[CANCER RESEARCH 45, 6296-6300, December 1985]

Basal Amino Acid Concentrations and the Response to Incremental Glucose

Infusion in Tumor Bearing Rats

Jeffrey M. Arbeit,1 Catherine M. Gorschboth, and Murray F. Brennan

Surgical Metabolism Section, Surgery Branch, National Cancer Institute, NIH, Bethesda, Maryland 20205

ABSTRACT

Plasma amino acid concentrations were measured in fastingnontumor bearing (NTB) and tumor bearing (TB; methylcholan-

threne induced sarcoma) male Fischer F344 rats during infusionof 0.9% NaCI solution or glucose at 3.72 or 13.05 ¿tmol/lOOgtotal body weight (TBW)/min. The animals were studied whenthe tumor comprised only 8% of the TBW at a time whendecreased food intake and weight loss were not manifest.

During 0.9% NaCI infusion there were no significant differencesbetween NTB or TB animals in the concentration of alanine (NTB:152.6 ±20.1; TB: 150.3 ±19.0 /IM; mean ±SD), branchedchain amino acids (BCAA) (NTB: 343.3 ±48.7; TB: 344.2 ±20.5tiM), essential amino acids, aromatic amino acids, or total aminoacids. During infusion of glucose at 3.72 jumol/100 g TBW/minthe alanine levels rose (NTB: 283.6 ±33.4; TB: 286.7 ±43.3fiM), and the BCAA levels fell (NTB: 215.9 ±19.4; TB: 228.7 ±43.4 UM) to similar concentrations in both NTB and TB animals.Glucose infusion at 13.05 ^mol/100 g TBW/min resulted in anadditional increase in the alanine concentration (NTB: 344.5 ±28.7; TB: 382.8 ± 116.6 MM), and a further decrease in theBCAA concentration (NTB: 166.4 ±30.8; TB: 160.7 ±30.5 ^M)without significant differences between NTB and TB animals.Paired analysis for each animal prior to and during glucoseinfusion demonstrated a similar absolute micromolar change inalanine and BCAA concentration during both glucose infusionrates in both NTB and TB animals. The levels of aromatic aminoacids and total amino acids were unchanged and the essentialamino acid concentrations were decreased only at the higherglucose infusion rate in both NTB and TB groups.

Basal amino acid metabolism appears similar in the NTB andTB animals, prior to the onset of anorexia and weight loss.During exogenous glucose infusion the reciprocal changes in theplasma alanine and BCAA concentrations support the conceptof a glucose-alanine-BCAA cycle at the whole body level that

appears to respond to a similar extent in NTB and TB animals.

INTRODUCTION

Certain patterns of plasma amino acid concentrations havebeen demonstrated to exist in various metabolic states; postprandial (1), prolonged fasting (2), kwashiorkor (3), marasmus(4), diabetes (5), hepatic encephalopathy (6), after elective operation (7, 8), fever (9), and sepsis (10). To date there has beenlittle work on the impact of tumor bearing on plasma amino acidlevels (11,12). Those reports demonstrating decreases in theconcentrations of the gluconeogenic amino acids (11,12), and

Received 5/30/85; revised 9/4/85; accepted 9/5/85.1To whom requests for reprints should be addressed, at Memorial Sloan-

Kettering Cancer Center, Department of Surgery, 1275 York Ave., New York, NY10021.

increases in the branched chain amino acids (11, 12), in tumorbearing, and nontumor bearing patients have been complicatedby antecedent weight loss and cachexia in the TB2 groups (11,

12). Since both plasma and tissue amino acids can be affectedby prior food intake (13, 14), the exact cause of the alteredplasma amino acid levels is obscure.

In addition, abnormalities in glucose metabolism have beenshown to exist in both TB animals (15-18) and humans (19, 20).

Increased Cori cycle activity has been demonstrated in tumorbearers in the basal state (21, 22), and blood lactate levels havebeen reported to rise disproportionately in TB animals (16, 18)and humans (23) given exogenous glucose.

