Mitochondria in Obesity and Type 2 Diabetes: Comprehensive Review on Mitochondrial Functioning and Involvement in Metabolic Diseases

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Part I

Educational Chapters

Chapter 1

Mitochondria: Ultrastructure, Dynamics, Biogenesis and Main Functions

Anne Devin⁎; Cyrielle Bouchez⁎; Thibaut Molinié⁎,†; Claudine David⁎; Stéphane Duvezin-Caubet⁎; Manuel Rojo⁎; Arnaud Mourier⁎; Nicole Averet⁎; Michel Rigoulet⁎    ⁎ Institut de Biochimie et Génétique Cellulaires, UMR 5095, CNRS, Université de Bordeaux, Bordeaux, France

† Université Bordeaux, IBGC, UMR 5095, Bordeaux, France

Abstract

Mitochondria are double membrane-bound organelles that possess their own genome encoding essential but minute part of mitochondrial proteome. The vast majority of mitochondrial proteins are nuclear-encoded and several complementary processes ensure proper targeting, import, processing, and assembly of these proteins into mitochondria. Coordination between the two genomes expression is essential for proper assembly and activity of the oxidative phosphorylation system, which play a key role in energy metabolism in the vast majority of eukaryotic cells. In vivo, mitochondria display a high degree of connectivity and mobility. Mitochondrial dynamics is an essential physiological process ensuring the proper localization of mitochondria at intracellular sites of high-energy demand. Beyond their role in energy metabolism, mitochondria are also involved in lipid, amino acid and nucleic acid syntheses and are essential for biosynthesis of iron-sulfur clusters, heme, and other prosthetic groups. Mitochondrial respiratory chain is an important site of ROS production and ROS level must be tightly controlled by various enzymes. Unbalance between ROS production and detoxification plays a key role in metabolic disorders. Mitochondrial activity is therefore essential to cell fate, and mitochondrial dysfunctions have been associated with metabolic disorders. This chapter gives a general introduction on mitochondrial properties and functions that will be developed further in subsequent specialized chapters.

Keywords

Mitochondria; Structure; Biogenesis; Dynamics; OXPHOS; Energy metabolism; ROS production and detoxification; Tissue specificity

Acknowledgments

The authors received continuous support from the CNRS (Centre National de la Recherche Scientifique), the Comité de Dordogne & Gironde de la Ligue Nationale Contre le Cancer, The Fondation ARC pour la recherche sur le Cancer, the Plan cancer 2014-2019 No BIO 2014 06, the ANR-16-CE14-0013, and Bordeaux University. This work is dedicated to the memory of professor Xavier Leverve, whose exceptional contribution and friendly collaboration were continuous throughout the years.

1 Introduction

Mitochondria are double-membrane-enclosed organelles that ensure a number of pivotal functions in the vast majority of eukaryotic cells. These include key steps in lipid and amino acid metabolism, biosynthesis of iron-sulfur clusters, heme, and further prosthetic groups, regulation of programmed cell death, and, most prominently, the final transformation of proteins, fats, and sugars into the cellular energy currency adenosine-5′-triphosphate (ATP) by oxidative phosphorylation (OXPHOS). Severe mitochondrial dysfunctions are associated with so-called mitochondrial diseases that appear linked to specific or broad OXPHOS defects. In addition, alterations of mitochondrial properties have been associated with further diseases linked to other metabolic and/or physiologic alterations. This chapter gives a general introduction on mitochondrial properties and functions that will be developed further in subsequent specialized chapters.

2 Mitochondrial Ultrastructure

Mitochondria display a unique and highly characteristic ultrastructure that allows their unambiguous identification in electron microscopy sections. Mitochondria are enveloped by an outer and an inner membrane enclosing a narrow intermembrane space. The outer membrane represents the border to the cytosol, and the inner membrane separates the intermembrane space from the matrix. The inner membrane is not limited to the mitochondrial boundary, but forms protrusions into the matrix―known as cristae―that provide a tremendous increase in surface.¹ The direct contacts between outer and inner membranes, as well as the transition between inner boundary and inner cristae membranes, occur at specific structures known as contact sites and cristae junctions, respectively (Fig. 1A). These structures are made and regulated by specific protein complexes known as MICOS (mitochondrial contact site and cristae organizing system) that are essential for several mitochondrial functions, including the maintenance of mitochondrial ultrastructure and the transport and exchange of proteins and lipids (reviewed in Ref. 2).

Fig. 1 Mitochondrial ultrastructure. (A) Mitochondria are enveloped by outer and inner membranes enclosing a narrow intermembrane space. The inner membrane encloses the mitochondrial matrix and forms invaginations called cristae. The direct contact between outer and inner membranes, as well as the transition between inner boundary and inner cristae membranes, occur at specific structures known as contact sites and cristae junctions, respectively. Outer mitochondrial membranes and the endoplasmic reticulum are in close or direct contact at sites termed MAMs (mitochondria associated membranes). (B) Mitochondria form filaments containing numerous mtDNA nucleoids in wild-type cells (WT) and display fragmented mitochondria in fusion-defective cells lacking fusion factor Mfn1 (MFN1 KO). Morphology and mitochondrial DNA distribution in cultured cells was visualized with antibodies against outer membrane Tom20 (red) and DNA (green) , respectively. The inset represents an enlargement of the indicated area. Bar = 20 μm.

These ultrastructural characteristics are common to the mitochondria of all eukaryotes. The topology, size, and relative abundance of the different membranes and subcompartments, however, vary significantly among species and tissues and as a function of the metabolic or physiological state.³ This is in accordance with the fact that each mitochondrial membrane and compartment hosts specific functions and activities.

