Serotonin receptor 2C and insulin secretion

Abstract

Type 2 diabetes mellitus (T2DM) describes a group of metabolic disorders characterized by defects in insulin secretion and insulin sensitivity. Insulin secretion from pancreatic β-cells is an important factor in the etiology of T2DM, though the complex regulation and mechanisms of insulin secretion from β-cells remains to be fully elucidated. High plasma levels of serotonin (5-hydroxytryptamine; 5-HT) have been reported in T2DM patients, though the potential effect on insulin secretion is unclear. However, it is known that the 5-HT receptor 2C (5-HT(2C)R) agonist, mCPP, decreases plasma insulin concentration in mice. As such, we aimed to investigate the expression of the 5-HT(2C)R in pancreatic islets of diabetic mice and the role of 5-HT(2C)R signaling in insulin secretion from pancreatic β-cells. We found that 5-HT(2C)R expression was significantly increased in pancreatic islets of db/db mice. Furthermore, treatment with a 5-HT(2C)R antagonist (SB242084) increased insulin secretion from pancreatic islets isolated from db/db mice in a dose-dependent manner, but had no effect in islets from control mice. The effect of a 5-HT(2C)R agonist (mCPP) and antagonist (SB242084) were further studied in isolated pancreatic islets from mice and Min-6 cells. We found that mCPP significantly inhibited insulin secretion in Min-6 cells and isolated islets in a dose-dependent manner, which could be reversed by SB242084 or RNA interference against 5-HT(2C)R. We also treated Min-6 cells with palmitic acid for 24 h, and found that the expression of 5-HT(2C)R increased in a dose-dependent manner; furthermore, the inhibition of insulin secretion in Min-6 cells induced by palmitic acid could be reversed by SB242084 or RNA interference against 5-HT(2C)R. Taken together, our data suggests that increased expression of 5-HT(2C)R in pancreatic β-cells might inhibit insulin secretion. This unique observation increases our understanding of T2DM and suggests new avenues for potential treatment.

Figure 1. Expression of 5-HT_2CR in pancreatic islets of db/db mice.

A: mRNA level of 5-HT_2CR was significantly higher in pancreatic islets of db/db mice compared to control mice. B: Protein level of 5-HT_2CR was significantly higher in pancreatic islets of db/db mice compared to control mice. C: Relative protein level of 5-HT_2CR. Graphs are shown as a ratio of 5-HT_2CR to β-actin and compared to controls. All bands were normalized as percentages of the control values. Shown are representative results (average of duplicates) of at least three independent experiments. (^** P<0.01).

Figure 2. Effect of SB242084 on insulin secretion from pancreatic islets of control and db/db mice.

After treatment with 1 to 10 µmol/l of SB242084 for 12 h, insulin secretion increased in pancreatic islets of db/db mice (A), but not in pancreatic islets of control mice (B; islets per well = 8; wells per group = 6; ^** P<0.01).

Figure 3. Effect of mCPP on insulin secretion from pancreatic β-cells.

A: After treatment with 1 to 100 µmol/l mCPP for 12 h, insulin secretion from Min-6 cells under stimulus of 16.7 mM glucose decreased in a mCPP dose-dependent manner (n = 6; ^* P<0.05; ^** P<0.01). B: After treatment with 1 to 100 µmol/l mCPP for 12 h, insulin secretion from isolated mouse pancreatic islets under stimulus of 16.7 mM glucose decreased in a mCPP dose-dependent manner (islets per well = 8; wells per group = 6; ^* P<0.05; ^** P<0.01).

Figure 4. Effect of SB242084 on mCPP-induced inhibition of insulin secretion from pancreatic β-cells.

