The effect of different grazing conditions on the insulin and incretin response to the oral glucose test in ponies.

BACKGROUND: The oral glucose test (OGT) is a useful tool for diagnosing insulin dysregulation (ID) and is somewhat repeatable in ponies under consistent management. This study aimed to determine whether the insulin and incretin responses to an OGT in ponies differed after short-term access to fertilised pasture, compared to unfertilised pasture, by using a randomised, repeated measures study design. Sixteen mixed-breed ponies were classified as severely insulin-dysregulated (SD; post-prandial insulin ≥80 µIU/mL) or not severely insulin-dysregulated (NSD; post-prandial insulin < 80 µIU/mL) using an OGT prior to the study. The ponies accessed pasture that was fertilised, or unfertilised, for 5 days (4 h/day, with supplemental hay provided at 0.7% bodyweight), with a 10 day period between phases. An OGT was performed after each phase. Glucose, insulin, active glucagon-like peptide-1 (aGLP-1), and glucose-dependent insulinotropic polypeptide (GIP) were measured in post-prandial blood samples. RESULTS: The volume of fertilised pasture was five-fold greater than unfertilised pasture, with % non-structural carbohydrates (NSC) similar between all forages. Consuming fertilised pasture increased (P = 0.018) the serum insulin response to an OGT, compared to grazing unfertilised pasture. A limitation of the study was that pasture intake was unable to be quantified. Insulin responses were greater in SD, compared to NSD, ponies (P < 0.001) and remained well above the test cut-off at all times. A subset of ponies, initially screened as NSD, became (more) insulin-dysregulated after pasture access. Further, aGLP-1 was a significant predictor of insulin concentration in this cohort. CONCLUSIONS: Whereas some insulin-dysregulated ponies were comparatively resistant to dietary intervention, others showed markedly different OGT responses following subtle changes in their forage-based diet. This implies that mild/early ID might be unmasked by dietary change, and that dietary management is important in these ponies. However, dietary management alone may not be adequate for all cases of ID.

Evaluation of locking compression plate fixation of the distal phalanx to the hoof wall as a potential therapy for laminitis.

BACKGROUND: Surgical stabilisation of the distal phalanx (DP) is a potential therapeutic strategy for severe acute laminitis. OBJECTIVE: To evaluate the effects of locking compression plate (LCP) fixation of the DP to the dorsal hoof wall. STUDY DESIGN: Ex vivo and in vivo experiments. METHODS: A T-shaped LCP was applied to one limb per pair in six pairs of cadaver forelimbs subjected to a combination of thermally induced lamellar failure and vertical load to simulate severe acute laminitis. Standard radiographic measurements were used to compare DP displacement. The LCP was then applied to one forefoot in 12 healthy Standardbred horses either standing (n = 6) or under general anaesthesia (n = 6). Lameness was evaluated daily, then horses were euthanised (day 8) and lamellar tissue analysed using light microscopy, histomorphometery and molecular markers of apoptosis. RESULTS: In the cadaver limb model, LCP fixation prevented the significant changes in hoof-distal phalanx distance, coronary extensor process distance and sole depth that characterised DP displacement in untreated limbs (p < 0.05). Application of the construct in vivo was well tolerated with minimal lameness (10/12 horses were sound at the trot on day 8); however, histology revealed dorsal lamellar pathology consistent with laminitis, but with extensive keratinocyte apoptosis. Adjacent to the LCP, caspase-3 positive cell counts were approximately 20-fold higher than control (p < 0.001). MAIN LIMITATIONS: Pathology was evaluated at a single time point. Microvascular perfusion was not evaluated. CONCLUSIONS: Rigid fixation of the DP to the hoof capsule was achieved with the LCP construct in a cadaver limb laminitis model. In live horses, LCP fixation caused regional lamellar pathology with extensive apoptosis, likely due to disturbed lamellar microvascular perfusion and/or mechanostasis. Understanding these mechanisms is critical for refinement of the technique in order to avoid iatrogenic lamellar damage.

Characterization of extracellular matrix macromolecules in primary cultures of equine keratinocytes.

