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Effect of different fatty acids on lipogenesis in rat and sheep adipose tissue in vitro
EFFECT OF DIFFERENT FATTY ACIDS ON LIPOGENESIS IN RAT AND SHEEP ADIPOSE TISSUE IN VITRO R. G. VERNON The Hannah
Research
Institute.
Ayr. Scotland
KA6 5HL,
U.K.
(Rw(~iwd 15 Decernht~r 1976) Abstract-l. The effect of different fatty acids in vitro on the rate of lipogenesis in rat and sheep adipose tissue slices has been investigated. 2. Both palmitic and linoleic acids, chosen as representative saturated and polyunsaturated fatty acids, reduced the rate of lipogenesis in rat and sheep adipose tissue by inhibiting the activity of one or more of the enzymes of lipogenesis. 3. In both species, stearic acid was the most effective inhibitor of lipogenesis; palmitic and oleic acids and mixtures of fatty acids, the compositions of which were based on that of the unesterified fatty acid fraction of rat and sheep plasma, were equally effective inhibitors of hpogenesis; linoleic acid was a less effective inhibitor of hpogenesis than palmitic acid. 4. The results are comoared with the effects of dietary saturated and polyunsaturated fatty acids on lipogenesis in rat and sheep adipose tissue.
INTRODUCTION
Radiochemical Centre. Amersham, U.K.; E. Merck Ltd., Darmstadt. Germany; Koch-Light Ltd.. Colnbrook, U.K. Bovine serum albumin was partially alysed (Hanson & Ballard. 1968). The had an unesterified fatty acid content albumin.
Lipogenesis in rat and sheep adipose tissue is inhibited by both the amount and the nature of dietary fat (Du & Kruger, 1972; Waterman et al.. 1975: Vernon, 1976). Lipogenesis in ovine adipose tissue was inhibited more by dietary saturated fatty acids than polyunsaturated fatty acids (Vernon, 1976). For lipogenesis in rat adipose tissue the picture is less clear; an earlier study (Du & Kruger. 1972) suggested that adipose tissue lipogenesis was inhibited to a greater extent by dietary polyunsaturated fatty acids than saturated fatty acids, thus resembling rat liver in its response to different dietary fatty acids (Bartley & Abraham, 1972). However. a more recent study (Waterman rt ul. 1975) has indicated that lipogenesis in rat adipose tissue was more inhibited by dietary saturated fatty acids than polyunsaturated fatty acids. and thus resembled ovine adipose tissue in its response to different dietary fatty acids. The mechanisms by which dietary fatty acids exert their differential effects on adipose tissue lipogenesis are unknown. Palmitic acid, however, has been shown to inhibit lipogenesis in rat adipose tissue irr citro (Saggerson & Tomassi, 1971; Soorana & Saggerson, 1975). Thus the purpose of this study was to compare the effect of different fatty acids. in particular palmitic and linoleic acids, chosen as representative saturated and polyunsaturated fatty acids. respectively, on lipogenesis in sheep and rat adipose tissue in dro to see if the
varied
effects
of dietary
fatty
acids
defatted and difinal preparation of 4.3 nmole/mg
Female Wistar rats. weighing about 300 g. were obtained from A. Tuck & Sons Ltd.. Rayleigh, Essex. U.K. Food (diet 41B. Oxoid Ltd.) and water were supplied ad libiturn. Sheep were pure bred Cheviot ewes. 556 years old; they were maintained on a diet of hay given at 7 a.m. and 4 p.m. each day. Rats were killed by cerebral dislocation and sheep were killed with a captive-bolt humane killer between IO and 11 a.m. Parametrial adipose tissue (rats) or perirenal adipose tissue (sheep) was removed immediately and kept in isotonic saline at 37
coditiorls
Samples (6&80mg) of adipose tissue slices. prepared free-hand. were incubated in 2.5 ml of Krebs-Ringer bicarbonate buffer. pH 7.4 with half the original calcium concentration (1.27 mM) and containing 5 mM glucose and 40 mg/ml bovine serum albumin.
Following a pre-incubation period of 15 min. either 0.25 PCi of (I-‘Qacetate or 0.05 $Zi of (I-“C)-acetate plus 2.5 mCi 3H,0 was added to the incubation medium: the final acetate concentration was 2 mM in each case. The tissue slices were then incubated for 2 hr at 37 C in an atmosphere of Oz:CO, (95:4v/v) in a shaking water bath. Incubation was terminated by removing the tissue slices with fine forceps and homogenising them in chloroform:methanol:0.15 M KC1 (I : 1: 1 by vol). Lipids were extracted and their fatty acids were transesterified using methanolic HCI as described previously (Vernon, 1975). The fatty acid methyl esters were extracted with hexane. their weight was determined gravimetrically, and following solution in a toluene scintillation fluid, their radioactivity was measured with an efficiency of 90”, in a Packard Tricarb 2425 liquid scintillation spectrometer. Samples of the
were
due to differential, direct effects of the fatty acids on adipose tissue lipogenesis. MATERIALS AND
silica gel G from Unisolve I from
METHODS
Materials Insulin (24 unit/mg), unlabelled fatty acids and bovine serum albumin (fraction V) were purchased from Sigma (London) Chemical Co. Ltd.: ( I-‘4C)-acetic acid. 59 mCi/ m-mole. (I-“Ctpalmitic acid. 59 mCi.!m-mole, (1-‘4C)linoleic acid. 57 mCi!m-mole and 3Hz0. 5 Ci’ml from the 517
R. G.
518
aqueous residue left after extraction of the fatty acid methyl esters with hexane were mixed with Unisolve I and their radioactivity was measured with an efficiency of 80?,. This aqueous fraction contained the glycerol derived from glycerides and other water-soluble components of the lipids released during transesterification. These components were not further separated and for the purpose of this study it was assumed that most of the radioactivity of the samples was associated with glycerol derived from glycerides. Addition of‘furty u&is
Fatty acids were taken up in 0.1 mt of 0.1 N KOH containing 4mg albumin and-were added to the incubation medium immediatelv before the addition of (1-‘%-acetate. KOH (0.1 ml of 0.1 6I) containing 4 mg albumin was added to control flasks. (‘~C~labelled fatty acids were added to the standard incubation medium. containing 2 mM unlabelied acetate to give a final concentration of palmitic or linoleic acid of 1 mM, O.O4~Ci/ml. Incubations and extraction of lipids were performed as described above. Samples of extracted lipid were separated into the major lipid-classes by thinlaver chromatogranhv on silica gel G (Vernon. 1975). The appropriate baids’of’silica gel w&e transferred to scintillation vials, and a gel was formed by the addition of H,O and Unisolve I (4: 10 v/v). The radioactivity of the gels was measured with an eficiency of 75”,;. Expression
of results
In view of the wide variation in the rate of lipogenesis found between animals, results for each individual experiment were expressed as a percentage of the control value (rate of lipogenesis found when tissue was incubated for 2 hr in the absence of fatty acids). These figures were pooled with corresponding values from other experiments to obtain the mean and S.E.M. values quoted. Statistical significance was calculated on the basis of Student’s t-test using a test for paired observations where appropriate. RESULTS Preliminary experiments with sheep adipose tissue slices showed that although glucose stimulated the rate of incorporation of (I-14C)-acetate into fatty acids, insulin had no effect on the rate. either in the presence or absence of glucose (Table 1). Addition of albumin to the incubation medium resulted in a significant (P < 0.05) increase in the rate of (l-14C)acetate incorporation into fatty acids in the presence of glucose and insulin in sheep adipose tissue slices (Table 1). but not rat adipose tissue slices (results not shown). It was also found that for both sheep and rat adipose tissue slices, less than lg/, of the
