Effect of a protein-free diet on muscle protein turnover and nitrogen conservation in euthyroid and hyperthyroid rats.

Although protein turnover in skeletal muscle is increased in hyperthyroidism and decreased in hypothyroidism, a deficient protein intake tends to increase serum T3 (tri-iodothyronine) while decreasing muscle protein turnover. To determine whether this diet-induced decrease in protein turnover can occur independent of thyroid status, we have examined muscle protein turnover and nitrogen conservation in hyperthyroid rats fed on a protein-free diet. After inducing hyperthyroidism by giving 20 micrograms of T3/100g body wt. daily for 7 days, groups of euthyroid and hyperthyroid animals were divided into subgroups fed on basal and protein-free diets. Muscle protein turnover was measured by N tau-methylhistidine excretion and [14C]tyrosine infusion. Urinary nitrogen output of euthyroid and hyperthyroid animals fed on the protein-free diet was also measured. Although hyperthyroidism increased the baseline rates of muscle protein synthesis and degradation, it did not prevent a decrease in these values in response to protein depletion. Furthermore, hyperthyroid rats showed greatly decreased nitrogen excretion in response to the protein-free diet, although not to values for euthyroid rats. These findings suggest that protein depletion made the experimental animals less responsive to the protein-catabolic effects of T3.     

Effect of leucine-rich dietary protein on in vitro protein synthesis in porcine muscle

Experiments were conducted to determine the effect of feeding diets containing leucine-rich proteins on in vitro protein synthesis in porcine muscle. Swine (10 kg initial weight) were fed for 4 weeks diets composed mainly of corn gluten meal, corn and soybean meal, and containing a total of 2.00, 2.33, 2.92, 3.12, 3.53, and 4.01% leucine. At the end of the growing period, six swine fed each diet were killed and samples of biceps femoris, longissimus dorsi, and triceps brachii were excised. Incorporation of [14C]phenylalanine into newly synthesized protein was measured using a cell-free in vitro system following recombination of purified soluble protein and ribosomal fractions. The feeding of diets containing increasing amounts of leucine-rich protein increased the free leucine concentration in plasma and skeletal muscle. There was no significant effect of diet on incorporation of [14C]phenylalanine into muscle protein following simple recombination of soluble protein and ribosomal fractions from the same tissues. Combination of muscle soluble protein from animals fed 2.00% leucine with ribosomal fractions of animals fed increasing quantities of leucine-rich protein, however, indicated increased protein synthetic activity of the ribosomal fraction in all muscles tested. Protein synthetic activity of the soluble protein fraction was not affected by diet. It was concluded that the feeding of leucine-rich dietary proteins beyond requirements for maximal rate of growth can increase the protein synthetic potential of porcine muscle cells although whole body growth is depressed.     

The effect of dietary protein on the excretion of alpha2u, the sex-dependent protein of the adult male rat.

Adult male rats were maintained on normal (20% casein), protein-free (0% casein), high protein (50% casein), decicient protein (20% zein), and a supplemented, deficient protein (20% zein plus L-lysine and L-tryptophan) diets. Rats on a protein-free diet excreted approximately 1 mg alpha2u/24 h compared with a normal of 10-15 mg/24 h. Depleted rats placed on a 20% casein diet showed a rapid restoration of the normal alpha2u excretion as well as total urinary proteins. Accumulation of alpha2u in the blood serum was measured in nep-rectomized rats. Rats on a 0% casein diet accumulated only 30% of the alpha2u compared to normals. On a 50% casein diet, rats excreted 30-50 mg alpha2u/24 h. However, the accumulation was normal in the serum of nephrectomized rats. A high protein diet did not stimulate alpha2u synthesis but probably increased the renal loss of all urinary proteins. The excretion of alpha2u on a zein diet was reduced to the same degree as with the protein-free diet. Supplementation with lysine and tryptophan restored the capacity to eliminate alpha21 to near normal levels. Accumulation of alpha2u in the serum of nephrectomized rats kept on the zein diets showed that the effect to suppress the synthesis of the ahpha2u. Supplementation restored the biosynthesis of alpha2u. We conclude that the effect of dietary protein on the excretion of urinary proteins in the adult male rat is caused in large part by an influence on the hepatic biosynthesis of alphay2u. The biosynthesis of this protein, which represents approximately 30% of the total urinary proteins, is dependent on an adequate supply of dietary protein.     

