Inhibition of carnitine palmitoyl-CoA transferase activity and fatty acid oxidation by lactate and oxfenicine in cardiac muscle

j Mol Cell Cardiol 17,619 625 (1985)

Inhibition of Carnitine Palmitoyl-CoA Transferase A c t i v i t y a n d Fatty Acid O x i d a t i o n by L a c t a t e a n d O x f e n i c i n e in C a r d i a c M u s c l e David R. Bielefeld, Thomas C. Vary and James R. Neely* Department of Physiology, The Milton S Hershey Medical Center, The Pennsylvania State University, Hershey, PA 17033, USA (Received 30 May 1984, accepted in revisedform 22 August 1984) D. R. BIELEFEI.D,T. C. VARYANnJ. R. NEELY. Inhibition of Carnitine Palmitoyl-CoA Transferase Activity and Fatty Acid Oxidation by Lactate and Oxfenicine in Cardiac Muscle. Journalof Molecularand CellularCardiology (1985), 17, 619 625. High concentrations of lactate and oxfenicine inhibit fatty acid oxidation in cardiac muscle. The site of this inhibition was investigated in isolated perfused rat hearts. In hearts perfused with glucose (11 mM) and [U-14C]palmitate (1.0 mM), addition of 5 mM lactate caused a 38% reduction in 14CO2 production. Tissue levels of long-chain acyl carnitine decreased suggesting that inhibition occurred at either fatty acyl CoA synthetase or carnitine-acyl CoA transferase. Cytosolic levels of acyl-CoA are low compared with mitochondrial levels and changes in acyl-CoA within the cytosolic compartment cannot be estimated directly. Consequently, the rate of conversion of 14C-palmitate to neutral lipids was used as an indicator of cytosolic acyl CoA levels. Lactate caused a 100% increase in 14C-fatty acid conversion to triglycerides suggesting that cytosolic levels of acyl-CoA increased in association with decreased acyl-carnitine. This indicates that lactate inhibited FFA oxidation at the level of carnitine-acyl CoA transferase. Oxfenicine (2 raM) reduced fatty acid oxidation by 45%, decreased acyl-carnitine levels by 80%, and increased conversion of 14C-palmitate to neutral lipids by 44%, suggesting that oxfenicine also inhibits fatty acid oxidation at the level of carnitine-acyl CoA transferase. These data further indicate that carnitine-acyl CoA transferase is an important site of control in the pathway of fatty acid oxidation. KEY WORDS: Carnitine; Carnitine palmitoyl-CoA transferase; Coenzyme A; Fatty acid oxidation; Lactate; Oxfenicine; Triglycerides.

Introduction T h e e l e v a t i o n of a r t e r i a l l a c t a t e c o n c e n t r a tion, e i t h e r b y the infusion of l a c t a t e [26] or t h r o u g h the process of h e m o r r h a g i c [27] or e n d o t o x i c [24] shock, has b e e n d e m o n s t r a t e d to d e c r e a s e the e x t r a c t i o n r a t i o o f fatty acids a n d the f r a c t i o n of C O 2 p r o d u c t i o n d e r i v e d f r o m free f a t t y acid o x i d a t i o n . H o w e v e r , the m e c h a n i s m b y w h i c h l a c t a t e exerts c o n t r o l o v e r free fatty acid m e t a b o l i s m r e m a i n s to be elucidated. P r e l i m i n a r y o b s e r v a t i o n s in this l a b o r a t o r y demonstrated that elevated lactate concentrations c a u s e d a d e c r e a s e in l o n g c h a i n acyl carn i t i n e levels in rat hearts perfused w i t h fatty a c i d [3-]. T h i s w o u l d suggest t h a t l a c t a t e inhibits f a t t y a c i d o x i d a t i o n p r i o r to t r a n s p o r t o f the acyl u n i t i n t o m i t o c h o n d r i a . A similar effect was o b s e r v e d by the a d d i t i o n o f oxfeni0022-2828/85/060619 + 07 $03.00/0

cine to perfusion solutions. O x f e n i c i n e (L-4h y d r o x y p h e n y l g l y c i n e ) is an a g e n t t h a t decreases m y o c a r d i a l free fatty acid e x t r a c t i o n and oxygen consumption while increasing e x t r a c t i o n of lactate, p y r u v a t e a n d glucose [1,

