Impact of ergot alkaloid and steroidal implant on whole-body protein turnover and expression of mTOR pathway proteins in muscle of cattle.

Introduction: Holstein steers (n = 32) were used to determine if the ergot analog, bromocriptine decreases muscle protein synthesis through inhibitory action on the mTOR pathway via a direct effect on signal proteins, and if these negative effects can be alleviated with anabolic agents. Methods: Steers were treated with intramuscular administration of bromocriptine (vehicle or 0.1 mg/kg BW) and a subdermal commercial steroidal implant containing trenbolone acetate (TBA) and estradiol 17ß (with or without), in a 2×2 factorial design. During the 35 day experiment, intake was restricted to 1.5 times maintenance energy requirement. On days 27 through 32, steers were moved to metabolism stalls for urine collection, and whole-body protein turnover was determined using a single pulse dose of [15N] glycine into the jugular vein on day 28. On day 35, skeletal muscle samples were collected before (basal state) and 60 min after (stimulated state) an i.v. glucose challenge (0.25 g glucose/kg). Blood samples were collected at regular intervals before and after glucose infusion for determination of circulating concentrations of glucose and insulin. Results: Bromocriptine reduced insulin and glucose clearance following the glucose challenge, indicating decreased insulin sensitivity and possible disruption of glucose uptake and metabolism in the skeletal muscle. Conversely, analysis of whole-body protein turnover demonstrated that bromocriptine does not appear to affect protein synthesis or urea excretion. Western immunoblot analysis of skeletal muscle showed that it did not affect abundance of S6K1 or 4E-BP1, so bromocriptine does not appear to inhibit activation of the mTOR pathway or protein synthesis. Estradiol/TBA implant decreased urea excretion and protein turnover but had no effect on protein synthesis, suggesting that steroidal implants promote protein accretion through unchanged rates of synthesis and decreased degradation, even in the presence of bromocriptine, resulting in improved daily gains. Implanted steers likely experienced increased IGF-1 signaling, but downstream activation of mTOR, S6K and 4E-BP1, and thus increased protein synthesis did not occur as expected. Conclusions: Overall, this data suggests that bromocriptine does not have a negative impact on muscle protein synthetic pathways independent of DMI.

Effects of Bromocriptine on Glucose and Insulin Dynamics in Normal and Insulin Dysregulated Horses.

The objectives of the study were to study the effects of the synthetic ergot alkaloid (EA), bromocriptine, on glucose and lipid metabolism in insulin dysregulated (ID, n = 7) and non-ID (n = 8) mares. Horses were individually housed and fed timothy grass hay and two daily concentrate meals so that the total diet provided 120% of daily DE requirements for maintenance. All horses were given intramuscular bromocriptine injections (0.1 mg/kg BW) every 3 days for 14 days. Before and after 14 days of treatment horses underwent a combined glucose-insulin tolerance test (CGIT) to assess insulin sensitivity and a feed challenge (1 g starch/kg BW from whole oats) to evaluate postprandial glycemic and insulinemic responses. ID horses had higher basal plasma concentrations of insulin (P = 0.01) and triglycerides (P = 0.02), and lower concentrations of adiponectin (P = 0.05) compared with non-ID horses. The CGIT response curve showed that ID horses had slower glucose clearance rates (P = 0.02) resulting in a longer time in positive phase (P = 0.03) and had higher insulin concentrations at 75 min (P = 0.0002) compared with non-ID horses. Glucose (P = 0.02) and insulin (P = 0.04) responses to the feeding challenge were lower in non-ID compared to ID horses. Regardless of insulin status, bromocriptine administration increased hay intake (P = 0.03) and decreased grain (P < 0.0001) and total DE (P = 0.0002) intake. Bromocriptine treatment decreased plasma prolactin (P = 0.0002) and cholesterol (P = 0.10) and increased (P = 0.02) adiponectin concentrations in all horses. Moreover, in both groups of horses, bromocriptine decreased glucose clearance rates (P = 0.02), increased time in positive phase (P = 0.04) of the CGIT and increased insulin concentrations at 75 min (P = 0.001). The postprandial glycemic (P = 0.01) and insulinemic (P = 0.001) response following the oats meal was lower after bromocriptine treatment in all horses. In conclusion, in contrast to data in humans and rodents, bromocriptine treatment reduced insulin sensitivity in all horses, regardless of their insulin status. These results indicate that the physiological effects of EA might be different in horses compared to other species. Moreover, because bromocriptine shares a high degree of homology with natural EA, further investigation is warranted in horses grazing endophyte-infected grasses.

