Sirpiglenastat (DRP-104) Induces Antitumor Efficacy through Direct, Broad Antagonism of Glutamine Metabolism and Stimulation of the Innate and Adaptive Immune Systems.

Glutamine is a conditionally essential amino acid consumed by rapidly proliferating cancer cells, which deprives the same fuel from immune cells and contributes to tumor immune evasion. As such, the broad antagonism of glutamine in tumors and the tumor microenvironment may lead to direct antitumor activity and stimulation of antitumoral immune responses. DRP-104 (sirpiglenastat) was designed as a novel prodrug of the broad-acting glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON). DRP-104 is an inactive form that is preferentially converted to DON within tumors. Metabolomic profiling of tumors treated with DRP-104 revealed widespread changes indicative of the disruption of tumor anabolism and canonical cancer metabolism pathways; including altered glutamine metabolism while several immunosuppressive metabolites were decreased. Gene expression profiling revealed broad immunological modulation, confirmed by flow cytometry indicating that DRP-104 treatment resulted in substantial and broad changes in various immune cell infiltrates, such as increased TIL, T, NK, and NK T cells. Functionally, T cells became more proliferative and less exhausted; tumor-associated macrophages were polarized to the M1 phenotype; MDSCs and protumorigenic proteins were decreased in TME. Finally, DRP-104 demonstrated significant antitumor activity as a monotherapy, which was further enhanced in combination with checkpoint blockade therapies, leading to improved survival and long-term durable cures. In summary, DRP-104 broadly remodels the tumor microenvironment by inducing extensive tumor metabolism effects and enhancing the infiltration and function of multiple immune cells distinct from those obtained by checkpoint inhibitor therapy. This unique mechanism of action supports the ongoing clinical development of DRP-104 alone and in combination with checkpoint inhibitors.

Dietary essential amino acids restore liver metabolism in ovariectomized mice via hepatic estrogen receptor α.

In female mammals, the cessation of ovarian functions is associated with significant metabolic alterations, weight gain, and increased susceptibility to a number of pathologies associated with ageing. The molecular mechanisms triggering these systemic events are unknown because most tissues are responsive to lowered circulating sex steroids. As it has been demonstrated that isoform alpha of the estrogen receptor (ERα) may be activated by both estrogens and amino acids, we test the metabolic effects of a diet enriched in specific amino acids in ovariectomized (OVX) mice. This diet is able to block the OVX-induced weight gain and fat deposition in the liver. The use of liver-specific ERα KO mice demonstrates that the hepatic ERα, through the control of liver lipid metabolism, has a key role in the systemic response to OVX. The study suggests that the liver ERα might be a valuable target for dietary treatments for the post-menopause.

Sulfur sequestration promotes multicellularity during nutrient limitation.

The behaviour of Dictyostelium discoideum depends on nutrients1. When sufficient food is present these amoebae exist in a unicellular state, but upon starvation they aggregate into a multicellular organism2,3. This biology makes D. discoideum an ideal model for investigating how fundamental metabolism commands cell differentiation and function. Here we show that reactive oxygen species-generated as a consequence of nutrient limitation-lead to the sequestration of cysteine in the antioxidant glutathione. This sequestration limits the use of the sulfur atom of cysteine in processes that contribute to mitochondrial metabolism and cellular proliferation, such as protein translation and the activity of enzymes that contain an iron-sulfur cluster. The regulated sequestration of sulfur maintains D. discoideum in a nonproliferating state that paves the way for multicellular development. This mechanism of signalling through reactive oxygen species highlights oxygen and sulfur as simple signalling molecules that dictate cell fate in an early eukaryote, with implications for responses to nutrient fluctuations in multicellular eukaryotes.

The Effect of Leucine-Enriched Essential Amino Acid Supplementation on Anabolic and Catabolic Signaling in Human Skeletal Muscle after Acute Resistance Exercise: A Randomized, Double-Blind, Placebo-Controlled, Parallel-Group Comparison Trial.

