Nutrient-induced thermogenesis (NIT) following amino acid infusion

Clmmzl Nurr-itron c 19941 13 II&l?2 @ Longman Group Ltd 1993

Nutrient-induced thermogenesis amino acid infusion M. SAKAUE, T. TSUJINAKA, Y. KIDO, Y. HAYASHIDA, K. KAN, T. EBISUI and T. MORI

(NIT) following

M. YANO, T. HOMMA,

Department of Surgery II, Osaka University Medical School, 2-2 Yamadaoka Japan (Reprint requests and correspondence to 77).

S&a-city

S. IIJIMA, Osaka 565,

ABSTRACT-Nutrient-induced thermogenesis (NIT) induced by parenteral infusion of amino acid (AA) mixtures of different composition and of the same AA mixtures given via different routes (parenteral or intraportal infusion) were investigated in rats using a small animal indirect calorimeter. When 8 different AA solutions of differing composition but with the same total concentration were infused parenterally, both standard NIT (each AA is assumed to generate 3.28 kcal/g) and specific NIT (heat energy of each AA is calculated assuming that it is oxidized to carbon dioxide and water, and metabolised to urea and sulphuric acid) values of the leucine (Leu)-rich and the glycine (Gly)-rich solutions were significantly greater than those of the control solution. Removal of Leu or Gly from the respective AA solutions reversed the increase of both NIT values down to control levels. When the parenteral and portal infusion routes were used in one rat, both NIT values for parenteral infusion of the Leu-rich solution were again significantly greater than those of the control. Likewise, both NIT values for intraportal infusion of the Leu-rich solution were also significantly greater than those of the control. However, no difference in NIT values was found between parenteral and portal infusion of either solution. The result of this study indicated that Leu and Gly may be thermogenic AAs, and the thermogenic effect of Leu is not dependent upon the route of infusion.

Introduction

thermogenic effects, and that intraportal infusion of the same AA mixtures, bypassing intestinal absorption, may have the same thermogenic effect as parenteral infusion. This study was designed to examine this hypothesis.

Increased energy expenditure (NIT) occurs after ingestion or intravenous infusion of nutrients (l-3), and consumes 5-10% of the ingested calories for carbohydrates, O--3% for fat, 20-30% for protein, and 10% for regular food (4, 5). Jequier et al reported that the increment in energy expenditure following the intravenous administration of glucose and lipid is 6-8% and 24% of the infused energy (6). Using a sensitive indirect calorimeter in a rat model, we demonstrated that parenteral administration of an AA solution induces a dose-dependent thermogenic response of 1 l-12%. However, intragastric administration of the same AA mixture induces a higher thermogenic effect of 22-23%. This may be due to oxidation of AAs in the intestine, since the portal concentrations of most AAs (except branched-chain amino acids (BCAA)), particularly glutamine and lysine, are lower with enteral compared with parenteral AA administration (7). From these results, we postulated that the parenteral infusion of AA mixtures of different composition may induce different

Materials and methods Animal

model

Male Wister rats (Nihon Animal Co.), weighing 180-220 g, were housed in cages and allowed free access to standard laboratory chow and water. After intraperitoneal injection of pentobarbital(3.5 mg/lOO g of body weight), in Experiment 1 (Fig.), a silicon rubber tube (0.5 mm inner-diameter, Dow Coming, Midland, MI) for parenteral administration was inserted into the superior vena cava through the external jugular vein and fixed according to the method of Steiger et al (8). In Experiment 2 (Fig.), in addition to the parenteral tube, another tube (0.3 mm innerdiameter) for intraportal administration was inserted into the portal vein in the same rat through the 116

CLINICAL NUTRITION

Experiment1

(NIT

of eight

different

amino acid

jcannulation c -48

(10 keel/kg/h)

Experiment

2 (NIT

1) Parenteral

0

by different

half-se1

infusion

(10 kcal/kg/h)

lntreportal

0

half

9(h)

amino acids-4 4

(10 ml/kg/h)

9(h)

route

bindi rect TPN

(10 kcaI/kg/h) Experimental

I

half

b

(10 ml/kg/h)

calorimetry

sal inb----$---intraportal

4

amino acids-4

(10 ml/kg/h)

9(h)

design.

ileocaecal vein. The total parenteral nutrition (TPN) solution contained 1 kcal/ml with a calorie to nitrogen ratio of 136.7, and was composed of 20.6% glucose, 3.78% AA and multivitamins. It was infused at a rate of 240 ml/kg/day and was maintained for 48 h before each experimental run in order to allow recovery from operative stress and sustain a stable nutritional state. Experimental

(10 ml/kg/h)

calorimetry

sal ine-arenteral

(10 ml/kg/h)

jcannulation

Fig.

