Essential Amino Acids and Protein Synthesis: Insights into Maximizing the Muscle and Whole-Body Response to Feeding.

Ingesting protein-containing supplements and foods provides essential amino acids (EAA) necessary to increase muscle and whole-body protein synthesis (WBPS). Large variations exist in the EAA composition of supplements and foods, ranging from free-form amino acids to whole protein foods. We sought to investigate how changes in peripheral EAA after ingesting various protein and free amino acid formats altered muscle and whole-body protein synthesis. Data were compiled from four previous studies that used primed, constant infusions of L-(ring-2H5)-phenylalanine and L-(3,3-2H2)-tyrosine to determine fractional synthetic rate of muscle protein (FSR), WBPS, and circulating EAA concentrations. Stepwise regression indicated that max EAA concentration (EAACmax; R2 = 0.524, p < 0.001), EAACmax (R2 = 0.341, p < 0.001), and change in EAA concentration (ΔEAA; R = 0.345, p < 0.001) were the strongest predictors for postprandial FSR, Δ (change from post absorptive to postprandial) FSR, and ΔWBPS, respectively. Within our dataset, the stepwise regression equation indicated that a 100% increase in peripheral EAA concentrations increases FSR by ~34%. Further, we observed significant (p < 0.05) positive (R = 0.420-0.724) correlations between the plasma EAA area under the curve above baseline, EAACmax, ΔEAA, and rate to EAACmax to postprandial FSR, ΔFSR, and ΔWBPS. Taken together our results indicate that across a large variety of EAA/protein-containing formats and food, large increases in peripheral EAA concentrations are required to drive a robust increase in muscle and whole-body protein synthesis.

Transepithelial transport across Caco-2 cell monolayers of angiotensin converting enzyme (ACE) inhibitory peptides derived from simulated in vitro gastrointestinal digestion of cooked chicken muscles.

Korat-chicken breast and thigh were subjected to heating at 70, 100 or 121 °C for 30 min and simulated in vitro gastrointestinal digestion. At 70 or 100 °C heating, digests of breast possessed higher ACE inhibitory activity than those of thigh. The highest ACE inhibitory activity was found in the digest of breast cooked at 70 °C (B/H-70), whereas breast heated at 121 °C (B/H-121) exhibited the lowest. The 1-kDa permeate of the B/H-70 digest revealed higher permeability through colorectal adenocarcinoma monolayers and ACE inhibitory activity than did B/H-121. Among nine transported peptides, APP derived from myosin showed the highest ACE inhibition, with a non-competitive characteristic (Ki 0.93 µM). Molecular docking showed that APP interacts with ACE via hydrogen bonds, electrostatic and van der Waals interactions. In conclusion, mild thermal treatment of chicken breast resulted in a higher amount of transported peptides, exerting higher ACE inhibitory activity, which could lead to potential health benefits.

Dietary protein structure affects endogenous ileal amino acids but not true ileal amino acid digestibility in growing male rats.

BACKGROUND: The amount of endogenous, as opposed to undigested dietary, protein in digesta is a measure of fundamental interest related to gut physiology and function. OBJECTIVE: The objective of this study was to determine whether alimentation with proteins having differing amino acid compositions influenced endogenous ileal amino acids (EIAAs) and true ileal amino acid digestibility (TIAAD) values. METHODS: Male rats (n = 8) were fed a purified diet containing 100 g/kg of 1 of 5 protein hydrolysates, each derived from a different semipurified intact protein source [gelatin, beef muscle (BM), casein, soy protein isolate (SPI), and lactalbumin] devoid of antinutritional factors or fiber. The rats were fed their respective hydrolysate-based diet for 1 d after receiving the same diet but containing the corresponding intact protein source for 7 d. Titanium dioxide was used as an indigestible marker. Ileal digesta were collected after the rats were killed, and EIAAs were determined (precipitate + retentate) after centrifugation and ultrafiltration of the digesta. The TIAAD values of the intact protein sources were determined using EIAA flows based on each protein hydrolysate. RESULTS: Mean EIAA flows differed (P < 0.05) across protein hydrolysates for most amino acids, with the mean ± SEM EIAA flows across amino acids being 262 ± 17, 253 ± 12, 248 ± 18, 226 ± 14, and 191 ± 20 mg/kg dry matter intake for the gelatin, BM, casein, SPI, and lactalbumin hydrolysates, respectively. The only difference (P < 0.05) for the mean EIAA flows across amino acids within each protein hydrolysate was observed between gelatin (262 ± 17 mg/kg) and lactalbumin (191 ± 20 mg/kg) hydrolysates. Except for Trp (P < 0.001) in gelatin and lactalbumin hydrolysates, EIAA flows determined using the casein hydrolysate were not different (P ≥ 0.05) from EIAA flows determined using the other protein hydrolysates. TIAAD values were not generally different (P ≥ 0.05) regardless of the hydrolysate used to determine the EIAA flows. CONCLUSIONS: Protein source affected EIAA flows, although the differences had little effect on TIAAD. Enzyme hydrolyzed casein is a suitable model hydrolysate for determining TIAAD with the enzyme-hydrolyzed protein-ultrafiltration technique.

