Glucose Deprivation Conditions Research Articles

Downregulation of miR-23b by transcription factor c-Myc alleviates ischemic brain injury by upregulating Nrf2

Ischemic brain injury (IBI) is a common acute cerebral vessel disease that occurs secondary to blockage in arteries, mainly characterized by insufficient blood supply to the brain. The transcription factor c-Myc in IBI continues to be implicated in numerous studies. This study was conducted with emphasis placed on the underlying mechanism of c-Myc in IBI. Clinical samples were collected from IBI patients. Middle cerebral artery occlusion (MCAO) was induced in mice by inserting a suture from the external carotid artery to the anterior cerebral artery through the internal carotid artery to mechanically block the blood supply at the origin of the middle cerebral artery, and cortical neurons from mice were exposed to oxygen glucose deprivation (OGD) conditions for IBI model in vitro construction. RT-qPCR was performed to determine microRNA-23b (miR-23b) expression. TUNEL staining and Western blot analysis was conducted to detect apoptosis. The regulatory relationship was analyzed by dual-luciferase reporter gene assay. After loss- and gain-of-function assays, triphenyltetrazolium chloride staining was carried out to detect the area of cerebral infarction, after which the spatial memory in mice was evaluated with Morris water maze test. As per our findings, miR-23b was upregulated in the serum of IBI patients and OGD-treated murine primary neurons. Silencing of miR-23b resulted in reduced OGD-induced neuronal apoptosis. miR-23b inversely targeted nuclear factor erythroid 2-related factor 2 (Nrf2) and c-Myc negatively regulated miR-23b expression. Overexpression of c-Myc and inhibition of miR-23b led to reduced neurological scores, infarction area, neuronal apoptosis, shortened platform arrival time and significantly increased the time spent on the platform quadrant and the times of crossing the platform in vivo. Collectively, downregulated miR-23b by c-Myc might alleviate IBI by upregulating Nrf2.

Metabolic Switch Under Glucose Deprivation Leading to Discovery of NR2F1 as a Stimulus of Osteoblast Differentiation.

Poor survival of grafted cells is the major impediment of successful cell-based therapies for bone regeneration. Implanted cells undergo rapid death in an ischemic environment largely because of hypoxia and metabolic stress from glucose deficiency. Understanding the intracellular metabolic processes and finding genes that can improve cell survival in these inhospitable conditions are necessary to enhance the success of cell therapies. Thus, the purpose of this study was to investigate changes of metabolic profile in glucose-deprived human bone marrow stromal/stem cells (hBMSCs) through metabolomics analysis and discover genes that could promote cell survival and osteogenic differentiation in a glucose-deprived microenvironment. Metabolomics analysis was performed to determine metabolic changes in a glucose stress metabolic model. In the absence of glucose, expression levels of all metabolites involved in glycolysis were significantly decreased than those in a glucose-supplemented state. In glucose-deprived osteogenic differentiation, reliance on tricarboxylic acid cycle (TCA)-predicted oxidative phosphorylation instead of glycolysis as the main mechanism for energy production in osteogenic induction. By comparing differentially expressed genes between glucose-deprived and glucose-supplemented hBMSCs, NR2F1 (Nuclear Receptor Subfamily 2 Group F Member 1) gene was discovered to be associated with enhanced survival and osteogenic differentiation in cells under metabolic stress. Small, interfering RNA (siRNA) for NR2F1 reduced cell viability and osteogenic differentiation of hBMSCs under glucose-supplemented conditions whereas NR2F1 overexpression enhanced osteogenic differentiation and cell survival of hBMSCs in glucose-deprived osteogenic conditions via the protein kinase B (AKT)/extracellular signal-regulated kinase (ERK) pathway. NR2F1-transfected hBMSCs significantly enhanced new bone formation in a critical size long-bone defect of rats compared with control vector-transfected hBMSCs. In conclusion, the results of this study provide an understanding of the metabolic profile of implanted cells in an ischemic microenvironment and demonstrate that NR2F1 treatment may overcome this deprivation by enhancing AKT and ERK regulation. These findings can be utilized in regenerative medicine for bone regeneration. © 2022 American Society for Bone and Mineral Research (ASBMR).

Hydrogen Sulfide Overproduction Is Involved in Acute Ischemic Cerebral Injury Under Hyperhomocysteinemia.

