Ketone body metabolism
Ketone body metabolism includes ketone body synthesis (ketogenesis) and breakdown (ketolysis). When the body goes from the fed to the fasted state the liver switches from an organ of carbohydrate utilization and fatty acid synthesis to one of fatty acid oxidation and ketone body production. This metabolic switch is amplified in uncontrolled diabetes. In these states the fat-derived energy (ketone bodies) generated in the liver enter the blood stream and are used by other organs, such as the brain, heart, kidney cortex and skeletal muscle. Ketone bodies are particularly important for the brain which has no other substantial non-glucose-derived energy source. The two main ketone bodies are acetoacetate (AcAc) and 3-hydroxybutyrate (3HB) also referred to as β-hydroxybutyrate, with acetone the third, and least abundant. Ketone bodies are always present in the blood and their levels increase during fasting and prolonged exercise. After an over-night fast, ketone bodies supply 2–6% of the body's energy requirements, while they supply 30–40% of the energy needs after a 3-day fast. When they build up in the blood they spill over into the urine. The presence of elevated ketone bodies in the blood is termed ketosis and the presence of ketone bodies in the urine is called ketonuria. The body can also rid itself of acetone through the lungs which gives the breath a fruity odour. Diabetes is the most common pathological cause of elevated blood ketones. In diabetic ketoacidosis, high levels of ketone bodies are produced in response to low insulin levels and high levels of counter-regulatory hormones.
Figure 1. Ketone bodies [Click to enlarge]The term ‘ketone bodies’ refers to three molecules, acetoacetate (AcAc), 3-hydroxybutyrate (3HB) and acetone (Figure 1). 3HB is formed from the reduction of AcAc in the mitochondria. These two predominant ketone bodies are energy-rich compounds that transport energy from the liver to other body tissues. Acetone is a minor product, generated by spontaneous decarboxylation of AcAc and is responsible for the sweet odor on the breath of individuals with ketoacidosis.
Ketone bodies are present in small amounts in the blood of healthy individuals during fasting or prolonged exercise and play a key role in sparing glucose utilization and reducing proteolysis. Unlike most other tissues, the brain cannot utilize fatty acids for energy when blood glucose levels become compromised. In this case, ketone bodies provide the brain with an alternative source of energy, amounting to nearly 2/3 of the brain's energy needs during periods of prolonged fasting and starvation.
Abnormally large quantities of ketone bodies are found in the blood of individuals who are experiencing diabetic ketoacidosis, alcoholic ketoacidosis, salicylate poisoning, and other rare conditions. Ketone bodies stimulate insulin release in vitro, generate oxygen radicals and cause lipid peroxidation. Lipid peroxidation and the generation of oxygen radicals may play a role in vascular disease in diabetes.
Ketogenesis is the process by which fatty acids are transformed into acetoacetate (AcAc) and 3-hydroxybutyrate (3HB) . This process takes place in the liver in specialized organelles called mitochondria. Under normal aerobic conditions glucose and fatty acids are metabolized to acetyl CoA by glycolysis and β-oxidation respectively. The acetyl CoA is then further metabolized to two molecules of CO2 by the tricarboxylic acid cycle (TCA cycle) which comprises eight sequential enzymic reactions. The energy released by each turn of the cycle is stored either as high energy phosphate in one molecule of GTP, or as high energy electrons in three molecules of NADH + H+ and one molecule of the reduced cofactor, coenzyme Q (QH2) via FADH2. The three NADH and one FADH2 produced by each turn of the cycle are re-oxidised and generate ATP in a process called oxidative phosphorylation. The theoretical yield is 3 ATP per molecule of NADH and 2 ATP per molecule of FADH2 making a total per cycle of 11 ATP and 1 GTP. Inefficiencies in oxidative phosphorylation reduce the actual yield to ~2.5 per NADH and 1.5 per FADH2 (10 ATP equivalents) per cycle. Energy production via the TCA cycle and oxidative phosphorylation takes place in mitochondria, the same organelles where ketogenesis occurs.
