Cellular binding and uptake of fluorescent glucose analogs 2-NBDG and 6-NBDG occurs independent of membrane glucose transporters.

The classical methods for determining glucose uptake rates in living cells involve the use of isotopically labeled 2-deoxy-d-glucose or 3-O-methyl-d-glucose, which enter cells via well-characterized membrane transporters of the SLC2A and SLC5A families, respectively. These classical methods, however, are increasingly being displaced by high-throughput assays that utilize fluorescent analogs of glucose. Among the most commonly used of these analogs are 2-NBDG and 6-NBDG, which contain a bulky 7-nitro-2,1,3-benzoxadiazol-4-yl-amino moiety in place of a hydroxy group on d-glucose. This fluorescent group significantly alters both the size and shape of these molecules compared to glucose, calling into question whether they actually enter cells by the same transport mechanisms. In this study, we took advantage of the well-defined glucose uptake mechanism of L929 murine fibroblasts, which rely exclusively on the Glut1/Slc2a1 membrane transporter. We demonstrate that neither pharmacologic inhibition of Glut1 nor genetic manipulation of its expression has a significant impact on the binding or uptake of 2-NBDG or 6-NBDG by L929 cells, though both approaches significantly impact [3H]-2-deoxyglucose uptake rates. Together these data indicate that 2-NBDG and 6-NBDG can bind and enter mammalian cells by transporter-independent mechanisms, which calls into question their utility as an accurate proxy for glucose transport.

Permeation of Molecules through Astroglial Connexin 43 Hemichannels Is Modulated by Cytokines with Parameters Depending on the Permeant Species.

Recent studies indicate that connexin hemichannels do not act as freely permeable non-selective pores, but they select permeants in an isoform-specific manner with cooperative, competitive and saturable kinetics. The aim of this study was to investigate whether the treatment with a mixture of IL-1ß plus TNF-α, a well-known pro-inflammatory condition that activates astroglial connexin 43 (Cx43) hemichannels, could alter their permeability to molecules. We found that IL-1ß plus TNF-α left-shifted the dye uptake rate vs. dye concentration relationship for Etd and 2-NBDG, but the opposite took place for DAPI or YO-PRO-1, whereas no alterations were observed for Prd. The latter modifications were accompanied of changes in Kd (Etd, DAPI, YO-PRO-1 or 2-NBDG) and Hill coefficients (Etd and YO-PRO-1), but not in alterations of Vmax. We speculate that IL-1ß plus TNF-α may distinctively affect the binding sites to permeants in astroglial Cx43 hemichannels rather than their number in the cell surface. Alternatively, IL-1ß plus TNF-α could induce the production of endogenous permeants that may favor or compete for in the pore-lining residues of Cx43 hemichannels. Future studies shall elucidate whether the differential ionic/molecule permeation of Cx43 hemichannels in astrocytes could impact their communication with neurons in the normal and inflamed nervous system.

A Warburg effect targeting vector designed to increase the uptake of compounds by cancer cells demonstrates glucose and hypoxia dependent uptake.

Glycoconjugation to target the Warburg effect provides the potential to enhance selective uptake of anticancer or imaging agents by cancer cells. A Warburg effect targeting group, rationally designed to facilitate uptake by glucose transporters and promote cellular accumulation due to phosphorylation by hexokinase (HK), has been synthesised. This targeting group, the C2 modified glucose analogue 2-(2-[2-(2-aminoethoxy)ethoxy]ethoxy)-D-glucose, has been conjugated to the fluorophore nitrobenzoxadiazole to evaluate its effect on uptake and accumulation in cancer cells. The targeting vector has demonstrated inhibition of glucose phosphorylation by HK, indicating its interaction with the enzyme and thereby confirming the potential to facilitate an intracellular trapping mechanism for compounds it is conjugated with. The cellular uptake of the fluorescent analogue is dependent on the glucose concentration and is so to a greater extent than is that of the widely used fluorescent glucose analogue, 2-NBDG. It also demonstrates selective uptake in the hypoxic regions of 3D spheroid tumour models whereas 2-NBDG is distributed primarily through the normoxic regions of the spheroid. The increased selectivity is consistent with the blocking of alternative uptake pathways.

Imaging cellular pharmacokinetics of 18F-FDG and 6-NBDG uptake by inflammatory and stem cells.

