Isocitrate Dehydrogenase Activity Research Articles

Overcoming glutamate auxotrophy in Escherichia coli itaconate overproducer by the Weimberg pathway

Biosynthesis of itaconic acid occurs through decarboxylation of the TCA cycle intermediate cis-aconitate. Engineering of efficient itaconate producers often requires elimination of the highly active isocitrate dehydrogenase to conserve cis-aconitate, leading to 2-ketoglutarate auxotrophy in the producing strains. Supplementation of glutamate or complex protein hydrolysate then becomes necessary, often in large quantities, to support the high cell density desired during itaconate fermentation and adds to the production cost. Here, we present an alternative approach to overcome the glutamate auxotrophy in itaconate producers by synthetically introducing the Weimberg pathway from Burkholderia xenovorans for 2-ketoglutarate biosynthesis. Because of its independence from natural carbohydrate assimilation pathways in Escherichia coli, the Weimberg pathway is able to provide 2-ketoglutarate using xylose without compromising the carbon flux toward itaconate. With xylose concentration carefully tuned to minimize excess 2-ketoglutarate flux in the stationary phase, the final strain accumulated 20 g/L of itaconate in minimal medium from 18 g/L of xylose and 45 g/L of glycerol. Necessity of the recombinant Weimberg pathway for growth also allowed us to maintain multi-copy plasmids carrying in operon the itaconate-producing genes without addition of antibiotics. Use of the Weimberg pathway for growth restoration is applicable to other production systems with disrupted 2-ketoglutarate synthesis.

Isocitrate dehydrogenase of Bacillus cereus is involved in biofilm formation.

Isocitrate dehydrogenase (IDH), a key enzyme in the TCA cycle, participates in the formation of biofilms in Staphylococcus aureus, but it remains to be clarified whether it is involved in the formation of Bacillus cereus biofilms. In this study, we scanned the genome of B. cereus 0-9 and found a gene encoding isocitrate dehydrogenase (FRY47_22620) named icdH. The IcdH protein was expressed and purified. The enzyme activity assay showed that the protein had IDH activity dependent on NADP+, indicating that this gene encoded an IDH. The ΔicdH mutant and its complemented strains were obtained by a homologous recombination strategy, and crystal violet data and CLSM were measured. The results showed that the biofilm yield of the mutant ΔicdH decreased, and the biofilm morphology also changed, while the growth of ΔicdH was not affected. The extracellular pH and citric acid content results showed that the ΔicdH mutant exhibited citric acid accumulation and acidification of the extracellular matrix. In addition, the addition of excess Fe3+ restored the biofilm formation of the ΔicdH mutant. It is speculated that IDH in B. cereus may regulate biofilm formation by modulating intracellular redox homeostasis. In addition, we found that the icdH deletion of B. cereus 0-9 could result in a reduced sporulation rate, which was significantly different from sporulation in B. subtilis caused by interruption of the stage I sporulation process due to icdH loss. All the above results provide us with new insights for further research on IDH.

Dietary α-lipoic acid requirement and its effects on antioxidant status, carbohydrate metabolism, and intestinal microflora in oriental river prawn Macrobrachium nipponense (De Haan)

Alpha lipoic acid (α-LA) is not only a natural antioxidant, but also a key cofactor for the enzymes pyruvate dehydrogenase and α-ketoglutarate dehydrogenase. The purpose of this study was to investigate the effects of dietary α-lipoic acid on the growth, antioxidant status, carbohydrate metabolism, and intestinal microflora of prawn, Macrobrachium nipponense. A total of 1250 juvenile M. nipponense with an average body weight of 0.050 ± 0.003 g were randomly divided into five groups with three replicates (50 prawns per tank) for an 8-week feeding trial. The groups were fed with basal semi-purified diets containing α-lipoic acid at five different concentrations: 0 mg/kg (LA1), 500 mg/kg (LA2), 1000 mg/kg (LA3), 2000 mg/kg (LA4) and 4000 mg/kg (LA5). The results showed that the weight gain (WG), specific growth rate (SGR) and the survival rate were significantly lower in LA5 than in other groups. Dietary α-lipoic acid at a concentration below 2000 mg/kg (LA4) significantly reduced the malondialdehyde (MDA) content and increased the glutathione (GSH) content in the hepatopancreas of M. nipponense, and significantly increased the activities of glutathione reductase (GR), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD). However, the MDA content in the hepatopancreas was higher in LA5 than in LA1. The prawns in the LA3 group showed the highest activities of isocitrate dehydrogenase (ICDH) and key enzymes in glycolysis (pyruvate kinase, PK; 6-phosphofructokinase, PFK; Hexokinase, HK), but lower activity of lactate dehydrogenase (LDH) compared with those in LA1 and LA2. Analyses of gut microflora at the operational taxonomic unit (OTU) level indicated that abundance (Chao index) and diversity (Simpson index) were lower in LA5 than in LA1 and LA3. The Shannon index's index was highest in LA5. The intestinal flora of M. nipponense comprised five main phyla and five main bacteria. The proportions of Proteobacteria and Citrobacter were higher in LA3 and LA5 than in LA1. The proportions of Actinobacteria, Bacteroidetes Aeromonas, Tyzzerella_3 and Mycobacterium were significantly higher in LA5 than in LA3 and LA1. In summary, dietary α-lipoic acid at an appropriate level (<2000 mg/kg) can improve the antioxidant capacity and carbohydrate metabolism of M. nipponense and modulate its intestinal microbiota without reducing growth performance. Excessive α-lipoic acid (4000 mg/kg) negatively affected the growth performance and antioxidant activity of M. nipponense. Calculations based on WG and SGR indicate that the optimal addition amount of α-lipoic acid for juvenile M. nipponense is 1354.8 mg/kg and 1329.92 mg/kg, respectively.

