Culture conditions necessary for optimizing albumin secretion were studied in rat hepatocytes maintained in a chemically defined, serum-free medium. Amino acid analysis of the culture medium, which was based on a 1:1 mixture of Ham's F12:Dulbecco's modified Eagle's medium (unsupplemented medium), revealed that certain essential amino acids were depleted from this medium over a 24-h incubation. Rates of albumin secretion were significantly higher and better maintained when the medium was supplemented with additional amino acids (supplemented medium). Moreover, selective removal of an essential amino acid resulted in an immediate decrease in total protein and albumin synthesis and after 48 h a further selective decrease in albumin synthesis. Linear rates of albumin secretion were observed over a wide variety of experimental conditions, but secretion was not strictly proportional to cell number. Maximal rates of secretion were obtained at plating densities of 2-3 x 106 cells/60 mm culture dish. Albumin secretion also increased with time in culture reaching a maximum on days 3 and 4. When added singly, either insulin or dexamethasone increased rates of albumin secretion in a dose-dependent manner, but both hormones and an adequate supply of amino acids were necessary for maximal rates of secretion as well as long-term maintenance of the hepatocytes (>3-4 days). In the presence of dexamethasone dose-response curve for insulin was shifted toward physiological concentrations. Changes in rates of albumin secretion in response to added hormones in supplemented media were found to parallel changes in albumin synthesis and relative amounts of albumin mRNA. Changes in gene transcription were probably involved.
|American Journal of Physiology - Endocrinology and Metabolism
|Published - 1987
All Science Journal Classification (ASJC) codes
- Endocrinology, Diabetes and Metabolism
- Physiology (medical)