Activities Of Glycolytic Enzymes Research Articles

Potential Roles of α-amylase in Alzheimer's Disease: Biomarker and Drug Target.

Alzheimer's disease (AD), the most common form of dementia, is pathologically characterized by the deposition of amyloid-β plaques and the formation of neurofibrillary tangles. In a neurodegenerative brain, glucose metabolism is also impaired and considered as one of the key features in AD patients. The impairment causes a reduction in glucose transporters and the uptake of glucose as well as alterations in the specific activity of glycolytic enzymes. Recently, it has been reported that α-amylase, a polysaccharide-degrading enzyme, is present in the human brain. The enzyme is known to be associated with various diseases such as type 2 diabetes mellitus and hyperamylasaemia. With this information at hand, we hypothesize that α-amylase could have a vital role in the demented brains of AD patients. This review aims to shed insight into the possible link between the expression levels of α-amylase and AD. Lastly, we also cover the diverse role of amylase inhibitors and how they could serve as a therapeutic agent to manage or stop AD progression.

Pseudoislet Aggregation of Pancreatic β-Cells Improves Glucose Stimulated Insulin Secretion by Altering Glucose Metabolism and Increasing ATP Production.

Appropriate glucose-stimulated insulin secretion (GSIS) by pancreatic β-cells is an essential component of blood glucose homeostasis. Configuration of β-cells as 3D pseudoislets (PI) improves the GSIS response compared to 2D monolayer (ML) culture. The aim of this study was to determine the underlying mechanisms. MIN6 β-cells were grown as ML or PI for 5 days. Human islets were isolated from patients without diabetes. Function was assessed by GSIS and metabolic capacity using the Seahorse bioanalyser. Connexin 36 was downregulated using inducible shRNA. Culturing MIN6 as PI improved GSIS. MIN6 PI showed higher glucose-stimulated oxygen consumption (OCR) and extracellular acidification (ECAR) rates. Further analysis showed the higher ECAR was, at least in part, a consequence of increased glycolysis. Intact human islets also showed glucose-stimulated increases in both OCR and ECAR rates, although the latter was smaller in magnitude compared to MIN6 PI. The higher rates of glucose-stimulated ATP production in MIN6 PI were consistent with increased enzyme activity of key glycolytic and TCA cycle enzymes. There was no impact of connexin 36 knockdown on GSIS or ATP production. Configuration of β-cells as PI improves GSIS by increasing the metabolic capacity of the cells, allowing higher ATP production in response to glucose.

Overexpression of Heat Shock Protein 70 Ameliorates Meat Quality of Broilers Subjected to Pre-Slaughter Transport at High Ambient Temperatures by Improving Energy Status of Pectoralis Major Muscle and Antioxidant Capacity.

The induction of heat shock protein 70 (HSP70) potentially mediates meat-quality development under stress conditions. To investigate the effects and mechanism of HSP70 on the meat quality of the pectoralis major (PM) muscles of broilers exposed to pre-slaughter transport, a total of 168 broilers were intraperitoneally injected with L-glutamine (Gln) or saline. Twenty-four hours later, broilers were subjected to transport or held under normal living conditions. The results indicated that acute Gln supplementation significantly increased HSP70 expression in the PM of transported broilers (p < 0.05). The overexpression of HSP70 significantly alleviated the decreases in muscle pH and water-holding capacity and improved the shrinking of muscle fibers induced by transport (p < 0.05). HSP70 induction increased ATP content, decreased the activities of glycolytic enzymes, and lowered the phosphorylation level of AMP-activated protein kinase in transported broilers (p < 0.05). In addition, the overexpression of HSP70 greatly increased total superoxide dismutase and the total antioxidant capability and decreased the levels of reactive oxygen species, malonaldehyde, and carbonyls in the PM of transported broilers (p < 0.05). Overall, this work indicated that HSP70 could effectively improve the meat quality of transported broilers by improving the energy status, inhibiting glycolytic influx, and restoring redox homeostasis.

