Hyperglycemia alters the schwann cell mitochondrial proteome and decreases coupled respiration in the absence of superoxide production
Liang Zhang, Cuijuan Yu, Francisco E Vasquez, Nadya Galeva, Isaac Onyango, Russell H Swerdlow, Rick T Dobrowsky, Liang Zhang, Cuijuan Yu, Francisco E Vasquez, Nadya Galeva, Isaac Onyango, Russell H Swerdlow, Rick T Dobrowsky
Abstract
Hyperglycemia-induced mitochondrial dysfunction contributes to sensory neuron pathology in diabetic neuropathy. Although Schwann cells (SCs) also undergo substantial degeneration in diabetic neuropathy, the effect of hyperglycemia on the SC mitochondrial proteome and mitochondrial function has not been examined. Stable isotope labeling with amino acids in cell culture (SILAC) was used to quantify the temporal effect of hyperglycemia on the mitochondrial proteome of primary SCs isolated from neonatal rats. Of 317 mitochondrial proteins identified, about 78% were quantified and detected at multiple time points. Pathway analysis indicated that proteins associated with mitochondrial dysfunction, oxidative phosphorylation, the TCA cycle, and detoxification were significantly increased in expression and over-represented. Assessing mitochondrial respiration in intact SCs indicated that hyperglycemia increased the overall rate of oxygen consumption but decreased the efficiency of coupled respiration. Although a glucose-dependent increase in superoxide production occurs in embryonic sensory neurons, hyperglycemia did not induce a substantial change in superoxide levels in SCs. This correlated with a 1.9-fold increase in Mn superoxide dismutase expression, which was confirmed by immunoblot and enzymatic activity assays. These data support that hyperglycemia alters mitochondrial respiration and can cause remodeling of the SC mitochondrial proteome independent of significant contributions from glucose-induced superoxide production.
Figures
K0 and K6 primary SCs were cultured for 16 days in 30 mM glucose, lysates were prepared and mixed together in a 1:1 mass ratio. Nuclear, mitochondrial and cytosolic fractions were isolated and 75 µg of protein was separated by SDS-PAGE for proteomic analysis (A) or 3 µg was subjected to immunoblot analysis (B) for the indicated organelle marker.
SCs were incubated in medium containing 5.5 mM (K0) or 30 mM (K6) medium for 2, 6 or 16 days and nuclear and cytoplasmic fractions were analyzed by GeLC-LTQ-FT MS/MS. (A) Venn diagram of number and percent of annotated nuclear proteins identified at each time point. To compare the effect of 2, 6, or 16 days hyperglycemia on the nuclear (B,C) or cytoplasmic (D,E) proteomes, the expression ratios were plotted versus protein number (B,D) or binned by 0.2 units and expressed as a percent of the total quantified proteins (C,E). Line indicates three times the standard deviation of the analytic variability and proteins between the lines did not show a significant change.
SCs were incubated in medium containing 5.5 mM (K0) or 30 mM (K6) medium for 2, 6 or 16 days and nuclear and cytoplasmic fractions were analyzed by GeLC-LTQ-FT MS/MS. (A) Venn diagram of number and percent of annotated nuclear proteins identified at each time point. To compare the effect of 2, 6, or 16 days hyperglycemia on the nuclear (B,C) or cytoplasmic (D,E) proteomes, the expression ratios were plotted versus protein number (B,D) or binned by 0.2 units and expressed as a percent of the total quantified proteins (C,E). Line indicates three times the standard deviation of the analytic variability and proteins between the lines did not show a significant change.
A) Quantified proteins were grouped based upon their annotated function. The expression ratios of all proteins associated with a function were averaged and the line indicates three times the standard deviation of the analytic variability associated with the quantitation. Categories above or below these limits define a significant change in expression. The number of quantified proteins per category is indicated in the legend. Significant differences between time points were determined using a one-way ANOVA and Tukey’s post hoc test. *, p< 0.05 compared to 2 days, ^, p < 0.05 compared to 6 days. B) The top two molecular networks affected after 2 days of hyperglycemia were identified using Ingenuity Pathway Analysis, merged and graphically presented to highlight the increase in proteins regulating mRNA processing and the decrease in proteins regulating protein synthesis, metabolism and transport. Gene names are identified in Supplementary Table 4.
A) Quantified proteins were grouped based upon their annotated function. The expression ratios of all proteins associated with a function were averaged and the line indicates three times the standard deviation of the analytic variability associated with the quantitation. Categories above or below these limits define a significant change in expression. The number of quantified proteins per category is indicated in the legend. Significant differences between time points were determined using a one-way ANOVA and Tukey’s post hoc test. *, p< 0.05 compared to 2 days, ^, p < 0.05 compared to 6 days. B) The top two molecular networks affected after 2 days of hyperglycemia were identified using Ingenuity Pathway Analysis, merged and graphically presented to highlight the increase in proteins regulating mRNA processing and the decrease in proteins regulating protein synthesis, metabolism and transport. Gene names are identified in Supplementary Table 4.
SCs were incubated in medium containing 5.5 mM (K0) or 30 mM (K6) medium for 2, 6 or 16 days and mitochondrial fractions were analyzed by GeLC-LTQ-FT MS/MS. (A)Venn diagram of number and percent of annotated mitochondrial proteins identified at each time point. To compare the effect of 2, 6, or 16 days hyperglycemia on the mitochondrial proteome, the expression ratios were plotted versus protein number (B) or binned by 0.2 units and expressed as a percent of the total quantified proteins (C). Reverse label indicates results from an experiment where control cells were labeled in K6 medium. Line indicates three times the standard deviation of the analytic variability and proteins between the lines did not show a significant change.
