Endoplasmic reticulum stress contributes to prediabetic peripheral neuropathy

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Experimental Neurology 247 (2013) 342–348

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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

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Article history: Received 14 June 2012 Revised 29 October 2012 Accepted 2 November 2012 Available online 8 November 2012 Keywords: Diabetic peripheral neuropathy Endoplasmic reticulum stress Eukaryotic initiation factor-2α High-fat diet fed mouse Motor nerve conduction velocity Prediabetic peripheral neuropathy Salubrinal Sensory nerve conduction velocity Streptozotocin Trimethylamine oxide Unfolded protein response Zucker (fa/fa) rat

a b s t r a c t Growing evidence suggests that prediabetes and metabolic syndrome are associated with increased risk for the development of microvascular complications including retinopathy, nephropathy, and, most commonly, peripheral painful neuropathy and/or autonomic neuropathy. The etiology of these disabling neuropathies is unclear, and several clinical and experimental studies implicated obesity, impaired fasting glycemia/impaired glucose 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 leading to the impairment of metabolism, transcriptional regulation, and gene expression, is emerging as a key mechanism of metabolic diseases including obesity and diabetes. We evaluated the role for this phenomenon in prediabetic neuropathy using two animal models i.e., Zucker (fa/fa) rats and high-fat diet fed mice which displayed obesity and impaired glucose tolerance in the absence of overt hyperglycemia. Endoplasmic reticulum stress manifest in upregulation of the glucose-regulated proteins BiP/GRP78 and GRP94 of unfolded protein response was identified in the sciatic nerve of Zucker rats. A chemical chaperone, trimethylamine oxide, blunted endoplasmic reticulum stress and alleviated sensory nerve conduction velocity deficit, thermal and mechanical hypoalgesia, and tactile allodynia. A selective inhibitor of eukaryotic initiation factor-2α dephosphorylation, salubrinal, improved glucose intolerance and alleviated peripheral nerve dysfunction in high-fat diet fed mice. Our findings suggest an important role of endoplasmic reticulum stress in the neurobiology 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 patients with both Type 1 and Type 2 diabetes, and is a leading cause of foot amputation (Boulton et al., 2005; Sinnreich et al., 2005; Tesfaye et al., 2010; Veves et al., 2008). Several groups reported a higher incidence of diabetes-like neuropathic changes in human subjects 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 of an association between impaired fasting glucose or impaired glucose tolerance and neuropathy is not uniformly accepted (Dyck et al., 2007). The etiology of neuropathy developing prior to the overt hyperglycemia is not well understood, and a number of clinical and experimental studies implicate obesity, impaired fasting glycemia/ impaired glucose tolerance, elevated triglyceride, cholesterol, and non-esterified fatty acids, as well as oxidative–nitrative stress (Coppey et 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 et al., 2003; Vincent et al., 2009; Watcho et al., 2010; Ziegler et al., 2009). ⁎ Corresponding author at: Pennington Biomedical Research Center, Louisiana State University System, 6400 Perkins Road, Baton Rouge, LA 70808, USA. Fax: +1 225 763 0274. E-mail address: [email protected] (I.G. Obrosova). 0014-4886/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.expneurol.2012.11.001

Endoplasmic reticulum (ER) stress is emerging as an important mechanism 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). ER stress results from damage to ER, an organelle playing a pivotal role in the folding and processing of newly synthesized proteins. ER stress leads to aberrant transcriptional regulation and gene expression, ion channel failure, dysmetabolism, impaired signaling, oxidative stress, and inflammation (Eizirik et al., 2008; Hotamisligil, 2010a,b). To counteract ER stress, the ER mounts the unfolded protein response (UPR). Three canonical arms of UPR include 1) PKR-like eukaryotic initiation factor 2A kinase (PERK) which phosphorylates eukaryotic initiation factor-2α (eIF2α) to suppress general protein translation; 2) inositolrequiring enzyme-1 (IRE1) involved in recruitment of several signaling molecules, splicing and production of an active transcription factor called X box-binding protein 1 (XBP-1), ER chaperones such as glucoseregulated protein BiP/GRP78 (BiP) and glucose-regulated protein 94 (GRP94), as well as CCAAT/enhancer-binding protein homologous protein (CHOP) and other components of the ER-associated degradation process; and 3) activating transcription factor-6 (ATP-6) which translocates to the Golgi apparatus and produces there an active transcription factor ATP-6N stimulating expression of chaperones and XBP-1. These three canonical arms of the UPR act together to reduce general protein synthesis, facilitate protein degradation, and increase folding capacity to resolve ER stress (Eizirik et al., 2008; Hotamisligil, 2010a,b).

