Volume 9, number 3

MOLECULAR & CELLULAR BIOCHEMISTRY

December 31, 1975

A S P E C T S OF K E T O G E N E S I S : C O N T R O L AND M E C H A N I S M O F K E T O N E - B O D Y F O R M A T I O N IN I S O L A T E D RAT-LIVER M I T O C H O N D R I A Matthijs L O P E S - C A R D O Z O , Ids MULDER, Frits van V U G T , Paul G. C. H E R M A N S & Simon G. van den B E R G H with technical assistance of Wies K L A Z I N G A and Elly de V R I E S - A K K E R M A N Laboratory of Veterinary Biochemistry, State University of Utrecht, Biltstraat 172, Utrecht, The Netherlands (Received January 10, 1975)

Summary The synthesis of ketone bodies by intact isolated rat-liver mitochondria has been studied at varying rates of acetyl-CoA production and of acetyl-CoA utilization in the Krebs cycle. Factors which enhanced the rate of acetyl-CoA production caused an increase in the fraction of acetyl-CoA which was incorporated into ketone bodies. On the other hand, it was found that factors which stimulated the formation of citrate lowered the relative rate of ketogenesis. It is concluded that acetyl-CoA is preferentially used for citrate synthesis, if the level of oxaloacetate in the mitochondrial matrix space is adequate. The intramitochondrial level of oxaloacetate, which is determined by the malate concentration and the ratio of N A D H over NAD +, is the main factor controlling the rate of citrate synthesis. The A T P / A D P ratio per se does not affect the activity of citrate synthase in this in vitro system. Ketogenesis can be described as an overflow of acetyl-groups: Ketone-body formation is stimulated only when the rate of acetyl-CoA production increases beyond the capacity for citrate synthesis. The interaction between fatty acid oxidation and pyruvate metabolism and the effects of longchain acyl-CoA on mitochondrial metabolism are discussed. Ketone bodies which were generated during the oxidation of [1-14C] fatty acids were

preferentially labelled in their carboxyl group. This carboxyl group had the same specific activity as the acetyl-CoA pool, whereas the specific activity of the acetone moiety of acetoacetate was much lower, especially at low rates of ketone-body formation. The activities of acetoacetyl-CoA deacylase and the hydroxymethylglutaryl-CoA (HMG-CoA) pathway were compared in soluble and mitochondrial fractions of rat- and cow-liver in different ketotic states. In rat-liver mitochondria, both pathways of acetoacetate synthesis were stimulated upon starvation or in alloxan diabetes. In cow liver, only the H M G - C o A pathway was increased during ketosis in the mitochondrial as well as in the soluble fraction.

1 Introduction One of the most important functions of the liver is to sustain an adequate supply of substrates in the blood. In an interplay with other tissues the levels of glucose, fatty acids, triglycerides, ketone bodies and amino acids in the circulation are adapted to the metabolic needs 1. It is wellknown 1 that glucose is used preferentially as a fuel in animals fed on a carbohydrate-rich diet and that excess carbohydrate is converted to triglycerides which are stored in fat depots. During shortage of carbohydrate, however, fatty acids and ketone bodies can replace glucose as a

Dr. W. Junk b.v. Publishers - The Hague, The Netherlands

155

fuel in most tissues z. The idea that ketogenesis is instrumental in achieving caloric homeostasis 3 in the various tissues has been emphasized by KREBS and coworkers 2'4. A pathological condition m a y develop, however, when the production of ketone bodies exceeds their utilization. In such conditions the level of ketone bodies in the blood increases until a balance with renal excretion is reached. Dairy cattle, in which the provision of c a r b o h y d r a t e is very delicate, are especially p r o n e to this metabolic imbalance 5'6. A l t h o u g h the events occurring at the level of the total organism have been delineated, the regulatory mechanisms underlying the changes in flow rate t h r o u g h the ketogenic p a t h w a y are still matter of debate 1'2'4'7 16 This paper is concerned with the control of ketogenesis in liver, which is the main source of ketone bodies in monogastric animals. (In ruminants the r u m e n m u c o s a contributes significantly to the p r o d u c t i o n of ketone bodies). Figure 1 summarizes the quantitatively important reactions which generate or c o n s u m e acetyl-CoA in a liver cell. Rat-liver m i t o c h o n d r i a contain the enzymatic equipment to catalyse the conversions depicted in Figure 1. Hence, these organelles are a useful system for the in vitro study of hepatic ketogenesis. In general, two hypotheses concerning the control of ketogenesis can be formulated - bearing in mind that they are not mutually exclusive and that both m a y in fact contribute to the regulation of k e t o n e - b o d y formation: (1) The rate of k e t o n e - b o d y formation is determined by the rate of acetyl-CoA formation and the rate of acetyl-CoA c o n s u m p t i o n in non-ketogenic pathways. (2) The rate of k e t o n e - b o d y formation is determined by the activity of the rate-limiting enzyme in the ketogenic pathway. Changes in the rate of ketogenesis are effected by m o d u l a t i o n of this activity.

