5‐Aminolevulinic acid and sodium ferrous citrate ameliorate muscle aging and extend healthspan in Drosophila

Declines in mitochondrial functions are associated with aging. The combination of 5‐aminolevulinic acid (5‐ALA) and sodium ferrous citrate (SFC) improves mitochondrial functions in cultured cells. In this study, we investigated the effects of dietary supplementation with 5‐ALA and SFC (5‐ALA/SFC) on the healthspan and life span of Drosophila  melanogaster. Adult Drosophila fruit flies were fed cornmeal food containing various concentrations of 5‐ALA/SFC. Locomotor functions, life span, muscle architecture, and age‐associated changes in mitochondrial function were analyzed. We found that feeding 5‐ALA/SFC mitigated age‐associated declines in locomotor functions and extended organismal life span. Moreover, 5‐ALA/SFC preserved muscle architecture and maintained the mitochondrial membrane potential in aged animals. Since 5‐ALA phosphate/SFC is used as a human dietary supplement, our results suggest that it could be used to slow the age‐related declines in muscle functions, prevent age‐associated clinical conditions such as frailty, and extend healthspan and life span.

Declines in mitochondrial functions are associated with aging. The combination of 5-aminolevulinic acid (5-ALA) and sodium ferrous citrate (SFC) improves mitochondrial functions in cultured cells. In this study, we investigated the effects of dietary supplementation with 5-ALA and SFC (5-ALA/SFC) on the healthspan and life span of Drosophila melanogaster.
Adult Drosophila fruit flies were fed cornmeal food containing various concentrations of 5-ALA/SFC. Locomotor functions, life span, muscle architecture, and age-associated changes in mitochondrial function were analyzed. We found that feeding 5-ALA/SFC mitigated age-associated declines in locomotor functions and extended organismal life span. Moreover, 5-ALA/SFC preserved muscle architecture and maintained the mitochondrial membrane potential in aged animals. Since 5-ALA phosphate/ SFC is used as a human dietary supplement, our results suggest that it could be used to slow the age-related declines in muscle functions, prevent age-associated clinical conditions such as frailty, and extend healthspan and life span.
Aging can be defined as the progressive deterioration of the functional properties of cells, tissues, and organs over a life span [1]. Cumulative declines in multiple physiological systems perturb homeostasis and adaptability to internal and external stresses, resulting in increased vulnerability to disease and mortality [2]. Among the problematic features of aging, frailty is a common clinical syndrome characterized by increased vulnerability to disease and mortality due to a decline in the functions of physiological systems. The ability to cope with daily and acute stressors is compromised, which increases the risk of poor health outcomes such as falls, injuries, and mortality [3]. The physical aspects of the frailty phenotype include low grip strength, gait speed, and muscle mass, which overlap with the symptoms of sarcopenia [4]. Although fragility is of increasing concern as populations age, therapeutic interventions for frailty and other age-related conditions are limited.
The age-associated physiological decline is linked with mitochondrial dysfunctions [1,5,6]. In aged tissues, the activity of mitochondrial enzymes is reduced, respiratory capacity is lower, and there is increased production of reactive oxygen species (ROS). A reduction in mitochondrial function is particularly prominent in highly oxidative tissues such as skeletal muscle [7].
Mitochondrial respiratory chain complexes II, III, and IV, and cytochrome c contain heme; heme deficiency may be an underlying cause of age-associated mitochondrial dysfunction [8]. The biosynthesis of heme, which is a porphyrin ring complexed with ferrous iron and protoporphyrin IX, starts in mitochondria with the condensation of succinyl-CoA with the amino acid glycine to generate 5-aminolevulinic acid (5-ALA). This process is mediated and rate-limited by ALA synthase [9]. Heme synthesis, ALA synthase expression, and 5-ALA levels decline with increasing age [10,11]. Treatment of cultured mouse cells and human fibroblasts with 5-ALA and sodium ferrous citrate (SFC) increases the activity of cytochrome c oxidase, the expression of oxidative phosphorylation complexes III, IV, and V, the oxygen consumption rate, and the production of ATP [12][13][14]. Thus, we hypothesized that 5-ALA treatment would protect muscle function by increasing mitochondrial ATP synthesis during aging.
In this study, we investigated the effects of 5-ALA/ SFC on age-related declines in physical activity and life span using the fruit fly Drosophila melanogaster. Drosophila is a genetically tractable model system with the relatively short life span and is used to study human diseases and aging. Age-associated dysfunctions are prominently observed in the muscle [15]. Here, we report that supplementation of food with 5-ALA/SFC significantly mitigated age-associated locomotor decline, extended the organismal life span, improved sarcomere structure, and maintained the mitochondrial membrane potential in aged muscle tissue.

