ExerciseBiochemistryOnline.pdf

EXERCISE BIOCHEMISTRY 1 STUDY GUIDE Energy RER Phases of Metabolism Catabolism Anabolism REDOX NAD NADP FAD 2 STUDY GUIDE Adenosine Triphosphate (ATP) Energy Systems ATP-PCr System Glycolytic System Control of Glycolysis Phosphofructokinase (PFK) Control by Lactate Dehydrogenase (LDH) Control by Pyr...

EXERCISE BIOCHEMISTRY 1 STUDY GUIDE Energy RER Phases of Metabolism Catabolism Anabolism REDOX NAD NADP FAD 2 STUDY GUIDE Adenosine Triphosphate (ATP) Energy Systems ATP-PCr System Glycolytic System Control of Glycolysis Phosphofructokinase (PFK) Control by Lactate Dehydrogenase (LDH) Control by Pyruvate Dehydrogenase (PDH) Control by Glycogenolysis 3 STUDY GUIDE Exercise Speeds up Glycolysis in Muscle Increased Substrates Phosphofructokinase Activation Pyruvate Kinase Activation Oxidative System Oxidative Glycolysis Krebs Cycle Electron Transport Chain 4 STUDY GUIDE Oxidative System (cont) Oxidation of Fat Esterification and Hydrolysis Utilization of Lipids During Exercise Mobilization from Adipose Circulation and Uptake Beta-Oxidation Mitochondrial Oxidation 5 STUDY GUIDE Substrate Shunt During Exercise Protein Metabolism Effects of Training on Protein Metabolism Oxygen Deficit/EPOC Lactate Threshold 6 Many biological factors that depend on production and liberation of energy. Energy: capacity or ability to perform work. Work is the application of a force through a distance. As a result, energy and work are inseparable. Amount of energy released in biological reaction is calculated from amount of heat produced (byproduct of metabolic activity is heat). Energy in biological systems measured in kilocalories (kcals). 1 kcal = amount heat needed to raise 1 kg or 1 liter of water 1oC. 7 Measuring Energy Release Can be calculated from heat produced 1 calorie (cal) = heat energy required to raise 1 g of water from 14.50C to 15.50C 1,000 cal = 1 kcal = 1 Calorie (dietary) 8 BMR measured by amount of heat being produced. Can also measure VO2 by amount of heat production. To support the BMR of average person, takes approximately 3.5 ml/kg/min of O2. Translates into absolute amount of approximately 250 ml/min. Food provides potential energy to maintain normal body function. Potential energy refers to energy of position (where is the energy located). When that energy is released it becomes kinetic energy. Kinetic energy results from metabolic activity. Food sources of carbohydrates, lipids, and proteins provide us with energy needed for metabolic activity. Where: CHO = 4.1 kcal Lipids = 9.4 kcal Protein = 4.1 kcal 9 Substrates: Fuel for Exercise Carbohydrate, fat, protein Carbon, hydrogen, oxygen, nitrogen Energy from chemical bonds in food stored in high-energy compound ATP Resting: 50% carbohydrate, 50% fat Exercise (short): more carbohydrate Exercise (long): carbohydrate, fat 10 Carbohydrate All carbohydrate converted to glucose 4.1 kcal/g; ~2,500 kcal stored in body Primary ATP substrate for muscles, brain Extra glucose stored as glycogen in liver, muscles Glycogen converted back to glucose when needed to make more ATP Glycogen stores limited (2,500 kcal), must rely on dietary carbohydrate to replenish 11 Fat Efficient substrate, efficient storage 9.4 kcal/g +70,000 kcal stored in body Energy substrate for prolonged, less intense exercise High net ATP yield but slow ATP production Must be broken down into free fatty acids (FFAs) and glycerol Only FFAs are used to make ATP 12 Table 2.1 13 Protein Energy substrate during starvation 4.1 kcal/g Must be converted into glucose (gluconeogenesis) Can also convert into FFAs (lipogenesis) For energy storage For cellular energy substrate 14 With activity, possible to identify primary substrate being used. Accomplished by calculating the respiratory exchange ratio (RER). Ratio is the relationship of amount of CO2 produced to amount of O2 consumed. RER = VCO2/VO2 Amount of oxygen consumed, proportional to amount of carbon in molecule. During complete oxidation of a glucose molecule, 6O2 are consumed and 6CO2 molecules produced: 6CO2/6O2 = 1 glucose With lipids, 16 molecules of CO2 are produced for every 23 molecules of O2 consumed: 16CO2/23O2 = 0.7 fats Range of 0.7 – 1.0 represents combination of fat and glucose. At rest, normal RER values approximately 0.75 – 0.81. 15 Phases of Metabolism Metabolism is divided into two phases: Catabolism Anabolism 16 17 Catabolism This includes degradation processes, a series of reactions where biological molecules are broken down into smaller molecules. These degradation processes have double utility: 1. They produce raw materials for the synthesis of our macromolecules 2. They release energy, part of which is used to synthesize ATP Catabolism is favored over anabolism because physical activity requires increased amounts of ATP which is synthesized during catabolic processes. 18 Energy is obtained by the breakdown of foodstuffs. Foodstuffs include: Carbohydrate Fats Proteins 19 There are three stages to extract energy from foodstuffs: 1.Macromolecules in food are broken down to their building blocks. Carbohydrates are converted to glucose and related compounds, lipids are converted to fatty acids and glycerol, and proteins are converted to amino acids. These reactions occur in the digestive tract, part of digestion, or in the cytosol. No ATP is synthesized in stage 1 this is a preparatory stage for ATP synthesis. 20 2 and 3. Occur solely in the cells. Stage 2 products of stage1 are degraded to simpler units. Glucose is catabolized through glycolysis, which accommodates glycerol from breakdown of lipids as well. Fatty acids are subjected to β oxidation, amino acids follow individual routes. Most of the compounds entering stage 2 end up in the acetyl group, which is part of acetyl coenzyme A. A small amount of ATP is produced in stage 2. 