The Regulation of Triglyceride Synthesis and Fatty Acid Synthesis in Rat Epididymal Adipose Tissue EFFECTS of ALTERED DIETARY and HORMONAL CONDITIONS

Biochem. J. (1970) 119, 221-242 221 Printed in Great Britain

The Regulation of Triglyceride Synthesis and Fatty Acid Synthesis in Rat Epididymal Adipose Tissue EFFECTS OF ALTERED DIETARY AND HORMONAL CONDITIONS

By E. D. SAGGERSON and A. L. GREENBAUM Department of Biochemistry, University College London, Gower Street, London W.C. 1, U.K. (Received 9 March 1970)

1. Epididymal adipose tissues obtained from rats that had been previously starved, starved and refed a high fat diet for 72h, starved and refed bread for 144h or fed a normal diet were incubated in the presence of insulin+glucose or insulin+glucose+acetate. 2. Measurements were made of the whole-tissue concentrations of hexose phosphates, triose phosphates, glycerol 1-phosphate, 3-phosphoglycerate, 6-phosphogluconate, adenine nucleotides, acid-soluble CoA, long-chain fatty acyl-CoA, malate and citrate after 1 h of incubation. The release of lactate, pyruvate and glycerol into the incubation medium during this period was also determined. 3. The rates of metabolism of glucose in the hexose mono- phosphate pathway, the glycolytic pathway, the citric acid cycle and into glyceride glycerol, fatty acids and lactate+ pyruvate were also determined over a 2 h period in similarly treated tissues. The metabolism of acetate to CO2 and fatty acids in the presence ofglucose was also measured. 4. The activities of acetyl-CoA carboxylase, fatty acid synthetase and isocitrate dehydrogenase were determined in adipose tissues from starved, starved and fat-refed, and alloxan-diabetic animals and also in tissues from animals that had been starved and refed bread for up to 96h. Changes in these activities were compared with the ability of similar tissues to incorporate [14C]glucose into fatty acids in vitro. 5. The activities of acetyl-CoA carboxylase and fatty acid synthetase roughly paralleled the ability of tissues to incorporate glucose into fatty acids. 6. Rates of triglyceride synthesis and fatty acid synthesis could not be correlated with tissue concentrations of long-chain fatty acyl-CoA, citrate or glycerol 1-phosphate. In some cases changes in phosphofructokinase flux rates could be correlated with changes in citrate concentration. 7. The main lesion in fatty acid synthesis in tissues from starved, starved and fat-refed, and alloxan-diabetic rats appeared to reside at the level of pyruvate utilization and to be related to the rate of endogenous lipolysis. 8. It is suggested that pyruvate utilization by the tissue may be regulated by the metabolism of fatty acids within the tissue. The significance of this in directing glucose utilization away from fatty acid synthesis and into glyceride-glycerol synthesis is discussed.

