Biochemistry, Department of
Date of this Version
Obesity is correlated with insulin resistance and elevated levels of glucose, triglycerides and free fatty acids in blood. This affects overall metabolism and leads to disease. In the obese state, fat also accumulates in non-adipose tissue including liver, muscle and pancreas, where it can lead to cellular dysfunction and death. Currently, only a limited number of drugs are available to combat obesity and it is clear that new drugs, which more narrowly target the metabolic pathways involved, are required. Fatty Acid Transport Proteins (FATPs) are bifunctional proteins involved in the uptake and activation of fatty acids by esterification with coenzyme A. Inhibition of uptake of fatty acids in non-adipose tissues seem an attractive mechanism for treatment of lipotoxicity and obesity related diseases. In this study, we have investigated a newly identified fatty acid uptake inhibitor called CB5/Grassofermata (2-benzyl-3-(4-chlorophenyl)-5-(4-nitrophenyl)pyrazolo[1,5-a]pyrimidin-7(4H)-one) that presumably works through interaction with Fatty Acid Transport Protein (FATP)-2.
In the present study we demonstrate the ability of CB5/Grassofermata to inhibit uptake of fatty acids in cell lines that are models for liver, small intestines, pancreas and muscle in low micro-molar ranges (IC50: 8-11μM). The inhibition was less effective in human adipocytes, model for adipose tissue (IC50: 58μM). Also, we show that CB5 specifically inhibits the uptake of long-chain (C12-C20) and very long-chain (≥C22) fatty acids and protects liver and pancreatic cells from lipotoxicity generated by palmitic acid. Pharmacokinetic analysis of CB5 demonstrated its ability to limit absorption of labeled oleate across intestinal epithelium after 6hrs of oral administration (300mg/kg). Finally, we used whole body knockout mice of FATP2 gene on high fat and low fat (60% and 12% energy from fat, respectively) diets to understand the implications of loss of FATP2 gene on lipid metabolism. Loss of FATP2 gene resulted in ~35% reduction in plasma triglycerides levels and demonstrated increased steatosis after 12 weeks on diets. Hepatic steatosis was likely caused by the expression of FATP5, ACSL1 and ACSL5 transport proteins causing increased accumulation of hepatic triglycerides reflected in the amount of saturated and monounsaturated fatty acid. These diet studies are important prior of using CB5 in diet-induced obesity mice models.
Advisor: Concetta C. DiRusso
A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy, Major: Biochemistry, Under the Supervision of Professor Concetta C. DiRusso. Lincoln, Nebraska: October, 2015
Copyright (c) 2015 Nipun Saini