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Effect of the fatty acid oxidation inhibitor 2-tetradecylglycidic acid (TDGA) on glucose and fatty acid oxidation in isolated rat soleus muscle
Int. J. Biochem. Vol. 20, No. 2, pp. 155-160, 1988
0020-711X/88 $3.00+0.00 Copyright © 1988PergamonJournals Ltd
Printed in Great Britain. All rights reserved
EFFECT OF THE FATTY ACID OXIDATION INHIBITOR 2-TETRADECYLGLYCIDIC ACID (TDGA) ON GLUCOSE AND FATTY ACID OXIDATION IN ISOLATED RAT SOLEUS MUSCLE ROBERT W. TUMAN*, JOHN M. JOSEPH, HENRY J. BRENTZEL and GENE F. TUTWILER Department of Biological Research, Endocrinology and Metabolism Section, McNeil Pharmaceutical, Spring House, PA 19477, U.S.A. [Tel. (215) 628-5000] (Received 15 M a y 1987)
A~tract--1. The effect of 2-tetradecylglycidicacid (TDGA), a potent, specific inhibitor of long-chain fatty acid oxidation, on fatty acid and glucose oxidation by isolated rat soleus muscle was studied. 2. TDGA inhibited [1-~4C]palmitateoxidation by soleus muscle in a concentration-dependent manner. 3. TDGA inhibited the activity of soleus muscle mitochondrial carnitine palmitoyltransferase A (CPT-A). 4. Added palmitate (0.5 mM) significantlyinhibited D-[U-~4C]glucoseoxidation and, under conditions where TDGA inhibited palmitate oxidation, the oxidation of D-[U-t4C]glucoseby isolated soleus muscle was significantly stimulated. 5. TDGA stimulation of glucose oxidation was reversed by octanoate, a medium-chain fatty acid whose oxidation is not inhibited by TDGA. 6. When nondiabetic rats were treated with TDGA (10mg/kg p.o./day x 3 days), fasting plasma glucose was significantly lowered and the ability of isolated contralateral soleus muscles to oxidize palmitate was inhibited while glucose oxidation was significantly stimulated.
INTRODUCTION
More than 20yr ago, Randle et al. (1963, 1964) proposed that increased oxidation of fatty acids and ketones restrained glucose utilization by muscle and thus might be a key factor underlying the decreased glucose tolerance in diabetes mellitus. An inhibitory effect of nonesterified fatty acids and ketone bodies on glucose metabolism has generally been demonstrable using isolated rat heart and in some cases using diaphragm muscle (Randle et al., 1963, 1964; Garland et al., 1964; Newsholme and Randle, 1964; Williamson, 1965; Neely et al., 1969). However, conflicting results have been reported for skeletal muscle, which, being approx. 40% of the body mass in man, is the major site for glucose disposal (Katz and McGarry, 1984). In the present studies, this question has been readdressed by studying isolated rat soleus muscle, a slow-twitch red oxidative skeletal muscle (Gould, 1973; Kugelberg, 1973) shown to have a greater capacity to oxidize fatty acids compared with white muscle (Havel, 1974; Okano and Shimojo, 1982). 2-Tetradecylglycidic acid (TDGA), a potent specific, irreversible inhibitor of mitochondrial carnitine palmitoyltransferase A (CPT-A) (Tutwiler and Ryzlak, 1980; Kiorpes et al., 1984), has been used to specifically inhibit the oxidation of long-chain fatty acids by isolated soleus muscle. Previous studies have
*To whom all correspondence should be addressed. TDGA--2-tetradecylglyeidic acid (McN3802); CPT-A--earnitine palmitoyltransferase A; KRB--Krebs-Ringer bicarbonate buffer.
Abbreviations:
indicated that this compound is a specific inhibitor of long-chain fatty acid oxidation in a variety of tissues including liver, diaphragm and cardiac muscle, where evidence has been presented that TDGA can also inhibit the oxidation of endogenous stores of fatty acid (Tutwiler et al., 1979, 1981). TDGA is a potent oral antiketonemic and hypoglycemic agent in animals (Tutwiler et al., 1978, 1981) and man (Mandarino et al., 1984), thus a stimulatory effect on skeletal muscle glucose utilization could contribute to its in vivo hypoglycemic activity. MATERIALS AND METHODS
Animals
Male Sprague-Dawley rats weighing 60-100 g (Charles River Breeding Laboratories, Wilmington, DE) were housed with free access to Purina Laboratory Chow and water. Materials
Bovine serum albumin (fatty acid free, Fraction V), L-carnitine, ATP, CoA, palmitoyl CoA, octanoate, and the salts used to prepare Krebs-Ringer bicarbonate buffer (KRB) were purchased from Sigma Chemical Co. (St Louis, MO). Radiolabeled substrates, o-[U-14C]glucose (4.4mCi/mmol), [l-14C]palmitic acid (6.7-9.9mCi/mmol), and [1-14C]octanoate (4.4mCi/mmol) were obtained from New England Nuclear Corporation (Boston, MA). O,L-[Methyl-3H]carnitine (2.0mCi/mmol) was purchased from Amersham Corporation. Blood glucose was determined by the glucose oxidase method (Glucostat, Worthington Biochemical or Autoflo, Boehringer-Mannheim). 2-Tetradecylglycidic acid (TDGA, MEN-3802) and 2-tetradecyloxiranemethanol (McN-3841) were synthesized at McNeil Pharmaceutical as described previously (Tutwiler et al., 1981).
