Non-thermal stress-induced modifications of fatty acids profiles in rat brown adipose tissue

J. therm. Biol. Vol. 17, No. 4/5, pp. 251-256, 1992 Printed in Great Britain. All rights reserved

0306-4565/92 $5.00+ 0.00 Copyright ~) 1992Pergamon Press Ltd

N O N - T H E R M A L STRESS-INDUCED MODIFICATIONS OF FATTY ACIDS PROFILES IN RAT BROWN ADIPOSE TISSUE ToMm OHNO,t KOJ~ OGAWA, 2 HIROSHI OHINATA, 3 TSUKASANOZU3 and AKIHIRO KUROSHIMA3. 1Laboratory of Nutrition Physiology, Hokkaido University of Education, 2Laboratory of Physical Education, Hokkaido Tokai University and 3Department of Physiology, Asahikawa Medical College, Nishikagura, Asahikawa 078, Japan (Received 25 January 1992; accepted in revised form 30 April 1992) Aia~-aet--l. The changes in fatty acids (FA) composition were studied in triglyceride and phospholipid fractions of interscapular brown adipose tissue (BAT) in cold-acclimated (5°C, 4 wk) (CA), repetitively immobilized (3 hr/day, 4 wk) (IM) and treadmill-running trained (T) (30 m/min, 30 min/day under 8° inclination, 4 wk) rats. 2. Fatty acids composition in triglyceride: in CA rats the saturated FA (SA) and polyunsaturated FA (PU) were higher, while monotmsaturated FA (MU) was lower. In IM rats SA was lower, while MU and PU were higher. Unsaturation index (UI) and PU/SA were higher in IM rats. In T rats SA and MU were lower, while PU was higher, UI and PU/SA were higher in T rats. 3. Fatty acids composition in phospholipid: in CA rats SA and PU were higher, while MU was lower. UI and PU/SA were higher. In IM rats SA and MU were lower, while PU was higher. UI and PU/SA were higher. In T rats MU was lower, but no changes were observed in UI and PU/SA. 4. These findings have indicated that non-thermal stress such as immobilization caused similar changes in FA composition of rat BAT, especially an increase in the extent of unsaturation in FA of phospholipid, to those previously reported and presently confirmed in CA animals. On the other hand, exercise training did not affect the extent of unsaturation in FA of phospholipid as assessed by UI and PU/SA. Key Word Index: Brown adipose tissue; fatty acids composition; triglyceride; phospholipid; repetitive immobilization stress; exercise training; cold acclimation

INTRODUCTION

stimulated in both stressand coldexposed animals (Bloom et al., 1973; Jansk~', 1978; Kuroshima et al., 1978, 1981). Moreover, physical exercise is known as another type of non-thermal stress which activates the sympathetic nervous system and glucagon secretion (Galbo, 1983), and exercise training has been shown to improve cold tolerance (Chin et al., 1973). Therefore, it is expected that exercise training could increase the non-shivering thermogenic capacity in BAT as cold acclimation. These findings suggest that non-thermal stress such as immobilization and exercise causes significant changes in the F A composition of BAT. This study was therefore carried out to find out whether repetitive intermittent immobilization or exercise training could modify the F A composition of BAT.

Brown adipose tissue (BAT) is the principal site for non-shivering thermogenesis, especially in coldacclimated small mammals such as rats (Foster and Frydman, 1979). This also seems to be the case for human neonates (Dawkins and Scopes, 1965). The main substrates for thermogenesis in BAT is fatty acid (FA) derived from endogenous triglyceride stores (Nichols and Locke, 1984). Several reports have indicated significant changes in F A composition of triglyeeride as well as phospholipid in BAT in coldacclimated rats (Moriya and Itoh, 1969; Ogawa et al., 1987, 1992; Riquier et al., 1976), suggesting a preferential utilization of specific F A and a significant role of F A composition of cellular membranes in BAT functions. Recently this tissue has also been shown to be activated by prolonged non-thermal stress such as immobilization, causing an increase in cold tolerance through enhanced non-shivering thermogenesis (NST) in this tissue (Kuroshima et al., 1984; Nozu et al., 1992b). The positive cross adaptation between cold and immobilization stress is suggested to be mediated by multiple common neuroendocrine factors such as catecbolamines, glucoeorticoids and glucagon, whose secretions have been shown to be

