A high protein meal affects plasma insulin concentrations and amino acid metabolism in horses with equine metabolic syndrome.

Equine metabolic syndrome (EMS) is characterized by an abnormal insulin response to a glycemic challenge but despite the known insulinotropic effects of certain amino acids, there is a paucity of data evaluating the impact of dietary protein on insulin dynamics in these horses. The objective was therefore to assess insulin and amino acid responses following intake of a high protein meal in healthy horses and those with EMS. Six mature horses diagnosed with EMS and six age-matched control horses without EMS were used. Horses were fed 2g/kg body mass (BM) of a high protein pellet (31% crude protein) at time 0 and 30min, for a total of 4g/kg BM, following an overnight fast. Blood samples collected during a 4h period were analysed for plasma glucose, insulin, amino acids and urea concentrations. Glucose concentrations were not different between groups (P=0.2). Horses with EMS had a 9-fold greater insulinemic response to the consumption of a high protein meal compared with controls (P=0.046). Post-prandial levels of histidine, citrulline, tyrosine, valine, methionine, isoleucine, leucine and ornithine were higher in horses with EMS (P<0.05). Baseline urea nitrogen concentrations were not significantly different between groups (P=0.1). Knowing that certain amino acids are insulin secretagogues, these results illustrate that consumption of a high protein meal caused a hyperinsulinemic response and affected amino acid dynamics in horses with EMS. These findings suggest that dietary protein content should be taken into consideration in the management of horses with insulin dysregulation.

Controlled trial of whole body protein synthesis and plasma amino acid concentrations in yearling horses fed graded amounts of lysine.

Lysine has been reported as the first limiting amino acid in typical equine diets. Indicator amino acid oxidation (IAAO) has become the standard method for determining amino acid requirements in other species, but prior to this study, it has not been used to determine equine requirements. The aim of this study was to evaluate whole body protein synthesis and plasma and muscle amino acid concentrations in response to graded levels of lysine intake in yearling horses. Six Thoroughbred colts (358 ± 5 kg) were fed each of six treatment lysine intakes ranging from 76 to 136 mg/kg body weight/day. Blood samples were taken before and 90 min after the morning concentrate meal. Gluteal muscle biopsies were taken ~100 min after the morning concentrate meal. The next day, whole body phenylalanine kinetics were determined using a 2 h primed, constant infusion of [(13)C] sodium bicarbonate followed by a 6 h primed, constant infusion of [1-(13)C] phenylalanine. Plasma lysine concentrations increased linearly (P <0.05) at both the 0 and 90 min time points with increasing lysine intakes. Free muscle asparagine, aspartate, arginine, glutamine, lysine, taurine and tryptophan concentrations responded quadratically to lysine intake (P <0.05). Phenylalanine kinetics did not differ between treatment intakes (P > 0.10). A broken line analysis of lysine intake and phenylalanine oxidation failed to yield a breakpoint from which to determine a lysine requirement. These diets may have been limiting in an amino acid other than lysine, underscoring the lack of data concerning amino acid requirements and bioavailability data in the horse.

Splanchnic extraction of phenylalanine in mature mares was not affected by threonine supplementation.

This study determined splanchnic extraction of phenylalanine at two intakes of threonine. Six Thoroughbred mares were supplemented with isonitrogenous amounts of either threonine or glutamate. Dietary threonine intakes were 119 (+Thr) and 58 (Basal) mg/kg/day, respectively. Each horse received each diet twice and each was studied once with an oral and once with an intravenous (IV) infusion of [1-(13)C]phenylalanine. A 2-h primed, constant IV infusion of [(13)C]sodium bicarbonate and a 4-h primed, constant infusion of [1-(13)C]phenylalanine, either orally or IV, were used to measure isotopic enrichments. Phenylalanine kinetics were not affected by diet (P > 0.05). Values for the splanchnic extraction of phenylalanine were 26 ± 5% and 27 ± 3% for the +Thr and Basal supplemented diets, respectively. These values will improve the accuracy of future equine indicator amino acid oxidation studies.

