Ketoglutarate Dehydrogenase Research Articles

Integration of metabolomic and transcriptomic analyses reveals novel regulatory functions of the ChREBP transcription factor in energy metabolism.

Carbohydrate Response Element-Binding Protein (ChREBP) is a transcription factor that activates key genes involved in glucose, fructose, and lipid metabolism in response to carbohydrate feeding, but its other potential roles in metabolic homeostasis have not been as well studied. We used liver-selective GalNAc-siRNA technology to suppress expression of ChREBP in rats fed a high fat/high sucrose diet and characterized hepatic and systemic responses by integrating transcriptomic and metabolomic analyses. GalNAc-siChREBP-treated rats had lower levels of multiple short-chain acyl CoA metabolites compared to rats treated with GalNAc-siCtrl containing a non-targeting siRNA sequence. These changes were related to a sharp decrease in free CoA levels in GalNAc-siChREBP treated-rats, accompanied by lower expression of transcripts encoding enzymes and transporters involved in CoA biosynthesis. These activities of ChREBP likely contribute to its complex effects on hepatic lipid and energy metabolism. While core enzymes of fatty acid (FA) oxidation are induced by ChREBP knockdown, accumulation of liver acylcarnitines and circulating ketones indicate diversion of acetyl CoA to ketone production rather than complete oxidation in the TCA cycle. Despite strong suppression of pyruvate kinase and activation of pyruvate dehydrogenase, pyruvate levels were maintained, likely via increased expression of pyruvate transporters, and decreased expression of lactate dehydrogenase and alanine transaminase. GalNAc-siChREBP treatment increased hepatic citrate and isocitrate levels while decreasing levels of distal TCA cycle intermediates. The drop in free CoA levels, needed for the 2-ketoglutarate dehydrogenase reaction, as well as a decrease in transcripts encoding the anaplerotic enzymes pyruvate carboxylase, glutamate dehydrogenase, and aspartate transaminase likely contributed to these effects. GalNAc-siChREBP treatment caused striking increases in PRPP and ZMP/AICAR levels, and decreases in GMP, IMP, AMP, NaNM, NAD(P), and NAD(P)H levels, accompanied by reduced expression of enzymes that catalyze late steps in purine and NAD synthesis. ChREBP suppression also increased expression of a set of plasma membrane amino acid transporters, possibly as an attempt to replenish TCA cycle intermediates. In sum, combining transcriptomic and metabolomic analyses has revealed regulatory functions of ChREBP that go well beyond its canonical roles in control of carbohydrate and lipid metabolism to now include mitochondrial metabolism and cellular energy balance.

Long-term exposure to polystyrene microplastics induces hepatotoxicity by altering lipid signatures in C57BL/6J mice

It is estimated that the life of plastics is hundreds to thousands of years, their lasting properties making plastic debris absorbing toxic chemicals and degrading into microplastics (MPs). The purpose of this study was to explore the effects of exposure to different size (0.08 and 0.5 μm) polystyrene (PS) in mice. After 16 weeks of exposure, it was found that PS-MPs could be identified in the liver. No effect of PS-MPs treatment on body weight was observed. PS-MPs exposure disturbed lipids and lipid-like molecule metabolisms and perturbed the citrate cycle and oxidative phosphorylation. Meanwhile, isocitrate dehydrogenase (ICDHc), nicotinamide adenine dinucleotide -malate dehydrogenase (NAD-MDH), succinate dehydrogenase (SDH), α ketoglutarate dehydrogenase (α-KGDH) activities and adenosine triphosphate (ATP) level were obviously affected by PS-MPs treatment. In addition, significant differences were recorded in catalase (CAT) and malondialdehyde (MDA) levels, indicating that PS-MPs exposure induced an oxidative stress in the liver. In conclusion, our present study provided the first evidence of: (a) long-term exposure to PS-MPs lead to PS-MPs accumulated in the liver and results in liver injury; (b) long-term exposure to PS-MPs disturbs lipids and lipid-like molecule metabolisms; (c) long-term exposure to PS-MPs perturbs citrate cycle and oxidative phosphorylation and leads to oxidative stress in the liver.

Abstract P3179: Tristetraprolin Regulates Metabolic And Epigenetic Remodeling In Cardiomyocytes Post Myocardial Infarction

