Expression Of Glutamate Dehydrogenase Research Articles

HIF-1α interacting with miR-2013-3p regulates pathogen-induced energy production changes via targeting glutamate dehydrogenase in the sea urchin Strongylocentrotus intermedius

ABSTRACT To investigate the functions of hypoxia inducible factor-1α (HIF-1α) homologs, we cloned and characterized the full-length cDNA of a novel HIF-1α homolog (SinHIF-1α) from the sea urchin Strongylocentrotus intermedius. We then explored the functions of SinHIF-1α and its regulatory factor (miR-2013-3p) in the pathogen-induced metabolic response of S. intermedius. The results showed the following: 1) The full-length cDNA of SinHIF-1α was 4265 bp, with a high level of sequence conservation across the phylum Echinodermata. 2) MiR-2013-3p could bind to the 3’-UTR of SinHIF-1α and negatively regulate the expression of SinHIF-1α in S. intermedius. 3) Both SinHIF-1α silencing and miR-2013-3p overexpression suppressed the relative content of adenosine triphosphate (ATP) in coelomocytes as well as the relative expression (mRNA and protein) and total enzyme activity of glutamate dehydrogenase (GDH) in S. intermedius. 4) The miR-2013-3p/SinHIF-1α axis may regulate ATP production at 6–24 h and 48–72 h post-pathogen infection through accompanied by alterations in the relative expression and enzyme activity of GDH in S. intermedius. In summary, all observations from this study indicated that the pathogen utilizes the miR-2013-3p/SinHIF-1α axis to reduce ATP production and thereby attenuate the antimicrobial capability of the host via targeting GDH.

Dietary protein load affects the energy and nitrogen balance requiring liver glutamate dehydrogenase to maintain physical activity

Provision of amino acids to the liver is instrumental for gluconeogenesis while it requires safe disposal of the amino group. The mitochondrial enzyme glutamate dehydrogenase (GDH) is central for hepatic ammonia detoxification by deaminating excessive amino acids towards ureagenesis and preventing hyperammonemia. The present study investigated the early adaptive responses to changes in dietary protein intake in control mice and liver-specific GDH knockout mice (Hep-Glud1-/-). Mice were fed chow diets with a wide coverage of protein contents; i.e. suboptimal 10%, standard 20%, over optimal 30%, and high 45% protein diets; switched every 4 days. Metabolic adaptations of the mice were assessed in calorimetric chambers before tissue collection and analyses. Hep-Glud1-/- mice exhibited impaired alanine induced gluconeogenesis and constitutive hyperammonemia. The expression and activity of GDH in liver lysates were not significantly changed by the different diets. However, applying an in situ redox-sensitive assay on cryopreserved tissue sections revealed higher hepatic GDH activity in mice fed the high-protein diets. On the same section series, immunohistochemistry provided corresponding mapping of the GDH expression. Cosinor analysis from calorimetric chambers showed that the circadian rhythm of food intake and energy expenditure was altered in Hep-Glud1-/- mice. In control mice, energy expenditure shifted from carbohydrate to amino acid oxidation when diet was switched to high protein content. This shift was impaired in Hep-Glud1-/- mice and consequently the spontaneous physical activity was markedly reduced in GDH knockout mice. These data highlight the central role of liver GDH in the energy balance adaptation to dietary proteins.

Effect of the mitochondrial transaminase (GOT2) on membrane potential-sensitive respiration in mitochondria of differentiated C2C12 muscle cells.

