Obesity and insulin resistance are established risk factors for development of laminitis in horses and ponies,1–3 and the term EMS has been adopted to describe equids with generalized or regional adiposity, insulin resistance, and laminitis.4 In human medicine, metabolic syndrome defines a set of risk factors, including obesity and insulin resistance, that predict the development of cardiovascular disease.5 In contrast, laminitis is of primary importance in horses with EMS. Insulin resistance is linked to cardiovascular disease in humans through its effects on the vascular endothelium,6 and this mechanism is also being examined in horses.7
Resting hyperglycemia is not a common component of EMS in horses,4 but abnormal glucose homeostasis might still play a role in the development of laminitis. A recent study8 identified increased fructosamine concentrations in laminitic horses, compared with findings in nonlaminitic horses, although the study population was not limited to animals with EMS. Hyperglycemia increases the production of reactive oxygen species and the formation of advanced glycation end products, which damage tissues and are important in the pathogenesis of cardiovascular disease in diabetic humans.9 Interstitial glucose concentrations in obese, insulin-resistant horses are variable throughout the day and sometimes exceed reference range.10 Transient periods of hyperglycemia may be important in the development of laminitis because in humans, mild fasting hyperglycemia increases cardiovascular risk, and postprandial hyperglycemia that is less severe than that considered the cutoff for diabetes mellitus causes endothelial dysfunction if it occurs over a prolonged period of time.11,12 Gradual development of microvascular dysfunction in response to repeated hyperglycemic episodes can be related to laminitis because horses with EMS often have mild laminitis that goes unnoticed by owners but develops over time after those horses graze on soluble carbohydrate-rich pasture.4
The development of laminitis in horses with EMS in association with grazing on soluble carbohydrate-rich pasture has suggested that inflammation contributes to the development of disease.4,13 Pasture grazing increases the risk of intestinal carbohydrate overload and translocation of gut-derived toxins such as LPS, exotoxins, and vasoactive amines into systemic circulation.13 Horses could therefore develop mild episodes of systemic inflammation while on pasture, which might further impact glucose and insulin dynamics. Both insulin resistance and hyperglycemia develop acutely during systemic inflammation because inflammatory cytokines and stress hormones such as glucocorticoids, catecholamines, and glucagon inhibit postreceptor insulin signaling pathways and promote hepatic glucose output.14,15 Lipopolysaccharide infusion decreases insulin sensitivity in clinically normal horses,16–18 but this situation has not been examined in horses with EMS. It must therefore be determined whether preexisting derangements in glucose and insulin homeostasis are exacerbated by inflammation in these animals, which could increase their risk of developing laminitis.
Clinical experience suggests that horses with EMS are at greater risk of developing laminitis when systemic illnesses such as bacterial enterocolitis develop. Obesity is associated with increased morbidity and mortality rates in critically ill humans,19,20 and obese patients are at increased risk of developing hyperglycemia.21 Laminitis shares many similarities with sepsis-associated organ failure, including microvascular dysfunction,22,23 and endothelial function is further compromised in obese individuals during sepsis.24 It is therefore our overall hypothesis that, in horses, EMS increases the risk of laminitis development in association with systemic inflammation. The purpose of the study of this report was to determine whether glucose and insulin dynamics differ between horses with EMS and healthy horses in response to inflammation. We hypothesized that EMS impacts alterations in glucose and insulin homeostasis induced by LPS infusion in horses.
Materials and Methods
Animals—Six healthy adult mares and 6 horses with EMS (3 mares and 3 geldings) from the University of Tennessee teaching and research herd were included in the study; a complete description of the horses is reported elsewhere.25 Healthy horses had no history of laminitis and after feed withholding, serum insulin concentrations were < 20 μU/mL. The study protocol was approved by the University of Tennessee Institutional Animal Care and Use Committee.
Study design—Glucose and insulin dynamics were assessed concurrently with inflammatory responses to LPS infusion, which are described separately.25 Three pairs of horses (1 healthy horse and 1 horse with EMS/pair) were randomly assigned to receive an IV infusion of LPSa (20 ng/kg diluted in 60 mL of sterile saline [0.9% NaCl] solution) in the first week and an IV infusion of sterile saline solution in the second week. Treatment order was reversed for the remaining 3 pairs of horses. The dose of LPS was chosen because it perturbs insulin sensitivity when administered to clinically normal horses.16,17 Testing was performed over 2 consecutive weeks between February and April 2010. Horses were simultaneously used to investigate inflammatory cytokine responses25 and alterations in glucose and insulin dynamics induced by LPS infusion.