fattyacid~ pyruvate~ ~

~acetoacetate acetyI-CoA > oxaloacetate >

citrate

Fig. 1. Production and utilization of acetyl-CoA in rat-liver mitochondria. 156

Since little is k n o w n about the properties of the enzymes involved in the synthesis of acetoacetate most authors have perforce a d o p t e d the first hypothesis4,V-a 1. In section 2 it will be shown that most of the observed variations in ketogenesis can be satisfactorily explained by the first hypothesis. This does not exclude a possible regulatory role of the enzymes in the ketogenic pathway. Therefore, we shall attempt to evaluate the second hypothesis as well (section 3).

2 Ketone-Body Synthesis in Rat-Liver Mitochondria : an Overflow of Acetyl-Coenzyme A In the first hypothesis it is implicitly assumed that acetyl-groups are preferentially taken up in the Krebs cycle. Hence, appreciable rates of ketoneb o d y p r o d u c t i o n can occur only when the rate of acetyl-CoA formation exceeds the capacity for citrate synthesis. According to this theory, ketogenesis is essentially an overflow p h e n o m e n o n and the rate of acetoacetate synthesis is considered to be determined only by the relative rates of/%oxidation and citrate synthesis. The capacity of the Krebs cycle for the uptake of acetyl-CoA will depend on the mitochondrial oxaloacetate leveP '7'1°'11, which is calculated to be lower than the K m of citrate synthase for oxaloacetate I v. In addition, long-chain acylC o A 7, A T P 9, succinyl-CoA 18 and citrate is have been implicated in the control of citrate synthesis, because these c o m p o u n d s modify the activity of citrate synthase in vitro*. Most evidence, however, is in favour of the concept * It has been postulated 65 that the uptake of acetyl-CoA in the Krebs cycle is limited by the activity of citrate synthase, implicating that the mass-action ratio of citrate synthesis can deviate largely from thermodynamic equilibrium. On the other hand the possibility has been considered~6 that citrate synthase has a high activity in comparison with the flow through this reaction and thus can establish near-equilibrium between its reactants. Recent evidence67 7o favours the latter hypothesis. If this is correct, it would invalidate the theory that adenine nucleotides or other metabolites regulate citrate synthesis by modulating the activity of citrate synthase. This is because enzyme modifiers can change the flow rate through a pathway only in the case of a 'non-equilibrium' enzyme69'71. if citrate synthase catalyses an equilibrium reaction, the [AcCoA]/ [CoASH] ratio is dictated by the relative amounts of oxaloacetate and citrate in the matrix 7°. In that case it is clear that oxaloacetate is one of the main regulators of ketogenesis.