We previously showed that the transaminase inhibitor, aminooxyacetic acid, reduced respiration energized at complex II (succinate dehydrogenase, SDH) in mitochondria isolated from mouse hindlimb muscle. The effect required a reduction in membrane potential with resultant accumulation of oxaloacetate (OAA), a potent inhibitor of SDH. To specifically assess the effect of the mitochondrial transaminase, glutamic oxaloacetic transaminase (GOT2) on complex II respiration, and to determine the effect in intact cells as well as isolated mitochondria, we performed respiratory and metabolic studies in wildtype (WT) and CRISPR-generated GOT2 knockdown (KD) C2C12 myocytes. Intact cell respiration by GOT2KD cells versus WT was reduced by adding carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) to lower potential. In mitochondria of C2C12 KD cells, respiration at low potential generated by 1 µM FCCP and energized at complex II by 10 mM succinate + 0.5 mM glutamate (but not by complex I substrates) was reduced versus WT mitochondria. Although we could not detect OAA, metabolite data suggested that OAA inhibition of SDH may have contributed to the FCCP effect. C2C12 mitochondria differed from skeletal muscle mitochondria in that the effect of FCCP on complex II respiration was not evident with ADP addition. We also observed that C2C12 cells, unlike skeletal muscle, expressed glutamate dehydrogenase, which competes with GOT2 for glutamate metabolism. In summary, GOT2 KD reduced C2C12 respiration in intact cells at low potential. From differential substrate effects, this occurred largely at complex II. Moreover, C2C12 versus muscle mitochondria differ in complex II sensitivity to ADP and differ markedly in expression of glutamate dehydrogenase.NEW & NOTEWORTHY Impairment of the mitochondrial transaminase, GOT2, reduces complex II (succinate dehydrogenase, SDH)-energized respiration in C2C12 myocytes. This occurs only at low inner membrane potential and is consistent with inhibition of SDH. Incidentally, we observed that C2C12 mitochondria compared with muscle tissue mitochondria differ in sensitivity of complex II respiration to ADP and in the expression of glutamate dehydrogenase.

Gefitinib Induces Apoptosis in NSCLC Cells by Promoting Glutaminolysis and Inhibiting the MEK/ERK Signaling Pathway.

Over 80% of lung cancer cases constitute non-small cell lung cancer (NSCLC), making it the most prevalent type of lung cancer globally and the leading cause of cancer-related deaths. The treatment of NSCLC patients with gefitinib has demonstrated promising initial efficacy. However, the underlying mechanism remains unclear. This study aims to investigate how gefitinib affects the mitogen-activated protein kinase kinase (MEK)/extracellular regulated protein kinases (ERK) signaling pathway-mediated growth and death of NSCLC cells. In this study, the NSCLC cell line A549 was cultured in vitro and divided into a control group and a gefitinib group. The viability of the A549 cells was assessed using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Flow cytometry was employed to detect apoptosis in A549 cells, and the expression of glutamate dehydrogenase (GDH1) mRNA in these cells was determined using real-time quantitative PCR (RT-PCR). Western blotting was utilized to evaluate the protein expression levels of key components in the MEK/ERK signaling pathway, including phospho-MEK1/2, MEK1/2, phospho-ERK1/2, and ERK1/2. Additionally, intracellular glutamine content in A549 cells was measured using a colorimetric method. In contrast to the control group, the proliferation of A549 cells, the transcription level of glutamate dehydrogenase (GDH1), the intracellular glutamine content, and the protein expression levels of phospho-MEK1/2 and phospho-ERK1/2 were significantly lower in the gefitinib group. Moreover, apoptosis markedly increased. Gefitinib expedites apoptosis and diminishes proliferation in the NSCLC cell line A549 by downregulating the epidermal growth factor receptor (EGFR)/MEK/ERK signaling pathway. This effect is accomplished by fostering the expression of GDH1 to augment glutaminolysis in A549 cells.

Mulberry leaf and konjac flour compound dietary fiber improves digestion and metabolism in elderly mice with high-fish-protein diet by regulating gut microbiota structure and intestinal tissue repair

Ensuring sufficient protein intake, efficient digestion, and optimal absorption are crucial for the elderly. This study aims to investigate the potential of a compound dietary fiber, consisting of mulberry leaf and konjac flour (CMK), to enhance the digestion and absorption of a high-fish-protein diet in elderly mice. Results showed that CMK effectively reduced the number of unique peptide segments, generated short-chain fatty acids (SCFA) in feces, improved the content of glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), amino acid, and urea nitrogen in serum, activated the contents of pepsin, trypsin, and erepsin, and enhanced the expression of glutamate dehydrogenase (GDH), peptide transporter 1 (PepT1), and aminopeptidase N (APN). Furthermore, CMK demonstrated its ability to decrease the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-10 (IL-10), lipopolysaccharide (LPS), and lipopolysaccharide binding protein (LBP), while increase the abundance of beneficial bacteria, such as Lactobacillus and Blautia. In conclusion, CMK proved effective in enhancing the digestion and metabolism of protein in elderly mice through the regulation of gut microbiota structure and intestinal tissue repair.

