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Background: The phosphorylation of eukaryotic initiation factor 2 (p-eIF2) during dietary amino acid insufficiency reduces protein synthesis and alters gene expression via the integrated stress response (ISR).Objective: We explored whether a Met-restricted (MR) diet activates the ISR to reduce body fat and regulate protein balance.Methods: Male and female mice aged 3-6 mo with either whole-body deletion of general control nonderepressible 2 (Gcn2) or liver-specific deletion of protein kinase R-like endoplasmic reticulum kinase (Perk) alongside wild-type or floxed control mice were fed an obesogenic diet sufficient in Met (0.86%) or an MR (0.12% Met) diet for ≤5 wk. Ala enrichment with deuterium was measured to calculate protein synthesis rates. The guanine nucleotide exchange factor activity of eIF2B was measured alongside p-eIF2 and hepatic mRNA expression levels at 2 d and 5 wk. Metabolic phenotyping was conducted at 4 wk, and body composition was measured throughout. Results were evaluated with the use of ANOVA (P < 0.05).Results: Feeding an MR diet for 2 d did not increase hepatic p-eIF2 or reduce eIF2B activity in wild-type or Gcn2-/- mice, yet many genes transcriptionally regulated by the ISR were altered in both strains in the same direction and amplitude. Feeding an MR diet for 5 wk increased p-eIF2 and reduced eIF2B activity in wild-type but not Gcn2-/- mice, yet ISR-regulated genes altered in both strains similarly. Furthermore, the MR diet reduced mixed and cytosolic but not mitochondrial protein synthesis in both the liver and skeletal muscle regardless of Gcn2 status. Despite the similarities between strains, the MR diet did not increase energy expenditure or reduce body fat in Gcn2-/- mice. Finally, feeding the MR diet to mice with Perk deleted in the liver increased hepatic p-eIF2 and altered body composition similar to floxed controls.Conclusions: Hepatic activation of the ISR resulting from an MR diet does not require p-eIF2. Gcn2 status influences body fat loss but not protein balance when Met is restricted.

We conducted two experiments to evaluate the flooding dose method for measuring intestinal and liver protein synthesis in sheep. Experiment 1 showed that large doses of phenylalanine did not cause marked metabolic disturbances. Experiment 2 examined the effectiveness of flooding with phenylalanine and the time dependency of the protein synthesis calculation. Rams were injected with 1.2 MBq L-[ring 2,6-3H]phenylalanine/kg body wt and slaughtered 20, 40 or 60 min later. Plasma specific radioactivity reached a plateau within 2.5 min and did not change significantly (P0.05) throughout the experiment. Tissue intracellular free pool specific radioactivity also remained constant from 20 to 60 min postinfusion. Flooding conditions were achieved in the intracellular free pool of intestinal tissues (specific radioactivity 70-96% of plasma specific radioactivity), although liver flooding was less successful (57-67%). Protein synthesis rates measured after 20 min were significantly (P0.05) higher in the liver, jejunum and ileum than those measured at 60 min. Protein synthesis rates also tended to decline with time in the duodenum and colon (P0.05). There were no significant differences (P0.05) between protein synthesis rates calculated using the intracellular specific radioactivity vs. plasma specific radioactivity in the duodenum, ileum or colon. Therefore, this method represents an improvement over continuous infusion methods for measurements of protein synthesis in visceral tissues.

International audience; Epidemiologic and experimental studies showed that folate deficiency is associated with increased risk of degenerative diseases by enhancing abnormal one-carbon metabolism. We studied the changes in the proteome of liver, the main tissue of folate storage and metabolism, in a rat model of dietary folate depletion. Four-month-old rats were fed for 4 wk an amino acid-defined diet without folate and compared with pair-fed rats given the same diet adequately supplemented with folic acid. Folate deprivation decreased plasma and hepatic folate concentrations dramatically, while increasing homocysteinemia significantly. Using 2-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight MS, we identified 9 spots corresponding to differentially expressed proteins in the liver of folate-deficient rats compared with controls. Among those spots, 4 had a significantly increased volume, whereas the volume of the 5 other spots was decreased. Upregulated proteins included glutathione peroxidase (GPx) 1 and peroxiredoxin 6, 2 enzymes involved in the response to oxidative stress, and MAWD binding protein (MAWDBP), which has been associated with cancer. MAWDBP was simultaneously identified as a second spot with a lower isoelectric point (pI) that vanished almost completely after folate deficiency. Decreased abundance was also observed for cofilin 1, a protein linked to tumorigenesis, and for the GRP 75 precursor and preproalbumin, both of which are responsive to oxidative stress and/or inflammation. Moreover, an enzyme activity assay and/or Western blot analysis of GPx-1 and MAWDBP confirmed the proteomic findings. Our results show that folate deficiency modifies the abundance of several liver proteins consistently with adaptive tissue responses to oxidative and degenerative processes.

