Development of a Web App to Convert Blood Insulin Concentrations among Various Immunoassays Used in Horses.

The measurement of the blood insulin concentration, and comparison to cut-offs, is essential in diagnosing insulin dysregulation, a common equine endocrinopathy. However, different insulin assays provide disparate results. We aimed to ease comparison between assays by compiling original and published data into a web app to convert insulin measurements from one assay to another. Data were available for ADVIA Centaur insulin chemiluminescent immunoassay (CLIA), Beckman Coulter insulin radioimmunoassay (RIA), Immulite 1000 CLIA, Immulite 2000 CLIA, Immulite 2000 XPi CLIA, Mercodia equine insulin enzyme-linked immunosorbent assay (ELISA), and Millipore porcine insulin RIA. Linear models were fitted for 13 assay pairs using non-decreasing splines, and integrated into this app. Assay comparisons including data from several studies showed a lower performance. This indicates technical variation between laboratories, which has not been described before, but is relevant when diagnostic measurements and cut-offs are provided by different laboratories. Nevertheless, the models' overall high performance (median r2 = 0.94; range 0.57-1.00) supports their use to interpret results from diagnostic insulin measurements when the reference assay is unavailable, and to compare values obtained from different assays.

Palatability, glycemic, and insulinemic responses to various carbohydrate formulations: Alternatives for the diagnosis of insulin dysregulation in horses?

BACKGROUND: Oral glycemic challenge (GC) tests are recommended for diagnosis of insulin dysregulation (ID). Various protocols are used, but all have limitations in terms of palatability, ease of use, variable composition, geographic availability, or some combination of these. HYPOTHESIS/OBJECTIVE: To evaluate newly developed formulations with defined carbohydrate composition for use as oral GCs. ANIMALS: Thirty-four horses and ponies in various metabolic states. METHODS: Our objectives were carried out in 2 separate cross-over experiments. First, the palatability and acceptance of various GCs (2 syrups, 1 granulate) offered for free intake were compared to glucose mixed in a chaff-based diet. Subsequently, syrups were administered by syringe and compared to an oral glucose test using naso-gastric tubing (tube OGT) to investigate the glycemic and insulinemic responses. Second, these variables were compared in the best performing GC-formulations (granulate further optimized to pelleted formulation and 1 syrup) and a tube OGT. All GCs were administered with equivalent amounts of 0.5 g glycemic carbohydrates per kg body weight. RESULTS: Only the GC pellets were consumed completely by all horses (consumption time 5 ± 2 min). When administered by syringe, the GC syrup also was well accepted. The insulin concentrations at 120 min correlated significantly between tube OGT and GC pellets (r = .717; P < .001) or GC syrup (r = .913; P < .001). The new GC syrup and GC pellets discriminate between healthy and ID horses. CONCLUSIONS AND CLINICAL SIGNIFICANCE: The GC pellets (DysChEq)™ and GC syrup can be used as palatable and well-accepted oral GC tests for assessment of ID in horses.

Carbohydrate pellets to assess insulin dysregulation in horses.

BACKGROUND: A glycemic challenge test is used for the diagnosis of insulin dysregulation (ID) in horses and ponies. Different forms of the test exist where the administrative route and dose of glucose vary, which makes interpretation of results challenging. HYPOTHESIS/OBJECTIVES: To evaluate the palatability of, and blood glucose and insulin responses to, carbohydrate pellets fed as an oral glucose test (OGT), and to establish the diagnostic threshold for ID when using the pellets. ANIMALS: University and privately-owned horses and ponies (n = 157) comprised of 31 breeds and both sexes. METHODS: Multicenter cohort study. A custom-produced glycemic pellet was offered for free intake at 0.5 g/kg BW soluble carbohydrate and serum insulin and blood glucose concentrations measured before and after (60, 120, and 180 minutes) the pellets were offered. Pellet acceptance and intake time (those that finished within 10 minutes) were determined to assess palatability. RESULTS: The pellets were palatable to 132/157 animals, and ponies found the pellets more (P = .004) palatable than horses. The median intake time (4 [3-6] minutes) was positively correlated with acceptance grade (r = .51; P < .0001). Consumption of the pellets elicited peak blood glucose (6.6 [5.8-7.8] mmol/L) and serum insulin (40.5 [19-99.8] µIU/mL) responses at 120 minutes. At 120 minutes the optimal cut-off was 83 µIU/mL (95% CI: 70-99 µIU/mL) for the IMMULITE 2000XPi assay. CONCLUSIONS AND CLINICAL IMPORTANCE: The pellets were palatable and a suitable, novel carbohydrate source for the OGT.

