We developed a panel of non-obese diabetic (NOD) mice deficient in major lysosomal cysteine proteases (cathepsins S, L and B) to identify protease enzymes essential for autoimmune diabetes. Null alleles for cathepsins (Cts) S, L or B were introgressed onto the NOD genetic background with 19 Idd markers at homozygosity. Diabetes onset was determined among females aged up to 6 months. We evaluated insulitis and sialadenitis in tissues using histology and computer assisted morphology. NOD mice deficient in Ctss or Ctsb were partially protected from diabetes with incidence at 33% and 28%, respectively, versus wild-type NOD (69%; p<0.00001). NODs lacking cathepsin L (Ctsl −/−) are completely protected from IDDM, as originally shown by others. Ctsl, Ctss, or Ctsb heterozygous mice were able to develop IDDM, although incidence levels were significantly lower for Ctsb+/− (50%) and Ctsl+/− (55%) as compared to NODs (69%; p<0.03). Ctsl−/− mice contain functional, diabetogenic T-cells and an enriched Foxp3+ regulatory T-cell population, and diabetes resistance was due to the presence of an expanded population of regulatory T-cells. These data provide additional information about the potency of the diabetogenic T-cell population in Ctsl−/− mice which were comparable in potency to wild-type NOD mice. These data illustrate the critical contribution of each of these proteases in determining IDDM in the NOD mouse and provide a useful set of models for further studies.

Metabolic diseases such as obesity and atherosclerosis result from complex interactions between environmental factors and genetic variants. A panel of chromosome substitution strains (CSSs) was developed to characterize genetic and dietary factors contributing to metabolic diseases and other biological traits and biomedical conditions. Our goal here was to identify quantitative trait loci (QTLs) contributing to obesity, energy expenditure and atherosclerosis. Parental strains C57BL/6 and A/J together with a panel of 21 CSSs derived from these progenitors were subjected to chronic feeding of rodent chow and atherosclerotic (females) or diabetogenic (males) test diets, and evaluated for a variety of metabolic phenotypes including several traits unique to this report, namely fat pad weights, energy balance and atherosclerosis. A total of 297 QTLs across 35 traits were discovered, two of which provided significant protection from atherosclerosis, and several dozen QTLs modulated body weight, body composition and circulating lipid levels in females and males. While several QTLs confirmed previous reports, most QTLs were novel. Finally, we applied the CSS quantitative genetic approach to energy balance, and identified three novel QTLs controlling energy expenditure and one QTL modulating food intake. Overall, we identified many new QTLs and phenotyped several novel traits in this mouse model of diet-induced metabolic diseases.

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.

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.