This research addresses two important aspects of fatty acid and associated triacylglycerol (TAG) metabolism, namely the mechanisms by which: I. VLDL assembly occurs within the secretory system of the hepatocyte and how these processes are regulated. VLDL, synthesized by the liver, is a major carrier of TAG in the blood and is the precursor of LDL, a major carrier of plasma cholesterol; and II. conversion of saturated fatty acids into mono- unsaturated fatty acids is regulated by feed-back control of expression of the mRNA encoding stearoyl-CoA desaturase (SCD). Desaturation of fatty acids is essential for maintenance of the functional properties of cellular membranes and storage triacylglycerol. Using hepatocytes in culture we discovered that assembly of TAG with apolipoproteins to form nascent VLDL occurs in the Golgi. Recently, we developed a cell-free (i.e. permeabilized cell) system with which to investigate the mechanisms of VLDL assembly and secretion.
The SPECIFIC AIMS are to: * determine the rate of transit of cholesterol and phospholipids through organelles of the secretory system of the hepatocyte. The site(s) of assembly of cholesterol and phospholipids with the nascent VLDL particle or apolipoproteins will be identified. The control by cholesterol of apo B synthesis and VLDL assembly will be determined. * establish the requirements (eg. cytosolic factors, ATP, metal ions, etc.) of the permeabilized hepatocyte system for translocation of VLDL components, ie. apolipoproteins, TAG, cholesterol (and ester) and phospholipids from the ER to/through Golgi elements and secretory vesicles. * purify and characterize the cytosolic factor(s) required for transit of TAG and phospholipids (possibly apoproteins, cholesterol) through the secretory system and for VLDL assembly in the permeabilized hepatocyte. * determine the mechanism(s) by which the apolipoproteins are """"""""held-up"""""""" in the Golgi prior for assembly with TAG. The possible role of fatty acylation-deacylation will be further explored. Cells must regulate the unsaturated fatty acid content of their membrane phospholipids and stored triacylglycerol. Without exogenous unsaturated fatty acids, fatty acid """"""""desaturation"""""""" occurs via stearoyl-CoA desaturase (SCD). SCD level is under rigorous feed-back control via expression of SCD mRNA. We have cloned, determined the structures and sequenced the 5' flanking regions of the SCD1 and SCD2 genes, which are expressed in specific cell types. Negative feed-back control of expression by unsaturated fatty acids is both gene- and cell type-specific.
The SPECIFIC AIMS are to determine: * the mechanism of """"""""negative feed-back' control by unsaturated fatty acids of expression of SCD mRNA. """"""""Positive feed-forward"""""""" control will also be investigated. * the molecular basis for tissue-specific expression and regulation of the SCD1 and SCD2 genes.
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| Bamberger, M J; Lane, M D (1988) Assembly of very low density lipoprotein in the hepatocyte. Differential transport of apoproteins through the secretory pathway. J Biol Chem 263:11868-78 |
