Equine Metabolic Syndrome Horses Research Articles

Focus on the epidemiology, pathophysiology, diagnosis, and management of insulin dysregulation in horses

Because there are various inter-related disorders of insulin metabolism that all may contribute to the final result of hyperinsulinaemia, the phrase "insulin dysregulation" has become the most acceptable term to use when describing equine metabolic syndrome (EMS). Insulin dysregulation is widespread in horses, and it appears to be more prevalent in physically sedentary horses. Laminitis and hyperinsulinaemia have been linked, as have other components of insulin dysregulation. Diagnosis of insulin dysregulation should be done in stages, beginning with basal testing, which includes baseline values of insulin and glucose, and concluding with dynamic tests, which detect tissue insulin resistance and hyperinsulinaemia. Dietary adjustments are the key to weight loss in the majority of EMS horses. To encourage weight loss, the horse should be given around 1.25% of its body weight every day. Exercise not only aids equid weight loss but also improves insulin sensitivity. The exercise should be tailored to the specific horse, taking into account its breed, fitness level, and owner's resources. When an obese horse is unable to exercise owing to laminitis or when obesity persists after extensive exercise and dietary management, levothyroxine sodium and metformin hydrochloride can be given to help accelerate weight loss and increase insulin sensitivity. Insulin dysregulation is a defining feature of equine metabolic syndrome and has garnered attention due to its direct link to laminitis. There is need for an in-depth understanding of ID in order to prevent the development of clinical laminitis. The current study discusses the epidemiology, pathophysiology, diagnosis, and management of insulin dysregulation in horses.

Sex hormone-binding globulin improves lipid metabolism and reduces inflammation in subcutaneous adipose tissue of metabolic syndrome-affected horses.

Equine metabolic syndrome (EMS) is a steadily growing endocrine disorder representing a real challenge in veterinary practice. As a multifactorial condition, EMS is characterized by three main metabolic abnormalities including insulin resistance, increased adiposity or obesity and hoof laminitis. Adipose tissue dysfunction is recognized as a core pathophysiological determinant of EMS, as it strongly participates to lipotoxicity and systemic metaflammation, both of which have been closely linked to the development of generalized insulin resistance. Besides, sex hormone binding globulin (SHBG) is an important sex steroids transporters that has been recently proposed as an important metabolic mediator. Therefore, the aim of this study was to verify whether SHBG treatment may ameliorate subcutaneous adipose tissue metabolic failure under EMS condition in terms of lipidome homeostasis, lipid metabolism programs, insulin signalling and local inflammation. Subcutaneous adipose tissue (SAT) biopsies were collected post-mortem from healthy (n = 3) and EMS (n = 3) slaughtered horses. SHBG protein has been applied to SAT samples from EMS horses for 24h at a final concentration of 50nM, while control groups (healthy and untreated EMS) were cultured in the presence of SHBG-vehicle only. Tissues from all groups were afterwards secured for downstream analysis of gene expression using RT-qPCR, protein levels by Western blot and ELISA assay and lipidomics through GC-MS technique. Obtained results showcased that SHBG intervention efficiently normalized the altered fatty acids (FAs) profiles by lowering the accumulation of saturated and trans FAs, as well as the pro-inflammatory arachidonic and linoleic acids. Moreover, SHBG showed promising value for the regulation of adipocyte lipolysis and engorgement by lowering the levels of perilipin-1. SHBG exerted moderated effect toward SCD1 and FASN enzymes expression, but increased the LPL abundance. Interestingly, SHBG exhibited a negative regulatory effect on pro-adipogenic stimulators and induced higher expression of KLF3, IRF3 and β-catenin, known as strong adipogenesis repressors. Finally, SHBG protein showed remarkable ability in restoring the insulin signal transduction, IR/IRS/Pi3K/AKT phosphorylation events and GLUT4 transporter abundance, and further attenuate pro-inflammatory response by lowering IL-6 tissue levels and targeting the PDIA3/ERK axis. Overall, the obtained data clearly demonstrate the benefice of SHBG treatment in the regulation of adipose tissue metabolism in the course of EMS and provide new insights for the development of molecular therapies with potential translational application to human metabolic disorders.

The Differences in Histoarchitecture of Hoof Lamellae between Obese and Lean Draft Horses.