11, 19]. T h e p r e s e n t i n v e s t i g a t i o n was i n i t i a t e d to e v a l u a t e the site in the p a t h w a y of f a t t y acid o x i d a t i o n w h i c h is i n h i b i t e d by l a c t a t e a n d oxfenicine.

Materials and Methods Perfusion technique M a l e , albino, S p r a g u e - D a w l e y rats (250 to 300 g) w e r e p u r c h a s e d f r o m C h a r l e s R i v e r B r e e d i n g L a b o r a t o r i e s . T h e rats w e r e either fed ad libitum or fasted for 48 h p r i o r to use. 9 1985 Academic Press Inc. (London) Limited

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The isolated working heart preparation was utilized [21]. The perfusate consisted of a Krebs-Henseleit bicarbonate buffer supplemented with 11 mM glucose and various concentrations of [U-14C]palmitate (New England Nuclear, 54 nCi/ml), [1-14C] octanoate (New England Nuclear, 43 nCi/ml), lactate, and oxfenicine (a gift from Pfizer, Ltd). Palmitate or octanoate was bound to 3% bovine serum albumin (Pentex Fraction V) and the concentration of palmitate was determined on the final perfusate [5]. Fatty acids were bound to albumin by converting the free acid to the sodium salt and adding the salt to a stirred, warm albumin solution. This solution was dialyzed overnight against a large volume of buffer and filtered through a 1.2/~m Millipore filter before use. Buffers (pH 7.4) were maintained at 37~ and gassed with 95% O 2 : 5 % CO 2. After an initial 10 min perfusion as a Langendorff preparation at an aortic pressure of 60 cm H20 , the working heart mode was initiated with a left atrial filling pressure of 10 cm H20. When used, lactate or oxfenicine were added to the perfusate during both the initial 10 rain perfusion and the working perfusion periods. Aortic pulse pressure and heart rate were monitored via a fluid-filled sidearm on the aortic cannula connected to a Statham pressure transducer and recorded on a Beckman dynograph.

was to be determined. The tissue was frozen using Wollenberger clamps precooled to the temperature of liquid nitrogen. The total frozen wet weight of each heart was recorded. For measurements of 14C-O2 production 1 ml samples ofperfusate were injected into a 25 ml stoppered, Erlenmeyer flasks containing 1 ml of 9N H2SO4. The 14CO2 released was collected in a center well containing 0.4 ml of 1 MH y a m i n e hydroxide. After 2 h of gentle shaking at room temperature, the wells were removed, placed in Oxifluor-CO 2 scintillation cocktail (New England Nuclear) and counted. Rates of fatty acid oxidation were calculated from rates of 14CO2 production and the specific activity of the perfusate fatty acid.