VERNON
labelled fatty acids produced during incubation with labelled precursors were released into the incubation medium; this was not affected by the addition of fatty acids to the incL~bation medium. For initial experiments in which the effects of fatty acids on the rate of fatty acid biosynthesis were examined, palmitic and linoleic acids were used as representative saturated and polyunsaturated fatty acids respectively. In the absence of insulin. addition of either 1 mM palmitic or linoleic acids signi~cantly (P < 0.001) reduced the rate of incorporation of both (l-‘4C)-acetate and 3H,0 into fatty acids in rat adipose tissue slices (Table 3). Palmitic acid resulted in a greater degree of inhibition of both ( l-‘4C)-acetate and 3H20 incorporation into fatty acids than did linoleic acid, but the difference was small and was only significant (P < 0.05) for the incorporation of 3H,0 into fatty acids. The incorporation of (I-14C>acetate into fatty acids was always inhibited to a greater extent by added fatty acids than was the incorporation of 3Hz0. but the ratio of 14C:3H incorporated was the same when either palmitic or linoleic acids were added (Table 2). Both fatty acids stimulated the incorporation of 3H,0 into glyce~de-~ly~rol (Table 2). Palmitic acid was slightly, but significantly (P < 0.05) more effective than linoleic acid. When insulin was included in the incubation medium the rates of incorporation of both (l-14C)acetate and 3H,0 into fatty acids in rat adipose tissue slices were significantly (P < 0.01) increased in both the presence and absence of added fatty acids (Table 3). In the presence of insulin, palmitic acid, but not linoleic acid. still significantly (P < 0.05) reduced the rate of incorporation of ( I-14C)-acetate into fatty acids (Table 3). Also, the rate of incorporation of both labelled precursors into fatty acids was significantly (P < 0.05) lower in the presence of 1 mM palmitic than I mM linoleic acid (Table 3). Insulin had no effect on the rate of incorporation of 3Hz0 into glyceride glycerol in the presence of added fatty acids (Table 3). however, in their absence. insulin resulted in a small, but significant (P < 0.05) decrease in the rate of incorporation of 3HZ0. In analogous experiments using sheep adipose tissue slices. addition of 1 mM palmitic or linoleic acids to the incubation medium again resulted in a significant (P < 0.001) decrease in the rate of incorporation of (I-‘“Ctacetate into fatty acids (Table 3); again, I mM palmitic acid was significantly (P < 0.05) more effective than 1 mM linoleic acid (Table 2). However, in contrast to rat adipose tissue slices, inclusion of insulin in the incubation medium had no effect on
Table 1. Effects of glucose, insulin and albumin on the rate of incorporation of (l-14C)acetate into fatty acids in sheep adipose tissue slices
Results are mean + S.E.M. of 3 observations. Concentrations: insulin 0.1 unit/ml: glucose 5 mM; albumin 4”” (w/v)
Effect of different fatty acids on lipogenesis
519
Table 2. Effect of palmitic and linoleic acids on the rate of incorporation of (1-‘4 C)-acetate into fatty acids and 3Hz0 into fatty acids and glyceride-glycerol in rat and sheep adipose tissue slices
Labelled
precursor
Relatwe rate of incorporation (“,, control value)
Rate of mcorporatton (control *alue: pmole:hr per g wet wt tissue)
Species
Lmoleic
Palmttic acid il mMl
acid
(1 mMl
lncorporat~on 1”~ fatty
actdc ,I-1Tl.acetate %I10 Ratio ‘H:“C (I-“C>acetate Incorporatmn mto glycerideglywol ‘H,O
Rat Rat Rat Sheep
0.65 * OlO(lbl
54 9 60.1 1’3.2 54.0
0.67 It 0.07 110)
144.7 + b.3
0.30 * no5 (251 0.25 i: (107 (I
II I?‘)_t0081111
Rat
* * f I
3.0 3.X h I 2.2
hl x 74,4 176.6 606
* * * k
3.7 6.4” 4.0 2.9”
136.9 & 5.5’
Control values represent the rate of incor~ration observed in the absence of added fatty acids (rat and sheep) and the absence of insulin (rat). Results are mean + S.E.M. with the number of observations in parenthesis after the control values. “Value si~ifi~antly different (P < 0.05) from corres~slding value obtained with palmitic acid. Table 3. Effect of insulin on the relative rates of incorporation of (I-‘%Z)-acetate into fatty acids and 3H,0 into fatty acids and glyceride-glycerol in rat and sheep adipose tissue slices in the presence and absence of added fatty acids
Rat adtpasc __..-__
Addition Fatty acrd 11 mM1 Pnlmittc Lmolcic NOIE Palmitic Lnmletc
Ftttt! .__
lnsulm (0
55.1 + 5 R(8p” 67.3 z 7.2”.“,’ 123.1) + 33 O”,”
acid actd + * +
acid acid
Gljcende-glycerol
acrds -I___
._-
(I-‘w-acetate
I unit/mll
1li15; 126’~“~~ v7 -
0 ; 2S.P’.6
Sheep adtpose tissue Fatty acrds
tissue
‘H>O
il-“Qacetate
%,O
59 5 * 4.7 l4F” 80 5 f X 3a.b.c
1399 137.3 906 1416
7Yiii- 44,6~~d 1s.:,s ;
I 2l? 70X.4 * ?O.Xl,‘.d
* i + I
44.5 66.3 105.6 60.5 71.6
52(4P 3.P 2.6” &I*.”
t35.7 * 8.P”
+ z f + +
5.9 (3)4,’ 6.6@ 42 3.2“~” 7.P”
Results are expressed as a percentage of the rate observed in the absence of added fatty acids and insulin and are mean f S.E.M. with the number of observations in parenthesis after the first value of each column. Value differs significantly (P <: 0.05) from: ‘Value obtained in absence of added fatty acids and insulin. b Value obtained in absence of added fatty acids and presence of insulin. ‘Corresponding value obtained with palmitic acid. ‘Corresponding value obtained in absence of added insulin. Table 4. Effect of palmitic and iinoieic acid concentration on the rate of incorporation of 3H,0 into fatty acids and glyceride-~y~erol in rat adipose tissue slices.
Fatty fnsuiin (0.1 uoiUm1) Incorporatmn
(I-“Cl-acetate
Incorporation >H,O
0
l6:O IX:1 16:O IX:? lb:0 1X-3 16-O 18 2
IW 1cQ I00 loo loo 100 loo
97.4 103.6 99.0 943 lOS.9 109.0 110.3 107.0
l6:O
loo
1X:2
loo
131 7 f 11.213) 115.6 + 10.7(3)
acid =~~~~“t~~tt~~
IW
(PM) t*Ml
300
ho0
i + ?I f * i A f
5.3 (3) 0.6131 7.0 (6) 3 9 16) 4.2 (3) 99.4 i_ 8.2 (3) 2.3 (3) 117.3 II6 3 rt 3.3 (3) 5
64.1 66.3 57.6 54.5 90.5 99.4 130.3
157.0 * 4713) 134.1 * 15.613)
169.X _t 7.813) 154.4 f 3.9 (3)
into fatty acids
‘H,O
Ratio of ‘H fatty acids
Fatty acid
and (l-‘%‘)-acetate
“C
incorporated
mto
+ +
* 8.3 13) i 4 5 (31 + 17.7 I61 li_ 28161 + I.3 (3) f 9.913) + r 1.4 170)(3)
Y7.0 X3.6 71 5 81.2 1102 96 I 117.7 II50
4.6 (3) 5.4 (3) X0(6) 5.4(6l 7413) 4.413) 4.713) 2.3131
71.X 16.5 63.4 59.3
t i k or
110.5 _t
It + ir t * i t C
5.5(3) 8.1 (3) 6.1 16) 3.7 16) 3.5 (31 0.6131 5.4(3) 6.0(3)
into glye~ride-glycerol 140.2 + 4.0(31 124.4 * i 1131
The rates of incorporation of (l-‘4C)-acetate and ‘Hz0 into fatty acids and glyceride-g~yccrol in the presence of added fatty acids are expressed as percentages of the rates observed with controls incubated in the absence of added fatty acids. Results are mean + S.E.M. with the number of observations in parenthesis. the rate of incorporation of (I-14C)-acetate into fatty acids in the absence or presence of 1 mM added fatty acids (Table 3). The degree of inhibition of incorporation of (l-14C)-acetate into fatty acids in both rat and sheep
adipose tissue slices, and ‘Hz0 into fatty acids in rat adipose tissue slices, was dependent on the concentration of fatty acid added to the incubation medium (Tables 4 and 5). As the concentration of fatty acid in the control flasks was 170~M due to
R. G.
520
VERNON
Table 5. Effect of palmitic and linoleic acid concentration on the rate of incorporation of (I-14C)-acetate into fatty acids in sheep adipose tissue slices
acids in the presence of added fatty acids was expressed as a percentage of the rate observed in the absence of added fatty acids. Results are mean + S.E.M. of 3 ohservntions.
