Glucose uptake and transport is a vital process for all mammalian cells and tissues. The proposed studies are directed at defining and characterizing the major glucose transporters involved in uptake by intestine, colon and liver and to elucidate the regulation of their expression. The major transporters are the Na+ -dependent transporter (NaGT), associated with lumenal surface of intestinal and colonic mucosa and the facilitated glucose transporter (GT: Na+ -independent) associated with the basolateral membranes of these enterocytes. The liver differs in that it not only takes up glucose (and stores it as glycogen) but also releases glucose to maintain normoglycemia. Recent evidence by our laboratory suggests that there is more than one hepatocyte G and more likely a family of related GTs. Our goal is to characterize these transporters and to study their regulation at the molecular level under normal conditions, environmental stresses (such as calorie deprivation, starvation, and altered lumenal conditions) and in association with malignancy (colon and liver). We will be able to study transporter regulation and gene expression using rat and human intestinal and colon cell lines (IEC-6, HT-29, Caco2), primary colon cells in culture, as well as in isolated rat hepatocytes and hepatoma cell lines. We will attempt to purify NaGT and clone as well as sequence NaGT cDNAs will also study the GT of enterocyte basolateral membranes to compare its structure with the GT of fibroblasts, adipocytes and red cells. In the case of the facilitated transporter (GT), we are in a position to clone its genomic sequences and to identify the regulatory sites located upstream (5-prime) from the start site of the coding sequences. We also look for trans-acting factors that may be involved in altering GT gene transcription under various exogenous and endogenous conditions associated with altered cellular GT levels. The liver GT system clearly differs and, in fact, GT transcription is enhanced in hepatoma cells and cell lines, but is not detectable in normal liver. It will be important to 1) study the factors involved in regulating GT expression in liver, 2) to characterize, molecularly clone and sequence the related GTs, and 3) to transduce adult hepatocytes with proto-oncogenes to assess the effect on GT gene expression and phenotypic alterations. These studies on the characterization and regulation of expression of the glucose transporters of enterocytes and liver are important to our understanding of the basic process of glucose transport and its alterations under a variety of stress conditions and especially with malignant transformation.
