The JOUSNAL or BIOLOGICAL CHEMISTRY Vol. 253, No. 3, Issue of February 10, pp. 189-799, 1378

Printed in U.S.A.

Activation of Mitochondrial Fatty Acid Oxidation by Calcium CONVERSION TO THE ENERGIZED STATE*

(Received for publication, May 25, 1977, and in revised form, September 29, 1977)

DAVID A. OTTO AND JOSEPH A. ONTKO

From The Laboratory of Lipid and Lipoprotein Studies, Oklahoma Medical Research Foundation, and The Department of Biochemistry and Molecular Biology, University of Oklahoma College of Medicine, Oklahoma City, Oklahoma 73104

The oxidation of [l-Wlpalmitate by isolated rat liver mitochondria was increased by calcium chloride, and was quantitatively accounted for by an increased production of ketone bodies facetoacetate plus /3-hydroxybutyrate). ‘%02 production was depressed by Ca2+. These changes were associated with an elevated /3-hydroxybutyrate:acetoacetate ratio, reflecting an increased mitochondrial NADH:NAD+ ratio. Calculations reveal that the additional NADH pro- duced via the increase in fatty acid oxidation was more than required for Ca 2+ transport and for the increased reduction of acetoacetate. The results indicate that the effect of Ca2+ on fatty acid oxidation was responsible for the elevation of the mitochondrial NADH:NAD+ ratio. The effects of Ca’+ on palmitate oxidation and the pyridine nucleotide oxidation-reduction state showed an absolute requirement for carnitine, CoA, ATP, and Mg2+. These effects exhibited half-maximal response under the standard incubation conditions, at approximately 80 nmol of Ca2+/ mg of protein, a value which proved to be a function of the pH of the system. These Ca*+-dependent changes were accompanied by an elevated steady state rate of oxygen consumption, measured after the completion of Ca2+ accu- mulation, and increased ATP production.

Ca*+ uptake studies in the presence of palmitate, CoA, carnitine, phosphate, Mg2+, and ATP showed that the initial rapid transport of Ca2+ was completed in 2 min. The fi- hydroxybutyrate:acetoacetate ratio decreased during this initial period of Ca*+ transport and subsequently increased. The Ca2+-dependent decline and subsequent elevation of the ATP concentration paralleled the temporal changes in the P-hydroxybutyrate to acetoacetate ratio. However, an ele- vation in the rate of ketone body production was observed at the onset of Ca2+ addition. This rate decreased slightly after 2 min, but remained greater than the control. The Ca*+ transport inhibitor ruthenium red, added at the time of Ca2+ addition, completely blocked the Ca*+-dependent elevations of ketone body production and the P-hydroxybu-

* This work was supported by Research Grant HL 13302 from the United States Public Health Service. The costs of nublication of this article were defrayed in part by the payment of iage charges. This article must therefore be hereby marked “Wuertisemenf’ in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

tyrate:acetoacetate ratio. I f ruthenium red was added after the completion of Ca*+ uptake, it did not prevent the further development of these effects. Procaine did not prevent the effects of Ca2+ on ketogenesis and the pyridine nucleotide oxidation-reduction state. These results indicate that Ca2+ accumulation, not Ca*+ transport per se, was prerequisite for the development of a steady state characterized by enhanced fatty acid oxidation and an elevated NADH:NAD+ ratio.

These observations provide evidence for a specific regula- tory action of calcium in the mitochondrial oxidation of fatty acids, which consequently influences the mitochon- drial energy state.

We have previously reported that Ca2+ ions exert a marked influence on the mitochondrial pyridine nucleotide oxidation- reduction state in intact hepatocytes (1, 2). This action of Ca2+ is characterized by an elevation in the mitochondrial NADH:NAD+ ratio, as reflected by an increased P-hydroxy- butyrate to acetoacetate ratio (1) and increased pyridine nucleotide fluorescence (2). This effect was most prominent when the isolated liver cells were provided with a long chain fatty acid substrate. The cytosolic NADH:NAD+ ratio, as

reflected by the 1actate:pyruvate ratio, was unchanged by Ca2+. Increased ketone body production accompanied the ele- vated mitochondrial NADH:NAD+ ratio. Several years ago Mellanby and Williamson (3) observed that Ca2+ elevated the P-hydroxybutyrate:acetoacetate ratio in rat liver slices, al- though they found no correlation between the effects of Ca2+ on this ratio and on ketone body production. Streffer and Williamson (4) later suggested that the increased ketogenesis, observed when liver slices derived from fasted rats were incubated with Ca2+, was via action of Ca2+ ions on plasma membrane permeability, preventing the leakage of cytosolic factors into the medium.

In isolated mitochondria supplied with a permeant anion, Ca2+ addition is followed by reversible oxidation of pyridine nucleotides with concurrent reversible increase in the oxida- tion of various substrates such as succinate and glutamate. These changes are a result of the energy-dependent Ca2+ uptake by mitochondria (5, 6) and are proportional to the

789

by guest on September 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

790 Ca2+, Fatty Acid Oxidation, and Mitochondrial Energy State

amount of Ca’+ added. Our fluorescence measurements with isolated liver cells demonstrate this initial oxidation (2); however, the cells treated with Ca”+ subsequently develop a greater state of reduction of the mitochondrial pyridine nu- cleotides (1, 2). This effect of Ca2+ in liver cells therefore appears to be caused by Ca2+ after its accumulation by the mitochondria and not associated with the transport process itself. In the intact cell system the involvement of the mito- chondrial Ca”+ transport process cannot be ruled out, since calcium uptake by intact hepatocytes is slow, reportedly only 5% of the rate obtained with cells treated with digitonin (7).

The effects of Ca2+ on ketogenesis and the mitochondrial NADH:NAD+ ratio in isolated liver cells (1, 2) might be explained by a stimulation of fatty acid oxidation with a subsequent rise in NADH production. This hypothesis is in contrast to the conclusions made concerning the effects of Ca2+ on rat liver slices (3, 4). The purpose of this study, therefore, was to investigate the effects of Ca2+ on the oxida- tion of fatty acids and the oxidation-reduction state in an isolated mitochondrial system, in which the effects of CaZ+ on the plasma membrane and cytosolic components are elimi- nated. The following observations implicate Ca2+ as a regula- tory factor in the mitochondrial oxidation of fatty acids and the associated pyridine nucleotide oxidation-reduction state in the mitochondrial matrix. A preliminary account of this work has been presented (8).

EXPERIMENTAL PROCEDURES

Methods -Adult male Holtzman rats were maintained on Purina Laboratory Chow and water ad Zibitum. Food was removed between 9 and 11 a.m. and 24 h later liver mitochondria were isolated. Rats were decapitated, exsanguinated, and the livers rapidly placed in ice-cold isolation medium (0.25 M sucrose, 3 mM Trislchloride, and 1 mM EGTA,’ pH 7.4, at 0”) and cut into small pieces. Subsequent mitochondrial isolation procedures were carried out at O-4”. Then 4- to 6-g portions of the chopped tissue were gently homogenized in 50 ml of isolation medium with three passes of a loose Teflon pestle in a glass Potter-Elvehjem homogenizer. The homogenate was centri- fuged at 500 x g for 10 min. The supernatant was then centrifuged at 9000 x g for 10 min. The resulting supernatant was discarded. The mitochondrial pellet was gently resuspended with a Teflon rod in the original volume of 0.25 M sucrose, 3 mM Trislchloride (without EGTA), and recentrifuged under the same conditions. After removal of the supernatant and any fluffy layer, the mitochondria were washed once more in this manner. The final pellet was resuspended in 0.25 M sucrose, 3 mM Tris/chloride (pH 7.4 at 0”) to provide a concentration of 30 to 40 mg of protein/ml. It was noticed that the Caz+-induced changes reported herein were of greater magnitude when the time between the isolation and incubation of mitochondria was 1 to 2 h, than when the mitochondria were incubated 0 to 1 h after isolation. This difference was related to a somewhat lower control rate of fatty acid oxidation at 1 to 2 h; the Caz+-induced rate did not differ. All mitochondrial preparations in these experiments had a respiratory control ratio ranging from 4.0 to 8.0 with the substrates 5 mM glutamate plus 5 mM malate.