that oxaloacetate plays a crucial role in the regulation of citrate synthesis 2'7'9-a¢. Unfortunately, tissue levels of oxaloacetate especially in the mitochondrial c o m p a r t m e n t are extremely low, and as a consequence the evidence is mainly indirect. In this section the following topics will be discussed in connection with the rate of k e t o n e - b o d y p r o d u c t i o n by isolated rat-liver m i t o c h o n d r i a : (i) Effects of Krebs-cycle intermediates and the mitochondrial energy state on the rate of citrate synthesis. (ii) The rate of acetyl-CoA production. (iii) The interaction between fatty-acid and pyruvate metabolism. The experimental a p p r o a c h is briefly discussed in the next paragraph. 2.1 The acetyl-ratio In order to study the control of a pathway, an u n a m b i g u o u s m e t h o d to determine the flow rate t h r o u g h this p a t h w a y is required. In the presence of added substrates, the oxidation of endogenous substrates by isolated m i t o c h o n d r i a is negligible. Therefore, a quantitative balance can be made up of the oxygen uptake and the various products which accumulate during the oxidation of added substrates (see refs 11 & 12 for a detailed discussion). P r o d u c t s of fatty acid oxidation in the presence of added malate are acetoacetate, 3hydroxybutyrate, citrate and C O z. Malate equilibrates with fumarate via the mitochondrial fumarate hydratase reaction. Succinate and phosp h o e n o l p y r u v a t e accumulate only slowly 19. W h e n carnitine is added, acetylcarnitine also is a reaction product. F r o m the oxygen uptake, after correction for the oxygen c o n s u m e d in the production of the various products, the a m o u n t of fatty acid which is oxidized to CO2 can be calculated 11. The total flux t h r o u g h the acetylC o A pool (EAcCoA) is the sum of the acetylgroups recovered in the various products and the calculated a m o u n t of acetyl-CoA oxidized to C O 2. Finally, the relative rate of ketogenesis is expressed in the acetyl-ratio: Acetyl-ratio = acetyl-CoA converted to ketone bodies total flux t h r o u g h the acetyl-CoA pool A complication m a y arise from the fact that malate can also be converted to acetyl-CoA via oxidation of pyruvate, which is in turn p r o d u c e d

by decarboxylation of oxaloacetate. It has, however, been pointed out 14 that this contribution of malate to the acetyl-CoA pool is low in a high-energy state and during a rapid generation of acetyl-CoA by/?-oxidation. 2.2 Effects of added malate on the acetyl-ratio Figure 2 shows the effect of malate on the acetylratio for State-3 oxidation of various substrates. W i t h o u t added malate the acetyl-ratio is high: most of the acetyl-CoA is converted to ketoneb o d y synthesis. In the presence of added malate, citrate synthesis is stimulated by the increased availability of oxaloacetate and ketogenesis is relatively diminished, as reflected by the decrease

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concentration for the oxidation of various substrates by ratliver mitochondria in a low-energy state (State 3). The mitochondria (6 8 mg protein; see legend to Fig. 3) were incubated at 25 °C in a standard reaction medium, containing 50 mM sucrose, 5 mM MgC12, 2 mM EDTA, 15 mM KC1 and 50 mM Tris-HC1 (pH 7.5), supplemented with 20 mM potassium phosphate (pH 7.5) and 1 mM AMP. Other additions: 1 mM pyruvate, 1 mM butyrate, 0.5 mM octanoate or 0.10 ml of a neutralized aqueous suspension of potassium palmitate containing 4 #moles per ml. Final volume, 2 ml. Oxygen uptake was recorded polarographically, using a vibrating platinum electrode (Gilson Oxygraph). Just before the oxygen was depleted (2-3 min after addition of the mitochondria), the reactants were rapidly transferred by suction into a centrifuge tube containing 0.5 ml ice-cold 2 MHC104. After centrifugation, the neutralized supernatant was used for the combined enzymatic assays of acetoacetate and citrate 11,64 Control experiments showed that 3-hydroxybutyrate did not accumulate under these conditions. The oxygen content of the reaction medium (0.45 0.50 patom/ml) was estimated by the catalase method 62. 157