Similar to the Cori cycle, a glucose-alanine-BCAA cycle has

been proposed to explain the elevation in plasma alanine anddepression in BCAA concentrations during exogenous glucoseinfusion reported in several studies (24-28). Gluconeogenesis

from alanine has been reported to be elevated in cancer patients(29, 30) and in several animal studies (16-18). This study ex

amines basal amino acid levels in a tumor bearing animal modelsystem in an early growth stage (31), when food intake is similarto NTB controls (18). The concentrations of individual or groupsof amino acids during two rates of exogenous glucose infusionare measured to determine whether there is a preferential response in the glucose-alanine-BCAA cycle or in individual amino

acids in TB compared to NTB animals.

MATERIALS AND METHODS

Preparation of Animals. Male Fischer F344 rats were obtained fromCharles River Breeding Laboratories, maintained on rat chow (NIH No.5018, Ralston Purina Co., St. Louis, MO) and water ad libitum, and keptin a 12-h light, 12-h dark cycle. Rats weighing 230-250 g were anesthe

tized with i.p. sodium pentobarbital, 50 mg/kg, and using aseptic technique, the left common carotid artery and right external jugular vein werecannulated (18), and the animals were placed in individual plastic metabolic cages (Model 1100, Maryland Plastics, Federalsburg, MD). Fourdays after cannulation the animals that were to be tumor bearing weregiven injections s.c. in the right flank with a suspension of 1 x 106 viable

cells (by trypan blue exclusion) (32) of a methylcholanthrene inducedsarcoma which has been serially passaged in our laboratory (31). BothNTB and TB animals were used only when they had regained theirpostoperative weight loss, and the tumors were 2-3 cm in diameter

(Table 1).Experimental Procedure. At 8:00 a.m. food was removed from the

cages, the animals were fasted with water given ad libitum, and urinewas collected in containers filled with 1.0 ml of 2 N H2SO4, and analyzedfor total nitrogen by the micro-Kjeldahl method (33). After 24 h, a base

line arterial blood sample was obtained, and the venous cannula wasconnected to a constant infusion (Harvard Apparatus, South Natick, MA)of either 0.9% NaCI solution or glucose at 3.72 or 13.05 jumol/100 g

2The abbreviations used in this paper are: TB, tumor bearing; NTB, nontumor

bearing; TBW, total body weight; BCAA, branched chain amino acids; AAA,aromatic amino acids; EAA, essential amino acids; TAA, total amino acids; ANOVA,analysis of variance.

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AMINO ACID METABOLISM IN TB RATS

TBW/min. These glucose infusion rates were the glucose turnover rateand 3.2 times the glucose turnover rate of a NTB rat as determined byD-[3-3H]glucose (18). Six h later, four arterial blood samples were ob

tained at 5, 20, 45, and 90 min posttracer injection (18). A preterminalsample of arterial blood was obtained for insulin levels, the animals werekilled by air embolization, and the TBWs were determined.

Preparation and Analysis of Blood Samples. The samples (220 /¿I)were placed in chilled tubes containing 22 n\ of sodium heparin (1000units/ml; Upjohn, Kalamazoo, Ml), centrifugea, and the plasma wasdeproteinized by membrane centrifugaron (Centriflo Cones Type CF50A;Amicon, Lexington, MA). A 35-/J aliquot of filtrate was diluted with 60 /il

of 0.15 N lithium citrate buffer, pH 2.20 (34) (Beckman Instruments, PaloAlto, CA), and 10 M! of norleucine (Sigma Chemical Co., St. Louis, MO)were added as an internal standard and placed on an amino acid analyzer(Beckman Instruments Model 121 MB). Variability in the sample aspiration was corrected by multiplying each amino acid concentration by anormalization factor obtained from the known quantity of added internalstandard.

