Metabolic Syndrome and Cardiovascular Disease

By T. Barry Levine and Arlene Bradley Levine

Trends indicate that the metabolic syndrome will become the leading risk factor for heart disease. Now more than ever you need an all-in-one reference that provides the tools and practical advice you need to:

• Identify at-risk patients
• Explain individual contributing factors
• Aid in patient education and motivation
• Direct comprehensive care and
• Choose the most appropriate interventions

Comprehensively revised to reflect leading-edge research and now organized to facilitate easy access to essential information and clinically-relevant guidance, Metabolic Syndrome and Cardiovascular Disease, 2e offers this and more. Not only will you receive a solid understanding of the pathophysiology underlying the metabolic syndrome and cardiovascular disease but also the rationale for today’s most effective treatments.

What’s new?

Filled with timely new content, this updated edition covers:

• New discoveries that have changed our understanding of the pathogenesis and interrelationship of metabolic syndrome, cardiovascular disease (CHD), and type 2 diabetes mellitus (DM)
• The relevance of mitochondria and telomeres
• Sleep and its impact on cardiometabolic health
• The pivotal interplay between insulin and forkhead transcriptionfactors
• Calorie restriction research
• Bariatric surgery experiences and outcomes

In addition, each chapter includes essential information on comorbidities, interventions, and pharmacotherapeutic options – an exclusive feature found only in the second edition!

• Language: English
• Publisher: Wiley
• Release date: Jul 5, 2012
• ISBN: 9781118480076

Book preview

Chapter 1

The Metabolic Syndrome: A Relevant Concept?

The concept of the metabolic syndrome arose from a research perspective. Epidemiologically, the term captures a confluence of clinical risk factors that tend to occur together, raising the question of whether these conditions have a single underlying cause.

Several different definitions of the syndrome have been proposed by various organizations, such as the International Diabetes Federation (IDF), World Health Organization (WHO), European Group for the Study of Insulin Resistance (EGIR), and the U.S. National Cholesterol Education Program (NCEP) Adult Treatment Panel (ATP), with various different constellations of risk factors. Although the detailed definitions differ among these organizations, the metabolic syndrome is generally diagnosed when a person presents with any three of the following findings: a generous waist circumference, ­elevated blood pressure, high triglyceride levels, low high-density lipoprotein (HDL) levels, or elevated fasting blood glucose.

However, beyond minor differences about specific components that make up the various definitions of the syndrome, there are significant disagreements as to the validity of naming this risk factor cluster as a separate condition and using it as a diagnostic tool for treatment. This controversy about the relevance of the metabolic syndrome has pitted diabetologists against cardiologists. In 2005, the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD) issued a statement discouraging the use of the term metabolic syndrome. In contrast, a few weeks later, the American Heart Association (AHA) and the National Heart, Lung, and Blood Institute (NHLBI) released statements encouraging the clinical use of that term. The controversy continues.

The EASD posits that no additional benefit derives from identifying the metabolic syndrome risk factor cluster over measuring and treating the individual risk factors. The EASD claims that there are no data to confirm that the metabolic syndrome is a true syndrome rather than a collection of co-aggregating cardiovascular risk factors; and that the collective association with cardiovascular disease is no more than the sum of its parts, much of the risk being linked to obesity, hypertension, glucose intolerance, and hyperglycemia. In short, diabetologists disagree with Aristotle that the whole is greater than the sum of its parts.

Since the syndrome may apply to 25 to 33% of the population, the organization also objects to applying a disease label to too many people. The 2005 EASD statement concluded: There is much fundamental, clinically important, and critically missing information about the metabolic syndrome to warrant a more serious examination of whether medical science is doing any good by drawing attention to and labeling millions of people with a presumed disease that does not stand on firm ground.

Other criticisms leveled at the concept of the metabolic syndrome are that there is no single therapy for such a syndrome. Rather, each risk factor has to be managed ­separately.

In truth, the metabolic syndrome concept is intellectually not rigorous and pathophysiologically not logical. One has a risk factor for inflammation (overweight) linked to a single manifestation of endothelial dysfunction (hypertension), associated with a manifestation of hepatic insulin resistance (dyslipidemia), coupled to pancreatic beta-cell failure (hyperglycemia). Furthermore, the Framingham Risk Score will perform better as a predictor of heart disease than the metabolic syndrome.

However, although there may be no synergy among the individual components of the metabolic syndrome on the risk of coronary outcomes, the risk of stroke and all-cause mortality associated with the metabolic syndrome appears to be significant, independent of its components. Also, as cardiologists, we find the metabolic syndrome a helpful concept: it is so readily recognized. How often do we not wonder if a person on the street or in the elevator has the metabolic syndrome? How often can we not just tell that an individual entering our office has the ­metabolic syndrome, only to confirm the diagnosis with easy, inexpensive testing? The most common presentation of the metabolic syndrome is in people with visceral fat, who are sedentary, and have poor dietary habits.

The metabolic syndrome is not a disease. It is individuals or people that have the metabolic syndrome, not patients. However, over a lifetime, the metabolic syndrome is itself a powerful predictor for the incidence of chronic disease – not only of vascular disease, for which the Framingham Risk Score would serve well, but also of cardiomyopathy, diabetes mellitus (DM), cancer, renal disease, and dementia, that will turn people into patients. It is alarming that almost 40 million Americans have DM, that more than twice that number have prediabetes, and that by 2050 one-third of Americans will be diabetic. It is potentially devastating that in the U.S. at least half the population is overweight and 40% will have the metabolic syndrome and be at risk for such diseases. It is, therefore, of tremendous value to be able to easily identify people with this cluster of risk factors. It enables us to target this population for more aggressive lifestyle advice, and for therapy, if needed.

The construct of the metabolic syndrome may not be intellectually pleasing, but it is simple, and it works. Those at-risk individuals who are sedentary, eat unhealthily and excessively, and have visceral and/or ectopic fat, also develop mitochondrial dysfunction, telomere attrition, inflammation, endothelial dysfunction, and insulin resistance. Such individuals typically have elevated triglycerides, and rather than carry a laundry list of diagnoses, many cardiologists prefer to follow Pythagoras: Do not say a little in many words but a great deal in a few.

Aside from saying much with little, does it make a difference in clinical practice? We would argue that it does.

Physicians diagnosing only traditional risk factors will likely neglect borderline abnormalities as not relevant or not requiring attention. A slightly generous waist or mildly depressed HDL may not be addressed on a hypertension follow-up visit. Traditional risk factors will fail to capture those at risk in the population.

Physicians treating individual risk factors will prescribe their preferred treatments for each. One might choose a beta-blocker for hypertension; fibrates or ezetimibe might be prescribed for abnormal lipid findings, or a sulfonylurea for hyperglycemia.

In contrast, a physician thinking of the metabolic ­syndrome will focus on all abnormalities, even if they are of borderline concern. He/she will be aware of the common pathophysiology underlying the individual’s presentation: the role that inflammation, oxidative stress, mitochondrial dysfunction, endothelial dysfunction, and insulin resistance all play.

Finding the metabolic syndrome allows the physician to elucidate modifiable factors that contribute to the pathophysiology: Is the person stressed or sleep deprived? Does the individual suffer from some chronic inflam­matory process? In this context, overweight is no longer a cosmetic issue but a significant source of systemic inflammation; inactivity or an unhealthy diet are no longer ­lifestyles but factors that engender endothelial and mitochondrial dysfunction and insulin resistance.

The therapeutic approach chosen will be holistic, addressing the underlying pathophysiology. While tailored to an individual’s need, interventions will be chosen to synergistically impact on all components. The emphasis will be on aggressive therapeutic lifestyle changes: they do have a major impact on all factors underlying the metabolic syndrome, thus improving all individual risk factors.

