Enzyme Of Pentose Phosphate Pathway Research Articles

Abstract 4333: Identification of glucose-6-phosphate dehydrogenase dependency in IDH1 mutant glioma cells using functional genomics

Abstract Astrocytoma and oligodendroglioma are most commonly initiated by missense mutations at the arginine 132 codon of isocitrate dehydrogenase 1 (IDH1). Biochemically the mutant IDH1 protein acquires neomorphic activity: reductive NADPH-dependent catalysis of α-ketoglutarate (αKG) to (R)-2-hydroxyglutarate [(R)-2-HG]. (R)-2-HG interferes with the function of αKG-dependent epigenetic modifiers, resulting in extensive epigenetic remodeling that impairs cellular differentiation and promotes gliomagenesis. Strategies for selectively targeting IDH1-mutant cells offer promise for improved treatment over the standard modalities of surgery, chemotherapy and radiotherapy. Whole-genome CRISPR/Cas9 knockout screens were conducted to identify gene dependencies in isogenic IDH1-mutant and wild-type (WT) U-87 MG cells. Cells were transduced with the Brunello single guide RNA (sgRNA) library, grown for 21 days, then sgRNA distributions were assessed by amplicon sequencing. Gene dependencies were identified using CRISPRcleanR and BAGELR pipelines. We discovered that glucose-6-phosphate dehydrogenase (G6PD), a pentose phosphate pathway (PPP) enzyme, was needed for viability of IDH1-mutant cells, but was dispensable for U-87 MG WT cells. The PPP is the main producer of cytosolic NADPH that is required for de novo lipogenesis, maintenance of antioxidants and production of (R)-2-HG by mutant IDH1. Previous reports demonstrated that IDH1-mutant cells increase PPP flux to support these processes. We have validated this dependency using G6PDi-1, a cell-active inhibitor of G6PD. Growth inhibition (IC50) assays demonstrated that IDH1-mutant U-87 MG cells were threefold more sensitive to G6PDi-1 with IC50 of 23 μM and enhanced cell death (increased propidium iodide uptake), compared with WT U-87 MG cells with IC50 of 67 μM. G6PD knockout was then conducted to further validate this dependency. Nucleofection of IDH1 WT U-87 MG cells with G6PD multi guide RNA-Cas9 ribonucleoproteins resulted in highly efficient knockout of G6PD at day 5 (99-100%), which was maintained at day 21 (96-98%). In contrast, efficient G6PD knockout in IDH1-mutant U-87 MG cells at day 5 (98-100%) was lost following 21 days culture (0-5%), indicating death of almost all edited cells within the culture. Finally, Kaplan-Meier analysis of IDH1-mutant patients in the TCGA lower grade glioma dataset demonstrated that higher G6PD expression correlated with worse median overall survival (7.3 versus 9.5 years; log-rank p=0.01), but was not prognostic in the IDH1 WT TCGA glioblastoma multiforme dataset. These findings demonstrate that a cell line model of IDH1-mutant glioma is highly dependent on G6PD for survival. This observation builds on the recognized importance of the PPP in the metabolic biology of IDH1-mutant malignancy by establishing G6PD as a targetable dependency in this disease. Citation Format: Kim M. Paras, Pooja Patel, Paul Choi, William R. Wilson, Tet-Woo Lee, Stephen M. Jamieson, Dean C. Singleton. Identification of glucose-6-phosphate dehydrogenase dependency in IDH1 mutant glioma cells using functional genomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4333.

Some pyrroles as inhibitors of the pentose phosphate pathways enzymes: An in vitro and molecular docking study.

Glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) are pentose phosphate pathway enzymes. Compounds with a heterocyclic pyrrole ring system containing this atom can be derivatized with various functional groups into highly effective bioactive agents. In this study, pyrrole derivatives on these enzyme's activity were investigated. The IC50 values of different concentrations of pyrrole derivatives for G6PD were found in the range of 0.022-0.221 mM Ki values 0.021 ± 0.003-0.177 ± 0.021 and for 6PGD IC50 values 0.020-0.147, mM Ki values 0.013 ± 0.002-0.113 ± 0.030 mM. The 2-acetyl-1-methylpyrrole (1g) showed the best inhibition value for G6PD and 6PGD enzymes. In addition, in silico molecular docking experiments were performed to elucidate how these pyrrole derivatives (1a-g) interact with the binding sites of the target enzymes. The study's findings on pyrrole derivatives could be used to create innovative therapeutics that could be a treatment for many diseases, especially cancer manifestations.

