Effects of a Low-Protein Diet on Plasma Amino Acid and Homocysteine Levels and Oxidative Status in Rats

Background/Aims: Transmethylation reactions and antioxidant metabolism are linked by transsulfuration, where homocysteine (Hcy) is converted to cysteine and reduced glutathione (GSH). Low protein intake can modulate the balance of this metabolic reaction. The aim of the present investigation was to study the effect of a low-protein diet on Hcy metabolism by monitoring levels of the amino acids involved in these pathways, and relating these levels to GSH levels and lipid peroxidation in rats. Methods: Sixteen rats were divided into 2 groups: control (C; standard AIN-93 diet, 20% protein) and low-protein diet (LPD; 8% protein diet). Rats in both groups were placed on the diets for 28 days. Results: A significant reduction (p < 0.05) in plasma Hcy concentration was found in LPD rats (0.16 +/- 0.04 mu mol/mg protein) versus C rats (0.25 +/- 0.03 mu mol/mg protein). Methionine levels were not significantly different between the 2 groups (C: 1.24 +/- 0.22 mu mol/mg protein; LPD: 1.03 +/- 0.27 mu mol/mg protein). A significant reduction (p ! 0.05) in hepatic GSH concentrations (C: 44 8 10 mu mol/mg protein; LPD: 17.4 +/- 4.3 mu mol/mg protein) was accompanied by an increase in lipid peroxidation (C: 0.13 +/- 0.01 mu mol/mg protein; LPD: 0.17 +/- 0.02 mu mol/mg protein; r = -0.62, p < 0.01). Conclusion: Hcy levels were reduced under a low-protein diet, resulting in modulated methyl balance and reduced GSH formation leading to increased susceptibility of hepatic cells to oxidative events. Copyright (C) 2009 S. Karger AG, Basel

Amino acid identity and/or position determines the proteasomal cleavage of the HLA-A*0201-restricted peptide tumor antigen MAGE-3271-279.

The proteasome plays a crucial role in the proteolytic processing of antigens presented to T cells in the context of major histocompatibility complex class I molecules. However, the rules governing the specificity of cleavage sites are still largely unknown. We have previously shown that a cytolytic T lymphocyte-defined antigenic peptide derived from the MAGE-3 tumor-associated antigen (MAGE-3(271-279), FLWGPRALV in one-letter code) is not presented at the surface of melanoma cell lines expressing the MAGE-3 protein. By using purified proteasome and MAGE-3(271-279) peptides extended at the C terminus by 6 amino acids, we identified predominant cleavages after residues 278 and 280 but no detectable cleavage after residue Val(279), the C terminus of the antigenic peptide. In the present study, we have investigated the influence of Pro(275), Leu(278), and Glu(280) on the proteasomal digestion of MAGE-3(271-285) substituted at these positions. We show that positions 278 and 280 are major proteasomal cleavage sites because they tolerate most amino acid substitutions. In contrast, the peptide bond after Val(279) is a minor cleavage site, influenced by both distal and proximal amino acid residues.

Proline- and acidic amino acid-rich basic leucine zipper proteins modulate peroxisome proliferator-activated receptor alpha (PPAR alpha) activity.

In mammals, many aspects of metabolism are under circadian control. At least in part, this regulation is achieved by core-clock or clock-controlled transcription factors whose abundance and/or activity oscillate during the day. The clock-controlled proline- and acidic amino acid-rich domain basic leucine zipper proteins D-site-binding protein, thyrotroph embryonic factor, and hepatic leukemia factor have previously been shown to participate in the circadian control of xenobiotic detoxification in liver and other peripheral organs. Here we present genetic and biochemical evidence that the three proline- and acidic amino acid-rich basic leucine zipper proteins also play a key role in circadian lipid metabolism by influencing the rhythmic expression and activity of the nuclear receptor peroxisome proliferator-activated receptor α (PPARα). Our results suggest that, in liver, D-site-binding protein, hepatic leukemia factor, and thyrotroph embryonic factor contribute to the circadian transcription of genes specifying acyl-CoA thioesterases, leading to a cyclic release of fatty acids from thioesters. In turn, the fatty acids act as ligands for PPARα, and the activated PPARα receptor then stimulates the transcription of genes encoding proteins involved in the uptake and/or metabolism of lipids, cholesterol, and glucose metabolism.

Discovery of amino acid variants in the human glucose-dependent insulinotropic polypeptide (GIP) receptor: the impact on the pancreatic beta cell responses and functional expression studies in Chinese hamster fibroblast cells.

