GLUT3-mediated cigarette smoke-induced epithelial-mesenchymal transition in chronic obstructive pulmonary disease through the NF-kB/ZEB1 pathway.

BACKGROUND: Airway remodelling plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Epithelial-mesenchymal transition (EMT) is a significant process during the occurrence of airway remodelling. Increasing evidence suggests that glucose transporter 3 (GLUT3) is involved in the epithelial mesenchymal transition (EMT) process of various diseases. However, the role of GLUT3 in EMT in the airway epithelial cells of COPD patients remains unclear. METHODS: We detected the levels of GLUT3 in the peripheral lung tissue of COPD patients and cigarette smoke (CS)-exposed mice. Two Gene Expression Omnibus GEO datasets were utilised to analyse GLUT3 gene expression profiles in COPD. Western blot and immunofluorescence were used to detect GLUT3 expression. In addition, we used the AAV9-GLUT3 inhibitor to reduce GLUT3 expression in the mice model. Masson's staining and lung function measurement were used detect the collagen deposition and penh in the mice. A cell study was performed to confirm the regulatory effect of GLUT3. Inhibition of GLUT3 expression with siRNA, Western blot, and immunofluorescence were used to detect the expression of E-cadherin, N-cadherin, vimentin, p65, and ZEB1. RESULTS: Based on the GEO data set analysis, GLUT3 expression in COPD patients was higher than in non-smokers. Moreover, GLUT3 was highly expressed in COPD patients, CS exposed mice, and BEAS-2B cells treated with CS extract (CSE). Further research revealed that down-regulation of GLUT3 significantly alleviated airway remodelling in vivo and in vitro. Lung function measurement showed that GLUT3 reduction reduced airway resistance in experimental COPD mice. Mechanistically, our study showed that reduction of GLUT3 inhibited CSE-induced EMT by down-regulating the NF-κB/ZEB1 pathway. CONCLUSION: We demonstrate that CS enhances the expression of GLUT3 in COPD and further confirm that GLUT3 may regulate airway remodelling in COPD through the NF-κB/ZEB1 pathway; these findings have potential value in the diagnosis and treatment of COPD. The down-regulation of GLUT3 significantly alleviated airway remodelling and reduced airway resistance in vivo. Our observations uncover a key role of GLUT3 in modulating airway remodelling and shed light on the development of GLUT3-targeted therapeutics for COPD.

Tumor-associated macrophages drive glycolysis through the IL-8/STAT3/GLUT3 signaling pathway in pancreatic cancer progression.

Glycolytic metabolism is a hallmark of pancreatic ductal adenocarcinoma (PDAC), and tumor-associated stromal cells play important roles in tumor metabolism. We previously reported that tumor-associated macrophages (TAMs) facilitate PDAC progression. However, little is known about whether TAMs are involved in regulating glycolysis in PDAC. Here, we found a positive correlation between CD68+ TAM infiltration and FDG maximal standardized uptake (FDG SUVmax) on PET-CT images of PDAC. We discovered that the glycolytic gene set was prominently enriched in the high TAM infiltration group through Gene Set Enrichment Analysis using The Cancer Genome Atlas database. Mechanistically, TAMs secreted IL-8 to promote GLUT3 expression in PDAC cells, enhancing tumor glycolysis both in vitro and in vivo, whereas this effect could be blocked by the IL-8 receptor inhibitor reparixin. Furthermore, IL-8 promoted the translocation of phosphorylated STAT3 into the nucleus to activate the GLUT3 promoter. Overall, we demonstrated that TAMs boosted PDAC cell glycolysis through the IL-8/STAT3/GLUT3 signaling pathway. Our cumulative findings suggest that the abrogation of TAM-induced tumor glycolysis by reparixin might exhibit an antitumor impact and offer a potential therapeutic target for PDAC.

Glut-3 Gene Knockdown as a Potential Strategy to Overcome Glioblastoma Radioresistance.

