Amino Acid Signaling Pathways Research Articles

Investigation of guanidino acetic acid and rumen-protected methionine induced improvements in longissimus lumborum muscle quality in beef cattle

This study examined the impact of dietary guanidino acetic acid (GAA) and rumen-protected methionine (RPM) on beef quality in Simmental bulls. For 140 days, forty-five bulls (453.43 ± 29.05 kg) were randomly divided into control (CON), 0.1% GAA (GAA), and 0.1% GAA + 0.1% RPM (GAM) groups with 15 bulls in each group and containing 3 pen with 5 bulls in each pen. Significant improvements in eye muscle area, pH48h, redness (a*) value, and crude protein (CP) content of longissimus lumborum (LL) muscles were observed in the GAA and GAM groups (P < 0.05). Conversely, the lightness (L*) value, drip loss, cooking loss, and moisture contents decreased (P < 0.05). Additionally, glutathione (GSH) and glutathione peroxidase (GSH-PX) concentrations of LL muscles in GAM were higher (P < 0.05), while malondialdehyde (MDA) content of LL muscles in GAA and GAM groups were lower (P < 0.05). Polyunsaturated fatty acids (PUFA) profiles were enriched in beef from GAM group (P < 0.05). The addition of GAA and RPM affected the expression of genes in LL muscle, such as HMOX1, EIF4E, SCD5, and NOS2, which are related to hypoxia metabolism, protein synthesis, and unsaturated fatty acid synthesis-related signaling pathways. In addition, GAA and RPM also affected the content of a series of metabolites such as L-tyrosine, L-tryptophan, and PC (O-16:0/0:0) involved in amino acid and lipid metabolism-related signaling pathways. In summary, GAA and RPM can improve the beef quality and its nutritional composition. These changes may be related to changes in gene expression and metabolic pathways related to protein metabolism and lipid metabolism in beef.

Effects of sodium selenite, yeast selenium, and nano-selenium on toxicity, growth, and selenium bioaccumulation in Lucilia sericata maggots.

In this study, we investigated the effects of different types of selenium (Se) (sodium selenite [SS], yeast selenium [YS], and nano-selenium [NS]) on the toxicity, growth, Se accumulation, and transformation of Lucilia sericata maggots (LSMs). We found that the 50% lethal concentration of LSMs exposed to SS was 2.18 and 1.96 times that of YS and NS, respectively. LSM growth was significantly promoted at exposure concentrations of 10-50mgkg-1 in group SS and 10-30mgkg-1 in group YS, whereas NS inhibited LSMs growth at all concentrations (p < 0.05). Total Se content in LSMs, conversion efficiency to organic and other forms of Se, and bioaccumulation factor of Se were the highest in the SS group when exposed to 50mgkg-1 (81.6mgkg-1, 94.6%, and 1.63, respectively). Transcriptomic results revealed that LSMs significantly upregulated the amino acid (alanine, aspartate, glutamic, and tyrosine) and tricarboxylic acid cycle signaling pathways (p < 0.05) on exposure to Se, resulting in a significant increase in LSMs biomass and quality. In conclusion, our study indicates that LSMs exhibit good tolerance to SS and can convert it into bioorganic or other forms of Se.

Pulsatile Leucine Administration during Continuous Enteral Feeding Enhances Skeletal Muscle Mechanistic Target of Rapamycin Complex 1 Signaling and Protein Synthesis in a Preterm Piglet Model

