Free Fatty Acid Effects on Mitochondrial Permeability: An Overview

Archives of Biochemistry and Biophysics Vol. 386, No. 1, February 1, pp. 52– 61, 2001 doi:10.1006/abbi.2000.2195, available online at http://www.idealibrary.com on

Free Fatty Acid Effects on Mitochondrial Permeability: An Overview Aya Sultan and Patricia M. Sokolove 1 Department of Pharmacology & Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland 21201

Received July 21, 2000, and in revised form October 25, 2000

A variety of experimental conditions elicit increases in mitochondrial permeability that can be differentiated from the classic cyclosporin A (CsA)-sensitive mitochondrial permeability transition (MPT). For example, butylated hydroxytoluene, signal peptides, and the hormone thyroxine have been shown to promote increases in mitochondrial permeability that are CsAinsensitive. Our laboratory has recently demonstrated that palmitic acid, a saturated 16-carbon free fatty acid (FFA), can also open a CsA-insensitive pore. This nonclassic permeability transition (NCPT) is further distinguished by a nonselective dependence on divalent cations and by spontaneous closure. To determine if induction of the NCPT is specific to palmitic acid and to resolve conflicting reports as to the mechanisms by which FFAs alter mitochondrial permeability, we examined in detail mitochondrial swelling induced by FFAs that differ in chain length and degree of saturation. The following results were obtained: (1) In the presence of modest Ca 2ⴙ concentrations (75 nmol/mg protein), medium-chain FFAs (C12–C18) were more effective in eliciting mitochondrial swelling than were shorter or longer FFAs; medium-chain alkanols and amines had no effect. (2) Under these conditions, saturated FFAs induced CsA-insensitive swelling with all the characteristics of the NCPT, while unsaturated FFAs triggered the MPT. (3) When matrix Ca 2ⴙ concentration was further elevated, unsaturated FFAs triggered the NCPT. (4) Mitochondrial swelling induced by saturated FFAs was inhibited by unsaturated FFAs but not by other saturated FFAs or medium-chain alkanols. These results suggest that ambient conditions can greatly influence the nature of the increase in mitochondrial permeability induced by FFAs. They are also consistent with our earlier proposal that Ca 2ⴙ 1

Correspondence should be addressed to Dr. Patricia Sokolove, c/o The Graduate School, 621 West Lombard Street, Suite 201, Baltimore, MD 21201. Fax: (410) 706-0265. E-mail: [email protected]. 52

(or Sr 2ⴙ) binding to FFAs in the inner leaflet of the inner mitochondrial membrane underlies the NCPT. © 2001 Academic Press

Key Words: rat liver mitochondria; permeability transition; cyclosporin A; free fatty acids.

Free fatty acids (FFAs) 2 exert a diverse array of biological effects. Some of the physiological roles ascribed to FFAs include uncoupling of oxidative phosphorylation (for reviews see 1, 2), functioning as second messengers (3), regulating Ca 2⫹ efflux and Ca 2⫹ channels (4, 5), modulating cytochrome c oxidase (6, 7), and potentiating NMDA currents (8). FFAs have also been implicated in a number of pathophysiological conditions such as ischemia–reperfusion injury (9 –11), Refsum disease (12), apoptosis (13, 14), obesity (14, 15), and diabetes (14, 16). Thus, it appears that the actions of FFAs are not only diverse, they are also complex. In addition to the above effects, FFAs are among a number of endogenous agents that are known to increase mitochondrial permeability. Induction of mitochondrial swelling by these agents has usually been credited to the opening of the mitochondrial permeability transition (MPT) pore (for reviews see 17, 18). The MPT is a well-characterized process that results in a nonselective increase in the permeability of the inner mitochondrial membrane to solutes smaller than 1500 Da. Criterial attributes of the MPT include an absolute 2 Abbreviations used: A 540, apparent absorbance at 540 nm; BHT, butylated hydroxytoluene; BSA, bovine serum albumin; CsA, cyclosporin A; EGTA, ethylene glycol bis(␤-aminoethyl ether) N,N⬘-tetraacetic acid; FFAs, free fatty acids; MW, molecular weight; MPT, mitochondrial permeability transition; NCPT, nonclassic permeability transition; PA, palmitic acid; PEG, polyethylene glycol; P i, inorganic phosphate; RLM, rat liver mitochondria; RR, ruthenium red; S, succinate; SR, sarcoplasmic reticulum; MCC, multiconductance channel; mOsM, milliosmolar; NMDA, N-methyl-D-aspartate.

