Salicylic acid stimulation of palmitic acid oxidation by rat skeletal muscle mitochondria

120 Biochimica et Biophysics Acta, 666 (1981) 120-I 26 Elsevier/North-Holland Biomedical Press

BBA 57900

SALICYLIC ACID STIMULATION OF PALMITIC ACID OXIDATION BY RAT SKELETAL MUSCLE

MITOCHONDRIA *

ROBERT E. JONES, E. WAYNE ASKEW, ARTHUR L. HECKER ** and FRED D. HOFELDT

Endocrinology Service, Fitzsimons Army Medical Center, Aurora, CO 80045 and Department of Nutrition, Letterman Army Institute of Research, Presidio of San Francisco, CA 94129 (U3.A.)

(Received October 8th, 1980) (Revised manuscript received June 19th, 1981)

Key words: Salicyclic acid; Palmitic acid oxidation; Oxidative phosphorylation; Fatty acid : CoASH ligase [AMP); (Rat mitochondria)

The effects of saIicyIic acid on pahnitic acid oxidation were studied using rat skeletal muscle mitochondria. Salicylic acid, in concentrations that exerted no effect on mitochondrial coupling (0.1 mM), significantly

stimulated mitochondrial pahnitic acid oxidation, with maximal stimulation occurring at subsaturating concentra- tions of substrate. In the same preparation, saiicylate had no effect on the oxidation of palmitoylcamitine or pahnitoyl-CoA. SaIicylate appears to augment the initial step of pahnitic acid oxidation by lowering the apparent Michaelii constant (K,) of long chain fatty acid : CoASH ligase (AMP) (EC 6.2.1.3) for pahnitic acid.

Introduction

Salicylates have diverse effects on intermediary metabolism. They uncouple oxidative phosphoryla- tion [l--4] and decrease the phosphorylation state of cellular adenine nucleotides [5] in a fashion similar to 2,4dinitrophenol [ 1,6,7 J . Salicylates also cause alterations in carbohydrate metabolism which include an increased peripheral utilization of glucose and decreased glycogen synthesis [8,9].

There have been suggestions that salicylates alter lipid metabolism, however, information concerning this interaction has been incomplete and contradic- tory. Brody [IO] initially demonstrated that 0.1 and 1.0 mM concentrations of salicylic acid suppressed

* A preliminary report of a portion of this work has been published in abstract form: Jones, R.E., Askew, E.W., and Hecker, A.L. (1975) Carnitine-Dependent Stimulation of Rat Skeletal Muscle Mitochondrial Respiration by Sali- cylic Acid, Fed. Proc. 34, 938.

** Present address: Medical Department, Ross Laboratories, Inc., Columbus, OH 43216, U.S.A.

octanoate oxidation in rat liver slices by 10 and 60%, respectively. In disagreement with these findings, Whitehouse and Bostrom [ 111 showed a 12% average increase in octanoate oxidation when 4.0 mM sali- cylic acid was added to their incubation medium.

The purpose of this study was 2-fold: (1) To deter- mine if salicylates do indeed influence fatty acid oxi- dation; and (2) if they do, to elucidate their site of action. Skeletal muscle was selected as the experi- mental tissue because skeletal muscle collectively comprises the largest fat oxidizing tissue of the body, and because skeletal muscle mitochondria possess a high capacity to oxidize fatty acids. The quadriceps femoris muscle group was used because it represents a typical mixed fiber muscle containing both red and white fibers [12].

Materials and Methods

Experimental animals and tissue preparation. Male Carworth CFN rats approximately lo-15 weeks of age were fed Purina Lab Chow ad libitum. They were killed by decapitation, and both quadriceps femoris

121

muscle groups were cleanly dissected and rinsed in ice- cold 0.15 M Xl. Mitochond~a were isolated using the method of Ernster and Nordenbrand [ 131, An 500 pi aliquot was removed from the mitochondrial preparation for total protein determination as described by Guance and D’Iorio [ 141; the remainder of the mitochondria was immediately utilized in fatty acid and pyruvate-malate oxidation studies as well as long chain fatty acid : CoA ligase (AMP) assays. Mitochondria employed in the equilibrium dialysis studies were isolated as described above and were frozen until required.

