Lactisole

Mouse GMCs (SV40 MES 13) and human proximal tubular cells (HK-2) were purchased from the China Center for Type Culture Collection (CCTCC) and were cultured in Dulbecco's modified Eagle's medium (DMEM, Glbco) containing 5.6 mmol/L glucose and 10% fetal bovine serum (FBS, Glbco) at 37°C and 5% CO_2.Initially, to determine proper concentrations of high glucose and lactisole, cells were randomly divided into the following groups: (1) normal control group (NC group, 5.6 mmol/L glucose); (2) high-glucose treatment group (HG group; cells were treated with culture medium containing 10 mmol/L, 20 mmol/L, and 30 mmol/L glucose); (3) osmotic pressure group (OP group, with medium that contained 5.6 mmol/L glucose + 24.4 mmol/L mannitol); (4) lactisole (Sigma-Aldrich, MO, USA) at 3, 5, and 10 mmol/L as an inhibitor of STR intervention group.
The cells in each group were induced for 6, 12, or 24 h before the culture supernatant was collected, and the protein and mRNA were extracted for further study. Viability of GMCs was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). For MTT analysis, absorbance at 570 nm was measured for the experimental groups using a microplate reader according to the manufacturer's instruction. Each experiment was performed in duplicate and repeated thrice.

The following compounds and toxin were purchased from Sigma-Aldrich (St. Louis, MO, United States): Acesulfame K, aspartame, D-fructose, sucrose, sodium saccharin, monellin, thaumatin, rebaudioside A, lactisole, somatostatin, epidermal growth factor (EGF), SFLLR, Pertussis toxin (PTx), latrunculin A, wortmannin, GW583340, Tyrphostin AG 1478 and GF109203x. Stevioside was from Emperors Herbologists (Jacksonville, FL, United States). P-4000, SC-45647 and superaspartame were gifts from Grant Dubois (the Coca-Cola Company, Atlanta, GA). Sucralose was from Toronto Research Chemicals, Inc. (North York, Ontario, Canada). Alitame (Aclame™) was a generous gift from Danisco (Terre Haute, IN, United States). Neotame was from the NutraSweet Company (Chicago, IL, United States). Dulcin was from Maybridge Chemical Company (Cornwall, United Kingdom). S1P, U1026 and GSK269962 were from Tocris Biosciences (Minneapolis, MN). S819, S5227 (Zhang et al., 2008 (link)), S679 (Tachdjian et al., 2009 ) and S1313 (Tachdjian et al., 2010 ) were synthesized in house.

On the basis of the method of Kato et al. [20 (link)], the synthesis of 3DG was performed as previously described [22 (link), 23 ]. UV, IR, ¹H-NMR, ¹³C-NMR, MS, and HPLC-ELSD were used to identify the synthetic 3DG [23 ]. Phloretin, lactisole, and phloridzin dihydrate were purchased from Sigma-Aldrich (St. Louis, MO). phloridzin dihydrate, Phloretin, and lactisole were prepared as a stock solution in DMSO, and the final DMSO concentration was adjusted to 0.05%.