Neurochemistry Lecture I PDF

Body Function II Lecture 1 Lectures: Tue (13:00; 14:00), Thr (13:00; 14:00; 15:00) Location: 4-12 (New building) Textbook: Slides and provided material (follow the portal) Instructor: TSU Assistant Professor George Burjanadze, PhD Office: TSU Building XI, room #608 E-mail: [email protected]...

Body Function II Lecture 1 Lectures: Tue (13:00; 14:00), Thr (13:00; 14:00; 15:00) Location: 4-12 (New building) Textbook: Slides and provided material (follow the portal) Instructor: TSU Assistant Professor George Burjanadze, PhD Office: TSU Building XI, room #608 E-mail: [email protected] Office hours: just about any time by appointment Prerequisite: General Biochemistry and Cell Biology Brain metabolism (Overview)  Objectives Know the major source for brain energy and what limits its supply Know the main aspects of metabolic pathways  Key points Brain is command center: always functioning and requires a large amount of energy to keep it operational Glucose is the main source for energy (80 g/day). 25% of total body glucose consumption TCA cycle functions at near maximum capacity Glycolysis functions at 20% capacity Ketone bodies in starvation but CAN NOT REPLACE GLUCOSE  Why brain needs energy? Maintain ionic gradients across the plasma membranes Various storage and transport processes Synthesis of neurotransmitters Synthesis of other cellular components Copyright © 2012, American Society for Neurochemistry. Published by Elsevier Inc. All rights reserved.  Lipids are used in brain to maintain membrane integrity rather than in metabolic roles Brain proteins are rapidly turned over (degraded) than other body proteins (Why…….?) TABLE 11-2: Cerebral Metabolic Rates (CMR) Vary Regionally and Are Activity Dependent Copyright © 2012, American Society for Neurochemistry. Published by Elsevier Inc. All rights reserved. TABLE 11-3: Selective Distribution of Brain Enzymes in Neurons and Astrocytes Copyright © 2012, American Society for Neurochemistry. Published by Elsevier Inc. All rights reserved. Aspects of Brain Metabolism Carbohydrate Metabolism Amino Acids Metabolism Lipid Metabolism  Carbohydrate Metabolism Normally, the brain energy source is aerobic oxidation of glucose Brain glycogen stores are small (about 1/10th of muscle) Glucose crosses Blood Brain Barrier (BBB) by facilitated diffusion (insulin-independent) Therefore, brain is very susceptible to hypoglycemia  When glucose phosphorylation is limited by the low brain glucose concentration, astrocytic glycogenolysis can provide the necessary glucosyl units to maintain ATP synthesis in the glial compartment (black lines). Glycogen can provide fuel to neurons, presumably in the form of lactate, during hypoglycemia and thus reduce the energy deficit in the neuronal compartment. When the glucose supply is sufficient, glucose is stored in glial glycogen (blue lines).  Under ordinary conditions The basic substrate for brain energy metabolism is glucose Brain glucose levels depend on: – Blood glucose levels – Uptake across BBB Glycogen levels vary and depends on plasma glucose levels; thus, Brain is very susceptible to hypoglycemia Glucose crosses BBB and taken up by brain cells by a specific transporters (Glut 1 & Glut 3) – Glut 1: expressed by glial cells – Transport glucose into the endothelial cells of the barrier – Glut 3: located on neurons facilitate glucose transport from ECF into neurons FIGURE 11-1: Cellular localization of specific isoforms of the glucose and monocarboxylic acid transporters in brain. Note that specific transporters are localized on different types of brain cells. (Courtesy of Ian Simpson and Susan Vannucci.) Copyright © 2012, American Society for Neurochemistry. Published by Elsevier Inc. All rights reserved.  Carbohydrate Metabolism During prolonged starvation (> 48 h) and in neonate, ketone bodies can be used as brain energy source When ketone bodies levels in blood are high, specific transporters are upregulated FIGURE 11-2: Glucose has multiple metabolic fates in brain. Glucose is the main substrate for energy production via glycolysis and TCA cycle metabolism. Furthermore, glucose metabolism is closely connected to carbohydrate, amino sugar, neuromodulators (D-serine and glycine), amino acids, and neurotransmitter biosynthesis via glycolytic and TCA cycle intermediates. Glucose is the main precursor for glycogen synthesis, which is localized mainly in astrocytes. Metabolism of glucose via the pentose phosphate shunt provides ribose-5- phosphate for synthesis of nucleotides and NADPH for lipid biosynthesis and maintenance of reduced glutathione. Copyright © 2012, American Society