Cellular Oxygen Consumption Rate Research Articles

Cellular oxygen consumption in patients with diabetic ketoacidosis

BackgroundDiabetic ketoacidosis (DKA) is a potentially life-threatening disorder associated with severe alterations in metabolism and acid–base status. Mitochondrial dysfunction is associated with diabetes and its complications. Thiamine and coenzyme Q10 (CoQ10) are important factors in aerobic metabolism. In this study, we measured cellular oxygen consumption rates (OCRs) and the effects of in vitro administration of thiamine and CoQ10 on OCRs in patients with DKA versus healthy controls.MethodsBlood samples were collected from a prospective cohort of patients with DKA and from controls. Cellular OCRs were measured in peripheral blood mononuclear cells (PBMC) without treatment and after treatment with thiamine, CoQ10, or both. The mitochondrial profile was measured using an XFe96 Extracellular Flux Analyzer and XF Cell Mito Stress Test Kit (Seahorse Bioscience). A linear quantile mixed model was used to compare OCRs and estimate treatment effects.ResultsA total of 62 patients with DKA and 48 controls were included in the study. The median basal and maximal OCRs were lower in the DKA group than in the control group (basal: 4.7 [IQR: 3.3, 7.9] vs. 7.9 [5.0, 9.5], p = 0.036; maximal: 16.4 [9.5, 28.1] vs. 31.5 [20.6, 46.0] pmol/min/µg protein, p < 0.001). In DKA samples, basal and maximal OCRs were significantly increased when treated with thiamine, CoQ10, or both. In controls, basal and maximal OCR were significantly increased only with thiamine treatment.ConclusionMitochondrial metabolic profiles of patients with DKA demonstrated lower cellular oxygen consumption when compared to healthy controls. Oxygen consumption increased significantly in cells of patients with DKA treated with thiamine or CoQ10. These results suggest that thiamine and CoQ10 could potentially have therapeutic benefits in DKA via their metabolic effects on mitochondrial cellular respiration.

PRKAA2-mediated mitophagy regulates oxygen consumption in yak renal tubular epithelial cells under chronic hypoxia

Hypoxic environments are significant factors in the induction of various kidney diseases and are closely associated with high oxygen consumption in the kidneys. Yaks live at high altitude for a long time, exhibit a unique ability to regulate kidney oxygen consumption, protecting them from hypoxia-induced damage. However, the mechanisms underlying the regulation of oxygen consumption in yak kidneys under hypoxic conditions remain unclear. To explore this hypoxia adaptation mechanism in yak kidneys, this study analyzed the oxygen consumption rate (OCR) of renal tubular epithelial cells (RTECs) under hypoxia. We found that the OCR and apoptosis rates of RTECs under chronic hypoxia (> 24 h) were lower than those under acute hypoxia (≤ 24 h). However, when oxygen consumption was promoted under chronic hypoxia, the apoptosis rate increased, indicating that reducing the cellular OCR is crucial for maintaining RTECs activity under hypoxia. High-throughput sequencing results showed that the mitophagy pathway is likely a key mechanism for inhibiting OCR of yak RTECs, with protein kinase AMP-activated catalytic subunit alpha 2 (PRKAA2) playing a significant role in this process. Further studies demonstrated that chronic hypoxia activates the mitophagy pathway, which inhibits oxidative phosphorylation (OXPHOS) while increasing glycolytic flux in yak RTECs. Conversely, when the mitophagy pathway was inhibited, there was an increase in the activity of OXPHOS enzymes and OCR. To further explore the role of PRKAA2 in the mitophagy pathway, we inhibited PRKAA2 expression under chronic hypoxia. Results showed that the downregulation of PRKAA2 decreased the expression of mitophagy-related proteins, such as p-FUNDC1/FUNDC1, LC3-II/LC3-I, BNIP3 and ULK1 while upregulating P62 expression. Additionally, there was an increase in the enzyme activities of Complex II, Complex IV, PDH, and SDH, which further promoted oxygen consumption in RTECs. These findings suggest that PRKAA2 mediated mitophagy under chronic hypoxia is crucial mechanism for reducing oxygen consumption in yak RTECs.

Inducing Immunogenic Cancer Cell Death through Oxygen‐Economized Photodynamic Therapy with Nitric Oxide‐Releasing Photosensitizers

AbstractPhotodynamic therapy (PDT) utilizes reactive oxygen species (ROS) for eradication of cancer cells. Its effectiveness is governed by the oxygen content, which is scarce in the hypoxic tumor microenvironment. We report herein two zinc(II) phthalocyanines substituted with two or four nitric oxide (NO)‐releasing moieties, namely ZnPc‐2NO and ZnPc‐4NO, which can suppress the mitochondrial respiration, thereby sparing more intracellular oxygen for PDT. Using HT29 human colorectal adenocarcinoma cells and A549 human lung carcinoma cells, we have demonstrated that both conjugates release NO upon interaction with the intracellular glutathione, which can reduce the cellular oxygen consumption rate and adenosine triphosphate generation and alter the mitochondrial membrane potential. They can also relieve the hypoxic status of cancer cells and decrease the expression of hypoxia‐inducible factor protein HIF‐1α. Upon light irradiation, both conjugates can generate ROS and induce cytotoxicity even under a hypoxic condition, overcoming the oxygen‐dependent nature of PDT. Interestingly, the photodynamic action of ZnPc‐2NO elicits the release of damage‐associated molecular patterns, inducing the maturation of dendritic cells and triggering an antitumor immune response. The immunogenic cell death caused by this oxygen‐economized PDT has been demonstrated through a series of in vitro and in vivo experiments.

