Fatty Acid Metabolism

Under normal conditions, fatty acids and triglycerides are the preferred substrate for the normal heart. Fatty acids are taken up by the cardiac myocyte through facilitative transport via fatty acid translocase (FAT/CD36).⁴ Once inside the myocyte, fatty acids are esterified to fatty acyl-CoA derivatives through a reaction mediated by fatty acyl-CoA synthetase, which utilizes the hydrolysis of ATP to adenosine monophosphate (AMP) to drive the reaction. This energy-requiring step may be of great importance in understanding the decrease in accumulation of the fatty acid analog, [¹²³I]β-methyl-iodophenyl pentadecanoic acid (BMIPP), seen in ischemic myocardium. After this activation, fatty acyl-CoA may be transported into the mitochondria following transesterification with carnitine by carnitine palmitoyltransferase 1 (CPT-1). This step represents the rate-limiting step of fatty acid oxidation and is regulated by cytosolic concentrations of malonyl-CoA; this important regulatory step of fatty acid metabolism is discussed in detail later in the chapter. Once inside the mitochondria, CPT-2 converts the fatty acylcarnitine back into fatty acyl-CoA for entry into the β-oxidation pathway. Another metabolic fate of the cytosolic fatty acyl-CoA is incorporation into triglycerides. It is this fate that predominates for BMIPP because the β-methyl group inhibits it entry into β-oxidation in the mitochondria.⁵

β-Oxidation represents a cycle of reactions that remove sequential 2-carbon acetyl-CoA units from the long-chain fatty acyl-CoA for entry into the TCA cycle. Not only is this set of four reactions necessary for breaking long-chain fatty acids up into smaller units, it also produces reducing equivalents in the form of NADH₂ and FADH₂ with each turn of the β-oxidation spiral. Along with NADH₂ and FADH₂ produced by the TCA cycle, these reducing equivalents drive the electron transport chain of the inner mitochondrial membrane, which is coupled to ATP synthesis and is discussed in detail later. The β-oxidation cycle is regulated by feedback inhibition through the accumulation of NADH₂ and FADH₂, and therefore the activity of this pathway is decreased by ischemia because the NADH₂ and FADH₂ cannot be oxidized to NAD and FAD, owing to decreased flux through the electron transport chain.

Glucose Metabolism

Glucose represents the other major fuel of the heart. The initial transport of glucose across the cell surface membrane represents the rate-limiting step of glucose metabolism and is mediated by facilitative glucose transporters (GLUTs).⁶ Of the 13 described GLUTs, only two, GLUT1 and GLUT4, are expressed to a significant degree in the heart. GLUT1 is present mostly on the cardiomyocyte cell surface and is responsible for basal glucose uptake. In contrast, GLUT4 exists both on the cell surface and in an intracellular pool of membrane vesicles that can translocate to the cell surface in response to insulin (Fig. 2-3). It is therefore GLUT4 translocation that is responsible for insulin-stimulated glucose uptake in the insulin-sensitive tissues of the heart, skeletal muscle, and adipose tissue. The translocation of GLUT4 is also responsible for the enhanced glycolysis observed during ischemia, although the mechanism of this translocation is independent of the insulin signaling pathway as described later in this chapter.^7–9

Figure 2-3 Insulin-stimulated translocation of the facilitative glucose transporter, GLUT4. Binding of insulin to the insulin receptor results in activation of insulin receptor substrates (IRS) 1 and 2. Activation of IRS increases the association of phosphatidylinositol-3 kinase (PI-3 K), which results in the phosphorylation and activation of Akt, ultimately resulting in translocation of GLUT4 from intracellular storage vesicles to the cell surface, increasing the transport of glucose into the cell (top panel). Immunofluorescent microscopy in heart muscle demonstrates diffuse intracellular distribution of GLUT4 under basal (Control) conditions; however, with insulin stimulation (+Insulin), there is redistribution of immunofluorescence to the cell surface (bottom panel).

Once inside the cardiac myocyte, glucose enters into the glycolytic pathway. Although the glycolytic pathway includes 10 separate enzymatic reactions, three reactions play critical roles in regulating glycolytic flux in the heart. The first is the phosphorylation of glucose by hexokinase; glucose-6-phosphate cannot be transported back out of the cell by the glucose transporters and therefore is trapped in the cell. This initial step in the glycolytic pathway requires energy from the hydrolysis of ATP to Adenosine diphosphate (ADP). It is also this reaction that is at the center of viability assessment, which is discussed later in detail. The glucose-6-phosphate that is produced by the hexokinase reaction sits at a branch point and either may continue in the glycolytic pathway or may be shunted into glycogen synthesis. In times of adequate provision of myocardial substrates, glycogen is synthesized for use during metabolic and hemodynamic stress.

