Hormonal regulation of multiple promoters of the rat mitochondrial glycerol‐3‐phosphate dehydrogenase gene

Abstract

Rat mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) is regulated by multiple promoters in a tissuespecific manner. Here, we demonstrate that thyroid hormone (3,5,3 H -tri-iodo-l-thyronine) and steroid hormone but not the peroxisome proliferator clofibrate and retinoic acid stimulate the activation of the ubiquitous promoter B in a receptor-dependent manner, whereas the more tissuerestricted promoters A and C are not inducible by these hormones. Thyroid hormone action is mediated by a direct repeat 14 (DR14) hormone-response element as identified by deletion and mutation analyses of promoter B in transient transfection analyses. The DR14 element was able to bind to an in vitro translated thyroid hormone receptor in band-shift and supershift experiments. The hormone-response element comaps with a recognition site for the transcription factor Sp1, suggesting complex regulation of this sequence element. Mutation of this Sp1-recognition site reduces the basal promoter B activity dramatically in HepG2 and HEK293 cells in transient transfection and abolishes the binding of Sp1 in band-shift experiments. As demonstrated by Western-blot experiments, administration of tri-iodothyronine to euthyroid rats increases hepatic mGPDH protein concentrations in vivo. As it has recently been reported that human mGPDH promoter B is not regulated by tri-iodothyronine, this is the first example of a differentially tri-iodothyronine-regulated orthologous gene promoter in man and rat.Keywords: gene regulation; glycerol phosphate shuttle; mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH); promoter; thyroid hormone.The FAD-dependent, mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) is located on the outer surface of the inner mitochondrial membrane [1] and is an essential component of the glycerol phosphate shuttle. In conjunction with the cytoplasmic glycerol-3-phosphate dehydrogenase (cGPDH), this shuttle uses the interconversion of glycerol-3-phosphate to dihydroxyacetone phosphate to transfer reduction equivalents from cytosolic NADH to the enzymebound FAD of mGPDH. Together with the aspartate± malate shuttle, the glycerol phosphate shuttle transfers reduction equivalents from the cytoplasm to the mitochondria to modulate the cytosolic NADH pool [2±4].High levels of mGPDH enzyme activity have been measured in tissues with a high rate of ATP generation, such as insect flight muscle [5] [18,19] and mGPDH inhibition studies [20,21], it has been demonstrated that the transfer of reduction equivalents into mitochondria is an essential requirement for glucoseinduced insulin secretion in pancreatic b-cells.Elevated mGPDH enzyme activities have been measured after treatment with thyroid hormone (3,5,3 H -tri-iodo-lthyronine) and the peroxisome proliferator clofibrate in various rat tissues [22,23]. However, the thyroid hormone responsiveness of enzyme activities and transcript levels is controversial, depending on the tissue environment. Administration of tri-iodothyronine to hypothyroid rats leads to dramatically incr...

A Refined Analysis of Superoxide Production by Mitochondrial sn-Glycerol 3-Phosphate Dehydrogenase

“…Expression and activity of mGPDH are known to differ widely between different tissues and in different environmental and hormonal states (Fig. 4, a and b) (3,(5)(6)(7). Therefore, depending on metabolic conditions, mGPDH not only coordinates cellular energy production but also probably participates in oxidative signaling both within mitochondria and beyond.…”

“…This shuttle activity is thought to coordinate glycolytic and mitochondrial metabolism in highly active tissues, and fittingly, mGPDH shows the highest expression in thermogenic brown fat, type II skeletal muscle fibers, brain, sperm, and pancreatic beta cells (2)(3)(4). Tissues with characteristically low expression of mGPDH, such as liver, kidney, and heart, show increased enzyme activity in response to thyroid and steroid hormones, whereas those with high expression are generally insensitive to this regulation (3,(5)(6)(7). Variations in mGPDH expression, activity, or genetic sequence have been associated with increased plasma levels of glycerol and free fatty acids (8), mental retardation (9), cancers (5, 10 -12), and diabetes (13,14).…”

Hepatic gene expression patterns in thyroid hormone-treated hypothyroid rats

“…Transient transfection experiments in human hepatocarcinoma HepG2 cells were conducted as described previously (Weitzel et al 2001a). Eight promoter fragments of six late T3-induced genes (as indicated in Table 2) were ligated either into pGL2 or pGL3 vectors (Promega) upstream of the luciferase gene.…”

“…This delayed response is surprising since nongenomic effects of T3 are already detectable within minutes (Davis et al 2002) and genomic effects of T3 are typically detectable within a few hours (Weitzel et al 2003). For many of the latter genes functional thyroid hormone response elements (TREs) have been reported (Zilz et al 1990,Weitzel et al 2001a, indicating a direct mechanism via binding of thyroid hormone receptors (TRs), which are ligand-regulated transcription factors. TRs modulate gene activity through alternately silencing or activating transcription by recruitment of either corepressor or coactivator complexes depending on the absence or presence of thyroid hormone (Robyr et al 2000, Zhang & Lazar 2000.…”

Germ Cell Nuclear Factor Relieves cAMP-response Element Modulator τ-mediated Activation of the Testis-specific Promoter of Human Mitochondrial Glycerol-3-phosphate Dehydrogenase

“…In conjunction with the cytosolic glycerol-3-phosphate dehydrogenase, this shuttle uses the interconversion of glycerol-3-phosphate to dihydroxyacetone phosphate to oxidize cytosolic NADH by transferring reduction equivalents from the cytosol to the mitochondrion (1,2). The expression of human and rat mGPDH is regulated by two somatic promoters in a tissue-specific manner (3)(4)(5)(6)(7), but rat mGPDH is additionally regulated by a third testis-specific promoter (8). The usage of the testis-specific promoter of mGPDH results in a shortened transcript length of ϳ2.4 kb containing an alternate first exon at its 5Ј end and a shortened 3Ј-untranslated region; however, the open reading frame remains unaffected (3,8).…”