Myocardial infarction (MI) triggers profound remodeling in the infarcted, border (BZ), and remote zones of the myocardium. Here, we studied the metabolic remodeling and epigenetic modifications that occur in response to inflammation in the BZ, focusing the role of an mRNA-binding protein that causes degradation of its target mRNA molecules, tristetraprolin (TTP). Inflammatory cells were observed to increase in the BZ, associated with the release of IL-1B and IFN-γ. Among proteins that are altered in the BZ cardiomyocytes, TTP displayed a significant increase in its mRNA and protein levels in the BZ. Isotope tracing using U-13C6 glucose in TTP-overexpressing cells showed that TTP overexpression results in an increase in TCA metabolites upstream of alpha ketoglutarate dehydrogenase (αKGDH), and a decrease of downstream metabolites, suggesting blocked activity at the level of αKGDH. We then identified the mRNA of the E2 component of αKGDH, Dihydrolipoamide S-Succinyltransferase (DLST), to be a target of TTP. Consistent with these findings, DLST mRNA and protein levels are decreased in BZ. DLST needs to be lipoylated to become active, and our data indicate that the mRNA for a key enzyme in DLST lipoylation (lipoic acid synthase, LIAS) is also a target of TTP. Overexpression of TTP led to a decrease in the level of DLST lipoylation. Epigenetic studies showed that, overexpression of TTP resulted in significant decrease in acetylation and methylation of H4K20. Finally, we demonstrated that heart specific TTP KO mice are protected against ischemic injury compared to WT mice, indicating that deletion of TTP alters the remodeling of the BZ, through its role in the regulation of αKGDH and production of αKG in response to infiltration of inflammatory cells.

Metabolite Cross-Feeding Promoting NADH Production and Electron Transfer during Efficient SMX Biodegradation by a Denitrifier and S. oneidensis MR-1 in the Presence of Nitrate.

Antibiotics often coexist with other pollutants (e.g., nitrate) in an aquatic environment, and their simultaneous biological removal has attracted widespread interest. We have found that sulfamethoxazole (SMX) and nitrate can be efficiently removed by the coculture of a model denitrifier (Paracoccus denitrificans, Pd) and Shewanella oneidensis MR-1 (So), and SMX degradation is affected by NADH production and electron transfer. In this paper, the mechanism of a coculture promoting NADH production and electron transfer was investigated by proteomic analysis and intermediate experiments. The results showed that glutamine and lactate produced by Pd were captured by So to synthesize thiamine and heme, and the released thiamine was taken up by Pd as a cofactor of pyruvate and ketoglutarate dehydrogenase, which were related to NADH generation. Additionally, Pd acquired heme, which facilitated electron transfer as heme, was the important composition of complex III and cytochrome c and the iron source of iron sulfur clusters, the key component of complex I in the electron transfer chain. Further investigation revealed that lactate and glutamine generated by Pd prompted So chemotactic moving toward Pd, which helped the two bacteria effectively obtain their required substances. Obviously, metabolite cross-feeding promoted NADH production and electron transfer, resulting in efficient SMX biodegradation by Pd and So in the presence of nitrate. Its feasibility was finally verified by the coculture of an activated sludge denitrifier and So.

Investigating the changes of the components of the Krebs cycle in patients with type 2 diabetes treated with squalene

Background: Type 2 diabetes mellitus is a chronic disease that impairs the body’s ability to regulate glucose. Recent studies have shown that squalene, a bioactive compound, has shown promising potential in increasing ATP levels for diabetic patients and aged individuals.Objective: Our main goal was to evaluate the cellular effects of different doses of squalene on the intermediates and enzymes of Krebs cycle, in order to determine if squalene increases ATP production among groups of people with type 2 diabetes. The intermediates and enzymes that are being studied are acetyl coenzyme (A-CoA), alpha ketoglutarate dehydrogenase (AKGDH), calcium ion (Ca2+), citrate synthase (CS), isocitrate dehydrogenase, oxaloacetate, and pyruvate dehydrogenase complex component (PDH).Methods: In this study, 30 healthy volunteers were selected as the healthy control group (group 1) and 120 volunteers with type 2 diabetes mellitus were selected. Subjects with diabetes were randomly divided into 4 groups. Group 2 was untreated with squalene and groups 3, 4, and 5 were treated with 200, 400 and 600 mg of squalene, respectively for 84 days. Intermediates and enzymes of the Krebs cycle as well as calcium ion were assayed on days 1, 14, 28, 56, and 84 according to the relevant protocols in all groups.Results: The squalene-treated diabetic groups were compared to group 2 that was not treated any squalene to determine the differences of the parameters. Throughout these 84 days, it was observed that only calcium levels increased in the diabetic patients with high statistical difference (P < 0.05). The other parameters: acetyl coenzyme, alpha ketoglutarate dehydrogenase, citrate synthase, isocitrate dehydrogenase, oxaloacetate, and pyruvate dehydrogenase did not have a significant difference (P > 0.05). Conclusion: Based on the findings of this study, the addition of various doses of squalene to a diabetic patient's diet increases the amount of calcium found in their metabolic process in relation to the Krebs cycle. As calcium is responsible for stimulating the Krebs Cycle, it is evident that squalene plays an important part in ATP production.Keywords: squalene, type 2 diabetes, Krebs cycle, calcium, ATP

Pathogenic variants in GCSH encoding the moonlighting H-protein cause combined nonketotic hyperglycinemia and lipoate deficiency.

