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Diabetic peripheral neuropathy (DPN) is the most common complication of type 2 diabetes (T2D). Although the cellular and molecular mechanisms of DPN are poorly understood, we and others have shown that altered gene expression and DNA methylation are implicated in disease pathogenesis. However, how DNA methylation might functionally impact gene expression and contribute to nerve damage remains unclear. Here, we analyzed genome-wide transcriptomic and methylomic profiles of sural nerves from T2D patients with DPN.Unbiased clustering of transcriptomics data separated samples into groups, which correlated with HbA1c levels. Accordingly, we found 998 differentially expressed genes (DEGs) and 929 differentially methylated genes (DMGs) between the groups with the highest and lowest HbA1c levels. Functional enrichment analysis revealed that DEGs and DMGs were enriched for pathways known to play a role in DPN, including those related to the immune system, extracellular matrix (ECM), and axon guidance. To understand the interaction between the transcriptome and methylome in DPN, we performed an integrated analysis of the overlapping genes between DEGs and DMGs. Integrated functional and network analysis identified genes and pathways modulating functions such as immune response, ECM regulation, and PI3K-Akt signaling.These results suggest for the first time that DNA methylation is a mechanism regulating gene expression in DPN. Overall, DPN patients with high HbA1c have distinct alterations in sural nerve DNA methylome and transcriptome, suggesting that optimal glycemic control in DPN patients is an important factor in maintaining epigenetic homeostasis and nerve function.

Background Diabetic peripheral neuropathy (DPN) is the most common complication of type 2 diabetes (T2D). Although the cellular and molecular mechanisms of DPN are poorly understood, we and others have shown that altered gene expression and DNA methylation are implicated in disease pathogenesis. However, how DNA methylation might functionally impact gene expression and contribute to nerve damage remains unclear. Here, we analyzed genome-wide transcriptomic and methylomic profiles of sural nerves from T2D patients with DPN. Results Unbiased clustering of transcriptomics data separated samples into groups, which correlated with HbA1c levels. Accordingly, we found 998 differentially expressed genes (DEGs) and 929 differentially methylated genes (DMGs) between the groups with the highest and lowest HbA1c levels. Functional enrichment analysis revealed that DEGs and DMGs were enriched for pathways known to play a role in DPN, including those related to the immune system, extracellular matrix (ECM), and axon guidance. To understand the interaction between the transcriptome and methylome in DPN, we performed an integrated analysis of the overlapping genes between DEGs and DMGs. Integrated functional and network analysis identified genes and pathways modulating functions such as immune response, ECM regulation, and PI3K-Akt signaling. Conclusion These results suggest for the first time that DNA methylation is a mechanism regulating gene expression in DPN. Overall, DPN patients with high HbA1c have distinct alterations in sural nerve DNA methylome and transcriptome, suggesting that optimal glycemic control in DPN patients is an important factor in maintaining epigenetic homeostasis and nerve function.

