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Reciprocal effects of 5-(tetradecyloxy)-2-furoic acid on fatty acid oxidation
ARCHIVES Vol.
OF BIOCHEMISTRY
242, No. 1, October,
AND
BIOPHYSICS
pp. 23-31,1985
Reciprocal Effects of 5-(tetradecyloxy)-2-furoic Acid on Fatty Acid Oxidation’ DAVID
A. OTT0,*p2 CHRIS CHATZIDAKIS,* AND GEORGE A. COOKT3
*Department of Nutrition, and TDepartment Center Received
EVA KASZIBA,*
Cook College, Rutgers University, New Brunswick, New Jersey of Pharmacology, College of Medicine, University of Tennessee for the Health Sciences, Memphis, Tennessee 38163 February
11,1985,
and in revised
form
May
08903,
30,1985
Under certain incubation conditions 5-(tetradecyloxy)-2-furoic acid (TOFA) stimulated the oxidation of palmitate by hepatocytes, as observed by others. A decrease in malonylCoA concentration accompanied the stimulation of oxidation. Under other conditions, however, TOFA inhibited fatty acid oxidation. The observed effects of TOFA depended on the TOFA and fatty acid concentrations, the cell concentration, the time of TOFA addition relative to the addition of fatty acid, and the nutritional state of the animal (fed or starved). The data indicate that only under limited incubation conditions may TOFA be used as an inhibitor of fatty acid synthesis without inhibition of fatty acid oxidation. When rat liver mitochondria were preincubated with TOFA, ketogenesis from palmitate was slightly inhibited (up to 20%) at TOFA concentrations that were less than that of CoA, but the inhibition became almost complete (up to 90%) when TOFA was greater than or equal to the CoA concentration. TOFA had only slight or no inhibitory effects on the oxidation of palmitoyl-CoA, palmitoyl(-)carnitine, or butyrate. Since TOFA can be converted to TOFyl-CoA, the data suggest that the inhibition of fatty acid oxidation from palmitate results from the decreased availability of CoA for extramitochondrial activation of fatty acids. These data, along with previous data of others, indicate that inhibition of fatty acid oxidation by CoA sequestration is a common mechanism of a group of carboxylic acid inhibitors. A general caution is appropriate with regard to the interpretation of results when using TOFA in studies of fatty acid oxidation. 0 1985 Academic
Press, Inc.
The hypolipidemic agent 5-(tetradecyloxy)-2-furoic acid (TOFA)4 is a potent inhibitor of fatty acid synthesis (l-3). Acti-
vation of this compound to TOFyl-CoA occurs in isolated microsomes and mitochondrial membranes (4, 5). McCune and Harris (4) indicated that the inhibition of fatty acid synthesis by TOFA occurs through an accumulation of TOFyl-CoA and inhibition of acetyl-CoA carboxylase (EC 6.4.1.2) by this compound. The result of this inhibition is a decrease in the concentration of malonyl-CoA (4, 6, i’), which is both a substrate for fatty acid synthesis and an inhibitor of the outer carnitine palmitoyltransferase (EC 2.3.1.21) (8, 9). On the basis of this latter observation, others have used TOFA to elevate the rate of fatty
’ New Jersey Agricultural Experiment Station Publication D-14131-1-84; supported by State funds, Biomedical Research Support Grant (PHS RR 0705818), and Rutgers University Research Council Award. a To whom correspondence should be addressed. a Recipient of an Established Investigatorship from the American Heart Association with funds contributed in part by the A.H.A., Tennessee Affiliate. ’ Abbreviations used: TOFA, 5-(tetradecyloxy)-2furoic acid; TOFyl-CoA, 5-(tetradecyloxy)-2-furoylcoenzyme A; DTT, dithiothreitol; Mes, 2-(N-morpholino)ethanesulfonic acid. 23
0003-9861185 Copyright All rights
$3.00
0 1985 by Academic Press, Inc. of reproduction in any form reserved.
24
OTTO
acid oxidation (4,6,7, lo), which correlates with a decrease in malonyl-CoA in hepatocytes from fed rats (6, 7). However, in preliminary experiments we observed that the rate of palmitate oxidation was decreased when we preincubated hepatocytes from starved rats with 200 PM TOFA. Because of the importance of TOFA as a metabolic inhibitor of fatty acid synthesis and its frequent use in metabolic studies, we conducted the following study to understand the unexpected inhibitory effect of TOFA on fatty acid oxidation. MATERIALS
AND
METHODS
Animals. Male Wistar rats weighing 200-250 g at the time of use were maintained on Purina rat chow with free access to food and water. All animals were housed in rooms with controlled temperature and with a 12/12, light/dark cycle (light from 7 AM to 7 PM). Refed rats refer to animals that were deprived of food for 48 h and refed ad l&turn for 48 h. Starved rats were deprived of food for 48 h. Isolation and incubation of hepatcxytes. Hepatocytes were prepared by the method of Berry and Friend (ll), modified as described by others (12, 13). To determine whether hepatocytes satisfied the criteria defined by Krebs (13) for intact, metabolically active hepatocytes, ATP was measured (14) on the cell preparations. Hepatocytes were preincubated in a total volume of 1.625 ml for 45 min at 37°C in Krebs-Henseleit medium (15) containing 1.2% albumin, 12.3 mM glucose, 12.3 mM lactate, 2.5 mM pyruvate, and 1.2 mM L-carnitine under an atmosphere of 95% 0215% COz. The purpose of preincubation was to elevate the malonyl-CoA concentration of the cells [(6); also see Table I] and to raise the carnitine content to nonlimiting levels (16). Following preincubation, 0.3’75 ml of palmitate/albumin solution in Krebs-Henseleit medium was added to a final concentration of either 0.5 mM/d% or 1.5 mM/d% as indicated, bringing the final incubation volume to 2.0 ml. Incubations after the addition of fatty acid were carried out for various times up to 20 min. TOFA at various concentrations was added either at the beginning of preincubation (as an acetone solution and dried before other additions to the flasks, which were then shaken for at least an hour at 37°C in the presence of albumin before the addition of cells) or at the time of fatty acid addition (bound to albumin in the palmitate/albumin solution). Isolation and incubation of liver mitochondria Rat liver mitochondria were isolated from 48 h starved rats as previously described (17, 18). The final pellet was resuspended in 0.25 M sucrose, 3 mrd Tris/HCl (pH 7.4 at 4OC) to provide a concentration of approximately 20 mg of mitochondrial protein/ml. All mi-
ET
AL. preparations had a respiratory control above 5.0 with the substrates 5 mM glutamate pIUS 5 mM UIahte. Mitochondria (approximately 5 mg of protein) were preincubated for 20 min at 37°C in a system containing final concentrations of 94 mM sucrose (includes sucrose from mitochondria addition), 60 mM KCI, 10 mM potassium phosphate, 5 mM MgClz, 3 mM ATP, 20 or 100 NM CoA (as indicated), 1 mM DTT, 0.4 mM L-carnitine, and 1.5% albumin in a total volume of 2.0 ml at a pH of 7.1-7.2. Following preincubation, fatty acid substrates in a minimal volume were added and the incubations were continued for 5 min. When included, TOFA was present at the beginning of the preincubation. Analytical procedures. From numerous hepatocyte experiments conducted in this laboratory using a variety of incubation conditions and hepatocytes from fed or starved rats, we determined that ketone body synthesis (acetoacetate plus 3-hydroxybutyrate) adequately reflects the total oxidation of fatty acids to i4COz plus acid-soluble radioactivity (correlation coefficient of 0.897 by linear regression analysis with n = 84) and was therefore used to monitor changes in fatty acid oxidation in these experiments. For analyses of ketone bodies and malonyl-CoA, the incubations were terminated with cold HClOd as previously described (18). Acetoacetate (20), 3-hydroxybutyrate (21), and malonyl-CoA (6) were determined enzymatically on neutralized (pH 5.0-6.0 with 4 M KOH, 0.25 M Mes) perchloric acid extracts. Protein was measured according to Lowry et al. (22), with modifications (23). The data in the figures and tables represent the mean of three or more separate experiments except for Fig. 4, which is a representative experiment. Statistical significance was determined using Student’s t test for paired data. Source ofmaterials. Most chemicals, biochemicals, and enzymes were obtained from sources previously given (18,24). Bovine albumin (CRG-7, fatty acid free) was from Armour Pharmaceutical Company. L-Carnitine was generously provided by Dr. Yuzo Kawashima of the Otsuka Pharmaceutical Factory, Inc. (Tokushima, Japan). TOFA (MDL 14,514) was a gift of Dr. Alfred Richardson, Jr. of Merrell Dow Pharmaceuticals Inc. (Cincinnati, Ohio).