the presence of residual unesterified fatty acids in the albumin, the true range of initial fatty acid copcentrations was from 170 to 1170 PM, corresponding to a fatty acid:albumin molar ratio of 0.3-2.0. Changes in the medium unesterified fatty acid concentration during the incubation period were not measured, but from the rates of fatty acid esterification given in Table 8. it can be calculated that after a 2 hr-incubation with either 1 mM palmitic or linoleic acids, approx 12”” and 4”, of the added fatty acid would have been utilised by rat and sheep adipose tissue slices, respectively. Palmitic and linoleic acids had similar effects on the rate of incorporation of (I-‘“Clacetate and 3Hz0 into fatty acids in rat adipose tissue slices over the range of concentrations used. Addition of insulin relieved the inhibition of rat adipose tissue fatty acid biosynthesis at all fatty acid concentrations tested (Table 4). Both the ratio of 3H : ’ 'C incorporated into fatty acids and the incorporation of 3H into glyceride-glycerol in rat adipose tissue slices increased as the concentration of added fattv acids was increased (Table 4). At all concentrations. palmitic and linoleic acids had essentially an identical effect on the ratio of 3H:‘JC incorporated into fattv acids. Analysis of variance showed that palmitic acid resulted in a significantly (P < 0.01) greater rate of incorporation of 3H into glyceride glycerol than did linoleic acid over the concentration range used. With regard to sheep adipose tissue. addition of palmitic acid resulted in a lower rate of fatty acid
Table
Rat
Sheep
6. Effecectof incubation
None Ih:0 IX.7 h”llC Ih ,, IX:7
time on the incorporation tissue slices in the presence
+ + +
synthesis than did addition of linoleic acid at all concentrations but an analysis of variance showed that the difference was not statistically significant (Table 5). The rate of lipogenesis in rat adipose tissue was linear with respect to time over the 2 hr-incubation period (Table 6). Inhibition by palmitic and linoleic acids was statistically significant (P < 0.05) at 10 and 30 min. respectively, after their addition. In contrast, the rate of lipogenesis in sheep adipose tissue increased with time during the experimental period. Inhibition by fatty acids was significant (P < 0.05) at all times except 30min after their addition. The reversibility of the inhibition of lipogenesis by fatty acids was examined by incubating adipose tissue slices in the presence of fatty acids (1 mM) for 60 min following which the slices were transferred to flasks containing medium devoid of fat.ty acids. These experiments showed that the rate of lipogenesis, previously reduced by incubation in the presence of fatty acids, was readily restored to the control value when the tissue slices were incubated in medium containing no added fatty acids (Table 7). The rate of uptake of palmitic and linoleic acids by both rat and sheep adipose tissue slices, as reflected by their rates of incorporation into lipids, was measured at IO, 60. 120 and 180 min after the addition of labelled fatty acids, and was found to be linear with respect to time over the whole experimental period (data not shown). There was no significant difference in the rate of uptake of palmitic and linoleic acids. Insulin had no effect on the rate of incorporation of fatty acids into lipids (Table 8). The effects of palmitic and linoleic acids on fatty acid biosynthesis in rat and sheep adipose tissue were compared with those of other fatty acids and also of mixtures of fatty acids, the composition of which were based on that of the unesterified fatty acid fraction of plasma from rats (Rose et al.. 1964) and sheep (Noble et al.. 1971) (Table 9). At a concentration of 1 mM, stearic was a significantly (P < 0.05) better inhibitor of both rat and sheep fatty acid biosynthesis than palmitic acid. With the exception of linolenic acid, the other fatty acids tested and also the fatty acid mixtures had similar effects on the rate of fatty acid biosynthesis to palmitic acid. The rate of incorporation of 3H10 into fatty acids in rat adipose tissue was not inhibited by the presence of 1 mM linolenic acid. Also, 1 mM linolenic acid did not stimulate the
of (1-“Q-acetate into fatty acids and absence of added fatty acids
7x * JO + hJ+2, 45 * 27 + 29 *
20 I)‘)” 11’1 115” 0 6”
26hi_13 IJK * ,5x i-0’)” IJI i-l!, ,,,7 +,15
17”
Ii 6 i_0.6
in rat and
r3.2 f JY
766+ 3.3” 27 I * 36” ?Y7+4? 77x * 07”
2x9* lb”
sheep
adipose
IUI 51.7 + 15.2 ii.9 + 9 6” 100 55 2 * 2.0” 6U.3 k 4.0”
Amounts of (I-“Qacetate incorporated into fatty acids are expressed as a percentage of the amount of ( 1-14C)-acetate incorporated into fatty acids in 120 min in the absence of added fatty acids. Results are mean + S.E.M. of 3 observations in each case. a Value significantly different (P < 0.05) from corresponding value obtained in the absence of added fatty acids.
Effect of different Table 7. Effect on the rate of incorporation incubating the adipose tissue slices in fatty
521
fatty acids on lipogenesis
of (I-“C)-acetate acid-free medium
into fatty acids in rat and sheep adipose tissue of after incubation in the presence of added fatty acids
Amounts of (I-“C)-acetate incorporated into fatty acids during each period are cxpressed as a percentage of the amount of (i-‘4C)-acetate incorporated into fatty acids in 2 hr in the absence of added fatty acids. Results arc mean k S.E.M. of 3 observations in each case. “Value significantly different (P < 0.05) from corresponding value obtained during OWN min.
incorporation of 3H,0 into glyceride-glycerol in rat adipose tissue slices. as did the other fatty acids; both rates were significantly (P < 0.05) different from those found using 1 mM palmitic acid. DISCC’SSION
A comparison of the effects of different fatty acids or their acyl-CoA esters on the activities of the enzymes of fatty acid biosynthesis is complicated. as, Table 8. Rate of incorporation of palmitic and linoleic acids into lipids in adipose tissue slices from rats and sheep
Insulin
Species Rat
(0 I unlt,ml)
* I,“)”
L,nolc,c
.ud
I1 mMI
11ur Ill50
+
acld
II mMI
+
Rat Sheep
Pahmt~
102tI II Y
*
45
YY 4 + 5 2 llh.,
+ xx
” 100% Corresponds to rates of fatty acid incorporation into lipids of 1.91 & 0.24 and 0.59 + 0.14 nmole!hr per g wet wt tissue for rat and sheep adipose tissue slices. respectively. Results are expressed as a percentage of the value obtained with 1 mM palmitic acid in the presence of insulin are mean + S.E.M. of three observations in each case.
being detergents. fatty acids and their acyl-CoA esters can inhibit enzymic activities by non-physiological processes (St-et-c. 1965: Taketa & Pogcll. 1966; Dorsey & Porter. 196X: Pande & Meade. 196X: Parvin & Dakshinamarti, 1970). Furthermore, both fatty acids and their acyl-CoA esters are probably bound to proteins or membranes within the cell so that their effective concentration is uncertain (Ockner tjt LI/., 1972: Mishkin t’t LII., 1972. 1974: Sumper. 1974). Thus it seemed preferable to compare the effects of different fatty acids on lipogenesis using intact cells; tissue slices in fact were used rather than isolated cells as it has not as yet proved possible to make satisfactory preparations of adipocytes from adult sheep adipose tissue. For such a system to be meaningful. it is clearly important that the concentration of the components of the incubation medium should be in the physiological range. Of the incubation medium components. the concentration of insulin added was in excess of the normal plasma concentration. but as some of the insulin would be bound to the glass surface of the incubation flask, its actual concentration in the incubation medium was uncertain. The range of fatty acid concentrations used in this study. 17&l 170 /tM (including the contribution of the albumin fatty acids) was in the physiological range for both rat (?O&l700 /tM. SCOW & Chernik.
Table 9. Relative rates of incorporation of (I-r’C)-acetate into fatty acids and acids and glyceride-glycerol in rat and sheep adipose tissue slices in the presence acids
Results are expressed as a percentage of the rate observed in the absence of and are mean + S.E.M. with the number of observations in parenthesis after each column. “Value differs significantlv (P < 0.05) from that for palmitic acid. ‘Composition of fatty acid mixture (wt percentage): palmitic 34; stcaric I?; 20 (used with rat adipose tissue); palmitic 26: stearic 30: oleic 39: linoleic 5 adipose tissue).