Electron microscopy analysis often depicts close proximity or even direct contacts between outer mitochondrial membranes and the endoplasmic reticulum (ER) (Fig. 1A) that are termed MAMs (mitochondrial associated membranes) or ERMES (ER-mitochondria encounter structure). The molecular composition of yeast ERMES complex is well established,⁴ but that of mammalian MAMs remain highly debated.⁵ These contacts mediate the interorganellar exchange of calcium and lipids, are involved in mitochondrial fission, and play a role in insulin signaling, autophagy, and apoptosis (see also Chapter 2). Consequently, alterations of MAMs properties and of MAM-dependent functions have been associated with human diseases, including diabetic syndromes.⁶, ⁷

3 Mitochondrial Morphology, Distribution, and Dynamics

Mitochondria are mobile structures that do not distribute homogeneously throughout cells, but localize to intracellular sites (e.g., apical membranes of secretary cells, synapses of neurons) where their function (e.g., ATP-synthesis by OXPHOS) is required. Mitochondrial distribution and mobility are ensured by adaptors and motors that mediate interactions with the microtubule and actin cytoskeletons.⁸, ⁹

Electron microscopy of ultrathin sections often has depicted mitochondria as isolated punctate structures that resemble bacteria (their phylogenetic ancestors) in form and in size. This appearance and the resemblance to that of their prokaryotic ancestors led to the longstanding concept that the mitochondrial compartment is composed of a collection of numerous small and independent organelles. Work in the last decades, however, has revealed that mitochondria continuously fuse and divide and that the equilibrium between antagonizing fusion and fission determine their morphology: Mitochondria appear as a network of interconnected filaments at high fusion/fission ratios and as a collection of small punctate structures when the fusion/fission ratio shifts toward fission (Fig. 1B). Fusion enables molecular exchanges between mitochondria (proteins, RNA, DNA) and leads to the existence of a single mitochondrial compartment within cells. Division enables severing of mitochondrial filaments and segregation of functional and defective mitochondria, a requisite for the selective degradation of individual mitochondria by autophagy or mitophagy (see Chapter 2).

Fusion and fission are energy consuming processes that depend on the hydrolysis of nucleotides by specific GTPases of the dynamin-superfamily. In addition, the fusion of inner membranes (but not of outer membranes) also depends on the inner membrane electrical potential difference.¹⁰ It is now well established that mitochondrial dynamics and bioenergetics are linked: The fusion/fission steady state is modulated by the bioenergetic status of cells and mitochondria and reciprocally, fusion/fission dynamics can modulate mitochondrial bioenergetics.¹¹, ¹² Accordingly, conditions leading to mitochondrial dysfunction and/or depolarization, such as the permeabilization of the outer membrane at the onset of apoptosis, are accompanied by mitochondrial fragmentation.¹³ Numerous pathological situations have been associated with alterations of the fusion/fission steady state, and mitochondrial fragmentation has become a hallmark of mitochondrial or cellular dysfunctions,¹⁴ including diabetes and related metabolic disorders.¹⁵

Several components of the mitochondrial fusion and fission machineries have been identified and many of them, notably some GTP-binding proteins of the dynamin superfamily, are conserved between animals and fungi.¹⁶ In mammals, outer membrane fusion is governed by mitofusins MFN1 and MFN2, two GTPases anchored to the outer mitochondrial membrane. Further reports reveal that mitofusin MFN2 can modulate contacts between ER and mitochondria, but the precise role of MFN2 in MAM biogenesis/modulation remains controversial.¹⁷, ¹⁸ Inner membrane fusion depends on OPA1, a GTPase that localizes to the intermembrane space. The size, membrane association, and activity of OPA1 is regulated by mitochondrial metalloproteases that generate isoforms of different lengths. Proteolytic processing of OPA1 is modulated by bioenergetics¹⁹ and altered by mitochondrial dysfunctions.²⁰ Mitochondrial fission is modulated by a machinery that relies on two cytosolic GTPases (DRP1/DNM1L and dynamin 2) and their complex interactions with outer membrane receptors, ER, MAMs, and the actin cytoskeleton.²¹–²³ To date, no specific machinery for inner membrane fission has been identified.

Mitochondrial fusion and fission are essential processes that are required for embryonic development of mice.²⁴ Further work has revealed that alterations of mitochondrial fusion/fission dynamics provoke defects in cellular bioenergetics and/or physiology²⁵ and that mutations of genes encoding fusion/fission proteins provoke hereditary neuropathies with highly variable symptoms.²⁴ While mutations of a fusion factor of the outer membrane (MFN2) are associated with a peripheral sensory and motor neuropathy (Charcot–Marie-Tooth disease of type 2A, CMT2A), that of an inner membrane fusion factor (OPA1) or of a fission factor (DNM1L) have been linked to optic nerve atrophy (dominant optic atrophy, DOA).²⁶ Alteration of fusion/fission dynamics, however, also has been associated with metabolic diseases and/or diabetes-related syndromes, including the response to high fat diet and/or the function of pancreatic beta cells.²⁷–²⁹