21 3. The acetyl group is oxidized to carbon dioxide through the citric acid cycle. Electrons released by this oxidation are taken up by NAD+ and FAD initially. Next, electrons are transferred to oxygen (O2 gets there from the lungs through the bloodstream). O2 is reduced to water in series of exergonic reactions (reaction loses energy during reaction) forming the electron transport chain. Coupled with this is oxidative phosphorylation, which is part of the energy of electron transport is used to synthesize most of a cell’s ATP. 22 Because carbohydrates, lipids, and proteins, are finally burned by O2 to CO2 to produce energy, they are referred to as fuels for cells and therefore for the processes of the human body, and therefore for exercise. 23 24 Anabolism Anabolism includes biosynthetic processes, in which cells form molecules from smaller units. Cells need anabolism to grow and divide, to replace molecules that wear out, and to create energy depots. Biosynthetic reactions utilize intermediate products of catabolism as starting materials. 25 Oxidation-Reduction Reactions (REDOX) Metabolic reactions often involve oxidations and reductions of biological substances. Involves the transfer of electrons. Transfer of electrons not always evident in a reaction, more helpful to look at transfer of hydrogen or oxygen atoms to determine if substance is oxidized or reduced. 26 A substance is oxidized when it loses one or more electrons or hydrogen or it accepts oxygen. A substance is reduced when it accepts one or more electrons or hydrogen or it loses oxygen. 27 When a compound is oxidized another is being reduced. Therefore we speak of oxidation-reduction reactions. 28 Catabolic processes usually include oxidations of metabolites and removal of hydrogen. These oxidations are called dehydrogenations. Anabolic processes include reductions of metabolites by the addition of hydrogen. 29 These reductions are called hydrogenations. These hydrogen transactions are performed by specialized compounds, the most common being nicotinamide adenine dinucleotide or NAD. 30 NAD consists of an AMP unit and a similar unit containing nicotinamide instead of adenine. Nicotinamide is a form of niacin (Vitamin B3, helps turn food into energy). 31 NAD exists in two forms, oxidized and reduced. Oxidized bears one positive charge. This one positive charge means we symbolize this form of NAD as NAD+ 32 When a metabolite participating in an oxidation-reduction reaction along with NAD+ is oxidized, it loses a hydrogen, which is detached. Thus, H- (called hydride ion) is removed. H- is then transferred to the nicotinamide ring, and converts NAD+ to NADH. NADH is the reduced form of NAD. 33 NADP Nicotinamide adenine dinucleotide phosphate. Differs from NAD in having an additional phosphoryl group attached. NADP comes in two interconvertible forms: NADP+ NADPH 34 Flavin adenine dinucleotide, a H acceptor in metabolite oxidations. Consists of an AMP unit and a flavin mononucleotide (FMN). FMN derived from vitamin B2 (riboflavin). 35 FAD accepts two H from the compound being oxidized, therefore converting FAD to FADH2, the reduced form of FAD. By accepting H, NAD+, NADP+, and FAD serve as oxidizing agents or oxidants. By donating H to metabolites, NADH, NADPH, and FADH2 serve as reducing agents. 36 Additionally, NADH and FADH2 donate H to oxygen. This donation of H to oxygen marks the end of oxidation of metabolites within cells. This renders oxygen the ultimate oxidant in the body, whereas NAD+ and FAD act as intermediates in this oxidationreduction relay. 37 38 ADENOSINE TRIPHOSPHATE (ATP) Energy liberated during breakdown of food not directly used to do work. Used to manufacture ATP which is stored in muscle cells. 39 Structure of ATP is adenosine and 3 phosphate groups. Bonds between the two terminal phosphate groups are high energy bonds. When one of high energy bonds is broken, liberate 7-12 KCALS of energy, and adenosine diphosphate (ADP) plus inorganic phosphate (Pi) are formed. 40 Energy released during breakdown of ATP represents immediate source of energy that can be used by muscle cell to perform work. 41 Figure 2.4 42 Can use water to break high energy phosphate bond. Process referred to as hydrolysis. Reverse this reaction to reform ATP, high energy source phosphocreatine (PCr) is required. 43 When breakdown of ATP occurs without oxygen, is called anaerobic metabolism. Breakdown occurs in presence of oxygen, called aerobic metabolism. Aerobic conversion of ADP to ATP is oxidative phosphorylation (add a phosphate). 44 Three methods which cells generate ATP: ATP-PCr system Glycolytic system Oxidative system 45 Bioenergetics: Basic Energy Systems ATP storage limited Body must constantly synthesize new ATP Three ATP synthesis pathways ATP-PCr system (anaerobic metabolism) Glycolytic system (anaerobic/aerobic metabolism) Oxidative system (aerobic metabolism) 46 ATP-PCr System Simplest of energy systems. Stores ATP and PCr. PCr used to rebuild ATP and help maintain relatively constant supply. Energy transfer from PCr crucial during transitions from low to high energy demand such as beginning of exercise. At this time energy requirements exceed energy provided from breakdown of stored nutrients. Concentration of PCr in cell approximately 4 to 6 times greater than ATP. Function for PCr provide energy for ATP resynthesis. 47 Release of energy from PCr facilitated by enzyme creatine kinase (CK). Enzyme acts on PCr to form separate Pi and creatine. Equation: CK PCr + ADP ATP + Cr Energy released can be used to couple Pi to ADP molecule forming ATP. This is a reversible reaction. 48 Figure 2.5 49 With ATP-PCr system, energy released from ATP by splitting of phosphate group, cells can prevent ATP depletion by reducing PCr, providing energy to form more ATP. Is a rapid process and can be accomplished without special structures within the cell. This process does not require presence of O2, said to be anaerobic. Process of reducing PCr occurs at first few seconds of exercise. At exhaustion both ATP and PCr levels quite low and unable to provide energy for further contractions and relaxations. 