‘H,O into of different
fatty fatty
added fatty acids the first value in
oleic 34; linoleic (used with sheep
522
R. G. VERNON
1970) and sheep plasma (20&19OOpM, Noble et al., 1971; Jarrett et al., 1974). Thus, the results clearly show that physiological concentrations of fatty acids can modulate the rate of lipogenesis in both rat and sheep adipose tissue. It was previously shown that incubation of rat adipose tissue or adipocytes with palmitic acid inhibited the incorporation of (14C) from (2-‘4Cbpyruvate, (2-l&C)-lactate, (U-14Cbfructose and 3H from 3H,0 into fatty acids (Saggerson & Tomassi, 1971; Soorana (a: Saggerson, 1975). However, Saggerson (1972u,~) did not observe inhibi~on of fatty acid biosynthesis from glucose in the presence of palmitic acid. Although it is not certain that fatty acids were inhibiting fatty acid synthesis from glucose in the present study, it would seem probable as they did inhibit the incorporation of ‘H from 3H,0 into fatty acids which is a measure of the total rate of lipogenesis (Jungas, 1968). This apparent difference in the effect of palmitic acid on lipogenesis from glucose could be due to dissimilarities in the incubation media used. in the two studies or perhaps in the age of the animals, 1%18Og rats used by Saggerson compared with 280-320g used in this study. It is pertinent to note that the degree of inhibition observed with the various fatty acid concentrations used in this study was very similar to that found when rat liver was perfused with oleic acid (Mayes & Topping, 1974) and when rat hepatocytes were incubated with palmitic or oleic acids (Nilsson et al.. 1973); in both these studies with rat liver. glucose was present in the incubation medium; also the rats weighed between 250 and 35Og, so the effects of exogenous fatty acids on lipogenesis may vary with age. Elucidation of the mechanism by which exogenous fatty acids inhibit fatty acid biosynthesis was not a primary aim of this study. However, the rapidity with which added fatty acids exerted their effects and also the fact that the inhibition was readily reversed on removal of the fatty acids indicate inhibition of the activity of one or more of the enzymes of fatty acid biosynthesis as suggested by Soorana and Saggerson (1975). Also, the ease with which the inhibition was reversed excludes enzyme inactivation. The particular enzyme or enzymes affected remains to be elucidated, but as acetate was found to be essentially the only precursor of lipogenesis in the adipose tissue from Cheviot sheep (Vernon, 1976). it would appear that for sheep adipose tissue the fatty acids were inhibiting acetyl-CoA carboxylase activity. Furthermore. incubating rat adipose tissue with insulin irz citro has been shown to reduce the concentration of long-chain fatty acyl-CoA esters and activate acetylCoA carboxylase (Halestrap & Denton, 1973) providing a possible explanation for the ability of insulin to prevent the inhibition of fatty acid biosynthesis by fatty acids in rat adipose tissue. Although glucose plus insulin have been shown to stimulate the incorporation of (I -14C)-acetate into fatty acids in ovine adipose tissue slices (Hanson & Ballard, 1967; Ingle et at., 1973) the effect of insulin itself does not seem to have been reported previously. The reason for the tissue’s lack of sensitivity to the hormone is not known but it would not appear to be due to the age of the sheep. as insulin, in the presence or absence of glucose, also had no effect on the
rate of fatty acid biosynthesis in adipose tissue slices from either &day old or 3-month old lambs (Vernon, unpublished observations). The results suggest that both palmitic and linoleic acids inhibited fatty acid biosynthesis in adipose tissue by the same mechanism. Assuming that these two fatty acids were representative of the others tested, it would appear that stearic acid was the most effective inhibitor of fatty acid biosynthesis in both sheep and rat adipose tissue. Stearic acid has also been found to be a more effective inhibitor of fatty acid biosynthesis in rat (Nilsson et al., 1974) and chick (Goodridge, 1972) hepatocytes than other fatty acids. In contrast. the polyunsaturated fatty acids, linoleic and, in particular, linolenic acid, were less effective inhibitors of lipogenesis in adipose tissue than palmitic acid. The lack of effect of linolenic acid on lipogenesis in rat adipose tissue could be due to a relatively low rate of uptake compared with other fatty acids as it did not stimulate the rate of glyceride glycerol synthesis. This makes the usual assumption that the rate of esterification, as measured by either the rate of fatty acid incorporation into lipids or the stimulation of glyceride glycerol synthesis by added fatty acids, reflects the rate of fatty acid uptake as suggested by the observation that essentially all the fatty acids taken up by adipose tissue from fed animals were esterified (Shapiro. 1965). Thus, it would appear that the differential effects of stearic, pahnitic and linoleic acids on the rate of lipogenesis were not due to differences in the rate of uptake. Also, the effect of insulin, reducing the degree of inhibition of lipogenesis, would not appear to be due to a change in the rate of fatty acid uptake. However, although stearic acid was a more potent inhibitor of fatty acid biosynthesis in vitro than the other fatty acids tested, this would not appear to account for the effects of different dietary fats on fatty acid bios~thesis in adipose tissue in uiuo. Dietary tallow resulted in a si~ifi~tly greater decrease in the rate of fatty acid biosynthesis in sheep adipose tissue than did a mixture of sunflower and soyabean oil (Vernon, 1976). but both diets increased the plasma unesterified stearic acid level to the same extent relative to that of control animals receiving a low-fat diet (Noble R. C., unpublished ob~rvation). The only fatty acid whose plasma concentration was changed differently by the two dietary fats was linoleic acid (Noble R. C., unpublished observation), but the changes were small and although linoleic acid was a less effective inhibitor of ovine lipogenesis than palmitic acid, the difference would appear to be too slight to offer a reasonable m~hanism. For rat adipose tissue the situation is complicated by the apparently conflicting reports of the relative effectiveness of dietary saturated and polyunsaturated fatty acids on fatty acid biosynthesis in adipose tissue (Du & Kruger, 1972; Waterman et al., 1975). The reason for these different findings is not known, but, the rats used by Du and Kruger (1972) showed signs of essential fatty acid deficiency prior to the administration of the various fatty acids whereas Waterman et al. (1975) avoided this complication by always including some polyunsaturated fatty acids in the diet. It is pertinent to note that the rats used by Waterman et al. (1975) showed a similar response to different
523
Effect of different fatty acids on lipogenesis dietary fatty acids to the sheep used by Vernon (1976).
again received adequate levels of essential fatty acids in the diet. Unfortunat~iy. the composition and concentration of the plasma unesterified fatty acid fraction was not reported in either study with rats. However, stearic acid is a relatively minor component (12% by weight) of the rat plasma unesterified fraction (Rose et al., 1964). and at this concentration its effect on fatty acid biosynthesis was swamped by the other fatty acids (Table 9). so it would appear that the relative concentration of stearic acid would have to increase several fold before it would have a significant effect on the degree of inhibition of fatty acid biosynthesis. Furthermore. although palmitic acid was a more effective inhibitor of lipogenesis than linoleic acid. the difference was small. Thus, saturated fatty acids seem to be more effective inhibitors of lipogenesis than ~iyun~turated fatty acids in both sheep and rat adipose tissue, but although this could contribute to the differential effects of dietary fatty acids on lipogenesis itr tliz~, it would appear most unlikely that this difference is the major mechanism. However. in this study, tissue was exposed to fatty acids for only short periods and the effects of the fatty acids were probably due to inhibition of enzyme activity. so the possibility remains that over longer periods the various fatty acids may have differential effects on the rates of enzyme synthesis or degradation. all of which
ketogenesis 111~114.
and
gluconeogenesis.
Biochem.
J.
140.
MISHZ;INS., STEIN
L., GRATMAITAN Z. & ARIASI. M. (1972) The binding of fatty acids to cytoplasmic proteins: binding to 2 protein in liver and other tissues of the rat. Biochem. hiophys. Res. Commurt. 47. 99771003. MISHKINS. & TLIRTOTTER. (1974), The bindine of long chain fatty acyl-CoA to Z, a cytoplasmic pro& present in liver and other tissues of the rat. ~iac~e~~. bja~~~~s. Rrs. Comrrnm. 57. 9 18-926. NILS~ONA.. SI;NDL~RR. & A~ESSONB. (1973) Biosynthesis of fatty acids and cholesterol in isolated rat liver parenchvmal cells: effect of albumin bound fattv acids. Eur.