The availability of oxaloacetate (OAA) is critical to the oxidation of acetyl CoA. If intracellular glucose levels become too low - as during fasting or as a result of low insulin levels in diabetes - then in the liver, oxaloacetate is depleted owing to its preferentially utilization in the process of gluconeogenesis. Thus OAA is unavailable for condensing with acetyl CoA for the latter’s oxidative metabolism via the TCA cycle. Instead, in the liver, acetyl CoA is diverted to ketone body formation (ketogenesis). Liver also lacks one of the key enzymes required for ketone body utilization - acetoacetyl succinyl CoA transferase - (see below). The unavailability of OAA and the lack of the above transferase explain why ketone bodies are synthesized in the liver but utilized in the peripheral tissues.
Figure 2. Ketogenesis and ketolysis pathways. Click for enlarged view. Fatty acid oxidation in liver mitochondria generates acetyl CoA. Under conditions of low glucose availability, the acetyl CoA cannot be oxidized via the TCA cycle because, in the liver, the oxaloacetate required for the first step (its condensation with acetyl CoA to form citrate), is unavailable being redirected to the process of glucose production (gluconeogenesis). Consequently the acetyl CoA is converted into ketone bodies which are used by non-liver tissues for energy (ATP) production. See text for details. Note the first and last enzymes in both processes are reversible and operate in both processes. In healthy adults, the liver is capable of producing up to 185g of ketone bodies per day. The process includes the following steps shown in Figure 2:
- β-oxidation of fatty acids to acetyl CoA,
- formation of acetoacetyl CoA from two molecules of acetyl CoA
- conversion of acetoacetyl CoA to 3-hydroxy-3-methylglutaryl CoA (HMG CoA)
- conversion of 3-hydroxy-3-methylglutaryl CoA to acetoacetate (AcAc)
- reduction of AcAc to 3-β-hydroxybutyrate (3HB) and
- the spontaneous decarboxylation of acetoacetate to acetone
The conversion of 2 molecules of acetyl CoA to acetoacetyl CoA and free CoA is catalyzed by the reversible enzyme acetoacetyl CoA thiolase. 3-hydroxy-3-methylglutaryl CoA (HMG CoA) is formed from acetoacetyl CoA by mitochondrial HMG CoA synthase (Figure 2). This step is stimulated by starvation, low levels of insulin, and the consumption of a high-fat diet. HMG CoA is also produced from ketogenic amino acids such as leucine, lysine, and tryptophan via a separate enzymatic process during amino acid catabolism. HMG CoA is then cleaved to liberate acetoacetate in a step mediated by 3-hydroxy-3-methylglutaryl CoA lyase (HMG CoA lyase). The reduction of acetoacetate (AcAc) to 3-hydroxybutyrate (3HB) is catalyzed by 3-hydroxybutyrate dehydrogenase, a phosphatidyl choline-dependent enzyme (Figure 2). During this step, NADH is oxidized to NAD+. The ultimate ratio of 3HB to AcAc in the blood is dependent on the redox potential (i.e. the NADH/NAD+ ratio) within liver mitochondria.
Acetoacetate and 3-hydroxybutyrate are short-chain (4-carbon) organic acids (Figure 1) that can freely diffuse across cell membranes. Therefore, ketone bodies can serve as a source of energy for the brain (which does not utilize fatty acids) and the other peripheral organs mentioned above. Ketone bodies are filtered and reabsorbed in the kidney. At physiologic pH, these organic acids dissociate completely. The large hydrogen-ion load generated during their pathologic production, in diabetic ketoacidosis, for example, rapidly overwhelms the normal buffering capacity and leads to a metabolic acidosis with an increased anion gap.
