Original Article Proteomic quantitative study of dorsal root ganglia and sciatic nerve in type 2 diabetic mice Marc Leal-Julià 1, 2, 6, Jorge J. Vilches 1, 3, Andrea Onieva 1, 2, 6, Sergi Verdés 1, 2, 6, Ángela Sánchez 1, 2, 6, Miguel Chillón 1, 2, 4, 6, Xavier Navarro 1, 3, 5, Assumpció Bosch 1, 2, 5, 6, * ABSTRACT Objective: Peripheral neuropathy is the most common and debilitating complication of type 2 diabetes, leading to sensory loss, dysautonomia, hyperalgesia, and spontaneous noxious sensations. Despite the clinical and economic burden of diabetic neuropathy, no effective treatment is available. More preclinical research must be conducted in order to gain further understanding of the aetiology of the disease and elucidate new therapeutic targets. Methods: The proteome of lumbar dorsal root ganglia and sciatic nerve of BKS-db/db mice, which contain a mutation of the leptin receptor and are an established type 2 diabetes model, was characterized for the first time by tandem mass tag labelling and mass spectrometry analysis. Results: Proteomic analysis showed differentially expressed proteins grouped into functional clusters in db/db peripheral nerves compared to control mice, underlining reduced glycolytic and TCA cycle metabolism, higher lipid catabolism, upregulation of muscle-like proteins in DRG and downregulation in SCN, increased cytoskeleton-related proteins, a mild dysregulation of folding chaperones, activation of acute-phase and inflammatory response, and alterations in glutathione metabolism and oxidative stress related proteins. Conclusions: Our data validate previous transcriptomic and metabolomic results and uncover new pathways altered in diabetic neuropathy. Our results point out that energetic deficiency could represent the main mechanism of neurodegeneration observed in diabetic neuropathy. These findings may provide important information to select appropriate targets to develop new therapeutic strategies. Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Keywords Diabetes; Obesity; Neuropathy; Proteomics; Dorsal root ganglia; Sciatic nerve 1. INTRODUCTION The exact mechanism that leads to the development of DN in type 2 diabetes is not completely known yet, but there is evidence that Diabetic complications represent the main reasons of disability, connects hyperglycaemia and dyslipidaemia with the activation of reduced life expectancy, and economic cost associated with diabetes multiple downstream pathways, like polyol pathway (hyperglycaemia), [1]. Diabetic neuropathy (DN) is one of the most common and debili- increased reactive oxygen species (ROS) formation and inflammation, tating complications of diabetes, affecting 50e60% of patients with glycation, and metabolic flux dysregulation, collectively resulting in type 1 and type 2 diabetes, although neurophysiological alterations are endoplasmic reticulum (ER) and mitochondrial dysfunction, impaired found in a majority of patients. It may eventually result in foot ulcer- transport, and abnormal neurotrophic signalling in dorsal root ganglia ations and lower limb amputations, accounting for up to 15% of this (DRG), and autonomic ganglia neurons, axons and Schwann cells [5]. type of intervention [2]. There is no cure for DN other than strict glycaemic control, dietary There are different forms of DN, but the most common presentation is changes, and exercise and management of neuropathic pain [3]. a chronic sensorimotor polyneuropathy. Patients develop abnormal However, glucose control alone in type 2 diabetic patients has shown sensory perception, including paraesthesia, reduced sensitivity to modest or no effect on preventing the progression of DN in large touch and temperature, and pain hypersensitivity and spontaneous clinical trials [6]. On the other hand, studies involving more than burning sensation in a “stocking-and-glove” pattern [2,3]. DN is 11,000 individuals from different countries consistently demonstrated characterized by a progressive distal-to-proximal degeneration of that obesity is independently associated with neuropathy [7]. Multiple peripheral nerves, comprising axonal dying-back degeneration, fibre molecular abnormalities may contribute to DN, and its treatment may loss, segmental demyelination, remyelination, and abnormal regen- require multiple strategies, including neuroprotection and repair. erative sprouts. Diabetic nerves develop vascular abnormalities, Over the past decade, with the introduction of high-throughput gene leading to reduced blood flow and hypoxia [4]. expression profiling assays and, more recently, metabolomics, the molecular landscape of the peripheral nervous system (PNS) of 1 Institute of Neurosciences, Univ. Autònoma de Barcelona, Spain 2Department of Biochemistry and Molecular Biology, Univ. Autònoma de Barcelona, Spain 3Department of Cell Biology, Physiology and Immunology, Univ. Autònoma de Barcelona, Spain 4Institut Català de Recerca i Estudis Avançats (ICREA), Barcelona, Spain 5Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas (CIBERNED), Spain 6Unitat Mixta UAB-VHIR, Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain *Corresponding author. Institute of Neurosciences, Edifici H, Universitat Autònoma de Barcelona, E-08193, Bellaterra, Spain. E-mail: assumpcio.bosch@uab.cat (A. Bosch). Received July 8, 2021  Revision received October 27, 2021  Accepted November 26, 2021  Available online 30 November 2021 https://doi.org/10.1016/j.molmet.2021.101408 MOLECULAR METABOLISM 55 (2022) 101408 Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1 www.molecularmetabolism.com Original Article diabetic murine models [8e12] and diabetic patients [13] has been sweat glands (aSG) and the estimated sweat output (SO) per secreting studied. To our knowledge, there are no studies exploring the effect of gland [18]. type 2 diabetes on the nervous system proteome. Poor correlation between transcriptomics and proteomics results has been demon- 2.4. Morphometry of tibial nerve strated, supporting the need for a multi-strategy approach [14]. In the Tibial nerve segments taken above the ankle were fixed in current study, we used the BKS-db/db mice which bear a mutation in glutaraldehyde-paraformaldehyde (3%:3%) in cacodylate buffer 0.1 M, the leptin receptor and show the typical characteristics of type 2 post-fixed with 1% OsO4 þ 0.8% potassium hexacyanoferrate in PBS, diabetes. It is one of the few type 2 diabetes mouse strains that dehydrated with acetone, and embedded in SPURR resin (Sigma- spontaneously develops DN [15]. We characterized the proteome of Aldrich, St. Louis, MO, US). Light microscopy images were acquired on lumbar DRG and sciatic nerve (SCN) of these mice at the beginning of 0.7-mm-semithin sections stained with toluidine blue, under a Nikon DN development by TMT labelling and LC/MS/MS analysis to gain ECLIPSE 80i microscope. Three to four fields, covering at least 300 further understanding of the effects of the disease and to elucidate new myelinated fibres, were randomly selected for morphometrical ana- targets for the development of advanced therapeutic strategies. Pro- lyses using ImageJ and ObjectJ plugin [17]. teomic results were validated by western blot, selecting relevant differentially expressed proteins. 2.5. Cutaneous innervation evaluation Plantar pads were fixed overnight with 4% PFA-PBS and cryoprotected 2. RESEARCH DESIGN AND METHODS in 30% PBS-sucrose for 48 h. Fifty mm thick cryotome pad sections were incubated with PGP9.5 antibody, and intraepidermal nerve fibres Experimental design is presented in Supplementary Figure 1. (IENFs) were counted under fluorescence microscopy [17]. Repre- sentative images from footpads were taken under a Nikon Eclipse 90i 2.1. Animals microscope to quantify the sweat gland area (SGA) and the percentage Male BKS-Leprdb/db (Envigo, Horst, The Netherlands) and Lean litter- of immunoreactive area using ImageJ software [19]. mate (BKS-Leprwt/wt) mice were used. We chose males over females since male db/db mice develop a more severe form of neuropathy [16]. 