Protein synthesis, in particular peptide-chain elongation, consumes cellular energy. Anoxia activates AMP-activated protein kinase (AMPK, see ), resulting in the inhibition of biosynthetic pathways to conserve ATP. In anoxic rat hepatocytes or in hepatocytes treated with 5-aminoimidazole-4-carboxamide (AICA) riboside, AMPK was activated and protein synthesis was inhibited. The inhibition of protein synthesis could not be explained by changes in the phosphorylation states of initiation factor 4E binding protein-1 (4E-BP1) or eukaryotic initiation factor 2alpha (eIF2alpha). However, the phosphorylation state of eukaryotic elongation factor 2 (eEF2) was increased in anoxic and AICA riboside-treated hepatocytes and in AICA riboside-treated CHO-K1 cells, and eEF2 phosphorylation is known to inhibit its activity. Incubation of CHO-K1 cells with increasing concentrations of 2-deoxyglucose suggested that the mammalian target of the rapamycin (mTOR) signaling pathway did not play a major role in controlling the level of eEF2 phosphorylation in response to mild ATP depletion. In HEK293 cells, transfection of a dominant-negative AMPK construct abolished the oligomycin-induced inhibition of protein synthesis and eEF2 phosphorylation. Lastly, eEF2 kinase, the kinase that phosphorylates eEF2, was activated in anoxic or AICA riboside-treated hepatocytes. Therefore, the activation of eEF2 kinase by AMPK, resulting in the phosphorylation and inactivation of eEF2, provides a novel mechanism for the inhibition of protein synthesis.

The ability of insulin to promote the phosphorylation of some proteins and the dephosphorylation of others is paradoxical. An insulin-stimulated protein kinase is shown to activate the type-1 protein phosphatase that controls glycogen metabolism, by phosphorylating its regulatory subunit at a specific serine. Furthermore, the phosphorylation of this residue is stimulated by insulin in vivo. Increased and decreased phosphorylation of proteins by insulin can therefore be explained through the same basic underlying mechanism.

Argininosuccinate synthetase (ASS, EC 6.3.4.5) catalyses the condensation of citrulline and aspartate to form argininosuccinate, the immediate precursor of arginine. First identified in the liver as the limiting enzyme of the urea cycle, ASS is now recognized as a ubiquitous enzyme in mammalian tissues. Indeed, discovery of the citrulline–NO cycle has increased interest in this enzyme that was found to represent a potential limiting step in NO synthesis. Depending on arginine utilization, location and regulation of ASS are quite different. In the liver, where arginine is hydrolyzed to form urea and ornithine, the ASS gene is highly expressed, and hormones and nutrients constitute the major regulating factors: (a) glucocorticoids, glucagon and insulin, particularly, control the expression of this gene both during development and adult life; (b) dietary protein intake stimulates ASS gene expression, with a particular efficiency of specific amino acids like glutamine. In contrast, in NO‐producing cells, where arginine is the direct substrate in the NO synthesis, ASS gene is expressed at a low level and in this way, proinflammatory signals constitute the main factors of regulation of the gene expression. In most cases, regulation of ASS gene expression is exerted at a transcriptional level, but molecular mechanisms are still poorly understood.

