Effects of training and of acute exercise in trained rats

Effects of training and of acute exercise in trained rats

Effects of Training and of Acute Exercise in Trained Rats By SVEN 0. Three groups of rats were studied. Two groups of rats were trained by running for...

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Effects of Training and of Acute Exercise in Trained Rats By SVEN 0. Three groups of rats were studied. Two groups of rats were trained by running for 2 mo. One group served as trained resting controls (T), the other group was given exercise immediately before sacrifice (T+E), the third group served as untrained resting controls (C). In plasma tbe concentration of free fatty acids (FFA) and glycerol was decreased in trained (T) compared to untrained rats (C) at rest. Training also lowered the content of triglycerides in the liver and the concentration of triglycerides in myocardium and skeletal muscle tissue. In exercised rats (T +E), the concentration of triglycerides was lowered in plasma and red skeletal muscle tissue; the content of triglycerides

I

FR~BERG in the liver increased. The results indicate that training influenced the mechanisms regulating metabolism of triglyceride fatty acids in tissues at rest. The possibility that reduced availability of plasma FFA for esterification was responsible for the lowered triglyceride “pools” in tissue was discussed. It was suggested that training adapted the lipid metabolism toward increased utilization of circulating fatty acids and decreased the role of endogenous muscle triglycerides as a source for fatty acids during exercise. Training also lowered the blood glucose concentration, which was further reduced during exercise.

N

A PREVIOUS STUDY the effect of acute exercise on plasma and tissue lipids in rat was investigated. 1 The postexercise concentration of plasma free fatty acids (FFA) was increased as is usually seen in man2 dog,3 and horse.4 A new finding was that the concentration of triglycerides in myocardium and red skeletal muscle tissue decreased during the exercise,l which suggests that the triglycerides in these tissues contributed with fatty acids to the energy metabolism during the exercise. Exercise was, however, without effect on the concentration of plasma triglycerideql which has been reported to decrease during exercise in man5,6 and rat7 subjected to physical training before the exercise. This suggests that training may have adapted the metabolism of the plasma triglycerides toward increased utilization of circulating triglyceride fatty acids during exercise. We have recently observed that training induced a lowering of the triglyceride concentration in plasma, liver, and skeletal muscle of adult rats, 12-13 mo of age.s Training may thus have iniluenced the factors that regulate metabolism of circulating as well as endogenous triglycerides also at rest in these rats. The purpose of this study was to measure the effect of training on the concentration of plasma and tissue lipids in younger rats and on the response of From the Department of Geriatrics, University of Uppsala and King Gustaf Vth Research Institute, Stockholm, Sweden. Received for publication June 28, 1971. Supported by grants from the Swedish Research Council (19X-204-04 and 19X-204-05) and Konung Gastaf Vth 80-&fond and Karolinska lnstitutets forskningsfonder. SVEN 0. FE&ERG, M.D.: Research Fellow, Department of Geriatrics, University of Uppsala, Uppsala, Sweden. 1044

METABOLISM, VOL. 20, No.

11 (NOVEMBER), 1971

TRAINING AND ACUTE EXERCISE IN RATS

1045

exercise on lipid metabolism in blood and tissues. The effect of exercise on the metabolism of triglycerides in plasma and muscle tissue in trained rats has been briefly discussed in comparison to findings in untrained rats.l MATERIALS