0003-9861/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

FREE FATTY ACIDS INCREASE MITOCHONDRIAL PERMEABILITY

dependence on matrix Ca 2⫹ and inhibition by the immunosuppressant cyclosporin A (CsA) at very low stoichiometries (19 –21). The MPT has generally been regarded as the sole mechanism responsible for nonselective increases in mitochondrial permeability. Recently, however, this assumption has been questioned. Several laboratories have demonstrated the existence of pores of multiple sizes (22–26) and pores with both low- and high-conductance states (27–30). In addition, butylated hydroxytoluene (BHT; Refs. 24, 26, 31), signal peptides (32), and thyroxine (33) have all been shown to elicit CsA-insensitive mitochondrial swelling. It has been proposed (34) that signal peptide-induced swelling and the multiconductance channel (MCC), a large (⬎1 nS conductance) channel with multiple substates (for reviews see 18, 35), reflect opening of the translocation pore involved in protein import rather than opening of the MPT pore. Although it is well established that FFAs elicit mitochondrial swelling, there is little consensus as to the mechanism of this FFA effect. Several laboratories have demonstrated that very low concentrations of free fatty acids can stimulate the CsA-sensitive swelling induced by a variety of MPT triggers (23, 36 – 41). On the other hand, recent work has indirectly implicated free fatty acids in mitochondrial swelling that is CsAinsensitive (23, 26, 36, 42). Reconciliation of the contrasting reports has been difficult due to the variations in experimental protocol and conditions. We have recently demonstrated that, in the absence of additional triggers, relatively low concentrations (20 – 60 ␮M) of the 16-carbon saturated FFA (16:0) palmitic acid (PA) induce mitochondrial swelling that differs dramatically from swelling associated with opening of the MPT pore (43). PA-promoted increases in the permeability of the inner mitochondrial membrane were characterized by an insensitivity to CsA, a requirement for mitochondrial energization, a generalized dependence on matrix cations (Sr 2⫹ ⫽ Ca 2⫹), rapid swelling occurring without a lag, and the opening of a pore that spontaneously closed. We have proposed that the failure of other authors to detect the CsA-insensitive swelling induced by palmitic acid was due to the use of low PA concentrations, establishment of an unfavorable ratio of PA/Ca 2⫹, or simultaneous inclusion of a trigger of the classic MPT. Here we report an extensive examination of the mitochondrial swelling induced by FFAs of different chain lengths and degrees of saturation aimed at resolving apparent discrepancies among earlier reports and generating a comprehensive overview of the effects of FFAs on mitochondrial permeability. We have used cyclosporin A sensitivity as an indicator of MPT involvement in the increases in membrane permeability seen with the various FFAs. Our data demonstrate

