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Thyroid hormones and lipolysis in physically trained rats
Thyroid Hormones
and Lipolysis
Alfred Wirth, G&an In rats
a single
bout
triiodothyronine In trained TSH
rats
T3/rT3
in TSH
of moderate training
should
and
induced
of rT3
triiodothyronine The
with
effect
exert
hormone
more
food
30,
No.
3 (March).
freely
The eating
in T4,
(T4).
and triiodothyronine/reverse
on lipolysis T3/rT3
rats.
groups
ratio
in vitro
as well
Moderate
T3 or rT3
restriction
and Per Bjiirntorp
thyroxine
not
animals.
was
potentiated.
as basal and stimulated
food
restriction
concentrations
were
and food-restricted
an effect
on peripheral
concentrations.
restriction.
The
results
1981
hormonal
Physical suggest
to produce
but caused
a
a decrease
different.
T3
aging
the serum
concentration
while
have different
With
in vitro
in
caused
a
responses
training that
seems
changes
of lipolysis,
to have
effects
in peripheral
they
in this
effects
regard
similar
of thyroid
to
hormones
attention.
THLETES decrease in body weight during periods of physical training; this is the result of a decrease in body fat mass.’ During continuous training the amount of body fat is reduced.’ This reduction in body fat has been observed during physical training programs of long duration.’ It has been suggested that this relatively steady level of body fat depends on the si7e of fat cells in adipose tissue. since these cells seem to decrease and remain at a certain limited size’ with training. The decrease of body fat observed during physical training might be due either to a decreased energy intake, and/or an increased energy expenditure. The tirst alternative could be achieved by regulation of the appetite, previously suggested.” The increased energy expenditure, caused by the increased work during physical training sessions, is usually not sufficient to explain the weight decrease,3 therefore, other mechanisms have to be examined, such as changes in thermogenesis associated with physical training. Adaptations of thermogenesis in the regulation of energy balance have been reported. Environmental temperature. food intake. and exercise in combination with food intake. as well as overeating are all examples where adaptation in thermogenesis have been suggested. Such regulation may be mediated by changes in the conversion of thyroxine (T4) to the biologically active 3,5.3’-triiodothyronine (T3) or to the biologically inactive 3,3’5’-triiodothyronine (rcverse T3. rT3) (for review, see 5, 6, 7). Variations in tissue sensitivity of T3 could be an alternate explanation. This possibility has been studied by measuring thyroid hormones, as well as by examining the sensitivity of the peripheral adipose tissue to T3 in the rat during a single bout of exercise, and following more prolonged exercise training. The design of these experiments also allowed us to study the role of aging on the concentration of thyroid hormones in the rat. A
Merabobsm,Vol.
(T3).
concentrations.
no changes
in trained
Lundberg,
of norepinephrine
sedentary,
caused
moderate
lipolysis
Per Arne
Trained Rats
increased.
both
intake
attract
Lindstedt,
in T4, T3 or rT3
animals
Training
on thyroid energy
exercise.
in comparison
exercise
effects
found
of trained
levels.
and that
and chronic
and opposite during
to that
of catecholamine
of T3 decreased Acute
were
GSran
in increased
20 hr after
decreased
similar
and
potentiation
resulted
ratio
no changes
concentrations
body weight
those
of exercise
(T3/rT3)
Holm,
in Physically
MATERIALS
AND
METHODS
Animals Male Sprague-Dawlcy
rats (168
IX5 g) were studied at 6 wk of
age. They were housed in individual cages with tap water supplied ad libitum. and fed commercial carbohydrates,
rat pellets containing
vitamins (EWOS,
Siidertalje,
Sweden).
In the acute exercise experiment (E) ran on a rodent treadmill from 15 22 m/min.
(Experiment
body weight)
after pentobarbital
20 hr following their exercise.
rats (S) were also studied fasting for the same period
prior to exsanguination
(12-16
hr). At exsanguination
In the training
expenment
ran on a rodent treadmill
(Experiment
during
T) exercising
rats (T)
the training
for
was set at an angle of 8”. The
and the speed of each training
increased
ofage)
(42-l 5. Quinton, Seattle. Washington)
8 wk. 5 days a wk. The treadmill duration
(7 wk
b 5 g (SEM).
the average weight in each group was 240
m/min,
E) exercising rats
for 75 min while increasing the speed
They were exsanguinatcd
injection (150 mg/kg Sedentary
5’s fat. 55%
22.5% protein by weight and suthcienr minerals and
period.
session was progressively Initially
the speed was 20
and the rats were exercised I5 min twice daily. During the
last two weeks of training
the rats were able to run for a single
period of 90 min every day at a speed of 37 m/min.
Twu sedentary
groups. one group freely-eating
food-restricted
(FE)
and another
(PF) to the training animals to equal body weight were studied. This regimen resulted in a 23” r restriction of food intake between groups FE and PF during exsanguinated
the last 4 wk of training.
Th~~rolihrrin Stilrtulation The
pituitary
examined
were anaesthetized TRH
with pentobarbrtal
(Rochc.
the Clinical
Metabolic
Hospital.
linib’ersity
Address
reprint requerts
mg/kg
was
Animals
body weight).
was injected intraperi-
Luhorutor~~
o/ the Department
o/‘C‘linica/
C‘hemistr,*.
of Giiteborg,Giitrhorg.
publication I. Sahlgren’r
(TRH)
dose of 500 ng/kg body weight. Blood was
1. and the Department
of Medicine
(50
Basel, Switzerland)
Medicine
Receivedfbr
thyroliberin
T 2 wk prior to exsanguination.
toneally in a submaximal
From
T rats were
Test
response to exogenous
in experiment
Synthetic
The
20 hr after the last bout of exercise.
of
Sahlgrenk
Sweden.
June I I. 1979. to ProJessor Hospital.