2.6. Immunohistochemistry The db/db mouse develops obesity from 4e5 weeks of age, insulin Anesthetized animals were perfused with 4% PFA-PBS buffer. DRG resistance and hyperglycaemia at 4e8 weeks of age. Mice were fed and SCN were cryoprotected with 30% sucrose and embedded in ad libitum with a standard diet (2014 Teklad Global; Envigo) and kept Tissue-Tek OCT compound (Sakura, Alphen aan den Rijn, Netherlands) under a lightedark cycle of 12 h. Diabetes was assessed by to obtain 10-mm-thick sections. Fluorescence signal was detected with measuring fasting blood glucose weekly with a Glucometer ContourÒ a laser confocal microscope ZEISS LSM 700. Antibodies list is in XT (Bayer, Leverkusen, Germany). Animals with glycaemia below Supplementary Table 1. 250 mg/dL were not included in further studies. Biochemical param- eters were measured by the Veterinary Clinical Facility (UAB, Bellaterra, 2.7. Relative protein quantification using TMT-LC/MS/MS and Spain). Animal care and experimental procedures were approved by bioinformatics analysis the Biosafety and the Ethical Committees of the Universitat Autònoma Proteomic study was performed independently with DRG and SCN at de Barcelona. 15 weeks of diabetes by CSIC/UAB Proteomics Facility. Six pools were prepared using two samples in each pool, thus obtaining three pools 2.2. Electrophysiology testing for each condition. Eighty mg of protein in RIPA buffer was digested by Nerve conduction tests were performed after 12, 17, 21, and 25 weeks Filter Aided Sample Preparation (FASP) method [20] using 200 mM of diabetes. Under anaesthesia with pentobarbital (50 mg/kg i.p.), the triethylamonium bicarbonate buffer. Each digested pool was labelled sciatic nerve was stimulated percutaneously through a pair of needle using different TMTsixplexÔ reagents (ThermoFisher, Waltham, MA, electrodes placed first at the sciatic notch and then at the ankle, US). Amine-reactive Thermo Scientific TMTÔ Isobaric Mass Tagging delivering pulses of 0.05 ms up to 25% above the intensity that gave a reagents enable multiplex quantitation of proteins using tandem mass maximal response. The compound muscle action potential (CMAP) was spectrometry. Sample fractionation using a SCX ion-exchange column recorded from the third interosseus plantar muscle with microneedle was performed to reduce sample complexity. Samples were injected electrodes. The sensory compound nerve action potential (CNAP) was using a C18 column and an analytic column connected on-line to the recorded from caudal nerves by stimulating at a distal site and high-resolution mass spectrometer LTQ-Orbitrap XL (ThermoFisher). recording with microneedles placed at the base of the tail. Evoked Peptide identification was performed using the Proteome Discoverer action potentials were amplified and displayed on a storage oscillo- v1.4 software (ThermoFisher) under 1% false discovery rate (FDR) scope (Tektronix 420, Berkshire, UK). The nerve conduction velocity criterion and using Uniprot 2017-10 database from Mus musculus. (NCV) was calculated for each segment tested: motor NCV (MNCV) for Protein quantification was done based on the intensity of reporter ions the sciatic notch-ankle segment and sensory NCV (SNCV) for a 35-mm labelled with TMT and considering only the unique peptides. Venn segment from the tail. During electrophysiological tests, the animals diagram was generated using Venny [21]. Characterization of protein were placed over a warm flat steamer to maintain body temperature localization was done using WebGestalt platform [22], whereas func- [17]. tional enrichment analysis was performed by DAVID bioinformatics resources 6.8 [23]. Functional data was validated with String v11.0 2.3. Sweating test [24] and GeneRanker (Genomatix, Munich, Germany) software. Mice were immobilized in a plastic tube and sweating was stimulated by subcutaneous injection of pilocarpine nitrate (5 mg/kg), which in- 2.8. Real time PCR duces a maximal sweating response. A silicone mould of the plantar Lumbar DRG and SCN were homogenized separately with Tissue Lyser surface of the hindpaw (Silasoft Normal, Detax, Ettlingen, Germany) LT at 50 Hz using QIAzol (Qiagen, Hilden, Germany) to obtain total RNA. was done after 10 min of stimulation to determine the number of active Messenger RNA was retrotranscribed using iScriptÔ cDNA Synthesis 2 MOLECULAR METABOLISM 55 (2022) 101408 Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). www.molecularmetabolism.com Table 1 e Summary of metabolic, electrophysiological, and morphometric parameters, sweat output test and IENFD and SG innervation in male db/db mice compared to lean control. Parameter Metabolic parameters Lean Db/Db Weeks of age 21 21 Weeks of diabetes - 15 Body weight (g) 27.85  0.80 37.83  1.48d Fasting blood glucose (mg/dL) 177.78  9.87 HI (>600 mg/dL) Fructosamine (mmol/L) 271.14  3.90 372.53  13.53c Free fatty acids (mmol/L) 0.84  0.06 1.09  0.06a Weeks of age 31 33 Weeks of diabetes - 25 Body weight (g) 30.33  2.12 29.93  3.47 ns Fasting blood glucose (mg/dL) 188.6  6.39 523.4  17.46d Fructosamine (mmol/L) 258.9  17.75 361.2  29.50c Free Fatty acids (mmol/L) 0.65  0.07 1.05  0.11b #Parameter Electrophysiological parameters Db/Db Weeks of diabetes 12 17 21 25 CMAP 86.57  2.84a 70.83  4.55b 71.64  5.89b 67.89  9.38b MNCV 85.16  6.20a 72.25  6.94b 78.42  8.76a 84.19  4.68a CNAP 96.29  7.97 ns 91.31  20.87 ns 56.23  9.43c 39.68  7.82d SNCV 85.00  1.62c 92.51  3.93 ns 77.74  2.71d 66.01  1.02d #Percentage of change compared to lean mice Parameter Morphometric parameters Lean Db/Db Weeks of diabetes - 25 G-ratio (perimeter) 0.626  0.004 0.666  0.008b Myelin thickness (mm) 1.729  0.062 1.579  0.046a Myelinated fibres/mm2 46,947  4105 58,953  1454a Parameter Sweat output test Lean Db/Db Weeks of diabetes - 12 Number of active SG 328.7  8.53 272.0  15.35a SO/active SG 0.49  0.04 0.24  0.08b Parameter Footpad PGP9.5 staining Lean Db/Db Weeks of diabetes - 15 SGA (mm2) 16723.14  1186.37 12094.98  1037.85b % innervated SGA 32.99  1.93 24.97  2.80a IENFD (NNF/mm) 50  2.30 24  7.68b n ¼ 6e9 animals per group for metabolic parameters, n ¼ 3e5 animals per group for morphometric parameters and n ¼ 7e13 animals per group for electrophysiology, sweating test and immunohistochemistry. a p < 0.05. b p < 0.01. c p < 0.001. d p < 0.0001, by Student’s T test. Kit, and the analysis of expression was performed by CFX96 TouchÔ 10% SDS-polyacrylamide gel electrophoresis. Immunoreactive pro- Real-Time PCR Detection System with SYBRÒ Green Master Mix (Bio- teins were detected by ETA C Ultra 2.0 ECL (Cyanagen, Bologna, Italy) Rad, Hercules, CA, US). Amplifications were as follows: heat activation and the ChemiDoc MP Imaging System (Bio-Rad). Protein bands were (3 min, 95  C), followed by 39 cycles of 95  C (10 s) and 58  C (30 s). quantified by densitometry using ImageJ and the results were Quantification relative to Rplp0 and Ywhaz controls was calculated normalized by actin or tubulin. Antibodies list is in Supplementary using the Pfaffl method. Primers’ list is in Supplementary Table 2. Table 3. 