Mitochondria in the elderly: is acetylcarnitine a rejuvenator?

Abstract

Endogenous acetylcarnitine is an indicator of acetyl-CoA synthesized by multiple metabolic pathways involving carbohydrates, amino acids, fatty acids, sterols, and ketone bodies, and utilized mainly by the tricarboxylic acid cycle. Acetylcarnitine supplementation has beneficial effects in elderly animals and humans, including restoration of mitochondrial content and function. These effects appear to be dose-dependent and occur even after short-term therapy. In order to set the stage for understanding the mechanism of action of acetylcarnitine, we review the metabolism and role of this compound. We suggest that acetylation of mitochondrial proteins leads to a specific increase in mitochondrial gene expression and mitochondrial protein synthesis. In the aged rat heart, this effect is translated to increased cytochrome b content, restoration of complex III activity, and oxidative phosphorylation, resulting in amelioration of the age-related mitochondrial defect.

1. Introduction

With people living longer, the number of aged individuals in the population in most industrialized countries is increasing and has important socio-economic and health consequences. Although medical progress has delayed death, improvements in alleviating the aging process lag behind; as a consequence, degenerative diseases, such as cardiovascular disease, Alzheimer, and cancer have increased [1]. The need for rational strategies to forestall the negative consequences of aging is one of the most important challenges for scientists in the 21st century.

Defects in oxidative phosphorylation during aging are now recognized as central players in impaired cellular and organ function (reviewed by Lesnefsky and Hoppel [2]). Impaired mitochondrial function not only affects energy production, but also increases the production of reactive oxygen species, further contributing to the aging process. Therapeutic agents targeting the mitochondrial defect constitute a meaningful way to fight aging.

Our review focuses on acetylcarnitine as a potential player in preventing age-related defects. Understanding the mechanism of action and the target of an agent that largely obviates age-related mitochondrial dysfunction is a rational approach for development of novel therapeutic agents. Such understanding leads to hope for improving health in the elderly. Why acetylcarnitine?— especially since this agent has failed in clinical trials for Alzheimer's disease [3], diabetic neuropathy [4, 5], and fatigue syndrome [6]. In fact, the literature contains little explanatory information concerning metabolism, metabolic effect, and potential mechanisms of the putative beneficial effect of acetylcarnitine in aging.

Herein we present acetylcarnitine as the avatar of metabolism, influenced by synthesis and utilization of acetyl-CoA through multiple metabolic pathways. By dissecting the effects of acetylcarnitine reported to occur in experimental studies, this review proposes a mechanism of action of the compound in the prevention of mitochondrial aging-related defects.

2. Endogenous acetylcarnitine

The equilibrium between acetyl-CoA (plus carnitine) and CoA (plus acetylcarnitine) (acetyl-CoA/CoA ratio) is crucial for mitochondrial metabolism. The mitochondrial content of endogenous acetylcarnitine is an indicator of mitochondrial metabolism of acetyl-CoA (Figure 1). Acetyl-CoA, derived from pyruvate, amino acids, and fatty acids, is reversibly converted to acetylcarnitine and CoA in the presence of carnitine by the carnitine acetyltransferase (CAT), a mitochondrial matrix enzyme attached to the inner membrane. This process regenerates free CoASH, which allows fatty acid oxidation and the tricarboxylic acid (TCA) cycle to proceed. Acetylcarnitine is transported to the cytosol through the mitochondrial inner membrane in exchange with carnitine by the antiport carnitine acylcarnitine translocase (CACT), thus providing acetyl groups for the synthesis of sterols, fatty acids, and ketone bodies.

Figure 1. Metabolic fate of acetyl-CoA derived from acetylcarnitine.

The supplemented acetylcarnitine (AcetylC) is transported into the mitochondria via the inner membrane carnitine acylcarnitine transferase (CACT). Acetyl-CoA formed through the mass-action of carnitine acetyltransferase (CAT) or synthesized de novo from acetate becomes available for tricarboxylic acid cycle (TCA), lipoic acid synthesis, and mitochondrial protein acetylation. Cytosolic acetyl-CoA is derived from transported mitochondrial citrate or peroxisomal acetate. Nuclear acetyl-CoA is either directly imported from the cytosol or synthesized from acetate by acetyl-CoA synthetase or via the potential nuclear CAT using acetylcarnitine. Nuclear acetyl-CoA controls gene expression via acetylation of histone and non-histone proteins.

Total carnitine and acetylcarnitine concentrations are maintained within normal ranges of 23–73 μmol/L and 3–14 μmol/L, respectively, in adult human plasma [7]. Plasma concentration and urinary excretion of acetylcarnitine vary with physiological and pathological states, reflecting mainly the degree of acetyl-CoA synthesis. In normal human subjects under fasting conditions, acetylcarnitine excretion in the urine increased to 78% of the excreted acylcarnitines [8]. In contrast, in obese human subjects under fasting conditions, the increase in urinary excretion of acetylcarnitine was markedly slower and at a lower level, suggesting a slower formation of acetyl-CoA derived from fat oxidation. In diabetic patients with ketosis, acetylcarnitine represented 61% of the total urine acylcarnitines, and decreased dramatically upon insulin treatment [8]. The content of acetylcarnitine in tissues depends upon the presence of the mitochondrial enzyme, CAT. Because of the limited amount of mitochondrial CAT, the transport of acetylcarnitine in liver is extremely restricted and the predominant transport of acetyl-CoA units out of hepatic mitochondria to the cytosol is as citrate (Figure 1).