Resistance exercise transiently activates anabolic and catabolic systems in skeletal muscle. Leucine-enriched essential amino acids (LEAAs) are reported to stimulate the muscle anabolic response at a lower dose than whey protein. However, little is known regarding the effect of LEAA supplementation on the resistance exercise-induced responses of the anabolic and catabolic systems. Here, we conducted a randomized, double-blind, placebo-controlled, parallel-group comparison trial to investigate the effect of LEAA supplementation on mechanistic target of rapamycin complex 1 (mTORC1), the ubiquitin-proteasome system and inflammatory cytokines after a single bout of resistance exercise in young men. A total of 20 healthy young male subjects were supplemented with either 5 g of LEAA or placebo, and then they performed 10 reps in three sets of leg extensions and leg curls (70% one-repetition maximum). LEAA supplementation augmented the phosphorylation of mTORSer2448 (+77.1%, p < 0.05), p70S6KThr389 (+1067.4%, p < 0.05), rpS6Ser240/244 (+171.3%, p < 0.05) and 4EBP1Thr37/46 (+33.4%, p < 0.05) after resistance exercise. However, LEAA supplementation did not change the response of the ubiquitinated proteins, MuRF-1 and Atrogin-1 expression. Additionally, the mRNA expression of IL-1ß and IL-6 did not change. These data indicated that LEAA supplementation augments the effect of resistance exercise by enhancing mTORC1 signal activation after exercise.

Seryl-tRNA synthetase-mediated essential amino acids regulate ß-casein synthesis via cell proliferation and mammalian target of rapamycin (mTOR) signaling pathway in bovine mammary epithelial cells.

Essential amino acids (EAA) play an important role in promoting milk protein synthesis in primary bovine mammary epithelial cells (BMEC). However, the regulatory mechanisms involved in the relationship between EAA and milk protein synthesis have not been fully explored. This study examined the effects of seryl-tRNA synthetase (SARS) on EAA-stimulated ß-casein synthesis, cell proliferation, and the mammalian target of rapamycin (mTOR) system in BMEC. First, BMEC were cultured in medium either lacking all EAA (-EAA) or that included all EAA (+EAA) for 12 h. The BMEC were then supplemented with the opposing treatments (-EAA supplemented with +EAA and vice versa) for 0 h, 10 min, 0.5 h, 1 h, 6 h, or 12 h, respectively. After the treatment-specific time allotment, proteins were collected for Western blotting. Subsequently, a 2 × 2 factorial design was used to evaluate the interactive of SARS inhibition (control or SARS inhibited) and EAA supply (+EAA or -EAA) on gene and protein abundance, cell viability, and cell cycle in BMEC. Based on the data obtained in the first experiment, the changes in protein abundance of ß-casein and SARS depended on EAA treatment time in similar patterns. The protein abundance of ß-casein, SARS, and mammalian target of rapamycin (mTOR)-related proteins, cell viability, cell cycle progression, and the mRNA abundance of cyclin D1 (CCND1, cell cycle progression marker) and marker of proliferation Ki-67 (MKI67, cell proliferation marker) were stimulated by the presence of EAA. Correspondingly, when cells were deprived of EAA, cell proliferation and abundance of these proteins and genes were reduced overall. Moreover, the decreases in these aspects were further exacerbated by inhibiting SARS, suggesting that an interaction between EAA and SARS is important for regulating protein synthesis. The results indicated that SARS stimulated the mTOR signaling pathway when EAA were present, enhanced EAA-stimulated cell proliferation, and contributed to increased ß-casein production in BMEC.

Muscle Protein Anabolic Resistance to Essential Amino Acids Does Not Occur in Healthy Older Adults Before or After Resistance Exercise Training.

