BCH). Affinity for MeAlB was greater than for BCH (Km = 3.2 2 0.2 and 8.7 -+ 0.2 mm, respectively). Rate of transport of MeAlB (Vmax = 104 f 3 pmol/pL/min) was slower than for BCH (970 2 12 pmol/pL/min), but accumulation was far more concentrative; transport of BCH, but not MeAIB, rapidly reached a steady-state level. MeAlB transport was reduced in the presence of SNAA; BCH transport was reduced to a lesser extent only by LNAA. Mixtures of AA at concentrations resembling those in plasmas of rats fed either a 8% or 50% casein diet reduced transport of MeAIB, whereas BCH transport was low only with the latter mixture. Only MeAlB transport was stimulated by insulin. Preincubation of muscles for 5 hours in a AA-free medium stimulated subsequent MeAlB uptake by about twofold to fourfold; this effect was suppressed by inhibitors of protein synthesis. Selective differences were thus observed in transport by skeletal muscle of model AA for the A and L systems: increased transport resulting from various stimuli was limited to the model for the A system, and transport of either model was depressed with mixtures containing physiological levels of AA. Changes in dietary protein or AA intake may thus alter transport of certain neutral AA into skeletal muscle via changes in plasma AA pools. Copyright 0 1991 by W.B. Saunders Company

I

N STUDIES in which rats were fed a low protein diet containing analogs of the large neutral amino acids (LNAA), the ratios of muscle to plasma concentrations of the branched-chain amino acids (BCAA) were lower than for the corresponding controls; these ratios were also low when rats were fed a high protein meal.’ At the same time, ratios for small neutral (SNAA) or basic amino acids (AA) were little affected by the treatments with the analogs. These results suggested the possibility that plasma levels of LNAA or of the tested analogs were elevated enough under these conditions to compete with plasma BCAA for entry into muscle, whereas transport of SNAA or basic AA, occurring primarily by systems insensitive to LNAA, was unaffected. Competition studies have provided evidence that the neutral AA share a common transport system for uptake into brain. In fact, the close correspondence between Km values of individual neutral amino acids for transport across the blood-brain barrier and their usual plasma concentrations results in entry of such AA into brain being controlled in large measure by competition among them for this common transport system.’ As Km values for AA transport into muscle and liver are believed to be much higher than usual blood concentrations, it has been assumed that competitive effects will not influence transport of AA into such tissues in vivo.2 In only a few studies, usually only with pairs of AA with one in high concentration, has competition for transport into muscle been examined.3.4 Nonmetabolizable AA analogs have been used exten-

From the Departments of Nutritional Sciences and Biochemistty, College of Agriculrural and Life Sciences, University of WisconsinMadison, Madison, WI. Supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison, and by grants from the National Institutes of Health, Bethesda, MD (DK-10747); CONACYT, Mexico; and Clintec Nutrition Inc, Deerfield, IL. Reprints are not available. Copyright 0 I991 by WB. Saunders Company 0026-0495l91t4004-0013$03.OOiO 410

sively to identify characteristics of different transport systems in tissues. Christensen et al have described analogs that serve as specific models for two different systems for neutral amino acids: 2-(methylamino)isobutyric acid (MeAIB) is specific for the A system, which transports neutral amino acids with small side chains (SNAA), while 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) is a specific probe for the L system, which transports neutral amino acids with large, nonpolar side chains (LNAA).s.6 Despite its importance to the organism, AA transport into muscle, as emphasized by Lewis and Goldspink’ and Hundal et al? has received little attention. Therefore, the present work was undertaken to obtain further information on the transport into soleus muscle of MeAIB and especially of BCH, a model that apparently has not been examined in isolated muscle. In particular, we have determined the potential for various natural and analog AA to compete with MeAIB or BCH for transport into muscle, and have investigated accumulation of these model AA in the presence of insulin or under conditions known to permit adaptive regulation in other preparations in which transport is stimulated by AA deprivation.8-‘” METHODS Animals Male rats of the Sprague-Dawley strain, weighing 40 to 50 g and with access to water and commercial rat food, were used upon arrival (approximately 1090 AM) from the supplier (Harlan SpragueDawley, Madison, WI). Transport Assay Rats were decapitated and their soleus muscles, weighing approximately 12 to 20 mg, dissected out.” The muscles were immediately incubated at 37°C in 3 mL of Krebs-Ringer bicarbonate (KRB) buffer, pH 7.4, as described for the individual experiments. The medium contained glucose (10 mmol/L), MeAIB or BCH at the desired concentrations (usually 0.2 mmol/L, within the normal range of plasma concentrations of many of the neutral AA), and 0.3 @i of [1-‘4C]MeAIB (40-60 Ci/mol) or [carboxyl%]BCH (2 Ci/mol), respectively. Incubations were carried out Metabolism,

