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Sweet taste receptor expressed in pancreatic beta-cells activates the calcium and cyclic AMP signaling systems and stimulates insulin secretion.

Nakagawa Y, Nagasawa M, Yamada S, Hara A, Mogami H, Nikolaev VO, Lohse MJ, Shigemura N, Ninomiya Y, Kojima I - PLoS ONE (2009)

Bottom Line: The effect of sucralose on [Ca(2+)](c) was inhibited by gurmarin, an inhibitor of the sweet taste receptor, but not affected by a G(q) inhibitor.Sucralose also induced sustained elevation of [cAMP](c), which was only partially inhibited by removal of extracellular calcium and nifedipine.Sweet taste receptor is expressed in beta-cells, and activation of this receptor induces insulin secretion by Ca(2+) and cAMP-dependent mechanisms.

View Article: PubMed Central - PubMed

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

ABSTRACT

Background: Sweet taste receptor is expressed in the taste buds and enteroendocrine cells acting as a sugar sensor. We investigated the expression and function of the sweet taste receptor in MIN6 cells and mouse islets.

Methodology/principal findings: The expression of the sweet taste receptor was determined by RT-PCR and immunohistochemistry. Changes in cytoplasmic Ca(2+) ([Ca(2+)](c)) and cAMP ([cAMP](c)) were monitored in MIN6 cells using fura-2 and Epac1-camps. Activation of protein kinase C was monitored by measuring translocation of MARCKS-GFP. Insulin was measured by radioimmunoassay. mRNA for T1R2, T1R3, and gustducin was expressed in MIN6 cells. In these cells, artificial sweeteners such as sucralose, succharin, and acesulfame-K increased insulin secretion and augmented secretion induced by glucose. Sucralose increased biphasic increase in [Ca(2+)](c). The second sustained phase was blocked by removal of extracellular calcium and addition of nifedipine. An inhibitor of inositol(1, 4, 5)-trisphophate receptor, 2-aminoethoxydiphenyl borate, blocked both phases of [Ca(2+)](c) response. The effect of sucralose on [Ca(2+)](c) was inhibited by gurmarin, an inhibitor of the sweet taste receptor, but not affected by a G(q) inhibitor. Sucralose also induced sustained elevation of [cAMP](c), which was only partially inhibited by removal of extracellular calcium and nifedipine. Finally, mouse islets expressed T1R2 and T1R3, and artificial sweeteners stimulated insulin secretion.

Conclusions: Sweet taste receptor is expressed in beta-cells, and activation of this receptor induces insulin secretion by Ca(2+) and cAMP-dependent mechanisms.

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Related in: MedlinePlus

Effect of sucralose on [Ca2+]c and [cAMP]c in MIN6 cells.(A) MIN6 cells expressing Epac1-camps were loaded with fura-2, and changes in [Ca2+]c (○) and [cAMP]c (•) were monitored. Cells were stimulated by 50 mM sucralose. Note that 50 mM mannitol did not affect [Ca2+]c or [cAMP]c indicating that the effect of sucralose was not simply due to changes in osmolarity. (B) Dose-response relationship for the effect of sucralose. Cells were stimulated with various concentrations of sucralose, and the area under the curve (AUC) for [Ca2+]c and [cAMP]c was calculated. Values are the mean±S.E. for five experiments. (C) Epac1-camps-expressing cells loaded with fura-2 were stimulated with 25 mM glucose, and changes in [Ca2+]c (○) and [cAMP]c (•) were monitored. (D) Cells were stimulated with 50 µM carbachol and changes in [Ca2+]c (○) and [cAMP]c (•) were monitored.
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pone-0005106-g003: Effect of sucralose on [Ca2+]c and [cAMP]c in MIN6 cells.(A) MIN6 cells expressing Epac1-camps were loaded with fura-2, and changes in [Ca2+]c (○) and [cAMP]c (•) were monitored. Cells were stimulated by 50 mM sucralose. Note that 50 mM mannitol did not affect [Ca2+]c or [cAMP]c indicating that the effect of sucralose was not simply due to changes in osmolarity. (B) Dose-response relationship for the effect of sucralose. Cells were stimulated with various concentrations of sucralose, and the area under the curve (AUC) for [Ca2+]c and [cAMP]c was calculated. Values are the mean±S.E. for five experiments. (C) Epac1-camps-expressing cells loaded with fura-2 were stimulated with 25 mM glucose, and changes in [Ca2+]c (○) and [cAMP]c (•) were monitored. (D) Cells were stimulated with 50 µM carbachol and changes in [Ca2+]c (○) and [cAMP]c (•) were monitored.