Horses were transported to the veterinary teaching hospital and housed in 3.7 × 3.7-m box stalls at the start of each testing week. After horses had a minimum 24-hour acclimatization period, a 14-gauge polypropylene IV catheterb was aseptically placed in a jugular vein of each horse, and a baseline FSIGTT was performed at 8:30 am on day 1 (−27 hours). Lipopolysaccharide or saline solution was infused slowly as a bolus over a 30-minute period beginning at 11:00 am on day 2. Conclusion of the IV infusion was designated as 0 hours. An FSIGTT was repeated at 0.5 hours (noon on day 2) and 21 hours (8:30 am on day 3). A liver biopsy was performed under standing sedation between 4 and 6 hours (relative to IV infusion of LPS or saline solution) as part of the concurrent study25 to assess inflammatory responses. A physical examination was performed 30 minutes before, hourly for 9 hours, and at 15 and 21 hours following LPS or saline solution administration. Each horse had ad libitum access to grass hay and water at all times. The IV catheter was removed after completion of the 21-hour FSIGTT, and each horse was returned to the research farm. Procedures were repeated the following week, and the alternate treatment (LPS or saline solution) was administered to each horse (total of 2 treatments/horse).
FSIGTT procedure—Testing was performed as described by Hoffman et al26 and modified by Tóth et al.27 A blood sample (20 mL) was collected via the IV catheter into a tube containing sodium fluoride–potassium oxalate (to obtain a plasma sample) and a tube containing no anticoagulant (to obtain a serum sample) at −10, −5, and 0 minutes relative to infusion of dextrose solution; mean values were calculated to obtain the baseline glucose and insulin values. An IV bolus of 50% dextrose solutionc was then administered (0 minutes) at a dose of 150 mg/kg. Blood samples were obtained at 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, and 19 minutes after bolus administration for measurement of blood glucose and insulin concentrations. A bolus of regular insulind (30 mU/kg) was administered IV at 20 minutes. Blood samples were subsequently obtained at 22, 23, 24, 25, 27, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, and 180 minutes after administration of the dextrose solution bolus. Blood samples in tubes containing sodium fluoride-potassium oxalate were immediately cooled on ice, and blood samples in tubes containing no anticoagulant were allowed to clot at room temperature (approx 22°C) for 1 hour. All tubes were centrifuged at 1,000 × g for 10 minutes, and plasma or serum samples were harvested and stored at −20°C until analyzed.
Assessment of plasma glucose and serum insulin concentrations—Glucose concentrations in plasma samples were measured in duplicate with a colorimetric assaye on an automated discrete analyzer.f Insulin concentrations in serum samples were measured in duplicate with a radioimmunoassayg that has been validated for use with equine plasma in our laboratory and by others.28 Intra-assay coefficients of variation < 5% and < 10% were required for acceptance of plasma glucose assay results and serum insulin assay results, respectively.
Interpretation of FSIGTT data with the minimal model—Minimal model29 parameters for SI, Sg, AIRg, and DI were calculated as previously described26,30 with computer software.h,i Disposition index was calculated by multiplying AIRg × SI.
Inflammatory cytokine gene expression in whole blood samples—As part of the concurrent study,25 additional blood samples (2.5 mL) for quantitation of IL-1β, IL-6, IL-8, IL-10, TNF-α, and B-Gus gene expression were collected at before (–30 minutes; baseline) and at 30, 60, 90, 120, 180, and 240 minutes after infusion of LPS or saline solution into whole blood RNA collection tubes. Tubes were allowed to incubate at room temperature (approx 22°C) for 8 hours, then stored at −20°C until analyzed. Total RNA extraction and real-time PCR assays were performed as described.25 The comparative cycle threshold (ΔΔCt) method was used to determine fold changes in inflammatory cytokine gene expression. Cytokine expression was normalized to the housekeeping gene B-Gus, and each horse's baseline value was used as the calibrator. Fold changes in gene expression were therefore determined at the level of the individual animal, and baseline gene expression was equal to a 1-fold change for all cytokines. Peak cytokine gene expressions were determined.
Statistical analysis—Mixed-model ANOVA for repeated measures was performed with computer softwarej to determine effects of group (EMS-affected horses vs healthy horses), treatment (LPS vs saline solution), time, and main effect interactions. Area under the curve values for glucose and insulin were calculated via the trapezoidal method and computer software.k Area under the curve for insulin and baseline insulin values required logarithmic transformation to fit the ANOVA's normal distribution assumptions. Minimal model parameters required square root transformation. Transformed data are reported as geometric means with 95% CI. All other data are reported as least squares means ± SEM. The autoregressive correlation parameter was excluded from the final model for all analyses. Mean separation was performed via a Fisher protected least significant difference test, and significance was set at a value of P < 0.05.
Associations among indices of glucose and insulin homeostasis and peak blood cytokine gene expression values25 were assessed via Spearman rank correlation coefficients. Correlation analysis was performed at the level of the individual animal. Significance was set at a value of P < 0.05 for all correlations.
Results
Response to LPS—Ten of 12 horses developed transient signs of depression, anorexia, pawing, yawning, head shaking, stretching, or muscle fasciculations following LPS infusion. Two horses (1 healthy horses and 1 EMS-affected horse from the same pair) had no clinical response to LPS infusion. Because physical examination variables did not change and leukopenia was not detected, all data collected after LPS infusion from these 2 nonresponders were excluded from further analyses.