of the a c e t y l - r a t i o . These results confirm the classic w o r k of LEHNINGER2°, who s h o w e d t h a t the level of K r e b s - c y c l e i n t e r m e d i a t e s is an imp o r t a n t p a r a m e t e r in the d i s t r i b u t i o n of acetylg r o u p s b e t w e e n the K r e b s cycle a n d k e t o n e - b o d y synthesis in i s o l a t e d rat-liver m i t o c h o n d r i a . It is clear f r o m F i g u r e 2 t h a t the r e l a t i o n s h i p b e t w e e n the a c e t y l - r a t i o a n d the m a l a t e c o n c e n t r a t i o n is d e p e n d e n t on the s u b s t r a t e used. In the presence of i mM m a l a t e , only 2 % of the oxidized p y r u v a t e is c o n v e r t e d to k e t o n e bodies, whereas with o c t a n o a t e ~ a l m o s t 40 % of the a c e t y l - C o A goes into the k e t o g e n i c p a t h w a y . T h e different a c e t y l - r a t i o s o b s e r v e d with the v a r i o u s prec u r s o r s of a c e t y l - C o A can be largely e x p l a i n e d by the different rates of a c e t y l - C o A p r o d u c t i o n (see 2.5). H o w e v e r , the preferential i n c o r p o r a t i o n of the co-terminal C H 3 - C H z - g r o u p of fatty acids into k e t o n e b o d i e s m a y also p l a y a role (see 3.4). 2.3 Effect of the mitochondrial energy state on the

acetyl-ratio T h e o x i d a t i o n e x p e r i m e n t shown in F i g u r e 2 was c a r r i e d out in State-3 c o n d i t i o n s : glucose a n d excess h e x o k i n a s e were present to c o n v e r t all A T P , f o r m e d in o x i d a t i v e p h o s p h o r y l a t i o n , i m m e d i a t e l y to A D P . In vivo, however, o x i d a t i o n r e a c t i o n s are p r o b a b l y c o n t r o l l e d b y a limited s u p p l y of A D P . T h u s the energy state in liver is p r o b a b l y in b e t w e e n State 3 a n d State 4 (see ref. 21) a n d for t h a t r e a s o n it is of interest to see w h a t h a p p e n s to the a c e t y l - r a t i o u p o n t r a n s i t i o n of State 3 to State 4. The energy state can be shifted from State 3 to State 4 b y a d d i n g decreasing a m o u n t s of hexok i n a s e to the m e d i u m 11. This causes an increase of the m i t o c h o n d r i a l A T P / A D P r a t i o , which in t u r n will induce an increased r e d u c t i o n of the p y r i d i n e nucleotides. As can be seen from F i g u r e 3, the m i t o c h o n d r i a l N A D H / N A D + r a t i o ( m o n i t o r e d b y the degree of r e d u c t i o n of the p r o d u c e d k e t o n e b o d i e s 17) rises when the hexokinase in the m e d i u m is lowered. T h e i m p o r t a n t p o i n t is t h a t the a c e t y l - r a t i o rises as well. In F i g u r e 4 the m e t a b o l i c events u p o n transitions b e t w e e n State 3 a n d State 4 are analysed. F i g u r e 4 A shows a t i m e course of p a l m i t a t e o x i d a t i o n , in w h i c h a State-4 --, State-3 t r a n s i t i o n is b r o u g h t a b o u t b y the a d d i t i o n of h e x o k i n a s e . In F i g u r e 4B a State-3 --* State-4 t r a n s i t i o n occurs after d e p l e t i o n of the a d d e d glucose. (It s h o u l d be n o t e d t h a t an a p p r e c i a b l e a m o u n t of acetyl158