Comprehensive Interrogation of Metabolic and Bioenergetic Responses of Early-Staged Zebrafish (Danio rerio) to a Commercial Copper Hydroxide Nanopesticide.

The use of copper hydroxide nanopesticide can pose exposure risks to aquatic organisms. In this study, the toxicity of a copper hydroxide nanopesticide, compared to conventional copper sulfate at environmentally relevant doses, was evaluated using metabolomics and bioenergetic assays in embryonic zebrafish. At a copper concentration of 100 μg/L, the nanopesticide caused higher mortality and deformity compared to copper ions alone; despite higher copper accumulation, increased metallothionein and elevated ATP-binding cassette (ABC) transporter activity in zebrafish exposed to copper ions were observed. Both nanopesticide and copper ions reduced the abundance of metabolites of glycolysis and induced energetic stress in zebrafish. The nanopesticide also increased concentrations of several organic acids involved in the tricarboxylic acid (TCA) cycle and elevated the activity of isocitrate dehydrogenase and α-ketoglutarate dehydrogenase, suggesting enhanced TCA cycle activity. Nanopesticide exposure depleted both glutamate and glutamine parallel to the upregulation of the TCA cycle. In addition, zebrafish exposed to the nanopesticide appeared to shift metabolism toward amino acid catabolism and lipid accumulation based upon altered expression profiles of glutaminase, glutamate dehydrogenase, fatty acid synthase, and acetyl-CoA carboxylase. Lastly, the ability of the ions to increase oxidative phosphorylation to alleviate energetic stress was reduced in the case of the nanopesticide. We hypothesize that, unlike copper ions alone, the nanopesticide induces higher toxicity to zebrafish because of increased protein catabolism. This study provides a comprehensive understanding of the risks of copper hydroxide nanopesticide exposure in relation to metabolic activity and mitochondrial function.

Loss of the mitochondrial phosphate carrier SLC25A3 induces remodeling of the cardiac mitochondrial protein acylome.

Mitochondria are recognized as signaling organelles, because under stress, mitochondria can trigger various signaling pathways to coordinate the cell's response. The specific pathway(s) engaged by mitochondria in response to mitochondrial energy defects in vivo and in high-energy tissues like the heart are not fully understood. Here, we investigated cardiac pathways activated in response to mitochondrial energy dysfunction by studying mice with cardiomyocyte-specific loss of the mitochondrial phosphate carrier (SLC25A3), an established model that develops cardiomyopathy as a result of defective mitochondrial ATP synthesis. Mitochondrial energy dysfunction induced a striking pattern of acylome remodeling, with significantly increased posttranslational acetylation and malonylation. Mass spectrometry-based proteomics further revealed that energy dysfunction-induced remodeling of the acetylome and malonylome preferentially impacts mitochondrial proteins. Acetylation and malonylation modified a highly interconnected interactome of mitochondrial proteins, and both modifications were present on the enzyme isocitrate dehydrogenase 2 (IDH2). Intriguingly, IDH2 activity was enhanced in SLC25A3-deleted mitochondria, and further study of IDH2 sites targeted by both acetylation and malonylation revealed that these modifications can have site-specific and distinct functional effects. Finally, we uncovered a novel cross talk between the two modifications, whereby mitochondrial energy dysfunction-induced acetylation of sirtuin 5 (SIRT5), inhibited its function. Because SIRT5 is a mitochondrial deacylase with demalonylase activity, this finding suggests that acetylation can modulate the malonylome. Together, our results position acylations as an arm of the mitochondrial response to energy dysfunction and suggest a mechanism by which focal disruption to the energy production machinery can have an expanded impact on global mitochondrial function.

