Myocardial utilization of carbohydrate and lipids

SPECIAL

ARTICLE

Myocardial

Utilization J. R. Neely.

of Carbohydrate M. J. Rovetto,

and

Lipids

and J. F. Oram

ARDIAC MUSCLE is capable of utilizing a variety of substrates as sources of energy.1-6 Glucose and fatty acids are the major fuels, but lactate, pyruvate, ketone bodies, and, to a lesser extent, amino acids are all potential sources of energy. The rate at which anyone of these substrates is utilized by the heart depends upon (1) the concentration of the substrate in the plasma, (2) the availability of alternative substrates, (3) the degree of mechanical activity of the heart, (4) the rate of oxidative respiration, and (5) the plasma levels of certain hormones. Mitochondria make up about 35% of the total volume of cardiac muscle,’ and under normal conditions oxidative phosphorylation accounts for almost all of the ATP that is produced. In a well-oxygenated heart, all of the substrates listed above can be completely oxidized in the citric acid cycle, and the NADH produced either from the cycle or from p-oxidation of fatty acids serves as the immediate source of energy for ATP production by oxidative phosphorylation. Glycolysis becomes an important source of ATP only when oxidative metabolism is limited. The importance of fatty acids in myocardial metabolism is well known. Their oxidation under some conditions may account for as much as 100% of oxidative phosphorylation.5,s+1s Under most conditions, fatty acids are oxidized in preference to carbohydrate and their oxidation normally accounts for about 60-70% of the oxidative metabolism. When present in the perfusate of isolated hearts, fatty acids remained the most used substrate for oxidative metabolism at high levels of cardiac work. 14.15In hearts that were perfused with glucose as the only exogenous substrate, glucose oxidation could account for only about 40% of the oxygen consumption at low cardiac workloads suggesting that endogenous lipids were also used in preference to carbohydrate. At high levels of work in these hearts, however, oxidation of glucose could account for as much as 80% of the total oxygen consumed.16 The normally low rate of carbohydrate utilization was also increased several-fold when oxidative metabolism was reduced.” The acceleration of substrate utilization under some conditions, and the preferential utilization of fatty acids over carbohydrate under normal conditions, involves a complicated system of regulatory interactions between the various metabolic pathways. These interactions bring about an integrated control of all the pathways and adjust the rate of each to meet the metabolic needs of the heart as a whole. The regulation of individual steps within the pathways of carbohy-

C

Front the Department oJPhvsiology, The Milton S Hershex Medical C‘enter. The Pennsvit’anio .Starr University. Hershey. Pa. Supported by USPHS grants HE 13028 and NIH-IVHLI-71-2499. and hi the Pennsylvania Hear: .Associalion. J. R. Neely, Ph.D.: Assi.~ranr ProJe.uor oJ Physiolog!,. The Pennsylvania Stare Univerrir~. Her.rhey, Pa. M. J. Rovetto, Ph.D.: Postdoctoral Fellow. The Pennsylvania Stale Universiry. Hershey. Pa J. F. Oram, Ph.D.: Postdocloral Fellow,. The Penns,,l\,ania Stare Universir~~. Hershe\,. Pa. Progress

m Cardiovascular

Diseases,

Vol

XV. No. 3 (November/December).

1972

289

290

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drate and lipid metabolism subjects of this review. GENERAL

and the interactions

PROPERTIES

OF

ROVETTO.

between these pathways

METABOLIC

AND

ORAM

are the

REGULATION

Flow of substrates through a metabolic pathway may be controlled by several factors.‘* (1) Concentration of the substrate for the first reaction may under some conditions control the overall rate of the pathway. For example, the rate of glucose utilization by the heart is dependent to a large extent on the concentration of glucose in the blood.‘“.2” At any concentration of glucose, the rate of utilization can be increased by insulin,‘“-“’ by increased cardiac work.‘” or by decreased oxidative metabolism,” Indicating that the overall rate is also controlled by a number of hormonal and metabolic factors. (2) A major factor that functions to regulate a metabolic pathway is the strategic location within the pathway of enzymes that are under allosteric control. 22 These regulatory enzymes can control both the overall rate of a pathway and the concentrations of intermediates within the pathway. Phosphofructokinase is a classic example of a regulatory enzyme and its activity often controls the overall rate of glycolysis as well as the concentration of glucose-6-P and fructose-6-P.Z:s-Z5 Control of a metabolic pathway often occurs through feedback control of the regulatory enzymes of the pathway. A regulator of an enzyme may be the immediate product of the reaction or a more distant product of the pathway. For example, pyruvate dehydrogenase is inhibited by acetyl CoA and NADH,‘” the immediate products of the reaction, as well as by ATP, 27 the final product of pyruvate oxidation. (3) Another common means of regulating the activity of an enzyme is through conversion of the enzyme from a form that is normally inactive, due to the presence of allosteric inhibitors in the cell, to a form that is not inhibited by these regulators.‘” Phosphorylase,“Y glycogen synthetase,“” pyruvate dehydrogenase’” and perhaps phosphofructokinase x are all examples of enzymes that are regulated in this matter. Conversion of an enzyme that is physiologically inactive to a form that has activity in the cell is frequently a major component in the hormonal regulation of metabolic pathways via cyclic 3’- 5’ AMP and, therefore, represents a ma.jor model for the mechanism of aceffect in Sutherland’s “Second Messenger” tion of hormones.“” (4) Another type of control of metabolic pathways involves variations in the absolute amount of an enzyme rather than a change in the activity of the existing enzyme.“’ This mechanism, however, does not appear to be very important in heart muscle, since the total activities of the regulatory enzymes that have been studied do not change under conditions where large alterations in the rates of reactions do occur.:“‘.:” REGULATION

OF

CARBOHYDRATE

UTILIZATION

Glucose represents one of the important fuels for respiration in aerobic hearts and its metabolism through glycolysis becomes the major source of ATP in hypoxic tissue. The major steps of glucose utilization that are known to be regulated are glucose transport, hexokinase, phosphofructokinase, pyruvate dehydrogenase and both glycogen synthetase and phosphorylase. The pathways of glucose and glycogen utilization are shown in Fig. I.

UTILIZATION

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Fig. 1. Major steps in pathways of glucose and glycogen metabolism in heart muscle.

Glucose

Transport

Transport of glucose across the cell membrane occurs by a process known as carrier-mediated transport or facilitated diffusion. For a detailed discussion of the carrier model and the properties of glucose transport, the reader is referred to a recent review.37 Briefly, the model includes a mobile protein component of the membrane (the “carrier”), which can move through the membrane and is freely accessible to glucose on either side. Free glucose combines with the carrier on one side of the membrane, and this combination renders glucose sufficiently lipid soluble to allow the sugar-carrier complex to passthrough the membrane. The complex then dissociates, releasing free glucose on the opposite side of the membrane. The process is freely reversible, it does not use metabolic energy and it serves only to equilibrate intracellular and extracellular concentrations of glucose. In the absenceof insulin, transport is the rate-limiting step for glucose utilization by heart muscle.3R.39Under this condition, phosphorylation of glucose by hexokinase is limited by the low concentration of intracellular free glucose. When transport in isolated rat hearts was stimulated by insulin, free glucose accumulated in the cells and the limiting step for glucose utilization was shifted from transport to the hexokinase reaction. 4’1Sugar transport in heart muscle is regulated by a number of factors in addition to insulin. Increased cardiac work accelerated the rate of glucose utilization, and stimulation of transport was dem-

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onstrated as a primary component of this effect.‘” Glucose utilization by rat hearts was inhibited by the presence of alternative substrates, such as long-chain fatty acids12.‘3 or ketone bodies.‘n.41 This effect was demonstrated better when transport was stimulated by insulin. Inhibition of transport was later demonstrated as a component of this effect. Oxidation of fatty acids inhibited transport of nonmetabolized sugars when transport was stimulated by insulin42 or by increased cardiac work.14 A decrease in oxidative metabolism accelerated glucose transport in heartI and skeletal muscle.43 The presence of palmitate did not inhibit transport in anaerobic hearts, which suggests that the fatty acid must be oxidized before transport is inhibited.‘” These studies indicate that sugar transport is regulated by insulin, cardiac work, availability of alternative substrates and degree of oxygenation of the heart. In vivo, all of these factors interact to produce a finely adjusted control of sugar transport. The molecular mechanisms involved in control of sugar transport are unknown. Insulin is thought to bind to the outer surface of the cell membrane where it modifies membrane components in such a way that the glucose-carrier complex can move through the membrane at a faster rate.37 The effects of hypoxia,“3 muscular work44 and oxidation of fatty acids’4,45 are thought to be mediated by changes in intracellular levels of specific activators or inhibitors of transport. In addition to acceleration of transport in the absence of insulin, increased cardiac work rendered the transport process more sensitive to stimulation by suboptimal concentrations of the hormone?’ This combination of effects of muscular work may explain the improved utilization of glucose by diabetics during exercise.46 Glycol.vsis Intracellular free glucose must be phosphorylated to glucose-6-P before it can be metabolized further. Phosphorylation is catalyzed by hexokinase. Isolated hexokinase is inhibited by glucose-6-P. 47 A correlation between the rate of glucose phosphorylation and the intracellular concentration of glucose-6-P has been demonstrated in heart muscle under a variety of conditions.4°.4x Thus, when the supply of hexose monophosphate is more than adequate to meet the needs of the cell, the concentration of glucose-6-P increases and regulates its own production by feedback inhibition of hexokinase. Glucose-6-P occupies the first branch point in glucose metabolism. It may be converted to acetyl CoA through the main glycolytic pathway, it may be used for glycogen synthesis, or it may be utilized by the pentose phosphate cycle. Control of the main glycolytic pathway occurs primarily at the levels of phosphofructokinase and pyruvate dehydrogenase. Phosphofructokinase catalyzes the first irreversible step in glycolysis and its activity controls the overall rate of this pathway under many conditions. 4g~s1The enzyme is subject to allosteric regulation by a variety of small molecules. Its activity is inhibited by ATPS2 and citrate,5”-“” and it is accelerated by Pi, fructose diphosphate, 5’ AMP, ADP and cyclic AMP.5’jJ7 This enzyme is one of the major sites of glycolytic inhibition by aerobic respiration. In well-oxygenated hearts, the tissue level of ATP was high, the levels of ADP. AMP and Pi were low, and phosphofructokinase was inhibited:5x,“” Inhibition of glycolysis by oxidation of fatty acids at both high and

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293

low levels of ventricular pressure development involved high tissue levels of citrate and ATP and low levels of AMP and Pi! ?3- j5v60When oxidative metabolism in muscle was limited, phosphofructokinase was activated by decreased levels of ATP and increased levels of 5’ AMP and Pi .4°.61-63Increased cardiac work accelerated the rate of this enzyme. 60.64The mechanism of this effect of cardiac work is not known, but it does not appear to involve changes in the tissue levels of adenine nucleotides. The conversion of pyruvate to acetyl CoA in the mitochondria is catalyzed by pyruvate dehydrogenase. After phosphofructokinase, this enzyme is the next major step in glycolysis to be regulated. Pyruvate dehydrogenase is subject to allosteric inhibition by the products of pyruvate metabolism, NADH, acetyl CoAZ” and ATP.2’ Oxidation of fatty acids resulted in inhibition of pyruvate dehydrogenase due to increased levels of NADH and acetyl C0A.G” Inhibition of pyruvate dehydrogenase by high levels of NADH in anaerobic hearts may function to divert pyruvate to formation of lactate, which would oxidize NADH produced at glyceraldehyde phosphate dehydrogenase and would allow glycolytic production of ATP to continue. In addition to allosteric control, pyruvate dehydrogenase is regulated by a phosphorylation-dephosphorylation mechanism.“’ Phosphorylation inactivates the enzyme while dephosphorylation activates it. There is evidence that this conversion of pyruvate dehydrogenase between active and inactive forms is under hormonal control and may function to regulate the activities of the enzyme in hearp” as well as other tissues.“’ Glwogen