ObjectivesThis study aimed to identify the involvement of hydrogen sulfide overproduction in acute brain injury under ischemia/reperfusion and hyperhomocysteinemia.MethodsIn vitro and in vivo experiments were conducted to determine: the effect of sodium hydrosulfide treatment on the human neuroblastoma cell line (SH-SY5Y) under conditions of oxygen and glucose deprivation; the changes of hydrogen sulfide levels, inflammatory factors, energetic metabolism, and mitochondrial function in the brain tissue of rats under either ischemia/reperfusion alone or a combination of ischemia/reperfusion and hyperhomocysteinemia; and the potential mechanism underlying the relationship between homocysteine and these changes through the addition of the related inhibitors. Furthermore, experimental technologies, including western blot, enzyme-linked immunosorbent assay, immunofluorescence, reverse transcription polymerase chain reaction, and flow cytometry, were used.ResultsOur study found that high concentration of sodium hydrosulfide treatment aggravated the decrease in mitochondrial membrane potential, the increase in both mitochondrial permeability transition pore and translocation of cytochrome C, as well as the accumulation of reactive oxygen species in oxygen and glucose deprived SH-SY5Y cells. As a result, neurological deficit appeared in rats with ischemia/reperfusion or ischemia/reperfusion and hyperhomocysteinemia, and a higher water content and larger infarction size of cerebral tissue appeared in rats combined ischemia/reperfusion and hyperhomocysteinemia. Furthermore, alterations in hydrogen sulfide production, inflammatory factors, and mitochondria morphology and function were more evident under the combined ischemia/reperfusion and hyperhomocysteinemia. These changes were, however, alleviated by the addition of inhibitors for CBS, CSE, Hcy, H2S, and NF-κB, although at different levels. Finally, we observed a negative relationship between the blockage of: (a) the nuclear factor kappa-B pathway and the levels of cystathionine β-synthase and hydrogen sulfide; and (b) the hydrogen sulfide pathway and the levels of inflammatory factors.ConclusionHydrogen sulfide overproduction and reactive inflammatory response are involved in ischemic cerebral injury under hyperhomocysteinemia. Future studies in this direction are warranted to provide a scientific base for targeted medicine development.

Proteomic Analysis Reveals the Effect of Trichostatin A and Bone Marrow-Derived Dendritic Cells on the Fatty Acid Metabolism of NIH3T3 Cells under Oxygen-Glucose Deprivation Conditions.

Fibroblasts mediate acute wound healing and long-term tissue remodeling with scarring after tissue injury. Following myocardial infarction (MI), necrotized cardiomyocytes become replaced by secreted extracellular matrix proteins produced by fibroblasts. Dendritic cells (DCs) can migrate from the bone marrow to the infarct areas and infarct border areas to mediate collagen accumulation after MI. Trichostatin A (TSA) is known to regulate apoptosis and proliferation in fibroblasts and affect the functions of DCs under oxygen-glucose deprivation (OGD) conditions. In this study, we used label-free quantitative proteomics to investigate the effects of TSA and bone marrow-derived dendritic cells (BMDCs) on NIH3T3 fibroblasts under OGD conditions. The results showed that the fatty acid degradation pathway was significantly upregulated in NIH3T3 cells under OGD conditions and that the fatty acid synthesis pathway was significantly downregulated in NIH3T3 cells treated with conditioned media (CM) from BMDCs treated with TSA under OGD conditions [BMDCs-CM(TSA)]. In addition, BMDCs-CM(TSA) significantly decreased the levels of triglycerides and free fatty acids and mediated fatty acid metabolism-related proteins in NIH3T3 cells under OGD conditions. In summary, this proteomics analysis showed that TSA and BMDCs affect fatty acid metabolism in NIH3T3 cells under OGD conditions.

The Effect of IDO on Neural Progenitor Cell Survival Under Oxygen Glucose Deprivation.