At 6 wk of age, male Fischer F344 rats were fed a purified, casein-based diet either ad libitum or in the amount of 60% of the diet consumed by the rats fed ad libitum (restricted diet). Hepatocytes were isolated from the rats between 2.5 and 19 mo of age. The protein content of the hepatocytes isolated from the rats fed the restricted amount of diet was significantly lower than that of hepatocytes isolated from rats fed ad libitum. The DNA and RNA content of the hepatocytes were similar for the rats fed the two dietary regimens. The absolute rate of protein synthesis for hepatocytes isolated from rats fed ad libitum decreased 55 % between 2.5 and 19 mo of age. However, the rate of protein synthesis by hepatocytes from rats fed the restricted amount of diet decreased only slightly with increasing age. At 19 mo of age, the rate of protein synthesis by hepatocytes from the rats fed the restricted amount of diet was significantly higher than the rate of protein synthesis for hepatocytes from rats fed ad libitum. Therefore, dietary restriction retards the age-related decline in liver protein synthesis. Reproduced by permission. Maria C. Birchenall-Sparks, Michael S. Roberts, Jeffrey Staeker, James E. Hardwick, Arlan Richardson, Effect of Dietary Restriction on Liver Protein Synthesis in Rats. J. Nutr. 115 , 944-950 (1985).

Effects of protein and energy intake on tissue protein synthesis and degradation were studied in rats fed 12, 24 and 36% protein diets at 100, 70, 50 and 30% of ad libitum intake begining on d 7 of lactation. Rates of protein synthesis were measured in liver, mammary, abdominal viscera and remaining carcass of rats on d 10 of lactation. Tissue protein loss was estimated as the difference in tissue protein from d 7 to 14. Data were analyzed using stepwise multiple quadratic regression to define response surfaces of protein loss, synthesis and degradation to intake of protein and energy. Protein loss increased with restriction of energy and protein intake in all tissues. However, carcass protein synthesis and degradation did not vary systematically with either energy or protein intake. Energy intake restriction increased visceral protein turnover while protein intake restriction decreased rate of protein turnover. Liver protein loss increased with decreased dietary protein due to increased liver protein degradation. Decreased energy intake also increased liver protein loss without directly affecting rate of synthesis or degradation. Mammary protein synthesis decreased very slightly with energy restriction while decreased protein intake sharply inhibited protein synthesis.

We investigated the effect of non-protein calorie source on the repletion by total parenteral nutrition (TPN) of protein-depleted rats. Rats were depleted by feeding them a protein free diet for 4 weeks. Control (non-depleted) and depleted rats were given one of three isocaloric, isonitrogenous TPN regi mens for 6 days. They differed in the proportion of non-protein calories given as glucose and fat: diet 1, 100% glucose; diet 2, 75% glucose, 25% fat; and diet 3, 100% fat. The liver, skeletal muscle and whole body protein synthesis and break down rates were measured by using (15N)glycineas a tracer. The fatty acid distri bution in the liver was measured by gas chromatography-mass spectrometry. Results: (a) Omission of fat leads to a decrease in the fraction of linoleate in the liver, (b) Giving all the non-protein calories as a fat emulsion causes fatty acid accumulation in the liver and a significant increase in the liver protein fractional synthesis rate in the control rats and killed about half the depleted rats, (c) For TPN regimens 1 and 2 both the muscle and whole body synthesis rates were in creased during repletion, (d) Of the three TPN regimens, the second one which contained both fat and glucose was superior to the other two. J. Nutr. Ill: 154-165, 1981.

Two groups of rat pups were gastrostomized at 24 hours of age and formula-fed each hour or once every 6 hours a daily intake equal to 33% of their body weight. They were killed at age 5 to 7 days when body weight had reached twice that at time of gastrostomy; organ weights, body composition and hepatic activity of glucose-6-phosphate-dehydrogenase (G-6-PD) and alpha-glycerophosphate dehydrogenase (alpha-GPD) were determined. The stomach, small intestine, liver and spleen of pups fed 4 times each day were significantly larger than those of pups fed hourly. Food efficiency (gain/kcal) of pups fed each hour was significantly greater than that of pups fed every 6 hours; however, because pups fed each hour gained more water and less fat, an estimate of the percentage of energy intake utilized for growth was significantly less for these animals than for those fed every 6 hours. Concentration of calcium was greater and concentration of sodium was less in the carcasses of pups fed each hour than in the carcasses of pups fed 4 times daily. Total liver activities of G-6-PD and alpha-GPD were significantly less in pups fed each hour but the activity of these enzymes expressed per milligram of liver protein did not differ significantly between the two feeding groups.

