Calcium release channel RyR2 regulates insulin release and glucose homeostasis

Abstract

The type 2 ryanodine receptor (RyR2) is a Ca2+ release channel on the endoplasmic reticulum (ER) of several types of cells, including cardiomyocytes and pancreatic β cells. In cardiomyocytes, RyR2-dependent Ca2+ release is critical for excitation-contraction coupling; however, a functional role for RyR2 in β cell insulin secretion and diabetes mellitus remains controversial. Here, we took advantage of rare RyR2 mutations that were identified in patients with a genetic form of exercise-induced sudden death (catecholaminergic polymorphic ventricular tachycardia [CPVT]). As these mutations result in a "leaky" RyR2 channel, we exploited them to assess RyR2 channel function in β cell dynamics. We discovered that CPVT patients with mutant leaky RyR2 present with glucose intolerance, which was heretofore unappreciated. In mice, transgenic expression of CPVT-associated RyR2 resulted in impaired glucose homeostasis, and an in-depth evaluation of pancreatic islets and β cells from these animals revealed intracellular Ca2+ leak via oxidized and nitrosylated RyR2 channels, activated ER stress response, mitochondrial dysfunction, and decreased fuel-stimulated insulin release. Additionally, we verified the effects of the pharmacological inhibition of intracellular Ca2+ leak in CPVT-associated RyR2-expressing mice, in human islets from diabetic patients, and in an established murine model of type 2 diabetes mellitus. Taken together, our data indicate that RyR2 channels play a crucial role in the regulation of insulin secretion and glucose homeostasis.

Figure 6. Model of RyR2-mediated ER Ca²⁺ leak in the pancreatic β cell.

Chronic intracellular Ca²⁺ leak via RyR2 channels causes store depletion, triggering ER stress and mitochondrial dysfunction, leading to reduced ATP synthesis and eventually decreased glucose-stimulated insulin release, indicating an altered metabolism-secretion coupling. Impaired mitochondrial fitness also leads to increased production of ROS, which trigger redox modifications of RyR2 alongside calstabin2 dissociation, thereby exacerbating the Ca²⁺ leak. GLUT2, glucose transporter 2.

Figure 5. Fixing the RyR2-mediated ER Ca²⁺ leak restores calstabin2 binding to RyR2 in human diabetic islets and improves ER stress response, glucose tolerance, and insulin secretion in diabetic mice.

(A) Representative immunoblots of pancreatic islets from healthy subjects (nondiabetic) and patients with T2DM. Islets were treated with or without S107 (10 μM for 4 hours). (B–D) Quantification (from triplicate experiments, donors’ clinical information is given in Supplemental Table 4) of RyR2 oxidation (B), RyR2 nitrosylation (C), and levels of calstabin2 bound to RyR2 (D). *P < 0.05 vs. nondiabetic, ANOVA. (E–H) Immunoblots of pancreatic islets from ob/ob mice treated with or without S107 (50 mg/kg/d in drinking water for 4 weeks); representative immunoblots (E) and quantification (triplicate experiments) of RyR2 oxidation (F), RyR2 nitrosylation (G), and calstabin2 bound to RyR2 (H). *P < 0.05 vs. untreated WT, ANOVA. (I) ER stress response in murine islets evaluated in terms of Bip, total and spliced Xbp1, and Chop by real-time RT-qPCR analysis of total RNA, relative to untreated WT mice (horizontal dashed line), using β actin as internal standard. Primer sequences are reported in Supplemental Table 3. Each bar represents mean ± SEM of 4 independent experiments, each performed in triplicate. *P < 0.05 vs. ob/ob plus vehicle; ^#P < 0.05 vs. WT, 2-tailed Student’s t test. (J and K) Effects of chronic S107 treatment (50 mg/kg/d in drinking water for 4 weeks) in ob/ob mice on glucose tolerance in vivo (J) and insulin release from isolated pancreatic islets (K). All data are shown as mean ± SEM. n ≥ 6 animals/group. *P < 0.05 vs. ob/ob plus vehicle, 2-tailed Student’s t test.

Figure 4. Intracellular Ca²⁺ leak via RyR2 causes decreased glucose-stimulated mitochondrial Ca²⁺ uptake, which is restored by Rycal treatment.