Metabolism

Glycogen synthesis and degradation occur by two separate pathways. Both pathways, however, are integrated through common control mechanisms in such a way that when glycogen synthesis is accelerated, glycogenolysis is inhibited and vice versa. UDPG-glycogen transferase is the site of both hormonal and nonhormonal control of glycogen synthesis,BR-70 and glycogen phosphorylase is the site of hormonal and nonhormonal control of glycogenolysis.“-‘” Both enzymes exist in physiologically active and inactive forms. The D form of UDPG-glycogen transferase is dependent upon the presence of glucose-6-P for its activity, whereas the I form is active in the absence of glucose6-P.H8,1i9Under normal conditions, the glucose-6-P concentration in the tissue is high enough to fully activate transferase D but its activity was found to be inhibited by the normally high tissue levels of ATPT4 Variations in the concentrations of glucose-6-P and ATP can serve to regulate the activity of this form of the enzyme. An important mechanism that functions to control the rate of glycogen transferase is the interconversion of the D and I forms .75 Transferase I is phosphorylated to the D form and rendered less active by a cydic AMP dependent protein kinase.76 Dephosphorylation of the D form by transferase D phosphatase converts it back to the I form. Regulation of these interconverting enzymes accounts for much of the control of glycogen synthesis. For example, epinephrine or glucagon decrease glycogen synthesis through increased intracellular levels of cyclic AMP. Increased cyclic AMP activates the protein kinase. which in turn converts

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transferase I to the less active D form. Insulin increases glycogen synthesis in cardiac muscle directly by promoting conversion of transferase D to the I form. The hormone exerts its effect on conversion of transferase D to the I form by increasing the dependence of protein kinase on cyclic AMP and it does not appear to change the tissue levels of this nucteotide. Insulin increases the activity of transferase D indirectly by its effect on glucose transport and increased tissue levels of glucose-6-P. The transferase interconverting enzymes are also subject to control by nonhormonal factors. Glycogen inhibits transferase phosphatase, which reduces the amount of transferase in the I form. Thus, increased levels of glycogen can inhibit its own synthesis and help set an upper limit on the amount of glycogen that can accumulate in cells. Glycogen phosphorylase is controlled by many of the same hormonal and nonhormonal factors that regulate glycogen synthetase. Phosphorylase h is active only in the presence of 5’ AMP, while the a form has activity in the absence of the nucleotide.” The b form is converted to phosphorylase a by a specific phosphorylase kinase that requires Ca++ for activity.7x,7” Phosphorylase a is converted back to the b form by phosphorylase phosphatase.x” The phosphorylase kinase also exists as active and inactive forms. x’ x2 Inactive phosphorylase kinase is phosphorylated (activated) by a cyclic AMP dependent protein kinase. The active form of phosphorylase kinase is dephosphorylated (inactivated) by phosphorylase kinase phosphatase. The same cyclic AMP dependent protein kinase that activates phosphorylase kinase and stimulates glycogenolysis also converts glycogen transferase from the I to the D form and inhibits glycogen synthesis?” Hormonal control of glycogenolysis is mediated through changes in the activities of this protein kinase. Epinephrine promotes net glycogenolysis through its action on adenyl cyclase and increased tissue levels of cyclic AMP. 71 In addition to activation of glycogenolysis and inhibition of glycogen synthesis, increased cyclic AMP also activates phosphofructokinase and accelerates the rate of hexose monophosphate removal through glycolysisP’ Nonhormonal control of glycogenolysis occurs primarily through allosteric regulation of phosphorylase h. Phosphorylase b is activated by 5’ AMP and in the presence of high levels of 5’ AMP is capable of as much activity per unit of enzyme protein as is phosphorylase u.~:$This activity, however, is normally inhibited by high tissue levels of ATP and glucose-6-P. The rate of glycogenolysis can be increased without conversion of phosphorylase h to CI by either increased levels of 5’ AMP or decreased levels of ATP and glucose-6-P. A correlation between the tissue levels of glucose-6-P and the rate of glycogen utilization has been demonstrated in rat hearts under a variety of conditions.“0.“4 The net effects of increased tissue levels of glucose-6-P on glycogen metabolism, therefore, result from both an inhibition of phosphorylase h and an activation of glycogen synthetase. Physiologically, interconversion of phosphorylase a and b is an important regulatory mechanism. Activation of phosphorylase h by allosteric regulators occurs only after a low energy state has developed within the cell, whereas conversion of the b to the a form can increase glycogenolysis before an actual energy deficit has developed. Release of epinephrine or norepinephrine, for ex-

UTILIZATION

OF CARBOHYDRATE

ample, can increase muscular work. INTEGRATED

AND

LIPIDS

295

the rate of glycogenolysis

CONTROL

OF AND

CARBOHYDRATE PATHOLOGICAL

simultaneously

UTILIZATION

with increased

UNDER

NORMAL

CONDITIONS

The rates of glucose and glycogen utilization depend upon (1) the plasma concentrations of glucose, insulin, and alternative substrates; (2) the degree of mechanical activity of the heart; and (3) the rate of oxidative metabolism. Overall control of carbohydrate metabolism under a variety of conditions has been shown to result from integrated control of the regulatory enzymes that were discussed above. Interaction

ofGlucose

and Fatty .4cid Metaholist~~

The preferential utilization of fatty acids by heart muscle involves inhibition of carbohydrate utilization at the levels of glucose transport, phosphofructokinase, hexokinase, glycogen phosphorylase, and pyruvate dehydrogenase and stimulation of glycogen synthetase. The mechanisms of these effects of fatty acid oxidation have been partially elucidated over the past decade. The inhibition of transport in the isolated rat hearts was demonstrated with either short- or longchain fatty acids or ketone bodies. 14*42The mechanism of this effect is not known. Inhibition of phosphofructokinase by fatty acids resulted from increased tissue levels of citrate?“+“” Decreased flow of substrate through this step resulted in elevated levels of fructose-6-P and glucose-6-P. This increase in glucose-6-P inhibited hexokinase and resulted in the accumulation of intracellular free glucose which further restricted sugar uptake. Increased glucose-6-P also inhibited glycogen utilization through inhibition of phosphorylase b and stimulation of glycogen transferase. The net effect of phosphofructokinase inhibition was decreased utilization of glucose through glycolysis and increased conversion of glucose to glycogen. Glucose utilization in the presence of fatty acids or ketone bodies is further restricted by inhibition of pyruvate dehydrogenase. Inhibition of this enzyme resulted from increased tissue levels of acetyl CoA and NADH when fatty acids are oxidized.X5 Both of these compounds have been shown to inhibit pyruvate dehydrogenase in vitro. Myocardial metabolism in diabetic or fasting animals is characterized by a low rate of glucose utilization, high levels of tissue glycogen and triglycerides. almost exclusive oxidation of fatty acids and ketone bodies, and by an increased resistance to insulin. Much of the abnormal carbohydraze metabolism by hearts of diabetic or fasted animals can be explained by the effects of fatty acid oxidation on glucose utilization as discussed above. Under these conditions, the rate of glucose transport is low, due to low plasma levels of insulin and high levels of free fatty acids and ketone bodies. Tissue glycogen stores are high due to inhibition of phosphofructokinase and increased levels of glucose-6-P. The insulin sensitivity of hearts from diabetic animals was improved by either in vivo treatment with insulin or by hypophysectomy or adrenalectomy.xfi The decreased insulin sensitivity of hearts from diabetic or fasted animals was later shown to result in part from increased tissue levels of triglycerides and a faster rate of lipolysis.xS The improved insulin sensitivity of hearts taken from diabetic animals

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treated with insulin or from hypophysectomized animals was explained by a slower rate of fatty acid mobilization from adipose tissue and reduced myocardial content of triglycerides. E$ect of Increased

Ventricular

Pressure

Development

on Glucose Utilization

In hearts perfused in the absence of insulin with buffer containing glucose as the only exogenous substrate, the rate of glucose utilization was estimated to be about 20 Fmole/g dry wt/hr at zero mm Hg peak systolic pressure.‘” This low rate was increased to about 100 in beating hearts that were developing 60 mm Hg peak systolic pressure and to a maximum of about 600 in hearts that were developing 120 mm Hg. These data indicated that over 95% of the maximum rate of glucose utilization by isolated hearts could be controlled by metabolic factors that were related to ventricular pressure development. This effect of pressure development involved an acceleration of sugar transport as well as an activation of phosphofructokinase.fi0*e4 Addition of palmitate to the perfusate inhibited the stimulated rate of glucose utilization at higher cardiac work loads but failed to reduce the slow rate of utilization at low work loads. Inhibition of glucose uptake at the higher levels of ventricular pressure development involved decreased rates of both sugar transport and flux through phosphofructokinase. Increased pressure development accelerated the rate of glycogen utilization in hearts perfused with glucose as the only exogenous substrate. This effect was prevented by including either insulin or palmitate in the perfusate. The rate of glycogen utilization was inversely related to the tissue level of glucose-6-P under these conditions. Increased cardiac work had a dual effect on glucose transport. In addition to a direct stimulation, increased cardiac work rendered the tissue more sensitive to stimulation by suboptimal concentrations of insulin. Effects of tschemia and Hypoxia on Mpocardial

Metabolisnl

Several reviews on anaerobic myocardial metabolism have been published recently.3~“~87-Yo Metabolism of the heart during ischemia and hypoxia is characterized by a decreased rate of ATP production from oxidative phosphorylation and an increased rate of glycolysis. The rate of synthesis of acetyl CoA from both pyruvate and P-oxidation of fatty acids is reduced by anoxia and the tissue levels of acetyl CoA and certain citric acid cycle intermediates decrease. Fatty acids are diverted from P-oxidation to deposition as tissue lipids4;g1 and pyruvate is diverted to formation of lactate.“4.62 In both hypoxic and ischemic hearts the initial limiting factor in ATP production is reduced oxygen supply and decreased oxidative phosphorylation. In addition to oxidative phosphorylation, ATP can be produced by substrate level phosphorylation in glycolysis and at the succinate thiokinase reaction in the citric acid cycle. Fumarate-dependent oxidation of NADH by reversed electron how has been reported to contribute to ATP production in anoxic hearts that were perfused with buffer containing fumarate.y2 Evidence on the relative contribution of the mitochondrial reactions to anaerobic ATP production in the absence of exogenous citric acid cycle intermediates is not available. The rate of glycolytic ATP production was increased four-fivefold by anoxia in isolated rat hearts., w However, this increased rate of

