Dietary polyunsaturated fats suppress the high-sucrose-induced increase of rat liver pyruvate dehydrogenase levels

126

BBAUP 54184

Dietary polyunsaturated fats suppress the ~~~ue~~se-i~d~~~d increase of rat liver pyruvate dehydrogenwe levels Luis A. Da Silva, Olga L. De Marcucci and Zulay R. bhnle Institutede Medicina hperimental, C&h

di Biqubnica, Facultad de Medkina, Unive&$ad Cennal:& VcnaUea, Caracas (Venezuela) &c&ed

Key word8: L&gene&

12 February 1993)

Pyruvate dehydrogenase compkq High-sucrom diet; L&genie enyme; @‘%I oil)

Pymvate dehydrogenase complex @DC) has a key role in the regulation of hepatic lipogenesis by dietary.factors. We have investigated the effects of dietary carbohydrateand fat on hepatic PDC Sucrose-based.or starch-baseddiets i;e& administered for ,15 days. A positive [email protected] between PDC activity and the lipogenic potential of. the ~et.w.~;~~d~ A ague, fat-free diet caused a 3-fold increase in total activitywhereas a high-starch,fat-free ,diet caused a 1.5~foldincrease,.as .coinpai%d with chow-fed rats. Dietary polyunsaturatedfat (PUPI caused a marked inhibitory effect’on total and active PDC; ,fisb osi being more effective than corn oil. Dietary saturated’fat(butter) failed to inhibit the sucrose-inducedelevation in tot& activity,but i&s ahnost as effective as fish oil in depressing percent active enzyme. Changes iu total PDC, a&vi& e@ely- &r$ated with modifications in the content of enqme qua&Wed by inmumoblotting, indicating that increased. enzyme ckitgnt : imd II@ activationis the predominant mechanism underlying the adaptive response to high-sucrose,feeding. This response&suppi-e’ssed by dietary PUF. Inhibition of hepatic lipogenesis by PUP involves a reduction of PDC c&tent as we&its that of several‘&og&ic enzymes. The relevant me&anisms remain to be established.

High carbohydrate intake usually leads to hypertriglyc&ridemia in experimental animals and in man. Triglyceridemia is influenced by the type and level of carbol&drate intake; monosaccharides or disaccharides usually having a larger effect than starch, and fructose intake having a more pronounced h~e~~~~~c effect than glucose [1,2]. The differential effect of fructose has been attri%uted, among other factors, to increased hepatic ~~~1 synthesis and secre&on. Fructose has been shown to be preferentially used, as compared to glucose, as a carbon source for hepatic @pogen&is [3,43. lotion, the capacity of the liver to $ilize this ketohexose is enhanFd by prolonged &take. This effect is due to the induction of several key enzymes

Correspondence to: 0.L De Me Institute de Me@cina Ekperimental, CStedra de Bioquimica, Facu@d de Medicina. Uoiversidad Chtfal.deV enemeki, Apiutdo 54031. ~U~.~ 1051-A, Venezuela Abbreviations: PDC, pymvate dehydrogenase complex;PDCs,achive pyruvatedehydrogenase; PEG, polyethylen&&col; Pup; $yunsaturated fat.

involved in fructose rne~~~ NADPH bin, fatty acid and triacylgllicerol &thesis [$$I. On the other ‘,band, carbohydrate @ip@ation of hepatiq lipid sy@hesis is’ mod&ted hy the ty& and amount of dietary fat. It ti piielldocument& t&iii dietary polienoic lipids qe more effe,ctive thari sat& rated fat in inhibiting hepatic libr?ge’nesis+nd iq low& ~QJh~e~~~d~~ wberee oils rich izi’n-3 f& acids are tiore potent than ni6 lipids ti l@s re%skd Dietm fat suppression df liver and .adip&e ti@ie lip&genesis occuq in .twopEjq?s: an .e&: .(2-4 l$ response pos@bly involving a red* ‘in.t&. p@po& tkm of active pyruvafie dehydrogeiike., @DCa) a$ inactivation of a&tjl @A ca&oxil~;‘%i&a l.or&&& response (2-3 days)’ Mich @ails a r&&io~ ,c$ JC pogenic enzyme levels. Polyuns&rat& aqt &l&eater efficacy than saturates at loWeri& the rates Of’&nq@e synthesis and reducixig .th& amo&t ‘Sf *A &i&g for these enzymes @viewed. hi.&zfs.~7+.33 &td Z$%;.:” Pyruvate ‘dehidrogenasi cOmpltix’.(PDc)’@ +’m# tienzyme aggregate tit I+ potetitial sig@&ance * regulating the rate of Ji@genesiS a&d:&e’ovq&ll car%& hydrate u@izat@n pf tlie J&er. ‘Itt..he+eti pq~js&i that this enzjme playsan Cnportant ’role in tlie re,*,ation of hepatic’$&&nesis by &et& factti%@,9]..PI’X. is regulated bj; ph~pho~lation i&I de~h~~~o~l~~~~

