Beyond the Mitochondrial Tune Up: Part III

Part III: Restoring Mitophagy – the Key To Mitochondrial Rejuvenation

Introduction

Mitochondrial decay resulting from oxidative damage accumulates with age and is universally recognized as a major contributing factor to the whole range of functional decline and tissue deterioration associated with aging.^{1 2 3 4 5 6 7} Part I of this review discussed Bruce Ames’ application of the Michaelis constant (KM) concept to the ramifications of age-associated oxidative damage to proteins. Aging-associated increases in oxidative damage to key enzymes results in their structural deformation and decreased binding affinity for the co-enzyme, causing a decrease in enzyme funtion.⁸ Ames’ research has demonstrated that increasing the availability of acetyl L-carnitine and α-lipoic acid, two nutrients that serve as mitochondrial enzyme co-factors, restores the velocity of the reactions (KM) in the related enzymes, and thus restores aging mitochondria’s ability to produce youthful levels of ATP.^{9 10 11 12 13} Part II focused on the inter-relationships among the folate, methylation and transsulfuration pathways, whose dysfunction results in increased free radical production coupled with disruption of glutathione (GSH) synthesis, thus accelerating mitochondrial decay and aging. Ensuring adequacy of all the nutrient co-factors necessary to restore KM in these pathways, as well as in mitochondrial oxidative phosphorylation, is first line therapy for promoting healthy aging.

However, a protocol whose end goal is restoration of KM, while certainly helpful in delaying mitochondrial decay, does not address the more fundamental issue – why does human physiology shift from a homeostasic state that repairs and balances itself to one that allows decay to accumulate? Researcher Wulf Dröge has called this shift “the first cause of death,” and his insight into its likely causes may both explain why calorie restriction increases longevity and provide a much less onerous way to opt out of the vicious cycle responsible for the age-associated transition from a state of youthful homeostatic repair to one that promotes mitochondrial decay. This is the topic of Part III of this review.

“Biological aging is no longer an unsolved problem”

In press releases for a symposium that took place at the International Association of Gerontology and Geriatrics 19th World Congress, held in Paris, July 5-9, 2009, the father of the oxidative theory of aging, Dr. Leonard Hayflick, is quoted as saying,

Aging occurs because the complex biological molecules of which we are all composed become dysfunctional over time as the energy necessary to keep them structurally sound diminishes, thus our molecules must be repaired or replaced frequently by our own extensive repair systems. These repair systems, which are also composed of complex molecules, eventually suffer the same molecular dysfunction. The time when the balance shifts in favor of the accumulation of dysfunctional molecules is determined by natural selection — and leads to the manifestation of age changes that we recognize are characteristic of an old person or animal. It must occur after both reach reproductive maturity, otherwise the species would vanish. These fundamental molecular dysfunctional events lead to an increase in vulnerability to age-associated disease therefore, the study, and even the resolution of age-associated diseases, will tell us little about the fundamental processes of aging.

Perhaps the key point made by Hayflick, both above and in a recent paper titled, “Biological aging is no longer an unsolved problem,” (and generally accepted among leading gerontology researchers) is that the body’s repair and maintenance systems are the primary determinants of longevity.¹⁴ The question then becomes, “What causes these systems to lose their ability to outpace the damage of daily life?” Hayflick asserts the shift is determined by natural selection, suggesting little can be done to extend lifespan. Wulf Dröge has proposed an alternate, much more optimistic, explanation: the shift from a homeostasis of repair to one of snowballing damage is due to the age-related onset of aberrant insulin signaling, which prevents autophagic recycling.^{15 16 17 18}

Autophagic mitochondrial recycling – key to longevity

Aging is a waste-full process. In youth, autophagy—controlled self-destruction, literally “self-eating”—removes and recycles cellular waste that would otherwise compromise cellular function and regeneration of replacement structures. In the mitochondria, which are easily damaged due to their role as the energy production factories in cells and can themselves become a key source of cellular damage, adequate autophagic waste recycling is critical for rejuvenation but diminishes in aging cells. Since several longevity mutants (such as long-lived C. elegans or Drosophilia mutants^{19 20}) manifest defects in a signaling mechanism that normally slows the rate of autophagic waste recycling, Dröge and other researchers have postulated that the aging-associated decline of mitochondrial autophagy or “mitophagy” may be the first most limiting mechanism that determines maximum life span in most animal species, including man.^{21 22}

Insulin signaling inhibits autophagy

In humans, the signaling mechanism that slows autophagic recycling is identical to the signaling mechanisms that control our response to insulin and insulin-like growth factor, thus it can also be called the insulin mechanism. Bottomline: aberrant insulin signaling that inhibits autophagy, particularly mitophagy, is the first cause of death.

