Comparison among the euglycemic-hyperinsulinemic clamp, insulin-modified frequently sampled intravenous glucose tolerance test, and oral glucose tolerance test for assessment of insulin sensitivity in healthy Standardbreds.

OBJECTIVE: To compare, in horses, estimates of insulin sensitivity obtained from minimal model analysis (MMA) of a frequently sampled IV glucose tolerance test (FSIGTT) with estimates from the euglycemic-hyperinsulinemic clamp (EHC) and to evaluate the validity of surrogate estimates of insulin sensitivity derived from an oral glucose tolerance test (OGTT). ANIMALS: 18 mature Standardbreds (mean ± SD body weight, 428.9 ± 35.9 kg; mean ± SD body condition score, 4.4 ± 1.0 [on a scale of 1 to 9]). PROCEDURES: All horses underwent at least 2 of the 3 procedures (EHC [n = 15], insulin-modified FSIGTT [18], and OGTT [18]) within a 10-day time frame to evaluate insulin sensitivity. RESULTS: Insulin sensitivity variables derived from the EHC and FSIGTT were strongly correlated (r = 0.88). When standardized to the same units of measure, these measures were still strongly correlated (r = 0.86) but were not equivalent. Area under the curve, peak insulin concentration, insulin concentration at 120 minutes, and 2 calculated indices from glucose and insulin data from the OGTT were significantly correlated with the EHC- and FSIGTT-derived estimates of insulin sensitivity. CONCLUSIONS AND CLINICAL RELEVANCE: In healthy Standardbreds with moderate body condition score, insulin sensitivities from the EHC and FSIGTT were strongly correlated but not equivalent. Estimates derived from an OGTT also may be useful to estimate insulin sensitivity.

Effects of acute hyperinsulinemia on inflammatory proteins in horses.

Laminitis is a painful, inflammatory disease of the equine hoof that often results in euthanasia. Elevated plasma insulin concentrations are a predictive factor for laminitis, and in previously healthy horses and ponies, laminitis was induced by infusion of insulin. Thus, we chose to determine if an infusion of insulin would increase plasma concentrations of inflammatory cytokines and cytokine mRNA abundance in subcutaneous adipose tissue, skeletal muscle, and white blood cells. Ten mature Thoroughbred mares received an insulin infusion that elevated plasma insulin concentrations for 6h or an equivalent volume of isotonic saline in a switchback design. Insulin infusion altered plasma concentrations of both TNF (P=0.037) and IL-6 (P=0.044), but did not result in consistent changes to either skeletal muscle or adipose tissue cytokine mRNA. Insulin may be involved in the production of inflammatory cytokines, and this could be a mechanism for insulin increasing the risk of laminitis.

Glucose and insulin dynamics associated with continuous rate infusion of dextrose solution or dextrose solution and insulin in healthy and endotoxin-exposed horses.

OBJECTIVE: To investigate the effects of a continuous rate infusion (CRI) of dextrose solution or dextrose solution and insulin on glucose and insulin concentrations in healthy and endotoxin-exposed horses. ANIMALS: 9 adult mares. PROCEDURES: During phase 1, treatments consisted of saline (0.9% NaCl) solution (control group; n = 4) or 20% dextrose solution (group 1; 4) administered IV as a 360-minute CRI. During phase 2, treatments consisted of 360-minute CRIs of 20% dextrose solution and insulin administered simultaneously at 367.6 mg/kg/h (30 kcal/kg/d) and 0.07 U/kg/h, respectively, in healthy horses (group 2; n = 4) or horses administered 35 ng of lipopolysaccharide/kg, IV, 24 hours before starting the dextrose solution and insulin CRIs (group 3; 4). A balanced crossover study design was used in both phases. Blood samples were collected for measurement of plasma glucose and insulin concentrations. RESULTS: Infusion of dextrose solution alone resulted in hyperglycemia for most of the 360-minute CRI. Insulin concentration increased significantly in group 1, compared with that in the control group. Mean insulin concentration of group 2 was significantly higher throughout most of the infusion period, compared with concentrations of the control group and group 1. Mean glucose concentration did not differ significantly between groups 2 and 3. CONCLUSIONS AND CLINICAL RELEVANCE: Insulin infusion at a rate of 0.07 U/kg/h was found to be effective for the prevention of hyperglycemia when administered concurrently with dextrose solution. This rate was considered to be safe because horses did not become hypoglycemic during infusions of dextrose solution.

Effect of feeding a high-protein diet following an 18-hour period of feed withholding on mammalian target of rapamycin-dependent signaling in skeletal muscle of mature horses.

