Protective Effect of Astaxanthin on Mitochondria As an Antioxidant

Many studies have observed various cellular and molecular changes in response to Astaxanthin treatment. Consequently, it can be challenging to determine which of these effects may be attributed to the direct mechanisms of action, such as its direct antioxidant activity or indirect downstream effects in response to chronic astaxanthin treatment. We focus below on the early changes resulting from acute exposure to astaxanthin to address this.

The mitochondrion is an organelle that produces energy by electron transport chain (ETC)/oxidative phosphorylation, and oxygen is consumed in this process. Most of the oxygen molecules entering the ETC are reduced to water, but a significant amount escapes in the form of ROS byproducts. Astaxanthin can significantly inhibit the lipid peroxidation of biological membranes. It has also been reported that astaxanthin added to cultured cells was transported to the mitochondria. Since most of the important components of the mitochondrial ETC are located within the inner membrane of mitochondria, astaxanthin is expected to protect mitochondrial membranes against oxidative damage caused by ROS. This is particularly relevant under conditions where ROS are overproduced, such as during states of metabolic stress caused by metabolic diseases and senescence. For example, astaxanthin was reported to be nephroprotective in a mouse model of diabetes mellitus and inhibit the generation of mitochondrial-derived ROS in human renal mesangial cells induced by hyperglycemic insults in vitro.

Astaxanthin inhibited the damaging effects of mitochondrial overload, including reduced muscle damage in rodents after heavy exercise, reduced oxidative modification of skeletal muscle proteins, and reduced inflammatory markers after treadmill exercise in mildly obese mice given a high-fat diet. These results suggest that astaxanthin may protect mitochondria from oxidative damage caused by ROS production when mitochondria are overloaded under conditions of physiological stress.

To investigate the antioxidant effect of astaxanthin on mitochondria, scientists examined PC12 cells, which are highly responsive to oxidative stress. This report challenged PC12 cells with antimycin A (AnA), which inhibits Complex III triggering ROS overproduction, resulting in cytotoxicity. Astaxanthin pre-treatment showed a time- and dose-dependent protective effect of AnA-treated PC12 cells, using sub-nanomolar amounts of astaxanthin. This treatment did not cause cell death in HeLa or Jurkat cells, which can utilize the glycolytic pathway, bypassing the mitochondrial ETC.

These results suggest that the addition of sub-nanomolar astaxanthin has a protective effect against oxidative damage caused by mitochondrial dysfunction in these cells. Interestingly, when organelle-localized redox-sensitive fluorescent proteins (roGFPs) were expressed in the cells, astaxanthin treatment did not change the cytoplasmic-reduced state under basal conditions or hydrogen peroxide (H2O2) treatment. Still, astaxanthin maintained a mitochondrial-reduced form under oxidative stress.

In addition, when evaluated by the fluorescence of MitoSOX, a dihydroethidium (DHE)-derived mitochondrial-selective superoxide probe, there was no decrease in the production of mitochondrial-derived superoxide in the presence of AnA. The lack of evidence for the direct scavenging of AnA-mediated superoxide by astaxanthin in this in vitro experimental model may be due to superoxide being diffused into the aqueous space. In contrast, astaxanthin remains in the mitochondrial inner membrane. Despite not observing the direct antioxidant activity of astaxanthin in this model, astaxanthin has exhibited physiological antioxidant activity or other physiological activities in some other studies, as will be discussed in later sections.

Concerning that consideration, although the addition of astaxanthin did not increase the membrane potential of basal cells, it was helpful in maintaining the membrane potential, which gradually decreased with incubation. These results suggest that although astaxanthin does not inhibit ROS formation, it could be effective in improving mitochondrial function by neutralizing ROS to curtail the downstream effect on mitochondrial membranes.

In a recent report from another group, skeletal muscle cells (Sol8 myotubes) derived from mouse soleus muscle were challenged by the addition of succinate, a substrate of Complex II and AnA that triggers the accumulation of ROS. ROS generated in the cells were observed using a fluorescent whole-cell superoxide probe (DHE), following the addition of AnA. Astaxanthin decreased the ROS-induced fluorescence in a concentration-dependent manner. Mitochondrial membrane potential was evaluated using JC-1 dye, which accumulates in mitochondria in the presence of mitochondrial membrane potential. Using JC-1 as an indicator of mitochondrial health and membrane integrity showed that the addition of astaxanthin alone did not change the basal mitochondrial membrane potential but did inhibit the decrease in membrane potential resulting from AnA-induced ROS accumulation.