We examined the role of the mammalian target of rapamycin (mTOR) in hepatic cell growth. To dissociate cell growth from cell proliferation, we employed an in vivo model of nonproliferative liver growth in rats, refeeding after 48 h of food deprivation. Starvation resulted in a decrease in liver mass, liver protein, and cell size, all of which were largely restored after 24 h of refeeding. Administration of the mTOR inhibitor, rapamycin, before the refeeding period partially inhibited the restoration of liver protein content. Refeeding was also associated with an increase in ribosomal protein S6 phosphorylation and phosphorylation of the eukaryotic initiation factor (eIF) 4E binding protein 1 (4E-BP1). 4E-BP1 phosphorylation was accompanied by a decrease in the abundance of the complex containing 4E-BP1 with eIF4E. These changes were prevented by rapamycin administration. However, association of eIF4E and eIF4G and eIF2alpha phosphorylation, both of which are stimulated by refeeding, were insensitive to rapamycin. The functional importance of these observations was confirmed by polysome fractionation, which showed that translation initiation of 5' oligopyrimidine tract-containing mRNAs, which encode ribosomal proteins, was inhibited by rapamycin, whereas translation of signal transducer and activator of transcription 1 (STAT1), a cap-dependent mRNA, was unaffected. The abundance of ribosomal proteins paralleled total protein content during refeeding in both control and rapamycin-injected rats. We conclude that accretion of liver protein during refeeding is dependent on mTOR-mediated activation of the translation of ribosomal proteins but not dependent on mTOR-mediated activation of cap-dependent translation initiation.

Whole-body and splanchnic metabolism of dietary amino acids derived from casein (CAS) or the corresponding crystalline L-amino acid mixture (AA) were compared. Male adult rats were adapted for 9 d to two isoenergetic, isonitrogenous diets (15 g/100 g protein, 5 g/100 g fat) containing either CAS or AA. On d 10, the rats were fed a single mixed meal (3 g dry mass) containing either intrinsically (13)C-labeled goat casein or the amino acid mixture containing [U-(13)C(6)] leucine and [alpha-(15)N] lysine. Rats were killed before and 1, 3, 5 and 7 h after meal ingestion and samples of plasma, stomach wall and contents, small intestine and liver were collected. (13)C and (15)N enrichments of free and protein-bound amino acids in plasma and tissues were analyzed by gas chromatography-combustion isotope ratio mass spectrometry. Urinary nitrogen excretion was higher (P < 0.05) and weight gain lower (P < 0.05) in rats given the AA diet, indicating a lower whole-body net protein synthesis. Free (13)C-leucine from the AA diet appeared in the intestinal mucosa free pool more rapidly (P < 0.05) than the CAS-(13)C-leucine, probably due to the faster transit through the stomach of the AA group. However, the incorporation of dietary leucine into plasma and liver proteins was higher in the CAS group 7 h after the meal (P < 0.05), whereas lysine incorporation into liver protein was higher in the AA group (P < 0.05). We conclude that whole-body protein homeostasis is better supported by dietary casein-bound than crystalline free amino acids, and that protein-bound leucine, but not lysine, is used more efficiently for liver protein synthesis than dietary free leucine.

High rumen-degradable starch (RDS) diets decrease milk fat. The increase of LPS in plasma associated with increased RDS impairs liver function, immune response and lipid metabolism, which depress the precursors for milk fat.This study investigated the mechanism of depression of milk fat precursors in the liver and small intestine of dairy goats fed different RDS diets.Eighteen Guanzhong lactating goats (second lactation, 45.8 ± 1.54 kg) and 6 ruminally cannulated dairy goats (aged 2-3 y, 54.0 ± 2.40 kg) were fed 3 different diets with low dietary RDS concentrations of 20.52% (LRDS), medium RDS of 22.15% (MRDS), and high RDS of 24.88% (HRDS) for 36 and 21 d, respectively, in experiments 1 and 2. The liver metabolites and jejunal microbiota in experiment 1 and LPS concentrations in rumen fluid and plasma in experiment 2 were measured. One-way ANOVA was used to analyze the biochemical parameters and mRNA or protein expression. The MIXED procedure was used to analyze LPS concentrations.In experiment 1, the HRDS diet showed increased activity of alkaline phosphatase (27.4 to 41.4 U/L) in plasma (P 0.05) compared with LRDS treatment. The HRDS diet significantly increased the hepatic concentrations of l-carnitine (129%), l-palmitoylcarnitine (306%), taurochenodeoxycholate (856%), and taurodeoxycholic acid (588%) in liver (variable importance in the projection 1, P 0.10) compared with the LRDS treatment. Goats fed the HRDS diet had 33.6% greater liver protein expression of carnitine palmitoyltransferase-1 (P 0.05), and greater relative abundance of Firmicutes and Ruminococcus 2 in the jejunal content (linear discriminant analysis 2.0, P 0.05) than did goats fed LRDS diet. In experiment 2, goats fed the HRDS diet had greater LPS concentrations in rumen fluid (7.57 to 13.6 kEU/mL) and plasma (0.037 to 0.179 EU/mL) (P 0.05) than did goats fed LRDS diet.Feeding the HRDS diet promoted hepatic lipid β-oxidation and disrupted phospholipid and bile acids metabolisms in liver, thereby reducing the supply of lipogenic precursors to the mammary gland in dairy goats.