Sphingolipidome of plasma, liver, and adipose tissues and its association with insulin response to oral glucose testing in Icelandic horses.

Insulin dysregulation (ID) is a determinant of equine metabolic syndrome. Among the sphingolipids, ceramides contribute to the development of ID; however, the cross talk between the liver and adipose tissue (AT) depots and the variation among AT depots in terms of ceramide metabolism are not well understood. We aimed to characterize the sphingolipidome of plasma, liver, and AT (nuchal, NUAT; subcutaneous, SCAT; omental, OMAT; retroperitoneal, RPAT) and their associations with insulin response to oral glucose testing (OGT) in normoinsulinemic and hyperinsulinemic horses. Plasma, liver, and AT samples were collected from 12 Icelandic horses upon euthanasia and analyzed by liquid chromatography-mass spectrometry. Eighty-four targeted compounds were effectively quantified. Comparing the AT depots, greater (false discovery rate, FDR < 0.05) ceramide, dihydroceramide, and sphingomyelin concentrations and lower glucosyl- and galactosyl-ceramides were found in RPAT and OMAT than in NUAT and SCAT. Hyperinsulinemic response to OGT was associated with sphingolipidome alterations primarily in the RPAT and OMAT, whereas the NUAT sphingolipidome did not show signs of ceramide accumulation, which was inconsistent with the previously proposed role of nuchal adiposity in ID. The plasma sphingolipidome was not significantly associated with the liver or AT sphingolipidomes, indicating that plasma profiles are determined by an interplay of various organs. Furthermore, hepatic sphingolipid profiles were not correlated with the profiles of AT depots. Finally, statistically valid partial least square regression models predicting insulin response were found in the plasma (Q2 = 0.58, R2 = 0.98), liver (Q2 = 0.64, R2 = 0.74), and RPAT (Q2 = 0.68, R2 = 0.79) sphingolipidome, but not in the other adipose tissues.

Evaluation of Inner Exposure of Horses to Zearalenone (ZEN), Deoxynivalenol (DON) and Their Metabolites in Relation to Colic and Health-Related Clinical-Chemical Traits.

Mycotoxin contaminated feed has been associated with colic of horses caused by intestinal disorders. Whether such disease conditions alter the intestinal toxin metabolism and transfer across a compromised mucosal barrier is unknown. A screening approach was used to relate blood residue levels of DON, ZEN and their metabolites to the status of the horses (sick vs. healthy). A total of 55 clinically healthy horses from 6 different farms with varying feeding background served as control for sick horses (N = 102) hospitalized due to colic. ZEN, alpha-zearalenol (ZEL), beta-ZEL and DON were detectable in peripheral blood as indicators for the inner exposure with significant farm effects for alpha- and beta-ZEL. However, the levels in sick horses were similar to all farms. Moreover, the proportion of beta-ZEL of all detected ZEN metabolites as an indicator for the degree of metabolism of ZEN was not different for sick horses but differed amongst the control farms. Although the incidence of DON in blood was generally low and not significantly different amongst healthy and sick horses, the positive samples were nearly exclusively found in sick horses suggesting either a higher toxin transfer, an association of DON with the development of colic or a different feeding background.

Associations of plasma sphingolipid profiles with insulin response during oral glucose testing in Icelandic horses.