4 Biogenesis, Maintenance, and Quality-Control

Present-day mitochondria are the descendants of prokaryotic endosymbionts that were internalized by the ancestor of today’s eukaryotic cells; they carry their own genome that represents a relic of this evolutionary past.³⁰ The mitochondrial genome encodes a few proteins (13 in humans) that are essential components of the OXPHOS machinery, as well as ribosomal and transfer RNAs (rRNAs, tRNAs) essential for their intramitochondrial translation. All human mtDNA-encoded genes give rise to hydrophobic, transmembrane subunits of the respiratory complexes and the ATP synthase. Besides complex II, the respiratory complexes and the ATP synthase that catalyze the OXPHOS reactions in the inner mitochondrial membrane, therefore, have a double genetic origin: although the majority of their components are encoded by the nuclear genome, a few essential subunits are encoded by a relatively small mitochondrial genome (16.5 kb in humans) that is present in thousands of copies per cell and localizes to hundreds of nucleoprotein complexes termed nucleoids.³¹, ³² The mitochondrial proteome comprises more than 1000 different proteins.³³ The vast majority of these proteins (> 99%) is encoded by the nuclear genome, synthesized on cytosolic ribosomes, and imported into the organelle. The proper biogenesis of mitochondria requires a number of processes ensuring, for example, the maintenance of the mitochondrial genome, the coordination of the two genomes’ expression, the import of nuclear-encoded components, as well as the maturation, folding, membrane insertion, and the final assembly in functional molecular complexes. The biogenesis of mitochondria, therefore, is a challenge for the cell with the coordination of two genomes, the membrane compartmentalization, the elaboration of multisubunit complexes containing co-factors/prosthetic groups. Furthermore, mitochondria are the main source of reactive oxygen species (ROS) and their components and thus are the first targets of ROS-induced damage. An array of proteins with chaperone and protease functions are required to ensure the protein quality control and proteostasis of mitochondria. For example, no less than 40 proteases are identified in mitochondria; 20 are resident and 20 are transient proteases translocated to mitochondria only in certain conditions performing additional proteolytic activities essentially related to apoptosis or autophagy.³⁴, ³⁵

4.1 Mitochondrial Protein Import and Sorting (Fig. 2)

The vast majority of mitochondrial proteins are translated on cytosolic ribosomes and subsequently targeted to and imported into mitochondria. Targeting to the matrix, the inner membrane and the intermembrane space mainly relies on the presence of cleavable targeting signals: 60% of all mitochondrial proteins have a N-terminal mitochondrial targeting signal (MTS) on their precursor that is cleaved upon/after mitochondrial import. Other proteins of the inner membrane and intermembrane space have internal targeting signals that, in some cases, have not yet been identified or characterized. The targeting of outer membrane protein follows somewhat different paths. Highly hydrophobic channels/beta-barrels are imported/inserted by a dedicated machinery, but the machineries allowing for the targeting of proteins with a single membrane anchor remain elusive.³⁶ It is important to note that targeting, folding, processing, and assembly appear exquisitely interlinked and thus, that translocases, proteases, and chaperones fulfill complementary, linked, and partially overlapping functions that are difficult to dissociate/describe separately. In addition, several of these protein import machineries interact with each other and with other complexes such as MICOS or ERMES. Therefore, all these machineries must be considered as a dynamic network rather than individual complexes, and interorganellar interactions have to be considered. Most of the genes encoding these proteins are essential and/or linked to a variety of diseases, showing that these functions are not dispensable. In the following sections, we present a succinct view about the main machineries mediating import and sorting of mitochondrial proteins (see Refs. 37, 38 for more extensive reviews).

Fig. 2 Overview of the main machineries for protein import and quality control in mitochondria. Most mitochondrial proteins are nuclear-encoded and imported into mitochondria. Several types of targeting signals determine the routes and final destination of precursor proteins. Main protein import machineries have been represented on this scheme: TOM, the translocase of inner membrane 23 (TIM23) coupled to presequence-associated motor (PAM), the sorting and assembly machinery complex (SAM), mitochondrial import and assembly machinery (MIA) composed by Mia40 and the sulfhydryl oxidase augmenter of liver regeneration (ALR), small TIMs and the carrier translocase TIM22. A subset of precursor proteins is subjected to proteolytic maturation, for example by the mitochondrial processing peptidase (MPP) for matrix proteins. To acquire a proper folding to become functional, mitochondrial proteins can be assisted by molecular chaperones, such as HSP60/HSP10 and mtHSP70. At last, damaged, unfolded, or unassembled proteins are degraded by quality-control proteases such as the Lon protease homolog (Lon) and the ATP-dependent Clp protease (ClpXP) in the matrix, and the intermembrane-oriented (i-AAA) or the matrix-oriented (m-AAA) proteases of the AAA family in the inner membrane.

4.1.1 Translocase of Outer Mitochondrial Membrane (TOM) Complex

The translocase of outer membrane represents the main entry gate for proteins bearing mitochondrial targeting signals. Precursors of mitochondrial proteins are chaperoned by combinations of cytosolic factors that are determined by the sequence/nature of their targeting signal, hydrophobicity, and many other factors. These precursors are recognized by the receptor proteins of the TOM complex. When exiting the TOM complex, precursor proteins follow different routes, depending on their targeting signals and final destination within mitochondria.

4.1.2 TIM23 Presequence Translocase

TIM23 is the presequence pathway. It resides in the inner membrane (TIM: translocase of inner membrane) and recognizes precursor proteins with cleavable amphipathic N-terminal targeting sequences emerging from the TOM complex. The inner membrane electrical potential difference is required to insert the positively charged N-terminal presequence precursors into the TIM23 pore, but it is not sufficient for a complete translocation across the IM. The mitochondrial HSP70 chaperone protein (mtHSP70), together with its regulating factors, form the presequence- associated motor (PAM) that interacts with the TIM23 core components and hydrolyses ATP as a secondary driving force to pull the proteins entirely inside the matrix. In most cases, the presequence is processed in the matrix by the mitochondrial processing peptidase (MPP) and/or additional matrix proteases. Alternatively, the TIM23 ensures the lateral insertion of precursors into the inner membrane. In this process, named stop-transfer, the translocation is interrupted when a hydrophobic stretch reaches the membrane pore and the protein is released in the inner membrane. Some of these precursors are processed further by the inner membrane protease IMP to release the mature protein in the intermembrane space. Consequently, TIM23 allows proteins to be directed either to the matrix, the inner membrane, or the intermembrane space.

