Na(+)-dependent transport of alanine and serine by liver plasma-membrane vesicles from rats fed a low-protein or a high-protein diet.

Plasma-membrane vesicles prepared from the liver of rats fed either a low-(LP) or a high-protein (HP) diet exhibited Na(+)-dependent active transport of alanine and serine. The process gave apparent kinetic parameters compatible with a single saturable component for both amino acids. Na,K-ATPase (EC 3.6.1.37), marker of the basolateral domain of the hepatocyte plasma-membrane, was chosen as reference for the expression of amino acid transport in vesicle preparations. The high-protein diet induced a significant increase in liver Na,K-ATPase activity also found in corresponding plasma-membrane preparations, in parallel with an increase in the capacity towards amino acid transport. This suggests that in rats fed the high protein diet, transcellular Na+ exchange, although increased, remains well balanced. N-Methylaminoisobutyric acid (MeAIB), due to its poor velocity, proved unsuitable to distinguish between systems A and ASC in the experimental model. Comparing Na(+)- and Li(+)-driven transport, a family of carriers with strict Na(+)-dependency (A-like) was evidenced in LP vesicles but not in HP vesicles. The sensitivity to the lowering of the pH from 7.5 to 6.5 in the external medium was similar in both type of vesicles when Na+ was the driving ion. In the HP vesicles the Li(+)-tolerant, pH-insensitive component (ASC-like) was increased in parallel with overall Na(+)-dependent transport. These functional properties suggest that the carriers involved in the stimulation of transport in HP vesicles are composite in nature. Increasing concentrations of an amino acid mixture mimicking the changes of portal aminoacidemia inhibited the transport of alanine and of serine. The degree of inhibition was correlated with the relative concentration of substrate and was independent of the nutritional treatment.

Regulation of hepatic synthesis of proteins by the chronology of protein ingestion.

Circadian variations in liver protein synthesis were were assessed in control rats fed a mixed 10% protein diet and in rats fed proteins as a separate meal either at 09:00 (SF 09) or at 21:00 (SF 21) and provided with a protein-free diet ad libitum. Protein synthesis was measured by incorporation of labelled leucine over a short period of time (15 min) at time-points regularly spaced over 24 h. In controls, the circadian variations observed were of moderate amplitude (from 2.75 mg/h per g at 09:00 to 5.77 mg/h per g at 06:00) correlated with increased protein and RNA contents of the liver. In separately fed animals ingestion of the protein meal triggered a 300% increase in protein synthesis within 1 h while the feeding pattern was unaltered. In the SF 09 group, high synthetic activity was not followed by an increase of hepatic protein content while hepatic urea concentrations were sharply increased and glucogenic amino acid pools were greatly depleted. It is suggested that the high influx of amino acids consecutive to the absorption of the dietary proteins is the key factor stimulating protein synthesis, while synchronisation with the energetic metabolism controls the degree of degradation. The possible involvement of variations in the insulin to glucagon ratio is discussed.

Induction of multiple forms of tyrosine aminotransferase by amino acid mixtures of different compositions.

Hepatic tyrosine aminotransferase (EC 2.6.1.5) was induced in rats by intubation of amino acid mixtures (complete or tryptophan-free). Enzyme activity was increased 4-fold by the complete mixture and 8-fold by the tryptophan-free mixture. The enzyme was analyzed by chromatography on CM-Sephadex. Chromatographic patterns were characteristic of the type of inducer rather than of the chronology of the induction cycle: after induction by the complete amino acid mixture the three forms of the enzyme were equally increased whereas after induction by the tryptophan-free mixture Form I was preferentially increased.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 June 2010 - 31 July 2010.

This article documents the addition of 205 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Bagassa guianensis, Bulweria bulwerii, Camelus bactrianus, Chaenogobius annularis, Creontiades dilutus, Diachasmimorpha tryoni, Dioscorea alata, Euhrychiopsis lecontei, Gmelina arborea, Haliotis discus hannai, Hirtella physophora, Melanaphis sacchari, Munida isos, Thaumastocoris peregrinus and Tuberolachnus salignus. These loci were cross-tested on the following species: Halobaena caerulea, Procellaria aequinoctialis, Oceanodroma monteiroi, Camelus ferus, Creontiades pacificus, Dioscorea rotundata, Dioscorea praehensilis, Dioscorea abyssinica, Dioscorea nummularia, Dioscorea transversa, Dioscorea esculenta, Dioscorea pentaphylla, Dioscorea trifida, Hirtella bicornis, Hirtella glandulosa, Licania alba, Licania canescens, Licania membranaceae, Couepia guianensis and 7 undescribed Thaumastocoris species.

Effect of separate feeding of proteins and lipids on pancreatic adaptation in the rat.