Mentions: To investigate the intracellular signaling system activated by the sweet taste receptor, we measured the effects of sucralose on [Ca2+]c and [cAMP]c. We monitored [Ca2+]c and [cAMP]c simultaneously using fura-2 and Epac1-camps. As shown in Figure 3A, sucralose induced increases in both [Ca2+]c and [cAMP]c. Sucralose actually induced biphasic elevation of [Ca2+]c. The initial rapid peak was followed by a gradual decrease in [Ca2+]c, and [Ca2+]c remained slightly elevated for a long period. The effect of sucralose was dose-dependent and detected at a concentration of 10 mM (Figure 3B). Note that the effect of sucralose on [Ca2+]c was not affected by diazoxide, an opener of the ATP-sensitive potassium channel (data not shown). The patterns of changes in [Ca2+]c and [cAMP]c were quite different from that induced by the high concentration of glucose (Figure 3C). As depicted, 25 mM glucose increased [Ca2+]c after a lag period of 2 to 5 min, which was accompanied by an elevation of [cAMP]c. The effect of sucralose was also different from that induced by carbachol, a muscarinic agonist which activates PLC-β. As depicted in Figure 3D, carbachol induced a transient increase in [Ca2+]c. In contrast, [cAMP]c was reduced by carbachol. We then examined the involvement of the sweet taste receptor in the action of sucralose. As shown in Figure 4A, 3 µg/ml gurmarin, an inhibitor of the sweet taste receptor in the taste buds [12] attenuated sucralose-induced elevation of [Ca2+]c. Quantitatively, the effect of gurmarin was statistically significant (Figure 4B). Higher concentration of gurmarin did not further inhibit the sucralose action. Gurmarin did not affect [cAMP]c response to sucralose (data not shown). We then examined the involvement of Gq/11 in the action of sucralose. As shown in Figure 4C, YM254890, an inhibitor of Gq/11 [13], barely inhibited the elevation of [Ca2+]c and [cAMP]c induced by sucralose. In contrast, the effects of carbachol on [Ca2+]c and [cAMP]c were completely blocked by YM254890 (Figure 4D). We then examined the dependency of [Ca2+]c and [cAMP]c responses on extracellular calcium. As shown in Figure 5A, removal of extracellular calcium markedly reduced the [Ca2+]c response to sucralose. In the absence of extracellular calcium, the effect of sucralose on [Ca2+]c was small and only transient. Furthermore, the effect of sucralose on [cAMP]c was attenuated. Similarly, [Ca2+]c response to sucralose was markedly inhibited by nifedipine, an inhibitor of the L-type voltage-dependent calcium channel. Again, nifedipine also reduced elevation of [cAMP]c induced by sucralose (Figure 5B). When extracellular sodium was removed, the effect of sucralose on [Ca2+]c was greatly reduced (Figure 5C). We also examined the effect of an inhibitor of inositol(1,4,5)-trisphosphate (Ins-P3) receptor 2-aminoethoxydiphenyl borate (2APB) [14]. As shown in Figure 5D, 2APB nearly completely blocked sucralose-induced elevation of [Ca2+]c. Both initial and sustained phases of the [Ca2+]c response were blocked. In this condition, [cAMP]c response was also reduced but not blocked completely. Quantitative analyses of the effects of these agents are summarized in Figure 5E. In the taste buds, artificial sweeteners activate PLC-β2 via gustducin, and resultant elevation of [Ca2+]c leads to an activation of TRPM5, which induces sodium entry and depolarizes the plasma membrane [15]. We therefore examined the expression of the channels belonging to the TRPM family. As shown in Figure 5F, TRPM5 was not detected in our experimental condition. Instead, TRPM4, which resembles TRPM5 in many respects, and TRPV2 were expressed.