4.1.3 The Carrier Pathway Into the Inner Membrane

The TIM22 complex (translocase of the inner mitochondrial membrane 22) is required for the import of a subset of multispanning hydrophobic proteins of the inner membrane that do not exhibit any cleavable presequence but rather several internal targeting elements. It also is called the carrier translocase as it mediates the import of a large family of carriers that contain six transmembrane domains and catalyzes the exchange of metabolites across the inner membrane.³⁹ TIM22 substrates first are taken up at the TOM exit by soluble chaperones of the small TIM chaperone family in the intermembrane space. The precursor proteins then are delivered to the TIM22 complex from the intermembrane space side and inserted into the inner membrane.

4.1.4 The Intermembrane Space Mitochondrial Import and Assembly Machinery (MIA)

The intermembrane space contains a number of proteins with characteristic cysteine motifs. The MIA complex couples oxidative protein folding to the biogenesis of intermembrane space proteins. It mediates the import and oxidative folding of substrates with conserved cysteine motifs (CX3C, CX9C). Key components are the oxidoreductase Mia40 (CHCHD4) and the sulfhydryl oxidase ALR (augmenter of liver regeneration). Mia40 engages in an intermolecular disulfide bond with the incoming substrate. Following oxidation of the substrate, MIA40 is reoxidized by ALR. In yeast, it has been shown that reduced FAD in Erv1 (yeast ALR) is reoxidized either by transferring the electrons directly to molecular oxygen, or to cytochrome c.⁴⁰–⁴³

4.1.5 Import of Mitochondrial Outer Membrane Proteins

Integral outer mitochondrial membrane proteins are of two types: beta-barrel proteins that are anchored in the outer membrane by multiple transmembrane beta strands and that are evolutionary conserved from Gram-negative bacteria (mitochondria’s ancestor); and proteins that are anchored in the outer membrane by one or more alpha-helical segments. A large variety of import pathways has been reported over the last years for mitochondrial outer membrane proteins and the best described system is the sorting and assembly machinery complex (SAM) for beta-barrel proteins. Several beta-barrel proteins such as VDAC (voltage dependent anion channel), TOM40 or SAM50 (channel forming proteins) are in the outer mitochondrial membrane. The members of this family contain an array of multiple beta strands arranged as transmembrane segments in the outer membrane. The mitochondrial targeting signal in these proteins consists of the last beta strands and the loop in between arranged as a hairpin. This signal is recognized by the TOM complex, which translocates the precursors. The interaction between the TOM complex and the SAM complex promotes the release of beta-barrel proteins into the membrane.

4.2 Protein Quality-Control (Fig. 2)

4.2.1 Protein Trafficking, Processing, and Activation: Processing Peptidases

Most mitochondrial proteins are imported from the cytosol thanks to targeting signals. The majority of these proteins require further processing of this targeting signal once they reach their final location in order to be active. Processing peptidases in the different mitochondrial compartments perform this maturation. In the matrix, MPP (mitochondrial processing peptidase) is in charge of removing the N-terminal targeting peptides from precursor proteins. Likewise, IMMPL1/2 (mitochondrial inner membrane protease) removes hydrophobic sorting signals from proteins sorted to the intermembrane space. Several mitochondrial proteins undergo additional proteolytic cleavages in order to regulate their activities (ribosome assembly and consequently synthesis of mitochondrially encoded subunits of the respiratory chain (AFG3L2), regulation of import (YME1L1), control of mitochondrial membrane composition (YME1L1), regulation of apoptotic resistance (YME1L1), mtDNA maintenance, and gene expression (LONP1, HTRA2), mitochondrial dynamics (YME1L1 and OMA1).³⁴

4.2.2 Chaperones Involved in the Correct Folding of Proteins Within Mitochondria

During import, precursor proteins must be maintained soluble and later must be folded and assembled (reviewed in Ref. 44). As stated previously, MIA40 and small TIM in the intermembrane space and mtHSP70 (HSPA9) together with HSP60 (HSPD1) and HSP10 (HSPE1) in the mitochondrial matrix fulfill chaperone functions. MIA40 and small TIM function in an ATP- independent manner, whereas in the mitochondrial matrix, chaperones are ATP-dependent. The mtHSP70 has an essential role in protein import by acting in the import motor to translocate proteins in the matrix. It also has a concomitant role in maintaining the preproteins unfolded during import to stabilize them and to avoid irregular associations. The mtHSP70 has a further role in the correct folding of polypeptides in the matrix together with HSP60, not only for incoming polypeptides but also for the biosynthesis and assembly of mtDNA-encoded subunits of the OXPHOS system. HSP60 is a member of the chaperonins/GroEL family also located in the matrix. It forms large complexes that interact with HSP10. ATP hydrolysis driven conformational changes allow the folding of the substrate proteins. Both mtHSP70 and HSP60 are essential proteins highlighting their unique and important roles in cellular metabolism.