It is well known that prior manipulation of the changes in the pattern of metabolism, namely a dietary or hormonal status of rats will influence the decreased utilization of glucose by the tissue and subsequent metabolism in vitro of adipose tissues an increased rate of lipolysis of endogenous gly- obtained from these animals. Previous starvation, cerides (Randle, Garland, Hales & Newsholme, fat feeding, or induction of alloxan-diabetes have 1963; Froesch, 1965). These changes can be reversed all been shown to lead to considerable decreases in in tissues from starved rats by refeeding and, in the rate of fatty acid synthesis from glucose and tissues from diabetic rats, by administration of other precursors by adipose tissues in vitro (Haus- insulin to the donor animals (Hausberger et al. 1954; berger, Milstein & Rutman, 1954; Hausberger & Katz, Landua & Bartsch, 1966). Dietary or Milstein, 1955; Winegrad & Renold, 1958; Jean- hormonally induced changes in the rate of fatty renaud & Renold, 1960; Leveille & Hanson, 1966). acid synthesis by adipose tissues in vitro, despite the Under conditions of starvation or alloxan-diabetes, importance of these tissues as a major site of lipo- decreased rates of fatty acid synthesis by adipose genesis, appear to have been considered to a far tissues in vitro are generally accompanied by other lesser extent than those in liver where such effects 222 E. D. SAGGERSON AN:]D A. L. GREENBAUM 1970 are well documented (Korchak & Masoro, 1962; the fatty acid synthetase complex show adaptive Wieland & Eger-Neufeldt, 1963; Bortz, Abraham changes in their activities with the intention of & Chaikoff, 1963). attempting to correlate changes in the contents Several factors could influence the metabolism of these, and other known adaptive enzymes, with of hexose to fatty acids by adipose tissues in vitro. altered rates of lipogenesis in vitro and with altered As discussed by Winegrad (1965), it is possible that metabolite concentrations in vitro in adipose tissues the capacity for uptake of glucose by tissues in vitro obtained from animals of varied hormonal and may be influenced by their previous dietary and dietary status. hormonal status, with consequent changes occurring Experiments involving measurements of the in the pathways of hexose utilization. Alterations tissue contents of metabolites in normal adipose in dietary and hormonal status are known to pro- tissues maintained in vitro in a range of steady duce changes in the activities of several enzymes of states through the inclusion of various hormones the lipogenic pathways which usually parallel or metabolites in the incubation medium, and changes in the lipogenic capacity in vitro. ATP- correlation of these contents with altered rates of citrate lyase, 'malic enzyme', hexokinase and the triglyceride synthesis from glucose have been hexose monophosphate pathway dehydrogenases all undertaken previously in attempts to elucidate exhibit apparently adaptive behaviour (Young, points of control in the metabolism of glucose to Shrago & Lardy, 1964; Moore, Chandler & Tetten- triglyceride (Denton, Yorke & Randle, 1966; horst, 1964; Wise & Ball, 1964; Kornacker & Ball, Denton & Halperin, 1968; Halperin & Denton, 1965; Hollifield & Parson, 1965; Anderson & 1969; Saggerson & Greenbaum, 1970). In the pre- Hollifield, 1966; McLean, Brown, Greenslade & sent study use is made of prior dietary or hormonal Brew, 1966; Katzen, 1966; Borrebaek, 1966) manipulations, rather than addition of substances similar to that found in liver. It does not appear to to the incubation medium, to vary the steady states have been established, however, whether the studied in vitro. activities of acetyl-CoA carboxylase and the fatty acid synthetase complex of adipose tissue show adaptive responses to changes in dietary and MATERIALS AND METHODS hormonal status as is the case for the liver enzymes (Gibson & Hubbard, 1960; Hubbard, Allman, Chemicals. Reagents and enzymes were obtained or pre- McLain & Gibson, 1961; Numa, Matsuhashi & pared as described by Saggerson & Greenbaum (1970). In Lynen, 1961; Wieland, Neufeldt, Numa & Lynen, addition, trisodium DL-isocitrate was obtained from the 1962; Bortz et al. 1963; Allman, Hubbard & Gibson, Sigma Chemical Co., St Louis, Mo., U.S.A. Acetyl-CoA tissues from rats of varied was synthesized by the method of Simon & Shemin (1953) 1965). Adipose dietary and standardized by the method of Stadtman (1957) by or hormonal status may also have altered intra- using phosphotransacetylase (EC 2.3.1.8). Malonyl-CoA cellular metabolite concentrations which may be was prepared by the reaction of CoASH and S-malonyl- critical in determining rates of fatty acid and tri- N-octanoyl thioethanolamine as described by Lynen glyceride synthesis. In particular, citrate has been (1962). S-Malonyl-N-octanoyl thioethanolamine (m.p. shown to be both an activator of adipose tissue 69-70°C) was prepared by Dr P. B. Bunyan. The malonyl- acetyl-CoA carboxylase (Martin & Vagelos, 1962; CoA was standardized by the hydroxamic acid procedure Vagelos, Alberts & Martin, 1963) and an inhibitor of of Stadtman (1957). Acetyl-CoA and malonyl-CoA were adipose tissue phosphofructokinase (Denton & stored in solution at pH 5.5 and -20°C. in Animals. Male albino rats of an inborn strain were used Randle, 1966) purified systems.The concentrations in all experiments. The animals were maintained on cube of long-chain fatty acyl-CoA derivatives, free fatty diet 41B (Bruce & Parkes, 1949) and were supplied with acids and glycerol 1-phosphate have also been water ad libitum. proposed to be of importance in the regulation of Treatment of animals. Animals used for measurements lipogenesis (Bortz & Lynen, 1963; Numa, Bortz & of enzyme activities weighed between 145 and 155g at Lynen, 1964; Korchak & Masoro, 1964; Kipnis & the commencement of treatment. Animals weighing Kalkoff, 1965; Howard & Lowenstein, 1964, 1965). between 150 and 180g were selected for measurement of It was the intention of the present study to tissue metabolite contents and for measurement of carbon attempt to correlate changes in fatty acid synthesis fluxes. The methods of starvation, starvation and refeed- from glucose and from acetate over a range of ing with high-fat or bread diets, the induction of alloxan- physiological states with changes in the intracellular diabetes and the treatment of alloxan-diabetic animals with insulin were as described by Saggerson & Greenbaum concentrations of possible regulatory metabolites. (1969). Animals designated 'controls' or 'normal' were Studies of metabolite concentrations may also per- maintained on cube diet 41B throughout. mit the location of possible regulatory enzymes Determination of tissue metabolite contents in vitro. The (Rolleston & Newsholme, 1967; Newsholme & incubation of fat pads, the extraction and assay of meta- Gevers, 1967). Measurements have also been made bolic intermediates in them, the analysis of incubation to determine whether acetyl-CoA carboxylase and media and the determination of tissue ['4C]sorbitol, [3H]- Vol. 119 EFFECTS OF DIET ON LIPOGENESIS IN FAT PAD 223 water and glucose spaces were all performed as described l jumol of NADP+ and 0.1-0.2ml of dialysed high-speed by Saggerson & Greenbaum (1970). supernatant. Extraction of acetyl-CoA carboxyla8e,fatty acid 8yntheta8e A88ay of acetyl-CoA carboxylaae (EC. 6.4.1.2). The and isocitrate dehydrogenase (NA DP). Animals were killed method used was basically that of Martin & Vagelos (1962) by cervical dislocation. The epididymal fat pads were and measured the incorporation of [14C]bicarbonate into immediately excised, trimmed free of blood vessels as far acid-stable material. Samples (0.1 or 0.2ml) of dialysed as possible, rinsed and homogenized in ice-cold 0.25M- high-speed supernatants were placed in 10ml glass centri- sucrose containing 0.5mM-dithiothreitol and 2mM-EDTA fuge tubes at 37°C. After 2min the following was added (5ml/g of tissue). In all cases the homogenate was made to give a final volume in each tube of 1.0ml: 40,umol of from the entire epididymal adipose tissue obtained from triethanolamine buffer, pH7.4, 80,umol of potassium one animal, except for a small piece of tissue (20-30mg) citrate, pH7.4, 20,tmol of MgCI2, 8temol of MnCl2, which was set aside for determination of tissue nitrogen. 