Additional studies examined the ability of astaxanthin to protect mitochondrial membranes under various conditions triggering oxidative stress. Another study reported that astaxanthin helped save mitochondrial respiratory chain activity against Fe2+-induced lipid peroxidation in mitochondria isolated from vitamin E-deficient rats. Astaxanthin also had a protective effect against ROS-mediated angiotensin II (Ang II)-induced mitochondrial dysfunction in vascular smooth muscle cells (VSMCs) and normalized mitochondrial respiratory parameters in the presence of ROS.

Mitochondria can initiate programmed cell death in response to oxidative stress, also known as apoptosis. Oxidative stress disturbs intracellular Ca2+ homeostasis, resulting in excessive Ca2+ efflux from the endoplasmic reticulum and an influx into mitochondria, which subsequently triggers mitochondrial membrane permeabilization, loss of mitochondrial membrane potential and the release of mitochondrial pro-apoptotic factors.

It has been widely reported that astaxanthin prevents the ROS-induced Ca2+ influx into mitochondria, protects against mitochondrial dysfunction, and inhibits apoptosis. The role of astaxanthin in modulating mitochondrial-mediated activation of apoptosis is beyond the scope of this review. However, the authors acknowledge that there has been extensive research on this subject, which merits its dedicated literature review.

Although the effects of astaxanthin differ slightly depending on the cell type, detection system, and mitochondrial substrate and condition, all reports have indicated that astaxanthin has a protective effect on mitochondria, especially on membrane components. Thus, the antioxidant effects of astaxanthin on membranes are not isolated to a single cell strain.

Astaxanthin could act to maintain and protect the integrity of the mitochondrial ETC and oxidative phosphorylation against oxidative stress. However, the cells used in these studies underwent relatively long-term astaxanthin treatments, possibly to overcome the slow intracellular uptake of astaxanthin.

Thus, it is unclear whether the observed mitochondrial protective effects were due to the direct antioxidant action of astaxanthin, induction of antioxidant enzymes via the Nrf2-Keap1 pathway, or remodeling of mitochondria-related genes.

Therefore, the presence of astaxanthin-mediated regulation of mitochondria-related gene expression and its putative mechanisms are presented in the following sections.

1. Aggressive Enhancement of Mitochondrial Activity and Metabolism via Gene Expression by Astaxanthin

Astaxanthin improves glucose and lipid metabolism and muscle strength, mainly by correcting abnormal gene expression or protein modification in the mitochondria, which is altered during oxidative injury. These effects are mainly attributed to the antioxidant effects of astaxanthin.

ROS production due to decreased activity of the mitochondrial ETC is thought to be involved in energy overload and metabolic disturbances. Paradoxically, it is widely recognized that at physiological levels, ROS generated from mitochondria are also beneficial in improving metabolism in response to exercise.

Unfortunately, it is practically difficult to distinguish between the physiological levels of ROS and levels resulting in oxidative stress. Furthermore, the pharmacological effects of astaxanthin were considered too complicated to be explained by only its antioxidant effects as a single compound. Thus, the authors deemed other mechanisms of action of astaxanthin outside of its antioxidant activity.

2. Nrf2 Pathway

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that plays an important role in maintaining redox status and modulating inflammation and mitochondrial biogenesis and function. Nrf2 interacts with target genes at DNA binding sites called antioxidant response elements (AREs). Nrf2 activity is modulated by the Kelch-like ECH-associated protein 1 (Keap1)/Nrf2, epigenetic DNA elements, PI3K/Akt pathway, and other transcription factors. Nrf2 dissociates from Keap-1 and is translocated from the cytoskeleton in the cytosol into the nucleus, where it can induce gene expression in response to ROS. Dissociation of Nrf-2 from Keap-1 is facilitated by ROS and strong electrophilic compounds, like polyphenols and isothiocyanates.

Early studies of carotenoids showed that lycopene significantly activated Nrf2 via Nrf2/Keap1 dissociation, and later it was shown that the degradation products of lycopene were the primary active forms. Lycopene metabolite is a potent electrophilic compound and could be considered an inducer of Nrf2. The impact of astaxanthin on the Nrf2 pathway for various cell types and disease models has been described in other good review papers. However, it should be noted that it is unclear whether this is a canonical pathway via dissociation of Keap1 or the result of some indirect non-canonical activation pathway. Indeed, astaxanthin increases the expression of Nrf2 in specific pathological models and certain tissues.

