The movement of amphipathic molecules within cells is one of the last frontiers for transport physiology. Included are many important molecules involved in energy metabolism (e.g., long chain fatty acids), signal transduction (thyroid and steroid hormones) and intermediary metabolism (bile acids, heme, cholesterol and retinoids). Our laboratory has assembled the necessary experimental and theoretical tools to study this process in depth. These include two methods of measuring cytoplasmic transport rates, one based on fluorescence recovery after laser photobleaching (FRAP) of cultured liver cells and the other based on the multiple indicator dilution (MID) method applied to the isolated perfused rat liver model. In addition, an artificial cytoplasm model will permit testing ideas with more freedom than is possible with living cells. Finally, sophisticated mathematical models will allow simulation of intracellular events that cannot be observed directly. Combined with genetic manipulation of cytoplasmic fatty acid binding protein (FABP) levels, these new approaches will allow us to determine the rate and mechanism of cytoplasmic transport and to define its importance in regulating the uptake and metabolism fatty acids. We hypothesize that cytoplasmic transport is a Crucial step in regulating the hepatic clearance and metabolism of fatty acids, hydrophobic bile acids, bilirubin, protoporphyrin, and numerous other endogenous metabolites and exogenous toxins. We believe that this process is regulated by soluble cytoplasmic binding proteins, which promote cytoplasmic mobility by reducing binding to cytoplasmic membranes. We suggest that disorders of cytoplasmic transport may contribute to clinically important conditions such as cholestasis and obesity.
Our specific aims are to: 1. Determine the rates of convection and diffusion of selected amphipathic molecules within liver cytoplasm 2. Define the mechanism and of cytoplasmic fatty acid transport, including the rates of each component step. 3. Assess the role of binding proteins in regulating cytoplasmic transport of amphipaths. 4. Determine if cytoplasmic transport is rate-limiting to fatty acid utilization by the liver. These studies will define the fundamental mechanisms that regulate fatty acid metabolism in normal liver, a necessary step to understanding how these processes become disordered in disease states including hepatic steatosis, cirrhosis, diabetes and obesity. Preliminary data have demonstrated that the proposed studies are feasible. Results should have important implications for the regulation of energy metabolism and hepatic clearance of amphipathic drugs and toxins.
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