30,umol of KH14CO3 (2,tCi), 10,emol of ATP, lumol of Homogenization was in a glass Potter-Elvehjem homo- dithiothreitol and 0.2,umol ofacetyl-CoA. The incubation genizer cooled in ice, fitted with a motor-driven Teflon was carried out for 10min at 37°C and was then pestle with a clearance of 0.5 mm. The homogenate was stopped by the addition of 0.2ml of acetic acid-HC1 centrifuged at 105000g for 30min at 2-40C in a no. 50 (10:1, v/v). The tubes were placed in ice, and 0.1ml rotor ofa Spinco model L preparative ultracentrifuge. The samples of the acidified solutions were spread on to circles resulting aqueous fraction was decanted off and dialysed of Whatman no. 1 filter paper (3cm diam.), suspended in for 2h at 4°C in 8/32in Visking seamless dialysis tubing a hot-air blast, and evaporated to dryness. The paper that had been previously boiled in 10mm-EDTA and well circles were immersed in 15ml of a scintillator consisting rinsed with water. The dialysis medium was 100vol. of of 4g of 2,5-bis-(5-tert.-butylbenzoxazol-2-yl)thiophen/ 50mM-triethanolamine buffer, pH 7.4, containing 2mM- litre oftoluene, and counted for radioactivity in a Nuclear- EDTA and 0.25mM-dithiothreitol. The extracts were Chicago liquid-scintillation counter. Observed count rates found to increase in volume by approx. 25% during were corrected to d.p.m. by the 'channels ratio' method dialysis. All dialysed high-speed supernatants were kept in of Baillie (1960). Corrections were also made for the ice until used, and all assays were performed within 6h, background count rate. It was observed that the area of during which time no loss ofenzyme activity was observed. the paper circle, at least up to 8cm2, did not affect the Control experiments, involving the re-extraction ofthe fat counting, nor did the orientation of the paper with respect plug produced by the centrifugation, showed that 7% of to the phototubes of the counter. However, only approx. the total extractable fatty acid synthetase activity and 80% of the d.p.m. of a standard "4C-containing sample only about 1% ofthe acetyl-CoA carboxylase and isocitrate could be detected when counted on these papers. A dehydrogenase (NADP) activities were retained in the fat correction factor was therefore used in the calculation of plug. Other control experiments indicated that the 0.25M- results. Control experiments showed that there was no sucrose medium was slightly better than a 0.15M-KCI- retention of "4C-containing material on the papers in based medium (Saggerson & Greenbaum, 1969) in incubations lacking the addition of dialysed high-speed extraction of acetyl-CoA carboxylase and fatty acid supernatant. Acid-stable radioactivity was taken to re- synthetase from the tissues. No correction was made for present malonyl-CoA (Martin & Vagelos, 1962; Levy, the contribution ofthe very small amount ofadipose tissue 1963). water (Crofford & Renold, 1965) to the total volumes of The concentrations of substrates and cofactors used in the homogenates. the assay were those that gave maximum, or near maxi- Assay offatty acid syntheta8e and isocitrate dehydrogena8e mum, incorporation of [14C]bicarbonate. Although it was (NADP). The enzyme measurements were made by re- essential that citrate be present in this assay, little was cording rates of change in extinction at 340nm with a gained, in terms of extra activation, from preincubating Unicam SP. 800 recording spectrophotometer at 25°C. All the enzyme with citrate, unlike the systems of Martin & reactions were carried out in 3 ml volumes in cuvettes of Vagelos (1962) or Levy (1963). This lack of an appreciable 1 cm light-path. In all cases the rates ofreaction were con- time-dependence of citrate activation is presumably due stant over the measured period (3-5min). Preliminary to the far higher citrate concentrations used in the present experiments indicated that in all cases the rates of re- study. Fang & Lowenstein (1967) have demonstrated that action were proportional to the amounts of dialysed high- the extent of citrate activation of acetyl-CoA carboxylase speed supernatant used, and that the concentrations of in rat liver extracts is dependent on the citrate con- substrates and pH of buffers used gave maximal activities. centration and on the time of activation in an inter- Reactions were started by the addition of the necessary dependent manner. Control experiments indicated that amount ofdialysed high-speed supernatant. Simultaneous the incorporation of [14C]bicarbonate into acid-stable blanks were carried out by addition of tissue extract to an material was proportional to the amount of dialysed high- identical cuvette from which a substrate was omitted. speed supernatant used, and that this incorporation was The methods used were basically those of Gibson & linear with time. Hubbard (1960) and Ochoa (1955). The components ofthe Determination of 14C flux rates in incubated tissue seg- assay mixtures were as follows. ment8. Incubation of tissue segments with various sub- Fatty acid synthetase. 100jumol of potassium phos- strates, extraction of tissues, measurements of the yields phate buffer, pH6.5, 0.5jHmol of NADP+, 75nmol of of 14C in various products and calculation of flux rates malonyl-CoA, 50nmol of acetyl-CoA and 0.1-0.4ml of were performed as described by Saggerson & Greenbaum dialysed high-speed supernatant. (1970). Isocitrate dehydrogenase (NADP) (EC 1.1.1.42). Determination of tissue nitrogen. This was performed as 100,umol of tris-HCl buffer, pH7.4, 101Imol of MgCI2, described by Saggerson & Greenbaum (1969), either on 224 E. D. SAGGERSON AND A. L. GREENBAUM 1970

4. 20-30mg samples of powdered frozen fat pads in experi- --I ments to determine tissue metabolite contents, or in 20-30mg samples of fresh tissue in experiments to deter- 4Q 0 .4 e -1-4F- mine enzyme activities or carbon fluxes. O =g bo 4 = eq

RESUJLTS bO (Lc Measurements of metabolic intermediates in incuba- 4. 0; 04.E t tion media and in adipose tissues, from rats of varied C.) 4- 00 (2 4 dietary status, incubated in the presence and absence 4.4a~- of acetate. Four experiments were performed in 10 10 which whole-tissue concentrations of metabolites 4Q. -H * were measured. Basic data concerning the animals used in these experiments are shown in Table 1. Table 1 also records data concerning the release of lactate, pyruvate and glycerol by whole, pooled 0'4 tissues incubated for h. Similar data on lactate, F04 o 4.ce pyruvate and glycerol release by single fat pad ,. C) 0 E segments incubated for 2 h are also shown in Tables sb 4B -4. -00 7 and 9. The results of Tables 1 and 7 are in agree- -4 F4. 0 4-4C ment in showing that glycerol release is increased CA) _ _ in tissues from starved rats with respect to those O C) C) CO 0 from normal rats, and that refeeding with a high- o - -H -f _ _ fat diet does not decrease the release of this meta- r -4 bolite, whereas refeeding with bread supresses the c3 increased glycerol release incurred on starvation * within 24h (Table 7). In all three experimental CO 4Q4 0 O O O o o 00 0 C-H -H treatments shown in Table 1, the release oflactate + .: o o 0 C; C; .4.a H H pyruvate by the incubated tissues was increased 1" 45~ -4 14 o o -; CO CO CO CoICo above normal; this is also shown in Tables 7 and 4-4. V 04 O .n Q 5- 9. Incubation in the presence of sodium acetate -4. was found to decrease this elevated lactate+pyru- 4.4*-Co a m 0' C 00 *- _ vate efflux in the case of tissues from starved and (a . X °4 cq cq es cq al _. d : O O starved, fat-refed rats (Tables 1 and 9), but to 4) C4- * . * . . . _ co _ esC increase it in the case of tissues from starved, c0J +0+ c; -4c; C; t 4514 Ca 4 bread-refed rats. Similarly, though to a less signi- 0 COC(-Ii C-li1.. liCC ,0o 4. ficant extent, incubation of tissues from starved, 4oQI- ( 545; 14 V or starved, fat-refed rats with acetate was found to ++ 4. -~ -- C 411 O Co o release 0 decrease the release of glycerol, whereas the 04 0) of glycerol was increased when tissues from starved, °Q c c bread-refed rats were incubated with acetate (Table ceC- e Co Co P4- * i. 4e 04 C> 1). The effects of sodium acetate on the metabolism 545-4-4VCCa CO C> O oftissues from alloxan-diabetic rats was not studied; however, it may be seen in Table 7 that, as in the *S' + + + case of from starved rats, the release of 0 tissues a 50 * 4a S.a * *. *. *5 1---f* * C 4t o~.fl *~ w n-v p Ca¢Cd- lactate+pyruvate and glycerol is considerably in- 0 ,e Cd creased in tissues from alloxan-diabetic rats. ~4.5cePO Treatment of alloxan-diabetic animals with insulin 0 p-S> G for 24h before incubation of the tissues led to a a e 0B~C0a2c ZS a , O O C c3O Z normalization of glycerol release and lowering of 00 . . . c the lactate+pyruvate production to near-normal 4. levels. Lo;0q Whole-tissue concentrations of various phos- -4a ce ~O phorylated sugars in incubated tissues of varied dietary status are shown in Table 2. In general, the concentrations of these intermediates per mg of 4. tissue N did not vary greatly between the experi- mental groups. However, tissues from starved rats EH Vol. 119 EFFECTS OF DIET ON LIPOGENESIS IN FAT PAD 225

