GLUT1 and GLUT9 as major contributors to glucose influx in HepG2 cells identified by a high sensitivity intramolecular FRET glucose sensor

Genetically encoded FRET glucose nanosensors have proven to be useful for imaging glucose flux in HepG2 cells. However, the dynamic range of the original sensor was limited and thus it did not appear optimal for high throughput screening of siRNA populations for identifying proteins involved in regulation of sugar flux. Here we describe a hybrid approach that combines linker-shortening with fluorophore-insertion to decrease the degrees of freedom for fluorophore positioning leading to improved nanosensor dynamics. We were able to develop a novel highly sensitive FRET nanosensor that shows a 10-fold higher ratio change and dynamic range (0.05-11 mM) in vivo, permitting analyses in the physiologically relevant range. As a proof of concept that this sensor can be used to screen for proteins playing a role in sugar flux and its control, we used siRNA inhibition of GLUT family members and show that GLUT1 is the major glucose transporter in HepG2 cells and that GLUT9 contributes as well, however to a lower extent. GFP fusions suggest that GLUT1 and 9 are preferentially localized to the plasma membrane and thus can account for the transport activity. The improved sensitivity of the novel glucose nanosensor increases the reliability of in vivo glucose flux analyses, and provides a new means for the screening of siRNA collections as well as drugs using high-content screens.     

Differential sensitivity of GLUT1- and GLUT2-expressing beta cells to streptozotocin.

Streptozotocin injection in animals destroys pancreatic beta cells, leading to insulinopenic diabetes. Here, we evaluated the toxic effect of streptozotocin (STZ) in GLUT2(-/-) mice reexpressing either GLUT1 or GLUT2 in their beta cells under the rat insulin promoter (RIPG1 x G2(-/-) and RIPG2 x G2(-/-) mice, respectively). We demonstrated that injection of STZ into RIPG2 x G2(-/-) mice induced hyperglycemia (>20 mM) and an approximately 80% reduction in pancreatic insulin content. In vitro, the viability of RIPG2 x G2(-/-) islets was also strongly reduced. In contrast, STZ did not induce hyperglycemia in RIPG1 x G2(-/-) mice and did not reduce pancreatic insulin content. The viability of in vitro cultured RIPG1 x G2(-/-) islets was also unaffected by STZ. As islets from each type of transgenic mice were functionally indistinguishable, these data strongly support the notion that STZ toxicity toward beta cells depends on the expression of GLUT2.     

Osmotic induction of calcium accumulation in human embryonic kidney cells detected with a high sensitivity FRET calcium sensor

Calcium serves as a second messenger in glucose-triggered insulin secretion of pancreatic cells. Less is known about sugar signaling in non-excitable cells. Here, the high sensitivity FRET calcium sensor TN-XXL was used to characterize glucose-induced calcium responses in non-excitable human embryonic kidney HEK293T cells. HEK293T cells responded to perfusion with glucose with a sustained and concentration-dependent increase in cytosolic calcium levels. Sucrose and mannitol triggered comparable calcium responses, suggesting that the increase of the calcium concentration was caused by osmotic effects. HEK293T cells are characterized by low endogenous glucose uptake capacity as shown with a high sensitivity glucose sensor. Consistently, when glucose influx was artificially increased by co-expression of GLUT glucose transporters, the glucose-induced calcium increase was significantly reduced. Neither calcium depletion, nor gadolinium or thapsigargin were able to inhibit the calcium accumulation. Taken together, membrane impermeable osmolytes such as sucrose and mannitol lead to an increase in calcium levels, while the effect of glucose depends on the cell's glucose uptake capacity and will thus vary between cell types in the body that differ in their glucose uptake capacity.     