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Fig. 3. Effects of the energy state of the mitochondria on the reduction of the ketone bodies and on the acetyl-ratio. The standard reaction medium (Fig. 2) was supplemented with 0.4 mM palmitate (complexed with albumin in a molar ratio of 5 : 1), 25/~M CoASH, 0.25 mM GSH, 0.5 mM L-carnitine, 0.5 mM L-malate, 0.5 mM ADP, 20 mM glucose, 20 mM potassium phosphate (pH 7.5) and hexokinase as indicated. Mitochondria (9.3 mg protein) were incubated during 16 min at 25 °C. Final volume, 2 ml. The oxygen uptake was measured manometrically in a Gilson Respirometer. Mitochondria were isolated from livers of Wistar rats (~, 150-200 g) essentially according to Myers and Slater 72. The homogenate was centrifuged during 5 min at 775 x g to remove nuclei, erythrocytes, intact liver cells and debris. Centrifugation of the supernatant for 10 rain at 4500 × g gave a mitochondrial pellet and a supernatant ($2). The mitochondrial fraction was washed once by suspension in 0.25 M sucrose and centrifugation for 10 min at 12,600 × g. Centrifugations were carried out at 0-5 °C in the SS-34 rotor of a Sorvall RC 2 B centrifuge. The relative centrifugal forces refer to the bottom of the centrifuge tube. The degree of reduction of the ketone bodies is expressed as {[hydroxybutyrate]/([acetoacetate] + [hydroxybutyrate])}. 100%. c a r n i t i n e a c c u m u l a t e s d u r i n g this e x p e r i m e n t : after 18 m i n m o r e t h a n 50 % of the a d d e d c a r n i t i n e was acetylated). The following a r g u m e n t s s u p p o r t the view t h a t the increased acetyl-ratio, o b s e r v e d in a State-3 State-4 t r a n s i t i o n , is caused b y a decreased a v a i l a b i l i t y of o x a l o a c e t a t e to citrate synthase r a t h e r t h a n b y an i n h i b i t i o n of citrate synthase b y the elevated level of A T P 9: (i) A c r o s s - o v e r is o b s e r v e d in the levels of m a l a t e plus f u m a r a t e a n d citrate on transition from State 4 to State 3 (Fig. 4A) or from State 3 to State 4 (Fig. 4B), i n d i c a t i n g t h a t citrate synthesis is a r a t e - l i m i t i n g step in State 4. (ii) T h e r a t i o of h y d r o x y b u t y r a t e over acetoacetate is a b o u t tenfold higher in State 4 t h a n in State 3 (Fig. 4), If the m i t o c h o n d r i a l m a l a t e

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The standard reaction medium (Fig. 2) was supplemented with 30 mM (A) or 15 mM (B) glucose, 1 mM ATP, 30 mMpotassium phosphate (pH 7.5), 0.9 mM palmitate (complexed to albumin in a 5 : 1 molar ratio), 50 #N CoASH, 0.5 mM GSH, 1 mM L-carnitine and 0.5 mM L-malate. Hexokinase (2.6 units/ml) was included in the medium (B) or added at t = 8 min (A). Mitochondria, 6.0 mg (A) or 5.6 mg (B) protein per ml, were incubated in 20 ml medium in 125-ml Erlenmeyer flasks, vigorously shaken at 25 °C. At the times indicated 2-ml samples were withdrawn for enzymatic assays11'64 of: acetoacetate (AcAc, D--N), citrate (Cit, © ©), 3-hydroxybutyrate (HB, I - - i ) , malate plus fumarate (M + F, e--O) and acetylcarnitine (AcCn, • •). Phosphoenolpyruvate (PEP, x - - x ) was determined fluorimetrically64. In the upper part of Figure 4B the phosphate concentration (P~)is plotted, showing a transition at t = 16 min, which can be attributed to the complete conversion of the added glucose to glucose 6-phosphate. level r e m a i n s c o n s t a n t a n d malate deh y d r o g e n a s e establishes e q u i l i b r i u m between its reactants, the increased N A D H / N A D + ratio in State 4 will effect a tenfold drop in the level of m i t o c h o n d r i a l oxaloacetate. (iii) The rate of p h o s p h o e n o l p y r u v a t e p r o d u c t i o n (Fig. 4A) is always low, but increases significantly u p o n a State-4 --~ State-3 t r a n s i t i o n z2. This indicates that the availability of oxaloacetate is increased in State 3. (iv) OLSON a n d WmLIAMSON1° did n o t find a relationship between the m i t o c h o n d r i a l A T P level a n d the rate of citrate synthesis. (v) The acetyl-ratio is n o t e n h a n c e d by the a d d i t i o n of A T P plus oligomycin to u n coupled m i t o c h o n d r i a oxidizing palmitoylcarnitine 11. F r o m these a r g u m e n t s we conclude t h a t the