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However, the excessive and long-term upregulation of UPR, and, in particular, 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 chronic diabetic complications such as nephropathy (Wu et al., 2010), early retinopathy (Zhong et al., 2012), as well as cognitive decline (SimsRobinson et al., 2012). In the present study, we evaluated the role for ER stress in neuropathic changes associated with prediabetes and obesity. We used a pharmacological approach with two agents, a non-specific chemical chaperone and protein stabilizer, trimethylamine oxide (TMAO), counteracting ER stress in toto, and the specific inhibitor of eukaryotic initiation factor-2α (eIF2α) dephosphorylation, salubrinal (Boyce et al., 2005). Materials and methods Reagents Unless otherwise stated, all chemicals were of reagent-grade quality, and were purchased from Sigma-Aldrich Chemical Co., St. Louis, MO. Salubrinal, a selective inhibitor of eIF2α dephoshorylation (Boyce et al., 2005), was purchased from Santa Cruz Biotechnology, Santa Cruz, CA. For Western blot analysis, rabbit polyclonal anti-GRP78/BiP and anti-GRP94 antibodies and mouse monoclonal HRP-conjugated anti-β-actin antibody were obtained from Abcam, Cambridge, MA. Rabbit polyclonal anti-phospho-eIF2α (ser51) and anti-eIF2α antibodies were obtained from Cell Signaling, Danvers, MA. Animals Background Exploration of the mechanisms of neuropathic changes preceding overt diabetes is complicated by the lack of animal models that develop prediabetes and obesity first and then spontaneously transit to overt diabetes. For this reason, the mechanisms underlying prediabetes per se as well as end-organ damage associated with this condition 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 (Coppey et al., 2011; Longo et al., 2011; Shevalye et al., 2012b; Sparks et al., 2005; Zawalich et al., 1995) that maintain metabolic abnormalities characteristic for prediabetes i.e., hyperinsulinemia, impaired glucose tolerance in the absence of overt hyperglycemia, hypertriglyceridemia and/or increased non-esterified fatty acid abundance, as well as hypercholesterolemia, during their whole life span. Both models exhibit nerve conduction deficit, small sensory nerve fiber dysfunction, and biochemical abnormalities in the peripheral nerve, spinal cord, and vasa nervorum (Coppey et al., 2011; Lupachyk et al., 2012; Obrosova et al., 2007; Oltman et al., 2005, 2008; Vincent et al., 2009; Watcho et al., 2010), and are, therefore, suitable for dissection of relative contribution of these phenomena to peripheral neuropathy in prediabetes. The experiments were performed in accordance with regulations specified by the Guide for the Care and Handling of Laboratory Animals (National Institutes of Health publication 85-23) and Pennington Biomedical Research Center Protocol for Animal Studies. To reduce the number of animals in our studies, the TMAO-treated Zucker fatty and Zucker lean rats described below in Experiment 1 and acipimoxtreated Zucker fatty and Zucker lean rats (Lupachyk et al., 2012) were compared to the same untreated controls. The six groups (four treated and two untreated) were studied in the same experiment. The blood chemistry and nerve function data for these untreated Zucker fatty and Zucker lean rats have been published previously (Lupachyk et al., 2012). In a similar fashion, a part of the C57Bl6/J mice fed with HFD for 16 weeks in our nephropathy-related study (Shevalye et al., 2012a) were treated with salubrinal as described herein (experiment 2 below). Initial body weights, blood glucose