Maintaining protein lipoylation is vital for cell metabolism. The H-protein encoded by GCSH has a dual role in protein lipoylation required for bioenergetic enzymes including pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase, and in the one-carbon metabolism through its involvement in glycine cleavage enzyme system, intersecting two vital roles for cell survival. Here, we report six patients with biallelic pathogenic variants in GCSH and a broad clinical spectrum ranging from neonatal fatal glycine encephalopathy to an attenuated phenotype of developmental delay, behavioral problems, limited epilepsy and variable movement problems. The mutational spectrum includes one insertion c.293-2_293-1insT, one deletion c.122_(228 + 1_229-1) del, one duplication of exons 4 and 5, one nonsense variant p.Gln76*and four missense p.His57Arg, p.Pro115Leu and p.Thr148Pro and the previously described p.Met1?. Via functional studies in patient's fibroblasts, molecular modeling, expression analysis in GCSH knockdown COS7 cells and yeast, and in vitro protein studies, we demonstrate for the first time that most variants identified in our cohort produced a hypomorphic effect on both mitochondrial activities, protein lipoylation and glycine metabolism, causing combined deficiency, whereas some missense variants affect primarily one function only. The clinical features of the patients reflect the impact of the GCSH changes on any of the two functions analyzed. Our analysis illustrates the complex interplay of functional and clinical impact when pathogenic variants affect a multifunctional protein involved in two metabolic pathways and emphasizes the value of the functional assays to select the treatment and investigate new personalized options.

Exploring the effects of artesunate and fuzheng huayu decoction on mitochondria in the treatment of schistosomiasis liver fibrosis

Objective: To compare the effects of artesunate (Art) and fuzheng huayu decoction on mitochondrial autophagy in the treatment of schistosomiasis liver fibrosis. Methods: Eighty C57BL/6 female mice were randomly divided into healthy control group, infection group, Art treatment group and Fuzheng Huayu Decoction treatment group, with 20 mice in each group. Mice in the infection group and treatment group were infected with 16 Schistosoma japonicum cercariae. After 6 weeks, praziquantel (300 mg/kg) was used for 2 days to kill the worms. The Art treatment group was treated with intraperitoneal injection of 100 mg/kg/day, while the Fuzheng Huayu Decoction treatment group was fed 16g of fuzheng huayu decoction per 1kg per day. After 6 weeks, fresh liver tissues of the four groups were collected. Masson staining and Western blot were used to observe the succinate dehydrogenase subunit A (SDHA) and malate dehydrogenase (MDH2), citrate synthase (CS), ketoglutarate dehydrogenase (OGDH), and target of rapamycin 1 (mTORC1) pathway involved in mitochondrial tricarboxylic acid cycle in liver tissues. The relative expression levels of adenylate activated protein kinase (AMPK) and mitochondrial autophagy pathway kinase (PINK1) were detected. Liver tissue samples were extracted from each group to detect the mitochondrial oxygen consumption rate. Two-way ANOVA was used to compare the significance and difference between two sets of samples. Results: Masson staining showed that the infection group mice had significantly higher liver fibrosis area than the healthy control group, while the Art treatment group and Fuzheng Huayu Decoction treatment group mice had lower liver fibrosis area than the infection group. Western blot analysis showed that the infection group (0.82 ± 0.05) had significantly lower relative expression of SDHA protein than the healthy control group (1.00 ± 0.05) (t = 11.23, P = 0.0035), while the Art treatment group (0.73 ± 0.05) had significantly higher relative expression of SDHA protein than the infection group (t = 10.79, P = 0.0073). However, there was no significant change in Fuzheng Huayu Decoction treatment group (0.98±0.05) (t = 1.925, P = 0.1266). The relative expression of p-AMPK protein was significantly higher in the infection group (1.15 ±0.05) than in the healthy control group (0.98 ± 0.07, t = 12.18, P = 0.0029), and the expression of p-AMPK in the Art treatment group (0.50 ± 0.05) was significantly lower than the infection group (t = 11.78, P = 0.0032). The relative protein expression of AMPK was significantly lower in the infection group (0.80 ± 0.05) than in the healthy control group (1.00 ± 0.05, t = 10.53, P = 0.0046). The expression of AMPK was significantly lower in the Art treatment group (0.54 ± 0.05) than in the infection group (T = 13.98, P = 0.0036). The relative expression of p-mTORC1 protein (0.93 ± 0.08) was not significantly different in the infection group than in the healthy control group (t = 2.28, P = 0.065), while the Art treatment group (0.63 ± 0.05) had significantly lower relative expression of p-mTORC1 protein than the infection group (t = 10.58, P = 0.029). The expression of p-mTORC1/ m-TORC1 was not significantly different in the infection group (0.98 ± 0.03) than in the healthy control group (0.97 ± 0.03, t = 0.98, P = 0.085), while the Art treatment group (0.63 ± 0.05) had significantly lower relative expression of p-mTORC1/ m-TORC1 than the infection group (t = 14.58, P = 0. 009). The relative protein expression of PINK1 was significantly lower in the infection group (0.55 ± 0.05) than in the healthy control group (1.00 ± 0.03, t = 13.49, P = 0.0011), while the Art treatment group (1.21 ± 0.05, t = 9.98, P = 0.0046) and Fuzheng Huayu Decoction treatment group (1.31 ±0.35, t = 6.98, P = 0.027) had significantly higher relative protein expression of PINK1 than the infection group. Mitochondrial function tests showed that after adding substrate complex II, the oxygen consumption of the infection group was lower than the healthy control group, while the Art treatment group and the Fuzheng Huayu Decoction treatment group had higher oxygen consumption than the infection group. The oxygen consumption was significantly lower after adding the substrate complex III in the infection group than the healthy control group, while the Art treatment group and Fuzheng Huayu Decoction treatment group had higher oxygen consumption than the infection group. Conclusion: Art can alleviate schistosomiasis liver fibrosis by inhibiting AMPK/mTORC1 signaling pathway activity and enhancing mitochondrial oxygen consumption, autophagy and SDHA expression.