increased acetyl-ratio a n d the observed i n h i b i t i o n of citrate synthesis in State 4 are n o t caused by i n h i b i t i o n of citrate synthase by the elevated level of A T P , b u t by the increased N A D H / N A D + ratio, which brings a b o u t a decrease of mitoc h o n d r i a l oxaloacetate. 2.4 Effect of palmitoyl-CoA on ketogenesis CoA-esters of l o n g - c h a i n fatty acids are p o t e n t i n h i b i t o r s of the t r a n s l o c a t i o n of A D P a n d A T P t h r o u g h the m i t o c h o n d r i a l i n n e r m e m b r a n e (see 23 for refs). I n h i b i t i o n of a d e n i n e - n u c l e o t i d e t r a n s l o c a t i o n increases the m i t o c h o n d r i a l energy state. It m a y be expected, therefore, that a d d i t i o n of l o n g - c h a i n acyl-CoA has a similar effect o n ketogenesis as a State-3 --, State-4 t r a n s i t i o n 23 (Fig. 3). Table 1 shows that p a l m i t o y l - C o A indeed has a 159

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F r o m F i g u r e 9 it is clear t h a t the specific activities of t h e t w o halves of a c e t o a c e t a t e f o r m e d f r o m a specifically l a b e l l e d fatty acid c a n p r o v i d e i n f o r m a t i o n o n t h e relative c o n t r i b u t i o n s of the v a r i o u s p a t h w a y s of a c e t o a c e t a t e synthesis. F u r t h e r c o n c l u s i o n s c a n be d r a w n if the specific activity of the a c e t y l - C o A p o o l c a n be determ i n e d . I n o r d e r to m a k e clear, b e f o r e h a n d , w h a t k i n d of i n f o r m a t i o n c a n be g a t h e r e d f r o m this type of e x p e r i m e n t , T a b l e 4 s u m m a r i z e s a h y p o t h e t i c a l case. T h r e e m a i n c o n c l u s i o n s c a n be d r a w n f r o m this T a b l e a n d f r o m F i g u r e 9: (i) It is p o s s i b l e to p r o v e the c o n t r i b u t i o n of a d e a c y l a s e to k e t o n e - b o d y synthesis o n l y if A c A c - C o A (type |) c o n t r i b u t e s to acetoacetate synthesis. (ii) It is p o s s i b l e to p r o v e the c o n t r i b u t i o n of A c A c - C o A (type I) to a c e t o a c e t a t e synthesis o n l y if a d e a c y l a s e c o n t r i b u t e s to k e t o n e b o d y synthesis. (iii) T h e specific a c t i v i t y of the c a r b o n y l - m o i e t y

Table 4 The interpretation of experiments with specifically labelled fatty acids, giving rise to asymmetrically labelled ketone bodies. An even-numbered fatty acid, labelled in an uneven carbon atom (but not in its co-terminal C4-moiety ) e.g. [1-14C~ octanoate, is oxidized by rat-liver mitochondria in the presence of malate and fluorocitrate. Under these conditions ketone bodies and citrate are the only labelled products which accumulate. The specific activities of the carbonyl- and carboxyl-group of acetoacetate (CO and CO2H, resp.) and the specific activity of citrate (CIT) are measured. Assuming that CIT represents the specific activity of the acetyl-CoA pool, the results can be qualitatively interpreted as follows. (A quantitative interpretation of these experiments will be presented elsewhere). Result CO ~< COzH < CIT

CO < CO2H = CIT

CO = zero

CO = CO2H = CIT

168

Intrepretation --Ketone bodies of the type AcAc are formed; therefore : - - Unlabelled AcAc-CoA (Fig. 9, Type I) is directly hydrolysed to AcAc, hence: - - A deacylase activity must contribute to ketone-body synthesis. --Ketone bodies of the type AcAc are not formed. --Only the CH3-CH 2- end of the fatty acid does not equilibrate with the acetyl-CoA pool, resulting in asymmetric labelling of the ketone bodies formed. --Ketone bodies of the type AcAc are not formed, hence: --AcAc-CoA (Fig. 9, Type III) does not contribute to acetoacetate synthesis. - - I t cannot be decided whether a deacylase contributes to ketone-body synthesis. - - O n l y ketone bodies of the type AcAc are found, hence: --AcAc-CoA (Fig. 9, Type I) and AcAc-CoA (Fig. 9, Type II) do not contribute to acetoacetate synthesis. - - I t cannot be decided whether a deacylase contributes to ketone-body synthesis.