The insulin-stimulated protein kinase (ISPK) was purified over 50 000-fold from extracts of rabbit skeletal muscle by a procedure involving chromatography on phosphocellulose, fractionation with ammonium sulphate, and further chromatography on DEAE-cellulose, phenyl-Superose, Mono S and Mono Q. About 10 pg enzyme was isolated from 800 g muscle (one rabbit) in four days with an overall recovery of 5%. The purified enzyme showed a single protein-staining band of apparent molecular mass 91 kDa when analysed by SDS/polyacrylamide gel electrophoresis. The ISPK comigrated during SDS/polyacrylamide gel electrophoresis with the enzyme S6 kinase I1 from Xenopus eggs, and was recognised in immunoblotting and immunoprecipitation experiments by antibodies raised against S6 kinase 11. The substrate specificities of ISPK and S6 kinase I1 were also very similar and like S6 kinase 11, ISPK that had been inactivated by protein phosphatase 2A could be reactivated by incubation with mitogen-activated protein kinase and MgATP. ISPK was distinct from an insulin-stimulated 70-kDa S6 kinase from rat liver in both substrate specificity and immunological cross reactivity. It is concluded that ISPK is closely related in structure to S6 kinase I1 and may be a mammalian equivalent of this enzyme. The possibility that ISPK is involved in mediating a number of the actions of insulin is discussed.We have recently identified a protein kinase in rabbit skeletal muscle whose activity is stimulated several-fold within 15 min of an acute injection of insulin [I]. This enzyme, termed the insulin-stimulated protein kinase (ISPK) activates the glycogen-associated form of protein phosphatase 1 (PPlG) by phosphorylating its glycogen-targetting subunit (G-subunit) on a particular serine residue, termed site-I . This increases by 2.5 -3-fold the rate at which PPlG dephosphorylates (activates) glycogen synthase and dephosphorylates (inactivates) phosphorylase kinase. This appears to represent the mechanism by which insulin stimulates glycogen synthesis and inhibits glycogenolysis in skeletal muscle, since the phosphorylation of site-1 on the glycogen-targetting subunit has also been shown to increase in vivo in response to insulin [I].The ISPK is inactivated by incubation with protein phosphatase 2A (PP2A) and cannot be reactivated by incubation with MgATP [l]. This indicates that a distinct kinase kinase is required to activate ISPK and that insulin probably exerts its effect by activating the kinase kinase. Here, we have purified ISPK to homogeneity and examined its specificity and regulatory properties. These and other experiments demonstrate that ISPK is very similar to an enzyme, termed S6 kinase I1 that was first identified in Xenopus eggs [2, 31. MATERIALS AND METHODS MaterialsPPlG [4], glycogen synthase [5] and the catalytic subunit of PP2A [6] from rabbit skeletal muscle, the 70-kDa S6 kinase from cycloheximide-treated rat liver [7], and S6 kinase I [8] and S6 kinase I1 [2] from Xenopus eggs were homogeneous preparations isolated as described pr...

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chelotte. Enteral glutamine stimulates protein synthesis and decreases ubiquitin mRNA level in human gut mucosa. Am J Physiol Gastrointest Liver Physiol 285: 266-G273, 2003. First published April 17, 2003 10.1152/ ajpgi.00385.2002.-Effects of glutamine on whole body and intestinal protein synthesis and on intestinal proteolysis were assessed in humans. Two groups of healthy volunteers received in a random order enteral glutamine (0.8 mmol ⅐ kg body wt Ϫ1 ⅐ h Ϫ1 ) compared either to saline or isonitrogenous amino acids. Intravenous [ 2 H5]phenylalanine and [ 13 C]leucine were simultaneously infused. After gas chromatography-mass spectrometry analysis, whole body protein turnover was estimated from traced plasma amino acid fluxes and the fractional synthesis rate (FSR) of gut mucosal protein was calculated from protein and intracellular phenylalanine and leucine enrichments in duodenal biopsies. mRNA levels for ubiquitin, cathepsin D, and m-calpain were analyzed in biopsies by RT-PCR. Glutamine significantly increased mucosal protein FSR compared with saline. Glutamine and amino acids had similar effects on FSR. The mRNA level for ubiquitin was significantly decreased after glutamine infusion compared with saline and amino acids, whereas cathepsin D and m-calpain mRNA levels were not affected. Enteral glutamine stimulates mucosal protein synthesis and may attenuate ubiquitin-dependent proteolysis and thus improve protein balance in human gut. intestine; protein metabolism; nutrients GLUTAMINE IS THE MAJOR FUEL for enterocytes, and it promotes intestinal growth and metabolism and maintains the structure and function of intestinal mucosa, especially in situations of gut injury (38) or after gut resection in animals (37). In vitro, glutamine stimulated intestinal cell proliferation (33). Numerous studies have reported the benefits of a glutamine enteral or parenteral nutritional supplementation on gut barrier (20,42). In animal studies, glutamine supply decreased the alterations of gut mucosa induced by prolonged starvation (20) or an experimental enterocolitis (2) by increasing the weight of the mucosa, the height of villi, and DNA and protein content. In humans, glutamineenriched parenteral nutrition maintained villus height and limited the increase of gut permeability (42). Treatment of patients with glutamine, growth hormone, and diet modifications after gut resection was reported to be beneficial on water and electrolyte absorption in an early uncontrolled study (5), but this has not been confirmed by more recent controlled studies (36).Additionally, enteral infusion of a high glutamine load in volunteers altered whole body leucine fluxes in a manner indicating a reduced protein oxidation and an increased protein synthesis (16). The beneficial effects of glutamine on gut mucosa could be partly due to a stimulation of protein synthesis as shown in animal studies, in vitro (17) and in vivo (41). In previous studies, we have shown that glutamine was well absorbed in human intestine (13) and stimulated ϳ40% duoden...