Estimation of tissue levels of metabolic intermediates

The frozen ventricular tissue was pulverized using a mortar and pestle cooled to the temperature of liquid nitrogen. Approximately 200 mg of frozen, powdered tissue was used to estimate the dry : wet ratio and 500 mg was stored for later analysis of tissue lipids. The remainder (800 mg) of the frozen tissue was extracted in 2 ml of ice cold perchloric acid (6% w/v) using a mortar and pestle, and centrifuged at 0~ A portion of the supernatant was neutralized with K O H and used for estimation of tissue levels of free CoA and carnitine. Another 0.5 ml portion of the supernatant was hydrolyzed at pH 11.5 for Estimation offatty acid oxidation 15 min at 55~ in the presence of 15 m~ The working heart preparation was perfused dithiothreitol for estimates of total acidin a closed recirculating system using an oxy- soluble CoA and carnitine (free plus short genator with a large surface area in constant chain esters). The perchloric acid precipitate was washed contact with a 95% 0 2 : 5 % CO 2 gas mixture. The gas exited through an outlet with 1.2% perchloric acid and divided into tube and was bubbled through a 1 M two portions. Long chain acyl CoA was Hyamine hydroxide trap for the collection of hydrolyzed at pH 11.5 to 12.0 for 15 min at gaseous CO 2 . Perfusate samples (3 ml) were 55~ in the presence of 15 m~ dithiothreitol. removed directly from the system via a syringe Long chain acyl carnitine was hydrolyzed at and were injected under mineral oil for tem- pH 12.5 to 13.0 for 1 h at 70~ The pH of the porary storage prior to assaying for 14CO2 as hydrolysates was decreased to one with 70% bicarbonate and dissolved CO z . Hearts were H C 1 0 4 to precipitate any residual protein. perfused for 30 min; perfusate samples were After centrifugation at 0~ the supernatants taken at 20, 25, and 30 min. At the end of the were neutralized with K O H and assayed for experiment, the hearts were either rapidly free CoA and carnitine. Free CoA was determined fluorometrically frozen for metabolite assays or were washed out with an ice cold saline solution for 5 min using the ~-ketoglutarate dehydrogenase reacbefore freezing to remove extracellular tion [8]. Free carnitine was assayed by a labeled fatty acid when triglyceride synthesis radioisotope procedure [16]. Acetyl CoA and

Lactate Inhibition o f Fatty Acid O x i d a t i o n carnitine, or more appropriately the short chain acyl esters of CoA a n d carnitine, were calculated by subtracting the concentrations of free CoA a n d carnitine from the total acidsoluble CoA a n d carnitine measured as the total free cofactor after hydrolysis of acyl esters.

Measurements of tissue lipids Tissue lipids were extracted a n d separated as described by Bowyer a n d K i n g [4]. A k n o w n weight of frozen, powdered tissue (approximately 500 mg) was extracted in 20 ml of ice cold c h l o r o f o r m : m e t h a n o l ( 2 : 1 v/v) using a Polytron homogenizer. A n additional 3 ml of m e t h a n o l was added, stirred, a n d allowed to stand in the cold for 1 h. After centrifugation, the sample was evaporated u n d e r nitrogen at 50~ T h e total lipids of the dried residue were dissolved in chloroform a n d q u a n t i t a t i v e l y transferred to a silicic acid c o l u m n (Bio-Sil HA, 325 mesh). T h e n e u t r a l lipid plus free fatty acid fraction was eluted with 20 ml of chloroform. T h e chloroform fraction was evaporated to dryness u n d e r nitrogen at 50~ and used for d e t e r m i n i n g total radioactivity and glyceride content. A portion was also used for thin layer chromatographic separation of n e u t r a l lipids using W h a t m a n L K 5 0 F linear K silica gel plates a n d a solvent system that consisted of isooctane : diethyl ether : acetic acid (74 : 24 : 2, v/v/v). Excellent resolution of monoglycerides, diglycerides-cholesterol, free fatty acids, triglycerides, and cholesterol esters was achieved with Rf values of 0.06, 0. t9, 0.29, 0.45 a n d 0.54 respectively. T h e T L C plates were air dried at room t e m p e r a t u r e and spots were visualized with iodine vapor. T h e separated lipids were scraped into c o u n t i n g vials to d e t e r m i n e i n c o r p o r a t i o n of label from [ U - l ' t C ] p a l m i t a t e . T h e n e u t r a l glycerides were saponified and their c o n t e n t was estimated by measuring glycerol release [6]. All d a t a are expressed per g r a m of dry heart weight a n d shown as means • S.E. Significance was determined using Student's t-test, a n d differences were considered significant w h e n P < 0.05. Results V e n t r i c u l a r function of hearts from fasted rats (as indicated by PSP x heart rate) was not