UTILIZATION

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297

glycolysis was not sufficient to maintain normal tissue concentrations of high energy phosphate. Estimates of the glycolytic contribution to anaerobic ATP production range from lo%-30% of the total energy requirements.” The maximum activities of most glycolytic enzymes in anaerobic hearts was estimated to be only l%-10% of their in vitro activity. 87 Factors responsible for limiting the activities of glycolytic enzymes in the intact heart are not known. The increased rate of glycolysis that occurs during the early phases of hypoxia and ischemia is not maintained upon continued exposure to decreased oxygen s~pply.~ The metabolic alterations that occur during the early periods of anoxia (i.e., prior to onset of irreversible tissue damage), however, hold the greatest interest from the standpoint of possible therapeutic interventions and this review will be concerned primarily with the early phase of oxygen deprivation. Changes that occur during the early transition from aerobic to anaerobic metabolism involves activation of carbohydrate utilization at the levels of glucose transport, hexokinase, phosphofructokinase, and glycogen phosphorylase. The rate of glucose uptake in isolated rat hearts that were perfused for 30 min with buffer containing 5 mM glucose was increased threefold by anoxia.93 This rate was increased fourfold by a combination of anoxia and insulin. Under both conditions, glucose transport was the rate-limiting step for glucose utilization as indicated by low levels of intracellular free glucose. The rate of transport was stimulated maximally by raising the concentration of glucose to 15 mM in the anoxic hearts, and intracellular free glucose accumulated indicating that phosphorylation became the rate-limiting step. At the higher glucose concentrations, addition of insulin had little effect on the anaerobic rate of transport. These data indicate that the accelerated rate of glucose transport in anaerobic hearts can be increased an additional 20%-30% by including insulin or by raising the concentration of glucose. These manipulations shifted the rate-limiting step from transport to phosphorylation. In the experiments cited above, the rate of glucose phosphorylation by hexokinase was increased fourfold by anoxia. Activation of hexokinase during anoxia can be accounted for by decreased intracellular levels of glucose-6-P and increased levels of inorganic Pi.46.58.62 Glucose-6-P inhibition of hexokinase may be relieved by increased levels of inorganic Pi?” Phosphofructokinase was rapidly activated by anoxia, as indicated by a faster flux through this step in association with decreased levels of hexose monophosphate and increased levels of fructose diphosphate. 45 ‘.’ 5x.B2Activation of phosphofructokinase was accounted for by decreased tissue levels of ATP and increased levels of fructose diphosphate, 5’ AMP and inorganic Pi. A large increase in the levels of inorganic Pi in the first 20 set of ischemiarg or anoxiac2 seemed to play a major role in activating glycolysis. The initial high rates of glycolysis were not maintained upon continued perfusion under anaerobic conditions. w The factors and glycolytic reactions that limit glycolysis after 2-3 min of anoxia are not fully understood. It has been suggested that phosphofructokinase may be limited due to a greater inhibitory effect of ATP at a pH below neutrality. 62.g5Thus, with increased tissue levels of lactate, flux through glycolysis may be limited by decreased pH. Although glycolysis can produce only limited amounts of ATP. the beneficial

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effects of glucose in hypoxic hearts is well documented. The rate of beating of isolated anoxic rat hearts was maintained longer when glucose was available;!“’ glucose loading of in situ dog hearts before anoxia improved postanoxic recovery; .q7.YHhigh levels of glucose helped prevent morphologic changes in the mitochondria of anoxic rat hearts and improved postanoxic recovery.9C1 Glycogen breakdown also contributes to the flow of glucose through glycolysis during anoxia. High glycogen levels in goldfish may make this species more resistant to anoxia.‘“” The progressive fall in cardiac glycogen levels throughout gestation and early neonatal periods appear to be inversely related to resistance to anoxia in the rat lol and fetal lambs,‘“” and increased glycogen stores appear to have a protective effect during anoxia in the fetal rabbit heart.‘03 Schuer and Stezoski”‘4 found that higher initial cardiac glycogen levels produced by reserpiniration of rats helped maintain cardiac performance and ATP levels over a 5-min period of anoxia. Lactate production in these hearts was 2.5 times as great as in nonreserpinized hearts exposed to anoxia. The entire glycogen store was not mobilized during anaerobiosis’04 and the rate of glycogen breakdown appeared to be independent of initial tissue levels. 1”5.1(‘RUtilization of glycogen rather than exogenous glucose during the anaerobic state would have the advantage of producing an additional mole of ATP per mole of glucose. Anaerobic utilization of glycogen through activation of giycogen phosphorylase has been adequately demonstrated. Activation of phosphorylase involves both a conversion of phosphorylase u to h and activation of the h form by decreased levels of ATP and glucose-6-P and increased levels of 5’ AMP and inorganic Pi.7” Wollenberger and Krause”” showed that conversion of phosphorylase h to a occurred within 7 set of ischemia in the in situ dog heart. Early activation of phosphorylase was shown to involve an adrenergic component,“” but release of endogenous catecholamine is not required for activation as demonstrated in hearts isolated from reserpinized animals.l~lx Anaerobic production of ATP by substrate level phosphorylation and reversed electron flow has been demonstrated in broken mitochondrial preparations.‘Og, 1”’ The performance of anaerobic rat hearts was improved by including substrates for these mitochondrial reactions in the perfusate.“’ The increased transport of glucose, mobilization of glycogen, and activation of PFK all increased glycolysis in oxygen-deficient states. The accelerated rate of carbohydrate utilization, however, was not enough to maintain the levels of high energy phosphate. The tissue levels of ATP and creatine phosphate during early ischemia in an isolated working rat preparation are shown in Fig. 2. Within I min. the level of creatine phosphate had decreased 30%, and by 2 min, the level had fallen to 70% of its initial value. On the other hand, ATP decreased by only 25% during the first 2 min of ischemia and remained near this level during the next IO min. Ventricular failure occurred in these hearts by 12 min of ischemia, although the level of ATP remained quite high. These results are similar to those reported by Gudbjarnason et al.“’ Cessation of contractile activity in ischemic dog myocardium occurred when ATP levels had declined only 20%. Creatine phosphate had decreased by 75% within 223 min of ischemia and remained at this low level. In contrast, nonischemic portions of these hearts continued contracting after nearly 70% of the ATP was depleted.

UTILIZATION

OF CARBOHYDRATE

AND

LIPIDS

Fig. 2. Effect of ischemia on tissue levels of ATP and creatine phosphate in isolated working rat heart preparations. Perfusate was Krebs-lienseleit bicarbonate buffer that contained 11 mM glucose and was gassed with Oz:CO1. 95:5. Control hearts (solid line) were maintained at a left atrial filling pressure of 7 mm Hg and a peak ventricular pressure of 90 mm Hg. Level of peak ventricular pressure was adjusted by varying the aortic resistance. Coronary flow averaged 14 ml/ min in these hearts, and ventricular performance was well maintained. lschemia (dashed lines) was produced by decreasing coronary flow to 5 ml/min at zero time on the figure. Coronary flow was decreased by closing the aortic outflow tube so that all of the cardiac output passed through the coronary vessels and by adjusting left atrial inflow to equal the desired rate of coronary flow (5 ml/min). With this manipulation, left atrial pressure was 3 mm Hg after 1 min. and ventricular pressure development was 60 mm Hg due to the high aortic resistance. Ventricular performance was maintained for only about 8 min in the ischemic hearts. Peak ventricular pressure decreased from 60-l 5 mm Hg between 8 and 12 min of perfusion, and coronary flow decreased from 6-l ml/min. Each value represents the mean f SEM for eight determinations.

299

E 9 k-, ::c, L \” ej g E 2 .Z ?i 2

p 2

^, “0 6 H p ; 4 E t = E IJ

PERFUSION

TIME

( min.)

Failure of the myocardium to maintain ventricular function when the content of ATP had declined by only 25% suggests either (I) that much of the ATP in ischemic hearts is not available for muscle contraction, or (2) that the contractile process is inhibited. The rapid decline in cardiac performance with either ischemia or anoxia could be closely correlated with the depletion of 25% of the total ATP. The pool of ATP that is not used by anoxic hearts may be located in the mitochondria, where the rate of translocation to the cytoplasm may be slower than normal. A model of how the mitochondria might serve as a functional compartment during ischemia has been proposed.“’ Another possible explanation of the dissociation of performance from ATP levels during ischemia has been proposed.1’p This model proposes that increased concentration of hydrogen ions interferes with Ca+’ binding on troponin. This would effectively decrease actin-myosin interaction. The result would be ‘decreased performance and a sparing of ATP and CP. However, creatine phosphate declines very rapidly during ischemia, therefore, a shift in the equilibrium of creatine kinase would also have to occur. CONTROL

OF

MYOCARDIAL

LIPID

UTILIZATION

Fatty acids have a number of effects on cardiac metabolism and function. As discussed earlier, fatty acids are oxidized in preference to carbohydrate and represent the major source of fuel for myocardial energy production. The tissue content of high energy phosphates was maintained at a higher level in isolated rat hearts that were oxidizing fatty acids than in those oxidizing glucose.“” The presence of exogenous palmitate was effective in maintaining initial rates of protein synthesis in isolated hearts that were perfused for 3 hr.“’ These beneficial

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effects have been observed with physiologic concentrations of fatty acids. High levels of FFA, on the other hand. have been shown to have several detrimental effects on myocardial function. At very high concentrations, FFAs were found to increase the rate of myocardial oxygen consumption,““~“” and in ischemic heart patients, high plasma FFAs were associated with the development of cardiac arrythmias. II7 In the isolated rat heartl’x,‘lY and the in situ dog heart,lZO.lZ1 abnormally high concentrations of FFA resulted in decreased force of muscle contraction and development of arrythmias. In contrast to carbohydrate metabolism, regulation of fatty acid metabolism in heart muscle is poorly understood. This portion of the review will discuss some of the current views on regulation of lipid metabolism and will present some data obtained in recent studies concerned with the intracellular mechanisms that are involved. Figure 3 illustrates the major steps in the pathways of fatty acid metabolism. This pathway involves (1) transfer of plasma FFA into the cells. (2) intracellular activation, (3) acyl transfer from cytosol to the mitochondrial matrix, (4) /I-oxidation, and (5) oxidation of acetyl CoA through the citric acid cycle. Plasma

Fatty Acids

Fatty acids are synthesized and stored in the liver and adipose tissue and must be transported through the blood to other tissues where they are oxidized. Since FFAs are practically insoluble in H,O, their transport in blood is accomplished by (I) binding of the acids to albumin and (2) by synthesis of triglyBLOOD

UTERSTITIAL SPACE

CYTOSOL

MITOCHONDRIAL OUTER

SPACES

MITOCHONDRIAL

MEMBRANE

INTERMEMBRANE INNER

SPACE MITOCHONDRIAL

MEMBRANE

MATRIX

ALB:FFA

‘ROTEIN:FFA

4TP

-AL

;=yL

SPACE

AMP + PiPt CoA-

-ION PROTEIN .IPASE GLYCEROL

a-GP L TG+FFA’

-

-I GLYCEROL

)/ACETY? ’ CARNlT’NE

1 1

/ /pyR

ACI C

Fig. 3.

Pathways

Cl ITRATE /

of fatty

acid

metabolism.

