Flooding Dose Of Phenylalanine Research Articles

25-Hydroxycholecalciferol Enhances Male Broiler Breast Meat Yield through the mTOR Pathway1–3

Background: In recent years, there has been a growing body of evidence indicating that replacing cholecalciferol (vitamin D3) with 25-hydroxycholecalciferol [25(OH)D3] through dietary supplementation enhances breast meat yield in broiler chickens. However, the underlying molecular mechanisms are still unknown.Objective: We investigated the effect of 25(OH)D3 on male broiler growth performance (body weight, feed intake, feed conversion ratio, and breast meat yield), muscle protein synthesis, and the potential underlying molecular mechanisms.Methods: Male Cobb 500 broiler chickens were divided into 4 body weight–matched groups and received a control diet with normal cholecalciferol (2760 IU/kg feed) for 42 d, a diet with high concentrations of cholecalciferol (5520 IU/kg feed) for 42 d, or a diet with 25(OH)D3 (5520 IU/kg feed) for 42 d (HyD-42). A fourth group consumed the HyD-42 for 21 d and then control feed for 21 d (HyD-21) (n = 360 birds, 12 replicates/treatment). Food and clean water were available for ad libitum consumption. At the end of the 42-d experiment, protein turnover was measured by phenylalanine flooding dose. Breast muscle tissues were collected and protein synthesis–related gene and protein expression were measured by real time polymerase chain reaction and Western blot, respectively. Functional studies were performed in vitro with the use of a quail myoblast (QM7) cell line. QM7 cells were treated with 2 doses (1 nM and 10 nM) of cholecalciferol or 25(OH)D3 alone or in combination with 100 nM rapamycin, and cell proliferation was determined by cell proliferation assay. Protein synthesis–related gene and protein expression were also determined.Results: The HyD-42 increased 25(OH)D3 circulating concentrations by 126% (P < 0.05), enhanced breast meat yield (P < 0.05), and increased the fractional rate of protein synthesis by 3-fold (P < 0.05) compared with the control diet. Molecular analyses revealed that breast muscle from chickens consuming the HyD-42 expressed significantly higher concentrations of vitamin D receptor (VDR), phospho mechanistic target of rapamycinSer2481, phospho ribosomal P70 S6 kinase (RPS6K)Thr421/Ser424, and antigen Ki-67 (Ki67) compared with the other groups. In line with the in vivo data, in vitro functional studies showed that cells treated with 25(OH)D3 for 24 h had increased VDR expression, and activated the mechanistic target of rapamycin (mTOR)/S6 kinase (S6K) pathway, enhanced Ki67 protein concentrations, and induced QM7 cell proliferation compared with untreated or cholecalciferol-treated cells. Blocking the mTOR pathway with rapamycin reversed these effects.Conclusion: Taken together, our findings provide evidence that the effects of 25(OH)D3 on male broiler breast muscle are likely mediated through the mTOR-S6K pathway.

Sarcopenia associated with portosystemic shunting is reversed by follistatin

The distinct role of portosystemic shunting (PSS) in the pathogenesis of sarcopenia (skeletal muscle loss) that occurs commonly in cirrhosis is unclear. We have previously shown increased expression of myostatin (inhibitor of skeletal muscle mass) in the portacaval anastamosis (PCA) rat model of sarcopenia of PSS. The present study was performed to examine the mechanisms of sarcopenia following PCA. In PCA and sham operated pair fed control rats, the phenylalanine flooding dose method was used to quantify the fractional and absolute protein synthesis rates in the skeletal muscle over time and in response to follistatin, a myostatin antagonist. The expression of myostatin and markers of satellite cell (myocyte precursors) proliferation and differentiation were quantified by real-time PCR and Western blot analyses. The absolute synthesis rate (ASR) was lower at 2, 4, and 6 weeks (p<0.05) and the fractional synthesis rate (FSR) of skeletal muscle protein was significantly lower (p<0.05) at week 2 in the PCA rats compared to control rats. Expression of myostatin was elevated while markers of satellite cell proliferation and differentiation were lower at 4 and 6 weeks after PCA. Follistatin increased skeletal muscle mass, muscle FSR and ASR, decreased expression of myostatin protein, and increased expression of markers of satellite cell function. Sarcopenia associated with PSS is caused by impaired protein synthesis and reduced satellite cell function due to increased myostatin expression. Confirming these alterations in human patients with cirrhosis will provide novel therapeutic targets for sarcopenia of liver disease.