In agreement with what is known of pancreatic adaptation to dietary composition, a progressive lowering of amylase-to-chymotrypsinogen ratio was observed in the pancreases of rats fed mixed diets of increasing casein and decreasing carbohydrate contents. The same changes in ratio were found when casein alone was increased in otherwise isonitrogenous meals fed to rats provided ad libitum with a protein-free diet. Pancreatic accumulation of chymotrypsinogen was increased while that of amylase was decreased in spite of low-protein and high-carbohydrate intakes. Lowering the lipid content of the protein-free diet reduced the effectiveness of casein meals in lowering the amylase-to-chymotrypsinogen ratio. It is concluded that 1) the capacity of protein-rich diets to induce adaptative changes in pancreatic secretion does not depend on the absolute amount of proteins passing through the duodenum but rather on the increased protein concentration in the alimentary bolus, 2) protein concentration affects both amylase and chymotrypsinogen, and 3) lipids have a cooperative effect.

Urea concentration and ornithine decarboxylase in liver of female rats.

In virgin female rats thioacetamide administration (1 mg/100 g body wt) induced a 16-fold increase in liver ornithine decarboxylase (ODC) activity and a significant decrease (19%) in hepatic urea concentration. The ornithine-metabolizing enzymes, ornithine-oxo-acid aminotransferase and ornithine carbamoyltransferase, were not modified by the treatment; only carbamoyltransferase, were not modified by the treatment; only carbamoyl-phosphate synthetase I activity was significantly reduced. In 19-day pregnant rats DL-alpha-difluoromethylornithine treatment inhibited the expression of enhanced ODC activity occurring normally at this stage of pregnancy. Concomitantly an inhibition of the usual decrease in hepatic urea was observed. This increase of ureagenesis occurred without any increase in liver N-acetylglutamate or ornithine concentrations, which remained as low as in normal pregnant rats.

[Rapid modifications of the rat hepatic lysosomal system as a function of the nutritional state]. / Modifications rapides du système lysosomal hépatique du rat en fonction de l'état nutritionnel.

Adult rats fed proteins as a meal given during the daytime exhibit alterations of liver protein metabolism characterized by simultaneous stimulations of protein synthesis and degradation, particularly during the hours following protein ingestion. The purpose of the present work was to determine if the stimulation of liver protein breakdown could be related to biophysical alterations of the lysosomal system. There is a growing amount of evidence to suggest that the lysosomal vacuolar system is involved in the physiological regulation of overall proteolytic rate. Rats, trained to eat a protein meal 2 hrs after the onset of light, were killed 6, 9, 18, 21 and 24 hrs after protein intake. Three fractions were isolated from 0.25 M sucrose liver homogenates after differential centrifugation. The mitochondrial-lysosomal fraction was further analyzed by isopycnic centrifugation in sucrose gradients. Three specific lysosomal enzyme activities were assessed: N-acetyl-beta-D glucosaminidase (marker), cathepsin D and cathepsin C (proteolytic enzymes). Total activities remained unchanged at all time-points, but the distributions between the different fractions recovered after differential centrifugation were altered 6 and 9 hrs after protein intake. A significantly higher percentage of N-acetyl-beta-D-glucosaminidase, cathepsin D and cathepsin C activities were recovered in the M + L fraction, suggesting a shift towards lysosomal forms of lighter density. This was confirmed by density gradient analysis. Thus, even in adapted rats, acute administration of protein during the daytime quickly induced biophysical alterations in the lysosomal system. The lysosomal distribution pattern observed at 6 and 9 hrs after protein intake might be due to lysosome enlargement by active autophagy and/or by the sequestration of lighter cellular material.

Liver ornithine decarboxylase in pregnant rats fed two levels of casein.

Liver ornithine decarboxylase (ODC) and tyrosine aminotransferase (TAT) activities were assessed at 2200 h (prandial phase) and at 1000 h (postprandial phase) in virgin and in pregnant (day 13-20) rats fed on different levels of casein and carbohydrate. In virgin rats, ODC levels were higher at 2200 h after resumption of eating than at 1000 hours, the inductive effect being greater with the high-casein than with the low-casein diet. Rapid deinduction followed termination of eating, resulting in equally low enzyme levels at 1000 h with both diets. On the contrary, prandial and postprandial levels of TAT were always greater with the high-protein diet. In pregnant rats, there was a progressive stimulation of ODC that reached a maximum on day 19. However, the inductive capacity of the high-protein diet was lower than that of the low-casein diet. Prandial rest was not followed by enzyme deinduction at 1000 h. In contrast, TAT stimulation remained dependent on overall casein ingestion. At constant casein but restricted carbohydrate intake, pregnant females exhibited a reduction in ODC stimulation. Thus, whereas in virgin females proteins are determinant in the regulation of ODC, during pregnancy there determinant in the regulation of ODC, during pregnancy there is a shift toward modulation by carbohydrates. Levels of liver urea and ornithine were found to vary in inverse proportion with the magnitude of ODC stimulation.