Consumption of a moderately Zn-deficient and Zn-supplemented diet affects soluble protein expression in rat soleus muscle

Zinc deficiency negatively affects muscle function, but there are limited biochemical data identifying the cause of this reduction in function. The objective of the present study was to identify soluble proteins in rat soleus muscle that were responsive to different levels of dietary zinc. Rats (n=21) were fed diets containing three concentrations of zinc: 5, 30 and 200 ppm for 42 days. There was no difference in body weights of the rats consuming the 5-ppm zinc diet compared to the rats consuming the 30- or 200-ppm zinc diets; however, bone zinc levels were significantly decreased in the 5-ppm dietary zinc group. Individual soluble protein fractions were isolated from these muscles and the samples were prepared for two-dimensional polyacrylamide gel electrophoresis. The expression levels of four proteins were significantly depressed by dietary Zn depletion and supplementation, S-glutathiolated carbonic anhydrase, myosin light polypeptide 3, heat shock protein 20 and heart fatty acid binding protein. This is the first report that indicates that both Zn depletion and supplementation result in protein expression profiles that may negatively affect skeletal muscle function. These results indicate that there are specific signaling pathways that require proper Zn nutriture for maintaining optimal muscle function and suggest that the consumption of pharmacologic doses of Zn may be detrimental to muscle function.     

Influence of dietary composition on growth and energy reserves in tench (Tinca tinca)

The effects of different protein, lipid and carbohydrate diets on growth and energy storage in tench, Tinca tinca L., were studied. Over a 2-month period fish were fed four different diets: control, protein-enriched, carbohydrate-enriched and lipid-enriched. The best growth rates were obtained with the control and protein-enriched diets; the carbohydrate diet produced the worst results (lowest specific growth rate, weight gain, nutritional index and hepatosomatic index). These results suggest that it is not advisable to reduce dietary fish protein below 35%, and that it is not possible to obtain a protein-sparing effect of either lipids or carbohydrates, at least in our experimental conditions. The high-protein diet resulted in the storage of energy excess as muscle proteins and hepatic glycogen. Tench fed the high-carbohydrate diet stored carbohydrates as muscle glycogen and reduced plasma triglycerides. Finally, both liver and muscle lipid content were in positive correlation to dietary lipid.     

Effect of essential fatty acid deficiency on G-proteins, cAMP-dependent protein kinase activity and mucin secretion in the rat submandibular salivary glands

Studies were conducted to determine whether β-adrenergic cell signalling is altered in submandibular salivary glands (SMSG) is essential fatty acid (EFA) deficiency. Three groups of rats were fed diets which were deficient in EFA (EFAD), marginally deficient in EFA (MEFAD) or contained sufficient amount of EFA (Control). Rats were killed after 20 wk on diets, SMSG were dissected out and cyclic AMP-dependent protein kinase (PKA) activity was measured. The specific enzyme activities were higher in the homogenates and supernatant fractions of the gland from EFAD and MEFAD rats compared with the controls. The relative levels of guanine nucleotide-binding regulatory proteins (Gs and Gi) were also measured in the SMSG membranes of rats fed the 3 diets. The levels of Gs were significantly higher in the EFAD and MEFAD groups than in the controls. No significant differences were observed in the secretion of trichloroacetic acid-phosphotungstic acid (TCA-PTA) precipitable glycoproteins from the SMSG slices among the 3 dietary groups.     

Studies on the Metabolic Activity of Muscle Proteins

The time-course of changes in total amount of proteins of sarcoplasmic, myofibrillar and stromal fractions in muscle of the rats fed a protein-free diet for 8, 16, 24 and 32 days, together with the referential data of those changes in the rats fed a protein-free diet up to time of death and a 60% casein diet for 12 days was determined respectively. The results were as follows: (1) The sarcoplasmic and the myofibrillar fractions decreased much more than the stromal fraction in the earlier stages of protein depletion following the same pattern as seen in reserve proteins. (2) The sarcoplasmic fraction decreased slightly more than the myofibrillar fraction as early as 8 days of the depletion, but the relative proportion between these two fractions was thereafter almost the same as that of the standard diet group. (3) In rats fed a 60% casein diet, the sarcoplasmic fraction increased markedly than the others.     

