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Enhanced glucose tolerance by SK4 channel inhibition in pancreatic beta-cells.

Düfer M, Gier B, Wolpers D, Krippeit-Drews P, Ruth P, Drews G - Diabetes (2009)

Bottom Line: SK4 channels were found to substantially contribute to K(slow) (slowly activating K(+) current).Deficiency of SK4 current induces elevated beta-cell responsiveness and coincides with improved glucose tolerance in vivo.Therefore, pharmacologic modulation of these channels might provide an interesting approach for the development of novel insulinotropic drugs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacy, the Department of Pharmacology, University of Tübingen, Tübingen, Germany.

ABSTRACT

Objective: Ca(2+)-regulated K(+) channels are involved in numerous Ca(2+)-dependent signaling pathways. In this study, we investigated whether the Ca(2+)-activated K(+) channel of intermediate conductance SK4 (KCa3.1, IK1) plays a physiological role in pancreatic beta-cell function.

Research design and methods: Glucose tolerance and insulin sensitivity were determined in wild-type (WT) or SK4 knockout (SK4-KO) mice. Electrophysiological experiments were performed with the patch-clamp technique. The cytosolic Ca(2+) concentration ([Ca(2+)](c)) was determined by fura-2 fluorescence. Insulin release was assessed by radioimmunoassay, and SK4 protein was detected by Western blot analysis.

Results: SK4-KO mice showed improved glucose tolerance, whereas insulin sensitivity was not altered. The animals were not hypoglycemic. Isolated SK4-KO beta-cells stimulated with 15 mmol/l glucose had an increased Ca(2+) action potential frequency, and single-action potentials were broadened. These alterations were coupled to increased [Ca(2+)](c). In addition, glucose responsiveness of membrane potential, [Ca(2+)](c), and insulin secretion were shifted to lower glucose concentrations. SK4 protein was expressed in WT islets. An increase in K(+) currents and concomitant membrane hyperpolarization could be evoked in WT beta-cells by the SK4 channel opener DCEBIO (100 micromol/l). Accordingly, the SK4 channel blocker TRAM-34 (1 micromol/l) partly inhibited K(Ca) currents and induced electrical activity at a threshold glucose concentration. In stimulated WT beta-cells, TRAM-34 further increased [Ca(2+)](c) and broadened action potentials similar to those seen in SK4-KO beta-cells. SK4 channels were found to substantially contribute to K(slow) (slowly activating K(+) current).

Conclusions: SK4 channels are involved in beta-cell stimulus-secretion coupling. Deficiency of SK4 current induces elevated beta-cell responsiveness and coincides with improved glucose tolerance in vivo. Therefore, pharmacologic modulation of these channels might provide an interesting approach for the development of novel insulinotropic drugs.

