Fatty acid synthesis and ketone body utilization by brown adipose tissue of the rat

Fatty acid synthesis and ketone body utilization by brown adipose tissue of the rat

244 Biochrmica et Blophysica Acta, 753 (1983) 244-248 Elsevier BBA 5 1485 FA’ITY ACID SYNTHESIS OF THE RAT RESPONSE JOHN WRIGHT TO COLD OR NUTRIT...

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244

Biochrmica et Blophysica Acta, 753 (1983) 244-248 Elsevier

BBA 5 1485

FA’ITY ACID SYNTHESIS OF THE RAT RESPONSE JOHN

WRIGHT

TO COLD OR NUTRITIONAL and LORANNE

Department of Clinical Biochemistry NE1 4LP (U.K.) (Received

May

AND KETONE

AGIUS

BODY UTILIZATION

BY BROWN

ADIPOSE

TISSUE

STATE?

*

and Metabolic

Medicine,

Royal

Victoria Infirmary,

Queen Victoria Road, Newcastle- upon -Tyne

1lth, 1983)

Key words: Brown adipose tissue; Fatty acid synthesis; Ketone body; Cold adaptation; Nutrition;

(Rat)

The effects of cold adaptation (exposure to 2-4°C for 2-3 weeks) on the incorporation of ‘H from 3H,0 and of D-3-hydroxy [3-‘4CIbutyrate into fatty acid in vivo in interscapular brown adipose tissue of the rat were examined. Fatty acid synthesis, measured with 3H,0, was increased in brown adipose tissue of cold-adapted rats pair-fed ad libitum but not in cold-adapted rats pair-fed to the same food intake as rats maintained at 1%20°C. Incorporation of D-3-hydroxybutyrate into fatty acid was increased in brown adipose tissue of cold-adapted rats, both when rats were fed ad libitum and when they were deprived of food for 48 h. Ketone bodies may be an important substrate for brown adipose tissue in the cold-adapted rat.

Introduction Brown adipose tissue of the rat is an important site of cold-induced thermogenesis in rats adapted to low ambient temperatures [l] and diet-induced thermogenesis in rats with an increased energy intake on a palatable diet [2]. The rate of fatty acid synthesis measured in vivo in brown adipose tissue is increased in cold-adapted rats [3] and it has been suggested that the increased rate of fatty acid synthesis may be related to thermogenesis [3,4]. In brown adipose tissue of rats maintained at warm ambient temperatures the rate of fatty acid synthesis is very sensitive to the nutritional state of the rat, being increased several-fold after administration of an oral load of glucose to rats fed ad libitum [5]. Rats increase their food intake when they are transferred to low ambient temperatures * To whom correspondence OOOS-2~/6U/83/$03.00

should be addressed.

0 1983 Elsevier Science

Publishers

B.V.

and allowed access to food ad libitum. A key question is whether the increased rate of fatty acid synthesis in cold-adapted rats is related to the ambient temperature per se or to the increased food intake in these rats. Brown adipose tissue is an important site of ketone body incorporation into fatty acid in both the rat fetus [6] and the adult rat maintained at warm ambient temperatures [7]. The turnover rate of ketone bodies increases during cold-adaptation in the rat [8]. This raises the question as to which tissues are responsible for the increased utilization of ketone bodies in the cold-adapted rat. The aims of the present study were: (1) to examine whether the increased rate of fatty acid synthesis in brown adipose tissue of cold-adapted rats is dependent on the increased food intake; (2) to investigate the effects of cold-adaptation on ketone-body incorporation into fatty acid by brown adipose tissue of rats in different nutritional states.