Objective: Indoleamine 2,3-dioxygenase (IDO) activity plays an important role in many neurological disorders in the central nervous system, which may be associated with immunomodulation or anti-inflammatory activity. However, the action of IDO in the ischemic condition is still poorly understood. The purpose of the present study is to explore the expression and action of IDO in stem cell culture under oxygen and glucose deprivation.Methods: Neural progenitor cells were obtained from the human embryonic stem cell line BG01. These cells underwent oxygen and glucose deprivation. We examined the IDO expression at 3 and 8 h of oxygen and glucose deprivation and then examined neuronal progenitor cell viability in the normal and oxygen and glucose deprivation condition using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. In addition, we studied the effect of IDO inhibition and the expression of TNF-α, IGF-1, VEGF, IL-6, FGFβ, TGFβ, EGF, and Leptin to explore the mechanism of IDO under the oxygen and glucose deprivation.Results: IDO expression in neural progenitor cells increased under oxygen and glucose deprivation, which is closely associated with cell death (p < 0.05). Inhibiting IDO did not affect cell survival in normal neural progenitor cells. However, inhibiting IDO could attenuate cell viability under oxygen and glucose deprivation (p < 0.05). Further study demonstrated that IDO expression was closely associated to the growth factor’s leptin expression.Conclusions: Our results demonstrated that an increase of IDO under oxygen and glucose deprivation was associated with cell death, suggesting that inhibiting IDO could be a target for neuroprotection.

Dexmedetomidine inhibits apoptosis of astrocytes induced by oxygen-glucose deprivation via targeting JAK/STAT3 signal pathway

ObjectiveThere is an increasing interest concerning the contribution of astrocytes to the intrinsic bioremediation of ischemic brain injury. The aim of this work was to disclose the effects and mechanism of dexmedetomidine (DEX) on the apoptosis of astrocytes under oxygen glucose deprivation (OGD) condition. MethodsPrimary cultured astrocytes separated from Sprague-Dawley (SD) rats were subjected to OGD treatment. Astrocytes were transfected with si-JMJD3 or pcDNA3.1-JMJD3 and then treated with DEX or JAK/STAT inhibitor (WP1066) before cell apoptosis was detected by TUNEL apoptosis kit. Western blot was applied to assess the level of apoptosis-related proteins Caspase-3, Bax and Bcl-2. Astrocyte cell viability was assessed by measuring the lactate dehydrogenase (LDH) level using a LDH assay kit. ResultsAstrocytes received OGD treatment had increased LDH and elevated apoptotic rate (P < 0.05). DEX could suppress OGD induced cytotoxic effect on astrocytes, as evidenced by decreased LDH release and suppressed cell apoptosis rate (P < 0.05). Meanwhile, DEX and WP1066 treatment were also found to inhibit the phosphorylation level of STAT1 and STAT3 (P < 0.05), indicating the DEX could suppress the activation of JAK/STAT signal pathway. JMJD3 overexpression in astrocytes could suppress the anti-apoptotic function of WP1066 in OGD treated astrocytes and hamper the protective effect of DEX in cell apoptosis (P < 0.05), suggesting that DEX and JAK/STAT signal pathway inhibits OGD induced apoptosis in astrocytes by down-regulating JMJD3. ConclusionDEX protects astrocytes against apoptosis via inhibiting JAK2/STAT3 signal pathway and downregulating JMJD3 expression in vitro.

Genotype, biofilm formation ability and specific gene transcripts characteristics of endodontic Enterococcus faecalis under glucose deprivation condition

ObjectiveTo study the relationship between the specific gene and biofilm formation ability of seven wild type Enterococcus faecalis (E. faecalis) under glucose deprivation conditions. DesignWild type E. faecalis (3RC, 5RC, 25RC, 31RC, 33RC, 37RC, 58RC) extracted from the teeth with persistent apical periodontitis were cultured under glucose deprivation conditions and then resequenced. The biofilm formation ability was compared using primary adherence assay, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The transcriptional level of biofilm formation–related genes (ace, gelE, efa, esp and fsrB) were detected. ResultsGenomic resequencing showed that 3RC and 58RC (Class B) were similar, while 5RC, 25RC, 31RC, 33RC and 37RC (Class A) were similar. Based on primary adherence assay, CLSM and SEM results, biofilm formation ability of Class B strains was lower, while Class A strains were higher when compared with control group (0.25 % glucose). Furthermore, compared with control group (0.25 % glucose), the transcriptional levels of ace, efa and fsrB genes were upregulated in all strains; the transcriptional levels of gelE were downregulated in Class B strains, upregulated in Class A strains; the transcriptional levels of esp of Class B strains were downregulated, while upregulated in 25RC, 31RC and 37RC (Class A), and not observed in 5RC and 33RC. ConclusionThe genotypes of wild type E. faecalis of different persistent periapical periodontitis teeth are different. The genotype differences and the transcription levels of related virulence genes (ace, gelE, efa, esp and fsrB) are related to the biological phenotype.

