Amino Acid Stimulation Research Articles

Context-dependent impact of the dietary non-essential amino acid tyrosine on Drosophila physiology and longevity.

Dietary protein intake modulates growth, reproduction, and longevity by stimulating amino acid (AA)-sensing pathways. Essential AAs are often considered as limiting nutrients during protein scarcity, and the role of dietary non-essential AAs (NEAAs) is less explored. Although tyrosine has been reported to be crucial for sensing protein restriction in Drosophila larvae, its effect on adult physiology and longevity remains unclear. Here, using a synthetic diet, we perform a systematic investigation of the effect of single NEAA deprivation on nutrient-sensing pathways, reproductive ability, starvation resistance, feeding behavior, and life span in adult female flies. Specifically, dietary tyrosine deprivation decreases internal tyrosine levels and fecundity, influences AA-sensing machineries, and extends life span. These nutritional responses are not observed under higher total AA intake or in infertile female flies, suggesting a context-dependent influence of dietary tyrosine. Our findings highlight the unique role of tyrosine as a potentially limiting nutrient, underscoring its value for dietary interventions aimed at enhancing health span.

Sodium disilicate pretreatment enhancing methane production from anaerobic digestion of waste activated sludge

Improving the efficiency of digestion is a major focus of waste activated sludge (WAS) anaerobic digestion (AD) research. This study investigates the viability and mechanism of sodium disilicate (SD) in enhancing the digestive efficacy of WAS. Batch AD experiments for the sludges pretreated by varied amounts of SD were carried out, and the results revealed that a dosage of 90 mmol/L resulted in the highest methane yield (247.9 mL/g·VS), marking a 90.0 % increase compared to the raw sludge. Mechanistic investigations highlighted that SD pretreatment facilitated EPS degradation, cell lysis, and modulation of interfacial interaction energy, ultimately enhancing sludge disintegration and methane generation. Microbial community analysis unveiled an enrichment of crucial functional microorganisms associated with hydrolysis, acidification, and methanation. The heightened presence of acetotrophic and hydrogenotrophic methanogens significantly boosted methane production. Furthermore, metagenomic analysis corroborated that the SD pretreatment stimulated amino acid metabolism, energy metabolism (specifically ABC transporter proteins), and carbohydrate metabolism, thereby enhancing methanogenic activity through increased microbial metabolic activities and upregulated key enzyme expression. This research underscores the potential of SD-assisted pretreatment strategies in augmenting WAS digestion, introducing supplementary pretreatment avenues for optimizing anaerobic biotechnology.

Cancer EV stimulate endothelial glycolysis to fuel protein synthesis via mTOR and AMPKα activation.

Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells that induce therapy resistance and has profound effects on cellular metabolism. As such, hypoxia is an important contributor to cancer progression and is associated with a poor prognosis. Metabolic alterations in cells within the tumour microenvironment support tumour growth via, amongst others, the suppression of immune reactions and the induction of angiogenesis. Recently, extracellular vesicles (EV) have emerged as important mediators of intercellular communication in support of cancer progression. Previously, we demonstrated the pro-angiogenic properties of hypoxic cancer cell derived EV. In this study, we investigate how (hypoxic) cancer cell derived EV mediate their effects. We demonstrate that cancer derived EV regulate cellular metabolism and protein synthesis in acceptor cells through increased activation of mTOR and AMPKα. Using metabolic tracer experiments, we demonstrate that EV stimulate glucose uptake in endothelial cells to fuel amino acid synthesis and stimulate amino acid uptake to increase protein synthesis. Despite alterations in cargo, we show that the effect of cancer derived EV on recipient cells is primarily determined by the EV producing cancer cell type rather than its oxygenation status.

