ABSTRACT
Plant-sourced proteins offer environmental and health benefits, and research increasingly includes them in study formulas. However, plant-based proteins have less of an anabolic effect than animal proteins due to their lower digestibility, lower essential amino acid content (especially leucine), and deficiency in other essential amino acids, such as sulfur amino acids or lysine. Thus, plant amino acids are directed toward oxidation rather than used for muscle protein synthesis. In this review, we evaluate the ability of plant- versus animal-based proteins to help maintain skeletal muscle mass in healthy and especially older people and examine different nutritional strategies for improving the anabolic properties of plant-based proteins. Among these strategies, increasing protein intake has led to a positive acute postprandial muscle protein synthesis response and even positive long-term improvement in lean mass. Increasing the quality of protein intake by improving amino acid composition could also compensate for the lower anabolic potential of plant-based proteins. We evaluated and discussed four nutritional strategies for improving the amino acid composition of plant-based proteins: fortifying plant-based proteins with specific essential amino acids, selective breeding, blending several plant protein sources, and blending plant with animal-based protein sources. These nutritional approaches need to be profoundly examined in older individuals in order to optimize protein intake for this population who require a high-quality food protein intake to mitigate age-related muscle loss.
Subject(s)
Anabolic Agents , Dietary Proteins/administration & dosage , Muscle Proteins/administration & dosage , Muscle, Skeletal/physiology , Plant Proteins/administration & dosage , Adult , Aged , Aged, 80 and over , Amino Acids/analysis , Animals , Dietary Proteins/pharmacology , Digestion , Female , Humans , Male , Middle Aged , Muscle Proteins/analysis , Muscle Proteins/metabolism , Nutritional Physiological Phenomena , Plant Proteins/analysis , Plant Proteins/metabolism , Protein Biosynthesis/physiologyABSTRACT
KEY POINTS: It has been suggested that leucine is primarily responsible for the increase in muscle protein synthesis after protein ingestion because leucine uniquely activates the mTOR-p70S6K signalling cascade. We compared the effects of ingesting protein or an amount of leucine equal to that in the protein during a hyperinsulinaemic-euglycaemic clamp (to eliminate potential confounding as a result of differences in the insulinogenic effect of protein and leucine ingestion) on muscle anabolic signalling and protein turnover in 28 women. We found that protein, but not leucine, ingestion increased muscle p-mTORSer2448 and p-p70S6KThr389 , although only protein, and not leucine, ingestion decreased muscle p-eIF2αSer51 and increased muscle protein synthesis. ABSTRACT: It has been suggested that leucine is primarily responsible for the increase in muscle protein synthesis (MPS) after protein ingestion because leucine uniquely activates the mTOR-p70S6K signalling cascade. We tested this hypothesis by measuring muscle p-mTORSer2448 , p-p70S6KThr389 and p-eIF2αSer51 , as well as protein turnover (by stable isotope labelled amino acid tracer infusion in conjunction with leg arteriovenous blood and muscle tissue sampling), in 28 women who consumed either 0.45 g protein kg-1 fat-free mass (containing 0.0513 g leucine kg-1 fat-free mass) or a control drink (n = 14) or 0.0513 g leucine kg-1 fat-free mass or a control drink (n = 14) during a hyperinsulinaemic-euglycaemic clamp procedure (HECP). Compared to basal conditions, the HECP alone (without protein or leucine ingestion) suppressed muscle protein breakdown by â¼20% and increased p-mTORSer2448 and p-p70S6KThr389 by >50% (all P < 0.05) but had no effect on p-eIF2αSer51 and MPS. Both protein and leucine ingestion further increased p-mTORSer2448 and p-p70S6KThr389 , although only protein, and not leucine, ingestion decreased (by â¼35%) p-eIF2αSer51 and increased (by â¼100%) MPS (all P < 0.05). Accordingly, leg net protein balance changed from negative (loss) during basal conditions to equilibrium during the HECP alone and the HECP with concomitant leucine ingestion and to positive (gain) during the HECP with concomitant protein ingestion. These results provide new insights into the regulation of MPS by demonstrating that leucine and mTOR signalling alone are not responsible for the muscle anabolic effect of protein ingestion during physiological hyperinsulinaemia, most probably because they fail to signal to eIF2α to initiate translation and/or additional amino acids are needed to sustain translation.
