Suppression of fatty acids synthesis in brown adipose tissue of mice fed diets rich dm long chain fatty acids

602

Biochimica et Btiphysica, 665 ( 1981) 602-607 Elsevier/North-Holland Biomedical Press

BBA 57898

SUPPRESSION OF FATTY ACIDS SYNTHESIS IN BROWN ADIPOSE TISSUE OF MICE FED DIETS RICH IN LONG CHAIN FATTY ACIDS PIET A. VAN DEN BRANDT Dunn Nutrition Laboratory,

(Received

April 27th,

and PAUL TRAYHURN

*

University of Cambridge and Medical Research Council, Milton Road. Cambridge CB4 IXJ (U.K.)

1981)

Key words: Fatty acid synthesis: Cold acclimation; Long chain fatty acid; (Brown adipose tissue)

The influence of dietary lipid on fatty acid synthesis in brown adipose tissue has been investigated by feeding different high-fat diets to cold-acclimated mice for a period of 2 weeks. Fatty acid synthesis was measured in vivo with ‘HzO, and the fats used in the study were maize oil, beef tallow and medium chain triacylglycerol oil. In the mice fed the maize oil and the beef tallow diets fatty acid synthesis was inhibited in al1 tissues examined - interscapular brown adipose tissue, epididymal white adipose tissue, the liver and the carcass. Synthesis was more inhibited, however, in brown adipose tissue than in other fissues, and the inhibition was greater on the maize oil diet than on the beef tallow.‘The medium chain triacylglycerol oil had no inhibitory effect on fatty acid synthesis in any tissue, and hepatic synthesis was even elevated on this diet. It is concluded that fatty acid synthesis in brown adipose tissue, as in other lipogenic tissues, is subject to strong suppression by dietary long chain fatty acids, and particularly by linoleic acid.

Introduction Brown adipose tissue is the main site of nonshivering thermogenesis in hibernating animals, in many newborn mammals, and in cold-exposed adult rodents [l-S]. Recent evidence has also indicated that the tissue may be responsible for ‘regulatory’ diet-induced thermogenesis [6-81. The main substrates for thermogenesis in brown adipose tissue are fatty acids [9] and several studies with both rats and mice have now shown that the tissue has a very high capacity for fatty acid synthesis [lo-IS]. In other highly lipogenic tissues - the liver, white adipose tissue, and the lactating mammary gland - fatty acid synthesis is strongly influenced by the quantity and nature of the dietary lipid [16-271. An indication that lipogenesis in brown ,adipose tissue may also be subject to strong dietary regulation comes from the observation that fatty acid synthesis in the tissue is

* To whom

correspondence

OOOS-2760/81/0000-0000/$02.50

should

be addressed.

0 1981 Elsevier/North-Holland

very low in suckling mice [ 121, where the fat intake from milk is high. In contrast, a rapid increase in fatty acid synthesis in brown adipose tissue has been observed in rats following a single oral administration of medium chain triacylglycerol oil, while no effect was noted with long chain triacylglycerol [ 14,281. In the present experiments we have investigated the long-term dietary regulation of fatty acid synthesis in brown adipose tissue by feeding high-fat diets of widely differing fatty acid composition to cold-acclimated mice for a period of 2 weeks. The fats used were beef tallow, maize oil (predominantly linoleic acid) and medium chain triacylglycerol oil. The experiments were performed on cold-acclimated mice because of the very high rates of fatty acid synthesis which occur in brown adipose tissue during chronic exposure of rodents to the cold [lo- 131. Materials and Methods Animals. Male mice of the C57BL/lOScSn strain were obtained from the Department of Pathology,

Biomedical

Press

603

University of Cambridge, and used at 4-5 months of age. At the start of the experiments the mice were caged singly in a room at 4 2 l”C, with a 12 h light/ 12 h dark cycle (light period from 07.00 to 19.00 h). For the first week of the acclimation period all the animals were given the ‘control’ low-fat diet (SpillersSpratts Rodent Breeding Diet 1, Spratts Patent Ltd., Barking, U.K.) After 1 week the mice were divided into four groups of equal mean weight, and one group was continued on the low-fat diet while the other three groups were transferred to the high-fat experimental diets. The animals were given the experimental diets for 2 weeks, during the last 5 days of which food intake was measured (with a correction for spillage) and faeces were collected. Diets. The control diet contained 3.4% lipid (manufacturer’s data) with the major fatty acids being c16:

0

(21%),

c18:

0

(15%),

cl8

: 1 (38%)

and

Cra : a (20%). The high-fat diets were prepared as described by Kirtland et al. [29] and Cryer et al. [30]; 200 g fat, 100 g casein and 100 mg vitamin E were mixed with 700 g of the powdered control diet. The final lipid content of the experimental diets was 22.4%. The medium chain triacylglycerol oil was obtained from Cow and Gate Ltd. (Trowbridge, U.K.), and the main fatty acids in the oil were Ca : o (56%) and Cl0 : o (40%). The fatty acid composition of the maize oil and beef tallow was determined by gasliquid chromatography [31] using a Pye Unicam Series 204 chromatograph (Pye Unicam, Cambridge, U.K.) The major fatty acids in the beef tallow were cl6

: 0 + cl6

: 1 (30%),

c18 : 0 (19%)

and

cl8

: 1

while the maize oil contained Cre : o + cl6 : 1 (11%) Crs : 1 (26%) and Crs : 2 (61%). The energy content of the diets and faeces was determined using an Adiabatic Bomb Calorimeter, Model CB-100 (Gallenkamp Ltd., London, U.K.) Fatty acid synthesis. Rates of fatty acid synthesis were determined in vivo by measuring the incorporation into fatty acids of 3H from 3Hz0 [32-361, essentially as described previously [ 11,371. Each mouse was injected intraperitoneally, between 08.45 and 09.45 h, with 500 @i of 3Hz0 (Radiochemical Centre, Amersham, U.K.) in 50 1.11 saline, and returned to the 4°C room. After 60 mm blood was collected from the jugular vein, and interscapular brown adipose tissue (trimmed free of white adipose tissue), the liver and the epididymal fat pads rapidly (41%),

removed. The dissected tissues and the carcass were then frozen in liquid nitrogen. Tissue samples were saponified in ethanolic KOH, acidified, and fatty acids extracted with light petroleum according to the method of Stansbie et al. [38]. The carcass was homogenised first before sampling for saponification [37]. Blood was centrifuged, and the plasma then deproteinised with 10% (W/V) trichloracetic acid. Measurement of radioactivity. Deproteinised plasma and tissue fatty acids were dissolved in a toluene/Triton X-100 (2 : 1, v/v) -based scintillation solution, and radioactivity measured in a Packard TriCarb 2425 liquid scintillation counter with corrections for both quench and background counts. Students’ unpaired t-test was used to assess the statistical significance of differences between groups. Results The mean body weight of each group of mice was initially the same, and no substantial change in weight occurred on any of the dietary regimes (Table I). The food intake in g per day was generally lower on the high-fat diets than on the low-fat diet. However, since the energy density as well as the ‘digestibility’ of all three high-fat diets was greater than that of the lowfat diet, the ‘digestible energy intake’ of the mice fed the high fat diets was higher than with the controls (Table I). Mice on the maize oil and medium chain triacylglycerol oil diets showed changes relative to the animals on the control diet in the weight of the main tissues in which fatty acid synthesis was measured (Table I). The weight of the interscapular brown adipose tissue was decreased in the animals fed the maize oil, while both interscapular brown adipose tissue and the epididymal fat pads were smaller in the medium chain triacylglycerol group. The mice fed the medium chain triacylglycerol oil diet had, however, a significant increase in liver weight. No changes in tissue weight were obtained on the beef tallow diet. Table II shows the rates of fatty acid synthesis obtained on the various diets. In the animals fed the lowfat diet the highest rate of synthesis was found in brown adipose tissue, and, as observed previously in cold-acclimated mice of a different strain [12], the rate in this tissue was very much higher than in other tissues. The mice fed the maize oil and beef tallow diets had substantially lower rates of synthesis com-

604 TABLE I BODY WEIGHT, TISSUE WEIGHTS AND FOOD INTAKE OF COLD-ACCLIMATED MICE FED DIFFERENT HIGH-FAT DIETS Mice were acclimated at 4*C for 3 weeks, and during the last 2 weeks they were fed the high-fat experimental diets: measurements of food intake and digestible energy intake were made for the last 5 days on the diets. For full experimental details see the text. The results are expressed as mean values f S.E. for seven animals on each diet. Low fat

Body wt. (g) Interscapular brown adipose tissue wt. (mg) Liver wt. (g) Epididymal white adipose tissue wt. (mg) Food intake (g/day) Digestible energy intake

@J/day)