OBJECTIVES: Myocardial infarction (MI) causes significant loss of cardiomyocytes, myocardial tissue damage, and impairment of myocardial function. The inability of cardiomyocytes to proliferate prevents the heart from self-regeneration. The treatment for advanced heart failure following an MI is heart transplantation despite the limited availability of the organs. Thus, stem-cell-based cardiac therapies could ultimately prevent heart failure by repairing injured myocardium that reverses cardiomyocyte loss. However, stem-cell-based therapies lack understanding of the mechanisms behind a successful therapy, including difficulty tracking stem cells to provide information on cell migration, proliferation and differentiation. In this study, we have investigated the interaction between different types of stem and inflammatory cells and cell-targeted imaging molecules, 18F-FDG and 6-NBDG, to identify uptake patterns and pharmacokinetics in vitro. METHODS: Macrophages (both M1 and M2), human induced pluripotent stem cells (hiPSCs), and human amniotic mesenchymal stem cells (hAMSCs) were incubated with either 18F-FDG or 6-NBDG. Excess radiotracer and fluorescence were removed and a 100 µm-thin CdWO4 scintillator plate was placed on top of the cells for radioluminescence microscopy imaging of 18F-FDG uptake, while no scintillator was needed for fluorescence imaging of 6-NBDG uptake. Light produced following beta decay was imaged with a highly sensitive inverted microscope (LV200, Olympus) and an Electron Multiplying Charge-Couple Device (EM-CCD) camera. Custom-written software was developed in MATLAB for image processing. RESULTS: The average cellular activity of 18F-FDG in a single cell of hAMSCs (0.670±0.028 fCi/µm2, P = 0.001) was 20% and 36% higher compared to uptake in hiPSCs (0.540±0.026 fCi/µm2, P = 0.003) and macrophages (0.430±0.023 fCi/µm2, P = 0.002), respectively. hAMSCs exhibited the slowest influx (0.210 min-1) but the fastest efflux (0.327 min-1) rate compared to the other tested cell lines for 18F-FDG. This cell line also has the highest phosphorylation but exhibited the lowest rate of de-phosphorylation. The uptake pattern for 6-NBDG was very different in these three cell lines. The average cellular activity of 6-NBDG in a single cell of macrophages (0.570±0.230 fM/µm2, P = 0.004) was 38% and 14% higher compared to hiPSCs (0.350±0.160 fM/µm2, P = 0.001) and hAMSCs (0.490±0.028 fM/µm2, P = 0.006), respectively. The influx (0.276 min-1), efflux (0.612 min-1), phosphorylation (0.269 min-1), and de-phosphorylation (0.049 min-1) rates were also highest for macrophages compared to the other two tested cell lines. CONCLUSION: hAMSCs were found to be 2-3× more sensitive to 18F-FDG molecule compared to hiPSCs/macrophages. However, macrophages exhibited the most sensitivity towards 6-NBDG. Based on this result, hAMSCs targeted with 18F-FDG could be more suitable for understanding the mechanisms behind successful therapy for treating MI patients by gathering information on cell migration, proliferation and differentiation.

Mechanism of transdermal permeation promotion of lipophilic drugs by ethosomes.

Ethosomes can promote the penetration of lipophilic drugs into the skin, but the underlying mechanism is still unknown. The purpose of this study was to investigate the mechanism of transdermal permeation promotion of lipophilic drugs by ethosomes. The formulation of ethosomes was optimized using the Box-Behnken experimental design, in which Rhodamine B and 1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3-phosphocholine were used to simulate a model lipophilic drug and act as a fluorescent tracer of ethosomal phospholipids, respectively. Liposomes with the same phospholipid concentration and a hydroethanolic solution with the same ethanol concentration were also prepared as controls. The percutaneous progression of the above fluorescent preparations was observed by confocal laser scanning microscopy, and the fluorescence intensity of the images was analyzed. The optimized ethosome formulation consisted of 2.45% yolk phospholipids, 30% ethanol, and 67.55% distilled water. The percutaneous permeation of Rhodamine B in the optimized ethosomes was superior to that in hydroethanolic solution (P<0.05) and liposomes (P<0.05). The ethosomes could penetrate the skin via the percutaneous pathway of the hair follicle and stratum corneum, while during the process of penetration, the vesicles were broken and the phospholipids were retained in the upper epidermis, with the test compounds penetrating gradually. The superior percutaneous penetration of ethosomes was linked to the synergistic effects of their ingredients. The percutaneous pathways of ethosomes included open hair follicles and stratum corneum pathways. In addition, the vesicles might break up during percutaneous penetration in the superficial layer of the skin, allowing the test compounds to keep permeating into the deeper layer alone, while the phospholipid was retained in the upper epidermis.

