Preventive effects of Chlorella on skeletal muscle atrophy in muscle-specific mitochondrial aldehyde dehydrogenase 2 activity-deficient mice
© Nakashima et al.; licensee BioMed Central Ltd. 2014
Received: 23 May 2014
Accepted: 7 October 2014
Published: 11 October 2014
Oxidative stress is involved in age-related muscle atrophy, such as sarcopenia. Since Chlorella, a unicellular green alga, contains various antioxidant substances, we used a mouse model of enhanced oxidative stress to investigate whether Chlorella could prevent muscle atrophy.
Aldehyde dehydrogenase 2 (ALDH2) is an anti-oxidative enzyme that detoxifies reactive aldehydes derived from lipid peroxides such as 4-hydroxy-2-nonenal (4-HNE). We therefore used transgenic mice expressing a dominant-negative form of ALDH2 (ALDH2*2 Tg mice) to selectively decrease ALDH2 activity in the muscles. To evaluate the effect of Chlorella, the mice were fed a Chlorella-supplemented diet (CSD) for 6 months.
ALDH2*2 Tg mice exhibited small body size, muscle atrophy, decreased fat content, osteopenia, and kyphosis, accompanied by increased muscular 4-HNE levels. The CSD helped in recovery of body weight, enhanced oxidative stress, and increased levels of a muscle impairment marker, creatine phosphokinase (CPK) induced by ALDH2*2. Furthermore, histological and histochemical analyses revealed that the consumption of the CSD improved skeletal muscle atrophy and the activity of the mitochondrial cytochrome c oxidase.
This study suggests that long-term consumption of Chlorella has the potential to prevent age-related muscle atrophy.
KeywordsMuscle atrophy Chlorella Aldehyde dehydrogenase 2 Mitochondrial cytochrome c oxidase Oxidative stress
Excessive production of reactive oxygen species (ROS) causes oxidative damage to DNA, proteins, and lipids. This damage accumulates with age in various organs, including the skeletal muscle, as observed in both humans [1–4] and animal models [5, 6]. Thus, it is hypothesized that some antioxidants may aid in preventing age-related disorders, including muscle atrophy, such as sarcopenia.
We have sought to develop animal models with enhanced oxidative stress and various impairments that are affected by age [7, 8]. Aldehyde dehydrogenases (ALDH) catalyze the conversion of reactive aldehydes to carboxylates . Mitochondrial ALDH2 is known to oxidize acetaldehyde produced from ethanol into acetate , and a single nucleotide polymorphism in this gene found in Asian populations, ALDH2*2, produces a dominant-negative protein that prevents this activity. We have previously revealed, through a molecular epidemiological analysis, that a higher concentration of lipid peroxides are present in the sera of ALDH2-deficient females than in those expressing active ALDH2 . Furthermore, we demonstrated that ALDH2 deficiency is a risk factor for late-onset Alzheimer’s disease , suggesting a role for this polymorphism in human diseases. Recently, ALDHs have emerged as an important enzyme in a variety of human pathologies. ALDH2 dysfunction contributes to a variety of human diseases including diabetes, cancer, cardiovascular diseases [13–15], neurodegenerative diseases, stroke, Fanconi anemia, pain, osteoporosis, and the aging process .
In previous examinations of ALDH2*2, we showed that mitochondrial ALDH activity was repressed when murine ALDH2*2 was stably expressed in the neuronal cell line PC12. Cells expressing ALDH2*2 were also vulnerable to 4-hydroxy-2-nonenal (4-HNE), and treatment with 4-HNE or antimycin A was shown to induce cell death [17, 18]. Additionally, ALDH deficiency enhanced oxidative stress through a vicious cycle .
A Tg mouse model expressing ALDH2*2 specifically in the brain decreased the ability of mice to detoxify 4-HNE in cortical neurons and accelerated the accumulation of 4-HNE in the brain . Consequently, these mice presented with age-related neurodegeneration accompanied by memory loss after maturation. Mice deficient in muscle-specific mitochondrial ALDH2 activity were also developed by inducing the transgenic expression of ALDH2*2 under the control of the actin promoter . These model animals will be helpful in investigating the antioxidant properties of health foods in vivo, as well as in studies examining the prevention of oxidative stress-related muscle atrophy.