BACKGROUND The neonatal methionine requirement must consider not only the high demand for rapid tissue protein expansion but also the demands as the precursor for a suite of critical transmethylation reactions. However, methionine metabolism is inherently complex because upon transferring its methyl group during transmethylation, methionine can be reformed by the dietary methyl donors choline (via betaine) and folate. OBJECTIVE We sought to determine whether dietary methyl donors contribute to methionine availability for protein synthesis in neonatal piglets. METHODS Yucatan miniature piglets aged 4-8 d were fed a diet that provided 38 μg folate/(kg·d), 60 mg choline/(kg·d), and 238 mg betaine/(kg·d) [methyl-sufficient (MS); n = 8] or a diet devoid of these methyl precursors [methyl-deficient (MD); n = 8]. After 5 d, dietary methionine was reduced from 0.30 to 0.20 g/(kg·d) in both groups. On day 6, piglets received a constant [1-13C]phenylalanine infusion to measure whole-body protein kinetics, and on day 8 they received a constant [3H-methyl]methionine infusion to measure tissue-specific protein synthesis in skeletal muscle, the liver, and the jejunum. RESULTS Whole-body phenylalanine flux, protein synthesis, and protein breakdown were 13%, 12%, and 22% lower, respectively, in the MD group than in the MS group (P < 0.05). Reduced whole-body protein synthesis in the MD piglets was attributed to 50% lower protein synthesis in skeletal muscle and the jejunum than in the MS piglets (P < 0.05). Furthermore, methionine availability in skeletal muscle was halved in piglets fed the MD diet (P < 0.05), and the specific radioactivity of methionine was doubled in the jejunum of MD piglets (P < 0.05), suggesting lower intestinal remethylation. Liver protein synthesis did not significantly differ between the groups, but secreted proteins were not measured. CONCLUSIONS Dietary methyl donors can affect whole-body and tissue-specific protein synthesis in neonatal piglets and should be considered when determining the methionine requirement.

The incorporation of phenylalanine-U-14C into liver, muscle protein and expired CO was determined in rats fed either carbohydrate or fat in order to elucidate the biochemical mechanism underlying the "protein sparing" action of dietary carbohy drate and fat. The rate of incorporation of this radioactive amino acid into these tissue proteins in vivo was compared by calculating (i) relative specific activity: the ratio of specific activity of the protein to that of the acid-soluble fraction in muscle and liver and (ii) recovery ratio: the ratio of radioactivity recovered in total muscle and/or liver protein versus that recovered in expired CO». The incorporation of phenylalanine-U-"C into protein increased in both liver and muscle of rats fed either carbohydrate or fat and the ratio of radioactivity recovered in muscle protein to that in liver protein in creased. Feeding either carbohydrate or fat caused a significant increase in rate of incor poration of this radioactive amino acid by liver slices as well as isolated diaphragm compared with that in fasted rats. The overall results indicate that feeding either carbohydrate or fat to fasted rats causes an increase in reutilization of endogenous amino acids and that this may be one of the possible mechanisms underlying the "protein sparing" action of dietary carbohydrate and fat. J. Nutr. 104: 264-271, 1974.

Eight-week old atherosclerosis-resistant Show Racer (SR) and -susceptible White Carneau (WC) pigeons were fed one of four puri fied diets as follows: a basal (dextrin) diet (B), a basal-cholesterol diet ( BC ), a sucrose diet ( S), or a sucrose-cholesterol diet ( SC ). Blood choles terol and triglycA©ride concentrations, liver malate dehydrogenase activity (MDHA), liver cholesterol and triglycA©rideconcentrations, liver protein and total lipid levels, and plasma lipoprotein lipase activity (LPLA) were assayed and evaluated statistically to determine their relationships to dietary modification and their significance in the pathogenesis of aortic athero sclerosis. Cholesterol supplementation increased the prevalence of fatty streaking in the aortas of the WC over those of the SR, and it also increased the severity of fatty streaking in both the WC (with one exception) and SR pigeons compared to their counterparts fed diets without cholesterol. Sucrose feeding did not consistently alter the levels of blood cholesterol and triglycA©rides. The MDHA of all the SR, when expressed on the basis of g of liver protein were significantly higher than those of the WC fed the same diet except those fed the basal diet. No significant differences were found in LPLA between the two breeds fed the various diets. All tissue lipid levels were positively correlated while the liver MDHA negatively correlated with the severity of the disease. For the SR, MDHA was also negatively correlated with the prevalence of fatty streaking. A larger num ber of SR developed the disease when subjected to elevated blood triglyc A©ride levels while a larger number of WC developed the disease when subjected to elevated blood cholesterol levels. J. Nutr. 108: 944-953, 1978.