127 of the alpha subunit of the pyruvate dehydrogenase component El). This tetrameric enzyme is tightly bound to the dihydrolipoyl acetyltransferase (E2) core, to which the other catalytic components (protein X and the lipoamide dehydrogenase, E3) and two regulatory enzymes (a protein kinase and a phoshatase) are attached [lO,ll]. Pyruvate is an inhibitor of PDC kinase and increased hepatic pyruvate concentration (likely to be present after fructose intake) may result iu activation of the complex, causing an increased flux of carbons from pyruvate to acetyl CoA and fatty acids. Increased flux through PDC may explain the acute effects of fructose on fatty acid biosynthesis [4,9,121. There are few reports on the long-term regulation of PDC, in particular the effects of high-carbohydrate and high-fat diets. In animals fed for l-3 weeks on high-sucrose or fructose diets, a Zfold increase in hepatic PDCa and total activity has been found [9,13,14].However, data on the effects of high-fat diets are conflicting, and their correlation with lipogenic rates is.uncertain [15-B]. We have recently reported [14] that hepatic PDC activity is reduced by high-PUF diets. In the present work, we further investigated the effects of various types of carbohydrate and fats on the enzyme activity. The data presented in this study demonstrate that hepatic PDC levels are influenced by the type and amotmt of dietary fat and carbohydrates in a similar way as has been reported for other enzymes related to fatty acid biosynthesis. These results support the proposal that this multienzyme complex plays an important role in the regulation of hepatic lipogenesis by dietary factors. Materialsand Methods ChemicaLF

The following chemicals were purchased from Sigma (USti substrates and cofactors for the enzymatic assays, reagents for imnumoblotting and polyethyleneglyco1 6000. Nitrocelhdose paper (0.45 pm) was obtained from Schleicher and !&hull (Easel, Germany). P_(paminophenyl)azobenzenosulphonic acid (AABS) was from Aldrich Chemical (MI, USA).

expense of reducing the carbohydrate or protein content. Protein varied between 20 and 25%. All diets were suplemented with cellulose (5%), AIN mineral mix (35%) and ATN Vitamin mix (l%b), choline bitartrate (0.2%), D,L-methioniue (0.3%) and Vitamin E (O.Ol%), supplied by TEKLAD Diets (Madison, WI, USA). The following materials were obtained locally: maize oil from COPOSA &uigua, Venezuela), cornstarch (Pandock, Caracas, Venezuela) and butter from ZARCO @&ado Miranda, Venezuela). Fish oil was generously provided by Schering Labs (Caracas, Venezuela). A group of animals was maintained with powdered standard laboratory chow (Ratarina from Protinal, Valencia, Venezuela). Water was provided ad hbitum. Rats were subjected to a 12 h light/l2 h dark cycle (06-18 h). Weight gain was recorded every 2 days. Final body weights varied between 180-200 g in all groups. of tisme c?xtracts Ehtperiments were initiated at 10.00 h. Animals were anesthetized with sodium pentobarbital (60 mg/kg body wt.). A portion of the hepatic right lobe was freeze-clamped and stored under liquid nitrogen until the day of extraction. Tissue extracts were prepared by sonication of powdered liver in an extraction btier according to [15], with minor modifications [14]. Clarified extracts were stored at -70°C.

Pnpuatim

Enzyme activity measurements The active form of pyruvate dehydrogenase complex was measured spectrophotometricahy according to [19]. Total PDC activity was assayed after a lo-15 min incubation in the presence of 5-10 ~1 of pigeon liver phosphatase (fraction 60% ammonium sulphate saturation) as described in 1141.Pigeon liver phosphoproteiu phosphatase and arylamine acetyltransferase were prepared as described previously 120,211.Citrate synthase activity was assayed according to 11191. Enzyme activity is defined as the amount of enzyme which catalizes the production of 1 pmol of substrate per min at 30°C. PDC activities were expressed per g wet weight, mg protein, per organ, or relative to citrate synthase units. Preparation of antigen and

