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Prevalence and risk factors for hyperinsulinaemia in ponies in Queensland, Australia RA Morgan,a TW McGowanb and CM McGowana,c*
Objectives To determine the prevalence of hyperinsulinaemia in a population of ponies in Queensland, Australia, and identify associated factors. Methods Breeders or traders of ponies within a 100 km radius of Gatton, Queensland, were recruited for study using an internet database. Clinical and management details were obtained, including body condition score, fat deposition and history or evidence of laminitis. Blood samples were analysed for serum insulin and triglyceride concentrations and plasma adrenocorticotrophic hormone (ACTH) and leptin concentrations following short-term removal from pasture and withholding of supplementary food for at least 12 h. Results Of 23 pony studs identified, 22 were available for visit. The study population consisted of 208 ponies: 70 Australian Ponies; 67 Welsh Mountain Ponies or Cobs; 51 Connemara Ponies; 20 Shetland ponies. We excluded 20 with suspected pituitary pars intermedia dysfunction (>15 years, ACTH >50 pg/mL). In total, 27% of the ponies (51/188) were hyperinsulinaemic (insulin >20 μIU/mL). The final multivariable model revealed increasing age, supplementary feeding and increased leptin and triglyceride concentrations to be associated with hyperinsulinaemia. Conclusions Hyperinsulinaemia was prevalent and associated with age and evidence of metabolic disturbance, including elevated leptin and triglyceride concentrations, in this population. A significant number of ponies were at risk of hyperinsulinaemia, which has implications for strategies to reduce the risk of laminitis in this population. Keywords
endocrinopathy; insulin; laminitis; ponies
Abbreviations ACTH, adrenocorticotrophic hormone; BCS, body condition score; CI, confidence interval; EMS, equine metabolic syndrome; IR, insulin resistance; OR, odds ratio; PPID, pituitary pars intermedia dysfunction Aust Vet J 2014;92:101–106
doi: 10.1111/avj.12159
H
orses with equine metabolic syndrome (EMS) have been defined as those with obesity, insulin resistance (IR) or hyperinsulinaemia and a predisposition to laminitis.1 The syndrome has emerged during the past decade as a disease of considerable importance because of the close association between EMS and *Corresponding author. a Philip Leverhulme Equine Hospital, University of Liverpool, Leahurst Campus, Neston, CH64 7TE, United Kingdom; [email protected] b Acorn Veterinary Clinic, Willows Veterinary Group, West Kirby, UK c Institute of Aging and Chronic Disease, University of Liverpool, Neston, UK
© 2014 Australian Veterinary Association
management, in particular over-nutrition and obesity, and because the resulting clinical disease, laminitis, is such a painful and crippling disorder.1 The high prevalence of obesity in the general horse population is of increasing concern, with one study identifying 45% of horses studied as fat or very fat.2 Another predisposing factor appears to be breed, with the majority of published cases or research herds used for the study of EMS consisting of British pony breeds or their crosses.3,4 British breed ponies in Finland were overrepresented in cases of endocrinopathic laminitis compared with a hospital population.5 Part of the breed effect may be that native British and Irish ponies have evolved to be ‘thrifty’, surviving periodic under-nutrition, for which relative IR is an advantage.6 In the Finnish study, the more common native breeds (Nordic cold bloods, Norwegian Fjords and Icelandic horses) were not as frequently represented with endocrinopathic laminitis, even though these breeds are also ‘thrifty’ and prone to obesity.5 Ponies derived from native British or Irish breeds can be considered as at-risk and therefore were specifically recruited for the present study. Insulin resistance and hyperinsulinaemia are defining factors for EMS and have been closely linked with the development or predisposition to laminitis in both field3,7 and experimental8 studies. In the horse, it is typically compensated IR, with hyperinsulinaemia compensating for the reduction in insulin function.9 Hyperinsulinaemia has been diagnosed in 86% of primary cases of laminitis.5 Research in equine and human populations has shown that hyperinsulinaemia is a good predictor of underlying metabolic disease.10,11 Basal hyperinsulinaemia is a good indicator of IR and has been advocated for use in epidemiological studies,1,12 yet there is little information on the prevalence of hyperinsulinaemia in the general horse or pony population.1,13 The prevalence of hyperinsulinaemia has not been determined in epidemiological studies and has been restricted to studies of single herds.3,7 It should be noted that IR in the horse does not always manifest as hyperinsulinaemia and in some cases dynamic testing with a combined glucose–insulin tolerance test is required to detect IR. In this study, we defined hyperinsulinaemia as insulin concentration greater than 20 μIU/mL, as suggested by American College of Veterinary Internal Medicine consensus.1 Several factors, including body condition score (BCS), regional adiposity, hyperleptinaemia and elevated triglyceride concentrations, have been associated with hyperinsulinaemia and metabolic disease in selected groups of horses or ponies,1,3,13,14 but the prevalence of such metabolic disturbances in the general populations is currently unknown. Most of the studies have, in fact, been carried out in single, closed herds3,7 In people, insulin, leptin and triglyceride concentrations and obesity are closely linked.15 Leptin and triglyceride concentrations have been used to differentiate metabolically healthy