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Soleus muscle isolation Rats, fasted for 24 hr, were killed by cervical dislocation. Soleus muscles were rapidly excised from the hindlimbs as described in detail by Maizels et al. (1977). Intact muscles were quickly rinsed in ice-cold 0.9% NaCI, gently blotted, and weighed with tendons and ligatures (3/0 silk surgical thread) on a Mettler HK-160 electronic balance. The muscles were tied across a V-shaped stainless steel holder at a length approximating the resting length of the muscle and then transferred directly to preincubation flasks. Unless otherwise indicated, contralateral muscles were used as paired controls. Since glucose metabolism of intact soleus muscles weighing more than 60 mg has been reported to be limited by inadequate substrate diffusion (Chaudry and Gould, 1969; Goldberg et al., 1975), only muscles weighing less than 50 mg wet weight were used. Incubation conditions Isolated muscles were preincubated for 30 min at 37°C in a Dubnoff shaker (80-100 cycles/min) with continuous gassing (95:5 O2~5~O2). Preincubation vessels (25ml Erlenmeyer flasks) contained 9.0 ml of KRB buffer (pH 7,4) with 2% bovine serum albumin (fatty acid free) and either unlabeled glucose (5.5 mM), palmitate (0.1 or 0.5 mM), or octanoate (0.5mM), depending on the substrate being measured. Following preincubation, muscles were quickly transferred to glass scintillation vials containing 3 ml of well-gassed media identical in composition to the preincubation buffer but containing either D-[U-m4C]glucose (0.5/zCi/ml), [1J4C]palmitate (0.167/z Ci/ml) or [IJ4Cloctanoate (0.167 #Ci/ml). The vials were gassed for 5 min with O2-CO2 (95: 5), sealed with rubber stoppers, and incubated for an additional 30-180 min. Stock solutions of [IJ4C]palmitate (1.5 and 7.5mM, 2.5/z Ci/ml) and [ I-14C]octanoate (7.5 mM, 2.5 ~tCi/ml) complexed to bovine serum albumin were prepared using the method of Spector et aL (I 965). TDGA and MEN-3841 were dissolved in dimethyl sulfoxide (DMSO) and added (I00/~1) to each vial. Drug was present during both the preincubation (final 15min) and incubation periods unless otherwise indicated. Control muscles were incubated with an equivalent volume of DMSO without drug; DMSO by itself did not significantly affect muscle substrate oxidation. Determination o f ~4C0 2 production Oxidation of D-[U-14C]glucose, [IJ4C]palmitate, and [lJ4C]octanoate was estimated from the production of ~4CO2. At the end of the incubation period, the vials were briefly opened to remove the muscle and then quickly resealed with a rubber serum stopper from which was suspended a plastic center well cup (Kontes) containing a 2 × 5 cm strip of Whatman filter paper saturated with 0.2 ml of Hyamine hydroxide. The incubation medium was acidified by injecting 1.0 ml of 10% H2SO4 through the vial closure and the vials were incubated at 37°C with shaking for an additional 60 min to quantitatively trap the liberated ~4CO2. The filter strips were removed from the wells, and the Hyamine-trapped 14CO2 was quantitated by liquid scintillation spectrometry using Biofluor (New England Nuclear) cocktail. The yield of ~4CO2 by this method (>80%) was determined by recovery of NaH~4CO3 added to 3.0ml of incubation media. Control vials without muscles were simultaneously incubated and treated identically to correct for nonspecific production of ~4CO:. The results are expressed as /amol substrate oxidized/g wet wt tissue per 3 hr. Measurement o f mitochondrial carnitine palmitoyltransferase activity Soleus muscles were removed from 24-hr fasted rats and homogenized in glass turbes on ice with a Polytron homogenizer (setting 5, 15-sec bursts). Mitochondria were isolated and carnitine palmitoyltransferase (CPT-A) activity was determined as previously described (Tutwiler et al.,
1981). Mitochondria were preincubated for 5min with TDGA, CoA, and ATP, a step previously found necessary for detection of TDGA's inhibitory activity (Tutwiler and Ryzlak, 1980).
Ex rivo studies Vehicle or TDGA (I0 mg/kg), suspended in 0.5% methylcellulose, were administered orally by gavage once-a-day for three consecutive days to two groups of rats. Rats were fasted for 18 hr prior to the last dose. Two hr after the last dose of TDGA or vehicle, whole blood was collected in heparinized tubes for determination of plasma glucose. Contralateral soleus muscles were removed from each rat and immediately incubated for measurement of glucose and palmitate oxidation.
Statistical methods Data for paired muscles were statistically analyzed using the paired Student's t-test. In some experiments muscles were randomly distributed among the experimental groups and the results were analyzed for statistical significance using the unpaired Student's t-test or, where appropriate, analysis of variance and Dunnett's 2-tailed t-test for multiple comparisons. A P-value of 0.05 or less was considered to be significant. RESULTS
Effects o f T D G A on p a l m i t a t e a n d glucose oxidation in vitro As shown in Fig. 1, [lJ4C]palmitate conversion to ~4CO2 by isolated soleus muscles was linear between 30 and 180 rain o f incubation, and T D G A (0.1 raM) significantly inhibited (70-90%) palmitate oxidation at 60, 90, and 180rain. The inhibitory effect o f
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Fig. 1. Effect of TDGA on [lJ4C]palmitate oxidation by soleus muscles isolated from fasted rats. Paired contralateral muscles were incubated for various times in the presence of [IJ4C]palmitate (0.5mM) and TDGA (0.1 mM) and the rate of oxidation determined by the amount of 1 4 C O 2 liberated into the media. Details are as described in Materials and Methods, except that drug was not present during the preincubation period. Results are presented as the mean 4-SEM of (N) muscles per group: [] . . . . . [] control muscles, (3 . . . . . © TDGA-treated muscles. Statistical significance compared with respective control group was determined by the paired Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001.
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TDGA effects in soleus muscle Table 1, Concentration-dependent effect of TDGA on [l-14C]palmitate oxidation by isolated rat soleus muscle
Table 2. Effect of palmitate and TDGA on D-[U-~4C]glucoseoxidation by soleus muscles isolated from fasted rats
Concentration Palmitate oxidation Addition (mM) (pmol/g wet wt per 3 hr) % I* None -0.254 ___0.013 (17)t -TDGA 0.1 0.024 __0.008 (10)* 91 TDGA 0.01 0.059 4-_0.011 (4):[: 77 TDGA 0.001 0.182 + 0.008 (3)~: 28 *Per cent inhibition relative to control group. fResults are mean values +_SEM for (N) muscles per group. Paired contralateral muscles were incubated with [l)4C]palmitate (0.1 mM) and either vehicle or various concentrations of TDGA as described in Materials and Methods. Control rates of oxidation were not significantlydifferent between muscle groups (ANOVA), therefore the data were pooled. Statistical significancevs control muscles determined by analysis of variance and Dunnett's 2-tailed t-test for multiple comparisons: :~P < 0.05 or greater.
Glucose oxidation Additions 0tmol/g wet wt per 3 hr) % Change None 0.62 + 0.04 (26) -Palmitate (0.5 mM) 0.42 +_0.03 (13)* -32 TDGA (0.1 raM) 1.48 _ 0.09 (16)* + 139 Palmitate (0.5 mM) 1.63 +_0.14(6)* + 163 + TDGA (0.1 mM) Incubation conditions and determination of D-[U-14Clglucose oxidation are described in Materials and Methods. Results are mean values +_SEM of (N) muscles in each group. Statistical significancevs control muscles (no additions) was determined by analysis of variance and Dunnett's 2-tailed t-test for multiple comparisons: *P < 0.05 or greater.
