A Novel TGR5 Activator WB403 Promotes GLP-1 Secretion and Preserves Pancreatic β-Cells in Type 2 Diabetic Mice

<div><p>The G protein-coupled receptor TGR5 is a membrane receptor for bile acids. Its agonism increases energy expenditure and controls blood glucose through secretion of glucagon-like peptide-1 in enteroendocrine cells. In this study, we explored the therapeutic potential of WB403, a small compound activating TGR5 which was identified by combining TGR5 targeted luciferase assay and active GLP-1 assay, in treating type 2 diabetes. After confirmation of TGR5 and GLP-1 stimulating activities in various cell systems, WB403 was examined in oral glucose tolerance test, and tested on different mouse models of type 2 diabetes for glycemic control and pancreatic β-cell protection effect. As a result, WB403 exhibited a moderate TGR5 activation effect while promoting GLP-1 secretion efficiently. Interestingly, gallbladder filling effect, which was reported for some known TGR5 agonists, was not detected in this novel compound. <i>In vivo</i> results showed that WB403 significantly improved glucose tolerance and decreased fasting blood glucose, postprandial blood glucose and HbA1c in type 2 diabetic mice. Further analysis revealed that WB403 increased pancreatic β-cells and restored the normal distribution pattern of α-cell and β-cell in islets. These findings demonstrated that TGR5 activator WB403 effectively promoted GLP-1 release, improved hyperglycemia and preserved the mass and function of pancreatic β-cells, whereas it did not show a significant side effect on gallbladder. It may represent a promising approach for future type 2 diabetes mellitus drug development.</p></div

WB403 preserved the mass of pancreatic β-cells and normal distribution of α and β-cells.

<p>(A) H&E staining of pancreas from <i>db/db</i> mice, and statistical result. Islets were sized by the Image J analysis software on alternated pancreatic sections spaced each by 100 μm. (B) Immunohistochemical analysis of pancreatic sections by anti-insulin antibody or anti-glucagon antibody. (C) H&E staining of pancreas from HFD/STZ mice, and statistical result. Results are representative islets from each group. 1–5 in the column graph on the right represents normal mice, diabetic mice treated with vehicle, WB403 50 mg/kg, WB403 100 mg/kg, sitagliptin 100 mg/kg respectively (n = 5). *p<0.05, **p<0.01, ***p<0.001 vs. diabetes-vehicle group.</p

Effect of WB403 on GPCRs related to GLP-1 secretion.

<p>(A) GPCR expression in human NCI-H716 and mouse MIN6 cells. In NCI-H716 cells, the amplified fragment is 457bp, 190bp, 452bp, and 290bp for hGPR40, hGPR119, hGPR120, hTGR5 respectively. In MIN6 cells, the amplified fragment is 332bp, 190bp, 470bp, and 190bp for mGpr40, mGpr119, mGpr120, mTgr5 respectively. (B) WB403 at 10, 20 μmol/l had no significant stimulation effect on hGPR119 dependent cAMP accumulation. (C) WB403 at 5, 10, 50 μmol/l had no significant effect on hGPR40-Ca²⁺ activation. (D) WB403 at 10 or 100 μmol/l had no significant effect on hGPR120-Ca²⁺ activation. GSK1292263 (1 μmol/l), TAK875 (0.1 μmol/l) and linoleic acid (LA, 10 μmol/l), TUG891 (10 μmol/l) were used as positive agonist of GPR119, GPR40 and GPR120 respectively. Data are representative of three experiments.</p

