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Excessive production of reactive oxygen species (ROS) contributes to progression of atherosclerosis, at least in part by causing endothelial dysfunction and inflammatory activation. The class III histone deacetylase SIRT1 has been implicated in extension of lifespan. In the vasculature,SIRT1 gain-of-function using SIRT1 overexpression or activation has been shown to improve endothelial function in mice and rats via stimulation of endothelial nitric oxide (NO) synthase (eNOS). However, the effects of SIRT1 loss-of-function on the endothelium in atherosclerosis remain to be characterized. Thus, we have investigated the endothelial effects of decreased endogenous SIRT1 in hypercholesterolemic ApoE-/- mice. We observed no difference in endothelial relaxation and eNOS (Ser1177) phosphorylation between 20-week old male atherosclerotic ApoE-/- SIRT1+/- and ApoE-/- SIRT1+/+ mice. However, SIRT1 prevented endothelial superoxide production, inhibited NF-κB signaling, and diminished expression of adhesion molecules. Treatment of young hypercholesterolemic ApoE-/- SIRT1+/- mice with lipopolysaccharide to boost NF-κB signaling led to a more pronounced endothelial expression of ICAM-1 and VCAM-1 as compared to ApoE-/- SIRT1+/+ mice. In conclusion, endogenous SIRT1 diminishes endothelial activation in ApoE-/- mice, but does not affect endothelium-dependent vasodilatation.

Nuclear receptors (NRs) are transcription factors activated by a multitude of hormones, other endogenous substances, and exogenous molecules. These proteins modulate the regulation of target genes by contacting their promoter or enhancer sequences at specific recognition sites. The identification of these response elements is the first step toward detailed insight into the regulatory mechanisms affecting a gene. We have developed NUBIScan, a computer algorithm to predict DNA recognition sites for NRs in the regulatory regions of genes. The algorithm is based on weighted nucleotide distribution matrices and combines scores from both half-sites necessary for NR dimer binding. It provides more specific identification of functional sites than previous in silico approaches, as evidenced by scanning published regulatory regions of drug-inducible genes and comparing the obtained predictions with experimental results. In prospective analyses, NUBIScan consistently identified new functional NR binding sites in sets of large sequences, which had eluded previous analyses. This is exemplified by the detailed functional analysis of the flanking region of two genes. This approach therefore facilitates the selection of likely sites of gene regulation for subsequent experimental analysis.

Ketone bodies (KBs) are crucial energy substrates during states of low carbohydrate availability. However, an aberrant regulation of KB homeostasis can lead to complications such as diabetic ketoacidosis. Exercise and diabetes affect systemic KB homeostasis, but the regulation of KB metabolism is still enigmatic. In our study in mice with either knockout or overexpression of the peroxisome proliferator-activated receptor-g coactivator (PGC)-1a in skeletal muscle, PGC-1a regulated ketolytic gene transcription in muscle. Furthermore, KB homeostasis of these mice was investigated during withholding of food, exercise, and ketogenic diet feeding, and after streptozotocin injection. In response to these ketogenic stimuli, modulation of PGC-1a levels in muscle affected systemic KB homeostasis. Moreover, the data demonstrate that skeletal muscle PGC-1a is necessary for the enhanced ketolytic capacity in response to exercise training and overexpression of PGC-1a in muscle enhances systemic ketolytic capacity and is sufficient to ameliorate diabetic hyperketonemia in mice. In cultured myotubes, the transcription factor estrogen-related receptor-a was a partner of PGC-1a in the regulation of ketolytic gene transcription. These results demonstrate a central role of skeletal muscle PGC-1a in the transcriptional regulation of systemic ketolytic capacity.-Svensson, K., Albert, V., Cardel, B., Salatino, S., Handschin, C. Skeletal muscle PGC-1a modulates systemic ketone body homeostasis and ameliorates diabetic hyperketonemia in mice. FASEB J. 30, 1976FASEB J. 30, -1986FASEB J. 30, (2016 During prolonged starvation, when carbohydrate availability is low, the ketone bodies (KBs) b-hydroxybutyrate (bOHB) and acetoacetate (AcAc) are necessary metabolic fuels that help maintain energy homeostasis (1). KBs are produced in the liver and are subsequently metabolized to acetyl-CoA in extrahepatic organs. Most KB metabolism occurs in the mitochondria and is catalyzed by the enzymes 3-hydroxybutyrate dehydrogenase, type 1 (BDH1), succinyl-CoA:3-ketoacid-coenzyme A transferase 1 (OXCT1), and acetyl-CoA acetyltransferase 1 (ACAT1) (2). Mutations of genes encoding these enzymes are associated with exacerbated ketosis in humans (3). Moreover, knockout of the rate-limiting ketolytic enzyme OXCT1 leads to severe hyperketonemia and lethality in mice (4). Hyperketonemia is a common complication in diabetes that can lead to severe and possibly lethal ketoacidosis (5) and has been attributed in part to impaired peripheral KB oxidation (6). However, relatively little is known about the transcriptional regulation of ketolytic enzymes (7). The peroxisome proliferator-activated receptor g coactivator (PGC)-1a is an essential transcriptional coactivator and has a well-established role in the regulation of mitochondrial metabolic processes such as oxidative phosphorylation and the tricarboxylic acid (TCA) cycle (8). Although these metabolic pathways are crucial for complete oxidation of KBs, it is not known whether PGC-1a directly affects the ...

Chicken xenobiotic receptor, pregnane X receptor, and constitutive androstane receptor are orphan nuclear receptors that have recently been discovered to regulate drug- and steroid-mediated induction of hepatic cytochromes P450 (CYP). This induction is part of an adaptive response involving numerous genes to exposure to drugs and chemicals and has major clinical and toxicological implications. Here we report experiments in the chicken hepatoma cell line LMH that suggest evolutionary conservation of the signaling pathways triggered by pregnane X receptor, constitutive androstane receptor, and chicken xenobiotic receptor. Thus, the phenobarbital-inducible enhancer units of the mouse Cyp2b10, rat CYP2B2, and human CYP2B6 genes were activated in reporter gene assays by the same compounds that activate the chicken CYP2H1 phenobarbital-inducible enhancer units. Chicken xenobiotic receptor, pregnane X receptor, and constitutive androstane receptor all bound to the CYP2H1 phenobarbital-inducible enhancer units in gel-shift experiments. In CV-1 cell transactivation assays, mammalian pregnane X receptors activate the chicken phenobarbital-inducible enhancer units to the same extent as does chicken xenobiotic receptor, each receptor maintaining its species-specific ligand spectrum. To assess the reported role of protein phosphorylation in drug-mediated induction, we treated LMH cells with okadaic acid and observed increased mRNA of delta-aminolevulinate synthase and CYP2H1 whereas expression of CYP3A37 was decreased. The effects of okadaic acid and other modifiers of protein phosphorylation in LMH cells are comparable to those seen on CYP2Bs and CYP3As in mammalian primary hepatocyte cultures. These results indicate that closely related nuclear receptors, transcription factors, and signaling pathways are mediating the transcriptional activation of multiple genes by xenobiotics in chicken, rodents, and man.