ERN1 knockdown modifies the effect of glucose deprivation on homeobox gene expressions in U87 glioma cells.

The aim of the present investigation was to study the expression of genes encoding homeobox proteins ZEB2 (zinc finger E-box binding homeobox 2), TGIF1 (TGFB induced factor homeobox 1), SPAG4 (sperm associated antigen 4), LHX1 (LIM homeobox 1), LHX2, LHX6, NKX3-1 (NK3 homeobox 1), and PRRX1 (paired related homeobox 1) in U87 glioma cells in response to glucose deprivation in control glioma cells and cells with knockdown of ERN1 (endoplasmic reticulum to nucleus signaling 1), the major pathway of the endoplasmic reticulum stress signaling, for evaluation of it possible significance in the control of glioma growth through ERN1 signaling and chemoresistance. The expression level of homeobox family genes was studied in control (transfected by vector) and ERN1 knockdown U87 glioma cells under glucose deprivation condition by real-time quantitative polymerase chain reaction. It was shown that the expression level of ZEB2, TGIF1, PRRX1, and LHX6 genes was up-regulated in control glioma cells treated by glucose deprivation. At the same time, the expression level of three other genes (NKX3-1, LHX1, and LHX2) was down-regulated. Furthermore, ERN1 knockdown of glioma cells significantly modified the effect glucose deprivation condition on the expression almost all studied genes. Thus, treatment of glioma cells without ERN1 enzymatic activity by glucose deprivation condition lead to down-regulation of the expression level of ZEB2 and SPAG4 as well as to more significant up-regulation of PRRX1 and TGIF1 genes. Moreover, the expression of LHX6 and NKX3-1 genes lost their sensitivity to glucose deprivation but LHX1 and LHX2 genes did not change it significantly. The results of this investigation demonstrate that ERN1 knockdown significantly modifies the sensitivity of most studied homeobox gene expressions to glucose deprivation condition and that these changes are a result of complex interaction of variable endoplasmic reticulum stress related and unrelated regulatory factors and contributed to glioma cell growth and possibly to their chemoresistance.

Abstract IA16: ER stress responses shape the ovarian cancer immune-microenvironment

Abstract Harnessing the intrinsic ability of our immune system to eliminate malignant cells represents the most promising new anticancer strategy since the development of chemotherapy. While cancer immunotherapy has been designated a scientific breakthrough, tumor-induced immunosuppression represents a major impediment to the success of this new approach in ovarian cancer. We uncovered that ovarian cancer inhibits the development of protective antitumor immunity by inducing a state of persistent “endoplasmic reticulum (ER) stress” in intratumoral dendritic cells (DCs) and T cells. First, we identified that sustained activation of the IRE1-XBP1 arm of the ER stress response in DCs was necessary for the aggressive and accelerated progression of primary and metastatic ovarian cancers in various preclinical models of disease. Mechanistically, intratumoral DCs exhibited constitutive IRE1-XBP1 activation, which was fueled by lipid peroxidation byproducts that caused ER stress by modifying ER-resident chaperones. Persistent XBP1 activation induced a comprehensive triglyceride biosynthetic program promoting accumulation of lipid droplets that subsequently impaired DC antigen presentation to T cells. Conditional deletion or therapeutic silencing of IRE1 or XBP1 in DCs restored their immunogenic function in the tumor and delayed ovarian cancer progression by eliciting protective antitumor T-cell responses. In addition, we recently demonstrated that ovarian cancer also triggers abnormal IRE1-XBP1 activation in T cells to control their mitochondrial function and antitumor activity. XBP1 upregulation in T cells isolated from human ovarian cancer specimens was associated with decreased intratumoral T-cell infiltration and reduced IFNG expression. Ascites fluid from ovarian cancer patients inhibited glucose uptake and caused N-linked protein glycosylation defects in T cells, leading to XBP1-driven mitochondrial dysfunction. Mechanistically, maladaptive XBP1 signaling limited the influx of glutamine necessary to sustain mitochondrial respiration under glucose-deprived conditions. Hence, ovarian cancer hosts lacking XBP1 selectively in T cells demonstrated superior antitumor immunity, delayed malignant progression, and increased overall survival. Taken together, our studies revealed that ovarian cancer provokes persistent IRE1-XBP1 signaling in immune cells to shape the tumor microenvironment and evade immune control. These findings provide a new mechanistic understanding of immunosuppression in ovarian cancer and offer novel avenues for immunotherapeutic intervention. Citation Format: Juan R. Cubillos-Ruiz. ER stress responses shape the ovarian cancer immune-microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr IA16.