5-ALA/SFC feeding
Flies were raised on the regular cornmeal. After eclosion, male flies were maintained on regular cornmeal food mixed with 5-ALA/SFC at the indicated concentration. Flies were placed at 30 flies/vial, and food vials were changed every 2-3 days. Quantification of 5-ALA levels in the fly food 5-ALA levels were analyzed as previously described with a modification [16]. Briefly, fly food was mixed with 8 times volume of water and centrifuged (2655 g, 10 min at 4°C). The supernatant was collected and the process was repeated, and subjected to fluorometric analysis as described in Ref. [16].

Climbing assay
The climbing assay was performed as previously described [17]. Approximately 30 flies were placed in an empty plastic vial (2.5 cm in diameter 9 8 cm in length). The vial was gently tapped to knock the flies to the bottom, and the height that the flies climbed in 10 s after tapping to the bottom of the vials was measured. Experiments were repeated more than three times, and a representative result was shown. Food vials were changed every 2-3 days.

Phalloidin staining
The thoracic muscles of male flies were dissected in cold Schneider's Drosophila medium (SDM; Thermo Fisher Scientific). First, an incision was made in the middle of the thorax, and then, indirect flight muscles were dissected from the thorax. The muscles were then fixed with 4% paraformaldehyde (Wako) for 30 min at room temperature, incubated with 2 µgÁmL À1 phalloidin/tetramethylrhodamine B isothiocyanate peptide (Sigma-Aldrich) in PBS overnight on a shaker at 4°C, and imaged under a laser confocal microscope (Zeiss LSM 710, Oberkochen, Germany) and analyzed with IMAGEJ [18].

Blue NativePAGE
Blue NativePAGE to analyze mitochondrial respiratory chain protein was carried out as described with a modification [19,20]. Thorax from 35 flies was homogenized in 1 ml of chilled mitochondrial isolation medium (250 mM sucrose, 10 mM Tris/HCl (pH 7.4), 0.15 mM MgCl 2 ). The samples were centrifuged twice for 10 min at 600 g at 4°C to remove debris. The supernatant was centrifuged again for 10 min at 7000 g at 4°C. For BN-PAGE analyses, the Nati-vePAGE Novex Bis-Tris Gel System (Life Technologies, Carlsbad, CA, USA) was used according to the manufacturer's protocol. Mitochondrial fractions were solubilized in sample buffer (50 mM NaCl, Tris-HCl (pH 7.4) and 1% Triton X-100). After centrifugation for 5 min at 17,800 g at 4°C, the supernatants were collected. Mitochondrial protein levels were determined using a BCA assay. 30 µg mitochondrial protein was mixed with 109 loading dye solution (5% Coomassie Blue G, 1 M aminohexanoic acid, 100 mM Bis-Tris) and separated on 3%-12% NativePAGE gels. Gels were stained with Coomassie Blue.

Western Blot
10 µg of mitochondrial protein was separated by 4-15% polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes using the Trans-BlotÒ Turbo Transfer System (Bio-Rad Laboratories, Hercules, CA, USA), and incubated with antibodies. Immunolabeled proteins were detected using a chemiluminescence kit (ImmunoStar LD) and a lumino-image analyzer [ChemiDoc MP System (Bio-Rad Laboratories)]. The primary antibodies used were rabbit anticitrate synthase. The secondary antibodies used were antirabbit IgG HRP-Linked Whole Ab Donkey (GE Healthcare).

Bilirubin assay
Twenty thoraxes were homogenized, and bilirubin levels were measured by using Bilirubin Assay Kit (Cell Biolabs, Inc., San Diego, CA, USA) according to the manufacturer's instruction.

qRT-PCR
Total RNA of the 20 thoraxes was purified according to the protocol provided with the RNeasy Plus Universal Mini Kit (Qiagen, Hilden, Germany). The amount and purity of the total RNA were measured using the NanoDrop One Spectrophotometer (Thermo Scientific). cDNA was synthesized from 0.5 lg of total RNA using the High Capacity RNA-to-cDNA Kit (Life Technologies). The expression level of each gene was measured using the PowerUp SYBR Green Master Mix (Life Technologies) and the StepOnePlus Real-Time PCR System (Life Technologies). Expression of genes of interest was standardized relative to rp49. Primers were designed using DRSC FlyPri-merBank (Harvard Medical School, Boston, MA, USA). Primer sequences are shown in Table S1.