Distinct regulation of glucose transport and GLUT1/GLUT3 transporters by glucose deprivation and IGF-I in chromaffin cells

Effects of prolonged metabolic (glucose deprivation) and hormonal [insulin-like growth factor I (IGF-I)] challenge on regulation of glucose transporter (GLUT) expression, glucose transport rate and possible signaling pathways involved were studied in the neuroendocrine chromaffin cell. The results show that bovine chromaffin cells express both GLUT1 and GLUT3. Glucose deprivation and IGF-I activation led to an elevation of GLUT1 and GLUT3 mRNA, the strongest effect being that of IGF-I on GLUT3 mRNA. Both types of stimulus increased the GLUT1 protein content in a cycloheximide (CHX)-sensitive manner, and the glucose transport rate was elevated by 3- to 4-fold after 48 h under both experimental conditions. IGF-I-induced glucose uptake was totally suppressed by CHX. In contrast, only approximately 50% of transport activation in glucose-deprived cells was sensitive to the protein synthesis inhibitor. Specific inhibitors of mTOR/FRAP and p38 MAPK each partially blocked IGF-I-stimulated glucose transport, but had no effect on transport rate in glucose-deprived cells. The results are consistent with IGF-I-activated transport being completely dependent on new GLUT protein synthesis while the enhanced transport in glucose-deprived cells was partially achieved independent of new synthesis of proteins, suggesting a mechanism relying on preexisting transporters.     

Amino acids influence the glucose uptake through GLUT4 in CHO-K1 cells under high glucose conditions

According to studies earlier, amino acids have proven to be antidiabetic, antiglycating, and anticataractogenic. The present study was to explore whether amino acids as mixtures could enhance glucose uptake in CHO-K1 cells specifically. The cells in F-12K1 serum-free medium were exposed to normal (7 mM) and high glucose (12, 17 and 27 mM) in the presence and absence of amino acids mixture (AAM) in varying concentration (2.5, 5 and 10 mM). The mixture 5 and 10 mM AAM increased the 2-deoxyglucose (2DG) uptake at all glucose concentration significantly. There was also a significant increase in the GLUT4 (glucose transporter) translocation as revealed by flow cytometer. Addition of a mixture of amino acids was found to improve cell viability, which got altered by high glucose in the CHO-K1 cells. Amino acids as mixture had a beneficial effect in improving the net utilization of glucose as an additive effect with insulin.     

Localization of erythrocyte/HepG2-type glucose transporter (GLUT1) in human placental villi

Summary The syncytiotrophoblast covering the surface of the placental villi contains the machinery for the transfer of specific substances between maternal and fetal blood, and also serves as a barrier. Existence of a facilitated-diffusion transporter for glucose in the syncytiotrophoblast has been suggested. Using antibodies to erythrocyte/HepG2-type glucose transporter (GLUT1), one isoform of the facilitated-diffusion glucose transporters, we detected a 50 kD protein in human placenta at term. By use of immunohistochemistry, GLUT1 was found to be abundant in both the syncytiotrophoblast and cytotrophoblast. Endothelial cells of the fetal capillaries also showed positive staining for GLUT1. Electron-microscopic examination revealed that GLUT1 was concentrated at both the microvillous apical plasma membrane and the infolded basal plasma membrane of the syncytiotrophoblast. Plasma membrane of the cytotrophoblast was also positive for GLUT1. GLUT1 at the apical plasma membrane of the syncytiotrophoblast may function for the entry of glucose into its cytoplasm, while GLUT1 at the basal plasma membrane may be essential for the exit of glucose from the cytoplasm into the stroma of the placental villi. Thus, GLUT1 at the plasma membranes of syncytiotrophoblast and endothelial cells may play an important role in the transport of glucose across the placental barrier.     

PYK2 as a mediator of endothelin-1/G alpha 11 signaling to GLUT4 glucose transporters