The hypoxic pattern of glioblastoma (GBM) is known to be a primary cause of radioresistance. Our study explored the possibility of using gene knockdown of key factors involved in the molecular response to hypoxia, to overcome GBM radioresistance. We used the U87 cell line subjected to chemical hypoxia generated by CoCl2 and exposed to 2 Gy of X-rays, as single or combined treatments, and evaluated gene expression changes of biomarkers involved in the Warburg effect, cell cycle control, and survival to identify the best molecular targets to be knocked-down, among those directly activated by the HIF-1α transcription factor. By this approach, glut-3 and pdk-1 genes were chosen, and the effects of their morpholino-induced gene silencing were evaluated by exploring the proliferative rates and the molecular modifications of the above-mentioned biomarkers. We found that, after combined treatments, glut-3 gene knockdown induced a greater decrease in cell proliferation, compared to pdk-1 gene knockdown and strong upregulation of glut-1 and ldha, as a sign of cell response to restore the anaerobic glycolysis pathway. Overall, glut-3 gene knockdown offered a better chance of controlling the anaerobic use of pyruvate and a better proliferation rate reduction, suggesting it is a suitable silencing target to overcome radioresistance.

Altered Regulation of the Glucose Transporter GLUT3 in PRDX1 Null Cells Caused Hypersensitivity to Arsenite.

Targeting tumour metabolism through glucose transporters is an attractive approach. However, the role these transporters play through interaction with other signalling proteins is not yet defined. The glucose transporter SLC2A3 (GLUT3) is a member of the solute carrier transporter proteins. GLUT3 has a high affinity for D-glucose and regulates glucose uptake in the neurons, as well as other tissues. Herein, we show that GLUT3 is involved in the uptake of arsenite, and its level is regulated by peroxiredoxin 1 (PRDX1). In the absence of PRDX1, GLUT3 mRNA and protein expression levels are low, but they are increased upon arsenite treatment, correlating with an increased uptake of glucose. The downregulation of GLUT3 by siRNA or deletion of the gene by CRISPR cas-9 confers resistance to arsenite. Additionally, the overexpression of GLUT3 sensitises the cells to arsenite. We further show that GLUT3 interacts with PRDX1, and it forms nuclear foci, which are redistributed upon arsenite exposure, as revealed by immunofluorescence analysis. We propose that GLUT3 plays a role in mediating the uptake of arsenite into cells, and its homeostatic and redox states are tightly regulated by PRDX1. As such, GLUT3 and PRDX1 are likely to be novel targets for arsenite-based cancer therapy.

GLUT3 promotes macrophage signaling and function via RAS-mediated endocytosis in atopic dermatitis and wound healing.

The facilitative GLUT1 and GLUT3 hexose transporters are expressed abundantly in macrophages, but whether they have distinct functions remains unclear. We confirmed that GLUT1 expression increased after M1 polarization stimuli and found that GLUT3 expression increased after M2 stimulation in macrophages. Conditional deletion of Glut3 (LysM-Cre Glut3fl/fl) impaired M2 polarization of bone marrow-derived macrophages. Alternatively activated macrophages from the skin of patients with atopic dermatitis showed increased GLUT3 expression, and a calcipotriol-induced model of atopic dermatitis was rescued in LysM-Cre Glut3fl/fl mice. M2-like macrophages expressed GLUT3 in human wound tissues as assessed by transcriptomics and costaining, and GLUT3 expression was significantly decreased in nonhealing, compared with healing, diabetic foot ulcers. In an excisional wound healing model, LysM-Cre Glut3fl/fl mice showed significantly impaired M2 macrophage polarization and delayed wound healing. GLUT3 promoted IL-4/STAT6 signaling, independently of its glucose transport activity. Unlike plasma membrane-localized GLUT1, GLUT3 was localized primarily to endosomes and was required for the efficient endocytosis of IL-4Rα subunits. GLUT3 interacted directly with GTP-bound RAS in vitro and in vivo through its intracytoplasmic loop domain, and this interaction was required for efficient STAT6 activation and M2 polarization. PAK activation and macropinocytosis were also impaired without GLUT3, suggesting broader roles for GLUT3 in the regulation of endocytosis. Thus, GLUT3 is required for efficient alternative macrophage polarization and function, through a glucose transport-independent, RAS-mediated role in the regulation of endocytosis and IL-4/STAT6 activation.

Environmentally relevant perinatal exposure to DBP accelerated spermatogenesis by promoting the glycolipid metabolism of Sertoli cells in male offspring mice.