tochondrial ratio (19)
RESULTS
AND
DISCUSSION
Effects of 200 PM TOFA on the rate of fatty acid oxidation in hepatocytes. When 200 PM TOFA was preincubated for 45 min with hepatocytes from starved rats (Fig. lA), prior to addition of 1.5 mM palmitate/ 4% albumin, there was a significant inhibition of palmitate oxidation to ketone bodies (approximately 20%) at each time
5-(TETRADECYLOXY)-2-FUROIC
ACID
AND
FATTY
ACID
25
OXIDATION
thus later experiments looked at a single time point. Factors controlling the eflects of TOFA on fatty acid oxidation in hepatocytes. In another group of experiments (Fig. 2), hepatocytes from starved or refed rats were incubated for 15 min with either 0.5 or 1.5 mM palmitateN% albumin in the presence of various concentrations of TOFA. (The binding of TOFA to albumin is necessary to solubilize this hydrophobic compound. Therefore, it is expected that the free concentration of TOFA is much less than the added level.) TOFA was either present during the 45-min preincubation or added at the time of palmitate addition. When added simultaneously with 1.5 mM palmitate, TOFA, at the lower concentrations,
5
IO
I5
20
INCUBATION TIME ImInI
FIG. 1. Effects of 200 ELM TOFA on the rate of palmitate oxidation in hepatocytes from (A) starved and (B) refed rats. Following a 45min preincubation, hepatocytes were incubated for various times at 37°C with 1.5 mM palmitate/4% albumin as described under Materials and Methods. Points on the figure are the mean from four hepatocyte preparations. (0), Control; (a), TOFA, added with palmitate after preincubation; (H), TOFA, present during preincubation.
point relative to the control (at P < 0.05 or less, n = 4; n = number of hepatocyte preparations). If the same concentration of TOFA was added after preincubation, simultaneously with palmitate addition, ketone body synthesis was only slightly depressed. When hepatocytes from refed rats (Fig. 1B) were preincubated with TOFA, ketogenesis was not different from the control. In contrast, ketone body synthesis was significantly greater than the control at points after 5 min, when TOFA was added simultaneously with fatty acid. Thus, the effects of TOFA on fatty acid oxidation (stimulation or inhibition) are dependent on the nutritional state of the animal (fed or starved) and the timing of TOFA addition relative to fatty acid addition. The data of Fig. 1 indicate that the rates of fatty acid oxidation were linear during the 20-min incubation period and
I.oo
0 50
0.25
40 80 120 160 TOFA CONCENTRATION IpMl
200
FIG. 2. Effects of TOFA concentration on the oxidation of 0.5 and 1.5 mM palmitate in hepatocytes from (A) starved and (B) refed rats. Following a 45-min preincubation, hepatocytes were incubated for 15 min at 37°C with either 0.5 or 1.5 mM palmitate/4% albumin in the presence of various concentrations of TOFA as described under Materials and Methods. Points on the figure are the means from four hepatocyte preparations. (0, l ), Control; (0, n ), TOFA added with palmitate after preincubation; (a, A), TOFA present during preincubation; open symbols, 0.5 mM palmitate; closed symbols, 1.5 mM palmitate.
26
OTTO
increased ketone body synthesis in hepatocytes from both nutritional groups. In hepatocytes from starved rats (Fig. 2A), maximal ketone body synthesis was observed at 5-10 PM TOFA, but as the concentration of TOFA was increased to 200 PM, the rate of synthesis gradually decreased to a level that was slightly, but significantly, less than the control (P < 0.05; n = 4). In the refed group (Fig. 2B), maximal stimulation was observed at 50100 PM. At 200 PM TOFA, the rate of synthesis remained higher than the control, but was significantly less (P < 0.025; n = 4) than the maximal rate observed at the lower concentrations. If TOFA was preincubated with the cells from starved rats before 1.5 IIIM palmitate addition (Fig. 2A), there was an inhibition of ketogenesis, reaching a maximum at 50100 PM TOFA. Under the same conditions in hepatocytes from refed rats (Fig. 2B), there was slight elevation of ketone body synthesis up to 5-10 I.~M TOFA, which was followed by a decline in rates at the higher concentrations to a level no different from the control. In these experiments, the cell concentrations ranged from 35 to 75 mg wet wt cells/ml incubation volume (the average wet wt was 55-60 mg/ml for cells from starved or refed rats). It was noted that within each nutritional group, the concentration of TOFA required to inhibit fatty acid oxidation relative to the maximally stimulated rate was decreased as the cell concentration decreased (data not shown). In the same group of experiments, the effect of decreasing the fatty acid concentration to 0.5 mM palmitate on the effects of TOFA was investigated (Fig. 2). When TOFA was added after the 45-min preincubation, simultaneously with 0.5 InM palmitateN% albumin, there was an elevation of ketogenesis, reaching a maximum at 20 and 50 ~.JM in hepatocytes from starved (Fig. 2A) and refed (Fig. 2B) rats, respectively. As the concentration of TOFA was increased further, the rate of oxidation remained at this elevated level at all concentrations in hepatocytes from refed rats. In cells from starved rats, there was a slight but significant decline from the maximum
ET
AL.
rate of ketogenesis (observed at 20 PM TOFA) when 100 or 200 PM TOFA was added (P < 0.05; n = 4); however, the rates of fatty acid oxidation at these high TOFA concentrations were still greater than the control. At the low fatty acid concentration, when TOFA was present during the preincubation, there was a significant stimulation (P < 0.05; n = 4) of fatty acid oxidation in hepatocytes from starved rats at 5 PM TOFA (Fig. 2A). As the TOFA concentration was increased further, however, this rate declined from the maximum until reaching the control level at 100 and 200 I.LM TOFA. In hepatocytes from refed rats, maximal elevation of ketone body synthesis was observed at 10 PM TOFA (P < 0.05; n = 4) and was maintained at this level at all higher concentrations (Fig. 2B). To further investigate the importance of fatty acid concentration on the effects of TOFA, we measured the oxidation of endogenous fatty acids during the 45-min preincubation as a function of TOFA concentration (Fig. 3). The rate of ketone body synthesis from endogenous fatty acids was markedly stimulated by TOFA, reaching a
E Y 1
,
40
,
80
,
120
TOFA CONCENTRATION
,
160
.I
200
IphI)
FIG. 3. Effects of TOFA concentration on endogenous fatty acid oxidation in hepatocytes from starved and refed rats. Hepatocytes were incubated for 45 min at 37°C (preincubation conditions) in the absence of added fatty acid substrate in the presence of various concentrations of TOFA (present at the start of incubation) as described under Materials and Methods. Points on the figure are the means from four hepatocyte preparations. (0), Starved; (O), refed.
