The IR Phenotype EMS & Risk of PAL

The IR Phenotype EMS & Risk of PAL
Early work indicated ponies grazing high NSC pastures and having characteristics such as IR and obesity Were at greater risk of laminitis. Resting hyperinsulinaemia predicts recurrence of laminitis in ponies on pasture. A genetic component was identified , and a comparison to human metabolic syndrome was made. Hyperinsulinemia itself is the direct cause of endocrinopathic laminitis rather than IR as suggested previously.

Human MetS involves obesity , IR , cardio vascular effects like high blood pressure and risk of heart attack and type 2 diabetes mellitus. While ponies did not show heart attacks the other characteristics were there . One study actually showed ponies on high nsc diets did have increased blood pressure as well as being IR and obese. This was initially called the Pre-Laminitic Metabolic Syndrome , and now called the Equine Metabolic Syndrome.

The argument –originally thought - PAL is triggered by CHO overload of the hind gut and absorption of substances that initiate lamellar failure. But why are only certain horses and ponies at risk.

Hyperinsulinemia- laminitis has been experimentally induced in healthy ponies and horses by infusing exogenous insulin with glucose i.v. Glucose was kept at normal levels. Hyperinsulinaemia has been shown to be a feature in the EMS phenotype, along with obesity and IR/insulin dysregulation. Wide variation in values among studies.

EMS phenotype-likely IR and/or associated hyper insulinaemia an important role. HI has induced laminitis in healthy trim horses and ponies within 2 days and 55 hours later respectively. Glucose was kept at normal levels. Testing for IR/Hi not practical for some methods. Simpler methods ?

Recognition of significant Breed differences vs definition of IR/HI.Current concensus -A fasting insulin >20 mIU/L indicates IR. 18% of 300 healthy non laminitic horses had a basal HI and 28% of Australian ponies. The latter showed age, BCS supplementary feeding and a history of laminitis were all associated with HI in the latter study.

Lack of consensus of the metabolic phenotype of laminitis prone horses and ponies. Partly due to study design And the complexity of the EMS phenotype.

The metabolic phenotypes routinely measured (insulin, insulin responses, adipokines, adiposity etc ) are influenced by the environment, and vary due to age, breed, and sex, even in normal individuals. Furthermore not all components of the syndrome (eg obesity) may be present in individuals with underlying metabolic derangements.

Compensated IR Fasting HI is usually accompanied by normoglycemia, suggests compensated IR in EMS. Eg. two studies in ponies And no difference in glucose tolerance when comparing laminitis prone to non laminitic ponies. In early type 2 diabetes of humans HI may be a compensatory response to IR. However, there is data suggesting HI can induce IR.

Laminitis –failure of the bond between the inner hoof wall and the distal phalynx.
Leads to separation or stretching of laminae, due to pull of DDFT, and pressure on the dorsal aspect of hoof wall during breakover. Mechanical weight effects. Collapse of the foot occurs. Rotation and sinking of P3 and Injury to other structures and causes unrelenting pain. Endocrine rather than sepsis associated laminitis is probably predominant for PAL. Naturally occurring endocrine diseases associated with laminitis include equine metabolic syndrome and pituitary pars intermedia dysfunction (PPID). EMS affects young to middle aged horses and ponies and is characterized by hyperinsulinemia & insulin resistance, with obesity a major risk factor.

Laminitis is a syndrome
Multiple inciting causes, similar results. 1-Sepsis associated with inflammation 2.-Endocrine or metabolic with IR , obesity and/or PPID. Usually laminitis is more associated with endocrine disorders than sepsis related. Not usually associated with markers of inflammation. No marked separation of the basement membrane as per SIRS. Endocrinopathis laminitis as elevated basal insulin(>20 mIU/l) , with or without a concurrent elevated (seasonally adjusted ) basal ACTH concentration (>29 pg/ml from Nov to July and > 47 pg/ml from Aug.-Oct. Or vet guidelines). 3.-Mechanical over load e.g.SLL, concussion 4.-BW Extract/toxin types

Endocrine associated laminitis
The lesions have not included the extensive basement membrane (BM) separation previously proposed as the fundamental (primary) histopathological change , based on models associated with systemic inflammatory disease (i.e, CHO overload), with prominance of epidermal apoptosis and mitotic activity as well as minimal neutrophil infiltration Inflammatory markers are not usually present as in carbohydrate overload models. Hyper glycaemia may not be present ie compensated IR/HI. Has focused attention on looking at the effects of diet and environment and genetics and other stresses that can affect laminitis through effects on insulin. Endocrine issues include PPID and EMS. Does pasture grazing affect insulin dynamics particularly in IR animals. PAL probably more associated with endocrine based laminitis.

Incidence of laminitis-lack of data-Overall 1-3% of all horses.
The most common cause of foot lameness at approximately 16% of all cases of lameness. A large uk survey showed over 6 years 23.5% of all horses had at least one episode of veterinary diagnosed laminitis and 46% of all cases were attributed to PASTURE grazing, with 7% to grain overload. Overall incidents seemed associated with hours of sunlight and many had repeat episodes 24% in the same year. It seems some animals are prone to repeated episodes of laminitis and suggests the possibility of phenotypic or genetic factors associated with susceptibility. Mares seem at greater risk

With PAL risk factors include Ponies and Obesity
Previous research showed signs of Obesity E.G. Generalised or regional such as the cresty neck , HI , (insulin> 32 mU/L) on winter pasture, or HL >7.3 ng/ml (winter pasture), plus hyperinsulinaemia and a history of laminitis were useful predictors of laminitis PAL seemed to occur when ponies were exposed to high CHO pasture. Ponies are naturally IR. IR and hypertension also seemed related in at least one study with ponies Expression of this metabolic phenotype was only noted in summer so environmental effects like summer pasture are important. This is called the Equine Metabolic Syndrome (EMS)

Equine Metabolic Syndrome-a cluster of abnormalities associated with laminitis
McCue et al JEVS 35 (2015) In 2002 it was recognized that primary features of a laminitis –prone phenotype (i.e. Obesity , insulin resistance were analogous to those described in human metabolic syndrome , including obesity, dyslipidaemia, glucose intolerance, and hypertension, associated with increased risk of cardiovascular disease and perhaps type 2 diabetes. Work has documented similarities . In 2010 the ACVIM listed criteria for EMS The 3 main criteria included documented or suspected insulin resistance, that is hyperinsulinaemia and/or abnormal glycemic and insulinaemic responses to oral or IV glucose or insulin challenges ;

Equine Metabolic Syndrome-a cluster of abnormalities associated with laminitis
Generalized obesity and/or increased adiposity in specific locations (regional adiposity ) including the nuchal ligament (“cresty neck”), tail head, behind the shoulder, in the prepuce or mammary gland area; And predisposition towards laminitis that develops in the absence of other recognized causes such as grain overload, retained placenta, colitis , colic or pleuropneumonia. Additional components suggested included hyper triglyceridemia, dyslipidemia, and increased low-density lipoprotein concentrations, hyperleptinemia, arterial hypertension, altered reproductive cycling in mares, and increased systemic markers of inflammation associated with obesity.

The understanding of EMS pathophysiology and risk factors for laminitis
The metabolic phenotypes routinely measured e.g. Insulin, insulin responses, adipokines, are highly influenced by the environment and vary due to physiologic factors such as age, breed, and sex, even in normal individuals. Furthermore not all components of the syndrome (e.g. Obesity) may be present in individuals with underlying metabolic derangements. The variability in biochemical measurements and inconsistency in clinical signs between individuals at risk of laminitis makes it difficult to establish clear diagnostic criteria or consensus phenotype.

EMS Phenotype It seems likely that IR/and or associated hyper insulinemia play an important role. IR-usually defined as the reduced ability of a given concentration of insulin to lower blood glucose levels. Very few studies on insulin sensitivity and other aspects of glucose and insulin in EMS or laminitis affected animals.

The EMS Phenotype Assessing SI cannot be applied in clinical practice easily. There are significant breed differences in SI , fasting insulin concentrations , and insulin responses during an oral sugar test (OST) further complicate a definition of a universal clinical definition in horses. Most have used fasting or resting measures of insulin and glucose and/or indices derived from these as surrogate indicators of IR.