Tissue Expressions of Regulatory Enzymes of the Krebs Cycle in Low- and High-grade Gliomas.

To compare tissue levels of the regulatory enzymes related to the Krebs cycle between low, and high-grade supratentorial gliomas. Forty patients who underwent surgery for supratentorial gliomas (19 with low-grade and 21 with high-grade gliomas) were evaluated. The regulatory enzymes directly involved in the Krebs cycle, namely pyruvate dehydrogenase, citrate synthase, ?-ketoglutarate dehydrogenase, and isocitrate dehydrogenase, and two enzymes that indirectly regulate the Krebs cycle, namely glutamate dehydrogenase and glutaminase, were quantitatively studied in tumor tissues using ELISA. The results were compared between the two groups. The levels of all enzymes were higher in the high-grade glioma group but only pyruvate dehydrogenase, citrate synthase, and isocitrate dehydrogenase levels showed statistical significance. Moreover, all enzymes showed higher tissue levels in grade- II compared to grade-I gliomas, but only two enzymes, glutamate dehydrogenase and glutaminase, reached significantly higher levels. In the high-grade glioma group, all enzymes again showed higher tissue levels in grade-IV gliomas than in grade-III gliomas, but none showed statistical significance. Regulatory enzymes of the Krebs cycle are increased in high-grade gliomas compared to low-grade gliomas. Glutaminolysis enzymes, namely glutamate dehydrogenase and glutaminase, which are required for resupplying the Krebs cycle, are also increased in order to meet the high energy demand in high-grade gliomas.

Complete Remission with Devimistat (CPI-613) in Refractory Burkitt Lymphoma

Complete Remission with Devimistat (CPI-613) in Refractory Burkitt Lymphoma

13C-Labeled Diethyl Ketoglutarate Derivatives as Hyperpolarized Probes of 2-Ketoglutarate Dehydrogenase Activity.

The TCA cycle is a central metabolic pathway for energy production and biosynthesis. A major control point of metabolic flux through the cycle is the decarboxylation of 2-ketoglutarate by the TCA cycle enzyme 2-ketoglutarate dehydrogenase (2-KGDH). In this project, we developed 13C labeled 2-ketoglutarate derivatives to monitor 2-KGDH activity in vivo. 13C NMR analysis of liver extracts revealed that uniformly 13C labeled 2-ketogutarate, in its cell permeable ester form, was rapidly taken up and hydrolyzed in liver and underwent extensive metabolism to produce labeled glutamate, succinate, lactate and other metabolites. Diethyl [1,2-13C2]-2-ketoglutarate was successfully polarized by dynamic nuclear polarization and within seconds after injection into rats, the probe produced hyperpolarized [13C]bicarbonate in the liver reflecting flux through the TCA cycle. These experiments demonstrate that this tracer offers the possibility of directly monitoring flux through 2-KGDH in vivo.