Parental dietary sucrose affects metabolic and antioxidant enzyme activities in Drosophila

ABSTRACTThe influence of parental nutrition as a key environmental factor that can influence offspring performance has become a hot topic for modern investigations. In the current study using fruit flies, Drosophila melanogaster, we found the sucrose concentration of the diet in the parental generation had a significant influence on the metabolism and antioxidant defense of the next generation, even when that subsequent generation had been shifted to a standard diet. We found that low concentrations of dietary carbohydrate in the parental generation led to higher activities of lactate dehydrogenase, malate dehydrogenase (MDH) and alanine transaminase (ALT) enzymes and increased urea content in their progeny. Moreover, higher activities of the first‐ and second‐line antioxidant defense enzymes were detected in F1 males generated from parents fed a low‐carbohydrate diet. Low sucrose concentration in the parental diet resulted in increased levels of protein thiols but decreased the low‐molecular‐weight thiol group content in F1 males. The influence of different dietary sucrose concentrations also resulted in sex‐dependent differences, where MDH, ALT, glutathione‐S‐transferase, glucose‐6‐phosphate dehydrogenase and isocitrate dehydrogenase activities and levels of thiol groups were unaffected in F1 females. The results highlight the transgenerational influence that specific dietary macronutrients provided in the parental diet, namely carbohydrates, can have on the next generation, influencing enzyme activities, metabolic pathways and antioxidant defenses in progeny Drosophila.

Protective effects of vitexin on cadmium-induced renal toxicity in rats.

Cadmium (Cd) is an industrial contaminant that poses severe threats to human and animal health. Vitexin (VIT) is a polyphenolic flavonoid of characteristic pharmacological properties. We explored the curative role of vitexin on Cd-induced mitochondrial-dysfunction in rat renal tissues. Twenty-four rats were equally divided into four groups and designated as control, Cd, Cd + vitexin and vitexin treated groups. The results showed that Cd exposure increased urea and creatinine levels while decreased creatinine clearance. Cd reduced the activities of antioxidant enzymes, i.e., catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione content in the Cd exposed group. Cd exposure significantly (p < 0.05) elevated the reactive oxygen species (ROS) and Thiobarbituric acid reactive substances (TBARS) levels in rat kidney. Cd also caused a significant (p < 0.05) reduction in the mitochondrial TCA-cycle enzymes, including isocitrate dehydrogenase, succinate dehydrogenase, alpha-ketoglutarate dehydrogenase, and malate-dehydrogenase activities. Besides, mitochondrial respiratory chain enzymes, including NADH-dehydrogenase, coenzyme Q-cytochrome reductase, succinic-coenzyme Q, and cytochrome c-oxidase activities were also decreased under Cd exposure. Cd exposure also damaged the mitochondrial membrane potential (MMP). However, VIT treatment potentially reduced the detrimental effects of Cd in the kidney of rats. In conclusion, our study indicated that the VIT could attenuate the Cd-induced renal toxicity in rats.

Redox metabolism in mussels (Brachidontes solisianus) under the influence of tides in a rocky beach in Southern Brazil

Aquatic invertebrates use antioxidant systems to endure intertidal environments, where temperature, oxygen availability and radiation incidence vary greatly. The upregulation of antioxidants during oxygen-limiting conditions, such as intertidal aerial exposure (AE), is known as preparation for oxidative stress (POS). Preparation for oxidative stress has been commonly reported for animals exposed to hypoxia in the lab, but few studies have investigated it under ecologically relevant natural settings. Here, we studied metabolic enzymes, antioxidants, and oxidative stress markers in Brachidontes solisianus mussels exposed to a natural tidal cycle in a rocky beach in Southern Brazil. Animals were collected at 6 time-points throughout the tidal cycle: after 2 h of AE; then after 1 h, 3 h, and 7 h of immersion; followed by 0.5 h and 4 h of AE. The activities of malate dehydrogenase, isocitrate dehydrogenase, and pyruvate kinase were unaffected by aerial exposure. Similarly, the acitivities of enzymes of the antioxidant system, catalase, glutathione transferase, glutathione reductase, and glucose 6-phosphate dehydrogenase did not change significantly throughout the tidal cycle. In contrast, total and reduced glutathione levels significantly increased at 4 h AE by 47% and 64%, respectively, compared with those in submerged mussels (1 h, 3 h, and 7 h immersion groups combined). Lipid peroxidation and carbonyl protein levels significantly increased after 4 h of AE compared with those in mussels underwater for 7 h. Our results indicate the occurrence of POS in B. solisianus mussels exposed to air under natural conditions during tidal cycles.