MATERIALS AND METHODS

*All correspondence should be addressed to: Akihiro Kuroshima, Department of Physiology, Asahikawa Medical College, Nishikagura, Asahikawa 078, Japan. 251

Study animals Adult male Wistar strain rats, weighing about 180g, were obtained from Shizuoka Laboratory Animal Centre, Hamamatu. Cold-acclimated (CA) rats were housed at 5 +_ I°C for 4 weeks. The stressed (IM) rats were subjected to stress for 4 weeks by 3 h daily immobilization with soft wire mesh on wooden board as described elsewhere (Butterfield and Rasche, 1975). The control (C) animals to these groups were housed at 25 + I°C and about 50% relative humidity for 4 weeks. The exercise-trained (T) rats were forced to run on a treadmill with electrical stimulation

252

Tome OHNOet al.

Table I. Effects of cold acclimation (CA) and repetitive immobilization (IM) on brown adipose tissue (BAT) weight, triglyceride (TG) and phospholipid (PL) in BAT Body weight (g) Initial C(11) CA (12) IM (10)

176±3.1 176±2.4 183 ± 3.0

BAT

Final

mg

278±3.8 250+3.0a 215 ± 4.5 ab

TG

per 100g body weight

193±7.8 475±17.8" 207 ± 11.5b

70±3.1 191±6.8" 96 ± 4.6 ab

mg/pad 104+6.9 165+9.6" 100 ± 7.7 b

PL per 100mg BAT 53+2.5 35±1.5' 48 ± 2.2 b

per 100mg BAT

mg/pad 4.6±0.22 15.3+0.63' 5.2 ± 0.57 b

2.4±0.06 3.2±0.08" 2.5 ± 0.14 b

Mean ± SEM C, controls. Numbers in parentheses indicate number of animals. "Significantly different vs C. bSignificantly different vs CA.

(Arakawa Kogyo Ltd, Tokyo) for 4 weeks (6 days/ week). Initial running time and speed was 10 rain and 10 m/min, respectively. The extent of exercise was gradually increased until 10 days, when the animals ran continuously for 30 min at 30 m/min under an 8° inclination. This running regime was continued for 4 weeks until end of training. The control for this group (TC) was placed on the treadmill without running for the same period as the trained group. All the experimental animals were placed under artificial lighting from 07.00 to 19.00 h and provided ad libitum with laboratory chow (Oriental MF, Oriental Yeast Co. Ltd, Tokyo) and tap water. Sampling o f tissues The animals were killed by decapitation without anaesthesia, taking care not to cause stress. IM and T animals were sacrificed on the day following the last immobilization or running. The interscapular BAT were removed, cleaned of any adhering tissue and kept at -70°C until analysed. Analytical methods. The tissue lipids were extracted according to the method of Islam et al. (1980); extraction ( x 2) by chloroform-methanol (2:1 v/v). The amounts of triglyceride and phospholipid in the extract were determined using the commercial kits of TG-Test Wako and PL-Test Wako (Wako Pure Chemicals Industries Ltd, Osaka), respectively. The same extract was used for FA analysis. Briefly, 4-5 ml of the extract were evaporated to dryness under nitrogen in a water bath at 30°C, then taken up

in 50/zl of extraction solution. 10/~1 of the solution was used directly for transesterification of triglyceride FA (Morrison and Smith, 1964). The remaining solution was applied to thin layer chromatography for separating phospholipid (Skipski and Barclay, 1969), and its FA was transesterified (Morrison and Smith, 1964). These transesterified FA solutions were dried under N2 in a water bath at 40°C, then taken up in 30/~1 of extraction solution for PL-FA, and 150300/al for TG, and analysed by a Hitachi Model 66330 gas chromatograph equipped with Hitachi Model 883A data processor. A 2 m glass column packed with Chromosorb W-AW-DMCS-FFAP (Gaskuro Kogyo Inc., Tokyo) was used as stationary phase and operated at 220°C. Quantitative standardization of the chromatograph was based on analysis of FA methyl esters standard mixture from NU-Chek-Prep, Inc. Calculations. FA composition was expressed as mol% for FA compositions and weight % for quantitative analysis. Unsaturation index (UI): the average number of double bonds per FA molecule as indicated by EMiNi/100 (Mi, mol% of each FA; Ni, number of double bonds of each FA). Arachidonate index (AI): arachidonate mol%/linoleate mol%. AI is equated with an effect on the elongation-desaturation pathway (Mak et al., 1983). Statistics. Statistical significance was determined by analysis of variance and Student's t-test, and the difference was assumed to be significant at P < 0.05. Multiple comparisons between the means after Table 2. Fatty acids compositions (mol o%) of BAT from