BackgroundContinuous feeding does not elicit an optimal anabolic response in skeletal muscle but is required for some preterm infants. We reported previously that intermittent intravenous pulses of leucine (Leu; 800 μmol Leu·kg–1·h–1 every 4 h) to continuously fed pigs born at term promoted mechanistic target of rapamycin complex 1 (mTORC1) activation and protein synthesis in skeletal muscle. ObjectivesThe aim was to determine the extent to which intravenous Leu pulses activate mTORC1 and enhance protein synthesis in the skeletal muscle of continuously fed pigs born preterm. MethodsPigs delivered 10 d preterm was advanced to full oral feeding >4 d and then assigned to 1 of the following 4 treatments for 28 h: 1) ALA (continuous feeding; pulsed with 800 μmol alanine·kg–1·h–1 every 4 h; n = 8); 2) L1× (continuous feeding; pulsed with 800 μmol Leu·kg–1·h–1 every 4 h; n = 7); 3) L2× (continuous feeding; pulsed with 1600 μmol Leu·kg–1·h–1 every 4 h; n = 8); and 4) INT (intermittent feeding every 4 h; supplied with 800 μmol alanine·kg–1 per feeding; n = 7). Muscle protein synthesis rates were determined with L-[2H5-ring]Phenylalanine. The activation of insulin, amino acid, and translation initiation signaling pathways were assessed by Western blot. ResultsPeak plasma Leu concentrations were 134% and 420% greater in the L2× compared to the L1× and ALA groups, respectively (P < 0.01). Protein synthesis was greater in the L2× than in the ALA and L1× groups in both the longissimus dorsi and gastrocnemius muscles (P < 0.05) but not different from the INT group (P > 0.10). Amino acid signaling upstream and translation initiation signaling downstream of mTORC1 largely corresponded to the differences in protein synthesis. ConclusionsIntravenous Leu pulses potentiate mTORC1 activity and protein synthesis in the skeletal muscles of continuously fed preterm pigs, but the amount required is greater than in pigs born at term.

Metabolomics-Based Investigation on the Metabolic Changes in Crassostrea gigas Experimentally Exposed to Galvanic Anodes.

Cathodic protection is widely used to protect metal structures from corrosion in marine environments using sacrificial galvanic anodes. These anodes, either in Zinc, or preferentially nowadays in Al-Zn-In alloys, are expected to corrode instead of the metal structures. This leads to the release of dissolved species, Zn2+, Al3+, and In3+, and solid phases such as Al(OH)3. Few studies have been conducted on their effects on marine organisms, and they concluded that further investigations are needed. We therefore evaluated the effects of Zn and Al-Zn-In anodes on oysters stabulated in tanks, under controlled conditions defined through a comparison with those prevailing in a given commercial seaport used as reference. We analyzed the entire metabolome of gills with a non-targeted metabolomic approach HRMS. A modelling study of the chemical species, corresponding to the degradation products of the anodes, likely to be present near the exposed oysters, was also included. We identified 16 and two metabolites modulated by Zn- and Al-Zn-In-anodes, respectively, that were involved in energy metabolism, osmoregulation, oxidative stress, lipid, nucleotide nucleoside and amino acid metabolisms, defense and signaling pathways. The combination of chemical modelling and metabolomic approach, used here for the first time, enlightened the influence of Zn present in the Al-Zn-In anodes.

Elevated temperature decreases cardiovascular toxicity of nanoplastics but adds to their lethality: A case study during zebrafish (Danio rerio) development

To highlight the key role of global warming on the toxicity of contaminants, the cardiovascular toxicity of nanoparticles (NPs) was estimated in developing zebrafish (Danio rerio) at different exposure temperatures, and the toxicity mechanisms were explored via multi-omic analyses. Polystyrene NPs (50 nm) at 0.1 mg·L−1 entered zebrafish embryos at 24 h post-fertilization and caused cardiovascular toxicity in the developing zebrafish at 27 ℃. This was explained by the down-regulation of the branched-chain amino acid and insulin signaling pathways owing to induced oxidative stress. Elevated exposure temperatures promoted the accumulation of NPs in developing zebrafish, increased the levels of oxidative stress and enhanced the oxidative phosphorylation rate in mitochondria, thus resulting in an additive effect on the mortality of zebrafish larvae. Notably, elevated exposure temperatures reduced the cardiovascular toxicity of NPs, as the effective concentration of NPs for inhibiting embryonic heartbeat rate increased from 0.1 mg·L−1 at 27 ℃ to 1.0 mg·L−1 at 30 ℃. Experiments of transgenic zebrafish Tg(myl7:GFP) and multi-omic analyses revealed that elevated temperatures enhanced the myocardial contractility of larvae, thus reducing the cardiovascular toxicity of NPs. However, the health risks of enhanced myocardial contraction caused by NP exposure at elevated temperatures requires further consideration.

Coordinated Regulation of Myelination by Growth Factor and Amino-acid Signaling Pathways.

Myelin-forming oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS) are essential for structural and functional homeostasis of nervous tissue. Albeit with certain similarities, the regulation of CNS and PNS myelination is executed differently. Recent advances highlight the coordinated regulation of oligodendrocyte myelination by amino-acid sensing and growth factor signaling pathways. In this review, we discuss novel insights into the understanding of differential regulation of oligodendrocyte and Schwann cell biology in CNS and PNS myelination, with particular focus on the roles of growth factor-stimulated RHEB-mTORC1 and GATOR2-mediated amino-acid sensing/signaling pathways. We also discuss recent progress on the metabolic regulation of oligodendrocytes and Schwann cells and the impact of their dysfunction on neuronal function and disease.