4) = o o C0to C; *I . -H - I I 00 oo 0t r- lo ~400 '4 C; (a H -H o . . . C> C> P- .d

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COO ,o4 +D -0 CO 0 w *00 , N CO CO 0-H 20 - c-I 04~ ~4CO 0 t] * eb o eq -H -H bi- 4) ~ ~ ce~ ~ ~ ~ CO 0-44 CZ4 - -q C> XO - - -H -H -H _; cli ¢ c-I*r- 4) to0 0 0 P4 - N ,0 c-I - - +. CO -H - - - o o ce M -H,I -H00 .~ 0 . Q CO t- CO (CO 6 r; *: ,s: 4) ¢too H- -q 00 0 0 c -H H 5 11 -0- o> es c -0 c.s-H-H- o 0) -i CO rO

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*- cddZX UDQ z 4)4 )$- 024 4 44 4 0 ce ocd ce4 o ._5 0,P tu'* Z cc_ 4"Ca = m _Ca 544.X )B '4 °.°*4a +Dii=a 1.0 COCzzZ > p4GQ 3 _ z ~ ~ ~ ic. 90 119 Ca B

-B 019 226 E. D. SAGGERSON AND A. L. GREENBAUM 1970

o V- exhibited significantly decreased concentrations of e614 14 e 6-phosphogluconate (in fact this represents the sum + + + + of 6-phosphogluconate +6-phosphogluconolactone) Go IC g- cc t- and tissues from starved, bread-refed rats exhibited rd ... greatly elevated concentrations of this metabolite. Go 4 cdgoa Significant alterations from normal in the con- centrations of glucose 6-phosphate, fructose 6- $ 4*P c aq x phosphate, fructose 1,6.diphosphate and 6-phos- 0r -40 -H -H -H o :; ce 1; phogluconate were not observed for any of the a + H + H experimental groups, although the concentrations X o6 c6 o6 -= of glucose 6-phosphate and fructose 6-phosphate in Ca)~ 4 appeared to be consistently greater than normal ..p P -ci 04. D tissues from starved, fat-refed and starved, bread- "~-H -H-H refed rats, and to be consistently below normal in $ ooo o from starved rats. The concentrations of ci e C9 tissues Ca ci c:II dihydroxyacetone phosphate and glycerol I-phos- 4a0 0 ( -H -H + -H phate were quite significantly elevated in tissues $0 C) a: P,. from starved, fat-refed rats, whereas the concentra- " v o elevated _> ° . q aacs tion of dihydroxyacetone phosphate was o B4 in tissues from starved, bread-refed rats. Incuba- 4-c4 0~ 0 tion with sodium acetate produced no statistically * V~ ccs significant changes in the concentrations of any - -0 of the phosphorylated sugars, although 6-phospho- -H-H -H C) 4a o o gluconate concentrations were slightly increased -H-H -H+ -H of acetate in all three e in the presence experimental O _ 4 to q o 4cN groups and the concentrations of glucose 6-phos- phate, fructose 6-phosphate, fructose 1,6-diphos- -SC "0ci] phate, dihydroxyacetone phosphate and 3- phosphoglycerate, were generally decreased by incubation with acetate. Table 3 shows that all the experimental groups a .6 ~00 xo had lower AMP concentrations than normal, 0. - ) -H -H so in the refed oi (6-H-H(6 -HC though only significantly starved, 01.3 0 a), t- t- *4 Incubation with acetate produced no oo oo co ¢: <,0 cs groups. *s oet O 0 -"~ noticeable effect on the adenine nucleotide pattem. ,0 t*C Table 4 shows that the three experimental groups of tissues had concentrations of long-chain fatty 14c ci S * acyl-CoA similar to normal tissues, but that the CoA were decreased in oa) ,0~ o acid-soluble concentrations 0 -4.;o tissues from starved rats, and were greater than , + + normal in tissues from bread-refed rats. 0.- .D starved, ce The values of total CoA were thus decreased and 44, - ) 4.m~ .3 .~o.6 3 0 8 Q elevated in these tissues respectively. Tissues from 6I starved and starved, fat-refed rats had normal malate concentrations, but the concentration of ~0 this metabolite was elevated in tissues from zea)Q starved, bread-refed rats. On the other hand, -4 , OD citrate concentrations were elevated in tissues from <,,> > tic e tstarved rats and in those from starved, fat-refed d20 e~z t animals, but were not significantly altered from 44. -+., E = tp u * normal in from starved, bread-refed rats. 0 ci O 2 q 0 tissues V rQ 6@ 'td &0 0 .10 ' Incubation with acetate produced no significant ci se a Zcc ci . 4 changes in the concentrations of the metabolites in Table 4 in any of the experimental groups, although A ao .36 the concentrations ofcitrate were consistently raised in the presence of acetate. Intracellular water epacs and glucose-[I4C]sorbitol spaces in incubated adipose tissuesfrom rats of varied Vol. 119 EFFECTS OF DIET ON LIPOGENESIS IN FAT PAD 227

cH dietary status. The results of Table 5 indicate that, *S 0 s s- :- expressed per mg of tissue N, the total intracellular - .: _ water space of the three groups of experimentally 0) + + + treated tissues were not significantly different from .) .5 + cl normal. Significant variations between the experi- --4 0 s mental groups in the value of glucose-[ 4C]sorbitol space were, however, found. Enzyme mass-action ratios in incubated adipose O -H 00 tissueefrom rats of varied dietary status. Mass-action 0), ratios (r) for the phosphofructokinase, adenylate Rt ho co kinase and phosphoglucose isomerase reactions are 0J + -H -H presented in Table 6. These ratios in the experi- GO

Go Go + normal tissues (Saggerson & Greenbaum, 1970). 0 _O -C) With tissues from starved, bread-refed rats it was 0) 0oeE o H + + found possible to assay glyceraldehyde 3-phosphate o o X t-1: 1 - (Table 2), therefore allowing an estimate of the S 4 01 separate r values for aldolase and triosephosphate

OD isomerase to be made for these tissues. The mass- ,oV action ratio for triosephosphate isomerase in the tissues from starved, bread-refed rats was found 0)0).X X az to be 6.21 ± 2.13 for tissues incubated with insulin c)o O0) O;+-O =+-: alone, and 5.55 ± 1.13 for the same tissues incubated R . with insulin+acetate. These values may be com- GO 0 Ca 0 o o 0 o pared with values for the apparent equilibrium constant of this reaction which range from 20 to 28 (Burton, 1957; Lowry & Passonneau, 1964). Mass-action ratios for the aldolase reaction in tissues from starved, bread-refed rats were - +- c3 .- 1.4x1O-4M in the presence of insulin alone and X0 ' X8.7 x 1O-5M in the presence of insulin+acetate. 4 V These values may be compared with published 255V values of the apparent equilibrium constant of the a reaction which range from 6.8x10-5M to 1.33x 5f;- W"a 10-4M (Herbert, Gordon, Sabrahmanyan & Green, 1940; Meyerhof & Junowicz-Kocholaty, 1943; 0;-O + + + Krebs & Kornberg, 1957; Hess, 1963; Lowry & c)0 oo -H C cl Passonneau, 1964). - - [Lactate]/[pyruvate] ratios in incubation and -C)-4a media, Q-., W 4- calculated extramitochondrialfree [NAD+]/[NADH] c; O ratios in epididymal adipose tissues8from rats ofvaried dietary status, incubated in the presence and absence Co of acetate. When incubated in the presence of a-- a: a insulin alone, for either 1 h as whole tissues (Table 6), - - - or for 2 h as small segments of tissue (Table 7), tissues from starved rats demonstrated a slightly

0 - increased lactate/pyruvate ratio compared with 4- 0 -a .a = normal tissues. Refeeding starved animals with cq cq cq ce bread led first to a significant increasing of the "5 a2 Sa o.4 o-4 t[lactate]/[pyruvate] ratio compared with normal fc ce tt ; + t ; ;: tissues after 24h of refeeding (Table 7). However, Ca Ca c >t S after 144h of with bread the ;0;- .3 "a refeeding [lactate]/ 121 0- ; ea .> o < o [pyruvate] ratio was generally lower than in normal CL e I . e; tissues (Tables 6, 7 and 9). Tissues from starved, 228 E. D. SAGGERSON AND A. L. GREENBAUM 1970