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EQUINE from metabolically unhealthy obese people.16 Identification of animals that are susceptible to laminitis because of metabolic disturbances is critical if the correct interventions are to be applied.9 In order to determine this, epidemiological studies on wider subpopulations of horses are necessary. Our hypothesis was that evidence of metabolic abnormality would be prevalent in pony breeds under normal management conditions. We therefore aimed to determine the prevalence of hyperinsulinaemia in the general pony population of Queensland, Australia, and to identify factors that may be associated with hyperinsulinaemia in this population. Materials and methods The study was approved by the University of Queensland Animal Ethics Committee. Listed pony studs and traders within 100 km of Gatton, south-east Queensland were identified using an internet listing (http:// australianhorsedirectory.com.au/). Each stud identified was contacted via telephone to request permission to visit. During the initial telephone conversation, the location and number of ponies were ascertained and a standard set of open-ended questions was asked concerning the owners’ management of the stud, their knowledge of pasture-associated laminitis and perceived risk or protective factors. The premises were visited during a 4-week period in February–March 2007. Data on the individual ponies, including age, sex, use, history of laminitis, current laminitis and any supplementary food given, were recorded. Each pony underwent a clinical examination and the following information was collected: height, BCS,17 presence of a cresty neck, the presence of bulging supraorbital fat pads and the presence of divergent rings on the hooves. A blood sample was collected following informed consent from the owner. Pastured animals were removed from pasture for 1–3 h prior to blood collection as per the protocol of previous studies.3,7 For housed ponies, managers were instructed that the morning feed be withheld prior to the visit. All visits took place in the morning. Blood samples were obtained by venipuncture of the left jugular vein and placed in 10-mL vacutainer tubes (Vacutainer systems, Becton-Dickinson, NJ, USA) containing either EDTA or no anticoagulant. The samples were immediately placed on ice. Plasma and serum was separated within 3 h by centrifugation and stored on ice. Samples were frozen at −80°C within 12 h of collection and remained so until analysis. Serum samples were analysed for insulin concentration using a radioimmunoassay (Diagnostic Systems Laboratories Inc., TX, USA) previously validated for use in horses.18 Plasma adrenocorticotrophic hormone (ACTH) concentrations were determined using a commercially available assay (Immunite 1000, Vetpath Laboratory Services, Ascot, WA, Australia) previously validated for use in horses.19 Serum triglyceride concentrations were analysed by commercial biochemistry analyser (Olympus AU400 Automated Chemistry Analyser, Olympus American Inc., Diagnostic Systems Division, USA) and plasma leptin concentrations were measured using a radioimmunoassay (Linco Research, St Louis, MO, USA) validated for use in horses.20
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Ponies were excluded from this study if there was a suspicion of pituitary pars intermedia dysfunction (PPID), defined as ponies that were 15 years or older with an ACTH measurement >50 pg/mL.19 Hyperinsulinaemia was defined as serum insulin concentration >20 μU/mL.1 Statistical analysis Prestudy sample size calculations were performed; assuming a pony population size of 100,000 and a frequency of hyperinsulinaemia of 10–20% (Minitab® Statistical Software 16, State College, PA, USA), a sample of 200 ponies gave a power of 99.9% at the level of 95% confidence. Descriptive statistics were performed using Mintab 16. The variables were also tested for correlations using the Pearson’s rank correlation. The data that were not normally distributed (Kolmogorov-Smirnov test) were log-transformed prior to testing for correlations and transformation was shown to improve the normality of the data. Explanatory variables included in the logistic regression were age, sex, breed, use, BCS, cresty neck, increased supraorbital fat, presence of laminitic rings, owner-reported history of laminitis, supplementary feeding and plasma leptin and serum triglyceride concentrations. All variables associated with the outcome with a change in deviance (log-likelihood ratio) at P < 0.25 were offered forward to a forward multivariable logistic regression. The results were then verified using a backward stepwise regression model. The overall fit of the model was ascertained using the Hosmer–Lemeshow goodness-of-fit test.21 the criterion for inclusion was a change in deviance at P < 0.05. Results are presented as mean ± SD where applicable. Results We identified 26 studs or traders from internet listings and published records. Of those, 23 were able to be contacted and 22 available to take part in the study (response rate, 96%). The 208 ponies on these premises were assessed for inclusion in the study; 20 were excluded following a suspicion of PPID, leaving a final study population of 188 ponies. The study population consisted of Australian Ponies (59, 31%), Welsh Mountain Ponies (60, 32%), Connemara Ponies (51, 27%) and Shetland Ponies (18, 10%). There were 143 mares (76%), 19 geldings (10%) and 26 stallions (14%). The mean age was 9 years (range 1–26 years). Their uses included breeding (99, 53%; 26 stallions and 73 mares), riding or showing (23, 12%) and dry or young stock not in work (66, 35%). The majority of the ponies (152/188, 81%) were kept entirely at pasture and the rest also received supplementary feeding (35/188, 19%). Supplementary feeds were non-grain based and typically high-fat based on coconut, soyabean meal or rice bran. All 22 owners in the study identified laminitis as an important issue in pony breeds and all reported numerous risk factors for its development as well as management strategies to avoid it. The most common owner-reported risk factors were the ponies being overweight (n = 12), too much feeding of grain (n = 9), lush grass, particularly after a period of drought (n = 10) and feeding grass with seed heads (n = 4). Other factors reported included stress, genetics and too much protein. Management practices implemented in order to prevent laminitis
