A large number of studies have evaluated rates and translation initiation of total protein synthesis in skeletal muscles in response to feeding during recent decades [13, 21]. However, such studies, mainly based on incorporation of labeled amino acids, suffer from uncertainties and complex assumptions for calculation of protein synthesis rate [10, 11, 22–24]. Difficulties occur particularly at rapid alterations in bio-dynamics during non-steady state conditions [10, 14]. Therefore, alternative and tracer independent methods have recently been applied in both animal and clinical experiments, complementary to tracer based methods. Such techniques are mainly based on assessment of phosphorylation/de-phosphorylation of regulatory proteins or protein complexes related to translation initiation of proteins where advantages are straight forward assessment of protein phosphorylation status in cells and tissues under evaluation without the need of steady state [6, 15]. Tissue sampling and processing are comparatively easy and analytical principles are robust at standardized conditions . However, limitations are that results reflect only initiation of overall protein bio-synthesis and do not reflect alterations of defined proteins. Determinations of the amount of a particular protein(s) in skeletal muscle tissue should in part resolve this problem, but is only applicable in long-term experiments, since it would be practically difficult to correctly assess quantitative alterations of defined proteins in skeletal muscle cells during short-term responses. Therefore, it should be possible to obtain relevant information by assessment of tissue transcript levels of defined myofibrillar proteins in response to feeding as applied in studies on orally refed healthy volunteers . Unexpectedly, it was then observed that oral refeeding caused a decline of myofibrillar transcripts in skeletal muscles, at conditions otherwise associated with anabolic metabolism [16, 26–28]. Such transcript information was seen in the light of observations that stimulation of gene transcription is usually reflected by increased tissue levels of transcripts for defined proteins aimed at subsequent translation to meet cellular requirements. [29–32]. Therefore, expected findings should be that net efflux of amino acids from skeletal muscles, due to increased net protein breakdown, should be associated with postprandial down-regulation in transcription of myofibrillar proteins. Normal oral feeding which is leading to rapid and pronounced activation of skeletal muscle protein synthesis, should then be characterized by increased transcription of required proteins . Based on this simplistic view, we decided to re-evaluate effects on transcripts of myofibrillar proteins as Myosin heavy chain 2A (myosin) and acta 1 (α-actin) in skeletal muscle tissue in response to refeeding, particularly with focus on effects by amino acids in both patient and animal experiments.
Myosin heavy chains contributes to 20-25% of overall muscle protein synthesis in humans [34, 35] while actin may display both lower and higher turnover compared to mixed muscle proteins [35, 36]. Muscle tissue is however composed of many different proteins where sarcoplasmatic and myofibrillar proteins have different basal turnover and synthesis rates at feeding . Adult human muscle tissue expresses three different isoforms of myosin heavy chain (MHC-I, MHC-IIa and MHC-IIx), where MHC-IIa is highly expressed in humans, while rodents express one additional form (MHC-IIb) .The myosin gene family is located in a cluster on chromosome 17 in humans and on chromosome 11 in mice . Studies have indicated that mRNA content of different myosin isoforms correlates to the relative content of various MHC proteins present in skeletal muscle tissue [40, 41] Changes in expression patterns of myosin heavy chain proteins exist in skeletal muscles during hypertrophy in the control of net muscle mass subsequent to loading [42–46], but less is known in response to feeding, although Mhc 2X mRNA is reported to unexpectedly increase after 7 days at reduced oral intake in rats . Our present findings show that that transcripts of myosin heavy chain 2A and actin appeared to decrease during continuous TPN administration in agreement with previous findings showing decreased MHC 2X mRNA levels at 3 hours after oral meal intake ;conditions that provide increased formation of eIF4G·eIF4E complex and decreased association of 4E-BP1·eIF4E .
There may be several reasons why myosin transcripts do not clear-cut reflect transcriptional activities and translational needs in cells during continuous long term nutrition exposure, although Rennie and coworkers  have reported transient changes in myofibrillar protein synthesis suggesting that muscle cells become refractory to amino acids in response to oral bolus feeding. However, long term provision of intravenous nutrition to patients leads to both time-proportional increases in muscle mass and continuously increased incorporation of labeled amino acids during the presence of high amino acid provision as seen in our present cell experiment (Figure 5). Therefore, it appears that transcript cellular levels of actin and myosin are influenced by a variety of factors that possibly determine absolute levels in both short and long term perspectives at nutrition.
It has never been finally assessed how amino acids signal across cell membranes to elicit triggers for induction of translation initiation, although it is assumed there are extracellular/intracellular amino acid sensors since muscle cells are sensitive to alterations of amino acid concentrations . Recently, amino acid transporter proteins gained increased interest based on their ability to sense amino acid changes and influence intracellular signaling . Regulation of expression of amino acid transporters may thus be an important part of the cell machinery in control of protein synthesis secondary to amino acids availability [49, 51]. Therefore, we investigated how transcription of the transporter protein Snat 2 (encoded by the gene slc38a2) was affected by refeeding in our models. Snat 2 is a transporter of neutral amino acids belonging to system A . Several amino acids in the refeeding medium (glutamine, histidine, cysteine, methonine) are transported by Snat 2, while branched chain- and aromatics are transported by system L across cell membranes [51, 52]. Amino acid transporting by system A increased following amino acid deprivation . Accordingly, we found that Snat 2 mRNA was lower in refed L6 cells compared to starved cells, although such alterations were not evident in vivo. Concentrations of Snat 2 mRNA were also decreased in refed cells by a group of amino acids (Gln, His, Lys, Arg, Thr). Refeeding L6 cells by branched chain amino acids decreased Snat 2 mRNA, although transported by system L, which operates by 1:1 amino acid exchange, which may couple influx of branched chain amino acids to efflux of cytoplasmatic amino acids such as glutamine . It is possible that refeeding cells with branched chain amino acids caused either efflux or influx of other amino acids, which may alter Snat 2 mRNA levels. If so, Snat 2 should be influenced by extra cellular concentrations or transmembrane fluxes of either Gln or His, since it was not changed by refeeding of cysteine or methione which are Snat 2 substrates.
Our microarray data on cultured cells confirm that amino acids have pronounced effects on steroid and lipid metabolism in skeletal muscle cells. Only GO categories/pathways related to lipid and steroid metabolism showed significant enrichment, although microarray experiments indicated that a large number of individual transcripts (30%) were changed following amino acid provision. It has been reported earlier that skeletal muscle cells are capable of local synthesis of sex steroid hormones , and there are several ways for cells to provide cholesterol for use in steroid synthesis, such as the mevalonate pathway, where cholesterol is synthesized through a series of enzyme reactions from Acetyl CoA and HMG-CoA [56, 57]. Thus, it was interesting to find that transcripts of all enzymes in this pathway were increased following amino acid provision to L6 cells. The expression of steroids and enzymes increases after exercise and may therefore represent an important part of anabolism following physical training in skeletal muscles . Thus, results in the present study confirm that amino acids have profound metabolic effects upstream to initiation of protein synthesis in cultured isolated skeletal muscle cells, as observed in animal and human skeletal muscle tissue [12, 59–64], in part related to individual groups of amino acids [65–69], as also observed in human biopsy specimens [17, 61].