Mechanism underlying effects of cellulose-degrading microbial inoculation on amino acid degradation and biosynthesis during composting

Amino acids are essential organic compounds in composting products. However, the mechanism underlying the amino acid metabolism during composting remains unclear. This study aims at exploring the impacts of inoculating cellulose-degrading microbes on amino acid metabolism during composting with mulberry branches and silkworm excrements. Cellulose-degrading microbial inoculation enhanced amino acid degradation by 18%–43% by increasing protease and sucrase activities and stimulating eight amino acid degradation pathways from the initial to thermophilic phases, with Enterococcus, Saccharomonospora, Corynebacterium being the dominant bacterial genera, but stimulated amino acid production by 54% by increasing sucrase and urease activities, decreasing β-glucosidase activities, and stimulating twenty-two amino acid synthesis pathways at the mature phase, with Thermobifida, Devosia, and Cellulosimicrobium being the dominant bacterial genera. The results suggest that cellulose-degrading microbial inoculation enhances amino acid degradation from the initial to thermophilic phases and biosynthesis at the mature phase, thereby improving the quality of organic fertilizer.

Exploring the potential nutritional benefits of Arthrospira maxima and Chlorella vulgaris: A focus on vitamin B12, amino acids, and micronutrients

Arthrospira (Limnospira) maxima (A. maxima) and Chlorella vulgaris (Ch. vulgaris) are among the approved microalgae and cyanobacteria (MaC) in the food industry that are known to be safe for consumption. However, both organisms are controversial regarding their vitamin B12 content, due to the possible occurrence of pseudo-cobalamin. Concurrently, their nutrition profiles remain understudied. The main purpose of the present study was to identify their nutrition profiles, focusing mainly on vitamin B12, amino acids, and micronutrients under iron-induced hormesis (10 mg/L Fe in treated samples). Our findings indicate a higher B12 content in A. maxima compared to Ch. vulgaris (both control and treated samples). Using liquid chromatography with tandem mass spectrometry (LC-MS/MS), the cyanocobalamin content was determined as 0.42 ± 0.09 μg/g dried weight (DW) in the A. maxima control and 0.55 ± 0.02 μg/g DW in treated A. maxima, resulting in an insignificant difference. In addition, the iron-enriched medium increased the amount of iron in both tested biomasses (p < 0.01). However, a more pronounced (approximately 100×) boost was observed in Ch. vulgaris, indicating a better absorption capacity (control Ch. vulgaris 0.16 ± 0.01 mg/g Fe, treated Ch. vulgaris 15.40 ± 0.34 mg/g Fe). Additionally, Ch. vulgaris also showed a higher micronutrient content. Using both tested microalgae, meeting the sufficient recommended daily mineral allowance for an adult is possible. By combining biomass from A. maxima and Ch. vulgaris in a ratio of 6:1, we can fulfill the recommended daily allowance of vitamin B12 and iron by consuming 6 tablets/6 g. Importantly, iron hormesis stimulated amino acid composition in both organisms. The profile of amino acids may suggest these biomasses as promising potential nutrition sources.

OTUD3 suppresses the mTORC1 signaling by deubiquitinating KPTN.

Background: Ubiquitination and deubiquitination modifications play pivotal roles in eukaryotic life processes, regulating protein dynamics via the ubiquitin-proteasome pathway. Dysregulation can impact disease development, including cancer and neurodegenerative disorders. Increasing evidence highlights their role in tumorigenesis, modulating key proteins. OTUD3, a deubiquitinase, stabilizes PTEN, suppressing tumor growth by inhibiting PI3K-AKT signaling. Yet, further OTUD3 substrates remain underexplored. Methods: We employed the In vivo ubiquitination assay to investigate the ubiquitination role of OTUD3 on KPTN within the cellular context. Additionally, CRISPR/Cas9 editing and Immunofluorescence were utilized to study the impact of OTUD3 on the mTOR signaling pathway in cells. Furthermore, Cell proliferation assay and NMR were employed to explore the effects of OTUD3 on cellular growth and proliferation. Results: OTUD3 serves as a deubiquitinase for KPTN. OTUD3 interacts with KPTN, facilitated by the OTU domain within OTUD3. Further investigations confirmed KPTN's ubiquitination modification, primarily at lysine residue 49. Ubiquitination experiments demonstrated OTUD3's ability to mediate KPTN's deubiquitination without affecting its protein levels. This suggests KPTN's ubiquitination is a function-regulated, non-degradable modification. Under various amino acid starvation or stimulation conditions, overexpressing OTUD3 reduces mTORC1 signaling activation, while knocking out OTUD3 further enhances it. Notably, OTUD3's regulation of mTORC1 signaling relies on its deubiquitinase activity, and this effect is observed even in PTEN KO cells, confirming its independence from PTEN, a reported substrate. OTUD3 also promotes GATOR1's lysosomal localization, a process requiring KPTN's involvement. Ultimately, OTUD3 affects cellular metabolic pool products by downregulating the mTORC1 pathway, significantly inhibiting tumor cell growth and proliferation. Discussion: Our experiments shed light on an alternative perspective regarding the intrinsic functions of OTUD3 in inhibiting tumor development. We propose a novel mechanism involving KPTN-mediated regulation of the mTORC1 signaling pathway, offering fresh insights into the occurrence and progression of tumor diseases driven by related genes. This may inspire new approaches for drug screening and cancer treatment, potentially guiding future therapies for relevant tumors.

