Experimental Neurology xxx (2013) xxx–xxx

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Experimental Neurology

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Endoplasmic reticulum stress contributes to prediabetic peripheral neuropathy

Sergey Lupachyk, Pierre Watcho, Alexander A. Obrosov, Roman Stavniichuk, Irina G. Obrosova ⁎Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, LA, USA

⁎ Corresponding author at: Pennington Biomedical ReUniversity System, 6400 Perkins Road, Baton Rouge, LA 708

E-mail address: obrosoig@pbrc.edu (I.G. Obrosova).

0014-4886/$ – see front matter © 2013 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.expneurol.2012.11.001

Please cite this article as: Lupachyk, S., et al., Ehttp://dx.doi.org/10.1016/j.expneurol.2012.1

a b s t r a c t

a r t i c l e i n f o

Article history:Received 14 June 2012Revised 29 October 2012Accepted 2 November 2012Available online xxxx

Keywords:Diabetic peripheral neuropathyEndoplasmic reticulum stressEukaryotic initiation factor-2αHigh-fat diet fed mouseMotor nerve conduction velocityPrediabetic peripheral neuropathySalubrinalSensory nerve conduction velocityStreptozotocinTrimethylamine oxideUnfolded protein responseZucker (fa/fa) rat

Growing evidence suggests that prediabetes and metabolic syndrome are associated with increased risk forthe development of microvascular complications including retinopathy, nephropathy, and, most commonly,peripheral painful neuropathy and/or autonomic neuropathy. The etiology of these disabling neuropathies isunclear, and several clinical and experimental studies implicated obesity, impaired fasting glycemia/impairedglucose tolerance, elevated triglyceride and non-esterified fatty acids, as well as oxidative–nitrative stress.Endoplasmic reticulum stress resulting from abnormal folding of newly synthesized proteins and leadingto the impairment of metabolism, transcriptional regulation, and gene expression, is emerging as a key mech-anism of metabolic diseases including obesity and diabetes. We evaluated the role for this phenomenon inprediabetic neuropathy using two animal models i.e., Zucker (fa/fa) rats and high-fat diet fed mice whichdisplayed obesity and impaired glucose tolerance in the absence of overt hyperglycemia. Endoplasmic retic-ulum stress manifest in upregulation of the glucose-regulated proteins BiP/GRP78 and GRP94 of unfoldedprotein response was identified in the sciatic nerve of Zucker rats. A chemical chaperone, trimethylamineoxide, blunted endoplasmic reticulum stress and alleviated sensory nerve conduction velocity deficit, thermaland mechanical hypoalgesia, and tactile allodynia. A selective inhibitor of eukaryotic initiation factor-2αdephosphorylation, salubrinal, improved glucose intolerance and alleviated peripheral nerve dysfunction inhigh-fat diet fed mice. Our findings suggest an important role of endoplasmic reticulum stress in the neuro-biology of prediabetic peripheral neuropathy, and identify a new therapeutic target.

© 2013 Elsevier Inc. All rights reserved.

Introduction

Diabetic peripheral neuropathy (DPN) affects at least 50% of pa-tients with both Type 1 and Type 2 diabetes, and is a leading causeof foot amputation (Boulton et al., 2005; Sinnreich et al., 2005;Tesfaye et al., 2010; Veves et al., 2008). Several groups reported ahigher incidence of diabetes-like neuropathic changes in human sub-jects with impaired glucose tolerance (Smith et al., 2001, 2006;Sumner et al., 2003; Ziegler et al., 2009) and metabolic syndrome(Bonadonna et al., 2006; Costa et al., 2004; Isomaa et al., 2001;Pittenger et al., 2005; Smith et al., 2008), although the existence ofan association between impaired fasting glucose or impaired glucosetolerance and neuropathy is not uniformly accepted (Dyck et al.,2007). The etiology of neuropathy developing prior to the overt hy-perglycemia is not well understood, and a number of clinical andexperimental studies implicate obesity, impaired fasting glycemia/impaired glucose tolerance, elevated triglyceride, cholesterol, andnon-esterified fatty acids, as well as oxidative–nitrative stress (Coppeyet al., 2011; Costa et al., 2004; Lupachyk et al., 2012; Obrosova et al.,2007; Oltman et al., 2005, 2008; Smith et al., 2006, 2008; Sumner etal., 2003; Vincent et al., 2009; Watcho et al., 2010; Ziegler et al., 2009).

search Center, Louisiana State08, USA. Fax:+1 225 763 0274.