Endothelin-1 (ET-1) signaling through G alpha(q/11) stimulates translocation of intracellular GLUT4 glucose transporters to the plasma membrane of 3T3-L1 adipocytes by an unknown mechanism that requires protein tyrosine phosphorylation and ADP-ribosylation factor 6 (ARF6) but is independent of phosphatidylinositol 3 (PI3)-kinase. In contrast, insulin action on this process requires PI3-kinase but not ARF6. Here we report the identification of two proteins selectively tyrosine-phosphorylated in response to ET-1 but not insulin: the Ca(2+)-activated tyrosine kinase PYK2 and its physiological substrate, the adhesion scaffold protein paxillin. Endogenous paxillin as well as expressed Myc-tagged PYK2 or a Myc-tagged kinase-deficient PYK2 protein were acutely directed to F-actin-rich adhesion sites from the adipocyte cytoplasm in response to ET-1 but not insulin. CADTK-related non-kinase (CRNK) is a dominant negative form of PYK2 containing the C-terminal portion of the protein, which binds paxillin but lacks the PYK2 autophosphorylation site (Tyr(402)). CRNK expression in 3T3-L1 adipocytes inhibited ET-1-mediated F-actin polymerization and translocation of Myc-tagged GLUT4-enhanced green fluorescent protein (EGFP) to the plasma membrane without disrupting insulin action on these processes. These data reveal the tyrosine kinase PYK2 as a required signaling element in the regulation of GLUT4 recycling in 3T3-L1 adipocytes by ET-1, whereas insulin signaling is directed through a different pathway.     

Differential subcellular distribution of glucose transporters GLUT1-6 and GLUT9 in human cancer: ultrastructural localization of GLUT1 and GLUT5 in breast tumor tissues

It has been proposed that the enhanced metabolic activity of tumor cells is accompanied by an increased expression of facilitative hexose transporters (GLUTs). However, a previous immunohistochemical analysis of GLUT1 expression in 154 malignant human neoplasms failed to detect the GLUT1 isoform in 87 tumors. We used 146 normal human tissues and 215 tumor samples to reassess GLUT1 expression. A similar number of samples were used to compare the expression of GLUT2-6 and 9. The classical expression of GLUT1-5 in different normal human tissues was confirmed, however, we were unable to detect GLUT2 in human pancreatic islet cells. GLUT6 was principally detected in testis germinal cells and GLUT9 was localized in kidney, liver, heart, and adrenal. In tumor samples, GLUT1, 2, and 5 were the main transporters detected. GLUT1 was the most widely expressed transporter, however, 42% of the samples had very low-to-negative expression levels. GLUT2 was detected in 31% of the samples, being mainly expressed in breast, colon, and liver carcinoma. GLUT5 was detected in 27% of breast and colon adenocarcinoma, liver carcinoma, lymphomas, and testis seminoma samples. In situ RT-PCR and ultrastructural immunohistochemistry confirmed GLUT5 expression in breast cancer. GLUT6 and 9 are not clearly over-expressed in human cancer. The extensive expression of GLUT2 and 5 (glucose/fructose and fructose transporters, respectively) in malignant human tissues indicates that fructose may be a good energy substrate in tumor cells. Our functional data obtained in vitro in different tumor cells support this hypothesis. Additionally, these results suggest that fructose uptake could be used for positron emission tomography imaging and, may possibly represent a novel target for the development of therapeutic agents in different human cancers.     

Hypothalamic ependymal-glial cells express the glucose transporter GLUT2, a protein involved in glucose sensing

The GLUT2 glucose transporter and the K-ATP-sensitive potassium channels have been implicated as an integral part of the glucose-sensing mechanism in the pancreatic islet beta cells. The expression of GLUT2 and K-ATP channels in the hypothalamic region suggest that they are also involved in a sensing mechanism in this area. The hypothalamic glial cells, known as tanycytes alpha and beta, are specialized ependymal cells that bridge the cerebrospinal fluid and the portal blood of the median eminence. We used immunocytochemistry, in situ hybridization and transport analyses to demonstrate the glucose transporters expressed in tanycytes. Confocal microscopy using specific antibodies against GLUT1 and GLUT2 indicated that both transporters are expressed in alpha and beta tanycytes. In addition, primary cultures of mouse hypothalamic tanycytes were found to express both GLUT1 and GLUT2 transporters. Transport studies, including 2-deoxy-glucose and fructose uptake in the presence or absence of inhibitors, indicated that these transporters are functional in cultured tanycytes. Finally, our analyses indicated that tanycytes express the K-ATP channel subunit Kir6.1 in vitro. As the expression of GLUT2 and K-ATP channel is linked to glucose-sensing mechanisms in pancreatic beta cells, we postulate that tanycytes may be responsible, at least in part, for a mechanism that allows the hypothalamus to detect changes in glucose concentrations.     

