Equine Insulin Dysregulation Research Articles

Studies in vitro of equine intestinal glucagon-like peptide-2 secretion

Equine insulin dysregulation (ID) is a significant metabolic problem because the hyperinsulinaemia that develops increases the animal's risk of developing laminitis, a debilitating foot condition. The role of gastrointestinal factors, such as incretin hormones, in the pathogenesis of ID and hyperinsulinaemia in horses is poorly understood, particularly in comparison to other species. Glucagon-like peptide-2 (GLP-2) is an intestinotrophic peptide released from L cells in the gastrointestinal tract and is implicated in metabolic dysfunction in other species. The aim of this study in vitro was to establish basic physiological understanding about intestinal secretion of GLP-2 in horses. Basal and glucose-stimulated GLP-2 secretion was measured in post-mortem tissue samples from the duodenum, jejunum, and ileum. We observed that GLP-2 secretion was minimal in samples from the duodenum compared to the jejunum and ileum (5-9-fold higher; P < 0.05). Furthermore, GLP-2 secretion was not responsive to glucose stimulation in the ileum or duodenum but was responsive to glucose in the jejunum. This effect in the jejunum was inhibited by 30 % (P = 0.02) using phlorizin, a selective sodium-glucose cotransporter-1 (SGLT-1) inhibitor, and by 38 % (P = 0.04) using phloretin, a non-selective SGLT-1/GLUT-2 inhibitor. The localisation of glucose-responsive GLP-2 secretion in the jejunum might be relevant to the development of post-prandial hyperinsulinaemia. This study has provided data on GLP-2 secretion from the equine small intestine that will enable more complex and dynamic studies on the pathogenesis of ID.

Expression of the GCG gene and secretion of active glucagon-like peptide-1 varies along the length of intestinal tract in horses.

Active glucagon-like peptide-1 (aGLP-1) has been implicated in the pathogenesis of equine insulin dysregulation (ID), but its role is unclear. Cleavage of proglucagon (coded by the GCG gene) produces aGLP-1 in enteral L cells. The aim in vivo was to examine the sequence of the exons of GCG in horses with and without ID, where aGLP-1 was higher in the group with ID. The aims in vitro were to identify and quantify the expression of GCG in the equine intestine (as a marker of L cells) and determine intestinal secretion of aGLP-1. Genomic studies were case-control studies. Expression and secretion studies in vitro were cross-sectional. The GCG gene sequence of the exons was determined using a hybridisation capture protocol. Expression and quantification of GCG in samples of stomach duodenum, jejunum, ileum, caecum and ascending and descending colon was achieved with droplet digital PCR. For secretory studies tissue explants were incubated with 12 mM glucose and aGLP-1 secretion was measured with an ELISA. Although the median [IQR] post-prandial aGLP-1 concentrations were higher (p = 0.03) in animals with ID (10.2 [8.79-15.5]), compared with healthy animals (8.47 [6.12-11.7]), there was 100% pairwise identity of the exons of the GCG sequence for the cohort. The mRNA concentrations of GCG and secretion of aGLP-1 differed (p < 0.001) throughout the intestine. Only the exons of the GCG gene were sequenced and breeds were not compared. The horses used for the study in vitro were not assessed for ID and different horses were used for the small, and large, intestinal studies. Differences in post-prandial aGLP-1 concentration were not due to a variant in the exons of the GCG gene sequence in this cohort. Both the large and small intestine are sites of GLP-1 secretion.

