Targeting valine catabolism to inhibit metabolic reprogramming in prostate cancer.

Metabolic reprogramming and energetic rewiring are hallmarks of cancer that fuel disease progression and facilitate therapy evasion. The remodelling of oxidative phosphorylation and enhanced lipogenesis have previously been characterised as key metabolic features of prostate cancer (PCa). Recently, succinate-dependent mitochondrial reprogramming was identified in high-grade prostate tumours, as well as upregulation of the enzymes associated with branched-chain amino acid (BCAA) catabolism. In this study, we hypothesised that the degradation of the BCAAs, particularly valine, may play a critical role in anapleurotic refuelling of the mitochondrial succinate pool, as well as the maintenance of intracellular lipid metabolism. Through the suppression of BCAA availability, we report significantly reduced lipid content, strongly indicating that BCAAs are important lipogenic fuels in PCa. This work also uncovered a novel compensatory mechanism, whereby fatty acid uptake is increased in response to extracellular valine deprivation. Inhibition of valine degradation via suppression of 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) resulted in a selective reduction of malignant prostate cell proliferation, decreased intracellular succinate and impaired cellular respiration. In combination with a comprehensive multi-omic investigation that incorporates next-generation sequencing, metabolomics, and high-content quantitative single-cell imaging, our work highlights a novel therapeutic target for selective inhibition of metabolic reprogramming in PCa.

Valine aminoacyl-tRNA synthetase promotes therapy resistance in melanoma.

Transfer RNA dynamics contribute to cancer development through regulation of codon-specific messenger RNA translation. Specific aminoacyl-tRNA synthetases can either promote or suppress tumourigenesis. Here we show that valine aminoacyl-tRNA synthetase (VARS) is a key player in the codon-biased translation reprogramming induced by resistance to targeted (MAPK) therapy in melanoma. The proteome rewiring in patient-derived MAPK therapy-resistant melanoma is biased towards the usage of valine and coincides with the upregulation of valine cognate tRNAs and of VARS expression and activity. Strikingly, VARS knockdown re-sensitizes MAPK-therapy-resistant patient-derived melanoma in vitro and in vivo. Mechanistically, VARS regulates the messenger RNA translation of valine-enriched transcripts, among which hydroxyacyl-CoA dehydrogenase mRNA encodes for a key enzyme in fatty acid oxidation. Resistant melanoma cultures rely on fatty acid oxidation and hydroxyacyl-CoA dehydrogenase for their survival upon MAPK treatment. Together, our data demonstrate that VARS may represent an attractive therapeutic target for the treatment of therapy-resistant melanoma.

Valsa mali PR1-like protein modulates an apple valine-glutamine protein to suppress JA signaling-mediated immunity.

Apple Valsa canker (AVC) is a devastating disease of apple (Malus × domestica), caused by Valsa mali (Vm). The Cysteine-rich secretory protein, Antigen 5, and Pathogenesis-related protein 1 (CAP) superfamily protein PATHOGENESIS-RELATED PROTEIN 1-LIKE PROTEIN c (VmPR1c) plays an important role in the pathogenicity of Vm. However, the mechanisms through which it exerts its virulence function in Vm-apple interactions remain unclear. In this study, we identified an apple valine-glutamine (VQ)-motif-containing protein, MdVQ29, as a VmPR1c target protein. MdVQ29-overexpressing transgenic apple plants showed substantially enhanced AVC resistance as compared with the wild type. MdVQ29 interacted with the transcription factor MdWRKY23, which was further shown to bind to the promoter of the jasmonic acid (JA) signaling-related gene CORONATINE INSENSITIVE 1 (MdCOI1) and activate its expression to activate the JA signaling pathway. Disease evaluation in lesion areas on infected leaves showed that MdVQ29 positively modulated apple resistance in a MdWRKY23-dependent manner. Furthermore, MdVQ29 promoted the transcriptional activity of MdWRKY23 toward MdCOI1. In addition, VmPR1c suppressed the MdVQ29-enhanced transcriptional activation activity of MdWRKY23 by promoting the degradation of MdVQ29 and inhibiting MdVQ29 expression and the MdVQ29-MdWRKY23 interaction, thereby interfering with the JA signaling pathway and facilitating Vm infection. Overall, our results demonstrate that VmPR1c targets MdVQ29 to manipulate the JA signaling pathway to regulate immunity. Thus, this study provides an important theoretical basis and guidance for mining and utilizing disease-resistance genetic resources for genetically improving apples.