F&y acid oxidar~u~. The oxidation of fatty acid substrates was performed polaro~ap~c~y with an oxygraph (Gilson Medical Electronics, Middleton, WI) equipped with a Clark electrode using the tech- nique of Dohm et al. [ 151 with the following modifi- cations. After equilibration at ambient atmospheric oxygen tension and temperature (3O”C), 0.9 ml of Dohm et al’s [ I.51 incubation mixture was added to a 1 .O ml water-jacketed cell. The reaction vessel was capped and salicylic acid was introduced into the incubation chamber. This was followed by the addi- tion of MgCls to a &al concentration of 1 .O mM and substrate (pahnitic acid, palmitoyl CoA or p~itoyl- o,I.carnitine). Finally, 0.1 ml of mitochondrial sus- pension (approximately 0.5 mg protein/assay) was added to the chamber bringing the total volume to 1 .O ml. The reaction for palmitic acid or palmitoyl- CoA oxidation was initiated by the addition of Lcarnitine (foal concentration 1 .O mM), and oxygen consumption of the preparation was recorded. Final calculations of the data included subtraction of endo- genous rates and an atmospheric correction factor to compensate for ~uctuations in a~osphe~c pressure. Results were expressed as pgatoms oxygen/h per mg protein. Each animal served as its own control with controi conditions being defined as absence of sali- cyclic acid.

Pyruvate-malate oxidation. The effect of salicylate on mitochondrial pyruvate-malate oxidation was tested by the use of the method of Dohm et al. [ 161. Salicyclic acid L-carnitine or 2,4-dinitrophenol in appropriate concentration was added to the test prep- aration. After equilibration, 0.1 mg of mitochondrial suspension was ~troduced into the oxygraph cell. The reaction was initiated by the addition of 0.3 mM ADP and oxygen consumption was measured as

described under ‘fatty acid oxidation’. The endoge- nous rate was subtracted and an atmosphe~c correc- tion factor was applied to the data. The ADP : oxy- gen ratio and respiratory control index (RCI) were calculated according to the method of Chance and Williams [ 171.

Equilibrium dialysis. A multicavity equilibrium dialysis cell (1.0 ml) (Laboratory Apparatus Com- pany, Cleveland, OH) was employed with a single thickness cellulose dialysis membrane having an average pore size of 24 _& (?Jan Waters and Rogers, Salt I&e City, UT). The ~cuba~on mixture com- mon to both sides of the membrane consisted of 0.8 ml buffer containing 154 mM NaCl, 0.15 mM KCl, 1 .O mM MgCls and a combination of aqueous penicil- lin G (2 units) and gentamicin (0.1 pg). Recently thawed mitochondrial suspension (0.5 mg protein/ assay) was added as the protein source to both sides. The solution placed on side 1 included varying con- centrations of palmitic acid, each of which contained 0.02 @i of [9,10-3H]palmitic acid (New England Nuclear; specific activity, 20 Ci/nmol). After equili- bration with the mitochondri~ proteins for I h via con- tinuous agitation, the dialysis was initiated by placing 1 .O ml of a E3H] pahnitic acid solution on side 1 of the membrane and 1 .O ml of the fatty acid free solu- tion on side 2. 0.1 mM salicylic acid was added to both sides of the membrane in the test cells. The cells were incubated for 24 h at 37°C in a shaking water bath. Controls were run in duplicate and test condi- tions were run in triplicate. After 24 h, a SO-@ aliquot was removed from each compartment, placed in 1.5 ml of a toluenelliquifluor counting solution and the radioactivity was measured in a Packard liquid- satiation counter. Results were expressed as bound to free ratios (dpm 3H side 1 : dpm 3H side 2).

Fatty acid : C5ASH &use (AMP] a&vi@. Long chain fatty acid : CoASH ligase (AMP) (EC 6.2.1.3) was studied using a modification of the radio-active ligand assay as proposed by Polokoff and Bell [ 18 1. Salicylic acid, ranging in concentration from 0.01 to 3.0 mM, was added only to the test incubations, with each animal serving as its own control. The final reac- tion volume was 0.4 ml, and all assays were run in triplicate. The reaction was initiated by the addition of appro~ately 0.25 mg prote~/assay of intact mitochond~a and allowed to incubate at 25°C for 30 s. The reaction was terminated by adding fatty acid-

122

poor bovine serum albumin followed by acid precipi- tation with chilled 03 M trichloroacetic acid. This mixture was then placed in an ice bath for 5 min and filtered over a Metricel filter (25 mm; pore size 0.45 pm). The filter was washed, dissolved in 10 ml of aqeous counting scintillant (Amersham) and counted. Results were expressed as nmol palmitoyl-CoA formed/min per mg protein.

