Sterol regulatory element-binding proteins are regulators of the rat thyroid peroxidase gene in thyroid cells.

Sterol regulatory element-binding proteins (SREBPs)-1c and -2, which were initially discovered as master transcriptional regulators of lipid biosynthesis and uptake, were recently identified as novel transcriptional regulators of the sodium-iodide symporter gene in the thyroid, which is essential for thyroid hormone synthesis. Based on this observation that SREBPs play a role for thyroid hormone synthesis, we hypothesized that another gene involved in thyroid hormone synthesis, the thyroid peroxidase (TPO) gene, is also a target of SREBP-1c and -2. Thyroid epithelial cells treated with 25-hydroxycholesterol, which is known to inhibit SREBP activation, had about 50% decreased mRNA levels of TPO. Similarly, the mRNA level of TPO was reduced by about 50% in response to siRNA mediated knockdown of both, SREBP-1 and SREBP-2. Reporter gene assays revealed that overexpression of active SREBP-1c and -2 causes a strong transcriptional activation of the rat TPO gene, which was localized to an approximately 80 bp region in the intron 1 of the rat TPO gene. In vitro- and in vivo-binding of both, SREBP-1c and SREBP-2, to this region in the rat TPO gene could be demonstrated using gel-shift assays and chromatin immunoprecipitation. Mutation analysis of the 80 bp region of rat TPO intron 1 revealed two isolated and two overlapping SREBP-binding elements from which one, the overlapping SRE+609/InvSRE+614, was shown to be functional in reporter gene assays. In connection with recent findings that the rat NIS gene is also a SREBP target gene in the thyroid, the present findings suggest that SREBPs may be possible novel targets for pharmacological modulation of thyroid hormone synthesis.

Sterol regulatory element-binding proteins are regulators of the NIS gene in thyroid cells.

The uptake of iodide into the thyroid, an essential step in thyroid hormone synthesis, is an active process mediated by the sodium-iodide symporter (NIS). Despite its strong dependence on TSH, the master regulator of the thyroid, the NIS gene was also reported to be regulated by non-TSH signaling pathways. In the present study we provide evidence that the rat NIS gene is subject to regulation by sterol regulatory element-binding proteins (SREBPs), which were initially identified as master transcriptional regulators of lipid biosynthesis and uptake. Studies in FRTL-5 thyrocytes revealed that TSH stimulates expression and maturation of SREBPs and expression of classical SREBP target genes involved in lipid biosynthesis and uptake. Almost identical effects were observed when the cAMP agonist forskolin was used instead of TSH. In TSH receptor-deficient mice, in which TSH/cAMP-dependent gene regulation is blocked, the expression of SREBP isoforms in the thyroid was markedly reduced when compared with wild-type mice. Sterol-mediated inhibition of SREBP maturation and/or RNA interference-mediated knockdown of SREBPs reduced expression of NIS and NIS-specific iodide uptake in FRTL-5 cells. Conversely, overexpression of active SREBPs caused a strong activation of the 5'-flanking region of the rat NIS gene mediated by binding to a functional SREBP binding site located in the 5'-untranslated region of the rat NIS gene. These findings show that TSH acts as a regulator of SREBP expression and maturation in thyroid epithelial cells and that SREBPs are novel transcriptional regulators of NIS.

The mouse gene encoding the carnitine biosynthetic enzyme 4-N-trimethylaminobutyraldehyde dehydrogenase is regulated by peroxisome proliferator-activated receptor α.

Genes involved in carnitine uptake and synthesis, such as organic cation transporter-2 (OCTN2) and γ-butyrobetaine dioxygenase (BBD), have been shown to be regulated by peroxisome proliferator-activated receptor (PPAR)α directly. Whether other genes encoding enzymes involved in the carnitine synthesis pathway, such as 4-N-trimethylaminobutyraldehyde dehydrogenase (TMABA-DH) and trimethyllysine dioxygenase (TMLD), are also direct PPARα target genes is less clear. In silico-analysis of the mouse TMLD promoter and first intron and the TMABA-DH promoter revealed several putative peroxisome proliferator response elements (PPRE) with high similarity to the consensus PPRE. Luciferase reporter gene assays using either a 2kb TMLD promoter or a 4kb TMLD first intron reporter constructs revealed no functional PPRE. In contrast, reporter gene assays using wild-type and mutated 5´-truncation TMABA-DH promoter reporter constructs showed that one PPRE located at position -132 in the proximal promoter is probably functional. Using gel shift assays we observed in vitro-binding of PPARα to this PPRE. Moreover, using chromatin immunoprecipitation assays we found that PPARα also binds in vivo to a nucleotide sequence spanning the PPRE at -132, which confirms that this PPRE is functional. In conclusion, the present study shows that the mouse TMABA-DH gene is a direct PPARα target gene. Together with the recent identification of the mouse BBD and the mouse OCTN2 genes as PPARα target genes this finding confirm that PPARα plays a key role in the regulation of carnitine homeostasis by controlling genes involved in carnitine synthesis and carnitine uptake.