&(TETRADECYLOXY)-2-FUROIC
ACID
AND
TABLE EFFECT
OF TOFA
AND EXOGENOUS IN HEPATOCYTES
FATTY
ACID
I
FATTY ACIDS ON THE MALONYL-COA FROM STARVED AND REFED RATS’
Malonyl-CoA
concentration
After
0 10 20 50*
Starved 8.8 6.1 3.4 2.2
f f + +
0.6 0.5 0.9 0.2
Refed (9) (4) (3) (3)
20.1 10.9 6.2 2.5
f f + +
2.7 2.0 1.7 0.4
CONCENTRATION
(nmol/g
After 45-min preincubation with glucose, lactate, and pyruvate [TOFA] (PM)
27
OXIDATION
cells) preincubation plus lo-min incubation with 1.5 mM palmitate/4% albumin Refed
Starved (9) (3) (4) (4)
6.1 4.2 3.6 2.0
f k f t
0.5 0.5 0.5 0.2
7.6 4.8 4.8 2.4
(9) (3) (3) (4)
f + * k
0.8 1.3 1.3 0.8
(9) (3) (3) (5)
a Hepatocytes were preincubated for 45 min at 37°C in the presence of various concentrations of TOFA, followed by a lo-min incubation in the presence of palmitate as described under Materials and Methods. Values are the means f SE (number of hepatocyte preparations). “Under all conditions tested, 50 pM TOFA maximally lowered malonyl-CoA to approximately 2 nmol/g cells.
maximum at lo-20 and 20-50 PM in hepatocytes from starved and refed rats, respectively. In the experiments of Figs. 2 and 3, the stimulatory effects of TOFA on fatty acid oxidation were accompanied by a depression of the malonyl-CoA concentration, reaching maximum reduction at 50 PM TOFA to approximately 2 nmol/g cells in hepatocytes from both starved and refed rats (Table I). However, in some of the experiments of Fig. 2, especially when TOFA was present during preincubation, the depression of fatty acid oxidation from the observed maximum level occurred at a TOFA concentration below 50 PM. In these specific cases, it is likely that fatty acid oxidation had not reached the maximum rate before inhibition occurred. The data from these studies indicate that the effects of TOFA on fatty acid oxidation are dependent, not only on the nutritional state of the animal and timing of addition but also on the fatty acid, TOFA, and cell concentrations. Inhibition of fatty acid oxidation bg TOFA in isolated mitochondria: Determination of mechanism. With rat liver mitochondria (from starved rats) preincubated for 20 min with TOFA, ketogenesis from 0.45 mM palmitate/l.5% albumin was
slightly inhibited (up to 20%) when the TOFA concentration was less than the CoA concentration, but the inhibition became maximal (up to 90%)when the TOFA concentration was greater than or equal to the CoA concentration (Fig. 4). Preincubation
I
I
40
60
TOFA CONCENTRATION
120
160
I
f,,M)
FIG. 4. Effects of TOFA concentration on palmitate oxidation in isolated liver mitochondria. Mitochondria from starved rats were preincubated for 20 min at 37°C in the absence of substrate with various concentrations of TOFA, followed by a 5-min incubation in the presence of 0.45 mM palmitate/l.5% albumin as described under Materials and Methods. Points on the figure are from a representative experiment. (0). 20 /.LM CoA; (0). 100 @M CoA.
28
OTTO
ET
of mitochondria with TOFA before addition of palmitate was required to observe the inhibitory effects. When compared with other fatty acid substrates which enter at points further in the oxidation pathway, 50 PM TOFA inhibited palmitate oxidation in the presence of 20 PM CoA by 80% (Table II), yet had minimal or no effects on the oxidation of palmitoyl-CoA, palmitoyl(-)carnitine, and butyrate. If 50 PM TOFA was added in the presence of 100 PM CoA, the inhibition of palmitate oxidation was almost completely prevented. The data of Table II were initially unexplainable on the basis that octanoate oxidation (in the presence of CoA and carnitine) was also inhibited 40% by TOFA (data not shown). It is generally believed that octanoate is activated in the mitochondria (25). The conditions of these experiments required preincubation of mitochondria with TOFA before fatty acid addition. We recently determined that under these conditions about 60% of octanoate oxidation is via a carni-
AL.
tine-dependent route, requiring extramitochondrial activation (18). It is clear from these data that the site of inhibition of fatty acid oxidation by TOFA is at extramitochondrial activation of the fatty acid. It has been shown by others (4) that TOFyl-CoA accumulates in hepatocytes incubated with TOFA and that this is associated with reduced levels of CoA and acetyl-CoA. Considered with our observations, these data indicate that inhibition of fatty acid oxidation by TOFA results from a decreased availability of cytosolic CoA for long-chain fatty acid activation. This conclusion is consistent with our findings that lower TOFA concentrations were required to inhibit ketogenesis under conditions giving higher control rates. Under these conditions, the CoA requirement for maximal fatty acid oxidation would be greater and therefore less CoA would need to be sequestered by TOFA to become limiting for fatty acid activation (compare endogenous, 0.5 mM, and 1.5 mM palmitate
TABLE
II
SITE OF THE INHIBITORY EFFECT OF TOFA ON THE OXIDATION IN ISOLATED LIVER MITOCHONDRIA’ Ketone
body
synthesis
OF FATTY
(nmol/mg
protein)
20 PM CoA Substrate
Control
100 50 PM TOFA
0.45 mM Paimitate, 1.5% albumin
124 + 13 (6)
0.45 mM Palmitoyl-CoA, 1.5% albumin
119 f
8 (11)
110*
0.45 mM Palmitoyl(-) carnitine, 1.5% albumin
152 +
8 (6)
10 mM Butyrate, 1.5% albumin
96 + 22 (7)
ACIDS
Control
PM
CoA 50 PM TOFA
6 (6)*
111 f 12 (5)
103 + 13 (5)**
9(11)
-
-
122 + 12 (6)**
-
-
91 + 17 (7)
-
-
24 +
’ Mitochondria from starved rats were preincubated with TOFA for 20 min at 3’7°C in the absence of substrate, followed by a 5 min incubation in the presence of the respective fatty acid substrate, as described under Materials and Methods. Values are the mean + S.E. (number of mitochondrial preparations). * Significantly different from the respective control value determined using Student’s t test for paired data; P < 0.001.