Valine and nonessential amino acids affect bidirectional transport rates of leucine and isoleucine in bovine mammary epithelial cells.

A more complete understanding of the mechanisms controlling AA transport in mammary glands of dairy cattle will help identify solutions to increase nitrogen feeding efficiency on farms. It was hypothesized that Ala, Gln, and Gly (NEAAG), which are actively transported into cells and exchanged for all branched-chain AA (BCAA), may stimulate transport of BCAA, and that Val may antagonize transport of the other BCAA due to transporter competition. Thus, we evaluated the effects of varying concentrations of NEAAG and Val on transport and metabolism of the BCAA Ala, Met, Phe, and Thr by bovine mammary epithelial cells. Primary cultures of bovine mammary epithelial cells were assigned to treatments of low (70% of mean in vivo plasma concentrations of lactating dairy cows) and high (200%) concentrations of Val and NEAAG (LVal and LNEAAG, HVal and HNEAAG, respectively) in a 2 × 2 factorial design. Cells were preloaded with treatment media containing [15N]-labeled AA for 24 h. The [15N]-labeled media were replaced with treatment media containing [13C]-labeled AA. Media and cells were harvested from plates at 0, 0.5, 1, 5, 15, 30, 60, and 240 min after application of the [13C]-labeled AA and assessed for [15N]- and [13C]-AA label concentrations. The data were used to derive transport, transamination, irreversible loss, and protein-synthesis fluxes. All Val fluxes, except synthesis of rapidly exchanging tissue protein, increased with the HVal treatment. Interestingly, the rapidly exchanging tissue protein, transamination, and irreversible-loss rate constants decreased with HVal, indicating that the significant flux increases were primarily driven by mass action with the cells resisting the flux increases by downregulating activity. However, the decreases could also reflect saturation of processes that would drive down the mass-action rate constants. This is supported by decreases in the same rate constants for Ile and Leu with HVal. This could be due to either competition for shared transamination and oxidation reactions or a reduction in enzymatic activity. Also, NEAAG did not affect Val fluxes, but influx and efflux rate constants increased for both Val and Leu with HNEAAG, indicating an activating substrate effect. Overall, AA transport rates generally responded concordantly with extracellular concentrations, indicating the transporters are not substrate-saturated within the in vivo range. However, BCAA transamination and oxidation enzymes may be approaching saturation within in vivo ranges. In addition, System L transport activity appeared to be stimulated by as much as 75% with high intracellular concentrations of Ala, Gln, and Gly. High concentrations of Val antagonized transport activity of Ile and Leu by 68% and 15%, respectively, indicating competitive inhibition, but this was only observable at HNEAAG concentrations. The exchange transporters of System L transport 8 of the essential AA that make up approximately 40% of milk protein, so better understanding this transporter is an important step for increased efficiency.

Valine promotes milk synthesis by regulating PKM2 nuclear accumulation and histone H3 acetylation through the TAS1R1-mTOR-DDX39B signaling pathway.

Valine, a branched-chain amino acid found in dairy cows, has been recognized for its critical role in milk synthesis. However, the precise effect of valine on lactation in dairy cows remains an area of investigation. In our study, bovine mammary epithelial cells (BMECs) were isolated to explore the mechanism through which valine enhances milk synthesis. The results showed that 100 µM valine significantly boosted the milk synthesis via TAS1R1-mTOR-DDX39B signaling pathway in BMECs. Subsequent investigations revealed that DDX39B governs the accumulation of PKM2 in the nuclei of BMECs. This nuclear buildup of PKM2 weakened the interaction between HDAC3 and histone H3, leading to an increase in the acetylation levels of histone H3. In an vivo context, the 0.25 % valine-enriched drinking water notably elevated in the expression of milk protein and fat in these mice. Further examination showed that 0.25 % valine drinking water considerably augmented the protein expression levels of DDX39B, PKM2, and p-mTOR in the mice mammary glands. In summary, our results suggest that valine, by modulating the TAS1R1-mTOR-DDX39B signaling pathway, directs the accumulation of PKM2 in the nucleus. This, in turn, escalates the acetylation levels of histone H3, promoting the synthesis of both milk protein and fat.

Low-Molecular-Weight Fish Collagen Peptide (Valine-Glycine-Proline-Hydroxyproline-Glycine-Proline-Alanine-Glycine) Prevents Osteoarthritis Symptoms in Chondrocytes and Monoiodoacetate-Injected Rats.