20 Can a common cereal grain be used to help indicate the presence of insulin dysregulation

Equine insulin dysregulation (ID) comprises a combination of basal and/or postprandial hyperinsulinemia and/or tissue insulin resistance. ID increases the risk of hyperinsulinemia-associated laminitis (HAL) and equids with ID need to be managed appropriately with a low non-structural carbohydrate (NSC) diet. Diagnosis is therefore crucial. The oral sugar test (OST) or the oral glucose test are currently the recommended practical diagnostic tests for basal and post prandial hyperinsulinemia. The Karo syrup orally administered in the OST is not available in all countries and glucose powder added to feed is not accepted by all equids. Recent work has shown ID animals have an augmented response to small amounts of even moderate NSC feedstuffs. This study evaluated the potential to use this response as a possible alternative indicator of ID status. The postprandial insulin responses to small amounts of the cereal grain, dehulled oats (DO, no further processing), were therefore evaluated in healthy (non-insulin dysregulated; NID) and ID horses (categorized by their results to an OST, 0.15 mL/kg BW). Twelve non-insulin dysregulated (573.2 + 59.9 kg; 16.1 + 3.6 yrs.) and 18 ID horses (594.7 + 93.3 kg; 15.2 + 2.6 yrs.) from 2 separate studies were examined. Horses were grain fasted and blood was collected via jugular venipuncture before (T0)offering DO (1.25 kg/BW study 1, S1; 1.0 kg/BW study 2, S2) and 60-min postprandially (T60). Insulin was analyzed by RIA and delta insulins were calculated (T60-T0). Statistics were analyzed on GraphPad 9.4.0 using ANOVA and Tukey post-hoc. Significance taken as P < 0.05. NID horses’ showed no significant change (P = 0.8) from basal insulin levels following both the DO and the OST (∼8 μIU/mL delta insulin). The ID horses showed a similar augmented mean insulin response to both the DO and the OST which was significantly higher than the NID response (P < 0.05). There was, however, more individual variation in the response to the DO than to the OST (T60 mean + SD [delta insulin]: S1–140.1 + 45.5 [82.0 + 10.1] μIU/mL OST, 170.4 + 92.1 [100.6 + 21.7] μIU/mL DO; S2–123.9 + 37.0 [76.0 + 11.3] μIU/mL OST, 158.6 + 64.4 [98.7 + 19.2] μIU/mL DO). The work suggests that feeding 1g/kg BW of DO might be a possible alternative method of identifying an ID animal when the currently recommended methods are not possible. However, more work is needed to define indicative cutoffs and to evaluate the individual variability.

Equine insulin dysregulation causes tissue specific alterations of proinflammatory cytokines and acute phase proteins in a NF-kB independent manner

Similar to human diabetes, equine metabolic syndrome (EMS) causes insulin dysregulation leading to debilitating sequela including laminitis. The pathophysiological mechanisms underlying EMS and laminitis are not well known. Therefore, using an insulin-resistant equine model, we hypothesized that insulin dysregulation induces an increased expression of inflammatory proteins in a tissue specific manner. Two groups of horses (n = -5/group) were categorized as insulin-resistant (IR) or insulin-sensitive (IS), using a frequently sampled intra-venous glucose tolerance test. Biopsies from skeletal muscle, and visceral and subcutaneous adipose tissues were collected in both groups. Protein expression was quantified via Western blotting in order to investigate HSP90, α 2 macroglobulin (A2M), Fibrinogen α, β, γ isoforms as well as cytokines, including interleukin-1β (IL-1β) and interleukin-6 (IL-6), in muscle and adipose tissues. Protein expression of HSP90, A2M and IL1-β was significantly greater in visceral adipose tissue of IR horses compared to IS horses. Fibrinogen (α and γ) expression was only significantly increased in subcutaneous adipose tissue of IR group compared to IS group. In contrast, no statistically significant difference in protein expression of proinflammatory cytokines and acute phase proteins was reported in skeletal muscle of IR vs. IS horses. Relative protein expression of total and phospho-NFκB protein expression was not statistically significantly changed in adipose tissues of IR horses compared to IS horses. In conclusion, proinflammatory cytokines and acute phase proteins were upregulated in adipose tissue, but not in skeletal muscle, through an NF-kB independent pathway. Insights from this study could reveal novel biomarkers and potential therapeutic targets for EMS.