Loss of expression of the glutamate dehydrogenase (gdh) of Streptococcus suis serotype 2 compromises growth and pathogenicity

Streptococcus suis serotype 2 is a zoonotic agent that causes substantial economic losses to the swine industry and threatens human public health. Factors that contribute to its ability to cause disease are not yet fully understood. Glutamate dehydrogenase (GDH) is an enzyme found in living cells and plays vital roles in cellular metabolism. It has also been shown to affect pathogenic potential of certain bacteria. In this study, we constructed a S. suis serotype 2 GDH mutant (Δgdh) by insertional inactivation mediated by a homologous recombination event and confirmed loss of expression of GDH in the mutant by immunoblot and enzyme activity staining assays. Compared with the wild type (WT) strain, Δgdh displayed a different phenotype. It exhibited impaired growth in all conditions evaluated (solid and broth media, increased temperature, varying pH, and salinity) and formed cells of reduced size. Using a swine infection model, pigs inoculated with the WT strain exhibited fever, specific signs of disease, and lesions, and the strain could be re-isolated from the brain, lung, joint fluid, and blood samples collected from the infected pigs. Pigs inoculated with the Δgdh strain did not exhibit any clinical signs of disease nor histologic lesions, and the strain could not be re-isolated from any of the tissues nor body fluid sampled. The Δgdh also showed a decreased level of survival in pig blood. Taken together, these results suggest that the gdh is important in S. suis physiology and its ability to colonize, disseminate, and cause disease.

Circular RNA Dipeptidyl Peptidase 4 (circDPP4) Stimulates the Expression of Glutamate Dehydrogenase 1 to Contribute to the Malignant Phenotypes of Prostate Cancer by Sponging miR-497-5p.

Circular RNA dipeptidyl peptidase 4 (circDPP4) has been confirmed as a novel oncogene in prostate cancer (PCa). In this study, we aimed to explore the underlying mechanism of circDPP4 in PCa progression. Levels of circDPP4, microRNA (miR)-497-5p, glutamate dehydrogenase 1 (GLUD1), proliferating cell nuclear antigen (PCNA), BCL2 associated X, apoptosis regulator (Bax), E-cadherin and Ki67 were gauged by a quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, or immunohistochemical method. We assessed the roles of variables in PCa cell phenotypes by measuring cell growth, apoptosis, motility and invasiveness. We performed RNA immunoprecipitation (RIP) and dual-luciferase reporter assays to confirm the interactions of circDPP4/miR-497-5p and miR-497-5p/GLUD1. A xenograft model was established to gauge the effect of circDPP4 in the tumorigenicity of PCa cells. PCa tumor tissues and cell lines revealed higher levels of circDPP4 and GLUD1 and a lower expression of miR-497-5p than controls. CircDPP4 silencing hindered the growth, motility and invasiveness of PCa cells. Conversely, silencing circDPP4 enhanced PCa cell apoptosis. Mechanistic analysis showed that circDPP4 functioned as a miR-497-5p sponge to reduce the suppressive action of miR-497-5p on GLUD1, which was validated as a direct miR-497-5p target. Furthermore, circDPP4 knockdown weakened the tumorigenicity of PCa cells. CircDPP4 facilitated PCa process by mediating the miR-497-5p/GLUD1 axis, providing a possible therapy target for PCa.

Targeting glutamine metabolism in hepatic stellate cells alleviates liver fibrosis

Glutamine metabolism plays an essential role in cell growth, and glutamate dehydrogenase (GDH) is a key enzyme. GDH promotes the metabolism of glutamate and glutamine to generate ATP, which is profoundly increased in multiple human cancers. Through in vitro and in vivo experiments, we verified that the small-molecule GDH inhibitor EGCG slowed the progression of fibrosis by inhibiting GDH enzyme activity and glutamine metabolism. SIRT4 is a mitochondrial enzyme with NAD that promotes ADP ribosylation and downregulates GDH activity. The role of SIRT4 in liver fibrosis and the related mechanisms are unknown. In this study, we measured the expression of SIRT4 and found that it was downregulated in liver fibrosis. Modest overexpression of SIRT4 protected the liver from fibrosis by inhibiting the transformation of glutamate to 2-ketoglutaric acid (α-KG) in the tricarboxylic acid cycle (TCA), thereby reducing the proliferative activity of hepatic stellate cells (HSCs). Collectively, our study reveals that SIRT4 controls GDH enzyme activity and expression, targeting glutamine metabolism in HSCs and alleviating liver fibrosis.