2.9. Western blot 2.10. Preparation of BSA-conjugated palmitate DRG and SCN, contralateral to those used in proteomic analysis, were Conjugation protocol from Seahorse Bioscience (Agilent, Santa Clara, sonicated and homogenized in RIPA lysis buffer with Tergitol NP-40 CA, US) was followed using Ultra Fatty Acid Free BSA (Roche, Merck) (Sigma-Aldrich) and Protease Inhibitor Cocktail (Merck, Darmstadt, and sodium palmitate (Sigma-Aldrich) to obtain 6:1 M ratio Palmita- Germany). Protein concentration was determined by PierceÔ BCA te:BSA. NSC-34 (motoneuron) and ND7/23 (DRG) hybrid cell lines were Assay (ThermoFisher). Ten to 20 mg of the sample was separated on selected to study the effect of palmitate in vitro. As negative controls, MOLECULAR METABOLISM 55 (2022) 101408 Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 3 www.molecularmetabolism.com Original Article Figure 1: Peripheral neuropathy profiling. A: G-ratio (axonal diameter/fibre diameter) versus axonal diameter (mm) plot from the whole set of analysed nerve fibres showing higher g-ratio due to thinner myelin in db/db compared with lean mice. B: Photographs of representative silicone moulds of one footpad with retained impressions of sweat droplets at 10 min after pilocarpine administration, showing that the number of active sweat glands (SG) and the sweat output per gland (SO/active SG) was lower in db/db mice. C: Fluorescence images of PGP9.5-immunostained plantar pads showing that intraepidermal nerve fibers and innervation of sweat glands were reduced in db/db mice. Scale bar ¼ 100 mm. BSA and methyl palmitate (non-metabolisable form of palmitate, 62 o C (30 s) and 72 o C (30 s), and a final step of 72 o C for 10 min. Sigma-Aldrich) were produced following the same protocol, and Primers used enabled the amplification of both transcript variants, thapsigargin (Sigma-Aldrich) was used as a positive inductor of ER unspliced Xbp1 (uXbp1, 330 bp) and spliced Xbp1 (sXbp1, 304 bp): stress. Fwd, 50 -TGAGAACCAGGAGTTAAGAACACGC-30 ; Rv, 50 -TTCTGGGTA- GACCTCT GGGAGTTCC-30 . PCR products were run into a 7% acryl- 2.11. Xbp1 alternative splicing amide gel. RNA from cell lines was obtained following the same protocol as for tissue samples. Endoplasmic reticulum stress was evaluated by qPCR 2.12. Statistical analysis using primers list in Supplementary Table 2 and by Xbp1 mRNA Data are expressed as mean  SEM. Electrophysiological, functional alternative splicing. Xbp1 splicing was assessed by conventional PCR: and histological results were analysed using Student’s t test comparing heat activation (95 o C, 5 min), followed by 30 cycles of 95 o C (1 min), db/db to lean mice, or two-way ANOVA followed by Bonferroni post- 4 MOLECULAR METABOLISM 55 (2022) 101408 Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). www.molecularmetabolism.com hoc test. For proteomic analysis, differentially expressed proteins were db/db mice, whereas values were maintained in the lean mice. Sen- determined using DanteR software [25] performing one-way ANOVA sory caudal nerve conduction also showed a progressive decline with a test followed by Benjamini & Hochberg correction to control the FDR. marked reduction at 25 weeks. Morphometric characterization of tibial Differences were considered significant when p < 0.05. nerves showed a higher g-ratio due to thinner myelin in db/db at 25 weeks compared with lean mice, corresponding to the decline of NCV. 3. RESULTS Morphometric analysis at previous time points did not detect any difference (data not shown). We also observed higher myelinated fibre 3.1. Peripheral neuropathy profile density in tibial nerves from diabetic mice as well as a shift towards Ninety four percent of db/db males spontaneously developed dia- smaller diameter fibres in db/db mice (Figure 1A). These changes may betes at 9 weeks of age as a result of severe obesity. Db/db mice indicate a degeneration of large fibres followed by a regenerative showed progressive increase in weight and glycaemia, reaching process that increases the number of smaller myelinated fibres values over 50 g and 600 mg/dL, respectively, until 12 weeks of [26,27]. Disorders of sweating, caused by degeneration of post- diabetes, then started to lose weight, despite sustained hyper- ganglionic sudomotor axons, are common in DN [28]. In agreement, glycaemia and high levels of serum free fatty acids and fructosamine the number of aSG and the SO per gland was lower in db/db than in (Table 1). This phenomenon was observed in multiple batches of lean mice already at 13 weeks of diabetes (Figure 1B, Table 1), animals. indicating full denervation of some SG and partial denervation of still We characterized DN in these animals by electrophysiological and responsive glands. Loss of innervation of SG was confirmed by sweating tests, nerve histology and cutaneous innervation (Figure 1, immunolabelling against PGP9.5 of plantar pads (Figure 1C). Diabetic Table 1). We observed a consistent decline in CMAP amplitude and mice showed a significant reduction of SGA innervation and of the MNCV from the first time point analysed (12 weeks of diabetes) in the IENFD, confirming a distal autonomic and sensory neuropathy [29]. Figure 2: Proteomic dataset characterization. A, B: Cellular component categories plot of DRG and SCN data obtained using WebGestalt software and gene ontology database. C, D: Volcano plot showing statistical significance (log10 p-value) versus magnitude of change (log2 fold change) of the whole dataset from DRG and SCN. In a volcano plot, the most upregulated genes are towards the right, the most downregulated genes are towards the left, and the most statistically significant genes are towards the top. Differentially expressed proteins with a fold change 1.5 or 1.5 and a p-value < 0.01 are highlighted in red. MOLECULAR METABOLISM 55 (2022) 101408 Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 5 www.molecularmetabolism.com Original Article Figure 3: Functional annotation analysis. A, B: Most relevant functional clusters obtained by DAVID 6.8 analysis are represented for DRG and sciatic nerve data. In red, proteins upregulated and, in green, downregulated proteins for each biological term are shown. Functional clusters are ordered according to an enrichment score for the group, based on the geometric mean of annotation terms’ p-values. 3.2. Functional enrichment those genes with larger fold changes that were also statistically sig- Based on electrophysiological and morphometric data, we selected db/ nificant (Figure 2). We used DAVID 6.8 to identify the most significant db mice with 15 weeks of diabetes to characterize the DRG and SCN biological functions in the datasets. Functional annotation clustering proteome before morphometric changes were detected. A proteomic allowed to pool similar biological processes, focussing on enrichment study of DRG identified 866 proteins, whereas in SCN we found 843. scores higher than 1.3 (p < 0.05). Figure 3 shows the most relevant DanteR software determined 286 and 312 differentially expressed functional clusters obtained by DAVID analysis and the number of proteins for DRG and SCN, respectively between db/db and lean males proteins found up or downregulated for each biological term. (one-way ANOVA following by Benjamini & Hochberg correction for To generate a pathway network of interconnected proteins, we used multiple testing). Regarding DRG, 185/286 proteins were upregulated String software (Supplementary Figure 3 and Supplementary Tables 4 and 101 downregulated. Of those, 68 proteins showed ratios higher and 5). In DRG, the network consisted in 221 nodes and 539 edges than 1.2, whereas 31 proteins were downregulated below ratios of 0.8. with an enrichment p-value <1.0e-16, indicating that these proteins Concerning SCN, 217/312 proteins were increased, 117 of which with are at least partially biologically interconnected. The protein associa- a ratio higher than 1.2. 95 were downregulated, 57 with a fold change tion network from SCN involved 308 nodes connected through 654 lower than 0.8. edges, also with significantly more interactions than expected. The differentially expressed proteins from DRG and SCN were We analysed datasets using GeneRanker software to perform a similar compared using a Venn diagram, showing a 34.3% of proteins shared analysis as DAVID but in an automated way, using conventional da- between both data sets (Supplementary Figure 2). tabases and existing bibliography as data source. We selected bio- We used WebGestalt to characterize protein localization based on logical functions derived from GO-terms as the most relevant subcellular categories from Gene Ontology and volcano plot to identify information (Supplementary Tables 6 and 7). 