The efficacy and safety of velagliflozin over 16\u2009weeks as a treatment for insulin dysregulation in ponies

BackgroundA previous six-week (wk) study demonstrated the potential of the sodium-glucose linked transport inhibitor velagliflozin as a novel treatment for equine insulin dysregulation. The present study examined the safety and efficacy of velagliflozin over 16 wk. of treatment, and over 4 wk. of withdrawal. Twenty-four insulin dysregulated ponies were selected, based on their hyper-responsiveness to a diet challenge meal containing 3.8 g non-structural carbohydrates (NSC)/kg bodyweight (BW). Ponies with serum insulin > 90 μIU/mL either 2 or 4 h after feeding were enrolled, and randomly allocated to receive either velagliflozin (0.3 mg/kg BW orally once daily, n = 12), or a placebo (n = 10–12) for 16 wk. The subjects were fed 7.5 g NSC/kg BW/day to maintain a fat body condition. Safety was assessed through daily monitoring, veterinary examination, and the measurement of fasting blood glucose, biochemistry and haematology. Efficacy at reducing post-prandial hyperinsulinemia was assessed using a diet challenge every 8 wk. during treatment and 4 wk. after withdrawal.ResultsVelagliflozin was well accepted by all subjects and caused no adverse effects or hypoglycaemia. Post-prandial serum insulin (insulin Cmax) did not change significantly in the control animals over the entire study period (P = 0.101). In contrast, insulin Cmax (mean ± SE) concentrations fell over time in the velagliflozin-treated group from 205 ± 25 μIU/mL in wk. 0, to 119 ± 19 μIU/mL (P = 0.015) and 117 ± 15 μIU/ml (P = 0.029) after 8 and 16 wk. of treatment, respectively. Although the insulin Cmax in this group was not significantly lower than in controls at wk-8 (P = 0.061), it was lower at wk-16 (P = 0.003), and all 12 treated ponies were below the previously-determined risk threshold for laminitis at this time. After 4 wk. withdrawal, the insulin Cmax returned to 199 ± 36 μIU/mL in the treated group, with no rebound effect.ConclusionsVelagliflozin appears to be a promising and safe treatment for equine insulin dysregulation, bringing post-prandial insulin concentrations below the laminitis risk threshold, albeit without normalising them.

Sweet taste receptor inhibitors: Potential treatment for equine insulin dysregulation

Hyperinsulinemia is a major risk factor for equine laminitis, a debilitating and painful foot condition. Sweet taste receptor (T1R2/3) inhibitors have been used to reduce the insulin and glucose responses to oral carbohydrates in other species. However, their effect in horses has not been investigated. It would be useful to be able to attenuate the large post-prandial insulin response that typically occurs when a carbohydrate-rich meal is fed to insulin-dysregulated horses. Here we have determined the efficacy of two T1R2/3 inhibitors, lactisole and Gymnema sylvestre, for reducing glucose uptake by the equine small intestine in vitro; and post-prandial insulin secretion in ponies in vivo, following a carbohydrate-based meal. We used gas chromatography-mass spectrometry to measure 2-deoxyglucose uptake by explants of small intestine, in the presence and absence of the T1R2/3 inhibitors. Lactisole and G sylvestre reduced 2-deoxyglucose uptake by the intestinal explants by 63% (P = 0.032) and 73% (P = 0.047), respectively, compared to control samples. The study in vivo investigated the effect of the inhibitors on the blood glucose and serum insulin responses to a meal containing D-glucose. Three doses of each inhibitor were tested using a Latin square design, and each dose was compared to a meal with no inhibitor added. Lactisole had no effect on glucose and insulin concentrations, whereas G sylvestre was partially effective at reducing post-prandial blood glucose (by ~10%) and serum insulin concentrations (~25%) in seven ponies, with a most effective dose of 10 mg/kg bodyweight. These data provide preliminary support that T1R2/3 inhibitors may be a useful therapeutic strategy for the management of equine insulin dysregulation and the prevention of laminitis. However, further optimisation of the dose and delivery method for these compounds is required, as well as a direct investigation of their activity on the equine sweet taste receptor.