Proton export upregulates aerobic glycolysis

IntroductionAggressive cancers commonly ferment glucose to lactic acid at high rates, even in the presence of oxygen. This is known as aerobic glycolysis, or the “Warburg Effect.” It is widely assumed that this is a consequence of the upregulation of glycolytic enzymes. Oncogenic drivers can increase the expression of most proteins in the glycolytic pathway, including the terminal step of exporting H+ equivalents from the cytoplasm. Proton exporters maintain an alkaline cytoplasmic pH, which can enhance all glycolytic enzyme activities, even in the absence of oncogene-related expression changes. Based on this observation, we hypothesized that increased uptake and fermentative metabolism of glucose could be driven by the expulsion of H+ equivalents from the cell.ResultsTo test this hypothesis, we stably transfected lowly glycolytic MCF-7, U2-OS, and glycolytic HEK293 cells to express proton-exporting systems: either PMA1 (plasma membrane ATPase 1, a yeast H+-ATPase) or CA-IX (carbonic anhydrase 9). The expression of either exporter in vitro enhanced aerobic glycolysis as measured by glucose consumption, lactate production, and extracellular acidification rate. This resulted in an increased intracellular pH, and metabolomic analyses indicated that this was associated with an increased flux of all glycolytic enzymes upstream of pyruvate kinase. These cells also demonstrated increased migratory and invasive phenotypes in vitro, and these were recapitulated in vivo by more aggressive behavior, whereby the acid-producing cells formed higher-grade tumors with higher rates of metastases. Neutralizing tumor acidity with oral buffers reduced the metastatic burden.ConclusionsTherefore, cancer cells which increase export of H+ equivalents subsequently increase intracellular alkalization, even without oncogenic driver mutations, and this is sufficient to alter cancer metabolism towards an upregulation of aerobic glycolysis, a Warburg phenotype. Overall, we have shown that the traditional understanding of cancer cells favoring glycolysis and the subsequent extracellular acidification is not always linear. Cells which can, independent of metabolism, acidify through proton exporter activity can sufficiently drive their metabolism towards glycolysis providing an important fitness advantage for survival.

Disruption of O-GlcNAcylation Homeostasis Induced Ovarian Granulosa Cell Injury in Bovine

O-linked β-N-acetylglucosamine (O-GlcNAc) modification is a ubiquitous, reversible, and highly dynamic post-translational modification, which takes charge of almost all biological processes examined. However, little information is available regarding the molecular regulation of O-GlcNAcylation in granulosa cell function and glucose metabolism. This study focused on the impact of disrupted O-GlcNAc cycling on the proliferation and apoptosis of bovine granulosa cells, and further aimed to determine how this influenced glucose metabolism. Pharmacological inhibition of OGT with benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside (BADGP) led to decreased cellular O-GlcNAc levels, as well as OGT and OGA protein expressions, whereas increasing O-GlcNAc levels with the OGA inhibitor, O-(2-acetamido-2-deoxy--gluco-pyranosylidene) (PUGNAc), resulted in elevated OGA protein expression and decreased OGT protein expression in granulosa cells. Dysregulated O-GlcNAc cycling reduced cell viability, downregulated the proliferation-related genes of CDC42 and PCNA transcripts, upregulated the pro-apoptotic genes of BAX and CASPASE-3 mRNA and the ratio of BAX/BCL-2, and increased the apoptotic rate. Glycolytic enzyme activities of hexokinase and pyruvate kinase, metabolite contents of pyruvate and lactate, mitochondrial membrane potential, ATP levels, and intermediate metabolic enzyme activities of succinate dehydrogenase and malate dehydrogenase involved in the tricarboxylic acid cycle, were significantly impaired in response to altered O-GlcNAc levels. Moreover, inhibition of OGT significantly increased the expression level of thioredoxin-interacting protein (TXNIP), but repression of OGA had no effect. Collectively, our results suggest that perturbation of O-GlcNAc cycling has a profound effect on granulosa cell function and glucose metabolism.