Fasting or resting measures of insulin or glucose re IR
Hyperinsulinaemia has been shown to be a feature in the EMS phenotype in ponies and Morgan horses for example. Although there is wide variation in values among studies that may in part be explained by differences in sampling conditions (e.g. Feed withholding, vs pasture grazing before collection of blood samples. The current consensus view is that fasting insulin >20 mIU/L indicates IR or veterinarians use their own criteria. Insulin vs IGF-1 vs MAPK vs AMPK

Insulin dysregulation –excessive I responses to sugar, fasting hyperinsulinemia, and IR all components of EMS Generalised obesity and/or regional adiposity accompany insulin dysregulation in many equids with EMS. Horses and ponies with EMS should be evaluated for evidence of laminitis , including divergent hoof growth rings, (founder lines) or 3rd phalanx rotation. Mild laminar pathology does not always cause overt lameness and can go unrecognised by horse owners. Increased adiposity is evident before laminitis in most animals , but a lean EMS phenotype also exists, with insulin dysregulation and laminitis occurring in horses and ponies with normal appearance

The lengthening of the lammelae-IGF-1

Hyperinsulinemia-fasting prior to sampling now recommended
Fasting hyperinsulinemia results from persistent stimulation of pancreatic B cells and in obese humans, increased insulin secretion during the interprandial period is attributed to elevated fatty acid concentrations. HI has also been associated with obesity in equids and higher fatty acid concentrations were detected in obese insulin-resistant horses in one study, although hay was available at the time of sampling. Beta-cell hyperplasia is also a potential cause of fasting hyperinsulinamia . Several hormones and nutrients act as insulin secretagogues and increase insulin concentrations , including incretin hormones, glucagon, glucose, fatty acids and arginine. Insulinomas can occur.

Post prandial hyperinsulinemia-a concern
Feeding almost continuous basis when grazing on pasture. Feeds vary in composition and this makes it difficult to establish reference ranges. Standardised tests are therefore recommended to diagnose HI in horses. OST-corn syrup, 15 ml per 100 kg bw. Blood sample at min . Serum insulin over 50 mU/l (60 at 60?) indicates an abnormal response. Not a specific indicator of laminitis risk. In feed oral glucose test with 1gkg bw glucose in a chaff based feed with samples collected 2 hours later. Or combined glucose –insulin test. 150 mg/kg bw 50% dextrose iv plus 0.10 u/kg bw insulin. IR is evidenced by blood glucose that remains above baseline at 45 minutes (and/or insulin >100 mU/l at this time point).

Fasting Hyperinsulinemia and IR is it the cart or the horse???
HI-in EMS , Usually accompanied by normo glycemia Suggests compensated IR-an increase in pancreatic insulin secretion occurs in response to reduced tissue SI, resulting in maintenance of glucose homeostasis. In the early stages of type 2 diabetes of humans, hyperinsulinemia is also considered to be a compensatory response to IR. However, there also is some data suggesting that hyperinsulinaemia can induce IR.

Other mechanisms may contribute to the fasting hyperinsulinemia and exaggerated response to glucose administration observed in EMS horses. Insulin concentration is determined by both rate of secretion and rate of clearance, with approximately 80% of endogenous Insulin removed by the liver. In humans and other species there is evidence of decreased insulin clearance in IR states (eg diabetes mellitus, obesity, nonalcoholic Fatty Liver Disease. Hyper insulinemia is caused by increased insulin secretion or delayed insulin clearance and this problem may be a cause or a consequence of IR. EG HI accepted explanation is that insulin secretion increases as a consequence of decreased tissue insulin sensitivity and referred to as compensated IR.

Fasting hyperinsulinemia and exaggerated response to glucose administration
Insulin secreted from B cells is normally cleared from the portal blood before reaching the peripheral circulation in horses. A reduction in Insulin clearance is thought to be a mechanism to preserve B-cell function and also to maintain peripheral insulin levels in the face of IR. Possibly a factor in EMS. One recent study in horses shows that reduced insulin clearance contributes to higher blood insulin concentrations in obesity. Measured by using low connecting C peptide insulin ratios . Since it is released in equal amounts, concentrations reflect insulin secretion and hepatic clearance

The cause and effect relationship between IR and hyperinsulinaemia-Frank N. And E.M. Tadros EVJ 46 (2014) Appears straightforward, but we have horses and ponies with postprandial hyperinsulinaemia that have normal glucose and insulin responses during the combined glucose-insulin test (CGIT), which is a test for insulin sensitivity. Other causes of postprandial hyperinsulinaemia following ingestion of sugars should therefore be considered, including increased stimulation by incretin hormones. These 2 hormones are secreted from L and K cells , respectively , within the small intestine in response to ingestion of sugars, amino acids and fats. The activity of both hormones is regulated through degradation by the enzyme DPP 4. Metabolites now appear capable of suppressing hepatic glucose production and exerting insulin like actions. Individuals vary. Identified in equine plasma after oral administration of glucose. Limits post prandial hyperglycemia by inducing insulin secretion as glucose (and other sugars, fats and amino acids) enters the small intestine

Incretin Hormones may contribute to an upregulation of i secretion in EMS
Labeled incretin hormones because they potentiate glucose -mediated insulin secretion and account for the higher insulin secretory response by oral vs IV glucose. GLP and GIP. Inhibits glucagon release in response to feeding and they slow gastric emptying and intestinal motility and induce satiety thereby minimizing postprandial hyperglycemia. In humans. Conflicting info re effects of obesity and type-2 diabetes on incretin hormone secretion and the incretin effect. Decreased GLP-1 baseline measures and responses to oral glucose have been associated with oral glucose intolerance. During OST was detected in EMS horses. Requires research.

Incretin Hormones may contribute to an upregulation of Insulin secretion in EMS
GLP-1 has a short half life due to rapid inactivation by an enzyme DPP4. Both GLP-1 analogues and DPP4 inhibitors are currently used therapeutics for the management of type 2 diabetes Postprandial HI with EMS might therefore reflect increased incretin hormone secretion or decreased degradation by dipeptidyl peptidase 4. Furthermore, incretins increase pancreatic islets in rodents by stimulation of B cell proliferation, induction of islet neogenesis, and inhibition of B cell apoptosis. Chronic incretin hormone stimulation may contribute to B cell hyperplasia, and HI in horses with EMS as per increased GLP-1 in human MetS.

Incretin Hormones may contribute to an upregulation of i secretion in EMS
Differences in total and active GLp-1 in horses due to breed, sex, and obesity status, and microbial micro flora of the stomach may influence sugars arriving at L cells in the small intestine and glucose bioavailability. Effects of specific sugars on active GLP-1 have not been determined in horses and this is relevant as studies in rats and dogs have shown that aGLP-1 varies according to food type. Chronic ingestion of high-fermentable fiber in dogs significantly increased GLP-1 vs low fermentable fiber. A decrease was noted for rats fed high fat. Rich pasture???? Chameroy K.A. JEVS 40 (2016) 16-25

Horses here were obese at first but lost fat mass with management.
Incretin Hormones may contribute to an upregulation of i secretion in EMS 2 Higher fasting GLP-1 has been associated with obesity and increased rates of fat oxidation in humans. Horses here were obese at first but lost fat mass with management. NAFLD –non alcoholic fatty liver disease is associated with obesity in humans , and this lowers aGLP-1 . This group has detected mild hepatic liver lipidosis in obese horses post mortem , so fatty infiltration of the liver may contribute to individual variability in aGLP-1 . (or insulin clearance). Horses and ponies may not respond like other species.

EMS If the first manifestation of insulin dysregulation in genetically predisposed horses is postprandial HI, then IR would raise insulin levels even further. Obesity, systemic inflammation, and concurrent endocrinopathies such as PPID are potential causes of IR that can exacerbate HI, by slowing insulin clearance. Inflammaging? HI might even induce IR itself through the process of homologous desensitisation , a process by which tissue sensitivity to insulin decreases as blood hormone levels increase. Acute increases in insulin concentration can still stimulate glucose uptake but chronic HI results in down regulation of the insulin receptor and downstream signalling.

Dyslipidemia-and other issues
In humans, MetS , IR, and/ or Hyperinsulinemia are thought to contribute to other components of the syndrome including dyslipidemia. Increased de-novo hepatic lipogenesis is a feature of IR and Hyperinsulinemia in human and animal models. The presence of more than 3 clinical findings associated with EMS in one study correctly predicted the development of laminitis in 11/13 ponies on high starch pastures.

Dyslipidemia-2 Increased Plasma triglyceride concentrations were a feature of the EMS phenotype in a closed herd of Welsh ponies, and also in outbred ponies with a history of recurrent laminitis, although in the latter study the relative hypertriglycemia was evident during summer but not winter. Horses and ponies may develop severe hypertriglyceridaemia secondary to endocrine disorders that are associated with IR. Hypertriglyceridaemia can resolve with treatment of the endocrinopathy. Although biochemical evidence of hepatic compromise was present, clinical abnormalities were not noted in these animals. EVJ 46 (2014)

Dyslipidemia-2 An increase in nonesterified fatty acid (NEFA) delivery to the liver may be an alternative or additional factor underlying lipid concentrations. In a small mixed breed group of obese, insulin resistant horses, increased serum NEFA concentrations ( but not Triglycerides) were detected , Whereas Serum NEFA’s ’s were not useful in the differentiation of an EMS phenotype in ponies Increased Serum NEFA ’s has not been a feature of the EMS phenotype in our recent study of >600 horses. One study showed increased very low –density lipoprotein triglyceride concentrations were observed in horses with EMS.