Sarcopenia is the decline of muscle mass and strength that occurs with aging . Since the progression of sarcopenia induces significant physical depression [20–22], this condition increases the risk of fractures due to fall and the possibility of becoming bedridden in elderly people. A central mechanism in the pathogenesis of sarcopenia is oxidative stress , which has been detected by the accumulation of several oxidative stress markers. These aldehyde species, which primarily include malondialdehyde (MDA) and 4-HNE, are spontaneously generated from lipid peroxides . Interestingly, 4-HNE is a strong electrophile that rapidly reacts with most biomolecules .
Chlorella, a unicellular green alga, contains a variety of nutritional components that are rich in protein, fatty acids, dietary fiber, chlorophylls, minerals, vitamins, and carotenoids. Thus, dried Chlorella powder or extracts in hot water have long been used as a health supplement in Asia. It has been reported that Chlorella elicits various immunopharmacological effects [26–28] and functions as an antioxidant in vitro and in vivo[29–37]. This is likely because it is rich in carotenoids and other antioxidants, including lutein, β-carotene, α-tocopherol, and ascorbic acid. We have already demonstrated that long-term consumption of Chlorella did not significantly affect the weight of any organs in wild type rats , and that long-term Chlorella consumption prevents oxidative stress, age-dependent cognitive decline, and central nervous system disorders in Tg mice expressing ALDH2*2 in the brain . Thus, Chlorella supplements have displayed antioxidant effects in a variety of animal experiments. However, it is unknown whether the consumption of Chlorella has an antioxidant effect in muscle tissues. Importantly, inflammation may cause sarcopenia in addition to oxidative stress. Since Chlorella extract slight decreased the expression of the pro-inflammatory cytokine IL-6 in mice , we here focused on the effects of oxidative stress.
In this study, we fed Chlorella to mice expressing a dominant negative, muscle-specific form of mitochondrial ALDH2 for 6 months. Our findings suggest that this supplement has the potential to mitigating skeletal muscle atrophy.
Animals and treatment
Composition of experimental diets supplemented with Chlorella powder
Control and wild type groups
Dextrinized corn starch
Vitamin mix (AIN-93-VM)a
Mineral mix (AIN-93-MX)a
Total weight (g)
Chlorella powder composition
Chlorella powder (per 100 g powder)
All mice were anesthetized and sacrificed prior to blood sample collection from the hepatic portal vein. Plasma was subsequently obtained by centrifugation at 3000 × g for 15 min at 4°C. The liver, kidneys, heart, lung, spleen, right gastrocnemius muscle, and adipose tissue (epididymal) of each mouse were quickly excised and weighed. For histological and histochemical studies, the left gastrocnemius muscle was frozen in hexane with Optimal Cutting Temperature (OCT) Compound (Sakura Finetek Japan, Tokyo, Japan) at -80°C. The quadricep muscle was washed with cold phosphate buffered saline (pH 7.4) and frozen in liquid nitrogen. All samples were stored at -80°C until use.
The plasma activities of creatine phosphokinase (CPK) and creatine phosphokinase-MB (CKMB) were measured using a DRI-CHEM autoanalyzer (FUJIFILM, Tokyo, Japan).
Bone density measurement
For computed tomography analysis of bone density, the whole bodies of the mice were scanned using a LaTheta LCT-100 experimental animal computed tomography system (Aloka, Tokyo, Japan). Contiguous 1-mm thick slices were used for quantitative assessment using LaTheta software (ver 1.00). Bone density was evaluated quantitatively.
Analysis of urinary oxidative stress
In order to measure urinary oxidative stress markers, mice were placed in a urine-sampling cage for 12 h at 2 and 4 months after the experiment began, and urine was collected. Urinary isoprostane levels were determined using a urinary isoprostane F2t ELISA kit (JaICA, Fukuroi, Japan), according to the manufacturer’s instructions. The creatinine concentration of each sample was measured using a LabAssay Creatinine kit (Wako, Osaka, Japan). Urinary isoprostane levels were normalized against creatinine concentration.