The second regulatory step of glycolysis is catalyzed by phosphofructokinase 1 (PFK-1), which converts fructose 6-phosphate to fructose 1,6-bisphosphate and, as with the hexokinase reaction, requires the hydrolysis of ATP to ADP. The activity of PFK-1 is decreased by increases in the cytosolic content of ATP. Therefore, when the energy charge of the cytosol is high, that is, there is abundant ATP, PFK-1 inactivation will decrease glycolysis. The end result is a shunting of glucose to storage as glycogen for use when ATP stores fall.

PFK-1 is also inhibited by citrate, which increases when there is sufficient TCA cycle flux to meet the energetic needs of the cell. This inhibition of glycolysis at the level of PFK-1 by ATP and citrate is the basis of a critical aspect of myocardial metabolism that regulates substrate selection: the glucose/fatty acid, or Randle, cycle. The oxidation of fatty acids in the mitochondria results in an increase in both ATP and citrate, which inhibits PFK-1 and thereby reduces glucose uptake.¹⁰ The operation of the Randle cycle has important implications with respect to myocardial substrate utilization under physiologic conditions such as the transition from the postprandial state, in which insulin stimulation and abundant circulating glucose lead to increased reliance on glucose, to the fasting state, in which the greater concentration of free fatty acids increases fatty acid metabolism, and disease states such as diabetes, in which there is a persistent increase in the free fatty acid concentration.

PFK-1 is also inhibited by decreases in the intracellular pH, which is important in the setting of myocardial ischemia. Specifically, with profound myocardial ischemia (i.e., a > 95% reduction in myocardial blood flow), the lactate and hydrogen ions produced by anaerobic glycolysis cannot be washed out of the myocyte, and the intracellular pH drops dramatically, resulting in cellular damage. The inhibition of PFK-1 by such a drop in pH during severe ischemia slows the production of hydrogen ions. However, this comes at the cost of diminished generation of ATP by anaerobic glycolysis. Because ATP cannot be generated by oxidative metabolism in this setting, this degree of ischemia represents a critical metabolic state in which irreversible myocyte damage can occur if adequate blood flow is not restored.

The third step of glycolysis that contributes to the regulation of glucose uptake and its ultimate conversion to pyruvate is catalyzed by glyceraldehyde 3-phosphate dehydrogenase, which converts glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate through an oxidation-reduction reaction. While glyceraldehyde 3-phosphate is oxidized to 1,3-bisphosphoglycerate, NAD is reduced to NADH₂. Like many of the reactions of the glycolytic pathway, the reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase can be inhibited by the accumulation of its end products. Under normal conditions, the majority of NADH₂ that is formed is transported to the mitochondria through the malate/aspartate shuttle to drive the electron transport chain and does not cause inhibition of glyceraldehyde 3-phosphate dehydrogenase. With ischemia, in which glycolysis is enhanced but the NADH₂ that is produced by the glyceraldehyde 3-phosphate dehydrogenase reaction is not utilized by the mitochondria, cytosolic NADH₂ can accumulate. With mild to moderate ischemia, when there is sufficient blood flow to remove the end products of glycolysis, lactate dehydrogenase will convert pyruvate to lactate with the concomitant oxidation of NADH₂ back to NAD. Under these conditions, glyceraldehyde 3-phosphate dehydrogenase will not be inhibited. However, with severe ischemia, there is insufficient washout of metabolic end products, and pyruvate cannot be converted to lactate. The resulting accumulation of NADH₂ will inhibit glyceraldehyde 3-phosphate dehydrogenase and thereby inhibit anaerobic glycolysis.