6 MOLECULAR METABOLISM 55 (2022) 101408 Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). www.molecularmetabolism.com Table 2 e DRG proteins highlighted in the results section are shown with the corresponding fold change ratio and grouped by functional clusters. DRG Oxidative stress 60S ribosomal proteins Acute-phase response (positive) GSTM1 (1.32) P2 (0.89) Haptoglobin (2.23) GSTM2 (1.10) L4 (0.83) Orosomucoid 1 (1.52) GTSM5 (1.37) L7 (0.88) Fibrinogen gamma chain (1.55) IDH2 (1.35) L7a (0.87) Complement C3 (1.68) SOD1 (1.13) L10a (0.79) Hemopexin (1.63) Glyoxalase I (1.12) L13 (0.88) Inter-alpha-inhibitor heavy chain 4 (1.64) Peroxiredoxin-1 (1.08) L17 (0.82) Peroxiredoxin-6 (1.11) L21 (0.75) Acute-phase response (negative) L30 (0.86) Serum albumin (0.91) Glycolitic pathway L31 (0.79) Alpha-2-HS-glycoprotein (0.91) Beta enolase (0.79) L32 (0.82) Phosphoglycerate mutase 2 (0.76) L34 (0.78) Inflammatory markers L35a (0.82) S100-A8 (1.32) Lipid catabolism L38 (0.87) S100-A9 (1.34) Trifunctional enzyme (1.19) Ig light chain kappa (C region) (1.79) Acetyl-CoA acetyltransferase (1.10) 40S ribosomal proteins Ig heavy chain gamma 2B (C region) (1.84) S2 (0.9) Lipid biosynthesis S3 (0.87) Protein folding 17-beta-HSD 12 (0.76) S4 (0.82) PPIase A (1.15) Fatty acid synthase (0.74) S5 (0.7) Alpha(B)-crystallin (1.32) NADP-dependent malic enzyme (0.87) S7 (0.79) HSP84 (0.82) S13 (0.79) Protein disulfide-isomerase A3 (0.91) Muscle-type and cytoskeleton S16 (0.86) Heat shock cognate 71 kDa protein (0.91) Myosin heavy chain-4 (2.13) S19 (0.88) HSP86 (0.81) Myosin light chain-1 (1.95) Endoplasmin (0.89) Tropomyosin-2 (1.91) Translation factors Chaperonin 10 (0.86) Myosin regulatory light chain 2 (1.68) eIF-4A-II (0.85) Chaperonin 60 (0.89) Actin (1.39) EF-1-gamma (0.84) Hsc70-interacting protein (0.91) Neurofilament light polypeptide (1.22) EF-1-alpha-2 (0.93) TCP-1-delta (0.77) Neurofilament heavy polypeptide (1.20) Calreticulin (0.88) Tubulin beta-5 chain (1.25) Highlighted proteins Calnexin (0.86) Tubulin beta-2A chain (1.24) SERCA1 (1.67) GRP-78 (0.95) Tubulin alpha-4A chain (1.21) ANT1 (0.73) Tubulin beta-4B chain (1.18) Creatine kinase M-type (0.88) Tubulin beta-4A chain (1.18) V-type ATPase subunit H (0.67) Tubulin beta-3 chain (1.16) MUP7 (0.24) Tubulin alpha-1A chain (1.12) Spectrin beta chain (1.08) Spectrin alpha chain (1.07) Moesin (1.09) Different software obtained similar biological conclusions from our of superoxide dismutase 1 and many peroxiredoxins isoforms, a proteomic datasets, concluding that db/db DRG and SCN develop highly-conserved family of antioxidant proteins that scavenge cellular metabolic dysregulation related to glucose and lipid metabolism, tissue ROS. Other antioxidant proteins, such as thioredoxin, glutaredoxin-1, scaffold abnormalities based on alterations in structural proteins, a and glyoxalase I, were found augmented. mild dysregulation of folding chaperones, an upregulation of acute- Lipid and carbohydrate metabolism were also altered in both tissues. phase response and inflammation, and an alteration of oxidative Several enzymes implicated in glycolytic pathway (phosphoglucose stress-related proteins. isomerase, phosphofructokinase, ß-enolase, phosphoglycerate mutase 2, phosphoglycerate kinase 1, GAPDH and fructose biphos- 3.3. Validation of the proteomic results phate aldose 1) were downregulated. In SCN, we observed a decline DRG and SCN proteins highlighted in this section are shown in Tables 2 in the content of aconitase, a TCA cycle enzyme. In SCN we also and 3 grouped by functional clusters with the corresponding fold detected decreased levels of OGDC-E2, which catalyses the con- change. We found multiple proteins involved in glutathione metabolism version of 2-oxoglutarate to succinyl-CoA and CO2 in the TCA cycle and antioxidant activity in both tissues. In DRG, several isoforms (Table 3). Regarding lipid metabolism, we found an upregulation in belonging to Mu class of Glutathione S enzymes (GSTM) were upre- lipid catabolism-related proteins (trifunctional enzyme, acetyl-CoA gulated, whereas in SCN, some of these isoforms were reduced. These acetyltransferase, perilipin-1, monoglyceride lipase, Ras-related opposite results were confirmed by western blot in both tissues protein Rab-7a and Enoyl-CoA delta isomerase I), whereas a (Figure 4AeD). Conjugated with glutathione, GSTM participates in the reduction in proteins implicated in lipid biosynthesis (17-ß-HSD 12, detoxification of electrophilic compounds, including products of fatty acid synthase, NADH-cytochrome b5 reductase 3 and NADP- oxidative stress. Concomitantly, we observed that mitochondrial iso- dependent malic enzyme). citrate dehydrogenase 2, which promotes regeneration of reduced Translation was downregulated in DRG as demonstrated by a decrease glutathione by supplying NADPH [30], was increased in DRG (Table 2) in proteins that constitute the ribosomal subunits 60S and 40S and the but not in SCN. On the other hand, in both tissues we found an increase translation-related factors eIF-4A-II, EF-1-gamma and EF-1-alpha-2 MOLECULAR METABOLISM 55 (2022) 101408 Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 7 www.molecularmetabolism.com Original Article Table 3 e SCN proteins highlighted in the results section are shown with the corresponding fold change ratio and grouped by functional clusters. SCN Oxidative stress Muscle-type and cytoskeleton Acute-phase response (positive) GSTM2 (0.69) Myosin heavy chain-4 (0.51) Haptoglobin (1.81) IDH2 (0.9) Myosin light chain-1 (0.54) Orosomucoid 1 (1.51) SOD1 (1.17) Tropomyosin-1 (0.66) Complement C3 (1.59) Thioredoxin (1.22) Tropomyosin-2 (0.52) Hemopexin (1.41) Glutaredoxin-1 (1.19) Myosin regulatory light chain 2 (0.56) Inter-alpha-inhibitor heavy chain 4 (1.44) Glyoxalase I (1.26) Actin (0.34) Peroxiredoxin-1 (1.06) Neurofilament light polypeptide (1.11) Acute-phase response (negative) Peroxiredoxin-6 (1.2) Neurofilament heavy polypeptide (1.11) Serum albumin (0.75) Peroxiredoxin-5 (1.23) Tubulin beta-5 chain (1.25) Alpha-2-HS-glycoprotein (0.89) Peroxiredoxin-2 (1.09) Tubulin beta-2A chain (1.74) Tubulin alpha-4A chain (1.56) Inflammatory markers Glycolitic pathway Tubulin beta-4B chain (1.36) S100-A9 (1.35) Phosphoglucose isomerase (0.81) Tubulin beta-4A chain (1.18) Ig light chain kappa (C region) (1.89) Phosphofructokinase (0.87) Tubulin beta-3 chain (1.23) Ig heavy chain gamma 2B (C region) (1.89) Beta enolase (0.46) Tubulin alpha-1A chain (1.29) Ig heavy chain gamma 2A (C region) (1.71) Phosphoglycerate mutase 2 (0.54) Spectrin beta chain (1.10) Ig heavy chain gamma 1 (C region) (1.46) Phosphoglycerate kinase 1 (0.86) Spectrin alpha chain (1.17) GAPDH (0.85) Moesin (1.21) Protein folding Fructose biphosphate aldose 1 (0.71) PPIase A (1.15) Highlighted proteins Alpha(B)-crystallin (1.18) TCA cycle SERCA1 (0.63) Prostaglandin E synthase 3 (1.19) Aconitase (0.89) ANT1 (0.73) HspB1 (1.09) Dihydrolipoamide succinyltransferase (0.72) Creatine kinase M-type (0.39) Heat shock cognate 71 kDa protein (1.06) MUP17 (0.31) TCP-1-alpha (1.27) Lipid catabolism Protein disulfide-isomerase A3 (0.91) Acetyl-CoA acetyltransferase (1.18) PDI (0.93) Perilipin-1 (0.76) HSP84 (0.88) Monoglyceride lipase (1.52) GRP-78 (0.88) Ras-related protein Rab-7a (1.10) Enoyl-CoA delta isomerase I (1.76) Lipid biosynthesis Fatty acid synthase (0.50) NADH-cytochrome b5 reductase 3 (0.83) NADP-dependent malic enzyme (0.68) (Table 2), but not in SCN. Another discrepancy between SCN and DRG translocation of ATP to the cytoplasm, was downregulated in both was the upregulation of muscle-related proteins in DRG, such as tissues (Tables 