Formation of cholinergic synapse-like specializations at developing murine muscle spindles.

Muscle spindles are complex stretch-sensitive mechanoreceptors. They consist of specialized skeletal muscle fibers, called intrafusal fibers, which are innervated in the central (equatorial) region by afferent sensory axons and in both polar regions by efferent γ-motoneurons. We show that AChRs are concentrated at the γ-motoneuron endplate as well as in the equatorial region where they colocalize with the sensory nerve ending. In addition to the AChRs, the contact site between sensory nerve ending and intrafusal muscle fiber contains a high concentration of choline acetyltransferase, vesicular acetylcholine transporter and the AChR-associated protein rapsyn. Moreover, bassoon, a component of the presynaptic cytomatrix involved in synaptic vesicle exocytosis, is present in γ-motoneuron endplates but also in the sensory nerve terminal. Finally, we demonstrate that during postnatal development of the γ-motoneuron endplate, the AChR subunit stoichiometry changes from the γ-subunit-containing fetal AChRs to the ε-subunit-containing adult AChRs, similar and approximately in parallel to the postnatal subunit maturation at the neuromuscular junction. In contrast, despite the onset of ε-subunit expression during postnatal development the γ-subunit remains detectable in the equatorial region by subunit-specific antibodies as well as by analysis of muscle spindles from mice with genetically-labeled AChR γ-subunits. These results demonstrate an unusual maturation of the AChR subunit composition at the annulospiral endings and suggest that in addition to the recently described glutamatergic secretory system, the sensory nerve terminals are also specialized for cholinergic synaptic transmission, synaptic vesicle storage and exocytosis.

Syncoilin is an intermediate filament protein in activated hepatic stellate cells.

Hepatic stellate cells (HSCs) play an important role in several (patho)physiologic conditions in the liver. In response to chronic injury, HSCs are activated and change from quiescent to myofibroblast-like cells with contractile properties. This shift in phenotype is accompanied by a change in expression of intermediate filament (IF) proteins. HSCs express a broad, but variable spectrum of IF proteins. In muscle, syncoilin was identified as an alpha-dystrobrevin binding protein with sequence homology to IF proteins. We investigated the expression of syncoilin in mouse and human HSCs. Syncoilin expression in isolated and cultured HSCs was studied by qPCR, Western blotting, and fluorescence immunocytochemistry. Syncoilin expression was also evaluated in other primary liver cell types and in in vivo-activated HSCs as well as total liver samples from fibrotic mice and cirrhotic patients. Syncoilin mRNA was present in human and mouse HSCs and was highly expressed in in vitro- and in vivo-activated HSCs. Syncoilin protein was strongly upregulated during in vitro activation of HSCs and undetectable in hepatocytes and liver sinusoidal endothelial cells. Syncoilin mRNA levels were elevated in both CCl4- and common bile duct ligation-treated mice. Syncoilin immunocytochemistry revealed filamentous staining in activated mouse HSCs that partially colocalized with α-smooth muscle actin, ß-actin, desmin, and α-tubulin. We show that in the liver, syncoilin is predominantly expressed by activated HSCs and displays very low-expression levels in other liver cell types, making it a good marker of activated HSCs. During in vitro activation of mouse HSCs, syncoilin is able to form filamentous structures or at least to closely interact with existing cellular filaments.

The influence of clonidine co-administration on the extent of lidocaine protein binding to rat serum and tissues.

Lidocaine is an amide local anesthetic and clonidine is an antihypertensive (α2-adrenergic agonist). The use of these two drugs in combination is recommended to enhance the analgesic effect of lidocaine. The aim of this study was to investigate the influence of clonidine co-administration on the extent of lidocaine binding to rat serum, heart and maxillofacial tissues in vivo and in vitro. Thirty-two Wistar rats received either lidocaine alone, or lidocaine and clonidine, in the masseter muscle, and were then sacrificed 15 or 30 min after treatment. Serum, masseter, mandible and heart samples were then isolated and incubated in 0.9% NaCl solution for 12 h at 8°C. The extent of binding in the incubation medium and the serum was estimated by ultrafiltration, and the free lidocaine fraction was determined by the radioscopic method in a ß-counter. An in vitro procedure was also performed. Serum, heart, masseter and mandible samples were incubated at 37°C for 15 or 30 min in Ringer's solution containing either lidocaine or lidocaine and clonidine, and the samples were similarly subjected to ultrafiltration. The percentage binding of lidocaine was again estimated by the radioscopic method. Lidocaine levels were found to be increased by clonidine co-administration in vivo and the free lidocaine fraction was enhanced in vitro as well in the examined tissues, obviously through mechanisms related to protein binding alterations.

