Human Protein Metabolism Research Articles

Compartmental analysis: a new approach to estimate protein breakdown and meal response in health and critical illness.

This study aimed to discuss the use of the pulse stable isotope tracer approach to study changes in metabolism in healthy individuals and critically ill patients. We found that in the postabsorptive state and healthy condition, intracellular protein breakdown and net intracellular protein breakdown, when calculated using the pulse tracer approach, are about double what has previously been reported using the more traditional primed-constant and continuous stable isotope approaches (600 versus 300 grams of protein/day). In critically ill patients, protein breakdown is even higher and calculated to be approximately 900 grams of protein/day, using the pulse tracer approach. Based on these data, we hypothesize that reducing protein breakdown in the postabsorptive state is key when trying to improve the condition of critically ill patients. Moreover, we also used the pulse tracer approach during feeding to better estimate the intracellular metabolic response to feeding. Our first observation is that endogenous protein breakdown does not seem to be reduced during feeding. We also have shown that when consuming a meal with a certain amount of protein, the biological value of that protein meal can be calculated with the pulse tracer approach. In conclusion, using the pulse stable isotope tracer approach to study protein kinetics in the postabsorptive state and during feeding expands our understanding of how dietary proteins can affect human protein metabolism. The intracellular protein synthesis stimulatory effect of a meal is an important factor to consider when calculating the exact protein requirements and needs, particularly in critical illness.

Comparison of the precursor, amino acid oxidation, and end-product methods for the evaluation of protein turnover in senior dogs.

Stable isotope methods have been used to study protein metabolism in humans; however, there application in dogs has not been frequently explored. The present study compared the methods of precursor (13C-Leucine), end-products (15N-Glycine), and amino acid oxidation (13C-Phenylalanine) to determine the whole-body protein turnover rate in senior dogs. Six dogs (12.7 ± 2.6 years age, 13.6 ± 0.6 kg bodyweight) received a dry food diet for maintenance and were subjected to all the above-mentioned methods in succession. To establish 13C and 15N kinetics, according to different methodologies blood plasma, urine, and expired air were collected using a specifically designed mask. The volume of CO2 was determined using respirometry. The study included four methods viz. 13C-Leucine, 13C-Phenylalanine evaluated with expired air, 13C-Phenylalanine evaluated with urine, and 15N-Glycine, with six dogs (repetitions) per method. Data was subjected to variance analysis and means were compared using the Tukey test (P<0.05). In addition, the agreement between the methods was evaluated using Pearson correlation and Bland-Altman statistics. Protein synthesis (3.39 ± 0.33 g.kg-0,75. d-1), breakdown (3.26 ± 0.18 g.kg-0.75.d-1), and flux estimations were similar among the four methods of study (P>0.05). However, only 13C-Leucine and 13C-Phenylalanine (expired air) presented an elevated Pearson correlation and concordance. This suggested that caution should be applied while comparing the results with the other methodologies.

Combined enteral infusion of glutamine, carbohydrates, and antioxidants modulates gut protein metabolism in humans

Background:Available data suggest that nutrients can affect intestinal protein metabolism, which contributes to the regulation of gut barrier function. Objective:We aimed to assess whether an oral nutritional supplement (ONS) containing glutamine (as the dipeptide Ala-Gln), carbohydrates, and antioxidants would modulate duodenal protein metabolism in healthy humans. Design:Thirty healthy control subjects were included and, over a period of 5 h, received by nasogastric tube either saline or ONS providing 11.7 kcal/kg as 0.877 g Ala-Gln/kg, 3.9 g carbohydrates/kg, and antioxidants (29.25 mg vitamin C/kg, 9.75 mg vitamin E/kg, 195 μg β-carotene/kg, 5.85 mg Se/kg, and 390 μg Zn/kg) or glutamine (0.585 g/kg, 2.34 kcal/kg). Simultaneously, a continuous intravenous infusion of l-[1-13C]-leucine was done until endoscopy. Leucine enrichment was assessed by using gas chromatography–mass spectrometric analysis, and mucosal fractional synthesis rate was calculated by using intracellular amino acid enrichment as precursor. Mucosal proteolytic pathways were also evaluated. Results:ONS infusion resulted in a doubling increase (P < 0.01) of duodenal fractional synthesis rate and a significant (P < 0.05) decrease in cathepsin D–mediated proteolysis compared with saline, whereas proteasome and Ca2+-dependent activities were unaffected. ONS infusion significantly (P < 0.01) decreased duodenal glutathione but not glutathione disulfide concentrations or the ratio of glutathione to glutathione disulfide. Insulinemia increased after ONS infusion, whereas plasma essential amino acids decreased. Infusion of glutamine alone did not reproduce ONS effects. Conclusions:ONS infusion improves duodenal protein balance in healthy humans. Further investigations are needed to study the origin of these effects and to evaluate ONS supply in stressed persons.

