Fatty acid synthesis from amino acids in sheep adipose tissue

Fatty acid synthesis from amino acids in sheep adipose tissue

Comp. Biochem. Physiol. Vol. 82R, No. 1, pp. 133-136, 1985 Printed in Great Britain 0305-0491/85 $3.00+0.00 © 1985 Pergamon Press Ltd F A T T Y ACID...

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Comp. Biochem. Physiol. Vol. 82R, No. 1, pp. 133-136, 1985 Printed in Great Britain

0305-0491/85 $3.00+0.00 © 1985 Pergamon Press Ltd

F A T T Y ACID SYNTHESIS F R O M A M I N O ACIDS IN SHEEP ADIPOSE TISSUE RICHARD G. VERNON, ERIC FINLEY and ELEANOR TAYLOR The Hannah Research Institute, Ayr, Scotland KA6 5HL, UK (Tel: 0292-76013) (Received 20 February 1985)

Abstract--1. The rates of incorporation of ~4C from ~4C labelled acetate, glucose, alanine, leucine, isoleucine and valine into fatty acids has been measured in perirenal adipose tissue from foetal lambs and 8-month-old sheep, and into both fatty acids and acylglycerol glycerol in adipose tissue from 3-year-old sheep and 220-240 g female rats. 2. Rates of incorporation of ~4C from amino acids into fatty acids were much lower in adipose tissue from sheep (at all three ages) than from rats, whereas rates of incorporation of ~4C into acylglycerol glycerol were either greater in sheep adipose tissue or the same as in rat adipose tissue. 3. The rate of incorporation of ~4C from amino acids into fatty acids decreased in the order leucine > alanine > isoleucine > valine in adipose tissue from rats and foetal lambs, and in the order leucine > alanine = isoleucine > valine in adipose tissue from 8-month- and 3-year-old sheep. 4. Amino acids make a very small contribution to fatty acid synthesis in adipose tissue from sheep at all stages of development examined while fatty acids are a minor product of amino acid metabolism in sheep adipose tissue. 5. The study provides further evidence for an important role for ATP-citrate lyase in restricting the utilization of acetyl-CoA generated in the mitochondria for fatty acid synthesis.

INTRODUCTION Although glucose is the major source of carbon for fatty acid synthesis in rat adipocytes, a variety of other substances can also provide carbon for this purpose, including amino acids (Feller, 1965). Sheep, and other ruminants, differ from rats in that acetate is the major source of carbon for fatty acid synthesis with minor contributions from other precursors including glucose, lactate, propionate and /3-hydroxybutyrate (Vernon, 1980). The use of amino acid carbon for fatty acid synthesis has not been investigated in sheep adipose tissue. In this study we compare the rate of fatty acid synthesis from alanine, leucine, isoleucine and valine with that from glucose and acetate in adipose tissue from foetal lambs, growing sheep and mature sheep and also young adult rats. The three groups of sheep were selected to cover three stages in development. In the foetus, placental transfer of amino acids exceeds their requirement for protein synthesis (Battaglia and Meschia, 1978; Girard et al., 1979) leaving a surplus for oxidation or other purposes; also, glucose and lactate are more important as precursors of fatty acid synthesis at this stage of development (Robertson et al., 1981). The 8-month-old sheep were still growing so amino acids might be expected to be used preferentially for protein synthesis in muscle whereas the 3-year-old sheep had reached full size with respect to muscle and skeletal growth. The rats used were roughly equivalent in age to the 8-month-old sheep.

100 days of pregnancy gradually increasing to 600 g per day by 125 days of pregnancy. The pregnant ewes and their foetuses were killed with a captive-bold humane killer at about 120 days of gestation while sheep at about 8 months and about 3 years of age were anaesthetised via a jugular catheter (Vernon et al., 1981) and samples of tissue removed prior to slaughter. Samples of perirenal adipose tissue were taken from the sheep and placed immediately in 0.15M NaC1 at 37°C. Pieces of adipose tissue weighing about 5 mg were cut with scissors and preincubated for about 20 min in Medium 199 (Gibco, Biocult Ltd, Paisley, Scotland) containing insulin (100ng/ml; Sigma UK Ltd), antibiotics (Robertson et al., 1982) and buffered with 25 mM Hepes (4-(2-hydroxymethyl)-l-piperazine-ethane sulphonic acid) pH 7.3. Medium 199 is a tissue culture medium and its constituents include glucose, acetate and amino acids (for concentrations see Table 1). The pieces of adipose tissue were incubated in Medium 199 for 2 hr at 37°C in a shaking water bath in the presence of either [l-14C]acetate; [U14C]glucose; [U-L4C]L-leucine; [U-14C]L-isoleucine; [U~4C]L-valine or [U-14C]L-alanine (Amersham International, UK). Following incubation, the pieces of adipose tissue were removed and the amount of ~4Cincorporated into fatty acid and acylglycero', glycerol was determined as described previously (Vernon et al., 1981a). The volume and number of adipocytes per g of tissue were determined on other pieces of adipose tissue as described previously for foetal (Vernon et al., 1981b) and adult sheep (Robertson et al., 1982). Results were analysed using Student's t-test for paired and unpaired observations as appropriate.

