Regulation by insulin of amino acid release and protein turnover in the perfused rat hemicorpus

Leonard "Jim" Jefferson, J. B. Li, S. R. Rannels

Research output: Contribution to journalArticle

148 Citations (Scopus)

Abstract

Net changes in the concentrations of 18 amino acids in perfusate and skeletal muscle were followed during perfusion of hemicorpus preparations from fed rats. Perfusate levels of 16 amino acids showed little change from their initial concentrations during the 1st h, but increased dramatically during the 2nd and 3rd h. Aspartate and glutamate levels decreased continuously throughout the perfusion. Release of alanine and glutamine accounted for approximately 50% of the total change in perfusate amino acids. The increase in perfusate amino acids was derived from net breakdown of muscle proteins and not from leakage from the intracellular pool as evidenced by elevated concentrations of intracellular amino acids in perfused muscle. Addition of insulin to the perfusate did not change the pattern of amino acid release during the 1st h of perfusion. However, during the 2nd and 3rd h the hormone completely prevented the net release of most amino acids and maintained intracellular concentrations of most amino acids at levels found in unperfused tissue. Effects of time of perfusion and insulin on amino acid release were accounted for by changes in the rate of protein turnover. Protein synthesis in gastrocnemius and psoas muscles in control perfusions decreased after 1 h to approximately 50% of the initial rate. This decrease was accompanied by a 2 fold increase in the level of ribosomal subunits, indicating development of a block in peptide chain initiation. Addition of insulin maintained the initial rate of synthesis and the in vivo level of ribosomal subunits, demonstrating that the hormone prevented the block in peptide chain initiation from forming. Addition of insulin after 2 h reversed the perfusion induced block in initiation. Synthesis of the specific muscle protein myosin was increased 45% over the control rate in the presence of insulin. Insulin also produced a 50% decrease in the rate of protein degradation during the 2nd and 3rd h of perfusion. A similar effect was noted when protein synthesis was inhibited by addition of cycloheximide. Higher concentrations of insulin were required to maximally inhibit protein degradation than to increase protein synthesis. Involvement of lysosomal proteases in the effect of insulin on protein degradation was evaluated by measuring cathepsin D activity in psoas muscle homogenates. 'Free' enzyme activity increased as a result of perfusion while addition of insulin maintained this activity at the unperfused level. Neither perfusion nor insulin had any effect on total cathepsin D activity. Alterations in protein degradation and lysosomal enzyme activity were not due to changes in levels of adenine nucleotides, GTP, or creatine phosphate.

Original languageEnglish (US)
Pages (from-to)1476-1483
Number of pages8
JournalJournal of Biological Chemistry
Volume252
Issue number4
StatePublished - Jan 1 1977

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Rats
Insulin
Perfusion
Amino Acids
Proteins
Proteolysis
Muscle
Psoas Muscles
Ribosome Subunits
Degradation
Cathepsin D
Muscle Proteins
Enzyme activity
Skeletal Muscle
Hormones
Peptides
Phosphocreatine
Adenine Nucleotides
Enzymes
Cycloheximide

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "Regulation by insulin of amino acid release and protein turnover in the perfused rat hemicorpus",
abstract = "Net changes in the concentrations of 18 amino acids in perfusate and skeletal muscle were followed during perfusion of hemicorpus preparations from fed rats. Perfusate levels of 16 amino acids showed little change from their initial concentrations during the 1st h, but increased dramatically during the 2nd and 3rd h. Aspartate and glutamate levels decreased continuously throughout the perfusion. Release of alanine and glutamine accounted for approximately 50{\%} of the total change in perfusate amino acids. The increase in perfusate amino acids was derived from net breakdown of muscle proteins and not from leakage from the intracellular pool as evidenced by elevated concentrations of intracellular amino acids in perfused muscle. Addition of insulin to the perfusate did not change the pattern of amino acid release during the 1st h of perfusion. However, during the 2nd and 3rd h the hormone completely prevented the net release of most amino acids and maintained intracellular concentrations of most amino acids at levels found in unperfused tissue. Effects of time of perfusion and insulin on amino acid release were accounted for by changes in the rate of protein turnover. Protein synthesis in gastrocnemius and psoas muscles in control perfusions decreased after 1 h to approximately 50{\%} of the initial rate. This decrease was accompanied by a 2 fold increase in the level of ribosomal subunits, indicating development of a block in peptide chain initiation. Addition of insulin maintained the initial rate of synthesis and the in vivo level of ribosomal subunits, demonstrating that the hormone prevented the block in peptide chain initiation from forming. Addition of insulin after 2 h reversed the perfusion induced block in initiation. Synthesis of the specific muscle protein myosin was increased 45{\%} over the control rate in the presence of insulin. Insulin also produced a 50{\%} decrease in the rate of protein degradation during the 2nd and 3rd h of perfusion. A similar effect was noted when protein synthesis was inhibited by addition of cycloheximide. Higher concentrations of insulin were required to maximally inhibit protein degradation than to increase protein synthesis. Involvement of lysosomal proteases in the effect of insulin on protein degradation was evaluated by measuring cathepsin D activity in psoas muscle homogenates. 'Free' enzyme activity increased as a result of perfusion while addition of insulin maintained this activity at the unperfused level. Neither perfusion nor insulin had any effect on total cathepsin D activity. Alterations in protein degradation and lysosomal enzyme activity were not due to changes in levels of adenine nucleotides, GTP, or creatine phosphate.",
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Regulation by insulin of amino acid release and protein turnover in the perfused rat hemicorpus. / Jefferson, Leonard "Jim"; Li, J. B.; Rannels, S. R.