Production of Large Amounts of [13C]Leucine-Enriched Milk Proteins by Lactating Cows

During protein metabolism kinetic studies, oral tracers are administered as labeled free amino acids and do not necessarily represent the metabolic fate of amino acids ingested as proteins. However, sufficient quantities of 13C-labeled proteins are not currently available. We here present a new methodology for producing large amounts of milk proteins intrinsically labeled with [13C]leucine. After surgical preparation, two lactating cows were infused with 80–90 g of L-[1-13C]leucine for 24–32 h, and milk was collected during and after the infusion. Casein and whey protein fractions were purified by membrane separation techniques. Arteriovenous balance across the udder indicated a very efficient extraction of leucine by the mammary gland. Five batches of pure casein and whey proteins, totaling 3854 g of protein of excellent bacteriological quality, were obtained. Two thirds of these proteins had [13C]leucine enrichments ranging from 10.5 to 19.4% ([13C]leucine atom percent excess). The overall tracer recoveries were 22 and 27% (cows 1 and 2, respectively). Thus, pure milk proteins were produced in large amounts with sufficient 13C enrichment to be used in human protein metabolism studies.

Dileucine ingestion is more effective than leucine in stimulating muscle protein turnover in young males: a double blind randomized controlled trial.

Leucine is regarded as an anabolic trigger for the mTORC1 pathway and the stimulation muscle protein synthesis rates. More recently, there has been an interest in underpinning the relevance of branched-chain amino acid (BCAA)-containing dipeptides and their intact absorption into circulation to regulate muscle anabolic responses. We investigated the effects of dileucine and leucine ingestion on postprandial muscle protein turnover. Ten healthy young men (age: 23 ± 3 yr) consumed either 2 g of leucine (LEU) or 2 g of dileucine (DILEU) in a randomized crossover design. The participants underwent repeated blood and muscle biopsy sampling during primed continuous infusions of l-[ring-13C6]phenylalanine and l-[15N]phenylalanine to determine myofibrillar protein synthesis (MPS) and mixed muscle protein breakdown rates (MPB), respectively. LEU and DILEU similarly increased plasma leucine net area under the curve (AUC; P = 0.396). DILEU increased plasma dileucine AUC to a greater extent than LEU (P = 0.013). Phosphorylation of Akt (P = 0.002), rpS6 (P < 0.001), and p70S6K (P < 0.001) increased over time under both LEU and DILEU conditions. Phosphorylation of 4E-BP1 (P = 0.229) and eEF2 (P = 0.999) did not change over time irrespective of condition. Cumulative (0-180 min) MPS increased in DILEU (0.075 ± 0.032%·h-1), but not in LEU (0.047 ± 0.029%·h-1; P = 0.023). MPB did not differ between LEU (0.043 ± 0.030%·h-1) and DILEU conditions (0.051 ± 0.027%·h-1; P = 0.659). Our results showed that dileucine ingestion elevated plasma dileucine concentrations and muscle protein turnover by stimulating MPS in young men.NEW & NOTEWORTHY The role of dipeptides as anabolic agents remains unresolved in humans. We show that the ingestion of 2 g dileucine increased plasma dileucine concentrations and resulted in an enhancement of muscle protein turnover by stimulating an increase in muscle protein synthesis rates in healthy young males. The ingestion of 2 g leucine, however, did not stimulate an increase in muscle protein turnover. Our work provides the first insights into the effects of dipeptides on human protein metabolism.

