Introduction

The concept of equine metabolic syn- drome was first suggested by Johnson (John- son, 2002). In human medicine, metabolic syndrome refers to a set of risk factors that predict the risk of cardiovascular disease, in- cluding obesity, glucose intolerance, insulin resistance, dyslipidemia,  microalbuminuria and hypertension (Landsberg et al., 2013). Increased adiposity, hyperinsulinemia, and insulin resistance are the three principal com- ponents of equine metabolic syndrome. It is difficult to separate those factors from one
another. Hyperinsulinemia is detected in most insulin resistant horses and affected animals are usually obese or exhibit regional adiposity (Frank and Tadros, 2014). Normal actions of insulin include inhibition of gluconeogenesis and lipolysis and stimulation of glycogen syn- thesis. Insulin resistance is defined as a reduc- tion in the action of insulin on target tissues. Mechanisms of insulin resistance include de- fects in the insulin receptor, insulin signaling pathways or glucose transporter 4 (GLUT4) synthesis, translocation or  function  (Samuel et al., 2016). There are different suggested methods to identify the insulin resistant hors- es. Screening tests are the practical methods and dynamic diagnostic tests containing com- bined glucose-insulin test and oral sugar test are also other suggested methods (Frank and Tadros, 2014).

Equine metabolic syndrome and insulin re- sistance occur most commonly in pony, Mor- gan, Paso Fino, Arabian, Saddlebred, Quarter and Tennessee Walking breeds. Most horses and ponies with equine metabolic syndrome are obese and owners often describe them as easy keepers. Environmental issues such as overfeeding and lack of exercise  contribute to obesity and these problems are increasing with  modern  management  practices  (Frank

and Tadros, 2014).

To the best of the author’s knowledge, there is no information about equine metabolic syn- drome and insulin resistance phenomenon in Darehshori horses as an originated breed from Iran. Hence, the present study was carried out by the screening method to evaluate the pres- ence of insulin resistance and its probable re- lation with age, sex, season and obesity in this breed, during one year.

Materials and Methods

This project was carried out from January till November 2015 on 26 Darehshori hors- es (12 adult stallions and 14 non-pregnant, non-lactating adult mares) at several eques- trian clubs around Shiraz, southwest Iran. Fourteen horses were under and others were above 10 years old. All of them were in rest- ing period, kept in individual stalls and fed with balanced rations including alfalfa hay, corn silage and barley grain. The horses were clinically healthy, had no history of debilitat- ing diseases, laminitis and were free from in- ternal and external parasites based on labora- tory parasitic examinations and routine anti parasitic programs. Body condition scores of the animals were estimated based on Hen- neke et al. (1983), 1 to 9 scoring system. The Hennekes’ horse body condition scoring sys- tem is a numerical scale used to evaluate the amount of fat on a horse’s body. It was first published by Henneke et al. at 1983 to cre- ate a universal scale to assess horses’ body- weight. It is a standardized system that  can be used across all breeds without specialized equipment; condition is  assessed  visually and by palpation. Scores range from 1 to 9 which refer to poor, very thin, thin, moderate- ly thin, moderate, moderately fleshy, fleshy, fat and extremely fat, hence, one being  poor

and nine being extremely fat. The ideal range for most horses is from 4 to 6. The system is based on both visual appraisal and palpable fat which cover the six major points of the horse including neck, shoulder, ribs, withers, lumbar vertebrae and tail head. Body condi- tion score of 12 horses was under 5 and the others were above 5. The horses were stud- ied during one year, every 45 days and blood sampling and body condition scoring were performed at each time. The first sampling was carried out at 1 January 2015 and was continued to 15 November 2015.

Insulin resistance phenomenon was as- sessed based on screening test (Frank and Tadros, 2014). According to this method, we left only one flake of hay after 10:00 PM for each horse and blood was collected at 06:00 AM the next morning. Evaluating the se- rum fasting glucose, insulin and leptin levels could reflect the insulin resistance or sensi- tivity. According to this procedure, if fasting glucose, insulin and leptin were respectively higher than 110 mg/dL, 20 µU/mL and 7 ng/ mL, it could be concluded that the horse was insulin resistant (Frank and Tadros,  2014).

Blood samples were collected via  jugu- lar  venipuncture  from  all  animals,  every 45 days, in plain tubes. Immediately after blood collection, sera were separated by cen- trifugation for 10 minutes at 3000 rpm and stored at -22 °C until assayed. Glucose was assayed by an enzymatic (glucose oxidase) colorimetric method (ZistChem®, Tehran, Iran). Insulin and leptin were measured by equine ELISA kits (Eastbiopharm®, Chi-  na). Reference number of equine insulin and leptin ELISA kits were E20160801045 and E20160801046. These analyses were per- formed by Stat Fax® 2100.