Obesity-a component of EMS but hyper insulinemia is also detected in lean animals
Some obese animals have normal insulin concentrations. These inconsistencies can be explained by considering obesity as a modifying factor , with the genetics of the individual animal determining the magnitude of insulin dysregulation. Hyper insulinaemia itself might even pro mote obesity through the anabolic effects of insulin on lipid metabolism, and contribute to the easy keeper phenotype. Horses and ponies with this phenotype readily become obese when overfed , and obesity can develop when grass is the only energy source. Eg with mice HI alters lipid metabolism and promotes obesity. With KO mice with genetic manipulations affecting the brain and pancreas not to produce insulin in excess, were protected from obesity and hepatic lipid infiltration.

Hypertension - Insulin Controls the NO and ET-1 systems in vascular tissue
In other species NO synthesis is impaired in IR, whereas hyperinsulinemia itself stimulates increased ET-1 production. There are insulin receptors in the endothelial tissue in the blood vessels of the foot, but not the lammellar tissue itself. The lammellar tissue does not depend on insulin for glucose uptake. The imbalance favors vaso constriction and is thought to contribute to hypertension in insulin resistant states such as MetS and type two diabetes mellitus. Arterial hypertension has been detected in mixed-breed , recurrent laminitic ponies during summer but not winter, whereas in other studies , mean blood pressure did not differ between groups of EMS vs non-EMS ponies in early spring, summer and fall. No concrete evidence of vascular disease has been demonstrated as associated with EMS. However, these animals are at increased risk for the development of laminitis. Could vascular effects of insulin be involved?

A vicious cycle-AT function is disrupted which disrupts insulin signalling in cells.
The result is IR or insulin dysregulation but not inflammation as per sepsis. Due to IR there is a need for increased secretion of insulin by pancreatic b cells. In humans the B-cells become exhausted following prolonged IR, and glucose rises with lower insulin. T2DM In equids, with EMS, for unknown reasons B-cell exhaustion does not occur so they persist in a chronic state of hyperinsulinaemia (or at least exaggerated insulin response to a CHO meal) and euglycaemia. Rather than CV disease, laminitis occurs HI, obesity and hepatic IR might therefore represent a vicious cycle also if lipid infiltration of the liver slows insulin clearance and higher fatty acid concentrations induce fasting hyperinsulinaemia.

EMS-Aberrant or excessive growth factor signalling in the lamellae :
(likely involving the activation of insulin –like growth factor -1 (IGF-1) receptors in the lammellar epithelial cells by insulin at supra physiological concentrations has recently been documented in both obese ponies fed a high energy diet and in the HEC model of laminitis. Additionally, the demonstrated decrease in lamellar concentration of the activated form of AMP –activated protein kinase (AMPK) , although most commonly studied in the setting of energy metabolism , may result in the disruption of lamellar epithelial cytoskeletal dynamics proposed to occur in EMSAL There are no insulin receptors in lammelar tissue but there are IGF-1 receptors in lammelar tissue.

Obesity- as in humans the prevalence of obesity in horses and ponies is on the rise
There is growing concern regarding the adverse health effects of an expanded fat mass One definition BCS > 7 over weight –with studies showing that body fat represents approximately 20-25% of total mass in the animals with a BCS above this cutoff. BCS > 8 obese Or simply BCS > 7 is obese Prevalence of obesity defined as >7 BCS has varied between 20-50% in recent studies, Obesity has been proposed as a risk factor for PAL in ponies.

Obesity-and/or the presence of one or more enlarged subcutaneous fat depots
High BCS and (regional adiposity) depots have been regarded as defining characteristics of EMS. Including the nuchal ligament region (cresty neck), behind the shoulder, (Unilateral or bilateral), around the tail head, in the preputial or mammary gland regions. The presence of abnormal swelling around the prepuce or mammary glands due to adipose tissue deposits and associated edema is often the presenting complaint. However, obesity has not been a consistent finding across studies and definitions of obesity vary.

Obesity two -A recent study of over 600 horses and ponies
with (PL) and without (NL) a history of laminitis (previous 12 months) has provided further insight into EMS including the association with obesity, Across breeds, fasting insulin , insulin 75 minutes after corn syrup admin (post-ost) and fasting Serum triglycerides were the variables most consistently elevated in animals with a history of laminitis. This confirms previous reports in ponies, but in contrast to previous reports demonstrates that elevated Serum triglycerides might also be key feature of EMS phenotype in horses, but gross lipaemia has not been described in EMS or PPID Unlike previous reports, morphometric measures e.g. NC/height ratios and Girth C to height ratios and BCS did not discriminate between PL and NL groups here?????

Prior wt loss in the PL group may have confounded the effects
But note- in the PL group, certain metabolic variables did not differ between obese (bcs>7) and non obese (bcs<7) animals, suggesting the metabolic derangement persists even after weight loss. In the NL group , however, serum triglycerides , fasting insulin, and post-ost insulin were higher in obese vs non obese animals. One interpretation is that obesity per se is not a requisite feature of the laminitis prone (ie EMS) phenotype , but components of the phenotype such as IR may be exacerbated when affected animals become obese.

Obesity-Metabolically Healthy Obese ?
In humans-The presence of obesity –related metabolic disturbances , including IR, hyperlipidemia, inflammation, and hypertension varies widely among obese individuals. Up to 30-35% of obese adult humans have a metabolically healthy phenotype?? - “The metabolically healthy obese”. Conversely up to 40% of adults with normal weight and BMI have metabolic perturbations typically associated with obesity and MetS, such as dyslipidemia, Hyperinsulinemia, and non alcoholic Fatty Liver Disease. ? Although the mechanisms underlying these different phenotypes are not fully understood, it is believed that differences in visceral fat accumulation and the response of adipocytes to increased lipid accumulation are important factors.

EMS horses have differences in their plasma lipodome S
EMS horses have differences in their plasma lipodome S. Elzinga et al JEVS 40 (2016) Conflicting results for laminitis vs inflammation. Eg PAL no difference in markers of inflammation? Comparing 14 EMS horses with 9 controls. The EMS horses had naturally occurring HI. TG and leptin were significantly higher in EMS and had differences in lipid profiles. These differences may have implications for cell signalling , membrane fluidity, and inflammation. In humans and animals, increases in the lipids appear to affect not only inflammation but insulin resistance. Certain ones can act as intracellular messengers And chemo attractants. Changes in nutrition can decrease markers of inflammation and increase IS Inflammation may not show in EMS because the immune system is compromised re lipids and cell health and function? EMS horses have differences in their plasma lipidome compared to controls similar to what has been observed in humans with metabolic symdrome.

Adipose Tissue –produces many proteins termed adipokines and cytokines
Including proinflammatory cytokines and other hormone-like proteins termed adipokines that exert local (paracrine) and systemic (endocrine) effects. Animal models have indicated that obesity results in a progressive dysregulation of adipose tissue function, including marked pro-inflammatory signaling that leads to the development of a systemic inflammatory response that, in turn, results in the development of IR and other metabolic abnormalities. However, not all excess fat carries equal risk, with visceral adiposity most strongly associated with systemic inflammation and IR. Preliminary observations that obesity per se is not a defining feature of EMS mirrors findings in humans –IR and Hyper triglyceridaemia can occur in non obese horses , whereas , conversely, the metabolic profile of some obese horses does not differ from that of lean individuals.

One hypothesis-Obesity is not a primary cause of EMS, but instead a marker
It may be a marker of an underlying metabolic dysfunction that, depending on other environmental factors (e.g. Diet, exercise, drives adipose tissue accretion and development of obesity. If so two important implications. First, the presence or absence of obesity cannot be used as a diagnostic criterion. Second , although dietary restriction and weight loss may result in some improvement in SI and so forth in affected animals , the underlying metabolic dysfunction is likely to persist. Further studies are required re impact of obesity on metabolic health and its role in EMS. Studies have shown an inverse relationship between BCS and SI, and positive relationships between apparent adiposity, resting insulin and blood markers of inflammation (SAA, TNF-A, mRNA encoding for IL-1B, and TNF-alpha.