Equivalent servings of free-range reindeer promote greater net protein balance compared to commercial beef

ABSTRACT Wild game consumption has been associated with health benefits but the acute influence on human protein metabolism remains unknown. We compared feeding-induced responses of equivalent amounts of free-range reindeer (FR) and commercial beef (CB) on protein kinetics using stable isotope methodology. Seven participants (age: 40 ± 14 years; body mass index: 24 ± 3 kg/m2) completed two randomised studies, ingesting 2 oz of FR or CB. L-[ring 2H5]phenylalanine & L-[ring 2H2]tyrosine were delivered via primed, continuous intravenous infusion. Blood samples were collected during the basal period and following consumption of FR or CB. Feeding-induced changes in whole-body protein synthesis (PS), protein breakdown (PB), and net protein balance (NB) were determined via plasma sample isotope enrichment analysis by gas chromatography-mass spectrometry; plasma essential amino acid (EAA) concentrations were determined by liquid chromatography-electrospray ionisation-mass spectrometry. Plasma post-prandial EAA concentrations were higher with FR compared to CB (P < 0.05). The acute feeding-induced PS response was not different, but PB was reduced and contributed to a superior level of NB (P < 0.00001) in FR compared to CB. Our results demonstrate that FR may influence more favourable protein metabolism than CB. These data support potential health benefits of wild game onf whole-body protein. Abbreviations: BMI: body mass index; DIAAS: digestible indispensable amino acid score; CB: commercial beef; EAA: essential amino acids; FR: free-range reindeer; Ra: rate of appearance; UAF: University of Alaska Fairbanks; USDA: USA Department of Agriculture

Superior Influence of Reindeer compared to Beef Ingestion on Protein Metabolism in Humans

Wild game consumption has been associated with health benefits, but the immediate influence on protein metabolism remains unknown. In order to address the direct effects on protein kinetics, we compared the feeding‐induced response to free‐range reindeer versus commercial beef using stable isotope methodology in humans. Eight male and female participants (age: 38±12 years; body mass index: 24±3 kg/m2) completed two studies using a randomized, crossover design in which they ingested reindeer or beef. L‐[2H5]phenylalanine &amp; L‐[2H2]tyrosine were delivered via primed, continuous intravenous infusion. Blood samples were collected during a 3‐hour basal period and for 4 hrs after consumption of reindeer and beef. A priming dose of L‐[2H4]tyrosine was also delivered. Feeding‐induced changes in whole body protein synthesis (PS), protein breakdown (PB), and net protein balance (NB) were determined via analysis of plasma samples by liquid chromatography‐electrospray ionization‐tandem mass spectrometry (LC‐ESI‐MS). Post‐prandial essential amino acid (EAA) concentrations were higher with reindeer ingestion (P=0.02). PS was not different but PB was reduced with reindeer compared to beef ingestion (P=0.02), contributing to superior level of NB (P=0.001). Reindeer promoted higher plasma EAA levels and NB compared to beef. The amino acid and fatty acid profiles of reindeer may have an immediate and greater benefit on protein metabolism as compared to beef. These data support the potential health benefits of wild game in the preservation of whole‐body protein.Support or Funding InformationResearch reported in this publication was supported by an Institutional Development Award (IDeA) under grant number P20GM103395 and the Biomedical Learning and Student Training Program (UL1GM118991, TL4GM118992, or RL5GM118990) from the National Institute of General Medical Sciences from the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

A four-compartment compartmental model to assess net whole body protein breakdown using a pulse of phenylalanine and tyrosine stable isotopes in humans.

The stable isotopes of phenylalanine (Phe) and tyrosine (Tyr) are often used to study whole body protein metabolism in humans. Noncompartmental approaches give limited physiological insight in the compartmental characteristics. We therefore developed a compartmental mathematical model of Phe/Tyr metabolism to describe protein fluxes by using stable tracer dynamic data in plasma following intravenous bolus of l-[ring-13C6]Phe and l-[ring-2H4]Tyr in healthy subjects. The model consists of four compartments describing Phe/Tyr kinetics. Because the model is a priori nonidentifiable, it is quantified in terms of two uniquely identifiable submodels representing two limit case scenarios, based on known physiology. The two submodels, identified by using the software SAAM II, fit well the experimental data of all individuals and provide an unbiased overview of the metabolic pathway in terms of intervals of validity of the non-uniquely identifiable variables. The model provides estimates of the flux from Phe to Tyr [4.1 ± 1.0 µmol·kg fat-free mass (FFM)-1·h-1 (mean ± SE)] and intervals of validity of the flux and pool estimates. Our preferred submodel yielded protein breakdown flux (50.5 ± 5.2 µmol·kg FFM-1·h-1), net protein breakdown (4.1 ± 1.0 µmol·kg FFM-1·h-1), Tyr from Phe hydroxylation (~12%), hydroxylated Phe (~8%), and flux ratio of Tyr to Phe arising from protein catabolism (0.68), consistent with available literature. The other submodel suggest that the assumptions made by noncompartmental analysis are consistently underestimated. Our accurate and detailed model for estimating Phe/Tyr metabolic pathways in humans might be essential to applications in a variety of scenarios describing whole body protein synthesis and breakdown in health and disease.

