Chronic Elevation of Skeletal Muscle [Ca2+]i Impairs Glucose Uptake. An in Vivo and in Vitro Study

Abstract

Skeletal muscle is the primary site of insulin-mediated glucose uptake through the body and, therefore, an essential contributor to glucose homeostasis maintenance. We have recently provided evidence that chronic elevated intracellular Ca²⁺ concentration at rest [(Ca²⁺)_i] compromises glucose homeostasis in malignant hyperthermia muscle cells. To further investigate how chronic elevated muscle [Ca²⁺]_i modifies insulin-mediated glucose homeostasis, we measured [Ca²⁺]_i and glucose uptake in vivo and in vitro in intact polarized muscle cells from glucose-intolerant RYR1-p.R163C and db/db mice. Glucose-intolerant RYR1-p.R163C and db/db mice have significantly elevated muscle [Ca²⁺]_i and reduced muscle glucose uptake compared to WT muscle cells. Dantrolene treatment (1.5 mg/kg IP injection for 2 weeks) caused a significant reduction in fasting blood glucose levels and muscle [Ca²⁺]_i and increased muscle glucose uptake compared to untreated RYR1-p.R163C and db/db mice. Furthermore, RYR1-p.R163C and db/db mice had abnormal basal insulin levels and response to glucose-stimulated insulin secretion. In vitro experiments conducted on single muscle fibers, dantrolene improved insulin-mediated glucose uptake in RYR1-p.R163C and db/db muscle fibers without affecting WT muscle fibers. In muscle cells with chronic elevated [Ca²⁺]_i, GLUT4 expression was significantly lower, and the subcellular fraction (plasma membrane/cytoplasmic) was abnormal compared to WT. The results of this study suggest that i) Chronic elevated muscle [Ca²⁺]_i decreases insulin-stimulated glucose uptake and consequently causes hyperglycemia; ii) Reduced muscle [Ca²⁺]_i by dantrolene improves muscle glucose uptake and subsequent hyperglycemia; iii) The mechanism by which chronic high levels of [Ca²⁺]_i interfere with insulin action appears to involve the expression of GLUT4 and its subcellular fractionation.

Keywords: GLUT4; calcium; dantrolene; diabetes; skeletal muscle.

FIGURE 1

Abnormal glucose tolerance in RYR1-p.R163C and db/db. GTTs were carried out in fasting WT, RYR1-p.R163C, and db/db mice. Basal blood glucose levels were elevated in RYR1-p.R163C and db/db compared to WT mice. A notable difference in average peak amplitude at 30 min was found in RYR1-p.R163C and db/db compared to WT. The glucose tolerance was expressed as the AUC (Insert), which was calculated using the trapezoid rule over the 120-min test duration (GraphPad Prism software 9.0). Values are expressed as mean ± S. D; experiments were conducted in n _mice = 6 per genotype. Statistical analysis was performed using a one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons. *** = p < 0.001.

FIGURE 2

Dantrolene improves glucose tolerance in RYR1-p.R163C and db/db mice. GTT was carried out before and after dantrolene treatment (1.5 mg/kg intraperitoneally daily for 2 weeks) in WT, RYR1-p.R163C, and db/db mice. Dantrolene significantly reduced fasting glycemia in RYR1-p.R163C (A) and db/db mice (B) and improved response to glucose load at all times studied in RyR1-p.R163C and db/db compared to untreated mice. The insets in Figures (A, B) represent the area under the curve before and after dantrolene treatment for 120 min. The results represent the mean ± S. D from n _mice = 5 per genotype. Statistical analysis was performed as described above. ns = non significative; *** = p < 0.001.

FIGURE 3

Abnormal insulin levels in response to glucose challenge. Insulin levels were determined in WT, RYR1-p.R163C, and db/db mice after a glucose load. The basal level of insulin was higher in RyR1-p.R163C and db/db than in WT mice and during the test duration of 120 min. The insert shows the AUC calculated using the trapezoid rule of the insulin determination (GraphPad Prism software 9.0). Values are expressed as mean ± S.D. Experiments were conducted in n _mice = 6 per genotype. Statistical analysis was performed as described above. ** = p < 0.01, *** = p < 0.001.

