Naturally occurring mutations in the mouse represents a rich for identification of novel genes affecting lipid metabolism. In this project, we will isolate the genes for two mouse mutations affecting triglyceride metabolism, fatty liver dystrophy (fld) and combined lipase deficiency (cld). The fld/fld mutant mice exhibit a transient hypertriglyceridemia and fatty liver during neonatal development. We recently determined that these animals subsequently develop insulin resistance and are grossly deficient in stores of white adipose tissue, and hypothesize that the fld mutation affects a component of the insulin signal transduction pathway. The hallmark feature of cld/cld mice is hypertriglyceridemia that is fatal within 2-3 days of birth, caused by the virtual absence of lipoprotein lipase and hepatic lipase activities. Recently we have determined that the basis for the defect is misfolding of newly synthesized lipase molecules in the endoplasmic reticulum. Our two aims will focus on isolation of the fld and cld genes and characterization of the cellular processes disrupted by the mutations.
In aim 1 we will identify the two genes using positional and functional cloning strategies. In the case of fld, we have narrowed the critical region to approximately kb using genetic and physical strategies. In the case of fld, we have narrowed the critical region to approximately kb using genetic and physical mapping; analysis of this region via exon trapping has identified an fld gene candidate that appears to be rearranged in the fld genome and is not expressed in tissues from the mutant mouse. We will now confirm this candidate by sequence analysis; additional candidate genes that we have isolated from this region by exon trapping will be analyzed if needed. In the case of cld, the gene critical region has been isolated as a series of over-lapping yeast and bacterial artificial chromosome clones spanning a distance of approximately 2 megabases. We are now poised to identify the clone containing the cld gene by complementation in tissue culture cell line, followed by gene identification using methods that have proved successful for the fld mutation.
In Aim 2, key questions concerning the molecular basis of the mutant phenotypes will be investigated. For fld these include determining the underlying defect in impaired glucose uptake by fld tissues, utilizing the isolated gene to explore protein function and evaluating the role of this gene in human diseases characterized by insulin resistance (e.g., FCHL, diabetes, atherosclerosis). For cld, we will investigate whether reduced levels of a specific chaperone, calnexin, contribute to the lipase deficiency that is the hallmark of this mutation. Once the gene is isolated, studies will focus on cld protein function and its potential role in human subjects exhibit combined lipase deficiency.
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