TDGA on [1-1aC]palmitate oxidation was c o n c e n t r a t i o n - d e p e n d e n t between 0.001 a n d 0.1 m M (Table 1). As previously d e m o n s t r a t e d in a variety o f o t h e r tissues (Tutwiler et al., 1981), the fl-oxidation o f [1-~4C]octanoate, whose m e t a b o l i s m does n o t require c a r n i t i n e - d e p e n d e n t t r a n s p o r t into the mitochondria, was n o t significantly inhibited by 0.1 m M T D G A (results n o t shown). However, soleus muscle carnitine palmitoyltransferase (CPT-A), was significantly inhibited (Fig. 2) following i n c u b a t i o n in vitro in the presence o f 10 t t M T D G A . The effect o f a d d e d palmitate (0.5 m M ) o n glucose oxidation by soleus muscles was tested. As s h o w n in Table 2, exogenous palmitate p r o d u c e d a small (32%), b u t statistically significant ( P < 0.05) suppression of glucose oxidation by isolated soleus muscles. U n d e r experimental conditions in which 0 . 1 m M T D G A significantly inhibited ( 8 0 - 9 0 % ) [1-~4C]palmitate oxidation, D-[U-~4C]glucose oxid a t i o n was significantly stimulated (139-163%) in soleus muscles i n c u b a t e d either in the absence or presence of exogenous palmitate (Table 2).
A d d i t i o n of exogenous o c t a n o a t e (2 m M ) , whose oxidation is n o t significantly inhibited by T D G A , reversed the stimulatory effect o f T D G A o n glucose oxidation (Fig. 3). Furthermore, McN-3841 ( 0 . 1 m M ) , a close structural, nonhypoglycemic a n a l o g of T D G A previously s h o w n (Tutwiler et al., 1979) not to inhibit palmitate oxidation in hemidiaphragms, did not significantly stimulate glucose oxidation in i n c u b a t e d soleus muscle (control, 0.89 + 0.03 vs McN-3841, 1.01 ___0.18/~mol glucose oxidized/g per 3 hr, N = 3/group, P = 0.55), comp a r e d with T D G A - t r e a t e d muscles i n c u b a t e d simultaneously (1.49 + 0 . 2 0 p m o l glucose oxidized/g per 3 h r , N = 3 , P <0.01).
Effect o f pretreatment o f rats with TDGA on the ability o f soleus muscles to oxidize palmitate and glucose ex vivo As s h o w n in Fig. 4, the plasma glucose of rats treated with T D G A (10 m g / k g p.o. per day x 3 days) was significantly lowered at 2 hr after the last dose c o m p a r e d with vehicle-treated rats. F u r t h e r m o r e , the ability o f their isolated soleus muscles to oxidize
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Fig. 2. Effect of TDGA on carnitine palmitoyltransferase (CPT-A) activity of soleus muscle mitochondria isolated from fasted rats. Intact mitochondria were isolated and CPT-A activity was measured by the incorporation of methyl-[3H]carnitine into palmitoyl-[3H]methyl-carnitine as described in Materials and Methods. Results are presented as the mean + SEM of (N) replicates. Statistical significance vs control was tested using the unpaired Student's t-test: ***P < 0.001. B.C 2 0 1 2 ~
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Fig. 3. Effect of octanoate on TDGA-stimulated glucose oxidation by soleus muscles isolated from fasted rats. Paired contralateral muscles were randomly assigned to receive vehicle or TDGA (0.1 raM) in the presence or absence of octanoate (2 mM). Glucose oxidation was determined as described in Materials and Methods, Results are presented as the mean + SEM of (N) muscles per group. Statistical significance vs control was assessed by the paired Student's t-test: **P < 0.01, N.S. P < 0.05.
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Fig. 4, Effect of pretreatment of rats with TDGA on fasting plasma glucose and ability of isolated soleus muscles to oxidize [1J4C]palmitate and D-[U-14C]glucoseex vivo. Rats were treated with either vehicle or TDGA (10 mg/kg p.o.) for 3 consecutive days. Rats were fasted 18 hr prior to dosing on day 3 and sacrificed 2 hr later for determination of the parameters shown below. Contralateral muscles from each rat were used for measurement of glucose (5.5 mM) and palmitate (0.1 mM) oxidation as described in Materials and Methods. Results are presented as the mean + SEM of (N) muscles for each group. Statistical significance was assessed using the paired Student's t-test: *P < 0.05: **P < 0.01; ***P < 0.001.
[l-]4C]palmitate was significantly inhibited D-[U-14C]glucose was significantly accelerated.
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DISCUSSION
Whereas inhibition of glucose utilization by exogenous fatty acids is well established in isolated perfused rat heart (Randle et al., 1964; Newsholme and Randle, 1964; Garland et al., 1964; Williamson, 1965; Neely et aL, 1969), this interaction has not been consistently demonstrated in voluntary skeletal muscle, including the perfused rat hindquarter (Houghton and Ruderman, 1971; Jefferson et al., 1972; Goodman et al., 1974; Berger et al., 1976; Ruderman et al., 1979, 1980; Richter et al., 1982), rat diaphragm (Garland et al., 1964; Randle et al., 1964; Shonfield and Kipnis, 1968) and in sartorius muscle fibers (Beatty and Bocek, 1971; Cassens et al., 1969). Supportive evidence for an inhibitory effect of fatty acids on glucose metabolism has been obtained in well-oxygenated perfused skeletal muscle (Rennie
and Holloszy, 1977), in exercising rats (Baldwin, 1972; Rennie et al., 1976), in dogs (Paul et al., 1966; Seyffert and Madison, 1967; Balasse, 1971), and in man (Ferrannini et al., 1983). Furthermore, we have reported (Tutwiler et al., 1978) using hemidiaphragms from fasted normal and diabetic rats, that selective inhibition of long-chain fatty acid oxidation by methyl-2-tetradecylglycidate (Me-TDGA, methyl palmoxirate) enhanced the ability of the tissue to oxidize glucose. More recently, several investigators have studied the interaction between fatty acids and glucose metabolism in isolated rat soleus muscle, a skeletal muscle composed almost entirely of red oxidative fibers (Gould, 1973; Kugelberg, 1973) which utilizes fatty acids as the primary metabolic fuel (Havel, 1974; Okano and Shimojo, 1982). In two such studies, addition of acetoacetate (Maizels et aL, 1977) and palmitate (Pearce and Connett, 1980) to isolated soleus muscles in vitro significantly depressed glucose utilization and glycolysis, while a third study (Zorzano et al., 1985)
159