BACKGROUND: Sphingolipids modulate insulin sensitivity in mammals. Increased synthesis of ceramides is linked to decreased insulin sensitivity of tissues. Conversely, activation of the insulin signaling pathway can downregulate ceramide synthesis. Elucidating the association between sphingolipid metabolism and insulin response during oral glucose testing may help explain the pathophysiology of insulin dysregulation in horses. HYPOTHESES: Horses with insulin dysregulation will have a plasma sphingolipid profile characterized by increased ceramide concentrations. The plasma sphingolipid profile will have decreased ceramide concentrations after acute activation of the insulin signaling pathway by oral glucose testing. ANIMALS: Twelve Icelandic horses. METHODS: Horses were subjected to an oral glucose test (0.5 g/kg body weight glucose), with plasma insulin concentrations measured at 0, 30, 60, 120, 180, and 240 minutes postglucose administration. Plasma samples were collected at 0 and 120 minutes for sphingolipid profiling using a liquid chromatography-mass spectrometry-based metabolomics analysis. Eighty-three species of sphingolipids were detected, including 3-ketosphinganines, dihydroceramides, ceramides, dihydrosphingomyelins, sphingomyelins, galatosylceramides, glucosylceramides, lactosylceramides, and ceramide-1-phosphates. RESULTS: Glucose administration did not significantly alter plasma sphingolipid profiles. C22:0-ceramide, C24:1-ceramide, C23:0-ceramide, C16:1-sphingomyelin, C22:0-dihydroceramide, and C24:0-ceramide were positively correlated with the insulin response (area under the curve). CONCLUSION AND CLINICAL IMPORTANCE: Positive correlation between the insulin response and sphingolipid concentrations implies upregulated sphingolipid metabolism in insulin dysregulated horses. A high plasma ceramide concentration can indicate insulin dysregulation in horses.

CT myelographic diagnosis of ligamentum flavum hypertrophy in a Warmblood Gelding with progressive ataxia.

An 8-year-old Warmblood gelding presented with a history of progressive ataxia for 6 weeks. Intra- and intervertebral ratios measured from lateral radiographs of the cervical spine were within normal limits. Computed tomographic myelography of the cervical spine revealed focal compression of the dorsal and the ventral contrast column as well as a ventral displacement of the spinal cord within the spinal canal due to a bulging of soft tissue attenuating material in the dorsal half of the intervertebral junction of C6 and C7. Post-mortem histopathological examination confirmed chondroid metaplasia of the ligamentum flavum at C6-C7.

Metabolic impact of weight variations in Icelandic horses.

BACKGROUND: Insulin dysregulation (ID) is an equine endocrine disorder, which is often accompanied by obesity and various metabolic perturbations. The relationship between weight variations and fluctuations of the insulin response to oral glucose tests (OGT) as well as the metabolic impact of ID have been described previously. The present study seeks to characterize the concomitant metabolic impact of variations in the insulin response and bodyweight during repeated OGTs using a metabolomics approach. METHODS: Nineteen Icelandic horses were subjected to five OGTs over one year and their bodyweight, insulin and metabolic response were monitored. Analysis of metabolite concentrations depending on time (during the OGT), relative bodyweight (rWeight; defined as the bodyweight at one OGT divided by the mean bodyweight across all OGTs) and relative insulin response (rAUCins; defined accordingly from the area under the insulin curve during OGT) was performed using linear models. Additionally, the pathways significantly associated with time, rWeight and rAUCins were identified by rotation set testing. RESULTS: The results suggested that weight gain and worsening of ID activate distinct metabolic pathways. The metabolic profile associated with weight gain indicated an increased activation of arginase, while the pathways associated with time and rAUCins were consistent with the expected effect of glucose and insulin, respectively. Overall, more metabolites were significantly associated with rWeight than with rAUCins.

Metabolic profile distinguishes laminitis-susceptible and -resistant ponies before and after feeding a high sugar diet.

