Nutr Res Pract.  2022 Feb;16(1):14-32. 10.4162/nrp.2022.16.1.14.

Gynostemma pentaphyllum extract and Gypenoside L enhance skeletal muscle differentiation and mitochondrial metabolism by activating the PGC-1α pathway in C2C12 myotubes

• Kim YH
• Jung JI
• Jeon YE
• Kim SM
• Oh TK
• Lee J
• Moon JM
• Kim TY
• Kim EJ

• KMID: 2525509
• DOI: http://doi.org/10.4162/nrp.2022.16.1.14

Abstract

BACKGROUND/OBJECTIVES
Peroxisome proliferator-activated receptor-gamma co-activator-1α (PGC-1α) has a central role in regulating muscle differentiation and mitochondrial metabolism. PGC-1α stimulates muscle growth and muscle fiber remodeling, concomitantly regulating lactate and lipid metabolism and promoting oxidative metabolism. Gynostemma pentaphyllum (Thumb.) has been widely employed as a traditional herbal medicine and possesses antioxidant, anti-obesity, anti-inflammatory, hypolipemic, hypoglycemic, and anticancer properties. We investigated whether G. pentaphyllum extract (GPE) and its active compound, gypenoside L (GL), affect muscle differentiation and mitochondrial metabolism via activation of the PGC-1α pathway in murine C2C12 myoblast cells.
MATERIALS/METHODS
C2C12 cells were treated with GPE and GL, and quantitative reverse transcription polymerase chain reaction and western blot were used to analyze the mRNA and protein expression levels. Myh1 was determined using immunocytochemistry. Mitochondrial reactive oxygen species generation was measured using the 2′7′-dichlorofluorescein diacetate assay.
RESULTS
GPE and GL promoted the differentiation of myoblasts into myotubes and elevated mRNA and protein expression levels of Myh1 (type IIx). GPE and GL also significantly increased the mRNA expression levels of the PGC-1α gene (Ppargc1a), lactate metabolismregulatory genes (Esrra and Mct1), adipocyte-browning gene fibronectin type III domaincontaining 5 gene (Fndc5), glycogen synthase gene (Gys), and lipid metabolism gene carnitine palmitoyltransferase 1b gene (Cpt1b). Moreover, GPE and GL induced the phosphorylation of AMP-activated protein kinase, p38, sirtuin1, and deacetylated PGC-1α. We also observed that treatment with GPE and GL significantly stimulated the expression of genes associated with the anti-oxidative stress response, such as Ucp2, Ucp3, Nrf2, and Sod2<>/i>.
CONCLUSIONS
The results indicated that GPE and GL enhance exercise performance by promoting myotube differentiation and mitochondrial metabolism through the upregulation of PGC-1α in C2C12 skeletal muscle.

Keyword

Gynostemma pentaphyllum; peroxisome proliferator-activated receptor gamma coactivator 1-alpha; muscle; skeletal

Figure

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  Fig. 1 Cell-toxicity analysis result. C2C12 cells were incubated in differentiation medium with GPE, GL, or CrM for 4 days. MTT assays showed no cytotoxic effects from the GPE, GL, and CrM treatments in C2C12 cells. All data represent five replicates (n = 5), and values are shown as means ± SE.GPE, Gynostemma pentaphyllum extract; GL, gypenoside L; CrM; creatine monohydrate.

• 

  Fig. 2 Effect of GPE and GL on the mRNA expression of MyHC genes in C2C12 myotubes. C2C12 cells were incubated in differentiation medium with GPE, GL, or CrM for 4 days. The mRNA expression levels of Myh1 (A), Myh7 (B), Myh2 (C), and Myh4 (D) were determined by qRT-PCR. All data represent five replicates (n = 5), and values are shown as means ± SE.GPE, Gynostemma pentaphyllum extract; GL, gypenoside L; CrM, creatine monohydrate.*P < 0.05 and **P < 0.01 indicate a significant difference from the control group.

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  Fig. 3 Effect of GPE and GL on the differentiation of myoblasts into myotubes. (A) C2C12 cells were incubated in differentiation medium with GPE, GL, or CrM for 4 (immunofluorescence staining) and 7 (western blot) days. (B) Protein expression of Myh1 was assessed by western blotting analysis. GAPDH was used as a loading control. All data represent five replicates (n = 5), and values are shown as means ± SE. (C) Immunofluorescence staining of Myh1 in C2C12 myotubes. The cells were fixed and immunostained for Myh1 (green), MitoTracker (red), and DAPI (blue). Myotube formation was observed by fluorescence microscopy (original magnification, 200×) (scale bar = 50 μm).GPE, Gynostemma pentaphyllum extract; GL, gypenoside L; CrM, creatine monohydrate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; DAPI, 4′,6-diamidino-2-phenylindole.**P < 0.01 indicate a significant difference from the control group.