Example Adipose Tissue (nuchal crest fat) and systemic markers of inflammation were unchanged in AA geldings after a 20% weight gain (increase in BCS from 6 of 9 to 8 of 9) In addition SI was unchanged in TB geldings after a 15% increase in body weight, Another recent study of horses and ponies reported no change in SI after a 20% increase in fat mass. These observations reinforce the idea that factors other than BCS and Adipose Tissue mass contribute to variance in SI.

Adipokines-leptin , a link between fat mass, food intake and energy expenditure
Leptin –mediating its effects by decreasing food intake and increasing Energy expenditure. It may reduce hepatic fatty acid synthesis. In humans and rodents, circulating leptin levels are typically proportional to fat mass, although leptin levels are also affected by gender, estrus cycle, and SI. Concentrations increase in obese and IR horses as a result of increased fat mass and leptin resistance. A hyper leptinaemia is also detected in normal BCS horses? One of the Major roles of leptin in peripheral tissues is the prevention of lipid accumulation , which is thought to promote Sensitivity to insulin (SI). In their studies, however, serum Leptin was not useful for the identification of laminitis prone animals although , consistent with previous studies, leptin concentrations were strongly associated with BCS.

Adipokines-leptin , a link between fat mass, food intake, and energy expenditure
In obese humans and rats, however, high leptin concentrations do not induce the expected response suggesting a resistance to the effects of leptin. Similar to humans, in horses there is a positive correlation between leptin concentrations and fat mass and/ or BCS., and leptin concentrations increase with weight gain and decrease with dietary restriction and weight loss. Leptin might serve as an indirect measure of adipocyte pathology and insulin dysfunction with a cut off of 4 ug/l for hyperleptinaemia. The correlation between leptin and Insulin in humans and horses has led to interest in measurement of leptin in EMS suspects. High leptin has been observed in IR horses and ponies, including non obese animals. In their studies, however, serum Leptin was not useful for the identification of laminitis prone animals although , consistent with previous studies, leptin concentrations were strongly associated with BCS.

Adiponectin-Conversely, Inversely proportional to body mass – an insulin sensitizer.
With pleotropic effects that include enhancement of fatty acid oxidation, and improvement of glucose tolerance. Possesses anti-inflammatory properties and counteracts IR caused by cytokines such as TNF-alpha on the endothelium. Leptin is generally proinflammatory and exacerbates endothelial activation and the generation of free radicals that damage the endothelium.

Adiponectin-Conversely, Inversely proportional to body mass - an insulin sensitizer.
In Humans adiponectins (both types) are correlated with several phenotypic characteristics in MetS including BMI, waist circumference, blood pressure, fasting glucose, and insulin concentrations and Plasma triglycerides. Inflammation a questionable role in metabolic/endocrine laminitis Similarly, an inverse relationship between blood adiponectin (total or high-molecular wt ) and BCS has been reported in horses. In contrast to leptin, low adiponectin concentrations were correlated with laminitis in this recent study. A study found no difference in inflammatory markers between pasture Pl ponies and phenotypic matched controls , however levels of adiponectin were found to be lower in PL ponies. Wray EVJ 45 (5)

Laminitis A current or recurrent episode The most common reason for evaluation of EMS.
Most laminitis cases in practice are secondary to EMS. The severity of EMS-associated laminitis is variable, ranging from subclinical to mild lameness. Clinical observations suggest EMS often develop laminitis when grazing at pasture (PAL), especially under conditions that favor increased accumulation of WSC in pasture forages. Eg in spring and early summer, or after rainfall in the summer or fall or when grass is stressed . See safer grass .org It has been Suggested EMS –associated laminitis begins as a seasonal problem with changes coincident with pasture changes in WSC , (I would add intakes) but then becomes a year round problem as the disease progresses. Previous studies have shown Incident laminitis in horses or ponies with suspected EMS was associated with pasture grazing in approximately 55% of cases.

Prolonged hyperinsulinemia can induce laminitis in healthy horses and ponies.
Important implications for understanding of EMS –and pasture associated laminitis. Studies of grazing horses have shown a positive relationship between pasture NSC content and circulating insulin concentrations. (NSC=sugars, fructans, and starches) and marked exacerbation of Hyperinsulinaemia has been observed in ponies with an EMS phenotype when grazing spring pasture (nsc 15-18% DM). Moreover, the hyperinsulinaemia coincided with episodes of laminitis in these animals. Whether Hyperinsulinemia plays an essential or exclusive role in the development of EMS-associated laminitis remains to be determined.

Laminitis- The association between grazing NSC –rich pasture and incident laminitis in EMS
Raises the possibility of hind gut CHO (carbohydrate) overload as a triggering mechanism? The experimental starch and oligofructose models elicits marked systemic and lamellar inflammatory responses and may not mimic PAL. In a recent study of IR (insulin resistant ) ponies, however, the feeding of a high NSC diet for 7 days that mimicked exposure to spring pasture had no effect on lamellar pro-inflammatory signalling but did result in exacerbating hyperinsulinemia.

Proposed Model for EMS Susceptibilty
Although environmental factors including excess nutrition have been linked to EMS, obesity, IR and laminitis, the high planes of nutrition and/or changes in the pasture do not result in metabolic derangements and laminitis in all horses. Recent studies indicate this individual variability may be due to an underlying genetic predisposition. They propose that the EMS and laminitis risk are Complex Genetic Traits that are the result of one or more inherited genetic alleles (dominant in the population) that are influenced by the environment. Precedent for this model comes from work in human and rodent models.

Genetic Risk Model

Genetics and Risk of Laminitis

EMS as a complex genetic trait.
Genetic alleles (one of two or more alternative forms of a gene that arise by mutation and are found at the same place on a chromosome). Regulation of insulin, glucose, and lipid metabolism in health and disease is complex and not completely understood; However, multiple primary genetic risk alleles that result in alterations in insulin, glucose, and/or lipid metabolism have been identified. Many of these genes interact in large common pathways and result in a related group of phenotypes (obesity, IR, dyslipidemia) that increase the risk of cardiovascular disease and type 2 diabetes mellitus.

Genetic Risk These genetic alleles and genetic interactions are often further influenced by the environment and gene- by- environment interactions , which range from straightforward to epigenetic in nature. (non genetic influences on gene expression). Similar to this scenario, an individual`s genetic risk for EMS likely comes from alleles at one or many genetic loci. An individual`s unique combination of genetic alleles , and the interactions between these alleles , determines the individual`s genetic risk. The alleles at a locus can either increase (risk allele) or decrease (protective allele) disease risk. In an additive genetic model, the effect at a given locus is determined by multiplying the estimated effect of an allele by the number of copies of the allele (zero, one or two ) at the locus.

Genetic Risk The sum of the effects at all loci and the effects of any allelic interactions define the estimated genetic risk for an individual. These genetic alleles work in concert with environmental influences to determine overall disease risk. Similar to the genetic risk, the environmental risk for an individual is the summation of the environmental risk factors , including any interaction between these factors. Metabolic disease risk is then the summation of genetic and environmental risk factors and the effects of any gene by environmental interactions. Fig. 1C.

Genetic Disease Risk Although complex genetic disease risk can be a summation of all risk factors (and the effects of their interactions), it is important to remember that the relative importance of each risk factor can vary between individuals or groups of individuals. Two individual horses may have a similar (moderate) risk for developing EMS due to genetics and environment. However, in one individual , this risk may be the summation of a few genetic risk alleles and many environmental factors (Fig.2A ), whereas another individual may have many risk genetic factors but be housed in a more optimal environment. Fig. 2b The ideal is 2C where the individual has few genetic and environmental risks.

Genetic model for breed differences in metabolic traits and EMS susceptibilty
This complex model of EMS susceptibility can also be used to explain the differences in breed susceptibility to EMS. Several reviews have raised the possibility of breed predisposition to EMS, with Welsh and Dartmoor ponies, Morgans, TWH, Saddlebreds, Arabians and Paso Fino breeds thought to be more susceptible. Affected horses and ponies appear to have high metabolic efficiency. “Easy keepers.”

Genetic model for breed differences in metabolic traits and EMS susceptibilty.
Breeds share key metabolic features of EMS , such as HI, exaggerated response to oral glucose, and elevation in serum triglycerides. Breeds can differ in the magnitude of these responses or other features, such as fasting insulin, Triglyceride , NEFA, and adipokine levels. For example, ponies in general have lower SI and a greater prevalence of hyperinsulinaemia (HI) when compared to horses. Ponies also tend to consume more feed than horses when provided AL access which may contribute to exacerbation of HI and risk of laminitis especially in animals maintained at pasture.

