Insulin effects on protein synthesis and secretion in primary cultures of amphibian hepatocytes

J. E. Stanchfield, James D Yager

Research output: Contribution to journalArticle

Abstract

The objective of the present study was to determine the effects of insulin on amphibian hepatocytes in primary culture. Hepatocytes were isolated from adult bullfrogs by collagenase perfusion and maintained as monolayers in serum-free medium. Cells cultured in the continuous presence of insulin exhibited a relatively constant rate of protein secretion over the first four to five days, whereas controls showed an almost three-fold decrease over the same time period. The decline in secreted proteins was equally represented in most exported proteins, except that serum albumin secretion showed twice as much of a decrease relative to the other proteins. The maintenance of protein secretion by insulin was the result of its effect on protein synthesis. The rate of protein synthesis was measured by the incorporation of (3H)-leucine into protein using culture medium containing 0.5 mM leucine, a condition where the specific radioactivity of leucyl-tRNA was shown to be equal to that of (3H)-leucine in the medium. Cultures maintained with insulin for 60 hours synthesized protein at two to three times the rate found in non-insulin treated controls whose rate of protein synthesis was first detectably decreased after nine hours of culture in the insulin-free medium. Sedimentation profiles of polyribosomes from hepatocytes maintained for 60 hours without insulin showed proportionately fewer ribosomes in large polysomes and more in monosomes and free ribosomal subunits than ribosomes from cells cultured with insulin. This result suggests that the decrease in protein synthesis found in the agsence of insulin is due to a defect in initiation. Insulin does not exert its effect by regulating cellular levels of ATP; no change in ATP content was found in cells maintained with or without insulin. The results show that insulin maintains high levels of protein synthesis and secretion in amphibian hepatocytes. The hepatocytes in monolayer culture provide a system to study the molecular mechanisms involved in the translational control of protein synthesis by insulin.

Original languageEnglish (US)
Pages (from-to)279-289
Number of pages11
JournalJournal of Cellular Physiology
Volume100
Issue number2
StatePublished - 1979
Externally publishedYes

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Amphibians
Hepatocytes
Insulin
Proteins
Leucine
Polyribosomes
Ribosomes
Monolayers
Cultured Cells
Adenosine Triphosphate
Ribosome Subunits
Rana catesbeiana
Serum-Free Culture Media
Radioactivity
Collagenases
Transfer RNA
Sedimentation
Serum Albumin
Culture Media
Perfusion

ASJC Scopus subject areas

  • Cell Biology
  • Clinical Biochemistry
  • Physiology

Cite this

Insulin effects on protein synthesis and secretion in primary cultures of amphibian hepatocytes. / Stanchfield, J. E.; Yager, James D.

In: Journal of Cellular Physiology, Vol. 100, No. 2, 1979, p. 279-289.

Research output: Contribution to journalArticle

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