Obesity is a common problem in horses. The associations between obesity and equine metabolic syndrome (EMS) and between EMS and laminitis are known. However, there is a lack of data on whether obesity itself can affect hoof lamellae. Forelimbs and blood from 12 draft horses (six obese and six lean) from a slaughterhouse were acquired. To exclude laminitis and EMS horses, insulin concentration was measured, and hooves were radiographed. Histological evaluation was performed. The shape of the primary and secondary epidermal lamellae (PEL and SEL) was evaluated, and the length of the keratinized and total primary epidermal lamellae was measured (KPEL and TEL). All horses showed pathological changes in lamellae. In the lean group, the changes were longer SELs, more proliferated and separated PDLs, and less standard PDLs. In the obese group, the changes were a lower number of club-shaped and standard SELs and significantly more tapered SELs. No difference in the shape of PELs and the length of KPELs was noticed. The research did not confirm the effects of obesity on lamellar failure. The measurements taken indicate that the lamellae are much longer compared to other research studies; this could indicate that the length of the PEL depends on the hoof size.

Systemic Administration of Rejuvenated Adipose-Derived Mesenchymal Stem Cells Improves Liver Metabolism in Equine Metabolic Syndrome (EMS)- New Approach in Veterinary Regenerative Medicine

Equine metabolic syndrome (EMS) is characterized by adiposity, insulin dysregulation and increased risk for laminitis. Increased levels of specific liver enzymes in the peripheral blood are typical findings in horses diagnosed with EMS. Current management of EMS is based on caloric restriction and increased physical activity. However, new potential treatment options are arising such as the transplantation of autologous adipose stem cells (ASC). However, cytophysiological properties of ASC derived from EMS horses are impaired which strongly limits their therapeutic potential. We hypothesized, that in vitro pharmacotherapy of those cells with 5-azacytidine (AZA) and resveratrol (RES) before their clinical application can reverse the aged phenotype of those cells and improve clinical outcome of autologous therapy. A 9 year old Dutch Warmblood Horse used for driving, was presented with severe obesity, insulin resistance. After EMS diagnosis, the animal received three intravenous injections of autologous, AZA/RES treated ASCs at weekly intervals. The therapeutic effect was assessed by the analysis of liver specific enzymes in the blood. ASC-transplantation reduced levels of glutamate dehydrogenase (GLDH), gamma-glutamyltransferase (GGT), lactate dehydrogenase (LDH) and aspartate transaminase (AST). This case report demonstrates the therapeutic potential of this intervention for EMS as well as apt utility of autologous, rejuvenated ASC injections.

The effect of insulin infusion on heart rate and systemic blood pressure in horses with equine metabolic syndrome.

There is little evidence that horses with equine metabolic syndrome (EMS) have higher resting blood pressures than horses with normal insulin sensitivity. However, there are indications that EMS horses have an altered dynamic response to the cardiovascular effects of insulin. To examine heart rate and the systemic blood pressure response in EMS and control horses during insulin infusion. Cross-sectional study. Horses were examined with an oral sugar test (OST) and a euglycaemic hyperinsulinaemic clamp (EHC). Based on the results from the OST, the horses were classified as either EMS or control. Blood pressure was measured before the start of the EHC and at 60, 120 and 180min of the EHC. There were 16 EMS and 12 control horses. Mean resting heart rate was higher for EMS horses compared with control (35.6±5.1 vs. 30.3±3.5beats/min). Systolic, diastolic and mean arterial blood pressure did not differ between groups at the start of the EHC. Blood pressure decreased gradually during the EHC in the control group, whereas the EMS group showed no decline in systolic, diastolic or mean arterial pressure. Limited number of horses. Horses with EMS have cardiovascular changes that affect resting heart rate and systemic blood pressure during insulin infusion.