AMP-activated protein kinase mediates glycolysis in post-mortem breast muscle of broilers

The aim of this study was to confirm the role of AMP-activated protein kinase (AMPK) in the regulation of post-mortem breast muscle glycolysis in the broiler, we evaluated the effects of intraperitoneal injection of the specific AMPK activator (5-aminoimidazole-4-carboxamide ribonucleoside, AICAR) and inhibitor (6-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a] pyrimidine, Compound C) on AMPK phosphorylation (p-AMPK) level, meat quality, glycolytic metabolites and glycolytic enzymes activities in post-mortem breast muscle of broilers. Compared to the CON group, intraperitoneal injection of AIRCR increased p-AMPK level, glycolytic enzyme activities and lactate content in post-mortem breast muscle, increased lightness (L*) and drip loss, but decreased pH45min and pH24h. However, intraperitoneal injection of Compound C, decreased p-AMPK level, glycolytic enzyme activities, glucose-6-phosphate and lactate content in post-mortem breast muscle, and increased pH24h. Based on these results, we conclude that AMPK played a vital role in regulating post-mortem glycolysis in the breast muscle of broilers. HIGHLIGHTS Injection of AIRCR promoting muscle glycolysis. Injection of Compound C alleviating muscle glycolysis. AMPK played a vital role in regulating post-mortem muscle glycolysis.

Comprehensive investigations of key mitochondrial metabolic changes in senescent human fibroblasts.

There is a paucity of detailed data related to the effect of senescence on the mitochondrial antioxidant capacity and redox state of senescent human cells. Activities of TCA cycle enzymes, respiratory chain complexes, hydrogen peroxide (H2O2), superoxide anions (SA), lipid peroxides (LPO), protein carbonyl content (PCC), thioredoxin reductase 2 (TrxR2), superoxide dismutase 2 (SOD2), glutathione peroxidase 1 (GPx1), glutathione reductase (GR), reduced glutathione (GSH), and oxidized glutathione (GSSG), along with levels of nicotinamide cofactors and ATP content were measured in young and senescent human foreskin fibroblasts. Primary and senescent cultures were biochemically identified by monitoring the augmented cellular activities of key glycolytic enzymes including phosphofructokinase, lactate dehydrogenase, and glycogen phosphorylase, and accumulation of H2O2, SA, LPO, PCC, and GSSG. Citrate synthase, aconitase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, and complex I-III, II-III, and IV activities were significantly diminished in P25 and P35 cells compared to P5 cells. This was accompanied by significant accumulation of mitochondrial H2O2, SA, LPO, and PCC, along with increased transcriptional and enzymatic activities of TrxR2, SOD2, GPx1, and GR. Notably, the GSH/GSSG ratio was significantly reduced whereas NAD+/NADH and NADP+/NADPH ratios were significantly elevated. Metabolic exhaustion was also evident in senescent cells underscored by the severely diminished ATP/ADP ratio. Profound oxidative stress may contribute, at least in part, to senescence pointing at a potential protective role of antioxidants in aging-associated disease.

Chronic intake of nutritive sweeteners and saccharin increases levels of glycolytic and lipogenic enzymes in rat liver

There are doubts about the impact of non-nutritive sweeteners consumption on lipogenic and glycolytic metabolism. Therefore, the objective was to determine the effects of chronic consumption of sweeteners on the activity levels of the enzymes glucokinase (GK), phosphofructokinase-1 (PFK-1), pyruvate kinase (PKL), acetyl coenzyme A carboxylase (ACC), and fatty acid synthase (FAS) in livers’ extracts. Groups of male and female Wistar rats drank solutions of sweeteners for 480 days: Sucrose 10%, glucose 14%, fructose 7%, acesulfame K 0.05%, aspartame:acesulfame mixture 1.55%, sucralose 0.017%, saccharin 0.033%, and a control group. The enzymatic activity in livers’ extracts was determined. Likewise, the levels of glucose, triglycerides, insulin, glucagon, and leptin were determined. In both genders, there were significant differences in the levels of enzymatic activity, hormonal, and biochemical parameters due to sweeteners consumption. The highest glycolytic and lipogenic enzyme activity levels were observed in the groups that ingested nutritive sweeteners and saccharin.