Since Sertoli cells (SCs) play an essential role in providing energy for spermatogenesis, the present study aimed to investigate the effects of maternal exposure to plasticizer Dibutyl phthalate (DBP) on the onset of spermatogenesis in male offspring through the metabolism pathway as well as the underlying molecular mechanism. Here, pregnant mice were treated with 0 (control), 50, 250, or 500 mg/kg/day DBP in 1 mL/kg corn oil administered daily by oral gavage from gestation day (GD) 12.5 to parturition. The in vivo results showed that 50 mg/kg/day DBP exposure could promote the expression of glucose metabolism-related proteins (GLUT3, LDHA, and MCT4) in the testis of 22 days male offspring. The in vitro results demonstrated that 0.1 mM monobutyl phthalate (MBP, the active metabolite of DBP) promoted the lactate production, glucose consumption, and glycolytic flux of immature SCs, which was paralleled by the upregulated expression of glucose metabolism-related proteins (GLUT1, GLUT3, LDHA, and MCT4). On the other hand, DBP/MBP increased fatty acid (FA) uptake, FA ß-oxidation, and ATP production by promoting the expression of CD36 in immature SCs, which might accelerate the maturity of SCs to support the onset of spermatogenesis. Therefore, our findings provided a new perspective on glycolipid metabolism to explain prenatal DBP exposure leading to earlier onset of spermatogenesis in male offspring mice.

Effect of glucose depletion and fructose administration during chondrogenic commitment in human bone marrow-derived stem cells.

BACKGROUND: Bone marrow mesenchymal stromal cells (BMSCs) are promising for therapeutic use in cartilage repair, because of their capacity to differentiate into chondrocytes. Often, in vitro differentiation protocols employ the use of high amount of glucose, which does not reflect cartilage physiology. For this reason, we investigated how different concentrations of glucose can affect the chondrogenic differentiation of BMSCs in cell culture pellets. Additionally, we investigated how fructose could influence the chondrogenic differentiation in vitro. METHODS: BMSC were isolated from six donors and cultured in DMEM containing glucose at either 25 mM (HG), 5.5 mM (LG) or 1 mM (LLG), and 1% non-essential amino acids, 1% ITS+, in the presence of 100 nM dexamethasone, 50 µg/ml ascorbic acid-2 phosphate and 10 ng/ml TGF-ß1. To investigate the effect of different metabolic substrates, other groups were exposed to additional 25 mM fructose. The media were replaced every second day until day 21 when all the pellets were harvested for further analyses. Biochemical analysis for glycosaminoglycans into pellets and released in medium was performed using the DMMB method. Expression of GLUT3 and GLUT5 was assayed by qPCR and validated using FACS analysis and immunofluorescence in monolayer cultures. Chondrogenic differentiation was further confirmed by qPCR analysis of COL2A1, COL1A1, COL10A1, ACAN, RUNX2, SOX9, SP7, MMP13, and PPARG, normalized on RPLP0. Type 2 collagen expression was subsequently validated by immunofluorescence analysis. RESULTS: We show for the first time the presence of fructose transporter GLUT5 in BMSC and its regulation during chondrogenic commitment. Additionally, decreasing glucose concentration during chondrogenesis dramatically decreased the yield of differentiation. However, the use of fructose alone or together with low glucose concentrations does not limit cell differentiation, but on the contrary it might help in maintaining a stable chondrogenic phenotype comparable with the standard culture conditions (high glucose). CONCLUSION: This study provides evidence that BMSC express GLUT5 and differentially regulate GLUT3 in the presence of glucose variation. This study gives a better comprehension of BMSCs sugar use during chondrogenesis.

Enforcing GLUT3 expression in CD8+ T cells improves fitness and tumor control by promoting glucose uptake and energy storage.

Despite the tremendous success of adoptive T-cell therapies (ACT) in fighting certain hematologic malignancies, not all patients respond, a proportion experience relapse, and effective ACT of most solid tumors remains elusive. In order to improve responses to ACT suppressive barriers in the solid tumor microenvironment (TME) including insufficient nutrient availability must be overcome. Here we explored how enforced expression of the high-affinity glucose transporter GLUT3 impacted tumor-directed T cells. Overexpression of GLUT3 in primary murine CD8+ T cells enhanced glucose uptake and increased glycogen and fatty acid storage, and was associated with increased mitochondrial fitness, reduced ROS levels, higher abundance of the anti-apoptotic protein Mcl-1, and better resistance to stress. Importantly, GLUT3-OT1 T cells conferred superior control of B16-OVA melanoma tumors and, in this same model, significantly improved survival. Moreover, a proportion of treated mice were cured and protected from re-challenge, indicative of long-term T cell persistence and memory formation. Enforcing expression of GLUT3 is thus a promising strategy to improve metabolic fitness and sustaining CD8+ T cell effector function in the context of ACT.

