Designed protein-protein association

The analysis of natural contact interfaces between protein subunits and between proteins has disclosed some general rules governing their association. We have applied these rules to produce a number of novel assemblies, demonstrating that a given protein can be engineered to form contacts at various points of its surface. Symmetry plays an important role because it defines the multiplicity of a designed contact and therefore the number of required mutations. Some of the proteins needed only a single side-chain alteration in order to associate to a higher-order complex. The mobility of the buried side chains has to be taken into account. Four assemblies have been structurally elucidated. Comparisons between the designed contacts and the results will provide useful guidelines for the development of future architectures.     

Evaluation of genetic and metabolic predispositions and nutritional risk factors for pasture-associated laminitis in ponies

OBJECTIVE: To evaluate genetic and metabolic predispositions and nutritional risk factors for development of pasture-associated laminitis in ponies. DESIGN: Observational cohort study. ANIMALS: 160 ponies. PROCEDURES: A previous diagnosis of laminitis was used to differentiate 54 ponies (PL group) from 106 nonlaminitic ponies (NL group). Pedigree analysis was used to determine a mode of inheritance for ponies with a previous diagnosis of laminitis. In early March, ponies were weighed and scored for body condition and basal venous blood samples were obtained. Plasma was analyzed for glucose, insulin, triglycerides, nonesterified fatty acids, and cortisol concentrations. Basal proxies for insulin sensitivity (reciprocal of the square root of insulin [RISQI]) and insulin secretory response (modified insulin-to-glucose ratio [MIRG]) were calculated. Observations were repeated in May, when some ponies had signs of clinical laminitis. RESULTS: A previous diagnosis of laminitis was consistent with the expected inheritance of a dominant major gene or genes with reduced penetrance. A prelaminitic metabolic profile was defined on the basis of body condition, plasma triglyceride concentration, RISQI, and MIRG. Meeting > or = 3 of these criteria differentiated PL- from NL-group ponies with a total predictive power of 78%. Determination of prelaminitic metabolic syndrome in March predicted 11 of 13 cases of clinical laminitis observed in May when pasture starch concentration was high. CONCLUSIONS AND CLINICAL RELEVANCE: Prelaminitic metabolic syndrome in apparently healthy ponies is comparable to metabolic syndromes in humans and is the first such set of risk factors to be supported by data in equids. Prelaminitic metabolic syndrome identifies ponies requiring special management, such as avoiding high starch intake that exacerbates insulin resistance.     

Insulin sensitivity and glucose dynamics during pre-weaning foal development and in response to maternal diet composition

Nutritional management of animals during pregnancy can affect glucose and insulin dynamics in the resulting offspring through influences on fetal development. Additionally, high starch feeding in mature horses is associated with reduced insulin sensitivity and an increased risk for diseases such as obesity and laminitis. However, no study has yet evaluated the effect of feeding a high starch diet to pregnant mares on glucose and insulin dynamics in their offspring. Twenty late-gestation mares maintained on pasture were provided two-thirds of digestible energy requirements from isocaloric, isonitrogenous low starch (LS, n = 10) or high starch (HS, n = 10) feed. Their foals were assessed with an insulin-modified frequently sampled intravenous glucose tolerance test at 5, 40, 80, and 160 d of age. Baseline glucose concentrations, insulin sensitivity, and insulin-independent glucose clearance in 5-d foals were all greater than values observed in mature horses and declined towards mature values as foals reached 160 d of age. Baseline glucose concentrations were all within normal range, but higher in foals born from HS mares through 80 d of age. Insulin sensitivity was not different between dietary groups until a trend for lower insulin sensitivity in HS foals emerged at 160 d of age. These data are the first to characterize decreasing insulin sensitivity and glucose tolerance in Thoroughbred foals from 5 to 160 d of age. This study also presents the first data examining glucose and insulin dynamics in developing foals in response to maternal high starch diet.     

Comparison of Insulin Sensitivity of Horses Adapted to Different Exercise Intensities

Diets high in concentrates and soluble carbohydrates are associated with reduced insulin sensitivity in horses. Exercise training could protect against diet-induced insulin resistance. The objective of this study was to determine the intensity of exercise training required to affect insulin sensitivity in stabled horses fed a diet high in concentrates but moderate in soluble carbohydrates. In all, 31 stabled horses underwent three different exercise regimens: turnout, light exercise, and moderate exercise, while being fed a diet containing 60% concentrate. Blood was sampled monthly and analyzed for insulin. Insulin sensitivity was assessed using basal insulin concentrations and calculated insulin sensitivity (reciprocal of the square root of insulin) and compared across months by analysis of variance with repeated measures. Insulin sensitivity (reciprocal of the square root of insulin) was higher during periods of moderate and light physical activity as compared with turnout. These results indicate that turnout alone may not be adequate to improve insulin sensitivity in horses fed high amounts of concentrate.     