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P.- -c H~~~~~~~~~~~c M1 o 00 o Vol. 119 EFFECTS OF DIET ON LIPOGENESIS IN FAT PAD 229

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+ ~~~bl oc c) P CO o -H 0H H - O O 0 00 H -H +- -H -H -H ~~~~~o z ~~~~ CO O CO O CO 0 00C EHr_Ocos 0t ¢ O r ce *_ .4j O_ :_ ; m% CQ Nr > CO o

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Ca0C 01

- 00 -H -H -H -H -H-H

.@ w rw r cs r r u:~~~~~~~~~~~~Ulb > V:i V:g4 0 Ca 0 O N0

;> r

g r ) r z CQ0; C4)

1-4 ;14

- 4 a Ca ce t- 400t-4t14. 14~0.- .tC 44.t- 9 rQr

-4- A- 00 N > 40 CDtp)lipids starvation (Table 7). The medium [lactate]/ was also routinely monitored, and the total radio- [pyruvate] ratio was not significantly elevated in activity incorporated into these two fractions and the case of alloxan-diabetic tissues (Table 7), but into the fatty acid fraction was compared with the was significantly elevated if the donor diabetic incorporation into total lipids, which was also animals had been pretreated with insulin for 24h monitored. Table 7 shows that the total percentage before incubation of tissues. Incubation of whole recovery of lipid fractions on saponification and or segmented tissues from normal, starved, fat- extraction was good (-90%) except in the experi- refed, or starved, bread-refed rats with acetate in ment with alloxan-diabetic tissues. the presence of insulin considerably decreased the Table 8 shows that both the total fat pad activities medium [lactate]/[pyruvate] ratio compared with and the activities per mg of tissue N of acetyl-CoA the same tissues incubated with insulin alone carboxylase and fatty acid synthetase were con- (Tables 6 and 9). However, no significant alterations siderably decreased in tissues from starved and in this ratio with starved animals were observed in alloxan-diabetic rats. The concentrations in tissues the presence of acetate. from starved rats were restored by refeeding bread Values are presented in Table 6 for the extra- over a period of up to 4 days, but not by refeeding mitochondrial free [NAD+]/[NADH] ratios in the the high-fat diet. These considerable fluctuations in incubated tissues as calculated independently from activity may be compared with the noticeable the medium [lactate]/[pyruvate] ratio (making the constancy of the activity per mg of tissue N of arbitrary assumption that the ratio in the incubation isocitrate dehydrogenase (NADP) which was medium at the end of the incubation is the same routinely assayed with the other two enzymes. It as that in the tissues at the time of sampling) and was decided that for comparison ofenzyme activities from the [glycerol 1-phosphate]/[dihydroxyacetone between the adipose tissues from rats of varied phosphate] ratio. Reasonable agreement, both in dietary status and fat content, the expression of terms of the absolute values ofthe ratios, and of the activities on a nitrogen basis was more suitable than direction of change of these ratios in the presence expression of activities per pair of fat pads or per g of sodium acetate, is obtained with tissues from wet wt. of tissue. starved and starved, fat-refed rats. In the case of Measurements of carbon fluxes through metabolic the tissues from starved, bread-refed rats however, pathways in incubated segments of adipose tissues. In there is considerable disagreement between the two Tables 9 and 10 measurements are presented of the estimates as to the direction of change of the rates of flux of glucose-derived carbon through [NAD+]/[NADH] ratio on incubation with acetate. various metabolic pathways in the presence and Also the agreement between the estimates of this absence of acetate. The tables also indicate the ratio for normal tissues is poor, although both magnitude of the flux of acetate-derived carbon methods of assessment indicated that the extra- into CO2 and into fatty acids in the presence of mitochondrial [NAD+]/[NADH] ratio in normal glucose. It may be seen that in tissues from starved tissues incubated with insulin + acetate is more and starved, fat-refed rats glucose utilization by oxidized than that in normal tissues incubated the glycolytic pathway, and more particularly by with insulin alone (Saggerson & Greenbaum, 1970). the hexose monophosphate pathway, is consider- Rates of whole-tissue fatty acid synthesis from ably decreased below the normal level. In tissutes glucose in vitro and activities offatty acid synthesizing from starved, bread-refed rats on the other hand, enzymes in adipose tissues from rats of varied dietary glucose utilization by both pathways was above status. The results in Table 7 indicate that [14C]- normal. Rates of citric acid cycle CO2 formation glucose incorporation into fatty acids by segments from acetate or from glucose did not, however, of adipose tissue in vitro is severely curtailed by appear to be affected by the type ofdietary manipu- starvation. On refeeding bread to the donor animals, lation of the donor animal (Table 10). In all the ability to incorporate glucose into fatty acid cases, addition of acetate to the incubation is regained and becomes greater than normal after medium led to an increased rate of citric acid 144h of refeeding. On the other hand, the refeeding cycle CO2 production from glucose. As shown in of a high-fat diet after starvation did not appear Table 7, previous starvation or starvation and fat- to elicit an appreciable restoration of fatty acid refeeding of the donor animals led to greatly synthesis. Feeding a high-fat diet and induction of diminished rates offatty acid synthesis from glucose. alloxan-diabetes also led to considerably diminished Table 10 shows that fatty acid synthesis from capacities of the tissues to synthesize fatty acids acetate was also diminished under these conditions. from glucose. Insulin therapy of alloxan-diabetic On the other hand, rates of fatty acid synthesis Vol. 119 EFFECTS OF DIET ON LIPOGENESIS IN FAT PAD 231

eq I -4 10 1H -H -H -H -4 X) C s4 t-. z t- 0 '6 co CO r-. CO

_) +D -HX -H-H

-H -H + -H +~- -H t- C C O Q OZ+444OC-H _ 10 _-4 _ - _ 4

t- t- -D 1- 0 00C v Ca r c

0 eq 10 .,q co o eI .o R <+ --H -H +H -H 0 eq 0-H CO COz P- C9ce -4 v4), 4) -H -H-Hl e ;;- 0 0 aq 4 0 1 00 -H Co 4)-4 Nq CO 10 CB0 04 -- cc to N t-4 06 eq -HH-H

'4 0 eC 00 *q t-. 0O4)4 -H H H -H -H ,° e tO 10 ep O eC 00 Ca CO4 C4)eq o Cei cl o6 o a. fCDD . HC *0 .9 C o 4)c * -I-- r t

-H H -H -H -H Cf) C* r"

C-- to eq eq CO i*Fa0+ + ++ --H -H * C;

* 0 t- o CO CO 10

'.40 H-H X-H + -H 00 10 CZ --: 10 -4 eC CO C; C; e 000 +F sC _A C* P- *O S eq CC CO 4^ _++ -* C4s 4 ";a -H -H -H -HC* -H.4 CO0Z C 0 t < c CC

4 0

OD~~~~~~~~~~~~~~~~~~~~~~~~~C

.< r.r Q C n ==t, , J , ,< e = .4. . CB 4) 4.2 .0H 4)4

i 5 44.02M , d,, 22 c3 - Coe 'R 232 E. D. SAGGERSON AND A. L. GREENBAUM 1970

0

o *4= O tCi C 01 ~Ol N1 oco Ca 4-HH NC O )0-)- -H -H H 0) -H-fl -H -H c+

4- NO CO _10

100 10000 0)) xo aq q Q 0". '0 +0) in 11O It + -O S rO Ca Ca Ca P -H -H -H -H - CO C .t 10 C o o~~~~~~~~~~~~~1 Goo 00 0)<10 < CO oe co 0 + -H -H -° Ca 0 0 0 4- Cr Cl= C-i c.) .H .Q m CD -H -H -HO -H_Q co 04- OO rq i 00 X CO= ce oq 0) d4 Ca o 4-Hi 00-H. *= -4 * ~ 0-O 100 O I-Hs 0r * o ce C CO CO CO

i- - . e o -H -H ~01-H -H -H -H -H-H - 01 01 CO - 0C . C; ti -H- W o CO b *I*Q I,,0 6 c; _O ~w*~0) 0V IH-HC1 *4 0 ° 0, C00 C1 C-i -I COO 0) CO C4 C;

-H -H CC - -H m cq ktl l 01 'It O O Ci o- E iC li co q c0 0 "P 0) 0 .0 0c; r- r- O * 04 c Ci C o o= w00 o 0 t 001 4+F J Z 0 4Q P-4 A 0 CO c-aq 01 -H

PC 75 -H -H -H -H .@ O O -H o> v eH-H01 Cq Xo . 01 CO

0)a 0

0 Ca~ 0 0 o Pa .f. - 100 _ Q W CO 00 00 0_ + + + o~~~~~~~~~~~~~~~~~~C+ 0 0 Ca cd . o 0) 0 00 o~~~~~~.tC +w CO) 00 -f -H -H 9 cs ~~~0 CO C0 00 +3 r.9 C)W 0) 00 CO It C 3 O z 0 0 X =R = = H4Q~iC3H ~C 00C 0 V X e -H4 -H.t (60100 CO0,-4 tPz V 4a 0 .0 .d

CO -H -H -HIdH N 1C CO COC 00 c= COOX 0 -a O~*0 w O; 1 O.O 0 O .)* 0a- Oa- km to 4)l zo M c30bQ 51Q a- _ 10_ c3e S.e0 * C) 0)4d -H -H -H aw.2 g + + + + -_ -* O s . z p c3 ° ak = Q s

