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Nitric oxide and mitochondrial biogenesis | Journal of Cell Science

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Nitric oxide and mitochondrial biogenesis | Journal of Cell Science

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Summary

The characteristic structural organization of mitochondria is the product of synthesis of macromolecules within the mitochondria together with the import of proteins and lipids synthesized outside the organelle. Synthetic and import processes are required for mitochondrial proliferation and might also facilitate the growth of pre-existing mitochondria. Recent evidence indicates that these events are regulated in a complex way by several agonists and environmental conditions, through activation of specific signaling pathways and transcription factors. A newly discovered role of this organelle in retrograde intracellular signaling back to the nucleus has also emerged. This is likely to have far-reaching implications in development, aging, disease and environmental adaptation. Generation of nitric oxide (NO) appears to be an important player in these processes, possibly acting as a unifying molecular switch to trigger the whole mitochondrial biogenesis process. High levels of NO acutely inhibit cell respiration by binding to cytochrome c oxidase. Conversely, chronic, smaller increases in NO levels stimulate mitochondrial biogenesis in diverse cell types. NO-induced mitochondrial biogenesis seems to be linked to proliferation and differentiation of normal and tumor cells, as well as in aging.

Introduction

Metabolically active cells, such as liver, kidney, muscle and brain cells, contain hundreds or thousands of mitochondria, which make up ∼40% of the cytoplasm. The egg cell (oocyte) passes on ∼100,000 mitochondria to the next generation. By contrast, blood cells and skin cells have very few or no mitochondria; sperm usually have <100. There are said to be 10 million billion mitochondria in an adult human (i.e. ∼10% of our body weight).

Mitochondria first captured the attention of cell physiologists some 50 years ago. The elucidation of their role in energy production - the passing of electrons along the series of respiratory enzyme complexes in the inner mitochondrial membrane, and the ensuing build up of a transmembrane proton gradient that drives ATP synthase - is one of the most fascinating enterprises in the history of science ( Mitchell, 1993). Recent evidence suggests that this process occurs in organelles that are not static. Mitochondria are in constant movement within cells, and numerous fusion and/or fission events take place. These are accompanied by variations in mitochondrial size, number and mass, which are triggered by a variety of physiological stimuli and differentiation states. More than 1000 genes and ∼20% of cellular proteins are involved, and a complex regulatory network ( Attardi and Schatz, 1988; Kelly and Scarpulla, 2004), including factors such as the transcription factors peroxisome-proliferator-activated receptor γ coactivator 1α (PGC-1α), nuclear respiratory factors (NRF-1 and NRF-2) and mitochondrial transcription factor A (Tfam), coordinates their behavior ( Kelly and Scarpulla, 2004).

The energetic role of mitochondria is central to the origin of the eukaryotic cell and the development of complex organisms; it is also involved in birth, aging-related diseases and cell death. Interestingly, the endogenous signaling molecule nitric oxide (NO) and other free radicals seem to play an important part in mitochondrial biology. Here, we discuss their roles in the different life processes under mitochondrial control, from birth to metabolism, aging and disease.

Mitochondrial biogenesis and morphology

The word biogenesis has been used to describe both the formation of new mitochondria in cells ( Attardi and Schatz, 1988; Leaver and Lonsdale, 1989) and their phylogenesis ( Roodyn and Wilkie, 1968; Shepard et al., 1998). Comprehension of its evolutionary origin is a prerequisite for understanding any biological structure or process. Here, we would only remark that the endosymbiotic hypothesis ( Margulis, 1981; Gray et al., 1999; Gray et al., 2001) postulated over a century ago ( Altmann, 1890) draws much support from the discovery of the unique genome of this organelle, which is presumed to be a relic of its evolutionary past. Studies of mitochondrial DNA (mtDNA) and its expression have amply affirmed the eubacterial roots of this genome ( Gray, 1992); knowledge of mitochondrial gene sequences has enabled researchers to trace back to a single ancestor that existed around the time of the division of the Proteobacteria ( Yang et al., 1985). Members of the rickettsial subdivision of the α-Proteobacteria are considered to be among the closest known eubacterial relatives of mitochondria ( Gray and Spencer, 1996).

Mitochondria divide during mitosis, providing daughter cells with a normal complement of mitochondria. There are also instances in which mitochondrial divisions are not tied to the cell cycle. For example, muscle mitochondria proliferate during myogenesis and following exercise ( Brunk, 1981; Moyes et al., 1997). Mitochondrial division can be induced by a wide range of substances, including benzodiazepine, inhibitors of oxidative phosphorylation, phorbol esters and Ca ²⁺ fluxes ( Vorobjev and Zorov, 1983; Muller-Hocker et al., 1986; Kawahara et al., 1991; Bereither-Hahn and Voth, 1994). In vertebrates, the number of mitochondria, or rather the volume of mitochondrial mass per cell, is further controlled by thyroid hormones, such as T ₃, which broadly influence metabolic rates in vertebrates and might specifically induce mitochondrial division ( Goglia et al., 1999). In addition, exposure of mammals to a low-temperature environment for prolonged periods of time induces a marked increase in mitochondrial mass in brown adipocytes; this provides an important mechanism for maintaining body energy balance and core temperature ( Klaus et al., 1991).

Mitochondria in cells of most tissues are tubular, but dynamic changes in morphology are driven by fission, fusion and translocation ( Bereiter-Hahn, 1990; Bereiter-Hahn and Voth, 1994; Nunnari et al., 1997; Yaffe, 2003; Meeusen et al., 2004; Okamoto and Shaw, 2005) ( Fig. 1). Mitochondria branch, stretch, retract and may even roll and unroll ( Rube and van der Bliek, 2004). At any given moment, they can be considered to be in a state of transition ( Yoon, 2005). Circumstantial evidence suggests that there is an intricate structure-function relationship between mitochondrial morphology and energy-producing activity ( Mannella et al., 1997; Nakada et al., 2001; Bach et al., 2003). Thus, it is intriguing to speculate that cells may change the shape of their mitochondria to alter ATP production ( Yoon, 2005). Furthermore, since long tubular mitochondria have been hypothesized to serve as cellular energy-transmitting cables ( Chen, 1988), morphological changes could disrupt or restore the local energy supply ( Yoon, 2005).

The dynamic nature of mitochondria might protect them by ensuring that regional losses of membrane potential, caused perhaps by local depletion of metabolic substrates or mtDNA, are always transient. In particular, mitochondrial fusion enables intermitochondrial cooperation by allowing exchange of membrane and matrix components; it might therefore help to restore local depletions and maintain mitochondrial function ( Nakada et al., 2001). An exciting recent paper reports that mitochondria and mtDNA can even move between cells along cytoplasmic projections ( Spees et al., 2006), which might rescue aerobic respiration in mammalian cells lacking functional mitochondria.

Finally, mitochondria also depend on the import transport of numerous proteins from the cytosol. This process relies on mitochondrial chaperones including heat shock protein 70 (Hsp70), as well as a specific import machinery including the TOM and TIM translocases ( Neupert, 1997; Wickner and Schekman, 2005). Taking into account the features of mitochondrial biology discussed above, we propose that an `operational definition' of mitochondrial biogenesis encompasses: (1) the ability to increase oxidative phosphorylation and ATP production in response to energy demands; (2) increased synthesis of new organelle constituents and their integration into the pre-existing mitochondrial reticulum; and (3) the import of nuclear-encoded proteins and fusion of single organelles to form a network with increased metabolic function.

NO as a regulator of mitochondrial functions

NO is synthesized from l-arginine and O ₂ by NO synthase (NOS) in almost all mammalian cells ( Moncada et al., 1991; Alderton et al., 2001). Three distinct cellular isoforms of NOS have been identified: the endothelial (eNOS) and neuronal (nNOS) isoforms are regulated by second messengers; and inducible NOS (iNOS) is induced by cytokines and bacterial products. All three isoforms can in fact be regulated by transcriptional and post-transcriptional mechanisms and are constitutively expressed in certain tissues ( Moncada et al., 1991; Alderton et al., 2001). Moreover, a mitochondrial NOS isoform (mtNOS) has been described as a constitutive protein of the mitochondrial inner membrane that generates NO in a Ca ²⁺-dependent reaction (reviewed by Ghafourifar and Cadenas, 2005), although its existence has been doubted by some ( Lacza et al., 2006). It is notable that eNOS is attached to the outer mitochondrial membrane in neurons and endothelial cells ( Henrich et al., 2002; Gao et al., 2004), which indicates that mitochondria might regulate NOS activity and, conversely, that eNOS might regulate mitochondrial function.

NO could act on mitochondria at several levels. Because of its vasodilating properties, it regulates blood flow to tissues; thus, indirectly, it supplies respiratory substrates to mitochondria and redistributes heat generated by respiring mitochondria. In addition, NO directly regulates the binding to and release of O ₂ from hemoglobin ( Wolzt et al., 1999) and thus the supply of O ₂ to mitochondria. NO also regulates mitochondrial function by binding to cytochrome c oxidase, the terminal enzyme in the electron-transport chain. It competes with O ₂, inhibiting the activity of the enzyme ( Cleeter et al., 1994; Brown and Cooper, 1994; Clementi et al., 1998) and thus negatively regulating mitochondrial oxidative phosphorylation - particularly at the low O ₂ concentrations usually found in tissues ( Clementi et al., 1999). This also leads to redistribution of O ₂ to neighboring cells ( Hagen et al., 2003). Moreover, the NO-dependent inhibition of cell respiration can change as part of the adaptive response to stress - for example, in response to alcohol toxicity ( Venkatraman et al., 2004) and cardiac failure ( Brookes et al., 2001). Together with hypoxia inducible factor 1α (HIF)-α, the NO-cytochrome c oxidase system helps fine-tune cell metabolism ( Semenza, 1999). Such a sensing mechanism might, for example, allow cardiac myocytes to adapt their metabolic function to hypoxia (Budinger, 1996). Finally, the recent finding that mitochondria have a form of NOS themselves is consistent with the idea that NO regulates mitochondrial functions directly ( Giulivi et al., 1998; Ghafourifar and Cadenas, 2005).

NO generated by eNOS: a key player in mitochondrial biogenesis?

Treatment of various cells with NO donors increases their mtDNA content, and this is sensitive to removal of NO by the NO scavenger oxyhemoglobin ( Nisoli et al., 2003). This effect occurs through increased expression of PGC-1α - the principal regulator of mitochondrial biogenesis - NRF-1, NRF-2 and Tfam ( Kelly and Scarpulla, 2004). It depends on the second messenger cGMP, through which NO frequently acts. Such NO-dependent mitochondrial biogenesis occurs in numerous cell types and is not restricted to a specific cell lineage or species ( Nisoli et al., 2003; Nisoli et al., 2004).

An important aspect of this effect is that it generates functionally active mitochondria capable of generating ATP through oxidative phosphorylation ( Nisoli et al., 2004). The significance of this profound change in energy metabolism remains to be investigated. However, mitochondrial activity is known to play crucial roles in various processes, such as the switch of skeletal muscle fibres from glycolytic to oxidative metabolism ( Lin et al., 2002), and the regeneration of cardiac and skeletal muscles ( Lehman et al., 2000; Stamler and Meissner, 2001).

Studies of eNOS -/- mice have demonstrated an obligatory role of eNOS in mitochondrial biogenesis. Brown fat from these mice is functionally inactive, and exposure of the animals to cold has been found to blunt mitochondrial biogenesis and (unlike in wild-type animals) results in a steep decline in core temperature ( Nisoli et al., 2003). In addition, deletion of eNOS is sufficient to reduce mitochondrial mass even in tissues that have basal expression levels of nNOS, and possibly iNOS, such as the brain, liver, muscle and heart. This is accompanied by a reduction in both basal O ₂ consumption and steady-state ATP levels, which occurs both in tissues dependent on oxidative metabolism and in glycolytic tissues, indicating that the effect is a general phenomenon.

The importance of NO as a mitochondrial biogenetic stimulus has broad implications for pathology. Impairment of mitochondrial function is associated with neurodegenerative diseases, neuromuscular disorders, liver and heart failure, and type 2 diabetes ( Kopecky et al., 1995; Hansford et al., 1999; Lehman et al., 2000; Patti et al., 2003; Mootha et al., 2003). The potential role of NO in diabetes and obesity is particularly relevant. In eNOS -/- mice, O ₂ consumption, an indicator of metabolic rate, is decreased. In genetic models of obesity, defective energy expenditure is linked to increased food intake and body-weight gain. eNOS -/- mice show similar food consumption to, but weigh more than, wild-type mice. Their increased body weight could be a result of their higher feed efficiency (i.e. ratio of weight gain: food intake) caused by defective energy expenditure ( Nisoli et al., 2003). Generating new, metabolically active mitochondria might therefore be an approach to the treatment of disorders in which impaired energy expenditure is evident.

Signaling through NO

The mechanisms by which NO and/or cGMP increase expression of PGC-1α and genes encoding mitochondrial proteins have not been analyzed in detail. Interestingly, in brown adipocytes, which exhibit several responses to NO (proliferation, differentiation, respiration and mitochondrial biogenesis), eNOS, NO and cGMP are located in both the cytoplasm and the nucleus. Their levels and activity are dynamically modulated by noradrenaline (NA) ( Giordano et al., 2002) (E.N., C. Tonello and M.O.C., unpublished results). In animals acclimatized to cold, in which high levels of NA are released from sympathetic nerve terminals that innervate richly brown adipocytes, elevations of eNOS, NO and cGMP levels in both the cytosol and nucleus of these cells correlates with an increase in the level of uncoupling protein 1 (UCP1). UCP1 is an inner mitochondrial membrane proton channel that dissipates the inner transmembrane potential (Δψ m) to produce heat ( Matthias et al., 1999; Nedergaard et al., 2001). Interestingly, NA induces expression of PGC-1α, which binds to the UCP1 promoter and increases UCP1 expression ( Lowell and Spiegelman, 2000). Thus, eNOS expression and NO and/or cGMP levels in brown adipocytes are linked to cell activation, mitochondrial biogenesis and heat production. Further investigation is needed to understand whether all these NA responses involve the nuclear NO system.

eNOS activity is modulated by protein-protein interaction and phosphorylation at specific serine or threonine residues. Phosphorylation at Ser114 inhibits its activity, whereas phosphorylation at Ser1177 stimulates it. Klinz et al. showed that eNOS phosphorylated at Ser114 is heavily enriched in the nucleus of proliferating mesenchymal stem cells (MSCs), whereas eNOS phosphorylated at Ser1177 is localized at filamentous structures in the cytosol ( Klinz et al., 2005). NO present in the nuclei of these and other cells might directly or indirectly affect gene expression. Remarkably, several transcription factors possess heme moieties that can bind NO. For example, Reinking et al. found that the fruit fly Drosophila melanogaster E75 nuclear receptor contains heme in its binding pocket and binds NO to control insect steroid biosynthesis ( Reinking et al., 2005). The vertebrate ortholog of E75, Rev-Erbα (which is expressed mainly in fat cells) is a crucial component of the mammalian circadian clock, an elegant molecular circuit in which NO and heme are key regulatory molecules ( Pardee et al., 2004). In addition, the enhancer binding protein NOR, which contains a mononuclear non-heme iron center, serves exclusively as an NO-responsive transcription factor in enteric bacteria ( Gardner et al., 2003). Binding of NO stimulates the ATPase activity of NOR, enabling the activation of transcription by RNA polymerase. Such transcription factors could play important roles in the response to NO, in addition to cGMP-dependent mechanisms.

One gene induced by NO is that encoding Hsp70 ( Kim et al., 1997; Nisoli et al., 2001). Hsp70 is an ATP-driven chaperone that binds each segment of imported mitochondrial protein chains as they enter the inner mitochondrial membrane, thereby restricting net movement to import ( Neupert and Brunner, 2002). Key components of the protein import machinery, including other chaperones and the translocases, have been reported to be upregulated in response to stimuli that induce mitochondrial biogenesis, including exercise and thyroid hormone ( Chabi et al., 2005). This is associated with an elevated import rate of nuclear-encoded proteins of the respiratory chain into mitochondria ( McNew and Goodman, 1994). The role of NO in these processes needs to be further investigated.

Other physiological roles

NO-induced mitochondrial biogenesis also has a role in the physiological phases of the rat ovarian cycle ( Navarro et al., 2005). A moderately increased rate of NO production in the proliferative phase is associated with mitochondrial biogenesis, whereas a high rate of NO generation (probably produced by mtNOS) at pro-estrus phase appears to trigger mitochondria-dependent apoptosis ( Navarro et al., 2005). Thus, the roles of mitochondria as ATP provider, and as a source of NO to signal for mitochondrion proliferation and mitochondria-dependent apoptosis, appear well adapted to serve the proliferation-apoptosis sequence of the ovarian cycle.

NO and mitochondrial biogenesis in `cell disorders'

As mentioned above, reduced NO levels might play a part in several disorders, including diabetes. However, there is also evidence of links between increased mitochondrial biogenesis induced by NO and cell dysfunction. NO mediates CD3-CD28 costimulation-induced mitochondrial hyperpolarization (MHP) and production of reactive O ₂ species (ROS), as well as sustained Ca ²⁺ fluxing in human T lymphocytes ( Nagy et al., 2003). Persistent MHP is associated with increased mitochondrial mass and Ca ²⁺ content in T cells of patients with systemic lupus erythematosus (SLE). Mitochondria can take up, store and release Ca ²⁺; therefore increased mitochondrial mass might account for altered Ca ²⁺ handling in SLE. Indeed, activation of T cells through CD3-CD28 costimulation initiates a biphasic elevation in cytosolic free Ca ²⁺ concentration: while rapid fluxing is enhanced, the plateau phase is diminished in lupus T cells ( Nagy et al., 2004), which exhibit both enhanced spontaneous apoptosis and defective activation-induced cell death. Moreover, monocytes of patients with SLE exhibit significantly higher amounts of NO than normal monocytes ( Nagy et al., 2004). Activated monocytes play a crucial role in the autoimmune response in SLE. In comparison with control monocytes, lupus monocytes increase Δψ m, mitochondrial mass and rapid Ca ²⁺ fluxing, but reduce sustained elevation of intracellular Ca ²⁺ in response to CD3-CD28 costimulation of normal T cells after co-culture ( Nagy et al., 2004). Increased NO production by monocytes might therefore be responsible for mitochondrial dysfunction in SLE ( Nagy et al., 2004).

Large-scale gene-expression analysis demonstrates that, in oncocytomas, which are large-cell tumors characterized by an abnormal proliferation of mitochondria, numerous nuclear genes encoding proteins involved in mitochondrial biogenesis, such as NRF-1 and eNOS, and components of the respiratory chain are upregulated ( Baris et al., 2004). Remarkably, primary B-cell chronic lymphocytic leukemia (CLL) cells contain significantly more mitochondria than do normal lymphocytes and their mitochondrial mass correlates with endogenous NO levels ( Carew et al., 2004). Expression of NRF-1 and Tfam is elevated in most CLL specimens examined and appears to be related to cellular NO levels. Furthermore, treatment of B cells with exogenous NO causes a substantial increase in mitochondrial mass ( Carew et al., 2004). NO-induced mitochondrial biogenesis thus appears to participate in the pathophysiology of multiple disorders. In particular, it might cause primary alterations of the cell cycle and/or apoptotic mechanisms that lead to malignant cell development. Alternatively, NO-induced mitochondrial biogenesis might compensate for a primary defect in mitochondrial ATP production by a feedback mechanism in tumor cells in which oxidative phosphorylation is decreased and substituted for by high rates of aerobic glycolysis ( Warburg, 1956).

NO and mitochondrial biogenesis in aging

Denham Harman, pioneer of free radical biology, first proposed the mitochondrial theory of aging in 1972 ( Harman, 1972). In this theory, the rate of aging and the onset of degenerative diseases is postulated to be determined by the rate of free-radical leakage and attacks on the adjacent mtDNA, which cause mutations that undermine mitochondrial function. As mitochondria decay, the performance of the cell as a whole declines, which leads to the traits observed in aging. Although this idea has been supported by much indirect evidence, the inability of antioxidants to prolong life has weakened support for it (see Lane, 2005; Trifunovic et al., 2005). Additionally, the accumulation of mitochondrial mutations with age has not been generally observed. Several studies have shown that most mitochondria in aging tissues have basically normal DNA, except perhaps in the control region and, moreover, are capable of virtually normal respiration ( Lightowlers et al., 1999; Trifunovic et al., 2005). Unexpectedly, a homoplasmic C150T transition near an origin of mtDNA replication has even been associated with greater longevity in an Italian population ( Zhang et al., 2003).

However, ROS can also function as second messengers that regulate signal transduction pathways and gene expression in the nucleus, and thus are not purely detrimental. Examples of signaling pathways modulated by ROS include: serine/threonine, tyrosine and mitogen-activated protein kinases; growth factors and transcription factors, such as nuclear factor (NF)-κB; soluble guanylate cyclase; intracellular Ca ²⁺; and the K⁺ channel (for reviews, see Forman et al., 2002; Landar and Darley-Usmar, 2003; Wolin et al., 2005; Hool, 2006). In addition, several genes are regulated by ROS in mammalian cells, including protooncogenes ( Fos, Myc and Jun), heme oxygenase, CL100 phosphatase, interleukin-8, catalase, glutathione peroxidase, mitochondrial mangano-superoxide dismutase, natural killer-enhancing factor-B, mitogen-activated protein kinase and γ-glutamyl transpeptidase (reviewed by Pryor et al., 2006).

