Thyroid hormone (T3) is essential for normal development, differentiation and metabolic balance. Only a limited number of T3-target genes have been identified so far and their complex regulation pattern is poorly understood. We performed cDNA expression array hybridisation to identify T3-regulated genes and to investigate their expression pattern after various time points in vivo. Radioactively labelled cDNA was prepared from hepatic RNA of hypothyroid and hyperthyroid rats 6, 24 and 48 h after the administration of T3. Labelled cDNA probes were hybridised to rat Atlas Arrays. Twenty-three of 588 genes were shown to be differentially regulated, 18 of which were previously not known to be regulated by T3. The expression of 19 genes was verified by independent northern blot hybridisation. Two different expression time courses of T3 expression were observed. In a first expression profile ('early' expression) the transcription level of the target genes rises within 6 h, drops by 24 h and increases again within 48 h after the administration of T3. In a second expression profile ('late' expression) the mRNA level rose in the first 6 h and rose further by 48 h, indicating an additional regulation mechanism. Nuclear respiratory factor (NRF)-1 and peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1), but not NRF-2, were up-regulated within 6 h after T3 administration, suggesting NRF-1 and/or PGC-1 as key regulators for mediating the 'late' expression pattern.

Thyroid hormone (T3) is essential for normal development, differentiation and metabolic balance. We have performed DNA microarray experiments using hepatic RNA from hypothyroid and T3-treated hypothyroid rats in order to characterize T3-induced gene expression patterns after various time points (6, 24 and 48 h after the administration of the hormone). Sixty-two of 4608 different genes displayed a reproducible T3-response, and cluster analysis divided these differentially regulated genes into six expression patterns. Thirty-six genes were not significantly regulated within the first 24 h. Transient transfection experiments of eight late-induced gene promoters failed to detect a thyroid hormone response element within their regulatory elements, suggesting an indirect activation mechanism(s). In search for an intermediate factor of T3 action, we examined whether various rather ubiquitous transcription factors, peroxisome proliferator-activated receptors (PPARs) and coactivators of the PPAR coactivator 1 family (PGC-1) are regulated by T3. Only PPAR and PERC/PGC-1 exhibit a significant T3-response within the first 6 h after treatment, identifying these factors as candidate components for mediating the late-induced expression pattern. Regulation of early-induced genes within the first 6 h after administration of T3 on transcript levels correlates with altered protein levels after 24 and 48 h in vivo.

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...

Mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) is regulated by multiple promoters in a tissue-specific manner. We characterized the testis-specific promoter C of the mGPDH gene and investigated the cellular localization of mGPDH within the testis. Electrophoretic mobility shift experiments identified a cAMP-response element (CRE) site at -57 that was active in the testis. An in vitro-translated CRE modulator (CREM) protein was able to bind this CRE site, and an anti-CREM antibody interfered with this complex. Ectopic expression of the testis-specific transcriptional activator CREMtau and protein kinase A in human hepatocarcinoma HepG2 cells activated a promoter C-driven luciferase construct in transient transfection experiments. Furthermore, mGPDH expression was undetectable in testis of CREM-deficient mice. The cellular localization of mGPDH expression and translation in adult rat testis was determined by in situ hybridization and immunohistochemistry techniques. The mGPDH transcripts were detected solely in postmeiotic germ cells. Expression of mGPDH was restricted from round spermatids to early elongating spermatids. The mGPDH protein was delayed in postmeiotic germ cells, restricted from late elongating spermatids to mature spermatids. Our results indicate that rat mGPDH is expressed by a testis-specific promoter from haploid male germ cells in a stage-specific manner.