Subject(s)
Anabolic Agents/administration & dosage , Eating , Glucose Clamp Technique/methods , Hyperinsulinism/metabolism , Leucine/administration & dosage , Muscle Proteins/administration & dosage , Signal Transduction , Female , Glucose/metabolism , Humans , Insulin/metabolism , Middle Aged , TOR Serine-Threonine Kinases/metabolismABSTRACT
The natriuretic peptide (NP) system is a critical endocrine, autocrine, and paracrine system and has been investigated for potential use against cardiovascular and metabolic diseases. The clearance of NPs is regulated by the proteolysis of neutral endopeptidase (NEP) and by endocytosis via natriuretic peptide receptor-3 (NPR3). A linear NPR3-selective peptide, [Cha8]-ANP(7-16)-NH2 (1), showed potent binding affinity for NPR3 but poor predicted chemical stability due to its free thiol group. A 12-mer peptide (9) without a thiol group was designed by the hybridization of two NPR3-binding peptides: a linear ANP fragment peptide analog and musclin, a murine member of the bHLH family of transcription factors, possessed high binding affinity and strict selectivity for NPR3. To increase the proteolytic resistance of 9, amino acid substitutions at the cleavage sites led to hydroxyacetyl-[d-Phe5,d-Hyp7,Cha8,d-Ser9,Hyp11,Arg(Me)14]-ANP(5-15)-NHCH3 (23), showing high and selective binding affinity for NPR3 over NPR1 and excellent stability in mouse serum. Compound 23 increased intracellular cGMP concentrations in primary cultured adipocytes, and continuous administration induced substantial plasma cGMP elevation in mice, suggesting its potential to clarify the physiological role of NPR3 and its therapeutic application.
Subject(s)
Atrial Natriuretic Factor/pharmacology , Muscle Proteins/pharmacology , Peptide Fragments/pharmacology , Receptors, Atrial Natriuretic Factor/antagonists & inhibitors , Transcription Factors/pharmacology , Amino Acid Sequence , Animals , Atrial Natriuretic Factor/administration & dosage , Atrial Natriuretic Factor/blood , Atrial Natriuretic Factor/chemistry , Mice , Mice, Inbred C57BL , Muscle Proteins/administration & dosage , Muscle Proteins/blood , Muscle Proteins/chemistry , Peptide Fragments/administration & dosage , Peptide Fragments/blood , Peptide Fragments/chemistry , Receptors, Atrial Natriuretic Factor/metabolism , Transcription Factors/administration & dosage , Transcription Factors/blood , Transcription Factors/chemistryABSTRACT
Dietary protein intake should be optimized in all athletes to ensure proper recovery and enhance the skeletal muscle adaptive response to exercise training. In addition to total protein intake, the use of specific proteincontaining food sources and the distribution of protein throughout the day are relevant for optimizing protein intake in athletes. In the present study, we examined the daily intake and distribution of various proteincontaining food sources in a large cohort of strength, endurance and team-sport athletes. Well-trained male (n=327) and female (n=226) athletes completed multiple web-based 24-hr dietary recalls over a 2-4 wk period. Total energy intake, the contribution of animal- and plant-based proteins to daily protein intake, and protein intake at six eating moments were determined. Daily protein intake averaged 108±33 and 90±24 g in men and women, respectively, which corresponded to relative intakes of 1.5±0.4 and 1.4±0.4 g/kg. Dietary protein intake was correlated with total energy intake in strength (r=0.71, p <.001), endurance (r=0.79, p <.001) and team-sport (r=0.77, p <.001) athletes. Animal and plant-based sources of protein intake was 57% and 43%, respectively. The distribution of protein intake was 19% (19±8 g) at breakfast, 24% (25±13 g) at lunch and 38% (38±15 g) at dinner. Protein intake was below the recommended 20 g for 58% of athletes at breakfast, 36% at lunch and 8% at dinner. In summary, this survey of athletes revealed they habitually consume > 1.2 g protein/kg/d, but the distribution throughout the day may be suboptimal to maximize the skeletal muscle adaptive response to training.