206-LB: Increase of Hexokinase-2 Protein without Increase of Other Glycolytic Enzymes in Myotubes with High Glucose Concentration Supports the Hypothesis of Hexokinase-2-Linked Unscheduled Glycolysis as a Driver of Insulin Resistance

Insulin resistance in skeletal muscle is the major metabolic defect leading to the development of type 2 diabetes. The initiating dysfunction driving development of insulin resistance has not been disclosed. Recently, stabilization of hexokinase-2 (HK2) to proteolysis by increased glucose concentration, increasing flux through early-stage glycolysis without increase in expression and activity of other glycolytic enzymes, was identified as the initiator of metabolic dysfunction in hyperglycemia. This creates a wave of increased glycolytic intermediates activating hexosamine and protein kinase C pathways and increasing formation of methylglyoxal. Increased glucose-6-phosphate displaces HK2 from mitochondria, producing mitochondrial hyperpolarization and dysfunction. This is termed HK2-linked unscheduled glycolysis. Skeletal muscle exposed to increased fasting plasma glucose may stimulate this. To test this, we studied glycolytic enzyme abundance in murine C2C12 myotubes incubated with 5.5 and 25 mM glucose for 5 days, determining HK2 protein changes by Western blotting, normalized to ß-actin. Protein abundances in C2C12 myotube cultures with 5.5, 11.1 and 25.0 mM glucose determined by label-free quantitative mass spectrometry proteomics were mined from published data. In C2C12 myotubes, HK2 protein increased 46 ± 5 % in 25 mM glucose (P&amp;lt;0.001, n = 3; Western blotting) . Proteomics corroborated this with 1.5 and 1.8 fold increase of HK2 in 11.1 and 25.0 mM glucose cultures, compared to 5.5. mM glucose control (P&amp;lt;0.01, n = 3) . Abundances of other glycolytic enzymes, hexokinase-1, glucose-6-phosphate isomerase, phosphofructokinase, aldolase, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase, were unchanged. This establishes the conditions for HK2-linked glycolytic overload. We conclude that high glucose concentration in C2C12 myotubes in vitro exhibits conditions producing hexokinase-2-linked unscheduled glycolysis Disclosure M. Xue: None. N. Rabbani: None. P. Thornalley: None. Funding Qatar Foundation, QB14Qatar University, QUHI-CMED-21_22-1

Podocyte specific deletion of PKM2 ameliorates LPS-induced podocyte injury through beta-catenin

BackgroundAcute kidney injury (AKI) is associated with a severe decline in kidney function caused by abnormalities within the podocytes' glomerular matrix. Recently, AKI has been linked to alterations in glycolysis and the activity of glycolytic enzymes, including pyruvate kinase M2 (PKM2). However, the contribution of this enzyme to AKI remains largely unexplored.MethodsCre-loxP technology was used to examine the effects of PKM2 specific deletion in podocytes on the activation status of key signaling pathways involved in the pathophysiology of AKI by lipopolysaccharides (LPS). In addition, we used lentiviral shRNA to generate murine podocytes deficient in PKM2 and investigated the molecular mechanisms mediating PKM2 actions in vitro.ResultsSpecific PKM2 deletion in podocytes ameliorated LPS-induced protein excretion and alleviated LPS-induced alterations in blood urea nitrogen and serum albumin levels. In addition, PKM2 deletion in podocytes alleviated LPS-induced structural and morphological alterations to the tubules and to the brush borders. At the molecular level, PKM2 deficiency in podocytes suppressed LPS-induced inflammation and apoptosis. In vitro, PKM2 knockdown in murine podocytes diminished LPS-induced apoptosis. These effects were concomitant with a reduction in LPS-induced activation of β-catenin and the loss of Wilms’ Tumor 1 (WT1) and nephrin. Notably, the overexpression of a constitutively active mutant of β-catenin abolished the protective effect of PKM2 knockdown. Conversely, PKM2 knockdown cells reconstituted with the phosphotyrosine binding–deficient PKM2 mutant (K433E) recapitulated the effect of PKM2 depletion on LPS-induced apoptosis, β-catenin activation, and reduction in WT1 expression.ConclusionsTaken together, our data demonstrates that PKM2 plays a key role in podocyte injury and suggests that targetting PKM2 in podocytes could serve as a promising therapeutic strategy for AKI.Trial registrationNot applicable.66jzd3p7x7ZtqB-vdEamwaVideo abstract

