J. Biol. Chem., Vol. 268, Issue 34, 25277-25284, Dec, 1993
Regulation of rDNA transcription by insulin in primary cultures of rat hepatocytes
DA Antonetti, SR Kimball, RL Horetsky and LS Jefferson
Department of Cellular and Molecular Physiology, College of Medicine, Pennsylvania State University, Hershey 17033.
The studies presented herein were designed to investigate the role of
insulin in maintaining the steady-state number of ribosomes in primary
cultures of rat hepatocytes. The RNA content of hepatocytes maintained in
the presence of insulin did not change during the 7 days of culture. In
contrast, the RNA content declined significantly following 2 days of
insulin deprivation and after 4 days without insulin reached a new
steady-state value that was approximately 60% of that observed for
hepatocytes maintained in the presence of the hormone. Following addition
of insulin, the RNA content of insulin-deprived hepatocytes started to
increase within 6 h and was restored to the control value within 48 h. The
amounts of 18 and 28 S RNA, and thus the number of ribosomes, changed in
concert with the total RNA content. Furthermore, synthesis of total
cellular protein responded in parallel to the changes in RNA content,
suggesting that the number of ribosomes was the primary determinant of
protein synthesis under the conditions of these experiments. About one-half
of the increase in RNA content following the addition of insulin to
insulin-deprived cells could be accounted for by a reduction in the rate of
degradation of ribosomes. The remainder of the change in RNA content must
have resulted from an increase in ribosome biogenesis. This possibility was
confirmed by measuring [3H]uridine incorporation into ribosomal RNA present
in mature ribosomes. The rate of synthesis of ribosomal RNA was reduced in
parallel with the fall in RNA content in insulin-deprived hepatocytes and
was restored to the control value within 3 h of the addition of insulin.
Alterations in the synthesis of ribosomal RNA in response to insulin
deprivation and replacement were associated with parallel changes in
transcription of rDNA as measured in nuclear run-on assays using a DNA
probe corresponding to the external transcribed spacer region of rat rDNA.
The data demonstrate that insulin regulates the number of ribosomes in
primary cultures of rat hepatocytes by accelerating the rate of
transcription of rDNA and by slowing the rate of ribosome degradation.
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