Prematurity Negatively Alters Activation of the Amino Acid Signaling Pathway That Regulates Protein Synthesis in Muscle of a Preterm Piglet Model

ObjectivesPreterm birth is often associated with reduced postnatal lean mass gain. Recently we found that postnatal weight gain and postprandial skeletal muscle protein synthesis in prematurely born piglets were blunted compared to term born pigs. This anabolic resistance is associated with reduced activation of mTORC1, a central regulator of protein synthesis. To identify the responsible mechanisms, we evaluated key regulatory components involved in the amino acid (AA)-induced activation of mTORC1 in skeletal muscle in preterm compared to term pigs. MethodsPiglets were delivered by cesarean section 11 d (preterm/PT, equivalent to 28–32 wk gestation in humans) or 2 d (term/T) before term; a jugular vein catheter was placed for delivery of total parenteral nutrition. On day 3, after 4 h fasting, piglets were fasted one additional h or fed an elemental diet (31.5 kcal/kg, 1.3 g/kg lactose, 2.7 g/kg AA, and 1.6 g/kg lipid), via oral gavage. Longissimus dorsi muscle was collected following euthanasia (fasted state or 60 min after feeding). The abundance and activation of AA signaling components were determined by immunoprecipitation and immunoblotting assays. ResultsThe abundances of AA transporters SNAT2/SLC38A2 (glutamine), LAT1/SLC7A5 (leucine), PAT1/SLC36A1 (H+AA), and SLC38A9 (arginine) were not affected by prematurity. Lysosomal-associated protein abundances (GATOR2, LAMTOR1/2, V-ATPase, Rag A/C, and Rheb) were similar in PT and T. Although the abundance of leucine sensors (Sestrin 2 and LRS) and the Sestrin2-GATOR2 complex were similar, the association of mTOR with RagA and RagC were lower post-feeding in PT than in T pigs (P < 0.05). Consequently, the activation of mTORC1, indicated by phosphorylation of Ser2448, was lower in PT than in T pigs after a meal (P < 0.05). ConclusionsThese results showed that the postprandial AA-induced activation of mTORC1 is reduced following preterm birth. Although AA transporter abundance is not altered by prematurity, the postprandial activation of the Rag signaling components that regulate mTORC1 activation is impaired by prematurity. The reduced AA-induced activation of mTORC1 likely contributes to the extrauterine growth faltering of prematurity. Funding SourcesNIH and USDA.

GATOR2 complex–mediated amino acid signaling regulates brain myelination

Amino acids are essential for cell growth and metabolism. Amino acid and growth factor signaling pathways coordinately regulate the mechanistic target of rapamycin complex 1 (mTORC1) kinase in cell growth and organ development. While major components of amino acid signaling mechanisms have been identified, their biological functions in organ development are unclear. We aimed to understand the functions of the critically positioned amino acid signaling complex GAP activity towards Rags 2 (GATOR2) in brain development. GATOR2 mediates amino acid signaling to mTORC1 by directly linking the amino acid sensors for arginine and leucine to downstream signaling complexes. Now, we report a role of GATOR2 in oligodendrocyte myelination in postnatal brain development. We show that the disruption of GATOR2 complex by genetic deletion of meiosis regulator for oocyte development (Mios, encoding a component of GATOR2) selectively impairs the formation of myelinating oligodendrocytes, thus brain myelination, without apparent effects on the formation of neurons and astrocytes. The loss of Mios impairs cell cycle progression of oligodendrocyte precursor cells, leading to their reduced proliferation and differentiation. Mios deletion manifests a cell type-dependent effect on mTORC1 in the brain, with oligodendroglial mTORC1 selectively affected. However, the role of Mios/GATOR2 in oligodendrocyte formation and myelination involves mTORC1-independent function. This study suggests that GATOR2 coordinates amino acid and growth factor signaling to regulate oligodendrocyte myelination.

Branched-Chain Amino Acid Supplementation Alters the Abundance of Mechanistic Target of Rapamycin and Insulin Signaling Proteins in Subcutaneous Adipose Explants from Lactating Holstein Cows.

