Reevaluation of amino acid stimulation of protein synthesis in murine- and human-derived skeletal muscle cells assessed by independent techniques

Abstract

Iresjö, Britt-Marie, Elisabeth Svanberg, and Kent Lundholm. Reevaluation of amino acid stimulation of protein synthesis in murineand human-derived skeletal muscle cells assessed by independent techniques. Am J Physiol Endocrinol Metab 288: E1028 -E1037, 2005. First published December 14, 2004; doi:10.1152 doi:10. /ajpendo.00295.2004 and human rhabdomyosarcoma cells were cultured standardized in low (0.28 mM) and normal (9 mM) amino acid (AA) concentrations to reevaluate by independent methods to what extent AA activate initiation of protein synthesis. Methods used were incorporation of radioactive AA into proteins, distribution analysis of RNA in density gradient, and Western blots on initiation factors of translation of proteins in cultured cells as well as in vivo (gastrocnemius, C57Bl mice) during starvation/refeeding. Incorporation rate of AA gave incorrect results in a variety of conditions, where phenylalanine stimulated the incorporation rate of phenylalanine into proteins, but not of tyrosine, and tyrosine stimulated incorporation of tyrosine but not of phenylalanine. Similar problems were observed when [35 S]methionine was used for labeling of fractionated cellular proteins. However, the methods entirely independent of labeled AA incorporation indicated that essential AA activate initiation of translation, whereas nonessential AA did not. Branched-chain AA and glutamine, in combination with some other AA, also stimulated initiation of translation. Starvation/refeeding in vitro agreed qualitatively with results in vivo evaluated by initiation factors. Insulin at physiological concentrations (100 M/ml) did not stimulate global protein synthesis at low or normal AA concentrations but did so at supraphysiological levels (3 mU/ml), confirmed by independent methods. Our results reemphasize that labeled AA should be used with caution for quantification of protein synthesis, since the precursor pool(s) for protein synthesis is not in complete equilibrium with surrounding AA. "Flooding" tracee experiments did not overcome this problem. amino acids; insulin; initiation of translation; protein synthesis; muscle cells AROUND 45% OF THE BODY WEIGHT in adult humans is skeletal muscles, an important nitrogen reserve in different stress conditions such as trauma, infection, and starvation with balanced regulation of muscle protein synthesis and degradation to maintain muscle mass at functional levels (51). Feeding stimulates synthesis, whereas acute and chronic starvation will increase and decrease degradation, respectively (35). Mechanisms behind controlled protein balance in skeletal muscles over time have been extensively described in humans and animals based on studies in a variety of models from subcellular to cellular and tissue to organ levels (51). Yet, integrated signals behind protein balance in skeletal muscles are not fully understood, although several studies suggest amino acids in combination with hormones (insulin, IGF-I, and growth hormone) as key factors (1) communicated by intracellular phosphoprotein...

Initiation factors for translation of proteins in the rectus abdominis muscle from patients on overnight standard parenteral nutrition before surgery

“…Thus results may not always be indicative of what has been concluded, as emphasized by discrepancies between the incorporation rates of amino acids and phosphorylation/dephosphorylation of regulatory proteins [28]. Both under-and over-estimation of protein synthesis rates may occur when labelling techniques are used [13], and measurements of the net balance of amino acids across resting or exercising muscles may not fully account for exchanges between various tissue components in organ compartments [3,29]. This situation may appear particularly uncertain if extracellular amino acids are controlling initiation of translation without prior equilibration with intracellular amino acids [30,31].…”

“…Accordingly, most studies have relied on measurements of the net balance of amino acids across vascular beds or on labelling of mixed muscle proteins following a bolus or primed constant infusion of labelled amino acids [26,27], techniques that suffer from both advantages and limitations. Results from dynamic and static evaluations of protein synthesis should therefore be interpreted with caution, as required prerequisites are not always fulfilled [13]. Thus results may not always be indicative of what has been concluded, as emphasized by discrepancies between the incorporation rates of amino acids and phosphorylation/dephosphorylation of regulatory proteins [28].…”

“…Accordingly, direct and indirect evidence have been reported in numerous studies on amino acid stimulation of muscle protein synthesis in a variety of clinical conditions [9][10][11][12]. However, we [13] and others [12] have also reported that classical experiments with the labelling of proteins in assessing protein synthesis at steady-state conditions may sometimes give erroneous results, even in the presence of flooding tracee conditions. Therefore there is a need for additional methods to be used to analyse the activation of protein synthesis in vivo [14][15][16].…”

Measurement of human mixed muscle protein fractional synthesis rate depends on the choice of amino acid tracer

“…Since the difference in FSR obtained with the leucine and phenylalanine tracers was independent of the phenylalanine isotopomer infused, we can rule out differences due to potential errors in the preparation of enrichment standards and speculate that the discrepancy must be attributed to the amino acid itself. In fact, Iresjö et al (15) recently described a phenomenon that would fit this notion in cell culture, where phenylalanine stimulated the incorporation of a phenylalanine but not a tyrosine tracer into protein and vice versa. This was observed when large amounts of these amino acids (405 M) were in the medium and could therefore be related to amino acid competition for transporters.…”

Two‐dimensional gel electrophoresis and mass spectrometry for biomarker discovery

“…Pulse-chase experiments and 2-DE has been taken to study protein turnover in adipocyte-derived cell cultures [180], skeletal muscle-derived cells in culture [181] and in articular cartilage proteins [182].…”