C12

C14

C16

C16-1

C18

C18-1

C18-2

C18-3

C20

C20-1

C

0.30 ±0.015

4.50 ±0.123

33.93 ±0.412

6.78 ±0.426

5.88 ±0.166

30.20 ±0.730

15.32 ± 1.013

0.74 ±0.048

--

1.24 ±0.071

CA

0.16 +0.004"

2.70 +0.033 a

38.79 +0.339'

1.33 ± 0.04(P

10.69 ± 0.151Y

22.03 ±0.185 a

20.26 ±0.294 a

0.71 ±0.011

0.01 ±0.013

1.77 _+0.041 a

IM

0.18 +0.011'

2.41 +0.135 a'b

25.11 -t-0.568a'b

1.37 ±0.172'

5.85 ±0.105 b

24.47 +0.208 a'b

32.51 +0.602 a'b

1.24 ±0.025 a'b

--

3.54 ±0.110 ''b

C

0.46 ±0.049

1.20 ±0.124

25.77 ±0.454

4.99 ±0.532

16.98 _+0.406

20.10 ±0.956

16.45 ± 1.149

0.25 ±0.036

--

0.51 ±0.058

CA

0.25 ±0.044'

0.40 ±0.042"

19.48 +0.456 a

0.32 +0.028 a

29.60 +0.342 a

8.72 +0.210 a

26.06 +0.513 a

0.22 -+0.006

--

0.24 _+0.020 ~

IM

0.29 ±0.056'

0.66 ±0.099 a

20.87 +0.078 a'b

0.59 +0.056 a

20.41 +0.310 ~'b

12.55 ±0.235 "b

27.38 ±0.475"

0.32 +0.016 "b

--

0.90 ±0.050 ''b

TG-FA

PL-FA

Cl4, myristate; C16, palm±rate; C16-1, palmitoleate; CI8, stearate; C18-1, oleate; C18-2, linoleate; C18-3, linolenate; C20-1, gadoleate; C20-2, PU, polyunsaturated FA; AI, arachidonate index (C20-4/C i 8-2); UI, unsaturation index. Each value consists of 10-12 samples. Mean ± SE. C, controls housed at 25°C for 4 weeks; TG, triglycerides; PL, phospholipids; FA, fatty acid. aSignificantly different vs C. bSignificantly different vs CA. - - N o t detected

Fatty acids profiles in rat BAT

253

Table 3. Effectof treadmill exercisetraining (T) on BAT weight, triglyceride(TG) and phospholipid (PL) in BAT Body weight (g) BAT TG PL per 100g per 100g per 100g Initial Final mg body weight mg/pad BAT mg/pad BAT TC ( 1 0 ) 161±2.7 283±2.7 175±11.2 61±3.4 116±11.5 65±2.6 4.1±0.28 2.4±0.13 T(8) 158±3.2 223 ± 6.4a 108 + 14.7a 48±5.9 51±9.0" 42_+2.7a 2.4+0.36 ~ 2.2±0.21 TC, controls for T. Legends are the same as in Table 1.

analysis of variance were performed by Ryan's test (Ryan, 1960). Abbreviations. C14, myristate; C16, palmitate; C16-1, palmitoleate; C18, stearate; C18-1, oleate; C18-2, linoleate; C18-3, linolenate; C20, arachidate; C20-1, gadoleate; C20-2, eicosadienoate; C203,bishomo-~-linolenic acid; C20-4, arachidonate; C22, behenate; C24, lignocerate; SA, saturated FA; M U , m o n o u n s a t u r a t e d FA; PU, polyunsaturated FA.