BACKGROUND: Most research to date involving laminins and extracellular matrix protein function in both normal and pathological conditions involves in vitro culture of keratinocytes. Few methods are established to allow for prolonged propagation of keratinocytes from equine tissues, including the hoof lamellae. In this study we modified cell isolation and culture techniques to allow for proliferation and sub-culturing of equine lamellar keratinocytes. Additionally, the production and processing of extracellular matrix molecules by skin and lamellar keratinocytes were studied. RESULTS: Physical and proteolytic tissue separation in combination with media containing a calcium concentration of 0.6 mM in combination with additional media supplements proved optimal for proliferation and subculture of equine lamellar keratinocytes on collagen coated substratum. Immunofluorescence and immunoblotting studies confirmed that equine skin and lamellar keratinocytes produce Ln-332 in vitro and processing of this molecule follows that of other species. As well, matrix components including integrin alpha-6 (alpha 6) and the hemidesmosome proteins, bullous pemphigoid antigen 1 (BP180) bullous pemphigoid antigen 2 (BP230) and plectin are also expressed. CONCLUSIONS: Isolation of equine keratinocytes and study of the matrix and adhesion related molecules produced by them provides a valuable tool for future work in the veterinary field.

The anatomy and physiology of the suspensory apparatus of the distal phalanx.

The equine hoof capsule protects the softer, more sensitive, structures within. Failure of the connection between hoof and bone (suspensory apparatus of the distal phalanx or SADP) results in the crippling lameness of laminitis. Active basal cell proliferation occurs principally in tubular hoof and proximal and distal lamellae. The remaining lamellae are virtually non-proliferative and the hoof wall moves past the stationary distal phalanx, by controlled activation and inhibition of constituent proteases. The lamellar corium derives most of its blood supply from the branches of the terminal arch which perforate the distal phalanx. Valveless veins within the foot can be exploited clinically for retrograde venous therapy or contrast radiography (venography). The basement membrane (BM) forms the interface between the lamellar epidermis and the adjacent dermis and the plasma membrane of each lamellar basal cell is attached to the BM by numerous electron dense adhesion plaques or hemidesmosomes the ultimate attachment unit of the SADP. Laminitis destroys and dislocates the BM and its components and without an intact, functional BM, the structure and function of the lamellar epidermis is pathologically compromised. Transcription and activation of constituent proteases occurs in normal hoof lamellae but in increased amounts during laminitis.

Carbohydrate alimentary overload laminitis.

In acute laminitis, the suspensory apparatus of the distal phalanx fails at the lamellar dermal/epidermal interface. A grading system for the histopathology of laminitis is based on the consistent pattern of histologic changes to the secondary epidermal lamellae, basal cells, and basement membrane that occur as carbohydrate-induced laminitis develops. The actual trigger factors of carbohydrate-induced laminitis remain unidentified.

Progression of venographic changes after experimentally induced laminitis.

Venography (retrograde venous angiography) is a relatively simple and practical method for vascular assessment of the digits in the standing horse. The technique is a useful adjunct to routine radiography. The clinical use of the laminitis venogram has resulted in a more comprehensive understanding of the collateral pathology associated with distal phalanx displacement and abnormal hoof growth. The effectiveness of therapeutic procedures such as hoof wall resection, coronary band grooving, deep digital flexor tenotomy, and therapeutic shoeing can be assessed by serial venography. This article discusses the venographic appearance during the transition from the clinically normal hoof to the severe chronic laminitis cases similar to those seen in practice.

Laminitic pain: parallels with pain states in humans and other species.

Laminitis poses a threat to all horses, and is widely considered as being one of the most important diseases of horses and a global equine welfare problem. The effects of laminitis lead to debilitation, development of pronounced digital pain, and great suffering in the afflicted animal. The precise pathophysiological processes that result in laminitic pain are poorly defined, and hence the delivery of effective palliative care is clinically challenging. Knowledge and understanding of pain states in other animal species may further aid the elucidation of equine laminitic pain mechanisms, guide the search for treatable causes of this multifactorial problem, and thereby help achieve enhanced therapeutic and palliative care. However, parallels drawn from pain states in other animals must consider species differences in both anatomy and physiology, and the specific nature of the laminitic disease process.

Supporting limb laminitis.

Supporting limb laminitis poses a threat to all horses suffering from severe unilateral lameness. Despite its devastating effects, relatively little is known about the precise pathologic processes that lead to its development. This article reviews the potential mechanisms of supporting limb laminitis, and the authors present some preliminary data based on advanced imaging and computer-based modeling techniques aimed at further elucidating the etiology of this unique form of laminitis. Gaining a better understanding of the pathologic processes that lead to supporting limb laminitis is essential to enable the development of appropriate countermeasures to safeguard horses at risk of the disease.