AND METHODS

Male Sprague-Dawley rats fed ad lib. were used. Two studies were done with 4.5 rats in each. Fifteen animals served as controls; the others were divided into two groups with 15 rats in each, and each group was trained simultaneously on separate treadmills. Details pertaining to the experimental design and weights of the rats before and after the training period are given in Table 1. The training consisted of running 3 hr/day, 5 days/wk for about 9 wk. The training period was between 10 a.m. and 1 p.m. at a speed of 25 cm/set in the first study; in the second study training was (II) between 1 p.m. and 4 p.m. at a speed of 33 cm/see. During training the control rats (C) were in the same room as the rats subjected to running. In study 1 the control rats were kept in their cages and in study 2 were kept on a nonmoving treadmill deprived of food and water. Off training, the rats were freely fed a pellet chow with fat content of 3.7%, carbohydrate content of 46%, and protein content of 20.8 % (AB Anticimex, Sollentuna, Sweden). During the experiment starting at 9 a.m., untrained rats (C) and trained control rates (T) were put on a nonmoving belt, and trained rats (T+E) were put on a moving belt with an interval of 10 min between each rat. In study 1 the rats ran for 3 hr at 25 cm/set and in study 2 for 3 hr at 33 cm/set. Each study was performed on 3 consecutive days with five rats from each of the three groups (C), (T), and (T+E). The remaining rats had their daily training as usual after the sampling was completed. Sampling was done between noon and 2 p.m. The rats were stunned by a blow at the head and killed by exsanguination, withdrawing blood into heparinized syringes from the bifurcation of the abdominal aorta. The whole liver, heart, and both gastrocnemii muscles were removed, immediately frozen in liquid nitrogen, and stored at -20°C. Before lipid analysis the muscle was thawed and separated into “red” and “white” muscle tissue as described earlier.9 Blood was immediately pipetted off and precipitated for determination of glucose.10 After and centrifugation the concentration of FFA,rrJs glycerol ,la cholesterol,14 phospholipids, triglyceridesls,ls in plasma was determined. The plasma protein concentration was also determined according to the biuret reaction. Myocardium, red and white skeletal muscle were homogenized with 5 ml of methanol, and the concentration of cholesterol,14 phospholipids, and triglycerideslsZls was determined on the chloroform extract of the methanol homogenate. The whole liver was homogenized with 100 ml of methanol, and the lipid fractions were determined on 5-ml aliquots of the methanol homogenate. The DNA concentration of the liver was determined according to Schneider17 on aliquots of the liver methanol homogenate after evaporation of the methanol. Statistical analysis was done according to Snedecor. 1s The trained group (T) has been compared to the untrained controls (C). and the exercised group (T+E) to the trained group (T). The mean values, given in the results, were calculated from 15 rats and include values obtained on the 3 days of sampling. No consistent changes were found when values for separate days of sampling were compared. RESULTS

Plasma

The FFA level was generally lower in study 2 than in study 1 (Table 2). The plasma FFA concentration was decreased after training in both studies 1 and 2. The decrease for group means was 25% and 45%) respectively. The acute exercise caused an increase in the plasma FFA concentration in both studies. The increase for group means was 50% and 260%, respectively. The plasma glycerol concentration was determined only in the second study. The per cent

2

1

15 15

15 15

15 15

Number of Rats C T T+E

25 33

,c%&

74 3

61 2

< 0.05 < 0.05

88 4

72* 1

69 2 > 0.05 > 0.05

71’ 2

>

7.55 0.16

0.05 > 0.05

7.42 0.10 _ > 0.05 > 0.05

2

89 t 2

between

< 0.05

153* 4

138 f 3

398 k 8 368 -c 4

C

> 0.05

0.47 t 0.05 < 0.001

0.85 0.04

< 0.001

0.58 ? 0.03

FFA,

387 f 7 366 + 7

T+E

E

< 0.001 compared.

< 0.001

0.254 $ 0.128 5 0.335 q 0.018 0.011 0.024

Glycer?l C (mmole/hter) T TiE

Levels

392 5 7 360 + 6

After Tyng

and T and T+

< 0.001

0.16 0.01

0.28 0.02

FFA (mec+/literT) + E

0.96* 0.08

C

0.54 t 0.40 t 0.61’ 0.03 0.04 0.06 < 0.001 < 0.001

Triglycerides (mmo$/hter) T+E

1 .os* 1.07’ 0.65 0.06 0.08 0.05 > 0.05 < 0.001

C

w:?ht

205 -+- 1 201 + 3

T+E

group means C and T compared,

> 0.05

142 3

91’ 3

7.51 0.07

88

149’ 130 3 6 4 > 0.05 > 0.05

131*

90’ 93* 83 2 4 2 > 0.05 < 0.05

7.57’ 7.48’ 7.50 0.07 0.05 0.06 > 0.05 > 0.05

Protein (g/l? mljT + E

205 +- 2 199 + 2

Befor;Training

Design

of Blood Glucose and on Plasma in Studies 1 and 2

205 f 2 199 f 2

on Concentration and Triglycerides

Oct., Nov. Feb., March

C

to Experimental

Phospholipids C Cmg/,$OOml) TtE

C

* n= 14; t n=12; $ n=5; I n=7; ( n=4. M, mean; SEM, standard error of the mean; p, degree of significance

P

M SEM

P

M SEM

67 63

and Acute Exercise After Training Glycerol, Cholesterol, Phospholipids,

trained rats.

