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Originally published In Press as doi:10.1074/jbc.M004078200 on August 22, 2000
J. Biol. Chem., Vol. 275, Issue 48, 38012-38021, December 1, 2000
Functional Analysis of Two Promoters for the Human Mitochondrial
Glycerol Phosphate Dehydrogenase Gene*
Qiuming
Gong,
Laura J.
Brown, and
Michael J.
MacDonald
From the Children's Diabetes Center, University of Wisconsin,
Madison, Wisconsin 53706
Mitochondrial glycerol phosphate dehydrogenase
(mGPD) is abundant in the normal pancreatic insulin cell, but its level
is lowered 50% by diabetes. To evaluate mGPD expression, we cloned and
characterized the 5'-flanking region of the human mGPD gene. The gene
has two alternative first exons and two promoters. The downstream
promoter (B) is 10 times more active than the upstream promoter (A) in
insulin-secreting cells (INS-1) and HeLa cells. Promoter B has higher
activity in INS-1 than in non- cells. Deletion and mutation analysis
suggested that a NRF-2 binding site at 94 to 101 and an E2F binding
site at 208 to 215 are important regulatory cis elements in
promoter B. Gel mobility shift assays indicated that the 94 to 101
region binds the NRF-2 protein. When INS-1 cells were maintained in the
presence of high glucose (25 mM) for 7 days, mGPD was
the only 1 of 6 enzyme activities lowered (53%). mGPD promoter B
activity was reduced by 60% in INS-1 cells by the high glucose, but in
HepG2 cells and HeLa cells, promoter B activity was unchanged or
slightly increased. Deletion analysis indicated the glucose
responsiveness was distributed across the region from 340 to 260 in
promoter B. The results indicate that mGPD gene transcription in the
beta cell is regulated differently from other cells and that decreased
mGPD promoter B transcription is at least in part the cause of the
decreased beta cell mGPD levels in diabetes.
*
This work was supported by National Institutes of Health
Grant DK 28348 and gifts from the Oscar C. Rennebohm Foundation and the
Robert Wood Johnson Family Trust.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 350772.
To whom correspondence should be addressed: Rm. 3459, University of Wisconsin Medical School, 1300 University Ave., Madison, WI 53706. Tel.: 608-262-1195; Fax: 608-262-9300; E-mail:
mjmacdon@facstaff.wisc.edu.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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Copyright © 2000 by the American Society for Biochemistry and Molecular Biology.
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