Genome-Wide Association Analyses of Equine Metabolic Syndrome Phenotypes in Welsh Ponies and Morgan Horses.

Equine metabolic syndrome (EMS) is a complex trait for which few genetic studies have been published. Our study objectives were to perform within breed genome-wide association analyses (GWA) to identify associated loci in two high-risk breeds, coupled with meta-analysis to identify shared and unique loci between breeds. GWA for 12 EMS traits identified 303 and 142 associated genomic regions in 264 Welsh ponies and 286 Morgan horses, respectively. Meta-analysis demonstrated that 65 GWA regions were shared across breeds. Region boundaries were defined based on a fixed-size or the breakdown of linkage disequilibrium, and prioritized if they were: shared between breeds or across traits (high priority), identified in a single GWA cohort (medium priority), or shared across traits with no SNPs reaching genome-wide significance (low priority), resulting in 56 high, 26 medium, and seven low priority regions including 1853 candidate genes in the Welsh ponies; and 39 high, eight medium, and nine low priority regions including 1167 candidate genes in the Morgans. The prioritized regions contained protein-coding genes which were functionally enriched for pathways associated with inflammation, glucose metabolism, or lipid metabolism. These data demonstrate that EMS is a polygenic trait with breed-specific risk alleles as well as those shared across breeds.

Evaluation of an HMGA2 variant for pleiotropic effects on height and metabolic traits in ponies.

BACKGROUND: Ponies are highly susceptible to metabolic derangements including hyperinsulinemia, insulin resistance, and adiposity. HYPOTHESIS/OBJECTIVES: Genetic loci affecting height in ponies have pleiotropic effects on metabolic pathways and increase the susceptibility to equine metabolic syndrome (EMS). ANIMALS: Two hundred ninety-four Welsh ponies and 529 horses. METHODS: Retrospective study of horses phenotyped for metabolic traits. Correlations between height and metabolic traits were assessed by Pearson's correlation coefficients. Complementary genome-wide analysis methods were used to identify a region of interest (ROI) for height and metabolic traits, determine the fraction of heritability contributed by the ROI, and identify candidate genes. RESULTS: There was an inverse relationship between height and baseline insulin (-0.26) in ponies. Genomic signature of selection and association analyses for both height and insulin identified the same ~1.3 megabase region on chromosome 6 that contained a shared ancestral haplotype between these traits. The ROI contributed ~40% of the heritability for height and ~20% of the heritability for insulin. High-mobility group AT-hook 2 was identified as a candidate gene, and Sanger sequencing detected a c.83G>A (p.G28E) variant associated with height in Shetland ponies. In our cohort of ponies, the A allele had a frequency of 0.76, was strongly correlated with height (-0.75), and was low to moderately correlated with metabolic traits including: insulin (0.32), insulin after an oral sugar test (0.25), non-esterified fatty acids (0.19), and triglyceride (0.22) concentrations. CONCLUSIONS AND CLINICAL IMPORTANCE: These data have important implications for identifying individuals at risk for EMS.

Case-control study of risk factors for pasture-and endocrinopathy-associated laminitis in North American horses.

OBJECTIVE To investigate risk factors for the development of pasture- and endocrinopathy-associated laminitis (PEAL) in horses and ponies in North America. DESIGN Case-control study. ANIMALS 199 horses with incident cases of PEAL and 351 horses from 2 control populations (healthy horses [n = 198] and horses with lameness not caused by laminitis [153]) that were evaluated in North America between January 2012 and December 2015 by veterinarian members of the American Association of Equine Practitioners. PROCEDURES North American members of the American Association of Equine Practitioners were contacted to participate in the study, and participating veterinarians provided historical data on incident cases of PEAL, each matched with a healthy control and a lameness control. Conditional logistic regression analysis was used to compare data on PEAL-affected horses with data on horses from each set of controls. RESULTS Horses with an obese body condition (ie, body condition score ≥ 7), generalized or regional adiposity (alone or in combination), preexisting endocrinopathy, or recent (within 30 days) glucocorticoid administration had increased odds of developing PEAL, compared with horses that did not have these findings. CONCLUSIONS AND CLINICAL RELEVANCE The present study identified several risk factors for PEAL that may assist not only in managing and preventing this form of laminitis, but also in guiding future research into its pathogenesis.

Comparison of two methods for measurement of equine adrenocorticotropin.

