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The visceral yolk sac is a fetal membrane with essential placental functions. It is the major site of synthesis of alpha-fetoprotein (AFP), the most abundant plasma protein in the fetus. We developed a system of rat yolk sac explants in serum-free culture medium to study the regulation of endodermal gene expression in yolk sac. The explanted yolk sac tissues retained their double-sided morphology for up to 48 hours. The epithelial cells of both layers remained tightly joined on a basement membrane as seen by light and electron microscopy. This probably accounts for the continued expression of several endodermal cell-specific markers. The levels of mRNA encoding AFP, vitamin D-binding protein (DBP), hepatocyte nuclear factor 1alpha and beta transcription factors did not change during the 48-hour culture period. This reflects the stability of the differentiation state of the yolk sac endodermal cells. Dexamethasone and phorbol ester (TPA) specifically reduced the AFP mRNA level without affecting that of DBP. This suggests that these transduction pathways are functional in the yolk sac during this period of gestation and could be involved in the physiological down-regulation of AFP gene expression before birth. All these results show that this serum-free culture of rat yolk sac explants is a valuable system for further investigating the action of natural compounds and pharmacological drugs on endodermal gene expression during the embryonic and fetal periods.

The oncodevelopmentally regulated alpha-fetoprotein (AFP) gene offers a very good model system to better understand the molecular mechanisms which dictate the specificity of gene expression in liver and control its tight modulation in the course of development and carcinogenesis. Transcription factors of the CCAAT/enhance-binding protein (C/EBP), hepatocyte nuclear factor-1 (HNF-1), and nuclear factor-1 (NF-1) families can bind in vitro to the promoter of the rat AFP gene, which makes the expression of the AFP gene specific to the liver. We have evaluated the influence of some of these factors on the activity of the AFP promoter by transfection of HepG2 hepatoma cells with the appropriate expression vector plus a CAT plasmid under the control of the AFP promoter. A similar plasmid bearing the rat albumin promoter was used as a control. C/EBP alpha, and C/EBP beta acted as transactivators on the AFP promoter, while LIP, a truncated form of C/EBP beta, was a potent negative regulator of the promoter. Interestingly, HNF-1 beta was found to be more potent than HNF-1 alpha in activating the AFP promoter in the HepG2 cells. This effect was highly promoter and cell specific since it did not occur with the rat albumin promoter or in Chinese hamster ovary cells. HNF-1 beta, which is produced earlier than HNF-1 alpha during liver development, would thus have the greater influence on the AFP promoter in early development. Our results pointed to a key role that NF1 might play in the functioning of the AFP promoter. Indeed, overexpression of NF1 induced a specific decrease in the activity of the AFP promoter. Competition between NF1 and HNF-1 for binding to their overlapping binding sites on the AFP promoter would be critical for modulating its activity.

Energy intake, weight gain, carcass composition, plasma fuels, hepatic metabolites and lipogenic enzyme activities were studied in adult rats fed either a low fat, high carbohydrate (LF) diet or one of two fat-containing diets in which 32% of the metabolizable energy was constituted by long-chain triglycerides (LCT) or medium-chain triglycerides (MCT). Compared with the LF diet, the MCT diet did not depress food and energy intake, weight gain, energy and nitrogen retention or lipid deposition and did not produce ketogenesis. The weight gain of rats fed LCT was 25% higher, and increased lipid deposition was observed. Lower lipogenic enzyme activities were observed in rats fed the LF diet containing 4% corn oil than in rats fed the MCT diet containing 1% corn oil. This effect disappeared when rats previously adapted to the LCT diet were fed LF or MCT diets containing 1% corn oil for 21 d. By d 21, in both groups, hepatic malic enzyme, ATP-citrate lyase, acetyl CoA carboxylase and fatty acid synthase activities were 2.2-, 2.0-, 2.3- and 1.8-fold higher than those of rats fed LCT. Intermediate hepatic glucose-6-phosphate dehydrogenase activities were observed in rats fed the MCT diet, compared with LCT (40% lower) and LF (1.6-fold higher) diets. These data show that in rats fed a diet in which MCT supplies 32% of metabolizable energy, a high activity of lipogenic enzymes is observed, suggesting that MCT had no inhibitory effect on the activity of these enzymes.