/ OAA/

t ISOClTkATE NADt,r

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301

cerides and lipoprotein in the liver. The majority of plasma FFAs are bound to albumin.‘22 Only about 0.01% of these acids are free in solution in the unbound form. Interstitial and intracellular FFAs are likewise bound to soluble proteins and to proteins located on cellular membranes.123,‘24 Fatty acid extraction by tissues is thought to occur by cellular binding sites competing with binding sites on plasma albumin for available fatty acids.lz5 The albumin binding sites have a wide range of affinities, and as the amount of FFAs per mole of albumin is increased, binding sites of lesser affinity are occupied.*22*L26 With an increase in FFAs bound to the lower affinity albumin binding sites, the equilibrium is shifted toward cellular binding, which establishes a more favorable condition for fatty acid uptake by the tissue. Cellular uptake of FFA has been shown to depend upon the albumin:FFA molar ratio.124.1+-l~’ Since the physiologic concentration of albumin in the serum rarely varies, the rate of uptake under most conditions is proportional to the concentration of plasma FFA.5.‘27,129 FFAs can also be supplied to the heart from plasma triglycerides or lipoproteins.‘32-‘“4 Lipoprotein lipases located on the capillary endothelium and muscle cell membranes break down glycerides to FFAs and glycerol (Fig. 3). These enzymes are under hormonal control and their activities increase during states of increased fatty acid mobilization from adipose tissue and states of increased myocardial dependence upon FFA, such as during fasting or diabetes.‘35-1:‘7 Since the rate of FFA extraction by the heart is proportional to the concentration of plasma FFA, the rate of FFA metabolism can be controlled to some extent through regulation of the plasma concentration of FFAs and triglycerides. The plasma levels of these compounds are in turn regulated by dietary intake of lipids and hormonal regulation of fatty acid synthesis, storage and mobilization in adipose tissue.lsx Fatt?) Acid Activation The FFAs taken up by the heart, whether derived from plasma FFA or hydrolysis of triglycerides, must be activated before they can be metabolized further. Activation involves the formation of fatty acyl CoA thioesters from FFA, CoASH and ATP (Fig. 3). In the liver, activation of fatty acids occurs at three separate locations: on the endoplasmic reticulum, on the outer mitochondrial membrane, and within the mitochondrial matrix. 13g.140In most cases, fatty acids activated within the mitochondrial matrix are oxidized directly by the fl-oxidation system. I41 Fatty acids activated outside the mitochondrial matrix must be transferred to the site of p-oxidation within the matrix by a carnitine-dependent process (to be discussed later). The carnitine-dependent process appears to account for the major portion of fatty acid oxidation in liver. In heart muscle, the activating enzymes are associated with the sarcoplasmic reticulum and outer mitochondrial membrane.‘40+14’ Oxidation of long-chain fatty acid by the heart is thought to be completely dependent on the presence of carnitine.142 These observations suggest that activation of long-chain fatty acids occurs outside the inner mitochondrial membrane. However, oxidation of short and medium chain length fatty acids is carnitine independent, suggesting that activation of these compounds may occur within the matrix.

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The extent to which the activity of acyl CoA synthetase is regulated is not clear at the present time. The activity of ATP-dependent synthetase was found to be inhibited by AMP and adenosine.‘l:’ A soluble protein was isolated from the cytoplasm of liver cells which increased the activity of long-chain acyl CoA synthetase. I44 These data suggest that their activity may be regulated in vivo. However, the intracellular levels of long-chain acyl CoA and acyl carnitine in both heart and liver were found to vary directly with the the concentration of circulating FFA, suggesting that the rate of activation is directly related to the availability of intracellular FFA.‘“‘-‘I” The acyl CoA synthetase must compete with other reactions for available CoASH and under some conditions availability of substrate may limit the rate of long-chain CoA synthesis. Such a process has been described in liver mitochondria for the intramitochondrial activating enzymes.‘“’ Activated fatty acids can be utilized in the cytosol for synthesis of triglycerides, phospholipids and longer chain fatty acids. Activated fatty acids must be converted to long-chain acyl carnitine derivatives before they are transferred across the inner mitochondrial membrane to the site of/I-oxidation. Fatty’ Acid

TransJer

Between

Cytosol

and Mitochorldrial

Matrik

The role of carnitine in facilitating transfer of fatty acids from the cytosol to the inner mitochondrial matrix is well known. 14XThe experimental observations that have helped to identify this role of carnitine have been discussedfully in recent reviews.141~‘44~‘*y Th ese observations can be outlined as follows: (I) Isolated heart mitochondria did not oxidize FFA except in the presence of added CoASH, ATP and carnitine; (2) longchain acyl CoA was oxidized only when carnitine was added; (3) long-chain acyl carnitine was oxidized freely; and (4) disrupted mitochondria oxidized acyl CoA in the absence of carnitine. Collectively, these observations suggest that activated acyl units are transferred from CoA to carnitine before they can be oxidized by the mitochondria. The transfer of acyl units between CoASH and carnitine is catalyzed by carnitine:acyl CoA transferases.“” Heart muscle contains several of these enzyme systems, which are specific for different chain length fatty acids. Carnitine:acyl CoA transferases that are specific for long-, medium-, and short-chain fatty acids have been identified and characterized.‘““,‘“’ These reactions are thought to be freely reversible with equilibrium constants near unity. The data indicating that formation of long-chain acyl carnitine facilitates the oxidation of fatty acids by mitochondria is well established. The exact mechanism of this facilitation, however, is not clear. The confusing issues involve the mechanisms of translocation of the acyl unit through the inner mitochondrial membrane and the compartmentalization of CoASH and acyl CoA between the cytosol and mitochondria. The inner membrane appears to be impermeable to CoASH, acyl CoA, acetyl carnitine, and free carnitine.‘““,‘“’ Because of the impermeability of the inner membrane to acetyl carnitine, it was assumedthat the membrane is also impermeable to long-chain acyl carnitine derivatives. Yet, the fatty acid moiety of acyl carnitine is transferred to intramitochondrial CoA,

UTILIZATION

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where it is freely oxidized by the p-oxidation system. The outer mitochondrial membrane is freely permeable to all the metabolites mentioned previously. Based on these observations, a model for translocation of the acyl units was proposed by Fritz and Yue’“’ and later modified by Yates and Garland.‘“” This mode1 proposes the existence of two carnitine: long-chain acyl CoA transferases: one located between the inner and outer mitochondrial membranes (transferase I in Fig. 3) and the other closely associated with the inner membrane (transferase II in Fig. 3). Two different mitochondrial transferases have recently been identified.‘5n.‘“4 One enzyme is soluble and is believed to be transferase I. The other enzyme is tightly bound to the inner mitochondrial membrane and is thought to be identical to transferase 11 in the model. Functionally, transferase I would catalyze the transfer of acyl units between CoASH and carnitine in the cytosol. Transferase II would function to transfer acyl units from acyl carnitine in the cytosol to CoASH in the mitochondrial matrix. A similar mechanism is thought to occur for translocation of acetyl units between the mitochondrial matrix and cytosol (Fig. 3). Evidence for regulation of the activities of these acyl transfer enzymes is inconclusive. The activity of long-chain acyl transferases was increased in livers. but not in hearts, from fasted or fat-fed rats.‘“; The rates of these reactions may be influenced by the relative concentration of their substrates.150 Palmityl CoA has been shown to be a competitive inhibitor of the long-chain acyl transferase with respect to its other substrate, carnitine. ‘56 Physiologically, this type of regulation could divert excess long-chain acyl CoA away from oxidation toward synthesis of triglycerides and phospholipids. The function of carnitine:acetyl CoA transferase is not clear. In most mammals, short-chain fatty acids do not represent a major source of substrate for energy metabolism. One function that has been proposed for this enzyme is the transfer of acetyl units from intramitochondrial acetyl CoA to extramitochondrial CoA where the acetyl units are used for synthesis of long-chain fatty acids.‘“’ This function could be important in tissues such as the liver where de novo synthesis of fatty acids is a primary function of the tissue. Acetyl transfer in rat liver, however. can be accomplished through transfer of citrate out of the mitochondria and lysis of citrate to oxaloacetate and acetyl CoA in the cytoplasm.‘“’ Heart muscle has a higher level of carnitine:acetyl CoA transferase than liver.‘5!’ Since fatty acid synthesis is a less significant process in the heart than in liver, transfer of acetyl units for this function would not appear to be of primary importance in the heart. It has been proposed that acetyl carnitine may represent a storage form of high energy acetyl units which can function to buffer large changes in acetyl COA.‘~” This function would be analogous to the role of creatine phosphate in buffering changes in ATP. Since carnitine is restricted to the extramitochondrial space, the function of carnitine:acetyl CoA transferase may be to transfer acetyl units from acetyl CoA inside the mitochondria. where it is formed to its storage form as acetyl carnitine in the cytosol. This would provide free CoASH inside the mitochondria for participation in @-oxidation of long-chain acids and in the citric acid cycle for succinyl C‘oA formation. About ten times more acetyl units are present in the heart as

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acetyl carnitine than as acetyl CoA. 145The tissue levels of both acetyl carnitine and acetyl CoA increased with increased concentration of exogenous FFA. P-Oxidation The enzymes of the P-oxidation system are located in the mitochondrial matrix. These enzymes catalyze the stepwise degradation of long and medium chain length fatty acyl CoA to acetyl COA.‘~O P-Oxidation is an energy yielding process. For each acetyl CoA formed, one NADH and one FADH is produced, which can be used by oxidative phosphorylation to produce five molecules of ATP. Little is known about the regulation of individual enzymes in this process. However, the overall rate may be determined by the energy state of the cell (NADH/NAD ratio) and by the amount of intramitochondrial CoASH present as derivatives other than long-chain acyl CoA (e.g., acetyl CoA and succinyl COA).‘~’ The major fate of acetyl CoA in cardiac muscle is oxidation through the citric acid cycle. Other processes that use acetyl CoA (ketogenesis, gluconeogenesis, and fatty acids synthesis) are either absent or operate at very low rates. Therefore, the rate of P-oxidation in the heart is determined by the rate of supply of long-chain acyl CoA, the rate of removal of acetyl CoA through the citric acid cycle, and the rate of oxidative phosphorylation. Integrated

Control ofFattv

Acid Utilization in the Intact Heart

As discussedpreviously, the overall rate of fatty acid utilization is determined (1) by the supply of exogenous FFA and (2) by the energy demands of the tissue. At a constant rate of energy utilization, increased supply of fatty acids would be expected to have a limited ability to accelerate fatty acid uptake. The upper limit would be reached when the supply of fatty acids exceeds the capacity of the cells to bind free fatty acids and to convert acyl units to CO*, to complex lipids, or to metabolic intermediates. At a constant level of exogenous fatty acid, increased cardiac work would be expected to increase fatty acid utilization due to an increased rate of ATP hydrolysis. The extent to which the concentration of exogenous FFA and the level of cardiac work influences the rate of fatty acid uptake by the isolated rat heart is illustrated in Fig. 4. This figure shows that the rate of palmitate uptake by the heart increased as the concentration of FFA bound to 3% serum albumin was raised. The data indicate that the rate of palmitate uptake increased in proportion to the concentration of FFA

t.5 PERFUSATE

PALMITATE

(rnt.4)

-

Fig. 4. Effects of perfusete palmitate and ventricular pressure development on the rate of palmitate uptake. Hearts were perfused by the Langendorff technique and the peak systolic pressure development was adjusted to 60 (solid line) or 100 mm Hg (dashed line) as described by Neely et al.‘“’ Palmitate uptake was estimated by measuring disappearance of palmitate from perfusate during 20 min of perfusion. Thirty ml of buffer containing glucose (11 mM), albumin (3%) and the concentration of palmitate shown in the figure were recirculated through the heart for the 20.min perfusion period. Each value represents the mean i SEM for six hearts. Palmitate uptake is expressed as pmoles/g dry wt/hr.