Per BjBrntorp.
i:niwrrir,’
Lkpartment
r!f Giitehor,~.
G&e-
horg, Sweden.
237
WIRTH ET AL.
238
for the separation of free and bound radioligand (Diagnostic Products Corp., Los Angeles, California, USA). Plasma 3,3’5’-triiodothyronine was analyzed by a single-antibody radioimmunoassay with polyethyleneglycol separation (Serono-Biodata, Coisins, Switzerland). The intraassay coefficients of variation which were calculated from the differences between duplicate analyses, were in two assay series: 2.3 and 2.6% (thyroxine), 2.7% (triiodothyronine), 6.2 and 11%(reverse triiodothyronine). Lipids from the fat pads were extracted by use of Dole’s extraction mixture.‘The lipid content was measured gravimetrically after evaporation of the solvent. Fat cell size was determined by a microscopic method.‘O The number of fat cells was calculated from the mean fat cell size of the fraction of the lipid content. Mean cell volume was calculated as described by Goldrick.”
drawn from the tail veins into heparinized syringes immediately before the injection of TRH and at 15and 30 min intervals after the injection.
Fat Pad Incubation The distal ends of the epididymal pads were removed and placed in Parker medium 199 buffer at room temperature and minced into pieces of about 50 mg. These were then incubated in 2 ml of Parkers medium 199, which contained 4% of bovine serum albumin (batch YE 2170. Armour, Eastbourne, England) and I mM glucose at pH 7.4. Incubations were carried out at 37” and in a gas phase of air. Additions were made from beginning of incubation as indicated in the test. Norepinephrine (Noradrenaline) was obtained from Astra, Siidertalje. Sweden, and T, (Liothyronin-natrium) from Nyegaard & Co, Oslo, Norway.
Statistics
Analytical Methods
Student’s t test was applied for group differences in experiment E. and analysis of variance for the calculation of group differences in experiment T. Significant differences in the latter experiment were further examined with linear contrasts according to Scheffe.” Student’s t test was used for paired values and conventional linear regression analysis for correlation. Values are presented as mean k SEM.
Glycerol was determined fluorometrically.* Plasma thyrotropin was analyzed by a double-antibody radioimmunoassay using rat thyrotropin, a rat thyroptropin standard (NIAMDD-RAT TSHRP-I), a rabbit anti-rat thyrotropin antiserum (The National Institute of Arthritis, Metabolism and Digestive Diseases Rat Pituitary Hormone Distribution Program through dr A.F. Parlow) and a swine anti-rabbit IgG antiserum (Dakopatts A/S, Copenhagen, Denmark). Labeling of rat thyrotropin was performed with chloramine-T. and the products were fractionated by filtration through a column (I .6 x 80 cm) of Sephadex G-75 (superfine) using a IO mM phosphate buffer at pH 7.4 with 20 g/liter of bovine albumin, 150 mmol/liter of sodium chloride and 15 mmol/liter of sodium azide. The material in the beginning of the descending part of the thyrotropin monomer peak was used. Immunoreactivity, with a fivefold excess of antibody, was 60%. The specific activity was calculated to about 300 Ci/g. Incubations were performed at 4°C for 5 + I days using bovine albumin (50 g/liter) in both the standards and the incubation buffer. The results were evaluated by a computer using a logit-log linearization procedure with correction of blank values for optimal curve fitting (Lindstedt, Lundberg and Norberg. unpublished). Correlation coefficients for the standard curves were better than 0.997 in four assay series. The intraassay coefficient of variation was 6.5% as calculated from the difference between duplicates (mean: 199 fig/liter, range 32-660 fig/liter, n = 195 duplicates). Plasma thyroxine and 3,5,3’-triiodothyronine were analyzed by a double-antibody radioimmunoassay. Polyethyleneglycol was used Table
1.
Plasma
T,, T,,
rT,,
Basal and TRH
Body Weight Experiment
E
(91
Stimulated
TSH
15
240
+ 5
64.5
+ 1.6
1.72
Sedentary
n=
15
240
+ 5
59.7
i- 1.6
1.43+0.02
n.?.. Experiment
in Acute
Exercise
Experiment
rT, hVnol/l)
T, (nmol/l)
n=
(S)
Table I shows that one period of exercise resulted in an increase in T4 and T3 concentrations as well as in T3/rT3 ratio, but no changes in rT3 or basal TSH concentrations. Exercise-trained animals in comparison with sedentary, freely-eating animals were lighter, but not different in T4, T3, and rT3 concentrations, although the T3/rT3 ratio was lower in the trained animals. Basal and stimulated TSH values were lower in the trained animals. The slight energy deprivation (23% restriction) to obtain animals of the same weight as those trained caused exactly the same changes as physical training, namely a decrease in T3/rT3 ratio and in TSH values, but no changes in the concentrations of the other thyroid hormones, also in harmony with the effects of (El and Training
to.05
+
0.07
0.22 0.21
(T)
15min
basal
rT,
t 0.01
8.09
I
kO.01
6.71
-to.19
n.s.
1
Experiment TSH (ug/llter)
T, T, (nmollll
Exercised (E)
E vs. S
RESULTS
0.44
30 ml”
107 k 5 115
t
17
n.s.
-co.01
T
Trained
(T)
n=
13
330
+
7
53.6
+ 2.5
1.16
+ 0.04
0.30
+ 0.02
3.97
k 0.28
81
5 5
378
5 31’
289
i
Pair-fed
(PF)
n =
14
332
?
7
56.4
+_ 0.7
1.19
+ 0.03
0.31
f
0.02
3.81
k 0.18
86
k 6
430
k 41*
332
k 27’
n=
15
395
k 8
61.1
k
1.26
+ 0.03
0.30
k 0.02
4.48
+ 0.31
128
k
561
? 56*
437
+ 40+
Freelv-eatinq
-
(FE)
1.6
10
T vs. FE
n.s.
