Date of Graduation

5-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Yan Huang

Committee Member

Navam Hettiarachchy

Second Committee Member

Guillermo Tellez-Isaias

Third Committee Member

Young Min Kwon

Fourth Committee Member

Jackson Lay Jr

Keywords

Adipogenesis; Fatty acids; Metformin; Myogenesis; Pregnancy; Stem cells

Abstract

The impacts of omega-3 (ω-3) fatty acid and metformin hydrochloride (Met-HCl) on muscle stem cells differentiation, mimicking the fetal muscle development under gestational diabetes mellitus (GDM), were investigated. ω-3 fatty acids have been known for their ability to regulate adipogenic transcription factors such as Sterol Regulatory Element-Binding Protien (SREBPs) and Peroxisome Proliferator-Activated Receptors (PPARs). This regulation resulted in gene expression modification for lipid metabolism and adipocyte differentiation, thus preventing excessive fat accumulation. Additionally, emerging evidence suggested that ω-3 fatty acids may also exert direct effects on myogenic processes that influence muscle stem cells proliferation, differentiation, and maturation. Meanwhile, Met-HCl, an antidiabetic drug, has been known for its metabolic effects on various stages of regulatory stem cells growth, particularly within myogenic models. This current study focused on C2C12 stem cells, a well-established model for skeletal muscle differentiation in vitro. The focus of this study was to elucidate the effects of omega-3 fatty acids on myogenesis and adipogenesis, as well as to evaluate the impact of metformin on myogenesis that was focused on stem cells commitment, proliferation, differentiation, and maturation. The results of the studies demonstrated the individual impacts of ω-3 fatty acids and metformin on muscle stem cell differentiation and explored potential genetic and molecular changes. It was hypothesized that ω-3 fatty acids and metformin may improve muscle growth in vitro by affecting the cell signaling pathways of myogenesis and adipogeneses. The hypothesis was evaluated by exploring the molecular and cellular mechanism of the influences of ω-3 and Met-HCl on skeletal muscle during pregnancy in vitro using C2C12 mouse muscle stem cell line. The results of our in vitro studies using mouse stem cell C2C12 cells indicated that significant morphological alterations occurred during myoblast differentiation, as confirmed by Oil Red O staining. Furthermore, RT-PCR and Oil Red O staining results validated the trans-differentiation of C2C12 cells into white adipocyte-like cells in the PUFA groups, characterized by the absence of multinucleated myotubes and the presence of lipid droplet-containing clusters of rounded cells compared to the CON groups. These observations suggest a potential link between excessive maternal exposure to EPA and DHA, contributing to the trans-differentiation of myoblasts and impacting the development, mass, and quality of offspring muscle tissue. Additionally, our investigation revealed that myogenesis was suppressed by metformin treatment, indicating its potential regulatory role in modulating muscle development. Notably, among the adipogenic marker genes, PPARγ expression was stimulated by metformin treatments in a concentration-dependent manner. Overall, our in vitro studies demonstrated morphological changes in myoblasts during differentiation, indicating trans-differentiation into adipocyte-like cells with PUFA exposure. This suggests a potential impact of maternal EPA and DHA on offspring muscle development. Additionally, our studies revealed metformin's inhibitory effect on myogenesis and its stimulation of adipogenic marker gene expression. These findings call for further research to explore the precise mechanisms underlying the observed effects and to investigate potential therapeutic interventions involving metformin in the context of muscle development and metabolic disorders.

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