In addition to its role in clearing glucose from the bloodstream, insulin stimulates protein synthesis in skeletal muscle and other tissues (provided sufficient amounts of free amino acids are available). Thus, most protein synthesis takes place during the day when food consumption raises blood levels of amino acids and insulin. Through the same signaling mechanism, the increase in amino acid and insulin concentrations triggers suppression of autophagic activity in the fed state, i.e., for most of the day.

Autophagic waste recycling happens mainly in the fasted state—in man, mainly at night—when amino acid and insulin concentrations in the blood drop off, protein synthesis largely shuts down, and autophagy is allowed to proceed. In young individuals, the resulting nightly autophagic protein breakdown is normally so strong that it causes skeletal muscle tissues to release substantial amounts of free amino acids, including the cysteine needed for glutathione synthesis and defense against oxidative stress. In the fed state during the daytime, glutathione biosynthesis is maintained by cysteine from dietary protein.

Excessive autophagic destruction is avoided at night through a feedback mechanism that halts autophagic clearance when its products, free amino acids, reach a sufficient concentration. Autophagy converts proteins from damaged mitochondria and other forms of cellular waste into free amino acids. The resulting increase in free amino acid concentrations activates the serine/threonine kinases, Akt1 and Akt2, which mobilize the signaling protein mTOR (the mammalian target of rapamycin), which then suppresses autophagic activity. The same mechanism operates during the day when dietary protein and carbohydrate are consumed; the resulting increase in plasma amino acid and insulin concentrations results in activation of mTOR, which stimulates protein synthesis while suppressing autophagic activity.

Oxidative stress causes aberrant insulin signaling in older individuals

The weak point in the homeostatic control of cysteine supplies for glutathione synthesis is that autophagic activity is regulated not only by the free amino acid levels, but also by the insulin receptor signaling cascade, which even in the absence of insulin in the post-absorptive (fasting) state, can be abnormally triggered by oxidative stress.¹⁶

Activation of the insulin receptor involves its autophosphorylation, which is followed by phosphorylation of several target proteins in the signaling cascade. The insulin signaling cascade is inhibited by several phosphatases, including protein tyrosine phosphatase 1B (PTB 1B), phosphatase, tensin homologue on chromosome 10 (PTEN), and SH2-domain-containing inositol phosphatase (SHIP2), all of which are inactivated under moderately oxidative conditions. Thus the age-related increase in oxidative stress sets up a vicious cycle in which the mechanisms that maintain adequate levels of cysteine in the fasted state are overcome, leaving older adults (and specifically, aging mitochondria) increasingly susceptible to oxidative damage and less able to remove damaged cellular components via autophagy.

Aberrant insulin signaling drains glutathione reserves. PTP 1B, a key redox-sensitive signaling phosphatase in the insulin receptor signaling pathway, is inhibited by hydrogen peroxide (H2O2), which converts PTB 1B’s catalytically necessary cysteine moiety into cysteine sulfenic acid (Cys-SOH), which then interacts spontaneously with glutathione to form glutathione disulfide (GSSH), the inactive form of glutathione. GSSH can also convert PTP 1B’s redox-sensitive cysteine residue into the inactive disulfide. Changes in the thiol/disulfide redox status (discussed in Part II of this review in relation to the connections among homocysteine, methylation and transsulfuration) can also significantly inhibit the activity of the redox-sensitive phosphatases that regulate insulin signaling.

