Exploring the Trpv1 and Pgc-1α Crosstalk Mechanisms Mediating the Effects of Plasticizers on Muscle Cell Differentiation and Metabolism( I )

Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer leached out from polyvinyl chloride (PVC) products and isquickly metabolized to its monoester equivalent mono(2-ethylhexyl) phthalate (MEHP) once entersorganisms. Previous studies have shown that DEHP/MEHP can serve as xenobiotic proliferators ofperoxisomes, which are involved in various metabolic pathways, including β-oxidation of fatty acids (FA),detoxification of hydrogen peroxide, and synthesis of cholesterol. DEHP/MEHP promotes the proliferationof peroxisomes through functioning as the ligands of the nuclear hormone receptors called peroxisomeproliferator receptors (PPARs). Apart from functioning as the ligand of intracellular PPARs, DEHP can alsobind to the transient receptor potential channel-vanilloid sub-family member 1 (TRPV1) to regulate its Ca+2channel activity. Skeletal muscle (SKM) is closely associated with systemic metabolic disorders such asinsulin resistance, type II diabetes, and obesity, and its metabolism is highly regulated by the oxidativemetabolism-promoting transcription coactivator PGC-1α. Besides, SKM contains high level of stem cells,called satellite cells, for coping with the continuous cycle of damage and regeneration. TRPV1 in SKM ismainly located in the sarcoplasmic reticulum (SR) responding for load signal induced Ca+2 release andcontraction. Interestingly, long-term activation of TRPV1 by capsaicin through food intake promotes SKMoxidative metabolism and exercise endurance, which suggests that SR TRPV1 is important to theintracellular relay of oxidative metabolic signals. Due to the continuous and inevitable exposure toDEHP/MEHP, further delineating the influence of DEHP on SKM metabolism and stem cell poolmaintenance is required. We will firstly examine whether DEHP affects the survival, proliferation, anddifferentiation abilities of satellite cells (derived from wildtype, TRPV1-/-, db/db, and mdx mice) and culturedC2C12 myoblasts (together called as SKM cells here) for the maintenance of muscle mass in the presenceand absence of DEHP so the influence of DEHP on this differentiation can be revealed. Secondly, theeffects of DEHP on SKM cell metabolism, including uptake and catabolism of glucose, fatty acid, andprotein, and the energy generation (such as ATP and NADPH) and utilization (such as O2 consumption) willbe carefully checked. Thirdly, the amount, biogenesis, and function of the cellular power houses,mitochondrion and peroxisome, will be examined to explore the possible pathways affected by DEHP.Fourthly, the genes mediating the effects of DEHP will be identified by microarray assay and their roles willbe verified by gain and loss of function assays. Finally, the crosstalk between TRPV1 and PGC-1α onDEPH targeted gene promoters will be discussed. The effects of DEPH on promoters of PGC-1α target gene,including its own, will also be examined in TRPV1-/- cells to reveal the relationship between these twopathways. The proposed works described above can be summarized as the following specific aims:1. Examining DEHP effects on SKM cell proliferation, differentiation, and survival.2. Metabolic function profiling of DEHP treated SKM cells and animals.3. Examining DEHP effects on peroxisome and mitochondrion biology4. Identifying and verifying the DEHP target genes in SKM.5. Analyzing the crosstalk between TRPV1 and PGC-1α upon DEPH treatment.