Hypothalamic ependymal cells express the glucose transporter GLUT2, a protein involved in glucose sensing

Kinetic analysis of vitamin C uptake has demonstrated that specialized cells take up ascorbic acid (AA), the reduced form of vitamin C, through sodium-AA cotransporters. Recently, two different isoforms of sodium-vitamin C cotransporters (SVCT 1, 2) that mediate high affinity Na⁺-dependent l -ascorbic acid have been cloned. SVCT2 was detected mainly in choroid plexus cells and neurons, however, there are no evidences of SVCT2 expression in glial cells. High concentrations of vitamin C has been demonstrated in brain hypothalamic area. The hypothalamic glial cells, known as alpha and beta tanycytes, are specialized ependymal cells that bridge the cerebrospinal fluid and the portal blood of the median eminence. Our hypothesis postulates that tanycytes take up reduced vitamin C from the portal blood and cerebrospinal fluid generating an high concentration of this vitamin in brain hypothalamic area. In situ immunohistochemical analyses demonstrated that SVCT2 transporter is selectively expressed in apical region of tanycytes. A newly developed primary culture of mouse hypothalamic tanycytes was used to confirm the expression and function of SVCT2 isoform in these cells. Reduced vitamin C uptake was temperature and sodium dependent. Kinetic analysis showed an apparent Km of 20 μ m and a Vmax of 45 pmol/min per million cells for the transport of ascorbic acid. The expression of SVCT2 was confirmed by immunoblots and RT–PCR. Tanycytes may perform a neuroprotective role concentrating the vitamin C in the hypothalamic area.
Acknowledgements:   Supported by Grands FONDECYT 1010843 and DIUC-GIA 201.034.006-1.4 from Concepción University.     

GLUT1-mediated glucose transport and its regulation by IGF-I in cultured bovine chromaffin cells

Regulation of glucose transport was studied in primary cultures of bovine chromaffin cells (BCC) using the glucose analogue 2-deoxyglucose (DOG) as a model substrate. The glucose transporter in freshly isolated and cultured BCC was identified as GLUT1 by Western immunoblots. The level of GLUT1 increased by time in culture and was followed by an enhancement in uptake of DOG. The DOG uptake was stimulated by insulin-like growth factor I (IGF-I) with an EC₅₀ of 1 nM and a maximal response (∼2-fold) was obtained at 10–100 nM IGF-I. Insulin was at least 100-fold less potent than IGF-I. Exposure to 10⁻⁸ M IGF-I also caused a redistribution of GLUT1 from an intracellular compartment to a plasma membrane-enriched fraction. Our results demonstrate a GLUT1-mediated glucose uptake in adrenomedullary cells. An enhanced glucose transport in response to IGF-I appears to be coupled to activation of IGF receptor type 1 and GLUT1 translocation. © 1996 Wiley-Liss, Inc.     

Glypican 3 binds to GLUT1 and decreases glucose transport activity in hepatocellular carcinoma cells

Glypican 3 (GPC3), a member of heparin sulfate proteoglycans, is attached to the cell surface by a glycosylphosphatidylinositol anchor and is reported to be overexpressed in liver cancers. In order to identify GPC3 binding proteins on the cell surface, we constructed a cDNA containing the C‐terminal cell surface‐attached form of GPC3 (GPC3c) in a baculoviral vector. The GPC3c bait protein was produced by expressing the construct in Sf21 insect cells and double purified using a His column and Flag immunoprecipitation. Purified GPC3c was used to uncover GPC3c‐interacting proteins. Using an LC–MS/MS proteomics strategy, we identified glucose transporter 1 (GLUT1) as a novel GPC3 interacting protein from the HepG2 hepatoma cell lysates. The interaction was confirmed by immunoprecipitation (IP)–WB analysis and surface plasmon resonance (SPR). SPR result showed the interaction of GLUT1 to GPC3c with equilibrium dissociation constants (K_D) of 1.61 nM. Moreover, both incubation with GPC3c protein and transfection of Gpc3c cDNA into HepG2 cells resulted in reduced glucose uptake activity. Our results indicate that GPC3 plays a role in glucose transport by interacting with GLUT1. J. Cell. Biochem. 111: 1252–1259, 2010. © 2010 Wiley‐Liss, Inc.     