2 and 3). We also observed lower levels of creatine myosin heavy chain-4, myosin light chain-1, tropomyosin-1, kinase M-type. This enzyme catalyses the reversible interconversion tropomyosin-2, myosin regulatory light chain 2, and actin (Table 2), but of creatine into phosphocreatine, which is used to keep constant their downregulation in SCN (Table 3). However, cytoskeleton proteins global and local ATP/ADP ratio [31]. ATP exhaustion is probably the were augmented in both tissues (neurofilament polypeptide, tubulin, cause of the downregulation of V-type proton ATPase subunit H in spectrin and moesin). DRG proteome (Table 2), which was validated by western blot in both Our data shows differentially expressed positive (haptoglobin, oroso- DRG and SCN tissues (Figure 4AeD). This multisubunit enzyme is mucoid 1, fibrinogen gamma chain, complement C3, hemopexin and responsible for the establishment of the acidic pH of endocytic and inter-a-inhibitor heavy chain 4) and negative, acute-phase proteins secretory organelles, pumping cytosolic Hþ into their lumen. (serum albumin and alpha-2-HS-glycoprotein) (Tables 2 and 3). C3 MUP (major urinary protein) was the most depleted protein in DRG and overexpression was confirmed by western blot in both tissues the second most downregulated in SCN. MUP is a lipocalin family (Figure 4AeD). Many of these proteins can modulate one or more member secreted to circulation by the liver and filtered by the kidneys stages of the inflammatory response. Associated with the acute-phase into the urine. However, we detected Mup mRNA both in DRG and SCN response activation, inflammatory markers like S100-A8/9 in DRG and by real time PCR and its expression was much lower in db/db S100-A9 in SCN were enriched, while levels of immunoglobulin light compared to lean mice, in agreement with our proteomic results. The chain kappa and heavy chain gamma were higher in both tissues. decrease in expression was dependent on the time of diabetes in DRG. In addition to the detailed functional clusters, some interesting proteins In SCN, Mup expression is almost absent already at 1 week of dia- were highlighted, such as SERCA1, ANT1, V-ATPase subunit H and betes, pointing at a very early effect of metabolic syndrome (Figure 5A). MUP. SERCA1 (Sarcoplasmic/endoplasmic reticulum calcium ATPase This result was corroborated by western blot in both tissues 1) was increased in DRG, but downregulated in SCN (Tables 2 and 3). (Figure 4AeD). MUP does not colocalize with neuronal somas nor In agreement with this observation, several molecular chaperones satellite cells in DRG (Figure 5B), and neither axons nor Schwann cells were moderately dysregulated in DRG and SCN such as PPIase A, in SCN (Figure 5C). Alternatively, we detected MUP in the extracellular alpha(B)-crystallin, HSP84, protein disulfide-isomerase A3, heat shock space, colocalizing with connective tissue in DRG and with endoneu- cognate 71 kDa protein, and GRP-78. rium in SCN, as observed by collagen IV colocalization (Figure 5D), Related to energy depletion, ANT1 (ADP/ATP translocase 1), which which adds relevance to the important changes found in proteins forms a channel in the inner mitochondrial membrane that allows the related to extracellular space (Figure 3). 8 MOLECULAR METABOLISM 55 (2022) 101408 Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). www.molecularmetabolism.com Figure 4: Western blot validation of proteomic data. A, B: MUP, ATP6V1H, GSTM5 and C3 proteins were selected to validate proteomic results using blot immunodetection based on their relevance in oxidative stress, inflammation or due to its new potential role in the peripheral nerve. Representative western blots of these proteins are shown for DRG and SCN at 15 weeks of diabetes. C, D: We found statistically significant differences between db/db and lean mice using a sample size of 4e7 mice (*p < 0.05, **p < 0.01 by Student’s t test) and, moreover, these correlate with the results obtained by mass spectrometry. 4. DISCUSSION proteomic data regarding fold change directionality that may be due in part to differences between both studies, as we used animals with Proteomic studies are of interest in deciphering the pathophysiological shorter duration of diabetes, and lean instead of heterozygous (db/þ) mechanisms and guiding the search for preventive strategies of dis- mice as controls. eases. We identified several altered pathways in the PNS of db/db mice, with most relevance in those related to oxidative stress, meta- 4.1. Oxidative and ER stress bolic dysregulation, ribosomal proteins, muscle-type and cytoskeleton Several proteins involved in glutathione metabolism and antioxidant proteins, and acute phase and inflammatory and UPR responses. activity are altered in our study, supporting the hypothesis that No other proteomic studies have been reported so far for DN in type 2 oxidative stress has an important role in DN aetiology [5]. In the diabetes. Using microarray technology, Pande et al. [8] characterized same model, Hinder et al. [9] described increased levels of 3- the transcriptional profile of the SCN from db/db mice at 20 weeks of nitrotyrosine and HODEs in DRG and SCN, markers of protein diabetes. They identified several pathways enriched in diabetic ani- oxidation and lipid peroxidation, respectively. It is also remarkable mals: carbohydrate and lipid metabolism, glutathione metabolism, cell the increase of glyoxalase I, as it participates in the elimination of development, cell adhesion and extracellular matrix-receptor interac- methylglyoxal, a toxic compound derived from glucose metabolism tion, and signal transduction pathways. We found similar biological in hyperglycaemic conditions [32]. In diabetes, oxidative stress and annotations but also some discrepancies between transcriptomic and saturated fatty acids reduced SERCA activity [33,34]. In agreement MOLECULAR METABOLISM 55 (2022) 101408 Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 9 www.molecularmetabolism.com Original Article Figure 5: MUP localization in PNS. A: Mup gene expression was evaluated by qPCR in DRG and SCN at three different time points (one, six and seventeen weeks after diabetes onset) observing a progressive decline in its expression inversely correlating with the time of diabetes. Data was normalized to lean mice (n ¼ 3e4 animals per group, *p < 0.05, **p < 0.01, by two-way ANOVA followed by Bonferroni’s Post hoc test). B: Satellite cells from lumbar DRG were labelled against glutamine synthetase (green) concluding that MUP (red) does not colocalize with this cell type. Scale bar ¼ 100 mm. C: Schwann cells from SCN were immunodetected using an antibody against P0 (green) indicating that MUP protein (red) was not part of the myelin. Scale bar ¼ 25 mm. D: Extracellular matrix was immunolabelled with collagen type IV (green) in DRG and SCN tissues. Colocalization of collagen type IV and MUP (red) are observed in the magnification (16) of the composite pictures demonstrating that MUP protein is located in the extracellular matrix of the PNS. Scale bars ¼ 100 mm (DRG) and 25 mm (SCN). with these data, we found downregulated levels of SERCA1 in SCN, previous bibliography with cultured Schwann cells [36]. Using the cell whereas increased in DRG. SERCA1 is a membrane-bound enzyme lines ND7/23 and NSC-34 as in vitro models of sensory and moto- that transports Ca2þ from the cytosol into the ER. Since protein neurons, respectively, we tested the activation of unfolded protein folding is Ca2þ-dependent, calcium depletion in the ER leads to response upon palmitate exposure for 12 h. What we observed was a protein misfolding, resulting in the activation of UPR to recover doseeresponse effect of palmitate on UPR markers such as Hspa5 homoeostasis [34]. A mild dysregulation in several molecular (BiP) and Ddit3 (CHOP) mRNA upregulation and Xbp1 splicing activation chaperones was detected in both tissues, which might result in ER (Supplementary Figures. 