Human papillomavirus16 E6 but not E7 upregulates GLUT1 expression in lung cancer cells by upregulating thioredoxin expression

Background and objective: E6 and E7 proteins in human papillomavirus (HPV) 16 are major oncogenes in several types of tumors, including lung cancer. Previous studies have demonstrated that both E6 and E7 oncoproteins can upregulate GLUT1 protein and mRNA expression levels in lung cancer cells. Thus, the present study aimed to investigate the main differences in the molecular mechanisms of GLUT1 expression regulated by E6 and E7. Methods: The double directional genetic manipulation and immunofluorescence were performed to explore the molecular mechanism of E6 or E7 upregulating the expression of GLUT1 in H1299 and A549 cell lines. Results: The overexpression of E6 in well-established lung cancer cell lines upregulated thioredoxin (Trx) protein expression. Notably, plasmid transfection or small interfering RNA transfection with E7 had no regulatory effect on Trx expression. As an important disulfide reductase of the intracellular antioxidant system, Trx plays important role in maintaining oxidative stress balance and protecting cells from oxidative damage. The overexpression of Trx increased the activation of NF-κB by upregulating p65 expression and promoting p65 nuclear translocation, and further upregulated GLUT1 protein and mRNA expression levels. The results of the present study demonstrated that E6, but not E7, upregulated GLUT1 expression in lung cancer cells by activating NF-κB due to the participation of Trx. Conclusion: These results suggest that Trx plays an important role in the pathogenesis of HPV-associated lung cancer, and propose a novel therapeutic target for HPV-associated lung cancer.

Cbl Negatively Regulates NLRP3 Inflammasome Activation through GLUT1-Dependent Glycolysis Inhibition

Activation of the nod-like receptor 3 (NLRP3) inflammasomes is crucial for immune defense, but improper and excessive activation causes inflammatory diseases. We previously reported that Cbl plays a pivotal role in suppressing NLRP3 inflammasome activation by inhibiting Pyk2-mediated apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization. Here, we showed that Cbl dampened NLRP3 inflammasome activation by inhibiting glycolysis, as demonstrated with Cbl knockout cells and treatment with the Cbl inhibitor hydrocotarnine. We revealed that the inhibition of Cbl promoted caspase-1 cleavage and interleukin (IL)-1β secretion through a glycolysis-dependent mechanism. Inhibiting Cbl increased cellular glucose uptake, glycolytic capacity, and mitochondrial oxidative phosphorylation capacity. Upon NLRP3 inflammasome activation, inhibiting Cbl increased glycolysis-dependent activation of mitochondrial respiration and increased the production of reactive oxygen species, which contributes to NLRP3 inflammasome activation and IL-1β secretion. Mechanistically, inhibiting Cbl increased surface expression of glucose transporter 1 (GLUT1) protein through post-transcriptional regulation, which increased cellular glucose uptake and consequently raised glycolytic capacity, and in turn enhanced NLRP3 inflammasome activation. Together, our findings provide new insights into the role of Cbl in NLRP3 inflammasome regulation through GLUT1 downregulation. We also show that a novel Cbl inhibitor, hydrocortanine, increased NLRP3 inflammasome activity via its effect on glycolysis.

OR14-07 Association Between Placental Glucose Uptake and Protein O-Glcnacylation and Birth Outcomes in Obese Non-Diabetic Mothers