Vol40.No4

(April~,1991:pp410-419

COMPETITION

FOR BCH AND MeAlB TRANSPORT

continuous shaking at 70 cycles per minute; flasks were continuously gassed with humidified O,/CO, (95:5). Muscles for control and experimental groups were always incubated simultaneously in order to control for variability in individual experiments. After the incubations were completed, muscles were rinsed with ice-cold buffer, blotted, weighed, transferred to vials containing 1 mL of NCS tissue solubilizer (Amersham, Arlington Heights, IL), and heated for 5 to 6 hours at 50°C. The resulting solution was neutralized with 30 FL of glacial acetic acid and 10 mL of organic counting scintillant (OCS, Amersham) was added. These samples and portions of the incubation media were counted with an external standard for quench correction (LKB Rackbeta, Model 1217, Gaithersburg, MD).

with

Competition Studies Natural AA and analogs were usually added individually to the medium at a concentration of 10 mmol/L; when other concentrations of competitors were used, these are indicated in the appropriate figures and tables. Dithiothreitol (DTT) was included when cysteine was tested as a competitor; preliminary studies showed that D’M alone did not affect the transport of either MeAIB or BCH.

Other Studies The effect of insulin was tested by including the hormone in the transport assay. Transport of MeAIB or BCH was also measured after preincubation of muscles for various lengths of time in the absence of AA. Muscles were then transferred to fresh medium and incubated with radioactive MeAIB or BCH as described above. In some of these studies, an inhibitor of protein or RNA synthesis (cycloheximide. puromycin, tunicamycin, or actinomycin D) was present during the preincubation and incubation periods. Release of natural AA into the medium after incubation of muscles with either model was measured by determining by ion exchange chromatography the concentrations of individual amino acids in sulfosalicylic acid extracts of the medium (Beckman Model 119CL. Palo Alto, CA).

Calculations The transport tion ratio (DR) given time:

of MeAIB or BCH was expressed as the distribuor as pmol/kL of intracellular water (ICF) for a

DR=

dpm/pL dpm/kL

ICF medium

’

where dpm/kL ICF = pmol/kL

[dpmimg muscle - dpm/kL medium][ECF/lOO] [TWCilOO] - [ECFDOO]

ICF = DR x concentration

(FmoliL)

411

IN SOLEUS MUSCLE

in medium,

where ECF represents the extracellular space and TWC the total water content of the muscle. The extracellular space was measured by incubating muscles with [‘4C]sucrose for selected periods. Values were 28.1% 2 0.3% at 10 minutes (n = 4), 31.2% 2 1.2% at both 30 and 60 minutes (combined n = 15), and 33.8% ? 1.9% at 240 minutes (n = 4). The total water content of the muscles was measured by drying the tissue in a vacuum oven at 100°C until a constant weight was reached (75.0% 2 2.1%; n = 20).

Materials L-amino acids and AA analogs. insulin (porcine, 26.2 IU/mg) and antibiotics were obtained from Sigma, St Louis, MO. Radioactive

MeAIB and BCH were (DuPont, Boston, MA).

obtained

from

New England

Nuclear

Statistics The results were evaluated by Student’s t test or by ANOVA followed by Fisher’s protected least significant difference (LSD) test to determine the significance of differences among groups. Kinetic constants were calculated by weighted nonlinear regression.” RESULTS

Accumulation, transport, and uptake have been used as interchangeable terms; accumulation is probably more accurate, as both influx and efflux are presumably occurring in experiments lasting more than 10 to 15 minutes. We have not included detailed results on the requirements for sodium, the presence of glucose, or the effects of anaerobiosis. Our findings were similar to those obtained for numerous other preparations: transport of the A system model MeAIB was reduced in the absence of sodium, glucose or oxygen, whereas transport of the L system model BCH was unaffected by these treatments.5

Effect of Time or of Substrate Concentration MeAIB was clearly accumulated in soleus muscle, as its concentration in the intracellular fluid was almost threefold higher than in the medium after 4 hours of incubation (Fig IA). The distribution ratio continued to increase throughout the incubation period. Uptake of BCH followed a different pattern (Fig 1B). The system reached a plateau within 2 hours, and the highest ratio obtained was 1.9, well below the value for MeAIB. Initial uptake of BCH was faster than that of MeAIB. A ratio of approximately 1.0 was achieved within 30 minutes for BCH, but not until 1 hour for MeAIB. Uptake of MeAIB was linear for at least 15 minutes, while BCH uptake was linear for only about 10 minutes; these results imply that little efflux occurred during these brief time periods. Effects of different concentrations of MeAIB and BCH on rates of uptake were measured by incubating muscles in the different media for 10 minutes, the linear phase of transport. Transport of MeAIB was saturable (Fig 2A). The calculated Km was 3.2 ? 0.2 mmol/L and the Vmax was 104 ~fr3 pmol/pL ICF/min. Transport of BCH (Fig 2B), was also saturable, but affinity (Km = 8.7 * 0.2 mmol/L) was lower than for MeAIB and Vmax was much higher (Vmax = 970 c 12 pmol/pL ICF/min). Similar values were obtained when these constants were calculated with a nonsaturable component included, indicating that little diffusion occurred under our conditions, as in perfused muscle.4