With therapeutic interventions, the clinician dealing with the metabolic syndrome will identify therapies that make sense physiologically, that lower inflammation and oxidative stress, that improve mitochondrial and ­endothelial function, and that reduce insulin resistance. The aim is to have every drug chosen help the entire ­syndrome: thus a renin–angiotensin–aldosterone system (RAAS) antagonist will be more appropriate than a calcium channel blocker, an HDL-raising statin will be more beneficial than ezetimibe, an AMP-activated protein kinase (AMPK) activator will be more helpful than a ­sulfonylurea. In the presence of the metabolic syndrome, a clinician may consider prescribing aspirin.

Yes, the metabolic syndrome targets a large segment of the population; however, identifying the many affected individuals is a benefit. These individuals are not diseased. They are simply at higher risk of developing DM, heart disease, cancer, and dementia. The metabolic syndrome allows easy diagnosis and targeting of people for aggressive lifestyle advice. It is an early time in the pathophysiological process when lifestyle interventions are still very effective. Diet and exercise continue to be the cor­nerstone of any metabolic syndrome prevention-and-­treatment strategy, and individuals and society at large will benefit from a timely preventive intervention.

Chapter 2

Mitochondria

Mitochondria have traditionally been viewed as cellular organelles for energy production in response to changes in energy demand. However, mitochondria also function as active signaling organelles in a number of important intracellular pathways [1]. As such, mitochondria have a dichotomous role in controlling both life and death processes by playing a critical part in cellular function, stress response, cytoprotection, and apoptosis, as well as in reactive radical biology and calcium (Ca²+) ­homeostasis [2, 3].

Intact mitochondrial function is central to good health and lifespan. Mitochondrial dysfunction and attenuation of cellular bioenergetics underlie a variety of diseases. Impaired mitochondrial function is thus closely associated with insulin resistance and contributes to the progression of diabetes mellitus (DM). Mitochondrial dysfunction plays a pivotal role in heart disease, diseases of the central nervous system, and aging [2, 4].

Background

Derivation

In many respects, mitochondria are akin to prokaryotic cells like bacteria. In fact, mitochondria have a unique evolutionary origin [5]. Whole-genome analyses suggest that mitochondria are descended from formerly free-living bacteria. Atmospheric oxygen appeared approximately 2.3 billion years ago. Prokaryotic cells evolved to harness the energy in oxygen. Mitochondria may be the evolutionary descendants of such oxygen-scavenging prokaryotes that established an endosymbiotic relationship within the cytosol of eukaryotes one-and-a-half to two billion years ago. Over time, mitochondria evolved into primary ­control centers for energy production and cellular life-and-death processes in eukaryotes. In effect, the history of eukaryotic development entails the fusion and coevolution of host and endosymbiont genomes [6–8].

Implications of life with mitochondria

The symbiotic relationship of mitochondria with eukaryotic cells may have been a turning point in the evolution of life, enabling the development of complex organisms [9].

Since mitochondria provide the energy for living, the enhanced mitochondrial supply of energy has permitted organisms to develop from single-celled entities into complex and sophisticated life forms. On the other hand, the requirements of mitochondria have modulated anatomy and physiology. As 98% of inhaled oxygen is consumed by mitochondria, their oxygen requirement has driven the need for the development of oxygen uptake membranes, such as lungs or gills. Mitochondrial fuel needs have also driven the development of gastrointestinal organs. Blood and the circulatory system serve to disseminate oxygen and energy substrates to the mitochondria within the cells of all tissues [2].

Structure

Mitochondria are membrane-enclosed, subcellular organelles distributed throughout the cytosol of most eukaryotic cells. Their shape is quite variable, ranging from small and spherical in adipocytes to oblong in hepatocytes, cylindrical, or thread-like and interconnected, depending on the cell type. They are approximately 0.5 µm wide and from 0.5 to several micrometers long, approximating the size of bacteria [6].

Mitochondria are highly organized structures. Different enzymes and reactions are confined to discrete membranes and aqueous compartments [10]. Specifically, in the mitochondria:

the outer membrane separates the mitochondrion from the cellular cytosol;

the inner membrane, subjacent to the outer membrane, encloses the interior compartment or matrix;

invaginations of the inner membrane, the cristae mitochondrialis, project into the mitochondrial interior, the matrix;

the matrix is of gel-like consistency, containing about 50% protein in a reticular network attached to the inner membrane; it also holds deoxyribonucleic acid (DNA) and ribosomes;

the intermembrane space separates the outer membrane from the inner membrane [6].

Mitochondria contain about 2000 proteins. Many of these are hydrophobic membrane-based proteins. Mitochondrial proteins derive from the synthesis of macromolecules within the mitochondria, together with the import of proteins and lipids synthesized outside the organelle [11].

The outer membrane

Proper cell function is contingent on the integrity of the outer mitochondrial membrane separating cytosolic from mitochondrial factors. The outer mitochondrial membrane is smooth, but it contains a number of proteins that can form channels to facilitate the transmembrane movement of ions and molecules [6]. Passage of metabolites through the outer mitochondrial membrane seems to occur through a voltage-dependent anion-selective channel. This channel exhibits gating between a non­conducting state and various subconductance states controlling the permeability of molecular species via ­differing cutoff sizes [12].

The inner membrane

The inner mitochondrial membrane is often highly folded. Those folds, which project into the mitochondrial matrix, are termed cristae [13]. In healthy cells, the inner mitochondrial membrane is relatively impermeable, which allows it to maintain the proton gradient driving respiration and the osmotic gradient created by the high concentration of metabolites in the mitochondrial matrix [14] (see below). The inner membrane has five insoluble integral membrane protein complexes:

1 Complex I: NADH dehydrogenase or NADH-ubiquinone oxidoreductase.

2 Complex II: succinate dehydrogenase or succinated ubiquinone oxidoreductase.

3 Complex III: cytochrome c reductase, the bc1 complex or ubiquinole-cytochrome c oxidoreductase.

4 Complex IV: cytochrome c oxidase (CcOX) or reduced cytochrome c: oxygen oxidoreductase.

5 Complex V: F0F1–ATPase (adenosine triphosphate [ATP] synthase) or proton-translocating ATP synthase [6, 15].

These complexes are highly multifaceted, intricate ­compounds. For example, Complex I is approximately 900 kDa, with over 45 subunits and more than 12 prosthetic electron transfer groups. The inner membrane phospholipid cardiolipin is associated with the respiratory complexes situated on the inner membrane. It is involved in cytochrome c insertion, retention, and electron transport function [16].

The mitochondrial matrix

The mitochondrial inner compartment, or matrix, ­contains soluble enzymes that oxidize fatty acids and catalyze the respiration of pyruvic acid and other small organic molecules. Here pyruvic acid undergoes both oxidation and decarboxylation. The inner membrane plus matrix are also termed mitoplast [6].

The mitochondrial genome

Mitochondria have their own DNA as well as their own ribosomes and transfer (t) ribonucleic acid (RNA). Although much smaller than the nuclear genome, the mitochondrial genome is equally important and may play a crucial role in aging and carcinogenesis [17].

Mitochondrial DNA (mtDNA) resembles that of a bacterium in its basic structure. The mitochondrial genome size is species specific; however, the genome map is consistent between species. The mtDNA is a circular, double-stranded molecule. It lies within the matrix, configured in punctate structures termed nucleoids. Each nucleoid may contain four to five identical copies of the mtDNA, which is present in thousands of copies per cell [18]. Although the mtDNA has no associated, protective histones, it is covered by the ­histone-like protein mitochondrial transcription factor A (TFAM) [19].

Mitochondrial genes

mtDNA molecules have 16,569 base pairs that encode 37 genes encompassing two mitochondrial rRNA genes, 22 tRNA genes, and 13 critical polypeptide subunits of the inner membrane mitochondrial ETC [20, 21].

Although mitochondria have their own DNA, many of the genes needed for other subunits of these complexes, and for mitochondrial function in general, are actually located within the nuclear genome. Complexes I, III, IV, and V encompass subunits encoded both by nuclear and mtDNA [6, 15]. In fact, more than 98% of the total protein complement of the organelle is encoded by the nuclear genome [48]. Of 82 structural subunits that make up the oxidative phosphorylation system in the mitochondria, nuclear DNA encodes 69. These are synthesized in the cytosol as preproteins containing a mitochondria-import sequence. Preproteins traverse the outer mitochondrial membrane in an unfolded state and translocate through the inner membrane into the matrix via import machinery that includes mitochondrial heat shock protein 70 (mtHSP70] [20, 21].