Simple SummaryBranched-chain amino acids (BCAAs) are import regulators of mechanistic target of rapamycin (mTOR). In humans and rodents, increased circulating BCAA levels are positively associated with changes in protein abundance of insulin and amino acid (AA) signaling pathways in organs such as skeletal muscle and adipose. Unlike aspects of fatty acid metabolism (e.g., lipolysis, lipogenesis), it is unknown if BCAA directly affect subcutaneous adipose tissue (SAT) AA metabolism and insulin signaling. We propose that BCAA availability within SAT could enhance aspects of AA and insulin function by promoting increases in the abundance of key proteins.The objective of this study was to investigate changes in protein abundance of mTOR and insulin signaling pathway components along with amino acid (AA) transporters in bovine s.c. adipose (SAT) explants in response to increased supply of Leu, Ile, or Val. Explants of SAT from four lactating Holstein cows were incubated with high-glucose serum-free DMEM, to which the 10 essential AAs were added to create the following treatments: ideal mix of essential AA (IPAA; Lys:Met 2.9:1; Lys:Thr 1.8:1; Lys:His 2.38:1; Lys:Val 1.23:1; Lys:Ile 1.45:1; Lys:Leu 0.85:1; Lys:Arg 2.08:1) or IPAA supplemented with Ile, Val, or Leu to achieve a Lys:Ile of 1.29:1 (incIle), Lys:Val 1.12:1 (incVal), or Lys:Leu (incLeu) 0.78:1 for 4 h. Compared with IPAA, incLeu or incIle led to greater activation of protein kinase B (AKT; p-AKT/total AKT) and mTOR (p-mTOR/total mTOR). Total EAA in media averaged 7.8 ± 0.06 mmol/L across treatments. Incubation with incLeu, incIle, or incVal led to greater protein abundance of solute carrier family 38 member 1 (SLC38A1), a Gln transporter, and the BCAA catabolism enzyme branched-chain α-keto acid dehydrogenase kinase (BCKDK) compared with IPAA. Activation of eukaryotic elongation factor 2 (eEF2; p-eEF2/total eEF2) was also greater in response to incLeu, incIle, or incVal. Furthermore, compared with incLeu or incIle, incVal supplementation led to greater abundance of SLC38A1 and BCKDK. BCKDK is a rate-limiting enzyme regulating BCAA catabolism via inactivation and phosphorylation of the BCKD complex. Overall, data suggested that enhanced individual supplementation of BCAA activates mTOR and insulin signaling in SAT. Increased AA transport into tissue and lower BCAA catabolism could be part of the mechanism driving these responses. The potential practical applications for enhancing post-ruminal supply of BCAA via feeding in rumen-protected form support in vivo studies to ascertain the role of these AAs on adipose tissue biology.

Intermittent Bolus Compared With Continuous Feeding Enhances Insulin and Amino Acid Signaling to Translation Initiation in Skeletal Muscle of Neonatal Pigs

BackgroundNutrition administered as intermittent bolus feeds rather than continuously promotes greater protein synthesis rates in skeletal muscle and enhances lean growth in a neonatal piglet model. The molecular mechanisms responsible remain unclear. ObjectivesWe aimed to identify the insulin- and/or amino acid-signaling components involved in the enhanced stimulation of skeletal muscle by intermittent bolus compared to continuous feeding in neonatal pigs born at term. MethodsTerm piglets (2–3 days old) were fed equal amounts of sow milk replacer [12.8 g protein and 155 kcal/(kg body weight · d)] by orogastric tube as intermittent bolus meals every 4 hours (INT) or by continuous infusion (CTS). After 21 days, gastrocnemius muscle samples were collected from CTS, INT-0 (before a meal), and INT-60 (60 minutes after a meal) groups (n = 6/group). Insulin- and amino acid-signaling components relevant to mechanistic target of rapamycin complex (mTORC) 1 activation and protein translation were measured. ResultsPhosphorylation of the insulin receptor, IRS-1, PDK1, mTORC2, pan-Akt, Akt1, Akt2, and TSC2 was 106% to 273% higher in the skeletal muscle of INT-60 piglets than in INT-0 and CTS piglets (P < 0.05), but phosphorylation of PTEN, PP2A, Akt3, ERK1/2, and AMPK did not differ among groups, nor did abundances of PHLPP, SHIP2, and Ubl4A. The association of GATOR2 with Sestrin1/2, but not CASTOR1, was 51% to 52% lower in INT-60 piglets than in INT-0 and CTS piglets (P < 0.05), but the abundances of SLC7A5/LAT1, SLC38A2/SNAT2, SLC38A9, Lamtor1/2, and V-ATPase did not differ. Associations of mTOR with RagA, RagC, and Rheb and phosphorylation of S6K1 and 4EBP1, but not eIF2α and eEF2, were 101% to 176% higher in INT-60 piglets than in INT-0 and CTS piglets (P < 0.05). ConclusionsThe enhanced rates of muscle protein synthesis and growth with intermittent bolus compared to continuous feeding in a neonatal piglet model can be explained by enhanced activation of both the insulin- and amino acid-signaling pathways that regulate translation initiation.

