Novel Expression of GLUT3, GLUT6 and GLUT10 in Equine Gluteal Muscle Following Glycogen-Depleting Exercise: Impact of Dietary Starch and Fat.

Horses have a slow rate of muscle glycogen repletion relative to other species for unknown reasons. Our aim was to determine the expression of glucose transporters (GLUT) and genes impacting GLUT4 expression and translocation in the gluteal muscle. Five fit Thoroughbred horses performed glycogen-depleting exercises on high-starch (HS, 2869 g starch/day) and low-starch, high-fat diets (LS-HF, 358 g starch/d) with gluteal muscle biopsies obtained before and after depletion and during repletion. Muscle glycogen declined by ≈30% on both diets with little increase during repletion on LS-HF. Transcriptomic analysis identified differential expression (DE) of only 2/12 genes impacting GLUT4 translocation (two subunits of AMP protein kinase) and only at depletion on LS-HF. Only 1/13 genes encoding proteins that promote GLUT4 transcription had increased DE (PPARGC1A at depletion LS-HF). GLUT4 comprised ≈30% of total GLUT mRNA expression at rest. Remarkably, by 72 h of repletion expression of GLUT3, GLUT6 and GLUT10 increased to ≈25% of total GLUT mRNA. Expression of GLUT6 and GLUT10 lagged from 24 h of repletion on HS to 72 h on LS-HF. Lacking an increase in GLUT4 gene expression in response to glycogen-depleting exercise, equine muscle increases GLUT3, GLUT6 and GLUT10 expression potentially to enhance glucose transport, resembling responses observed in resistance trained GLUT4-null mice.

Impact of Coenzyme Q10 Supplementation on Skeletal Muscle Respiration, Antioxidants, and the Muscle Proteome in Thoroughbred Horses.

Coenzyme Q10 (CoQ10) is an essential component of the mitochondrial electron transfer system and a potent antioxidant. The impact of CoQ10 supplementation on mitochondrial capacities and the muscle proteome is largely unknown. This study determined the effect of CoQ10 supplementation on muscle CoQ10 concentrations, antioxidant balance, the proteome, and mitochondrial respiratory capacities. In a randomized cross-over design, six Thoroughbred horses received 1600 mg/d CoQ10 or no supplement (control) for 30-d periods separated by a 60-d washout. Muscle samples were taken at the end of each period. Muscle CoQ10 and glutathione (GSH) concentrations were determined using mass spectrometry, antioxidant activities by fluorometry, mitochondrial enzyme activities and oxidative stress by colorimetry, and mitochondrial respiratory capacities by high-resolution respirometry. Data were analyzed using mixed linear models with period, supplementation, and period × supplementation as fixed effects and horse as a repeated effect. Proteomics was performed by tandem mass tag 11-plex analysis and permutation testing with FDR < 0.05. Concentrations of muscle CoQ10 (p = 0.07), GSH (p = 0.75), and malondialdehyde (p = 0.47), as well as activities of superoxide dismutase (p = 0.16) and catalase (p = 0.66), did not differ, whereas glutathione peroxidase activity (p = 0.003) was lower when horses received CoQ10 compared to no supplement. Intrinsic (relative to citrate synthase activity) electron transfer capacity with complex II (ECII) was greater, and the contribution of complex I to maximal electron transfer capacity (FCRPCI and FCRPCIG) was lower when horses received CoQ10 with no impact of CoQ10 on mitochondrial volume density. Decreased expression of subunits in complexes I, III, and IV, as well as tricarboxylic acid cycle (TCA) enzymes, was noted in proteomics when horses received CoQ10. We conclude that with CoQ10 supplementation, decreased expression of TCA cycle enzymes that produce NADH and complex I subunits, which utilize NADH together with enhanced electron transfer capacity via complex II, supports an enhanced reliance on substrates supplying complex II during mitochondrial respiration.

Long-chain polyunsaturated fatty acid supplementation increases levels in red blood cells and reduces the prevalence and severity of squamous gastric ulcers in exercised Thoroughbreds.

