Labeled amino acid infusion studies of in vivo protein synthesis with stable isotope tracers and gas chromatography—mass spectrometry

1991 ◽  
Vol 247 (2) ◽  
pp. 255-263 ◽  
Author(s):  
Jann Arends ◽  
Dennis M. Bier
1988 ◽  
Vol 254 (2) ◽  
pp. 579-584 ◽  
Author(s):  
P J Garlick ◽  
I Grant

Rates of muscle protein synthesis were measured in vivo in tissues of post-absorptive young rats that were given intravenous infusions of various combinations of insulin and amino acids. In the absence of amino acid infusion, there was a steady rise in muscle protein synthesis with plasma insulin concentration up to 158 mu units/ml, but when a complete amino acids mixtures was included maximal rates were obtained at 20 mu units/ml. The effect of the complete mixture could be reproduced by a mixture of essential amino acids or of branched-chain amino acids, but not by a non-essential mixture, alanine, methionine or glutamine. It is concluded that amino acids, particularly the branched-chain ones, increase the sensitivity of muscle protein synthesis to insulin.


1993 ◽  
Vol 264 (6) ◽  
pp. E958-E965 ◽  
Author(s):  
P. H. McNulty ◽  
L. H. Young ◽  
E. J. Barrett

Whether insulin, at physiological concentrations, stimulates net muscle protein synthesis in vivo remains unresolved. To examine this, we infused either saline, insulin (2.8 mU.kg-1.min-1, euglycemic clamp), an amino acid solution, or insulin plus amino acids for 4 h into awake overnight-fasted rats. Heart and skeletal muscle protein synthesis was measured by either a continuous tracer infusion method, using L-[1-14C]leucine, L-[2,5-3H]leucine, or L-[ring-2,6-3H]phenylalanine or by injection of L-[ring-2,6-3H]phenylalanine with a pool-flooding bolus of unlabeled phenylalanine. In heart, synthesis rates obtained using the arterial plasma specific activity of [3H]phenylalanine administered as either a tracer infusion or flooding bolus were comparable in saline-treated rats (range 10.9 +/- 1.2 to 12.2 +/- 0.9%/day) and were not affected by infusion of insulin or amino acids. Estimates using continuous infusion of L-[1-14C]leucine were significantly lower (P < 0.001), except when unlabeled amino acids were given also. In skeletal muscle, rates estimated using the flooding bolus (6.7 +/- 0.8%/day) were also not affected by insulin or amino acids. Estimates using continuous infusion of [3H]leucine (2.6 +/- 0.3%/day) or [3H]phenylalanine (2.8 +/- 1.0%/day) were lower and were still lower using [14C]leucine (1.6 +/- 0.6%/day), but increased toward those estimated with the flooding bolus during amino acid infusion. We conclude that, in heart muscle of the mature rat in vivo, neither insulin nor amino acids affect protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)


2000 ◽  
Vol 279 (2) ◽  
pp. E301-E306 ◽  
Author(s):  
W. Long ◽  
L. Saffer ◽  
L. Wei ◽  
E. J. Barrett

Refeeding reverses the muscle protein loss seen with fasting. The physiological regulators and cellular control sites responsible for this reversal are incompletely defined. Phosphorylation of phosphorylated heat-acid stabled protein (PHAS-I) frees eukaryotic initiation factor 4E (eIF4E) and stimulates protein synthesis by accelerating translation initiation. Phosphorylation of p70 S6-kinase (p70S6k) is thought to be involved in the regulation of the synthesis of some ribosomsal proteins and other selected proteins with polypyrimidine clusters near the transcription start site. We examined whether phosphorylation of PHAS-I and p70S6k was increased by feeding and determined the separate effects of insulin and amino acids on PHAS-I and p70S6k phosphorylation in rat skeletal muscle in vivo. Muscle was obtained from rats fed ad libitum or fasted overnight ( n = 5 each). Other fasted rats were infused with insulin (3 μU · min−1 · kg−1, euglycemic clamp), amino acids, or the two combined. Gastrocnemius was freeze-clamped, and PHAS-I and p70S6k phosphorylation was measured by quantifying the several phosphorylated forms of these proteins seen on Western blots. We observed that feeding increased phosphorylation of both PHAS-I and p70S6k ( P < 0.05). Infusion of amino acids alone reproduced the effect of feeding. Physiological hyperinsulinemia increased p70S6K ( P< 0.05) but not PHAS-I phosphorylation ( P = 0.98). Addition of insulin to amino acid infusion was no more effective than amino acids alone in promoting PHAS-I and p70S6kphosphorylation. We conclude that amino acid infusion alone enhances the activation of the protein synthetic pathways in vivo in rat skeletal muscle. This effect is not dependent on increases in plasma insulin and simulates the activation of protein synthesis that accompanies normal feeding.


1988 ◽  
Vol 118 (2) ◽  
pp. 301-305 ◽  
Author(s):  
K. Gréen ◽  
O. Vesterqvist ◽  
V. Grill

Abstract. The in vivo synthesis of thromboxane A2 and prostacyclin was estimated in 23 diabetics through measurements of the major urinary metabolites 2,3-dinor-thromboxane B2 and 2,3-dinor-6-keto-PGF1α utilizing gas chromatography-mass spectrometry. Mean excretion was similar to that in non-diabetic subjects. The possible influence of hyperglycemia on the excretion of 2,3-dinor-thromboxane B2 and 2,3-dinor-6-keto-PGF1α was evaluated in three ways: by measuring excretion before and during an acute 9-h normalization of hyperglycemia through an artificial pancreas (Biostator) as well as by comparing excretion before and 7–12 days or 40–180 days after the initiation of insulin treatment. Despite significant reducing effects on hyperglycemia or on levels of hemoglobin A1c, no effects on the excretion of the thromboxane and prostacyclin metabolites could be found. Abnormal formation of thromboxane or prostacyclin is not a generalized feature of the diabetic state.


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