Glucose metabolism in rat lymphatic tissues: Effects of acute and chronic exercise

Life Sciences Vol. 8, Part 1, pp . 459-468, 1969 . Printed in Great Britain.

GLUCOSE

Pergamon Press

METABOLISM IN RAT LYMPHATIC TISSUES:

EFFECTS OF ACUTE AND CHRONIC EXERCISE Michael P . Dieter National Institutes of Health, äIAMD-LPB, Bethesda, Maryland 20014

(Received 1 November 1968 ; in final form 3 February 1969)

During the early proliferative phases of lymphoid organ development the cellular requirements for energy and biosynthesis are derived mainly from glycolysis (17,18,19) and the hexose monophosphate shunt (7) .

Stress

interrupts normal development, depresses cellular metabolises and causes lymphoid organ involution (8) .

It is evident from biochemical studies that

many of the stress effects result from activation pf the hypothalamopituitary-adrenal macis and an attendant rise in circulating corticosteroids . One of the earliest demonstrable lymphatic tissue responses to corticosteroids is an impairment of glucose uptake and utilization (14,15) that presumably leads to secondary responses such as altered enzyme activity (5,7,20) . Serum enzyme studies have shown that compensatory changes occur in glucose metabolizing enzymes after prior conditioning of animals to stress (2,10) .

Apparently similar compensatory changes occur in lymphatic tissue

enzyme activity .

It was shown that initial decreases in lymphatic organ

weight and metabolism after corticosteroid administration were followed with a recovery phase characterized by increases in organ weight and tissue glucose oxidation (6) . In the present study the detailed nature of these compensatory changes were examined .

Endogenous adrenocortical activity of trained or untrained

rats was altered by severe exercise stress (13)

and correlated with the

pattern of lymphatic tissue enzyme responses .

Emphasis was placed on

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maasurament of those enzymes directing the flow of metabolites through glycolysis or the hexose monophosphate shunt, since these pathways of glucose utilization are critically important during the early stages of lymphatic tissue development (7) . Materials and Methods lour groups of 28 day old aale Sprague-Dwley rats, 100-121 gn body weight, were maintained on Purina Lab Chow and water, id

ibitum .

One

group served as unexercised, untrained controls, a second was untrained but exercised 10 hours, &'third was first trained and then exercised 10 hours, and a fourth group trained but not given the final 10 hour exercise stress (the third group was allowed 12 hours rest prior to the prolonged exercise) . The untrained rats were accustomed to handling throughout the training period .

Training consisted of 21 daily four hour exercise periods on a

rotating cylindrical mesh cage, turning at 4 .53 rpm (equivalent to 6 .9 m/min), with five minute rest periods on the half hour .

Immediately at the end of

the experiment, at 50 days of age, all rata were killed by chloroform anaesthesia .

The animals were quietly removed from the exerciser or holding

cage and individually placed into a chloroform jar .

The ambulatory movement

of the rats usually ceased within 20 seconds and breathing within 60 seconds . By use of such care the quantitative error in the estimate of adrenocortical activity due to chloroform (23) was minimized . adrenals were removed and frozen in dry ice.

Their thymus, spleen, and Blood was withdrawn by cardiac

puncture, the plasma separated, and frozen . Corticosteroids were determined in the adrenals and plasma on an AmincoBowman spectrophotofluoroaeter by the method of Guillemin, et al . (9) . Adrenal ascorbic acid was determined by the indophenol method (11) on a Bausch and Lomb Spectronic 20 at 520 am . Portions of the thymus and spleen (2Z w/v) were homogenized for two minutes using a Lapine cone drive stirrer,

teflon pestles, and glass tubes

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in ice cold 0 .05 M Tris buffer (pH 7 .4) with added EDTA ( .005 M) .

The

activity of glutamic-pyruvic transaainase (GPT) was determined in whole homogenates since 50% activity was lost after high speed centrifugation ; activities of glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconic dehydrogenase (6-PGD),

"malic enzyme", HADP decarboxylating (MDR), a-glycero-

phosphate dehydrogenase (GPD), and lactic dehydrogenase (LDH) were determined in supernatant fractions prepared by centrifugation for twenty minutes at 21,000 x g and OOC .

Enzyme activity was determined at room temperature

(250C) on a recording Beckman model DU spectrophotometer at 340 nm by following absorbante changes of respective coenzymes (4) .

Reactions were

initiated by the addition of excess substrate and followed at pH 8 .0 (GPD and LDH) or pH 7 .4 (G6PD, 6-PGD, MDR, GPT) ; it was shown that reactant concentrations yielded a velocity that was directly proportional to the amount of tissue extract .

Protein concentration of the supernatant fractions

was determined by the phenol method (12,16) and each tissue enzyme activity expressed in milliunits (mNM substrate transformed/mg protein/min) .