OBJECTIVE: To assess the relationship between plasma and RBC fatty acid composition and incidence and severity of squamous gastric ulcers when altered by short-chain (SC) or long-chain (LC) polyunsaturated fatty acid (PUFA) supplementation. ANIMALS: 13 fit Thoroughbred horses in training. PROCEDURES: Horses were evaluated by gastroscopy for squamous ulcer score, gastric pH, and blood fatty acid composition prior to supplementation (UNSUPP) and after 3 months of supplementation with a corn-flax oil blend of alpha-linolenic acid and linoleic acid (SC-PUFA) or a gamma-linolenic acid (GLA)-fish oil blend of GLA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA; LC-PUFA) in a crossover design. Prior to gastroscopy and blood collection, horses performed a 4,600-m standardized exercise test on the racetrack as a stressor. RESULTS: Three months of supplementation with LC-PUFAs increased RBC levels of GLA, dihomo-gamma-linolenic acid (DGLA), arachidonic acid (AA), EPA, and DHA, and reduced severe ulcer prevalence (38% UNSUPP vs 8% LC-PUFA with a severe ulcer score of grade 3 to 4). Short-chain PUFA supplementation did not effectively elevate RBC GLA, DGLA, AA, EPA, or DHA and severe ulcer incidence was not different (38% UNSUPP vs 23% SC-PUFA with a severe ulcer score of grade 3 to 4). Lower levels of RBC GLA, DGLA, AA, and EPA correlated with severe squamous gastric ulceration (grade 3 to 4). CLINICAL RELEVANCE: Equine gastric ulcer syndrome is prevalent in high-performance horses and is a concern to owners and trainers. Long-chain PUFA supplementation increased levels of GLA, DGLA, AA, EPA, and DHA, unlike SC-PUFA supplementation, and was associated positively with prevention or resolution of severe squamous gastric ulceration. Further studies are needed to evaluate different management styles and exercise intensities.

The Impact of N-Acetyl Cysteine and Coenzyme Q10 Supplementation on Skeletal Muscle Antioxidants and Proteome in Fit Thoroughbred Horses.

Horses have one of the highest skeletal muscle oxidative capacities amongst mammals, which, combined with a high glycolytic capacity, could perturb redox status during maximal exercise. We determined the effect of 30 d of oral coenzyme Q10 and N-acetyl-cysteine supplementation (NACQ) on muscle glutathione (GSH), cysteine, ROS, and coenzyme Q10 concentrations, and the muscle proteome, in seven maximally exercising Thoroughbred horses using a placebo and randomized cross-over design. Gluteal muscle biopsies were obtained the day before and 1 h after maximal exercise. Concentrations of GSH, cysteine, coenzyme Q10, and ROS were measured, and citrate synthase, glutathione peroxidase, and superoxide dismutase activities analyzed. GSH increased significantly 1 h post-exercise in the NACQ group (p = 0.022), whereas other antioxidant concentrations/activities were unchanged. TMT proteomic analysis revealed 40 differentially expressed proteins with NACQ out of 387 identified, including upregulation of 13 mitochondrial proteins (TCA cycle and NADPH production), 4 Z-disc proteins, and down regulation of 9 glycolytic proteins. NACQ supplementation significantly impacted muscle redox capacity after intense exercise by enhancing muscle glutathione concentrations and increasing expression of proteins involved in the uptake of glutathione into mitochondria and the NAPDH-associated reduction of oxidized glutathione, without any evident detrimental effects on performance.

Omeprazole Reduces Calcium Digestibility in Thoroughbred Horses.

Proton pump inhibitors such as omeprazole reduce nutrient digestibility in humans. This study determined the effect of omeprazole on the digestibility of diets containing limestone or marine-derived calcium (BMC) and to assess changes in blood parameters associated with gastric acid production and calcium status in horses. Thoroughbreds were used to evaluate the digestibility of diets containing different calcium sources with or without omeprazole over four 21-day periods. Each 21-day period had a 15-day diet adaptation phase followed by a 6-day collection phase, consisting of a 5-day total fecal collection period and a final day for gastroscopy and blood sampling. Horses were fed the same diet with either 60 g/d BMC or 50 g/d limestone, so the total diet provided ∼45 g calcium. Horses on omeprazole were given GastroGard once daily for the final 14 day of each 21-day period, which supplied 3.91 ± 0.17 mg/kg BW/d of omeprazole. On day 21, blood samples were taken and gastric fluid pH was measured 8 hour after omeprazole administration. Omeprazole had a profound effect on gastric fluid pH in omeprazole-treated horses compared with nontreated horses. Serum gastrin doubled in omeprazole-treated horses compared with nontreated horses. Omeprazole and calcium source did not affect digestibility of phosphorus, magnesium, potassium, sodium, iron, copper, zinc, or manganese but did affect calcium digestibility. Omeprazole reduced apparent calcium digestibility from 52.0% to 41.4% in limestone and from 55.1% to 46.5% in BMC, equalling a 20.3% and 15.6% decrease in calcium digestibility in the limestone and BMC, respectively. Mineral source had a significant effect on calcium digestibility with BMC at 50.8% and limestone at 46.7%.