Comprehensive Analysis of the Role of SLC2A3 on Prognosis and Immune Infiltration in Head and Neck Squamous Cell Carcinoma.

Background: SLC2A3 is upregulated in various cancer types and promotes proliferation, invasion, and metabolism. However, its role in the prognosis and immune regulation of head and neck squamous cell carcinoma (HNSCC) is still obscure. This study is aimed at exploring the prognostic and immunotherapeutic potential of SLC2A3 in HNSCC. Methods: All data were downloaded from TCGA database and integrated via R software. SLC2A3 expression was evaluated using R software, TIMER, CPTAC, and HPA databases. The association between SLC2A3 expression and clinicopathologic characteristics was assessed by R software. The effect of SLC2A3 on survival was analyzed by R software and Kaplan-Meier Plotter. Genomic alterations in SLC2A3 were investigated using the cBioPortal database. Coexpression of SLC2A3 was studied using LinkedOmics and STRING, and enrichment analyses were performed with R software. The relationship between SLC2A3 expression and immune infiltration was determined using TIMER and TISIDB databases. Immune checkpoints and ESTIMATE score were analyzed via the SangerBox database. Results: SLC2A3 expression was upregulated in HNSCC tissues compared to normal tissues. It was significantly related to TNM stage, histological grade, and alcohol history. High SLC2A3 expression was associated with poor prognosis in HNSCC. Coexpression analysis indicated that SLC2A3 mostly participated in the HIF-1 signaling pathway and glycolysis. Furthermore, SLC2A3 expression strongly correlated with tumor-infiltrating lymphocytes in HNSCC. Conclusion: SLC2A3 could serve as a potential prognostic biomarker for tumor immune infiltration in HNSCC.

ß-Carotene regulates glucose transport and insulin resistance in gestational diabetes mellitus by increasing the expression of SHBG.

The aim of this work was to study the effect and mechanism of ß-carotene on insulin resistance and glucose transport in gestational diabetes mellitus (GDM). Placental tissue and venous blood of 26 GDM patients and 18 normal women were collected. Mice fed a high-fat diet were established as GDM models and treated with ß-carotene, from which peripheral blood and placenta tissue were collected. HTR-8/SVneo cells were treated with 10-7  mol/L insulin for 48 h and established as insulin resistance cell models. The expression of SHBG, GLUT1, GLUT3, GLUT4, IRS-1, IRS-2, PI3Kp85α, and p-CREB/CREB in placental tissues and HTR-8/SVneo cells was detected. Insulin resistance index was calculated from the values of fasting blood glucose and fasting insulin. The glucose consumption of insulin-resistant cells was calculated by detecting the glucose content of the supernatant. The cyclic adenosine monophosphate (cAMP) kit was applied to measure the concentration of cAMP in cells. SHBG was lowly expressed in GDM. ß-Carotene treatment upregulated SHBG in the placenta of GDM mice and in insulin-resistant HTR-8/SVneo cells. Overexpression of SHBG upregulated GLUT3, GLUT4, and IRS-1 and enhanced glucose consumption in insulin-resistant cells. ß-Carotene treatment promoted the expression of SHBG, GLUT4 and IRS-1 and increased glucose consumption in insulin-resistant cells underexpressing SHBG. Silencing of SHBG decreased the levels of cAMP and pCREB/CREB but ß-carotene treatment increased their expression despite SHBG silencing in insulin-resistant cells. ß-Carotene promotes glucose transport and inhibits insulin resistance in GDM by increasing the expression of SHBG.

[Effect of Rehmanniae Radix Praeparata on energy metabolism in prefrontal cortex of rats with attention deficit hyperactivity disorder based on "static Yin and dynamic Yang" theory].