TDGA effects in soleus muscle presented evidence, based upon metabolite measurements, for selective operation of the glucose-fatty acid cycle in soleus muscle. The results of the present studies support an inverse relationship between long-chain fatty acid oxidation and glucose oxidation in rat soleus muscle. We observed a significant depression of glucose oxidation by soleus muscles incubated in the presence of added palmitate; however, addition of exogenous octanoate did not significantly reduce glucose oxidation (Fig. 3) despite being adequately oxidized. Under experimental conditions where TDGA inhibited [1-14C]palmitate oxidation and CPT-A enzyme activity by greater than 90%, the ability of rat soleus muscles to oxidize glucose was significantly enhanced (Table 2) both in the presence and absence of exogenous palmitate. The observation that TDGA stimulated glucose oxidation even in the absence of exogenous palmitate suggests that oxidation of endogenous fatty acid fuels during standard incubation conditions was sufficient to suppress glucose utilization, and that inhibition of the oxidation of endogenous long-chain fatty acids by TDGA was sufficient to reverse the restraint of glucose oxidation. The specificity of this effect is supported by the observations that a close structural, nonhypoglycemic analog of TDGA (McN-3841), which failed to inhibit fatty acid oxidation in vitro or to produce hypoglycemia in vivo (Tutwiler et al., 1979), did not stimulate glucose oxidation, and octanoate, whose oxidation is not inhibited by TDGA, reversed the stimulatory effect of TDGA on glucose oxidation. Furthermore, when nondiabetic rats were treated with TDGA, plasma glucose was significantly reduced and isolated soleus muscles were found to oxidize less palmitate and more glucose than muscles from vehicle-treated rats. These observations support a specific interaction between long-chain fatty acid oxidation and glucose metabolism in a red fiber skeletal muscle. This conclusion is further supported by preliminary studies in isolated in situ perfused canine gracilis muscle in which a statistically significant stimulation of glucose uptake, which was reversible with octanoate, was observed following addition of TDGA (87 #M) to the buffer perfusate (Bowden, Brentzel and Tutwiler, unpublished resuits). Lawrence et al. (1986) presented histochemical evidence for, and Stearns et al. (1979) reported an increase in, fat utilization in skeletal muscle of diabetic rats. Similar results on glucose oxidation with T D G A were found in preliminary studies using soleus muscles isolated from streptozotocin-induced diabetic rats (data not presented). Previously reported studies (Tutwiler and Ryzlak, 1980; Kiorpes, 1984) have demonstrated the site of action of TDGA and Me-TDGA for inhibiting fatty acid oxidation to be inhibition of the mitochondrial enzyme carnitine palmitoyltransferase (CPT-A). In these studies, soleus muscle mitochondrial CPT-A was significantly inhibited following incubation with TDGA, suggesting that this enzyme is the locus for the inhibitory effects of TDGA on long-chain fatty acid oxidation in soleus muscle as in other tissues. The finding of an interrelationship between longchain fatty acid oxidation and glucose oxidation in
soleus muscle suggests that effects on peripheral skeletal muscle glucose utilization might also contribute to the hypoglycemic activity of TDGA. This suggestion is further supported by in vivo studies in nondiabetic dogs using euglycemic clamp techniques (Bowden et al., 1982) and in diabetic dogs (Tutwiler et al., 1983) and man (Mandarino et al., 1984) using hepatic balance procedures. Acknowledgements--Special thanks are due to Maria Wagner and Melissa Muskas for their help in the preparation of this manuscript.
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Katz J. and McGarry J. D. (1984) The glucose paradox. Is glucose a substrate for liver metabolism? J. clin. Invest. 74, 1901-1909. Kiorpes T. C., Hoerr D., Ho W., Weaner L. E., Inman M. G. and Tutwiler G. F. (1984) Identification of 2-tetradecylglycidyl coenzyme A as the active form of methyl-2-tetradecylglycidate (methyl palmoxirate) and its characterization as an irreversible, active site-directed inhibitor of carnitine palmitoyltransferase A in isolated rat liver mitochondria. J. biol. Chem. 259, 9750-9755. Kugelberg E. (1973) Histochemical composition, contraction speed and fatiguability of rat soleus motor units. J. neurol. Sci. 20, 177 198. Lawrence G. M., Walker D. G. and Trayer I. P, (1986) Histochemical evidence of changes in fuel metabolism induced in red, white and intermediate muscle fibers of streptozotocin-treated rats. Histochem. J. 18, 203 212. Maizels E. Z., Ruderman N. B., Goodman M. N. and Lau D. (1977) Effect of acetoacetate on glucose metabolism in the soleus and extensor digitorum longus muscles of the rat. Biochem. J. 162, 557-568. Mandarino L., Tsalikian E., Bartold S., Marsh H., Carney A., Buerklin E., Tutwiler G., Haymond M., Handwerger B. and Rizza R. (1984) Mechanism of hyperglycemia and response to treatment with an inhibitor of fatty acid oxidation in a patient with insulin resistance due to anti-insulin receptor antibodies. J. clin. Endoer. Metab. 59, 658~564. Neely J. R., Bowman R. H. and Morgan H. E. (1969) Effects of ventricular pressure development and palmitate on glucose transport. Am. J. Physiol. 216, 804-81 I. Newsholme E. A. and Randle P. J. (1964) Regulation of glucose uptake by muscle. 7. Effects of fatty acids, ketone bodies and pyruvate, and of alloxan-diabetes, starvation, hypophysectomy and adrenalectomy, on the concentrations of hexose phosphates, nucleotides, and inorganic phosphate in perfused rat heart. Biochem. J. 93, 641~551. Okano G. and Shimojo T. (1982) Utilization of long-chain free fatty acids in white and red muscle of rats. Biochim. biophys. Acta 710, 122-127. Paul P. B., lssekutz B. Jr. and Miller H, I. (1966) Interrelationship of free fatty acids and glucose metabolism in the dog. Am. J. Physiol. 211, 1313-1320. Pearce F. J. and Connett R. J. (1980) Effect of lactate and palmitate on substrate utilization of isolated rat soleus. Am. J. Physiol. 238 (Cell Physiol. 7), C149~C159. Randle P. J., Garland P. B., Hales C. N. and Newsholme E. A. (1963) The glucose-fatty acid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet i, 785-790. Randle P. J., Newsholme E. A. and Garland P. B. (1964) Regulation of glucose uptake by muscle. 8. Effects of fatty acids, ketone bodies and pyruvate, and of alloxandiabetes and starvation, on the uptake and metabolic fate of glucose in rat heart and diaphragm muscles. Biochem. J. 93, 652~65. Rennie M. J. and Hossoszy J, O. (1977) Inhibition of glucose uptake and glycogenolysis by availability of oleate in well-oxygenated perfused skeletal muscle. Biochem. J. 168, 161-170.