** P < 0.025.
5-(TETRADECYLOXY)-%FlJROIC
ACID
and refed versus starved in Figs. 2 and 3). Sequestration of CoA has also been suggested as a possible mechanism for inhibition of fatty acid oxidation by other carboxylic acid compounds, such as hypoglycin and 4-pentenoate (26), benzoic acid and derivatives (27), and valproic acid (28), all of which appear to require activation to the CoA ester for inhibition of fatty acid oxidation. It is likely that the numerous inhibitory effects of these carboxylic acid compounds on hepatic glucose and fatty acid metabolism are the result of two operative mechanisms, a direct effect of the CoA ester on specific enzymes and CoA sequestration. Neither mechanism alone appears to account for all the effects. It has been reported that TOFA also inhibits cholesterol synthesis, in hepatocytes (1) and perfused liver (10). The concentration required for half-maximal inhibition was at least four times greater than that needed to inhibit fatty acid synthesis (1). Even though TOFA has been shown to inhibit tricarboxylate transport (29), the site of inhibition of cholesterol synthesis appears to be after citrate transport since synthesis from 3H20 and [14C]acetate were equally inhibited (1). The primary site of inhibition of fatty acid synthesis by TOFA has been shown to be at acetyl-CoA carboxylase (4) which is not in common with cholesterol synthesis. Considering these observations and the present data, it is reasonable to suggest that cholesterol synthesis is also inhibited by a limitation of cytosolic CoA as a result of sequestration by TOFA. If this were the case, it would be expected from the data of Fig. 2 and Table I that inhibition of cholesterol synthesis would require higher TOFA concentrations than inhibition of fatty acid synthesis. The site of inhibition would likely be at ATPcitrate lyase (EC 4.1.3.8), which requires CoA. Cholesterol synthesis from acetate should also be inhibited since acetate activation can occur in the cytosol. In this regard, CoA sequestration may also contribute to the inhibition of triglyceride synthesis and secretion (10) by TOFA and to the reduction of plasma triglyceride levels (30).
AND
FATTY
ACID
OXIDATION
29
From what is known about the kinetic constants and location of the enzyme(s) which convert TOFA to TOFyl-CoA (4,5), it has been suggested that the long-chain acyl-CoA synthetase (EC 6.2.1.3) is responsible for this formation. If so, it would be expected that palmitate and TOFA would compete for activation. It is likely that in the presence of added fatty acid only small amounts of TOFA would be converted to TOFyl-CoA. This could explain the minimal inhibition of palmitate oxidation when TOFA is added simultaneously with palmitate, as opposed to the marked inhibition when TOFA is added before the addition of palmitate in either hepatocyte (Figs. 1 and 2) or mitochondrial experiments (Fig. 4). Also note the difference in concentration dependence when TOFA is added with or before palmitate addition (Fig. 2). Similar effects are observed under both conditions, but at a lower concentration when TOFA is added before fatty acid, suggesting that under this condition more TOFyl-CoA is available at a particular concentration. Since it has been shown that acetyl-CoA carboxylase is inhibited 90% by 2.5 pM TOFyl-CoA (4), it would not be expected that this competition would significantly interfere with the effects of TOFA on malonyl-CoA levels. In this regard, it is reasonable to suggest a situation in which the rate of fatty acid oxidation in the presence of TOFA is a balance between the stimulation resulting from decreasing malonyl-CoA concentrations and the inhibition caused by CoA sequestration. TOFA is probably the most potent and effective inhibitor of fatty acid synthesis available, causing approximately 90% inhibition in hepatocytes at 20-50 PM (1). However, if the drug also inhibits fatty acid oxidation, the usefulness of this compound as a metabolic inhibitor would be severely limited. From the present data it is possible to choose conditions that would essentially eliminate this problem: (a) A concentration of TOFA no higher than 20-50 pM should be used in hepatocyte studies since fatty acid synthesis (1) and malonyl-CoA levels (Table I) are maximally lowered at these
30
OTTO
concentrations. However, since the free concentration of TOFA depends on the concentration of albumin, the total concentrations indicated above are only relevant to the conditions used in these studies. In additional experiments (data not shown) conducted under otherwise identical conditions to the experiments of Figs. 2 and 3 (including the presence of 4% albumin), the concentration curves were shifted to the left when TOFA was added dissolved in dimethyl sulfoxide. This would be expected if the solubility of TOFA and thus the free concentration was increased. Therefore, the mode of TOFA addition should also be considered (e.g., albumin concentration used in incubations and whether added bound to albumin or dissolved in a hydrophobic vehicle). (b) If TOFA is added at the time of fatty acid addition, the competition for activation should keep TOFylCoA at minimal levels, thus maintaining the free CoA supply necessary for maximal fatty acid oxidation but still at a concentration that would inhibit acetyl-CoA carboxylase. (c) The actual concentration used would depend on the nutritional state of the animal (fed or starved), as the data indicate a greater TOFA requirement for maximal stimulation of oxidation in hepatocytes from refed rats (Figs. 2 and 3). This observation is likely related to the higher malonyl-CoA concentrations (after preincubation; Table I) and to the greater sensitivity of fatty acid oxidation to malonyl-CoA-dependent inhibition in hepatocytes from refed rats (32), such that malonyl-CoA must be depressed to a greater extent to observe maximum stimulation of fatty acid oxidation. (d) Within a nutritional group, the TOFA concentration at which inhibition occurred was directly proportional to the cell concentration in a particular experiment (see text). TOFA probably enters the cells by a passive mechanism. However, it has been shown that TOFyl-CoA accumulates in hepatocytes incubated with TOFA (4), probably because TOFyl-CoA is not appreciably metabolized. It is possible, therefore, that the lower the cell concentration, the greater would be the TOFyl-CoA concentration per
ET
AL.
cell resulting in greater CoA sequestration, and thus less TOFA would be required to cause inhibition. (e) Finally, the added fatty acid concentration in hepatocyte experiments should be as low as possible in order to decrease the CoA requirement. If these guidelines are followed, it is possible to use TOFA as an inhibitor of fatty acid synthesis without inhibitory effects on fatty acid oxidation. However, a general caution is appropriate with regard to the interpretation of results when using TOFA in isolated hepatocytes or perfused livers, especially when investigating cytosolic CoA-requiring reactions. REFERENCES 1. PANEK, E., COOK, G. A., AND CORNELL, N. W. (1977) Lipids l&814-818. 2. KARIYA, T., AND WILLIE, L. J. (1978) B&hem, Bio-
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J. (1974)
in
Methods in Enzymatic Analysis (Bergmeyer, H. II., ed.), 2nd English ed., pp. 1836-1839, Academic Press, New York. 22.
LOWRY, AND
0.
H.,
RANDALL,
265-275.
ROSEBROUGH, R. J. (1951)
N.
J., FARR,
J. Biol.
Chem
FATTY
MARKWELL, AND
488,249-262.
20.
AND
A. L.,
193,
ACID
M. A. K., HAAS, TOLBERT,
31
OXIDATION S. M., BIEBER,
Anal.
N. E. (1978)
L. L.,
B&hem.
87,
206-210. 24. OTTO, D. A., AND ONTKO, J. A. (1982) Eur. B&hem. 129,479-485. 25. AAS, M. (1971) B&him. Biophys. Acta 231,32-47. 26.
BRESSLER,
27.
(1969) PharmucoL Rev. 21,105-130. MCCUNE, S. A., DURANT, P. J., FLANDERS, AND HARRIS, R. A. (1982) Arch. B&hem.
phys. 28.
BECKER,
Arch. 29.
R.,
CORREDOR,
C.,
AND
J.
BRENDEL,
K. L. E.,
Bic-
214,124-133. CORD-MICHAEL,
B&hem.
RIBREAU-GAYON,
AND
Biophys. G. (1976)
HARRIS,
R. A. (1983)
223,381-392. FEBS L&t.