The metabolic syndrome, prediabetes, and type 2 diabetes are increasingly common conditions worldwide, which predispose patients to the development of significant complications in both the central and peripheral nervous systems. Recently, our integrated lipidomics and transcriptomics study revealed an important role for lipid signaling pathways in peripheral nerves of mouse models of prediabetes and type 2 diabetes. While these data point to possible mechanisms of nerve damage peripherally, less is known about damage due to prediabetes and diabetes in the brain. This is of particular interest as growing evidence indicates that these conditions negatively affect cognition. Therefore, we performed RNA-Seq on hippocampi, a major site of learning and memory in the brain. Samples were isolated from a model of prediabetes and cognitive decline, wherein mice are fed a 60% high-fat diet (HFD). We identified various differentially expressed genes (DEGs) due to HFD, particularly after 11 wks (n=886), with fewer after 19 wks (n=111). Pathway analysis showed enrichment of pathways previously identified in the peripheral nervous system in response to HFD, including inflammatory and MAPK signaling pathways. Of interest, ∼31% of DEGs due to 11 wks of HFD overlapped with DEGs due to age (control animals only, comparing 19 to 11 wks of diet). These overlapping DEGs included genes such as fatty acid elongase 6 and hyccin (leukodystrophy protein FAM126A), which have roles in lipid metabolism and myelination, respectively. Enrichment analysis revealed multiple metabolic and protein processing pathways, such as oxidative phosphorylation and the proteasome. These data suggest that, like the peripheral nervous system, HFD effects similar pathways in the brain and may accelerate age-related gene expression changes in the hippocampus. However, more robust studies are needed to fully understand the influence of HFD on possible premature brain aging and how gene expression changes may contribute to cognitive decline. Disclosure S. Elzinga: None. R. Henn: None. P.D. O’Brien: None. K. Guo: None. S. Eid: None. A.E. Rumora: None. L.M. Hinder: None. J.M. Hayes: None. F. Mendelson: None. E.L. Feldman: Consultant; Self; Novartis Pharmaceuticals Corporation. J. Hur: None. Funding Program for Neurology Research and Discovery (T32DK007245, T32DK101357); National Institutes of Health (R24DK082841)

Alzheimer’s disease (AD) is a growing problem worldwide, and there are currently no effective treatments for this devastating disease. The neurotrophic growth factors insulin and insulin-like growth factor-I (IGF-I) are currently being investigated as potential therapeutic approaches for AD in preclinical and clinical studies. However, given that the metabolic syndrome (MetS) and diabetes are risk factors for AD, it is unknown how associated insulin resistance (IR) in the brain may impact the effectiveness of these therapies for AD. In this report, we therefore investigated the mechanisms underlying the effects of insulin and IGF-I on AD-associated pathology in the context of IR, with particular emphasis on phosphorylation of amyloid precursor protein (APP), a key step in promoting amyloid plaque formation in AD. Both insulin and IGF-I decreased APP phosphorylation in cultured primary cortical neurons, supporting their therapeutic use in AD. Induction of IR blocked the beneficial effect of insulin and reduced the effect of IGF-I on APP dephosphorylation. These effects were mediated by the phosphatidylinositol 3- kinase (PI3-K)/protein kinase B (Akt) pathway, as inhibition of this pathway during IR restored the effect of IGF-I on APP dephosphorylation. Finally, we explored the translational relevance of these results in vivo by demonstrating that high fat diet fed mice, a robust model of IR and MetS, exhibited the expected increased brain APP phosphorylation. Overall, these data suggest that the beneficial therapeutic effect of insulin and IGF-I on APP phosphorylation is negatively impacted by IR, and suggest that insulin and IGF-I alone may not be appropriate therapies for AD patients with IR, MetS, or diabetes.

Obesity is an increasing problem in the equine population with recent reports indicating that the percentage of overweight horses may range anywhere from 20.6-51%. Obesity in horses has been linked to more serious health concerns such as equine metabolic syndrome (EMS). EMS is a serious problem in the equine industry given its defining characteristics of insulin dysregualtion and obesity, as well as the involvement of laminitis. Little research however has been conducted to determine the effects of EMS on routine healthcare of these horses, in particular how they respond to vaccination. It has been shown that obese humans and mice have decreased immune responses to vaccination. EMS may have similar effects on vaccine responses in horses. If this is the case, these animals may be more susceptible to disease, acting as unknown disease reservoirs. Therefore, we investigated the effects of EMS on immune responses to routine influenza vaccination. Twenty-five adult horses of mixed-sex and mixed-breed (8-21 years old) horses; 13 EMS and 12 non-EMS were selected. Within each group, 4 horses served as non-vaccinate saline controls and the remaining horses were vaccinated with a commercially available equine influenza vaccine. Vaccination (influenza or saline) was administered on weeks 0 and 3, and peripheral blood samples taken on week 0 prior to vaccination and on weeks 1, 2, 3, 4, and 5 post vaccination. Blood samples were used to measure hemagglutination inhibition (HI) titers and equine influenza specific IgGa, IgGb, and IgGT levels. Blood samples were also used to isolate peripheral blood mononuclear cells (PBMCs) for analysis of cell mediated immune (CMI) responses via real-time polymerase chain reaction (RT-PCR). All horses receiving influenza vaccination responded with significant increases (P 0.05) in HI titers, and IgGa and IgGb equine influenza specific antibodies following vaccination compared to saline controls. EMS did not significantly affect (P 0.05) humoral immune responses as measured by HI titers or IgG antibody isotypes to influenza vaccination. There was an effect of metabolic status on CMI responses, with influenza vaccinated EMS horses having lower gene expression of IFN-γ (P = 0.02) and IL-2 (P = 0.01) compared to vaccinated non-EMS control horses. Given these results, it appears that while metabolic status does not influence humoral responses to an inactivated influenza vaccine in horses, horses with EMS appear to have a reduced CMI response to vaccination compared to metabolically normal, non-EMS control horses.