To address the question of the Nrf2-mediated activation of antioxidant enzymes in response to astaxanthin, we used obese mice to evaluate the expression of antioxidant enzymes downstream of Nrf2 and other genes in various tissues. We found that even in epididymal adipose tissue, which was most affected by oxidative stress, gene expression of several Nrf2 targets was altered. Still, there was no significant change in the gene expression status of Gclc or Nqo1. An important finding was that when bone marrow-derived macrophages (BMDMs) isolated from wild-type and Nrf2-knockout mice were stimulated with lipopolysaccharide (LPS), astaxanthin reduced the accumulation of intracellular ROS, regardless of genotype. Thus, Nrf2 is unlikely to be involved in the reduction of intracellular ROS by astaxanthin.

Therefore, these results were confounding effects of other transcription factors, such as the peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1α), and it is doubtful whether the Nrf2/Keap1 pathway mediated this. In other words, we cannot deny the possibility that this is the result of enhancements in gene expression due to activation of the PGC-1α/Sirtuins pathway by astaxanthin and that Nrf2 is transferred to the nucleus as a result of oxidative stress rather than by the action of astaxanthin by the canonical Nrf2/Keap1 pathway.

In addition, it was recently reported that mouse carotene-9′,10′-oxygenase is a functionally palmitoylated enzyme that, upon binding to xanthophylls in the mitochondria, can be translocated into the nucleus via depalmitoylation. Once in the nucleus, it may attach to AREs, possibly associated with other transcription factors such as Nrf2, and then regulate downstream gene expression. It has been reported that mice with whole-body knockout of BCDO2 function developed metabolic dysfunction derived from the peripheral and hypothalamus, even when fed a diet thought to be free of carotenoids.

Importantly, failure of gene expression related to the antioxidant response, such as Nrf2, was observed frequently in the knockout mice used in these studies. In conclusion, although the level of influence of astaxanthin on this pathway is not known, it is suggested that carotenoids may activate Nrf-2 in a different way to the commonly known Nrf2/Keap1 pathway.

3. Nuclear Receptors

In rodents and primates, including humans, obesity caused by a high-fat diet is believed to induce insulin resistance, deteriorate glucose and lipid metabolism, and induce metabolic syndrome and type 2 diabetes (T2DM).

In contrast, it has also been reported that, in a high-fat diet, skeletal muscle mitochondria and their component proteins are increased, likely as a compensatory mechanism, causing mitochondrial dysfunction. It is strongly suggested that oxidative stress due to mitochondrial dysfunction is also involved in insulin resistance in adipose tissue and the liver. It has been reported that insulin resistance could be improved by astaxanthin. Although most anti-diabetic drugs target the liver or adipose tissue for their pharmacological action, research has shown in a hyperinsulinemic-euglycemic clamp study in obese mice that astaxanthin exerts its function not in the liver but in skeletal muscle and adipose tissue.

The skeletal muscle is the largest glucose metabolizing organ in the whole body and has plasticity, responding to exercise quality and quantity. We looked at the gastrocnemius muscle in astaxanthin-administrated mice; we found that gene expression was strongly altered in favor of glucose and lipid metabolism with or without obesity. This resulted in remodeling muscles to increase slow-twitch fibers containing more mitochondria and blood vessels.

This change in the quality of the skeletal muscle improved the endurance of the mice, which was consistent with other reports. Possibly, these changes may indicate that the reported effects of astaxanthin on capillary regression in immobilized muscle atrophy may be due, in part, to effects other than the antioxidant activity of astaxanthin.

Furthermore, the expression of mitochondria-related transcription factors was altered in this skeletal muscle. These effects were mainly found in the gastrocnemius muscle, with more minor changes in other skeletal muscles (unpublished data). Of particular interest was the upregulation of gene expressions of a series of members of the peroxisome proliferator-activated receptor family and estrogen receptor-related family of genes such as Ppargc1a. Interestingly, the gene expression of mitochondria-associated Sirtuins was also significantly increased.