C I c n c3 <10 O CO -D

r 4a 1.0 404 o . . 0. .0 . 0B .H -0 , a S c dSce - N a-

a-- cc a-a-. 100 O-4 ° Y CO/2c0.O ce ca Q) t Vol. 119 EFFECTS OF DIET ON LIPOGENESIS IN FAT PAD 233 from glucose and from acetate were greater than approaching maximal. It should be stressed that normal in tissues from starved, bread-refed rats. several factors, not included in the assays recorded In all cases the inclusion of acetate in the incubation in Table 8, alter the rate ofthe acetyl-CoA carboxyl- medium elicited increased rates of fatty acid syn- ase reaction. For example, there are several reports thesis from glucose together with increased rates ofliver cytoplasmic particles activatingor inhibiting of hexose monophosphate pathway CO2 production soluble preparations of liver and adipose tissue and, in the case of tissues from normal, starved and acetyl-CoA carboxylase (Masoro, Korchak & starved, bread-refed rats, increased rates of glucose Porter, 1962; Korchak & Masoro, 1963; Howard & utilization by glycolysis. With tissues from starved, Lowenstein, 1964; Martin & Pittman, 1966), and fat-refed rats however, the rate ofglucose utilization in fact Margolis & Baum (1966) have suggested that by glycolysis was in fact decreased when acetate pigeon liver acetyl-CoA carboxylase may be was added and instead, more glucose carbon became associated with membrane material in the cell. available for fatty acid synthesis through a decrease The possible complexity of the behaviour of acetyl- in the rate of release of lactate +pyruvate. CoA carboxylase has also been indicated by the Table 10 also shows that the ratios of glucose findings of Gregolin, Ryder, Warner, Kleinschmidt carbon to acetate carbon appearing in the products & Lane (1966), Greenspan & Lowenstein (1967) and (fatty acids and citric acid cycle C02) were less in Fang & Lowenstein (1967). Comparison of the the case of tissues from starved and starved, fat- measared enzyme activities with whole-tissue rates refed rats than in those from normal and starved, of fatty acid synthesis is also complicated by the bread-refed rats, suggesting that in tissues from fact than an appreciable, but unknown, proportion starved and starved, fat-refed animals glucose is of the tissue fatty acid synthesizing capacity, in discriminated against, compared with acetate, as a liver at least, appears to reside in the mitochondrial supplier of acetyl-CoA. It is also noteworthy that and microsomal fractions of centrifuged tissue the glucose carbon/acetate carbon ratio was found homogenates. The results presented in this study to be much higher in fatty acids than in citric acid refer only to measurements made on high-speed cycle CO2 in all four experimental groups. supernatants. However, the far higher dilutions used in preparation of adipose tissue homogenates compared with liver homogenates may act to re- DISCUSSION move enzymes adhering to membrane structures (Margolis & Baum, 1966). Hubbard, McCaman, From the results presented above, it is apparent Smith & Gibson (1961) have demonstrated the that previous fasting, fasting and fat-refeeding, existence of a [1,3 1 4C]malonyl-CoA decarboxylase fat-feeding and induction of alloxan diabetes in in rat liver high-speed supernatant preparations. rats all bring about a lesion in fatty acid synthesis This activity was not greatly affected by starving from glucose in vitro in tissues taken from these and refeeding. If a similar activity is present in animals. The results presented may offer a partial adipose tissue, the results for acetyl-CoA carboxyl- explanation of this lesion. ase shown in Table 8 may well be an underestimation Activities of acetyl-CoA carboxylase and fatty acid of the activity of that enzyme, particularly in synthetase. Although the rat adipose tissue acetyl- those physiological states where the activity is CoA carboxylase and fatty acid synthetase have lowest. It should also be pointed out that phos- been purified and extensively characterized by phorylated sugar activators offatty acid synthetase Martin & Vagelos (1962), Vagelos et al. (1963), (Wakil, Goldman, Williamson & Toomey, 1966) Martin, Horning & Vagelos (1961) and Horning, were not included in the assay of that enzyme Martin, Karmen & Vagelos (1961) there appear, ex- complex in this study. However, the assay was cept for a paper by Dakshinamurti & Desjardins performed in an orthophosphate buffer which is (1969), to be no reports in the literature concerning itself a slight activator. variations of the activities of these two enzymes Although the ineasured activities of acetyl-CoA with physiological state in adipose tissue. carboxylase and fatty acid synthetase cannot be It is probably not valid to compare in a quantita- quantitatively correlated with other physiological tive manner the maximal activities ofthese enzymes parameters, it is probably reasonable to attempt as measured in the extracts prepared in the present qualitatively to correlate changes in the measured study (Table 8) with measured rates of fatty acid activities of these two enzymes with other physio- synthesis in vivo or in intact tissues in vitro. This logical changes. The results of this study suggested is particularly true for acetyl-CoA carboxylase. This that the adipose tissue acetyl-CoA carboxylase and enzyme appears to have such a complex set of fatty acid synthetase were quite adaptive in re- substrate and activator requirements that, at sponse to changes in dietary status, and far more present, it is not possible to assess whether any so than the isocitrate dehydrogenase, which was combination of these in vitro wouild give an activity assayed in the same extracts as a non-adaptive 234 E. D. SAGGERSON AND A. L. GREENBAUM 1970 control (Young et al. 1964; Leveille & Hanson, 1966) of lipogenesis in vitro are accompanied by lowered and also more so than several adipose tissue contents of malate dehydrogenase (NADP), ATP- glycolytic enzymes (Saggerson & Greenbaum, citrate lyase and hexose monophosphate pathway 1969). The alteration of the adipose tissue con- dehydrogenases in tissues from animals refed a tents of acetyl-CoA carboxylase and fatty acid high-carbohydrate, protein-free diet. In adipose synthetase in response to starvation, alloxan- tissue therefore, the significance to be attached to diabetes and high-carbohydrate refeeding appeared prior alterations in enzyme contents in explaining to be qualitatively the same as the responses altered rates of lipogenesis in vitro is unclear. of the liver enzymes to such treatments (Gibson Correlations of whole-tissue metabolite concentra- & Hubbard, 1960; Hubbard, McCaman et al. 1961; tions with carbon fluxes. In normal adipose tissues Numa et al. 1961; Wieland et al. 1962; Korchak incubated in the presence of glucose together with & Masoro, 1962; Bortz et al. 1963; Allman et al. other agents that lead to alterations in the rates of 1965), although the responses of the adipose triglyceride and fatty acid synthesis there appears tissue enzymes under the conditions used in to be no meaningful correlation between these this study did not appear to be as swift, or of such parameters and the concentration in the tissues of a magnitude, as those of the liver. The failure in metabolites such as citrate, long-chain fatty acyl- adipose tissue of rats fed the high-fat diet (mainly CoA and glycerol 1-phosphate (Denton & Halperin, unsaturated fatty acid) to restore the acetyl-CoA 1968; Saggerson & Greenbaum, 1970). Similar carboxylase and fatty acid synthetase contents correlations for the results obtained in this study after starvation has also been observed in liver were attempted (Table 11). Denton & Halperin (Allman et al. 1965). It is noteworthy, however, (1968) pointed out that in attempting to draw that administration of the high-fat diet could conclusions from such correlations it is necessary restore the content of isocitrate dehydrogenase to assume that changes in whole-tissue concentra- (NADP) (Table 8) and the contents of several tions of metabolites reflect changes in relevant cell glycolytic enzymes (Saggerson & Greenbaum, 1969). compartments. The validity of this assumption In general, alterations in the measured activities cannot be easily established. In Table 11 the of adipose tissue acetyl-CoA carboxylase and fatty metabolite concentrations are, in fact, presented acid synthetase in response to dietary and hormonal per mg of tissue N. However, it would seem to be manipulation (Table 8) paralleled alterations in the as valid to correlate these values with the flux ability of tissues from similarly treated rats to rates as to correlate whole-tissue concentrations incorporate glucose or acetate into fatty acids in since significant alterations in the volume of intra- vitro (Tables 7 and 10) in that starvation and cellular water per mg of tissue N were not encount- induction of alloxan-diabetes resulted in a lowering ered between the dietary treatments (Table 5). of the activities of the two enzymes and of the rate Table 11 shows that rates oftriglyceride synthesis of fatty acid synthesis in vitro. Refeeding with bread were essentially similar in all the dietary conditions after starvation resulted in an increase in these studied. No conclusions concerning the control of three parameters, whereas refeeding with the high- fatty acid esterification can therefore be drawn from fat diet did not. A similar parallelism has been correlations of these flux rates with the tissue drawn between the lipogenic capacity of adipose concentrations of long-chain fatty acyl-CoA and tissues in vitro and their contents of malate de- glycerol 1-phosphate. In fact, as was noted by hydrogenase (NADP) and ATP-citrate lyase (Wise Saggerson & Greenbaum (1970), under the con- & Ball, 1964; Young et al. 1964; Kornacker & Ball, ditions used in this study, i.e. high, non-physiological 1965; Brown & McLean, 1965). However, the concentrations of glucose and insulin in the incuba- demonstration of such a parallelism does not, by tion media, the fatty acid esterifying system of the itself, indicate whether altered rates of lipogenesis tissue is probably always near to saturation with in vitro are wholly or partially dependent on, or glycerol 1-phosphate. Whole-tissue concentrations independent of, altered enzyme contents of the of this metabolite, from the results of Tables 2 and adipose tissues. In rat liver there does not appear 5 of the present study, ranged from 2.1 mm for to be any correlation between the resumption of tissues from normal rats to 5.OmM for tissues from lipogenesis in vitro and the adaptive response of starved, fat-refed rats. Also, the concentrations of lipogenic enzymes on refeeding a high-carbo- glycerol 1-phosphate in the tissues did not appear hydrate, protein-free diet after starvation (Tepper- to be related to the glycolytic rate. However, the man, De la Garza & Tepperman, 1968). Srere & extramitochondrial free [NAD+]/[NADH] ratio is Foster (1967) have also demonstrated a lack of probably of importance in governing the concentra- correlation between the liver content ofATP-citrate tion of this metabolite (Halperin & Denton, 1969; lyase and the lipogenic capacity of liver slices. Saggerson & Greenbaum, 1970) since the tissues However, Jomain & Hanson (1969) have demon- from starved, fat-refed rats contained the highest strated that in adipose tissue, unlike liver, low rates concentrations of glycerol 1-phosphate and were Vol. 119 EFFECTS OF DIET ON LIPOGENESIS IN FAT PAD 235 also associated with the highest [lactate]/[pyruvate] C) ratios in the incubation medium (Tables 1, 7 and 9).