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of acetoacetate provides a measure for the contribution of A c A c - C o A (type III, formed by recombination of two molecules of acetylCoA from the pool) to acetoacetate synthesis. Table 5 shows the results of a number of representative experiments in which various [1-1~C] fatty acids were incubated with rat-liver mitochondria in the presence of fluorocitrate and excess malate. Ketone bodies and citrate, produced during the incubation, were separated by column chromatography 6° and their specific activities were determined. The distribution of 14C over the carbonyl- and carboxyl-moieties of acetoacetate was also determined 61. The specific activity of citrate was assumed to reflect the specific activity of the acetyl-CoA pool. The following observations in Table 5 are relevant to the mechanism of acetoacetate synthesis: (i) The relative specific activity (RSA, see legend to Table 5) of the carbonyl-group of acetoacetate is always lower than that of citrate, indicating that the co-terminal C2-unit of the fatty acid substrate does not equilibrate with the acetyl-CoA pool and is preferentially incorporated into ketone bodies. (ii) The carboxyl-group of acetoacetate has about the same RSA as citrate under all conditions tested. F r o m this we may conclude that incorporation of intact CA-units of the fatty acid into acetoacetate (deacylation of AcAc-CoA, type I of Fig. 9) does not occur and, hence, that positive evidence for the operation of a deacylase is not obtained. On the other hand we may recall that these results neither exclude a contribution of the deacylase pathway (see Table 4). It may be noted that with [1-14C] butyrate as substrate the RSA of the carboxylgroup is slightly higher than that of citrate. More experiments are needed, however, to establish whether this difference is significant, in which case it can be taken as evidence for the contribution of a deacylase to acetoacetate synthesis. (iii) The most interesting phenomenon is the increase of the asymmetry ratio going from State 3 to State 4. As the asymmetry ratio is the quotient of the (specific) activities of the carbonyl- and the carboxyl-groups of acetoacetate, an increased asymmetry ratio is indicative of an increased contribution of the reverse 170

thiolase reaction to ketogenesis. The simplest hypothesis to explain this result is that the rate of recombination of acetyl-CoA to A c A c - C o A (type III of Fig. 9) depends on the AcCoA2/ CoASH ratio which is in its turn determined by the rates of/?-oxidation and of citrate synthesis. Figure 10 corroborates the above hypothesis. When palmitate oxidation is stimulated by L-carnitine, the acetyl-ratio increases (cf. Fig. 5). A parallel increase of the asymmetry ratio can be observed. In short, the asymmetry ratio, which is diagnostic for the contribution of the reverse thiolase reaction, and the acetyl-ratio may both depend on the ratio of acetyl-CoA over CoASH, which increases when the rate of/%oxidation exceeds the capacity of the Krebs cycle for uptake of acetyl-groups. HUTH et al. 16 have also reported an increase of the asymmetry ratio when palmitate oxidation was stimulated. They consider this result to be evidence for a regulatory role of thiolase (see 3.1). 0.3

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15 20 2~5 rote of ocety/-CoA production (nmoles/min per rag) Fig. 10. Parallel increases of the acetyl- and the asymmetry ratio with increasing rates of palmitate oxidation. Rat-liver mitochondria (5.2 mg protein) were incubated for 60 min at 25 °C in the standard reaction medium (Fig. 2) supplemented with 40 m~ glucose, 40 mM phosphate (pH 7.5), 1 mM ATP, 5 mM L-malate, 0.1 mM fluorocitrate, 2.6 units hexokinase, 20 #M CoASH, 0.2 mM GSH, 0.9 mM [I-1'~C] palmitate (0.33 Ci/mole) complexed to 0.15 mM albumin in a final volume of 2 ml. The rate of palmitate oxidation was varied by changing the concentration of L-carnitine in the medium (0-0.25 m~). After addition of perchloric acid (0.4 M, final concentration) most (> 99 %) of the radioactive palmitate is found in the protein pellet TM. The acid supernatant was used for the assays of citrate and ketone bodies and of the asymmetry ratio 61. The acetyl-ratio was calculated as A(ketone bodies)/A(ketone bodies + citrate).