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ndoplasmic reticulum stress1.001

Endoplasmic reticulum (ER) stress is emerging as an importantmechanism of metabolic diseases including obesity and diabetes(Eizirik et al., 2008; Fu et al., 2011, 2012; Hummasti and Hotamisligil,2010; Kars et al., 2010; Kharroubi et al., 2004; Maris et al., 2012). ERstress results from damage to ER, an organelle playing a pivotal role inthe folding and processing of newly synthesized proteins. ER stressleads to aberrant transcriptional regulation and gene expression, ionchannel failure, dysmetabolism, impaired signaling, oxidative stress,and inflammation (Eizirik et al., 2008; Hotamisligil, 2010a,b). To coun-teract ER stress, the ER mounts the unfolded protein response (UPR).Three canonical arms of UPR include 1) PKR-like eukaryotic initiationfactor 2A kinase (PERK) which phosphorylates eukaryotic initiationfactor-2α (eIF2α) to suppress general protein translation; 2) inositol-requiring enzyme-1 (IRE1) involved in recruitment of several signalingmolecules, splicing and production of an active transcription factorcalled X box-binding protein 1 (XBP-1), ER chaperones such as glucose-regulated protein BiP/GRP78 (BiP) and glucose-regulated protein 94(GRP94), as well as CCAAT/enhancer-binding protein homologous pro-tein (CHOP) and other components of the ER-associated degradationprocess; and 3) activating transcription factor-6 (ATP-6)which translo-cates to the Golgi apparatus and produces there an active transcriptionfactor ATP-6N stimulating expression of chaperones and XBP-1. Thesethree canonical arms of the UPR act together to reduce general pro-tein synthesis, facilitate protein degradation, and increase folding ca-pacity to resolve ER stress (Eizirik et al., 2008; Hotamisligil, 2010a,b).

contributes to prediabetic peripheral neuropathy, Exp. Neurol. (2013),

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However, the excessive and long-term upregulation of UPR, and, in par-ticular, XBP-1, CHOP, ATF-4, has been shown to result in cell injury(Eizirik et al., 2008; Hotamisligil, 2010a,b).

Recent reports implicate ER stress in the development of chronicdiabetic complications such as nephropathy (Wu et al., 2010), earlyretinopathy (Zhong et al., 2012), as well as cognitive decline (Sims-Robinson et al., 2012). In the present study, we evaluated the rolefor ER stress in neuropathic changes associated with prediabetes andobesity. We used a pharmacological approach with two agents, anon-specific chemical chaperone and protein stabilizer, trimethylamineoxide (TMAO), counteracting ER stress in toto, and the specific inhibitorof eukaryotic initiation factor-2α (eIF2α) dephosphorylation, salubrinal(Boyce et al., 2005).

Materials and methods

Reagents

Unless otherwise stated, all chemicals were of reagent-grade qual-ity, and were purchased from Sigma-Aldrich Chemical Co., St. Louis,MO. Salubrinal, a selective inhibitor of eIF2α dephoshorylation (Boyceet al., 2005), was purchased from Santa Cruz Biotechnology, SantaCruz, CA. For Western blot analysis, rabbit polyclonal anti-GRP78/BiPand anti-GRP94 antibodies and mouse monoclonal HRP-conjugatedanti-β-actin antibodywere obtained from Abcam, Cambridge, MA. Rab-bit polyclonal anti-phospho-eIF2α (ser51) and anti-eIF2α antibodieswere obtained from Cell Signaling, Danvers, MA.