In addition to causing the functional inactivation of these phosphatases, low concentrations of H2O2 or an oxidative shift in the GSH:GSSH redox status strongly increases the activity of the basic insulin receptor tyrosine kinase in the absence of insulin. Since the activity of the insulin receptor signaling pathway is determined by a balance between kinase and phosphatase activities, oxidative activation of the kinase combined with the simultaneous inactivation of phosphatases synergistically upregulates the insulin signaling pathway. The end result is an age-associated decrease in autophagy and the body’s ability to maintain adequate post-absorptive (night time or fasting) cysteine levels. The resulting drop in glutathione production and intracellular glutathione concentrations compromises the mitochondria’s ability to scavenge reactive oxygen species (ROS), producing a vicious cycle that drives the progressive increase in ROS-mediated structural damage and its corollary, the progressive decline in energy production and repair that accompanies aging.

Aberrant insulin signaling inhibits key recycling and repair proteins

If this were not harmful enough, aberrant insulin signaling also results in inhibition of the forkhead transcription factor, FOXO 1, and peroxisome proliferator-activated receptor-coactivator 1 (PGC-1α).15

Activation of the insulin receptor leads to sequential activation of a number of protein and lipid kinases, including the serine/threonine kinases Akt1 and Akt2, which not only stimulate mTOR and thus downregulate autophagic protein catabolism (and thus cysteine supplies), but elicit phosphorylation (inhibition) of FOXO1, a transcription factor that induces expression of proteins involved in both of the proteolysis recycling pathways: the autophagic/lysosomal pathway and the ubiquitin-proteasomal pathway. (In contrast to the role of autophagy in removal of defective organelles, the ubiquitin-proteasomal pathway is responsible for recycling damaged, long-lived muscular proteins that are not degraded through the autophagic/lysosomal pathway.) FOXO genes also interact with SIRT1, a sirtuin protein that is the human homologue of the yeast sirtuin, silent information regulator 2 (SIR2), which assists in DNA repair and regulates genes that undergo alteration with age. Sirtuin activators, notably resveratrol (which activates both SIRT1 and PGC-1α, and improves the functioning of the mitochondria) ^{23 24} have been shown to inhibit gene expression profiles associated with muscle aging and age-related cardiac dysfunction in mice and may extend lifespan.^{25 26}

Akt2 also phosphorylates and inhibits the transcriptional coactivator PGC-1α, a regulator of post-absorptive hepatic metabolism that works in close association with FOXO1. In mice, PGC-1α is required for the expression of several mitochondrial genes in the liver, skeletal muscle, heart, brain, and brown fat. Strongly induced in the liver upon fasting, PGC-1α promotes hepatic fatty acid oxidation and shifts fuel usage from glucose to fat in the post-absorptive period. Thus, ROS-related aberrant activation of Akt2 may contribute to the development of hyperlipidemia and obesity. This may be the mechanism behind the association noted between low plasma cysteine concentrations and both hyperlipidemia and obesity. In support of this hypothesis, the redox sensitivity of the basic insulin receptor signaling activity in vivo has been confirmed in a placebo-controlled clinical study of non-diabetic obese persons in whom basic (i.e., post-absorptive) insulin receptor signaling decreased after supplementation with relatively small doses of N-acetyl cysteine (200 mg NAC t.i.d. for a total daily dose of 0.6 g for 8 weeks).²⁷

In young healthy individuals, the role played by oxygen radicals and H2O2 in enhancing insulin signaling is helpful, and so important that almost all cells and tissues contain special enzymes that produce H2O2, and O2- at a well-regulated rate. But in older individuals in whom basal levels of oxidative stress are already increased, aberrant insulin signaling during the fasting state that militates against mitophagy and further decreases glutathione stores, becomes the final straw. A vicious cycle is initiated. Lessened mitophagy results in a decrease in plasma cysteine concentrations, which causes a decrease in intracellular glutathione, which causes a corresponding increase in H2O2 and O2-, which leads to further insulin-independent upregulation of the basic insulin signaling mechanism, which further represses mitophagy, continuing the cycle.

Calorie restriction extends lifespan by promoting autophagy

The relationship between FOXO1 and the sirtuins, both of which are inhibited by insulin receptor signaling, may be the key mechanism behind the life-extending effects of calorie restriction with adequate nutrition (CRAN). CRAN reduces mitochondrial ROS production, lessening aberrant insulin signaling (and thus inhibition of FOXO1 and SIRT1), and promotes mitochondrial renewal via autophagy.²⁸ Preliminary research indicates that even moderate CRAN (a reduction in caloric intake of just 8% rather than the traditional 30-40%) may promote muscle mitochondrial biogenesis in middle-aged human subjects and may therefore both delay onset and mitigate progression of sacropenia in older adults.^{29 30 31}

The insulin receptor signaling cascade is inhibited by several phosphatases, including protein tyrosine phosphatase 1B (PTB 1B), phosphatase and tensin homolog on chromosome 10 (PTEN) and SH2-domain-containing inositol phosphatase (SHIP2), all of which are inactivated by ROS. In addition, activity of the basic insulin receptor tyrosine kinase is strongly increased by low concentrations of H2O2 or by an oxidative shift in the GSH redox status.