for Neurochemistry. Published by Elsevier Inc. All rights reserved. FIGURE 11-3: Glycolysis produces pyruvate and requires regeneration of NAD+. A schematic representation of aerobic (A1 and A2) and anaerobic (B) glycolysis. Glucose is phosphorylated to glucose-6-phosphate and subsequently to fructose-1,6-bisphosphate via fructose-6-phosphate and phosphofructokinase 1, the main regulatory enzymes in brain glycolysis. NADH is produced in the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. Either the NADH produced is oxidized in the lactate dehydrogenase reaction by reduction of pyruvate to lactate (B), or the reducing equivalent from NADH is transferred to the mitochondria via the malate aspartate shuttle to be oxidized in the electron transport chain for oxidative phosphorylation (A1). Pyruvate from aerobic glycolysis is subsequently metabolized via the tricarboxylic acid (TCA) cycle (A2). Note that lactate can also be produced under aerobic conditions, e.g., when glycolytic flux exceeds that of the TCA cycle. Copyright © 2012, American Society for Neurochemistry. Published by Elsevier Inc. All rights reserved.  Pentose Phosphate Pathway Under basal conditions 5% of brain glucose is metabolized via the pentose phosphate shunt (PPS), also termed the hexose monophosphate pathway (HMP shunt), a pathway active in both neurons and astrocytes. The PPS has relatively high activity in developing brain, reaching a peak during myelination. Its main contribution is probably to produce the NADPH needed for reductive reactions in lipid synthesis. NADPH is also necessary for the maintenance of reduced glutathione, which is required for the inactivation of reactive oxygen species. Thus, the activity of the PPS responds to oxidative stress. The shunt also provides pentose for nucleotide synthesis; however, only a small fraction of the activity of this pathway would be required. Pentose phosphate flux apparently is regulated by the concentrations of Glc-6-P, NADP+, glyceraldehyde-3-phosphate and fructose-6-phosphate. Since transketolase, an enzyme in this shunt, requires thiamine pyrophosphate as a cofactor, poor myelin maintenance in thiamine deficiency may reflect failure of this pathway to provide sufficient NADPH for lipid synthesis  Glycerol phosphate dehydrogenase Glycerol phosphate dehydrogenase (GPDH) is indirectly associated with glycolysis and reduces dihydroxyacetone phosphate to glycerol-3- phosphate, oxidizing NADH in the process. Under hypoxic conditions, glycerol-3-phosphate and lactate increase initially at comparable rates, although the amount of lactate produced greatly exceeds that of glycerol-3-phosphate.  Malate-Aspartate Shuttle The malate–aspartate shuttle is the most important pathway for transferring reducing equivalents from the cytosol to the mitochondria in brain and involves both the cytosolic and mitochondrial forms of aspartate aminotransferase and malate dehydrogenase, the mitochondrial aspartate–glutamate carrier and the dicarboxylic acid carrier in brain. Two Ca2+-sensitive aspartate–glutamate carriers have been identified; citrin (AGC2) is found primarily in liver and kidney, and aralar1 (AGC1) is found in skeletal muscle and brain. Aralar1 is highly enriched on neuronal mitochondria and is often found in areas with high levels of cytochrome oxidase. A considerably lower level of aralar1 and malate–aspartate shuttle activity is present in astrocytes. The activity of the malate–aspartate shuttle increases during development in parallel with synaptogenesis  The malate–aspartate shuttle has a role in linking metabolic pathways in brain. It is essential for coordinating the activity of the glycolytic pathway and the TCA cycle by maintaining a low redox state (NADH/NAD+ ) essential for continuation of glycolysis Activity of the shuttle is much higher in neurons than astrocytes, which is consistent with the involvement of the shuttle in the synthesis of neurotransmitter glutamate and the enrichment of aralar1 on neuronal mitochondria The activity of the malate–aspartate shuttle is impaired in pathological conditions including hypoxic/ischemic brain damage. Impaired shuttle activity limits the ability of neurons to oxidize lactate for energy. The malate–aspartate shuttle transfers reducing equivalents from cytosol to mitochondria. NADH is produced in glycolysis and in the conversion of lactate (Lac) to pyruvate (Pyr) via lactate dehydrogenase (LDH). The reducing equivalents from NADH are transferred via the malate aspartate shuttle to be oxidized via electron transport to support oxidative phosphorylation. Oxaloacetate (OAA) is converted to malate (Mal) by