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that, when Ca 2⫹ concentrations in the assay reagent are modest, saturated and unsaturated free fatty acids induce dramatically different types of mitochondrial swelling. Saturated FFAs triggered the CsA-insensitive swelling we refer to as the nonclassic permeability transition (NCPT), whereas unsaturated FFAs produced the CsA-sensitive swelling characteristic of the classic MPT. However, experimental conditions can be selected under which unsaturated free fatty acids elicit increases in mitochondrial permeability that are similar to those seen in the presence of saturated free fatty acids. It is proposed that regulation of mitochondrial permeability by FFAs is a complex phenomenon and reflects multiple distinct processes. FFAs may act as physiological regulators of mitochondrial permeability, but the nature of the FFA-induced increase in mitochondrial permeability is highly dependent on and influenced by cytosolic conditions. MATERIALS AND METHODS Reagents. Polyethylene glycols (PEGs) were obtained from Fluka (Milwaukee, WI) and Chelex-100 (100 –200 mesh) was obtained from Bio-Rad (Richmond, CA). Cyclosporin A (OL 27– 400) was the generous gift of Sandoz Research Institute (East Hanover, NJ). Other biochemicals were from Sigma Chemical Company (St. Louis, MO) and all reagents were of the highest grade available. Mitochondrial isolation and assay conditions. Mitochondria (RLM) were isolated from the livers of large (⬎250 g), male Sprague– Dawley rats by standard differential centrifugation techniques in low-salt buffers (44). Assays were carried out at 30°C in a resin (Chelex-100)-treated reagent containing 210 mM mannitol, 70 mM sucrose, and 10 mM Hepes–KOH, pH 7.4, supplemented with 0.8 ␮M rotenone, except when noted otherwise. The total reaction volume was 0.7 ml and the final mitochondrial protein concentration was 0.4 mg/ml, determined according to Lowry et al. (45) using bovine serum albumin as standard. Results are representative of multiple experiments (n ⱖ 3), except where indicated. Increases in mitochondrial permeability were followed via mitochondrial swelling by monitoring apparent absorbance at 540 nm ( A 540 ; Ref. 46) with an LKB Ultrospec II UV-Visible or Pharmacia Biotech Ultrospec 2000 UV-Visible spectrophotometer. Data are quantified in terms of the maximal rate of decrease of absorbance at 540 nm (⌬A 540 /min/mg) by drawing a tangent to the plot of absorbance vs time at its steepest point. Mitochondria were incubated for 3 min in the basic assay reagent. Free fatty acids, alkanols, P i, and potential inhibitors were added prior to mitochondria, except where specifically noted otherwise. When used, Ca 2⫹ or Sr 2⫹ was added to the assay solution at 3 min and mitochondria were energized upon addition of 5 mM succinate at 3.5 min, unless otherwise noted. Throughout, the combination of 1 mM P i plus 100 ␮M Ca 2⫹ was used to induce the classic MPT for the purpose of comparison. Assessment of pore status (open or closed) was accomplished as follows. At Time 0, mitochondria were added to 0.56 ml of the assay solution. After swelling was completed, 0.14 ml of 300 mOsM PEG (6000 MW) was added to bring the final reaction volume to 0.7 ml and the final protein concentration to 0.4 mg/ml. Mitochondrial shrinkage (an increase in A 540 ) subsequent to PEG addition was taken as an indication that the pore remained open (43).

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FIG. 1. In the absence of classic MPT triggers, saturated free fatty acids (A) induce rapid, CsA-insensitive mitochondrial swelling while unsaturated free fatty acids (B) induce slower, CsA-sensitive mitochondrial swelling. RLM were incubated in assay reagent supplemented with 30 ␮M free fatty acid ⫾ 1 ␮M CsA. Swelling was induced by adding 30 ␮M Ca 2⫹ at 3 min and 5 mM succinate at 3.5 min. Maximal rate of swelling (V max) was determined as described under Materials and Methods. Data show means ⫾ SD (n ⫽ 3; except C15:1 and C17:1, n ⫽ 2). To determine whether CsA substantially inhibited swelling induced by the various free fatty acids, statistical significance was assessed by unpaired t tests for experiments in which n ⫽ 3 (* indicates statistically significant inhibition, P ⬍ 0.05).

RESULTS

Saturated and unsaturated free fatty acids induce characteristically different types of mitochondrial swelling. The effects of free fatty acid saturation and chain length on maximal rate of mitochondrial swelling (V max) and CsA sensitivity were examined (Fig. 1). In all assays, FFAs and Ca 2⫹ were present at 30 ␮M (75 nmol/mg mitochondrial protein). Figure 1A depicts the