concentrations, and glucose tolerance data in C57Bl6/J mice fed with normal chow or HFD for 16 weeks were published previously (Shevalye et al., 2012a). Experiment 1 Zucker fatty and Zucker lean rats were purchased from Charles River, Wilmington, MA. They were fed a standard rat chow (PMI Nutrition International, Brentwood, MO) and had access to water ad libitum throughout the experiment. 16 week-old rats were weighed. Blood samples for glucose measurements were taken from the tail vein. Zucker fatty and Zucker lean rats were randomly divided into groups maintained with or without TMAO treatment, 110 mg kg −1 d −1, in the drinking water, for another 4 weeks. Glucose tolerance test (2 g glucose, i.p., after 12-h fasting), and measurements of serum insulin, total cholesterol, VLDL/LDL cholesterol, triglyceride, and NEFA, as well as MNCV, SNCV, thermal and mechanical algesia, and tactile response thresholds were conducted in 16 week-old Zucker fatty and Zucker lean rats before TMAO treatment, and in 20 week-old untreated and TMAO-treated Zucker fatty and Zucker lean rats at the end of experiment. Experiment 2 Mature male C57Bl6/J mice were purchased from Jackson Laboratories, Bar Harbor, ME, and had access to water ad libitum throughout the experiment. The mice were assigned to receive normal or high-fat diets (D12328, 10.5 kcal% fat, and D 12330, 58 kcal% fat with corn starch, respectively, Research Diets, Inc., New Brunswick, NJ), for 16 weeks. Then the mice were maintained with or without treatment with salubrinal for another 4 weeks. We used salubrinal at 1 mg kg−1 d−1, i.e., the dose previously employed and shown effective in chronic studies in rodents (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 administration) and at 20 weeks. Evaluation of MNCV, SNCV, thermal and mechanical algesia, and tactile response thresholds was performed at a baseline (prior to high-fat diet feeding), at 16 weeks (prior to salubrinal administration), 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 cervical dislocation. Sciatic nerves (experiments 1 and 2) and spinal cords (experiment 2) were rapidly isolated, immediately frozen in liquid nitrogen, and stored at –80 °C prior to assessment of variables of UPR by Western blot analysis. Serum insulin, lipids, and non-esterified fatty acids Rat serum insulin concentrations were measured with the Ultra Sensitive Rat Insulin ELISA Kit from Crystal Chem, Downers Grove, IL, and mouse serum insulin concentrations with the Rat/Mouse Insulin ELISA Kit, Millipore, Billerica, MA. Rat and mouse serum total cholesterol concentrations were quantified with the Cholesterol Quantification Kit, MBL International, Woburn, MA, and rat and mouse serum triglyceride concentrations with the Triglyceride Quantification Kit, Abcam, Cambridge, MA. Rat VLDL/LDL concentrations were measured with 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 measurements were performed according to the manufacturer's instructions. Nerve functional studies Nerve functional studies included measurements of sciatic motor nerve conduction velocity (MNCV) and hind-limb digital sensory