Circadian rhythms were studied in ad libitum- and meal-fed Zucker rats. Lean and obese female rats were fed ad libitum or in a single meal from 1400-1800 for 12 d. Both groups of ad libitum-fed rats exhibited a nocturnal feeding pattern; obese rats ate more than the lean rats during the light and dark parts of the cycle. After 12 d, groups of rats were killed at 4-h intervals over a 24-h period, inclusively, and the presence of circadian rhythms was evaluated for the serum metabolites, liver constituents and the liver lipogenic enzymes. The obese ad libitum-fed rats demonstrated rhythms for serum triglycerides, serum cholesterol, liver protein and glycogen, and the activities of fatty acid synthetase, citrate cleavage enzyme (EC 4.1.3.8), and glucose-6-P dehydrogenase (EC 1.1.1.49). Meal feeding the obese rats modified the rhythms for serum cholesterol, liver glycogen and the activities of glucose-6-P dehydrogenase, and citrate cleavage enzyme and a rhythm was established for serum glucose. The feeding schedule did not cue a rhythm for serum triglycerides and phosphogluconate dehydrogenase (EC 1.1.1.44) or modify the rhythm for liver protein. The lean ad libitum-fed rats displayed rhythms for all the measured parameters except serum glucose. Feeding-cued rhythms for all metabolites except serum cholesterol were observed when the lean rats were meal-fed. These data suggest that the rhythms of the ad libitum-fed obese rats may not be similar to those observed in the lean rat. Yet, changing the feeding schedule may be important in establishing rhythm patterns that will alter the anabolic profile characteristic of the Zucker obese rat.

The effect of diets containing as their protein component wheat gluten or casein, fed ad libitum to adult male rats for 1 to 6 days, after 6 days of protein deprivation, on the liver levels of glucose-6-phosphate dehydrogenase (G8PD) and 6-phosphogluconate dehydrogenase (6PGD) were studied. In rats fed the gluten diet the values of body weight gain, liver size, liver protein, liver xanthine oxidase, G«PD and 6PGD activities were lower in comparison with rats fed the casein diet. Amino acid supplements to the gluten diet improved the nutritive value of this diet, as shown b> the enhancement of body weight gain, liver size, liver protein and xanthine oxidase activity to levels found in rats fed the casein diet, but failed to increase GePD and 6PGD activities. Essential fatty acids added to the casein diet, in order to bring their level up to that of the gluten diet, proved incapable of decreasing the levels of G«PD and 6PGD activities found in rats fed the casein diet..It was concluded that the differ ential effect of gluten and casein on liver GoPD and 6PGD is independent of differences in the nutritive value and in the essential fatty acid content of these proteins, and is dependent upon one or more factors different from those known to affect the liver HMP shunt dehydrogenases. J. Nutr. 104: 819-827, 1974.

The effect of increasing nitrogen intake with constant calorie supply was studied in protein-depleted rats. Animals were randomized into three paired groups to receive either total parenteral nutrition (TPN) or enteral (EF) isocaloric feedings (240 kcal X kg-1 X day-1) differing only in their amount of amino acids. The diets, composed of dextrose, a safflower oil emulsion and crystalline amino acids, were infused continuously for 5 days. Daily nitrogen intakes were for controls (EF, n = 7; TPN, n = 6) 0.25 g/kg; for groups 1 (EF, n = 7; TPN, n = 8) 1.4 g/kg and for groups 2 (EF, n = 7; TPN, n = 7) 2.5 g/kg. The rats in groups 1 and 2 obtained significant protein repletion in terms of body weight, nitrogen retention, liver protein and serum albumin regardless of the route of nutrient delivery. However, rats in groups 2 (higher nitrogen intake) presented a more rapid and greater recovery despite a reduction in nitrogen utilization. Whole-body leucine kinetics studied at the end of controlled feeding periods were similar for EF and TPN rats. However, increasing nitrogen intake (1 and 2) increased whole-body leucine flux and incorporation into protein resulting in a better balance, since leucine release from protein breakdown was comparable to controls. Liver protein fractional synthetic rates were reduced in EF while remaining at a high rate in TPN group 2 and still more elevated in TPN group 1, suggesting a preferential support of liver protein by enteral feeding or the effect of TPN as a non-physiologic route.

A double isotope technique was used to evaluate the effects of a synthetic antimicrobial compound (carbadox) upon the synthesis and degradation of rat liver and muscle proteins. Trials 1 and 2 used, respectively, 20 and 12 male Sprague-Dawley rats with average initial weights of 75 and 81 g. A 16% crude protein corn-soybean diet without or with carbadox (55 ppm) was fed. Rats were randomly assigned to diets which were fed 10 days prior to the initial isotope (14C-lysine) injection. The second isotope injection (3H-lysine) was made 3 days after the 14C-lysine injection in trial 1, 6 days in trial 2. Four hours after the second injection, the rats were killed and the right and left quadriceps and a sample of liver were removed. Total protein from the right quadriceps was prepared via a boiling trichloroacetic acid (TCA) extraction procedure. The left quadriceps was separated into myofibrillar and sarcoplasmic protein fractions via a KCl salt gradient. In both trials, gains were similar across treatments, but markedly reduced during the injection periods. Gain/feed prior to injection was greater (P less than 0.05) for carbadox-fed rats. In both trials right quadriceps 3H-incorporation (expressed as 3H cpm/mg protein) and relative degradation (3H cpm/14C cpm) were not affected by dietary carbadox. The sarcoplasmic and myofibrillar protein fractions were also not significantly affected. However, in both trials liver protein 3H-incorporation was enhanced (P less than 0.025) by carbadox, while liver protein degradation was affected (P less than 0.001) only in trial 2.