4

routes)

*indirect

I

-48

amino acids-4

route

MI

2)

calorimetry

inb+parenterel

(10 ml/kg/h)

/cannulation

-48

solutions)

-indirect 1

TPN

I I7

protocol

In Experiment 1 (Fig.), the increment in resting energy expenditure (REE) was measured during parenteral infusion of AA solutions of 8 different compositions with the same total concentration, as shown in Table 1. The alanine (Ala)-rich and glycine (Gly)-rich solutions were selected as representative glucogenic AA rich solutions and the BCAA rich solutions were selected because only BCAA increased in the portal vein after intragastric infusion, as compared with parenteral infusion of the same AA mixture in our previous study (7). The concentrations of these 5 AAs were relatively reduced in the control solution to enhance the additive effect of each AA. To confirm the thermogenic effects of Leu and Gly, a Leu or Gly-depleted solution was made. At first, the basal REE at 4 h after half-saline infusion at a rate of 1.67 x lOA mI/g/min was determined by indirect

calorimetry, and then the average values of REE for 5 h after infusion of 8 different compositions of AA solutions at the same infusion rate were obtained in 6 rats each. The increment in REE was calculated by substracting the basal REE value from the average REE value during AA infusion. In Experiment 2-l (Fig.), the increment in REE during parenteral infusion of two kinds of AA solutions (Control and Leu-rich) was obtained in 6 rats each, and in Experiment 2-2 (Fig.) the increment in REE during intraportal infusion of the same AA solutions (Control and Leu-rich) was obtained in 6 rats each. The method and calculation of energy expenditure in indirect calorimetry were described in (9). Briefly, the basal REE was measured at 4 h after half-saline infusion at 1.67 x 10-4ml/g/min. In an open-circuit indirect calorimeter, animals breathe inside a translucent plastic hood into which dry, compressed air is constantly blown at a rate of 0.8 l/min. After passing through the animal cage, the expired gas was completely dried with an electric cooler and CaCl? to absorb water vapour. The concentrations of O2 and CO, were then measured with an Expired Gas Monitor IH2tB (Sanyo). The REE and respiratory quotient (RQ) were successively calculated with an on-line computer using the table deviced by Lusk (10).

Total concentration (mg/dl) Total energy (kcal/dl) Standard Specific 36.08 46.94

36.07 42.97

36.09 45.41

11003

11001

10998

3. Leucine-rich solution (Leu-rich) 561 1400 648 736 407 915 473 176 771 1472 1155 96 490 998 190 22 439 54

630 211 728 827 457 1028 532 197 866 1654 1298 108 551 1121 213 24 493 60

5.9 5.9 5.3 4.5 4.8 6.3 2.3 5.8 3.5 2.1 3.4 3.1 2.6 4.1 2.3 3.7 2.5 5.0

lsoleucine Leucine Valine Lysine Methionine Phenylalanine Threonine Tryptophan Alanine Glycine Arginine Glutamic acid Histidine Proline Aspartic acid Cystine Swine Tyrosine

2. Isoleucine-rich solution (Ile-rich) 2500 173 597 678 375 843 436 162 710 1356 1064 88 452 919 175 20 404 49

1. Control solution (Control)

of 8 different amino acid solutions

Biological heat production (kcab)

Compositions

Amino acids

Table 1

36.08 50.10

11001

337 113 5500 443 245 550 285 106 464 886 695 58 295 601 114 13 264 32

4. Valine-rich solution (Val-rich)

36.09 41.16

11002

373 125 431 490 271 609 315 117 5000 980 769 64 326 664 126 14 292 36

5. Alanine-rich solution (Ala-rich)

36.10 35.90

11008

405 136 468 532 294 660 342 127 557 5000 834 69 354 721 137 16 317 39

6. Glycine-rich solution (Gly-rich)

36.11 40.84

11010

642 742 844 466 1048 542 201 883 1687 1334 110 62 1144 217 25 502 61

7. Leucine-depleted solution (Leu-depleted)

36.08 48.07

11001

1391 114 586 1193 227 26 524 64

670 277 774 880 486 1093 565 201 921

8. Glycine-depleted solution (Gly-depleted)

CLINICAL NUTRITION

Before each measurement, standard gas mixtures, NZ02-C02, 78.5-21-M%, N2-C02, 99-l%, were introduced for calibration of O2 and CO2 concentrations. The calorimetry started at 07:OO and the infusion continued for 9 h. During the experiment, rats were generally resting and their behaviour was inspected continuously. Data, obtained when rats were active, were not adopted for REE measurement. NIT value was expressed as the increment in REE over the percentage of infused energy in kcal per body weight (g) per minute. The energy of each infused AA solution was calculated in two ways. In the first, standard NIT was calculated, assuming that 1 g of AA contained in each solution generates a mean of 3.28 kcal. In the second, calculating specific NIT, the biological heat energy of each AA was calculated on the assumption that the carbon skeleton of each AA was oxidised to carbon dioxide, the hydrogen skeleton to water, the nitrogen skeleton to urea and the sulphur skeleton to sulphuric acid (11). The total energies of each solution calculated by the two methods were then obtained as shown in Table 1. Statistical analysis All data are expressed as mean + SD. Statistical comparisons were computed using the ANOVA and Dunnett test for Experiment 1, and the unpaired Student t-test adjusted by Boneferroni’s method for Experiment 2. A p value less than 0.05 was considered significant.

