Toll-like receptor and pro-inflammatory cytokine expression during prolonged hyperinsulinaemia in horses: Implications for laminitis

Equine laminitis, a disease of the lamellar structure of the horse’s hoof, can be incited by numerous factors that include inflammatory and metabolic aetiologies. However, the role of inflammation in hyperinsulinaemic laminitis has not been adequately defined. Tolllike receptor (TLR) activation results in up-regulation of inflammatory pathways and the release of pro-inflammatory cytokines, including interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-�), and may be a pathogenic factor in laminitis. The aim of this study was to determine whether TLR4 expression and subsequent pro-inflammatory cytokine production is increased in lamellae and skeletal muscle during equine hyperinsulinaemia. Standardbred horses were treated with either a prolonged, euglycaemic hyperinsulinaemic clamp (p-EHC) or a prolonged, glucose infusion (p-GI), which induced marked and moderate hyperinsulinaemia, respectively. Age-matched control horses were treated simultaneously with a balanced electrolyte solution. Treated horses developed clinical (p-EHC) or subclinical (p-GI) laminitis, whereas controls did not. Skeletal muscle and lamellar protein extracts were analysed by Western blotting for TLR4, IL-6, TNF-� and suppressor of cytokine signalling 3 (SOCS3) expression. Lamellar protein expression of TLR4 and TNF-�, but not IL-6, was increased by the p-EHC, compared to control horses. A significant positive correlation was found between lamellar TLR4 and SOCS3. Skeletal muscle protein expression of TLR4 signalling parameters did not differ between control and p-EHC-treated horses. Similarly, the p-GI did not result in up-regulation of lamellar protein expression of any parameter. The results suggest that insulin-sensitive tissues may not accurately reflect lamellar pathology during hyperinsulinaemia. While TLR4 is present in the lamellae, its activation appears unlikely to contribute significantly to the developmental pathogenesis of hyperinsulinaemic laminitis. However, inflammation may have a role to play in the later stages (e.g., repair or remodelling) of the disease

Continuous intravenous infusion of glucose induces endogenous hyperinsulinaemia and lamellar histopathology in Standardbred horses

Endocrinopathic laminitis is frequently associated with hyperinsulinaemia but the role of glucose in the pathogenesis of the disease has not been fully investigated. This study aimed to determine the endogenous insulin response to a quantity of glucose equivalent to that administered during a laminitis-inducing, euglycaemic, hyperinsulinaemic clamp, over 48. h in insulin-sensitive Standardbred racehorses. In addition, the study investigated whether glucose infusion, in the absence of exogenous insulin administration, would result in the development of clinical and histopathological evidence of laminitis. Glucose (50% dextrose) was infused intravenously at a rate of 0.68 mL/kg/h for 48. h in treated horses (n = 4) and control horses (n = 3) received a balanced electrolyte solution (0.68 mL/kg/h). Lamellar histology was examined at the conclusion of the experiment. Horses in the treatment group were insulin sensitive (M value 0.039 ± 0.0012. mmol/kg/min and M-to-I ratio (100×) 0.014 ± 0.002) as determined by an approximated hyperglycaemic clamp. Treated horses developed glycosuria, hyperglycaemia (10.7 ± 0.78. mmol/L) and hyperinsulinaemia (208 ± 26.1. μIU/mL), whereas control horses did not. None of the horses became lame as a consequence of the experiment but all of the treated horses developed histopathological evidence of laminitis in at least one foot. Combined with earlier studies, the results showed that laminitis may be induced by either insulin alone or a combination of insulin and glucose, but that it is unlikely to be due to a glucose overload mechanism. Based on the histopathological data, the potential threshold for insulin toxicity (i.e. laminitis) in horses may be at or below a serum concentration of ∼200. μIU/mL