Statistical analysis. With the exception of the equilibrium dialysis data where an unpaired t-test [ 191 was employed, Student’s t analysis [ 191 for paired data was used throughout the study. Michaelis constants and maximal velocities were calculated

The salicylate dose response curve for ligase activ-

using the method of Cleland [20].

ity was performed in a fashion identical to that listed above, except 0.002 mM palmitic acid was used and 0.1 mg of fatty acid poor bovine serum albumin (1 mg/ml) was added at the termination of the reaction. These modifications were utilized to study the effects of salicylic acid at the approximate Michaelis con- stant concentration of palmitic acid for the reaction and to obviate any possible binding competition between salicylic acid and palmitoyl-CoA for the hydrophobic binding sites on albumin. Percent stimulation was calculated as (salicylate fortified incubation-control)/control X 100%.

Results

Fatty acid oxidation. As shown in Fig. 1, salicylic acid exerted a significant stimulatory effect on palmitic acid oxidation, with the most profound actions being manifest at lower concentrations of palmitic acid. Salicyclic acid, at a concentration of 0.1 mM, produced a 28 + 7% (mean iS.E.) stimula- tion over control (P < 0.01). At concentrations of 0.4 mM salicylic acid or greater, this stimulatory effect was lost (4 ? 5%; P> 0.3). When palmitoyl-D,L- carnitine and palmitoyl-CoA were used as the sub- strate for oxidation (Table I), salicylic acid exerted no effect on the oxidative rate.

Pyruvate-malate oxidation. The effect of salicylic acid on oxidative phosphorylation coupled to pyru- vate-malate oxidation is illustrated in Table II. Sali- cylic acid at concentrations used in the fatty acid oxidation experiments (0.1 mM) failed to exert any significant effect on mitochondrial coupling as indi-

15

1

,,O 1 CONTROL1 ,.’

5 IO 50

f IPALMilIC ACID mM)-’

Fig. 1. Effect of 0.1 mM salicylic acid on palmitic acid oxi- dation. Values shown are the reciprocals of the mean for oxidative rates versus the reciprocal of substrate (palmitic acid) concentrations (n = 6). With the exception of the highest palmitic acid concentration, all velocities (control vs. salicylate) are statistically different (paired t; P < 0.01).

cated by unchanged ADP : oxygen ratios and respira- tory control indices. However, the mitochondrial preparation was progressively uncoupled by increas-

Equilibrium dialysis. The results of palmitic acid equilibrium dialysis with mitochondrial proteins are

ing concentrations of salicylic acid (0.5 and 1 .O mM)

depicted in Fig. 2. As can be seen, salicylate did not

and completely uncoupled by 0.1 mM 2,4-dinitro- phenol (data not shown).

CONTROL 7

i __

-------i:::::i::___

-* 1

SILICILIC AClD

0 I

0.02 0.1 0.2

IPALMITIC ACID, In”

Fig. 2. Effect of 0.1 mM salicylic acid on protein-binding of palmitic acid. Results are expressed as mean +S.E. of six experiments. The ratios of bound : free for all points were not statistically different (unpaired t; P > 0.1).

123

TABLE I

EFFECTS OF SALICYLLC ACID ON SUBSTRATES FOR FATTY ACID OXIDATION

Values are oxygen uptake in ngatoms oxygen/h per mg protein and basal refers to the oxidation rate in the absence of added sub- strate.

Expt. Basal

Control

0.1 mM 0.1 mM 0.05 mM Palmitic acid Palmitoyl-CoA Palmitoylcarnitine

0.1 mM Salicylic acid Control 0.1 mM Control 0.1 mM Control 0.1 mM

Salicylic acid a Salicylic acid Salicylic acid

1 0.7 1.1 2 1.1 1.0 3 1.0 0.9 4 0.9 1.2 5 0.9 1.2 6 0.8 0.6

ap < 0.001 compared to control.