00 ._ In this study the rate of triglyceride synthesis was calculated from the rate of flow of [U-14C]glucose- 4-400 Ca derived carbon into glyceride glycerol. The validity C CC of this approach has been discussed by Denton & cv Randle (1967) and by Denton & Halperin (1968). Ca We&.4w Considerable variations in the flux of glucose PR -o A4Ca carbon into fatty acids were encountered in the PSQ- . C 0F. treatments studied. However, these cannot be C meaningfully related to the proposed roles of long- PI 00) 0 ._0c chain fatty acyl-CoA as an inhibitor of fatty acid 'c C 0; -o synthesis or of citrate as an activator. Concentra- '4. tions of long-chain fatty acyl-CoA were remarkably 0 bo constant in the four cases studied, although their

w proportion of the measured CoA pool (total CoA) 00 varied considerably in the four physiological states. 0-00O caOD C OD The apparent depletion of the total CoA pool on E-4 starvation and its replenishment on refeeding is a o3 Q o e in notable observation. Considerable variations in '0 the concentrations of citrate were observed; how- '4. Cd Q4 ._ ever, the highest concentrations of citrate w re 0 encountered in tissues with the lowest rates of 000; X fatty acid synthesis. Denton et al. (1966) reported 00 4, E elevated citrate concentrations in tissues from

*_ alloxan-diabetic rats incubated in the presence of 00Ca 55 S :>,;4cvOa O a) glucose+insulin. Ballard & Hanson (1969) have o 44 also recorded the absence of any direct relationship EH between the total citrate concentration in epididy- mal fat pads in vivo and rates offatty acid synthesis. Ocv X .4 It is possible, however, as discussed by Saggerson _ _ & Greenbaum (1970), that acetyl-CoA carboxylase 00o r may be saturated with citrate at the concentrations _ P4 4- present in these tissues. It should be pointed out W o Ccv.- that in assessment of the rate of fatty acid syn- thesis it is assumed that the specific activity of cO the immediate fatty acid precursor, acetyl-CoA, ,O 0X ._4. 0g is the same as that of glucose. Since adipose ._ tissues appear to be able to metabolize free fatty eo acids at appreciable rates under certain circum- C4Q c)0$ stances (Milstein & Driscoll, 1959; Bally, Cahill, co Le Boeuf & Renold, 1960), dilution of this acetyl- *- o CoA be o00 .0.4~ pool might expected, particularly in the 0~~~~~~~~0 starved and starved, fat-refed states where the rate of endogenous lipolysis, as indicated by gly- 00cv cerol release, is increased. However, Tables 9 and _e tt 10 indicate that the rates of glyceride-glycerol formation in all four dietary states generally slightly exceed the rates of formation required to E- 44c re-esterify all the fatty acids released by endogenous InV~~~~~~~~~~~~~~& lipolysis (assuming three fatty acid molecules 0' C~~~~~~~~~~~~~~~~C; released/glycerol molecule) and the fatty acids synthesized de novo from glucose (assuming an of W average chain length 17 carbon atoms; Flatt & 4Qo -Ca Ca.-- -ct' Ball, 1964). In this case dilution of the fatty acid E._ precursor pool may not lead to serious under- O t A estimation of the rate of fatty acid synthesis, .OCO o5 4 0 although any error introduced by the metabolism 236 E. D. SAGGERSON AND A. L. GREENBAUM 1970 of fatty acids present in the tissue at the commence- the synthesis of fatty acids from triose phosphates ment of incubation cannot be assessed. are considered below. It should be pointed out that Table 11 indicates that the low rates of glucose- citrate regulation of phosphofructokinase is not carbon flux to fatty acids in tissues from starved and consistent with the elevated concentrations of starved, fat-refed rats are not accompanied by citrate and the increased glycolytic flux in tissues decreased contents of glucose 6-phosphate. Table 2 from starved, bread-refed rats unless other factors shows that the size of the tissue content of glucose are involved. Presumably the elevated steady-state 6-phosphate is indicative of the content of the concentrations of citrate in tissues from starved and other glycolytic intermediates measured. When starved, fat-refed animals reflect a decrease in the tissues from normal animals are incubated with ability of tissues to utilize citrate relative to its glucose in the absence of insulin, or in the presence synthesis, thus establishing a new steady state. The of anti-insulin serum, tissue contents of glycolytic reasons for a decreased ability to utilize citrate in intermediates are depleted compared with insulin- these tissues is unclear; however, the fact that treated tissues and the fluxes of glucose-carbon tissues from starved and starved, fat-refed rats through the glycolytic pathway and into fatty contain decreased contents of ATP-citrate lyase, acids are decreased to approximately the same acetyl-CoA carboxylase and fatty acid synthetase, extent (Denton et al. 1966; Halperin & Denton, as discussed above, should be borne in mind. A 1969; Saggerson & Greenbaum, 1970). The absence control mechanism whereby the cytoplasmic con- of such a depletion of glycolytic intermediates, and centration of citrate regulates the activity of phos- the fact that glycolytic flux is not decreased by an phofructokinase so that its production keeps pace amount proportional to the decrease in the rate of with its utilization would appear to be reasonable, fatty acid synthesis in tissues from starved and but would not be consistent with the proposed role starved, fat-refed rats suggests that, unlike normal of citrate as an activator of acetyl-CoA carboxylase tissues incubated without insulin, the lesion in fatty unless there were a considerable difference between acid synthesis cannot be explained by a decreased the cytoplasmic concentrations at which citrate ability to transport glucose. exerted its effects on phosphofructokinase and Although not of the same magnitude as the acetyl-CoA carboxylase. Citrate regulation of decrease in fatty acid synthesis, the approximate phosphofructokinase would presumably be trans- halving in the flux of glucose-derived carbon from mitted to glucose uptake via a glucose 6-phosphate- hexose phosphate to triose phosphates in tissues mediated inhibition of adipose tissue hexokinases from starved and starved, fat-refed rats would (Grossbard & Schimke, 1966), thus accounting for indicate the possibility of metabolic control at the the observations that glucose uptake in vitro is low phosphofructokinase level. A diminished activity in tissues from diabetic, starved and starved, fat- of phosphofructokinase under these conditions refed rats (Denton et al. 1966; E. D. Saggerson & would correlate with the observed increases in the A. L. Greenbaum, unpublished work). concentrations of citrate, a known inhibitor of Enzyme mass-action ratios. As was considered adipose tissue phosphofructokinase (Denton & above, the results in Table 6 indicate that in all the Randle, 1966). That phosphofructokinase may be experimental conditions studied, the phospho- capable of regulatory behaviour in the tissues in fructokinase reactants appear to be considerably this study is indicated by the fact that the mass- displaced from equilibrium (Hess, 1963) and this action ratio for this reaction is considerably less displacement is similar in each dietary state. In than the apparent equilibrium constant (Table 6). all cases the reactants of phosphoglucose isomerase Evidence for regulatory behaviour of phospho- were very close to their equilibrium values, as fructokinase in normal adipose tissues in vitro has found in other tissues (Hess, 1962; Lowry & been obtained by Halperin & Denton (1969) and Passonneau, 1964; Williamson, 1965; Minakami & by Saggerson & Greenbaum (1970). Unfortunately, Yoshikawa, 1966; Rolleston & Newsholme, 1967) since the contents of glycolytic enzymes are and in normal adipose tissues over a range of different in the various physiological states under incubation conditions (Saggerson & Greenbaum, study here (Saggerson & Greenbaum, 1969), cor- 1970). This near-equilibration of the phospho- relations between alterations in the tissue concentra- glucose isomerase reactants is not unexpected since tions of fructose 6-phosphate and alterations in the the activity of this enzyme is high in all the physio- rate ofglycolytic flux cannot be made in attempts to logical states studied (Saggerson & Greenbaum, clarify the possibility of phosphofructokinase 1969). The mass-action ratio for the combined regulation through citrate inhibition. Such an reaction aldolase x triose phosphate isomerase was inhibition would however, only partially explain the usually one-fifth to one-tenth of the product of the lesion in fatty acid synthesis occurring in tissues apparent equilibrium constants for the two re- from starved and starved, fat-refed rats. Other actions, which varies between 1.4 x 10-3 M and possible points of metabolic regulation governing 3.7 x 10-3M, depending on the combination of Vlol. 119 EFFECTS OF DIET ON LIPOGENESIS IN FAT PAD 2'37 published apparent equilibrium constants used acid cycle CO2 from endogenous precursors. The (Herbert et al. 1940; Meyerhof & Junowicz- fact that the incorporation of acetate relative to Kocholaty, 1943; Krebs & Kornberg, 1957; Hess, glucose into citric acid cycle CO2 is better than into 1963; Lowry & Passonneau, 1964). Inmostcasesthe fatty acids is apparent from Table 10. In each r values for the separate aldolase and triose phos- dietary state there is a difference of approximately phate isomerase reactions could not be ascertained threefold between the glucose carbon/acetate since the tissue concentrations of glyceraldehyde carbon ratios for citric acid cycle CO2 and fatty acids. 3-phosphate were too low to measure. However, A result of this nature would suggest the existence reasonable estimates of the concentration of this of mitochondrial and extramitochondrial pools of metabolite were made in tissues from starved, acetyl-CoA of different specific radioactivities when bread-refed rats and the results indicated that, for glucose and [14C]acetate are used together as incubated tissues of this dietary group at least, the substrates. A bimodal intracellular localization of aldolase reactants were present in near-equilibrium adipose tissue acetyl-CoA synthetase, as found in proportions, whereas those of the triose phosphate liver by Aas & Bremer (1968) might account for the isomerase reaction were not (KaIr 5). riesults of this study. The results presented here are, Effects of acetate on glucose metabolism. In all however, not consistent with those presented by four dietary states the addition of acetate led to Rognstad & Katz (1966) or by Flatt & Ball (1966). increased total rates of fatty acid synthesis. In It is also necessary to consider the possibility that tissues from normal and starved rats the increases 14CO2 is produced from [U-14C]acetate by pathways were 63% and 144% respectively, results very other than the citric acid cycle. Leveille (1967) has similar to those reported by Flatt & Ball (1966). demonstrated incorporation of [14C]acetate into In tissues from starved, fat-refed and starved, glyceride glycerol in the presence of pyruvate. bread-refed rats the increases were 65% and 34% 14CO2 would be produced in this pathway by the respectively. These results suggest that the ob- phosphoenolpyruvate carboxykinase reaction. served rates of fatty acid synthesis in the presence However, in the presence of glucose, [14C]acetate of glucose alone in all four dietary states are not incorporation into glyceride glycerol appears to be governed by the altered maximal activities of lipo- very small. The glucose carbon/acetate carbon genic enzymes. However, in tissues from starved ratios in citric acid cycle CO2 and in fatty acids are and starved, fat-refed rats, the total rates of fatty decreased in tissues from starved and starved, fat- acid synthesis and the rates of synthesis solely from refed rats, indicating that there may be a metabolic acetate were greatly diminished compared with block in these tissues between the uptake of glucose those in normal tissues whereas those in tissues from and the formation of acetyl-CoA compared with starved, bread-refed rats were elevated. Whether tissues from normal and starved, bread-refed rats. altered enzyme contents contribute to these differ- It is noteworthy that Haft (1964) has reported a ences cannot be ascertained. In all four dietary similar discrimination against glucose compared states the increases in fatty acid synthesis in the with acetate as a lipogenic precursor in perfused presence of acetate were accompanied by increases hearts from diabetic rats. In general, the altered in the calculated flux of glucose-carbon through the metabolic rates in the presence of acetate were not hexose monophosphate pathway, suggesting that accompanied by significant alterations in the tissue altered rates of fatty acid synthesis accompanying concentrations of metabolites (Tables 2, 3 and 4). dietary manipulations cannot be accounted for by No conclusions concerning the nature of the effect the possibility that altered contents of the hexose of acetate can therefore be drawn from these monophosphatepathway dehydrogenases (Hollifield measurements. & Parson, 1965; Leveille & Hanson, 1966; McLean, Metabolism of pyruvate and its dependence on Brown & Greenbaum, 1968) limit the supply of endogenous lipolysis. Table 11 shows that although NADPH. rates of fatty acid synthesis from glucose were In all four dietary states the flux of glucose- greatly decreased in tissues from starved and carbon into citric acid cycle CO2 was slightly starved, fat-refed rats, glycolytic rates were not increased in the presence of acetate, and three- to correspondingly altered. In these tissues pyruvate five-fold increases in the total flux of glucose+ acet- decarboxylation was, however, decreased to approx- ate carbon into citric acid cycle CO2 were observed. imately the same extent as fatty acid synthesis Similar increases in measured citric acid cycle flux while lactate +pyruvate release was disproportion- in the presence of acetate in normal tissues were ately high. In tissues from starved, bread-refed rats reported by Rognstad & Katz (1966). This effect the glycolytic rate, lactate +pyruvate release, was not observed by Flatt & Ball (1966). It should pyruvate decarboxylation and fatty acid synthesis be stressed that the citric acid cycle results of this were all elevated to approximately the same extent. study and of Rognstad & Katz (1966) do not in- The results indicate that in tissues from starved clude any assessment of the production of citric and starved, fat-refed rats the diminution in fatty 238 E. D. SAGGERSON AND A. L. GREENBAUM 1970 12 effects on pyruvate utilization similar to those 10 demonstrated in heart and in liver (Evans, Opie

