Jeffery Escobar

Isoleucine and Leucine Independently Regulate mTOR Signaling and Protein Synthesis in MAC-T Cells and Bovine Mammary Tissue Slices

Understanding the regulatory effects of individual amino acids (AA) on milk protein synthesis rates is important for improving protein and AA requirement models for lactation. The objective of this study was to examine the effects of individual essential AA (EAA) on cellular signaling and fractional protein synthesis rates (FSR) in bovine mammary cells. Omission of L-arginine, L-isoleucine, L-leucine, or all EAA reduced (P < 0.05) mammalian target of rapamycin (mTOR; Ser2448) and ribosomal protein S6 (rpS6; Ser235/236) phosphorylation in MAC-T cells. Phosphorylation of mTOR and rpS6 kinase 1 (S6K1; Thr389) decreased (P < 0.05) in the absence of L-isoleucine, L-leucine, or all EAA in lactogenic mammary tissue slices. Omission of L-tryptophan also reduced S6K1 phosphorylation (P = 0.01). Supplementation of L-leucine to media depleted of EAA increased mTOR and rpS6 and decreased eukaryotic elongation factor 2 (Thr56) phosphorylation (P < 0.05) in MAC-T cells. Supplementation of L-isoleucine increased mTOR, S6K1, and rpS6 phosphorylation (P < 0.05). No single EAA considerably affected eukaryotic initiation factor 2-α (eIF2α; Ser51) phosphorylation, but phosphorylation was reduced in response to provision of all EAA (P < 0.04). FSR declined when L-isoleucine (P = 0.01), L-leucine (P = 0.01), L-methionine (P = 0.02), or L-threonine (P = 0.07) was depleted in media and was positively correlated (R = 0.64, P < 0.01) with phosphorylation of mTOR and negatively correlated (R = -0.42, P = 0.01) with phosphorylation of eIF2α. Such regulation of protein synthesis will result in variable efficiency of transfer of absorbed EAA to milk protein and is incompatible with the assumption that a single nutrient limits protein synthesis that is encoded in current diet formulation strategies.

Essential Amino Acids Regulate Both Initiation and Elongation of mRNA Translation Independent of Insulin in MAC-T Cells and Bovine Mammary Tissue Slices

Current nutrient requirement models assume fixed efficiencies of absorbed amino acid (AA) conversion to milk protein. Regulation of mammary protein synthesis (PS) potentially violates this assumption by changing the relationship between AA supply and milk protein output. The objective of this study was to investigate the effects of essential AA (EAA) and insulin on cellular signaling and PS rates in bovine mammary cells. MAC-T cells were subjected to 0 or 100% of normal EAA concentrations in DMEM/F12 and 0 or 100 μg insulin/L in a 2 × 2 factorial arrangement of treatments. Lactogenic bovine mammary tissue slices (MTS) were subjected to the same treatments, except low-EAA was 5% of normal DMEM/F12 concentrations. In MAC-T cells, EAA increased phosphorylation of mammalian target of rapamycin (mTOR; Ser2448), ribosomal protein S6 kinase 1 (S6K1; Thr389), eIF4E binding protein 1 (4EBP1; Thr37/46), and insulin receptor substrate 1 (IRS1; Ser1101), and reduced phosphorylation of eukaryotic elongation factor 2 (eEF2; Thr56) and eukaryotic initiation factor (eIF) 2-α (Ser51). In the presence of insulin, phosphorylation of Akt (Ser473), mTOR, S6K1, 4EBP1, and IRS1 increased in MAC-T cells. In MTS, EAA had similar effects on phosphorylation of signaling proteins and increased mammary PS rates. Insulin did not affect MTS signaling, perhaps due to inadequate levels. Effects of EAA and insulin were independent and additive for mTOR signaling in MAC-T cells. EAA did not inhibit insulin stimulation of Akt phosphorylation. PS rates were strongly associated with phosphorylation of 4EBP1 and eEF2 in MTS. EAA availability affected translation initiation and elongation control points to more strongly regulate PS than insulin.

Leucine and α-Ketoisocaproic Acid, but Not Norleucine, Stimulate Skeletal Muscle Protein Synthesis in Neonatal Pigs