Extracts derived from the leaves of the stevia plant (stevioside) are commonly used as sweeteners for humans and horses. Stevioside appears to be safe for human consumption, including for individuals with insulin dysregulation. In the horse, the safety or metabolic effects of stevioside on normal animals or on those with metabolic dysfunction are unknown. Furthermore, the inflammatory response to a glycemic challenge or to stevioside in horses is not well defined. Therefore, the objective of this study was to measure the effects of stevioside and a glycemic challenge on insulin, glucose, and inflammatory responses in horses with a common metabolic dysfunction (equine metabolic syndrome or EMS) compared with non-EMS controls. To accomplish this, 15 horses were selected; 8 EMS and 7 age-matched controls. An oral sugar test was performed using Karo corn syrup (karo) or stevioside in a random crossover design. Horses were given 0.15 mL/kg body weight of karo or its equivalent grams of sugar in stevia dissolved in water. Blood samples were collected by jugular venipuncture before administration of either stevia or karo and at 60 and 240 min after administration. Serum was used for glucose and insulin determination and plasma for isolation of peripheral blood mononuclear cells (PBMCs) for inflammatory cytokine analysis via flow cytometry and reverse transcription PCR (RT-PCR). Stevia appeared to stimulate lower glycemic and insulinemic responses when compared to karo, in particular in EMS horses. EMS and control horses had inverse inflammatory responses to administration of either stevia or karo with EMS horses having a proinflammatory response (P ≤ 0.05). These data provide evidence as to why horses with EMS may be predisposed to developing laminitis, potentially as a result of an exaggerated inflammatory response to glycemic and insulinemic responses. Furthermore, the data provide new avenues for exploring mechanisms behind the syndrome, in particular when using a glycemic challenge.

Obesity and metabolic disorders are associated with systemic low-grade chronic inflammation, both in humans and animals. The aim of the study is to assess the effects of obesity and hyperinsulinemia on individual components of the acute-phase reaction in equine metabolic syndrome (EMS) horses. Eight mixed-breed EMS and six control, age-matched horses of both sexes were included in the study. Animals were classified as EMS or control based on the assessment of BCS, cresty neck score, and basal insulin50 μU/mL and/or insulin responses to the oral sugar test (OST)60 μU/mL. Peripheral venous blood was collected. The expression of proinflammatory cytokines, the concentration of circulating cytokines, and acute-phase proteins (serum amyloid A, C-reactive protein, haptoglobin, activin A, and procalcitonin) were measured. The data were analyzed using the Mann-Whitney test, whereas correlations were examined using Spearman's correlation coefficient. The tests were statistically significant if P ≤ 0.05. There were no differences in cytokine gene expression, circulating cytokine concentrations, or concentrations of acute-phase proteins between the EMS and the control groups. There was a strong correlation between the basal concentration of insulin and the serum concentrations of IL-6 (r = 0.71, P0.05). Activin A was positively correlated with post-OST insulin concentrations (r = 0.707, P = 0.05), indicating that this marker of inflammation could warrant further investigation in horses with EMS.