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<p>GPR54, Kisspeptin-1 receptor (KISS1R), a member of rhodopsin family, plays a critical role in puberty development and has been proposed to be involved in regulation of energy metabolism. This study aims to explore the function of GPR54 in adipogenesis, lipid metabolism, and obesity in addition to its effect through hormones. Results showed that when fed a high-fat diet, the weight growth of castrated or ovariectomized Gpr54^−/− mice was significantly slower than that of WT control, together with a lower triglyceride concentration. The ratio of white adipose tissue was lower, and average size of adipocytes was smaller in Gpr54^−/− mice. Meanwhile, there were less adipose tissue macrophages (ATMs), especially pro-inflammatory macrophages. Expression of inflammatory related genes also indicated that inflammatory response caused by obesity was not as drastic in Gpr54^−/− mice as in WT mice. Liver triglyceride in Gpr54^−/− mice was reduced, especially in female mice. On the other hand, oil drop formation was accelerated when hepatocytes were stimulated by kisspeptin-10 (Kp-10). Primary mesenchymal stem cells (MSCs) of Gpr54^−/− mice were less likely to differentiate into adipocytes. When stimulated by Kp-10, 3T3-L1 cell differentiation into adipocytes was accelerated and triglyceride synthesis was significantly promoted. These data indicated that GPR54 could affect obesity development by promoting adipocyte differentiation and triglyceride accumulation. To further elucidate the mechanism, genes related to lipid metabolism were analyzed. The expression of genes involved in lipid synthesis including PPARγ, ACC1, ADIPO, and FAS was significantly changed in Gpr54^−/− mice. Among them PPARγ which also participate in adipocyte differentiation displayed a marked reduction. Moreover, phosphorylation of ERK, which involved in GPR54 signaling, was significantly decreased in Gpr54^−/− mice, suggesting that GPR54 may promote lipid synthesis and obesity development by activating MAP kinase pathway. Therefore, in addition to the involvement in hormone regulation, our study demonstrated that GPR54 directly participates in obesity development by promoting adipocyte differentiation and fat accumulation. This provided evidence of involvement of GPR54 in lipid metabolism, and revealed new potentials for the identification and development of novel drug targets for metabolic diseases.</p

WB403 improved hyperglycemia of HFD/STZ mice.

<p>Mice were treated with different concentration of WB403, vehicle or sitagliptin. FBG (A) and PBG (B) were measured every week during the 8-week treatment period. (C, D) HbA1c levels in serum. Values are mean ± SEM (n = 8). *p<0.05, **p<0.01, ***p<0.001 vs. vehicle group.</p

Effects of WB403 on glucose tolerance during OGTT in normal and diabetic mice.

<p>OGTT time course and AUC in normal mice (A, B), HFD/STZ mice (C, D) and <i>db/db</i> mice (E, F). Values represent mean ± SD (n = 10). *p<0.05, **p<0.01, ***p<0.001 vs. vehicle group. Asterisks in A, C and E reflect significance between WB403 100 mg/kg and vehicle group.</p

Effects of WB403 treatment in <i>db/db</i> mice.

<p>(A, B) FBG and PBG measured every week during the 4-week treatment period. (C, D) Serum levels of HbA1c, measured at the end of intervention by Adicon Clinical Laboratories (Shanghai, China) using an immunoturbidimetric assay on Beckman AU680 biochemical analyzer. (E) Average daily food intake per mouse during the treatment period. Data was collected three times per week. (F) Average daily water intake per mouse. (G) Body weight from different groups of mice. (H) Serum levels of insulin were measured at the end of intervention. Values are mean ± SEM (n = 5). *p<0.05, **p<0.01, ***p<0.001 vs. vehicle group.</p

WB403 activated TGR5 and promoted active GLP-1 secretion.

<p>(A) WB403 stimulated hTGR5 in CRE-luciferase report system at the concentration range of 1–20 μmol/l. (B) WB403 stimulated hTGR5 specific cAMP accumulation at the concentration range of 1–50 μmol/l. n = 3. **p<0.01, ***p<0.001 vs. DMSO (+TGR5) group. (C) The hTGR5 targeted CRE-luciferase activity of WB403 and ZY403. EC₅₀ was 5.5 μmol/l and 1.3 μmol/l for WB403 and ZY403 respectively. (D) Effect of WB403 and ZY403 on gallbladders of mice. Normal mice were fasted overnight and treated with compounds (200 mg/kg) or vehicle (DMSO) by ip injection. Gallbladders (GB) were removed 30 min later and volumes measured then normalized to body weight (B.W.). n = 10. **p<0.01 vs. vehicle group. (E) Structure of WB403 and ZY403. WB403 promoted GLP-1 secretion in NCI-H716 cells (F), primary enterocytes (G), and MIN6 cells (H). n = 3. *p<0.05, **p<0.01, ***p<0.001 vs. vehicle group. (I) WB403 promoted GLP-1 in ICR mice. n = 5. **p<0.01 vs. vehicle group. Values represent mean ± SEM.</p