Development of Intrinsically Labeled Eggs and Poultry Meat for Use in Human Metabolic Research

Background: Stable isotope amino acids are regularly used as tracers to examine whole-body and muscle protein metabolism in humans. To accurately assess in vivo dietary protein digestion and absorption kinetics, the amino acid tracer is required to be incorporated within the dietary protein food source (i.e., intrinsically labeled protein).Objective: We assessed the practicality of producing eggs and poultry meat intrinsically labeled with L-[5,5,5-2H3]leucine through noninvasive oral tracer administration.Methods: A specifically formulated diet containing 0.52% leucine was supplemented with 0.3% L-[5,5,5-2H3]leucine and subsequently fed to 3 laying hens (Lohmann LSL Whites) for 55 d. On day 55, the hens were slaughtered and their meat, bones, and organs were harvested to determine tissue labeling. In Expt. 1, 2 healthy young men [mean ± SEM age: 22 ± 1.5 y; mean ± SEM body mass index (BMI; in kg/m2): 23.7 ± 0.5] ingested 18 g L-[5,5,5-2H3]leucine–labeled egg protein. In Expt. 2, 2 healthy young men (mean ± SEM age: 20.0 ± 0.0 y; mean ± SEM BMI: 26.4 ± 3.1) ingested 28 g L-[5,5,5-2H3]leucine–labeled poultry meat protein. Plasma samples (Expts. 1 and 2) and muscle biopsies (Expt. 1) were collected before and after labeled-food ingestion.Results: High tracer labeling [>20 mole percent excess (MPE)] in the eggs was obtained after 7 d and maintained throughout the feeding protocol (P < 0.05). Over a 55-d period, ∼850 g egg protein (145 eggs) was produced, with a mean ± SEM tracer enrichment of 22.0 ± 0.8 MPE. Mean ± SEM L-[5,5,5-2H3]leucine enrichment in the meat was 9.6 ± 0.1 MPE. In Expts. 1 and 2, the consumption of labeled eggs and poultry meat protein increased plasma L-[5,5,5-2H3]leucine enrichment, with mean ± SEM peak values of 6.7 ± 0.1 MPE and 4.0 ± 0.9 MPE, respectively. The mean ± SEM 5-h postprandial increase in myofibrillar L-[5,5,5-2H3]leucine enrichment after egg ingestion in healthy young men was 0.051 ± 0.008 MPE (Expt. 1).Conclusion: We demonstrated the feasibility of producing intrinsically labeled eggs and poultry meat for use in human metabolic research.

NOVEL INSIGHTS ON INTAKE OF MEAT AND PREVENTION OF SARCOPENIA: ALL REASONS FOR AN ADEQUATE CONSUMPTION.

sarcopenia is defined as a syndrome characterized by progressive and generalized loss of muscle mass and strength. The main cause of sarcopenia is the alteration of protein metabolism, in which the proteolytic processes are not accompanied by an appropriate protein synthesis and muscle cells lose progressively the sensitivity to the anabolic stimulus. The most rational approach to delay the progression of sarcopenia and counteract the anabolic resistance is proper nutrition. Meat contains biologically active compounds, such as creatine, carnitine, Conjugated Linoleic Acid (CLA) which have significant impacts upon human protein metabolism. we performed a narrative literature review to evaluate the till-now evidence regarding: 1. adequate intake of meat in elderly as a topic for prevention of sarcopenia; 2. the correct intake of biologically active compounds contain in meat, which have significant impacts upon human protein metabolism and so have beneficial effects on prevention of sarcopenia. This review included 62 eligible studies. the results demonstrated that in elderly the optimum diet therapy for the sarcopenia prevention and treatment, which must aim at achieving specific metabolic goals, must recommend the consumption of 113 g of meat (220 kcal; 30 g protein) five time a week. in a varied and balanced diet, for preventing sarcopenia, it is recommended to assume meat 4-5 times a week (white meat 2 times per week, lean red meat less than 2 times per week, processed meat less than 1 time per week), as suggested in the diet pyramid for elderly.

