This report describes a significant breakthrough in laminitis research. Many of the predisposing factors are explained and a number of triggers can now be understood, through the identification of a common causative agent. This report is designed to inform horse owners, clinical veterinarians and research scientists about this discovery and its immediate implications for prevention and treatment, as well as informing future research aimed at new therapeutic options.

Background
This project was conceived after an earlier study showing that horses with Equine Cushing’s Syndrome are more likely to develop laminitis and survive less than two years after diagnosis, if they also have high serum concentrations of insulin. Equine Cushing’s Syndrome is a disease more often occurring in ponies and older horses, and is characterised by the excess production of cortisol. Although this is not the most common cause of laminitis, there is a likely link between this chronic disease and the short-term use of synthetic cortisol-like drugs (to treat inflammation), which can also trigger laminitis causing great concern to owners and veterinarians.

To an endocrinologist, the most obvious connection between cortisol and insulin is glucose. Following a stressful stimulus, high concentrations of cortisol act in concert with adrenaline to provide fuel for the brain to coordinate a ‘flight or fight’ response. This is achieved by increasing the synthesis of glucose from other energy substrates, releasing glucose from storage sites, and preventing tissues other than the brain from taking glucose out of the circulation. In this regard, cortisol and insulin oppose one another. The main role of insulin is to maintain normal blood glucose concentrations by stimulating the uptake of excess glucose by many tissues, such as skeletal muscle. To achieve this, most cells contain a specific type of glucose transport protein (GLUT4-type) which depends on insulin for its activation.

In a healthy horse, a short-term increase in glucose caused by stress, exercise, or a meal, would be followed by an increase in insulin, so that blood glucose concentrations do not rise too high, the brain is not overloaded with glucose, and the tissues are able to take up this vital energy source (Figure 1a). However, when the glucose transport proteins are overworked (e.g. through chronically elevated cortisol concentrations or chronic overfeeding), they become less responsive to insulin, such that glucose intolerance/insulin resistance develops. The defining indication of this condition is that individuals have much higher insulin concentrations than normal following a glucose challenge (Figure 1b).

This project was based on the theory that insulin resistance is the factor that predisposes horses to laminitis, and that damage to the hoof is a direct consequence of glucose deprivation. The hoof is known to have an unusually high metabolic demand for glucose, and previous work using isolated tissue had demonstrated that the lamellae become separated if the glucose supply is blocked. 

Aims and objectives
The aim of the project was to test the hypothesis that laminitis is caused by impaired glucose uptake in the hoof, as a result of cortisol and/or adrenaline action, coupled with insulin resistance. Establishing this fact would provide a clear direction for future research into appropriate treatments. However, before the hypothesis could be tested in tissue from horses with laminitis, it was necessary to determine the normal rate of glucose uptake and confirm the mechanisms which control this in healthy tissue. In particular, it was important to confirm that the hoof contains GLUT4-type glucose transporters that are dependent on insulin for their activation, and that hormones such as adrenaline and cortisol are able to oppose insulin action in the short-term and long-term, respectively.
 
Methods
Samples of hoof tissue were collected from horses soon after they had been killed at a local abattoir. The samples were cut into thin slices and incubated in a warm nutrient medium allowing them to remain intact for several days. A modified form of glucose that had been synthesised with a radioactive marker was used to measure the rate of glucose uptake into the tissue, and various drugs and hormones were added to study their effects. In complementary studies, the number of ß-type adrenaline receptors (ß-adrenoceptors) was identified, also through the use of radio-labelled compounds.
 
Results

• The dominant type of adrenaline receptor in the hoof was identified as the ß2-adrenoceptor, similar to that found in skeletal muscle. A small proportion of ß-adrenoceptors (10%) were of the ß1-type, similar to those found in the heart;
• we confirmed that the ß-adrenoceptors in lamellar tissue have a functional effect, showing that their activation with a ß-agonist drug can reduce glucose uptake by 20 to 40%;
• we also noted that the number of ß-adrenoceptors in lamellar tissue varies markedly between horses, and that only half the animals tested showed a glucose response to a ß-agonist drug. This variation did not show a strong association to breed, age, or gender of the horses;
• the most significant but unexpected finding, was that hoof tissue is not dependent on insulin for glucose uptake, nor does it show a glucose response to insulin, even at high insulin concentrations;
• further studies showed that the glucose transport proteins in hoof tissue do not become saturated at low glucose concentrations (unlike typical GLUT4 transporters), but that they behave in a similar manner to the insulin-independent GLUT1 transporters usually found in the brain;
• finally, a technique was developed to measure the expression of genes for equine glucose transporters. This was used to confirm the presence of GLUT1 transporters in brain and lamellar tissue, whereas GLUT4 transporters were found in skeletal muscle, but not in lamellar tissue.