BACKGROUND: Insulin dysregulation (ID) is a key risk factor for equine endocrinopathic laminitis, but in many cases ID can only be assessed accurately using dynamic tests. The identification of other biomarkers could provide an alternative or adjunct diagnostic method, to allow early intervention before laminitis develops. The present study characterised the metabolome of ponies with varying degrees of ID using basal and postprandial plasma samples obtained during a previous study, which examined the predictive power of blood insulin levels for the development of laminitis, in ponies fed a high-sugar diet. Samples from 10 pre-laminitic (PL - subsequently developed laminitis) and 10 non-laminitic (NL - did not develop laminitis) ponies were used in a targeted metabolomic assay. Differential concentration and pathway analysis were performed using linear models and global tests. RESULTS: Significant changes in the concentration of six glycerophospholipids (adj. P ≤ 0.024) and a global enrichment of the glucose-alanine cycle (adj. P = 0.048) were found to characterise the response of PL ponies to the high-sugar diet. In contrast, the metabolites showed no significant association with the presence or absence of pituitary pars intermedia dysfunction in all ponies. CONCLUSIONS: The present results suggest that ID and laminitis risk are associated with alterations in the glycerophospholipid and glucose metabolism, which may help understand and explain some molecular processes causing or resulting from these conditions. The prognostic value of the identified biomarkers for laminitis remains to be investigated in further metabolomic trials in horses and ponies.

Metabolic changes induced by oral glucose tests in horses and their diagnostic use.

BACKGROUND: Little is known about the implications of hyperinsulinemia on energy metabolism, and such knowledge might help understand the pathophysiology of insulin dysregulation. OBJECTIVES: Describe differences in the metabolic response to an oral glucose test, depending on the magnitude of the insulin response. ANIMALS: Twelve Icelandic horses in various metabolic states. METHODS: Horses were subjected to 3 oral glucose tests (OGT; 0.5 g/kg body weight glucose). Basal, 120 and 180 minutes samples were analyzed using a combined liquid chromatography tandem mass spectrometry and flow injection analysis tandem mass spectrometry metabolomic assay. Insulin concentrations were measured using an ELISA. Analysis was performed using linear models and partial least-squares regression. RESULTS: The kynurenine : tryptophan ratio increased over time during the OGT (adjusted P-value = .001). A high insulin response was associated with lower arginine (adjusted P-value = .02) and carnitine (adjusted P-value = .03) concentrations. A predictive model using only baseline samples performed well with as few as 7 distinct metabolites (sensitivity, 86%; 95% confidence interval [CI], 81%-90%; specificity, 88%; 95% CI, 84%-92%). CONCLUSIONS AND CLINICAL IMPORTANCE: Our results suggest induction of low-grade inflammation during the OGT. Plasma arginine and carnitine concentrations were lower in horses with high insulin response and could constitute potential therapeutic targets. Development of screening tools to identify insulin-dysregulated horses using only baseline blood sample appears promising.

Weight loss is linearly associated with a reduction of the insulin response to an oral glucose test in Icelandic horses.

BACKGROUND: Insulin dysregulation (ID) goes along with lasting or transient hyperinsulinemia able to trigger equine laminitis, a painful and crippling foot condition. Promoting weight loss through dietary changes and physical activity is currently the main option to prevent this disease. This study aimed at describing the relationship between weight variations and the level of ID as determined by oral glucose tests (OGT). Therefore, the insulin response of 19 Icelandic horses to repeated OGTs was retrospectively analysed considering the variations in their body weight. RESULTS: There was a strong linear relationship between variations in body weight and variations in the total insulin response to OGT as approximated by the area under the curve of insulin (p < 0.001). As indicated by a weighted least squares model, the insulin response decreased by 22% for 5% weight loss on average. However some horses did not respond to weight loss with a reduction of their insulin response to OGT. Additionally, a high correlation between 120 min serum insulin concentration and total insulin response was observed (r = 0.96, p < 0.001). CONCLUSIONS: The results corroborate that weight loss is effective against ID and allow for a better quantification of the expected improvement of the insulin response after weight loss. However, it is unclear why some horses did not respond as expected. The high correlation between the 120 min insulin concentration and total insulin response suggests that insulin status can be accurately determined and monitored with only few samples in a practical setting.

Lower plasma trans-4-hydroxyproline and methionine sulfoxide levels are associated with insulin dysregulation in horses.

