Developmental decline in components of signal transduction pathways regulating protein synthesis in pig muscle

Our previous studies showed that the feeding-induced stimulation of protein synthesis in skeletal muscle of neonatal pigs is accompanied by enhanced phosphorylation of the eukaryotic initiation factor (eIF)4E-binding protein (4E-BP1) and the ribosomal protein S6 kinase (S6K1). These effects of feeding are substantially reduced with development. The goal of the present investigation was to delineate the basis for the reduced responsiveness to feeding observed in the older animals. In these studies, the content and activity of protein kinases located upstream of S6K1 and 4E-BP1 in signal transduction pathways activated by amino acids, insulin, and insulin-like growth factor I were examined in 7- and 26-day-old pigs that were either fasted overnight or fed porcine milk after an overnight fast. Feeding stimulated phosphatidylinositol (PI) 3-kinase activity to the same extent in muscle of 7- and 26-day-old pigs, suggesting that PI 3-kinase is not limiting in muscle of older animals. In contrast, protein kinase B (PKB) activity was significantly less in muscle from 26- vs. 7-day-old pigs, regardless of nutritional status, suggesting that its activity is regulated by mechanisms distinct from PI 3-kinase. In part, the reduced PKB responsiveness can be attributed to a developmental decline in PKB content. Likewise, muscle content of the protein kinase termed mammalian target of rapamycin (mTOR) in 26-day-old pigs was <25% of that in 7-day-old animals. Finally, in agreement with our earlier work showing that S6K1 phosphorylation is reduced in older animals, S6K1 activity was stimulated to a lesser extent in 26- compared with 7-day-old pigs. Overall, the results suggest that the blunted protein synthetic response observed in 26- vs. 7-day-old neonatal pigs is due in part to decreased content and/or activity of signaling components downstream of PI 3-kinase, e.g., PKB, mTOR, and S6K1.     

Contribution of insulin to the translational control of protein synthesis in skeletal muscle by leucine

Enhanced protein synthesis in skeletal muscle after ingestion of a balanced meal in postabsorptive rats is mimicked by oral leucine administration. To assess the contribution of insulin to the protein synthetic response to leucine, food-deprived (18 h) male rats (approximately 200 g) were intravenously administered a primed-constant infusion of somatostatin (60 microg + 3 microg.kg(-1).h(-1)) or vehicle beginning 1 h before administration of leucine (1.35 g L-leucine/kg) or saline (control). Rats were killed 15, 30, 45, 60, or 120 min after leucine administration. Compared with controls, serum insulin concentrations were elevated between 15 and 45 min after leucine administration but returned to basal values by 60 min. Somatostatin maintained insulin concentrations at basal levels throughout the time course. Protein synthesis was increased between 30 and 60 min, and this effect was blocked by somatostatin. Enhanced assembly of the mRNA cap-binding complex (composed of eukaryotic initiation factors eIF4E and eIF4G) and hyperphosphorylation of the eIF4E-binding protein 1 (4E-BP1), the 70-kDa ribosomal protein S6 kinase (S6K1), and the ribosomal protein S6 (rp S6) were observed as early as 15 min and persisted for at least 60 min. Somatostatin attenuated the leucine-induced changes in 4E-BP1 and S6K1 phosphorylation and completely blocked the change in rp S6 phosphorylation but had no effect on eIF4G small middle dot eIF4E assembly. Overall, the results suggest that the leucine-induced enhancement of protein synthesis and the phosphorylation states of 4E-BP1 and S6K1 are facilitated by the transient increase in serum insulin. In contrast, assembly of the mRNA cap-binding complex occurs independently of increases in insulin and, by itself, is insufficient to stimulate rates of protein synthesis in skeletal muscle after leucine administration.     

