From Table to Stable: A Comparative Review of Selected Aspects of Human and Equine Metabolic Syndrome

Journal of Equine Veterinary Science 79 (2019) 131e138

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Review Article

From Table to Stable: A Comparative Review of Selected Aspects of Human and Equine Metabolic Syndrome Valentina M. Ragno a, *, Gordon A. Zello b, Colby D. Klein a, Julia B. Montgomery a a b

Department of Large Animal Clinical Sciences, WCVM, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada Section of Nutrition, College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon SK S7N 5E5, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 March 2019 Received in revised form 6 June 2019 Accepted 8 June 2019 Available online 20 June 2019

Obesity data in people and companion animals are depicting a future of increasing morbidity, cost for society, and significant health and welfare concerns. Between 25 and 50% of cats, dogs, and horses in developed countries are overweight or obese, which mirrors the situation in humans. Equine metabolic syndrome (EMS) was named after human metabolic syndrome (MetS), which has about 30 years of lead in research efforts. Even though the complications of the two syndromes seem to grossly differ (cardiac vs. laminitis risk), a number of similar disease mechanisms are worthy of investigation. Since the first EMS consensus statement by the American College of Veterinary Internal Medicine in 2010, numerous studies have confirmed the link between insulin dysregulation and laminitis, even though the mechanisms are not fully understood. After the discovery of the role of adipokines in MetS, evidence about inflammatory mechanisms related to adiposity in rodent models, companion animals, horses, and humans is constantly increasing. Oxidative and dicarbonyl stress have been correlated with insulin dysregulation, obesity, and recently with laminitis. Vascular actions of insulin through nitric oxide, endothelin-1, and other mechanisms are being studied in horses and can provide a better understanding of laminitis pathophysiology. More research is needed on neuropathic mechanisms in insulindysregulated horses, which could be important in the pathogenesis of laminitis and laminitic pain. Human literature can provide viable material for novel studies in areas that have received limited attention, in addition to being valuable information for clients about the consequences of unhealthy management of their horses. © 2019 Elsevier Inc. All rights reserved.

Keywords: Equine metabolic syndrome Insulin dysregulation Obesity Metaflammation Endocrinopathic laminitis

1. Introduction In humans, the concept of metabolic syndrome (MetS) was first introduced in the 1970s, and described as a combination of obesity, abnormal glucose regulation, dyslipidemia, and hypertension [1]. The modern definition evolved over the years and presents slight variations based on statements from different medical associations (World Health Organization [2], European Group for the Study of Insulin Resistance [3], American College of Endocrinology [4],

Animal welfare/ethical statement: The authors declare that no animals or client data were used in the preparation of this review manuscript, therefore no ethical approval was needed. Conflict of interest statement: The authors declare no conflicts of interest. * Corresponding author at: Valentina M. Ragno, Department of Large Animal Clinical Sciences, WCVM, University of Saskatchewan, 52 Campus Drive, Saskatoon, SK S7N 5B4, Canada. E-mail address: [email protected] (V.M. Ragno). https://doi.org/10.1016/j.jevs.2019.06.003 0737-0806/© 2019 Elsevier Inc. All rights reserved.

American Diabetes Association [5], International Diabetes Federation [6], etc.). Based on these definitions, MetS describes a cluster of risk factors that predispose patients to type II diabetes mellitus (T2DM) and cardiovascular disease (CVD): obesity, insulin resistance (IR), hypertension, and dyslipidemia. The prevalence of MetS is continuously increasing worldwide, affecting on average around 20%e25% of the population, with socioeconomic, ethnic, and age variations [7]. Obesity appears to be the earliest sign in most cases, and its prevalence is ever rising [8]. The World Health Organization reported that in 2016, among the adult worldwide population, 39% were overweight (i.e., body mass index [BMI]  25 kg/m2), with a third (13%) being obese (BMI  30 kg/m2) [1]. In parallel with the obesity epidemic in people, companion animals have been experiencing a similar escalation over the past few decades. A multicenter retrospective study published in 2006 showed that, based on data from 1995, 29% of dogs were overweight and 5.1% were obese in the United States [9]. From the same data set, 35% of adult cats were scored as overweight or obese [10],

8.3% (24) 28.6% (83) na 290 Veterinarian Abbreviations: BCS, body condition score; na, data not available.