4.2 4.9 4.9 5.0 6.2 5.6 4.2 5.1 2.4 2.6 5.3 5.2 3.8 4.2 4.8 5.6 7.7 7.4 1.6 2.5 2.8 2.9 4.5 5.0 2.4 2.9 3.1 3.0 5.6 5.7 1.5 2.1 - - 4.1 4.9

E I

1 0.003 0.01 0.05

5 PALMITIC ACID hM1

3.0 -

2.0 -

1.0 -

Km (mM) V

CONTROL 0.05 InM !3A XMwnBw*nct

CONTROL

0/ 1

I I I

200 1000 2000

% (PALMITIC ACID) mM

Fig. 3. Effect of 0.05 m&f saiicylate on Long chain fatty acid : CoASH ligase (AMP). Values showb are the reciprocals of the mean for palmitoylCoA formation versus the reciprocal of the patmitic acid concentmtions (n = 6). Inset figure shows initial velocity as a function of substrate. The points along the curves f ----,controivs.------ P < 0.025) except for those corresponding to 0.05 mM palm&ate.

, salicylic acid) are statisti~~y different (paired t;

(SA)

T -L-

Fig, 4. Effect of increasing concentrations of salicyfic acid on fatty acid : CoASH (AMP). Values are expressed as mean *SE. percent stimulation over control (n = 5). AlI values are statistically different from control (P < 0.01). Substrate was 0.002 mM palmitic acid.

~g~~c~~y shift the bound to free ratio of pahni- tate, thus ~dicat~g no competitive binding actions between either compound.

Long chain fatty acid : CoASH ligase (AMPj, As can be seen in Fig. 3, salicylic acid enhanced the rate of ligase-mediated thioesterification of palmitate to palmitoyl CoA by lowering the apparent K,,, of palmitic acid from 0.0042 to 0.0029 mM (P = 0.035) without altering the maximal velocity of reaction (P> 0.1). Salicylate, when added with albumin at the end of the reaction, had no effect on ligase activity (data not shown).

At endogenous (no added substrate) and saturating (0.05 mM or greater) concentrations of palmitic acid, 0.05 mM salicylic acid produced little or no stimula- tion over control values (3 + 2 and 0.3 + 5%, respec- tively; P> 0.1). However, when subsaturating con- centrations (0.005-0.01 mM) of pahnitate were present, salicylate would stimulate the reaction up to 50% (P<O.Ol).

The effects of increasing concentrations of sali- cylic acid on ligase rate is shown in Fig. 4. The maxi- mal stimulatory effect of salicylate appeared to occur at concentrations of 0.05 mM salicyclic acid or greater.

Discussion

The results of this study suggest that salicyiic acid stimulates mitochondrial palmitic acid oxidation by enhancing the rate of the activation step of fatty acid oxidation, fatty acid : CoASH ligase (AMP).

This conclusion is supported by our data which demonstrate that salicyclic acid, in concentrations capable of promoting free pahnitic acid oxidation, has no effect on the oxidative rate of either palmit- oyl-CoA or pahnitoylcarnitine (Table I). These ob- servations localize the action of salicyclic acid to the outer mitochondrial membrane or cristal space and exlcudes p~itoylcarnitine U-acyl transferase, mem- brane transport of fatty acyl carnitines,electron trans- port and matrix functions such as P-oxidation and the citric acid cycle. This data tentatively pinpoint the site of stimulation as prior to the formation of palmitoyl-CoA. Long chain fatty acid : CoASH ligase (AMP) fulfills both of these criteria in that this enzyme has been localized to the outer mitochondrial membrane/cristal space 121,221 and is responsible for the biosynthesis of paimitoyl-CoA as the initial step in fatty acid oxidation (23).

The studies on palmitic acid oxida~on (Fig. 1) imply that salicyclic acid has no effect on the maxi- mal velocity of pahnitate-linked mitochondrial respiration; however, salicylate lowers the K, of palmitic acid at some critical step in the overall reac- tion sequence of fatty acid oxidation. The kinetic studies on long chain fatty acid : CoASH ligase (AMP) (Fig. 3) demonstrate the salicylate-induced kinetic alterations in ligase activity conform to those pre- dicted by the double-reciprocal plot of palmitate oxi- dation such that salicylate lowers the ligase apparent K, ~almitic acid) by 42% compared to controls without a significant alteration in maximal velocity.