OBJECTIVE: To determine the effect of refeeding following an 18-hour period of feed withholding on the phosphorylation of translation initiation factors in the skeletal muscle of mature horses. ANIMALS: 8 adult horses. PROCEDURES: Following an 18-hour period of feed withholding, horses either continued to have feed withheld (postabsorptive state) or were fed 2 g/kg of a high-protein feed (33% crude protein) at time 0 and 30 minutes (postprandial state). Blood samples were taken throughout the experimental period. At 90 minutes, a biopsy specimen was taken from the middle gluteal muscle to measure the phosphorylation of translation initiation factors and tissue amino acid concentrations. Plasma glucose, insulin, and amino acid concentrations were also measured. RESULTS: Horses in the postprandial state had significantly higher plasma insulin, glucose, and amino acid concentrations than did those in the postabsorptive state at the time of biopsy. Refeeding significantly increased the phosphorylation state of riboprotein S6 and eukaryotic initiation factor 4E binding protein 1. CONCLUSIONS AND CLINICAL RELEVANCE: In mature horses, feeding resulted in increased mammalian target of rapamycin signaling and the mechanism appeared to be independent of an increase in Akt phosphorylation at Ser47³. Results indicate that adult horses may be able to increase rates of muscle protein synthesis in response to feeding and that dietary amino acids appear to be the main mediators of this effect.

Effects of exercise training on adiposity, insulin sensitivity, and plasma hormone and lipid concentrations in overweight or obese, insulin-resistant horses.

OBJECTIVE: To determine effects of exercise training without dietary restriction on adiposity, basal hormone and lipid concentrations and glucose and insulin dynamics in overweight or obese, insulin-resistant horses. ANIMALS: 12 overweight or obese (body condition score > or = 7), insulin-resistant (insulin sensitivity < or = 1.2 x 10(-4) L/min/mU) geldings. PROCEDURES: 4 horses remained sedentary, and 8 horses were exercised for 4 weeks at low intensity and 4 weeks at higher intensity, followed by 2 weeks of detraining. Prior to and after each training period, frequently sampled IV glucose tolerance tests with minimal model analysis were performed and baseline plasma insulin, glucose, triglycerides, non-esterified fatty acids, and leptin concentrations were analyzed. Adiposity was assessed by use of morphometrics, ultrasonic subcutaneous fat thickness, and estimation of fat mass from total body water (deuterium dilution method). RESULTS: Body weight and fat mass decreased by 4% (mean +/- SD, 20 +/- 8 kg) and 34% (32 +/- 9 kg), respectively, compared with pre-exercise values, with similar losses during low- and higher-intensity training. There was no effect of exercise training on subcutaneous fat thickness, plasma hormone and lipid concentrations, or minimal model parameters of glucose and insulin dynamics. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that moderate exercise training without concurrent dietary restriction does not mitigate insulin resistance in overweight or obese horses. A more pronounced reduction in adiposity or higher volume or intensity of exercise may be necessary for improvement in insulin sensitivity in such horses.

Effects of diet-induced weight gain on insulin sensitivity and plasma hormone and lipid concentrations in horses.

OBJECTIVE: To determine the effects of diet-induced weight gain on glucose and insulin dynamics and plasma hormone and lipid concentrations in horses. ANIMALS: 13 adult geldings. PROCEDURES: Horses were fed 200% of their digestible energy requirements for maintenance for 16 weeks to induce weight gain. Frequently sampled IV glucose tolerance tests were performed before and after weight gain to evaluate glucose and insulin dynamics. Adiposity (assessed via condition scoring, morphometric measurements, and subcutaneous fat depth) and plasma concentrations of insulin, glucose, nonesterified fatty acids, triglycerides, and leptin were measured on a weekly or biweekly basis. RESULTS: Mean + or - SD body weight increased by 20% from 440 + or - 44 kg to 526 + or - 53 kg, and body condition score (scale, 1 to 9) increased from 6 + or - 1 to 8 + or - 1. Plasma glucose, triglyceride, and nonesterified fatty acid concentrations were similar before and after weight gain. Leptin and insulin concentrations increased with weight gain. Mean + or - SD insulin sensitivity decreased by 71 + or - 28%, accompanied by a 408 + or - 201% increase in acute insulin response to glucose, which resulted in similar disposition index before and after weight gain. CONCLUSIONS AND CLINICAL RELEVANCE: Diet-induced weight gain in horses occurred concurrently with decreased insulin sensitivity that was effectively compensated for by an increase in insulin secretory response. Obesity resulted in hyperinsulinemia and hyperleptinemia, compared with baseline values, but no changes in lipid concentrations were apparent. Preventing obesity is a potential strategy to help avoid insulin resistance, hyperinsulinemia, and hyperleptinemia in horses.