245

Materials and Methods 3H 2O and D-3-hydroxy [ 3-14C]butyrate were from Amersham International PLC (Amersham, Bucks, U.K.). Enzymes were from Boehringer (Mannheim, F.R.G.). Cofactors were from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Measurement of fatty acid synthesis with ‘H,O The rate of fatty acid synthesis in vivo was measured by the incorporation of 3H from 3H,0 into saponifiable fatty acid. The rats were injected with 3H,0 (5 mCi in 0.5 ml) intraperitoneally and they were anaesthetised and dissected after 60 min. A sample of aorta1 blood was taken for determination of plasma specific radioactivity and blood ketone bodies. The interscapular brown adipose tissue, epididymal white adipose tissue, liver, intestine and kidney were removed and weighed and tissue lipid was saponified and extracted as described in Ref. 9. The dried lipid extract was weighed and the radioactivity determined. The rates are expressed as pmol of 3H incorporated into tissue fatty acid/h per g wet wt. of tissue. Within the experimental period (60 min) only a small proportion of the triacylglycerols synthesized in liver is transported to peripheral tissues [lo]. Incorporation of D-3-hydroxy [3-‘4C]butyrate into lipid The incorporation of D-3-hydroxy [3-14C]butyrate into lipid in vivo was measured as described in Ref. 7. The rats were anaesthetized with diethyl ether for 3 min and D-3-hydroxy [3-14C]butyrate (0.11 pmol, 2.5 PCi in 0.3 ml) was injected into a femoral vein. Blood (0.3 ml) was collected from the tip of the tail at 2-min intervals for 12 rnin for determination of plasma radioactivity. The rats were anaesthetised and dissected at 20 min. Aorta1 blood was taken for determination of ketone bodies. The tissues were removed and weighed and tissue lipid was saponified and extracted as described in Ref. 9. The radioactivity in the dried lipid extract was expressed as a percentage of the dose injected per 100 g body weight incorporated per g wet wt. of tissue. Blood ketone bodies were determined in neutralized perchloric acid extracts [ 111.

Rats Male rats (body wt. 200-260 g) of the Albino Wistar strain bred in The Animal House of the University of Newcastle Medical School were used. They were fed on standard chow diet containing 4.3% crude oil, 22.7% crude protein and 55% carbohydrate, obtained from Special Diet Services (Witham, Essex, U.K.). In this study ‘warmadapted rats’ refers to rats that were maintained at 18-20°C throughout, ‘cold-adapted rats’ refers to rats that were maintained at 2-4°C for 2-3 weeks before the experiment. The food intake of the cold-adapted rats allowed access to food ad libiturn was between 2 and 2.5 times that of the warm-adapted rats. To investigate the effects of ambient temperature and food intake on lipogenesis measured in vivo with 3H,0, rats of the same body wt. were divided into three groups. One group was maintained at 18-20°C for a further 3 weeks and allowed access to food ad libitum (warm-adapted, fed ad libitum). The rats were caged singly and food intake was monitored daily. The second group was maintained for 3 weeks at 2-4°C and allowed access to food ad libitum (cold-adapted, fed ad libitum). The third group was maintained for 3 weeks at 2-4°C the rats were allowed access to food ad libitum during the first 2 weeks, and during the third week they were pair-fed to the same intake as the warm-adapted rats.

Results Incorporation of ‘H20 into fatty acid Fatty acid synthesis was measured by the incorporation of 3H from 3H,0 into fatty acid in vivo in warm-adapted and cold-adapted rats fed ad libitum and in cold-adapted rats pair-fed to the same intake as the warm-adapted rats. The rate of incorporation of 3H into fatty acid expressed per g wet wt. of tissue in the warm-adapted rats was 6-7-fold higher in brown adipose tissue than in liver, white adipose tissue or intestine and 13-fold higher than in kidney (Table I). In the cold-adapted rats fed ad libitum the rates of fatty acid synthesis in brown adipose tissue and liver were higher than in the warm-adapted rats (Table I). However, in the cold-adapted rats that had been pair-fed to the same food intake as the warm-adapted rats, the

246

TABLE I RATE OF INCORPORATION OF 3H,0 INTO FATTY ACID IN VIVO IN COLD-ADAPTED RATS FED AD LIBITUM OR PAIR-FED WITH WARM-ADAPTED RATS Rates of fatty acid synthesis are expressed as nmol of ‘H incorporated into saponified fatty acid/h per g wet wt. The results are mean +S.E. of the numbers of rats shown in parentheses. Values that are significantly different by Student’s f-test from those of the warm-adapted rats are shown: * P < 0.01 Tissue

Kidney Intestine White adipose tissue Brown adipose tissue Liver

State of rats Warm-adapted, fed ad libitum

Cold-adapted, fed ad libitum

Cold-adapted, pair-fed

(3)