3 3

Time of Year

Pertaining

Cholesterol C Cmg/+OO n$)+ E

of Training

Blood GIucose C (mg/l$O ml) T+E

Table Z.-Effect

Training

of Data

Duration (hr) (days)

l.-Summary

C, control rats; T, trained rats; T f E, exercised * Mean f. standard error of the mean.

1 2

___.

Study

Table

“c

crl P ^_

P

!z

1047

TRAINING AND ACUTE EXERCISE IN RATS

Table 3.-E&t of Training and Acute Exercise After Training on Liver Weight and Content of DNA and Triglycerides in Liver in Studies 1 and 2 C

Study 1

Study 2 _

W&f

M SEM P

14.0

M

14.8

(9)

14.3

T+E

13.0

0.4 0.4 0.2 > 0.05 < 0.01 14.8

C

8.25

DNAT(mg) 7.98

TEiglycer$es T+E

7X59*

0.19 0.27 0.20 > 0.05 > 0.05

12.9

9.72 10.07 9.58

P SEM 0.3 > 0.050.4 < 0.01 0.4 * n = 14. Symbols as in Table 2.

0.55 > 0.05 0.66 > 0.05 0.57

116

117

(gmole) T+E

124

: 0.05 “> o.os6 102

70

93

<8 0.0014< 0.016

change closely followed the corresponding change in the plasma FFA concentration. Training was without effect on the plasma concentration of cholesterol and triglycerides, but a small decrease was observed in the concentration of phospholipids in study 2. Exercise was followed by a pronounced decrease in the plasma triglyceride concentration in both studies and a slight decrease of the cholesterol concentration in study 1. The blood glucose concentration decreased during training in study 2, and a further decrease was observed in this study in response to the acute exercise. Liver

The liver weight and the total amount of triglycerides and DNA in the liver are given in Table 3. The triglyceride content was decreased in study 2 after training. Since the liver weights were uneffected by training, this implies that triglyceride concentration had also been lowered by the more intensive training. Training induced no changes in the DNA content. The acute exercise was followed by an increase in the triglyceride content of the liver in study 2, but no change was observed in the DNA content in either of the two studies. On a wet weight basis the concentration of triglycerides was increased after exercise. This, together with the decrease in the liver weight, suggests that the water content of the liver was reduced during exercise. Muscle

Tissue

In red muscle the triglyceride concentration was significantly decreased after training (Table 4). The per cent decrease of the mean values was about 20% in both studies. The phospholipid concentration was increased after training. The cholesterol concentration increased only in study 2. The acute exercise caused no change in the concentration of cholesterol and phospholipids. The triglyceride concentration was, however, lowered after acute exercise in study 2 where the exercise intensity was highest. In the myocardium (Table 4) the triglyceride concentration was decreased after training in study 2. A similar, although not significant, change was found in study 1. The exercise was without effect on the concentration of triglycerides in the myocardium. From Table 4 it is seen that the DNA concentration was unchanged after training, as well as after acute exercise.

C

* N=14; Symbols

1.38 1.26* 1.36* 0.05 0.02 0.02 < 0.01 > 0.05

M SEM P

2

t N=13. as in Table

2.

12.4

12.8

14.9 0.2 0.05

0.2 0.2 < 0.001 > 0.05

T-FE

T

Red M+e PhTz.+plds

15.0* 14.0* o.oP~‘> 0.2 <

0.2

11.3

1.40

0.05 0.04 > 0.05 > 0.05

0.05

C

T+E

M SEM P

1.41

mT

cp1;sfge;o1

1.28

0.07 0.08 < 0.05 > 0.05

1.40

T+E

1.50* 1.19 t 0.92* 0.12 0.09 0.10 < 0.05 < 0.05

0.13

1.71*

C

Triglycerides Wqe/g)

0.02

0.72

C

2.54 0.24

1.74

1.60

T+E

in

1.66 1.64 0.08 0.07 < 0.01 > 0.05

0.12 0.17 > 0.05 > 0.05

2.04

0.66

0.03

C

T+E

Triglycerides Wm+e/g)

and Triglycerides

Myocardium

0.03 0.05 > 0.05 > 0.05

0.68*

DNA Cm+/)

of Training and Acute Exercise After Training on Concentration of Cholesterol, Phospholipids, Red Muscle Tissue, and of DNA and Triglycerides in Myocardium in Studies 1 and 2