GDH promotes isoprenaline-induced cardiac hypertrophy by activating mTOR signaling via elevation of α-ketoglutarate level.

Numerous studies reveal that metabolism dysfunction contributes to the development of pathological cardiac hypertrophy. While the abnormal lipid and glucose utilization in cardiomyocytes responding to hypertrophic stimuli have been extensively studied, the alteration and implication of glutaminolysis are rarely discussed. In the present work, we provide the first evidence that glutamate dehydrogenase (GDH), an enzyme that catalyzes conversion of glutamate into ɑ-ketoglutarate (AKG), participates in isoprenaline (ISO)-induced cardiac hypertrophy through activating mammalian target of rapamycin (mTOR) signaling. The expression and activity of GDH were enhanced in cultured cardiomyocytes and rat hearts following ISO treatment. Overexpression of GDH, but not its enzymatically inactive mutant, provoked cardiac hypertrophy. In contrast, GDH knockdown could relieve ISO-triggered hypertrophic responses. The intracellular AKG level was elevated by ISO or GDH overexpression, which led to increased phosphorylation of mTOR and downstream effector ribosomal protein S6 kinase (S6K). Exogenous supplement of AKG also resulted in mTOR activation and cardiomyocyte hypertrophy. However, incubation with rapamycin, an mTOR inhibitor, attenuated hypertrophic responses in cardiomyocytes. Furthermore, GDH silencing protected rats from ISO-induced cardiac hypertrophy. These findings give a further insight into the role of GDH in cardiac hypertrophy and suggest it as a potential target for hypertrophy-related cardiomyopathy.

Elongating porcine conceptuses can utilize glutaminolysis as an anaplerotic pathway to maintain the TCA cycle†.

During the peri-implantation period of pregnancy, the trophectoderm of pig conceptuses utilize glucose via multiple biosynthetic pathways to support elongation and implantation, resulting in limited availability of pyruvate for metabolism via the TCA cycle. Therefore, we hypothesized that porcine trophectoderm cells replenish tricarboxylic acid (TCA) cycle intermediates via a process known as anaplerosis and that trophectoderm cells convert glutamine to α-ketoglutarate, a TCA cycle intermediate, through glutaminolysis. Results demonstrate: (1) that expression of glutaminase (GLS) increases in trophectoderm and glutamine synthetase (GLUL) increases in extra-embryonic endoderm of conceptuses, suggesting that extra-embryonic endoderm synthesizes glutamine, and trophectoderm converts glutamine into glutamate; and (2) that expression of glutamate dehydrogenase 1 (GLUD1) decreases and expression of aminotransferases including PSAT1 increase in trophectoderm, suggesting that glutaminolysis occurs in the trophectoderm through the GLS-aminotransferase pathway during the peri-implantation period. We then incubated porcine conceptuses with 13C-glutamine in the presence or absence of glucose in the culture media and then monitored the movement of glutamine-derived carbons through metabolic intermediates within glutaminolysis and the TCA cycle. The 13C-labeled carbons were accumulated in glutamate, α-ketoglutarate, succinate, malate, citrate, and aspartate in both the presence and absence of glucose in the media, and the accumulation of 13C-labeled carbons significantly increased in the absence of glucose in the media. Collectively, our results indicate that during the peri-implantation period of pregnancy, the proliferating and migrating trophectoderm cells of elongating porcine conceptuses utilize glutamine via glutaminolysis as an alternate carbon source to maintain TCA cycle flux.