The present study investigated the effect of Rehmanniae Radix Praeparata(RRP) on the energy metabolism of prefrontal cortex(PFC) of spontaneously hypertensive rats with attention deficit hyperactivity disorder(ADHD) based on the "static Yin and dynamic Yang" theory.Thirty spontaneously hypertensive male rats aged 3 weeks were randomly divided into a model group, a methylphenidate(MPH) group(2 mg·kg~(-1)), and an RRP group(2.4 g·kg~(-1)).Wistar-Kyoto(WKY) male rats of the same age were assigned to the normal group.Rats were treated with corresponding drugs twice per day, and those in the model group and the normal group received the same volume of 0.9% sodium carboxymethyl cellulose(CMC-Na) solution by gavage.The open-field test was performed to evaluate the spontaneous and impulsive behaviors of rats before treatment and on the 4~(th) week after treatment.Four weeks after treatment, PFC was isolated and mitochondria were prepared.The content of adenosine triphosphate(ATP), adenosine diphosphate(ADP), and adenosine monophosphate(AMP) in the PFC was determined by high-performance liquid chromatography(HPLC), and energy charge(EC) was calculated.The parameters related to mitochondrial respiratory function were measured by the Clark oxygen electrode, specifically, state 3 respiration(ST3), state 4 respiration(ST4), and respiratory control rate(RCR).Enzymatic activities of succinate dehydrogenase(SDH), cytochrome C oxidase(COX), Na~+-K~+-ATPase, and Ca~(2+)-Mg~(2+)-ATPase were measured by chemical colorimetry.Mitochondrial permeability transition pore(mPTP) opening was measured by spectrophotometry.Protein expression of glucose transporter 1(GLUT1) and GLUT3 in PFC was tested by Western blot.Compared with the results in the model group, RRP could significantly reduce the total distance of movement, vertical times, and distance in the central area in the open field test(P<0.05 or P<0.01), increase the content of ATP and EC, decrease the content of AMP(P<0.05), elevate ST3 and RCR(P<0.05), potentiate activities of SDH, COX, Na~+-K~+-ATPase, and Ca~(2+)-Mg~(2+)-ATPase(P<0.05 or P<0.01), inhibit the opening of mPTP, and increase the expression levels of GLUT1 and GLUT3 proteins(P<0.05).It was inferred that RRP could inhibit hyperacti-vity and impulsivity by improving the energy metabolism disorder in PFC of ADHD rats, and its mechanism may be related to the improvement of mitochondrial respiratory function, potentiation of Na~+-K~+-ATPase, Ca~(2+)-Mg~(2+)-ATPase, and mitochondrial respiratory enzymes, inhibition of the opening of mPTP, and up-regulation of the expression of glucose transporter proteins.This study initially reveals the biological connotation of the "static Yin and dynamic Yang" theory in ADHD.

Effect of Hypoxia on Glucose Transporter 1 and 3 Gene Expression in Placental Mesenchymal Stem Cells Derived from Growth-Restricted Fetuses.

(1) Background: Glucose is transferred from maternal blood to the fetus by glucose transporters. What is the effect of hypoxia on the gene expression of placenta glucose transporter 1 (GLUT1) and glucose transporter 3 (GLUT3) in growth-restricted fetus is interesting. (2) Methods: The gene expression of GLUT1 and GLUT3 and the protein expression of HIF-1α were evaluated under nonhypoxic conditions and after 4 and 8 h under hypoxic conditions in placental mesenchymal stem cells derived from monochorionic twin pregnancies with selective intrauterine growth restriction. (3) Results: The gene expressions of GLUT1 and GLUT3 under hypoxia conditions were higher in placental mesenchymal stem cells derived from appropriate-for-gestational-age fetuses than in those from selective intrauterine growth-restricted fetuses. However, the protein expression of hypoxia induced factor-1 α (HIF-1α) at hypoxia condition was not lower in placenta mesenchymal stem cells from selective intrauterine growth-restricted fetuses than in placental mesenchymal stem cells from appropriate-for-gestational-age fetuses. (4) Conclusions: Hypoxia-induced upregulation of GLUT1 and GLUT3 expression was decreased in placental mesenchymal stem cells from selective intrauterine growth-restricted fetuses but not due to decreased HIF-1α expression. Selective growth-restricted fetuses have less capacity for hypoxia-induced upregulation of placental glucose transport.

RIP140 inhibits glycolysis-dependent proliferation of breast cancer cells by regulating GLUT3 expression through transcriptional crosstalk between hypoxia induced factor and p53.

Glycolysis is essential to support cancer cell proliferation, even in the presence of oxygen. The transcriptional co-regulator RIP140 represses the activity of transcription factors that drive cell proliferation and metabolism and plays a role in mammary tumorigenesis. Here we use cell proliferation and metabolic assays to demonstrate that RIP140-deficiency causes a glycolysis-dependent increase in breast tumor growth. We further demonstrate that RIP140 reduces the transcription of the glucose transporter GLUT3 gene, by inhibiting the transcriptional activity of hypoxia inducible factor HIF-2α in cooperation with p53. Interestingly, RIP140 expression was significantly associated with good prognosis only for breast cancer patients with tumors expressing low GLUT3, low HIF-2α and high p53, thus confirming the mechanism of RIP140 anti-tumor activity provided by our experimental data. Overall, our work establishes RIP140 as a critical modulator of the p53/HIF cross-talk to inhibit breast cancer cell glycolysis and proliferation.