Proteomic sensitivity to dietary manipulations in rainbow trout

Changes in dietary protein sources due to substitution of fish meal by other protein sources can have metabolic consequences in farmed fish. A proteomics approach was used to study the protein profiles of livers of rainbow trout that have been fed two diets containing different proportions of plant ingredients. Both diets control (C) and soy (S) contained fish meal and plant ingredients and synthetic amino acids, but diet S had a greater proportion of soybean meal. A feeding trial was performed for 12 weeks at the end of which, growth and protein metabolism parameters were measured. Protein growth rates were not different in fish fed different diets; however, protein consumption and protein synthesis rates were higher in the fish fed the diet S. Fish fed diet S had lower efficiency of retention of synthesised protein. Ammonia excretion was increased as well as the activities of hepatic glutamate dehydrogenase and aspartate amino transferase (ASAT). No differences were found in free amino acid pools in either liver or muscle between diets. Protein extraction followed by high-resolution two-dimensional electrophoresis, coupled with gel image analysis, allowed identification and expression of hundreds of protein. Individual proteins of interest were then subjected to further analysis leading to protein identification by trypsin digest fingerprinting. During this study, approximately 800 liver proteins were analysed for expression pattern, of which 33 were found to be differentially expressed between diets C and S. Seventeen proteins were positively identified after database searching. Proteins were identified from diverse metabolic pathways, demonstrating the complex nature of gene expression responses to dietary manipulation revealed by proteomic characterisation.     

Use of different combinations of macronutrients in diets for dentex (Dentex dentex): Effects on intermediary metabolism

The influence of the dietary macronutrient balance on the intermediary metabolism of common dentex (Dentex dentex L.) was evaluated. Four experimental diets combining high and low levels of macronutrients were formulated. Dentex fed on 43% protein had higher liver and muscle lipid content, corresponding with an increased hepatic G6PDH activity. This “excess” of hepatic lipids at higher protein levels could be used to obtain energy as would be reflected by hepatic HOAD. In the liver, 43% of dietary protein induced higher AlaAT and FBPase activities. Similarly, dentex fed on the P₄₃C₂₈ and P₃₈C₂₈ diets showed an increased hepatic and muscular gluconeogenic pathways (higher FBPase activity) from amino acids (elevated AlaAT) and/or glycerol (elevated GK). However, changes in glycemia were not observed among dietary treatments. At coronary level, the use of lower dietary protein induced an increase in the activity of glycolytic (PK and HK-IV) and lipolytic (HOAD) enzymes. Considering the overall results and the experimental conditions, it could be suggested that dietary protein could be reduced until 38% without affecting negatively the normal physiology of dentex. Moreover, high dietary carbohydrate levels could not be used efficiently by dentex given that gluconeogenesis occurs.     

Paradoxical protection of both protein-free and high protein diets against acute ammonium intoxication

Rats were fed standard (20% protein), protein-free or high protein (80%) diets for 15 days and then injected intraperitoneally with ammonium acetate (7 mmol/Kg). Survival was 6%, 75% and 100%, respectively, for rats fed standard, protein-free and high protein diets. After injection of 6 mmol/Kg of ammonium acetate, blood ammonia reached a peak (at ca. 2 mM) after 7, 25 and 30 min for rats fed high protein, protein-free and standard diets, respectively. The results presented indicate that protection in the high protein group is due to faster detoxication of ammonia via a more active urea cycle while the tolerance of the protein-free group to higher levels of ammonia remains to be clarified.     

Influence of dietary protein on insulin-like growth factor binding proteins in the chicken