The two incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), are insulinotropic factors released from the small intestine to the blood stream in response to oral glucose ingestion. The insulinotropic effect of GLP-1 is maintained in patients with Type II (non-insulin-dependent) diabetes mellitus, whereas, for unknown reasons, the effect of GIP is diminished or lacking. We defined the exon-intron boundaries of the human GIP receptor, made a mutational analysis of the gene and identified two amino acid substitutions, A207 V and E354Q. In an association study of 227 Caucasian Type II diabetic patients and 224 matched glucose tolerant control subjects, the allelic frequency of the A207 V polymorphism was 1.1% in Type II diabetic patients and 0.7% in control subjects (p = 0.48), whereas the allelic frequency of the codon 354 polymorphism was 24.9% in Type II diabetic patients versus 23.2% in control subjects. Interestingly, the glucose tolerant subjects (6% of the population) who were homozygous for the codon 354 variant had on average a 14% decrease in fasting serum C-peptide concentration (p = 0.01) and an 11% decrease in the same variable 30 min after an oral glucose load (p = 0.03) compared with subjects with the wild-type receptor. Investigation of the function of the two GIP receptor variants in Chinese hamster fibroblasts showed, however, that the GIP-induced cAMP formation and the binding of GIP to cells expressing the variant receptors were not different from the findings in cells expressing the wildtype GIP receptor. In conclusion, amino acid variants in the GIP receptor are not associated with random Type II diabetes in patients of Danish Caucasian origin or with altered GIP binding and GIP-induced cAMP production when stably transfected in Chinese hamster fibroblasts. The finding of an association between homozygosity for the codon 354 variant and reduced fasting and post oral glucose tolerance test (OGTT) serum C-peptide concentrations, however, calls for further investigations and could suggest that GIP even in the fasting state regulates the beta-cell secretory response.

Effects of exaggerated amino acid and protein supply in man.

A general update review of the dynamic aspect of protein metabolism is presented. The effect of excess protein level on protein metabolism has been the object of a limited number of studies in man. From the information available, it appears that the primary regulatory pathway for body protein homeostasis is the process of amino acid (protein) oxidation.

A specific protein-enriched enteral formula decreases cortisolemia and improves plasma albumin and amino acid concentrations in elderly patients

Background: Old age is associated with an involuntary and progressive but physiological loss of muscle mass. The aim of this study was to evaluate the effects of exclusive consumption for 6 months of a protein-enriched enteral diet with a relatively high content of branched-chain amino acids on albuminemia, cortisolemia, plasma aminoacids, insulin resistance, and inflammation biomarkers in elderly patients. Methods: Thirty-two patients from the Clinical Nutrition Outpatient Unit at our hospital exclusively consumed a protein-enriched enteral diet for 6 months. Data were collected at baseline and at 3 and 6 months on anthropometric and biochemical parameters and on plasma concentrations of amino acids, cortisol,adrenocorticotropic hormone, urea, creatinine, insulin resistance, and inflammation biomarkers. Results: The percentage of patients with albumin concentration below normal cut-off values decreased from 18% to 0% by the end of the study. At 6 months, concentrations of total plasma (p = 0.008) and essential amino acids(p = 0.011), especially branched-chain amino acids (p = 0.031), were higher versus baseline values, whereas 3-methylhistidine (p = 0.001), cortisol (p = 0.001) and adrenocorticotropic hormone (p = 0.004) levels were lower. Conclusions: Regular intake of specific protein-enriched enteral formula increases plasma essential amino acids, especially branched-chain amino acids, and decreases cortisol and 3-methylhistidine, while plasma urea and creatinine remain unchanged.

A trap system for the isolation of amino acid sequences inducing GPI anchor addition.

We designed a trap system to isolate different amino acid sequences which could target proteins to the cell surface via GPI anchor transfer. This selection procedure is based on the insertion of various sequences which regenerate a functional GPI anchor signal sequence and therefore provoke re-expression at the surface of a reporter molecule. Using this trap for cell surface targeting sequences, we could show the importance of the defined elements essential for GPI anchor addition. Such a system could be used for an exhaustive analysis of the carboxyl terminus structural requirements for GPI membrane anchoring.

The complete amino acid sequence for brain beta spectrin (beta fodrin): relationship to globin sequences.