A?lim&anddlets

Male Sprague-Dawley rats (initial weight 90-100 B), bred in our local animal house, were fed ad hbitum for 2 weeks one of the following starch-based diets: 65% starch, fat-free; 60% starch, 5% corn oil; 52% starch, 5% corn oil plus 10% fish oil; 45% starch, 22% corn oil; or one of the following sucrose-based diets: 65% sucrose, fat-free, 60% sucrose, 5% corn oil, 60% sucrose, 10% corn oil; 60% sucrose, 10% fish oil; 60% sucrose, 10% butter. Fat content was increased at the

antisenun

H.ighly purified PDC from bovine heart and polyclonal rabbit antiies against the whole complex were prepared as described in [22]. SDS po~acrylamide-gcl electmphomia

ting

and lmmunoblot-

1 ml tissue extract was incubated with 0.15-0.18 vol. 35% PEG at pH 6.4 for 30 min at 4°C with occasional stirring. Fully assembled PDC was precipitated by this treatment L23,241.Samples were centrifuged at 10000

128 TABLE I Fatty acid wmposition of dietary fats Fatty acid composition was determihed as described in the Materials and Methods section. Fatty acid

Butter

< 12:o 12:o 14:o 16:0 16 : 1 (n-7) 18:0 18 : 1 (n-9) 18: 2 (n-6) 18:3 (n-3) 20:5.(n-3) 22:6(n-3)

Fish oil

corn oil

8.2 3.0 12.6 38.9 3.8 11.5 28.4

14.8 15.3 12.8 0.6 16.0

1” -

4.9 21.6 5.8

15.2

29.9 54.9

x g for 3 min and total enzyme activity was assayed in the supematants to estimate recoveries. Rtiery’ of total PDC activity after PEG preci&tatioy was above 97% in liver extracts from the v?rious groups of animals used in this study. Pellets were dissolved in 1 ml 5%’ SDS by a 10 s sonication at room temperature. Samples containing up to 150 pg proteinwere loaded on 10% polyacrilamide slab gels. Electrophoresis was cariied out with the discontinuous btier system of Lae& [WI using pyronin Y as a tracking dye. The resolved proteins were then transferred ‘on to nitrocellulose paper essentially as described in 1261using a Tram+Blot Cell (Bio-Rad Labs.) at 40 mA foi 16-20 h. SDS was added to 0.02% to the transfer buffer. The nitrocellulose membranes were incubated for 1 h at room temperature with rabbit anti-PDC diluted 1: $0 or 1: 100 in blocking buffer [26,ifl. Nititicellulose paper was then washed and incubated for 1 h with protein A-alkaline phosphatase conj@ate (diluted

1: 10000). Binding of the .antibodies was assayed with nitroblue tetrazolium and 5-bromo-4 &Ioro-3 $Iolylphosphate. Purple stained-bands usually became visible within 5-10 min. Blots were dried, cut into strips and made tfanspar; ent by immersion in cedarwood oti. &ips w&?‘deiisitometrically scanned ai 550 nm with a afore 240 spectrophotomett?r. The areas under the, pe& c&esponding to E2 and Ela polypeptid& of.‘PrjC weti estimated by triangulation. Analytical methods

Protein tintent was determined using bovine .serum albumin as a standard:[28]. Fatty acid qalysis of. dietary .fat yas dete,+inedafter transmethylation as descriied [29].-.,&tty a&d methyl esters were qu+ntitated by gas ‘chr@atografi?.& in a glass column (length 1.83 112and’int@nal d&+&r 4 mm), packed with 4% PEG adipate’on Ch@m&orb AW (80-100 mesh) using a F&tile&Pack&d.58@-A chromatograph. Gvcn .temp&ttie w& ‘WC .aiid Carrier gas @Q flow rate was 60 ml/e.Inj+tor la&i detector tempeiatures were both 25@‘C..&My aci+ were identified by retention .tinhe in tiparis& *iti known standards. Statistical methods

Statistical evaluatioq of the resultswascarried out by one-way ANOVA u&g the ~Du&a&~&$ificance test. ResuIts Eficts of dietary fat on PDC activity in animal fed starch-based diets

Table II shows the effects of dietary..PUF qn PDC activity of rats fed starch-based diets for ‘2 weeks.