The mitochondrial suspension, 0.25 ml, was incubated with 1.0 ml of 0.25 M sucrose, 3 mM Tris/chloride, 0.5 ml of 3 mM palmitate- 6% bovine albumin (fatty acid free), and 0.25 ml of mixed medium (pH 7.0 to 7.1) to provide a final concentration of 0.45 mM ATP, 0.8 mM m-carnitine, 60 mM KCl, 10 mM potassium phosphate, 20 PM

CoA, 5 mM MgCl,, and 1 mM dithiothreitol in a 2 ml final incubation volume. The above concentrations of ATP, m-carnitine, and CoA were used except when the effect of the respective cofactor was being investigated. When octanoate was the substrate, the incuba- tion was in the absence of carnitine, CoA, and dithiothreitol. Incubations were carried out in triplicate at 37” for 15 min except where indicated otherwise.

For analyses of ketone bodies and ATP the incubations were terminated with perchloric acid as described earlier (11. Acetoacetate

- 1 The abbreviation used is: EGTA, ethylene glycol bis(P-amino-

ethyl ether)-Nfl’-tetraacetic acid.

(9), P-hydroxybutyrate (101, and ATP (11) were determined enzy- matically in neutralized perchloric acid extracts. Incubations for analyses of 14C0, and [“Clacetoacetate were carried out in 25-ml Erlenmeyer flasks with center wells containing folded filter paper and 0.2 ml of 10% KOH. These were determined as previously described (12). [l-‘4ClPalmitate utilization was measured by lipid extraction with chloroform/methanol, 2:l (v/v), washing the lipid extracts according to Folch et al. (131, and subsequent thin layer chromatographic isolation of the fatty acid band (12). The amount of U-14Clpalmitate utilized is the calculated difference between the added [l-14Clpalmitate and that recovered unused at the end of the incubation period. Oxygen consumption was measured with a Clark- type electrode (14).

Incubations utilizing added albumin-bound palmitate had a final concentration of 0.75 mM palmitate-1.5% albumin. The specific activity of the added [l-L4Clpalmitate used in these experiments was 785 dpm/nmol of palmitate. When octanoate was the substrate (0.375 mM, final concentration), the incubation medium did not contain albumin.

Ca*+ uptake under the above experimental conditions was deter- mined by measuring the radioactivity of 45CaZ+. At the end of the incubation period Ca2+ uptake was quenched by the addition of 2 ml of ice-cold quenching solution (0.25 M sucrose, 3 mM Tris/chloride, and 10 mM EGTA) to the 2 ml incubation system, according to the method of Reed and Bygrave (15). This mixture (0.5 ml) was filtered through the Millipore filter (HAWP, pore size 0.45 pm), followed by an additional 0.5 ml of ice-cold quenching solution to wash the filter. Both the filter and an aliquot of the filtrate were counted with a Packard liquid scintillation counter. The scintillation mix- ture previously described (161 was used for counting of i4C and 45Caz+.

Ruthenium red was recrystallized from a commercial preparation (Sigma Chemical Co.; approximately 20% pure) according to Fletcher et al. (171, yielding a 65% pure sample as analyzed spectro- photometrically at 532 nm (es32 nm = 6.15 x IO4 M-I cm-‘; M, = 858.3 (17)). A stock solution was prepared (13 mM) from the recrystallized preparation and stored in the dark at 4”. On the day of the experiment, an aliquot of the stock solution was filtered through a Millipore filter (0.22 pm) and diluted to the working concentration, based upon the above molar absorption coefficient.

ADP (la), AMP (181, and NAD+ (191 were measured by fluoromet- ric procedures in perchloric acid extracts. Since NADH is unstable at low pH, it was extracted under alkaline conditions. For the fluorometric measurement of NADH (19) the incubation was termi- nated with 0.5 ml of 1 N alcoholic KOH, followed by heating for 5 min at 90” to destroy NAD+. The extract was cooled and then adjusted to pH 7.8. A mixture of a-ketoglutarate, NH&l, and glutamate dehydrogenase was then added to convert NADH to NAD+. After standing for 15 min at room temperature, 0.2 ml of 3 M perchloric acid/ml of extract was added and centrifuged to remove protein. The extract was finally adjusted to pH 7.2 with 1 M KOH. The measured value of NAD+ in this extract was equated to the level of NADH in the original sample.

Materials - ATP, ADP, m-carnitine, dithiothreitol, EGTA, Tris (Trizma base), palmitic acid, octanoic acid, ruthenium red, procaine hydrochloride, and butacaine hemi-sulfate were purchased from the Sigma Chemical Co. Pentex bovine albumin, Fraction V (fatty acid- free) from Miles Laboratory, was used for the preparation of albu- min-bound palmitate. The coenzymes NADH, NAD+, and coanzyme A were supplied by P-L Biochemicals. cr-Ketoglutarate was from Calbiochem. Sucrose was a Baker-analyzed reagent. All enzymes used in the analyses were obtained from Boehringer Mannheim. [l- i4ClPalmitate was from New England Nuclear. *CaCl, was obtained from Amersham/Searle.

RESULTS

Effects of Ca2+ on Oxidation of [l-WlPalmitate-The ef- fects of Ca2+ on the oxidation of [l-14C1palmitate to its prod- ucts, CO, and ketone bodies, are shown in Table I. Ca2+ (400 pM, corresponding to 80 nmol/mg of protein) increased palmi- tate conversion to ketone bodies 282 nmol and decreased conversion to CO, 9 nmol. It is reasonable to use ketone body production as an index of the change in fatty acid oxidation under these experimental conditions in which CO, production is a small fraction of the total oxidation products. The stimu-

by guest on September 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

CaZ+, Fatty Acid Oxidation, and Mitochondrial Energy State 791

latory effect of Ca2+ on fatty acid oxidation was associated with an elevation in the @hydroxybutyrate:acetoacetate ratio. The measured concentrations of ATP and ADP in this experi- ment were unchanged by Ca2+, indicating sufficient NADH production in the control system (P-hydroxybutyrate:acetoac- etate ratio = 2.19) to maintain a high ATP:ADP ratio. Calculations made from the experiment of Table I demon- strate that the theoretical amount of NADH plus FADHz produced from the Ca2+-dependent increase in fatty acid oxidation to ketone bodies is in excess of the energy deficit due to Krebs cycle inhibition, fatty acid activation, and Ca2+ uptake, as well as the additional NADH required to reduce acetoacetate (Table II). Thus, activation of fatty acid oxidation by Ca2+ could readily account for the observed alterations in the mitochondrial energy state. The results are consonant with the concept that the Ca2+-induced elevation of the mito- chondrial NADH:NAD+ ratio is secondary to a primary effect of Caz+ on fatty acid oxidation.

The question “Does an effect of Caz+ on endogenous lipids contribute to the increased ketogenesis?” was addressed early in this investigation. This is an important point in view of Ca2+-stimulated phospholipase activity in many tissues (20, 21). The observed specific radioactivity of the ketone bodies produced by mitochondria incubated with [l-14Clpalmitate in the presence of Ca2+ very closely approximated the theoretical yield, namely [l-Wlpalmitate specific activity + 4. Therefore,

the Ca2+-induced increase in ketogenesis was derived from increased oxidation of the added palmitate. The effect of Caz+ on ketogenesis by mitochondria in the absence of added fatty acid substrate was also examined. It was found that the addition of Ca2+ in the absence of added substrate caused a slight decrease in total ketone body production, from 59 to 39 nmol, while the P-hydroxybutyrate:acetoacetate ratio did not change from the control value of 0.26. In this same experiment ketone body production in the presence of 0.75 mM palmitate- 1.5% albumin was increased by Caz+ addition from 448 to 1473 nmol, and the /?-hydroxybutyrate:acetoacetate ratio increased from 0.57 to 7.82. It is therefore clear that endogenous fatty acid oxidation accounts for only a small percentage of the total ketone body production and that endogenous lipids are not involved in the observed effects of Ca2+.