Inducing Immunogenic Cancer Cell Death through Oxygen-Economized Photodynamic Therapy with Nitric Oxide-Releasing Photosensitizers.

Photodynamic therapy (PDT) utilizes reactive oxygen species (ROS) for eradication of cancer cells. Its effectiveness is governed by the oxygen content, which is scarce in the hypoxic tumor microenvironment. We report herein two zinc(II) phthalocyanines substituted with two or four nitric oxide (NO)-releasing moieties, namely ZnPc-2NO and ZnPc-4NO, which can suppress the mitochondrial respiration, thereby sparing more intracellular oxygen for PDT. Using HT29 human colorectal adenocarcinoma cells and A549 human lung carcinoma cells, we have demonstrated that both conjugates release NO upon interaction with the intracellular glutathione, which can reduce the cellular oxygen consumption rate and adenosine triphosphate generation and alter the mitochondrial membrane potential. They can also relieve the hypoxic status of cancer cells and decrease the expression of hypoxia-inducible factor protein HIF-1α. Upon light irradiation, both conjugates can generate ROS and induce cytotoxicity even under a hypoxic condition, overcoming the oxygen-dependent nature of PDT. Interestingly, the photodynamic action of ZnPc-2NO elicits the release of damage-associated molecular patterns, inducing the maturation of dendritic cells and triggering an antitumor immune response. The immunogenic cell death caused by this oxygen-economized PDT has been demonstrated through a series of in vitro and in vivo experiments.

Resveratrol suppresses liver cancer progression by downregulating AKR1C3: targeting HCC with HSA nanomaterial as a carrier to enhance therapeutic efficacy.

The enzyme AKR1C3 plays a crucial role in hormone and drug metabolism and is associated with abnormal expression in liver cancer, leading to tumor progression and poor prognosis. Nanoparticles modified with HSA can modulate the tumor microenvironment by enhancing photodynamic therapy to induce apoptosis in tumor cells and alleviate hypoxia. Therefore, exploring the potential regulatory mechanisms of resveratrol on AKR1C3 through the construction of HSA-RSV NPs carriers holds significant theoretical and clinical implications for the treatment of liver cancer. The aim of this study is to investigate the targeted regulation of AKR1C3 expression through the loading of resveratrol (RSV) on nanomaterials HSA-RSV NPs (Nanoparticles) in order to alleviate tumor hypoxia and inhibit the progression of hepatocellular carcinoma (HCC), and to explore its molecular mechanism. PubChem database and PharmMapper server were used to screen the target genes of RSV. HCC-related differentially expressed genes (DEGs) were analyzed through the GEO dataset, and relevant genes were retrieved from the GeneCards database, resulting in the intersection of the three to obtain candidate DEGs. GO and KEGG enrichment analyses were performed on the candidate DEGs to analyze the potential cellular functions and molecular signaling pathways affected by the main target genes. The cytohubba plugin was used to screen the top 10 target genes ranked by Degree and further intersected the results of LASSO and Random Forest (RF) to obtain hub genes. The expression analysis of hub genes and the prediction of malignant tumor prognosis were conducted. Furthermore, a pharmacophore model was constructed using PharmMapper. Molecular docking simulations were performed using AutoDockTools 1.5.6 software, and ROC curve analysis was performed to determine the core target. In vitro cell experiments were carried out by selecting appropriate HCC cell lines, treating HCC cells with different concentrations of RSV, or silencing or overexpressing AKR1C3 using lentivirus. CCK-8, clone formation, flow cytometry, scratch experiment, and Transwell were used to measure cancer cell viability, proliferation, migration, invasion, and apoptosis, respectively. Cellular oxygen consumption rate was analyzed using the Seahorse XF24 analyzer. HSA-RSV NPs were prepared, and their characterization and cytotoxicity were evaluated. The biological functional changes of HCC cells after treatment were detected. An HCC subcutaneous xenograft model was established in mice using HepG2 cell lines. HSA-RSV NPs were injected via the tail vein, with a control group set, to observe changes in tumor growth, tumor targeting of NPs, and biological safety. TUNEL, Ki67, and APC-hypoxia probe staining were performed on excised tumor tissue to detect tumor cell proliferation, apoptosis, and hypoxia. Lentivirus was used to silence or overexpress AKR1C3 simultaneously with the injection of HSA-RSV NPs via the tail vein to assess the impact of AKR1C3 on the regulation of HSA-RSV NPs in HCC progression. Bioinformatics analysis revealed that AKR1C3 is an important target gene involved in the regulation of HCC by RSV, which is associated with the prognosis of HCC patients and upregulated in expression. In vitro cell experiments showed that RSV significantly inhibits the respiratory metabolism of HCC cells, suppressing their proliferation, migration, and invasion and promoting apoptosis. Silencing AKR1C3 further enhances the toxicity of RSV towards HCC cells. The characterization and cytotoxicity experiments of nanomaterials demonstrated the successful construction of HSA-RSV NPs, which exhibited stronger inhibitory effects on HCC cells. In vivo, animal experiments further confirmed that targeted downregulation of AKR1C3 by HSA-RSV NPs suppresses the progression of HCC and tumor hypoxia while exhibiting tumor targeting and biological safety. Targeted downregulation of AKR1C3 by HSA-RSV NPs can alleviate HCC tumor hypoxia and inhibit the progression of HCC.