It has been reported that changes in the muscle expression of these genes can lead to enhanced lipid utilization, vascularization, and improved insulin resistance in obesity.

In addition, 2-thiobarbituric acid reactive substances (TBARS), a marker of oxidative stress, were unchanged from the control mice. There were no systematic changes in the expression of inflammatory cytokine genes, suggesting that they probably did not depend on antioxidant activity.

Considering this, it is possible that astaxanthin and its derivatives directly regulate nuclear transcription factors as ligands. For example, astaxanthin is known to regulate the gene expression of peroxisome proliferator-activated receptor (PPAR) family members and is often recognized as a ligand. It was revealed that astaxanthin bound to PPARγ by CoA-BAP assays in a dose-dependent manner, acting as partial inhibitors to regulate parts of the genes of PPARγ targets in vitro studies, using PPARγ reporter assays in adipocytes and macrophages.

It has been reported that astaxanthin regulates the gene expression of ATP-binding cassette transporters (ABC) A1 and G1, which are key molecules in cholesterol efflux from macrophages, the first step in reverse cholesterol transport a major anti-atherosclerotic property of high-density lipoprotein (HDL). This effect is mainly due to activation of the liver X receptor (LXR) complexes with PPARγ or other nuclear receptors, such as all-trans retinoic acid receptors (RARs) and retinoid X receptors (RXRs), then transcriptional regulation by binding to LXR-responsive elements. Intriguingly, when a human ABCA1/G1 promoter-reporter assay was performed, astaxanthin activated both promoters with or without LXR-responsive elements, indicating LXR-independence in these activations. This raises the possibility that astaxanthin, or its metabolites, partially bind to nuclear receptors such as RARs, RXRs, and PPARs, but not their full activation (such as full-agonist/antagonist), thus partially regulating their activity (such as partial agonist/antagonist).

Apocarotenoids, the primary metabolites of carotenoids by BCDO2 and oxidation, have also been shown to affect these nuclear receptors. A few pieces of information are available to shed some light on this putative pathway. Apo-canthaxanthinoic acids are metabolites of canthaxanthin that possess an astaxanthin-like 4-keto group. One canthaxanthin metabolite, 4-oxoretinoic acid, significantly enhances connexin 43 mRNA stability by binding to its 3′-UTR, which upregulates the expression of this component of gap junctions that mediates intercellular communication.

Moreover, 4-oxoretinoic acid also activates the retinoic acid-beta2 receptor (RXRβ2) to stimulate gap junction communication. Regarding astaxanthin, it is known that astaxanthin derivatives regulate the expression of connexin 43 and that astaxanthin itself enhances the expression of connexin 43. However, dependent on phosphorylation-mediated modifications by astaxanthin dose, connexin 43 activity is affected. Since 3-hydroxy-4-oxo-β-ionone and its reduced form 3-hydroxy-4-oxo-7,8 dihydro-β-ionone were found in human plasma as metabolites of astaxanthin, they may be responsible for mediating this activity. These results suggest that astaxanthin may also be a partial agonist of RARs and RXRs, although it is much weaker than all-trans retinoic acid.

Interestingly, we have also shown an effect of carotenoids, including astaxanthin, on retinoic acid-related orphan receptor gamma t (RORγt) as a receptor mediating CD4+ T cell differentiation into Th17 cells.

In summary, when naïve mouse T cells were treated with IL-1β, IL-6, IL-23, and anti-IFN-γ antibodies to induce pathogenic Th17, astaxanthin suppressed pathological Th17 maturation. It reduced the gene expression of IL-17A, which plays a vital role in the development of pathogenicity.

However, it does not affect the expression of IL-17F, which is involved in intestinal biological defense.

In other reports of Th17 induction by addition of TGF-β and IL-6, including non-pathogenic Th17, only fucoxanthin among various carotenoids exhibited significant inhibition of secretion of IL-17, which may be found both as IL-17A and IL-17F. Focusing on the differences between the two studies, our study was more affected by the RORγt induction of Th17 cells, suggesting that perhaps carotenoids or their derivatives, including astaxanthin, can function as antagonists of RORγt. The activity itself is probably weak, but it may impact chronic inflammation and immunity in tissues with high exposure, such as in the intestine.

In mice, astaxanthin significantly accumulated in adipose tissue and liver, indicating that the activities showed above probably contribute to the pharmacological effects of astaxanthin on nuclear receptors.