Subject(s)
Athletes , Athletic Performance , Dietary Proteins/administration & dosage , Exercise , Feeding Behavior , Physical Exertion , Sports Nutritional Physiological Phenomena , Adolescent , Adult , Child , Cohort Studies , Feeding Behavior/ethnology , Female , Humans , Internet , Male , Middle Aged , Milk Proteins/administration & dosage , Muscle Proteins/administration & dosage , Muscle Strength , Netherlands , Nutrition Surveys , Physical Endurance , Plant Proteins, Dietary/administration & dosage , Sports Nutritional Physiological Phenomena/ethnology , Young AdultABSTRACT
BACKGROUND: Reported associations between protein intake from different sources and type 2 diabetes (T2D) have been inconsistent. OBJECTIVE: We prospectively examined the relations of total, animal, and plant protein intakes with incident T2D. DESIGN: We followed 21,523 participants (women: 61.7%) between 1990 and 2007 from the Melbourne Collaborative Cohort Study who were free of diabetes, cardiovascular disease, cancer, and kidney stones at baseline. We also conducted a meta-analysis that included the results from our cohort and from 10 previous prospective studies. RESULTS: A total of 929 new cases (4.3%) of T2D were documented during a mean of 11.7 y of follow-up. Multivariate-adjusted ORs for incident T2D in the highest compared with lowest quintiles of total and animal protein intakes as percentages of energy were 1.23 (95% CI: 0.96, 1.56; P-trend = 0.029) and 1.29 (95% CI: 0.99, 1.67; P-trend = 0.014), respectively. These associations appeared to be greater in men and in participants with normal baseline plasma glucose, body mass index, or blood pressure. Plant protein intake was inversely associated with incident T2D in women only (OR; 0.60; 95% CI: 0.37, 0.99). In the meta-analysis of 11 prospective cohort studies with 505,624 participants and 37,918 T2D cases (follow-up range: 5-24 y), pooled RRs for the comparison of the highest with lowest categories of total, animal, and plant protein intakes were 1.09 (95% CI: 1.06, 1.13), 1.19 (95% CI: 1.11, 1.28), and 0.95 (95% CI: 0.89, 1.02), respectively. Associations between animal protein intake and T2D were similar across sex, geographic region, length of follow-up, study quality, and method of expressing protein intake. An inverse association between plant protein intake and T2D was observed in women (RR: 0.93; 95% CI: 0.85, 1.00) and in US populations (RR: 0.91; 95% CI: 0.84, 0.97). CONCLUSION: Higher intakes of total and animal protein were both associated with increased risks of T2D, whereas higher plant protein intake tended to be associated with lower risk of T2D.
Subject(s)
Diabetes Mellitus, Type 2/epidemiology , Dietary Proteins/administration & dosage , Blood Glucose/metabolism , Blood Pressure , Body Mass Index , Diabetes Mellitus, Type 2/blood , Energy Intake , Humans , Incidence , Muscle Proteins/administration & dosage , Nutrition Assessment , Plant Proteins/administration & dosage , Risk FactorsABSTRACT
Long-term consumption of red meat has been considered a potential risk to gut health, but this is based on clinic investigations, excessive intake of fat, heme and some injurious compounds formed during cooking or additions to processed meat products. Whether intake of red meat protein affects gut bacteria and the health of the host remains unclear. In this work, we compared the composition of gut bacteria in the caecum, by sequencing the V4-V5 region of 16S ribosomal RNA gene, obtained from rats fed with proteins from red meat (beef and pork), white meat (chicken and fish) and other sources (casein and soy). The results showed significant differences in profiles of gut bacteria between the six diet groups. Rats fed with meat proteins had a similar overall structure of caecal bacterial communities separated from those fed non-meat proteins. The beneficial genus Lactobacillus was higher in the white meat than in the red meat or non-meat protein groups. Also, rats fed with meat proteins and casein had significantly lower levels of lipopolysaccharide-binding proteins, suggesting that the intake of meat proteins may maintain a more balanced composition of gut bacteria, thereby reducing the antigen load and inflammatory response in the host.
Subject(s)
Cecum/microbiology , Dietary Proteins/metabolism , Gastrointestinal Microbiome/physiology , Milk Proteins/metabolism , Muscle Proteins/metabolism , Plant Proteins/metabolism , Animal Feed/microbiology , Animals , Cecum/metabolism , Dairy Products/microbiology , Male , Meat/microbiology , Milk Proteins/administration & dosage , Muscle Proteins/administration & dosage , Rats , Rats, Sprague-DawleyABSTRACT
Bioactive peptides are short amino acid sequences, that upon release from the parent protein may play different physiological roles, including antioxidant, antihypertensive, antimicrobial, and other bioactivities. They have been identified from a range of foods, including those of animal origin, e.g., milk and muscle sources (with pork, beef, or chicken and various species of fish and marine organism). Bioactive peptides are encrypted within the sequence of the parent protein molecule and latent until released and activated by enzymatic proteolysis, e.g. during gastrointestinal digestion or food processing. Bioactive peptides derived from food sources have the potential for incorporation into functional foods and nutraceuticals. The aim of this paper is to present an overview of the muscle-derived bioactive peptides, especially those of fermented meats and the potential benefits of these bioactive compounds to human health.