PRELIMINARY ACTIVITY OF DEVIMISTAT (CPI 613) IN PATIENTS WITH RELAPSED OR REFRACTORY BURKITT LYMPHOMA/LEUKEMIA

Introduction: Patients (pts) with primary refractory or relapsed Burkitt lymphoma/leukemia (BL) or high-grade B-cell lymphoma with MYC and BCL2 rearrangements (double hit, DHL) and/or BCL6 (triple hit, THL) have a dismal prognosis. A dysregulated MYC oncogene has downstream effects on proliferation and highly glycolytic metabolism with tricarboxylic acid (TCA) cycle intermediates as biosynthetic precursors. Devimistat is a non-redox active analogue of lipoic acid, a required cofactor for two key TCA cycle mitochondrial enzymes: pyruvate dehydrogenase and alpha ketoglutarate dehydrogenase. Disruption results in a shutdown of ATP and biosyntheticintermediate production leading to cancer cell death. In a phase I trial (n = 26) a pt with multiply refractory BL had a partial remission sustained over one year prior to resection. She remains in remission 7 years later. As of March 2021, 20 clinical studies in over 700 pts with various cancers have been conducted (ongoing/completed) with devimistat. We initiated a phase II trial to further explore efficacy. Methods: NCT03793140 is a multicenter study enrolling 17 patients on each of two cohorts BL or DHL/THL. Pts must have had one prior therapy or are refusing standard of care, measurable disease or isolated bone marrow involvement, and must not be within 3 months of a prior stem cell transplant. Active central nervous system (CNS) parenchymal disease is excluded, but leptomeningeal disease is allowed if the CSF is negative for more than 4 weeks and the maintenance intrathecal/intraOmmaya therapy is ongoing. Devimistat is given by central line over 2 hours daily x 5 days for two 14-day cycles and then as maintenance every 21 days. Pts are evaluable for response if they receive at least 4/5 days of the first cycle. At least 1response in the first 9 pts by cycle 3 was needed to expand to 17 pts in total in that cohort. Results: 9 pts were enrolled in the DHL/THL arm. Number of prior therapies was 3(1-6). No responses and only 1 stable disease resulted in cohort closure. Thus far, 8 BL pts were enrolled. Number of prior therapies was 3 (2-4). 2 pts were inevaluable. 1/6 pts had a response. This HIV+ pt had a thigh mass having received 4 prior therapies. He had a near complete metabolic remission after 3 cycles of devimistat. PET/CT assessment: Thigh lesion baseline October 19, 2020: 10.8 x 6.5 cm SUV 24. After cycle 3: unmeasurable, SUV 2. He is currently in cycle 7, having had a 4-week treatment delay of cycle 5 due to CoVID 19 infection. ECOG improved from 3 to 0. Adverse events (AE): As of March 03, 2021, no serious AEs related to study drug. 4 pts had grade 3 events at least possibly related: 2 neutropenia, 1 thrombocytopenia and 1 elevated bilirubin. 1 patient had a dose reduction for grade 2 alanine aminotransferase increase. Conclusions: Although our results are preliminary, the near complete remission in this patient is promising in a disease where no viable treatment options exist.

Artesunate alleviates schistosomiasis-induced liver fibrosis by downregulation of mitochondrial complex Ⅰ subunit NDUFB8 and complex Ⅲ subunit UQCRC2 in hepatic stellate cells

Hepatic stellate cells (HSCs) play a key role in the pathogenesis of hepatic fibrosis. Inhibition of the HSCs activity is an ideal strategy in the treatment of fibrosis, but there is no drug yet for this strategy. Artesunate (ART) has been shown to protect liver from fibrosis through inhibition of HSCs activity. However, the mechanism of ART activity remains to be fully uncovered. In this study, we tested ART in a mouse model of hepatic fibrosis established in the schistosomiasis-infected mice. The mechanism of ART action was investigated in the HSC cell line LX-2. ART significantly inhibited hepatic fibrosis. In LX-2 cells, ART efficiently inhibited the cell activity in proliferation and mRNA expression of fibrosis marker genes including Col1a1 and Col3a1. An impact of ART on mitochondria was observed for suppression of enzymes in the citric acid cycle (TCA), such as citrate synthase (CS), isocitrate dehydrogenase (IDH2), and alpha ketoglutarate dehydrogenase (OGDH) in a dose-dependent manner. ART decreased the mitochondrial oxygen consumption rate (OCR) and the protein levels of mitochondrial complex Ⅰ subunit NDUFB8 and complex Ⅲ subunit UQCRC2 in HSCs. All of these alterations were observed with an increase in HSC apoptosis. This study suggests that ART may alleviate liver fibrosis by downregulation of HSC activity through suppression of NDUFB8 and UQCRC2 in mitochondria. This study provides a new insight into the mechanism of the ART activity in the inhibition of schistosomiasis-induced liver fibrosis.

Influence of some cinnamic acid derivatives on changes of the tricarboxylic acid cycle enzymes activity in rats under brain ischemia conditions

