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Nuclear respiratory factor 1: a novel inhibitor of the human gene promoter of the cardiac isoform of acetyl-coenzyme a carboxylase
Introduction: Nuclear respiratory factor-1 (NRF-1) is a pivotal transcriptional modulator controlling the expression of nuclear genes encoding mitochondrial proteins. Activation of 5'-AMP-activated protein kinase (AMPK) has been associated with enhanced NRF-1 gene promoter binding activity. Furtherm...
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| Format: | Thesis |
| Language: | English English |
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Department of Medicine
2025
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| _version_ | 1867613316241686528 |
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| access_status_str | Open Access |
| author | Adam, Tasneem |
| author2 | Hatter Cardiovascular, Essop, M F |
| author_browse | Adam, Tasneem Hatter Cardiovascular, Essop, M F |
| author_facet | Hatter Cardiovascular, Essop, M F Adam, Tasneem |
| author_sort | Adam, Tasneem |
| collection | Thesis |
| description | Introduction: Nuclear respiratory factor-1 (NRF-1) is a pivotal transcriptional modulator controlling the expression of nuclear genes encoding mitochondrial proteins. Activation of 5'-AMP-activated protein kinase (AMPK) has been associated with enhanced NRF-1 gene promoter binding activity. Furthermore, NRF-1 has been linked with increased gene expression of carnitine palmitoyltransferase-1 (CPT-1), the rate-limiting mitochondrial fatty acid (FA) transfer enzyme. Upstream, the cardiac-enriched isoform of acetyl-CoA carboxylase (ACCl3) synthesises malonyl-CoA, a potent inhibitor of CPT-1 and FA 13-oxidation. Since ACCl3 induction elevates malonyl-CoA levels, we hypothesised that AMPK activates NRF-1 thereby inhibiting ACCl3 expression in the heart to ultimately increase FA !3-oxidation. Methods: A 1,317 bp human ACCl3 gene promoter-luciferase construct (pPll13-1317/+65-Luc) was transiently transfected into rat neonatal cardiomyocytes ± the following expression constructs: NRF-1 (pSG5-NRF-1) and dominant negative NRF-1 (pCMV-HA-dnNRF-1). To elucidate whether AMPK is involved in the transcriptional regulation of ACCl3 gene, the transfected cardiomyocytes were treated with AMPK activators, i.e. AICAR or metformin for 24 hours. Transfections were also performed with upstream stimulatory factor 1 (USF1), a known transactivator of the human ACCl3 gene promoter. In addition, the effect of NRF-1 on endogenous USF1 transcriptional activity was evaluated with a luciferase construct containing multiple copies of USF1-specific enhancer elements (pUSF1-Luc). Results: NRF-1 overexpression reduced ACCl3 gene promoter activity by 56 ± 8.8% (p<0.001 vs. pPlll3-1317/+65-Luc). Cotransfection with the dnNRF-1 construct abrogated this effect. Addition of AICAR dose-dependently decreased ACCl3 gene promoter activity (p<0.05 vs. untreated control). In contrast, NRF-1 attenuation of ACCl3 gene promoter activity was not altered by AICAR treatment. Exposure to metformin did not affect the ACCl3 gene promoter activity at baseline, and in response to NRF-1. NRF-1 inhibited the USF1-dependent upregulation of ACCl3 gene promoter activity by 58 ± 7.5% (p<0.001 vs. pPlll3-1317/+65-Luc + USF1), an effect reversed with the dnNRF-1 construct. In addition, NRF-1 reduced endogenous USF1 transcriptional activity by 55 ± 6.2% (p<0.001 vs. pUSF1-Luc). This effect was abolished with the dnNRF-1 construct. The NRF-1-mediated decrease in endogenous USF1 transcriptional activity was independent of AMPK activation with AICAR. The ACC~ mRNA expression level was unaffected at baseline, and by NRF-1 overexpression. However, AICAR (2 mM) treatment markedly reduced the ACC~ mRNA expression level (p<0.05 vs. vehicle) at baseline, but had no effect in response to NRF-1 overexpression. Conclusion: Our data reveal a unique, inhibitory role for NRF-1 in the transcriptional regulation of human ACC~ gene in the mammalian heart, independent of AMPK activation. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/41985 |
| institution | University of Cape Town (South Africa) |
| language | English eng |
| last_indexed | 2026-06-10T12:34:10.861Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2025 |
| publishDateRange | 2025 |
| publishDateSort | 2025 |
| publisher | Department of Medicine |