There have been numerous postulates regarding the rate-limiting step in fatty acid oxidation, which have included the relative concentration ratios of long chain fatty acyl CoA thioesters to ADP [24], free CoASH regeneration [25] and acyl transfer from CoA to carnitine [26]. Through modification of a single step in a multi-enzyme sequence and having this alteration affect the entire pathway of palmitic acid oxidation, our results would implicate the Iigase reaction as the ran-l~iting step of long chain fatty acid oxidation in skeletal muscle mitochondria un-

der the conditions present in our study. The effect of salicylate on the ligase reaction is

concentration-dependent and appears to be maximal at levels of greater than 0.05 mM (Fig. 4). However, the peak stimulatory effect of salicylate on whole mitochondria fatty acid oxidation occurs at approxi- mately 0.1 mM, and, at 0.4 mM salicylic acid,palmi- tate oxidation is no longer promoted by the presence of salicylates. This apparent dicotomy is easily rationalized in view of the decreased ability of un- coupled mitochondria to oxidize fatty acids [27,28]. In our preparation, the uncoupling properties of sali- cylic acid become evident at concentrations of 0.5 mM or greater (Table II). Thus, salicylate stimulation of palmitic acid oxidation is confined to a narrow concentration range, balancing the augmentation of long chain fatty acid activation to fatty acyl-CoA with uncoupling of oxidative phosphorylation.

As an alternate explanation, salicylates could directly or indirectly increase the concentration of substrate available for oxidation or thioesterifrcation by either activating a lipase in the preparation or by competitively displacing fatty acids from protein- binding sites. Our results do not support these pos- sibilities. The equilibrium dialysis experiments (Fig. 2) demonstrate that salicylate does not appreciably affect the binding of palmitic acid to mitochondrial protein and, thereby, excludes competitive displace- ment as an artifact of the system. The lack of sali- cylate stimulation of ligase activity at endogenous levels of fatty acids is compelling evidence against the activation of a latent mitochondrial lipase.

TABLE II

EFFECTS OF SALICYLIC ACID ON OXIDATIVE PHOS- PHORYLATION

Substrate: 8 mM pyruvate plus 8 mM L-malate. Values shown are the means + S.E. of six experiments. Salicylic acid incubations were identical to control except salicylic acid was added with the mitochondrial preparation.

Condition ADP : oxygen ratio

RCI

Control 2.56 + 0.04 0.1 mM Salicylic acid 2.54 ? 0.05 a 0.5 mM Salicylic acid 2.45 + 0.04 b

13.34 + 1.07 11.21 ? 2.06 a 5.04 + 0.22 b

1 .O mM Salicylic acid 2.14 _+ 0.05 c 3.30 _+ 0.11 c

a Not significant (P > 0.25); b P < 0.05; c P < 0.001.

125

Killenberg et al. [29] have provided evidence for the existence of an enzyme in bovine liver mitochon- dria that has the ability to form a salicyl-CoA thio- ester. If a reaction of this type could occur in our preparation, the kinetics of the ligase assay would be altered to show an enhanced rate at lower concen- trations of substrate and factitiously lower the apparent K, for palmitic acid. This likelihood is un- tenable for two reasons. First, the degree of enhance- ment of ligase activity is dependent upon palmitic acid concentration when the level of salicylate is held constant; and second, if salicylate serves as substrate for the reaction, the greatest increment in activity would be expected at the lowest concentration of competing substrate (i.e., endogenous palmitic acid). However, the data presented are inconsistent with this prediction.

It appears that salicylic acid stimulates the rate of skeletal muscle mitochondrial long chain fatty acid oxidation by lowering the apparent K, of fatty acid : CoASH ligase (AMP) for palmitic acid and, at higher salicylate concentrations, this augmentation of enzymatic fatty acid activation is, in part, offset by the uncoupling capabilities of salicylates.

The exact mechanism of salicylate-induced enhancement of ligase activity is unclear. Could sali- cylic acid directly activate the enzyme complex or is this action due to a secondary effector? Further studies are needed to define these possibilities.

Acknowledgements

We are grateful to Dr. A. Wayne Meikle for assis- tance in performing equilibrium dialysis, to Drs. T. Phil O’Barr and David T. Zolock for their advice and manuscript review, to Dr. Struther Walker for his statistical recommendations, and to Ms. Kae Robson for her secretarial assistence.

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