Competition Studies Transport of MeAIB into isolated soleus muscle was not depressed in the presence of high concentrations of lysine or aspartic acid, typical basic and acidic AA (Table 1). Typical LNAA produced small, but statistically nonsignificant, decreases in mean values for uptake of MeAIB. Threonine, histidine and glutamine significantly depressed

TOVAR ET AL

A 0

60

120 Time

Time

180

240

(min)

(min)

Fig 1. Effect of time on transport into soleus muscle of (A) MeAlB (1 mmol/L) and (B) BCH (1 mmol/L). Results are reported as the DR 5 SE; n = 4.

MeAIB accumulation (by 17%, 27%, and 40%, respectively), but the most effective competitors for MeAIB transport were SNAA such as serine, proline, and alanine (accumulation depressed by 43%, 44%, and 55%, respectively). Typical analogs of SNAA, especially 2-aminoisobutyric acid (AIB) and homoserine, were also effective competitors for MeAIB transport. The presence of cold MeAIB produced a significant inhibition, again providing evidence of saturability of the transport system for this AA as in Fig 2A. Acidic or basic AA did not depress transport of BCH into soleus muscle (Table 2). BCH transport, unlike that of MeAIB, was not clearly depressed in the presence of SNAA such as proline and alanine. Although BCH uptake was inhibited to about the same extent by alanine, cysteine, threonine, and serine, only the effect of serine was statistically significant. The most effective competitors for BCH transport were typical LNAA such as the branched-chains, where isoleucine, the strongest competitor, reduced BCH transport by 37%. Aromatic AA such as tryptophan and phenylalanine significantly reduced BCH transport by approximately 30%, an effect similar to that produced by histidine. Norleucine and norvaline reduced BCH uptake to approximately the same extent but, because of high variability with norvaline, only the effect of norleucine was statistically significant; norleucine appeared to be a less effective competitor than any of the tested natural LNAA. SNAA analogs that were tested did not compete with BCH for transport. Addition of 10 mmol/L BCH reduced the uptake of radioactive BCH by 30%, consistent with the results shown in Fig 2B. In further studies, the transport of MeAIB or BCH was measured in the presence of different concentrations of serine and phenylalanine as competitors for MeAIB and BCH, respectively. Serine at a concentration of 40 mmol/L depressed uptake of MeAIB by 60% (Fig 3A), while phenylalanine (40 mmol/L) decreased BCH uptake by 48% (Fig 3B). Accumulation of MeAIB or BCH was also measured in media containing mixtures of AA at concentrations found in plasma after rats, previously adapted to consuming S-hour meals of an 18% casein diet, had access for 4 to 7 hours to a 6% or 50% casein diet.‘j MeAIB uptake was reduced 39% and 43%, respectively, below control values when muscles were incubated with AA mixtures simulating plasma concentrations of rats fed a 6% or 50% casein diet (Table 3). In contrast, BCH uptake was unaffected when muscles were incubated in the mixture simulating plasma from rats fed the 6% casein diet; however, its uptake was reduced by 23% when the medium contained AA, as in plasma from rats fed a 50% casein diet. As would be expected, release into the medium of natural AA from individual muscles occurred during the incubation period. However, after 1 hour of incubation with only MeAIB in the medium initially, concentrations of SNAA in the medium ranged from only 0.008 mmol/L (threonine) to 0.029 mmol/L (glycine); proline was not detected. Except for glutamine (0.026 mmol/L), concentra-

COMPETITION

FOR BCH AND MeAlB TRANSPORT

IN SOLEUS MUSCLE

800 80

0

2

4

6

MeAlB

(mM)

8

10

0

5

10

15

20

BCH (mM)

Fig 2. Effect of initial concentration on rates of uptake into soleus muscle of (A) MeAlB and (B) BCH. Incubations were carried out for 10 minutes. Results are expressed as pmol/min/yL ICF + SE: n = 4.

tions of LNAA in the medium were even lower than those of the SNAA (0.002 mmol/L for methionine and leucine; others undetectable). AA release after incubation with BCH (30 minutes) was either similar to, or lower than, that after the longer incubation with MeAIB. The amounts released were so low that resultant competition for transport of either MeAIB or BCH would be highly improbable. Thus, our studies are unlikely to be compromised by endogenous AA present in the medium as a result of their efflux from the muscle. Effect of Insulin