A comparison of 2H2O and phenylalanine flooding dose to investigate muscle protein synthesis with acute exercise in rats

The primary objective of this investigation was to determine whether (2)H(2)O and phenylalanine (Phe) flooding dose methods yield comparable fractional rates of protein synthesis (FSR) in skeletal muscle following a single bout of high-intensity resistance exercise (RE). Sprague-Dawley rats were assigned by body mass to either 4-h control (CON 4 h; n = 6), 4-h resistance exercise (RE 4 h; n = 6), 24-h control (CON 24 h; n = 6), or 24-h resistance exercise (RE 24 h; n = 6). The RE groups were operantly conditioned to engage in a single bout of high-intensity, "squat-like" RE. All rats were given an intraperitoneal injection of 99.9% (2)H(2)O and provided 4.0% (2)H(2)O drinking water for either 24 (n = 12) or 4 h (n = 12) prior to receiving a flooding dose of l-[2,3,4,5,6-(3)H]Phe 16 h post-RE. Neither method detected an effect of RE on FSR in the mixed gastrocnemius, plantaris, or soleus muscle. Aside from the qualitative similarities between methods, the 4-h (2)H(2)O FSR measurements, when expressed in percent per hour, were quantitatively greater than the 24-h (2)H(2)O and Phe flooding in all muscles (P < 0.001), and the 24-h (2)H(2)O was greater than the Phe flooding dose in the mixed gastrocnemius and plantaris (P < 0.05). In contrast, the actual percentage of newly synthesized protein was significantly higher in the 24- vs. 4-h (2)H(2)O and Phe flooding dose groups (P < 0.001). These results suggest that the methodologies provide "qualitatively" similar results when a perturbation such as RE is studied. However, due to potential quantitative differences between methods, the experimental question should determine what approach should be used.

Impaired translation initiation activation and reduced protein synthesis in weaned piglets fed a low-protein diet

Weanling mammals (including infants) often experience intestinal dysfunction when fed a high-protein diet. Recent work with the piglet (an animal model for studying human infant nutrition) shows that reducing protein intake can improve gut function during weaning but compromises the provision of essential amino acids (EAA) for muscle growth. The present study was conducted with weaned pigs to test the hypothesis that supplementing deficient EAA (Lys, Met, Thr, Trp, Leu, Ile and Val) to a low-protein diet may maintain the activation of translation initiation factors and adequate protein synthesis in tissues. Pigs were weaned at 21 days of age and fed diets containing 20.7, 16.7 or 12.7% crude protein (CP), with the low-CP diets supplemented with EAA to achieve the levels in the high-CP diet. On Day 14 of the trial, tissue protein synthesis was determined using the phenylalanine flooding dose method. Reducing dietary CP levels decreased protein synthesis in pancreas, liver, kidney and longissimus muscle. A low-CP diet reduced the phosphorylation of eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1) in skeletal muscle and liver while increasing the formation of an inactive eIF4E·4E-BP1 complex in muscle. Dietary protein deficiency also decreased the phosphorylation of mammalian target of rapamycin (mTOR) and the formation of an active eIF4E·eIF4G complex in liver. These results demonstrate for the first time that chronic feeding of a low-CP diet suppresses protein synthesis in animals partly by inhibiting mTOR signaling. Additionally, our findings indicate that supplementing deficient EAA to low-protein diets is not highly effective in restoring protein synthesis or whole-body growth in piglets. We suggest that conditionally essential amino acids (e.g., glutamine and arginine) may be required to maintain the activation of translation initiation factors and optimal protein synthesis in neonates.