“.S.
n.s.
<0.05
CO.00
PF vs. FE
10.01
n.s.
n.s.
n.s.
CO.05
<0.05
n.s.
“.*.
“.S.
“.S.
n.s.
“.!%.
T vs. PF S (experiment
E)
vs. FE (experwnent Significance
1
n.s.
T) for increase
in TSH:
l
= p < 0.001.
Means
k SEM.
1
10.00
1
1
“.a.
“.S.
“.S.
40’
THYROID
AND
TRAINING
IN
239
RATS
training. Consequently, when comparing the exercise trained group with the moderately energy-deprived group, no differences were found. Table 2 shows the results of measurement of glycerol release from the epididymal fat pads of the groups. Rats subjected to one period of exercise showed an increased norepinephrine response of lipolysis. This increase was further accentuated in the presence of T3 (1 .I x lO_‘M). Exercise-trained animals showed smaller fat cells and lower basal lipolysis than sedentary, freely-eating animals. Norepinephrine caused a stimulation in both groups, more pronounced in relative terms in the trained group. Addition of T3 had no effect on basal lipolysis but caused a further increase in the norepinephrine-stimulated lipolysis in the trained group, but not in the sedentary group. Moderate food-restriction again showed essentially the same effects as training, namely smaller fat cells. lower basal lipolysis. and a permissive effect of T3 on norepinephrine-stimulated lipolysis. Consequently, there were no differences between the trained and moderately food-restricted groups in any of the measured variables. Comparing the effects of acute exercise and exercise training it is found that the lipolytic response of norepinephrinc was relatively higher after acute exercise (+ 242%,) than after training (+71%). The permissive role of T3 on norepinephrine-induced lipolysis was also possibly higher in the acute exercise group ( 4 32%) than in the trained group (+22%,). The freely-eating, sedentary rats in the acute exercise experiment were 7 weeks old and those in the exercise-training experiment 14 wk old. Comparing the results obtained in these both groups allows an analysis of the effect of aging. Aging then was found to be followed by an increase in rT3 concentrations and a decrease in T3/rT3 ratio (Table I). This has previously been reported.” Table
2.
Fat
Cell
Size
and
Glycerol
Release
From and
6 8
i
“23
9 -2
Fig. T,
1.
Correlations
exercise
(El experiment. between
i
J.6
1.7
l.8
T3hml/l)
between
on noradrenaline
difference
’
1.5
m
.
i
effect
aline
14 .
.
._ 0
.. . ” ” ’
T,.
T, concentrations
stimulated Glycerol
incubations
Triangles
glycerol release
with
rats.
and
in the
is expressed
noradrenaline
exercising
in plasma
release
acute
as percent and
noradren-
Squares-freely-eating
controls.
There were significant. positive correlations between T3 concentrations and the net T3 effect on lipolysis (permissive etrcct on norepinephrine-induced lipolysis) in the acute exercise experiment (Fig. I ) but not in the exercise-training experiment. T3/rT3 and the T3 effect was also signilicantly correlated (r: 0.38. p < 0.05, not shown). DISCUSSION
The present work was designed to allow an analysis of the effects of acute cxcrcise and exercise-training on thyroid hormone concentrations, as well as the effects of T3 on adipose tissue lipolysis as a selected test of peripheral sensitivity of thyroid hormones. Acute exercise caused an increase in T4 and T3 concentrations as well as an increase in the ratio of T3/rT3. This was
Epididymal
Fat
Experiment
Pads
of Rats
in Experiment
E (exhaustive
exercise)
T (training) Glycerol Release (nmol/ 1 O5 cells/Z
hi
Fat 1
Cell
2 + NA(2
SlZe Expenment Exercise
(E)
Sedentary
(S)
+ T&l
1 \
5
4
1 NA(2
_
‘g/ml)
10
5M)
v 10
- T,(l
1 .
‘g/ml)
10
n _
15
61.9
t
1.2
16.5
+
1.5
56.4
i
3.8$
17.1
+
1.8
55.8
i
n
15
63.7
+_ 1.1
17.4
f
1.4
37.4
+ 2.4%
21.1
-
1.6
39.8
+ 2.6
n.s.
“.s.
E vs. s Experiment
Y 10 _~
(urn)
E
3
n.s.
-:O.OOl
7 5
“MI
NA(2
s 10
- T,tl
‘g/ml)
1 x 10
74.1
+ 5.5$
41.2
-
2 6
0.001
. 0.05
T
TraIned
(T)
” =
13
63.7
+
1.7
23.0
+
1.8
39.3
f
3.9*
24.1
i
1.4
38.1
+ 3.7
48.6
+ 4.0*
Pawfed
(PF)
n = 14
68.2
r
1.6
28.5
k 2.3
36.0
i
3.6$
27.8
i
2.4
40.3
14.6
49.4
I
” = 15
78.8
L
1.9
46.7
+ 3.6
60.2
i
5.9f
47.5
f
4.7
59.4
*
Freely-eating
(FE)
T vs. FE
<0.001
-co.00
1
. 0.05
PF vs. FE
<-0.00
1
I 0.05
T vs. PF Statistical
n.s. slgniflcance
of pair differences
“.S.
“.S.
of glycerol
release:
l
= p < 0.05,
“.S.
t
= p c: 0.01.
t
“.S.
= p < 0.001
for
5 5
70.9
5.4t + 6.3
-.O.Ol 0.05 “.S.
1 vs. 2. 2 vs. 4, and 2 vs. 5.