Baseline glucose and insulin concentrations—Baseline plasma glucose and serum insulin concentrations from each FSIGTT were summarized (Table 1). Values represent resting plasma glucose and serum insulin concentrations. Administration of LPS significantly increased baseline (resting) serum insulin (treatment × time; P < 0.001) and plasma glucose (treatment × time; P < 0.001) concentrations at 21 hours, compared with baseline findings following the saline solution treatment; however, differences between horses with EMS and healthy horses were not detected. Compared with healthy horses, horses with EMS had higher (P < 0.007) baseline (resting) insulin concentrations overall.
Baseline plasma glucose and serum insulin concentrations determined during each of 3 FSIGTTs performed in 6 healthy horses and 6 horses with EMS following IV bolus administration of LPS (20 ng/kg [in 60 mL of sterile saline {0.9% NaCl} solution]) or saline solution, followed by the other treatment after a 7-day washout period.
| FSIGTT | P value | ||||||
|---|---|---|---|---|---|---|---|
| Variable | Horse group | Treatment | −27 hours | 0.5 hours | 21 hours | Group* | Treatment × time† |
| Baseline glucose (mg/dL) | EMS affected | LPS | 89.0 ± 2.9 | 85.7 ± 2.8 | 112.2 ± 7.0 | 0.371 | 0.001 |
| Saline solution | 92.7 ± 4.5 | 92.5 ± 2.4 | 94.9 ± 4.1 | ||||
| Healthy | LPS | 90.7 ± 3.0 | 85.2 ± 4.0 | 103.7 ± 3.1 | |||
| Saline solution | 90.2 ± 4.3 | 90.5 ± 3.1 | 90.1 ± 2.1 | ||||
| Baseline insulin (μU/mL) | EMS affected | LPS | 13.7 (9.8–19.2) | 8.0 (6.9–9.3) | 31.4 (19.4–51.0) | 0.007 | 0.001 |
| Saline solution | 13.9 (8.0–23.9) | 25.8 (12.1–55.1) | 24.1 (11.9–49.2) | ||||
| Healthy | LPS | 7.1 (4.1–12.3) | 6.1 (2.9–12.9) | 11.5 (5.9–22.6) | |||
| Saline solution | 5.4 (3.8–7.7) | 6.0 (3.2–11.4) | 5.3 (3.7–7.5) | ||||
An FSIGTT was performed in each horse before (−27 hours) and at 0.5 and 21 hours after conclusion of each infusion (0 hours). Two horses (1 healthy horse and 1 horse with EMS) had no clinical response to LPS infusion, and those data were not used in the analyses. Baseline insulin concentrations are reported as geometric mean and 95% CI, and baseline glucose concentrations are reported as mean ± SEM; values represent resting plasma glucose and serum insulin concentrations.
Insulin concentrations were higher overall in horses with EMS than in healthy horses.
For each variable, values were increased significantly (P < 0.05) at 21 hours in the LPS groups, compared with the pretreatment value, but differences between EMS-affected and healthy horses were not detected. Changes were not significant (P ≥ 0.05) in horses after administration of saline solution infusions.
Prior to the testing period (at −27 hours) during 1 or both weeks of the study, baseline (resting) hyperinsulinemia (> 20 μU/mL)l was detected in 2 of 6 horses with EMS and 1 of 6 healthy horses. Lipopolysaccharide infusion induced hyperinsulinemia in 5 of 5 horses with EMS and 1 of 6 healthy horses at 21 hours; this healthy horse was not the one that was hyperinsulinemic at −27 hours. Hyperinsulinemia was detected during saline solution infusion in 4 of 6 and 3 of 6 horses with EMS at 0.5 and 21 hours, respectively. Resting hyperinsulinemia was not detected in healthy horses when they received saline solution.
Baseline (resting) hyperglycemia (> 110 mg/dL)m was not detected in any horse at −27 or 0.5 hours, but LPS infusion induced hyperglycemia in 4 of 5 horses with EMS and 1 of 6 healthy horses at 21 hours. One of 6 horses with EMS became hyperglycemic 21 hours after saline solution infusion.
AUCg and AUCi—Area under the curve values for plasma glucose and serum insulin concentrations were summarized (Table 2). Significant group × treatment × time interactions were detected for AUCg from 0 to 180 minutes (P = 0.039) and AUCi during the first 19 minutes (before IV insulin administration; P = 0.016). Treatment × time interactions were significant for AUCi from 0 to 180 minutes (P = 0.014; Figure 1) and AUCg for the first 19 minutes (P < 0.001; Figure 2). When group and group × time effects were examined, AUCi (group, P = 0.005) and AUCg (group, P = 0.030; group × time, P = 0.011) from 0 to 180 minutes were higher in horses with EMS.
Treatment × time interactions (P = 0.014) for AUCi (from 0 to 180 minutes during an FSIGTT) following IV bolus administration of LPS (20 ng/kg [in 60 mL of sterile saline {0.9% NaCl} solution]; dark bars) or saline solution (light bars) in 6 healthy horses and 6 horses with EMS. Horses received 1 treatment followed by the other treatment after a 7-day washout period. Two horses (1 healthy horse and 1 horse with EMS) had no clinical response to LPS infusion, and those data were not used in the analyses. Pretreatment data were obtained 27 hours before each infusion; posttreatment data were obtained at 0.5 and 21 hours after conclusion of the infusion (0 minutes). Data are expressed as geometric mean and 95% CI and are displayed on a linear scale. a–cDifferent letters denote significant (P < 0.05) differences among means.