62, 309-
312. 30.
PARKER,
R. A., KARIYA,
T., GRISAR,
J. M., AND
PE-
TROW, V. (1977) J. Med. Chem. 20, 781-791. 31. VAN TOL, A. (1975) Mol. Cell Biochem. 7,19-31. 32. OTTO, D. A., COOK, G. A., AND REISS, P. D. (1983) in Isolation, Characterization, and Use of Hepatocytes (Harris, R. A., and Cornell, N. W., eds.), pp. 41-48, Elsevier, New York.
OF BIOCHEMISTRY
242, No. 1, October,
AND
BIOPHYSICS
pp. 23-31,1985
Reciprocal Effects of 5-(tetradecyloxy)-2-furoic Acid on Fatty Acid Oxidation’ DAVID
A. OTT0,*p2 CHRIS CHATZIDAKIS,* AND GEORGE A. COOKT3
*Department of Nutrition, and TDepartment Center Received
EVA KASZIBA,*
Cook College, Rutgers University, New Brunswick, New Jersey of Pharmacology, College of Medicine, University of Tennessee for the Health Sciences, Memphis, Tennessee 38163 February
11,1985,
and in revised
form
May
08903,
30,1985
Under certain incubation conditions 5-(tetradecyloxy)-2-furoic acid (TOFA) stimulated the oxidation of palmitate by hepatocytes, as observed by others. A decrease in malonylCoA concentration accompanied the stimulation of oxidation. Under other conditions, however, TOFA inhibited fatty acid oxidation. The observed effects of TOFA depended on the TOFA and fatty acid concentrations, the cell concentration, the time of TOFA addition relative to the addition of fatty acid, and the nutritional state of the animal (fed or starved). The data indicate that only under limited incubation conditions may TOFA be used as an inhibitor of fatty acid synthesis without inhibition of fatty acid oxidation. When rat liver mitochondria were preincubated with TOFA, ketogenesis from palmitate was slightly inhibited (up to 20%) at TOFA concentrations that were less than that of CoA, but the inhibition became almost complete (up to 90%) when TOFA was greater than or equal to the CoA concentration. TOFA had only slight or no inhibitory effects on the oxidation of palmitoyl-CoA, palmitoyl(-)carnitine, or butyrate. Since TOFA can be converted to TOFyl-CoA, the data suggest that the inhibition of fatty acid oxidation from palmitate results from the decreased availability of CoA for extramitochondrial activation of fatty acids. These data, along with previous data of others, indicate that inhibition of fatty acid oxidation by CoA sequestration is a common mechanism of a group of carboxylic acid inhibitors. A general caution is appropriate with regard to the interpretation of results when using TOFA in studies of fatty acid oxidation. 0 1985 Academic
Press, Inc.
The hypolipidemic agent 5-(tetradecyloxy)-2-furoic acid (TOFA)4 is a potent inhibitor of fatty acid synthesis (l-3). Acti-
vation of this compound to TOFyl-CoA occurs in isolated microsomes and mitochondrial membranes (4, 5). McCune and Harris (4) indicated that the inhibition of fatty acid synthesis by TOFA occurs through an accumulation of TOFyl-CoA and inhibition of acetyl-CoA carboxylase (EC 6.4.1.2) by this compound. The result of this inhibition is a decrease in the concentration of malonyl-CoA (4, 6, i’), which is both a substrate for fatty acid synthesis and an inhibitor of the outer carnitine palmitoyltransferase (EC 2.3.1.21) (8, 9). On the basis of this latter observation, others have used TOFA to elevate the rate of fatty
’ New Jersey Agricultural Experiment Station Publication D-14131-1-84; supported by State funds, Biomedical Research Support Grant (PHS RR 0705818), and Rutgers University Research Council Award. a To whom correspondence should be addressed. a Recipient of an Established Investigatorship from the American Heart Association with funds contributed in part by the A.H.A., Tennessee Affiliate. ’ Abbreviations used: TOFA, 5-(tetradecyloxy)-2furoic acid; TOFyl-CoA, 5-(tetradecyloxy)-2-furoylcoenzyme A; DTT, dithiothreitol; Mes, 2-(N-morpholino)ethanesulfonic acid. 23
0003-9861185 Copyright All rights
$3.00
0 1985 by Academic Press, Inc. of reproduction in any form reserved.
24
OTTO
acid oxidation (4,6,7, lo), which correlates with a decrease in malonyl-CoA in hepatocytes from fed rats (6, 7). However, in preliminary experiments we observed that the rate of palmitate oxidation was decreased when we preincubated hepatocytes from starved rats with 200 PM TOFA. Because of the importance of TOFA as a metabolic inhibitor of fatty acid synthesis and its frequent use in metabolic studies, we conducted the following study to understand the unexpected inhibitory effect of TOFA on fatty acid oxidation. MATERIALS
AND
METHODS
Animals. Male Wistar rats weighing 200-250 g at the time of use were maintained on Purina rat chow with free access to food and water. All animals were housed in rooms with controlled temperature and with a 12/12, light/dark cycle (light from 7 AM to 7 PM). Refed rats refer to animals that were deprived of food for 48 h and refed ad l&turn for 48 h. Starved rats were deprived of food for 48 h. Isolation and incubation of hepatcxytes. Hepatocytes were prepared by the method of Berry and Friend (ll), modified as described by others (12, 13). To determine whether hepatocytes satisfied the criteria defined by Krebs (13) for intact, metabolically active hepatocytes, ATP was measured (14) on the cell preparations. Hepatocytes were preincubated in a total volume of 1.625 ml for 45 min at 37°C in Krebs-Henseleit medium (15) containing 1.2% albumin, 12.3 mM glucose, 12.3 mM lactate, 2.5 mM pyruvate, and 1.2 mM L-carnitine under an atmosphere of 95% 0215% COz. The purpose of preincubation was to elevate the malonyl-CoA concentration of the cells [(6); also see Table I] and to raise the carnitine content to nonlimiting levels (16). Following preincubation, 0.3’75 ml of palmitate/albumin solution in Krebs-Henseleit medium was added to a final concentration of either 0.5 mM/d% or 1.5 mM/d% as indicated, bringing the final incubation volume to 2.0 ml. Incubations after the addition of fatty acid were carried out for various times up to 20 min. TOFA at various concentrations was added either at the beginning of preincubation (as an acetone solution and dried before other additions to the flasks, which were then shaken for at least an hour at 37°C in the presence of albumin before the addition of cells) or at the time of fatty acid addition (bound to albumin in the palmitate/albumin solution). Isolation and incubation of liver mitochondria Rat liver mitochondria were isolated from 48 h starved rats as previously described (17, 18). The final pellet was resuspended in 0.25 M sucrose, 3 mrd Tris/HCl (pH 7.4 at 4OC) to provide a concentration of approximately 20 mg of mitochondrial protein/ml. All mi-
ET
AL. preparations had a respiratory control above 5.0 with the substrates 5 mM glutamate pIUS 5 mM UIahte. Mitochondria (approximately 5 mg of protein) were preincubated for 20 min at 37°C in a system containing final concentrations of 94 mM sucrose (includes sucrose from mitochondria addition), 60 mM KCI, 10 mM potassium phosphate, 5 mM MgClz, 3 mM ATP, 20 or 100 NM CoA (as indicated), 1 mM DTT, 0.4 mM L-carnitine, and 1.5% albumin in a total volume of 2.0 ml at a pH of 7.1-7.2. Following preincubation, fatty acid substrates in a minimal volume were added and the incubations were continued for 5 min. When included, TOFA was present at the beginning of the preincubation. Analytical procedures. From numerous hepatocyte experiments conducted in this laboratory using a variety of incubation conditions and hepatocytes from fed or starved rats, we determined that ketone body synthesis (acetoacetate plus 3-hydroxybutyrate) adequately reflects the total oxidation of fatty acids to i4COz plus acid-soluble radioactivity (correlation coefficient of 0.897 by linear regression analysis with n = 84) and was therefore used to monitor changes in fatty acid oxidation in these experiments. For analyses of ketone bodies and malonyl-CoA, the incubations were terminated with cold HClOd as previously described (18). Acetoacetate (20), 3-hydroxybutyrate (21), and malonyl-CoA (6) were determined enzymatically on neutralized (pH 5.0-6.0 with 4 M KOH, 0.25 M Mes) perchloric acid extracts. Protein was measured according to Lowry et al. (22), with modifications (23). The data in the figures and tables represent the mean of three or more separate experiments except for Fig. 4, which is a representative experiment. Statistical significance was determined using Student’s t test for paired data. Source ofmaterials. Most chemicals, biochemicals, and enzymes were obtained from sources previously given (18,24). Bovine albumin (CRG-7, fatty acid free) was from Armour Pharmaceutical Company. L-Carnitine was generously provided by Dr. Yuzo Kawashima of the Otsuka Pharmaceutical Factory, Inc. (Tokushima, Japan). TOFA (MDL 14,514) was a gift of Dr. Alfred Richardson, Jr. of Merrell Dow Pharmaceuticals Inc. (Cincinnati, Ohio).
