Short-chain fatty acid (SCFA) levels in the colon are greater than 200 mM, derived from fermentation of dietary fiber and other dietary components. These SCFAs play critical roles in colonic cell physiology. First, they are the principal energy source for colonic epithelial cells via their metabolism by beta-oxidation in the colonic cell mitochondria. Second, they are natural inducers of colonic cell differentiation both in vivo, and of colonic carcinoma cell lines in vitro, and a differentiation program that they initiate has a component pathway that results in colonic cell apoptosis. Our previous work, and that of others, has demonstrated a clear link among the metabolism of SCFAs, mitochondrial gene expression and function, growth arrest and the entry of cells into a pathway which results in differentiation and/or apoptosis. Moreover, recent reports have provided compelling data for a direct role of mitochondria in apoptosis in other systems. This application is to use novel genetic mouse models, and genetic and biochemical manipulation in culture, to define the pathways and mechanisms that link SCFAs, their metabolism, mitochondrial function, and normal cellular development in the colonic mucosa, and how perturbations of this pathway modulate genetic and carcinogen initiated colon tumorigenesis. There are 3 specific aims: 1) To utilize a genetic mouse model in which there is a homozygous deletion of the nuclear gene for short-chain acyl dehydrogenase (SCAD), which encodes the mitochondrial enzyme that catalyzes the first step in mitochondrial beta oxidation of SCFAs, to determine the role of SCFA metabolism in the effects of tributyrin, the triglyceride of butyrate, on both chemically (AOM) and genetically (Apc) initiated colon cancer, and on the intermediate end-points of aberrant crypt focus formation, cell proliferation, and apoptosis; 2) based on our data, to determine the role of mitochondrial function and interaction with the waf1/cip1 gene in mechanisms of SCFA induced cell cycle arrest and entry into a pathway of apoptosis in SW620 colonic epithelial cells in culture; 3) to use waf1/cip1 null mice, and a mouse strain with a conditional targeted inactivation of the nuclear gene for mitochondrial cytochrome C, to test the following model: that SCFA inhibition of Apc initiated gastrointestinal tumors is waf1/cip1 dependent; and that this inhibition requires mitochondrial function, and not only metabolism of SCFAs.