Role of the mitochondrial citrate-oxoglutarate carrier in lipid accumulation in the oleaginous fungus Mortierella alpina.

The transport of citrate from the mitochondria to the cytoplasm is essential during lipid accumulation. This study aimed to explore the role of mitochondrial citrate-oxoglutarate carrier in lipid accumulation in the oleaginous fungus Mortierella alpina. Homologous MaYHM (the gene encoding the mitochondrial citrate-oxoglutarate carrier) was overexpressed in M. alpina. The fatty acid content of MaYHM-overexpressing recombinant strains was increased by up to 30% compared with the control. Moreover, the intracellular α-ketoglutarate level in recombinant strains was increased by 2.2 fold, together with a 23-35% decrease in NAD+-isocitrate dehydrogenase activity compared with the control. The overexpression of MaYHM altered the metabolic flux in the glutamate dehydrogenase shunt and 4-aminobutyric acid shunt during metabolic reprogramming, supplying more carbon to synthesize fatty acids. Overexpression of MaYHM resulted in more efflux of citrate from mitochondria to the cytoplasm and enhanced lipid accumulation. These findings provide new perspectives for the improvement of industrial lipid production in M. alpina.

Modulation in isocitrate dehydrogenase activity under citrate enrichment affects carbon and nitrogen fixations in the cyanobacterium Nostoc muscorum Meg 1

The enzyme isocitrate dehydrogenase (IDH) converts isocitrate synthesized from citrate to α-ketoglutarate in the TCA cycle. In cyanobacteria, α-KG has an additional role where it donates its carbon skeleton for ammonium assimilation in the GS-GOGAT pathway thereby linking carbon and nitrogen metabolisms. Looking at this crucial function of IDH that makes α-KG available for both carbon and nitrogen assimilation, changes brought about in its activity under excess availability of citrate in a cyanobacterium was evaluated. Further, how these changes are transmitted downstream affecting carbon and nitrogen metabolisms were also evaluated. A 100 μM citrate supplementation induced IDH activity. Consequently, there was an increase in concentrations of photosynthetic pigments, D1 protein and RuBisCO as well as in PSII activity. Heterocyst differentiation was initiated and an upsurge in the activities of nitrogenase and GS were recorded. An enhancement in the total protein and carbohydrate content reiterated the positive influence of citrate enrichment on carbon and nitrogen fixation. The increase in the mRNA contents of IDH, D1 protein, RuBisCO, nitrogenase and GS indicated their induction at the genetic level. Finally, there was augmentation in total biomass production by ∼28%. Interestingly as citrate concentration was increased to 500 μM, both C- and N- fixations were highly compromised suggesting that even though citrate is an essential metabolite in the cells, it became toxic beyond a certain concentration to the organism. SEM and TEM studies showed no changes in the organism's morphology and ultra-structure in presence of 100 μM citrate while adverse changes were noticed in presence of 500 μM citrate.

IDH knockdown alters foraging behavior in the termite Odontotermes formosanus in different social contexts.

Foraging, as an energy-consuming behavior, is very important for colony survival in termites. How energy metabolism related to glucose decomposition and adenosine triphosphate (ATP) production influences foraging behavior in termites is still unclear. Here, we analyzed the change in energy metabolism in the whole organism and brain after silencing the key metabolic gene isocitrate dehydrogenase (IDH) and then investigated its impact on foraging behavior in the subterranean termite Odontotermes formosanus in different social contexts. The IDH gene exhibited higher expression in the abdomen and head of O. formosanus. The knockdown of IDH resulted in metabolic disorders in the whole organism. The dsIDH-injected workers showed significantly reduced walking activity but increased foraging success. Interestingly, IDH knockdown altered brain energy metabolism, resulting in a decline in ATP levels and an increase in IDH activity. Additionally, the social context affected brain energy metabolism and, thus, altered foraging behavior in O. formosanus. We found that the presence of predator ants increased the negative influence on the foraging behavior of dsIDH-injected workers, including a decrease in foraging success. However, an increase in the number of nestmate soldiers could provide social buffering to relieve the adverse effect of predator ants on worker foraging behavior. Our orthogonal experiments further verified that the role of the IDH gene as an inherent factor was dominant in manipulating termite foraging behavior compared with external social contexts, suggesting that energy metabolism, especially brain energy metabolism, plays a crucial role in regulating termite foraging behavior.