621

TABLE 1. The effect of lactate or oxfenicine on CO 2 production from palmitate in hearts from fasted rats perfused with 11 mM glucose and 1.0 mM palmitate Addition to perfusate

a4CO z production (#tool FA/g dry/rain)

None 5 mM Lactate None 2 m~ Oxfenicine

0.50 0.31 0.51 0.28

• + • •

0.03 0.03" 0.04 0.028

13 10 10 10

The hearts received a 10 min washout perfusion with buffer containing 11 mM glucose with or without the inclusion of 5 mM lactate or 2 mM oxfenicine.The hearts were switched to perfusion with buffer containing 11 mMglucose and 1.0 mM [U-14C]pahnitate bound to 3% bovine serum albumin with or without the addition of 5 mM lactate or 2 mM oxfenicine. The data are expressed as the means _ S.E. " P < 0.05 to correspondingcontrol values. altered by a d d i t i o n of 5 mM lactate or 2 mM oxfenicine to the s t a n d a r d perfusion m e d i u m of 11 mM glucose a n d 1 mM palmitate (data not shown). However, addition of lactate or oxfenicine to the perfusate reduced t 4 C O 2 production from [ U - 1 4 C ] p a l m i t a t e by 38% a n d 45%, respectively (Table 1). This effect of lactate on fatty acid oxidation was not altered by the n u t r i t i o n a l state of the animals. Lactate inhibited palmitate oxidation by approximately 30% in hearts from either fed or fasted animals (Table 2). I n association with reduced fatty acid oxidation, lactate decreased long chain acyl carnitine by 34% while oxfenicine lowered it by 80% (Table 3). TABLE 2. The effect of lactate on the COz production from palmitate in hearts from fasted or fed rats perfused with l 1 mM glucose and 1.3 mM palmitate Condition

Addition to perfusate

14CO2 production (#tool FA/g dry/min)

Fed Fed Fasted Fasted

None 5 mM Lactate None 5 mM Lactate

0.45 _+ 0.04 0.32 _ 0.04a 0.54 i- 0.05 0.38 __.0.04~

The hearts received a 10 min washout perfusion with buffer containing 11 mM glucose with or without the inclusion of 5 mM lactate, The hearts were switched to perfusion with buffer containing 11 mM glucose and 1.3 m~ [U-14C]pahnitate bound to 3% bovine serum albumin with or without the addition of 5 mM lactate. The data are expressed as the means + S.E.,n ~ 5. a p < 0.05 to correspondingcontrol values.

622

D . R . B i e l e f e l d et aL

T A B L E 3. T h e effect of lactate or oxfenicine on the levels of carnitine derivatives in hearts from fasted rats perfused with l 1 mM glucose a n d 1.0 mM palmitate Addition to perfusate None 5 m M Lactate None 2mM Ox~nicine

Free carnitine 2875 4118 3163 4608

• • • •

274 227 ~ 296 294"

Short-chain acyl carnitine 2279 1630 1895 1146

• • • •

277 226 380 229"

Acid-soluble carnitine 5176 5747 5067 5754

• • • •

139 165 173 317

Long-chain acyl carnitine 761 495 975 202

• • • •

58 43 a 111 14"

Total carnitine 5938 6244 5982 5957

• • • •

136 167 189 318

22 21 10 10

The hearts received a 10 rain washout perfusion with buffer containing 11 mM glucose with or without the inclusion of 5 mM lactate or 2 mM oxfenicine. The hearts were switched to perfusion with buffer containing 11 mM glucose and 1.0 mM [U-14C]palmitate bound to 3% bovine serum albumin with or without the addition of 5 mM lactate or 2 mM oxfenicine. The data are expressed as nmol/g of dry heart tissue and represent the means -t- S.E. for the number of hearts shown. " P < 0.05 to corresponding control values.

