Amla Enhances Mitochondrial Spare Respiratory Capacity by Increasing Mitochondrial Biogenesis and Antioxidant Systems in a Murine Skeletal Muscle Cell Line

Abstract

Amla is one of the most important plants in Indian traditional medicine and has been shown to improve various age-related disorders while decreasing oxidative stress. Mitochondrial dysfunction is a proposed cause of aging through elevated oxidative stress. In this study, we investigated the effects of Amla on mitochondrial function in C2C12 myotubes, a murine skeletal muscle cell model with abundant mitochondria. Based on cell flux analysis, treatment with an extract of Amla fruit enhanced mitochondrial spare respiratory capacity, which enables cells to overcome various stresses. To further explore the mechanisms underlying these effects on mitochondrial function, we analyzed mitochondrial biogenesis and antioxidant systems, both proposed regulators of mitochondrial spare respiratory capacity. We found that Amla treatment stimulated both systems accompanied by AMPK and Nrf2 activation. Furthermore, we found that Amla treatment exhibited cytoprotective effects and lowered reactive oxygen species (ROS) levels in cells subjected to t-BHP-induced oxidative stress. These effects were accompanied by increased oxygen consumption, suggesting that Amla protected cells against oxidative stress by using enhanced spare respiratory capacity to produce more energy. Thus we identified protective effects of Amla, involving activation of mitochondrial function, which potentially explain its various effects on age-related disorders.

Figure 1

Amla treatment stimulated mitochondrial bioenergetic function. C2C12 myotubes were pretreated with two doses of Amla (100 μg/mL or 200 μg/mL) for 48 h and subjected to mitochondrial function analysis. ((a), left panel) Schematic figure analyzing mitochondrial function using an extracellular flux analyzer. After measuring basal OCR, oligomycin, FCCP, and rotenone/antimycin A were sequentially injected to measure OCR from proton leak, maximal respiratory capacity, and nonmitochondrial respiration, respectively. OCR from nonmitochondrial respiration was subtracted from OCR at each stage to calculate the net OCR for basal (Base), proton leak (Leak), and maximal respiratory capacity (Max) values. Mitochondrial spare respiratory capacity and ATP-transratio were calculated by the formula shown. ((a), right panel) OCR measurements over time (n = 10 or 11). (b) Basal, Max, and Leak OCRs represented as average values from multiple measurements. (c) Mitochondrial spare respiratory capacity was calculated from Max and Basal OCRs. (d) ATP-transratio was calculated from Basal and Leak OCRs. ^∗ p < 0.05; ^∗∗ p < 0.01 as compared with control (n = 10 or 11).

Figure 2

Amla treatment stimulated mitochondrial biogenesis by AMPK activation. C2C12 myotubes were incubated with Amla (200 μg/mL) for 48 h. Cell lysates were prepared for western blotting, RT-qPCR, and mtDNA analysis. (a) Relative mtDNA content was determined by qPCR using specific primer sets for the mitochondrial and nuclear genome. ^∗∗ p < 0.01; n = 8. (b) Phosphorylated AMPKα to total AMPKα ratios were determined by western blot. ^∗∗ p < 0.01; n = 6. (c) Relative contents of PGC1α, NRF1, and mtTFA mRNAs were determined by RT-qPCR. ^∗ p < 0.05 and ^∗∗ p < 0.01; n = 5. 18S rRNA was used as an internal control for RT-qPCR.

Figure 3

Amla treatment stimulated antioxidant systems by Nrf2 activation. (a) Activation of Nrf2 was analyzed using an ARE luciferase assay. Data are expressed as relative activities (reporter luciferase activity/control luciferase activity) as compared with data from control cells. ^∗∗ p < 0.01; n = 5. (b and c) C2C12 myotubes were incubated with Amla (200 μg/mL) for 48 h. (b) Nuclear lysates were analyzed by western blot, and YY-1 was used an internal control for nuclear protein. ^∗ p < 0.05; n = 6. (c) Relative levels of mRNA for antioxidant system related genes were analyzed by RT-qPCR. ^∗ p < 0.05; ^∗∗ p < 0.01; n = 5. 18S rRNA was used as an internal control for RT-qPCR.

Figure 4

Amla treatment exhibited a cytoprotective effect against oxidative stress and concomitantly increased oxygen consumption. C2C12 myotubes were pretreated with Amla (200 μg/mL) for 48 h and then treated with t-BHP (250 μM  or 500 μM). (a) Schematic showing the time points for three experiments performed to evaluate the cytoprotective effects of Amla treatment. (b) Cell viability was analyzed by MTT assay at 6 h after t-BHP treatment. ^∗∗ p < 0.01 versus t-BHP-untreated cells; ^†† p < 0.01 versus Amla-untreated cells treated with each t-BHP concentration; n = 20. (c) Relative ROS levels in cells were analyzed at 2 h after t-BHP stimulation. ^∗∗ p < 0.01 versus t-BHP-untreated cells; ^†† p < 0.01 versus Amla-untreated cells treated with each t-BHP concentration; n = 12. (d) OCR after t-BHP stimulation was analyzed following t-BHP injection after three basal OCR measurements. OCR was measured every 8 min for a total of 160 min. Data are represented as relative-OCR values divided by the basal OCR values measured prior to t-BHP treatment (n = 10).

Figure 5

Schematic figure showing the effects of Amla treatment resulting in reduced oxidative stress.