Deletion of endothelial IGFBP5 protects against ischaemic hindlimb injury by promoting angiogenesis.

Angiogenesis is critical for forming new blood vessels from antedating vascular vessels. The endothelium is essential for angiogenesis, vascular remodelling and minimisation of functional deficits following ischaemia. The insulin-like growth factor (IGF) family is crucial for angiogenesis. Insulin-like growth factor-binding protein 5 (IGFBP5), a binding protein of the IGF family, may have places in angiogenesis, but the mechanisms are not yet completely understood. We sought to probe whether IGFBP5 is involved in pathological angiogenesis and uncover the molecular mechanisms behind it. IGFBP5 expression was elevated in the vascular endothelium of gastrocnemius muscle from critical limb ischaemia patients and hindlimb ischaemic (HLI) mice and hypoxic human umbilical vein endothelial cells (HUVECs). In vivo, loss of endothelial IGFBP5 (IGFBP5EKO) facilitated the recovery of blood vessel function and limb necrosis in HLI mice. Moreover, skin damage healing and aortic ring sprouting were faster in IGFBP5EKO mice than in control mice. In vitro, the genetic inhibition of IGFBP5 in HUVECs significantly promoted tube formation, cell proliferation and migration by mediating the phosphorylation of IGF1R, Erk1/2 and Akt. Intriguingly, pharmacological treatment of HUVECs with recombinant human IGFBP5 ensued a contrasting effect on angiogenesis by inhibiting the IGF1 or IGF2 function. Genetic inhibition of IGFBP5 promoted cellular oxygen consumption and extracellular acidification rates via IGF1R-mediated glycolytic adenosine triphosphate (ATP) metabolism. Mechanistically, IGFBP5 exerted its role via E3 ubiquitin ligase Von Hippel-Lindau (VHL)-regulated HIF1α stability. Furthermore, the knockdown of the endothelial IGF1R partially abolished the reformative effect of IGFBP5EKO mice post-HLI. Our findings demonstrate that IGFBP5 ablation enhances angiogenesis by promoting ATP metabolism and stabilising HIF1α, implying IGFBP5 is a novel therapeutic target for treating abnormal angiogenesis-related conditions.

Effects of HMGCR deficiency on skeletal muscle development.

Pathogenic variants in HMGCR were recently linked to a limb-girdle muscular dystrophy (LGMD) phenotype. The protein product HMG CoA reductase (HMGCR) catalyzes a key component of the cholesterol synthesis pathway. The two other muscle diseases associated with HMGCR, statin-associated myopathy (SAM) and autoimmune anti-HMGCR myopathy, are not inherited in a Mendelian pattern. The mechanism linking pathogenic variants in HMGCR with skeletal muscle dysfunction is unclear. We knocked down Hmgcr in mouse skeletal myoblasts, knocked down hmgcr in Drosophila, and expressed three pathogenic HMGCR variants (c.1327C>T, p.Arg443Trp; c.1522_1524delTCT, p.Ser508del; and c.1621G>A, p.Ala541Thr) in Hmgcr knockdown mouse myoblasts. Hmgcr deficiency was associated with decreased proliferation, increased apoptosis, and impaired myotube fusion. Transcriptome sequencing of Hmgcr knockdown versus control myoblasts revealed differential expression involving mitochondrial function, with corresponding differences in cellular oxygen consumption rates. Both ubiquitous and muscle-specific knockdown of hmgcr in Drosophila led to lethality. Overexpression of reference HMGCR cDNA rescued myotube fusion in knockdown cells, whereas overexpression of the pathogenic variants of HMGCR cDNA did not. These results suggest that the three HMGCR-related muscle diseases share disease mechanisms related to skeletal muscle development.

Modulation of mitochondrial function by extracellular acidosis in tumor cells and normal fibroblasts: Role of signaling pathways

In many tumors pronounced extracellular acidosis resulting from glycolytic metabolism is found. Since several environmental stress factors affect the mitochondrial activity the aim of the study was to analyze the impact of acidosis on cellular oxygen consumption and which signaling pathways may be involved in the regulation.In two tumor cell lines and normal fibroblasts cellular oxygen consumption rate (OCR) and mitochondrial function were measured after 3 h at pH 6.6. Besides the activation of ERK1/2, p38 and PI3K signaling in the cytosolic and mitochondrial compartment, the mitochondrial structure and proteins related to mitochondria fission were analyzed.The acidic extracellular environment increased OCR in tumor cells but not in fibroblasts. In parallel, the mitochondrial membrane potential increased at low pH. In both tumor lines (but not in fibroblasts), the phosphorylation of ERK1/2 and PI3K/Akt was significantly increased, and both cascades were involved in OCR modulation. The activation of signaling pathways was located predominantly in the mitochondrial compartment of the cells. At low pH, the mitochondrial structure in tumor cells showed structural changes related to elongation whereas mitochondria fragmentation was reduced indicating mitochondria fusion. However, these morphological changes were not related to ERK1/2 or PI3K signaling.Acidic stress seems to induce an increased oxygen consumption, which might further aggravate tumor hypoxia. Low pH also induces mitochondria fusion that is not mediated by ERK1/2 or PI3K signaling. The mechanism by which these signaling cascades modulate the respiratory activity of tumor cells needs further investigation.