cytosolic malate dehydrogenase (cMDH) and NADH is oxidized to NAD+. Malate is transported into the mitochondrial matrix by a carrier and converted into OAA, and NAD + is reduced to NADH via mitochondrial malate dehydrogenase (mMDH). Within mitochondria, oxaloacetate is converted to aspartate, and α- ketoglutarate (α-KG) is formed from glutamate (Glu) via mitochondrial aspartate aminotransferase (mAAT). Aspartate leaves the mitochondrial matrix in exchange for a molecule of glutamate + H +. The irreversible aspartate–glutamate carrier favors glutamate uptake, as the transport is driven in the direction of the mitochondrial membrane potential. In the cytosol, aspartate is converted back to oxaloacetate via cytosolic aspartate aminotransferase (cAAT). Copyright © 2012, American Society for Neurochemistry. Published by Elsevier Inc. All rights reserved.  Lactate metabolism Lactate is formed under both aerobic and anaerobic conditions in brain. Under anaerobic conditions or upregulation of glycolysis, considerable lactate is produced, presumably in astrocytes, via glycolysis to provide ATP. Conversion of lactate to pyruvate by lactate dehydrogenase (LDH) is necessary for further metabolism of lactate through the tricarboxylic acid (TCA) cycle. Lactate dehydrogenase exists in different isoforms named LDH1–5, consisting of tetramers formed from two distinct subunits. The A and B subunits of LDH are the products of separate genes and are differentially regulated. These isoforms have slightly different kinetic properties regarding rates of catalysis and affinities for pyruvate. LDH4 and LDH5 are the predominant isoforms in astrocytes, and the LDH1 and LDH2 in neurons  upregulation of LDH may contribute to the cerebral lactate accumulation seen in thiamine deficiency. Lactate dehydrogenase functions in the cytoplasm to oxidize NADH, which accumulates as a result of the activity of glyceraldehyde-3- phosphate dehydrogenase in glycolysis. This permits glycolytic ATP production to continue under anaerobic conditions. Lactate dehydrogenase also functions under aerobic conditions because NADH cannot penetrate mitochondrial membranes. Therefore, oxidation of NADH in the cytoplasm depends on this reaction and on the activity of the malate–aspartate shuttle, and to a much lesser extent the glyceraldehyde-3-phosphate shuttle that transfers reducing equivalents to mitochondria. Lactate is an advantageous substrate for neurons since energy is obtained as NADH without the expense of ATP The carbon skeleton from lactate is readily incorporated into the neurotransmitters glutamate and GABA in neurons  It is important to recognize the complexity of lactate balance since changes in the lactate level can be the net result of many processes, including: plasma lactate level, altered transport into and release from brain cells, changes in the glycolytic rate, alterations in the rates of the malate–aspartate shuttle activity, activity of the pyruvate dehydrogenase complex, TCA cycle activity, the cytoplasmic pH and redox state, the rate of glycogenolysis in astrocytes and the rate of lactate efflux from brain  Oxidative Decarboxylation of Pyruvate pyruvate formed in glycolysis needs to enter the TCA cycle that is accomplished via the pyruvate dehydrogenase (PDH) complex localized in the mitochondria, which controls the rate of pyruvate entry into the TCA cycle as acetyl coenzyme A (acetylCoA). Pyruvate dehydrogenase is a mitochondrial multienzyme complex consisting of pyruvate decarboxylase and two other enzymes, lipoate acetyltransferase and lipoamide dehydrogenase, as well as the coenzymes thiamine pyrophosphate, lipoic acid, CoA, FAD and NAD+.  PDH is inactivated by being phosphorylated at the decarboxylase moiety by a tightly bound Mg2+ /ATP4 -dependent protein kinase. PDH is activated by being dephosphorylated by a loosely bound Mg2+ - and Ca2+-dependent phosphatase. Pyruvate protects the complex against inactivation by inhibiting the kinase. ADP is a competitive inhibitor of Mg2+ for the inactivating kinase. Under conditions of greater metabolic demand, increases in pyruvate and ADP and decreases in acetyl-CoA and ATP make the complex more active. Pyruvate dehydrogenase is inhibited by NADH, thereby decreasing formation of acetyl-CoA during hypoxia and allowing more pyruvate to be reduced by lactate dehydrogenase, thus forming the NAD+ necessary to sustain glycolysis.  