Insulin (200 nmol/L) significantly raised uptake of MeAIB to approximately 20% above the control value (Table 4). As expected for an A system substrate, there was a significant reduction in MeAIB uptake when muscles were incubated in a sodium-free medium, and no stimulatory effect of insulin on MeAIB transport occurred in the absence of sodium. BCH transport was not affected by sodium, or by insulin (200 nmol/L) regardless of the presence of sodium (Table 4) nor was there any effect on BCH transport of several higher concentrations of insulin, up to 6,400 nmol/L (not shown). Preincubation with insulin for 1 hour before incubation with both insulin and BCH also did not stimulate BCH uptake, whereas MeAIB transport was again clearly increased by 32% over the corresponding control preincubated without insulin (Table 4).

Adaptive Regulation

In order to study the effects of AA deprivation, muscles were preincubated in buffer alone for periods of time up to 5 hours before being transferred to flasks containing radioactive MeAIB or BCH (Fig 4). After only 30 minutes of preincubation, the subsequently measured accumulation of MeAIB did not differ from control values; however, as the preincubation periods were extended, MeAIB uptake measured subsequently increased steadily so that after 5 hours of preincubation, MeAIB accumulation was almost fourfold above that for the untreated controls. In contrast, preincubation for 2 hours was needed to induce even a minor, although statistically significant, stimulation of BCH accumulation of about 25%; no further increase occurred when the preincubation periods were extended (Fig 4). MeAIB transport was stimulated much less by AA deprivation if specific antibiotics were included in the preincubation medium (Table 5A). In the presence of cycloheximide or puromycin, inhibitors of protein synthesis, MeAIB accumulation measured after 5 hours of preincubation was less than 50% above that for the nonpreincubated control, a statistically nonsignificant increase. Actinomycin D, an inhibitor of RNA synthesis, also strongly inhibited the adaptive response, whereas tunicamycin, an inhibitor of protein glycosylation, was somewhat less effective at the tested concentration (Table 5A). When addition of cycloheximide was delayed until after the first hour of the S-hour

414

TOVAR ET AL

Table 1. Effects of Natural and Analog AA on MeAlB Accumulation in

Soleus Muscle Control

Competitor

(pmol MeAIWrL

% of Control

ICF)

NaturalAA Lvs

352 264

4OOk

28

113

Asp Phe

352 k64

390 + 48

111

352k64

350 k 36

99

Tvr Ide

332 lr46

306 k 30

92

350 2 8

314k18

90

Trp Val

350 + 8

310230

89

350? 8

310 + 14

89

Leu

352? 64

3062

12

87

Thr

350 ?a

290 214x

83

His

350 + 8

254i 8*

73

Gln

332 -t48

198k 101

60

cvs Ser

332 + 48

192 +10*

58

350 + 8

198?10*

57

Pro

332 -c48

186-c 12*

56

Ala

35Ok8

156 + 22*

45

Analog AA Norleu

352 k 64

232 -t 20

66

AABA

332 2 48

216 f 8*

65

NoNal

332 k48

206 f 28*

62

AIB

352 -t64

202 + lo*

57

Homoser

352 *64

1925 20*

55

MeAlB

332 24%

172 t4*

52

DISCUSSION

NOTE. Incubations were carried out in Krebs-Ringer bicarbonateglucose buffer containing radioactive MeAlB (0.2 mmol/L) and competitors at 10 mmol/L (L forms). Incubations were for 60 minutes, the time required for intracellular

MeAlB to reach the concentration

in the

medium (taken to be more representative of conditions in vivo than are initial values). Values are means 2 SEfor four muscles per treatment. Abbreviations: AABA, 2-amino-n-butyrate;

competitors for MeAIB transport as shown in Table 1) during only the 5-hour preincubation period also suppressed the adaptive response of MeAIB transport observed consistently following extended preincubation without amino acids in the medium (Table 5B). Under the conditions of this experiment, concentrations of SNAA in the final incubation medium would have depended on SNAA efflux from the preincubated muscle and would have been far less than 10 mmol/L; thus, the depression of MeAIB uptake is not due to the competition effects shown in Table 1. The results for the nonpreincubated groups again illustrate competition by SNAA for MeAIB transport as in Table 1. MeAIB uptake, however, was unaffected when muscles, preincubated with an equimolar mixture of LNAA (10 mmol/L; leucine, isoleucine, valine, phenylalanine, and tryptophan), were subsequently incubated with labeled MeAIB. MeAIB uptake by these muscles was 95% of the value for the corresponding control sample preincubated without LNAA (Table SC).