© 2014 Australian Veterinary Association
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included monitoring weight (n = 8), avoiding lush pastures or grass with seed heads (n = 6), avoiding overfeeding of grain (n = 3) and keeping mares in foal (n = 6). The mean BCS (out of 5)17 of the whole population was 3.97 ± 0.57 (median 4, interquartile range 3.5–4.5). Clinical examination revealed a cresty neck score >322 in 113 (60%), bulging supraorbital fat in 82 (43.6%) and laminitic rings in 42 (22.3%); 23 (12.2%) of the ponies had a history of laminitis and all of them had laminitic rings on clinical examination, although not all ponies with clinical evidence of laminitic rings had an owner-reported history of laminitis. Only two ponies had current laminitis during sampling and both were hyperinsulinaemic (110 and 373 μU/mL, respectively). The mean serum insulin concentration was 25.4 μU/mL (± 55.2, median 10.9, range 4–520) (Figure 1). The mean triglyceride concentration was 0.55 ± 1.2 mmol/L (median 0.4, range 0.0–15.8) and mean plasma leptin concentration was 5.4 ± 4.2 ng/mL (median 4.3, range 1.1–24.9). In total, 52 (27.7%, 95% confidence interval (CI) 21.4–34.6) ponies were hyperinsulinaemic (insulin concentration > 20μU/mL). The mean BCS (out of 5) did not differ between ponies with or without hyperinsulinaemia. Data for insulin, leptin and triglyceride concentrations were not normally distributed, so they were log-transformed prior to investigation of correlations. There was a moderate correlation between log serum insulin and log plasma leptin (r = 0.41; P < 0.001) (Figure 2). There was a mild correlation between log serum insulin concentration and log serum triglyceride concentration (r = 0.37; P < 0.001) (Figure 3) and between log plasma leptin concentration and log serum triglyceride concentration (r = 0.35, P < 0.001). There was a weak correlation between age and log serum insulin (r = 0.24, P = 0.001). No other correlations were identified. Several variables were found to be significant on univariable analysis (Tables 1, 2) and these were offered forward into a multivariable model. The final model showed that increasing age (odds ratio (OR) 1.14, 95% CI 1.06–1.22, P < 0.001), supplementary feeding (OR 4.89, 95% CI 1.96–12.17, P = 0.001), increasing triglyceride concentration (OR 4.19, 95% CI 1.40–12.50, P = 0.01) and increasing plasma leptin
© 2014 Australian Veterinary Association
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Figure 2. Relationship between log insulin concentration and log leptin concentration (r = 0.41; P < 0.001; n = 188).
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Figure 1. Distribution of serum insulin concentrations across the study population (n = 188).
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Table 1. Univariable analysis of continuous variables put forward into the multivariable model for the presence of hyperinsulinaemia in 188 Australian ponies
Variable
Odds ratio
95% confidence interval
P value
Age (years) Triglycerides (mmol/l) Leptin (ng/mL)
1.08 5.63 1.19
1.03–1.14 2.09–15.16 1.09–1.30
0.004 0.001 1000 acres), so a possible explanation is that they were naturally exercising more than the other ponies in the study. It has been shown that wild horses will walk up to 15 km/day, even when forage and water are available.38 Exercise has been shown to reduce IR and this possibility requires further investigation.39 In humans, familial and genetic links to obesity and metabolic syndrome have been identified, although these are often complicated by factors such as lifestyle and epigenetic factors.40,41 A familial predisposition to EMS has been postulated,3 but a genetic link has not yet been determined in horses.
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The 12% of ponies with a history of laminitis in this study is consistent with previous reports,42 although lower than in the single pony herds used in other studies.3,7 A history of laminitis was significantly associated with hyperinsulinaemia at the univariable level, but was not retained in the final model. It is possible that the ponies with a history of laminitis were more carefully managed than those without and thus it was not identified as a risk factor for current hyperinsulinaemia in the final model. Significant hyperinsulinaemia (>70 μU/mL) has been associated with an increased Obel grade laminitis score.43 Insulin concentration has been shown to be a good prognostic indicator in horses with PPID such that animals with hyperinsulinaemia are more likely to develop laminitis and survive for less than 2 years after diagnosis.44 The creation of a hyperinsulinaemic state has been shown to induce laminitis in experimental animals.8 The exact mechanism by which hyperinsulinaemia results in laminitis is yet to be elucidated, but the results of this study demonstrate the high prevalence of hyperinsulinaemia in the general pony population. Conclusion Hyperinsulinaemia was prevalent in this sample of the general pony population of Queensland, despite drought conditions at the time of the study. Hyperinsulinaemia was associated with increasing age, supplementary feeding and indicators of metabolic dysfunction; that is, increased leptin and triglyceride concentrations. The findings of this study help to characterise the metabolic abnormalities associated with IR in pony populations in which many individuals may be at increased risk of laminitis. Hyperinsulinaemia associated with hyperleptinaemia and hypertriglyceridaemia may represent a state of metabolic disturbance that indicates IR and adipose dysfunction. Acknowledgments The authors are grateful to the University of Queensland for their funding of this project, the Horse Trust who kindly sponsored the residency of RA Morgan and all the owners and breeders included in the study.
