Kobunshi Ronbunshu

Glutamine is well recognized as critical to the growth of most cell types. Within mitochondria glutamine is converted to glutamate by glutaminase. Oxaloacetate and glutamate then react to form alpha-ketoglutarate (α-KG) and aspartate catalyzed by glutamic-oxaloacetic transaminase (GOT2) or directly converted to α-KG by glutamate dehydrogenase (GDH). We investigated the role of GOT2 in mediating glutamate metabolism and cell growth in undifferentiated C2C12 cells. CRISPR mediated GOT2 knockout (KO) impaired cell growth, partially overcome by higher concentrations of glutamine. Mitochondrial respiration did not differ between KO and wildtype (WT) cells. Metabolite profiling showed that GOT2KO decreased aspartate by about 50% in KO versus WT cells. In contrast, α-KG increased. Metabolites reflecting the pentose phosphate pathway were significantly increased in KO cells. Metabolic pathway analyses revealed alteration of the TCA cycle, the pentose phosphate pathway, and amino acid metabolism. Glutamine 13C-tracing revealed decreased generation of aspartate, increased ribulose phosphate and evidence for reductive carboxylation of α-KG to isocitrate in KO cells. GDH expression was detected in C2C12 cells but did not differ between WT and GOT2KO mitochondria. GDH is not or barely expressed in adult muscle, however, we observed clear expression in pre-weanling mice. Cytosolic glutamic-oxaloacetic transaminase, GOT1, expression did not differ between GOT2KO and WT cells. In summary, GOT2 is necessary for glutamate flux and generation of downstream metabolites needed for the growth of C2C12 myoblasts. Although respiration did not differ, lack of aspartate and other compounds needed for cell proliferation may have been major factors impairing growth.

Abstract Lithium is used in the long-term treatment of bipolar disorder, exhibiting a beneficial effect on the neuronal cells. The concentration of lithium in the blood serum can vary and can easily approach a level that is related to cardiotoxic adverse effects. This is due to its narrow therapeutic index. In this study, we investigated the effect of higher than therapeutic dose of lithium. Rat cardiomyoblast cells were treated with 2 mM LiCl for 48 h, after which the mitochondrial parameters of the cells were analyzed. Lithium exposure reduced maximal respiratory capacity by diminishing reserve respiratory capacity (RRC), linked to a decrease in complex I (NADH dehydrogenase) activity and elevated superoxide radical levels. In addition, lithium treatment altered the composition of cellular membranes, including mitochondrial cardiolipin, a lipid essential for mitochondrial function. These findings suggest that impaired complex I activity, oxidative stress, and cardiolipin depletion collectively impair the ability of cells to meet high energy demands.

Fibrillation of the amyloid beta (Aβ) peptide has often been associated with neurodegenerative pathologies such as Alzheimer’s disease. In this study we examined the influence of several potential compositions of the lipid membrane on Aβ fibrillation by using liposomes as a basic model membrane. Firstly, it was revealed that Aβ fibrillation kinetics were enhanced and had the potential to occur at a faster rate on more fluid membranes compared to solid membranes. Next, the extent of fibril-related damage to membranes was examined with analysis of membrane polarity via the steady-state emission spectra of 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan). It was revealed that there was slight hydration behavior of the membrane during the lag phase (tlag) of the kinetic process, possibly coinciding with Aβ monomer binding. However, as the fibrillation kinetic process continued the membrane gradually dehydrated. Hydration states of membranes during and after Aβ fibrillation processes were further examined via deconvolution analysis of the obtained Laurdan spectra. This allows a mapping of membrane hydration from the interior to exterior regions of the lipid membrane. Results revealed slight but definitive variations in deeper region membrane polarity during the time course of Aβ fibrillation, suggesting Aβ aggregation impacts not only the surface level aggregating region but also the inner regions of the membrane. These results can ultimately contribute to the future investigations of the nature of the membrane damage caused by Aβ aggregation.