Adequate nutrition and probiotics have each been shown to reduce the severity of colitis, but their impact on hepatic and gastrointestinal protein metabolism has not been studied. Our objective was to determine whether maintaining adequate nutrition compared with administering probiotics affected protein synthesis, colon histopathology, and oxidative stress in our macronutrient-restricted piglet model of colitis. Piglets (n = 8/group) receiving dextran sulfate to induce colitis were randomized to 3 treatment groups: macronutrient restricted (MR); macronutrient restricted with VSL #3 probiotics (MRP), or well nourished (WNC). An additional 8 piglets served as healthy references for comparative purposes given the unique nature of the experimental model. A primed, constant infusion of the tracer L-[ring-(2)H(5)]phenylalanine was performed in colitis piglets after 14 d to determine the fractional synthesis rates of proteins in small intestinal mucosa, colon, and liver and of plasma proteins (total protein, fibrinogen, albumin). Colon histopathology and oxidative stress were also assessed. Compared with MR piglets, both WNC and MRP piglets had higher protein synthesis rates in liver and plasma protein pools. However, only adequate nutrition increased protein synthesis in the colon and decreased colitis severity. Whereas probiotics did not stimulate gastrointestinal protein synthesis or reduce colitis severity, a still-unidentified signaling mechanism between the gut and liver seems to be responsible for the probiotic-induced increase in liver protein and plasma protein synthesis. These data underscore the importance of maintaining nutrient intake in pediatric patients with gastrointestinal disease. A strategy for correcting compromised nutrition seems to be more beneficial for reducing damage during colitis than using probiotics only.

Protein synthesis has been measured in vitro in postmitochondrial extracts from livers of rats fed levels of casein ranging from 0 to 40% by weight. The maximal capacity for protein synthesis per milligram of RNA, measured with each amino acid added at 250 mumol/L, was 40-60% higher in rats fed a protein-free diet than in those fed 6 or 15% casein. Our results suggest that the livers of rats fed a protein-free diet are primed for the synthesis of tissue proteins and, given an adequate supply of amino acids, the rate of protein synthesis would be as high as or higher than the rate in protein-replete animals. When amino acids were added to the in vitro system at concentrations found in plasma of rats fed 0, 6, 15 or 40% casein the rate of protein synthesis increased by three- to fourfold over this range, with the highest rate observed for the 15% dietary casein level. We conclude that when protein intake is below the requirement level, the rate of liver protein synthesis may be limited by amino acid supply, by the capacity of the system for protein synthesis or by both.

This investigation was conducted to evaluate the changes in the total content of protein and RNA in liver and muscles of rat dams before and after acute separation from their litters. Groups of 8-12 rats fed ad libitum were killed on the 12th (group L12) and 20th d (group L20) of lactation and on the 1st (group W1) and 7th d (group W7) after weaning. Nonpregnant, nonlactating rats paired for age served as controls. Dry matter, protein, DNA and RNA levels of the mammary gland, liver and muscles of the right hindlimb were determined. Wet weight and total organ or tissue protein, DNA and RNA were higher in mammary glands of L12 and L20 than in age-matched controls. These values were lower in groups W1 and W7 than in the lactating groups. No changes were noted in the total liver protein or DNA content, but total liver RNA was greater in groups L12 and L20 than in controls or group W1. Total muscle dry matter, DNA and RNA were significantly lower in groups L12 and L20 than in groups W1 and W7. Muscle protein content increased progressively from the 12th to 20th d of lactation to a peak in group W1, and it decreased to values found in age-matched controls in group W7. Although the muscle protein mass of the hindlimb during peak lactation (group L12) was only 63% of that in nonlactating control rats, within 1 d of weaning it was significantly higher than in nonlactating rats. Similar changes in RNA suggest that these changes in protein content are related to an adaptative mechanism designed to handle the surplus of plasma amino acids not used by the mammary gland after weaning.

Neonates deposit protein at a very high rate and efficiently utilize dietary amino acids for protein deposition. This high efficiency is associated with an elevated stimulation of tissue protein synthesis by feeding. Our recent studies have focused on identification of the factors that mediate this response in the neonate. A positive curvilinear relationship between skeletal muscle protein synthesis and plasma insulin concentration was identified in fasted and fed suckling pigs; the relationship changes with development. To test the specific effects of insulin on protein metabolism in the neonate, a procedure to clamp amino acids, under hyperinsulinemic conditions, was developed. By using this technique, we showed that insulin-stimulated whole-body amino acid disposal is elevated in the neonate, and this response may account for the efficient use of dietary amino acids for protein accretion. More recent studies suggest that the enhanced stimulation of skeletal muscle protein synthesis by feeding in the neonate is primarily insulin mediated; however, the stimulation of liver protein synthesis by feeding seems to be largely a function of amino acid concentration.