A machine learning analysis of a \u201cnormal-like\u201d IDH-WT diffuse glioma transcriptomic subgroup associated with prolonged survival reveals novel immune and neurotransmitter-related actionable targets

BackgroundClassification of primary central nervous system tumors according to the World Health Organization guidelines follows the integration of histologic interpretation with molecular information and aims at providing the most precise prognosis and optimal patient management. According to the cIMPACT-NOW update 3, diffuse isocitrate dehydrogenase-wild type (IDH-WT) gliomas should be graded as grade IV glioblastomas (GBM) if they possess one or more of the following molecular markers that predict aggressive clinical course: EGFR amplification, TERT promoter mutation, and whole-chromosome 7 gain combined with chromosome 10 loss.MethodsThe Cancer Genome Atlas (TCGA) glioma expression datasets were reanalyzed in order to identify novel tumor subcategories which would be considered as GBM-equivalents with the current diagnostic algorithm. Unsupervised clustering allowed the identification of previously unrecognized transcriptomic subcategories. A supervised machine learning algorithm (k-nearest neighbor model) was also used to identify gene signatures specific to some of these subcategories.ResultsWe identified 14 IDH-WT infiltrating gliomas displaying a “normal-like” (NL) transcriptomic profile associated with a longer survival. Genes such as C5AR1 (complement receptor), SLC32A1 (vesicular gamma-aminobutyric acid transporter), MSR1 (or CD204, scavenger receptor A), and SYT5 (synaptotagmin 5) were differentially expressed and comprised in gene signatures specific to NL IDH-WT gliomas which were validated further using the Chinese Glioma Genome Atlas datasets. These gene signatures showed high discriminative power and correlation with survival.ConclusionNL IDH-WT gliomas represent an infiltrating glioma subcategory with a superior prognosis which can only be detected using genome-wide analysis. Differential expression of genes potentially involved in immune checkpoint and amino acid signaling pathways is providing insight into mechanisms of gliomagenesis and could pave the way to novel treatment targets for infiltrating gliomas.

The Rag GTPase Regulates the Dynamic Behavior of TSC Downstream of Both Amino Acid and Growth Factor Restriction.

The dysregulation of the metabolic regulator TOR complex I (TORC1) contributes to a wide array of human pathologies. Tuberous sclerosis complex (TSC) is a potent inhibitor of TORC1. Here, we demonstrate that the Rag GTPase acts in both the amino-acid-sensing and growth factor signaling pathways to control TORC1 activity through the regulation of TSC dynamics in HeLa cells and Drosophila. We find that TSC lysosomal-cytosolic exchange increases in response to both amino acid and growth factor restriction. Moreover, the rate of exchange mirrors TSC function, with depletions of the Rag GTPase blocking TSC lysosomal mobility and rescuing TORC1 activity. Finally, we show that the GATOR2 complex controls the phosphorylation of TSC2, which is essential for TSC exchange. Our data support the model that the amino acid and growth factor signaling pathways converge on the Rag GTPase to inhibit TORC1 activity through the regulation of TSC dynamics.