Resveratrol regulates neuronal glucose uptake and insulin sensitivity via P21-activated kinase 2 (PAK2).

We have recently reported P21-activated kinase 2 (PAK2), a serine/threonine kinase as a negative regulator of neuronal glucose uptake and insulin sensitivity. Resveratrol (RSV), a natural polyphenol with anti-oxidative, anti-inflammatory and anti-diabetic properties, regulates PAK2 activity in HepG2 and ESC-B5 cell apoptosis. However, regulation of PAK2 by RSV in neuronal insulin signaling pathway, if any, is still unknown. In the present study, RSV treatment significantly increased PAK2 activity under insulin-sensitive and insulin-resistant condition, along with a marked decrease in glucose uptake in differentiated N2A cells. Pretreatment with AMPK inhibitor, followed by RSV treatment resulted in reduction in PAK2 activity whereas glucose uptake showed an increase. However, pretreatment with Akt inhibitor and then RSV exposure significantly increased PAK2 activity, with a corresponding decrease in glucose uptake. RSV treatment increased AMPK activity and decreased Akt activity. In conclusion, RSV negatively regulates neuronal glucose uptake and insulin sensitivity via PAK2.

Central alarin ameliorated insulin resistance of adipocytes in type 2 diabetic rats.

Alarin, a regulatory peptide, belongs to the galanin family and plays the same regulatory roles as galanin in orexigenic activity and energy metabolism. Our previous studies had found that galanin might facilitate insulin sensitivity via activation of its central receptors. To date, little is known about whether central alarin may exert similar effects on insulin sensitivity. In order to investigate this, alarin and its specific antagonist, alarin 6-25Cys, were administered into the cerebral ventricles of type 2 diabetic rats (T2DR) to evaluate the changes in insulin resistance. The results indicated that central treatment with alarin significantly increased the body weight of animals, the 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose uptake, the plasma adiponectin levels, the glucose infusion rates in hyperinsulinemic-euglycemic clamp tests, the vesicle-associated membrane protein 2 as well as glucose transporter 4 (GLUT4 (SLC2A4)) protein and mRNA levels, and the ratios of GLUT4 contents in plasma membranes to total cell membranes in adipocytes, but reduced blood glucose and plasma retinol-binding protein 4 levels. These effects of alarin may be inhibited by pretreatment with alarin 6-25Cys. The above-mentioned results suggest that the central alarin projective system may facilitate insulin sensitivity and glucose uptake via the increase in GLUT4 content and GLUT4 translocation from intracellular pools to plasma membranes in T2DR.

Fiber-optic system for dual-modality imaging of glucose probes 18F-FDG and 6-NBDG in atherosclerotic plaques.

BACKGROUND: Atherosclerosis is a progressive inflammatory condition that underlies coronary artery disease (CAD)-the leading cause of death in the United States. Thus, the ultimate goal of this research is to advance our understanding of human CAD by improving the characterization of metabolically active vulnerable plaques within the coronary arteries using a novel catheter-based imaging system. The aims of this study include (1) developing a novel fiber-optic imaging system with a scintillator to detect both 18F and fluorescent glucose probes, and (2) validating the system on ex vivo murine plaques. METHODS: A novel design implements a flexible fiber-optic catheter consisting of both a radio-luminescence and a fluorescence imaging system to detect radionuclide 18F-fluorodeoxyglucose (18F-FDG) and the fluorescent analog 6-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6-Deoxyglucose (6-NBDG), respectively. Murine macrophage-rich atherosclerotic carotid plaques were imaged ex vivo after intravenous delivery of 18F-FDG or 6-NBDG. Confirmatory optical imaging by IVIS-200 and autoradiography were also performed. RESULTS: Our fiber-optic imaging system successfully visualized both 18F-FDG and 6-NBDG probes in atherosclerotic plaques. For 18F-FDG, the ligated left carotid arteries (LCs) exhibited 4.9-fold higher radioluminescence signal intensity compared to the non-ligated right carotid arteries (RCs) (2.6 × 10(4) ± 1.4 × 10(3) vs. 5.4 × 10(3) ± 1.3 × 10(3) A.U., P = 0.008). Similarly, for 6-NBDG, the ligated LCs emitted 4.3-fold brighter fluorescent signals than the control RCs (1.6 × 10(2) ± 2.7 × 10(1) vs. 3.8 × 10(1) ± 5.9 A.U., P = 0.002). The higher uptake of both 18F-FDG and 6-NBDG in ligated LCs were confirmed with the IVIS-200 system. Autoradiography further verified the higher uptake of 18F-FDG by the LCs. CONCLUSIONS: This novel fiber-optic imaging system was sensitive to both radionuclide and fluorescent glucose probes taken up by murine atherosclerotic plaques. In addition, 6-NBDG is a promising novel fluorescent probe for detecting macrophage-rich atherosclerotic plaques.