Animals

BackgroundExploration of the mechanisms of neuropathic changes preceding

overt diabetes is complicated by the lack of animal models that de-velop prediabetes and obesity first and then spontaneously transitto overt diabetes. For this reason, the mechanisms underlying predia-betes per se as well as end-organ damage associated with this condi-tion are studied in Zucker fatty (fa/fa) rats (Henriksen et al., 2011;Muellenbach et al., 2008; Oltman et al., 2005, 2008; Tong et al.,2010; Zhou et al., 1998) and high-fat diet (HFD) fed mice (Coppeyet al., 2011; Longo et al., 2011; Shevalye et al., 2012b; Sparks et al.,2005; Zawalich et al., 1995) that maintain metabolic abnormalitiescharacteristic for prediabetes i.e., hyperinsulinemia, impaired glucosetolerance in the absence of overt hyperglycemia, hypertriglyceridemiaand/or increased non-esterified fatty acid abundance, as well as hy-percholesterolemia, during their whole life span. Both models exhibitnerve conduction deficit, small sensory nerve fiber dysfunction, andbiochemical abnormalities in the peripheral nerve, spinal cord, andvasa nervorum (Coppey et al., 2011; Lupachyk et al., 2012; Obrosovaet al., 2007; Oltman et al., 2005, 2008; Vincent et al., 2009; Watchoet al., 2010), and are, therefore, suitable for dissection of relative con-tribution of these phenomena to peripheral neuropathy in prediabe-tes. The experiments were performed in accordance with regulationsspecified by the Guide for the Care and Handling of Laboratory Ani-mals (National Institutes of Health publication 85-23) and PenningtonBiomedical Research Center Protocol for Animal Studies. To reducethe number of animals in our studies, the TMAO-treated Zucker fattyand Zucker lean rats described below in Experiment 1 and acipimox-treated Zucker fatty and Zucker lean rats (Lupachyk et al., 2012)were compared to the same untreated controls. The six groups (fourtreated and two untreated) were studied in the same experiment.The blood chemistry and nerve function data for these untreatedZucker fatty and Zucker lean rats have been published previously(Lupachyk et al., 2012). In a similar fashion, a part of the C57Bl6/Jmice fed with HFD for 16 weeks in our nephropathy-related study(Shevalye et al., 2012a) were treated with salubrinal as describedherein (experiment 2 below). Initial body weights, blood glucose

Please cite this article as: Lupachyk, S., et al., Endoplasmic reticulum stresshttp://dx.doi.org/10.1016/j.expneurol.2012.11.001

concentrations, and glucose tolerance data in C57Bl6/J mice fedwith normal chow or HFD for 16 weeks were published previously(Shevalye et al., 2012a).

Experiment 1Zucker fatty and Zucker lean rats were purchased from Charles

River,Wilmington,MA. Theywere fed a standard rat chow (PMI Nutri-tion International, Brentwood,MO) and had access towater ad libitumthroughout the experiment. 16 week-old rats were weighed. Bloodsamples for glucose measurements were taken from the tail vein.Zucker fatty and Zucker lean rats were randomly divided into groupsmaintained with or without TMAO treatment, 110 mg kg−1 d−1, inthe drinking water, for another 4 weeks. Glucose tolerance test (2 gglucose, i.p., after 12-h fasting), and measurements of serum insulin,total cholesterol, VLDL/LDL cholesterol, triglyceride, and NEFA, as wellas MNCV, SNCV, thermal and mechanical algesia, and tactile responsethresholds were conducted in 16 week-old Zucker fatty and Zuckerlean rats before TMAO treatment, and in 20 week-old untreated andTMAO-treated Zucker fatty and Zucker lean rats at the end of experiment.

Experiment 2Mature male C57Bl6/J mice were purchased from Jackson Laborato-

ries, Bar Harbor, ME, and had access to water ad libitum throughout theexperiment. Themice were assigned to receive normal or high-fat diets(D12328, 10.5 kcal% fat, and D 12330, 58 kcal% fat with corn starch, re-spectively, Research Diets, Inc., New Brunswick, NJ), for 16 weeks. Thenthe mice were maintained with or without treatment with salubrinalfor another 4 weeks. We used salubrinal at 1 mg kg−1 d−1, i.e., thedose previously employed and shown effective in chronic studies in ro-dents (Pallet et al., 2008; Saxena et al., 2009;Wu et al., 2011; Zhu et al.,2008). Measurements of serum insulin, total cholesterol, triglyceride,and NEFA were performed at 16 weeks (prior to salubrinal administra-tion) and at 20 weeks. Evaluation of MNCV, SNCV, thermal and me-chanical algesia, and tactile response thresholds was performed at abaseline (prior to high-fat diet feeding), at 16 weeks (prior to salubrinaladministration), and at 20 weeks.

Anesthesia, euthanasia and tissue sampling

At the end of both experiments, the animals were sedated by CO2.Rats were immediately sacrificed by decapitation, and mice by cervi-cal dislocation. Sciatic nerves (experiments 1 and 2) and spinal cords(experiment 2) were rapidly isolated, immediately frozen in liquid ni-trogen, and stored at –80 °C prior to assessment of variables of UPRby Western blot analysis.