The amino acid sequence of mouse brain beta spectrin (beta fodrin), deduced from the nucleotide sequence of complementary DNA clones, reveals that this non-erythroid beta spectrin comprises 2363 residues, with a molecular weight of 274,449 Da. Brain beta spectrin contains three structural domains and we suggest the position of several functional domains including f-actin, synapsin I, ankyrin and spectrin self association sites. Analysis of deduced amino acid sequences indicated striking homology and similar structural characteristics of brain beta spectrin repeats beta 11 and beta 12 to globins. In vitro analysis has demonstrated that heme is capable of specific attachment to brain spectrin, suggesting possible new functions in electron transfer, oxygen binding, nitric oxide binding or heme scavenging.

Identification of amino acid residues in the alpha, beta, and gamma subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation.

The amiloride-sensitive epithelial Na channel (ENaC) is a heteromultimeric channel made of three alpha beta gamma subunits. The structures involved in the ion permeation pathway have only been partially identified, and the respective contributions of each subunit in the formation of the conduction pore has not yet been established. Using a site-directed mutagenesis approach, we have identified in a short segment preceding the second membrane-spanning domain (the pre-M2 segment) amino acid residues involved in ion permeation and critical for channel block by amiloride. Cys substitutions of Gly residues in beta and gamma subunits at position beta G525 and gamma G537 increased the apparent inhibitory constant (Ki) for amiloride by > 1,000-fold and decreased channel unitary current without affecting ion selectivity. The corresponding mutation S583 to C in the alpha subunit increased amiloride Ki by 20-fold, without changing channel conducting properties. Coexpression of these mutated alpha beta gamma subunits resulted in a non-conducting channel expressed at the cell surface. Finally, these Cys substitutions increased channel affinity for block by external Zn2+ ions, in particular the alpha S583C mutant showing a Ki for Zn2+ of 29 microM. Mutations of residues alpha W582L, or beta G522D also increased amiloride Ki, the later mutation generating a Ca2+ blocking site located 15% within the membrane electric field. These experiments provide strong evidence that alpha beta gamma ENaCs are pore-forming subunits involved in ion permeation through the channel. The pre-M2 segment of alpha beta gamma subunits may form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We propose that amiloride interacts with Na+ ions at an external Na+ binding site preventing ion permeation through the channel pore.

Activities of amino acid metabolizing enzymes in the stomach and small intestitine of developing rats

The activities of aspartate and alanine transaminase, serine dehydratase, arginase, glutamate dehydrogenase, adenylate deaminase and glutamine synthetase were determined in the stomach and small intestine of developing rats. Despite the common embryonic origin of the intestine and stomach, their enzymes showed quite different activity levels and patterns of development, depending on their roles. Most enzyme activities were low during late intrauterine life and after birth, attaining adult levels with the change of diet at weaning. No arginase activity was found in the stomach and no changes were detected in adenylate deaminase in the stomach or intestine throughout the period studied. Alanine transaminase, serine dehydratase and, to some extent, glutamine synthetase levels, significantly higher in late intrauterine life, decreased after birth, suggesting that the foetal stomach has a transient ability to handle amino acids.

Effect of glucose on ureagenesis during exercise in amino acid-infused dogs

The purpose of this study was to investigate the effect of glucose administered with amino acids before and during exercise on hepatic ureagenesis. Eight mongrel dogs subjected to treadmill running for 150 minutes at 10 km/h on a 12% incline were intravenously infused with either a mixture of amino acids and glucose (AAG) or amino acids alone (AA). The infusion was started 60 minutes before exercise and continued until the end of exercise. The rate of urinary urea excretion increased after infusion of both AAG and AA. However, the rate of urinary urea excretion was significantly lower in the AAG group versus the AA group during the first 1.5 hours of the recovery period ([R0 to R90] 514+/-24 v 637+/-24 mg/h, mean+/-SE, P < .05). Moreover, hepatic urea output was decreased during AAG versus AA infusion (229+/-62 v 367+/-55 microg/kg/min, P < .05). Hepatic glucose production during exercise was also significantly lower in AAG versus AA infusion (354+/-54 v 589+/-56 mg/kg, P < .05). On the other hand, no difference was observed in hepatic total amino acid uptake between the groups. Thus, these results indicate that AAG administered before and during exercise appears to reduce hepatic ureagenesis due to reduced hepatic gluconeogenesis as compared with administration of AA alone. These findings also suggest that nitrogen retention is enhanced by glucose administered during exercise.

Adaptation of canine distemper virus to canine footpad keratinocytes modifies polymerase activity and fusogenicity through amino acid substitutions in the P/V/C and H proteins.

