Three primer pair sequences for siRNA–GLP-1R and negative control (Stealth Negative) were purchased from Invitrogen as shown in Table 1. Huh7 cells were transfected using Lipofectamine RNAiMAX reagent (Invitrogen)

following the manufacturer’s reverse transfection protocol. Cells were plated at 50% confluency and transfected with the siRNA sequences at 30 nM and maintained for 48 CH5424802 chemical structure hours. GLP-1R knockdown was confirmed by way of immunoblot analysis. Cell lysates were prepared and subjected to immunoblot analysis for GLP-1R, PDK1, AKT, and PKC-ζ. All data are presented as the mean ± standard error (SE). Statistical analysis was performed using Graphpad Instat 3 software (http://www.graphpad.com). Groups were compared using parametric tests

(paired Student t test or one-way analysis of variance with posttest following statistical standards). P < 0.05 was considered statistically significant. SCH 900776 purchase Western blot analysis revealed the presence of GLP-1R in Huh7 cells and primary human hepatocytes (Fig. 1A). As shown in Fig. 1B, there was a multifold increase of GLP-1R in Huh7 cells compared with preimmune serum-treated controls (P < 0.05). GLP-1R is internalized on stimulation by GLP-1 or exendin-4 (Fig. 2). This was first demonstrated by way of cell surface expression analysis (bioluminescence assay) (Fig. 2A). We then confirmed the microscopic findings by way of subcellular fractionation (Fig. 2B). This demonstrated that following MCE公司 GLP-1R exposure to its agonist, the membrane-bound fraction was reduced. Upon stimulation with either GLP-1 or exendin-4, there was a decrease in the amount of receptor seen on the cell membrane under confocal microcopy (Fig. 2C). These data suggest that there is loss of the receptor from the cell membrane. Both confocal and fluorescent imaging confirmed that GLP-1R is internalized. Fig. 2C (left panel) shows untreated cells in which GLP-1R (in green) is seen lining the cell membrane. On treatment with GLP-1 or exendin-4, the receptor (Fig. 2C, right panel)

was detected primarily in the cytoplasm rather than on the plasma membrane (yellow arrows). These data support the detection of internalization of the receptor by way of bioluminescence assay, which was also confirmed by subcellular fractionation analysis. To determine whether a physiologic endpoint of putative GLP-1 receptor signaling could be achieved, we used several approaches to explore whether there was a significant reduction in the cellular TG content following exendin-4 treatment. As seen on Oil Red O staining (Fig. 3A), following engorgement of Huh7 cells with palmitate and oleate, exendin-4 greatly reduced TG stores; this was further corroborated by TG quantitation (Fig. 3B).