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nerve conduction velocity (SNCV), thermal response latency, and mechanical and tactile response thresholds. Sciatic MNCV and hind-limb SNCV were recorded as we described previously (Obrosova et al., 2004). Temperature probe and HL-1, Heat Lamp (Physitemp Instruments, Inc., Clifton, NJ) was used to maintain body and hind-limb temperature at 37 °C. To determine the sensitivity to noxious heat (Hargreaves method), rats or mice were placed within a plexiglass chamber on a transparent glass surface and allowed to acclimate for at least 20 min. A thermal stimulation meter (IITC model 336 TG Combination Tail Flick & Paw algesia meter, IITC Life Science, Woodland Hills, CA) was used. The device was activated after placing the stimulator directly beneath the plantar surface of the hindpaw. The paw withdrawal latency in response to the radiant heat (a heating rate of ~ 1.3 °C per s, cut-off time 35 s for rats and 30 s for mice) was recorded. Floor temperature was set at ~ 32–33 °C (manufacturer's setup). Individual measurements were repeated four to five times and the mean value calculated. Paw (rats) and tail (mice) pressure thresholds were registered with a Paw/Tail Pressure Analgesia meter for the Randall–Selitto test (37215; Analgesy-Meter, UGO-Basile, Comerio VA, Italy). Tactile responses were evaluated by quantifying the withdrawal threshold of the hindpaw in response to stimulation with flexible von Frey filaments. We described those methods in detail in our previous publications (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 levels were evaluated by Western blot analysis. We employed 10% sodium dodecyl sulfate (SDS)–polyacrylamide gels, and the electrophoresis was conducted for 2 h. After blocking free binding sites as described below, primary antibodies against phosphorylated eIF2α, GRP78/BiP, and GRP94 were applied overnight, at 4 °C. Then secondary antibody was applied at room temperature for 1 h. Protein bands detected by the antibodies were visualized with Amersham ECL TM Western Blotting Detection Reagent (Little Chalfont, Buckinghamshire, UK). The membranes used for detection of phosphorylated eIF2α were stripped and reprobed with primary antibody against total eIF2α. After incubation with secondary antibody and visualization of total eIF2α protein band as described above, the membranes were stripped again and reprobed 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 (total eIF2α), all diluted in 20 mmol/l Tris–HCl buffer, pH 7.5, containing 150 mmol/l NaCl and 0.05% Tween 20, for 1 h. Stripping was conducted in 25 mmol/l glycine–HCl, pH 2.5 buffer containing 2% SDS. Statistical analysis The results are expressed as mean ± standard errors. Individual two-group comparisons (experiment 1: Zucker fatty and Zucker lean rats, before TMAO treatment; experiment 2: mice fed normal and high-fat diets, before salubrinal treatment) were made using the unpaired two-tailed Student's t-test or Mann–Whitney rank sum test where appropriate. Significance was defined at P ≤0.05. For multiple group comparisons, data were subjected to equality of variance F test, and then to log transformation, if necessary, before one-way analysis of variance. Where overall significance (Pb 0.05) was attained, individual between group comparisons were made using the Student–Newman– Keuls multiple range test. Significance was defined at P≤0.05. When between-group variance differences could not be normalized by log transformation (datasets for final body weights and plasma glucose), the data were analyzed by the nonparametric Kruskal–Wallis one-way analysis of variance, followed by the Bonferroni/Dunn or Fisher's PLSD tests for multiple comparisons.

Results Experiment 1 Inhibition of ER stress did not affect glycemia or impaired glucose tolerance, but alleviated hyperinsulinemia, hypertriglyceridemia, fatty acidemia, and peripheral nerve dysfunction associated with prediabetic neuropathy. Both 16 week-old and 20 week-old Zucker rats displayed obesity, slightly elevated non-fasting blood glucose concentrations, hyperinsulinemia, increased serum total and VLDL/LDL cholesterol concentrations, hypertriglyceridemia, fatty acidemia, impaired glucose tolerance, SNCV deficit, as well as thermal and mechanical hypoalgesia and tactile allodynia (Table 1, Figs. 1A, B). MNCVs were indistinguishable between 16 week-old and 20 week-old Zucker rats and the age-matched Zucker lean rats. The molecular chaperone, BiP and GRP94, levels were increased by 39% and 23% in the sciatic nerve of 20 week-old Zucker rats, compared with the age-matched Zucker lean rats (Pb 0.01 andb 0.05, respectively, Figs. 1C–F). This increase is indicative of activation of UPR and ER stress, associated with prediabetic neuropathy. TMAO-treated Zucker rats displayed normal sciatic nerve BiP and GRP94 levels. Inhibition of ER stress by TMAO abolished SNCV deficit and alleviated small sensory nerve fiber dysfunction. TMAO reduced hyperinsulinemia, hypertriglyceridemia, and fatty acidemia, without any effect on non-fasting glycemia, glucose tolerance, or serum total and VLDL/LDL cholesterol concentrations. TMAO treatment did not affect sciatic nerve BiP and GRP94 levels or any variables of peripheral nerve function in Zucker lean rats. Experiment 2 Salubrinal, a selective inhibitor of eIF2α dephosphorylation, improved glucose tolerance, normalized serum triglyceride concentrations, and alleviated hypercholesterolemia, fatty acidemia, and peripheral nerve 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 hyperglycemia (Figs. 2A,B), and reduced phospho-eIF2α level (42%) and phospho-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 SNCV deficits, thermal and mechanical hypoalgesia, and tactile allodynia (Table 2). Salubrinal did not affect weight gain and non-fasting blood glucose concentrations, but improved glucose tolerance (Fig. 2B), normalized serum triglyceride concentrations, and alleviated hyperinsulinemia, hypercholesterolemia, and fatty acidemia. Sciatic nerve phospho-eIF2α level and phospho-eIF2α/total eIF2α ratio remained unchanged (Figs. 2C, D). The agent increased spinal cord phosphoeIF2α content and phospho-eIF2α/total eIF2α ratio to the levels that were significantly different from those in mice fed normal chow (Pb 0.01 for both comparisons, Figs. 2E, F). Salubrinal alleviated MNCV and SNCV deficits and small sensory nerve fiber dysfunction induced by HFD consumption (Table 2). The agent did not affect any variables of ER stress/UPR or peripheral nerve function in mice fed normal chow. Discussion The findings reported herein implicate ER stress, manifested in upregulation of UPR, in the development of nerve conduction deficit and small sensory nerve fiber dysfunction associated with prediabetes and obesity. Alleviation of both ER stress and peripheral nerve dysfunction in the absence of improvement of glucose tolerance in TMAO-treated Zucker rats suggests that ER stress in the peripheral nervous system (PNS), rather than in insulin-resistant tissues and resultant postprandial hyperglycemia, contributes to neuropathic changes associated with prediabetes. Note, however, that administration of either