0 4a 8 & Renold, 1963; Garland, Newsholme & Randle, 1964; Bremer, 1965, 1966; von Jagow, Westermann lc cd tt 6 & Wieland, 1968). However, the extent to which fatty acids are metabolized by the tissues in this o0 1z 4

M 0 study is unknown. The increased lipolysis of tissue 2 0 o + glycerides occurring in normal adipose tissues . incubated with adrenaline+insulin compared with :s 0 0.2 0.4 0.6 0.8 1.0 _ Ca bi tissues incubated with insulin alone is also accom- Glycerol release (,umol/h per mg of tissue N) panied by increased rates of lactate + pyruvate release and decreased rates of fatty acid synthesis Fig. 1. Relationship between the rate of release ofglycerol from glucose (Denton & Halperin, 1968; Saggerson and the relative rates of production of lactate + pyruvate & Greenbaum, 1970). Cahill, LeBoeuf & Flinn and fatty acids by incubated adipose tissues from rats of (1960) observed that adipose tissues incubated with varied dietary status. palmitic acid showed alterations in glucose meta- bolism similar to those found in tissues incu- bated with adrenaline and therefore suggested that changes in glucose metabolism observed acid synthesis rate is to some extent brought about in the presence of lipolytic hormones may be by a decreased ability ofthe tissues to decarboxylate secondary to elevations of intracellular free fatty pyruvate. Table 7 suggests that this situation also acid concentrations resulting from an accelerated holds for tissues subjected to other dietary and lipolysis. Cahill et al. (1960), however, did not hormonal manipulation as indicated by the high determine whether incubation with palmitate value of the lactate +pyruvate released/fatty acid resulted in altered rates of lactate+pyruvate re- synthesis ratio in some, but not all, tissues exhibiting lease. It appears, in fact, that incubation of adipose low rates of lipogenesis. In all cases Table 7 shows tissues with palmitate or oleate in the presence of that high values of this ratio were encountered in glucosc +insulin does not result in elevated rates tissues displaying an elevated rate of endogenous of lactate +pyruvate release or in decreased rates of lipolysis (as monitored by glycerol release). A fatty acid synthesis from glucose (Denton & relationship between the relative utilization of Halperin, 1968; Saggerson & G-reenbaum, 1970) and glucose carbon for lactate + pyruvate formation or only mimics the effect of adrenaline in that glucose fatty acid synthesis and free fatty acid release metabolism to glyceride glycerol and citric acid within the tissue is illustrated in Fig. 1. Although cycle CO2 is increased (Saggerson & Greenbaum, this relationship may be fortuitous, it may have 1970). However, these findings do not necessarily metabolic significance. Presumably free fatty acids contradict the suggestion that metabolism of fatty released inside the adipose tissues by breakdown of acids by adipose tissues may decrease the utilization tissue glycerides will either be re-esterified or of pyruvate for fatty acid synthesis, since Dole metabolized by the tissue. Under the conditions (1961) showed that, under conditions of incubation employed in this study, release of free fatty acids similar to those employed in this study i.e. insulin into the surrounding medium can be discounted and high concentrations of glucose present, extern- since, in order to demonstrate free fatty acid release ally supplied 14C-labelled fatty acids do not equilib- from adipose tissue in vitro, albumin must be present rate with the intracellular pool(s) of fatty acids. in the incubation medium (Reshef, Shafrir & Under the conditions used by Dole (1961) however, Shapiro, 1958). Adipose tissues influenced by considerable esterification of the supplied l4C- factors such as prior fasting, alloxan-diabetes or labelled fatty acids occurred, suggesting that treatment with lipolytic hormones are known to esterification of medium fatty acids keeps pace with have elevated intracellular concentrations of free their uptake. These results, and the demonstration fatty acids (White & Engel, 1958; Dole, 1961; by Bally et al. (1960) that the oxidation ofexternally Vaughan, 1961; Buckle, 1963). Whether increased supplied fatty acids by adipose tissues in vitro is intracellular concentrations of free fatty acids are extremely sensitive to the presence of glucose, accompanied by increased rates of their metabolism suggests that under the conditions used only free is unknown, although Bally et al. (1960) have shown fatty acids generated inside the tissue by lipolysis of that oxidation of palmitate to CO2 by adipose tissue glycerides may be able to penetrate to the tissues in vitro is linearly related to its concentration intracellular site(s) of a metabolism that could in the incubation medium in the absence of glucose. influence the utilization of pyruvate. The sites of It would seem reasonable to speculate that meta- such metabolism should presumably be spatially bolism of fatty acids by adipose tissue would have removed from the sites of fatty acid esterification Vol. 119 EFFECTS OF DIET ON LIPOGENESIS IN FAT PAD 239 within the tissue. The very high tissue concentra- pyruvate is decreased, less intramitochondrial tions in vitro of glycerol 1-phosphate encountered acetyl-CoA is expected to be available for export under the incubation conditions used in this study to the cytoplasm for fatty acid synthesis, and hence and elsewhere in the presence of insulin (Denton the utilization of NADPH and ATP in that process et al. 1966; Denton & Halperin, 1968; Saggerson & will be diminished. Flatt & Ball (1966) suggested Greenbaum, 1970) indicates that esterification of that a major factor controlling the rate of fatty acid externally supplied free fatty acids may be extreme- synthesis from glucose is the rate at which NADH ly rapid. In vivo, however, it is quite possible that produced at the two dehydrogenation steps in the esterification of externally supplied free fatty acids pathway between triose phosphate and acetyl-CoA may not keep pace with their uptake under certain can be reoxidized. In adipose tissue the major conditions, thus leading to an elevation of intra- means of reoxidizing this NADH are reductive cellular concentrations of free fatty acids and an synthesis of lactate and glycerol 1-phosphate, increased rate of a mode of their metabolism likely transhydrogenation via malate dehydrogenase and to influence the utilization of pyruvate. Concentra- 'malic enzyme' to yield NADPH for reductive tions of glycerol 1-phosphate in adipose tissues synthesis of fatty acids, and reoxidation by the freeze-clamped in situ and taken from anaesthetized respiratory chain at a rate presumably coupled to animals are in fact considerably lower than those the ATP demand for fatty acid synthesis. Although found in vitro (Saggerson & Greenbaum, 1970). A not fully established, it has been suggested that control mechanism may therefore exist in vivo NADH produced in the cytoplasm of adipose tissue that could divert the utilization of carbohydrate from the oxidation of triose phosphates is used sources available to adipose tissues away from fatty solely in that compartment under normal conditions acid synthesis and into glycerol 1-phosphate pro- and that no transfer of hydrogen equivalents from duction in response to increased uptake of dietary the cytoplasm to the mitochondria takes place fatty acids by the tissue. Under the conditions used (Rognstad & Katz, 1966; Schmidt & Katz, 1969). in vitro a control mechanism of this nature may This is also discussed by Saggerson & Greenbaum not be apparent due to high potential rates of (1970). If these concepts are correct, it is most esterification resulting from the unusually high con- likely that demands for ATP for fatty acid synthesis centrations of glycerol 1-phosphate unless the effect from glucose, over and above the tissue's basal is mimicked by the intracellular introduction of metabolic needs, will be met by respiration of fatty acids to the tissue through lipolysis of tissue pyruvate dehydrogenase-derived NADH, the pro- glycerides. At present it is only possible to speculate duction of which varies in a manner approximately on the way in which fatty acid metabolism might proportional to the supply of acetyl-CoA for fatty possibly affect pyruvate utilization in adipose acid synthesis through control ofpyruvate dehydro- tissues. However, a possible mechanism might genation. It is of course possible that if fatty acid involve, as in other tissues, an inhibition of the synthesis were inhibited at some point beyond pyruvate dehydrogenase enzyme complex associ- acetyl-CoA production from pyruvate, mitochondri- ated with increased values of the mitochondrial al NADH might be expected to accumulate due to a [acetyl-CoA]/[CoASH] and [NADH]/[NAD+] ratios decreased demand for ATP for fatty acid synthesis resulting from increased rates ofmitochondrial fatty and, through inhibition of pyruvate dehydrogena- acid metabolism (Garland & Randle, 1964; Randle tion, set a new rate ofpyruvate utilization. A control et al. 1966; Hansen & Henning, 1966; Nicholls, mechanism of this nature would not be dependent Shepherd & Garland, 1967). Denton, Halperin & on the involvement offatty acid oxidation discussed Randle (1969) have also considered the possibility above and would be consistent with the observed that adipose tissue pyruvate dehydrogenase may increases in citrate concentrations and depletions be subject to a control similar to that found in liver in lipogenic enzyme contents in tissues from starved, and heart. Further experiments to elucidate the starved and fat-refed, and diabetic rats. However, effects of fatty acid metabolism on pyruvate a mechanism of this nature would still have to be utilization by adipose tissues and adipose tissue consistent with the observed increases in lipolytic mitochondria and to elucidate the control char- rate in these tissues, although a direct, inhibitory acteristics of adipose tissue pyruvate dehydro- action of free fatty acids on lipogenic enzymes genase are required. might explain this. The existence of a block in the Fate of cytoplasmic NADH. Whatever its mech- lipogenic pathway after pyruvate utilization is anism, and whether it is or is not brought about supported by the finding in this study that acetate through fatty acid metabolism, any restriction incorporation into fatty acids, though not as on glucose-derived pyruvate metabolism, as is seriously decreased as that of glucose, is still low encountered in tissues from starved, starved, fat- in tissues from starved and starved, fat-refed rats. refed, and diabetic rats, will have important meta- Also it has been found that acetate incorporation bolic consequences. If oxidative decarboxylation of into fatty acids is decreased in tissues incubated 240 E. D. SAGGERSON AND A. L. GREENBAUM 1970 with adrenaline (Orth, Odell & Williams, 1960; E. D. Saggerson & A. L. Greenbaum, unpublished work). It is thus unclear whether a decrease in pyruvate utilization, or some step subsequent to

'3)0 that, might be the primary site of the lesion in fatty acid synthesis considered in the present work. 0 a In the cytoplasm, a decrease in fatty acid syn- C) thesis, whether primarily dependent on diminished pyruvate utilization or on some other mechanism, must result in a decreased utilization of NADPH

0 and hence of NADH transhydrogenation with the 43 120 r-4 result that glycolytic NADH production will be 64 decreased to match its utilization. This therefore C) 5,. 3OD introduces a control point into the glycolytic sequence besides the possible citrate control of phosphofructokinase discussed above. This is C)O illustrated by the fact that glucose-carbon flux from triose phosphates to pyruvate is decreased in tissues from starved and starved, fat-refed rats and elevated in those from starved, bread-refed animals (Table O"C 9). It is also noteworthy that, in normal tissues and 0 Ca in those from starved, bread-refed rats, reductive 5-0 synthesis of lactate and glycerol 1-phosphate account for quite a small proportion (<30%) of the .0(I utilization of cytoplasmic NADH (Table 12), whereas in tissues from starved and starved, fat- refed rats these two processes become responsible for the utilization ofmost ofthe cytoplasmic NADH os o q o 1 4 0 (>80%) when fatty acid synthesis rates, and there- -:C ci4 ~c 0IA'C fore NADH transhydrogenation rates, are low. In this respect glycolysis in adipose tissues from rats o o starved and starved, fat-refed in vitro is similar to that encountered in anaerobic muscle or yeast in 03 ~) m.Ca O d that in these systems the process is to a large extent maintained through the reductive synthesis of lactate and ethanol respectively. 4) In vivo, the linking of triose phosphate oxidation to fatty acid synthesis may be of considerable importance. For example, on ingestion offat by the parent organism, pyruvate utilization for fatty acid m +D .0t ceD CaXC )~Ca) C))- synthesis by adipose tissues may be decreased as g±t + + discussed above. However, the content of AMP in these tissues may be increased under these con- o fi ditions as a result of esterification of incoming fatty 5-m "._ acids (Saggerson & Greenbaum, 1970), leading to

3t _ e an activation of phosphofructokinase. A second point of control at the level of triose phosphate oxidation would ensure that metabolism of glucose C)zz through an activated phosphofructokinase stage was diverted into glycerol 1-phosphate production m and that the activation of phosphofructokinase did waad not lead to a squandering of carbohydrate as lactate and pyruvate which would mainly be released by the

OO4Qw .C tissue. In this context it may be noted that, unlike other fat pad glycolytic enzymes, not only does the ll. triosephosphate dehydrogenase not recover its activity on refeeding a high-fat diet after starvation, ;O Ca,a3 but also the activity of this enzyme is decreased on Vol. 119 EFFECTS OF DIET ON LIPOGENESIS IN FAT PAD 241 refeeding the same fat diet without prior starvation Garland, P. B. & Randle, P. J. (1964). Biochem. J. 91, (Saggerson & Greenbaum, 1969). 6c. Gibson, D. M. & Hubbard, D. D. (1960). Biochem. We are grateful to the Science Research Council for the biophy8. Re8. Commun. 3, 531. grant of a studentship to E.D.S. that was held during the Greenspan, M. & Lowenstein, J. M. (1967). Arch8 earlier part of this investigation. We also thank the Biochem. Biophys. 118, 260. Medical Research Council for the purchase of a Unicam Gregolin, C., Ryder, E., Warner, R. C., Kleinschmidt, SP. 800 spectrophotometer and the Central Research Fund A. K. & Lane, M. D. (1966). 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