Milk fat globule membrane (MFGM) protein is known to have several health benefits, including an anti-sarcopenia effect; however, its mechanism is unclear. The aim of this study was to investigate the potential mechanism of action of the MFGM protein. The MFGM protein was extracted and separated into 4 fractions, and Fraction 2 (57% of total MFGM) demonstrated the greatest effect on C2C12 cell proliferation. Milk fat globule-EGF factor 8 (MFG-E8) accounted for 82.35% of the MFGM protein. The effects of whole Fraction 2 (100μg/mL, 200μg/mL and 300μg/mL) on cell proliferation and morphology were measured. Using qRT-PCR or a Western blot assay, several regulatory factors, e.g., PI3K P85α, p-pI3K p85α (Tyr 508), Akt, p-Akt (Ser 473), mTOR and p-mTOR (Ser 2448), were measured in cells incubated with 200μg/mL of Fraction 2 with or without wortmannin. The results demonstrated that Fraction 2 induced C2C12 cell proliferation in a dose-dependent manner, upregulated the mRNA expression of mTOR and p70S6K, and activated PI3K, Akt, mTOR and P70S6K phosphorylation; however, Fraction 2 inhibited FOXO3a and 4E-BP. The results demonstrate that the MFGM protein, predominantly MFG-E8, promotes cell proliferation through the PI3K/Akt/mTOR signaling pathway. This study elucidated the molecular mechanism of the MFGM protein, primarily MFG-E8, in promoting C2C12 cell proliferation via the PI3K/Akt/mTOR/P70S6K signal pathway.

Milk fat globule-EGF factor 8 (MFG-E8) is one of the major proteins in milk fat globule membrane. In this study, mouse-derived C₂C₁₂ myoblast cells were served as an experimentally tractable model system for investigating the molecular basis of skeletal muscle cell specification and development. To examine the biochemical adaptations associated with myocytes formation comprehensively, a liquid chromatography coupled with tandem mass spectrometry label-free semi-quantitative approach was used to analyse the myogenic C₂C₁₂ proliferation program. Over 1987 proteins were identified in C₂C₁₂ cells. The MFG-E8 (200 μg/mL) and MFG-E8 (500 μg/mL) with significant differences were compared based on the relative abundance. The result profiles of regulation of MFG-E8 to the expression of proteins in C₂C₁₂ cells revealed that differential waves of expression of proteins linked to intracellular signaling, transcription, cytoarchitecture, adhesion, metabolism, and muscle contraction across during the C₂C₁₂ cell proliferation process. Based on the analysis of KEGG and STRING database, further to verification the expression of PI3K and ERK phosphorylation levels by Western blot. This study found that the data of proteomic was complementary to recent MFG-E8 studies of protein expression patterns in developing myotubes and provided a holistic framework for understanding how diverse biochemical processes are coordinated at the cellular level during skeletal muscle development.

Milk fat globule epidermal growth factor VIII (MFG-E8) and whey protein have emerged as promising bionutrient supplements for enhancing skeletal muscle mass and function. In the present study, aging-related sarcopenia rat model was employed to elucidate the effects of the combined administration of MFG-E8 and whey protein on the catabolism and anabolism of gastrocnemius protein. Combined intervention led to notable enhancements in the anti-oxidative stress status and mitochondrial biogenesis capacity of gastrocnemius muscle fibers in the aging rats, concomitant with a significant inhibition of lipid accumulation. Moreover, the synergistic effect of MFG-E8 and whey protein was found to exert modulatory effects on key signaling pathways, including PI3K/Akt/PGC-1α pathway and MAPK/ERK signaling pathways in the gastrocnemius muscle of the aging rats. Specifically, this combined intervention was observed to promote mitochondrial biogenesis and regulate the expression of protein anabolism and catabolism-related regulators, thereby facilitating the alleviation of mitochondrial oxidative stress and enhancing biogenesis in gastrocnemius tissues. The findings of our study provide compelling evidence for the potential of MFG-E8 as a promising dietary supplement with anti-sarcopenic properties to ameliorate muscle protein metabolism disorders and mitigate mitochondrial-mediated myoblast apoptosis induced by oxidative stress.

Sarcopenia has high prevalence in the elderly population, but the genes and pathways related to aging in elderly patients with sarcopenia are poorly understood. Milk fat globule epidermal growth factor 8 (MFG-E8) is a peripheral membrane glycoprotein isolated from the milk fat globule membrane (MFGM). It has been found to exhibit various nutritional effects, including antibacterial, anti-cancer, anti-oxidant, anti-sarcopenia, and improving brain development and cognitive effects. This study aimed to investigate key differentially expressed genes (DEGs) and pathways associated with the progression of sarcopenia using bioinformatics analysis and in vitro myoblast experiment. The gene expression profiles of GSE8479 and GSE9676, which includes 40 young normal samples and 55 elderly samples, were downloaded from the Gene Expression Omnibus Database (GEO). Over 3253 DEGs were identified in the young and elderly samples (adjusted p value <0.05). A total of 213 co-expressed significantly DEGs were identified with Venn diagrams, including 82 up-regulated DEGs and 131 down-regulated DEGs. Based on the analysis of Gene Ontology (GO), protein–protein interaction (PPI) networks and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, 10 hub genes screened by our study have been proved to play a role in regulating the occurrence and development of aging-related sarcopenia mainly via metabolic pathways, Huntington's disease, Parkinson's disease, oxidative phosphorylation and non-alcoholic fatty liver disease pathways. To further verify the protective effect of MFG-E8 on oxidative stress injured myoblasts, the cell cycle distribution, cell viability and reactive oxygen species (ROS) production were measured. The protein and mRNA levels of Akt, extracellular regulated protein kinases (ERK), p21Cip1, p27Kip1, cyclin D1, cyclin E1, cyclin-dependent kinase (CDK) 2 and 4 were quantified using qRT-PCR and western blot analysis. The results indicated that MFG-E8 has potential anti-sarcopenia effects by promoting ERK and Akt activation-mediated cell proliferation and cell cycle progression in myoblasts.