Intracellular Energy-Transfer Networks and High-Resolution Respirometry: A Convenient Approach for Studying Their Function

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Abstract

Compartmentalization of high-energy phosphate carriers between intracellular micro-compartments is a phenomenon that ensures efficient energy use. To connect these sites, creatine kinase (CK) and adenylate kinase (AK) energy-transfer networks, which are functionally coupled to oxidative phosphorylation (OXPHOS), could serve as important regulators of cellular energy fluxes. Here, we introduce how selective permeabilization of cellular outer membrane and high-resolution respirometry can be used to study functional coupling between CK or AK pathways and OXPHOS in different cells and tissues. Using the protocols presented here the ability of creatine or adenosine monophosphate to stimulate OXPHOS through CK and AK reactions, respectively, is easily observable and quantifiable. Additionally, functional coupling between hexokinase and mitochondria can be investigated by monitoring the effect of glucose on respiration. Taken together, high-resolution respirometry in combination with permeabilization is a convenient approach for investigating energy-transfer networks in small quantities of cells and tissues in health and in pathology.

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Isozymes of mammalian hexokinase: structure, subcellular localization and metabolic function.

John E. Wilson (2003)

The first step in metabolism of glucose (Glc) is usually phosphorylation, catalyzed by hexokinase. However, the Glc-6-P produced can then enter one or more of several alternative pathways. Selective expression of isozymic forms of hexokinase, differing in catalytic and regulatory properties as well as subcellular localization, is likely to be an important factor in determining the pattern of Glc metabolism in mammalian tissues/cells. Despite their overall structural similarity, the Type I, Type II and Type III isozymes differ in important respects. All three isozymes are inhibited by the product, Glc-6-P, but with the Type I isozyme, this inhibition is antagonized by P(I), whereas with the Type II and Type III isozymes, P(i) actually causes additional inhibition. Reciprocal changes in intracellular levels of Glc-6-P and P(i) are closely associated with cellular energy status, and it is proposed that the response of the Type I isozyme to these effectors adapts it for catabolic function, introducing Glc into glycolytic metabolism for energy production. In contrast, the Type II, and probably the Type III, isozymes are suggested to serve primarily anabolic functions, e.g. to provide Glc-6-P for glycogen synthesis or metabolism via the pentose phosphate pathway for lipid synthesis. Type I hexokinase binds to mitochondria through interaction with porin, the protein that forms channels through which metabolites traverse the outer mitochondrial membrane. Several experimental approaches have led to the conclusion that the Type I isozyme, bound to actively phosphorylating mitochondria, selectively uses intramitochondrial ATP as substrate. Such interactions are thought to facilitate coordination of the introduction of Glc into glycolysis, via the hexokinase reaction, with the terminal oxidative stages of Glc metabolism occurring in the mitochondria, thus ensuring an overall rate of Glc metabolism commensurate with cellular energy demands and avoiding excessive production of lactate. The Type II isozyme also binds to mitochondria. Whether such coupling occurs with mitochondrially bound Type II hexokinase in normal tissues, and how it might be related to the proposed anabolic role of this isozyme, remain to be determined. The Type III isozyme lacks the hydrophobic N-terminal sequence known to be critical for binding of the Type I and Type II isozymes to mitochondria. Immunolocalization studies have indicated that, in many cell types, the Type III has a perinuclear localization, the possible metabolic consequences of which remain unclear.

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Hexokinase-2 bound to mitochondria: cancer's stygian link to the "Warburg Effect" and a pivotal target for effective therapy.

Saroj P Mathupala, Young H Ko, Peter Pedersen (2009)

The most common metabolic hallmark of malignant tumors, i.e., the "Warburg effect" is their propensity to metabolize glucose to lactic acid at a high rate even in the presence of oxygen. The pivotal player in this frequent cancer phenotype is mitochondrial-bound hexokinase [Bustamante E, Pedersen PL. High aerobic glycolysis of rat hepatoma cells in culture: role of mitochondrial hexokinase. Proc Natl Acad Sci USA 1977;74(9):3735-9; Bustamante E, Morris HP, Pedersen PL. Energy metabolism of tumor cells. Requirement for a form of hexokinase with a propensity for mitochondrial binding. J Biol Chem 1981;256(16):8699-704]. Now, in clinics worldwide this prominent phenotype forms the basis of one of the most common detection systems for cancer, i.e., positron emission tomography (PET). Significantly, HK-2 is the major bound hexokinase isoform expressed in cancers that exhibit a "Warburg effect". This includes most cancers that metastasize and kill their human host. By stationing itself on the outer mitochondrial membrane, HK-2 also helps immortalize cancer cells, escapes product inhibition and gains preferential access to newly synthesized ATP for phosphorylating glucose. The latter event traps this essential nutrient inside the tumor cells as glucose-6-P, some of which is funneled off to serve as carbon precursors to help promote the production of new cancer cells while much is converted to lactic acid that exits the cells. The resultant acidity likely wards off an immune response while preparing surrounding tissues for invasion. With the re-emergence and acceptance of both the "Warburg effect" as a prominent phenotype of most clinical cancers, and "metabolic targeting" as a rational therapeutic strategy, a number of laboratories are focusing on metabolite entry or exit steps. One remarkable success story [Ko YH, Smith BL, Wang Y, Pomper MG, Rini DA, Torbenson MS, et al. Advanced cancers: eradication in all cases using 3-bromopyruvate therapy to deplete ATP. Biochem Biophys Res Commun 2004;324(1):269-75] is the use of the small molecule 3-bromopyruvate (3-BP) that selectively enters and destroys the cells of large tumors in animals by targeting both HK-2 and the mitochondrial ATP synthasome. This leads to very rapid ATP depletion and tumor destruction without harm to the animals. This review focuses on the multiple roles played by HK-2 in cancer and its potential as a metabolic target for complete cancer destruction.