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References
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1. Frank N, Geor RJ, Bailey SR et al. Equine metabolic syndrome. J Vet Intern Med 2010;24:467–475. 2. Wyse CA, McNie KA, Tannahil VJ et al. Prevalence of obesity in riding horses in Scotland. Vet Rec 2008;162:590–591. 3. Treiber KH, Kronfeld DS, Hess TM et al. Evaluation of genetic and metabolic predispositions and nutritional risk factors for pasture-associated laminitis in ponies. J Am Vet Med Assoc 2006;228:1538–1545. 4. McGowan CM. Endocrinopathic laminitis. Vet Clin North Am Equine Pract 2010; 26:233–237.2. 5. Karikoski NP, Horn I, McGowan TW et al. The prevalence of endocrinopathic laminitis among horses presented for laminitis at a first-opinion/referral equine hospital. Domest Anim Endocrinol 2011;41:111–117. 6. Neel JV. Diabetes mellitus: a “thrifty” genotype rendered detrimental by “progress”? Am J Hum Genet 1962;14:353–362. 7. Carter RA, Treiber KH, Geor RJ et al. Prediction of incipient pasture-associated laminitis from hyperinsulinaemia, hyperleptinaemia and generalised and localised obesity in a cohort of ponies. Equine Vet J 2009;41:171–178. 8. Asplin KE, Sillence MN, Pollitt CC et al. Induction of laminitis by prolonged hyperinsulinaemia in clinically normal ponies. Vet J 2007;174:530–535. 9. Kronfeld DS, Treiber KH, Hess TM et al. Metabolic syndrome in healthy ponies facilitates nutritional countermeasures against pasture laminitis. J Nutr 2006;136: 2090S–2093S. 10. Nadeau JA, Frank N, Valipe SR et al. Blood lipid, glucose, and insulin concentrations in Morgan horses and Thoroughbreds. J Equine Vet Sci 2006;26:401–405. 11. Alberti KGMM, Zimmet P, Shaw J. Metabolic syndrome: a new world-wide definition. A consensus statement from the International Diabetes Federation. Diabet Med 2006;23:469–480. 12. Geor RJ. Metabolic predispositions to laminitis in horses and ponies: obesity, insulin resistance and metabolic syndromes. J Equine Vet Sci 2008;28:753–759. 13. Frank N, Elliott SB, Brandt LE et al. Physical characteristics, blood hormone concentrations, and plasma lipid concentrations in obese horses with insulin resistance. J Am Vet Med Assoc 2006;228:1383–1390. 14. Pratt-Phillips SE, Owens KM, Dowler LE et al. Assessment of resting insulin and leptin concentrations and their association with managerial and innate factors in horses. J Equine Vet Sci 2010;30:127–133. 15. Labruna G, Pasanisi F, Nardelli C et al. High leptin/adiponectin ratio and serum triglycerides are associated with an at-risk phenotype in young severely obese patients. Obesity 2011;19:1492–1496. 16. Primeau V, Coderre L, Karelis AD et al. Characterizing the profile of obese patients who are metabolically healthy. Int J Obes 2011;35:971–981. 17. Carroll CL, Huntington PJ. Body condition scoring and weight estimation of horses. Equine Vet J 1988;20:41–45. 18. McGowan T, Geor R, Evans H. Comparison of 4 assays for serum insulin analysis in the horse (Abstract). J Vet Intern Med 2008; 22:692. 19. Copas VEN, Durham AE. Circannual variation in plasma adrenocorticotropic hormone concentrations in the UK in normal horses and ponies, and those with pituitary pars intermedia dysfunction. Equine Vet J 2012; 44:440–443 20. Kearns CF, McKeever KH, Roegner V et al. Adiponectin and leptin are related to fat mass in horses. Vet J 2006;172:460–465. 21. Hosmer DW, Lemeshow S. Applied logistic regression. Wiley, New York. 1989. 22. Carter RA, Geor RJ, Burton Staniar W et al. Apparent adiposity assessed by standardised scoring systems and morphometric measurements in horses and ponies. Vet J 2009;179:204–210. 23. Muno J, Gallatin L, Geor RJ. Prevalence and risk factors for hyperinsulinemia in clinically normal horses in central Ohio (Abstract). J Vet Intern Med 2009;23:721.
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24. Kronfeld DS, Treiber KH, Geor RJ. Comparison of nonspecific indications and quantitative methods for the assessment of insulin resistance in horses and ponies. J Am Vet Med Assoc 2005;226:712–719. 25. George LA, Staniar WB, Cubitt TA et al. Evaluation of the effects of pregnancy on insulin sensitivity, insulin secretion, and glucose dynamics in Thoroughbred mares. Am J Vet Res 2011;72:666–674. 26. Aguilera CM, Gil-Campos M, Cañete R et al. Alterations in plasma and tissue lipids associated with obesity and metabolic syndrome. Clin Sci 2008;114:183– 193. 27. Caltabilota TJ, Earl LR, Thompson DL Jr et al. Hyperleptinemia in mares and geldings: assessment of insulin sensitivity from glucose responses to insulin injection. J Anim Sci 2010;88:2940–2949. 28. Carter RA, McCutcheon LJ, George LA et al. Effects of diet-induced weight gain on insulin sensitivity and plasma hormone and lipid concentrations in horses. Am J Vet Res 2009;70:1250–1258. 29. Bruce KD, Cagampang FR. Epigenetic priming of the metabolic syndrome. Toxicol Mech Methods 2011;21:353–361. 30. Storer WA, Thompson DL Jr, Waller CA et al. Hormonal patterns in normal and hyperleptinemic mares in response to three common feeding-housing regimens. J Anim Sci 2007;85:2873–2881. 31. Alford P, Geller S, Richardson B et al. A multicenter, matched case-control study of risk factors for equine laminitis. Prev Vet Med 2001;49:209–222. 32. Gupte AA, Bomhoff GL, Geiger PC. Age-related differences in skeletal muscle insulin signaling: the role of stress kinases and heat shock proteins. J Appl Physiol 2008;105:839–848. 33. Hoffman RM, Boston RC, Stefanovski D et al. Obesity and diet affect glucose dynamics and insulin sensitivity in Thoroughbred geldings. J Anim Sci 2003;81: 2333–2342 34. Van de Woestijne AP, Monajemi H, Kalkhoven E et al. Adipose tissue dysfunction and hypertriglyceridemia: mechanisms and management. Obesity Rev 2011;12:2333–2342. 