‘M)
240
WIRTH ET AL.
associated with an augmented norepinephrine response of lipolysis, as well as with an increased permissive effect of T3 on norepinephrine-stimulated lipolysis, possibly indicating an increased peripheral sensitivity of this system to T3. Furthermore, correlations between peripheral effects and plasma concentration changes of thyroid hormones could be demonstrated. In the experiments with exercise-training the effects on hormone concentrations were largely the opposite, viz. a decrease in the T3/rT3 ratio. Furthermore, TSH levels in the basal state and after TRH-stimulation were lower than in sedentary, freely eating controls. In these observations then physical training had the sbme general effects as energy intake restriction, previously described.6.7.13.14.24.25It was also found in the present work that a sedentary group, subjected to a moderate food-restriction to match the body-weight of the trained group, showed the same changes as the training rats. The lipolytic process was also changed in parallel between trained and food-restricted rats, except an increased norepinephrine sensitivity in the trained group, previously described.‘7,‘8 The parallel effects were smaller fat cells, lower basal lipolysis and a permissive effect of T3 on norepinephrine-stimulated lipolysis. The effects of exercise on thyroid hormones have previously been analysed in a variety of experimental
designs, and with varying resu1ts.‘5”6.23 The present report indicates that it is of fundamental importance to analyse the acute exercise situation separate from that after physical training, because the associated changes are not only different, but actually opposite. Furthermore, definition of the energy balance is of importance for the results because negative energy balance and training seems to have similar effects. The present studies indicate that when starting an exercise training program there is an initial phase where T4 and T3 concentrations increase. This seems to be associated with an augmentation of peripheral sensitivity to norepinephrine. Eventually this thyroid hormone concentration increase is disappearing while at least some of the effects on peripheral sensitivity are remaining. This suggests that the peripheral effects are the more consistent ones, and that these should attract more interest for detailed studies in the future. It is known previously that T3 produces a permissive effect on norepinephrine-induced lipolysis.‘9~‘o~2’ It seems possible that the previously reported enhancing effect of exercise on lipolysis is actually mediated via peripheral T3-effects as seen in the present work. This possibility is suggested by recent findings where physical training and T3 effect lipolysis regulation in an identical, specific way, enhancing /%adrenergic sensitivity without changing adrenergic receptors.‘h.27
REFERENCES 1. Parizkova J: Impact of age, diet and exercise on man’s body composition. Ann NY Acad Sci I 10:661-674, 1973 2. Bjijrntorp P, Fahltn M, Grimby G. et al: Carbohydrate and lipid metabolism in middle-aged, physically well-trained men. Metabolism 21:1037, 1972 3. Bjiirntorp P: Exercise in the treatment of obesity. Clin Endocr Metab 5:43 I, 1976 4. Mayer J, Marshall NB, Vitale JJ, et al: Exercise, food intake and body weight in normal rats and genetically obese adult mice. Amer J Physiol 177:544-548, 1954 5. Miller DS: Thermogenesis in every day life. Proc Sot Congr of Energy Balance in Man. Excerpta Medica, 1974 6. Westgren U: On the production of thyroxine, active and inactive triiodothyronine in man 1977. Thesis. Studentlitteratur, Lund, Sweden 7. Danforth E. Burger AC, Goldman RF, et al: Thermogenesis during weight gain. Recent Adv Obes Res II. Chapter V. 1977, London, Newman Pub1 Co 8. Laurel1 G, Tibbling G: An enzymatic fluorometric micromethod for the determination of glycerol. Clin Chim Acta 13:317. 1966 9. Dole VP: A relation between nonesterified fatty acids in plasma and the metabolism of glucose. J Clin Invest 35:150-153, 10. SjSstr8m L. Bjiirntorp P, Vrana J: Microscopic fat cell size measurements on frozen-cut adipose tissue in comparison with automatic determinations of osmium fixed cells. J Lipid Res 12:52 1, 1971 I I. Goldrick RB: Morphological changes in the adipocyte during fat deposition and mobilization. Amer J Physiol 212:777, 1967
12. SchelTi HA: The analysis of variance. New York, John Wiley & Sons, Inc., 1959, p 66 13. Palmblad J, Levi L, Burger A, et al: Effects of total energy withdrawal (fasting) on the levels of growth hormone, thyrotropin, cortisol, adrenaline, noradrenaline. T,. T, and rT, in healthy males. Acta Med Stand 201: 15-22, 1977 14. Glass AR, Mellitt R, Burman KD. et al: Serum triiodothyronine in undernourished rats: Dependence on dietary composition rather than total calorie or protein intake. Endocrinology 102: 1925% 1928, 1978 15. Berchtold P. Berger M, Herrmann J. et al: Thyroid hormones and TSH during physical exercise in healthy and diabetic subjects. Europ J Clin Invest. 7:4-5. 1977 (abstract) 16. Balsam A, Leppo LE: Effect of physical training on the metabolism of thyroid hormones in man. J Appl Physiol 38:212215, 1975 17. Askew EW, Huston RL. Plopper CC, et al: Adipose Tissue Cellularity and Lipolysis. Response to exercise and cortisol treatment. J Clin Invest 56:521-529, 1975 18. Holm G, Jacobsson B, Holm J, et al: Effects of submaximal physical exercise on adipose tissue metabolism in man. lnt J Obesity 11249-257, 1977 19. Vaughan M: An in Vitro Effect of Triiodothyronine on Rat AdiposeTissue. J Clin Invest 46:1482Zl491, 1967 20. Goodman HM, Bray GA: Role of thyroid hormones in lipolysis. Amer J Physiol 210:1053~1058, 1966 21. Kaciuba-UScilko H, Greenleaf JE, Kozlowski S, et al: Thyroid hormone induced changes in body temperature and metabolism during exercise in dogs. Am J Physiol 229:260-264, 1975
THYROID AND TRAINING
22. Westgren
241
IN RATS
U, Burger A, lngemansson N, et al: Blood levels of
3.5.3’.triiodothyronine
and thyroxine:
Differences between children,
adults, and elderly subjects. Acta Med Stand 200:493-495, 23. Winder
WW.