Increased transport of nutrients such as glucose, across the placenta, has been linked to abnormal fetal growth and pregnancy complications in obese non-diabetic mothers (1); however, the underlying mechanisms are poorly understood. We hypothesized that in placenta, the metabolic and nutrient sensor O-GlcNAc transferase (OGT), highly sensitive to glucose flux through the hexosamine biosynthetic pathway (HBP), responds to maternal obesogenic environment by increasing O-GlcNAc post-translational modification of nucleocytoplasmic proteins in the placenta altering fetal growth trajectories. Tissue biopsies were isolated from placentas collected at term from 26 non-diabetic mothers alongside routine biochemistry and anthropometric data (maternal fasting glucose, glycated hemoglobin (HbA1c), early pregnancy body weight (BMI) and birth weight). OGT and glucose transporter 1 (GLUT1) protein expression as well as tissue levels of O-GlcNAcylation were determined by immunoblotting using specific antibodies. The BeWo choriocarcinoma cell line was also used as an in vitro model of trophoblast to test the effect of high glucose and GLUT1 silencing on the OGT activity by immunoblotting. Maternal BMI was positively correlated to birth weight centile (BWC) (p=0.0130, R2=0.231), maternal fasting glucose (p=0.0177, R2=0.221) and HbA1c levels (p= 0.0156, R2=0.229) as well as placental OGT protein expression (p=0.0294, R2=0.183). The latter was positively associated to the levels of protein O-GlcNAcylation (p=0.0023, R2=0.326). Interestingly, GLUT1 protein levels were positively correlated to BWC (p=0.0056, R2=0.279) and strongly correlated to protein O-GlcNAcylation (p<0.0001, R2=0.507), suggesting an increase in the placental flux of glucose. In agreement with findings in placenta biopsies, in BeWo cells total protein O-GlcNAcylation levels were altered by cell exposure to different glucose levels (5 mM vs 15 mM, p<0.01). This was prevented by downregulating OGT or GLUT1 expression (p<0.001) using gene silencing. In addition, OGT protein levels were negatively associated to AMP-activated protein kinase (AMPK) activation (p=0.0005, R2=0.402) in placenta biopsies identifying a novel cross-talk between two placental nutrient sensors, OGT and AMPK, previously shown in other tissues (2). Accordingly, the silencing of OGT promoted the activation of AMPK (p<0.01) and its downstream target acetyl-CoA carboxylase (ACC) (p<0.01) in BeWo cells, as demonstrated by increased phosphorylation of residues Thr172 and Ser79 for AMPK and ACC respectively. Such obesity-associated cross talk between metabolic and nutrient sensors might disrupt multiple cellular pathways involved in fetal development and growth. References: (1) Acosta et al. Am J Obstet Gynecol. 2015 Feb;212(2):227. (2) Bullen et al. J Biol Chem. 2014 Apr 11;289(15):10592-606.

Distinct roles of ezrin, radixin and moesin in maintaining the plasma membrane localizations and functions of human blood–brain barrier transporters

The purpose of this study was to clarify the roles of ERM proteins (ezrin/radixin/moesin) in the regulation of membrane localization and transport activity of transporters at the human blood–brain barrier (BBB). Ezrin or moesin knockdown in a human in vitro BBB model cell line (hCMEC/D3) reduced both BCRP and GLUT1 protein expression levels on the plasma membrane. Radixin knockdown reduced not only BCRP and GLUT1, but also P-gp membrane expression. These results indicate that P-gp, BCRP and GLUT1 proteins are maintained on the plasma membrane via different ERM proteins. Furthermore, moesin knockdown caused the largest decrease of P-gp and BCRP efflux activity among the ERM proteins, whereas GLUT1 influx activity was similarly reduced by knockdown of each ERM protein. To investigate how moesin knockdown reduced P-gp efflux activity without loss of P-gp from the plasma membrane, we examined the role of PKCβI. PKCβI increased P-gp phosphorylation and reduced P-gp efflux activity. Radixin and moesin proteins were detected in isolated human brain capillaries, and their protein abundances were within a 3-fold range, compared with those in hCMEC/D3 cell line. These findings may mean that ezrin, radixin and moesin maintain the functions of different transporters in different ways at the human BBB.

PO-278 The Mechanism Of LIL combined with AZD8055 On The Glutamine Addiction Of CT26 Cells