Sweet taste receptor expressed in pancreatic beta-cells activates the calcium and cyclic AMP signaling systems and stimulates insulin secretion.

Nakagawa Y, Nagasawa M, Yamada S, Hara A, Mogami H, Nikolaev VO, Lohse MJ, Shigemura N, Ninomiya Y, Kojima I - PLoS ONE (2009)

Effect of sucralose on [Ca2+]c and [cAMP]c in MIN6 cells.(A) MIN6 cells expressing Epac1-camps were loaded with fura-2, and changes in [Ca2+]c (○) and [cAMP]c (•) were monitored. Cells were stimulated by 50 mM sucralose. Note that 50 mM mannitol did not affect [Ca2+]c or [cAMP]c indicating that the effect of sucralose was not simply due to changes in osmolarity. (B) Dose-response relationship for the effect of sucralose. Cells were stimulated with various concentrations of sucralose, and the area under the curve (AUC) for [Ca2+]c and [cAMP]c was calculated. Values are the mean±S.E. for five experiments. (C) Epac1-camps-expressing cells loaded with fura-2 were stimulated with 25 mM glucose, and changes in [Ca2+]c (○) and [cAMP]c (•) were monitored. (D) Cells were stimulated with 50 µM carbachol and changes in [Ca2+]c (○) and [cAMP]c (•) were monitored.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2663034&req=5

pone-0005106-g003: Effect of sucralose on [Ca2+]c and [cAMP]c in MIN6 cells.(A) MIN6 cells expressing Epac1-camps were loaded with fura-2, and changes in [Ca2+]c (○) and [cAMP]c (•) were monitored. Cells were stimulated by 50 mM sucralose. Note that 50 mM mannitol did not affect [Ca2+]c or [cAMP]c indicating that the effect of sucralose was not simply due to changes in osmolarity. (B) Dose-response relationship for the effect of sucralose. Cells were stimulated with various concentrations of sucralose, and the area under the curve (AUC) for [Ca2+]c and [cAMP]c was calculated. Values are the mean±S.E. for five experiments. (C) Epac1-camps-expressing cells loaded with fura-2 were stimulated with 25 mM glucose, and changes in [Ca2+]c (○) and [cAMP]c (•) were monitored. (D) Cells were stimulated with 50 µM carbachol and changes in [Ca2+]c (○) and [cAMP]c (•) were monitored.
Mentions: To investigate the intracellular signaling system activated by the sweet taste receptor, we measured the effects of sucralose on [Ca2+]c and [cAMP]c. We monitored [Ca2+]c and [cAMP]c simultaneously using fura-2 and Epac1-camps. As shown in Figure 3A, sucralose induced increases in both [Ca2+]c and [cAMP]c. Sucralose actually induced biphasic elevation of [Ca2+]c. The initial rapid peak was followed by a gradual decrease in [Ca2+]c, and [Ca2+]c remained slightly elevated for a long period. The effect of sucralose was dose-dependent and detected at a concentration of 10 mM (Figure 3B). Note that the effect of sucralose on [Ca2+]c was not affected by diazoxide, an opener of the ATP-sensitive potassium channel (data not shown). The patterns of changes in [Ca2+]c and [cAMP]c were quite different from that induced by the high concentration of glucose (Figure 3C). As depicted, 25 mM glucose increased [Ca2+]c after a lag period of 2 to 5 min, which was accompanied by an elevation of [cAMP]c. The effect of sucralose was also different from that induced by carbachol, a muscarinic agonist which activates PLC-β. As depicted in Figure 3D, carbachol induced a transient increase in [Ca2+]c. In contrast, [cAMP]c was reduced by carbachol. We then examined the involvement of the sweet taste receptor in the action of sucralose. As shown in Figure 4A, 3 µg/ml gurmarin, an inhibitor of the sweet taste receptor in the taste buds [12] attenuated sucralose-induced elevation of [Ca2+]c. Quantitatively, the effect of gurmarin was statistically significant (Figure 4B). Higher concentration of gurmarin did not further inhibit the sucralose action. Gurmarin did not affect [cAMP]c response to sucralose (data not shown). We then examined the involvement of Gq/11 in the action of sucralose. As shown in Figure 4C, YM254890, an inhibitor of Gq/11 [13], barely inhibited the elevation of [Ca2+]c and [cAMP]c induced by sucralose. In contrast, the effects of carbachol on [Ca2+]c and [cAMP]c were completely blocked by YM254890 (Figure 4D). We then examined the dependency of [Ca2+]c and [cAMP]c responses on extracellular calcium. As shown in Figure 5A, removal of extracellular calcium markedly reduced the [Ca2+]c response to sucralose. In the absence of extracellular calcium, the effect of sucralose on [Ca2+]c was small and only transient. Furthermore, the effect of sucralose on [cAMP]c was attenuated. Similarly, [Ca2+]c response to sucralose was markedly inhibited by nifedipine, an inhibitor of the L-type voltage-dependent calcium channel. Again, nifedipine also reduced elevation of [cAMP]c induced by sucralose (Figure 5B). When extracellular sodium was removed, the effect of sucralose on [Ca2+]c was greatly reduced (Figure 5C). We also examined the effect of an inhibitor of inositol(1,4,5)-trisphosphate (Ins-P3) receptor 2-aminoethoxydiphenyl borate (2APB) [14]. As shown in Figure 5D, 2APB nearly completely blocked sucralose-induced elevation of [Ca2+]c. Both initial and sustained phases of the [Ca2+]c response were blocked. In this condition, [cAMP]c response was also reduced but not blocked completely. Quantitative analyses of the effects of these agents are summarized in Figure 5E. In the taste buds, artificial sweeteners activate PLC-β2 via gustducin, and resultant elevation of [Ca2+]c leads to an activation of TRPM5, which induces sodium entry and depolarizes the plasma membrane [15]. We therefore examined the expression of the channels belonging to the TRPM family. As shown in Figure 5F, TRPM5 was not detected in our experimental condition. Instead, TRPM4, which resembles TRPM5 in many respects, and TRPV2 were expressed.