The objective of this study was to investigate the effect of low-molecular-weight fish collagen (valine-glycine-proline-hydroxyproline-glycine-proline-alanine-glycine; LMWCP) on H2O2- or LPS-treated primary chondrocytes and monoiodoacetate (MIA)-induced osteoarthritis rat models. Our findings indicated that LMWCP treatment exhibited protective effects by preventing chondrocyte death and reducing matrix degradation in both H2O2-treated primary chondrocytes and cartilage tissue from MIA-induced osteoarthritis rats. This was achieved by increasing the levels of aggrecan, collagen type I, collagen type II, TIMP-1, and TIMP-3, while simultaneously decreasing catabolic factors such as phosphorylation of Smad, MMP-3, and MMP-13. Additionally, LMWCP treatment effectively suppressed the activation of inflammation and apoptosis pathways in both LPS-treated primary chondrocytes and cartilage tissue from MIA-induced osteoarthritis rats. These results suggest that LMWCP supplementation ameliorates the progression of osteoarthritis through its direct impact on inflammation and apoptosis in chondrocytes.

[Analysis of the difference in metabolites and gene expressions between pre-receptive and receptive endometria].

OBJECTIVE: To analyze the difference in the gene expression, amino acid and carnitine levels in the cervical secretions between the endometria of pre-receptive and receptive stages, with an aim to provide clues for identifying new molecular markers for endometrial receptivity. METHODS: Fifty nine infertile women treated at the Department of Reproductive Medicine of Linyi People's Hospital from January 6, 2020 to January 31, 2022 were selected as as the study subjects, which were matched with 3 pairs (6 cases) of infertile women preparing for embryo transfer based on factors such as age, body mass index, and length of infertility. Endometrial tissue samples were collected for gene transcription and expression analysis. Twenty five women who had become pregnant through assisted reproductive technology were selected as the control group, and 28 non-pregnant women receiving ovulation monitoring at the Outpatient Department were enrolled as the case group. Status of endometrial receptivity was determined by ultrasonography. In the former group, endometrial tissues were sampled for sequencing, and GO and KEGG database enrichment analysis of differentially expressed genes was carried out. In the latter group, cervical secretions were collected, and amino acid and carnitine levels were measured by mass spectrometry. Statistical analysis was carried out using rank sum test, t test and chi-square test with SPSS v25.0 software. RESULTS: No difference was found in the clinical data of the patients with regard to age, body mass index, infertility years, AMH, FSH, LH, E2, and type of infertility. Compared with the receptive endometrial tissues, there were 100 significantly up-regulated genes and 191 significantly down-regulated genes in the pre-receptive endometrial tissue, with the most significantly altered ones being HLA-DRB5 and MMP10. The biological processes, molecular functions and pathways enriched by more differentially expressed genes in GO and KEGG were mainly immune regulation, cell adhesion and tryptophan metabolism. Analysis of secretion metabolism also revealed a significant difference in the levels of amino acids and carnitine metabolites between the two groups (P < 0.05), in particular those of Alanine, Valine, 3-hydroxybutyrylcarnitine (C4OH) + malonylcarnitine (C3DC)/captoylcarnitine (C10). CONCLUSION: A significant difference has been discovered in the levels of gene transcription and protein expression in the endometrial tissues from the pre-receptive and receptive stages. The levels of amino acids and carnitine, such as Alanine, Valine, 3-hydroxybutyryl carnitine (C4OH)+malonyl carnitine (C3DC)/caproyl carnitine (C10), may be associated with the receptive status of the endometrium, though this need to be verified with larger samples.

Branched-chain amino acid modulation of lipid metabolism, gluconeogenesis, and inflammation in a finishing pig model: targeting leucine and valine.

Branched-chain amino acids (BCAAs) play a regulatory role in adipogenesis and energy balance. Therefore, this study aimed to investigate the impact of BCAA supplements, especially leucine (Leu) and valine (Val) supplementation, on lipid metabolism and related disorders in a finishing pig model. The results demonstrated that Leu (1%) and Val decreased serum as well as hepatic lipid accumulation. Moreover, metabolomics and lipidomics analyses revealed that Leu and Val markedly downregulated the level of various lipid species in the liver. This outcome may be explained by Leu and Val promoting cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/hormone-sensitive triglyceride lipase (HSL) signaling pathways. Leu and Val altered the fatty acid composition in distinct adipose tissues and decreased the levels of inflammatory factors. Additionally, they significantly decreased back fat thickness, and the results of the fatty acid profiles demonstrated that Leu and Val significantly increased the levels of monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) while decreasing those of saturated fatty acids (SFAs), especially in back fat and abdominal fat. Besides, Leu and Val restored glucose homeostasis by suppressing gluconeogenesis through the serine/threonine protein kinase (AKT)/transcription factor forkhead box O1 (FOXO1) signaling pathway in the liver and back fat. In summary, these results suggest that Leu and Val may serve as key regulators for modulating lipid metabolism and steatosis.