Objective The photobiomodulation of low-intensity lasers can activate many pathways and molecules, which involved in tumor growth, invasion, metastasis, and resistance to drugs or radiation therapy, but studies of low-intensity lasers on tumor cell addiction, have not been reported. Numerous studies have shown that changes in plasma and muscle glutamine levels can occur when the body undergoes prolonged exercise (>1 hour) or overtraining. AZD8055 is a dual inhibitor of mammalian rapamycin complex (mTORC)1/mTORC2 that inhibits phosphorylation of mTORC1 (p70S6K and 4E-BP1), mTORC2 (AKT) and downstream proteins. Based on the experimental model of glutamine addiction in laboratory, we chose the worst conditions to cause super-proliferation of CT26 cells and simulate the drug resistance of solid tumors. The purpose is to study tumor deterioration from the perspective of tumor cell function. Under the state, the effect of low-intensity laser combined with AZD8055 on the proliferation of glutamine addiction in CT26 cells is also the development of exercise intervention tumors, and the experimental basis is proposed from the perspective of nutrient metabolism. Methods This experiment used a pre-experimental glutamine addiction model to culture mouse colon cancer CT26 cells after horse serum shock, and used different intensity 640±15 nm low-intensity lasers from light-emitting diode arrays (red light at 640±15 nm from Light emitting diode array, RLED), 15 min/day；different concentrations of AZD8055 (0 ~ 100μM) on different proliferative CT26 cells at different times. Cell proliferation was detected by MTT assay; mRNA levels of proliferation-related genes were detected by RT-PCR; protein levels were detected by western blot. The data were analyzed by SPSS21.0 statistical software and self-similarity index analysis. Among them, the statistical significance level was set at 0.05, P<0.05 was different; P<0.01 was significant difference. In the self-similarity index analysis method, l>0.8 is a significant difference. Results 1.CT26 cells were incubated at Gln7.94 mM until the 8th day, and AZD8055 was added at different concentrations. Compared with no inhibitor , and at 6 h, the cells were significantly increased when the concentration of AZD8055 was 0.001 μM, 1 μM, 10 μM and 100 μM. (P<0.01); at 12h, the cells were significantly inhibition when the of AZD8055>0.5μM (P<0.01). 2.From the growth inhibition rate analysis, at 48 h and 72 h, the inhibition rate exceeded 50%,when the concentration of AZD8055 was 100 μM (61.01% and 87.46%), when< 0.1 μM, the cells were proliferation occurred at 48 h and 72 h, and it is drug resistance. 3.When AZD8055 combined with low-intensity laser treatment of CT26 cells, CT26 cells had different degrees of proliferative effects at different time points: 12h, 24h, 48h and 72h. Especially at 48h, the light intensity of LIL2 to LIL8 had a significant proliferative effect on CT26 cells (l≥0.8). 4.Compared with the control group (0 hours without inhibitor), the CT26 cells were treatment with AZD8055 1μM , the mRNA level of the gene GLUT1 was significantly down-regulated in all time periods (P<0.01). When the LIL(2.17 mW/cm2) combined AZD8055 (1μM) significantly promoted the proliferation of CT26 cells at 24h and 48h. It is related to up-regulated of protein expression in all time points: at 6h and 12h, the pMEK protein and the pMEK/ MEK were significantly up-regulated(P<0.01). At 24h, the expression of GLUT1 protein was up-regulated(P<0.01), MEK protein, pERK protein and ERK/ pERK were significantly up-regulated (P<0.01). At 48h, the GLUT1 protein, MEK protein , pERK protein and pERK/ERK were up-regulated (P<0.01). Conclusions 1.The minimum inhibitory concentration of AZD8055 on super-proliferative CT26 cells was 0.5 μM, and the super-proliferative state showed more obvious drug resistance. 2.When low-intensity lasers combined with AZD8055 treated CT26 super-proliferating cells, CT26 cells are not sensitive to AZD8055, while low-intensity lasers promote CT26 cells proliferation by up-regulating MEK/ERK signaling pathways.

Sesquiterpenes from Baizhu Stimulate Glucose Uptake by Activating AMPK and PI3K

Baizhu, the dried rhizome of Atractylodes Macrocephala Koidz (Compositae), is one of the most important traditional Chinese herbal medicines. Baizhu is generally used to treat digestive disorders and diabetes in Asian countries. This study investigates the activity of two sesquiterpenes isolated from Baizhu, atractylenolide I (AT-I) and atractylenolide II (AT-II), for their effects on glucose uptake in mouse skeletal muscle C2C12 cells, and the corresponding mechanism. These compounds show a significant stimulatory effect on glucose uptake in C2C12 myotubes. Both AT-I and AT-II significantly increased GLUT4 but not GLUT1 protein levels, and promoted GLUT4 translocation to the plasma membrane. The increased glucose uptake induced by these compounds is associated with activation of AMP-activated protein kinase (AMPK) and PI3K/Akt pathways in these cells. Further studies have indicated that AT-I and AT-II ameliorate TNF-[Formula: see text]-induced insulin resistance in C2C12 myotubes. In summary, our findings highlight the insulin mimetic activity of Baizhu in myotubes, and provide insights into the action mechanism underlying these effects. Our findings may also prove highly relevant to the development of novel therapeutic applications for these compounds.