Hyperinsulinemic laminitis.

Laminitis occurring in association with hyperinsulinemia is frequently encountered in today's equine population. New evidence suggests that hyperinsulinemia is the direct cause of this form of laminitis, rather than insulin resistance per se. The mechanism by which elevated serum insulin concentrations result in lamellar dysfunction is currently under investigation by many researchers and the use of a new insulin infusion model for investigating the pathogenesis of insulin-associated laminitis will doubtless enhance progress in this field of research. By focusing on the metabolic and vascular actions of insulin in the lamellar microenvironment, our research group is trying to gain an insight into the pathophysiological processes involved in this complex problem, in order to better understand the disease.

Microbial events in the hindgut during carbohydrate-induced equine laminitis.

Equine laminitis is the most serious foot disease of the horse, often resulting in death or euthanasia. Laminitis has long been recognized as an affliction of horses, as has the association of this condition with the ingestion of carbohydrates. Research into the pathophysiology of this condition has been facilitated by the development of reliable models for experimentally inducing laminitis, and DNA-based techniques for profiling complex microbiomes have dramatically increased the knowledge of the microbiology of this disease. Recent studies have provided substantial evidence showing equine hindgut streptococcal species to be the most likely causative agent. Although these studies are not definitive, they provide the foundations for future work to determine the source of laminitis trigger factors and their mechanisms of action.

The lamellar wedge.

In horses with chronic laminitis, an abnormal horn structure called the lamellar wedge develops within the lamellar region of the foot. This pathologic structure adversely affects normal foot function, and influences return to previous performance levels. Understanding the pathologic process that leads to the development of this structure is essential for correct supportive foot management of the horse with chronic laminitis. The ability to prevent or reduce the formation of the lamellar wedge may eventually lead to better outcomes in cases of laminitis.

The effect of continuous digital hypothermia on lamellar energy metabolism and perfusion during laminitis development in two experimental models.

BACKGROUND: Continuous digital hypothermia (CDH) prevents lamellar failure in the euglycaemic hyperinsulinaemic clamp (EHC) and oligofructose (OF) laminitis models, but the mechanisms remain unclear. OBJECTIVES: To evaluate the effects of CDH on lamellar energy metabolism and perfusion in healthy horses and during EHC and OF laminitis models. STUDY DESIGN: In vivo experiment. METHODS: Archived samples were used from Standardbred geldings that received no treatment (CON) (n = 8) or underwent EHC (n = 8) or OF (n = 6) laminitis models. Both forelimbs were instrumented with a lamellar microdialysis system, and one forelimb was cooled (CDH) with the other maintained at ambient temperature (AMB). Microdialysate was collected every 6 hours and analysed for glucose, lactate and pyruvate concentrations and lactate to pyruvate ratio (L:P). Microdialysis urea clearance was used to estimate lamellar tissue perfusion. Data were analysed using a mixed-effects linear regression model. RESULTS: Glucose did not change in CDH limbs relative to AMB in CON (P = .3), EHC (P = .3) or OF (P = .6) groups. There was a decrease in lactate (P < .001) and pyruvate (P < .01) in CDH limbs relative to AMB in all groups. L:P decreased in CON CDH relative to CON AMB (P < .001) but was not different in EHC (P = .6) and OF (P = .07) groups. Urea clearance decreased in CDH limbs relative to AMB in CON (P = .002) and EHC (P < .001), but not in OF (P = .4). MAIN LIMITATIONS: The EHC model may not mimic natural endocrinopathic laminitis. CONCLUSIONS: CDH caused a marked decrease in lamellar glucose metabolism (CON, EHC and OF) and perfusion (CON and EHC) without affecting lamellar glucose concentration. Although cellular energy failure is not a primary pathophysiological event in EHC and OF laminitis models, CDH may act by limiting energy supply to pathologic cellular processes whilst preserving those critical to lamellar homoeostasis.

Lamellar energy metabolism and perfusion in the euglycaemic hyperinsulinaemic clamp model of equine laminitis.