Heninger
RW:
Effect
of exercise
levels of thyroid hormones in the rat. Am J Physiol 22
I976
Harris ARC,
27:1074-1083.
Fang SL, Azizi thyroid
suppressed TSH and refeeding Metabolism
KD,
Smallridge
secretion during on TSH
29:46-52.
RC. Osborne
I: I 139-l
R, et al: Nature
undernutrition:
responses to prolonged 1980
143, of
effect of fasting TRH
in the
rat.
on
Metabolism
1978
26.
Holm G, Jacobsson B. Toss L. et al: The etfect of physical
exercise on the regulation Burman
F et al: Effect of starvation
function
on tissue
1971 24.
25.
hypothalamic-pituitary
of beta adrenergic
late cyclasc in rat adipocytes.
Proc III
receptors and adeny-
Int Congr Obesity
Oct. 9-l I) 1980 (in print)
infusions. 27. Jacobsson B: 1980. Personal communrcation
(Rome
and Lipolysis
Alfred Wirth, G&an In rats
a single
bout
triiodothyronine In trained TSH
rats
T3/rT3
in TSH
of moderate training
should
and
induced
of rT3
triiodothyronine The
with
effect
exert
hormone
more
food
30,
No.
3 (March).
freely
The eating
in T4,
(T4).
and triiodothyronine/reverse
on lipolysis T3/rT3
rats.
groups
ratio
in vitro
as well
Moderate
T3 or rT3
restriction
and Per Bjiirntorp
thyroxine
not
animals.
was
potentiated.
as basal and stimulated
food
restriction
concentrations
were
and food-restricted
an effect
on peripheral
concentrations.
restriction.
The
results
1981
hormonal
Physical suggest
to produce
but caused
a
a decrease
different.
T3
aging
the serum
concentration
while
have different
With
in vitro
in
caused
a
responses
training that
seems
changes
of lipolysis,
to have
effects
in peripheral
they
in this
effects
regard
similar
of thyroid
to
hormones
attention.
THLETES decrease in body weight during periods of physical training; this is the result of a decrease in body fat mass.’ During continuous training the amount of body fat is reduced.’ This reduction in body fat has been observed during physical training programs of long duration.’ It has been suggested that this relatively steady level of body fat depends on the si7e of fat cells in adipose tissue. since these cells seem to decrease and remain at a certain limited size’ with training. The decrease of body fat observed during physical training might be due either to a decreased energy intake, and/or an increased energy expenditure. The tirst alternative could be achieved by regulation of the appetite, previously suggested.” The increased energy expenditure, caused by the increased work during physical training sessions, is usually not sufficient to explain the weight decrease,3 therefore, other mechanisms have to be examined, such as changes in thermogenesis associated with physical training. Adaptations of thermogenesis in the regulation of energy balance have been reported. Environmental temperature. food intake. and exercise in combination with food intake. as well as overeating are all examples where adaptation in thermogenesis have been suggested. Such regulation may be mediated by changes in the conversion of thyroxine (T4) to the biologically active 3,5.3’-triiodothyronine (T3) or to the biologically inactive 3,3’5’-triiodothyronine (rcverse T3. rT3) (for review, see 5, 6, 7). Variations in tissue sensitivity of T3 could be an alternate explanation. This possibility has been studied by measuring thyroid hormones, as well as by examining the sensitivity of the peripheral adipose tissue to T3 in the rat during a single bout of exercise, and following more prolonged exercise training. The design of these experiments also allowed us to study the role of aging on the concentration of thyroid hormones in the rat. A
Merabobsm,Vol.
(T3).
concentrations.
no changes
in trained
Lundberg,
of norepinephrine
sedentary,
caused
moderate
lipolysis
Per Arne
Trained Rats
increased.
both
intake
attract
Lindstedt,
in T4, T3 or rT3
animals
Training
on thyroid energy
exercise.
in comparison
exercise
effects
found
of trained
levels.
and that
and chronic
and opposite during
to that
of catecholamine
of T3 decreased Acute
were
GSran
in increased
20 hr after
decreased
similar
and
potentiation
resulted
ratio
no changes
concentrations
body weight
those
of exercise
(T3/rT3)
Holm,
in Physically
MATERIALS
AND
METHODS
Animals Male Sprague-Dawlcy
rats (168
IX5 g) were studied at 6 wk of
age. They were housed in individual cages with tap water supplied ad libitum. and fed commercial carbohydrates,
rat pellets containing
vitamins (EWOS,
Siidertalje,
Sweden).
In the acute exercise experiment (E) ran on a rodent treadmill from 15 22 m/min.
(Experiment
body weight)
after pentobarbital
20 hr following their exercise.
rats (S) were also studied fasting for the same period
prior to exsanguination
(12-16
hr). At exsanguination
In the training
expenment
ran on a rodent treadmill
(Experiment
during
T) exercising
rats (T)
the training
for
was set at an angle of 8”. The
and the speed of each training
increased
ofage)
(42-l 5. Quinton, Seattle. Washington)
8 wk. 5 days a wk. The treadmill duration
(7 wk
b 5 g (SEM).
the average weight in each group was 240
m/min,
E) exercising rats
for 75 min while increasing the speed
They were exsanguinatcd
injection (150 mg/kg Sedentary
5’s fat. 55%
22.5% protein by weight and suthcienr minerals and
period.
session was progressively Initially
the speed was 20
and the rats were exercised I5 min twice daily. During the
last two weeks of training
the rats were able to run for a single
period of 90 min every day at a speed of 37 m/min.
Twu sedentary
groups. one group freely-eating
food-restricted
(FE)
and another
(PF) to the training animals to equal body weight were studied. This regimen resulted in a 23” r restriction of food intake between groups FE and PF during exsanguinated
the last 4 wk of training.