In the presence of H2O2 and ATP, the insulin receptor kinase domain is phosphorylated at its catalytic site and thereby rendered catalytically active in the absence of insulin. The phosphatases, in turn, contain a redox-sensitive cysteine moiety in their catalytic site, which is converted by H2O2 into a sulfenic acid moiety, which renders the phosphatase inactive. The balance between kinase and phosphatase activities determines the rate of phosphorylation of the insulin receptor kinase domain and several downstream targets including the phosphatidylinositol phosphates, the serine/ threonine kinase Akt1 and the mTOR. Ultimately, this balance controls the aging-related functions of autophagy and sirtuin proteins.

The insulin-independent oxidative upregulation of insulin receptor signaling activity (basic IRS) results in inhibition of autophagic removal of damaged cell structures (Autophagy) and ability to maintain post-absorptive plasma cysteine concentrations. This causes a decrease in intracellular glutathione (GSH) and a corresponding increase in ROS (H2O2, O2-) concentrations that further upregulates the insulin-independent insulin receptor signaling, perpetuating the cycle.

These changes occur in primarily during the night and early morning hours, i.e., in the post-absorptive/fasting condition. In the postprandial (fed) state, the insulin receptor plays a positive role as a key regulator of glucose clearance, protein synthesis, etc. Its negative effects are related to aberrant (insulin-independent) insulin signaling in the post-absorptive (fasting) condition.

Mitochondria – key players in initiating the first cause of death

Mitochondria are widely believed to play the pivotal role in aging because they are a major source of oxygen radicals and arguably their most important targets. Thus stressed mitochondria are a preferred target for autophagy. However, it is a common finding in old age that structurally abnormal mitochondria accumulate in post mitotic cells, mitochondrial oxidative phosphorylation decreases, and mitochondrial H2O2 production increases—the latter indicating that at least some of the abnormal mitochondria have defective electron transport chains and produce abnormally high amounts of O2-, H2O2, and other ROS.

This happens because mitochondrial DNA is particularly vulnerable to oxidative damage and shows a more than ten-fold greater mutation rate than nuclear DNA. O2- and other ROS induce mitochondrial DNA deletion mutants, which have been hypothesized to out replicate the wild-type (normal) mitochondrial DNA genome.³² The DNA of these mutated mitochondria may then code for abnormal electron transport chain complex enzymes, resulting in increased electron loss, increased production of H2O2 and O2-, and the initiation of the vicious cycle of progressively increasing oxidative stress coupled with progressively decreasing autophagic recycling. ROS may also cause lipofuscin deposits that further compromise autophagic removal of defective mitochondria.

Breaking the vicious cycle with N-acetyl cysteine

A pivotal factor in the shift from damage repair to damage accumulation in the aging adult is that during the night and early morning hours, older individuals are producing less glutathione (GSH). Fortunately, this can be ameliorated or even reversed. Supplementation with oral N-acetylcysteine (in doses ranging from 0.6 to 8 grams/day) has been shown to replenish intracellular GSH levels even in a wide range of infections, genetic defects and metabolic disorders, including HIV infection, cystic fibrosis, COPD, cardiac dysfunction, diabetes, colon cancer and Alzheimer’s disease.³³ (Since cysteine is easily oxidized, most clinical studies have used either the relatively stable synthetic cysteine derivative, N-acetylcysteine (NAC), or a naturally derived cysteine-rich undenatured whey protein isolate, both of which have been shown to decrease insulin responsiveness in the fasted state.)¹⁵

