PDF hosted at the Radboud Repository of the Radboud University Nijmegen The following full text is a publisher's version. For additional information about this publication click this link. http://hdl.handle.net/2066/19330 Please be advised that this information was generated on 2021-10-05 and may be subject to change. The role of Adenylate kinase 1 in energy transfer A study of mice lacking Adenylate kinase 1 Edwin Janssen The role of Adenylate kinase 1 in energy transfer A study of mice lacking Adenylate kinase 1 een wetenschappelijke proeve op het gebied van de Medische Wetenschappen Proefschrift ter verkrijging van de graad van doctor aan de Katholieke Universiteit Nijmegen, op gezag van de Rector Magnificus Prof. Dr. C.W.P.M. Blom, volgens besluit van het college van Decanen in het openbaar te verdedigen op Donderdag 2 oktober 2003 des namiddags om 1.30 uur precies door Edwinus Everardus Wilhelmus Janssen geboren op 25 januari 1971 te Nijmegen Promotor: Prof. Dr. B. Wieringa Manuscriptcommissie: Prof. Dr. J.A.M. Smeitink Prof. Dr. P.N.R. Dekhuijzen Prof. Dr. G.J. van der Vusse (Universiteit Maastricht) The studies presented in this thesis were performed at the Department of Cell Biology, Nijmegen Center for Molecular Life Sciences, University Medical Center St. Radboud, Nijmegen, The Netherlands. Financial support was obtained from the Dutch Organization for Scientific Research (NWO-GMW, 901-01-095) and the Dutch Cancer Society (KWF, KUN 98-1808) ISBN: 90-9017093-6 © 2003 by Edwin E.W. Janssen Printed by PrintPartners Ipskamp, Enschede The energetic state o f mind is influenced by the rhythm o f life. Drum your own rhythm. Aan Sam en Noor Contents Introduction 9 Generation of metabolic fuel 11 The AK isoenzyme family 13 The cell physiological role of adenylate kinase 14 1.3.1 AMP deaminase 17 1.3.2 AMP-activated protein kinase 18 1.3.3 Regulation of ATP sensitive K+ channels 19 The metabolic network for high-energy phosphoryl transfer 20 1.4.1 The creatine kinase/creatine phosphate system 20 1.4.2 Guanylate kinase 21 1.4.3 The NDPK circuit for high-energy phosphoryl transfer 22 1.4.4 Glycolytic ATP generation 23 Bioenergetics of skeletal and cardiac muscle and brain 24 1.5.1 Skeletal muscle design and energetics 24 1.5.2 Molecular factors regulating muscle design 27 1.5.3 Cardiac muscle design and energetics 27 1.5.4 Brain energetics 29 Experimental approaches 30 1.6.1 Animal models to study the function of gene products in vivo 30 1.6.2 How to study the adaptive response to gene inactivation? 31 -Transcriptome analysis 31 -Quantitative protein and activity analysis 32 -Metabolic flux measurements by 18O metabolic labeling 32 Aim and outline of this thesis 34 Adenylate kinase 1 gene deletion disrupts muscle energetic 41 economy despite metabolic rearrangement EMBO Journal 19, 6371-6381 (2000) Compromised energetics in the adenylate kinase AK1 gene 63 knockout heart under metabolic stress JBiol Chem 275, 41424-41429 (2000) Adenylate kinase 1 deficiency induces molecular and 79 structural adaptations to support muscle energy metabolism J Biol Chem 278, 12937-12945 (2003) Impaired intracellular energetic communication in muscles 103 from creatine kinase and adenylate kinase (M-CK/AK1) double knockout mice J Biol Chem 278, 30441-30449 (2003) Chapter 6 Two structurally distinct and spatially compartmentalized 125 adenylate kinases are expressed from the AK1 gene in mouse brain. Mol Cell Biochem (2003), in press Chapter 7 Summarizing discussion / Samenvatting en discussie 147 Abbreviations 159 Dankwoord 163 Curriculum vitae 165 Publications 167 Chapter 1 Introduction Introduction 1.1 Generation of metabolic fuel All living cells require energy to fulfil their basic biological processes necessary for life. This energy is (mainly) generated by the oxidation of metabolic substrates and is represented by the universal energy molecule, ATP. Hydrolytic cleavage of this ATP yields the energy for cell division, cell motility, transport of cellular constituents, and active transport of ions and various other cellular processes. Substrates to drive the production of ATP are obtained via the intake and digestion of food, distributed through the blood stream, and delivered to cells by specialized transport across the cellular membrane. Oxidative phosphorylation (OXPHOS) together with glycolysis and fatty acid oxidation are the principal pathways for the production of ATP. The net conversion of glucose into pyruvate through glycolysis, followed by the conversion of pyruvate and fatty acids into acetyl-CoA and acyl- CoA form the prelude to the tricarboxic acid (TCA) cycle, P-oxidation reactions, and OXPHOS in mitochondria (1). The TCA cycle and P-oxidation pathways produce the reducing equivalents NADH and FADH2 that ultimately