3. Evidence for the protective effect of acetylcarnitine against the aging defect

Aging is accompanied by a progressive decline of physiological function, that leads to an increased rate of disease [9]. At the cellular level, aging is characterized by structural disorganization, disturbances in protein synthesis, decreased enzyme activity, and progressive impairment of the functions of cellular organelles [10]. The weight of the accumulating evidence indicates that the age-related damage is an ineluctable consequence of normal oxygen metabolism associated with a relentless formation of reactive oxygen species (ROS; [10]). Much of ROS production occurs in the mitochondria [11, 12], making this organelle both the source and the target of oxidative stress in advanced age [11–14]. Defects of mitochondrial metabolism in the elderly have been observed in heart [15–18], skeletal muscle [19, 20], liver [21–24], and brain [21, 25, 26], as reviewed by Lesnefsky and Hoppel [2].

3.1 The aging defect

3.1.1 Heart under standard and ischemic conditions

The mitochondrial defects in the aging heart are localised specifically in the interfibrillar mitochondrial (IFM) population [15], which is located between the myofibrils [27]. Mitochondrial oxidative phosphorylation in the IFM isolated from elderly rat heart decreased with substrates feeding electrons into complexes I (glutamate; [28]), III (duroquinol; [16, 28]), and IV (ascorbate and TMPD; [15, 28]). Because the aging defect was not relieved by uncoupling, the electron transport chain (ETC), rather than the phosphorylation system, was identified as the site of the defect. Furthermore, measurement of enzyme activities showed decreases in complexes III [16] and IV [15] and no change in complexes I and II. Defects in fatty acid oxidation [30] or Complex IV in a mixed population of mitochondria isolated from aging heart [31–33] were localised specifically to the IFM [15].

Electron flow through complex III involves the oxidation of ubiquinol followed by the simultaneous transfer of electrons to both cytochromes b and c1 in a bifurcated fashion mediated by motion of the iron-sulfur protein [34–36]. The subunit peptides (subunits VIII and X) and catalytic centers (cytochrome b, c₁, and iron-sulfur protein) were not changed with aging in IFM [16]. Similarly, the content of cytochromes b and c₁ is preserved in combined populations of heart mitochondria [25] as well as in individual populations [37] from aged rat compared to young rats. The aging defect resides within the myxothiazol-binding domain in the vicinity of heme b_L of cytochrome b (Qo center) [18].

Mitochondrial dysfunction contributes to myocardial injury during ischemia/reperfusion injury [17, 38]. When exposed to a stress such as ischemia, mitochondria undergo damage, such as changes in ultrastructure [39] and functional impairment [40]. Cardiac ischemia results in damage to the mitochondrial ETC [37, 41, 42]. Short term ischemia (10–20 min) causes a decrease in complex I activity [41–43], as well as a reduction in phosphorylation activity involving complex V [42] and adenine nucleotide translocase [44, 45]. After a longer ischemic period, damage occurs at the level of complex III [46] and then complex IV [41]. It needs to be emphasized that the mitochondrial damage occurs during ischemia, rather than during reperfusion [17, 37, 47].

The aged heart exposed to ischemia sustains greater injury compared to the adult heart both in patients [48] and in animal models [49–52]. Compared to the adult heart, oxidative damage (oxidative protein modification [49]) and calcium-mediated damage [53] are increased, indicating mitochondria-driven mechanisms of injury. Ischemic-induced defects in complex III and IV are added to the aging-induced defect at the same complexes. Furthermore, because complex III is recognized as a source of ROS [54], the complex III defect in the aging heart provides an explanation for the enhanced oxidative damage occurring during ischemia [37]. By paramagnetic resonance signal, the ischemic defect was localized at the iron-sulfur protein in both populations of mitochondria, and is due to a functional decrease rather than a loss of subunit peptide [37].

To focus on the importance of complex III in the oxidative damage in mitochondria and during aging, we undertook studies to limit electron flow into complex III and thus decrease ROS production. The blockade of mitochondrial respiration with the irreversible inhibitor rotenone [55] or the reversible inhibitor amobarbital [56] at complex I before ischemia protects the distal ETC against ischemic damage. Protection of the ETC during ischemia by the reversible blockade of electron transport with amobarbital markedly decreases myocardial injury measured after reperfusion, supporting the premise that ischemic damage to mitochondria is a key factor in myocardial injury [57, 58]. The ischemic damage to the ETC increased both the capacity and the net production of H₂O₂ from complexes I and III and sets the stage for an increase in ROS production during reperfusion as a mechanism of cardiac injury [47].

In conclusion, at the onset of reperfusion in the aging heart, complex III in IFM exhibits the additive damage of aging and of ischemia; the combination is a likely mechanism of enhanced ischemic injury in the aging heart [37]. These defects act in concert to further slow electron flow within complex III, increase the reduction of cytochrome b, enhance production of ROS, and finally lead to cell death by necrosis or by apoptosis [17].

3.1.2 Skeletal muscle

In aged human and rat skeletal muscle, a reduction of muscle mass (sarcopenia; [59]) and performance occurs [60, 61]. The decreased mitochondrial content in muscle indicates an important role of mitochondrial biogenesis, defined as growth and division of pre-existing mitochondria [62], in the aging process. In the muscle from young animals, the mitochondrial content is maintained by production of new mitochondria. In elderly rats, in contrast, the content and the yield of total mitochondria decrease [19]. Additionally, an inverse relationship was shown between mitochondrial content and age [20]. Mitochondrial performance in aged skeletal muscle, however, is not affected, as shown by similar rates of OXPHOS with different substrates in young and old rats [19, 63] and in humans [64–66]. In contrast, other studies have reported defects in oxidative phosphorylation in the presence of substrates feeding electrons into complex I or complex II in skeletal muscle from aged humans [67] or mice [68]. Other studies have also shown aging-defects in activities of complexes I [69–71], II [70], III [70], IV [67, 69, 70], and V [70, 71].