CD8+ T-cell metabolism is related to cerebrovascular reactivity in middle-aged adults.

Cerebrovascular reactivity (CVR) decreases with advancing age, contributing to increased risk of cognitive impairment; however, the mechanisms underlying the age-related decrease in CVR are incompletely understood. Age-related changes to T cells, such as impaired mitochondrial respiration, increased inflammation, likely contribute to peripheral and cerebrovascular dysfunction in animals. However, whether T-cell mitochondrial respiration is related to cerebrovascular function in humans is not known. Therefore, we hypothesized that peripheral T-cell mitochondrial respiration would be positively associated with CVR and that T-cell glycolytic metabolism would be negatively associated with CVR. Twenty middle-aged adults (58 ± 5 yr) were recruited for this study. T cells were separated from peripheral blood mononuclear cells. Cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR, a marker of glycolytic activity) were measured using extracellular flux analysis. CVR was quantified using the breath-hold index (BHI), which reflects the change in blood velocity in the middle-cerebral artery (MCAv) during a 30-s breath-hold. In contrast to our hypothesis, we found that basal OCR in CD8+ T cells (β = -0.59, R2 = 0.27, P = 0.019) was negatively associated with BHI. However, in accordance with our hypothesis, we found that basal ECAR (β = -2.20, R2 = 0.29, P = 0.015) and maximum ECAR (β = -50, R2 = 0.24, P = 0.029) were negatively associated with BHI in CD8+ T cells. There were no associations observed in CD4+ T cells. These associations appeared to be primarily mediated by an association with the pressor response to the breath-hold test. Overall, our findings suggest that CD8+ T-cell respiration and glycolytic activity may influence CVR in humans.NEW & NOTEWORTHY Peripheral T-cell metabolism is related to in vivo cerebrovascular reactivity in humans. Higher glycolytic metabolism in CD8+ T cells was associated with lower cerebrovascular reactivity to a breath-hold in middle-aged adults, which is possibly reflective of a more proinflammatory state in midlife.

Exercise-inducible circulating extracellular vesicle irisin promotes browning and the thermogenic program in white adipose tissue.

Exercise can reduce body weight and promote white fat browning, but the underlying mechanisms remain largely unknown. This study investigated the role of fibronectin type III domain-containing protein 5 (FNDC5)/Irisin, a hormone released from exercising muscle, in the browning of white fat in circulating extracellular vesicles (EVs). Mice were subjected to a 4 weeks of running table exercise, and fat browning was analyzed via histology, protein blotting and qPCR. Circulating EVs were extracted by ultrahigh-speed centrifugation, and ELISA was used to measure the irisin concentration in the circulating EVs. Circulating EVs that differentially expressed irisin were applied to adipocytes, and the effect of EV-irisin on adipocyte energy metabolism was analyzed by immunofluorescence, protein blotting, and cellular oxygen consumption rate analysis. During sustained exercise, the mice lost weight and developed fat browning. FNDC5 was induced, cleaved, and secreted into irisin, and irisin levels subsequently increased in the plasma during exercise. Interestingly, irisin was highly expressed in circulating EVs that effectively promoted adipose browning. Mechanistically, the circulating EV-irisin complex is transported intracellularly by the adipocyte membrane receptor integrin αV, which in turn activates the AMPK signaling pathway, which is dependent on mitochondrial uncoupling protein 1 to cause mitochondrial plasmonic leakage and promote heat production. After inhibition of the AMPK signaling pathway, the effects of the EV-irisin on promoting fat browning were minimal. Exercise leads to the accumulation of circulating EV-irisin, which enhances adipose energy metabolism and thermogenesis and promotes white fat browning in mice, leading to weight loss.

SMAD4 inhibits glycolysis in ovarian cancer through PI3K/AKT/HK2 signaling pathway by activating ARHGAP10.

ARHGAP10 is a tumor-suppressor gene related to ovarian cancer (OC) progression; however, its specific mechanism is unclear. To investigate the effect of ARHGAP10 on OC cell migration, invasion, and glycolysis. Quantitative real-time PCR (qRT-PCR) quantified mRNA and protein expressions of AKT, p-AKT, HK2, and SMAD4 were tested by Western blot. EdU, Wound healing, and Transwell assay were utilized to evaluate OC cell proliferation, migration, and invasion. We used a Seahorse XF24 Extracellular Flux Analyzer to monitor cellular oxygen consumption rates (OCR) and extracellular acidification rates (ECAR). Chromatin immunoprecipitation (ChIP) was used to analyze the transcriptional regulation of ARHGAP10 by SMAD4. ARHGAP10 expression in OC tissues was detected by immunohistochemistry. Our results showed that ARHGAP10 expression was negatively related to lactate levels in human OC tissues. ARHGAP10 overexpression can inhibit the migration, proliferation, and invasion of OC cells, and this function can be blocked by 2-Deoxy-D-glucose. Moreover, we found that ARHGAP10 expression can be rescued with the AKT inhibitor LY294002. This study revealed that the antitumor effects of ARHGAP10 in vivo and in vitro possibly suppress oncogenic glycolysis through the PI3K/AKT/HK2-regulated glycolysis metabolism pathway.