Dietary protein/carbohydrate ratio and feeding frequency affect feed utilization, intermediary metabolism, and economic efficiency of gilthead seabream (Sparus aurata) juveniles

To evaluate the effects of dietary protein/carbohydrate (P/CHO) ratio and feeding frequency (FF) on growth, intermediary metabolism, and economic efficiency of gilthead seabream (Sparus aurata) juveniles, two practical isolipidic (17%) diets were formulated to include high protein (50%)/ low starch (10%) (diet P50/CHO10) or low protein (40%)/ high starch (20%) (diet P40/CHO20). Triplicate groups of fish with 9.1 ± 0.01 g were fed for 60 days with these diets until visual satiation at three FF: one (9:00), two (9:00 and 17:00), or three (9:00, 13:00, and 17:00) meals per day. Dietary P/CHO ratios did not affect growth performance while feeding 2 or 3 meals per day improved fish growth. Fish fed diet P40/CHO20 had increased feed intake (FI), protein efficiency ratio (PER), and nitrogen retention (NR), and lower feed efficiency (FE), nitrogen intake (NI), and economic conversion ratio (ECR). Feeding 2 or 3 meals per day increased FI, NI, ECR, and economic profit index, and decreased FE, PER, and NR. Fish fed diet P40/CHO20 presented increased hepatic lipid and glycogen content, hepatocyte area covered by lipid vacuoles, and glucokinase (gk) gene expression, and decreased glutamate dehydrogenase expression. Fish fed 3 meals per day had decreased plasma triglycerides and total protein levels, while fish fed 2 or 3 meals per day presented decreased hepatic growth hormone receptor-i (ghr-i), gk, and fatty acid synthase gene expression. Interaction between P/CHO ratio and FF was only observed in plasmatic glucose, cholesterol, and total lipids levels, and insulin-like growth factor-1, and ghr-ii gene expression. Overall, glycogenesis, glycolysis, and economic efficiency seemed to be increased while the amino acid catabolism was reduced in fish fed the P40/CHO20 diet. Higher FF increased growth and economic efficiency, and reduced glycolysis and lipogenesis pathways. In conclusion, a diet with P40/CHO20 ratio fed twice a day appears to be the most adequate strategy regarding feed utilization and economic efficiency for gilthead seabream juveniles in order to achieve optimum sustainable aquaculture.

Sodium butyrate reduces ammonia emissions through glutamate metabolic pathways in cecal microorganisms of laying hens

Ammonia emission is an important problem that needs to be solved in laying hen industries. Sodium butyrate (SB) is considered to have potential for reducing ammonia production because of its ability to improve nitrogen metabolism. In this in vitro fermentation study, we presented a correlation analysis of the metatranscriptome and metaproteome of lay hen cecal microorganisms, in order to identify important proteins and pathways involved in ammonia production reduction due to sodium butyrate supplementation. The results showed that sodium butyrate supplement decreased the production of ammonia by 26.22% as compared with the non-sodium butyrate supplementation (CK) group. The SB group exhibited a lower concentration of ammonium nitrogen (NH4+-N) and a decreased pH. Sodium butyrate promoted the uric acid concentration and lowered the uricase activity in the fermentation broth of laying hens cecal content. Notably, the ‘alanine, aspartate and glutamate metabolism’ category was more abundant in the SB group. The addition of sodium butyrate increased the expression of glutamate dehydrogenase (GDH) gene in cecal microbiota (e.g., Ruminococcus sp. and Bacteroides sp.) in vitro. The metaproteome analysis results showed that the expression of GDH with NADPH as coenzyme (NADPH-GDH) was up-regulated in cecal microbiota by sodium butyrate supplement. Our results indicate that sodium butyrate can affect glutamate metabolism through regulating the expression of glutamate dehydrogenase in cecal microorganisms, thereby reducing ammonia production. This study reveals that glutamate dehydrogenase-mediated glutamate metabolism play a key role in ammonia emission reduction in laying hen and provide theoretical basis for further developing ammonia production reduction approach.