Changes in mtDNA may represent a molecular clock on a time scale that is relevant for human evolution [13]. mtDNA is more susceptible to damage. It mutates faster than nuclear DNA, and this higher rate of mutation underlies certain congenital and genetic diseases and contributes to age-related dysfunction [9].

Mitochondrial inheritance

The presence of mtDNA allows for the non-Mendelian, cytoplasmic inheritance of genetic information. mtDNA is inherited maternally, since few of the mitochondria in sperm survive in the fertilized egg.

Spermatocytes have only approximately 100 mitochondria in a portion of the tail. In contrast, oocytes have around 100,000 mitochondria. As somatic cells develop, male mtDNA is increasingly diluted out such that only 0.01% of the mtDNA is paternal [6].

Number

Somatic cells contain hundreds of mitochondria. The number of mitochondria per cell varies as a function of the cell type, being higher in metabolically active cells. Rat hepatocytes contain about 800 mitochondria. Osteocytes have less than 400 mitochondria per cell, erythrocytes have none, while ova have 2000–20,000 per cell [13]. Mitochondria take up 20% of cell volume in hepatocytes, over 50% in cardiomyocytes, and 67% in oxidative skeletal muscle such as the soleus. Skeletal myocytes typically carry between 400 and 4000 mitochondria [13, 22].

Location

Mitochondria are nonrandomly distributed in the cytosol as a reticulum, anchored to the actin cytoskeleton. They are highly motile and constantly redistribute via interaction with cytoskeletal motors along cytoskeletal tracks in response to the metabolic needs of the cell [13].

Mitochondria are positioned either near the source of fuel on which they depend, i.e. in proximity to the plasma membrane; or adjacent to energy-requiring structures, such as the contractile myofilaments in skeletal myocytes. In actively dividing cells, mitochondria are close to the nucleus and the ribosomes. In adult cardiomyocytes, mitochondria are arranged longitudinally along the myofibrils at the sar­comeric A-band, providing most of the ATP needed for myocardial contraction and ion homeostasis. Adult cardiomyocytes are often binucleate, and their nuclei are ­surrounded by densely grouped mitochondria [6].

Dynamics

The hundreds of mitochondria within a typical cell undergo continual cycles of fusion and fission. As a result, the identity of any individual mitochondrion is transient [23].

Mitochondrial fusion and fission is regulated by members of the dynamin superfamily of large conserved guanosine triphosphatases (GTPases). Mitofusin, an integral mitochondrial membrane protein, is one such mitochondrial GTPase, which is required on adjacent mitochondria to mediate fusion. Fusion entails the coordinated joining of the outer lipid membranes as well as the inner ones. In the process, mitochondrial contents, including mtDNA within the matrix, merge. Conversely, a fission event causes a single mitochondrion to split into two. The steady-state shape of mitochondria is the result of a careful balance between fusion and fission [23].

Mitochondrial dynamics are physiologically important and are linked to apoptosis and life-span control [24]. Fission followed by selective fusion segregates dysfunctional mitochondria and permits their removal by autophagy [25]. Mitochondrial fusion appears to be important for the maintenance of a healthy, homogeneous mitochondrial population. Fission processes have widespread functions essential to life, as fission precedes apoptosis. Conceivably, mitochondrial fragmentation via fission may facilitate the release of mitochondrial proteins and metabolites that trigger cell death. In yeast, such as Podospora anserina and Saccharomyces cerevisiae, reduced mitochondrial fission extends life span by increasing ­cellular resistance to the induction of apoptosis [24].

Function

More than 50 years ago, mitochondria were identified as the principal intracellular site of oxidative energy metabolism. Mitochondria play a crucial role in intermediary metabolism, energy conversion, and energy homeostasis by metabolizing nutrient fuels and producing bioenergy in the form of ATP and heat in eukaryotic cells [148]. They house the key enzyme systems required for intermediary metabolism. In an average animal cell, more than 95% of ATP is produced within these organelles via oxidative reactions [26].

Mitochondrial energy production is central for most cellular processes, such as the maintenance of ionic gradients within cells, cell movement and division, the synthesis and secretion of messenger molecules, or the contraction of cardiac, skeletal, and smooth muscles. The energy demands of the postnatal mammalian heart are met primarily by ATP produced in mitochondria [27]. Mitochondrial energy sustains the order of life over and above the entropy of death and decay [2].

Mitochondria also serve functions more remotely related to provisioning the energy for life:

In pancreatic beta-cells, mitochondria transduce the secretory pathway for insulin in response to glucose.

Mitochondria may serve as central sensors of oxygen in a number of tissues, such as the vasculature and the myocardium. For example, mitochondria may mediate the assessment of oxygen tension in the carotid body and the pulmonary vasculature [28].

In addition, some mitochondrial functions extend far beyond even indirect energy provision. Mitochondria also play a key role in cell signaling. They perform numerous biosynthetic and degradative reactions that are fundamental to a variety of cellular functions:

Mitochondria contain the rate-limiting enzymes in ­steroid biosynthesis, the synthesis of heme, and the carbonic anhydrase required for gastric acid secretion.

There are mitochondrially localized steps in the synthetic pathways for purines and Fe-S clusters [26].

In certain cell types, by accumulating Ca²+ when cytosolic Ca²+ levels are high, mitochondria play a role in Ca²+ homeostasis and in the coordination of complex intracellular Ca²+-signaling pathways.

In young mammals, mitochondria contribute to nonshivering thermogenesis via the physiological uncoupling of mitochondria [2].

Anchored by the actin cytoskeleton, mitochondria may function as mechanotransducers in endothelial cells [29].

The production of free radical species by mitochondria may act as a signal transduction mechanism in the regulation of ion-channel activities and in the initiation of cytoprotective mechanisms in stressed cells [2].

Mitochondria are key regulators of cell viability and are crucial to triggering and mediating the cytoprotective responses to stressors [16].

Mitochondria provide antioxidant responses to prooxidant stresses [16].

Mitochondria regulate pivotal early events in apoptosis [16] and play a role in necrotic cell death [30].

Mitochondria are required for the regulation of cell cycle control, development, the sculpting of embryonic shape, sexual differentiation, menopause, antiviral responses, and aging [9, 31].

Mitochondrial-cell communications

Mitochondria do not operate as independent cellular organelles. They serve as a central platform in the execution of diverse cellular events and have an important role in cell signaling. For example, mitochondrial capacity adjusts exquisitely to cellular metabolic demand. Alternatively, changes in the functional state of mitochondria, due to physiologic and pathologic stimuli, alter cellular responses, for example, reconfiguring metabolism or cell survival programs [32].

To effect such changes, nuclei and mitochondria have to communicate in order to coordinate nuclear and mitochondrial genome replication, gene expression, and cell signaling. There is also bidirectional communication between the mitochondrial reticulum and the rest of the cell. Proteins, such as GTPases, kinases, and phosphatases, link and regulate mitochondrial and cellular functions and dynamics [5]. When mitochondria communicate directly with the nucleus to orchestrate changes in nuclear gene expression, the process is termed retrograde signaling [3].

Targets of retrograde signaling

There are elaborate intercommunications between mitochondria and the nucleus under normal and pathophysiological conditions, coordinating not only mitochondrial gene expression and genome maintenance but also nuclear gene expression [1]. The retrograde response responds in a continuous manner to the changing metabolic needs of the cell, affecting a variety of cellular states and processes such as:

carbohydrate and nitrogen metabolism

altered cytoplasmic Ca²+ levels

production of reactive oxygen species (ROS)

altered stress kinase pathway activation

altered nuclear gene transcription

regulation of cell cycle progression and proliferation [33]

cell growth, morphogenesis, development, and environmental adaptation [34]

aging, life-span regulation, tumorigenesis, and disease [11, 32, 33].