3.1.3 Brain

In the brain from aged compared to young animals, the mitochondrial mass did not vary [21], but the function of mitochondria is affected. OXPHOS in the presence of substrates feeding electrons through complexes I and II decrease in the brain from aged compared to young rats [25]. The activities of complex I [25, 26, 72, 73], complex I+III [21, 74] and complex IV [21, 26, 72–78], and the content in cytochromes c and aa3 [25] also were reported to decrease during aging.

3.1.4 Liver

In the liver from aged compared to young animals, the mitochondrial mass is preserved but the function of mitochondria is affected [21]. The rate of OXPHOS in the presence of substrates feeding electron into complexes I, II, or III decreased in aged animals [22–24, 79, 80] and humans [81]. The decrease in OXPHOS in rat liver mitochondria was explained by a defect in complex III, whereas complex II [72, 79, 80], II+III [21], and IV [79, 80] were unaltered by age. An additional defect in complex IV activities was observed in the liver from aged mice [21, 72, 74].

3.2 Protection by acetylcarnitine: Experimental evidence

3.2.1 Organism, organ functions

The improvement of mitochondrial OXPHOS during aging has an important impact on preservation of organ function, as well as sensitivity of organs following a stress event, such as ischemia. With bolus administration of acetylcarnitine, three hours before an ischemic event, cardiac recovery improved in the aged heart [28]. The ischemic injury experienced by the aged heart treated with acetylcarnitine was neutralized to a level comparable to that in the adult heart in terms of myocardial damage and contractile recovery after ischemia. In the adult heart, acetylcarnitine treatment, however, does not alter the degree of injury occurring during ischemia; these data suggest that acetylcarnitine does not alter the basic mechanisms of ischemic damage common to adults and aged rat. From a central nervous system perspective, oral administration of acetylcarnitine in old rats has been shown to improve cognitive function [82] and ambulatory activity [83].

3.2.2 Mitochondrial structure

The improvement of cognitive function and ambulatory activity following acetylcarnitine supplementation [82], was related to restored mitochondrial cristae [83] and reduction of oxidized RNA, likely mitochondrial [84], in hypocampus neurons. A dose-response study showed that lower doses (0.5% in drinking water) ameliorated the age-related decline in mitochondrial cristae in the dentate gyrus of the hypocampus more effectively than did the higher doses (1.5%). In contrast to the lower doses, higher doses of acetylcarnitine did not decrease lipid peroxidation products in the brain, but caused an increase in protein carbonyl content [83]. These data suggest that an increase in oxidative stress in the brain is a side effect of high doses of acetylcarnitine.

3.2.3 Integrity of mitochondrial inner membrane lipid environment

The mitochondrial lipid environment contains the unique phospholipid, cardiolipin. The inner membrane tetra-linoleoyl cardiolipin (L₄CL) preserves the physical properties of the mitochondrial inner membrane [85, 86], mitochondrial oxidative phosphorylation [87, 88], and activities of the mitochondrial transporters [89], ETC enzymes [33, 90–92], and the phosphorylation apparatus [93]. Additionally, cardiolipin is involved in the respirasome assembly of the mitochondrial complexes [94] and anchors cytochrome c to the inner mitochondrial membrane [95]. Cardiolipin depletion results in reduced complex I, III [90], and IV [96] activity along with the dissociation of subunits VI_a and IV_b from the complex IV.

Loss of cardiolipin has been purported as the mechanism for the aging defect in mitochondrial respiration and ETC activities (Table 1), and by restoring cardiolipin content, acetylcarnitine reverses the aging mitochondrial defect in rat heart [97, 98], skeletal muscle [99], and liver [100]. In contrast to these studies, we found that the content of total cardiolipin did not decrease and the molecular cardiolipin species did not change during aging [101]. Important differences in methodology may explain these discrepancies. The previously reported differences in cardiolipin content in adult versus aged rats were based on an HPLC-UV absorption method, without an internal standard. When using a balanced study approach to measure cardiolipin [101], the results showed that the content was not affected by aging. Since cardiolipin content is not decreased in aging, the mechanism by which acetylcarnitine improved mitochondrial function does not involve modification of cardiolipin content (Table 1).

Table 1.

Different proposed mechanisms of acetylcarnitine effect during supplementation for short term (ST) or long term (LT) periods, with experimental evidences supporting or rejecting the mechanisms.