Skeletal muscle plays an important role in maintaining the stability of blood AA concentrations.‘4 Rate of entry of an individual AA into muscle increases when blood concentration of that AA is increased.15 Hence, excess free AA may be stored, at least temporarily, in muscle through expansion of its free AA pools. Muscle protein degradation contributes to maintenance of blood AA pools, especially during periods of food or protein deprivation.16 In addition,

AIB, 2-aminoisobutyrate;

Homoser homoserine; Norleu, norleucine; Norval, norvaline.

Table 2. Effects of Natural and Analog AA on BCH Accumulation in Soleus Muscle

*P < .05 when compared with the corresponding control.

Control preincubation period, subsequent uptake of MeAIB (682 & 44 pmol/pL ICF) was intermediate between values for the nonpreincubated control and those for samples preincubated without antibiotics. Omission of actinomycin D during the first hour only of the 5-hour preincubation period did not significantly alter MeAIB uptake (680 -t 80 pmol/pL ICF) from that observed after 5 hours of preincubation with this agent. Once stimulation of MeAIB accumulation had occurred in response to extended amino acid deprivation, the stimulatory effect declined if the preincubation period was continued in the presence of 0.2 mmol/L MeAIB (Fig 5). Thus, after exposure of 5-hour preincubated muscles to 0.2 mmol/L cold MeAIB for various further periods up to 2 hours, subsequent uptake of radioactive MeAIB from fresh medium was reduced to a value less than halfway between those for the samples preincubated without cold MeAIB and the standard controls. In contrast, when control muscles previously incubated in the amino acid-free medium for 5 hours were incubated for another 2 hours under the same conditions, further stimulation of MeAIB uptake occurred. The presence of a mixture of SNAA (10 mmol/L; equimolar in serine, proline, and alanine, the most effective

Competitor

% of Control

(pm01 BCH/pL ICF)

Natural AA Lvs

236 f 8

278 + 14’

117

ASP

236 k8

256 +6

108

Pro

280 + 18

2762

14

99

Gln

280 f 18

268 ? 28

96

Ala

226 2 26

194 + 4

86

Cvs

280 + 18

234 ? 12

84

Thr

226 2 26

190 2 8

84

Ser

226 % 26

188 f IO’

83

Leu

236 + 8

180% IO*

76

Trp

226 f 26

164+10*

72

His

226 f 26

162?6*

72

Phe

236 f 8

166 2 6*

JO

Val

226 + 26

154 r 6’

68

Ile

226 2 26

142 2 2*

63

Analog AA MeAlE AIB

280 2 18

286 -+ 32

102

236 + 8

240214

102

AABA

280 +- 18

262 r 26

94

Homoser

236 + 8

214 t 6

91

Norleu

236 -t 8

198 % 2*

84

Norval

280 + 18

220 & 14

79

NOTE. incubations with labeled BCH (0.2 mmol/L) were carried out for 30 minutes, the time required for intracellular BCH to reach the concentration in the medium. Other details are described in Table 1.

415

COMPETITION FOR BCH AND MeAlB TRANSPORT IN SOLEUS MUSCLE

400

s 300 Ti zl a Ez v) 200 0) s E Q_ 100

50 -

A

0 0

10

20

Concentration

of Serine

0

40

30

.

0

(mM)

10 of

40

30

20

Concentration

B

Phenylalanine

(mM)

Fig 3. Competition for transport into isolated soleus muscle of (A) MeAlB in the presence of increasing concentrations of serine, and (B) BCH in the presence of increasing concentrations of phenylalanine. Muscles were incubated in buffer containing labeled MeAlB or BCH (0.2 mmol/L) for 60 or 30 minutes, respectively, the approximate times required for intracellular concentrations of these AA to reach that in the medium. Each point represents the mean + SE (n = 4).

muscle is an active site of metabolism of AA and contributes particularly to the removal of BCAA and the release of alanine and glutamine into the circulation.” As the rates of all of these processes respond to changes in AA suppl~,‘~

the kinetic and regulatory properties of systems for the transport of AA between blood and muscle have the potential to influence the concentrations of AA in both the extracellular fluid and muscle compartments.

Table 3. Accumulation of MeAlB or BCH in Soleus Muscles Incubated

Table 4. Effect of Preincubation With Insulin or Sodium Ion on the

in Media Containing Mixtures of AA in Concentrations

Comparable

Ability of Insulin to Stimulate Accumulation of MeAlB or BCH in

to Those in Plasma

Soleus Muscle

MeAlB

BCH

MeAlB BCH pmolfpl ICF

(pmol/kL ICF)

Trcatlllent Control

462 c 22”

236 + 6”

6%

282 2 10D

234 )_ 22”