The investigation of insulin-induced laminitis in horses

Equine laminitis is a common, complex and insidious disease affecting the lamellar structures of the horse’s foot. The lamellae are located between the inner hoof wall and the distal phalanx and damage results in lameness and loss of function. Many disparate, inciting factors are responsible for lamellar failure, including hyperinsulinaemia. The pathophysiology of the condition has been pursued by researchers but remains largely undescribed. The pathogenesis of hyperinsulinaemic laminitis has proved difficult to study. However, the discovery of an experimental model that induced laminitis with a prolonged euglycaemic, hyperinsulinaemic clamp (p-EHC) within 72 h in ponies, has enabled research on the ‘laminitogenic’ potential of insulin. This thesis aimed to investigate the mechanism of insulin-induced laminitis in horses by: 1) determining the role of increased (or decreased) blood flow to the hoof in the developmental and acute stages of the disease, 2) determining whether hyperinsulinaemic laminitis occurs due to a whole body or local digital mechanism, 3) describing the lamellar lesion caused by hyperinsulinaemia and comparing this to laminitis caused by other factors and 4) examining the role of degradative enzymes, glucose, advanced glycation end products, reactive oxygen species and the insulin-like growth factor (IGF-1) receptor in insulin-induced laminitis. Initially, the p-EHC was examined in healthy, Standardbred horses, with laminitis occurring within 48 h in the treatment group (n = 4), but not in controls (n = 4). Based on hoof wall surface temperatures, persistent vasodilation appeared to be a feature of the developmental and acute stages of the disease. Insulin is a vasodilator, so to test the hypothesis that persistent digital vasodilation is the principal mechanism of insulin-induced laminitis, a separate study was performed. A regional intra-osseous infusion of the potent vasodilator ATP-MgCl2 induced digital vasodilation in the absence of hyperinsulinaemia. Persistent digital vasodilation was achieved for 48 h, but this did not induce laminitis. Thus, insulin does not cause laminitis solely through a vascular mechanism. To determine whether insulin acts directly in the lamellar microenvironment, or whether a systemic mechanism is required, insulin was administered intra-osseously into the lamellar region without concurrent glucose. Unfortunately, significant digital hyperinsulinaemia did not result and the opportunity to determine whether insulin acts at a local level was thwarted. Further work on local induction techniques for the study of laminitis pathophysiology is required. Adaption of the whole body p-EHC model, to allow examination of lamellar tissue obtained during the developmental (6 h, 12 h, 24 h) phase of insulin-induced laminitis, enabled temporal analysis of laminitis pathology. Samples from the acute and developmental stages of the disease were examined with histology, morphometry, immunohistochemistry and transmission electron microscopy (TEM). The hyperinsulinaemic lamellar lesion was described, and compared to the alimentary carbohydrate overload (ACO) model of laminitis histopathology. Structurally, the lesions of insulin-induced laminitis did not differ significantly from ACO. However, insulin-induced lesions were characterised by less lamellar inflammation, as evidenced by more subdued calprotectin immunoreactivity. Significant lamellar pathology, including secondary epidermal lamellar (SEL) lengthening and narrowing and epidermal cell apoptosis, commenced early in the developmental phase (6 h). Cell proliferation was increased late in the developmental (24 h), and in the acute (48 h) phase. Increased cell proliferation and changes in SEL cell morphology may contribute to significant lamellar lengthening by 48 h, leading to lamellar dysfunction and lameness. Lamellar basement membrane (BM) damage commenced late in the developmental phase (24 h) and became more generalised by the acute phase (48 h), suggesting that it was a downstream event. Ultrastructural analysis revealed decreased hemidesmosome density and significant widening of the BM zone. Gelatin zymography and quantitative real time-PCR studies of lamellar tissue demonstrated that minimal metalloproteinase activity occurred in the lamellae during the developmental and acute stages of insulin-induced laminitis, thus discounting their having a primary pathogenic role in BM proteolysis. Advanced glycation end products (AGEs) were up-regulated in the acute phase of insulin-induced laminitis, compared with control horses, but their receptor, RAGE, was not. However, the AGE increase occurred after significant lamellar pathology was underway. Thus, while a pathogenic role for AGEs is unlikely, they may be an important factor in chronic disease. Reactive oxygen species were not up-regulated in laminitic horses, indicating that oxidative stress is not a feature of acute, hyperinsulinaemic laminitis. However, it may occur in chronic disease secondary to AGE formation. The role of glucose in the pathogenesis of the disease was further investigated. A final experiment determined the endogenous pancreatic response to, and the effect on the lamellae of, a 48 h infusion that delivered an equivalent quantity of glucose to that administered during a p-EHC, but without exogenous insulin. The endogenous insulin response to glucose overload was 5-fold lower (~ 200 µIU/mL) than the serum insulin concentration achieved during the p-EHC. Clinical laminitis did not occur, discounting a primary role for glucose in disease pathogenesis. The resultant endogenous hyperinsulinaemia did induce histological lamellar pathology similar to lesions seen during the developmental stages of the disease. Thus, a possible threshold for disease onset was identified. This suggests that hyperinsulinaemic equids are at considerable risk of subclinical disease in the field, especially during periods of prolonged hyperinsulinaemia. Down-regulation of both the insulin and IGF-1 receptors was observed during the developmental and acute phases of insulin-induced laminitis, suggesting that both receptors were activated. Overstimulation of the IGF-1 system leads to cell proliferation and survival, and this may be the mechanism responsible for the increased cell proliferation and decreased apoptosis occurring in the late developmental (24 h) and acute phases of the disease. Overall, this thesis has contributed significant knowledge about hyperinsulinaemic laminitis and provides clear directions for future research. The findings have largely demonstrated which factors are not involved in the pathogenesis of insulin-induced laminitis, and this work has discounted several theories about the disease mechanism. Further work needs to be directed towards the cellular and intra-cellular consequences of insulin and, in particular, how the interaction between insulin and the IGF-1 system may lead to lamellar failure