Subject(s)
Antihypertensive Agents/analysis , Antioxidants/analysis , Dietary Proteins/analysis , Meat Products/analysis , Meat/analysis , Muscle Proteins/analysis , Peptide Fragments/analysis , Animals , Antihypertensive Agents/administration & dosage , Antioxidants/administration & dosage , Antioxidants/metabolism , Dietary Proteins/administration & dosage , Dietary Proteins/metabolism , Fermentation , Functional Food/analysis , Humans , Meat/microbiology , Meat Products/microbiology , Muscle Proteins/administration & dosage , Muscle Proteins/metabolism , Peptide Fragments/administration & dosage , Peptide Fragments/metabolism , ProteolysisABSTRACT
BACKGROUND: The protein leverage hypothesis requires specific evidence that protein intake is regulated more strongly than energy intake. OBJECTIVE: The objective was to determine ad libitum energy intake, body weight changes, appetite profile, and nitrogen balance in response to 3 diets with different protein-to-carbohydrate + fat ratios over 12 consecutive days, with beef as a source of protein. DESIGN: A 3-arm, 12-d randomized crossover study was performed in 30 men and 28 women [mean ± SD age: 33 ± 16 y; body mass index (in kg/m²): 24.4 ± 4.0] with the use of diets containing 5%, 15%, and 30% of energy (En%) from protein, predominantly from beef. RESULTS: Energy intake was significantly lower in the 30En%-protein condition (8.73 ± 1.93 MJ/d) than in the 5En%-protein (9.48 ± 1.67 MJ/d) and 15En%-protein (9.30 ± 1.62 MJ/d) conditions (P = 0.001), stemming largely from lower energy intake during meals (P = 0.001). Hunger (P = 0.001) and desire to eat (P = 0.001) ratings were higher and fullness ratings were lower (P = 0.001) in the 5En%-protein condition than in the 15En%-protein and 30En%-protein conditions. Nitrogen excretion was lower in the 5En%-protein condition (4.7 ± 1.5 g/24 h; P = 0.001) and was higher in the 30En%-protein condition (15.3 ± 8.7 g/24 h; P = 0.001) compared with the 15En%-protein condition (10.0 ± 5.2 g/24 h). Nitrogen balance was maintained in the 5En%-protein condition and was positive in the 15En%- and 30En%-protein conditions (P = 0.001). CONCLUSIONS: Complete protein leverage did not occur because subjects did not consume to a common protein amount at the expense of energy balance. Individuals did underconsume relative to energy requirements from high-protein diets. The lack of support for protein leverage effects on a low-protein diet may stem from the fact that protein intake was sufficient to maintain nitrogen balance over the 12-d trial.
Subject(s)
Appetite Regulation , Dietary Proteins/administration & dosage , Energy Intake , Meat , Muscle Proteins/administration & dosage , Adolescent , Adult , Animals , Biomarkers/urine , Cattle , Cross-Over Studies , Diet, Protein-Restricted/adverse effects , Dietary Proteins/adverse effects , Dietary Proteins/metabolism , Female , Humans , Male , Meat/adverse effects , Middle Aged , Muscle Proteins/adverse effects , Muscle Proteins/metabolism , Nitrogen/urine , Single-Blind Method , Weight Loss , Young AdultABSTRACT
Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is anchored to the synaptic basal lamina via a triple helical collagen Q (ColQ) in the form of asymmetric AChE (AChE/ColQ). The C-terminal domain of ColQ binds to MuSK, the muscle-specific receptor tyrosine kinase, that mediates a signal for acetylcholine receptor (AChR) clustering at the NMJ. ColQ also binds to heparan sulfate proteoglycans including perlecan. Congenital defects of ColQ cause endplate AChE deficiency. A single intravenous administration of adeno-associated virus serotype 8 (AAV8)-COLQ to Colq-/- mice rescued motor functions, synaptic transmission, and the ultrastructure of NMJ. We also injected AAV1-COLQ-IRES-EGFP to the left tibialis anterior and observed colocalization of AChE/ColQ at all the examined NMJs of the non-injected limbs. Additionally, injection of purified recombinant AChE/ColQ protein complex into gluteus maximus accumulated AChE in non-injected forelimbs. These observations suggest that the tissue-targeting signal of ColQ can be exploited to specifically deliver the transgene product to the target tissue. MuSK antibody-positive myasthenia gravis (MG) accounts for 5-15% of autoimmune MG. As AChR deficiency is typically mild and as cholinesterase inhibitors are generally ineffective or worsen myasthenic symptoms, we asked if the patient's MuSK-IgG interferes with binding of ColQ to MuSK. In vitro overlay of AChE/ColQ to muscle sections of Colq-/- mice revealed that MuSK-IgG blocks binding of ColQ to the NMJ. In vitro plate-binding of MuSK to ColQ disclosed that MuSK-IgG exerts a dose-dependent block of MuSK-ColQ interaction. In addition, passive transfer of MuSK-IgG to mice reduced the size and density of ColQ to â¼10% of controls and had a lesser effect on the sizes and densities of AChR and MuSK. Elucidation of molecular mechanisms of specific binding of ColQ to the NMJ enabled us to ameliorate devastating myasthenic symptoms of Colq-/- mice and to reveal bases of anti-MuSK MG.