To investigate the role of silent information regulator 6 (SIRT6) in regulating podocyte injury in diabetic nephropathy (DN) through autophagy mediated by Notch signaling pathway. A blank control group (group A), a diabetic nephropathy group (group B), and a Sirt6 intervention group (group C) were established. The group A cells were human normal glomerular podocyte cell lines (HGPCs) without any treatment. In group B, the cells were cultivated in glucose medium containing 30 mmol/L and a 10 µmol/L anti-LSirt6 antibody solution. Three sets of cells were tested for their capacity to proliferate via CCK8, for protein expression via Western blot, for associated mRNA expression levels via qPCR, and for cell migration and invasion ability via Transwell. The podocyte proliferation and migration activity in group B were reduced compared to group A, while these properties in group C were elevated compared to group B (DN). B Group is diabetes nephropathy. Compared with those in group B, the number of invading podocytes in group C were greater than those in group A, and the overall apoptosis rate in group C was lower than that in group B. The expression levels of apoptotic proteins in the podocytes in group C were greater than those in group B, and the bcl-2 level was lower than those in group B. The Notch1 and Jagged1 mRNA and protein levels in the podocytes in group B were greater than those in group A, whereas those in the podocytes in group C were lower than those in group B. Sirt6 can protect against podocyte autophagy injury in DN by regulating the Notch1 signaling pathway.

Cholestasis caused by impaired bile secretion in the liver is associated with the accumulation of primary bile acids (BA): cholic acid (CA) and chenodeoxycholic acid (CDCA) in the cells of this organ. The paper studies the uncoupling effect of the CA and CDCA on the succinate-fueled rat liver mitochondria under conditions of ΔpH to Δψ conversion by nigericin. It has been established that without nigericin, the dependence of the resting-state (state 4) respiration rate on the concentrations of these BA is nonlinear and is described by a parabolic equation. Under these conditions, the specific inhibitor of the ADP/ATP-antiporter – carboxyatractylate and the substrate of the aspartate/glutamate-antiporter – glutamate do not affect the state 4 respiration of mitochondria stimulated by these BA. It is suggested that without nigericin, the protonophore action of BA is due to the formation of a dimeric complex of their anion with the acid. In the presence of nigericin, the dependence of state 4 respiration rate on BA concentration is linear. Under these conditions, carboxyatractylate inhibits BA-stimulated respiration. Unlike the CDCA, the uncoupling action of CA is also suppressed by the substrates of the aspartate/glutamate-antiporter. The obtained results are considered as evidence that in the presence of nigericin, uncoupling action of CDCA is carried out primarily with the participation of ADP/ATP-antiporter. Both ADP/ATP-antiporter and aspartate/glutamate-antiporter are involved in the uncoupling action of CA. It is concluded that nigericin modifies the mechanism of the uncoupling action of BA in liver mitochondria by converting ΔpH to Δψ.

The main therapeutic strategy for the treatment of patients with toxic liver failure is the elimination of the toxic agent in combination with the targeted mitigation of pathological processes that have been initiated due to the toxicant. In the current research we evaluated the strategy of metabolic supplementation to improve mitochondrial bioenergetics during acute liver intoxication. In our study, we have shown that acute CCl4-induced intoxication negatively affects Complex I (in the presence of glutamate-malate as energy substrates) based respiration, generation of mitochondrial membrane potential (ΔΨm), mitochondrial NAD(P)H pool and NADH redox index, mitochondrial calcium retention capacity (CRC) and structure and functions of the liver. Boosting of mitochondrial bioenergetics through the complex II, using succinate as metabolic substrate in vitro, significantly improved mitochondrial respiration and generation of ΔΨm, but not mitochondrial CRC. Co-application of rotenone along with succinate, to prevent possible reverse electron flow, didn’t show significant differences compared to the effects of succinate alone. Treatment of animals with acute liver failure, using a metabolic supplement containing succinate, inosine, methionine and nicotinamide improved Complex I based respiration, generation of ΔΨm, mitochondrial NAD(P)H pool and NADH redox index, mitochondrial CRC and slightly decreased the level of oxidative stress. These changes resulted in averting destructive and dystrophic changes in the structure of rat liver tissue caused by CCl4 intoxication, concomitantly enhancing hepatic functionality. Thus, we propose that metabolic supplementation targeting complex II could serve as a potential adjunctive therapy in the management of acute liver intoxication.