Triggers of retrograde regulation

Retrograde signaling from the mitochondria to the nucleus and cytosol adjusts the transcription of target genes in response to the respiratory state of the cell [35]. It appears to occur partially via ROS and Ca²+ signaling [1, 6]. Signaling events can be modulated by alterations in the mitochondrial membrane potential [3] or by insufficiency in the ETC with mitochondrial dysfunction [34, 37].

Pathways of retrograde regulation

A number of pathways mediate the retrograde response.

The RTG pathway initiates readjustments of carbo­hydrate and nitrogen metabolism through nuclear accumulation of the heterodimeric transcription factor complex composed of the bHLH/Zip proteins Rtg1p and Rtg3p [38].

In both yeast and animal cells, retrograde signaling is linked to the energy-sensing Target of Rapamycin (TOR) signaling [35, 39].

In both plants and animals, there is a retrograde ­mitochondria-to-nucleus-dependent expression of a heat shock protein (hsp) gene network that may facilitate cell defense and survival [37].

Mitochondrial genetic and metabolic stress and mitochondrial dysfunction alter Ca²+ dynamics and cause Ca²+-dependent activation of calcineurin, nuclear factor of activated T-cells (NFAT), activating transcription factor (ATF)2, and nuclear factor (NF) kappaB/Rel factors, which collectively alter the expression of an array of nuclear genes [36, 39].

Cellular respiration

The high-energy phosphate bonds of ATP constitute the chemical energy currency that drives all energy-consuming cellular functions. Aerobic organisms consume oxygen in order to extract chemical energy from nutrient fuel. Respiration is the process whereby food molecules are oxidized to generate energy, with carbon dioxide (CO2) and water (H2O) as byproducts, in a process diametrically opposed to ­photosynthesis. In eukaryotic cells, mitochondria link oxidative respiration with the metabolism of nutrients, using >90% of total body oxygen consumption, to generate ATP.

Oxidative phosphorylation entails a process wherein the mitochondrion rapidly generates chemical energy currency through the complete oxidation of fatty acids, glucose, and lactate. Specifically, the mitochondrion creates a proton motive force across the mitochondrial inner membrane akin to a capacitor. This capacitor traps and stores the chemical energy that is released during cellular respiration. This potential energy is then used to generate the high-energy phosphate bonds of ATP from adenosine 5′-diphosphate (ADP) and inorganic phosphate [15].

Cellular respiration takes place in three stages:

1 Glycolysis

2 The tricarboxylic acid (TCA) cycle

3 The ETC.

Metabolic phenotype

The metabolic phenotype of mitochondria is cell and tissue specific. The contribution of specific mitochondrial metabolic pathways and nutrient substrates for the generation of ATP via oxidative phosphorylation varies among tissue types. Mitochondria in

muscle and heart have a high capacity for ATP production via oxidation of fatty acids;

brain produce ATP via glucose oxidation [27].

Accordingly, the expression of enzymes for fatty acid oxidation is markedly higher in heart mitochondria than in the brain. In the heart, fatty acids are the chief mitochondrial energy substrate. They provide the greatest yield of ATP per mole relative to other substrates, such as glucose or lactate (analogous to the higher energy yield from gasoline versus ethanol) [27].

Consistent with the tissue-specific mitochondrial energy-substrate preferences, the expression of metabolic enzymes differs markedly between myocyte and neuronal mitochondria [27].

Similarly, mitochondria may engage in:

tissue-specific coupled respiration, which efficiently converts the chemical energy of food molecules into ATP, as in muscle, brain, and heart; and

tissue-specific uncoupled respiration, which allows for the release of chemical energy as heat, as in brown fat [27].

As an aside, not only do mitochondrial structure and physiology vary between different organs and tissues; mtDNA repair ability appears to differ between tissues, even between pulmonary arterial, venous, and microvascular endothelial cells [40].

Respiration of glucose

This is the first stage of cellular respiration of a nutrient molecule, such as glucose. It occurs in two phases, within the cytosol and the mitochondria:

1 Cytosol: Anaerobic glycolysis entails the breakdown of glucose into two pyruvic acid molecules. Pyruvic acid, a three-carbon intermediate, can then be converted to lactate outside the mitochondrion. This process yields two ATP for each glucose molecule and two molecules of nicotinamide adenine dinucleotide (NADH).

2 Mitochondria: Pyruvic acid enters mitochondria via a special transport system located on the inner aspect of the mitochondrial membrane. Upon entry into the mitochondrial matrix, pyruvic acid is oxidized via NAD+, in the process producing NADH + H+. The subsequent decarboxylation of oxidized pyruvic acid yields acetyl coenzyme A (CoA), a two-carbon molecule that can enter the TCA cycle for further oxidation. The oxidation of pyruvic acid also produces CO2 and H2O [6].

Respiration of fatty acids

Fatty acids are catabolized in mitochondria via the fatty acid beta-oxidation pathway generating

acetyl CoA for further oxidation in the mitochondrial TCA cycle; as well as

reducing equivalents for the mitochondrial ETC.

Fatty acids require a transport mechanism to cross the mitochondrial membrane from the cytoplasm. In the presence of carnitine, carnitine palmitoyl-transferase 1 (CPT-1) condenses fatty acyl groups from the acyl CoA with carnitine, forming acyl carnitine. Acyl carnitine is then able to enter the mitochondria. Within the mitochondrial matrix, acyl carnitine is converted back to fatty acyl CoA for beta-oxidation. Fatty acid beta-oxidation produces acetyl CoA as well as reducing equivalents (NADH and FADH2). Acetyl CoA then enters the TCA cycle [41].

The tricarboxylic acid cycle

The TCA cycle is the second stage of cellular respiration. The enzymes for this stage are soluble within the mitochondrial matrix. Alternative names for the tricarboxylic acid cycle are Krebs or citric acid cycle. Citric acid is formed as acetyl CoA is donated to oxaloacetic acid. Citric acid undergoes a number of enzymatic steps, which regenerate oxaloacetic acid at the completion of the cycle, allowing oxaloacetic acid to be repeatedly recycled for the processing of further molecules of acetyl CoA. The interim net outcome of pyruvic acid oxidation yields:

three CO2 molecules derived from the three carbon atoms of pyruvic acid

one ATP molecule from the conversion of alpha-­ketoglutaric acid to succinic acid

four pairs of electrons removed and transferred to NAD+, reducing the latter to NADH + H+

one pair of electrons, derived from succinic acid, reducing FAD to FADH2.

In effect, as substrates for mitochondrial oxidation are processed through the TCA cycle, they shift the NADH/NAD+ and FADH2/FAD couples to a reduced state. NADH and FADH2 then transfer their electrons to the ETC [2, 6].

The electron transport chain

The ETC is the third stage of cellular respiration. It produces ATP through the process of oxidative phosphorylation. The respiratory chain or ETC is a sequence of the Complexes I–IV located in the mitochondrial inner membrane and arrayed in a supramolecular organization [4].

Complex IV, the hemoprotein CcOX, is the terminal electron acceptor of the mitochondrial ETC. It is the primary site of cellular oxygen consumption and, as such, is central to oxidative phosphorylation and the generation of ATP. There are also two diffusible electron-transfer molecules that shuttle electrons from one complex to the next:

1 ubiquinone or coenzyme Q 10

2 cytochrome c [6].

The production of ATP via oxidative phosphorylation comprises two linked major steps:

1 the oxidation of the reduced NADH and/or FADH2 generated during the glycolysis, beta-oxidation, and TCA cycle stages, and

2 the phosphorylation of ADP to form high-energy ATP [22].

Oxidation

Typically, oxidation, or burning, is an energy-releasing process. This step extracts the energy derived from nutrient fuel. Instead of just dissipating the energy as heat, as would occur in a fire, the energy is effectively stored for later use.