UTILIZATION

OF CARBOHYDRATE

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305

.,,--=----8---, /---l)-----------o Fig. 5. Effects of fatty acid concentration and _ 70ventricular pressure development on the rate of -E :,:.--------._ _____ oxygen consumption. Hearts were perfused for ‘, GO10 min with buffer containing glucose (11 mM) L2 If as the only exogenous substrate prior to switchE 50ing to a perfusate that contained either glucose z 1 40alone (solid circles), glucose plus 0.4 mM palmir..nof tate (open circles), or glucose plus 1.2 mfvl palmi5 . 0 l u: _ -8 .I 4 tate (triangles). Perfusion with these substrates E 30. . was continued for 16 min at 60 mm Hg peak 4 ventricular pressure development (solid lines). 2 zo8 After 6 min at 60 mm Hg, ventricular ‘pressure development was increased to 120 mm Hg and o” IOthe rate of oxygen consumption was followed 1 for an additional 10 min (dashed lines). Oxygen 0 2 4 6 6 IO 12 14 16 consumption was estimated by measuring the PERFUSION TIME I ml”.) difference in POz of the arterial and venous perfusate and the rate of coronary flow and is expressed as pmoles/g dry wt/min. Coronary effluent was collected by cannulating the pulmonary artery and collecting the effluent from this artery under heptane. In the Langendorff preparation, coronary effluent, which is returned to the right atrium, is the only fiuid pumped out the pulmonary artery. Each value represents the mean of from 6-12 determinations.

over the range of 0 to about 0.6 mM palmitate. The rate of uptake did not increase further as the concentration of palmitate was raised to 1.2 mM. This leveling off in the rate of uptake at the higher concentrations of palmitate indicated that intracellular processes became limiting for fatty acid utilization. Increased cardiac work accelerated the rate of fatty acid uptake at all concentrations studied. This acceleration resulted from an increased rate of oxidative phosphorylation as indicated by a greater than twofold increase in the rate of oxygen consumption (Fig. 5). The rate of oxygen consumption was also increased in hearts oxidizing fatty acids as compared to those oxidizing glucose. This effect of fatty acids was seen at both levels of cardiac work. The theoretical ratios of molecules of ATP produced per atom of oxygen consumed is 3.17 for glucose and 2.8 for palmitate. If these ratios are operative in the tissue, the expected total ATP yield would be approximately the same for hearts perfused with glucose alone as those perfused with glucose + palmitate. The ATP yield in pmoles/g/min was 166, 174, and 185 in hearts developing 60 ‘mm Hg systolic pressure and 380, 360, and 381 at 120 mm Hg for hearts perfused with buffer containing glucose, glucose + 0.4 mM palmitate and glucose + 1.2 mM palmitate, respectively. The data shown in Fig. 4 indicates that palmitate uptake by the isolated rat heart was proportional to the albumin: FFA molar ratio and the level of cardiac work. The solid lines in Figs. 6, 7, and 8 show the changes that occurred in the tissue content of key intermediates in the pathway of fatty acid oxidation as the concentration of paimitate bound to 3% albumin was raised from O-l.2 mM in hearts that were developing 60 mm Hg peak systolic pressure. The tissue levels of fatty acyl CoA and fatty acyl carnitine (Fig. 6), acetyl CoA (Fig. 7), and acetyl carnitine (Fig. 8) increased with increased prefusate palmitate. This rise in the levels of acyl CoA and carnitine derivatives was associated with a fall in

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306

I

200

1

1

J

1.0

1.2

r

0

I 0.2

L

0.4

PERFUSATE

0.6

0.8

MLMITATE

,

(mM1

Fig. 7. Effects of perfusate palmitate and ventricular pressure development on tissue levels of ecetyl CoA and free CoASH. Experimental conditions were same as described for Fig. 6. The hearts were developing either 60 (solid lines1 or 120 mm Hg (dashed lines) peak ventricular prassure. Each value represents the mean & SEM for 6-l 2 determinations.

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Fig. 6. Effects of increased concentration of exogenous palmitate and increased ventricular pressure development on tissue content of fatty acyl CoA and fatty acyl carnitine. All hearts received a lo-min preliminary perfusion at 60 mm Hg aortic perfusion pressure with buffer containing glucose (11 mMI. At this time, the hearts were switched to a perfusate that contained glucose (11 mM), albumin (3%) and the concentration of palmitate shown in the figure. Ventricular pressure development was adjusted to either 60 mm Hg (solid line) or 120 mm Hg (dashed lines), and perfusion was continued for 6 min. Six min of perfusion with fatty acid was selected, since this was the time required for citric acid cycle intermediates to reach a new steady state following addition of fatty acid to the perfusate of isolated rat hearts. “’ Each value represents the mean & SEM for 6-8 determinations.

100 02 = & ::

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f

p’j

50

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if-i_---

7 0

&--‘--I I 0.2

I 0.4

PERFIJSATE

I 0.6 PALMITATE

I 0.8

1 1.0 (mH)

1 1.2

UTILIZATION

OF

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AND

LIPIDS

307

4

Fig. 8. Effects of perfusate palmitate and ventricular pressure development on tissue levels of acetyl carnitine and free carnitine. Experimental conditions were same as described for Fig. 6. Hearts developing 60 mm Hg peak systolic pressure are represented by solid lines, and those developing 120 mm Hg are represented by dashed lines. Each value represents the mean f SEM for 6-12 determinations.

Or

PERFUSATE

PALMITATE

( mh4)

the tissue levels of free CoASH and carnitine. As the concentration of exogenous fatty acid was raised from O-0.4 mM, the rate of palmitate uptake was proportional to its concentration in the perfusate, and there was only a slight rise in tissue content of long-chain acyl CoA, long-chain acyl carnitine and acetyl CoA. The level of acetyl carnitine increased by about 100%. The increase in acyl carnitine derivatives was proportionally greater than the increase in acyl CoA derivatives. As the concentration of palmitate was raised from 0.4-1.2 mM, the tissue content of long-chain acyl CoA and acetyl CoA increased by 50% and 400% respectively. Both long-chain acyl carnitine increased by about 200%. There was no further increase in the levels of these metabolites when the palmitate concentration was raised above 1.2 mM. The large increase in metabolic intermediates at high levels of exogenous fatty acid was associated with a leveling off in the rate of palmitate uptake. These results indicate that at low concentrations of exogenous palmitate, the rate of its utilization was limited by the avaiiability of FFA. At high concentration, palmitate utilization was limited by the rate of acetyl CoA oxidation through the citric acid cycle and acyl intermediates accumulated in the tissue. The extent of intracellular accumulation of acyl derivatives may have been limited by the large decrease in free CoA (Fig. 7). The 300% rise in the level of long-chain acyl carnitine compared to only a 50% increase in long-chain acyl CoA indicated that the rate of long-chain acyl transfer from CoASH to carnitine by transferase I (Fig. 3) exceeded the rate of fatty acid activation. The high ratio of long-chain acyl carnitine to acyl CoA also suggest that the rate of transferase I exceeded that of transferase II. The rate of

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Fig. 9. Time relationship between palmitate uptake and changes in tissue levels of CoASH, acetyl CoA. and fatty acyl CoA. Hearts were perfused for 10 min with buffer containing glucose as the only substrate and then switched to a perfusate that contained 11 mM glucose plus 1.2 mM palmitate at zero time on the figure. Peak systolic pressure development was maintained at 60 mm Hg. The rate of palmitate uptake (open triangles) and the tissue levels of fatty acyl CoA isolid circles). acetyl CoA lopen circles) and free CoA (solid triangles) were measured at 2.min intervals during 10 min of perfusion in the presence of the fatty acid. Palmitate uptake was measured as disappearance of label from U-C,” palmitate. Each value is mean of 6 determinations. Values are expressed per g of dry tissue.

activation at low concentration of palmitate appeared to be limited by the availability of FFA and at high exogenous palmitate by the availability of free CoASH. The suggestion that the rate of activation was influenced by availability of substrate is supported by the data shown in Fig. 9. In these experiments, hearts were perfused for 10 min with buffer containing glucose (11 mM) as the only substrate, and then at zero time in the figure they were switched to perfusion with buffer containing glucose and 1.2 mM palmitate. The rate of palmitate uptake and the tissue levels of long-chain acyl CoA, acetyl CoA and free CoA were measured at the times indicated. During the first 2 min of perfusion with palmitate, the rate of fatty acid uptake increased to a maximum and then declined to a steady-state level after 10 min. The tissue content of long-chain acyl CoA was also maximal during the first 2 min and declined to a new steadystate level after 6 min. The tissue level of acetyl CoA, on the other hand, did not reach a maximum until 6 min. The content of free CoA dropped to its lowest level within 2 min and remained there for the duration of the perfusion. These data suggest that when free fatty acids were first made available and when the tissue levels of free CoA were high (O-2 min), the rate of fatty acid activation exceeded the rate of its oxidation to acetyl CoA. During this time, a larger portion of the free CoA was esterified as long-chain acyl CoA. As more of the available CoASH accumulated as acetyl CoA, the rate of palmitate activation and uptake declined. Transient changes in the levels of acetyl carnitine (not shown here) were similar to those for acetyl CoA indicating that acetyl units were rapidly translocated from the mitochondrial matrix to the cytosol. It may be assumed that much of the increase in acetyl CoA represented extramitochondrial accumulation by transfer from acetyl carnitine. This would utilize the same CoASH pool that is available for fatty acid activation, which would account for the decline in long-chain acyl CoA as acetyl CoA accumulated. Limited availability of free CoASH within the mitochondrial matrix would also influence the rate of

UTILIZATION

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LIPIDS

309

transferase II (Fig. 3) which could partially account for the proportionally greater increase in acyl carnitine as compared to acyl CoA (Fig. 6). The dashed lines in Figs. 6, 7, and 8 illustrate the effects of increasing ventricular pressure development from 60-120 mm Hg on the tissue content of longchain acyl CoA and carnitine, acetyl CoA and carnitine, and free CoASH and carnitine. With increased cardiac work, the rate of acetyl CoA oxidation through the citric acid cycle was accelerated as indicated by much lower tissue levels of acetyl CoA and acetyl carnitine (Figs. 7 and 8). This effect was very pronounced at the higher concentrations of palmitate. A faster rate of the citric acid cycle with increased cardiac work was also demonstrated by an increased rate of C’“O, production from U-C14-palmitate (Fig. 10). The levels of free carnitine were consistently higher with increased cardiac work except at very low concentrations of palmitate. This is the change that would be expected to result from a faster rate of removal of acetyl units by an acceleration of the citric acid cycle. Changes in the tissue levels of free CoASH, however, did not follow the expected parttern. When the perfusate palmitate was less than 0.6 mM, increased cardiac work lowered the levels of free CoASH at the same time that the level of acetyl CoA was reduced. At palmitate concentrations greater than 0.6 mM, free CoASH increased with increased cardiac work but the rise was not in proportion to the decrease in acetyl CoA. These data indicate that with increased concentrations of palmitate, CoASH accumulated as acetyl CoA at the expense of some other CoA ester and with increased cardiac work the other CoA ester accumulated at the expense of acetyl CoA. As will be discussed in the section on the citric acid cycle, these changes in CoA can be accounted for by lower levels of succinyl CoA at the higher concentrations of palmitate and by higher levels of succinyl CoA with increased cardiac IOO-

60-

Fig. 10. Effects of increasing exogenous palmitate and ventricular pressure development on the rate of C”O? production from U-C” palmitate. Hearts ware perfused for 10 min with buffer containing glucose (11 mM). At zero time on the figure, they were switched to a buffer containing glucose plus 0.4 mM palmitate (circles) or 1.2 mM palmitate (triangles). Ventricular pressure development was adjusted to 60 mm Hg (solid lines) or 120 mm Hg (dashed lines) at this time. Each value represents the mean rate of C’lO, production as palmitate equivalents per g of dry tissue per hr for eight hearts.