Serum insulin, lipids, and non-esterified fatty acids

Rat serum insulin concentrations were measured with the UltraSensitive Rat Insulin ELISA Kit from Crystal Chem, Downers Grove, IL,and mouse serum insulin concentrations with the Rat/Mouse InsulinELISA Kit, Millipore, Billerica, MA. Rat and mouse serum total choles-terol concentrations were quantified with the Cholesterol Quantifica-tion Kit, MBL International, Woburn, MA, and rat and mouse serumtriglyceride concentrations with the Triglyceride Quantification Kit,Abcam, Cambridge, MA. Rat VLDL/LDL concentrations were measuredwith the HDL and LDL/VLDL Cholesterol Assay Kit, Abcam, Cambridge,MA, and serum NEFA concentrations with the HR Series NEFA-HR(2)Kit, Wako Pure Chemical Industries, Osaka, Japan. All the measure-ments were performed according to the manufacturer's instructions.

Nerve functional studies

Nerve functional studies included measurements of sciatic motornerve conduction velocity (MNCV) and hind-limb digital sensory

contributes to prediabetic peripheral neuropathy, Exp. Neurol. (2013),

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nerve conduction velocity (SNCV), thermal response latency, and me-chanical and tactile response thresholds.

Sciatic MNCV and hind-limb SNCV were recorded as we describedpreviously (Obrosova et al., 2004). Temperature probe and HL-1, HeatLamp (Physitemp Instruments, Inc., Clifton, NJ) was used to maintainbody and hind-limb temperature at 37 °C.

To determine the sensitivity to noxious heat (Hargreavesmethod),rats or mice were placed within a plexiglass chamber on a transparentglass surface and allowed to acclimate for at least 20 min. A thermalstimulation meter (IITC model 336 TG Combination Tail Flick & Pawalgesia meter, IITC Life Science, Woodland Hills, CA) was used. Thedevice was activated after placing the stimulator directly beneaththe plantar surface of the hindpaw. The pawwithdrawal latency in re-sponse to the radiant heat (a heating rate of ~1.3 °C per s, cut-off time35 s for rats and 30 s for mice) was recorded. Floor temperature wasset at ~32–33 °C (manufacturer's setup). Individual measurementswere repeated four to five times and the mean value calculated.

Paw (rats) and tail (mice) pressure thresholds were registeredwith a Paw/Tail Pressure Analgesia meter for the Randall–Selitto test(37215; Analgesy-Meter, UGO-Basile, Comerio VA, Italy). Tactile re-sponses were evaluated by quantifying the withdrawal threshold ofthe hindpaw in response to stimulation with flexible von Frey fila-ments. We described those methods in detail in our previous publica-tions (Drel et al., 2007; Obrosova et al., 2007; Shevalye et al., 2012b).

Western blot analysis of UPR components in sciatic nerve and spinal cord

Total and phosphorylated eIF2α, BiP/GRP78, and GRP94 levelswere evaluated by Western blot analysis. We employed 10% sodiumdodecyl sulfate (SDS)–polyacrylamide gels, and the electrophoresiswas conducted for 2 h. After blocking free binding sites as describedbelow, primary antibodies against phosphorylated eIF2α, GRP78/BiP,and GRP94 were applied overnight, at 4 °C. Then secondary antibodywas applied at room temperature for 1 h. Protein bands detected bythe antibodies were visualized with Amersham ECLTM Western Blot-ting Detection Reagent (Little Chalfont, Buckinghamshire, UK). Themembranes used for detection of phosphorylated eIF2αwere strippedand reprobedwith primary antibody against total eIF2α. After incuba-tion with secondary antibody and visualization of total eIF2α proteinband as described above, the membranes were stripped again andreprobed with β-actin antibody to confirm equal protein loading.Free binding sites were blocked in 5% (w/v) BSA (phospho-eIF2α),3% (w/v) BSA (BiP/GRP78, GRP94), or 5% (w/v) non-fat dry milk (totaleIF2α), all diluted in 20 mmol/l Tris–HCl buffer, pH 7.5, containing150 mmol/l NaCl and 0.05% Tween 20, for 1 h. Strippingwas conductedin 25 mmol/l glycine–HCl, pH 2.5 buffer containing 2% SDS.

