Abstract
The origin of corticosteroid-binding globulin (CBG) and its evolution in comparison with alpha-fetoprotein (AFP) and albumin synthesis, during early development of rat liver (days 13 and 15 of fetal life), have been investigated using cultured fetal hepatocytes. Synthesis and secretion of CBG, AFP, and albumin is evidence by cycloheximide-sensitive [14C]leucine incorporation into immunoprecipitable polypeptides secreted by cultured hepatocytes into the medium, two-dimensional immunoelectrophoretic and autoradiographic identification of newly synthesized labeled proteins, corticosterone and estradiol-17 beta binding to CBG and AFP, respectively, and indirect immunofluorescence localization of AFP, albumin, and CBG in cultured fetal hepatocytes. CBG, albumin, and AFP accounted for 6, 11, and 25% (in 13-day-old rat fetuses) and 5, 15, and 28% (15-day-old rat fetuses), respectively, of the total secreted proteins in the culture medium. The rates of CBG, AFP, and albumin (counts/minute of secretion [14C]leucine incorporated per milligram of cell protein/hour of culture) in the hepatocytes of 15-day-old rat fetuses were 1.48-, 2.1-, and 2.57-fold higher, respectively, than in the 13-day-old rat fetuses. These results indicate that fetal liver is also active in CBG synthesis, along with AFP and albumin, as early as day 13 of fetal life and that the synthetic rates of these secretory proteins depend upon the developmental stage of the fetal liver. This developmental related change in the rate of synthesis of CBG by the fetal hepatocytes may regulate the level of free (active) glucocorticoid in the fetal circulation and thereby the initiation and regulation of glucocorticoid-dependent processes during the crucial stages of the differentiation of fetal liver and other developing tissues.
Highlights
The origin of corticosteroid-binding globulin (CBG) Corticosteroid-binding globulin (CBG’ or transcortin), the andits evolution in comparison witha-fetoprotein major high affinity serum glycoprotein binder for natural (AFP) and albumin synthesis, during early develop- corticoids, has been envisaged as the protein controlling the ment of rat liver, have availability of free corticoid concentrabeen investigated using culturedfetal hepatocytes. tions and therebeyxpression of corticoid-dependent processes
These results indicatethat fetal nantly express AFP, wheras albumin synthesis and its level liver is active in CBG synthesis, along with AFP in the fetal serum are low throughout the prenatal developand albumin, as early as day 13 of fetal life and that ment [15]
Our studies provide conclusive evidence that developing hepatocytes are thesite of CBG synthesis and secretion; that fetal liver is capable of synthesizing CBG, AFP, and albumin as early as day 13 of fetal life; and that the rateosf synthesis of these secretory proteins depend upon the developmental stage of the hepatocyte
Summary
24 h and analyzed by 2-DIEP and autoradiography as described in the legend of Fig. 3. The culture media were quantitatively analyzed for CBG, AFP, albumin (using the specific immunoprecipitation procedure), and total proteins. Electrophoretograms (a, c, and e ) represent secretory protein patterns of cultured hepatocytes after 24 h in serum-free (a and c) and 10% FCS-supplemented ( e ) media. Secretion of total proteins (tricarboxylic acid-insoluble) by cultured hepatocytes displayed an increase in both serum-free or -supplemented medium during 3 days of the culture period (Fig. 6a). Serum-supplemented cultures secreted approximately 8% more protein (during the third day) than the cultures grown diate gel containing anti-CBG. CBGshowed little variation with respect to FCS presence and remained 5-6% in both FCS-free and -supplemented media It may beconcludedthat in vitro maintenance of the hepatocyte in culture may result inselectiveloss of specificgene expression, as evident by albumin synthesis in our studies. The rates of synthesis of proteins like CBG, AFP, and albumin as observed
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