Altered gut microbiome, bile acid composition and metabolome in sarcopenia in liver cirrhosis.

BACKGROUND: Sarcopenia in liver cirrhosis is associated with low quality of life and high mortality risk. The pathogenesis has yet to be fully understood. We hypothesized that gut microbiome, bile acid (BA) composition and metabolites differ between cirrhotic patients with and without sarcopenia and contribute to pathogenesis. METHODS: Cirrhotic patients with (n = 78) and without (n = 38) sarcopenia and non-cirrhotic controls with (n = 39) and without (n = 20) sarcopenia were included in this study. Faecal microbiome composition was studied by 16S rDNA sequencing, serum and faecal BA composition by ultra-high-performance liquid chromatography-tandem mass spectrometry, and metabolite composition in serum, faeces and urine by nuclear magnetic resonance. RESULTS: Bacteroides fragilis, Blautia marseille, Sutterella spp. and Veillonella parvula were associated with cirrhotic patients with sarcopenia, whereas Bacteroides ovatus was more abundant in cirrhotic patients without sarcopenia. We observed significantly elevated secondary BAs, deoxycholic acid (DCA; P = 0.01) and lithocholic acid (LCA; P = 0.02), and the ratios of deoxycholic acid to cholic acid (DCA:CA; P = 0.04), lithocholic acid to chenodeoxycholic acid (LCA:CDCA; P = 0.03) and 12 alpha-hydroxylated to non-12 alpha-hydroxylated BAs (12-α-OH:non-12-α-OH BAs; P = 0.04) in serum of cirrhotic patients with sarcopenia compared with cirrhotic patients without sarcopenia, indicating an enhanced transformation of primary to secondary BAs by the gut microbiome. CA (P = 0.02) and the ratios of CA:CDCA (P = 0.03) and total ursodeoxycholic acid to total secondary BAs (T-UDCA:total-sec-BAs, P = 0.03) were significantly reduced in the stool of cirrhotic patients with sarcopenia compared with cirrhotic patients without sarcopenia. Also, valine and acetate were significantly reduced in the serum of cirrhotic patients with sarcopenia compared with cirrhotic patients without sarcopenia (P = 0.01 and P = 0.03, respectively). Multivariate logistic regression further confirmed the association of B. ovatus (P = 0.01, odds ratio [OR]: 12.8, 95% confidence interval [CI]: 168.1; 2.2), the ratios of 12-α-OH:non-12-α-OH BAs (P = 0.03, OR: 2.54, 95% CI: 0.99; 6.55) and T-UDCA:total-sec-BAs (P = 0.04, OR: 0.25, 95% CI: 0.06; 0.98) in serum and stool CA:CDCA (P = 0.04, OR: 0.79, 95% CI: 0.62; 0.99), and serum valine (P = 0.04, OR: 1.00, 95% CI: 1.02; 1.00) with sarcopenia in cirrhosis after correcting for the severity of liver disease and sex. CONCLUSIONS: Our study suggests a potential functional gut microbiome-host interaction linking sarcopenia with the altered gut microbiomes, BA profiles and amino acids pointing towards a potential mechanistic interplay in understanding sarcopenia pathogenesis.

Dehydroepiandrosterone supplementation and the impact of follicular fluid metabolome and cytokinome profiles in poor ovarian responders.