0

0

4

2 PERFUSION

TIME

6 (min 1

310

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work and acceleration of the citric acid cycle. Since succinyl CoA is formed in the citric acid cycle, it shares the intramitochondrial pool of CoASH with both the P-oxidation system and transferase II. Changes in the rate of succinyl CoA synthesis would, therefore, be expected to influence the rate of P-oxidation and vice versa. Increased cardiac work resulted in lower tissue levels of long-chain acyl CoA and higher levels of long-chain acyl carnitine at all the concentrations of exogenous fatty acid that were studied. The decrease in long-chain acyl CoA was more pronounced at the lower concentrations. These changes in the tissue levels of acyl CoA and carnitine derivatives associated with a faster rate of fatty acid uptake and oxidation indicated that the rate of long-chain acyl carnitine production by transferase I (Fig. 3) was stimulated with increased cardiac work. The extent of this stimulation was sufficient to result in increased levels of long-chain acyl carnitine even though the levels of acetyl CoA, the end product of@-oxidation, were drastically reduced. Interpretation of these data from intact tissue is complicated by the existence of at least two noninterchangeable pools of free CoA, cytosolic and intramitochondrial. CoASH in the cytosolic pool is thought to be accessible to the fatty acid activation system, to transferase I, and to carnitine:acetyl CoA transferases (Fig. 3). The intramitochondrial pool is thought to be accessible to transferase II, to carnitine:acetyl CoA transferases, to the P-oxidation enzymes and to citric acid cycle enzymes. One explanation of increased tissue levels of long-chain acyl carnitine would be that the level of free CoASH decreased with increased cardiac work to a larger extent in the intramitochondrial pool than it did in the cytosolic pool. This would result in relatively low intramitochondrial free CoASH to function as acceptor of acyl units from acyl carnitine at the transferase 11 reaction and relatively high cytosolic CoASH, which would accelerate fatty acid activation. The net result would be a faster rate of acyl transfer from cytosolic acyl CoA to carnitine than from acyl carnitine to intramitochondrial CoASH resulting in a lower intramitochondrial acyl CoA content. Increased levels of long-chain acyl carnitine indicate that the rate of transferase I was in fact faster than the rate of transferase II. As mentioned earlier, increased levels of succinyl CoA at the higher cardiac work load suggest that a large part of the intramitochondrial CoASH was esterified as succinyl CoA and this could account for lower levels of intramitochondrial CoASH. This explanation implies that the small decrease in total long-chain acyl CoA at 120 mm Hg systolic pressure occurred primarily within the mitochondrial matrix. The decline in the level of intramitochondrial long-chain acyl CoA could be explained by the faster rate of P-oxidation that occurred with increased cardiac work due to removal of NADH and FADH by increased oxidative phosphorylation and by the removal of acetyl CoA due to an acceleration of the citric acid cycle. Since the level of acetyl carnitine also decreased with increased work, the large decrease in acetyl CoA probably represented a decline in both the cytosolic and the mitochondrial pools. Free CoASH released from acetyl CoA in the mitochondrial matrix could be used for both P-oxidation and synthesis of succinyl CoA whereas the CoASH released in the cytosol would be available to accelerate fatty acid activation. Acceleration of fatty acid activation

UTILIZATION

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311

and/or transferase I was demonstrated by a decrease in the level of intracellular FFA (data not shown) and by increased levels of fatty acyl carnitine. The decrease in intracellular free fatty acid indicated that the mechanism of increased fatty acid uptake with increased cardiac work involved a faster rate of fatty acid removal from tissue binding sites and thus the establishment of a larger concentration gradient for diffusion of free fatty acids from the perfusate to the tissue. Increased tissue levels of fatty acyl carnitine at the higher workload indicated that the rates of fatty acid activation and transferase I were faster than translocation of acyl units into the mitochondria by transferase II. As mentioned above, this could result from reduced levels of intramitochondrial free CoASH to act as acceptor of acyl units from acyl carnitine. It would also be explained by the rise in free carnitine that resulted from a faster rate of acetyl carnitine utilization. Higher levels of free carnitine would stimulate the rate of transferase I, increase the level of acyl carnitine and decrease the level of cytosolic acyl CoA and FFA. In either case, however, translocation of acyl units across the inner mitochondrial membrane appeared to restrict fatty acid oxidation with increased cardiac work. This was indicated by the large decrease in acetyl CoA at the time fatty acyl carnitine was increased. Whether this restriction was due to decreased intramitochondrial CoASH or to a limited rate of translocation through the membrane by transferase II is not clear. This restriction can be overcome by supplying octanoate as the fatty acid substrate. Since mitochondrial oxidation of octanoate does not depend on the presence of carnitine, this fatty acid should bypass the carnitine dependent translocation step across the inner mitochondrial membrane. The data shown in Table 1 illustrate that the tissue levels of acetyl CoA were not decreased by increased cardiac work even though the rate of octanoate oxidation increased twofold. With palmitate as substrate, however, the tissue levels of acetyl CoA Table

Substrate

1.

Effects

Tissue

Levels

of Pressure of Acetyl

Development CoA

in the

and Isolated

Substrate Rat

on

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Added

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were greatly reduced. even when the concentration of palmitate was abnormally high. The rate of fatty acid utilization (as acetate equivalents) was about the same with either palmitate and octanoate as substrate. These data indicate that production of acetyl CoA from palmitate proceeded at a slower rate than it did from octanoate. At high levels of cardiac work, production from palmitate could not keep pace with oxidation and the level of acetyl CoA decreased. With octanoate, on the other hand, production of acetyl CoA could proceed as fast as its oxidation even at the higher cardiac work load. These results, along with those presented in Figs. 6, 7. and 8, suggest that translocation of palmityl units through the inner mitochondrial membrane represents the rate-limiting step for acetyl CoA production at high cardiac workloads. The data discussed thus far in this section indicate that the rate of fatty acid uptake and oxidation at a low cardiac work load was limited by the rate of acetyl CoA oxidation through the citric acid cycle. The accumulation of acetyl CoA and the decrease in free CoASH limited the rate of fatty acid activation and poxidation. A shift in esterified CoA from succinyl CoA to acyl CoA at high concentrations of exogenous palmitate would also influence the rate of the citric acid cycle. With increased cardiac work, the rate of fatty acid uptake and oxidation was accelerated at every concentration of exogenous palmitate studied. This effect resulted from a faster rate of ATP hydrolysis and oxidative phosphorylation as indicated by an increased rate of oxygen consumption. Acceleration of the citric acid cycle resulted in a large decrease in the tissue content of acetyl CoA and acetyl carnitine and a shift of CoASH from acetyl CoA to succinyl CoA. P-Oxidation was accelerated by the reduced level of acetyl CoA and by a faster rate of removal of NADH by oxidative phosphorylation. The faster rate of P-oxidation and the shift of intramitochondrial CoASH to succinyl CoA resulted in lower tissue levels of long-chain acyl CoA. The level of long-chain acyl carnitine increased and tissue FFA decreased due to acceleration of fatty acid activation. Activation was stimulated by increased extramitochondrial levels of free CoA and carnitine as acetyl units were transferred from cytosolic acetyl CoA to carnitine and then translocated to intramitochondrial CoA at a faster rate. The rate of translocation of long-chain acyl units from cytosolic acyl carnitine to intramitochondrial CoASH, although proceeding at a faster rate, appeared to restrict the overall rate of fatty acid utilization at the higher level of cardiac work. This was indicated by increased levels of long-chain acyl carnitine and reduced levels of long-chain acyl CoA and acetyl CoA. At very low levels of exogenous palmitate, the rate of fatty acid uptake was limited by availability of FFA at both levels of cardiac work. Control of the Citric Acid Cycle Production of ketone bodies in heart muscle is almost nonexistent, and de novo synthesis of fatty acids, although present, is not a primary function of the tissue. Therefore, the major fate of acetyl CoA is oxidation through the citric acid cycle.163 the rate of the citric acid cycle is ultimately determined by the rates of ATP hydrolysis and oxidative phosphorylation. Feedback regulation of the cycle by changes in the ATP/ADP and NADH/NAD ratios ensure that the rate of the cycle is geared to the rate of energy expenditure by muscle

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contraction. Regulation of the cycle in intact tissue and isolated mitochondria has been demonstrated to occur at the levels of citrate synthetase, isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase. Citrate synthetase catalyzes the first irreversible (11 Citrate Synthetase: reaction in the citric acid cycle and this step has been proposed as a major site of control of the cycle. The isolated enzyme is regulated by changes in the ATP/ADP ratio.16*.16” ATP is a competitive inhibitor with respect to acetyl CoA. This direct regulation by ATP, however, does not appear to control the rate of citrate synthesis in the intact tissue,‘6fl~‘6s or in isolated mitochondria.‘“g Most of the control of citrate synthesis in heart muscle appears to occur through changes in the availability of its substrates (acetyl CoA and oxaloacetate). Availability of substrates was the major factor controlling citrate synthetase in isolated perfused rat hearts. 168.17vIn hearts perfused with glucose as the only exogenous substrate, the tissue levels of acetyl CoA and citrate were low suggesting that availability of acetyl CoA controlled the rate of citrate synthesis. Addition of either fatty acids or ketone bodies to the perfusate increased the tissue content of acetyl CoA and citric acid cycle intermediates and, under this condition, availability of oxaloacetate became limiting for citrate synthesis. Anoxia, even in the presence of fatty acids, resulted in low tissue levels of acetyl CoA and citrate suggesting that the rate of citrate synthesis was determined by availability of acetyl CoA under this condition.‘7n Presumably, production of acetyl CoA from both pyruvate and P-oxidation of fatty acids would be inhibited by a high NADH/NAD ratio in anoxic tissue. The rate of citrate synthesis by isolated heart mitochondria during state 4 respiration (no added ADP) was limited by decreased availability of oxaloacetate. 1Rg.171Acceleration of citrate synthesis during the state 4 to state 3 transition was accounted for by increased availability of oxaloacetate. Although availability of substrates appears to be a major factor in the control of citrate synthesis, changes in the affinity of citrate synthetase for each of its substrates may be an important regulatory mechanism in some conditions. In addition to the effects of ATP, crystalline citrate synthetase was competitively inhibited by CoASH and succinyl CoA (with respect to acetyl CoA) and by citrate (with respect to oxaloacetate). 17z Variation in the levels of citrate and succinyl CoA accounted for much of the control of citrate synthetase in uncoupled mitochondria when the NADH/NAD ratio was low and the level of oxaloacetate was high.lSg (21 NAD-specijic Isocitrate Dehydrogenase: This is known to be a regulatory enzyme in heart muscle. This enzyme is inhibited by NADH and ATP and is activated by ADP.173-175 The ADP activation is modified by Ca*2. At low levels of Ca+2 ( ltQM), ADP had no effect on the activity of the enzyme, whereas at higher Ca+2 (1FM) increasing the concentration of ADP to 0.1 mM increased its activity.‘76 Thus, uptake and release of Ca+2 by mitochondria during the cardiac cycle may influence the activity of isocitrate dehydrogenase. Regulation of the cycle by changes in NADH/NAD ratio has been demonstrated in a number of preparations including both perfused organs and isolated mitochondria. Isocitrate dehydrogenase was inhibited by a high NADH/NAD ratio in perfused livers and in isolated liver mitochondria that were oxidizing palmityl