Citation: American Journal of Veterinary Research 74, 7; 10.2460/ajvr.74.7.1020
Treatment × time interactions (P < 0.001) for AUCg (from 0 to 19 minutes during an FSIGTT) following IV bolus administration of LPS (20 ng/kg) or saline solution infusion in the 6 healthy horses and 6 horses with EMS in Figure 1. Data are expressed as mean ± SEM and are displayed on a linear scale. a,bDifferent letters denote significant (P < 0.05) differences among means. See Figure 1 for key.
Citation: American Journal of Veterinary Research 74, 7; 10.2460/ajvr.74.7.1020
Area under the curve values for plasma glucose and serum insulin concentrations determined during each of 3 FSIGTTs performed in the 6 healthy horses and 6 horses with EMS in Table 1 following IV bolus administration of LPS (20 ng/kg) or saline solution.
| FSIGTT | |||||||
|---|---|---|---|---|---|---|---|
| Period | Variable | Horse group | Treatment | −27 hours | 0.5 hours | 21 hours | |
| 0–180 min | AUCg | EMS affected | LPS | 18.7 ± 0.2f | 20.1 ± 0.5c,d,e,f | 29.8 ± 1.01a | 0.039 |
| ([mg/dL × min] × 103) | Saline solution | 21.6 ± 1.0c,d,e | 21.8 ± 0.8c | 21.8 ± 0.9c,d | |||
| Healthy | LPS | 19.0 ± 0.9f | 19.2 ± 0.5e,f | 25.3 ± 1.5b | |||
| Saline solution | 20.0 ± 1.1c,d,e,f | 19.4 ± 0.6e,f | 19.5 ± 0.4d,e,f | ||||
| AUCi | EMS affected | LPS | 8.6 (7.1–10.3) | 12.3 (6.9–22.1) | 20.1 (12.9–31.3) | 0.185 | |
| ([μU/mL × min] × 103) | Saline solution | 10.8 (8.4–14.0) | 15.4 (9.4–25.2) | 13.2 (7.4–23.6) | |||
| Healthy | LPS | 5.4 (4.5–6.4) | 6.8 (5.6–8.2) | 8.5 (6.4–11.2) | |||
| Saline solution | 6.0 (4.7–7.5) | 4.8 (3.7–6.3) | 6.1 (4.4–8.4) | ||||
| 0–19 min | AUCg | EMS affected | LPS | 3.6 ± 0.1 | 3.7 ± 0.2 | 4.4 ± 0.1 | 0.226 |
| ([mg/dL × min] × 103) | Saline solution | 3.8 ± 0.1 | 3.9 ± 0.1 | 3.9 ± 0.1 | |||
| Healthy | LPS | 3.7 ± 0.1 | 3.8 ± 0.1 | 4.1 ± 0.1 | |||
| Saline solution | 3.7 ± 0.1 | 3.8 ± 0.1 | 3.8 ± 0.1 | ||||
| AUCi | EMS affected | LPS | 2.3 ± 0.3a,b,c | 1.4 ± 0.6d | 2.5 ± 0.4a,b | 0.016 | |
| ([μU/mL × min] × 103) | Saline solution | 2.1 ± 0.3b,c,d | 3.2 ± 0.6a | 3.0 ± 0.6a | |||
| Healthy | LPS | 1.3 ± 0.2c,d | 1.1 ± 0.1d | 1.7 ± 0.3b,c,d | |||
| Saline solution | 1.4 ± 0.2c,d | 1.3 ± 0.2c,d | 1.3 ± 0.1c,d | ||||
Values for AUCi (0 to 180 minutes) are reported as geometric mean and 95% CI, and all other data are reported as mean ± SEM.
Within each variable, different superscript letters indicate significant (P < 0.05) differences among means.
See Table 1 for remainder of key.
Minimal model analysis—Minimal model results were summarized (Table 3). Insulin sensitivity decreased in response to LPS infusion (treatment × time; P < 0.001; Figure 3), and significant (P = 0.016) group × time interactions were also detected. Baseline mean SI values for healthy horses and horses with EMS did not differ, but SI was lower overall in horses with EMS (group; P < 0.001). Glucose effectiveness in the horses with EMS decreased over time (group × time; P = 0.002; Figure 4), and treatment × time effects were significant for AIRg (P < 0.001; Figure 5). Disposition index decreased in response to LPS infusion (treatment × time, P < 0.001; Figure 6) and in horses with EMS over time (group × time, P = 0.044).