Furthermore, mitochondria are better protected against ROS damage than was once assumed. Not only are 5-10 copies of the mtDNA genome present in every mitochondrion, but also recent work shows that mitochondria are reasonably efficient at repairing damage to their genes and that recombination can fix mitochondrial genetic damage ( Kraytsberg et al., 2004; Spees et al., 2006). Thus, they might operate a sensitive feedback system, in which the leaked ROS themselves act as signals to calibrate and adjust mitochondrial performance. This does not mean that they are not toxic - just less than previously supposed. Mutations that impair electron transport and consequently lead to ROS production might thus be able to signal the synthesis of new respiratory chain components encoded in the nucleus. Such a `retrograde response' would enable the cell to compensate for a defect.

Mitochondrial retrograde pathways in mammalian cells have been extensively studied (reviewed by Butow and Avadhani, 2004). In particular, a retrograde signaling mechanism involving increased cytosolic free Ca ²⁺ concentration exists in skeletal myoblasts, human lung carcinoma cells and rat pheochromocytoma cells ( Butow and Avadhani, 2004). Ca ²⁺ released by mitochondria leads to the activation of calcineurin, which in turn activates the transcription factors NFAT and NFκB. One target gene for this retrograde signaling that is activated by these transcription factors may be the gene encoding eNOS. In addition, NO is known to induce the production of ROS and trigger redox signaling ( Brookes et al., 2002). The NO produced in mitochondria under physiological conditions might therefore induce retrograde signaling through altered Δψ m, cytosol cGMP production, or translocation of NO and/or cGMP to the nucleus, where they could induce mitochondrial biogenesis. Yeast, which do not depend on their mitochondria to survive, actually live longer when such retrograde signaling is active. In the eukaryotic cells that depend on mitochondria to survive, the purpose of this retrograde signaling could be to correct mitochondrial deficiencies and, without it, they might live less long. Note that ROS can of course be generated in various cells in response to a variety of stimuli, including growth factors, cytokines and physicochemical stress, which greatly extends their role as signaling molecules beyond mitochondrial retrograde responses ( Zmijewski et al., 2005; Watanabe et al., 2006).

Numerous studies have demonstrated that calorie restriction (CR) extends lifespan in organisms from yeast to rodents and possibly primates ( Masoro, 2003). In mammals, CR delays the onset of age-associated diseases including cancer, atherosclerosis and diabetes ( Masoro, 2003). The molecular mechanisms underlying this effect are not known. Although recently questioned ( Kaeberlein et al., 2005), CR is proposed to extend the lifespan of budding yeast by increasing the activity of Sir2 ( Lin et al., 2000; Lin et al., 2004; Kaeberlein et al., 2002; Anderson et al., 2003), a member of the conserved sirtuin family of nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylases ( Hekimi and Guarente, 2003). Recent evidence indicates that CR induces eNOS and that the resulting surge of NO activates synthesis of a broad array of mitochondrial proteins and increases production of mtDNA, respiration and ATP levels in several different tissues and organs, including white and brown fat, brain, liver and heart ( Nisoli et al., 2005). Intriguingly, NO also activates expression of the mammalian Sir2 ortholog SIRT1, which is induced in mouse tissues by CR in wild-type mice but not in eNOS -/- mice ( Nisoli et al., 2005). Because SIRT1 is known to mediate resistance to cellular stress by a variety of mechanisms ( Luo et al., 2001), increases in its levels might be essential for greater longevity of the organism. These findings lead to a model in which CR induces eNOS, which results in mitochondrial biogenesis through increased PGC-1α expression and upregulation of SIRT1 and perhaps other longevity-promoting agents ( Fig. 2). Preliminary results suggest that SIRT1 mediates mitochondrial biogenesis in fat cells by increasing PGC-1α expression (E.N., C. Tonello and M.O.C., unpublished results) and, recently, López-Lluch et al. confirmed that CR induces mitochondrial biogenesis and bioenergetic efficiency both in vitro and in vivo ( López-Lluch et al., 2006).

The increase in both PGC-1α and SIRT1 levels after CR is also relevant for fat metabolism ( Bordone and Guarente, 2005). PGC-1α coordinately regulates genes involved in mitochondrial biogenesis and β-oxidation of fatty acids ( Lin et al., 2005) and downregulation of adipogenesis ( Picard et al., 2004). During CR, NO can thus increase β-oxidation of fatty acids and lipolysis and inhibit adipocyte differentiation by acting through SIRT1, PGC-1α and mitochondrial biogenesis. This should reduce fat accumulation, which is known to have an impact on lifespan ( Bluher et al., 2003; Chiu et al., 2004). Thus, the NO-mediated mitochondrial biogenesis seems to play a role in slowing aging.

"If we wish to live longer and, then, to rid ourselves of the diseases of old age, we will need more mitochondria", writes Nick Lane in his book ( Lane, 2005). In principle, this could be achieved pharmacologically, with NO donors for example. Such an approach would have the potential to cure all diseases of old age at once, rather than trying to tackle each independently, a tack that has so far failed to deliver meaningful clinical breakthroughs.

Perspectives

Mitochondria are important dynamic organelles for cell survival and function. Their biogenesis might be involved in the control of cell metabolism and signal transduction, and requires the choreographed expression of diverse transcription activators, including PGC-1α and NRF-1. We now know that NO acts as a key messenger to activate the mitochondrial biogenesis program in various cell types. However, many issues remain to be elucidated. These include: (1) the precise mechanism(s) by which NO activates PGC-1α and/or NRF-1 to trigger mitochondrial biogenesis; (2) the nature of its effects on mitochondrial dynamics; (3) the relationship between mitochondrial NO and cytosolic and/or nuclear NO in the modulation of gene expression in the nucleus; (4) the links between NO, mitochondrial biogenesis and apoptosis; and (5) the true relevance of NO-induced mitochondrial biogenesis for prevention of aging-related diseases. Future research on the role of NO in mitochondrial biology will give us important insights into the evolution of eukaryotic life and, perhaps, the treatment of multiple diseases.

Acknowledgements

We thank E. Clementi and S. Moncada for discussion, and members of our laboratories for their enthusiastic work on the NO-mitochondria connection. We apologize for the many seminal contributions that have not been cited for lack of space. This work was supported by grants from the Ministero dell'Istruzione, dell'Università e della Ricerca cofinanziamento 2005 to E.N., and the University of Milan and the Italian Ministry of Health to E.N.

View Abstract

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Nitric oxide and mitochondrial biogenesis

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The characteristic structural organization of mitochondria is the product of synthesis of macromolecules within the mitochondria together with the import of proteins and lipids synthesized outside the organelle. Synthetic and import processes are required for mitochondrial proliferation and might also facilitate the growth of pre-existing mitochondria.

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ABSTRACT

Betaine is distributed widely in animals, plants, and microorganisms, and rich dietary sources include seafood, especially marine invertebrates (≈1%); wheat germ or bran (≈1%); and spinach (≈0.7%). The principal physiologic role of betaine is as an osmolyte and methyl donor (transmethylation). As an osmolyte, betaine protects cells, proteins, and enzymes from environmental stress (eg, low water, high salinity, or extreme temperature). As a methyl donor, betaine participates in the methionine cycle-primarily in the human liver and kidneys. Inadequate dietary intake of methyl groups leads to hypomethylation in many important pathways, including 1) disturbed hepatic protein (methionine) metabolism as determined by elevated plasma homocysteine concentrations and decreased S-adenosylmethionine concentrations, and 2) inadequate hepatic fat metabolism, which leads to steatosis (fatty accumulation) and subsequent plasma dyslipidemia. This alteration in liver metabolism may contribute to various diseases, including coronary, cerebral, hepatic, and vascular diseases. Betaine has been shown to protect internal organs, improve vascular risk factors, and enhance performance. Databases of betaine content in food are being developed for correlation with population health studies. The growing body of evidence shows that betaine is an important nutrient for the prevention of chronic disease.

INTRODUCTION

Betaine is found in microorganisms, plants, and animals and is a significant component of many foods ( 1- 10), including wheat, shellfish, spinach, and sugar beets. Betaine is a zwitterionic quaternary ammonium compound that is also known as trimethylglycine, glycine betaine, lycine, and oxyneurine. It is a methyl derivative of the amino acid glycine with a formula of (CH ₃) ₃N⁺CH ₂COO − and a molecular weight of 117.2, and it has been characterized as a methylamine because of its 3 chemically reactive methyl groups ( 11). Betaine was first discovered in the juice of sugar beets ( Beta vulgaris) in the 19th century and was subsequently found in several other organisms. The physiologic function of betaine is either as an organic osmolyte to protect cells under stress or as a catabolic source of methyl groups via transmethylation for use in many biochemical pathways ( 12).

Humans obtain betaine from foods that contain either betaine or choline-containing compounds. Betaine is present in foods in variable amounts that are generally related to growing and osmotic stress conditions. Some examples of food with high betaine content are shown in , and we estimate (SAS Craig, Danisco USA Inc, unpublished observations, 2004) that dietary intake of betaine ranges from an average of 1 g/d to a high of 2.5 g/d (for a diet high in whole wheat and seafood). The principle metabolic fate of choline is via irreversible oxidation to betaine in the liver and kidney ( 28- 32) via a two-step process (). First, choline is oxidized to betaine aldehyde by the enzyme choline dehydrogenase. This enzyme can also convert betaine aldehyde to betaine in the presence of NAD⁺ ( 33). Choline dehydrogenase activity occurs in the mitochondria, on the matrix side of the inner membrane ( 34- 36). Betaine aldehyde is then oxidized to betaine by the NAD⁺-dependent enzyme betaine aldehyde dehydrogenase both in mitochondria and in the cytosol ( 37). The remainder of dietary choline is used to make acetylcholine and phospholipids such as phosphatidylcholine. A diet of normal foods is estimated to deliver 1 g choline/d ( 38).

INTESTINAL ABSORPTION AND METABOLISM

Cellular absorption of betaine has been described in many organisms from bacteria to vertebrates ( 43- 50). Accumulation is via active Na⁺ or Cl − coupled and passive Na⁺ independent transport systems. As an N-methylated amino acid, betaine is transported by amino acid transport systems-mainly by betaine γ-aminobutyric acid transport and amino acid transport system A ( 51- 54). Animal studies showed rapid postprandial absorption of betaine in the small intestine via the duodenum ( 22, 50).

Human studies on betaine intake showed rapid absorption and distribution, with a peak increase in serum betaine at ≈1-2 h ( 55- 57). Resting concentrations of betaine in human serum range from ≈20 to 70 μmol/L ( 55- 60), with concentrations similar in diabetic subjects but lower in subjects with renal disease ( 60). High concentrations of betaine are found in human and animal neonates, probably because of choline metabolism ( 61). Betaine is mainly eliminated by metabolism, not excretion, even at relatively high doses of 100 mg/kg body wt ( 56). However, urinary excretion of betaine is elevated in subjects with renal disease or diabetes ( 60, 62- 64). Subacute and subchronic rat studies determined that betaine is nontoxic at all doses studied (0-5% of the diet) ( 65). At high doses, red blood cell physiology was mildly perturbed, and variations in findings between studies were explained as a difference in chow formulation. The authors concluded that betaine is safe at a daily intake of 9-15 g (average of 12 g).

Betaine is catabolized via a series of enzyme reactions that occur mainly in the mitochondria of liver and kidney cells. These transmethylation reactions involve the transfer of methyl (one-carbon) groups via the methionine cycle ( Figure 1) in vital biological processes ( 66- 76). The conversion of homocysteine to methionine is important to conserve methionine, detoxify homocysteine, and produce S-adenosylmethionine (SAM) ( 77). Elevated total homocysteine concentrations and low SAM concentrations have been associated with chronic disease (discussed in detail later). The formation of methionine from homocysteine can occur either via betaine or via 5-methyltetrahydrofolate (CH ₃-THF). Animal studies have shown that both pathways are equally important ( 78, 79) and that betaine is a vital methylating agent ( 80, 81). Betaine transfers a methyl group via the enzyme betaine homocysteine methyl transferase (BHMT) to become dimethylglycine. The CH ₃-THF reaction utilizes the enzyme methionine synthase to transfer a methyl group to the cofactor cobalamin (vitamin B-12), forming methylcobalamin. Then methylcobalamin transfers the methyl to homocysteine to form methionine. Methylenetetrahydrofolate reductase (MTHFR) is involved in reforming CH ₃-THF from methylene-THF. In addition, homocysteine can be metabolized via transulfuration to form cystathionine. This is catalyzed by cystathionine β-synthase (CBS), which utilizes vitamin B-6 (pyridoxine) as a cofactor. BHMT, formerly called betaine transmethylase, is a zinc metalloenzyme in a distorted barrel shape ( 82- 84) with an optimum pH of 7.8 ( 85) that occurs in the human liver, kidney, and optic lens ( 85- 89). Liver BHMT concentrations increase when rats are fed diets supplemented with betaine or choline ( 90), showing an adaptive change in the catabolism of betaine. BHMT is expressed in the kidney cortex and in liver hepatocytes in humans, pigs, and rats ( 91).

Betaine increases serum methionine, transmethylation rate, homocysteine remethylation, and methionine oxidation in healthy adults ( 92). Animals injected with betaine ( 93) show a dose-dependent increase in red blood cell SAM-a direct methyl donor in many important pathways, including synthesis of proteins, creatine, phospholipids, hormones, polyamines, carnitine, adrenaline, and DNA methylation. Thus, transmethylation metabolic pathways closely interconnect choline, betaine, methionine, CH ₃-THF, and vitamins B-6 and B-12, intersecting at the formation of methionine from homocysteine. Perturbing the metabolism of one of these pathways results in compensatory changes in the others ( 94). For example, methylene-THF can be regenerated via serine or the betaine metabolites dimethylglycine, sarcosine, and glycine ( 55, 95).

OSMOLYTE

If betaine is not catabolized, it is used as an organic osmolyte. The regulation of cellular hydration state, and therefore cell volume, is important for maintenance of cell function. Sensitive metabolic pathways include protein turnover, amino acid and ammonia metabolism, carbohydrate and fatty acid metabolism, plasma membrane transport, bile excretion, pH control, and gene expression ( 96). Cells adapt to external osmotic stress by accumulating low-molecular-weight inorganic ions (eg, sodium, potassium, and chloride) and organic osmolytes (eg, methylated amines, certain amino acids, and sugar alcohols). However, the increase in intracellular concentrations of inorganic ions is limited because of their destabilizing effect on protein structure and enzyme function ( 43, 97- 100). Betaine is nonperturbing to cellular metabolism, highly compatible with enzyme function, and stabilizes cellular metabolic function under different kinds of stress ( 7, 11, 37) in various organisms and animal tissues ( 101- 103). Mechanistic studies showed that there is little or no binding of betaine to protein surfaces, allowing cells to control the surface tension of water without affecting the ionic strength of the environment, eg, stabilization of lipase ( 104). Betaine is the most effective osmolyte studied for hydration of albumin ( 17), forming almost a complete monolayer of water around the protein, and betaine can maintain hemoglobin solvation ( 105).

There are many examples of the osmolyte function of betaine. It accumulates in the kidney ( 106- 111) from exogenous origin rather than de novo synthesis ( 47, 112, 113) to protect cells from high concentrations of electrolytes ( 114) and urea ( 115). Betaine modulates immune function in osmotically stressed liver macrophages (Kupffer cells) via tumor necrosis factor α release ( 116), phagocytosis ( 117), and suppression of prostaglandin formation and cyclooxygenase 2 expression ( 118). It regulates water balance and movement across the intestinal epithelium ( 119). Betaine regulates erythrocyte (red blood cell) membrane ATPases via conformational changes, which results in cell volume control ( 120). It protects skeletal muscle myosin ATPases and prevents myosin structural changes due to urea ( 121). Betaine also protects early preimplantation embryos in vitro ( 122) and prevents apoptosis (programmed cell death) due to hypertonic stress in porcine pulmonary arterial endothelial cells ( 123). Therefore, this osmotic adaptation to stress helps a variety of cells and organs continue to function and protects against premature apoptosis.

LIVER HEALTH

Nonalcoholic fatty liver disease (NAFLD) is a relatively recent term that describes a progressive range of liver pathologies from simple steatosis (fat accumulation) to steatohepatitis (fatty inflammation), fibrosis (excessive fibrous tissue), and cirrhosis (serious liver damage) ( 124). NAFLD affects ≈20% of the general global population, ≈50% of diabetic subjects, >50% of obese persons, and 90% of morbidly obese persons ( 125). It is now recognized to be the hepatic manifestation of the metabolic syndrome ( 126). Alterations in hepatic transmethylation may contribute to various diseases, including coronary, cerebral, hepatic, and vascular diseases ( 127). One manifestation, hepatic steatosis (fatty liver), is a common result of obesity, consumption of a high-fat diet, insulin resistance, diabetes, alcohol consumption, and other liver damage ( 128).

Betaine is a lipotrope-something that prevents or reduces accumulation of fat in the liver. Studies on healthy and diabetic animals show that a high-fat diet leads to hepatic steatosis that is prevented by ingestion of betaine or choline ( 129- 131). Hepatic steatosis also occurs as result of a sucrose-only diet ( 130) or a diet low in both lipotrope and fat ( 132). Betaine can prevent and cure cirrhosis in rats ( 133, 134), mobilize hepatic cholesterol and phospholipids in rats fed a high-cholesterol diet ( 135), treat hyperlipidemia ( 136), and be utilized in the synthesis of carnitine ( 137). Dietary betaine enhances the secretion of VLDL via methylation of phosphatidylethanolamine ( 138) to form phosphatidylcholine. Phosphatidylethanolamine N-methyltransferase knockout mice ( 139) develop hepatic steatosis and abnormal choline metabolite concentrations despite ingesting a recommended dietary intake of choline. This highlights the importance of the SAM route to maintain liver health. One hypothesis regarding the lipotropic properties of betaine is that it contains an electrophilic methyl group that ameliorates pathologic states induced by reductive and oxidative stress ( 140).

Humans with diabetes exhibit hepatic steatosis, and early studies showed that dietary betaine improves liver function ( 141- 143), including a slight decrease in plasma cholesterol and lipids, a substantial decrease in plasma bilirubin, a reduction in liver size, and better diabetic control. Several reviews ( 125, 128, 144- 149) recommend using betaine to treat NAFLD, including nonalcoholic steatohepatitis (NASH). A 1-y pilot study ( 150) showed that betaine (20 g/d) is safe and well tolerated and leads to significant biochemical and histologic improvement in patients with NASH. This includes improvements in serum concentrations of liver enzymes (aspartate aminotransferase and alanine aminotransferase), in steatosis, in necroinflammatory grade, and in stage of fibrosis. Betaine glucuronate supplementation (in combination with diethanolamine glucuronate and nicotinamide ascorbate) of patients with NASH lowers hepatic steatosis and significantly reduces liver transaminases (alanine aminotransferase, aspartate aminotransferase, and γ-glutamyltransferase) ( 151).

Betaine protects the rat liver from toxins such as chloroform ( 54, 152, 153), lipopolysaccharide ( 154), methotrexate ( 155, 156), and carbon tetrachloride ( 157- 159). Specific effects include reduced hepatic lipidosis and necrosis; improved morphology of mitochondria, rough endoplasmic reticulum, Golgi complexes, and nuclear DNA; increased BHMT and SAM; and decreased alanine aminotransferase. Betaine also protects against bile-induced apoptosis via inhibition of the proapoptotic mitochondrial pathway ( 160). Coadministration of equimolar doses of betaine may alleviate the hepatotoxic risk associated with niacin therapy ( 161) because niacin catabolism leads to a high demand for methyl groups, leading to a reduction in hepatic concentrations of SAM. A methyl group deficiency may also contribute to niacin-induced insulin resistance by altering the membrane properties of skeletal muscle.

Ethanol feeding can affect several hepatic enzymes in animals, including decreasing methionine synthetase activity ( 162). This leads to increased BHMT activity to maintain hepatic SAM at normal concentrations ( 163- 165). Betaine supplementation of alcohol-fed animals prevents and partially reverses alcoholic fatty liver ( 165- 167); decreases homocysteine and S-adenosylhomocysteine (SAH) concentrations, endoplasmic reticulum stress, and liver injury ( 168- 170); prevents the ethanol-induced accumulation of CH ₃-THF (methyl-folate trap) ( 171); protects erythrocyte membranes ( 172); and prevents vitamin A depletion and peroxidative damage ( 173). Ethanol-induced elevations in hepatic concentrations of SAH inhibit the activity of phosphatidylethanolamine methyltransferase, which is necessary to achieve adequate concentrations of phosphatidylcholine ( 139). This inhibition may reduce VLDL synthesis and transport of triacylglycerols in the liver ( 174) and thus at least partly explain the hepatic steatosis due to ethanol feeding. Elevations in the ratio of SAM to SAH by betaine may improve the altered signaling and genomic hypomethylation caused by ethanol and a high-fat diet ( 175). SAM is currently used successfully to treat liver disease and other maladies, and betaine may be an alternative therapy ( 77).

Elevated serum homocysteine is frequent in patients with chronic liver disease, and high dosages of folate, cobalamin, and vitamin B-6 frequently fail to normalize homocysteine concentrations ( 176). Also, plasma concentrations of betaine metabolites (dimethyl glycine and sarcosine) increase due to increased activity of BHMT ( 177). However, BHMT enzyme activity is reduced in cirrhotic rat livers, which may explain the elevated plasma homocysteine concentrations in cirrhosis ( 178). A recent editorial ( 87) suggests that "Future studies should focus on the dietary content of choline and the possibility that supplementation with choline or betaine will restore the intracellular concentrations of betaine necessary for homocysteine methylation."