NILS~ON A.,
SUNDLERR. & AKESSONB. (1974) Effect of different albumin-bound fatty acids on fatty acid and cholesterol biosynthesis in rat hepatocytes. FE&S Lett. 45. 282-285. NOBLER. C.. STEELEW. & M~~KE J. H. (1971) The plasma lipids of the ewe during pregnancy and lactation. Res. vet. Sci. 12. 47-53. OCKNERR. K., MANNINGJ. A., POP~ENHAUSER R. B. & Ho W. K. L. (1972) A binding protein for fatty acids in cgtosol of intestinaf mucosa, liver. my~arditlm and other tissues. Science 177, 56-58. PANDE S. V. & MEADEJ. F. (1968) Inhibition of enzyme activities by free fatty acids. J. hiol. Chem. 243. 6180-6185. PARVINR. & DA~SH~NA~RTIK. (1970) Inhibition of gIuconeogenic enzymes by free fatty acids and palmitoyl coenzyme A. J. hiol. C’hrm. 245, 5733-5778. ROSEH., VAUGFIANM. & STEINBERG D. (1964) Utilization of fatty acids by rat liver slices as a function of medium concentration. Am. J. Physioi. 206, 3455350. AckJlO~I~~~rr,lf,~zf-The author wishes to thank Mrs. E. SAFGEKSONE. D. (197%) The regulation of glvceride synTaylor for skilled technical assistance. thesis in isolated fat cells: the-effects of paimitate and lipolytic agents. Biocizem. J. 128. 1057-1067. REFERENCES SA&G&ON E. D. (197%) The regulation of glyceride synthesis in isolated fat cells: the effects of acetate, pyruvate, BARTLEY J. C. & ABRAHAM S. (1973 Hepatic lipogenesis lactate, paimitate, electron-acceptors, uncoupling agents in fasted. re-fed rats and mice: response to dietary fats and oligomycin. Biockan, J. 128. 1069-l 078. of differing fatty acid composition. Biochim. hiophys. Acta 280, 258-266. SAGGERSONE. D. & TOMASS~ G. (19711 The regulation of glyceride synthesis from nvruvate in isoiated fat cells: DORSEY J. A. & PORTERJ. W. (1968) The effect of paimitvl ihe elfects bf palmitate ani alteration of dietary status. coenzyme A on pigeon liver fatty acid synthetase. J. hi& Eur. J. Biochetn. 23. 10%I t 7. thrm. 243. 35 12.-3516. Dn T. J. & KRUGER F. A. (1977) Effect of essential fatty Scow R. 0. & CHERNICX S. S. (1970) In Comprehensive acids on fatty acid synthesis in epididymal fat cells of Biochrrtktry (Edited by FLORKINM. & STOTZ E. H.) the rat. J. Ntctr. 102, 1033-103X. Vol. IX. pp. 20-49. Elsevier. Amersterdam. GC~~RI~X.XA. G. (1972) Regulation of the activity of acetyl SHAPIROB. ( 1965) In Harldhook qf Physiology (Edited by RENQLUA. E. & CAH~I_LG. E.. JR.) Section 5, pp. coenzyme A carboxylase by palmityl coenzyme A and 217-223. Williams & Wilkins. Baltimore. MD. citrate. J. hiof. Cheni. 247, 6946-6952. HALESTRAPA. P. & DENTOWR. M. (1973) Insulin and the %ORANAS. R. & SAGGERWNE. D. (1975) Studies on the regulation of adipose tissue acetyl coenzyme A carboxyrole of insulin in the regulation of glyceride synthesis lase. Biochem. J. 132, 5099517. in rat epididymal adipose tissue. Biodirm. d. 150, HANSONR. W. & BALLARDF. J. (1967) The relative signifr441-451. cance of acetate and glucose as precursors for lipid synSREREP. A. (1965) Palmityl coenzyme A inhibition of the thesis in liver and adipose tissue from ruminants. Biocitrate-condensing enzyme. Biochim. hiophys. Acta 106, them. J. 105, 519-536. 445-455. HANSON R. W. & BALLARD F. J. (1968) Citrate, pyruvate SLTMPER M. (1974) Control of fatty acid biosynthesis by and lactate contan~inants of commercial serum albumin. long-chain acyi-CoAs and bv liuid membranes. Eur. J. J. Lipid Rrs. 9, 667-668. BiO&i?l. 49, 469-475. _ . INGLED. L.. BAUU~ND. E. & GARRIGUSU. S. (1972) LipoTA~ETAK. & POGELLB. M. (1966) The effect of palmityl genesis in the ruminant: in vitro study of tissue sites, coenzyme A on glucose 6-phosphate dehydrogenase and carbon source and reducing equivalent generation for other enzymes. J. hiol. Client. 241, 720-726. fatty acid synthesis. J. NW. 102, 609616. VERNONR. G. (1975) Effect of dietarv safflower oil uuon JARRE-~T 1. G.. FILSELL0. H. & BALLARDF. J. (19’74)Metalipogenesis in neonataf lamb. ~~~~~~10, 284-289. ’ bolic and endocrine interrelationships in normai and VERNONR. G. (1976) Etfect of dietary fats on ovine adipose diabetic sheep. Harm. Merah. Res. Supof. Ser. 4. 1I 1-i 16. tissue metabolism. Lipids 11. 662-669. JUNGASR. L. (1968) Fatty acid synthesis in adipose tissue WATERMAN R. A., ROMEOS D. R.. TSAI A. C., MILLER E. R. incubated in tritiated water. ~~oc~~}ii~.~~r r 7, 3708-37 16. & LEVULLE G. A. (1975) Intluence of dietarv safflower MAYES P. A. & TOPPINGD. L. (1974) Regulation of hepatic oil and tallow on growth, plasma lipids and Iipogenesis lipogenesis by plasma free fatty acids: simultaneous in rats. pigs and chicks. Proc. Sot. esn. Biol. Med. 150, studies on lipoprotein secretion. cholesterol synthesis. 3477351: -
Research
Institute.
Ayr. Scotland
KA6 5HL,
U.K.
(Rw(~iwd 15 Decernht~r 1976) Abstract-l. The effect of different fatty acids in vitro on the rate of lipogenesis in rat and sheep adipose tissue slices has been investigated. 2. Both palmitic and linoleic acids, chosen as representative saturated and polyunsaturated fatty acids, reduced the rate of lipogenesis in rat and sheep adipose tissue by inhibiting the activity of one or more of the enzymes of lipogenesis. 3. In both species, stearic acid was the most effective inhibitor of lipogenesis; palmitic and oleic acids and mixtures of fatty acids, the compositions of which were based on that of the unesterified fatty acid fraction of rat and sheep plasma, were equally effective inhibitors of hpogenesis; linoleic acid was a less effective inhibitor of hpogenesis than palmitic acid. 4. The results are comoared with the effects of dietary saturated and polyunsaturated fatty acids on lipogenesis in rat and sheep adipose tissue.
INTRODUCTION
Radiochemical Centre. Amersham, U.K.; E. Merck Ltd., Darmstadt. Germany; Koch-Light Ltd.. Colnbrook, U.K. Bovine serum albumin was partially alysed (Hanson & Ballard. 1968). The had an unesterified fatty acid content albumin.
Lipogenesis in rat and sheep adipose tissue is inhibited by both the amount and the nature of dietary fat (Du & Kruger, 1972; Waterman et al.. 1975: Vernon, 1976). Lipogenesis in ovine adipose tissue was inhibited more by dietary saturated fatty acids than polyunsaturated fatty acids (Vernon, 1976). For lipogenesis in rat adipose tissue the picture is less clear; an earlier study (Du & Kruger. 1972) suggested that adipose tissue lipogenesis was inhibited to a greater extent by dietary polyunsaturated fatty acids than saturated fatty acids, thus resembling rat liver in its response to different dietary fatty acids (Bartley & Abraham, 1972). However. a more recent study (Waterman rt ul. 1975) has indicated that lipogenesis in rat adipose tissue was more inhibited by dietary saturated fatty acids than polyunsaturated fatty acids. and thus resembled ovine adipose tissue in its response to different dietary fatty acids. The mechanisms by which dietary fatty acids exert their differential effects on adipose tissue lipogenesis are unknown. Palmitic acid, however, has been shown to inhibit lipogenesis in rat adipose tissue irr citro (Saggerson & Tomassi, 1971; Soorana & Saggerson, 1975). Thus the purpose of this study was to compare the effect of different fatty acids. in particular palmitic and linoleic acids, chosen as representative saturated and polyunsaturated fatty acids. respectively, on lipogenesis in sheep and rat adipose tissue in dro to see if the
varied
effects
of dietary
fatty
acids
defatted and difinal preparation of 4.3 nmole/mg
Female Wistar rats. weighing about 300 g. were obtained from A. Tuck & Sons Ltd.. Rayleigh, Essex. U.K. Food (diet 41B. Oxoid Ltd.) and water were supplied ad libiturn. Sheep were pure bred Cheviot ewes. 556 years old; they were maintained on a diet of hay given at 7 a.m. and 4 p.m. each day. Rats were killed by cerebral dislocation and sheep were killed with a captive-bolt humane killer between IO and 11 a.m. Parametrial adipose tissue (rats) or perirenal adipose tissue (sheep) was removed immediately and kept in isotonic saline at 37
coditiorls
Samples (6&80mg) of adipose tissue slices. prepared free-hand. were incubated in 2.5 ml of Krebs-Ringer bicarbonate buffer. pH 7.4 with half the original calcium concentration (1.27 mM) and containing 5 mM glucose and 40 mg/ml bovine serum albumin.
Following a pre-incubation period of 15 min. either 0.25 PCi of (I-‘Qacetate or 0.05 $Zi of (I-“C)-acetate plus 2.5 mCi 3H,0 was added to the incubation medium: the final acetate concentration was 2 mM in each case. The tissue slices were then incubated for 2 hr at 37 C in an atmosphere of Oz:CO, (95:4v/v) in a shaking water bath. Incubation was terminated by removing the tissue slices with fine forceps and homogenising them in chloroform:methanol:0.15 M KC1 (I : 1: 1 by vol). Lipids were extracted and their fatty acids were transesterified using methanolic HCI as described previously (Vernon, 1975). The fatty acid methyl esters were extracted with hexane. their weight was determined gravimetrically, and following solution in a toluene scintillation fluid, their radioactivity was measured with an efficiency of 90”, in a Packard Tricarb 2425 liquid scintillation spectrometer. Samples of the
were
due to differential, direct effects of the fatty acids on adipose tissue lipogenesis. MATERIALS AND
silica gel G from Unisolve I from
METHODS
Materials Insulin (24 unit/mg), unlabelled fatty acids and bovine serum albumin (fraction V) were purchased from Sigma (London) Chemical Co. Ltd.: ( I-‘4C)-acetic acid. 59 mCi/ m-mole. (I-“Ctpalmitic acid. 59 mCi.!m-mole, (1-‘4C)linoleic acid. 57 mCi!m-mole and 3Hz0. 5 Ci’ml from the 517