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

Zucker lean

16 week-old rats (before treatment) Body weight (g) Blood glucose (mmol/l) Insulin, ng/ml Total cholesterol, mg/dl VLDL/LDL cholesterol, mg/dl Triglycerides, mmol/l NEFA, mEq/l MNCV (ms−1) SNCV (ms−1) Thermal response latency (s) Mechanical withdrawal thresholds (g) Tactile response thresholds (g)

431 ± 5 6.1 ± 0.2 0.51 ± 0.07 147.0 ± 43.5 27.2 ± 1.3 2.1 ± 0.1 0.24 ± 0.03 53.9 ± 0.8 41.5 ± 0.4 14.6 ± 0.9 116 ± 6 16.0 ± 1.9

20 week-old rats (after treatment) Body weight (g) Blood glucose (mmol/l) Insulin, ng/ml Total cholesterol, mg/dl VLDL/LDL cholesterol, mg/dl Triglycerides, mmol/L NEFA, mEq/l MNCV (ms−1) SNCV (ms−1) Thermal response latency (s) Mechanical withdrawal thresholds (g) Tactile response thresholds (g)

463 ± 11 6.2 ± 0.1 0.34 ± 0.07 161.8 ± 7.5 24.2 ± 1.3 2.1 ± 0.2 0.15 ± 0.02 55.1 ± 0.7 45.7 ± 1.0 10.7 ± 0.2 105 ± 6 18.7 ± 0.8

Zucker lean + TMAO

Zucker 670 ± 8 8.9 ± 0.7** 3.70 ± 0.48** 424.4 ± 33.3** 70.0 ± 11.3** 9.3 ± 0.7 ** 1.19 ± 0.21** 53.6 ± 1.0 36.7 ± 0.9** 21.2 ± 1.4** 178 ± 10** 8.4 ± 0.8**

466 ± 10 6.5 ± 0.2 0.49 ± 0.06 170.6 ± 7.0 29.0 ± 2.3 1.8 ± 0.2 0.21 ± 0.04 54.3 ± 1.2 45.9 ± 0.8 11.1 ± 0.2 110 ± 2 21.0 ± 1.5

720 ± 28** 8.8 ± 1.1 2.00 ± 0.40** 589.5 ± 10.9** 62.7 ± 25.9* 10.6 ± 2.3** 1.40 ± 0.28** 54.4 ± 0.7 37.8 ± 0.9** 22.8 ± 1.2** 163 ± 7** 7.1 ± 0.5**

Data are expressed as Mean ± SEM. n= 5–11 per group. TMAO — trimethylamine oxide. *, ** P b 0.05 b 0.01 vs non-diabetic controls. maintained without TMAO treatment. a The data for untreated controls (Zucker lean and Zucker rats) have been published previously (Lupachyk et al., 2012).