Muscle Fractional Protein Synthesis Is Higher in Iberian than in Landrace Growing Pigs Fed Adequate or Lysine-Deficient Diets1–3

Protein deposition in Iberian pigs is low and the reasons for this are unknown. We investigated differences in protein synthesis rate in tissues of 30 Iberian and Landrace gilts fed 2 diets with adequate amino acid composition containing 160 or 120 g crude protein (CP)/kg, or a lysine-deficient diet (containing 120 or 160 g CP/kg for Iberian and Landrace pigs, respectively). Pigs were infused with a flooding dose of phenylalanine (15% as [(2)H(5)]-phenylalanine). Blood samples were taken from 12 to 40 min after the start of infusion, and samples from longissimus dorsi (ld), biceps femoris (bf), and semimembranosus (sm) muscles, liver, and duodenum were taken after slaughter. Body weights (BW) were 22.9 +/- 0.37 and 27.1 +/- 0.64 kg for Iberian and Landrace pigs, respectively. Iberian pigs fed the adequate diets had higher muscle fractional protein synthesis rates (FSR, %/d) than Landrace pigs. The FSR were 7.9 +/- 0.34 vs. 6.3 +/- 0.29%/d; 8.3 +/- 0.36 vs. 6.3 +/- 0.21%/d, and 7.7 +/- 0.23 vs. 6.4 +/- 0.36%/d for ld, bf, and sm muscles in Iberian and Landrace breeds, respectively (P < 0.01). However, muscles were between 20 and 32% smaller in the Iberian pigs (P < 0.01). Dietary protein level did not affect muscle FSR or size in either breed. Lysine deficiency reduced muscle FSR (46-49%, P < 0.001). Visceral tissues had greater relative weights in Iberian pigs (P < 0.001) with no breed differences in FSR. These findings might explain the low efficiency of protein and energy utilization by Iberian pigs compared with conventional pig breeds.

The effect of a flooding dose of phenylalanine on indicators of metabolic status in pigs

The metabolic effect of infusing growing pigs with a large dose of phenylalanine was investigated. Packed cell volume and plasma glucose levels were not affected (P &gt; 0.05), while plasma insulin concentration was increased at some time periods compared to a saline control (P &lt; 0.05). For amino acids, only phenylalanine and tyrosine concentrations were increased (P &lt; 0.05) by phenylalanine infusion. A flooding dose of phenylalanine may not significantly alter the metabolic status of the pig and could therefore be used in measuring protein synthesis in pigs. Key words: Amino acid, flooding dose, glucose, insulin, phenylalanine, pig

Influence of dietary protein, energy and corticosteroids on protein turnover, proteoglycan sulphation and growth of long bone and skeletal muscle in the rat.

1. We report here the extent to which changes in protein turnover contribute to the previously described inhibition of growth of rat tibial length and skeletal muscle mass in response to protein deficiency [1], energy restriction and corticosterone treatment [2]. Measurements of 35S uptake in vivo also enabled the qualitative pattern of changes in proteoglycan synthesis in bone and muscle to be established. 2. Protein deficiency was examined by ad libitum feeding of 20%, 7%, 3.5% and 0.5% protein diets with measurements at 1, 3 and 7 days (all diets), and 14 and 21 days (0.5% protein). In bone this induced delayed inhibition of tibial growth with parallel inhibition of protein synthesis, as measured by the phenylalanine flooding dose method. This was mediated by reductions in both ribosomal capacity (RNA/protein ratio) and activity (protein synthesis/RNA) in the 0.5% protein group. The pattern of inhibition of proteoglycan sulphation, measured as 35S uptake 60 min after injection of a tracer dose of labelled sulphate, was similar to that of protein synthesis. 3. In muscle there was an intermediate graded inhibition of protein synthesis by protein deficiency, mediated by reductions in both ribosomal capacity and activity in the 0.5% protein group, which preceded growth inhibition in the 7% and 3.5% groups, and which was progressive with time. Transient increases in proteolysis contributed to the growth inhibition is some groups, but the rate fell eventually in the 0.5% group. The pattern of response of proteoglycan sulphation differed from protein synthesis with a delayed inhibition, but with subsequent marked reduction. 4. Energy restriction was induced by diets fed for 4 or 8 days at 75%, 50% and 25% ad libitum intakes with protein intakes held constant, and corticosterone treatment involved a dose of 10 mg day-1 100-1 g (subcutaneous) with ad libitum feeding. In bone this induced a pattern of length growth inhibition which was dissociated from inhibition of protein synthesis in the moderately restricted (75% and 50%) groups. Only in the 25% group and in the 8 day corticosterone group was protein synthesis inhibited, through reductions in ribosomal capacity and activity. 35S uptake was also dissociated from growth inhibition, with reduced 35S uptake observed only after corticosterone treatment or 8 days of the 50% or 25% diets. 5. In muscle the energy restriction and corticosterone treatment induced parallel inhibitions of growth and protein synthesis, mediated by similar graded reductions in the RNA/protein ratios and in the 25% group in the KRNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Phenylalanine Flooding Dose Procedure Is Effective in Measuring Intestinal and Liver Protein Synthesis in Sheep ,