fuel OXPHOS via the activity of enzymes in the complexes of the electron transport chain (Fig. 1). In this process the free energy in NADH and FADH2 is used to create a proton electrochemical gradient across the mitochondrial inner membrane. When protons move down this gradient into the matrix of the mitochondrion this energizes the F0F1-ATPase and ATP is formed. In parallel, water is produced following the reduction of molecular oxygen in the cytochrome oxidase reaction sequence (complex IV) of the respiratory chain. It is of note here that maintenance of the appropriate redox concentrations of the reducing equivalents NADH and FADH2 inside and outside the mitochondria is of crucial importance for efficiency in the process. These concentrations are regulated by numerous enzymatic steps and by exchange of electrons from these compounds across the mitochondrial membrane via the glycerol phosphate and malate­ aspartate shuttles (2, 3). Under conditions of extreme workload, the supply of oxygen for OXPHOS may become rate limiting and a metabolic insult may result. Under such condition, the glycolytic machinery has the ability to supply ATP (in a process called substrate-level phosphorylation). This situation is characterized by the accumulation of H+ and lactate. Conley et al. have suggested that accumulation of H+, and subsequent decrease in intracellular pH, disturbs the metabolic steady-state and hence OXPHOS (4). A reduced rate of oxidation of glycolytic products by mitochondria activates lactate dehydrogenase (LDH), which regenerates NAD+ and lactate to prevent accumulation of glycolytically produced NADH and pyruvate (Fig. 1). However, production of lactate has been found also under well-oxygenated conditions, which demonstrates that glycolysis can provide a high and sustained supply of ATP irrespective of the oxidative status of mitochondria (5, 6). In fact, lactate seems to be an intermediate between glucose and complete oxidation into mitochondria: the LDH isoform within a locale of high ATPase activity (e.g. a myofibril) converts pyruvate to lactate 11 Chapter 1 Glucose Lactate Fatty acids Figure 1. Schematic representation of general aspects of cellular energy metabolism. Glucose is transported into the cytosol via specific glucose transporters at the membrane and utilized by glycolysis to produce ATP and metabolic substrates for the tricarboxic acid (TCA) cycle and oxidative phosphorylation. The end product of glycolysis, pyruvate, is converted into lactate if not transported into the mitochondrial matrix (see text). The TCA cycle, together with the p-oxidation of fatty acids in the mitochondrial matrix, produce the NADH and FADH2 required for the reduction by O2 in the production of ATP via the reaction chain of oxidative phosphorylation reactions. ATP and ADP are transported in or out the mitochondrial matrix via specific adenosine nucleotide translocators, present at the mitochondrial inner membrane. Inorganic phosphate (Pi) is transported into the mitochondrial matrix by specific inorganic phosphate carriers. 12 Introduction and this can be partly oxidized to pyruvate by an LDH-isoform in close vicinity of the mitochondrial membrane (7). ADP and Pi, the products resulting from hydrolytic cleavage of ATP, are required as substrates for the formation of ATP in mitochondria and glycolysis (Fig. 1). The molecular mechanism of the delivery of ADP from the cytosol to the matrix of mitochondria is poorly understood but is likely under the control of high-energy phosphate transfer circuits. One of these circuits is formed by the members of the family of creatine kinases (CKs; see paragraph 1.4.1.), which have been considered key players in the regulation and equilibration of adenine nucleotides between different cellular locales (8, 9). Along with CK adenylate kinase (AK) equilibrates and distributes high-energy phosphates among adenine nucleotides in order to attain adenine nucleotide homeostasis (8, 10, 11). Recent studies on AKs have suggested that AKs, in parallel with CKs, are active integral components of a high-energy transfer network that warrants effective handling and distribution of high-energy phosphates (12-15). 1.2 The AK isoenzyme family AKs (EC 2.7.4.3) belong to the family of nucleoside monophosphate kinases (NMKs) that catalyze the reversible exchange of P- and y-phosphoryls among adenine nucleotides, via the reaction: 2ADP o ATP + AMP (11, 16, 17). In this way, AKs equilibrate and regulate the pool of intracellular adenine nucleotides and contribute to homeostasis of adenine nucleotide metabolism (10). At present, 5 different AK isoenzymes have been identified in mammalian cells (18-20). These AK-isoforms are encoded by separate genes and each have their own distinct tissue distribution