Returning to Table 5, it will be clear that the RSA of citrate should equal unity when (i) the added fatty acid is the only precursor of acetylCoA; (ii) all acetyl-groups are equivalent: acetoacetate is only s~cnthesized from the acetylCoA pool. If the latter condition is not fulfilled, the RSA of citrate will rise above one if the substrate is a [ 1 - 1 4 C ] fatty acid. RSA values for citrate significantly lower than one were observed in the case of butyrate oxidation in State 3, indicating that the former condition is not fulfilled and that the acetyl-CoA pool is diluted with unlabelled acetyl-CoA. In fact, from the disappearance of malate and the formation of fumarate and citrate the contribution of the pathway malate --. oxaloacetate --. pyruvate acetyl-CoA to the pool can be calculated 14 (column Mal ~ AcCoA of Table 5). The finding that this pathway is suppressed in State 4 and at higher rates of fl-oxidati0n confirms previous results14. Finally, it should be noted that also in the fluorocitrate-inhibited system of Table 5 the acetyl-ratio depends on the mitochondrial energy-state (2.3) and to a lesser extent on the rate of fl-oxidation (2.5).

4 Conclusion Ketogenesis by isolated rat-liver mitochondria can be described as an overflow process (section 2, hypothesis (1)). The rate of ketone-body synthesis is determined by the rates of production of acetyl-CoA and of its utilization in citrate synthesis (2.5). The malate concentration in the medium (2.2), the rate of pyruvate carboxylation (2.6) and the mitochondrial N A D H / N A D + ratio (2.3) are important parameters in the regulation of citrate synthesis, pointing to the availability of oxaloacetate as the main factor controlling the uptake of acetyl-CoA in the Krebs cycle (cf footnote to p. 157). An elevated level of palmitoyl-CoA, generated at the outside of the mitochondrial inner-membrane, causes increases in the relative rate of ketogenesis and in the ratio of 3-hydroxybutyrate over acetoacetate (2.4). These events can be explained by an inhibition of adenine-nucleotide translocation by long-chain acyl-CoA esters. When the rate of acetyl-CoA production is increased beyond the capacity for citrate synthesis, the ratio of acetyl-CoA over CoASH in

the matrix space rises and triggers an enhanced rate of acetoacetate synthesis, probably due to the increased availability of acetoacetyl-CoA to H M G - C o A synthase (3.1). With [1-~4C] fatty acids as substrates an enhanced rate of acetoacetate synthesis brings about a more symmetrical distribution of t4C between the two halves of acetoacetate (Table 5, Fig. 10). This shows that the reverse thiolase reaction is stimulated under these Conditions. No evidence for the operation of an acetoacetyl-CoA deacylase in intact rat-liver mitochondria could be obtained (3.4). On the other hand, deacylase activity has been demonstrated to be present both in the cytoplasm and in the mitochondrial compartment of rat and cow liver (3.3). The capacities of the deacylase and the H M G - C o A pathways in rat-liver mitochondria are increased upon starvation or in alloxan diabetes, whereas in cow-liver mitochondria as well as in the cytoplasm only the H M G - C o A pathway is increased during ketosis (Table 3). Finally, it should be emphasized that the significance of these results and conclusions is restricted to the in vitro system under study: isolated liver mitochondria. In our opinion, however, a detailed knowledge of the regulatory mechanisms operating at the level of these organelles is required to unravel the pattern of control in the intact hepatocyte.

Acknowledgements We are indebted to Dr. E. LAGERWEYof the Institute for Veterinary Surgery, State University of Utrecht, for cow-liver samples taken by a biopsy technique. Furthermore we would like to express our appreciation to Mrs. J. A. H . VERWEIJ -- VAN" DIJL for typing the manuscript and to Mr. W. D. BRANDS for drawing the figures. This investigation was supported in part by the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).

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Aspects of ketogenesis: control and mechanism of ketone-body formation in isolated rat-liver mitochondria.

The synthesis of ketone bodies by intact isolated rat-liver mitochondria has been studied at varying rates of acetyl-CoA production and of acetyl-CoA ...
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