Ketolysis is the process by which ketone bodies produced in the liver are converted (in non-liver tissues), into acetyl CoA which, on complete oxidation via the tricarboxylic acid cycle and oxidative phosphorylation, provides energy for various intracellular metabolic activities (Figure 3). Ketolysis occurs in the mitochondria of many extrahepatic organs. The central nervous system is particularly dependent on the delivery of ketone bodies produced in the liver for the process of ketolysis, since ketogenesis occurs very slowly if at all in the central nervous system.
Figure 3. Interplay between ketone body production (ketogenesis) in the liver and ketone body utilization utilization (ketolysis) in non-hepatic tissue such as skeletal muscle. Click to enlarge. Free fatty acids are released into the circulation by lipolysis and broken down into multiple copies of acetyl CoA by β-oxidation. Under conditions of low glucose availability ketogenesis occurs in the liver producing the three ketone bodies, 3-hydroxybutyrate, acetoacetate and acetone. The production of the first two is catalysed by four enzymes: acetoacetyl CoA thiolase (denoted by 1), HMG CoA synthase (2), HMG CoA lyase (3) and 3 hydroxy butyrate dehydrogenase (4). The acetone is formed by non-enzymic decarboxylation of acetoacetate and cannot be used as an energy source. Acetoacetate and 3-hydroxybutyrate pass from the liver to the general circulation and are absorbed by non-hepatic tissues where they can be used as fuel. The 3-hydroxybutyrate is oxidized to acetoacetate by 3 hydroxy butyrate dehydrogenase (denoted as I in the figure) and then converted to acetoacetyl CoA by acetoacetyl succinyl CoA transferase (II). The acetoacetyl CoA is then split by acetoacetyl CoA thiolase (III) into two molecules of acetyl CoA which are metabolized into CO2 and H2O via the TCA cycle and oxidative phosphorylation generating many molecules of ATP. Based on slide 25 in Chaudhuri.As summarized in Figures 2 and 3, ketolysis involves three steps, two of which are reversible reactions carried out by two (3-hydroxy butyrate dehydrogenase and acetoacetyl CoA thiolase) of the four enzymes involved in ketogenesis. The first step in ketolysis is the oxidation of 3-hydroxybutyrate to acetoacetate by the reversible enzyme 3-hydroxy butyrate dehydrogenase followed by the reconstitution of acetoacetyl CoA from acetoacetate by the enzyme acetoacetyl succinyl CoA transferase (also called succinyl CoA: 3-oxoacid CoA transferase (SCOT)). This enzyme uses succinyl CoA, an intermediate product of the tricarboxylic acid cycle, as the CoA donor. The third and final step in ketosis is the generation of 2 molecules of acetyl CoA from CoA and acetoacetyl CoA by the reversible enzyme acetoacetyl CoA thiolase (Figures 2 and 3). The acetyl CoA formed is then oxidized in nonhepatic tissues via the TCA cycle.
Acetoacetyl succinyl CoA transferase is the rate-determining step in ketolysis. Its activity is highest in the heart and kidney, followed by the central nervous system and skeletal muscle. Due to the sheer mass of skeletal muscle, this tissue accounts for the highest fraction of total ketone body utilization in the resting state. Acetoacetyl succinyl CoA transferase activity is down-regulated by high (>5 mM) intracellular levels of acetoacetate (AcAc). This phenomenon is responsible for the observed increase in circulating levels of ketone bodies during the early phases (3 days to 2 weeks) of starvation, despite relatively constant rates of hepatic ketogenesis during this period. Acetoacetyl succinyl CoA transferase activity is also present, but at very low levels, in the liver.
Acetoacetyl CoA thiolase, the enzyme responsible for the final key step in ketolysis in extrahepatic tissues, tends to enhance the production of acetyl CoA from acetoacetyl CoA. Acetoacetyl CoA thiolase is also present in the liver – the primary locus of ketogenesis – where it plays a key role as the first step in ketogenesis - the creation of acetoacetyl CoA from two molecules of acetyl CoA. Acetoacetyl CoA thiolase is a multipurpose enzyme that participates in several other metabolic pathways including fatty acid metabolism and the degradation of some amino acids.
