male sprague dawley rats were made diabetic with a single intraperitoneal injection of 75 mg / kg stz ( sigma , st louis , mo ) . insulin implants ( two linplant implants ; linshin canada , toronto , on , canada ) were placed subcutaneously into stz - diabetic rats after 18 weeks of diabetes and kept in place for 4 weeks . animal procedures followed the guidelines of the university of manitoba animal care committee using the canadian council of animal care rules . immortalized sc16 schwann cells were cultured in low glucose dulbecco 's modified eagle 's medium containing 125 mg / l c6-lysine ( k6 ) and 84 mg / l c6,n4-arginine ( r10 ) ( cambridge isotopes , andover , ma ) , 10% dialyzed fcs ( atlas biologicals , fort collins , co ) , and antibiotics ( 22 ) . crude mitochondria from labeled cells were obtained by differential centrifugation and purified through a discontinuous nycodenz gradient ( 23 ) . for quantitative analysis of the drg mitochondrial proteome , the k6r10-labeled mitochondria were used as a source of culture - derived isotope tags to serve as internal standards ( 20 ) . after assessing the protein concentration of the preparations , the unlabeled mitochondrial protein ( k0r0 ) obtained from each of the control ( n = 4 ) , diabetic ( n = 3 ) , and diabetic + insulin ( n = 4 ) treatments were mixed in a 2:1 ratio with k6r10 labeled mitochondria . total protein ( 6070 g ) was subjected to sds - page , the gel was stained with colloidal coomassie blue , and lanes were cut into 5  1 cm pieces . a detailed description of these parameters is provided in supplementary table 1 in the online appendix 2 at http://diabetes.diabetesjournals.org/cgi/content/full/db10-0818/dc1 . lumbar drg sensory neurons from adult male sprague - dawley rats were isolated and dissociated as described ( 6,16 ) . cells were plated onto poly - d - l - ornithine hydrobromide and laminin - coated 25-mm glass cover slips ( german glass # 1 , electron microscopy sciences , hatfield , pa ) . neurons were grown in defined hams f-12 medium with n2 additives ( no insulin ) , supplemented with neurotrophic factors : 0.1 ng / ml nerve growth factor , 1.0 ng / ml glial cell line - derived neurotrophic factor , and 0.1 ng / ml neurotrophin-3 ( all obtained from sigma ) . neurons from control rats were cultured with 10 mmol / l d - glucose and 10 nmol / l insulin and neurons from diabetic rats were plated with 25 mmol / l d - glucose and no insulin for 1 to 3 days . cultured neurons from control or diabetic rats were either 1 ) imaged in real time for intracellular ros by loading with 1.2 mol / l 5-(and-6)-chloromethyl-27-dichlorodihydrofluorescein diacetate acetyl ester ( cm - h2dcfda ) , or 2 ) fixed and stained for adducts of 4-hydroxy-2-nonenal ( 4-hne ) ( a product of lipid peroxidation ) , as previously described ( 16 ) . to study the role of polyol pathway in ros production , cultures were treated acutely with the specific sorbitol dehydrogenase ( sdh ) inhibitor , sdi-158 ( 24 ) ( 10 mol / l ; a gift from dr . cultured drg neurons were loaded with 3.0 nmol / l tetramethyl rhodamine methyl ester ( tmrm ; molecular probes , eugene , or ) for 1 h and the fluorescence signal in the axons detected with a carl zeiss lsm510 confocal inverted microscope ( x100 objective ; excitation at 540 nm and emission > 560 nm ) . the tmrm was used in subquench mode  in which decreased fluorescence intensity indicates reduced mitochondrial inner membrane potential ( 25 ) . antimycin a and oligomycin ( sigma ) were injected into the culture media to a final concentration of 10 mol / l and 1 mol / l , respectively , at 1 min after baseline fluorescence measurements . all axons in each field were assessed as the average of fluorescence pixel intensity per axon length using the carl zeiss software package ( 16 ) . intramitochondrial ros generation , mainly superoxide , was detected using the fluorescent mitosox red dye ( molecular probes , catalog # m36008 ) . lumbar drg neurons were loaded with 400 nmol / l of mitosox red ( in 100% anhydrous dmso ; molecular probes ) for 15 min with or without 1.0 mol / l oligomycin at 37c , and then washed three times with f-12 and excited at 514 nm and emission > 560 nm . oxygen consumption was determined at 37c using the oroboros oxygraph-2k ( oroboros instruments gmbh , innsbruck , austria ) ( 8) . mitochondria from freshly isolated and intact lumbar drg were resuspended in kcl medium ( 80 mmol / l kcl , 10 mmol / l tris - hcl , 3.0 mmol / l mgcl2 , 1.0 mmol / l edta , 5.0 mmol / l potassium phosphate , ph 7.4 ) . various substrates and inhibitors of the mitochondrial respiratory chain complexes were used as described ( 8) . enzymatic activities in lumbar drg mitochondrial preparations were