In Situ Assessment of Hepatic Glutamate Dehydrogenase Activity Provides Novel Insights into Amino Acid Metabolism

The liver serves as a hub for amino acid metabolism, in particular concerning ammonia detoxification. The mitochondrial enzyme glutamate dehydrogenase (GDH) plays a central role this pathway, bridging amino acid deamination and glutamate handling toward urea formation. Numerous studies have investigated the function of GDH in the liver, pointing to complex allosteric regulations. However, typical measurements of GDH expression and activity in tissue extracts do not allow the assessment of the actual in situ enzymatic activity that relies on the preservation of intracellular organization. In this study, we developed an in situ redox-sensitive nitro blue tetrazolium (NBT) assay to investigate the hepatic GDH activity in cryopreserved liver sections. On the same section series, immunohistochemistry provided corresponding mapping of the expression of GDH and associated enzymes. The study was conducted in control floxed mice as well as in liver-specific GDH knockout mice (Hep-Glud1-/-). We evaluated the responses to different dietary protein intakes, i.e. mice fed a standard 20% protein diet versus 30% and 45% protein contents as high protein diets. Tissues of interest were collected from control floxed mice and Hep-Glud1-/- mice after 4 hours of fasting. Measured in liver lysates of control floxed mice, expression and activity of GDH were not significantly changed by the high protein diets. However, measured in situ, the NBT-based assay revealed that GDH activity was markedly increased by the high protein diets. In vivo, this was accompanied by an increase in plasma urea levels, while plasma ammonia levels remained unchanged. Surprisingly, Hep-Glud1-/- mice lacking liver GDH exhibited hyperammonemia, even under the standard 20% protein diet. In control floxed mice, immunohistochemistry on liver cryosections showed that GDH was evenly distributed within the tissue. However, the NBT assay performed on the same cryosections revealed hot spots of in situ GDH activity predominantly in hepatic perivenous area. These readouts were absent in Hep-Glud1-/- mice. Overall, our study shows that commonly used assessments of GDH function do not reflect in situ activity. Such activity is increased by a high protein diet, while expression levels are unchanged; measurements of GDH activity in liver extracts missing the in situ adaptation of the enzyme uncovered by the NBT assay.

Application of a dissolved oxygen control strategy to increase the expression of Streptococcus suis glutamate dehydrogenase in Escherichia coli.

The accumulation of acetate in Escherichia coli inhibits cell growth and desired protein synthesis, and cell density and protein expression are increased by reduction of acetate excretion. Dissolved oxygen (DO) is an important parameter for acetate synthesis, and the accumulation of acetate is inversely correlated to DO level. In this study, the effect of DO levels on glutamate dehydrogenase (GDH) expression was investigated, and then different DO control strategies were tested for effects on GDH expression. DO control strategy IV (50% 0-9h, 30% 9-18h) provided the highest cell density (15.43g/L) and GDH concentration (3.42g/L), values 1.59- and 1.99-times higher than those achieved at 10% DO. The accumulation of acetate was 2.24g/L with DO control strategy IV, a decrease of 40.74% relative to that achieved for growth at 10% DO. Additionally, under DO control strategy IV, there was lower expression of PoxB, a key enzyme for acetate synthesis, at both the transcriptional and translational level. At the same time, higher transcription and protein expression levels were observed for a glyoxylate shunt gene (aceA), an acetate uptake gene (acs), gluconeogensis and anaplerotic pathways genes (pckA, ppsA, ppc, and sfcA), and a TCA cycle gene (gltA). The flux of acetate with DO strategy IV was 8.4%, a decrease of 62.33% compared with the flux at 10% DO. This decrease represents both lower flux for acetate synthesis and increased flux of reused acetate.

Inoculation with Clariodeoglomus etunicatum improves leaf food quality of tea exposed to P stress