Apigenin sensitizes radiotherapy of mouse subcutaneous glioma through attenuations of cell stemness and DNA damage repair by inhibiting NF-κB/HIF-1α-mediated glycolysis

We used radioresistant SU3-5R stem cells-inoculated subcutaneous glioma model to investigate the radiosensitization effect of apigenin. After treatment of glioma with apigenin 20 mg/kg for 12 days, irradiation 8 Gray twice or their combination, the tumor volume and weight were decreased, especially in the combination group. Apigenin inhibited the activities of glycolytic enzymes and expressions of nuclear factor kappa B (NF-κB) p65, hypoxia inducible factor-lα (HIF-1α), glucose transporter (GLUT)-1/3 and pyruvate kinase isozyme type M2 (PKM2) proteins in tumor tissues. After treatment of SU3-5R cells with apigenin 7.5 µM, the fluorescence intensity of CD133 positive cells was decreased, the percentage of cells with comet tails was increased, and the expressions of lipopolysaccharide-induced NF-κB p65, HIF-1α, GLUT-3 and PKM2 proteins were reduced. These results demonstrate that apigenin can sensitize the radiotherapy of glioma via the attenuations of cell stemness and DNA damage repair by inhibiting NF-κB/HIF-1α-mediated glycolytic enzymes and protein expressions.

Study on the HIF-1α regulated by glycolytic pathways and mitochondrial function in yaks of different altitudes during postmortem aging.

The study investigated the glycolysis pathway mediated by hypoxia-inducible factor-1α (HIF-1α) and the mechanism of its regulation. The results indicated that HIF-1α expression initially increased before subsequently decreasing with aging time during postmortem (p < .01). Glucose transporter-1 (GLUT-1), lactate dehydrogenase (LDH), and hexokinase (HK) displayed a similar trend with aging time (p < .01) while pyruvate dehydrogenase kinase 1 (PDK-1) increased gradually within the first 12 hr before decreasing at 24-120 hr. However, after treatment with a HIF-1α inhibitor, no significant differences were observed in the mitochondrial morphology. Furthermore, lactate content decreased, along with LDH, HK, and F0F1-ATP activities as well as GLUT-1 and PDK-1 expression (p < .01). The shear force for all groups also increased during postmortem aging (p < .01), with that of the controls being significantly higher compared with the treatment groups (p < .01). These findings confirmed that, after slaughter, the hypoxic environment within the muscles provided essential conditions for HIF-1α expression, which, in turn, activated the glycolysis pathway by mediating changes in the activities of glycolytic enzymes and mitochondrial function. Moreover, in accelerating glycolysis rate, the expression of HIF-1α further played a negative role in meat tenderization during postmortem aging. This, it was concluded that HIF-1α expression plays a significant role in postmortem yak meat tenderization by regulating the glycolysis pathway. PRATICAL APPLICATIONS: While converting muscle into meat through hypoxic glycolysis during postmortem aging is undeniable, the biochemical mechanism of this process mediated remains quite obscure. However, the meat quality difference which impact muscle regulation mechanism during postmortem aging has not been reported. The study investigated the HIF-1α played a major role in both the glycolytic pathway and as well as meat tenderness during the postmortem aging of yak meat. The glycolysis pathway is mediated by hypoxia-inducible factor-1α (HIF-1α), the mechanism of its regulation, and meat tenderness during the postmortem aging of yak meat.