performed spectrophotometrically as previously described ( 8) . when appropriate , data were subjected to one - way anova with post hoc comparison using the tukey test or regression analysis with a one - phase exponential decay parametric test with the fisher parameter ( graphpad prism 4 , graphpad software , san diego , ca ) . in all other cases , two - tailed student t tests were performed . to determine the threshold for statistical significance for the proteomic data , proteins showing at least a 20 or 25% increase or decrease were grouped and compared with the entire dataset using a kruskal - wallis nonparametric anova and the dunn multiple comparison test . this analysis indicated that a minimum difference of 25% was necessary for a value to be considered statistically different from the dataset . male sprague dawley rats were made diabetic with a single intraperitoneal injection of 75 mg / kg stz ( sigma , st louis , mo ) . insulin implants ( two linplant implants ; linshin canada , toronto , on , canada ) were placed subcutaneously into stz - diabetic rats after 18 weeks of diabetes and kept in place for 4 weeks . animal procedures followed the guidelines of the university of manitoba animal care committee using the canadian council of animal care rules . mitochondrial preparations from drg were isolated as described ( 8,21 ) . immortalized sc16 schwann cells were cultured in low glucose dulbecco 's modified eagle 's medium containing 125 mg / l c6-lysine ( k6 ) and 84 mg / l c6,n4-arginine ( r10 ) ( cambridge isotopes , andover , ma ) , 10% dialyzed fcs ( atlas biologicals , fort collins , co ) , and antibiotics ( 22 ) . crude mitochondria from labeled cells were obtained by differential centrifugation and purified through a discontinuous nycodenz gradient ( 23 ) . for quantitative analysis of the drg mitochondrial proteome , the k6r10-labeled mitochondria were used as a source of culture - derived isotope tags to serve as internal standards ( 20 ) . after assessing the protein concentration of the preparations , the unlabeled mitochondrial protein ( k0r0 ) obtained from each of the control ( n = 4 ) , diabetic ( n = 3 ) , and diabetic + insulin ( n = 4 ) treatments were mixed in a 2:1 ratio with k6r10 labeled mitochondria . total protein ( 6070 g ) was subjected to sds - page , the gel was stained with colloidal coomassie blue , and lanes were cut into 5  1 cm pieces . a detailed description of these parameters is provided in supplementary table 1 in the online appendix 2 at http://diabetes.diabetesjournals.org/cgi/content/full/db10-0818/dc1 . lumbar drg sensory neurons from adult male sprague - dawley rats were isolated and dissociated as described ( 6,16 ) . cells were plated onto poly - d - l - ornithine hydrobromide and laminin - coated 25-mm glass cover slips ( german glass # 1 , electron microscopy sciences , hatfield , pa ) . neurons were grown in defined hams f-12 medium with n2 additives ( no insulin ) , supplemented with neurotrophic factors : 0.1 ng / ml nerve growth factor , 1.0 ng / ml glial cell line - derived neurotrophic factor , and 0.1 ng / ml neurotrophin-3 ( all obtained from sigma ) . neurons from control rats were cultured with 10 mmol / l d - glucose and 10 nmol / l insulin and neurons from diabetic rats were plated with 25 mmol / l d - glucose and no insulin for 1 to 3 days . cultured neurons from control or diabetic rats were either 1 ) imaged in real time for intracellular ros by loading with 1.2 mol / l 5-(and-6)-chloromethyl-27-dichlorodihydrofluorescein diacetate acetyl ester ( cm - h2dcfda ) , or 2 ) fixed and stained for adducts of 4-hydroxy-2-nonenal ( 4-hne ) ( a product of lipid peroxidation ) , as previously described ( 16 ) . to study the role of polyol pathway in ros production , cultures were treated acutely with the specific sorbitol dehydrogenase ( sdh ) inhibitor , sdi-158 ( 24 ) ( 10 mol / l ; a gift from dr . cultured drg neurons were loaded with 3.0 nmol / l tetramethyl rhodamine methyl ester ( tmrm ; molecular probes , eugene , or ) for 1 h and the fluorescence signal in the axons detected with a carl zeiss lsm510 confocal inverted microscope ( x100 objective ; excitation at 540 nm and emission > 560 nm ) . the tmrm was used in subquench mode  in which decreased fluorescence intensity indicates reduced mitochondrial inner membrane potential ( 25 ) . antimycin a and oligomycin ( sigma ) were injected into the culture media to a final concentration of 10 mol / l and 1 mol / l , respectively , at 1 min after baseline fluorescence measurements . all axons in each field were assessed as the average of fluorescence pixel intensity per axon length using the carl zeiss software package ( 16 ) . intramitochondrial ros generation , mainly