Direct measurement of the diphosphopyridine nucleotide content of the mitochondrial system incubated with 0.75 mM palmitate-1.5% albumin + Ca*+ showed that the NAD+ con- centration decreased from 19.01 ? 0.82 to 13.19 2 0.33 nmol/ 10 mg of protein (mean + SE., p < 0.001, n = 6) and the NADH concentration increased from 2.62 f 0.26 to 5.71 ? 0.26 nmol/lO mg of protein (mean + S.E.,p < 0.001, n = 6) in the presence of Ca2+ (80 nmol/mg of protein). Ca2+ increased the NADH:NAD+ ratio from 0.14 to 0.41. Based upon the known equilibrium constant for fi-hydroxybutyrate dehydro- genase (4.93 x 1OW (22)), it may be calculated from the p-

TABLE I

Effect of Ca*+ on oxidation of palmitate by isolated liver mitochondria

Mitochondria (10.0 mg of protein) were incubated with 0.75 rn~ [1-Wlpalmitate-1.5% albumin as described under “Experimental Procedures.” WO, formation, W ketone body production (acetoacetate plus fi-hydroxybutyrate), and [lJ4Clpalmitate utilization (the calculated difference between the [l-‘4Clpalmitate added and that recovered unused at the end of the incubation period) were measured. Values are means of three determinations ? S.E.

Control + CaCI, (400 p&d A P

Palmitate utilized (nmol) 413.8 2 12.8 690.9 f 25.6 i277.1 <O.OOl Palmitate oxidized to CO, (nmol) 16.4 ? 1.2 7.1 f 0.1 -9.3 <O.OOl Palmitate oxidized to ketone bodies (nmol) 116.1 ? 18.6 398.3 + 13.8 +282.2 <O.OOl &Hvdroxvbutvrate:Acetoacetate ratio 2.19 f 0.23 7.29 k 0.98 +5.20 <0.005

TABLE II

Production and utiEization of mitochondrial reducing equivalents: changes caused by Caz+ estimated from Table I Ca*+-induced chance Calculations Reducing equivalents

A. Energy production 1. Reducing equivalents produced from

additional fatty acid oxidized B. Energy utilization

1. Deficit in formation of reducing equivalents via Krebs cycle

2. ATP expended to activate additional fatty acid oxidized

277.1 nmol palmitate oxidized x 7 INADH + FADHJ/palmitate oxidized

9.3 nmol less palmitate oxidized x 8 acetyl-CoA/palmitate oxidized

74.4 nmol acetyl-CoA x I3 NADH + FADH, + ATP”/acetyl-CoA entering the Krebs cycle1

277.1 nmol palmitate activated x 2 ATP/palmitate activatedb

+I940 nmol NADH + 1940 nmol FADH,

-248 nmol NADH -74 nmol FADH,

- 183 nmol NADH

554.2 nmol ATP x 0.33 NADH consumed/ATP generated

3. Required for Ca*+ uptake 800 nmol of Can+c + 6 Ca2+ transported/NADH oxi- - 133 nmol NADH dized

4. To reduce acetoacetate 760 nmol of additional acetoacetate reduced” -760 nmol NADH C. Calculated excess of NADH + FADH, 1940 nmol NADH - 1324 nmol NADH +616 nmol NADH

1940 nmol FADH, - 74 nmol FADH, + 1866 nmol FADH,

a ATP is derived from substrate level phosphorylation and is c 400 ELM CaCl, in a 2 ml incubation volume. equivalent to 0.33 NADH. d Values of P-hydroxybutyrate from the experiment of Table I,

b Two high energy phosphate bonds are utilized for the activation obtained by enzymatic analysis, went from 544 nmol in the control process, since AMP and PP, are formed. to 1304 nmol in the presence of 400 FM CaCl,.

by guest on September 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

792 Ca2+, Fatty Acid Oxidation, and Mitochondrial Energy State

hydroxybutyrate to acetoacetate ratio (which was increased by CaZ+ from 0.10 to 2.64) that the ratio of NADH to NAD+ of the free mitochondrial diphosphopyridine nucleotides in- creased from 0.005 to 0.130. This difference in the total and free NADH:NAD+ ratio is consistent with the reported differ- ential binding of these nucleotides to protein; more NADH than NAD+ is protein-bound (22-24).

Carnitine and CoA Requirements of Ca2+-dependent Zn- creases in Fatty Acid Oxidation and Mitochondrial NADH:NAD+ Ratio-Since the effect of Ca2+ on the mitochon- drial pyridine nucleotide oxidation-reduction state appeared to be related to an increase in fatty acid oxidation, the cofactor requirements for the Ca*+-dependent changes were analyzed. The effect of carnitine concentration is illustrated in Fig. 1. As the m-carnitine concentration was increased from 0 to 50 PM there was a great elevation of ketone body production in the presence of Ca2+ (Fig. 1) with no further change above 50 pM carnitine. The control rate of ketone body production increased slightly by comparison and it also reached a maximum change at 50 PM m-carnitine. The results demonstrate that the effect of Ca2+ on ketogenesis was completely dependent upon the presence of catalytic amounts of carnitine. Similar changes occurred in the P-hydroxybutyr- ate:acetoacetate ratio (Fig. IS) except that the Ca*+-dependent increase in the ratio was less responsive than the elevation of ketogenesis at carnitine concentrations of 0 to 20 PM, support- ing the concept that the effect on the ratio was secondary to the increase in fatty acid oxidation. The ratio with Ca*+ at 30 pM DL-CaITIitine began to rise, reaching a plateau at 100 PM

DL-Cardine. The P-hydroxybutyrate:acetoacetate ratio in the absence of Ca2+ did not change over the range of carnitine concentrations tested. To ensure the presence of adequate concentrations of carnitine, 0.8 mM m-carnitine was used throughout these experiments.

&enzyme A, also a cofactor required for long chain fatty acid oxidation, was analyzed as above for effects of its concen- tration on the Caz+-dependent stimulation of fatty acid oxida- tion and the associated NAD+ oxidation-reduction state (Fig. 2). In the absence of CoA, Ca2+ was totally ineffective in elevating either ketone body production (Fig. 2A) or the ratio of P-hydroxybutyrate to acetoacetate (Fig. 281. However, as CoA was added in increasing amounts, Ca2+ caused elevations

\ 7

DL-CARNITINE (mM) FIG. 1. Effect of m-carnitine concentration on the Ca*+-depend-

ent elevation of (A) total ketone body production and (I?) the p- hydroxybutyrate:acetoacetate ratio. Mitochondria (10.0 mg of pro- tein) were incubated with 0.75 mr.r palmitate-1.5% albumin for 15 min at 37” as described under “Experimental Procedures.” The DL-

camitine concentration was varied from 0 to 1.4 mM. Analyses were in triplicate; mean values are shown. O-O, control; O-O, plus 300 +LM CaCl, (60 nmol of Ca2+/mg of protein).

in both ketone body production and the NADH:NAD+ ratio, reaching a maximum difference from the control at 40 pM

CoA. At concentrations of CoA above 40 PM the control rate of ketone body production and the control NADH:NAD+ ratio also increased. This was presumably the result of increased palmitoyl-CoA content and the resultant inhibition of adenine nucleotide translocation (25-29). In order to avoid inhibitory concentrations of CoA, 20 pM was chosen for this study.

Relation between Ca2+-induced Changes and Ca2+ Concen- tration- Ca2+ at low concentrations (0 to 100 pM) decreased ketone body production slightly (Fig. 3A 1. This was paralleled by a decrease in the ATP content of the system (this measure- ment represents total ATP in the system), which suggests the activation of some energy-utilizing reaction. As the Ca2+ concentration was increased above 100 pM the reverse oc-

COENZYME A (uM)

FIG. 2. Effect of coenzyme A concentration on the Ca*+-dependent elevation of (A) total ketone body production and (B) the P-hydrox- ybutyrate:acetoacetate ratio. Mitochondria (11.63 mg of protein) were incubated with 0.75 rnM palmitate-1.5% albumin for 15 min at 37” as described under “Experimental Procedures.” The coenxyme A concentration was varied from 0 to 100 ELM. Analyses were in triplicate; mean values are shown. O-O, control; O-0, plus 400 PM CaCl, (69 nmol of Ca*+/mg of protein).