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

BackgroundInsulin 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.ResultsOral 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.ConclusionOral 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.

Glucagon-like peptide-2: A potential role in equine insulin dysregulation.

Equine insulin dysregulation (ID) is a common and poorly understood disorder that increases the risk of laminitis. Recent data show that the condition may be associated with alteration of the enteroinsular axis and enhanced glucose bioavailability. Upregulation of glucagon-like peptide-2 (GLP-2), an intestinotrophic peptide, leads to enhanced nutrient uptake and metabolic dysfunction in other species. The study aimed to 1) determine whether GLP-2 is differentially expressed in insulin-dysregulated ponies, compared with healthy ponies, and 2) confirm intestinal expression of the GLP-2 receptor in horses (eGLP-2R). Cohort study. Fasting and post-prandial GLP-2 concentrations were measured in archived plasma samples obtained from 25 mixed-breed ponies during two feeding studies. Measurements were undertaken with an ELISA that was validated for equine use as part of the current study. Ponies were designated as healthy or insulin-dysregulated based on an oral glucose test, and the results were compared between groups. The gene expression of the eGLP-2R was determined with polymerase chain reaction. Basal, fasted plasma GLP-2 concentrations were higher in ponies with ID, compared with healthy ponies. Grazing increased GLP-2 in healthy, but not in insulin-dysregulated, ponies. The eGLP-2R gene was expressed in the small intestine and pancreas. The study was performed with a relatively small sample size. The specificity of the GLP-2 assay could not be determined due to the lack of equine-specific assay standards. This study has demonstrated that GLP-2 may be important in the pathogenesis of equine ID and suggests that the eGLP-2R may be a novel therapeutic target for the treatment of equine ID.

Equine glucagon-like peptide-1 receptor physiology.

BackgroundEquine metabolic syndrome (EMS) is associated with insulin dysregulation, which often manifests as post-prandial hyperinsulinemia. Circulating concentrations of the incretin hormone, glucagon-like peptide-1 (GLP-1) correlate with an increased insulin response to carbohydrate intake in animals with EMS. However, little is known about the equine GLP-1 receptor (eGLP-1R), or whether GLP-1 concentrations can be manipulated. The objectives were to determine (1) the tissue localisation of the eGLP-1R, (2) the GLP-1 secretory capacity of equine intestine in response to glucose and (3) whether GLP-1 stimulated insulin secretion from isolated pancreatic islets can be attenuated.MethodsArchived and abattoir-sourced tissues from healthy horses were used. Reverse transcriptase PCR was used to determine the tissue distribution of the eGLP-1R gene, with immunohistochemical confirmation of its pancreatic location. The GLP-1 secretion from intestinal explants in response to 4 and 12 mM glucose was quantified in vitro. Pancreatic islets were freshly isolated to assess the insulin secretory response to GLP-1 agonism and antagonism in vitro, using concentration-response experiments.ResultsThe eGLP-1R gene is widely distributed in horses (pancreas, heart, liver, kidney, duodenum, digital lamellae, tongue and gluteal skeletal muscle). Within the pancreas the eGLP-1R was immunolocalised to the pancreatic islets. Insulin secretion from pancreatic islets was concentration-dependent with human GLP-1, but not the synthetic analogue exendin-4. The GLP-1R antagonist exendin 9-39 (1 nM) reduced (P = 0.08) insulin secretion by 27%.DiscussionThe distribution of the eGLP-1R across a range of tissues indicates that it may have functions beyond insulin release. The ability to reduce insulin secretion, and therefore hyperinsulinemia, through eGLP-1R antagonism is a promising and novel approach to managing equine insulin dysregulation.