TABLE II Hepaiic PDC

activiiyin lulsjkl starchy

diets szglpla?lmtcdwith imiabk?ainou?mof Jloiywmtumtedfifrrt

Animals were fed ad libitum for 2 vlcs

on puribied’starch-based dieta supplemented or,not with PUF at the levels. i$icakd. ,,m, tot+ F and citrate synthase (Cs) activities wem measured in the extracts as described in the Materials and M&hods secti&. Rcmlt$cire &e m&Anf.S,E., A = 4-6. Values in the same column with di&rent mperscript lettei are signkantiy different (P< 0.05). N.D., not deter&n&. Type of diet

Expt. No. 1 Fat-free S%Comoil EX!$Yo. 2 Fat-& 5%c!Lnnoil 5% C!omoil+ 10% Fish oil 22%ckmloiI

PDCa mU/g wet wt.

mujg wet wt.

mU/U CS

U/organ

PDca %

159*14 * 82flOb 105* 5b

1186* 95a 83Of 36b 751f 76b

77*7 a 50*3b 45f5b

11.7*.1.3 a 8.7kO.7 b 7.4*0.6 b

16& TX ’ .8.9 f l..CC! 10.9v 2.4 b

N.D. 13Ok 6’

1524*146 * 1100* 9Ob

N.D. 51*3a

14.9*0.9 a 13.2k2.0 ’

12;0* 1.0 p

8Of 6b 76* 8b 68* 4b

1176f105 b 785* 35= 1181* 41b

44&4’ 34k2 b 46*3.

13.1 f 1.0 a &OiO.O b lo.o*~.o b

6.9yO.f$: 9.6* 1.0 = &o-j0;6 b

Total activity

129 There was a 3040% increase in total PDC activity per g wet wt. in the rats fed the high-starch, fat-free diet as compared with chow (expt. 1). The addition of 5% corn oil to the diet caused a signiiicant reduction of about 30% in total PDC activity, approaching the vahres from chow-fed animals. When corn oil content was raised from 5% to 22%, an additional 30% reduction of enzyme activity was observed (Table II, expt. 2). Since higher fat content rendered diets not easily accepted by the animals, rats were fed a diet containing a lower PUP level (15%) but with a large proportion of n-3 fatty acids. The combination of 5% corn oil plus 10% fish oil did not cause a further decrease in total activity as compared with 5% corn oil (T.able II, expt. 2). The effects of the different types of diets on total PDC activity can also be appreciated when values were expressed in terms of other parameters, such as units of citrate synthase or per organ. The active enzyme (PDCa) increased 15-fold in the rats fed the fat-free diet as compared with animals fed on chow, and decreased about 50% when the fat content was raised to 5% (Table II, expt. 1). A similar pattern was observed for % PDCa. Higher fat contents (15 or 22%) produced a further decrease in PDCa (Table II, expt. 2). Effects of die&myfats on PDC activity in animals fed suc?me-baseddiets The effects of dietary fat on PDC activity of rats fed sucrosebased diets are shown in Table III. Total PDC activity per g wet wt. increased about 3-fold in animals fed the fat-free diet as compared to those fed chow. The addition of PDF caused a significant reduction in total PDC activity as compared to the group fed the fat-free diet. Saturated fat (butter) failed to elicit the

same response (expt. 2). When the diet was supplemented with 5% corn oil, PDC activity dropped by 33%. Activity values further decreased, although not significantly, when corn oil or fish oil was raised to 10% by wt. However, these values were higher than those obtained from rats fed chow. The magnitude of the changes in PDC activity caused by the high-sucrose diets and by the addition of different amounts of fat are also observed when the values were expressed as a ratio to citrate synthase activity or per mg protein (Table III). The effects of the fat-free diet and dietary PDF on PDCa were similar to those observed for total activity. Percent PDCa was not signilicantly altered in the animals fed the fat-free diet for 2 weeks, and increased in those fed 5% corn oil as compared with chow-fed rats. Fish oil (10% by wt.) was more effective at inhibiting total and active enzyme than 10% corn oil, consequently, a very low proportion of active enzyme was present in the liver of those animals. Saturated fat (butter) did not suppress the sucrose-induced increase in total PDC activity, but it was ahnost as. effective as fish oil at reducing PDCa and percent PDCa (Table III, expt. 2). Total PDC activity values (Units/g wet wt.) obtained after 7 days of diet were significantly higher (70%) than those from chow-fed rats, but lower (25%) than in animals maintained on the fat-free diet for 15 days. A similar conclusion can be drawn by examining the results expressed in terms of citrate synthase activity or per rug protein. Effeccts of high-sucrose feeding on immumdetectable hepatic PDC levels Immunodetection using specific antibodies directed against bovine heart PDC was used as a probe to