RESULTS

MATERIALS AND

METHODS

Sheep used were Cheviots. The non-pregnant animals were fed 400 g of a cereal mix per day plus hay ad libitum; the pregnant ewes were grazing in an open field and were given a supplement of the cereal mix of 200 g per day from

The concentration of the various precursors used in Medium 199 and in sheep plasma are given in Table 1. Adipocyte mean cell volumes were 6 + 2, 279 + 84, 1276 _+ 90 and 417 _ 25 pl for foetal lambs, 8-month-old sheep, 3-year-old sheep and adult rats, 133

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Table 1. Concentration of precursors used in Medium 199 and sheep plasma Precursor Acetate Glucose L-Alan±he L-Leucine L-Isoleucine t-Valine

Medium 199 (mM) 0.60 5.50 0.28 0.46 0.15 0.21

Sheep plasma (mM) 0.6-1.0"~ 3-4 J 0.10 1 0.15 0.10 [ 0.27 J

r e s p e c t i v e l y (results are m e a n + S E M o f 4 o r 5 o b s e r vations). T h e rate o f f a t t y acid s y n t h e s i s f r o m a c e t a t e v a r i e d with age, t h e rate b e i n g h i g h e s t in a d i p o s e t i s s u e f r o m 8 - m o n t h - o l d a n i m a l s ( T a b l e 2). A c e t a t e w a s b y f a r t h e b e s t p r e c u r s o r for f a t t y acid s y n t h e s i s in a d i p o s e tissue f r o m b o t h 8 - m o n t h - o l d a n d 3 - y e a r - o l d s h e e p ( T a b l e s 2 a n d 3); a c e t a t e a n d g l u c o s e c a r b o n m a d e s i m i l a r c o n t r i b u t i o n s to f a t t y acid s y n t h e s i s in a d i p o s e tissue f r o m foetal l a m b s , d u e to t h e m u c h l o w e r rate o f f a t t y acid s y n t h e s i s f r o m a c e t a t e a t this s t a g e of development compared with older animals (Table 2). F o r b o t h 8 - m o n t h - o l d a n d 3 - y e a r - o l d s h e e p t h e rate o f f a t t y acid s y n t h e s i s f r o m t h e o t h e r p r e c u r s o r s decreased in the order glucose > leucine > a l a n i n e = i s o l e u c i n e > valine. F o r t h e foetal l a m b s t h e s e q u e n c e o f p r e f e r e n c e (in d e c l i n i n g o r d e r ) w a s g l u c o s e > a l a n i n e = l e u c i n e > i s o l e u c i n e > valine: at this s t a g e o f d e v e l o p m e n t t h e relative c o n t r i b u t i o n s o f g l u c o s e a n d a l a n i n e were s i g n i f i c a n t l y h i g h e r t h a n for o l d e r s h e e p ( T a b l e 3). W i t h t h e e x c e p t i o n o f a l a n i n e , t h e rate o f f a t t y acid s y n t h e s i s f r o m a m i n o a c i d s w a s s i g n i f i c a n t l y ( P < 0.05) l o w e r in a d i p o s e t i s s u e f r o m foetal t h a n o l d e r s h e e p ( T a b l e 2). F o r rat a d i p o s e tissue, t h e rate o f f a t t y acid s y n t h e s i s f r o m a c e t a t e w a s s i m i l a r to t h a t o f

References Vernon (1980) Bergman and Heitmann (1978)

