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Regulation of the pyruvate dehydrogenase complex

Naik, Sharon S.
http://rave.ohiolink.edu/etdc/view?acc_num=case1062509957

Abstract Details

1995, Doctor of Philosophy, Case Western Reserve University, Biochemistry.
The mammalian pyruvate dehydrogenase complex (PDC) is subjected to both short-term (product inhibition and covalent modification) and long term (increases in total activity and protein mass) regulation mediated by dietary and hormonal treatments. Initial research efforts were focused on the isolation and characterization of the human E1β gene. The 2.3 kb E1β promoter region lacked a TATA box homology but contained initiator sequences (two) and Sp1 sites (three) frequently found in TATA-less promoters that may play a role in defining the start site of transcription. Transient expression of chloramphenicol acetyltransferase (CAT) activity of nested deletions of the E1β 5′-flanking region (-2316 to +32 base pairs) measured in human hepatoma (HepG2) cells, suggested the possible presence of repressor elements located between sequences -2316 to -930 base pairs. Deletion constructs containing sequences from -929 to +32 and -98 to +32 base pair showed approximately 7- and 20-fold increases in CAT over that containing -2316 to +32 base pairs of the E1β promoter region. The presence of a repressor element between -2316 to -929 base pairs was further confirmed by measuring CAT activity from a heterologous thymidine kinase promoter. Similar studies on the measurement of promoter activity of a 796 base pair E1α promoter fragment (-763 to +33 bp) generated by polymerase chain reaction (PCR) from normal human skin fibroblasts was measured in HepG2 cells. Several sequences showing consensus with known functional elements have been identified within the 5′-flanking region of the human E1α gene. They include the following: activator protein AP-1, cAMP-responsive element (CRE), two activator proteins (AP-2), CCAAT/enhancer binding protein (C/EBP) and an insulin-responsive sequence (IRS). It was observed that a construct containing the 796 base pair fragment (-763 to +33) resulted in very high CAT expression comparable to that derived from pSV2CAT (positive control). The smallest construct, containing only 102 base pairs of the promoter region resulted in a significant decrease in CAT activity, but was still capable of efficiently promoting CAT expression. The E1α promoter (-763 to +33) resembles facultative promoters in having appropriately positioned "TATA" and "CAAT" boxes with several upstream potential enhancer sequences which allows for efficient expression of CAT in HepG2 cells. Diet-induced long-term regulation of PDC appears to involve coordinated modulation of all the component proteins of the complex. The present study was carried out to evaluate the long-term effects of high-sucrose and high-fat diets on PDC activity in the liver. Rats fed a high-sucrose diet for two and three weeks resulted in a 2-fold increase in total activity compared to chow-fed rats. Changes in total PDC activity closely correlated with alterations in the amount of proteins quantified by immunoblotting, suggesting that increased enzyme content is the predominant mechanism underlying the adaptive response to high-sucrose feeding. There was no significant change in the total PDC activity in rats fed a high fat diet for two weeks as compared to animals fed the chow diet. The levels of RNA determined by Northern analysis showed an increase of approximately 1.5 fold in the high-sucros e group as compared to the chow fed animals. This indicates that the increase in the amount of PDC proteins observed in the liver upon feeding a high-sucrose diet may be mediated by transcriptional and/or translational mechanism
Mulchand Patel (Advisor)
156 p.
Biology, Molecular
Pyruvate dehydrogenase complex (PDC)

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Citations

  • Naik, S. S. (1995). Regulation of the pyruvate dehydrogenase complex [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1062509957

    APA Style (7th edition)

  • Naik, Sharon. Regulation of the pyruvate dehydrogenase complex. 1995. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1062509957.

    MLA Style (8th edition)

  • Naik, Sharon. "Regulation of the pyruvate dehydrogenase complex." Doctoral dissertation, Case Western Reserve University, 1995. http://rave.ohiolink.edu/etdc/view?acc_num=case1062509957

    Chicago Manual of Style (17th edition)

case1062509957
432
© 1995, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.