Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M/PCK2), phosphoglycerate dehydrogenase (PHGDH) and muscle cell growth
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/P...
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| Format: | Thesis (University of Nottingham only) |
| Language: | English |
| Published: |
2018
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| Online Access: | https://eprints.nottingham.ac.uk/52138/ |
| _version_ | 1848798656607551488 |
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| author | Brearley, Madelaine C. |
| author_facet | Brearley, Madelaine C. |
| author_sort | Brearley, Madelaine C. |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | 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. |
| first_indexed | 2025-11-14T20:23:14Z |
| format | Thesis (University of Nottingham only) |
| id | nottingham-52138 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T20:23:14Z |
| publishDate | 2018 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-521382025-02-28T14:08:47Z https://eprints.nottingham.ac.uk/52138/ Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M/PCK2), phosphoglycerate dehydrogenase (PHGDH) and muscle cell growth Brearley, Madelaine C. 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. 2018-07-13 Thesis (University of Nottingham only) NonPeerReviewed application/pdf en arr https://eprints.nottingham.ac.uk/52138/1/Brearley%20-%20PhD%20Thesis%20-%20Final.pdf Brearley, Madelaine C. (2018) Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M/PCK2), phosphoglycerate dehydrogenase (PHGDH) and muscle cell growth. PhD thesis, University of Nottingham. PEPCK-M; PCK2: PHGDH; C2C12; muscle cell growth; dbcAMP; 3-Mercaptopicolinic acid |
| spellingShingle | PEPCK-M; PCK2: PHGDH; C2C12; muscle cell growth; dbcAMP; 3-Mercaptopicolinic acid Brearley, Madelaine C. Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M/PCK2), phosphoglycerate dehydrogenase (PHGDH) and muscle cell growth |
| title | Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M/PCK2), phosphoglycerate dehydrogenase (PHGDH) and muscle cell growth |
| title_full | Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M/PCK2), phosphoglycerate dehydrogenase (PHGDH) and muscle cell growth |
| title_fullStr | Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M/PCK2), phosphoglycerate dehydrogenase (PHGDH) and muscle cell growth |
| title_full_unstemmed | Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M/PCK2), phosphoglycerate dehydrogenase (PHGDH) and muscle cell growth |
| title_short | Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M/PCK2), phosphoglycerate dehydrogenase (PHGDH) and muscle cell growth |
| title_sort | mitochondrial phosphoenolpyruvate carboxykinase (pepck-m/pck2), phosphoglycerate dehydrogenase (phgdh) and muscle cell growth |
| topic | PEPCK-M; PCK2: PHGDH; C2C12; muscle cell growth; dbcAMP; 3-Mercaptopicolinic acid |
| url | https://eprints.nottingham.ac.uk/52138/ |