64 Culture of bovine oocytes and embryos with metabolic hormones concentrations associated with equine metabolic syndrome

Maternal metabolic status could affect fertility and early embryo development due to altered concentrations of metabolic hormones. Equine metabolic syndrome (EMS) is a condition in horses associated with obesity and insulin resistance. Equine metabolic syndrome is accompanied by increased concentrations of insulin and leptin and decreased concentrations of adiponectin, in ovarian follicular fluid (FF) and in systemic circulation (SYST). We sought to determine how altered concentrations of insulin, leptin, and adiponectin (ILA), consistent with those in mares with EMS (EMS) or normal mares (normal), would affect blastocyst formation rates, blastocyst gene expression for metabolism and inner cell mass formation (OCT4, SOX2, COX2, DNMT3a1, HK2, LDH, PDH, and GLUT1), and metabolite uptake from culture media. Because equine oocytes are not available for large-scale study, a bovine model was used in this preliminary study to determine the impact of altered ILA on oocytes and embryos. Bovine ovaries were obtained from an abattoir and embryos produced as previously described using chemically defined media (CDM; Barcelo-Fimbres and Seidel 2007Mol. Reprod. Dev. 74, 1406-1418). Briefly, oocytes were cultured in in vitro maturation medium (IVM), fertilized in FCDM, presumptive zygotes were placed into CDM-1 for ~56h. Cleavage rates were assessed, and embryos were moved to CDM-2 for ~122 additional hours. Treatments consisted of 5 groups: (1) standard oocyte IVM, FCDM and embryo production (EP) system (control), (2) IVM with normal FF ILA and control FCDM and EP, (3) IVM with normal FF ILA and FCDM and EP with normal SYST ILA, (4) IVM with EMS FF ILA and control FCDM and EP, and (5) IVM with EMS FF ILA and FCDM and EP with EMS SYST ILA. Seven days after fertilization, blastocysts were pooled in groups of 5 and placed into 50mL of CDM-2 for 24h. Embryos were removed, and medium was frozen and stored at −80°C to determine metabolite usage via gas chromatography mass spectroscopy. Pooled embryos were washed and placed into RNA lysis solution for relative quantitative PCR. Statistical comparisons were performed using ANOVA with a post-hoc Tukey test. Blastocyst formation rates and gene expression of viability markers were not significantly different among groups. However, aspartate was lower (P=0.02) in spent media from Group 3 (normal FF and SYST ILA) and tended (P=0.09) to be lower in media from Group 5 (EMS FF and SYST ILA) when compared with controls (Group 1). The ILA during early embryo development but not oocyte maturation appeared to be associated with increased uptake of aspartate, a nonessential amino acid, thought to be involved in osmoregulation, cellular signalling, and in mouse embryos, facilitate the metabolism of lactate. In conclusion, the addition of ILA in concentrations observed in normal horses and EMS horses did not affect blastocyst formation rates or markers of embryo viability, although embryo metabolism could have been altered.

Immunomodulatory Properties of Adipose-Derived Stem Cells Treated with 5-Azacytydine and Resveratrol on Peripheral Blood Mononuclear Cells and Macrophages in Metabolic Syndrome Animals.

Endocrine disorders, including equine metabolic syndrome (EMS), are a serious issue in veterinary medicine and horse breeding. Furthermore, EMS was shown to affect the cytophysiological properties of adipose-derived stem cells, reducing their therapeutic potential. However, it was shown that those cells can be rejuvenated while using a combination of two chemicals: 5-azacytydine (AZA) and resveratrol (RES). In the present study, we decided to evaluate the immunomodulatory properties of AZA/RES-treated adipose-derived stem cells (ASC) isolated from EMS horses (ASCEMS). Thus, we co-cultured ASC with peripheral blood mononuclear cells (PBMC) and RAW264.7 macrophages. Most attention was placed on regulatory T lymphocytes (TREG), as well as the messenger RNA (mRNA) and protein levels of several cytokines (tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-10, and IL-1β). Moreover, we also investigated the expression of genes related to auto- and mitophagy in both PBMCs and ASCs. PBMCs were obtained from healthy and EMS-suffering individuals and were co-cultured with ASCs that were isolated from healthy and EMS horses cultured in control conditions and with AZA/RES. We discovered that cells treated with AZA/RES increase the TREG number while co-cultured with PBMCs. Moreover, the co-culture of PBMCs with AZA/RES-treated ASCEMS induced mitophagy in PBMCs. Furthermore, ASCEMS pre-treated with AZA/RES displayed anti-inflammatory properties, as decreased levels of TNF-α, nitric oxide (NO), and IL-6 were observed in those cells in comparison with their untreated counterparts in the co-culture with RAW264.7 macrophages. In summary, we demonstrated that ASCEMS treated with AZA/RES displayed increased anti-inflammatory properties, and was able to regulate and activate the TREG-related anti-inflammatory response.