35. Vick MM, Adams AA, Murphy BA et al. Relationships among inflammatory cytokines, obesity, and insulin sensitivity in the horse. J Anim Sci 2007;85:1144– 1155. 36. Dugdale AHA, Curtis GC, Harris PA et al. Assessment of body fat in the pony. Part I: relationships between the anatomical distribution of adipose tissue, body composition and body condition. Equine Vet J 2011; 43:552–561 37. Blüher M. The distinction of metabolically ‘healthy’ from ‘unhealthy’ obese individuals. Curr Opin Lipidol 2010;21:38–43. 38. Hampson BA, De laat MA, Mills PC et al. Distances travelled by feral horses in ‘outback’ australia. Equine Vet J 2010;42:582–586. 39. Powell DM, Reedy SE, Sessions DR et al. Effect of short-term exercise training on insulin sensitivity in obese and lean mares. Equine Vet J Suppl 2002;34:81–84. 40. Monda KL, North KE, Hunt SC et al. The genetics of obesity and the metabolic syndrome. Endocr Metab Immune Dis Drug Targets 2010;10:86–108. 41. Choquet H, Meyre D. Genetics of obesity: what have we learned? Curr Genomics 2011;12:169–179. 42. US Department of Agriculture. Lameness and laminitis in US horses. Publication no. N318.0400. USDA National Animal Health Monitoring System, Fort Collins, CO, 2000. 43. Walsh DM, McGowan CM, McGowan T et al. Correlation of plasma insulin concentration with laminitis score in a field study of equine Cushing’s disease and equine metabolic syndrome. J Equine Vet Sci 2009;29:87–94. 44. McGowan CM, Frost R, Pfeiffer DU, Neiger R. Serum insulin concentrations in horses with equine Cushing’s syndrome: response to a cortisol inhibitor and prognostic value. Equine Vet J 2004;36:295–298. (Accepted for publication 25 October 2013)
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Prevalence and risk factors for hyperinsulinaemia in ponies in Queensland, Australia RA Morgan,a TW McGowanb and CM McGowana,c*
Objectives To determine the prevalence of hyperinsulinaemia in a population of ponies in Queensland, Australia, and identify associated factors. Methods Breeders or traders of ponies within a 100 km radius of Gatton, Queensland, were recruited for study using an internet database. Clinical and management details were obtained, including body condition score, fat deposition and history or evidence of laminitis. Blood samples were analysed for serum insulin and triglyceride concentrations and plasma adrenocorticotrophic hormone (ACTH) and leptin concentrations following short-term removal from pasture and withholding of supplementary food for at least 12 h. Results Of 23 pony studs identified, 22 were available for visit. The study population consisted of 208 ponies: 70 Australian Ponies; 67 Welsh Mountain Ponies or Cobs; 51 Connemara Ponies; 20 Shetland ponies. We excluded 20 with suspected pituitary pars intermedia dysfunction (>15 years, ACTH >50 pg/mL). In total, 27% of the ponies (51/188) were hyperinsulinaemic (insulin >20 μIU/mL). The final multivariable model revealed increasing age, supplementary feeding and increased leptin and triglyceride concentrations to be associated with hyperinsulinaemia. Conclusions Hyperinsulinaemia was prevalent and associated with age and evidence of metabolic disturbance, including elevated leptin and triglyceride concentrations, in this population. A significant number of ponies were at risk of hyperinsulinaemia, which has implications for strategies to reduce the risk of laminitis in this population. Keywords
endocrinopathy; insulin; laminitis; ponies
Abbreviations ACTH, adrenocorticotrophic hormone; BCS, body condition score; CI, confidence interval; EMS, equine metabolic syndrome; IR, insulin resistance; OR, odds ratio; PPID, pituitary pars intermedia dysfunction Aust Vet J 2014;92:101–106
doi: 10.1111/avj.12159
H
orses with equine metabolic syndrome (EMS) have been defined as those with obesity, insulin resistance (IR) or hyperinsulinaemia and a predisposition to laminitis.1 The syndrome has emerged during the past decade as a disease of considerable importance because of the close association between EMS and *Corresponding author. a Philip Leverhulme Equine Hospital, University of Liverpool, Leahurst Campus, Neston, CH64 7TE, United Kingdom; [email protected] b Acorn Veterinary Clinic, Willows Veterinary Group, West Kirby, UK c Institute of Aging and Chronic Disease, University of Liverpool, Neston, UK
© 2014 Australian Veterinary Association
management, in particular over-nutrition and obesity, and because the resulting clinical disease, laminitis, is such a painful and crippling disorder.1 The high prevalence of obesity in the general horse population is of increasing concern, with one study identifying 45% of horses studied as fat or very fat.2 Another predisposing factor appears to be breed, with the majority of published cases or research herds used for the study of EMS consisting of British pony breeds or their crosses.3,4 British breed ponies in Finland were overrepresented in cases of endocrinopathic laminitis compared with a hospital population.5 Part of the breed effect may be that native British and Irish ponies have evolved to be ‘thrifty’, surviving periodic under-nutrition, for which relative IR is an advantage.6 In the Finnish study, the more common native breeds (Nordic cold bloods, Norwegian Fjords and Icelandic horses) were not as frequently represented with endocrinopathic laminitis, even though these breeds are also ‘thrifty’ and prone to obesity.5 Ponies derived from native British or Irish breeds can be considered as at-risk and therefore were specifically recruited for the present study. Insulin resistance and hyperinsulinaemia are defining factors for EMS and have been closely linked with the development or predisposition to laminitis in both field3,7 and experimental8 studies. In the horse, it is typically compensated IR, with hyperinsulinaemia compensating for the reduction