We determined the effect of dietary protein on the distribution of insulin-like growth factor (IGF) binding proteins in chicken plasma. Three groups of male broilers (n=6 per group) were fed (ad libitum) isocaloric diets containing 12, 21 or 30% dietary protein. Birds were fed respective diets beginning at 7 days of age and killed at 28 days. No differences were observed between adequate (21%) and high (30%) protein intakes for any of the parameters investigated (growth criteria, plasma levels of IGF-I, growth hormone or IGF-binding proteins). Feeding protein deficient diets (12%) resulted in a 34% decrease in body weight, 17% decrease in feed intake and a 39% increase in feed/gain ratio. IGF-binding proteins in plasma samples were separated by SDS-PAGE and transferred to nitrocellulose sheets. Nitrocellulose blots were probed with [¹²⁵I]chicken IGF-II. Four regions of binding activity corresponding to 70, 43, 30 and 24 kDa were observed in all samples. Birds consuming 12% dietary group protein had less than 50% of the 43-kDa binding activity of birds consuming 21 or 30% dietary protein. The 30-kDa binding activity was 42% lower in the 12% dietary protein group compared to birds consuming adequate protein. In contrast, 70- and 24-kDa binding activities were not influenced by dietary protein. Chickens consuming 12% dietary protein had higher levels of growth hormone and lower levels of IGF-I than those consuming 21 or 30% dietary protein. These data indicate that in chickens, the circulating levels of at least two independent IGF-binding proteins are influenced by dietary protein.     

The nature of the dietary protein impacts the tissue-to-diet 15N discrimination factors in laboratory rats

Due to the existence of isotope effects on some metabolic pathways of amino acid and protein metabolism, animal tissues are (15)N-enriched relative to their dietary nitrogen sources and this (15)N enrichment varies among different tissues and metabolic pools. The magnitude of the tissue-to-diet discrimination (Δ(15)N) has also been shown to depend on dietary factors. Since dietary protein sources affect amino acid and protein metabolism, we hypothesized that they would impact this discrimination factor, with selective effects at the tissue level. To test this hypothesis, we investigated in rats the influence of a milk or soy protein-based diet on Δ(15)N in various nitrogen fractions (urea, protein and non-protein fractions) of blood and tissues, focusing on visceral tissues. Regardless of the diet, the different protein fractions of blood and tissues were generally (15)N-enriched relative to their non-protein fraction and to the diet (Δ(15)N>0), with large variations in the Δ(15)N between tissue proteins. Δ(15)N values were markedly lower in tissue proteins of rats fed milk proteins compared to those fed soy proteins, in all sampled tissues except in the intestine, and the amplitude of Δ(15)N differences between diets differed between tissues. Both between-tissue and between-diet Δ(15)N differences are probably related to modulations of the relative orientation of dietary and endogenous amino acids in the different metabolic pathways. More specifically, the smaller Δ(15)N values observed in tissue proteins with milk than soy dietary protein may be due to a slightly more direct channeling of dietary amino acids for tissue protein renewal and to a lower recycling of amino acids through fractionating pathways. In conclusion, the present data indicate that natural Δ(15)N of tissue are sensitive markers of the specific subtle regional modifications of the protein and amino acid metabolism induced by the protein dietary source.     

Cell wall glycoproteins and polysaccharides of mature runner beans

A novel use of chlorite-HOAc treatment (delignification procedure) for the isolation of hydroxyproline (HP) rich “glycoproteins” from the depectinated cell wall material of mature runner beans is described. This procedure can be used for the isolation of wall proteins even from heavily lignified tissues. Its main disadvantage is that some of the constituent amino acids are either destroyed or modified; the nature of these changes was studied using gelatine, lysozyme and “cytoplasmic proteins” of mature beans. The main amino acids to be affected were tyrosine, cystine, methionine and lysine. The chlorite-HOAc solubilized proteins were separated by PhOH-H₂O fractionation into two distinct “glycoprotein fractions”. The major fraction (isolated from the aqueous layer) contained most of the HP of the solubilized proteins. The sugars obtained on hydrolysis of both “glycoproteins” were galactose, arabinose, glucose, xylose, rhamnose and uronic acid. Most of the proteins remaining in the holocellulose could readily be extracted with cold alkali and were relatively poor in HP.     

Regulation of muscle protein degradation,not synthesis,by dietary leucine in rats fed a protein-deficient diet

The aim of this study was to elucidate the effects of long-term intake of leucine in dietary protein malnutrition on muscle protein synthesis and degradation. A reduction in muscle mass was suppressed by leucine-supplementation (1.5% leucine) in rats fed protein-free diet for 7 days. Furthermore, the rate of muscle protein degradation was decreased without an increase in muscle protein synthesis. In addition, to elucidate the mechanism involved in the suppressive effect of leucine, we measured the activities of degradation systems in muscle. Proteinase activity (calpain and proteasome) and ubiquitin ligase mRNA (Atrogin-1 and MuRF1) expression were not suppressed in animals fed a leucine-supplemented diet, whereas the autophagy marker, protein light chain 3 active form (LC3-II), expression was significantly decreased. These results suggest that the protein-free diet supplemented with leucine suppresses muscle protein degradation through inhibition of autophagy rather than protein synthesis.     