The serum insulin concentration was determined by a radioimmunoas-

say technique (35).Data Analysis. The individualamino acids were organized into groups;

TAA, BCAA (leucine, isoleucine, and valine), AAA (phenylalanine, tyro-

sine, and tryptophan), and EAA (BCAA, lysine, arginine, histidine, phenylalanine, tryptophan, methionine, and threonine) to facilitate recognitionof patterns of response (28).

The concentration of each amino acid or group of amino acids is themean ±SD of each of the sample time points of each animal duringinfusion; after preliminary analysis of variance (36) showed no statisticaldifferences between time points. Statistical significance between groupsof animals was determined by the two-tailed Student's i test for paired

and unpaired data and two-way ANOVA where appropriate (36).

RESULTS

The characteristics of the animal model are displayed in Table1. The TB animals were used in an early stage of tumor growthwhen the tumor burden was only about 8% of the total bodyweight (Table 1). Food intake measured during the 48-h periodprior to the 24-h fast was similar, but the carcass weight was

still significantly decreased in the TB compared to the NTBanimals (Table 1). The significantly decreased urinary nitrogenexcretion exhibited by the TB animals suggested that the decrement in carcass weight was not due to accelerated nitrogenloss in this group (Table 1).

During 0.9% NaCI infusion there were small but statisticallysignificant lower levels of aspartate, citrulline, and serine in theTB compared to the NTB animals (Table 2). The concentrationsof alanine, glutamine, BCAA, AAA, EAA, and TAA were similarin the NTB and TB groups (Table 2).

In the animals infused with glucose at 3.72 ^mol/100 g TBW/min, the concentrations of arginine, asparagine, proline, glycine,

lysine, tyrosine, and taurine rose to a similar degree in both NTBand TB groups, while the level of serine which was also elevatedin both groups was persistently lower in the TB animals (Table2). The alanine level was 86 and 90% higher, and the concentrations of leucine, isoleucine, and valine each were significantlydecreased to a similar degree in NTB and TB animals (Table 2),yielding BCAA levels that were 46 and 34% lower in the NTBand TB groups, respectively, compared to values during 0.9%NaCI infusion (Table 2). Paired analysis for each animal of thedifference in alanine and BCAA concentrations at base line andduring glucose infusion at 3.72 ^mol/100 g TBW/min showedthat the alanine concentration rose 85.5 ±35.7 and 130.5 ±45.3 MM,while the BCAA fell 186.8 ±52.10 and 118.2 ±112.2MM in the NTB and TB animals, respectively (Chart 1). Therewere no significant differences between the absolute micromolarvalue of the alanine rise or the BCAA fall in either NTB or TBanimals (two-way ANOVA, P > 0.05). The concentration of

glutamine (Table 2) and the levels of AAA, EAA, and TAA weresimilar in both NTB and TB animals to values during 0.9% NaCIinfusion (Table 2).

Infusion of glucose at 13.05 ^mol/100 g TBW/min resulted inconcentrations of arginine, asparagine, proline, glycine, lysine,tyrosine, serine, and taurine that were at a level closer to thatseen during infusion of 0.9% NaCI in both NTB and TB animals,with serine still significantly lower in the TB group (Table 2). Thealanine concentration was an additional 40% and 65% higher inthe NTB and TB groups, respectively, compared to the groupsinfused with glucose at the lower rate, without a significantdifference between NTB and TB animals (Table 2). The individualBCAA each fell significantly to a similar degree (Table 2), andthere was an additional 15 and 20% decrease in the total BCAAlevels in the NTB and TB animals compared to levels in thesegroups at the lower glucose infusion rate (Table 2). Pairedanalysis for each animal demonstrated that the alanine concentration rose 162.2 ±56.6 and 221.8 ±93.8 HIM, and that theBCAA fell 251.5 ±34.4 and 198.5 ±48.3 MMin the NTB and TBanimals (Chart 1). There were again no significant differenceseither in the absolute micromolar alanine concentration rise compared to the micromolar fall in the BCAA concentration, orbetween NTB and TB animals (two-way ANOVA, P > 0.05). The

concentration of glutamine was 35% lower in both NTB and TBanimals compared to levels seen in the groups during 0.9% NaCIinfusion (Table 2). The AAA and TAA concentrations were againsimilar to the values during the 0.9% NaCI and lower glucoseinfusion rate (Table 2).