Circular RNA circVAMP3 promotes aerobic glycolysis and proliferation by regulating LDHA in renal cell carcinoma

Metabolic dysfunction is seen in cancer cells where increased glycolysis provides energy for growth. Circular RNAs (circRNAs) are thought to assist in glucose metabolism and the switch to glycolysis. Through screening, we found that circVAMP3 was necessary for both glycolytic and proliferative activities in renal cell carcinoma (RCC). Furthermore, circVAMP3 expression was elevated in RCC patients in correspondence with TNM stage. Mechanistically, circVAMP3 was observed to interact directly with lactate dehydrogenase A (LDHA) and modulate its activity. The circVAMP3–LDHA interaction facilitated LDHA phosphorylation at tyrosine 10 (Y10) catalyzed by the upstream kinase fibroblast growth factor receptor type 1 (FGFR1). Therefore, this study reveals a novel molecular mechanism by which circVAMP3 promotes glycolysis and proliferation through regulating the enzymatic activity of glycolytic enzyme, suggesting that circVAMP3 may represent an RCC biomarker and treatment target.

Ethanol Shifts Myoblast Bioenergetic Phenotype: Implications for Muscle Regeneration

At‐risk alcohol use is nearly twice as prevalent among people living with HIV (PLWH) as the general population. Alcohol and HIV can independently and synergistically increase risk for metabolic diseases, including myopathy. Decreased functional skeletal muscle (SKM) mass is associated with morbidity and all‐cause mortality. A major focus of our research is to investigate the independent and combined effects of alcohol and HIV on SKM regenerative capacity and its contribution to myopathy development. Previously, we showed that in vitro ethanol decreases myoblast extracellular acidification rate (ECAR), indicative of decreased glycolytic function, and that the decrease in ECAR was associated with decreased myoblast differentiation. The objective of this study was to determine effects of prior and recent alcohol exposure and simian immunodeficiency virus (SIV) on bioenergetic function in myoblasts from naïve and SIV‐infected rhesus macaques. Female rhesus macaques (6‐10 years of age) were administered chronic binge alcohol (CBA; peak blood alcohol concentration=50‐60 mM, 5 days per week) or isovolumetric water vehicle (VEH) beginning 3 months prior to infection with SIVmac251. Daily antiretroviral therapy (ART) was initiated 2.5 months after SIV infection, and macaques were humanely euthanized 9 months later. SKM samples were collected for myoblast isolation prior to CBA or VEH (naïve, N=6) and at study end point (VEH/SIV, N=6; CBA/SIV, N=6). Myoblasts from each group were incubated with ethanol (0 and 50 mM) for 3 days prior to Mito Stress Test, Glycolysis Stress Test, and ATP Rate Assay using an XFe96 Extracellular Flux Analyzer. There were no significant differences due to CBA or SIV on mitochondrial or glycolytic function for any measure. During the Mito Stress Test, ethanol exposure significantly (p&lt;0.05) decreased ECAR at baseline and after the addition of oligomycin (ATP synthase inhibitor), indicative of decreased glycolytic function, without significant changes in mitochondrial function. Similarly, ethanol exposure decreased all parameters in the Glycolysis Stress Test (non‐glycolytic acidification, glycolysis, glycolytic reserve, and glycolytic capacity). An ATP rate assay confirmed that ethanol exposure significantly decreased glycolytic ATP production, contributing to a decrease in total ATP production. These results indicate that recent exposure to an intoxicating concentration of ethanol shifts myoblast bioenergetic phenotype, impairs glycolysis, and produces a bioenergetic deficit regardless of prior alcohol exposure or SIV/ART. The shift away from glycolysis occurs under basal and stressed conditions. We propose that altered glycolytic enzyme activity underlies the alcohol‐mediated decrease in glycolytic function, negatively impacting myoblast differentiation and SKM regenerative capacity.