Analysis of mitochondrial morphology
Male flies were anesthetized, and the indirect flight muscle was dissected in cold SDM. The muscles were then fixed for 30 min with 4% paraformaldehyde at room temperature. After fixation, samples were washed three times with 0.1% PBST (0.1% triton in 19 PBS) for 5 min each time.
The samples were blocked with 1% normal goat serum (NGS)/0.1% PBST for 30 min at room temperature. The samples were then incubated with anti-ATP5A antibody (1 : 250; Abcam) in 1% NGS/0.1% PBST overnight at 4°C. The next day, the samples were washed three times for 5 min in 0.1% PBST and then incubated in 1% NGS with 0.1% PBST containing Alexa Fluor 488 anti-mouse IgG (Thermo Fisher) at room temperature for 3 h. Subsequent to this, the samples were washed three times in 0.1% PBST and mounted in VectaShield mounting medium (Vector Laboratories, Burlingame, CA, USA). Images were captured using a laser confocal microscope (Zeiss LSM 710) and analyzed with ImageJ [18].

Mitochondrial membrane potential assay
Male flies were anesthetized, and the indirect flight muscles were dissected in cold SDM.

4-HNE staining
Male flies were anesthetized, and the indirect flight muscles were dissected in cold SDM. The muscles were then fixed for 40 min with 4% paraformaldehyde at room temperature. After fixation, samples were washed three times with 0.1% PBST (0.1% triton in 19 PBS) for 5 min each time.
The samples were blocked with 1% NGS/0.1% PBST for 5 h at room temperature. The samples were then incubated with anti-4-HNE antibody (1 : 100; Abcam) in 1% NGS/ 0.1% PBST overnight at 4°C. The next day, the samples were washed three times for 15 min in 0.1% PBST. Alexa Fluor 488 anti-rabbit IgG (Thermo Fisher) and phalloidin were then added to the samples, and kept at room temperature for 3 h. Subsequent to this, the samples were washed three times in 0.1% PBST and mounted in VectaShield mounting medium (Vector Laboratories). Images were captured using a laser confocal microscope (Zeiss LSM 710) and analyzed with IMAGEJ [18]. , and R (R Core Team, 2021) [21]. Differences were assessed using one-way ANOVA or logrank test followed by Dunnett's test or Tukey's HSD. P values < 0.05 were considered statistically significant.

5-ALA is stable in fly food for up to 2 weeks
To examine the effect of 5-ALA on aging and life span, we maintained flies on a diet of cornmeal food containing 5-ALA and SFC. The addition of 5-ALA (100 µM or higher) and SFC to the culture medium of cells significantly increases the cellular oxygen consumption rate [12]. However, it is not clear whether 5-ALA/SFC mixed with food is efficiently absorbed and distributed to fly tissues. Since the concentration of dietary chemicals used in most fly studies is higher than the concentrations of chemicals used in cultured cells [22][23][24][25][26], we prepared food containing 0.05, 0.5, 5, and 50 mM 5-ALA hydrochloride combined with SFC at a ratio of 20 : 1.
We analyzed the stability of 5-ALA in the food media with or without flies. 5-ALA was stable for at least two weeks in fly food stored at 25 or 4°C (Table 1). Moreover, the 5-ALA concentration did not reduce when flies were raised in food (Table 2), indicating that the presence of fly excrement does not affect the 5-ALA concentration in food.

5-ALA/SFC ameliorates the age-related decline in locomotor activity
We used a climbing assay [27], which is a robust behavioral assay that takes advantage of innate negative geotaxis behavior, to analyze the effects of 5-ALA/SFC on the age-related declines in locomotor function. Flies were raised in regular cornmeal food and after eclosion were maintained in cornmeal food containing various concentrations of 5-ALA and SFC. Flies maintained on regular food begin to show an age-dependent decline in climbing ability around 3 weeks of age [27]. Supplementation of the diet with 5-ALA/SFC significantly improved the locomotor activity of 3-week-old flies in a dose-dependent manner (Fig. 1). Locomotor function was significantly improved by 0.05 mM 5-ALA and Table 1. 5-ALA is stable in fly food for up to 2 weeks. Fly food was mixed with 50 mM 5-ALA and 2.5 mM SFC. The concentration of 5-ALA was analyzed on the day on which the food was prepared (day 0) and after it had been stored for 1 week (day 7) or 2 weeks (day 14) at 4 or 25°C. The concentration of 5-ALA in food stored at 25°C was higher due to water evaporation (see the column headed 'relative weight of food'). The expected concentrations of 5-ALA without water evaporation are shown in brackets.
Amount of 5-ALA in 100 g of food (g) Relative weight of food (%) 5-ALA concentration (mM)  Table 2. 5-ALA is stable in food containing cultured flies. Flies were cultured in food containing 5-ALA at 25°C. When nextgeneration flies eclosed after 10 days (day 10), flies and larvae were removed from the food, and the 5-ALA concentration was analyzed.
Amount of 5-ALA in 100 g of food (g) 0.0025 mM SFC and was further improved by higher concentrations (Fig. 1). These results suggest that dietary supplementation with 5-ALA/SFC counteracts an age-related physiological decline.  (Fig. 2). These results indicate that 0.5 mM 5-ALA/0.025 mM SFC extends healthspan, and 5 mM 5-ALA/0.25 mM SFC or higher concentrations extend both healthspan and life span.