Assessment of Owner and Veterinarian Awareness of Equine Insulin Dysregulation and Available Treatments in Southeastern United States

Insulin dysregulation (ID) refers collectively to horses afflicted with fasting hyperinsulinemia and insulin resistance. Although there is no current data indicating the percentage of equines afflicted with ID, it appears to be a common subject among equine enthusiasts. In order to gauge public awareness of ID in horses and its available treatments, 122 horse owners and 18 veterinarians, representing 25 states, participated in a self-administered online survey. Questions were postulated to ascertain participant's awareness of the symptoms, management techniques, current treatments and medications, and knowledge acquisition associated with ID. The study revealed more than half of participants were “moderately aware” (34%) to “very aware” (25%) of ID in horses; however, when identifying symptoms of ID, some reported symptoms of pituitary pars intermedia dysfunction as those of ID. Many owners recognized current management strategies for ID; however, actual implementation strategies did not reflect standard management methods. Treatments mentioned for ID include supplements (14% of owners), thyroid supplement Thyro-L (13% and 72%), and pergolide/Prascend (21% and 61%) of owners and veterinarians, respectively. Veterinarians recommended diet and exercise as their main management strategy. Both owners and veterinarians reported using published research and veterinarians as a main source of information. The findings of this survey suggest that although awareness and recognition of the condition is high, proper identification, treatment, and management of ID varies. Moreover, research addressing the efficacy of pergolide as a treatment for ID is lacking. Additional education and research is needed to address these shortcomings.

The diagnosis of equine insulin dysregulation.

Insulin dysregulation is the hallmark of equine metabolic syndrome and has received attention because of its direct association with laminitis. In the absence of an adequate treatment for laminitis, a focus on prophylaxis is needed, making early detection of individuals at risk of developing laminitis one of the main challenges in equine endocrinology. Recent studies have shown that insulin dysregulation goes beyond tissue insulin resistance and it is now demonstrated that the equine enteroinsular axis plays a major role in insulin secretion and equine hyperinsulinaemia. In this review, we discuss the different tests currently available to diagnose insulin dysregulation in horses: the ones investigating tissue insulin resistance and those investigating the enteroinsular axis, detailing their goals, practicalities and limitations. This review supports the contention that the diagnosis of equine insulin dysregulation should now be based on the investigation of both tissue insulin resistance and the equine enteroinsular axis. Regardless of the tests used many factors of variation, such as breed, diet, fasting state or season, have been identified and could potentially confound the results of a specific test. Therefore, careful interpretation of the results of a given test in each individual situation is required to optimise the detection of horses at risk of laminitis.

Owner and veterinarian awareness of equine insulin dysregulation and available treatments

Owner and veterinarian awareness of equine insulin dysregulation and available treatments

Comparison of Three Methods for Evaluation of Equine Insulin Regulation in Horses of Varied Body Condition Score

Multiple dynamic field tests are used for assessment of equine insulin resistance or altered insulin regulation. However, the relationship between markers of glucose homeostasis and insulin disposal obtained by different testing protocols is unknown. We hypothesized that two recently developed field tests for evaluation of equine insulin dysregulation, the insulin response to dexamethasone test (IRDT) and oral sugar test (OST), would yield comparable results to the hyperinsulinemic euglycemic clamp (HEC). Fifteen light breed horses with body condition scores (BCS) 3 of 9 to 8 of 9 were used in this study. Eight horses (BCS, 5 of 9 to 7 of 9) underwent an OST under two different housing conditions, pasture, and stall (experiment 1). These eight horses also underwent an HEC and IRDT over a 4-week period (experiment 2), and results were compared with the OST stall. Finally, eight horses (BCS, 3 of 9 to 8 of 9), including one horse from experiments 1 and 2, underwent an OST on pasture three times over a 14–16-week period during the summer and the fall (experiment 3). The HEC did not correlate with either the OST or IRDT. The OST was not different when performed in the pasture compared within a stall but did change significantly over time on pasture. These results suggest that in insulin-sensitive horses, the OST and IRDT results are not primarily determined by tissue insulin sensitivity in horses of varying BCS. Furthermore, OST results may vary depending on pasture composition or season.