The aim of the study was to assess the effect of certain derivatives of cinnamic acids on changes of the tricarboxylic acid cycle enzymes activity under experimental cerebral ischemia.
 Materials and methods. Brain ischemia was modeled by irreversible right-sided coagulation of the middle cerebral artery. Test compounds: 4-hydroxy-3,5-ditretbutyl cinnamic acid, coumaric, coffee, synapic, cinnamic, 4-hydroxycinnamic and ferulic acids, as well as a reference drug succinic acid was administered at a dose of 100 mg / kg per os for 3 days after the reproduction of ischemia. Then, changes in the activity of aconitase, citrate synthase, and -ketoglutarate dehydrogenase were evaluated in the supernatant of the brain.
 Results. The use of all the studied compounds and the reference drug helped to restore the activity of enzymes of the tricarboxylic acid cycle. The most pronounced results were obtained when animals were treated by 4-hydroxy-3,5-ditretbutyl cinnamic acid, against the background of which the activity of citrate synthase was higher than in animals treated by succinic, coumaric, coffee, synapic and ferulic acids by 1.53 (p 0.05), 1.41 (p 0.05), 1.4 (p 0.05), 1.46 (p 0.05) and 1.41 (p 0.05) times, respectively. Also, with the administration of 4-hydroxy-3,5-ditretbutyl cinnamic acid, the activity of aconitase was higher compared to rats that were administered with succinic, coumaric, coffee, synapic and ferulic acids by 2.47 (p 0.05), 2.49 (p 0.05), 3.44 (p 0.05), 2.59 (p 0.05) and 1.9 (p 0.05) times, respectively.
 Conclusion. The administration of the studied in this work cinnamic acid derivatives helps to restore the activity of citrate synthase, aconitase, and -ketoglutarate dehydrogenase in rats under conditions of cerebral ischemia. The most pronounced changes in the activity of enzymes were obtained with the iadministration of 4-hydroxy-3,5-ditretbutyl cinnamic acid.

An "energy-auxotroph" Escherichia coli provides an in vivo platform for assessing NADH regeneration systems.

An efficient in vivo regeneration of the primary cellular resources NADH and ATP is vital for optimizing the production of value-added chemicals and enabling the activity of synthetic pathways. Currently, such regeneration routes are tested and characterized mainly in vitro before being introduced into the cell. However, in vitro measurements could be misleading as they do not reflect enzyme activity under physiological conditions. Here, we construct an in vivo platform to test and compare NADH regeneration systems. By deleting dihydrolipoyl dehydrogenase in Escherichia coli, we abolish the activity of pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase. When cultivated on acetate, the resulting strain is auxotrophic to NADH and ATP: acetate can be assimilated via the glyoxylate shunt but cannot be oxidized to provide the cell with reducing power and energy. This strain can, therefore, serve to select for and test different NADH regeneration routes. We exemplify this by comparing several NAD-dependent formate dehydrogenases and methanol dehydrogenases. We identify the most efficient enzyme variants under in vivo conditions and pinpoint optimal feedstock concentrations that maximize NADH biosynthesis while avoiding cellular toxicity. Our strain thus provides a useful platform for comparing and optimizing enzymatic systems for cofactor regeneration under physiological conditions.

255-LB: Impaired Gsa/cAMP Signaling Exclusive to Pancreatic ß-Cells Exhibits a Unique Urinary Metabolome via Nontargeted Metabolomics in a Murine Model of Diabetes In Vivo

Background: β-cell dysfunction is a hallmark feature of diabetes mellitus. G protein a-subunit (Gsa) mediates receptor-stimulated intracellular cAMP production In β cells in order to increase glucose-stimulated insulin secretion, cell proliferation and survival. Growing interest in urinary metabolome has emerged as many drugs aim to improve outcomes by targeting kidney function. Given clinical significance of the Gsa pathway, we aimed to study changes of urinary metabolome as a result of Gsa deficiency. Methods: Mice with Gsα deficiency limited to β cells (Gsa βcell-/-) were generated by mating Gsα-floxed mice with MIPcreERT mice in order to directly examine Gsα/cAMP signaling role in β cells, in a similar method to Xie et.al. Urine was collected from (Gsa βcell-/-) mice and controls and stored at -80C until processed for non-targeted metabolomics using GCMS. Results: In a small cohort, 17 metabolites were significantly altered between the two groups (P<0.05). While pathway analysis somewhat echoed findings described in other models of diabetes (urea cycle, Malate-Aspartate shuttle, and amino acid metabolism), 4 metabolites were completely undetectable in (Gsa βcell-/-) urine including 3-Oxoglutaric acid and Methylglutarate. In addition, some changed in a different direction to what was previously described in other models of diabetes (e.g: Gsa βcell-/- mice exhibited decreased urinary adipic acid). Enrichment analysis of disease associated metabolite sets of the urine mimicked changes seen in enzymatic defects of 3-Hydroxy-3-Methylglutaryl-Coa Lyase, Kynureninase Deficiency, 2-Ketoglutarate Dehydrogenase and Wolcott-Rallison Syndrome (P<0.05). Conclusion: Gsa pathway deficiency in β cells produced a model of diabetes with unique metabolome. Studying changes of urine metabolome after treatment with antiglycemics in the future might highlight the drugs renal impact in the setting of Gsa deficiency. Disclosure A. Ilaiwy: None. M. Capozzi: None. J.L. Brown: None. D. D’Alessio: Advisory Panel; Self; Eli Lilly and Company. Consultant; Self; Intarcia Therapeutics. Research Support; Self; Ansh Labs, Eli Lilly and Company, Merck Sharp & Dohme Corp. Other Relationship; Self; Novo Nordisk A/S. J. Campbell: Research Support; Self; Eli Lilly and Company, Novo Nordisk Inc. Speaker’s Bureau; Self; Merck Sharp & Dohme Corp.