in insulin function.9 Hyperinsulinaemia has been diagnosed in 86% of primary cases of laminitis.5 Research in equine and human populations has shown that hyperinsulinaemia is a good predictor of underlying metabolic disease.10,11 Basal hyperinsulinaemia is a good indicator of IR and has been advocated for use in epidemiological studies,1,12 yet there is little information on the prevalence of hyperinsulinaemia in the general horse or pony population.1,13 The prevalence of hyperinsulinaemia has not been determined in epidemiological studies and has been restricted to studies of single herds.3,7 It should be noted that IR in the horse does not always manifest as hyperinsulinaemia and in some cases dynamic testing with a combined glucose–insulin tolerance test is required to detect IR. In this study, we defined hyperinsulinaemia as insulin concentration greater than 20 μIU/mL, as suggested by American College of Veterinary Internal Medicine consensus.1 Several factors, including body condition score (BCS), regional adiposity, hyperleptinaemia and elevated triglyceride concentrations, have been associated with hyperinsulinaemia and metabolic disease in selected groups of horses or ponies,1,3,13,14 but the prevalence of such metabolic disturbances in the general populations is currently unknown. Most of the studies have, in fact, been carried out in single, closed herds3,7 In people, insulin, leptin and triglyceride concentrations and obesity are closely linked.15 Leptin and triglyceride concentrations have been used to differentiate metabolically healthy
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EQUINE from metabolically unhealthy obese people.16 Identification of animals that are susceptible to laminitis because of metabolic disturbances is critical if the correct interventions are to be applied.9 In order to determine this, epidemiological studies on wider subpopulations of horses are necessary. Our hypothesis was that evidence of metabolic abnormality would be prevalent in pony breeds under normal management conditions. We therefore aimed to determine the prevalence of hyperinsulinaemia in the general pony population of Queensland, Australia, and to identify factors that may be associated with hyperinsulinaemia in this population. Materials and methods The study was approved by the University of Queensland Animal Ethics Committee. Listed pony studs and traders within 100 km of Gatton, south-east Queensland were identified using an internet listing (http:// australianhorsedirectory.com.au/). Each stud identified was contacted via telephone to request permission to visit. During the initial telephone conversation, the location and number of ponies were ascertained and a standard set of open-ended questions was asked concerning the owners’ management of the stud, their knowledge of pasture-associated laminitis and perceived risk or protective factors. The premises were visited during a 4-week period in February–March 2007. Data on the individual ponies, including age, sex, use, history of laminitis, current laminitis and any supplementary food given, were recorded. Each pony underwent a clinical examination and the following information was collected: height, BCS,17 presence of a cresty neck, the presence of bulging supraorbital fat pads and the presence of divergent rings on the hooves. A blood sample was collected following informed consent from the owner. Pastured animals were removed from pasture for 1–3 h prior to blood collection as per the protocol of previous studies.3,7 For housed ponies, managers were instructed that the morning feed be withheld prior to the visit. All visits took place in the morning. Blood samples were obtained by venipuncture of the left jugular vein and placed in 10-mL vacutainer tubes (Vacutainer systems, Becton-Dickinson, NJ, USA) containing either EDTA or no anticoagulant. The samples were immediately placed on ice. Plasma and serum was separated within 3 h by centrifugation and stored on ice. Samples were frozen at −80°C within 12 h of collection and remained so until analysis. Serum samples were analysed for insulin concentration using a radioimmunoassay (Diagnostic Systems Laboratories Inc., TX, USA) previously validated for use in horses.18 Plasma adrenocorticotrophic hormone (ACTH) concentrations were determined using a commercially available assay (Immunite 1000, Vetpath Laboratory Services, Ascot, WA, Australia) previously validated for use in horses.19 Serum triglyceride concentrations were analysed by commercial biochemistry analyser (Olympus AU400 Automated Chemistry Analyser, Olympus American Inc., Diagnostic Systems Division, USA) and plasma leptin concentrations were measured using a radioimmunoassay (Linco Research, St Louis, MO, USA) validated for use in horses.20
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Ponies were excluded from this study if there was a suspicion of pituitary pars intermedia dysfunction (PPID), defined as ponies that were 15 years or older with an ACTH measurement >50 pg/mL.19 Hyperinsulinaemia was defined as serum insulin concentration >20 μU/mL.1 Statistical analysis Prestudy sample size calculations were performed; assuming a pony population size of 100,000 and a frequency of hyperinsulinaemia of 10–20% (Minitab® Statistical Software 16, State College, PA, USA), a sample of 200 ponies gave a power of 99.9% at the level of 95% confidence. Descriptive statistics were performed using Mintab 16. The variables were also tested for correlations using the Pearson’s rank correlation. The data that were not normally distributed (Kolmogorov-Smirnov test) were log-transformed prior to testing for correlations and transformation was shown to improve the normality of the data. Explanatory variables included in the logistic regression were