Serum insulin was barely detectable in both NTB and TBgroups during 0.9% NaCI infusion (Chart 2). Glucose infusion at3.72 Mmol/100 g TBW/min resulted in a small elevation, while

Table 1

Body weights and food intake in NTB and TB animals

GroupNTB

TBn15 14TBW8

(g)21

6.4 ±9.0e

225.7 ±7.0Carcass

wt°(g)21

6.4 ±9.0208.3 ±10.3'Tumor

wta-"(g)0

17.3 ±8.3%

of TBWoccupiedby tumor*0

7.7 ±3.748-h

foodintakec(g)30.2

±3.128.1 ±4.524-h

nitrogenbalance''(mg)-21

2.8 ±11.9-194.1 ±21.9'

8 Determinedat the end of the experiment after the 24-h fast and 6-h infusion.b Tumor was implanted via a s.c. injection of a suspensionof 1 x 10ecells 4 days postcannulation.c Measured 16-18 days after cannulation when the animalshad regainedtheir postoperative weight loss and just prior to the 24-h fast.'' Measured during the 24-h fast prior to the start of the infusions.e Mean ±SD.'P< 0.05; Student's t test.

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AMINO ACID METABOLISM IN TB RATS

Table 2Individual amino acid concentrationsduring 0.9% NaCIinfusion and incrementalglucose infusion

0.9% NaCI Glucose (fimol/100 g TBW/min)

Amino acid(MM)AlanineArginineAspartateAsparagineCitrullineCysteineGlutamineGlutamateGlycineHistidineIsoleucineLeucineLysineMethionineOmithinePhenylalanineProlineSerineTaurineThreonineTryptophanTyrosineValineNTB(n =5)1

52.6 +20.1s68.6

±12.130.0±7.14.5±0.426.1±3.161.2+8.8392.8±54.5100.4±

14.9120.8±31.646.2±6.965.0±6.8104.0±13.1280.0±45.235.3±4.924.9±5.847.8±6.264.6±7.0111.8±