Once glucose is metabolized to pyruvate, it is transported into the mitochondria, where it is converted to acetyl-CoA through the action of pyruvate dehydrogenase (PDH). PDH is a multienzyme complex that is regulated by the metabolic status of the cell. Specifically, PDH is inhibited by increased [NADH₂]/[NAD] and [acetyl-CoA]/[CoASH] ratios, both of which occur when there is a relative overabundance of NADH₂ and acetyl-CoA that outstrips the ability of the mitochondria to utilize these metabolites.¹¹ This regulation of PDH activity is mediated by PDH kinase, which phosphorylates and thereby inactivates the PDH complex. In the setting of enhanced fatty acid oxidation, flux through PDH is inhibited by increased PDH kinase activity,¹² providing another level of regulation of substrate selection in the heart. Conversely, PDH activity can be increased by dephosphorylation, which occurs in response to insulin stimulation.¹³ In addition, PDH can be activated by increases in workload through a calcium-dependent mechanism.

In addition to fatty acids and glucose, lactate can be a significant source of ATP production in the myocardium. This is especially true during exercise because the lactate that is released by exercising muscle is avidly taken up by myocardium through the monocarboxylic acid transporter. This exogenous lactate is converted to pyruvate through the action of lactate dehydrogenase, which now will produce additional NADH₂ through the reverse of the reaction described earlier. Because of the high content of lactate dehydrogenase in the myocardium, this enzyme is not rate limiting for lactate metabolism. Rather, it is the regulation of PDH that determines the utilization of lactate by the heart.

Tricarboxylic Acid Cycle Metabolism and the Electron Transport Chain

β-Oxidation of fatty acids, glycolysis of both exogenous glucose and endogenous glycogen, and uptake of exogenous lactate result in the conversion of these fuels to a common energetic currency, namely acetyl-CoA, which enters the TCA cycle by condensing with oxaloacetate to form citrate. The citrate that is formed undergoes subsequent oxidative and decarboxylating reactions in the TCA cycle, which results in the generation of five important compounds that not only help to drive mitochondrial ATP synthesis but are also important with respect to metabolic imaging. The first is the ultimate conversion of the 6-carbon citrate to the 4-carbon oxaloacetate, which is then available for another “turn” of the TCA. This is linked to the second important product, carbon dioxide (CO₂), which is produced through two decarboxylation steps, one mediated by isocitrate dehydrogenase and the other mediated by α-ketoglutarate dehydrogenase. This CO₂ is released from the cell and ultimately leaves the body through the lungs. The third is the production of NADH₂ by isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and malate dehydrogenase. This NADH₂ is used by the electron transport chain to generate the mitochondrial membrane potential required to power the mitochondrial F₀F₁-ATPase that converts ADP to ATP (Fig. 2-4). In addition, FADH₂ is produced by succinate dehydrogenase and also contributes electrons to the electron transport chain for the conversion of oxygen to water, but because of the location of succinate dehydrogenase in the inner aspect of the inner mitochondrial membrane, it does not contribute to the mitochondrial membrane potential. Finally, the high-energy phosphate, guanosine triphosphate (GTP), is generated through substrate-level phosphorylation by succinyl-CoA synthetase, a reaction that becomes important to energy production during ischemia because, like glycolysis, it does not require oxygen to produce high-energy phosphates.¹⁴

Figure 2-4 Coupling of tricarboxylic acid (TCA) cycle flux with adenosine triphosphate (ATP) synthesis. Reducing equivalents in the form of nicotinamide adenine dinucleotide (NADH₂) and flavin adenine dinucleotide (FADH₂) are generated by the TCA cycle and provide electrons for complexes of the electron transport chain. This results in an oxidation back to NAD and FAD and transport of protons from the mitochondrial matrix to the space between the inner (IMM) and outer mitochondrial membrane (OMM), creating a membrane potential. This membrane potential drives ATP synthesis by F₀F₁-ATPase, although the membrane potential can also be dissipated by uncoupling proteins (UCPs), without the production of ATP.

Of great importance to the evaluation of myocardial mitochondrial function by nuclear methods is the fact that there is a direct relationship between the entry of acetyl-CoA into the tricarboxylic acid cycle and the conversion of oxygen to water through the electron transport chain. Because of this coupling of acetyl-CoA metabolism to oxygen consumption, it is possible to determine rates of myocardial oxygen consumption noninvasively using the positron emission tomography (PET) tracer [1-¹¹C]-acetate.¹⁵ Furthermore, a coupling between energy demand and energy production translates to the coupling of TCA flux to ATP synthesis. Specifically, increases in workload result in an increase in cytosolic calcium; this increased cytosolic calcium concentration increases mitochondrial calcium content, which activates not only PDH but the calcium-dependent enzymes, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase, that are part of the TCA cycle.