R. G.
518
aqueous residue left after extraction of the fatty acid methyl esters with hexane were mixed with Unisolve I and their radioactivity was measured with an efficiency of 80?,. This aqueous fraction contained the glycerol derived from glycerides and other water-soluble components of the lipids released during transesterification. These components were not further separated and for the purpose of this study it was assumed that most of the radioactivity of the samples was associated with glycerol derived from glycerides. Addition of‘furty u&is
Fatty acids were taken up in 0.1 mt of 0.1 N KOH containing 4mg albumin and-were added to the incubation medium immediatelv before the addition of (1-‘%-acetate. KOH (0.1 ml of 0.1 6I) containing 4 mg albumin was added to control flasks. (‘~C~labelled fatty acids were added to the standard incubation medium. containing 2 mM unlabelied acetate to give a final concentration of palmitic or linoleic acid of 1 mM, O.O4~Ci/ml. Incubations and extraction of lipids were performed as described above. Samples of extracted lipid were separated into the major lipid-classes by thinlaver chromatogranhv on silica gel G (Vernon. 1975). The appropriate baids’of’silica gel w&e transferred to scintillation vials, and a gel was formed by the addition of H,O and Unisolve I (4: 10 v/v). The radioactivity of the gels was measured with an eficiency of 75”,;. Expression
of results
In view of the wide variation in the rate of lipogenesis found between animals, results for each individual experiment were expressed as a percentage of the control value (rate of lipogenesis found when tissue was incubated for 2 hr in the absence of fatty acids). These figures were pooled with corresponding values from other experiments to obtain the mean and S.E.M. values quoted. Statistical significance was calculated on the basis of Student’s t-test using a test for paired observations where appropriate. RESULTS Preliminary experiments with sheep adipose tissue slices showed that although glucose stimulated the rate of incorporation of (I-14C)-acetate into fatty acids, insulin had no effect on the rate. either in the presence or absence of glucose (Table 1). Addition of albumin to the incubation medium resulted in a significant (P < 0.05) increase in the rate of (l-14C)acetate incorporation into fatty acids in the presence of glucose and insulin in sheep adipose tissue slices (Table 1). but not rat adipose tissue slices (results not shown). It was also found that for both sheep and rat adipose tissue slices, less than lg/, of the
VERNON
labelled fatty acids produced during incubation with labelled precursors were released into the incubation medium; this was not affected by the addition of fatty acids to the incL~bation medium. For initial experiments in which the effects of fatty acids on the rate of fatty acid biosynthesis were examined, palmitic and linoleic acids were used as representative saturated and polyunsaturated fatty acids respectively. In the absence of insulin. addition of either 1 mM palmitic or linoleic acids signi~cantly (P < 0.001) reduced the rate of incorporation of both (l-‘4C)-acetate and 3H,0 into fatty acids in rat adipose tissue slices (Table 3). Palmitic acid resulted in a greater degree of inhibition of both ( l-‘4C)-acetate and 3H20 incorporation into fatty acids than did linoleic acid, but the difference was small and was only significant (P < 0.05) for the incorporation of 3H,0 into fatty acids. The incorporation of (I-14C>acetate into fatty acids was always inhibited to a greater extent by added fatty acids than was the incorporation of 3Hz0. but the ratio of 14C:3H incorporated was the same when either palmitic or linoleic acids were added (Table 2). Both fatty acids stimulated the incorporation of 3H,0 into glyce~de-~ly~rol (Table 2). Palmitic acid was slightly, but significantly (P < 0.05) more effective than linoleic acid. When insulin was included in the incubation medium the rates of incorporation of both (l-14C)acetate and 3H,0 into fatty acids in rat adipose tissue slices were significantly (P < 0.01) increased in both the presence and absence of added fatty acids (Table 3). In the presence of insulin, palmitic acid, but not linoleic acid. still significantly (P < 0.05) reduced the rate of incorporation of ( I-14C)-acetate into fatty acids (Table 3). Also, the rate of incorporation of both labelled precursors into fatty acids was significantly (P < 0.05) lower in the presence of 1 mM palmitic than I mM linoleic acid (Table 3). Insulin had no effect on the rate of incorporation of 3Hz0 into glyceride glycerol in the presence of added fatty acids (Table 3). however, in their absence. insulin resulted in a small, but significant (P < 0.05) decrease in the rate of incorporation of 3HZ0. In analogous experiments using sheep adipose tissue slices. addition of 1 mM palmitic or linoleic acids to the incubation medium again resulted in a significant (P < 0.001) decrease in the rate of incorporation of (I-‘“Ctacetate into fatty acids (Table 3); again, I mM palmitic acid was significantly (P < 0.05) more effective than 1 mM linoleic acid (Table 2). However, in contrast to rat adipose tissue slices, inclusion of insulin in the incubation medium had no effect on
Table 1. Effects of glucose, insulin and albumin on the rate of incorporation of (l-14C)acetate into fatty acids in sheep adipose tissue slices
Results are mean + S.E.M. of 3 observations. Concentrations: insulin 0.1 unit/ml: glucose 5 mM; albumin 4”” (w/v)
Effect of different fatty acids on lipogenesis
519
Table 2. Effect of palmitic and linoleic acids on the rate of incorporation of (1-‘4 C)-acetate into fatty acids and 3Hz0 into fatty acids and glyceride-glycerol in rat and sheep adipose tissue slices
Labelled
precursor
Relatwe rate of incorporation (“,, control value)
Rate of mcorporatton (control *alue: pmole:hr per g wet wt tissue)
Species
Lmoleic
Palmttic acid il mMl
acid
(1 mMl
lncorporat~on 1”~ fatty
actdc ,I-1Tl.acetate %I10 Ratio ‘H:“C (I-“C>acetate Incorporatmn mto glycerideglywol ‘H,O
Rat Rat Rat Sheep
0.65 * OlO(lbl
54 9 60.1 1’3.2 54.0
0.67 It 0.07 110)
144.7 + b.3
0.30 * no5 (251 0.25 i: (107 (I
II I?‘)_t0081111
Rat
* * f I
3.0 3.X h I 2.2
hl x 74,4 176.6 606
* * * k
3.7 6.4” 4.0 2.9”
136.9 & 5.5’
Control values represent the rate of incor~ration observed in the absence of added fatty acids (rat and sheep) and the absence of insulin (rat). Results are mean + S.E.M. with the number of observations in parenthesis after the control values. “Value si~ifi~antly different (P < 0.05) from corres~slding value obtained with palmitic acid. Table 3. Effect of insulin on the relative rates of incorporation of (I-‘%Z)-acetate into fatty acids and 3H,0 into fatty acids and glyceride-glycerol in rat and sheep adipose tissue slices in the presence and absence of added fatty acids
Rat adtpasc __..-__
Addition Fatty acrd 11 mM1 Pnlmittc Lmolcic NOIE Palmitic Lnmletc
Ftttt! .__
lnsulm (0
55.1 + 5 R(8p” 67.3 z 7.2”.“,’ 123.1) + 33 O”,”
acid actd + * +
acid acid
Gljcende-glycerol
acrds -I___
._-
(I-‘w-acetate
I unit/mll
1li15; 126’~“~~ v7 -
0 ; 2S.P’.6
Sheep adtpose tissue Fatty acrds
tissue
‘H>O
il-“Qacetate
%,O
59 5 * 4.7 l4F” 80 5 f X 3a.b.c
1399 137.3 906 1416
7Yiii- 44,6~~d 1s.:,s ;
I 2l? 70X.4 * ?O.Xl,‘.d
* i + I
44.5 66.3 105.6 60.5 71.6
52(4P 3.P 2.6” &I*.”
t35.7 * 8.P”
+ z f + +
5.9 (3)4,’ 6.6@ 42 3.2“~” 7.P”
Results are expressed as a percentage of the rate observed in the absence of added fatty acids and insulin and are mean f S.E.M. with the number of observations in parenthesis after the first value of each column. Value differs significantly (P <: 0.05) from: ‘Value obtained in absence of added fatty acids and insulin. b Value obtained in absence of added fatty acids and presence of insulin. ‘Corresponding value obtained with palmitic acid. ‘Corresponding value obtained in absence of added insulin. Table 4. Effect of palmitic and iinoieic acid concentration on the rate of incorporation of 3H,0 into fatty acids and glyceride-~y~erol in rat adipose tissue slices.