BACKGROUND: Poor ovarian responders (POR) are women undergoing in-vitro fertilization who respond poorly to ovarian stimulation, resulting in the retrieval of lower number of oocytes, and subsequently lower pregnancy rates. The follicular fluid (FF) provides a crucial microenvironment for the proper development of follicles and oocytes through tightly controlled metabolism and cell signaling. Androgens such as dehydroepiandrosterone (DHEA) have been proposed to alter the POR follicular microenvironment, but the impact DHEA imposes on the FF metabolome and cytokine profiles is unknown. Therefore, the objective of this study is to profile and identify metabolomic changes in the FF with DHEA supplementation in POR patients. METHODS: FF samples collected from 52 POR patients who underwent IVF with DHEA supplementation (DHEA +) and without (DHEA-; controls) were analyzed using untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics and a large-scale multiplex suspension immunoassay covering 65 cytokines, chemokines and growth factors. Multivariate statistical modelling by partial least squares-discriminant regression (PLSR) analysis was performed for revealing metabolome-scale differences. Further, differential metabolite analysis between the two groups was performed by PLSR ß-coefficient regression analysis and Student's t-test. RESULTS: Untargeted metabolomics identified 118 FF metabolites of diverse chemistries and concentrations which spanned three orders of magnitude. They include metabolic products highly associated with ovarian function - amino acids for regulating pH and osmolarity, lipids such fatty acids and cholesterols for oocyte maturation, and glucocorticoids for ovarian steroidogenesis. Four metabolites, namely, glycerophosphocholine, linoleic acid, progesterone, and valine were significantly lower in DHEA + relative to DHEA- (p < 0.05-0.005). The area under the curves of progesterone glycerophosphocholine, linoleic acid and valine are 0.711, 0.730, 0.785 and 0.818 (p < 0.05-0.01). In DHEA + patients, progesterone positively correlated with IGF-1 (Pearson r: 0.6757, p < 0.01); glycerophosphocholine negatively correlated with AMH (Pearson r: -0.5815; p < 0.05); linoleic acid correlated with estradiol and IGF-1 (Pearson r: 0.7016 and 0.8203, respectively; p < 0.01 for both). In DHEA- patients, valine negatively correlated with serum-free testosterone (Pearson r: -0.8774; p < 0.0001). Using the large-scale immunoassay of 45 cytokines, we observed significantly lower MCP1, IFNγ, LIF and VEGF-D levels in DHEA + relative to DHEA. CONCLUSIONS: In POR patients, DHEA supplementation altered the FF metabolome and cytokine profile. The identified four FF metabolites that significantly changed with DHEA may provide information for titrating and monitoring individual DHEA supplementation.

Targeted Amino Acids Profiling of Human Seminal Plasma from Teratozoospermia Patients Using LC-MS/MS.

Identifying the metabolome of human seminal plasma (HSP) is a new research area to screen putative biomarkers of infertility. This case-control study was performed on HSP specimens of 15 infertile patients with teratozoospermia (defined as normal sperm morphology < 4%) and 12 confirmed fertile normozoospermic men as the control group to investigate the seminal metabolic signature and whether there are differences in the metabolome between two groups. HSPs were subjected to LC-MS-MS analysis. MetaboAnalyst5.0 software was utilized for statistical analysis. Different univariate and multivariate analyses were used, including T-tests, fold change analysis, random forest (RF), and metabolite set enrichment analysis (MSEA). Teratozoospermic samples contained seventeen significantly different amino acids. Upregulated metabolites include glutamine, asparagine, and glycylproline, whereas downregulated metabolites include cysteine, γ-aminobutyric acid, histidine, hydroxylysine, hydroxyproline, glycine, proline, methionine, ornithine, tryptophan, aspartic acid, argininosuccinic acid, α-aminoadipic acid, and ß-aminoisobutyric acid. RF algorithm defined a set of 15 metabolites that constitute the significant features of teratozoospermia. In particular, increased glutamine, asparagine, and decreased cysteine, tryptophan, glycine, and valine were strong predictors of teratozoospemia. The most affected metabolic pathways in teratozoospermic men are the aminoacyl-tRNA, arginine, valine-leucine, and isoleucine biosynthesis. Altered metabolites detected in teratozoospermia were responsible for various roles in sperm functions that classified into four subgroups as follows: related metabolites to antioxidant function, energy production, sperm function, and spermatogenesis. The altered amino acid metabolome identified in this study may be related to the etiology of teratozoospermia, and may provide novel insight into potential biomarkers of male infertility for therapeutic targets.

Phenylalanine diminishes M1 macrophage inflammation.

Emerging evidence suggests that amino acids dictate the effector functions of immune cells; however, whether and how phenylalanine (Phe) orchestrates the polarization of macrophages is not understood. Here, we determined that Phe attenuated lipopolysaccharide (LPS) and P. multocida serotype A strain CQ2 (PmCQ2) infection-induced inflammation in vivo. Furthermore, we demonstrated that Phe inhibited the production of interleukin (IL)-1ß and tumor necrosis factor (TNF)-α in proinflammatory (M1) macrophages. Phe reprogrammed the transcriptomic and metabolic profiles and enhanced oxidative phosphorylation in M1 macrophages, which reduced the activation of caspase-1. Notably, the valine-succinyl-CoA axis played a critical role in Phe-mediated inhibition of IL-1ß production in M1 macrophages. Taken together, our findings suggest that manipulating the valine-succinyl-CoA axis provides a potential target for preventing and/or treating macrophage-related diseases.