relationship between saturated free fatty acid chain length and mitochondrial swelling. The short-chain saturated FFA capric acid (C10:0) was unable to elicit mitochondrial swelling. A progressive increase in V max was observed with the medium-chain saturated FFAs, reaching a maximal rate of swelling with pentadecanoic acid (C15:0); increasing chain length beyond C15:0 resulted in a progressive decrease in V max. CsA partially inhibited mitochondrial swelling induced by undecanoic acid (C11:0) and lauric acid (C12:0). However, CsA had no effect on mitochondrial swelling induced by the saturated FFAs C13:0 –C18:0. Long-chain FFAs (C19:0 –C20:0) produced minimal mitochondrial swelling. These results are in stark contrast with those obtained with the unsaturated free fatty acids (Fig. 1B). Medium-chain unsaturated FFAs (C14:1–C18:3) induced markedly slower mitochondrial swelling than did their saturated counterparts. Furthermore, CsA completely inhibited the mitochondrial swelling induced by each of these unsaturated FFAs. CsA sensitivity and rates of swelling were similar irrespective of the degree of unsaturation: compare oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3). The long-chain unsaturated FFA arachidonic acid (20:4), however, failed to elicit mitochondrial swelling under analogous conditions. Several alkanols were also examined for their ability to induce mitochondrial swelling. Neither short-chain saturated (C10:OH), medium-chain saturated (C16: OH), nor medium-chain unsaturated (C18:2:OH) alkanols were able to produce any swelling, regardless of concentration used (data not shown). The characteristics of the mitochondrial swelling induced by a saturated FFA, an unsaturated FFA, a medium-chain alkanol, and the classic MPT triggers, P i ⫹ Ca 2⫹, are compared in Fig. 2. In all panels, trace 1 is a control recorded in the absence of triggers and Ca 2⫹. Palmitic acid (C16:0) induced rapid mitochondrial swelling that occurred without a lag (Fig. 2A, trace 2). This swelling was CsA-insensitive (trace 3) and was supported by Ca 2⫹ (trace 2) or Sr 2⫹ (trace 4). Both palmitoleic acid (C16:1, Fig. 2B) and the classic MPT trigger, P i ⫹ Ca 2⫹ (Fig. 2D), produced relatively slower mitochondrial swelling that occurred after a lag (trace 2), although the lag observed with P i and Ca 2⫹ is seen as a very brief increase in A 540 that is obscured by traces 3 and 4 in this figure. This swelling was characterized by a dramatic CsA sensitivity (trace 3) and an absolute requirement for Ca 2⫹ (compare traces 2 and 4). On the other hand, medium-chain alkanols, such as C16:OH, produced minimal swelling (Fig. 2C, trace 2) that was further decreased by CsA (trace 3). The medium-chain amine, stearylamine (C18), behaved like the alkanols (data not shown). Therefore, it appears that under these conditions (30 ␮M FFA, 30 ␮M Ca 2⫹),

FREE FATTY ACIDS INCREASE MITOCHONDRIAL PERMEABILITY

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FIG. 2. Saturated free fatty acids and unsaturated free fatty acids elicit distinct types of mitochondrial swelling. RLM were incubated in assay reagent supplemented with the trigger (either FFA or P i) ⫾ 1 ␮M CsA. Swelling was triggered by (A) 30 ␮M C16:0 ⫹ 30 ␮M Ca 2⫹, (B) 30 ␮M C16:1 ⫹ 30 ␮M Ca 2⫹, (C) 30 ␮M C16:OH ⫹ 30 ␮M Ca 2⫹, or (D) 1 mM P i ⫹ 100 ␮M Ca 2⫹. The arrows indicate the times of additions. In each panel, the numbered traces represent (1) succinate alone, (2) ⫹ trigger, (3) ⫹ trigger ⫹ CsA, and (4) ⫹ trigger ⫹ 30 ␮M Sr 2⫹ in lieu of Ca 2⫹. Data are representative of multiple (n ⱖ 3) experiments (note: the four panels are not taken from the same experiment).

medium-chain saturated FFAs induce a NCPT while medium-chain unsaturated FFAs induce the classic MPT. Unsaturated free fatty acids can produce either type of mitochondrial swelling depending on experimental conditions. To further investigate the influence of saturation on the type of mitochondrial swelling elicited, we varied the experimental conditions under which unsaturated FFAs were used as triggers (Fig. 3). As depicted previously, in the presence of 30 ␮M Ca 2⫹, 30 ␮M palmitoleic acid (C16:1) induced relatively slow swelling (Fig. 3A, trace 1) that was completely abrogated by CsA (trace 2). If the Ca 2⫹ concentration was raised to 100 ␮M, however, a change in the kinetics of swelling was observed (Fig. 3B). Under these conditions, C16:1-induced swelling was rapid and occurred without a lag (trace 1); CsA remained a potent inhibitor of this swelling (trace 2). We have previously demonstrated that palmitic acid (C16:0) requires the presence of matrix Ca 2⫹ to elicit maximal CsA-insensitive swelling (43). We hypothesized that the increased rapidity of mitochondrial swelling triggered by palmitoleic acid (C16:1) and 100 ␮M Ca 2⫹ might actually be due to more Ca 2⫹ ions