superoxide , was detected using the fluorescent mitosox red dye ( molecular probes , catalog # m36008 ) . lumbar drg neurons were loaded with 400 nmol / l of mitosox red ( in 100% anhydrous dmso ; molecular probes ) for 15 min with or without 1.0 mol / l oligomycin at 37c , and then washed three times with f-12 and excited at 514 nm and emission > 560 nm . oxygen consumption was determined at 37c using the oroboros oxygraph-2k ( oroboros instruments gmbh , innsbruck , austria ) ( 8) . mitochondria from freshly isolated and intact lumbar drg were resuspended in kcl medium ( 80 mmol / l kcl , 10 mmol / l tris - hcl , 3.0 mmol / l mgcl2 , 1.0 mmol / l edta , 5.0 mmol / l potassium phosphate , ph 7.4 ) . various substrates and inhibitors of the mitochondrial respiratory chain complexes were used as described ( 8) . enzymatic activities in lumbar drg mitochondrial preparations were performed spectrophotometrically as previously described ( 8) . when appropriate , data were subjected to one - way anova with post hoc comparison using the tukey test or regression analysis with a one - phase exponential decay parametric test with the fisher parameter ( graphpad prism 4 , graphpad software , san diego , ca ) . in all other cases , two - tailed student t tests were performed . to determine the threshold for statistical significance for the proteomic data , proteins showing at least a 20 or 25% increase or decrease were grouped and compared with the entire dataset using a kruskal - wallis nonparametric anova and the dunn multiple comparison test . this analysis indicated that a minimum difference of 25% was necessary for a value to be considered statistically different from the dataset . stz - diabetic rats did not suffer weight loss during the study , but they showed reduced weight gain after 22 weeks of stz - diabetes compared with age - matched controls ( table 1 ) . persistence of diabetes was indicated by elevated nonfasting blood glucose and glycated hemoglobin levels ( table 1 ) . stz - diabetic rats that received insulin supplementation for the final 4 weeks of a 22-week period of diabetes showed a partial , but statistically significant , recovery of body weight , blood glucose , and glycated hemoglobin levels . body weights , plasma glucose , and glycated hemoglobin ( hba1c of treatment groups values are means  sd . starting weights for the groups were 293  8.8 g ( mean  sd ; n = 38 ) . nonfasting blood sugar concentration was measured using the accu - chek compact plus glucometer ( roche , laval , quebec city , canada ) and blood glycated hemoglobin ( hba1c ) levels by the a1cnow + system ( bayer healthcare , sunnyvale , ca ) . * p < 0.001 vs. other groups ; * * p < 0.001 vs. diabetic + insulin ( one - way anova with tukey 's test ) . to quantitatively assess the effect of diabetes and insulin therapy on the mitochondrial proteome of lumbar drg , we labeled sc16 immortalized schwann cells with isotopic forms of lysine ( k6 ) and arginine ( r10 ) and isolated labeled mitochondria to serve as internal standards ( fig . we examined the quantitative accuracy of this approach by mixing the k6r10:k0r0 mitochondria in ratios of 0.75:1 , 1.5:1 , and 3:1 . the k6/k0 or r10/r0 ratios for individual peptides were obtained from maxquant analysis , and a linear response was observed after plotting the average peptide ratio obtained from each mixture against the known mixing ratio ( supplementary fig . 1 in online appendix 1 ) . unlabeled ( k0r0 ) drg mitochondria from the three treatments were then individually mixed in a 2:1 ratio with the k6r10 mitochondria prior to sds - page and ltq - ft ms / ms analysis . from more than 43,600 identified peptides , after culling out contaminants ( n = 30 ) , reverse - decoy hits ( n = 13 ) , and proteins identified by only a single unique peptide , we identified 672 proteins , of which 334 ( 49.6% ) were quantified by at least one unique peptide identified in samples from at least two animals ( supplementary online table 1 ) . the median number of quantified ratios for the three treatments was : control ( n = 5 ) , diabetic ( n = 8) , and diabetic + insulin ( n = 7 ) . of the total proteins identified , 206 ( 30% ) were annotated as mitochondrial / glycolytic and 151 were quantified ( 73% ) . unlabeled ( k0r0 ) mitochondrial fractions were prepared from the lumbar drgs obtained from each animal in the three treatment groups . each k0r0 mitochondrial fraction was mixed in a 2:1 ratio with k6r10-labeled mitochondria obtained from sc16 cells that had been metabolically labeled with c6 lysine ( k6 ) and c6,n4 arginine ( r10 ) for 10 days . the proteins were resolved by sds - page , digested with trypsin , and analyzed by rp - hplc / ltq - ft ms / ms . for