Multiple Henneke Canada (SK) 2017 CVJ Kosolofski et al [21]

2012e2013 2016 Aus Vet J Potter et al [20]

na Act Vet Sc 2016 Jensen et al [19]

2014

23.1% (53) 37.1% (85) 3e33

4e26

24% (61)

10.2% (26)

Scale 1-6 Questionnaire Overweight 5 Overweight BCS 7 Obese BCS 8e9 Overweight BCS 6 Owners underestimating; Obese BCS 7 Ponies obesity 3 Overweight BCS 7 Obese BCS 8-9 1e35

Multiple modified Carroll Owner 792 and Huntington Denmark Icelandic Henneke Experienced person 254 horse Australia (VIC) Multiple Henneke Researcher 229 UK 2015 EVJ Robin et al [18]

2009e2011

Multiple Henneke UK 2014 Peer J Giles et al [17]

2011

31.2% (247)

27.1% (26) winter 35.4% (34) summer na na 1e28 96

18.7% (56) 51% (153) 4e20 300

Two independent scorers Veterinarian Multiple Henneke 2006 2012

USA (VA)

na 20.6% (33) na 160 Owner Multiple Anon 2008 2011

UK

20% (73) 48% (176) 3e36 366 Trained personnel Multiple Henneke 2007 2010

Pratt-Phillips JEVS et al [14] Stephenson Vet Rec et al [15] Thatcher et al [16] JVIM

USA (NC)

10% (32) 45% (144) 4e25 319 Veterinarian Multiple Webb and Weaver UK (Scotland) Vet Rec Wyse et al [13]

2005 2008

Journal

Insulin dysregulation and its diagnosis in the horse have been reviewed in recent publications [26,27]. Despite the current lack of a single “gold standard” test, insulin dysregulation is the most consistent and objectively measurable factor among the triad of EMS features (i.e., increased generalized or regional adiposity, IR, predisposition for laminitis). Interestingly, a lean phenotype of horses with insulin dysregulation and laminitis has also been observed, as well as obese horses that do not present insulin dysregulation [28]. In human medicine, this last phenotype is defined “metabolically healthy obese” [29e31]. This subgroup of obese humans feature a nondysfunctional adipose tissue, characterized by higher adiponectin levels [30], lower inflammatory markers, and different fatty acid composition [31], accompanied by the absence of insulin dysregulation and a supposedly more favorable prognosis for cardiovascular disease, cancer and all-cause mortality compared with obese, and insulin-dysregulated subjects [29]. However, over time, if obesity is not addressed, a significant proportion of these individuals develop insulin dysregulation and other features of MetS [32,33]. In horses, hyperinsulinemia is now one of the models used in the induction of laminitis for research purposes [34,35], but the mechanisms of experimental and naturally occurring endocrinopathic laminitis are still not fully understood [36]. Dietary composition has long been recognized as an important factor in the

Table 1 Summary of overweight and obesity prevalence in recent equine studies.

2. Insulin Dysregulation in MetS and EMS

Year Year (Study) Country (Area) Breed (Published)