Metagenomic analysis revealed the evolution of microbial communities, metabolic pathways, and functional genes in the heterotrophic nitrification-aerobic denitrification process under La3+ stress

Trivalent lanthanum (La3+) exists widely in ammonia nitrogen (NH4+-N) tailing water from ionic rare earth mines; however, its effect on heterotrophic nitrification-aerobic denitrification (HN-AD) is unknown, thereby limiting the application of the HN-AD process in this field. In this study, we conducted an HN-AD process using a sequencing batch reactor (5 L) that was continuously operated to directly treat acidic (NH4)2SO4 wastewater (influent NH4+-N concentration of approximately 110 mg/L and influent pH of 5) containing different La3+ concentrations (0–100 mg/L). The NH4+-N removal efficiency of the reactor reached 98.25 % at a La3+ concentration of 100 mg/L. The reactor was in a neutral-to-alkaline environment, which favored La3+ precipitation and complexation. Metagenomic analysis revealed that the relative abundance of Thauera in the reactor remained high (88.62–92.27 %) under La3+ stress. The relative abundances of Pannonobacter and Hyphomonas significantly increased, whereas that of Azoarcus significantly decreased. Metabolic functions in the reactor were mainly contributed by Thauera, and the abundance of metabolic functions under low La3+ stress (≤5 mg/L) significantly differed from that under high La3+ stress (≥10 mg/L). The relative abundance of ammonia assimilation-related genes in the reactor was high and significantly correlated with ammonia removal. However, traditional ammonia oxidation genes were not annotated, and unknown ammonia oxidation pathways may have been present in the reactor. Moreover, La3+ stimulated amino acid biosynthesis and translocation, the citrate cycle, sulfur metabolism, and oxidative phosphorylation and promoted the overproduction of extracellular polymeric substances, which underwent complexation and adsorbed La3+ to reduce its toxicity. Our results showed that the HN-AD process had a strong tolerance to La3+, stable NH4+-N removal efficiency, the potential to recover La3+, and considerable application prospects in treating NH4+-N tailing water from ionic rare earth mines.

Long Noncoding RNA Gpr137b-ps Promotes Advanced Atherosclerosis via the Regulation of Autophagy in Macrophages.