Altered turnover of calcium regulatory proteins of the sarcoplasmic reticulum in aged skeletal muscle.

We have measured the in vivo protein turnover for the major calcium regulatory proteins of the sarcoplasmic reticulum from the skeletal muscle of young adult (7 months) and aged (28 months) Fischer 344 rats. From the time course of the incorporation and decay of protein-associated radioactivity after a pulse injection of [14C]leucine and correcting for leucine reutilization, in young rats, the apparent half-lives for calsequestrin, the 53-kDa glycoprotein, and ryanodine receptor are 5.4 +/- 0.4, 6.3 +/- 1.3, and 8.3 +/- 1.3 days, respectively. A half-life of 14.5 +/- 2.5 days was estimated for the Ca-ATPase isolated from young muscle. Differences in protein turnover associated with aging were determined using sequential injection of two different isotopic labels ([14C]leucine and [3H]leucine) to provide an estimate of protein synthesis and degradation within the same animal. The Ca-ATPase and ryanodine receptor isolated from aged muscle exhibits 27 +/- 5% and 25 +/- 3% slower protein turnover, respectively, relative to that from young muscle. In contrast, the 53-kDa glycoprotein exhibits a 25 +/- 5% more rapid turnover in aged SR, while calsequestrin exhibits no age-dependent alteration in turnover. Statistical analysis comparing the sensitivity of various methods for discriminating different rates of protein turnover validates the approach used in this study and demonstrates that the use of two isotopic labels provides at least a 6-fold more sensitive means to detect age-related differences in protein turnover relative to other methods.

Erinacin, an antihaemorrhagic factor from the European hedgehog, Erinaceus europaeus.

An antihaemorrhagic factor named erinacin was purified from the skeletal muscle extract of the European hedgehog, Erinaceus europaeus, by ammonium sulfate precipitation followed by various steps of ion-exchange (DEAE-cellulose), absorption chromatography (hydroxylapatite), and gel filtration (cellofine gel). A 625-fold purification was achieved with an overall yield of 19% antihaemorrhagic activity. The protein effectively inhibited the activity of Bothrops jararaca venom haemorrhagin and did not inhibit the enzymatic activity of trypsin and chymotrypsin. Erinacin is a large molecule (about 1,000,000 mol. wt). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of two subunits: one with an apparent mol. wt of 35,000 forming a larger subunit (350,000) by cross-linking with disulfide bridges, and a second with a mol. wt of 39,000 without disulfides. Dissociation of erinacin into its subunits resulted in complete loss of its antihaemorrhagic activity.

Coexistence of slow and fast isoforms of contractile and regulatory proteins in human skeletal muscle fibres induced by endurance training.

The distribution of fast and slow isoforms of troponin C, I, and T components and myosin heavy chains was investigated in histochemically typed myofibrillar ATPase intermediate (IM) fibres, that is, fibres that stain after both acid and alkaline preincubation in stainings for myofibrillar ATPase. In addition to the previously described IM fibres of types IIC and IB, fibres that displayed staining characteristics between types IIC and IB were observed and termed type IIC-IB. The IM fibres constitute less than 1% of the fibres in normal human limb and abdominal muscles. The IM fibres studied here resulted from extensive endurance training of human triceps brachii muscle (n = 6) and were induced by conversion of a proportion (13%) of type II fibres. The immunohistochemical stains of serial sections with antibodies to slow isoforms of troponin I, T, C and myosin heavy chain showed no staining of type II fibres but intense staining of types I and IB fibres, whereas type IIC fibres stained with intermediate intensity. The antibodies to fast isoforms of the troponin components and myosin heavy chain did not give rise to staining of type I fibres but dark staining of type II fibres. Type IB fibres stained with intermediate intensity and type IIC was either as dark as type II or slightly lighter. Type IIC-IB fibres showed staining intensities intermediate between those observed for types IB and IIC in the immunohistochemical stains. It is therefore concluded that training-induced myofibrillar ATPase intermediate human skeletal muscle fibres are characterized by the coexistence of slow and fast isoforms of contractile and regulatory proteins.(ABSTRACT TRUNCATED AT 250 WORDS)