Background: The muscle protein anabolic response to contraction and feeding may be blunted in older adults. Acute bouts of exercise can improve the ability of amino acids to stimulate muscle protein synthesis (MPS) by activating mechanistic target of rapamycin complex 1 (mTORC1) signaling, but it is not known whether exercise training may improve muscle sensitivity to amino acid availability. Objective: The aim of this study was to determine if muscle protein anabolism is resistant to essential amino acids (EAAs) and whether resistance exercise training (RET) improves muscle sensitivity to EAA in healthy older adults. Methods: In a longitudinal study, 19 healthy older adults [mean ± SD age: 71 ± 4 y body mass index (kg/m2): 28 ± 3] were trained for 12 wk with a whole-body program of progressive RET (60-75% 1-repetition maximum). Body composition, strength, and metabolic health were measured pre- and posttraining. We also performed stable isotope infusion experiments with muscle biopsies pre- and posttraining to measure MPS and markers of amino acid sensing in the basal state and in response to 6.8 g of EAA ingestion. Results: RET increased muscle strength by 16%, lean mass by 2%, and muscle cross-sectional area by 27% in healthy older adults (P < 0.05). MPS and mTORC1 signaling (i.e., phosphorylation status of protein kinase B, 4E binding protein 1, 70-kDa S6 protein kinase, and ribosomal protein S6) increased after EAA ingestion (P < 0.05) pre- and posttraining. RET increased basal MPS by 36% (P < 0.05); however, RET did not affect the response of MPS and mTORC1 signaling to EAA ingestion. Conclusion: RET increases strength and basal MPS, promoting hypertrophy in healthy older adults. In these subjects, a small dose of EAAs stimulates muscle mTORC1 signaling and MPS, and this response to EAAs does not improve after RET. Our data indicate that anabolic resistance to amino acids may not be a problem in healthy older adults. This trial was registered at www.clinicaltrials.gov as NCT02999802.

Leucine-Enriched Essential Amino Acids Augment Muscle Glycogen Content in Rats Seven Days after Eccentric Contraction.

Eccentric contractions induce muscle damage, which impairs recovery of glycogen and adenosine tri-phosphate (ATP) content over several days. Leucine-enriched essential amino acids (LEAAs) enhance the recovery in muscles that are damaged after eccentric contractions. However, the role of LEAAs in this process remains unclear. We evaluated the content in glycogen and high energy phosphates molecules (phosphocreatine (PCr), adenosine di-phosphate (ADP) and ATP) in rats that were following electrically stimulated eccentric contractions. Muscle glycogen content decreased immediately after the contraction and remained low for the first three days after the stimulation, but increased seven days after the eccentric contraction. LEAAs administration did not change muscle glycogen content during the first three days after the contraction. Interestingly, however, it induced a further increase in muscle glycogen seven days after the stimulation. Contrarily, ATP content decreased immediately after the eccentric contraction, and remained lower for up to seven days after. Additionally, LEAAs administration did not affect the ATP content over the experimental period. Finally, ADP and PCr levels did not significantly change after the contractions or LEAA administration. LEAAs modulate the recovery of glycogen content in muscle after damage-inducing exercise.

Postexercise essential amino acid supplementation amplifies skeletal muscle satellite cell proliferation in older men 24 hours postexercise.

Aged skeletal muscle has an attenuated and delayed ability to proliferate satellite cells in response to resistance exercise. The mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway is a focal point for cell growth, however, the effect of postexercise mTORC1 activation on human skeletal muscle satellite cell (SC) proliferation is unknown. To test the proliferative capacity of skeletal muscle SC in aging muscle to a potent mTORC1 activator (i.e., EAA; essential amino acids) we recruited older (~72y) men to conduct leg resistance exercise (8setsx10reps) without (-EAA; n = 8) and with (+EAA: n = 11) ingestion of 10 g of EAA 1 h postexercise. Muscle biopsies were taken before exercise (Pre) and 24 h postexercise (Post) for assessment of expression and fiber type-specific Pax7+ SC, Ki67+Pax7+ SC and MyoD+ SC -EAA did not show an increase in Pax7+ satellite cells at Post(P > 0.82). Although statistical significance for an increase in Pax7 +  SC at 24 h post-RE was not observed in +EAA versus -EAA, we observed trends for a treatment difference (P < 0.1). When examining the change from Pre to Post trends were demonstrated (#/myofiber: P = 0.076; and %/myonuclei: P = 0.065) for a greater increase in +EAA versus -EAA Notably, we found an increase SC proliferation in +EAA, but not -EAA with increase in Ki67+ SC and MyoD+ cells (P < 0.05). Ki67+ SC also exhibited a significant group difference Post (P < 0.010). Pax7+ SC in fast twitch myofibers did not change and were not different between groups (P > 0.10). CDK2, MEF2C, RB1 mRNA only increased in +EAA (P < 0.05). Acute muscle satellite cell proliferative capacity may be partially rescued with postexercise EAA ingestion in older men.