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  Fig. 4 Quantification of MitoTracker and Myh1 staining intensity in C2C12 cells. Differentiated C2C12 cells at day 4 were stained with MitoTracker (A) and Myh1 (B). Fluorescence intensity of immunofluorescence sections was quantified using Image J software. The results are expressed as means ± SEM (n = 5).SEM, standard error of the mean; GPE, Gynostemma pentaphyllum extract; GL, gypenoside L; CrM, creatine monohydrate.*P < 0.05, **P < 0.01, and ***P < 0.001 indicate a significant difference from the control group.

• 

  Fig. 5 Effects of GPE and GL on lactate metabolism in C2C12 myotubes. Differentiated C2C12 myotubes were treated with/without GPE, GL, or CrM. The mRNA expression levels of Ppargc1a (A), Ldhb (B), Esrra (C), and Mct1 (D) were evaluated by qRT-PCR. Target gene expression levels were normalized to the Gapdh expression level. Data are expressed as means ± SE (n = 5).GPE, Gynostemma pentaphyllum extract; GL, gypenoside L; CrM, creatine monohydrate; qRT-PCR, quantitative reverse transcription polymerase chain reaction.*P < 0.05 indicate a significant difference from the control group.

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  Fig. 6 Effects of GPE and GL on adipocyte browning, glycogen synthesis, and lipid metabolism in C2C12 myotubes. Differentiated C2C12 myotubes were treated with GPE, GL, or CrM for 4 days. The mRNA expression levels of genes related to adipocyte browning (Fndc5) (A), glycogen synthesis (Gys) (B), and lipid metabolism (Cpt1b) (C) were determined by qRT-PCR. Target gene expression levels were normalized to the Gapdh expression level. Each value represents the mean ± SE (n = 5).GPE, Gynostemma pentaphyllum extract; GL, gypenoside L; CrM, creatine monohydrate; Fndc5, fibronectin type III domain-containing protein 5 gene; Gys, glycogen synthase gene; qRT-PCR, quantitative reverse transcription polymerase chain reaction.*P < 0.05 and **P < 0.01 indicate a significant difference from the control group.

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  Fig. 7 Effect of GPE and GL on the AMPK/p38 MAPK/SIRT1/PGC-1α signaling pathway. C2C12 cells were cultured for 4 days in differentiation medium supplemented with/without GPE, GL, or CrM. The ratios of p-AMPK/AMPK (A), p-p38/p38 (B), p-SIRT1/SIRT1(C), and PGC-1α/GAPDH (D) were determined. Each bar represents the mean ± SE (n = 5) relative to the control group.GPE, Gynostemma pentaphyllum extract; GL, gypenoside L; CrM, creatine monohydrate; AMPK, AMP-activated protein kinase; PGC-1α, peroxisome proliferator-activated receptor-gamma co-activator 1α; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.*P < 0.05 and **P < 0.01 indicate a significant difference from the control group.

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  Fig. 8 Effect of GPE and GL on the relative mRNA expression level of oxidative stress-related genes in C2C12 myotubes. C2C12 cells were treated with GPE, GL, or CrM for 4 days. The mRNA levels of Ucp2 (A), Ucp3 (B), Sod2 (C), and the protein levels of phosphorylated NRF2 and NRF2 (D) were analyzed by qRT-PCR and western blot assay, respectively. Gapdh served as an internal control. The ratio of p-NRF2/NRF2 was determined. (E) Effects of GPE and GL on ROS levels in C2C12 myotubes. Cells were studied under normal conditions (Veh), treatment with tBHP for 3 h (Control), and combinations of tBHP with GPE, GL or CrM. ROS levels are presented as fold changes from control cells. All values are presented as the mean ± SE (n = 5).GPE, Gynostemma pentaphyllum extract; GL, gypenoside L; CrM, creatine monohydrate; NRF, nuclear respiratory factor; qRT-PCR, quantitative reverse transcription polymerase chain reaction; ROS, reactive oxygen species; tBHP, tert-butyl hydrogen peroxide.*P < 0.05, **P < 0.01, and ***P < 0.001 indicate a significant difference from control group. ### P < 0.001 Indicate significant difference from tBHP alone.

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  Fig. 9 Tentative mechanisms by which GPE and GL stimulate muscle differentiation and mitochondrial metabolism in C2C12 myotubes.GPE, Gynostemma pentaphyllum extract; GL, gypenoside L; AMPK, AMP-activated protein kinase; PGC-1α, proliferator-activated receptor-gamma coactivator 1α; ERRα, estrogen-related receptor α; MCT1, monocarboxylate transporter 1; UCP, uncoupling protein; NRF, nuclear respiratory factor; SOD, superoxide dismutase; CPT, carnitine palmitoyl transferase; GYS, glycogen synthase.

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