each protein , the ratio of k0r0 to k6r10 quantifies the endogenous protein relative to the internal standard . dividing the protein ratios obtained in the diabetic or diabetic + insulin treatment by those obtained from control animals cancels out the k6r10 internal standard and provides the fold control value . the protein expression ratios from the diabetic and diabetic + insulin treatments were binned and the number of proteins per bin counted . c : to determine the effect of diabetes and insulin therapy on mitochondrial versus nonmitochondrial proteins , the expression ratio for each protein was plotted against each treatment . solid and dotted lines demarcate the threshold necessary for proteins to show a significant change in the diabetic and diabetic + insulin treatments , respectively . proteins in between dotted and solid lines did not change with either treatment . yellow shading indicates proteins that were significantly up- or downregulated by diabetes and normalized by insulin therapy . blue shading indicates proteins that were increased by diabetes but not normalized by insulin therapy . green shading indicates proteins not affected by diabetes but increased by insulin therapy . to provide a global view of the effect of diabetes and insulin therapy on protein expression , insulin therapy induced a rightward shift toward normalizing expression and promoted a significant increase in protein expression . pathway analysis found that the proteins associated with mitochondrial dysfunction , oxidative phosphorylation , and ubiquinone biosynthesis ( primarily complex i proteins ) were the most significantly over - represented and showed the greatest percentage of proteins that underwent significant downregulation ( table 2 ) . consistent with the diabetic phenotype , proteins associated with ketone body biology were also over - represented and diabetes increased the expression of succinyl - coa:3-ketoacid - coa transferase one ( scot ) , which is critical in acetoacetate clearance . over - represented canonical pathways identified in the proteomic analysis significance provides the confidence of the association as identified by the p value of the fisher exact test . the ratio provides the percentage of proteins associated with the pathway that underwent a significant change . the total genes column refers to all known genes to be linked to the pathway ( not necessarily identified by the proteomic screen ) . to determine if diabetes and insulin therapy had a distinct effect on mitochondrial versus nonmitochondrial proteins , the expression ratios for each protein were plotted against each treatment ( fig . this analysis indicated that diabetes has little effect on the majority of mitochondrial and nonmitochondrial proteins that were quantified ( the region between solid and dotted lines ) . with rare exception , insulin therapy did not decrease protein expression , but led to a significant increase in the expression of numerous nonmitochondrial proteins ( green shading ) . enrichment analysis of proteins in this region using the biological networks gene ontology ( bingo ) plugin of cytoscape found that the cluster frequency for proteins annotated to the biologic process of translation was 36.1% , a sevenfold enrichment . we also noted a small group of proteins that were significantly increased by diabetes but whose expression was unchanged by insulin ( blue shading ) . bingo analysis of this subset of proteins indicated that small g protein signaling and protein transport were the enriched processes . diabetes caused a statistically relevant change in 27% of quantified mitochondrial proteins , and insulin therapy had an ameliorative effect that , in general , normalized this decrease ( fig . 1c , yellow shading ) . consistent with another proteomic study of heart mitochondria ( 14 ) , bioinformatic analysis found that proteins associated with canonical pathways of mitochondrial dysfunction and oxidative phosphorylation were over - represented and mainly decreased in expression ( table 3 ) . representative examples from a diabetic animal show a 51% decrease in the complex i protein , nadh dehydrogenase fe - s protein 3 ( ndufs3 ) and a 29% decline of mn superoxide dismutase ( mn - sod ) ( fig . however , insulin therapy improved the deficits in ndufs3 and mn - sod expression ( fig . 2c ) as previously characterized using western blotting ( 8,16 ) . on the other hand , diabetes did not alter the expression of atp synthase  ( supplementary fig . 