I 1

CALCIUM CHLORIDE (mM)

FIG. 3. Effect of CaZ+ concentration on (A) ketone body produc- tion and ATP content and (B) the P-hydroxybutyrate:acetoacetate ratio. Mitochondria (6.4 mg of protein) were incubated with 0.75 mM palmitate-1.5% albumin for 15 min at 37” as described under “Experimental Procedures.” CaCl* was added at the beginning of the incubation period at final concentrations of 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1.0, 1.5, and 2.0 mM, corresponding to 0, 15.6, 31.3, 62.5, 93.8, 125, 156, 234, 313, 469, and 625 nmol of Ca*+/mg of protein. Analyses were in triplicate; mean values are shown. O-O, total ketone body production in micromoles; O-O, ATP (micromoles); A-A, B-hydroxybutyrate to acetoacetate ratio.

by guest on September 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Ca2+, Fatty Acid Oxidation, and Mitochondrial Energy State 793

cm-red, a marked elevation in the rate of ketogenesis was observed, corresponding to a 750% increase at 750 pM CaCl,. This was accompanied by an increase in the ATP content, which attained a level slightly higher than in the absence of Ca*+. At Ca*+ concentrations of 1 mM and greater, ketogenesis relative to the maximum rate was progressively suppressed along with a complete exhaustion of ATP. The P-hydroxybu- tyrate:acetoacetate ratio, which was measured in the same experiment (Fig. 3B), was greatly affected by the Ca2+ concen- tration. This ratio increased at the same Ca2+ level at which ketogenesis was first elevated, namely 200 PM CaCl,. The maximum effect of Ca2+ on the ratio was observed at 400 pM

CaCI,. Further elevation of the Ca2+ concentration caused the P-hydroxybutyrate:acetoacetate ratio to decline. This decline occurred at a Caz+ concentration much lower than that which depressed total ketone body production.

In related experiments 200 pM and 400 pM Ca*+ (82 and 163 nmol/mg of protein) increased the steady state rate of oxygen consumption after 15 min of incubation 31% and 41%, respec- tively. Ketogenesis and the P-hydroxybutyrate:acetoacetate ratio were sequentially increased; the ATP content was main- tained. Higher concentrations of Ca2+, 600 pM and 800 pM, inhibited O2 consumption 18% and 30%, respectively, as keto- genesis, the P-hydroxybutyrate:acetoacetate ratio, and ATP content declined.

Since the Ca2+-dependent elevations of ketone body synthe- sis and the P-hydroxybutyrate:acetoacetate ratio are a func- tion of Ca*+ concentration, as shown in Fig. 3, and since Ca2+ is actively accumulated by mitochondria, the Ca2+ concentra- tion required to produce a half-maximal response as a function of the mitochondrial protein concentration was examined. This is demonstrated in Fig. 4. It is clear that higher concen- trations of Ca2+ were needed to produce a half-maximal increase in the rate of ketone body production and the p- hydroxybutyrate:acetacetate ratio as the protein concentra- tion was increased. It was calculated from these data, and from data obtained in other experiments of this design (not shown), that the Ca2+ concentration required to produce a half-maximal response is 82.4 ? 7.5 nmol of Ca’+/mg of

CALCIUM CHLORIDE (mhl) FIG. 4. Effect of mitochondrial protein concentration on the Ca*+-

dependent elevation of (A) ketone body production and (23) the p- hydroxybutyrate to acetoacetate ratio. Mitochondria at three differ- ent protein concentrations were incubated with 0.75 rnM palmitate- 1.5% albumin for 15 min at 37” as described under “Experimental Procedures.” CaCI, was added at the beginning of the incubation period at final concentrations from 0 to 1.5 mM. Analyses were in triplicate; mean values are shown. O-0, 6.8 mg of protein; A-A, 5.3 mg of protein; U--m, 3.9 mg of protein.

protein for stimulation of ketone body production and 84.7 * 6.3 nmol of Ca2+/mg of protein for elevation of the p-hydroxy- butyrate:acetoacetate ratio (mean f S.E., n = 12).

Mitochondrial CW-+ Transport - It is generally accepted that Ca2+ uptake into isolated liver mitochondria is rapid, in the presence of an adequate energy source, Mg2+, and a permeant anion such as phosphate (5). The incubation system used in our experiments contains (among other additions reported under “Experimental Procedures”) 0.75 mM palmi- tate, 0.45 mM ATP, 5 mM MgCl,, and 10 mM phosphate; therefore, one would expect mitochondrial Ca*+ accumulation. In order to define the rate of Ca2+ transport under these experimental conditions, Wa2+ uptake was measured under identical conditions used throughout this study (Fig. 5). When 360 PM Ca*+ was added, CaZ+ (101 nmol/mg of protein) was accumulated almost completely by 1.5 min and was main- tained within the mitochondria for at least 20 min (the last time point measured).

When 1 pM ruthenium red, a Ca*+ transport inhibitor (30, 31), was present in the incubation system from the start of the reaction (Table III), Ca2+ did not stimulate ketone body production or elevate the p-hydroxybutyrate to acetoacetate ratio. A study of Ca2+ uptake with this concentration of ruthenium red showed that the mitochondria were still able to slowly accumulate approximately 1.7 nmol of Ca2+/min/mg of protein at a constant rate (~5% of the uninhibited rate in these experiments), reaching 25 nmol of Ca*+/mg of protein by 15 min of incubation (figure not shown). This level of Ca*+ was shown in Fig. 3 to be ineffective in elevating ketone body production or the P-hydroxybutyrate:acetoacetate ratio. Ru- thenium red (1 PM) completely inhibited both the initial stimulation of respiration due to Ca2+ transport and the stimulation of the steady state respiration described above. However, the local anesthetic procaine (400 PM), which blocks

MINUTES FIG. 5. Rate of Ca2+ uptake. Mitochondria (7.1 mg of protein)

were incubated with 0.75 rnM palmitate-1.5% albumin at 37” as described under “Experimental Procedures.” WaCl, (360 PM) was added after 1 min of preincubation to reach temperature equilibra- tion. Ca*+ uptake was determined by the filtration method. Both the Wa*+ remaining in the filtrate and on the Millipore filter were measured. O-O, intramitochondrial Ca*+ on filter; O-O, extramitochondrial Ca*+ in filtrate.

by guest on September 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

794 Ca2+ , Fatty Acid Oxidation, and Mitochondrial Energy State

the binding of divalent cations to low affinity phospholipid sites (321, was totally ineffective in preventing the Ca*+- induced changes (Table III). Similar results were obtained with 400 pM butacaine (data not shown).

Rate of Ca2+-dependent Elevation ofFatty Acid Oxidation - A time study of the Ca’+-dependent changes in ketogenesis, the /3-hydroxybutyrate to acetoacetate ratio, and the ATP concentration show (Fig. 6) that the rate of ketogenesis (Fig. 6A) was stimulated at the point of Ca*+ addition. This elevated rate of ketogenesis continued throughout the entire period of incubation with only a slight decrease after 2 min. The p- hydroxybutyrate to acetoacetate ratio (Fig. 6B) was not ele- vated until 2 min after Ca2+ addition, which corresponds to completion of the initial energy-dependent Caz+ uptake (Fig. 5). Subsequent to this period of Caz+ accumulation, the /3- hydroxybutyrate:acetoacetate ratio showed a sharp increase over the control, reaching near maximum elevation after 10 min. Therefore, at 15 min (the incubation time used in most of these experiments), the mitochondrial NADH:NAD+ ratio was near the steady state.

Caz+ addition decreased the ATP concentration (Fig. EC) below the control level during the period of rapid uptake of Ca*+ (Fig. 5). After this initial drop, the ATP level in the presence of Ca2+ rapidly exceeded the control values. The elevated ATP concentration in the presence of Ca2+ was

maintained for the remainder of the incubation period. The degree of elevation of the ATP concentration by Ca2+ was found to be related to the variable energy state of the control system. It was observed in various experiments that when the P-hydroxybutyrate:acetoacetate ratio in the control sys- tem was less than about 0.5, the ATP:ADP ratio was de- pressed, relative to the Ca*+-supplemented system. However, in some preparations of mitochondria, in which the control p- hydroxybutyrat.e:acetacetate ratio was higher, the phospho- rylation state of the adenine nucleotide system was main- tained near maximum.

When ruthenium red was added after the completion of Ca2+ uptake (at 5 min) there was a slight inhibition of the Ca*+-induced elevation in ketone body production (Fig. 6A) which continued for the remainder of the 20-min incubation period; ruthenium red did not affect the control values. A similar alteration of low magnitude was seen in the Ca2+- dependent elevation of the P-hydroxybutyrate:acetoacetate ratio (Fig. 6B); ruthenium red added at 5 min retarded the increase in the ratio. The elevation in the ATP content by Ca2+ was slightly lowered by ruthenium red (Fig. 6C). We also observed that 1 PM ruthenium red, when added at 5 min, only partially blocks (-20% inhibition) the Ca2+-stimulated rate of oxygen consumption measured at 15 min.