Effect of dietary fats with odd or even numbers of carbon atoms on metabolic response and muscle damage with exercise in Quarter Horse-type horses with type 1 polysaccharide storage myopathy.

OBJECTIVE: To evaluate effects of fats with odd and even numbers of carbon atoms on muscle metabolism in exercising horses with polysaccharide storage myopathy (PSSM). ANIMALS: 8 horses with PSSM (6 females and 2 males; mean +/- SD age, 6.3 +/- 3.9 years). PROCEDURES: Isocaloric diets (grain, triheptanoin, corn oil, and high-fat, low-starch [HFLS] feed) were fed for 3 weeks each; horses performed daily treadmill exercise. Grain was fed to establish an exercise target, and HFLS feed was fed as a negative control diet. Daily plasma samples were obtained. For each diet, a 15-minute exercise test was performed, and gluteus medius muscle specimens and blood samples were obtained before and after exercise. RESULTS: Feeding triheptanoin, compared with the corn oil diet, resulted in exercise intolerance; higher plasma creatine kinase (CK) activity and concentrations of C3:0- and C7:0-acylcarnitine and insulin; and lower concentrations of nonesterified fatty acids (NEFA) and C16:0-, C18:1-, and C18:2-acylcarnitine, without changes in concentrations of plasma glucose or resting muscle substrates and metabolites. Feeding grain induced higher CK activity and insulin concentrations and lower NEFA concentrations than did corn oil or HFLS feed. Feeding grain induced higher glucose concentrations than did triheptanoin and corn oil. In muscle, feeding grain resulted in lower glucose-6-phosphate, higher citrate, and higher postexercise lactate concentrations than did the other diets. CONCLUSIONS AND CLINICAL RELEVANCE: Triheptanoin had detrimental effects, reflecting decreased availability of NEFA, increased insulin stimulation of glycogen synthesis, and potential inhibition of lipid oxidation. Long-chain fats are the best dietetic for PSSM.

Effect of triheptanoin on muscle metabolism during submaximal exercise in horses.

OBJECTIVE: To compare effects of corn oil or a 7-carbon fat (triheptanoin) on acylcarnitine, lipid, and carbohydrate metabolism in plasma or muscle of exercising horses. ANIMALS: 8 Thoroughbred geldings. PROCEDURES: Horses received isocaloric diets containing 650 mL of oil (triheptanoin or corn oil)/d for 18 or 25 days in a crossover design with a 26-day washout period. On day 17 or 24 of each feeding period, the respective oil (217 mL) was nasogastrically administered; 120 minutes later, horses performed a 90-minute submaximal exercise test (SET). Blood and muscle samples were obtained before oil administration and immediately before (blood only), during (blood only), immediately after, and 24 hours after SETs. RESULTS: Compared with values before oil administration, triheptanoin administration increased plasma insulin and C7:0-, C5:0- and C3:0-acylcarnitine concentrations, whereas corn oil administration increased plasma NEFA concentrations. During SETs, plasma C7:0-, C5:0-, and C3:0-acylcarnitine concentrations were higher when triheptanoin, rather than corn oil, was administered to horses. Plasma glucose, NEFA, and C2:0-, C18:1-, and C18:2-acylcarnitine concentrations increased during SETs similarly for both oils. Respiratory quotient and muscle lactate, citrate, malate, glycogen, and ATP concentrations changed similarly from before to after SETs for both oils. Compared with muscle concentrations immediately after SETs, those for glucose-6-phosphate and citrate 24 hours after SETs were lower and for glycogen were similar to values before SETs. CONCLUSIONS AND CLINICAL RELEVANCE: Fatigue was not associated with depletion of citric acid cycle intermediates for either oil. Triheptanoin induced a significantly higher insulin secretion and did not appear to enhance muscle glycogen repletion.

Effect of dietary starch, fat, and bicarbonate content on exercise responses and serum creatine kinase activity in equine recurrent exertional rhabdomyolysis.