Beef tallow

Maize oil

33.8 f

32.1 + 0.9

0.5

32.0

Medium chain triacylglycerol oil .--. 31.7 f

ir 0.6 a

0.5 b

170 +9 1.98 + 0.04

136 L 8b 1.89It 0.06

1.51 i I 1.84 _+ 0.07

f 4d 129 2.27 + 0.07c

219 + 25 9.2 i 0.3

219 C 16 7.9 k 0.4 b

i 23 254 8.2 t 0.2 c

_c19’ 113 8.8 + 0.2

153

154

129

t4

+ 7b

a P < 0.05, b P < 0.02, c P < 0.01, d P < 0.001, - compared with the control mice

pared with the control animals in all the tissues examined. On the beef tallow diet the fatty acid synthesis rates in the liver, epididymal fat pads and the carcass were decreased to apprbximately 40% of the rates in the same tissues of the low-fat-fed animals, while the synthesis rate in brown adipose tissue was decreased to only 24% of the control. The synthesis rates obtained on the maize oil diet were 67% of those on the low fat diet for the liver, 28% for the epididymal’ fat pads, and 42% for the carcass. The synthesis rate on the maize oil diet in brown adipose

_+ 3d

168

i

4d

fed the low-fatdiet.

tissue was, however, only 11% of that of the low fat control. The rates of fatty acid synthesis in the mice fed the medium chain triacylglycerol diet were similar to those obtained on the low-fat diet for all tissues except the liver; hepatic synthesis was substantially higher in the animals fed the medium chain triacylglycerol oil. The effect of each of the dietary regimes on whole-body fatty acid synthesis, and the total synthesis in each tissue is shown in Table III. Whole-body

TABLE II THE EFFECT OF FEEDING DIFFERENT HIGH-FAT DIETS ON THE RATE OF FATTY ACID SYNTHESIS IN BROWN ADIPOSE TISSUE AND OTHER TISSUES OF COLD-ACCLIMATED MICE Mice were acclimated at 4’C for 3 weeks, and during the last 2 weeks they were fed the high fat experimental diets. 3H20 (500 yCi) was injected intraperitoneally and tissues removed 60 min later. For full experimental details see the text. The results are expressed as mean values + S.E. for seven animals on each diet. Fatty acid synthesis (ggatom 3H incorporated/h

Interscapular brown adipose tissue Liver Epididymal white adipose tissue Carcass

Low fat

Maize oil

358.1 i: 95.6 42.3 _+ 4.0 24.9 I 6.9 17.92 4.0

39.8 28.4 6.9 7.5

-t 8.6 r_t2.1 rt 1.9 4 0.5

c c a a

per g)

Beef tallow

Medium chain triacylglycerol oil

84.4 17.1 9.8 7.3

335.1 109.1 28.5 19.5

t i f rt

16.9 b 2.1 d 2.4 0.7 a

a P < 0.05, b P < 0.02, c P < 0.01, d P < 0.001 - compared with the control mice fed the low-fat diet.

f 36.7 -’ 4.1 d -c 5.5 + 1.2

60.5

TABLE III THE EFFECT OF FEEDING DIFFERENT HIGH-FAT DIETS ON WHOLE:-BODY FATTY ACID SYNTHESIS, AND THE TOTAL SYNTHESIS IN INTERSCAPULAR BROWN ADIPOSE TISSUE AND OTHER TISSUES OF COLD-ACCLIMATED The total fatty acid synthesis was calculated for each tissue from the synthesis per g (Table II) and the tissue weight. The results are expressed as mean values -CSE. for seven animals on each diet. The percentage contribution of each tissue to whole-body synthesis is given in parentheses. Fatty acid synthesis (pgatom 3H incorporated/h Low fat

Interscapu~r brown adipose tissue Liver Epididymal white adipose tissue Carcass Whole-body

59.3 + 14.8 (7.9) 83.8 Z!Z 8.3 (13.3) 4.8 t 1.1 (0.6) 564.6 t 125.1 (78.2) 712.6 t 147.4 (100)

per tissue)

Maize oil

5.65 53.7 i

Beef tallow

1.3 c (1.9) 4.6 c (18.8)

13.2* 2.7 c (4.8) 31.7 2 4.4d (11.9)

1.5 c 0.5 a (0.5) 224.8 + 15.7 b (78.9) 285.6 -c 21.1 b (100)

2.4 i 0.6 (0.9) 219.0 + 23.9 b (82.4) 266.3 + 29.0 b (100)

~edlum chain triacylglycerol oil

42.8 + 4.0 (4.91 246.5 i 9.7 d (28.9) 2.9 + 0.5 569.2 + 37.0 861.4 k 43.4

(0.3) (65.8) (100)

a P < 0.05, bP < 0.02, cP < 0.01, d P < 0.001 - compared with the control mice fed the low-fat diet.