Effect of Extramitochondrial pH on Ca2+-dependent Ef-

TABLE III

Effects of ruthenium red and procaine on Ca 2+-dependent stimulation of ketone body production and elevation of /3 hydrorybutyrate:acetoacetate ratio

Mitochondria (Experiment 1, 7.3 mg of protein; Experiment 2, 6.8 mg of protein) were incubated with 0.75 rnM palmitate-1.5% albumin as described under “Experimental Procedures.” Ketone bodies were measured after 15 min of incubation at 37”. Values are the means of three determinations 2 S.E.

Ketone body production Ratio of P-hydroxybutyrate to acetoacetate

nmol nm01

Experiment 1 Control 667 lr 47 1045 + 12 <O.OOl +Ruthenium red (1 PM) 734 2 36 672 k 28 <N.S.”

Experiment 2 Control 514 k 29 1058 k 32 <O.OOl +Procaine (400 I.LM) 546 + 5 1104 k 15 <O.OOl

1.41 k 0.16 6.10 k 0.35 <O.OOl 1.48 ? 0.24 1.55 * 0.21 N.S.

0.95 5 0.02 4.79 + 0.02 <O.OOl 0.74 2 0.01 4.34 2 0.05 <O.OOl

o N.S., not significant.

MINUTES MINUTES

I 6 10 15 20

MINUTES

FIG. 6. Rate of the Ca2+-dependent elevation of (A) total ketone preincubation. Mean values of duplicate determinations are shown. body production, (B) P-hydroxybutyrate:acetoacetate ratio, and (C) O-O, control; A-A, plus 1 FM ruthenium red added aft.er 5 ATP content; effects of ruthenium red when added after the comple- min of incubation; O-O, plus 400 ELM CaCIZ (101 nmol of Ca*+/mg tion of Cay+ uptake. Mitochondria (7.9 mg of protein) were incubated of protein); A----A, plus 400 pM CaCl, and 1 ELM ruthenium red with 0.75 mM palmitate-1.5% albumin at 37” for the respective added after 5 min of incubation. times as described under “Experimental Procedures” after 5 min of

by guest on September 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Ca2+, Fatty Acid Oxidation, and Mitochondrial Energy State 795

fects- In the preliminary stages of this study it became apparent that the incubation pH was a critical factor in the demonstration of the above effects of Ca2+ on fatty acid oxidation and the &hydroxybutyrate:acetoacetate ratio. Therefore, the pH dependence was investigated (Fig. 7). The apparent pH optimum in the presence of 300 pM Ca2+ (60 nmol/mg of protein) was approximately 6.9. The effectiveness of Ca2+ decreased rapidly on either side of this optimum pH, having no effect below pH 6.6 and above pH 7.5. There was a direct relationship, both in the presence and absence of Ca2+ ions, between the level of ketone body production (Fig. 7A), the P-hydroxybutyrate:acetoacetate ratio (Fig. 7B), and the level of ATP in the system (Fig. 7C).

Additional studies revealed that the Ca2+ concentration directly determined the pH optimum. As the Ca*+ concentra- tion was decreased, the optimum pH likewise decreased. This relationship was investigated by Ca2+ concentration depend- ence experiments as a function of the pH. We found that lowering the incubation pH shifted the complete Ca2+ concen- tration dependence curve (the shape of the curve is illustrated in Fig. 3) toward the lower Ca2+ concentration range without changing the magnitude of the maximal response. Increasing the pH of the medium caused a shift in the opposite direction. These data, which show the Ca2+ concentration necessary for a half-maximal increase in the rate of ketogenesis and the p- hydroxybutyrate:acetoacetate ratio as a function of incubation pH, are summarized in Table IV. At more acidic pH values, the mitochondria appear to be more sensitive to CaZ+ with respect to elevation of fatty acid oxidation and mitochondrial pyridine nucleotide oxidation-reduction state. Therefore, the Ca2+ concentration required for a half-maximal response is a function of the extramitochondrial pH, as well as the mito- chondrial protein concentration. Results similar to those of Table IV were obtained when the medium chain fatty acid substrate octanoate (in the absence of albumin, CoA, dithio- threitol, and camitine) was used in place of palmitate.

Znvestigation of ATP and Mg2+ Requirements - During the development of the incubation conditions in the early stages of this study, ATP was routinely included in the incubation system. It was reasoned that since Ca2+ uptake was energy- dependent, this process might effectively compete for the energy necessary to activate the fatty acid substrate for

6.5 1.0 1.5 8.0 8.5 z

PH 9;

oxidation and thus inhibit fatty acid oxidation, instead of stimulating this process. This was supported by our finding that Ca*+ was totally ineffective in elevating ketone body production or the P-hydroxybutyrate:acetoacetate ratio in the absence of added ATP. In fact Ca’+ addition caused a total depletion of endogenous ATP. In order to explore the effects of Ca2+ on fatty acid oxidation and the energy state, 0.45 mM ATP was chosen for the incubation system. An ATP concentra- tion in this range was shown to optimally support ATP-driven Ca2+ transport in the presence of phosphate and Mg2+ (33).

In studying the effects of Ca2+, especially the concentration dependence shown in Fig. 3, the problem of Ca*+ complexes must be considered. The CaATP*- complex has a relatively high stability constant, 3.2 x lo4 M-’ (34). Therefore, the effect of ATP concentration in relation to the Ca*+ concentra- tion dependence was investigated. It was found that at 2.25 mM ATP and when the Ca2+ concentrations were 0, 50, 100, 200, 300, 400, 500, 750, 1000, 1500, and 2000 PM, rates of ketone body production were 0.55, 0.55, 0.38, 0.38, 0.61, 0.95, 1.18, 1.31, 1.36, 1.07, and 0.58 pmol, respectively (as compared to values from Fig. 3 with 0.45 mM ATP in the same experi-

TABLE IV

Effect ofpH on Caz+ concentration required to half-maximally elevate ketone body production and &hydroxybutyrate:acetoacetate

ratio

Mitochondria (5.0 mg of protein) were incubated with 0.75 rn~ palmitate-1.5% albumin for 15 min at 37” as described under “Exper- imental Procedures.” The effects of [Ca*+l (tested at 0, 10, 25, 50, 100, 200, 400, 500, 600, 800, and 1000 pM) on ketogenesis and the p- hydroxybutyrate to acetoacetate ratio were measured at the differ- ent incubation pH values. Analyses were in duplicate. The [Ca2+l 112 mm values were,determined from the respective Ca2+ con- centration curves.

PH

6.6 6.9 7.1 7.3 7.6

ICa*+l,,2 ma

Ketone b&z produc- Ratio of @hydroxybu- tyrate to aeetoacetat.?

(nmol Cd+lmg protein)

10 5 48 35 73 68

113 103 160 158

PH FIG. 7. Effect of extramitochondrial pH on the Ca’+-dependent for 15 min at 37” as described under “Experimental Procedures”

changes in (A) total ketone body production, (B) P-hydroxybutyr- except the pH of the incubation medium was varied from 6.2 to 8.5. ate:acetoacetate ratio, and (C) ATP content. Mitochondria (10.0 mg Analyses were in triplicate; mean values are shown. O-O, of protein) were incubated with 0.75 mM palmitate-1.5% albumin control; O-O, plus 300 PM CaCI, (60 nmol of Caz+/mg of protein).

by guest on September 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

796 Ca2+ > Fatty Acid Oxidation, and Mitochondrial Energy State

ment). The corresponding P-hydroxybutyrate:acetoacetate ra- tios were 0.64, 0.71, 0.12, 0.10, 0.25, 1.06, 2.80, 2.99, 1.57, 0.33, and 0.23, respectively. These maximum responses occur at higher Ca2+ concentrations than observed at the lower ATP concentration (0.45 mM, Fig. 3). Entman et al. (35) demon- strated that a ratio of Mg*+ to ATP of about 3:l is required for optimal Ca2+-stimulated ATPase activity in the sarcoplasmic reticulum. A Mg*+:ATP ratio of at least this value is required to decrease the CaATP2- complex. (The stability constant for the MgATP*- complex (7.3 x lo4 M-V is over 2-fold higher than for the CaATP2- complex (34).) In our system the Mg2+ concentration was 5 mm. With 0.45 mM ATP the Mg’+:ATP ratio is 11:l; therefore, complexing of Ca2+ by ATP should not be significant. However, at 2.25 mM ATP the Mg*+:ATP ratio is 2.5; under these conditions some of the Ca*+ is complexed by ATP and therefore is not available to interact with the mitochondria. This explains the shift observed with 2.25 mM ATP, as compared to the curves of Fig. 3, toward higher Ca2+ concentrations. It was concluded that minimum but adequate amounts of ATP (0.45 mM ATP) should be used in the present study to support both CaZ+ uptake and fatty acid oxidation.