Furthermore, in vitro, in vivo and clinical studies have all confirmed that the insulin-independent basal activity of the insulin receptor, while increased by ROS, can be downregulated by supplementation with NAC. In clinical trials, NAC supplementation has also been shown to improve a number of parameters associated with aging including an increase in skeletal muscle wasting and the ratio of body fat/lean body mass (sarcopenia), an increase in plasma levels of the inflammatory cytokine tumor necrosis factor alpha (TNF-α), and decreases in immune function and plasma albumin* levels.¹⁵

[ *Hypoalbuminaemia has been strongly correlated with functional decline and increased mortality risk in older individuals. Elderly subjects and patients with practically all types of catabolic conditions typically show a conspicuous decrease in the plasma albumin level. Albumin normally constitutes about 60% of human plasma protein and plays an important role in regulating blood volume by maintaining the oncoosmotic pressure of blood needed to avoid edema, and by serving as the carrier for hydrophobic molecules, including lipid soluble hormones, bile salts, unconjugated bilirubin, free fatty acids (apoprotein), calcium, ions (transferrin), and some drugs (e.g., warfarin, phenobutazone, clofibrate & phenytoin). Lowered albumin levels (normal range is 3.5 to 5 g/dL) may also increase risk of ADRs since competition among drugs for albumin binding sites can result in increased levels of the free fraction of one or more of the drugs, thereby affecting potency.]^{34 35 36 37 38}

In a placebo controlled double-blind study of frail elderly patients, treatment with NAC doubled the increase in knee extensor strength during a 6-week program of physical exercise and slowed the subsequent decline during a 6-week follow-up period. On average, the exercise program was completely ineffective in persons who had low plasma concentrations of cysteine under fasting conditions. Conversely, persons with low fasting cysteine levels showed the greatest benefits from supplementation. While the plasma level of TNF-α increased significantly during the exercise program (and an increase in TNF-α is typical following exercise, even in healthy, young individuals), cysteine supplementation completely prevented this increase.^{17 39}

Studies of both elderly subjects and cancer patients have found that the aging and disease related loss of skeletal muscle mass, or sarcopenia, is significantly related to the oxidative shift in the plasma thiol/disulfide redox status. Treatment with NAC has been shown to ameliorate muscle wasting both in senescence and in cancer patients.^{39 40}

As Dröge noted in a paper titled, “Oxidative stress and ageing: is ageing a cysteine deficiency syndrome?,” all of this suggests that loss of youth, health and quality of life may be partly explained by a age-related deficit in the body’s cysteine and glutathione reservoirs, and that aging may be postponed by supplementation with NAC.⁴¹

Optimizing NAC Supplementation

The goal is NAC supplementation sufficient to increase glutathione levels, prevent aberrant insulin signaling, and restore night-time mitophagic activity and amino acid homeostasis to a youthful level.

The typical dosage of NAC given as an oral supplement in clinical trials is 600 mg b.i.d., although 200 mg t.i.d. has been shown to provide benefit in just 8 weeks.²⁷ On the other hand, doses as high as 1 gram t.i.d. have been used in cystic fibrosis patients for 4-12 weeks with no adverse effects, and in AIDs patients, oral NAC doses up to 8 grams/day did not cause clinically significant adverse reactions.^{42 43 44 45 46 47}

For anti-aging purposes, 600 mg b.i.d. of NAC is likely to be adequate. Furthermore, unnecessarily high cysteine supplementation should be avoided. Glutathione biosynthesis proceeds up to a certain concentration, which in the liver is approximately 10 millimolar (mM), after which any excess cysteine is converted into cystine or catabolized into sulfate and protons (ie., metabolic acid).Metabolic acidification leads to dedgradation of skeletal muscle protein and loss of lean body mass. Ideally, the diet should include 75-85% alkali-producing foods (grains, vegetables, most fruits) and 15-25% acid-producing foods (meat, fish, dairy, eggs [specifically egg whites]). The Standard American Diet renders most people in the U. S. slightly acidic. The cost of buffering excess acid is initially levied on bones but compromises function in all cells and tissues. Excessive intake of cysteine may increase formation of metabolic acid in tissues. Urine pH can be checked to evaluate the patient’s metabolic pH. The urine should be approximately neutral, i.e., with a pH of 6.5-6.8.15