Galectin-3 protects against ischemic stroke by promoting neuro-angiogenesis via apoptosis inhibition and Akt/Caspase regulation

Post-stroke neurological deficits and mortality are often associated with vascular disruption and neuronal apoptosis. Galectin-3 (Gal3) is a potent pro-survival and angiogenic factor. However, little is known about its protective role in the cerebral ischemia/reperfusion (I/R) injury. We have previously shown significant up-regulation of Gal3 in the post-stroke rat brain, and that blocking of Gal3 with neutralizing antibody decreases the cerebral blood vessel density. Our current study demonstrates that intracerebral local delivery of the Gal3 into rat brain at the time of reperfusion exerts neuroprotection. Ischemic lesion volume and neuronal cell death were significantly reduced as compared with the vehicle-treated MCAO rat brains. Gal3 increased vessel density and neuronal survival after I/R in rat brains. Importantly, Gal3-treated groups showed significant improvement in motor and sensory functional recovery. Gal3 increased neuronal cell viability under in vitro oxygen–glucose deprivation conditions in association with increased phosphorylated-Akt, decreased phosphorylated-ERK1/2, and reduced caspase-3 activity. Gene expression analysis showed down regulation of pro-apoptotic and inflammatory genes including Fas-ligand, and upregulation of pro-survival and pro-angiogenic genes including Bcl-2, PECAM, and occludin. These results indicate a key role for Gal3 in neuro-vascular protection and functional recovery following ischemic stroke through modulation of angiogenic and apoptotic pathways.

SIRT7 activates p53 by enhancing PCAF-mediated MDM2 degradation to arrest the cell cycle

Sirtuin 7 (SIRT7), an NAD+-dependent deacetylase, plays vital roles in energy sensing, but the underlying mechanisms of action remain less clear. Here, we report that SIRT7 is required for p53-dependent cell-cycle arrest during glucose deprivation. We show that SIRT7 directly interacts with p300/CBP-associated factor (PCAF) and the affinity for this interaction increases during glucose deprivation. Upon binding, SIRT7 deacetylates PCAF at lysine 720 (K720), which augments PCAF binding to murine double minute (MDM2), the p53 E3 ubiquitin ligase, leading to accelerated MDM2 degradation. This effect results in upregulated expression of the cell-cycle inhibitor, p21Waf1/Cip1, which further leads to cell-cycle arrest and decreased cell viability. These data highlight the importance of the SIRT7–PCAF interaction in regulating p53 activity and cell-cycle progression during conditions of glucose deprivation. This axis may represent a new avenue to design effective therapeutics based on tumor starvation.

Pancastatin A and B Have Selective Cytotoxicity on Glucose-Deprived PANC-1 Human Pancreatic Cancer Cells.

Glucose deprivation and hypoxia frequently occur in solid tumor cells, including pancreatic cancer cells. Glucose deprivation activates the unfolded protein response (UPR) and causes the upregulation of glucose-regulated protein 78 (GRP78). Induction of GRP78 has been shown to protect cancer cells. Therefore, shutting down of GRP78 expression may be a novel strategy in anticancer drug development. Based on this understanding, a screening system established for anticancer agents that exhibit selective cytotoxicity on pancreatic cancer cells under glucose-deprived conditions. To test this hypothesis, the new compounds isolated, pancastatin A (PST-A) and B (PSTB), from Ponciri Fructus. PST-A and B were identified as glabretal triterpenoid moieties by electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopic methods. PST-A and B suppressed the accumulation of the UPR hallmark gene, GRP78, during glucose deprivation. Furthermore, PST-A and B showed selective cytotoxicity on PANC-1 pancreatic cancer cells under glucose deprivation. Interestingly, PST-A and B had no effect on these cells under normal growth conditions. Our results suggest that PST-A and B act as novel therapeutic agents to induce selective cell death in glucose-deprived pancreatic cancer cells.