Breed and Genetic Risk Factors leading to EMS and laminitis are shared across breeds
Although differences in the severity and secondary features of the EMS phenotype between breeds , or between individuals within a breed , result from modifying genetic risk factors with variable frequencies. Fig 3-depicts a hypothetical model for EMS or laminitis risk allele sharing across breeds.

Genetics and Risk of Laminitis

Breed and Genetic Risk Factors leading to EMS and laminitis are shared across breeds
In this model, Six alleles combine to confer EMS risk-circle sizes indicated relative frequency of each allele in each breed. All breeds share two major alleles resulting in Hyperinsulinemia (allele A) and hypertriglyceridemia –(allele C). Each breed also has a unique combination of modifying alleles. (B,D, E, and F) with different hypothetical effects explaining the breed differences in phenotypic means. For example , allele B, altered insulin release and/or clearance and increased fasting or OST insulin,

Example-major genetic risk factors-fig 3
Allele D altered tissue utilization or lipolysis and increased Triglycerides . Allele E enhanced SI or improved clearance and lower fasting insulin or OST insulin Allele F elevated NEFAs In this model allele B frequency in breed 3 is greater than breed 2 which is greater than breed 1., which could explain higher mean Serum insulin in breeds 2 and 3 compared with breed 1. In contrast allele E that is unique to breed 4 results in lower fasting insulin and lower insulin responses to glucose challenge . The high frequency of allele D and allele F (nearing 100%) in breed 2 could explain differences in breed 2 means for other measures such as triglycerides and NEFA`s.

This hypothetical model is similar to proposed models for MetS in humans
Where the relative importance of a given risk allele varies between populations and ethnic groups , explaining differences in MetS risk between populations. Conclusions The genetics of metabolic trait variation and EMS or laminitis risk in horses is complex, with variability in risk alleles, allele penetrance, environmental risk factors, gene by environment, and gene by gene interaction all playing a role in an individual`s risk. Furthermore the variable phenotypes across breeds likely reflect the alleles segregating within each breed and their relative importance.

This hypothetical model is similar to proposed models for MetS in humans
Although we have made progress in identifying the breed differences in metabolic phenotypes and work has begun to identify alleles associated with measured metabolic traits, dissecting out all the factors depicted in figure 1 and the allels depicted in Fig.3 will require several investigations. The genetic alleles underlying EMS risk may each be driving individual components of the EMS phenotype . Identification of each allele and understanding its impact on this system and identifying environmental factors that alter the allelic effects are the first steps in dissecting this complex disease.

Conclusions 2 However, indentifying the genes and alleles underlying individual variability in the measured hormonal and biochemical measures of EMS susceptibility, and unravelling the complexity of gene-by-environmental interactions and variable phenotype expression between Breeds , should lead to insights into EMS pathophysiology Similar to MetS in humans, we expect that the genetic alleles underlying EMS risk may each be driving individual components of the EMS phenotype. Identification of each allele and understanding its impact on this system and identifying environmental factors that alter the allelic effects are the first steps in dissecting this complex disease.

Recent research on obesity – a landmark obsevational case control study client owned
Key findings for PEAL –pasture and endocrinopathy associated laminitis. 199 cases-supporting a causal relationship of obesity and laminitis The Horse March 2017. Maybe obesity is a symptom of a bigger problem!!! Onset of signs was greater in spring and summer compared to fall and winter Grazing lush pastures Recent changes in stabling or diet BCS of 7 or higher, generalised and/or regional adiposity, (fat distribution), larger neck circumference, or decreased height (ponies) TB and WB were at a decreased risk. An endocrinopathic disease EMS or PPID were at greater risk. Horses with a recent glucocorticoid admon were at greater risk The first study of only acute or incident cases .

The biome EMS horses have differences in their fecal microbiota .
Gut microbiota vs inflammation vs metabolic dysfunction and characteristics of EMS. S.E. Elzinga et al jevs 44 (2016) 9-16 Biogenic amines could alter digital and lamellar function during laminitis. There may be a predisposition for venoconstriction within the vasculature of the equine digit. Why laminitis can result from a variety of pathologic systemic conditions. J.K. Belknap 2017

Cryo therapy-with of overload EVJ 46 (2014) 554-559
Laminitis occurred in more clinically compromised horses. Use of ICE reduced the incidence of clinical laminitis in the study population suggesting that digital cryotherapy is an effective prophylactic strategy for the prevention of laminitis in horses with colitis. The mechanisms are unknown, but may cause protective vasoconstriction, impeding the delivery of laminitis trigger factors to the lamina , hypothermia may slow the metabolic rate of the lamina, inhibit early inflammatory signalling, and/or diminish production of proteinases implicated in destruction of lamellar extra cellular matrix.

Adeno Associated Virus-J.B.Mason et al EVJ 49 (2017) 79-86
Transgenes can be successfully delivered to the Equine distal extremity using rAAV vectors and that serotypes can successfully transduce tissues of the equine foot. When the vector was diluted with surfactant containing saline, the level of transduction increased dramatically. The increased level of transduction due to the addition of surfactant also improved the distribution pattern of transduction. To develop a gene therapy approach for the prevention of laminitis in the contralateral foot of horses with major musculoskeletal injuries and non weight bearing lameness.

Trace Minerals in the Okanagan

Selenium levels Okanagan

Selenium Okanagan An area generally deficient but local levels may vary., usually less than 0.1 ppm in the Okanagan. Be aware alkaline soils are more conducive to selenium uptake and A major component of enzyme glutathione peroxidase. Protects cell membranes. Required for the control of thyroid metabolism. Deficiency more pronounced in foals with WMD at birth. Mature horses partly protected by other factors but these drop off with age. Vitamin E in green grass may mask a borderline deficiency. Affects keratinization , Acute excess =blind staggers or chronic excess =alkali disease. Foot and coat issues.gut, lung, heart and kidney issues. Se may replace sulfur in tissues May only see poor frog and chronic thrush in marginal excess.

Copper-average values are low in the Okanagan, often less than half NRC
In multiple cellular functions Mobilization of iron stores and haemoglobin formation. Myelination of nerves Keratin formation eg for cracked feet in the Okanagan , Part of the enzyme controlling connective tissue formation in the fetus and growing horse drives the formation of cross linkages in collagen fibers. The mare must provide needs for the foal as milk is low and foals rely on reserves when born. For bone formation. For melanin synthesis ie coat colour. Other trace minerals may interfere with copper absorption. Use 10 or ratios for iron, copper, zinc and manganese. Iron levels can be very variable. Suggest ppm for most classes of horse and broodmares respectively. Some suggest 25 ppm for broodmares.

Zinc- along with manganese most soils in Okanagan are low as are plants
Functions in many tissues a component of many enzymes. Highest in pancreatic tissue and hoof horn and liver. Intermediate levels in muscle. Required for replication of DNA/RNA and cell growth and gene function. For insulin function. For cognitive function Linked to skin health. Deficiency causes parakeratosis. Inappetance in foals, reduced growth rate, alopecia, and decreased horn strength and hardness. Oversupply may tie up copper causing OCD if extremely high . Ratio of 3:1 copper ideal as they share the same absorptive mechanism. as

Manganese-Okanagan soils are very low in manganese and zinc
Essential for many enzymes. For carbohydrate and lipid metabolism. For synthesis of chondroitin sulfate necessary for cartilage formation. Protects cells from oxidative damage. Involved with fertility and central nervous system function. There is no manganese storage in the body as per selenium which has no storage depot. It must be there every day. Milk levels are low. Suggested levels are 40 ppm NRC . Relatively safe to use at higher levels. Ratios as per other trace minerals

Prospective cohort study evaluating risk factors for the development of pasture associated laminitis in the United Kingdom N.J. Menzies-Gow et al EVJ (2016) 1-7 Certain individuals appear to be predisposed to recurrent pasture associated laminitis. Previous studies have predominantly investigated risk factors only after disease occurrence. This study was to investigate risk factors for PAL prior to disease occurrence. A metabolic phenotype with similarities to human MetS , including insulin dysregulation, dyslipidaemia and altered circulating adipokine concentrations with or without obesity appears to be associated with increased risk for laminitis. Thus the same pathologic mechanisms that underlie the Cardio Vascular diseases associated with human MetS , including changes in insulin signalling, inflammatory cytokines and endothelial dysfunction, may contribute to laminitis risk. Multiple variables have been evaluated previously as laminitis risk factors. But these studies have evaluated animals after disease occurrence and the differences detected may reflect the disease rather than a predisposition. Identifying risk factors prior to disease occurrence would allow the targeting of preventive management strategies.