Essential amino acid mixtures drive cancer cells to apoptosis through proteasome inhibition and autophagy activation.

Cancer cells require both energy and material to survive and duplicate in a competitive environment. Nutrients, such as amino acids (AAs), are not only a caloric source, but can also modulate cell metabolism and modify hormone homeostasis. Our hypothesis is that the environmental messages provided by AAs rule the dynamics of cancer cell life or death, and the alteration of the balance between essential amino acids (EAAs) and non-essential amino acids (NEAAs) (lower and higher than 50%, respectively) present in nutrients may represent a key instrument to alter environment-dependent messages, thus mastering cancer cells destiny. In this study, two AA mixtures, one exclusively consisting of EAAs and the other consisting of 85% EAAs and 15% NEAAs, were tested to explore their effects on the viability of both normal and cancer cell lines and to clarify the molecular mechanisms involved. Both mixtures exerted a cell-dependent anti-proliferative, cytotoxic effect involving the inhibition of proteasome activity and the consequent activation of autophagy and apoptosis. These results, besides further validating the notion of the peculiar interdependence and extensive crosstalk between the ubiquitin-proteasome system (UPS) and autophagy, indicate that variation in the ratio of EAAs and NEAAs can deeply influence cancer cell survival. Consequently, customization of dietary ratios among EAAs and NEAAs by specific AA mixtures may represent a promising anticancer strategy able to selectively induce death of cancer cells through the induction of apoptosis via both UPS inhibition and autophagy activation.

Whey protein, amino acids, and vitamin D supplementation with physical activity increases fat-free mass and strength, functionality, and quality of life and decreases inflammation in sarcopenic elderly.

BACKGROUND: Interventions to attenuate the adverse effects of age-related loss of skeletal muscle and function include increased physical activity and nutritional supplementation. OBJECTIVE: This study tested the hypothesis that nutritional supplementation with whey protein (22 g), essential amino acids (10.9 g, including 4 g leucine), and vitamin D [2.5 µg (100 IU)] concurrent with regular, controlled physical activity would increase fat-free mass, strength, physical function, and quality of life, and reduce the risk of malnutrition in sarcopenic elderly persons. DESIGN: A total of 130 sarcopenic elderly people (53 men and 77 women; mean age: 80.3 y) participated in a 12-wk randomized, double-blind, placebo-controlled supplementation trial. All participants concurrently took part in a controlled physical activity program. We examined body composition with dual-energy X-ray absorptiometry, muscle strength with a handgrip dynamometer, and blood biochemical indexes of nutritional and health status, and evaluated global nutritional status, physical function, and quality of life before and after the 12 wk of intervention. RESULTS: Compared with physical activity and placebo, supplementation plus physical activity increased fat-free mass (1.7-kg gain, P < 0.001), relative skeletal muscle mass (P = 0.009), android distribution of fat (P = 0.021), handgrip strength (P = 0.001), standardized summary scores for physical components (P = 0.030), activities of daily living (P = 0.001), mini nutritional assessment (P = 0.003), and insulin-like growth factor I (P = 0.002), and lowered C-reactive protein (P = 0.038). CONCLUSION: Supplementation with whey protein, essential amino acids, and vitamin D, in conjunction with age-appropriate exercise, not only boosts fat-free mass and strength but also enhances other aspects that contribute to well-being in sarcopenic elderly. This trial was registered at clinicaltrials.gov as NCT02402608.

Leucine does not affect mechanistic target of rapamycin complex 1 assembly but is required for maximal ribosomal protein s6 kinase 1 activity in human skeletal muscle following resistance exercise.