Current therapies cannot completely reverse advanced atherosclerosis. High levels of amino acids, induced by Western diet, stimulate mTORC1 (mammalian target of rapamycin complex 1)-autophagy defects in macrophages, accelerating atherosclerotic plaque progression. In addition, autophagy-lysosomal dysfunction contributes to plaque necrotic core enlargement and lipid accumulation. Therefore, it is essential to investigate the novel mechanism and molecules to reverse amino acid-mTORC1-autophagy signaling dysfunction in macrophages of patients with advanced atherosclerosis. We observed that Gpr137b-ps (G-protein-coupled receptor 137B, pseudogene) was upregulated in advanced atherosclerotic plaques. The effect of Gpr137b-ps on the progression of atherosclerosis was studied by generating advanced plaques in ApoE-/- mice with cardiac-specific knockout of Gpr137b-ps. Bone marrow-derived macrophages and mouse mononuclear macrophage cell line RAW264.7 cells were subjected to starvation or amino acid stimulation to study amino acid-mTORC1-autophagy signaling. Using both gain- and loss-of-function approaches, we explored the mechanism of Gpr137b-ps-regulated autophagy. Our results demonstrated that Gpr137b-ps deficiency led to enhanced autophagy in macrophages and reduced atherosclerotic lesions, characterized by fewer necrotic cores and less lipid accumulation. Knockdown of Gpr137b-ps increased autophagy and prevented amino acid-induced mTORC1 signaling activation. As the downstream binding protein of Gpr137b-ps, HSC70 (heat shock cognate 70) rescued the impaired autophagy induced by Gpr137b-ps. Furthermore, Gpr137b-ps interfered with the HSC70 binding to G3BP (Ras GTPase-activating protein-binding protein), which tethers the TSC (tuberous sclerosis complex) complex to lysosomes and suppresses mTORC1 signaling. In addition to verifying that the NTF2 (nuclear transport factor 2) domain of G3BP binds to HSC70 by in vitro protein synthesis, we further demonstrated that HSC70 binds to the NTF2 domain of G3BP through its W90-F92 motif by using computational modeling. These findings reveal that Gpr137b-ps plays an essential role in the regulation of macrophage autophagy, which is crucial for the progression of advanced atherosclerosis. Gpr137b-ps impairs the interaction of HSC70 with G3BP to regulate amino acid-mTORC1-autophagy signaling, and these results provide a new potential therapeutic direction for the treatment of advanced atherosclerosis.

The physiology of growth hormone (GH) in adults: translational journey to GH replacement therapy.

The fact that growth hormone (GH) plays an important role in health after the cessation of growth requiring replacement therapy in adult life has only been recognised in the last three decades. This has only been made possible by recombinant technology providing GH supplies required to undertake investigations in the physiology of GH action and the benefits of replacement therapy in patients identified by rigorously validated diagnostic tests for GH deficiency (GHD). Human studies have revealed important regulatory roles in substrate metabolism, sodium homeostasis, body composition, and physical function. GH-induced anabolism is achieved by stimulating amino acid incorporation into protein while reducing oxidative loss simultaneously enhancing lipid utilisation by stimulating fatty acid oxidation and reducing lipid storage. Sodium and fluid retention are enhanced by activating the renin-angiotensin system and distal renal tubular reabsorption. GH stimulates the aerobic and anaerobic energy systems that underpin muscle and cardiovascular function. These pleiotropic actions explain the clinical picture of increased adiposity, reduced lean mass, and impaired physical and psychological function in the GHD adult, all of which are reversed when GH is replaced. Women require a greater replacement dose of GH than men. This is because androgens enhance while oestrogens attenuate GH action. The oestrogen effect is route-dependent, occurring with oral delivery blunting the liver-mediated actions of GH by directly inhibiting GH receptor signalling, global experience spanning over 30 years has attested to the safety, efficacy, and benefits of replacement therapy for adults with GHD.

Analysis of differentially expressed genes related to cerebral ischaemia in young rats based on the Gene Expression Omnibus database.

The incidence rate of cerebral infarction in young people is increasing day by day, the age of onset tends to be younger, and its internal pathogenesis and mechanism are very complicated, which leads to greater difficulties in treatment. Therefore, it is essential to analyze the key pathway that affects the onset of cerebral infarction in young people from the perspective of genetics. To compare the differentially expressed genes in the brain tissue of young and aged rats with middle cerebral artery occlusion and to analyse their effect on the key signalling pathway involved in the development of cerebral ischaemia in young rats. The Gene Expression Omnibus 2R online analysis tool was used to analyse the differentially expressed genes in the GSE166162 dataset regarding the development of cerebral ischaemia in young and aged groups of rats. DAVID 6.8 software was further used to filter the differentially expressed genes. These genes were subjected to Gene Ontology (GO) function analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis to determine the key gene pathway that affects the occurrence of cerebral ischaemia in young rats. Thirty-five differentially expressed genes (such as Igf2, Col1a2, and Sfrp1) were obtained; 73 GO enrichment analysis pathways are mainly involved in biological processes such as drug response, amino acid stimulation response, blood vessel development, various signalling pathways, and enzyme regulation. They are involved in molecular functions such as drug binding, protein binding, dopamine binding, metal ion binding, and dopamine neurotransmitter receptor activity. KEGG pathway enrichment analysis showed a significantly enriched pathway: The cyclic adenosine monophosphate (c-AMP) signalling pathway. The c-AMP signalling pathway might be the key pathway in the intervention of cerebral infarction in young people.