Pro	Contra
Effect on integrity of the lipid environment of the mitochondrial inner membrane, principally the cardiolipin content (section 3.2.3)
↑ cardiolipin content in rat heart mitochondria; LT [159] [160]	No change in cardiolipin total content, acyl group or individual molecular species in mitochondria from aged heart [101]
Control of mitochondrial protein synthesis (biogenesis) by acetylation/deacetylation regulating mt translational activity (section 3.2.4, 5.3, 5.4, 5.5)
↑ OXPHOS and ETC activity in the aging heart; ST [28]	Need data on the specific protein acetylated
↑ cytochrome b and aa3 in heart ST [28] and brain; LT [25]
Preservation of nit content in skeletal muscle during aging [161] or inactive status [102], and in brain during aging; (LT) [103]
↑ mt RNA [162]
↑ nuclear transcripts for factors involved in nit biogenesis in skeletal muscle; LT [161]
Prevention of unloading-induced downregulation of mRNA levels of kinases to transduce metabolic and neuronal stimuli into mt biogenesis in skeletal muscle; LT [161]
Energy source (section 4.2.1)
↑ fat utilization as a metabolic fuel and ↑ protein deposition [161]	No change in total energy expenditure; LT [161]
Dual effect on mitochondrial oxidative stress due to antioxidant properties of the compound (section 5.1)
↓ ROS production in β-cells exposed to oleic-acid related with protection against mt dysfunction and maintenance of the glucose-stimulated insulin secretion [120]	None of the carnitine derivatives were able to scavenge peroxyl or superoxide radicals [130]
	The effect may occur as a consequence of preservation of mitochondrial function rather than a direct effect of acetylcarnitine
↓ oxidized RNA in hypocampus neurons associated with improved cognitive function and mitochondrial cristae formation [83]
↑ oxidative stress at high doses in brain, consistent with antioxidants effect [83]
Antiapototic effect (section 5.2)
Protect neurons [163] and hepatocytes [133] against methamphetamine-induced cellular death.	How can this increase mitochondrial proteins content or function?
Protect dopaminergic system against intraventricular injection of methamphetamine in rats [135]	The effect may occur as a consequence of preservation of mitochondrial function rather than a direct effect of acetylcarnitine

ST: Hours, LT: weeks, mt: mitochondria

3.2.4 Mitochondrial function and content of mitochondrial proteins

Several reports showed that treatment with acetylcarnitine may have impact on mitochondrial biogenesis (Table 1). Mitochondrial biogenesis is indicated by the increase in mitochondrial size, number, and mass. Change in mitochondrial biogenesis may affect the content of mitochondrial proteins or function by an increase in specific functional units.

Treatment of the aged rat heart by intraperitoneal administration of acetylcarnitine restored the mitochondrial respiration through complexes III and IV in IFM to the level of the adult heart. It reversed the age-related decrease in the activity of complex III. The treatment also increased cytochrome b content in both IFM and SSM and cytochrome aa3 specifically in SSM from old rats [28]. In the aging heart that has been pretreated with acetylcarnitine and exposed to ischemia-reperfusion, the mitochondrial defect due to aging is eliminated, and only the defect due to ischemia is still present, improving the recovery after ischemia-reperfusion to the level of the adult heart [28]. Those effects were observed only three hours after administration of the acetylcarnitine. The supplementation with N-acetylcysteine also has been shown to prevent the decrease in cytochromes c and aa3 in the aging brain [25], suggesting that the effect of acetylcarnitine on the content of mitochondrial proteins is due to the acetyl portion rather than the carnitine portion.

Long term administration of acetylcarnitine (1 month, in drinking water) increases the mitochondrial content in rat skeletal muscle [99], suggesting that acetylcarnitine stimulates mitochondrial proliferation, even if the latter is not detected after a short time of supplementation. Acetylcarnitine also helps in preserving the mitochondrial content during a long period of inactivity of skeletal muscle [102] and during aging in the brain [103].

4. Supplemental acetylcarnitine: pharmacokinetics

4.1. Absorption, distribution, excretion

Orally supplemented acetylcarnitine is taken up from the gastrointestinal tract into the blood [104]. Acetylcarnitine is deacetylated during or immediately after its uptake into intestinal cells, and a portion of the newly formed intracellular free carnitine is re-acetylated [104]. Whereas high doses of acetylcarnitine are well tolerated, the absorption of orally administered acetylcarnitine is poor. In elderly humans with senile dementia, daily oral administration of 2 g for 50 days slightly raised the plasma concentration of acetylcarnitine. Carnitine concentration in plasma was unchanged whereas total carnitine (carnitine plus acylcarnitines) rose modestly [105] due to increased level of acylcarnitines. In contrast, in the adult human, intravenously administered acetylcarnitine as a bolus (500 mg) led to a rapid increase in plasma concentration followed by a progressive decline reaching the base value in 12 hours. The administered acetylcarnitine was mostly excreted in urine as carnitine and acetylcarnitine during the first 24 h after administration. Conversion of acetylcarnitine to carnitine was higher than the average renal clearance of acetylcarnitine, suggesting that a large proportion of the acetyl moieties of the exogenously administered acetylcarnitine is either rapidly used in biosynthesis and stored (in a form other than acetylcarnitine), or hydrolyzed [106].

The body distribution of acetylcarnitine is determined by systems transporting acetylcarnitine into cells against a concentration gradient. The uptake of acetylcarnitine by cardiomyocytes in culture is saturable and inhibited by carnitine [107], suggesting that both compounds share the same transporter. Ohashi et al. found that acetylcarnitine is transported by the organic cation transporter 2 (OCTN2), a homolog of the OCTN1, with high affinity (K_m 8.50 μM) in a saturable manner [108]. The pH profile and Hill coefficient (0.989) of the OCTN2-mediated acetylcarnitine uptake were similar to those of carnitine transport. In heart, OCTN2 is highly expressed [109], suggesting a significant role in the uptake of acetylcarnitine in cardiomyocytes. A similar saturable sodium-dependent system exists at the blood-brain barrier; the transported acetylcarnitine is reported to improve neuronal energetics and to increase the synthesis of the neurotransmitter acetylcholine in the cholinergic system. The mechanism for acetylcarnitine export from the cell has not been characterized.

4.2. Effect of supplemental acetylcarnitine on metabolism

The question is: are the benefits of the supplemented acetylcarnitine due to 1) the acetyl unit as acetyl-CoA, 2) the carnitine derived from the parent compound, or 3) the intact acetylcarnitine?