Fatty fnsuiin (0.1 uoiUm1) Incorporatmn
(I-“Cl-acetate
Incorporation >H,O
0
l6:O IX:1 16:O IX:? lb:0 1X-3 16-O 18 2
IW 1cQ I00 loo loo 100 loo
97.4 103.6 99.0 943 lOS.9 109.0 110.3 107.0
l6:O
loo
1X:2
loo
131 7 f 11.213) 115.6 + 10.7(3)
acid =~~~~“t~~tt~~
IW
(PM) t*Ml
300
ho0
i + ?I f * i A f
5.3 (3) 0.6131 7.0 (6) 3 9 16) 4.2 (3) 99.4 i_ 8.2 (3) 2.3 (3) 117.3 II6 3 rt 3.3 (3) 5
64.1 66.3 57.6 54.5 90.5 99.4 130.3
157.0 * 4713) 134.1 * 15.613)
169.X _t 7.813) 154.4 f 3.9 (3)
into fatty acids
‘H,O
Ratio of ‘H fatty acids
Fatty acid
and (l-‘%‘)-acetate
“C
incorporated
mto
+ +
* 8.3 13) i 4 5 (31 + 17.7 I61 li_ 28161 + I.3 (3) f 9.913) + r 1.4 170)(3)
Y7.0 X3.6 71 5 81.2 1102 96 I 117.7 II50
4.6 (3) 5.4 (3) X0(6) 5.4(6l 7413) 4.413) 4.713) 2.3131
71.X 16.5 63.4 59.3
t i k or
110.5 _t
It + ir t * i t C
5.5(3) 8.1 (3) 6.1 16) 3.7 16) 3.5 (31 0.6131 5.4(3) 6.0(3)
into glye~ride-glycerol 140.2 + 4.0(31 124.4 * i 1131
The rates of incorporation of (l-‘4C)-acetate and ‘Hz0 into fatty acids and glyceride-g~yccrol in the presence of added fatty acids are expressed as percentages of the rates observed with controls incubated in the absence of added fatty acids. Results are mean + S.E.M. with the number of observations in parenthesis. the rate of incorporation of (I-14C)-acetate into fatty acids in the absence or presence of 1 mM added fatty acids (Table 3). The degree of inhibition of incorporation of (l-14C)-acetate into fatty acids in both rat and sheep
adipose tissue slices, and ‘Hz0 into fatty acids in rat adipose tissue slices, was dependent on the concentration of fatty acid added to the incubation medium (Tables 4 and 5). As the concentration of fatty acid in the control flasks was 170~M due to
R. G.
520
VERNON
Table 5. Effect of palmitic and linoleic acid concentration on the rate of incorporation of (I-14C)-acetate into fatty acids in sheep adipose tissue slices
acids in the presence of added fatty acids was expressed as a percentage of the rate observed in the absence of added fatty acids. Results are mean + S.E.M. of 3 ohservntions.
the presence of residual unesterified fatty acids in the albumin, the true range of initial fatty acid copcentrations was from 170 to 1170 PM, corresponding to a fatty acid:albumin molar ratio of 0.3-2.0. Changes in the medium unesterified fatty acid concentration during the incubation period were not measured, but from the rates of fatty acid esterification given in Table 8. it can be calculated that after a 2 hr-incubation with either 1 mM palmitic or linoleic acids, approx 12”” and 4”, of the added fatty acid would have been utilised by rat and sheep adipose tissue slices, respectively. Palmitic and linoleic acids had similar effects on the rate of incorporation of (I-‘“Clacetate and 3Hz0 into fatty acids in rat adipose tissue slices over the range of concentrations used. Addition of insulin relieved the inhibition of rat adipose tissue fatty acid biosynthesis at all fatty acid concentrations tested (Table 4). Both the ratio of 3H : ’ 'C incorporated into fatty acids and the incorporation of 3H into glyceride-glycerol in rat adipose tissue slices increased as the concentration of added fattv acids was increased (Table 4). At all concentrations. palmitic and linoleic acids had essentially an identical effect on the ratio of 3H:‘JC incorporated into fattv acids. Analysis of variance showed that palmitic acid resulted in a significantly (P < 0.01) greater rate of incorporation of 3H into glyceride glycerol than did linoleic acid over the concentration range used. With regard to sheep adipose tissue. addition of palmitic acid resulted in a lower rate of fatty acid
Table
Rat
Sheep
6. Effecectof incubation
None Ih:0 IX.7 h”llC Ih ,, IX:7
time on the incorporation tissue slices in the presence
+ + +
synthesis than did addition of linoleic acid at all concentrations but an analysis of variance showed that the difference was not statistically significant (Table 5). The rate of lipogenesis in rat adipose tissue was linear with respect to time over the 2 hr-incubation period (Table 6). Inhibition by palmitic and linoleic acids was statistically significant (P < 0.05) at 10 and 30 min. respectively, after their addition. In contrast, the rate of lipogenesis in sheep adipose tissue increased with time during the experimental period. Inhibition by fatty acids was significant (P < 0.05) at all times except 30min after their addition. The reversibility of the inhibition of lipogenesis by fatty acids was examined by incubating adipose tissue slices in the presence of fatty acids (1 mM) for 60 min following which the slices were transferred to flasks containing medium devoid of fat.ty acids. These experiments showed that the rate of lipogenesis, previously reduced by incubation in the presence of fatty acids, was readily restored to the control value when the tissue slices were incubated in medium containing no added fatty acids (Table 7). The rate of uptake of palmitic and linoleic acids by both rat and sheep adipose tissue slices, as reflected by their rates of incorporation into lipids, was measured at IO, 60. 120 and 180 min after the addition of labelled fatty acids, and was found to be linear with respect to time over the whole experimental period (data not shown). There was no significant difference in the rate of uptake of palmitic and linoleic acids. Insulin had no effect on the rate of incorporation of fatty acids into lipids (Table 8). The effects of palmitic and linoleic acids on fatty acid biosynthesis in rat and sheep adipose tissue were compared with those of other fatty acids and also of mixtures of fatty acids, the composition of which were based on that of the unesterified fatty acid fraction of plasma from rats (Rose et al.. 1964) and sheep (Noble et al.. 1971) (Table 9). At a concentration of 1 mM, stearic was a significantly (P < 0.05) better inhibitor of both rat and sheep fatty acid biosynthesis than palmitic acid. With the exception of linolenic acid, the other fatty acids tested and also the fatty acid mixtures had similar effects on the rate of fatty acid biosynthesis to palmitic acid. The rate of incorporation of 3H10 into fatty acids in rat adipose tissue was not inhibited by the presence of 1 mM linolenic acid. Also, 1 mM linolenic acid did not stimulate the
of (1-“Q-acetate into fatty acids and absence of added fatty acids
7x * JO + hJ+2, 45 * 27 + 29 *
20 I)‘)” 11’1 115” 0 6”
26hi_13 IJK * ,5x i-0’)” IJI i-l!, ,,,7 +,15
17”
Ii 6 i_0.6
in rat and
r3.2 f JY
766+ 3.3” 27 I * 36” ?Y7+4? 77x * 07”
2x9* lb”
sheep
adipose
IUI 51.7 + 15.2 ii.9 + 9 6” 100 55 2 * 2.0” 6U.3 k 4.0”
Amounts of (I-“Qacetate incorporated into fatty acids are expressed as a percentage of the amount of ( 1-14C)-acetate incorporated into fatty acids in 120 min in the absence of added fatty acids. Results are mean + S.E.M. of 3 observations in each case. a Value significantly different (P < 0.05) from corresponding value obtained in the absence of added fatty acids.
Effect of different Table 7. Effect on the rate of incorporation incubating the adipose tissue slices in fatty
521
fatty acids on lipogenesis
of (I-“C)-acetate acid-free medium
into fatty acids in rat and sheep adipose tissue of after incubation in the presence of added fatty acids
Amounts of (I-“C)-acetate incorporated into fatty acids during each period are cxpressed as a percentage of the amount of (i-‘4C)-acetate incorporated into fatty acids in 2 hr in the absence of added fatty acids. Results arc mean k S.E.M. of 3 observations in each case. “Value significantly different (P < 0.05) from corresponding value obtained during OWN min.
incorporation of 3H,0 into glyceride-glycerol in rat adipose tissue slices. as did the other fatty acids; both rates were significantly (P < 0.05) different from those found using 1 mM palmitic acid. DISCC’SSION
A comparison of the effects of different fatty acids or their acyl-CoA esters on the activities of the enzymes of fatty acid biosynthesis is complicated. as, Table 8. Rate of incorporation of palmitic and linoleic acids into lipids in adipose tissue slices from rats and sheep
Insulin
Species Rat
(0 I unlt,ml)
* I,“)”
L,nolc,c
.ud
I1 mMI
11ur Ill50
+
acld
II mMI
+
Rat Sheep
Pahmt~
102tI II Y
*
45
YY 4 + 5 2 llh.,
+ xx
” 100% Corresponds to rates of fatty acid incorporation into lipids of 1.91 & 0.24 and 0.59 + 0.14 nmole!hr per g wet wt tissue for rat and sheep adipose tissue slices. respectively. Results are expressed as a percentage of the value obtained with 1 mM palmitic acid in the presence of insulin are mean + S.E.M. of three observations in each case.
being detergents. fatty acids and their acyl-CoA esters can inhibit enzymic activities by non-physiological processes (St-et-c. 1965: Taketa & Pogcll. 1966; Dorsey & Porter. 196X: Pande & Meade. 196X: Parvin & Dakshinamarti, 1970). Furthermore, both fatty acids and their acyl-CoA esters are probably bound to proteins or membranes within the cell so that their effective concentration is uncertain (Ockner tjt LI/., 1972: Mishkin t’t LII., 1972. 1974: Sumper. 1974). Thus it seemed preferable to compare the effects of different fatty acids on lipogenesis using intact cells; tissue slices in fact were used rather than isolated cells as it has not as yet proved possible to make satisfactory preparations of adipocytes from adult sheep adipose tissue. For such a system to be meaningful. it is clearly important that the concentration of the components of the incubation medium should be in the physiological range. Of the incubation medium components. the concentration of insulin added was in excess of the normal plasma concentration. but as some of the insulin would be bound to the glass surface of the incubation flask, its actual concentration in the incubation medium was uncertain. The range of fatty acid concentrations used in this study. 17&l 170 /tM (including the contribution of the albumin fatty acids) was in the physiological range for both rat (?O&l700 /tM. SCOW & Chernik.