The present study aimed to evaluate the effect of an arbuscular mycorrhizal fungus (AMF), Clariodeoglomus etunicatum, on leaf food quality and relevant gene expression levels of tea (Camellia sinensis cv. ‘Fuding Dabaicha’) seedlings exposed to 0.5 μM P (P0.5) and 50 μM P (P50) levels. Twenty-four weeks later, the seedlings recorded higher root mycorrhizal fungal colonization in P50 than in P0.5. AMF-inoculated tea plants represented significantly higher leaf fructose and glucose contents and lower sucrose content than non-inoculated plants, irrespective of substate P levels. AMF treatment also increased total amino acids content in P0.5 and P50, accompanied with higher expression of glutamate dehydrogenase (CsGDH) and lower expression of glutamine synthetase (CsGS) and glutamine oxoglutarate aminotransferase (CsGOGAT). The total flavonoid content was higher in mycorrhizal versus non-mycorrhizal plants under P0.5 and P50, together with induced expression of phenylalanine ammonia-lyase (CsPAL) and cinnamic acid 4-hydroxylase (CsC4H). Mycorrhizal fungal inoculation improved catechins content, which is due to the up-regulated expression of flavanone 3-hydroxylase (CsF3H), flavonoid 3'-hydroxylase (CsF3'H), dihydroflavonol 4-reductase (CsDFR), leucoanthocyanidin reductase (CsLAR), anthocyanidin reductase (CsANR), and chalcone isomerase (CsCHI) under P0.5. However, under P50, the gene involved in catechins synthesis was not affected or down-regulated by mycorrhization, implying a complex mechanism (e.g. nutrient improvement). AMF also inhibited the tea caffeine synthase 1 (CsTCS1) expression regardless of P levels. Therefore, the results of this study concluded that inoculation with C. etunicatum improves leaf food quality of tea exposed to P stress, but the improved mechanisms were different between P0.5 and P50.

Inhibition of the mTORC1/NF-κB Axis Alters Amino Acid Metabolism in Human Hepatocytes

In addition to serving as the building blocks for protein synthesis, amino acids can be used as an energy source, through catabolism. The transamination, oxidative deamination, and decarboxylation processes that occur during amino acid catabolism are catalyzed by specific enzymes, including aspartate aminotransferase (AST), glutamate dehydrogenase (GDH), glutamic acid decarboxylase (GAD), and ornithine decarboxylase (ODC); however, the overall molecular mechanisms through which amino acid catabolism occurs remain largely unknown. To examine the role of mechanistic target of rapamycin complex 1 (mTORC1) on amino acid catabolism, mTORC1 was inactivated by rapamycin or shRNA targeting Raptor, versus activated by overexpressing Rheb or amino acids in human hepatocytes. The expression of amino acid catabolic genes and related transcription factor was investigated by RT/real-time PCR and western blot analysis. A few types of amino acid metabolite were examined by ELISA and HPLC analysis. The data showed that inactivated mTORC1 resulted in inhibition of NF-κB and the expression of AST, GDH, GAD, and ODC, whereas activated mTORC1 enhanced NF-κB activation and the expression levels of the catabolism-associated genes. Further, inhibition of NF-κB reduced the expression levels of AST, GDH, GAD, and ODC. mTORC1 upregulated NF-κB activation and the expression of AST and ODC in response to glutamate and ornithine treatments, whereas rapamycin inhibited the utilization of glutamate and ornithine in hepatocytes. Taken together, these results indicated that the mTORC1/NF-κB axis modulates the rate of amino acid catabolism by regulating the expression of key catabolic enzymes in hepatocytes.

Brain endothelial cells metabolize glutamate via glutamate dehydrogenase to replenish TCA-intermediates and produce ATP under hypoglycemic conditions.

The endothelial cells of the blood-brain barrier participate in the regulation of glutamate concentrations in the brain interstitial fluid by taking up brain glutamate. However, endothelial glutamate metabolism has not been characterized, nor is its role in brain glutamate homeostasis and endothelial energy production known. The aim of this study was to investigate endothelial glutamate dehydrogenase (GDH) expression and glutamate metabolism and probe its functional significance. The primary brain endothelial cells were isolated from bovine and mouse brains, and human brain endothelial cells were derived from induced pluripotent stem cells. GDH expression on the protein level and GDH function were investigated in the model systems using western blotting, confocal microscopy, 13 C-glutamate metabolism, and Seahorse assay. In this study, it was shown that GDH was expressed in murine and bovine brain capillaries and in cultured primary mouse and bovine brain endothelial cells as well as in human-induced pluripotent stem cell-derived endothelial cells. The endothelial GDH expression was confirmed in brain capillaries from mice carrying a central nervous system-specific GDH knockout. Endothelial cells from all tested species metabolized 13 C-glutamate to α-ketoglutarate, which subsequently entered the tricarboxylic acid (TCA)-cycle. Brain endothelial cells maintained mitochondrial oxygen consumption rates, when supplied with glutamate alone, whereas glutamate supplied in addition to glucose did not lead to additional oxygen consumption. In conclusion, brain endothelial cells directly take up and metabolize glutamate and utilize the resulting α-ketoglutarate in the tricarboxylic acid cycle to ultimately yield ATP if glucose is unavailable.