Techniques to monitor glycolysis.

An increased flux through glycolysis supports the proliferation of cancer cells by providing additional energy in the form of ATP as well as glucose-derived metabolic intermediates for nucleotide, lipid, and protein biosynthesis. Thus, glycolysis and other metabolic pathways that control cell proliferation may represent valuable targets for therapeutic interventions and diagnostic procedures. In this context, the measurement of glucose uptake and lactate excretion by malignant cells may be useful to detect shifts in glucose catabolism, while determining the activity of rate-limiting glycolytic enzymes can provide insights into points of metabolic regulation. Moreover, metabolomic studies can be used to generate large, integrated datasets to track changes in carbon flux through glycolysis and its collateral anabolic pathways. As discussed here, these approaches can reveal and quantify the metabolic alterations that underlie malignant cell proliferation.

Radiation induces aerobic glycolysis through reactive oxygen species.

BACKGROUND AND PURPOSE: Although radiation induced reoxygenation has been thought to increase radiosensitivity, we have shown that its associated oxidative stress can have radioprotective effects, including stabilization of the transcription factor hypoxia inducible factor 1 (HIF-1). HIF-1 is known to regulate many of the glycolytic enzymes, thereby promoting aerobic glycolysis, which is known to promote treatment resistance. Thus, we hypothesized that reoxygenation after radiation would increase glycolysis. We previously showed that blockade of oxidative stress using a superoxide dismutase (SOD) mimic during reoxygenation can downregulate HIF-1 activity. Here we tested whether concurrent use of this drug with radiotherapy would reduce the switch to a glycolytic phenotype. MATERIALS AND METHODS: 40 mice with skin fold window chambers implanted with 4T1 mammary carcinomas were randomized into (1) no treatment, (2) radiation alone, (3) SOD mimic alone, and (4) SOD mimic with concurrent radiation. All mice were imaged on the ninth day following tumor implantation (30 h following radiation treatment) following injection of a fluorescent glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG). Hemoglobin saturation was measured by using hyperspectral imaging to quantify oxygenation state. RESULTS: Mice treated with radiation showed significantly higher 2-NBDG fluorescence compared to controls (p=0.007). Hemoglobin saturation analysis demonstrated reoxygenation following radiation, coinciding with the observed increase in glycolysis. The concurrent use of the SOD mimic with radiation demonstrated a significant reduction in 2-NBDG fluorescence compared to effects seen after radiation alone, while having no effect on reoxygenation. CONCLUSIONS: Radiation induces an increase in tumor glucose demand approximately 30 h following therapy during reoxygenation. The use of an SOD mimic can prevent the increase in aerobic glycolysis when used concurrently with radiation, without preventing reoxygenation.

17ß-estradiol activates glucose uptake via GLUT4 translocation and PI3K/Akt signaling pathway in MCF-7 cells.