TABLE III HqIaticPLxactivilyinratsfed

sllLavm~dit?ts~withfats

Animalswerefedadlibitumfw2weeLs~erpt.1)arasindicated~expt.2)ons ucrtxwbased diets supplemented or not with corn oil fish oil or bu~ratthelcvelsindicated.PDCa,totalPDCandcitratesynthaae(CS)activitieswere&~~intheliver~esdescn’bedinthe Material9 and Methpds aection.Results repwent the mean* SB., n = S-10. Values in the same column with dBerent superscript letter are ignifhntly diKerent (P< 0.05). N.D., not determined. Qpeofdiet

Total activity mu/g wet wt.

EkptNo.1 Fat-free S%ChllOil 10% corn oil lO%Ffislloil 2:. 2 Fat-he (7 d) Fat-h (15 d) 10% Butter (15 d)

mu/g wet wt.

mU/U C8

mU/mg ProtgJh

189&47 ’ 243*43n 95f20b 31* 2f 63f10b

2289*147. 1532k124 b 1363* 73b 1228* %b 886* 90=

112* 88* 79f 60* 46*

13.9* 0.8 * 9.8 f 1.0 b 8.6kO.4 b,c 7.7*0.7 c 4.8kO.J d

113*14. 111*19. 53* 3b 63* 7b

1828*166. 2417& 146 b 2414* 133 b 1079* 77=

137*18 ’ N.D. 17Ok16. 62k 6b

6. sb 5b 6c 5O

12.4 f 1.2 ’ 14.4 f 1.4 * 17.5* 1.3 b 7.8kO.5 c

(%I

10.6 f 3.0 0 15.7&20 * 8.1 f 1.3 b 2.5*0.1 c 7.2* 1.0 b 5.9rtO.6 ’ 4.6rtO.7 ’ 22f0.1b 6.0 f 1.0 *

130 establish whether the changes observed in total PDC activity were due to alterations in the content of hepatic complex. Since PDC represents a small component of the total protein of the liver cell, its immunological detection and quantitation by immunoblotting in a crude extract was hindered by the fact that relatively large amounts of protein had to be loaded onto the gels, and at these high levels, color intensity did not increase linearly with protein concentration. PEG precipitation of the complex present in the extracts allowed the loading of smaller amounts of protein yielding a linear response (O-26 &. Analysis by immunoblotting with anti-PDC serum of the supematant fractions after PEG treatment showed only traces of PDC polypeptides. Quantitative precipitation with high recoveries of activity were only obtained when the prouzdure was performed at pH 6.4 [23,24]. A large increase in hepatic PDC levels was observed when animals were transferred from standard chow to high-sucrose, fat-free diets. Fii. 1 shows the PDC polypeptide pattern obtained by immunoblotting of liver extracts from rats fed either chow or high-sucrose, fat-free diet. Anti-PDC serum exhibited high reactivity against all the components of the rat complex, except the lipoamide dehydrogenase 0X3), as previously described [22]. The pattern of subunits was similar to that of the bovine heart enxyme although the component E2 from the rat complex migrated as a protein of smaller Mr 1301.The relative cross-reactivity against E2

HS tot

frw

and Ela was higher than that for El/?. Densitometric scanning of the blots under the highest peaks (E2 and Ela components) indicated a 2.0-3.0-fold increase in PDC levels in the liver of animals fed the fat-free diet as compared with chow (lane 8 and lane 2, respeo tively). This pattern of differences was co&med in four separate experiments. A similar ratio was obtained when the other PDC components were used ,to make the comparison. High-sucrose feeding did not alter the pattern and ratios of PDC polypeptides, as compared with chow. Effects of dietaryfat on the h& high-sucme-fed animals

of hepzticPDC in

Fig. 2, part A illustrates the effects of 10% corn oil on the content of hepatic PDC. There was a reduction in PDC levels in the rats fed the diet containing’ib% corn oil compared to the animals fed the fat-free diet; A IS-2.2-fold decrease in PDC levels was estimated from the ratio of the areas under the E2 and Ela components (3 determinations). In ‘a sin&u way, a decrease in PDC content (3.0-4.@fold) was obserired in extra&s obtained from animals fed 10% fish oil (Fig. ZB), whereas no significant differences in hepatic PDC levels were observed when animals fed 10% butter were compared with those fed the fat-free diet (Fig. ‘3, 3 determinations). Supplementation of diets with the different types of fat did not cause changes in the ratios of PDC compo-

Chow

E2 X

I

Elk

0.05

El/3

L (2) S

I2

34

3

6

7891011

I2

131 nents as compared with the respective controls. Differences in the ratios of PDC bands observed in Fig. 2A as compared with blots shown in Figs. 1,2B and 3 are due to the lower dilution of antiserum used.