PIGT promotes cell growth, glycolysis, and metastasis in bladder cancer by modulating GLUT1 glycosylation and membrane trafficking

BackgroundBladder cancer is very common worldwide. PIGT is a subunit of the glycosylphosphatidylinositol transamidase which involves in tumorigenesis and invasiveness. m6A modification of mRNA has been linked to cell proliferation, tumor progression and other biological events. However, how PIGT is regulated and what is the function of PIGT in bladder cancer remains to be elucidated.MethodsPIGT was silenced or overexpressed to study its role in regulating bladder cancer. Cell proliferation and invasion were examined with the Cell Counting Kit-8, colony formation and Transwell assay, respectively. Cellular oxygen consumption rates or extracellular acidification rates were detected by a XF24 Analyzer. Quantitative RT-PCR and immunoblots were performed to detect mRNA and protein levels.ResultsPIGT was overexpressed in bladder cancer. Silencing PIGT inhibited cell proliferation, oxidative phosphorylation, and glycolysis. Overexpressing PIGT promoted cell proliferation, oxidative phosphorylation, glycolysis in vitro and tumor metastasis in vivo by activating glucose transporter 1 (GLUT1). PIGT also promoted GLUT1 glycosylation and membrane trafficking. Wilms’ tumor 1-associated protein (WTAP) mediated PIGT m6A modification, and m6A reader, insulin-like growth factor 2 mRNA-binding protein (IGF2BP2), binds to the methylated PIGT to promote the stability of PIGT, leading to up-regulation of PIGT.ConclusionWTAP mediates PIGT m6A modification to increase the stability of PIGT via the IGF2BP2, which enhances cell proliferation, glycolysis, and metastasis in bladder cancer by modulating GLUT1 glycosylation and membrane trafficking.

The superiority of PMFs on reversing drug resistance of colon cancer and the effect on aerobic glycolysis-ROS-autophagy signaling axis.

Polymethoxylated flavones (PMFs) are compounds present in citrus peels and other Rutaceae plants, which exhibit diverse biological activities, including robust antitumor and antioxidant effects. However, the mechanism of PMFs in reversing drug resistance to colon cancer remains unknown. In the present study, we aimed to investigate the potential connection between the aerobic glycolysis-ROS-autophagy signaling axis and the reversal of PTX resistance in colon cancer by PMFs. MTT Cell viability assay and colony formation assay were used to investigate the effect of PMFs combined with PTX in reversing HCT8/T cell resistance ex vivo; the mRNA and protein levels of the target were detected by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), quantitative real-time fluorescence polymerase chain reaction (qRT-PCR) and Western blot protein immunoblotting (WB); An HCT8/T cell xenograft model was established to investigate the MDR reversal activity of PMFs in vivo; The extracellular acidification rate (ECAR) and the oxygen consumption rate (OCR) were detected to assess the cellular oxygen consumption rate and glycolytic process. HCT8/T cells demonstrated significant resistance to PTX, up-regulating the expression levels of ABCB1 mRNA, P-gp, LC3-I, and LC3-II protein, and increasing intracellular reactive oxygen species (ROS) content. PMFs mainly contain two active ingredients, nobiletin, and tangeretin, which were able to reverse drug resistance in HCT8/T cells in a concentration-dependent manner. PMFs exhibited high tolerance in the HCT8/T nude mouse model while increasing the sensitivity of PTX-resistant cells and suppressing tumor growth significantly. PMFs combined with PTX reduced extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) in HCT8/T cells. Additionally, PMFs reduced intracellular ROS content, down-regulated the expression levels of autophagy-related proteins LC3-I, LC3-II, Beclin1, and ATG7, and significantly reduced the number of autophagosomes in HCT8/T cells. The present study demonstrated that PMFs could potentially reverse PTX resistance in colon cancer by regulating the aerobic glycolysis-ROS-autophagy signaling axis, which indicated that PMFs would be potential potentiators for future chemotherapeutic agents in colon cancer.