AMPK-dependent phosphorylation of HDAC8 triggers PGM1 expression to promote lung cancer cell survival under glucose starvation.

Cancer cells undergo metabolic reprogramming to sustain their own survival under an environment of increased energy demand; however, the mechanism by which cancer cells ensure survival under glucose deprivation stressed conditions remains elusive. Here, we show that deprivation of glucose, dramatically activated the glycogen pathway, accompanied by elevated phosphoglucomutase 1 (PGM1) expression. We further identified that AMP-activated protein kinase (AMPK) stimulated PGM1 expression by inducing histone deacetylase 8 (HDAC8) phosphorylation. Moreover, we demonstrated that glucose deprivation-induced AMPK activation stimulated the translocation of HDAC8 from the nucleus to the cytoplasm, consequently disrupting the binding between HDAC8 and histone 3. PGM1 expression was also found to be critical for lung cancer glycolysis, the oxidative pentose phosphate pathway, and oxidative phosphorylation under glucose deprivation conditions, and further led to the aberrant expression of metabolic enzymes involved in glucose metabolism mediated by ERK1/2. Finally, PGM1 was found to be highly expressed in lung cancer tissues from patients, which correlated with a poor prognosis. Taken together, these results revealed that AMPK activation by glucose deprivation leads to enhanced PGM1 expression, an essential component of the metabolic switch, to facilitate cancer progression, suggesting PGM1 as promising anti-cancer treatment targets.

Astrocyte-derived exosomes transfer miR-190b to inhibit oxygen and glucose deprivation-induced autophagy and neuronal apoptosis

ABSTRACT Our previous work has verified that astrocytes (AS)-derived exosomes (AS-Exo) inhibited autophagy and ameliorated neuronal damage in experimental ischemic stroke. However, the mechanism of AS-Exo regulation of autophagy remains unclear. The aim of this study was to investigate the regulatory mechanism of AS-Exo on neuronal autophagy. The mouse hippocampal neuronal cell line HT-22 was cultured in oxygen and glucose deprivation (OGD) condition to mimic ischemic injury. The primary astrocytes were used to isolate exosomes. Exosome labeling and uptake by HT-22 cells were observed by confocal laser microscopy. miR-190b expression was determined by qRT-PCR. HT-22 cell vitality and apoptosis were determined by CCK-8 assay and TUNEL staining, respectively. Levels of TNF-α, IL-6 and IL-1β were analyzed by ELISA. Protein levels of apoptosis-related cleaved caspase-3, Bax, Bcl-2 and autophagy-related Beclin-1, LC3-I/II, Atg7, P62 were determined by western blot. A dual-luciferase reporter assay was performed to confirm the direct interaction between miR-190b and Atg7. miR-190b expression in AS-Exo was found to be significantly higher than that in AS. AS-Exo-mediated transfer of miR-190b attenuated OGD-induced neuronal apoptosis via suppressing autophagy. Moreover, Atg7 was identified as a target of miR-190b. AS-Exo-mediated transfer of miR-190b regulated autophagy by targeting Atg7. Collectively, our data indicated that AS-Exo transferred miR-190b to inhibit OGD-induced autophagy and neuronal apoptosis.

Impaired Vps34 complex activity-mediated autophagy inhibition contributes to endothelial progenitor cells damage in the ischemic conditions

AimsEndothelial progenitor cells (EPCs) are widely accepted to be applied in ischemic diseases. However, the therapeutic potency is largely impeded because of its inviability in these ischemic conditions. Autophagy is recognized to be vital in cell activity. Therefore, we explore the role and the mechanism of autophagy in ischemic EPCs. Methods and resultsWe applied 7d-cultured bone marrow EPCs to investigate the autophagy status under the oxygen and glucose deprivation (OGD) conditions in vitro, mimicking the in-vivo harsh ischemia and anoxia microenvironment. We found increased EPC apoptosis, accompanied by an impaired autophagy activation. Intriguingly, mTOR inhibitor Rapamycin was incapable to reverse this damped autophagy and EPC damage. We further found that autophagy pathway downstream Vps34-Beclin1-Atg14 complex assembly and activity were impaired in OGD conditions, and an autophagy-inducing peptide Tat-Beclin1 largely recovered the impaired complex activity and attenuated OGD-stimulated EPC injury through restoring autophagy activation. ConclusionsThe present study discovered that autophagy activation is inhibited when EPCs located in the ischemia and anoxia conditions. Restoration of Vps34 complex activity obtains sufficient autophagy, thus promoting EPC survival, which will provide a potential target and advance our understanding of autophagy manipulation in stem cell transplantation.