(A–C) Mitochondrial Ca²⁺ uptake in response to glucose was decreased in pancreatic β cells from 2 CPVT mouse models with leaky RyR2 (representative traces for RyR2-R2474S and RyR2-N2386I are shown in A and B, respectively). S107 treatment (50 mg/kg/d, 4 weeks) improved mitochondrial Ca²⁺ uptake. (D–F) Caffeine-induced mitochondrial Ca²⁺ uptake was also markedly reduced, reflecting depleted ER Ca²⁺ stores, and was improved with S107 treatment (representative traces for RyR2-R2474S and RyR2-N2386I are shown in D and E, respectively). n = 7–10 mice per group. Data are shown as mean ± SEM. *P < 0.05 vs. WT.

Figure 3. Leaky RyR2 channels lead to abnormal mitochondrial structure and function in pancreatic β cells.

(A) Representative transmission electron micrographs of pancreatic β cell mitochondria from 4-month-old WT, RyR2-R2474S, and RyR2-N2386I mice, treated for 4 weeks with S107 or vehicle. Original magnification, ×30,000; insets, ×65000. N, nucleus. Scale bars: 500 nm. (B–D) Morphometric analyses of mitochondria reveal ultrastructural abnormalities in CPVT mice. (B) Mitochondrial area, (C) cristae density, (D) percentage of abnormal mitochondria per cell (mitochondria were defined as abnormal when a loss of electron density was detectable in more than 20% of the area of a mitochondrion). (E) mtDNA/nDNA copy number, (F) ROS production, (G) UCP2 expression, (H) aconitase activity, and (I) mt-ATP6 expression in isolated pancreatic islets. (J and K) ATP production in isolated pancreatic islets challenged with glucose (at indicated concentrations, J) or pyruvate (10 mM, K). Islets were isolated from n = 7–10 mice per group. Data are shown as mean ± SEM (triplicate measurements per sample). Box plots in B indicate upper/lower quartiles, lines in the middle of each box are the medians, and the whiskers represent the range of minimum and maximum values of total mitochondrial area per section. *P < 0.05 vs. WT, ANOVA, Tukey-Kramer post hoc correction.

Figure 2. Leaky RyR2 channels cause impaired insulin secretion.

(A) Representative images of pancreatic islets from WT, RyR2-R2474S, and RyR2-N2386I mice stained for insulin (red) and glucagon (yellow). Original magnification, ×63. Scale bars: 200 μm. Pancreatic weight (B), islet density (C) and size (D), ratio of Σ insulin area/Σ islet area (E), β cell mass (F), and insulin content (G). Data are expressed as mean ± SEM. Additional details are given in Methods. (G–J) Insulin release in response to glucose (H), leucine plus glutamine (I), or glyburide (J) evaluated ex vivo in pancreatic islets isolated from WT and CPVT mice undergoing S107 (50 mg/kg/d, 4 weeks) or vehicle treatment. Data are expressed as mean ± SEM. n = 6–10 animals/group. *P < 0.05 vs. WT, ANOVA, Tukey-Kramer post hoc correction.

Figure 1. Humans and mice with leaky RyR2 channels exhibit glucose intolerance.

OGTT in healthy subjects (see Supplemental Table 1 for patient characteristics) and patients with CPVT due to RYR2 mutations (see Supplemental Table 2 for RYR2 mutations in CPVT patients) with determination of blood glucose (A) and serum insulin levels (B). *P < 0.05 compared with healthy subjects, 2-tailed Student’s t test. (C) Representative immunoblots of pancreatic islets from CPVT mice chronically (50 mg/kg/d, 4 weeks) treated with S107. 2,4 DNPH, 2,4-dinitrophenylhydrazone; Cys-NO, nitrosylation. (D–F) Quantification of data shown in C (from triplicate experiments). (G and H) Resting cytosolic [Ca²⁺]_cyt is elevated in pancreatic β cells from CPVT mice (pancreatic β cell Ca²⁺ imaged using acetoxy-methyl-ester fura-2, fura-2 AM). (G) ER Ca²⁺ stores measured using caffeine to release the RyR2 Ca²⁺ pool are markedly depleted in pancreatic β cells from CPVT mice (H); acute (10 μM, 4 hours) S107 treatment restores ER Ca²⁺ by inhibiting RyR2-mediated leak. (I and J) Blood glucose levels following i.p. glucose challenge in WT, RyR2-R2474S, and RyR2-N2386I mice chronically treated with S107. Insulin concentration measured in sera from WT, RyR2-R2474S (K), and RyR2-N2386I (L) mice, following i.p. glucose challenge, with and without chronic S107. (M) Plasma glucagon in fed and fasted (6 hours) conditions. (N) Blood glucose levels measured following i.p. insulin injection. Insets in I–L represent AUC. Data are expressed as mean ± SEM. n = 9–12/group. *P < 0.05 compared with WT, ANOVA, Tukey-Kramer post hoc correction. Additional details are given in Methods.