However, it is necessary to consider species differences in the effects on nuclear receptors, especially the PPAR family. For example, it is known that astaxanthin and its metabolites induce cytochrome P450 (CYPs), such as CYP1A1, CYP1A2, CYP3A4, and CYP2B6 in rodent hepatocytes, probably via PPARα activation by astaxanthin. However, this effect requires several tens fold higher concentrations in human hepatocytes than in rats. Furthermore, since the beneficial effects of astaxanthin on metabolisms and skeletal muscle function have been shown in human clinical trials, the actual contribution of PPARs might be minor.

It is suggested that there may be mechanisms of action that are less sensitive to species differences, such as specific antioxidant activities and other mechanisms. Based on this idea, we investigated the mechanism of action; as one of the astaxanthin targets, we have identified “AMP-activated protein kinase” (AMPK).

4. AMPK/Sirtuins/PGC-1α Pathway

AMPK is a key sensor of cellular energy status present in essentially all eukaryotes. It is a heterotrimer comprising a catalytic α subunit and regulatory β and γ subunits. AMPK plays a crucial role in energy metabolism, including lipid, glucose, and protein metabolism, and is important for mitochondrial biogenesis and quality control.

In recent years, AMPK has received much attention for its important role as a target of metformin, thiazolidinediones, and exercise therapy to treat T2DM and related metabolic diseases. In skeletal muscle, AMPK and SIRT1/PGC-1α work together to mediate metabolic adaptation during fasting and exercise. These reciprocal enhancements of activity result from the direct induction of Pgc1a gene expression by AMPK, the enhancement of activity via deacetylation of PGC-1α by SIRT1, and the increase in intracellular amounts nicotinamide adenine dinucleotide (NAD+) by the induction of Nampt gene expression by AMPK. These unique interactions are discussed in detail in another good review. Previous studies have shown that astaxanthin increases the levels of PGC-1α in skeletal muscle. To determine if the upregulation of PGC-1α in response to astaxanthin was mediated by AMPK, we examined PGC-1α expression using a mouse skeletal muscle cell line (C2C12 cells), following the knockdown of AMPKα1/2 expression.

We observed that AMPKα1/2 knockdown abolished the increased expression of PGC-1α in response to astaxanthin, indicating that astaxanthin directly stimulates AMPK. This suggests that the effect of astaxanthin in upregulating PGC-1α levels in skeletal muscle occurs via an AMPK-dependent pathway.

5. Astaxanthin Contributes to Mitochondrial Quality Control

Astaxanthin also probably has a beneficial effect on mitochondrial quality control, mainly through AMPK activation. It has been reported that astaxanthin can prevent pulmonary fibrosis by promoting myofibroblast apoptosis through dynamin-1-like protein (Drp1)-mediated mitochondrial fission.

Furthermore, AMPK phosphorylates and activates mitochondrial fission factor (MFF), which associates with Drp1, leading to mitochondrial fission. These reports use experimental models with mitochondrial dysfunction, such as cancer cells, which describe a beneficial aspect of astaxanthin mitochondrial quality control. In skeletal muscle, Drp1 is upregulated during acute phase exercise where mitochondrial fission is induced.

In addition, Drp1 may play an important role in processing exercise-impaired mitochondria since Drp1 deficiency reduced muscle endurance and running performance and altered muscle adaptations in response to exercise training.

On the other hand, astaxanthin has a protective effect on mitochondria against heat stress and Ang II-induced mitochondrial dysfunction. At this time, it normalizes the upregulation of the Drp1 gene expression caused by the damage. It has also been reported that astaxanthin activates autophagy and inhibits apoptosis in Helicobacter pylori-infected gastric epithelial cell line AGS via AMPK-mediated phosphorylation of Unc-51-like autophagy-activating kinase 1 (Ulk1).

In addition, during AngII-induced mitochondrial damage to VSMCs, astaxanthin treatment resulted in the mitophagy-mediated induction of Parkin, PTEN-induced kinase 1 (Pink1), and autophagosome activation. Astaxanthin induces the gene expression of sirt-3, probably via ERRα or ERRγ and PGC-1α. Sirt-3 plays a crucial role in mitochondrial dynamics and contributes to mitochondrial quality control. The quality control for dysfunctional mitochondria by astaxanthin seems to be achieved by AMPK and related signaling pathways.