Effects of dexamethasone administration on insulin resistance and components of insulin signaling and glucose metabolism in equine skeletal muscle.

OBJECTIVE: To determine the effects of dexamethasone treatment on selected components of insulin signaling and glucose metabolism in skeletal muscle obtained from horses before and after administration of a euglycemic-hyperinsulinemic clamp (EHC). ANIMALS: 6 adult Standardbreds. PROCEDURES: In a balanced crossover study, horses received either dexamethasone (0.08 mg/kg, IV, q 48 h) or an equivalent volume of saline (0.9% NaCl) solution, IV, for 21 days. A 2-hour EHC was administered for measurement of insulin sensitivity 1 day after treatment. Muscle biopsy specimens obtained before and after the EHC were analyzed for glucose transporter 4, protein kinase B (PKB), glycogen synthase kinase (GSK)-3alpha/beta protein abundance and phosphorylation state (PKB Ser(473) and GSK-3alpha/beta Ser(21/9)), glycogen synthase and hexokinase enzyme activities, and muscle glycogen concentration. RESULTS: Dexamethasone treatment resulted in resting hyperinsulinemia and a significant decrease (70%) in glucose infusion rate during the EHC. In the dexamethasone group, increased hexokinase activity, abrogation of the insulin-stimulated increase in glycogen synthase fractional velocity, and decreased phosphorylation of GSK-3alpha Ser(21) and GSK-3B Ser(9) were detected, but there was no effect of dexamethasone treatment on glucose transporter 4 content and glycogen concentration or on PKB abundance and phosphorylation state. CONCLUSIONS AND CLINICAL RELEVANCE: In horses, 21 days of dexamethasone treatment resulted in substantial insulin resistance and impaired GSK-3 phosphorylation in skeletal muscle, which may have contributed to the decreased glycogen synthase activity seen after insulin stimulation.

Effects of dexamethasone on glucose dynamics and insulin sensitivity in healthy horses.

OBJECTIVE: To determine effects of dexamethasone on glucose dynamics and insulin sensitivity in healthy horses. ANIMALS: 6 adult Standardbreds. PROCEDURES: In a balanced crossover study, horses received dexamethasone (0.08 mg/ kg, IV, q 48 h) or an equivalent volume of saline (0.9% NaCl) solution (control treatment) during a 21-day period. Horses underwent a 3-hour frequently sampled IV glucose tolerance test (FSIGT) 2 days after treatment. Minimal model analysis of glucose and insulin data from FSIGTs were used to estimate insulin sensitivity (Si), glucose effectiveness (Sg), acute insulin response to glucose (AIRg), and disposition index. Proxies for Si (reciprocal of the inverse square of basal insulin concentration [RISQI]) and beta-cell responsiveness (modified insulin-to-glucose ratio [MIRG]) were calculated from basal plasma glucose and serum insulin concentrations. RESULTS: Mean serum insulin concentration was significantly higher in dexamethasone-treated horses than control horses on days 7, 14, and 21. Similarly, mean plasma glucose concentration was higher in dexamethasone-treated horses on days 7, 14, and 21; this value differed significantly on day 14 but not on days 7 or 21. Minimal model analysis of FSIGT data revealed a significant decrease in Si and a significant increase in AIRg after dexamethasone treatment, with no change in Sg or disposition index. Mean RISQI was significantly lower, whereas MIRG was higher, in dexamethasone-treated horses than control horses on days 7, 14, and 21. CONCLUSIONS AND CLINICAL RELEVANCE: The study revealed marked insulin resistance in healthy horses after 21 days of dexamethasone administration. Because insulin resistance has been associated with a predisposition to laminitis, a glucocorticoid-induced decrease in insulin sensitivity may increase risk for development of laminitis in some horses and ponies.

Time course of insulin sensitivity and skeletal muscle glycogen synthase activity after a single bout of exercise in horses.