reaching the mitochondrial matrix. To test this hypothesis, we induced mitochondrial swelling with C16:1 under experimental conditions that would be predicted to further increase the matrix Ca 2⫹ accumulation: energization of mitochondria in the presence of Ca 2⫹ prior to addition of the FFA to eliminate negative effects of Ca 2⫹ chelation by the FFA on Ca 2⫹ uptake (Ref. 43, esp. Fig. 3); incubation of mitochondria with P i, which increases the membrane potential (47), thereby enhancing Ca 2⫹ uniporter function (17); and incubation of mitochondria with ruthenium red (after Ca 2⫹ uptake but prior to FFA addition), to decrease the Ca 2⫹ efflux through the Ca 2⫹ uniporter that would accompany the uncoupling effects of palmitoleic acid (17, 48). Under these conditions, palmitoleic acid (C16:1) elicited swelling that was rapid and occurred without a lag (Figs. 3C and 3D, trace 1). Most notable, however, was the partial (Fig. 3C, trace 2) or essentially complete loss of CsA sensitivity (Fig. 3D, trace 2). Thus, unsaturated FFAs appeared to trigger the same nonclassic permeability transition as saturated FFAs, provided that matrix Ca 2⫹ levels were sufficiently high. Although it is conceivable that P i might exert additional effects in this system, any action on the classic MPT is ruled out by

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FIG. 3. The mode of unsaturated FFA action is dependent on experimental conditions. RLM were incubated in assay reagent supplemented with C16:1 (excepted as noted in panel C), ⫾1 mM P i, and ⫾1 ␮M CsA. The arrows indicate the times of additions. Swelling was induced by (A) 30 ␮M C16:1 ⫹ 30 ␮M Ca 2⫹, (B) 30 ␮M C16:1 ⫹ 100 ␮M Ca 2⫹, (C) 100 ␮M Ca 2⫹ ⫹ 30 ␮M C16:1 added at 5 min, or (D) 30 ␮M C16:1 ⫹ P i ⫹ 100 ␮M Ca 2⫹. In each panel, the numbered traces represent (1) trigger ⫺ CsA and (2) trigger ⫹ CsA. Data are representative of multiple (n ⱖ 3) experiments.

the data in Fig. 2B, which demonstrate that the MPT triggered by the combination of 1 mM P i and 100 ␮M Ca 2⫹ is completely inhibited by CsA. This conclusion, namely, that unsaturated FFAs can induce the NCPT provided matrix Ca 2⫹ levels are sufficiently high, was further substantiated when we examined the effects of FFAs on pore status (Fig. 4). Pore status, i.e., whether the pore is in the open or closed state, is assessed by adding a high (6000) MW PEG to the assay after mitochondria complete swelling. We have recently shown that pore status is dependent on the triggers used to induce mitochondrial swelling (43). When mitochondrial swelling resulted from the induction of the MPT by Ca 2⫹ ⫹ P i, the pore remained open after swelling was complete. However, when mitochondrial swelling was triggered by palmitic acid (C16:0), the pore spontaneously closed. Figure 4 compares the swelling induced by palmitoleic acid (C16:1) at modest and elevated Ca 2⫹ concentrations with these two wellcharacterized swelling paradigms. In cases where the occurrence of the NCPT was suspected (Figs. 4C and 4D), CsA was added to the assay prior to the mitochondria to prevent the occurrence of the MPT. CsA was not present when swelling was triggered with palmitoleic acid ⫹ 100 ␮M Ca 2⫹ or P i ⫹ 100 ␮M Ca 2⫹ (Figs. 4A and

4B) since it had already been shown to inhibit such swelling completely or substantially (Figs. 3B and 2D, respectively). Addition of 6000 MW PEG to mitochondria that had undergone the classic MPT, upon exposure to Ca 2⫹ ⫹ P i (Fig. 4A) or palmitoleic acid (Fig. 4B) or other mediumchain unsaturated FFAs (data not shown), resulted in a substantial increase in absorbance. In contrast, no change in absorbance was detected upon addition of 6000 MW PEG to mitochondria that had undergone the NCPT, triggered either by palmitic acid (Fig. 4C) or other medium-chain saturated FFAs (data not shown). Under conditions that would result in high matrix Ca 2⫹, such as supplementation with 1 mM P i and 100 ␮M Ca 2⫹, the addition of 6000 MW to mitochondria that had been induced to swell by palmitoleic acid again brought about no change in absorbance (Fig. 4D); under these conditions, the pore opened by palmitoleic acid had closed spontaneously. Similar results, i.e., no change in absorbance, were obtained when 6000 MW PEG was added to mitochondria that were energized and incubated in 100 ␮M Ca 2⫹ prior to addition of C16:1 or when ruthenium red was added to mitochondria after Ca 2⫹ uptake but before triggering with C16:1 (other experimental conditions that result in high ma-