4.2.3 Additional Factors Required for the Biosynthesis of OXPHOS Complexes

In addition to classical chaperones with general functions in mitochondria, numerous substrate-specific factors are required to obtain functional complexes of the respiratory chain or functional ATP synthase. Indeed, the biogenesis of large hetero-oligomeric complexes such as OXPHOS complexes is a challenge for the cells because of the large number of subunits added to their dual genetic origin and the presence of metal cofactors. Therefore, these processes usually are assisted by dedicated chaperones and multiple quality-control checkpoints. A striking example is the biogenesis of the cytochrome-c-oxidase, the copper-heme a terminal oxidase of the respiratory chain (complex IV). This transmembrane complex consists of 14 subunits of which three are mtDNA-encoded. To date, 30 COX assembly factors already have been described, with roles ranging from mitochondrial mRNA stability and translation activation to metalation and assembly of hemes and of the different subunits in functional complexes and supercomplexes.⁴⁵

4.2.4 Protein Quality Control (PQC) (Table 1)

Protein quality control refers to the mechanisms and factors required for the maintenance of protein homeostasis in mitochondria in normal or under stress conditions. These factors include chaperones to take care of unfolded or misfolded proteins, as well as proteases to degrade proteins that are terminally damaged (oxidized, aggregated) and/or do not reach their final destination (not assembled properly). The mtHSP70 and HSP60 have important roles in preventing aggregation, refolding of misfolded proteins to an active conformation, or stabilization in a soluble state awaiting for degradation by proteases. They are strongly induced in stress conditions where damaged/unfolded proteins accumulate (elevated temperatures, exposure to toxic compounds, elevated ROS, mitochondrial unfolded protein response), and were accordingly initially recognized as HSPs (heat shock proteins).⁴⁴

Table 1

An array of mitochondrial proteins (molecular chaperones and proteases) are involved in the maintenance of protein homeostasis in mitochondria. The main components have been listed in this table together with their known implications in disease and in metabolism related to diabetes. IMS, inter-membrane space; AAA+, ATPases associated with diverse cellular activities.

Proteases involved in PQC are mostly chambered proteases of the AAA family (ATPases associated with diverse cellular activities) that hold a chaperone-like activity allowing for the necessary unfolding of substrate for their ATP-driven translocation inside the protease cavity and proteolytic cleavage: the Lon protease homolog (LONP1) and the ATP-dependent ClpP protease in the matrix, intermembrane-oriented i-AAA (YME1L1) and matrix-oriented m-AAA both anchored in the inner mitochondrial membrane.³⁴, ³⁵, ⁴⁴ They degrade membrane spanning and membrane-associated subunits of the electron transport chain that are damaged, oxidized, or nonassembled, and have regulatory functions (mitochondrial protein synthesis, dynamics, calcium homeostasis). LONP1 substrates are aconitase, succinate dehydrogenase subunit 5, and transcription factor A (TFAM), and several other OXPHOS complex subunits, such as heme-related enzymes.

Mitoproteases are also essential components of the mitochondrial stress response (for instance heat stress, elevated ROS levels, low inner membrane potential difference). UPRmt (mitochondrial unfolded protein response) is a good example of recently described mitochondrial stress response. It is triggered by mitochondrial proteotoxic stress (accumulation of unfolded/unassembled proteins) that induces the expression of nuclear genes, notably nuclear- encoded mitochondrial quality-control components (protease and chaperones) in order to restore mitochondrial protein homeostasis. There is evidence for the implication of CLPP and LONP1 in this process probably together with YME1L1. Another example is the inactivation of mitochondrial fusion through the proteolytic processing of OPA1 by the inner membrane metalloendopeptidase OMA1 in stress conditions.⁵²

Intricate links between mitochondrial quality control and energetic metabolism have been reported in human metabolic diseases or mouse models.⁵³–⁵⁵ For example, a knockout mouse model of the stress-induced metalloprotease OMA1 alters the metabolic function. Oma1-deficient mice show an obesity phenotype, characterized by an increase in body weight and hepatic steatosis, in addition to defective thermogenesis because of reduced energy expenditure. These alterations were especially significant under stress conditions (high-fat diet or cold-shock).⁵¹, ⁵⁶ Surprisingly, however, a recent report showed that loss of another mitochondrial protease participating in quality-control (ClpP) in mice leads to reduced adiposity and improved insulin sensitivity, increased whole-body energy expenditure, and upregulated mitochondrial biogenesis in white adipose tissue. Moreover, the absence of ClpP protects mice from diet-induced obesity and insulin resistance.⁵⁰

5 Mitochondrial Functions

5.1 Mitochondrial Oxidative Phosphorylations and Mitochondrial Carriers

Mitochondrial outer membrane has a high permeability, thanks to porine (VDAC) which, on isolated mitochondria, allows the free diffusion of molecules up to 5000 Da. It has been shown that in situ, however, permeability of this channel could be regulated,⁵⁷ leading to subtle regulations of mitochondrial oxidative phosphorylations and regulation of metabolic channeling processes.⁵⁸ In eukaryotic cells, oxidative phosphorylations take place in the inner membrane of mitochondria. According to the chemiosmotic theory initially formulated by Peter Mitchell,⁵⁹ this process converts a redox energy―from food conversion processes―into a proton osmotic gradient (electrochemical proton potential difference that has an electric component ∆ Ψ and a chemical component ∆ pH) that is itself converted into another kind of chemical energy usable for various cell functions, that is, the phosphate potential (ATP/ADP.Pi). Since Mitchell’s proposal of the chemiosmotic theory, numerous studies have validated this hypothesis, which is now well accepted. Oxidative phosphorylations require the mitochondrial respiratory chain, the ATPsynthase, ATP/ADP antiporter, and the Pi−/H+ symporter. In contrast to the outer membrane, the mitochondrial inner membrane is impermeable, particularly for molecules that harbor a positive or negative charge. Metabolites exchanges between the intermembrane space and mitochondrial matrix are ensured by a family of proteins called mitochondrial carriers. The main carriers are listed in Table 2.⁶⁰, ⁶¹ These carriers can be classified in regards to their dependence to the components of the inner membrane potential difference (∆ Ψ and ∆ pH) and the transport mechanism. Of interest for the purpose of this chapter is the ATP/ADP carrier (ANT) that catalyzes the electrogenic exchange (dependent on ∆ Ψ) of ATP⁴ − with ADP³ −; another electrogenic exchanger is the glutamate/aspartate exchanger that catalyzes the electrogenic exchange of aspartate with glutamate + H+. This mechanism is responsible for the vectorial aspect of the exchange (i.e., glutamate import and aspartate export), which is mandatory for the transfer of reduced equivalents in the mitochondrial matrix by the malate aspartate shuttle. The Pi−/H+ and pyruvate/H+ symporters are electroneutral but dependent on the ∆ pH, which favors Pi and pyruvate import. Di- and tricarboxylate carriers are electroneutral. Calcium import/export to/from mitochondrial matrix involves three distinct carriers. Calcium import goes through mitochondrial calcium uniport, which is electrogenic, and calcium release from the matrix requires an electrogenic exchanger (Ca² +/3–4Na+). It also has been proposed a Ca² +/2K+ and a Ca² +/2H+. Their relevance, however, is still controversial.⁶¹

