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dc.contributor.advisorThompson, Mary P
dc.contributor.authorRae-Whitcombe, Sharleen Margaret
dc.date.available2010-07-14T02:58:38Z
dc.date.copyright2010
dc.identifier.citationRae-Whitcombe, S. M. (2010). Regulation of the human adiponutrin promoter, PNPLA3 (Thesis, Master of Science). University of Otago. Retrieved from http://hdl.handle.net/10523/356en
dc.identifier.urihttp://hdl.handle.net/10523/356
dc.description.abstractThe adiponutrin gene (Adpn) and its human homologue PNPLA3 code for a protein which is highly expressed in adipose tissue and liver, and appears to be involved in regulation of energy homeostasis. The nutritional regulation and patterns of tissue expression exhibited by Adpn/PNPLA3 are reminiscent of lipogenic genes (Baulande et al., 2001, Polson and Thompson, 2004), yet regions of the gene sequence are homologous to those of functional significance in lipolytic proteins such as adipocyte triaclyglycerol lipase (ATGL) (Lake et al., 2005). Experimental evidence has been provided in vitro that Adpn/PNPLA3 can catalyse both lipogenic and lipolytic reactions (Jenkins et al., 2004; Lake et al., 2005), but its precise biological function remains unclear. Previously, 3.1 kb of the 5´ upstream region (USR) of the human adiponutrin gene PNPLA3 was cloned into a pGL4.10 luc2 reporter vector (Dr. Mary Thompson, University of Otago), and transfected into Chinese hamster ovary (CHO) cells (Kennedy, 2007). This was demonstrated to significantly upregulate luc2 expression over the basic pGL4.10 vector. The current project aimed to investigate whether the PNPLA3 5´ USR is regulated in response to glucose and/or insulin, or the chemical compounds forskolin, rosiglitazone, or α-lipoic acid. Possible involvement of the regulatory protein upstream stimulatory factor 2 (USF2) in stimulating PNPLA3 transcription was also investigated, as this factor is known to be involved in glucose-dependent upregulation of other genes. The pADPN reporter plasmid was transiently transfected into three mammalian cell lines; Huh-7, HepG2 and CHO-IR. Cells were then subjected to various experimental treatments, and the activity of the promoter was assayed by measurement of luciferase activity in harvested cell lysates. Endogenous levels of PNPLA3 mRNA in the human hepatoma cell line Huh-7 were also assayed for mRNA levels in response to these treatments, using reverse transcription qPCR. The 3.1 kb PNPLA3 5´ USR upregulated baseline transcription of the luc2 reporter gene in low glucose media up to 46 fold in Huh-7 cells, 16 fold in CHO-IR cells and 6 fold in HepG2 cells, demonstrating cell-line specific regulation of the promoter. High glucose (12.5- 25 mM) upregulated PNPLA3 promoter activity in HepG2 (up to 3.5 fold) and Huh-7 (up to 2 fold), but upregulation was not clearly demonstrated in CHO-IR cells. Insulin upregulated PNPLA3 promoter activity up to 2 fold in CHO-IR cells in the presence of glucose, but this was not observed in Huh7 or HepG2 cells. The regulatory factor USF2 also upregulated PNPLA3 promoter activity in CHO-IR cells up to 1.6 fold, and appeared to be operating independently of insulin. Rosiglitazone produced a 1.3 fold increase in PNPLA3 promoter activity in Huh-7 cells, and the endogenous PNPLA3 mRNA in Huh-7 cells was also observed to be upregulated 1.4 fold by rosiglitazone. Forskolin was observed to downregulate the endogenous PNPLA3 mRNA in Huh-7 cells by 30%. Alpha lipoic acid maintained endogenous PNPLA3 mRNA at low levels compared to controls although, in contrast, it caused upregulation of the promoter in a dose dependent manner. In conclusion, the PNPLA3 3.1 kb 5´ region contains elements which have been demonstrated to upregulate the transcription of the luc2 reporter gene in a cell-line specific manner. This upregulation is further enhanced by glucose, insulin, rosiglitazone, α-lipoic acid and USF2 in some, but not all cell culture types. In the liver carcinoma cell line Huh-7, endogenous mRNA was downregulated by forskolin and α-lipoic acid and upregulated slightly by rosiglitazone, but no effect of insulin or glucose was observed.en_NZ
dc.format.mimetypeapplication/pdf
dc.publisherUniversity of Otago
dc.rightshttp://www.otago.ac.nz/administration/policies/otago003228.htmlen_NZ
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.rights.urihttp://www.otago.ac.nz/administration/policies/otago003228.html
dc.subjectGene promoter regulationen_NZ
dc.subjectMammalian cell cultureen_NZ
dc.titleRegulation of the human adiponutrin promoter, PNPLA3en_NZ
dc.typeThesis
dc.date.updated2010-07-14T01:51:26Z
thesis.degree.disciplineBiochemistryen_NZ
thesis.degree.nameMaster of Scienceen_NZ
thesis.degree.grantorUniversity of Otago
thesis.degree.levelMasters Theses
otago.openaccessOpen
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