Upregulation of skeletal muscle PGC-1α through the elevation of cyclic AMP levels by Cyanidin-3-glucoside enhances exercise performance

Abstract

Regular exercise and physical training enhance physiological capacity and improve metabolic diseases. Skeletal muscles require peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) in the process of their adaptation to exercise owing to PGC-1α's ability to regulate mitochondrial biogenesis, angiogenesis, and oxidative metabolism. Cyanidin-3-glucoside (Cy3G) is a natural polyphenol and a nutraceutical factor, which has several beneficial effects on human health. Here, the effect of Cy3G on exercise performance and the underlying mechanisms involved were investigated. ICR mice were given Cy3G (1 mg/kg, orally) everyday and made to perform weight-loaded swimming exercise for 15 days. The endurance of mice orally administered with Cy3G was improved, enabling them to swim longer (time) and while the levels of exercise-induced lactate and fatigue markers (urea nitrogen, creatinine and total ketone bodies) were reduced. Additionally, the expression of lactate metabolism-related genes (lactate dehydrogenase B and monocarboxylate transporter 1) in gastrocnemius and biceps femoris muscles was increased in response to Cy3G-induced PGC-1α upregulation. In vitro, using C2C12 myotubes, Cy3G-induced elevation of intracellular cyclic AMP levels increased PGC-1α expression via the Ca²⁺/calmodulin-dependent protein kinase kinase pathway. This study demonstrates that Cy3G enhances exercise performance by activating lactate metabolism through skeletal muscle PGC-1α upregulation.

Figure 1. Regulation of blood lactate and glucose levels enhanced swimming time.

Mice were trained to perform a swimming exercise and performed the exercise every other day for 14 days, and a swimming-until-exhaustion test was carried out on day 15. On days 13 and 15, (A,C) blood lactate levels before and after exercise (0 and 60 min) and (B,D) blood glucose levels after exercise (0 min) were evaluated. (E) On day 15, swimming time to exhaustion was measured. Values are expressed as the mean ± standard deviation. *P ≤ 0.05 and **P ≤ 0.01 indicate a significant difference from the control group.

Figure 2. Lactate metabolism-related genes expression in the gastrocnemius and biceps femoris were controlled in response to Cyanidin-3-glucoside (Cy3G)-induced PGC-1α upregulation.

Expression levels of PGC-1α and PGC-1α-targeted genes in the gastrocnemius (A,C) and biceps femoris (B,D) were evaluated. (A,B) Expression levels of mRNA were normalised to the β-actin expression level. (C,D) Protein expression levels were normalised to the expression of GAPDH. All gels were run under the same experimental conditions and the representative blots were shown. Values are expressed as the mean ± standard deviation and relative to the no exercise group. *P ≤ 0.05 and **P ≤ 0.01 indicate a significant difference from the no exercise group. ^#P ≤ 0.05 and ^##P ≤ 0.01 indicate significant difference from the control group.

Figure 3. Lactate metabolism of C2C12 myotubes was enhanced in response to Cyanidin-3-glucoside (Cy3G).

Differentiated C2C12 myotubes were treated with or without Cy3G for 24 h. After that, cell viability (A) and mRNA expression levels of LDHa, LDHb and MCT1 (B) were evaluated. (B) Gene expression levels were normalised to the β-actin expression level. (C) After Cy3G treatment (24 h), C2C12 myotubes were cultured with serum- and glucose-free DMEM containing 20 mM lactate for 15 min. Intracellular ATP production in C2C12 myotubes was then evaluated. Values are expressed as the mean ± standard deviation of triplicate experiments. **P ≤ 0.01 indicates a significant difference from the control group. ^#P ≤ 0.05 and ^##P ≤ 0.01 indicate a significant difference from the lactate-treated control group.

Figure 4. Mitochondrial content of skeletal muscle cells was increased by Cyanidin-3-glucoside (Cy3G)-induced PGC-1α upregulation.

Differentiated C2C12 myotubes and HSMM were treated with or without Cy3G for 6 h (A,C) or 24 h (B,D), after which, the gene expression of PGC-1α, TFAM, CPT-1β, and UCP-3 (A,C) and the mitochondria content (B,D) were evaluated. Differentiated C2C12 myotubes were transfected with PGC-1α siRNA or Control siRNA for 48 h and then, treated with or without Cy3G for (E) 6 h or (F) 24 h. Following treatment, PGC-1α mRNA levels (E) and mitochondria content (F) were evaluated. (A,C,E) Expression levels of mRNA were normalised to the β-actin expression level and expressed relative to the control. Values are expressed as the mean ± standard deviation of triplicate experiments. *P ≤ 0.05 and **P ≤ 0.01 indicate a significant difference from the control group. ^##P ≤ 0.01 indicates a significant difference from the Cy3G group.

Figure 5. CaMKK–AMPK pathway and intracellular cAMP levels were involved in Cyanidin-3-glucoside (Cy3G)-induced PGC-1α upregulation.

C2C12 myotubes were treated with or without Cy3G (10 μM) for 0–6 h and then phosphorylated AMPK levels were evaluated and values were normalised to the β-actin expression level. All gels were run under the same experimental conditions and the representative blots were shown (A). C2C12 myotubes were pre-treated with or without STO-609 (1 μg/ml) for 30 min. Cy3G (10 μM) treatment was then carried out with or without STO-609 for 6 h. PGC-1α mRNA levels were evaluated and values were normalised to the β-actin expression level (B). C2C12 myotubes were pre-incubated with Fluo4 AM for 30 min and subsequently treated with or without Cy3G for 15–90 min (C). C2C12 myotubes were treated with or without Cy3G (10 μM) and with or without PDE inhibitors (500 μM IBMX and 100 μM Ro20-1724) for 15 min. The intracellular cAMP level was then measured (D). Values are expressed as the mean ± standard deviation of triplicate experiments. *P ≤ 0.05 and **P ≤ 0.01 indicate a significant difference from the control group. ^##P ≤ 0.01 indicates a significant difference from the Cy3G group. N.S. indicates that the mean value is not significantly different from that of the STO-609-treated control group.

Figure 6. Skeletal muscle PGC-1α upregulation by Cyanidin-3-glucoside (Cy3G) enhances exercise performance.

Increase of lactate metabolism in response to Cy3G-induced PGC-1α upregulation enhanced exercise performance and reduced fatigue. This PGC-1α upregulation is modulated by CaMKK–AMPK pathway via the elevation of intracellular cAMP levels.