Bottom Line: The effect of sucralose on [Ca(2+)](c) was inhibited by gurmarin, an inhibitor of the sweet taste receptor, but not affected by a G(q) inhibitor.Sucralose also induced sustained elevation of [cAMP](c), which was only partially inhibited by removal of extracellular calcium and nifedipine.Sweet taste receptor is expressed in beta-cells, and activation of this receptor induces insulin secretion by Ca(2+) and cAMP-dependent mechanisms.

View Article: PubMed Central - PubMed

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

ABSTRACT

Background: Sweet taste receptor is expressed in the taste buds and enteroendocrine cells acting as a sugar sensor. We investigated the expression and function of the sweet taste receptor in MIN6 cells and mouse islets.

Methodology/principal findings: The expression of the sweet taste receptor was determined by RT-PCR and immunohistochemistry. Changes in cytoplasmic Ca(2+) ([Ca(2+)](c)) and cAMP ([cAMP](c)) were monitored in MIN6 cells using fura-2 and Epac1-camps. Activation of protein kinase C was monitored by measuring translocation of MARCKS-GFP. Insulin was measured by radioimmunoassay. mRNA for T1R2, T1R3, and gustducin was expressed in MIN6 cells. In these cells, artificial sweeteners such as sucralose, succharin, and acesulfame-K increased insulin secretion and augmented secretion induced by glucose. Sucralose increased biphasic increase in [Ca(2+)](c). The second sustained phase was blocked by removal of extracellular calcium and addition of nifedipine. An inhibitor of inositol(1, 4, 5)-trisphophate receptor, 2-aminoethoxydiphenyl borate, blocked both phases of [Ca(2+)](c) response. The effect of sucralose on [Ca(2+)](c) was inhibited by gurmarin, an inhibitor of the sweet taste receptor, but not affected by a G(q) inhibitor. Sucralose also induced sustained elevation of [cAMP](c), which was only partially inhibited by removal of extracellular calcium and nifedipine. Finally, mouse islets expressed T1R2 and T1R3, and artificial sweeteners stimulated insulin secretion.

Conclusions: Sweet taste receptor is expressed in beta-cells, and activation of this receptor induces insulin secretion by Ca(2+) and cAMP-dependent mechanisms.

Show MeSH
Related in: MedlinePlus