4.2.1 Fate of acetyl-CoA

Exogenous acetylcarnitine is transported via CACT through the inner membrane to the mitochondrial matrix where it is exposed to CAT, which reversibly transforms acetylcarnitine to acetyl-CoA. The substrates used by the enzyme in one direction are competitive inhibitors of the reaction products obtained in that direction [110]. Therefore, if the intramitochondrial concentration of acetylcarnitine is higher than the K_m of the enzyme (350 μM), one would expect that the exogenous acetylcarnitine facilitates the continuous generation of acetyl-CoA. An increased acetyl-CoA/CoA ratio decreases the activity of pyruvate dehydrogenase and inhibits mitochondrial fatty acid β-oxidation and the TCA cycle. Therefore, the increase in mitochondrial acetyl-CoA would have negative effects on mitochondrial metabolism. However, the mitochondrial content of acetyl-CoA during acetylcarnitine supplementation is the result of acetyl-CoA formation, due to the mass-action of CAT, and utilization of the former. The consumption of acetyl-CoA in irreversible metabolic pathways, i.e., lipoic acid and citrate synthesis within the mitochondria followed by biosynthetic processes within the cytosol (Figure 1), drives the process and maintains the mitochondrial content of acetyl-CoA within a safe range.

Catabolism of acetyl moieties via TCA cycle was suggested [111, 112] to stimulate energy production (Table 1). A Positron Emission Tomography study of the incorporation of acetate from acetylcarnitine in the brain indicated an accumulation and rapid metabolism of the acetate moiety of acetylcarnitine, but not of its carnitine part [113]. In mammalian sperm and in insect flight muscle, the endogenous cytosolic acetylcarnitine serves as an available source of acetyl-CoA used by the TCA for energy production [110]. In contrast, oral supplementation with acetylcarnitine for one month in old rats does not increase the total energy expenditure, despite the increase in fat utilization as metabolic fuel [99]. Coupled with the limited bioavailability of 10% of orally-supplemented acetylcarnitine, the administration of 2 g acetylcarnitine (10 mmoles) and its incorporation in the TCA cycle would give rise to 12 mmoles ATP per day. This value represents only 0.01% of the total amount of ATP turned over in an adult human organism per day (65 kg/day, 118 moles/day). Therefore, supplemental acetylcarnitine is not a major energy provider.

Anabolism of acetyl-CoA derived from the supplemented acetylcarnitine refers to the entrance of acetyl moieties into biosynthetic pathways. Acetylcarnitine provides acetyl-CoA for the synthesis of acetylcholine in the brain and fatty acids in lipogenic tissues that contain fatty acid synthase, i.e., brain, liver, and adipose tissue. Citrate generated by the condensation of acetyl-CoA and oxaloacetate in the TCA cycle is transported into the cytosol where it is cleaved by citrate lyase to re-generate cytosolic acetyl-CoA. One-third of the acetyl-CoA that reaches the cytosol via citrate in the brain is used for acetylcholine synthesis [114]. ¹³C NMR spectroscopy following 4 hour [(1,2-¹³C₂)acetyl]-L-carnitine intravenous infusion showed that in the liver the acetyl groups enter the TCA cycle since ¹³C was found in liver glutamate, glutamine, and glutathione and was incorporated into cholesterol and 3-hydroxybutyrate [115]. Farrell et al. confirmed the incorporation of these acetyl groups into the TCA cycle since ¹⁴CO₂ was a major product of IV injected [1-¹⁴C]-acetylcarnitine in rats [116]. Additionally, substantial radioactivity was found in fatty acids of phospholipids and triacylglycerols in the liver, with smaller amounts in the heart, brain, skeletal muscle and kidney [116]. Furthermore, the bulk of radioactivity was in fatty acids of phospholipids in the brain after intraventricular injection of [1-¹⁴C]-acetylcarnitine [112]. Similar results were obtained in cultured cells, where added acetylcarnitine (2 mM) contributed about 10% of the acetyl-CoA used for de novo synthesis and for elongation of fatty acids, as well as 6% for ketogenesis [117].

The concept that fatty acid synthesis is limited to the cytosol of lipogenic cells was recently revised. We recently provided evidence for the occurrence of fatty chain elongation in rat heart [118]. Also, several nuclear-encoded components of a putative mitochondrial pathway for de novo fatty acid synthesis were identified and characterized within mitochondria. Since lipoic acid synthase was found in mammalian mitochondria [119], and lipoic acid (LA) reversed age-associated mitochondrial decay [82, 120], it is reasonable to hypothesize that the supplemented acetylcarnitine exerts part of its protective effect via providing acetyl groups for LA synthesis within the mitochondria. Bovine heart mitochondria are able to elongate a C2 primer and synthesize de novo octanoyl precursor as the substrate for lipoic acid synthase, the enzyme that introduces the sulfur atoms into the octanoyl precursor for LA synthesis [121]. In addition to being an antioxidant, LA is an essential cofactor in mitochondrial metabolism. Acetylcarnitine in association with LA is a more effective supplemental regimen than acetylcarnitine alone to protect mitochondria [100].

4.2.2 Effect of carnitine

The metabolic effects of acetylcarnitine supplementation in aged animals might be due to carnitine. Aging is associated with carnitine insufficiency which may compromise function during stress conditions when carnitine is needed. A decrease in carnitine content has been described in the brain and myocardium of aged animals [122]. An age-related decrease in carnitine content in the brain and plasma is associated with an increase in carnitine content in the liver, possibly induced by an impaired transport of carnitine from the liver to the blood in old animals [83]. Chronic oral supplementation with acetylcarnitine in rats reverses the age-related decline in carnitine content in the brain, plasma [83], skeletal muscle, and heart [123]. Acetylcarnitine is reported to be better absorbed and to cross the blood-brain barrier more efficiently compared to carnitine [124, 125]. The recovery of brain, heart, and plasma carnitine content in the old animals treated with acetylcarnitine indicates that its intestinal absorption and tissue uptake are unchanged during aging.