Insulin sensitivity index (determined during FSIGTTs) derived for the 6 healthy horses and 6 horses with EMS in Figure 1 following IV bolus administration of LPS (20 ng/kg) or saline solution infusion. Data are expressed as geometric mean and 95% CI and are displayed on a linear scale. A—Values of SI for all horses following administration of LPS (dark bars) or saline solution (light bars). Treatment × time interactions were significant (P < 0.001). B—Values of SI for horses with EMS (dark bars) and healthy horses (light bars). Group × time interactions were significant (P = 0.016). a–dDifferent letters denote significant (P < 0.05) differences among means. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 74, 7; 10.2460/ajvr.74.7.1020
Group × time interactions (P = 0.002) for Sg (determined during FSIGTTs) derived for the 6 healthy horses (light bars) and 6 horses with EMS (dark bars) in Figure 1 following IV bolus administration of LPS (20 ng/kg) or saline solution infusion. Data are expressed as geometric mean and 95% CI and are displayed on a linear scale. a,bDifferent letters denote significant (P < 0.05) differences among means. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 74, 7; 10.2460/ajvr.74.7.1020
Treatment × time interactions (P < 0.001) for AIRg (determined during FSIGTTs) derived for the 6 healthy horses and 6 horses with EMS in Figure 1 following IV bolus administration of LPS (20 ng/kg [dark bars]) or saline solution (light bars) infusion. Data are expressed as geometric means and 95% CI and are displayed on a linear scale. a–cDifferent letters denote significant (P < 0.05) differences among means. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 74, 7; 10.2460/ajvr.74.7.1020
Disposition index (determined during FSIGTTs) derived for the 6 healthy horses and 6 horses with EMS in Figure 1 following IV bolus administration of LPS (20 ng/kg) or saline solution infusion. Data are expressed as geometric mean and 95% CI and are displayed on a linear scale. A—Values of DI for all horses following administration of LPS (dark bars) or saline solution (light bars). Treatment × time interactions were significant (P < 0.001). B—Values of DI for horses with EMS (dark bars) and healthy horses (light bars). Group × time interactions were significant (P = 0.044). a–dDifferent letters denote significant (P < 0.05) differences among means. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 74, 7; 10.2460/ajvr.74.7.1020
Minimal model analysis results for the 6 healthy horses and 6 horses with EMS in Table 1 following IV bolus administration of LPS (20 ng/kg) or saline solution.
| FSIGTT | ||||||
|---|---|---|---|---|---|---|
| Variable | Horse group | Treatment | −27 hours | 0.5 hours | 21 hours | |
| SI | EMS affected | LPS | 2.28 (1.24–3.64) | 0.93 (0.34–1.82) | 0.22 (0.11–0.38) | 0.919 |
| ([L × min−1 × mU−1] × 10−4) | Saline solution | 1.03 (0.59–1.59) | 0.86 (0.48–1.35) | 0.70 (0.33–1.20) | ||
| Healthy | LPS | 3.25 (2.24–4.45) | 3.40 (1.90–5.33) | 0.98 (0.15–2.55) | ||
| Saline solution | 1.81 (0.86–3.11) | 3.99 (2.39–6.01) | 2.43 (1.41–3.74) | |||
| Sg ([min−1] × 10−2) | EMS affected | LPS | 2.64 (1.44–4.19) | 3.25 (1.92–4.94) | 0.64 (0.22–1.27) | 0.200 |
| Saline solution | 2.58 (1.89–3.38) | 2.15 (1.68–2.69) | 1.42 (0.65–2.48) | |||
| Healthy | LPS | 2.66 (2.10–3.29) | 3.10 (1.74–4.84) | 2.74 (1.41–4.52) | ||
| Saline solution | 2.01 (1.53–2.56) | 2.61 (1.81–3.54) | 2.36 (1.77–3.04) | |||
| AIRg (mU × min × L−1) | EMS affected | LPS | 660 (357–1,055) | 309 (10–1,021) | 423 (206–719) | 0.114 |
| Saline solution | 533 (309–820) | 883 (518–1,345) | 821 (458–1,291) | |||
| Healthy | LPS | 255 (143–400) | 108 (22–258) | 333 (206–493) | ||
| Saline solution | 212 (87–394) | 260 (128–439) | 278 (147–453) | |||
| DI (× 10−2) | EMS affected | LPS | 1479 (603–2,743) | 250 (0.34–1,037) | 61 (25–116) | 0.487 |
| Saline solution | 622 (182–1,327) | 819 (285–1,628) | 522 (286–829) | |||
| Healthy | LPS | 770 (423–1,220) | 379 (45–1,039) | 339 (22–1,035) | ||
| Saline solution | 372 (108–796) | 923 (521–1,439) | 603 (341–942) | |||
Data are reported as geometric mean and 95% CI.
See Table 1 for remainder of key.
Correlations—Peak cytokine gene expression in the collected blood samples was negatively correlated with minimal model parameters at 0.5 and 21 hours. However, peak blood cytokine gene expression was positively correlated with AUCi at 21 hours (Table 4).
Spearman rank correlation coefficients for associations among peak blood cytokine gene expression values and minimal model analysis parameters (determined via FSIGTT) for the 6 healthy horses and 6 horses with EMS in Table 1 following IV bolus administration of LPS (20 ng/kg) or saline solution.
| Blood cytokine gene | SI (determined at 0.5 h after infusion) | SI (determined at 21 h after infusion) | Sg (determined at 21 h after infusion) | AUCi (determined at 21 h after infusion) |
|---|---|---|---|---|
| Interleukin-1β | –0.762 (0.028) | — | — | — |
| Interleukin-8 | –0.810 (0.015) | — | –0.762 (0.028) | 0.714 (0.047) |
| Interleukin-10 | –0.786 (0.021) | — | — | — |
| Tumor necrosis factor-α | — | –0.714 (0.047) | — | — |
Significance was set at P < 0.05, and P values are displayed in parentheses.