The branched-chain amino acid, leucine, acts as a nutrient signal to stimulate protein synthesis in skeletal muscle of young pigs. However, the chemical structure responsible for this effect has not been identified. We have shown that the other branched-chain amino acids, isoleucine and valine, are not able to stimulate protein synthesis when raised in plasma to levels within the postprandial range. In this study, we evaluated the effect of leucine, alpha-ketoisocaproic acid (KIC), and norleucine infusion (0 or 400 micromol kg(-1) h(-1) for 60 min) on protein synthesis and activation of translation initiation factors in piglets. Infusion of leucine, KIC, and norleucine raised plasma levels of each compound compared with controls. KIC also increased (P < 0.01) and norleucine reduced (P < 0.02) plasma levels of leucine compared with controls. Administration of leucine and KIC resulted in greater (P < 0.006) phosphorylation of eukaryotic initiation factor (eIF) 4E binding protein-1 (4E-BP1) and eIF4G, lower (P < 0.04) abundance of the inactive 4E-BP1.eIF4E complex, and greater (P < 0.05) active eIF4G.eIF4E complex formation in skeletal muscle compared with controls. Protein synthesis in skeletal muscle was greater (P < 0.02) in leucine- and KIC-infused pigs than in those in the control group. Norleucine infusion did not affect muscle protein synthesis or translation initiation factor activation. In liver, neither protein synthesis nor activation of translation initiation factors was affected by treatment. These results suggest that the ability of leucine to act as a nutrient signal to stimulate skeletal muscle protein synthesis is specific for leucine and/or its metabolite, KIC.

Effects of jugular-infused lysine, methionine, and branched-chain amino acids on milk protein synthesis in high-producing dairy cows

In addition to lysine and methionine, current ration-balancing programs suggest that branched-chain amino acid (BCAA) supply may also be limiting in dairy cows. The objective of this study was to investigate whether BCAA, leucine, isoleucine, and valine become limiting for milk protein synthesis when methionine and lysine supply were not limiting. Nine multiparous Holstein cows with an average milk production of 53.5±7.1 kg/d were randomly assigned to 7-d continuous jugular infusions of saline (CTL), methionine and lysine (ML; 12 g and 21 g/d, respectively), or ML plus leucine, isoleucine, and valine (ML+BCAA; 35 g, 15 g, and 15 g/d, respectively) in a 3×3 Latin square design with 3 infusion periods separated by 7-d noninfusion periods. The basal diet consisted of 40% corn silage, 14% alfalfa hay, and a concentrate mix, and respectively supplied lysine, methionine, isoleucine, leucine, and valine as 6.1, 1.8, 4.7, 8.9, and 5.3% of metabolizable protein. Dry matter intake (23.9 kg/d), milk yield (52.8 kg/d), fat content (2.55%), fat yield (1.33 kg/d), lactose content (4.77%), lactose yield (2.51 kg/d), and milk protein efficiency (0.38) were similar across treatments. Protein yield and protein content were not significantly different between ML (1.52 kg/d and 2.88%, respectively) and ML+BCAA (1.51 kg/d and 2.83%, respectively), but they were significantly greater than that of CTL (1.39 kg/d and 2.71%). Cows that received ML+BCAA had less milk urea nitrogen content (10.9 mg/dL) compared with milk of CTL cows (12.4 mg/dL) and ML cows (11.8 mg/dL). Whereas high-producing cows responded positively to methionine and lysine supplementation, no apparent benefits of BCAA supplementation in milk protein synthesis were found. Infusion of BCAA may have stimulated synthesis of other body proteins, probably muscle proteins, as evidenced by decreased milk urea nitrogen.

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.

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 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.

Energy Dense, Protein Restricted Diet Increases Adiposity and Perturbs Metabolism in Young, Genetically Lean Pigs

Animal models of obesity and metabolic dysregulation during growth (or childhood) are lacking. Our objective was to increase adiposity and induce metabolic syndrome in young, genetically lean pigs. Pre-pubertal female pigs, age 35 d, were fed a high-energy diet (HED; n = 12), containing 15% tallow, 35% refined sugars and 9.1–12.9% crude protein, or a control corn-based diet (n = 11) with 12.2–19.2% crude protein for 16 wk. Initially, HED pigs self-regulated energy intake similar to controls, but by wk 5, consumed more (P<0.001) energy per kg body weight. At wk 15, pigs were subjected to an oral glucose tolerance test (OGTT); blood glucose increased (P<0.05) in control pigs and returned to baseline levels within 60 min. HED pigs were hyperglycemic at time 0, and blood glucose did not return to baseline (P = 0.01), even 4 h post-challenge. During OGTT, glucose area under the curve (AUC) was higher and insulin AUC was lower in HED pigs compared to controls (P = 0.001). Chronic HED intake increased (P<0.05) subcutaneous, intramuscular, and perirenal fat deposition, and induced hyperglycemia, hypoinsulinemia, and low-density lipoprotein hypercholesterolemia. A subset of HED pigs (n = 7) was transitioned back to a control diet for an additional six weeks. These pigs were subjected to an additional OGTT at 22 wk. Glucose AUC and insulin AUC did not improve, supporting that dietary intervention was not sufficient to recover glucose tolerance or insulin production. These data suggest a HED may be used to increase adiposity and disrupt glucose homeostasis in young, growing pigs.