BACKGROUND: Hyperinsulinaemia is associated with the development of endocrinopathic laminitis; however, the mechanisms remain unclear. OBJECTIVES: Evaluate the effects of hyperinsulinaemia on lamellar energy metabolism and perfusion during laminitis development. STUDY DESIGN: In vivo experiment. METHODS: Eight Standardbred horses were instrumented with a microdialysis probe in the lamellae of a forelimb. A 24 hours baseline period (BASELINE) was followed by 48 hours of a continuous euglycaemic hyperinsulinaemic clamp (EHC) from 24 to 72 hours (CLAMP). Microdialysate was collected every 6 hours and analysed for glucose, lactate and pyruvate concentrations and lactate-to-pyruvate ratio (L:P). Microdialysis urea clearance was used to estimate lamellar tissue perfusion. Archived microdialysis samples from six identically instrumented Standardbred horses served as controls (CON). Variables were compared over time and between EHC and CON horses using a mixed-effects linear regression model. RESULTS: Glucose concentration decreased during the CLAMP period in CON and EHC horses (P < .001), but there was no difference between CON and EHC (P > .9). Lactate concentration increased during the CLAMP period in CON and EHC horses (P < .001), however, the rate of increase was significantly higher in EHC horses relative to CON (P = .014). There was a relative increase in pyruvate concentration in EHC horses compared with CON during the CLAMP period (P = .03). L:P increased significantly in CON horses during the CLAMP period (P < .001) but not in EHC (P = .1). Urea clearance did not change in CON (P = .9) or EHC (P = .05) during the CLAMP, but did increase in EHC relative to CON (P = .02). MAIN LIMITATIONS: The effects of microdialysis probe implantation on perfusion and metabolism remain unclear. The EHC model may not mimic natural endocrinopathic laminitis. CONCLUSIONS: Laminitis developed without evidence of lamellar hypoperfusion or energy stress. Therapies to improve perfusion are unlikely to affect the initial development of endocrinopathic laminitis.

Effects of an anti-IGF-1 receptor monoclonal antibody on laminitis induced by prolonged hyperinsulinaemia in Standardbred horses.

Currently, there are no registered veterinary drugs for the treatment of endocrinopathic equine laminitis, and although this form of the disease is known to be caused by prolonged hyperinsulinaemia, the mechanism of insulin toxicity is unclear. One possibility is that high concentrations of insulin activate IGF-1 receptors (IGF-1R) in lamellar tissue, leading to uncontrolled cell proliferation and epidermal lamellar dysregulation. An equinized version of a human anti-IGF-1R therapeutic monoclonal antibody (mAb11) was generated to test this theory, using a modification of the prolonged euglycaemic-hyperinsulinaemic clamp technique. Healthy Standardbred horses were infused for 48 h with 0.9% saline (negative-control, n = 6), a combination of insulin (4.5 mIU/kgBW/min) and a variable infusion of 50% glucose to maintain euglycaemia (positive-control, n = 6), or insulin and glucose, preceded by a low dose of mAb11 (20 mg), designed to treat one foot only and delivered by retrograde infusion into one forelimb (mAb-treated, n = 7). Maximum insulin concentrations were 502 ± 54.4 and 435 ± 30.4 µIU/mL in the positive-control and mAb11-treated groups, respectively (P = 0.33). While the control group remained healthy, all the insulin-treated horses developed laminitis within 30 h, as judged by clinical examination, foot radiographs and histological analysis. Some effects of insulin were not attenuated by the antibody, however, relative to the positive-control group, horses treated with mAb11 showed less sinking of the distal phalanx (P < 0.05) and milder histological changes, with markedly less elongation at the tips of the secondary epidermal lamellae (P < 0.05). These differences were apparent in both front feet and were statistically significant when the values for both feet were combined. The results confirm that IGF-1R may have a role in insulin-induced laminitis and suggest that mAb11 warrants further research as a potential agent to prevent or treat the disease.

An investigation of the equine epidermal growth factor system during hyperinsulinemic laminitis.

Equine laminitis is a disease of the digital epidermal lamellae typified by epidermal cell proliferation and structural collapse. Most commonly the disease is caused by hyperinsulinemia, although the pathogenesis is incompletely understood. Insulin can activate the epidermal growth factor (EGF) system in other species and the present study tested the hypothesis that upregulation of EGF receptor (EGFR) signalling is a key factor in laminitis pathophysiology. First, we examined lamellar tissue from healthy Standardbred horses and those with induced hyperinsulinemia and laminitis for EGFR distribution and quantity using immunostaining and gene expression, respectively. Phosphorylation of EGFR was also quantified. Next, plasma EGF concentrations were compared in healthy and insulin-infused horses, and in healthy and insulin-dysregulated ponies before and after feeding. The EGFR were localised to the secondary epidermal lamellae, with stronger staining in parabasal, rather than basal, cells. No change in EGFR gene expression occurred with laminitis, although the receptor showed some phosphorylation. No difference was seen in EGF concentrations in horses, but in insulin-dysregulated ponies mean, post-prandial EGF concentrations were almost three times higher than in healthy ponies (274 ± 90 vs. 97.4 ± 20.9 pg/mL, P = 0.05). Although the EGFR does not appear to play a major pathogenic role in hyperinsulinemic laminitis, the significance of increased EGF in insulin-dysregulated ponies deserves further investigation.