4 and 5). homoeostasis disruption [35]. A possible explanation for these results could be related to the dif- We did not expect the lack of activation of the unfolded protein ference between acute and chronic exposure to a particular insult. response in diabetic PNS. Some folding chaperones exhibited lower Acute exposure to palmitate may trigger a marked response to levels compared to lean animals. These findings are opposite to those counteract the ER stress, whereas chronic exposure may eventually observed in proof of concept studies we performed in vitro as well as overwhelm the cell capacity leading to blunted UPR. 10 MOLECULAR METABOLISM 55 (2022) 101408 Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). www.molecularmetabolism.com 4.2. Metabolic deficiency member of the immunocalin family that modulates immune and in- Several enzymes implicated in glycolytic pathways and the TCA cycle flammatory responses [47], and it is increased in adipose tissue of db/ were downregulated, coinciding with a previous study reporting the db mice and in plasma of obese and/or diabetic patients. Higher AGP1 reduction of glycolytic metabolites and lower levels of citrate and levels were beneficial in db/db mice as it improved insulin tolerance isocitrate in db/db mice, which may indicate energetic deficiency [9]. In with the suppression of proinflammatory and prooxidative profiles in parallel, we found an upregulation in lipid catabolism whereas a their adipose tissue [48]. Agp1 mRNA upregulation in db/db DRG has decline in lipid biosynthesis, as an effort to increase cellular ATP. The also been described [11]. On the other hand, we found overexpression enrichment of genes related to lipid metabolism has also been of complement C3, one of the most versatile complements in host described by Pande et al. [8], which hypothesized that it could be defense and inflammation. Activated complement is present in the associated to increased amounts of adipose tissue in the epineurium of microvessels of sural nerve from diabetic patients [49] as well as in the db/db mice. Furthermore, Sas et al. [37] described an increase in murine DRG after spinal nerve ligation [45], suggesting a potential role metabolic flux towards lipid oxidation in SCN from db/db mice after 20 in myelin clearance after injury [50]. Hemopexin, which may be weeks with diabetes. involved in oxidative stress protection, was also increased in DRG and In this regard, lower ATP availability in the PNS of db/db mice is also SCN of db/db mice in this study. In injured peripheral nerves, hemo- represented by downregulation of ANT1 and creatine kinase M-type pexin mRNA is expressed by fibroblasts, Schwann cells, and invading (M-CK), in both tissues. Furthermore, ANT1, which is responsible for macrophages, accumulating in the extracellular matrix of SCN and DRG 50e66% of the basal proton conductance (BPC), has been proposed to [51]. All these data suggest that the higher levels of acute-phase protect from oxidative damage by partial dissipation of Dp by mild proteins could be a mechanism of protection against excessive uncoupling and limiting the formation of ROS [38]. Since ANT1 content inflammation and oxidative damage in DN. We observed the enrich- is lower, poor BPC may increase superoxide production. The down- ment of inflammatory markers such as S100-A8/9 with a role in regulation of V-ATPase subunit H in DRG may also be related to the activation of NADPH oxidase and advanced glycation end products decrease in ATP concentration. V-ATPase has a role in protein pro- (AGEs) receptors, which are involved in the pathogenesis of compli- cessing and degradation by lysosomes, endocytosis, and vesicular cations of diabetes [5,52,53]. S100-A8/9 are also associated with a traffic, as well as synaptic vesicle loading, driven by the electro- higher risk of developing DN in type 1 diabetic patients [54]. In addi- chemical gradient [39]. tion, higher levels of immunoglobulin light chain kappa and heavy Transcriptomic and metabolomic studies in type 1 diabetes drew chain gamma were also detected. Higher expression of Ig g chain at similar conclusions regarding metabolic deficiency in diabetic PNS as RNA level was found in DRG from db/db mice, suggesting that upre- type 2 diabetes. Akude et al. [40] demonstrated lower levels of proteins gulation at protein level must be at least partially of neuronal origin related to TCA cycle and mitochondrial respiratory chain in DRG sen- [11]. sory neurons from STZ-induced diabetic rats after 22 weeks. Rojas et al. [41] detected an evident reduction of some TCA metabolites in 4.6. Major urinary protein sciatic nerve of type 1 diabetic C57 Bl6/j mouse after 22 weeks with Of note, the most downregulated protein in DRG and the second most diabetes. Overall, it seems that the long-term outcome in type 1 dia- downregulated in SCN was MUP, a lipocalin family member that binds betes models may be a metabolic deficiency as described in type 2 to pheromones and other lipophilic molecules and regulates its diabetes models by previous bibliography and our own data. transport and release into the air from urine marks [55]. Mup1 mRNA was reduced by 30-fold in the liver of db/db mice and decreased in 4.3. Translational machinery circulation of both high-fat diet (HFD) and db/db mice [56]. Recom- In DRG, we found a notable decrease in proteins related to translational binant MUP1 suppresses the gluconeogenic and lipogenic programs in machinery, especially those that constitute the ribosomal subunits 60S the liver [57] and promotes mitochondrial biogenesis and oxidative and 40S. This could also be related to ATP depletion or even to insulin phosphorylation in skeletal muscles, thus increasing energy expendi- resistance, which may decrease the insulin mediated protein synthesis ture and insulin sensitivity [56]. Furthermore, rosiglitazone and [42]. Ribosomal proteins were not altered in SCN. resveratrol, which decrease hyperglycaemia and glucose intolerance in diabetic mice, also stimulate Mup1 expression in the liver [56,58]. 4.4. Cytoskeleton and muscle-like proteins Mup1 is also expressed in mouse skeletal muscle, and it markedly The upregulation of muscle-related proteins in DRG was corroborated decreases with HFD but increases with exercise training. Moreover, it by other studies reporting the presence of myosin and troponin in DRG improves insulin-stimulated GLUT4 translocation in cultured muscle neurons [43,44], while actin and tropomyosin isoforms, the major cells [59]. Here, we demonstrate that MUP is expressed by both SCN components of neuronal microfilaments, were increased in DRG and DRG and located in the extracellular matrix of PNS. Its expression following spinal nerve ligation, suggesting that muscle-type enzymes inversely correlates with the time of diabetes in DRG and it is a very could be upregulated to uphold functional and structural stability of early event in SCN, which needs further studies. Despite structural cells under stress [45]. Overall, it seems that there is an alteration of similarities with the acute-phase protein AGP1, as observed by the cell scaffold considering also the upregulation of cytoskeleton proteins pairwise alignment of both three-dimensional structures, and both in both tissues that could be related with injury or inflammation. belonging to the lipocalin family, we were not able to demonstrate in vitro a role of MUP in oxidative stress protection (data not shown). It 4.5. Inflammatory response would be interesting to study the overexpression or downregulation of Acute-phase response proteins increase or decrease in plasma during MUP in the PNS of T2DM and wild-type animals. inflammatory disorders, modulating the inflammatory response. We found both, positive and negative