To determine the effect of dietary starch, bicarbonate, and fat content on metabolic responses and serum creatine kinase (CK) activity in exercising Thoroughbreds with recurrent exertional rhabdomyolysis (RER), 5 RER horses were fed 3 isocaloric diets (28.8 Mcal/d [120.5 MJ/d]) for 3 weeks in a crossover design and exercised for 30 minutes on a treadmill 5 days/wk. On the last day of each diet, an incremental standardized exercise test (SET) was performed. The starch diet contained 40% digestible energy (DE) as starch and 5% as fat: the bicarbonate-starch diet was identical but was supplemented with sodium bicarbonate (4.2% of the pellet): and the fat diet provided 7% DE as starch and 20% as fat. Serum CK activity before the SET was similar among the diets. Serum CK activity (log transformed) after submaximal exercise differed dramatically among the diets and was greatest on the bicarbonate-starch diet (6.51 +/- 1.5) and lowest on the fat diet (5.71 +/- 0.6). Appreciable differences were observed in the severity of RER among individual horses. Postexercise plasma pH, bicarbonate concentration, and lactate concentration did not differ among the diets. Resting heart rates before the SET were markedly lower on the fat diet than on the starch diet. Muscle lactate and glycogen concentrations before and after the SET did not differ markedly among the diets. A high-fat, low-starch diet results in dramatically lower postexercise CK activity in severely affected RER horses than does a low-fat, high-starch diet without measurably altering muscle lactate and glycogen concentrations. Dietary bicarbonate supplementation at the concentration administered in this study did not prevent increased serum CK activity on a high-starch diet.

Comparison of volumetric urine collection versus single-sample urine collection in horses consuming diets varying in cation-anion balance.

OBJECTIVE: To determine daily variation in urinary clearance and fractional excretion (FE) of electrolytes and minerals within and between horses and to compare volumetric and single-sample urine collection for determining FE values of diets with a range of dietary cation-anion balance (DCAB). ANIMALS: 5 Thoroughbred and 6 mixed-breed mares. PROCEDURE: 3 isocaloric diets with low, medium, and high DCAB values (85, 190, and 380 mEq/kg of dry matter, respectively) were each fed for 14 days. Daily blood samples, single urine samples collected by using a urinary catheter (5 mares), and volumetric urine collections (6 mares) were obtained during the last 72 hours of each diet. RESULTS: Urine and plasma pH values, plasma concentrations, and FE values of sodium, chloride, potassium, magnesium, phosphorus, and calcium were altered by varying the DCAB. Noticeable variation in clearance and FE values was detected within horses from day-to-day on the same diet as well as between horses. Fractional excretion values were not significantly different between single-sample and volumetric methods, except for magnesium in the high DCAB diet. Volumetric and single-sample collections revealed similar patterns of change in urinary FE values with varying DCAB, except for calcium and magnesium. CONCLUSIONS AND CLINICAL RELEVANCE: Substantial variation in clearance and FE of electrolytes and minerals are evident within horses between 24-hour periods as well as between horses fed a specific diet. Three daily urine samples provide similar information regarding dietary-induced changes in clearance and FE values (excluding calcium and magnesium) as that obtained by volumetric urine collection.

Plasma and urine electrolyte and mineral concentrations in Thoroughbred horses with recurrent exertional rhabdomyolysis after consumption of diets varying in cation-anion balance.

OBJECTIVE: To determine whether plasma, urine, and fecal electrolyte and mineral concentrations differ between clinically normal horses and Thoroughbreds with recurrent exertional rhabdomyolysis (RER) after consumption of diets varying in cation-anion balance. ANIMALS: 5 Thoroughbred mares with RER and 6 clinically normal mixed-breed mares. PROCEDURE: Each of 3 isocaloric diets designated as low, medium, and high on the basis of dietary cation-anion balance (DCAB) values of 85, 190, and 380, respectively, were fed to horses for 14 days. During the last 72 hours, 3 horses with RER and 3 control horses had daily urine and fecal samples obtained by total 24-hour collection. Remaining horses had urine samples collected daily by single catheterization. RESULTS: For each diet, no differences existed between horses with RER and control horses in plasma pH, electrolyte concentrations, and creatine kinase activity or in urine pH and renal fractional excretion (FE) values. Plasma pH, strong ion difference, bicarbonate and total carbon dioxide concentrations, and base excess decreased and plasma chloride and ionized calcium concentrations increased with decreasing DCAB. Urine pH decreased with decreasing DCAB. The FE of chloride and phosphorus were greatest for horses fed the low diet. The FE values for all electrolytes exept magnesium did not differ between urine samples obtained by single catheterization and total 24-hour collection. Daily balance of calcium, phosphorus, sodium, chloride, and potassium did not differ significantly among horses fed the various diets. CONCLUSIONS: In clinically normal horses and in horses with RER, the DCAB strongly affects plasma and urine pH and the FE of sodium, potassium, chloride, and phosphorus.