Acetyl-CoA Acetyl coenzyme A formed from glucose is the precursor for acetylcholine. Although acetylcholine synthesis normally is controlled by the rate of choline uptake and choline acetyltransferase activity, the supply of acetyl coenzyme A (acetyl-CoA) can be limiting under adverse conditions. The mitochondrial membrane is not permeable to the acetyl-CoA produced within it, but there is efflux of its condensation product, citrate. Acetyl-CoA can subsequently be formed from citrate in the cytosol by ATP citrate lyase. The acetyl moiety of acetylcholine is formed in a compartment, presumably representing the synaptic terminal, having a rapid glucose turnover. The cytosol of cholinergic nerve endings is rich in citrate lyase, and it is likely that citrate shuttles the acetyl-CoA from the mitochondrial compartment to the cytosol. During hypoxia or hypoglycaemia, acetylcholine synthesis can be inhibited by failure of the acetyl-CoA supply.  Pyruvate Carboxylase Pyruvate carboxylation in astrocytes is the major anaplerotic pathway in brain. Pyruvate carboxylase, which has been shown to be primarily, if not exclusively, an astrocytic enzyme, fulfills this ‘anaplerotic’ or refilling function in brain by adding a CO2 to pyruvate forming oxaloacetate which is then combined with acetyl-CoA to form citrate. Although malic enzyme or the combined action of phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate kinase can fix CO2, they do not perform this function to a significant extent in brain. To export net amounts of citrate, α-ketoglutarate or oxaloacetate from the TCA cycle, the supply of dicarboxylic acids must be replenished. The CO2 fixation rate sets the upper limit at which biosynthetic reactions can occur. The pyruvate carboxylase reaction in astrocytes is essential for neurons because they are continuously releasing amino acid neurotransmitters and thus require metabolites from astrocytes for the replenishment of the neuronal TCA cycle and neurotransmitter biosynthesis.  Citrate The steady-state concentration of citrate in brain is relatively high compared to other TCA cycle and glycolytic intermediates, reaching 0.4 mmol/l in the cerebrospinal fluid. This is compatible with the ability of astrocytes to synthesize and release a considerable amount of citrate in vitro at a rate reflecting the activity of pyruvate carboxylase. Citrate synthesis is compartmentalized in astrocytes; pyruvate carboxylation has a specific role in the formation of a releasable pool of citrate, but has less of a role in synthesis of the smaller intracellular pool. Bicarbonate stimulates the release of citrate, which is threefold greater from cerebellar astrocytes than from cortical astrocytes. This difference reflects the functional specialization of astrocytes in different brain regions, possibly in response to the neuronal specialization.  It has been shown that citrate can attenuate the inhibitory action of Zn2+ on NMDA- receptor-mediated glutamate release. The ability of astrocytes to release large amounts of citrate may be related to the ability of these cells to significantly upregulate glycolysis under certain conditions. This may be so since the release of citrate would decrease its intracellular concentration, thereby relieving the inhibitory action of citrate on PFK1 in the glycolytic pathway  TCA Cycle Activity of the TCA cycle is subject to control at several enzymatic steps of the cycle and by the local ADP concentration, which is a prime activator of the mitochondrial respiration. As in other tissues, there are two isocitrate dehydrogenases in brain. One is active primarily in the cytoplasm and requires nicotinamide adenine dinucleotide phosphate (NADP+) as cofactor; the second is the mitochondrial isocitrate dehydrogenase that requires NAD+ and participates in the TCA cycle α-Ketoglutarate dehydrogenase, which oxidatively decarboxylates α-ketoglutarate, requires the same cofactors as pyruvate dehydrogenase Succinate dehydrogenase is the enzyme that catalyses the oxidation of succinate to fumarate and is also part of the respiratory chain. Isocitrate and succinate concentrations in brain are affected little by changes in the flux of the TCA cycle as long as an adequate glucose supply is available. rapid removal of oxaloacetate. The latter is maintained at a very low concentration under steady-state conditions by the condensation reaction with acetyl-CoA catalyzed by citrate synthase  Malate dehydrogenase is one of several enzymes in the TCA cycle present in both the cytoplasm and mitochondria. The MDH catalysed pathway is influenced by the concentration of key metabolites in the mitochondria (e.g. oxaloacetate, α-ketoglutarate and citrate)  Mitochondria are distributed with varying densities throughout the central nervous system with the more vascular parts containing most of the mitochondria. Under certain metabolic conditions