Subject(s)
Acetylcholinesterase/metabolism , Collagen/metabolism , Muscle Proteins/metabolism , Myasthenic Syndromes, Congenital/therapy , Neuromuscular Junction/metabolism , Acetylcholinesterase/administration & dosage , Acetylcholinesterase/deficiency , Acetylcholinesterase/genetics , Animals , Collagen/administration & dosage , Collagen/deficiency , Collagen/genetics , Dependovirus/genetics , Female , GPI-Linked Proteins/metabolism , Genetic Therapy , Humans , Immunoglobulin G/administration & dosage , Injections, Intramuscular , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle Proteins/administration & dosage , Muscle Proteins/deficiency , Muscle Proteins/genetics , Myasthenia Gravis, Autoimmune, Experimental/genetics , Myasthenia Gravis, Autoimmune, Experimental/metabolism , Myasthenia Gravis, Autoimmune, Experimental/therapy , Myasthenic Syndromes, Congenital/genetics , Myasthenic Syndromes, Congenital/metabolism , Receptor Protein-Tyrosine Kinases/immunology , Receptor Protein-Tyrosine Kinases/metabolism , Recombinant Proteins/administration & dosage , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Synaptic TransmissionABSTRACT
During ageing, immobilization periods increase and are partially responsible of sarcopaenia by inducing a muscle atrophy which is hardly recovered from. Immobilization-induced atrophy is due to an increase of muscle apoptotic and proteolytic processes and decreased protein synthesis. Moreover, previous data suggested that the lack of muscle mass recovery might be due to a defect in protein synthesis response during rehabilitation. This study was conducted to explore protein synthesis during reloading and leucine supplementation effect as a nutritional strategy for muscle recovery. Old rats (2224 months old) were subjected to unilateral hindlimb casting for 8 days (I8) and allowed to recover for 1040 days (R10R40). They were fed a casein (±leucine) diet during the recovery. Immobilized gastrocnemius muscles atrophied by 20%, and did not recover even at R40. Amount of polyubiquitinated conjugates and chymotrypsin- and trypsin-like activities of the 26S proteasome increased. These changes paralleled an 'anabolic resistance' of the protein synthesis at the postprandial state (decrease of protein synthesis, P-S6 and P-4E-BP1). During the recovery, proteasome activities remained elevated until R10 before complete normalization and protein synthesis was slightly increased. With free leucine supplementation during recovery, if proteasome activities were normalized earlier and protein synthesis was higher during the whole recovery, it nevertheless failed in muscle mass gain. This discrepancy could be due to a 'desynchronization' between the leucine signal and the availability of amino acids coming from casein digestion. Thus, when supplemented with leucine-rich proteins (i.e. whey) and high protein diets, animals partially recovered the muscle mass loss.
Subject(s)
Aging/physiology , Leucine/administration & dosage , Muscle Fibers, Skeletal/physiology , Muscle Proteins/administration & dosage , Muscular Atrophy/diet therapy , Muscular Atrophy/physiopathology , Amino Acids/blood , Animals , Diet , Dietary Supplements , Hindlimb Suspension/methods , Leucine/metabolism , Male , Milk Proteins/metabolism , Muscle Fibers, Skeletal/metabolism , Muscle Proteins/metabolism , Muscular Atrophy/metabolism , Proteasome Endopeptidase Complex/metabolism , Rats , Rats, Wistar , Ubiquitin/metabolism , Whey ProteinsABSTRACT
BACKGROUND: We have previously shown that deficiency for the z-disc protein calsarcin-1 (CS1) sensitizes the heart to calcineurin signaling and to stimuli of pathological hypertrophy. In the present study we asked whether overexpression of CS1 might exhibit antihypertrophic effects, and therefore we tested this hypothesis both in vitro and in vivo. METHODS AND RESULTS: Adenoviral gene transfer of CS1 into neonatal cardiomyocytes inhibited hypertrophy as a result of Gq-agonist stimulation, including angiotensin-II (Ang-II), endothelin-1, and phenylephrine. Consistently, Adenoviral gene transfer of CS1 also led to the reduction of increased levels of atrial natriuretic factor (mRNA) and the calcineurin-sensitive gene MCIP1.4, suggesting that CS1 inhibits calcineurin-dependent signaling. Furthermore, we generated CS1-overexpressing transgenic mice (CS1Tg). Unchallenged CS1Tg mice did not exhibit a pathological phenotype as assessed by echocardiography and analysis of cardiac gene expression. Likewise, when subjected to long-term infusion of Ang-II, both CS1Tg and wild-type mice developed a similar degree of arterial hypertension. Yet, in contrast to wild-type mice, Ang-II-treated CS1Tg animals did not display cardiac hypertrophy. Despite the absence of hypertrophy, both fractional shortening and dP/dt(max) were preserved in CS1Tg Ang-II-treated mice as assessed by echocardiography and cardiac catheterization, respectively. Moreover, induction of the hypertrophic gene program (atrial natriuretic factor, brain natriuretic peptide) was markedly blunted, and expression of the calcineurin-dependent gene MCIP1.4 was significantly reduced in CS1Tg mice, again consistent with an inhibitory role of CS1 on calcineurin. CONCLUSIONS: The sarcomeric protein CS1 prevents Ang-II-induced cardiomyocyte hypertrophy at least in part via inhibition of calcineurin signaling. Thus, overexpression of CS1 might represent a novel approach to attenuate pathological cardiac hypertrophy.