356 Meal feeding compared with continuous feeding enhances insulin and amino acid signaling to translation initiation in skeletal muscle of pigs

Abstract Objectives: Meal feeding enhances skeletal muscle protein synthesis and lean growth more than continuous feeding in piglets. This enhanced muscle protein synthesis with meal feeding is associated with increased activation of mTORC1-dependent translation initiation. The mechanism underlying this response is unknown. We aimed to identify insulin and amino acid signaling components involved in the enhanced lean growth that results from meal feeding vs. continuous feeding in term-born pigs. Methods: Newborn piglets were fed for 21 d an equal amount of sow milk replacer (12.8 g protein and 155 kcal/(kg BW.d)) by gastrostomy tube either as intermittent bolus meals every 4 h (MEAL) or by continuous infusion (CON). After 21 d, gastrocnemius muscle was collected from CON, and before (MEAL-0) or 60 min after a meal (MEAL-60). Components of the insulin and amino acid signaling pathways up- and downstream of mTORC1 that regulate protein translation were measured. Results: Phosphorylation of AKT and TCS2 was greater in MEAL-60 than in MEAL-0 and CON (P &amp;lt; 0.05). The association of Sestrin2 with GATOR2 was similar in CON and MEAL-0 but was lower in MEAL-60 (P &amp;lt; 0.05). The abundances of RagA-mTOR, RagC-mTOR, and Rheb-mTOR, but not CASTOR1-GATOR2, complexes were higher in MEAL-60 than in CON and MEAL-0 (P &amp;lt; 0.05). The phosphorylation of S6K1 and 4EBP1 was higher in MEAL-60 than CON and MEAL-0 (P &amp;lt; 0.05). The abundances of Sestrin2, GATOR2, CASTOR1, RagA, RagC, and Rheb and the phosphorylation of eIF2alpha, eEF2, ERK1/2 and AMPK were unaffected by treatments. Conclusions: Our results demonstrate that the enhanced rate of skeletal muscle protein synthesis and lean growth with meal feeding compared with continuous feeding are due to the enhanced activation of both insulin and amino acid signaling pathways that result in the greater stimulation of translation initiation. Support: NIH HD085573, USDA CRIS 6250-51000-055, NIH HD072891, USDA NIFA 2013-67015-20438.

Dihydrocapsaicin Inhibits Epithelial Cell Transformation through Targeting Amino Acid Signaling and c-Fos Expression.

Chili peppers are one of the most widely consumed spices worldwide. However, research on the health benefits of chili peppers and some of its constituents has raised controversy as to whether chili pepper compounds possess cancer-promoting or cancer-preventive effects. While ample studies have been carried out to examine the effect of capsaicin in carcinogenesis, the chemopreventive effect of other major components in chili pepper, including dihydrocapsaicin, capsiate, and capsanthin, is relatively unclear. Herein, we investigated the inhibitory effect of chili pepper components on malignant cell transformation. Among the tested chili pepper compounds, dihydrocapsaicin displayed the strongest inhibitory activity against epidermal growth factor (EGF)-induced neoplastic transformation. Dihydrocapsaicin specifically suppressed EGF-induced phosphorylations of the p70S6K1-S6 pathway and the expression of c-Fos. A reduction in c-Fos levels by dihydrocapsaicin led to a concomitant downregulation of AP-1 activation. Further analysis of the molecular mechanism responsible for the dihydrocapsaicin-mediated decrease in phospho-p70S6K1, revealed that dihydrocapsaicin can block amino acid-dependent mechanistic targets of rapamycin complex 1 (mTORC1)-p70S6K1-S6 signal activation. Additionally, dihydrocapsaicin was able to selectively augment amino acid deprivation-induced cell death in mTORC1-hyperactive cells. Collectively, dihydrocapsaicin exerted chemopreventive effects through inhibiting amino acid signaling and c-Fos pathways and, thus, might be a promising cancer preventive natural agent.

Intermittent Bolus Compared with Continuous Feeding Enhances Insulin and Amino Acid Signaling to Translation Initiation in Skeletal Muscle of Pigs Born at Term (P08-071-19)