BACKGROUND: Insulin dysregulation in horses is a metabolic condition defined by high insulin concentrations in the blood and peripheral insulin resistance. This hyperinsulinemia is often associated with severe damage in the hooves, resulting in laminitis. However, we currently lack detailed information regarding the potential involvement of particular metabolic pathways in pathophysiological causes and consequences of equine insulin dysregulation. This study aimed to assess the dynamic metabolic responses given to an oral glucose test (OGT) in insulin-sensitive and insulin-dysregulated horses by a targeted metabolomics approach to identify novel metabolites associated with insulin dysregulation. RESULTS: Oral glucose testing triggered alterations in serum insulin (26.28 ± 4.20 vs. 422.84 ± 88.86 µIU/mL, p < 0.001) and plasma glucose concentrations (5.00 ± 0.08 vs. 9.43 ± 0.44 mmol/L, p < 0.001) comparing basal and stimulated conditions after 180 min. Metabolome analyses indicated OGT-induced changes in short-chain acylcarnitines (6.00 ± 0.53 vs. 3.99 ± 0.23 µmol/L, p < 0.001), long-chain acylcarnitines (0.13 ± 0.004 vs. 0.11 ± 0.002 µmol/L, p < 0.001) and amino acids (2.18 ± 0.11 vs. 1.87 ± 0.08 µmol/L, p < 0.05). Kynurenine concentrations increased (2.88 ± 0.18 vs. 3.50 ± 0.19 µmol/L, p < 0.01), whereas spermidine concentrations decreased during OGT (0.09 ± 0.004 vs. 0.08 ± 0.002 µmol/L, p < 0.01), indicating proinflammatory conditions after oral glucose load. Insulin dysregulation was associated with lower concentrations of trans-4-hydroxyproline (4.41 ± 0.29 vs. 6.37 ± 0.71 µmol/L, p < 0.05) and methionine sulfoxide (0.40 ± 0.06 vs. 0.87 ± 0.13 µmol/L, p < 0.01; mean ± SEM in insulin-dysregulated vs. insulin-sensitive basal samples, respectively), two metabolites which are related to antioxidant defense mechanisms. CONCLUSION: Oral glucose application during OGT resulted in profound metabolic and proinflammatory changes in horses. Furthermore, insulin dysregulation was predicted in basal samples (without OGT) by pathways associated with trans-4-hydroxyproline and methionine sulfoxide, suggesting that oxidative stress and oxidant-antioxidant disequilibrium are contributing factors to insulin dysregulation. The present findings provide new hypotheses for future research to better understand the underlying pathophysiology of insulin dysregulation in horses.

Retrospective analysis of insulin responses to standard dosed oral glucose tests (OGTs) via naso-gastric tubing towards definition of an objective cut-off value.

BACKGROUND: Insulin dysregulation (ID) with basal or postprandial hyperinsulinemia is one of the key findings in horses and ponies suffering from the equine metabolic syndrome (EMS). Assessment of ID can easily be performed in clinical settings by the use of oral glucose challenge tests. Oral glucose test (OGT) performed with 1 g/kg bodyweight (BW) glucose administered via naso-gastric tube allows the exact administration of a defined glucose dosage in a short time. However, reliable cut-off values have not been available so far. Therefore, the aim of the study was to describe variations in insulin response to OGT via naso-gastric tubing and to provide a clinical useful cut-off value for ID when using the insulin quantification performed with an equine-optimized insulin enzyme-linked immunosorbent assay. RESULTS: Data visualization revealed no clear separation in the serum insulin concentration of insulin sensitive and insulin dysregulated horses during OGT. Therefore, a model based clustering method was used to circumvent the use of an arbitrary limit for categorization. This method considered all data-points for the classification, taking into account the individual insulin trajectory during the OGT. With this method two clusters were differentiated, one with low and one with high insulin responses during OGT. The cluster of individuals with low insulin response was consistently detected, independently of the initialization parameters of the algorithm. In this cluster the 97.5% quantile of insulin is 110 µLU/mL at 120 min. We suggest using this insulin concentration of 110 µLU/mL as a cut-off value for samples obtained at 120 min in OGT. CONCLUSION: OGT performed with 1 g/kg BW glucose and administration via naso-gastric tubing can easily be performed under clinical settings. Application of the cut-off value of 110 µLU/mL at 120 min allows assessment of ID in horses.