By restoring tissue carnitine in the elderly, acetylcarnitine supplementation may facilitate the elimination of potentially toxic acyl-CoA metabolites derived from fatty acid oxidation. This would be required when acyl groups accumulate, i.e., increased fatty acid oxidation, and is accomplished by elimination of specific acylcarnitines [126].

4.2.3 Effect of intact acetylcarnitine

High concentrations of acetylcarnitine may normalize age-related abnormalities in the kinetic properties of CAT. An age-related decrease in CAT activity occurs in soleus, diaphragm, and heart from rat [122], and brain microvessels and cerebellum from humans with Alzheimer's disease [127]. Liu et al. [128] found a moderate decrease in CAT activity and a marked increase in the K_m for both substrates, acetylcarnitine and CoA, in the brain of old rats due to alteration at the active site of CAT by malondialdehyde, a product of lipid peroxidation. Feeding old rats high amounts of acetylcarnitine restored brain CAT activity, as well as CAT-binding affinity for both substrates. In addition, the inhibitory effect of malondialdehyde on in vitro CAT activity was inhibited by acetylcarnitine, suggesting a competitive mechanism for the protective effect of acetylcarnitine.

5. New proposed protective mechanisms for an old compound

Acetylcarnitine has been considered a “mitochondrial nutrient” [120, 125], that reverses both aging-related mitochondrial dysfunction and the reaction of elderly mitochondria to challenge [28]. We propose that the rejuvenating effect of acetylcarnitine on mitochondria is through mechanisms in addition to the aforementioned metabolic effects. The next section reviews recent discoveries about the effects of acetylcarnitine on cellular signalling pathways, that may explain how elderly mitochondria are converted to a more youthful state (Figure 2).

Figure 2.

Signaling pathways for the protective effects of acetylcarnitine.

5.1. Dual effect on mitochondrial oxidative stress

The antioxidant properties of acetylcarnitine have been both popularized and advertized. Although unexpected from its chemical structure, the antioxidant properties of acetylcarnitine were reported in in vitro experiments (summarized in Table 1) and explained by its iron-chelating properties [129]. In contrast, when administered via the perfusate to ischemia-reperfused rat hearts, none of the carnitine derivatives were able to scavenge peroxyl or superoxide radicals [130].

In cell culture studies, the decrease in oxidative stress by acetylcarnitine occurred by 1) exerting a protective effect on mitochondrial structure and function, making the ETC less prone for electron leak and superoxide production, and 2) stimulating the endogenous cellular antioxidant defence mechanisms. The effect on mitochondria is supported by pretreatment of pancreatic β-cells with micromolar concentrations of acetylcarnitine, which protected the cells from oleic acid-induced mitochondrial dysfunction and decreasing ROS production [120]. Secondly, acetylcarnitine supplementation stimulated endogenous cellular antioxidant defence mechanisms. Treatment of astrocytes with acetylcarnitine (10–100 μM for 6 hours) increased the amount and activity of heme oxygenase-1 (HO-1) [131]. In addition, pre-incubation of astrocytes with acetylcarnitine before the initiation of a nitrosative stress with lipopolysacharide and interferon, prevented the decrease in complex IV activity, protein nitration and restored the reduced glutathione/oxidized glutathione ratio [131]. HO-1, the rate-limiting enzyme in the production of bilirubin, catalyzes the oxidative cleavage of the heme molecule to form biliverdin and carbon monoxide (CO). Therefore, the beneficial effect of HO-1 is due to both the antioxidant property of biliverdin/bilirubin and the increase in CO availability.

5.2. Antiapoptotic effect

High concentrations of acetylcarnitine (1 mM) protect neurons [132] and hepatocytes [133] against cellular death induced by methamphetamine, that is mediated via cardiolipin peroxidation, cytochrome c release, induction of mitochondrial transition pore and apoptosis [134]. Also, acetylcarnitine protected the dopaminergic system against the intraventricular injection of methamphetamine in rats [135] (Table 1).

The antiapoptotic effect of acetylcarnitine may be related to the overexpression and activation of HO-1, which increases the level of antiapoptotic bcl-2 protein and inactivates the pro-apoptotic transcription factor p53 in neurons [136]. It was suggested that co-localization of increased HO-1 with senile plaques [137] and neurofibrilar tangles [138] in human brains with Alzheimer disease reflects the adaptive reaction of neurons in order to limit neurodegeneration.

Orally-supplemented acetylcarnitine in rats was reported to decrease caspase activation by increasing the level of X-linked inhibitor of apoptosis protein (XIAP), thus limiting the mitochondrial-induced apoptosis in peripheral neurons [139]. Neither a protective effect on apoptosis induction nor a decrease in XIAP level was observed by these authors after carnitine administration, suggesting that the acetyl groups of acetylcarnitine have a fundamental role in protecting against mitochondrial-induced apoptosis.