Results In Experiment 1, after parenteral infusion of 8 different AA solutions, RBE rapidly increased and reached a plateau in approximately 30 min. This plateau was maintained during the following infusion period. Standard and specific NIT values of the Leu-rich (17.8 f 1.2%, 13.8 + 0.9%) and the Gly-rich (15.3 + 1.9%, 16.1 + 2.0%) solutions were significantly higher than those of the control (12.4 + 0.6%, 10.2 If: 0.5%), but NIT values of other AA solutions were similar to the control levels. On the other hand, the NIT values in the Leu (12.9 + 0.6%, 11 .O f 0.5%) or Gly-depleted (13.7 +_1.5%, 11.1 +_1.2%) solutions decreased to the control levels (Table 2). Though leucine is an essential amino acid, the infusion of the Leu-depleted solution did not cause any temporal change in calorimetry at least during the experimental period. Based on these results, both Leu and Gly may be thermogenic AAs. In Experiment 2 (Table 3), in parenteral infusion

119

with the portal route clamped, the NIT values of the Leu-rich solution (22.2 f 2.9%, 17.6 f 2.3%) were also significantly higher than those of the control (14.3 + 1.7%, 12.1 + 1.4%). In addition, parenteral infusion of the Leu-rich and the Control AA solutions by the portal route caused higher NIT in Experiment 2, compared to the parenteral infusion of the same AA solutions in Experiment 1, though the reason for this is unclear. During intraportal infusion of the two different AA solutions (control and Leu-rich), with the parenteral route clamped, RBE increased and reached a plateau in approximately 1 h. This plateau was maintained during the following infusion period. The NIT values of the Leu-rich solution (22.4 + 8.7%, 17.8 f 6.9%) were significantly higher than those of the control (15.7 +_ 2.3%, 13.0 f 1.9%). No differences in standard and specific NIT values were found between the two infusion routes with either solution.

Discussion Intravenous infusion of AA is known to induce a thermogenic response (6,7, 12). Though the different metabolism of individual AA may induce different thermogenic effects, few reports have been available to clarify this point. Our present results indicated that Leu and Gly may be more thermogenic than other AAs. It is generally accepted that AA catabolism is not the dominant factor determining postprandial thermogenesis (13, 14). The NIT of AA is probably more related to protein synthesis or non-oxididative disposal. Gly is utilized mainly for collagen synthesis (15), and Leu is dispersed, not only in the muscle, but also in the adipose tissue for conversion particularly to cholesterol, which is regarded as a significant physiological function of the adipose tissue (16). The role of Leu in protein metabolism has been elucidated. Sreekuman et al (17) reported that the infusion of a 2% Leu solution in humans spares oxidation of other substrates and decreases protein degradation, and Yagasaki et al (18) reported, in an in-vitro experiment, that Leu and a-keto isocapronic acid (KIC) stimulate protein synthesis. Thus the thermogenic effect of Leu may be due to increased utilization of AA for protein synthesis. On the other hand, NIT values of the alanine (Ala)-rich, the vaiine (Val)-rich and the isoleucine (Ile)-rich solutions were not different from those in the control. It may be speculated that Ala and Val are glucogenic AA and mainly utilized as an energy fuel. Ile is a glucogenic and ketogenic AA, and also utilized as an energy fuel. Pitkanen recently reported (12) that infusion of a

6 6 6 6 6 6 6 6

1. 2. 3. 4. 5. 6. 7. 8.

7.5 f 0.4 7.4 + 1.0 10.7 f 0.7 7.6f 1.1 7.1 kO.6 9.7 f 1.2 7.8 + 0.4 8.2 * 0.9

(x 10c’kcal/g/min)

AREE

60.12 60.14 60.15 60.14 60.16 60.18 60.19 60.14

Standard infused energy (x 10dkcal/g/min)

6

2. Leu-rich Intraportal 32.95 + 1.12

33.00 + 0.32

33.24 32.52 + rt 1.20 1.21

basal REE (kcal/day)

36.60 f 1.43

35.93 f 0.16

35.78 36.35 + f 0.97 1.23

REE (kcahday)

Values are expressed as mean + SD. *: P < 0.001, unpaired red Student’s t-test adjusted by Boneferroni’s NS: not significant

6

Control

infusion

66

infusion

Control Leu-rich

n

test.