In vitro inhibition potency of malononitrile derivatives on the activity of two pentose phosphate pathway enzymes: accompanied by molecular docking evaluation.

Many disorders, including cancer and malaria, could be targeted via the pentose phosphate pathway (PPP),whose products are key in biosynthetic reactions incells. The goal of this study was to find new PPP inhibitors. The inhibition effects of malononitrile derivatives on Glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) were analyzed through invitro experiments. Besides, molecular docking studies were performed to predict the interactions having role in inhibition of compounds. K i constants of derivatives were found between 4.24±0.46-69.63±7.75 µM for G6PD and 1.91±0.12-95.07±11.08 µM for 6PGD. Derivatives indicated non-competitive inhibition on both enzymes except forcompound 4. The findings of the molecular docking studies revealed that free-binding energy estimations agreed with invitro data. The structure of these malononitrile derivatives may guide for drug discovery in targeting the PPP.

MiR-375 impairs breast cancer cell stemness by targeting the KLF5/G6PD signaling axis.

Recurrence of breast cancer may be due to the presence of breast cancer stem cells (BCSC). Abnormal tumor cell growth is closely associated with increased reactive oxygen species (ROS) and disruption of redox homeostasis, and BCSCs exhibit low levels of ROS. The detailed mechanism between the low levels of ROS in BCSCs and their maintenance of stemness characteristics has not been reported. A growing number of studies have shown that tumor development is often accompanied by metabolic reprogramming, which is an important hallmark of tumor cells. As the first rate-limiting enzyme of pentose phosphate pathway (PPP), the expression of G6PD is precisely regulated in tumor cells, and there is a certain correlation between PPP and BCSCs. MiR-375 has been shown to inhibit stem cell-like properties in breast cancer, but the exact mechanism is not clear. Here, KLF5, as a transcription factor, was identified to bind to the promoter of G6PD to promote its expression, whereas miR-375 inhibited the expression of KLF5 by binding to the 3'UTR region of KLF5 mRNA and thus reduced the expression of G6PD expression, inhibits PPP to reduce NADPH, and increases ROS levels in breast cancer cells, thereby weakening breast cancer cell stemness. Our study reveals the specific mechanism by which miR-375 targets the KLF5/G6PD signaling axis to diminish the stemness of breast cancer cells, providing a therapeutic strategy against BCSCs.

The Impact of G6PD Deficiency on Liver Function in Children Under 15

Glucose-6-phosphate dehydrogenase (G6PD) is involved in the first phase of the pentose phosphate pathway (PPP), entailing the reduction of nicotinamide adenine dinucleotide phosphate (NADP+) and the oxidation of glucose-6-phosphate to 6-phosphogluconolactone. A deficiency in the G6PD activity can result in numerous illnesses, including liver diseases. The first three enzymes in PPP are responsible for the oxidative phase. They make NADPH, which is needed to deal with oxidative stress, especially in people who do not have enough G6PD. The study included (90) samples from people with hemolytic anemia (Favzim) and control to determine the levels of G6PD, albumin, total protein, liver function, alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and bilirubin. The results showed that there was a significant decrease (P<0.05) in the serum levels of G6PD in the patients group when compared with the control group. Also, there was a significant increase (P<0.05) in the serum levels of albumin, total protein, ALP, ALT, AST and total bilirubin in patients with the G6PD deficiency when compared with the control group. In addition, this study revealed a strong positive correlation between some biochemical parameters studied, such as: Total protein with TSB (r2 = 0.897), TSB with ALT (r2 = 0.893), AST with ALT (r2 = 0.623) and total protein with ALT (r2 = 0.804).

Abstract WMP83: The Pentose Phosphate Pathway is Crucial for Combating Heme-Induced Ferroptosis in Red Blood Cells After ICH