314

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carnitine.177~‘7” Activation of isocitrate dehydrogenase was demonstrated in isolated heart mitochondria during the state 4 to state 3 transition.17’ Under this condition, activation of the enzyme was associated with a decreased NADH/ NAD ratio and with higher levels of ADP. Oxidation of octanoate by isolated rat hearts resulted in inhibition of isocitrate dehydrogenase as indicated by increased tissue levels of isocitrate and decreased levels ofa-ketoglutarate.17” On the other hand, evidence for control of isocitrate dehydrogenase was not obtained in isolated rat hearts that were oxidizing acetate.‘“’ In this instance, control of a-ketoglutarate dehydrogenase appeared to be an important factor in regulating the citric acid cycle. A greater degree of control at the level of isocitrate dehydrogenase in hearts that were oxidizing long-chain fatty acid, as compared to those oxidizing acetate, may have resulted from the extra NADH that is produced through P-oxidation of the long-chain fatty acids. Acetate is converted to acetyl CoA without any prior oxidation, and the only NADH produced from oxidation of acetate is from within the cycle itself. (31 a-Ketoglutarate Deh!ldrogena.se. Isolated n-ketoglutarate dehydrogenase is inhibited by succinyl CoA and NADH, both products of the reaction.‘R” The enzyme is also inhibited by acetyl CoA and palmityl COA.‘~‘,‘~~ These acyl CoA derivatives appear to compete with free CoASH for binding sites on the enzyme. Regulation of this step appeared to be the dominant control mechanism of the cycle in isolated hearts that were oxidizing acetate.“‘X In the steady state, the rate of this reaction limited the rate of citrate synthetase through decreased availability of oxaloacetate. Flux through the a-ketoglutarate dehydrogenase step was inhibited in isolated heart mitochondria during state 4 respiration, and it was accelerated during the state 4 to state 3 transition.‘“” Activation of the enzyme. under these conditions, was associated with decreased mitochondrial levels of both NADH and succinyl CoA. (41 Integrated Control oJ’ the Citric Acid Q~le in Intact Tissue: As indicated in the discussion of individual enzymes, the mechanism and the site of control of the citric acid cycle appears to depend upon the type of substrate that is oxidized and upon the metabolic state of the tissue. When glucose was the only exogenous substrate available, the tissue levels of acetyl CoA and citric acid cycle intermediates were low and the rate of the cycle was limited by the availability of acetyl CoA. IBX.li’l Addition of either fatty acids or ketone bodies resulted in increased tissue levels of acetyl CoA and in stimulation of citrate synthesis. With high levels of acetyl CoA, citrate and other cycle intermediates accumulated in the tissue. The rate of citrate synthesis and the extent to which citrate accumulated was limited by the availability of oxaloacetate. In heart muscle, oxaloacetate is produced either from within the cycle by oxidation of malate or from outside the cycle by transamination from aspartate. Conversion of aspartate to oxaloacetate was the major source of extra carbon for accumulation of cycle intermediates in hearts that were oxidizing fatty acids or ketone bodies.16”.‘7’JTransamination between aspartate and a-ketoglutarate will result in net input of carbon into the cycle only if this reaction is coupled to glutamate. pyruvate aminotransferase’7’ (see Fig. 3). Coupling of these two transamination reactions appeared to be the mechanism

UTILIZATION

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of net input or removal of carbon from the total pool of cycle intermediates under several conditions.‘6”.‘70.1R3--IX5 As discussed previously, the rate of the citric acid cycle was a major limiting factor for fatty acid utilization at low cardiac workloads. Acceleration of the cycle with increased cardiac work resulted in a faster rate of FFA uptake and oxidation. The mechanisms of these effects were studied in hearts that were perfused with various levels of exogenous palmitate at both 60 and 120 mm Hg ventricular pressure development. (ai Eflects workloads:

of increased

concentrations

of e,xogenous fatty* acid at low cardiac

The solid lines in Fig. 11 illustrate the effects of increasing the concentration of exogenous paimitate on the tissue levels of citric acid cycle intermediates in hearts that were developing 60 mm Hg peak systolic pressure. The 4000

ACETYL

CARNITINE

400

ACETYL

CoA

r

300 200 *-A 100

- c.

l=d!!c-

400

ISOCITRATE

a-

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l

;-

KETOGLUTARATE

SUCCINYL

400

CoA

f

aor

2000 ,533

OXALOACETATE

ASPARTATE

zooor

ALANINE ,

,

0.4

0.e

PYAUVATE

IO00 b

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PALMITATE

1.2

1.4

0

04

0.6

1.2

16

(mhtl

Fig. 11. Effects of increasing perfusate palmirate and ventricular pressure development levels of intermediates of the citric acid cycle. Experimental conditions were same as Solid lines represent hearts developing 60 m m Hg ventricular pressure and dashed lines those developing 120 m m Hg. Each value represents the mean of 6-12 determinations.

on tissue for Fig. 6. represent

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sequence of events appeared to be as follows: Over the range of O&O..5 mM palmitate, the rate of acetyl CoA production remained the major restriction on fatty acid utilization, and there was only a slight accumulation of acetyl CoA. During this time, however, a major reshuffling of carbon occurred among the intermediates of the citric acid cycle. There was a net shift of carbon into citrate and isocitrate, at the expense of the intermediates in the portion of the cycle from a-ketoglutarate through oxaloacetate. The levels of succinate and fumarate were not measured but based on the decrease in succinyl CoA, malate and oxaloacetate. it may be inferred that the levels of these intermediates also decreased. Aspartate decreased by about 650 mpmole/g dry wt, while glutamate increased to about the same extent, indicating that net input of carbon by transamination from amino acids did not occur. For each oxaloacetate formed from aspartate, one cr-ketoglutarate was converted to glutamate. Thus, coupling of the glutamate:pyruvate aminotransferase reaction to aspartate:a-ketoglutarate aminotransferase did not appear to account for input of carbon into the cycle. The level of alanine actually decreased rather than increased as would be expected if coupling had occurred. The decrease in pyruvate, which would be expected by coupling of the two transamination reactions, probably resulted from inhibition of glycolysis due to increased levels of citrate rather than increased conversion to alanine. Under these conditions, transamination between aspartate and a-ketoglutarate appeared to function only in short circuiting the cycle from cu-ketogiutarate to formation of oxaloacetate resulting in a net shift of carbon from the span of the cycle between a-ketoglutarate through oxaloacetate into the span consisting of citrate and isocitrate. The decrease in succinyl CoA, malate and oxaloacetate, however, could account for only about 305’4 of the net increase in citrate and isocitrate indicating that much of the extra carbon must have come from other metabolites, perhaps from decreased levels of succinate and fumarate. At fatty acid concentrations above 0.5 mM, the large increase in acetyl CoA indicated that the rate of the citric acid cycle limited the rate of fatty oxidation. At these levels of exogenous fatty acid, the major restriction on the cycle occurred at the level of isocitrate dehydrogenase. This conclusion was based on the large increases in citrate and isocitrate and a decrease in cu-ketoglutarate. In the presence of high levels of acetyl CoA, accumulation of citrate was limited by the availability of oxaloacetate. No further changes occurred in the levels of aspartate as the concentration for fatty acid was raised from 0.5 to 1.2 mM indicating that transamination reactions did not participate in the additional shift of carbon within the cycle. The additional increase in citrate and isocitrate appeared to occur through further decreases in the levels of intermediates in the cycle between cY-ketoglutarate and oxaloacetate, principally through decreased a-ketoglutarate. This shift in carbon could be accounted for by inhibition of isocitrate dehydorgenase, which would restrict the cycle at this level and allow the intermediates from a-ketoglutarate through oxaloacetate to decrease. Inhibition of this enzyme could have resulted from an increased NADH/NAD ratio. An increased NADH/NAD ratio. as the concentration of palmitate was increased, was indicated by a rise in the malate/oxaloacetate, glutamate/a-ketoglutarate and isocitratela-ketoglutarate ratios (Fig. 12).

UTILIZATION

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317

LIPIDS MALATE

/ OXALOACETATE

ISOCITRATE

/a-KETOGLUTARATE

GLUTAMATE

/ a- KETOGLUTARATE

250Fig. 12. Effects of fatty acid concentration and ventricular pressure development on malate/ and oxaloacetate, isocitrate/a-ketoglutarate. glutamate/a-ketoglutarate ratios. These ratios were calculated from data shown in Fig. 11. Solid lines indicate effects of raising concentration o! exogenous palmitate in hearts developing 60 mm Hg peak systolic pressure. Dashed lines represent hearts that were developing 120 mm Hg ventricular pressure.

200150-

IJO-

0

.-

---.---1 0.4 PERFUSATE

/M

1 0.6 PALMITATE

1 1.2

1 1.6 (mM)

Another explanation for the shift in carbon from the latter portion of the cycle into citrate and isocitrate would be that a-ketoglutarate was transported out of the mitochondrial matrix at a faster rate and converted to isocitrate in the cytosol by the NADP-linked isocitrate dehydrogenase. This would effectively short circuit the cycle and account for the accumulation of citrate and isocitrate. It is known that most of the increase in cycle intermediates when FFAs are oxidized occurs outside the mitochondrial matrix. If the high levels of NADH also reflected high levels of NADPH as the concentration of palmitate was raised, then conversion of cY-ketoglutarate to isocitrate in the cytosol would be increased. Increased car(b) Eflects of increased cardiac work on fhe citric acid cycle: diac work accelerated the rate of palmitate uptake (Fig. 4) and oxidation as indicated by a fourfold increase in the rate of CZ40, production from U-C14-palmitate (Fig. 10). A major factor in this effect of cardiac work was an acceleration of the citric acid cycle and increased oxidation of acetyl CoA (dashed lines, Fig. 11). The decreased levels of acetyl CoA that were associated with a fourfold increase in the rate of CO, production indicated that citrate synthetase was stimulated by increased cardiac work. Stimulation of this enzyme appeared to result from higher tissue levels of oxaloacetate at all concentrations of perfusate palmi-

318

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tate. Increased levels of oxaloacetate resulted from both a stimulation of reactions within the cycle and from transamination of glutamate to a-ketoglutarate. The specific reactions that were stimulated in the cycle by increased cardiac work depended upon the concentration of exogenous palmitate. At low levels of fatty acids, increased cardiac work accelerated production of oxaloacetate by stimulation of a cr-ketoglutarate dehydrogenase and malate dehydrogenase as indicated by the decreased levels of a-ketoglutarate and malate and by the increased levels of succinyl CoA and oxaloacetate (Fig. I I). At higher levels of exogenous palmitate, on the other hand, stimulation of isocitric dehydrogenase appeared to be the major factor involved in increased production of oxaloacetate. This conclusion was based on decreased levels of citrate and isocitrate and increased levels ofa-ketoglutarate, succinyl CoA, malate. and oxaloacetate. At all concentrations of perfusate palmitate, increased cardiac work resulted in lower levels of glutamate and higher levels of aspartate, indicating a reversal in the direction of the transamination reaction with increased cardiac work, as compared to increased fatty acid at low levels of cardiac work. In other experiments, not shown here, the tissue levels of alanine remained essentially unchanged with increased cardiac work. This indicates that coupling of glutamate:pyruvate aminotransferase played only a minor role in the effects of increased cardiac work. The increased levels of pyruvate at the higher cardiac workload probably resulted from increased production from glycolysis. The rate of glycolysis was accelerated with increased cardiac work even in the presence of 5 mM acetate.64 In hearts that were perfused with glucose alone, increased cardiac work stimulated the rates of a-ketoglutarate dehydrogenase and malate dehydrogenase. When both glucose and palmitate were present as substrates, increased work stimulated isocitrate dehydrogenase (the major restraint on the cycle with high ievels of fatty acids), as well as malate dehydrogenase. Stimulation of these enzymes resulted from a decreased NADH/NAD ratio as indicated by a large decrease in the malate/oxaloacetate, glutamate/a-ketoglutarate and isocitrate/cr-ketoglutarate ratios (Fig. 12). A lower NADH/NAD ratio resulted from a faster rate of oxidative phosphorylation as shown by a two- to three-fold increase in the rate of oxygen consumption (Fig. 5). Tissue Lipids As mentioned earlier, activated fatty acids are used for synthesis of triglycerides and phospholipids as well as for oxidation by mitochondria. The tissue content of complex lipids appeared to be related to the circulating level of free fatty acids.“.14.15.85.1R6.1K7 The triglyceride content of hearts removed from fasted or diabetic animals was higher than normal.“” This effect was attributed to elevated levels of plasma fatty acids and increased tissue content of acyl CoA derivatives. In addition, the activity of lipoprotein lipase was increased in hearts from diabetic or fasted animals which would facilitate uptake of plasma triglycerides.13”.18X In hearts perfused with C4-labeled fatty acid, the proportion of label that appeared as CO, was decreased and incorporation into triglyceride was increased during hypoxia. 4*9LThis effect of hypoxia probably resulted from reduced rates of P-oxidation and increased levels of long-chain acyl CoA. The