Table 2

Mitochondrial carriers allow an exchange of varied compounds between the inter-membrane space and the mitochondrial matrix. These carriers can be separated in two categories: the electrogenic carriers, such as adenine nucleotide translocator (ANT), aspartate/glutamate carrier (AGC) and mitochondrial Ca² + uniporter (MCU), and the electroneutral carriers, such as phosphate inorganic carrier (PiC), oxoglutarate carrier (OGC), citrate carrier (CIC), dicarboxylate carrier (DIC), tricarboxylate carrier and mitochondrial pyruvate carrier (MPC).

The respiratory chain comprises four complexes: I–IV. Complexes I, III, and IV couple electron transfer to proton extrusion whereas complex II only transfers electrons to the quinone pool. Complex I (NADH-CoQ oxidoreductase) catalyzes the electron transfer from NADH to quinone. Complex II (succinate-CoQ oxidoreductase) couples succinate oxidation to quinone reduction. Complex III (quinol-cytochrome c oxidoreductase) couples quinol oxidation to cytochrome c reduction. Complex IV (cytochrome c oxidase) catalyzes the last step of electron transfer from reduced cytochrome c to oxygen. Several other enzymes exist in the inner mitochondrial membrane, which can feed the respiratory chain with electrons such as glycerol-3-phosphate (G3P) dehydrogenase, electron transfer flavoprotein dehydrogenase, dihydroorotate dehydrogenase, and choline dehydrogenase. Complexes I, III, and IV use the redox span along the respiratory chain (Gibbs energy of the redox reaction) to pump out protons against the electrochemical proton gradient (see Fig. 3). The inner membrane electrical potential difference is used by some mitochondrial carriers and mainly by the ATPsynthase, which is able to convert passive proton transfer into ATP synthesis from ADP and Pi (Fig. 3). The structure of the respiratory chain as a succession of complexes relies on both functional studies and biochemical isolation.⁶² Electron transfer in between the complexes depends on both the quinone pool and cytochrome c, which are electrons mobile carriers. Use of nonionic detergents allowed the isolation of more organized structures, called supercomplexes, in which some of the previously described complexes are associated with specific stoechiometries.⁶³ The composition of these supercomplexes has been shown to be variable and to depend on physiological conditions. It has been proposed that these supercomplexes would be an advantage to the functioning of the respiratory chain, such as electrons channeling, kinetic efficiency, decrease in ROS production. These proposals, however, are still debated to this day.⁶⁴–⁶⁶

Fig. 3 Scheme of the mammalian oxidative phosphorylation (OXPHOS) system. The OXPHOS system can be described as two functional entities: the respiratory chain (RC) and the phosphorylating system. The RC is composed by holoenzymes as the NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), cytochrome-c reductase (complex III), and cytochrome-c oxidase (complex IV). Other dehydrogenases, such as the glycerol-3-phosphate dehydrogenase (G3PDH), can be connected to the RC. The RC also is composed by mobile electrons carrier such as coenzyme Q and cytochrome-c. The RC is functionally coupled to the phosphorylating system composed by the ATP synthase, adenine nucleotide transporter (ANT), and phosphate inorganic carrier (PiC).