Rennie M. J., Winder W. W. and Holloszy J. O. (1976~ A sparing effect of increased plasma fatty acids on muscle and liver glycogen content in the exercising rat. Bi~whem. J. 156, 647~55. Richter E. A., Ruderman N. B., Gavras H., Belur E. R. and Galbo H. (1982) Muscle glycogenolysis during exercise: dual control by epinephrine and contractions. Am. J. Physiol. 242, E25-E32. Ruderman N. B., Goodman M. N., Conover C. A. and Berger M. (1979) Substrate utilization in perfused skeletal muscle. Diabetes 28, Suppl. 1, 13 17. Ruderman N. B., Kemmer F. W., Goodman M. N. and Berger M. (1980) Oxygen consumption in perfused muscle: effect of perfusion with aged, fresh, and agerejuvenated erythrocytes on oxygen consumption, tissue metabolites and inhibition of glucose utilization by acetoacetate. Biochem. J. 190, 57 64. Seyffert W. A. and Madison L. L. (1967) Physiologic effects of metabolic fuels on carbohydrate metabolism. 1. Acute effect of elevation of plasma free fatty acids on hepatic glucose output, peripheral glucose utilization, serum insulin and plasma glucagon levels. Diabetes 16, 765 776. Shonfeld G. and Kipnis D. (1968) Effects of fatty acids on carbohydrate and fatty acid metabolism of ral diaphragm. Am. J. Physiol. 215, 513 522. Spector A., Steinberg D. and Tanaka A. (1965) Uptake of free fatty acids by Ehrlich ascites tumor cells. J. biol. Chem. 240, 1032 1041. Stearns S. B., Tepperman H. M. and Tepperman J. (1979) Studies on the utilization and mobilization of lipid in skeletal muscles from streptozotocin-diabetic and control rats. J. Lipid Res. 20, 654-662. Tutwiler G. F., Ho W. and Mohrbacher R. J. (1981) 2-Tetradecylglycidic acid. In Methods in Enzymology (Edited by Lowenstein G.), Vol. 73, pp. 533-551. Academic Press, New York. Tutwiler G. F., Joseph J, M. and Tuman R. W. (1983) Effect of chronic dosing with methyl palmoxirate (McN-3716) on plasma glucose and ketone levels of streptozotocin/alloxan-diabetic dogs. Diabetes 31, Suppl. 1, 69A. Tutwiler G. F., Kirsch T., Mohrbacher R. J. and Ho W. (1978) Pharmacologic profile of methyl 2-tetradecylglycidate (McN-3716)--an orally effective hypoglycemic agent. Metabolism 27, 153%1556. Tutwiler G. F., Mohrbacher R. and Ho W. (1979) Methyl 2-tetradecylglycidate, an orally effective hypoglycemic agent that inhibits log-chain fatty acid oxidation selectively. Diabetes 28, 242 248. Tutwiler G. F. and Ryzlak M. T. (1980) Inhibition of mitochondrial carnitine palmitoyl transferase by 2-tetradecylglycidic acid (McN-3801). Lt~, Sci. 26, 393-397. Williamson J. R. (1965) Glycolytic control mechanisms. I. Inhibition of glycolysis by acetate and pyruvate in the isolated, perfused rat heart. J. biol. Chem. 240, 2308 1321. Zorzano A., Balon T. W., Brandy L. J,, Rivera P., Garetto L. P., Young J. C., Goodman M. N. and Ruderman N. B. (1985) Effects of starvation and exercise on concentrations of critrate, hexose phosphates and glycogen in skeletal muscle and heart. Biochem. J. 232, 585 591.
0020-711X/88 $3.00+0.00 Copyright © 1988PergamonJournals Ltd
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EFFECT OF THE FATTY ACID OXIDATION INHIBITOR 2-TETRADECYLGLYCIDIC ACID (TDGA) ON GLUCOSE AND FATTY ACID OXIDATION IN ISOLATED RAT SOLEUS MUSCLE ROBERT W. TUMAN*, JOHN M. JOSEPH, HENRY J. BRENTZEL and GENE F. TUTWILER Department of Biological Research, Endocrinology and Metabolism Section, McNeil Pharmaceutical, Spring House, PA 19477, U.S.A. [Tel. (215) 628-5000] (Received 15 M a y 1987)
A~tract--1. The effect of 2-tetradecylglycidicacid (TDGA), a potent, specific inhibitor of long-chain fatty acid oxidation, on fatty acid and glucose oxidation by isolated rat soleus muscle was studied. 2. TDGA inhibited [1-~4C]palmitateoxidation by soleus muscle in a concentration-dependent manner. 3. TDGA inhibited the activity of soleus muscle mitochondrial carnitine palmitoyltransferase A (CPT-A). 4. Added palmitate (0.5 mM) significantlyinhibited D-[U-~4C]glucoseoxidation and, under conditions where TDGA inhibited palmitate oxidation, the oxidation of D-[U-t4C]glucoseby isolated soleus muscle was significantly stimulated. 5. TDGA stimulation of glucose oxidation was reversed by octanoate, a medium-chain fatty acid whose oxidation is not inhibited by TDGA. 6. When nondiabetic rats were treated with TDGA (10mg/kg p.o./day x 3 days), fasting plasma glucose was significantly lowered and the ability of isolated contralateral soleus muscles to oxidize palmitate was inhibited while glucose oxidation was significantly stimulated.
INTRODUCTION
More than 20yr ago, Randle et al. (1963, 1964) proposed that increased oxidation of fatty acids and ketones restrained glucose utilization by muscle and thus might be a key factor underlying the decreased glucose tolerance in diabetes mellitus. An inhibitory effect of nonesterified fatty acids and ketone bodies on glucose metabolism has generally been demonstrable using isolated rat heart and in some cases using diaphragm muscle (Randle et al., 1963, 1964; Garland et al., 1964; Newsholme and Randle, 1964; Williamson, 1965; Neely et al., 1969). However, conflicting results have been reported for skeletal muscle, which, being approx. 40% of the body mass in man, is the major site for glucose disposal (Katz and McGarry, 1984). In the present studies, this question has been readdressed by studying isolated rat soleus muscle, a slow-twitch red oxidative skeletal muscle (Gould, 1973; Kugelberg, 1973) shown to have a greater capacity to oxidize fatty acids compared with white muscle (Havel, 1974; Okano and Shimojo, 1982). 2-Tetradecylglycidic acid (TDGA), a potent specific, irreversible inhibitor of mitochondrial carnitine palmitoyltransferase A (CPT-A) (Tutwiler and Ryzlak, 1980; Kiorpes et al., 1984), has been used to specifically inhibit the oxidation of long-chain fatty acids by isolated soleus muscle. Previous studies have
*To whom all correspondence should be addressed. TDGA--2-tetradecylglyeidic acid (McN3802); CPT-A--earnitine palmitoyltransferase A; KRB--Krebs-Ringer bicarbonate buffer.