2 in supplementary online appendix 1 ) . effect of diabetes and insulin therapy on representative proteins annotated to oxidative phosphorylation and mitochondrial dysfunction values shown are the mean and the asterisk ( * ) indicates proteins that showed a significant change in expression . percent change represents effect of insulin treatment on the protein expression ratio measured from diabetic rats . representative peptide mass spectra showing the effect of diabetes and insulin therapy on ndufs3 and mnsod . a : the upper spectrum shows the doubly - charged ion of the unlabeled ( m / z 743.90 ) and r10 labeled ( m / z 748.90 ) vvaepvelaqefr peptide of ndufs3 from a control animal . since the peptide is doubly charged , the mass difference is 5 atomic mass units ; and the other peaks represent the isotopic envelope of the monoisotopic peak . the lower spectrum shows the doubly - charged ion of the unlabeled ( m / z 720.91 ) and k6 labeled ( m / z 723.91 ) gdvttqvalqpalk peptide of mn - sod from a control animal . since the peptide is doubly charged , the mass difference is 3 atomic mass units ; and the other peaks represent the isotopic envelope of the monoisotopic peak . b : upper and lower spectra show the same ndufs3 and mn - sod peptides , but from a diabetic animal . the k0r0/k6r10 ratios for each peptide are indicated and the diab / control ratio were obtained after dividing by the control ratios from panel a. c : upper and lower spectra show the same ndufs3 and mn - sod peptide , but from a diabetic + insulin - treated animal . the k0r0/k6r10 ratios for each peptide are indicated and the diab / control ratio were obtained after dividing by the control ratios from panel a. note that the intensity of the k6 and r10 peptides are very similar between the treatments ( a  c ) , indicating that the changes in protein expression are minimally influenced by the internal standard . lumbar drg from age - matched control and 22-week - old stz - diabetic rats were analyzed for rates of oxygen consumption as shown in fig . respiratory chain activity , whether coupled or uncoupled , was significantly depressed in diabetic samples . in agreement with the proteomic data and oxygen consumption results , the enzymatic activities of rotenone - sensitive nadh - cytochrome c reductase ( complex i ) and cytochrome c oxidase ( cox ; complex iv ) , as well as the krebs cycle enzyme , citrate synthase , were significantly decreased in stz - diabetic rats compared with control ( supplementary table 2 ) . a : oxygen consumption was assessed in freshly isolated mitochondria from lumbar drg of age - matched control and 22-week - old diabetic rats using an oroboros oxygraph 2k . coupled respiration rates were measured in the presence of pyruvate ( p ) ( 10 mmol / l ) , malate ( m ) ( 5.0 mmol / l ) , and adp ( 2.0 the addition of fccp ( 0.5 mol / l ) permits a measure of uncoupled respiratory chain activity . addition of ascorbate ( asc ) ( 5.0 mmol / l ) and tmpd ( 0.5 mmol / l ) permit an analysis of complex iv activity that was verified by specific inhibitors . values are mean  sem ; n = 5 . * p < 0.05 vs. controls ; * * p < 0.001 vs. controls . b : images of fluorescence confocal microscopy using tmrm in live cultures of drg neurons isolated from control adult rats showing effect of antimycin a and oligomycin . c : trace of tmrm fluorescence signal in the axons of cultured drg neurons isolated from age - matched controls and stz - diabetic rats . d : shows the area under the tmrm fluorescence trace ( area under the curve ) for control ( open bar ) and diabetic ( filled bar ) neurons . the area under the curve was estimated from the baseline ( at the point of injection ) to a fluorescence level of 0.2 and between time points 1.0 min and 6 min using sums of squares ( shown by dotted line ) . values are the means  sem , n = 6580 axons ; * p < 0.001 compared with control , t test . the rate constant of decay ( k ) = 0.013  0.0004 ( control ) and 0.006  0.0001 ( diabetic ) . half - life of decay = 54.19 s ( control ) and 108.7 s ( diabetic ) . the fisher parametric ( f ) ratio = 409.5 , p < 0.0001 , control vs. diabetic . ( a high - quality digital representation of this figure is available in the online issue . ) adult sensory neurons were cultured for 1 day from age - matched controls and 22-week - old stz - diabetic rats and loaded with tmrm . this dye was used at a subquench concentration where a decrease in fluorescence signal intensity indicated reduced mitochondrial inner membrane potential ( 25 ) . the live neurons were exposed to a combination of antimycin a ( inhibitor of complex iii ) and oligomycin ( inhibitor of atp synthase ) , and the fluorescence signal in axons detected by confocal microscopy . antimycin a blocks electron transfer leading to mitochondrial depolarization , whereas oligomycin inhibits the atpase and prevents reverse pumping of protons and associated generation of a proton gradient . therefore , the mitochondrial