Abstract 5363: Genotoxicity of trace level of hexavalent chromium existing in city water

Abstract Tens of millions of Americans consume trace amounts of potentially toxic hexavalent chromium (Cr(VI)) through drinking city water every day. According to the studies conducted by the Environmental Working Group, 89% of the water samples from U.S. cities are contaminated with Cr(VI) at levels ranging from 0.03 to 12.9 μg/L. While trivalent chromium (Cr(III)) is among the essential trace elements for normal carbohydrate, lipid, and protein metabolism in humans, Cr(VI) has been extensively studied as a toxic heavy metal ion. Cr(VI) is a well-known human carcinogen by inhalation; in contrast, it is a rodent carcinogen by ingestion of extremely high doses (57,300 μg/L or higher) in drinking water. The United States Environmental Protection Agency (USEPA) and other regulatory agencies in the world have set the maximum contaminant levels (MCL) for total Cr or Cr(VI) in human drinking water at 50 or 100 μg/L, although the USEPA is currently re-evaluating these regulations. The mechanism by which Cr(VI) causes genotoxicity is well-characterized at concentrations equivalent to current regulatory levels or higher. The genotoxic mode of action of Cr(VI) appears to be due to the intracellular reduction of Cr(VI) to Cr(III) leading to the formation of a ternary complex between DNA, Cr(III), and intracellular reducing molecules. However, little information is available regarding the biological effects of Cr(VI) at doses lower than 100 μg/L. In this study, we investigated the effects of Cr(VI) at nanomolar ranges (1 μg/L or higher) on the DNA damage response in isogenic cultured cells deficient in various DNA repair genes. Here we show that the DNA damage response is observed after a three-day incubation with Cr(VI) at 2-5 μg/L (ppb), which is lower than the Cr(VI) concentrations detected in some of city water in the US. Strikingly, the genotoxicity of Cr(VI) decreased ∼3000 times depending on its incubation time, ranging from three days to ten minutes. Our dose- and time-dependent Cr(VI) genotoxicity results, combined with the estimated transit time of Cr(VI) in the digestive tracts of humans, strongly suggest that trace amounts of Cr(VI) in drinking water at levels 12.9 μg/L or lower are unlikely to cause a significant impact on inducing carcinogenicity in the oral cavity and small intestine. Citation Format: Xu Tian, Keyur Patel, John R. Ridpath, James A. Swenberg, Jun Nakamura. Genotoxicity of trace level of hexavalent chromium existing in city water. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5363. doi:10.1158/1538-7445.AM2014-5363

The crystal structure of human Aldehyde Oxidase in native and inhibited forms

Aldehyde oxidases (AOX; E.C. 1.2.3.1) are molybdo-flavoenzymes with broad substrate specificity, oxidizing aldehydes and N-heterocycles. AOX belongs to the xanthine oxidase (XO) family of Mo-containing enzymes. The true physiological function of AOX is still unknown, although it is recognized to play a role in the metabolism of compounds with medicinal and toxicological relevance [1]. AOX importance has increased in recent years since it is substituting Cyt-P450 as the central drug-metabolizing system in humans. We have solved the 3D structure of mouse AOX3 to 2.9 Å resolution [2] that was the first structure of an aldehyde oxidase, providing important evidences on substrate and inhibitor specificities between AOX and XO. The complement of AOX proteins in mammals varies from one in humans (hAOX1) to four in rodents (mAOX1, mAOX3, mAOX4 and mAOX3L1) as a result of evolutionary genetic events. Due to this unusual complement of AOX genes in different animal species, conclusions regarding protein metabolism in humans cannot be taken exclusively from the mouse model. Using the human aldehyde oxidase (hAOX1) purified after heterologous expression in Escherichia coli we were able to crystallize it and solve its 3D structure to 2.7 Å resolution (submitted). In addition to the native protein we also solved the structure of an inhibited form of the enzyme to 2.6Å resolution. Analysis of the protein active site and comparison with the structure of the mouse isoform (mAOX3) allowed us to identity, for the first time, the unique features that characterize hAOX1 as an important drug-metabolizing enzyme. In spite of the similarities of both enzymes, they show marked and relevant differences at the Mo active site, substrate tunnel as well as at the FAD site. The ensemble of these structures provides important insights into the role of aldehyde oxidases, contributing to elucidate the clinical metabolism implications of hAOX1 in humans which has particular relevance for novel drug design studies.