Nutrient-Sensitising Drug Repurposing Screen Identifies Lomerizine As a Mitochondrial Metabolism Inhibitor in Chronic Myeloid Leukaemia

In chronic myeloid leukaemia (CML), the persistence of leukaemic stem cells (LSCs) following first line treatment with tyrosine kinase inhibitors (TKIs) such as imatinib, can lead to disease relapse. We have previously demonstrated that therapy-resistant CML LSCs rely on oxidative phosphorylation (OXPHOS) for survival and that targeting mitochondrial metabolism sensitises CML LSCs to imatinib treatment. However, current OXPHOS inhibitors have demonstrated limited efficacy, or are associated with adverse effects in clinical trials, highlighting that identification of clinically safe oxidative pathway inhibitors is warranted. To identify clinically applicable compounds that target cells dependent on mitochondrial metabolism, we used a nutrient-sensitised repurposing screening strategy. Upon replacement of glucose with galactose as the only sugar source in cell culture media, there is a shift in cellular metabolism towards OXPHOS (as cells are unable to effectively use galactose for glycolysis). Therefore, CML cells grown in galactose-containing media are more sensitive to inhibition of mitochondrial respiration than glucose-grown cells. Using this approach, we screened total of &amp;gt;1,100 unique and structurally diverse FDA-approved drugs in CML cells, cultured in the presence of 11mM glucose or 11mM galactose. This uncovered lomerizine dihydrochloride, a L-type Ca2+ channel blocker used as an anti-migraine drug, as a candidate compound effective against OXPHOS-dependent CML cells. Subsequently, patient derived CML CD34+ cells were shown to have increased ER mass (the main intracellular Ca2+ store) and an increase in mitochondrial Ca2+ levels compared with normal cells. Moreover, transcriptional analysis revealed that Ca2+ channel genes such as voltage-gated CACNA1D and receptor-activated TRPC6 are significantly upregulated in CML LSCs (CD34+CD38-) when compared with normal haematopoietic stem cells (HSCs), which was not reverted following imatinib treatment. Therefore, we hypothesised that the observed increase in mitochondrial metabolism in CML LSCs was dependent on CACNA1D and TRPC6 activity, and the increase in mitochondrial Ca2+ levels, which could be inhibited by lomerizine treatment. Notably, CRISPR-Cas9-mediated CACNA1D or TRPC6 knockout significantly reduced cytoplasmic and mitochondrial Ca2+ levels, and decreased cellular oxygen consumption rate, indicative of reduction in OXPHOS. Similarly, while imatinib treatment did not affect intracellular Ca2+ levels in primary CML CD34+ cells, lomerizine treatment significantly reduced cytoplasmic and mitochondrial Ca2+ levels, and mitochondrial respiration/OXPHOS. Applying liquid chromatography-mass spectrometry and isotope assisted metabolomics, we traced labelled nutrients (glucose and palmitate) in CD34+ cells. This revealed a significant reduction in activity of mitochondria-located Ca2+ dependent dehydrogenases and overall reduction in tricarboxylic acid (TCA) cycle activity in primary CML cells following lomerizine treatment. Of clinical relevance, using combination of cell surface markers, cell division trackers and functional assays, including long-term culture initiating cell (LTC-IC) assay, we demonstrate that lomerizine treatment, alone or in combination with imatinib, targeted CML LSCs when cultured in physiological medium in vitro, without affecting normal HSCs. Notably, combination treatment with imatinib and lomerizine reduced CML tumour burden, targeted CML LSCs and extended survival in xenotransplantation model of human CML. In summary, we reveal for the first time that Ca2+ regulation is disrupted in CML stem cells and influx via CACNA1D and TRPC6 is crucial for maintaining high mitochondrial Ca2+ levels and enhanced mitochondrial OXPHOS. This renders CML stem cells sensitive to lomerizine, an FDA-approved Ca2+ inhibitor identified following metabolism-specific drug-repurposing screening.

Inhibiting the endoplasmic reticulum stress response enhances the effect of doxorubicin by altering the lipid metabolism of liver cancer cells

Hepatocellular carcinoma (HCC) is characterized by a low and variable response to chemotherapeutic treatments. One contributing factor to the overall pharmacodynamics is the activation of endoplasmic reticulum (ER) stress pathways. This is a cellular stress mechanism that becomes activated when the cell's need for protein synthesis surpasses the ER's capacity to maintain accurate protein folding, and has been implicated in creating drug-resistance in several solid tumors. ObjectiveTo identify the role of ER-stress and lipid metabolism in mediating drug response in HCC. MethodsBy using a chemically-induced mouse model for HCC, we administered the ER-stress inhibitor 4μ8C and/or doxorubicin (DOX) twice weekly for three weeks post-tumor initiation. Histological analyses were performed alongside comprehensive molecular biology and lipidomics assessments of isolated liver samples. In vitro models, including HCC cells, spheroids, and patient-derived liver organoids were subjected to 4μ8C and/or DOX, enabling us to assess their synergistic effects on cellular viability, lipid metabolism, and oxygen consumption rate. ResultsWe reveal a pivotal synergy between ER-stress modulation and drug response in HCC. The inhibition of ER-stress using 4μ8C not only enhances the cytotoxic effect of DOX, but also significantly reduces cellular lipid metabolism. This intricate interplay culminates in the deprivation of energy reserves essential for the sustenance of tumor cells. ConclusionsThis study elucidates the interplay between lipid metabolism and ER-stress modulation in enhancing doxorubicin efficacy in HCC. This novel approach not only deepens our understanding of the disease, but also uncovers a promising avenue for therapeutic innovation. The long-term impact of our study could open the possibility of ER-stress inhibitors and/or lipase inhibitors as adjuvant treatments for HCC-patients.