Insulin signaling in various equine tissues under basal conditions and acute stimulation by intravenously injected insulin.

The aim of the study was to analyze key proteins of the equine insulin signaling cascade and their extent of phosphorylation in biopsies from muscle tissue (MT), liver tissue (LT), and nuchal AT, subcutaneous AT, and retroperitoneal adipose tissues. This was investigated under unstimulated (B1) and intravenously insulin stimulated (B2) conditions, which were achieved by injection of insulin (0.1 IU/kg bodyweight) and glucose (150 mg/kg bodyweight). Twelve warmblood horses aged 15 ± 6.8 yr (yr), weighing 559 ± 79 kg, and with a mean body condition score of 4.7 ± 1.5 were included in the study. Key proteins of the insulin signaling cascade were semiquantitatively determined using Western blotting. Furthermore, modulation of the cascade was assessed. The basal expression of the proteins was only slightly influenced during the experimental period. Insulin induced a high extent of phosphorylation of insulin receptor in LT (P < 0.01) but not in MT. Protein kinase B and mechanistic target of rapamycin expressed a higher extent of phosphorylation in all tissues in B2 biopsies. Adenosine monophosphate protein kinase, as a component related to insulin signaling, expressed enhanced phosphorylation in MT (P < 0.05) and adipose tissues (nuchal AT P < 0.05; SCAT P < 0.01; retroperitoneal adipose tissue P < 0.05), but not in LT at B2. Tissue-specific variations in the acute response of insulin signaling to intravenously injected insulin were observed. In conclusion, insulin sensitivity in healthy horses is based on a complex concerted action of different tissues by their variations in the molecular response to insulin.

Effects of deoxynivalenol (DON), zearalenone (ZEN), and related metabolites on equine peripheral blood mononuclear cells (PBMC) in vitro and background occurrence of these toxins in horses.

Both deoxynivalenol (DON), zearalenone (ZEN), and their metabolites are known to modulate immune cells in various species whereby viability and proliferation are influenced. Such effects were rarely examined in horses. Therefore, one aim of the present study was to titrate the inhibitory concentrations of DON, 3-acetyl-DON (3AcDON), de-epoxy-DON (DOM-1), ZEN, and α- and ß-zearalenol (ZEL) at which viability and proliferation of equine PBMC were reduced by 50 % (IC50) and 10 % (IC10) in vitro. For evaluation of practical relevance of the in vitro findings, a further aim was to screen horses for the background occurrence of DON, ZEN, and their metabolites in systemic circulation and to relate toxin residues both to the inhibitory toxin concentrations and to hematological and clinical-chemical characteristics.The IC50 (µM) for DON, 3AcDON, ß-ZEL, α-ZEL, and ZEN were determined at 3.09, 25.90, 75.44, 97.44, and 98.15 in unstimulated cells, respectively, while in proliferating cells, the corresponding IC50 values were 0.73, 6.89, 45.16, 75.96, and 82.51. Neither viability nor proliferation was influenced by DOM-1 up to a concentration of 100 µM.The in vivo screening (N = 49) revealed the occurrence of ZEN (N = 24), α-ZEL (N = 3), ß-ZEL (N = 37), DON, and DOM-1 (N = 2). The detected concentrations were much lower than the corresponding IC50 while the IC10 of DON and ß-ZEL for proliferating PBMC corresponded to approximately 26 and 35 ng/mL which might be relevant when contaminated diets are fed.Clinical-chemical and hematological traits were not related to mycotoxin residue levels excepting blood urea nitrogen which was positively correlated to the sum of ß-ZEL, α-ZEL, and ZEN concentration. Whether this reflects simply the feeding history of the horses or renal failures giving rise to a prolonged half-life of the toxins needs to be clarified further.