The relationship between estrogen and some types of breast cancer has been clearly established. However, although several studies have demonstrated the relationship between estrogen and glucose uptake via phosphatidylinositol 3-kinase (PI3K)/Akt in other tissues, not too much is known about the possible cross talk between them for development and maintenance of breast cancer. This study was designed to test the rapid effects of 17ß-estradiol (E2) or its membrane-impermeable form conjugated with BSA (E2BSA) on glucose uptake in a positive estrogen receptor (ER) breast cancer cell line, through the possible relationship between key components of the PI3K/Akt signaling pathway and acute steroid treatment. MCF-7 human breast cancer cells were cultured in standard conditions. Then 10 nM E2 or E2BSA conjugated were administered before obtaining the cell lysates. To study the glucose uptake, the glucose fluorescent analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose was used. We report an ER-dependent activation of some of the key steps of the PI3K/Akt signaling pathway cascade that leads cells to improve some mechanisms that finally increase glucose uptake capacity. Our data suggest that both E2 and E2BSA enhance the entrance of the fluorescent glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose, and also activates PI3K/Akt signaling pathway, leading to translocation of glucose transporter 4 to the plasma membrane in an ERα-dependent manner. E2 enhances ER-dependent rapid signaling triggered, partially in the plasma membrane, allowing ERα-positive MCF-7 breast cancer cells to increase glucose uptake, which could be essential to meet the energy demands of the high rate of proliferation.

Noninvasive photoacoustic computed tomography of mouse brain metabolism in vivo.

We have demonstrated the feasibility of imaging mouse brain metabolism using photoacoustic computed tomography (PACT), a fast, noninvasive and functional imaging modality with optical contrast and acoustic resolution. Brain responses to forepaw stimulations were imaged transdermally and transcranially. 2-NBDG, which diffuses well across the blood-brain-barrier, provided exogenous contrast for photoacoustic imaging of glucose response. Concurrently, hemoglobin provided endogenous contrast for photoacoustic imaging of hemodynamic response. Glucose and hemodynamic responses were quantitatively decoupled by using two-wavelength measurements. We found that glucose uptake and blood perfusion around the somatosensory region of the contralateral hemisphere were both increased by stimulations, indicating elevated neuron activity. While the glucose response area was more homogenous and confined within the somatosensory region, the hemodynamic response area had a clear vascular pattern and spread wider than the somatosensory region. Our results demonstrate that 2-NBDG-enhanced PACT is a promising tool for noninvasive studies of brain metabolism.

The Monascus metabolite monascin against TNF-α-induced insulin resistance via suppressing PPAR-γ phosphorylation in C2C12 myotubes.

Chronic inflammation in muscle tissue causes insulin resistance and type-2 diabetes. Peroxisome proliferator-activated receptor (PPAR) ligands are reported to activate the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, including pioglitazone, which belong to the thiazolidinedione (TZD). Monascin (MS), a Monascus metabolite, has been reported to exert anti-inflammatory activity in our recent study. Therefore, the alleviating mechanism of MS on tumor necrosis factor-α (TNF-α; 20ng/mL) induced insulin resistance in C2C12 cells was investigated in this study. Results showed that MS increased the uptake of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) in C2C12 myotubes. This result was associated with both PPAR-γ activity and PI3K/Akt pathway caused by MS inhibited p-JNK activity and prevented PPAR-γ phosphorylation. Moreover, we found that MS may act a PPAR-γ agonist to improve insulin sensitivity, and this issue was further confirmed by PPAR-γ antagonist (GW9662). Briefly, MS as pioglitazone, stabilized PPAR-γ structure and diminished PPAR-γ phosphorylation thereby improving insulin resistance.

A fluorescence method for measurement of glucose transport in kidney cells.

BACKGROUND: Diabetes may alter renal glucose reabsorption by sodium (Na(+))-dependent glucose transporters (SGLTs). Radiolabeled substrates are commonly used for in vitro measurements of SGLT activity in kidney cells. We optimized a method to measure glucose uptake using a fluorescent substrate, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG). METHODS: Uptake buffers for 2-NBDG were the same as for (14)C-labeled α-methyl-d-glucopyranoside ([(14)C]AMG). Cell lysis buffer was optimized for fluorescence of 2-NBDG and Hoechst DNA stain. Uptake was performed on cultures of primary mouse kidney cells (PMKCs), the LLC-PK(1) proximal tubule cell line, or COS-7 cells transiently overexpressing mouse SGLT1 or SGLT2 by incubating cells at 37°C in buffer containing 50-200 µM 2-NBDG. Microscopy was performed to visualize uptake in intact cells, while a fluorescence microplate reader was used to measure intracellular concentration of 2-NBDG ([2-NBDG](i)) in cell homogenates. RESULTS: Fluorescent cells were observed in cultures of PMKCs and LLC-PK(1) cells exposed to 2-NBDG in the presence or absence of Na(+). In LLC-PK(1) cells, 2-NBDG transport in the presence of Na(+) had a maximum rate of 0.05 nmol/min/µg of DNA. In these cells, Na(+)-independent uptake of 2-NBDG was blocked with the GLUT inhibitor, cytochalasin B. The Na(+)-dependent uptake of 2-NBDG decreased in response to co-exposure to the SGLT substrate, AMG, and it could be blocked with the SGLT inhibitor, phlorizin. Immunocytochemistry showed overexpression of SGLT1 and SGLT2 in COS-7 cells, in which, in the presence of Na(+), [2-NBDG](i) was fivefold higher than in controls. CONCLUSION: Glucose transport in cultured kidney cells can be measured with the fluorescence method described in this study.

