Loss of high-frequency glucose-induced Ca2+ oscillations in pancreatic islets correlates with impaired glucose tolerance in Trpm5-/- mice

Abstract

Glucose homeostasis is critically dependent on insulin release from pancreatic beta-cells, which is strictly regulated by glucose-induced oscillations in membrane potential (V(m)) and the cytosolic calcium level ([Ca(2+)](cyt)). We propose that TRPM5, a Ca(2+)-activated monovalent cation channel, is a positive regulator of glucose-induced insulin release. Immunofluorescence revealed expression of TRPM5 in pancreatic islets. A Ca(2+)-activated nonselective cation current with TRPM5-like properties is significantly reduced in Trpm5(-/-) cells. Ca(2+)-imaging and electrophysiological analysis show that glucose-induced oscillations of V(m) and [Ca(2+)](cyt) have on average a reduced frequency in Trpm5(-/-) islets, specifically due to a lack of fast oscillations. As a consequence, glucose-induced insulin release from Trpm5(-/-) pancreatic islets is significantly reduced, resulting in an impaired glucose tolerance in Trpm5(-/-) mice.

Fig. 1.

Expression of TRPM5 protein in pancreatic islets of Langerhans immunostaining for TRPM5 and insulin of pancreatic islets from WT and Trpm5^−/− mice.

Fig. 2.

Characterization of a TRPM5-dependent calcium-activated nonselective cation current in single pancreatic islet cells. (A) Representative current traces in response to voltage ramps from −125 mV to +125 mV in WT cells dialyzed with either 0 or 1.5 μM Ca²⁺ in a bath solution containing 150 mM Na⁺ or 150 mM NMDG⁺. (B) Representative current traces as in A in Trpm5^−/− cells. (C) Mean current densities at +80 mV and −80 mV in WT and Trpm5^−/− cells (n = 12–14) in response to 0 Ca or 1.5 μM Ca²⁺ as in A. (D) Representative example of a current measured in a WT cell in the whole cell configuration during a step at +80 mV showing the activation kinetics of the calcium-activated current. After 60 ms, flash photolysis of caged Ca²⁺ was performed (arrow). [Ca²⁺]_cyt was measured simultaneously. (E) Representative example of a current measured as in D in a Trpm5^−/− cell. (F) Ca²⁺ dependency of the current activated after uncaging of Ca²⁺ at +80 mV in WT and Trpm5^−/− cells. The response is the difference in current density before and after the flash photolysis and is obtained from experiments as shown in D and E. *P < 0.05.

Fig. 3.

Overall reduced frequency of intracellular Ca²⁺ oscillations in isolated islets from Trpm5^−/− mice as a result of a lack of fast oscillations. (A) Effect of glucose (10 mM) on the [Ca²⁺]_cyt in islets from WT and Trpm5^−/− mice. Initially, the islets were bathed in a solution containing 3 mM glucose. Arrows indicate application of 10 mM glucose. (B) Average increase in ratio (F₃₅₀/F₃₈₀) after stimulation with 10 mM glucose in islets from WT and Trpm5^−/− mice (n = 28–34 from five to seven mice; P = 0.23). (C) Frequency of oscillations in individual experiments from WT and Trpm5^−/− islets, counted as the number of peaks per min (n = 28–34 from five to seven mice). [Ca²⁺]_cyt increase of 15% was considered to be an oscillation, when 100% is the amplitude between the baseline and the highest level reached in 10 mM glucose. ***P < 0.001. (D) Proportion of islets showing slow, mixed, and fast oscillation patterns according to Fourier analysis in WT (38.2%, 38.2%, and 23.6%, respectively) and Trpm5^−/− islets (89.3%, 10.7%, and 0%, respectively): n = 28 to 34 from five to seven mice; χ² analysis WT vs. Trpm5^−/−: P = 0.0006.

Fig. 4.

V_m measurements in WT and Trpm5^−/− islets stimulated with 10 mM glucose. (A) Representative examples of V_m measurements in WT islets showing fast (Left) and slow (Middle) oscillation patterns and in Trpm5^−/− islets displaying slow oscillations (Right). (B) Comparison of duration of the interburst interval, threshold potential for burst initiation, and slope of the depolarization during the interburst interval in WT fast- and slow-oscillating islets and in Trpm5^−/− slow-oscillating islets (n = 8–9 from five to six mice). *P < 0.05; ***P < 0.001.

Fig. 5.

Reduced glucose-induced insulin release leads to a disturbed glucose tolerance in Trpm5^−/− mice. (A) Insulin secretion from WT and Trpm5^−/− islets. Islets were challenged with different glucose concentrations as indicated (n = 8 per group). Insulin release was normalized to islet insulin content. *P < 0.05. (B) Plasma insulin levels after an i.p. glucose injection in overnight fasted WT and Trpm5^−/− mice (n = 4–5 mice; *P < 0.05; **P < 0.01). (C) OGTT and IPGTT glucose tolerance tests in WT and Trpm5^−/− mice. (n = 7 WT and n = 7 Trpm5^−/− mice for OGTT, n = 4 WT and n = 4 Trpm5^−/− mice for IPGTT. *P < 0.05; **P < 0.01.)