Subject(s)
Angiotensin II/pharmacology , Calcineurin/metabolism , Cardiomegaly/therapy , Carrier Proteins/pharmacology , Genetic Therapy/methods , Muscle Proteins/pharmacology , Animals , Animals, Newborn , Atrial Natriuretic Factor/drug effects , Atrial Natriuretic Factor/genetics , Calcium-Binding Proteins , Cardiomegaly/etiology , Carrier Proteins/administration & dosage , Intracellular Signaling Peptides and Proteins/drug effects , Intracellular Signaling Peptides and Proteins/genetics , Mice , Mice, Transgenic , Muscle Proteins/administration & dosage , Muscle Proteins/drug effects , Muscle Proteins/genetics , RNA, Messenger/analysis , RNA, Messenger/drug effects , Signal Transduction/drug effectsABSTRACT
Recent studies show that overexpression of small heat shock protein 20 (Hsp20) in mouse hearts reduces infarct size and improves cardiac performance. However, it is not known whether Hsp20 exerts its protective action through improved calcium handling or chaperone activity. The C-terminal extensions of small heat shock proteins, such as alphaB-crystallin and Hsp25, are implicated in chaperoning activity. Through adenovirus mediated overexpression of Hsp20 with C-terminal extension substitution, we delineated the mechanism of protection. Neonatal and adult rat cardiomyocytes overexpressing either the full-length Hsp20 or Hsp20 with a C-terminal extension substitution were subjected to simulated ischemia for 14-16 h followed by reperfusion 6-8 h. Overexpressing Hsp20 with a C-terminus extension substitution did not protect against simulated ischemia/reperfusion in either adult (98+/-8.8% LDH release of control) or neonatal cardiomyocytes (103+/-1.8% CK release of control) as measured by creatine kinase (CK) and lactate dehydrogenase (LDH) cell viability assays (n=4, P<0.05). However, this Hsp20 C-terminal substitution mutant increased calcium transients 33+/-11% and cell contraction amplitude 60+/-15% as quantified through epifluorescence microscopy (n=16 to 34 cells per heart from 4 to 5 hearts, P<0.05). In contrast, overexpression of the full-length Hsp20 protected cultured adult (53+/-8.5% LDH release of control) and neonatal rat (57+/-8.3% CK release of control) cardiomyocytes from simulated ischemia/reperfusion injury. This overexpression also increased calcium transients 30+/-10% and cell contraction amplitude 50+/-10%. These novel data suggest that the C-terminal extension of Hsp20 is essential for cardioprotection. Hsp20 renders this protection through its C-terminal extension protein domain, while this part of the protein is not involved in the Hsp20 ability to increase both calcium transients and cell contraction.
Subject(s)
Cardiotonic Agents/pharmacology , Genetic Therapy , HSP20 Heat-Shock Proteins/pharmacology , Muscle Proteins/pharmacology , Myocardial Reperfusion Injury/prevention & control , Adenoviridae/genetics , Animals , Animals, Newborn , Calcium/metabolism , Cardiotonic Agents/administration & dosage , Creatine Kinase/metabolism , HSP20 Heat-Shock Proteins/administration & dosage , HSP20 Heat-Shock Proteins/genetics , L-Lactate Dehydrogenase/metabolism , Muscle Proteins/administration & dosage , Muscle Proteins/genetics , Myocytes, Cardiac/drug effects , Rats , Rats, Sprague-Dawley , Transgenes/physiologyABSTRACT
In recent years, several groups have reported a variety of strategies for developing biological pacemakers whose ultimate function would be to supplement/replace electronic pacemakers. Strategies have included gene therapy using naked plasmids or viral vectors and cell therapy for which both adult human mesenchymal stem cells (hMSCs) and human embryonic stem cells have been employed. This article reviews the various approaches and summarizes our own research in which the pacemaker gene, HCN2, is administered via viral vector or in an hMSC platform to produce pacemaker function in the intact canine heart.