Use of proxies and reference quintiles obtained from minimal model analysis for determination of insulin sensitivity and pancreatic beta-cell responsiveness in horses

OBJECTIVE: To develop proxies calculated from basal plasma glucose and insulin concentrations that predict insulin sensitivity (SI; L.min(-1) x mU(-1)) and beta-cell responsiveness (ie, acute insulin response to glucose [AIRg]; mU/L x min(-1)) and to determine reference quintiles for these and minimal model variables. ANIMALS: 1 laminitic pony and 46 healthy horses. PROCEDURE: Basal plasma glucose (mg/dL) and insulin (mU/L) concentrations were determined from blood samples obtained between 8:00 AM and 9:00 AM. Minimal model results for 46 horses were compared by equivalence testing with proxies for screening SI and pancreatic beta-cell responsiveness in humans and with 2 new proxies for screening in horses (ie, reciprocal of the square root of insulin [RISQI] and modified insulin-to-glucose ratio [MIRG]). RESULTS: Best predictors of SI and AIRg were RISQI (r = 0.77) and MIRG (r = 0.75) as follows: SI = 7.93(RISQI) - 1.03 and AIRg = 70.1(MIRG) - 13.8, where RISQI equals plasma insulin concentration(-0.5) and MIRG equals [800 - 0.30(plasma insulin concentration 50)(2)]/(plasma glucose concentration - 30). Total predictive powers were 78% and 80% for RISQI and MIRG, respectively. Reference ranges and quintiles for a population of healthy horses were calculated nonparametrically. CONCLUSIONS AND CLINICAL RELEVANCE: Proxies for screening SI and pancreatic beta-cell responsiveness in horses from this study compared favorably with proxies used effectively for humans. Combined use of RISQI and MIRG will enable differentiation between compensated and uncompensated insulin resistance. The sample size of our study allowed for determination of sound reference range values and quintiles for healthy horses.     

Insulin resistance and compensation in Thoroughbred weanlings adapted to high-glycemic meals

Insulin resistance has been suggested to increase the risk of certain diseases, including osteochondrosis and laminitis. Our objective was to evaluate the effect of adaptation to high-glycemic meals on glucose-insulin regulation in healthy Thoroughbred weanlings. Twelve Thoroughbred foals were raised on pasture and supplemented twice daily with a feed high in either sugar and starch (SS; 49% nonstructural carbohydrates, 21% NDF, 3% crude fat on a DM basis) or fat and fiber (FF; 12% nonstructural carbohydrates, 44% NDF, 10% crude fat on a DM basis). As weanlings (age 199 +/- 5 d; BW 274 +/- 5 kg) the subjects underwent a modified frequently sampled i.v. glucose tolerance test. A series of 39 blood samples was collected from -60 to 360 min, with a glucose bolus of 300 mg/kg BW injected at 0 min and an insulin bolus of 1.5 mIU/kg BW at 20 min. All samples were analyzed for glucose and insulin, and basal samples also were analyzed for plasma cortisol, triglyceride, and IGF-I. The minimal model of glucose and insulin dynamics was used to determine insulin sensitivity (SI), glucose effectiveness, acute insulin response to glucose (AIRg), and disposition index (DI). Insulin sensitivity was 37% less (P = 0.007) in weanlings fed SS than in those fed FF; however, DI did not differ (P = 0.65) between diets because AIRg tended to be negatively correlated with SI (r = -0.55; P = 0.067). This finding indicates that the SI decrease was compensated by AIRg in the weanlings adapted to SS. This compensation was further demonstrated by greater insulin concentrations in SS-adapted weanlings compared with FF-adapted weanlings at 11 of 36 sample points (P < 0.055) and greater (P = 0.040) total area under the insulin curve in SS than in FF weanlings. Plasma cortisol and triglycerides did not differ between dietary groups, but IGF-I was greater (P = 0.001) in SS weanlings. Despite appearing healthy, horses adapted to high-glycemic feeds may exhibit changes in altered insulin sensitivity and compensation that increase the risk of diseases involving insulin resistance. These changes seem to be partially amenable to dietary management.