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Regulation of calcium-permeable TRPV2 channel by insulin in pancreatic beta-cells.

Hisanaga E, Nagasawa M, Ueki K, Kulkarni RN, Mori M, Kojima I - Diabetes (2008)

Bottom Line: Calcium-permeable cation channel TRPV2 is expressed in pancreatic beta-cells.In MIN6 cells, TRPV2 was observed mainly in cytoplasm in an unstimulated condition.Likewise, cell growth induced by serum and glucose was inhibited by tranilast or by knockdown of TRPV2.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan.

ABSTRACT

Objective: Calcium-permeable cation channel TRPV2 is expressed in pancreatic beta-cells. We investigated regulation and function of TRPV2 in beta-cells.

Research design and methods: Translocation of TRPV2 was assessed in MIN6 cells and cultured mouse beta-cells by transfecting TRPV2 fused to green fluorescent protein or TRPV2 containing c-Myc tag in the extracellular domain. Calcium entry was assessed by monitoring fura-2 fluorescence.

Results: In MIN6 cells, TRPV2 was observed mainly in cytoplasm in an unstimulated condition. Addition of exogenous insulin induced translocation and insertion of TRPV2 to the plasma membrane. Consistent with these observations, insulin increased calcium entry, which was inhibited by tranilast, an inhibitor of TRPV2, or by knockdown of TRPV2 using shRNA. A high concentration of glucose also induced translocation of TRPV2, which was blocked by nefedipine, diazoxide, and somatostatin, agents blocking glucose-induced insulin secretion. Knockdown of the insulin receptor attenuated insulin-induced translocation of TRPV2. Similarly, the effect of insulin on TRPV2 translocation was not observed in a beta-cell line derived from islets obtained from a beta-cell-specific insulin receptor knockout mouse. Knockdown of TRPV2 or addition of tranilast significantly inhibited insulin secretion induced by a high concentration of glucose. Likewise, cell growth induced by serum and glucose was inhibited by tranilast or by knockdown of TRPV2. Finally, insulin-induced translocation of TRPV2 was observed in cultured mouse beta-cells, and knockdown of TRPV2 reduced insulin secretion induced by glucose.

Conclusions: TRPV2 is regulated by insulin and is involved in the autocrine action of this hormone on beta-cells.

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Assessment of the role of the insulin receptor in insulin-induced translocation of TRPV2. A: Effect of shRNA on the expression of the insulin receptor. MIN6 cells were transfected with Ad-shLacZ or Ad-shIR, and the expression of IR was quantified by real-time RT-PCR. Values are the mean ± SE for four experiments. B: Effect of insulin in shRNA-treated cells. MIN6 cells transfected with Ad-shIR were incubated for 30 min in the absence (a) or presence (b) of 10 nmol/l insulin or 15% FBS (c), and cell surface expression of c-myc–TRPV2 was measured. C: Quantification of the results of B. *P < 0.01 vs. none. D: Effect of insulin and FBS on cell-surface expression of c-myc–TRPV2 in βIRKO cells. βIRKO cells (a–c) and control βWT cells (d–f) expressing c-myc–TRPV2 were incubated for 30 min in the absence (a and d) and presence (b and e) of 10 nmol/l insulin or 15% FBS (c and f), and cell surface expression of c-myc–TRPV2 was measured. E: Quantification of the results of D. a: βIRKO cells. b: Control βWT cells. *P < 0.05 vs. none.
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f4: Assessment of the role of the insulin receptor in insulin-induced translocation of TRPV2. A: Effect of shRNA on the expression of the insulin receptor. MIN6 cells were transfected with Ad-shLacZ or Ad-shIR, and the expression of IR was quantified by real-time RT-PCR. Values are the mean ± SE for four experiments. B: Effect of insulin in shRNA-treated cells. MIN6 cells transfected with Ad-shIR were incubated for 30 min in the absence (a) or presence (b) of 10 nmol/l insulin or 15% FBS (c), and cell surface expression of c-myc–TRPV2 was measured. C: Quantification of the results of B. *P < 0.01 vs. none. D: Effect of insulin and FBS on cell-surface expression of c-myc–TRPV2 in βIRKO cells. βIRKO cells (a–c) and control βWT cells (d–f) expressing c-myc–TRPV2 were incubated for 30 min in the absence (a and d) and presence (b and e) of 10 nmol/l insulin or 15% FBS (c and f), and cell surface expression of c-myc–TRPV2 was measured. E: Quantification of the results of D. a: βIRKO cells. b: Control βWT cells. *P < 0.05 vs. none.

Mentions: We next addressed whether or not the effect of insulin was mediated by IR. We decreased the expression of IR by introducing shRNA. In cells infected with Ad-shIR, the expression of IR was markedly reduced (Fig. 4A). Under this condition, insulin was unable to induce translocation of c-myc–TRPV2 on the cell surface (Fig. 4B, b). In contrast, serum increased the expression of TRPV2 (Fig. 4B, c). Figure 4C shows quantitative analysis of the cell surface expression of c-myc in IR knocked-down cells. We also examined the effect of insulin in βIRKO cells, a cell line derived from β-cells of βIRKO mice. Insulin was unable to increase the expression of c-myc-TRPV2 in the plasma membrane (Fig. 4D, b; Fig. 4E, a), whereas serum significantly increased the cell surface expression of c-myc–TRPV2 (Fig. 4D, c; Fig. 4E, a). Note that insulin increased the expression of c-myc–TRPV2 in the plasma membrane in the control β-cell line βWT (Fig. 4D, e; Fig. 4E, b).