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Glucose responsiveness of SK4-KO β-cells is shifted to lower glucose concentrations. A: β-Cells were stimulated with either 6 or 8 mmol/l glucose, respectively. The diagram illustrates the fraction of cells showing Ca2+ action potentials in response to the indicated glucose concentration. In this series of experiments, 12 WT and 15 SK4-KO β-cells were tested. B: The concentration-response curve was determined by perifusing isolated β-cells with different glucose concentrations. Cells in which [Ca2+]c increased or displayed oscillations within 15 min of perifusion were regarded as glucose responsive. The number of cells tested with each glucose concentration was as follows (WT/SK4-KO): 0.5 mmol/l glucose, 49 WT/55 SK4-KO cells; 3 mmol/l glucose, 46/51; 5 mmol/l glucose, 49/54; 6 mmol/l glucose, 74/73; 8 mmol/l glucose, 56/68; 15 mmol/l glucose, 16/36. The cells were obtained from preparations of three to nine animals per condition. To avoid overlapping, the data points for WT and SK4-KO β-cells are shifted to left and right within the graph. C: Insulin secretion was compared in islets incubated with 3 or 6 mmol/l glucose (6 G) for 1 h. The diagram shows the percentage of islet preparations with significant increase in insulin release by 6 mmol/l glucose (eight independent preparations for each genotype). D and E: TRAM-34 induces electrical activity in WT β-cells treated with substimulatory glucose concentrations but not in SK4-KO β-cells. In this series of experiments, glucose concentration was lowered from 10 to 6 mmol/l or 5 mmol/l glucose. After termination of electrical activity, TRAM-34 (1 μmol/l) was added and action potentials reoccurred in four of five WT cells tested. In SK4-KO β-cells, TRAM-34 was without depolarizing effect. The experiment is representative of three.
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Figure 3: Glucose responsiveness of SK4-KO β-cells is shifted to lower glucose concentrations. A: β-Cells were stimulated with either 6 or 8 mmol/l glucose, respectively. The diagram illustrates the fraction of cells showing Ca2+ action potentials in response to the indicated glucose concentration. In this series of experiments, 12 WT and 15 SK4-KO β-cells were tested. B: The concentration-response curve was determined by perifusing isolated β-cells with different glucose concentrations. Cells in which [Ca2+]c increased or displayed oscillations within 15 min of perifusion were regarded as glucose responsive. The number of cells tested with each glucose concentration was as follows (WT/SK4-KO): 0.5 mmol/l glucose, 49 WT/55 SK4-KO cells; 3 mmol/l glucose, 46/51; 5 mmol/l glucose, 49/54; 6 mmol/l glucose, 74/73; 8 mmol/l glucose, 56/68; 15 mmol/l glucose, 16/36. The cells were obtained from preparations of three to nine animals per condition. To avoid overlapping, the data points for WT and SK4-KO β-cells are shifted to left and right within the graph. C: Insulin secretion was compared in islets incubated with 3 or 6 mmol/l glucose (6 G) for 1 h. The diagram shows the percentage of islet preparations with significant increase in insulin release by 6 mmol/l glucose (eight independent preparations for each genotype). D and E: TRAM-34 induces electrical activity in WT β-cells treated with substimulatory glucose concentrations but not in SK4-KO β-cells. In this series of experiments, glucose concentration was lowered from 10 to 6 mmol/l or 5 mmol/l glucose. After termination of electrical activity, TRAM-34 (1 μmol/l) was added and action potentials reoccurred in four of five WT cells tested. In SK4-KO β-cells, TRAM-34 was without depolarizing effect. The experiment is representative of three.

Mentions: Neither SK4-KO nor TRAM-34 influenced the resting membrane potential, which was −77 ± 1 mV in 0.5 mmol/l glucose and −76 ± 1 mV with TRAM-34 (1 μmol/l, n = 3) compared with −75 ± 1 mV in SK4-KO β-cells (n = 7, not shown). To find out whether ablation of SK4 channels affects glucose responsiveness, we investigated whether stimulation of SK4-KO β-cells was shifted to lower glucose concentrations. Cells were perifused with bath solution containing 6 or 8 mmol/l glucose. In WT β-cells, no electrical activity was observed with 6 mmol/l glucose (n = 7), whereas 37.5% of the cells were depolarized and Ca2+ APs occurred with 8 mmol/l glucose (n = 8). By contrast, in SK4-KO mice, 63.6% of the β-cells were already stimulated by 6 mmol/l glucose (n = 11) and all cells (100%) by 8 mmol/l glucose (n = 5) (Fig. 3A). Consistent with the higher fraction of electrically active β-cells, we observed a significant left shift of the glucose concentration–response curve of [Ca2+]c in SK4-KO versus WT β-cells (Fig. 3B). In these experiments, isolated β-cells were perifused with bath solutions containing 0.5–15 mmol/l glucose. Cells were considered to be glucose responsive if they displayed an increase in [Ca2+]c and/or Ca2+ oscillations. The D50 value (50% probability for glucose responsiveness) was 6.37 mmol/l (95% CI 6.09–6.68) for WT β-cells and was reduced to 5.67 mmol/l (5.29–6.05) for SK4-KO β-cells. SK4-KO also affected insulin secretion. Islets were incubated in 3, 6, or 8 mmol/l glucose for 60 min. WT and SK4-KO islets had similar insulin content (WT: 29 ± 3 ng/islet; SK4-KO: 29 ± 1 ng/islet, n = 8 different preparations for both genotypes), and there was no significant change in insulin release under basal conditions (3 mmol/l glucose) (WT: 33 ± 7 pg/[islet h], SK4-KO: 33 ± 10 pg/[islet h], n = 8 for both genotypes). Compared with basal secretion in 3 mmol/l, glucose stimulation of secretion occurred in all experiments when glucose was elevated to 8 mmol/l irrespective of the genotype (n = 8). However, in agreement with a left shift in glucose responsiveness of Vm and [Ca2+]c, only 38% of the WT but 75% of the SK4-KO islet preparations displayed an increase in secretion with 6 mmol/l glucose (Fig. 3C). These data clearly demonstrate that genetic ablation of SK4 channels sensitizes the β-cells to glucose stimulation.