DNA methylation is an epigenetic mechanism important for the regulation of gene expression, which plays a vital role in the interaction between genetic and environmental factors. Aberrant epigenetic changes are implicated in the pathogenesis of diabetes and diabetic complications, but the role of DNA methylation in diabetic peripheral neuropathy (DPN) is not well understood. Therefore, our aim in this study was to explore the role of DNA methylation in the progression of DPN in type 2 diabetes. We compared genome-wide DNA methylation profiles of human sural nerve biopsies from subjects with stable or improving nerve fibre counts to biopsies from subjects with progressive loss of nerve fibres. Nerve fibre counts were determined by comparing myelinated nerve fibre densities between an initial and repeat biopsy separated by 52 weeks. Subjects with significant nerve regeneration (regenerators) and subjects with significant nerve degeneration (degenerators) represent the two extreme DPN phenotypes. Using reduced representation bisulfite sequencing, we identified 3,460 differentially methylated CpG dinucleotides between the two groups. The genes associated with differentially methylated CpGs were highly enriched in biological processes that have previously been implicated in DPN such as nervous system development, neuron development, and axon guidance, as well as glycerophospholipid metabolism and mitogen-activated protein kinase (MAPK) signalling. These findings are the first to provide a comprehensive analysis of DNA methylation profiling in human sural nerves of subjects with DPN and suggest that epigenetic regulation has an important role in the progression of this prevalent diabetic complication.

Equine metabolic syndrome (EMS) is an ever-increasing problem in the equine industry, especially considering that it is a risk factor for the development of laminitis. Equine metabolic syndrome is similar to metabolic syndrome in humans, which has been associated with alterations in intestinal microbiota. However, no work to date has been published to characterize the fecal microbiota in the EMS horse to determine differences, if any, from the metabolically normal animal. Therefore, our objective was to characterize the fecal microbiota of horses with EMS compared with non-EMS controls. Ten horses were classified as having EMS, and 10 non-EMS controls were selected for this work. Equine metabolic syndrome was determined as: insulin resistance, general or regional adiposity, and a history of or predisposition to laminitis. Blood collection via jugular venipuncture was performed, along with an oral sugar challenge. Concurrent with blood collection, phenotypic measurements and fecal grab samples were taken. Fecal samples were used to extract DNA for next generation sequence-based analysis of the bacterial microbiota. Equine metabolic syndrome horses exhibited a decrease in fecal microbial diversity, and there were differences in overall community structure between EMS horses and controls. The operational taxonomic unit with the highest linear discriminant analysis effect size in association with EMS horses was a member of subdivision 5 of Verrucomicrobia incertae sedis (uncertain classification). Interestingly, the control group had an overrepresentation of genus Fibrobacter . These data demonstrate that EMS horses have differences in their fecal microbiota compared with controls.

Equine metabolic syndrome (EMS) is a growing problem in the equine industry, particularly considering it is a risk factor for the development of laminitis. Equine metabolic syndrome is similar to metabolic syndrome in humans, which is associated with abnormal circulating plasma lipid concentrations. Thus, our objectives were to characterize the plasma lipid profiles, or lipidome, of horses with EMS compared to non-EMS controls and to further characterize the inflammatory state of these horses. Twenty-three horses of mixed breed and sex were selected. Of these, 14 were classified as EMS and 9 as non-EMS controls. Equine metabolic syndrome was determined by insulin resistance, general or regional adiposity, and a history of or predisposition to laminitis. Fasting serum and plasma samples were collected via jugular venipuncture. Serum samples were used to determine insulin, leptin, triglyceride, cholesterol, and nonesterified fatty acid concentrations. Heparinized plasma samples were used to isolate peripheral blood mononuclear cells for inflammatory cytokine determination and ethylenediaminetetraacetic acid plasma to analyze lipidomes. Equine metabolic syndrome horses had increased serum leptin and triglycerides. Plasma lipidomic analysis indicated that EMS horses had elevated triacylglycerides, diacylglycerides, monoacylglycerides, and ceramide compared to control horses. They had lower plasma sphingomyelins, suflatide, and choline plasmalogens. Peripheral blood mononuclear cell analysis for cytokine protein concentration via flow cytometry and gene transcription via real-time polymerase chain reaction showed no differences between the two groups; however, high variability may have influenced results. These data demonstrate that EMS horses have differences in their plasma lipidome compared to controls, similar to what has been observed in humans with metabolic syndrome.