Reining in equine metabolic syndrome: A gluttony of challenges [Editorial]

We humans are complicated creatures. Despite remarkable intellect, a fearsome ability to push boundaries and superior survival mechanisms, we are at times our own worst enemy. Metabolic syndrome continues to be a premier health problem in developed, and now increasingly in undeveloped, nations. It is spreading across the planet like an infectious disease and is costing us millions. Metabolic disease remains an important focus both for medical research and for governments desperate to ease the burden on already over-taxed health systems. Unlike some previous worldwide health epidemics, obesity-related diseases will require more than a single, silver bullet. A simple vaccine or treatment cannot overcome a lack of education, awareness and in some cases sheer determination; the human element of these diseases. Undeniably, these ‘human elements’ also complicate our ability, as veterinarians, to effectively manage the growing incidence of equine obesity and metabolic disease..

Science in brief: Progress in endocrinopathic laminitis research: Have we got a foothold?

Free to read on publisher's website This month, Equine Veterinary Journal has compiled an online collection ‘Understanding and managing endocrinopathic laminitis’. Research on the endocrinopathic form of laminitis has accelerated in the past decade. Once experimental laminitis induction studies 1 confirmed clinical and field suspicions 2 that hyperinsulinaemia was the key initiating factor for this unique form of the disease, targeted research on the pathophysiological mechanisms of the condition prospered. Much data has now been gathered to show that insulin dysregulation (ID; disturbed glucose and insulin regulation) lies at the heart of endocrinopathic laminitis pathophysiology. It is also clear that laminitis associated with endocrine factors is distinct from sepsis‐related and supporting limb variants of the disease. ..