Inhibition of the ketolytic acetyl CoA supply to tumors could be their “Achilles heel”

Inhibition of the ketolytic acetyl <scp>CoA</scp> supply to tumors could be their “Achilles heel”

Exclusive neuronal detection of KGDHC-specific subunits in the adult human brain cortex despite pancellular protein lysine succinylation

The ketoglutarate dehydrogenase complex (KGDHC) consists of three different subunits encoded by OGDH (or OGDHL), DLST, and DLD, combined in different stoichiometries. DLD subunit is shared between KGDHC and pyruvate dehydrogenase complex, branched-chain alpha-keto acid dehydrogenase complex, and the glycine cleavage system. Despite KGDHC’s implication in neurodegenerative diseases, cell-specific localization of its subunits in the adult human brain has never been investigated. Here, we show that immunoreactivity of all known isoforms of OGDHL, OGDH, and DLST was detected exclusively in neurons of surgical human cortical tissue samples identified by their morphology and visualized by double labeling with fluorescent Nissl, while being absent from glia expressing GFAP, Aldhl1, myelin basic protein, Olig2, or IBA1. In contrast, DLD immunoreactivity was evident in both neurons and glia. Specificity of anti-KGDHC subunits antisera was verified by a decrease in staining of siRNA-treated human cancer cell lines directed against the respective coding gene products; furthermore, immunoreactivity of KGDHC subunits in human fibroblasts co-localized > 99% with mitotracker orange, while western blotting of 63 post-mortem brain samples and purified recombinant proteins afforded further assurance regarding antisera monospecificity. KGDHC subunit immunoreactivity correlated with data from the Human Protein Atlas as well as RNA-Seq data from the Allen Brain Atlas corresponding to genes coding for KGDHC components. Protein lysine succinylation, however, was immunohistochemically evident in all cortical cells; this was unexpected, because this posttranslational modification requires succinyl-CoA, the product of KGDHC. In view of the fact that glia of the human brain cortex lack succinate-CoA ligase, an enzyme producing succinyl-CoA when operating in reverse, protein lysine succinylation in these cells must exclusively rely on propionate and/or ketone body metabolism or some other yet to be discovered pathway encompassing succinyl-CoA.

A Phase II Clinical Trial of Cpi-613 (devimistat) in Patients with Relapsed or Refractory Burkitt Lymphoma/Leukemia or High-Grade B-Cell Lymphoma with Rearrangements of MYC and BCL2 and/or BCL6

Background: Patients with primary refractory or relapsed Burkitt lymphoma/leukemia (BL) or high-grade B-cell lymphoma with rearrangements of MYC and BCL2 (double hit, DHL) and/or BCL6 (triple hit, THL) have a dismal prognosis with patients rarely achieving meaningful remissions following second line therapy. No standard therapeutic approach exists for this group. The characteristic hallmark of these diseases is a dysregulated MYC oncogene with downstream effects on both proliferation and highly glycolytic metabolism which use tricarboxylic acid (TCA) cycle intermediates as biosynthetic precursors. CPI 613® (devimistat) is a non-redox active analogue of lipoic acid, a required cofactor for two key mitochondrial enzymes of the TCA cycle: pyruvate dehydrogenase and alpha ketoglutarate dehydrogenase. Disruption of these enzyme activities results in a shutdown of ATP and biosynthetic-intermediate production leading to cancer cell death by apoptosis or necrosis. In the initialphase I trial a patient with multiply refractory BL had a partial remission on CPI 613 sustained for over one year prior to surgical resection. Given the rarity of these types of responses in multiply relapsed BL we initiated a phase II trial to further explore efficacy. CPI-613® has FDA orphan status for BL. Study Design and Methods: NCT03793140 is a multicenter study enrolling 17 patients on each of two cohorts BL or DHL/THL. Patients must have had one prior therapy or are refusing standard of care, measurable disease or isolated bone marrow involvement, and must not be within 3 months of a prior stem cell transplant. Patients with active central nervous system (CNS) parenchymal disease are excluded, but those with leptomeningeal disease are eligible if the CSF is negative for lymphoma for more than 4 weeks and the maintenance intrathecal/intraOmmaya therapy is ongoing . CPI 613 is given by central line over 2 hours daily x 5 days for two 14-day cycles and then in 21 day cycles. With a primary objective of overall response, treatment can be used as a bridge to transplant. Secondary endpoints include duration of response, progression-free survival (PFS) and overall survival (OS). Primary and secondary outcomes will be correlated with pre-treatment biomarkers including variances in serum metabolites, immunohistochemistry staining for PDH, KGDH, PDKs1-4, SOD2 and pretreatment cytokine profiles. Biostatistics include a Simon minimax two-stage design for efficacy after the first 10 patients in each of the BL and DHL/THL cohorts separately analyzed. Additionally, an interim analysis for toxicity will be conducted after the first 10 study participants have completed two complete cycles or have come off study. Figure Disclosures Noy: Raphael Pharma: Research Funding; Pharamcyclics: Research Funding; Janssen: Consultancy; Medscape: Honoraria; Prime Oncology: Honoraria; NIH: Research Funding. Pardee:Rafael Pharmaceuticals: Consultancy, Research Funding; Karyopharm: Research Funding; Pharmacyclics/Janssen: Speakers Bureau; Celgene: Speakers Bureau; Amgen: Speakers Bureau; CBM Bipharma: Membership on an entity's Board of Directors or advisory committees; Spherix Intellectual Property: Research Funding. Abramson:AbbVie Inc, Amgen Inc, Bayer HealthCare Pharmaceuticals, Celgene Corporation, EMD Serono Inc, Genentech, Gilead Sciences Inc, Janssen Biotech Inc, Juno Therapeutics, a Celgene Company, Karyopharm Therapeutics, Kite Pharma Inc, Merck, Novartis, Seattle Gen: Consultancy. Dunleavy:Pharmacyclics: Membership on an entity's Board of Directors or advisory committees. Luther:Raphael: Employment. OffLabel Disclosure: CPI 613 devimistat is a is a non-redox active analogue of lipoic acid, a required cofactor for two key mitochondrial enzymes of the TCA cycle