8 - m o n t h - o l d sheep; h o w e v e r , r a t e s f r o m all o t h e r p r e c u r s o r s were m u c h h i g h e r t h a n in s h e e p a d i p o s e tissue, a l t h o u g h t h e s e q u e n c e o f p r e f e r e n c e (in decreasing order), glucose > leucine > alanine > i s o l e u c i n e > v a l i n e w a s t h e s a m e as t h a t for foetal l a m b s . F a t t y acid s y n t h e s i s w a s g r e a t e s t f r o m g l u c o s e in r a t a d i p o s e tissue. T h e rate o f i n c o r p o r a t i o n o f ~4C f r o m ~4C-labelled p r e c u r s o r s i n t o a c y l g l y c e r o l glycerol w a s s i m i l a r for a c e t a t e , a l a n i n e , i s o l e u c i n e a n d v a l i n e in a d i p o s e t i s s u e f r o m b o t h r a t s a n d s h e e p w h e r e a s t h e rate o1 i n c o r p o r a t i o n f r o m l e u c i n e w a s g r e a t e r ( P < 0.05) in s h e e p t h a n r a t a d i p o s e t i s s u e ( T a b l e 4). G l u c o s e w a s b y far t h e best p r e c u r s o r for a c y l g l y c e r o l glycerol s y n t h e s i s in b o t h species ( T a b l e 4). DISCUSSION T h e relative r a t e s o f u t i l i z a t i o n o f g l u c o s e a n d a c e t a t e c a r b o n for f a t t y acid s y n t h e s i s by a d i p o s e t i s s u e f r o m s h e e p a n d r a t s a r e in a c c o r d w i t h p r e v i o u s s t u d i e s ( B a l l a r d et al., 1969; V e r n o n , 1980), while the relatively low rate o f f a t t y acid s y n t h e s i s f r o m a c e t a t e in a d i p o s e t i s s u e f r o m foetal l a m b s c o m p a r e d with o l d e r a n i m a l s is in a g r e e m e n t w i t h earlier s t u d i e s ( V e r n o n et al., 1981b; R o b e r t s o n et al., 1981). Feller

Table 2. Fatty acid synthesis from acetate, glucose and amino acids in per±renal adipose tissue from sheep and rats ng atoms of carbon incorporated into fatty acid/2 hr per 106 cells Sheep Precursor Acetate Glucose Alan±he Leucine lsoleucine Valine

120 days gestation 170 ± 34a 146 ± 65a 2.7 ± 0.8 ~ 3.9 ± 1.3~ 0.5 ± 0.2~ 0.1 ± 0.0~

8-months old 2225 ± 205b 164 ± 107~ 13.4 ± 6.6~ 92.5 ± 18.7b 13.4 ± 4.8 b 1.1 ± 0.4b

Rat 3-years old 791 ± 242c 34 _+ 13a 2.2 ± 0.6a 17.9 ± 5.7~ 2.7 ± 0.9~ 0.9 +_0.1 b

220-240 g body wt 2242 ± 369b 8656 ± 3031h 335 _+ 130b 609 ± 76c 70 ± 11.5c 30 ± 5.5c

Results are mean ± SEM of 4 observations except for 3-year-old sheep for which results are mean ± SEM of 5 observations. a, b, c, d: in each row, values with different subscripts differ significantly (P < 0.05). Table 3. Relative contributions of carbon from acetate, glucose and amino acids to fatty acid synthesis in adipose tissue from sheep and rats Rate of fatty acid synthesis (ng atoms incorporated) as a percentage of rate from acetate or glucose Sheep Rats 120 days 8-months 3-years 220-240 g gestation old old body wt Precursor 100 100 100 100 34.2 + 9.5 Acetate 78.1 + 21.0a 6.9 ± 4.0 b 4.7 + 0.8 b 353.0 ± 76.0c 100 Glucose 1.6+_0.3~ 0.6+_0.2 b 0.4 ± 0.1 b 13.6±2.9 c 4.1 +0.6 Alanine 2.2 +_0.4~ 4.0 _+0.5 b 2.2 + 0.2~ 27.6 4- 1.7c 9.4 + 2.5 Leucine 0.3 ± 0.1 ~ 0.6 ___0.2~ 0.4 ___0.1 a 3.0 + 0.4 b 1.0 ± 0.2 lsoleucine 0.1 ±0.0 a 0.1 +_0.0a 0.2+_0.1" 1.3 ±0.1 b 0.4±0.1 Valine Results are mean ± SEM of 4 or 5 observations (see Table 2). a, b, c: in each row, mean values with different subscripts differ significantly (P < 0.05).

