Morphometric, metabolic, and inflammatory markers across a cohort of client-owned horses and ponies on the insulin dysregulation spectrum
Introduction
The pathophysiology of equine metabolic syndrome (EMS) entails an impaired insulin and glucose metabolism [1], commonly defined as insulin dysregulation (ID). This central feature of EMS can present as hyperinsulinemia, insulin resistance, and/or abnormal insulin response to meals [2]. In laboratory mammals and humans, a hyperglycemic state has been associated with elevations of the highly reactive dicarbonyl methylglyoxal (MG) and of metabolites of the glyoxalase system, which catalyzes the conversion of MG to D-lactate to limit formation of advanced glycation end-products [3]. Both were elevated in rodent models of spontaneous hypertension [4], metabolic syndrome (MetS) [5], obesity, and type II diabetes mellitus (T2DM) [6]. Human studies have confirmed the role of MG in obesity [6,7], diabetic ketoacidosis [8], and diabetes-related conditions [6,[9], [10], [11]]. Circulating D-lactate is increased in ketoacidotic and non-ketoacidotic cats with T2DM [12]. While by definition horses with EMS are not persistently hyperglycemic, even intermittent mild to moderate elevations in glycemia (e.g. in the post-prandial state) [13,14] or other unexplored mechanisms may affect MG and D-lactate concentrations, which to the authors’ knowledge have not been evaluated in EMS. Selected comparative aspects of human and equine pathophysiology of metabolic syndrome have been recently reviewed in further detail by the authors [15].
Research in humans has shown a link between metabolism, obesity, and inflammation, defined “metaflammation” [16]. Pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6, have been implicated in impaired glucose uptake in people who are obese [16] and in horses [17]. In horses with natural or induced ID, cytokines were increased in plasma [18] and adipose tissue [19]. In particular, TNF-α was more consistently elevated in EMS horses compared to healthy controls [20], [21], [22], [23], [24], although in other studies there was no difference [25], [26], [27] or it was even decreased [28]. Interleukin-6 has also been found increased in horses with ID [23,27], although its mRNA expression was downregulated in EMS horses in other studies [20,25] and recently found not to be significantly different [26,28]. Monocyte chemoattractant protein-1 (MCP-1) mediates inflammation through macrophage recruitment and is significantly increased in dysfunctional adipose tissue of obese rodents and humans [29]; a recent study has demonstrated its increased expression in peri-renal and visceral adipose tissue of horses with advanced EMS [24]. Due to conflicting findings, the extent of inflammation and whether it is an underlying cause or consequence of obesity and ID in horses are still subject of debate. This is complicated by suspected metabolic differences between generalized and regional adiposity in horses [30], which have not been completely clarified although research is ongoing in determining the role of adipose stores in different anatomical locations [24,31].
The objective of the present study was to determine if ID in horses could be predicted by one or a combination of morphometric [body condition score (BCS), cresty neck score (CNS)], metabolic (MG, D-lactate, L-lactate, triglycerides, glucose, insulin), or inflammatory (TNF-α, IL-6, MCP-1) markers. The primary hypothesis was that blood D-lactate and MG concentrations of horses with ID would differ from those of healthy controls. Secondly, it was hypothesized that CNS and serum inflammatory markers TNFα, IL-6, MCP-1 would differ between horses with ID and healthy controls.
Section snippets
Animals
Clients voluntarily enrolled horses and ponies in May to mid-September, with 81% of samples collected in the summer months. Volunteers were recruited through referral or advertisement through social media, fliers, and word-of-mouth (Table 1) shows the breeds represented. Both normal and over-conditioned horses were included. Owners preferably transported horses to the veterinary teaching hospital (n = 17), or the research team travelled to the farm (n = 15 horses, 4 farms) when multiple horses
Results
37 horses were initially evaluated. 2 were excluded due to abnormal resting ACTH (PPID with concomitant ID), and 3 due to missing data. Of the 32 horses that met the inclusion criteria, mean age (years ± SD) was 13 ±4 (range 5-24). Individual breeds had a small sample size and were not included in statistical analysis (Table 1). 12 horses (38%; 95% CI: 21 – 56%) were classified as ID and 20 (62%; 95% CI: 44 – 79%) as IS by the CGIT. Of the 12 ID horses, 3 (25%) were detected by a glucose
Discussion
D-lactate and the other biomarkers tested were not good predictors of ID in this population of horses. Differences in equine vs. human or rodent metabolism, inconsistent hyperglycemia in horses with EMS, and the effects of sample storage and handling may explain why D-lactate was not different between groups [38]. Immediate analysis of MG and D-lactate through a different method, such as high-performance liquid chromatography, may have been more accurate [11]. Additionally, due to its toxicity,
Limitations
This study had several limitations. By testing client-owned horses, different management practices could affect the presence of ID. Identification of risk factors was not an objective of this study, however, and testing location was not statistically significantly associated with the presence of ID. To reduce nutritional influence on the rest results, all horses in the study were managed identically by only receiving one flake of hay after 10pm the night before sampling and no concentrates.
Conclusions
In this population of horses, no significant evidence of an increase in D-lactate or MG was shown that could justify their use as early biomarkers for ID. Moreover, inflammation is a dynamic mechanism, and a 1-time test of circulating biomarker levels may not be sufficient to detect low-grade metaflammation in EMS. A strength of this study was examining a cohort of client-owned horses across a variety of obesity and insulin dysregulation stages, as opposed to a research herd of severely insulin
Acknowledgment
The authors gratefully acknowledge Deborah Michel for the invaluable technical knowledge of laboratory assays, Dr. Sarah Parker from the WCVM's Centre for Applied Epidemiology for help with the statistical analysis, the students Harry Hayes and Ekaterina Charles-Dudko, and all the owners who enrolled their horses in this study.
Author statement
V. Ragno, F. Uehlinger, G. Zello, K. Robinson and J. Montgomery contributed to study conception and design. V. Ragno, C. Klein and N. Sereda performed acquisition of data and laboratory analysis. All authors were involved in data analysis, writing and or/revision, and final approval of the manuscript.
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Ethical statement: All experimental procedures were approved by the University of Saskatchewan's Animal Care and Use Committee, Animal Research Ethics Board and followed the Canadian Council on Animal Care (CCAC) guidelines. Owner consent was obtained upon enrolment of horses in the study.