50 Figure 2.6 51 Capacity to maintain ATP levels with energy from PCr is limited. These stores can only maintain energy needs for about 3 – 15 seconds. Only means which PCr can be reformed from Pi and Cr is from energy released by breakdown of ATP. Occurs during recovery from exercise. Because we can only rely on ATP-PCr system for energy over short period of time, we must rely on other processes for ATP formation. 52 GLYCOLYTIC SYSTEM 53 54 Involves breakdown of glucose for liberation of energy. Process is glycolysis, breakdown of glucose is via glycolytic enzymes. Blood glucose comes from digestion of carbohydrate and breakdown of liver glycogen. Glycogen is synthesized by glucose via glycogenesis. To use glucose or glycogen for energy, must be broken down into glucose-6-phosphate (G6P). conversion of glucose into G6P requires 1 ATP. However, glycogen to G6P does not require ATP. Once the G6P is formed, glycolysis begins. 55 56 Glycolysis involves 10 enzymatically controlled reactions. Rate limiting enzyme in glycolysis is phosphofructokinase (PFK). This helps determine the speed of glycolysis. In initial stages of glycolysis must invest 2 ATP to allow process to continue. After investing 2 ATP we split fructose-6-phosphate into fructose 1,6-diphosphate. 57 58 Through degredation of fructose 1,6-diphosphate to pyruvate or lactic acid, is a total of 4 ATP which are generated. Since 2 ATP were invested early on, the net gain of ATP is 2 when glucose is used. (1 ATP needed to breakdown glucose) If glycogen is used, only 1 ATP is invested early on, and net gain is 3 ATP. 59 60 During glycolysis two pairs of hydrogen atoms are removed from substrate and their electrons are passed to NAD+ to form NADH (the substrate is oxidized and NAD is reduced). If processed directly through the respiratory chain, oxidation of NADH will produce 3 ATP. In some instances we pass electrons to FAD forming FADH2. When this is oxidized, 2 ATP are generated. Within glycolysis, the ultimate end product is pyruvate/pyruvic acid. This compound is the end result during moderate levels of exercise, and in the presence of oxygen is converted into acytel coenzyme A (acetyl CoA) to enter into oxidative metabolism. During strenuous exercise, the energy demands exceed either the oxygen demands, or rate of utilization. Result, the rate of production of hydrogen joined to NADH exceeds that rate that it can be processed through the respiratory chain (ETS). 61 62 Excess hydrogen ions combine with pyruvate and ultimately form lactic acid. This is catalyzed by lactate dehydrogenase (LDH). Demands of glycolytic system are higher than its rate. The NADH cannot transport the hydrogen ion through the respiratory chain. Although tend to think of lactic acid as bad, or metabolic waste, can be used as fuel in some instances. Formation of lactate with strenuous exercise can be used for synthesis of glucose via Cori Cycle. This is via a process known as gluconeogenesis, the formation of new glucose (Cori Cycle takes place in the liver). Energy pathway of anaerobic glycolysis is primary metabolic pathway for events lasting from 1-3 minutes. 63 64 The Control of Glycolysis Two kind of control predominate glycolysis: Feed forward Feedback 65 Feed forward mechanisms set the rough gain in metabolic regulation. Feedback mechanisms accomplish the fine-tuning of energy supply to demand. Feed-forward and feedback controls are illustrated by their various effects on the level and movement of molecules through the G6P pool. 66 67 In feed-forward control of glycolysis, factors that increase G6P levels tend to stimulate glycolysis. Feed-forward factors include stimulation of glycogenolysis (by epinephrine and contractions) and glucose uptake (by contractions and insulin). Therefore, with exercise of moderate to high intensity, blood glucose rises because of the stimulation of hepatic (liver) glucose production which increases faster than the increase in muscle glucose uptake. Feedback controls involve changes in levels of metabolites that result from glycolysis or from muscle contraction. A decline in blood glucose concentration such as occurs at the end of exercise is probably the most important feedback control in normal healthy subject. Feedback control usually resides at the PFK step and can either 68 speed (stimulate) or slow (inhibit) regulatory enzymes. Phosphofructokinase (PFK) A dominant factor in the regulation of glycolysis is the activity of PFK. Two forms of PFK exist: PFK-1 (for glycolysis) PFK-2 (for glycogenolysis) In muscle PFK-1 predominates. 69 PFK is probably the rate-limiting in muscle when glycolysis is rapid during exercise, but factors such as ATP, PCr, and citrate PFK to slow its activity during rest. When exercise starts, immediate changes in the relative concentrations of PFK modulators increase its activity. Pyruvate kinase (helps make pyruvate), hexokinase (helps make G6P), and LDH are other important controlling enzymes of glycolysis. 70 71 Control by Lactate Dehydrogenase (LDH) The terminal enzyme of glycolysis, which results in the formation of lactic acid from pyruvic acid, is LDH. When glycolysis is slow, LDH is in competition with mitochondria for pyruvate. However, LDH is an enzyme of significant content in muscle, especially type II muscle. Because the free energy yield of LDH is large, the reaction to LDH proceeds actively to completion. Therefore lactate is always formed and as a consequence resting muscle always produces and releases lactate on a 72 net basis. There are two basic types of LDH: Muscle Heart These LDH types are found predominantly in white skeletal muscle and heart. 73 Control by Pyruvate Dehydrogenase (PDH) Although a mitochondrial and not a glycolytic enzyme, PDH is a key enzyme whose activity can affect the rate of lactate production. When PDH is active, pyruvate can be diverted to the mitochondria for oxidation. By competing with LDH for pyruvate, PDH indirectly affects the NADH/NAD+ ratio ( NADH; NAD) and therefore the rate of glycolysis (slows glycolysis). 