Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M/PCK2), phosphoglycerate dehydrogenase (PHGDH) and muscle cell growth
Publication Date
July 13, 2018
Creator
Abstract
Our group reported upregulation of a novel group of genes was associated with beta-adrenergic agonist (BA)-induced muscle hypertrophy in pigs. The aim of this PhD was to investigate the expression of these genes, and particularly the role of mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M/PCK2) and phosphoglycerate dehydrogenase (PHGDH), in muscle cell growth.
A significant (p<0.01) increase in mRNA transcript abundance was detected at day 2 of differentiation in C2C12 cells for PEPCK-M, PHGDH, phosphoserine aminotransferase-1, phosphoserine phosphatase, asparagine synthetase, sestrin-2 and activating transcription factor-5. This novel peak coincided with the peak in myogenin mRNA, connecting these genes with a crucial point of myogenic differentiation. Hypertrophy was induced in C2C12 myotubes treated with dibutyryl-cAMP (dbcAMP), mimicking the BA response in vivo, however mRNA expression of these genes were unaffected. The porcine myosin heavy chain (MyHC)-IIB promoter-reporter C2C12 cell assay demonstrated similar in vivo responses to known anabolic and catabolic agents. Thus, C2C12 cells were utilised to determine the role of PEPCK-M and PHGDH in myogenic differentiation.
Firstly, C2C12 cells were treated with a PEPCK inhibitor, 3-Mercaptopicolinic acid (3-MPA). 3-MPA induced differentiation, resulting in a hypertrophic response comparable to dbcAMP treatment. However, it was unclear whether 3-MPA inhibited PEPCK-M enzyme activity as 3-MPA interfered with the in vitro assay. Next, C2C12 cells were transfected with either PCK2 or PHGDH overexpression construct. No obvious phenotype was observed, but PHGDH and PEPCK-M overexpression both increased MyHC-IIB mRNA. The reoccurring induction of the same group of genes along with MyHC-IIB supports the hypothesis that co-ordinated upregulation of these genes may drive hypertrophic growth.
To conclude, PEPCK-M, along with other genes upregulated with BA-induced hypertrophy and C2C12 differentiation, show co-ordinate regulation in times of high biosynthetic demand. PEPCK-M appears to sit at an intersection that allows metabolic flux to be largely altered by diverting intermediates during energy metabolism.
Item Type
ethesis
Thesis Type
PhD
Supervisors
Subjects (LC)
Associated Schools / Departments
School of Biosciences (UK)
eprints ID
52138
UoN Repository URI
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Name
Brearley - PhD Thesis - Final.pdf
Type
Full-text
Description
Examined
Size
8 MB
Format
Adobe PDF
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