B o t h s u b s t a n c e s r e d u c e d t h e levels o f a c e t y l c a r n i t i n e a n d i n c r e a s e d t h e c o n t e n t o f free canitine. The total cellular carnitine content remained unchanged. Neither substance had a p r o f o u n d effect o n levels o f C o A d e r i v a t i v e s ( T a b l e 4). T h e s e results s u g g e s t t h a t i n h i b i t i o n o f f a t t y acid oxidation by lactate or oxfenicine occurred either at fatty acid activation or at c a r n i t i n e acyl C o A t r a n s f e r a s e I. I f c a r n i t i n e a c y l C o A t r a n s f e r a s e I w a s i n h i b i t e d , cytosolic levels o f acyl C o A s h o u l d i n c r e a s e w h e r e a s if f a t t y a c i d a c t i v a t i o n w a s i n h i b i t e d , cytosolic acyl C o A levels s h o u l d d e c r e a s e . W i t h p r e s e n t t e c h n i q u e s , it is m o s t difficult to m e a s u r e t h e s m a l l f r a c t i o n o f t o t a l acyl C o A p r e s e n t i n t h e cytosol s e p a r a t e l y f r o m t h e m u c h l a r g e r m i t o -

c h o n d r i a l f r a c t i o n [12], t h e r e f o r e , a n i n d i r e c t e s t i m a t e o f t h e d i r e c t i o n o f c h a n g e i n cytosolic acyl C o A w a s o b t a i n e d b y d e t e r m i n i n g t h e incorporation of ~4C-palmitate into neutral lipids. B o t h l a c t a t e a n d o x f e n i c i n e i n c r e a s e d t h e c o n v e r s i o n o f [ U - 1 4 C ] p a l m i t a t e to trig l y c e r i d e s a n d c a u s e d a n e t i n c r e a s e in t h e e n d o g e n o u s t r i g l y c e r i d e p o o l ( T a b l e 5). O x i d a t i o n o f o c t a n o a t e does n o t r e q u i r e t h e c a r n i t i n e a c y l - C o A t r a n s f e r a s e r e a c t i o n for t r a n s p o r t i n t o t h e m i t o c h o n d r i a [7]. T h e r e fore, t h e effect o f l a c t a t e a n d o x f e n i c i n e o n octanoate oxidation was determined. Lactate caused only a 17% inhibition of CO2 prod u c t i o n f r o m o c t a n o a t e ( T a b l e 6). O x f e n i c i n e not only did not inhibit, but actually increased the oxidation ofoctanoate by 32%.

T A B L E 4. T h e effect of lactate or oxfenicine on the levels of CoA derivatives in hearts from fasted rats perfused with 11 mM glucose and 1.0 mM p a h n i t a t e A d d i t i o n to perfusate None 5 mM Lactate None 2 mM Oxfenicine

Free CoA 238 282 226 252

_ 18 • 10a __+ 19 • i0

Short-chain acyl CoA 160 132 137 I03

__ 19 • 14 _+ 22 • 6

Acid-soluble CoA 398 414 363 355

__ 17 • 17 • 16 __ 9

Long-chain Acyl CoA 173 I62 173 152

+ 8 ___8 • 18 • 5

Total CoA 571 576 537 507

__ 18 • 20 __+21 + 13

22 2I 5 5

The hearts received a l0 min washout perfusion with buffer containing 11 mM glucose with or without the inclusion of 5 mM lactate or 2 mM oxfenicine. The hearts were switched to perfusion with buffer containing 11 mM glucose and 1.0 mM [U-t'~C]palmitate bound to 3% bovine serum albumin with or without the addition of 5 mM lactate or 2 mM oxfenicine. The data are expressed as nmol/g of dry heart tissue and represent the means _+ s.E. for the number of hearts shown. a p < 0,05 to corresponding control values.