— = Not significant.
See Table 1 for remainder of key.
Discussion
In the present study, an IV infusion of LPS lowered insulin sensitivity but did not induce laminitis in healthy horses and horses with EMS. Horses with EMS had a transient decrease in pancreatic insulin secretion and greater loss of glycemic control following LPS administration. Our hypothesis that EMS impacts alterations in glucose and insulin dynamics induced by LPS administration was therefore supported.
Two methods were used to assess glucose and insulin dynamics from FSIGTT data in the present study. Area under the curve values for plasma glucose and serum insulin concentrations were calculated for each full FSIGTT procedure and for the first 19 minutes of each curve. Area under the curve values provide an estimation of insulin sensitivity and have been shown to be increased in insulin-resistant horses.31 Examination of insulin responses during the first 19 minutes of the FSIGTT procedure (before administration of exogenous insulin) provides information on the pancreatic response to a glucose challenge, and increased insulin secretion suggests pancreatic compensation for peripheral insulin resistance. Minimal model analysis was the other method used in this study. This is a compartmental model of glucose and insulin dynamics that partitions glucose disposal into glucose-dependent and insulin-dependent components.26,32 The SI is an index of peripheral insulin sensitivity and describes the ability of insulin to mediate glucose disposal. Glucose effectiveness describes the ability of glucose to stimulate its own disposal through mass effect and becomes increasingly important to glucose homeostasis when insulin-dependent glucose disposal is impaired.33,34 The AIRg provides an assessment of the pancreatic response to a glucose challenge and is calculated from values obtained in the first 10 minutes of the FSIGTT procedure, prior to administration of exogenous insulin. Disposition index is an assessment of whether pancreatic insulin secretion is adequately compensating for alterations in peripheral insulin sensitivity and is calculated by multiplying SI with AIRg.35
Mean baseline SI values for healthy horses in the present study compared favorably with reported values for lean light-breed horses.17,26,36 Baseline SI values for horses with EMS, however, did not differ from those for healthy horses and were higher than the means of 0.39 × 10−4 L × min−1 × mU−1 to 0.62 × 10−4 L × min−1 × mU−1 previously reported for a cohort of Arabian and Arabian-cross geldings in which obesity was induced through dietary manipulation.36 Values were also higher than those reported for obese Thoroughbred geldings (mean SI, 0.37 × 10−4 L × min−1 × mU−1) but were comparable to values obtained for moderately obese animals (mean SI, 1.47 × 10−4 L × min−1 × mU−1).26
One reason for this discrepancy in mean baseline SI values is that horses with EMS were part of an intensively managed research herd, and several horses had lost weight prior to enrollment in the present study. Weight loss has been shown to improve insulin sensitivity in horses and ponies.37,38 Although a cutoff value for determining insulin resistance in horses has not been established, SI values < 0.78 × 10−4 L × min−1 × mU−1 correspond to the lowest quintile reported for a group of 46 healthy horses and can be used as a guideline for interpreting results.39 Variability in baseline SI in both healthy horses and horses with EMS was evident between the first and second weeks of the study, and this might be a result of inherent variability in glucose and insulin homeostasis attributable to factors such as stress. Although mean baseline SI values did not differ between healthy horses and horses with EMS, a significant group effect was detected, indicating that SI was lower overall in horses with EMS as treatments were administered and procedures performed.
In the present study, AUCi (from 0 to 180 minutes) increased and SI values decreased in both healthy horses and horses with EMS at 21 hours after LPS infusion. These findings indicated that insulin resistance developed in response to endotoxin infusion and are consistent with previous studies16,17 by our research group that revealed insulin resistance in clinically normal horses at 20 to 24 hours after LPS administration at the same dose. In those studies,16,17 values of 0.9 × 10−4 L × min−1 × mU−1 and 0.63 × 10−4 L × min−1 × mU−1 were detected at 20 and 24 hours, respectively. In another report, Vick et al18 detected insulin resistance with a hyperinsulinemic-euglycemic clamp procedure in clinically normal mares 24 hours after endotoxin infusion, and inflammatory cytokine expression was also increased in blood and adipose tissue samples. Blood cytokine gene expression was also measured for 4 hours following LPS administration in the present study, and negative correlations were detected between peak cytokine expression and indices of glucose and insulin homeostasis at 21 hours.
Development of insulin resistance following LPS administration can be explained by the cross-talk that occurs between inflammatory cytokine and insulin signaling pathways, such that insulin sensitivity decreases during systemic inflammation.14 Serine kinases such as protein kinase C, c-Jun NH2-terminal kinase, and inhibitor of nuclear factor κ-B kinase are integral to inflammatory signaling and also induce insulin resistance via phosphorylation of serine residues in key components of the insulin signaling pathway.40 Although systemic inflammation might be expected to affect insulin sensitivity to a greater extent in horses with EMS, differences in SI between healthy horses and horses with EMS in the present study were not significant. Following LPS infusion, however, resting hyperinsulinemia was detected in all horses with EMS but only 1 healthy horse. Horses with EMS also had significantly higher AUCg (0 to 180 minutes) values than did healthy horses at 21 hours after LPS infusion.