Effect of dietary lysine on muscle protein turnover in growing chickens.

Day-old male chickens were fed ad libitum isoenergetic diets containing 20% crude protein but differing in their lysine content (from 6.5 up to 11.3 g/kg). At 3 weeks of age, protein fractional synthesis rates in the pectoralis major muscle were determined using a large dose injection of 120 mumol per kg body weight of L-[4-3H] phenylalanine. Protein gain in the pectoralis major was measured between 19 and 23 days of age. Protein breakdown was obtained by calculating the difference between protein synthesis and deposition. Weight gain varied curvilinearly with dietary lysine intake and was maximum for 11.3 g lysine/kg of diet. In birds fed an adequate lysine intake (10.1-11.3 g/kg) protein fractional synthesis and breakdown rates were 23.6-25.9 and 17.8-19.8%/d respectively. Increasing lysine supplementation in the diet resulted in an impairment of protein fractional breakdown rates. By contrast, protein fractional synthesis rates remained unchanged owing mainly to an improvement in the synthesis efficiency (kRNA), until birds were fed an adequate lysine intake. These data suggest that the growth rate reduction of chickens fed lysine deficient diets was due to alterations in both rates of protein synthesis and breakdown in skeletal muscle. A maximum protein deposition is achieved when kRNA was optimal, ie for a dietary lysine content of about 9 g/kg, a value close to the requirement.     

Tryptophan Metabolism of Rats Fed on an Amino Acid Diet Devoid of One Branched Chain Amino Acid

In an attempt to study on metabolic changes in rats fed on an amino acid diet devoid of one branched chain amino acid and of niacin, rats were force-fed a leucine-free, isoleucine-free, valine-free or complete amino acid diet for 3 or 4 days and killed 3 hr after the feeding on day 4 or 5 to observe the body weight changes, the urinary nitrogen and N¹-methylnicotinamide (MNA), and liver tryptophanpyrrolase (TPase) and tyrosine-α-keto-glutarate transaminase (TKase) activities.

The excretion of the urinary nitrogen and MNA, TPase and TKase activities, and fat content of livers of rats force-fed these amino acid deficient diets were higher than those fed the complete amino acid diet. It was further confirmed in the present study that changes in TPase activity of rats given diets devoid of one essential amino acid were in the same direction with changes in urinary MNA which was observed in the previous studies on rats given threonine-free, tryptophan-free, methionine-free, lysine-free and complete amino acid diets. However, such metabolic changes in rats fed the leucine-free diet were not so remarkable, compared with those of rats fed the other amino acid deficient diets.     

Effect of cell age on erythrocyte fatty acid composition in rats on different dietary regimes

1. Rat erythrocytes were fractionated into young, mature and old cell fractions by centrifugation. The fatty acid composition of each fraction was determined by gas-liquid chromatography, under four different dietary conditions: with adequate linoleic acid in the diet, with a diet deficient in linoleic acid, and with the deficient diet supplemented with corn oil for 3 and 12 days. 2. Significant differences were observed in the fatty acid composition of cells of different ages on all diets. The pattern of fatty acid distribution depended on the particular acid in question, on its concentration in the total erythrocyte sample and on the nature of the dietary fat. 3. When corn oil was fed to rats that had been fed previously on a deficient diet, the changes in fatty acid composition that occurred depended on the acid and on the cell age. For example, young cells were more active in incorporating palmitic acid and arachidonic acid but incorporated linoleic acid at a slightly lower rate than older cells. 4. These results are believed to indicate the presence in the erythrocyte of transacylases with different specificities, and to show that transacylase activity changes as the cells age.     