was lower, but its content was greater in C A than in I M rats. Phospholipid was greater in C A than in C rats. It was the same between C and IM rats, and greater in C A than in IM rats. FA composition in BAT triglyceride (Table 2). In C A rats SA and P U were higher, and M U was lower than in C rats. In I M rats PU, U I and P U / S A were greater, and SA and M U were lower than in C rats. M U , PU, U I and P U / S A were greater in IM than in C A rats. FA composition in BAT phospholipid (Table 2). In C A rats SA, PU, U I and P U / S A were higher, and M U and AI were lower than in C rats. In IM rats these values, except SA which did not change, showed similar changes to those in C A rats, as compared to those in C rats. M U and P U / S A were higher, and AI was lower in IM than in C A rats.

RESULTS

Effects of cold acclimation and immobilization stress Body and tissue weights (Table 1). The initial body weights did not differ a m o n g C, C A and IM rats. The final body weights were smaller in C A and I M rats as compared with C rats, indicating a suppressed weight gain in C A and IM rats as previously reported (Kuroshima et al., 1984; K u r o s h i m a and Yahata, 1985). B A T weight was markedly greater in C A rats, being 2.7 times greater in terms of per 100 g body weight, as compared with that in C rats. In IM rats B A T weight in terms of whole tissue pad did not differ from that in C rats, but the relative weight to 100 g body weight was significantly greater in IM rats than in C rats.

Effects of exercise training Body and tissue weights (Table 3). Initial body

Triglyceride and phospholipid in BAT (Table 1).

weight did not differ between T C and T rats; final body weight was smaller in T than in T C rats. This indicates that the exercise training suppressed the weight gain, as did C A and IM. B A T weight was smaller in T than in T C rats, but the relative weight to the body weight did not differ between T and T C rats.

Triglyceride level per 100 mg B A T was lower in C A rats than in C rats, but its content in whole tissue pad was greater in C A ones. This was due to the greater tissue weight in C A rats. There were no differences in triglyceride between C and IM rats. Triglyceride level

Triglyceride was smaller in T than in T C rats. Phospholipid content was smaller in T than T C rats, but its level did not differ between T and T C rats.

cold-acclimated(CA) and repetitivelyimmobilized(IM) rats C20-2 C20-3 C20-4 C22 C24

Triglyceride and phospholipid in BAT (Table 2).

SA

MU

PU

AI

UI

PU/SA

0.15 ±0.013 0.11 ±0.003' 0.31 __.0.007l'b

0.03 _+0.003 0.02 -_k_0.00l 0.16 ± 0.010-.b

0.44 ±0.030 0.80 +0.031a 1.01 + 0.040-'b

0.04 ±0.005 0.31 ±0.034a 0.31 + 0.007'

0.45 45.09 38.23 ±0.051 ±0.290 ±1.046 0.32 52.97 25.13 __.0.012a _0.393a +0.207i 1.59 35.39 29.38 ± 0.057~'b ± 0.626Lb ± 0.236''b

16.68 ±1.101 21.90 +0.318' 35.22 ± 0.627''b

0.03 ±0.001 0.04 _+0.00P 0.03 ± 0.001b

0.73 ±0.013 0.71 ±0.007 1.03 ± 0.013~'b

0.37 ±0.026 0.41 ±0.009 1.00 + 0.036a'b

0.34 +0.015 0.11

0.52 +0.048 0.16

8.05 ±0.393 11.46

0.15 ±0.014 0.17

4.24 ±0.188 2.81

48.79 ±0.710 52.71

25.59 ± 1.444 9.28

25.61 ± 1.557 38.01

0.50 +0.014 0.44

0.94 ±0.027 1.09

0.53 ±0.035 0.72

±0.006'

±0.021 m

±0.252'

±0.015

±0.100-

±0.520-

± 0.246"

±0.602'

±0.011'

±0.014 a

±0.018'

0.22

0.56

9.88

0.18

5.21

47,62

14.03

38.35

0.36

I.II

0.81

±0.007''b ± 0.034b ±0.271"b ±0.018 ±0.126a'b ±0.515b ±0.300-'b ±0.700- 4:-0.006Lb ±0.017' _+0.023Lb eicosadienoate; C20-3, bishomo-ydinolenic acid; C20-4, arachidonate; C24, lignoccrate; SA, saturated FA; MU, monounsaturated FA;

TOMmOrtsO et al.