ObjectivesOur recent study in a piglet model of the human neonate born at full term showed that intermittent bolus feeding promotes greater rates of protein synthesis in skeletal muscle than continuous feeding, leading to an increase in lean growth. This enhanced rate of muscle protein synthesis with intermittent bolus feeding is associated with an increased activation of mTORC1-dependent translation initiation. However, the mechanism underlying this response is unknown. In this study, we aimed to identify the insulin and/or amino acid signaling components involved in the enhanced stimulation of lean growth by intermittent bolus compared to continuous feeding in term-born pigs. MethodsTerm piglets (2–3 d old) were fed for 21 d an equal amount (240 ml/kg body weight [BW]/d) of sow milk replacer containing 12.8 g protein and 175 kcal/kg BW/d. Feedings were administered by gastrostomy tube either as intermittent bolus meals every 4 h (INT) or by continuous infusion (CON). After 21 d, gastrocnemius muscle was collected from CON, INT-0 (before a meal) and INT-60 (60 min after a meal) groups. Upstream and downstream insulin and amino acid signaling components of relevance to mTORC1 activation and protein translation were measured. ResultsPhosphorylation of AKT and TCS2 was greater in INT-60 than in INT-0 and CON groups (P < 0.05). There was no significant difference between CON and INT groups in the phosphorylation of ERK 1/2 and AMPK. The association of Sestrin2, a leucine sensor, with GATOR2 was similar in CON and INT-0 but was lower in INT-60 (P < 0.05). The abundances of RagA-mTOR, RagC-mTOR, and Rheb-mTOR complexes were higher in INT-60 than in CON and INT-0 (P < 0.05). The phosphorylation of S6K1 and 4EBP1 was higher in INT-60 than CON and INT-0 groups (P < 0.05). Phosphorylation of eIF2alpha and eEF2 were not affected by treatments. ConclusionsOur results demonstrate that, following a full-term birth, the enhanced rate of skeletal muscle protein synthesis and lean growth with intermittent bolus compared to continuous feeding is at least in part due to the enhanced activation of both insulin and amino acid signaling pathways leading to greater stimulation of translation initiation. Funding SourcesNIH HD085573, USDA CRIS 6250-51000-055, NIH HD072891, USDA NIFA 2013-67015-20438.

Interventions Targeting Glucocorticoid-Krüppel-like Factor 15-Branched-Chain Amino Acid Signaling Improve Disease Phenotypes in Spinal Muscular Atrophy Mice

The circadian glucocorticoid-Krüppel-like factor 15-branched-chain amino acid (GC-KLF15-BCAA) signaling pathway is a key regulatory axis in muscle, whose imbalance has wide-reaching effects on metabolic homeostasis. Spinal muscular atrophy (SMA) is a neuromuscular disorder also characterized by intrinsic muscle pathologies, metabolic abnormalities and disrupted sleep patterns, which can influence or be influenced by circadian regulatory networks that control behavioral and metabolic rhythms. We therefore set out to investigate the contribution of the GC-KLF15-BCAA pathway in SMA pathophysiology of Taiwanese Smn−/−;SMN2 and Smn2B/− mouse models. We thus uncover substantial dysregulation of GC-KLF15-BCAA diurnal rhythmicity in serum, skeletal muscle and metabolic tissues of SMA mice. Importantly, modulating the components of the GC-KLF15-BCAA pathway via pharmacological (prednisolone), genetic (muscle-specific Klf15 overexpression) and dietary (BCAA supplementation) interventions significantly improves disease phenotypes in SMA mice. Our study highlights the GC-KLF15-BCAA pathway as a contributor to SMA pathogenesis and provides several treatment avenues to alleviate peripheral manifestations of the disease. The therapeutic potential of targeting metabolic perturbations by diet and commercially available drugs could have a broader implementation across other neuromuscular and metabolic disorders characterized by altered GC-KLF15-BCAA signaling.

Spatial Activation of TORC1 Is Regulated by Hedgehog and E2F1 Signaling in the Drosophila Eye

Target of rapamycin complex 1 (TORC1) regulates cell growth in response to nutrients and growth factors. Although TORC1 signaling has been thoroughly studied at the cellular level, the regulation of TORC1 in multicellular tissues and organs has remained elusive. Here we found that TORC1 is selectively activated in the second mitotic wave (SMW), the terminal synchronous cell division, of the developing Drosophila eye. We demonstrated that Hedgehog (Hh) signaling regulates TORC1 through E2F1 and the cyclin D/Cdk4 complex in the SMW, and this regulation is independent from insulin and amino acid signaling pathways. TORC1 is necessary for the proper G1/S transition of the cells, and the activation of TORC1 rescues the cell-cycle defect of Hhsignaling-deficient cells in the SMW. Based on this evolutionarily conserved regulation of TORC1 by Hh signaling, we propose that Hh-dependent developmental signaling pathways spatially regulate TORC1 activity in multicellular organisms.

