In mammals, there are two different genes encoding for glutaminase isoforms, named liver (LGA) and kidney (KGA) types. LGA has long been believed to be present only in liver mitochondria from adult animals. However, we have recently reported the presence of LGA mRNA in human brain. We now describe the expression of LGA mRNA in the brain of other mammals (cow, mouse, rabbit, and rat) and in different areas of human brain as assessed by Northern blot analysis. The presence of mRNA encoding for this isoform in rat brain was further confirmed by reverse transcriptase-PCR cloning and sequencing. Although it has been well accepted that glutaminase is a mitochondrial enzyme, using newly generated isoform-specific antibodies, we have found a differential intracellular immunolocalization of both glutaminase isoforms in rat and monkey brain. In both species, KGA protein was present in mitochondria, whereas LGA protein was localized in nuclei. Furthermore, subcellular fractionation and Western blot analysis revealed that brain LGA was enriched in nuclei where it was catalytically active. Nuclear glutaminase exhibited a kinetic behavior that resembles that of the liver-type enzyme with regard to the low phosphate concentration requirement; however, nuclear glutaminase was susceptible to glutamate inhibition, a property that is absent in the rat liver enzyme.

Phosphate-activated glutaminase (GA) is overexpressed in certain types of tumour but its exact role in tumour cell growth and proliferation is unknown. Here we describe the isolation of a full-length cDNA clone of human breast cancer ZR75 cells, by a combination of lambdagt10 cDNA library screening and the rapid amplification of cDNA ends ('RACE') technique. The cDNA of human GA is 2408 nt with a 1806-base open reading frame encoding a 602-residue protein with a predicted molecular mass of 66309 Da. The deduced amino acid sequence contains a putative mitochondrial import presequence of 14 residues at the N-terminal end. Heterologous expression and purification in Escherichia coli yielded a product of the expected molecular size that was recognized by using antibodies against the recombinant human GA. Sequence analyses showed that human GA was highly similar to the rat liver enzyme. Northern gel analysis revealed that the gene is present in human liver, brain and pancreas, in which a major transcript of 2.4 kb was demonstrated, but not in kidney, heart, skeletal muscle, lung or placenta. These results strongly suggest that the first human GA cloned, the GA from ZR-75 breast cancer cells, and presumably those from human liver and brain, are liver-type isoenzymes, in sharp contrast with the present view that considers the kidney type as the isoform expressed in all tissues with GA activity, with the exception of postnatal liver.

Cholesterol 7-␣-hydroxylase (CYP7A1) is the key enzyme that commits cholesterol to the neutral bile acid biosynthesis pathway and is highly regulated. In the current studies, we have uncovered a role for the transcriptional co-activator PGC-1␣ in CYP7A1 gene transcription. PGC-1␣ plays a vital role in adaptive thermogenesis in brown adipose tissue and stimulates genes important to mitochondrial function and oxidative metabolism. It is also involved in the activation of hepatic gluconeogenesic gene expression during fasting. Because the mRNA for CYP7A1 was also induced in mouse liver by fasting, we reasoned that PGC-1␣ might be an important co-activator for CYP7A1. Here we show that PGC-1␣ and CYP7A1 are also co-induced in livers of mice in response to streptozotocin induced diabetes. Additionally, infection of cultured HepG2 cells with a recombinant adenovirus expressing PGC-1␣ directly activates CYP7A1 gene expression and increases bile acid biosynthesis as well. Furthermore, we show that PGC-1␣ activates the CYP7A1 promoter directly in transient transfection assays in cultured cells. Thus, PGC-1␣ is a key activator of CYP7A1 and bile acid biosynthesis and is likely responsible for the fasting and diabetes dependent induction of CYP7A1. PGC-1␣ has already been shown to be a critical activator of several other oxidative processes including adaptive thermogenesis and fatty acid oxidation. Our studies provide further evidence of the fundamental role played by PGC-1␣ in oxidative metabolism and define PGC-1␣ as a link between diabetes and bile acid metabolism. The CYP7A11 enzyme converts cholesterol into 7-␣-hydroxycholesterol, which is the first specific intermediate in the neutral bile acid biosynthesis pathway in the liver (1). This is a crucial enzyme in mammalian cholesterol metabolism as diversion into the bile acid pathway is the main route for eliminating excess cholesterol from the body. Because of its key role in cholesterol metabolism, the CYP7A1 enzyme and its gene have been studied as an important model for dietary regulation for several years. These studies have revealed that there is a significant amount of regulation at the level of transcription initiation (2); however, post-transcriptional mechanisms for control also occur (1, 3).The CYP7A1 promoter has been extensively evaluated by several groups and the proximal regions of both the mouse and rat promoters contain two direct repeat type elements that bind several nuclear receptors, some of which are indicated in Fig. 1. There is also a binding site for the monomeric orphan receptor LRH-1 that overlaps the DR-1 element. The DR-4 is a target site for the nuclear receptor LXR, which confers positive regulation by cholesterol to the CYP7A1 promoter in mice and rats (2). However, the DR-4 is not conserved in the human gene which is not subject to feed forward regulation by cholesterol (4).CYP7A1 is also activated in livers of fasted mice (5). Similarly, the transcriptional co-activator PGC-1␣ is induced by fasting in liver where it activates transcrip...

Two key regulatory enzymes in the bile acid biosynthesis pathway are cholesterol 7␣-hydroxylase/CYP7A1 (7␣-hydroxylase) and sterol 12␣-hydroxylase/CYP8B1 (12␣-hydroxylase). It has been shown previously that hepatocyte nuclear factor-4␣ (HNF-4) and the ␣ 1 -fetoprotein transcription factor (FTF) are activators of 7␣-and 12␣-hydroxylase transcription and that the small heterodimer partner (SHP) suppresses bile acid biosynthesis by heterodimerizing with FTF. However, the role of FTF in bile acid biosynthesis has been studied only in tissue culture systems. In heterozygous FTF knockout mice, 7␣-and 12␣-hydroxylase genes were expressed at 5-7-fold higher levels than in wild-type mice, an apparent direct contradiction to previous in vitro observations. This higher expression of the 7␣-and 12␣-hydroxylase genes resulted in a 33% higher bile acid pool in their gallbladders, bile more enriched in cholic acid, and a 13% decrease in plasma cholesterol levels. Adenovirusmediated FTF overexpression in wild-type mice resulted in 10-fold lower expression of the 7␣-and 12␣-hydroxylase genes and up to 8-fold higher SHP expression, highlighting the dual role that FTF plays in different promoters. Shorter overexpression times still resulted in lower 7␣-and 12␣-hydroxylase expression, but unchanged SHP expression, suggesting that two different mechanisms are involved in the FTF-mediated suppression of 7␣-and 12␣-hydroxylase expression. This FTF-mediated suppression of the expression of two bile acid biosynthesis genes resulted in a 3-fold lower rate of bile acid synthesis in a rat bile fistula animal model. Based on these observations and on protein binding studies performed in vitro and by chromatin immunoprecipitation, we hypothesize that FTF has two synergetic effects that contribute to its role in bile acid biosynthesis: 1) it has the ability to activate the expression of SHP, which in turn heterodimerizes and suppresses FTF transactivation activity; and 2) it occupies the FTF/ HNF-4 recognition site within the 7␣-and 12␣-hydroxylase promoters, which can otherwise be occupied by a factor (HNF-4) that cannot be suppressed by SHP.

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