acute-phase proteins differentially 4.7. DRG and SCN comparison expressed in db/db PNS. As in our study, haptoglobin, orosomucoid 1, Despite the Venn diagram showing a low percentage of common and C-reactive protein were increased in diabetic patients, whereas differentially expressed proteins between these two sets, further serum albumin was also decreased [46]. Orosomucoid 1 (AGP1) is a functional analysis with bioinformatic resources clearly demonstrated MOLECULAR METABOLISM 55 (2022) 101408 Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 11 www.molecularmetabolism.com Original Article small differences between DRG and SCN proteome under diabetes. [3] Pop-Busui, R., Boulton, A.J., Feldman, E.L., Bril, V., Freeman, R., Malik, R.A., Most functional clusters are shared between tissues. To highlight some et al., 2017. Diabetic neuropathy: a position statement by the American dia- differences, the antioxidant enzymes like GSTM, the sarco/ER Ca2þ betes association. Diabetes Care 40(1):136e154. ATPase, and the muscle-related proteins are all increased in DRG but [4] Pasnoor, M., Dimachkie, M.M., Kluding, P., Barohn, R.J., 2013. Diabetic not in SCN, which may indicate a higher response to stress by the DRG neuropathy part 1: overview and symmetric phenotypes. Neurologic Clinics compared to the SCN. Translational machinery was found decreased in 31(2):425e445. DRG, whereas no changes were detected for SCN. These results [5] Feldman, E.L., Callaghan, B.C., Pop-Busui, R., Zochodne, D.W., Wright, D.E., correlate with a previous publication that compared transcriptional Bennett, D.L., et al., 2019. Diabetic neuropathy. Nature Reviews Disease network from DRG and SCN in type 2 diabetes using microarray Primers 5(1):41. analysis without any relevant difference highlighted between them [6] Callaghan, B.C., Little, A.A., Feldman, E.L., Hughes, R.A., 2012. Enhanced [60]. glucose control for preventing and treating diabetic neuropathy. Cochrane Database of Systematic Reviews 6:CD007543. 4.8. Conclusions [7] Callaghan, B.C., Gallagher, G., Fridman, V., Feldman, E.L., 2020. Diabetic In conclusion, our proteomic data uncover new pathways altered in DN neuropathy: what does the future hold? Diabetologia 63(5):891e897. and validate previous transcriptomic and metabolomic results, con- [8] Pande, M., Hur, J., Hong, Y., Backus, C., Hayes, J.M., Oh, S.S., et al., 2011. firming that both DRG and SCN present molecular alterations even Transcriptional profiling of diabetic neuropathy in the BKS db/db mouse: a before morphometric changes developed. It is worth mentioning that model of type 2 diabetes. Diabetes 60(7):1981e1989. many efforts have been made to develop therapeutic approaches that [9] Hinder, L.M., Vivekanandan-Giri, A., McLean, L.L., Pennathur, S., counteract oxidative stress and inflammation with low or moderate Feldman, E.L., 2013. Decreased glycolytic and tricarboxylic acid cycle in- effect in clinical practice. Our results, in conjunction with other pub- termediates coincide with peripheral nervous system oxidative stress in a lications, point out that low ATP bioavailability due to metabolic murine model of type 2 diabetes. Journal of Endocrinology 216(1):1e11. impairment could represent the main mechanism of neuro- [10] Ma, J., Pan, P., Anyika, M., Blagg, B.S., Dobrowsky, R.T., 2015. Modulating degeneration observed in diabetic neuropathy. These findings may molecular chaperones improves mitochondrial bioenergetics and decreases provide important information to select appropriate targets to develop the inflammatory transcriptome in diabetic sensory neurons. ACS Chemical new therapeutic strategies for DN. Neuroscience, 1637e1648. It would be interesting to compare the proteomic data from DbDb with [11] Hur, J., Dauch, J.R., Hinder, L.M., Hayes, J.M., Backus, C., Pennathur, S., obese and prediabetic HFD models, since obesity itself represents an et al., 2015. The metabolic syndrome and microvascular complications in a independent risk factor for diabetic neuropathy. This comparison may murine model of type 2 diabetes. Diabetes 64(9):3294e3304. enable to elucidate the role of obesity before hyperglycaemia develops. [12] O’Brien, P.D., Hur, J., Hayes, J.M., Backus, C., Sakowski, S.A., Feldman, E.L., 2015. BTBR ob/ob mice as a novel diabetic neuropathy model: neurological ACKNOWLEDGEMENTS characterization and gene expression analyses. Neurobiology of Disease 73: 348e355. M.L.J researched data and wrote the manuscript. J.J.V researched data. A.O [13] Hur, J., Sullivan, K.A., Pande, M., Hong, Y., Sima, A.A., Jagadish, H.V., et al., contributed to animal experiments. S.V contributed to animal experiments and dis- 2011. The identification of gene expression profiles associated with pro- cussion. A.S reviewed/edited manuscript. M.C contributed to discussion and gression of human diabetic neuropathy. Brain 134(Pt 11):3222e3235. reviewed/edited manuscript. X.N researched data, contributed to discussion and [14] Nie, L., Wu, G., Culley, D.E., Scholten, J.C., Zhang, W., 2007. Integrative reviewed/edited manuscript. A.B wrote the manuscript. We thank Meritxell Puig, analysis of transcriptomic and proteomic data: challenges, solutions and ap- Ángel Vázquez and Dr. Jesús Ruberte (UAB) for technical assistance. A.B is the plications. Critical Reviews in Biotechnology 27(2):63e75. guarantor of this work and, as such, had full access to all the data in the study and [15] O’Brien, P.D., Sakowski, S.A., Feldman, E.L., 2014. Mouse models of diabetic takes responsibility for the integrity of the data and the accuracy of the data analysis. neuropathy. ILAR Journal 54(3):259e272. This work was supported by the Fundació Marató TV3 (grant 201607.10) and 2017 [16] Fan, B., Liu, X.S., Szalad, A., Wang, L., Zhang, R., Chopp, M., et al., 2018. SGR1468 to M.C. M.L.J, A.O and S.V are recipients of predoctoral fellowships from Influence of sex on cognition and peripheral neurovascular function in diabetic Generalitat de Catalunya (2019FI_B2 00061; 2020FI_B2 00037 and 2020FI_B1 mice. Frontiers in Neuroscience 12:795. 00054, respectively). The authors declare that there is no conflict of interest. [17] Homs, J., Ariza, L., Pages, G., Verdu, E., Casals, L., Udina, E., et al., 2011. Comparative study of peripheral neuropathy and nerve regeneration in NOD APPENDIX A. SUPPLEMENTARY DATA and ICR diabetic mice. Journal of the Peripheral Nervous System 16(3):213e 227. Supplementary data to this article can be found online at https://doi.org/10.1016/j. [18] Vilches, J.J., Navarro, X., Verdú, E., 1995. Functional sudomotor responses to molmet.2021.101408. cholinergic agonists and antagonists in the mouse. Journal of the Autonomic Nervous System 55(1e2):105e111. DECLARATIONS OF INTEREST [19] Vilches, J.J., Wynick, D., Kofler, B., Lang, R., Navarro, X., 2012. Sudomotor function and sweat gland innervation in galanin knockout mice. Neuropeptides None. 46(4):151e155. [20] Wisniewski, J.R., Zougman, A., Nagaraj, N., Mann, M., 2009. Universal sample REFERENCES preparation method for proteome analysis. Nature Methods 6(5):359e362. [21] Oliveros, J.C., 2007e2015. Venny. An interactive tool for comparing lists with Venn’s diagrams. [1] International Diabetes Federation, 2019. IDF diabetes atlas, 9th ed. Brussels, [22] Liao, Y., Wang, J., Jaehnig, E.J., Shi, Z., Zhang, B., 2019. WebGestalt 2019: Belgium: International Diabetes Federation. gene set analysis toolkit with revamped UIs and APIs. Nucleic Acids Research [2] Forbes, J.M., Cooper, M.E., 2013. Mechanisms of diabetic complications. 47(W1):W199eW205. Physiological Reviews 93(1):137e188. 