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Brearley, Madelaine C. (2018) Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M/PCK2), phosphoglycerate dehydrogenase (PHGDH) and muscle cell growth. PhD thesis, University of Nottingham.

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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:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Brameld, John M. Parr, Tim
Keywords:	PEPCK-M; PCK2: PHGDH; C2C12; muscle cell growth; dbcAMP; 3-Mercaptopicolinic acid
Subjects:	Q Science > QP Physiology > QP501 Animal biochemistry
Faculties/Schools:	UK Campuses > Faculty of Science > School of Biosciences
Item ID:	52138
Depositing User:	Brearley, Madelaine C.
Date Deposited:	29 Oct 2018 14:54
Last Modified:	13 Jul 2022 04:30
URI:	https://eprints.nottingham.ac.uk/id/eprint/52138

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