Primary Cultures Of Fetal Hepatocytes Research Articles

In vitro and in vivo regulation of hepatic mitogen-activated protein kinases in fetal rats.

We have studied the role of mitogen-activated protein (MAP) kinases in fetal hepatocyte growth in vitro and in vivo. With myelin basic protein (MBP) as the phosphate acceptor, kinase activity in cultured fetal hepatocyte lysates increased fourfold after exposure to transforming growth factor-alpha (TGF-alpha) for 10 min. This TGF-alpha-responsive MBP kinase activity was accounted for by five distinct MAP kinase isoforms detected by Western immunoblotting. All had negligible activity in cultured fetal hepatocytes under basal conditions. Treatment of fetal hepatocytes with hepatocyte growth factor led to activation of the predominant isoforms, relative molecular weight (M(r)) = 42,000 and 44,000 in a manner indistinguishable from TGF-alpha, whereas insulin had no effect. All five of the immunoreactive MAP kinases were present in both fetal and adult liver homogenates. The M(r) = 42,000 and 44,000 isoforms were only minimally activated in vivo. We conclude that the mitogen-independent growth exhibited by fetal hepatocytes in primary culture is not associated with tonic activation of the MAP kinase system. Our data support the possibility that fetal hepatic growth may be, in part, independent of the action of growth factors as mediated via the MAP kinase system.

Hepatocyte Growth Factor Up-Regulates MET Expression in Rat Fetal Hepatocytes in Primary Culture

Hepatocyte growth factor (HGF) is a potent mitogen for primary cultured fetal hepatocytes. In the present study, we have analyzed the c-met/HGF receptor expression in fetal hepatocytes and its modulation by growth factors and hormones. 20-day old fetal liver showed a barely expression of c-met mRNA levels. However, when fetal hepatocytes were incubated in the presence of HGF, a 10-fold increase in c-met mRNA levels was observed 30 min after addition of the factor. This HGF-induced effect on c-met expression was transient, losing its up-regulatory effect after 24 hours and returning to the initial levels at 48 hours. Transforming growth factor-beta, a negative regulator of fetal liver growth, increased c-met mRNA levels 48 hours after the addition of the factor, whereas glucocorticoids had a negative effect.

Development of primary culture of ovine fetal hepatocytes for studies of amino acid metabolism and insulinlike growth factors

We report the development and characterization of a system of primary culture of ovine fetal hepatocytes to aid in the understanding of the cellular regulation of fetal growth and metabolism with emphasis on amino acid metabolism and insulinlike growth factor gene expression and to allow comparison to in vivo studies. Hepatocytes were isolated from late gestation fetal lambs by in situ perfusion and collagenase digestion utilizing occlusion of the ductus venosus to limit intrahepatic shunting. Hepatocytes were cultured in media modified to mimic fetal concentrations of glucose, lactate, and amino acids. Ovine fetal hepatocytes in primary culture maintain the pattern of fetal amino acid production and utilization seen across the fetal liver in vivo. Specifically, there is a net production of serine and a net utilization of glycine. Cultured ovine fetal hepatocytes specifically increase tritiated thymidine incorporation in response to insulin and insulinlike growth factor II (IGF-II). IGF-II mRNA abundance is high and IGF-I mRNA is low in cultured ovine fetal hepatocytes as in the fetal sheep liver in vivo. These data demonstrate the successful isolation of ovine fetal hepatocytes that retain some of the characteristics of the ovine fetal liver while maintained in short-term culture.

Studies on the expression of liver-specific functions of human fetal hepatocytes in primary culture.

Biochemical functions of human livers were studied using fetal hepatocytes in primary culture. Immunocytochemical staining showed that albumin was not expressed in any fetal hepatocytes, whereas alpha-fetoprotein was detected in almost all the cells. Tryptophan 2,3-dioxygenase (TO, EC 1.13.11.11.) activity was not induced in the presence of 10(-7) M dexamethasone and 10(-7) M glucagon, but the activity of tyrosine aminotransferase (TAT, EC 2.6.1.5.) was elevated about 35 fold under the same conditions. These results suggest that the TAT and alpha-fetoprotein genes are activated in human fetal liver at 14 to 20 weeks of gestation.

Primary cultures of fetal hepatocytes from the genetically obese Zucker rat: protein synthesis.

Primary fetal hepatocytes derived from Zucker rats with expected fa gene frequencies of 0.0 and 0.75 have been established and can be used to detect early effects of the fa gene on hepatocellular metabolism. Paired incubation experiments demonstrate that protein synthesis in 0.75 fa gene cultures is significantly less than in 0.0 fa gene cultures under basal conditions. Insulin stimulates protein synthesis in 0.0 fa gene cultures but has no effect on 0.75 fa gene cultures. Cycloheximide inhibits protein synthesis in both types of culture. NH4Cl inhibits protein synthesis in 0.0 but not in 0.75 fa gene cultures. These data suggest that fetal hepatocytes bearing the fa gene have in vitro a generally sluggish anabolic capacity and a blunted capacity to respond to insulin compared to fetal hepatocytes without the fa gene. These diminished capacities may be expression of a genetic error in lysosomal function.

Primary cultures of fetal hepatocytes from the genetically obese Zucker rat: characterization and total lipogenesis.

Primary fetal hepatocyte cultures derived from Zucker rats and with expected fa-gene frequencies of 0.0 and 0.75 have been established and can be used to detect early effects of the fa gene on hepatocellular metabolism. Proliferative capacity is similar in both types of culture. Changes of the growth media significantly decrease total lipogenesis in both 0.0 and 0.75 fa-gene culture grown in arginine-free DME medium. Paired incubation experiments demonstrate that total lipogenesis in 0.75 fa gene cultures is significantly less than in 0.0 fa-gene cultures under basal conditions. Stimulation of total lipogenesis by pharmacological doses of insulin and excess substrate (glucose) is significantly less in the 0.75 fa gene than in the 0.0 fa-gene cultures. These data suggest that the development of obesity in the Zucker rat cannot be attributed to elevated hepatic lipogenesis in the fetus.

Development of glycogen storage ability under cortisol control in primary cultures of rat fetal hepatocytes

Cortisol has been shown to induce glycogen storage function in primary cultures of fetal hepatocytes. The method we describe provides a homogeneous population of hepatocytes by elimination of hematopoietic cells. Hepatocytes transplanted from 15-day-old fetuses were grown in the absence or presence of cortisol (10 −5 M) for periods of up to 4 days. In the presence of cortisol, after a lag period (24 hr), the glycogen content increased sharply, regardless of whether the medium was replaced or not. Incorporation of radioactivity from (U) 14C-glucose into glycogen paralleled glycogen accumulation, but the specific activity of the stored glycogen was lower than the final specific activity of the glucose in the medium. This result shows that free glucose is a good precursor of glycogen but not the only one. Data from chase and labeling experiments prove that the hormone acts on the synthetic pathway. If cortisol was removed the glycogen content dropped, suggesting that glycogen synthesis depends on the continuous presence of the hormone. The in vitro maturation of hepatocyte can be provoked by the hormone before the normal in vivo maturation stage of the onset of glycogen accumulation. Other studies of the same in vivo phenomenon have demonstrated that accumulation of glycogen in the liver prior to birth is corticosteroid dependent, but only an in vitro study could clearly show that the hormone acts at the cellular level.