Determination of chromium(III) in aqueous solution using CePO4:Tb3+ nanocrystals in a fluorescence resonance energy transfer system

Trivalent chromium is an essential element required for normal carbohydrate, lipid and protein metabolism in humans and animals. This article describes an efficient fluorescence resonance energy transfer (FRET) system between CePO4 :Tb(3+) nanocrystals as the donor and chromium(III) as the acceptor. CePO4 :Tb(3+) nanocrystals were synthesized in aqueous solution, and characterized by transmission electron microscopy. Under optimum conditions, a linear calibration graph was obtained (R(2) = 0.996). The linear range and detection limit of chromium(III) were 0.01-2.2 μM, and 9.1 nM, respectively. The proposed method had a wide linear range and proved to be very sensitive, rapid and simple. Moreover, the method was applied successfully to the determination of chromium(III) in synthetic samples and tap water.

Enteral delivery of proteins stimulates protein synthesis in human duodenal mucosa in the fed state through a mammalian target of rapamycin–independent pathway

Glutamine modulates duodenal protein metabolism in fasted healthy humans, but its effects in a fed state remain unknown. We aimed to assess the effects of either glutamine or an isonitrogenous protein mixture on duodenal protein metabolism in humans in the fed state. Twenty-four healthy volunteers were randomly included in 2 groups. Each volunteer was studied on 2 occasions in a random order and received, during 5 h, either an enteral infusion of maltodextrins alone (0.25 g · kg⁻¹ · h⁻¹; both groups) that mimicked a carbohydrate fed state or maltodextrins with glutamine (group 1) or an isonitrogenous (22.4 mg N · kg⁻¹ · h⁻¹) protein powder (group 2). Simultaneously, a continuous intravenous infusion of ¹³C-leucine and ²H₅-phenylalanine (both 9 μmol · kg⁻¹ · h⁻¹) was performed. Endoscopic duodenal biopsies were taken. Leucine and phenylalanine enrichments were assessed by using gas chromatography-mass spectrometry in duodenal proteins and the intracellular free amino acids pool to calculate the mucosal fractional synthesis rate (FSR). Proteasome proteolytic activities and phosphokinase expression were assessed by using specific fluorogenic substrates and macroarrays, respectively. The FSR and proteasome activity were not different after the glutamine supply compared with after maltodextrins alone. In contrast, the FSR increased (1.7-fold increase; P < 0.05) after protein-powder delivery without modification of total proteasome activity. The protein powder increased insulinemia, PI3 kinase, and erk phosphorylation but did not affect the mammalian target of rapamycin (mTOR) pathway and mitogen-activated protein kinase signal-integrating kinase 1 phosphorylation. A trend for an increase of eukaryotic translation initiation factor 4E phosphorylation was observed (P = 0.07). In the carbohydrate fed state, enteral proteins but not glutamine increased duodenal protein synthesis through an mTOR independent pathway in humans.

Dietary omega-3 fatty acid supplementation increases the rate of muscle protein synthesis in older adults: a randomized controlled trial