BCS Method

Evaluator

Horses (n) Age (y) Overweight % Obese % (n) Cutoffs (BCS) (n) Including Obese

Comments

and recent reviews report a feline combined overweight and obesity prevalence of 25%e40% [11,12]. More recent prevalence data for obesity in companion animals are lacking, but anecdotally the practitioners’ perception is that overweight and obesity are now well above those ranges and still increasing. As horses have shifted from transportation and farm work to sport and leisure companions, their life span and tendency to become overweight have both increased. As displayed in Table 1, recent studies show an alarming prevalence of overweight horses when scored by experienced investigators in several areas of the world, ranging from 24% to 51%, with an obesity prevalence of 8.3%e23.1% [13e21]. In all reports in which owners were requested to assess the body condition of their horses, they tended to underestimate it compared with the researcher [13,15,20]. Ponies and cobs tended to be more overweight in several studies [17,18,20]. A certain proportion of over conditioned horses can present a cluster of symptoms, in which insulin dysregulation is preponderant and represents a strong risk factor for laminitis. This group of risk factors was compared with the human metabolic syndrome in 2002 by Johnson, who suggested that it should be called “equine metabolic syndrome” (EMS) [22]. In 2010, the consensus statement of the American College of Veterinary Internal Medicine (ACVIM) [23] defined the most common EMS phenotype as having increased generalized or regional adiposity, IR, and a predisposition for laminitis. Since then, equine endocrinopathies have become the most commonly diagnosed cause of laminitis [24], identifying EMS as a source of major welfare concerns. Consequently, scientists have been investigating the syndrome from many different viewpoints, culminating in the publication of a new consensus statement by the European College of Equine Internal Medicine (ECEIM) [25]. The objective of the present review was to summarize recent advances in the characterization of EMS, with particular focus on the inflammatory, oxidative, cardiovascular, and neuropathic mechanisms and to compare these findings with the current knowledge about MetS. The literature search was performed between September 2017 and March 2019 on PubMed, Google Scholar, and the University of Saskatchewan library database.

Fair owner agreement Scale 1e6 (underestimation) Overweight 5e6 Obese 6 Overweight BCS 6 Obese BCS 8e9 Scale 0e5 Questionnaire (owners Overweight 3 underestimating) Overweight BCS 7 Obese BCS 8e9 Obese BCS7

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Author

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development and management of MetS. In a recent equine study, obesity achieved by a high cereal diet provoked insulin dysregulation. In the group where weight gain was induced by a high fat diet, obesity was not accompanied by alterations in insulin sensitivity [37]. These results are similar to those of a previous study, in which horses on a high sugar diet showed a decrease in insulin sensitivity that was not seen on a high fat diet [38]. In fact, some of the scientific literature in humans suggests that reducing digestible carbohydrate intake and introducing more unsaturated fats in the diet may have some benefit in nutritional therapy for overweight and diabetes in people [35,36]. Certain types of carbohydrates (e.g., simple sugars) in the human diet are known to contribute to IR and overweight, whereas unsaturated fats have been shown to ameliorate metabolic profiles [39,40] and to decrease inflammation levels [41]. Further studies are needed to better understand the effect of dietary composition on insulin metabolism and inflammatory mediators in horses, which could lead to improved dietary management of at-risk and affected equines. 3. Metaflammation The term metaflammation defines a low-grade, chronic inflammatory state originated by an aberrant immune response to excessive availability of nutrients and propagated by the consequent reaction of tissues involved in metabolism, in particular the adipose tissue [42]. 3.1. Inflammatory Cytokines in Obesity More than two decades ago, tumor necrosis factor alpha (TNF-a) was the first inflammatory cytokine found to be increased both systemically and in the adipose tissue of obese mice compared with controls, in correlation with IR [43]. Shortly after, increased gene expression of TNF-a was demonstrated in human fat from obese subjects, by the same research group, again with a positive correlation with hyperinsulinemia [44]. The discovery of leptin dates back to the same period and since then, the synthesis of more than 600 molecules with hormonal and inflammatory actions, collectively defined as adipokines, has been found in human and animal adipose tissue [45,46]. In obesity, adipose tissue can become dysfunctional and alter the secretion of adipokines, with systemic consequences that affect several organs, including the cardiovascular system, liver, pancreas, brain, and the immune system. Activation of adipose tissue resident macrophages, local hypoxia due to tissue hyperplasia and other mechanisms add to obesity-related inflammation and contribute to the decrease in insulin sensitivity of adipose and other tissues [45,47]. In human subjects, abdominal visceral fat seems to predominantly contribute to metaflammation. A similar role has been postulated for the fat deposits along the nuchal ligament in the horse [48], which supports the clinical impression that cresty neck score (CNS) [49] correlates with insulin dysregulation more than body condition score (BCS). Another fat deposition site, the tail head, has been investigated; however, available studies do not support a role of that particular adipose tissue in metaflammation [50]. The utility of adipokines as diagnostic biomarkers and as targets for pharmacologic treatments is an active area of research in humans and more recently also in horses (Table 2) [51e58]. Initial evidence of a link between obesity, IR and increased inflammatory response in horses was provided by Vick et al [51], who demonstrated that TNF-a and IL-1 expression and TNF-a plasma concentration increased in correlation with increased adiposity and decreased insulin sensitivity, whereas IL-6 expression decreased in the same conditions. The discordant findings about IL-6 may be explained by human and murine studies, where IL-6 produced by