5.2 Thermokinetic Control and Yield of Oxidative Phosphorylations

According to the chemiosmotic hypothesis,⁶⁷, ⁶⁸ the energy transduction between redox free energy and phosphate potential is allowed by inner mitochondrial membrane proton pumps structurally independent but functionally connected by an inner membrane electrical potential difference between two bulk phases: intermembranal space and mitochondrial matrix (∆ μH+). By analogy with the functioning of an electric battery, Mitchell introduced the term protonmotive force (∆ p = ∆ μH+/F), which is used largely in bioenergetics. Two main questions to be taken into account are the yield of energy conversion (ATP/O) and the relative fluxes involved in this process where both ATP synthesis and NADH oxidation are important. It is necessary, therefore, to understand how the values of the different coupled fluxes are determined in an integrated system, such as oxidative phosphorylations. Obviously, the coupling of fluxes is mediated by forces, but, at first sight, the quantitative relationships between fluxes might depend on the properties of the whole system. In a complex metabolic network like mitochondrial oxidative phosphorylations, a very simple quantitative analysis is the determination of the yield of the overall reaction, as is performed by measuring ATP synthesis flux over oxygen consumption flux (ATP/O). With this approach, the yield can vary and several mechanisms possibly involved have been proposed. The first mechanism decreasing the coupling efficiency, the proton leak, is a direct consequence of the nature of the energetic intermediary, the protonmotive force. Biological membranes always present some proton conductance (LH), and the resulting proton flux is strictly dependent on protonmotive force (JH = LH·∆p). This membrane conductance is a specific property of the membrane itself, but is not entirely independent from the protonmotive force. The size of this proton leak can modulate the yield of oxidative phosphorylations (ATP/O), and it has been shown that protonophores uncouple oxidative phosphorylations by increasing proton membrane conductance.⁶⁹–⁷⁵ In this uncoupling process, the first event is a dissipation of protonmotive force, which leads to an increase in respiratory rate and a decrease in the ATP synthesis rate, thus leading to a decrease in ATP/O ratio. Even if much experimental work has established strong evidence that some protonophoric action can quantitatively account for the uncoupling of oxidative phosphorylations,⁷⁶ no definitive proof that the uncoupling is exclusively and quantitatively because of an increase in cation membranal conductance has been obtained. In fact, from a growing amount of data, it is evident that the question of the multiplicity of uncoupling mechanisms is still open. The first member of the uncoupling protein (UCP) family, brown adipose tissue uncoupling protein 1 (UCP1), was identified in 1976.⁷⁷ Its uncoupling activity has been demonstrated largely, as well as the regulators of this activity. It is activated by fatty acids and inhibited by GDP. Its activation ensures thermogenesis. Based on sequence homology, closely related proteins were described in mammals. The actual function of these proteins (i.e., UCP2 and UCP3) is still a matter of debate.⁷⁸, ⁷⁹ The definition of the function of these proteins is crucial in terms of bioenergetics in the sense that a slight uncoupling would lead to a decrease in protonmotive force, in ATP synthesis and in mitochondrial ROS production.

Among the multiplicity of uncoupling mechanisms, two kinds of experimental evidence can be noted: It has been shown that some uncoupling effects are not linked to a significant decrease in protonmotive force, which indicates that the decrease in oxidative phosphorylations efficiency is not, in this case, the consequence of an increase in membranal proton conductance⁸⁰–⁸⁶; and direct or indirect estimations of the coupled flow through different proton pumps indicate that their intrinsic stoichiometry, that is, the H+/2e− stoichiometry of the respiratory chain and the H+/ATP stoichiometry of the ATPsynthase, might vary as a function of many physical parameters or some drug addition.⁸³, ⁸⁴, ⁸⁶–⁹⁷, This led Azzone et al.⁹⁸, ⁹⁹ to propose another mechanism causing a loss of oxidative phosphorylations yield. Such a new possibility called slip is a decrease in the efficiency of a proton pump because of partial and variable decoupling of chemical reaction and proton transport. We have described a new energy wastage mechanism of interest. In isolated yeast mitochondria, the membrane proton conductance is shown to be strictly dependent on external dehydrogenases activity. An increase in their activity leads to an increase in the membrane proton conductance. This proton permeability is independent of the respiratory chain and ATP synthase proton pumps. This mechanism is an active proton leak. In such a process, the high cellular redox constraints can be alleviated without affecting much the ATP synthesis flux and allows a decrease in mitochondrial ROS production.¹⁰⁰

5.3 Mitochondrial ROS and Their Detoxification

5.3.1 Dioxygen: A Poison for Life

Dioxygen, owning two unpaired electrons each located in a different antibonding orbital, is a biradical. These two electrons have the same spin quantum number or, as often is written, they have a parallel spin. This constitutes the most stable state, that is, ground state, of dioxygen. If a diatomic oxygen molecule attempts to oxidize another atom or molecule by accepting a pair of electrons, both of these electrons must have an antiparallel spin, so as to fit the vacant space in the orbitals. In most cases, electrons forming a pair in an orbital have opposite spins in accordance with Pauli’s principle. This imposes a restriction on electron transfer, which leads dioxygen to accept electrons one at a time. Consequently, dioxygen reacts only very slowly with all nonradicals. Even if the redox potential of the O2/H2O couple is extremely positive, oxygen in the air cannot immediately burn living organisms because of the structure of their organic compounds. This spin restriction of dioxygen reactivity allowed the complex evolution of organisms and a metabolic adaptation, that is, the use of dioxygen as last electron acceptor during catabolism leading to an efficient energy transfer in cells.¹⁰¹

−. This compound is a very active electron donor, potentially able to generate a toxic cascade of electron transfer reactions. At physiological pH, however, the main reason for superoxide disappearance in aqueous solution is its dismutation catalyzed by the superoxide dismutase enzyme. It often is said that spontaneous dismutation is very rapid, but it is worth noting that such a reaction becomes slower as the pH rises and yeast mutant devoid of superoxide dismutases (sod 1 and sod 2) cannot survive when cells switch from fermentation to growth fueled by respiration.− (i.e., the Fenton reaction). In biological systems, this product can induce a cascade of radical reactions potentially involving any cell compound.

5.3.2 ROS Production by the Respiratory Chain

It generally is believed that the main superoxide producer in the cell is the respiratory chain (Fig. 4). Two of the respiratory chain complexes (I and III) have been, for a long time, recognized as involved in superoxide production.¹⁰⁴ Under physiological conditions, predictions estimate that the superoxide production is about 0.1% of the respiratory rate.¹⁰⁵, ¹⁰⁶ One should keep in mind, however, that such a production is highly dependent on the respiratory state/rate of the mitochondria. At the level of complex III, it has been shown that the Q-cycle can produce superoxides on the inner and outer surfaces of the inner mitochondrial membrane.¹⁰⁷, ¹⁰⁸ Because superoxides do not cross this membrane, it is important to know the size of the relative fluxes in the intermembranal space and into the matrix.