age, sex, breed, use, BCS, cresty neck, increased supraorbital fat, presence of laminitic rings, owner-reported history of laminitis, supplementary feeding and plasma leptin and serum triglyceride concentrations. All variables associated with the outcome with a change in deviance (log-likelihood ratio) at P < 0.25 were offered forward to a forward multivariable logistic regression. The results were then verified using a backward stepwise regression model. The overall fit of the model was ascertained using the Hosmer–Lemeshow goodness-of-fit test.21 the criterion for inclusion was a change in deviance at P < 0.05. Results are presented as mean ± SD where applicable. Results We identified 26 studs or traders from internet listings and published records. Of those, 23 were able to be contacted and 22 available to take part in the study (response rate, 96%). The 208 ponies on these premises were assessed for inclusion in the study; 20 were excluded following a suspicion of PPID, leaving a final study population of 188 ponies. The study population consisted of Australian Ponies (59, 31%), Welsh Mountain Ponies (60, 32%), Connemara Ponies (51, 27%) and Shetland Ponies (18, 10%). There were 143 mares (76%), 19 geldings (10%) and 26 stallions (14%). The mean age was 9 years (range 1–26 years). Their uses included breeding (99, 53%; 26 stallions and 73 mares), riding or showing (23, 12%) and dry or young stock not in work (66, 35%). The majority of the ponies (152/188, 81%) were kept entirely at pasture and the rest also received supplementary feeding (35/188, 19%). Supplementary feeds were non-grain based and typically high-fat based on coconut, soyabean meal or rice bran. All 22 owners in the study identified laminitis as an important issue in pony breeds and all reported numerous risk factors for its development as well as management strategies to avoid it. The most common owner-reported risk factors were the ponies being overweight (n = 12), too much feeding of grain (n = 9), lush grass, particularly after a period of drought (n = 10) and feeding grass with seed heads (n = 4). Other factors reported included stress, genetics and too much protein. Management practices implemented in order to prevent laminitis
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included monitoring weight (n = 8), avoiding lush pastures or grass with seed heads (n = 6), avoiding overfeeding of grain (n = 3) and keeping mares in foal (n = 6). The mean BCS (out of 5)17 of the whole population was 3.97 ± 0.57 (median 4, interquartile range 3.5–4.5). Clinical examination revealed a cresty neck score >322 in 113 (60%), bulging supraorbital fat in 82 (43.6%) and laminitic rings in 42 (22.3%); 23 (12.2%) of the ponies had a history of laminitis and all of them had laminitic rings on clinical examination, although not all ponies with clinical evidence of laminitic rings had an owner-reported history of laminitis. Only two ponies had current laminitis during sampling and both were hyperinsulinaemic (110 and 373 μU/mL, respectively). The mean serum insulin concentration was 25.4 μU/mL (± 55.2, median 10.9, range 4–520) (Figure 1). The mean triglyceride concentration was 0.55 ± 1.2 mmol/L (median 0.4, range 0.0–15.8) and mean plasma leptin concentration was 5.4 ± 4.2 ng/mL (median 4.3, range 1.1–24.9). In total, 52 (27.7%, 95% confidence interval (CI) 21.4–34.6) ponies were hyperinsulinaemic (insulin concentration > 20μU/mL). The mean BCS (out of 5) did not differ between ponies with or without hyperinsulinaemia. Data for insulin, leptin and triglyceride concentrations were not normally distributed, so they were log-transformed prior to investigation of correlations. There was a moderate correlation between log serum insulin and log plasma leptin (r = 0.41; P < 0.001) (Figure 2). There was a mild correlation between log serum insulin concentration and log serum triglyceride concentration (r = 0.37; P < 0.001) (Figure 3) and between log plasma leptin concentration and log serum triglyceride concentration (r = 0.35, P < 0.001). There was a weak correlation between age and log serum insulin (r = 0.24, P = 0.001). No other correlations were identified. Several variables were found to be significant on univariable analysis (Tables 1, 2) and these were offered forward into a multivariable model. The final model showed that increasing age (odds ratio (OR) 1.14, 95% CI 1.06–1.22, P < 0.001), supplementary feeding (OR 4.89, 95% CI 1.96–12.17, P = 0.001), increasing triglyceride concentration (OR 4.19, 95% CI 1.40–12.50, P = 0.01) and increasing plasma leptin
© 2014 Australian Veterinary Association
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Figure 2. Relationship between log insulin concentration and log leptin concentration (r = 0.41; P < 0.001; n = 188).
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Figure 1. Distribution of serum insulin concentrations across the study population (n = 188).
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Table 1. Univariable analysis of continuous variables put forward into the multivariable model for the presence of hyperinsulinaemia in 188 Australian ponies
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0.004 0.001 1000 acres), so a possible explanation is that they were naturally exercising more than the other ponies in the study. It has been shown that wild horses will walk up to 15 km/day, even when forage and water are available.38 Exercise has been shown to reduce IR and this possibility requires further investigation.39 In humans, familial and genetic links to obesity and metabolic syndrome have been identified, although these are often complicated by factors such as lifestyle and epigenetic factors.40,41 A familial predisposition to EMS has been postulated,3 but a genetic link has not yet been determined in horses.