74 Glycolytic flux and the competition between cytoplasmic LDH and mitochondrial PDH for pyruvate affect the ratio of pyruvate to lactate as well as the ratio of NADH to NAD+. In general cytoplasmic reduction (NADH) slows glycolysis whereas oxidation (NAD) speeds glycolysis. 75 However, during exercise, glycogen breakdown is greatly accelerated and glycogen not glucose is the major precursor for glycolysis. During steady state exercise at 65% of max, glycogen breakdown can exceed glucose uptake by four to five times. Therefore glycolysis is said to be under feed-forward control. 76 EXERCISE SPEEDS UP GLYCOLYSIS IN MUSCLE Exercise can augment the glycolytic rate in a muscle by hundreds of times and by more than one mechanism. 77 Increased Substrates First substrate availability increases. As glycogenolysis is accelerated, there is a rise in the concentration of glucose 6-phosphate. 78 Furthermore, the exercising muscles take up more glucose from the blood mainly because of two factors: Enhanced blood flow to the active muscles (up to 20 times the flow at rest) Increased number of glucose transporters in the plasma membrane 79 Cells take up glucose through integral proteins of the plasma membrane called glucose transporters and abbreviated as GLUT. Muscle fibers contain several forms of these proteins which perform passive transport of glucose to the cytosol. GLUT4 the most abundant form is distinct in that it is not always present in the plasma membrane. Instead it commutes between a population of intracellular vesicles and the sarcolemma or the transverse tubule membrane. 80 81 The movement of GLUT4 from the intracellular vesicles to the plasma membrane provides a means of controlling glucose uptake by a muscle fiber, the more GLUT4 molecules are present in the plasma membrane, the more glucose will enter the cell. Exercise augments the movement of GLUT4 to the plasma membrane, resulting in higher glucose uptake. The biochemical mechanism of this event is not known, but it appears to be triggered by calcium release from the SR. Insulin also augments glucose uptake by muscle fibers through the movement of GLUT4 to the plasma membrane. 82 Phosphfructokinase Activation The second way in which exercise speeds up glycolysis is through other substances binding to PFK that provide regulation of PFK, the enzyme catalyzing the third reaction. PFK is inhibited by ATP ( ATP; PFK) The inhibition is enhanced by PCr and relieved by AMP ( AMP; PFK) This means that the enzyme is relatively inactive in a resting muscle in which the ATP and PCr are high, while the AMP is low. However when the ATP and PCr begin to decrease at the onset of exercise, whereas AMP increases, PFK is activated. 83 Finally the enzyme is inhibited at the acidic pH. The muscle pH becomes acidic during exercise because of the anaerobic breakdown of carbohydrates, most of which is glycolysis. It has been proposed that a decrease in glycolytic rate because of PFK inhibition at low pH may protect the muscle fibers and the blood from an excessive and hazardous fall in pH. 84 Pyruvate Kinase (helps make pyruvate) Activation The regulation of pyruvate kinase is of interest, the enzyme catalyzing the last reaction of glycolysis. ATP and PCr inhibit pyruvate kinase but ADP activates it. The changes in the concentrations of the three compounds (ATP, PCr, ADP) during exercise favor the activation of the enzyme. 85 86 Oxidative System The oxidative system final system of cellular energy production. Is most complex of 3 energy systems. In contrast to anaerobic ATP production, oxidative system has much energy yield. Therefore aerobic metabolism is primary method of energy production during endurance events (also predominately used at rest). Oxidative production of ATP involves 3 processes: Glycolysis Krebs cycle (Citric Acid Cycle and/or TCA cycle) Electron transport chain 87 Glycolosis Plays a a role in both anaerobic and aerobic metabolism. Occurs outside mitochondria. Process is same with or without oxygen, is the presence of oxygen which determines the fate of pyruvic acid. With oxygen, pyruvic acid is converted to Acetyl CoA by losing CO2. Results in pyruvic acid becoming an acetyl group which combines with co-enzyme A to form acetyl CoA, before entering Krebs. 88 KREBS CYCLE 89 Figure 2.9 90 Occurs within mitochondria. CO2 produced, oxidation and reduction occur, ATP produced. CO2 removed from pyruvic acid and all CO2 produced is diffused into blood and carried to lungs where it is eliminated from body. Oxidation in Krebs, electrons are removed in the form of hydrogen atoms. At 4 different sites in Krebs, hydrogen is removed and passed through electron transport chain where end result is formation of water and ATP units. 91 Figure 2.9 92 Most important function of Krebs is generation of electrons for transfer to respiratory chain by NAD and in one instance FAD. At end of Krebs, 2 ATP have been formed and substrate (CHO) has been broken down into carbon and hydrogen. Events in Krebs happen twice since 2 molecules of pyruvate are formed from 1 glucose molecule in glycolysis. 93 Figure 2.9 94 Electron Transport Chain Hydrogen that is released during Krebs and combined with NAD or FAD is carried to electron transport chain or respiratory chain. Hydrogen ions and electrons enter electron transport chain through FADH2 and NADH and are transported to oxygen via electron carriers, end product is water. Electron carriers often referred to as cytochomes. They have iron as an important part of structure. 95 Because iron is important in steps leading to oxidative phosphorylation, people with iron deficiencies have little energy and are lethargic. 96 During aerobic metabolism, most of total ATP generated in electron transport chain. Is total of 32-33 ATP generated with complete breakdown of a glucose molecule. Range in ATP generation due to whether an NAD or FAD is used for electron transport. Oxidative metabolism used in events lasting longer than 3 minutes. 