Intracerebral hemorrhage (ICH) occurs upon blood vessel rupture, allowing blood into brain tissue. Red blood cells (RBCs) make up 99%+ of blood cells. Brain-trapped RBCs undergo phagocytosis; however, many RBCs lyse prior to being phagocytosed due to their abundance. RBC lysis releases hemoglobin and toxic heme, driving oxidative stress especially in lipid-rich plasma membranes (PM), causing tissue damage and ferroptosis. Few studies have explored RBC fate in ICH, but unraveling the factors governing RBC lysis may yield ICH therapies.In in vitro human and murine RBC cultures, we observed RBCs are exquisitely sensitive to heme-induced death with increased oxidized fatty acids (FAs), a ferroptosis marker (Fig 1A-B). The ferroptosis inducer Erastin validated RBC susceptibility to ferroptosis (Fig 1C). Heme-induced cell death was countered by β-mercaptoethanol (β-ME), N-acetylcysteine (NAC), ferrostatin-1 (Fer-1) and Vitamin E (Vit. E), ferroptosis inhibitors, confirming RBC death was due to ferroptosis (Fig 1D-G). Heme and Erastin induced RBC ferroptosis differs from eryptosis, RBC specific programmed cell death, as it isn't Ca++ dependent (Fig1H&I). Unlike other cells, RBCs solely rely on the Pentose Phosphate Pathway (PPP) for NADPH production for oxidation defense. PPP inhibition with Dehydroisoandrosterone (DHEA) and treatment with ThioNa, an NADP+ synthesis inhibitor, both heightened heme-induced RBC ferroptosis (Fig 1J-L). These data support the PPP's role against heme-driven ferroptosis. Heme induced RBC ferroptosis may trigger a chain reaction of RBC lysis and ongoing heme release. Notably, 400 million people face G6PD deficiency, the rate-limiting PPP enzyme, leading to NADPH depletion in RBCs with enhanced susceptibility to oxidation. Hence, in addition to identifying the PPP in RBCs resistance to heme toxicity, our findings could aid in identifying a population that is particularly vulnerable to the devastating effects of ICH.

Glucose 6-phosphate dehydrogenase variants increase NADPH pools for yeast isoprenoid production.

Isoprenoid biosynthesis has a significant requirement for the co-factor NADPH. Thus, increasing NADPH levels for enhancing isoprenoid yields in synthetic biology is critical. Previous efforts have focused on diverting flux into the pentose phosphate pathway or overproducing enzymes that generate NADPH. In this study, we instead focused on increasing the efficiency of enzymes that generate NADPH. We first established a robust genetic screen that allowed us to screen improved variants. The pentose phosphate pathway enzyme, glucose 6-phosphate dehydrogenase (G6PD), was chosen for further improvement. Different gene fusions of G6PD with the downstream enzyme in the pentose phosphate pathway, 6-phosphogluconolactonase (6PGL), were created. The linker-less G6PD-6PGL fusion displayed the highest activity, and although it had slightly lower activity than the WT enzyme, the affinity for G6P was higher and showed higher yields of the diterpenoid sclareol in vivo. A second gene fusion approach was to fuse G6PD to truncated HMG-CoA reductase, the rate-limiting step and also the major NADPH consumer in the pathway. Both domains were functional, and the fusion also yielded higher sclareol levels. We simultaneously carried out a rational mutagenesis approach with G6PD, which led to the identification of two mutants of G6PD, N403D and S238QI239F, that showed 15-25% higher activity in vitro. The diterpene sclareol yields were also increased in the strains overexpressing these mutants relative to WT G6PD, and these will be very beneficial in synthetic biology applications.

A feedback loop of PPP and PI3K/AKT signal pathway drives regorafenib-resistance in HCC

BackgroundHepatocellular carcinoma (HCC) is a principal type of liver cancer with high incidence and mortality rates. Regorafenib is a novel oral multikinase inhibitor for second-line therapy for advanced HCC. However, resistance to regorafenib is gradually becoming a dilemma for HCC and the mechanism remains unclear. In this study, we aimed to reveal the metabolic profiles of regorafenib-resistant cells and the key role and mechanism of the most relevant metabolic pathway in regorafenib resistance.MethodsMetabolomics was performed to detect the metabolic alteration between drug-sensitive and regorafenib-resistant cells. Colony formation assay, CCK-8 assay and flow cytometry were applied to observe cell colony formation, cell proliferation and apoptosis, respectively. The protein and mRNA levels were detected by western blot and RT-qPCR. Cell lines of Glucose-6-phosphate dehydrogenase(G6PD) knockdown in regorafenib-resistant cells or G6PD overexpression in HCC cell lines were stably established by lentivirus infection technique. G6PD activity, NADPH level, NADPH/NADP+ ratio, the ratio of ROS positive cells, GSH level, and GSH/GSSG ratio were detected to evaluate the anti-oxidative stress ability of cells. Phosphorylation levels of NADK were evaluated by immunoprecipitation.ResultsMetabonomics analysis revealed that pentose phosphate pathway (PPP) was the most relevant metabolic pathway in regorafenib resistance in HCC. Compared with drug-sensitive cells, G6PD enzyme activity, NADPH level and NADPH/NADP+ ratio were increased in regorafenib-resistant cells, but the ratio of ROS positive cells and the apoptosis rate under the conditions of oxidative stress were decreased. Furthermore, G6PD suppression using shRNA or an inhibitor, sensitized regorafenib-resistant cells to regorafenib. In contrast, G6PD overexpression blunted the effects of regorafenib to drug-sensitive cells. Mechanistically, G6PD, the rate-limiting enzyme of PPP, regulated the PI3K/AKT activation. Furthermore, PI3K/AKT inhibition decreased G6PD protein expression, G6PD enzymatic activity and the capacity of PPP to anti-oxidative stress possibly by inhibited the expression and phosphorylation of NADK.ConclusionTaken together, a feedback loop of PPP and PI3K/AKT signal pathway drives regorafenib-resistance in HCC and targeting the feedback loop could be a promising approach to overcome drug resistance.