Discussion

The results presented in this study demonstrate that hepatic PDC activity and total content are modulated

HS corn dl HSfat frea --

HS fat free IA

B

E2 X El& E’P

HS flsh oil

b

(51

I

0.05 [

I

(I)

i FGg.2. Immunologlcd detection of PDc in liver kcun MB fed high-sucrose diets supplemented with polyunsaturated fats. Crude extmcts were prepared from rats fed on highdiets kupplemented or not with 10% corn oil (Part A) or 10% fish oil (Part B) and precipitated with PEG as described in the Materials and Methods section. Elcctrophomis and immunoblotting were performed as desaibed in legend toTig. 3, except thatfortheblotshowninPartA,thean~erumwssdiluted1:50.PartA:lanerrland5,40pgprotein,lanes2and6,U)pg;lanes3and7,10 ~g;lanes4and8,5p&S~comspondatobovineheartPDC(50ne)usedasastsndard.PartB:lanes1and5,25pgprotein;lanes2and6,50pg; lanes 3 and 7,75 pg; and lanes 4 and 8,100 pg. The arrow indicates the direction of ekctrophore-tic migration.

132

r-

HS butter

I2

34

5 678

9 1011 12s

Fig.3.lllmmoblottingoflumplesfromlivcrofr8tBfcdlligll-suau6c diets applemcntcd or not with saturated fat. PEGliver cxhmts wcrcpluparedfmalMimabfadhigll~ diets mppkmontcd or not with 1096butter. Other dota& u daicrii iy kgend to Fig. 1. Lauui 1, ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

by fatty acid composition and amount of dietary fat, as well as the type of carbohydrate present in the diet. Omission of fat caused a large stimulation of total and active enzyme in animals fed sucrose (about 3.0fold), and a moderate increase in PDCa and total activity in the starch-fed group (1.5fold), as compared to rats fed chow. The addition of 5% corn oil to the starch-based diet caused a reduction of the total activity of the complex to values which were similar to those obtained in chow-fed a&u&, whereas addition of PUP up to 10% (either corn or fish oil) to the highsucrose diet-depressed enzyme activity to values which were still above those found in rats fed chow. Thus, the potent stimulatory effect of sucroseuxUning diets rendered PDC activity more resistant to inbibition by dietary fat than starch-containing diets. Dietaq oils rich in polyunsaturated fatty acids suppressed the high&hydrate-induced increase in total PDC activity, whereas saturated fat (butter) had no effecLBothtypesofPUPaswellassaturatedfat produced a significant decrease in PDCain the animals fed sucrose, but fish oil had the greatest inhiiitory effect.

There was a positive correlation between PDC a.ctivk ity and the lipogenic potential of the diet. Dietary conditions which maximally stimulate lipogemxis WIh+ucrose, fat-free) caused the largest increase in the complex activity, whereas the addition of PUP, particularly marine oil which ‘effectively inhibits hepatic fatty acid synthesis, reduced .the complex~actihy despite the high-carbohydrate level .of the diet. The incapability of the diet amtahkg saturate&fat (10% by weight) in producing an inhibitory effect on total PDC correlates with the rower ability of this type of fat in depressing hepatic lipogenesis and lipopmc enzyme activities in rats [7,38]. Nevertheless, the 50% de&a@ of PDCa and percent. .PDCa U’able ‘III, expt. 2) suggests that saturated fat may also @hibit fbtx. through PDC and the lipogenic pathway., Decreased P,DCa activity by fatty acidg has been. attributed to elevation of the ratio acetylCoA/CoA and/or NADH/NAD resuljing from increased fat oxidatioc Michstinmktes PDC kinark activity and reduces percent act&enzyme [31-l.This may explain the inhiibitoky effects of samrated fat and PUP on the active e!n!@e. It has been previously reported that d&try satu-