The wild-type canine distemper virus (CDV) strain A75/17 induces a non-cytocidal infection in cultures of canine footpad keratinocytes (CFKs) but produces very little progeny virus. After only three passages in CFKs, the virus produced 100-fold more progeny and induced a limited cytopathic effect. Sequence analysis of the CFK-adapted virus revealed only three amino acid differences, of which one was located in each the P/V/C, M and H proteins. In order to assess which amino acid changes were responsible for the increase of infectious virus production and altered phenotype of infection, we generated a series of recombinant viruses. Their analysis showed that the altered P/V/C proteins were responsible for the higher levels of virus progeny formation and that the amino acid change in the cytoplasmic tail of the H protein was the major determinant of cytopathogenicity.

Protein turnover, amino acid requirements and recommendations for athletes and active populations

Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers (13C-lysine, 15N-glycine, ²H5-phenylalanine) and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes) compared to resistance exercise, which induces fiber hypertrophy (myofibrils). Nitrogen balance (difference between protein intake and protein degradation) for athletes is usually balanced when the intake of protein reaches 1.2 g·kg-1·day-1 compared to 0.8 g·kg-1·day-1 in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins) and carbohydrate (± 30 g maltodextrine) drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h.

Amino acid transport : the special case of a H/L-glutamate cotransport system in Asparagus sprengeri mesophyll cells /

The addition of L-Glutamate (L-GLU) and L-Hethionine ~ulfoximine (L-HSO) to mechanically isolated. photosynthetically competent, Asparagus sprengeri mesophyll cells ~u~pended in 1mM CaS04 cau~ed an immediate transient alkalinization of the cell su~pension medium in both the light and dark. The alkalinization response was specific and stereospecific as none of the L-isomers of the other 19 protein amino acids tested or D-GLU gave this response. Uptake of 14C-L-GLU was stimulated by the light. The addition of non-radioactive L-GLU. or L-GLU analogs together with 14C-L-GLU showed that only L-GLU and L-HSO stimulated alkalinization whilst inhibiting the uptake of 14C-L-GLU. Both the L-GLU dependent alkalinization and the upt~ke of 14C-L-GLU were stimulated when the external pH was decreased from 6.5 to 5.5. Increasing external K+ concentrations inhibited the uptake of 14C-L-GLU. Fusicoccin (FC) stimulated uptake. The L-GLU dependent alkalinization re~ponse exhibited monophasic saturation kinetics while the uptake of 14C-L-GLU exhibited biphasic saturation kinetics. In addition to a saturable component. the uptake kinetics also showed a linear component of uptake. Addition of L-GLU and L-MSO caused internal acidification of the cell as measured by a change in the distribution of 14C-DMO. There was no change in K+ efflux when L-GLU was added. A H+ to L-GLUinflux stoichiometry of 3:1 wa~ mea~ured at an external I.-GLU concentration of O.5mM and increased with increasing external 13 L-QLU concentration. Metabolism of L-GLU was detected manometrlcally by observing an increase in COa evolution upon the addition of L-QLU and by detection of i*C02 evolution upon the addition of »*C-L-GLU. »*C02 evolution was higher in the dark than in the light. The data are consistent with the operation of a H+/L-QLO cotransport system. The data also show that attempts to quantify the stoichlometry of the process were complicated by the metabolism of L-GLU.

A model of net amino acid absorption and utilization by the portal-drained viscera of the lactating dairy cow

A more complete understanding of amino acid ( AA) metabolism by the various tissues of the body is required to improve upon current systems for predicting the use of absorbed AA. The objective of this work was to construct and parameterize a model of net removal of AA by the portal-drained viscera (PDV). Six cows were prepared with arterial, portal, and hepatic catheters and infused abomasally with 0, 200, 400, or 600 g of casein daily. Casein infusion increased milk yield quadratically and tended to increase milk protein yield quadratically. Arterial concentrations of a number of essential AA increased linearly with respect to infusion amount. When infused casein was assumed to have a true digestion coefficient of 0.95, the minimum likely true digestion coefficient for noninfused duodenal protein was found to be 0.80. Net PDV use of AA appeared to be linearly related to total supply (arterial plus absorption), and extraction percentages ranged from 0.5 to 7.25% for essential AA. Prediction errors for portal vein AA concentrations ranged from 4 to 9% of the observed mean concentrations. Removal of AA by PDV represented approximately 33% of total postabsorptive catabolic use, including use during absorption but excluding use for milk protein synthesis, and was apparently adequate to support endogenous N losses in feces of 18.4 g/d. As 69% of this use was from arterial blood, increased PDV catabolism of AA in part represents increased absorption of AA in excess of amounts required by other body tissues. Based on the present model, increased anabolic use of AA in the mammary and other tissues would reduce the catabolic use of AA by the PDV.