FA synthesis in adipose tissue Table 4. Rate of incorporationof ~4Cfrom variousprecursors into acylglycerol glycerol in adipose tissue from 3-year-oldsheep and 220--240g femalerats ng atoms carbon incorporated/2hr per 104 cells Precursor Sheep Rats Glucose 952 +__293 2462 + 343* Acetate 27.5 _ 3.0 19.7_ 1.7 Alanine 2.8 _+0.2 3.6 + 0.4 Leucine 16.1 ___2.5 6.7 +0.9* Isoleucine 6.4 + 1.3 2.9 + 0.6 Valine 3.6 + 0.2 2.4 ---Z-0.8 _ Resultsare mean+ SEM of 5 observations(sheep)or 4 observations (rats). *Values significantlydifferent from that for sheep adipose tissue (P < 0.05).

(1965) also found that the rate of fatty acid synthesis from amino acids in rat adipose tissue decreased in the order leucine > alanine > isoleucine > valine (see also Rosenthal et al., 1974). The rate of fatty acid synthesis from leucine in rat adipose tissue relative to that from glucose is in agreement with a previous report (Rosenthal et al., 1974). Adipose tissue is thought to be an important site of amino acid metabolism in the rat, particularly with respect to the degradation of branch-chain amino acids (Rosenthal et al., 1974; Snell and Duff, 1977; Tischler and Goldberg, 1980). The metabolism of leucine has been studied in greatest detail and one of the major products is fatty acid (Meikle and Klain, 1972; Rosenthal et al., 1974; Goodman, 1977). The total contribution of amino acid carbon to fatty acid synthesis in rat adipose tissue has not been assessed but the present study suggests that the four amino acids chosen make a significant contribution (15Y/oof that of glucose carbon). The role of adipose tissue in amino acid metabolism in general in ruminants is not clear. The tissue appears to have a significant role in leucine metabolism (Wijayasinghe et al., 1983). The very similar rate of incorporation of ~4C from ~4C labelled amino acids and also acetate into acylglycerol glycerol in adipose tissue from rats and sheep would suggest that the rates of amino acid catabolism are probably similar in adipose tissue from both species. There is no net synthesis of acylglycerol glycerol from acetate or leucine in mammalian tissues hence the appearance of ~4C from 'aC-acetate in this moiety reflects randomization of taC-label amongst tricarboxylic acid cycle intermediates suggesting similar degrees of labelling of these intermediates in adipose tissue from the two species. Net glycerogenesis is possible from the glucogenic amino acids such as valine and alanine but again rates are very similar in adipose tissue from rats and sheep. Thus the major difference between the two species in the metabolism of amino acids in adipose tissue appears to be the much lower rate of fatty acid synthesis from amino acids in sheep. Recently, Wijayasinghe et al. (1984) have also shown that fatty acid synthesis from leucine is very low in sheep adipose tissue. Thus even if adipose tissue is found to be an important site of amino acid metabolism in sheep, it appears unlikely that their metabolism will make a significant contribution to fatty acid synthesis.

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Sheep adipose differs from rat adipose tissue in that it has relatively low activities of pyruvate dehydrogenase (Robertson et al., 1982) and ATP-citrate lyase (Ballard et al., 1969); the low activity of the former enzyme will restrict the utilization of glucose and alanine for fatty acid synthesis, whereas the low ATP-citrate lyase activity will also restrict the use of leucine and isoleucine (partly) as well as glucose and alanine for fatty acid synthesis, as their catabolism yields mitochondrial acetyl CoA. Thus the low ATPcitrate lyase activity of adipose tissue from adult and also foetal sheep (Vernon et al., 1981b) is probably a major reason for the low rate of fatty acid synthesis from these amino acids in the tissue. Valine, however, is metabolized to methylmalonyl-CoA while isoleucine catabolism yields equimolar amounts of acetyl CoA and propionyl CoA. Branch-chain fatty acids and fatty acids with odd numbers of carbon atoms are found in sheep adipose tissue (Christie, 1978) which could be derived from using methylmalonyl-CoA and propionyl CoA for fatty acid synthesis (Vernon, 1980), but catabolism of valine and isoleucine would not appear to be important sources of these compounds. Labelled carbon from these amino acids could also appear in fatty acid after randomization in the tricarboxylic acid cycle, but again, this would be restricted by the low ATPcitrate lyase activity. Thus the study shows that amino acid carbon makes very little contribution to fatty acid synthesis in sheep adipose tissue and also that fatty acids are a minor product of amino acid metabolism. In addition, the study provides further evidence for a key role of ATP-citrate lyase in restricting the use of acetyl CoA generated in the mitochondria for fatty acid synthesis in sheep adipose tissue. Acknowledgements--We thank Mr D. Fullarton and Mr C. Park for care of the sheep and Mr J. McDill for care of the rats. REFERENCES

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