HEART HEALTH

A series of papers ( 179- 182) showed that a combination of betaine and guanidinoacetate (glycocyamine) improves the symptoms of subjects with chronic illness, including heart disease, without toxicity. Betaine can provide a methyl group to guanidinoacetate, via methionine, for the formation of creatine ( 179). Overall, treatment led to an improved sense of well-being, less fatigue, greater strength and endurance, and increased desire for (and performance of) physical and mental work. Subjects with cardiac decompensation (arteriosclerosis or rheumatic disease) ( 180) and congestive heart failure ( 181) had improved cardiac function. Subjects gained weight (improved nitrogen balance) and reported lessened symptoms of arthritis and asthma and increased libido, and those with hypertension experienced transient reduced blood pressure. Glucose tolerance increased in both diabetic subjects and subjects without diabetes. It was therefore concluded in 1951 that "... further evaluation of these compounds in patients with heart disease is both desirable and worthwhile" ( 182).

In the same year, Morrison ( 183) concluded that atherosclerosis is a preventable metabolic error and that ingestion of lipotropic agents is part of a preventative strategy. He showed that betaine treatment of atherosclerosis improves the ratio of serum phospholipids to serum cholesterol, the sense of well-being, exercise and activity, appetite, angina pain, dyspnea (shortness of breath), and libido ( 184). Morrison found "... betaine to be the most valuable and most effective lipotropic agent tested for treatment in the human subject" ( 184). Finally, a betaine-lipotrope combination (betaine, choline, liver extract, vitamin B-12, and a low-fat, low-cholesterol diet) led to an impressive decrease in the mortality of subjects with atherosclerosis ( 185). There was a 25% mortality rate after 12 mo in the control group but no deaths in the lipotrope group.

HOMOCYSTEINEMIA

Epidemiologic studies showed that persons with an elevated serum homocysteine concentration, termed hyperhomocyst(e)inemia or homocyst(e)inemia, have increased risks of cardiovascular disease, stroke, Alzheimer disease, dementia, neural tube defects, and other metabolic disorders ( 190- 197). Elevated fasting serum homocysteine and an elevated response of homocysteine after methionine loading (postmethionine) are independent risk factors for cardiovascular disease ( 198). Betaine can improve both risk factors, and in humans with cardiovascular disease, there is a significant inverse relation between plasma betaine and homocysteine concentrations in fasting ( 199) and postmethionine ( 200) states. A betaine-rich diet might lower cardiovascular disease risk in healthy humans ( 201).

Homocysteinemia is caused by an imbalance in the methionine cycle because of either genetic or nongenetic (eg, nutritional) defects that result in elevated serum homocysteine. Chronic elevation of homocysteine results in parallel increases in intracellular SAH, and this, in combination with the SAM:SAH ratio, is predictive of cellular methylation status ( 202). The severity of clinical symptoms is correlated with the degree of biochemical abnormality. Prevention or treatment, such as activation of defective (mutant) enzyme activity with the relevant cofactor or its precursor (eg, folic acid or vitamins B-12 or B-6), is based on the cause and severity of the defect. If homocysteinemia does not respond to this treatment, pharmacologic doses of betaine or folic acid have been used to enhance the alternative pathway of homocysteine turnover ( 203, 204).

Homocystinuria

Severe homocysteinemia, which is caused by hereditary homozygous genetic dysfunction (inborn errors of metabolism), is often termed homocystinuria. The incidence varies geographically and is estimated as 1/200 000 worldwide ( 205). Symptoms associated with homocystinuria include mental retardation, seizures, psychiatric disturbances, mental retardation, displacement of the lens of the eye (ectopia lentis), skeletal abnormalities (osteoporosis and scoliosis), and arteriosclerosis and thromboembolism that may lead to life-threatening complications ( 205). Homocystinuria can be caused by a deficiency of CBS, MTHFR, or methylcobalamin synthesis ( Figure 1). Several investigators have determined that betaine can lower homocysteine concentrations and improve some clinical conditions with no adverse events ( 206). Some studies lasted for 13-16 y, and dosage was typically 6 g/d.

CBS condenses homocysteine and serine to form cystathionine. Vitamin B-6 is a cofactor for CBS, and ≈50% of subjects with homocystinuria are responsive to vitamin B-6 therapy. Homocystinuria that is not responsive to vitamin B-6 is much more difficult to treat ( 207), but betaine, either alone or in conjunction with other therapies, improves clinical symptoms, reduces homocysteine, and increases plasma methionine, serine, and cysteine concentrations ( 208- 221). Homocysteine also accumulates in the central nervous system in CBS deficiency and may play a role in neurologic complications. Betaine treatment in children significantly lowers homocysteine in cerebrospinal fluid and raises serine and SAM concentrations ( 222). Studies on MTHFR deficiency ( 223- 230) showed that betaine improves homocysteine remethylation, lowers plasma homocysteine, normalizes very low plasma methionine, elevates SAM, and leads to clinical improvement. A defect in cobalamin-C (vitamin B-12) activation results in methylmalonic acidemia and homocystinuria, and treatment with cobalamin, folate, or vitamin B-6 does not completely correct the biochemical defect. Addition of betaine leads to normalized homocysteine and clinical improvement ( 231- 234).

Betaine prevents seizures in animals induced by homocysteine ( 235), electroconvulsive shock and pentylenetetrazol ( 236, 237), and strychnine ( 238, 239) and delays the onset of neurologic impairment due to vitamin B-12 deficiency ( 240). Rett Syndrome is caused by mutations in the MECP2 gene ( 241), and a current clinical study is evaluating whether betaine and folic acid can increase the degree of methylation of some CpG sites on subject DNA and thereby improve symptoms ( 242). An open-label pilot study of patients with Alzheimer disease did not find a benefit from betaine treatment ( 243), although efficacy could not be determined because there was no control group.

Mild homocysteinemia

Mild homocysteinemia is much more common than is homocystinuria and is characterized by mildly elevated fasting or postmethionine homocysteine concentrations. Pooled data from many studies showed that mild homocysteinemia occurs in 9-42% of subjects under 50 y of age who have peripheral or cerebral occlusive arterial disease, myocardial infarction, or thromboembolism ( 244). Mild postmethionine homocysteinemia is present in 21% of young patients with coronary artery disease, 24% of patients with cerebrovascular disease, and 32% of patients with peripheral vascular disease ( 245).

Mild homocysteinemia can be reduced immediately and in the long term by ingesting a combination of vitamin B-6, folic acid, and betaine ( 244, 245) or betaine (1.5-6 g/d) alone ( 57, 189, 198, 201, 246). Betaine, but not folic acid, is effective at preventing an increase in postmethionine homocysteine concentrations ( 198, 201, 244, 247, 248). Betaine-dependent remethylation occurs mainly in the liver and kidney, whereas folate-dependent remethylation occurs in most cells. Serum homocysteine concentrations may be represented by the extrahepatic supply to the liver via folate remethylation and by the hepatic output via betaine remethylation ( 198). Therefore, combined ingestion of folic acid and betaine may be the most effective method of lowering homocysteine.

Genetic variants of the enzymes of homocysteine metabolism influence the risk of homocysteinemia and therefore of cardiovascular disease and other common disorders ( 249). Mild MTHFR deficiency, due to a polymorphism caused by a substitution (677C→T) in the gene that changes an alanine residue to valine, is common (≈10-15% homozygosity in North America) ( 250, 251). The resulting MTHFR is thermolabile, has decreased enzyme activity, and leads to moderate homocysteinemia if folate status is low. MTHFR-compromised mice with homocysteinemia are more sensitive to changes in betaine intake than are wild-type animals. In mice with mild or severe homocysteinemia due to heterozygosity or homozygosity for a disruption in the MTHFR gene, betaine supplementation decreases homocysteine, restores liver betaine and phosphocholine pools, and prevents severe steatosis ( 199). In mice with moderate homocysteinemia caused by a disruption in the CBS gene, betaine supplementation decreases homocysteine, increases liver betaine, and increases BHMT activity in both heterozygous and wild types ( 252). There are several mutations of the BHMT gene ( 253), and the QQ genotype, a substitution of glutamine for arginine (Q239R), is a common variant of BHMT in humans that occurs in ≈10-17% of the adult population ( 253, 254). The QQ genotype is associated with decreased risks of neural tube defects ( 254) and coronary artery disease ( 249).

KIDNEY HEALTH

BHMT is abundant in primate and pig kidneys, occurs in very low concentrations in rat kidneys, and is essentially absent from the other major organs of monogastric animals ( 91). BHMT activity in human, pig, and rat kidneys is expressed only in the proximal tubules of the cortex and is absent from the medulla. Immunohistochemical staining patterns show cytosolic expression in both the kidney and the liver. The concentration of betaine in the liver and the kidney is controlled by tonicity in vivo via regulation of BHMT ( 255). Under hypertonic conditions, renal and hepatic BHMT decreases to conserve betaine to be utilized as an osmoprotectant. Conversely, under hypotonic conditions, BHMT increases to reduce betaine and help maintain optimal cell turgor. Betaine accumulation in kidney medullary cells exposed to hypertonic stress is dependent on cyclooxygenase 2 activity ( 110). Betaine protects against the nephrotoxic effects of carbon tetrachloride in rats ( 256), and in patients with renal disease taking folic acid, betaine can reduce postmethionine homocysteine concentrations but not fasting homocysteine ( 248, 257- 259).

DNA METHYLATION

Incomplete DNA methylation may lead to genetic instability, senescence, and cancer ( 260- 262), and optimal "methylation diets," including betaine, have been suggested for the prevention and treatment of a variety of conditions ( 263). Synthesis of choline for packaging and secretion of fat from the liver consumes methyl groups and appears to compete with other methylation reactions. Thus, a high-fat diet exacerbates methyl deficiency, and a higher proportion of cofactors and methyl group sources relative to calories may explain the extended life span observed in caloric restriction experiments ( 260).

Epigenetics is the study of heritable traits that do not involve a change in gene sequence (ie, different phenotypes of identical genotypes). Yellow agouti mice have an epigenetic variation in coat color-more yellow is associated with altered metabolism, greater obesity, diabetes, cancer, and earlier mortality. In epigenetics, methylation is the addition of methyl groups to cytosine in DNA to form 5-methylcytosine by utilizing the enzyme DNA methyltransferase. In maternal yellow agouti mice, methyl supplementation, including supplementation with betaine, before and during pregnancy increases DNA methylation (5-methylcytosine) and changes the phenotype of their genetically identical offspring in the healthy, longer-lived direction, ie, less yellow coat ( 264- 267). These studies help address why a cohort of animals with very little genetic or environmental variation will still yield a considerable spread in longevity and disease. Epigenetic variations that affect adult health and life span may be established during embryonic and fetal development involving incomplete methylation of genes and endogenous retroviruses.

PERFORMANCE

As discussed earlier, betaine provides a methyl group to guanidinoacetate via methionine to produce creatine de novo ( 69, 179). A human poliomyelitis study ( 268) found that ingestion of a betaine-guanidinoacetate combination leads to an improved sense of well-being, less fatigue, and greater general strength and endurance during recovery. Whenever motor unit activity or residual power remained in a muscle, betaine-guanidinoacetate led to a substantial increase. A patent was granted ( 269) for this combination, or betaine alone, for the treatment of diseased and weak muscle tissues. A study of subjects with motor neuron disease ( 270) found that betaine-guanidinoacetate did not impart any objective benefit. However, some patients derived a slight subjective benefit, so the authors concluded that it might be useful for temporary psychological support. Betaine may improve athletic performance, because its addition to a carbohydrate-electrolyte fluid-replacement beverage results in improved mean sprint time to exhaustion and evidence of enhanced anaerobic and aerobic metabolism ( 57, 271). Rehydration with betaine also results in differential plasma volume changes and reduced LDL and homocysteine.

CONCLUSIONS

Betaine is an important human nutrient obtained from the diet from a variety of foods. It is rapidly absorbed and utilized as an osmolyte and source of methyl groups and thereby helps to maintain liver, heart, and kidney health. Betaine can reduce the elevated serum homocysteine concentrations associated with mild or severe hyperhomocystinuria via the methionine cycle and may play a role in epigenetics and athletic performance.

I thank Tony Barak and James Finkelstein of the Department of Veterans Affairs and Kirsti Tiihonen of Danisco Innovation for helpful contributions to the text.

REFERENCES

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Betaine in human nutrition | The American Journal of Clinical Nutrition | Oxford Academic

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Betaine is distributed widely in animals, plants, and microorganisms, and rich dietary sources include seafood, especially marine invertebrates (≈1%); wheat germ or bran (≈1%); and spinach (≈0.7%). The principal physiologic role of betaine is as an osmolyte and methyl donor (transmethylation).

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Whole Beets vs. Juice for Improving Athletic Performance | NutritionFacts.org

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Whole Beets vs. Juice for Improving Athletic Performance | NutritionFacts.org

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What is the latest science on the performance-enhancing qualities of nitrate-rich vegetables?

Nitrates, concentrated in green leafy vegetables and beets, underwent a great makeover a few years ago from inert substances to having profound effects on the power plants within all of our cells, reducing the oxygen cost during exercise-meaning we can bust out the same amount of work with less oxygen. So, one little shot of beet juice allows free divers to hold their breath for over four minutes; they get about a half-minute longer, and for others, this improved muscle efficiency allows athletes to exercise at a higher power output or running speed for the same amount of breath. I profiled this discovery in an unprecedented 17-part video series, the longest I think I've ever done-it was just so fascinating. But that was back in 2012; what's happened since? Well, this all led to many athletes-elite and amateur alike-consuming beetroot juice prior to competition. But what does the new science say?

Well, most of the studies were done on men; turns out it works on women too-even African-American women, an even more neglected research demographic. Same workload power outputs using significantly less oxygen after drinking beet juice. But forget beet juice; what about whole beets? Cheaper; healthier; can find them in any produce aisle. But there had never been any studies on actual beets, until now.

Whole beetroot consumption acutely improves running performance. They gave physically fit men and women a cup and a half of baked beets, which is equal to about a can of beets, 75 minutes before running a 5K. They started out the same, but during the last mile of the 5K race, the beet group pulled ahead, compared to the placebo group, who were given berries instead.

Though they were running faster, their heart rate wasn't any higher. If anything, the beet group reported less exertion. Faster time with less effort? They don't call them block-rockin' beets for nothing.

But if nitrates are so good, why not just take them in a pill? Nitrate supplements with names like "Hellfire"-although they can work, their long-term safety is questionable. Non-vegetable sources of nitrates may have detrimental health effects; so, if we want to improve our performance, we should ideally obtain nitrates from whole vegetables. The industry knows this, so instead markets an array of nitric oxide-stimulating supplements. However, there is little or no evidence of a performance improvement following supplementation with these so called NO boosters. The evidence is with the vegetables.

How much money can companies make selling beets though? So, how about a novel beetroot-enriched bread product? We've tried to get people to eat their fruits and vegetables, and where has that gotten us? But hey, lots of people eat white bread; why not have them eat red bread? And, indeed, it worked; red beet bread brought down blood pressures, and improved the ability of arteries to relax and dilate naturally. Bread, therefore, may be an effective vehicle to increase vegetable consumption without significant dietary changes, because heaven forbid people should have to change their diet to improve their health.
To see any graphs, charts, graphics, images, and quotes to which Dr. Greger may be referring, watch the above video. This is just an approximation of the audio contributed by Katie Schloer.
Please consider volunteering to help out on the site.

A M Jones. Influence of dietary nitrate on the physiological determinants of exercise performance: a critical review. Appl Physiol Nutr Metab. 2014 Sep;39(9):1019-28.

W T Clements, S R Lee, R J Bloomer. Nitrate ingestion: a review of the health and physical performance effects. Nutrients. 2014 Nov 18;6(11):5224-64.

F Wang, S Dai, M Wang, H Morrison. Erectile dysfunction and fruit/vegetable consumption among diabetic Canadian men. Urology. 2013 Dec;82(6):1330-5.

F J Larsen, T A Schiffer, S Borniquel, K Sahlin, B Ekblom, J O Lundberg, E Weitzberg. Dietary inorganic nitrate improves mitochondrial efficiency in humans. Cell Metab. 2011 Feb 2;13(2):149-59.

M Murphy, K Eliot, R M Heuertz, E Weiss. Whole beetroot consumption acutely improves running performance. J Acad Nutr Diet. 2012 Apr;112(4):548-52.

D A Hobbs, M G Goulding, A Nguyen, T Malaver, C F Walker, T W George, L Methven, J A Lovegrove. Acute ingestion of beetroot bread increases endothelium-independent vasodilation and lowers diastolic blood pressure in healthy men: a randomized controlled trial. J Nutr. 2013 Sep;143(9):1399-405.

D A Hobbs, N Kaffa, T W George, L Methven, J A Lovegrove. Blood pressure-lowering effects of beetroot juice and novel beetroot-enriched bread products in normotensive male subjects. Br J Nutr. 2012 Dec 14;108(11):2066-74.

V Bond Jr, B H Curry, R G Adams, R M Millis, G E Haddad. Cardiorespiratory function associated with dietary nitrate supplementation. Appl Physiol Nutr Metab. 2014 Feb; 39(2): 168-172.

H K Engan, A M Jones, F Ehrenberg, E Schagatay. Acute dietary nitrate supplementation improves dry static apnea performance. Respir Physiol Neurobiol. 2012 Jul 1;182(2-3):53-9.

Doctor's Note

17-part video series? My videos were much shorter back then, but still! Here you go if you want to put the whole discovery in context and get the detailed mechanism:

Wait, that's only 16. I think at the time I was also including Meat Additives to Diminish Toxicity.

How else can we support athletic performance? See:

On the other hand, Paleo Diets May Negate Benefits of Exercise.

It's great that we can improve athletic performance eating a few beets, but what about people who could really benefit from a more efficient use of oxygen? That's the subject of my next video, Oxygenating Blood With Nitrate-Rich Vegetables.

If you haven't yet, you can subscribe to my videos for free by clicking here.

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Whole Beets vs. Juice for Improving Athletic Performance | NutritionFacts.org

# data[].attributes.attachment_link.description
What is the latest science on the performance-enhancing qualities of nitrate-rich vegetables? Nitrates, concentrated in green leafy vegetables and beets, underwent a great makeover a few years ago from inert substances to having profound effects on the power plants within all of our cells, reducing the oxygen cost during exercise-meaning we can bust out the same amount of work with less oxygen.

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The Role of microRNAs in Mitochondria: Small Players Acting Wide

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The Role of microRNAs in Mitochondria: Small Players Acting Wide

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Signaling the mitochondrial unfolded protein response

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Signaling the mitochondrial unfolded protein response

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Signaling the mitochondrial unfolded protein response

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Mitochondria are compartmentalized organelles essential for numerous cellular functions including ATP generation, iron-sulfur cluster biogenesis, nucl...

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Pharmacological approaches to restore mitochondrial function.: EBSCOhost

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Pharmacological approaches to restore mitochondrial function.: EBSCOhost

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Mitochondrial biogenesis: pharmacological approaches. - PubMed - NCBI

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Mitochondrial biogenesis: pharmacological approaches. - PubMed - NCBI

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Among them creatine, Coenzyme Q10 and mitochondrial targeted antioxidants/peptides are reported to have the most remarkable effects in clinical trials.

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Mitochondrial biogenesis: pharmacological approaches. - PubMed - NCBI

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Curr Pharm Des. 2014;20(35):5507-9. Editorial; Introductory Journal Article

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Chronic AMP-activated protein kinase activation and a high-fat diet have an additive effect on mitochondria in rat skeletal muscle. - PubMed - NCBI

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Chronic AMP-activated protein kinase activation and a high-fat diet have an additive effect on mitochondria in rat skeletal muscle. - PubMed - NCBI

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Chronic AMP-activated protein kinase activation and a high-fat diet have an additive effect on mitochondria in rat skeletal muscle. - PubMed - NCBI

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Berberine: The World's Most Powerful Supplement?

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Berberine: The World's Most Powerful Supplement?

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Does it affect the mitochondria?

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A compound called berberine is one of the most effective natural supplements available.

It has very impressive health benefits, and affects your body at the molecular level.

Berberine has been shown to lower blood sugar, cause weight loss and improve heart health, to name a few.

It is one of the few supplements shown to be as effective as a pharmaceutical drug.

This is a detailed review of berberine and its health effects.

Berberine is a bioactive compound that can be extracted from several different plants, including a group of shrubs called Berberis ( 1).

Technically, it belongs to a class of compounds called alkaloids. It has a yellow color, and has often been used as a dye.

Berberine has a long history of use in traditional Chinese medicine, where it was used to treat various ailments.

Now, modern science has confirmed that it has impressive benefits for several different health problems ( 2).

Bottom Line: Berberine is a compound that can be extracted from several different plants. It has a long history of use in traditional Chinese medicine.

Berberine has now been tested in hundreds of different studies.

It has been shown to have powerful effects on many different biological systems ( 3).

After you ingest berberine, it gets taken in by the body and transported into the bloodstream. Then it travels into the body's cells.

Inside the cells, it binds to several different "molecular targets" and changes their function ( 4). This is similar to how pharmaceutical drugs work.

I am not going to get into much detail here, because the biological mechanisms are complicated and diverse.

However, one of the main actions of berberine is to activate an enzyme inside cells called AMP-activated protein kinase (AMPK) ( 5).

This enzyme is sometimes referred to as a "metabolic master switch" ( 6).

It is found in the cells of various organs, including the brain, muscle, kidney, heart and liver. This enzyme plays a major role in regulating metabolism ( 7, 8).

Berberine also affects various other molecules inside cells, and may even affect which genes are turned on or off ( 4).

Bottom Line: Berberine affects the body at the molecular level, and has a variety of functions inside cells. One of the main functions is activating an important enzyme called AMPK, which regulates metabolism.

Type 2 diabetes is a serious disease that has become incredibly common in recent decades, causing millions of deaths every year.

It is characterized by elevated blood sugar (glucose) levels, either caused by insulin resistance or lack of insulin.

Over time, high blood sugar levels can damage the body's tissues and organs, leading to various health problems and a shortened lifespan.