Th~~rolihrrin Stilrtulation The
pituitary
examined
were anaesthetized TRH
with pentobarbrtal
(Rochc.
the Clinical
Metabolic
Hospital.
linib’ersity
Address
reprint requerts
mg/kg
was
Animals
body weight).
was injected intraperi-
Luhorutor~~
o/ the Department
o/‘C‘linica/
C‘hemistr,*.
of Giiteborg,Giitrhorg.
publication I. Sahlgren’r
(TRH)
dose of 500 ng/kg body weight. Blood was
1. and the Department
of Medicine
(50
Basel, Switzerland)
Medicine
Receivedfbr
thyroliberin
T 2 wk prior to exsanguination.
toneally in a submaximal
From
T rats were
Test
response to exogenous
in experiment
Synthetic
The
20 hr after the last bout of exercise.
of
Sahlgrenk
Sweden.
June I I. 1979. to ProJessor Hospital.
Per BjBrntorp.
i:niwrrir,’
Lkpartment
r!f Giitehor,~.
G&e-
horg, Sweden.
237
WIRTH ET AL.
238
for the separation of free and bound radioligand (Diagnostic Products Corp., Los Angeles, California, USA). Plasma 3,3’5’-triiodothyronine was analyzed by a single-antibody radioimmunoassay with polyethyleneglycol separation (Serono-Biodata, Coisins, Switzerland). The intraassay coefficients of variation which were calculated from the differences between duplicate analyses, were in two assay series: 2.3 and 2.6% (thyroxine), 2.7% (triiodothyronine), 6.2 and 11%(reverse triiodothyronine). Lipids from the fat pads were extracted by use of Dole’s extraction mixture.‘The lipid content was measured gravimetrically after evaporation of the solvent. Fat cell size was determined by a microscopic method.‘O The number of fat cells was calculated from the mean fat cell size of the fraction of the lipid content. Mean cell volume was calculated as described by Goldrick.”
drawn from the tail veins into heparinized syringes immediately before the injection of TRH and at 15and 30 min intervals after the injection.
Fat Pad Incubation The distal ends of the epididymal pads were removed and placed in Parker medium 199 buffer at room temperature and minced into pieces of about 50 mg. These were then incubated in 2 ml of Parkers medium 199, which contained 4% of bovine serum albumin (batch YE 2170. Armour, Eastbourne, England) and I mM glucose at pH 7.4. Incubations were carried out at 37” and in a gas phase of air. Additions were made from beginning of incubation as indicated in the test. Norepinephrine (Noradrenaline) was obtained from Astra, Siidertalje. Sweden, and T, (Liothyronin-natrium) from Nyegaard & Co, Oslo, Norway.
Statistics
Analytical Methods
Student’s t test was applied for group differences in experiment E. and analysis of variance for the calculation of group differences in experiment T. Significant differences in the latter experiment were further examined with linear contrasts according to Scheffe.” Student’s t test was used for paired values and conventional linear regression analysis for correlation. Values are presented as mean k SEM.
Glycerol was determined fluorometrically.* Plasma thyrotropin was analyzed by a double-antibody radioimmunoassay using rat thyrotropin, a rat thyroptropin standard (NIAMDD-RAT TSHRP-I), a rabbit anti-rat thyrotropin antiserum (The National Institute of Arthritis, Metabolism and Digestive Diseases Rat Pituitary Hormone Distribution Program through dr A.F. Parlow) and a swine anti-rabbit IgG antiserum (Dakopatts A/S, Copenhagen, Denmark). Labeling of rat thyrotropin was performed with chloramine-T. and the products were fractionated by filtration through a column (I .6 x 80 cm) of Sephadex G-75 (superfine) using a IO mM phosphate buffer at pH 7.4 with 20 g/liter of bovine albumin, 150 mmol/liter of sodium chloride and 15 mmol/liter of sodium azide. The material in the beginning of the descending part of the thyrotropin monomer peak was used. Immunoreactivity, with a fivefold excess of antibody, was 60%. The specific activity was calculated to about 300 Ci/g. Incubations were performed at 4°C for 5 + I days using bovine albumin (50 g/liter) in both the standards and the incubation buffer. The results were evaluated by a computer using a logit-log linearization procedure with correction of blank values for optimal curve fitting (Lindstedt, Lundberg and Norberg. unpublished). Correlation coefficients for the standard curves were better than 0.997 in four assay series. The intraassay coefficient of variation was 6.5% as calculated from the difference between duplicates (mean: 199 fig/liter, range 32-660 fig/liter, n = 195 duplicates). Plasma thyroxine and 3,5,3’-triiodothyronine were analyzed by a double-antibody radioimmunoassay. Polyethyleneglycol was used Table
1.
Plasma
T,, T,,
rT,,
Basal and TRH
Body Weight Experiment
E
(91
Stimulated
TSH
15
240
+ 5
64.5
+ 1.6
1.72
Sedentary
n=
15
240
+ 5
59.7
i- 1.6
1.43+0.02
n.?.. Experiment
in Acute
Exercise
Experiment
rT, hVnol/l)
T, (nmol/l)
n=
(S)
Table I shows that one period of exercise resulted in an increase in T4 and T3 concentrations as well as in T3/rT3 ratio, but no changes in rT3 or basal TSH concentrations. Exercise-trained animals in comparison with sedentary, freely-eating animals were lighter, but not different in T4, T3, and rT3 concentrations, although the T3/rT3 ratio was lower in the trained animals. Basal and stimulated TSH values were lower in the trained animals. The slight energy deprivation (23% restriction) to obtain animals of the same weight as those trained caused exactly the same changes as physical training, namely a decrease in T3/rT3 ratio and in TSH values, but no changes in the concentrations of the other thyroid hormones, also in harmony with the effects of (El and Training
to.05
+
0.07
0.22 0.21
(T)
15min
basal
rT,
t 0.01
8.09
I
kO.01
6.71
-to.19
n.s.