Standard NIT value (%)

solutions

17.8 12.7 11.8 15.3 12.9 13.7

22.4 + 8.7

15.7k2.3

14.3 + 1.7 22.2 f 2.9

1.2 1.8 1.0 1.9 0.6 1.5

I* NS 1* ’

1

1*

1.9 17.8 + 6.9

13.0f

12.1 1.4 17.6 *+ 2.3

’

I* 1y

NS

1 NS

1*

*

Significance

Significance

10.2 f 0.5 8.9 + 1.2 13.8 + 0.9 8.8? 1.3 10.0 dz0.8 16.1 f 2.0 11.0+0.5 11.1 + 1.2

Specific NIT value (%)

Specific NIT value (%)

*

Significance

NS

Significance

k + f + f +

12.4 f 0.6 12.3 + 1.7

Standard NIT value (%)

NIT values by different infusion routes of control and leucine-rich

1. Parenteral

Solution

Table 3

71.62 78.23 75.68 83.50 68.61 59.83 68.07 80.12

Specific infused energy (x lO”kcal/g/min)

infusion of 8 different amino acid solutions

Values are expressed as mean f SD. AREE: Increase of REE *: P < 0.001, ANOVA and Dunnett test.

Control Ile-rich Leu-rich Val-rich Ala-rich Gly-rich Leu-depleted Gly-depleted

n

NIT values on parenteral

Solution

Table 2

5

8 --1

F

3 =

R

3

CLINICAL NUTRITION

high-BCAA mixture (89%) causes less thermogenic response than a balanced AA mixture ( 13 f 10 vs 22 rf: 9%), which is contrary to the present result. One of the reasons may be that infusion of an extraordinary unbalanced AA mixture as an 89% BCAA mixture induces a reduction in protein synthesis, which results in lowered thermogenic response. The extent of enrichment of feeds with specific amino acids is different in this study (the Val-rich solution has 5500 mg/dl of Val vs. 728 mg/dl in the control solution, the Leu-rich solution has 1400 mg/dl of leucine vs. 211 mg/dl in the control solution), because we chose the nearly maximum soluble concentration of each target AA in the respective specific solutions (i.e. valine is much soluble than leucine). On the other hand, the NIT values of the Leu or Gly depleted solution were not less than but equivalent to the control. Because of variations in the relative proportions of certain amino acids (Gly in the Leu depleted solution and Leu in the Gly depleted solution) this may have modulated the thermogenic response to the respective solutions. Because of these problems, the results of the present study can only indicate that Leu and Gly are probably but not certainly thermogenic. However, our further experiments have disclosed that the parenteral infusion of a single AA solution of Gly or Leu caused higher thermogenic response than other AA solutions with the same concentration (data not shown). Another interesting observation in this study was that the parenteral infusion of the Leu-rich or the control solution in Experiment 2 (portal route was constructed after laparotomy) causes a higher thermogenie response than that in Experiment 1 (no portal route). It was speculated that utilization of amino acids (especially leucine) may be augmented after surgical stress, which results in an increase in NIT. Further study is of course necessary to clarify this point. In the second part of our study, the NIT values of the AA mixtures, given through the different administration routes (parenteral and intraportal), were investigated to confirm our previous conclusion that the cost of intestinal metabolism contributes to the increase of NIT in intragastric infusion of the AA solution (7). When the intestine was bypassed by intraportal infusion, the NIT values of respective AA solutions were the same as those of parenteral infusion. Regarding thermogenic response, the composition of the AA mixture was a major determinant in the absence of intestinal absorption. Based on the findings of this study, we conclude that parenteral infusion of an AA mixture does induce a thermogenic response which differs depending upon

121

the composition of the AA mixture. The additional energy demands imposed by NIT needs to be taken into account to determine the energy requirements of any particular patient. Care must be taken not to neglect the influence of an infused AA mixture when indirect calorimetry is carried out in a patient receiving a BCAA rich AA solution (especially one rich in leucine) as in the ICU. In conclusion, 1) NIT induced by infusion of a AA mixture is influenced by differences in AA composition, and leucine and glycine may be thermogenic AAs. 2) Intraportal infusion of an AA mixture causes a similar thermogenic effect to that of parenteral infusion because of the absence of intestinal metabolic cost.

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13.

14.

15.

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NUTRIENT-INDUCED THERMOGENESIS (NIT) FOLLOWING AMINO ACID INFUSION

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on amino acid and glucose metabolism in humans. Metabolism 1992; 41: 643-648. 18. Yagasaki K, Saito K, Funabiki R. Involvement of arachidonic acid metabolism in insulin-stimulated protein synthesis in cultured L6 myocytes. Agricultural and Biological Chemistry 1991; 55: 1449-1453.

Submission date: 23 March 1993: Acceptedfor revision: 17 October 1993