Thyroid stimulating hormone stimulates the expression of glucose transporter 2 via its receptor in pancreatic β cell line, INS-1 cells

Abstract

Thyroid stimulating hormone (TSH) stimulates the secretion of thyroid hormones by binding the TSH receptor (TSHR). TSHR is well-known to be expressed in thyroid tissue, excepting it, TSHR has also been expressed in many other tissues. In this study, we have examined the expression of TSHR in rat pancreatic islets and evaluated the role of TSH in regulating pancreas-specific gene expression. TSHR was confirmed to be expressed in rodent pancreatic islets and its cell line, INS-1 cells. TSH directly affected the glucose uptake in INS cells by up-regulating the expression of GLUT2, and furthermore this process was blocked by SB203580, the specific inhibitor of the p38 MAPK signaling pathway. Similarly, TSH stimulated GLUT2 promoter activity, while both a dominant-negative p38MAPK α isoform (p38MAPK α-DN) and the specific inhibitor for p38MAPK α abolished the stimulatory effect of TSH on GLUT2 promoter activity. Finally, INS-1 cells treated with TSH showed increased protein level of glucokinase and enhanced glucose-stimulated insulin secretion. Together, these results confirm that TSHR is expressed in INS-1 cells and rat pancreatic islets, and suggest that activation of the p38MAPK α might be required for TSH-induced GLUT2 gene transcription in pancreatic β cells.

Figure 1

Characterization of TSHR expression in rat pancreatic islets and in INS-1 cells. (a–c) Protein isolated from rat thyroid, pancreas, pancreatic islet or INS-1 cells (shown on the top of each band) was detected by TSHR α antibody. Full-length blots are presented in Supplementary Figure 1. (d) By using TSHR specific primer, PCR products were generated based on template cDNA isolated from rat pancreatic islets and two dishes of INS-1. (e) Immunocytochemistry of INS-1 cells with the TSHR α subunit antibody (green). (f) Immunohistochemistry of TSHR and insulin (brown) in primary pancreatic islets from the rat. Bar = 20 µm.

Figure 2

Roles of TSH in the expression of GLUT2 in pancreatic β cells. (a), TSH increases the abundance of GLUT2 protein in a dose-dependent manner. The ratio of GLUT2 to GAPDH is shown as a percentage of the control ratio. Full-length blots are presented in Supplementary Figure 2. (b) and (c), The effect of TSH at 100 µIU/ml on GLUT2 mRNA expression by real-time PCR in INS-1 cells (b) and pancreatic islets (c). GAPDH is used as a control. A graph showing the mean ± SEM (n = 3) of separate experiments for each treatment group is indicated. The *denotes a significant difference (P < 0.05) compared to 0.

Figure 3

Roles of TSH in the promoter activity of GLUT2 in INS-1 cells. (a), TSH increases the promoter activity of GLUT2 in a dose-dependent manner. (b) Effects of a PKA inhibitor H-89 (H-89), a p38 MAPK inhibitor SB203580 (SB), or a CaMKK inhibitor STO609 (STO609) on TSH (100 µIU/ml)-induced GLUT2 promoter activity. (c) A plasmid containing pcDNA (empty vector), p38 MAPK or p38 MAPK-domain negative (p38 MAPK-DN) DNA was co-transfected into INS-1 cells, along with a plasmid containing the GLUT2 promoter. The cells were then treated with or without TSH at 100 µIU/ml for 24 h. (d) A plasmid containing pcDNA (empty vector) or DNA encoding different domain negative subunits (α-DN, β-DN, or γ-DN) of p38 MAPK was each separately co-transfected into INS-1 cells, along with a plasmid containing the GLUT2 promoter. (e) Effects of the α subunit of p38 MAPK inhibitor SCIO469 (SCIO469) on TSH (100 µIU/ml)-induced GLUT2 promoter activity. Percentage of promoter activity is relative to control activity level (0, pcDNA or DMSO without TSH), and is shown as the mean ± SEM (n = 3) of separate experiments in the graph. The *denotes a significant difference (P < 0.05) compared to 0, DMSO or pcDNA and the #denotes a significant difference (P < 0.05) compared to pcDNA plus TSH or DMSO plus TSH.

Figure 4

Inhibition of p38-MAPK blocks the effect of TSH on GLUT2 expression in pancreatic β cells a and c, protein expression of GLUT2 in INS-1 cells. (a) Or pancreatic islets (c) which were pre-treated with a p38 MAPK inhibitor SB203580 (SB) or a p38 MAPK inhibitor SCIO469 (SCIO) for 30 min after 6 h starvation and then were incubated with TSH for 24 h. The ratio of GLUT2 to GAPDH is shown as a percentage of the control ratio. (b) The effect of SB203580 or SCIO469 on mRNA expression of GLUT2 induced by TSH. GAPDH is used as a control. A graph showing the mean ± SEM (n = 3) of separate experiments for each treatment group is indicated. The *denotes a significant difference (P < 0.05) compared to DMSO; The #denotes a significant difference (P < 0.05) compared to DMSO plus TSH.

Figure 5

The effect of TSH on glucose uptake in INS-1 cells. After 6 h starvation, INS-1 cells were incubated with DMSO or an inhibitor of p38 MAPK (SB203580) for 30 min, and following treated by TSH at 100 µIU/ml for 24 h. Then the glucose uptake (green) was checked as per the manufacturer’s instruction. NC-Pal served as the negative control and cells treated with DMSO were used as a positive control. The magnification of third and fourth rows are 400 times while the magnification of first and second rows are 200 times. Bar = 40 µm.

Figure 6

The effect of TSH on the expression of glucokinase and insulin in pancreatic β cells. TSH increases the abundance of glucokinase (GK) protein (a) and insulin (b) in a dose-dependent manner. The ratio of GK or insulin to GAPDH is shown as a percentage of the control ratio. Full-length blots are presented in Supplementary Figure 3. c, e and g, glucose-stimulated insulin secretion (GSIS) in INS-1 cells treated with TSH (c) or SB + TSH (g) or in pancreatic islets treated with TSH (e). d, f and h, fold of insulin secretion from low glucose (3.3 mM) to high glucose (16.7 mM) in INS-1 cells treated with TSH (d) or SB + TSH (f) or in pancreatic islets treated with TSH (f). A graph showing the mean ± SEM (n = 3) of separate experiments for each treatment group is indicated. The *denotes a significant difference (P < 0.05) compared to 0.