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

Zucker + TMAO


745 ± 21** 9.8 ± 1.6** 1.33 ± 0.15**,# 527.6 ± 10.3** 50.3 ± 7.5 7.0 ± 0.5**,# 0.73 ± 0.17**,## 54.4 ± 1.7 44.1 ± 1.2## 15.0 ± 0.5**,## 127 ± 6**,## 12.2 ± 1.5**,## p b 0.05 and b 0.01 vs diabetic rats

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

Fig. 1. (A) Glucose tolerance curves in 16 week-old Zucker and Zucker lean rats. (B) Glucose tolerance curves, (C, E) representative Western blot analyses of sciatic nerve BiP/GRP78 and GRP94, and (D, F) sciatic nerve BiP/GRP78 and GRP94 contents, in 20 week-old Zucker and Zucker lean rats maintained with or without trimethylamine oxide treatment. TMAO — trimethylamine oxide. Mean±SEM, n=8–10 per group. *,** Pb 0.05 andb 0.01 vs Zucker lean rats; #, ## Pb 0.05 andb 0.01 vs Zucker rats maintained without trimethylamine oxide treatment.


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

Mice fed normal chow

16-week feeding (before treatment) Body weight (g) Blood glucose (mmol/l) Insulin, ng/ml Total cholesterol, mg/dl Triglycerides, mmol/l NEFA, mEq/l MNCV (ms−1) SNCV (ms−1) Thermal response latency (s) Mechanical withdrawal thresholds (g) Tactile response thresholds (g)

30.2 ± 0.5 7.5 ± 0.3 0.92 ± 0.19 121.9 ± 11.9 46.1 ± 6.7 0.28 ± 0.04 51.5 ± 1.0 38.7 ± 0.5 8.7 ± 0.2 115 ± 5 1.79 ± 0.24

20-week feeding (after salubrinal treatment) Body weight (g) Blood glucose (mmol/l) Insulin, ng/ml Total cholesterol, mg/dl Triglycerides, mmol/l NEFA, mEq/l MNCV (ms−1) SNCV (ms−1) Thermal response latency (s) Mechanical withdrawal thresholds (g) Tactile response thresholds (g)

32.1 ± 0.9 7.2 ± 0.2 0.94 ± 0.22 126.0 ± 13.9 46.1 ± 8.1 0.26 ± 0.05 51.9 ± 1.2 39.8 ± 1.4 9.2 ± 0.3 111 ± 5 1.63 ± 0.21

Mice fed normal chow + salubrinal

Mice fed high-fat diet

Mice fed high-fat diet + salubrinal

48.1 ± 0.7** 7.2 ± 0.2 2.42 ± 0.25** 268.2 ± 8.6** 68.6 ± 5.9* 1.10 ± 0.10** 45.4 ± 1.1** 31.9 ± 0.7** 14.8 ± 1.2** 163 ± 7** 0.87 ± 0.13**

32.1 ± 0.7 6.9 ± 0.2 1.00 ± 0.22 125.4 ± 9.3 38.6 ± 4.2 0.25 ± 0.03 50.6 ± 1.1 37.9 ± 0.7 10.1 ± 0.3 108 ± 3 1.51 ± 0.12

49.4 ± 0.5** 6.9 ± 0.1 2.61 ± 0.30** 290.0 ± 23.3** 66.8 ± 5.9* 1.02 ± 0.10** 46.6 ± 1.2** 32.3 ± 0.9** 15.5 ± 1.3** 151 ± 4** 0.98 ± 0.14**

49.6 ± 0.6** 6.5 ± 0.2 1.49 ± 0.13## 221.1 ± 15.3**,## 46.3 ± 3.6## 0.66 ± 0.06**,## 50.7 ± 0.8# 37.0 ± 0.3*,## 11.8 ± 0.3**,## 124 ± 2**,## 1.37 ± 0.12#

Data are expressed as Mean ± SEM. n = 7–23 per group. *,** P b 0.05 b 0.01 vs mice fed normal chow. #, ## p b 0.05 and b 0.01 vs high-fat diet fed mice maintained without salubrinal treatment. 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 nephropathyrelated study (Shevalye et al., 2012a).