A) Quantified proteins were grouped based upon their annotated function. The expression ratios of all proteins associated with a function were averaged and the line indicates three times the standard deviation of the analytic variability associated with the quantitation. Categories above or below these limits define a significant change in expression. The number of quantified proteins per category is indicated in the legend. Significant differences between time points were determined using a one-way ANOVA and Tukey’s post hoc test. #, p< 0.05 compared to 2 days, *, p < 0.05 compared to 2 and 6 days. FAM, fatty acid metabolism; Ox Phos, oxidative phosphorylation. B) The top over-represented toxicologic functions were identified using Ingenuity Pathway Analysis. Ordinate values are –log of the Benjamini-Hochberg False Discovery Rate p-value.
SCs were subjected to hyperglycemic stress for 3 days and the oxygen consumption rate (OCR) (A) and extracellular acidification rate (ECAR) (B) were measured in intact cells using an Extracellular Flux Analyzer. Oligomycin, FCCP and rotenone/myxothiazole were added at the indicated times to determine the rate of proton leak and O2 coupled ATP production. Respiration experiments were repeated three times and the results presented are four replicate measures per time point from one representative experiment.
SCs were incubated with 5.5 mM (K0) or 30 mM (K6) glucose for 6 days and mitochondrial and cytosolic fractions were isolated. Spectra show MS1 scans indicating an increase in intensity of a representative labeled peptide from mitochondrial MnSOD (A) but no change in intensity of a labeled peptide for cytosolic Cu/Zn SOD (B). The expression of MnSOD (C) or CuZn SOD (D) was determined by immunoblot analysis using a heavy mitochondrial fraction and cytosolic fraction prepared from SCs were treated for the indicated days with 30 mM glucose. (E) MnSOD expression was normalized to β-actin levels and the average of two separate experiments is expressed as a fold control.
SCs were incubated with 5.5 mM (K0) or 30 mM (K6) glucose for 6 days and mitochondrial and cytosolic fractions were isolated. Spectra show MS1 scans indicating an increase in intensity of a representative labeled peptide from mitochondrial MnSOD (A) but no change in intensity of a labeled peptide for cytosolic Cu/Zn SOD (B). The expression of MnSOD (C) or CuZn SOD (D) was determined by immunoblot analysis using a heavy mitochondrial fraction and cytosolic fraction prepared from SCs were treated for the indicated days with 30 mM glucose. (E) MnSOD expression was normalized to β-actin levels and the average of two separate experiments is expressed as a fold control.
(A) SCs were incubated with 5.5 mM or 30 mM glucose for the indicated time and a heavy mitochondrial fraction was isolated. MnSOD activity was determined after a 30 min preincubation with 2 mM NaCN to inhibit residual Cu/Zn SOD activity and results are the mean ± SEM from three separate experiments. Asterisks indicate p < 0.05 compared to control. (B) SCs were incubated with 5.5 mM or 30 mM glucose for the indicated time and cellular superoxide levels were determined after the addition of 3 µM dihydroethidine. Results are the mean of four experiments perform with 8 replicates each and are expressed as a percent of control. (C) SCs were incubated with 5.5 mM or 30 mM glucose for 6 days, treated with MitoSox Red and MitoTracker Green and mitochondrial levels of superoxide were visualized by confocal microscopy. Incubation with Antimycin A served as a positive control for mitochondrial superoxide generation. (D) Quantitative image analysis of superoxide levels. Results are from a representative experiment performed twice and are the mean ± SEM from at least 200 cells per treatment. Asterisk indicates p< 0.05 versus low and high glucose treatments.
(A) SCs were incubated with 5.5 mM or 30 mM glucose for the indicated time and a heavy mitochondrial fraction was isolated. MnSOD activity was determined after a 30 min preincubation with 2 mM NaCN to inhibit residual Cu/Zn SOD activity and results are the mean ± SEM from three separate experiments. Asterisks indicate p < 0.05 compared to control. (B) SCs were incubated with 5.5 mM or 30 mM glucose for the indicated time and cellular superoxide levels were determined after the addition of 3 µM dihydroethidine. Results are the mean of four experiments perform with 8 replicates each and are expressed as a percent of control. (C) SCs were incubated with 5.5 mM or 30 mM glucose for 6 days, treated with MitoSox Red and MitoTracker Green and mitochondrial levels of superoxide were visualized by confocal microscopy. Incubation with Antimycin A served as a positive control for mitochondrial superoxide generation. (D) Quantitative image analysis of superoxide levels. Results are from a representative experiment performed twice and are the mean ± SEM from at least 200 cells per treatment. Asterisk indicates p< 0.05 versus low and high glucose treatments.
(A) SCs were incubated with 5.5 mM or 30 mM glucose for the indicated time and a heavy mitochondrial fraction was isolated. MnSOD activity was determined after a 30 min preincubation with 2 mM NaCN to inhibit residual Cu/Zn SOD activity and results are the mean ± SEM from three separate experiments. Asterisks indicate p < 0.05 compared to control. (B) SCs were incubated with 5.5 mM or 30 mM glucose for the indicated time and cellular superoxide levels were determined after the addition of 3 µM dihydroethidine. Results are the mean of four experiments perform with 8 replicates each and are expressed as a percent of control. (C) SCs were incubated with 5.5 mM or 30 mM glucose for 6 days, treated with MitoSox Red and MitoTracker Green and mitochondrial levels of superoxide were visualized by confocal microscopy. Incubation with Antimycin A served as a positive control for mitochondrial superoxide generation. (D) Quantitative image analysis of superoxide levels. Results are from a representative experiment performed twice and are the mean ± SEM from at least 200 cells per treatment. Asterisk indicates p< 0.05 versus low and high glucose treatments.
Source: PubMed