Cistanoside F Ameliorates Lipid Accumulation and Enhances Myogenic Differentiation via AMPK-Dependent Signaling in C2C12 Myotubes

Abstract

Sarcopenic obesity (SO) is a metabolic disorder for which no effective pharmacological treatments are currently available. Cistanoside F (Cis), a phenoxyethanol-derived compound, remains relatively unexplored in the context of lipid metabolism regulation, as well as its potential mechanisms and therapeutic applications in metabolic disorders. Consequently, this study aimed to evaluate the potential of Cis in ameliorating the pathological manifestations of SO in C2C12 cells. Two classical adipogenic differentiation models using C2C12 cells were employed to quantitatively assess the ability of Cis to inhibit lipid droplet formation, utilizing Oil Red O staining coupled with high-content imaging analysis. Markers associated with adipogenic and myogenic differentiation were examined using quantitative real-time PCR and Western blotting. Our experimental findings demonstrated that Cis significantly attenuated lipid droplet accumulation and promoted muscle protein synthesis via the modulation of PPARγ, ATGL, CPT1b, and UCP1 expression during lipogenic differentiation of C2C12 cells. Cis significantly upregulated the phosphorylation and expression levels of key metabolic regulators, including p-AMPK/AMPK, p-ACC1/ACC1, and MHC. We identified a positive regulatory feedback mechanism between AMPK signaling and MHC expression in the adipogenic differentiation model, suggesting that Cis exerts its therapeutic effects through AMPK-dependent pathways. This is the first study to provide the first experimental evidence supporting the therapeutic potential of Cis for metabolic regulation, targeting adiposity reduction and muscle mass enhancement. Furthermore, Cis exhibited potent anti-inflammatory properties, as demonstrated by its ability to significantly downregulate proinflammatory mediators, including IL-6 and p-NF-κB/NF-κB, during adipogenic differentiation. These novel findings regarding the anti-inflammatory mechanisms of Cis will form the basis for our subsequent in-depth mechanistic investigations.

Keywords: AMPK pathway; C2C12 cells; adipogenesis; cistanoside F; sarcopenic obesity.

Figure 1

Cis treatment protocol. C2C12 cells were cultured in 10% FBS medium at a density of 80% (A) 0.5 mM isobutylmethylxanthine, 150 µM dexamethasone, 85 nM insulin, and 30 µM rosiglitazone (IB+R+IN+D) were used for 2 days, followed by 85 nM insulin and 30 µM rosiglitazone (IN+D) for 6 days. (B) Palmitic acid (PA) was used with 2% BSA, 1% FBS, and 1% penicillin/streptomycin for 48 h. (C) Lipopolysaccharide (LPS) was used for 24 h after 2% HOS for 3 days in C2C12 cells.

Figure 2

Muscle cell content and marker expression after Cis administration. (A,E) Muscle tube diameter was observed through bright-field microscopy and measured. (B) Cell viability was detected using the CCK8 assay for 24, 48, and 72 H. (C) qRT-PCR for mhc mRNA on days 2, 4, and 8. (D,F–I) MHC expression in the IB+R+IN+D model on days 2, 4, and 8 and in the PA model after 48 H. n = 6. ^## p < 0.01, compared with the vehicle group; ** p < 0.01, compared with the model group. ns, no significance.

Figure 3

Effects of Cis on lipid content in C2C12 cells. (A) Lipid droplet content of two lipid differentiation models of C2C12 cells, measured using oil red staining and light microscopy. (B) C2C12 intracellular lipid drop content detected using TG, TCHO, LDL, and HDL detection kits. (C,D) Lipid droplets in two models of significant time of lipid differentiation were measured by a high-content instrument. (E,F) CPT1b, PPARγ, ATGL, and UCP1 expression in the IB+R+IN+D model on day 8. n = 6. ^## p < 0.01, compared with the vehicle group; * p < 0.05, ** p < 0.01, compared with the model group. ns, no significance.

Figure 4

Expression of PGC-1α, MMP, and ROS in two adipogenic models after Cis administration. (A,B) PGC-1α expression. (C,D) Immunofluorescence of TMRE, the statistic was evaluated by fluorescence spectroscopy, at λ_excitation = 550 nm and λ_emission = 575 nm. (E,G) Immunofluorescence of JC-1, statistic was evaluated by fluorescence spectroscopy, JC-1 monomer at λ_excitation = 490 nm and λ_emission = 530 nm and JC-1 aggregate at λ_excitation = 525 nm and λ_emission = 590 nm. (F,H) Immunofluorescence of ROS, the statistic was evaluated by fluorescence spectroscopy, at λ_excitation = 488 nm and λ_emission = 525 nm. n = 6. ^## p < 0.01, compared with the vehicle group; * p < 0.05, ** p < 0.01, compared with the model group.

Figure 5

Expression of p-AMPK/AMPK and its downstream proteins p-ACC1/ACC1 after Cis administration. (A) Molecular docking for the AMPKα–Cis correlation. (B,C) p-AMPK/AMPK and p-ACC1/ACC1 expression in two lipogenic differentiation models. (D,E) ACC2 expression in the IB+R+IN+D model. n = 6. ^# p < 0.05, ^## p < 0.01, compared with the vehicle group; ** p < 0.01, compared with the model group. ns, no significance.

Figure 6

Relationship between p-AMPK/AMPK and MHC expression in vehicle groups after exposure to the (A,B) AMPK inhibitor AICAR and (C,D) AMPK agonist compound C. n = 6. ^## p < 0.01, compared with the vehicle group. ns, no significance. (E) Pearson correlation analysis with all dates in vehicle groups with r = 0.9507 and p < 0.0001.

Figure 7

Relationship between p-AMPK/AMPK and MHC expression in model groups after exposure to the (A,B) AMPK inhibitor AICAR and (C,D) AMPK agonist compound C. n = 6. * p < 0.05, ** p < 0.01, compared with the model group; ^&& p < 0.01, compared with the compound C group. ns, no significance. (E) Pearson correlation analysis with all dates in model groups with r = 0.6561 and p < 0.0001.

Figure 8

Expression of inflammation- and muscle-related markers after Cis addition in IB+R+IN+D and LPS models. (A,D) p-NF-κB/NF-κB expression in the IB+R+IN+D model. (B,C) Il6 mRNA and Elisa expression in the IB+R+IN+D model. (E) The relationship between sarcopenia, inflammation, and lipid metabolism. (F,G) Immunofluorescence localization of the inflammatory marker NF-κB (Red) and muscle quantity marker Desmin (Red) in the LPS model. Nuclei were stained with DAPI (Blue). n = 6. ^## p < 0.01, compared with the vehicle group; * p < 0.05, ** p < 0.01, compared with the model group. ns, no significance.