Sodium butyrate preserves aspects of the differentiated phenotype of normal adult rat hepatocytes in culture

Abstract

We have determined that sodium butyrate and, to a lesser extent, dimethylsulfoxide (DMSO) and 3-aminobenzamide (3-AB) preserve aspects of the differentiated phenotype of primary cultures of adult rat hepatocytes. The histone deacetylase inhibitor, butyrate, inhibits the increase in gamma-glutamyltranspeptidase (GGT) activity and the decrease in basal tyrosine aminotransferase (TAT) activity normally observed when hepatocytes are cultured under appropriate conditions. The effects of butyrate on GGT and TAT activities are accompanied by parallel changes in GGT and TAT mRNA levels. The poly(ADP)ribose-synthetase inhibitor, 3-aminobenzamide, has effects similar to butyrate on GGT activity and mRNA levels, while both 3-AB and DMSO increase basal TAT activity in cultured hepatocytes. Under appropriate conditions all three agents--butyrate, 3-AB, and DMSO--extend the length of time cultured hepatocytes can be maintained as confluent monolayers. However, under all the conditions studied, butyrate extended the length of time hepatocytes could be maintained as monolayers more than any other treatment used. Butyrate-treated hepatocytes maintained ultrastructural features that were more similar to those of hepatocytes in vivo than hepatocytes treated with any other of the agents tested. Histone acetylation levels of primary cultures of adult rat hepatocytes declined concomitant with the loss of the differentiated phenotype of the cells. These results suggest that histone acetylation may play a role in the changes in gene expression observed when hepatocytes are placed in culture.

The emergence of ErbB2 expression in cultured rat hepatocytes correlates with enhanced and diversified EGF-mediated signaling

“…Finally, we evaluated if HRG-␤ 1 or PMA stimulated ErbB2 expression. These agents have little endogenous growth stimulatory ability but can potentiate TGF-␣-mediated DNA synthesis (32). Figure 6C shows that neither HRG-␤ 1 nor PMA alone induced ErbB2; however, both increased the TGF-␣ induction of ErbB2, akin to their effects on DNA synthesis.…”

The current status of primary hepatocyte culture

“…Two factors -high amino acid concentrations (about 3fold compared to Dulbecco's modified Eagle's medium, DMEM) and the lack of a need for a CO 2 incubatorwere important reasons why some investigators chose L-15 medium for primary culture of adult rat hepatocytes. In fact, the medium has been used for many years to study the maintenance or induction, or both, of several differentiated hepatocyte functions (Michalopoulos et al 1976;Sawada et al 1987;Staecker et al 1988). Although primary hepatocytes can be satisfactorily maintained in L-15 medium in a 100% air incubator, they do not undergo mitosis and divide under these conditions to an appreciable extent (Sawada et al 1987).…”

Amino acid‐rich medium (Leibovitz L‐15) enhances and prolongs proliferation of primary cultured rat hepatocytes in the absence of serum

“…During cultures. This pattern of DNA synthesis of the hepatocytes cultured in modified L-15 medium was much different from that of the DNA synthesis in cells in DMEMiFl2 medium, which had been previously used in our laboratory (Sawada et al, 1986;Tomomura et al, 1987;Staecker et al, 1988) and by others (Bottenstein and Sato, 1979). We then compared the time course of DNA synthesis in hepatocytes cultured in modified L-15 medium to that of hepatocytes cultured in modified DMEM/F12 (Fig.…”

“…Leibovitz L-15 medium was originally designed for virus production in cell lines cultured in a 100% air incubator (Leibovitz, 1963). In our laboratory, L-15 medium in a 100% air incubator has proven to be quite effective for the maintenance of differentiated 1982; Nakamura et al, 1984; Houck and hf ichalopou-functions of adult hepatocytes in culture (Michalopoulos et al, 1976;Sirica et al, 1979;Sawada et al, 1987;Staecker et al, 1988), but unsatisfactory in sup orting cell division . In the stulies described herein, however, we show that primary cultured adult rat hepatocytes can go through multiple cell divisions in a modified L-15 medium when it contains sodium bicarbonate in a 5% coz/95% air atmosphere.…”