97 Figure 2.8 98 Oxidation of Fat Biological role of lipids is that they are energy storage forms held in reserve for use when carbohydrate energy availability is limited or usage not fully activated. For a given amount of oxygen CHO oxidation provides more energy than does lipid oxidation. (Less oxygen needed to oxidize CHO) However, per unit weight stored in the body, lipids are highly efficient storage forms that yield several times more energy than do carbohydrates. 99 CHO is the preferred fuel while dietary fat is the preferred energy storage form. Lipid is stored mainly in adipose tissue and the liver. Additionally lipid can be stored within muscle cells and to some extent in the blood. Despite the large quantity of lipid available as fuel, the processes of lipid utilization are slow to be activated and proceed at rates significantly slower than the processes controlling carbohydrate catabolism. As opposed to CHO, fat catabolism is purely an aerobic process, which is best developed in the heart, liver and type I muscle fibers. 100 The sparing of glucose and glycogen metabolism by fats during prolonged exercise in highly trained individuals slows the depletion of these essential nutrients. Thus increased fat oxidation capability due to training and genetic endowment greatly enhances endurance. Stored lipids most plentiful source of potential energy. Glycogen stores may be able to provide on average 1200 to 2000 KCAL of energy, lipids can provide about 70,000 to 75,000 KCAL of energy. Triglycerides, phospholipids, cholesterol, many classifications of lipids. Triglycerides are major energy source. Are stored in fat cells and skeletal muscle fibers, must be broken down to basic unit (glycerol and 3 FFA) to be used for energy. 101 Esterification and Hydrolysis The making of triglycerides is called esterification, it involves attachment of a fatty acid to glycerol by means of an oxygen atom. Lipolysis is the process of triglyceride hydrolysis. Esterification and lipolysis are essentially reversals of each other. 102 Triglycerides synthesized in plants or animals are consumed by humans and then digested in a process involving lipolysis or stored through the re-esterification of fatty acids delivered to adipose tissue. The mobilization of triglycerides stored in adipose tissue involves another lipolysis. In skeletal muscle, adipose tissue, and probably heart, αglycerolphosphate (not glycerol; which is also used to make triglyceride) is the backbone substrate for triglyceride synthesis. With triglyceride breakdown, the glycerol goes to the liver when not used to synthesize triglyceride in skeletal muscle, adipose tissue, and heart. 103 In these tissues (skeletal muscle, adipose tissue, and heart) α-glycerolphosphate is derived from glycolysis, that is, from CHO. 104 This knowledge can be helpful for building body fat reserves in ultraendurance events and also for understanding why a diet with too many calories and rich in simple carbohydrates (sugars and starches) plus fats makes people obese. (increased α-glycerolphosphate from CHO makes triglycerides). Muscle heart and adipose cells cannot reuse glycerol for triglyceride synthesis because the cells lack the enzyme glycerol kinase, the enzyme responsible for phosphorylation of glycerol to α-glycerolphosphate; reesterificaiton of glycerol to fatty acids to form triglyceride is not thought to be possible in myocytes, cardiocytes, or adipocytes. 105 Glycerol kinase converts glycerol to α-glycerolphosphate which can then synthesize triglyceride. 106 Due to the action of α-glycerolphosphate in tissues, reeserification is a process relegated to the liver. 107 THE UTILIZATION OF LIPIDS DURING EXERCISE Lipid utilization is a complicated process that usually begins at one site (adipose tissue) and ends at another (skeletal muscle mitochondria). The metabolism at each of these sites and at intervening sites is well controlled, so the ultimate process, lipid oxidation is a highly integrated process. 108 The processes of lipid metabolism during exercise can be summarized as follows: 1. Mobilization – the breakdown of adipose and intramuscular triglycerides 2. Circulation – the transport of FFA from adipose to muscle 3. Uptake – the entry of FFAs into muscles from blood 4. Activation – raising the energy level of fatty acids preparatory to catabolism 5. Translocation – the entry of activated fatty acids into the mitochondria 6. β-Oxidation – the production of acetyl-CoA from activated fatty acids and the production of reducing equivalents (NADH and FADH2) 7. Mitochondrial oxidation – Krebs cycle and electron transport chain activity 109 Mobilization from Adipose Lipoprotein Lipase (LPL) is stimulated by insulin and glucagon and promotes fat storage, hormone sensitive lipase (HSL) stimulates fat breakdown, is inhibited by insulin and is stimulated by other hormones including the catecholamines and growth hormone. 110 Two activators of the HSL system (epi and GH) reach adipose tissue via circulation, whereas norepi is released locally by sympathetic nerve endings within adipose tissue. Compared to the release of GH which is slow, the release of the catecholamines (epi and norepi) is relatively rapid. The catecholamines are therefore responsible for the initiation of lipolysis at exercise onset. It takes 10 to 15 minutes for blood levels of GH to increase during exercise. GH helps to maintain lipolysis during prolonged exercise. 111 An important issue is the source of blood glycerol during exercise. There has been no documented evidence of the presence of glycerol kinase in either adipocytes or myocytes. Glycerol kinase exists mainly in the liver, where in the presence of ATP, glycerol is phosphorylated to αglycerlphosphate (used to synthesize triglyceride). 