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. 2010 Jul;59(7):1686-93.
doi: 10.2337/db09-0630. Epub 2010 Apr 22.

Inhibition of beta-cell sodium-calcium exchange enhances glucose-dependent elevations in cytoplasmic calcium and insulin secretion

Kevin S C Hamming  1 , Daniel Soliman, Nicola J Webster, Gavin J Searle, Laura C Matemisz, David A Liknes, Xiao-Qing Dai, Thomas Pulinilkunnil, Michael J Riedel, Jason R B Dyck, Patrick E Macdonald, Peter E Light
Affiliations

Inhibition of beta-cell sodium-calcium exchange enhances glucose-dependent elevations in cytoplasmic calcium and insulin secretion

Kevin S C Hamming et al. Diabetes. 2010 Jul.

Abstract

Objective: The sodium-calcium exchanger isoform 1 (NCX1) regulates cytoplasmic calcium (Ca(2+)(c)) required for insulin secretion in beta-cells. NCX1 is alternatively spliced, resulting in the expression of splice variants in different tissues such as NCX1.3 and -1.7 in beta-cells. As pharmacological inhibitors of NCX1 splice variants are in development, the pharmacological profile of beta-cell NCX1.3 and -1.7 and the cellular effects of NCX1 inhibition were investigated.

Research design and methods: The patch-clamp technique was used to examine the pharmacological profile of the NCX1 inhibitor KB-R7943 on recombinant NCX1.3 and -1.7 activity. Ca(2+) imaging and membrane capacitance were used to assess the effects of KB-R7943 on Ca(2+)(c) and insulin secretion in mouse and human beta-cells and islets.

Results: NCX1.3 and -1.7 calcium extrusion (forward-mode) activity was approximately 16-fold more sensitive to KB-R7943 inhibition compared with cardiac NCX1.1 (IC(50s) = 2.9 and 2.4 vs. 43.0 micromol/l, respectively). In single mouse/human beta-cells, 1 micromol/l KB-R7943 increased insulin granule exocytosis but was without effect on alpha-cell glucagon granule exocytosis. KB-R7943 also augmented sulfonylurea and glucose-stimulated Ca(2+)(c) levels and insulin secretion in mouse and human islets, although KB-R7943 was without effect under nonstimulated conditions.

Conclusions: Islet NCX1 splice variants display a markedly greater sensitivity to pharmacological inhibition than the cardiac NCX1.1 splice variant. NCX1 inhibition resulted in glucose-dependent increases in Ca(2+)(c) and insulin secretion in mouse and human islets. Thus, we identify beta-cell NCX1 splice variants as _targets for the development of novel glucose-sensitive insulinotropic drugs for type 2 diabetes.

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Figures

FIG. 1.
FIG. 1.
KB-R7943 inhibits FM NCX1 activity in a splice variant–dependent manner. A: Representative current recordings of NCX1.1, -1.3, and -1.7 splice variants demonstrating that the β-cell NCX1 splice variants, NCX1.3 and -1.7, display FM inactivation, whereas the cardiac NCX1.1 splice variant does not. B–D: Representative current recordings illustrating that FM NCX1.1 activity is resistant to inhibition by 5 μmol/l KB-R7943, whereas NCX1.3 and -1.7 displayed marked KB-R7943 inhibition. E: KB-R7943 concentration-inhibition curves for NCX1.1, -1.3, and -1.7 FM currents. F: Diagrammatic representation of the alternative splicing region exon composition for various NCX1 splice variants. G: Grouped KB-R7943 (5 μmol/l) inhibition data for FM NCX1.1, -1.11, -1.3, -1.4, and -1.7 currents. *P < 0.05, n = 7–10 patches per group. 0 = <1 μmol/l Ca2+. NCX1 proteins were expressed in tsA201 cells.
FIG. 2.
FIG. 2.
The late FM NCX1.3 and -1.7 currents are more sensitive to KB-R7943 inhibition (3 μmol/l) compared with peak currents. Representative recordings of NCX1.3 (A) and -1.7 (C) FM currents before (control) and after the application of 3 μmol/l KB-R7943. Concentration-response curves for NCX1.3 (B) and -1.7 (D) late and peak KB-R7943 FM current inhibition demonstrating that KB-R7943 preferentially inhibits the late FM currents compared with peak FM currents. *P < 0.05, n = 4–10 patches per concentration. 0 = <1 μmol/l Ca2+. NCX1 proteins were expressed in tsA201 cells.
FIG. 3.
FIG. 3.
Calcium imaging of mouse islets. A: Application of 20 μmol/l tolbutamide (Tolb) to islets superfused with 2.8 mmol/l glucose increases Ca2+c levels that are lower upon a second tolbutamide application. B: In contrast, the addition of 1 μmol/l KB-R7943 significantly increases the magnitude of Ca2+c elevation observed during the second application of tolbutamide. KB-R7943 does not increase Ca2+c levels when applied alone in the presence of 2.8 mmol/l glucose. Ci: Grouped data showing that KB-R7943 significantly increases Ca2+c levels (*P < 0.05, n = 6–11 islets) in the presence of tolbutamide or KCl. Cii: Grouped data showing that in the absence of the tolbutamide or KCl stimulatory signal, KB-R7943 does not increase Ca2+c (n = 6–9 islets). D: Grouped data indicating that the rate at which Ca2+c returned to basal levels is slower in the presence of KB-R7943 for both tolbutamide and KCl (*P < 0.05, n = 6–9 islets). E: Increasing glucose from 2.8 to 11.1 mmol/l elicited increases in Ca2+c, whereas 1 μmol/l KB-R7943 did not increase Ca2+c at 2.8 mmol/l glucose but significantly increased Ca2+c in 11.1 mmol/l glucose when compared with 11.1 mmol/l glucose alone. Grouped data showing that 1 μmol/l KB-R7943 significantly increased the peak (Δpeak) (Fi) and average Ca2+c (AUC) in response to 11.1 mmol/l glucose (Fii). *P < 0.05, n = 9 islets per group.
FIG. 4.
FIG. 4.
NCX1 inhibition and Ca2+-dependent exocytosis in β- and α-cells. Representative capacitance measurements and grouped data of cumulative changes at individual pulse numbers from mouse and human β-cells with reduced NCX1 expression (Ad-shNCX1), scrambled control vector (Ad-scramble) (A), or in the presence of 1μmol/l KB-R7943 (C and E) in response to a depolarizing train of membrane potential pulses. B: Western blot analysis (i, ii) showing that NCX1 protein expression is only partially reduced as a result of Ad-shNCX1 infection in MIN6 cells (i–iii). β-actin (i) and total ACC (ii) were used as loading controls. D: Representative capacitance measurements and grouped data for calcium infusion experiments in the presence and absence of 1μmol/l KB-R7943. F: Representative capacitance measurements and grouped data from mouse α-cells in response to 1 μmol/l KB-R7943. *P < 0.05, n = 8–20 cells per group or three separate lysates (B).
FIG. 5.
FIG. 5.
Glucose-stimulated insulin secretion from mouse and human islets in response to elevations in glucose from 2.8 to 11.1 mmol/l (denoted by arrows). KB-R7943 (1 μmol/l) augments insulin secretion from mouse (A) and human (B) islets only in the presence of elevated glucose (11.1 mmol/l). *P < 0.05, n = 5–6 for mouse and 2–3 for humans.
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