The effects of exercise training and dietary chromium intake on rat liver and muscle glycogen metabolism, tissue and body weight and feed consumption were examined. After 16 wk of training, liver, gastrocnemius and biceps femoris glycogen concentrations were higher in the trained compared to sedentary groups, independent of dietary chromium. There was a chromium x training interaction on glycogen synthase activities in the liver and gastrocnemius muscle. Liver glycogen phosphorylase activities (expressed per g liver) were lower in the chromium-supplemented rats as compared to the non-supplemented rats after 5 wk of dietary treatment, but were similar after 8 wk and higher after 18 wk. Gastrocnemius phosphorylase activity (expressed per mg protein) was lower in the trained rats as compared to the sedentary rats after 16 wk, independent of dietary chromium. Biceps femoris phosphorylase activities were not altered due to training or dietary chromium. Total protein concentration increased in the liver but decreased in the gastrocnemius due to dietary chromium. In summary, liver glycogen synthase and phosphorylase activities were dependent upon dietary chromium. Dietary chromium altered gastrocnemius synthase, but not phosphorylase activities. Changes in enzyme activities may be related to the chromium-dependent effects on liver protein and the chromium and training-dependent effects on gastrocnemius total protein.

Young growing rats were used to evaluate the effect of dietary composition on growth rate, body and organ weights, biochemical composition of brain and liver, plasma growth hormone concentration, and plasma somatomedin ( SM) activity. Young male rats ( 10/group ) were fed four levels of dietary protein (casein: 2.5, 5, 10, and 20%) and three levels of dietary fat (corn oil: 1.2, 10, and 20%) over a 30-day period. Changes in total body and organ weights paralleled increasing dietary protein and were affected to a lesser extent by dietary fat. Liver protein paralleled in creasing dietary protein. Glycogen, protein, DNA, and RNA determinations on the brain were not significantly influenced by dietary manipulation. Plasma growth hormone concentrations were not significantly different due to dietary protein or fat. Plasma SM activity was positively correlated with dietary protein levels (0.80 units/2.5% protein for all fat levels to 1.35 units/20% protein for all fat levels). Plasma SM was suppressed approxi mately 25% with increasing dietary fat in rats fed the 207r protein diets. Furthermore, SM appears to be directly related to food efficiency ( g gain/ 100 kcal) and body weight change. These data suggest that dietary com position has a direct effect on plasma SM activity and that the severe growth retardation associated with protein malnutrition may be related to its additional effect on serum SM. J. Nutr. 109: 613-620, 1979.

Rate of protein synthesis in mammary tissue and liver as well as pup weight and milk yield were measured at d 15 of lactation in rats fed throughout gestation and lactation diets that varied in protein quality (wheat, wheat supplemented with lysine and threonine or casein supplemented with methionine), protein quantity [11 or 21% (wt/wt)] and feeding level (ad libitum or pair-fed to 100 or 85% of the consumption of dams fed wheat ad libitum). Improvement in dietary protein quality or quantity at the ad libitum feeding level produced large increases in mammary protein synthesis and pup weight; pair-feeding at 100 or 85% of the ad libitum wheat consumption level reduced or eliminated these increases. Increases in protein synthesis and pup weight associated with protein quality improvement were of similar magnitude to those associated with nearly doubling protein content of maternal diets. The responses of liver protein synthesis to dietary manipulation were qualitatively similar to those seen in mammary tissue. These data demonstrate that maternal dietary protein quality, protein quantity and feeding level all affect mammary protein synthesis and pup weight, and that, considered across all diets, changes in mammary protein synthesis are highly correlated with changes in pup weight and protein nutritional value.

Pregnant rats were used to determine effects of salicylamide on the sequential uptake and loss of radiosulfate by maternal and fetal tissues. Separate and combined effects of salicylamide and protein restrictions on radiosulfate retention by fetal rat tissues were determined. Rats were fed salicylamide-containing or control diets from the 4th or 6th day of gestation. Rats were killed between the 17th and 19th days of gestation, following intramuscular injection of sodium-35S-sulfate. Fetal and placental radiosulfate uptake was related to maternal serum levels. Salicylamide administration decreased radiosulfate uptake by maternal serum and liver, fetus and placenta--effects being dose-dependent. Differences in radiosulfate uptake by the fetus and placenta over time, induced by salicylamide, were also significant independently of maternal serum levels of radiosulfate. Major retention of 35S was found in the fetal cartilage with lower concentrations in the fetal skin, intestine, brain and liver. Protein restriction increased retention of 35S, and salicylamide administration caused a significant reduction in 35S retention by fetal tissues. Skeletal malformations were found in fetuses from dams receiving salicylamide. It is concluded that this drug lowers the availability of sulfate to the fetus from the dam, impairs the incorporation of sulfate by fetal tissues utilizing sulfates, and is teratogenic.

Although protein requirements have been shown to decrease in rats with age, very few studies have dealth with old animals. In addition, a model of protein deficiency in aged rats has not been established. Nineteen-month-old male Sprague-Dawley rats were fed a 25 (control), 5, or 3.5% casein diet for 1 week ad libitum. Liver protein and RNA content were reduced in the 3.5% casein group when compared to controls. No differences were observed between the control and the 5% casein group. Also, no differences were observed in incorporation of [14C]orotic acid into liver nuclear RNA between these two groups. However, this variable was significantly higher in the 3.5% casein group when compared to control. When comparing the results of this study with our previous studies in young adult rats, the 3.5% casein diet produced biochemical changes in liver that are qualitatively and quantitatively similar to the ones observed in young adult rats fed a 6% casein diet ad libitum, which allows a more direct comparison of the effect of protein deficiency on the aged rat.

