Murine phospholipid hydroperoxide glutathione peroxidase: cDNA sequence, tissue expression, and mapping.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx), also known as glutathione peroxidase 4 (GPX4), is a 19-kDa, monomeric enzyme that protects cells from lipid peroxide-mediated damage by catalyzing the reduction of lipid peroxides. PHGPx is synthesized in two forms, as a 194-amino acid peptide that predominates in gonadal tissue and localizes to mitochondria, and as a 170-amino acid protein that predominates in most somatic tissues and localizes to the cytoplasm. With the rapid amplification of cDNA ends (RACE) procedure, an 876-bp PHGPx cDNA was amplified from mouse testis, and a 767-bp PHGPx cDNA was amplified from mouse heart. The cDNA sequences were identical except that the testis cDNA contained an additional 109 bp at its 5' end. With a partial cDNA with complete homology to both the testis and myocardial PHGPx cDNAs, the murine tissue distribution of PHGPx mRNA expression was determined by Northern blotting. Highest level of PHGPx expression was found in the testis, followed by the kidney, heart and skeletal muscle, liver, brain, lung, and spleen. Northern blotting performed with a cDNA specific for the longer PHGPx transcript demonstrated that this longer PHGPx transcript was present only in the testis. A 1.4-kb PHGPx genomic fragment was amplified from murine kidney DNA and used to map the PHGPx gene by linkage analysis of restriction fragment length variants (RFLVs). The murine PHGPx gene (Gpx4) was mapped to a region of murine Chromosome (Chr) 10, located 43 cM from the centromere, that is syntenic with the human locus, which is located at the terminus of the short arm of human Chr 19. This information may be valuable in characterizing the role of PHGPx in modulating susceptibility to lipid peroxide-mediated injury in inbred murine strains and for targeted disruption of the gene.

Dietary antioxidants inhibit development of fatty streak lesions in the LDL receptor-deficient mouse.

Oxidized low density lipoprotein (LDL) promotes atherogenesis. Although pharmacological antioxidants such as probucol inhibit both LDL oxidation and atherosclerosis in hyperlipidemic animals, the effects of natural antioxidants such as vitamin E are inconclusive. To further determine the effects of supplemental dietary antioxidants in vivo, we evaluated whether combined dietary antioxidants (0.1% vitamin E, 0.5% beta-carotene, and 0.05% vitamin C) inhibit LDL oxidation and fatty streak lesion development in homozygous LDL receptor-null (LDLR-/-) mice fed a high-fat, high-cholesterol diet. An additional group of mice were fed black tea, which has been shown to inhibit LDL oxidation in vitro. After receiving a high-fat, high-cholesterol diet for 8 weeks, the combined antioxidant-supplemented (antioxidant) group (n=18), tea group (n=19), and control group (n=17) had equivalent plasma cholesterol levels. LDL oxidation, as measured by the lag phase of conjugated diene formation, was markedly inhibited in the antioxidant group compared with the tea or control groups [mean lag phases=143+/-7 (antioxidant), 100+/-5 (tea), and 84+/-4 (control) minutes; P<0.0001 antioxidant versus tea or control]. The cross-sectional surface area of fatty streak lesions in the aortic sinus was reduced by 60% in the antioxidant group compared with both the tea and control groups (P<0.0001 antioxidant versus tea or control). There was no difference in lesion area between tea and control groups. Although both LDL oxidation and atherosclerosis were significantly inhibited in the antioxidant group, no correlation between lag phase values and lesion size was observed among individual animals. Furthermore, black tea did not inhibit fatty streak development in LDLR-/- mice. These data suggest that combined natural dietary antioxidants inhibit both LDL oxidation and atherogenesis in animals with elevated LDL but that inhibition of LDL oxidation alone may not prevent the development of atherosclerosis.

Mouse glycosylphosphatidylinositol-specific phospholipase D (Gpld1) characterization.

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is an 110-kDa monomeric protein found in the circulation that is capable of degrading the GPI anchor utilized by dozens of cell-surface proteins in the presence of detergent. This protein is relatively abundant (5-10 microgram/ml in human serum), yet its sites of synthesis, gene structure, and overall function are unclear. It is our purpose to use the mouse system to determine its putative roles in lipid transport, pathogen control, and diabetes. We have isolated murine full-length cDNA for GPI-PLD from a pancreatic alpha cell library. The deduced amino acid sequence shows 74% homology to bovine and human GPI-PLD. There is a single structural gene (Gpld1) mapping to mouse Chromosome (Chr) 13, and among nine tissues, liver showed the greatest abundance of GPI-PLD mRNA. Genetic differences in serum GPI-PLD activity were seen among four mouse strains, and no correlation was seen between GPI-PLD activity and circulating levels of high density lipoproteins in these mice. This is the first report of map position and genetic regulation for Gpld1. This information will enable us to further study the expression and function of GPI-PLD in normal and pathological conditions.

Genetically determined body weight loss in mice fed diets containing salmon oil.

Several reports describe adverse effects of dietary fish oil. We examined the influence of dietary salmon oil (138 g/kg diet) fed without or with 5 g supplemental cholesterol/kg diet on body weight and plasma lipid concentrations of inbred mice. Salmon oil contained 0.17 g naturally occurring cholesterol/kg diet. Mice used were BALB/c, C57BL/6 and seven recombinant inbred strains derived from BALB/c and C57BL/6 (CXB). Parental strains BALB/c and C57BL/6 maintained or gained body weight when fed both salmon oil diets. Mice of recombinant inbred strains showed weight gain except for CXB-E and -H mice. Although CXB-E mice lost approximately 12% of initial body weight after 10 d of consuming either salmon oil diet, no further reductions in body weight were seen. CXB-H mice maintained or gained weight when fed the salmon oil-high cholesterol diet but showed a steady decline in body weight (up to 30% of initial weight) while consuming the salmon oil-low cholesterol diet. The biochemical basis for weight loss in CXB-H mice was studied and results suggest effects of diet on satiety and/or lipid utilization. Because nonparental body weight phenotypes were observed among recombinant inbred strains, body weight response to salmon oil feeding is controlled by multiple genes.