Accurate measurement of equine adrenocorticotropin (ACTH) is important for the diagnosis of equine pituitary pars intermedia dysfunction (PPID). Several radioimmunoassays (RIAs) and chemiluminescent immunoassays (CIAs) are used for measurement of ACTH concentration in horses; whether these methods yield similar results across a range of concentrations is not determined. We evaluated agreement between a commercial RIA and CIA. Archived plasma samples ( n = 633) were measured with both assays. Correlation between the 2 methods was moderate ( r = 0.49, p < 0.001). Bland-Altman analysis revealed poor agreement, with a proportional bias and widening limits of agreement with increasing values. Poor agreement between assays was also observed when evaluating plasma samples with concentrations at or below the recommended diagnostic cutoff value for PPID testing. The lack of agreement suggests that measurements obtained should not be considered interchangeable between methods.

Comparison among the euglycemic-hyperinsulinemic clamp, insulin-modified frequently sampled intravenous glucose tolerance test, and oral glucose tolerance test for assessment of insulin sensitivity in healthy Standardbreds.

OBJECTIVE: To compare, in horses, estimates of insulin sensitivity obtained from minimal model analysis (MMA) of a frequently sampled IV glucose tolerance test (FSIGTT) with estimates from the euglycemic-hyperinsulinemic clamp (EHC) and to evaluate the validity of surrogate estimates of insulin sensitivity derived from an oral glucose tolerance test (OGTT). ANIMALS: 18 mature Standardbreds (mean ± SD body weight, 428.9 ± 35.9 kg; mean ± SD body condition score, 4.4 ± 1.0 [on a scale of 1 to 9]). PROCEDURES: All horses underwent at least 2 of the 3 procedures (EHC [n = 15], insulin-modified FSIGTT [18], and OGTT [18]) within a 10-day time frame to evaluate insulin sensitivity. RESULTS: Insulin sensitivity variables derived from the EHC and FSIGTT were strongly correlated (r = 0.88). When standardized to the same units of measure, these measures were still strongly correlated (r = 0.86) but were not equivalent. Area under the curve, peak insulin concentration, insulin concentration at 120 minutes, and 2 calculated indices from glucose and insulin data from the OGTT were significantly correlated with the EHC- and FSIGTT-derived estimates of insulin sensitivity. CONCLUSIONS AND CLINICAL RELEVANCE: In healthy Standardbreds with moderate body condition score, insulin sensitivities from the EHC and FSIGTT were strongly correlated but not equivalent. Estimates derived from an OGTT also may be useful to estimate insulin sensitivity.

Effects of the insulin sensitizing drug, pioglitazone, and lipopolysaccharide administration on markers of systemic inflammation and clinical parameters in horses.

Equine metabolic syndrome (EMS) is a condition of obese horses characterized by insulin resistance, systemic inflammation, and an increased risk of laminitis. The pathogenesis of EMS is thought, in part, to be due to inflammatory proteins produced by adipose tissue. Reducing inflammation may decrease the incidence of laminitis in horses with EMS. Pioglitazone hydrochloride, a thiazolidinedione, has efficacy to reduce obesity associated inflammation in humans. Eight normal, adult, horses were administered 1mg/kg pioglitazone for 14 days, and eight horses served as controls. Physical examination and hematologic variables, transcript abundance of pro-inflammatory cytokines in skeletal muscle and adipose tissue, and circulating concentrations of the acute phase protein, serum amyloid A and pro-inflammatory cytokine, TNF-α were assessed prior to, and following, an LPS infusion (35 ng/kg). The objective was to determine if pre-treatment with pioglitazone would mitigate the development of inflammation and associated clinical markers of inflammation following LPS administration. Lipopolysaccharide administration induced systemic inflammation, as assessed by clinical and hematological aberrations, increased TNF-α, SAA and adipose tissue IL-6 mRNA abundance, however no mitigating effects of pioglitazone were detected. A longer treatment period or higher dose might be indicated for future experiments.

Effects of acute hyperinsulinemia on inflammatory proteins in horses.

Laminitis is a painful, inflammatory disease of the equine hoof that often results in euthanasia. Elevated plasma insulin concentrations are a predictive factor for laminitis, and in previously healthy horses and ponies, laminitis was induced by infusion of insulin. Thus, we chose to determine if an infusion of insulin would increase plasma concentrations of inflammatory cytokines and cytokine mRNA abundance in subcutaneous adipose tissue, skeletal muscle, and white blood cells. Ten mature Thoroughbred mares received an insulin infusion that elevated plasma insulin concentrations for 6h or an equivalent volume of isotonic saline in a switchback design. Insulin infusion altered plasma concentrations of both TNF (P=0.037) and IL-6 (P=0.044), but did not result in consistent changes to either skeletal muscle or adipose tissue cytokine mRNA. Insulin may be involved in the production of inflammatory cytokines, and this could be a mechanism for insulin increasing the risk of laminitis.