Changes in protein content and function were investigated in male Sprague-Dawley Crl:Cd rats that were either fasted, consumed a diet con taining 1.5 g fat/100 g initial body weight per day or received ad libitum an 85% carbohydrate, minimal nitrogen diet. Fasted rats became moribund after 9-10 days and were both hypoglycA©mie and hypoketonemic. In contrast, rats fed the fat-only and high carbohydrate, minimal nitrogen diets survived the 28-day study period. Although rats consuming the high carbohydrate, minimal nitrogen diet lost less body weight and nitrogen than either fasted animals or rats receiving the fat-only diet, they also had a loss of delayed hyper- sensitivity recall to tuberculin antigen and a 55% decrease in plasma essential amino acid concentrations. Rats fed the fat-only diet maintained delayed hy- persensitivity recall and near normal concentrations of plasma essential amino acids despite a greater loss of body nitrogen than was observed in terminally fasted animals. Plasma albumin concentrations and total liver protein content showed comparable decreases in rats consuming the two incomplete diets. We concluded that nonprotein calories prolong survival in the rat and fat or pre dominantly glucose calories are sufficient at preventing mortality. However, the source or quantity of nonprotein calories consumed during periods of protein deprivation can markedly alter individual tissue protein status and func tion. J. Nutr. Ill: 1287-1296, 1981.

The effects of magnesium deficiency on the in vivo incorporation of labeled precursors into tissue macromolecules were studied. In severe Mg deficiency developing over 4 months, total protein synthesis in spleen and thymus was depressed by 40% to 50%, while DNA synthesis was increased by as much as 350%. RNA synthesis was not significantly altered. Protein synthesis in kidney was also reduced. In the intact liver, DNA synthesis was increased and RNA synthesis reduced, but protein synthesis was unchanged. The regeneration of the liver after partial hepatectomy was impaired, however, which suggested that the capacity of the liver protein synthetic system was reduced. The effects of magnesium deficiency were unaltered by the ingestion of ethanol that comprised approximately 25% of total energy. Each of the organs enlarged in Mg deficiency, and the spleen was strikingly hyperplastic. The increase in splenic DNA synthesis in the presence of depressed protein synthesis may be an early stage in a lymphoproliferative process leading ultimately to neoplasia.

The effects of feeding a diet deficient in zinc (Zn) to male rats on histidine metabolism were studied. Results showed that significantly higher percentages of DL-histidine-carboxyl-14C and L-histidine-2-(ring)-14C were oxidized by Zn-deficient rats. The incorporation of L-histidine-2-(ring)-14C into the proteins of skin, muscle, and kidney were significantly reduced in Zn-deficient rats as compared to Zn-supplemented rats. Conversely, the radioactivity of liver protein of Zn-deficient rats was significantly increased. Zn deficiency increased the activities of liver histidase and urocanase but had no effect on the activity of liver histidine-pyruvate transaminase. The increases of enzymatic activities were not due to food intake and can be prevented upon Zn repletion. The liver of Zn-deficient rats contained normal amount of histidine but a reduced quantity of histamine. The results on urinary excretion indicated that Zn-deficient rats discharged the same amounts of one-methyl and three-methyl histidine as Zn-supplemented pair-fed rats. Overall findings support in principle the concept that Zn deficiency results in disturbances of protein metabolism and also indicate that Zn is an important factor in regulating histidine metabolism through the urocanic acid pathway.

Obese and lean Zucker rats were made diabetic by intracardiac injections of alloxan (65-72 mg/kg body weight) and then given daily injections of protamine zinc insulin [1.25 U/(100 g/d)] for 6, 9 and 12 d. Body weight, food intake, plasma glucose and immunoreactive insulin concentrations were not different for lean and obese diabetic rats of similar ages. Rates of increase in carcass protein, mixed muscle protein and myofibrillar protein were less in obese than in lean rats. However, rates of increase for the sarcoplasmic fraction were not different. Fractional rates of synthesis of total muscle protein and myofibrillar protein, as determined by continuous intravenous infusion of [14C]tyrosine, were comparable in the two genotypes. Fractional rate of myofibrillar protein degradation, as determined by urinary 3-methylhistidine excretion, was higher in obese than in lean rats. Differences in calculated absolute rates between genotypes did not parallel differences in the fractional rates, due mainly to a smaller protein mass in obese rats. As a consequence, absolute synthetic rates were lower in obese rats, while absolute degradation rates were similar in the two genotypes. In contrast, rates of liver protein synthesis were similar in obese and lean rats, whether expressed as fractional or absolute rates. These results indicate that decreased protein deposition in the obese animal is a consequence of both an absolute decrease in protein synthesis in muscle as well as a disproportionately elevated protein degradation in muscle. Hyperinsulinemia normally seen in obese rats may be an adaptive response to minimize the impaired balance between protein synthesis and degradation.