Mouse γ-butyrobetaine dioxygenase is regulated by peroxisome proliferator-activated receptor α through a PPRE located in the proximal promoter.

Convincing evidence from studies with peroxisome proliferator-activated receptor (PPAR)α-deficient mice suggested that the carnitine biosynthetic enzyme γ-butyrobetaine dioxygenase (BBD) is regulated by PPARα. However, the identification of BBD as a direct PPARα target gene as well as its exact regulation remained to be demonstrated. In silico-analysis of the mouse BBD promoter revealed seven putative peroxisome proliferator response elements (PPRE) with high similarity to the consensus PPRE. Luciferase reporter gene assays using mutated and non-mutated serial 5'-truncation BBD promoter reporter constructs revealed that one PPRE located at -75 to -87 relative to the transcription start site in the proximal BBD promoter is probably functional. Using gel shift assays we observed in vitro-binding of PPARα/RXRα heterodimer to this PPRE confirming that it is functional. In conclusion, the present study clearly shows that mouse BBD is a direct PPARα target gene and that transcriptional up-regulation of mouse BBD by PPARα is likely mediated by binding of the PPARα/RXR heterodimer to one PPRE located in its proximal promoter region. The results confirm emerging evidence from recent studies that PPARα plays a key role in the regulation of carnitine homeostasis by controlling genes involved in both, carnitine synthesis and carnitine uptake.

Fasting Upregulates PPARalpha Target Genes in Brain and Influences Pituitary Hormone Expression in a PPARalpha Dependent Manner.

PPARalpha is a lipid-activable transcription factor that mediates the adaptive response to fasting. Recent data indicate an important role of brain PPARalpha in physiological functions. However, it has not yet been shown whether PPARalpha in brain can be activated in the fasting state. Here we demonstrate that fasting of rats increased mRNA concentrations of typical PPARalpha target genes implicated in beta-oxidation of fatty acids (acyl-CoA oxidase, carnitine palmitoyltransferase-1, medium chain acyl-CoA dehydrogenase) and ketogenesis (mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase) in pituitary gland and partially also in frontal cortex and diencephalon compared to nonfasted animals. These data strongly indicate that fasting activates PPARalpha in brain and pituitary gland. Furthermore, pituitary prolactin and luteinizing hormone-beta mRNA concentrations were increased upon fasting in wild-type mice but not in mice lacking PPARalpha. For proopiomelanocortin and thyrotropin-beta, genotype-specific differences in pituitary mRNA concentrations were observed. Thus, PPARalpha seems to be involved in transcriptional regulation of pituitary hormones.

Carnitine synthesis and uptake into cells are stimulated by fasting in pigs as a model of nonproliferating species.

In rodents, fasting increases the carnitine concentration in the liver by an up-regulation of enzymes of hepatic carnitine synthesis and novel organic cation transporter (OCTN) 2, mediated by activation of peroxisome proliferator-activated receptor (PPAR) alpha. This study was performed to investigate whether such effects occur also in pigs which like humans, as nonproliferating species, have a lower expression of PPARalpha and are less responsive to treatment with PPARalpha agonists than rodents. An experiment with 20 pigs was performed, which were either fed a diet ad-libitum or fasted for 24 h. Fasted pigs had higher relative mRNA concentrations of the PPARalpha target genes carnitine palmitoyltransferase 1 and acyl-CoA oxidase in liver, heart, kidney, and small intestinal mucosa than control pigs, indicative of PPARalpha activation in these tissues (P<.05). Fasted pigs had a higher activity of gamma-butyrobetaine dioxygenase (BBD), enzyme that catalyses the last step of carnitine biosynthesis in liver and kidney, and higher relative mRNA concentrations of OCTN2, the most important carnitine transporter, in liver, kidney, skeletal muscle, and small intestinal mucosa than control pigs (P<.05). Fasted pigs moreover had higher concentrations of free and total carnitine in liver and kidney than control pigs (P<.05). This study shows for the first time that fasting increases the activity of BBD in liver and kidney and up-regulates the expression of OCTN2 in various tissues of pigs, probably mediated by PPARalpha activation. It is concluded that nonproliferating species are also able to cover their increased demand for carnitine during fasting by an increased carnitine synthesis and uptake into cells.