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.

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.

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.

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.

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.

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.

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.

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.

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.

FEBS LettersVolume 143, Issue 1 p. 81-85 Full-length articleFree Access Circadian variations of A-mediated transport in rat-liver plasma membrane vesicles Genevieve Bourdel, Genevieve Bourdel Centre de Recherches sur la Nutrition du CNRS, 9, rue Jules Hetzel, 92190 Meudon-Bellevue, FranceSearch for more papers by this authorMarguerite Forestier, Marguerite Forestier Centre de Recherches sur la Nutrition du CNRS, 9, rue Jules Hetzel, 92190 Meudon-Bellevue, FranceSearch for more papers by this author Genevieve Bourdel, Genevieve Bourdel Centre de Recherches sur la Nutrition du CNRS, 9, rue Jules Hetzel, 92190 Meudon-Bellevue, FranceSearch for more papers by this authorMarguerite Forestier, Marguerite Forestier Centre de Recherches sur la Nutrition du CNRS, 9, rue Jules Hetzel, 92190 Meudon-Bellevue, FranceSearch for more papers by this author First published: June 21, 1982 https://doi.org/10.1016/0014-5793(82)80278-0Citations: 5AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References 1 J.K. Tews, N.A. Woodcock, A.E. Harper, J. Biol. Chem., 245, (1970), 3026– 3032. 2 M. Fehlmann, A. Le Cam, P. Freychet, J. Biol. Chem., 254, (1979), 10431– 10437. 3 A. Le Cam, P. Freychet, Biochem. Biophys. Res. Commun., 72, (1976), 893– 901. 4 J.W. Edmondson, L. Lumeng, Biochem. Biophys. Res. Commun., 96, (1980), 61– 68. 5 M. Fehlmann, O. Morin, P. Kitbagi, P. Freychet, Endocrinology, 109, (1981), 253– 261. 6 A. Le Cam, P. Freychet, Diabetologia, 15, (1978), 117– 123. 7 D.S. Kelley, J.D. Shull, V.R. Potter, J. Cell. Physiol., 103, (1980), 159– 168. 8 R.F. Kletzien, M.W. Pariza, J.E. Becker, V.R. Potter, J. Biol. Chem., 251, (1976), 3014– 3020. 9 A. Le Cam, P. Freychet, Mol. Cell. Endocrinol., 9, (1977), 205– 214. 10 A. Le Cam, P. Freychet, Endocrinology, 102, (1978), 379– 385. 11 D.S. Kelley, V.R. Potter, J. Biol. Chem., 253, (1978), 9009– 9017. 12 J.K. Tews, N.A. Woodcock, A.E. Harper, J. Nutr., 102, (1972), 409– 418. 13 M. Fehlmann, A. Le Cam, P. Kitbagi, J.-F. Rey, P. Freychet, J. Biol. Chem., 254, (1979), 401– 407. 14 G. Bourdel, J. Kande, D. Robin, P. Robin, J. Nutr., 111, (1981), 1528– 1535. 15 V. Ehrhardt, Chronobiologia, 4, (1977), 205– 213. 16 M.S. Kilberg, H.N. Christensen, M.E. Handlogten, Biochem. Biophys. Res. Commun., 88, (1979), 744– 751. 17 M.S. Kilberg, M.E. Handlogten, H.N. Christensen, J. Biol. Chem., 256, (1981), 3304– 3312. 18 H.J. Sips, M.M. Van Amelsvoort, K. Van Dam, Eur. J. Biochem., 105, (1980), 217– 224. 19 H.O. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem., 193, (1951), 265– 275. 20 N.N. Aronson jr., O. Touster, Methods Enzymol., 31, (1974), 92– 21 J.M. Van Amelsfoort, H.J. Sips, K. Van Dam, Biochem. J., 174, (1978), 1083– 1086. 22 J.W. Edmondson, L. Lumeng, T.-K. Li, J. Biol. Chem., 254, (1979), 1633– 1658. 23 C. Jarousse, B. Lardeux, G. Bourdel, A. Girard-Globa, G. Rosselin, J. Nutr., 110, (1980), 1764– 1773. Citing Literature Volume143, Issue1June 21, 1982Pages 81-85 ReferencesRelatedInformation

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.

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.

Influence of protein ingestion on the isopycnic distribution pattern of rat liver lysosomes.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

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.