133 rated fat (up to 40% by weight) had no effect on hepatic PDC activity in animals fed starch-based diets [15]. However, other authors [16] have observed a substantial decrease in hepatic total and PDCa activity. The omission of dietary carbohydrate in the latter work may explain the discrepancies observed. In rats fed glucose- or fructose-based diets, the effects of different types of fat on hepatic PDC displayed a complex pattern with no evident correlation with the rates of lipogenesis or with changes in the activities of the lipogenic enzymes 117,181. We have quantitated PDC levels in liver by using specific polyclonal antiiies directed against the whole complex from bovine heart, and demonstrated that the diet-induced variations in PDC specific activity were due to changes in enzyme content. Dietary treatments produced no significant changes in the polypeptide pattern and ratio of the components of the multienxyme aggregate. Alterations of PDC levels, as detected by immunoblotting, cannot be attributed to differential precipitation of PDC by PEG, since recoveries of total activity were above 97% in all cases. Changes in enzyme content were of similar magnitude to changes in specific activity values (Table III). Stimulation of hepatic PDC by fructose- or sucrosecontaining diets has been previously found [9,13,14]. However, the results of the present work show that the predominant mechanism involved in the adaptive response to high-sucrose feeding is an increase of enxyme content and not activation (dephosphorylation) of existing enzyme. Furthermore, it was found little evidence of any change in the proportion of enzyme in the active form. Increased hepatic PDC levels during high-sucrose feeding could, at least partially, account for the increased triacylglycerol synthesis promoted by this sugar. PUF feeding suppressed the sucrose-induced increase in enxyme content. Fish oil had a more pronounced effect on PDC levels than corn oil. This could be attributed to the n-3 fatty acids present in the former (Table I). Unlike PUF, saturated fat-feeding (at 10% by wt.) showed to be uncapable of signiticantly modifying the mechanisms which regulate the levels of PDC in liver cells. Since the activity of citrate synthase - an exclusively mitochondrial enzyme - did not vary significantly among the groups studied (data not shown), we can conclude that the long-term (adaptive) response of PDC to dietary factors involved a specific modification of PDC levels per mitochondrion and is not due to a general alteration of mitochondrial protein content. Gur results indicate that PDC activity was modified by diet in a similar way to that reported for other lipogenic enzymes, but the response seems to be slower. In our work, the degree of stimulation by high-sucrose, fat-free diets was dependent on the length of treat-

ment. After a week, total activity was below the level achieved after 2 weeks of diet. This slower response may be explained by the relatively long half-life of hepatic PDC (8.1 days; [32D as compared to other lipogenic enzymes (1-3 days; [33,343).It has been shown that changes in total PDC activity of adipose tissue took place after l-2 weeks of diet [15,35]. Dietary treatments did not cause a preferential effect on a particular PDC component, but had a similar influence on the levels of the various PDC polypeptides. Thus, long-term regulation by sucrose or dietary fat (or their metabolites) appears to iuvolve coordinated changes in the turnover rate of all the components of the complex in liver of rats. Since PDC polypeptides are encoded in the nucleus and imported into the mitochondria [36], PDC levels may be regulated by mechanisms that modify the rates of synthesis and/or degradation of the polypeptides as well as the rates of import and assembly of the complex inside the organelles. A lysosomal protease which cleaves and dissociates the complex has been partially purified and characterized from rat liver [371.At present, it is not known whether this activity plays a role in regulating the rates of degradation of the whole complex Elucidation of the mechanisms involved in the control of the levels of PDC polypeptides in rat liver mitochondria, as well as the interactions of signals derived from nutrients and/or hormones with these mechanisms, awaits further investigation. Aclmowkdgements

The authors are grateful to the members of the Lipidology Section (this Institute), speciahy to Drs. Venezuela Axabache, Zuy Dminguez and Holger N. Grtix, for their expert help and support. Dr. Iv&r. Contreras’ help in discussing and revising the manuscript is greatly acknowledged. This work was supported by grants No. lo-33-1976-88/91 from the Consejo de Desarrollo Cientffico y Humams’ tico de la Universidad Central de Venezuela and Sl-2356 from the Consejo National de Investigaciones Cientfficas y Tecnol&icas (CONICIT). Rekrcmces 1 V&a, A. and Fbbry, P. (19831 Wld. Rev. Nutr. Diet 42,56-1011. 2 Vrba, A. and Kazdov& L (1986) Pro& Biochem. Warmaeol.21, 59-73. 3 Chevw, MM., Willey, J.R. and Lmeille., GA (1972) J. Nutr. 102,337-342. 4 thmona, A. and Freedland, RA (1989) J. Nutr. 119,1304-1310. 5 Heinz, F. (19721 Acta Med. !kand. Suppl. 542,27-36. 6 Znkim, D. (1972) Acta Med. Scmd. Suppl. 542,205-214. 7 Clarke, S.D. (1986) Me.tabolic adaptations to dietary fats in Diemy fat and Cancer Up, C, Birt, D., Rogem, RR. and Mettlin, C, eds.), pp. 531-553, A.R. Liss, New York.