112 In liver glycerol also serves a minor role as a gluconeogenic (making of new glucose) precursor during exercise. Alternatively, after glycerol conversion to αglycerlphosphate in liver, the carbon released from glycerol can be used in glycolysis. 113 Because they are insoluble in fluid, fatty acids must be carried in the blood. The blood protein albumin carries almost all the FFAs and therefore a majority of the total lipids transported in the blood. Even though the quantity of lipids existing as FFAs in the blood at any one time is only a small part of the total blood lipid content, the turnover of blood FFAs is very rapid (due to rapid uptake). Therefore, the contribution of FFAs to the fuel supply during rest and exercise far exceeds the contribution of other blood lipids such as triglycerides. 114 Circulation and Uptake Because the uptake of FFAs in lipid delivery depends to a large extent on the arterial fatty acid content, the rate of adipose lipolysis directly affects FFA uptake by muscle. A key to lipid oxidation during exercise is the arterial level of FFAs. Any factor that stimulates adipose lipolysis and raises blood FFA levels could promote exercise endurance if substrate supply is limiting. 115 The rate of blood flow through muscle is a major determinant of FFA uptake and utilization during exercise. The enhancement of cardiac output and muscle blood flow by endurance training is a major factor determining the ability to oxidize lipids during exercise. The greater the muscle blood flow, the greater the delivery, uptake, and utilization of FFAs by muscle during exercise. 116 The uptake of fatty acids into muscle from blood albumin is accomplished through a specific receptor site on the sarcolemma. The receptor is sarcolemmal fatty acid binding protein (SFABP), also known as plasma membrane fatty acid binding protein (FABPpm). Evidence suggests that S-FABP works in conjunction with a second protein that facilitates translocation of fatty acids across the sarcolemma. 117 This second protein is fatty acid transporter (FAT). The S-FABP is one of a family of FABP’s that exist throughout the muscle cell matrix. Together the muscle cell FABPs function to transport fatty acids throughout the cell. 118 As a result of there being a variable number of receptor binding sites in sarcolemmal membranes, the entry of fatty acids from blood into muscle cells is more rapid in the heart than in red or white skeletal muscle. However, type I muscle has more FABPs than white, and endurance training increases the number of S-FABPs. While there appears to be long-term adaptation and regulation of the number of S-FABPs, there is no evidence of acute regulation, such as that noted with GLUT-4 translocation. Therefore, acute regulation of FFA oxidation in heart and muscle is downstream of the FABPs, most likely at the level of the mitochondrial reticulum. 119 Activation and Translocation The first step in intracellular metabolism of lipids resembles the first step in glycolysis. The activation process raises the fatty acids to a higher energy level and involves ATP. 120 121 However the process differs from the activation in glycolysis in that the fatty acid is attached to coenzyme A (CoA) with the formation of a CoA derivative, termed fatty acyl-CoA. The site of fatty acyl-CoA formation is the outer mitochondrial membrane. However, the site of fatty acid oxidation is the mitochondrial matrix. Fatty acids gain entry into or exit from mitochondria by a transport mechanism. For fatty acids, the mechanism involves a carrier, carnitine, and the carnitine acyltransferase enzymes. 122 This mechanism involves stripping off CoA and its return to the cytosol, and the acceptance of the fatty acid by carnitine, with the formation of fatty acyl-carnitine. This process is catalyzed by a family of enzymes collectively called carnitine acyl transferase 1 (CAT1). The fatty acyl-carnitine complex is free to move across the mitochondrial membrane, where, on the inner side, carnitine transferase 2 catalyzes the reverse reaction, leaving carnitine within the membrane, and releasing fatty acyl-CoA into the mitochondrial matrix, where CoA is found. 123 124 β-Oxidation The β-oxidation cycle, located in the mitochondrial matrix serves several purposes. First it degrades the fatty acyl-CoA to acetyl-CoA by cleaving the carbon atoms two at a time. The acetyl-CoA formed as the result of β-oxidation can then enter the TCA (Krebs) cycle, each acetyl-CoA resulting in the formation of 12 ATPs. 125 The rate-limiting step in β-oxidation is probably the last step, which is catalyzed by the enzyme β-ketothiolase. This enzyme is inhibited by acetyl-CoA (high acetyl-CoA slows β-oxidation). Thus, when acetyl-CoA levels are elevated, such as after a meal rich in carbohydrates or during hard physical exercise; when PDH (helps make pyruvate) is activated; and acetyl-CoA is produced from CHO degradation fat utilization decreases. 126 When acetyl-CoA levels are depleted, such as from glycogen depletion following exhausting exercise, fat utilization is promoted. Like other metabolic enzyme systems, the β-oxidation pathway is also controlled by mitochondrial redox (i.e., the NADH/NAD ratio). Reduction (i.e., high NADH/NAD) slows β-oxidation, whereas oxidation stimulates β-oxidation. With reduction there is no need to use fat, glycolysis produces the pyruvate. 127 The second function of the β-oxidation pathway is to produce the high-energy reducing equivalents NADH and FADH2. For each cycle of the β-oxidation pathway, one each of NADH and FADH2 is formed. These are collectively worth 5 ATP (3 + 2). 128 129 Mitochondrial Oxidation After fatty acids are converted to acetyl-CoA, the metabolism of their residues is the same as that of the residues from sugar and carbohydrate. The formation of citrate in Krebs represents a common entry point for the metabolism of acetyl-CoA derived from the various fuel sources. 