tochondrial ratio (19)
RESULTS
AND
DISCUSSION
Effects of 200 PM TOFA on the rate of fatty acid oxidation in hepatocytes. When 200 PM TOFA was preincubated for 45 min with hepatocytes from starved rats (Fig. lA), prior to addition of 1.5 mM palmitate/ 4% albumin, there was a significant inhibition of palmitate oxidation to ketone bodies (approximately 20%) at each time
5-(TETRADECYLOXY)-2-FUROIC
ACID
AND
FATTY
ACID
25
OXIDATION
thus later experiments looked at a single time point. Factors controlling the eflects of TOFA on fatty acid oxidation in hepatocytes. In another group of experiments (Fig. 2), hepatocytes from starved or refed rats were incubated for 15 min with either 0.5 or 1.5 mM palmitateN% albumin in the presence of various concentrations of TOFA. (The binding of TOFA to albumin is necessary to solubilize this hydrophobic compound. Therefore, it is expected that the free concentration of TOFA is much less than the added level.) TOFA was either present during the 45-min preincubation or added at the time of palmitate addition. When added simultaneously with 1.5 mM palmitate, TOFA, at the lower concentrations,
5
IO
I5
20
INCUBATION TIME ImInI
FIG. 1. Effects of 200 ELM TOFA on the rate of palmitate oxidation in hepatocytes from (A) starved and (B) refed rats. Following a 45min preincubation, hepatocytes were incubated for various times at 37°C with 1.5 mM palmitate/4% albumin as described under Materials and Methods. Points on the figure are the mean from four hepatocyte preparations. (0), Control; (a), TOFA, added with palmitate after preincubation; (H), TOFA, present during preincubation.
point relative to the control (at P < 0.05 or less, n = 4; n = number of hepatocyte preparations). If the same concentration of TOFA was added after preincubation, simultaneously with palmitate addition, ketone body synthesis was only slightly depressed. When hepatocytes from refed rats (Fig. 1B) were preincubated with TOFA, ketogenesis was not different from the control. In contrast, ketone body synthesis was significantly greater than the control at points after 5 min, when TOFA was added simultaneously with fatty acid. Thus, the effects of TOFA on fatty acid oxidation (stimulation or inhibition) are dependent on the nutritional state of the animal (fed or starved) and the timing of TOFA addition relative to fatty acid addition. The data of Fig. 1 indicate that the rates of fatty acid oxidation were linear during the 20-min incubation period and
I.oo
0 50
0.25
40 80 120 160 TOFA CONCENTRATION IpMl
200
FIG. 2. Effects of TOFA concentration on the oxidation of 0.5 and 1.5 mM palmitate in hepatocytes from (A) starved and (B) refed rats. Following a 45-min preincubation, hepatocytes were incubated for 15 min at 37°C with either 0.5 or 1.5 mM palmitate/4% albumin in the presence of various concentrations of TOFA as described under Materials and Methods. Points on the figure are the means from four hepatocyte preparations. (0, l ), Control; (0, n ), TOFA added with palmitate after preincubation; (a, A), TOFA present during preincubation; open symbols, 0.5 mM palmitate; closed symbols, 1.5 mM palmitate.
26
OTTO
increased ketone body synthesis in hepatocytes from both nutritional groups. In hepatocytes from starved rats (Fig. 2A), maximal ketone body synthesis was observed at 5-10 PM TOFA, but as the concentration of TOFA was increased to 200 PM, the rate of synthesis gradually decreased to a level that was slightly, but significantly, less than the control (P < 0.05; n = 4). In the refed group (Fig. 2B), maximal stimulation was observed at 50100 PM. At 200 PM TOFA, the rate of synthesis remained higher than the control, but was significantly less (P < 0.025; n = 4) than the maximal rate observed at the lower concentrations. If TOFA was preincubated with the cells from starved rats before 1.5 IIIM palmitate addition (Fig. 2A), there was an inhibition of ketogenesis, reaching a maximum at 50100 PM TOFA. Under the same conditions in hepatocytes from refed rats (Fig. 2B), there was slight elevation of ketone body synthesis up to 5-10 I.~M TOFA, which was followed by a decline in rates at the higher concentrations to a level no different from the control. In these experiments, the cell concentrations ranged from 35 to 75 mg wet wt cells/ml incubation volume (the average wet wt was 55-60 mg/ml for cells from starved or refed rats). It was noted that within each nutritional group, the concentration of TOFA required to inhibit fatty acid oxidation relative to the maximally stimulated rate was decreased as the cell concentration decreased (data not shown). In the same group of experiments, the effect of decreasing the fatty acid concentration to 0.5 mM palmitate on the effects of TOFA was investigated (Fig. 2). When TOFA was added after the 45-min preincubation, simultaneously with 0.5 InM palmitateN% albumin, there was an elevation of ketogenesis, reaching a maximum at 20 and 50 ~.JM in hepatocytes from starved (Fig. 2A) and refed (Fig. 2B) rats, respectively. As the concentration of TOFA was increased further, the rate of oxidation remained at this elevated level at all concentrations in hepatocytes from refed rats. In cells from starved rats, there was a slight but significant decline from the maximum
ET
AL.
rate of ketogenesis (observed at 20 PM TOFA) when 100 or 200 PM TOFA was added (P < 0.05; n = 4); however, the rates of fatty acid oxidation at these high TOFA concentrations were still greater than the control. At the low fatty acid concentration, when TOFA was present during the preincubation, there was a significant stimulation (P < 0.05; n = 4) of fatty acid oxidation in hepatocytes from starved rats at 5 PM TOFA (Fig. 2A). As the TOFA concentration was increased further, however, this rate declined from the maximum until reaching the control level at 100 and 200 I.LM TOFA. In hepatocytes from refed rats, maximal elevation of ketone body synthesis was observed at 10 PM TOFA (P < 0.05; n = 4) and was maintained at this level at all higher concentrations (Fig. 2B). To further investigate the importance of fatty acid concentration on the effects of TOFA, we measured the oxidation of endogenous fatty acids during the 45-min preincubation as a function of TOFA concentration (Fig. 3). The rate of ketone body synthesis from endogenous fatty acids was markedly stimulated by TOFA, reaching a
E Y 1
,
40
,
80
,
120
TOFA CONCENTRATION
,
160
.I
200
IphI)
FIG. 3. Effects of TOFA concentration on endogenous fatty acid oxidation in hepatocytes from starved and refed rats. Hepatocytes were incubated for 45 min at 37°C (preincubation conditions) in the absence of added fatty acid substrate in the presence of various concentrations of TOFA (present at the start of incubation) as described under Materials and Methods. Points on the figure are the means from four hepatocyte preparations. (0), Starved; (O), refed.