134 8 Wieland, O.H. (1983) Rev. Physiul. Biochem. Phanuacol. 96, 123-178 9 Vrfma, A, Raulin, J., Loriette, C. and Kazdnv& L. (1983) Nutr. Rep. Mt. 28,1437-l&4. 10 Yeaman, S.J. (1989) Biiem. J. 257,625-632. 11 De Marcucci, 0.L and Lindsay, J.G. (1985) .Eur. J. Biochem. 149, 641-64811. 12 M&as, PA. and I..aher, ME (1986) Prog* B&hem. PharmacoL 21,33-58. 13 Chieco, A, Gutman, R and Basilicn, M.Z. (1986) Nutr. Rap. Int. 33,465-476. 14 Da SiIva, LA, De Marcucci, 0.L and Carmona, A. (1992) Camp. Bioahum. PhysioI. 103A, 407-411. 15 Stausbie, D., Denton, R.M., Br@es, BJ.. Pa& HT. and Randlc, PJ. (1976) B&hem. J. 154,225-236. 16 Be&m, M., Tepperman, H.M. and Tepperman, J. (1983) Endocrinology 112,50-59. 17 Hersberg, G.R. (1983)Adv. Nutr. Res. 5,221-253. 18 Henberg, G.R. and Rogerson, M. (1988) J. Nuts. 118,1061-1067. 19 &ore, HG., Demon, RM, Martin, B.R. and Randk, PJ. (1971) Biochem. J. 125,115-127. 20 Hiian, LM., Ksiezak-Reding, H., Baker, A.C. and Blass, J.P. (1986) Arch. Biuchem. Biophys. 246,381-390. 21 T&or, ‘H., MehIer, AH. and Stadtman, E.R. (1953) J. Biol. C&em. 204,127-138. 22 Da Mamu&, OL, Hunter, A and Lindsay, J.G. (1985) Biuchem. J. 226,509-517. 23 I&n, T.C, PelIey, J.W., Pet& PI-I., Hucho, I?, RandaIl, D.D. and Reed, LJ. (1972) Arch. Biochem. Biophys. 148,327-342.

24 Stanley, C.J. and Perham, RN. (1980) Biochem. J. 191,147-154. 25 Laemmh, U.K. (1970) Nature 227,680~685. 26 Towbin, A., Stahehn, T and Gordon, J. (1979) Proc. NatI. Adad. ti USA 76,4350-4354. 27 Batteiger, B., Nuwhall, W.J. and Jones, R.B. (1982) J. Immunol. Methods 55,297-307. 28 MarhsvelI, MA., Haas, SM., Bieber, LL and To&r& NE. (1978) Anal. Biochem. 87,206-210. 29 Stahl, E. (1969) Thm-layer chromatography. A laboratory bandbook. 2nd Ed, pp. 392-400. Sprin8er-Verlag, New York. 30 Matuda, S., Shrirahama, T., &h&ii T., ‘Miura, S. ‘and Mdri, M. (1983) Biochim. Biophys. Acta 741,86-93. 31 Rsndle, PJ. (1986) Biochem. Sue. Traus. Ip,799$6. 32 Weinberg, M.B. and Utter, M.oLp’. (lm) 1. Biol. &em 254, j&$92-9499. 33 Numa, S. and Yamashita, S. (1979) Curr. Top. Cell Rggul. $ 197-223. 34 Gibson, D.M., Lyons, R.T., Scott; D.F. and Muto, Y. (19721 Adv. Enzyme Regul. 18,187-204. 35 Begum, N., Teppekan, H&i. and. T~p~~~, J. fl98.21 .EndocrilIoiogy 114 1914-1921. 36 De Marcwci, OX,., gibe, G.M., Dii J. and L.indsay,%G. (19881 Biochem. J. 2$1,817-823. 37 Lynen, k, Sedlacze& E. and Wieland, O.H. (1978) Biochem. J. 169,321-328. 38 Clarke, S.D. and Abraham, S. (1992)ZASEB 3.6,3146-3152 39 kit&, N. (1%) Eur. 2. B&hem. 205,433_4i12.