The purpose of this project is to study the regulation of intestinal acyl CoA: cholesterol acyltransferase (ACAT) by plasma lipoproteins and by phosphorylation-dephosphorylation. This will be accomplished in two parts. To study the regulation of intestinal ACAT by lipoproteins, twenty pairs of inbred, littermate rats will be surgically joined to form a parabiontic union. With time, these animals will share a common circulation. One rat of a joined pair will be fed a diet containing cholesterol (Diet C). Its mate will be fed a diet devoid of cholesterol (Diet B). In another pair of parabiont rats, one animal will also be fed Diet B. Its mate will be fed a control diet (Diet A). The small intestine from animals fed Diet B will be assimilating similar amounts of cholesterol, however, some of these animals will have hyperlipidemia, i.e., those that have a joined mate ingesting Diet C. Microsomal ACAT and HMG CoA reductase activity will be determined in the proximal, middle, and distal thirds of the small intestine in 20 pairs of parabiont rats. Serum and microsomal cholesterol levels will be measured. Conclusions will be made regarding the capability of the intestine in taking up and degrading lipoproteins as a source of cholesterol. The second part of the project will involve experiments to investigate the regulation of intestinal ACAT by phosphorylation-dephosphorylation. This short-term mechanism of regulation will be studied in cell-free systems, in isolated intestinal cells, and with solubilized ACAT. Phosphoprotein phosphatase and protein kinase will be partially purified from intestinal cytosol. The activity state of ACAT in vivo will be determined by preparing intestinal microsomes in the presence of NaF, a phophatase inhibitor. ACAT activity will also be measured under conditions of phosphorylation. Microsomes will be incubated with increasing concentrations of MgCl2, ATP and protein kinase. Reversibililty of the reaction will be studied by incubating NaF- or Mg++/ATP-treated microsomes with phosphoprotein phosphatase. Similar experiments will be done on solubilized ACAT to determine if detachment from the membrane alters the conditions for either activation or deactivation. In isolated enterocytes, cAMP levels will be increased by incubating them with cAMP, glucagon, or xanthines. The effect of increased cAMP levels on cholesterol esterification will be measured. These studies will determine if intestinal ACAT, like intestinal HMG-CoA reductase, is regulated by phosphorylation-dephosphorylation.