synthesis on the maize oil and beef tallow diets was appro~ate~y 40% of that on the low-fat diet. The proportion of whole-body synthesis attributable to the liver and to the carcass was not substantially different in the maize oil, beef tallow and low-fat groups; in each case the carcass accounted for over three-quarters of the total synthesis. There was, however, a major difference between the groups in the proportion of whole-body synthesis attributable to brown adipose tissue. Interscapular brown adipose tissue accounted for 7.9% of whole-body synthesis on the low-fat diet, but for only 4,8 and l-9%, respectively, on the beef tallow and maize oil diets. The total fatty acid synthesis in interscapular brown adipose tissue of the mice fed the control diet was 4.5 times that of the same tissue on the beef tallow diet and 10.6 times that on the maize oil diet. hole-body fatty acid synthesis was not decreased by the medium chain triacylglycerol diet. The proportion of whole-body synthesis in the liver was, however, greater on this diet than on the low-fat diet (28.9 versus 13.3%), while the proportions in the carcass and in interscapular brown adipose tissue were decreased. Discussion The present experiments demonstrate that fatty acid synthesis in brown adipose tissue of coldaccli-

mated mice is markedly inhibited by feeding diets rich in long chain fatty acids. Similar results have been obtained in mice kept at normal environmental temperatures (Van den Brandt, P. and Trayhurn, P,, unpub~shed data), and the effects seem to be the result of a long-term adaptation since fatty acid synthesis in brown adipose tissue of both virgin and lactating rats has been shown not to be inhibited following a single oral administration of glycerol trioleate [ 14,283. The present experiments also indicate that fatty acid synthesis in brown adipose tissue is more sensitive to dietary lipid than in other tissues. Consequently, dietary lipid may be preferentially channelled to brown adipose tissue during high rates of ~e~ogenes~s, and this may be ~portant if the tissue is to play a major role in ‘regulatory’ dietinduced the~ogenesis [G-8]. The suppression of fatty acid synthesis in brown adipose tissue was greater on the maize oil than on the beef tallow diet, and this suggests that fatty acid synthesis in this tissue may be particularly sensitive to dietary linoleic acid, or perhaps to polyunsaturated fatty acids in general. Hepatic fatty acid synthesis is generally considered to be inhibited more by polyunsaturated than by other fatty acids [17-19,22,23,25, 271, but this was not observed in the present study either in the cold-acclimated mice or in mice maintamed under normal environmental conditions. The

606

possible explanations for this difference include species and strain differences in the animals, the length of time on the diets, and differences in the fatty acid composition of the various control diets. In contrast to the results obtained on the maize oil and beef tallow diets, a diet rich in medium chain triacylglycerol oil had no inhibitory effect on fatty acid synthesis in any tissue, and hepatic synthesis was even elevated in comparison with the low-fat diet. However, after a single ~tragast~c load of medium chain triacyiglycerol oil both virgin and lactating rats have been shown to exhibit a marked increase in fatty acid synthesis in brown adipose tissue [ 14,281; hepatic synthesis was also increased by medium chain triacylglycerol oil in the same experiments. Part of the explanation for the higher rates of fatty acid synthesis on the medium chain triacylglycerol diet, relative to the other high-fat diets, may be in some elongation of the medium chain fatty acids. Alternatively, dietary medium chain triacylglycerol oil may be largely oxidized in the liver, leading to a requirement for fatty acid synthesis in medium chain triacyiglycerol-fed mice which is generally similar to that of animals on a low-fat diet. It should be noted that the mice fed the high-fat diets consumed less carbohydrate than those given the low-fat diet, but this is unlikely to in~uence the interpretation .of the present experiments since other studies have shown that a decrease in carbohydrate intake is not a significant factor in the suppression of fatty acid synthesis by dietary lipid [24,2.5,27]. Furthermore, changes in carbohydrate intake cannot explain either the differences observed between the maize oil and beef tallow diets or the particular sensitivity of brown adipose tissue to dietary lipid. Acknowledgements

References 1

8 9 10 11 12 13 14 15 16

17 18 19 20

21 22

We are very grateful to Dr. M.I. Gurr (N.I.R.D., Reading, U.K.) for much helpful advice. We are also grateful to Dr. Gurr and to Drs. W.P.T. James and D.R. Fraser for commenting on the manuscript, and to Mrs. B.A. Sutcliffe and Mr. S.E. Olpin for help with the gas-liquid chromatography. P.V.D.B. was on leave from the Department of Human Nutrition, Agricultural University of Wageningen (The Netherlands).