In: Journal of Biological Chemistry, Vol. 252, No. 4, 01.01.1977, p. 1476-1483.

Research output: Contribution to journalArticle

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N2 - Net changes in the concentrations of 18 amino acids in perfusate and skeletal muscle were followed during perfusion of hemicorpus preparations from fed rats. Perfusate levels of 16 amino acids showed little change from their initial concentrations during the 1st h, but increased dramatically during the 2nd and 3rd h. Aspartate and glutamate levels decreased continuously throughout the perfusion. Release of alanine and glutamine accounted for approximately 50% of the total change in perfusate amino acids. The increase in perfusate amino acids was derived from net breakdown of muscle proteins and not from leakage from the intracellular pool as evidenced by elevated concentrations of intracellular amino acids in perfused muscle. Addition of insulin to the perfusate did not change the pattern of amino acid release during the 1st h of perfusion. However, during the 2nd and 3rd h the hormone completely prevented the net release of most amino acids and maintained intracellular concentrations of most amino acids at levels found in unperfused tissue. Effects of time of perfusion and insulin on amino acid release were accounted for by changes in the rate of protein turnover. Protein synthesis in gastrocnemius and psoas muscles in control perfusions decreased after 1 h to approximately 50% of the initial rate. This decrease was accompanied by a 2 fold increase in the level of ribosomal subunits, indicating development of a block in peptide chain initiation. Addition of insulin maintained the initial rate of synthesis and the in vivo level of ribosomal subunits, demonstrating that the hormone prevented the block in peptide chain initiation from forming. Addition of insulin after 2 h reversed the perfusion induced block in initiation. Synthesis of the specific muscle protein myosin was increased 45% over the control rate in the presence of insulin. Insulin also produced a 50% decrease in the rate of protein degradation during the 2nd and 3rd h of perfusion. A similar effect was noted when protein synthesis was inhibited by addition of cycloheximide. Higher concentrations of insulin were required to maximally inhibit protein degradation than to increase protein synthesis. Involvement of lysosomal proteases in the effect of insulin on protein degradation was evaluated by measuring cathepsin D activity in psoas muscle homogenates. 'Free' enzyme activity increased as a result of perfusion while addition of insulin maintained this activity at the unperfused level. Neither perfusion nor insulin had any effect on total cathepsin D activity. Alterations in protein degradation and lysosomal enzyme activity were not due to changes in levels of adenine nucleotides, GTP, or creatine phosphate.

AB - Net changes in the concentrations of 18 amino acids in perfusate and skeletal muscle were followed during perfusion of hemicorpus preparations from fed rats. Perfusate levels of 16 amino acids showed little change from their initial concentrations during the 1st h, but increased dramatically during the 2nd and 3rd h. Aspartate and glutamate levels decreased continuously throughout the perfusion. Release of alanine and glutamine accounted for approximately 50% of the total change in perfusate amino acids. The increase in perfusate amino acids was derived from net breakdown of muscle proteins and not from leakage from the intracellular pool as evidenced by elevated concentrations of intracellular amino acids in perfused muscle. Addition of insulin to the perfusate did not change the pattern of amino acid release during the 1st h of perfusion. However, during the 2nd and 3rd h the hormone completely prevented the net release of most amino acids and maintained intracellular concentrations of most amino acids at levels found in unperfused tissue. Effects of time of perfusion and insulin on amino acid release were accounted for by changes in the rate of protein turnover. Protein synthesis in gastrocnemius and psoas muscles in control perfusions decreased after 1 h to approximately 50% of the initial rate. This decrease was accompanied by a 2 fold increase in the level of ribosomal subunits, indicating development of a block in peptide chain initiation. Addition of insulin maintained the initial rate of synthesis and the in vivo level of ribosomal subunits, demonstrating that the hormone prevented the block in peptide chain initiation from forming. Addition of insulin after 2 h reversed the perfusion induced block in initiation. Synthesis of the specific muscle protein myosin was increased 45% over the control rate in the presence of insulin. Insulin also produced a 50% decrease in the rate of protein degradation during the 2nd and 3rd h of perfusion. A similar effect was noted when protein synthesis was inhibited by addition of cycloheximide. Higher concentrations of insulin were required to maximally inhibit protein degradation than to increase protein synthesis. Involvement of lysosomal proteases in the effect of insulin on protein degradation was evaluated by measuring cathepsin D activity in psoas muscle homogenates. 'Free' enzyme activity increased as a result of perfusion while addition of insulin maintained this activity at the unperfused level. Neither perfusion nor insulin had any effect on total cathepsin D activity. Alterations in protein degradation and lysosomal enzyme activity were not due to changes in levels of adenine nucleotides, GTP, or creatine phosphate.

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