Selective attenuation of metabolic branch of insulin receptor down-signaling by high glucose in a hepatoma cell line, HepG2 cells

The effects of a high concentration of glucose on the insulin receptor-down signaling were investigated in human hepatoma (HepG2) cells in vitro to delineate the molecular mechanism of insulin resistance under glucose toxicity. Treatment of the cells with high concentrations of glucose (15-33 mm) caused phosphorylation of serine residues of the insulin receptor substrate 1 (IRS-1), leading to reduced electrophoretic mobility of it. The phosphorylation of IRS-1 with high glucose treatment was blocked only by protein kinase C (PKC) inhibitors. The high glucose treatment attenuated insulin-induced association of IRS-1 and phosphatidylinositol 3-kinase and insulin-stimulated phosphorylation of Akt. A metabolic effect of insulin, stimulation of glycogen synthesis, was also inhibited by the treatment. In contrast, insulin-induced association of Shc and Grb2 was not inhibited. Treatment of the cells with high glucose promoted the translocation of PKCepsilon and PKCdelta from the cytosol to the plasma membrane but not that of other PKC isoforms. Finally, PKCepsilon and PKCdelta directly phosphorylated IRS-1 under cell-free conditions. We conclude that a high concentration of glucose causes phosphorylation of IRS-1, leading to selective attenuation of metabolic signaling of insulin. PKCepsilon and PKCdelta are involved in the down-regulation of insulin signaling, and they may lie in a pathway regulating the phosphorylation of IRS-1.     

DAMPs released by pyroptotic cells as major contributors and therapeutic targets for CAR-T-related toxicities

CAR-T transfer, recently well-developed immunotherapy, has offered substantial benefit to more and more patients with advanced cancers. However, along with growing experience in the clinical application comes the increasing awareness of the potentially fatal adverse effects, most notably cytokine release syndrome (CRS) and neurotoxicity. Understanding the mechanisms underlying these toxicities can help to improve therapeutic outcomes. Recent findings highlight the importance of monocyte/macrophage in CAR-T-related toxicities (CARTOX) and shed light on a novel mechanism mediated by damage-associated molecular patterns (DAMPs) released from pyroptotic cells. Therefore, this review summarizes these findings and provides practical guidance to the management of CARTOX.Subject terms: Cell death and immune response, Cancer immunotherapy, Inflammatory diseases     

Polymorphisms at the GLUT1 (HepG2) and GLUT4 (muscle/adipocyte) glucose transporter genes and non-insulin-dependent diabetes mellitus (NIDDM)

Summary In order to determine the possible contribution of the GLUT1 (HepG2) glucose transporter gene to the inheritance of non-insulin-dependent diabetes mellitus (NIDDM), two restriction fragment length polymorphisms (RFLPs) and the related haplotypes at this locus were studied in 48 Italian diabetic patients and 58 normal subjects. Genotype frequencies for the XbaI polymorphism were significantly different between patients and controls (XbaI: ² = 9.80, df= 2, P < 0.0079). A significant difference was also found in the allele frequencies between NIDDM patients and controls (² =9.39, df = 1, P < 0.0022), whereas no differences were found for the StuI RFLP. No linkage disequilibrium was detected between the XbaI and StuI RFLPs in this sample. The analysis of the four haplotype frequencies (X1S1, X1S2, X2S1, X2S2) revealed a significant difference between diabetic patients and controls (² = 14.26, df =3, P < 0.002). By comparing single haplotype frequencies, a significant difference between the two groups was found for the X1S1 and X2S2 haplotypes. A two-allele RFLP at the GLUT4 (muscle/adipocyte) glucose transporter gene, detected with the restriction enzyme KpnI, was also examined; no differences were found between patients and controls for this RFLP. The finding of an association between polymorphic markers at the GLUT1 transporter and NIDDM suggests that this locus may contribute to the inherited susceptibility to the disease in this Italian population.     