membrane potential ( and associated proton gradient ) is completely dissipated in the presence of both these drugs . in the presence of antimycin a + oligomycin , the rate of mitochondrial depolarization was more rapid in axons of normal neurons compared with diabetic neurons ( fig . this suggests that prior to the addition of antimycin a + oligomycin , the axonal mitochondria were more highly polarized in the normal neurons compared with the diabetic neurons . mitochondrial physiology was further investigated by treating cultured neurons from control and diabetic rats with oligomycin alone . blockade of the atpase results in a buildup of the transmembrane proton gradient and induces hyperpolarization of the mitochondrial inner membrane , as indicated by elevated tmrm fluorescence ( fig . a transient hyperpolarization was observed , followed by a recovery due to adaption of the respiratory chain . for example , uncoupling proteins become active and dissipate the proton gradient under a high inner membrane potential ( 27 ) . diabetic neurons exhibited a significantly greater level of hyperpolarization upon oligomycin application and the adaptive response was impaired . impaired respiratory function is associated with reduced ros generation in the mitochondrial matrix of cultured neurons isolated from stz - diabetic rats . a : tmrm fluorescence trace of oligomycin - induced mitochondrial inner membrane hyperpolarization in the axons of control and diabetic neurons . inset shows the area under the tmrm fluorescence trace ( auc ) for control ( open bar ) and diabetic ( filled bar ) neurons . the auc was estimated from the baseline ( at the point of injection , dotted line ) , and between time points of 1.0 min and 4 min using sums of squares . values are mean  sem , n = 6580 axons,*p < 0.01 compared with control , t test . b  e : images of mitosox red fluorescence in cultures of drg neurons showing the effect of 5.0 mol / l fccp . f : quantification of real - time mitosox red fluorescence levels in the axons of cultured drg neurons after 5.0 mol / l fccp treatment . f ratio = 32.48 , p < 0.0001 ( control vs. diabetic with or without oligomycin by one - way anova ) . values are mean  sem , n = 3573 axons ; * * p < 0.001 compared with diabetic or diabetic + oligomycin - treated cells by one - way anova . ( a high - quality digital representation of this figure is available in the online issue . ) we determined if the respiratory chain was contributing to oxidative stress in diabetic neurons by loading cells with the mitochondrially - targeted ros detector , mitosox red . a subset of diabetic neurons was pretreated with oligomycin to hyperpolarize the inner mitochondrial membrane and maximize loading of mitosox red into the mitochondrial matrix . neurons were treated with the uncoupler , carbonylcyanide - p - trifluoromethoxyphenylhydrazone ( fccp ) , to dissipate the transmembrane electrochemical gradient and enhance the rate of electron transfer . increased respiratory chain activity leads to augmented electron leakage and associated generation of ros , primarily superoxide . in normal neurons this was demonstrated with elevated fccp - induced mitosox red fluorescence ( fig . 4f and g ) . diabetic neurons , with or without prior oligomycin treatment , exhibited lower mitosox red fluorescence intensities indicative of reduced levels of superoxide being generated by the respiratory chain . parallel cultures demonstrated elevated oxidative stress in axons of diabetic neurons under 25 mmol / l glucose versus control neurons as exhibited by raised dichlorodihydrofluorescein ( dcf ) fluorescence and enhanced staining for adducts of 4-hne ( fig . 5a subsets of these cultures were treated acutely with the specific sdh inhibitor , sdi-158 , to investigate the role of the polyol pathway in ros production . figure 5 g and h shows that blockade of high glucose - dependent sorbitol production results in reduced ros generation in axons of diabetic neurons . axons of sensory neurons from stz - diabetic rats exhibit elevated oxidative stress that is ameliorated by the blockade of sdh . images of ros levels in axons at 24 h in adult drg neuron culture from ( a ) control and ( b ) stz - diabetic rats . cultures were stained for ros using cm - h2dcfda dye ( dcf is the fluorescent product resulting from oxidation ) . values are means  sem , n = 4457 axons , * p < 0.05 by t test . immunofluorescent images of accumulation of adducts of 4-hne in axons in sensory neuron cultures after 3 days ; ( c ) is control and ( d ) is diabetic culture . values are means  sem , n = 1927 axons , * * p < 0.01 by t test . g : trace of dcf - fluorescence signal in the axons of cultured drg neurons isolated from age - matched