To explore the regulatory mechanism of lncRNA UCA1 and NRF2 in cardiomyocyte aging. In this study, we explored how lncRNA UCA1 regulates NRF2 and its effect on cardiomyocyte aging. H9c2 cardiomyocytes were cultured and treated with H2O2 to simulate cardiomyocyte aging in vitro. The expression levels of lncRNA UCA1 and NRF2 in cells were detected using qRT-PCR. Cell viability was assessed using the CCK8 assay, and cell aging was detected via Sa-β-gal staining. The levels of oxidative stress markers (SOD, MDA, ROS) and the expressions of ferroptosis-related proteins (ACSL4, TFR1, FTH1, GPX4) were measured. The regulatory mechanism between UCA1 and NRF2 was investigated using RIP-qPCR. Additionally, changes in m6A modification levels and the expression of m6A modification-related proteins in cells after UCA1 overexpression were analyzed by western blot. Our results indicate that H2O2 treatment significantly downregulated the expression of lncRNA UCA1 and NRF2. UCA1 overexpression promoted H9c2 cell proliferation, inhibited cell aging, increased SOD activity and the expression of FTH1 and GPX4 proteins, and decreased MDA and ROS content as well as ACSL4 and TFR1 protein expression. RIP-qPCR verified that UCA1 can promote the expression of NRF2 in cells. Overexpression of UCA1 significantly increased the expression of the demethylase FTO, leading to a reduction in m6A modification levels. Furthermore, there was significant enrichment between FTO and NRF2, and overexpression of FTO improved the expression of NRF2 protein in cells. Taken together, lncRNA UCA1 inhibits oxidative stress and ferroptosis, thereby preventing cardiomyocyte aging. This protective effect is likely mediated by increasing the expression of demethylase FTO and reducing m6A modification, which promotes the expression of NRF2.

Oxidative stress-induced lens epithelial cells (LECs) death plays a pivotal role in pathogenesis of age-related cataract (ARC), causing significant visual impairment. Apoptosis of porcine granulosa cells mediated by MMP2 is linked to DNA damage. The current study aimed to investigate the potential mechanism of MMP2 in DNA damage, apoptosis and senescence of lens epithelial cells caused by oxidative stress. HLE-B3 cells were treated with different doses of H2O2 for 24 h, and CCK-8 was used to detect cell viability. Furthermore, western blotting was used to detect the expressions of MMP2, Bcl2, Bax, cleaved caspase3, γ-H2AX, p16, p21, and TIMP2. DCFH-DA staining was used to assess ROS levels. Moreover, EdU staining was used to detect cell proliferation, and flow cytometry was used to detect cell apoptosis. Then, 15A3 immunofluorescence staining and γ-H2AX staining were used to detect DNA damage. In addition, SA-β-gal staining was used to observe cell senescence. The present findings suggest that oxidative stress triggers damage to LECs viability and elevates the expression of MMP2. Furthermore, MMP2 interference attenuates H2O2-induced active damage, apoptosis, DNA damage, and cellular senescence in LECs. Additionally, TIMP2 expression is down-regulated in H2O2-induced LECs, which suppresses the expression of MMP2 induced by H2O2. These findings highlight the crucial role of MMP2 and TIMP2 in the modulation of oxidative stress-induced cellular responses in LECs. Collectively, TIMP2 alleviates H2O2-induced lens epithelial cell viability damage, apoptosis, DNA damage and cell senescence in LECs by inhibiting MMP2.

Influenza A (H1N1) virus is an acute respiratory infection responsible for enormous morbidity and mortality worldwide. The tripartite motif-containing protein 46 (TRIM46) has an antiviral function that inhibits various viral infections. This study is designed to explore the role and mechanism of TRIM46 in the progress of H1N1 infection. Herein, we infected A549 or 16HBE cells with the H1N1 virus at different times to assess TRIM46 and solute carrier family 7 member 11 (SLC7A11) expression. TRIM46 and Influenza A nucleoprotein mRNA levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). TRIM46, solute carrier family 7 member 11 (SLC7A11), and Nucleoprotein protein levels were detected using protein level were detected by western blot assay. Cell virulence was determined using Virulence assay (TCID50) assay. Cell viability was determined using Cell Counting Kit-8 (CCK-8) assay. Reactive oxygen species (ROS), intracellular iron content, Malondialdehyde (MDA), and Glutathione (GSH) levels were determined using special assay kits. The stability of SLC7A11 was assessed by Cycloheximide (CHX) assay. Interaction between TRIM46 and SLC7A11 was verified using Co-immunoprecipitation (CoIP) assay. The biological role of TRIM46 was assessed in H1N1 virus-challenged lung injury mice in vivo. TRIM46 level was significantly increased during H1N1 virus infection, and SLC7A11 expression was decreased. TRIM46 downregulation could suppress H1N1 virus replication and relieve H1N1 infection-induced ferroptosis and inflammation in A549 or 16HBE cells. Mechanistically, TRIM46 could promote SLC7A11 ubiquitination and decrease its stability. TRIM46 knockdown repressed H1N1 virus-induced lung injury in vivo. TRIM46 could contribute to influenza A H1N1 virus infection by promoting SLC7A11 ubiquitination in A549 cells, which indicates that targeting TRIM46 may improve the prognosis of patients.