Weaning of foals is a stressful event, and often, times can be associated with negative health consequences, including reduced immunity and increased susceptibility to gastrointestinal and respiratory disorders. Thus, a possible preventative immunomodulator of weaning-associated stress was studied in 21 pony foals (205 days ± 5 day old). Of these, n = 11 received the immunostimulant EqStim and n = 10 received a saline control on days −7, −3, and 0 (day of weaning). During these time points, as well as days 3, 7, 14, and 21, after weaning measurements were determined for the following parameters: clinical parameters as well as plasma cortisol concentrations, immune function using flow cytometry for protein determination and real-time polymerase chain reaction (PCR) for gene expression, and pathogen detection using nasal swabs (NSs) and PCR. Results showed an overall time effect of weaning for most parameters: decreased rectal temperature, increased nasal discharge and NS PCR detection of Streptococcus equi subsp. zooepidemicus , increased serum cortisol levels, and both decreased and increased production of cytokines (lymphocyte production interferon gamma and tumor necrosis factor alpha (TNF-α) and whole blood levels of IL-1β, TNF-α, and IL-10). In addition, there was an effect of treatment on cortisol concentrations and peripheral blood mononuclear cell production of IL-1β. Data suggest that, regardless of treatment, the weaning process produces significant changes in clinical and immunologic measures in foals. Although treatment with the immunostimulant EqStim had no effect on the incidences of nasal shedding of respiratory pathogens or other clinical parameters, it did appear to modulate rises in cortisol after weaning and possibly enhance innate immunity.

Age, neurodegenerative disorders, and dysfunction of insulin secretion may be correlated with increased systemic concentrations of acute phase markers. Thus, the study aimed to determine the effect of age, pituitary pars intermedia dysfunction (PPID), and insulin dysregulation (ID) associated with PPID, on markers of the acute phase reaction. Twenty-nine mix-breed horses of both sexes were classified into groups: (1) healthy adult controls, (2) healthy non-PPID geriatric horses, (3) PPID ID+ horses, and (4) PPID ID- horses. Whole blood proinflammatory cytokine gene expression and serum concentrations of pro- and anti-inflammatory cytokines and acute phase proteins were measured. The data were analyzed using the Mann-Whitney U-test, and correlations between groups of data were assessed using Spearman's correlation coefficient. The tests were statistically significant if P < 0.05. No differences in the whole blood cytokine gene expression, serum cytokine concentrations, or acute phase proteins were noted between the groups. In the PPID ID group, there was a strong correlation between the ACTH concentration after the administration of thyrotropin-releasing hormone and the expression of IL-8 (r = 0.941; P = 0.0321). In the PPID ID+ group, there was a strong correlation between basal insulin concentrations and serum amyloid A (SAA; r = 0.936; P = 0.0083) as well as between postprandial insulin concentrations and SAA (r = 0.965; P = 0.001). These data suggest that neurodegeneration in horses moderately affects circulating markers of inflammation and that ID in horses with PPID influences acute phase inflammatory markers.