controls and stz - diabetic rats and treated acutely with 10 mol / l sdi-158 . k = 0.09  0.02 ( control ) and 0.13  0.009 ( diabetic ) . half - life of decay = 7.5 min ( control ) and 5.5 min ( diabetic ) . h shows the area under the dcf fluorescence trace ( auc ) for control ( open bar ) and diabetic ( filled bar ) neurons . the auc was estimated from 0.2 to 1.6 on fluorescence axis and between time points 0 to 22 min using sums of squares ( dotted lines show upper and lower limits ) . values are means  sem , n = 4251 axons ; * * p < 0.01 compared with control by t test . ( a high - quality digital representation of this figure is available in the online issue . ) the results show that respiratory chain components of the mitochondrial proteome are downregulated in drg in diabetes and this phenotypic alteration was associated with impairment in respiratory chain activity . in addition , for the first time , this study demonstrates that altered mitochondrial proteome expression is linked to altered mitochondrial physiology in axons of diabetic neurons . and finally , although oxidative stress was present in axons of diabetic neurons , the results show that polyol pathway activity , not aberrant respiratory chain function , contributes to generation of ros . we used silac to provide a set of culture - derived isotope tags ( 20 ) to serve as internal standards for quantifying the effect of diabetes on the composition of the mitochondrial proteome from drg . one advantage of culture - derived isotope tags is the quantitative accuracy that can be achieved relative to label - free approaches ( 28 ) , especially for low abundant proteins . despite our small sample size , a 25% change in expression was sufficient to reach statistical significance , and the number of mitochondrial proteins that exceeded this threshold was similar to that previously reported in mitochondria isolated from heart tissue of diabetic akita mice ( 14 ) . further , the results of our unbiased quantification were in close agreement with those obtained by targeted immunoblot analyses for cox subunit iv , ndufs3 , atp synthase , and mn - sod in two prior studies ( 8,16 ) . similar to a previous gene array study delineating alterations in mrna expression in drg from diabetic rats ( 29 ) , the magnitude of changes observed in the mitochondrial proteins were rather modest and averaged 0.44  0.16-fold in either direction . interestingly , the gene array study reported that after 2 months of diabetes , no modification occurred in gene expression of enzymes associated with the tricarboxylic acid cycle . we also observed no changes in pyruvate dehydrogenase and in six of eight of the tricarboxylic acid cycle enzymes at the protein level , the exceptions being fumarate hydratase and succinyl co - a ligase . similarly , with the exception of hexokinase 1 , the remaining glycolytic enzymes were not altered , in agreement with mrna expression studies at 2 months of diabetes ( 29 ) . we previously reported that hexokinase 1 localizes to mitochondria of drg ( 30 ) and this protein was reproducibly detected in the heavy mitochondrial fraction of all animals . interestingly , although the hexokinase gene and protein expression were not modified after 23 months of diabetes in two studies ( 2930 ) , expression was decreased after 22 weeks of diabetes in the present study . since this was the only glycolytic enzyme that significantly changed , it is tempting to speculate that its lowered expression also has a function separate from glucose metabolism . in this regard , sensory neurons from diabetic adult animals have an impaired ability to support neurite development ( 16 ) and directly inhibiting hexokinase activity blocks neurotrophin - induced neuritogenesis ( 31 ) . in the cardiac system , diminished mitochondrial respiratory function caused by diabetes has also been identified by proteomic and gene array techniques ( 14,32 ) . these broad changes in gene expression could be triggered by altered activity of key upstream regulators . for example , in human skeletal muscle in type 2 diabetes , the transcriptional regulator nrf-1 and the transcriptional coactivator peroxisome proliferator - activated receptor- coactivator 1  ( pgc-1 ) were downregulated and corresponded with reduced expression of proteins that regulate cellular energy metabolism , including mitochondrial biogenesis and oxidative phosphorylation ( 33,34 ) . in fact , our preliminary data demonstrated a significant reduction in activity of amp kinase , a regulator of pgc1- , in drg in type 1 diabetic rodents ( manuscript in preparation ) . initial exposure to high glucose concentration over 1 to 4 weeks in drg in diabetic rats is linked to upregulation of glycolytic