Modulation of glucose metabolism through macrophage-membrane-coated metal-organic framework nanoparticles for triple-negative breast cancer therapy

The clinical presentation of triple-negative breast cancer signifies a pathophysiological trajectory characterized by an aggressive nature. To satisfy the rapid tumor cell proliferation, glycolysis emerges as the primary modality for energy production, consequently engendering the copious generation of lactic acid. This, in turn, fosters an acidic microenvironment within the tumor milieu, which facilitates angiogenesis, tumor metastasis, extracellular matrix remodeling, and immune evasion. If the glycolytic flux was attenuated, a heightened influx of glucose-6-phosphate (G6P) towards the pentose phosphate pathway (PPP) ensues, promoting the synthesis of nicotinamide adenine dinucleotide phosphate (NADPH) and reduced glutathione (GSH). This orchestrated metabolic cascade gives rise to reductive equivalents and biosynthetic substrates, emblematic of a salient hallmark in the landscape of cancer cell metabolism. To surmount the quandary presented by aberrant tumor metabolism, we developed macrophage-membrane-coated metal–organic framework nanoparticles (AP@ZIF-Mem) to deliver atorvastatin (an inhibitor of MCT4, a lactic acid transporter within glycolysis) and polydatin (an inhibitor of G6PD, the rate-limiting enzyme of PPP) to interfere the glucose metabolism for sensitizing triple-negative breast cancer treatment. After i.v. injection, AP@ZIF-Mem could be accumulated into the tumor site via macrophage-biomimetic behavior. Through orchestrated intervention, these nanoparticles could provoke the accumulation of lactic acid within tumor cells, thereby precipitating a pronounced intratumoral acidosis; In parallel, the abrogation of G6PD function occasioned a surge in intracellular reactive oxygen species (ROS), consequentially culminating in diminished ribulose-5-phosphate and NADPH production. This dual-pronged intervention not only disrupted the tumor's energy provisioning framework but also perturbed the aberrant redox state of the tumor milieu, thereby sensitizing tumor treatment and impeding the metastatic trajectory. This study provided a paradigm of treatment/adjuvant treatment via the manipulation of glucose metabolism for triple-negative breast cancer.

Activation of the pentose phosphate pathway in macrophages is crucial for granuloma formation in sarcoidosis.

Sarcoidosis is a disease of unknown etiology in which granulomas form throughout the body and is typically treated with glucocorticoids, but there are no approved steroid-sparing alternatives. Here, we investigated the mechanism of granuloma formation using single-cell RNA-Seq in sarcoidosis patients. We observed that the percentages of triggering receptor expressed on myeloid cells 2-positive (TREM2-positive) macrophages expressing angiotensin-converting enzyme (ACE) and lysozyme, diagnostic makers of sarcoidosis, were increased in cutaneous sarcoidosis granulomas. Macrophages in the sarcoidosis lesion were hypermetabolic, especially in the pentose phosphate pathway (PPP). Expression of the PPP enzymes, such as fructose-1,6-bisphosphatase 1 (FBP1), was elevated in both systemic granuloma lesions and serum of sarcoidosis patients. Granuloma formation was attenuated by the PPP inhibitors in in vitro giant cell and in vivo murine granuloma models. These results suggest that the PPP may be a promising target for developing therapeutics for sarcoidosis.

Human papillomavirus type 16 E6 promotes cervical cancer proliferation by upregulating transketolase enzymatic activity through the activation of protein kinase B.