Changes in hepatic levels of lactate, pyruvate, phosphoenolpyruvate (PEP), alpha-ketoglutarate, malate, oxaloacetate, ketone bodies, alanine, serine, glycine, aspartate, glutamate, glutamine, valine, urea, adenine nucleotides and inorganic phosphate were examined in rats consuming a high protein, carbohydrate-free diet for up to 40 days. While some components showed transient changes, others (pyruvate, malate, oxaloacetate, PEP, ketone bodies, alanine, glycine, glutamine, valine, urea, adenine nucleotides and inorganic phosphate) were permanently altered. The cytoplasmic and mitochondrial redox states were only transiently affected and by day 24 were not different from control values. In contrast, the cytoplasmic phosphorylation state was affected from day 1 on; this suggests a role for the latter in permanently reorienting metabolism toward gluconeogenesis and ureogenesis.

Pyruvate kinase activity and rates of gluconeogenesis and glycolysis in rat hepatocytes were evaluated by production of glucose and lactate + pyruvate from dihydroxyacetone during the first 48 hours after the shift from a low protein, high carbohydrate diet to a high protein, carbohydrate-free diet. The effect of glucagon was also studied. In the absence of glucagon, 11-17 hours after the dietary shift when glycogen was lowest, gluconeogenesis was maximal and glycolysis minimal. The concentration of fructose 1,6-biphosphate was high and did not change during the experiment. The activity ratio of pyruvate kinase measured with phosphoenolpyruvate (PEP) (V0.5 mM PEP/V4 mM PEP) was high in crude extracts and low in (NH4)2SO4-treated extracts, but remained unchanged during the whole experiment. There was no correlation of the rates of gluconeogenesis or glycolysis from dihydroxyacetone with the activity ratio of pyruvate kinase. With glucagon, gluconeogenesis from dihydroxyacetone was increased and a concurrent decrease in glycolysis was paralleled with a decrease in the fructose 1,6-bisphosphate concentration and in the activity ratio of pyruvate kinase. The activity ratio of pyruvate kinase in (NH4)2SO4-treated cells represented about 50% of that in the absence of the hormone. This difference may be related to glucagon-induced phosphorylation of pyruvate kinase.

Gluconeogenesis from dihydroxyacetone (DHA), glycerol, lactate, pyruvate or alanine was studied in the absence or in the presence of glucagon in hepatocytes isolated from starved rats or from rats fed a high protein diet for 2-48 h. In both groups, gluconeogenesis from DHA, glycerol, lactate and pyruvate exhibited similar changes over 48 h; the rates of glucose production increased progressively until 24 h and then plateaued. During the early phase (2-11 h), gluconeogenesis from DHA and glycerol were higher than gluconeogenesis from lactate and pyruvate. During the first 24 h of the experiment, gluconeogenesis from alanine displays a kinetic similar to that from lactate or pyruvate. After feeding a high protein diet for 24 to 48 h, gluconeogenesis from alanine was slightly higher than that in starved rats and paralleled the increase in alanine aminotransferase activity. Glucagon stimulated gluconeogenesis from DHA up to 48 h, but with glycerol this effect occurred only during the early phase (2-11 h). Glucagon stimulated gluconeogenesis from lactate, pyruvate or alanine by 1.35-fold throughout the experimental period. These findings suggest that the development of gluconeogenesis during starvation or after feeding a high protein diet displays different kinetics, depending on the substrate used and on the level of entry in the gluconeogenic pathway: triose phosphates or pyruvate.