Lactate Inhibition o f Fatty Acid Oxidation

623

T A B L E 5. The effect of lactate or oxfenicine on triglyceride production in hearts from rats perfused with 11 mM glucose and 1.0 mM palmitate

T i m e of perfusion 5 5 10 10 30 30 30

min min min min min min rain

[U- t 4 C] Palmi tate incorporation into triglycerides (/~mol FFA/g dry wt)

Addition to perihsate None 5 mM None 5 mM None 5 mM 2 mM

3.23 8.49 3.70 10.20 6.03 12.06 8.70

Lactate Lactate Lactate Oxfenicine

+ 0.58 __ 0.93" + 0.59 + 0.49" _+ 0.34 • 0.51" +_ 0.39"

n

Triglycerides (/~mol/g dry wt)

5 5 5 5 21 15 10

21.5 + 3.5 30,7 • 3.8 27,1 • 4.1

I1 8 4

The hearts received a 10 min washout perfusion with buffer containing 11 mM glucose with or without the inclusion of 5 mM lactate or 2 mM oxfenicine. The hearts were switched to perfusion with buffer containing 11 mM glucose and 1.0 m~ [UJ4C]palmitate bound to 3% bovine serum albumin with or without the addition of 5 mM lactate or 2 mM oxfenicine. The data represent the means 4- s.E. a p < 0.05 to corresponding control values.

T A B L E 6. The effect of lactate or oxfenicine on CO 2 production from octanoate in hearts from fasted rats perfused with 11 mM glucose and 2.0 mM octanoate Addition to perfusate

a4CO2 production (#tool FA/g/min)

None 5 mM Lactate None 2 mM Oxfenicine

1.98 1.64 1.92 2.53

+_ 0.08 + 0.08" + 0.14 + 0.16 a

The hearts received a 10 min washout perfusion with buffer containing 11 mM glucose with or without the inclusion of 5 mM lactate or 2 mM oxfenicine. The hearts were switched to perfusion with buffer containing 11 mM glucose and 2.0 mM [1-1*C]octanoate bound to 3% bovine serum albumin with or without the addition of 5 mM lactate or 2 mM oxfenicine. The data are expressed as the means + S.E., n = 4. a p < 0.05 to corresponding control values.

Discussion I n c r e a s e o f tissue l a c t a t e has b e e n d e m o n s t r a t e d to r e d u c e o x i d a t i o n of l o n g c h a i n fatty acids by the d o g h e a r t in situ [26], perfused rat h e a r t [14], a n d isolated m y o c y t e s [13, 18]. H o w e v e r , the m e c h a n i s m of this effect of l a c t a t e is u n k n o w n . T h e d a t a p r e s e n t e d in the p r e s e n t study suggest t h a t b o t h l a c t a t e a n d o x f e n i c i n e exert c o n t r o l o f f a t t y o x i d a t i o n by i n h i b i t i o n of carn i t i n e acyl C o A transferase I. T h i s c o n c l u s i o n is based on the o b s e r v a t i o n t h a t d e c r e a s e d 14