As the reduced NADH and FADH2 are oxidized to NAD+ and FAD, they supply electrons to the ETC. The constituent complexes provide a range of redox potentials from −280 mV for Complex I to +250 mV for Complex IV. As electrons enter Complex I and flow through the chain, they follow the redox hierarchy of the ETC complexes [42]. In stepwise fashion, electrons are transferred to ­flavoproteins, non-heme iron-sulfur centers, cytochromes, and finally to O2, in the process generating H2O via the addition of protons [6, 15].

These protons are also generated during the oxidation of NADH and FADH2. The protons are pumped into the intermembrane space through Complexes I, III, and IV. The pumped protons create an electrochemical gradient across the inner membrane, storing the chemical energy that has been released [22] (see below).

Phosphorylation

Since the phosphate bond of ATP is high-energy, this step inputs the energy previously extracted and stored from the oxidation (or burning) of nutrient fuel for the synthesis of high-energy phosphate bonds.

Oxidative phosphorylation generates approximately 90% of the ATP necessary for cellular function [43]. The phosphorylation step is carried out by the inner ­membrane Complex V, ATP synthase, which phosphorylates ADP to ATP [44]. The reaction of ATP synthase is the final step in oxidative phosphorylation. The energy driving this reaction derives from the mitochondrial ­electrochemical gradient. In general, each glucose molecule generates 36–38 molecules of ATP [6].

ATP is then transported into the cytosol via the adenine nucleotide translocase or translocator (ANT), also termed the ATP/ADP carrier, for use in cellular energy-requiring enzymatic reactions and specialized processes [2].

ATP synthase can respond rapidly to changes in cellular energy demand. Residues in ATP synthase can ­modulate enzymatic activity through regulation of the intramolecular rotation of the enzyme with the electrochemical potential, and through actual inhibitors of ATP synthase [45].

The mitochondrial membrane potential

The mitochondrial membrane potential stores the energy released from the burning of food calories for use in the ­synthesis of ATP.

Mitochondria capture the chemical energy released from the oxidation of substrates by generating a transmembrane proton gradient. The chemical energy released in the ETC process is harnessed by the three Complexes I, III, and IV of the chain. These inner membrane complexes, in particular Complex III, pump protons against the concentration ­gradient from the mitochondrial matrix across the inner membrane into the mitochondrial intermembrane space [46]. For four electrons reducing oxygen to water, 20 ­protons are pumped into the intermembrane space [6].

These transferred protons generate a large electro-chemical gradient across the mitochondrial inner membrane, consisting of

1 a chemical proton gradient (pH), which also expresses itself as

2 a transmembrane electrical potential [44].

The electrical potential across the inner mitochondrial membrane renders the mitochondrial matrix significantly more electronegative, negative inside by about –180 to 200 mV, when compared to the proton-containing mitochondrial intermembrane space. This mitochondrial membrane potential is the stored energy, akin to the creation of a capacitor or chemical battery, which drives the synthesis of ATP [2]. As the concentration of protons rises in the mitochondrial intermembrane space, it increases the electrochemical and diffusion gradient across the inner membrane.

The inner mitochondrial membrane potential reflects the composite measure of mitochondrial function. Maintenance of this electrochemical potential requires

1 the appropriate functioning of transport mechanisms linking the cytosol and the mitochondrial matrix;

2 the catalytic integrity of enzymes of beta-oxidation and the TCA cycle;

3 the functional integrity of electron transfer redox centers of oxidative phosphorylation [3] ;

4 an inner membrane that is relatively impermeant to ions [44].

The mitochondrial inner membrane potential is not static; rather, the modulation of this electrochemical gradient directly controls mitochondrial ATP generation, Ca²+ flux, and the production and control of ROS.

A modest modulation of the mitochondrial membrane potential appears to confer cytoprotective adaptations that enhance tolerance to ischemic and redox stress [3].

A serious reduction in mitochondrial potential is a common result of ETC inhibition [37].

There may be flickering of the mitochondrial membrane potential, particularly in response to stressful conditions [47].

Energy is released from the mitochondrial membrane potential when protons flow down the concentration gradient back into the mitochondrial matrix [2]. The principal way protons can reenter the matrix is through facilitated diffusion via the inner membrane Complex V, the ATP synthase complex. ATP synthase harnesses the energy of the electrochemical potential by phosphorylating ADP to ATP through the synthesis of the high-energy phosphate bond in ATP in a process termed chemiosmosis [6].

Modulation of mitochondrial metabolic activity

In health, the mitochondrial energy production and supply very closely match cellular energy demand. This energy demand/supply balance requires exquisite dynamic regulation of mitochondrial metabolic pathways. It occurs in complex ways and at many levels.

The oxidative capacity of mitochondria is determined by

1 the activity of each mitochondrion, i.e. the expression level of oxidative phosphorylation subunits, and by

2 the number and size of mitochondria [22].

The mitochondrial content in cells is variable and is adjusted to suit physiologically changing circumstances [26]. Mitochondrial functional capacity and number are dynamically regulated in accordance with cellular energy demands during developmental stages and in response to diverse physiologic conditions.

Factors that can modulate mitochondrial oxidative flux are:

several agonists and signaling pathways, such as exposure to thyroid hormone

environmental conditions, such as postnatal development, exercise training, hypoxia, or temperature

concentrations of substrates and metabolic intermediates, such as fatty acids

posttranslational modification of enzymes catalyzing key, rate-limiting reactions

gene transcription [10, 41, 48].

Particularly in the heart, the high-capacity mitochondrial system needs to match ATP production with functional demands by adjusting both cardiac mitochondrial number and activity.

Mitochondrial biogenesis

Mitochondria cannot be created de novo. They can only replicate from preexisting mitochondria. To that end, they recruit new proteins, which are added to preexisting subcompartments [48]. When mitochondria have sufficiently enlarged, mtDNA is replicated and they undergo fission like bacteria [6]. This process is termed mitochondrial biogenesis.

The biogenesis of mitochondria requires the expression of about 1000 genes, 95% of which are encoded by nuclear chromosomes, the remainder by mitochondrial genes [26]. Regulatory pathways transduce the changes in cellular energy requirements to the coordinate transcriptional control of nuclear and mitochondrial genes encoding mitochondrial proteins involved in electron transport and oxidative phosphorylation [27, 48].

Mitochondrial removal

Mitochondrial numbers are controlled by lysosomal autophagy. In the process, the membranes of the endoplasmic reticulum wrap around the mitochondrion. Vesicles from the Golgi complex containing hydrolases join with the autophagic vacuole. The lysosome forms as the pH drops and the content is degraded [6].

Mitochondrial uncoupling

In the macroscopic world, the burning or oxidation of fuel in order to energize electrical or mechanical processes is inefficient as some energy is always lost as heat. The same is true for mitochondria.

Although the mitochondrial inner membrane proton gradient and electrochemical potential are used to store energy for the synthesis of ATP, the membrane is not impermeable. Some protons leak from the intermembrane space back into the matrix. In effect, they uncouple the term oxidative from phosphorylation and short-­circuit the mitochondrial motive force. As a result, oxidative phosphorylation is less energy efficient. Less energy is funneled into ATP synthesis. This short-circuiting is not trivial. Up to 20% of the basal metabolic rate may be used to cover this basal proton leak [46].

The proton leak engendered by mitochondrial uncoupling dissipates the mitochondrial gradient as body heat rather than allowing it to be captured in ATP synthesis. This apparent coupling inefficiency has been adapted physiologically. Its degree is variable. It can be modulated in order to help regulate energy metabolism. Uncoupling has implications for thermoregulation and weight loss. It affects the generation of ROS by mitochondria. Uncoupled mitochondria reduce cell viability and can activate apoptosis [49].

Uncoupling proteins

The proteins that reduce the proton gradient and uncouple ATP synthesis are, not surprisingly, called "uncoupling proteins" (UCPs). UCPs belong to a large family of mitochondrial inner membrane anion carrier proteins that facilitate the exchange of substrates across the mitochondrial inner membrane. Members of this family play essential roles in the trafficking of intermediary metabolites into and out of the mitochondrial matrix [50].