The time course of insulin sensitivity, skeletal muscle glycogen and GLUT4 content, and glycogen synthase (GS) activity after a single bout of intense exercise was examined in eight horses. On separate days, a euglycemic-hyperinsulinemic clamp (EHC) was undertaken at 0.5, 4, or 24 h after exercise or after 48 h of rest [control (Con)]. There was no increase in mean glucose infusion rate (GIR) with exercise (0.5-, 4-, and 24-h trials), and GIR was significantly decreased at 0.5 h postexercise (GIR: 8.6 +/- 2.7, 6.7 +/- 2.0, 9.0 +/- 2.0, and 10.6 +/- 2.2 mg.kg(-1).min(-1) for Con and at 0.5, 4, and 24 h, respectively). Before each EHC, muscle glycogen content (mmol glucosyl units/kg dry muscle) was higher (P < 0.05) for Con (565 +/- 102) than for other treatments (317 +/- 84, 362 +/- 79, and 382 +/- 74 for 0.5, 4, and 24 h, respectively) and muscle GLUT4 content was unchanged. Pre-EHC active-to-total GS activity ratio was higher (P < 0.05) at 0.5, 4, and 24 h after exercise than in Con. Post-EHC active GS and GS activity ratio were higher (P < 0.05) in Con and at 24 h. There was a significant inverse correlation (r = -0.43, P = 0.02) between glycogen content and GS activity ratio but no relationship between GS activity and GIR. The lack of increase in insulin sensitivity, determined by EHC, after exercise that resulted in a significant reduction in muscle glycogen content is consistent with the slow rate of muscle glycogen resynthesis observed in equine studies.

Repeatability of 2 methods for assessment of insulin sensitivity and glucose dynamics in horses.

Both the euglycemic-hyperinsulinemic clamp (EHC) and minimal model analysis of the frequently sampled intravenous glucose tolerance test (FSIGT) have been applied for measurement of insulin sensitivity in horses. However, no published data are available on the reproducibility of these methods. Therefore, the objective of this study was to evaluate the variation and repeatability of measures of glucose dynamics and insulin sensitivity in horses derived from minimal model analysis of the FSIGT and from the EHC method. Six healthy horses underwent both the FSIGT and EHC on 2 occasions over a 4-week period, with a minimum of 5 days between tests. Coefficient of variation (CV) and intraclass correlation coefficient (ICC) were calculated for measures of glucose metabolism and insulin sensitivity derived from each test. In the EHC, insulin sensitivity, expressed as the amount of metabolized glucose (M) per unit of serum insulin (I) (M/I ratio), averaged 0.19 +/- 0.06 x 10(-4) mmol/kg/min x (pmol/L)(-1) with an average interday CV of 14.1 +/- 5.7% (range, 7-20%) and ICC of 0.74. Minimal model analysis of the FSIGT demonstrated mean insulin sensitivity (Si) of 0.49 +/- 0.17 x 10(-4)/min x (pmol/L)(-1) with an average interday CV of 23.7 +/- 11.2% (range, 9-35%) and ICC of 0.33. Mean CV and ICC for minimal model glucose effectiveness (Sg) and acute insulin response (AIRg) were, respectively, 26.4 +/- 11.2% (range 13-40%) and 0.10 and 11.7 +/- 6.5% (range 7-21%) and 0.98. Insulin sensitivity measured by the EHC has lower interday variation when compared with the minimal model estimate derived from the FSIGT.

Effects of adenosine pretreatment on detection of free radicals in ischemic and reperfused canine gracilis muscle flaps by use of spin-trapping electron paramagnetic resonance spectroscopy.

OBJECTIVE: To determine whether adenosine pretreatment attenuates free radical production and muscle damage in ischemic and reperfused canine skeletal muscle. ANIMALS: 9 healthy mixed-breed dogs. PROCEDURE: Dogs were anesthetized, and both gracilis muscles were isolated, leaving only the major vascular pedicle intact. Saline (0.9% NaCl) solution was injected into the artery supplying the control flap, whereas adenosine (10 mg) was injected into the contralateral artery. Ischemia was induced in both flaps for 4 hours. alpha-Phenyl-N-tert-butylnitrone was administered IV to each dog 1 hour prior to reperfusion. Following 15 minutes of reperfusion, effluent blood samples from each muscle flap were obtained and processed for spin-trapping electron paramagnetic resonance (EPR) spectroscopy. Muscle biopsy specimens were obtained for histologic evaluation, and dogs were euthanatized. RESULTS: EPR spectra of strong intensity were obtained from analysis of 5 of 9 paired samples. Signals identified were characteristic of oxygen- and carbon-centered free radical adducts. Signal intensity of spectra from adenosine-treated flaps was significantly less than that of control flaps; mean signal attenuation was 36% in the adenosine-treated group. Histologic evaluation of muscle flaps did not reveal significant differences between groups. CONCLUSIONS AND CLINICAL RELEVANCE: Treatment of canine muscle flaps with adenosine prior to a period of ischemia reduced but did not completely attenuate free radical production after reperfusion. However, adenosine pretreatment did not affect histologic abnormalities.