Differential effect of two dietary protein sources on time course response of muscle anabolic signaling pathways in normal and insulin dysregulated horses.

The objective of the study was to characterize the temporal changes of phosphorylation patterns of mTOR signaling proteins in response to two dietary protein sources in insulin dysregulated (ID, n = 8) and non-ID (n = 8) horses. Horses were individually housed and fed timothy grass hay and 2 daily concentrate meals so that protein was the first limiting nutrient and the total diet provided 120% of daily DE requirements for maintenance. On sample days, horses randomly received 0.25 g CP/kg BW of a pelleted alfalfa (AP) or commercial protein supplement (PS). Blood samples were collected before and 30, 60, 90, 120, 150, 180, 210, 240, 300, 360, 420, and 480 min post feeding and analyzed for plasma glucose, insulin and amino acid (AA) concentrations. Gluteus Medius muscle samples were obtained before and 90, 180, and 300 min after feeding and analyzed for relative abundance of phosphorylated mTOR pathway components using western immunoblot analysis. There was no effect of protein source on postprandial glucose and insulin responses (P ≥ 0.14) but consumption of PS elicited a 2 times larger AUC for essential AA (EAA), greater peak concentrations of EAA and a shorter time to reach peak EAA concentrations compared to AP. Abundance of phosphorylated mTOR (P = 0.08) and rpS6 (P = 0.10) tended to be ~1.5-fold greater after consumption of PS at 90 min compared to AP. Dephosphorylation patterns differed between protein sources and was slower for AP compared to PS. ID horses had a 2 times greater (P = 0.009) AUC and 3 times higher postprandial peak concentrations (P < 0.0001) for insulin compared to non-ID horses after consumption of both treatment pellets, but EAA responses were similar between groups (P = 0.53). Insulin status did not affect rpS6 or mTOR phosphorylation after consumption of either protein source (P ≥ 0.35), but phosphorylated rpS6 abundance was twice as high in ID compared to non-ID horses (P = 0.007). These results suggest that the consumption of higher quality protein sources may result in greater postprandial activation of the mTOR pathway compared to equal amounts of a forage-based protein source. Moreover, ID does not impair postprandial activation of mTOR and rpS6 proteins in horses following a protein-rich meal.

Pathways regulating equine skeletal muscle protein synthesis respond in a dose-dependent manner to graded levels of protein intake.

Activation of the mechanistic target of rapamycin (mTOR)-controlled anabolic signaling pathways in skeletal muscle of rodents and humans is responsive to the level of dietary protein supply, with maximal activation and rates of protein synthesis achieved with 0.2 to 0.4 g protein/kg body weight (BW). In horses, few data are available on the required level of dietary protein to maximize protein synthesis for maintenance and growth of skeletal muscle. To evaluate the effect of dietary protein level on muscle mTOR pathway activation, five mares received different amounts of a protein supplement that provided 0, 0.06, 0.125, 0.25, or 0.5 g of crude protein (CP)/kg BW per meal in a 5 × 5 Latin square design. On each sample day, horses were fasted overnight and were fed only their protein meal the following morning. A preprandial (0 min) and postprandial (90 min) blood sample was collected and a gluteus medius muscle sample was obtained 90 min after feeding the protein meal. Blood samples were analyzed for glucose, insulin, and amino acid concentrations. Activation of mTOR pathway components (mTOR and ribosomal protein S6 [rpS6]) in the muscle samples was measured by Western immunoblot analysis. Postprandial plasma glucose (P = 0.007) and insulin (P = 0.09) showed a quadratic increase, while total essential amino acid (P < 0.0001) concentrations increased linearly with the graded intake of the protein supplement. Activation of mTOR (P = 0.02) and its downstream target, rpS6 (P = 0.0008), increased quadratically and linearly in relation to the level of protein intake, respectively. Comparisons of individual doses showed no differences (P > 0.05) between the 0.25 and 0.5 g of protein intake for either mTOR or rpS6 activation, indicating that protein synthesis may have reached near maximal capacity around 0.25 g CP/kg BW. This is the first study to show that the activation of muscle protein synthetic pathways in horses is dose-dependent on the level of protein intake. Consumption of a moderate dose of high-quality protein resulted in near maximal muscle mTOR pathway activation in mature, sedentary horses.