Browsing by Author "Cohen, David E."
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Item Genetic ablation or chemical inhibition of phosphatidylcholine transfer protein attenuates diet?induced hepatic glucose production†‡(Hepatology, 2012-08) Shishova, Ekaterina Y; Stoll, Janis M.; Ersoy, Baran A.; Shrestha, Sudeep; Scapa, Erez F.; Li, Yingxia; Niepel, Michele W.; Su, Ya; Jelicks, Linda A.; Stahl, Gregory L.; Glicksman, Marcie A.; Gutierrez-Juarez, Roger; Cuny, Gregory D.; Cohen, David E.Phosphatidylcholine transfer protein (PC?TP, synonym StARD2) is a highly specific intracellular lipid binding protein that is enriched in liver. Coding region polymorphisms in both humans and mice appear to confer protection against measures of insulin resistance. The current study was designed to test the hypotheses that Pctp?/? mice are protected against diet?induced increases in hepatic glucose production and that small molecule inhibition of PC?TP recapitulates this phenotype. Pctp?/? and wildtype mice were subjected to high?fat feeding and rates of hepatic glucose production and glucose clearance were quantified by hyperinsulinemic euglycemic clamp studies and pyruvate tolerance tests. These studies revealed that high?fat diet?induced increases in hepatic glucose production were markedly attenuated in Pctp?/? mice. Small molecule inhibitors of PC?TP were synthesized and their potencies, as well as mechanism of inhibition, were characterized in vitro. An optimized inhibitor was administered to high?fat?fed mice and used to explore effects on insulin signaling in cell culture systems. Small molecule inhibitors bound PC?TP, displaced phosphatidylcholines from the lipid binding site, and increased the thermal stability of the protein. Administration of the optimized inhibitor to wildtype mice attenuated hepatic glucose production associated with high?fat feeding, but had no activity in Pctp?/? mice. Indicative of a mechanism for reducing glucose intolerance that is distinct from commonly utilized insulin?sensitizing agents, the inhibitor promoted insulin?independent phosphorylation of key insulin signaling molecules. Conclusion: These findings suggest PC?TP inhibition as a novel therapeutic strategy in the management of hepatic insulin resistance.Item Small-molecule inhibitors of phosphatidylcholine transfer protein/StarD2 identified by high-throughput screening(Analytical Biochemistry, 2009-12) Wagle, Neil; Xian, Jun; Shishova, Ekaterina Y.; Wei, Jie; Glicksman, Marcie A.; Cuny, Gregory D.; Stein, Ross L.; Cohen, David E.Phosphatidylcholine transfer protein (PC–TP, also referred to as StarD2) is a highly specific intracellular lipid-binding protein that catalyzes the transfer of phosphatidylcholines between membranes in vitro. Recent studies have suggested that PC–TP in vivo functions to regulate fatty acid and glucose metabolism, possibly via interactions with selected other proteins. To begin to address the relationship between activity in vitro and biological function, we undertook a high-throughput screen to identify small-molecule inhibitors of the phosphatidylcholine transfer activity of PC–TP. After adapting a fluorescence quench assay to measure phosphatidylcholine transfer activity, we screened 114,752 compounds of a small-molecule library. The high-throughput screen identified 14 potential PC–TP inhibitors. Of these, 6 compounds exhibited characteristics consistent with specific inhibition of PC–TP activity, with IC50 values that ranged from 4.1 to 95.0 ?M under conditions of the in vitro assay. These compounds should serve as valuable reagents to elucidate the biological function of PC–TP. Because mice with homozygous disruption of the PC–TP gene (Pctp) are sensitized to insulin action and relatively resistant to the development of atherosclerosis, these inhibitors may also prove to be of value in the management of diabetes and atherosclerotic cardiovascular diseases.