(3)

(3)

6.OkO.3 12.4_+ 3.2 9.5 f 2.0 69.8 f 6.7 9.4 f 2.5

10.9*0.3 * 15.2kO.4 l3.1& 1.0 141.3rt2.7 * 21.3k2.2 *

5.3 + 8.6* 8.4+ 70.1+ 11.8+

rate of fatty acid synthesis in brown adipose tissue was not significantly different from the rate in the warm-adapted rats (Table I). Incorporation of D-3-hydroxy[3-‘4C]butyrate into fatty acid In warm-adapted rats fed ad libitum the incorporation of radioactivity into fatty acid in vivo after injection of D-3-hydroxy[3-‘4C]butyrate expressed per g wet wt. of tissue was highest in interscapular brown adipose tissue and intestine and lowest in heart, diaphragm muscle and brain (Table II). The incorporation of label into brown adipose tissue was 16fold higher than in white adipose tissue (Table II). These experiments provide information on the percentage of the circulating ketone bodies incorporated into fatty acid in the various tissues, but not on the ‘actual rates of fatty acid synthesis from ketone bodies. In the cold-adapted rats fed ad libitum the percentage incorporation of radioactivity into fatty acid was increased in brown adipose tissue, but not in the other extrahepatic tissues (Table II). Incorporation of label into hepatic fatty acid was increased in the cold-adapted rats. Since the liver has only a low activity of 3-oxoacid-CoA transferase (EC 2.8.3.5) the incorporation of label into hepatic fatty acid probably represents cytosolic formation of acetoacetyl-CoA via acetoacetyl-CoA synthetase. The higher incorporation of label in the liver of cold-adapted rats may be related to the higher rate of lipogenesis (Table I).

0.3 1.2 1.3 6.4 1.9

In rats that had been deprived of food for 48 h, incorporation of D-3-hydroxybutyrate into fatty acid in warm-adapted rats was 3-fold higher in interscapular brown adipose tissue than in intestine (Table III). In the 48-h-starved rats that had been cold-adapted for 2-3 weeks or exposed to cold for 48 h only (cold-stressed) the percentage incorporation of radioactivity into brown adipose tissue fatty acid was higher (58%) than in the

TABLE II PERCENTAGE INCORPORATION OF D-3-HYDROXY[3- “C]BLJTYRATE INTO FATTY ACID IN VIVO IN WARM-ADAPTED AND COLD-ADAPTED RATS FED AD LIBITUM Incorporation of 14C label into fatty acid is expressed as % of injected dose per 100 g body wt. incorporated/g wet wt. of tissue. The results are mean &S.E. of the numbers of rats shown in parentheses. Values that are significantly different by Student’s r-test from those of the warm-adapted rats are shown: * P -c 0.01 Tissue

Brain Heart Lung Diaphragm Kidney Intestine White adipose tissue Brown adipose tissue Liver

State of rats Warm-adapted

Cold-adapted

(4)

(4)

0.034* 0.006 0.013*0.003 0.095 f 0.037 0.027 + 0.006 0.054*0.010 0.416 f 0.073 0.039 + 0.005 0.525 f 0.076 0.084 f 0.026

0.03 1 f 0.006 0.025 + 0.008 0.080*0.014 0.046 + 0.0 13 0.058+0.011 0.390 * 0.110 0.043 * 0.011 0.982 k 0.106 * 0.242 + 0.079 *

241 TABLE III PERCENTAGE INCORPORATION OF D-3-HYDROXY[314C]BUTYRATE INTO FATTY ACID IN VIVO IN WARMADAPTED AND COLD-ADAPTED RATS STARVED FOR 48 h Incorporation of 14C label into fatty acid is expressed as I&of injected dose per 100 g body wt. incorporated/g wet wt. of tissue. The results are mean + S.E. of the numbers of rats shown in parentheses. Values that are significantly different by Student’s r-test from those of the warm-adapted rats are shown: * P < 0.05 Tissue

State of rats Warm-adapted

Cold-adapted

48-h coldexposed

(3)

(3)

(3)