FREE FATTY ACIDS INCREASE MITOCHONDRIAL PERMEABILITY

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FIG. 4. The pore opened by saturated free fatty acids closes spontaneously while the pore opened by unsaturated free fatty acids remains open or closes spontaneously depending on experimental conditions. RLM were incubated in assay reagent supplemented with trigger (FFA or P i) ⫾ 1 ␮M CsA. Arrows indicate times of additions. Swelling was triggered by (A) 1 mM P i ⫹ 100 ␮M Ca 2⫹, (B) 30 ␮M C16:1 ⫹ 100 ␮M Ca 2⫹, (C) 30 ␮M C16:0 ⫹ 30 ␮M Ca 2⫹ ⫹ CsA, or (D) 30 ␮M C16:1 ⫹ 1 mM P i ⫹ 100 ␮M Ca 2⫹ ⫹ CsA. As described under Materials and Methods, 0.14 ml of 6000 MW PEG was added after swelling was complete. Data are representative of multiple (n ⱖ 3) experiments (data shown in the four panels were collected on different days).

trix Ca 2⫹, data not shown). These data confirm the suggestion that the pore opened by saturated FFAs under conditions of high matrix Ca 2⫹ is indeed the NCPT. The swelling induced by saturated free fatty acids is inhibited by unsaturated free fatty acids. Figure 5 depicts the effects of unsaturated FFAs (Fig. 5A), other saturated FFAs (Fig. 5B), and alkanols (Fig. 5B) on mitochondrial swelling induced by palmitic acid (C16: 0). In all experiments, 1 ␮M CsA was present to prevent interference from the classic MPT. As seen in Fig. 5A, complete inhibition of PA-induced mitochondrial swelling (trace 1) was observed with the unsaturated FFAs myristoleic acid (C14:1, trace 2), heptadecanoic acid (C17:1, trace 5), oleic acid (C18:1, trace 6), and linoleic acid (C18:2, trace 7). Partial inhibition was observed with pentadecanoic acid (C15:1, trace 3) and palmitoleic acid (C16:1, trace 4). Increasing the degree of saturation to ⱖ3 double bonds resulted in the partial (C18:3, compare trace 8 with traces 6 and 7) or complete (C20:4, trace 9) loss of inhibition; in fact, arachidonic acid (C20:4) had a slightly stimulatory effect. On

the other hand, as shown in Fig. 5B, saturated FFAs (traces 2 and 3) and alkanols (traces 4 and 5) were ineffective inhibitors of mitochondrial swelling induced by C16:0. A similar pattern of inhibition was obtained when mitochondrial swelling was induced by myristic acid (C14:0) or pentadecanoic acid (C15:0; data not shown). DISCUSSION

We have previously reported that the saturated free fatty acid palmitic acid (C16:0) triggers a nonclassic permeability transition (43). This NCPT could be clearly distinguished from the MPT by a number of characteristics. In contrast to the MPT, mitochondrial permeability due to the induction of the NCPT was rapid, CsA-insensitive, and supported by Ca 2⫹ and other divalents, and it resulted from the opening of a pore that spontaneously closed upon completion of swelling. This study further demonstrates that hydrocarbon effects on mitochondrial permeability are complex and depend on chain length, degree of saturation, charge, and matrix Ca 2⫹ concentration.

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FIG. 5. Unsaturated FFAs, but not saturated FFAs or alkanols, inhibit swelling induced by palmitic acid. RLM were incubated in assay reagent supplemented with 1 ␮M CsA, C16:0, and the additional FFAs indicated below. Arrows indicate times of additions of Ca 2⫹ and succinate. (A) Effect of unsaturated FFAs (30 ␮M). Swelling was induced by 30 ␮M C16:0 ⫹ 30 ␮M Ca 2⫹, in the presence of (1) succinate alone, (2) C14:1, (3) C15:1, (4) C16:1, (5) C17:1 (6) C18:1, (7) C18:2, (8) C18:3, or (9) C20:4. (B) Effect of saturated fatty acids or alkanols (30 ␮M). Swelling was induced by 30 ␮M C16:0 ⫹ 30 ␮M Ca 2⫹, in the presence of (1) succinate alone, (2) C10:0, (3) C15:0, (4) C10:OH, or (5) C16:OH. Data are representative of multiple (n ⱖ 3) experiments.