Genome-wide identification of the valine-glutamine motif containing gene family and the role of VQ25-1 in pollen germination in Brassica oleracea.

BACKGROUND: The plant-specific valine-glutamine (VQ) motif containing proteins tightly regulate plant growth, development, and stress responses. However, the genome-wide identification and functional analysis of Brassica oleracea (B. oleracea) VQ genes have not been reported. OBJECTIVE: To identify the VQ gene family in B. oleracea and analyze the function of Bo25-1 in pollen germination. METHODS: The Hidden Markov Model (HMM) of VQ family was used to query the BoVQ genes in the B. oleracea genome. The BoVQ genes preferentially expressed in anthers were screened by qRT-PCR. Subcellular localization of VQ25-1 was observed in Nicotiana benthamiana (N. benthamiana) leaves. To analysis the role of BoVQ25-1 in pollen germination, the expression of BoVQ25-1 was suppressed using antisense-oligonucleotides (AS-ODN). RESULTS: A total of 64 BoVQ genes were identified in the B. oleracea genome. BoVQ25-1 was found to be preferentially expressed in the B. oleracea anthers. BoVQ25-1 was cloned from the anthers of the B. oleracea cultivar 'Fast Cycle'. BoVQ25-1 is localized to the nucleus. The pollen germination rate significantly decreased after AS-ODN treatment. CONCLUSION: Sixty-four BoVQ genes were identified in the B. oleracea genome, of which BoVQ25-1 plays an important role in pollen germination.

Effects of branched-chain amino acids on Shiraia perylenequinone production in mycelium cultures.

BACKGROUND: Perylenequinones from Shiraia fruiting bodies are excellent photosensitizers and widely used for anti-cancer photodynamic therapy (PDT). The lower yield of Shiraia perylenequinones becomes a significant bottleneck for their medical application. Branched-chain amino acids (BCAAs) not only serve as important precursors for protein synthesis, but also are involved in signaling pathway in cell growth and development. However, there are few reports concerning their regulation of fungal secondary metabolism. In present study, the eliciting effects of BCAAs including L-isoleucine (L-Ile), L-leucine (L-Leu) and L-valine (L-Val) on Shiraia perylenequinone production were investigated. RESULTS: Based on the analysis of the transcriptome and amino acid contents of Shiraia in the production medium, we revealed the involvement of BCAAs in perylenequinone biosynthesis. The fungal conidiation was promoted by L-Val treatment at 1.5 g/L, but inhibited by L-Leu. The spore germination was promoted by both. The production of fungal perylenequinones including hypocrellins A (HA), HC and elsinochromes A-C (EA-EC) was stimulated significantly by L-Val at 1.5 g/L, but sharply suppressed by L-Leu. After L-Val treatment (1.5 g/L) in Shiraia mycelium cultures, HA, one of the main bioactive perylenequinones reached highest production 237.92 mg/L, about 2.12-fold than that of the control. Simultaneously, we found that the expression levels of key genes involved in the central carbon metabolism and in the late steps for perylenequinone biosynthesis were up-regulated significantly by L-Val, but most of them were down-regulated by L-Leu. CONCLUSIONS: Our transcriptome analysis demonstrated that BCAA metabolism was involved in Shiraia perylenequinone biosynthesis. Exogenous BCAAs exhibit contrasting effects on Shiraia growth and perylenequinones production. L-Val could promote perylenequinone biosynthesis via not only enhancing the central carbon metabolism for more precursors, but also eliciting perylenequinone biosynthetic gene expressions. This is the first report on the regulation of BCAAs on fungal perylenequinone production. These findings provided a basis for understanding physiological roles of BCAAs and a new avenue for increasing perylenequinone production in Shiraia mycelium cultures.

Valine Treatment Enhances Antimicrobial Component Production in Mammary Epithelial Cells and the Milk of Lactating Goats Without Influencing the Tight Junction Barrier.