All data are presented as mean ± standard deviation (SD). Differences between the  av-

erage concentrations of each serological fac- tor between groups (age, sex and body con- dition scores) on similar days were analyzed by repeated measures ANOVA. Repeated measures ANOVA was also used to evaluate the changing patterns of each factor during the year. The statistical analyses were per- formed by using SPSS 20 (SPSS Inc, Chi- cago, Illinois). The level of significance was set at P-value<0.05.

Results

The results of the  screening  test,  every 45 days during a year for insulin resistance based on age, sex and body condition score are presented in Fig. 1. Based on Frank and Tadros (2014), fasting glucose, insulin and leptin at 110 mg/dL, 20 µU/mL and 7 ng/mL, respectively, are the cut-off points and higher values indicate insulin resistant horses. Hor- izontal dotted lines at these limits were used to better identify the insulin resistance phe- nomenon at each sampling  day.

Glucose levels in aged horses (above 10 years old) were significantly higher than younger ones mainly in warmer  months (May till August; P<0.05; Fig. 1). Glucose values in older horses in warmer months were higher than the cut-off point for insu-  lin resistance. The glucose concentrations in younger horses were under the cut-off point during the period of the study. In the stal- lions, glucose was significantly higher than the mares in warmer months (P<0.05) and its levels were above the cut-off point of insulin resistance. Glucose in mares was under 110 mg/dL during the study (Fig. 1). The  fast- ing blood glucose in the horses  with  great- er body condition scores was significantly higher (P<0.05). The glucose levels follow- ing screening test in the horses with higher body condition scores were above the cut-off

Figure 1. The results of evaluating the serum glucose, insulin and leptin concentrations according to the insulin re- sistance screening test in Darehshori horses, based on age, sex and body condition score at every 45 day samplings, during one year. Fasting blood glucose, insulin and leptin at 110 mg/dL, 20 µU/mL and 7 ng/mL, are the cut-off points of insulin resistance, respectively. Higher values indicate insulin resistant horses. Stars indicate significant differences (P<0.05).

point of insulin resistance at all studied days (Fig. 1). Based on the fasting blood glucose assay for the screening testing of insulin re- sistance, it may be suggested that the stal- lions and older horses were insulin resistant in warmer months. However, obese Dare- hshori horses had insulin resistance during the study period (Fig. 1).

Insulin levels in older horses were signifi- cantly higher than younger ones in warmer months (P<0.05; Fig. 1). Its levels in older horses in warmer months were higher than cut-off point of insulin resistance. The in- sulin values in younger horses were under cut-off point during the  study.  The  results of the repeated measures ANOVA showed that insulin was increased significantly fol- lowing the climate becoming warmer in both age groups (P<0.05, Fig. 1). In stallions, in- sulin was significantly higher than mares in warmer months (P<0.05) and its levels  were

above the cut-off point of insulin resistance. Insulin in mares was under 20 µU/mL during the study (Fig. 1). Insulin levels in the horses with greater body condition scores were sig- nificantly higher than other ones (P<0.05). The insulin levels following screening test in the horses with high body condition scores were above the cut-off point of insulin re- sistance during the study (Fig. 1). Based on evaluating the insulin for the screening  test of insulin resistance, it may be  proposed  that the stallions and older Darehshori hors- es were insulin resistant in warmer months. However, obese horses had insulin resistance during the year.

The results of the repeated measures ANO- VA showed that leptin increased significantly following the the climate becoming warmer in both age groups (P<0.05, Fig. 1). There were no statistical differences between old and  young  horses  (P>0.05).  Leptin  in both

age groups in warmer months was higher, but its levels were under cut-off point of in- sulin resistance during the study (Fig. 1). In stallions, leptin was significantly higher than mares in warmer months (P<0.05) and its levels were above the cut-off point of insulin resistance (Fig. 1). Leptin in mares was un- der 7 ng/mL on all of the studied days (Fig. 1). Leptin levels in the horses with greater body condition scores were significantly higher than other ones (P<0.05). The leptin values following screening testing in horses with high body condition scores were above the cut-off point of insulin resistance at all times (Fig. 1). Based on the leptin assay for the screening test of insulin resistance, it may be suggested that the stallions and older hors- es were insulin resistant in warmer months and obese Darehshori horses had insulin re- sistance during the whole year.

Discussion

Insulin is synthesized by the pancreatic β cells and is primarily a glucose storage hor- mone because it facilitates cellular glucose uptake, promotes glycogenesis, and  inhib-  its gluconeogenesis (Sharabi et al., 2015). Insulin secretion increases in response to increased blood glucose concentrations to maintain normoglycemia by stimulating cel- lular glucose uptake, glycogenesis, and fatty acid synthesis, as well as reducing glucose production  by  decreasing   gluconeogene- sis, lipolysis and proteinolysis (Samuel and Shulman, 2016).