Characterization of Apoptosis, Autophagy and Oxidative Stress in Pancreatic Islets Cells and Intestinal Epithelial Cells Isolated from Equine Metabolic Syndrome (EMS) Horses.

Endocrine disorders are becoming an increasing problem in both human and veterinary medicine. In recent years, more and more horses worldwide have been suffering from equine metabolic syndrome (EMS). This metabolic disorder is characterized by pathological obesity, hyperinsulinaemia, hyperglycaemia and insulin resistance. Although metabolic disorders, including diabetes, have been extensively studied, there are still no data on the molecular effects of EMS in horses. Thus, the aim of this study was to evaluate apoptosis, oxidative stress, autophagy and microRNA (miR) expression in multipotent intestinal epithelial stem cells (IECs) and pancreatic islets (PIs) isolated post mortem form healthy and EMS diagnosed horses. Our group was the first to describe how EMS affects IEC and PI aging and senescence. First, we evaluated isolation and culture protocol for these cells and subsequently established their metabolic status in vitro. Both IECs and PIs isolated from EMS horses were characterized by increased apoptosis and senescence. Moreover, they accumulated elevated levels of reactive oxygen species (ROS). Here we have observed that autophagy/mitophagy may be a protective mechanism which allows those cells to maintain their physiological function, clear protein aggregates and remove damaged organelles. Furthermore, it may play a crucial role in reducing endoplasmic reticulum (ER) stress. This protective mechanism may help to overcome the harmful effects of ROS and provide building blocks for protein and ATP synthesis.

Humoral and cell-mediated immune responses to influenza vaccination in equine metabolic syndrome (EMS) horses

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.

Excessive Endoplasmic Reticulum Stress Correlates with Impaired Mitochondrial Dynamics, Mitophagy and Apoptosis, in Liver and Adipose Tissue, but Not in Muscles in EMS Horses.

Nowadays, endocrine disorders have become more frequent in both human and veterinary medicine. In horses, reduced physical activity combined with carbohydrate and sugar overload may result in the development of the so-called equine metabolic syndrome (EMS). EMS is characterized by insulin resistance, hyperinsulinemia, elevated blood triglyceride concentrations and usually obesity. Although the phenotypic features of EMS individuals are well known, the molecular mechanism underlying disease development remains elusive. Therefore, in the present study, we analyzed insulin-sensitive tissues, i.e., muscles, liver and adipose tissue in order to evaluate insulin resistance and apoptosis. Furthermore, we assessed mitochondrial dynamics and mitophagy in those tissues, because mitochondrial dysfunction is linked to the development of metabolic syndrome. We established the expression of genes related to insulin resistance, endoplasmic reticulum (ER) stress and mitochondria clearance by mitophagy using RT-PCR and Western blot. Cell ultrastructure was visualized using electron transmission microscopy. The results indicated that adipose tissue and liver of EMS horses were characterized by increased mitochondrial damage and mitophagy followed by triggering of apoptosis as mitophagy fails to restore cellular homeostasis. However, in muscles, apoptosis was reduced, suggesting the existence of a protective mechanism allowing that tissue to maintain homeostasis.

Comparison of the Fecal Microbiota in Horses With Equine Metabolic Syndrome and Metabolically Normal Controls Fed a Similar All-Forage Diet

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.

Plasma Lipidomic and Inflammatory Cytokine Profiles of Horses With Equine Metabolic Syndrome

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.

Comparison of Plasma Active Glucagon-Like Peptide 1 Concentrations in Normal Horses and Those With Equine Metabolic Syndrome and in Horses Placed on a High-Grain Diet