How to easily detect plant NADH-glutamate dehydrogenase (GDH) activity? A simple and reliable in planta procedure suitable for tissues, extracts and heterologous microbial systems

Plant NADH glutamate dehydrogenase (GDH) is an intriguing enzyme, since it is involved in different metabolic processes owing to its reversible (anabolic/catabolic) activity and due to the oligomeric nature of the enzyme, that gives rise to several isoforms. The complexity of GDH isoenzymes pattern and the variability of the spatial and temporal localization of the different isoforms have limited our comprehension of the physiological role of GDH in plants. Genetics, immunological, and biochemical approaches have been used until now in order to shed light on the regulatory mechanism that control GDH expression in different plant systems and environmental conditions. We describe here the validation of a simple in planta GDH activity staining procedure, providing evidence that it might be used, with different purposes, to determine GDH expression in plant organs, tissues, extracts and also heterologous systems.

Wolbachia and Sirtuin-4 interaction is associated with alterations in host glucose metabolism and bacterial titer.

Wolbachia is an intracellular bacterial symbiont of arthropods notorious for inducing many reproductive manipulations that foster its dissemination. Wolbachia affects many aspects of host biology, including metabolism, longevity and physiology, being described as a nutrient provisioning or metabolic parasite, depending on the host-microbe association. Sirtuins (SIRTs) are a family of NAD+-dependent post-translational regulatory enzymes known to affect many of the same processes altered by Wolbachia, including aging and metabolism, among others. Despite a clear overlap in control of host-derived pathways and physiology, no work has demonstrated a link between these two regulators. We used genetically tractable Drosophila melanogaster to explore the role of sirtuins in shaping signaling pathways in the context of a host-symbiont model. By using transcriptional profiling and metabolic assays in the context of genetic knockouts/over-expressions, we examined the effect of several Wolbachia strains on host sirtuin expression across distinct tissues and timepoints. We also quantified the downstream effects of the sirtuin x Wolbachia interaction on host glucose metabolism, and in turn, how it impacted Wolbachia titer. Our results indicate that the presence of Wolbachia is associated with (1) reduced sirt-4 expression in a strain-specific manner, and (2) alterations in host glutamate dehydrogenase expression and ATP levels, key components of glucose metabolism. We detected high glucose levels in Wolbachia-infected flies, which further increased when sirt-4 was over-expressed. However, under sirt-4 knockout, flies displayed a hypoglycemic state not rescued to normal levels in the presence of Wolbachia. Finally, whole body sirt-4 over-expression resulted in reduced Wolbachia ovarian titer. Our results expand knowledge of Wolbachia-host associations in the context of a yet unexplored class of host post-translational regulatory enzymes with implications for conserved host signaling pathways and bacterial titer, factors known to impact host biology and the symbiont’s ability to spread through populations.

Differences in carbon and nitrogen metabolism between male and female Populus cathayana in response to deficient nitrogen.

Sexual dimorphism occurs regarding carbon and nitrogen metabolic processes in response to nitrogen supply. Differences in fixation and remobilization of carbon and allocation and assimilation of nitrogen between sexes may differ under severe defoliation. The dioecious species Populus cathayana was studied after two defoliation treatments with two N levels. Males had a higher capacity of carbon fixation because of higher gas exchange and fluorescence traits of leaves after severe long-term defoliation under deficient N. Males had higher leaf abscisic acid, stomatal conductance and leaf sucrose phosphate synthase activity increasing transport of sucrose to sinks. Males had a higher carbon sink than females, because under N-deficient conditions, males accumulated >131.10% and 90.65% root starch than males in the control, whereas females accumulated >40.55% and 52.81%, respectively, than females in the control group. Males allocated less non-protein N (NNon-p) to leaves, having higher nitrogen use efficiency (photosynthetic nitrogen use efficiency), higher glutamate dehydrogenase (GDH) and higher leaf GDH expression, even after long-term severe defoliation under deficient N. Females had higher leaf jasmonic acid concentration and NNon-p. The present study suggested that females allocated more carbon and nitrogen to defense chemicals than males after long-term severe defoliation under deficient N.