Uptake of 2-NBDG as a method to monitor therapy response in breast cancer cell lines.

This study quantifies uptake of a fluorescent glucose analog, (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose) (2-NBDG), in a large panel of breast cancer cells and demonstrates potential to monitor changes in glycolysis caused by anticancer and endocrine therapies. Expressions of glucose transporter (GLUT 1) and hexokinase (HK I), which phosphorylates 2-NBDG, were measured via western blot in two normal mammary epithelial and eight breast cancer cell lines of varying biological subtype. Fluorescence intensity of each cell line labeled with 100 lM 2-NBDG for 20 min or unlabeled control was quantified. A subset of cancer cells was treated with anticancer and endocrine therapies, and 2-NBDG fluorescence changes were measured. Expression of GLUT 1 was necessary for uptake of 2-NBDG, as demonstrated by lack of 2-NBDG uptake in normal human mammary epithelial cells (HMECs). GLUT 1 expression and 2-NBDG uptake was ubiquitous among all breast cancer lines. Reduction and stimulation of 2-NBDG uptake was demonstrated by perturbation with anticancer agents, lonidamine (LND), and a-cyano-hydroxycinnamate (a-Cinn), respectively. LND directly inhibits HK and significantly reduced 2-NBDG fluorescence in a subset of two breast cancer cell lines. Conversely, when cells were treated with a-Cinn, a drug used to increase glycolysis, 2-NBDG uptake was increased. Furthermore, tamoxifen (tam), a common endocrine therapy, was administered to estrogen receptor positive and negative (ER?/-) breast cells and demonstrated a decreased 2-NBDG uptake in ER? cells, reflecting a decrease in glycolysis. Results indicate that 2-NBDG uptake can be used to measure changes in glycolysis and has potential for use in early drug development.

[Uptake of 2-NBDG by human breast cancer cells in vitro].

OBJECTIVE: The purpose of this study was to assess the feasibility of fluorescent 2-deoxyglucose analog, 2-[N-(7-nitrobenz-2-oxa-1, 3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG), that could be taken up by breast cancer cells highly expressing glucose transporter 1 (GLUT-1). The purpose of this study was to clarify if a fluorescent 2-deoxyglucose analog, 2-[N-(7-nitrobenz-2-oxa-1, 3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG), can be taken up by breast cancer cells highly expressing glucose transporter 1 (GLUT-1), and to assess whether it can be used as a targeting imaging agent. METHODS: The expressions of GLUT-1 mRNA and protein in breast cancer MDA-MB-231 cells were detected by RT-PCR and immunohistochemistry, respectively. The difference of GLUT-1 protein expression between breast cancer MDA-MB-231 cells and MCF-7 cells was compared by Western blot. Secondly, MDA-MB-231 cells which were grown in 6-well plates were incubated with 2-NBDG, and the result of 2-NBDG uptake was analyzed by fluorescence microscopy and flow cytometry. The difference of 2-NBDG absorption in MDA-MB-231 and MCF-7 cells was compared by flow cytometry. RESULTS: The results of RT-PCR and immunohistochemistry confirmed that MDA-MB-231 cells highly expressed GLUT-1. Furthermore, Western blot revealed that GLUT-1 expression of MDA-MB-231 cells (0.946 ± 0.007) was higher than that in the MCF-7 cells (0.833 ± 0.010). Fluorescence microscopic and flow cytometric analysis showed that 2-NBDG was uptaken rapidly by MDA-MB-231 cells. Addition of 50 mmol/L D-glucose to the media with 2-NBDG reduced its uptake by 46.0%. Moreover, flow cytometry indicated that the fluorescence intensity of MDA-MB-231 cells (25.10 ± 0.57) was higher than that of MCF-7 cells (10.12 ± 0.62) when incubated with 2-NBDG for 20 minutes. CONCLUSION: The preliminary data clearly demonstrate that 2-NBDG is taken up and accumulated in breast cancer cells that highly express GLUT-1, and may be used as an optical probe for glucose uptake in hypermetabolic malignant cells.