Kidney

0.056 f 0.011

0.053 fO.O1O

0.63kO.016

Intestine White adipose tissue Brown adipose tissue Liver

0.117+0.020 0.059 * 0.008 0.335 + 0.003 0.16850.054

0.129 0.070 0.526 0.181

0.108 f 0.05 1 0.049 * 0.011 0.530 f 0.045 * 0.153*0.043

warm-adapted rats. There was no significant difference between the rats that had been coldadapted for 3 weeks and the rats that had been exposed to cold for 48 h only (Table III). Blood ketone bodies There was no significant difference in the concentration of blood ketone bodies (acetoacetate and D-3-hydroxybutyrate) in the ad libitum-fed warm- and cold-adapted rats (warm-adapted, 0.39 k 0.05; cold-adapted, 0.43 + 0.05 mM, mean + S.D.). The ketone body concentration was increased in the cold-adapted rats pair-fed to the same intake as the warm-adapted rats (0.80 k 0.17 mM). In the 48-h-starved rats the blood ketone body concentration was lower in the cold-adapted than in the warm-adapted rats (warm-adapted, 2.84 * 0.13; cold-adapted, 1.97 f 0.10 mM). Discussion Studies on various species of rodents [3,12- 141 have demonstrated an increased rate of fatty acid synthesis (measured with 3H,0 in vivo) in brown adipose tissue after exposure of the animals to low ambient temperatures for a few weeks. It has been widely emphasised that in rats that have been cold-adapted, brown adipose tissue is a major contributor to total body fatty acid synthesis (for a review see Ref. 4). It is noteworthy also that in rats that have not been exposed to low ambient temperatures, administration of insulin or an oral load

k 0.070 i 0.043 + 0.039 * f0.062

of glucose produces the same high rates of ‘H incorporation into brown adipose tissue fatty acid [5] as in cold-adapted rats allowed to feed ad libitum [3]. The present study shows that an increased rate of fatty acid synthesis during coldadaptation occurred when the rats increased their food intake, but not when they were maintained on a pair-fed regime. This tends to suggest that the increased rate of fatty acid synthesis in coldadapted rats allowed access to food ad libitum may be related to the increased food intake rather than to the ambient temperature per se. If it is assumed that thermogenesis was increased in the cold-adapted pair-fed. rats relative to the warmadapted rats, then fatty acid synthesis may not be an indication of thermogenesis. One notable example of a physiological state associated with increased thermogenesis in brown adipose tissue but a low rate of fatty acid synthesis is the neonatal rat. Brown adipose tissue is an important site of thermogenesis in the suckling neonatal mammal [ 15,161; however, lipogenesis decreases after parturition when the neonate feeds on the maternal milk, which is essentially a high-fat diet, but increases when the rats are weaned to the standard chow high-carbohydrate diet [ 17-201. When adult rats are transferred from standard chow diet to a mixture of chow and a palatable high-fat diet, which results in a constant carbohydrate but increased lipid intake, fatty acid synthesis in brown adipose tissue is decreased [21]. It is tempting to suggest that the rate of lipogene-

248

sis in brown adipose tissue is dependent on the carbohydrate and lipid intake; although the physiological state of the rat, e.g., pregnancy and lactation, may have an overriding influence [5,22]. If thermogenesis is increased in the cold-adapted pair-fed rats then these rats would be expected to be in negative energy balance. This may explain the increase in concentration of circulating ketone bodies that was observed in cold-adapted pair-fed rats but not in cold-adapted rats allowed to feed ad libitum. Studies with perfused liver from 18-hstarved rats suggested that ketogenesis is increased in cold-adapted rats [8]. In the present study the ketonaemia observed after 48 h starvation was lower in the cold-adapted than in the warmadapted rats. This may be the result of increased peripheral utilization of ketone bodies in the coldadapted rats. Cold-adaptation increased the percentage of the circulating ketone bodies that was directed to brown adipose tissue lipid (expressed per g wet wt. of tissue) both in the ad libitum fed state and also after 48 h starvation. This observation together with the hypertrophy of brown adipose tissue [ 161 and the increased turnover rate of ketone bodies [8] during cold-adaptation suggest that ketone bodies may be an important substrate for brown adipose tissue in the cold-adapted rat. Acknowledgement This work was supported abetic Association.

by the British

Di-

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