Hyperinsulinemia is associated with laminitis in horses, and active glucagon-like peptide 1 (aGLP-1) stimulates insulin secretion. We hypothesized that plasma aGLP-1 concentrations measured during an oral sugar test (OST) would differ significantly between normal horses and those with equine metabolic syndrome (EMS) and that aGLP-1 concentrations would increase when EMS horses were placed on a high-grain diet. An enzyme-linked immunosorbent assay for aGLP-1 was validated with equine plasma and six horses with EMS, and 10 healthy Quarter horse crossbred mares were compared. Eleven months later, the same horses with EMS were placed on a high-grain diet for 8 weeks and aGLP-1 concentrations were measured during an oral glucose tolerance test at 0 and 8 weeks. Frequently sampled intravenous glucose tolerance tests were also performed. The assay was validated with equine plasma and concentrations increased during the OST. Area under the aGLP-1 curve did not differ between normal and EMS horses. There was a weak trend (P = .097) toward a higher maximum percentage increase in aGLP-1 concentrations during the OST in EMS horses compared with normal horses. Body weight increased (P = .031) after 8 weeks when EMS horses were placed on a high-grain diet. Resting (fasted) insulin concentrations significantly increased (P = .012), but plasma aGLP-1 concentrations did not change significantly. Our hypotheses were not supported because plasma aGLP-1 concentrations did not differ significantly between normal horses and those with EMS and did not increase when EMS horses were placed on a high-grain diet for 8 weeks. However, a trend toward higher percentage increases in aGLP-1 concentrations during the OST was detected in EMS horses, compared with normal horses, and this warrants further investigation.

Equine Metabolic Syndrome Affects Viability, Senescence, and Stress Factors of Equine Adipose-Derived Mesenchymal Stromal Stem Cells: New Insight into EqASCs Isolated from EMS Horses in the Context of Their Aging.

Currently, equine metabolic syndrome (EMS), an endocrine disease linked to insulin resistance, affects an increasing number of horses. However, little is known about the effect of EMS on mesenchymal stem cells that reside in adipose tissue (ASC). Thus it is crucial to evaluate the viability and growth kinetics of these cells, particularly in terms of their application in regenerative medicine. In this study, we investigated the proliferative capacity, morphological features, and accumulation of oxidative stress factors in mesenchymal stem cells isolated from healthy animals (ASCN) and horses suffering from EMS (ASCEMS). ASCEMS displayed senescent phenotype associated with β-galactosidase accumulation, enlarged cell bodies and nuclei, increased apoptosis, and reduced heterochromatin architecture. Moreover, we observed increased amounts of nitric oxide (NO) and reactive oxygen species (ROS) in these cells, accompanied by reduced superoxide dismutase (SOD) activity. We also found in ASCEMS an elevated number of impaired mitochondria, characterized by membrane raptures, disarrayed cristae, and vacuole formation. Our results suggest that the toxic compounds, accumulating in the mitochondria under oxidative stress, lead to alternations in their morphology and may be partially responsible for the senescent phenotype and decreased proliferation potential of ASCEMS.

99 Characterization of lipid and inflammatory profiles in horses with equine metabolic syndrome

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.

The Activity of IL-6 and TNF-α in Adipose Tissue and Peripheral Blood in horses Suffering from Equine Metabolic Syndrome (EMS)

Equine metabolic syndrome (EMS) is a metabolic disorder characterized by excessive obesity and or/regional adiposity, insulin resistance (IR) and prior or current laminitis. In the course of the EMS, an important role exert systemic inflammation caused by excessive expression of proinflammatory cytokines. The aim of the current investigation was to examine relationships between expression IL-6 and TNF-α in adipose tissue and their concentration in peripheral blood in horses with EMS. On the basis of range of research procedures, horses were divided into two groups: group A (EMS horses, n=8) and group B (healthy horses with overweight, n=8). The concentration of the proinflammatory cytokines in the peripheral blood and their expression in the adipose tissue were determined. The results of the EMS group showed numerous macrophages and lymphocytes infiltration and increased diameters of adipocytes (P<0.05). The concentration of TNF-α in the serum of insulin-resistant horses was statistically higher compared to the healthy individuals. No significant differences were observed between the EMS horses and the control horses in the concentration of IL-6 in the serum. Moreover, our research revealed expression of investigated cytokines in adipose tissue. The results shows a higher expression of TNF-α in the EMS groups. What is more, macrophages infiltration were observed. In the case of IL-6, were detected the similar arrangement of this cytokines in adipocytes between both test groups. The study indicates the importance of pro-inflammatory proteins TNFα in the equine metabolic syndrome.