We conducted two experiments to evaluate the flooding dose method for measuring intestinal and liver protein synthesis in sheep. Experiment 1 showed that large doses of phenylalanine did not cause marked metabolic disturbances. Experiment 2 examined the effectiveness of flooding with phenylalanine and the time dependency of the protein synthesis calculation. Rams were injected with 1.2 MBq L-[ring 2,6-3H]phenylalanine/kg body wt and slaughtered 20, 40 or 60 min later. Plasma specific radioactivity reached a plateau within 2.5 min and did not change significantly (P > 0.05) throughout the experiment. Tissue intracellular free pool specific radioactivity also remained constant from 20 to 60 min postinfusion. Flooding conditions were achieved in the intracellular free pool of intestinal tissues (specific radioactivity 70–96% of plasma specific radioactivity), although liver flooding was less successful (57–67%). Protein synthesis rates measured after 20 min were significantly (P < 0.05) higher in the liver, jejunum and ileum than those measured at 60 min. Protein synthesis rates also tended to decline with time in the duodenum and colon (P > 0.05). There were no significant differences (P > 0.05) between protein synthesis rates calculated using the intracellular specific radioactivity vs. plasma specific radioactivity in the duodenum, ileum or colon. Therefore, this method represents an improvement over continuous infusion methods for measurements of protein synthesis in visceral tissues.

Measurement of Protein Synthesis by the Phenylalanine Flooding Dose Technique: Effect of Phenylalanine and Anaesthesia on Plasma Electrolyte, Enzyme and Metabolite Levels

Ten minutes after an intravenous flooding dose of phenylalanine to rats, plasma sodium and calcium concentrations were slightly reduced (by 2-7%) but no effects on potassium or phosphate were observed. Creatine kinase activities were significantly increased by phenylalanine injection (by 39%), but alkaline phosphatase, alanine aminotransferase, lactate dehydrogenase and aspartate aminotransferase activities were unaltered. Plasma concentrations of total proteins, albumin, cholesterol, triglycerides, urea, creatinine and glucose were also unaffected. In the presence of anaesthesia, phenylalanine injection had almost identical effects, although the increase in creatine kinase activities did not reach statistical significance. Anaesthesia for 10 min reduced plasma potassium concentrations (by 27%), and calcium (by 5%), though phosphate and sodium were unaltered. The activities of lactate dehydrogenase, creatine kinase and aspartate aminotransferase were reduced by between 36-52%, but alkaline phosphatase and alanine aminotransferase activities were unaltered by anaesthesia. Plasma concentrations of total proteins and albumin were also reduced (both by 9%), but glucose concentrations were increased (by 33%). Anaesthesia had no other significant effects on cholesterol, triglycerides, urea or creatinine concentrations. The qualitative effects of anaesthesia in the presence of raised free phenylalanine concentrations were similar. It was concluded that, except for creatine kinase, determinations of plasma constituents in phenylalanine-injected rats could be made without overt interpretational errors. However, caution is required in interpreting data on plasma constituents from anaesthetized rats.