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the muscular tissue during exercise showed lipolytic, antiinflammatory, and insulin-sensitizing actions. Conversely, a chronic elevation of adipose-derived IL-6 is proinflammatory and promotes IR [59]. Unfortunately, the two sources cannot be discriminated in vivo, currently limiting the usefulness of IL-6 interpretation in IR studies. As evidenced in Table 2, discordant findings have been published since then, with approximately half of the studies supporting an inflammatory state in EMS [51e53,56] and half not having found a difference between EMS and healthy horses with regard to cytokines such as TNF-a, IL-1b, IL-4, IL-6, IL-8, IL-10, and interferon gamma (IFN-g) [54,55,57,58]. These discordant findings may be due to several differences between studies, such as fed or fasted state of the experimental animals, the inclusion of ponies or only horses, testing circulating cytokines or their expression in tissues. 3.2. Other Markers of Inflammation Another inflammatory marker that is widely utilized in humans, both as a diabetes and as a CVD predictor, is C-reactive protein (CRP) [47], but unfortunately it does not appear to be consistently elevated in inflammatory conditions of horses [60] and it has been defined as a moderate acute-phase protein in this species [61]. In two equine studies, no difference was found in plasma CRP concentrations between obese hyperinsulinemic horses and lean, normoinsulinemic controls [52,54]. Serum amyloid A has been associated with obesity and T2DM in humans [62e64], and it was found to correlate with hyperinsulinemia and obesity in one equine study [55], but not in subsequent research by different groups [57,64]. 3.3. Diet and Inflammation A recent crossover study comparing stevioside to Karo syrup for the oral sugar test (OST) in normal versus EMS horses highlighted an intriguing acute proinflammatory effect of both types of sugar in EMS horses, with an increase of TNF-a and IFN-g expression at 60 minutes after administration, whereas non-EMS horses had an opposite response after the same glycemic challenge [65]. In another study, healthy Thoroughbred mares showed an increase in IL-1b 60 minutes after a meal high (60%) in nonstructural carbohydrates (NSC), compared with mares eating a 10% NSC meal [66]. This warrants further investigation to determine if EMS horses tend to respond with an inflammatory reaction after every carbohydrate meal, and has potential relevance for designing a dietary management plan for these horses. Postprandial inflammation has been the focus of recent human studies, and foods with pro- and antiinflammatory properties have been identified [67]. Some of the proinflammatory foods are the ones rich in simple sugars or saturated fats, whereas anti-inflammatory foods identified are those that contain monounsaturated fatty acids, such as avocado, salmon, and extra virgin olive oil, or antioxidants, such as orange juice and berries [67]. In horses, according to one study, omega-3 supplementation may reduce airway inflammation associated with equine asthma [68]. Recent literature has investigated effects of omega-3 fatty acids on insulin sensitivity, highlighting potential beneficial effects [69], although not confirmed by a later study [70]. Further research is needed to test the supposed anti-inflammatory properties of certain vegetable products consumed by horses to confirm or disprove what feed and supplement companies may already be claiming as potential health benefits. Conflicting evidence about the role of inflammation in EMS emerged from several studies, with some experiments failing to highlight elevations in inflammatory markers with obesity and IR [54,58]. As evidenced in Table 2, these studies are difficult to

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Table 2 Inflammatory markers and adipokines and their relationship with EMS features in equine studies. Study, Subjects (n), ProInflammatory State in EMS Y/N

Statistically Significant (P < .05) Relationships Reported

% fat, BCS

Whole blood mRNA: IL-1, IL-6, TNFa (RT-PCR), serum TNF-a (ELISA)

BCS, CNS

Fibrinogen; plasma TNF-a and CRP (ELISA) TNF-a expression in muscle (SM), SC (nuchal ligament), and VIS adipose tissues (Western Immunoblotting) Plasma TNF-a and CRP (ELISA); leukocyte mRNA: TNF-a, IL-1, IL-6, IL-4, IL-8, IL-10 and IFN-g (qPCR)