De novo fatty acid synthesis and NADPH generation in equine adipose and liver tissue.

The lipogenic capacities of equine liver and adipose tissue explants were investigated in vitro. Preference for glucose or acetate as the primary carbon source for de novo fatty acid synthesis was determined using (14)C labeled substrates. Additional aims included determining the relative contribution of NADPH generating pathways to reducing equivalent generation and comparing the lipogenic activity of two adipose depots, mesenteric and subcutaneous harvested from the crest region of the neck. Mesenteric adipose tissue had greater lipogenic activity than subcutaneous adipose tissue, and liver tissue showed minimal (14)C incorporation into fatty acids, indicating a low hepatic lipogenic capacity. Acetate was found to be the primary carbon source for fatty acid synthesis due to both the appearance of the (14)C label in the lipid fraction and the low activity of ATP-citrate lyase. Finally, the pentose phosphate and isocitrate dehydrogenase enzymes contributed to NADPH production in equine adipose tissue.

Adaptive response of equine intestinal Na+/glucose co-transporter (SGLT1) to an increase in dietary soluble carbohydrate.

Experimental and epidemiological evidence suggests that consumption of hydrolyzable carbohydrate, hCHO (grain), by horses is an important risk factor for colic, a common cause of equine mortality. It is unknown whether the small intestinal capacity to digest hCHO and/or to absorb monosaccharides is limiting, or even if horses can adapt to increased carbohydrate load. We investigated changes in the brush-border membrane carbohydrate digestive enzymes and glucose absorptive capacity of horse small intestine in response to increased hCHO. Expression of the Na(+)/glucose co-transporter, SGLT1, was assessed by Western blotting, immunohistochemistry, Northern blotting, QPCR, and Na(+)-dependent D-glucose transport. Glucose transport rates, SGLT1 protein, and mRNA expression were all 2-fold higher in the jejunum and 3- to 5-fold higher in the ileum of horses maintained on a hCHO-enriched diet compared to pasture forage. Activity of the disaccharidases was unaltered by diet. In a well-controlled study, we determined SGLT1 expression in the duodenal and ileal biopsies of horses switched, gradually over a 2-month period, from low (<1.0 g/kg bwt/day) to high hCHO (6.0 g/kg bwt/day) diets of known composition. We show that SGLT1 expression is enhanced, with time, 2-fold in the duodenum and 3.3-fold in the ileum. The study has important implications for dietary management of the horse.

Inflammatory and redox status of ponies with a history of pasture-associated laminitis.

Inflammatory and redox signals could render lamellar tissue susceptible to damage and contribute to higher risk for laminitis in obese or insulin resistant ponies just as these factors contribute to health risks in humans with metabolic syndrome. This study evaluated circulating markers of inflammatory and redox status in ponies that had a history of recurrent bouts of pasture-associated laminitis (PL, n = 42) or had never developed clinical laminitis (NL, n = 34) under the current management conditions. There were no differences (P > 0.05) between PL and NL ponies for markers of antioxidant function (glutathione, glutathione peroxidase, superoxide dismutase) or increased oxidative pressure (malondialdehyde, apoptosis, 3-nitrotyrosine). Inflammatory status, as indicated by fibrinogen concentration, was also not different between pony groups (P = 0.84). However, PL ponies had higher (P < 0.001) plasma concentrations of the pro-inflammatory cytokine TNF-alpha than NL ponies. This suggests that a predisposition to laminitis is associated with increased circulating inflammatory cytokines. TNF-alpha could also represent a contributing factor to increased insulin resistance observed in laminitis prone ponies. These results provide new insight into potential mechanisms and risk factors underlying laminitis.

Dietary energy source affects glucose kinetics in trained Arabian geldings at rest and during endurance exercise.