We examined how the stimulatory effect of leucine on the mechanistic target of rapamycin complex 1 (mTORC1) pathway is affected by the presence of the remaining essential amino acids (EAAs). Nine male subjects performed resistance exercise on 4 occasions and were randomly supplied EAAs with leucine, EAAs without leucine (EAA-Leu), leucine alone, or flavored water (placebo; control). Muscle biopsies were taken from the vastus lateralis before and 60 and 90 min after exercise. Biopsies were analyzed for protein phosphorylation, kinase activity, protein-protein interactions, amino acid concentrations, and tracer incorporation. Leucine alone stimulated ribosomal protein s6 kinase 1 (S6K1) phosphorylation ∼280% more than placebo and EAA-Leu after exercise. Moreover, this response was enhanced by 60-75% after intake of EAAs compared with that of leucine alone (P < 0.05). Kinase activity of S6K1 reflected that of S6K1 phosphorylation; 60 min after exercise, the activity was elevated 3.3- and 4.2-fold with intake of leucine alone and with EAAs, respectively (P < 0.05). The interaction between mammalian target of rapamycin and regulatory-associated protein of mammalian target of rapamycin was unaltered in response to both resistance exercise and amino acid provision. Leucine alone stimulates mTORC1 signaling, although this response is enhanced by other EAAs and does not appear to be caused by alterations in mTORC1 assembly.

Dietary supplementation with essential amino acids boosts the beneficial effects of rosuvastatin on mouse kidney.

The effects of high-potency statins on renal function are controversial. To address the impact of statins on renal morpho-functional aspects, normotensive young mice were treated with rosuvastatin (Rvs). Moreover, because statins may impair mitochondrial function, mice received either dietary supplementation with an amino acid mixture enriched in essential amino acids (EAAm), which we previously demonstrated to increase mitochondrial biogenesis in muscle or an unsupplemented control diet for 1 month. Mitochondrial biogenesis and function, apoptosis, and insulin signaling pathway events were studied, primarily in cortical proximal tubules. By electron microscopy analysis, mitochondria were more abundant and more heterogeneous in size, with dense granules in the inner matrix, in Rvs- and Rvs plus EAAm-treated animals. Rvs administration increased protein kinase B and endothelial nitric oxide synthase phosphorylation, but the mammalian target of rapamycin signaling pathway was not affected. Rvs increased the expression of sirtuin 1, peroxisome proliferator-activated receptor γ coactivator-1α, cytochrome oxidase type IV, cytochrome c, and mitochondrial biogenesis markers. Levels of glucose-regulated protein 75 (Grp75), B-cell lymphoma 2, and cyclin-dependent kinase inhibitor 1 were increased in cortical proximal tubules, and expression of the endoplasmic reticulum-mitochondrial chaperone Grp78 was decreased. EAAm supplementation maintained or enhanced these changes. Rvs promotes mitochondrial biogenesis, with a probable anti-apoptotic effect. EAAm boosts these processes and may contribute to the efficient control of cellular energetics and survival in the mouse kidney. This suggests that appropriate nutritional interventions may enhance the beneficial actions of Rvs, and could potentially prevent chronic renal side effects.

Effects of AMP-activated protein kinase (AMPK) signaling and essential amino acids on mammalian target of rapamycin (mTOR) signaling and protein synthesis rates in mammary cells.

Regulation of mammary protein synthesis potentially changes the relationships between AA supply and milk protein output represented in current nutrient requirement models. Glucose and AA regulate muscle protein synthesis via cellular signaling pathways involving mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK). The objective of this study was to investigate the effects of essential AA (EAA) and acetate or glucose on mTOR and AMPK signaling pathways and milk protein synthesis rates. A bovine mammary epithelial cell line, MAC-T, was subjected to different media containing 0 or 3.5 mmol/L EAA concentrations with 0 or 5 mmol/L acetate or 0 or 17.5 mmol/L glucose in 2 separate 2 × 2 factorial studies. In a separate set of experiments, lactogenic bovine mammary tissue slices were subjected to the same treatments except that the low EAA treatment contained a low level of EAA (0.18 mmol/L). Supplementation of EAA enhanced phosphorylation of mTOR (Ser2448) and eukaryotic initiation factor 4E binding protein 1 (4EBP1, Thr37/46), and reduced phosphorylation of eukaryotic elongation factor 2 (eEF2, Thr56) in MAC-T cells. Concentration of ATP and phosphorylation of AMPK increased and decreased, respectively, in the presence of EAA in MAC-T cells. Acetate, EAA, or glucose numerically reduced AMPK phosphorylation by about 16% in mammary tissue slices. Provision of EAA increased phosphorylation of mTOR and 4EBP1, intracellular total EAA concentration, and casein synthesis rates in mammary tissue slices, irrespective of the presence of acetate or glucose in the medium. Phosphorylation of mTOR had a marginally negative association with AMPK phosphorylation, which was positively related to eEF2 phosphorylation. Casein synthesis rates were positively and more strongly linked to mTOR phosphorylation than the negative link between eEF2 phosphorylation and casein synthesis rates. A 100% increase in mTOR phosphorylation was associated with an increase in the casein synthesis rate of 0.74%·h(-1), whereas a 100% increase in eEF2 phosphorylation was related to a decline in the casein synthesis rate of 0.33%·h(-1). Although AMPK phosphorylation was responsive to cellular energy status and had a negative effect on mTOR-mediated signals in bovine mammary epithelial cells, its effect on milk protein synthesis rates appeared to be marginal compared with the mTOR-mediated regulation of milk protein synthesis by EAA.