1
Experiment TSH (ug/llter)
T, T, (nmollll
Exercised (E)
E vs. S
RESULTS
0.44
30 ml”
107 k 5 115
t
17
n.s.
-co.01
T
Trained
(T)
n=
13
330
+
7
53.6
+ 2.5
1.16
+ 0.04
0.30
+ 0.02
3.97
k 0.28
81
5 5
378
5 31’
289
i
Pair-fed
(PF)
n =
14
332
?
7
56.4
+_ 0.7
1.19
+ 0.03
0.31
f
0.02
3.81
k 0.18
86
k 6
430
k 41*
332
k 27’
n=
15
395
k 8
61.1
k
1.26
+ 0.03
0.30
k 0.02
4.48
+ 0.31
128
k
561
? 56*
437
+ 40+
Freelv-eatinq
-
(FE)
1.6
10
T vs. FE
n.s.
“.S.
n.s.
<0.05
CO.00
PF vs. FE
10.01
n.s.
n.s.
n.s.
CO.05
<0.05
n.s.
“.*.
“.S.
“.S.
n.s.
“.!%.
T vs. PF S (experiment
E)
vs. FE (experwnent Significance
1
n.s.
T) for increase
in TSH:
l
= p < 0.001.
Means
k SEM.
1
10.00
1
1
“.a.
“.S.
“.S.
40’
THYROID
AND
TRAINING
IN
239
RATS
training. Consequently, when comparing the exercise trained group with the moderately energy-deprived group, no differences were found. Table 2 shows the results of measurement of glycerol release from the epididymal fat pads of the groups. Rats subjected to one period of exercise showed an increased norepinephrine response of lipolysis. This increase was further accentuated in the presence of T3 (1 .I x lO_‘M). Exercise-trained animals showed smaller fat cells and lower basal lipolysis than sedentary, freely-eating animals. Norepinephrine caused a stimulation in both groups, more pronounced in relative terms in the trained group. Addition of T3 had no effect on basal lipolysis but caused a further increase in the norepinephrine-stimulated lipolysis in the trained group, but not in the sedentary group. Moderate food-restriction again showed essentially the same effects as training, namely smaller fat cells. lower basal lipolysis. and a permissive effect of T3 on norepinephrine-stimulated lipolysis. Consequently, there were no differences between the trained and moderately food-restricted groups in any of the measured variables. Comparing the effects of acute exercise and exercise training it is found that the lipolytic response of norepinephrinc was relatively higher after acute exercise (+ 242%,) than after training (+71%). The permissive role of T3 on norepinephrine-induced lipolysis was also possibly higher in the acute exercise group ( 4 32%) than in the trained group (+22%,). The freely-eating, sedentary rats in the acute exercise experiment were 7 weeks old and those in the exercise-training experiment 14 wk old. Comparing the results obtained in these both groups allows an analysis of the effect of aging. Aging then was found to be followed by an increase in rT3 concentrations and a decrease in T3/rT3 ratio (Table I). This has previously been reported.” Table
2.
Fat
Cell
Size
and
Glycerol
Release
From and
6 8
i
“23
9 -2
Fig. T,
1.
Correlations
exercise
(El experiment. between
i
J.6
1.7
l.8
T3hml/l)
between
on noradrenaline
difference
’
1.5
m
.
i
effect
aline
14 .
.
._ 0
.. . ” ” ’
T,.
T, concentrations
stimulated Glycerol
incubations
Triangles
glycerol release
with
rats.
and
in the
is expressed
noradrenaline
exercising
in plasma
release
acute
as percent and
noradren-
Squares-freely-eating
controls.
There were significant. positive correlations between T3 concentrations and the net T3 effect on lipolysis (permissive etrcct on norepinephrine-induced lipolysis) in the acute exercise experiment (Fig. I ) but not in the exercise-training experiment. T3/rT3 and the T3 effect was also signilicantly correlated (r: 0.38. p < 0.05, not shown). DISCUSSION
The present work was designed to allow an analysis of the effects of acute cxcrcise and exercise-training on thyroid hormone concentrations, as well as the effects of T3 on adipose tissue lipolysis as a selected test of peripheral sensitivity of thyroid hormones. Acute exercise caused an increase in T4 and T3 concentrations as well as an increase in the ratio of T3/rT3. This was
Epididymal
Fat
Experiment
Pads
of Rats
in Experiment
E (exhaustive
exercise)
T (training) Glycerol Release (nmol/ 1 O5 cells/Z
hi
Fat 1
Cell
2 + NA(2
SlZe Expenment Exercise
(E)
Sedentary
(S)
+ T&l
1 \
5
4
1 NA(2
_
‘g/ml)
10
5M)
v 10
- T,(l
1 .
‘g/ml)
10
n _
15
61.9
t
1.2
16.5
+
1.5
56.4
i
3.8$
17.1
+
1.8
55.8
i
n
15
63.7
+_ 1.1
17.4
f
1.4
37.4
+ 2.4%
21.1
-
1.6
39.8
+ 2.6
n.s.
“.s.
E vs. s Experiment
Y 10 _~
(urn)
E
3
n.s.
-:O.OOl
7 5
“MI
NA(2
s 10
- T,tl
‘g/ml)
1 x 10
74.1
+ 5.5$
41.2
-
2 6
0.001
. 0.05
T
TraIned
(T)
” =
13
63.7
+
1.7
23.0
+
1.8
39.3
f
3.9*
24.1
i
1.4
38.1
+ 3.7
48.6
+ 4.0*
Pawfed
(PF)
n = 14
68.2
r
1.6
28.5
k 2.3
36.0
i
3.6$
27.8
i
2.4
40.3
14.6
49.4
I
” = 15
78.8
L
1.9
46.7
+ 3.6
60.2
i
5.9f
47.5
f
4.7
59.4
*
Freely-eating
(FE)
T vs. FE
<0.001
-co.00
1
. 0.05
PF vs. FE
<-0.00
1
I 0.05
T vs. PF Statistical
n.s. slgniflcance
of pair differences
“.S.