12 MOLECULAR METABOLISM 55 (2022) 101408 Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). www.molecularmetabolism.com [23] Huang, d.W., Sherman, B.T., Lempicki, R.A., 2009. Systematic and integrative [43] Miller, M., Bower, E., Levitt, P., Li, D., Chantler, P.D., 1992. Myosin II distri- analysis of large gene lists using DAVID bioinformatics resources. Nature bution in neurons is consistent with a role in growth cone motility but not Protocols 4(1):44e57. synaptic vesicle mobilization. Neuron 8(1):25e44. [24] Szklarczyk, D., Gable, A.L., Lyon, D., Junge, A., Wyder, S., Huerta-Cepas, J., [44] Roisen, F.J., Wilson, F.J., Yorke, G., Inczedy-Marcsek, M., Hirabayashi, T., et al., 2019. STRING v11: protein-protein association networks with increased 1983. Immunohistochemical localization of troponin-C in cultured neurons. coverage, supporting functional discovery in genome-wide experimental Journal of Muscle Research & Cell Motility 4(2):163e175. datasets. Nucleic Acids Research 47(D1):D607eD613. [45] Komori, N., Takemori, N., Kim, H.K., Singh, A., Hwang, S.H., Foreman, R.D., [25] Polpitiya, A.D., Qian, W.J., Jaitly, N., Petyuk, V.A., Adkins, J.N., Camp, D.G., et al., 2007. Proteomics study of neuropathic and nonneuropathic dorsal root et al., 2008. DAnTE: a statistical tool for quantitative analysis of -omics data. ganglia: altered protein regulation following segmental spinal nerve ligation Bioinformatics 24(13):1556e1558. injury. Physiol Genomics 29(2):215e230. [26] Robertson, D.M., Sima, A.A., 1980. Diabetic neuropathy in the mutant mouse [46] McMillan, D.E., 1989. Increased levels of acute-phase serum proteins in [C57BL/ks(db/db)]: a morphometric study. Diabetes 29(1):60e67. diabetes. Metabolism 38(11):1042e1046. [27] Campero, M., Ezquer, M., Ezquer, F., 2015. Nerve excitability and structural [47] Hochepied, T., Berger, F.G., Baumann, H., Libert, C., 2003. Alpha(1)-acid changes in myelinated axons from diabetic mice. Experimental and Clinical glycoprotein: an acute phase protein with inflammatory and immunomodu- Endocrinology & Diabetes 123(8):485e491. lating properties. Cytokine & Growth Factor Reviews 14(1):25e34. [28] Kennedy, W.R., Navarro, X., 1989. Sympathetic sudomotor function in diabetic [48] Lee, Y.S., Choi, J.W., Hwang, I., Lee, J.W., Lee, J.H., Kim, A.Y., et al., 2010. neuropathy. Archives of Neurology 46(11):1182e1186. Adipocytokine orosomucoid integrates inflammatory and metabolic signals to [29] Liu, Y., Sebastian, B., Liu, B., Zhang, Y., Fissel, J.A., Pan, B., et al., 2017. preserve energy homeostasis by resolving immoderate inflammation. Journal Sensory and autonomic function and structure in footpads of a diabetic mouse of Biological Chemistry 285(29):22174e22185. model. Scientific Reports 7:41401. [49] Rosoklija, G.B., Dwork, A.J., Younger, D.S., Karlikaya, G., Latov, N., Hays, A.P., [30] Jo, S.H., Son, M.K., Koh, H.J., Lee, S.M., Song, I.H., Kim, Y.O., et al., 2001. 2000. Local activation of the complement system in endoneurial microvessels Control of mitochondrial redox balance and cellular defense against oxidative of diabetic neuropathy. Acta Neuropathologica 99(1):55e62. damage by mitochondrial NADPþ-dependent isocitrate dehydrogenase. [50] de Jonge, R.R., van Schaik, I.N., Vreijling, J.P., Troost, D., Baas, F., 2004. Journal of Biological Chemistry 276(19):16168e16176. Expression of complement components in the peripheral nervous system. [31] Schlattner, U., Tokarska-Schlattner, M., Wallimann, T., 2006. Mitochondrial Human Molecular Genetics 13(3):295e302. creatine kinase in human health and disease. Biochimica et Biophysica Acta [51] Camborieux, L., Bertrand, N., Swerts, J.P., 1998. Changes in expression and 1762(2):164e180. localization of hemopexin and its transcripts in injured nervous system: a [32] Maessen, D.E., Stehouwer, C.D., Schalkwijk, C.G., 2015. The role of meth- comparison of central and peripheral tissues. Neuroscience 82(4):1039e ylglyoxal and the glyoxalase system in diabetes and other age-related dis- 1052. eases. Clinical Science 128(12):839e861. [52] Lim, S.Y., Raftery, M.J., Goyette, J., Hsu, K., Geczy, C.L., 2009. Oxidative [33] Horáková, L., Strosova, M.K., Spickett, C.M., Blaskovic, D., 2013. Impairment modifications of S100 proteins: functional regulation by redox. Journal of of calcium ATPases by high glucose and potential pharmacological protection. Leukocyte Biology 86(3):577e587. Free Radical Research 47(Suppl 1):81e92. [53] Leclerc, E., Fritz, G., Vetter, S.W., Heizmann, C.W., 2009. Binding of S100 [34] Egnatchik, R.A., Leamy, A.K., Jacobson, D.A., Shiota, M., Young, J.D., 2014. ER proteins to RAGE: an update. Biochimica et Biophysica Acta 1793(6):993e calcium release promotes mitochondrial dysfunction and hepatic cell lipotoxicity 1007. in response to palmitate overload. Molecular Metabolism 3(5):544e553. [54] Jin, Y., Sharma, A., Carey, C., Hopkins, D., Wang, X., Robertson, D.G., et al., [35] Lupachyk, S., Watcho, P., Obrosov, A.A., Stavniichuk, R., Obrosova, I.G., 2013. 2013. The expression of inflammatory genes is upregulated in peripheral blood Endoplasmic reticulum stress contributes to prediabetic peripheral neuropathy. of patients with type 1 diabetes. Diabetes Care 36(9):2794e2802. Experimental Neurology 247:342e348. [55] Zhou, Y., Rui, L., 2010. Major urinary protein regulation of chemical [36] Padilla, A., Descorbeth, M., Almeyda, A.L., Payne, K., De Leon, M., 2011. communication and nutrient metabolism. Vitamins & Hormones 83:151e Hyperglycemia magnifies Schwann cell dysfunction and cell death triggered by 163. PA-induced lipotoxicity. Brain Research 1370:64e79. [56] Hui, X., Zhu, W., Wang, Y., Lam, K.S., Zhang, J., Wu, D., et al., 2009. Major [37] Sas, K.M., Kayampilly, P., Byun, J., Nair, V., Hinder, L.M., Hur, J., et al., 2016. urinary protein-1 increases energy expenditure and improves glucose intol- Tissue-specific metabolic reprogramming drives nutrient flux in diabetic erance through enhancing mitochondrial function in skeletal muscle of diabetic complications. JCI Insight 1(15):e86976. mice. Journal of Biological Chemistry 284(21):14050e14057. [38] Cadenas, S., 2018. Mitochondrial uncoupling, ROS generation and car- [57] Zhou, Y., Jiang, L., Rui, L., 2009. Identification of MUP1 as a regulator for dioprotection. Biochimica et Biophysica Acta e Bioenergetics 1859(9):940e glucose and lipid metabolism in mice. Journal of Biological Chemistry 284(17): 950. 11152e11159. [39] Maxson, M.E., Grinstein, S., 2014. The vacuolar-type H⁺-ATPase at a glance - [58] Baur, J.A., Pearson, K.J., Price, N.L., Jamieson, H.A., Lerin, C., Kalra, A., et al., more than a proton pump. Journal of Cell Science 127(Pt 23):4987e4993. 2006. Resveratrol improves health and survival of mice on a high-calorie diet. [40] Akude, E., Zherebitskaya, E., Chowdhury, S.K., Smith, D.R., Dobrowsky, R.T., Nature 444(7117):337e342. Fernyhough, P., 2011. Diminished superoxide generation is associated with [59] Kleinert, M., Parker, B.L., Jensen, T.E., Raun, S.H., Pham, P., Han, X., et al., respiratory chain dysfunction and changes in the mitochondrial proteome of 2018. Quantitative proteomic characterization of cellular pathways associated sensory neurons from diabetic rats. Diabetes 60(1):288e297. with altered insulin sensitivity in skeletal muscle following high-fat diet feeding [41] Rojas, D.R., Kuner, R., Agarwal, N., 2019. Metabolomic signature of type 1 and exercise training. Scientific Reports 8(1):10723. diabetes-induced sensory loss and nerve damage in diabetic neuropathy. [60] Hinder, L.M., Murdock, B.J., Park, M., Bender, D.E., O’Brien, P.D., Journal of Molecular Medicine (Berlin) 97(6):845e854. Rumora, A.E., et al., 2018. Transcriptional networks of progressive diabetic [42] Ashford, A.J., Pain, V.M., 1986. Effect of diabetes on the rates of synthesis and peripheral neuropathy in the db/db mouse model of type 2 diabetes: an in- degradation of ribosomes in rat muscle and liver in vivo. Journal of Biological flammatory story. Experimental Neurology 305:33e43. Chemistry 261(9):4059e4065. MOLECULAR METABOLISM 55 (2022) 101408 Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 13 www.molecularmetabolism.com