Diurnal variation in hepatic levels of lactate, pyruvate, phosphoenolpyruvate (PEP), alpha-ketoglutarate, malate, oxaloacetate, ketone bodies, alanine, serine, glycine, aspartate, glutamate, glutamine, valine, urea, adenine nucleotides and inorganic phosphate were studied in rats adapted to a high protein, carbohydrate-free diet for 24 days. Most circadian rhythms differed in relation to controls (10% protein diet); many merely had different amplitudes, some were inverted, and some exhibited drastically altered patterns. Cytoplasmic redox state exhibited nearly similar variations and phosphorylation state differed primarily in amplitude whereas mitochondrial redox state was highly depressed in the absorptive phase. The metabolic regulation implied by the results is discussed in relation to both circadian variations of plasma insulin and glucagon concentrations, and pyruvate kinase and phosphoenolpyruvate carboxykinase activities previously reported.

Pertinent hepatic metabolites and enzymes were examined in rats fed a high carbohydrate (HC) diet and during the first 24 h of either starvation or feeding a high protein (HP) diet. Consumption of the HC diet induced slight but definite 24-h oscillations in hepatic concentrations of cyclic AMP, glycogen, glucose 6-phosphate, fructose 2,6-bisphosphate, fructose 1,6-bisphosphate and phosphoenolpyruvate, as well as the activities of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase and phosphoenolpyruvate carboxykinase. The transition to starvation or the HP diet induced, within 12 h, concurrent increases in cyclic AMP and phosphoenolpyruvate and decreases in glycogen, glucose 6-phosphate, fructose 6-phosphate, fructose 2,6-bisphosphate and fructose 1,6-bisphosphate. These changes were associated with a decrease in the ratio of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and an increase in phosphoenolpyruvate carboxykinase. These results suggest that the activity of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle is similar during the first 24 h of starvation or HP consumption.

Plasma hormones, glucose and free fatty acids, liver glycogen and two key enzymes of glycolysis and gluconeogenesis were examined in adult rats during a 40-day period of high protein feeding. Plasma insulin fell within 1 day but returned to normal after 4 days. Glucagon changed more slowly, reaching a maximum on day 4 and declined to near the control value within 24 days. Consequently, the insulin to glucagon ratio was lower on days 1, 4 and 8 and was nearly normal on day 24. With respect to hepatic enzymes, phosphoenolpyruvate carboxykinase activity rose sharply on the 1st day and remained elevated for 40-day period; the L-isozyme of pyruvate kinase, although unchanged on the 1st day, decreased thereafter and from day 8 on represented 15--20% of control. Circadian variations in these parameters were also measured in rats adapted to the high protein diet. In such animals, the diurnal change in plasma hormones was less marked but tended to be inverted with respect to controls; the insulin/glucagon ratio was highest during daylight on high protein and in late night on the control diet. Over 24 hours, pyruvate kinase activity was related directly and phosphoenolpyruvate carboxykinase inversely to the hormone ratio. We concluded that in rats adapted to high protein, as in controls, the diurnal balance between glycolysis and gluconeogenesis is probably regulated by the same factor, namely the insulin/glucagon ratio.

Adult male rats were force-fed either a single dose of 200 mg of DL-methionine or an amino acid mixture with the pattern of casein. The animals were anesthetized with pentobarbital at 0, 1.5, 3 and 4 hours after intubation, and samples of liver were removed by a freeze-clamping technique at 0, 15 and 30 seconds of ischemia. Hepatic ATP, ADP, AMP, Pi, glucose-6-phosphate, glucose and certain nitrogenous compounds as well as plasma glucose were determined. After methionine was given by intubation, hepatic methionine and glutathione increased severalfold within 1.5 hours; cystathionine showed a transient rise at this time but returned to normal at 3 and 4 hours, when taurine was progressively increasing. Several nonessential amino acids decreased, suggesting that they may be utilized for energy. Methionine force-feeding did not modify the concentration of hepatic adenine nucleotides and probably did not change their turnover, as estimated from changes during ischemia. The level and production of Pi during ischemia was increased however. After force-feeding the amino acid mixture, hepatic methionine, cystathionine and taurine were unaffected, and glutathione increased only at hours 3 and 4; glycine and threonine were elevated by 1.5 hours. Hepatic adenine nucleotides, inorganic phosphate and glucose were not significantly affected by the feeding of amino acids by intubation.