6. Is the AMPK-Activating Effect of AstaxanthinIndependent of Its Antioxidant Effect?

From large-scale epidemiological studies, it is well known that moderate exercise increases energy expenditure and improves obesity, thereby preventing and improving T2DM. Interestingly, as an epidemiological intervention study, the actual incidence of T2DM was followed up for about three years in thousands of subjects diagnosed with a high risk of developing T2DM, and it was found that the incidence of T2DM was significantly reduced when metformin, an oral biguanide-derived antidiabetic, and AMPK activator, was taken before the onset of the disease. Collectively, moderate exercise-dependent or -independent activation of AMPK is a beneficial strategy for preventing the incidence of T2DM and improving energy metabolism.

The beneficial effects of moderate exercise on the metabolism are partially mediated by ROS, which involves the activation of AMPK by a physiological level of mitochondrial ROS. Exercise-induced activation of AMPK depends on the physiological mechanisms of muscle contraction, e.g., energy depletion, increasing influx of Ca2+, activation of MAPK pathways (e.g., p38) by mitochondrial ROS, and direct oxidative modification of AMPK.

Therefore, the administration of antioxidants may hurt exercise therapy for glucose or lipid intolerance in T2DM and metabolic syndrome. The chronic administration of certain antioxidants counteracts the glucose tolerance that is improved by exercise therapy and training-induced adaptations in endurance performance.

Thus, it is still controversial whether many other antioxidants, including astaxanthin, can help improve exercise performance. Astaxanthin may be beneficial for skeletal muscle damage after high-intensity endurance exercise. However, the influence of astaxanthin on mild to moderate-intensity aerobic exercise used to address ROS levels in T2DM and obesity is less clear.

Recently, it has been reported that the antioxidative hepatocyte selenoprotein P (SeP) caused a physiological effect in skeletal muscle called “exercise resistance”. Exercise resistance decreases the therapeutic effects of exercise as an intervention for glucose intolerance by inhibiting the ROS-mediated activation of AMPK. Exercise resistance has also correlated with the plasma concentration of SeP in humans.

Therefore, it is necessary to carefully judge whether the chronic administration of astaxanthin will cause exercise resistance. Astaxanthin further enhanced lipid utilization in obese mice with daily exercise training. We also evaluated glucose tolerance in obese mice that received daily exercise and found that astaxanthin, together with practice, improved glucose tolerance in an additive manner.

In further in vitro cell studies using C2C12 myotube, we evaluated the phosphorylation of Thr172 in AMPKα by adding H2O2, a ROS that activates AMPK. When comparing the effect of antioxidants on ROS-mediated AMPK phosphorylation, the H2O2 scavenger, N-acetylcysteine (NAC), inhibited AMPK phosphorylation, whereas astaxanthin did not.

Therefore, it was concluded that astaxanthin does not interfere with the beneficial effects of exercise therapy. This result may reflect that astaxanthin is less effective in neutralizing H2O2 than other kinds of ROS. Another explanation may be that astaxanthin and AMPK may be localized at different sites in the cell.

It has been reported that the phosphorylation of Thr172 residue of AMPKα is dephosphorylated by protein phosphatase 2A (PP2A), a serine-threonine protein phosphatase, resulting in the inhibition of AMPK activity. We also observed that astaxanthin increases PP2A phosphorylation and decreases its activity at the basal state, concurrent with the enhancement of insulin-induced Akt phosphorylation in L6 cells.

As PP2A itself is of low sensitivity to the ROS, it appears that the ROS-related activation of PP2A requires a cofactor such as the Src kinase family, which is also known to be resident in lipid raft, and activated under the change in redox balance. Astaxanthin may activate this kinase by modifying the redox balance at the membrane. Therefore, the antioxidant activity of astaxanthin may also affect the activation of AMPK by ROS, although this is complicated by the localization and type of ROS.

There are also two types of AMPKα subunit: AMPKα1, which is localized in the cytosol; and AMPKα2, which is localized in the mitochondria or nucleus. It is still unknown which one can be activated by astaxanthin. The functions of these two proteins are distinct. It has recently been shown that AMPKα2, but not AMPKα1, activates the transcription of the PGC-1α gene by translocating into the nucleus when activated.

The activation of AMPKα2 is required to maintain skeletal muscle integrity. AMPKα1 contributes to myogenesis’s middle stage and regulates the ACC-mediated mitochondrial incorporation of fatty acids and autophagy.