In addition, consumption of an excessively large amount of cysteine as one dose will result in a potentially significant portion not being quickly cleared from the oxidative environment of the blood for use in biosynthesis of proteins and glutathione. Cysteine remaining in the bloodstream will be oxidized to disulfide cystine, causing an undesirable shift in the thiol/disulfide status of the blood plasma, which in turn, will alter the set points of redox-sensitive signaling pathways. Excessive cysteine may also lower basal insulin receptor signaling activity to the point that glucose clearance after food intake is compromised to the level of mild diabetes—an effect that can be easily monitored by a routine test of glucose clearance.¹⁵

Since glutathione and cysteine concentrations are relatively low in the post-absorptive state, especially in older individuals, NAC is best taken early in the morning and before retiring for the night — several hours after consumption of the evening meal to ensure a postabsorptive state (i.e., that normal insulin signaling in response to food intake is not occurring).

Effectiveness of NAC dosage may be evaluated by measuring the early morning fasting plasma cysteine levels. Do not request total cysteine (tCys); this test reports only cystine levels since labs often oxidize the specimen, so all cysteine is converted to cystine.⁴⁸ Request cyst(e)ine; this terminology is used to indicate a combination of reduced (cysteine) and oxidized (cystine) forms. These will be in dynamic equilibrium. Normal reference range is 4.9 – 8.0 mmol/dL.⁴⁹

A functional assessment of glutathione production and cysteine adequacy to meet the need for GSH synthesis can be provided by a urinary organic acids test evaluating α-hydroxybutyrate and sulfate. Since α-hydroxybutyrate is a byproduct of GSH production, high levels indicate increased hepatic GSH synthesis due to chronic oxidative stress. Urinary sulfate levels reflect the adequacy of sulfur-containing amino acids (e.g., cysteine) and also GSH adequacy (low sulfate levels indicate low total body GSH and thus increased need for sulfur-containing amino acids, e.g., cysteine). Taken together, urinary α-hydroxybutyrate and sulfate provide a picture of the dynamics of GSH synthesis and whether the body’s GSH needs are being met.^{48 50}

Immediately before or after a bout of physical exercise, consumption of 10-30 grams of cysteine-rich whey protein (depending on body weight and exercise intensity) is a better option since this delivers amino acids preferentially to skeletal muscle while also providing cysteine to lessen oxidative stress.¹⁵

Night-time autophagic activity can be increased by drinking 3.5 to 16 ounces of water about 2-3 hours before breakfast. This will temporarily dilute blood plasma concentrations of leucine and other regulatory amino acids, triggering autophagy to the point where the original equilibrium of regulatory amino acids is restored. Since the cysteine and leucine content of muscle protein is comparable, an autophagy-mediated increase in plasma leucine will be accompanied by a similar increase in cysteine, thus facilitating glutathione biosynthesis. While for younger adults, waking at 4-5 a.m. to drink water will be problematic, it is less likely to be onerous for older individuals, who often wake at least once during the night and frequently have a reduced sensation of thirst, so do not consume enough water to optimally support cellular functions. Optimal daily water intake is estimated to be 3.7 liters (3.9 quarts) for men and 2.7 liters (2.8 quarts) for women.¹⁵

Conclusion

Autophagy, the body’s mechanism for the removal and recycling of cellular waste, is key to rejuvenation, especially in the mitochondria, and is allowed to proceed only in the postabsorptive state, in which insulin signaling ebbs to its lowest (basal) level. With age, ROS-induced aberrant triggering of the insulin signaling mechanism in the postabsorptive state inhibits autophagy, preventing maintenance of plasma cysteine and intracellular glutathione levels throughout the night and early morning hours, and initiating a vicious cycle of progressively increasing oxidative stress and mitochondrial dysfunction. Since accumulation of cellular waste, decreased cellular glutathione concentrations and increased oxidative stress are common hallmarks of aging in a broad range of species, while lowered insulin signaling that promotes autophagy, whether resulting from genetic mutation or caloric restriction (with adequate nutrition), has been shown to extend lifespan in virtually all species tested to date, it seems likely that human life- and health-span may be extended by a protocol designed to re-establish youthful levels of mitophagy and postabsorptive cysteine concentrations, such as the one proposed by Dröge and outlined in this article.

Read Part I: Delaying the Mitochondrial Decay of Aging

Read Part II: The Methylation – Transsulfuration Connection to Mitochondrial Decay

References