Functional Characterization of the Solute Carrier LAT-1 (SLC7A5/SLC2A3) in Human Brain Capillary Endothelial Cells with Rapid UPLC-MS/MS Quantification of Intracellular Isotopically Labelled L-Leucine.

The solute carrier L-type amino acid transporter 1 (LAT-1/SLC7A5) is a viable target for drug delivery to the central nervous system (CNS) and tumors due to its high abundance at the blood-brain barrier and in tumor tissue. LAT-1 is only localized on the cell surface as a heterodimer with CD98, which is not required for transporter function. To support future CNS drug-delivery development based on LAT-1 targeting, we established an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for stable isotopically labeled leucine ([13C6, 15N]-L-leucine), with a dynamic range of 0.1-1000 ng/mL that can be applied for the functional testing of LAT-1 activity when combined with specific inhibitors and, consequently, the LAT-1 inhibition capacity of new compounds. The assay was established in a 96-well format, facilitating high-throughput experiments, and, hence, can support the screening for novel inhibitors. Applicable recommendations of the US Food and Drug Administration and European Medicines Agency for bioanalytical method validation were followed to validate the assay. The assay was applied to investigate the IC50 of two well-known LAT-1 inhibitors on hCMEC/D3 cells: the highly specific LAT-1 inhibitor JPH203, which was also used to demonstrate LAT-1 specific uptake, and the general system L inhibitor BCH. In addition, the [13C6, 15N]-L-leucine uptake was determined on two human brain capillary endothelial cell lines (NKIM-6 and hCMEC/D3), which were characterized for their expressional differences of LAT-1 at the protein and mRNA level and the surface amount of CD98. The IC50 values of the inhibitors were in concordance with previously reported values. Furthermore, the [13C6, 15N]-L-leucine uptake was significantly higher in hCMEC/D3 cells compared to NKIM-6 cells, which correlated with higher expression of LAT-1 and a higher surface amount of CD98. Therefore, the UPLC-MS/MS quantification of ([13C6, 15N]-L-leucine is a feasible strategy for the functional characterization of LAT-1 activity in cells or tissue.

Altered glucose metabolism and its association with carbonic anhydrase 8 in Machado-Joseph Disease.

Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is an autosomal dominant neurodegenerative disease. This disorder is caused by polyglutamine (polyQ)-containing mutant ataxin-3, which tends to misfold and aggregate in neuron cells. We previously demonstrated a protective function of carbonic anhydrase 8 (CA8) in MJD disease models and a decreased glycolytic activity associated with down-regulated CA8 in a human osteosarcoma (OS) cell model. Given that a reduction in body weight accompanied by gait and balance instability was observed in MJD patients and transgenic (Tg) mice, in this study, we aimed to examine whether metabolic defects are associated with MJD and whether CA8 expression is involved in metabolic dysfunction in MJD. Our data first showed that glucose uptake ability decreases in cells harboring mutant ataxin-3, but increases in cells overexpressing CA8. In addition, the expressions of glucose transporter 3 (GLUT3) and phosphofructokinase-1 (PFK1) were significantly decreased in the presence of mutant ataxin-3. Consistently, immunohistochemistry (IHC) showed that GLUT3 was less expressed in cerebella of aged MJD Tg mice, indicating that the dysfunction of GLUT3 may be associated with late-stage disease. On the other hand, transient down-regulation of CA8 revealed decreased expressions of GLUT3 and PFK1 in HEK293 cells harboring wild-type (WT) ataxin-3, but no further reduction of GLUT3 and PFK1 expressions were observed in HEK293 cells harboring mutant ataxin-3. Moreover, immunoprecipitation (IP) and immunofluorescence (IF) demonstrated that interactions exist between ataxin-3, CA8 and GLUT3 in MJD cellular and Tg models. These lines of evidence suggest that CA8 plays an important role in glucose metabolism and has different impacts on cells with or without mutant ataxin-3. Interestingly, the decreased relative abundance of Firmicutes/Bacteroidetes (F/B) ratio in the feces of aged MJD Tg mice coincided with weight loss and metabolic dysfunction in MJD. Taken together, our results are the first to demonstrate the effects of CA8 on glucose metabolism and its involvement in the metabolic defects in MJD disease. Further investigations will be required to clarify the underlying mechanisms for the metabolic defects associated with MJD.