Regulation of ketogenesis
The rate of ketogenesis depends upon the activity of three enzymes. One is hormone-sensitive lipase (or triglyceride lipase), which is found in peripheral adipocytes. The other two are acetyl CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA synthase (HMG CoA synthase), which are found in the liver. Hormone-sensitive lipase catalyzes the conversion of triglycerides to diglycerides for further degradation to the free fatty acids (lipolysis) that serve as substrates for ketogenesis. On the other hand, acetyl CoA carboxylase catalyzes the conversion of acetyl CoA to malonyl CoA, increasing the hepatic level of the primary substrate of fatty acid biosynthesis. Malonyl CoA levels vary in the liver directly according to the rate of fatty acid synthesis and inversely with the rate of fatty acid oxidation. Therefore, malonyl CoA plays a pivotal role in the regulation of ketogenesis. Low levels of malonyl CoA stimulate transport of fatty acids into the mitochondria via the carnitine shuttle for oxidation to ketone bodies (see lipolysis and lipogenesis for details). Malonyl CoA normally inhibits carnitine palmitoyltransferase 1, the enzyme that transports fatty acyl CoA across the mitochondrial membrane.
Hormone-sensitive lipase and acetyl CoA carboxylase, are exquisitely controlled by the level of circulating insulin (which acts to inhibit ketogenesis), and epinephrine and glucagon (which act to stimulate ketogenesis). Thus in fasting or diabetes the high levels of glucagon and low levels of insulin favor ketogenesis through the promotion of lipolysis in the adipocyte and the stimulation of fatty acid oxidation in the liver.
Insulin inhibits lipolysis and ketogenesis and stimulates lipogenesis by triggering the inhibitory dephosphorylation of hormone-sensitive lipase and the activating dephosphorylation of acetyl CoA carboxylase. In the adipocytes, dephosphorylation of hormone-sensitive lipase inhibits the breakdown of triglycerides to fatty acids and glycerol, the rate-limiting step in the release of free fatty acids (lipolysis) from the adipocyte. This thereby reduces the amount of substrate that is available to generate acetyl CoA (via fatty acid oxidation) for ketogenesis. In addition, insulin-mediated dephosphorylation of inhibitory sites on hepatic acetyl CoA carboxylase increases the production of malonyl CoA and simultaneously reduces the rate at which fatty acids can enter hepatic mitochondria for oxidation and ketone body production.
Glucagon stimulates ketogenesis by doing the opposite of insulin. Glucagon triggers the phosphorylation of both hormone-sensitive lipase and acetyl CoA carboxylase by cyclic AMP-dependent protein kinase. In the adipocytes, phosphorylation of hormone-sensitive lipase by cyclic AMP-dependent protein kinase stimulates the release of fatty acids from triglycerides (see lipolysis). Glycerol freely diffuses out of the adipose tissue into the circulation for transport to the liver. Free fatty acids enter the circulation and travel (bound to albumin) for uptake and metabolism in other tissues such as the heart, skeletal muscle, kidney, and the liver. In hepatocytes, phosphorylation of acetyl CoA carboxylase by cyclic AMP-dependent protein kinase reduces the production of malonyl CoA which, in turn, stimulates fatty acid uptake by the mitochondria, and thus increases the amount of substrate available for ketogenesis.
Hepatic mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMG CoA synthase) is the third key enzyme involved in the control of ketogenesis. The activity of this enzyme is increased by starvation and a high-fat diet, and decreased by insulin. These factors modulate the activity of HMG CoA synthase by altering the production of mRNA and the post-translational phase of protein synthesis via reversible succinylation of the enzyme itself. Increasing the activity of HMG CoA synthase leads to the production of ketone bodies.
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