5-ALA/SFC maintains muscle architecture during aging
Muscle integrity is essential for locomotor function and declines during aging [28]. We analyzed the  The number, size, and arrangement of myonuclei are associated with muscle function [29]. Myonuclei in the center of the myofiber are called central nuclei; they are associated with myofibers that have degenerated and regenerated, and are a prominent feature of aged skeletal muscle in mice [11][12][13][14][15][16]. We found that the density of myonuclei was greater in aged flies than in young flies (Fig. 3B These results suggest that the muscle of aged flies treated with 5-ALA/SFC maintains a key feature, namely myonuclear density, which is usually associated with young muscle.

5-ALA/SFC maintains mitochondrial membrane potential
Mitochondrial dysfunctions occur in aged muscle tissues [15]. We sought to determine whether the protective effects of 5-ALA/SFC are accompanied by improved mitochondrial functions. Analyses of the expression of mitochondrial respiratory complexes by blue NativePAGE showed that the aged flies with or without 5-ALA/SFC expressed similar levels of mitochondrial respiratory complexes (Fig. 4A). The levels of mRNA coding respiratory chain proteins such as ND42, SdhA, UQCRC2, COX4, and ATP5A were further analyzed by qRT-PCR. 5-ALA/SFC increased the levels of mRNA coding SdhA, UQCRC2, and COX4 but not the levels of ND42 or ATP5A (Fig. 4B). The levels and activity of citrate synthase were similar with or without 5-ALA/SFC treatment (Fig. 4A,C), suggesting that 5-ALA/SFC does not alter mitochondrial activity. Mitochondrial dynamics, such as fission and fusions, act as quality control mechanisms and decline during aging [30]. We analyzed mitochondrial morphology by immunostaining with anti-ATP5A and found that 5-ALA/SFC feeding did not affect mitochondrial size in the indirect flight muscle in aged flies (Fig. 4D). We also investigated whether 5-ALA/SFC affects overall heme metabolism in flies. Treatment with 5-ALA/SFC feeding did not significantly change the levels of bilirubin, the catabolic product of heme metabolism (Fig. 4E).
Finally, we analyzed the mitochondrial membrane potential by staining dissected tissues with mitochondrial membrane potential-sensitive dyes. We used Mito-Tracker, whose accumulation in mitochondria is dependent upon their membrane potential and preserved after fixation. We found that the MitoTracker signal intensity was significantly increased in the muscles in aged flies and was reduced by 5-ALA/SFC (Fig. 4F, compare 0 mM 5-ALA/0 mM SFC and 0.5 mM 5-ALA/0.025 mM SFC or 50 mM 5-ALA/2.5 mM SFC). We also used MT-1, another cationic dye sequestered to mitochondria by their membrane potential [31]. MT-1 signals in young animals and those in aged animals were not significantly different, while 5-ALA/SFC significantly reduced them (Fig. 4F, compare 0 mM 5-ALA/0 mM SFC and 50 mM 5-ALA/2.5 mM SFC).
The rate of ROS production depends on mitochondrial membrane potential, and hyperpolarization of mitochondria could lead to an increase in ROS production [32]. 5-ALA/SFC may preserve the mild depolarization of the mitochondrial inner membrane to reduce ROS production. To test this hypothesis, we analyzed ROS levels by using 4-HNE, a product of lipid peroxidation and a biomarker of oxidative stress [33]. The aged flies exhibited significantly increased 4-HNE staining, indicating increased oxidative damage (Fig. 4G, compare 1 and 35 days with 0 mM 5-ALA/