Metabolic dysfunction and laminitis

Objective: This review focuses on laminitis that develops as a result of metabolic dysfunction and aims to provide a concise assessment of the current state of knowledge on this form of the disease. Outline: The most prevalent form of laminitis is associated with metabolic or endocrinopathic diseases, such as Equine Metabolic Syndrome and pituitary pars intermedia dysfunction, and the feeding of high-energy diets, particularly those rich in non-structural carbohydrates. Insulin dysregulation is the key hormonal imbalance implicated in causing this form of laminitis and hyperinsulinaemia is an important risk factor for the disease. Hyperinsulinaemia can occur in association with insulin resistance, obesity, regionalised adiposity, dysregulated cortisol metabolism and may also be related to other factors, such as breed and genetic predisposition. Recognition of hyperinsulinaemia is best achieved by using a dynamic oral glucose test that can be performed relatively easily under field conditions. Insulin produces a unique pathological lesion in the lamellae and the features of this lesion have informed investigations on the pathogenesis of the disease. Research into the mechanism of disease is continuing so that more targeted therapies than are currently available can be developed. However, dietary restriction and exercise remain effective management strategies for metabolic disease. Conclusions: Although the pathogenic mechanism/s of metabolic and endocrinopathic forms of laminitis remain the subject of intense research, ample data on risk factors for the disease are available. Efforts focussed on preventing the disease should aim to identify metabolic disease and reduce obesity and insulin resistance in at-risk individuals

Persistent digital hyperthermia over a 48 h period does not induce laminitis in horses

Persistent digital hyperthermia, presumably due to vasodilation, occurs during the developmental and acute stages of insulin-induced laminitis. The objectives of this study were to determine if persistent digital hyperthermia is the principal pathogenic mechanism responsible for the development of laminitis. The potent vasodilator, ATP-MgCl 2 was infused continuously into the distal phalanx of the left forefoot of six Standardbred racehorses for 48h via intra-osseous infusion to promote persistent digital hyperthermia. The right forefoot was infused with saline solution and acted as an internal control. Clinical signs of lameness at the walk were not detected at 0h, 24h or 48h post-infusion. Mean±SE hoof wall temperatures of the left forefoot (29.4±0.25°C) were higher (P<0.05) than those on the right (27.5±0.38°C). Serum insulin (15.0±2.89μIU/mL) and blood glucose (5.4±0.22mM) concentrations remained unchanged during the experiment. Histopathological evidence of laminitis was not detected in any horse. The results demonstrated that digital vasodilation up to 30 °C for a period of 48. h does not trigger laminitis in the absence of hyperinsulinaemia. Thus, although digital hyperthermia may play a role in the pathogenesis of laminitis, it is not the sole mechanism involved

Equine hyperinsulinemia: investigation of the enteroinsular axis during insulin dysregulation

Compared to other species insulin dysregulation in equids is poorly understood. Hyperinsulinemia causes laminitis, a significant and often lethal disease affecting the pedal bone/hoof wall attachment site. Until recently, hyperinsulinemia has been considered a counter-regulatory response to insulin resistance (IR), but there is growing evidence to support a gastrointestinal etiology. Incretin hormones released from the proximal intestine, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide, augment insulin secretion in several species, but require investigation in horses. This study investigated peripheral and gut-derived factors impacting insulin secretion by comparing the response to intravenous (IV) and oral D-glucose. Oral and IV tests were performed in 22 ponies previously shown to be insulin dysregulated, of which only 15 were classified as IR (IV test). In a more detailed study, nine different ponies received four treatments: D-glucose orally, D-glucose IV, oats and Workhorse-mix. Insulin, glucose and incretin concentrations were measured before and after each treatment. All nine ponies showed similar IV responses, but five were markedly hyper-responsive to oral D-glucose and four were not. Insulin responsiveness to oral D-glucose was strongly associated with blood glucose concentrations and oral glucose bioavailability, presumably driven by glucose absorption/distribution, as there was no difference in glucose clearance rates. Insulin was also positively associated with active GLP-1 following D-glucose and grain. This study has confirmed a functional enteroinsular axis in ponies which likely contributes to insulin dysregulation that may predispose them to laminitis. Further, IV tests for IR are not reliable predictors of the oral response to dietary non-structural carbohydrate