50%

262 + 12b

182 -t 6b

NOTE. Incubations with MeAl

or BCH (0.2 mmol/L) were carried out

in media containing AA in concentrations resembling those in plasma of rats fed a 6% or 50% casein diet.‘” For the mixture simulating plasma values for the 6% casein group, concentrations (pmol/L) in the medium were: Asp, 15; Thr, 1,340; Ser, 470; Glu, 110; Pro, 370; Gly, 290; Ala, 1,230; Citrulline, 80; Val, 170; Cys, 13; Met, 130; Be, 60; Leu, 100; Tyr, 10; Phe, 40; Trp, 60; Orn, 60; Lys, 500; His, 90.; and Arg, 40 (sum = 4,360

(A) Insulin _

Sodium +

298 -c 9”

1944

11

+ _

+ _

353 f 16”

188”

7

139 -c 15”

170 + 10

+

_

145 r 10

168 f 6

(B) Insulin _

Preincubation _

307 i 26”

163 _t 8

_

+

365 -c 6”

163 2 4

+

+

481 + 26”

167 2 18

NOTE. (A) Muscles were incubated with labeled MeAlB or BCH (0.2

kmol/L for neutral AA). For the mixture simulating plasma values for the

mmol/L) and porcine insulin (200 nmol/L) in bicarbonate-glucose

50% casein group, concentrations were: Asp, 30; Thr, 1,760; Ser. 810;

or in this buffer in which NaCl and NaHCO, were replaced by choline

buffer

Glu. 170; Pro, 1,450; Gly, 210; Ala, 1.010: Citrulline, 150; Val, 850; Cys,

chloride and l&CO,,

15: Met, 200; Ile, 280; Leu, 420; Tyr, 360; Phe, 110; Trp, 190; Orn, 100;

with insulin was 1 hour; muscles were then transferred to fresh medium

Lys, 880; His. 150; and Arg, 80 (sum = 7,820 p,mol/L for neutral AA).

without or with insulin and labeled MeAlB or BCH as indicated. Other

Other details are described in Tables 1 and 2.

details are described in Tables 1 and 2.

For MeAl

or BCH, respectively,

different

superscripts

indicate

significant differences (P < .05; ANOVA followed by the LSD test).

Different

respectively. (9) Preincubation period vyithout or

superscripts

indicate significant differences

for MeAlB

within each section (P < .05; ANOVA followed by the LSD test).

416

TOVAR ET AL

however, glutamine clearly reduced MeAIB, but not BCH, transport into isolated soleus muscle. Although histidine is also a substrate for the N” system,3 it reduced MeAIB uptake as well as that of BCH into soleus muscle; the latter effect on BCH transport may reflect its characteristics as a LNAA. The greater degree of competition with MeAIB than with BCH may be explained in part by the fact that the observed Km for MeAIB of 3.2 mmol/L was less than half of the value for BCH. A similar differential effect, especially at low concentrations of the competitors, was suggested by measurements of MeAIB and BCH transport in the presence of varying concentrations of serine and phenylalanine, respectively (Fig 3A and B). Competition studies with various muscle preparations have been reported for transport of AIB or other neutral AA, but seldom of MeAIB or BCH.%4,6.“‘23

o! 0.0

’

’

1.0

2.0

Preincubation

,

’

’

’

3.0

4.0

5.0

Time

(hr)

Fig 4. Effect of time of preincubetion in AA-free medium on uptake of MeAlB or BCH by soleus muscle. After the indicated preincubation times, muscles were transferred to flasks containing radioactive MeAlB or BCH (0.2 mmol/L) and incubated as described in the legend for Fig 3.

After consumption of a meal, the presence of increased amounts of AA in the blood may alter tissue concentrations of these nutrients by various means, including effects on transport mechanisms. A well-known example is that of brain in which Peng et alz4 originally showed that AA concentrations can be selectively altered depending on the plasma AA pattern, which in turn reflects the composition of the diet; this effect is probably prominent in brain because Km values for AA transport into brain do not differ greatly from AA concentrations in plasma.’ Our results show that MeAIB accumulation was reduced when muscles were incubated in media containing AA in amounts equivaTable 5. Stimulation of MeAlB Transport into Soleus Muscle by AA

The value of presumably specific nonmetabolizable, model AA for characterization of different AA transport systems is well recognized. Although studies in muscle with natural AA or nonspecific models such as AIB have been reported, there have been only limited investigations with MeAIB.‘8,‘9 Even less information is available for BCH.6 The present studies allow the first opportunity for comparisons in isolated soleus muscle of the transport characteristics of these recognized models. Our results indicate that the A system in muscle is able to maintain small neutral AA at levels well above those in the medium; this is supported by observations that concentrations of such amino acids in muscle can range from twofold to sevenfold above plasma values.‘,” Rapid equilibration of BCH between the medium and the tissue is consistent with observations that in other tissue preparations AA transported by the L system exchange rapidly between cells and their surrounding media. 5*9~Lo Thus, skeletal muscle content of LNAA generally reflects plasma concentrations of these nutrients.‘,15 Threonine competes relatively poorly with MeAIB for transport into muscle, suggesting that the ASC system for SNAA, such as alanine, serine, and cysteine, may contribute to threonine entry into muscle as it does in brait? and certain other tissues.” The presence of the ASC system in isolated skeletal muscle has been reported.” Glutamine transport into perfused muscle appears to occur by a specific system (designated N”) not inhibited by MeAIB’;