“.S.
of glycerol
release:
l
= p < 0.05,
“.S.
t
= p c: 0.01.
t
“.S.
= p < 0.001
for
5 5
70.9
5.4t + 6.3
-.O.Ol 0.05 “.S.
1 vs. 2. 2 vs. 4, and 2 vs. 5.
‘M)
240
WIRTH ET AL.
associated with an augmented norepinephrine response of lipolysis, as well as with an increased permissive effect of T3 on norepinephrine-stimulated lipolysis, possibly indicating an increased peripheral sensitivity of this system to T3. Furthermore, correlations between peripheral effects and plasma concentration changes of thyroid hormones could be demonstrated. In the experiments with exercise-training the effects on hormone concentrations were largely the opposite, viz. a decrease in the T3/rT3 ratio. Furthermore, TSH levels in the basal state and after TRH-stimulation were lower than in sedentary, freely eating controls. In these observations then physical training had the sbme general effects as energy intake restriction, previously described.6.7.13.14.24.25It was also found in the present work that a sedentary group, subjected to a moderate food-restriction to match the body-weight of the trained group, showed the same changes as the training rats. The lipolytic process was also changed in parallel between trained and food-restricted rats, except an increased norepinephrine sensitivity in the trained group, previously described.‘7,‘8 The parallel effects were smaller fat cells, lower basal lipolysis and a permissive effect of T3 on norepinephrine-stimulated lipolysis. The effects of exercise on thyroid hormones have previously been analysed in a variety of experimental
designs, and with varying resu1ts.‘5”6.23 The present report indicates that it is of fundamental importance to analyse the acute exercise situation separate from that after physical training, because the associated changes are not only different, but actually opposite. Furthermore, definition of the energy balance is of importance for the results because negative energy balance and training seems to have similar effects. The present studies indicate that when starting an exercise training program there is an initial phase where T4 and T3 concentrations increase. This seems to be associated with an augmentation of peripheral sensitivity to norepinephrine. Eventually this thyroid hormone concentration increase is disappearing while at least some of the effects on peripheral sensitivity are remaining. This suggests that the peripheral effects are the more consistent ones, and that these should attract more interest for detailed studies in the future. It is known previously that T3 produces a permissive effect on norepinephrine-induced lipolysis.‘9~‘o~2’ It seems possible that the previously reported enhancing effect of exercise on lipolysis is actually mediated via peripheral T3-effects as seen in the present work. This possibility is suggested by recent findings where physical training and T3 effect lipolysis regulation in an identical, specific way, enhancing /%adrenergic sensitivity without changing adrenergic receptors.‘h.27
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12. SchelTi HA: The analysis of variance. New York, John Wiley & Sons, Inc., 1959, p 66 13. Palmblad J, Levi L, Burger A, et al: Effects of total energy withdrawal (fasting) on the levels of growth hormone, thyrotropin, cortisol, adrenaline, noradrenaline. T,. T, and rT, in healthy males. Acta Med Stand 201: 15-22, 1977 14. Glass AR, Mellitt R, Burman KD. et al: Serum triiodothyronine in undernourished rats: Dependence on dietary composition rather than total calorie or protein intake. Endocrinology 102: 1925% 1928, 1978 15. Berchtold P. Berger M, Herrmann J. et al: Thyroid hormones and TSH during physical exercise in healthy and diabetic subjects. Europ J Clin Invest. 7:4-5. 1977 (abstract) 16. Balsam A, Leppo LE: Effect of physical training on the metabolism of thyroid hormones in man. J Appl Physiol 38:212215, 1975 17. Askew EW, Huston RL. Plopper CC, et al: Adipose Tissue Cellularity and Lipolysis. Response to exercise and cortisol treatment. J Clin Invest 56:521-529, 1975 18. Holm G, Jacobsson B, Holm J, et al: Effects of submaximal physical exercise on adipose tissue metabolism in man. lnt J Obesity 11249-257, 1977 19. Vaughan M: An in Vitro Effect of Triiodothyronine on Rat AdiposeTissue. J Clin Invest 46:1482Zl491, 1967 20. Goodman HM, Bray GA: Role of thyroid hormones in lipolysis. Amer J Physiol 210:1053~1058, 1966 21. Kaciuba-UScilko H, Greenleaf JE, Kozlowski S, et al: Thyroid hormone induced changes in body temperature and metabolism during exercise in dogs. Am J Physiol 229:260-264, 1975
THYROID AND TRAINING
22. Westgren
241
IN RATS
U, Burger A, lngemansson N, et al: Blood levels of
3.5.3’.triiodothyronine
and thyroxine:
Differences between children,
adults, and elderly subjects. Acta Med Stand 200:493-495, 23. Winder
WW.
Heninger
RW:
Effect
of exercise
levels of thyroid hormones in the rat. Am J Physiol 22
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Harris ARC,
27:1074-1083.
Fang SL, Azizi thyroid
suppressed TSH and refeeding Metabolism
KD,
Smallridge
secretion during on TSH
29:46-52.
RC. Osborne
I: I 139-l
R, et al: Nature
undernutrition:
responses to prolonged 1980
143, of
effect of fasting TRH
in the
rat.
on
Metabolism
1978
26.
Holm G, Jacobsson B. Toss L. et al: The etfect of physical
exercise on the regulation Burman
F et al: Effect of starvation
function
on tissue
1971 24.
25.
hypothalamic-pituitary
of beta adrenergic
late cyclasc in rat adipocytes.
Proc III
receptors and adeny-
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Oct. 9-l I) 1980 (in print)
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(Rome