Measurement of oxidative stress in skeletal muscle
MDA and 4-hydroxyalkenals (HAE) levels in the quadriceps muscles were determined using a Bioxytech LPO-586 assay kit (OxisResearch, Oregon, USA). Briefly, pieces of the quadriceps muscle were homogenized in phosphate-buffered saline (pH 7.4) containing 5 mM butylated hydroxytoluene. After homogenization, the samples were centrifuged at 3000 × g for 10 min at 4°C, and the clear supernatants were subjected to the LPO-586 assay. MDA and HAE levels were assayed using the methanesulfonic acid solvent procedure according to the manufacturer’s instructions. The LPO-586 assay is based on the reaction of a chromogenic reagent, N-methyl-2-phenylindole, with MDA and HAE at 45°C. These compounds react with N-methyl-2-phenylindole to yield a stable chromophore with a maximal absorbance at 586 nm. The absorbance of the resultant samples was measured at 586 nm. The protein concentration of each sample was measured using a Pierce BCA protein assay kit (Thermo Scientific, Rockford, USA). MDA and HAE levels were normalized against protein concentration.
Histological and histochemical studies
Frozen gastrocnemius muscle samples were sliced into sections (8-μm thick) and mounted on silane-coated glass slides. Frozen sections were dried and stained with hematoxylin and eosin (H&E). For enzymatic cytochrome c oxidase histochemical staining, frozen sections were dried and incubated in 0.1 mol/L sodium phosphate (pH 7.4), 0.5 mg/ml 3,3-diaminobenzidin (DAB, Wako), 130 μg/ml catalase (Nacalai tesque, Kyoto, Japan), and 1 mg/ml cytochrome c (Nacalai tesque) at room temperature for 60 min. The cross-sectional area of the gastrocnemius muscle cells and the area of stained cytochrome c oxidase were calculated using the Image J (ver1.41; National Institutes of Health, Bethesda, MD), and they are presented as the percent ratio (%) versus wild type from 3 different points for each mouse.
All values shown are the mean ± SD. One-way ANOVA (Fisher’s PLSD test) followed by contrast testing was used to compare the data from multiple groups. Relationships between given variables were examined by linear regression analysis and the Pearson correlation coefficient. All experiments were examined in a blinded fashion, and statistical significance was accepted as p <0.05.
Consumption of a Chlorella-supplemented diet reduces oxidative stress and reverses skeletal muscle impairment
Excess oxidative stress is known to impair muscle. To evaluate the extent of muscle impairment in the ALDH2*2 Tg mice, we measured the levels of CPK and CKMB expression in plasma. The ALDH2*2 Tg mice had increased levels of both proteins; however, CPK levels were significantly reduced after CSD administration (Figure 1c). A trend toward reduced of CKMB levels was also observed (Figure 1d). These results suggest that the consumption of Chlorella reduces oxidative stress and reverses skeletal muscle impairment in ALDH2*2 mice (Figure 1).
Effect of Chlorella-supplemented diet consumption on body size and skeletal muscle atrophy
Effects of Chlorella consumption on bone density and organ weights
Wild type group
Bone density (mg/cm3)
410.9 ± 24.0#
426.5 ± 21.8
442.0 ± 21.5
Organ weights (mg)
1230.5 ± 134.6##
1265.2 ± 140.0##
2082.4 ± 391.9
224.4 ± 37.6
238.4 ± 25.5
237.9 ± 32.6
129.3 ± 16.0##
135.1 ± 7.6##
165.5 ± 6.8
212.8 ± 23.3##
212.2 ± 27.0##
263.8 ± 50.9
54.0 ± 6.9##
60.5 ± 9.4##
80.1 ± 11.1
107.9 ± 10.8##
129.8 ± 19.6*,##
189.0 ± 8.4
521.7 ± 265.3##
824.2 ± 144.9*,##
1044.8 ± 174.4
Protective effects of Chlorella-supplemented diet consumption on mitochondrial dysfunction in ALDH2* 2 Tg mice
The present study demonstrated that long-term intake of Chlorella reduced oxidative stress, as determined by changes in oxidative stress markers, including urinary 15-isoprostane and muscle MDA and HAE in transgenic mice with enhanced oxidative stress. This improvement resulted in decreased disintegration of muscle, plasma CPK, and plasma CKMB, and increased bone density, organ weight, muscle cell size, and total body weight. Moreover, mitochondrial activity was improved by long-term intake of Chlorella in the oxidative stress-enhanced mice.