Statistical analysis

The results are expressed as mean±standard errors. Individualtwo-group comparisons (experiment 1: Zucker fatty and Zucker leanrats, before TMAO treatment; experiment 2: mice fed normal andhigh-fat diets, before salubrinal treatment) were made using theunpaired two-tailed Student's t-test or Mann–Whitney rank sum testwhere appropriate. Significance was defined at P≤0.05. For multiplegroup comparisons, data were subjected to equality of variance F test,and then to log transformation, if necessary, before one-way analysis ofvariance. Where overall significance (Pb0.05) was attained, individualbetween group comparisons were made using the Student–Newman–Keuls multiple range test. Significance was defined at P≤0.05. Whenbetween-group variance differences could not be normalized by logtransformation (datasets for final body weights and plasma glucose),the data were analyzed by the nonparametric Kruskal–Wallis one-wayanalysis of variance, followed by the Bonferroni/Dunn or Fisher's PLSDtests for multiple comparisons.

Please cite this article as: Lupachyk, S., et al., Endoplasmic reticulum stresshttp://dx.doi.org/10.1016/j.expneurol.2012.11.001

Results

Experiment 1

Inhibition of ER stress did not affect glycemia or impaired glucosetolerance, but alleviated hyperinsulinemia, hypertriglyceridemia,fatty acidemia, and peripheral nerve dysfunction associated with pre-diabetic neuropathy.

Both 16 week-old and 20 week-old Zucker rats displayed obesity,slightly elevated non-fasting blood glucose concentrations, hyperin-sulinemia, increased serum total and VLDL/LDL cholesterol concentra-tions, hypertriglyceridemia, fatty acidemia, impaired glucose tolerance,SNCV deficit, as well as thermal and mechanical hypoalgesia and tactileallodynia (Table 1, Figs. 1A, B). MNCVs were indistinguishable between16 week-old and 20 week-old Zucker rats and the age-matched Zuckerlean rats. Themolecular chaperone, BiP andGRP94, levelswere increasedby 39% and 23% in the sciatic nerve of 20 week-old Zucker rats, com-paredwith the age-matched Zucker lean rats (Pb0.01 andb0.05, respec-tively, Figs. 1C–F). This increase is indicative of activation of UPR and ERstress, associated with prediabetic neuropathy. TMAO-treated Zuckerrats displayed normal sciatic nerve BiP and GRP94 levels. Inhibition ofER stress by TMAO abolished SNCV deficit and alleviated small sensorynerve fiber dysfunction. TMAO reduced hyperinsulinemia, hypertriglyc-eridemia, and fatty acidemia, without any effect on non-fasting glycemia,glucose tolerance, or serum total and VLDL/LDL cholesterol concentra-tions. TMAO treatment did not affect sciatic nerve BiP and GRP94 levelsor any variables of peripheral nerve function in Zucker lean rats.

Experiment 2

Salubrinal, a selective inhibitor of eIF2α dephosphorylation, im-proved glucose tolerance, normalized serum triglyceride concentrations,and alleviated hypercholesterolemia, fatty acidemia, and peripheralnerve dysfunction in high-fat diet fed mice.

Mice fed HFD for 16 weeks displayed obesity, hyperinsulinemia,elevated serum cholesterol, triglyceride, and NEFA concentrations(Table 2), impaired glucose tolerance in the absence of overt hyper-glycemia (Figs. 2A,B), and reduced phospho-eIF2α level (42%) andphospho-eIF2α/total eIF2α ratio (31%) in sciatic nerve (Figs. 2C, D),but not spinal cord (Figs. 2E, F). After 16 weeks of HFD consumption,the mice exhibited neuropathic changes including MNCV and SNCVdeficits, thermal and mechanical hypoalgesia, and tactile allodynia(Table 2). Salubrinal did not affect weight gain and non-fasting bloodglucose concentrations, but improved glucose tolerance (Fig. 2B),normalized serum triglyceride concentrations, and alleviated hyper-insulinemia, hypercholesterolemia, and fatty acidemia. Sciatic nervephospho-eIF2α level and phospho-eIF2α/total eIF2α ratio remainedunchanged (Figs. 2C, D). The agent increased spinal cord phospho-eIF2α content and phospho-eIF2α/total eIF2α ratio to the levels thatwere significantly different from those in mice fed normal chow(Pb0.01 for both comparisons, Figs. 2E, F). Salubrinal alleviated MNCVand SNCV deficits and small sensory nerve fiber dysfunction inducedby HFD consumption (Table 2). The agent did not affect any variablesof ER stress/UPR or peripheral nerve function in mice fed normal chow.