The

difference between means were statistically analyzed by the Student-t test . Results Adrenocortical Responses Untrained rats stressed by acute exercise displayed statistically significant changes in all parameters of adrenocortical activity (Table I) . There was adrenal hypertrophy, a decrease in the concentration of adrenal ascorbic acid and corticosterone, and an increase in plasma corticosterone . Except for adrenal corticosterone, the response to prolonged exercise in trained rats was qualitatively similar, although quantitatively the per cent changes were only half as great . Lymphatic Organ Weights The relative and absolute weight of the spleen was significantly less after exercise of the untrained rats (Table II) .

There was a slight but

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TABLE I Adrenocortical Responses in Trained or Untrained Bats Exercised 10 Sours .

Means ± S .E .M ., z - 10 .

ADREMALS Treatment

Weight mg

PLASMA

Ascorbic Acid as/200 ga

Corticosterone NEm/Bm

Corticosterone Wgm/100 mi

Untrained Rats zone

34 .7 + 1 .3

381+ 25

18 .1 + 1.1

12 .3 + 1.9

Exercise

44 .1 + 1.9 a

272 + 131

11 .1 + 0 .51

40 .8 + 2 .01

Trained Rats zone

46 .8 + 2 .0

378 + 17

14 .3 + 0 .4

12 .0 + 0.9

Exercise

55 .6 + 2 .4b

338 +

14 .6 + 0 .9

25 .9 + 2 .3b

a b

9b

Significantly different than untrained controls, p < 0 .05. Significantly different than trained controls,

p < 0 .05.

not statistically significant decline in thymus weight .

In contrast the

relative and absolute weight of both the thymus and spleen decreased following exercise of the trained rats . Lymphatic Org an Enzymes Exercise of untrained rats induced a slight but statistically significant decrease in the specific activity of glutsmic-pyruvic transaminase and lactic dehydrogenase in the thymus (Table III) . 30Z increase in malic enzyme activity (Table IV) .

In the spleen there was a In trained rats exercise

resulted in & " significant decline In hexose monophosphate shunt enzyme activity (G6PD and 6 PGD) in thyme and spleen, a large increase in glutauicpyruvic transaminase activity in both tissues (75-85Z increase),

and a 40 and

80Z increase in malic enzyme activity of thymus and spleen, respectively .

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TABLE II Lymphatic Organ Weights in Trained or Untrained Rats Exercised 10 Hours .

Means ± S .E .M ., N - 10 .

SPLEEN WEIGHT

THYMUS WEIGHT Treatment

gm/100 gm

mg

mg

gm/100 gm

Untrained Rats None

581 ± 17

0.24 ± .01

663 ± 52

0.27 ± .02

Exercise

518 + 27

0 .21 + .01

466 + 32a

0 .19 ± .01a

Trained Rata None

438 ± 38

0.20 ± .02

623 + 45

0 .29 ± .02

Exercise

341 + 20

0.16 + .01b

487 ± 25b

0 .23 ± .01b

a

Significantly different than untrained controls, p < 0 .05 .

b

Significantly different than trained controls,

p < 0.05 .

Tables III and IV also illustrate the concentration of total protein in the supernatant fractions used for enzyme assay.

These levels remained

constant, validating the use of specific activity to express enzymatic change . Discussion A variety of atressors (exercise, altitude, cold exposure) stimulate the hypothalamo-adrenal-pituitary axis and elevate endogenous levels of corticosteroids (13,27,28) .

Prolonged exercise of untrained rats produced

a 230% increase in plasma corticosterone which is similar to that found in other studies (3,13) .

The growth and metabolism of two target organs, the

thymus and spleen, responded differently to the hormonal stimulus .

Thymi

weighed only slightly less than controls but there was a significant decrease in thymus glutamic-pyruvic transaminase and lactic dehydrogenase activity . There may be a temporal delay before the thymus responds to elevated corticosteroid secretion, or else the degree and (or) duration of adrenocortical

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TABLE III Enzymatic Changea in Thymus of Trained or Untrained Rata Exercised 10 Sours .

Means ± S .E .K ., ä - 10 .

UW12AIXED Unexerciaed

TRAI1=

Exercised

24 Me 28 .0 + 1.8b .8 + 0.7 6PGD

21 .4 + 1.1

19 .6 + 0.6

Unexercised

25 .2 + 0.8

22 .6+0 .0

19 .6 + 1.0

17 .8 + 1 .2

23 GPT 28 .7 + 2.5 21 .4 + 1.3c

mm

4.24+

GPD

33 .5 + 3 .9

.35

5.11 +

.39

42 .1 + 6.9

Exercised

.0 + 1.6 3.27 +

.25

41 .7 + 5.8

896 LUS 965 + 33 854 + 31c

43 .0 + 5 .8d

+ 26

4.61 + .29d 43 .4

+ 7 .2

828

+ 34

Protein Concentration of Tissue Supernatants (mg/ml) 1 .9

+ 0.1

2 .3

+ 0.2

2 .0

+ 0 .1

2 .2

a

See text for enzyme abbreviations .

b

Enzyme activity in milliunita .

c

Significantly different than untrained.controls, _p < 0 .05.

d

Significantly different than trained controls,

+ 0.1

p < 0.05.

activation by 10 hours exercise was insufficient to induce marked thymus atrophy.