By confirming that the hoof is not dependent on insulin for glucose uptake, we had rejected the null hypothesis that glucose deprivation due to insulin resistance is the cause of laminitis.

Additional findings and a new hypothesis
Despite eliminating impaired lamellar glucose uptake as a key trigger for laminitis, the question of how cortisol, adrenaline and insulin may interact to cause laminitis, remained. Furthermore, new evidence was published to show that high insulin concentrations are also a characteristic of ponies that are predisposed to pasture-induced laminitis, a condition in which there is an unusually high seasonal intake of non-structural carbohydrates.

By re-arranging the proposed relationship between cortisol, glucose, insulin and glucose transport, we developed a new hypothesis, which is outlined in Figure 1.

Figure 1. Proposed relationship between glucose, insulin resistance/glucose tolerance and laminitis. (a) moderate insulin production occurs in a healthy horse when glucose concentrations rise following a meal or stressful event; (b) Chronic overfeeding, Cushing’s Syndrome, overuse of corticosteroids and/or genetics can cause a horse to become intolerant to glucose (insulin-resistant); (c) A significant glucose challenge resulting from stress or the consumption of lush pasture in a glucose-intolerant horse, leads to a massive insulin response which is able to restore blood glucose concentrations, but precipitates laminitis.

According to this hypothesis, laminitis occurs as a consequence of insulin resistance, coupled with a significant glucose challenge. The underlying cause of insulin resistance may involve a number of factors including Cushing’s syndrome, excessive use of corticosteroid drugs, obesity, overfeeding, or a genetic component. However, the high concentrations of insulin occurring in these horses are not merely a sign of failing glucose transporters, but are the direct cause of laminitis. Accordingly, for this hypothesis to be correct, high concentrations of insulin would be expected to induce laminitis in any horse.

For our final experiment nine ponies were selected with no clinical signs of illness and no recorded history of laminitis. Five ponies received a constant infusion of insulin for up to 72 hours, together with an infusion of glucose to maintain normal resting glucose concentrations. The other four ponies received a saline solution and served as controls.

All the ponies given insulin developed laminitis within 58 to 72 hours, diagnosed by clinical signs and confirmed by histological examination of their hooves. None of the controls developed laminitis.

Implications
We have shown that high concentrations of insulin cause laminitis. This occurs as a result of a glucose challenge, combined with glucose intolerance (insulin resistance) which may be inherited, or developed over a long period of illness, corticosteroid use, or dietary mismanagement. Insulin toxicity may not account for all cases of laminitis, but the concept unites a number of major themes and explains several known triggers. It also provides a clear pathway towards reducing laminitis by:

• identifying animals at risk through the use of insulin measurements and a glucose tolerance test;
• avoiding the use of corticosteroids or high-energy diets in such animals;
• intervening to restore glucose tolerance through diet and exercise, or with the use of glucose-sensitising agents; and
• conducting further research to discover the mechanism of insulin action, leading to new avenues for therapy.

Recommendations
A simple, rapid and reliable glucose tolerance test should be employed to identify horses at risk of hyperinsulinaemia and laminitis.
It is important that all horse owners become aware of the risk associated with prolonged hyperinsulinaemia and take appropriate action to avoid this.

While some information about restoring insulin sensitivity/glucose tolerance is already available to guide horse owners, further research is needed to test novel pharmaceutical, neutraceutical and dietary approaches.

The techniques used by different laboratories to measure insulin concentrations in horses need to be standardised using a national or international reference standard, with appropriate quality controls, so that meaningful clinical comparisons can be made.

The potential of corticosteroid drugs to cause glucose intolerance should be investigated further.
Meanwhile, if corticosteroid drugs are indicated to treat inflammation or other conditions, the user should be mindful of the risks, particularly in horses that are intolerant to glucose. Monitoring insulin concentrations in horses that require corticosteroid therapy may be useful in detecting when insulin resistance becomes clinically significant.

The mechanism by which insulin causes laminitis should be investigated further, with a view to discovering new avenues for therapy.