Discussion
Administration of 5-ALA/SFC ameliorates mitochondrial dysfunction in fibroblasts from individuals with mitochondrial diseases [12] and in white adipose tissue from mice with diet-induced obesity [13]. Treatment with 5-ALA/SFC also improves muscle function in mice with sarcopenia and in a mouse model of chronic kidney disease [34]. Here, we report for the first time that 5-ALA/SFC protects against organismal aging. The muscle architecture in aged flies treated with 5-ALA/SFC was similar to that in young animals (Fig. 3); this effect of 5-ALA/SFC on muscle architecture may contribute to its protective effects on agedependent declines in locomotion (Fig. 1). Reactive oxygen species induce oxidative damage to cellular macromolecules due to their high chemical reactivity and are believed to be a significant cause of aging [35]. The respiratory chain is the primary production site of superoxide, and ROS are formed as a byproduct of oxidative phosphorylation [35]. The rate of ROS production depends on mitochondrial membrane potential and the activity of the respiratory complexes [32]. Dissipation of the mitochondrial membrane potential could increase ROS generation when respiration is inhibited [36]. On the contrary, hyperpolarization of mitochondria can also lead to ROS production [32,37]. In fact, mild depolarization of the mitochondrial inner membrane has been reported as a defense mechanism that prevents oxidant-mediated damage by reducing mitochondrial ROS generation through an ADPrecycling mechanism [36,[38][39][40]. This mild depolarization decreases during aging in the skeletal muscle of mice [41], suggesting that elevated oxidative damages with aging may be partly caused by the loss of mild depolarization of the mitochondrial inner membrane. In agreement with this, we found that 5-ALA/SFC treatment reduced the mitochondrial membrane potential and oxidative damages in the aged muscles (Fig. 4). These results suggest that 5-ALA/SFC has a novel mechanism of action that benefits physical functions. Further analyses of the molecular mechanisms by which 5-ALA/SFC regulates mitochondrial membrane potential would enhance our understanding of the roles of mitochondria in aging.
5-ALA is a natural delta amino acid widely present in nature [42]. In addition, 5-ALA/SFC is consumed as a dietary supplement in several countries [43,44]. Age-associated conditions such as frailty are caused by cumulative damage over a long time [45], so a dietary supplement might be a suitable format for preventing age-associated conditions. Our results suggest that 5-ALA/SFC is a possible therapeutic intervention for preventing frailty in elderly people.

Conclusions
Oral administration of 5-ALA/SFC mitigates agedependent declines in locomotor function, extends life span, and maintains muscle architecture and mitochondrial membrane potential in aged Drosophila. Our  , complex I; CV, complex V; CIII, complex III; CIV, complex IV; CII, complex II). (Bottom panel) Western blot analysis of the samples with an anti-citrate synthase antibody. n = 3 and a representative image is shown. (B) 5-ALA/SFC slightly increased the levels of mRNA of mitochondrial enzymes. qRT-PCR from the thorax of flies at 35 days old. Data are expressed as mean AE SE, n = 3; *P < 0.05, **P < 0.01; one-way ANOVA followed by Dunnett's test. (C) Treatment with 5-ALA/SFC did not alter citrate synthase activity. Citrate synthase activity was normalized to protein levels and shown as the ratio relative to flies not treated with 5-ALA/SFC. The data shown are from an average of two independent experiments. (D) 5-ALA/SFC did not affect mitochondrial sizes significantly. Indirect flight muscles were dissected from young (1 day old) or aged (35 days old) flies and stained with an anti-ATP5A antibody (green) to detect mitochondria. Representative images are shown on the left, and quantitation of the data is shown on the right. Data are expressed as mean, n = 3; P > 0.05; chi-squared test. Scale bar = 10 µm. (E) Treatment with 5-ALA/SFC did not alter bilirubin levels. Bilirubin levels were normalized with protein levels and expressed as the ratio relative to the flies not treated with 5-ALA/SFC treatment. Data are expressed as mean AE SE, n = 3; P > 0.05; one-way ANOVA followed by Dunnett's test. (F) Indirect flight muscles were stained with the mitochondrial dye MitoTracker Deep Red FM (green) and MT-1 (red). Representative images (left) and quantification of the signal intensity (right) are shown. Data are expressed as mean AE SE, and numbers in the bars indicate n; ****P < 0.0001; one-way ANOVA followed by Tukey's test. Scale bar = 10 µm. (G) The indirect flight muscles were stained with an anti-4-HNE antibody (green) and phalloidin (red). Representative images (left) and quantification of the areas of 4-HNE aggregates in the muscle (right) are shown. Data are expressed as mean AE SE, n = 25; *P < 0.05, ****P < 0.0001; one-way ANOVA followed by Tukey's test. Scale bar = 10 µm. results also suggest that 5-ALA/SFC supports the maintenance of the mitochondrial membrane potential and suppresses ROS generation. Together, our findings suggest that 5-ALA/SFC may be a possible therapeutic intervention for age-related declines in muscle functions and for preventing important symptoms of aging such as frailty.

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article. Table S1. Primer sequences (5 0 -3 0 ).