Fig. 4 Main enzymatic systems of detoxification of ROS generated by the OXPHOS system. NADH dehydrogenase (I), glycerol-3-phosphate dehydrogenase (G3PDH), succinate dehydrogenase (II), cytochrome c reductase (III) generate superoxides; CoQ , coenzyme Q; Cyt c , cytochrome c; GPX , glutathione peroxidase; Gred , glutathione reductase; GSH , reductive form of glutathione; GSSG , oxidative form of glutathione; TPX , thiol peroxidase; Tred , thiol reductase; Trx O 2 ˙ − , superoxide; SOD , superoxide dismutase; H 2 O 2 , hydrogen peroxide; TH , transhydrogenase.

Another site in the respiratory chain involved in superoxide formation is complex I. There is now general agreement that, in this large multisubunit complex, the electron transfer is working at near equilibrium and, therefore, superoxide production might be linked to both forward electron transport (FET) and reverse electron transport (RET).¹⁰⁴, ¹⁰⁹–¹¹⁵, It is likely that different sites of superoxide production exist in complex I and that the sites involved are different for FET or in RET.¹¹⁶, ¹¹⁷ The sites of complex I associated superoxide generation are still controversial. Three main sites have been proposed: the flavine mononucleotide (FMN),¹¹⁸–¹²⁰ the Fe-S clusters (more likely N1a or N2)¹¹², ¹²¹, ¹²² and a ubiquinone specifically linked to complex I (iQ site).¹²³ Rotenone inhibits electron transfer right upstream the quinone binding site. Consequently, superoxides that are produced by complex I in the presence of NADH are most likely because of the electron carriers (flavin or Fe/S clusters). ROS generation flux, however, seems mainly linked to reverse electron flux, and the iQ site could be a major player in this flux. It should be stressed that, unlike ROS production for FET, ROS production from RET can originate either at the actual RET site or at the dehydrogenase level.

As previously mentioned, the respiratory chain complexes are the most studied elements regarding the contribution of mitochondria to ROS production. The mitochondrial dehydrogenases, however, also have been shown to be involved in the following process.

The α-ketoglutarate dehydrogenase complex (αKGDHC) catalyzes the oxidation of α- ketoglutarate in succinyl-coA with the production of NADH in the Krebs cycle. It is composed of multiple copies of three enzymes: α-ketoglutarate dehydrogenase (E1), dihydrolipoamide succinyl-transferase (E2), and dihydrolipoyl dehydrogenase (Dld or LADH, E3).¹²⁴–¹²⁶ Starkov et al.¹²⁷ showed that this complex is able to produce ROS during its catalytic process in mammalian brain mitochondria. This event seems to be linked to the flavin cofactor of the Dld, which can generate superoxide anion.¹²⁸, ¹²⁹ These observations suggest the possibility of ROS generation by other mitochondrial flavoenzymes such as the pyruvate dehydrogenase complex (PDHC), which also is constituted of a Dld.¹²⁷

One way to reoxidize the NADH produced during glycolysis is the glycerol-3-phosphate shuttle. This shuttle is made up of two isoforms of the enzyme called glycerol-3-phosphate dehydrogenase (G3PDH), which differ by their localization and cofactors. The mitochondrial isoform¹³⁰ is on the external side of the mitochondrial inner membrane and is a FAD-linked enzyme that donates electrons to the respiratory chain via the ubiquinone pool. Its ROS production is supposed to be the consequence of the absence of a coenzyme Q binding site in the mitochondrial G3DPH, which would diminish the protection of ubisemiquinone produced during glycerol-3-phosphate oxidation.

Years ago, it was shown that pigeon and rat mitochondria respiration with palmitoyl-carnitine as substrate could lead to H2O2 release.− or from the semiquinone formed during ETF oxidation.¹⁴⁴–¹⁴⁶

Consequently, ETF and/or ETF-QO are supposed to generate ROS with palmitoyl-carnitine as substrate.

5.3.3 Control of ROS Production Flux by Mitochondria

The redox level of the respiratory chain electron carriers, including ubisemiquinone, is thermokinetically controlled. Thus, the forces directly associated with respiratory chain activity, that are the redox potential of the NADH/NAD+ couple and the protonmotive force, are powerful regulators of the steady-state concentration of the free ubisemiquinone radical. An increase in electron supply to the respiratory chain or in protonmotive force must lead to a rise in ubisemiquinone radical content. Moreover, kinetic constraints exerted downstream of the quinone pool (e.g., at the level of cytochrome oxidase) could further increase the level of free radical ubisemiquinones. Moderate uncoupling, however, will lead to a decrease in force and kinetic constraints and, therefore, effectively decrease superoxide production by the respiratory chain.¹⁴⁷ The opposite effect can be observed when the respiratory chain is inhibited.¹³², ¹⁴⁸ Other mechanisms that induce a decrease of the redox proton pump efficiency (such as slipping) are expected to be less effective because they do not affect the protonmotive force significantly.¹⁴⁹–¹⁵² It is well known that the transition nonphosphorylating/phosphorylating or the uncoupling state decrease ROS generation in isolated mitochondria.¹⁵³ Free fatty acids exert different effects on mitochondria; they inhibit electron flux at the complex I and probably complex III levels inducing an increase in ROS production associated with forward electron transport in isolated rat heart or liver mitochondria.¹⁵⁴ They also induce a slight uncoupling effect that seems responsible for a large decrease in ROS formation linked to reverse electron transfer. This illustrates the subtle ROS production response to changes in electron flux through the respiratory