When Mg*+ was omitted from the incubation medium Ca2+ had no stimulatory effect on either the rate of ketone body production or the elevation of the P-hydroxybutyrate: acetoacetate ratio. Ca*+ concentrations from 200 to 800 pM

were investigated, especially in light of the increased problem of CaATP*- complex formation in the absence of Mg2+.

DISCUSSION

The present experiments clearly demonstrate that Ca*+ increased the rate of long chain fatty acid oxidation by rat liver mitochondria (Table I), far in excess of that required to support Ca *+ transport (Table II). Of special significance is the finding that Ca’+ also elevated the p-hydroxy- butyrate:acetoacetat ratio. The observed inhibition of CO, production by Ca’+ may be explained by the known involve- ment of the mitochondrial NADH:NAD+ ratio in the regula- tion of the citric acid cycle (36-43). The calculations in Table II indicate that the NADH produced via the Ca?+-dependent increase in fatty acid oxidation was responsible for the rise in the NADH:NAD+ ratio. Additional evidence in support of this point is provided by the dependence of the Ca*+-induced reduction of NAD+ upon the cofactors (carnitine and CoA) of fatty acid oxidation (Figs. 1 and 2).

We previously found that the Ca”+-dependent elevation of [l-“Vlpalmitate oxidation by mitochondria was accompanied by a decrease in [l-‘4C]palmitoyl-carnitine content (44) indi- cating the action of Ca2+ at some site beyond acylcarnitine formation. We also reported (44) that, as observed with palmitate, Ca”+ stimulated oxidation of the medium chain fatty acid octanoate (in the absence of CoA, carnitine, and albumin) and concurrently increased the mitochondrial NADH:NAD+ ratio. Unlike long chain fatty acids, medium chain fatty acids traverse the inner mitochondrial membrane in a carnitine-independent process and are activated within the matrix compartment (45, 46). Our results with palmitate and octanoate, therefore, suggest that Caz+ is involved in regulating the rate of P-oxidation.

Even though long and medium chain fatty acids enter the mitochondria by different means, it remains possible that CaZ+ activates fatty acid oxidation by stimulating the uptake of both these fatty acids through the mechanism proposed by Levitsky and Skulachev (47). They postulated that when an

electrochemical potential, consisting of a transmembrane elec- trical potential (AUr) and a chemical potential (ApH), is imposed upon the inner mitochondrial membrane as a result of electron transport (48-501, the protonated (cationic) form of acylcarnitine is drawn from the positively charged (acidic) outer compartment into the negatively charged (alkaline) inner compartment. The Ca 2+ transport-induced ejection of H+ (6, 51-55) and alkalinization of the inner membrane- matrix (54, 56, 57) may facilitate the protonation of long chain acylcarnitine and increase the driving force for the inward movement of this cationic species. Protonation of octanoate could increase its uptake through the hydrophobic regions of the inner mitochondrial membrane, as well as promote the movement of the substrate into the alkaline matrix compartment,

Since albumin was not used in the earlier experiments with octanoate (44), the similar effects of Ca2+ with octanoate and palmitate, as well as the effect of Ca2+ on the palmitoyl- carnitine content (44), oppose an involvement of Ca2+ with the palmitate-albumin complex in the observed increases in fatty acid oxidation and the mitochondrial NADH:NAD+ ra- tio. Studies in our laboratory, utilizing the equilibrium parti- tion method described by Spector et al. (581, indicate no apparent effect of Ca2+ on the binding of palmitate to bovine serum albumin in the range of fatty acid/albumin molar ratios employed in these experiments.

The parallel increases in the rate of ketogenesis (Fig. 3A), the p-hydroxybutyrate:acetoacetate ratio (Fig. 3B), the ATP concentration (Fig. 3A), and the steady state rate of oxygen consumption (“Results”) with increasing Ca2+ concentrations up to 400 PM are consistent with the proposal that the primary action of Ca 2+ is the stimulation of fatty acid oxida- tion. Such an effect would result in a greater rate of NADH production via P-oxidation, causing an increase in the mito- chondrial NADH:NAD+ ratio. The consequent increase in the gradient of reducing equivalents fi-om NADH dehydrogenase to cytochrome oxidase provides greater rates of respiration and ATP synthesis. The Ca2+-dependent increase in fatty acid oxidation is not just a consequence of the additional energy demand imposed by Ca 2+ transport, since such a response should be accompanied by an oxidation of the pyridine nucleo- tides and a decrease in the concentration of ATP, whereas the opposite was observed, the calculations in Table II also oppose this proposal.

At 0.75 mM Ca*+ (Fig. 3), the ratio of Ca2+:mitochondrial protein was approaching the range of “massive loading” con- ditions, which causes irreversible functional and structural damage to the mitochondria (5). The loss of respiratory control under these conditions (5) explains the decrease in the p- hydroxybutyrate:acetoacetate ratio at high Ca2+ concentration (Fig. 3B). The parallel changes in fatty acid oxidation may be the result of decreased ATP content or defect(s) consequent to the loss of membrane integrity. Whichever the case, a de- crease in the steady state rate of oxygen consumption (“Re- suits”) would be expected since fatty acids are the major source of reducing equivalents in this system.

That Ca2+ was actively accumulated in this mitochondrial system is clear from the results shown in Fig. 5. Prevention of the Ca2+-dependent effects by the Ca*+ transport inhibitor ruthenium red (Table III) firmly supports the concept that the uptake and/or presence of Ca2+ within the mitochondria is prerequisite to effects of Ca2+ on the rate of fatty acid oxidation and on the mitochondrial NADH:NAD+ ratio. Re- sults with procaine (Table III) eliminate the possible media-

by guest on September 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Ca2+, Fatty Acid Oxidation, and Mitochondrial Energy State 797

tion of these effects by the binding of Ca2+ to low affinity phospholipid sites of the inner mitochondrial membrane (32).

It is clear from the data shown in Fig. 6 that the mecha- nisms by which Ca2+ is initially accumulated and by which it stimulates fatty acid oxidation are not directly related, since the stimulation of fatty acid oxidation continues after the completion of Ca2+ accumulation. It remains possible, how- ever, that a by-product of CaZ+ transport, for example aciditi- cation of the extramitochondrial medium via Ca2+ transport- dependent H+ ejection (6, 51-Xi), is linked to the stimulation of fatty acid oxidation.

The ruthenium red treatment (Fig. 6) was employed to test another possibility, that following initial uptake the slow energy-dependent accumulation of Ca2+, adjunct to the con- stant efllux of Ca2+ (51), may be related to the effects on fatty acid oxidation. Stucki and Ineichen (59) showed that ruthe- nium red, added to mitochondria afIer the accumulation of Ca*+, inhibited the reaccumulation of Ca2+ by mitochondria but did not prevent the continual efflux. The continued Ca2+- dependent elevation of fatty acid oxidation after ruthenium red addition, Fig. 6, demonstrates that the effect of Ca2+ on fatty acid oxidation was not a direct response to the slow energy-requiring process of Ca2+ recycling. The data indicate, therefore, that the presence of Ca*+ within the mitochondria or some by-product of the initial transport of Ca2+ was re- quired.

Since the concentration of ruthenium red used in these studies completely blocked the initial Ca2+-dependent stimu- lation of respiration (“Results”), ruthenium red added after the completion of Ca2+ uptake would be expected to block any increase in the steady state respiration associated with the constant et&x and reaccumulation of Ca2+. The observation that ruthenium red only slightly blocked the Ca*+-dependent increase in steady state respiration (“Results”) implies that only a small part of the increase in respiration was caused by the constant reaccumulation of Ca2+. It follows, therefore, that the major portion of the elevated state 4 respiration was a result of the Ca2+-dependent stimulation of fatty acid oxida- tion and the consequent increase in NADH production and associated ATP synthesis.

It is known that decreasing the pH decreases the binding of Ca2+ to low affinity phospholipid sites on the mitochondrial membrane (32, 60). This might explain the more effective action of Ca2+ at lower pH (Table IV), i.e. increasing the amount of divalent cation available at the lower pH for the stimulation of fatty acid oxidation. However, there are not enough low affinity binding sites (61) to account for the changes shown in Table IV. Also, the pH range investigated is actually above the pK, of most of the sites.