&(TETRADECYLOXY)-2-FUROIC
ACID
AND
TABLE EFFECT
OF TOFA
AND EXOGENOUS IN HEPATOCYTES
FATTY
ACID
I
FATTY ACIDS ON THE MALONYL-COA FROM STARVED AND REFED RATS’
Malonyl-CoA
concentration
After
0 10 20 50*
Starved 8.8 6.1 3.4 2.2
f f + +
0.6 0.5 0.9 0.2
Refed (9) (4) (3) (3)
20.1 10.9 6.2 2.5
f f + +
2.7 2.0 1.7 0.4
CONCENTRATION
(nmol/g
After 45-min preincubation with glucose, lactate, and pyruvate [TOFA] (PM)
27
OXIDATION
cells) preincubation plus lo-min incubation with 1.5 mM palmitate/4% albumin Refed
Starved (9) (3) (4) (4)
6.1 4.2 3.6 2.0
f k f t
0.5 0.5 0.5 0.2
7.6 4.8 4.8 2.4
(9) (3) (3) (4)
f + * k
0.8 1.3 1.3 0.8
(9) (3) (3) (5)
a Hepatocytes were preincubated for 45 min at 37°C in the presence of various concentrations of TOFA, followed by a lo-min incubation in the presence of palmitate as described under Materials and Methods. Values are the means f SE (number of hepatocyte preparations). “Under all conditions tested, 50 pM TOFA maximally lowered malonyl-CoA to approximately 2 nmol/g cells.
maximum at lo-20 and 20-50 PM in hepatocytes from starved and refed rats, respectively. In the experiments of Figs. 2 and 3, the stimulatory effects of TOFA on fatty acid oxidation were accompanied by a depression of the malonyl-CoA concentration, reaching maximum reduction at 50 PM TOFA to approximately 2 nmol/g cells in hepatocytes from both starved and refed rats (Table I). However, in some of the experiments of Fig. 2, especially when TOFA was present during preincubation, the depression of fatty acid oxidation from the observed maximum level occurred at a TOFA concentration below 50 PM. In these specific cases, it is likely that fatty acid oxidation had not reached the maximum rate before inhibition occurred. The data from these studies indicate that the effects of TOFA on fatty acid oxidation are dependent, not only on the nutritional state of the animal and timing of addition but also on the fatty acid, TOFA, and cell concentrations. Inhibition of fatty acid oxidation bg TOFA in isolated mitochondria: Determination of mechanism. With rat liver mitochondria (from starved rats) preincubated for 20 min with TOFA, ketogenesis from 0.45 mM palmitate/l.5% albumin was
slightly inhibited (up to 20%) when the TOFA concentration was less than the CoA concentration, but the inhibition became maximal (up to 90%)when the TOFA concentration was greater than or equal to the CoA concentration (Fig. 4). Preincubation
I
I
40
60
TOFA CONCENTRATION
120
160
I
f,,M)
FIG. 4. Effects of TOFA concentration on palmitate oxidation in isolated liver mitochondria. Mitochondria from starved rats were preincubated for 20 min at 37°C in the absence of substrate with various concentrations of TOFA, followed by a 5-min incubation in the presence of 0.45 mM palmitate/l.5% albumin as described under Materials and Methods. Points on the figure are from a representative experiment. (0). 20 /.LM CoA; (0). 100 @M CoA.
28
OTTO
ET
of mitochondria with TOFA before addition of palmitate was required to observe the inhibitory effects. When compared with other fatty acid substrates which enter at points further in the oxidation pathway, 50 PM TOFA inhibited palmitate oxidation in the presence of 20 PM CoA by 80% (Table II), yet had minimal or no effects on the oxidation of palmitoyl-CoA, palmitoyl(-)carnitine, and butyrate. If 50 PM TOFA was added in the presence of 100 PM CoA, the inhibition of palmitate oxidation was almost completely prevented. The data of Table II were initially unexplainable on the basis that octanoate oxidation (in the presence of CoA and carnitine) was also inhibited 40% by TOFA (data not shown). It is generally believed that octanoate is activated in the mitochondria (25). The conditions of these experiments required preincubation of mitochondria with TOFA before fatty acid addition. We recently determined that under these conditions about 60% of octanoate oxidation is via a carni-
AL.
tine-dependent route, requiring extramitochondrial activation (18). It is clear from these data that the site of inhibition of fatty acid oxidation by TOFA is at extramitochondrial activation of the fatty acid. It has been shown by others (4) that TOFyl-CoA accumulates in hepatocytes incubated with TOFA and that this is associated with reduced levels of CoA and acetyl-CoA. Considered with our observations, these data indicate that inhibition of fatty acid oxidation by TOFA results from a decreased availability of cytosolic CoA for long-chain fatty acid activation. This conclusion is consistent with our findings that lower TOFA concentrations were required to inhibit ketogenesis under conditions giving higher control rates. Under these conditions, the CoA requirement for maximal fatty acid oxidation would be greater and therefore less CoA would need to be sequestered by TOFA to become limiting for fatty acid activation (compare endogenous, 0.5 mM, and 1.5 mM palmitate
TABLE
II
SITE OF THE INHIBITORY EFFECT OF TOFA ON THE OXIDATION IN ISOLATED LIVER MITOCHONDRIA’ Ketone
body
synthesis
OF FATTY
(nmol/mg
protein)
20 PM CoA Substrate
Control
100 50 PM TOFA
0.45 mM Paimitate, 1.5% albumin
124 + 13 (6)
0.45 mM Palmitoyl-CoA, 1.5% albumin
119 f
8 (11)
110*
0.45 mM Palmitoyl(-) carnitine, 1.5% albumin
152 +
8 (6)
10 mM Butyrate, 1.5% albumin
96 + 22 (7)
ACIDS
Control
PM
CoA 50 PM TOFA
6 (6)*
111 f 12 (5)
103 + 13 (5)**
9(11)
-
-
122 + 12 (6)**
-
-
91 + 17 (7)
-
-
24 +
’ Mitochondria from starved rats were preincubated with TOFA for 20 min at 3’7°C in the absence of substrate, followed by a 5 min incubation in the presence of the respective fatty acid substrate, as described under Materials and Methods. Values are the mean + S.E. (number of mitochondrial preparations). * Significantly different from the respective control value determined using Student’s t test for paired data; P < 0.001.