An mTORC1-mediated negative feedback loop constrains amino acid-induced FLCN-Rag activation in renal cells with TSC2 loss

The mechanistic target of rapamycin complex 1 (mTORC1) integrates inputs from growth factors and nutrients, but how mTORC1 autoregulates its activity remains unclear. The MiT/TFE transcription factors are phosphorylated and inactivated by mTORC1 following lysosomal recruitment by RagC/D GTPases in response to amino acid stimulation. We find that starvation-induced lysosomal localization of the RagC/D GAP complex, FLCN:FNIP2, is markedly impaired in a mTORC1-sensitive manner in renal cells with TSC2 loss, resulting in unexpected TFEB hypophosphorylation and activation upon feeding. TFEB phosphorylation in TSC2-null renal cells is partially restored by destabilization of the lysosomal folliculin complex (LFC) induced by FLCN mutants and is fully rescued by forced lysosomal localization of the FLCN:FNIP2 dimer. Our data indicate that a negative feedback loop constrains amino acid-induced, FLCN:FNIP2-mediated RagC activity in renal cells with constitutive mTORC1 signaling, and the resulting MiT/TFE hyperactivation may drive oncogenesis with loss of the TSC2 tumor suppressor.

The NRF2 antagonist ML385 inhibits PI3K-mTOR signaling and growth of lung squamous cell carcinoma cells.

Lung squamous cell carcinoma (LUSC) currently has limited therapeutic options because of the relatively few validated targets and the lack of clinical drugs for some of these targets. Although NRF2/NFE2L2 pathway activation commonly occurs in LUSC, NRF2 has predominantly been studied in other cancer models. Here, we investigated the function of NRF2 in LUSC, including in organoid models, and we explored the activity of a small molecule NRF2 inhibitor ML385, which has not previously been investigated in LUSC. We first explored the role of NRF2 signaling in LUSC cancer cell line and organoid proliferation through NRF2 knockdown or ML385 treatment, both in vivo and in vitro. Next, we performed Western blot and immunofluorescence assays to determine the effect of NRF2 inhibition on PI3K-mTOR signaling. Finally, we used cell viability and clonogenic assays to explore whether ML385 could sensitize LUSC cancer cells to PI3K inhibitors. We find that downregulation of NRF2 signaling inhibited proliferation of LUSC cancer cell lines and organoids, both in vivo and in vitro. We also demonstrate that inhibition of NRF2 reduces PI3K-mTOR signaling, with two potential mechanisms being involved. Although NRF2 promotes AKT phosphorylation, it also acts downstream of AKT to increase RagD protein expression and recruitment of mTOR to lysosomes after amino acid stimulation. We also find that ML385 potentiates LUSC growth inhibition by a pan-PI3K inhibitor, which correlates with stronger inhibition of PI3K-mTOR signaling. Our data provide additional support for NRF2 promoting LUSC growth through PI3K-mTOR activation and support development of NRF2 inhibitors for the treatment of LUSC.

Numb activates the mTORC1 signaling pathway in proximal tubular epithelial cells by upregulating V1G1 expression

