A Single Amino Acid Mutation (R104P) in the E/DRY Motif of GPR40 Impairs Receptor Function.

Type 2 Diabetes Mellitus with insulin resistance, pancreatic β cell dysfunction, and hepatic glucose overproduction is increasing in epidemic proportions worldwide. G protein-coupled receptor 40 (GPR40), a clinically proven anti-diabetic drug target, is mainly expressed in pancreatic β cells and insulin-secreting cell lines. Long chain fatty acids (LCFA) increase intracellular calcium concentration and amplify glucose-stimulated insulin secretion by activating GPR40. Here we report that the arginine 104 (R104) is critical for the normal function of GPR40. Mutation of R104 to Proline (R104P) results in complete loss of the receptor function. Linoleic acid, ligand of GPR40, could not elicit calcium increase and ERK phosphorylation in cells expressing this mutant receptor. Further study indicated the R104P mutation reduces cell surface localization of GPR40 without affecting the expression of the protein. The small portion of GPR40 R104P mutant that is still located on the membrane has no physiological function, and does not internalize in response to linoleic acid stimulation. These data demonstrate that R104 in GPR40 is critically involved in the normal receptor functions. Interestingly, R104P is a registered single-nucleotide polymorphism of GPR40. The relationship of this GPR40 variant and type 2 diabetes warrants further investigation

Testosterone-induced benign prostatic hyperplasia rat and dog as facile models to assess drugs targeting lower urinary tract symptoms.

Benign prostatic hyperplasia (BPH) is an age-related disease, affecting a majority of elderly men worldwide. Medical management of BPH is an alternative to surgical treatment of this disease. Currently, α1-adrenergic receptor (α1-AR) antagonists are among the first line drugs to treat BPH by reducing the tension of urinary track and thus the obstructive symptoms in voiding. In drug development, old male dogs with spontaneous BPH are considered the golden standard of the animal models. However, old dogs (>6 years) are expensive and not all old dogs develop BPH. So it is necessary to develop more accessible animal models for drug efficacy evaluation. Here we describe the development of testosterone-induced BPH models in both rats and young adult dogs and their applications in the in vivo evaluation of α1-AR antagonist. The BPH rats and dogs induced by chronic testosterone treatment have significantly increased micturition frequency and reduced mean voided volume, very similar to the clinical symptoms of BPH patients. Silodosin, an α1-AR antagonist, significantly reduces the urinary frequency and increases the voided volume in BPH model animals in a dose-dependent manner. The results demonstrate that testosterone-induced BPH rat and dog models might provide a more efficient way to evaluate micturition behavior in anti-BPH drug studies

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

Engineering magnetically induced antibacterial organic/inorganic hybrid nanoparticles for the treatment of periodontitis

The global age-standardized prevalence rate of severe periodontitis has already reached 13.1%, which makes it the second most common cause of tooth loss after caries, significantly reducing the quality of life for patients. Due to the complexity in the oral environment, it is practically difficult for conventional antibacterial materials to enter the periodontal pocket directly to eliminate periodontal plaque and inactivate target bacteria, such as Porphyromonas gingivalis (P. gingivalis). To solve this problem, this study proposes a type of antibacterial magnetic nanoparticles (named as AMPs) capable of anchoring polyhexamethylene biguanidine (PHMB) to treat periodontitis through magnetic induction-targeted fixation. According to the results of in vitro antibacterial experiments, AMPs could effectively remove bacterial biofilms by taking advantage of magnetic inductivity and the nanosize effect of nanoparticles, as well as the antibacterial effect of PHMB, with a clearance rate approaching 80%. More importantly, the in vivo experiments conducted on periodontitis demonstrated the disappearance of gingival redness and suppuration after AMPs treatment. As revealed by the quantitative analysis of microCT, different from the periodontitis groups, the growth of alveolar bone was recorded in the AMPs groups. In addition, HE staining confirmed histologically that the AMPs treatment group had an increase in the height of the alveolar ridge and bone mass but a reduction in the infiltration of inflammatory factors, which substantiated the therapeutic effect of AMPs on periodontitis. Therefore, the AMPs proposed in this study are expected to be widely applied as drug carriers intended for the treatment of periodontitis

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 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