Prospective cohort study evaluating risk factors for the development of pasture associated laminitis in the United Kingdom EVJ (2016) 1-7 Potential risk factors requiring investigation include obesity, insulin dysregulation, inflammatory cytokines and markers of endothelial dysfunction. IV infusion methods for identification of insulin dysregulation are not practical in the field. The Dexamethasone suppression test (DST) is a potential dynamic test for insulin dysregulation as exogenous cortisol analogues antagonise the actions of insulin to result in increased endogenous insulin secretion. Previously laminitic ponies had a greater increase in insulin concentration post-DM compared with controls in spring and summer. Concentrations of the anti-inflammatory marker adiponectin were significantly lower and plasma triglyceride concentrations were significantly higher in previously laminitic ponies in late spring and winter.

Leptin-igf-1 Leptin is an adipose–derived hormone ; hyperleptinemia in humans is associated with human MetS, insulin dysregulation, vascular inflammation and endothelial dysfunction. IGF-1 has insulin like metabolic actions and lower IGF-1 concentrations are associated with human obesity, insulin dysregulation and human MetS. IGF binding proteins (IGFBPs) are an important link between the insulin and IGF systems and play important roles in human obesity and human MetS. Abnormal IGFBP expression is a sensitive marker of insulin dysregulation and used to identify Individuals with insulin dysregulation at high cardio vascular risk and as an early marker of human MetS. None of these factors or parameters has been assessed as a predictor of laminitis development prospectively.

Current reference-standard non-invasive test for assessing endothelial function
Using endothelial –dependent vasomotion in human subjects is flow mediated dilation; However, this is not suitable for use in equids, in which it lacks accuracy and precision. Additionally various circulating molecules are used as biomarkers of endothelial dysfunction, including von Willebrandt’s factor (vWF), soluble (s) E-selectin and P-selectin. Thus it would be logical to evaluate these in horses prior to the occurrence of laminitis. The aim of the present study - To investigate prospectively certain risk factors for the development of PAL in animals with no known history of laminitis. Potential risk factors included morphometric measures of obesity, and circulating concentrations of biomarkers of insulin dysregulation, adipokines and endothelial dysfunction

The uk study In order that the DST was performed for the benefit of the individual animal as a test for PPID and because the youngest animal with PPID to be reported in the scientific literature was aged 7 years, only animals aged greater than or equal to 7 were included. animals were required to detect a 5-10 fold increase in laminitis risk in animals with insulin dysregulation (assuming a 2% laminitis risk over 2 years in animals without insulin dysregulation and an exposed to unexposed ratio of 1:10). Thus to recruit 400 client owned ponies aged greater than or equal to 7 years with no known history of laminitis. Animals were not cases . They were healthy animals recruited directly from owners within 50 miles of the RVC. They were excluded if they displayed clinical signs of acute chronic or previous laminitis (including lameness affecting two or more limbs, increased digital pulses, the characteristic stance of leaning back on the heels, divergent growth rings ) or PPID (including hypertrichosis) at the time of recruitment. In addition ponies were excluded from the study if they were subsequently identified as having an abnormal cortisol response to dexamethasone suggestive of PPID.

Overnight DMST Measured height, determined weight, BCS, using six areas etc., and measured height and thickness of the crest of the neck above the nuchal ligament at the point halfway between the poll And withers. Blood collected before and 19 hours after the im administration of dexamethasone. Mediator analysis- Plasma adiponectin, and leptin, and Serum Insulin were measured. Plasma total IGF-1, IGFBP-1, IGF BP-3, C-reactive protein (CRP), P-Selectin and sE-selectin concentrations were measured. Plasma triglyceride concentrations were measured and Plasma vWF was measured. Follow up Owners were contacted after 12, 24, and 36 months re PAL diagnosed by a veterinarian in the preceding 12 months.

Results 446 ponies median age 15 (10-20 ) years.50.4% mares 49.6% geldings. A range of pony breeds were represented. Welsh 36.4% Shetland 17.0% Cob 9.4% New Forest 9.0% Cross breed 7.6% Other 20.6% The majority, 72.2%, were overweight or obese (bcs 7-9/9). 27.2% ideal weight BCS 4-6/9 0.5% underweight BCS 1-3/9 After 1,2 and 3 years cumulatively, 18 (4.0%), 30 (6.7%), and 44 (9.9%) animals , respectively , had developed pasture associated laminitis PAL. 416 (93.3%) , 374 (83.9%), and 348 (78.0%) respectively remained non –laminitic, and 12 (2.7%) , 42 (9.4%) and 54 (12.1%) had been euthaized for reasons other than laminitis. Table 1

Risk factors-most of the pair wise correlations were weak
Other than those for basal insulin and insulin post dexamethasone . The only significant interaction between risk factors was between basal insulin and post dexamethasone insulin concentration after 2 years and 3 years. For the data obtained after one year, the risk factors taken into the multivariate regression analysis included P adiponectin, Serum basal Insulin , and insulin post –dexamethasone and P-selectin concentrations. With the exception of P-selectin , these all proved to be significant risk factors for the development of laminitis and so remained in the multivariate analysis.

Risk factors-most of the pair wise correlations were weak
For the data obtained after 2 years, the risk factors taken into the multivariate regression analysis included Plasma or Serum CRP, adiponectin, IGF-1, basal Insulin and serum Insulin post-dexamethasone concentrations. . Those that proved to be significant risk factors for development of laminitis and so remained in the multivariate analysis were Plasma adiponectin, Plasma IGF-1, serum basal insulin and serum insulin post dexamethasone concentrations. Table 2 For the data obtained after 3 years, the risk factors taken into multivariate regression analysis included Plasma or Serum adiponectin, basal insulin, insulin post dexamethasone , and IGFBP-3 . Other than the IGFBP-3 all of these proved to be significant risk factors for the development of laminitis and so remained in the multivariate analysis.

Morphometric data None of the morphometric data proved to be significant risk factors for the development of laminitis over 1,2 or 3 years. *** There was no improvement in these values when variables were combined. Consistent risk factors for the future development of laminitis prior to the occurrence of the disease here, included low plasma adiponectin, and high basal and post-dexamethasone serum insulin concentrations.

Adiponectin has anti- atherogenic and anti-inflammatory properties
( in humans and rodents). Circulating concentrations are decreased in obese individuals and in patients with human MetS., type 2 diabetes, and cardiovascular disease. Despite being specifically secreted by adipocytes, a strong negative correlation exists between adiponectin concentrations and body mass index in human patients. And a similar observation has been made in horses. Previously laminitic ponies have been shown to have significantly lower Plasma adiponectin concentrations compared with non-laminitic ponies irrespective of season.

Adiponectin Lower plasma adiponectin concentrations may promote a decreased anti-inflammatory capacity in previously affected horses. The present study reports for the first time that lower adiponectin concentrations occur in animals prior to clinical signs of laminitis , indicating that hypoadiponectinaemia may be a risk factor for laminitis rather than solely a consequence of the disease. Analysis revealed the accuracy with which adiponectin can be used to distinguish animals that do from those that do not develop laminitis in 1,2 or 3 years was fair and a Cut off of 2.5 ug/ml gave acceptable sensitivity (78%) and specificity (79%). The link between hypoadiponectinaemia and risk for laminitis here was not attributable to obesity as obesity was not a risk factor for laminitis and there was no correlation between plasma adiponectin concentrations and morphometric measures of obesity . **** In other species , adiponectin promotes vasorelaxation through increased vascular expression of endothelial nitric oxide (NO) synthase and prostacylin (PGI2) synthase and through the opening of smooth muscle cell K+ channels.

Adiponectin Thus , low adiponectin may increase the risk for future laminitis through decreased vasorelaxation of the equine digital vasculature. Alternatively, there is evidence in humans of cross-talk between adiponectin and both the insulin (InsR) and IGF-1 (IGF-1R) receptors. Adiponectin in association with insulin is able to induce activation of InsR and IGF-1 R and activate the downstream intracellular signalling pathways .

Adeponectin Supra physiologic hyperinsulinemia causes laminitis in healthy equids; At high concentrations insulin can bind to and activate InsR , IGF-1R and InsR/IGF-1R, And IGF-1R and InsR have been detected In lamellar epithelial and endothelial cells respectively. Thus hypoadiponectinaemia and hyperinsulinaemia could potentially combine to alter lamellar epithelial and endothelial InsR and IGF-1R expression ,resulting in epithelial proliferation, and endothelial dysfunction and consequent laminitis. This hypothesis Requires research!