Addition of carbohydrate or alanine to an essential amino acid mixture does not enhance human skeletal muscle protein anabolism.

In humans, essential amino acids (EAAs) stimulate muscle protein synthesis (MPS) with no effect on muscle protein breakdown (MPB). Insulin can stimulate MPS, and carbohydrates (CHOs) and insulin decrease MPB. Net protein balance (NB; indicator of overall anabolism) is greatest when MPS is maximized and MPB is minimized. To determine whether adding CHO or a gluconeogenic amino acid to EAAs would improve NB compared with EAA alone, young men and women (n = 21) ingested 10 g EAA alone, with 30 g sucrose (EAA+CHO), or with 30 g alanine (EAA+ALA). The fractional synthetic rate and phenylalanine kinetics (MPS, MPB, NB) were assessed by stable isotopic methods on muscle biopsies at baseline and 60 and 180 min following nutrient ingestion. Insulin increased 30 min postingestion in all groups and remained elevated in the EAA+CHO and EAA+ALA groups for 60 and 120 min, respectively. The fractional synthetic rate increased from baseline at 60 min in all groups (P < 0.05; EAA = 0.053 ± 0.018 to 0.090 ± 0.039% · h(-1); EAA+ALA = 0.051 ± 0.005 to 0.087 ± 0.015% · h(-1); EAA+CHO = 0.049 ± 0.006 to 0.115 ± 0.024% · h(-1)). MPS and NB peaked at 30 min in the EAA and EAA+CHO groups but at 60 min in the EAA+ALA group and NB was elevated above baseline longer in the EAA+ALA group than in the EAA group (P < 0.05). Although responses were more robust in the EAA+CHO group and prolonged in the EAA+ALA group, AUCs were similar among all groups for fractional synthetic rate, MPS, MPB, and NB. Because the overall muscle protein anabolic response was not improved in either the EAA+ALA or EAA+CHO group compared with EAA, we conclude that protein nutritional interventions to enhance muscle protein anabolism do not require such additional energy.

Glutamine stimulates mTORC1 independent of the cell content of essential amino acids.

Glutamine and leucine are important mTORC1 modulators, although their roles are not precisely defined. In HepG2 and HeLa cells glutamine-free incubation lowers mTORC1 activity, although cell leucine is not decreased. mTORC1 activity, suppressed by amino acid-free incubation, is completely rescued only if essential amino acids (EAA) and glutamine are simultaneously restored, although cell leucine is higher in the absence than in the presence of glutamine. Thus, glutamine stimulates mTORC1 independent of cell leucine, suggesting the existence of two distinct stimulatory signals from either glutamine or EAA.

Direct sensing of systemic and nutritional signals by haematopoietic progenitors in Drosophila.