15.395.1±15.811

3.6 ±27.422.9±14.557.8

±6.6174.1±30.3TB

(n =5)150.3±

19.065.7±5.321.1

±1.0"4.3

±0.621.5±2.656.3

±14.9394.9±46.7124.5

±22.7110.3+11.044.9

±2.167.0±2.7104.0±4.0275.8+31.031

.3±2.327.1±3.547.9±4.367.4±6.789.1±12.4d93.3

±13.4111.9±12.120.5

+18.455.8±5.8173.0+

15.03.72NTB

(n =5)283.6+33.4"97.1±4.0"32.0

±6.96.0±0.930.8±6.166.8+8.6389.3±64.7136.2±32.0147

.2±7.846.6+5.332.6±4.3"58.1±7.1*353.3±50.0"38.7

±3.525.2±1.951.5

+5.486.6+6.9*132.4

±9.1"109.9

±67.9127.1±20.011.0+3.173.6±6.4"125.1±11.56TB

(n =4)286.7±43.3"97.7±16.2o27.2

+7.25.7±1.223.9

+2.461.8+3.8412.3±53.7153.8

±35.1145.2±20.2"48.1

±4.738.2±6.2"61.9+9.8°369.6±50.0640.9

±4.723.4±5.458.7±5.092.6±9.3"100.1±19.2o113.4±

10.5b137.1+20.717.7

+4.5d69.6±3.9o128.4

±27.513.05NTB

(n =4)344.5+ 28.7"'c79.9±3.8C34.2±4.9"5.3+0.6"20.6±1.7i>'c54.9

±5.8255.4±20.1ft-c127.2

+24.4140.4+12.749.2±3.224.5±2.6"-°44.2±5.9b-c304.7

±30.037.6±3.418.6±0.8C50.8

±5.981.3±3.2"111.7

+2.6°82.8

±12.4113.1±1.911.8±0.970.9

+11.897.6±24.56TB

(n =5)382.8+116.6"74.2

±8.8C20.7±8.1"5.7

±1.622.1±5.246.4+11.4c261.3+23.56'0149.2

±44.6131.0+17.945.2±6.522.4±2.7*-c38.5±5.1b-c31

0.5 ±61.638.8±3.41

6.8 + 2.9oc53.7

±6.586.6±20.278.8+

10.5d74.7±11.1i>-c114.8

±17.012.5±14.773.6

+10.8699.7±22.9"

" Mean ±SD.6P < 0.05; comparisonof values during each glucose infusion with levelsduring 0.9% NaCIinfusion within each group, Student's t test.c P < 0.05; comparison of values during glucose infusion at 13.05 /imol/100 g TBW/min and at 3.72 ^mol/100 g TBW/min.d P < 0.05; comparisonof TB to NTB during each infusion.

infusion at 13.05 /¿mol/I00 g TBW/min produced a marked andsignificant response (Chart 2). There were no significant differences in insulin levels between the NTB and TB animals duringany of the infusions.

DISCUSSION

At an early stage of tumor development, after a 24-h fast,

there are minor differences in basal plasma amino acid levelsbetween NTB and TB animals. At two different rates of exogenous glucose infusion, the pattern of response of individual andgroups of amino acids are similar in NTB and TB animals.

The arterial-venous cannulated animal model (18) utilized in

this study has been shown to be in good health with return topreoperative weight and resumption of normal food intake post-

cannulation (Table 1). The TB animals were chosen for use at apoint of tumor growth prior to the onset of marked cachexia(31), when the measured food intake was similar to, and thenitrogen retention more avid, compared to NTB animals.

The amino acid levels in NTB and TB animals during 0.9%NaCI infusion were lower than those found previously for fedrats (37), but similar to those reported for rats fasted for 24 h(13, 38), emphasizing the importance of antecedent food intakeon plasma amino acid metabolism (13).

The concentrations of the aromatic amino acids in both theNTB and TB groups were similar to values found for fasted ratswithout arterial-venous cannulas (39). Since phenylalanine andthe phenylalanine-tyrosine ratio have been reported to be ele

vated in shock (40), sepsis (9,10), and trauma (8), the low valuesin this study are further support for the physiologically basalstate of the animal model in general, and that in particular theTB animals lack this element of a stress pattern in their plasmaamino acid profiles.

Previous studies in our laboratory and others have showndecreased plasma alanine and glutamine levels (11, 12) andelevated rates of gluconeogenesis from alanine (16, 17, 29, 30)in TB compared to NTB animals and humans. Prior work in thisanimal model has shown that the percentage of glucose derivedfrom alanine is not increased in TB compared to NTB animals(18). These results, together with the similar levels of basalplasma alanine and glutamine in the NTB and TB groups in thisstudy, further suggest that gluconeogenesis from amino acidprecursors is not increased in TB animals without anorexia, sincethese amino acids account for the majority of skeletal musclerelease (41, 42), hepatic uptake and glucose production (43).

The biphasic concentration response of many of the nones-sential and some of the essential amino acids to the incrementalglucose infusions could be due to a combination of hormonelevel and substrate availability effects. Previous work has shownthat mild increases in serum insulin have little effect on skeletalmuscle amino acid release, while marked elevations inhibit thisrelease substantially (41). Thus at the low infusion rate exogenous glucose would provide additional substrate for nonessentialamino acid formation via stimulation of transamination reactions(44), while the low insulin levels seen during this infusion ratewould be inadequate to prevent release of those de novo andessential amino acids. At the high glucose infusion rate theelevated serum insulin levels would inhibit amino acid release(41), and tend to offset any rise due to stimulation of de novosynthesis (44). The similar response to exogenous glucose infusion displayed by NTB and TB animals implies that amino acid-insulin sensitivity is similar in both groups of animals, and is incontrast to previous reports demonstrating decreased glucosetolerance (45), elevated insulin levels (46), and decreased glucose-insulin sensitivity (47) in TB compared to NTB animals and

humans.