ISY as BCS and % fat [ IS Y as serum TNF-a and IL-1 mRNA [ [ BCS and % fat associated with [ TNF-a and IL-1 mRNA, and with Y IL-6 mRNA PL correlated [ BCS and IR plasma TNF-a [ in PL compared with NL IR had [ TNF-a in VIS

Laminitis Predisposition

Adiposity

Vick et al 2007 [51] (60) Y

HEC

na

Treiber et al 2009 [52] (76) Y

RISQI

Waller et al 2012 [53] (12) Y

FSIGTT

History of pastureassociated laminitis na

Holbrook et al 2012 [54] (21) N

Fasting hyperinsulinemia (30 mIU/mL)

na

BCS, BMI, generalized or regional adiposity, neck circumference

Suagee et al 2012 [55] (110) Y/N

Fasting hyperinsulinemia (20 mIU/mL)

na

BCS (Normal: 5e6; Overweight: 7; Obese: 8e9)

circulating TNF-a, IL-6, IL-1b and SAA (ELISA)

Basinska et al 2015 [56] (16) Y

Resting insulin (60 mIU/mL), CGIT, leptin (>7 ng/mL)

History, divergent growth rings, coffin bone rotation

BCS (Control: 7e8; EMS: 8e9), CNS (Control: 1e2; EMS: 3e5)

Serum IL-6, TNF-a, and leptin (ELISA); subcutaneous adipose tissue IL-6 and TNF-a (IHC)

Elzinga et al 2016 [57] (23) N

Fasting hyperinsulinemia (>20 mIU/mL), OST insulin > 60 mIU/mL

History of pastureassociated laminitis

BCS (EMS > 6.5), CNS (EMS > 2.5)

Banse et al 2016 [58] (35) N

Resting hyperinsulinemia (30 mIU/mL)

na

BCS (Ideal: 4e5; Obese: 7e9)

Serum leptin (RIA), triglycerides, cholesterol and NEFA (colorimetric assay); PBMC IFNg and TNFa; mRNA (IFNg, IL-6, IL-10 and TNFa) (RT-PCR) Plasma SAA and TNF-a, muscle TNFa (ELISA); muscle (SM) mRNA TNFa, IL-6 and IL-10

BCS

Neck circumference, BMI and fasting insulin [ in obese group IL-1 and IL-6 mRNA expression Y in hyperinsulinemic and obese group Plasma SAA [ HI and [ BCS TNF-a and IL-6 [ in mares compared with stallions and geldings IL-6 [ with age of all Serum TNF-a, IL-6, and leptin [ in EMS group [ expression of IL-6 in EMS pony adipocytes No differences for any inflammatory markers or mRNA TG and leptin [ in EMS compared with control Plasma TNFa and muscle TNFa Y with [ BCS (independent of HI)

Abbreviations: BCS, body condition score; BMI, body mass index; CNS, cresty neck score; CGIT, combined glucose insulin tolerance test; CRP, C-reactive protein; EMS, equine metabolic syndrome; FSIGTT, frequently sampled insulin glucose tolerance test; HEC, hyperinsulinemic euglycemic clamp; HI, hyperinsulinemia; IFN, interferon; IHC, immunohistochemistry; IR, insulin resistance; IS, insulin sensitivity; NL, never laminitis; OST, oral sugar test; PBMC, peripheral blood mononuclear cells; PL, pasture-associated laminitis; qPCR, quantitative PCR; RT-PCR, real-time PCR; RISQI, reciprocal inverse square of insulin; RIA, radioimmunoassay; SAA, serum amyloid A; SC, subcutaneous; SM, semimembranosus; TNF, tumor necrosis factor; TG, triglycerides; VIS, visceral.

