ABSTRACT
Alterations in fat metabolism, in particular elevated plasma concentrations of free fatty acids and triglycerides (TG), have been implicated in the pathogenesis of Type 2 diabetes, obesity, and cardiovascular disease. Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), a member of the large family of membrane-bound O-acyltransferases, catalyzes the final step in triacylglycerol formation. In the intestine, DGAT1 is one of the acyltransferases responsible for the reesterficiation of dietary TG. Following a single dose of a selective pharmacological inhibitor of DGAT1, PF-04620110, a dose-dependent inhibition of TG and vitamin A absorption postprandially was demonstrated in rodents and human subjects. In C57/BL6J mice, acute DGAT1 inhibition alters the temporal and spatial pattern of dietary lipid absorption. To understand the impact of DGAT1 inhibition on enterocyte lipid metabolism, lipomic profiling was performed in rat intestine and plasma as well as human plasma. DGAT1 inhibition causes an enrichment of polyunsaturated fatty acids within the TG class of lipids. This pharmacological intervention gives us insight as to the role of DGAT1 in human dietary lipid absorption.
Subject(s)
Diacylglycerol O-Acyltransferase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Intestinal Absorption/drug effects , Oxazepines/pharmacology , Adolescent , Adult , Animals , Case-Control Studies , Diacylglycerol O-Acyltransferase/genetics , Diacylglycerol O-Acyltransferase/metabolism , Dietary Fats/blood , Dietary Fats/metabolism , Dose-Response Relationship, Drug , Enterocytes/metabolism , Enzyme Inhibitors/pharmacokinetics , Fatty Acids, Unsaturated/blood , Fatty Acids, Unsaturated/metabolism , Female , Humans , Intestinal Mucosa/metabolism , Lipid Metabolism/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Oxazepines/pharmacokinetics , Postprandial Period , Rats , Rats, Sprague-Dawley , Triglycerides/blood , Triglycerides/metabolism , Vitamin A/metabolismABSTRACT
Human liver glycogen phosphorylase (HLGP) catalyzes the breakdown of glycogen to maintain serum glucose levels and is a therapeutic target for diabetes. HLGP is regulated by multiple interacting allosteric sites, each of which is a potential drug binding site. We used surface plasmon resonance (SPR) to screen for compounds that bind to the purine allosteric inhibitor site. We determined the affinities of a series of compounds and solved the crystal structures of three representative ligands with K(D) values from 17-550 microM. The crystal structures reveal that the affinities are partly determined by ligand-specific water-mediated hydrogen bonds and side chain movements. These effects could not be predicted; both crystallographic and SPR studies were required to understand the important features of binding and together provide a basis for the design of new allosteric inhibitors targeting this site.