Dissection of a contribution of individual components of UPR to pathological conditions including metabolic diseases is quite challenging because of the lack of specific, suitable for in vivo administration, inhibitors. Despite growing evidence that eIF2α phosphorylation is required for preservation of normal β-cell function (Kaufman et al., 2010), in vitro studies with the selective inhibitor of eIF2α dephosphorylation, salubrinal (Boyce et al., 2005), generated paradoxical results of potentiation of fatty acid-induced endoplasmic reticulum stress, cell dysfunction, and premature apoptosis in cultured pancreatic β-cells (Cnop et al., 2007). Note, however, that salubrinal was employed in several chronic in vivo studies (Pallet et al., 2008; Saxena et al., 2009; Zhu et al., 2008) in which the development of diabetes due to β-cell death, if it did occur, would not have remained unnoticed. In one of them (Saxena et al., 2009), salubrinal, employed for inhibition of ER stress in spinal cord motoneurons of FALS mice, alleviated manifestations of amyotrophic lateral sclerosis and delayed its progression. In another one (Sokka et al., 2007), salubrinal protected against rat brain hippocampal neuron excitotoxic injury, which accompanies neurological disorders, such as epilepsy and brain trauma. Salubrinal administration to HFD-fed mice in the current study did not affect phospho-eIF2α content and phospho-eIF2α/total eIF2α ratio in the sciatic nerve, and caused a modest increase of both variables in the whole spinal cord. The agent improved glucose tolerance, restored normal serum triglyceride concentrations, reduced hyperinsulinemia, hypercholesterolemia, and fatty acidemia, and, despite a minor effect on eIF2α phosphorylation in two tissue-sites for diabetes-like neuropathy, alleviated MNCV and SNCV deficits, thermal and mechanical hypoalgesia, and tactile allodynia. Note, that in our previous experiments in HFD-fed mice (Obrosova et al., 2007), an improvement of glucose tolerance by a 6-week consumption of normal chow did not increase MNCV. A beneficial effect of salubrinal on HFD-induced prediabetic neuropathy in the current study is likely mediated through both amelioration of the prediabetic condition per se, and the biochemical changes in the peripheral nervous system independent of the wholebody glucose homeostasis. Our findings are in line with several previous reports (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

pathogenetic mechanisms underlying prediabetes-associated end-organ damage, because pharmacological and genetic manipulations abrogating oxidative and now ER stress in this model interfere with the HFD-induced prediabetic phenotype per se. Evaluation of salubrinal on neuropathy in other models of prediabetes and in overt diabetes has never been performed. With consideration of observations of attenuated neurodegeneration in other neurological diseases in the two afore-mentioned salubrinal studies (Saxena et al., 2009; Sokka et al., 2007), it would be interesting to explore the effects of salubrinal in the models of diabetic peripheral neuropathy (DPN) exhibiting neurodegenerative changes. Streptozotocin-diabetic rats develop clearly manifest axonal atrophy of large myelinated fibers (Drel et al., 2010; Kato et al., 2000; Yagihashi et al., 1982) and intraepidermal nerve fiber loss (Lauria et al., 2005; Obrosova et al., 2008). Also note that they exhibit β-cell necrosis and irreversible hyperglycemia two-three days after induction of diabetes and would, therefore, be an ideal model for studying the potential of salubrinal to prevent or delay diabetes-induced neurodegenerative changes in PNS. In conclusion, our findings suggest the important role of ER stress in neuropathic changes associated with prediabetes, and identify a new therapeutic target. Further studies should evaluate the role of multiple individual components of ER stress/UPR in prediabetic neuropathy, as well as interactions of ER stress with other mechanisms such as oxidative–nitrative stress and 12/15-lipoxygenase activation implicated in this condition (Obrosova et al., 2007; Stavniichuk et al., 2010; Vincent et al., 2009). With consideration of similarity of many mechanisms underlying prediabetes-associated damage of different end-organs, this study may stimulate evaluation of the role for ER stress in other complications of prediabetes e.g., kidney disease (Shevalye et al., 2012a; Wei et al., 2004).

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

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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 sciatic nerve 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 diets for 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. *,** P b 0.05 and b 0.01 vs mice fed normal diet; ## P b 0.01 vs mice maintained without salubrinal treatment. Glucose tolerance 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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