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Cytokines/Adipokines Tested (Method)

EMS Criteria Insulin Dysregulation

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compare due to inconsistencies that have been found in the criteria and methodologies used to distinguish horses as insulin-resistant/ sensitive, or as obese/nonobese. In some cases, a low sample size and therefore a lack of statistical power might have contributed to the inability to identify differences. Furthermore, when animals were subdivided based on adiposity, “metabolically healthy obese” horses may have been inappropriately included in the EMS group, or lean EMS horses with only regional adiposity could have been missed based on the chosen EMS criteria. This discrepancy was evidenced in a recent study, where several overweight or obese horses had normal combined glucose insulin tolerance test results [71]. In that study, there was no significant difference in BCS between insulin dysregulated and healthy horses, whereas the CNS was a better predictor of insulin dysregulation. 3.4. Important Adipokines Without a Primary Inflammatory Role Leptin, the first adipokine to be characterized in the human species, is supposed to prevent fat accumulation and promote insulin sensitivity [46]. This adipokine has seen trends of excitement and disenchantment in the equine-scientific community. What has been discovered so far may suggest some degree of leptin resistance [71,72] in EMS because higher levels of leptin seem to positively correlate with obesity, insulin levels, and laminitis [72]. Very recently, a genomewide association study identified a locus involved in lipid metabolism that was associated with obesity in Arabian horses [73]. Adiponectin negatively correlates with BCS [74], enhances insulin sensitivity and has anti-inflammatory properties [46]. Its decrease has been correlated with development of laminitis in a recent prospective study [75]. High molecular weight adiponectin seems to be more relevant in equines [76] and humans alike [77] and may have a role as a biomarker in EMS screening. 4. Oxidative and Dicarbonyl Stress Oxidative stress, defined as an imbalance between antioxidant mechanisms and production of reactive oxygen species, has been observed in conjunction with obesity and insulin dysregulation in humans [78]. Lower levels or inadequate intake of vitamins, such as vitamin A, C, and E [79] and microelements, such as magnesium [80] and selenium [81] have been associated with reduced efficacy of antioxidant systems in obesity and MetS in humans. The adipokine leptin, which increases in adipose tissue dysfunction, has an effect of activation on NADPH oxidase with resulting increased generation of reactive oxygen species, in particular hydrogen peroxide (H2O2) and hydroxyl radicals [82]. Glutathione peroxidase (GPx) is involved in the detoxification of lipid hydroperoxides, which have been found in higher levels after exercise in correlation with increased oxygen consumption during exercise in obese humans as compared with nonobese [83]. In one study, horses with BCS  7/9 displayed decreased activity of erythrocyte GPx compared with horses with a BCS of 4e6/9 [84]. By contrast, another equine study found a positive correlation between antioxidant capacity in muscular tissue and BCS, without signs of oxidative damage [85]. Dicarbonyl stress is a common denominator of T2DM, aging, obesity, and cardiovascular disease, and also a hallmark of MetS. In T2DM, an increase in methylglyoxal (MG), a by-product of glycolysis, is observed, presumably due to excessive availability of glucose. Methylglyoxal is a highly reactive molecule, which triggers the glycation of proteins and DNA resulting in the formation of advanced glycation end products (AGEs), which can cause mutations and cell dysfunction, leading to dicarbonyl stress [86]. There are also theories about a contribution of MG to impaired glucose