Many studies show that berberine can significantly reduce blood sugar levels in individuals with type 2 diabetes ( 9).

In fact, its effectiveness is comparable to the popular diabetes drug metformin (Glucophage) ( 2, 10).

It seems to work via multiple different mechanisms ( 11):

Decreases insulin resistance, making the blood sugar lowering hormone insulin more effective.

Increases glycolysis, helping the body break down sugars inside cells.

Decrease sugar production in the liver.

Slows the breakdown of carbohydrates in the gut.

Increases the number of beneficial bacteria in the gut.

In one study of 116 diabetic patients, 1 gram of berberine per day lowered fasting blood sugar by 20%, from 7.0 to 5.6 mmol/L (126 to 101 mg/dL), or from diabetic to normal levels ( 12).

It also lowered hemoglobin A1c by 12% (a marker for long-term blood sugar levels), and also improved blood lipids like cholesterol and triglycerides ( 12).

According to a big review of 14 studies, berberine is as effective as oral diabetes drugs, including metformin, glipizide and rosiglitazone ( 13).

It works very well with lifestyle modifications, and also has additive effects when administered with other blood sugar lowering drugs ( 2).

If you look at discussions online, you often see people with sky-high blood sugars literally normalizing them just by taking this supplement.

This stuff really works, in both the studies and the real world.

Bottom Line: Berberine is very effective at lowering blood sugar and HbA1c, which are elevated in people with diabetes. It works as well as some pharmaceutical drugs.

Berberine may also be effective as a weight loss supplement.

So far, two studies have examined the effects on body weight.

In a 12-week study in obese individuals, 500 mg taken three times per day caused about 5 pounds of weight loss, on average. The participants also lost 3.6% of their body fat ( 14).

Another more impressive study was conducted in 37 men and women with metabolic syndrome. This study went on for 3 months, and the participants took 300 mg, 3 times per day.

The participants dropped their body mass index (BMI) levels from 31.5 to 27.4, or from obese to overweight in only 3 months. They also lost belly fat and improved many health markers ( 15).

The researchers believe that the weight loss is caused by improved function of fat-regulating hormones, such as insulin, adiponectin and leptin.

Berberine also appears to inhibit the growth of fat cells at the molecular level ( 16, 17).

However, more research is needed on the weight loss effects of berberine.

Bottom Line: Two studies have shown that berberine can cause significant weight loss, while improving all sorts of other health markers at the same time.

Heart disease is currently the world's most common cause of premature death.

Many factors that can be measured in the blood are associated with an increased risk of heart disease.

As it turns out, berberine has been shown to improve many of these factors.

According to a review of 11 studies, it can ( 18):

Lower total cholesterol by 0.61 mmol/L (24 mg/dL).

Lower LDL cholesterol by 0.65 mmol/L (25 mg/dL).

Lower blood triglycerides by 0.50 mmol/L (44 mg/dL).

Raise HDL cholesterol by 0.05 mmol/L (2 mg/dL).

It has also been shown to lower apolipoprotein B by 13-15%, which is a very important risk factor ( 19, 20).

According to some studies, berberine works by inhibiting an enzyme called PCSK9. This leads to more LDL being removed from the bloodstream ( 21, 22).

Keep in mind that diabetes, high blood sugar levels and obesity are also major risk factors for heart disease, all of which seem to be improved with this supplement.

Given the beneficial effects on all these risk factors, it seems likely that berberine could drastically reduce the risk of heart disease.

Bottom Line: Studies show that berberine reduces cholesterol and triglyceride levels, while raising HDL (the "good") cholesterol. It may lower the risk of heart disease in the long-term.

Berberine may also have numerous other health benefits:

Depression: Rat studies show that it may help fight depression (23, 24, 25).

Cancer: Test tube and animal studies have shown that it can reduce the growth and spread of various different types of cancer (26, 27).

Antioxidant and anti-inflammatory: It has been shown to have potent antioxidant and anti-inflammatory effects in some studies (28, 29, 30).

Infections: It has been shown to fight harmful microorganisms, including bacteria, viruses, fungi and parasites (31, 32, 33, 34).

Fatty liver: It can reduce fat build-up in the liver, which should help protect against non-alcoholic fatty liver disease (NAFLD) (35, 36).

Heart failure: One study showed that it drastically improved symptoms and reduced risk of death in heart failure patients (37).

Many of these benefits need more research before firm recommendations can be made, but the current evidence is very promising.

Bottom Line: Preliminary studies show that berberine may have benefits against depression, cancer, infections, fatty liver and heart failure. It also has potent antioxidant and anti-inflammatory effects.

Many of the studies cited in the article used dosages in the range of 900 to 1500 mg per day.

It is common to take 500 mg, 3 times per day, before meals (a total of 1500 mg per day).

Berberine has a half-life of several hours, so it is necessary to spread your dosage to several times per day to achieve stable blood levels.

If you have a medical condition or are on any medications, then it is recommended that you speak to your doctor before taking it.

This is especially important if you are currently taking blood sugar lowering medications.

Overall, berberine has an outstanding safety profile. The main side effects are related to digestion, and there are some reports of cramping, diarrhea, flatulence, constipation and stomach pain ( 10).

Bottom Line: A common dosage recommendation is 500 mg, 3 times per day, half an hour before meals. Berberine may cause digestive side effects in some people.

Berberine is one of very few supplements that are as effective as a drug.

It has powerful effects on various aspects of health, especially blood sugar control.

If you want to try a berberine supplement, then there is a good selection of high-quality supplements available on Amazon.

The people who stand to benefit the most are individuals with type 2 diabetes and metabolic syndrome.

However, it may also be useful as a general protection against chronic disease, as well as an anti-aging supplement.

If you use supplements, then berberine may be one of the top ones to include in your arsenal.

I've personally been taking it for a few weeks now, for general health improvement.

I plan to continue taking it, and I look forward to seeing more research on this promising health-promoting substance.

Healthline and our partners may receive a portion of the revenues if you make a purchase using a link above.

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Berberine: A Powerful Supplement With Many Benefits

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A compound called berberine is one of the most effective natural supplements available. It has very impressive health benefits, and affects your body at the molecular level. Berberine has been shown to lower blood sugar, cause weight loss and improve heart health, to name a few.

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Need to research the effects of shelled hemp seeds on mitochondrial function.

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PureHealingFoods.com helps you thrive by bringing together natures most powerful healing foods at the best prices to make it easy for you to get all the proteins, vitamins, antioxidants, minerals, trace minerals, pigments, essential fatty acids, that you need to be well.

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Non-profit foundation providing reliable, scientifically accurate, personalized information for convenient and enjoyable healthy eating.

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Minding your mitochondria | Dr. Terry Wahls | TEDxIowaCity - YouTube

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Minding your mitochondria | Dr. Terry Wahls | TEDxIowaCity - YouTube

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Minding your mitochondria | Dr. Terry Wahls | TEDxIowaCity

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Note from TED: This talk, which features health advice based on a personal narrative, has been flagged as potentially outside TED's curatorial guidelines. Viewer discretion advised. The guidelines we give our TEDx organizers are described in more detail here: http://www.ted.com/pages/tedx_curating_speakers ***** Dr. Terry Wahls learned how to properly fuel her body.

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Mechanisms of Mitochondrial Fission and Fusion

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Mechanisms of Mitochondrial Fission and Fusion

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Mechanisms of Mitochondrial Fission and Fusion

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A new type of review journal, featuring comprehensive collections of expert review articles on important topics in the molecular life sciences

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Exercise Training-Induced Regulation of Mitochondria Quality: Biogenesis

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Exercise Training-Induced Regulation of Mitochondria Quality: Biogenesis

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Biogenesis

Our laboratory has published a recent review on the topics of mitochondrial biogenesis, fiber type switching, and angiogenesis in skeletal muscle. Here, we will seek to summarize the current understanding of exercise-induced mitochondrial biogenesis and to evaluate new concepts related to the process of mitochondrial maintenance in this field. For decades, investigators have explored the adaptive mechanisms of endurance exercise training and observed increases in mitochondrial density either indirectly assessed by the expression of mitochondrial markers ( e.g., cytochrome c oxidase IV (COX IV)) or more directly observed through transmission electron microscopy and mitochondrial function measured by mitochondrial enzyme activity and oxygen consumption. Although it is well known that the process of mitochondrial biogenesis requires a coordinated regulation of both the nuclear and mitochondrial encoded genes, the primary mechanisms of adaptation remains unclear. The discovery of PGC-1α in brown adipose tissue, originally as a cold-inducible regulator of adaptive thermogenesis, has triggered mounting interest in elucidating the molecular and signaling mechanisms underlying exercise-induced mitochondrial biogenesis in skeletal muscle. Biochemically identified as a coactivator of PPAR-γ, PGC-1α is a coactivator of a great number of transcription factors, including the PPAR family transcription factors, nuclear respiratory factors (NRF), transcription factor of activated mitochondria, and others. PGC-1α is indeed capable of coactivating the required nuclear and mitochondrial genes for the organelle's synthesis. Because in large part of this common theme among the PGC-1α-inducible transcription factors, PGC-1α is now considered a "master regulator" of mitochondrial biogenesis. Recent data from Ugoccioni et al. suggest that other factors act in parallel to PGC-1α to improve mitochondrial viability and function. Nevertheless, only more recently, the questions of whether PGC-1α is induced in skeletal muscle by exercise and whether its induction is sufficient and necessary to promote mitochondrial biogenesis have been addressed. The short answer to these questions is now a resounding yes.

Overexpression of PGC-1α Enhances Mitochondrial Biogenesis, and PGC-1α Deficiency Blunts Endurance Exercise-induced Mitochondrial Biogenesis in Skeletal Muscle

Work by Leick et al. demonstrated that mice with global deletion of the PGC-1α gene exhibited normal wheel running activity and were capable of inducing oxidative gene expression in skeletal muscle. However, subsequent work using this same mouse model led to the conclusion that PGC-1α is indeed necessary for exercise training-induced protection from an age-related decline in oxidative function (citrate synthase activity) and antioxidant gene expression (superoxide dismutase 2). It is important to understand that such global gene deletion approaches may directly affect other metabolically active tissues, including the brown adipose and neural tissues. These multiorgan effects, coupled with enhanced adenosine monophosphate-activated protein kinase (AMPK) activity in resting skeletal muscle, may have prevented the delineation of the functional importance of PGC-1α in skeletal muscle, particularly under the condition of exercise training. More recently, skeletal muscle-specific PGC-1α knockout mice were generated, and data obtained from these mice showed that although exercise training-induced fiber type transformation appeared normal, exercise-induced mitochondrial biogenesis and angiogenesis in skeletal muscle were significantly attenuated. These results indicate the necessity of PGC-1α for normal exercise-induced metabolic adaptations, including mitochondrial biogenesis, in skeletal muscle. Considering the previous findings from Lin et al. that transgenic mice with a skeletal muscle-specific overexpression of PGC-1α have enhanced expression of mitochondrial genes, fatigue resistance, and fiber type transformation toward oxidative myofibers, we now postulate that enhanced PGC-1α activity is sufficient to induce mitochondrial biogenesis, fiber type transformation, and angiogenesis. However, PGC-1α activity is necessary only for the induction of mitochondrial biogenesis and angiogenesis.

PGC-1α Regulation by Exercise

A relative wealth of data has been published documenting the effects of acute exercise and exercise training on PGC-1α expression. A consensus now exists that exercise training, including both endurance and resistance exercise, enhances PGC-1α expression because of transcriptional regulation. More recent evidence from Perry et al. showed that a single bout of exercise was sufficient to induce PGC-1α protein and messenger ribonucleic acid (mRNA) expression in skeletal muscle, whereas increases in markers for mitochondrial biogenesis could be observed only after the third bout. Interestingly, PGC-1α mRNA level returned to baseline between each bout, and the induction of PGC-1α was lessened with each bout. These findings suggest that the signaling-transcription coupling machinery for the PGC-1α gene can accurately sense the stress induced by altered functional demands of exercise and provide appropriate instructive cues to induce adaptations in skeletal muscle to minimize the exercise-induced stress.

Exercise-induced mitochondrial biogenesis also may be controlled by targeted activation of PGC-1α, indicated by its translocation to the nucleus, and this adaptive process may begin before increased PGC-1α expression. Intriguingly, Safdar et al. showed that a single bout of exercise increases PGC-1α level not only in the nucleus but also in the mitochondria. Furthermore, the increased PGC-1α expression also was associated with enhanced interaction between PGC-1α and transcription factor of activated mitochondria at the mitochondrial DNA D-loop, as well as increased binding of PGC-1α to the NRF-1 promoter in the nucleus. Therefore, exercise is not only capable of inducing PGC-1α protein expression but also the translocation of PGC-1α to necessary compartments to stimulate the transcription of the nuclear and mitochondrial encoded mitochondrial genes, as well as the replication of mitochondrial DNA.

PGC-1α also is regulated by posttranslational modification, such as phosphorylation and deacetylation. For example, PGC-1α can be deacetylated by sirtuin (silent mating type information regulation 2 homolog) 1 (SIRT1). Although there is no correlation between the total muscle content of SIRT1 and PGC-1α or mitochondrial biogenesis, SIRT1 activation is associated with increases in PGC-1α target gene expression, consistent with SIRT1 activation of PGC-1α through deacetylation. Interestingly, this SIRT1-mediated PGC-1α activation requires phosphorylation of PGC-1α by AMPK. The fact that PGC-1α activity is controlled both transcriptionally and posttranscriptionally reveals an elegant regulatory system with great fidelity in integrating both contractile and metabolic cues in exercise-induced skeletal muscle adaptation.

PGC-1α expression and activity are largely regulated by upstream signaling pathways of protein kinases. The two primary protein kinases involved in the regulation of PGC-1α in skeletal muscle are AMPK and p38γ mitogen-activated protein kinase (p38γ MAPK). At least two AMPK phosphorylation sites have been identified on PGC-1α. AMPK not only activates PGC-1α but also promotes the transcription of the PGC-1α gene, controlling both PGC-1α expression and activity. It also has been shown that AMPK induces mitochondrial gene expression through PGC-1α. In fact, AMPK has been referred to as a "master metabolic switch" for acute regulation of energy metabolism and exercise training-induced adaptations. Acute exercise appears to activate AMPK by phosphorylation at Thr172. Although a single bout of exercise results in increased AMPK activity in skeletal muscle, long-term exercise training leads to an increase in AMPK protein content. However, neither acute exercise nor long-term exercise training is capable of increasing AMPK-α protein content in skeletal muscle of PGC-1α-deficient mice. This suggests that not only is PGC-1α reliant on AMPK but also, at least, the α subunit of AMPK is reliant on PGC-1α.

We have previously shown that a single bout of voluntary running in mice is sufficient to activate the p38 MAPK pathway. We further showed that p38 MAPK activity is sufficient to promote the PGC-1α promoter activity through myocyte enhancer factor 2 and activating transcription factor 2. In subsequent studies, we demonstrated that muscle-specific deletion of either p38α or p38β MAPK does not affect exercise-induced mitochondrial biogenesis and angiogenesis in skeletal muscle; however, mice with muscle-specific deletion of the p38γ MAPK or PGC-1α gene exhibit attenuated mitochondrial biogenesis and angiogenesis. These studies revealed for the first time that p38 MAPK stimulation of PGC-1α is specifically modulated by the γ isoform and led to the conclusion that the p38γ MAPK-PGC-1α regulatory axis is functionally required for the normal exercise-induced metabolic adaptations in skeletal muscle.

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Exercise Training-Induced Regulation of Mitochondria Quality

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Our laboratory has published a recent review on the topics of mitochondrial biogenesis, fiber type switching, and angiogenesis in skeletal muscle. Here, we will seek to summarize the current understanding of exercise-induced mitochondrial biogenesis and to evaluate new concepts related to the process of mitochondrial maintenance in this field.

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Abstract

Myocardial ischemia-reperfusion (IR) injury can cause ventricular cell death and is a major pathological event leading to morbidity and mortality in those with coronary artery disease. Interestingly, as few as five bouts of exercise on consecutive days can rapidly produce a cardiac phenotype that resists IR-induced myocardial injury. This review summarizes the development of exercise-induced cardioprotection and the mechanisms responsible for this important adaptive response.

Coronary artery disease remains a major cause of death worldwide ( 71). The major pathology associated with coronary artery disease is commonly the direct consequence of an ischemia-reperfusion (IR) insult. In this regard, the resultant myocardial damage due to IR injury is proportional to ischemic duration ( 98). Given the high incidence of coronary artery disease and the associated IR-induced cardiac injury, developing a countermeasure to protect the heart against IR-induced damage is important. In this regard, it is well established that exercise training provides robust cardioprotection against IR injury. For example, human epidemiological studies reveal that regular exercise reduces the risk of death during a myocardial IR insult (reviewed in Ref. 49). Importantly, numerous animal studies provide direct evidence that endurance exercise protects the heart from IR-induced injury (reviewed in Refs. 28, 56, 92).

This review summarizes the present understanding of the mechanisms responsible for exercise-induced cardioprotection against IR-induced injury. We begin with an introduction to IR-induced myocardial injury followed by a brief summary of the evidence demonstrating that exercise is cardioprotective. The remainder of this report focuses on the putative mechanisms responsible for exercise-induced cardioprotection with an emphasis on the important role that mitochondrial adaptations play in producing the protected phenotype.

Levels of IR-Induced Cardiac Injury

Depending on the duration of ischemia, three different levels of IR-induced cardiac injury ensue (). The lowest level of IR-induced injury is the occurrence of cardiac arrhythmias. In this scenario, rapid reperfusion occurring after 1-5 min of ischemia often promotes ventricular tachycardia or fibrillation without depressed myocardial contractile performance or cardiac cell death ( 25). Reperfusion following 5-20 min of ischemia produces a higher level of myocardial injury termed "myocardial stunning" ( 25). Myocardial stunning is characterized by impaired ventricular contractility that occurs in the absence of cardiac cell death ( 9). Typically, IR-induced myocardial stunning results in cardiac contraction deficits that last 24-72 h following the insult ( 9). The final and most severe level of IR injury occurs when the duration of ischemia exceeds 20 min. During this most severe level of IR injury, cardiac myocytes are irreversibly damaged, and death occurs due to both apoptosis and necrosis ( 25). Additional evidence indicates that apoptotic tissue death can be either mitigated or potentiated by autophagic processes within ventricular myocytes exposed to ischemia ( 61, 100).

Cellular Events Leading to IR-Induced Cardiac Injury

Despite the complexity of the events leading to IR-induced cardiac damage, the key factors responsible for IR-induced myocardial cell death are largely understood (for a review, see Refs. 33, 77, 91). During prolonged myocardial ischemia, numerous interrelated events occur resulting in decreased cellular [ATP], small increases in reactive oxygen species (ROS) production, accumulation of hydrogen ions, increased levels of cytosolic-free calcium, and activation of the calcium-activated protease calpain (). During reperfusion, ROS production is exacerbated, cellular calcium overload continues, calpain remains active, and activation of caspase-3 occurs ( 50, 77). Furthermore, leukocyte infiltration to the area of cardiac injury occurs following severe IR damage to the heart (i.e., ischemia longer than 20 min) ( 77). Collectively, these factors promote mitochondrial injury and subsequent cardiac myocyte death ( 33, 77).

It is established that mitochondrial ROS production is central in IR-induced myocardial injury ( 124). Indeed, mitochondrial ROS are produced during both ischemia and reperfusion, with the majority of ROS production occurring during reperfusion ( 4, 124). The importance of ROS-mediated damage to the heart during an IR event is confirmed by reports demonstrating that delivery of exogenous antioxidants provide protection against IR-induced cardiac injury ( 2, 18, 44, 59). Although IR-induced ROS production occurs in several cellular locations, mitochondrial production of ROS plays a dominant role in IR-mediated oxidant injury in the heart ( 2, 59).

Of the various pathological events that lead to IR injury, increased ROS production and increased cytosolic calcium levels play key roles. Furthermore, several lines of evidence indicate that cross talk occurs between ROS and calcium to magnify IR injury. For example, increased cytosolic calcium levels mediate cellular injury by activation of calpain, promotion of mitochondrial damage, and facilitating mitochondrial ROS production ( 33, 65, 86). Conversely, oxidative stress fosters cellular calcium overload in at least two ways. First, ROS-mediated formation of reactive aldehydes (i.e., 4-hydroxyl-2,3-trans-nonenal) attenuates plasma membrane calcium ATPase activity and diminishes calcium removal from the cell, resulting in increased cellular calcium levels ( 102). Second, ROS-mediated oxidation of the sarcoplasmic reticulum calcium release channel (i.e., ryanodine receptor) fosters release of calcium into the cytosol ( 3, 34).

Among the numerous factors that contribute to IR-induced cardiac cellular injury, mitochondria viability is a key arbitrator of cardiac cell life or death. Indeed, the degree to which aerobic ATP production is preserved in mitochondria following an IR insult determines whether the cell will live or die via necrosis or apoptosis ( 35, 36, 39, 65, 81). The main factor linking mitochondria to IR-induced cell death involves permeabilization of the mitochondrial outer membrane and/or opening of the mitochondrial permeability transition pore, which is linked to both apoptotic and necrotic cell death ( 81). Specifically, IR-induced apoptosis occurs during reperfusion by permeabilization of the outer mitochondrial membrane and the release of cytochrome c into the cytosol ( 65). Necrotic cell death can occur during both ischemia and reperfusion and involves the opening of the mitochondrial permeability transition pore followed by mitochondrial swelling and rupture ( 65).