Deprivation and its Suppression by Preincubation With Antibiotics of With AA Treatment

pmol MeAIWpL

ICF

(A) Antibiotics 326 k 19’

No preincubation

1,262 2 68

Preincubation +Puromycin (0.2 mmol/L)

476 -c 9”,’

+Cycloheximide

486 -t 44’,’

(0.1 mmol/L)

+Actinomycin D (0.05 mmol/L)

594 k 48”

+Tunicamycin

864 + 94’

(2.4 nmol/L)

(6) Preincubation with SNAA 404 k 34”

No preincubation

148 + 10

+ 10 mmol/L SNAA

798 f 44”

Preincubation

514 r 20d

+ 10 mmol/L SNAA (C) Preincubation with LNAA

334 + 21”

No preincubation

258 + 12b

+I0 mmol/L LNAA

785 + 40’

Preincubation

743 -t 54’

+ IO mmol/L LNAA

NOTE. (A) Antibiotics were present during both the preincubation (5 hours) and incubation (1 hour) periods. (B) SNAA, equimolar mixture of serine, proline, and alanine. (C) LNAA. equimolar mixture of leucine. isoleucine, valine, phenylalanine,

and tryptophan.

SNAA and LNAA

were included in the media during the 5-hour preincubation

period

only. Muscles without preincubation were immediately placed in buffer containing radioactive MeAlB (0.2 mmol/L). Other details are described in Table 1. Different superscripts indicate significant differences within sections A, B, or C (P c .05: ANOVA, followed by the LSD test).

COMPETITION

FOR BCH AND MeAlB TRANSPORT

IN SOLEUS MUSCLE

60

0 Preincubation

120 Time

(min)

Fig 5. Repression of MeAlB transport after its stimulation by preincubation of muscles in an AA-free medium. Muscles were preincubated for 5 hours as for experiments summarized in Fig 4, then with unlabeled MeAlB (0.2 mmol/L) for the indicated times up to 2 hours, and finally with radioactive MeAlB (0.2 mmol/L) for 60 minutes as described in Fig 3. 0, Preincubated with cold MeAIB; A, preincubated without cold MeAlB for a total of 7 hours; 0, no preincubation.

lent to those found in plasma after rats consumed a meal of a 6% or 50% casein diet.13 This observation may be explained by the fact that the total concentrations of the SNAA (threonine, serine, proline, glycine, and alanine) were 3.70 and 5.25 mmol/L, respectively, somewhat above the observed Km of 3.2 mmol/L for MeAIB (even though these SNAA were not equally competitive on a molar basis). The sum of the concentrations of the LNAA (branched-chains, methionine, phenylalanine, tyrosine, tryptophan, and histidine) were 0.66 and 2.56 mmol/L, respectively, for rats fed the 6% and 50% casein diets. The lower concentration of LNAA was less than 10% of the observed Km for BCH and no inhibition of its accumulation into soleus muscle was observed with this level of LNAA. However, in media containing the higher amount of LNAA, BCH uptake was significantly reduced. Hundal et al4 found evidence for competition among pairs of AA including the branched-chains and phenylalanine (Km for each was approximately 20 mmol/L), but not by high concentrations of other neutral AA. The sensitivity of BCH transport into soleus muscle to physiological concentrations of LNAA may reflect the relatively low Km for BCH under our conditions. Our results suggest that under physiological conditions, competition among SNAA for passage via the A system