Ablation of Proton/Glucose Exporter SLC45A2 Enhances Melanosomal Glycolysis to Inhibit Melanin Biosynthesis and Promote Melanoma Metastasis

Sequence variation in SLC45A2 are responsible for oculocutaneous albinism type 4 in many species and are associated with melanoma susceptibility, but the molecular mechanism is unclear. In this study, we used Slc45a2-deficient melanocyte and mouse models to elucidate the roles of SLC45A2 in melanogenesis and melanoma metastasis. We found that the acidified cellular environment impairs the activity of key melanogenic enzyme tyrosinase in Slc45a2-deficient melanocytes. SLC45A2 is identified as a proton/glucose exporter in melanosomes, and its ablation increases the acidification of melanosomal pH through enhanced glycolysis. Intriguingly, 13C-glucose-labeled metabolic flux and biochemical assays show that melanosomes are active glucose-metabolizing organelles, indicating that elevated glycolysis mainly occurs in melanosomes owing to Slc45a2 deficiency. Moreover, Slc45a2 deficiency significantly upregulates the activities of glycolytic enzymes and phosphatidylinositol 3-kinase/protein kinase B signaling to promote glycolysis-dependent survival and metastasis of melanoma cells. Collectively, our study reveals that the proton/glucose exporter SLC45A2 mediates melanin synthesis and melanoma metastasis primarily by modulating melanosomal glucose metabolism.

Glycolytic oscillations in HeLa cervical cancer cell spheroids.

Previous studies have unravelled glycolytic oscillations in cancer cells, such as HeLa cervical and DU145 prostate cancer cells, using a monolayer culture system. Here, we demonstrate glycolytic oscillations in HeLa cervical cancer cell spheroids. Experiments revealed that a small number of HeLa cells in spheroids exhibited heterogeneous oscillations with a higher frequency than those in monolayers. Model analyses and our previous experiments indicated that the higher frequencies of oscillations in spheroids were mostly due to the increase in glycolytic enzyme activity in the cells, and to the decrease in glucose concentration by diffusional transport of glucose from the surface to inside the spheroids, as well as the increase in cell density through spheroid formation. These results and our previous studies imply that more malignant cancer cells tend to exhibit glycolytic oscillations with higher frequencies than less malignant cells. Adjacent cells in spheroids oscillated within a 10% difference in frequency, but did not synchronize with each other. This suggests that weak cell-to-cell interactions might exist among HeLa cells connected with cadherins in the spheroid microenvironment; however, the interactions were not strong enough to induce synchronization of glycolytic oscillations.

MFN2 knockdown promotes osteogenic differentiation of iPSC-MSCs through aerobic glycolysis mediated by the Wnt/\u03b2-catenin signaling pathway

BackgroundMitofusin-2 (MFN2) is a kind of GTPase that participates in the regulation of mitochondrial fusion, which is related to a variety of physiological and pathological processes, including energy metabolism, cell differentiation, and embryonic development. However, it remains unclear whether MFN2 is involved in the metabolism and osteogenic differentiation of mesenchymal stem cells (MSCs).MethodsMFN2 knockdown (MFN2-KD) and MFN2-overexpressing (MFN2-OE) induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) were constructed by lentivirus. The commercial kits were utilized to detect the glycolysis and oxidative phosphorylation (OXPHOS) rate. Flow cytometry, Western blot, quantitative real-time polymerase chain reaction (qRT-PCR), RNA-seq, immunofluorescence, and immunoprecipitation were employed for phenotype and molecular mechanism assessment.ResultsWe demonstrated that MFN2 and Wnt/β-catenin signaling pathway regulated glycolysis of iPSC-MSCs. The lack of MFN2 promoted the osteogenic differentiation of iPSC-MSCs, and aerobic glycolysis in the presence of sufficient oxygen, which increased glucose consumption and lactic acid production, as well as the glycolytic enzyme activity and gene expression. Inhibiting the Wnt/β-catenin signaling pathway normalized the enhanced glycolytic rate and osteogenic differentiation of MFN2-KD iPSC-MSCs. MFN2-OE iPSC-MSCs displayed the opposite phenotype.ConclusionsDownregulating MFN2 promotes osteogenic differentiation of iPSC-MSCs through aerobic glycolysis mediated by the Wnt/β-catenin signaling pathway. Our research reveals the new function of MFN2 in regulating the osteogenic differentiation and energy metabolism of MSCs, which will provide a new therapeutic target and theoretical basis for alveolar bone repair and periodontal regenerative treatment.

