Pyruvate Administration Restores Impaired Nociception by Enhancing Neurite Outgrowth in Streptozotocin-Induced Diabetic Mice

Abstract

Diabetic peripheral neuropathy (DPN) is a chronic complication of diabetes mellitus for which effective treatments remain undeveloped. Metabolic changes and inflammation are proposed as primary mechanisms underlying DPN pathogenesis. Our previous studies demonstrate that exogenous pyruvate plays a crucial role in maintaining glycolysis-tricarboxylic acid cycle flux under high-glucose conditions and also exhibits anti-inflammatory properties. To evaluate its therapeutic potential, we assessed whether pyruvate administration could restore DPN in vivo and in vitro. We assessed casual blood glucose levels, body weight, motor and sensory nerve conduction velocities, mechanical sensitivity, and intraepidermal nerve fiber density in streptozotocin-induced diabetic C57/BL/6J mice that received drinking water with or without sodium pyruvate (10 mg/mL) from 2 to 13 weeks after diabetes induction. In addition, we evaluated neurite length in ND7/23 cells, a dorsal root ganglion neuron cell line, under high-glucose conditions. Pyruvate administration in diabetic mice alleviated mechanical sensitivity deficits and improved intraepidermal nerve fiber density. Additionally, neurite length in ND7/23 cells was inhibited under high-glucose conditions but was fully restored by supplementation with high concentrations (10 mM) of pyruvate. These findings suggest that exogenous pyruvate may be a promising therapeutic candidate for DPN.

Keywords: Diabetic peripheral neuropathy 2; Nociception 4; Pyruvate 1; dorsal root ganglion neurons 3.

Figure 1

Schematic representation of the animal study. Six-week-old mice were injected with streptozotocin (STZ) or citrate buffer. Following injection, the mice were provided ad libitum access to drinking water, in the presence or absence of sodium pyruvate, from 2 to 13 weeks post-injection. The experiments were conducted as shown in this figure. BW: body weight, Glc: casual blood glucose, VFT: von Frey test, NCV: motor and sensory nerve conduction velocity, IENFD: intraepidermal nerve fiber density.

Figure 2

Pyruvate administration did not affect body weight or blood glucose levels in both control and STZ-injected mice. Body weight (A) and casual blood glucose levels (B) were measured at 1, 3, 7, and 11 weeks after STZ injection. Data are presented for buffer-injected mice drinking water (CW, light blue, n = 8) or pyruvate (CP, yellow, n = 7) and for STZ-injected mice drinking water (SW, blue, n = 8) or pyruvate (SP, red, n = 8). Values represent the mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by post hoc comparisons with the Tukey HSD test. (A) a: p < 0.01 CW vs. SW and CP vs. SW, p < 0.05 CW vs. SP, and CP vs. SP, b: p < 0.01 CW vs. SW, CW vs. SP, CP vs. SW and CP vs. SP; and, c: p < 0.01 CW vs. SW, CP vs. SW and CP vs. SP, p < 0.05 CW vs. SP. (B) a: p < 0.01 CW vs. SW, CW vs. SP, CP vs. SW and CP vs. SP.

Figure 3

Pyruvate administration to STZ-induced diabetic mice did not restore sensory and motor nerve conduction velocities. Sensory nerve conduction velocity (SNCV; (A) and motor nerve conduction velocity (MNCV; (B) were measured at 4, 8, and 12 weeks after STZ injection. Data are presented for buffer-injected mice drinking water (CW, light blue, n = 8) or pyruvate (CP, yellow, n = 7) and for STZ-injected mice drinking water (SW, blue, n = 8) or pyruvate (SP, red, n = 8). Values represent the mean ± SEM with individual values depicted as circles. Statistical analysis was performed using the Kruskal-Willis test, followed by post hoc comparisons with the Steel-Dwass test. * p < 0.05, ** p < 0.01.

Figure 4

Pyruvate administration tended to improve impaired nociception in STZ-injected mice. The von Frey test was conducted at 4, 8, and 12 weeks after STZ injection. Data are presented for buffer-injected mice drinking water (CW, light blue, n = 8) or pyruvate (CP, yellow, n = 7) and for STZ-injected mice drinking water (SW, blue, n = 8) or pyruvate (SP, red, n = 8). Values represent the mean ± SEM, with individual values depicted as circles. Statistical analysis was performed using the Kruskal-Willis test, followed by post hoc comparisons with the Steel-Dwass test. * p < 0.05, ** p < 0.01.