A growing number of reports clearly demonstrate that amino acids are able to control physiological functions at different levels, including the initiation of protein translation, mRNA stabilization and gene transcription [1][2][3]. Although the molecular mechanisms involved in the control of gene expression by amino Molecular data rapidly accumulating on the regulation of gene expression by amino acids in mammalian cells highlight the large variety of mechanisms that are involved. Transcription factors, such as the basic-leucine zipper factors, activating transcription factors and CCAAT/enhancer-binding protein, as well as specific regulatory sequences, such as amino acid response element and nutrient-sensing response element, have been shown to mediate the inhibitory effect of some amino acids. Moreover, amino acids exert a wide range of effects via the activation of different signalling pathways and various transcription factors, and a number of cis elements distinct from amino acid response element/nutrient-sensing response element sequences were shown to respond to changes in amino acid concentration. Particular attention has been paid to the effects of glutamine, the most abundant amino acid, which at appropriate concentrations enhances a great number of cell functions via the activation of various transcription factors. The glutamine-responsive genes and the transcription factors involved correspond tightly to the specific effects of the amino acid in the inflammatory response, cell proliferation, differentiation and survival, and metabolic functions. Indeed, in addition to the major role played by nuclear factor-jB in the anti-inflammatory action of glutamine, the stimulatory role of activating protein-1 and the inhibitory role of C/EBP homology binding protein in growth-promotion, and the role of c-myc in cell survival, many other transcription factors are also involved in the action of glutamine to regulate apoptosis and intermediary metabolism in different cell types and tissues. The signalling pathways leading to the activation of transcription factors suggest that several kinases are involved, particularly mitogen-activated protein kinases. In most cases, however, the precise pathways from the entrance of the amino acid into the cell to the activation of gene transcription remain elusive.

Glutamine stimulated glycogen synthesis and lactate production in hepatocytes from overnight-fasted normal and diabetic rats. The effect, which was half-maximal with about 3 mM-glutamine, depended on glucose concentration and was maximal below 10 mM-glucose. beta-2-Aminobicyclo[2.2.1.]heptane-2-carboxylic acid, an analogue of leucine, stimulated glutaminase flux, but inhibited the stimulation of glycogen synthesis by glutamine. Various purine analogues and inhibitors of purine synthesis were found to inhibit glycogen synthesis from glucose, but they did not abolish the stimulatory effect of glutamine on glycogen synthesis. The correlation between the rate of glycogen synthesis and synthase activity suggested that the stimulation of glycogen synthesis by glutamine depended solely on the activation of glycogen synthase. This activation of synthase was not due to a change in total synthase, nor was it caused by a faster inactivation of glycogen phosphorylase, as was the case after glucose. It could, however, result from a stimulation of synthase phosphatase, since, after the addition of 1 nM-glucagon or 10 nM-vasopressin, glutamine did not interfere with the inactivation of synthase, but did promote its subsequent re-activation. Glutamine was also found to inhibit ketone-body production and to stimulate lipogenesis.