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The 12% of ponies with a history of laminitis in this study is consistent with previous reports,42 although lower than in the single pony herds used in other studies.3,7 A history of laminitis was significantly associated with hyperinsulinaemia at the univariable level, but was not retained in the final model. It is possible that the ponies with a history of laminitis were more carefully managed than those without and thus it was not identified as a risk factor for current hyperinsulinaemia in the final model. Significant hyperinsulinaemia (>70 μU/mL) has been associated with an increased Obel grade laminitis score.43 Insulin concentration has been shown to be a good prognostic indicator in horses with PPID such that animals with hyperinsulinaemia are more likely to develop laminitis and survive for less than 2 years after diagnosis.44 The creation of a hyperinsulinaemic state has been shown to induce laminitis in experimental animals.8 The exact mechanism by which hyperinsulinaemia results in laminitis is yet to be elucidated, but the results of this study demonstrate the high prevalence of hyperinsulinaemia in the general pony population. Conclusion Hyperinsulinaemia was prevalent in this sample of the general pony population of Queensland, despite drought conditions at the time of the study. Hyperinsulinaemia was associated with increasing age, supplementary feeding and indicators of metabolic dysfunction; that is, increased leptin and triglyceride concentrations. The findings of this study help to characterise the metabolic abnormalities associated with IR in pony populations in which many individuals may be at increased risk of laminitis. Hyperinsulinaemia associated with hyperleptinaemia and hypertriglyceridaemia may represent a state of metabolic disturbance that indicates IR and adipose dysfunction. Acknowledgments The authors are grateful to the University of Queensland for their funding of this project, the Horse Trust who kindly sponsored the residency of RA Morgan and all the owners and breeders included in the study.
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24. Kronfeld DS, Treiber KH, Geor RJ. Comparison of nonspecific indications and quantitative methods for the assessment of insulin resistance in horses and ponies. J Am Vet Med Assoc 2005;226:712–719. 25. George LA, Staniar WB, Cubitt TA et al. Evaluation of the effects of pregnancy on insulin sensitivity, insulin secretion, and glucose dynamics in Thoroughbred mares. Am J Vet Res 2011;72:666–674. 26. Aguilera CM, Gil-Campos M, Cañete R et al. Alterations in plasma and tissue lipids associated with obesity and metabolic syndrome. Clin Sci 2008;114:183– 193. 27. Caltabilota TJ, Earl LR, Thompson DL Jr et al. Hyperleptinemia in mares and geldings: assessment of insulin sensitivity from glucose responses to insulin injection. J Anim Sci 2010;88:2940–2949. 28. Carter RA, McCutcheon LJ, George LA et al. Effects of diet-induced weight gain on insulin sensitivity and plasma hormone and lipid concentrations in horses. Am J Vet Res 2009;70:1250–1258. 29. Bruce KD, Cagampang FR. Epigenetic priming of the metabolic syndrome. Toxicol Mech Methods 2011;21:353–361. 30. Storer WA, Thompson DL Jr, Waller CA et al. Hormonal patterns in normal and hyperleptinemic mares in response to three common feeding-housing regimens. J Anim Sci 2007;85:2873–2881. 31. Alford P, Geller S, Richardson B et al. A multicenter, matched case-control study of risk factors for equine laminitis. Prev Vet Med 2001;49:209–222. 32. Gupte AA, Bomhoff GL, Geiger PC. Age-related differences in skeletal muscle insulin signaling: the role of stress kinases and heat shock proteins. J Appl Physiol 2008;105:839–848. 33. Hoffman RM, Boston RC, Stefanovski D et al. Obesity and diet affect glucose dynamics and insulin sensitivity in Thoroughbred geldings. J Anim Sci 2003;81: 2333–2342 34. Van de Woestijne AP, Monajemi H, Kalkhoven E et al. Adipose tissue dysfunction and hypertriglyceridemia: mechanisms and management. Obesity Rev 2011;12:2333–2342. 35. Vick MM, Adams AA, Murphy BA et al. Relationships among inflammatory cytokines, obesity, and insulin sensitivity in the horse. J Anim Sci 2007;85:1144– 1155. 36. Dugdale AHA, Curtis GC, Harris PA et al. Assessment of body fat in the pony. Part I: relationships between the anatomical distribution of adipose tissue, body composition and body condition. Equine Vet J 2011; 43:552–561 37. Blüher M. The distinction of metabolically ‘healthy’ from ‘unhealthy’ obese individuals. Curr Opin Lipidol 2010;21:38–43. 38. Hampson BA, De laat MA, Mills PC et al. Distances travelled by feral horses in ‘outback’ australia. Equine Vet J 2010;42:582–586. 39. Powell DM, Reedy SE, Sessions DR et al. Effect of short-term exercise training on insulin sensitivity in obese and lean mares. Equine Vet J Suppl 2002;34:81–84. 40. Monda KL, North KE, Hunt SC et al. The genetics of obesity and the metabolic syndrome. Endocr Metab Immune Dis Drug Targets 2010;10:86–108. 41. Choquet H, Meyre D. Genetics of obesity: what have we learned? Curr Genomics 2011;12:169–179. 42. US Department of Agriculture. Lameness and laminitis in US horses. Publication no. N318.0400. USDA National Animal Health Monitoring System, Fort Collins, CO, 2000. 43. Walsh DM, McGowan CM, McGowan T et al. Correlation of plasma insulin concentration with laminitis score in a field study of equine Cushing’s disease and equine metabolic syndrome. J Equine Vet Sci 2009;29:87–94. 44. McGowan CM, Frost R, Pfeiffer DU, Neiger R. Serum insulin concentrations in horses with equine Cushing’s syndrome: response to a cortisol inhibitor and prognostic value. Equine Vet J 2004;36:295–298. (Accepted for publication 25 October 2013)
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