Over 99% of precancerous cervical lesions are associated with human papillomavirus (HPV) infection, with HPV types 16 and 18 (especially type 16) found in over 70% of cervical cancer cases globally. E6, a critical HPV gene, triggers malignant proliferation by degrading p53; however, this mechanism alone cannot fully explain the oncogenic effects of HPV16 E6. Therefore, we aimed to investigate new targets of HPV oncogenic mechanisms. Our results revealed significant changes in nonoxidative pentose phosphate pathway (PPP) metabolites in HPV16-positive cells. However, the role of nonoxidative PPP in HPV-associated cell transformation and tumor development remained unexplored. In this study, we investigated the impact and mechanisms of HPV16 E6 on cervical cancer proliferation using the HPV-negative cervical cancer cell line (C33A). HPV16 E6 was found to promote cervical cancer cell proliferation both in vitro and in vivo, activating the nonoxidative PPP. Transketolase (TKT), a key enzyme in the nonoxidative PPP, is highly expressed in cervical cancer tissues and associated with poor prognosis. HPV16 E6 promotes cervical cancer cell proliferation by upregulating TKT activity through the activation of AKT. In addition, oxythiamine (OT), a TKT inhibitor, hindered tumor growth, with enhanced effects when combined with cisplatin (DDP). In conclusion, HPV16 E6 promotes cervical cancer proliferation by upregulating TKT activity through the activation of AKT. OT demonstrates the potential to inhibit HPV16-positive cervical cancer growth, and when combined with DDP, could further enhance the tumor-suppressive effect of DDP.

The effects of morin and methotrexate on pentose phosphate pathway enzymes and GR/GST/TrxR enzyme activities: An in vivo and in silico study.

In this study, the mechanisms by which the enzymes glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), glutathione reductase (GR), glutathione-S-transferase (GST), and thioredoxin reductase (TrxR) are inhibited by methotrexate (MTX) were investigated, as well as whether the antioxidant morin can mitigate or prevent these adverse effects in vivo and in silico. For 10 days, rats received oral doses of morin (50 and 100 mg/kg body weight). On the fifth day, a single intraperitoneal injection of MTX (20 mg/kg body weight) was administered to generate toxicity. Decreased activities of G6PD, 6PGD, GR, GST, and TrxR were associated with MTX-related toxicity while morin treatment increased the activity of the enzymes. The docking analysis indicated that H-bonds, pi-pi stacking, and pi-cation interactions were the dominant interactions in these enzyme-binding pockets. Furthermore, the docked poses of morin and MTX against GST were subjected to molecular dynamic simulations for 200 ns, to assess the stability of both complexes and also to predict key amino acid residues in the binding pockets throughout the simulation. The results of this study suggest that morin may be a viable means of alleviating the enzyme activities of important regulatory enzymes against MTX-induced toxicity.

PLK1 regulating chemoradiotherapy sensitivity of esophageal squamous cell carcinoma through pentose phosphate pathway/ferroptosis

BackgroundEsophageal squamous cell carcinoma (ESCC) is the most common pathological type of esophageal cancer in China, accounting for more than 90 %. Most patients were diagnosed with advanced-stage ESCC, for whom new adjuvant therapy is recommended. Therefore, it is urgent to explore new therapeutic targets for ESCC. Ferroptosis, a newly discovered iron-dependent programmed cell death, has been shown to play an important role in carcinogenesis by many studies. This study explored the effect of Polo like kinase 1 (PLK1) on chemoradiotherapy sensitivity of ESCC through ferroptosis MethodsIn this study, we knocked out the expression of PLK1 (PLK1-KO) in ESCC cell lines (KYSE150 and ECA109) with CRISPR/CAS9. The effects of PLK1-knock out on G6PD, the rate-limiting enzyme of pentose phosphate pathway (PPP), and downstream NADPH and GSH were explored. The lipid peroxidation was observed by flow cytometry, and the changes in mitochondria were observed by transmission electron microscopy. Next, through the CCK-8 assay and clone formation assay, the sensitivity to cobalt 60 rays, paclitaxel, and cisplatin were assessed after PLK1-knock out, and the nude mouse tumorigenesis experiment further verified it. The regulation of transcription factor YY1 on PLK1 was evaluated by dual luciferase reporter assay. The expression and correlation of PLK1 and YY1, and their impact on prognosis were analyzed in more than 300 ESCC cases from the GEO database and our center. Finally, the above results were further proved by single-cell sequencing. ResultsAfter PLK1 knockout, the expression of G6PD dimer and the level of NADPH and GSH in KYSE150 and ECA109 cells significantly decreased. Accordingly, lipid peroxidation increased, mitochondria became smaller, membrane density increased, and ferroptosis was more likely to occur. However, with the stimulation of exogenous GSH (10 mM), there was no significant difference in lipid peroxidation and ferroptosis between the PLK1-KO group and the control group. After ionizing radiation, the PLK1-KO group had higher lipid peroxidation ratio, more cell death, and was more sensitive to radiation, while exogenous GSH (10 mM) could eliminate this difference. Similar results could also be observed when receiving paclitaxel combined with cisplatin and chemoradiotherapy. The expression of PLK1, G6PD dimer, and the level of NADPH and GSH in KYSE150, ECA109, and 293 T cells stably transfected with YY1-shRNAs significantly decreased, and the cells were more sensitive to radiotherapy and chemotherapy. ESCC patients from the GEO database and our center, YY1 and PLK1 expression were significantly positively-correlated, and the survival of patients with high expression of PLK1 was significantly shorter. Further analysis of single-cell sequencing specimens of ESCC in our center confirmed the above results. ConclusionIn ESCC, down-regulation of PLK1 can inhibit PPP, and reduce the level of NADPH and GSH, thereby promoting ferroptosis and improving their sensitivity to radiotherapy and chemotherapy. Transcription factor YY1 has a positive regulatory effect on PLK1, and their expressions were positively correlated. PLK1 may be a target for predicting and enhancing the chemoradiotherapy sensitivity of ESCC.