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AMINO ACID METABOLISM IN TB RATS

300

200

100

-100

-200

-300

INTB

DTB

[] ALANINE

HBCAA

0.9%NaCI

i3.72 13.05

INFUSIONS(u moleglucose/10Og TBW/min)

Chart 1. Change in alanine and BCAA concentrations during 0.9% NaCI andexogenous glucose infusions in NTB and TB animals determined from the differences between the preinfusionand the mean valueduring infusion for each animal.During 0.9% NaCIinfusion the change in alanineor BCAA concentrationswere notsignificantlydifferent from zero (P> 0.05 two-tailed paired Student's f test). During

exogenous glucose infusion at both 3.72 and 13.05 ^mol/100 g TBW/min theamount of increase in alanine concentration was similar to the absolute value ofthe decrease of BCAA concentration (P > 0.05 two-tailed paired Student's f test).

During either 0.9% NaCI infusion or either of the glucose infusions the change inalanine and BCAA concentrations was similar in both NTB and TB animals (P >0.05 two-way ANOVA).

0.9%NaCI

3.72 13.05

INFUSION(limole glucose/100g TBW/min)

Chart 2. Serum insulin during 0.9% NaCI and incremental glucose infusions.There was no significant difference between animals infused with glucose at 3.72fjmol/100 g TBW/min and 0.9% NaCI.There was a significant difference betweenthe above groups and the animals infused with glucose at 13.05 >imol/100g TBW/min. There were no significant differences between NTB and TB animals withineach infusion group.

The most prominent findings in this study were the elevatedalanine and decreased BCAA levels present during incrementalglucose infusion. These results have been demonstrated in anumber of studies at the whole body (24, 27, 28), limb (41, 42,48), and tissue (25, 26, 49) level. A glucose-alanine-BCAA cycle

has been proposed to explain these data, whereby glucoseproduced in the liver during fasting is converted to pyruvate inskeletal muscle, transaminated with amino nitrogen derived fromthe BCAA to produce alanine, and then converted back toglucose again in the liver by gluconeogenesis (26). Sixty to 90%of the alanine carbon has been estimated to be derived fromglucose (50), with the remainder being supplied from other aminoacids via the Krebs cycle (51, 52) or from proteolysis (52). Aminimum of 60% of the amino nitrogen has been demonstratedto be derived from the BCAA (53). In this study, the similarmicromolar rise in alanine and fall in BCAA plasma concentrationsis consistent with the theory of stoichiometric alanine transami-

nation by the BCAA (26). However, the 35% decrease in plasmaglutamine concentration at the high glucose infusion rate issuggestive of its additional role as an amino group alanine donorwhich is postulated to occur both at the tissue (48, 52) andinterorgan (54) level. Further, the whole body plasma alanineconcentration response to exogenous glucose may be evenmore complex as illustrated by work demonstrating depressedfractional hepatic uptake of this amino acid after p.o. glucosechallenge (55).

Given that the glucose-alanine-BCAA cycle accounts for a

major proportion of alanine metabolism, the similar plasma alanine and BCAA response suggests that this cycle is not preferentially increased in TB compared to NTB animals during exogenous glucose infusion. These results are in contrast to previouswork in this animal model, which showed significantly elevatedlactate levels indicative of increased Cori cycle activity in TBcompared to NTB animals during incremental glucose infusion(18).

In conclusion, basal plasma amino acid metabolism appearssimilar in NTB and TB animals prior to anorexia and markedcachexia. The similar pattern of plasma amino acid concentrationresponse to incremental glucose infusion in NTB and TB animalssuggests that whole body amino acid-insulin sensitivity is similar,and that the glucose-alanine-BCAA cycle is not preferentially

increased in the TB animals.

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CANCER RESEARCH VOL. 45 DECEMBER 1985

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