The critical role of glucose deprivation in epithelial-mesenchymal transition in hepatocellular carcinoma under hypoxia

Imaging with 18F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) is used to determine sites of abnormal glucose metabolism to predict high tumor grade, metastasis, and poor patient survival. However, not all tumors with increased 18F-FDG uptake show aggressive tumor biology, as evident from the moderate correlation between metastasis and high FDG uptake. We hypothesized that metastasis is likely attributable to the complexity and heterogeneity of the cancer microenvironment. To identify the cancer microenvironment that induces the epithelial-mesenchymal transition (EMT) process, tumor areas of patients with HCC were analyzed by immunostaining. Our data demonstrated the induction of EMT process in HCC cells with low proliferation under hypoxic conditions. To validate our finding, among HCC cell lines, HepG2 cells with highly increased expression of HIF1α under hypoxia were employed in vitro and in vivo. Major changes in EMT-associated protein expression, such as the up-regulation of N-cadherin and snail/slug are associated with decreased proliferation-related protein (PCNA) caused by glucose deprivation under hypoxia. Indeed, PCNA knockdown-HepG2 cells under hypoxia showed the induction of more EMT process compare to the control. Thus, HCC cells with low proliferative potential under glucose-deprived and hypoxic conditions show high probability for induced EMT process and promote cell invasion. This study investigates reasons as to why an EMT process cannot fully be predicted. Our observations indicate that rather than analyzing a single factor, an integrated analysis of hypoxia with low glucose metabolism and low cell proliferation might be helpful to predict the potential impact on induction of EMT process and promotion of cell invasion.

Enhanced expression of asparagine synthetase under glucose-deprived conditions promotes esophageal squamous cell carcinoma development.

Background: Cancer cells survive and develop under nutrient deficient microenvironment caused by low blood supply. Although anaerobic metabolism could function through the enhanced uptake of glucose, other mechanisms of tolerance to glucose deficient conditions might be required.Materials and Methods: Expression of asparagine synthetase (ASNS) under normal glucose and glucose-deprived conditions was examined. Cancer cell proliferation and migration were evaluated by in vitro and in vivo assays. In addition, the relationship between ASNS expression and cancer stages was also analyzed.Results: Expression of ASNS was enhanced under glucose deficient conditions. In vitro assays indicated that ASNS could promote the proliferation and migration abilities of esophageal squamous cell carcinoma (ESCC) cells under glucose deficient condition. In mechanism, 2 critical effectors during nutrient deprivation, NRF2 and ATF4, were upregulated and demonstrated to promote ASNS expression. Clinically, high level of ASNS was significantly associated with ESCC with advanced stages and metastasis. In vivo, ASNS could promote tumor growth and metastasis in mouse xenograft models.Conclusion: This study uncovered that glucose deprivation induces the overexpression of ASNS in ESCC cells, which in turn causes cancer cell tolerance to nutrient stress and promotes cancer development. The illustration of the mechanism sheds deep insight on how cell biology was regulated in response to the conditions of limited nutrient availability.

Recognition of the gluconeogenic enzyme, Pck1, via the Gid4 E3 ligase: An in silico perspective.

Gluconeogenesis, the reverse process of glycolysis, is a favorable mechanism at conditions of glucose deprivation. Pck1 is a rate-limiting gluconeogenic enzyme, where its deficiency or mutation contributes to serious clinical situations as neonatal hypoglycemia and liver failure. A recent report confirms that Pck1 is a target for proteasomal degradation through its proline residue at the penultimate position, recognized by Gid4 E3 ligase, but with a lack of informative structural details. In this study, we delineate the localized sequence motif, degron, that specifically interact with Gid4 ligase and unravel the binding mode of Pck1 to the Gid4 ligase by using molecular docking and molecular dynamics. The peptide/protein docking HPEPDOCK web server along with molecular dynamic simulations are applied to demonstrate the binding mode and interactions of a Pck1 wild type (SPSK) and mutant (K4V) with the recently solved structure of Gid4 ligase. Results unveil a distinct binding mode of the mutated peptide compared with the wild type despite having comparable binding affinities to Gid4. Moreover, the four-residue peptide is found insufficient for Gid4 binding, while the seven-residue peptide suffices for binding to Gid4. The amino acids S134, K135, and N137 in the loop L1 (between β1 and β2) of the Gid4 are essential for the stabilization of the seven-residue peptide in the binding site of the ligase. The presence of Val4 instead of Lys4 smashes the H-bonds that are formed between Lys4 and Gid4 in the wild type peptide, making the peptide prone to bind with the other side of the binding pocket (L4 loop of Gid4). The dynamics of Gid4 L3 loop is affected dramatically once K4V mutant Pck1 peptide is introduced. This opens the door to explore the mutation effects on the binding mode and smooth the path to target protein degradation by design competitive and non-competitive inhibitors.

