Background: A number of conditions are associated with laminitis in horses, such as corticosteroid administration, equine Cushing’s syndrome and equine metabolic syndrome. In common to these conditions are disturbed glucose and insulin metabolism and importantly, the development of insulin resistance. Insulin resistance is seen when the insulin-responsive glucose transport proteins (GLUTs) that are largely responsible for glucose disposal in tissues such as skeletal muscle begin to fail. Aims: 1. The aim of this thesis was to determine the relationship between disturbed carbohydrate metabolism and laminitis in horses and test the hypothesis that impaired glucose uptake in the hoof lamellae is involved in the pathogenesis of laminitis, by investigating the mechanisms that control glucose uptake in hoof lamellae. 2. Having determined that glucose uptake in the hoof occurs independently of insulin, the hypothesis was re-examined, with the aim of determining the effects of hyperinsulinaemia, in the absence of cortisol manipulation, dietary modification, or hyperglycaemia on lamellar integrity in the hooves of healthy ponies. Methods: 1. An in vitro lamellar explant model was used to investigate the effects of insulin on glucose uptake in hoof lamellae. The β-adrenoceptor (β-AR) populations in equine lamellae were characterised using the radioligand binding technique and the glucose uptake response to stimulation with a potent β-AR agonist (l-isoprenaline) in the hoof was investigated. Further, the mRNA expression of both GLUT1 and GLUT4 in lamellar tissue was determined via PCR analysis. 2. Clinically healthy ponies were randomly allocated to either treatment (n = 5) or control (n = 4) groups. Treatment involved a prolonged (72 h) euglycaemic-hyperinsulinaemic clamp technique while control ponies received an equivalent volume infusion of 0.9% saline. Ponies were euthanased at the onset of Obel grade 2 laminitis (treatment) or at 72 hours (controls). Lamellar tissue was obtained from all ponies and analysed via gelatin zymography, histopathology and immunohistochemistry. Results: 1. The predominant β-AR subtype in lamellae was the β2-AR (90%), with β1-AR expression less abundant (10%). Furthermore, stimulation with l-isoprenaline inhibited glucose uptake by up to 30% in lamellae. This is consistent with the known effects of isoprenaline in other species and tissues, and supports the hypothesis that stimulation with adrenaline could result in reduced glucose uptake in hoof lamellae, suggesting a possible mechanism by which impaired glucose metabolism may be involved in laminitis. Glucose uptake in lamellar explants was not affected by either acute (10-120 min) or long-term (24 h) stimulation with porcine insulin. These results do not support a glucose deprivation model for laminitis, in which reduced insulin sensitivity results in impaired glucose uptake in the hoof. Further, exposing lamellar explants to increasing concentrations of glucose resulted in a GLUT saturation point indicative of predominantly insulin-independent GLUT1 proteins. GLUT1 mRNA expression was strong in brain, coronary band and lamellar tissue and weak in skeletal muscle in control animals and was similar in ponies with insulin-induced laminitis. In contrast, mRNA expression of the insulin-dependent GLUT4 was strong in skeletal muscle and was either absent or barely detectable in coronary band and lamellar tissue. These results are consistent with a predominantly GLUT1-mediated glucose transport system, and suggest that it is unlikely that the GLUT4 gene plays a substantial role in glucose uptake in the hoof. 2. Treated ponies all developed laminitis within 55.4 5.5 hours, while no laminitis occurred in control ponies. Insulin-induced laminitis indicated elongated, collapsed secondary epidermal lamellae (SEL), as well as enlarged and increased numbers of basal cell nuclei, mitotic figures and increased keratinisation in SELs. However, in contrast to the histological appearance of tissue obtained from oligofructose-induced laminitis, basement membrane disintegration was not a major finding. There was no increase in either active or latent forms of MMP-2 or -9 in lamellar homogenates obtained from ponies with insulin-induced laminitis compared with controls, except in one pony that demonstrated increased MMP-2 activity, which was euthanased five days after developing laminitis. Conclusions: Collectively, the research outlined in this thesis indicates that glucose uptake in the equine hoof is independent of insulin, utilising a predominantly GLUT1-mediated glucose transport system. However, a more complete understanding of the metabolic processes within equine hoof lamellae would involve further characterising the effects of other hormones, such as cortisol and IGF-1 on glucose transport, as these results indicate a possible role for adrenaline in reducing glucose uptake in lamellar tissue. Nevertheless, the results presented in this thesis do not support a glucose deprivation model for laminitis, in which reduced insulin sensitivity results in impaired uptake of glucose into hoof lamellar tissue. This research demonstrates that prolonged hyperinsulinaemia induces laminitis in normal ponies, independent of changes in blood glucose concentration. Preliminary studies investigating the pathophysiology of insulin-induced laminitis suggest the possible involvement of increased cellular proliferation and inflammation, rather than MMP activation. However, the exact mechanism by which hyperinsulinaemia induces laminitis awaits further investigation.
| Identifer | oai:union.ndltd.org:ADTP/279157 |
| Creators | Katie Asplin |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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