Strategies to Apply Water-Deficit Stress: Similarities and Disparities at the Whole Plant Metabolism Level in Medicago truncatula.

Water-deficit stresses such as drought and salinity are the most important factors limiting crop productivity. Hence, understanding the plant responses to these stresses is key for the improvement of their tolerance and yield. In this study M. truncatula plants were subjected to 250 mM NaCl as well as reduced irrigation (No-W) and 250 g/L polyethylene glycol (PEG)-6000 to induce salinity and drought stress, respectively, provoking a drop to −1.7 MPa in leaf water potential. The whole plant physiology and metabolism was explored by characterizing the stress responses at root, phloem sap and leaf organ level. PEG treatment led to some typical responses of plants to drought stress, but in addition to PEG uptake, an important impairment of nutrient uptake and a different regulation of carbon metabolism could be observed compared to No-W plants. No-W plants showed an important redistribution of antioxidants and assimilates to the root tissue, with a distinctive increase in root proline degradation and alkaline invertase activity. On the contrary, salinity provoked an increase in leaf starch and isocitrate dehydrogenase activity, suggesting key roles in the plant response to this stress. Overall, results suggest higher protection of salt-stressed shoots and non-irrigated roots through different mechanisms, including the regulation of proline and carbon metabolism, while discarding PEG as safe mimicker of drought. This raises the need to understand the effect at the whole plant level of the different strategies employed to apply water-deficit stress.

Oxidative stress and hepatocellular mitochondrial dysfunction attenuated by asiatic acid in streptozotocin-induced diabetic rats

Oxidative stress produced by mitochondria is one of the main causes for diabetes associated complications. This study was planned to assess the efficacy of asiatic acid (AA) on oxidative stress mediated hepatocellular mitochondrial dysfunction in streptozotocin (STZ)-induced type-2 diabetic rats. Diabetic rats were generated by intraperitoneal injection of STZ administered as single dose at 40 mg/kg body weight (b. wt.). Then the rats were treated with 20 mg of AA or 600 μg of glibenclamide (Glib)/kg b. wt, once in a day for 45 days. The results of this study illustrated that diabetic rat liver mitochondrial malondialdehyde (MDA), reactive oxygen species (ROS) and protein carbonyl (PCO) levels were significantly increased at the same time antioxidant enzymes and non-enzymes status was markedly diminished when compared with the liver mitochondria of normal control rats. A marked reduction in ROS, MDA, PCO and significant amelioration in non-enzymatic and enzymatic antioxidants were observed in diabetic rats treated with AA or Glib as compared with untreated diabetic control rats. Tricarboxylic acid (TCA) cycle enzymes like isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase and electron transport chain (ETC) complexes activities were significantly diminished in diabetic rats. Besides, mitochondrial uncoupling protein-2 (UCP2) expression was significantly upregulated at the same time adenosine triphosphate (ATP) level and mitochondrial membrane potential (MMP) was markedly lowered in the mitochondria of diabetic rats. AA or Glib treatment with diabetic rats restored the TCA cycle enzymes activities, ETC complexes, UCP2, MMP and ATP level to near normal control rats as compared with untreated diabetic control rats. Hence, the results of the current study indicated that AA ameliorated mitochondrial function by attenuating oxidative stress in STZ-induced type-2 diabetic rats.

Therapeutic effect and mechanism of Shouhui Tongbian Capsules on slow transit constipation model mice