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metabolism, based on studies in mice [87]. Methylglyoxal is normally detoxified through the glyoxalase system, in which the ratelimiting enzyme is glyoxalase-1 (Glo-1). The resulting product is Dlactate, which is excreted in the urine [88]. Glyoxalase-1 is available in the cytosol in all mammalian cells [87]. In humans with T2DM, drugs able to induce the expression of Glo-1 have been tested in clinical trials against vascular complications of T2DM, with positive results on glucose and insulin dynamics [89]; further clinical development of these treatments is underway. In experimentally induced laminitis with the hyperinsulinemic model, AGEs were observed to accumulate in significant amounts at the hoof lamellar tissue level toward the acute phase of the pathologic process (48 hours after the start of hyperinsulinemia) [90]. Valle et al demonstrated increased plasma levels of pentosidine, a glycoxidative marker, in ponies with clinical EMS and concurrent or historical laminitis, compared with nonlaminitic ponies. In that group of ponies, pentosidine had a positive correlation with insulin levels, insulin-to-glucose ratio and triglycerides [91]. Further studies are needed to elucidate the role of AGEs in EMS and if these mechanisms could be pharmacologically manipulated. 5. Macrovascular and Microvascular Complications of Metabolic Syndrome A person with MetS has a threefold risk of cardiovascular disease compared with a healthy person, and 5 times greater chance to develop T2DM [7], which frequently has long-term cardiovascular consequences. In human medicine, cardiovascular complications are further classified as macro- and microvascular, with possible common pathogenic mechanisms that involve AGEs, oxidative stress, and low-grade chronic inflammation [92]. Macrovascular disease in humans encompasses ischemic heart disease, stroke, and peripheral vasculopathy, largely because of atherosclerosis [92]. Recent evidence points to local IR at the cardiac muscle level as an additional mechanism for the development of cardiomyopathy [93]. In horses, laminitis has been considered the corresponding vascular event in comparison with MetS [94] because of lack of evidence of other cardiovascular effects of EMS and to differences in diet and metabolism which seem to prevent the development of atherosclerosis. However, in a recent study, ponies with EMS had increased heart rates and hypertrophic left ventricles among other signs of cardiac remodeling, which were significantly correlated with insulin levels after an OST, autonomic nervous system tone, and blood pressure [95]. In the same study, the subgroup of Shetland ponies presented also significantly increased blood pressure. It is possible that cardiovascular disease in horses with EMS is more prevalent than previously thought, but currently underdiagnosed, and differences between horses and ponies may exist. Microvascular pathology involves arterioles, venules, and capillaries; therefore, it manifests itself as nephropathy, retinopathy [96], and neuropathy in people with MetS or T2DM [92]. Insulin acts on the microvasculature to regulate delivery of nutrients to tissues (e.g., glucose to muscle). Insulin can control both the release of vasodilating nitric oxide (NO) from the endothelium through a signaling pathway that involves phosphatidylinositol 3-kinase (PI3K), and the secretion of the potent vasoconstrictor endothelin-1 (ET-1) through the mitogen-activated protein kinase pathway. The PI3K pathway is also involved in insulin-dependent glucose uptake, which, similarly to NO release, is decreased during IR. Nitric oxide and ET-1 are only two of the many vasoactive molecules in the body. However, during insulin dysregulation, obesity, and MetS, a state of endothelial dysfunction develops, which involves inflammation, endothelial proliferation, and increased oxidative stress. In this situation, there is an imbalance

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between the two arms of insulin signaling, with decreased NO availability and normal to increased ET-1 action [95e97], further enhanced by compensatory hyperinsulinemia [97]; this disparity is known to correlate with hypertension and CVD in human patients [98,99]. Recently this was also demonstrated in patients with diabetes who had to undergo lower-limb amputation because of angiopathy [100]. In that study, amputees also showed increased lipid peroxidation, represented by malondialdehyde activity, and nuclear factor KB activation. Diabetic amputees also experience delayed wound healing due to altered immune and inflammatory responses [101]. Furthermore, a role of ET-1 has recently been postulated in the pathogenesis of retinal vein occlusion [96], for which T2DM is a known risk factor. Laminitis has been proposed to be the EMS “alter ego” of CVD in MetS and T2DM [94,102]. Equine digital blood vessels have been histologically described as degenerated in naturally occurring laminitis [103], and serial venograms are commonly used in vivo to assess the extent of the vascular damage and to determine prognosis and direction of therapy [104,105]. Even though histologically the vascular pathology at the lamellar level does not necessarily mirror the atherosclerotic changes observed in people, an increasing body of work has investigated the role of endothelial dysfunction and the effect of different mediators on lamellar circulation in healthy and pathologic states [106e108]. Gauff et al [107] demonstrated higher mean vascular resistance and more abundant expression of ET-1 in lamellar vessels of equine isolated limbs that were infused with insulin. Even in humans, the vascular mechanisms related to IR and MetS have not been completely elucidated. However, the currently proposed theory is that both micro- and macrovascular alterations, including endothelial dysfunction, oxidative and dicarbonyl stress, are intertwined in determining the clinical cardiovascular pathology associated with MetS and T2DM [92,109]. For example, the same mechanisms of endothelial dysfunction that affect small vessels are observed in myocardial cells, leading to myocardial hypertrophy, cellular death, and fibrosis [93]. 6. Neuropathy: Human Diabetic Foot and Equine Laminitis Oxidative stress and microvascular injury are the underlying mechanisms of nerve damage involved in sensorimotor and autonomic neuropathies. These pathologies have a significant prevalence as complications of type I and type II diabetes mellitus, but are also frequently found in prediabetic patients, where the neuropathy can be the first sign of disease [110]. MetS and obesity have been strongly associated as risk factors to some of these neuropathies, such as the distal symmetric sensory polyneuropathy (DSPN) [111]. Peripheral artery disease has been associated with DSPN as a comorbidity [111], which highlights how the vascular and nervous systems are interconnected. DSPN is a major risk factor for the development of diabetic foot syndrome, in many cases accompanied by peripheral arterial occlusive disease (atherosclerosis) [112]. In the pathogenesis of human “diabetic foot”, neuropathy [113] and microvascular disease [114] lead to troublesome nonhealing ulcers that can ultimately result in gangrene of large portions of the limb, especially if offloading and treatment is delayed, with the subsequent need to amputate the affected limb. Equine studies addressing neuropathic changes and related pain in laminitic horses are rare, but results are supportive of the contribution of neuropathic changes to the well-known chronic pain response in this disease. Jones et al [115] analyzed sensory nerve fibers and pain behavior in terminally laminitic horses, finding that unmyelinated and myelinated fibers were significantly decreased and peripheral nerve injury markers were increased compared with horses euthanized for other reasons. Another study