Table 9. Relative rates of incorporation of (I-r’C)-acetate into fatty acids and acids and glyceride-glycerol in rat and sheep adipose tissue slices in the presence acids
Results are expressed as a percentage of the rate observed in the absence of and are mean + S.E.M. with the number of observations in parenthesis after each column. “Value differs significantlv (P < 0.05) from that for palmitic acid. ‘Composition of fatty acid mixture (wt percentage): palmitic 34; stcaric I?; 20 (used with rat adipose tissue); palmitic 26: stearic 30: oleic 39: linoleic 5 adipose tissue).
‘H,O into of different
fatty fatty
added fatty acids the first value in
oleic 34; linoleic (used with sheep
522
R. G. VERNON
1970) and sheep plasma (20&19OOpM, Noble et al., 1971; Jarrett et al., 1974). Thus, the results clearly show that physiological concentrations of fatty acids can modulate the rate of lipogenesis in both rat and sheep adipose tissue. It was previously shown that incubation of rat adipose tissue or adipocytes with palmitic acid inhibited the incorporation of (14C) from (2-‘4Cbpyruvate, (2-l&C)-lactate, (U-14Cbfructose and 3H from 3H,0 into fatty acids (Saggerson & Tomassi, 1971; Soorana (a: Saggerson, 1975). However, Saggerson (1972u,~) did not observe inhibi~on of fatty acid biosynthesis from glucose in the presence of palmitic acid. Although it is not certain that fatty acids were inhibiting fatty acid synthesis from glucose in the present study, it would seem probable as they did inhibit the incorporation of ‘H from 3H,0 into fatty acids which is a measure of the total rate of lipogenesis (Jungas, 1968). This apparent difference in the effect of palmitic acid on lipogenesis from glucose could be due to dissimilarities in the incubation media used. in the two studies or perhaps in the age of the animals, 1%18Og rats used by Saggerson compared with 280-320g used in this study. It is pertinent to note that the degree of inhibition observed with the various fatty acid concentrations used in this study was very similar to that found when rat liver was perfused with oleic acid (Mayes & Topping, 1974) and when rat hepatocytes were incubated with palmitic or oleic acids (Nilsson et al.. 1973); in both these studies with rat liver. glucose was present in the incubation medium; also the rats weighed between 250 and 35Og, so the effects of exogenous fatty acids on lipogenesis may vary with age. Elucidation of the mechanism by which exogenous fatty acids inhibit fatty acid biosynthesis was not a primary aim of this study. However, the rapidity with which added fatty acids exerted their effects and also the fact that the inhibition was readily reversed on removal of the fatty acids indicate inhibition of the activity of one or more of the enzymes of fatty acid biosynthesis as suggested by Soorana and Saggerson (1975). Also, the ease with which the inhibition was reversed excludes enzyme inactivation. The particular enzyme or enzymes affected remains to be elucidated, but as acetate was found to be essentially the only precursor of lipogenesis in the adipose tissue from Cheviot sheep (Vernon, 1976). it would appear that for sheep adipose tissue the fatty acids were inhibiting acetyl-CoA carboxylase activity. Furthermore. incubating rat adipose tissue with insulin irz citro has been shown to reduce the concentration of long-chain fatty acyl-CoA esters and activate acetylCoA carboxylase (Halestrap & Denton, 1973) providing a possible explanation for the ability of insulin to prevent the inhibition of fatty acid biosynthesis by fatty acids in rat adipose tissue. Although glucose plus insulin have been shown to stimulate the incorporation of (I -14C)-acetate into fatty acids in ovine adipose tissue slices (Hanson & Ballard, 1967; Ingle et at., 1973) the effect of insulin itself does not seem to have been reported previously. The reason for the tissue’s lack of sensitivity to the hormone is not known but it would not appear to be due to the age of the sheep. as insulin, in the presence or absence of glucose, also had no effect on the
rate of fatty acid biosynthesis in adipose tissue slices from either &day old or 3-month old lambs (Vernon, unpublished observations). The results suggest that both palmitic and linoleic acids inhibited fatty acid biosynthesis in adipose tissue by the same mechanism. Assuming that these two fatty acids were representative of the others tested, it would appear that stearic acid was the most effective inhibitor of fatty acid biosynthesis in both sheep and rat adipose tissue. Stearic acid has also been found to be a more effective inhibitor of fatty acid biosynthesis in rat (Nilsson et al., 1974) and chick (Goodridge, 1972) hepatocytes than other fatty acids. In contrast. the polyunsaturated fatty acids, linoleic and, in particular, linolenic acid, were less effective inhibitors of lipogenesis in adipose tissue than palmitic acid. The lack of effect of linolenic acid on lipogenesis in rat adipose tissue could be due to a relatively low rate of uptake compared with other fatty acids as it did not stimulate the rate of glyceride glycerol synthesis. This makes the usual assumption that the rate of esterification, as measured by either the rate of fatty acid incorporation into lipids or the stimulation of glyceride glycerol synthesis by added fatty acids, reflects the rate of fatty acid uptake as suggested by the observation that essentially all the fatty acids taken up by adipose tissue from fed animals were esterified (Shapiro. 1965). Thus, it would appear that the differential effects of stearic, pahnitic and linoleic acids on the rate of lipogenesis were not due to differences in the rate of uptake. Also, the effect of insulin, reducing the degree of inhibition of lipogenesis, would not appear to be due to a change in the rate of fatty acid uptake. However, although stearic acid was a more potent inhibitor of fatty acid biosynthesis in vitro than the other fatty acids tested, this would not appear to account for the effects of different dietary fats on fatty acid bios~thesis in adipose tissue in uiuo. Dietary tallow resulted in a si~ifi~tly greater decrease in the rate of fatty acid biosynthesis in sheep adipose tissue than did a mixture of sunflower and soyabean oil (Vernon, 1976). but both diets increased the plasma unesterified stearic acid level to the same extent relative to that of control animals receiving a low-fat diet (Noble R. C., unpublished ob~rvation). The only fatty acid whose plasma concentration was changed differently by the two dietary fats was linoleic acid (Noble R. C., unpublished observation), but the changes were small and although linoleic acid was a less effective inhibitor of ovine lipogenesis than palmitic acid, the difference would appear to be too slight to offer a reasonable m~hanism. For rat adipose tissue the situation is complicated by the apparently conflicting reports of the relative effectiveness of dietary saturated and polyunsaturated fatty acids on fatty acid biosynthesis in adipose tissue (Du & Kruger, 1972; Waterman et al., 1975). The reason for these different findings is not known, but, the rats used by Du and Kruger (1972) showed signs of essential fatty acid deficiency prior to the administration of the various fatty acids whereas Waterman et al. (1975) avoided this complication by always including some polyunsaturated fatty acids in the diet. It is pertinent to note that the rats used by Waterman et al. (1975) showed a similar response to different
523
Effect of different fatty acids on lipogenesis dietary fatty acids to the sheep used by Vernon (1976).
again received adequate levels of essential fatty acids in the diet. Unfortunat~iy. the composition and concentration of the plasma unesterified fatty acid fraction was not reported in either study with rats. However, stearic acid is a relatively minor component (12% by weight) of the rat plasma unesterified fraction (Rose et al., 1964). and at this concentration its effect on fatty acid biosynthesis was swamped by the other fatty acids (Table 9). so it would appear that the relative concentration of stearic acid would have to increase several fold before it would have a significant effect on the degree of inhibition of fatty acid biosynthesis. Furthermore. although palmitic acid was a more effective inhibitor of lipogenesis than linoleic acid. the difference was small. Thus, saturated fatty acids seem to be more effective inhibitors of lipogenesis than ~iyun~turated fatty acids in both sheep and rat adipose tissue, but although this could contribute to the differential effects of dietary fatty acids on lipogenesis itr tliz~, it would appear most unlikely that this difference is the major mechanism. However. in this study, tissue was exposed to fatty acids for only short periods and the effects of the fatty acids were probably due to inhibition of enzyme activity. so the possibility remains that over longer periods the various fatty acids may have differential effects on the rates of enzyme synthesis or degradation. all of which
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and
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J.
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