Discussion

The findings reported herein implicate ER stress, manifested inupregulation of UPR, in the development of nerve conduction deficitand small sensory nerve fiber dysfunction associated with prediabe-tes and obesity. Alleviation of both ER stress and peripheral nervedysfunction in the absence of improvement of glucose tolerance inTMAO-treated Zucker rats suggests that ER stress in the peripheralnervous system (PNS), rather than in insulin-resistant tissues and resul-tant postprandial hyperglycemia, contributes to neuropathic changes as-sociated with prediabetes. Note, however, that administration of either

contributes to prediabetic peripheral neuropathy, Exp. Neurol. (2013),

Table 1Variables of peripheral nerve function in Zucker and Zucker lean rats maintained with or without TMAO treatmenta.

Group Variable Zucker lean Zucker lean+TMAO Zucker Zucker+TMAO

16 week-old rats (before treatment)Body weight (g) 431±5 670±8Blood glucose (mmol/l) 6.1±0.2 8.9±0.7**Insulin, ng/ml 0.51±0.07 3.70±0.48**Total cholesterol, mg/dl 147.0±43.5 424.4±33.3**VLDL/LDL cholesterol, mg/dl 27.2±1.3 70.0±11.3**Triglycerides, mmol/l 2.1±0.1 9.3±0.7 **NEFA, mEq/l 0.24±0.03 1.19±0.21**MNCV (ms−1) 53.9±0.8 53.6±1.0SNCV (ms−1) 41.5±0.4 36.7±0.9**Thermal response latency (s) 14.6±0.9 21.2±1.4**Mechanical withdrawal thresholds (g) 116±6 178±10**Tactile response thresholds (g) 16.0±1.9 8.4±0.8**

20 week-old rats (after treatment)Body weight (g) 463±11 466±10 720±28** 745±21**Blood glucose (mmol/l) 6.2±0.1 6.5±0.2 8.8±1.1 9.8±1.6**Insulin, ng/ml 0.34±0.07 0.49±0.06 2.00±0.40** 1.33±0.15**,#

Total cholesterol, mg/dl 161.8±7.5 170.6±7.0 589.5±10.9** 527.6±10.3**VLDL/LDL cholesterol, mg/dl 24.2±1.3 29.0±2.3 62.7±25.9* 50.3±7.5Triglycerides, mmol/L 2.1±0.2 1.8±0.2 10.6±2.3** 7.0±0.5**,#

NEFA, mEq/l 0.15±0.02 0.21±0.04 1.40±0.28** 0.73±0.17**,##

MNCV (ms−1) 55.1±0.7 54.3±1.2 54.4±0.7 54.4±1.7SNCV (ms−1) 45.7±1.0 45.9±0.8 37.8±0.9** 44.1±1.2##

Thermal response latency (s) 10.7±0.2 11.1±0.2 22.8±1.2** 15.0±0.5**,##

Mechanical withdrawal thresholds (g) 105±6 110±2 163±7** 127±6**,##

Tactile response thresholds (g) 18.7±0.8 21.0±1.5 7.1±0.5** 12.2±1.5**,##

Data are expressed as Mean ± SEM. n=5–11 per group. TMAO — trimethylamine oxide. *, ** Pb0.05b0.01 vs non-diabetic controls. #,## pb0.05 andb0.01 vs diabetic ratsmaintained without TMAO treatment.

a The data for untreated controls (Zucker lean and Zucker rats) have been published previously (Lupachyk et al., 2012).

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TMAO or salubrinal, i.e., two structurally unrelated agents counteractingER stress through different mechanisms, alleviated hypertriglyceridemiaand fatty acidemia known to play an important role in oxidative stress

Fig. 1. (A) Glucose tolerance curves in 16 week-old Zucker and Zucker lean rats. (B) Glucoseand GRP94, and (D, F) sciatic nerve BiP/GRP78 and GRP94 contents, in 20 week-old Zucker antrimethylamine oxide. Mean±SEM, n=8–10 per group. *,** Pb0.05 andb0.01 vs Zucker leatreatment.

Please cite this article as: Lupachyk, S., et al., Endoplasmic reticulum stresshttp://dx.doi.org/10.1016/j.expneurol.2012.11.001

and neuropathic changes associatedwith prediabetes and overt diabetes(Lupachyk et al., 2012; Tesfaye et al., 2005; Wiggin et al., 2009; Ziegleret al., 2004).

tolerance curves, (C, E) representative Western blot analyses of sciatic nerve BiP/GRP78d Zucker lean rats maintained with or without trimethylamine oxide treatment. TMAO—

n rats; #, ## Pb0.05 andb0.01 vs Zucker rats maintained without trimethylamine oxide

contributes to prediabetic peripheral neuropathy, Exp. Neurol. (2013),

Table 2Variables of peripheral nerve function in high-fat diet fed mice maintained with or without salubrinal treatmenta.