Pancreatic hydrolases in cold-induced hyperphagia of rats fed a low or high-fat diet.

Rats fed either a low (2p. 100) or high (40 p. 100)-fat diet were exposed to 22 or 5 degrees C. The resulting hyperphagia adequately compensated energy losses as judged from body weight. The cold-induced hyperphagia was accompanied by a non-parallel increase in pancreatic hydrolases. Amylase and lipase were not increased above the adaptive levels they had respectively reached in the heat with a high-starch or high-lipid diet. Chymotrypsinogen, on the contrary, responded to increased intake of both diets. It also responded to the higher protein concentration in the high-fat diet caused by isocaloric replacement of starch by fat. Colipase varied independently of lipase and was increased additively by fat and protein intakes. Consequently, although limiting for lipase in the warm, colipase rose to a 1:1 ratio in the cold. Increased intake had a consistent pleiotropic effect evidenced by an increase of amylase with the high-fat diet and of lipase with the low-fat diet. The net effect was a significant increase in the lipid-digesting potential of the organism of lipid-fed animals upon exposure to cold, while the starch-digesting potential remained unaffected in starch-fed animals.

Possible metabolic implications of pyruvate and lactate accumulation in the liver of pregnant rats.

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.

Alanine uptake by liver at midpregnancy in rats.

The participation of the liver to the increase in alanine utilization seen at midpregnancy was studied in 9- and 12-day pregnant rats. Liver fractional extraction of alanine was assessed in vivo from the changes in concentration in afferent and efferent vessels. Hepatic active transport of alanine was determined in vitro using isolated plasma-membrane vesicles. Compared with nonpregnant controls, alanine fractional extraction was significantly increased on day 12 but not on day 9 of pregnancy. Vesicles isolated from 9- and 12-day pregnant animals had a greater capacity for Na+-dependent transport than those from controls. Eadie-Hofstee plotting showed that this increase was due to an increase in Vmax with no change in Km. Both A and ASC systems contributed to the Vmax increase. These results indicate that, although by day 9 the liver has developed an increased capacity for alanine uptake, the actual extraction is seen only by day 12 of pregnancy. At this stage the liver participates actively in the turnover of alanine and the development of hypoalaninemia.

Quantitative and qualitative circadian variations of amino acid intestinal efflux in mixed-fed and in protein-meal-fed rats.

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.

Circadian feeding pattern in pregnant rats fed three levels of protein.

Food intake was measured at regular intervals over 24 h in pregnant and non-pregnant female rats fed diets of different protein content: 10, 16 and 32%. During the course of pregnancy, a first period of hyperphagia was observed (days 2-12) irrespective of the composition of the diet. A second phase of hyperphagia occurred later (days 16-19) which was more marked with the better balanced diet (16% protein). During the first half of pregnancy, the increase in intake occurred principally at the beginning of the night (compensatory reaction). Later on, the stimulation extended to the last part of the night (anticipatory reaction). The nocturnal predominance of feeding activity was maintained in pregnant females in spite of their increased metabolic requirements.

Differential regulation of lipase and colipase in the rat pancreas by dietary fat and proteins.

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.

Regulation of protein synthesis and enzyme accumulation in the rat pancreas by amount and timing of dietary protein.

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.

Activity of several enzymes of amino acid catabolism in the liver of rats fed protein as a meal.

Rats having a protein-free diet available ad libitum were fed a daily casein meal at the beginning of either the light- or the dark-phase of the day. A control group received a mixed-diet ad libitum. In all three groups, daily food ingestion was the same and casein corresponded to 12% of total intake. Liver activities of alanine, aspartate, ornithine and tyrosine aminotransferase, ornithine decarboxylase and serine dehydratase were assessed. In mixed-fed controls, all activities were low. Tyrosine aminotransferase and ornithine decarboxylase exhibited clear circadian rhythms of low amplitude. Feeding casein as a concentrated meal had no effect on aspartate aminotransferase. It depressed alanine aminotransferase and serine dehydratase activities. Tyrosine aminotransferase and ornithine decarboxylase exhibited rapid and strong stimulatory responses but, within 12 hours, returned to levels similar to those observed in mixed-fed controls. Ornithine aminotransferase was increased in the group receiving the casein meal during the light phase. It is concluded that the capacity for amino acid catabolism remains low in separately-fed animals, and that only tyrosine and especially ornithine, which may become limiting for urea synthesis, are actively metabolized. Thus, when high fluxes of amino acids reach the liver following the absorption of the casein meal, more amino acids are available for incorporation into newly synthesized proteins.

Circadian variations of liver free amino acid content in mixed-fed and protein-meal-fed rats.

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.

Methionine synthesis, aminoimidazole carboxamide excretion and folate levels in pregnant rats.

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.

Portal insulin and glucagon in rats fed proteins as a meal: immediate variations and circadian modulations.

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.