Impact of dietary components on phenotypic measurements for Equine Metabolic Syndrome

Previous studies in healthy horses have demonstrated that dietary adaptation has an impact on many of the phenotypic variables measured and used as diagnostic criteria for horses with presumptive equine metabolic syndrome (EMS). However, the relationship between dietary intake and measured responses in EMS horses is less understood. Our objectives were to evaluate the relationships between dietary components and phenotypic variables used as EMS criteria and todetermine if the relationship differs based on obesity or previous laminitis status. Biochemical and dietary data were collected from 634 horses from 167 farms located throughout the United States and Canada. Biochemical variables were measured in the fasted state (insulin, glucose, triglycerides, NEFA and leptin concentrations) and after an oral sugar test (glucose and insulin concentrations. For each individual, total daily dietary consumption was calculated by weighing the daily rations of hay and concentrate and estimating pasture consumption based on hours of pasture access per day. Concentrate, hay and pasture samples were analyzed (Equi-Analytical, Ithaca, NY). Total daily digestible energy, crude protein, neutral detergent fiber, water soluble carbohydrates, and starch were calculated as a percent body weight in dry matter intake. Correlations with dietary components adjusted for obesity, previous laminitis status, age, breed, sex, and exercise were determined using a mixed effects model with farm as a random effect. Interaction terms were included in the model to detect differences in the correlations between dietary components and EMS phenotypic

Seasonal Variation in Serum Concentrations of Selected Metabolic Hormones in Horses

Determination of adrenocorticotropic hormone (ACTH) concentration is a commonly used test in the evaluation of endocrine causes of equine laminitis, but the concentration in healthy horses can be high at certain times of year, which alters the specificity of the ACTH test. To determine if circulating concentrations of ACTH, cortisol, glucose, insulin, and thyroxine vary month to month in healthy horses and in horses with equine metabolic syndrome (EMS). Nine healthy adult horses were studied on their farm/stable over the course of 1 year. After the diagnosis of EMS, 10 laminitic horses residing at the same farm/stable were also studied. Prospective study of healthy and laminitic horses. Plasma/serum samples were analyzed for concentrations of hormones and glucose. ACTH was the only analyte to show a discrete seasonal pattern, with concentrations in healthy and EMS horses frequently outside of the reference range (9-35 pg/mL) in August through October. Insulin was elevated (>40 microIU/mL) in EMS horses during most months and median serum glucose was generally higher in EMS horses (100 mg/dL, range, 76-163 mg/ dL) than in controls (94 mg/dL, range, 56-110 mg/dL), but no seasonal patterns for insulin or glucose were found. An increased ACTH concentration in horses in late summer or autumn should be interpreted with caution. In contrast, insulin concentration is maintained within the reference range throughout the year in healthy horses, thus an increased insulin concentration at any time of year should raise suspicions of EMS, ECD, or both.

Correlation of Plasma Insulin Concentration with Laminitis Score in a Field Study of Equine Cushing's Disease and Equine Metabolic Syndrome

This study aimed to investigate endocrinologic test values and the response to treatment of two commonly encountered causes of endocrinopathic laminitis, equine Cushing's disease (ECD) and equine metabolic syndrome (EMS), in a veterinary practice setting. In particular, the study aimed to determine whether insulin concentration correlated to the severity of clinical laminitis in horses with EMS or ECD. Twenty-five horses were included in the study and assigned to one of three groups: ECD (n = 6), EMS (n = 10), and controls (n = 9). Blood samples were collected at an initial visit and then at regular intervals for the next 12 months. Plasma concentrations of adrenocorticotropin (ACTH), cortisol, and insulin and serum concentrations of glucose and total thyroxine (T4) were obtained. Horses with ECD had significantly higher plasma ACTH concentrations than EMS horses or controls. Horses with EMS and ECD both had significantly higher plasma insulin concentrations than control horses, which was correlated with the Obel grade of laminitis ( r = 0.63). After treatment, there was a trend for a reduction in plasma ACTH concentration in horses with ECD. A program of diet and exercise for horses with EMS resulted in reductions in both plasma insulin concentrations and bodyweight, which was variable, depending on the individual. There was a significant correlation between the change in plasma insulin concentration and Obel grade of laminitis ( r = 0.69). This study has highlighted the importance of baseline plasma insulin concentration as a potential indicator of the susceptibility of horses to laminitis and the response to a program of diet and exercise.