Uptake And Transport Characteristics Of Neuromedin U2 Receptor Agonists In Caco-2 Cell Line

Neuromedin U2 receptor (NMU2R) is one of the two neuromedin U (NMU) receptors, which is extensively expressed in the central nervous system (CNS). NMU2R plays an essential role in the regulation of feeding behavior and energy homeostasis, and has been proposed to be an important CNS target of metabolic diseases such as obesity and type 2 diabetes. We have previously discovered several small molecules (e.g., EUK2000 and EUK2010) that exhibited potent agonist properties of NMU2R in vitro. The results showed that oral administration of the NMU2R agonists to obese rats resulted in the reduction of body weight. Moreover, in those NMU2R agonist-treated rats, the levels of blood glucose and the insulin sensitivity were improved. These in vivo results provide evidence for the potential of these natural products (EUK2000 and EUK2010) or their structure-modified derivatives (e.g., EUK2030 and EUK2085) to be the lead compounds of the drugable NMU2R agonists.P-glycoprotein and the multidrug resistance-associated protein (MRP) provide an important drug resistance function in the body. The absorption, distribution, metabolism and excretion of many compounds in the body were influenced by the interaction with P-glycoprotein or MRP. It is still unclear that whether the EUK compounds could be absorbed in the body and how do they interact with P-glycoprotein or MRP. In the present study, we investigated the uptake and transport characteristics of these compounds (EUK2000, EUK2010, EUK2030 and EUK2085) in Caco-2 cells. Meanwhile, the interactions of these compounds with P-glycoprotein and MRP in the cell model were also evaluated at molecular as well as cellular levels by HPLC, calcein AM and RT-PCR techniques.1. Uptake characteristics of NMU2R agonists in Caco-2 cellsIn order to investigate the uptake characteristics of NMU2R agonists in Caco-2 cells, we placed different concentrations of NMU2R agonists in Caco-2 cells co-cultured with or without verapamil (a specific inhibitor of P-glycoprotein) within 60 minutes. The cells were then sonicated, and the amounts of the coumpounds being uptaken in the cells were measured using HPLC. The results indicated that EUK2000, EUK2030 and EUK2085 can be readily absorbed by Caco-2 cells in a concentration-dependent manner, suggesting that they were transported by passive way. The uptake of these compounds were significantly increased when co-culture with verapamil, suggested that they were substrate of P-glycoprotein. EUK2010, however, was found not to be absorbed by Caco-2 cells.2. Transport characteristics of NMU2R agonists across Caco-2 monolayersIn order to investigate the absorption characteristics of NMU2R agonists in human intestinal, we performed the transport experiments using Caco-2 cells cultured on transwell at least 21 days. We found that EUK2085 could easily transport across the Caco-2 monolayers with an apparent permeability coefficient (P_app) of 1.54×10^-5cm/s, while EUK2000, EUK2010 and EUK2030 couldn't transport across the Caco-2 cells.3. Effect of NMU2R agonists on MDR1, MRP1 and MRP2 mRNA expression in Caco-2 cellsIn order to investigate the effects of NMU2R agonists on MDR1, MRP1 and MRP2 mRNA expressions in Caco-2 cells, we treated the cells with different EUK compound for 48 and 72 hours, then extracted RNA and reversed them to cDNA, quantitated by RT-PCR technique. The results showed that EUK2000 down-regulated, while EUK2030 and EUK2085 up-regulated, EUK2010 showed no effect on the expression of these genes.4. Inhibitory effect of NMU2R agonist on the function of P-glycoprotein or MRPTo investigate whether the NMU2R agonists have inhibitory effect on the function of P-glycoprotein and/or MRP, we placed calcein AM and different EUK compound together in Caco-2 cells for 35 minutes, then detected the fluorescene value of calcein in Caco-2 cells. The results suggested that EUK2000 and EUK2085 had inhibitory effects on the activities of P-glycoprotein and/or MRP with EC50 values of 47μM and 40μM, respectively, compared to verapamil, a positive control with EC50 of 40μM. However, the data slao showed that EUK2010 and EUK2030 had no such inhibitory effects as compeared with verapamil.