Loss of neuronatin promotes “browning” of primary mouse adipocytes while reducing Glut1-mediated glucose disposal

Failure of white adipose tissue to appropriately store excess metabolic substrate seems to underpin obesity-associated type 2 diabetes. Encouraging “browning” of white adipose has been suggested as a therapeutic strategy to help dispose of excess stored lipid and ameliorate the resulting insulin resistance. Genetic variation at the DNA locus encoding the novel proteolipid neuronatin has been associated with obesity, and we recently observed that neuronatin expression is reduced in subcutaneous adipose tissue from obese humans. Thus, to explore the function of neuronatin further, we used RNAi to silence its expression in murine primary adipocyte cultures and examined the effects on adipocyte phenotype. We found that primary adipocytes express only the longer isoform of neuronatin. Loss of neuronatin led to increased mitochondrial biogenesis, indicated by greater intensity of MitoTracker Green staining. This was accompanied by increased expression of UCP1 and the key genes in mitochondrial oxidative phosphorylation, PGC-1α, Cox8b, and Cox4 in primary subcutaneous white adipocytes, indicative of a “browning” effect. In addition, phosphorylation of AMPK and ACC was increased, suggestive of increased fatty acid utilization. Similar, but less pronounced, effects of neuronatin silencing were also noted in primary brown adipocytes. In contrast, loss of neuronatin caused a reduction in both basal and insulin-stimulated glucose uptake and glycogen synthesis, likely mediated by a reduction in Glut1 protein upon silencing of neuronatin. In contrast, loss of neuronatin had no effect on insulin signaling. In conclusion, neuronatin appears to be a novel regulator of browning and metabolic substrate disposal in white adipocytes.

Expression of conventional and novel glucose transporters, GLUT1, -9, -10, and -12, in vascular smooth muscle cells

Intimal hyperplasia is characterized by exaggerated proliferation of vascular smooth muscle cells (VSMCs). Enhanced VSMC growth is dependent on increased glucose uptake and metabolism. Facilitative glucose transporters (GLUTs) are comprised of conventional GLUT isoforms (GLUT1–5) and novel GLUT isoforms (GLUT6–14). Previous studies demonstrate that GLUT1 overexpression or GLUT10 downregulation contribute to phenotypic changes in VSMCs. To date, the expression profile of all 14 GLUT isoforms has not been fully examined in VSMCs. Using the proliferative and differentiated phenotypes of human aortic VSMCs, the present study has determined the relative abundance of GLUT1–14 mRNAs by quantitative real-time PCR analysis. Twelve GLUT mRNAs excluding GLUT7 and GLUT14 were detectable in VSMCs. In the proliferative phenotype, the relative abundance of key GLUT mRNAs was GLUT1 (∼43%) > GLUT10 (∼26%) > GLUT9 (∼13%) > GLUT12 (∼4%), whereas in the differentiated phenotype the relative abundance was GLUT10 (∼28%) > GLUT1 (∼25%) > GLUT12 (∼20%) > GLUT9 (∼14%), together constituting 86–87% of total GLUT transcripts. To confirm the expression of key GLUT proteins, immunoblot and immunocytochemical analyses were performed using GLUT isoform-specific primary antibodies. The protein bands characteristic of GLUT1, -9, -10, and -12 were detected in VSMCs in parallel with respective positive controls. In particular, GLUT1 protein expression showed different molecular forms representative of altered glycosylation. While GLUT1 protein displayed a predominant distribution in the plasma membrane, GLUT9, -10, and -12 proteins were mostly distributed in the intracellular compartments. The present study provides the first direct evidence for GLUT9 and GLUT12 expression in VSMCs in conjunction with the previously identified GLUT1 and GLUT10.