Abbreviations:
indicated that this compound is a specific inhibitor of long-chain fatty acid oxidation in a variety of tissues including liver, diaphragm and cardiac muscle, where evidence has been presented that TDGA can also inhibit the oxidation of endogenous stores of fatty acid (Tutwiler et al., 1979, 1981). TDGA is a potent oral antiketonemic and hypoglycemic agent in animals (Tutwiler et al., 1978, 1981) and man (Mandarino et al., 1984), thus a stimulatory effect on skeletal muscle glucose utilization could contribute to its in vivo hypoglycemic activity. MATERIALS AND METHODS
Animals
Male Sprague-Dawley rats weighing 60-100 g (Charles River Breeding Laboratories, Wilmington, DE) were housed with free access to Purina Laboratory Chow and water. Materials
Bovine serum albumin (fatty acid free, Fraction V), L-carnitine, ATP, CoA, palmitoyl CoA, octanoate, and the salts used to prepare Krebs-Ringer bicarbonate buffer (KRB) were purchased from Sigma Chemical Co. (St Louis, MO). Radiolabeled substrates, o-[U-14C]glucose (4.4mCi/mmol), [l-14C]palmitic acid (6.7-9.9mCi/mmol), and [1-14C]octanoate (4.4mCi/mmol) were obtained from New England Nuclear Corporation (Boston, MA). O,L-[Methyl-3H]carnitine (2.0mCi/mmol) was purchased from Amersham Corporation. Blood glucose was determined by the glucose oxidase method (Glucostat, Worthington Biochemical or Autoflo, Boehringer-Mannheim). 2-Tetradecylglycidic acid (TDGA, MEN-3802) and 2-tetradecyloxiranemethanol (McN-3841) were synthesized at McNeil Pharmaceutical as described previously (Tutwiler et al., 1981).
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ROBERT W. TUMAN et al.
Soleus muscle isolation Rats, fasted for 24 hr, were killed by cervical dislocation. Soleus muscles were rapidly excised from the hindlimbs as described in detail by Maizels et al. (1977). Intact muscles were quickly rinsed in ice-cold 0.9% NaCI, gently blotted, and weighed with tendons and ligatures (3/0 silk surgical thread) on a Mettler HK-160 electronic balance. The muscles were tied across a V-shaped stainless steel holder at a length approximating the resting length of the muscle and then transferred directly to preincubation flasks. Unless otherwise indicated, contralateral muscles were used as paired controls. Since glucose metabolism of intact soleus muscles weighing more than 60 mg has been reported to be limited by inadequate substrate diffusion (Chaudry and Gould, 1969; Goldberg et al., 1975), only muscles weighing less than 50 mg wet weight were used. Incubation conditions Isolated muscles were preincubated for 30 min at 37°C in a Dubnoff shaker (80-100 cycles/min) with continuous gassing (95:5 O2~5~O2). Preincubation vessels (25ml Erlenmeyer flasks) contained 9.0 ml of KRB buffer (pH 7,4) with 2% bovine serum albumin (fatty acid free) and either unlabeled glucose (5.5 mM), palmitate (0.1 or 0.5 mM), or octanoate (0.5mM), depending on the substrate being measured. Following preincubation, muscles were quickly transferred to glass scintillation vials containing 3 ml of well-gassed media identical in composition to the preincubation buffer but containing either D-[U-m4C]glucose (0.5/zCi/ml), [1J4C]palmitate (0.167/z Ci/ml) or [IJ4Cloctanoate (0.167 #Ci/ml). The vials were gassed for 5 min with O2-CO2 (95: 5), sealed with rubber stoppers, and incubated for an additional 30-180 min. Stock solutions of [IJ4C]palmitate (1.5 and 7.5mM, 2.5/z Ci/ml) and [ I-14C]octanoate (7.5 mM, 2.5 ~tCi/ml) complexed to bovine serum albumin were prepared using the method of Spector et aL (I 965). TDGA and MEN-3841 were dissolved in dimethyl sulfoxide (DMSO) and added (I00/~1) to each vial. Drug was present during both the preincubation (final 15min) and incubation periods unless otherwise indicated. Control muscles were incubated with an equivalent volume of DMSO without drug; DMSO by itself did not significantly affect muscle substrate oxidation. Determination o f ~4C0 2 production Oxidation of D-[U-14C]glucose, [IJ4C]palmitate, and [lJ4C]octanoate was estimated from the production of ~4CO2. At the end of the incubation period, the vials were briefly opened to remove the muscle and then quickly resealed with a rubber serum stopper from which was suspended a plastic center well cup (Kontes) containing a 2 × 5 cm strip of Whatman filter paper saturated with 0.2 ml of Hyamine hydroxide. The incubation medium was acidified by injecting 1.0 ml of 10% H2SO4 through the vial closure and the vials were incubated at 37°C with shaking for an additional 60 min to quantitatively trap the liberated ~4CO2. The filter strips were removed from the wells, and the Hyamine-trapped 14CO2 was quantitated by liquid scintillation spectrometry using Biofluor (New England Nuclear) cocktail. The yield of ~4CO2 by this method (>80%) was determined by recovery of NaH~4CO3 added to 3.0ml of incubation media. Control vials without muscles were simultaneously incubated and treated identically to correct for nonspecific production of ~4CO:. The results are expressed as /amol substrate oxidized/g wet wt tissue per 3 hr. Measurement o f mitochondrial carnitine palmitoyltransferase activity Soleus muscles were removed from 24-hr fasted rats and homogenized in glass turbes on ice with a Polytron homogenizer (setting 5, 15-sec bursts). Mitochondria were isolated and carnitine palmitoyltransferase (CPT-A) activity was determined as previously described (Tutwiler et al.,
1981). Mitochondria were preincubated for 5min with TDGA, CoA, and ATP, a step previously found necessary for detection of TDGA's inhibitory activity (Tutwiler and Ryzlak, 1980).
Ex rivo studies Vehicle or TDGA (I0 mg/kg), suspended in 0.5% methylcellulose, were administered orally by gavage once-a-day for three consecutive days to two groups of rats. Rats were fasted for 18 hr prior to the last dose. Two hr after the last dose of TDGA or vehicle, whole blood was collected in heparinized tubes for determination of plasma glucose. Contralateral soleus muscles were removed from each rat and immediately incubated for measurement of glucose and palmitate oxidation.