Reversing transmembrane electron flow: the DsbD and DsbB protein families

DsbD and DsbB are two proteins that in Escherichia coli catalyze transmembrane electron flow in opposite directions, thereby allowing reversible oxidoreduction of periplasmic dithiol/disulfide-containing proteins. We have identified all recognizable homologues of these two proteins in the databases and have conducted structural and phylogenetic analyses of the two families. The larger DsbD family is more diverse in sequence, topology, function and organismal distribution than the smaller DsbB family. DsbB homologues are rarely found outside of the proteobacteria, although DsbD homologues are found in many bacterial kingdoms as well as archaea and plant chloroplasts. Few organisms with a fully sequenced genome and a DsbB homologue lack a DsbD homologue, and most of these DsbD homologues fall within two clusters in the DsbD tree, exhibiting phylogenetic relationships that are the same as those observed for the DsbB proteins. These observations suggest that a subset of the DsbD homologues evolved in parallel with the DsbB family to perform a single unified function involving reversible extracytoplasmic protein dithiol-disulfide bond interchange. DsbD family proteins are shown to have arisen by an internal gene duplication event, and this observation leads to prediction of the pathway taken for the evolutionary appearance of the different protein topological types found within this family.     

4E-BP1 and S6K1: translational integration sites for nutritional and hormonal information in muscle

Maintenance of cellular protein stores in skeletal muscle depends on a tightly regulated synthesis-degradation equilibrium that is conditionally modulated under an extensive range of physiological and pathophysiological circumstances. Recent studies have established the initiation phase of mRNA translation as a pivotal site of regulation for global rates of protein synthesis, as well as a site through which the synthesis of specific proteins is controlled. The protein synthetic pathway is exquisitely sensitive to the availability of hormones and nutrients and employs a comprehensive integrative strategy to interpret the information provided by hormonal and nutritional cues. The translational repressor, eukaryotic initiation factor 4E binding protein 1 (4E-BP1), and the 70-kDa ribosomal protein S6 kinase (S6K1) have emerged as important components of this strategy, and together they coordinate the behavior of both eukaryotic initiation factors and the ribosome. This review discusses the role of 4E-BP1 and S6K1 in translational control and outlines the mechanisms through which hormones and nutrients effect changes in mRNA translation through the influence of these translational effectors.     

Effect of sepsis on eIE4E availability in skeletal muscle

Chronic septic abscess formation causes an inhibition of protein synthesis in gastrocnemius that is not observed in rats with a sterile abscess. The inhibition is associated with an impaired translation initiation. The present study was designed to investigate the effects of sepsis on phosphorylation and availability of eukaryotic initiation factor (eIF)4E in gastrocnemius 5 days after induction of a sterile or septic abscess. Neither sepsis nor sterile inflammation altered the extent of eIF4E phosphorylation. Moreover, no changes in the amount of the binding protein 4E-BP1 associated with eIF4E or in the phosphorylation of 4E-BP1 were observed during sepsis or sterile inflammation. In contrast, sepsis and sterile inflammation caused a reduction in the relative amount of eIF4G bound to eIF4E compared with controls. The diminished amount of eIF4G bound to eIF4E was not the result of a reduced abundance of eIF4E. Sepsis, but not sterile inflammation, caused an increase in the cellular abundance of eIF4E. The results provide evidence that alterations in the eIF4E system are probably not rate controlling for the synthesis of total, mixed proteins in gastrocnemius during sepsis. Instead, on the basis of our previous studies, changes in eIF2B appear to be responsible for limiting protein synthesis in skeletal muscle during sepsis.     

Flavonoid Responses in Wheat Grown at Elevated CO2: Green Versus Senescent Leaves

We compared flavonoids in green, mature, and senescing flag leaves of wheat grown under ambient (AC - 370 mol mol^-1) and elevated (EC - 550 mol mol^-1) concentrations of CO₂ in a FACE (Free Air CO₂ Enrichment) system. The concentrations of flag leaf flavonoids (e.g., isoorientin and tricin) decreased to one third in mature leaves, and the majoritary isoorientin almost disappeared in senescing leaves. Flavonoid concentrations increased in green well-developed flag leaves under EC (46 % isoorientin and 55 % tricin), whereas the differences disappeared in mature and senescing flag leaves. Predictions of changes in litter phenolic concentrations and their effects on decomposition rates under EC based on changes in green leaves need to be revised.