A: For Min-6 cells, after treatment with 5 to 25 µmol/l mCPP with or without 5 µmol/l SB242084 for 12 h, the groups with SB242085 added showed higher insulin secretion under stimulus of 16.7 mM glucose, compared to control (n = 6; ^* P<0.05; ^** P<0.01). B: For isolated mouse pancreatic islets, after treatment with 5 to 25 µmol/l mCPP with or without 5 µmol/l SB242084 for 12 h, the groups with SB242085 added showed higher insulin secretion under stimulus of 16.7 mM glucose, compared to control (islets per well = 8; wells per group = 6; ^* P<0.05; ^** P<0.01).

Figure 5. Screening of siRNA efficacy.

Min-6 cells were transfected with si5-HT_2CR-1, si5-HT_2CR-2, si5-HT_2CR-3 and control siRNA separately. After 48 h in culture expression of 5-HT_2CR in each group was analyzed at the mRNA (A) and protein level (B). C: Relative protein level of 5-HT_2CR. Graphs are shown as a ratio of 5-HT_2CR to β-actin and compared to controls. All bands were normalized as percentages of the control values. Shown are representative results (average of duplicates) of at least three independent experiments. (^* P<0.05; ^** P<0.01).

Figure 6. Effect of RNA interference of 5-HT_2CR on mCPP-induced inhibition of insulin secretion from Min-6 cells.

Min-6 cells were transfected with si5-HT_2CR-3 or control siRNA. After 36 h in culture cells were treated with 5, 15 or 25 µmol/l mCPP for another 12 h. The si5-HT_2CR-3-treated groups showed higher insulin secretion under stimulus of 16.7 mM glucose compared to control (n = 6; ^* P<0.05; ^** P<0.01).

Figure 7. Effect of palmitic acid on expression of 5-HT_2CR in Min-6 cells.

Min-6 cells were treated with 0.1, 0.2 or 0.3 mM of palmitic acid for 24 h, and then the expression of 5-HT_2CR was analyzed at the mRNA (A) and protein level (B). C: Relative protein level of 5-HT_2CR. Graphs are shown as a ratio of 5-HT_2CR to β-actin and compared to controls. All bands were normalized as percentages of the control values. Shown are representative results (average of duplicates) of at least three independent experiments. (^* P<0.05; ^** P<0.01).

Figure 8. A: Effect of SB242084 on palmitic acid’s inhibition of insulin secretion from Min-6 cells.

Min-6 cells were treated with 0.1 to 0.3 mM palmitic acid for 12 h, and were then treated with vehicle or 5 µmol/l SB242084 for a further 12 h. The groups with SB242084 added showed higher insulin secretion under stimulus of 16.7 mM glucose, compared to control (n = 6; ^** P<0.01). B: Effect of RNA interference of 5-HT_2CR on palmitic acid mediated inhibition of insulin secretion from Min-6 cells. Min-6 cells were transfected with si5-HT_2CR-3 or control siRNA and cultured for 24 h, next 0.1 to 0.3 mM palmitic acid was added for a further 24 h. The si5-HT_2CR-3-treated groups showed higher insulin secretion under stimulus of 16.7 mM glucose compared to control (n = 6; ^** P<0.01).

Figure 9. A: Protein level of TPH in pancreatic islets of db/db mice compared to control mice.

B: Effect of palmitic acid on protein level of TPH in Min-6 cells. Min-6 cells were treated with 0.1, 0.2 or 0.3 mM of palmitic acid for 24 h, and then the expression of TPH were analyzed. C: Relative protein level of TPH1 in (A). D: Relative protein level of TPH2 in (A). E: Relative protein level of TPH1 in (B). F: Relative protein level of TPH2 in (B). G: mRNA level of TPH1 was significantly higher in pancreatic islets of db/db mice compared to control mice. H: mRNA level of TPH2 was significantly higher in pancreatic islets of db/db mice compared to control mice. I: Effect of palmitic acid on mRNA level of TPH1 in Min-6 cells. J: Effect of palmitic acid on mRNA level of TPH2 in Min-6 cells. (^* P<0.05, ^** P<0.01).