Statistical methods Data for paired muscles were statistically analyzed using the paired Student's t-test. In some experiments muscles were randomly distributed among the experimental groups and the results were analyzed for statistical significance using the unpaired Student's t-test or, where appropriate, analysis of variance and Dunnett's 2-tailed t-test for multiple comparisons. A P-value of 0.05 or less was considered to be significant. RESULTS
Effects o f T D G A on p a l m i t a t e a n d glucose oxidation in vitro As shown in Fig. 1, [lJ4C]palmitate conversion to ~4CO2 by isolated soleus muscles was linear between 30 and 180 rain o f incubation, and T D G A (0.1 raM) significantly inhibited (70-90%) palmitate oxidation at 60, 90, and 180rain. The inhibitory effect o f
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Fig. 1. Effect of TDGA on [lJ4C]palmitate oxidation by soleus muscles isolated from fasted rats. Paired contralateral muscles were incubated for various times in the presence of [IJ4C]palmitate (0.5mM) and TDGA (0.1 mM) and the rate of oxidation determined by the amount of 1 4 C O 2 liberated into the media. Details are as described in Materials and Methods, except that drug was not present during the preincubation period. Results are presented as the mean 4-SEM of (N) muscles per group: [] . . . . . [] control muscles, (3 . . . . . © TDGA-treated muscles. Statistical significance compared with respective control group was determined by the paired Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001.
157
TDGA effects in soleus muscle Table 1, Concentration-dependent effect of TDGA on [l-14C]palmitate oxidation by isolated rat soleus muscle
Table 2. Effect of palmitate and TDGA on D-[U-~4C]glucoseoxidation by soleus muscles isolated from fasted rats
Concentration Palmitate oxidation Addition (mM) (pmol/g wet wt per 3 hr) % I* None -0.254 ___0.013 (17)t -TDGA 0.1 0.024 __0.008 (10)* 91 TDGA 0.01 0.059 4-_0.011 (4):[: 77 TDGA 0.001 0.182 + 0.008 (3)~: 28 *Per cent inhibition relative to control group. fResults are mean values +_SEM for (N) muscles per group. Paired contralateral muscles were incubated with [l)4C]palmitate (0.1 mM) and either vehicle or various concentrations of TDGA as described in Materials and Methods. Control rates of oxidation were not significantlydifferent between muscle groups (ANOVA), therefore the data were pooled. Statistical significancevs control muscles determined by analysis of variance and Dunnett's 2-tailed t-test for multiple comparisons: :~P < 0.05 or greater.
Glucose oxidation Additions 0tmol/g wet wt per 3 hr) % Change None 0.62 + 0.04 (26) -Palmitate (0.5 mM) 0.42 +_0.03 (13)* -32 TDGA (0.1 raM) 1.48 _ 0.09 (16)* + 139 Palmitate (0.5 mM) 1.63 +_0.14(6)* + 163 + TDGA (0.1 mM) Incubation conditions and determination of D-[U-14Clglucose oxidation are described in Materials and Methods. Results are mean values +_SEM of (N) muscles in each group. Statistical significancevs control muscles (no additions) was determined by analysis of variance and Dunnett's 2-tailed t-test for multiple comparisons: *P < 0.05 or greater.
TDGA on [1-1aC]palmitate oxidation was c o n c e n t r a t i o n - d e p e n d e n t between 0.001 a n d 0.1 m M (Table 1). As previously d e m o n s t r a t e d in a variety o f o t h e r tissues (Tutwiler et al., 1981), the fl-oxidation o f [1-~4C]octanoate, whose m e t a b o l i s m does n o t require c a r n i t i n e - d e p e n d e n t t r a n s p o r t into the mitochondria, was n o t significantly inhibited by 0.1 m M T D G A (results n o t shown). However, soleus muscle carnitine palmitoyltransferase (CPT-A), was significantly inhibited (Fig. 2) following i n c u b a t i o n in vitro in the presence o f 10 t t M T D G A . The effect o f a d d e d palmitate (0.5 m M ) o n glucose oxidation by soleus muscles was tested. As s h o w n in Table 2, exogenous palmitate p r o d u c e d a small (32%), b u t statistically significant ( P < 0.05) suppression of glucose oxidation by isolated soleus muscles. U n d e r experimental conditions in which 0 . 1 m M T D G A significantly inhibited ( 8 0 - 9 0 % ) [1-~4C]palmitate oxidation, D-[U-~4C]glucose oxid a t i o n was significantly stimulated (139-163%) in soleus muscles i n c u b a t e d either in the absence or presence of exogenous palmitate (Table 2).
A d d i t i o n of exogenous o c t a n o a t e (2 m M ) , whose oxidation is n o t significantly inhibited by T D G A , reversed the stimulatory effect o f T D G A o n glucose oxidation (Fig. 3). Furthermore, McN-3841 ( 0 . 1 m M ) , a close structural, nonhypoglycemic a n a l o g of T D G A previously s h o w n (Tutwiler et al., 1979) not to inhibit palmitate oxidation in hemidiaphragms, did not significantly stimulate glucose oxidation in i n c u b a t e d soleus muscle (control, 0.89 + 0.03 vs McN-3841, 1.01 ___0.18/~mol glucose oxidized/g per 3 hr, N = 3/group, P = 0.55), comp a r e d with T D G A - t r e a t e d muscles i n c u b a t e d simultaneously (1.49 + 0 . 2 0 p m o l glucose oxidized/g per 3 h r , N = 3 , P <0.01).
Effect o f pretreatment o f rats with TDGA on the ability o f soleus muscles to oxidize palmitate and glucose ex vivo As s h o w n in Fig. 4, the plasma glucose of rats treated with T D G A (10 m g / k g p.o. per day x 3 days) was significantly lowered at 2 hr after the last dose c o m p a r e d with vehicle-treated rats. F u r t h e r m o r e , the ability o f their isolated soleus muscles to oxidize
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Fig. 2. Effect of TDGA on carnitine palmitoyltransferase (CPT-A) activity of soleus muscle mitochondria isolated from fasted rats. Intact mitochondria were isolated and CPT-A activity was measured by the incorporation of methyl-[3H]carnitine into palmitoyl-[3H]methyl-carnitine as described in Materials and Methods. Results are presented as the mean + SEM of (N) replicates. Statistical significance vs control was tested using the unpaired Student's t-test: ***P < 0.001. B.C 2 0 1 2 ~
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Fig. 3. Effect of octanoate on TDGA-stimulated glucose oxidation by soleus muscles isolated from fasted rats. Paired contralateral muscles were randomly assigned to receive vehicle or TDGA (0.1 raM) in the presence or absence of octanoate (2 mM). Glucose oxidation was determined as described in Materials and Methods, Results are presented as the mean + SEM of (N) muscles per group. Statistical significance vs control was assessed by the paired Student's t-test: **P < 0.01, N.S. P < 0.05.
