The studies outlined in this proposal have as a long-term objective to elucidate the mechanism underlying the cytoplasmic transport of amphipathic molecules like long-chain fatty acids and bile acids. Despite intense interest, understanding of the intracellular movement of these important biological substrates is still surprising limited. The investigator's hypothesis is that this transport occurs by diffusion and that this process is enhanced by co-diffusion with soluble binding proteins like fatty acid binding protein (FABP) or bile acid binding protein (BABP). Furthermore, the investigator suggests that the cytoplasmic transport of fatty acids and bile acids is so slow that it regulates the overall rate of uptake and metabolism of these important amphipathic molecules. The cytoplasmic transport of a fluorescent fatty acid analog,NBD-stearate, and a fluorescent bile acid analog dansyl taurocholate, will be measured by two dimensional fluorescence recovery after photobleaching (FRAP). This technique can determine estimates of the cytoplasmic transport rate of these fluorescent probes as well as their partition between soluble proteins and intracellular membranes. These measurements will be done in rat hepatocytes and hepatocyte couplets as well as in human hepatoma cells (HepG2/C3A cells). The use of two dimensional FRAP will allow the detection of """"""""directional"""""""" diffusion and/or convection as mechanisms of cytoplasmic transport.
The specific aims of the proposed studies are to: 1) develop a laser photobleaching system to accurately measure the spatial (directional) transport of fluorescent molecules; 2) characterize the intracellular transport of fatty acids and bile acids in the cytoplasm of liver cells; 3) determine the mechanism and specificity of protein facilitation of cytoplasmic transport of fatty acids and bile acids; and 4) determine the influence of cytoplasmic binding proteins such as fatty acid binding protein and bile acid binding protein on the intracellular transport of NBD-stearate and dansyl taurocholate. The failure of traditional enzymatic and molecular biological techniques to resolve the basis for the intracellular transport of fatty acids and bile acids reflects the many unique features of amphipathic molecules. These include extensive binding to cytosolic proteins and membranes and the resulting slow exchange between adjacent cellular compartments. By helping to define the molecular basis for the interaction between fatty acids and bile acids and their respective cytoplasmic binding proteins, these studies will generate crucial information needed to explain the pathogenesis of diseases as diverse as atherosclerosis, obesity, and gallstones. Insights and new methods developed in this proposal may apply to the intracellular transport of other biologically important molecules including cholesterol, thyroid and steroid hormones.
|Milliano, M T; Luxon, B A (2001) The peroxisomal proliferator clofibrate enhances the hepatic cytoplasmic movement of fatty acids in rats. Hepatology 33:413-8|
|Luxon, B A; Milliano, M T; Weisiger, R A (2000) Induction of hepatic cytosolic fatty acid binding protein with clofibrate accelerates both membrane and cytoplasmic transport of palmitate. Biochim Biophys Acta 1487:309-18|
|Luxon, B A; Milliano, M T (1999) Cytoplasmic transport of fatty acids in rat enterocytes: role of binding to fatty acid-binding protein. Am J Physiol 277:G361-6|
|Luxon, B A; Holly, D C; Milliano, M T et al. (1998) Sex differences in multiple steps in hepatic transport of palmitate support a balanced uptake mechanism. Am J Physiol 274:G52-61|
|Luxon, B A; Milliano, M T (1997) Cytoplasmic codiffusion of fatty acids is not specific for fatty acid binding protein. Am J Physiol 273:C859-67|