Advanced glycation endproducts in horses with insulin-induced laminitis

Advanced glycation endproducts (AGEs) have been implicated in the pathogenesis of cancer, inflammatory conditions and diabetic complications. An interaction of AGEs with their receptor (RAGE) results in increased release of pro-inflammatory cytokines and reactive oxygen species (ROS), causing damage to susceptible tissues. Laminitis, a debilitating foot condition of horses, occurs in association with endocrine dysfunction and the potential involvement of AGE and RAGE in the pathogenesis of the disease has not been previously investigated. Glucose transport in lamellar tissue is thought to be largely insulin-independent (GLUT-1), which may make the lamellae susceptible to protein glycosylation and oxidative stress during periods of increased glucose metabolism. Archived lamellar tissue from horses with insulin-induced laminitis (n=4), normal control horses (n=4) and horses in the developmental stages (6 h, 12 h and 24 h) of the disease (n=12) was assessed for AGE accumulation and the presence of oxidative protein damage and cellular lipid peroxidation. The equine-specific RAGE gene was identified in lamellar tissue, sequenced and is now available on GenBank. Lamellar glucose transporter (GLUT-1 and GLUT-4) gene expression was assessed quantitatively with qRT-PCR in laminitic and control horses and horses in the mid-developmental time-point (24 h) of the disease. Significant AGE accumulation had occurred by the onset of insulin-induced laminitis (48 h) but not at earlier time-points, or in control horses. Evidence of oxidative stress was not found in any group. The equine-specific RAGE gene was not expressed differently in treated and control animals, nor was the insulin-dependent glucose transporter GLUT-4. However, the glucose transporter GLUT-1 was increased in lamellar tissue in the developmental stages of insulin-induced laminitis compared to control horses and the insulin-independent nature of the lamellae may facilitate AGE formation. However, due to the lack of AGE accumulation during disease development and a failure to detect an increase in ROS or upregulation of RAGE, it appears unlikely that oxidative stress and protein glycosylation play a central role in the pathogenesis of acute, insulin-induced laminitis

Equine Laminitis: Comparative histopathology 48 hours after experimental induction with insulin or alimentary oligofructose in standardbred horses

Laminitis has many triggers and comparing the histopathology of lesions induced by different causes may help to establish whether a common mechanism or multiple pathologies are involved. The aim of this study was to describe the microscopical lesions and to quantify morphometric changes in the lamellae of horses with insulin-induced (n = 4) and oligofructose (OF) -induced laminitis (n = 4) compared with normal controls (n = 4). Archived lamellar samples collected during two previous studies were used. Laminitis was induced within 48 h in standardbred horses with either a euglycaemic, hyperinsulinaemic clamp (EHC) technique or, in a separate experiment, with an overdose of alimentary OF. Normal tissue was obtained from control horses in the EPIC experiment that received a balanced electrolyte solution intravenously for 48 h. Six measurements of lamellar length and width were recorded for each hoof. Leucocyte infiltration was assessed by immunolocalization of calprotectin. All control horses exhibited normal lamellar architecture, whereas treated horses developed clinical and histopathological changes consistent with laminitis. Laminitic samples displayed lengthening and narrowing of secondary epidermal lamellae (SELs), rounded epidermal basal cell (EBC) nuclei, mitosis and apoptosis. In the fore feet of laminitic horses, the length from the end of the keratinized axis to the axial tip of the primary epidermal lamellae (PELs) was increased (P < 0.05). SELs were significantly longer (P < 0.05) and narrower (P < 0.05) in the treated horses compared with controls. The two treated groups did riot differ from each other in SEL length or width. Calprotectin expression was absent in control horses, moderate in hyperinsulinaemic horses and marked in OF-treated horses. Laminitis induced experimentally with insulin or OF results in comparable lengthening and narrowing of the SELs and elongation of the axial end of the PELs at 48 h. Immunolocalization of calprotectin indicated that hyperinsulinaemia induces less leucocyte emigration than carbohydrate overload at 48 h. The microscopical lesion of laminitis is similar, but not identical in different forms of the disease. (C) 2011 Elsevier Ltd. All rights reserved