Insulin and incretin responses to grazing in insulin-dysregulated and healthy ponies.

BACKGROUND: Supraphysiological insulin and incretin responses to a cereal-based diet have been described in horses and ponies with insulin dysregulation (ID). However, the hormonal responses to grazing have not yet been described. OBJECTIVES: To determine if there is a difference in the insulin and incretin responses to grazing pasture between insulin-dysregulated and healthy ponies. ANIMALS: A cohort of 16 ponies comprising 5 with normal insulin regulation (NIR), 6 with moderate ID (MID), and 5 with severe ID (SID). METHODS: In this case-control study, an oral glucose test (OGT) was used to determine the insulin responsiveness of each pony to PO carbohydrate before grazing pasture (4 hours) for 3 consecutive days. Serial blood samples collected during grazing were analyzed for glucose, insulin, glucose-dependent insulinotropic peptide (GIP) and active glucagon-like peptide-1 (aGLP-1), and compared among pony groups and day of pasture access. RESULTS: The area under the insulin curve when grazing increased with ID severity (P < .03). The median (range) maximal insulin concentration was greater in the MID (72.5 [129] µIU/mL) and SID (255 [338.5] µIU/mL) groups, compared to the NIR (11.7 [24.9] µIU/mL) group (P < .03) and occurred within 2-4 hours of grazing. Postprandial OGT insulin concentration was positively correlated with 2 hours post-grazing insulin across all 3 grazing days (P ≤ .03). The aGLP-1 and GIP concentrations increased in response to grazing but did not differ among groups. CONCLUSIONS AND CLINICAL IMPORTANCE: Grazing pasture provoked an increased insulin and incretin response in insulin-dysregulated ponies within 4 hours of grazing. The pasture and OGT insulin concentrations were correlated.

A "modified Obel" method for the severity scoring of (endocrinopathic) equine laminitis.

BACKGROUND: Laminitis is a common equine disease characterized by foot pain, and is commonly diagnosed using a five-grade Obel system developed in 1948 using sepsis-related cases. However, endocrinopathic laminitis is now the most common form of the disease and clinical signs may be mild, or spread across two Obel grades. This paper describes a modified method which assigns scores to discreet clinical signs, providing a wider scale suitable for use in a research setting. METHODS: The "modified Obel" method was developed using an iterative process. First, a prototype method was developed during the detailed observation of 37 ponies undergoing a laminitis induction experiment. The final method was refined and validated using video footage taken during the induction study and from a clinical trial of naturally occurring endocrinopathic laminitis cases. The Obel method was deconstructed and key laminitis signs were evaluated to develop a three-stage, five criteria method that employs a severity scale of 0-12. Veterinarians (n = 28) were recruited to watch and assess 15 video recordings of cases of varying severity, using the Obel and "modified Obel" methods. The inter-observer agreement (reproducibility) was determined using Kendall's coefficient of concordance (Kendall W) and Krippendorf's alpha reliability coefficient. A total of 14 veterinarians repeated the exercise 2-4 weeks after their original assessment, to determine intra-observer agreement (repeatability), assessed using a weighted kappa statistic (kw). Agreement between methods was calculated by converting all "modified Obel" scores to Obel grades and calculating the mean and distribution of the differences. RESULTS: The "modified Obel" and Obel methods showed excellent and similar inter-observer agreement based on the Kendall W value (0.87, P < 0.001 vs. 0.85, P < 0.001) and Krippendorf's alpha (95% CI) value (0.83 [0.53-0.90] vs. 0.77 [0.55-0.85]). Based on the kw value, the "modified Obel" method also had substantial repeatability, although slightly less than the Obel method, (0.80 vs. 0.91). Excellent agreement between the methods was found, with the mean difference (95% CI), comparing the Obel grade, with the "modified Obel" score converted to an Obel grade, being -0.12 (-0.19 to -0.06) grades. The Obel and converted "modified Obel" grades were identical 62% of the time (259/420) and a difference of one grade (higher or lower) occurred in 35% of cases (148/420). CONCLUSION: Both methods show excellent agreement, reproducibility and repeatability when used to diagnose endocrinopathic laminitis. The "modified Obel" method is a three-step examination process for severity-scoring of endocrinopathic laminitis, initially proposed for use within a research setting. When using the modified method a diagnosis of laminitis also requires clinical acumen. The allocation of scores for specific clinical signs should be particularly useful in research trials monitoring laminitis recovery.