Subject(s)
Arrhythmias, Cardiac/therapy , Cardiac Pacing, Artificial , Genetic Therapy , Ion Channels/physiology , Mesenchymal Stem Cells/cytology , Muscle Proteins/physiology , Myocytes, Cardiac/cytology , Adenoviridae/genetics , Adult , Animals , Arrhythmias, Cardiac/genetics , Arrhythmias, Cardiac/pathology , Cells, Cultured/drug effects , Cells, Cultured/physiology , Cells, Cultured/transplantation , Coculture Techniques , Dogs , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels , Ion Channels/administration & dosage , Mesenchymal Stem Cells/drug effects , Muscle Proteins/administration & dosage , Myocytes, Cardiac/drug effects , Plasmids , Potassium Channels , Stem Cell TransplantationABSTRACT
Peptides of the trefoil factor family (TFF1, TFF2 and TFF3) are co-secreted with mucus in most organ systems and are believed to interact with mucins to produce high-viscosity, stable gel complexes. We have previously demonstrated that cells in the GI tract possess binding sites to TFF2 and that injected TFF2 ends up in the mucus layer. In the present study, tissue binding and metabolism of parenterally administered human TFF1 and TFF3 in rats were described and compared to the immunohistochemical localization of the TFF peptides. 125I-TFF1 monomer and 125I-TFF3 mono- and dimer were given intravenously to female Wistar rats. The tissue distribution was assessed by gamma counting of organ samples and by autoradiography of histological sections. The degradation of 125I-TFF3 was studied by means of trichloracetic acid (TCA) precipitation and the saturability of the binding by administration of excess unlabelled peptide. The TFF peptides were localized in histologic sections from the GI tract by immunohistochemistry. Injected TFF3 dimer (12%) was taken up by the GI tract. At autoradiography, grains were localized to the same cells that were immunoreactive to TFF2. The binding could be displaced by excess TFF3. Similar binding was observed for the TFF1 and TFF3 monomers apart from binding in the stomach, where the uptake was only 15% in comparison to the dimer. There was no specific binding outside the GI tract and no binding to TFF1 or TFF3 immunoreactive cells. In conclusion, the TFF2-binding cells in the gastrointestinal tract seem to have basolateral, receptor-like activity to all three TFF peptides. The mucous neck cells of the stomach predominantly take up TFFs with two trefoil domains, indicating a different receptor-like activity in the stomach compared to the rest of the GI tract.
Subject(s)
Digestive System/metabolism , Growth Inhibitors/metabolism , Mucins/metabolism , Muscle Proteins/metabolism , Neuropeptides , Peptide Fragments/pharmacology , Peptides/metabolism , Proteins/metabolism , Animals , Cells, Cultured , Estrogens/metabolism , Female , Growth Inhibitors/administration & dosage , Humans , Immunoenzyme Techniques , Injections, Intravenous , Iodine Radioisotopes , Mucins/administration & dosage , Muscle Proteins/administration & dosage , Proteins/administration & dosage , Rats , Rats, Wistar , Tissue Distribution , Trefoil Factor-1 , Trefoil Factor-2 , Trefoil Factor-3 , Tumor Suppressor ProteinsABSTRACT
It has been previously documented that the thermolysin-digest of "Katsuo-bushi", a Japanese traditional food processed from dried bonito possesses potent inhibitory activity against angiotensin I-converting enzyme (ACE). The present authors isolated eight kinds of ACE-inhibitory peptides from it. Of these isolated peptides, LKPNM (IC50 = 2.4 microM) was found to be hydrolyzed by ACE to produce LKP (IC50 = 0.32 microM) with 8-fold higher ACE-inhibitory activity relative to the parent peptide or LKPNM, suggesting that LKPNM can be regarded as a prodrug-type ACE-inhibitory peptide. For assessment of relative antihypertensive activities of LKPNM and LKP to that of captopril, they were orally administered to SHR rats to monitor time-course changes of blood pressures, whereby it was evidenced that both LKPNM and captopril showed maximal decrease of blood pressure 4 h after oral administration and their efficacies lasted until 6 h post-administration. In sharp contrast, however, maximal reduction of blood pressure occurred as early as 2 h after administration of LKP. Minimum effective doses of LKPNM, LKP and captopril were 8, 2.25 and 1.25 mg/kg, respectively. When compared on molar basis, antihypertensive activities of LKPNM and LKP accounted for 66% and 91% relative to that of captopril, respectively, whereas in vitro ACE-inhibitory activities of LKPNM and LKP were no more than 0.92% and 7.73% compared with that of captopril (IC50 = 0.022 microM). It is of interest to note that both of these peptides exert remarkably higher antihypertensive activities in vivo despite weaker in vitro ACE-inhibitory effects, which was ascertained by using captopril as the reference drug.