Effects of threonine supplementation on whole-body protein synthesis and plasma metabolites in growing and mature horses.

Current equine threonine requirement estimates do not account for probable use of threonine to maintain gut health and mucin synthesis. The objective of this study was to determine if threonine supplementation (+Thr) would increase whole-body protein synthesis (WBPS) in weanling colts (Study 1) and adult mares (Study 2). Both studies used a crossover design, where each of six animals was studied twice while receiving the isonitrogenous diets. The basal diets contained lower threonine levels (Basal) than the threonine (+Thr) supplemented diets. Threonine intakes in mg/kg BW/day were as follows: 79 (Basal) and 162 (+Thr) for Study 1 and 58 (Basal) and 119 (+Thr) for Study 2, in comparison to the NRC estimated requirements of 81 and 33 mg/kg BW/day for weanling and mature horses, respectively. Following 5 days of adaptation, blood samples were taken before and 90 min after the morning concentrate meal. The next day, whole-body phenylalanine kinetics were determined using a 2 h primed, constant infusion of [(13)C]sodium bicarbonate followed by a 4 h primed, constant infusion of [1-(13)C]phenylalanine. Most plasma amino acid (AA) concentrations were elevated post-feeding (P < 0.01). Lysine and valine plasma concentrations were lower (P <0.10), while methionine, threonine, and glycine plasma concentrations were greater (P <0.10) 90 min post concentrate meal feeding with +Thr in both studies. Phenylalanine flux, intake, oxidation and non-oxidative disposal were similar between treatments (P > 0.05). These findings suggest that supplementation of a single AA can affect the metabolism of several AAs and threonine was not a limiting AA in these diets.

Pituitary pars intermedia dysfunction does not necessarily impair insulin sensitivity in old horses.

Equine pituitary pars intermedia dysfunction (PPID) has been associated with reduced insulin sensitivity in comparison with younger adult horses; however, the difference in insulin sensitivity between horses with PPID and aged-matched controls has not been well characterized. The objective of the study was to determine if aged horses with PPID had reduced insulin sensitivity and alterations in the insulin-mediated signaling pathways in the skeletal muscle when compared with healthy aged horses. Isoglycemic hyperinsulinemic clamp procedures were conducted in 12 horses that were classified as either PPID (n = 6; age: 25.0 ± 2.5 yr; mean ± standard deviation) or non-PPID, aged-matched controls (control) (n = 6; age: 25.7 ± 2.0 yr). Blood samples were taken before and during the clamp procedures to measure plasma glucose, insulin, and amino acid concentrations, and 2 muscle biopsies were collected from the gluteus medius muscle, one in the basal state and the second at the end of the clamp procedure (insulin-stimulated state). Plasma insulin concentrations increased ∼9-fold during the clamp compared with basal conditions (P < 0.001) in both groups. During the last 30 min of the clamp, the rate of glucose infusion required to maintain isoglycemia in horses with PPID was similar to that in the control horses (P = 0.67). The plasma concentrations of most indispensible amino acids were lower in the insulin-stimulated state than the basal state (P < 0.05). PPID status did not have an effect on the activation of factors associated with protein synthesis and breakdown; however, factors associated with protein synthesis had increased phosphorylation in the insulin-stimulated state, compared with basal. The results from this study provide evidence that PPID is not always associated with impairments in insulin sensitivity.

Dietary crude protein intake influences rates of whole-body protein synthesis in weanling horses.