In summary, numerous factors interact to produce cardiac cell injury during an IR insult. Of the various events responsible for IR injury, mitochondrial damage plays a central role, since these organelles are vital gate keepers of life and death in the cardiac myocyte. It follows that preventing IR-induced mitochondrial injury protects cardiac myocytes during an IR insult. Given the wealth of evidence indicating that exercise training produces robust protection against IR injury, it has been postulated that beneficial mitochondrial adaptations underpin these observations. A brief summary of the evidence that exercise provides protection against IR-induced myocardial injury follows.

Exercise Promotes Cardioprotection

It is well established that repeated bouts of exercise improve myocardial tolerance to IR in animals, which is commonly referred to as exercise-induced cardioprotection (reviewed in Refs. 28, 56, 92). Similar to the ischemic preconditioning (PC) phenomenon produced by a brief period of surgically induced ischemia, exercise-induced cardioprotection is also biphasic. The first phase of cardioprotection is acquired rapidly following an acute exercise bout (i.e., 0.5 h after exercise). However, this initial protection is rapidly lost within 3 h postexercise. The mechanisms responsible for the early phase of preconditioning are not well characterized but likely include allosteric activation of the endogenous antioxidant enzyme superoxide dismutase (SOD2) located within mitochondria of ventricular myocytes ( 46, 118). Given the similar time course for the early window of protection in both exercise-induced cardioprotection and PC, it is tempting to speculate that common mechanisms could mediate both forms of cardioprotection, although this conclusion requires scientific verification. The second or late phase of exercise-induced cardioprotection is achieved within 24 h after the exercise bout and persists for at least 9 days following a 5-day exercise routine and is far more robust than the aforementioned early protective window ( 66). This review is focused on the mechanisms responsible for this second window of exercise-induced cardioprotection.

As mentioned earlier, repeated bouts of endurance exercise protect against IR-induced arrhythmias ( 29, 43, 75), myocardial stunning ( 11, 22, 23, 32, 42, 69, 70, 89, 113, 114), and myocardial infarction ( 29, 31, 95, 118). Interestingly, only 3-5 consecutive days of endurance exercise is required to achieve a significant level of cardioprotection against IR-induced myocardial infarction () ( 23, 31, 44). Complete details about the dose-response impact of aerobic exercise intensity on cardioprotection remain unknown. Findings from several studies provide insight into the influence of exercise intensity on resulting cardioprotection. One investigation into the role of intensity suggested that exercise below 55-60% V̇o 2max did not achieve IR injury resistance ( 107), whereas another study concluded that both moderate- (i.e., 50% V̇o 2max) and relatively high-intensity (i.e., 70% V̇o 2max) exercise appear to be equally protective against IR-induced myocardial stunning ( 67). Regardless, it is feasible that there may be an exercise intensity threshold above which cardioprotection is achieved. Predictably, exercise-induced cardioprotection in rodents is lost rapidly (i.e., within 9-18 days) following the cessation of exercise training ( 66).

Although it is clear that continuous aerobic exercise (e.g., 60 min) produces a cardioprotective phenotype, growing evidence suggests that high-intensity interval training (1 min of exercise at ≥V̇o 2max) also produces a cardioprotective phenotype ( 69, 70). Furthermore, a recent study suggests that 12 wk of resistance exercise training provides protection against IR-induced myocardial infarction in rats ( 24, 103).

What Are the Mechanisms Responsible for Exercise-Induced Cardioprotection?

The mechanism(s) responsible for exercise-induced myocardial protection against IR injury remains a debated issue as numerous putative mediators have been proposed. In theory, exercise-induced cardioprotection could be achieved by any physiological adaptation that attenuates one or more of the damaging events that occur during ischemia and/or reperfusion. For example, exercise-induced cardioprotection could be acquired by changes in the coronary arteries (i.e., increased collateral circulation) and/or intrinsic changes in the cardiac myocyte. Potential intrinsic changes in the cardiac myocyte that could provide cellular protection against IR injury include increased glycolytic flux, altered nitric oxide (NO) signaling, increased levels of heat shock proteins (HSPs), amplified myocardial cyclooxygenase-2 (COX-2) activity, elevated endoplasmic reticulum (ER) stress proteins, enhanced function of sarcolemmal and/or mitochondrial ATP-sensitive potassium channels, increased cytosolic antioxidant capacity, and/or altered mitochondrial antioxidant capacity (). An overview of the evidence to support or deny the role for each of these potential cardioprotective mediators is highlighted in the next segments.

Exercise-Induced Changes in Coronary Circulation Are Not Required for Cardioprotection

Endurance exercise-induced structural changes in the coronary circulation include increased conduit artery diameters and increased arteriolar densities and diameters (reviewed in Ref. 62). Specifically, in both young rats and humans, several months of endurance exercise increases the volume of the coronary vasculature because of increased conduit artery size ( 45, 68). Furthermore, regular aerobic exercise induces functional adaptations in the coronary circulation, including enhanced endothelium-dependent vasodilation ( 62). Although both of these training-induced adaptations within the coronary circulation could protect the heart during an IR event, at present, no experimental data exists to directly link these exercise-induced changes to short-term (e.g., 3-5 days) exercise-mediated cardioprotection. Indeed, a few consecutive days of exercise training can provide cardioprotection against IR injury ( 23, 42, 113), but structural changes in the coronary circulation do not occur within this short period of exercise training ( 118). Furthermore, evidence indicates that cardiac myocytes isolated from the heart of exercise-trained animals are protected against hypoxia-reoxygenation injury ( 55). Therefore, these results indicate that exercise can directly modify the cardiac myocyte to achieve a cardioprotective phenotype, and, consequently, a change in the coronary circulation is not required to achieve exercise-induced cardioprotection. Indeed, although exercise training has the potential to increase coronary flow during reperfusion ( 62), studies of isolated perfused hearts demonstrate that short-term exercise provides cardioprotection independent of improvements in coronary flow ( 11, 66, 93). Thus the remainder of this review will focus on exercise-induced endogenous changes in the cardiac myocyte that can contribute to cardioprotection.

Altered Glycolytic Flux as a Potential Mediator of Exercise-Induced Cardioprotection

Myocardial survival during an IR insult is dependent, at least in part, on cellular energy status. To limit the energy deficit during ischemia and hypoxia, cardiac energy production switches from the preferential use of fatty acids to carbohydrates; this switch in fuel utilization is essential to sustain ATP production via glycolysis ( 116). However, whether increased glycolytic flux is advantageous or detrimental during ischemia depends on the duration of the ischemic insult. Indeed, increased glycolysis in the ischemic heart is a double-edged sword since the accumulation of glycolytic end-products (e.g., lactate) during prolonged ischemia are detrimental to postischemic recovery ( 104). Hence, limiting glycolysis during long-duration myocardial ischemia could be a protective strategy to minimize IR injury ( 116). In this regard, it has been reported that PC results in a decrease in glycolytic flux in the ischemic heart ( 116). Similarly, evidence indicates that endurance exercise training decreases the rate of glycolysis in the rat heart during ischemia ( 14). Although the mechanism(s) by which exercise training alters the metabolic phenotype of the heart to produce this response is unknown, it is feasible that a reduction in glycolysis during ischemia could be cardioprotective. Nonetheless, to date, no direct evidence exists to mechanistically connect exercise-induced changes in myocardial glycolytic flux to cardioprotection.

Altered NO Signaling as a Facilitator of Exercise-Induced Cardioprotection

NO is produced in tissues from L-arginine, oxygen, and NADPH by NO synthase (NOS) enzymes ( 90). Numerous studies reveal that endurance exercise training results in increased phosphorylation and activity of endothelial NOS (eNOS) in both humans and animals ( 19, 38, 41). This exercise-induced rise in eNOS activity is associated with increased production of NO, as evidenced by augmented levels of both nitrite and nitrosothiols in tissue and blood. In this regard, nitrite is produced by the oxidation of NO in aerobic conditions ( 117), whereas nitrosothiols are formed when cysteine thiols in proteins are modified by NO via a process known as S-nitrosylation ( 27). It follows that circulating levels of nitrite and nitrosthiols are commonly used as biomarkers of NO availability ( 15). Importantly, nitrite is a potentially important storage form of NO in both blood and tissues because nitrite can be converted to NO by either acid reduction or nitrite reductases during ischemia ( 64).

A recent review summarized the possible role that NO metabolites play in the cardioprotective effects of exercise ( 15). In regard to the mechanisms linking NO to cardioprotection, increased NO levels can increase the S-nitrosylation of cardiac proteins during myocardial IR, which can attenuate injurious processes such as apoptosis ( 15). Indeed, caspase-3 activity can be inhibited via protein S-nitrosylation, and NO production of nitrosthiols can modify complex I of the mitochondrial electron transport chain, resulting in reduced mitochondrial ROS production during an IR event ( 15). Together, these changes could protect cardiac myocytes against IR-induced damage ( 117), and a recent study employing eNOS knockout mice concluded that increased production of NO is essential to achieve the cardioprotection associated with endurance exercise. However, it is also possible that some of the exercise-induced adaptations in the heart are not achieved in the eNOS knockout mouse. Indeed, a recent study reveals that several exercise-induced adaptations within the heart require eNOS expression ( 20). Therefore, additional experiments are required to confirm or deny whether NO or other NO metabolites are a requirement to achieve exercise-induced cardioprotection.

Elevated Myocardial Heat Shock Proteins Are Not Essential for Exercise-Induced Cardioprotection

It is clear that transgenic overexpression of heat shock protein 72 (HSP72) protects the heart against IR-induced injury ( 48, 51, 111). Furthermore, repeated bouts of endurance exercise result in a three- to fivefold increase in cardiac HSP72 levels ( 21, 44, 89, 113). In theory, elevated cellular levels of HSP72 can protect the myocardium against IR injury by augmenting myocardial antioxidant capacity, protecting mitochondria against IR injury, and preventing apoptosis ( 51, 109, 111).

Although exercise promotes the accumulation of HSP72 in the heart and HSP72 is cardioprotective, three independent studies demonstrate that an exercise-induced increase in HSP72 is not essential for exercise-induced cardioprotection ( 42, 95, 113). Therefore, although overexpression of HSP72 is sufficient to promote cardioprotection, increases in myocardial HSP72 are not a requirement to achieve exercise-induced cardioprotection.

Increased Myocardial Cyclooxygenase-2 is not Responsible for Exercise-Induced Cardioprotection

Cyclooxygenase-2 (COX-2) is the rate-limiting enzyme in prostaglandin biosynthesis catalyzing the conversion of arachidonic acid to prostaglandins. Over the past decade, COX-2 has emerged as an obligatory mediator of the late phase of ischemic preconditioning-induced cardioprotection, and it follows that COX-2 could also be a candidate molecule to explain exercise-induced cardioprotection ( 8, 10, 101).

The PC cardioprotective actions of COX-2 appear to spring from PGE ₂ and/or PGI ₂ production, since this elevated prostanoid production improves vasodilation within the coronary vasculature ( 1, 47). Nonetheless, several lines of evidence suggest that COX-2 is not required for exercise-induced cardioprotection. For example, exercise does not elevate COX-2 levels in the rat heart ( 94). Similarly, pharmacological inhibition of COX-2 does not prevent exercise-induced protection against IR-induced cardiac arrhythmias ( 78). Together, these findings suggest that an increase in COX-2 is not essential to achieve exercise-induced cardioprotection against IR injury.

Elevated Endoplasmic Reticulum Stress Proteins do not Contribute to Exercise-Mediated Cardioprotection

Endoplasmic reticulum (ER) stress proteins represent a group of cardioprotective proteins that could contribute to exercise-induced cardioprotection, since recent evidence indicates that ER stress contributes to IR-induced myocardial injury ( 80). Indeed, IR-induced ER dysfunction can promote both mitochondrial-dependent and -independent cell death resulting from a disturbance in calcium homeostasis and/or impaired protein folding ( 115).

Two proteins that could protect against ER stress are the glucose-regulated proteins Grp78 and Grp94. Both Grp78 and Grp94 function in ER protein folding and also exhibit calcium-binding properties, and overexpression of Grp94 and Grp78 can protect cardiomyocytes against both calcium overload and oxidative damage ( 115, 120, 121). Moreover, increased Grp78 and Grp94 expression is linked to a reduction in IR-induced necrosis and apoptosis in the heart ( 74). Nevertheless, exercise does not elevate Grp78 and Grp94 ( 76). Therefore, the existing evidence indicates that increased ER stress proteins are not a requirement for exercise-induced cardioprotection against IR injury.

Sarcolemmal and/or Mitochondrial ATP-Sensitive Potassium Channels May Participate in Exercise Trained-Mediated Cardioprotection

ATP-sensitive potassium channels located in the sarcolemma (sarcoK ATP) and mitochondria (mitoK ATP) of cardiac myocytes are important regulators of cardiac myocyte function ( 79, 84). Both sarcoK ATP and mitoK ATP channels are composed of two protein complexes, an inwardly rectifying potassium channel pore and an associated sulfonylurea receptor subunit that governs channel opening. These energy-sensing channels are named according to a classical understanding of their function, in which potassium ion currents are inhibited by an abundance of cellular ATP. Channel activation also occurs in response to other activating factors such as acute ischemia, increases in adenosine and MgADP, along with activation of protein kinase C-ε (PKC-ε) ( 99). Independent of the activating stimulus, opening either sarcoK ATP or mitoK ATP channels before an IR insult confers cardioprotection ( 40, 60). The means by which K ATP channels confer cardioprotection are not fully understood. Nonetheless, it is clear that K ATP channel opening prevents IR injury through multiple biochemical alterations within the ventricular myocyte. For instance, opening of sarcoK ATP channels is predicted to protect against IR injury by shortening the cardiac action potential duration via the acceleration of phase 3 repolarization ( 40). Shortening the cardiac action potential could inhibit Ca ²⁺ entry into the cell via L-type Ca ²⁺ channels and avert Ca ²⁺ overload ( 40). Furthermore, the slowing of depolarization could also reduce Ca ²⁺ entry by avoiding the reversal of the Na⁺/Ca ²⁺ exchanger. Together, these actions could protect the cardiac myocyte during IR by reducing the cytosolic Ca ²⁺ overload. Another role that sarcoK ATP channels play is triggering the opening of the mitoK ATP channels ( 84). The specific details of how mitoK ATP channels protect the heart against IR injury remains controversial, but it is believed that opening the mitoK ATP channels protects mitochondria against IR-induced Ca ²⁺ overload and damage ( 5, 40).

The role that sarcoK ATP channels play in exercise-induced protection against IR injury has received limited investigative attention, but two studies suggest that endurance exercise training increases the expression of sarcoK ATP channels in the cardiac myocyte ( 12, 122). Furthermore, other reports reveal that pharmacological blockage of the sarcoK ATP channels impairs the exercise-induced protective benefits against IR-induced myocardial necrosis ( 12, 96). Nonetheless, because of concerns associated with the pharmacological inhibitors used in these studies, it is difficult to form a firm conclusion regarding the mechanistic role that sarcoK ATP channels play in exercise-induced cardioprotection.

Finally, using pharmacological inhibitors of the MitoK ATP channel, it appears that MitoK ATP channel activation protects the heart against IR-induced ventricular arrhythmias ( 97) but does not protect against IR-induced infarction ( 12). Nonetheless, the inability to detect the molecular identity of the MitoK ATP channel and concerns associated with the specificity of the channel blocker used in these experiments does not permit firm conclusions regarding the role that the MitoK ATP channel plays in exercise-induced cardioprotection.

Increased Cytosolic Antioxidant Capacity May Play a Required Role in Exercise-Induced Cardioprotection

Although the etiology of cardiac IR injury involves numerous factors and varies as a function of the duration of ischemia, mitochondrial-produced ROS are significant contributors to the necrotic and apoptotic cell death following an IR insult (reviewed in Refs. 77, 91). There is abundant evidence that endurance exercise training increases several components of the antioxidant buffering system in the heart. In particular, many studies agree that endurance exercise increases SOD2, which is the SOD isoform located in the mitochondrial matrix (reviewed in Refs. 28, 56, 92). Another SOD isoform (SOD1) is located in both the cytosol and the mitochondrial intermembrane space ( 90). Emerging evidence indicates that exercise training also elevates SOD1 in the mitochondria ( 63). In contrast, the impact of exercise on the cytosolic isoform of SOD1 in the heart remains equivocal.

SOD is a first line of defense against superoxide in cells, and SOD-mediated dismutation of superoxide results in formation of the nonradical ROS hydrogen peroxide (H ₂O ₂). Cardiac myocytes are equipped to eliminate H ₂O ₂ via several routes, including the enzymatic removal by catalase, thioredoxins, and glutathione peroxidase ( 90). However, most studies report that the activities of catalase, thioredoxins, and glutathione peroxidase are not increased in the heart following exercise ( 28, 30, 31, 53, 97, 105, 112). Nonetheless, growing evidence suggests that exercise training increases the activity of glutathione reductase in the heart via posttranslational modifications ( 29, 30). An increase in glutathione reductase activity would amplify the heart's ability to replenish cardiac levels of glutathione that is required for glutathione peroxidase to remove H ₂O ₂. In this regard, a recent report concludes that increases in glutathione reductase activity play an essential role in exercise-induced cardioprotection ( 29). However, it is currently unclear whether this exercise-induced increase in glutathione reductase activity in cardiac myocytes is confined to the cytosolic compartment alone or whether glutathione reductase activity also increases in other cellular compartments such as the mitochondrion. Regardless of the cellular location of this enzyme, it appears likely that increases in myocardial glutathione reductase activity contribute to exercise-induced cardioprotection.

Exercise-Induced Alterations in Mitochondrial Proteins and Phenotype Are Central to Exercise-Induced Cardioprotection

Growing evidence reveals that endurance exercise training results in alterations in mitochondrial phenotype, and this adaptation is required to achieve exercise-induced cardioprotection. In the following segments, we discuss exercise-induced changes in mitochondrial phenotype and evaluate evidence that exercise-induced increases in key mitochondrial proteins are required to achieve cardioprotection against an IR insult.

Conclusions and New Frontiers

Myocardial IR injury is a major cause of morbidity and mortality around the world, and, therefore, protecting the heart against IR injury is important. Currently, the only pragmatic method of providing sustainable cardioprotection against IR-induced myocardial injury is endurance exercise training, and regular participation in endurance exercise protects the heart against all levels of IR-induced injury.

The precise cellular adaptations responsible for exercise-induced cardioprotection remain a topic of debate. Nonetheless, there is evolving evidence that exercise training promotes alterations in mitochondrial protein expression and a change in mitochondrial phenotype that resists IR-induced damage. Furthermore, convincing evidence indicates that elevated myocardial SOD2 plays a major role in exercise-induced cardioprotection by protecting mitochondria and the cardiac myocyte against IR-induced oxidative damage. Moreover, it is feasible that other cardioprotective proteins exist in mitochondria that contribute to exercise training-induced cardioprotection. Moreover, there is reason to believe that both physiological and biochemical mechanisms exert synergistic protection in the exercised heart. Relative to the anti-arrhythmic protection afforded by elevated SOD and MitoKATP content of exercised ventricles, there is motive to suspect that autonomic changes associated with exercise may also contribute to the observed cardioprotection. This matter of synergistic protection against IR injury could help to explain clinical observations that exercise appears to be more protective than can be accounted for statistically using traditional risk factor analyses ( 52).

Although much has been learned about exercise-induced cardioprotection, many unanswered questions remain. For example, the role that posttranslational modifications play in exercise-induced cardioprotection against IR-injury is unknown. Indeed, posttranslational modifications to proteins such as phosphorylation, methylation, and acetylation are essential regulators of cell signaling pathways, and their contributions to cardioprotection are poorly understood ( 87).

Finally, another unanswered question regarding the mechanisms of exercise-induced cardioprotection relates to the impact of exercise training on mitochondrial turnover in cardiac myocytes. This type of mitochondrial quality control depends on the dynamic balance between mitophagy and mitochondrial biogenesis along with fission and fusion processes ( 37, 119). Theoretically, increased mitophagy of damaged mitochondria that produce high levels of ROS would benefit the exercised heart by leaving behind only the healthiest mitochondria. Therefore, these remaining mitochondria should have a higher threshold for opening of the mitochondrial permeability transition pore ( 37). This raises the question of "What role does mitochondrial quality in the cardiac myocyte play in cardioprotection?" Clearly, there is much more to be learned about the mechanisms responsible for exercise-induced cardioprotection against IR injury.

Footnotes

This work was supported by a grant from the National Heart, Lung, and Blood Institute (R01 HL-067855) awarded to S. K. Powers.

No conflicts of interest, financial or otherwise, are declared by the author(s).

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References

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Myocardial ischemia-reperfusion (IR) injury can cause ventricular cell death and is a major pathological event leading to morbidity and mortality in those with coronary artery disease. Interestingly, as few as five bouts of exercise on consecutive days can rapidly produce a cardiac phenotype that resists IR-induced myocardial injury.

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The pathways of mitophagy for quality control and clearance of mitochondria

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The pathways of mitophagy for quality control and clearance of mitochondria

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Abstract

Selective autophagy of mitochondria, known as mitophagy, is an important mitochondrial quality control mechanism that eliminates damaged mitochondria. Mitophagy also mediates removal of mitochondria from developing erythrocytes, and contributes to maternal inheritance of mitochondrial DNA through the elimination of sperm-derived mitochondria. Recent studies have identified specific regulators of mitophagy that ensure selective sequestration of mitochondria as cargo. In yeast, the mitochondrial outer membrane protein autophagy-related gene 32 (ATG32) recruits the autophagic machinery to mitochondria, while mammalian Nix is required for degradation of erythrocyte mitochondria. The elimination of damaged mitochondria in mammals is mediated by a pathway comprised of PTEN-induced putative protein kinase 1 (PINK1) and the E3 ubiquitin ligase Parkin. PINK1 and Parkin accumulate on damaged mitochondria, promote their segregation from the mitochondrial network, and target these organelles for autophagic degradation in a process that requires Parkin-dependent ubiquitination of mitochondrial proteins. Here we will review recent advances in our understanding of the different pathways of mitophagy. In addition, we will discuss the relevance of these pathways in neurons where defects in mitophagy have been implicated in neurodegeneration.