UCPs uncouple mitochondrial respiration from ATP production by leaking protons into the mitochondrial matrix, thus dissipating the mitochondrial energy potential as heat [51]. In brown adipose tissue enriched in mitochondria, UCP gene expression is highly cold-­inducible through the activation of the sympathetic ­nervous system via beta-adrenoreceptor signaling and 3′-5′-cyclic ade­nosine monophosphate (cAMP) [51].

UCP1, or thermogenin, is the UCP homologue that is abundantly expressed in brown adipose tissue, where it plays a thermo- and metaboregulatory role. UCP1 mediates cold exposure-induced and diet-induced nonshivering thermogenesis [46, 50].

UCP2 and UCP3 typically do not regulate adaptive thermogenesis. They are stringently regulated and may serve to lower mitochondrial coupling efficiency [46]. As such, they appear to have an important role in regulating the production of ROS. Mitochondrial ROS production is exquisitely sensitive to membrane potential (see below). A high mitochondrial electrochemical gradient accelerates cellular ROS production. Even mild UCP-mediated uncoupling of oxidative phosphorylation, by increasing the proton leak across the mitochondrial inner membrane, effectively attenuates mitochondrial free radical pro­duction and ROS-induced damage [50]. It inhibits inflammation, protects against cellular damage, and inhibits cell death [46, 50, 52].

UCP2 is distributed in the pancreas, the immune system, white adipose tissue, and the brain. It has a role in lipid metabolism, mitochondrial bioenergetics, oxidative stress, apoptosis, insulin secretion, and even carcinogenesis [50,53]. By reducing oxidative stress, UCP2 may be both vasculo- and neuroprotective [54].

UCP3 is predominantly expressed in human skeletal muscle, a significant site of whole-body energy expenditure in lean individuals. It is also found in brown adipose tissue, cardiac muscle, and in certain areas of the brain [50]. It may transport fatty acids out of mitochondria, thereby protecting the mitochondria from fatty acid anions or peroxides [55]. UCP3 has a potential role in human metabolism and may be an important therapeutic target in type 2 DM [50].

Factors that affect mitochondrial number and activity

In general, mitochondrial oxidative metabolism is closely matched with mitochondrial content. Numerous regulatory factors and signals orchestrate this linkage.

Nuclear transcriptional regulators of mitochondrial function

A variety of the nuclear regulatory mediators control the transcription of nuclear and mitochondrial genes for key proteins that handle mitochondrial maintenance and proliferation. Some examples are:

1 DNA-binding transcription factors. These bind to specific DNA elements in the promoter region of genes in order to regulate the transcriptional activity and expression of a gene:

a ubiquitous transcription factors:

the zinc finger proteins Sp1, YY1

cAMP-responsive element binding protein (CREB), also a target for calmodulin-dependent kinase (CAMK)

myocyte-specific enhancer factors (MEF)-2/E-box [26]

peroxisome proliferator-activated receptor (PPAR)- alpha [56]

AMP-activated protein kinase (AMPK) [56].

b nuclear factors:

nuclear respiratory factor (NRF)-1 and -2

TFAM

MT1–4

REBOX/OXBOX [26].

2 nuclear transcriptional coactivators. These do not bind to DNA directly; instead, they work through direct protein-protein interactions with other transcription factors [58]:

PPAR-gamma coactivator (PGC)-1 (see below)

PGC-1–related coactivator (PRC), which shares structural and functional similarities with PGC-1 [26, 58].

Other factors

Calcium

Since several key mitochondrial enzymes are regulated by Ca²+, mitochondrial metabolism is responsive to physiological changes in mitochondrial Ca²+ concentration. An increase in mitochondrial Ca²+ concentration increases mitochondrial ATP production in cells [28] (see below).

Nitric oxide

Nitric oxide (NO) is a pivotal regulator of mitochondrial metabolism. It may modulate mitochondrial content and body energy balance in response to physiological stimuli, such as exercise or cold exposure, and may function as a unifying molecular switch to trigger the entire mitochondriogenic process [59] (see below).

Malonyl CoA

Malonyl CoA is a critical metabolic effector of fatty acid oxidation and obesity [60]. Malonyl CoA is a substrate for fatty acid biosynthesis. It is also a potent inhibitor of mitochondrial carnitine palmitoyltransferase (CPT) 1, an essential enzyme involved in mitochondrial fatty acid uptake. Accordingly, a reduction in cellular malonyl CoA levels and an increase in CPT1 activity contribute to an increase in mitochondrial fatty acid oxidation. Levels of malonyl CoA may be reduced via

an increase in malonyl-CoA degradation due to increased malonyl-CoA decarboxylase (MCD) activity;

the inhibition of acetyl CoA carboxylase (ACC) synthesis of malonyl CoA, due to AMPK phosphorylation of ACC [60].

Cell positioning

Mitochondrial respiration and metabolism may be spatially and temporally regulated by the architecture and positioning of the organelle [5]. For example, in cardiomyocytes, the spatial organization of mitochondria favors their interaction with the sarcoplasmic reticulum, thereby facilitating Ca²+-mediated crosstalk between these two organelles [61].

Hormones

Glucocorticoid and thyroid hormones affect energy metabolism, affecting sugar and fatty acid metabolism as well as oxidative phosphorylation in mitochondria. Upon binding to nuclear receptors, the ensuing complex activates or represses gene transcription by interacting with transcription factors, coactivators, or the transcription ­initiation complex. These hormones have rapid- and slow onset effects, respectively enhancing mitochondrial respiration as well as mitochondrial biogenesis [26].

Insulin

Insulin is an important regulator of mitochondrial ATP synthesis in skeletal muscle of healthy subjects [62]. In skeletal muscle, insulin infusion stimulates CcOX and citrate synthase enzyme activities [63].

Peroxisome proliferator–activated receptor gamma coactivator-1

As its name implies, PGC-1 is a coactivator. Coactivators have the ability to integrate the action of multiple transcription factors in order to orchestrate programs of gene expression essential to cellular energetics [58]. As coactivator, PGC-1 serves as an adaptor or scaffold to recruit other coactivator proteins in order to remodel chromatin. Chromatin remodeling by coactivator complexes enhances the probability that a gene will be transcribed by the RNA polymerase II complex [41].

PGC-1alpha and beta are inducible master regulators of mitochondrial biogenesis and oxidative phosphorylation gene expression in response to energy demands. They play a pivotal role coordinating nuclear and mitochondrial signals [27]. PGC-1 may also be a component of the regulatory communication between mitochondrial biogenesis and metabolic activity, linking the actions of NRF-1 and other regulators of mitochondrial biogenesis to the control of specific mitochondrial pathways, such as fatty acid oxidation and the TCA cycle [27].

PGC-1 is highly expressed in tissues with high energy demand and high-capacity mitochondrial content, such as:

the myocardium

the brain

the kidneys

slow-twitch skeletal muscle, and

brown adipose tissue [26].

PGC-1 is rapidly induced by physiological conditions that increase the demand for mitochondrial ATP production, particularly when mitochondrial reliance on fatty acids as a fuel is increased, such as:

during postnatal maturation

in response to short-term fasting

with cold exposure

with endurance exercise

by beta-agonists

by thyroid hormone [27].

PGC-1alpha is a strong coactivator of several nuclear receptors and key transcription factors, such as:

PPAR-gamma

PPAR-alpha

the retinoic acid receptor

the estrogen-related receptor

the mineralocorticoid receptor

the thyroid hormone receptor

NRF-1 and NRF-2.

The mRNA level of PGC-1 is positively associated with those of NRF-1 and TFAM [27, 51, 64]. PGC-1 is also a target of CREB and may mediate cAMP-related signaling [26].

PGC-1 can induce either uncoupled or coupled respiration, depending on the cell type, and it appears to play a role in determining the metabolic phenotype of mitochondria among specialized cell types. For example, PGC-1 is a ­regulator of mitochondrial function in thermogenic brown adipose tissue, where it induces predominantly uncoupled mitochondrial respiration by inducing UCP1 expression [27]. Conceivably, the availability of PGC-1 coactivation partners in any given tissue, such as NRF-1, PPAR-alpha, PPAR-gamma, and other transcription factors, dictates the level of enzyme and protein expression in specific mitochondrial pathways [27].