AbstractBacillus licheniformis can use cyanide as a nitrogen source for its growth. However, it can also carry out aerobic respiration in the presence of this compound, a classic inhibitor of mammalian cytochrome c oxidase, indicating that B. licheniformis has a branched respiratory chain with various terminal oxidases. Here, we studied the modifications in the respiratory chain of B. licheniformis when cells were cultured in Nutrient Broth, an alkaline medium with ammonium, or an alkaline medium with cyanide. Then, we measured oxygen consumption in intact cells and membranes, enzyme activities, carried out 1D and 2D-BN-PAGE, followed by mass spectrometry analysis of BN-PAGE bands associated with NADH, NADPH, and succinate dehydrogenase activities. We found that cell growth was favored in a nutrient medium than in an alkaline medium with cyanide. In parallel, respiratory activity progressively decreased in cells cultured in the rich medium, alkaline medium with ammonium, and the lowest activity was in the cells growing in the alkaline medium with cyanide. B. licheniformis membranes contain NADH, NADPH, and succinate dehydrogenases, and the proteomic analysis detected the nitrate reductase and the bc, caa3, aa3, and bd complexes. The succinate dehydrogenase migrated with a molecular mass of 375 kDa, indicating its association with the nitrate reductase (115 kDa + 241 kDa, respectively). The NADH dehydrogenase of B. licheniformis forms aggregates of different molecular mass.

N6-methyladenosine (m6A) modification is, a more common epigenetic modification, mainly found in mRNA. More and more researches have shown the important functions of m6A on human cancers. This study seeks to explore the role of hnRNPA2B1 and m6A-dependent mechanism in cervical cancer. Elevated hnRNPA2B1 indicated the poor prognosis of cervical cancer patients. Enforced hnRNPA2B1 reduced the apoptosis, and accelerated the proliferation and migration of cervical cancer cells in vitro. Besides, hnRNPA2B1 promoted the aerobic glycolysis of cervical cancer cells, including the lactate secretion, glucose uptake, ATP production, extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). LDHA was found as the downstream target of hnRNPA2B1 by m6A site. Moreover, hnRNPA2B1 enhanced the mRNA stability of LDHA through m6A-dependent manner. LDHA inhibitor (FX-11) could reverse the effect of hnRNPA2B1. Taken together, the data revealed that hnRNPA2B1 promoted the proliferation, migration and aerobic glycolysis of cervical cancer cells by m6A/LDHA-dependent manner. These findings might bring a new idea for cervical cancer treatment.

High IQ motif-containing GTPase activating protein 3 (IQGAP3) expression is considered to be associated with poor prognosis of colorectal cancer (CRC). However, its role in early-onset CRC (EOCRC) progress is unclear. The mRNA and protein levels of IQGAP3 and Parkin (PRKN) were examined by qRT-PCR and western blot. Cell proliferation, apoptosis and metastasis were determined by CCK8 assay, EdU assay, flow cytometry and transwell assay. ROS, MDA, GSH, Fe2+, ACSL4 and SLC7A11 levels were detected to assess cell ferroptosis. The interaction between PRKN and IQGAP3 was assessed by Co-IP assay and ubiquitination assay. Xenograft tumor models were constructed to explore the effect of PRKN and IQGAP3 on the tumorigenesis in vivo. IQGAP3 was upregulated, while PRKN was downregulated in EOCRC tissues and cells. IQGAP3 knockdown inhibited CRC cell proliferation, migration and invasion, while enhanced apoptosis and ferroptosis. PRKN ubiquitinated IQGAP3 to promote its degradation. PRKN overexpression suppressed CRC cell growth, metastasis and promoted ferroptosis, while these effects were reversed by upregulating IQGAP3. In animal study, upregulation of PRKN reduced CRC tumorigenesis by decreasing IQGAP3 expression in vivo. IQGAP3, ubiquitinated by PRKN, promoted EOCRC progression by enhancing cell proliferation, metastasis, repressing apoptosis and ferroptosis, which provided a novel target for EOCRC treatment.