Furthermore, the activation of AMPKα2 contributes to inducing the gene expression of ERRα and ERRγ, although the response varies by tissue. ERRα and ERRγ cooperate with PGC-1α to regulate the expression of a group of genes involved in mitochondria-related genes, muscle fiber, angiogenesis, hypoxic response, and myogenesis, which is beneficial for energy metabolism in mitochondria.

Moreover, in the skeletal muscles of astaxanthin-treated obese and lean mice, the gene expression levels of Sirtuins such as Sirt1 and Sirt3 were upregulated, and Nampt was also upregulated, which is closely related to AMPK activation. Intracellular NAD+ levels were significantly elevated when C2C12 myoblast cells were treated with astaxanthin, compared with the solvent vehicle group.

Therefore, astaxanthin likely activates Sirtuins via activating de novo synthesis of NAD+. It is reasonable to conclude that this is the result of the activation of both AMPKα1 and AMPKα2 because the activation of AMPKα1 by astaxanthin enhances the phosphorylation of ACC, resulting in increased lipid incorporation, and the activation of AMPKα2 also causes changes in gene expression in skeletal muscle.

However, the activation intensity was significant only in skeletal muscles with high plasticity or mixed muscle fibers, such as the gastrocnemius.

Meanwhile, skeletal muscle mass was not significantly increased, suggesting that it is reasonable to position astaxanthin as a partial exercise mimetic. Collectively, we believe that astaxanthin administration in skeletal muscle forms a positive feedback loop via AMPK activation on mitochondrial biogenesis and energy metabolism by

(1) activation of Sirtuins by regulating NAD+ levels;

(2) activation of PGC-1α by Sirtuins;

(3) induction of gene expression of Sirtuins by increasing ERRs expression; and

(4) their concerted action.

In addition to the above, there is another important mechanism of astaxanthin mitochondrial activation, including AMPK activation, which is believed to be through the action of adiponectin and its receptors. It has been reported that the administration of astaxanthin significantly increases the amount of adiponectin in the blood, and its gene expression in adipose tissue, in obese animals and humans. Adiponectin is an adipokine with beneficial aspects secreted by smaller adipocytes. Its gene expression is regulated by PPARγ, which plays a role in the beneficial effects of its agonist, antidiabetic drug thiazolidinediones.

Although astaxanthin is a partial modulator of PPARγ, it does not seem to have any inhibitory effects on adiponectin gene expression. The adiponectin receptors are AdipoR1 and AdipoR2, which are expressed at different levels in different tissues, and are involved in the regulation of glucose and fatty acid metabolism, mainly through the activation of Ca2+ signaling, AMPK/SIRT1, and PPARα signaling pathways. It has been reported that astaxanthin has an inhibitory effect on Ca2+ signaling, which is mainly involved in ROS.

Still, its effect on Ca2+/calmodulin-dependent protein kinase β (CaMKKβ), which activates AMPK is unknown.

Thus, to summarize what is currently known, oxidative stress decreases the amount of adiponectin and its receptors. In contrast, astaxanthin prevents or increases the amount of adiponectin and its receptors, possibly leading to the activation of AMPK.

In recent years, mitochondria have been implicated in various processes related to aging, including senescence, inflammation, and further age-related functional impairment of tissues and organs. In skeletal muscle, the relationship between mitochondrial dysfunction and insulin resistance during aging is confounded by many factors, suggesting some association, although this is complex.

For example, age-related mitochondrial dysfunction raises the level of ROS release from mitochondria, which induces phosphorylation of serine in IRS proteins and disturbs insulin signaling, resulting in insulin resistance. Astaxanthin regulates insulin signaling.

We have shown that astaxanthin inhibits the serine phosphorylation of the IRS-1 protein by ROS. However, we cannot eliminate the possibility that this effect is acute and unrelated to the mitochondria-mediated response.

Additionally, as shown many times, we have reported that astaxanthin potentially improves mitochondria function via AMPK/Sirtuins/PGC-1α pathway.

Furthermore, astaxanthin potentially elevates the level of NAD+ in cells. More importantly, it has been revealed that increasing the intracellular NAD+ concentration may improve age-related mitochondrial dysfunction and insulin resistance, which has attracted the researcher’s attention.