Shouhui Tongbian Capsules was used to explore the therapeutic effect and potential mechanism on slow transit constipation model mice induced by loperamide hydrochloride. In the experiment, loperamide hydrochloride-induced ICR mice were used as the model of slow transit constipation. Fifty ICR mice were divided into the blank group, model group and high, medium and low dose groups of Shouhui Tongbian Capsules extract(100, 200 and 400 mg·kg~(-1)). The model group and the administration groups were then modeled using loperamide hydrochloride intragastrically to obtain slow transit constipation. After successful modeling, high, medium and low doses of drugs were given to each drug group by intragastric administration. After 14 days of administration, the first defecation time, 6 h defecation grain number, 6 h defecation wet weight and dry weight, black feces discharged within 6 h and the fecal water content were measured. Intestinal tissues were taken for c-Kit and SCF immunohistochemical sections to detect the expression of c-Kit and SCF in the blank group, model group and high, medium and low dose groups of the medicinal extract of Shouhui Tongbian Capsules. The tissue changes in the intestinal wall of mice were detected by HE staining. At the same time, partial intestinal tissues were taken to test the activity of ATP synthase and isocitrate dehydrogenase in intestinal tissues of mice. RESULTS:: showed that Shouhui Tongbian Capsules effectively improved the symptoms of slow transit constipation in ICR mice and promoted intestinal movement. Shouhui Tongbian Capsules obviously shortened the time of discharging black stool for the first time, improved the intestinal propulsion rate, increased the water content and amount of feces, and improved the constipation symptoms. Mechanism study revealed that Shouhui Tongbian Capsules increased ATP synthase activity and mitochondrial isocitrate dehydrogenase activity in intestinal tissue, and up-regulated c-Kit/SCF signaling pathway to promote interstitial Cajal cells proliferation, intestinal nerve transmission, intestinal motility and transport capacity.

Molecular mechanism study of Shouhui Tongbian Capsules on promoting energy metabolism of gastrointestinal stromal cells

Mechanism study was performed to explore how Shouhui Tongbian Capsules promotes energy metabolism of gastrointestinal stromal cells. In this study, gastrointestinal stromal cells line GIST-882 was used as the model to explore energy metabolism regulation effects of Shouhui Tongbian Capsules extract(10, 20, 50 and 100 μg·mL~(-1)) by measuring the cell proliferation, ATP level, mitochondrial membrane potential, and mitochondrial isocitrate dehydrogenase activity. Meanwhile, Western blot was used to detect the proteins expression of SCF/c-Kit and CDK2/cyclin A signaling pathways. Our results showed that Shouhui Tongbian Capsules promoted cell proliferation and increased ATP level of gastrointestinal stromal cells. In addition, Shouhui Tongbian Capsules obviously improved mitochondrial structural integrity, and increased mitochondrial membrane potential in GIST-882 cells. Mechanism study revealed that Shouhui Tongbian Capsules increased mitochondrial isocitrate dehydrogenase activity and up-regulated the proteins expression of SCF/c-Kit and CDK2/cyclin A signaling pathways. Collectively, our study indicated that Shouhui Tongbian Capsules promoted the energy metabolism for gastrointestinal stromal cells proliferation by activating mitochondrial isocitrate dehydrogenase to induce ATP production, as well as activating SCF/c-Kit and CDK2/cyclin A signaling pathways.

Metabolic acclimation supports higher aluminium-induced secretion of citrate and malate in an aluminium-tolerant hybrid clone of Eucalyptus

BackgroundEucalyptus is the main plantation wood species, mostly grown in aluminized acid soils. To understand the response of Eucalyptus clones to aluminum (Al) toxicity, the Al-tolerant Eucalyptus grandis × E. urophylla clone GL-9 (designated “G9”) and the Al-sensitive E. urophylla clone GL-4 (designated “W4”) were employed to investigate the production and secretion of citrate and malate by roots.ResultsEucalyptus seedlings in hydroponics were exposed to the presence or absence of 4.4 mM Al at pH 4.0 for 24 h. The protein synthesis inhibitor cycloheximide (CHM) and anion channel blocker phenylglyoxal (PG) were applied to explore possible pathways involved in organic acid secretion. The secretion of malate and citrate was earlier and greater in G9 than in W4, corresponding to less Al accumulation in G9. The concentration of Al in G9 roots peaked after 1 h and decreased afterwards, corresponding with a rapid induction of malate secretion. A time-lag of about 6 h in citrate efflux in G9 was followed by robust secretion to support continuous Al-detoxification. Malate secretion alone may alleviate Al toxicity because the peaks of Al accumulation and malate secretion were simultaneous in W4, which did not secrete appreciable citrate. Enhanced activities of citrate synthase (CS) and phosphoenolpyruvate carboxylase (PEPC), and reduced activities of isocitrate dehydrogenase (IDH), aconitase (ACO) and malic enzyme (ME) were closely associated with the greater secretion of citrate in G9. PG effectively inhibited citrate and malate secretion in both Eucalyptus clones. CHM also inhibited malate and citrate secretion in G9, and citrate secretion in W4, but notably did not affect malate secretion in W4.ConclusionsG9 immediately secrete malate from roots, which had an initial effect on Al-detoxification, followed by time-delayed citrate secretion. Pre-existing anion channel protein first contributed to malate secretion, while synthesis of carrier protein appeared to be needed for citrate excretion. The changes of organic acid concentrations in response to Al can be achieved by enhanced CS and PEPC activities, but was supported by changes in the activities of other enzymes involved in organic acid metabolism. The above information may help to further explore genes related to Al-tolerance in Eucalyptus.