The sodium-glucose co-transporter 2 inhibitor velagliflozin reduces hyperinsulinemia and prevents laminitis in insulin-dysregulated ponies.

There are no registered veterinary drugs for treating insulin dysregulation and preventing insulin-associated laminitis in horses. Velagliflozin is a sodium-glucose co-transport 2 inhibitor that reduces renal glucose reabsorption, promotes glucosuria, and consequently, decreases blood glucose and insulin concentrations. This study aimed to determine if velagliflozin reduced hyperinsulinemia and prevented laminitis in insulin-dysregulated ponies fed a challenge diet high in non-structural carbohydrates (NSC). An oral glucose test (1 g dextrose/kg BW) was used to screen 75 ponies for insulin dysregulation, of which 49 ponies with the highest insulin concentrations were selected. These animals were assigned randomly to either a treated group (n = 12) that received velagliflozin (0.3 mg/kg BW, p.o., s.i.d.) throughout the study, or a control group (n = 37). All ponies were fed a maintenance diet of alfalfa hay for 3 weeks, before transferring to a challenge diet (12 g NSC/kg BW/d) for up to 18 d. Blood glucose and serum insulin concentrations were measured over 4 h after feeding, on d 2 of the diet. The maximum glucose concentration was 22% lower (P = 0.014) in treated animals, with a geometric mean (95% CI) of 9.4 (8.0-11.0) mM, versus 12.1 (10.7-13.7) mM in the controls. This was reflected by lower (45%) maximum insulin concentrations in the treated group (P = 0.017), of 149 (97-228) µIU/mL, versus 272 (207-356) µIU/mL for controls. The diet induced Obel grade 1 or 2 laminitis in 14 of the 37 controls (38%), whereas no velagliflozin-treated pony developed laminitis (P = 0.011). Velagliflozin was well-tolerated, with no hypoglycemia or any clinical signs of adverse effects. The main limitation of this study was the sample size. Velagliflozin shows promise as a safe and effective compound for treating insulin dysregulation and preventing laminitis by reducing the hyperinsulinemic response to dietary NSC.

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.

Induction of laminitis by prolonged hyperinsulinaemia in clinically normal ponies.

The purpose of this study was to determine the effects of prolonged administration of insulin, whilst maintaining normal glucose concentrations, on hoof lamellar integrity in vivo on healthy ponies with no known history of laminitis or insulin resistance. Nine clinically healthy, unrelated ponies were randomly allocated to either a treatment group (n =5; 5.9+/-1.7 years) or control group (n =4; 7.0+/-2.8 years). The treatment group received insulin via a euglycaemic hyperinsulinaemic clamp technique modified and prolonged for up to 72 h. Control ponies were infused with an equivalent volume of 0.9% saline. Ponies were euthanized at the Obel grade 2 stage of clinical laminitis and hoof lamellar tissues were harvested and examined for histopathological evidence of laminitis. Basal serum insulin and blood glucose concentrations were 15.7+/-1.8 microU/mL and 5.2+/-0.1 mmol/L, respectively (mean+/-SE) and were not significantly different between groups. Mean serum insulin concentration in treatment ponies was 1036+/-55 microU/mL vs. 14.6 microU/mL in controls. All ponies in the treatment group developed clinical and histological laminitis (Obel grade 2) in all four feet within 72 h (55.4+/-5.5h), whereas none of the control ponies developed laminitis. There was no clinical evidence of gastrointestinal involvement and the ponies showed no signs of systemic illness throughout the experiment. The data show that laminitis can be induced in healthy young ponies, with no prior history of laminitis, by maintaining prolonged hyperinsulinaemia with euglycaemia. This suggests a role for insulin in the pathogenesis of laminitis, independent of hyperglycaemia, or alterations in hind-gut fermentation. For the clinician, early detection and control of hyperinsulinaemia may facilitate management of endocrinopathic laminitis.