The objective of this study was to measure whole-body protein kinetics in weanling horses receiving forage and one of two different concentrates: (1) commercial crude protein (CCP) concentrate, which with the forage provided 4.1 g CP/kg bodyweight (BW)/day (189 mg lysine (Lys)/kg BW/day), and (2) recommended crude protein (RCP) concentrate which, with the same forage, provided 3.1 g CP/kg BW/day (194 mg Lys/kg BW/day). Blood samples were taken to determine the response of plasma amino acid concentrations to half the daily concentrate allocation. The next day, a 2 h-primed, constant infusion of [(13)C]sodium bicarbonate and a 4 h-primed, constant infusion of [1-(13)C]phenylalanine were used with breath and blood sampling to measure breath (13)CO2 and blood [(13)C]phenylalanine enrichment. Horses on the CCP diet showed an increase from baseline in plasma isoleucine, leucine, lysine, threonine, valine, alanine, arginine, asparagine, glutamine, ornithine, proline, serine, and tyrosine at 120 min post-feeding. Baseline plasma amino acid concentrations were greater with the CCP diet for histidine, isoleucine, leucine, threonine, valine, asparagine, proline, and serine. Phenylalanine, lysine, and methionine were greater in the plasma of horses receiving the RCP treatment at 0 and 120 min. Phenylalanine intake was standardized between groups; however, horses receiving the RCP diet had greater rates of phenylalanine oxidation (P = 0.02) and lower rates of non-oxidative phenylalanine disposal (P = 0.04). Lower whole-body protein synthesis indicates a limiting amino acid in the RCP diet.

Recovery of insulin sensitivity in mature horses after a 3 week course of dexamethasone therapy.

REASONS FOR PERFORMING THE STUDY: Dexamethasone is an anti-inflammatory drug commonly used in equine medicine. Insulin sensitivity decreases with prolonged dexamethasone administration, but little information is available about the duration of this side effect after long-term treatment ends. OBJECTIVES: To determine how long it takes for blood glucose, insulin and markers of insulin sensitivity to return to normal ranges after extended dexamethasone treatment has ceased. STUDY DESIGN: Experimental study. METHODS: Eight healthy, mature, mixed-breed horses received 0.04 mg/kg bwt/day oral dexamethasone for 21 days. Blood samples were taken weekly during dexamethasone treatment (Days -21, -14 and -7). Following the final dose of dexamethasone on Day 0, blood samples were taken on Days 1-6, 8, 10, 12, 15 and 22. Day -21 represents baseline or normal blood predexamethasone. RESULTS: On Day 1, plasma glucose and insulin concentrations and the modified insulin-to-glucose ratio (a proxy for pancreatic ß cell responsiveness) were higher and the reciprocal of the square root of insulin (a proxy for the estimate of insulin sensitivity) was lower, in comparison with Day -21 values. Blood glucose concentrations dropped and returned to Day -21 values by Day 2. Insulin concentrations remained elevated until Day 3. Values for the modified insulin-to-glucose ratio decreased and returned to Day -21 concentrations by Day 4. Values for the reciprocal of the square root of insulin did not return to Day -21 values until Day 15. CONCLUSIONS: These results indicate that, in contrast to blood glucose concentrations, which return to normal quickly (within 2 days after treatment ends), the pancreatic insulin-secreting response has a delayed recovery.

Effects of the rate of insulin infusion during isoglycemic, hyperinsulinemic clamp procedures on measures of insulin action in healthy, mature thoroughbred mares.

The objective of this study was to determine whether the rate of insulin infusion during isoglycemic hyperinsulinemic clamp procedures affected measures of insulin action, including glucose disposal and plasma non-esterified fatty acid, endothelin-1, and nitric oxide concentrations, in mature, healthy horses. Eight thoroughbred mares were studied during a 2-h hyperinsulinemic clamp procedure, conducted at each of 4 rates of insulin infusion: 0 (CON), 1.2 (LOWINS), 3 (MEDINS), and 6 (HIGHINS) mU · kg(-1) · min(-1). The infusion rate of a dextrose solution was adjusted throughout the clamp procedures to maintain blood glucose levels within 10% of baseline glucose concentrations. Plasma insulin concentrations were measured throughout the clamp procedures, and used with the rate of glucose infusion to calculate the plasma insulin concentration-to-rate of glucose infusion ratio, a measure of insulin action on glucose disposal. The rate of glucose infusion increased with rate of insulin infusion (P < 0.05). The plasma insulin concentration-to-rate of glucose infusion ratio was highest for the LOWINS treatment (P < 0.05) and decreased by 62% (P < 0.05) and 84% (P < 0.05) for the MEDINS and HIGHINS treatments, respectively. Although plasma non-esterified fatty acid concentrations were lower than baseline by t = 30 min of the clamp procedures in the LOWINS, MEDINS, and HIGHINS treatments (P < 0.05), the decline was similar for all 3 rates of insulin infusion. Jugular vein plasma nitric oxide and endothelin-1 concentrations were not affected by insulin infusion rate (P > 0.05). The data indicate that it is important to standardize insulin infusion rate if data are to be compared between hyperinsulinemic clamp studies.