The Drosophila lymph gland is a haematopoietic organ in which progenitor cells, which are most akin to the common myeloid progenitor in mammals, proliferate and differentiate into three types of mature cell--plasmatocytes, crystal cells and lamellocytes--the functions of which are reminiscent of mammalian myeloid cells. During the first and early second instars of larval development, the lymph gland contains only progenitors, whereas in the third instar, a medial region of the primary lobe of the lymph gland called the medullary zone contains these progenitors, and maturing blood cells are found juxtaposed in a peripheral region designated the cortical zone. A third group of cells referred to as the posterior signalling centre functions as a haematopoietic niche. Similarly to mammalian myeloid cells, Drosophila blood cells respond to multiple stresses including hypoxia, infection and oxidative stress. However, how systemic signals are sensed by myeloid progenitors to regulate cell-fate determination has not been well described. Here, we show that the haematopoietic progenitors of Drosophila are direct targets of systemic (insulin) and nutritional (essential amino acid) signals, and that these systemic signals maintain the progenitors by promoting Wingless (WNT in mammals) signalling. We expect that this study will promote investigation of such possible direct signal sensing mechanisms by mammalian myeloid progenitors.

Effects of essential amino acids or glutamine deprivation on intestinal permeability and protein synthesis in HCT-8 cells: involvement of GCN2 and mTOR pathways.

GCN2 and mTOR pathways are involved in the regulation of protein metabolism in response to amino acid availability in different tissues. However, regulation at intestinal level is poorly documented. The aim of the study was to evaluate the effects of a deprivation of essential amino acids (EAA) or glutamine (Gln) on these pathways in intestinal epithelial cells. Intestinal epithelial cell, HCT-8, were incubated during 6 h with 1/DMEM culture medium containing EAA, non EAA and Gln, 2/with saline as positive control of nutritional deprivation, 3/DMEM without EAA, 4/DMEM without Gln or 5/DMEM without Gln and supplemented with a glutamine synthase inhibitor (MSO, 4 mM). Intestinal permeability was evaluated by the measure of transepithelial electric resistance (TEER). Using [L-(2)H(3)]-leucine incorporation, fractional synthesis rate (FSR) was calculated from the assessed enrichment in proteins and free amino acid pool by GCMS. Expression of eiF2α (phosphorylated or not), used as marker of GCN2 pathway, and of 4E-BP1 (phosphorylated or not), used as a marker of mTOR pathway, was evaluated by immunoblot. Results were compared by ANOVA. Six-hours EAA deprivation did not significantly affect TEER and FSR but decreased p-4E-BP1 and increased p-eiF2α. In contrast, Gln deprivation decreased FSR and p-4E-BP1. MSO induced a marked decrease of TEER and FSR and an increase of p-eiF2α, whereas mTOR pathway remained activated. These results suggest that both mTOR and GCN2 pathways can mediate the limiting effects of Gln deprivation on protein synthesis according to its severity.

Essential amino acids regulate both initiation and elongation of mRNA translation independent of insulin in MAC-T cells and bovine mammary tissue slices.

Current nutrient requirement models assume fixed efficiencies of absorbed amino acid (AA) conversion to milk protein. Regulation of mammary protein synthesis (PS) potentially violates this assumption by changing the relationship between AA supply and milk protein output. The objective of this study was to investigate the effects of essential AA (EAA) and insulin on cellular signaling and PS rates in bovine mammary cells. MAC-T cells were subjected to 0 or 100% of normal EAA concentrations in DMEM/F12 and 0 or 100 µg insulin/L in a 2 × 2 factorial arrangement of treatments. Lactogenic bovine mammary tissue slices (MTS) were subjected to the same treatments, except low-EAA was 5% of normal DMEM/F12 concentrations. In MAC-T cells, EAA increased phosphorylation of mammalian target of rapamycin (mTOR; Ser2448), ribosomal protein S6 kinase 1 (S6K1; Thr389), eIF4E binding protein 1 (4EBP1; Thr37/46), and insulin receptor substrate 1 (IRS1; Ser1101), and reduced phosphorylation of eukaryotic elongation factor 2 (eEF2; Thr56) and eukaryotic initiation factor (eIF) 2-α (Ser51). In the presence of insulin, phosphorylation of Akt (Ser473), mTOR, S6K1, 4EBP1, and IRS1 increased in MAC-T cells. In MTS, EAA had similar effects on phosphorylation of signaling proteins and increased mammary PS rates. Insulin did not affect MTS signaling, perhaps due to inadequate levels. Effects of EAA and insulin were independent and additive for mTOR signaling in MAC-T cells. EAA did not inhibit insulin stimulation of Akt phosphorylation. PS rates were strongly associated with phosphorylation of 4EBP1 and eEF2 in MTS. EAA availability affected translation initiation and elongation control points to more strongly regulate PS than insulin.

