Linoleic acid induces internalization of GPR40.

<p>HEK293 cell lines stably expressing with myc-tagged wild type or R104P mutant, were incubated with 100 μM Linoleic acid for 0–60 min at 37°C and stained with permeabilized condition. And the cell-surface level of GPR40 was determined by measuring surface myc immunoreactivity with Immunofluorescence microscopy.</p

Mutants of GPR40.

<p>The point mutations of GPR40 tested in this study are represented as a transmembrane snake plot. The blocked area represents plasma membrane with seven putative transmembrane domains.</p

Effect of R104P mutant on cell surface localization of GPR40.

<p>(A) Translocation of GPR40 was studied with HEK293 cells transiently transfected with wild type or R104P mutant, and Cells were stained with non-permeabilized and permeabilized condition. GPR40 membranes were stained with myc-tagged GPR40 (red, arrows). Cell nuclei were stained with Hoechst 33342 (blue), 2 μg of DNA transfected. (B) Quantitative analysis of wild type and R104P mutant expression with plasmid of different amount as measured by membranes (M) and total protein (T), (shown in panel A). Data are means ± SEM (n = 3). *P < 0.05 and **P < 0.01, WT membranes versus R104P membranes. Scale bar, 10 μm.</p

Effect of R104P mutant on GPR40 mediated ERK phosphorylation.

<p>HEK293 cells transiently transfected with wild type (A) or R104P mutant (B) or empty vector (C) were stimulated with different ligands at indicated concentration at 10min or 100 μM LA for various durations (0, 5, 10, 15, 30 and 60 min) at 37°C, and ERK1/2 phosphorylation was detected with Western blot. ERK phosphorylation levels were normalized to the GAPDH level in the same sample. Data are means ± SEM (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001, versus vehicle control.</p

Calcium response in HEK293 cells expressing various GPR40 mutants in response to LA, DHA and AM8596.

<p>HEK293 cells were transfected with wild type or mutant GPR40 by electroporation. Cells were then loaded with Fluo-4 AM and intracellular calcium changes after stimulation with different compounds were monitored. (A) Intracellular calcium changes of empty vector, wild-type and R104P/Y202C/R211H mutant hGPR40. (B, C and D) Calcium mobilization in empty vector, wild-type, R104P/Y202C/R211H, R104P, Y202C, R211H and Y202C/R211H in response to LA (B), DHA (C) and AM8596 (D). Data are presented as the means ± SEM (n = 3).</p

Effect of expression of GPR40 in HEK 293 cells on calcium response.

<p>(A) HEK293 cells were transiently transfected with different amount of plasmids (wild type or R104P mutant), and GPR40 was detected with Western blot. (B and C) HEK-293 cells were transfected with wild type (B) or R104P mutant (C), then cells were loaded with Fluo-4 AM and intracellular calcium changes after stimulation with linoleic acid were monitored. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.</p

Voiding behavior in testosterone-induced BPH dogs.

<p>Prostatic dimensions were obtained with ultrasonic diagnosis instrument before castration (pre-BPH) and 8 weeks after testosterone propionate administration (BPH). (A-D) Representative ultrasound images of the prostates of a pre-BPH dog (A and B) and a testosterone-induced BPH dog (C and D). Line 1, line 2 and line 3 represented the prostate length, width and thickness, respectively. Dog prostate volume = length × width × thickness. The prostate volume (E) and prostate volume to body weight ratio (F) of pre-BPH and testosterone-induced BPH dogs. H&E stains of prostate in pre-BPH and BPH dogs (G and H). (I) Micturition frequency, (J) mean voided volume and (K) total voided volume in the first 2 hours after water load were measured at 8 weeks after the induction of BPH. *P < 0.05 and ***P < 0.001 versus pre-BPH control (Student t test). Scale bar, 50μm.</p

Voiding behavior in testosterone-induced BPH rats.

<p>The rat BPH model was generated by testosterone injections for 4 weeks after castration. Sham: sham operated + olive oil injection; BPH: castration + testosterone propionate injection (dissolved in olive oil, 25 mg/kg, once daily). (A) Prostate (containing the ventral, lateral and dorsal lobes) weight and (B) prostate weight to body weight ratios (PW/BW) of sham and BPH rats. H&E stains of dorsal lobe (C and D) and ventral lobe in shame rats and BPH rats (E and F). (G) Micturition frequency, (H) mean voided volume and (I) total voided volume were measured in sham and BPH rats in the first 2 hours after water load (30 ml/kg). **P < 0.01, ***P < 0.001 versus sham control (Student t test). Scale bar, 50μm.</p

Effects of silodosin on the micturition parameters in BOO rats.

<p>BOO rat model was established by partial ligature of the proximal urethra. Voiding behavior was studied after 4 weeks of recovery. Twenty minutes after oral administration of silodosin, distilled water (30 ml/kg) were given orally. (A) Micturition frequency, (B) mean voided volume and (C) total voided volume were measured for the first 2 hours after water load. ^##P < 0.01 versus sham control (Student t test). *P < 0.05, **P < 0.01 and ***P < 0.001 versus vehicle control (One-way ANOVA test).</p

Effects of silodosin on the micturition parameters in testosterone-induced BPH dogs.

<p>Twenty minutes after oral administration of silodosin, distilled water (30 ml/kg) were given orally. (A) Micturition frequency, (B) mean voided volume and (C) total voided volume in the first 2 hours after water load were measured in a metabolic cage. *P < 0.05, ***P < 0.001 versus vehicle control (One-way ANOVA test).</p