Insulin-induced gene 2 protects against hepatic ischemia–reperfusion injury via metabolic remodeling

BackgroundHepatic ischemia–reperfusion (IR) injury is the primary reason for complications following hepatectomy and liver transplantation (LT). Insulin-induced gene 2 (Insig2) is one of several proteins that anchor the reticulum in the cytoplasm and is essential for metabolism and inflammatory responses. However, its function in IR injury remains ambiguous.MethodsInsig2 global knock-out (KO) mice and mice with adeno-associated-virus8 (AAV8)-delivered Insig2 hepatocyte-specific overexpression were subjected to a 70% hepatic IR model. Liver injury was assessed by monitoring hepatic histology, inflammatory responses, and apoptosis. Hypoxia/reoxygenation stimulation (H/R) of primary hepatocytes and hypoxia model induced by cobalt chloride (CoCl2) were used for in vitro experiments. Multi-omics analysis of transcriptomics, proteomics, and metabolomics was used to investigate the molecular mechanisms underlying Insig2.ResultsHepatic Insig2 expression was significantly reduced in clinical samples undergoing LT and the mouse IR model. Our findings showed that Insig2 depletion significantly aggravated IR-induced hepatic inflammation, cell death and injury, whereas Insig2 overexpression caused the opposite phenotypes. The results of in vitro H/R experiments were consistent with those in vivo. Mechanistically, multi-omics analysis revealed that Insig2 is associated with increased antioxidant pentose phosphate pathway (PPP) activity. The inhibition of glucose-6-phosphate-dehydrogenase (G6PD), a rate-limiting enzyme of PPP, rescued the protective effect of Insig2 overexpression, exacerbating liver injury. Finally, our findings indicated that mouse IR injury could be attenuated by developing a nanoparticle delivery system that enables liver-targeted delivery of substrate of PPP (glucose 6-phosphate).ConclusionsInsig2 has a protective function in liver IR by upregulating the PPP activity and remodeling glucose metabolism. The supplementary glucose 6-phosphate (G6P) salt may serve as a viable therapeutic target for alleviating hepatic IR.

A fingerprint of 2-[18F]FDG radiometabolites – How tissue-specific metabolism beyond 2-[18F]FDG-6-P could affect tracer accumulation

Studies indicate that the radiotracer 2-[18F]fluoro-2-deoxy-D-glucose (2-[18F]FDG) can be metabolized beyond 2-[18F]FDG-6-phosphate (2-[18F]FDG-6-P), but its metabolism is incompletely understood. Most importantly, it remains unclear whether downstream metabolism affects tracer accumulation invivo. Here we present a fingerprint of 2-[18F]FDG radiometabolites over time in cancer cells, corresponding tumor xenografts and murine organs. Strikingly, radiometabolites representing glycogen metabolism or the oxPPP correlated inversely with tracer accumulation across all examined tissues. Recent studies suggest that not only hexokinase, but also hexose-6-phosphate dehydrogenase (H6PD), an enzyme of the oxidative pentose phosphate pathway (oxPPP), determines 2-[18F]FDG accumulation. However, little is known about the corresponding enzyme glucose-6-phosphate dehydrogenase (G6PD). Our mechanistic invitro experiments on the role of the oxPPP propose that 2-[18F]FDG can be metabolized via both G6PD and H6PD, but data from separate enzyme knockdown suggest diverging roles in downstream tracer metabolism. Overall, we propose that tissue-specific metabolism beyond 2-[18F]FDG-6-P could matter for imaging.