Galectin-1 Fuels Monocyte-Driven Hyperinflammation and Represents a Novel Therapeutic Target in Myeloproliferative Neoplasms

JDP and STO co-corresponding authors Previous studies by our group and others have demonstrated that monocytes play a key role in driving hyperinflammation in myeloproliferative neoplasms (MPNs), and that aberrant inflammatory cytokine signaling contributes to disease progression and poor prognosis in MPNs. To interrogate relationships between cellular sources and targets of inflammatory cytokines, we performed single cell RNA-seq (scRNA-seq) in PBMCs and progenitor cells from MPN patients and healthy controls. Monocytes from MPN patients displayed significant enrichment of inflammation-relevant genes as expected. Cell-cell communication networks inferred from expression of ligands and receptors predicted monocytes as a pivotal mediator of cell interactions. Further analysis revealed galectin signaling as one of the most robust input/output pathways for monocytes and significant enrichment of LGALS1 (galectin-1) expression in MPN monocytes. Galectins are a class of proteins that bind specifically to β-galactoside carbohydrates, such as N-linked or O-linked glycosylated proteins. Abnormal expression of galectins in various cancers has been found to mediate broad biological functions, including cell proliferation, apoptosis, adhesion, and inflammation. However, the mechanisms by which galectins contribute to MPN pathogenesis remain incompletely understood. To corroborate our sRNA-seq findings, we performed flow cytometry analysis which demonstrated elevated expression of galectin-1 in MPN patient CD14+ monocytes. Increased plasma levels of galectin-1 were also identified in MPN patients compared to healthy individuals. Higher galectin-1 protein levels in both Kit+ progenitor cells and CD11b+ myeloid cells from Jak2 V617F knock-in mice were also observed when compared to wild type mice. Additionally, galectin-1 expression was induced by MPL W515L and JAK2 V617F and inhibited by ruxolitinib in Ba/F3 cells. Thus, our results show enrichment of galectin-1 in both MPN patients and mouse models, with evidence of direct contribution from specific MPN driver mutations. Our scRNA-seq dataset analysis showed significant correlations of galectin-1 expression with inflammatory and oxidative phosphorylation (OXPHOS) genes in MPN patient monocytes. To investigate the effects of galectin-1 in monocyte activation, we incubated CD14+ monocytes from MPN patients with recombinant galectin-1 (rGal-1) and observed markedly stimulated secretion of inflammatory cytokines, such as IL-1α, IL-1β, IL-6, IL-8 and TNF-α. Furthermore, OTX008, a galectin-1 inhibitor, inhibited the transcription and secretion of inflammatory cytokines in MPN patient CD14+ monocytes and monocytic cell lines. Notably, both genetic and pharmacologic inhibition of galectin-1 reduced cellular ATP level and oxygen consumption rate, suggesting a metabolic reprogramming in MPNs. Although galectin-1 mediates broad biological functions, the molecular mechanisms by which galectin-1 regulates signaling pathways and transcription in MPN cells had not been delineated. Our scRNA-seq heterogeneity analysis suggested that genes in the PI3K-AKT-mTOR pathway were differentially expressed between galectin-1 high- and low-expressing monocytes. We therefore incubated both monocytic cell lines and monocytes from MPN patients with rGal-1 and observed stimulation of the PI3K-AKT-mTOR pathway, represented by increased levels of phosphorylated mTOR, AKT and S6. Consistently, genetic and pharmacologic inhibition of galectin-1 inhibited activation of the PI3K-AKT-mTOR pathway. In summary, our results demonstrate upregulation of galectin-1 in MPN monocytes, potentially due to activation of JAK2-driven signaling. We further demonstrate that galectin-1 fuels inflammation potentially via metabolic reprogramming of monocytes and activation of PI3K-AKT-mTOR signaling. Our data also suggest galectin-1 as a putative therapeutic target in MPNs.

BTN3A3 inhibits clear cell renal cell carcinoma progression by regulating the ROS/MAPK pathway via interacting with RPS3A

Butyrophilin subfamily 3 member A3 (BTN3A3) is a member of the immunoglobulin superfamily and functions as a tumor suppressor in multiple cancer types. Our study has revealed that in clear cell renal cell carcinoma (ccRCC), patients who express high levels of BTN3A3 experience longer survival times than those with lower expression. Further, we have observed that BTN3A3 inhibits the proliferation, migration, and invasion of ccRCC cells. Through the utilization of an immunoprecipitation assay followed by mass spectrometry, we have discovered that BTN3A3 binds directly to RPS3A. Knockdown of BTN3A3 led to increased cell proliferation, migration, and invasion. However, this effect was significantly reduced when RPS3A was simultaneously overexpressed. Previous reports have demonstrated that RPS3A positively regulates mitochondrial function and reactive oxygen species (ROS) levels. Our study has shown that overexpression of both BTN3A3 and RPS3A can increase cellular oxygen consumption rate (OCR) and ROS levels. Furthermore, we have observed that the addition of H2O2 can reverse the effects of BTN3A3 knockdown on cell proliferation and migration by increasing the cellular ROS level. ROS play a crucial role in regulating the MAPK pathway and tumor cell growth. To further explore this relationship, we examined RNA-Seq and immunoblotting data and found that BTN3A3 can negatively regulate the degree of activation of the MAPK signaling pathway. This finding suggests that the BTN3A3/RPS3A complex may regulate ccRCC progression by modulating MAPK pathways. Therefore, BTN3A3 could serve as both a prognostic marker and a potential therapeutic target for ccRCC patients.