Oxidative stress in skeletal muscle is associated with the atrophy, loss of muscle function, and fibers in sarcopenia [23, 42]. Thus, it is important to reduce oxidative stress in our daily lives. Indeed, epidemiological studies of community-dwelling older adults have demonstrated that the low carotenoid level in blood is associated with low skeletal muscle strength and the development of walking disabilities . These reports further indicate that dietary carotenoid intake is efficacious in the prevention age-related muscle disorders.
It is unknown why Chlorella was effective in reducing oxidative stress. As Chlorella contains various dietary antioxidant substances, including carotenoids and vitamins, it could be a potential dietary source of these compounds. Additionally, Chlorella also has a large chloroplast, in which plastoquinone substitutes for ubiquinone as an electron carrier in the photosynthetic electron-transport chain. Plastoquinone has been shown to have greater antioxidant properties than ubiquinone  and does not pose a danger for pro-oxidant effects within a range of concentrations . Therefore, the effect of Chlorella is likely synergistic between the plastoquinone and carotenoids provided in the CSD, thereby protecting against the impairments observed in ALDH2*2 Tg mice. Indeed, we have previously shown that Chlorella consumption reduces oxidative stress (4-HNE) in the dentate gyrus of the hippocampus . In the present study, we have further demonstrated that the consumption of a CSD markedly suppresses oxidative stress in the quadriceps muscle, and that there is a negative correlation between oxidative stress in quadriceps and gastrocnemius muscle atrophy.
The age-dependent accumulation of mitochondrial DNA (mtDNA) mutations, which lead to mitochondrial dysfunction, may be an important contributor to sarcopenia [46, 47]. A causal role for these age-related mtDNA deletion mutations and mitochondrial dysfunction in sarcopenia has been supported by findings that these alterations induce the loss of cytochrome c oxidase activity in aged rats, primates, and human skeletal muscle cross sections [48–52]. Conversely, since the stimulation of HNE degradation restored the decline in cytochrome c oxidase activity , HNE should inhibit cytochrome c oxidase activity through the formation of HNE adducts with cytochrome c oxidase subunits. These findings indicate that the protection of mitochondrial function, especially with regard to the cytochrome c oxidase activity, could be important to prevent sarcopenia. As ALDH2 activity is suppressed in ALDH2*2 Tg mice, they cannot efficiently degrade HNE in muscles . In this study, the consumption of a CSD maintained cytochrome c oxidase activity in the gastrocnemius muscle of ALDH2*2 Tg mice, and a negative correlation between cytochrome c oxidase activity and MDA and HAE was identified. Therefore, the consumption of Chlorella may act to prevent the accumulation of HNE, thereby preventing mitochondrial dysfunction through the protection of cytochrome c oxidase activity.
As a lack of protein intake and decreased amino acid muscle protein synthesis leads to a decline in muscle mass, the intake of dietary protein has been recommended to slow and prevent the progression of sarcopenia [54, 55]. With a protein content of approximately 65%, the continuous intake of Chlorella may therefore be useful in both enhancing muscle protein synthesis and preventing muscle atrophy. In particular, Chlorella contains essential amino acids such as the BCAA valine, leucine, and isoleucine, which are important components of actin and myosin-composing muscle, and may be beneficial in the prevention and treatment of sarcopenia [56, 57].
Since Chlorella supplement contains various useful substances, it may not be easy to identify a single substance that exhibits a beneficial effect against muscle atrophy. Supplements with multiple compounds, such as Chlorella, may be particularly beneficial because of their synergic effects.
Finally, the present study showed the usefulness of this ALDH2 deficient mouse for evaluating anti-oxidative supplements.
This study demonstrates that long-term consumption of Chlorella in the diet has beneficial effects on body weight, and prevents oxidative stress, muscle atrophy, and mitochondrial dysfunction in ALDH2*2 Tg mice. This suggests that Chlorella intake may be useful in the treatment of sarcopenia.
Aldehyde dehydrogenase 2
Dominant-negative form of ALDH2
- ALDH2*2 Tg mice:
Muscle-specific mitochondrial ALDH2 activity deficient mice
Reactive oxygen species
Hematoxylin and eosin
This research was supported by Chlorella Industry Co. Ltd.
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