254

C12

C14

C16

C16-1

C18

C18-1

Table4. Fattyacids compositions (tool%) C18-2 C18-3 C20 C20-1

29.16 _+0.811 25.31 _+0.586 a

18.26 0.85 _+0.960 _+0.040 29.34 1.27 _+1.028" _+0.041 a

--

18.54 _+1.010 23.12 +1.277a

--

TG-FA

TC T

0.32 _+0.016 0.22 _+0.008*

4.62 _+0.146 3.14 _+0.167a

32.08 _+0.438 27.39 +0.400~

5.59 6.26 _+0.486 _+0.205 1.87 6.35 -+0.367' -+0.173

--

1.57 _+0.067 2.78 +0.129°

PI.,-FA

0.35 1.00 25.24 3.06 _+0.038 _+0.081 + 0 . 5 9 3 -+0.535 0.56 1.23 26.07 0.59 T +0.132 + 0 . 2 0 8 -+1.043 -t-0.077 a Each valueconsistsof 6-10 samples. TC, controlsfor T rats. "Significantlydifferentvs TC. Legendsare the sameas in Table2. TC

19.08 18.30 -+0.658 +0.737 21.89 14.18 -+1.180 _+0.434 a

FA composition in B A T triglyceride (Table 4). In T rats PU, UI and PU/SA were higher, and SA, M U and AI were lower than in TC rats. FA composition in B A T phospholipid (Table 4). In T rats M U and AI were lower than in TC rats, but the indexes of unsaturation, UI and PU/SA, were not changed as compared to those in TC rats.

DISCUSSION

The present study shows that non-thermal stress such as repetitive intermittent immobilization causes less increase in body weight, greater mass of BAT, and increased arachidonate, PU, UI and PU/SA in the phospholipid fraction of BAT. We have previously reported similar changes in FA profiles of BAT phospholipid in cold-acclimated rats (Ogawa et al., 1992). Meantime, we have shown that the same non-thermal stress mentioned above causes positive cross adaptation to cold, enhancing cold tolerance through an increased thermogenic capacity in BAT (Kuroshima et al., 1984; Nozu et al., 1992b). BAT mitochondria in these animals resemble those in the cold-acclimated ones (Thomson et al., 1969; Kuroshima et al., 1984). In previous studies adopting the same experimental protocols we have observed that cold acclimation increases food intake (Kuroshima and Yahata, 1985), while repetitive immobilization (Kuroshima et al., 1984) as well as exercise training (Nozu et al., 1992a) does not affect food intake per 100 g body weight. We have also suggested that an enhanced NST in BAT during cold acclimation is closely related to cold itself but not the increased food intake (Kuroshima and Yahata, 1985). Thus, although the changes in food intake might affect the FA composition to some extent in the present study, it may be concluded that the changes in FA composition of BAT would result mainly from food-independent factors, i.e. cold and immobilization stress. It is well known that not only cold, but also non-thermal stressful stimuli stimulate sympathetic nerve activity, adrenocortical and glucagon secretions which are involved in BAT function (Felig et al., 1986; Jansk~, 1978; Kuroshima et al., 1978, 1981). Moreover, it was reported that noradrenaline could significantly stimulate phospholipid turnover in BAT (Senault et al., 1988). These findings suggest that noradrenaline is one of the factors which are responsible for changes in FA composition of