Much of the equine population is obese and therefore predisposed to the development of additional health concerns such as equine metabolic syndrome (EMS). However, pharmacologic treatments for EMS are limited. Omega-3 fatty acid supplementation is a therapeutic strategy in humans with metabolic dysfunction that improves insulin sensitivity and reduces inflammation, but the effects of omega-3 fatty acid supplementation in horses with EMS are unclear. Therefore, in this pilot study, 10 mixed-sex and mixed-breed horses with EMS were fed a docosahexaenoic acid (DHA)-rich microalgae containing 16 g DHA/horse/d or served as controls for 46 days. Inflammatory status was measured using serologic enzyme-linked immunosorbent assay and in peripheral blood mononuclear cells (PBMCs) using flow cytometry and reverse transcription polymerase chain reaction. Circulating fatty acids, triglyceride, leptin, and adiponectin concentrations were also determined. Insulin and glucose dynamics were assessed with oral sugar test (OST) and frequently sampled intravenous glucose tolerance testing. Postsupplementation, treated horses had an increase in many circulating fatty acids, including DHA (P < .001). Treated horses also had lower serum triglycerides postsupplementation (P = .02) and a trend (P = .07) for reduced PBMC tumor necrosis factor α. Interestingly, after 46 days, control horses had an increase in insulin responses to the OST (P = .01), whereas treated horses did not (P = .69). These pilot data indicate that DHA-rich microalgae supplementation alters circulating fatty acids, modulates metabolic parameters, and may reduce inflammation in horses with EMS.

Peripheral neuropathy (PN) is a common, morbid complication of diabetes and prediabetes. Dysfunction of toll like receptors (TLR) 2 and 4 has been implicated in PN pathogenesis, and blocking TLR4 is proposed as a treatment for neuropathic pain. WT and TLR 2/4 global KO (TLR2/4-/-; n=5) mice were placed on a 60% high fat diet (HFD, n=5), with 5 WT controls provided a standard diet (CON, n=5). Mice underwent metabolic, neuropathic, and immunological phenotyping (Table 1) at early and late disease time points. TLR2/4-/- mice weighed more (P ≤ 0.023) but did not have increased fasting blood glucose levels. They had reduced hind paw withdrawal latencies (HPL) compared to HFD mice (P ≤ 0.01) and were similar to CON in early disease, but worsened in later disease. TLR2/4-/- mice consistently had lower sural nerve conduction velocities than CON and worsened as disease progressed (P ≤ 0.048). Immunophenotyping during late disease showed differences in the number of peripheral blood Ly6C- myeloid cells (P ≤ 0.05) and F4/80+ expression (P ≤ 0.0001). Inflammatory profiles of the sural nerve indicated a lower gene expression of the chemoattractant CCL-12 chemokine in TLR2/4-/- compared to CON mice. These results suggest that TLR2/4 KO may be initially protective against HFD induced neuropathy, but as PN progresses this effect is lost. Differences in circulating immune cells and CCL-12 gene expression may indicate an anti-inflammatory phenotype in TLR2/4-/- mice.Table 1. Metabolic, neuropathic and immunological phenotyping.Phenotypic MetricCON (n=5)HFD (n=5)TLR2/4-/-(n=5)Late stage weight (g)26 ± 339 ± 351 ± 3Late stage fasting blood glucose (mg/dL)150 ± 22190 ± 22172 ± 22HPL (sec)Early: 3.6 ± 0.3Late: 3.5 ± 0.4Early: 5.7 ± 0.3Late: 5.5 ± 0.4Early: 4.4 ± 0.3Late: 6.1 ± 0.4Sural NCV (m/s)Early: 22.0 ± 0.7Late: 23.9 ± 0.7Early: 18.5 ± 0.7Late: 17.9 ± 0.7Early: 20.0 ± 0.7Late: 17.2 ± 0.9Late stage Ly6C- circulating myeloid cells (104 cells/mL)7.8 ± 2.614.0 ± 2.74.8 ± 2.7Late stage circulating myeloid F4/80 expression (mean florescent intensity)978 ± 9611383 ± 10199967 ± 1019Late stage CCL-12 sural nerve gene expression (relative quantity)1.16 ± 0.240.59 ± 0.280.32 ± 0.24 Disclosure S. Elzinga: None. B. Murdock: None. J.M. Hayes: None. M.A. Tabbey: None. E.L. Feldman: None.

Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes. Emerging evidence suggests that aberrant DNA methylation may play a critical role in the pathogenesis of diabetic complications. Yet, its involvement in DPN is not fully characterized. In this study, we performed a large-scale genome-wide methylation profiling of sural nerve samples obtained from subjects with type 2 diabetes mellitus (T2DM) using Reduced Representation Bisulfite Sequencing (RRBS). A total of 78 samples were sequenced and mapped to the human reference genome. Differential analysis was performed on two groups, identified by our previous transcriptomic analysis of the same samples, which showed significantly different HbA1c levels: Group 1 (=8.5%±1.6%) and Group 2 ( In summary, we demonstrate that type 2 diabetic patients with peripheral neuropathy and high HbA1c show distinct variations in sural nerve methylome, suggesting that DNA methylation and DPN are associated and that these associations are at least in part HbA1c-dependent. Our results provide new insights into the role of HbA1c in epigenetic variation, and identify candidate genes relevant to the pathogenesis of DPN in human sural nerves of subjects with T2DM. Disclosure K. Guo: None. S. Elzinga: None. S. Eid: None. C. Figueroa-Romero: None. B.A. McGregor: None. G. de Anda-Jáuregui: None. C. Pacut: None. E.L. Feldman: None. J. Hur: None.

Neuropathy is a common, morbid complication of the metabolic syndrome, prediabetes, and diabetes. Recent studies have indicated a potential role for the immune system in the development of neuropathy. In particular, toll-like receptors (TLR) 2 and 4 have been linked to metabolic dysfunction, and blocking TLR4 is proposed as a treatment for neuropathic pain. In the current study, we investigated the role of the immune system, particularly TLRs 2 and 4, in the pathogenesis and progression of neuropathy. Sural or sciatic nerve gene expression arrays from humans and murine neuropathy models of prediabetes and diabetes were first analyzed to identify differentially expressed TLR2- and TLR4-associated genes within the KEGG (Kyoto Encyclopedia of Genes and Genomes) database. We observed that genes associated with TLRs 2 and 4, particularly lipopolysaccharide binding protein (LPB) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta (PIK3CB), were dysregulated across species and across multiple murine models of prediabetic and diabetic neuropathy. To further understand the role of these pathways in vivo, TLR 2 and 4 global knockout mice placed on a 60% high fat diet (HFD-TLR2/4(−/−)) were compared with wild type (WT) mice on a high fat diet (HFD-WT) and WT controls on a standard diet (CON). Mice then underwent metabolic, neuropathic, and immunological phenotyping at two time points to assess the impact of TLR signaling on neuropathy and immunity during metabolic dysfunction over time. We found that HFD-TLR2/4(−/−) and HFD-WT mice weighed more than CON mice but did not have increased fasting blood glucose levels. Despite normal blood glucose levels, HFD-TLR2/4(−/−) mice eventually developed neuropathy at the later time point (28 wks of age) but were somewhat protected from neuropathy at the early time point (16 wks of age) as measured by shorter hind paw withdraw latencies. This is in contrast to HFD-WT mice which developed neuropathy within 11 wks of being placed on a high fat diet and were neuropathic by all measures at both the early and late time points. Finally, we immunophenotyped all three mouse groups at the later time point and found differences in the number of peripheral blood Ly6C-myeloid cells as well as F4/80+ expression. These results indicate that TLR signaling influences early development of neuropathy in sensory neurons, potentially via immune modulation and recruitment.

