Development of gluconeogenesis from various precursors in isolated rat hepatocytes during starvation or after feeding a high protein, carbohydrate-free diet.

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.

Comparison between starvation and consumption of a high protein diet in rats: hepatic metabolites and amino acid levels during the first 24 hours.

Changes in hepatic levels of lactate, pyruvate, phosphoenolpyruvate, alpha-ketoglutarate, malate, oxaloacetate, adenine nucleotides, inorganic phosphate, ketone bodies, alanine, serine, glycine, aspartate, glutamate, valine and urea were examined in adult rats during the first 24 h of either starvation or consumption of a high protein (HP) diet. No differences were found between these two conditions in the concentration of metabolites studied or the cytosolic redox state. Under both conditions, the cytosolic phosphorylation state decreased to a low 15 h into the experiment but the changes were more pronounced on the HP diet. Hepatic ketone bodies rose sharply after 12 h, with the increase 2.5 times greater for starved rats. In starvation, hepatic aspartate, valine, and urea were low and glycine was high, whereas the opposite was seen for the HP diet. In both groups, alanine fell within 9 h and remained low thereafter. These findings suggest that, in the first 24 h of starvation, the energy necessary for gluconeogenesis is obtained from fatty acid oxidation, while during HP feeding the energy for both gluconeogenesis and ureagenesis are derived from fatty acid oxidation and amino acid oxidation.

Studies on the early changes in rat hepatic fructose 2,6-bisphosphate and enzymes in response to a high protein diet.

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.

Comparison between starvation and consumption of a high protein diet: plasma insulin and glucagon and hepatic activities of gluconeogenic enzymes during the first 24 hours.

Plasma insulin, glucagon, glucose, free fatty acids and glycerol, hepatic cyclic AMP and glycogen, and liver phosphoenolpyruvate carboxykinase (PEPCK), fructose 1,6-bisphosphatase (FBPase), glucose 6-phosphatase (G6Pase) and alanine amino transferase (AAT) activities were examined in adult rats during the first 24 h of either starvation or consumption of a high protein, carbohydrate-free (HP) diet. Under both nutritional conditions, plasma insulin fell within 12 h and remained constant thereafter. Glucagon increased 12 h after the start of the experiment and peaked between 18-24 h. The insulin: glucagon ratio was lower during the last 12 h of the experiment. In both experimental groups, liver cyclic AMP increased progressively and peaked between 15-24 h, but it increase was higher on HP diet than on starvation. Whereas plasma glucose remained low on starvation for 24 h, it returned to normal on consumption of the HP diet. In both groups, liver glycogen fell within 12 h and remained low until the end of experiment. FBPase, G6Pase and AAT did not change on starvation, while they increased toward the end of 1 d HP consumption. During starvation or consumption of the HP diet, PEPCK increased progressively and peaked between 15-24 h, but the increase was greater with the HP diet than with starvation. These findings suggest that in the first 24 hours, the adaptative response of hepatic gluconeogenesis is higher with a HP diet than upon starvation.

Gluconeogenesis from dihydroxyacetone in rat hepatocytes during the shift from a low protein, high carbohydrate to a high protein, carbohydrate-free diet.

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.

Pyruvate kinase activity in isolated rat hepatocytes during a feeding cycle and during fasting.

The short-term regulation of pyruvate kinase in rat hepatocytes was studied during a feeding cycle or progressive fasting. In fed and fasted rats, the activity ratio of pyruvate kinase (V0.5-mMPEP/Vmax) in crude extracts was directly correlated with the concentration of glucose 1,6-bisphosphate++ in hepatocytes. Precipitation of the enzyme from homogenates with ammonium sulphate, which removes fructose 1,6-bisphosphate, induced in both groups of animals a low activity ratio of pyruvate kinase which remained unchanged during the whole experiment. These results show that in absence of added glucagon in hepatocytes, the activity of pyruvate kinase is mainly controlled by the intracellular level of fructose 1,6-bisphosphate. Addition of glucagon to hepatocytes from fed or fasted rats inactivated pyruvate kinase and decreased the concentration of fructose 1,6-bisphosphate in cells. However, in crude extracts both the activity ratio of pyruvate kinase and fructose 1,6-biphosphate levels were higher in fed rats than in starved rats. These findings suggest that glucagon-induced inactivation of pyruvate kinase also depends upon the concentration of fructose 1,6-biphosphate in hepatocytes.

Development of gluconeogenesis from dihydroxyacetone in rat hepatocytes during a feeding cycle and starvation.

Pyruvate kinase activity and the rates of gluconeogenesis and glycolysis in rat hepatocytes were evaluated by production of glucose and lactate + pyruvate from dihydroxyacetone during a feeding cycle or progressive starvation. In fed rats, during daylight (low food intake) and until darkness, gluconeogenesis progressively increased and glycolysis decreased slightly, but gluconeogenesis never exceeded glycolysis. During nocturnal feeding, gluconeogenesis and glycolysis returned to their morning rates. After 8 h starvation, an equal proportion of dihydroxyacetone was converted into glucose and into lactate + pyruvate. When glycogen was depleted (11 h of starvation), gluconeogenesis was maximal and glycolysis minimal. In fed and starved rats, the concentration of fructose 1,6-bisphosphate was the same. The activity ratio of pyruvate kinase (ratio of velocity at 0.5 mM-phosphoenolpyruvate to the maximum catalytic activity obtained with 4mM-phosphoenolpyruvate) was high in crude extracts of cells incubated with dihydroxyacetone and low in (NH4)2SO4-treated extracts, but remained unchanged during the whole experiment. There was no correlation between the rates of gluconeogenesis and glycolysis from dihydroxyacetone and the activity ratio of pyruvate kinase.