The production of antimicrobial components and the formation of less-permeable tight junctions (TJs) are important in the defense system of lactating mammary glands and for safe dairy production. Valine is a branched-chain amino acid that is actively consumed in the mammary glands and promotes the production of major milk components like ß-casein; additionally, branched-chain amino acids stimulate antimicrobial component production in the intestines. Therefore, we hypothesized that valine strengthens the mammary gland defense system without influencing milk production. We investigated the effects of valine in vitro using cultured mammary epithelial cells (MECs) and in vivo using the mammary glands of lactating Tokara goats. Valine treatment at 4 mM increased the secretion of S100A7 and lactoferrin as well as the intracellular concentration of ß-defensin 1 and cathelicidin 7 in cultured MECs. In addition, an intravenous injection of valine increased S100A7 levels in the milk of Tokara goats without influencing milk yield and milk components (i.e., fat, protein, lactose, and solids). In contrast, valine treatment did not affect TJ barrier function either in vitro or in vivo. These findings indicate that valine enhances antimicrobial component production without influencing milk production and TJ barrier function in lactating mammary glands; thus, valine contributes to safe dairy production.

A design of experiments screen reveals that Clostridium novyi-NT spore germinant sensing is stereoflexible for valine and its analogs.

Although Clostridium novyi-NT is an anti-cancer bacterial therapeutic which germinates within hypoxic tumors to kill cancer cells, the actual germination triggers for C. novyi-NT are still unknown. In this study, we screen candidate germinants using combinatorial experimental designs and discover by serendipity that D-valine is a potent germinant, inducing 50% spore germination at 4.2 mM concentration. Further investigation revealed that five D-valine analogs are also germinants and four of these analogs are enantiomeric pairs. This stereoflexible effect of L- and D-amino acids shows that spore germination is a complex process where enantiomeric interactions can be confounders. This study also identifies L-cysteine as a germinant, and hypoxanthine and inosine as co-germinants. Several other amino acids promote (L-valine, L-histidine, L-threonine and L-alanine) or inhibit (L-arginine, L-glycine, L-lysine, L-tryptophan) germination in an interaction-dependent manner. D-alanine inhibits all germination, even in complex growth media. This work lays the foundation for improving the germination efficacy of C. novyi-NT spores in tumors.

Comparison of amino acid digestibility and its additivity determined with slaughter or cecectomy method for yellow-feather chicken.

The objective of this experiment was to compare the slaughter and cecectomy methods to determine amino acid (AA) digestibility of corn and soybean meal and their additivity in a corn-soybean meal diet. A completely randomized design was adopted to determine endogenous AA losses (EAAL) and AA digestibility in each of corn, soybean meal, and a corn-soybean meal diet using either slaughter or cecectomy methods. Each treatment contained 6 replicates with 3 chickens per replicate. The endogenous loss (EL) of histidine and glycine was lower and the EL of methionine and phenylalanine was greater when determined by slaughter vs. cecectomy (P < 0.05). The EL of arginine, isoleucine, leucine, lysine, methionine, phenylalanine, valine, alanine, aspartic acid, glutamic acid, and serine determined by slaughter were 1.2 to 3.2 times of those from cecectomy. The standard error (SE) of EL of 14 AA (excluding histidine and glycine) obtained by slaughter method was 2.1 to 9.6 times of those by cecectomy method. The apparent and standardized digestibility was not affected by methods for most AA except apparent digestibility of methionine, phenylalanine and glycine, and standardized digestibility of glycine in corn. The apparent and standardized digestibility of most AA except apparent digestibility of glycine and standardized digestibility of lysine, cysteine and glycine were less for slaughter versus cecectomy methods in soybean meal (P < 0.05). Using slaughter method resulted in reduced apparent digestibility of 15 AA (except glycine) and reduced standardized digestibility of 7 AA (arginine, isoleucine, leucine, valine, aspartic acid, glutamic acid, and proline) relative to cecectomy method (P < 0.05), but the standardized digestibility of glycine was greater when determined by slaughter vs. cecectomy methods in corn-soybean meal diet (P < 0.05). The mean value of SE of 16 AA digestibility in slaughter method was 2.9 times of that by cecectomy method. The apparent digestibility of 2 and 9 of 16 AA and the standardized digestibility of 15 and 7 of 16 AA were additive when using slaughter and cecectomy determinations, respectively. In conclusion, compared to the slaughter method, cecectomy method had less SE and EAAL but greater apparent digestibility of methionine and phenylalanine in corn, and the apparent digestibility of 15 AA (except glycine) in soybean meal and corn-soybean meal diet. Additivity in apparent and standardized AA digestibility was more inconsistent when determined with slaughter vs. cecectomy methods. These findings suggest that the cecectomy method is more suitable than the slaughter method to determine the digestibility of AA.

Urinary Metabolomics Study on the Protective Role of Cocoa in Zucker Diabetic Rats via 1H-NMR-Based Approach.