Regulation of growth, invasion and metabolism of breast ductal carcinoma through CCL2/CCR2 signaling interactions with MET receptor tyrosine kinases

With over 60,000 cases diagnosed annually in the US, ductal carcinoma in situ (DCIS) is the most prevalent form of early-stage breast cancer. Because many DCIS cases never progress to invasive ductal carcinomas (IDC), overtreatment remains a significant problem. Up to 20% patients experience disease recurrence, indicating that standard treatments do not effectively treat DCIS for a subset of patients. By understanding the mechanisms of DCIS progression, we can develop new treatment strategies better tailored to patients. The chemokine CCL2 and its receptor CCR2 are known to regulate macrophage recruitment during inflammation and cancer progression. Recent studies indicate that increased CCL2/CCR2 signaling in breast epithelial cells enhance formation of IDC. Here, we characterized the molecular mechanisms important for CCL2/CCR2-mediated DCIS progression. Phospho-protein array profiling revealed that CCL2 stimulated phosphorylation of MET receptor tyrosine kinases in breast cancer cells. Co-immunoprecipitation and proximity ligation assays demonstrated that CCL2-induced MET activity depended on interactions with CCR2 and SRC. Extracellular flux analysis and biochemical assays revealed that CCL2/CCR2 signaling in breast cancer cells enhanced glycolytic enzyme expression and activity. CRISPR knockout and pharmacologic inhibition of MET revealed that CCL2/CCR2-induced breast cancer cell proliferation, survival, migration and glycolysis through MET-dependent mechanisms. In animals, MET inhibitors blocked CCR2-mediated DCIS progression and metabolism. CCR2 and MET were significantly co-expressed in patient DCIS and IDC tissues. In summary, MET receptor activity is an important mechanism for CCL2/CCR2-mediated progression and metabolism of early-stage breast cancer, with important clinical implications.

Relationship with Rice Taste of Rice Endosperm Glycolytic Enzyme Activity Amount and Viscoelasticity

本研究では, 酵素活性ならびに粘弾性と食味の関係性について検討を行い, 以下の知見が得られた.

Glucose Metabolism and Aging of Hematopoietic Stem and Progenitor Cells.

Comprehensive proteomics studies of human hematopoietic stem and progenitor cells (HSPC) have revealed that aging of the HSPC compartment is characterized by elevated glycolysis. This is in addition to deregulations found in murine transcriptomics studies, such as an increased differentiation bias towards the myeloid lineage, alterations in DNA repair, and a decrease in lymphoid development. The increase in glycolytic enzyme activity is caused by the expansion of a more glycolytic HSPC subset. We therefore developed a method to isolate HSPC into three distinct categories according to their glucose uptake (GU) levels, namely the GUhigh, GUinter and GUlow subsets. Single-cell transcriptomics studies showed that the GUhigh subset is highly enriched for HSPC with a differentiation bias towards myeloid lineages. Gene set enrichment analysis (GSEA) demonstrated that the gene sets for cell cycle arrest, senescence-associated secretory phenotype, and the anti-apoptosis and P53 pathways are significantly upregulated in the GUhigh population. With this series of studies, we have produced a comprehensive proteomics and single-cell transcriptomics atlas of molecular changes in human HSPC upon aging. Although many of the molecular deregulations are similar to those found in mice, there are significant differences. The most unique finding is the association of elevated central carbon metabolism with senescence. Due to the lack of specific markers, the isolation and collection of senescent cells have yet to be developed, especially for human HSPC. The GUhigh subset from the human HSPC compartment possesses all the transcriptome characteristics of senescence. This property may be exploited to accurately enrich, visualize, and trace senescence development in human bone marrow.