Insulin sensitivity index values decreased over time in the study horses with EMS independent of treatment. Stress from hospitalization and testing procedures could have contributed to insulin resistance in these horses because decreased insulin sensitivity has been attributed to stress during prolonged stall confinement.17 Stress hormones including glucocorticoids and epinephrine decrease insulin sensitivity through mechanisms that include downregulation of proteins in the insulin signaling cascade and inhibition of glucose uptake and storage.41,42 In contrast, insulin sensitivity increased transiently in healthy horses with either treatment at 0.5 hours. Transient increases in insulin sensitivity and glucose utilization following LPS administration have been described for humans43,44 and horses,18 but the increased insulin sensitivity in horses administered saline solution is difficult to explain. Insulin sensitivity varied markedly in horses with EMS throughout the present study, which suggests that stress, diet, housing, or other factors influenced this variable. Horses with EMS may therefore be less able to adapt to situations such as hospitalization, and insulin resistance may be exacerbated under these conditions.
Both AUCi (0 to 19 minutes) and AIRg provide assessments of pancreatic responses to a glucose challenge. Increased pancreatic insulin secretion indicates compensation for peripheral insulin resistance.16 In the present study, AUCi (0 to 19 minutes) did not change over time in healthy horses following either treatment but transiently decreased in horses with EMS after infusion of LPS. This finding suggested that pancreatic insulin output was acutely inhibited, and this response has been detected 12 hours after LPS administration in rats.45 Results indicated that horses with EMS are more susceptible to suppression of pancreatic insulin output caused by inflammation.
Values for AIRg in healthy horses in the present study were within ranges reported for lean light-breed horses, although considerable variability exists among reports.16,26,36 In previous studies, AIRg in clinically normal horses ranged from a mean value of 206 mU × min × L−1 reported by Carter et al36 to 520 mU × min × L−1 reported by our research group for a cohort of 16 healthy horses.16 These values overlap with AIRg reported for obese Thoroughbred geldings (mean, 408 mU × min × L−1),26 and the upper limit of the range is consistent with baseline values obtained for horses with EMS in the present study. Direct comparisons among studies may be hampered by variability in study populations or differences in methods. Although not significant, AIRg was approximately 2- to 3-fold as high in horses with EMS as in healthy horses in the present study, suggesting a degree of compensated insulin resistance.2,36 Values for AIRg increased in horses with EMS at 0.5 and 21 hours after saline solution infusion and were comparable to those reported for obese light-breed horses (mean, 804 mU × min × L−1 to 973 mU × min × L−1).36 This finding suggests that horses with EMS had greater derangements in glucose and insulin homeostasis, which were potentially stress induced. Of note, AIRg did not increase at 21 hours after LPS administration in either healthy horses or horses with EMS, which contrasts with the finding from a previous study16 that pancreatic compensation occurs 24 hours after LPS administration in horses.
Glucose effectiveness has been identified as an important determinant of glucose tolerance in obese horses,26 but baseline values did not differ between groups in the present study. A decrease in Sg was detected in horses with EMS at 21 hours after infusion, independent of treatment. This differs from previous studies, in which Sg in clinically normal horses remained unaltered following LPS infusion.16,17 Reductions in Sg in response to epinephrine infusion in dogs have been reported,46 which supports the hypothesis that horses with EMS are more affected by physiologic stress than are healthy horses.
Although insulin secretion increased as a result of insulin resistance in both healthy horses and horses with EMS in the present study, glucose homeostasis was not maintained as effectively in horses with EMS after LPS was administered. Higher AUCg (0 to 180 minutes) values were detected in both groups of horses at 21 hours after LPS administration, and the magnitude of this increase was greater in horses with EMS, suggesting greater loss of glycemic control. Resting hyperglycemia following LPS administration was also detected in 4 of 5 horses with EMS, but in only 1 of 6 healthy horses. Changes in DI also supported inadequate pancreatic compensation and loss of glycemic control. Hyperglycemia can develop as a result of reduced tissue insulin sensitivity or inadequate pancreatic insulin secretion; it can also result from increased hepatic glucose production, and a major source of glucose during sepsis is hepatic gluconeogenesis.14,21 Insulin normally inhibits hepatic glucose output, but sepsis hinders this control mechanism by inducing hepatic insulin resistance.21,47 In a recent study48 by our research group, reduced hepatic insulin clearance was evident in horses with EMS, and this may have been a consequence of hepatic insulin resistance. Horses with EMS might be more susceptible to hyperglycemia during systemic illness, and this could contribute to the development of laminitis.