23

24 25 26 21

Smith, R.E. and Horwitz, B.A. (1969) Physiol. Rev. 49, 330-425 Jansky, L. (1973) Biol. Rev. 4885-132 Foster, D.O. and Frydman, M.L. (1978) Can. J. Physiol. Pharmacol. 56,l lo-122 Foster, D.O. and Frydman, M.L. (1979) Can, J. Physiol. Pharmacol. 57,257-270 Tburlby, P.L. and Trayhurn, P. (1980) Pfliigcrs Arch, 385,193-201 Rothwell, N.J. and Stock, M.J. (1979) Nature (Lond.) 281,31-35 Brooks, S.L., Rothwell, N.J., Stock, M.J., Goodbody, A.E. and Trayhurn, P. (1980) Nature (Lond.) 286, 274216 Rothweil, N.J. and Stock, M.J. (1981) Pfliigers Arch. 389,237-242 Nicholls, D.G. (1979) B&him. Biophys. Acta 549, l-29 McCormack, J.G. and Denton. R.M. (1977) Biochem. J. 166,627-630 Trayhurn, P. (1979) FEBS Lett. 104, 13-16 Trayhurn, P. (1981) Biochim. Biophys. Acta 664, 549560 Ram, EA., Salmon, D.M.W. and Hems, D.A. (1979) FEBS Lett. 108,33-36 Agius, L. and Williamson, D.H. (1980) Biochem. J. 190, 477-480 Shimazu, T. and Takahashi, A. (1980) Nature (Lond.) 284,62-63 Romsos, D.R. and Leveille, G.A. (1974) in Advances in Lipid Research (Paoletti, R. and Kritchevsky, D., eds.), Vol. 12, pp. 97-146, Academic Press, New York Clarke, S.D., Romsos, D.R. and Leveihe, G.A. (1977) 3. Nutr. 107,1170-l 181 Clarke, S.D., Romsos, D.R. and Leveille, G.A. (1977) J. Nutr. 107.1277-l 287 Clarke, SD., Romsos, D.R. and LeveiBe, G.A. (1977) J. Nutr. 107,1468-l 476 Waterman, R.A., Romsos, D.R., Tsai, A.C., Miller, E.R. and Leveille, G.A. (1975) Proc. Sot. Exptl. Biol. Med. 150,347-351 Romsos, D.R., Muiruri, K.L., Lin, P.-Y. and Leveihe, G.A. (1978) Proc. Sot. Exptl. Biol. Med. 159,308312 Bartley, J.C. and Abraham, S. (1972) Biochim.Biophys. Acta 280,258-266 Jeffcoat, R. (1979) in Essays in Biochemistry (Campbell, P.N. and Marshall, R.D., eds.), Vol. 15, pp. l-36, Academic Press, London Cawthorne, M.A. and Cornish, S. (1979) Int. J. Obesity 3,83-90 Triscari, J., Hamilton, J.G. and Sullivan, A.C. (1978) J. Nutr. 108,815-825 Grigor, M.R. and Warren, S.M. (1980) Biochem. J. 188, 61-65 Herzberg, G.R. and Janmohamed, N. (1980) Br. J. Nub. 43,571-579

607 28 Agius, L. and WiLliamson, D.H. (1980) Biochem. J. 192, 361-364 29 Kirtland, J., Gurr, M.I. and Widdowson, E.M. (1976) Nutr. Metab. 20,338-350 30 Cryer, A., K&land, J., Jones, H.M. and Gurr, M.I. (1978) Biochem. J. 170,149-172 31 Hartman, L. and Lago, R.C.A. (1973) Lab. Practice 22, 47.5-477 32 Fain, J.N. and Scow, R.O. (1966) Am. J. Physiol. 210, 19-25

33 Windmueller, H.G. and Spaeth, A.E. (1966) J. Bioi. Chem.241,2891-2899 34 Windmueller, H.G. and Spaeth, A.E. (X967) Arch. Biothem. Biophys. 122,362-369 35 Jungas, R.L. (1968) Biochemistry 7,3708-3717 36 Lowenstein, J.M. (1971) J. Biol. Chem. 246,629-632 37 Trayhum, P. (1980) Biochim. Biophys. Acta 620, lo-17 38 Stansbie, D., Brownsey, R.W., Crettaz, M. and Denton, R.M. (1976) Biochem. J. 160,413-416