** P < 0.025.
5-(TETRADECYLOXY)-%FlJROIC
ACID
and refed versus starved in Figs. 2 and 3). Sequestration of CoA has also been suggested as a possible mechanism for inhibition of fatty acid oxidation by other carboxylic acid compounds, such as hypoglycin and 4-pentenoate (26), benzoic acid and derivatives (27), and valproic acid (28), all of which appear to require activation to the CoA ester for inhibition of fatty acid oxidation. It is likely that the numerous inhibitory effects of these carboxylic acid compounds on hepatic glucose and fatty acid metabolism are the result of two operative mechanisms, a direct effect of the CoA ester on specific enzymes and CoA sequestration. Neither mechanism alone appears to account for all the effects. It has been reported that TOFA also inhibits cholesterol synthesis, in hepatocytes (1) and perfused liver (10). The concentration required for half-maximal inhibition was at least four times greater than that needed to inhibit fatty acid synthesis (1). Even though TOFA has been shown to inhibit tricarboxylate transport (29), the site of inhibition of cholesterol synthesis appears to be after citrate transport since synthesis from 3H20 and [14C]acetate were equally inhibited (1). The primary site of inhibition of fatty acid synthesis by TOFA has been shown to be at acetyl-CoA carboxylase (4) which is not in common with cholesterol synthesis. Considering these observations and the present data, it is reasonable to suggest that cholesterol synthesis is also inhibited by a limitation of cytosolic CoA as a result of sequestration by TOFA. If this were the case, it would be expected from the data of Fig. 2 and Table I that inhibition of cholesterol synthesis would require higher TOFA concentrations than inhibition of fatty acid synthesis. The site of inhibition would likely be at ATPcitrate lyase (EC 4.1.3.8), which requires CoA. Cholesterol synthesis from acetate should also be inhibited since acetate activation can occur in the cytosol. In this regard, CoA sequestration may also contribute to the inhibition of triglyceride synthesis and secretion (10) by TOFA and to the reduction of plasma triglyceride levels (30).
AND
FATTY
ACID
OXIDATION
29
From what is known about the kinetic constants and location of the enzyme(s) which convert TOFA to TOFyl-CoA (4,5), it has been suggested that the long-chain acyl-CoA synthetase (EC 6.2.1.3) is responsible for this formation. If so, it would be expected that palmitate and TOFA would compete for activation. It is likely that in the presence of added fatty acid only small amounts of TOFA would be converted to TOFyl-CoA. This could explain the minimal inhibition of palmitate oxidation when TOFA is added simultaneously with palmitate, as opposed to the marked inhibition when TOFA is added before the addition of palmitate in either hepatocyte (Figs. 1 and 2) or mitochondrial experiments (Fig. 4). Also note the difference in concentration dependence when TOFA is added with or before palmitate addition (Fig. 2). Similar effects are observed under both conditions, but at a lower concentration when TOFA is added before fatty acid, suggesting that under this condition more TOFyl-CoA is available at a particular concentration. Since it has been shown that acetyl-CoA carboxylase is inhibited 90% by 2.5 pM TOFyl-CoA (4), it would not be expected that this competition would significantly interfere with the effects of TOFA on malonyl-CoA levels. In this regard, it is reasonable to suggest a situation in which the rate of fatty acid oxidation in the presence of TOFA is a balance between the stimulation resulting from decreasing malonyl-CoA concentrations and the inhibition caused by CoA sequestration. TOFA is probably the most potent and effective inhibitor of fatty acid synthesis available, causing approximately 90% inhibition in hepatocytes at 20-50 PM (1). However, if the drug also inhibits fatty acid oxidation, the usefulness of this compound as a metabolic inhibitor would be severely limited. From the present data it is possible to choose conditions that would essentially eliminate this problem: (a) A concentration of TOFA no higher than 20-50 pM should be used in hepatocyte studies since fatty acid synthesis (1) and malonyl-CoA levels (Table I) are maximally lowered at these
30
OTTO
concentrations. However, since the free concentration of TOFA depends on the concentration of albumin, the total concentrations indicated above are only relevant to the conditions used in these studies. In additional experiments (data not shown) conducted under otherwise identical conditions to the experiments of Figs. 2 and 3 (including the presence of 4% albumin), the concentration curves were shifted to the left when TOFA was added dissolved in dimethyl sulfoxide. This would be expected if the solubility of TOFA and thus the free concentration was increased. Therefore, the mode of TOFA addition should also be considered (e.g., albumin concentration used in incubations and whether added bound to albumin or dissolved in a hydrophobic vehicle). (b) If TOFA is added at the time of fatty acid addition, the competition for activation should keep TOFylCoA at minimal levels, thus maintaining the free CoA supply necessary for maximal fatty acid oxidation but still at a concentration that would inhibit acetyl-CoA carboxylase. (c) The actual concentration used would depend on the nutritional state of the animal (fed or starved), as the data indicate a greater TOFA requirement for maximal stimulation of oxidation in hepatocytes from refed rats (Figs. 2 and 3). This observation is likely related to the higher malonyl-CoA concentrations (after preincubation; Table I) and to the greater sensitivity of fatty acid oxidation to malonyl-CoA-dependent inhibition in hepatocytes from refed rats (32), such that malonyl-CoA must be depressed to a greater extent to observe maximum stimulation of fatty acid oxidation. (d) Within a nutritional group, the TOFA concentration at which inhibition occurred was directly proportional to the cell concentration in a particular experiment (see text). TOFA probably enters the cells by a passive mechanism. However, it has been shown that TOFyl-CoA accumulates in hepatocytes incubated with TOFA (4), probably because TOFyl-CoA is not appreciably metabolized. It is possible, therefore, that the lower the cell concentration, the greater would be the TOFyl-CoA concentration per
ET
AL.
cell resulting in greater CoA sequestration, and thus less TOFA would be required to cause inhibition. (e) Finally, the added fatty acid concentration in hepatocyte experiments should be as low as possible in order to decrease the CoA requirement. If these guidelines are followed, it is possible to use TOFA as an inhibitor of fatty acid synthesis without inhibitory effects on fatty acid oxidation. However, a general caution is appropriate with regard to the interpretation of results when using TOFA in isolated hepatocytes or perfused livers, especially when investigating cytosolic CoA-requiring reactions. REFERENCES 1. PANEK, E., COOK, G. A., AND CORNELL, N. W. (1977) Lipids l&814-818. 2. KARIYA, T., AND WILLIE, L. J. (1978) B&hem, Bio-
phys. Res. Commun 80,1022-1024. 3. HARRIS, R. A., MAPES, J. P., OCHS, R. S., CRABB, D. W., AND STROPES, L. (1979) in Hormones and Energy Metabolism (Klachko, D. M., Anderson, R. R., and Heimberg, M., eds.), Vol. 3, pp. 1742, Plenum, New York. 4. MCCUNE, S. A., AND HARRIS, R. A. (1979) J. Biol. chxm. 254,10095-10101. 5. HARRIS, R. A., AND MCCUNE, S. A. (1982) in Methods in Enzymology (Lowenstein, J. M., ed.), Vol. 72, pp. 552-559, Academic Press, New York. 6. COOK, G. A., KING, M. T., AND VEECH, R. L. (1979)
J. Biol. Chem. 53,2529-2531. 7. MCGARRY,
J. D., AND FOSTER, D. W. (1979)
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