Effects of Arbutin on Potassium Bromate-Induced Erythrocyte Toxicity in Rats: Biochemical Evaluation of Some Metabolic Enzyme Activities In Vivo and In Vitro.

In the present study, the inhibitory effect of potassium bromate on the pentose phosphate pathway and intracellular antioxidant systems enzymes (glucose 6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), glutathione reductase (GR), glutathione S-transferase (GST), and thioredoxin reductase (TrxR)) and the role of arbutin in ameliorating this inhibition were investigated. In the in vivo phase of the study, Wistar Albino rats (28 male adults) were randomly divided into four groups. Control (n = 7): isotonic serum (0.5 mL, i.p), potassium bromate group (n = 7): potassium bromate (100 mg/kg), arbutin group (n = 7): arbutin (i.p.) (50 mg/kg/day), potassium bromate + arbutin, and Group (n = 7): potassium bromate (100 mg/kg) + arbutin (50 mg/kg/day) (i.p). The results of in vivo study showed that the activities of G6PD, 6PGD, GR, and TrxR enzymes were strongly inhibited in potassium bromate groups (p < 0.05). It was determined that GST enzyme activity decreased in the potassium bromate group, but this decrease was not statistically significant compared to the control group (p ⩾ 0.05). A statistically significant increase was found in G6PD, 6PGD, GST, and TrxR enzyme activities in the arbutin group compared to the control group (p < 0.05). The increase in GR enzyme activity was not statistically significant (p ⩾ 0.05). The potassium bromate + arbutin group's enzyme activity increased in comparison to the potassium bromate group and was discovered to be closer to the control group. It was found that potassium bromate inhibited the 6PGD enzyme obtained from rat erythrocyte tissues with IC50 = 346 μM value and Ki = 434.4 μM ± 6.1 value, and the inhibition was noncompetitive.

Targeting Heat Shock Protein 27 and Fatty Acid Oxidation Augments Cisplatin Treatment in Cisplatin-Resistant Ovarian Cancer Cell Lines.

Most ovarian cancer patients develop recurrent cancers which are often resistant to commonly employed chemotherapy agents, such as cisplatin. We have previously shown that the inhibition of heat shock protein 27 (HSP27) or fatty acid oxidation (FAO) sensitizes cisplatin-resistant ovarian cancer cell lines to cisplatin and dual inhibition of both HSP27 and FAO induces substantial cell death in vitro. However, it is unclear how HSP27 and FAO promote cisplatin resistance, and if dual inhibition of both HSP27 and FAO would augment cisplatin treatment in vivo. Here we showed that HSP27 knockdown in two cisplatin-resistant ovarian cancer cell lines (A2780CIS and PEO4) resulted in more ROS production upon cisplatin treatment. HSP27-knockdown cancer cells exhibited decreased levels of reduced glutathione (GSH) and glucose6phosphate dehydrogenase (G6PD), a crucial pentose phosphate pathway enzyme. ROS depletion with the compound N-acetyl cysteine (NAC) attenuated cisplatin-induced upregulation of HSP27, FAO, and markers of apoptosis and ferroptosis in cisplatin-resistant ovarian cancer cell lines. Finally, inhibition of HSP27 and FAO with ivermectin and perhexiline enhanced the cytotoxic effect of cisplatin in A2780CIS xenograft tumors in vivo. Our results suggest that two different cisplatin-resistant ovarian cancer cell lines upregulate HSP27 and FAO to deplete cisplatin-induced ROS to attenuate cisplatin's cytotoxic effect.

Peroxyl radicals modify 6-phosphogluconolactonase from Escherichia coli via oxidation of specific amino acids and aggregation which inhibits enzyme activity.

