Inhibition of intestinal absorption of cholesterol by ezetimibe or bile acids by SC-435 alters lipoprotein metabolism and extends the lifespan of SR-BI/apoE double knockout mice.

SR-BI/apoE double knockout (dKO) mice exhibit many features of human coronary heart disease (CHD), including hypercholesterolemia, occlusive coronary atherosclerosis, cardiac hypertrophy, myocardial infarctions, cardiac dysfunction and premature death. Ezetimibe is a FDA-approved, intestinal cholesterol absorption inhibitor that lowers plasma LDL cholesterol in humans and animals and inhibits aortic root atherosclerosis in apoE KO mice, but has not been proven to reduce CHD. Three-week-ezetimibe treatment of dKO mice (0.005% (w/w) in standard chow administered from weaning) resulted in a 35% decrease in cholesterol in IDL/LDL-size lipoproteins, but not in VLDL- and HDL-size lipoproteins. Ezetimibe treatment significantly reduced aortic root (57%) and coronary arterial (68%) atherosclerosis, cardiomegaly (24%) and cardiac fibrosis (57%), and prolonged the lives of the mice (27%). This represents the first demonstration of beneficial effects of ezetimibe treatment on CHD. The dKO mice were similarly treated with SC-435 (0.01% (w/w)), an apical sodium codependent bile acid transporter (ASBT) inhibitor, that blocks intestinal absorption of bile acids, lowers plasma cholesterol in animals, and reduces aortic root atherosclerosis in apoE KO mice. The effects of SC-435 treatment were similar to those of ezetimibe: 37% decrease in ILD/LDL-size lipoprotein cholesterol and 57% prolongation in median lifespan. Thus, inhibition of intestinal absorption of either cholesterol (ezetimibe) or bile acids (SC-435) significantly reduced plasma IDL/LDL-size lipoprotein cholesterol levels and improved survival of SR-BI/apoE dKO mice. The SR-BI/apoE dKO murine model of atherosclerotic occlusive, arterial CHD appears to provide a useful system to evaluate compounds that modulate cholesterol homeostasis and atherosclerosis.

Intra- and interreader reproducibility of magnetic resonance imaging for quantifying the lipid-rich necrotic core is improved with gadolinium contrast enhancement.

PURPOSE: To test the hypothesis that intra- and interreader reproducibility for measuring the lipid-rich necrotic core (LR-NC) size is significantly improved with gadolinium (Gd) contrast-enhanced magnetic resonance imaging (CEMRI) compared to non-CEMRI. MATERIALS AND METHODS: Thirty-seven individuals with >50% carotid artery stenosis underwent carotid MRI at 1.5T (pre- and postcontrast T1-weighted (T1W), T2-weighted (T2W), proton density-weighted (PDW), and three-dimensional time-of-flight (TOF) sequences). Two independent readers measured the mean area of the LR-NC from the precontrast images only, followed by a second measurement using the additional postcontrast images. One reader repeated the measurements after an interval of five months. Intra- and interreader reproducibility was analyzed by means of the intraclass correlation coefficient (ICC), coefficient of variation (CV), and standard deviation (SD). RESULTS: The CV decreased from 33.7% to 8.8% for intrareader measurements of the LR-NC, and from 33.5% to 17.6% for interreader measurements. The SD was significantly smaller with CEMRI than with non-CEMRI (P = 0.003 and P = 0.006, respectively). The ICC increased from 0.94 to 0.99 and from 0.85 to 0.93 for the intra- and interreader measurements, respectively. CONCLUSION: Reader reproducibility for in vivo MRI quantification of LR-NC size is significantly improved by the addition of Gd contrast in individuals with >50% carotid stenosis.

A radiometric assay for glutamine:fructose-6-phosphate amidotransferase.

Glutamine:fructose-6-phosphate amidotransferase (GFAT) catalyzes the first step in the biosynthesis of amino sugars by transferring the amino group from l-glutamine to the acceptor substrate, fructose 6-phosphate, generating the products glucosamine 6-phosphate and glutamic acid. We describe a method for the synthesis and purification of the substrate, fructose 6-phosphate, and methods for a radiometric assay of human GFAT1 that can be performed in either of two formats: a small disposable-column format and a high-throughput 96-well-plate format. The method performed in the column format can detect 1 pmol of glucosamine 6-phosphate, much less than that required by previously published assays that measure GlcN 6-phosphate. The column assay demonstrates a broad linear range with low variability. In both formats, the assay is linear with time and enzyme concentration and is highly reproducible. This method greatly improves the sensitivity and speed with which GFAT1 activity can be measured and facilitates direct kinetic measurement of the transferase activity.

Kinetic characterization of human glutamine-fructose-6-phosphate amidotransferase I: potent feedback inhibition by glucosamine 6-phosphate.

Glutamine-fructose-6-phosphate amidotransferase (GFAT) catalyzes the first committed step in the pathway for biosynthesis of hexosamines in mammals. A member of the N-terminal nucleophile class of amidotransferases, GFAT transfers the amino group from the L-glutamine amide to D-fructose 6-phosphate, producing glutamic acid and glucosamine 6-phosphate. The kinetic constants reported previously for mammalian GFAT implicate a relatively low affinity for the acceptor substrate, fructose 6-phosphate (Fru-6-P, K(m) 0.2-1 mm). Utilizing a new sensitive assay that measures the production of glucosamine 6-phosphate (GlcN-6-P), purified recombinant human GFAT1 (hGFAT1) exhibited a K(m) for Fru-6-P of 7 microm, and was highly sensitive to product inhibition by GlcN-6-P. In a second assay method that measures the stimulation of glutaminase activity, a K(d) of 2 microm was measured for Fru-6-P binding to hGFAT1. Further, we report that the product, GlcN-6-P, is a potent competitive inhibitor for the Fru-6-P site, with a K(i) measured of 6 microm. Unlike other members of the amidotransferase family, where glutamate production is loosely coupled to amide transfer, we have demonstrated that hGFAT1 production of glutamate and GlcN-6-P are strictly coupled in the absence of inhibitors. Similar to other amidotransferases, competitive inhibitors that bind at the synthase site may inhibit the synthase activity without inhibiting the glutaminase activity at the hydrolase domain. GlcN-6-P, for example, inhibited the transfer reaction while fully activating the glutaminase activity at the hydrolase domain. Inhibition of hGFAT1 by the end product of the pathway, UDP-GlcNAc, was competitive with a K(i) of 4 microm. These data suggest that hGFAT1 is fully active at physiological levels of Fru-6-P and may be regulated by its product GlcN-6-P in addition to the pathway end product, UDP-GlcNAc.