Keywords: mitochondria, mitophagy, PINK1, Parkin, mitochondrial dynamics, neuron

Facts

Mitophagy mediates clearance of damaged mitochondria, and is also involved in the removal of mitochondria from maturing erythrocytes and eliminating sperm-derived mitochondria after fertilization.

In yeast, the mitochondrial outer membrane protein autophagy-related gene 32 (ATG32) recruits the core autophagic machinery to mitochondria for their selective removal.

A pathway containing PTEN-induced putative protein kinase 1 (PINK1) and Parkin responds to loss of mitochondrial membrane potential by targeting damaged mitochondria for clearance.

The PINK1/Parkin pathway is triggered when PINK1 is stabilized on the outer membrane of damaged mitochondria, where it facilitates recruitment of cytosolic Parkin.

Damaged mitochondria are prevented from moving and fusing with the mitochondrial reticulum in preparation for their mitophagic clearance.

Open Questions

How distinct are the mitophagy pathways triggered by different stimuli or conditions?

To what extent does Parkin activate mitophagy by causing the degradation of mitochondrial surface proteins or hyper-ubiquitinating the mitochondrial surface?

How do PINK1 and Parkin interact with one another and with substrates on the mitochondrial surface in causing mitophagy?

In contrast to the remarkable conservation of the core autophagic machinery, why are mitophagy-specific genes so little conserved between yeast and mammals?

How best should mitophagy be triggered by researchers probing physiologically relevant pathways?

The mitochondrion is an important actor in the life of a eukaryotic cell, but, like all good actors, a mitochondrion needs to exit the stage at the right time. Mitophagy removes mitochondria once they have played their part. This article will review the mechanism by which they are cleared and the cues that signal their removal. Alongside the critical metabolic functions of mitochondria in fatty acid oxidation, the Krebs cycle, and oxidative phosphorylation, mitochondria can also potentially damage cells. Reactive oxygen species (ROS), in particular superoxide anion (O ₂*−), hydrogen peroxide (H ₂O ₂), and hydroxyl radical ( *OH) are toxic byproducts of oxidative phosphorylation. ROS causes oxidative damage to mitochondrial lipids, DNA, and proteins, making mitochondria further prone to ROS production. In turn, damaged mitochondria release high levels of Ca ²⁺ and cytochrome c to the cytosol and thereby trigger apoptosis. Thus, like macroscopic energy sources and power plants, these microscopic power supplies are essential but prone to release hazardous materials, particularly when they have been compromised by damage or age. Accordingly, ensuring proper elimination of dysfunctional mitochondria is imperative to cellular survival, and mitochondrial damage has been implicated in aging, diabetes, and neurodegenerative diseases.

To prevent cellular damage by preserving a population of healthy mitochondria, several quality control mechanisms have evolved (). Mitochondria have their own proteolytic system, two AAA protease complexes in the inner membrane, whose function is to degrade unfolded membrane proteins. Cytosolic proteosomes have also been shown to mediate degradation of proteins on the inner and outer mitochondrial membrane. In addition to proteolytic and proteosomal degradation, recent evidence points to a lysosomal pathway in which vesicles bud from mitochondrial tubules, sequester selected mitochondrial cargos, and then deliver those mitochondrial components to the lysosome for degradation. This pathway is active under steady-state conditions and is further stimulated by oxidative stress. These mitochondrially derived vesicles may represent a mechanism for selective removal of oxidized mitochondrial proteins while leaving the whole organelle intact.

The aforementioned pathways account for degradation of only a subset of mitochondrial proteins, whereas radioisotope pulse-chase assays indicate that the entire protein contents of mitochondria are turned over within a few days. This observation implies that alternative mechanisms for bulk degradation of mitochondria exist. Accordingly, entire mitochondrial organelles were often observed in yeast vacuoles and mammalian lysosomes in electron microscopy studies. Bulk degradation of cellular contents occurs through a regulated process called autophagy (reviewed elsewhere in this issue); however, a selective form of autophagy, termed mitophagy, is chiefly responsible for elimination of damaged or superfluous mitochondria. In this process, mitochondria are sequestered in double-membrane vesicles and delivered to lysosomes for hydrolytic degradation. In addition, although mitochondrial turnover and clearance of damaged mitochondria may be the primary functions of mitophagy, there are other specialized cases of mitophagy: the complete removal of mitochondria during erythrocyte maturation and the selective destruction of sperm-derived mitochondria after oocyte fertilization. In this review, we will discuss the general features of mitophagy as a quality control pathway, and also the activation of specific types of mitophagy in response to different physiological triggers.

Overview of Autophagy

Autophagy (from Greek, meaning self-eating) is a broad term referring to different pathways for bulk degradation of cytosolic components and organelles, including mitochondria, through delivery to the lysosome. There are three distinct classes of autophagy: microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy. In microautophagy, invaginations of the lysosomal membrane directly engulf portions of the cytoplasm. In contrast, CMA involves the chaperone Hsc70 and its co-chaperones that recognize and unfold substrate proteins with a KFERQ amino-acid motif. The substrates bind to the lysosomal protein LAMP-2A and are translocated across the lysosomal membrane for degradation.

Macroautophagy is the major type of autophagy and it is well conserved from yeast to mammals. Non-selective or bulk autophagy is induced by starvation to provide the cell with essential nutrients. In contrast, selective autophagy occurs to clear unwanted or damaged organelles, including mitochondria. In this process, an isolation membrane (known as phagophore) surrounds a portion of the cytoplasm or an organelle, forming a double membranous structure called the autophagosome. The autophagosome then fuses with the lysosome to form an autolysosome, in which the hydrolytic degradation of contents of the autophagosome will occur. Mitochondria are removed by a form of macroautophagy (called mitophagy), in which the core machinery of bulk macroautophagy is harnassed for the selective clearance of a mitochondrion. We will therefore focus our discussion on macroautophagy, herein referred to as autophagy.

Autophagy can be mechanistically broken down into the following five steps: initiation of the isolation membrane, elongation, closure of the isolation membrane and autophagsome formation, autophagosome-lysosome fusion, and lysosomal degradation. These stages are similar whether invoked for the clearance of bulk cytosol or of a mitochondrion. Genetic screens for defects in this pathway in the yeast Saccharomyces cerevisiae have identified nearly 30 ATGs. The ATG genes that form the core machinery in yeast are ATG1-10, ATG12-16, and ATG18. These core genes are highly conserved in mammals and, except for ATG13 in some circumstances, are also essential for mitochondrial autophagy. In the case of starvation-induced autophagy, the process begins with a complex consisting of ATG1, a kinase, and ATG13, its regulatory subunit, that exist in a complex with other autophagy proteins. Target of rapamycin complex 1 (TORC1) normally restricts ATG13 from interacting with ATG1, but starvation removes this inhibition and allows the cascade to proceed. The mammalian ortholog of these proteins, the ULK1 complex, is similarly regulated by TORC1. In the case of stress response in mammalian cells, mitophagy can similarly be signaled by the Hsp90-Cdc37 complex acting through ULK1 and ATG13. Other pathways for activating mitophagy are discussed below, but all seem to require the ATG1/ULK1 complex for initiation, but do not necessarily require ATG13. The formation of the autophagosome, whether surrounding a mitochondrion or other cargo, proceeds by means of the ATG1/ULK1 complex activating a class III phosphatidylinositol 3-kinase (PI3K) complex that includes ATG14, Vps34 and Beclin1. PI3K-mediated formation of phosphatidyl inositol triphosphate (PIP3)-rich membrane domains is important for nucleation of the isolation membrane. In some cases, stress-induced bulk autophagy as well as mitophagy occurs independently of Beclin 1, suggesting that alternative pathways for production of PIP3 may exist. Elongation of the isolation membrane is then mediated by two ubiquitin-like conjugation systems. In the first system, the ubiquitin fold-containing protein ATG12 is conjugated to ATG5 and an ATG5-ATG12-ATG16L1 complex then localizes at the isolation membrane. ATG12 can also be conjugated to ATG3 and this complex, although not necessary for general autophagy, promotes mitophagy when the mitochondria are depolarized. The mechanism by which this additional ATG12 complex figures in selective mitophagy remains unknown. The second ubiquitin-like reaction that is shared by the general autophagy pathway and mitophagy employs the ubiquitin fold-containing protein microtubule-associated protein light chain 3 (LC3) (also called MAP1LC3 or LC3B, the ortholog of yeast ATG8). It is synthesized in the pro-LC3 form, cleaved by ATG4B to form LC3-I, and conjugated in ubiquitin-like reactions to phosphatidyl ethanolamine (PE) forming LC3-II. LC3-II localizes to isolation membranes and is required for elongation and closure of this membrane to form mature autophagosomes.

Although clearly essential for forming the defining double-membrane vesicle of the autophagosome, the origin of the membrane component remains controversial. De novo membrane assembly has been proposed; however, various studies have implicated the ER, Golgi, plasma membrane, and more recently mitochondria as possible membrane sources for the autophagosome. Yoshi and co-workers observed close association of the isolation membrane with rough ER during mitophagy, implicating ER in particular as a membrane source for mitophagy. On the basis of the evidence suggesting mitochondria as a membrane source, in general autophagy, it is intriguing to speculate that, in mitophagy, the mitochondrion could supply the autophogosomal membrane for its own degradation. Clearly, further work is required to unambiguously determine the membrane sources in both general and cargo-selective autophagy.

Mitophagy as a Selective Form of Autophagy

The extensive similarity of the pathways for general autophagy and mitophagy, as summarized above, raises an obvious question: How is an autophagosome directed selectively to mitochondria and how is that process triggered without the activation of bulk autophagy? Although mitochondria can be engulfed non-selectively along with other cytosolic contents during bulk autophagy, both yeast and mammalian cells can selectively degrade damaged or superfluous mitochondria by mitophagy. In yeast, mitophagy can be activated by mutations that impair mitochondrial electrochemical potential; two such mutations in Fmc1 and Mdm38 cause aggregation of the F ₀F ₁ ATPsynthase subunits and impairment of the mitochondrial H⁺/K⁺ exchanger, respectively. Similarly, knockdown of an inhibitor of the mammalian F ₀F ₁ ATPsynthase, IF1, causes a dramatic reduction in mitochondrial mass that correlates with increased autophagic activity. Depolarization of mitochondria with the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) or photo-irradiation induces autophagic clearance of mammalian mitochondria while leaving other organelles such as peroxisomes intact. Recently, it has been reported that mitochondria carrying deleterious mutations in mtDNA can be selectively eliminated through mitophagy, although additional activation of macroautophagy, such as rapamycin-mediated inhibition of TORC1, may be required for efficient clearance. Of course, mitochondria with DNA mutations are encountered in human patients and so the clearance mechanism cannot be perfectly efficient; presumably, some threshold of damage must be crossed before mitophagy is likely to occur. Mitophagy also serves as a mechanism to regulate organelle number in response to developmental or physiological cues. Yeast cells that are grown on a non-fermentable carbon source require mitochondria for oxidative phosphorylation. Under such conditions, mitochondria are spared from degradation even during starvation-induced bulk autophagy, indicating differential regulation of bulk autophagy and mitophagy. In contrast, when yeast are switched from growth on non-fermentable to fermentable media, mitophagy occurs to reduce mitochondrial mass as oxidative phosphorylation is no longer needed. Thus, mitophagy must be mechanistically separable from other forms of autophagy so as to eliminate selectively dysfunctional or superfluous mitochondria in response to cues particular to mitophagy. Mitochondrial-specific mechanisms are beginning to emerge.

Mitophagy-Specific Effectors

To identify factors that might account for the selectivity of mitophagy, two groups independently performed screens for yeast mutants defective in mitophagy. Taken together, they found approximately 40 genes that were distinct from known ATGs and required for mitophagy but not bulk autophagy. A previously identified gene, Uth1, had been shown to be required for mitochondrial removal, induced by rapamycin and starvation; conditions that would induce bulk autophagy. This gene was not isolated in the screens for selective mitophagy where mitophagy was induced by switching to fermentable media. From these screens, ATG11 emerged as an important factor in mitochondrial degradation. ATG11 has also been implicated in other forms of selective autophagy including pexophagy (selective autophagy of peroxisomes). ATG11 appears to function as an adaptor between ATG8 and the proteins that function as the receptor for ATG11 on each particular organelle or cargo to be selectively removed. In the case of mitochondria, the ATG11 receptor is ATG32, another protein uncovered in the yeast screen, which localizes to mitochondria and binds ATG11 and ATG8 upon induction of mitophagy. In this manner, ATG32 recruits the canonical autophagic machinery to the mitochondria. Surprisingly, however, loss of ATG32 did not affect cellular ROS levels or growth on a non-fermentable carbon source, which may imply that other quality control pathways independent of ATG32-mediated mitophagy exist.

Unfortunately, ATG11 and ATG32 do not have any identifiable homologs in higher eukaryotes. Mammals could, however, have functional homologs of these proteins. For example, the ubiquitin-binding adaptor p62 (SQSTM1) may have an ATG11-like role. It accumulates on damaged mitochondria and has been reported to recruit mitochondria to the autophagosome by binding to ATG8/LC3 in some studies but not others. Another protein, Nix, may serve the function of both ATG32 and ATG11. Nix is a resident protein on the mitochondria and also binds directly to LC3. Nix is induced in developing reticulocytes and is required for the subsequent clearance of mitochondria during eythrocyte development. Yet another pathway involves the ubiquitin ligase Parkin as an important player in recruiting autophagosomes to damaged mitochondria. We will discuss the roles of Nix and Parkin in more detail in the next sections.

Two puzzles remain. The first concerns the initiation of the autophagic pathway in selective mitophagy. Although the proteins just discussed may explain the selective recruitment of ATG8 or LC-3 to mitochondria, ATG8/LC3 represent a late stage in the autophagic pathway, downstream of several protein complexes known to be essential for mitophagy as well. It is not yet clear how conditions that trigger mitophagy, whether metabolic changes or mitochondrial damage, initiate the cascade. The second puzzle arises from a recent mammalian genomic screen that identified 96 new genes required for Parkin-mediated mitophagy. Lack of significant similarities in the genes identified by this screen and those in yeast may suggest extensive differences between the regulation of mitophagy in mammals and yeast. This may reflect the different physiological purposes of mitophagy in such different cell types. However, it should also be considered that the inducers of mitophagy also differed in these screens; those in yeast employed shifts in nutrient sources, while the mammalian screens used depolarization of the mitochondrial membrane potential. There may be multiple routes for the recruitment of the autophagosome to the mitochondria, all of which will converge on the core apparatus of autophagy.

Modulation of Mitochondrial Dynamics before the onset of Mitophagy

Mitochondria are dynamic organelles that move within the cell and frequently undergo fission and fusion. In many cells, a large fraction of the mitochondria at any given moment are in an interconnected network. Mitochondrial dynamics and mitophagy are closely related; a mitochondrion has to be separated from the mitochondrial network to be engulfed by the autophagosome (). In this section, we will briefly review the molecular machinery that regulates mitochondrial dynamics and discuss the interaction of this machinery with the mitophagic pathway.

Fission and fusion, as well as mitochondrial trafficking on actin (in yeast) or the microtubule cytoskeleton (in higher organisms) enable the cell to adjust mitochondrial distribution to changing local needs. Mitochondrial fission and fusion are regulated by members of a family of conserved large GTPases, initially identified in yeast. The dynamin-like GTPase, dynain-related protein 1 (Drp1) (Dnm1 in yeast), mediates fission by forming a multimeric complex that wraps around the outer membrane of mitochondrial tubules and exerts mechanical force to produce membrane scission. In contrast to fission where mitochondria are divided using only an outer membrane apparatus, two distinct machineries, Mitofusin 1 and 2 (Fzo in yeast) and optic atrophy 1 (Opa1) (Mgm1 in yeast), are required for fusion of the outer and inner membranes, respectively. Mitochondrial fusion is also regulated by mitochondrial motility, although less directly; stationary mitochondria are less likely to encounter another mitochondrion with which to fuse. A critical function of fusion and fission is to allow for efficient distribution of mtDNA and proteins throughout the mitochondrial network. Even brief fusion events can allow extensive exchanges of mitochondrial content. By following the fate of individual photo-labeled mitochondria, Twig and co-workers have shown that a fusion event is often followed by fission, creating daughter mitochondria with uneven mitochondrial potentials. Whereas the daughter mitochondrion with the healthier membrane potential will continue to participate in fusion and fission cycles with the mitochondrial reticulum, the depolarized daughter mitochondrion is unlikely to undergo fusion and is often degraded through mitophagy.

Depolarization causes the loss of Opa1 and Mitofusins (Mfn1 and 2), proteins necessary for the fusion of the inner and outer membranes of mitochondria, and thereby promotes mitochondrial fragmentation. In agreement with the essential role of fission in mitophagy, a yeast genetic screen revealed that Dnm1, the yeast homolog of Drp1, is essential for mitophagy. Similarly in mammals, genetic manipulations that cause excessive fusion, such as overexpression of Opa1 or dominant-negative Drp1, preclude autophagic degradation of mitochondria. This protection from autophagy by hyperfusion occurs physiologically during certain types of starvation-induced autophagy, in which a fused mitochondrial network protects mitochondria from degradation. In summary, a shift in the balance of mitochondrial dynamics towards increased fission and decreased fusion promotes segregation of damaged mitochondria and facilitates their clearance by mitophagy.

The PINK1/Parkin Pathway of Mitophagy

A recently identified pathway that has emerged as a paradigm for mammalian mitophagy is mediated by the PINK1 and the E3 ubiquitin ligase Parkin (). The genes encoding PINK1 and Parkin were found to be mutated in certain forms of autosomal recessive Parkinson's disease (PD). Accumulation of dysfunctional mitochondria in the brains of PD patients implied a link between PINK1 and Parkin and mitochondrial quality control. Genetic studies in Drosophila further suggested a role for PINK1 and Parkin in the regulation of mitochondrial integrity. Loss of either protein in flies results in mitochondrial dysfunction and thereby causes degeneration of flight muscles and dopaminergic neurons. Overexpression of Parkin ameliorates the mitochondrial phenotypes in PINK1-deficient flies. This interaction and other genetic experiments demonstrate that the two genes act in the same pathway, with PINK1 upstream of Parkin. Parkin overexpression in mammalian cytoplasmic hybrid cells containing both wild-type and mutant mtDNA leads to selective elimination of mitochondria carrying mutant mtDNA. PINK1 and Parkin also promote removal of mitochondria damaged by chemical uncouplers, such as CCCP. While a role for PINK1 and Parkin in the clearance of defective mitochondria has extensive evidence, certain controversies remain. Stable knockdown of PINK1 has been reported to induce, rather than inhibit, mitophagy in neuronal cell lines. One explanation for this unexpected result is that PINK1 deficiency leads to higher mitochondrial oxidative stress, which in turn might trigger compensatory PINK1-independent mitophagy. In another study, it was found that while Parkin overexpression stimulates CCCP-induced mitophagy, it appears to inhibit bulk autophagy under basal conditions. This observation suggests that Parkin may differentially regulate distinct types of autophagy.

As discussed in the previous section, segregation of mitochondria destined for mitophagy appears to precede their degradation. Accordingly, PINK1 and Parkin play central roles in regulating mitochondrial dynamics and the mechanistic basis of this aspect of their function is presently the best understood. A genetic interaction between the PINK1/Parkin pathway and the fission/fusion machinery was discovered in flies where overexpression of Drp1 led to suppression of mitochondrial defects in PINK1 and Parkin mutants. Although similar studies in mammalian systems have produced contradictory results, the findings in the fly indicate that PINK1 and Parkin promote fission. Consistent with this conclusion, mammalian and fly studies indicate that upon mitochondrial depolarization, Parkin translocates to mitochondria and causes polyubiquitination of Mitofusin, targeting it for removal from the mitochondrial membrane by the p97 AAA-ATPase and subsequent proteosomal degradation (). Degradation of Mitofusin prevents damaged mitochondria from fusing with the functional mitochondrial network and is essential for their subsequent degradation by mitophagy. However, although loss of Mitofusin is necessary for mitophagy to proceed, it is not by itself sufficient to trigger mitophagy and PINK1 and Parkin can initiate mitophagy in cells lacking Mitofusin. Mitochondrial fragmentation in the absence of Mitofusin thus appears to be a permissive rather than a triggering event.

The PINK1 and Parkin pathway of mitophagy is also closely associated with the regulation of mitochondrial movements. Mitochondrial depolarization causes PINK1 and Parkin to associate with mitochondria Rho-GTPase (Miro), a mitochondrial adaptor protein that anchors a kinesin motor complex to the mitochondrial surface. This association leads to the phosphorylation of Miro by PINK1 and the subsequent proteosomal degradation of Miro in a Parkin-dependent manner (). Loss of this adaptor protein releases kinesin from the mitochondrion and arrests mitochondrial motility. Because stationary mitochondria are less likely to undergo fusion, this arrest may serve as an additional quarantine mechanism to segregate damaged mitochondria from the rest of the mitochondrial reticulum before mitophagy. Although it remains to be seen whether degradation of Miro is a required event for mitochondrial elimination, the PINK1/Parkin-mediated degradation of both Miro and Mitofusin illustrate how regulation of mitochondrial dynamics is coupled to mitophagy and have provided insight into the mechanisms by which these enzymes act. In the following section, we will describe the molecular details of the PINK1/Parkin pathway of mitophagy.