FIGURE 4

[Ca ²⁺ ] _i in intact skeletal muscle cells. Effect of dantrolene. The records represent the specific potential of Ca²⁺ obtained from WT, RYR1-pR163C, and db/db muscle cells. [Ca²⁺]_i was calculated using the corresponding microelectrode calibration curve (Lopez et al., 1983). (A) Shows a representative record of the Ca²⁺ specific potential obtained from a WT muscle cell, [Ca²⁺]_i was 119 nM. The upper left and right arrows indicate Ca²⁺ microelectrode impalement and withdrawal, respectively; (B) Ca²⁺ specific potential recorded from RYR1-pR163C muscle fiber, [Ca²⁺]_i was 284 nM; (C) Ca²⁺ specific potential recorded from a db/db muscle fiber; [Ca²⁺]_i was 329 nM. (D) [Ca²⁺]_i was significantly higher in RYR1-pR163C and db/db than WT muscle cells. Dantrolene treatment (1.5 mg/kg IP daily for 2 weeks) reduced [Ca²⁺]_i in all genotypes; however, the reduction was greater in RYR1 p. R163C (2.8 times) and db/db (2.9 times) than in WT muscles (1.2 times). Values are expressed as mean ± S. D; experiments were carried out in n _mice = 6 per genotype; n _cells = 13–16/genotype group. Statistical analysis was performed as described above. *** = p < 0.001. Calibration bar = 1 min.

FIGURE 5

Increased [Ca²⁺]i impairs glucose uptake in muscle fibers. [Ca²⁺]_i and glucose uptake were recorded simultaneously in vivo in intact gastrocnemius fibers WT and RyR1-p.R163C and db/db using double-barreled selective Ca²⁺ microelectrodes and the 2-NBDG fluorescent glucose analog. (A) Left panel: Intracellular [Ca²⁺] was higher in RYR1-p.R163C and db/db than in the WT muscle. Right panel: Glucose uptake was significantly reduced in RYR1-p.R163C and db/db compared to WT muscle. All fluorescence signals were normalized to WT. (B) A positive correlation between muscle glucose uptake and [Ca²⁺]_i was found in RyR1-p.R163C and db/db (R = 0.63). An increase in muscle [Ca²⁺]_i caused a decrease in glucose uptake. (C) Show a positive correlation analysis between mice’s blood glucose (mg/dL) and muscle [Ca²⁺]_i (nM) (R = 0.68). Increased muscle [Ca²⁺]_i caused a more elevated blood glucose concentration. (D) Left panel: [Ca²⁺]_i was elevated in RYR1-p.R163C and db/db compared to WT muscle. Dantrolene treatment significantly reduced [Ca²⁺]_i and all genotypes. Right panel: insulin-dependent glucose uptake was reduced in RYR1-p.R163C and db/db compared to WT muscle. Dantrolene improved glucose uptake in the RYR1-p.R163C and db/db muscles, with no significant effect in WT. All fluorescence signals were normalized to WT. The results represent the mean ± S. D from n _cells = 14–52/genotype group, isolated from n _mice = 5–6 per genotype; n _cells = 14–52/genotype group. Statistical analysis was performed as described above. ** = p < 0.01; *** = p < 0.001.

FIGURE 6

Glucose uptake in WT, RYR1-p.R163C, and db/db single fibers. Effects of dantrolene. (A) Single muscle fiber recordings of 2-NBDG fluorescent signals from WT, RYR1-p.R163C, and db/db muscle. (B) Typical records of NBDG fluorescent signals show the effect of dantrolene on insulin-dependent glucose uptake in single muscle fibers WT, RYR1-p.R163C, and db/db. (C) Shows the summary of abnormal insulin-dependent glucose uptake in RYR1-p.R163C and db/db single muscle fibers and the effects of dantrolene. The florescent rate values were normalized to untreated WT and expressed as mean ± S. D; experiments were carried out in n _mice = 5 per genotype; n _fibers = 9–14/genotype group. Statistical analysis was performed as described above. *** = p < 0.001. Calibration bar = 5 min.

FIGURE 7

Expression and subcellular distribution of GLUT4 in skeletal muscle. The total expression of GLUT4 was significantly lower in RYR1-pR163C and db/db gastrocnemius muscle compared to age-matched WT muscle. Furthermore, the GLUT4 subcellular membrane fraction was significantly reduced in RYR1-pR163C and db/db muscles compared to WT; in contrast, the cytoplasmic fraction of GLUT4 was higher in RYR1-pR163C and db/db muscles than in WT. (A) Representative immunoblots collected from WT, RyR1-p.R163C, and the db/db muscle homogenates. (B) Densitometric quantification of the global and subcellular distribution of GLUT4 in WT, RYR1-pR163C, and db/db muscle. Values are expressed as mean ± S.D. Experiments were carried out in n _mice = 3 per genotype, n _WB = 5. Statistical analysis was performed as described above. ns = non significative, ** = p < 0.01, *** = p < 0.001.