Regulation of calcium-permeable TRPV2 channel by insulin in pancreatic beta-cells.

Hisanaga E, Nagasawa M, Ueki K, Kulkarni RN, Mori M, Kojima I - Diabetes (2008)

Assessment of the role of the insulin receptor in insulin-induced translocation of TRPV2. A: Effect of shRNA on the expression of the insulin receptor. MIN6 cells were transfected with Ad-shLacZ or Ad-shIR, and the expression of IR was quantified by real-time RT-PCR. Values are the mean ± SE for four experiments. B: Effect of insulin in shRNA-treated cells. MIN6 cells transfected with Ad-shIR were incubated for 30 min in the absence (a) or presence (b) of 10 nmol/l insulin or 15% FBS (c), and cell surface expression of c-myc–TRPV2 was measured. C: Quantification of the results of B. *P < 0.01 vs. none. D: Effect of insulin and FBS on cell-surface expression of c-myc–TRPV2 in βIRKO cells. βIRKO cells (a–c) and control βWT cells (d–f) expressing c-myc–TRPV2 were incubated for 30 min in the absence (a and d) and presence (b and e) of 10 nmol/l insulin or 15% FBS (c and f), and cell surface expression of c-myc–TRPV2 was measured. E: Quantification of the results of D. a: βIRKO cells. b: Control βWT cells. *P < 0.05 vs. none.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2606868&req=5

f4: Assessment of the role of the insulin receptor in insulin-induced translocation of TRPV2. A: Effect of shRNA on the expression of the insulin receptor. MIN6 cells were transfected with Ad-shLacZ or Ad-shIR, and the expression of IR was quantified by real-time RT-PCR. Values are the mean ± SE for four experiments. B: Effect of insulin in shRNA-treated cells. MIN6 cells transfected with Ad-shIR were incubated for 30 min in the absence (a) or presence (b) of 10 nmol/l insulin or 15% FBS (c), and cell surface expression of c-myc–TRPV2 was measured. C: Quantification of the results of B. *P < 0.01 vs. none. D: Effect of insulin and FBS on cell-surface expression of c-myc–TRPV2 in βIRKO cells. βIRKO cells (a–c) and control βWT cells (d–f) expressing c-myc–TRPV2 were incubated for 30 min in the absence (a and d) and presence (b and e) of 10 nmol/l insulin or 15% FBS (c and f), and cell surface expression of c-myc–TRPV2 was measured. E: Quantification of the results of D. a: βIRKO cells. b: Control βWT cells. *P < 0.05 vs. none.
Mentions: We next addressed whether or not the effect of insulin was mediated by IR. We decreased the expression of IR by introducing shRNA. In cells infected with Ad-shIR, the expression of IR was markedly reduced (Fig. 4A). Under this condition, insulin was unable to induce translocation of c-myc–TRPV2 on the cell surface (Fig. 4B, b). In contrast, serum increased the expression of TRPV2 (Fig. 4B, c). Figure 4C shows quantitative analysis of the cell surface expression of c-myc in IR knocked-down cells. We also examined the effect of insulin in βIRKO cells, a cell line derived from β-cells of βIRKO mice. Insulin was unable to increase the expression of c-myc-TRPV2 in the plasma membrane (Fig. 4D, b; Fig. 4E, a), whereas serum significantly increased the cell surface expression of c-myc–TRPV2 (Fig. 4D, c; Fig. 4E, a). Note that insulin increased the expression of c-myc–TRPV2 in the plasma membrane in the control β-cell line βWT (Fig. 4D, e; Fig. 4E, b).

Bottom Line: Calcium-permeable cation channel TRPV2 is expressed in pancreatic beta-cells.In MIN6 cells, TRPV2 was observed mainly in cytoplasm in an unstimulated condition.Likewise, cell growth induced by serum and glucose was inhibited by tranilast or by knockdown of TRPV2.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan.

ABSTRACT

Objective: Calcium-permeable cation channel TRPV2 is expressed in pancreatic beta-cells. We investigated regulation and function of TRPV2 in beta-cells.

Research design and methods: Translocation of TRPV2 was assessed in MIN6 cells and cultured mouse beta-cells by transfecting TRPV2 fused to green fluorescent protein or TRPV2 containing c-Myc tag in the extracellular domain. Calcium entry was assessed by monitoring fura-2 fluorescence.

Results: In MIN6 cells, TRPV2 was observed mainly in cytoplasm in an unstimulated condition. Addition of exogenous insulin induced translocation and insertion of TRPV2 to the plasma membrane. Consistent with these observations, insulin increased calcium entry, which was inhibited by tranilast, an inhibitor of TRPV2, or by knockdown of TRPV2 using shRNA. A high concentration of glucose also induced translocation of TRPV2, which was blocked by nefedipine, diazoxide, and somatostatin, agents blocking glucose-induced insulin secretion. Knockdown of the insulin receptor attenuated insulin-induced translocation of TRPV2. Similarly, the effect of insulin on TRPV2 translocation was not observed in a beta-cell line derived from islets obtained from a beta-cell-specific insulin receptor knockout mouse. Knockdown of TRPV2 or addition of tranilast significantly inhibited insulin secretion induced by a high concentration of glucose. Likewise, cell growth induced by serum and glucose was inhibited by tranilast or by knockdown of TRPV2. Finally, insulin-induced translocation of TRPV2 was observed in cultured mouse beta-cells, and knockdown of TRPV2 reduced insulin secretion induced by glucose.

Conclusions: TRPV2 is regulated by insulin and is involved in the autocrine action of this hormone on beta-cells.

Show MeSH
Related in: MedlinePlus