Accumulation of PINK1 and Parkin on damaged mitochondria

PINK1 is a serine/thereonine kinase that contains a mitochondrial targeting sequence allowing for its mitochondrial localization. In healthy mitochondria, PINK1 is constitutively imported, probably via the TIM/TOM complex, to the inner membrane where it is cleaved by several proteases including the mitochondrial-processing protease (MPP) and the inner membrane presenilin-associated rhomboid-like protease PARL, and ultimately proteolytically degraded (). Loss of mitochondrial membrane potential may be the event that acutely activates this pathway; loss of the potential gradient precludes import of PINK1 to the inner membrane, thereby stabilizing intact PINK1 on the mitochondrial outer membrane where it interacts with the TOM complex. In this manner, PINK1 serves as a sensor for mitochondrial damage. Accumulation of PINK1 on the mitochondrial surface induces translocation of Parkin from the cytosol to damaged mitochondria through a mechanism as yet unknown. Once recruited, Parkin promotes degradation of mitochondria through mitophagy. In mammalian cells, recruitment of Parkin to damaged mitochondria requires PINK1, although in fly studies Parkin overexpression was sufficient to rescue mitochondrial defects in PINK1-null mutants. Even in the absence of mitochondrial depolarization, a recombinant form of PINK1 that is stably localized on the mitochondrial outer membrane is sufficient for Parkin recruitment and induction of mitophagy. In addition, PINK1 artificially targeted to peroxisomal membranes can recruit Parkin and mediate autophagic degradation of the peroxisome. Thus, PINK1 and Parkin form the minimal machinery for recruitment of the canonical autophagy players to target organelles. Although the kinase activity of PINK1 is required for it to recruit Parkin, it is unclear as to how this occurs. One hypothesis is that PINK1 binding to and phosphorylation of Parkin serves to activate that enzyme and cause its translocation, but this remains controversial. Alternatively, it is possible that PINK1 phosphorylates an unknown substrate, and it is the primed substrate, not PINK1 per se, that binds to and recruits Parkin. This model is consistent with the sequential action of PINK1 and Parkin on the substrate Miro, but there is no evidence at present that Miro itself is essential for mitophagy.

Although PINK1 can clearly promote Parkin-dependent mitophagy, it is not necessarily the only means of activating the Parkin pathway. In Drosophila, Parkin overexpression rescues the mitochondrial morphology defects of a PINK1-null mutant, indicating that PINK1 is not essential for Parkin function. Parkin overexpression can also arrest mitochondrial motility in PINK1-deficient flies. Furthermore, Parkin mutants had more severe phenotypes than PINK1-null flies, suggesting PINK1-independent functions of Parkin. On the other hand, activation of mitophagy by CCCP was not observed in mammalian cells when PINK1 was knocked down. This leaves open the question of whether Parkin can be activated by pathways other than mitochondrial depolarization and PINK1 activation in mammalian cells.

Parkin promotes ubiquitination of mitochondrial proteins

Following its mitochondrial translocation, the E3 ubiquitinligase activity of Parkin appears to increase. Parkin mediates formation of two types of polyubiquitin chains: lysine (K) 48 linkage associated with proteosomal degradation of the substrate; and lysine (K) 63 linkage associated with autophagic degradation. Although Parkin-mediated polyubiquitination of mitochondrial substrates is likely to be the mechanism by which it triggers mitophagy, () the details of how this happens remain uncertain. In one model, Parkin mediates formation of K48 chains on specific mitochondrial outer membrane proteins and it is the proteosomal degradation of these proteins that triggers mitophagy. In support of this model, inhibition of proteosome activity prevents mitophagy of damaged mitochondria. A proteomic study in HeLa cells revealed Parkin- and proteosome-dependent degradation of Miro, Mitofusin, hFis1, and Tom70, as well as others, before mitophagy. However, as mentioned above, while proteosomal elimination of Mitofusin is required, it is not sufficient for initiation of mitophagy. Moreover, proteosome inhibition in Mitofusin-null cells prevents mitophagy, indicating that Mitofusin is not the key target. Therefore, it is not clear whether the degradation of these candidate proteins, if any, leads to activation of mitophagy or if they are merely permissive for mitophagy by, among other things, arresting motility and inducing fragmentation (). In a second model, Parkin causes formation of K63 (or 27)-linked ubiquitin chains on mitochondrial outer surface, and these ubiquitin chains, rather than inducing proteosomal degradation, initiate a signaling cascade that activates mitophagy (). In support of this model, Geisler et al. found abundant Parkin-dependent K63 and K27 ubiquitin chains and identified ubiquitination of the mitochondrial outer membrane protein VDAC, a component of the permeability transition pore, to be essential for mitophagy. However, the requirement of VDAC ubiquitination for mitophagy could not be replicated in a different study. In fact, it is possible that the mere presence of high levels of ubiquitin on the mitochondrial surface, rather than ubiquitination of a particular substrate, is sufficient for induction of mitophagy. This hypothesis is consistent with the ability of Parkin ectopically targeted to peroxisomes to mediate surface ubiquitination and autophagic degradation of these organelles. In summary, while the ubiquitin ligase activity of Parkin is well established, further work is required to determine how Parkin-mediated ubiquitination promotes mitophagy.

Interaction of PINK1 and Parkin with the core autophagy machinery

Parkin-mediated mitophagy requires core components of the autophagic machinery. Specifically, blocking the activities of ATG3, ATG5, ATG5, or ATG 7, and class III PI3K prevents degradation of CCCP-treated mitochondria. How does the PINK1/Parkin pathway interact with and activate the autophagic machinery? The ubiquitin-binding adaptor protein p62/SQSTRM1 accumulates on depolarized mitochondria and it has also been proposed to facilitate recruitment of damaged mitochondria to autophagosomes by binding to LC3 ( Figure 4bii ). However, it is not clear whether p62 is required for Parkin-mediated mitophagy, as mitochondrial elimination has been reported to occur in the absence of p62-mediated clustering. Nor would p62-mediated recruitment of damaged mitochondria to pre-existing isolation membranes explain how Parkin might stimulate the formation of new isolation membranes. Recent studies suggests that both PINK1 and Parkin can directly interact with the Beclin-1 PI3K complex. Parkin-dependent recruitment of Ambra1, an activator of the Beclin1 complex, to mitochondria upon depolarization could lead to activation of the Beclin-1 complex and nucleation of pre-autophagosomal membranes around damaged mitochondria. Therefore, both functions of Parkin, namely, ubiquitination of the mitochondrial outer membrane proteins and recruitment of Ambra1, may contribute to mitophagy of damaged mitochondria. The cytoplasmic E3 ubiquitin ligase SMURF1 has also been found to be required for Parkin-dependent mitophagy. The exact function of SMURF1 in mitophagy is not clear yet; however, the authors did find that its membrane targeting domain, and not its ubiquitin ligase activity, was required. On the basis of this finding, it is proposed that SMURF1 might be involved in the delivery of mitochondria to the autophagosome. In addition to its role in mitophagy of defective mitochondria, Parkin also stimulates mitochondrial biogenesis, presumably to replace damaged mitchondria with healthy and functional organelles. Upon depolarization, Parkin causes degradation of the trascriptional repressor PARIS. Loss of PARIS releases the mitochondrial biogenesis transcription factor PGC1α to activate its target genes.

In summary, the current body of evidence highlights the PINK1/Parkin pathway of mitophagy as an important regulator of mitochondrial homeostasis. Many questions, however, remain unanswered. How does PINK1 trigger Parkin recruitment? How does Parkin interact with various components of the core autophagic machinery and regulate mitophagy at different steps of autophagosome formation and maturation? How are the differential functions of Parkin in promoting mitophagy and mitochondrial biogenesis coordinated? Is the PINK1/Parkin pathway relevant only in cases of mitochondrial damage or does it also contribute to the steady-state turnover of mitochondria? It is noteworthy to mention that PINK1 and Parkin cannot be the only pathway for degradation of damaged mitochondria. Several observations support this conclusion. First, neither humans nor flies with mutations in the PINK1/Parkin pathway show a gross increase in their cellular mitochondrial mass. Second, PINK1 or Parkin knockout mice appear largely normal and have only subtle phenotypes, which would not be expected if turnover of damaged mitochondria was completely blocked. It remains to be seen what other parallel pathways account for mitochondrial turnover in the absence of PINK1 and Parkin.

What best models a physiological trigger of damage-induced mitophagy?

Because mitophagy is infrequent in healthy mammalian cells, researchers typically challenge cells to trigger the process, particularly to examine damage-induced mitophagy and the PINK1/Parkin pathway. The mitochondrial uncouplers, CCCP and FCCP, are most commonly used in this context. These drugs are nonspecific ionophores that cause a severe loss of mitochondrial membrane potential within minutes followed by recruitment of Parkin and LC3 to the mitochondria. For biochemical studies, the ability to depolarize all the mitochondria in a cell culture with CCCP has clear advantages. However, how similar is this phenomenon to the process that normally removes the sporadically damaged mitochondrion within an otherwise healthy cell? Presumably, the mitophagic apparatus in most cells was not designed to cope with the immediate loss of all mitochondria. In addition, CCCP is a nonspecific ionophore that affects cellular components other than the mitochondria. In fact, CCCP causes disruption of the microtubule cytoskeleton and prevents lysosomal acidification. For gentler disruption of mitochondrial function, several other approaches have been used. These include application of valinomycin, a K⁺ ionophore, and antimycin A, a blocker of complex III, and these also induce recruitment of Parkin. These compounds, although less drastic than CCCP, also affect the entire population of mitochondria. Damage to subsets of mitochondria has been achieved by photoirradiation and by the inducing mitochondrial DNA damage. These manipulations triggered selective removal of the damaged mitochondria and did so via Parkin recruitment. At present, therefore, these subtler perturbations of mitochondrial status largely confirm the conclusions from experiments with CCCP, but future work in this field may wish to focus, whenever possible, on milder techniques for induction of mitophagy that more closely resemble physiological conditions.

Mitophagy in Erythrocyte Maturation, Hypoxia, and Embryogenesis

Among the specializations that allow erythrocytes to mediate gas exchange between the lungs and peripheral tissues is the absence of internal organelles, including mitochondria. The lack of mitochondria may be important because erythrocytes experience high levels of oxidative stress during hemoglobin-mediated oxygen transport. Exposure to oxidative stress would increase mitochondrial ROS production and its consequent cellular damage. The transformation from a mitochondria-containing reticulocyte to a mature erythrocyte takes place over 2 to 3 days, during which time all their internal organelles, including mitochondria, are eliminated. The elimination of mitochondria in reticulocytes is mediated by a specialized form of extreme mitophagy that requires Nix (BNIP3L), a Bcl2-related mitochondria outer membrane protein with an atypical BH3 domain (). In fact, Nix−/− mouse retain mitochondria in their erythrocytes and develop anemia due to decreased survival of those cells. Nix is required specifically for elimination of reticulocyte mitochondria, but not other organelles that are lost during maturation such as ribosomes and the nucleus. Nix-dependent clearance of mitochondria is distinct from apoptotic pathways as it does not require other Bcl2 proteins such as Bak, Bax, Bcl-xl, Bim, or Puma. How then does Nix participate in mitophagy? Studies on reticulocyte maturation suggest that Nix is not required for the induction stage, as LC3-rich isolation membranes form in Nix−/− mice. Instead, Nix is required for incorporation of mitochondria into autophagosomes. Nix contains a conserved LC3-binding motif known as LIR (LC3-interacting region) and therefore may act as a receptor for targeting mitochondria to autophagosomes in a manner similar to the yeast ATG32/ATG11 pair. Despite the involvement of Nix, however, the upstream signal that activates mitophagy in these cells remains unknown.

The function of Nix may not be restricted to erythrocyte maturation; Nix could also be involved in depolarization-induced mitophagy, as CCCP treatment enhances Nix and LC3 interaction in HeLa cells. There is not yet consensus in these cases as to whether Nix functions upstream or downstream of mitochondrial depolarization. Unlike the clear requirement for Nix in erythrocyte maturation, treatment of reticulocytes with the uncoupling agent FCCP induces mitophagy in the absence of Nix. Thus, Nix can have at most a facilitating or parallel function in depolarization-induced mitophagy.

Nix, and another BH3-only protein Bnip3, are also involved in hypoxia-induced mitophagy in mammalian fibroblasts. Removal of mitochondria during hypoxia is important to reduce ROS production and maintain oxygen homeostasis. Activation of hypoxia-inducible factor under hypoxic conditions induces expression of Nix and Bnip3, which at high levels disrupt the interaction between Bcl2 and Beclin1. Once released, Beclin 1 is free to nucleate pre-autophagosomal membranes and induce ATG5-dependent autophagy. This mechanism does not explain why mitochondria are selectively degraded in hypoxia. A recent study, however, identified the mitochondrial outer membrane protein, FUNDC1, as a mitochondrial receptor for hypoxia-induced mitophagy. FUNDC1 binds to LC3 through a conserved LIR motif and this interaction is strengthened under hypoxic conditions, facilitating engulfment of mitochondria by autophagosomes. It is attractive to hypothesize that Nix, Bnip3, and FUNDC1 function in a coordinate manner during hypoxia with the consequence that FUNDC1 selectively targets mitochondria to autophagosomes formed through Nix- and Bnip3-mediated activation of Beclin 1.

Two groups have recently described a novel developmental role for mitophagy in the elimination of paternal mitochondria from fertilized oocytes (). In most eukaryotes, only maternal mitochondrial DNA is inherited, although sperms do contain mitochondria that are present in the oocyte immediately after fertilization. These paternal mitochondria are rapidly destroyed, although the evolutionary advantage this confers is unclear. Mechanistically it had not been known whether strict maternal inheritance was due to simple dilution of paternal mtDNA or active degradation of paternal mitochondria. The two studies in Caenorhabditis elegans show that an autophagic process accounts for selective degradation of sperm-derived mitochondria in early stages of embryogenesis. When autophagy is inhibited, paternal mitochondria persist but remain fragmented and unable to fuse with maternal mitochondria. While the fusion incompetence of paternal mitochondria may predispose them to mitophagy, the signal that activates this exceptional form of mitophagy selectively on sperm-derived mitochondria remains unknown.

Mitophagy in Neurons

Neurons represent a particularly interesting cell type for mitophagy because they combine high demand for mitochondria and high mitochondrial stress with a challenging cellular architecture. Neuronal demand for mitochondria is reflected in the high local density of mitochondria at presynaptic endings, post-synaptic densities, nodes of Ranvier, and in growth cones, where mitochondrial function is required to sustain neuronal activity. Indeed, it is estimated that the brain of a resting human is responsible for 25% of oxygen consumption, although it consists of only 2% of the volume. In addition to their importance for ATP production in neurons, neuronal mitochondria also serve important Ca ²⁺-buffering roles. Neurons are post-mitotic and non-proliferating cells; therefore, their mitochondria are particularly prone to accumulation of oxidative damage over time and high levels of Ca ²⁺ influx in neurons may be a further stressor. Not surprisingly, mitochondrial damage and dysregulation of mitophagy have been implicated in several neurodegenerative disease associated with aging, such as PD, Alzheimer's disease (AD), and Huntington's disease (HD). Significantly, it is not clear whether it is the upregulation or downregulation of mitophagy that contributes to the pathology of some of these neurodegenerative diseases. In addition, the neuronal process of mitophagy remains enigmatic as pathways of mitophagy have mainly been characterized in non-neuronal cell lines. While the role of PINK1 and Parkin in non-neuronal mitophagy is well established and mutations in these two genes are associated with neurodegeneration in hereditary PD, it remains to be seen whether our knowledge of damage-induced mitophagy can be applied to neurons. In fact, it has been reported that unlike other cell types, depolarization of neuronal mitochondria does not lead to Parkin recruitment or their autophagic clearance. On the other hand, recruitment of Parkin to depolarized neuronal mitochondria and their Parkin-dependent mitophagy have been observed in other studies. Clearly, further work is required to address the relevance of the PINK1/Parkin-mediated pathway of mitophagy in neurons.

Neurons face a unique challenge for mitochondrial turnover because a large fraction of the mitochondrial mass resides in distal axonal and dendritic processes, far away from the soma where most of the biogenesis is likely to occur and most of the lysosomes are thought to be located. The scarcity of lysosomes in axons may suggest that mitochondrial degradation preferentially occurs in the soma. While it may take days for axonal transport to deliver an organelle to the soma for degradation, rapid elimination of dysfunctional mitochondria is probably critical for protection of neurons against oxidative damage and degeneration. Not surprisingly, perhaps, neuronal autophagy plays a critical role in the maintenance of homeostasis in distal axons. Indeed, neurons are particularly sensitive to loss of autophagic genes. At basal levels, neuronal autophagy is highly efficient, and as a result, autophagosomes are rarely observed in neurons. In fact, multiple lines of evidence suggest that regulation of autophagy in neurons is distinct from other cell types.

The life cycle of damaged axonal mitochondria is poorly understood and it is not clear whether they need to be trafficked back to the cell body at any stage during their autophagic degradation. While preferential retrograde movement of depolarized mitochondria has been controversial, the destruction of Miro and arrest of mitochondrial movement that occurs upon mitochondrial damage will prevent the retrograde transport of mitochondria on which the PINK1/Parkin pathway has been activated. If retrograde transport is required for the completion of mitophagy, it is therefore not clear at which stage of the process defective mitochondria are retrieved from axons. It may be advantageous to the neuron to first sequester the damaged organelle locally within an autophagosome before transport to the soma. Retrograde transport of the autophagosome would be independent of Miro, a mitochondrion-specific adaptor. Indeed, autophagic markers have been detected in axons, suggesting that autophagosomes can form in neuronal processes. In addition, distal formation and retrograde transport of autophagosomes in axons of primary neurons have been reported and mitochondrial markers have been observed in these autophagosomes. Nevertheless, it is not certain that retrograde transport of an autophagosome to the soma is strictly required as lysosomal markers have also been found to colocalize with autophagosomes in distal axons, implying that lysosomal degradation can be completed outside the soma. It bears mentioning that the above studies have looked at the total population of autophagosomes, and the fate of mitophagosomes formed in response to damage in particular have not been analyzed. Understanding the dynamics of damaged neuronal mitochondria and the molecular players of neuronal mitophagy will help elucidate the contribution of mitochondrial dysfunction to neurodegenerative diseases.

Conclusion

How diverse will the pathways to mitophagy prove to be? From one perspective, considerable evidence in both yeast and mammalian cells indicates that all modes of mitophagy are harnessing the core machinery of autophagy. The important steps of autophagy, in which autophagosomes grow and surround their cargo before fusion with a lysosome, are equally pertinent to the engulfment and destruction of the mitochondria. However, the triggers that can activate this specialized and selective form of autophagy can be quite diverse, ranging from changes in the nutrient conditions of yeast media to the developmental transition from reticulocyte to erythrocyte and to damage or drug-induced loss of mitochondrial membrane potential. Several proteins are now identified that are critical in directing autophagosomes to the mitochondria, including ATG32, Nix, p62, PINK1, and Parkin. Ubiquitination of the mitochondrion, and/or the interaction of a mitochondrial receptor with the autophagosomal protein LC3 appear to be important in targeting of the autophagosome, but in each case the details of this recruitment remain unclear. More mysterious, perhaps, is the means by which the cues for mitophagy may initiate autophagosome formation before the association with mitochondrial membranes. Finally, there is interplay between control of mitochondrial dynamics - their movements, fissions, and fusions - and the progression of mitophagy; arrest and fragmentation of mitochondria after damage is likely to facilitate the selective clearance of damaged organelles. The existence of multiple pathways for mitophagy is likely to provide distinct control mechanisms for regulating constitutive turnover of mitochondria, rapid responses to damaged mitochondria, adjustments to mitochondrial status with nutrient availability, and highly specialized developmental needs.

Glossary

References

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Kanki T, Klionsky D, Okamoto K. Mitochondria autophagy in yeast. CORD Conf Proc. 2011;14:1989-2001.

Nowikovsky K, Reipert S, Devenish RJ, Schweyen RJ. Mdm38 protein depletion causes loss of mitochondrial K⁺/H⁺ exchange activity, osmotic swelling and mitophagy. Cell Death Differ. 2007;14:1647-1656.

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The pathways of mitophagy for quality control and clearance of mitochondria

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Selective autophagy of mitochondria, known as mitophagy, is an important mitochondrial quality control mechanism that eliminates damaged mitochondria. Mitophagy also mediates removal of mitochondria from developing erythrocytes, and contributes to maternal inheritance of mitochondrial DNA through the elimination of sperm-derived mitochondria. Recent studies have identified specific regulators of mitophagy that ensure selective sequestration of mitochondria as cargo.

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Mitophagy is triggered by mild oxidative stress in a mitochondrial fission dependent manner

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Mitophagy is triggered by mild oxidative stress in a mitochondrial fission dependent manner

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Mitophagy is triggered by mild oxidative stress in a mitochondrial fission dependent manner

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Mitochondrial dysfunction is linked to apoptosis, aging, cancer, and a number of neurodegenerative and muscular disorders. The interplay between mitop...

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Mitochondria are primarily responsible for providing the contracting cardiac myocyte with a continuous supply of ATP. However, mitochondria can rapidly change into death-promoting organelles. In response to changes in the intracellular environment, mitochondria become producers of excessive reactive oxygen species and release prodeath proteins, resulting in disrupted ATP synthesis and activation of cell death pathways.