Insulin resistance is defined as a  reduc- tion in the action of insulin on target tissues (Frank and Tadros, 2014). Mechanisms of in- sulin resistance include defects in the insulin receptor, insulin signaling pathways, or glu- cose transporter 4 (GLUT4) synthesis, trans- location,  or  function.  One  important action

of insulin is to stimulate glucose  transport into cells, and this occurs rapidly as GLUT4 proteins translocate to cell membranes. Re- sults of a recent study indicate that GLUT4 translocation is impaired in insulin-resistant horses (Samuel and Shulman, 2016). Insulin resistance screening testing is a simple and practical method to diagnose the equine met- abolic syndrome (Frank  and Tadros,  2014). In this method, fasting blood glucose and in- sulin concentrations should be measured to screen for hyperglycemia and hyperinsulin- emia, which serve as indicators of insulin re- sistance. Leptin measurements are also suit- able to evaluate the fat metabolism. During the screening tests, blood glucose concen- trations are within reference range in most insulin-resistant horses because  euglycemia is maintained through increased pancreatic insulin secretion. However, glucose concen- trations should always be measured to detect uncompensated insulin resistance or diabetes mellitus. Some of these patients can only be identified by detecting hyperglycemia be- cause insulin concentrations  have  returned  to reference range as a result of pancreatic insufficiency (Frank and Tadros,  2014).

At present, the most useful screening test for insulin resistance is the resting insulin concentration, which must be performed after a short fast to minimize the impact of feed- ing (Anhê et al., 2015). As with many tests, the result is more likely to be a true positive the further it falls outside of reference range. A markedly elevated fasting insulin concen- tration therefore serves as a good indication of insulin resistance. However, it is more difficult to interpret results that are closer to reference range and breed-specific ranges are needed to improve accuracy (Frank and Tad- ros, 2014). The present study tried to find out the insulin resistant Darehshori horses at  dif-

ferent situations such as age, sex, season and obesity. Higher resting serum insulin concen- trations were detected in aged stallions with obesity, suggesting that this value may be a useful screening parameter for insulin resis- tance. Hyperinsulinemia is a feature of insu- lin resistance in humans and occurs when in- sulin secretion from the pancreas increases to compensate for reduced response to insulin (Cerf, 2013).

Based on the results of the current research, insulin resistance was not found in younger horses in any of the seasons but older ones showed this phenomenon in warmer months (Fig. 1). Darehshori stallions were insulin re- sistant in warmer months only, but this met- abolic disorder was not found in Darehshori mares in any of the studied seasons (Fig. 1). Results of the Frank and Tadros (2014) study indicated that resting serum insulin and leptin concentrations are useful screening parame- ters for insulin resistance in horses, but oth- er factors including time of day and season must be considered when interpreting these values. Gordon and McKeever (2005) deter- mined that glucose and insulin responses to feeding are higher in the morning. Fitzgerald and McManus (2000) determined that serum leptin concentrations were highest in mature mares during the late summer and early fall and lowest during the winter months in Ken- tucky. They stated that the serum insulin con- centrations in obese mares across a 12-month period were also higher during the summer and early fall.

In the present study, the horses with low-  er body condition scores had no insulin re- sistance in any of the studied seasons but obese horses showed this phenomenon at all sampling months (Fig. 1). Frank and Tadros (2014) assembled a population of obese hors- es with  insulin  resistance,  and these  horses

had significantly greater resting glucose, in- sulin, and leptin concentrations than healthy non-obese horses.

Some horses are  genetically  predisposed to obesity because of adaptations to survival on poorer quality forages (Giles et al., 2014). According to this theory, consumption of concentrated feeds or grazing on rich pas- tures might therefore promote obesity in sus- ceptible horses. Genetic and environmental factors are likely to be important in the de- velopment of obesity in horses, and it is inter- esting that obese insulin resistant horses are aged, which suggests that time is required for environmental factors to alter glucose metab- olism (Frank and Tadros, 2014). Obesity was defined by body condition score in our study with the scale developed by Henneke et al. (1983). Obese horses in this study were insu- lin resistant and they had regional adiposity at those locations. An association between obe- sity and insulin resistance has been reported in horses (Hoffman et al.,  2003).  Hoffman  et al. (2003) studied obese Thoroughbred geldings by use of the frequently-sampled intravenous glucose tolerance test and they concluded that mean insulin sensitivity    was

< 20% of the value in non-obese geldings. Obesity and lack of exercise are primary risk factors for insulin resistance in humans, and the risk of developing type 2 diabetes mel- litus increases with the severity of obesity (Landsberg et al., 2013).

Adipokines are released from adipocytes and two adipokines have been examined to date in horses including leptin and adiponec- tin (Frank, 2018). Leptin is an adipocyte-de- rived hormone product of the obesity gene that influences food intake and energy use, and can be used as an indicator of energy balance. High levels of leptin increase ener- gy expenditure and decrease food intake  and