Although thymus weight loss under the present experimental

conditions was nominal,

lymphocytolysis or cellular emigration may account

for the decrease in tissue enzyme activity, e.g ., a loss of tissue enzyme protein to the serum as has been noted for tbymus LDH after whole body x-irradiation (1) .

Exercise of untrained rats caused a significant but

apparently temporary loss in spleen weight (see Table II) .

It may be

ascribed to the extrusion of splenic erythrocytes in response to relative hypoxia after exercise .

Increase in malic enzyme is perhaps related to

reductive requirements for subsequent erythropoetic activity in the spleen .

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TABLE IV Enzymatic Changes in Spleen of Trained or Untrained Rats Exercised 10 Hours .

Means ± S .E .M ., li - 10 .

UNTRAINED Unexercised

TRA131ED

Exercised

37 G6PDa 35 .9 + 1.5b .3 + 1.2

Unexercised

Exercised

33 .2 + 1 .5

27 .0 + 1 .Id

6PGD

21 .7 + 0.9

21 .1 + 1.0

20 .2 + 0 .8

15 .1 + 0 .5d

CPT

19 .8 + 1.1

19 .1 + 0 .8

20 .7 + 0 .9

36 .3 + 4.9d

2.51 + 1 MM

.14

3 .20 +

.16c

.76 +

.12

3 .50 +

.16d

GPD

19 .3 + 1.2

21 .4 + 1.6

19 .2 + 1 .2

17 .7 + 1 .4

LDH

590

625

634

603

+ 30

+ 21

+ 22

+ 23

Protein Concentration of Tissue Supernatants (mg/ml) 3 .0 + 0.1 a b

3 .3 + 0 .1

3 .2 + 0 .2

3 .5 + 0 .1

See text for enzyme abbreviations . Enzyme activity in milliunits .

c

Significantly different than untrained controls, p < 0.05 .

d

Significantly different than trained controls,

p < 0 .05.

Exercise of trained rats resulted in adrenocortical responses which, except for adrenal corticosterone, were qualitatively similar to those in untrained, exercised rats .

Quantitatively it was evident that trained rats

had partially adapted to the daily training regime . recently reported in animals trained by swimming (3) .

Similar findings were However that study

found no difference in the plasma corticosterone response of trained or untrained rats treated with exogenous ACTH .

In the present study analagous

treatment (prolonged exercise of trained animals) doubled the circulating plasma corticosterone level but did not affect the adrenal content . results (see Table I)

These

suggest that factors affecting the clearance rate of

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corticostarome Vere altered by exercise . Exercise of the trained rats resulted in a loss of spleen weight and also a loss in thyme weight .

Even though adremocortical responses were

ameliorated by training, the loss of organ weight (40% and 25% in thyme and spleen, respectively) is understandable in light of the known permissive effects of corticosteroids (24) .

Other hormones elaborated during exercise

stress, such as the catechol amines, inhibit the growth of lymphatic organs and are augmented by low levels of corticoids (25) .

The increases in

glutaic-pyruvic transaminase activity in thymus and spleen have been previously demonstrated to be a result of corticosteroid administration (20) . The depression of hexose monophosphate shunt activity in the thyme and spleen substantiates a previous contention that this metabolic pathway is hormonally influenced in the lymphatic organs (7) .

In addition the specific

activity of malic enzyme (KADP decarboxylating) increased in the thymus and spleen after exercise of the trained rats . Hormonally mediated changes in the activity of "group" enzymes have been considered as evidence of adaptive regulation of tissue metabolism (21,26) .

A recant in vitro study of rat lymphocytes showed that x-irredia

tion caused a magnitude increase in the flow-rate of substrate through glycolysis (22) .

The regulatory step was found to be phosphofructokinase .

Present in vivo results show that the thymus and spleen, during conditions of suboptimal growth, exhibit decreased hexose mnophosphate shunt activity as well as increased activity of enzymes related to glycolysis .

This

suggests that another hormonally mediated regulatory mechanism available to lymphatic organs may be the diversion of substrate (glucose-6-pbosphate) from the indirect to the direct oxidative pathway (glycolysis) of glucose oxidation .

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SUHKARY The pathways of glucose metabolism in lymphatic tissues of immature rats were examined enzymatically during periods of exercise-induced, elevated corticosteroid secretion .

Acute exercise (10 hours) of untrained

rats increased corticosterone levels 230% above controls, caused a moderate arrest of thyans and spleen growth, decreased thymus CPT and LDH activity 15-25% below controls, and increased spleen MDR activity 25% .

Acute

exercise of trained rats increased corticosterone secretion only 116% above controls, but caused a marked arrest of lymphatic tissue growth and a variety of changes in tissue enzyme activity .

These included increases in

CPT and MDR, reciprocal changes in LDH and a-GPD, and decreases in G6PD and 6-PGD activity .

The pattern of enzymatic response was interpreted as a

hormonally mediated regulatory mechanism to divert substrate (glucose-6phosphate) from the hexose monophosphate shunt to glycolysis during periods of suboptimal lymphatic tissue growth . ACINOWLEDGKENT The technical assistance of Mr . Wn . L . Conway is gratefully acknowledged . References 1.

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