1

1.31

Table 4.-Effect

TRAINING

AND ACUTE

EXERCISE

IN RATS

1049

In the untrained control rats (C) the content of cholesterol and phospholipids in the liver and the concentration of these lipid fractions in the myocardium and white skeletal muscle was of the same order of magnitude as previous1y.l Since neither training nor exercise caused any significant change in these lipid fractions, the values were omitted from the tables. In white muscle tissue, however, the triglyceride concentration was lowered from 1.46 i 0.01 pmole/g to 0.92 i: 0.07 pmole/g in trained rats (T). DISCUSSION

The main findings were that training induced a decrease in the concentration of triglycerides in liver, myocardium, and muscle tissue and in the concentration of FFA and glycerol in plasma at rest. Exercise caused a decrease in the concentration of triglycerides in plasma and muscle tissue. Although the effect of acute exercise on the metabolism of plasma FFA has been thoroughly studied,’ less information is available on physical conditioning on this lipid fraction. In the present study the plasma FFA concentration at rest was lowered in trained rats. This is at variance with previous findings where, however, the training program consisted of shorter daily runs,l”,“O shorter duration of the training period,R or swimming.’ The lowered plasma FFA concentration at rest suggests that training influenced the metabolism of the plasma FFA. The fractional turnover rate may have increased, or the mobilization of fatty acids from the adipose tissue may have decreased. The decrease in the plasma glycerol concentration after training supports the concept of a lowered rate of lipolysis in adipose tissue.‘l An increased uptake of glycerol in the liver may, however, also explain the lower glycerol values in plasma. The plasma FFA flux”,“‘,‘” has been reported to be of importance for the production as well as for the concentration of triglycerides in the liver. The triglyceride content of the liver was reduced in rats trained at the higher intensity (study 2) but not in rats trained at the lower intensity (study I), although the arterial level of the plasma FFA was depressed at rest in both studies. Furthermore, in elderly rats the liver triglyceride content was reduced after training without significant decrease in the arterial plasma FFA concentration.x These findings indicate that the arterial flow of FFA to the liver was not the only determinant for the metabolism of endogenous triglycerides in livers of trained rats. The total flow of plasma FFA to the liver also includes the amount of fatty acids transported to the liver from the omental adipose tissue via the portal vein. Training may have induced changes in the splanchnic blood flow as well as in the rate of lipolysis in omental adipose tissue, which has been reported to differ from the rate of lipolysis in the subcutaneous adipose tissue in vitro.!!*,?” The triglyceride concentration tended to decrease also in the striated muscle tissues of trained rats, which is in accordance with findings in elderly rats.8 The lowered muscle triglyceride concentration in trained rats may to some extent be due to reduced availability of plasma FFA for triglyceride synthesis in the muscle tissue.‘” Training may also have influenced the metabolic pathways for triglyceride fatty acids in the muscle tissue, e.g., the fractional turnover rate of the endogenous muscle triglyceride fatty acids may have increased with a subse-