Metagenomic analysis of relationships between the denitrification process and carbon metabolism in a bioaugmented full-scale tannery wastewater treatment plant.

The goal of this study was to investigate the relationship between the denitrification process and carbon metabolism in a full-scale tannery wastewater treatment plant bioaugmented with the microbial consortium BM-S-1. The metagenomic analysis of the microbial community showed that Brachymonas denitrificans, a known denitrifier, was present at a high level in the treatment stages of buffering (B), primary aeration (PA), and sludge digestion (SD). The occurrences of the amino acid-degrading enzymes alpha ketoglutarate dehydrogenase (α-KGDH) and tryptophan synthase were highly correlated with the presence of denitrification genes, such as napA, narG, nosZ and norB. The occurrence of glutamate dehydrogenase (GDH) was also highly paralleled with the occurrence of denitrification genes such as napA, narG, and norZ. The denitrification genes (nosZ, narG, napA, norB and nrfA) and amino acid degradation enzymes (tryptophan synthase, α-KGDH and pyridoxal phosphate dependent enzymes) were observed at high levels in B. This indicates that degradation of amino acids and denitrification of nitrate may potentially occur in B. The high concentrations of the fatty acid degradation enzyme groups (enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and β-ketothiolase) were observed together with the denitrification genes, such as napA, narG and nosZ. Phospholipase/carboxylesterase, enoyl-CoA hydratase/isomerase, acyl-CoA dehydrogenase, phenylacetate degradation enzyme and 3-hydroxyacyl-CoA dehydrogenase 2 were also dominant in B. All these results clearly indicate that the denitrification pathways are potentially linked to the degradation pathways of amino acids and fatty acids whose degradation products go through the TCA cycle, generating the NADH that is used as electron donors for denitrification.

Aggravated effects of coexisting marginal thiamine deficits and zinc excess on SN56 neuronal cells

ABSTRACT Objectives: Zinc excitotoxicity and thiamine pyrophosphate deficiency (TD) are known pathogenic signals contributing to mechanism of different encephalopathies through inhibition of enzymes responsible for energy metabolism such as pyruvate dehydrogenase, aconitase or ketoglutarate dehydrogenase. The aim of this work was to investigate whether subclinical Zn excess and TD, frequent in aging brain, may combine yielding overt neuronal impairment. Results: Clonal SN56 cholinergic neuronal cells of septal origin were used as the model of brain cholinergic neurons, which are particularly susceptible to neurodegeneration in the course of Alzheimer’s disease, hypoxia and other dementia-linked brain pathologies. Neither subtoxic concentration of Zn (0.10 mM) nor mild 20–25% TD deficits alone caused significant negative changes in cultured cholinergic neurons viability and their acetyl-CoA/acetylcholine metabolism. However, cells with mild TD accumulated Zn in excess, which impaired their energy metabolism causing a loss of neurons viability and their function as neurotransmitters. These negative effects of Zn were aggravated by amprolium which is an inhibitor of thiamine intracellular transport. Conclusion: Our data indicate that TD may amplify otherwise non-harmful border-line Zn excitotoxic signals yielding progress of neurodegeneration.