Link , insulin and laminitis
Hyperinsulinaemia and/or insulin dysregulation have been reported in previously laminitic animals in a number of studies. Hyperinsulinaemia and Insulin dysregulation may predispose to laminitis by triggering disturbances in vascular function through down regulation of the phosphatidylinositol 3-kinase (PI-3K) pathway and hence reduction in production of the vasodilator NO in the face of continued vaso constrictor production. Alternatively , as previously discussed, insulin may be excessively stimulating lamellar IGF-1 receptors , leading to inappropriate epithelial cell proliferation with lamellar weakening and consequent laminitis . Serum basal insulin concentrations of >32 uIU/ml were found to have good sensitivity (100%) and specificity (80%) for predicting clinical laminitis in the following 3 months in previously laminitic animals.

Insulin Similarly, here, increased Serum insulin in non-laminitic animals were significantly associated with the subsequent development of laminitis. Analysis revealed that The accuracy with which Serum basal insulin concentrations can be used to separate out those that do and do not go on to develop laminitis cumulatively in 1,2 or 3 years was good (after 2 years) to fair (after 1 and 3 years) and a cut off of 21.8 uIU/ml gave acceptable sensitivity (78%) and specificity ( 67%) values. This value is identical to that proposed above and consistent with previous findings in insulin dysregulation. However, these animals were not fasted before blood sampling caution.

Insulin –single measures of insulin are affected by a number of Factors
Including diet, exercise, stress and time of day. Thus, Dynamic tests of endocrine function are advocated to detect insulin dysregulation. The DST is a potential dynamic test for insulin dysregulation as exogenous cortisol analogues antagonize insulin resulting in increased endogenous Insulin secretion. Previouisly laminitic ponies had a greater increase in Serum insulin post-dexamethasone compared to controls, although this was seen only in spring and summer and a cutoff value of 75 uIU/ml was suggested to distinguish groups of previously laminitic from controls. Similarly, here, an exaggerated insulin response to dex amethasone was associated with the subsequent development of laminitis in non laminitic ponies. Analysis revealed the accuracy with which Serum Insulin post dexamethasone can be used to distinguish between those that do and do not go on to develop laminitis cumulatively in 1,2 or 3 years was fair to poor and a cut off value of uIU/ml gave fair sensitivity (69%) and specificity (68%) values for the development of laminitis over the next 12 months. The present study was designed prior to the recent increase in popularity of oral sugar or glucose tests as dynamic tests of insulin dysregulation. The insulin response to dexamethasone has not been directly compared to ost or ogt thus not possible to extrapolate these results to those tests.

Insulin –like growth factor -1 primarily produced in the liver from GH metabolism
Prior to secretion into the circulation. It has short term insulin-like metabolic actions and long term growth factor like effects on cell proliferation and differentiation . Lower IGF-1 are associated in other species with obesity, insulin resistance, human MetS, type 2 diabetes and increased risk of cv disease. The mechanism behind these inverse relationships is unknown. Median Plasma IGF-1 concentrations in those that remained non-laminitic here were similar to those previously reported for adult horses , whereas the median Plasma IGF-1 concentrations in those ponies that subsequently developed laminitis after 2 years were sig lower. Plasma IGF-1 concentrations have not been measured in previously laminitic ponies , however, season and bcs have been shown to have effects in other populations of horses.

Plasma IGF-1 concentrations
Sig higher in summer than winter and in over weight vs under weight mares. Here samples were collected from all ponies at the same time of year. August, and BCS was not a risk factor for the development of laminitis. ?? Whereas IGF-1 have been reported to decrease with age in man , no evidence ageing is a factor in changes of IGF-1 in adult horses, or here. Thus for unknown reasons, low IGF-1 were associated only with an increased risk for the development of laminitis after 2 years, and not after 1 or 3 years, which suggests IGF-1 concentration is not directly associated with increased risk.? Although IGFBP expression has been suggested to be useful, as a sensitive marker of insulin dysregulation, to identify individuals with insulin dysregulation at high cardiovascular risk and as an early marker of human MetS, concentrations of IGFBP-1 or IGFBP-3 were not useful in detection of animals at increased risk for the future development of laminitis.

Leptin –an Adipose Tissue derived hormone and hyper leptinaemia
Associated with Human MetS, IR, vascular inflammation, and endothelial dysfunction . In horses and ponies hyperleptinaemia is associated with hyperinsulinaemia , obesity, and previous laminitis in some but not all studies. In addition hyperleptinaemia was used to predict clinical laminitis in the subsequent 3 months in previously laminitic animals. However, although there was a weak positive correlation between Plasma leptin and bcs, no such correlation with serum insulin concentration emerged and there was no association between Plasma leptin concentrations and the subsequent development of laminitis here. Increased plasma Triglycerides are associated with hyperinsulinaemia , obesity, and previous laminitis . However, in agreement with here, such increases were not beneficial in the prediction of clinical laminitis in the following 3 months in previously laminitic animals.

C-reactive protein is an acute phase protein increased concentrations
Are associated with IR, human MetS , and cardio vascular disease. In horses, increases in CRP have been reported in induced inflammation and laminitis, pneumonia, enteritis, arthritis, and after castration. Other studies have however reported serum CRP concentrations to be unaffected by inflammatory disease . CRP have not been evaluated in association with naturally occurring laminitis , but did not differ significantly between control animals and hyperinsulinaemic obese horses. Here, CRP were similar to those reported in healthy horses in one study , but lower than those reported in another study. And were not found to be significant risk factors for the subsequent development of laminitis.

Plasma vWF antigen measured only in assoc with exercise or clotting disorders
In humans, vWF concentrations are increased in obesity, MetS , and IR. However, Could not be used to predict development of human MetS in patients with hypertension. P-selectin and sE-selectin are markers of endothelial dysfunction in other species. And the role of endothelial dysfunction is important in human MetS and insulin dysregulation and the development of associated cv diseases. Thus it is Logical to postulate that endothelial dysfunction may play a role in the pathogenesis of laminitis associated with insulin dysregulation . However, none of the biomarkers of endothelial dysfunction measured were associated with the subsequent development of laminitis here.

Although an association between Previous laminitis and generalised and/or regional adiposity has neen reported And generalised and/or regional adiposity was used to predict clinical laminitis in the subsequent 3 months in previously laminitic animals , Surprisingly, no such association with future laminitis was found here? But the majority of ponies (72.2%) HERE were overweight or obese, which reflects a proportion similar to those previously reported for obesity within the UK pony population in some studies. But much greater than other studies evaluating both horses and ponies (30-45%). This high % of obesity may have prevented it from being a discernible risk factor within the population stidied ; alternatively, obesity alone may not be a significant risk factor for the future development of laminitis..

Main Limitation Animals were only examined at a single time point. There is no current evidence for the longevity or stability of these biomarkers. Possibly both these and the morphometric data values changed considerably during the 3 years and had a significant impact on the risk of laminitis. In conclusion, risk factors for the development of laminitis in previously non laminitic animals here included low plasma adiponectin, as well as high basal insulin and serum insulin post dexamethasone concentrations. The accuracy of these parameters in distinguishing between animals that did and did not develop laminitis after 1,2 or 3 years was good (serum basal insulin ) after 1 year, fair (all others) or poor (serum insulin post dexamethasone) and generated cut-off values with acceptable sensitivities and specificities. Combinations of these biomarkers did not improve their predictive value. It should be acknowledged that these cut-off values were generated using samples obtained at a particular time of year (summer) and measured using single assays and that as no radiography was done, it is possible animals with pre-existing subclinical laminitis were included. Surprisingly. The development of laminitis was not associated with regional or generalised obesity, hyperleptinaemia, or hyper tri glyceridaemia. In addition there was no association with circulating CRP, IGF-1, IGFBP-1, IGFBP-3, sE-selectin, P-selectin or vWF antigen concentrations. Requires research re morphometric and metabolic variables.

Laminar Inflammatpry events in lean and obese ponies subjected to high carbohydrate feeding:Implications for pasture associated laminitis. T.A. Burns et al. EVJ 47 (2015) Acute massive enteral CHO overload is associated with laminar inflammation. It is unclear of this is true for more prolonged periods of moderate dietary carbohydrate intake. The objective was to characterise laminar inflammation in ponies exposed to a dietary CHO challenge meant to mimic acute pasture exposure. Ponies (n=22) were divided into either lean (n=11) or obese (n=11) BCS <4 or > 7 respectively. And further stratified to remain on low NSC vs high NSC diet At 7% and 42% dm (hay chop or hay chop plus sweet feed and OF), For 7 Days- Laminar samples were collected following euthanasia.