Figure 5

Pyruvate administration restored intraepidermal nerve fiber density (IENFD) in STZ-induced diabetic mice. IENFD (A,B) was estimated at 13 weeks after STZ injection. (A) Intraepidermal nerve fibers indicated by white arrows were detected by immunohistochemistry using an anti-PGP 9.5 antibody. The dot lines show epidermis. (B) IENFDs were quantified from the images shown (A). Data are presented for buffer-injected mice drinking water (CW, light blue, n = 8) or pyruvate (CP, yellow, n = 8) and for STZ-injected mice drinking water (SW, blue, n = 8) or pyruvate (SP, red, n = 8). Scale bar: 50 μm. Values represent the mean ± SEM, with individual values depicted as circles. Statistical analysis was performed using one-way ANOVA, followed by post hoc comparisons with the Tukey HSD test. * p < 0.05, ** p < 0.01.

Figure 6

High conditions of pyruvate prevented ND7/23 cells from hyperglycemia-induced inhibition of neurite length. Differentiated (A) and undifferentiated (C) ND7/23 cells were exposed to either 5 or 60 mM glucose with 0.1, 1, or 10 mM pyruvate. Cells exposed to 60 mM glucose in the presence of 0.1 mM pyruvate experienced cell death. Neurite length was visualized using a βIII tubulin antibody. Neurite length measurements of differentiated (B) and undifferentiated (D) ND7/23 cells were taken under normal glucose conditions (5 mM, blue colors) and high glucose conditions (60 mM, yellow colors), with 0.1, 1, or 10 mM pyruvate. Scale bar: 100 μm. Box and whisker plots represent data from 199 to 366 cells across three independent experiments. Statistical analysis was performed using one-way ANOVA, followed by post hoc comparisons with the Tukey HSD test. * p < 0.05, ** p < 0.01.

Figure 7

Measurement of cell viability, ROS production, and mitochondrial membrane potential of ND7/23 cells under differentiated and undifferentiated conditions. Differentiated (A–C) and undifferentiated (D–F) ND7/23 cells were exposed to either 5 (blue bars) or 60 mM (yellow bars) glucose with 0.1, 1, or 10 mM pyruvate. Cell viability (A,D), ROS production (B,E), and mitochondrial membrane potential (C,F) under each condition were assessed. Values represent the mean + SD, with individual values depicted as circles (A,D). Bar graph and box and whisker plots represent data from 3 wells (A,D), 61 to 125 cells (B,E) and 40 to 66 cells (C,F) across three independent experiments. Statistical analysis was performed using one-way ANOVA, followed by post hoc comparisons with the Tukey HSD test (C,E), or Kruskal-Wilis test, followed by post hoc comparisons with the Steel Dwass test (A,B,D,F). * p < 0.05, ** p < 0.01.

Figure 8

High levels of pyruvate prevented ND7/23 cells from hyperglycemia-induced neurite degeneration. Neurite outgrowth of ND7/23 cells was enhanced under differentiated conditions followed by maintenance under 60 mM glucose in the presence of 1 or 10 mM pyruvate under differentiated (A,B) and undifferentiated (C,D) conditions. Phase contrast images of ND7/23 cells under differentiated (A) and undifferentiated (C) conditions are shown. Neurite length measurements of differentiated (B) and undifferentiated (D) ND7/23 cells were taken under 60 mM glucose conditions with 1 mM (yellow dots), or 10 mM (orange dots) pyruvate. Scale bar: 100 μm. A combination of dot plots and bars of ±SD represent data from 79 to 103 cells across three independent experiments. Statistical analysis was performed using Student’s t test. ** p < 0.01.

Figure 9

High levels of pyruvate prevented ND7/23 cells from hyperglycemia-induced neurite degeneration. Neurite outgrowth of ND7/23 cells were enhanced under differentiated conditions and then exposed to 60 mM glucose in the presence of 1 or 10 mM pyruvate under differentiated (A–C) and undifferentiated (D,E) conditions. Bar graph and box and whisker plots exhibit the data of cell viability (A,D), ROS production (B,E), and mitochondrial membrane potential (C,F) under 60 mM glucose conditions with 1 mM (yellow bars), or 10 mM (orange bras) pyruvate. Values represent the mean + SD, with individual values depicted as circles (A,D). Bar graph and box and whisker plots represent data from 3 wells (A,D), 342 to 418 cells (B,E) and 57 cells (C,F) across three independent experiments. Statistical analysis was performed using student’s t test. ** p < 0.01.