PGC-1 serves as a master regulator of mitochondrial oxidative metabolism that coordinates the capacity of each step required for ATP synthesis [41]. In cultured cardiac ­myocytes, the forced expression of PGC-1 induces the coordinate expression of nuclear and mitochondrial genes involved in multiple mitochondrial energy-transduction/energy-production pathways in order to increase the capacity for

mitochondrial fatty acid oxidation

oxidative phosphorylation [27].

PGC-1 functions as a master regulator of mitochondrial content and drives mitochondrial biogenesis. In the process, PGC-1 coactivates nuclear-encoded transcription factors, like NRF-1. NRF effects are largely mediated secondarily through the induction of the mitochondrial transcription factor TFAM. NRF-responsive regulatory elements are present in the promoter region of the gene encoding TFAM [27]. TFAM, a nuclear-encoded protein, stimulates and is necessary for mtDNA replication, transcription, and mitochondrial biogenesis. It is the signal through which the nucleus regulates mitochondrial genome transcription and biogenesis, as well as the control of specific mitochondrial pathways, such as the fatty acid oxidation and the TCA [26, 27, 41, 51, 64, 65].

AMPK and CAMK may be cytosolic messengers that initiate adaptive mitochondrial biogenesis. AMPK and CAMK appear to rapidly activate PGC-1, which in turn orchestrates the well-organized expression of the multitude of proteins involved in these adaptations [56].

Mitochondrial production of prooxidant species

Reactive oxygen species have a Janus-like relationship to cell physiology:

1 on the one hand, high levels of ROS mediate cell damage and death;

2 on the other hand, ROS are crucial for cell signaling and cell protection [66].

Mitochondria are the main producers of free radical species in all cell types except leukocytes. They are also major targets for oxidative damage. Mitochondria play a critical role in disease and aging not only by virtue of being the major source but also by being the most proximal casualty of ROS [67].

Mitochondrial production of prooxidants

In aerobic cells, the mitochondrial ETC is the major source of ROS. ROS are the natural byproducts of normal cell respiration. Low cellular levels of superoxide, the hydroxyl radical, and hydrogen peroxide are continually being produced from the ETC [68]. At atmospheric oxygen concentrations, between 1 and 3% of the O2 reduced in the mitochondrial ETC during ATP production may form superoxide [1].

Respiration as source of oxidant stress

The generation of unpaired electrons is a natural consequence of mitochondrial respiration. Electrons ­inevitably leak from several ETC sites. Each one-electron transfer to molecular oxygen, O2, reduces O2 to the very highly reactive superoxide (O2·–) ion. Mitochondrial superoxide is produced on the matrix side of the ­organelle by the reduction of O2 at ETC Complexes I and III and at some components of the TCA cycle, such as alpha-­ketoglutarate dehydrogenase [54].

Complex I is a primary site for mitochondrial ROS generation [16]. The source of superoxide in Complex I may be the iron sulfur center N2 [4]. On the other hand, mitochondrial NO synthase (mtNOS) may also have a role in the generation of superoxide by Complex I [69]. Mitochondrial generation of prooxidant species also derives from an ­electron leak to oxygen at the ubiquinone site interface ­between Complexes II and III [1]. Approximately 1–5% of all electrons are transferred in this way [68].

Other ROS

Other radical species, such as hydroxyl ions (OH−) and hydrogen peroxide (H2O2] may also be present in mitochondria at considerable concentrations. Superoxide is dismutated to hydrogen peroxide either spontaneously or via superoxide dismutase. Hydrogen peroxide, which appears to be capable of crossing membranes, is the main vehicle through which mitochondrially generated ROS may escape from mitochondria to the cytosol and beyond [2]. Superoxide may react with NO to form peroxynitrite, a very potent oxidizing agent. The consequences of this reaction are complex inasmuch as it simultaneously causes the loss of molecules that may be protective (NO, low dose ROS) [28].

Increased mitochondrial ROS production

Mitochondrial ROS production rises as a result of

an increased mitochondrial electrochemical potential, as from excessive food intake

inflammation

mitochondrial dysfunction.

High mitochondrial potential

ROS production increases when excess electrons are provided to the mitochondrial ETC. Mitochondrial ­membrane potential is the principal parameter regulating the generation of mitochondrial ROS. As a general rule, the ­electron leak rises with an increase in mitochondrial potential and diminishes with mitochondrial depolarization [70]. Thus the mild depolarization of mitochondria by UCPs may serve as a protective mechanism to limit mitochondrial ROS generation in some instances. On the other hand, the highest rate of ROS production occurs when the proton gradient is high but oxygen consumption (ATP demand) is low. Typically, excessive nutrient input combined with inactivity engender a high proton-motive force, low ATP demand, and high ROS elaboration. In such circumstances, most ­electron ­carriers are occupied by electrons, and excess electrons are transferred to oxygen without ATP production. When exercise increases ATP demand, electron transfers are coupled to ATP production, reducing ROS production [22].

Inflammatory activation

During inflammatory processes, ROS may originate from many cell types and from various sources other than mitochondria. Activated neutrophils attracted to regions of tissue injury can generate huge quantities of ROS. There is crosstalk between some proinflammatory pathways and mitochondrial ROS production [28].

Tumor necrosis factor (TNF)-alpha binding to membrane receptors triggers complex signal transduction ­cascades, some of which also result in the excessive mitochondrial production of ROS. The mitochondrial ETC is the major source of TNF-alpha-induced ROS. ROS play a crucial role in TNF-alpha-mediated cytotoxicity [1].

Inflammatory activation of the nicotinamide adenine dinucleotide phosphate (NADH/NADPH) oxidases leads to crosstalk with the mitochondrion and increased ROS formation from the organelle [54].

Lipid oxidation products are generated both ­nonspecifically and through the cyclooxygenase and lipoxygenase pathways. These compounds interact with mitochondria and induce the formation of ROS. The mechanism whereby these oxidized lipids associate with mitochondria is unknown; however, the process is ­saturable and associated with increased stimulation of mitochondrial ROS formation [54].

Thus oxidized low-density lipoprotein (LDL) induces ­mitochondrial ROS formation. At low levels, these may be cytoprotective [54]. However, higher concentrations of oxidized LDL cause endothelial mitochondria to further increase ROS production with toxic effects [71].

Dysfunctional mitochondria

When mitochondrial respiratory proteins are damaged, uncontrolled ROS formation occurs pathologically [54]. In many types of cardiovascular diseases, ROS overwhelm antioxidant defenses and become damaging, engendering oxidative stress [28] (see below).

Regulation of mitochondrial ROS formation

Mitochondrial depolarizations, even small ones, trigger ROS release. In the vasculature, mitochondrial generation of ROS can occur in response to several stimuli, including flow, temperature, and carbon monoxide [72–74].

Mitochondrial ROS formation is regulated through ­several mechanisms:

The posttranslational modification of mitochondrial proteins by thiolation or by electrophilic lipids functions as a potential positive regulator of mitochondrial ROS formation.

Uncoupling proteins are potential negative regulators.

Mitochondrial NO formation may be linked with ­control of mitochondrial ROS production [54].

There is crosstalk between the thiol status and the ­controlled formation of ROS. The mitochondrial glutaredoxin 2 pathway modulates the redox couples that ­control the S-glutathionylation of proteins in the ETC. S-glutathionylation of the 70-kDa subunit of Complex I enhances superoxide formation [54].

Mitochondrial redox signaling pathways

ROS are not solely toxic byproducts of cellular metabolism. In the normal cell, low physiological levels of ROS ­generated from mitochondria play a critical part in diverse signaling ­pathways [72]. Some of the effects are mediated via posttranslational modification of redox-sensitive ­proteins [75].