Enhanced glucose tolerance by SK4 channel inhibition in pancreatic beta-cells.

Düfer M, Gier B, Wolpers D, Krippeit-Drews P, Ruth P, Drews G - Diabetes (2009)

Glucose responsiveness of SK4-KO β-cells is shifted to lower glucose concentrations. A: β-Cells were stimulated with either 6 or 8 mmol/l glucose, respectively. The diagram illustrates the fraction of cells showing Ca2+ action potentials in response to the indicated glucose concentration. In this series of experiments, 12 WT and 15 SK4-KO β-cells were tested. B: The concentration-response curve was determined by perifusing isolated β-cells with different glucose concentrations. Cells in which [Ca2+]c increased or displayed oscillations within 15 min of perifusion were regarded as glucose responsive. The number of cells tested with each glucose concentration was as follows (WT/SK4-KO): 0.5 mmol/l glucose, 49 WT/55 SK4-KO cells; 3 mmol/l glucose, 46/51; 5 mmol/l glucose, 49/54; 6 mmol/l glucose, 74/73; 8 mmol/l glucose, 56/68; 15 mmol/l glucose, 16/36. The cells were obtained from preparations of three to nine animals per condition. To avoid overlapping, the data points for WT and SK4-KO β-cells are shifted to left and right within the graph. C: Insulin secretion was compared in islets incubated with 3 or 6 mmol/l glucose (6 G) for 1 h. The diagram shows the percentage of islet preparations with significant increase in insulin release by 6 mmol/l glucose (eight independent preparations for each genotype). D and E: TRAM-34 induces electrical activity in WT β-cells treated with substimulatory glucose concentrations but not in SK4-KO β-cells. In this series of experiments, glucose concentration was lowered from 10 to 6 mmol/l or 5 mmol/l glucose. After termination of electrical activity, TRAM-34 (1 μmol/l) was added and action potentials reoccurred in four of five WT cells tested. In SK4-KO β-cells, TRAM-34 was without depolarizing effect. The experiment is representative of three.
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Figure 3: Glucose responsiveness of SK4-KO β-cells is shifted to lower glucose concentrations. A: β-Cells were stimulated with either 6 or 8 mmol/l glucose, respectively. The diagram illustrates the fraction of cells showing Ca2+ action potentials in response to the indicated glucose concentration. In this series of experiments, 12 WT and 15 SK4-KO β-cells were tested. B: The concentration-response curve was determined by perifusing isolated β-cells with different glucose concentrations. Cells in which [Ca2+]c increased or displayed oscillations within 15 min of perifusion were regarded as glucose responsive. The number of cells tested with each glucose concentration was as follows (WT/SK4-KO): 0.5 mmol/l glucose, 49 WT/55 SK4-KO cells; 3 mmol/l glucose, 46/51; 5 mmol/l glucose, 49/54; 6 mmol/l glucose, 74/73; 8 mmol/l glucose, 56/68; 15 mmol/l glucose, 16/36. The cells were obtained from preparations of three to nine animals per condition. To avoid overlapping, the data points for WT and SK4-KO β-cells are shifted to left and right within the graph. C: Insulin secretion was compared in islets incubated with 3 or 6 mmol/l glucose (6 G) for 1 h. The diagram shows the percentage of islet preparations with significant increase in insulin release by 6 mmol/l glucose (eight independent preparations for each genotype). D and E: TRAM-34 induces electrical activity in WT β-cells treated with substimulatory glucose concentrations but not in SK4-KO β-cells. In this series of experiments, glucose concentration was lowered from 10 to 6 mmol/l or 5 mmol/l glucose. After termination of electrical activity, TRAM-34 (1 μmol/l) was added and action potentials reoccurred in four of five WT cells tested. In SK4-KO β-cells, TRAM-34 was without depolarizing effect. The experiment is representative of three.
Mentions: Neither SK4-KO nor TRAM-34 influenced the resting membrane potential, which was −77 ± 1 mV in 0.5 mmol/l glucose and −76 ± 1 mV with TRAM-34 (1 μmol/l, n = 3) compared with −75 ± 1 mV in SK4-KO β-cells (n = 7, not shown). To find out whether ablation of SK4 channels affects glucose responsiveness, we investigated whether stimulation of SK4-KO β-cells was shifted to lower glucose concentrations. Cells were perifused with bath solution containing 6 or 8 mmol/l glucose. In WT β-cells, no electrical activity was observed with 6 mmol/l glucose (n = 7), whereas 37.5% of the cells were depolarized and Ca2+ APs occurred with 8 mmol/l glucose (n = 8). By contrast, in SK4-KO mice, 63.6% of the β-cells were already stimulated by 6 mmol/l glucose (n = 11) and all cells (100%) by 8 mmol/l glucose (n = 5) (Fig. 3A). Consistent with the higher fraction of electrically active β-cells, we observed a significant left shift of the glucose concentration–response curve of [Ca2+]c in SK4-KO versus WT β-cells (Fig. 3B). In these experiments, isolated β-cells were perifused with bath solutions containing 0.5–15 mmol/l glucose. Cells were considered to be glucose responsive if they displayed an increase in [Ca2+]c and/or Ca2+ oscillations. The D50 value (50% probability for glucose responsiveness) was 6.37 mmol/l (95% CI 6.09–6.68) for WT β-cells and was reduced to 5.67 mmol/l (5.29–6.05) for SK4-KO β-cells. SK4-KO also affected insulin secretion. Islets were incubated in 3, 6, or 8 mmol/l glucose for 60 min. WT and SK4-KO islets had similar insulin content (WT: 29 ± 3 ng/islet; SK4-KO: 29 ± 1 ng/islet, n = 8 different preparations for both genotypes), and there was no significant change in insulin release under basal conditions (3 mmol/l glucose) (WT: 33 ± 7 pg/[islet h], SK4-KO: 33 ± 10 pg/[islet h], n = 8 for both genotypes). Compared with basal secretion in 3 mmol/l, glucose stimulation of secretion occurred in all experiments when glucose was elevated to 8 mmol/l irrespective of the genotype (n = 8). However, in agreement with a left shift in glucose responsiveness of Vm and [Ca2+]c, only 38% of the WT but 75% of the SK4-KO islet preparations displayed an increase in secretion with 6 mmol/l glucose (Fig. 3C). These data clearly demonstrate that genetic ablation of SK4 channels sensitizes the β-cells to glucose stimulation.