417

from blood into skeletal muscle might occur normally, whereas competition among LNAA for transport via the L system is likely only when their total plasma concentrations are unusually high, eg, after the animal consumes a nigh protein diet with resultant several-fold increases in concentrations of certain LNAA as in Table 3, or one containing disproportionate amounts of AA, or in uncontrolled genetic disorders of AA metabolism. The high capacity and low affinity of the L system, as well as the occurrence of trans-stimulation, may help explain why concentrations of LNAA in skeletal muscle are seldom much above those in plasma.4,‘5 However, the modest competition by some LNAA for MeAIB transport suggests that the A system may be involved in the limited concentrative accumulation of LNAA often observed in vivo. Hormonal control of transport into tissues is important in regulation of intracellular AA po01s.~~‘”Insulin, for example, stimulates uptake of both natural and analogue AA such as AIB and MeAIB by various muscle types, especially diaphragm. We found that insulin significantly stimulated MeAIB uptake into soleus muscle, while BCH transport was unaffected by this hormone. These results are consistent with other studies in vitro, which show that insulin stimulates uptake of certain natural AA,‘5-‘7 as well as of analogs such as AIB”~‘“-” and MeAIB.‘*.‘” Some studies also indicate the inability of insulin to stimulate transport of primarily LNAA,L’~25~27 including BCH.6 Hundal et al4 found that AA transport in perfused muscle is not strongly responsive to insulin. Despite such observations, insulin has often been assumed to stimulate uptake into muscle of neutral AA in general, especially the branched-chains”.“; the apparent increase in transport may instead reflect the effects of insulin-stimulated synthesis of muscle proteins.” Stimulation of AA transport by extended exposure to an AA-free medium, as well as its prevention in the presence of antibiotics, was first demonstrated in liver.j4 Later studies showed that this adaptive regulation is usually restricted to those neutral AA transported by the A and probably the N systems (reviewed in refs 9,lO). Our results showing little effect on BCH but clear stimulation of MeAIB uptake into soleus muscle fit with this conclusion. Adaptive regulation has also been demonstrated by others in isolated skeletal muscle” or in cultured myocytes.23 Its prevention by inhibitors of protein or RNA synthesis implies that the effect is due to formation of new protein(s), as well as the corresponding RNA. Because of the inhibitory effect of tunicamytin on MeAIB transport under our conditions, protein glycosylation may also be an important aspect of adaptive regulation in muscle as in hepatocytes? Our studies have shown that MeAIB transport is clearly concentrative; is subject to competition by high concentrations of individual SNAA or by AA mixtures simulating plasma AA profiles, but not by most individual LNAA, can be stimulated by insulin; shows adaptive regulation upon AA deprivation; and has requirements for sodium, an energy source, and oxygen (results not shown). Generally, these features of MeAIB transport contrast strongly with observed characteristics of BCH transport. Uptake of this model is barely concentrative, with a higher Km and Vmax

418

TOVAR ET AL

than for MeAIB; is subject to competition by high concentrations of individual LNAA or by AA mixtures simulating only selected plasma AA profiles, but not by most individual SNAA, is not stimulated by insulin; shows little response to AA deprivation; and has no demonstrable requirement for sodium, an energy source, or oxygen (results not shown). Our observations provide a basis for a better understanding of AA transport into skeletal muscle in vivo. The extent to which the rate of entry of AA into tissues other than brain is controlled directly by regulation of transport is unclear. The high capacity, low concentrative ability, and lack of hormonal responsiveness of the L system for transport of LNAA into muscle suggest that rate of uptake of individual AA by this system is regulated in vivo mainly by their concentrations in the extracellular fluid and by metabolic removal through AA degradation or protein synthesis. That uptake of AA transported into muscle by

this system may be influenced significantly by mutual competition seems remote unless their extracellular fluid concentrations are unusually high as may occur after ingestion of a high protein meal, or disproportionate amounts of AA, or in individuals with certain genetic defects of AA metabolism. The lower capacity, higher affinity, and possible hormonal and adaptive responsiveness of the A system for transport of SNAA into muscle suggest that mutual competition could frequently occur among AA transported by this system. Despite the importance of transport in maintaining a reservoir of AA in muscle, the extent to which the supply is controlled directly by regulation of transport remains unresolved. ACKNOWLEDGMENT

We are grateful to Joyce Repa for performing

the amino

acid

analyses.

REFERENCES 1. Tews JK, Harper AE: Food intake, growth and tissue amino acids in rats fed amino acid analogues. J Nutr 115:1180-1195, 1985 2. Pardridge WM: Brain metabolism: A perspective blood-brain barrier. Physiol Rev 63:1481-1535,1983

from the

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COMPETITION

FOR BCH AND MeAlB TRANSPORT IN SOLEUS MUSCLE

30. Zorzano A, Balon TW, Garetto LP, et al: Muscle o-aminoisobutyric acid transport after exercise: Enhanced stimulation by insulin. Am J Physiol248:E546-E552,1985 31. Karlstad MD, Sayeed MM: cy-Aminoisobutyric acid transport in rat soleus muscle during endotoxic shock. Am J Physiol 248:R142-R146,1985 32. Munro HN, Fernstrom JD, Wurtman RJ: Insulin, plasma amino acid imbalance and hepatic coma. Lancet 1:722-726,1975

419

33. Wurtman RJ: Behavioural effects of nutrients. Lancet 1:11451147,1983 34. Tews JK, Woodcock NA, Harper AE: Stimulation of amino acid transport in rat liver slices by epinephrine, glucagon and adenosine 3’,5’-monophosphate. J Biol Chem 245:3026-3032, 1970 35. Barber EF, Handlogten ME, Kilberg MS: Induction of amino acid transport system A in rat hepatocytes is blocked by tunicamycin. J Biol Chem 258:11851-11855,1983