The goals of this project are (1) to understand the biochemical and molecular mechanisms that regulate preadipocyte determination and (2) to develop a specific marker or set of markers for the preadipocyte phenotype. Both of these goals are of great importance in human disease because abnormal preadipocyte formation may play a central role in massive obesity and lipodystrophy, life-threatening conditions involving adipose tissue. There are currently no molecular markers for the preadipocyte nor is there substantial data about how this cell type forms from mesenchymal precursors. cDNA clones specific for the preadipocyte phenotype will be isolated from a large cDNA phage library we have prepared from preadipose 3T3-F442A cells. This library will be screened differentially with cDNA from preadipocytes and non-preadipose 3T3 cells and by a subtractive-hybridization scheme using a modified cDNA labelling protocol we have developed. cDNA clones of interest will be further characterized by hybridization to mRNA from a battery of preadipose and non-preadipose mesenchymal cells. The identity of such sequences will be examined by cDNA sequence determination and computer assisted search of existing data banks. The intracellular localization of preadipose-specific proteins will be investigated by preparation of specific antibodies against the encoded protein using open-reading frame bacterial vectors, immunohistochemical procedures and Western blotting. The potential regulatory role of preadipose-specific mRNAs in cell determination will be investigated by the construction of viral- based vectors which can express these mRNAs in non-preadipose 3T3-cells and other cell types. The capacity of these cells to differentiate will subsequently be monitored at the morphological, RNA and protein levels. The effect of expression of anti-sense mRNA for preadipose-specific genes will be examined in fully determined preadipocytes. Because of the importance of the preadipocyte in human disease, the presence of preadipocyte-specific mRNA proteins will be quantitatively examined in cohorts of obese and lipodystrophic patients. Should these experiments indicate a possible defect in or overproduction of a particular gene product, we will examine the DNA of a cohort of such patients and normal subjects to assess the degree of allelic variation and restriction-fragments length polymorphisms in these populations.