UTILIZATION

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319

levels of acetyl CoA in hearts perfused with buffer containing octanoate or palmitate were greatly reduced by hypoxia indicating that P-oxidation was inhibited.‘70 Triglyceride lipase of heart muscle appeared to be under hormonal control in much the same way as adipose tissue lipase. 18g--Ig’ Both epinephrine and glucagon increased the tissue level of cyclic AMP and activated lipolysis. This activation was inhibited by (?-adrenergic blocking agents. Unlike its effect on adipose tissue, insulin did not have an antilipolytic action in heart muscle. However, when hearts were removed from fasting or diabetic animals and perfused in vitro, the rate of lipolysis was faster than normal.ss An increased rate of lipolysis in these hearts may have been related to the high tissue content of triglycerides. Triglycerides, but not phospholipids can be used to support energy metabolism in heart muscle.“. I4~15~85.187~192~ 193 There are about 50 pmoles of fatty acids/g dry tissue stored in the heart as triglycerides. If all of the triglycerides were used, enough fatty acids are present to support the total oxygen consumption of the heart for about 45 min. However, in hearts perfused without exogenous substrate, 30%50% of the triglyceride fatty acid remained in the tissue even after the heart stopped beating, indicating that only about half of the triglyceride fatty acids are available for energy metabolism. As discussed earlier, exogenous fatty acids are utilized in preference to either exogenous glucose or tissue glycogen. Endogenous lipid was also utilized in preference to exogenous carbohydrate. In hearts perfused with glucose as the only exogenous substrate, oxidation of glucose accounted for only about 40% of the total oxygen consumption at low levels (60 mm Hg) of ventricular pressure development.‘n.L4~8” The remainder of the oxygen consumed was presumably used to oxidize tissue lipid. With increased cardiac work, oxidation of glucose accounted for about 70% of the oxygen consumption.‘4 The rate of utilization of tissue lipid was accelerated by increased ventricular pressure development in rat hearts perfused with buffer containing either no substrate, glucose or a combination of glucose and acetate;1aJ”,1g4 but utilization was not increased in hearts that were supplied with a combination of glucose and palmitate. ‘*,x When palmitate was present, the tissue levels of long-chain acyl CoA derivatives were high, which may account for the low rates of lipid utilization with increased cardiac work. Myocardial

Fatty Acid .‘$vtzthesis

Organs such as the liver and adipose tissue are capable of synthesizing large amounts of fatty acids. The heart, on the other hand, is primarily an energy utilizing tissue, but apparently it is capable of both de novo synthesis of fatty acids and elongation of preexisting fatty acids.1g”-‘97 Several functions for fatty acid synthesis in heart muscle have been proposed. Fatty acid synthesis in heart muscle is exclusively a mitochondrial function. The enzymes responsible for elongation of preexisting fatty acids are located on the outer mitochondrial membrane and de novo synthesis is associated with the inner membrane and matrix. This arrangement of the synthetic systems led Whereatlg6 to suggest that one function of fatty acid synthesis in the heart might be to facilitate oxida-

320

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tion of cytoplasmic NADH. Elongation of fatty acids on the outer mitochondrial membrane would utilize cytoplasmic NADH and facilitate the transfer of cytoplasmic reducing equivalents into the mitochondria in the form of longer chain fatty acids. NADH was more effective than NADPH in stimulating fatty acid synthesis by heart mitochondria. lg8 De novo synthesis of C,, to C,, acids inside the mitochondria could reoxidize mitochondrial NADH during states of reduced respiratory chain activity (i.e., in ischemic tissue) and thereby facilitate substrate level phosphorylation. Recent studies have shown that about 50% of the fatty acids present in heart mitochondria are /3-hydroxy acids.lg9 At least 50% of the de novo synthesis that occurred in mitochondria resulted in production of P-hydroxy acids. It is thought that these hydroxy acids could act as a shuttle for reducing equivalents between the cytosol and mitochondrial matrix. Extramitochondrial elongation of fatty acid may utilize acetyl CoA formed by carnitine acetyl transferase activity, and translocation of the elongated acyl CoA ester into the mitochondrial matrix would function to shuttle acetyl units back into the mitochondria. The main role of fatty acid synthesis in heart may be production of structural lipids. g5 In hearts perfused with radioactive acetate, acetyl units were incorporated into C,, acids by de novo synthesis and into C,, acids by elongation. In these hearts, labeled fatty acids were distributed in phospholipids (78%) triglycerides (17%), and free fatty acids (4%). SUMMARY

Fatty acids represent a very important, if not the most important, substrate for myocardial energy metabolism. The heart derives fatty acids from circulating FFA bound to albumin and from plasma triglycerides. The rate of extraction of albumin bound FFA depends upon the albumin:FFA molar ratio and the metabolic state of the tissue. Since the albumin concentration in vivo is fairly constant, the albumin:fatty acid ratio is determined by the concentration of fatty acids in the serum. The fatty acid concentration is in turn regulated by dietary intake, de novo synthesis in the liver and adipose tissue, and the rate of fatty acid mobilization from adipose tissue. Mobilization from adipose tissue is increased during states of substrate deficiency such as fasting, diabetes or during the postabsorptive state and is decreased during periods of substrate excess. The rate of myocardial utilization of circulating triglycerides depends on the concentration of triglycerides and on the activity of lipoprotein lipases. The activity of these enzymes is under hormonal control and is increased by fasting or diabetes and is decreased by refeeding. Although the rate of FFA uptake by the heart is dependent upon the level of circulating FFA, the rate of uptake at any one concentration of exogenous FFA depends upon the metabolic state of the tissue. The rate of uptake and oxidation was increased by epinephrine (probably as a result of a positive inotropic effect) and by increased ventricular pressure development. The rate of uptake was decreased and the incorporation of fatty acids into tissue lipids was increased by reduced oxygen supply to the tissue. Regulation of fatty acid utilization by the heart is poorly understood. At low concentrations of exogenous fatty acid, the rate-limiting steps for uptake are located prior to formation of acetyl CoA. At 0.4 mM exogenous palmitate,

UTILIZATION

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LIPIDS

321

increased ventricular pressure development accelerated the rates of oxygen consumption, CO2 production from C14-palmitate and palmitate uptake. This faster rate of uptake was associated with an increased tissue content of longchain acyl carnitine and a decreased content of long-chain acyl CoA and FFA. These data suggest that fatty acid uptake was accelerated by increased cardiac work due to an acceleration of carnitine-palmityl CoA transferase and reduced levels of tissue FFA. The lower intracellular levels of FFA would establish a larger concentration gradient between intracellular binding sites and binding sites on plasma albumin, which would accelerate transfer of exogenous fatty acid into the myocardial cells. At higher levels of exogenous fatty acids, the citric acid cycle limited the rate of fatty acid oxidation and uptake. The rate of the citric acid cycle was limited at the level of isocitrate dehydrogenase (due to a high NADH/NAD ratio) and at the level of citrate synthetase (due to reduced availability of oxaloacetate). The limited rate of isocitric dehydrogenase resulted in increased tissue levels of citrate and isocitrate and decreased levels of a-ketoglutarate, succinyl CoA, malate, and oxaloacetate. Accumulation of citrate and isocitrate was limited by the availability of oxaloacetate and a limited rate of citrate synthetase resulted in accumulation of high levels of acetyl CoA. The carbon that accumulated within the cycle as citrate and isocitrate was derived from intermediates in the span of the cycle form a-ketoglutarate to oxaloacetate and by converting aspartate to oxaloacetate. Low levels of oxaloacetate resulted from a high NADH/ NAD ratio, a limited availability of malate, due to inhibition of isocitric dehydrogenase, a reduced level of aspartate and from increased utilization by citrate synthetase. Increased ventricular pressure development accelerated the rate of NADH oxidation as indicated by a faster rate of oxygen consumption, increased the rate of flux through the citric acid cycle as shown by a faster rate of CO, production, and decreased the level of acetyl CoA and increased the rate of fatty acid uptake. Increased cardiac work accelerated citrate synthesis by increased levels of oxaloacetate at all levels of exogenous fatty acids that were studied. At low levels of fatty acid, increased production of oxaloacetate resulted from a stimulation of cY-ketoglutarate dehydrogenase and malate dehydrogenase. At higher levels of exogenous palmitate, the primary effect of increased work was stimulation of isocitric dehydrogenase. The rate of malate dehydrogenase was also increased. These effects of cardiac work resulted from an increased rate of oxidative phosphorylation and a reduced NADH/NAD ratio. Data presented indicated that translocation of long-chain acyl groups from extramitochondrial acyl carnitine to intramitochondrial acyl CoA restricted fatty acid oxidation when the level of exogenous fatty acid was low or when the tissue content of acetyl CoA was decreased by increased cardiac work at high levels of exogenous fatty acid. This restriction was bypassed and a high tissue level of acetyl CoA was maintained in hearts that were perfused with octanoate even when the rate of the citric acid cycie and the rate of octanoate uptake was accelerated twofold by increased cardiac work. With palmitate as suhstrate, increased cardiac work resulted in lower levels of acetyl CoA, acetyl carnitine and long-chain acyl CoA and in higher levels of long-chain acyl

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carnitine, indicating that carnitine:acyl CoA transferase was stimulated. Higher levels of acyl carnitine would help overcome the restriction to acyl translocation across the inner mitochondrial membrane and facilitate fatty acid oxidation. Endogenous triglycerides represent a readily available supply of substrate for energy metabolism. Oxidation of the triglyceride fatty acids stored in the tissue could support normal rates of oxygen consumption for about 45 min. Only about 50% of these fatty acids appeared to be available for oxidation. The rate of triglyceride breakdown was accelerated by increased ventricular pressure development and this effect was reduced by the presence of exogenous long-chain fatty acids, but not by short-chain acids. The rate of triglyceride synthesis was increased by greater availability of circulating fatty acids and higher tissue levels of acyl CoA or by a reduced rate of oxidative metabolism. REFERENCES 1. McGinty. D. A.: Studies on the coronary circulation. 1. Absorption of lactic acid by the heart muscle. Amer. J. Physiol. 98:244, 193 I. In

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