| publisherStr | Department of Medicine |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/41985 Nuclear respiratory factor 1: a novel inhibitor of the human gene promoter of the cardiac isoform of acetyl-coenzyme a carboxylase Adam, Tasneem Hatter Cardiovascular, Essop, M F cardiac isoform Introduction: Nuclear respiratory factor-1 (NRF-1) is a pivotal transcriptional modulator controlling the expression of nuclear genes encoding mitochondrial proteins. Activation of 5'-AMP-activated protein kinase (AMPK) has been associated with enhanced NRF-1 gene promoter binding activity. Furthermore, NRF-1 has been linked with increased gene expression of carnitine palmitoyltransferase-1 (CPT-1), the rate-limiting mitochondrial fatty acid (FA) transfer enzyme. Upstream, the cardiac-enriched isoform of acetyl-CoA carboxylase (ACCl3) synthesises malonyl-CoA, a potent inhibitor of CPT-1 and FA 13-oxidation. Since ACCl3 induction elevates malonyl-CoA levels, we hypothesised that AMPK activates NRF-1 thereby inhibiting ACCl3 expression in the heart to ultimately increase FA !3-oxidation. Methods: A 1,317 bp human ACCl3 gene promoter-luciferase construct (pPll13-1317/+65-Luc) was transiently transfected into rat neonatal cardiomyocytes ± the following expression constructs: NRF-1 (pSG5-NRF-1) and dominant negative NRF-1 (pCMV-HA-dnNRF-1). To elucidate whether AMPK is involved in the transcriptional regulation of ACCl3 gene, the transfected cardiomyocytes were treated with AMPK activators, i.e. AICAR or metformin for 24 hours. Transfections were also performed with upstream stimulatory factor 1 (USF1), a known transactivator of the human ACCl3 gene promoter. In addition, the effect of NRF-1 on endogenous USF1 transcriptional activity was evaluated with a luciferase construct containing multiple copies of USF1-specific enhancer elements (pUSF1-Luc). Results: NRF-1 overexpression reduced ACCl3 gene promoter activity by 56 ± 8.8% (p<0.001 vs. pPlll3-1317/+65-Luc). Cotransfection with the dnNRF-1 construct abrogated this effect. Addition of AICAR dose-dependently decreased ACCl3 gene promoter activity (p<0.05 vs. untreated control). In contrast, NRF-1 attenuation of ACCl3 gene promoter activity was not altered by AICAR treatment. Exposure to metformin did not affect the ACCl3 gene promoter activity at baseline, and in response to NRF-1. NRF-1 inhibited the USF1-dependent upregulation of ACCl3 gene promoter activity by 58 ± 7.5% (p<0.001 vs. pPlll3-1317/+65-Luc + USF1), an effect reversed with the dnNRF-1 construct. In addition, NRF-1 reduced endogenous USF1 transcriptional activity by 55 ± 6.2% (p<0.001 vs. pUSF1-Luc). This effect was abolished with the dnNRF-1 construct. The NRF-1-mediated decrease in endogenous USF1 transcriptional activity was independent of AMPK activation with AICAR. The ACC~ mRNA expression level was unaffected at baseline, and by NRF-1 overexpression. However, AICAR (2 mM) treatment markedly reduced the ACC~ mRNA expression level (p<0.05 vs. vehicle) at baseline, but had no effect in response to NRF-1 overexpression. Conclusion: Our data reveal a unique, inhibitory role for NRF-1 in the transcriptional regulation of human ACC~ gene in the mammalian heart, independent of AMPK activation. 2025-10-08T07:07:16Z 2025-10-08T07:07:16Z 2008 2025-10-08T07:04:35Z Thesis / Dissertation Masters MSc http://hdl.handle.net/11427/41985 en eng application/pdf Department of Medicine Faculty of Health Sciences Universiy of Cape Town |
| spellingShingle | cardiac isoform Adam, Tasneem Nuclear respiratory factor 1: a novel inhibitor of the human gene promoter of the cardiac isoform of acetyl-coenzyme a carboxylase |
| thesis_degree_str | Master's |
| title | Nuclear respiratory factor 1: a novel inhibitor of the human gene promoter of the cardiac isoform of acetyl-coenzyme a carboxylase |
| title_full | Nuclear respiratory factor 1: a novel inhibitor of the human gene promoter of the cardiac isoform of acetyl-coenzyme a carboxylase |
| title_fullStr | Nuclear respiratory factor 1: a novel inhibitor of the human gene promoter of the cardiac isoform of acetyl-coenzyme a carboxylase |
| title_full_unstemmed | Nuclear respiratory factor 1: a novel inhibitor of the human gene promoter of the cardiac isoform of acetyl-coenzyme a carboxylase |
| title_short | Nuclear respiratory factor 1: a novel inhibitor of the human gene promoter of the cardiac isoform of acetyl-coenzyme a carboxylase |
| title_sort | nuclear respiratory factor 1 a novel inhibitor of the human gene promoter of the cardiac isoform of acetyl coenzyme a carboxylase |
| topic | cardiac isoform |
| url | http://hdl.handle.net/11427/41985 |
| work_keys_str_mv | AT adamtasneem nuclearrespiratoryfactor1anovelinhibitorofthehumangenepromoterofthecardiacisoformofacetylcoenzymeacarboxylase |