To investigate the role of Numb in regulating mammalian target of rapamycin (mTOR) complex 1 (mTORC1) signaling pathway. Male BALB/C mouse models of acute kidney injury (AKI) were subjected to intravenous injections of Numb-siRNA or NC-siRNA with or without intraperitoneal cisplatin injections. After the treatments, the expressions and distribution of Numb and megalin in the renal tissues of the mice were detected with immunohistochemistry, and the renal expressions of Numb, S6, p-S6, S6K1, p-S6K1, 4EBP1 and p-4EBP1 were examined with Western blotting. The proximal renal tubular epithelial cells were isolated from the mice transfected with Numb-siRNA for in vitro culture. In NRK-52E cells, the effects of amino acid stimulation, Numb knockdown, and V1G1 overexpression, alone or in combination, on expressions of Numb, S6 and p-S6 were detected with Western blotting; the expressions of AMPK and p-AMPK were also detected in transfected NRK-52E cells, mouse kidneys and cultured mouse renal tubular epithelial cells. In BALB/C mice, injection of Numb-siRNA caused significant reductions of Numb and p-S6 expressions without affecting megalin expression in the renal proximal tubules (P < 0.05). Cisplatin treatment obviously upregulated p-S6K1 and p-4EBP1 expressions in the kidneys of the mice (P < 0.05), and this effect was significantly inhibited by treatment with Numb-siRNA (P < 0.05). In NRK-52E cells, amino acid stimulation significantly upregulated the expression of p-S6 (P < 0.05), which was strongly suppressed by transfection with Numb-siRNA (P < 0.05). Numb knockdown inhibited AMPK activation in NRK-52E cells, mouse kidneys and primary proximal tubular epithelial cells (P < 0.05). Numb knockdown significantly downregulated V1G1 expression in NRK-52E cells (P < 0.05), and V1G1 overexpression obviously reversed the inhibitory effect of Numb-siRNA on S6 phosphorylation (P < 0.05). Numb promotes the activation of mTORC1 signaling in proximal tubular epithelial cells by upregulating V1G1 expression.

The role of dietary plant and animal protein intakes on mitigating sarcopenia risk.

Purpose of reviewTo highlight contemporary findings comparing the digestibility of animal and plant proteins, their stimulatory effects on muscle protein synthesis, and associations with sarcopenia.Recent findingsAnimal proteins are more digestible than plant proteins, resulting in greater amino acid availability and stimulation of muscle protein synthesis. However, isolated plant proteins, plant protein blends, and modified plant proteins enriched with indispensable amino acids can elicit comparable digestion and absorption kinetics to animal proteins. More research is needed to determine whether these modified plant protein sources can effectively mitigate sarcopenia risk.SummaryBoth animal and plant protein foods can be incorporated into a healthful eating plan that limits risk of age-related diseases, such as sarcopenia. Humans eat food rather than isolated nutrients; as such, considering the context of the overall diet and its impact on health, instead of solely focusing on individual nutrients in isolation, is important.

Alkaline intracellular pH (pHi) increases mTORC1 and mTORC2 signaling through PI3K to promote protein synthesis and cell survival