Palmitoylation of the carboxyl-terminal tail of dopamine D4 receptor is required for surface expression, endocytosis, and signaling

The amino acid sequences and signaling pathways of D2-like dopamine receptors (D2R, D3R, and D4R) are highly conserved. D4R has been suggested to be associated with novelty seeking, and binds with high affinity to an atypical antipsychotic drug with fewer motor function-related side effects than typical neuroleptics. A study comparing D2R and D3R reported that palmitoylation is important for the proper functioning of D3R. Although D4R is a member of the D2-like receptor family, its palmitoylation status and the functional significance of any such posttranslational modification are unknown. In this study, it was found that D4-4, an alternatively spliced form of D4R, was palmitoylated on Cys467, the terminal amino acid residue of the receptor. When palmitoylation of D4R was inhibited, by either mutation of the consensus site or by treatment with the palmitoylation inhibitor 2-bromopalmitate (2BP), D4R cell surface expression, signaling, and endocytosis were all impaired. Exchanging the carboxyl-terminal tails of D2R and D4R resulted in a switching of the palmitoylation phenotype, as well as concomitant changes in functionality, such as receptor surface expression, endocytosis, and signaling. Despite the high degree of homology in the carboxyl-terminal tails of D2-like receptors, palmitoylation occurs exclusively in the D3R and D4R family members, with the D4R tail being sufficient to endow D2R with palmitoylation-associated functionality. Thus, this study provides new insights into a consensus sequence for palmitoylation, as well as possible strategies for the regulation of D4R, which has implications for the treatment of various neurological and psychiatric disorders.

Crosstalk between cystine and glutathione is critical for the regulation of amino acid signaling pathways and ferroptosis.

Although essential amino acids regulate mechanistic target of rapamycin complex 1 (mTORC1) and the integrated stress response (ISR), the role of cysteine is unknown. We found that in hepatoma HepG2 cells, cystine (oxidized form of cysteine) activated mTORC1 and suppressed the ISR. Cystine deprivation induced GSH efflux and extracellular degradation, which aimed to restore cellular cysteine. Inhibition of γ-glutamyl transpeptidase (GGT) impaired the ability of GSH or cell-permeable GSH to restore mTORC1 signaling and the ISR, suggesting that the capacity of GSH to release cysteine, but not GSH per se, regulated the signaling networks. Inhibition of protein translation restored both mTORC1 signaling and the ISR during cystine starvation, suggesting the bulk of cellular cysteine was committed to the biosynthetic process. Cellular cysteine and GSH displayed overlapping protective roles in the suppression of ferroptosis, further supporting their cooperation in the regulation of cell signaling. Thus, cellular cysteine and its derivative GSH cooperate to regulate mTORC1 pathway, the ISR and ferroptosis.

Water Stress Modulates Soybean Aphid Performance, Feeding Behavior, and Virus Transmission in Soybean.

Little is known about how water stress including drought and flooding modifies the ability of plants to resist simultaneous attack by insect feeding and transmission of insect-vectored pathogen. We analyzed insect population growth, feeding behaviors, virus transmission, and plant amino acid profiles and defense gene expression to characterize mechanisms underlying the interaction between water stress, soybean aphid and aphid-transmitted, Soybean mosaic virus, on soybean plants. Population growth of non-viruliferous aphids was reduced under drought stress and saturation, likely because the aphids spent less time feeding from the sieve element on these plants compared to well-watered plants. Water stress did not impact population growth of viruliferous aphids. However, virus incidence and transmission rate was lowest under drought stress and highest under saturated conditions since viruliferous aphids took the greatest amount time to puncture cells and transmit the virus under saturated conditions and lowest time under drought stress. Petiole exudates from drought-stressed plants had the highest level of total free amino acids including asparagine and valine that are critical for aphid performance. Aphids did not benefit from improved phloem sap quality as indicated by their lower densities on drought-stressed plants. Saturation, on the other hand, resulted in low amino acid content compared to all of the other treatments. Drought and saturation had significant and opposing effects on expression of marker genes involved in abscisic acid (ABA) signaling. Drought alone significantly increased expression of ABA marker genes, which likely led to suppression of salicylic acid (SA)- and jasmonic acid (JA)-related genes. In contrast, ABA marker genes were down-regulated under saturation, while expression of SA- and JA-related genes was up-regulated. We propose that the apparent antagonism between ABA and SA/JA signaling pathways contributed to an increase in aphid densities under drought and their decrease under saturation. Taken together, our findings suggests that plant responses to water stress is complex involving changes in phloem amino acid composition and signaling pathways, which can impact aphid populations and virus transmission.