Bottom Line: SK4 channels were found to substantially contribute to K(slow) (slowly activating K(+) current).Deficiency of SK4 current induces elevated beta-cell responsiveness and coincides with improved glucose tolerance in vivo.Therefore, pharmacologic modulation of these channels might provide an interesting approach for the development of novel insulinotropic drugs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacy, the Department of Pharmacology, University of Tübingen, Tübingen, Germany.

ABSTRACT

Objective: Ca(2+)-regulated K(+) channels are involved in numerous Ca(2+)-dependent signaling pathways. In this study, we investigated whether the Ca(2+)-activated K(+) channel of intermediate conductance SK4 (KCa3.1, IK1) plays a physiological role in pancreatic beta-cell function.

Research design and methods: Glucose tolerance and insulin sensitivity were determined in wild-type (WT) or SK4 knockout (SK4-KO) mice. Electrophysiological experiments were performed with the patch-clamp technique. The cytosolic Ca(2+) concentration ([Ca(2+)](c)) was determined by fura-2 fluorescence. Insulin release was assessed by radioimmunoassay, and SK4 protein was detected by Western blot analysis.

Results: SK4-KO mice showed improved glucose tolerance, whereas insulin sensitivity was not altered. The animals were not hypoglycemic. Isolated SK4-KO beta-cells stimulated with 15 mmol/l glucose had an increased Ca(2+) action potential frequency, and single-action potentials were broadened. These alterations were coupled to increased [Ca(2+)](c). In addition, glucose responsiveness of membrane potential, [Ca(2+)](c), and insulin secretion were shifted to lower glucose concentrations. SK4 protein was expressed in WT islets. An increase in K(+) currents and concomitant membrane hyperpolarization could be evoked in WT beta-cells by the SK4 channel opener DCEBIO (100 micromol/l). Accordingly, the SK4 channel blocker TRAM-34 (1 micromol/l) partly inhibited K(Ca) currents and induced electrical activity at a threshold glucose concentration. In stimulated WT beta-cells, TRAM-34 further increased [Ca(2+)](c) and broadened action potentials similar to those seen in SK4-KO beta-cells. SK4 channels were found to substantially contribute to K(slow) (slowly activating K(+) current).

Conclusions: SK4 channels are involved in beta-cell stimulus-secretion coupling. Deficiency of SK4 current induces elevated beta-cell responsiveness and coincides with improved glucose tolerance in vivo. Therefore, pharmacologic modulation of these channels might provide an interesting approach for the development of novel insulinotropic drugs.

Show MeSH
Related in: MedlinePlus