Medium-chain saturated (C13:0 –C18:0) and unsaturated (C14:1–C18:3) FFAs induced significantly different types of mitochondrial swelling. Under standard conditions of 30 ␮M FFA and 30 ␮M Ca 2⫹, mediumchain saturated FFAs elicited rapid mitochondrial swelling (Fig. 1A) in the absence of a lag (Fig. 2A). This swelling was insensitive to CsA (Figs. 1A and 2A) and could be supported by Sr 2⫹ (Fig. 2A). Furthermore, in agreement with our previous results (43), the pore opened by these saturated FFAs closed spontaneously (Fig. 4C). In contrast, medium-chain unsaturated FFAs, under the same conditions, promoted increases in mitochondrial permeability that were relatively slow (Figs. 1B and 2B), occurred after a lag (Fig. 2B), and were inhibited by CsA (Fig. 1B). Sr 2⫹ could not substitute for Ca 2⫹ to support this swelling (Fig. 2B) and the pore remained open once swelling was complete (Fig. 4B). Thus, saturated FFAs triggered the NCPT, while unsaturated FFAs opened the MPT pore. Short-chain (C10:0) and long-chain (C19:0 –C20:0, Fig. 1A) saturated FFAs, long-chain unsaturated FFAs (C20:4, Fig. 1B), alkanols (Fig. 2C), and a medium-

chain amine (data not shown) were unable to produce significant mitochondrial swelling. Therefore, it appears that the presence of a carboxylic acid moiety on medium-chain-length hydrocarbons is necessary for the induction of substantial swelling and that the type of swelling elicited by free fatty acids depends on the degree of saturation. There is precedent for this pattern of FFA specificity. Most noteworthy are FFA effects on sarcoplasmic reticulum (SR). Palmitic acid (C16:0) enhanced Ca 2⫹ sequestration by SR whereas oleic acid (C18:1) was inhibitory (49, 50). Moreover, when FFA chain length and saturation were varied, pentadecanoic acid (C15:0) and palmitic acid (C16:0) most effectively promoted Ca 2⫹ sequestration, while myristic acid (C14:0) and heptadecanoic acid (C17:0) were less effective. Saturated FFAs, whose chain lengths were less than 14 carbons or greater than 17 carbons, and all unsaturated FFAs tested were ineffective (50). The authors attributed the distinct results they obtained with saturated and unsaturated FFAs to the specificity of a FFA transport protein in the SR membrane.

FREE FATTY ACIDS INCREASE MITOCHONDRIAL PERMEABILITY

In our experimental system, it is possible to “convert” the behavior of unsaturated free fatty acids to that characteristic of saturated free fatty acids. When Ca 2⫹ uptake occurred prior to unsaturated FFA addition (Fig. 3C) or was enhanced by P i (Fig. 3D), unsaturated FFAs produced mitochondrial swelling that was comparable to swelling obtained with saturated free fatty acids. This unsaturated FFA-induced swelling was rapid and occurred without a lag (Figs. 3C and 3D). Inhibition by CsA was either reduced (Fig. 3C) or eliminated (Fig. 3D), while Sr 2⫹ substituted effectively for Ca 2⫹ (data not shown). Furthermore, the pore opened under these conditions closed spontaneously. In other words, when matrix Ca 2⫹ levels were sufficiently high, unsaturated FFAs induced the NCPT. We therefore propose that the differences between saturated and unsaturated FFA effects on mitochondrial swelling result not from their differential uptake by mitochondria but from their disparate affinities for Ca 2⫹. Specifically, we attribute the differential effects on mitochondrial permeability of saturated and unsaturated FFAs to the higher affinity for Ca 2⫹ of the latter. Differences between the effects of saturated and unsaturated FFAs on Ca 2⫹ binding are well documented. For example, low concentrations (2–3 mol of FFA/mol of albumin) of unsaturated FFAs have been shown to enhance the affinity and maximum binding of Ca 2⫹ to human albumin, while 2.5- to 3.5-fold higher concentrations of saturated FFAs were required to observe the same effect (51). Unsaturated FFAs also bound, in a Ca 2⫹-dependent manner, to a site formed by the heterocomplex of two calcium-binding proteins, MRP8 and MRP14, isolated from human keratinocytes (52). This Ca 2⫹-dependent FFA-binding site was highly specific for unsaturated FFAs; saturated FFAs bound with low affinity and were poor competitors (52). Finally, the paramagnetic Ca 2⫹ analog Mn 2⫹ was used in a study of divalent cation binding to phospholipid vesicles (53). It was reported that unsaturated phosphatidylserine bound Mn 2⫹ with a much higher affinity than saturated phosphatidylserine. The authors concluded that cation affinity was associated with lipid fluidity and could be modulated by the interactions between FFA chains. Given the assumption of differential FFA affinity for Ca 2⫹, our data are consistent with the model we previously put forth to explain how FFA interaction with mitochondria might bring about swelling (43). Briefly, the model suggests that the accumulation of sufficient levels of a divalent cation–FFA complex in the inner leaflet of the inner membrane opens and stabilizes a pore in the inner membrane that is responsible for CsA-insensitive swelling. Since FFAs are capable of rapid flip–flop (54), the uncomplexed form will reach equilibrium in the two leaflets of the inner membrane. Energization of such mitochondria will result in FFAs