Advances in modeling and tracer techniques provide new perspective into glucose utilization and potential consequences to health or exercise performance. This study used stable isotope and compartmental modeling to evaluate how adaptation to a feed high in sugar and starch (SS) compared with a feed high in fat and fiber (FF) affects glucose kinetics at rest and during exercise in horses. Six trained Arabians adapted to each feed underwent similar tests at rest and while running approximately 4 m/s on a treadmill. For both tests, horses received 100 micromol/kg body weight [6,6-(2)H]glucose through a venous catheter. Circulating tracer glucose was described for 150 min by exponential decay curves and compartmental analysis. All parameters of glucose transfer increased with exercise (P < or = 0.004). Compared with FF horses, SS horses had higher circulating glucose (P = 0.022) and fractional glucose transfer rates (min(-1)) at rest (P = 0.055). Exercise increased glucose irreversible loss (mmol/min) more in SS horses (P = 0.037). Total glucose transfer during exercise tended to be greater in SS horses (0.027 +/- 0.002 mmol/min) compared with FF horses (0.023 +/- 0.002 mmol/min) (P = 0.109). This study characterized the effect of diet on glucose kinetics in resting and exercising horses using new modeling methods. Horses adapted to a fat-supplemented feed utilized less glucose during low-intensity exercise. Fat supplementation in horses may therefore promote greater flexibility in the selection of substrate to meet energy demands for optimal health and performance.

Evaluation of a laparoscopic technique for collection of serial full-thickness small intestinal biopsy specimens in standing sedated horses.

OBJECTIVE: To assess a technique for laparoscopic collection of serial full-thickness small intestinal biopsy specimens in horses. ANIMALS: 13 healthy adult horses. PROCEDURES: In the ex vivo portion of the study, sections of duodenum and jejunum obtained from 6 horses immediately after euthanasia were divided into 3 segments. Each segment was randomly assigned to the control group, the double-layer hand-sewn closure group, or the endoscopic linear stapler (ELS) group. Bursting strength and bursting wall tension were measured and compared among groups; luminal diameter reduction at the biopsy site was compared between the biopsy groups. In the in vivo portion of the study, serial full-thickness small intestinal biopsy specimens were laparoscopically collected with an ELS from the descending duodenum and distal portion of the jejunum at monthly intervals in 7 sedated, standing horses. Biopsy specimens were evaluated for suitability for histologic examination. RESULTS: Mean bursting strength and bursting wall tension were significantly lower in the ELS group than in the hand-sewn and control groups in both the duodenal and jejunal segments. Use of the hand-sewn closure technique at the biopsy site reduced luminal diameter significantly more than use of the stapling technique. In the in vivo part of the study, all 52 biopsy specimens collected during 26 laparoscopic procedures were suitable for histologic examination and no clinically important perioperative complications developed. CONCLUSIONS AND CLINICAL RELEVANCE: Laparoscopic collection of serial full-thickness small intestinal biopsy specimens with a 45-mm ELS may be an effective and safe technique for use in healthy adult experimental horses.

Effects of dexamethasone on glucose dynamics and insulin sensitivity in healthy horses.

OBJECTIVE: To determine effects of dexamethasone on glucose dynamics and insulin sensitivity in healthy horses. ANIMALS: 6 adult Standardbreds. PROCEDURES: In a balanced crossover study, horses received dexamethasone (0.08 mg/ kg, IV, q 48 h) or an equivalent volume of saline (0.9% NaCl) solution (control treatment) during a 21-day period. Horses underwent a 3-hour frequently sampled IV glucose tolerance test (FSIGT) 2 days after treatment. Minimal model analysis of glucose and insulin data from FSIGTs were used to estimate insulin sensitivity (Si), glucose effectiveness (Sg), acute insulin response to glucose (AIRg), and disposition index. Proxies for Si (reciprocal of the inverse square of basal insulin concentration [RISQI]) and beta-cell responsiveness (modified insulin-to-glucose ratio [MIRG]) were calculated from basal plasma glucose and serum insulin concentrations. RESULTS: Mean serum insulin concentration was significantly higher in dexamethasone-treated horses than control horses on days 7, 14, and 21. Similarly, mean plasma glucose concentration was higher in dexamethasone-treated horses on days 7, 14, and 21; this value differed significantly on day 14 but not on days 7 or 21. Minimal model analysis of FSIGT data revealed a significant decrease in Si and a significant increase in AIRg after dexamethasone treatment, with no change in Sg or disposition index. Mean RISQI was significantly lower, whereas MIRG was higher, in dexamethasone-treated horses than control horses on days 7, 14, and 21. CONCLUSIONS AND CLINICAL RELEVANCE: The study revealed marked insulin resistance in healthy horses after 21 days of dexamethasone administration. Because insulin resistance has been associated with a predisposition to laminitis, a glucocorticoid-induced decrease in insulin sensitivity may increase risk for development of laminitis in some horses and ponies.

Repeatability of 2 methods for assessment of insulin sensitivity and glucose dynamics in horses.