Feeding behavior toxicity in the marine rotifer Brachionus plicatilis caused by 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47): Characteristics and mechanisms

Polybrominated diphenyl ether contamination in marine environments has received special attention due to its accumulation and magnification in the marine food web and toxicity to organisms. In the present study, a series of short-term toxicological tests were conducted with the marine rotifer Brachionus plicatilis to assess the effects on ingestion and digestive performance after treatment with 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) at nonlethal concentrations under controlled laboratory conditions and to analyze the possible mechanism. The results showed that with accumulation in rotifers, BDE-47 caused a significant decline in the filtration and feeding rates in a concentration-dependent manner. Moreover, the activities of amylase (AMS) and protease were affected, indicating that BDE-47 impaired ingestion and digestion efficiency. BDE-47 exposure did not lead to abnormal microstructures in the main digestive tract (e.g., cilia around the corona, mastax, stomach, digestive gland and esophagus), but the gastric parietal cells shrank, suggesting nutritional deficiency. BDE-47 prominently induced the occurrence of irregular mitochondria at the cilia root, and mitochondrial and isocitrate dehydrogenase activity declined, indicating mitochondrial dysfunction. Furthermore, the activity of ATPase, which catalyzes ATP hydrolysis, decreased as the BDE-47 concentration rose, implying that BDE-47 retarded rotifer ATP dynamics, inevitably interfering with cilia movement to ingest food. Additionally, a significant decline in acetylcholine esterase activity was observed, which led to a hindrance in neurotransmission involved in food intake and digestion. Altogether, our results demonstrated that nonlethal concentrations of BDE-47 could induce feeding depression in rotifers, which is mainly attributed to stymied energy metabolism and nerve conduction.

Sestrin2 maintains OXPHOS integrity to modulate cardiac substrate metabolism during ischemia and reperfusion

Sestrin2 (Sesn2) is a stress-inducible protein that declines with aging in the heart. We reported that rescue Sesn2 levels in aged mouse hearts through gene therapy improves the resistance of aged hearts to ischemia and reperfusion (I/R) insults. We hypothesize that Sesn2 as a scaffold protein maintains mitochondrial integrity to protect heart from ischemic injury during I/R. Young C57BL/6 J (3–6 months), aged C57BL/6 J (24–26 months), and young Sesn2 KO (3–6 months, C57BL/6 J background) mice were subjected to in vivo regional ischemia and reperfusion. The left ventricle was collected for transcriptomics, proteomics and metabolomics analysis. The results demonstrated that Sesn2 deficiency leads to aging-like cardiac diastolic dysfunction and intolerance to ischemia reperfusion stress. Seahorse analysis demonstrated that Sesn2 deficiency in aged and young Sesn2 KO versus young hearts lead to impaired mitochondrial respiration rate with defects in Complex I and Complex II activity. The Sesn2 targeted proteomics analysis revealed that Sesn2 plays a critical role in maintaining mitochondrial functional integrity through modulating mitochondria biosynthesis and assembling of oxidative phosphorylation (OXPHOS) complexes. The RNA-Seq data showed that alterations in the expression of mitochondrial compositional and functional genes and substrate metabolism related genes in young Sesn2 KO and aged versus young hearts. Further immunofluorescence and immunoprecipitation analysis demonstrated that Sesn2 is translocated into mitochondria and interacts with OXPHOS components to maintain mitochondrial integrity in response to I/R stress. Biochemical analysis revealed that Sesn2 is associated with citrate cycle components to modulate pyruvate dehydrogenase and isocitrate dehydrogenase activities during I/R stress. Thus, Sesn2 serves as a scaffold protein interacting with OXPHOS components to maintain mitochondrial integrity under I/R stress. Age-related downregulation of cardiac Sesn2 fragilizes mitochondrial functional integrity in response to ischemic stress.