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in the outer leaflet of the inner membrane competing with the electrophoretic uniporter for available Ca 2⫹. If the Ca 2⫹ concentration is low, Ca 2⫹ binding to FFAs in the external leaflet of the inner membrane will predominate. Ca 2⫹ accumulation will be minimal and the equilibrium distribution of the FFA may be disrupted, resulting in movement of the FFAs from the inner to the outer leaflet. As the Ca 2⫹ concentration is increased (for a fixed FFA concentration), the uniporter will compete more and more successfully for Ca 2⫹ and matrix Ca 2⫹ levels will become sufficient to support formation and accumulation of a Ca 2⫹–FFA complex at the inner surface of the inner membrane, with the result that the NCPT occurs. The amount of Ca 2⫹ required to support occurrence of the NCPT will clearly reflect the affinity of the FFA for Ca 2⫹. This is exactly the pattern we observe if we compare saturated and unsaturated FFAs: more Ca 2⫹ is required to allow unsaturated FFAs to induce the NCPT. The model is also able to explain the fact that, for both saturated (43) and unsaturated FFAs (Fig. 3), opening of the NCPT pore is enhanced when Ca 2⫹ uptake is favored. Finally, the inhibition by unsaturated FFAs of the NCPT induced by saturated FFAs (Fig. 4) can be attributed to the ability of the former to lower free Ca 2⫹ concentrations. Although we have described the characteristics of FFA-induced pore opening, our data provide only limited insight into the underlying mechanism. Pore opening may result from membrane perturbation by the cation–FFA complex. Saturated FFAs are known to increase the membrane phase transition temperature (55–57) and order bilayer head groups (57, 58). Unsaturated FFAs decrease the membrane phase transition temperature (55–57), disorder the membrane interior (55, 57), and order bilayer head groups (57). Since unsaturated FFA-induced mitochondrial swelling can resemble saturated FFA-induced swelling under specific experimental conditions, it seems likely that the membrane perturbing effects these FFAs share would be responsible for pore opening. One of the effects on the membrane that both saturated and unsaturated FFAs have in common is ordering of bilayer head groups. Many membrane proteins are highly sensitive to their surrounding lipid environment; it is conceivable that a mitochondrial protein or proteins undergoes a conformational change in response to altered head group packing that leads to pore opening. Free fatty acids have numerous well-described biological properties, several of which relate directly to their effects on mitochondria. For example, FFAs uncouple oxidative phosphorylation (1, 2) and provide energy for cells via ␤-oxidation. The hormone thyroxine has been reported to increase FFA levels (18) and elicit CsA-insensitive mitochondrial swelling (33). This CsAinsensitive swelling may represent the induction of the

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NCPT and may provide a mechanism for the mitochondrial response to hormonal stimulation. Levels of FFAs are also observed to increase dramatically in a number of pathophysiological conditions that include ischemia/reperfusion injury (9 –11), Refsum disease (12), apoptosis (13, 14), obesity (14, 15), and diabetes (14, 16). Since mitochondrial swelling is often associated with these disorders, involvement of the MPT pore has been hypothesized. Here we demonstrate that the mitochondrial swelling elicited by FFAs can occur via two distinct mechanisms. The possibility must be considered that mitochondrial responses in cells are finely regulated by matrix conditions so that permeability increases can arise either by induction of the MPT or the NCPT. An accurate understanding of the processes involved in each instance of altered regulation of mitochondrial permeability will be required before appropriate therapeutic interventions can be developed. ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid (MDSG1797) from the American Heart Association, Mid-Atlantic Affiliate to PMS, and a Graduate Student Association Research Grant awarded to A.S. A.S. was supported by Training Grant T32GM08181 from the National Institutes of Health. We also thank Dr. Gary Fiskum, who graciously made his four-channel recording system available to us.

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