6-phosphogluconolactonase (6PGL) catalyzes the second reaction of the pentose phosphate pathway (PPP) converting 6-phosphogluconolactone to 6-phosphogluconate. The PPP is critical to the generation of NADPH and metabolic intermediates, but some of its components are susceptible to oxidative inactivation. Previous studies have characterized damage to the first (glucose-6-phosphate dehydrogenase) and third (6-phosphogluconate dehydrogenase) enzymes of the pathway, but no data are available for 6PGL. This knowledge gap is addressed here. Oxidation of Escherichia coli 6PGL by peroxyl radicals (ROO•, from AAPH (2,2'-azobis(2-methylpropionamidine) dihydrochloride) was examined using SDS-PAGE, amino acid consumption, liquid chromatography with mass detection (LC-MS), protein carbonyl formation and computational methods. NADPH generation was assessed using mixtures all three enzymes of the oxidative phase of the PPP. Incubation of 6PGL with 10 or 100mM AAPH resulted in protein aggregation mostly due to reducible (disulfide) bonds. High fluxes of ROO• induced consumption of Cys, Met and Trp, with the Cys oxidation rationalizing the aggregate formation. Low levels of carbonyls were detected, while LC-MS analyses provided evidence for oxidation of selected Trp and Met residues (Met1, Trp18, Met41, Trp203, Met220 and Met221). ROO• elicited little loss of enzymatic activity of monomeric 6PGL, but the aggregates showed diminished NADPH generation. This is consistent with in silico analyses that indicate that the modified Trp and Met are far from the 6-phosphogluconolactone binding site and the catalytic dyad (His130 and Arg179). Together these data indicate that monomeric 6PGL is a robust enzyme towards oxidative inactivation by ROO• and when compared to other PPP enzymes.

Hypoxia-induced reprogramming of glucose-dependent metabolic pathways maintains the stemness of human bone marrow-derived endothelial progenitor cells

The benefits of hypoxia for maintaining the stemness of cultured human bone marrow-derived endothelial progenitor cells (BM EPCs) have previously been demonstrated but the mechanisms responsible remain unclear. Growing evidences suggest that cellular metabolism plays an important role in regulating stem cell fate and self-renewal. Here we aimed to detect the changes of glucose metabolism and to explore its role on maintaining the stemness of BM EPCs under hypoxia. We identified the metabolic status of BM EPCs by using extracellular flux analysis, LC–MS/MS, and 13C tracing HPLC-QE-MS, and found that hypoxia induced glucose metabolic reprogramming, which manifested as increased glycolysis and pentose phosphate pathway (PPP), decreased tricarboxylic acid (TCA) and mitochondrial respiration. We further pharmacologically altered the metabolic status of cells by employing various of inhibitors of key enzymes of glycolysis, PPP, TCA cycle and mitochondria electron transport chain (ETC). We found that inhibiting glycolysis or PPP impaired cell proliferation either under normoxia or hypoxia. On the contrary, inhibiting pyruvate oxidation, TCA or ETC promoted cell proliferation under normoxia mimicking hypoxic conditions. Moreover, promoting pyruvate oxidation reverses the maintenance effect of hypoxia on cell stemness. Taken together, our data suggest that hypoxia induced glucose metabolic reprogramming maintains the stemness of BM EPCs, and artificial manipulation of cell metabolism can be an effective way for regulating the stemness of BM EPCs, thereby improving the efficiency of cell expansion in vitro.

Incidence of G6pd Deficiency in Young Asymptomatic Males in Pakistan

Background: NADPH is crucial for glutathione reduction. Deficiecy of G6PD leads to excessive oxidised glutathione that denatures hemoglobin and forms Heinz bodies. Heinz bodies damages RBC membrane and cause premature removal of cells by spleen. G6PD deficiency is the most common X-linked inherited red cell enzymopathy that leads to hemolytic anemia and neonatal jaundice in enzyme deficient patients under oxidative stress. G6PD is the rate limiting enzyme in pentose phosphate pathway, a dominant form of glycolysis in RBCs. G6PD converts glucose-6 phosphate into 6-phosphonogluconolactone and reduces NADP to NADPH. Objective: To determine incidence of G6PD deficiency in young healthy males in Pakistan. Study design: A descriptive cross sectional study Place and duration: Study was carried out at PAF Hospital Sargodha from Jan 2021 to Feb 2023 Methodology: G6PD levels were checked in young healthy male volunteers of 12 to 18 years of age, after taking informed consent from subjects and their parents. Results: Out of 1493 volunteers, only 61 were G6PD deficient and rest all had normal levels. This shows an incidence of 4.08% in our population. Conclusion: Comparing with previous studies suggests 2-4% incidence of G6PD deficiency in Pakistani population. A larger study including females and studying variants of disease is needed. Patients with lab confirmed P.Vivax should get screened for G6PD deficiency, so that they receive Primaquine. Keywords: Incidence, G6PD deficiency, Malaria, Primaquine.