Insulin infusion stimulates whole-body protein synthesis and activates the upstream and downstream effectors of mechanistic target of rapamycin signaling in the gluteus medius muscle of mature horses.

Little is known about the role insulin plays in regulating whole-body and muscle protein metabolism in horses. The objective of this study was to determine the effects of graded rates of insulin infusion on plasma amino acid concentrations and the activation of factors in the mechanistic target of rapamycin signaling pathway in the skeletal muscle of horses. Isoglycemic, hyperinsulinemic clamp procedures were conducted in 8 mature, thoroughbred mares receiving 4 rates of insulin infusion: 0 mU · kg(-1) · min(-1) (CON), 1.2 mU · kg(-1) · min(-1) (LOWINS), 3 mU · kg(-1) · min(-1) (MEDINS), and 6 mU · kg(-1) · min(-1) (HIGHINS). Blood samples were taken throughout the clamp procedures to measure plasma amino acid concentrations, and a biopsy from the gluteus medius muscle was collected at the end of the 2-h clamp to measure phosphorylation of protein kinase B, eukaryotic initiation factor 4E-binding protein 1, and riboprotein S6. Plasma concentrations of most of the essential amino acids decreased (P < 0.05) after 120 min of insulin infusion in horses receiving the LOWINS, MEDINS, and HIGHINS treatments, with the largest decreases occurring in horses receiving the MEDINS and HIGHINS treatments. Phosphorylation of protein kinase B, 4E-binding protein 1, and riboprotein S6 increased with all 3 rates of insulin infusion (P > 0.05), relative to CON, with maximum phosphorylation achieved with MEDINS and HIGHINS treatments. These results indicate that insulin stimulates whole-body and muscle protein synthesis in mature horses.

Effects of leucine or whey protein addition to an oral glucose solution on serum insulin, plasma glucose and plasma amino acid responses in horses at rest and following exercise.

REASONS FOR PERFORMING STUDY: Providing protein or amino acid mixtures in combination with glucose to post exercise in man has resulted in increases in the post feeding insulin response and in muscle glycogen and protein synthesis rates. However, whether protein and/or amino acids can modify the post exercise insulin responses in horses remains to be fully elucidated. OBJECTIVES: To determine whether whey protein or leucine addition to a glucose solution affects the post gavage plasma insulin, glucose and amino acid responses in horses and whether these responses are different following a period of exercise vs. rest. METHODS: Six mature, conditioned Thoroughbreds received a nasogastric gavage containing either 1 g/kg bwt glucose (G), G + 0.3 g/kg bwt whey protein (GW) or G + 0.3 g/kg bwt leucine (GL), following a period of either rest (R) or an exercise test on a high speed treadmill (EX). Each horse was studied under all 6 treatment conditions, separated by 10 day intervals. Blood samples were collected pre-exercise/rest, pregavage and at regular intervals up to 300 min post gavage. Plasma was analysed for glucose and amino acid concentrations and serum insulin concentrations were determined. RESULTS: There was a significantly (P < 0.05) greater insulin response in GL-R and GL-EX when compared to the other treatments. When compared to rest, post exercise plasma glucose responses were lower in G and GW but unchanged following GL administration. Plasma alanine concentrations were elevated post exercise in all EX treatments. With the exception of markedly elevated plasma leucine concentrations after GL-R and GL-EX, the plasma concentrations of all indispensable amino acids decreased during the post gavage period. CONCLUSIONS: Leucine but not whey protein augmented the serum insulin response to an oral glucose load. Leucine supplementation warrants further investigation as a means to increase the rate of post exercise muscle glycogen synthesis in horses.