Effect of insulin on triacylglycerol synthesis and secretion by chicken hepatocytes in primary culture

Abstract

A primary culture system of chicken hepatocytes was developed to study the effects of genetic, hormonal and nutritional factors on hepatic triglyceride (TG) secretion in the chicken, and the effect of insulin on TG synthesis and secretion examined. TG synthesis and secretion was measured using [ 3H]-glycerol incorporation into cellular and secreted TG. An additional step consisting of brief incubation of the monolayer with trypsin solution to improve harvesting of the medium immediately adjacent to the cell monolayer, is also proposed. In our culture system, TG secretion occurred at least up to 75 hr of culture, showing a maximum between 40 and 60 hr of culture. No significant effect of insulin could be observed after 24 hr of culture. A clear stimulatory effect was observed with the 10 −9 M insulin concentration after 48 hr. The mean ratio of the secretion rates in the presence or absence of insulin was 2.20 ± 0.30 (SEM, n = 4). In contrast, the 10 −6 M concentration of insulin had no effect on TG secretion. The use of an additional trypsinization step enhanced the findings obtained by simple removal of the culture medium: a clear stimulatory effect of insulin on TG synthesis was observed after both 24 and 48 hr of culture. Analysis of TG fatty acid composition showed an imbalance of monoene versus saturated fatty acids between cellular and secreted TG, secreted TG being more monounsaturated than cellular TG. These results were obtained in the absence of exogenous fatty acid. An oleic acid supplement in the culture medium did not significantly influence TG secretion. In summary, a primary culture system for chicken hepatocyte was developed. A high level of TG secretion was seen in these cells with or without exogenous fatty acid and this secretion was stimulated by insulin. It was concluded that chicken hepatocytes in primary culture provide a useful model for studying regulation of TG secretion.