Attempts to increase NAD+ levels in vivo, such as supplementation with nicotinamide mononucleotide (NMN), an intermediate in the biosynthesis of NAD+, have potentially ameliorated age-related insulin resistance and improved mitochondrial function.

Regarding the beneficial effects of astaxanthin on age-related metabolic changes, the authors preliminarily evaluated the effects of astaxanthin on insulin resistance and glucose intolerance with aging using male C57BL/6J mice.

In mice, glucose intolerance and insulin resistance occur with age, compensatory islet expansion, and hyperinsulinemia. However, this is still controversial depending on feeding conditions, genetic background, and sex. Astaxanthin was administered for about one year, corresponding to middle age, when metabolic abnormalities would occur in humans. Continuous administration of astaxanthin to mice on a standard chow diet significantly improved age-related insulin resistance and glucose intolerance.

It is also well known that prediabetes and Alzheimer’s disease increase in prevalence with age. Recently, a direct connection was revealed between peripheral hyperinsulinemia, as found in prediabetes, and age-related neurodegeneration and cognitive decline, such as Alzheimer’s disease. The role of astaxanthin in cognitive function is beyond the scope of this review.

However, the authors acknowledge that there has been extensive research on this subject, which merits its dedicated literature review. In human clinical trials and animal models, astaxanthin has been shown to be beneficial in cognitive decline associated with aging. Although most of the discussion of the mechanism of action is based on the antioxidant effects of astaxanthin, the authors would like to suggest that this should be expanded to include mechanisms such as described in this section.

Based on these findings, as a strategy for preventing age-related diseases and extending lifespan and supplementation with NAD+ intermediates such as NMN, astaxanthin may also have beneficial effects on age-related diseases. Even in clinical trials, astaxanthin has been shown to help extend the walking distance and increase muscle strength in the elderly. Therefore, astaxanthin is also an attractive potential candidate for anti-aging.

To summarize this section, astaxanthin treatment has been seen to (i) significantly ameliorates insulin resistance and glucose intolerance through AMPK activation; (ii) stimulate mitochondrial biogenesis in the muscle; (iii) enhance exercise tolerance and exercise-induced fatty acid metabolism.

7. Direct Effects of Astaxanthin on Mitochondria: Actions beyond Its Antioxidant Activity

It is still unclear how astaxanthin directly affects mitochondria, besides its antioxidant activity, including its redox-mediated PP2A inhibition of AMPK activation.

However, looking at astaxanthin’s pharmacological efficacy shown in the above section, it is very difficult to explain its physiological activities by only referencing astaxanthin’s antioxidant activity. Astaxanthin may physically affect mitochondrial membrane dynamics due to its rigid and long conjugated double bonds and terminal polar groups.

Astaxanthin may affect the function of mitochondrial membranes and membrane proteins by a mechanism similar to that described for insulin signaling, whereby astaxanthin modulated the association of adaptor proteins with insulin receptors in the plasma membrane. To support the notion that astaxanthin modulates mitochondrial membrane proteins, we can refer to a few reports on the effects of astaxanthin on the mitochondrial respiratory chain proteins.

The evaluation of the mitochondrial respiratory chain in HeLa cells showed that the addition of astaxanthin increased oxygen consumption in the basal condition, but did not observe any significant changes in the maximal oxygen consumption in the presence of the mitochondrial uncoupler, oligomycin.

Therefore, the baseline to uncoupled oxygen consumption ratio was significantly higher in the presence of astaxanthin. However, this effect may also be mediated by changes in gene expression in response to astaxanthin administration to the cells.

One report took a different approach to study mitochondrial protection, using rat heart mitochondria. They found that astaxanthin inhibited the opening of mitochondrial permeability transition pores (mPTPs), which are large-conductance channels formed after the overload of Ca2+, or in response to oxidative stress. Astaxanthin treatment in this model also slowed down the mitochondrial swelling associated with the opening of mPTPs.

When glutamate and malate were used as substrates in isolated rat heart mitochondria, and astaxanthin was added at a relatively high concentration of 5 μM, it decreased State 4 respiration with increased ADP-dependent State3 respiration conjugated with ATP synthesis was observed. The respiratory control index (RCI), which indicates the effectiveness of the mitochondria in promoting oxidative phosphorylation and coupling between oxygen consumption and ATP production, also increased with astaxanthin treatment.