Differences in portal flow rates of amino acids and liver composition between rats fed casein or lactalbumin

The portal appearance rates and net rates of amino acids' absorption were studied in rats fed semi-synthetic diets containing either casein or lactalbumin (CAS and LA, respectively) as the only protein sources. Rats were pre-adapted to the experimental diets for 5 days prior to the absorption studies. Rats fed the LA diet had higher (p < 0.05) portal vein concentrations of free essential amino acids than those fed the CAS diet at 0, 60, 105 and 150 min after feeding. Portal and arterial concentrations of arginine, leucine, tryptophan, lysine and methionine were higher (p < 0.05) in rats fed LA at most time points tested, while concentrations of tyrosine were higher (p < 0.05) in CAS fed rats. When portal flow rates were compared, values for arginine, threonine, alanine, leucine, tryptophan and lysine were higher (p < 0.05) in LA at most time points tested, while proline, tyrosine and valine were higher (p < 0.05) for CAS fed rats after 60 and 105 min feeding. Portal blood flow varied (p < 0.05) with time in rats fed protein-free or LA diets, and was higher (p < 0.05) than that of CAS at 105 min. Intestinal net rates of absorption of tyrosine, valine, leucine and lysine were higher (p < 0.05) for LA fed rats as compared to those fed CAS at most time points tested, while alanine and proline net rates were higher (p < 0.05) for CAS fed rats at 60, 105 and 150 min. Amounts of protein in stomach contents of rats fed the CAS diet were significantly higher (p < 0.05) than those in LA fed rats at 60, 105 and 150 min after feeding. The relative liver weight of the rats fed the CAS diet was lower (p < 0.05) than that of animals fed the LA diet. Lower (p < 0.05) liver glycogen and lipid contents were determined in rats fed CAS diet respect to LA or protein-free fed rats. Results indicate that dietary and plasma amino acids profile are only partially related, and that under normal feeding conditions amino acids from CAS and LA are absorbed at different rates, which is likely to affect liver composition and metabolism.     

Biochemical Studies on Indian Wild Legumes

Chemical screening of some profusely growing wild leguminous seeds of Acacia, Albizzia and Leucaena species, for their nutritional constituents suggested the possibility of their inclusion in animal nutrition. However, since the whole seeds proved to be unpalatable as well as toxic in some cases, it was thought of utility to employ their isolated proteins instead. Accordingly, with a view to explore newer and effective sources of dietary proteins, simple and effective solubility method was employed for their extraction, precipitation, fractionation and isolation from the undesirable wild seed constituents to obtain the protein fractions in the pure form which could be incorporated in animal nutrition. Distribution of total nitrogen and dispersion of nitrogenous constituents in the seeds have also been studied. Extraction of proteins of considerable purity is best effected with sodium chloride solution (5%, w/v) and subsequent dialysis of the extract. Proteins were isolated from seven varieties of leguminous seeds by acid-alkali extraction method and their protein efficiency ratio and the biological value have been evaluated by animal experiments with and without supplementing the isolated protein diets with the missing essential amino acids.     

Short-term, increasing dietary protein and fat moderately affect energy expenditure, substrate oxidation and uncoupling protein gene expression in rats

Macronutrient composition of diets can influence body-weight development and energy balance. We studied the short-term effects of high-protein (HP) and/or high-fat (HF) diets on energy expenditure (EE) and uncoupling protein (UCP1-3) gene expression. Adult male rats were fed ad libitum with diets containing different protein-fat ratios: adequate protein-normal fat (AP-NF): 20% casein, 5% fat; adequate protein-high fat (AP-HF): 20% casein, 17% fat; high protein-normal fat (HP-NF): 60% casein, 5% fat; high protein-high fat (HP-HF): 60% casein, 17% fat. Wheat starch was used for adjustment of energy content. After 4 days, overnight EE and oxygen consumption, as measured by indirect calorimetry, were higher and body-weight gain was lower in rats fed with HP diets as compared with rats fed diets with adequate protein content (P<.05). Exchanging carbohydrates by protein increased fat oxidation in HF diet fed groups. The UCP1 mRNA expression in brown adipose tissue was not significantly different in HP diet fed groups as compared with AP diet fed groups. Expression of different homologues of UCPs positively correlated with nighttime oxygen consumption and EE. Moreover, dietary protein and fat distinctly influenced liver UCP2 and skeletal muscle UCP3 mRNA expressions. These findings demonstrated that a 4-day ad libitum high dietary protein exposure influences energy balance in rats. A function of UCPs in energy balance and dissipating food energy was suggested. Future experiments are focused on the regulation of UCP gene expression by dietary protein, which could be important for body-weight management.