Metformin Induces Oxidative Stress-Mediated Apoptosis without the Blockade of Glycolysis in H4IIE Hepatocellular Carcinoma Cells.

Metformin, a widely prescribed anti-diabetic drug, also exerts anti-cancer effects in different types of cancers. Although a number of molecular mechanisms have been suggested, the metabolic features underlying metformin's anti-cancer activity is not fully understood enough. Because cancer cells have been known to prefer inefficient aerobic glycolysis to support their proliferation, it is important to clarify by which metformin affects metabolism to suppress the proliferation of cancer cells. Here, we report the metabolic changes induced by metformin and its relevance to the induction of apoptosis in H4II rat hepatocellular carcinoma cells. H4IIE cells were treated with metformin and other reagents in culture media with various nutritional compositions. Glutamine as well as pyruvate enhanced the viability of H4IIE cells in glucose-deprived conditions. Protective effects of glucose and pyruvate were comparable at same concentrations (5 mM). Metformin induced apoptosis irrespective of any nutritional conditions. Glucose consumption and lactate production were stimulated by metformin. Inhibition of glycolysis by 2-deoxyglucose suppressed the metformin-induced lactate production but additively enhanced metformin's pro-apoptotic effect. These results indicate that metformin does not interfere but accelerate glycolysis. Unexpectedly, the production of reactive oxygen species (ROS) was markedly stimulated by metformin. A potent antioxidant, N-acetylcysteine (NAC) suppressed all pro-apoptotic changes as well as ROS generation induced by metformin. Taken together, metformin does not interfere with glycolysis but promotes apoptosis by enhancing oxidative stress.

A substitution mutation in a conserved domain of mammalian acetate-dependent acetyl CoA synthetase 2 results in destabilized protein and impaired HIF-2 signaling.

The response to environmental stresses by eukaryotic organisms includes activation of protective biological mechanisms, orchestrated in part by transcriptional regulators. The tri-member Hypoxia Inducible Factor (HIF) family of DNA-binding transcription factors include HIF-2, which is activated under conditions of oxygen or glucose deprivation. Although oxygen-dependent protein degradation is a key mechanism by which HIF-1 and HIF-2 activity is regulated, HIF-2 is also influenced substantially by the coupled action of acetylation and deacetylation. The acetylation/deacetylation process that HIF-2 undergoes employs a specific acetyltransferase and deacetylase. Likewise, the supply of the acetyl donor, acetyl CoA, used for HIF-2 acetylation originates from a specific acetyl CoA generator, acetate-dependent acetyl CoA synthetase 2 (Acss2). Although Acss2 is predominantly cytosolic, a subset of the Acss2 cellular pool is enriched in the nucleus following oxygen or glucose deprivation. Prevention of nuclear localization by a directed mutation in a putative nuclear localization signal in Acss2 abrogates HIF-2 acetylation and blunts HIF-2 dependent signaling as well as flank tumor growth for knockdown/rescue cancer cells expressing ectopic Acss2. In this study, we report generation of a novel mouse strain using CRISPR/Cas9 mutagenesis that express this mutant Acss2 allele in the mouse germline. The homozygous mutant mice have impaired induction of the canonical HIF-2 target gene erythropoietin and blunted recovery from acute anemia. Surprisingly, Acss2 protein levels are dramatically reduced in these mutant mice. Functional studies investigating the basis for this phenotype reveal multiple protein instability domains in the Acss2 carboxy terminus. The findings described herein may be of relevance in the regulation of native Acss2 protein as well as for humans carrying missense mutations in these domains.