5.3. Potential control of gene transcription by reversible lysine acetylation

The transcription of nuclear DNA recently has been linked to acetylation and deacetylation of core histone tails at lysine residues [140]; acetylated histone tails are associated with active chromatin, whereas histone deacetylation is associated with transcriptional repression of genes, because the removal of acetyl groups from lysine residues limits accessibility of the DNA for transcription [141]. Therefore, histone acetyl-transferases and deacetylases are transcriptional co-regulators. All known acetyltransferases use acetyl-CoA as a donor for acetylation. The existence of distinct mitochondrial and nucleo-cytosolic acetyl-CoA pools has been unambiguously shown; the nuclear concentration of acetyl-CoA is the limiting factor for histone acetylation [142]. Can acetylcarnitine increase the nuclear acetyl-CoA pool and increase the transcription of nuclear DNA (Table 1)?

The nuclear content of acetyl-CoA following acetylcarnitine supplementation was not determined. The possible existence of a nuclear CAT (referred by [111]) and the discovery of the nuclear acetyl-CoA synthetase may explain the contribution of supplemental acetylcarnitine to the nuclear acetyl-CoA. It was recently found that the global transcription in yeast, mainly under the control of the histone acetylase activity rather than the histone deacetylase activity, is regulated by the steady-state level of acetyl-CoA supplied by the nuclear acetyl-CoA synthetase [142]. Also, the free passage of acetyl-CoA through the nuclear pore complex facilitates the traffic of cytosolic acetyl-CoA to the nucleus [142]. Since cytoplasm does not contain CAT, the origin of the cytosolic acetyl-CoA generated from acetylcarnitine required contributions by mitochondria and peroxisomes. Mitochondrial acetyl-CoA derived from acetylcarnitine is incorporated into TCA cycle and forms citrate which further generates acetyl-CoA into the cytosol of lipogenic cells. Peroxisomal CAT reversibly transforms both endogenous (derived from peroxisomal fatty acid β-oxidation) and supplemented acetylcarnitine into acetyl-CoA. In the liver, acetyl-CoA is hydrolyzed into acetate by the respective acetyl-CoA hydrolases and then reactivated by the nuclear acetyl-CoA synthetase.

The transcription of specific nuclear genes controls the replication and transcription of the mitochondrial genome. Nuclear respiratory factor-1 (NRF-1) and mitochondrial transcription factor A (TFAM) are required for mitochondrial DNA (mtDNA) replication; together with mitochondrial transcription factors B (TFB1 and TFB2), they stimulate the transcription of both light and heavy chains of mtDNA [143]. In addition to acetylation of histone proteins, site-specific acetylation of non-histone proteins plays an important role in transcriptional regulation. In particular, high mobility group (HMG)-box proteins are acetylated [144]. TFAM contain two HMG-box-like domains. The total amount of TFAM increases in the liver, cerebellum and kidney with aging [145].

The acetylation status of histone and non-histone nuclear proteins was not determined following acetylcarnitine supplementation. As noted in section 4.1, acetylcarnitine up-regulates genes involved in cellular antioxidant capacity and repair [131]. Could this regulation by acetylcarnitine occur by changing the acetylation status of histone and non-histone nuclear proteins?

5.4. Control of the activity of mitochondrial enzymes by reversible lysine acetylation

The mitochondrial presence of several nicotinamide adenine nucleotide (NAD⁺)-dependent deacetylase silent information regulators (sirtuins, SIRT3, 4, and 5) recently has been described [146–148]. A proteomic survey of protein acetylation identified 388 acetylation sites on 195 proteins in mitochondria from HeLa cells and mouse liver, representing 20% of mitochondrial proteins; these proteins included those involved in the TCA cycle, fatty acid β-oxidation, amino acid and carbohydrate metabolism, membrane transport, and ETC [144]. We found that long-chain acyl-CoA synthetase 1 is acetylated at the lysine residue 633 in rat liver mitochondria [149]. Lysine acetylation neutralizes the positive charge and increases the hydrophobicity of the lysine side chain. While the functional consequence of this postranslational modification is unknown, these modifications should lead to changes in protein conformation and function. Mitochondrial matrix acetyl-CoA synthetase is reversibly acetylated at a lysine residue in the active site of the enzyme, and the SIRT3-induced deacetylation activates the enzyme [150]. Glutamate dehydrogenase is the known mitochondrial target for both SIRT3 [146] and SIRT4 [147] enzymatic activity and is inhibited upon deacetylation. A recent report shows that SIRT3 reversibly binds, decreases the acetylation status, and augments the activity of mitochondrial complex I [151]. These data clearly show that acetylation can control the activity of mitochondrial enzymes, and possibly de novo synthesis of acetyl-CoA in mitochondria. Since acetyl-CoA is the acetylation donor for all known acetyltransferases, the concentration of mitochondrial acetyl-CoA could be a limiting factor in the acetylation reaction; in fact, the K_m for acetyl-CoA is high (330 μM) for the skeletal muscle mitochondrial acetyltransferase [152]). By increasing the acetyl-CoA content, supplemented acetylcarnitine should increase the acetylation status of mitochondrial proteins (Table 1).

5.5. Increase in mitochondrial biogenesis

Mitochondrial biogenesis relies on a spatiotemporally coordinated synthesis and import of approximately 1000 nuclear-encoded proteins, some of which are assembled with mitochondrial-encoded proteins within newly synthesized inner and outer mitochondrial phospholipid membranes. The replication of mitochondrial DNA, as well as the mitochondrial fusion and fission mechanisms, also must be synchronized with these processes.