CO 2 production from [UJ4C]palmitate is associated w i t h a d e c r e a s e in the tissue level of l o n g c h a i n acyl c a r n i t i n e a n d the rise in cytosolic a c y l - C o A a n d free carnitine. S i n c e 9 5 % of t o t a l c e l l u l a r c a r n i t i n e is l o c a t e d in the cytosol [12], the d e c r e a s e in acyl c a r n i t i n e a n d i n c r e a s e in free c a r n i t i n e most likely o c c u r r e d in the cytosolic space. T h i s d e c r e a s e in p r o d u c t c o u p l e d w i t h a large rise in the substrate (free c a r n i t i n e ) , i n d i c a t e s a slower r a t e o f the transferase I r e a c t i o n . O f course, this slower r a t e for transferase I c o u l d h a v e o c c u r r e d if the o t h e r substrate for the reaction, l o n g c h a i n acyl C o A , w e r e l i m i t i n g . I f l a c t a t e or oxfenicine i n h i b i t e d e i t h e r f a t t y acid u p t a k e or a c t i v a t i o n to acyl C o A , cytosolic levels of acyl C o A w o u l d be e x p e c t e d to d e c r e a s e a n d m i g h t l i m i t flux t h r o u g h the transferase. However, the rather large i n c r e a s e in f a t t y a c i d i n c o r p o r a t e d i n t o triglycerides (two to three-fold w i t h l a c t a t e a n d 5 0 % or m o r e w i t h oxfenicine) a n d the a c t u a l i n c r e a s e in the t r i g l y c e r i d e pool size w i t h b o t h c o m p o u n d s i n d i c a t e s t h a t cytosolic acyl C o A i n c r e a s e d w h e n l a c t a t e ~ o r oxfenicine w e r e present. A d i r e c t effect of l a c t a t e a n d oxfenicine on t r i g l y c e r i d e synthesis or lipolysis c a n n o t be r u l e d out, b u t g i v e n t h a t acyl C o A serves as a substrate for triglyceride synthesis a n d as a n i n h i b i t o r o f lipolysis [15, 25], it is m o s t likely t h a t the n e t increase in t r i g l y c e r i d e synthesis resulted f r o m an increase in acyl C o A levels in the cytosol. T h i s i n t e r p r e t a t i o n

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of the d a t a involves m a k i n g the a s s u m p t i o n t h a t : (1) the c o n c e n t r a t i o n of acyl C o A in the cytosol is n o r m a l l y lower t h a n the K m for triglyceride synthesis; (2) other substrates for lipid synthesis do not c h a n g e ; (3) the specific activity of cytosolic acyl CoA does not increase a n d (4) that substantial inhibition of lipolysis did not occur. Even with the assumptions, however, the a p p a r e n t rise in cytosolic acyl C o A and the rise in cytosolic free carnitine, b o t h substrates for carnitine acyl C o A transferase I, along with the decrease in acyl carnitine and decreased flux through the reaction strongly suggest inhibition at this site in the fatty acid oxidation p a t h w a y . O x i d a t i o n o f o c t a n o a t e does not require the carnitine acyl C o A transferase reactions, b u t does utilize the m i t o c h o n d r i a l fl-oxidation system. Since lactate a n d oxfenicine did not inhibit oxidation of octanoate, it would a p p e a r t h a t they do not have an effect on floxidation. This observation provides a d d i tional evidence that the inhibition of p a l m i t a t e oxidation involved the carnitine d e p e n d e n t reactions. T h e i n t r a c e l l u l a r m e t a b o l i t e responsible for transferase inhibition b y lactate is not known.

H o w e v e r , lactate is k n o w n to stimulate acetylC o A carboxylase activity in isolated rat hepatocytes [2] which m a y elevate m a l o n y l CoA, a n d m a l o n y l C o A is a strong i n h i b i t o r of transferase I from b o t h heart a n d liver [17, 23]. M c G a r r y et al. [23] recently d e m o n strated the presence of m a l o n y l C o A in h e a r t muscle. W e have confirmed the presence of m a l o n y l C o A and a d d i t i o n a l l y d e m o n s t r a t e d the presence of acetyl C o A carboxylase activity ( u n p u b l i s h e d observations). Thus, it is possible t h a t transferase I activity m a y be r e g u l a t e d b y m a l o n y l C o A in heart. A p p a r ently the active i n t r a c e l l u l a r form of oxfenicine is 4 - h y d r o x y p h e n y l glyoxylate [10]. This c o m p o u n d has structural similarities with m a l o n y l C o A and m a y mimic m a l o n y l C o A inhibition o f c a r n i t i n e acyl C o A transferase I.

Acknowledgements T h e authors t h a n k D e b b i e A. Berkich for her excellent technical assistance and Mrs Bonnie L. M e r l i n o for typing the paper. This work was s u p p o r t e d by grants from the J u v e n i l e Diabetes F o u n d a t i o n a n d N I H ( H L - 2 0 4 8 4 ) . D R B is a Fellow of the J u v e n i l e Diabetes Foundation.

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