5 research outputs found
ERK signaling through the GLP-1R.
<p>HEK-GLP-1R cells (<b>A and B</b>) or INS-1E cells (<b>C and D</b>) were challenged for 5 min with either vehicle (1% v/v DMSO), GLP-1 9–36 amide (1 or 10 µM as indicated) or compound 2 alone or GLP-1 9–36 amide (1 µM) and compound 2 together as indicated. Cells were also challenged with GLP-1 7–36 amide (10 nM). Levels of phospho-ERK were then determined by Western blotting. The intensity of the bands representing phospho-ERK was determined using ImageJ and the mean data are shown in the panels below the immunoblot with basal (0) levels subtracted. Data are either representative of 3 experiments or mean+s.e.m., n = 3. *, P<0.05 and ** P<0.01 by Student's test versus the numerical sum of both GLP-1 9–36 amide (1 µM) and compound 2 at the concentration indicated when used alone.</p
Functional interaction of compound 2 and GLP-1 9–36 amide at the GLP-1R in HEK-GLP-1R cells.
<p>The pEC<sub>50</sub> values of GLP-1 9–36 amide-mediated cAMP generation in the presence of increasing concentrations of compound 2. The pEC<sub>50</sub> values have been determined from the data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047936#pone-0047936-g003" target="_blank">Figure 3</a>. Data are mean±s.e.m., n = 4, ** P<0.01 and *** P<0.001 versus 0 µM compound 2 by Dunnett's multiple range test following oneway ANOVA.</p
Functional interaction between ligands on GLP-1R-mediated cAMP generation in HEK-GLP-1R cells.
<p>HEK-GLP-1R cells were pretreated (Pre-) for 10 min with 1 µM GLP-1 9–36 amide in the presence of IBMX before challenge for 15 min with the indicated concentrations of agonists. Where no pre-treatment is indicated, an equivalent volume of buffer (KHB) was added for 10 min in the presence of IBMX prior to ligand addition for 15 min. Levels of intracellular cAMP were then determined relative to the cellular protein content. The final concentration of DMSO (vehicle) for the 15 min treatment period was 5% v/v in all cases. Data are mean±s.e.m., n = 3.</p
Compound 2 potentiates GLP-1 9–36 amide-mediated cAMP generation by membranes from HEK-GLP-1R cells.
<p>Cell membranes prepared from HEK-GLP-1R cells were incubated with ligands as indicated for 5 min at 30°C in the presence of IBMX before determination of cAMP. A) Concentration-dependent cAMP generation in response to GLP-1 7-36 amide or to GLP-1 9–36 amide either alone or in combination with 3 µM compound 2. <b>B</b>) Responses to GLP-1 9–36 amide or compound 2 alone or the two in combination. The numerical sums of cAMP generation in response to GLP-1 9–36 amide and compound 2 are shown. Data are mean±/+s.e.m., n = 3. For * P<0.05 by Bonferroni's multiple range. For clarity only differences between ‘numerical’ and ‘co-addition’ conditions are shown.</p
Time course of cAMP generation in response to GLP-1 9–36 amide, compound 2 or co-stimulation in HEK-GLP-1R cells.
<p>HEK-GLP-1R cells were either untreated (Basal; not visible) or treated for the indicated times with GLP-1 9–36 amide (1 µM), compound 2 (1 µM) or the two in combination (Co-addition) in the presence of IBMX. The final concentration of DMSO (vehicle) was 5% v/v in all cases. In addition to the measured levels of cAMP generation, the numerical sum of cAMP generation in response to GLP-1 9–36 amide and compound 2 alone are presented (Numerical). Data are mean±s.e.m., n = 3, ** P<0.01 and *** P<0.001 by Bonferroni's multiple range test following oneway ANOVA. For clarity, only differences between ‘numerical’ and ‘co-addition’ conditions are shown.</p