Serotonin Influences Insulin Secretion in Rat Insulinoma INS-1E Cells

Abstract

Serotonin or 5-hydroxytryptamine (5-HT) is a monoamine that plays a critical role in insulin secretion, energy metabolism, and mitochondrial biogenesis. However, the action of serotonin in insulin production and secretion by pancreatic β cells has not yet been elucidated. Here, we investigated how exogenous nanomolar serotonin concentrations regulate insulin synthesis and secretion in rat insulinoma INS-1E cells. Nanomolar serotonin concentrations (10 and 50 nM) significantly increased insulin protein expression above the constant levels in untreated control cells and decreased insulin protein levels in the media. The reductions in insulin protein levels in the media may be associated with ubiquitin-mediated protein degradation. The levels of membrane vesicle trafficking-related proteins including Rab5, Rab3A, syntaxin6, clathrin, and EEA1 proteins were significantly decreased by serotonin treatment compared to the untreated control cells, whereas the expressions of Rab27A, GOPC, and p-caveolin-1 proteins were significantly reduced by serotonin treatment. In this condition, serotonin receptors, Gαq-coupled 5-HT2b receptor (Htr2b), and ligand-gated ion channel receptor Htr3a were significantly decreased by serotonin treatment. To confirm the serotonylation of Rab3A and Rab27A during insulin secretion, we investigated the protein levels of Rab3A and Rab27A, in which transglutaminase 2 (TGase2) serotonylated Rab3A but not Rab27A. The increases in ERK phosphorylation levels were consistent with increases in the expression of p-Akt. Also, the expression level of the Bcl-2 protein was significantly increased by 50 and 100 nM serotonin treatment compared to the untreated control cells, whereas the levels of Cu/Zn-SOD and Mn-SOD proteins decreased. These results indicate that nanomolar serotonin treatment regulates the insulin protein level but decreases this level in media through membrane vesicle trafficking-related proteins (Rab5, Rab3A, syntaxin6, clathrin, and EEA1), the Akt/ERK pathway, and Htr2b/Htr3a in INS-1E cells.

Keywords: Akt/ERK; insulin; serotonin; serotonin receptor.

Figure 1

The expression of insulin protein in cells and media in rat insulinoma INS-1E cells treated with serotonin. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% serotonin for 48 and 72 h at 37 °C with 5% CO₂. Insulin protein, LC3, and ubiquitin were then analyzed by Western blot (A). The relative amounts of insulin protein (B) and ubiquitinylation (C) were quantified as described in Materials and Methods. Data represent mean ± SD of three experiments. * p < 0.05, *** p < 0.001 vs. without serotonin in cells; ## p < 0.01, ### p < 0.001 vs. without serotonin in media; &&& p < 0.001, insulin in cells vs. insulin in media.

Figure 2

Levels of membrane vesicle trafficking-related proteins including Rab5, Rab3a, APPL1, syntaxin 6, clathrin, EEA1, Rab27a, GOPC, and p-caveolin-1 in rat insulinoma INS-1E cells treated with serotonin. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% serotonin for 48 h at 37 °C with 5% CO₂. Proteins were then analyzed by Western blot (A). The expressions of membrane vesicle trafficking-related proteins were then analyzed by Western blot (A). The relative amounts of Rab5 (B), Rab3a (C), APPL1 (D), syntaxin6 (E), clathrin (F), EEA1 (G), Rab27a (H), GOPC (I), and p-caveolin-1 (J), were quantified as described in Materials and Methods. Data represent mean ± SD of three experiments. ** p < 0.01, *** p < 0.001 vs. without serotonin in cells.

Figure 3

Levels of serotonin receptors Htr1d, Htr2b, and Htr3a in rat insulinoma INS-1E cells treated with serotonin. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% serotonin for 48 h at 37 °C with 5% CO₂. The expressions of Htr1d, Htr2b, and Htr3a proteins were then analyzed by Western blot (A). The relative amounts of Htr1d (B), Htr2b (C), and Htr3a (D) were quantified as described in Materials and Methods. Data represent mean ± SD of three experiments. ** p < 0.01, *** p < 0.001 vs. without serotonin.

Figure 4

The level of TGase2 protein and the serotonylation of Rab3a and Rab27a in rat insulinoma INS-1E cells. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% FBS with serotonin for 48 h at 37 °C with 5% CO₂. TGase2 protein and the serotonylation of Rab3a and Rab27a were then analyzed by Western blot (A). The relative amounts of TGase2 protein (B) and the serotonylation of Rab3a (C) and Rab27a (D) were quantified as described in Materials and Methods. Data represent mean ± SD of three experiments. * p < 0.05, *** p < 0.001 vs. without serotonin in no serotonylation condition; ### p < 0.001 vs. without serotonin in serotonylation condition; &&& p < 0.001, no serotonylation condition vs. serotonylation condition. Arrows are the serotonylation of Rab3a.

Figure 4

The level of TGase2 protein and the serotonylation of Rab3a and Rab27a in rat insulinoma INS-1E cells. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% FBS with serotonin for 48 h at 37 °C with 5% CO₂. TGase2 protein and the serotonylation of Rab3a and Rab27a were then analyzed by Western blot (A). The relative amounts of TGase2 protein (B) and the serotonylation of Rab3a (C) and Rab27a (D) were quantified as described in Materials and Methods. Data represent mean ± SD of three experiments. * p < 0.05, *** p < 0.001 vs. without serotonin in no serotonylation condition; ### p < 0.001 vs. without serotonin in serotonylation condition; &&& p < 0.001, no serotonylation condition vs. serotonylation condition. Arrows are the serotonylation of Rab3a.

Figure 5

The phosphorylations of Akt and ERK in serotonin-treated rat insulinoma INS-1E cells. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% FBS with serotonin for 48 h at 37 °C with 5% CO₂. p-Akt and p-ERK expressions were then analyzed by Western blot (A). The relative amounts of p-Akt (B) and p-ERK (C) were quantified as described in Materials and Methods. Data represent mean ± SD of three experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. without serotonin.

Figure 6

The expression of Bcl-2, Bax, Cu/Zn-SOD, Mn-SOD, and catalase proteins in rat insulinoma INS-1E cells treated with serotonin. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% FBS with serotonin for 48 h at 37 °C with 5% CO₂. Bcl-2, Bax, Cu/Zn-SOD, Mn-SOD, and catalase proteins were then analyzed by Western blot (A). The relative amounts of Bcl-2 (B), Bax (C), Cu/Zn-SOD (D), Mn-SOD (E), and catalase (F) proteins were quantified as described in Materials and Methods. Data represent mean ± SD of three experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. without serotonin.