with low levels of acetyl-CoA, such as in hypoglycaemia, only a part of the TCA cycle may operate, particularly in neurons and synaptic terminals. This truncated cycle, which enables utilization of glutamine/glutamate as energy substrates, consists of the steps from α- ketoglutarate to oxaloacetate and leads to aspartate production and sometimes accumulation. The majority of the reducing equivalents produced in the complete TCA cycle are derived from these reactions. The energy production from this truncated TCA cycle corresponds to 75% of that produced from the entire cycle ATP production in brain is highly regulated. However, it is not likely that there is a net production of 38 ATP equivalents per mole of glucose, since a fraction of the glucose taken up is converted to lactate, and there is a proton leak as well as specific non-ATP synthase uses of protons (e.g. malate–aspartate shuttle, phosphate transport and ATP transport) across the mitochondrial membrane. The steady-state concentration of ATP is high and represents the net sum of very rapid synthesis and utilization. On average, half of the terminal phosphate groups turn over in about 3 seconds; the turnover is probably much faster in certain regions.  The level of ~P is kept constant by regulation of ADP phosphorylation in relation to ATP hydrolysis. The active adenylyl kinase reaction, which forms equivalent amounts of ATP and AMP from ADP, prevents any great accumulation of ADP. Only a small amount of AMP is present under steady-state conditions; thus, a relatively small decrease in ATP may lead to a relatively large increase in AMP, which is a positive modulator of AMP kinase, which in turn regulates many reactions that lead to increased ATP synthesis. Such an amplification factor provides a sensitive control for maintenance of ATP levels. Phosphocreatine has a role in maintaining adenosine triphosphate levels in brain. The concentration of phosphocreatine (PCr) in brain is even higher than that of ATP, and creatine phosphokinase (CPK) is extremely active. The PCr level is exquisitely sensitive to changes in oxygenation, providing ~P for ADP phosphorylation and, thus, maintaining ATP levels. The CPK system also may function in regulating mitochondrial activity. In neurons with a very heterogeneous mitochondrial distribution, the creatine/phosphocreatine shuttle may play a critical role in energy transport. It was observed that knocking out the CPK enzymes in mouse brain had an effect on spatial learning but not on motor function  All of the necessary machinery for using monocarboxylic acids for energy is present in adult brain. In prolonged starvation, the carbohydrate stores of the body are exhausted and the rate of gluconeogenesis is insufficient to provide glucose fast enough to meet the requirements of the brain; Blood ketone concentrations rise as a result of the rapid fat catabolism. The brain then apparently turns to the ketone bodies as the source of its energy supply. Cerebral utilization of ketone bodies appears to follow passively their concentrations in arterial blood. In normal adults, ketone concentrations are very low in blood and cerebral utilization of ketones is negligible. In ketotic states resulting from starvation, fat- feeding or ketogenic diets, diabetes, or any other condition that accelerates the mobilization and catabolism of fat, cerebral utilization of ketones is increased more or less in direct proportion to the degree of ketosis. The enzymes responsible for their metabolism, D-β-hydroxybutyrate dehydrogenase, acetoacetate-succinyl-CoA transferase and acetoacetyl-CoA- thiolase, are present in brain tissue in sufficient amounts to convert them into acetyl-CoA  D-β-hydroxybutyrate is incapable of maintaining or restoring normal cerebral function in the absence of glucose in the blood. This suggests that, although it can partially replace glucose, it cannot fully satisfy the cerebral energy needs in the absence of some glucose consumption. As the first product of d-β-hydroxybutyrate oxidation, acetoacetate, is metabolized further by its displacement of the succinyl moiety of succinyl-CoA to form acetoacetyl-CoA. A certain rate of glucose utilization may be essential to drive the TCA cycle, to provide enough succinyl-CoA to permit the further oxidation of acetoacetate and, hence, to pull along the oxidation of d-β- hydroxybutyrate. Brain uses lactate and ketone bodies in the presence of glucose. Some short chain fatty acids can be used by brain astrocytes in vitro for energy. There is evidence that amino acids can be oxidized in vivo by the brain for energy. Thank you for your attention