Effect of Physical Training on the Activity of the Low Km Phosphodiesterase in Heart Ventricular and Skeletal Muscle Tissues of Normal and Diabetic Rats

&#x0D; &#x0D; &#x0D; &#x0D; This study assessed the effect of physical training on the low Michaelis constant cyclic AMP phosphodiesterase (low Km PDE) activity in heart ventricular tissue and three different skeletal muscle tissues of normal and diabetic rats. Rats were rendered diabetic with streptozotocin (45 mg/kg i.v.) and either kept sedentary (SD, n=16) or submitted to a progressive 10-week treadmill running program (TD, n=17). Two groups of nondiabetic rats served as trained (TC, n=17) and sedentary controls (SC, n=15). The activity of NAD-linked isocitrate dehydrogenase was significantly increased (p &lt; 0.001) in the gastrocnemius muscle of trained animals, confirming that they were adequately trained. Plasma glucose levels were elevated in SD rats (18.8 ± 1.7 mmol/l) compared to those in SC rats (7.7 ± 0.2 mmol/l ; mean ± standard error of the mean [SEM], p &lt; 0.001). These levels were partially normalized following training (12.7 ± 1.7 pmol/l; p &lt; 0.01 vs. SD rats). Plasma insulin levels were significantly reduced in TC rats (223 ± 16 pmol/l) compared with those in SC rats (306 ± 13 pmol/l; p &lt; 0.01). Similarly, the levels in SC rats were significantly different when compared with SD rats (155 ± 15 pmol/l; p &lt; 0.01). In TD rats, plasma insulin levels (156 ± 14 pmol/l) were similar to those of SD rats. This suggests that mild diabetes mellitus in the rat can be improved by endurance training and that improved glycemic control may be mediated by an increase in insulin sensitivity. The low Km PDE activity in the membranes prepared from the four different muscle tissues was not modified by diabetes. Similarly, physical training in normal and diabetic rats did not induce any significant changes in the low Km PDE activity in any of the tissues tested. This suggests that improvements in myocardial contractile function and in glucose homeostasis, as seen in diabetic rats submitted to endurance training, are not associated with changes in PDE.&#x0D; &#x0D; &#x0D; &#x0D;

Abstract PO-028: Investigating the contribution of CHAC1 to persistent mitochondrial respiratory function in NSCLC under cysteine deprivation

Abstract Solid tumors exhibit metabolic rewiring to enhance their energetic capacity while meeting the biosynthetic demands for rapid proliferation. Though this reprogramming supports robust tumor growth, it often imposes bottlenecks and metabolic dependencies that can be exploited clinically. Among the metabolic vulnerabilities that is of therapeutic interest is the sensitivity of many cancers to sustained deprivation of the non-essential amino acid cysteine (Cys). In certain malignant contexts, ferroptosis has been linked to mitochondrial dysfunction, where oxidative metabolism drives ROS production, the loss of mitochondrial membrane integrity and respiratory function, leading to the propagation of lipid radicals that cripple cell viability. We found that while non-small cell lung cancer (NSCLC) cells are susceptible to Cys-starvation induced ferroptosis, mitochondrial respiratory function robustly persists under prolonged Cys deprivation in these NSCLC cells. We also find that mitochondrial iron-sulfur (Fe-S) protein function (e.g. aconitase, lipoic acid synthase, and respiratory complexes I-III) critical to oxidative metabolism is unaltered by Cys deprivation despite the need for Cys-derived sulfur in the synthesis of Fe-S clusters. Under these Cys-restricted conditions, we observe an increase in the expression of glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1), a mitochondrial protein that catalyzes the degradation of the antioxidant glutathione (GSH). Because Cys is a constituent of the tripeptide antioxidant, enhanced mitochondrial GSH turnover could provide Cys for the generation of Fe-S clusters. Consistent with this, CRISPR-mediated knockout of CHAC1 diminished Fe-S protein function in Cys-starved NSCLC cells. Despite this decrease in oxidative capacity, CHAC1 knockout prolonged survival under Cys-deprivation, suggesting that intact mitochondrial function may contribute to Cys-starvation induced cell death. Enhanced GSH degradation by CHAC1 would also restrict GSH-based antioxidant capacity imposing a greater requirement for the NADPH-dependent thioredoxin system. Interestingly, we find that Cys deprivation also induces the expression of nicotinamide nucleotide transhydrogenase (NNT), a critical contributor of mitochondrial NADPH in lung cancer. Moreover, we find that the activity of the mitochondrial NADPH-producing isocitrate dehydrogenase (IDH2) is enhanced under Cys deprivation independent of a change in expression. Collectively, these data indicate a potential rewiring of mitochondrial antioxidant defenses for the maintenance of robust mitochondrial respiratory function in the absence of extracellular Cys. This ultimately may constitute a self-defeating response to nutrient stress in NSCLC, where mitochondria selfishly sustain functionality at the expense of cell viability. Citation Format: Nathan P. Ward, Gina M. DeNicola. Investigating the contribution of CHAC1 to persistent mitochondrial respiratory function in NSCLC under cysteine deprivation [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-028.