Both the euglycemic-hyperinsulinemic clamp (EHC) and minimal model analysis of the frequently sampled intravenous glucose tolerance test (FSIGT) have been applied for measurement of insulin sensitivity in horses. However, no published data are available on the reproducibility of these methods. Therefore, the objective of this study was to evaluate the variation and repeatability of measures of glucose dynamics and insulin sensitivity in horses derived from minimal model analysis of the FSIGT and from the EHC method. Six healthy horses underwent both the FSIGT and EHC on 2 occasions over a 4-week period, with a minimum of 5 days between tests. Coefficient of variation (CV) and intraclass correlation coefficient (ICC) were calculated for measures of glucose metabolism and insulin sensitivity derived from each test. In the EHC, insulin sensitivity, expressed as the amount of metabolized glucose (M) per unit of serum insulin (I) (M/I ratio), averaged 0.19 +/- 0.06 x 10(-4) mmol/kg/min x (pmol/L)(-1) with an average interday CV of 14.1 +/- 5.7% (range, 7-20%) and ICC of 0.74. Minimal model analysis of the FSIGT demonstrated mean insulin sensitivity (Si) of 0.49 +/- 0.17 x 10(-4)/min x (pmol/L)(-1) with an average interday CV of 23.7 +/- 11.2% (range, 9-35%) and ICC of 0.33. Mean CV and ICC for minimal model glucose effectiveness (Sg) and acute insulin response (AIRg) were, respectively, 26.4 +/- 11.2% (range 13-40%) and 0.10 and 11.7 +/- 6.5% (range 7-21%) and 0.98. Insulin sensitivity measured by the EHC has lower interday variation when compared with the minimal model estimate derived from the FSIGT.

Dietary composition influences short-term endurance training-induced adaptations of substrate partitioning during exercise.

The purpose of the current study was to examine the influence of dietary composition on short-term endurance training-induced adaptations of substrate partitioning and time trial exercise performance. Eight untrained males cycled for 90 min at approximately 54% aerobic capacity while being infused with [6,6(2)H]glucose before and after two 10-d experimental phases separated by a 2-week washout period. Time trial performance was measured after the 90-min exercise trials before and after the 2nd experimental phase. During the first 10-d phase, subjects were randomly assigned to consume either a high carbohydrate or high fat diet while remaining inactive (CHO or FAT). During the second 10-d phase, subjects consumed the opposite diet, and both groups performed identical daily supervised endurance training (CHO+T or FAT+T). CHO and CHO+T did not affect exercise metabolism. FAT reduced glucose flux at the end of exercise, while FAT+T substantially increased whole body lipid oxidation during exercise and reduced glucose flux at the end of exercise. Despite these differences in adaptation of substrate use, training resulted in similar improvements in time trial performance for both groups. We conclude that (a) 10-d high fat diets result in substantial increases in whole body lipid oxidation during exercise when combined with daily aerobic training, and (b) diet does not affect short-term training-induced improvements in high-intensity time trial performance.

Effect of dietary starch, fat, and bicarbonate content on exercise responses and serum creatine kinase activity in equine recurrent exertional rhabdomyolysis.

To determine the effect of dietary starch, bicarbonate, and fat content on metabolic responses and serum creatine kinase (CK) activity in exercising Thoroughbreds with recurrent exertional rhabdomyolysis (RER), 5 RER horses were fed 3 isocaloric diets (28.8 Mcal/d [120.5 MJ/d]) for 3 weeks in a crossover design and exercised for 30 minutes on a treadmill 5 days/wk. On the last day of each diet, an incremental standardized exercise test (SET) was performed. The starch diet contained 40% digestible energy (DE) as starch and 5% as fat: the bicarbonate-starch diet was identical but was supplemented with sodium bicarbonate (4.2% of the pellet): and the fat diet provided 7% DE as starch and 20% as fat. Serum CK activity before the SET was similar among the diets. Serum CK activity (log transformed) after submaximal exercise differed dramatically among the diets and was greatest on the bicarbonate-starch diet (6.51 +/- 1.5) and lowest on the fat diet (5.71 +/- 0.6). Appreciable differences were observed in the severity of RER among individual horses. Postexercise plasma pH, bicarbonate concentration, and lactate concentration did not differ among the diets. Resting heart rates before the SET were markedly lower on the fat diet than on the starch diet. Muscle lactate and glycogen concentrations before and after the SET did not differ markedly among the diets. A high-fat, low-starch diet results in dramatically lower postexercise CK activity in severely affected RER horses than does a low-fat, high-starch diet without measurably altering muscle lactate and glycogen concentrations. Dietary bicarbonate supplementation at the concentration administered in this study did not prevent increased serum CK activity on a high-starch diet.