T.A. Burns et al. EVJ 47 (2015) Laminar samples were collected following euthanasia and examined for CD163, MAC387calprotectin, and cyclo-oxygenase-2 (COX-2) Laminar mRNA concentrations of several proinflammatory molecules (interleukin -1B (IL-1B), IL-6, tumor necrosis factor –alpha TNF-a , IL-8, IL-10, monocyte chemo attractant protein-1 (MCP-1), MCP-2, inducible nitic oxide synthase (iNOS), intercellular adhesion molecule -1 (ICAM-1), E-selectin, plasminogen activator inhibitor -1 (PAI-1) and COX-2 were evaluated. Markers of inflammation were monitored. High carbohydrate feeding resulted in no increase in proinflammatory cytokine expression ; laminar COX-2 expression was increased by high carbohydrate feeding. No laminar leucocyte infiltration was observed in response to high carbohydrate feeding. Results suggest that the marked laminar inflammation observed in models of sepsis –associated laminitis may not play a central role in the pathophysiology of pasture associated laminitis .

Back ground Laminitis is a frequent (and often life and performance limiting ) sequelae to many inflammatory diseases , particularly those diseases characterised by Gram- Sepsis or endotoxemia (such as strangulating GIT disease , septic pleuropneumonia, septic metritis, and enterocolitis). Sepsis –associated laminitis has been suggested to share several pathophysiological characteristics in common with sepsis-associated end-organ injury in man. Most notably , among these commonalities , end-organ inflammation (characterised by robust proinflammatory cytokine and chemokine gene expression , adhesion molecule expression and leukocyte infiltration) is reliably observed both in human sepsis and in the well –characterised models of sepsis –associated laminitis , the BWE model, and enteral CHO overload. These inflammatory processes have been proposed to play a direct role in the laminar injury and subsequent failure that occurs in these models, as the onset and acceleration of laminar inflammatory responses have been shown to correlate temporally with the onset of clinical lameness.

Endocrine disease In addition to sepsis, laminitis also frequently accompanies endocrine disease of equids, such as EMS and PPID . Laminitis associated with endocrinopathy is reported to be the type most frequently encountered . Obesity in several species (rodents man and horses) has been reported to be associated with not only expansion of general AT mass, but also increased endocrine activity of that tissue; Obese individuals have increased numbers of infiltrating AT macrophages and increased circulating concentrations of several proinflammatory cytokines, including Tumor necrosis factor-alpha . It has been suggested by some that equine endocrinopathic laminitis may represent an end –organ Injury resulting from a more global proinflammatory state associated with obesity and insulin resistance and that laminitis in this setting is likely also inflammatory. However, the role of inflammatory events in the pathology of endocrinopathic laminitis has not been rigorously investigated and is poorly understood. The purpose of this study was to characterise the expression of proinflammatory cytokine and chemokine genes and the degree of leukocyte infiltration in the digital laminae of ponies subjected to CHO challenge designed to mimic abrupt exposure to pasture rich in NSC, a stimulus reported to be associated with new –onset laminitis in metabolically predisposed (i.e. Systemically insulin resistant ) equids.

Protocol Housed on dirt lots
Control fed 2.5% bw hay with 7% NSC as starch and ESC .dm Or sweet feed at 1.5% BW fed 3 x per day plus OF 2 gms per kg BW added to hay chop ration . Fed for 7 days Mean NSC consumption of ponies was 1.8 grams per kg BW vs 8.0 grams per kg BW for Controls and Challenge respectively. Basal serum insulin prior to (Day 0) and after completion (Day 7) were measured.

Results The systemic insulin response and weight changes demonstrated that the insulin sensitivity was lower in obese animals although no weight gain was observed. There was no observed effect of diet or BCS on laminar concentrations of markers of inflammation. High CHO feeding increased laminar mRNA concentrations of Cox-2 , but no effect of BCS was noted. Laminar E-selectin mRNA concentrations were decreased in obese ponies compared with lean ponies while CHO feeding did not significantly affect laminar E-selectin mRNA concentrations. COX-2-an enzyme responsible for inflammation and pain. E-selectin a cell adhesion molecule expressed only endothelial cells activated by cytokines.

Digital Laminar Immunohistochemistry
There was no observed effect of diet or BCS on the number of laminar CD-163 positive or calprotectin –positive cells CD163-positive cells were readily observed in all laminar sections . In contrast, very few calprotectin –positive cells were observed in any of the laminar tissue sections regardless of group assignment. Also, virtually all calprotectin-positive cells observed within the digital laminae were keratinocytes , very few calprotectin leukocytes were noted in any laminar section and these were all intravascular. Cyclo –oxygenase-2 positive cells were observed in both the laminar epidermis and vascular elements of the dermis as per reports., while the number of laminar COX-2 cells were not quantified, there did not appear to be any effect of diet or BCS on laminar COX-2 expression.

Discussion These results do not support a prominent role for inflammatory events in the pathogenesis of laminitis associated with high carbohydrate feeding in ponies, as no evidence of upregulation of laminar proinflammatory cytokine and chemokine gene expression or laminar leukocyte infiltration were noted following high carbohydrate feeding for 7 days. While a criticism might be that the protocol was not meant to create a laminitis model, per se, and the findings therefore would not be appropriately extrapolated to endocrinopathic laminitis in general, it is important to note that the ponies fed the high NSC diet did develop basal hyperinsulinaemia during the feeding protocol.

Hyperinsulinaemia has been previously described as a primary determinant of laminitis risk in the setting of endocrine disease and experimental hyperinsulinaemia has been shown to precipitate laminitis in normal ponies and light breed horses. This model may be more representative of naturally occurring disease than protocols of previous reports, as the challenge was enteral and more closely represented pasture exposure .

The vicious circle of laminitis prone horses and ponies.

Further , 4 of the ponies did develop clinical laminitis ; these individuals were not outliers in any of the categories evaluated describing laminar inflammatory responses (i.e. their laminae did not contain higher concentrations of cytokine mRNA or leucocytes than nonlaminitic individuals with the same diet exposure. Finally, additional investigations into the regulation of several signalling pathways within the digital laminae of these ponies suggest that this model does affect laminar metabolism , increased NSC resulted in decreased phosphorylation of AMPK and markedly increased phosphorylation of RPS6 (a protein downstream of growth factor signalling in the laminae , events which may be involved in the degree of adherence of laminar basal epithelial cells (LBEC) to their basement membrane.

Signalling pathways While the laminar mRNA concentrations of proinflammatory cytokines and chemokines were not observed to increase in response to high CHO feeding, Laminar Cox-2 mRNA concentrations were increased in the ponies of this study in response to high CHO feeding. (with no effect observed on BCS) Laminar Cox-2 expression was localised to multiple cell types (including epidermal keratinocytes, endothelial and fibroblast –like cells ) and the cellular localisation of laminar Cox-2 expression observed was in agreement with other reports. This finding may implicates several cell types in the pathophysiology of endocrinopathic laminitis ; however, the unique role of the LBEC in maintenance of laminar integrity (LBEC are responsible for maintaining adherence of the laminar epithelium to its basement membrane , dysadhesion of which is a hallmark of laminitis ) makes it a compelling target to investigate.

The vicious circle of laminitis prone horses and ponies.

Signalling pathways Regulation of COX-2 gene expression has most commonly been described in the setting of acute inflammation , in which activation of COX-2 transcription has been shown to be driven primarily through activation of nuclear factor kappa B (NFkB) downstream of proinflammatory cytokine signalling . While the results of the study are consistent with a role for COX-2 in the laminar pathophysiology that occurs secondary to high carbohydrate feeding, the increased laminar COX-2 expression observed in high NSC fed ponies does not appear to be associated with other changes characteristic of inflammation such as laminar proinflammatory cytokine elaboration and leokocyte infiltration. It may be that alternative signalling pathways known to increase COX -2 expression such as growth factor signalling through the extra cellular signalling pathways (ERK) may be more important in the pathogenesis of endocrinopathic laminitis. This warrants further research as Inhibitiors of several growth factor receptors are commercialy available . Equioxx?

Cox-2 Recent studies in molecular carcinogenesis and clinical oncology suggest an important role for Cox-2 activity in promoting epithelial to mesenchymal transition, (EMT) a phenomema known to be involved in normal development , wound healing, and metastasis of neoplasms. During EMT, mature epithelial cells can de-differentiate and adopt a more motile /invasive phenotype. A process that involves coordinated expression of several transcription factors and activation of multiple enzymatic cascades. Because of its role in potentitating EMT, COX-2 may represent a therapeutic target in the treatment and prevention of laminitis in at risk horses and ponies, due to the availability of relatively COX-2 selective inhibitors approved eg Equioxx). The signalling is different from early stages of acute enetral CHO laminitis requiring research.

The IR Phenotype EMS & Risk of PAL

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The IR Phenotype EMS & Risk of PAL

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