Group Variable Mice fed normal chow Mice fed normal chow+salubrinal Mice fed high-fat diet Mice fed high-fat diet+salubrinal

16-week feeding (before treatment)Body weight (g) 30.2±0.5 48.1±0.7**Blood glucose (mmol/l) 7.5±0.3 7.2±0.2Insulin, ng/ml 0.92±0.19 2.42±0.25**Total cholesterol, mg/dl 121.9±11.9 268.2±8.6**Triglycerides, mmol/l 46.1±6.7 68.6±5.9*NEFA, mEq/l 0.28±0.04 1.10±0.10**MNCV (ms−1) 51.5±1.0 45.4±1.1**SNCV (ms−1) 38.7±0.5 31.9±0.7**Thermal response latency (s) 8.7±0.2 14.8±1.2**Mechanical withdrawal thresholds (g) 115±5 163±7**Tactile response thresholds (g) 1.79±0.24 0.87±0.13**

20-week feeding (after salubrinal treatment)Body weight (g) 32.1±0.9 32.1±0.7 49.4±0.5** 49.6±0.6**Blood glucose (mmol/l) 7.2±0.2 6.9±0.2 6.9±0.1 6.5±0.2Insulin, ng/ml 0.94±0.22 1.00±0.22 2.61±0.30** 1.49±0.13##

Total cholesterol, mg/dl 126.0±13.9 125.4±9.3 290.0±23.3** 221.1±15.3**,##

Triglycerides, mmol/l 46.1±8.1 38.6±4.2 66.8±5.9* 46.3±3.6##

NEFA, mEq/l 0.26±0.05 0.25±0.03 1.02±0.10** 0.66±0.06**,##

MNCV (ms−1) 51.9±1.2 50.6±1.1 46.6±1.2** 50.7±0.8#

SNCV (ms−1) 39.8±1.4 37.9±0.7 32.3±0.9** 37.0±0.3*,##

Thermal response latency (s) 9.2±0.3 10.1±0.3 15.5±1.3** 11.8±0.3**,##

Mechanical withdrawal thresholds (g) 111±5 108±3 151±4** 124±2**,##

Tactile response thresholds (g) 1.63±0.21 1.51±0.12 0.98±0.14** 1.37±0.12#

Data are expressed as Mean ± SEM. n=7–23 per group. *,** Pb0.05b0.01 vs mice fed normal chow. #, ## pb0.05 and b0.01 vs high-fat diet fed mice maintained without salubrinaltreatment.

a Body weight and blood glucose concentration data in C57Bl6/J mice fed normal or high-fat diets for 16 weeks have been published previously in our diabetic nephropathy-related study (Shevalye et al., 2012a).

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Dissection of a contribution of individual components of UPR topathological conditions including metabolic diseases is quite chal-lenging because of the lack of specific, suitable for in vivo administra-tion, inhibitors. Despite growing evidence that eIF2α phosphorylationis required for preservation of normal β-cell function (Kaufman et al.,2010), in vitro studies with the selective inhibitor of eIF2α dephos-phorylation, salubrinal (Boyce et al., 2005), generated paradoxicalresults of potentiation of fatty acid-induced endoplasmic reticulumstress, cell dysfunction, and premature apoptosis in cultured pancre-atic β-cells (Cnop et al., 2007). Note, however, that salubrinal wasemployed in several chronic in vivo studies (Pallet et al., 2008; Saxenaet al., 2009; Zhu et al., 2008) in which the development of diabetesdue to β-cell death, if it did occur, would not have remained unnoticed.In one of them (Saxena et al., 2009), salubrinal, employed for inhibitionof ER stress in spinal cord motoneurons of FALS mice, alleviated mani-festations of amyotrophic lateral sclerosis and delayed its progression.In another one (Sokka et al., 2007), salubrinal protected against ratbrain hippocampal neuron excitotoxic injury, which accompanies neu-rological disorders, such as epilepsy and brain trauma. Salubrinaladministration to HFD-fed mice in the current study did not affectphospho-eIF2α content and phospho-eIF2α/total eIF2α ratio in thesciatic nerve, and caused a modest increase of both variables in thewhole spinal cord. The agent improved glucose tolerance, restorednormal serum triglyceride concentrations, reduced hyperinsulinemia,hypercholesterolemia, and fatty acidemia, and, despite a minor effecton eIF2α phosphorylation in two tissue-sites for diabetes-like neuro-pathy, alleviated MNCV and SNCV deficits, thermal and mechanicalhypoalgesia, and tactile allodynia. Note, that in our previous experi-ments in HFD-fed mice (Obrosova et al., 2007), an improvement ofglucose tolerance by a 6-week consumption of normal chow did notincrease MNCV. A beneficial effect of salubrinal on HFD-induced predi-abetic neuropathy in the current study is likely mediated through bothamelioration of the prediabetic condition per se, and the biochemicalchanges in the peripheral nervous system independent of the whole-body glucose homeostasis. Our findings are in linewith several previousreports (Hoehn et al., 2009; Shertzer et al., 2008;Weisberg et al., 2008)indicating that a HFD-fed mouse is not an ideal model for dissection of