Mild chronic hyperglycemia can cause endothelial damage,11,12 which may explain why horses with EMS are at greater risk of developing laminitis during systemic inflammation. Hyperglycemia causes mitochondrial overproduction of reactive oxygen species in endothelial cells, leading directly to endothelial dysfunction.9 Reactive oxygen species incite inflammation and the production of inflammatory cytokines; they also promote vasoconstriction by reacting with and depleting nitric oxide9,49 and exert prothrombotic effects on vessels.9,14 Hyperglycemia augments the production of inflammatory cytokines in humans with sepsis50 and has been shown to increase matrix metalloproteinase-1, −2, and −9 expression in cultured endothelial cells and macrophages.51 Inflammation, vascular dysfunction, and increased matrix metalloproteinase activity are key features of acute laminitis22,23 and might therefore be exacerbated by hyperglycemia.
Insulin sensitivity was measured in the present study because microvascular dysfunction caused by insulin resistance plays an important role in the pathogenesis of cardiovascular disease in humans6 and could also contribute to development of laminitis in horses with EMS. However, studies52,53 have also revealed that hyperinsulinemia is detrimental to laminar tissues independently of insulin resistance, and exposure to supra-physiologic insulin concentrations for approximately 2 days induces laminitis in clinically normal horses and ponies. In the present study, resting hyperinsulinemia was detected in all horses with EMS and 1 healthy horse after LPS infusion. Although mean serum insulin concentrations used to induce laminitis under experimental conditions exceed 1,000 μU/mL and are not commonly encountered in clinical cases, it is possible that moderate increases in serum insulin concentration adversely affect the laminae after prolonged exposure. Histopathologic evidence of subclinical laminitis was recently detected in clinically normal horses receiving a 48-hour-long IV glucose infusion that maintained serum insulin concentrations between 100 and 300 μU/mL; these findings support a role for hyperinsulinemia, alone or in combination with hyperglycemia, in the development of pathological changes in the laminae of horses.54
One limitation of the present study was that the horses with EMS and the healthy horses did not differ significantly with respect to body condition score or baseline insulin sensitivity, although most horses with EMS retained abnormal characteristics such as generalized or regional adiposity. It is possible that greater differences would have been detected between groups if EMS-affected horses had been in an exacerbated state of insulin resistance or obesity. Small group sizes also limited the power of the study. Another limitation of this study was that stress was not assessed. Plasma ACTH, cortisol, and epinephrine concentrations can be measured to quantify stress, but these measurements were not performed at the time of the study, and samples were not retained for subsequent analysis. Sources of stress in this study included confinement, blood sample collection, and the liver biopsy procedure.
Despite limitations in study design, differences were detected between healthy horses and horses with EMS, particularly with regard to glycemic control and potentially their responses to stress. Equine metabolic syndrome is thought to have an underlying genetic basis,2,55 and development of the syndrome may rely upon interactions between individual horses and their environment. For example, laminitis-prone ponies have exaggerated insulin responses and decreased insulin sensitivity in response to high dietary carbohydrate intake.56,57 In humans, hyperglycemia during sepsis is sometimes the first indication of occult diabetes mellitus.21 Endotoxin challenge may therefore have unmasked inherent defects in insulin and glucose regulation in horses with EMS.
On the basis of the results of the present study, we conclude that EMS modulates glucose and insulin responses and adversely impacts glycemic control in horses following administration of LPS. Horses with EMS may also be more susceptible to derangements in glucose and insulin homeostasis as a result of stress, which could increase the risk of laminitis in stressful situations such as hospitalization. Further studies are required to examine the role of stress in the pathogenesis of laminitis.
ABBREVIATIONS
| AIRg | Acute insulin response to glucose |
| AUCg | Area under the curve for plasma glucose concentration |
| AUCi | Area under the curve for serum insulin concentration |
| B-Gus | β-glucuronidase |
| CI | Confidence interval |
| DI | Disposition index |
| EMS | Equine metabolic syndrome |
| FSIGTT | Insulin-modified frequently sampled IV glucose tolerance test |
| IL | Interleukin |
| LPS | Lipopolysaccharide |
| Sg | Glucose effectiveness |
| SI | Insulin sensitivity index |
| TNF | Tumor necrosis factor |
Escherichia coli O55:B5 LPS solution, Sigma Aldrich Inc, St Louis, Mo.
Abbocath-T, 14 gauge × 140 mm, Abbott Laboratories, North Chicago, Ill.
Vedco Inc, St Joseph, Mo.
Humulin R, Eli Lilly and Co, Indianapolis, Ind.
Glucose, Roche Diagnostic Systems Inc, Somerville, NJ.
Cobas Mira, Roche Diagnostic Systems Inc, Somerville, NJ.
Coat-A-Count Insulin, Diagnostic Products Corp, Los Angeles, Calif.
MinMod Millennium, version 6.10, Raymond Boston, University of Pennsylvania, Kennett Square, Pa.
Stata, version 9.2, Stata Corp, College Station, Tex.
PROC MIXED, SAS, version 9.2, SAS Institute Inc, Cary, NC.
SAS, version 9.2, SAS Institute Inc, Cary, NC.
Coat-A-Count insulin radioimmunoassay, Diagnostic Products Corp, Los Angeles, Calif.
Clinical Pathology Laboratory, University of Tennessee, Knoxville, Tenn.
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