The conserved kinase mTOR (mechanistic target of rapamycin) regulates cell metabolism and promotes cell growth, proliferation, and survival in response to diverse environmental cues (e.g., nutrients; growth factors; hormones). mTOR forms the catalytic core of two multiprotein complexes, mTORC1 and mTORC2, which possess unique downstream targets and cellular functions. While mTORC1 and mTORC2 often respond to distinct upstream cues, they share a requirement for PI3K in their activation by growth factors. While many studies agree that amino acids activate mTORC1 but not mTORC2, several studies reported paradoxical activation of mTORC2 by amino acids. We noted that stimulating amino acid starved cells with a commercial mixture of amino acids increased mTORC2‐dependent Akt S473 phosphorylation rapidly while re‐feeding cells with complete DMEM containing amino acids failed to do so. Interestingly, we found the pH of the commercial amino acid mixture to be ~ pH 10. Upon controlling for pH, stimulating starved cells with amino acids at pH 10 but not 7.4 increased mTORC2 signaling. Moreover, DMEM at alkaline pH was sufficient to increase mTORC2 catalytic activity and signaling. Using a fluorescent pH‐sensitive dye (cSNARF‐1‐AM) coupled to ratio‐metric live cell imaging, we confirmed that alkaline extracellular pH (pHe) translated into a rapid increase in intracellular pH (pHi). Moreover, blunting this increase with a pharmacological inhibitor of an H+ transporter attenuated the increase in mTORC2 signaling by pHe. Alkaline pHi also activated AMPK, a canonical sensor of energetic stress that promotes mTORC2 signaling, as reported previously by us. Functionally, we found that alkaline pHi attenuated apoptosis caused by growth factor withdrawal through activation of AMPK‐mTORC2 signaling. These results indicate that alkaline pHi augments mTORC2 signaling to promote cell survival, in part through AMPK. In the course of this work, we noted that pHi increased phosphorylation of several downstream targets of PI3K (e.g., Akt P‐T308 and P‐S473; S6K1 P‐T389 and P‐T229; PRAS40 P‐T246; Tsc2 P‐S939), suggesting that PI3K itself responds to changes in pHi. Indeed, alkaline pHi increased PI‐3’,4’,5’‐P3 levels in a manner sensitive to the PI3K inhibitor BYL‐719. Thus, alkaline pHi elevates PI3K activity, which increases both mTORC1 and mTORC2 signaling. Mechanistically, we found that activation of PI3K by alkaline pHi induced dissociation of Tsc2 from lysosomal membranes, thereby relieving TSC‐mediated suppression of Rheb, a mTORC1‐activating GTPase. Functionally, we found that activation of PI3K by alkaline pHi increased mTORC1‐mediated 4EBP1 phosphorylation, which initiates cap‐dependent translation by eIF4E. Alkaline pHi also increased mTORC1‐driven protein synthesis. Taken together, these findings reveal alkaline pHi as a previously unrecognized activator of PI3K‐mTORC1/2 signaling that promotes protein synthesis and cell survival. As elevated pHi represents an under‐appreciated hallmark of cancer cells, these findings suggest that by alkaline pHi sensing by the PI3K‐mTOR axis and AMPK‐mTORC2 axes may contribute to tumorigenesis.

Pyruvate kinase and GLP-1 receptor signaling converge on cAMP production to enhance insulin secretion

Pyruvate kinase (PK), by converting ADP and phosphoenolpyruvate (PEP) to ATP and pyruvate, initiates insulin secretion via KATP closure and enhances insulin secretion via mitochondrial PEP production. The physiological drivers of mitochondrial PEP production include amino acids, which also work on neighboring α-cells to stimulate insulin secretion through activation of β-cell glucagon and GLP-1 receptors. We therefore hypothesized that PK would augment α-cell paracrine signaling upon amino acid (AA) stimulation.

Nitrogen modulates strontium uptake and toxicity in Hypericum perforatum plants

Strontium is an unavoidable element occurring in plants due to its abundance in the soil and similarity with calcium. To mimic natural conditions, impacts of additional inorganic (nitrate) or organic (urea and allantoin) nitrogen sources (1 mM of each N form in addition to 3.53 mM N in the basic cultivation solution) or N deficit on strontium-induced changes (100 µM Sr) in the widely used medicinal plant Hypericum perforatum L. were studied. Though various effects of Sr on primary (stimulation of amino acids but depression of most Krebs acids, ascorbic acid and thiols) and secondary metabolites (stimulation of phenols but no change of pseudo/hypericin) or mineral elements were observed (reduction of Ca amount in both shoots and roots), organic N forms often mitigated negative action of Sr or even combined stimulatory impact was observed. Organic N forms also elevated shoot accumulation of Sr while N deficit reduced it. Additional N forms, rather than Sr itself, modulated reactive oxygen species and nitric oxide formation in the root tissue. Germination experiment showed no toxicity of Sr to H. perforatum up to 1 mM Sr and even stimulated accumulation of amino acids and phenols, indicating similar ontogenetic-related responses.

A novel amino acid signaling process governs glucose-6-phosphatase transcription

SummaryEmerging evidence has shown that amino acids act as metabolic regulatory signals. Here, we showed that glucose-6-phosphatase (G6Pase) mRNA levels in cultured hepatocyte models were downregulated in an amino-acid-depleted medium. Inversely, stimulation with amino acids increased G6Pase mRNA levels, demonstrating that G6Pase mRNA level is directly controlled by amino acids in a reversible manner. Promoter assay revealed that these amino-acid-mediated changes in G6Pase mRNA levels were attributable to transcriptional regulation, independent of canonical hormone signaling pathways. Metabolomic analysis revealed that amino acid starvation induces a defect in the urea cycle, decreasing ornithine, a major intermediate, and supplementation of ornithine in an amino-acid-depleted medium fully rescued G6Pase mRNA transcription, similar to the effects of amino acid stimulation. This pathway was also independent of established mammalian target of rapamycin complex 1 pathway. Collectively, we present a hypothetical concept of “metabolic regulatory amino acid signal,” possibly mediated by ornithine.