In addition to increasing the sensitivity of the mitochondria to Ca*+ with respect to stimulating fatty acid oxidation, decreased pH also elevated the control rate of ketone body production, the /%hydroxybutyrate:acetoacetate ratio, and ATP concentration (Fig. 7). The data from Fig. 7 and Table IV taken together, imply that endogenous Ca2+ may become increasingly more effective as the pH is decreased, but it is equally possible that decreased pH acts similar to, or is involved in, the mechanism of Ca2+-stimulated fatty acid oxidation in such a manner that the two effects are additive or synergistic. This implicates the Ca2+ transport-induced ejection of H+ (6, 51-55) in the effect of CaZ+ on fatty acid oxidation, a possibility discussed above.

Consequent to CaZ+ accumulation, interaction of Ca2+ and phosphate in the alkaline mitochondrial matrix may decrease

the concentration of inorganic phosphate in this compartment via calcium phosphate formation and thereby contribute to the development of an energized state by increasing the [ATFWATPI [Pi].

The physiological concentration of Ca*+ within the mito- chondria is not accurately known; however, our measure- ments together with reports in the literature suggest it may be between 5 and 20 nmol/mg of protein. Also, the cytosolic pH is probably relatively more acidic than that within the mitochondrial matrix due to the H+ ejection associated with electron transport (48-50). Therefore, it is reasonable to sug- gest from the data of Table IV that these Ca2+-dependent effects may be demonstrated at the physiological concentra- tion of Ca2+.

Our observation that Mg2+ is required for the effects of Ca2+ on fatty acid oxidation is not surprising, since Mg*+ is required for the activation of fatty acids and for the uptake of Ca*+. Neither is it surprising to find that ATP is necessary for these effects of Ca*+. Fatty acid oxidation requires initial conversion of the substrate to the CoA ester at the expense of ATP. The endogenous ATP would be insufficient for maxi- mum acyl-CoA formation; in addition Ca*+ transport utilizes endogenous ATP preferentially (62), depleting it before utiliz- ing the energy derived from substrate oxidation. We did find, however, that ATP can be totally replaced by ADP, providing similar elevations of ketogenesis and the NADH:NAD+ ratio by Ca2+. Added ADP was recovered almost completely as ATP at the end of the 15-min incubation period in both the control system and in the presence of Ca2+, indicating opera- tional coupling of oxidation and phosphorylation. It appears, therefore, that the size of the adenine nucleotide pool is important to the degree that it allows for potential ATP synthesis to support both Ca2+ transport and acyl-CoA forma- tion.

The data indicate that Ca2+ stimulates fatty acid oxidation in excess of the energy requirements of the in vitro system, generating a more energized state. The data support a se- quence of events in which activation of fatty acid oxidation is the primary site of Ca*+ action, resulting in the greater rate of reducing equivalent production, and consequently a higher steady state rate of oxygen consumption and ATP synthesis. Ca2+ accumulation is a prerequisite. The exact mechanism of the Ca2+ effect on fatty acid oxidation is not known. We suggest alternative hypotheses, that Ca*+ activates fatty acid oxidation (a) directly through specific interactions of Ca2+ with an enzyme(s) of P-oxidation, (5) indirectly through actions of Ca2+ on the respiratory chain or on mitochondrial structure (limitation of fatty acid oxidation by the respiratory chain (63) and influence of mitochondrial structure on acyl- carnitine oxidation (64) have been presented), or (c) by in- creasing the uptake of both long and medium chain fatty acids into the mitochondria by similar mechansims. In support of an indirect action of Ca2+ on fatty acid oxidation, we have observed with electron microscopy that after 15 min of incu- bation with palmitate-albumin plus Ca2+ the mitochondria were in the orthodox state as compared to the condensed state of the control (minus Ca*+) mitochondria. This ultrastructural change was accompanied by an elevation of ketone body production and the /3-hydroxybutyrate:acetoacetate ratio, thus indicating a structure/function relationship. Similar Cal+-in- duced morphological changes have been observed by others (65-67). It is equally possible however, that the observed transition to the orthodox state may be the result of the energization of the mitochondria via fatty acid oxidation

by guest on September 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

798 Ca2+, Fatty Acid Oxidation, and Mitochondrial Energy State

rather than the cause of the increased oxidation. The similarity between the present results with isolated

mitochondria and our earlier observations with isolated liver cells (1, 2, 44) suggest a physiological correlate te our in vitro findings. Hormone-induced redistribution of Ca2+ (68-72) may regulate the rate of fatty acid oxidation during different metabolic states, and thereby influence the partition of fatty acids between the pathways of oxidation and esterification (12, 44, 73, 74). Thus, Ca2+ may be involved in the ketogenic actions of glucagon (44, 75-79) and insulin deficiency (79-81). The present findings might also explain the stimulation of gluconeogenesis in rat liver cell systems by Ca*+ (82) as well as the Ca’+-dependent stimulation by glucagon and cyclic AMP (83, 841, with enhanced fatty acid oxidation providing an elevated mitochondrial NADH:NAD+ ratio and the energy required for glucose synthesis. Further investigations on the molecular mechanisms of Ca2+ action in liver cell and mito- chondrial systems are in progress.

Acknowledgments -The technical assistance of Anna Hen- ley, Siu-Big Leung, and Janet Reich is gratefully acknowl- edged.

REFERENCES

1.

2.

3.

4.

5.

6. 7.

8. 9.

10.

11.

12. 13.

14. 15. 16. 17.

18.

19.

20.

21.

22.

23.

24. 25.

26.

Otto, D. A., and Ontko, J. A. (1974) B&hen. Biophys. Res. Commun. 61, 743-750

Ontko, J. A., Otto, D. A., Oshino, N., and Chance, B. (1975) FEBS Lett. 53, 297-301

Mellanby, J., and Williamson, D. H. (1963) Biochem. J. 88, 440- 444

Streffer, C., and Williamson, D. H. (1965) Biochem. J. 95, 552- 560

Lehnineer. A. L.. Carafoli. E.. and Rossi. C. S. (1967) Ado. Enzymoi. 29, 259-320 ’ ’

Chance. B. (1965) J. Biol. Chem. 240. 2729-2748 Dubinsky, W. P., and Cockrell, R. S: (1975) FEBS Lett. 59, 39-

43 Otto, D. A., and Ontko, J. A. (1976) Fed. Proc. 35, 1502 Mellanby, J., and Williamson, D. H. (1974) in Methods of

Enzymatic Analysis (Bergmeyer, H. II., ed) 2nd English Ed, pp. 1840-1843, Academic Press, New York

Williamson, D. H., and Mellanby, J. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. II., ed) 2nd English Ed, UP. 1836-1839. Academic Press. New York

Lamprecht, W.,. and Trautschold, I. (1974) in Methods of Envy- matic Analysis (Bergmeyer, H. U., ed) 2nd English Ed, pp. 2101-2109, Academic Press, New York

Ontko, J. A. (1972) J. Biol. Chem. 247, 1788-1800 Folch, J.. Lees, M.. and Sloane Stanlev. G. H. (1957) J. Biol.

Chem. i26, 497-569 --

Estabrook, R. W. (1967) Methods Enzymol. 10. 41-47 Reed, K. C., and Bygrave, F. L. (1974) B&hem. J. 140,143-155 Ontko, J. A. (1970) J. Lipid Res. 11, 367-375 Fletcher, J. M., GreenReId, B. F., Hardy, C. J., Scargill, D.,

and Woodhead, J. L. (1961) J. Chem. Sot. London, 2000-2006 Williamson, J. R., and Corkey, B. E. (19691 Methods Enzymol.

13, 434-513 Klingenberg, M. (1974) in Methods ofEnzymatic Analysis (Berg-

meyer, H. U., ed) 2nd English Ed, pp. 20452059, Academic Press, New York

Pieterson. W. A.. Volwerk. J. J.. and de Haas. G. H. (1974) Biochemistry 13, 1439-1445 ’

Garcia. A., Newkirk. J. D.. and Mavis. R. D. (1975) Biochem. Biophys.‘Res. Corn&n. 64, 128-135

Williamson, D. H., Lund, P., and Krebs, H. A. (1967) B&hem. J. 103, 514-527

Winer, A. D., Schwert, G. W., and Millar, D. B. S. (1959) J. Biol. Chem. 234. 1149-1154

Raval, D. N., andwolfe, R. C. (1962) Biochemistry 1, 263-269 Shug, A. L., Lerner, E., Elson, C., and Shrago, E. (19711

B&hem. Biophys. Res. Commun. 43, 557-563 McLean, P., Gumaa, K. A., and Greenbaum, A. L. (1971) FEBS

Lett. 17, 345-350 67.