130 Figure 2.9 131 THE SUBSTRATE SHUNT DURING EXERCISE It appears as though during exercise that active and inactive tissue beds reciprocally alter their demands for carbohydrates during exercise. It is recognized that even though working muscles utilize mainly carbohydrate-derived fuels, the rest of the body is free to utilize lipids. However, during hard exercise, inactive tissues become glucose resistant, thus leaving glucose to be shunted to and taken up by active muscles. 132 Protein Metabolism Even though carbohydrates and fats are preferred fuels, can also use protein or rather the amino acids that form them. Some amino acids can be converted into glucose by gluconeogenesis, and some converted into various intermediates of oxidative metabolism, such as pyruvate or acetyl CoA to enter oxidative process. Proteins’ energy yield not as easily determined as carbohydrate or fat. Protein also contains nitrogen. When amino acids broken down, some of the released nitrogen used to form new amino acids. The remaining nitrogen cannot be oxidized by the body. This is converted into urea and excreted primarily in the urine. This conversion requires use of ATP, so some energy spent in this process. 133 EFFECTS OF TRAINING ON PROTEIN METABOLISM Regular physical activity is the most powerful factor controlling muscle protein metabolism. 134 Different kinds of training elicit different adaptations. Most research on the adaptations of protein metabolism refer to resistance training. When combined with adequate protein intake, resistance training increases the amount of proteins, particularly the contractile proteins (myosin, actin, etc) in the trained muscles. This causes a bulging of the myofibrils in the muscle fibers resulting in the longitudinal splitting of the myofibrils and an increase in their number. 135 Along with the number of myofibrils the volumes of the cytosol and sarcoplasmic reticulum also increase. As a result of these changes, the cross sectional area of the muscle fibers increases (hypertrophy). Then the cross sectional area of the entire muscle and maximal strength increase. This adaptation is measurable a few weeks after the beginning of training and proceeds at a rate of 1 to 3% per week Muscle hypertrophy reaches a plateau after about 6 months of hard resistance training, then a alteration in training program is necessary to continue gains. 136 Sprint training also causes muscle hypertrophy although hypertrophy in this case is smaller and requires about two months to appear. The number of muscle fibers does not change in hypertrophy. A rise in number of muscle fibers is hyperplasia which has been observed mainly in animal models with little evidence in humans. 137 In the case of atrophy the cross sectional area of human muscle fibers decreases but their number does not seem to change. Endurance training is usually not accompanied by muscle hypertrophy unless it employs high intensities in which case it may cause some increase in muscle proteins and a small degree of hypertrophy. The characteristic adaptation of muscle protein metabolism to endurance training is different, the increase in mitochondrial proteins leading to an increase in mitochondrial number and size. The generation of new mitochondria is termed mitochondrial biogenesis. 138 The biochemical mechanisms mediating the effects of training on protein metabolism are largely unknown. They appear to begin with mechanical, neural, hormonal, and metabolic stimuli that modify gene expression. 139 140 141 Figure 2.13 142 OXYGEN DEFICIT/EPOC (Excess Post-exercise Oxygen Consumption) With aerobic activity, oxygen uptake dramatically increases during first minute of exercise. Without increase in intensity, after third or fourth minute, oxygen uptake reaches plateau. Oxygen uptake remains for the rest of exercise bout, provided no increase in exercise intensity. Plateau considered steady state, reflects a balance between energy required by working muscles and ATP production via aerobic metabolism. (energy supply = energy demand) At onset of exercise, do not instantaneously go to steady state, must have transition phase. At conclusion of exercise, transition phase to decrease oxygen uptake to resting levels. Transition phases known as: Oxygen deficit EPOC 143 Oxygen Deficit Initial stages of exercise, increase in oxygen uptake always lag with energy expenditure. Oxygen important in energy-transfer reactions when serves as electron acceptor and combines with H + generated in glycolysis, β-oxidation, or Krebs. Within several minutes of submaximal exercise, H+ production proportional to exercise intensity and oxygen uptake, steady-state is achieved in aerobic metabolism. 144 Energy provided during oxygen deficit is nonaerobic energy, used until a steady-state is reached between oxygen uptake in active tissues and energy demands of exercise. To continue exercise at steady-state, must continually replenish ATP via glycolysis or aerobic metabolism. Before deplete high energy phosphates (PCr) during exercise, lactic acid begins to increase in exercising muscle. Indicates that anaerobic glycolysis does contribute to energy demands in early stages of vigorous activity. 145 Energy for exercise is not a result of turning on or off an energy system, there is considerable overlap from one mode of energy transfer to another. If in a trained state, more efficient oxygen uptake early in exercise. 146 EPOC (Excess Post-exercise Oxygen Consumption) Initial minutes of recovery, oxygen demand does not immediately decrease. EPOC has been identified as having 2 components: Fast Slow 147 Fast component of EPOC thought to represent rebuilding of ATP and PCr used, especially in initial stages. Slow component thought to result from removal of accumulated lactate from tissues through conversion to glycogen (Cori Cycle) or oxidation to CO2 and H2O, thus providing energy to restore glycogen stores. 148 Both fast and slow components of EPOC thought to reflect anaerobic activity that had occurred during exercise. Belief was that by examining post-exercise oxygen consumption, could estimate amount of anaerobic activity that had occurred. This explanation is too simplistic. 