Subject(s)
Angiotensin-Converting Enzyme Inhibitors/pharmacology , Fish Products/analysis , Muscle Proteins/pharmacology , Oligopeptides/pharmacology , Prodrugs/pharmacology , Administration, Oral , Angiotensin-Converting Enzyme Inhibitors/administration & dosage , Angiotensin-Converting Enzyme Inhibitors/metabolism , Animals , Antihypertensive Agents/administration & dosage , Antihypertensive Agents/metabolism , Antihypertensive Agents/pharmacology , Captopril/administration & dosage , Chickens , Humans , Injections, Intravenous , Muscle Proteins/administration & dosage , Muscle Proteins/metabolism , Oligopeptides/administration & dosage , Oligopeptides/metabolism , Prodrugs/administration & dosage , Prodrugs/metabolism , Rats , Rats, Inbred SHR , Thermolysin/metabolismABSTRACT
Tumor metastases are extremely rare in striated muscles. This is surprising given the fact that this tissue constitutes 60% of body weight. The present study focuses on small molecules produced and secreted by muscle cells which possess anti-cancer activity in vivo. Recently we have shown that a low molecular weight fraction (< 1000 Dalton) of skeletal muscle cell conditioned medium (muscle factor-MF), markedly inhibits the proliferation of carcinoma, sarcoma or melanoma cell lines in vitro. The MF exerts a cytostatic effect on tumor cell growth and arrests the cells in the G0/G1 of the cell cycle. However, normal cell proliferation, such as bone marrow and fibroblasts, was stimulated following incubation with MF. In this study, the effect of orally administered MF on melanoma and sarcoma growth was examined in mice. The administration of MF to mice inoculated intravenously with melanoma (B16-F10) or sarcoma (MCA-105) cells, resulted in a statistically significant inhibition of metastatic lung foci. In a different model, melanoma was induced in the foot pad and after development of a local lesion, the leg was amputated. A prolonged survival time was observed in the MF treated groups. Since the MF stimulated bone marrow cell proliferation in vitro, we decided to test its efficacy as an inhibitor of the myelotoxic effect exerted by chemotherapy, in vivo. MF, administered after chemotherapy, restored the number of white blood cells and yielded an increased percentage of neutrophils compared with the decline in these parameters after administration of chemotherapy alone. Thus, it is indicated that MF exerted a systemic anti tumor and chemoprotective effect when given orally. It can be concluded that it is bioavailable and is not biodegradable in the digestive system. MF may be considered as a potential therapy for the prevention of tumor spread.
Subject(s)
Lung Neoplasms/prevention & control , Lung Neoplasms/secondary , Muscle Proteins/administration & dosage , Sarcoma, Experimental/pathology , Administration, Oral , Animals , Antineoplastic Agents/adverse effects , Bone Marrow Cells/drug effects , Bone Marrow Cells/pathology , Cell Division/drug effects , Cell Line , Lung Neoplasms/drug therapy , Male , Mice , Mice, Inbred C57BL , Muscle Proteins/pharmacology , Sarcoma, Experimental/drug therapyABSTRACT
The biological value of 3 concentrates of sarcoplasmatic proteins from industrial fish (minthal, cod, putassu) was assayed in the 4-week experiment on growing male Wistar rats by the methods of counting the coefficients of protein effectiveness and pure protein effectiveness that comprised 100, 110 and 110% in relation to the corresponding parameters obtained with casein. The actual assimilation of the fish product proteins proved to be significantly higher than that of casein. Hypocholesterolemic and hypolipidemic effects of the protein products studied are, probably, caused by the presence of pectin.
Subject(s)
Animal Nutritional Physiological Phenomena , Dietary Proteins/metabolism , Fish Products , Growth/physiology , Lipids/blood , Muscle Proteins/metabolism , Sarcoplasmic Reticulum , Animal Feed , Animals , Dietary Proteins/administration & dosage , Male , Muscle Proteins/administration & dosage , Nutritive Value , Rats , Rats, Inbred StrainsABSTRACT
Immunization of rats with a myocyte antigen results in the formation and accumulation of the precipitating antibodies presented mainly by beta-globulins in the blood titre. Under conditions of the animals' immunization and simultaneous physical training the formation of specific antibodies is, mainly, due to gamma-globulins.