EFFECT OF INSULIN ON TRIACYLGLYCEROL SYNTHESIS AND SECRETION BY CHICKEN HEPATOCYTES IN PRIMARY CULTURE

A primary culture system of chicken hepatocytes was developed to study the effects of genetic, hormonal and nutritional factors on hepatic t riglyceride (TG) secretion in the chicken, and the effect of insulin o n TG synthesis and secretion examined. TG synthesis and secretion was measured using [H-3]-glycerol incorporation into cellular and secreted TG. An additional step consisting of brief incubation of the monolaye r with trypsin solution to improve harvesting of the medium immediatel y adjacent to the cell monolayer, is also proposed. In our culture sys tem, 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 insu lin could be observed after 24 hr of culture. A clear stimulatory effe ct was observed with the 10(-9) M insulin concentration after 48 hr. T he mean ratio of the secretion rates in the presence or absence of ins ulin was 2.20 +/- 0.30 (SEM, n = 4). In contrast, the 10(-6) M concent ration of insulin had no effect on TG secretion. The use of an additio nal trypsinization step enhanced the findings obtained by simple remov al of the culture medium: a clear stimulatory effect of insulin on TG synthesis was observed after both ZA and 48 hr of culture. Analysis of TG fatty acid composition showed an imbalance of monoene versus satur ated fatty acids between cellular and secreted TG, secreted TG being m ore monounsaturated than cellular TG. These results were obtained in t he absence of exogenous fatty acid. An oleic acid supplement in the cu lture 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 exogeno us fatty acid and this secretion was stimulated by insulin. It was con cluded that chicken hepatocytes in primary culture provide a useful mo del for studying regulation of TG secretion. Copyright (C) 1996 Elsevi er Science Ltd.