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Fig. 4, Effect of pretreatment of rats with TDGA on fasting plasma glucose and ability of isolated soleus muscles to oxidize [1J4C]palmitate and D-[U-14C]glucoseex vivo. Rats were treated with either vehicle or TDGA (10 mg/kg p.o.) for 3 consecutive days. Rats were fasted 18 hr prior to dosing on day 3 and sacrificed 2 hr later for determination of the parameters shown below. Contralateral muscles from each rat were used for measurement of glucose (5.5 mM) and palmitate (0.1 mM) oxidation as described in Materials and Methods. Results are presented as the mean + SEM of (N) muscles for each group. Statistical significance was assessed using the paired Student's t-test: *P < 0.05: **P < 0.01; ***P < 0.001.
[l-]4C]palmitate was significantly inhibited D-[U-14C]glucose was significantly accelerated.
and
DISCUSSION
Whereas inhibition of glucose utilization by exogenous fatty acids is well established in isolated perfused rat heart (Randle et al., 1964; Newsholme and Randle, 1964; Garland et al., 1964; Williamson, 1965; Neely et aL, 1969), this interaction has not been consistently demonstrated in voluntary skeletal muscle, including the perfused rat hindquarter (Houghton and Ruderman, 1971; Jefferson et al., 1972; Goodman et al., 1974; Berger et al., 1976; Ruderman et al., 1979, 1980; Richter et al., 1982), rat diaphragm (Garland et al., 1964; Randle et al., 1964; Shonfield and Kipnis, 1968) and in sartorius muscle fibers (Beatty and Bocek, 1971; Cassens et al., 1969). Supportive evidence for an inhibitory effect of fatty acids on glucose metabolism has been obtained in well-oxygenated perfused skeletal muscle (Rennie
and Holloszy, 1977), in exercising rats (Baldwin, 1972; Rennie et al., 1976), in dogs (Paul et al., 1966; Seyffert and Madison, 1967; Balasse, 1971), and in man (Ferrannini et al., 1983). Furthermore, we have reported (Tutwiler et al., 1978) using hemidiaphragms from fasted normal and diabetic rats, that selective inhibition of long-chain fatty acid oxidation by methyl-2-tetradecylglycidate (Me-TDGA, methyl palmoxirate) enhanced the ability of the tissue to oxidize glucose. More recently, several investigators have studied the interaction between fatty acids and glucose metabolism in isolated rat soleus muscle, a skeletal muscle composed almost entirely of red oxidative fibers (Gould, 1973; Kugelberg, 1973) which utilizes fatty acids as the primary metabolic fuel (Havel, 1974; Okano and Shimojo, 1982). In two such studies, addition of acetoacetate (Maizels et aL, 1977) and palmitate (Pearce and Connett, 1980) to isolated soleus muscles in vitro significantly depressed glucose utilization and glycolysis, while a third study (Zorzano et al., 1985)
159
TDGA effects in soleus muscle presented evidence, based upon metabolite measurements, for selective operation of the glucose-fatty acid cycle in soleus muscle. The results of the present studies support an inverse relationship between long-chain fatty acid oxidation and glucose oxidation in rat soleus muscle. We observed a significant depression of glucose oxidation by soleus muscles incubated in the presence of added palmitate; however, addition of exogenous octanoate did not significantly reduce glucose oxidation (Fig. 3) despite being adequately oxidized. Under experimental conditions where TDGA inhibited [1-14C]palmitate oxidation and CPT-A enzyme activity by greater than 90%, the ability of rat soleus muscles to oxidize glucose was significantly enhanced (Table 2) both in the presence and absence of exogenous palmitate. The observation that TDGA stimulated glucose oxidation even in the absence of exogenous palmitate suggests that oxidation of endogenous fatty acid fuels during standard incubation conditions was sufficient to suppress glucose utilization, and that inhibition of the oxidation of endogenous long-chain fatty acids by TDGA was sufficient to reverse the restraint of glucose oxidation. The specificity of this effect is supported by the observations that a close structural, nonhypoglycemic analog of TDGA (McN-3841), which failed to inhibit fatty acid oxidation in vitro or to produce hypoglycemia in vivo (Tutwiler et al., 1979), did not stimulate glucose oxidation, and octanoate, whose oxidation is not inhibited by TDGA, reversed the stimulatory effect of TDGA on glucose oxidation. Furthermore, when nondiabetic rats were treated with TDGA, plasma glucose was significantly reduced and isolated soleus muscles were found to oxidize less palmitate and more glucose than muscles from vehicle-treated rats. These observations support a specific interaction between long-chain fatty acid oxidation and glucose metabolism in a red fiber skeletal muscle. This conclusion is further supported by preliminary studies in isolated in situ perfused canine gracilis muscle in which a statistically significant stimulation of glucose uptake, which was reversible with octanoate, was observed following addition of TDGA (87 #M) to the buffer perfusate (Bowden, Brentzel and Tutwiler, unpublished resuits). Lawrence et al. (1986) presented histochemical evidence for, and Stearns et al. (1979) reported an increase in, fat utilization in skeletal muscle of diabetic rats. Similar results on glucose oxidation with T D G A were found in preliminary studies using soleus muscles isolated from streptozotocin-induced diabetic rats (data not presented). Previously reported studies (Tutwiler and Ryzlak, 1980; Kiorpes, 1984) have demonstrated the site of action of TDGA and Me-TDGA for inhibiting fatty acid oxidation to be inhibition of the mitochondrial enzyme carnitine palmitoyltransferase (CPT-A). In these studies, soleus muscle mitochondrial CPT-A was significantly inhibited following incubation with TDGA, suggesting that this enzyme is the locus for the inhibitory effects of TDGA on long-chain fatty acid oxidation in soleus muscle as in other tissues. The finding of an interrelationship between longchain fatty acid oxidation and glucose oxidation in
soleus muscle suggests that effects on peripheral skeletal muscle glucose utilization might also contribute to the hypoglycemic activity of TDGA. This suggestion is further supported by in vivo studies in nondiabetic dogs using euglycemic clamp techniques (Bowden et al., 1982) and in diabetic dogs (Tutwiler et al., 1983) and man (Mandarino et al., 1984) using hepatic balance procedures. Acknowledgements--Special thanks are due to Maria Wagner and Melissa Muskas for their help in the preparation of this manuscript.
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