Oral acetylcarnitine supplementation in rats increases soleus muscle mitochondrial content, nuclear transcripts of factors involved in mitochondrial biogenesis (PGC-1α, NRF-1, TFAM), as well as the level of mitochondrial transcripts (COX I, ATP6, ND6, 16 S rRNA), and prevents the unloading-induced downregulation of mRNA levels of kinases able to transduce metabolic (AMPK) and neuronal stimuli (CaMKIIβ) [153]. Acetylcarnitine enhances the activity and amount of HO-1 in cell culture in a dose- and time-dependent manner [131]. Further, HO-1 was shown to increase mitochondrial biogenesis in cardiomyocytes via the transcriptional control of the nuclear respiratory factor-1 (NRF-1) [154]. According to the model proposed by Piantadosi et al. [154], endogenous CO generated by HO-1 binds the reduced a3 heme in cytochrome c oxidase and increases the superoxide production from complex III [155]. Superoxide induces MnSOD overexpression and is dismutated to hydrogen peroxide, which activates protein kinase B (Akt) [156]. Akt deactivates glycogen synthase kinase-3β, allowing the nuclear translocation of Nrf2 [157]. Nuclear Nrf2 binds to antioxidant response elements in the HO-1, MnSOD, and NFR-1 gene promoters, thus amplifying the initial signal and driving the transcription of TFAM and other genes that have promoter binding sites for NRF-1. These genes are involved in the control of mitochondrial transcription and protein synthesis, mitochondrial protein import, and oxidative phosphorylation [154].

A study from our laboratory shows that acetylcarnitine reverses the age-related decrease in the activity of complex III and oxidative phosphorylation through complex III and IV, and increases the amount of cytochrome b and aa3 hemes in cardiac mitochondria isolated from old rats [28]. Of interest, both cytochrome b and aa3 proteins are encoded by the mitochondrial genome, suggesting that acetylcarnitine enhances either mtDNA transcription, the stability of mitochondrial mRNA, or mitochondrial protein synthesis. Since the transcription of mtDNA is polycistronic, other proteins encoded by mtDNA -subunits of complexes I, IV and V- would be expected to increase. Because the yield of mitochondria was unchanged, this suggests that mitochondrial replication of mtDNA, which is under similar nuclear control and parallels the polycistronic transcription of mtDNA, was not increased upon acetylcarnitine supplementation. These data suggest that short-term administration of acetylcarnitine enhances the stability of mitochondrial transcripts or mitochondrial protein synthesis (Table 1). These data also are supported by the observation that the decreased levels of a ribosomal RNA (12S rRNA) and a messenger RNA (mRNA for the subunit I of complex IV) were reversed in the brain and cardiac muscle of old rats one hour after acetylcarnitine administration [103]. Furthermore, the increase in the content of TFAM, which controls mitochondrial DNA transcription and translation, remains in skeletal muscle one month after the withdrawal from chronic acetylcarnitine supplementation, indicating the long-lasting effect of acetylcarnitine on mitochondria [158]. In the adult heart, similar acute acetylcarnitine treatment did not increase OXPHOS or the activity of ETC enzymes, suggesting that a selective age-related signalling pathway, possible initiated by the complex III-generated superoxide [18], is essential for the effect of acetylcarnitine in the aged heart.

6. Conclusion and future perspectives

Age-related decreases in mitochondrial oxidative capacity contribute to cardiac pathology in the elderly. Protecting mitochondria should forestall the decrease in age-related decay of these organelles (decrease in gene expression and mitochondrial biogenesis, oxidative stress, apoptosis) and consequent organ failure. We review acetylcarnitine as a therapeutic agent with the ability to reverse the age-associated mitochondrial defect and its impact on age-related diseases, such as cardiac ischemia.

Two common mechanisms have been proposed for improvement of mitochondrial function in aging. Although cardiolipin has been touted as the target of acetylcarnitine, because the content of mitochondrial cardiolipin does not decrease with aging, this proposed mechanism is not plausible. In contrast, the experimental evidence is compelling and convincing that acetylcarnitine reverses the age-related decrease in mitochondrial DNA transcription and translation in brain and cardiac muscle. In addition, in the heart, similar data support the increased content of the mitochondrial-encoded ETC subunits, normalization of ETC complex activities, and restoration of oxidative phosphorylation, indicating that acetylcarnitine acts by increasing mitochondrial protein synthesis.

Since 20% of the mitochondrial proteins are lysine-acetylated, the reversible acetylation of the mitochondrial proteome represents an underappreciated, but fundamental, mechanism to affect the activity of mitochondrial proteins. While the functional effect of acetylation/deacetylation has been shown for only three mitochondrial proteins, this mechanism of regulation has tremendous potential for linking the altered mitochondrial proteome with mitochondrial function. Such a link could lead to the mapping of mitochondrial metabolic pathways affected by acetylation, particularly in aging. Further studies are necessary to define if factors governing mitochondrial protein synthesis, i.e., mitochondrial ribosomal proteins, are acetylated following acetylcarnitine supplementation, and if this specific postranslational modification increases the mitochondrial translational activity and the content of mitochondrial-encoded ETC subunits. Deciphering the mitochondrial mechanisms of the protective effects of acetylcarnitine will set a stage to translate these events into therapeutic strategies to improve the clinical picture of the elderly.

Figure 3. Proposed mechanism for the effect of acetylcarnitine on aged mitochondria.

The supplemented acetylcarnitine is transported into the cardiac mitochondria where the acetyl group favors the production of acetyl-CoA both directly and by activation of carnitine acetyltransferase (CAT), which is decreased in aging. The acetyl-CoA acts on the acetylation status of mitochondrial proteins, that increases mitochondrial transcription and protein synthesis. As a result, cytochrome b content increases, leading to increased activity of electron transport chain (ETC) complexes, and stimulating the oxidative phosphorylation (OXPHOS). This sequence of events leads to the restoration of the aging-related mitochondrial defect.