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Warburg, me and Hexokinase 2: Multiple discoveries of key molecular events underlying one of cancers' most common phenotypes, the "Warburg Effect", i.e., elevated glycolysis in the presence of oxygen.

Nancy Pedersen (2007)

As a new faculty member at The Johns Hopkins University, School of Medicine, the author began research on cancer in 1969 because this frequently fatal disease touched many whom he knew. He was intrigued with its viscous nature, the failure of all who studied it to find a cure, and also fascinated by the pioneering work of Otto Warburg, a biochemical legend and Nobel laureate. Warburg who died 1 year later in 1970 had shown in the 1920s that the most striking biochemical phenotype of cancers is their aberrant energy metabolism. Unlike normal tissues that derive most of their energy (ATP) by metabolizing the sugar glucose to carbon dioxide and water, a process that involves oxygen-dependent organelles called "mitochondria", Warburg showed that cancers frequently rely less on mitochondria and obtain as much as 50% of their ATP by metabolizing glucose directly to lactic acid, even in the presence of oxygen. This frequent phenotype of cancers became known as the "Warburg effect", and the author of this review strongly believed its understanding would facilitate the discovery of a cure. Following in the final footsteps of Warburg and caught in the midst of an unpleasant anti-Warburg, anti-metabolic era, the author and his students/collaborators began quietly to identify the key molecular events involved in the "Warburg effect". Here, the author describes via a series of sequential discoveries touching five decades how despite some impairment in the respiratory capacity of malignant tumors, that hexokinase 2 (HK-2), its mitochondrial receptor (VDAC), and the gene that encodes HK-2 (HK-2 gene) play the most pivotal and direct roles in the "Warburg effect". They discovered also that like a "Trojan horse" the simple lactic acid analog 3-bromopyruvate selectively enters the cells of cancerous animal tumors that exhibit the "Warburg effect" and quickly dissipates their energy (ATP) production factories (i.e., glycolysis and mitochondria) resulting in tumor destruction without harm to the animals.

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Journal

Journal ID (nlm-ta): Int J Mol Sci

Journal ID (iso-abbrev): Int J Mol Sci

Journal ID (publisher-id): ijms

Title: International Journal of Molecular Sciences

Publisher: MDPI

ISSN (Electronic): 1422-0067

Publication date (Electronic): 26 September 2018

Publication date Collection: October 2018

Volume: 19

Issue: 10

Electronic Location Identifier: 2933

Affiliations

Laboratory of Chemical Biology, National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, 12618 Tallinn, Estonia; kersti.tepp@ 123456kbfi.ee (K.T.); a.klepinin1989@ 123456gmail.com (A.K.); ljudmila.ounpuu@ 123456gmail.com (L.K.); igor@ 123456chemnet.ee (I.S.) tuuli.kaambre@ 123456kbfi.ee (T.K.)

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[* ]Correspondence: marju.puurand@ 123456kbfi.ee ; Tel.: +372-6-398-365

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Marju Puurand https://orcid.org/0000-0002-0083-3390

Kersti Tepp https://orcid.org/0000-0001-7367-6632

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Publisher ID: ijms-19-02933

DOI: 10.3390/ijms19102933

PMC ID: 6213097

PubMed ID: 30261663

SO-VID: ef8258c6-a494-4b8d-a155-65642a4b257a

License:

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

Intracellular Energy-Transfer Networks and High-Resolution Respirometry: A Convenient Approach for Studying Their Function

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Most cited references 77

Isozymes of mammalian hexokinase: structure, subcellular localization and metabolic function.

Hexokinase-2 bound to mitochondria: cancer's stygian link to the "Warburg Effect" and a pivotal target for effective therapy.

Warburg, me and Hexokinase 2: Multiple discoveries of key molecular events underlying one of cancers' most common phenotypes, the "Warburg Effect", i.e., elevated glycolysis in the presence of oxygen.

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