The purpose of this study was to evaluate whether aged horses demonstrate statistically higher fecal egg counts (FECs) compared with middle-aged horses and to investigate systemic expression of proinflammatory cytokines in old and middle-aged horses treated with moxidectin compared with horses treated with pyrantel pamoate. Old horses (n = 21) and middle-aged horses (n = 19) were blocked by levels of inflammation and randomly allocated to one of the following treatment groups: group 1 (n = 8) old treated with moxidectin gel; group 2 (n = 9) old treated with pyrantel pamoate paste; group 3 (n = 4) old receiving no treatment; group 4 (n = 8) middle-aged treated with moxidectin gel; group 5 (n = 7) middle-aged treated with pyrantel pamoate paste, and group 6 (n = 4) middle-aged receiving no treatment. Fecal samples were collected on day 0 and again 14 days after treatment to determine FECs and presence of Strongylus vulgaris by polymerase chain reaction. Peripheral blood was also collected on day 0 and again at days 3, 5, and 14 after deworming for inflammatory cytokine analysis, along with routine hematological analyses. Results indicated that old horses have significantly higher FEC than middle-aged adults. Fecal egg counts declined significantly after anthelmintic treatment in both age groups of horses. Inflammatory markers exhibited differences between age groups and by anthelmintic treatment. In summary, this study provided evidence of different inflammatory and immunologic reactions to anthelmintic treatment in old horses. Moreover, the higher FECs found in the old horse group may have practical implications for parasite management routines on farms with representation of this age group.

s / Journal of Equine Veterinary Science 35 (2015) 418e436 426 99 Characterization of lipid and inflammatory profiles in horses with equine metabolic syndrome S.E. Elzinga*, P.L. Wood , and A.A. Adams 1 University of Kentucky, Lexington, KY, USA; 2 Lincoln Memorial University, Harrogate, TN, USA Equine metabolic syndrome (EMS) is a growing problem in the equine industry that is linked to the potentially devastating condition laminitis. One of the defining characteristics of EMS is obesity. In humans and mice, a relationship between obesity, lipid profiles, and inflammation has been observed. As such, focus has shifted to using lipidomics to classify lipid profiles of individuals with metabolic dysfunction, but this technique has yet to be utilized in the horse. Moreover, previous work has yielded conflicting results about the inflammatory state of EMS animals. Considering that abnormal lipid profiles may contribute to altered cell signaling or increased inflammation, our goal was to examine possible differences in plasma lipid profiles of EMS horses compared with controls, as well as better understand their inflammatory state. To accomplish this, 23 age-matched mixed sex and breed horses (13 ± 3 years old) were selected in the spring of 2014 from the University of Kentucky’s herd. Of the horses, 9 were classified controls and 14 EMS. EMS was determined by the presence of hyperinsulinemia, general or regional obesity, and history of laminitis. All horses were maintained on a diet of mixed grass hay and had access to water and a mineral block ad libitum. Prior to blood sample collection horses were weighed, and average body condition (BCS) and cresty neck score (CNS) was calculated from 3 trained investigators. Following fasting, serum and plasma sample collection was performed via jugular venipuncture and an oral sugar test was administered. Serum was analyzed for insulin, leptin, triglyceride, cholesterol, and nonesterified fatty acid (NEFA) concentrations. Plasma samples were used to isolate peripheral blood mononuclear cells for cytokine protein concentration via flow cytometry and gene transcription via RT-PCR. Plasma was utilized for lipidomic analysis. Data were analyzed using SIGMASTAT version 3.1 by 2 way ANOVAwith fixed effects set as EMS vs control and age. As expected, EMS horses had increased BCS, CNS, basal insulin, and insulin post administration of oral sugar (P 0.05) differences between the 2 groups, however high variability may have influenced results. Lipidomics analysis revealed significantly elevated (P < 0.05) plasma tri-, di-, mono-acylglycerides, and ceramide in EMS horses, but lower sphingomyelins, suflatides, and choline plasmalogens. Thus, EMS horses have differences in their lipid profiles compared with controls.