Loss of muscle mass with aging is a major public health concern. Omega-3 (n-3) fatty acids stimulate protein anabolism in animals and might therefore be useful for the treatment of sarcopenia. However, the effect of omega-3 fatty acids on human protein metabolism is unknown. The objective of this study was to evaluate the effect of omega-3 fatty acid supplementation on the rate of muscle protein synthesis in older adults. Sixteen healthy, older adults were randomly assigned to receive either omega-3 fatty acids or corn oil for 8 wk. The rate of muscle protein synthesis and the phosphorylation of key elements of the anabolic signaling pathway were evaluated before and after supplementation during basal, postabsorptive conditions and during a hyperaminoacidemic-hyperinsulinemic clamp. Corn oil supplementation had no effect on the muscle protein synthesis rate and the extent of anabolic signaling element phosphorylation in muscle. Omega-3 fatty acid supplementation had no effect on the basal rate of muscle protein synthesis (mean ± SEM: 0.051 ± 0.005%/h compared with 0.053 ± 0.008%/h before and after supplementation, respectively; P = 0.80) but augmented the hyperaminoacidemia-hyperinsulinemia-induced increase in the rate of muscle protein synthesis (from 0.009 ± 0.005%/h above basal values to 0.031 ± 0.003%/h above basal values; P < 0.01), which was accompanied by greater increases in muscle mTOR(Ser2448) (P = 0.08) and p70s6k(Thr389) (P < 0.01) phosphorylation. Omega-3 fatty acids stimulate muscle protein synthesis in older adults and may be useful for the prevention and treatment of sarcopenia. This trial was registered at clinical trials.gov as NCT00794079.

Combined enteral infusion of glutamine, carbohydrates, and antioxidants modulates gut protein metabolism in humans

Combined enteral infusion of glutamine, carbohydrates, and antioxidants modulates gut protein metabolism in humans

Lack of Effect of Acute Enteral Arginine Infusion on Whole-Body and Intestinal Protein Metabolism in Humans

Arginine is a conditionally essential amino acid and exerts anabolic effects. We studied the effects of enteral arginine on whole-body and duodenal protein metabolism. Eight healthy fasted volunteers received randomly a 5-hr enteral infusion of either arginine (Arg; 20 g) or an isonitrogenous amino acid mixture (AA) and an IV infusion of [13C]leucine. Duodenal biopsies were performed. Whole-body protein turnover and duodenal protein synthesis (FSR) were calculated from GC/MS-assessed enrichment. The mRNA levels for major components of proteolytic pathways, ubiquitin, cathepsin D, and m-calpain, were evaluated by RT-PCR. Results were compared using paired Wilcoxon test. Endogenous, oxidative, and nonoxidative leucine fluxes were not different after Arg and AA infusions, respectively. Duodenal mucosal protein FSR (71% +/- 26% vs 81% +/- 30%/day) and mRNA levels of ubiquitin, cathepsin D, and m-calpain were also similar after Arg and AA infusions. We conclude that in healthy subjects, arginine infusion exerts no effect on whole-body and duodenal protein metabolism. Whether arginine might specifically affect these parameters in catabolic or inflammatory situations remains to be determined.

Contribution of Research with Farm Animals to Protein Metabolism Concepts: A Historical Perspective1

The roles of proteins, carbohydrates, fats, and micronutrients in animal and human nutrition were broadly described during the late 18th and 19th centuries, and knowledge in protein nutrition evolved from work with all species. Although much of the fundamental and theoretical research in protein metabolism during the 20th century was conducted with laboratory animals, basic protein nutrition research in farm animals complemented those efforts and led to the development and use of new investigative methods (particularly in amino acid nutrition) as well as use of animal models in furthering the understanding of human protein metabolism. All these efforts have led to a contemporary hybrid model of protein nutrition and metabolism applicable to both humans and animal species. Now in the 21st century, farm animals are used in fetal and pediatric nutrition research, and data accruing for excess amino acid feeding in research with farm animals provide direction for assessment of pharmacological effects of amino acids when consumed in excessive quantities. Thus, as nutritional science is moving forward into nutrigenomics, nutriproteomics, and metabolomics, farm animal and human nutrition research interactions will likely continue with genetically modified farm animals produced for agricultural reasons (improved function and product quality) or those produced with human genes introduced to generate even better models of human protein metabolism.

