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The influence of protein ingestion on liver albumin synthesis and albumin content was investigated in rats fed protein as a meal (90% casein) given apart from the other dietary components provided ad libitum. In this condition, protein ingestion rapidly stimulates liver total protein synthesis. Separately fed rats were studied 6 and 20 h after the protein meal. Control rats fed mixed diets containing 13 or 80% casein were killed either during the absorptive (night) or postabsorptive (light) periods. The ratio of hepatic albumin synthesis to total protein synthesis remained fairly constant (12-15%) in all groups, indicating that albumin synthesis paralleled total protein synthesis. Liver albumin content measured in microsomes by immunonephelometry was significantly higher in separately fed rats killed 6 h postmeal than in those killed after 20 h. In rats fed 13% casein, the liver albumin content remained high regardless of the time of killing. In rats fed 80% casein, the albumin content was higher during the absorptive period than during the postabsorptive period. Immunoperoxidase staining of the hepatocyte organelles involved in albumin synthesis, especially the Golgi apparatus, was more intense for separately fed rats killed 6 h postmeal than for those killed after 20 h. Livers of rats fed 13% casein also exhibited a pattern indicative of high hepatocyte albumin content, whereas livers of rats fed 80% casein contained less. These results show that, in separate feeding, wide circadian variations of albumin synthesis run parallel to changes in liver albumin content.

Experiments were designed to investigate whether the metabolic responses of pregnant females are in keeping with the known state of gestational hyperinsulinemia. Groups of female rats fed a 32% protein diet were killed on days 13, 15, 17, 19 and 21 of pregnancy, during either daytime or during night-time. Liver pyruvate kinase and glucose-6-phosphate dehydrogenase activities were increased over nonpregnant values from day 13 onward in agreement with what can be expected as a result of the gestational hyperinsulinemia. Liver malate dehydrogenase (NADP) activity was increased to lesser extent and later. Pyruvate and lactate accumulated in maternal liver from day 13 onward. The fact that this accumulation could not be related to any further increase of food intake during this time and that it correlated at day 21 with litter size was taken as indication of a probable contribution of the conceptus to maternal pyruvate and lactate accumulation in late pregnancy. Liver alanine amino-transferase activity decreased as pregnancy progressed. No change in serine dehydratase activity was found. Cytosolic aspartate aminotransferase activity remained unchanged. Mitochondrial activity increased as pregnancy progressed.

The magnitude and composition of amino acid intestinal efflux was followed over a light-dark cycle in rats ingesting the same daily amount of protein administered either in a mixed diet (12% casein) or as a separate meal (70% casein concentrate) fed 2 hours after the onset of the light period with a protein-free diet available at all times. Intestinal efflux was determined by an instant porto-aortic difference measured on pooled samples from six rats at time-points spaced every 3 hours over a light-dark cycle. During protein digestion (dark for the mixed-fed rats) and (light for the separately-fed ones), essential amino acid composition of intestinal output fell into line with that of the protein ingested (casein) while non-essential amino acid composition did not. The discrepancy bore on alanine and glycine which were released in excess and on aspartic and glutamic acids, glutamine and serine which were released in deficit from their content in casein. From the follow-up of individual amino acid release and uptake, we concluded that intestinal efflux reflects the composition of the dietary protein only with respect to the amino acids, mostly essential, that are not metabolized by the intestinal wall.

The capacity for tetrahydrofolate regeneration through folate-linked methionine synthesis and for purine-ring closure through formylation of aminoimidazole carboxamide ribotide was studied in pregnant female rats fed diets containing either methionine or homocystine with or without folic acid. Plasma and liver folates, serine transhydroxymethylase, 5,10-methylene tetrahydrofolate dehydrogenase and glutamate formiminotransferase activities were also assayed. Pregnancy proceeded normally in all groups. Hypotrophic fetuses were observed only with the diet containing homocystine and no folic acid. Plasma folates were severely depleted at the end of pregnancy even when folic acid was present in the diet. Hepatic stores of folate were twice as high in the methionine as in the homocystine-fed pregnant females supplemented with folic acid. This favorable effect of methionine was not observed in folic acid-deficient females. No change in levels of serine transhydroxymethylase, 5,10-methylenetetrahydrofolate dehydrogenase, glutamate formimino-transferase activities was observed. Pregnancy did not stimulate methionine synthetase activity, the level of which was primarily affected by the nutritional conditions. Because of its low output and narrow range of adaptativity, methionine synthetase cannot be the sole regulatory factor of THF regeneration. Urinary excretion of aminoimidazole carboxamide was enhanced in folic acid-deficient pregnant females and was not prevented by supplying methionine.