27. Lenartowicz, E., Winter, C., Kunz, W., and Wojtczak, A. B. (1976) Eur. J. Biochem. 67, 137-144

28. Vaartjes, W. J., Lopes-Cardoso, M., and van den Bergh, S. G. (1972) FEBS Lett. 26, 117-122

29. Wilson, D. F., Erecinska, M., and Dutton, P. L. (1974) Annu. Rev. Biophys. Bioeng. 3, 203-230

30. Moore, C. L. (1971) B&hem. Biophys. Res. Commun. 42, 298- 305

31. Vasington, F. D.. Gazzotti, P., Tiozzo. R., and Carafoli, E.

32.

33

34.

35.

36

37. 38.

39.

(1972) Biochim. Biophys. Acta’256, 43-54 Scarpa, A., and Azzi, A. (1968) B&him. Biophys. Acta 150,

473-481 Bielawski, J., and Lehninger, A. L. (1966) J. Biol. Chem. 241,

4316-4322 O’Sullivan, W. J., and Perrin, D. D. (1964) Biochemistry 3, 18-

26 Entman, M. L., Snow, T. R., Freed, D., and Schwartz, A. (1973)

J. Biol. Chem. 248, 7762-7772 Smith, C. M., B&a, J., and Williamson, J. R. (1974) J. Biol.

Chem. 249. 1497-1505 Ontko, J. A. (1973) J. Lipid Res. 14, 78-86 Safer, B., and Williamson, J. R. (1973) J. Biol. Chem. 248,

2570-257s

40.

41.

42.

Ontko, J. A. (1974) in Alcohol and Aldehyde Metabolizing Systems (Thurman, R. G., Yonetani, T., Williamson, J. R., and Chance, B., eds) pp. 299-314, Academic Press, New York

Hansford, R. G., and Johnson, R. N. (1975) J. Biol. Chem. 250, 8361-8375

LaNoue, K. F., Walajtys, E. I., and Williamson, J. R. (1973) J. Biol. Chem. 248, 7171-7183

Lopes-Cardozo, M., Mulder, I., van Vugt, F., Hermans, P. G. C., and van den Bergh, S. G. (1975) Mol. Cell. Biochem. 9, 155-173

43.

44.

45. 46. 47.

46.

49.

Estabrook, R. W., and Holowinsky, A. (1961) J. Biophys. Bio- them. Cytol. 9, 19-28

Ontko, J. A., and Otto, D. A. (1978) inBiochemical and Clinical Aspects of Ketone Body Metabolism (Soling, H. D., and Ssu- fert, C. D., eds) Goorg Thieme Verlag, Stuttgart, in press

Aas, M. (1971) Biochim. Biophys. Acta 231, 32-47 Van Tol, A. (1975) Mol. Cell. Biochem. 7, 19-31 Levitsky, D. O., and Skulachev, V. P. (1972) Biochim. Biophys.

Acta 275.33-50 Mitchell, P. (19681 Chemiosmotic Coupling and Energy Trans-

duction, Glynn Research Ltd., Bodmin Skulaehev, V. P. (1971) in Current Topics in Bioenergetics

(Sanadi, D. R., ed) Vol. 4, pp. 127-190, Academic Press, New York

50.

51.

52.

53.

Chance, B., Lee, C. P., and Mela, L. (19671 Fed. Proc. 26, 1341- 1354

54.

55. 56.

57. 58.

59.

Drahota, Z., Carafoli, E., Rossi, C. S., Gamble, R. L., and Lehninger, A. L. (1965) J. Biol. Chem. 240, 2712-2720

Rasmussen, H., Chance, B., and Ogata, E. (1965) Proc. Natl. Acad. Sci. U. S. A. 53, 1069-1076

Chappell, J. B., Cohn, M., and Greville, G. D. (1963) in Energy- linked Functions of Mitochondria (Chance, B., ed) pp. 219- 231, Academic Press, New York

Rossi, C. S., Bielawski, J., and Lohninger, A. L. (1966) J. Biol. Chem. 241, 1919-1921

Wenner, C. E. (1966) J. Biol. Chem. 241, 2810-2819 Gear, A. R. L., Rossi, C. S., Reynafarje, B., and Lehninger, A.

L. (1967) J. Biol. Chem. 242, 3403-3413 Chance, B., and Mela, L. (1966) J. Biol. Chem. 241,4588-4599 Spector, A. A., John, K., and Fletcher, J. E. (1969) J. Lipid

Res. 69, 56-67 Stucki, J. W., and Ineichen, E. A. (1974) Eur. J. Biochem. 48,

365-375 60. Seimiya, T., and Ohki, S. (1973) Biochim. Biophys. Actu 298,

546-561 61. Carafoli, E. (1974) B&hem. Sot. Symp. 39, 89-109 62. Brand, M. D., and Lehninger. A. L. (1975) J. Biol. Chem. 250. -

7958-7960 63. 64. 65.

Pande, S. V. (1971) J. Biol. Chem. 246, 5384-5390 Osmundsen, H., and Bremer, J. (1976) FEBS Lett. 69, 221-224 Hackenbrock, C. R., and Caplan, A. I. (1969) J. Cell. Biol. 42,

221-234 66. Hunter, D. R., Haworth, R. A., and Southard, J. H. (1976) J.

Biol.Chem. 251,5069-5077 Pfeiffer, D. R., Kuo, T. H., and Tchen, T. T. (1976) Arch.

by guest on September 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Ca2+, Fatty Acid Oxidation, and Mitochondrial Energy State 799

Biochem. Biophys. 176, 556-563 68. Rasmussen, H.: and Tenenhouse, A. (1968) Proc. Natl. Acud.

Sci. U. S. A. 59, 1364-1370 69. Friedmann, N., and Rasmussen, H. (1970) &whim. Biophys.

Acta 222, 41-52 70. Borle, A. B. (1974) J. Membr. Biol. 16, 221-236 71. Dorman, D. M., Barritt, G. J., and Bygrave, F. L. (1975)

Biochem. J. 150, 389-395 72. McDonald, J. M., Bruns. D. E.. and Jarett. L. (1976) Biochem.

Biophys: Res. donm&. 71, 114-121 ’ ’ 73. Mayes, P. A., and Felts. J. M. (1967) Nature 215. 716-718 74. M&a&y, J. D., Meier, J. M., and Foster, D. W.‘(1973) J. Biol.

Chem. 248,270-278 75. Struck, E., Ashmore, J., and Wieland, 0. (1965) Biochem. 2.

343- 107-110 76. Williamson, J. R., Browning, E. T., Thurman, R. G., and

Scholz, R. (1969) J. Biol. Chew 244, 5055-5064

77.

78.

79.

80.

81.

82.

83.

84.

Heimberg, M., Weinstein, I., and Kohout, M. (1969) J. Biol. Chem. 244, 5131-5139

Exton, J. H., Corbin, J. G., and Harper, S. C. (1972) J. Biol. Chem. 247, 4996-5003

McGarry, J. D., Wright, P. H., and Foster, D. W. (1975) J. Clin. Znoest. 55, 1202-1209

Bieberdorf, F. A., Chernick, S. S., and Scow, R. 0. (1970) J. CZin. Zmest. 49, 16851693

Woodside, W. F., and Heimberg, M. (1976) J. Biol. Chem. 251, 13-23

Zahlten, R. N., Kneer, N. M., Stratman, F. W., and Lardy, H. A. (1974) Arch. Biochem. Bioohvs. 161. 528-535

Tolbert, M. E. M., and Fain, J. N. (1974) J. BioE. Chem. 249, 1162-1166

Pilkis, S. J., Claus, T. H., Johnson, R. A., and Park, C. R. (1975) J. Biol. Chew 250, 6328-6336

by guest on September 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

D A Otto and J A Ontkoenergized state.

Activation of mitochondrial fatty acid oxidation by calcium. Conversion to the

1978, 253:789-799.J. Biol. Chem. 

  http://www.jbc.org/content/253/3/789.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/253/3/789.citation.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on September 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from