Essential amino acids improve insulin activation of AKT/MTOR signaling in soleus muscle of aged rats.

Essential amino acids (EAA) improve basal muscle protein synthesis in the elderly. Nevertheless, in settings of prolonged supplementation, putative signal pathways of EAA are currently unknown. The purpose of this study was to test the effects of prolonged supplementation of EAA enriched mixture (12-L-Amin) on Insulin/Insulin-like Growth Factor-1 (IGF1) pathway by measuring total and phosphorylated Akt (Ser473) and its upstream (IRS1 at Ser636) and downstream (mTOR at Ser2448, p70S6K at Thr389) targets in basal conditions and following acute insulin (0.1 U/L) incubation in vitro. To this aim, soleus muscles were dissected from male Wistar rats divided in three groups of 7 each: adults (AD, 10 mo of age), elderly (EL, 22 mo of age) and elderly supplemented (EL-AA, 12-L-Amin 1.5gr/Kg die in drinking water for 3 mo). EL showed reduced basal and post-insulin mTOR and p70S6K activation and reduced post-insulin IRS1 degradation relative to AD. EL-AA showed an increase of post-insulin Akt activation, no change in basal and post-insulin phospho-mTOR, lower reduction of phospho-p70S6K and increased post-insulin IRS1 degradation relative to AD. These results demonstrate that chronic 12-LAmin administration exerts anti-ageing effects on the activation/inactivation of the Insulin/IGF1/mTOR pathway which is identified as putative target of EAA in the elderly.

Chronic improvement of amino acid nutrition stimulates initiation of global messenger ribonucleic acid translation in tissues of sheep without affecting protein elongation.

Initiation of mRNA translation and elongation of the polypeptide chain are 2 regulated processes responsible for the short-term postprandial acceleration of protein synthesis in animal tissues. It is known that a chronic increase in the absorptive supply of AA stimulates protein synthesis in ruminant animals, but effects on translation initiation and elongation are unknown. To determine whether initiation or elongation phases of global mRNA translation are affected by chronic elevation of AA supply, 24 ewe lambs of 25.9 +/- 2.5 kg of BW were randomly allocated to 4 treatment groups of 6 lambs each. All lambs received a basal diet of barley and hay at 1.2 times maintenance ME intake. Treatments were an intravenous (i.v.) saline infusion as a control, i.v. infusion of 6 essential AA (EAA; Arg, Lys, His, Thr, Met, Cys) for 10 d, i.v. infusion of the same EAA excluding Met and Cys (EAA-SAA) for 10 d, and an oral drench of fishmeal twice daily for 17 d. Fishmeal supplementation supplied an extra 719 mg of N x kg(-0.75) x d(-1) and N retention was increased 519 mg x kg(-0.75) x d(-1) over the control. The EAA treatment supplied an extra 343 mg of N x kg(-0.75) x d(-1) directly into the blood, and N balance was increased by 268 mg x kg(-0.75) x d(-1). Deletion of Met plus Cys from EAA had no effect on N balance. The results indicate that Met plus Cys did not limit body protein gain on the basal diet alone or the basal diet plus 6 AA. Protein fractional synthesis rates in liver, duodenum, skin, rumen, semimembranosus, and LM were measured by a flooding dose procedure using L-[ring-2,6-(3)H]-Phe. Ribosome transit times were estimated from the ratio of nascent to total protein-bound radioactivities. Fishmeal and EAA treatments had no effect on RNA, DNA, or protein contents of tissues, but fractional synthesis rate, translational efficiency, and concentrations of active ribosomes were consistently elevated. Ribosome transit time was not affected by long-term AA supply. We conclude that the chronic stimulation of protein synthesis by long-term i.v. infusion of EAA or supplementation with an undegradable protein source is brought about by an improvement in the rate of initiation of mRNA translation with no change in the rate of polypeptide chain elongation.