Why muscle stops building when it's working

About 25 years ago some colleagues and I carried out long and complicated studies on ourselves to attempt to find out what happened to human protein metabolism during exercise (Rennie et al. 1981). We dosed ourselves with 15N-glycine and measured the production of labelled ammonia in urine before and during nearly 4 h of uphill walking on a treadmill and during the 24 h afterwards. What we found suggested that whole-body protein synthesis was depressed during exercise and that although there was an increase in the utilization of protein as a metabolic fuel this was almost insignificant compared with the use of fat and carbohydrate. We also found some evidence, somewhat to our surprise, that muscle protein breakdown was not increased during exercise but we lacked the appropriate techniques to make direct measurements of protein synthesis and breakdown in human muscle although that did not stop us speculating that muscle protein turnover was probably depressed during exercise: teleologically, muscle's metabolic machinery had more important things to do than worry about maintenance during contractile activity. (In fact no convincing fall in human muscle protein synthesis has yet been demonstrated during resistance exercise – probably for technical reasons although Bob Wolfe and colleagues did show that treadmill walking at about 40% of V˙O2max resulted in a downward trend in quadriceps protein synthesis (Carraro et al. 1990). Nevertheless measurements made over periods of time which include a period of exercise and a period post-exercise, suggest that the average rate of protein synthesis is depressed (Cuthbertson et al. 2002). In the middle 1980's Ann-Christin Bylund-Fellenius, on sabbatical with Jim Jefferson at Penn State, did experiments in perfused rat hind limb demonstrating that contractile activity was associated with marked depression of muscle protein synthesis and that the effect appeared to be associated with a fall in the ATP/ADP ratio (Bylund-Fellenius et al. 1984). This suggested that muscular contraction imposed some energetic limitation – or at least flipped some allosteric switches which might turn off protein synthesis. However, the rapid report by Rose et al. (2005) in this issue of The Journal of Physiology suggests a much more direct and elegant explanation for why muscle protein synthesis would fall during exercise. What Rose has demonstrated is that one of the factors which controls the rate of translation of pre-existing mRNA in muscle – the eukaryotic elongation factor eEF2 – shows increased phosphorylation (indicating decreased activity) in human muscle during dynamic exercise; the effect is apparently not mediated via what Graham Hardie, its discoverer, has described as the fuel gauge of the cell, AMP kinase but instead by a Ca2+–calmodulin-dependent process activating eEF2 kinase. This is a more direct way of shutting off protein synthesis than the mechanism which would operate through alteration of the ATP/ADP ratio. It is a pity that the authors did not simultaneously measure protein synthesis (which with modern methods is now feasible) during their studies and indeed, nowadays, I would submit that it is becoming mandatory that studies of signalling should include measurement of the physiological readout as well as indirect measures of signalling activity such as phosphorylation. Nevertheless, these results raise a number of interesting questions such as what other control mechanisms in muscle protein turnover are regulated by Ca2+–calmodulin-dependent processes? Do any of the processes stay switched off immediately after exercise even though there is an immediate fall in calcium and presumably a decrease in the Ca2+–calmodulin activity as sarcoplasmic calcium concentration falls? What about gene transcription? We are in the middle of a very exciting period of dissection of the control mechanisms of muscle protein metabolism which ought to lead us to a better understanding of alterations in composition (myosin type variation, mitochondrial biogenesis, anabolic hypertrophy, etc.). This work, although of major interest to athletes and sports scientists, is likely to have its pay-off in identifying therapeutic targets for drugs which could revolutionize treatment of muscle wasting in a variety of acute and chronic diseases.

Effect of Short-Term Prednisone Use on Blood Flow, Muscle Protein Metabolism, and Function

Glucocorticoids can cause muscle atrophy, but the effect on muscle protein metabolism in humans has not been adequately studied to know whether protein synthesis, breakdown, or both are altered. We tested the effect of 6 d of oral prednisone (Pred, 0.5 mg/kg.d) on muscle protein metabolism and function. Six healthy subjects (three men/three women, 22-41 yr) completed two trials (randomized, double-blind, cross-over) with Pred and placebo. Fasting glucose, insulin, IGF-I, and glucagon were higher on Pred vs. placebo, whereas IGF-II and IGF binding protein-1 and -2 were lower. Whole-body amino acid fluxes, blood urea nitrogen, and urinary nitrogen loss were not statistically different between trials. Leg blood flow was 25% lower on Pred leading to 15-30% lower amino acid flux among the artery, vein, and muscle. However, amino acid net balance and rates of protein synthesis and breakdown were unchanged, as were synthesis rates of total mixed, mitochondrial, sarcoplasmic, and myosin heavy chain muscle proteins. Muscle mitochondrial function, muscle strength, and resting energy expenditure were also unchanged. These results demonstrate that a short-term moderate dose of prednisone affects glucose metabolism but has no effect on whole-body or leg muscle protein metabolism or muscle function.