Lowering Plasma 1-Deoxysphingolipids Improves Neuropathy in Diabetic Rats
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Figures
- Figure 1
Schematic diagram showing the flow of the experiment. For the preventive schedule, a serine-enriched (Ser) or a standard diet (Std) was given from day 0 after STZ injection and the animals were followed for 18 weeks until they were killed. For the therapeutic schedule, animals received an initial standard diet for 8 weeks after STZ injection, followed by random separation into either a serine-enriched or a standard diet group. The animals then were followed until the end of the study period at week 24 after STZ.
- Figure 2
Effect of the serine-enriched diet on plasma concentrations of glucose, TGs, and serine. Line plots show the time course concentrations of plasma glucose (A and B), TGs (D and E), and serine (G and H) of the animals used in the study in the preventive (left) and therapeutic schedules (middle) for the different groups. Scatter plots show the values of plasma glucose (C), TGs (F), and serine (I) at week 16 after STZ injection for the preventive group and week 24 after STZ injection for the therapeutic group. The values are expressed as mean ± SEM. P values were calculated using ANOVA followed by Bonferroni correction. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. CTRL Ser, control rats on serine diet; CTRL Std, control rats on standard diet; STZ Ser, STZ rats on serine diet; STZ Std, STZ rats on standard diet.
- Figure 3
Effect of serine-enriched diet on plasma concentrations of typical sphingolipids. Line plots show the plasma concentrations of C18SO (A and B), C18SA (D and E), C18PhytoSO-based sphingolipids (G and H), and C18SAdiene (J and K) over the entire period of the preventive (left) and therapeutic schedules (middle) for the different groups. Scatter plots show the values for C18SO (C), C18SA (F), C18PhytoSO (I), and C18SAdiene (L) at week 17 after STZ for the preventive group and week 24 after STZ for the therapeutic groups. The values are expressed as mean ± SEM. P values were calculated using ANOVA followed by Bonferroni correction. *P < 0.05, **P < 0.01, ****P < 0.0001. CTRL Ser, control rats on serine diet; CTRL Std, control rats on standard diet; STZ Ser, STZ rats on serine diet; STZ Std, STZ rats on standard diet.
- Figure 4
Effect of serine-enriched diet on plasma concentrations of 1-deoxysphingolipids. Line plots show plasma concentrations of 1-deoxySA (A and B) over the entire period of the preventive (left) and therapeutic schedules (middle) for the different groups. Scatter plots show the values for 1-deoxySA (C) at week 17 after STZ for the preventive group and week 24 after STZ for the therapeutic groups. The values are expressed as mean ± SEM. P values were calculated using ANOVA followed by Bonferroni correction. ****P < 0.0001. CTRL Ser, control rats on serine diet; CTRL Std, control rats on standard diet; STZ Ser, STZ rats on serine diet; STZ Std, STZ rats on standard diet.
- Figure 5
Effect of L-serine on of mechanical and thermal nociception. Force withdrawal threshold (A and B) and thermal response latency (D and E) were assessed for the preventive and the therapeutic groups at the beginning of the study and over time until the end the study. Black arrows refer to the time when the serine-enriched diet was introduced to the respective groups in the therapeutic schedule. Scatter plots show the force withdrawal threshold (C) and thermal response latency (F) for the preventive groups at week 14 after STZ injection and week 23 after STZ injection for the therapeutic groups. The values are expressed as mean ± SEM. P values were calculated using ANOVA followed by Bonferroni correction. For the force withdrawal threshold, the values were log-transformed before the P values were calculated. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. CTRL Ser, control rats on serine diet; CTRL Std, control rats on standard diet; STZ Ser, STZ rats on serine diet; STZ Std, STZ rats on standard diet.
- Figure 6
Effect of serine on sensory NCV (A), Na+K+-ATPase activity (B), and IENF density (C). Scatter plots for the preventive (week 14 after STZ injection) and the therapeutic groups (week 23 after STZ injection). The values are expressed as mean ± SEM. P values were calculated using ANOVA followed by Bonferroni correction. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. CTRL Std, control rats on standard diet; CTRL Ser, control rats on serine diet; STZ Std, STZ rats on standard diet; STZ Ser, STZ rats on serine diet. D: Scatter plot showing the correlation between NCV and plasma 1-deoxySA concentrations. A highly significant inverse correlation between plasma 1-deoxySA concentrations and NCV was observed of the whole group of animals (P = 5.23 × 10−12). Variables were log-transformed because the control groups skewed the normal distribution to the right. Pearson correlation coefficient and the asymptomatic P value are shown.
- Figure 7
Effect of serine-enriched diet on axon, nerve fiber diameter. Distribution of axon (A and B) and nerve fiber diameters (C and D) shows a significant difference between the control and STZ rats (more axons and fibers with large diameters and fewer with shorter diameters in the control group). There is a significant improvement in the serine-supplemented STZ rats [STZ Ser] in the preventive group but not the therapeutic group (more axons and fibers with larger diameters and fewer with smaller diameters compared with the STZ rats on the standard diet [STZ Std]). Scatter plots show the mean ± SEM for the frequency distribution of axons or nerve fibers (three replicates from each rat and three rats per group). The solid lines represent the fitted Gaussian distribution model. Nerve fiber diameter shows two Gaussian peaks in the control rats, denoting two different fiber types; this pattern is lost in the STZ rats. P values were calculated comparing the fitted Gaussian distribution of each group. CTRL Std, control rats on standard diet.
- Figure 8
Effect of a serine-enriched diet on the morphometry of DRG neurons in the preventive group. A: Light microcopy of sections of DRG neurons in the preventive group stained with toluidine blue. DRG neurons of STZ-treated animals are smaller in size compared with control animals. No evidence of cell damage was observed. Column plots show the average size of soma (B), nuclei (C), and nucleoli (D) of DRG neurons in the control (CTRL) and STZ rats on a standard diet (Std) and a serine diet (Ser). The values are expressed as mean ± SD. P values were calculated using ANOVA followed by the Tukey post hoc test. **P < 0.01, ***P < 0.001.
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