149 During initial stage of exercise, some oxygen borrowed from oxygen stores (hemoglobin and myoglobin). During recovery, that oxygen must be recovered. Respiration during recovery remains elevated temporarily in effort to clear CO2 that has accumulated in tissues as byproduct of metabolism. 150 Body temperature also elevated, keeps metabolic and respiratory rates high, requiring more oxygen. Greater part of slow component of recovery oxygen consumption accounted for the effect of temperature on metabolism. 151 Figure 5.5 152 Lactate Threshold Believed to be best indicator of potential for endurance exercise. Defined as: point at which blood lactate begins to accumulate above resting levels during exercise of increasing intensity. 153 Light to moderate activity, lactate remains slightly above resting levels. More intense effort, lactate accumulates more rapidly. 154 Accumulation may be based on assumed relative tissue hypoxia during heavy exercise. With glycolysis being predominant energy system, NADH production not enough to shuttle H+ down respiratory chain. Therefore, pyruvate accepts excess H+ and lactic acid is formed in the presence of LDH. 155 Some evidence to indicate continual production of lactic acid. Under aerobic conditions, production matched by removal, keeping concentrations stable. Known as turnover. Higher intensity activity, appears production exceeds removal, consequently lactate accumulates in tissues. 156 Also speculation that LDH activity in type II muscles favors conversion of pyruvic acid to lactic acid, while in slow-twitch favors conversion of lactic acid to pyruvic acid. (LDH enzyme that catalyzes these reactions). Recruitment of type II fibers favor lactate formation. 157 Lactate threshold been thought to reflect interaction of aerobic and anaerobic energy systems. Some suggest that lactate threshold represents significant shift towards anaerobic glycolysis. Consequently, threshold also been referred to as anaerobic threshold. 158 Controversy regarding relationship of lactate threshold to anaerobic metabolism. Well before threshold reached, lactate being produced and removed. Additionally does not appear to be clear breakpoint of accumulation. 159 Lactate threshold usually expressed in terms of percentage of VO2max at which occurs. Ability to exercise at high intensity without accumulating lactate beneficial to athlete, due to lactic acid formation associated with fatigue. 160 Lactate threshold at 80% VO2max suggest greater exercise tolerance than threshold at 60% VO2max. In 2 individuals with same maximal oxygen uptake, person with highest lactate threshold exhibits best endurance performance. 161 Anaerobic Energy Expenditure: Lactate Threshold Lactate threshold: point at which blood lactate accumulation  markedly Lactate production rate > lactate clearance rate Interaction of aerobic and anaerobic systems Good indicator of potential for endurance exercise Usually expressed as percentage of VO2max (continued) 162 Figure 5.6 163 Anaerobic Energy Expenditure: Lactate Threshold (continued) Higher lactate threshold = better endurance performance For two athletes with same VO2max higher lactate threshold predicts better performance 164 UTILIZING LACTATE Lactate produced in hardworking muscle cannot be converted to anything other than pyruvate by a reversal of the conversion of pyruvate to lactate since it participates in no other reaction in the body. 165 However, this conversion cannot be reversed in the cytosol of contracting muscle fibers since there is a shortage of NAD+. Besides if NAD+ were abundant lactate production would not have increased in the first place. Therefore, what lactate does is leave the muscle fibers. 166 Their (muscle fibers) plasma membrane is highly permeable to lactate, which thus diffuses to the extracellular fluid (down its concentration gradient) and from there to the bloodstream, which disperses it all over the body. 167 The exit of lactate is facilitated by proteins spanning the sarcolemma and transverse tubule membrane which are the MCT’s (monocarboxylate transport proteins). 168 Because it crosses the membrane easily, lactate can enter organs in which its concentration is lower than in the blood. Such organs are primarily the skeletal muscles that did not participate in the exercise, the heart, and the liver. Small quantities of lactate are also taken up by the brain and kidneys. 169 It is even possible for fibers in a muscle to take up lactate from other fibers of the same muscle. This can happen because muscles contain a mixture of the three fiber types, and that they differ among other respects in the ability to resynthesize ATP through anaerobic processes. 170 Upon entering a cell lactate can be oxidized to pyruvate via the reaction: Lactate + NAD+ pyruvate + NADH + H This conversion is favored by a high NAD/NADH ratio (so NAD can combine with lactate) in the cytosol of the cells that take up lactate. 171 The pyruvate thus formed follows primarily one of two alternative routes: 1. It is fully oxidized to three CO2 through the reaction catalyzed by the pyruvate dehydrogenase complex and then through the citric acid cycle. Most of the lactate produced during hard exercise follows this route in the heart and skeletal muscles, releasing a substantial amount of energy. 172 Each lactate molecule yields one cytosolic NADH upon conversion to pyruvate. This NADH generates ATP through the electron transport chain and oxidative phosphorylation. The conversion of pyruvate to acetyl CoA produces one mitochondrial NADH. 173 The oxidation of acetyl CoA in the citric acid cycle yields three NADH, one FADH2 and one GTP. In all, lactate oxidation produces 15 or 14 ATP. 174 2. Pyruvate can be used to resynthesize glucose. This happens primarily in the liver which hosts the metabolic pathway of gluconeogenesis. 175 176