Progesterone partially recovers placental glucose transporters in dexamethasone-induced intrauterine growth restriction.

RESEARCH QUESTION: How does progesterone improve fetal outcome and change the expression of placental glucose transporters (GLUT) in dexamethasone-induced intrauterine growth restriction (IUGR)? DESIGN: A total of 64 rats were divided randomly into four different treatment groups based on daily i.p. injections of either saline or dexamethasone in the presence or absence of progesterone. Injections started on the 15th day of gestation (15dg) and lasted until the day of sacrifice at 19dg or 21dg. Maternal plasma progesterone concentrations were measured by enzyme-linked immunosorbent assay. The gene and protein expression of placental GLUT1 and GLUT3 were evaluated in the placental labyrinth and basal zones by real-time polymerase chain reaction and Western blotting, respectively. The localization of GLUT1 and GLUT3 was evaluated by immunohistochemistry. RESULTS: Dexamethasone induced significant decreases in maternal serum progesterone concentrations (P = 0.029) and placental (P < 0.001) and fetal body (P = 0.009) weights. Dexamethasone also reduced the expression of GLUT1 in the labyrinth zone (P = 0.028) and GLUT3 in both the labyrinth (P = 0.002) and basal zones (P = 0.026). Coadministration of dexamethasone and progesterone prevented the reduction in fetal body weight, placental weight and placental GLUT expression compared with that seen in dexamethasone-treated groups. CONCLUSION: These results suggest that progesterone prevents the significant reduction in fetal and placental weights in dexamethasone-induced IUGR, possibly through improving the expression of placental GLUT.

miR-3189-targeted GLUT3 repression by HDAC2 knockdown inhibits glioblastoma tumorigenesis through regulating glucose metabolism and proliferation.

BACKGROUND: Epigenetic regulations frequently appear in Glioblastoma (GBM) and are highly associated with metabolic alterations. Especially, Histone deacetylases (HDACs) correlates with the regulation of tumorigenesis and cell metabolism in GBM progression, and HDAC inhibitors report to have therapeutic efficacy in GBM and other neurological diseases; however, GBM prevention and therapy by HDAC inhibition lacks a mechanism in the focus of metabolic reprogramming. METHODS: HDAC2 highly express in GBM and is analyzed in TCGA/GEPIA databases. Therefore, HDAC2 knockdown affects GBM cell death. Analysis of RNA sequencing and qRT-PCR reveals that miR-3189 increases and GLUT3 decreases by HDAC2 knockdown. GBM tumorigenesis also examines by using in vivo orthotopic xenograft tumor models. The metabolism change in HDAC2 knockdown GBM cells measures by glucose uptake, lactate production, and OCR/ECAR analysis, indicating that HDAC2 knockdown induces GBM cell death by inhibiting GLUT3. RESULTS: Notably, GLUT3 was suppressed by increasing miR-3189, demonstrating that miR-3189-mediated GLUT3 inhibition shows an anti-tumorigenic effect and cell death by regulating glucose metabolism in HDAC2 knockdown GBM. CONCLUSIONS: Our findings will demonstrate the central role of HDAC2 in GBM tumorigenesis through the reprogramming of glucose metabolism by controlling miR-3189-inhibited GLUT3 expression, providing a potential new therapeutic strategy for GBM treatment.

GLUT3 inhibitor discovery through in silico ligand screening and in vivo validation in eukaryotic expression systems.

The passive transport of glucose and related hexoses in human cells is facilitated by members of the glucose transporter family (GLUT, SLC2 gene family). GLUT3 is a high-affinity glucose transporter primarily responsible for glucose entry in neurons. Changes in its expression have been implicated in neurodegenerative diseases and cancer. GLUT3 inhibitors can provide new ways to probe the pathophysiological role of GLUT3 and tackle GLUT3-dependent cancers. Through in silico screening of an ~ 8 million compounds library against the inward- and outward-facing models of GLUT3, we selected ~ 200 ligand candidates. These were tested for in vivo inhibition of GLUT3 expressed in hexose transporter-deficient yeast cells, resulting in six new GLUT3 inhibitors. Examining their specificity for GLUT1-5 revealed that the most potent GLUT3 inhibitor (G3iA, IC50 ~ 7 µM) was most selective for GLUT3, inhibiting less strongly only GLUT2 (IC50 ~ 29 µM). None of the GLUT3 inhibitors affected GLUT5, three inhibited GLUT1 with equal or twofold lower potency, and four showed comparable or two- to fivefold better inhibition of GLUT4. G3iD was a pan-Class 1 GLUT inhibitor with the highest preference for GLUT4 (IC50 ~ 3.9 µM). Given the prevalence of GLUT1 and GLUT3 overexpression in many cancers and multiple myeloma's reliance on GLUT4, these GLUT3 inhibitors may discriminately hinder glucose entry into various cancer cells, promising novel therapeutic avenues in oncology.