Please cite this article as: Lupachyk, S., et al., Endoplasmic reticulum stresshttp://dx.doi.org/10.1016/j.expneurol.2012.11.001

pathogenetic mechanisms underlying prediabetes-associated end-organdamage, because pharmacological and genetic manipulations abrogatingoxidative andnowER stress in thismodel interferewith theHFD-inducedprediabetic phenotype per se.

Evaluation of salubrinal on neuropathy in other models of prediabe-tes and in overt diabetes has never been performed.With considerationof observations of attenuated neurodegeneration in other neurologicaldiseases in the two afore-mentioned salubrinal studies (Saxena et al.,2009; Sokka et al., 2007), it would be interesting to explore the effectsof salubrinal in the models of diabetic peripheral neuropathy (DPN)exhibiting neurodegenerative changes. Streptozotocin-diabetic rats de-velop clearly manifest axonal atrophy of large myelinated fibers (Drelet al., 2010; Kato et al., 2000; Yagihashi et al., 1982) and intraepidermalnervefiber loss (Lauria et al., 2005; Obrosova et al., 2008). Also note thatthey exhibit β-cell necrosis and irreversible hyperglycemia two-threedays after induction of diabetes and would, therefore, be an idealmodel for studying the potential of salubrinal to prevent or delaydiabetes-induced neurodegenerative changes in PNS.

In conclusion, our findings suggest the important role of ER stressin neuropathic changes associated with prediabetes, and identify anew therapeutic target. Further studies should evaluate the role ofmultiple individual components of ER stress/UPR in prediabetic neu-ropathy, as well as interactions of ER stress with other mechanismssuch as oxidative–nitrative stress and 12/15-lipoxygenase activationimplicated in this condition (Obrosova et al., 2007; Stavniichuk et al.,2010; Vincent et al., 2009). With consideration of similarity of manymechanisms underlying prediabetes-associated damage of differentend-organs, this studymay stimulate evaluation of the role for ER stressin other complications of prediabetes e.g., kidney disease (Shevalye etal., 2012a; Wei et al., 2004).

Acknowledgments

S.L., P.W., A.A.O., R.S., and I.G.O. were partially supported by theNational Institutes of Health grants RO1DK074517, RO1DK077141,and RO1DK081147 (all to I.G.O.).

contributes to prediabetic peripheral neuropathy, Exp. Neurol. (2013),

Fig. 2. (A) Glucose tolerance curves in C57Bl6/J mice fed normal or high-fat diets for 16 weeks. (B) Glucose tolerance curves, (C, E) representative Western blot analyses of sciaticnerve and spinal cord total and phosphorylated eIF2α, and (D, F) sciatic nerve and spinal cord total and phosphorylated eIF2α contents, in C57Bl6/J mice fed normal or high-fat dietsfor 20 weeks and maintained with or without salubrinal treatment for 4 weeks after initial 16 weeks without treatment. Mean±SEM, n=8–12 per group. *,** Pb0.05 andb0.01 vsmice fed normal diet; ## Pb0.01 vs mice maintained without salubrinal treatment. Glucose tolerance data in C57Bl6/J mice fed normal or high-fat diets for 16 weeks have beenpublished previously in our diabetic nephropathy-related study (Shevalye et al., 2012a).

6 S. Lupachyk et al. / Experimental Neurology xxx (2013) xxx–xxx

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