Detection of MMP activity in living cells by a genetically encoded surface-displayed FRET sensor

Matrix metalloproteinases (MMPs) are secretory endopeptidases. They have been associated with invasion by cancer-cell and metastasis. Previous studies have demonstrated that proteolytic activity could be detected using fluorescence resonance energy transfer (FRET) with mutants of GFP. To monitor MMP activity, we constructed vectors that encoded a MMP Substrate Site (MSS) between YFP and CFP. In vitro, YFP-MSS-CFP can be used to detect MMP activity and 1,10-phenathroline inhibition of MMP activity. In living cells, MMPs are secreted proteins and act outside of the cell, and therefore YFP-MSS-CFPdisplay was anchored on the cellular surface to detect extracellular MMP. A pDisplay-YC vector expressing the YFP-MSS-CFPdisplay on the cellular surface was transfected into MCF-7 cells that expressed low levels of MMP. Efficient transfer of energy from excited CFP to YFP within the YFP-MSS-CFPdisplay molecule was observed, and real-time FRET was declined when MCF-7 was incubated with MMP2. However, no such transfer of energy was detected in the YFP-MSS-CFPdisplay expressing MDA-MB 435s cells, in which high secretory MMP2 were expressed. The FRET sensor YFP-MSS-CFPdisplay can sensitively and reliably monitor MMP activation in living cells and can be used for high-throughput screening of MMP inhibitors for anti-cancer treatments.     

Adenovirus mediated overexpression of CYP2E1 increases sensitivity of HepG2 cells to acetaminophen induced cytotoxicity

To study the biochemical and toxicological properties of cytochrome P450 2E1 (CYP2E1), an adenovirus containing human CYP2E1 cDNA (Ad-CYP2E1) was constructed and was shown to successfully mediate the overexpression of CYP2E1 in HepG2 cells. Acetaminophen (APAP) toxicity to HepG2 cells infected with Ad-CYP2E1 was characterized as a preliminary proof of principle experiment to validate the functionality of the CYP2E1 adenovirus. Compared with cells infected with Ad-LacZ, HepG2 cells infected with Ad-CYP2E1 were more sensitive to APAP induced necrosis and apoptosis when the cells were depleted of intracellular reduced glutathione (GSH). The APAP cytotoxicity was dependent on both the concentration of APAP and the multiplicity of infection of the Ad-CYP2E1 virus. Apoptosis induced by APAP in HepG2 cells overexpressing CYP2E1 was caspase dependent and could be inhibited by the pan-caspase inhibitor Z-VAD-fmk. After treatment with APAP, mitochondrial membrane potential was dramatically decreased in the CYP2E1-expressing cells. APAP protein adducts were elevated in HepG2 cells infected with Ad-CYP2E1 compared with that in cells infected with Ad-LacZ; two bands around 90 KD were found only in the CYP2E1-expressing cells. These results demonstrate that adenovirus-mediated overexpression of human CYP2E1 activates APAP to reactive metabolites which damage mitochondria, form protein adducts, and result in toxicity to HepG2 cells. The Ad-CYP2E1 may be useful for studies designed to investigate the role of CYP2E1 in APAP and alcoholic liver injury and to further characterize the actions and effects of CYP2E1.     

9-Anthroylnitrile binding to serine-181 in myosin subfragment 1 as revealed by FRET spectroscopy and molecular modeling.

It has been shown that one of the 12 serine residues within the 23 kDa segment of myosin subfragment 1 can be covalently modified with a fluorescent probe 9-anthroylnitrile (ANN) [Hiratsuka, T. (1989) J. Biol. Chem. 264 (30), 18188-18194]. To identify the exact binding site of the probe, the distances between the bound ANN as donor and acceptors in known positions (Lys-553 or Cys-707) of the myosin head were determined by using fluorescence resonance energy transfer. Comparison of the spectroscopic results with distances obtained from the atomic model of subfragment 1 revealed that ANN binds to Ser-181. The result was in good agreement with the assumptions of Andreev and co-workers [Andreev, O. A., et al. (1995) J. Muscle Res. Cell Motil. 16 (4), 353-367]. This conclusion was further supported by protein modeling calculations. The results presented herein might bring ANN into the focus when the molecular mechanism and effects of the binding of ATP and its subsequent hydrolysis are studied.