0.24 -+0.007 0.28 -+0.025

--

0.60 +0.089 0.73 +0.164

BAT phospholipid by cold exposure and immobilization stress. Although the physiological meaning of such changes in FA of BAT phospholipid remains to be explained, it has been claimed that a rise in unsaturated FA in phospholipid of tissue would exert beneficial changes in the cellular membrane functions such as ion transport, membrane permeability, activity of membrane-bound enzymes and resistance to cell damage (Spector and Yorek, 1985; Thomas et al., 1977). Therefore, the cold- and stress-induced changes in FA compositions of BAT phospholipid may not be accidental, but adaptive ones. Cold acclimation increased triglyceride content and decreased its level in BAT, while immobilizationstress did not affect either. In contrast to the similarity of the changes in the FA composition of BAT phospholipid between the cold-acclimated and stressed rats, those of BAT triglyceride differed between these groups; MU decreased and PU increased in both groups, while SA changed in the opposite direction. This may result from a different preference in utilization of MU and SA fraction of BAT triglyceride between the groups. FA profiles in triglyceride would also be modified by lipogenesis, since it has been shown that cold acclimation markedly enhances the rate of fatty acid synthesis in BAT (Trayhurn, 1979). Moreover, BAT has been shown to release a considerable amount of free fatty acids into circulation (Nedergaard and Lindberg, 1979), suggesting that BAT supplies free fatty acids as energy substrates for other tissues, as well as heat (McKee and Andrews, 1990). It is thus possible that a different triglyceride metabolism causes different FA profiles in BAT triglyceride between the groups. Physical exercise is also known as another type of non-thermal stress which activates the sympathetic nervous system and glucagon secretion (Galbo, 1983). Therefore, it is expected that exercise training would increase the NST capacity in BAT as cold acclimation. In contrast to immobilization stress, treadmill exercise has shown no effect (Wickler et al., 1987; Richard et al. 1986) or suppressive effect (Gohil et al., 1984) on the NST capacity in BAT. The same regime of exercise training as that in the present study also showed suppressed in vitro thermogenic responses of BAT (Nozu et al., 1992a). In the present study, exercise training decreased BAT weight, and both triglyceride and phospholipid content. The training changed FA composition in BAT triglyceride to a certain degree, but did not alter the indexes of

Fatty acids profiles in rat BAT of BAT from exercise-trained(T) rats C20-2 C20-3 C20-4

255

C22

C24

SA

MU

PU

AI

UI

PU/SA

0.17 +0.021 0.26

0.04 +0.004 0.08

0.51 ±0.025 0.71

0.03 ±0.003 0.22

0.55 ±0.047 1.08

43.86 ±0.349 38.40

36.32 ± 1.127 29.95

19.82 ± 1.033 31.65

0.03 ± 0.001 0.02

0.78 + 0.011 0.96

0.45 ±0,023 0.83

± 0.012"

± 0.005"

+ 0.045"

± 0.046"

± 0.069"

+ 0.652'

+ 0.848"

± I.119"

_ 0.001"

+ 0.017a

_+0,04(P

0.31 ± 0.017 0.22 + 0.015"

0.58 _+0.057 0.32 ± 0.032a

8.79 _+0.385 7.64 _+0.61 !

0.13 ± 0.016 0.19 + 0.029

3.78 ± 0.423 3.06 ± 0.259

49.58 +_0.896 52.91 ± 1.839

21.95 + 1.056 15.51 _+0.637"

28.47 _+1.310 31.58 ± 1.750

0.48 + 0.025 0.33 + 0.022"

0.97 + 0.027 0.95 _+0.046

0.58 _+0,034 0,61 _+0.051

unsaturation such as U I and P U / S A in B A T phospholipid. The decrease in B A T trigiyceride from the exercised animals may be a result o f the increased release and utilization o f triglyceride F A as an energy substrate for exercise, but not for an enhanced B A T thermogenesis. Judging from these results it is inferred that exercise training suppresses B A T thermogenesis to spare energy substrates for physical work. Other energy shortage-inducing conditions, such as food restriction (Rothwell and Stock, 1982) and lactation (Trayhurn and Richard, 1985), have been found to cause suppressed B A T thermogenesis that would permit an improved metabolic efficiency and provide obvious advantages for functions other than heat production. The mechanism involved in such changes in the B A T function remains obscure. An elevation o f body temperature during treadmill running may suppress the sympathetic stimulation of B A T (Banet et al., 1978; Fuller et al., 1977). It is also possible that during exercise when heat loss must be facilitated and blood flow to the skeletal muscle increases, the blood flow through B A T would be reduced. In spite o f reduced B A T thermogenesis in exercise-trained rats, it is interesting to note that treadmill exercise induces a greater cold tolerance by means o f an increased capacity to elevate the metabolic rate (Stremme and Hammel, 1967). Since there seems to be no enhancing effect of exercise training upon N S T , it is inferred that the enhanced capability to elevate metabolic rate in the cold is mainly due to an improved shivering capacity. REFERENCES

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