STAT3 mediated regulation of glucose metabolism in leukemia cells.

Cancer cells rewire metabolic pathways as they demand more ATP and building blocks for proliferation. Glucose is the most consumed nutrient by cancer cells and metabolized to lactate even in the presence of oxygen. This phenomenon is called 'aerobic glycolysis'. Also, glucose level is found lower in tumor environment. Leukemia is characterized by abnormal proliferation of hematopoietic cells. STAT3 a transcription factor and an oncogene is upregulated in many tumor types. Despite its well-defined functions, STAT3 has also been proposed as a metabolic regulator. In this study, we aimed to determine the role STAT3 activation in glucose limitation, in leukemia cell lines. K562, NB-4 and HL-60 cells were found sensitive to glucose limitation. In low glucose conditions, total and nuclear STAT3 protein was decreased in all cells. In mitochondria, S727 phosphorylated STAT3 (mitochondrial form) was determined slightly increased in K562 and NB-4 cells. On the other side, ectopically STAT3 expressing cells had increased glucose consumption and less proliferated in low glucose medium. This data suggests that aerobic glycolysis might be upregulated upon STAT3 expression in leukemia cells, in glucose limitation. Furthermore, in this study, it was found that GLUT3 expressing cells did not reduce STAT3 expression in low glucose medium. GLUT3 was previously determined as a molecular marker for cell sensitivity to glucose limitation, therefore, it could be hypothesized as GLUT3 expressing cells might not need to alter STAT3 expression in low glucose level. Overall, our data suggest that leukemia cells rewire glucose metabolism via STAT3 expression in glucose limitation. Elucidating pathways that cause differential phosphorylation of STAT3 and its interaction with other energy regulating pathways in cellular response to glucose limitation might be beneficial to design new drug targets such as STAT3 inhibitors for leukemia treatment.

LINC00667 Promotes Progression of Esophageal Cancer Cells by Regulating miR-200b-3p/SLC2A3 Axis.

BACKGROUND: Recently, more and more evidence indicated that the long non-coding RNA was strictly related to the occurrence and progression of human cancers, including esophageal cancer (EC). We observed that LINC00667 was increased in EC, but the function of LINC00667 was unclear. Therefore, the function and potential molecular mechanism of LINC00667 in the progression of EC need to be further studied. METHODS: Quantitative real-time PCR was used to investigate the levels of LINC00667, miR-200b-3p, and SLC2A3. The levels of protein involved in cell cycle, cell apoptosis, epithelial-mesenchymal transition, as well as SLC2A3 were quantitatived by western blot. The role of LINC00667 in the proliferative, migratory and invasive capabilities of EC cells were measured by cell counting kit-8 assay, EdU assay, flow cytometry assay, wound healing assay and transwell assay, respectively. Interaction between LINC00667 and miR-200b-3p or miR-200b-3p and SLC2A3 were confirmed using a luciferase reporter assay. RESULTS: In this work, we found that LINC00667 expression was up-regulated in EC cell lines, and LINC00667 knockdown inhibited cell proliferation, migration, and invasion in EC cells. In addition, it showed that LINC00667 functioned as competitive endogenous RNA for miR-200b-3p by the DIANA-LncBase database. Moreover, we used targetscan online software to predict SLC2A3 as a target gene of miR-200b-3p. Subsequently, rescue experiments confirmed that knocking out SLC2A3 could reverse the inhibitory effect of miR-200b-3p on EC cells transfected with sh-LINC00667. CONCLUSION: Herein, we revealed the novel mechanism of LINC00667 on regulating metastasis-related gene by sponge regulatory axis during EC metastasis. Our results demonstrated that LINC00667 plays a critical role in metastatic EC by mediating sponge regulatory axis miR-200b-3p/SLC2A3. To explore function of LINC00667/miR-200b-3p/SLC2A3 axis may provide an informative biomarker of malignancy and a highly selective anti-EC therapeutic target.