1050

SVEN 0. FRijBERG

quent lowering of the muscle triglyceride concentration. It cannot be excluded, however, that the lowered level of muscle triglycerides after training was due to reduced amount of adipose tissue interspersed between the muscle cells. In the present study exercise lowered the concentration of triglycerides in plasma and red skeletal muscle tissue of trained rats. The lowered plasma triglyceride concentration after exercise is in accordance with findings in mar9 and .‘i In untrained rats, however, the same amount of exercise as in study 2 (Table 1) was without effect on the plasma triglyceride concentration. This suggests that training influenced factors that regulate the metabolism of the plasma triglycerides during exercise. The lipoprotein lipase enzyme system (clearing factor lipase) is believed to be of major importance for the removal of plasma triglycerides,2” and exercise has been reported to increase the activity of this enzyme system.“’ Training may have increased the amount of enzyme “units” and thus the capacity to remove triglyceride fatty acids from the blood during exercise. The increased content of liver triglycerides in trained compared to untrained rats1 does, however, not exclude that the secretion of triglycerides from the liver to the blood was inhibited during exercise. In red muscle tissue the triglyceride concentration decreased with 0.27 pmole/g (Table 4) during exercise in trained rats. The corresponding decrease was 0.65 /*mole/g in untrained rats1 (1.47 in control and 0.82 rlmole/g in exercised rats p < 0.001). The smaller decrease in response to the exercise was apparently due to the lower triglyceride concentration in trained rats at rest. It may be assumed that with reduced availability of triglyceride fatty acids in local stores, the working muscle covered its metabolic need of fat by increased utilization of circulating fatty acids. This hypothesis is to some extent supported by findings in exercise studies on mar9Js and dog?O where training appears to have increased the utilization of plasma FFA during exercise. It should be emphasized that the decrease in muscle triglyceride concentration during exercise, if due to oxidation of the disappeared triglycerides, could have contributed only a small fraction of the fat oxidized in the present study. REFERENCES 1. FrBberg,S. 0.: Effect of acute exercise on tissue lipids in rats. Metabolism 20:714, 1971. 2. Carlson, L. A., Boberg, J., and Hogstedt, B.: Some physiological and clinical implications of lipid mobilization from adipose tissue. (In Renold, A. E., and Cahill, G. F. (Eds.): Handbook of Physiology, Adipose Tissue. 63:625, 1965. 3. Miller, H., Issekutz, B., and Rodahl, K.: Effect of exercise on the metabolism of fatty acids in the dog. Amer. J. Physiol. 205:167, 1963. 4. Carlson, L. A., Friiberg, S., and Persson, S.: Concentration and turnover of the free fatty acids of plasma and concentration of blood glucose during exercise in horses. Acta Physiol. Stand. 63:434, 1965.

5. Carlson, L. A., and Mossfeldt, F.: Acute effects of prolonged, heavy exercise on the concentration of plasma lipids and lipoproteins in man. Acta Physiol. Stand. 62:51, 1964. 6. Young, D. R., Adachi, R. R., and cose oxidation and longed exercise in 23~734, 1967.

Pelligra, R., Shapira, J., Skrettingland. K.: Glureplacement during proman. .I. Appl. Physiol.

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C. M., exercise lipopro26:760, S. 0.: plasma

TRAINING

AND ACUTE

EXERCISE

IN RATS

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1051 on the relationship between the concentration of plasma free fatty acids and glycerol in vivo. Metabolism 12:132, 1963. 22. -, and Nye, E. R.: Acute effects of nicotinic acid in the rat. I. Plasma and liver lipids and blood glucose. Acta Med. Stand. 179.453, 1966. 23. -, Friiberg, S. O., and Nye, E. R.: Acute effects of nicotinic acid on plasma, liver, heart and muscle lipids. Nicotinic acid in the rat II. Acta Med. Stand. 180:571, 1966. 24. -, and Hallberg, D.: Basal lipolysis and effects of norepinephrine and prostaglandin E, on lipolysis in human subcutaneous and omental adipose tissue. J. Lab. Clin. Med. 71:368, 1968. 25. Micheli, H., Carlson, L. A., and Hallberg, D.: Comparison of lipolysis in human subcutaneous and omental adipose tissue with regard to effects of noradrenaline, theophylline, prostaglandin E, and age. Acta Chir. Stand. 135:663, 1969. 26. Robinson, D. S., and French, J. E.: Heparin, the clearing factor lipase and fat transport. Pharmacol. Rev. 12:241, 1960. 27. Nikkils, E. A., Torsti, P., and PenttilP, 0.: The effect of exercise on lipoprotein lipase activity of rat heart, adipose tissue and skeletal muscle. Metabolism 12:863, 1963. 28. Have], R. J., Naimark, A., and Borcbgrevink, C. F.: Turnover rate and oxidation of free fatty acids of blood plasma in man during exercise: Studies during continuous infusion of palmitate-Cl% J. Clin. Invest. 42:1054, 1963. 29. -, Carlson, L. A., Ekelund, L-G., and Holmgren, A.: Turnover rate and oxidation of different free fatty acids in maa during exercise. J. Appl. Physiol. 19:613, 1964. 30. Issekutz, B., Jr., Miller, H. I., and Rodahl, K.: Lipid and carbohydrate metabolism during exercise. Fed. Proc. 25:1415, 1966.