For example, physiological levels of mitochondrial-derived ROS may

participate in mitochondria-to-nucleus signaling, ­acting as the second messenger in the signal transduction pathway [1]

modulate intracellular calcium [76]

serve as a metabolic sensor, linking mitochondrial ­respiration with signal changes in vascular function and growth, and establishing mitochondria as regulators of tissue perfusion [74, 77]

participate in the activation of endothelial NOS (eNOS) and NO generation by engendering Ca²+ sparks from sub-plasmalemmal endoplasmic reticulum in close proximity to caveolae-based eNOS [77, 78]

function as the small-vessel endothelium-derived hyperpolarizing factor [73, 77]

stimulate flow-mediated vasodilation [73]

stimulate cell growth, differentiation, and migration [68, 77, 79]

induce growth factor receptor transactivation, including receptors for vascular endothelial growth factor (VEGF)-2, platelet-derived growth factor (PDGF), angiotensin II, epidermal growth factor (EGF), transforming growth factor (TGF)-beta, and TNF-alpha [54, 77, 80]

activate protein kinases, such as Akt, extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK) [79]

cause the redox activation of c-Jun N-terminal kinase (JNK), which inhibits mitochondrial metabolic enzymes [81]

mediate cytoprotection mechanisms [1, 54], counteract apoptotic stimuli [68], and integrate cell death and survival signaling pathways.

Mechanism of ROS signal transduction

There are at least four possibilities for superoxide signal transduction [54]:

1 Superoxide itself is detected by iron–sulfur proteins such as aconitase. This receptor for superoxide may then release iron into the mitochondrion, which may ­promote lipid peroxidation and the consequent formation of electrophilic lipids capable of modifying protein thiols.

2 Superoxide is converted to hydrogen peroxide by the action of superoxide dismutases, which are present in both the mitochondrial matrix and intermembrane space. Hydrogen peroxide can readily cross membranes and regulate cytosolic redox-sensitive signaling ­pathways.

3 Superoxide may competitively react with NO to form peroxynitrite, which may have a role in signal transduction.

4 The combined interaction of hydrogen peroxide and reactive nitrogen species (RNS) with peroxidase enzymes can lead to posttranslational modification of proteins. Thus tyrosine residues can be nitrated by myeloperoxidase in the presence of both nitrite and hydrogen ­peroxide. Protein targets may include fibrinogen and ­apolipoprotein A1 [54].

MAPKs, such as both ERKs 1 and 2, JNK, and p38 MAPK, mediate the downstream effects of mitochondrial ROS/RNS. The specific effects of MAPK activation are a function of cell type and conditions [54].

Targets of mitochondrial prooxidant damage

High levels of ROS elaborated by mitochondria are detrimental.

They can exert cytocidal effect either directly or via downstream signaling events leading to cell death [1].

They can also cause nonlethal damage by reacting with and modifying cell membranes, lipids, proteins, RNA, and DNA.

Mitochondrial lipids, enzymes, and DNA are themselves major targets for mitochondrial ROS. This is why mitochondria are the main source and the main target of cellular free radicals.

Mitochondrial DNA

The production of free radicals by mitochondria sustains reactions that may selectively damage mtDNA within the organelle [2]. There are different types of oxidative DNA lesions, ranging from base modifications to single- and double-strand breaks [82].

mtDNA, in particular, is vulnerable to oxidative insult for several reasons:

1 mtDNA has no associated histones, which can protect it from oxidative damage [1];

2 mtDNA is in close proximity to the site of ROS/RNS production at the inner mitochondrial membrane [43];

3 mtDNA has a relative deficiency in repair mechanisms inasmuch as mitochondrial polymerases lack specificity for base excision repair and are themselves modified by ROS, which can potentially lead to changes in polymerase function and increased mutation rates in mtDNA [54];

4 mtDNA is constituted only of coding sequences, whereas nuclear DNA contains noncoding sequences [22].Thus the level of oxidized bases is two to three times higher in mitochondrial than in nuclear DNA [83]. The extent of mtDNA damage reflects the total exposure to oxidative stress.

Other ROS targets

Other ROS targets are:

the mitochondrial ETC proteins [28]

the mitochondrial voltage-dependent anion channel (VDAC) [67]

telomeres

redox signaling pathways [28]

calcium homeostasis [28].

Mitochondrial antioxidant defense

Mitochondria have significant antioxidant defenses in order to neutralize their prooxidant threat [2]. Intramitochondrial antioxidants play a critical role in ­preventing oxidative damage to existing mitochondrial proteins [54]. An additional important factor for protein maintenance in the presence of oxidative stress is the enzymatic reversal of oxidative modifications and/or protein degradation [1]. Mitochondrial metabolic potential is thus maintained by the combined actions of

1 antioxidant defenses and

2 molecular repair mechanisms [84].

Failure of these protein maintenance systems is likely a critical component of the loss in mitochondrial viability and the aging process as oxidized mitochondrial matrix proteins accumulate during aging [2].

Antioxidants

Mitochondria contain high concentrations of the antioxidants:

glutathione peroxidase-1 and mitochondrial S-nitro­soglutathione

catalase

a variant of superoxide dismutase (SOD), manganese-dependent superoxide dismutase (MnSOD) [2], and

lipoic acid [69].

MnSOD and glutathione peroxidase-1 are the primary mitochondrial enzymatic defensive mechanisms. They play a critical role in the cellular defense against super­oxide produced by mitochondria during normal ­cellular metabolism [84,85]. These antioxidants are ­situated in the matrix of the mitochondria, in close proximity to the production site of ROS in the ETC [85]. This location allows for the intramitochondrial neutralization of ROS [16]. MnSOD converts superoxide to hydrogen peroxide, which is then further degraded by catalase and per­oxiredoxins [84].

Increased levels of MnSOD are cytoprotective [1]. Similarly, overexpression of catalase in the mitochondria reduces oxidative damage, delays the onset of cardiovascular aging, and extends murine lifespan [86].

In contrast, a reduction or deficiency of MnSOD ­promotes cytotoxicity under conditions of oxidant stress [1]. In apo E-/-mice, decreased MnSOD activity promotes atherosclerotic lesion development and increases aortic mtDNA damage [54]. Decreased levels of lipoic acid ­disturb the overall antioxidant defense ­network, causing increased inflammation, insulin resistance, and mitochondrial dysfunction [87].

Molecular clean-up

The ATP-stimulated mitochondrial Lon protease plays an important role in the degradation of oxidized mitochondrial matrix proteins. It is a highly conserved protease found in prokaryotes and the mitochondrial compartment of eukaryotes. There is an age-dependent decline in the activity and regulation of this proteolytic system that may underlie the accumulation of oxidatively modified and dysfunctional proteins with age [88].

Mitochondria and nitric oxide

Nitric oxide affects mitochondrial energy metabolism, O2 ­consumption, and O2 free radical formation [89].

Nitric oxide (NO·) is a free radical that is physiologically generated in virtually all cell types. NO is a small signaling molecule, which may diffuse from its site of synthesis to different intra- and extracellular compartments.

Three types of NO-dependent signaling pathways appear to mediate NO effects, those involving:

1 NO itself via activation of soluble guanylate cyclase with the generation of 3′-5′-cyclic guanosine monophosphate (cGMP)

2 S-nitrosation of proteins, including the inhibition of caspases, and

3 autocrine signaling with the intracellular formation of peroxynitrite and the activation of the MAPKs [90].

NO can exert both cytoprotective and cytotoxic actions. NO, or its RNS derivatives, have multiple effects on mitochondria that impact on cell physiology and cell death, such as:

1 the stimulation of mitochondrial biogenesis in diverse cell types by chronic, small increases in NO/cGMP [10, 11];

2 the reversible inhibition of mitochondrial respiration at cytochrome c oxidase, CcOX, by high levels of NO;

3 the stimulation of mitochondrial production of superoxide, hydrogen peroxide, and peroxynitrite by NO;

4 the irreversible inhibition of mitochondrial respiration at multiple sites by RNS;

5 the induction of mitochondrial permeability transition and apoptosis (see