Pike-perch larvae growth in response to administration of lactobacilli-enriched inert feed during first feeding

This study evaluated whether inert feed enriched with Lb. paracasei subsp. paracasei BGHN14 may be used as a weaning diet for first feeding pike-perch larvae. Three experimental groups were weaned from the start of exogenous feeding: two groups were given inert feed enriched with BGHN14 either via 12 h incubation with live BGHN14 cells or via coating with homogenized BGHN14 cells and one group was supplemented non-enriched inert feed. In all three groups Artemia was co-fed with inert feed during weaning. Control group larvae were fed Artemia exclusively during the treatment period. Treatment lasted fourteen days, starting from the 6th day post-hatch (DPH). Larval sampling was performed on the 20th DPH for gene expression and enzyme activity analysis. Larvae were also sampled on the 32nd DPH for morphometric and body composition analysis. Our results showed that weaning of first feeding pike-perch larvae was associated with an increase of fish condition (0.72 ± 0.12–0.77 ± 0.11 versus 0.67 ± 0.11 in controls), but it suppressed skeleton development, according to Col1 mRNA expression (1 ± 0.51–1.06 ± 0.36 versus 2.07 ± 0.53 in controls) and reduced fat deposition (1.25 ± 0.23–1.49 ± 0.33 versus 1.84 ± 0.31% in controls). This presumably reflected lower availability of soluble proteins in microdiet as opposed to live food, along with high leaching rate of amino acids from solid feed particles, as reported in our previous studies. However, skeleton differentiation was not impaired in group weaned on BGHN14 homogenate coated feed (Col1 mRNA expression: 2.68 ± 0.72), which was enriched in skeleton building and taste stimulating amino acids. These larvae were also presented with substantially higher length (15.28 ± 2.55 versus 13.93 ± 2.31 mm in controls) and weight (26.56 ± 13.83 versus 21.03 ± 11.25 mg in controls), which correlated with lower trypsin activity (1.06 ± 0.13 versus 1.43 ± 0.26 mU/mg of proteins in controls) and an increase of PLA2 to trypsin activity ratio (453.12 ± 109.36 versus 264.84 ± 69.03 in controls). Present study suggests that weaning of first feeding pike-perch larvae using BGHN14 homogenate coated microdiet supports skeleton development and improves fish growth.

Cancer-secreted miRNAs regulate amino-acid-induced mTORC1 signaling and fibroblast protein synthesis.

Metabolic reprogramming of non-cancer cells residing in a tumor microenvironment, as a result of the adaptations to cancer-derived metabolic and non-metabolic factors, is an emerging aspect of cancer-host interaction. We show that in normal and cancer-associated fibroblasts, breast cancer-secreted extracellular vesicles suppress mTOR signaling upon amino acid stimulation to globally reduce mRNA translation. This is through delivery of cancer-derived miR-105 and miR-204, which target RAGC, a component of Rag GTPases that regulate mTORC1 signaling. Following amino acid starvation and subsequent re-feeding, 13 C-arginine labeling of de novo synthesized proteins shows selective translation of proteins that cluster to specific cellular functional pathways. The repertoire of these newly synthesized proteins is altered in fibroblasts treated with cancer-derived extracellular vesicles, in addition to the overall suppressed protein synthesis. In human breast tumors, RAGC protein levels are inversely correlated with miR-105 in the stroma. Our results suggest that through educating fibroblasts to reduce and re-prioritize mRNA translation, cancer cells rewire the metabolic fluxes of amino acid pool and dynamically regulate stroma-produced proteins during periodic nutrient fluctuations.