Hydroxy-α-sanshool from the fruits of Zanthoxylum bungeanum Maxim. promotes browning of white fat by activating TRPV1 to induce PPAR-γ deacetylation

BackgroundAccumulating evidence suggested increasing energy expenditure is a feasible strategy for combating obesity, and browning of white adipose tissue (WAT) to promote thermogenesis might be one of the attractive ways. Hydroxy-α-sanshool (HAS), a natural amide alkaloid extracted from the fruits of Zanthoxylum bungeanum Maxim, possesses lots of benefits in lipid metabolism regulation. MethodsThe anti-obesity effect of HAS was investigated by establishing an animal model of obesity and a 3T3-L1 differentiation cell model. Effects of HAS on the whole-body fat and liver of obese mice, and the role of HAS in inducing browning of white fat were studied by Micro CT, Metabolic cage detection, Cell mitochondrial pressure detection, transmission electron microscopy and cold exposure assays. Furthermore, the Real-time PCR (qPCR), digital PCR (dPCR), western blot, Co-immunoprecipitation (Co-IP), molecular docking, drug affinity responsive target stability (DARTS), Cellular thermal shift assay (CETSA) and other methods were used to investigate the target and mechanisms of HAS. ResultsWe found that treatment with HAS helped mice combat obesity caused by a high fat diet (HFD) and improve metabolic characteristics. In addition, our results suggested that the anti-obesity effect of HAS is related to increase energy consumption and thermogenesis via induction of browning of WAT. The further investigations uncovered that HAS can up-regulate UCP-1 expression, increase mitochondria number, and elevate the cellular oxygen consumption rates (OCRs) of white adipocytes. Importantly, the results indicated that browning effects of HAS is closely associated with SIRT1-dependent PPAR-γ deacetylation through activating the TRPV1/AMPK pathway, and TRPV1 is the potential drug target of HAS for the browning effects of WAT. ConclusionsOur results suggested the HAS can promote browning of WAT via regulating AMPK/SIRT-1/PPARγ signaling, and the potential drug target of HAS is the membrane receptor of TRPV1.

Accumulation of Prion Triggers the Enhanced Glycolysis via Activation of AMKP Pathway in Prion-Infected Rodent and Cell Models.

Mitochondrial dysfunction is one of the hallmarks in the pathophysiology of prion disease and other neurodegenerative diseases. Various metabolic dysfunctions are identified and considered to contribute to the progression of some types of neurodegenerative diseases. In this study, we evaluated the status of glycolysis pathway in prion-infected rodent and cell models. The levels of the key enzymes, hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK) were significantly increased, accompanying with markedly downregulated mitochondrial complexes. Double-stained IFAs revealed that the increased HK2 and PFK distributed widely in GFAP-, Iba1-, and NeuN-positive cells. We also identified increased levels of AMP-activated protein kinase (AMPK) and the downstream signaling. Changes of AMPK activity in prion-infected cells by the AMPK-specific inhibitor or activator induced the corresponding alterations not only in the downstream signaling, but also the expressions of three key kinases in glycolysis pathway and the mitochondrial complexes. Transient removal or complete clearance of prion propagation in the prion-infected cells partially but significantly reversed the increases of the key enzymes in glycolysis, the upregulation of AMPK signaling pathway, and the decreases of the mitochondrial complexes. Measurements of the cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) showed lower OCR and higher ECAR in prion-infected cell line, which were sufficiently reversed by clearance of prion propagation. Those data indicate a metabolic reprogramming from oxidative phosphorylation to glycolysis in the brains during the progression of prion disease. Accumulation of PrPSc is critical for the switch to glycolysis, largely via activating AMPK pathway.

Targeted deletion of von-Hippel-Lindau in the proximal tubule conditions the kidney against early diabetic kidney disease.

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease. Glomerular hyperfiltration and albuminuria subject the proximal tubule (PT) to a subsequent elevation of workload, growth, and hypoxia. Hypoxia plays an ambiguous role in the development and progression of DKD and shall be clarified in our study. PT-von-Hippel-Lindau (Vhl)-deleted mouse model in combination with streptozotocin (STZ)-induced type I diabetes mellitus (DM) was phenotyped. In contrary to PT-Vhl-deleted STZ-induced type 1 DM mice, proteinuria and glomerular hyperfiltration occurred in diabetic control mice the latter due to higher nitric oxide synthase 1 and sodium and glucose transporter expression. PT Vhl deletion and DKD share common alterations in gene expression profiles, including glomerular and tubular morphology, and tubular transport and metabolism. Compared to diabetic control mice, the most significantly altered in PT Vhl-deleted STZ-induced type 1 DM mice were Ldc-1, regulating cellular oxygen consumption rate, and Zbtb16, inhibiting autophagy. Alignment of altered genes in heat maps uncovered that Vhl deletion prior to STZ-induced DM preconditioned the kidney against DKD. HIF-1α stabilization leading to histone modification and chromatin remodeling resets most genes altered upon DKD towards the control level. These data demonstrate that PT HIF-1α stabilization is a hallmark of early DKD and that targeting hypoxia prior to the onset of type 1 DM normalizes renal cell homeostasis and prevents DKD development.