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Introduction

Endurance exercise has long been recognized to not only enhance physical performance, but also bring about beneficial health outcomes. The most studied phenotypic adaptations in skeletal muscle to chronic exercise are increased vascularization (angiogenesis), fiber type transformation toward oxidative myofibers and increased mitochondrial content/function. Extensive effort has been afforded to the pursuit of understanding the underlying molecular mechanisms that regulate mitochondrial biogenesis, the process by which new mitochondria are formed. These efforts have culminated with the discovery of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) ( 38) and the unveiling of its function in exercise-induced mitochondrial biogenesis in skeletal muscle. Interestingly, data from Krieger et al. ( 20) suggest that exercise improves function of the subsarcolemmal portion of mitochondria. Therefore, exercise seems to not only increase the number of organelles, but also to improve the function/efficiency of the mitochondrial pool/network. The resulting functional improvement in the mitochondrial network most likely results from increased rates of mitochondrial biogenesis and efficient removal of dysfunctional/damaged mitochondria. In other words, mitochondrial biogenesis is critical, but may not be the only regulatory event that leads to the improved function of the mitochondrial network in skeletal muscle. Although a discussion of the many facets of mitochondrial function is beyond the scope of this review, it is known that this organelle is not only responsible for the aerobic synthesis of adenosine triphosphate (ATP), but may also affect calcium (Ca ²⁺) homeostasis and redox state in muscle cells. In fact, exercise training seems to positively impact all of these aspects in skeletal muscle. However, mitochondria, like other organelles, are subject to damages, and the mitochondrial DNA is especially susceptible to deletions caused by oxidative stress and aging compared with nuclear DNA ( 26). Therefore, it is imperative that muscle cells have means not only to generate new mitochondria, but also maintain the healthy ones and remove the damaged/dysfunctional ones. The regulation of this mitochondrial life cycle, from the biogenesis of new mitochondria to the removal of damaged/dysfunctional mitochondria, ultimately determines the overall quantity, and most importantly, quality and function of mitochondria in skeletal muscle, which are the determinants of metabolic function and physical performance (). These processes allow for a program to replace old, unhealthy mitochondria with new, healthy mitochondria; analogous to the idea of replacing old cars of low fuel efficiency for new cars of high fuel efficiency to clean the environment. In this review, we present the evidence suggesting that exercise training stimulates not only the biogenesis of mitochondria but also the removal of old and unhealthy mitochondria through mitochondrial dynamics and autophagy.

Biogenesis

Our laboratory has published a recent review on the topics of mitochondrial biogenesis, fiber type switching and angiogenesis in skeletal muscle ( 50). Here, we will seek to summarize the current understanding of exercise-induced mitochondrial biogenesis and to evaluate new concepts related to the process of mitochondrial maintenance in this field. For decades, investigators have explored the adaptive mechanisms of endurance exercise training and observed increases in mitochondrial density, either indirectly assessed by the expression of mitochondrial markers (e.g. cytochrome c oxidase-IV [COX-IV]), or more directly observed through transmission electron microscopy, and mitochondrial function, measured by mitochondrial enzyme activity and oxygen consumption. Although it is well known that the process of mitochondrial biogenesis requires a coordinated regulation of both the nuclear and mitochondrial encoded genes, the primary mechanisms of adaptation remained unclear. The discovery of PGC-1α in brown adipose tissue, originally as a cold-inducible regulator of adaptive thermogenesis, has triggered mounting interest in elucidating the molecular and signaling mechanisms underlying exercise-induced mitochondrial biogenesis in skeletal muscle. Biochemically identified as a co-activator of peroxisome proliferator-activated receptor-γ (PPARγ), PGC-1α is a coactivator of a great number of transcription factors, including the PPAR family transcription factors, nuclear respiratory factors (NRFs), transcription factor of activated mitochondria (TFAM) and others ( 28, 37). PGC-1α is indeed capable of co-activating the required nuclear and mitochondrial genes for the organelle's synthesis. Due in large part to this common theme among the PGC-1α inducible transcription factors, PGC-1α is now considered a "master regulator" of mitochondrial biogenesis. Recent data from Ugoccioni et al. ( 45) suggest that other factors act in parallel to PGC-1α to improve mitochondrial viability and function. Nevertheless, only more recently the questions of whether PGC-1α is induced in skeletal muscle by exercise and whether its induction is sufficient and necessary to promote mitochondrial biogenesis have been addressed. The short answer to these questions is now a resounding yes.

Overexpression of PGC-1α enhances mitochondrial biogenesis and PGC-1α deficiency blunts endurance exercise-induced mitochondrial biogenesis in skeletal muscle

Work by Leick et al. ( 22) demonstrated that mice with global deletion of the Pgc-1α gene exhibited normal wheel running activity and were capable of inducing oxidative gene expression in skeletal muscle. However, subsequent work using this same mouse model led to the conclusion that PGC-1α is indeed necessary for exercise training-induced protection from age-related decline in oxidative function (citrate synthase activity) and antioxidant gene expression (superoxide dismutase 2 [SOD2]) ( 21). It is important to understand that such global gene deletion approaches may directly affect other metabolically active tissues, including the brown adipose and neural tissues. These multi-organ effects, coupled with enhanced AMP-activated protein kinase (AMPK) activity in resting skeletal muscle, may have prevented the delineation of the functional importance of PGC-1α in skeletal muscle, particularly under the condition of exercise training ( 25). More recently, skeletal muscle-specific Pgc-1α knockout mice were generated ( 15), and data obtained from these mice showed that while exercise training-induced fiber type transformation appeared normal, exercise-induced mitochondrial biogenesis and angiogenesis in skeletal muscle were significantly attenuated ( 11). These results indicate the necessity of PGC-1α for normal exercise-induced metabolic adaptations, including mitochondrial biogenesis, in skeletal muscle. Considering the previous finding from Lin et al. ( 24) that transgenic mice with a skeletal muscle-specific overexpression of PGC-1α have enhanced expression of mitochondrial genes, fatigue resistance, and fiber type transformation toward oxidative myofibers; we now postulate that enhanced PGC-1α activity is sufficient to induced mitochondrial biogenesis, fiber type transformation, and angiogenesis. However, PGC-1α activity is necessary only for the induction of mitochondrial biogenesis and angiogenesis.

PGC-1α regulation by exercise

A relative wealth of data has been published documenting the effects of acute exercise and exercise training on PGC-1α expression. A consensus now exists that exercise training, including both endurance and resistance exercise, enhances PGC-1α expression due to transcriptional regulation ( 3, 7, 10). More recent evidence from Perry et al. ( 35) showed that a single bout of exercise was sufficient to induce PGC-1α protein and messenger ribonucleic acid (mRNA) expression in skeletal muscle while increases in markers for mitochondrial biogenesis could be observed only after the third bout. Interestingly, PGC-1α mRNA level returned to baseline between each bout, and the induction of PGC-1α was lessened with each bout. These findings suggest that the signaling-transcription coupling machinery for the PGC-1α gene can accurately sense the stress induced by altered functional demands of exercise and provide appropriate instructive cues to induce adaptations in skeletal muscle to minimize the exercise-induced stress.

Exercise-induced mitochondrial biogenesis may also be controlled by targeted activation of PGC-1α indicated by its translocation to the nucleus ( 43), and this adaptive process may begin prior to increased PGC-1α expression ( 48). Intriguingly, Safdar et al. ( 41) showed that a single bout of exercise increases PGC-1α level not only in the nucleus, but also in the mitochondria. Furthermore, the increased PGC-1α expression was also associated with enhanced interaction between PGC-1α and TFAM at the mitochondrial DNA D-loop, as well as increased binding of PGC-1α to the NRF-1 promoter in the nucleus. Therefore, exercise is not only capable of inducing PGC-1α protein expression, but also the translocation of PGC-1α to necessary compartments to stimulate the transcription of the nuclear and mitochondrial encoded mitochondrial genes, as well as the replication of mitochondrial DNA.

PGC-1α is also regulated by post-translational modification, such as phosphorylation and deacetylation. For example, PGC-1α can be deacetylated by sirtuin (silent mating type information regulation 2 homolog) 1 (SIRT1). Although there is no correlation between the total muscle content of SIRT1 and PGC-1α or mitochondrial biogenesis, SIRT1 activation is associated with increases in PGC-1α target gene expression, consistent with SIRT1 activation of PGC-1α through deacetylation ( 14). Interestingly, this SIRT1-mediated PGC-1α activation requires phosphorylation of PGC-1α by AMPK ( 4). The fact that PGC-1α activity is controlled both transcriptionally and post-transcriptionally reveals an elegant regulatory system with great fidelity in integrating both contractile and metabolic cues in exercise-induced skeletal muscle adaptation.

PGC-1α expression and activity are largely regulated by upstream signaling pathways of protein kinases. The two primary protein kinases involved in the regulation of PGC-1α in skeletal muscle are AMPK ( 18) and p38γ mitogen-activated protein kinase (p38γ MAPK) ( 1, 36). At least two AMPK phosphorylation sites have been identified on PGC-1α ( 18). AMPK not only activates PGC-1α, but also promotes the transcription of the PGC-1α gene ( 17), controlling both PGC-1α expression and activity. It has also been shown that AMPK induces mitochondrial gene expression through PGC-1α ( 18). In fact, AMPK has been referred to as a "master metabolic switch" for acute regulation of energy metabolism and exercise training-induced adaptations. Acute exercise appears to activate AMPK by phosphorylation at Thr172 ( 22). While a single bout of exercise results in increased AMPK activity in skeletal muscle, long-term exercise training leads to an increase in AMPK protein content ( 6, 43). However, neither acute exercise nor long-term exercise training are capable of increasing AMPKα protein content in skeletal muscle of Pgc-1α deficient mice ( 22). This suggests that not only is PGC-1α reliant upon AMPK, but also at least the α subunit of AMPK is reliant upon PGC-1α.

We have previously shown that a single bout of voluntary running in mice is sufficient to activate the p38 MAPK pathway ( 1). We further showed that p38 MAPK activity is sufficient to promote the Pgc-1α promoter activity through myocyte enhancer factor-2 (MEF2) and activating transcription factor 2 (ATF2) ( 1). In subsequent studies, we demonstrated that muscle-specific deletion of either p38α or p38β MAPK does not affect exercise-induced mitochondrial biogenesis and angiogenesis in skeletal muscle; however, mice with muscle-specific deletion of the p38γ MAPK or Pgc-1α gene exhibit attenuated mitochondrial biogenesis and angiogenesis ( 11, 36). These studies revealed for the first time that p38 MAPK stimulation of PGC-1α is specifically modulated by the γ isoform ( 36) and led to the conclusion that the p38γ MAPK-PGC-1α regulatory axis is functionally required for the normal exercise-induced metabolic adaptations in skeletal muscle.

Maintenance and Mitophagy

While exercise-induced addition of new mitochondria is of extreme importance, the maintenance of a healthy population of mitochondria may be of equal or greater value. Mitochondrial damage induced by reactive oxygen species (ROS) ( 2) can lead to the accumulation of metabolic intermediates ( 19), which in turn further impair mitochondrial function and trigger a vicious cycle. These pathological changes ultimately hinder the ability of mitochondria to function properly. It is conceivable that efficient removal of damaged mitochondria is critical in maintaining overall mitochondrial function in a tissue/organ like skeletal muscle. Furthermore, an accumulation of damaged mitochondria, associated with sedentary lifestyle and/or high-fat diets, may impair skeletal muscle contractile and metabolic functions. For example, mitochondrial dysfunction has been implicated in the development of insulin resistance ( 39), likely the result of excessive production of ROS and accumulation of byproducts of lipid metabolism. Therefore, it is imperative for the muscle to be able to both recognize and selectively remove damaged mitochondria.

Maintenance and Mitochondrial Dynamics

Mitochondria form tubular networks in mammalian cells, which extend through the cytosol and exist in close proximity to other important organelles and structures, such as the nucleus, endoplasmic reticulum and cytoskeleton. Interestingly, skeletal muscle fibers contain two distinct mitochondrial populations (i.e. subsarcolemmal and intermyofibrillar). While the intermyofibrillar mitochondria maintain relatively high respiratory capacity, the subsarcolemmal mitochondria more readily adapt to exercise training ( 20). Mitochondria are dynamic organelles that move about the cell joining and separating as necessary. However, due to the physical limitation posed by the dense contractile apparatus in skeletal muscle it is unlikely that such processes occur frequently across different mitochondrial populations in skeletal muscle. This joining and separating of mitochondria from the network is referred to as fusion and fission, respectively, and allows healthy, metabolically active cells to form a large interconnected network of mitochondria for sharing of components (proteins, substrates, mitochondrial DNA [mtDNA]) and removal of dysfunctional regions. Therefore, the balance between these processes and the signals responsible for their regulation are of extreme importance to the maintenance of the mitochondrial network.

The machinery involved in mitochondrial dynamics requires the participation of several proteins. Mitochondrial fusion involves mitofusins 1 and 2 (MFN1 and MFN2), which control fusion of the mitochondrial outer membrane, and optic atrophy type 1 (Opa1), which controls fusion of the mitochondrial inner membrane. Fission of the mitochondrial outer membrane in mammalian species is largely controlled by dynamin-related protein 1 (Drp1), which may be recruited by Fission1 (Fis1) or mitochondrial fission factor (MFF). Westermann has previously published a more complete review on the machinery of mitochondrial dynamics ( 46).

Genetic manipulation of the genes encoding the machinery of mitochondrial dynamics reveals the importance of a balance between the fusion and fission processes. For example, knockdown of Opa1 produced fragmented mitochondrial structure as the cells might be capable of performing fission but incapable of fusion ( 12). In contrast, animals with cardiac-specific deletion of MFN2 demonstrated abnormally enlarged subsarcolemmal mitochondria, but not intermyofibrillar mitochondria in cardiomyocytes ( 32). These findings demonstrate the importance of mitochondrial dynamics in maintaining a normal, healthy mitochondrial network. However, certain conditions favor one process over the other for the overall maintenance of this network. For example, mitochondrial fusion appears to be shut down in depolarized mitochondria, whereas mitochondria undergoing a fission event are often depolarized ( 44). The depolarization and fission events appear to precede the removal of those mitochondria by mitophagy ( 44), providing a possible link from the regulation of mitochondrial dynamics to the removal of damaged portions of the mitochondrial network.

Exercise training appears to regulate both mitochondrial fusion and fission processes. For example, seven sessions of high intensity interval training have been shown to progressively increase the protein content of MFN1 and Fis1 ( 35). Ding et al. ( 8) further showed that a single bout of treadmill running in rats induced increased MFN1 and MFN2 mRNA 24 h post-exercise while both MFN1 and MFN2 proteins remained at baseline. Cartoni et al. ( 5) demonstrated that MFN1 and MFN2 mRNA content was enhanced in human skeletal muscle at 24 h following a single bout of cycling exercise, concurrent with increased in COX IV mRNA. It is of note that cycling exercise induced increases in PGC-1α and estrogen-related receptor α (ERRα) mRNA at 2 h post-exercise prior to the changes in MFN1, MFN2 and COX IV. Interestingly, subsequent in vitro studies showed that MFN1 and MFN2 transcription was regulated by PGC-1α through ERRα ( 5). Together, these findings suggest that PGC-1α plays an important function in regulating the expression of the machinery for at least the mitochondrial fusion process in skeletal muscle under the conditions of exercise. Therefore, not only may mitochondrial biogenesis be regulated by PGC-1α but also the dynamics of mitochondrial fusion and fission.

Mitophagy

Autophagy is an evolutionarily conserved process for lysosome-dependent degradation of organelles and macromolecules. Two major forms of autophagy are known: non-selective autophagy, which is often stimulated by starvation, and selective autophagy, which may be triggered by and functions to remove aggregate/misfolded proteins and damaged organelles (e.g., mitochondria) ( 27). Autophagy begins when a pre-autophagosomal structure of unknown origin giving rise to phagophore, which elongates and engulfs the target, forming a double-membrane structure known as autophagosome. Autophagosome then fuses with lysosome to form autophagolysosome for execution of degradation ( 33). The whole process requires a family of proteins termed "autophagy related genes" (Atgs), including Atg1 (also known as Ulk1), Atg5, Atg6 (also known in mammalian systems as Beclin 1), Atg7, and Atg14. Specifically, the autophagic removal of mitochondria (mitophagy) may be a critical control step in the maintenance of mitochondrial quality, which presumably occurs following selection of dysfunctional, depolarized mitochondria through the fission/fusion processes. Indeed, inhibition of autophagy (deletion of the Atg7 gene) resulted in reduced mitochondrial respiration in mouse skeletal muscle and increased oxidative stress in cell culture ( 49).

The regulation and impact of endurance exercise on autophagy in skeletal muscle is only starting to emerge. Wohlgemuth et al. has shown that mild (8%) life-long calorie restriction (CR) either alone or in combination with exercise prevented aging-induced reduction in basal autophagy in skeletal muscle in rats ( 47). Smuder et al. ( 42) recently showed that 5 days of treadmill exercise blunted the global induction of autophagy markers in the soleus muscle of rats treated with the anti-tumor agent doxorubicin, but that it also resulted in moderate increases in Atg6 mRNA and protein and Atg7 mRNA in non-doxorubicin treated rats. Altogether, these results suggest that exercise training helps maintain the expression of autophagy proteins with aging and may even promote the expression of autophagy proteins in skeletal muscle, which may mediate the beneficial effects of exercise training. In this context, recent studies demonstrate that autophagy is induced by acute exercise in skeletal muscle ( 13, 16, 31) and other peripheral tissues ( 13, 16). More importantly, He et al. reported that acute exercise-induced increase in autophagy flux was inhibited in mice lacking three conserved phosphorylation residues in B-cell lymphoma 2 (Bcl-2 [mice termed ), which disables the required dissociation of Atg6 from the Bcl-2 protein for the induction of autophagy. The authors further observed that exercise training-induced improvements in glucose tolerance and blood triglycerides and cholesterol profiles in mice on a high-fat diet were blunted. In addition, deficient activation of autophagy by exercise also prevented normal phosphorylation and activation of AMPK and its downstream target Acetyl CoA Carboxylase (ACC), as well as translocation of glucose transporter 4 (GLUT4) to the cell membrane in skeletal muscle, which is a potential mechanism for the lack of metabolic adaptation in mice. What regulatory mechanism is responsible for acute-exercise induced autophagy and whether long-term exercise training affects autophagy in skeletal muscle remain to be ascertained.

Mitophagy in skeletal muscle has received growing attention, especially in the context of muscle atrophy ( 40, 49), but very little is known about the key proteins involved and the signals required for the regulation of basal levels of mitophagy. Much of our current understanding of mitophagy comes from studies in neural tissues from animal models of neural diseases, such as Parkinson's disease. We now know that among regulatory factors with key functional roles in the regulation of mitophagy Bcl-2 anchors to the membranes of both the endoplasmic reticulum (ER) and the mitochondria ( 34). Bcl-2, a key anti-apoptotic factor that binds to the mitochondrial outer membrane, also binds to and tethers Atg6 to the mitochondrial and ER membranes. Atg6 forms a complex with Atg14 to induce the formation of phagophore ( 23), playing a critical role in the induction and flux of autophagy. Additionally, Parkin and phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-induced putative kinase protein 1 (Pink1) have been demonstrated to relay the signals associated with mitochondrial damage to the induction of mitophagy ( 29).

Other proteins that have been implicated in mitophagy are mitochondrially localized BNIP3 (Bcl-2 and 19 kD interacting protein-37) and BNIP3-like protein (BNIP3L) ( 30, 40). Indeed forced expression of BNIP3 in adult fibers induces massive mitophagy ( 40), and BNIP3L seems to be involved in the recruitment of the autophagy machinery to the mitochondria ( 30).

Finally two classic exercise-induced pathways, AMPK, the "master metabolic switch", and mammalian target of rapamycin (mTOR), perhaps the primary cog of protein synthesis and therefore muscle hypertrophy, have been shown to play important roles in autophagy regulation. In concert with the generally opposed nature of AMPK and mTOR, previous evidence supports that activation of AMPK induces stimulatory phosphorylation of Ulk1 for induction of autophagy, whereas mTOR results in an inhibitory phosphorylation of Ulk1 ( 9). Since these kinases are differentially regulated depending on the mode and intensity of exercise, they may help to fine-tune mitophagy following acute exercise and as an adaptation to exercise training. However, their specific role in skeletal muscle autophagy has yet to be explored. This is an exciting new area of research, and future studies should look into the proteins involved in the mitophagy process, as well as their regulation in the context of exercise.

Summary

Adaptations to exercise training are broad and span multiple organ systems. In skeletal muscle, one of the most important adaptations is enhanced metabolic capacity, which helps improve performance and health. The primary underlying mechanisms involve the regulation of the mitochondrial network. Great attention has been paid in recent years to the mechanisms involved in the generation and addition of new mitochondria, which is now known to be regulated by PGC-1α. Here, we propose that remodeling of the mitochondrial network through fusion and fission, and elimination of damaged/dysfunctional mitochondria through mitophagy are all of importance in exercise-induced adaptation. This dynamic process of replacing old, unhealthy mitochondria with new, healthy mitochondria underscores enhanced quantity and quality of mitochondria in skeletal muscle following exercise training ().

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Endurance exercise has long been recognized to not only enhance physical performance, but also bring about beneficial health outcomes. The most studied phenotypic adaptations in skeletal muscle to chronic exercise are increased vascularization (angiogenesis), fiber type transformation toward oxidative myofibers and increased mitochondrial content/function.

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Dr. Max Testa: Debunking the Myths of Lactic Acid

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Max Testa's Lactic Acid Talk at Signature Cycle's Passoni Night Max Testa is a sports medicine physician whose methodology emphasizes the importance of a fine-tuned human engine to help athletes reach peak performance.

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ATP Synthase: The power plant of the cell - YouTube

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ATP Synthase: The power plant of the cell - YouTube

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Molecular Machines - ATP Synthase: The power plant of the cell

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Maximizing Your Mitochondria with Magnesium - YouTube

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Maximizing Your Mitochondria with Magnesium - YouTube

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Maximizing Your Mitochondria with Magnesium