Cocoa constitutes one of the richest sources of dietary flavonoids with demonstrated anti-diabetic potential. However, the metabolic impact of cocoa intake in a diabetic context remains unexplored. In this study, metabolomics tools have been used to investigate the potential metabolic changes induced by cocoa in type 2 diabetes (T2D). To this end, male Zucker diabetic fatty rats were fed on standard (ZDF) or 10% cocoa-rich diet (ZDF-C) from week 10 to 20 of life. Cocoa supplementation clearly decreased serum glucose levels, improved glucose metabolism and produced significant changes in the urine metabolome of ZDF animals. Fourteen differential urinary metabolites were identified, with eight of them significantly modified by cocoa. An analysis of pathways revealed that butanoate metabolism and the synthesis and degradation of branched-chain amino acids and ketone bodies are involved in the beneficial impact of cocoa on diabetes. Moreover, correlation analysis indicated major associations between some of these urine metabolites (mainly valine, leucine, and isoleucine) and body weight, glycemia, insulin sensitivity, and glycated hemoglobin levels. Overall, this untargeted metabolomics approach provides a clear metabolic fingerprint associated to chronic cocoa intake that can be used as a marker for the improvement of glucose homeostasis in a diabetic context.

Branched-chain amino acids regulate intracellular protein turnover in porcine mammary epithelial cells.

Dietary supplementation with branched-chain amino acids (BCAAs) to lactating sows has been reported to enhance their milk production, but the underlying mechanisms remain largely unknown. This study was conducted with porcine mammary epithelial cells (PMECs) to test the hypothesis that individual BCAAs or their mixture stimulates protein synthesis and inhibit proteolysis in PMECs. Cells were cultured at 37 °C in customized Dulbecco's modified Eagle medium containing 5 mmol/L D-glucose, 1 mmol/L L-phenylalanine, L-[ring-2,4-3H]phenylalanine, 0.1 (control), 0.25, 0.5, 1, or 2 mmol/L L-leucine, L-isoleucine or L-valine or an equimolar mixture of the three BCAAs. The culture medium also contained physiological concentrations of other amino acids found in the plasma of lactating sows. Proliferation, protein synthesis, proteolysis, ß-casein production, the mechanistic target of rapamycin (mTOR) signaling, and the ubiquitin-proteasome pathway were determined for PMECs. Cell proliferation and abundances of phosphorylated mTOR, eukaryotic translation initiation factor 4E-binding protein 1, and ribosomal protein S6 kinase ß-1 proteins increased (P < 0.05), but abundances of ubiquitinated protein and 20S proteasome decreased (P < 0.05) when extracellular concentrations of L-leucine, L-isoleucine, L-valine, or an equimolar mixture of BCAAs were increased from 0.1 to 2 mmol/L. Compared with the control, 0.25, 0.5, 1 or 2 mmol/L BCAAs enhanced (P < 0.01) protein (including ß-casein) synthesis, while decreasing (P < 0.05) proteolysis in PMECs in a dose-dependent manner. Collectively, our results indicate that physiological concentrations of BCAAs regulate protein turnover in mammary epithelial cells to favor net protein synthesis through stimulating the mTOR signaling pathway and inhibiting the ubiquitin-proteasome pathway.

Enhancement of BCAT2-Mediated Valine Catabolism Stimulates ß-Casein Synthesis via the AMPK-mTOR Signaling Axis in Bovine Mammary Epithelial Cells.

Valine, a kind of branched-chain amino acid, plays a regulatory role beyond that of a building block in milk protein synthesis. However, the underlying molecular mechanism through which valine stimulates ß-casein synthesis has not been clarified. Therefore, our study aimed to evaluate the effect of valine on ß-casein synthesis and shed light into the molecular mechanism using an in vitro model. Results showed that valine supplementation significantly increased ß-casein synthesis in bovine mammary epithelial cells (BMECs). Meanwhile, the supplementation of valine resulted in high levels of branched-chain aminotransferase transaminase 2 (BCAT2), TCA-cycle intermediate metabolites, and ATP, AMP-activated protein kinase (AMPK) inhibition, and mammalian target of rapamycin (mTOR) activation. Furthermore, the inhibition of BCAT2 decreased the ß-casein synthesis and downregulated the AMPK-mTOR pathway, with similar results observed for AMPK activation. Together, the present data indicate that valine promotes the synthesis of ß-casein by affecting the AMPK-mTOR signaling axis and that BCAT2-mediated valine catabolism is the key target.