[116] found the tunica media of the palmar digital vein to be thinner in chronically laminitic horses and identified expression of the purinergic P2X receptor, previously associated with chronic pain, in the media of the palmar digital artery and epidermal cells of the secondary lamellae (subtypes 3 and 7, respectively). So far, no direct link between laminitis-associated neuropathy and EMS or insulin dysregulation has been established, even though diabetes in humans has been mentioned as a comparison, together with other causes of neuropathy [117]. More studies are needed to determine if some of the mechanisms found in T2DM apply to laminitis pathophysiology in the insulin dysregulated horse. 7. Conclusions In light of the expanding trans-species obesity epidemic, a shift has occurred in what owners (and parents/caregivers) perceive as a “normal” body condition of their animals (and children) [118]. As in humans, this shift increasingly affects pets' and horses’ welfare and promotes future negative health outcomes. Without being an exhaustive review of the complex mechanisms involved in EMS, the aim of this article was to collect recent work that has been produced in human and equine medicine since the publication of the ACVIM EMS consensus statement in 2010 on specific aspects of MetS/EMS pathophysiology that can be approached in a comparative fashion. The recently published ECEIM consensus statement provided brief updates on EMS pathophysiological mechanisms, while focusing more on much needed recommendations for diagnosis and management [25]. A comparative approach with MetS can give veterinarians an instrument to facilitate clients’ understanding of the health implications, in addition to providing useful starting points for future primary research studies. Acknowledgments This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Authors' contributions: V.M.R. contributed to conceptualization, investigation, writingeoriginal draft, review and editing. G.A.Z. contributed to conceptualization, resources, writingereview and editing, supervision. C.D.K. contributed to investigation, data curation, writingereview and editing, visualization. J.B.M. contributed to conceptualization, resources, writingereview and editing, supervision. References [1] Haller H, Hanefeld M. Synoptische betrachtung metabolischer risikofaktoren. € rungen In: Haller H, Hanefeld M, Jaross W, editors. Lipidstoffwechselsto diagnostik, klinik und therapie. 1st ed. Jena: Fischer; 1975. p. 254e64. [2] Alberti KG, Zimmet PZ. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation. Diabet Med 1998;15: 539e53. [3] Balkau B, Charles MA. Comment on the provisional report from the WHO consultation. European group for the study of insulin resistance (EGIR). Diabet Med 1999;16:442e3. [4] Einhorn D. American College of Endocrinology position statement on the insulin resistance syndrome. Endocr Pract 2003;9:5e21. [5] Kahn R, Buse J, Ferrannini E, Stern M, American Diabetes Association, European Association for the Study of Diabetes. The metabolic syndrome: time for a critical appraisal: joint statement from the American diabetes association and the European association for the study of diabetes. Diabetes Care 2005;28:2289e304. [6] Alberti KG, Zimmet P, Shaw J. Metabolic syndrome-a new world-wide definition. A consensus statement from the international diabetes federation. Diabet Med 2006;23:469e80.

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