pathway expression ( mrna ) ( 29 ) . studies in endothelial or schwann cells demonstrate that acute exposure to glucose elevates ros and respiratory chain activity , respectively ( 2,23 ) . therefore , in the short term , hyperglycemia triggers enhanced glycolysis and associated respiratory chain activity ( and possibly ros ) . however , in the longer term , the high intracellular glucose concentration provides an ample supply of atp via several nonmitochondrial - dependent pathways . consequently , the metabolic phenotype of the cell adapts and functions in the absence of a dependence on the tricarboxylic acid cycle and oxidative phosphorylation for atp production , possibly by initiating a process homologous to the thus , rates of electron donation to the respiratory chain are suboptimal in neurons in long - term diabetic rats and may predispose to lower rates of mitochondrial respiratory chain activity and oxidative phosphorylation . key metabolic activity sensors and/or regulators such as ampk and nrf-1 are putative candidates for this modulation , although it is unlikely that subsequently adapted metabolism of glucose is channeled through glycolysis in isolation . in this regard , elevated glucose flux through the aldehyde and aldose reductase pathway could be critical ( 24,36 ) . for example , studies in lens ( 37 ) , retina ( 5 ) , and cardiac tissue ( 38 ) in medium- to long - term animal models of type 1 diabetes show that parts of the glycolytic pathway function are depressed . ros production linked to enhanced electron leakage from the respiratory chain induced by uncoupling , and theoretically comprising superoxide , was lower in the axons of neurons from diabetic rats ( fig . 4 and 5 g provide preliminary evidence that the sources of ros in axons of diabetic neurons are not from aberrant respiratory chain function , but in part from polyol pathway activity . the latter pathway has been proposed as a source of ros through a putative sorbitol accumulation - dependent nadph oxidase route in previous studies ( 24,36 ) . downregulation of the respiratory chain machinery would be predicted to lead to depolarization of the mitochondrial membrane ( fig . 3c was not the result of resistance to uncoupling in the diabetic neurons since complementary measures of respiratory chain activity show lower rates of electron transfer in diabetic neurons ; also , see fig . 1 in chowdhury et al . ( 8) . these findings differ from those in cultured endothelial cells where high glucose concentration enhanced mitochondrial membrane potential and induced elevated ros ( 23 ) . figure 4a reveals that oligomycin treatment caused a greater level of mitochondrial inner membrane hyperpolarization above baseline in diabetic neurons compared with normal neurons , further highlighting that adult sensory neurons with a history of diabetes and under high glucose concentration behave differently to endothelial cells . the adaption of mitochondria in normal neurons to hyperpolarization was not observed in diabetic neurons , again stressing the aberrant phenotype of mitochondrial physiology . uncoupling proteins such as adenine nucleotide transporters ( ant1/2 ) contribute to the dissipation of a high mitochondrial membrane potential ( 27 ) and expression was depressed in diabetic mitochondria ( supplementary table 1 ) . in conclusion , our proteomics data reveal a range of altered expression profiles in the mitochondrial proteome of drg from diabetic rats . this modified expression pattern was associated with aberrant mitochondrial respiratory chain physiology and function . under high glucose concentration , the neuron cell body perceives that mitochondrial function can be downgraded ; however , this may ignore the unique high - energy requirements of the nerve ending and contribute to distal axon degeneration . for example , growth cone motility that underpins axonal plasticity and regeneration in the skin has an exceedingly high demand for atp because of significant levels of actin treadmilling ( 39 ) . impaired respiratory chain function did not elevate ros generation , even though oxidative stress was observed in axons . in fact , the lower rates of respiratory chain activity were linked to mitochondrial membrane depolarization , improper adaption to oligomycin - induced membrane hyperpolarization , and reduced levels of superoxide derived from electron leakage during electron transport . in axons of neurons from long - term diabetic rats , sites of ros production colocalize with the mitochondrial compartment ( 16,40 ) . for example , nadph oxidase has been localized to the mitochondrial compartment of kidney cortex and mesanglial cells and mediates elevated ros under high glucose concentration ( 41 ) .