The adaptative response of the exocrine pancreas to diets rich in lipids and the influence of dietary proteins are reconsidered taking into account colipase. This was particularly necessary in the rat since colipase is often limiting with respect to lipase in this species. In a first experiment, adult male rats received diets containing 10, 15, 18, 40 or 86% casein together with 2, 8 or 40% lard. Under these conditions, lipase was found to be sensitive to variations in the lipid contents of the diet and was two-fold higher with 40% than with 8% lipid, in diets containing 40% casein. Proteins exerted a permissive effect since no response was recorded with diets containing less than 18% casein. Colipase responded to protein intake, even in lipid-poor diets (2% lard), and was increased by a factor 3 when casein was raised from 18 to 40%. In a second experiment, proteins were fed as a separate meal, and the remainder of the diet (provided ad libitum) contained either 8 or 40% lard. In a diurnal study, lipase and colipase were followed every 3 hours over 24 hours. Both lipase and colipase were found to accumulate after the protein meal. Colipase was found to accumulate much faster than lipase in all cases and reach 3 times basal levels 9 hours after the protein meal. This resulted in important diurnal variations in the ratio of colipase to lipase which modulates lipolytic activity. It is concluded that colipase is particularly sensitive to protein intake, perhaps more than to lipid intake and may become and limiting factor of lipid digestion.

In adult rats, proteins fed as a meal apart from the remainder of the diet induce alterations of protein metabolism characterized by the simultaneous stimulation of protein synthesis and breakdown. These alterations occur in parallel with an acceleration of glycogenolysis. The purpose of this work was to investigate whether these metabolic changes are related to variations in portal insulin and glucagon levels or to insulin-glucagon balance. Portal hormone concentrations, aortic glycemia and aminoacidemia, liver glycogen contents were followed over a day-night cycle in rats adapted either to mixed feeding (10% protein) or to separate feeding (protein meal given 2 hours after the onset of the light phase). Insulin and glucagon were assayed by radioimmunoassay, glucagon with antibody K 964 specific for 3500 MW glucagon. During the 3 hours following the protein meal, the portal ratio of insulin to glucagon decreased; liver glycogenolysis and glucogenic amino acid catabolism were enhanced. This glucagonotropic and glucogenic response to a protein meal administered during daytime is consistent with the increase in protein turnover previously observed. Separate feeding did not alter the overall circadian pattern of portal insulinemia which rose at night but it did alter the overall circadian pattern of portal insulinemia which rose at night but it did alter that of portal glucagonemia by maintaining it at a low level during the nightly prandial period. No correlation could be evidenced between portal insulin concentrations and the aortic levels of any amino acid in either mixed-fed or separately-fed animals. Portal glucagonemia appeared to be weakly correlated with the aortic level of arginine in both experimental groups. In the separately fed group, highly significant correlation could be evidenced between portal insulin concentrations and the aortic levels of any amino acid in either mixed-fed or separately-fed animals. Portal glucagonemia appeared to be weakly correlated with the aortic level of arginine in both experimental groups. In the separately fed group, highly significant correlations were found between portal glucagonemia and aortic concentrations of the three branched and the two aromatic amino acids.

A 24-hour study in rats evidenced a clear rhythmicity of both synthesis and storage of pancreatic hydrolases. Synthesis measured by incorporation of 3H leucine into proteins was maximal during the night, reaching 21.3 mg/g tissue at 2400 hours against 4.1 mg/g at 0900 hours. Amylase and chymotrypsinogen contents, on the contrary, were 2-fold higher during the day (resting period) than at night (feeding period), while trypsinogen did not vary significantly. The diametrical opposition between the variations in synthesis and enzyme contents shows that, during periods of active feeding, stimulated synthesis merely balances excretion, while during periods of spontaneous fasting, basal synthesis is greater than basal secretion resulting in a preprandial accumulation of hydrolases. The effect of dietary proteins was investigated by feeding them as a separate meal at different times of the day while providing a protein-free diet ad libitum. In this case the general pattern of synthesis was biphasic. Rates of protein synthesis increased rapidly 2- to 3-fold after the protein meal, while tissue amino acids concentrations dropped. This first peak was tentatively attributed to the action of digestive hormones released after protein ingestion. The second peak occurred 15-18 hours, later together with a rise in tissue amino-acids due to limited endogenous proteolysis. This suggests that digestive hormones and amino-acid supply act independently to stimulate the synthesis of hydrolases in the pancreas. The amount of enzyme stored depends on the timing of the protein meal with respect to the period of most intense feeding, i.e. on the timing of maximal synthesis with respect to maximal secretion.

Hepatic amino acid contents were determined at time-points regularly spaced over a light-dark cycle in rats fed either a 12% casein diet or a single daily meal given 2 hours after the onset of the light phase with a protein-free diet and libitum. In mixed-fed rats, non-essential amino acid hepatic content remained stable over 24 hours while that of essential amino acids rose during the early part of the night in connection with the onset of prandial activity, but long before portal levels increased. The possibility of factors related to food intake (insulin, glucagon, gastrointestinal hormones) or to its chronology (corticoids) stimulating active transport is discussed. In separately-fed rats, amino acid pools increased in the 30 minutes following protein administration in connection with rising portal levels. During the rest of the light phase, a general depletion of non-essential amino acids occurred. It was most rapid for glutamine, alanine and aspartic acid and was followed by accumulation during the night phase, a pattern fitting well with gluconeogenesis and ureogenesis stimulation following protein ingestion. Essential amino acid decrease was linear and spanned over both the light and dark phases in correlation with decreasing portal levels.