NBDT (3-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-3-toluene)--a novel fluorescent dye for studying mechanisms of toluene uptake into vital bacteria.

Uptake of small hydrophobic substances such as toluene into bacteria is widely assumed to occur by passive diffusion. Some toluene degrading bacteria, however, are described to contain uptake systems which may be involved in the transport of this compound. In this study, a fluorescently labeled toluene analogue dye (3-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-3-toluene; NBDT), flow cytometry, and shot gun proteome analysis were used to follow toluene uptake into bacteria in more detail. The new dye has excitation peaks at 444 and 475 nm and an emission peak at 537 nm. The toluene-degraders P. putida mt-2 and P. putida F1 as well as P. putida KT2440 and E. coli K12 as negative controls were included. To enable quantification of NBDT uptake, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was added to inactivate NBDT efflux pumps. The porin inhibitor cadaverine was added to study the porin-mediated influx of toluene. Cadaverine reduced NBDT uptake by toluene-grown P. putida mt-2 and F1 by 25% and 42%, respectively, thus revealing an involvement and possibly a regulatory function of porins in the uptake of the toxic substrate toluene. Shot gun proteome measurements gave evidence for the presence of toluene transporting porins in P. putida mt-2 grown on toluene but not when grown on glucose.

Molecular MRI of cardiomyocyte apoptosis with simultaneous delayed-enhancement MRI distinguishes apoptotic and necrotic myocytes in vivo: potential for midmyocardial salvage in acute ischemia.

BACKGROUND: A novel dual-contrast molecular MRI technique to image both cardiomyocyte apoptosis and necrosis in vivo within 4 to 6 hours of ischemia is presented. The technique uses the annexin-based nanoparticle AnxCLIO-Cy5.5 (apoptosis) and simultaneous delayed-enhancement imaging with a novel gadolinium chelate, Gd-DTPA-NBD (necrosis). METHODS AND RESULTS: Mice with transient coronary ligation were injected intravenously at the onset of reperfusion with AnxCLIO-Cy5.5 (n=7) or the control probe Inact_CLIO-Cy5.5 (n=6). T2*-weighted MR images (9.4 T) were acquired within 4 to 6 hours of reperfusion. The contrast-to-noise ratio between injured and uninjured myocardium was measured. The mice were then injected with Gd-DTPA-NBD, and delayed-enhancement imaging was performed within 10 to 30 minutes. Uptake of AnxCLIO-Cy5.5 was most prominent in the midmyocardium and was significantly greater than that of Inact_CLIO-Cy5.5 (contrast-to-noise ratio, 8.82+/-1.5 versus 3.78+/-1.1; P<0.05). Only 21+/-3% of the myocardium with accumulation of AnxCLIO-Cy5.5 showed delayed-enhancement of Gd-DTPA-NBD. Wall thickening was significantly reduced in segments with delayed enhancement and/or transmural accumulation of AnxCLIO-Cy5.5 (P<0.001). Fluorescence microscopy of AnxCLIO-Cy5.5 and immunohistochemistry of Gd-DTPA-NBD confirmed the presence of large numbers of apoptotic but potentially viable cardiomyocytes (AnxCLIO-Cy5.5 positive, Gd-DTPA-NBD negative) in the midmyocardium. CONCLUSIONS: A novel technique to image cardiomyocyte apoptosis and necrosis in vivo within 4 to 6 hours of injury is presented and reveals large areas of apoptotic but viable myocardium in the midmyocardium. Strategies to salvage the numerous apoptotic but potentially viable cardiomyocytes in the midmyocardium in acute ischemia should be investigated.

ATP8B1 deficiency disrupts the bile canalicular membrane bilayer structure in hepatocytes, but FXR expression and activity are maintained.

BACKGROUND & AIMS: Progressive familial intrahepatic cholestasis 1 (PFIC1) results from mutations in ATP8B1, a putative aminophospholipid flippase. Conflicting hypotheses have been proposed for the pathogenesis of PFIC1. The aim of this study was to determine whether ATP8B1 deficiency produces cholestasis by altering the activity of the farnesoid X receptor (FXR) or by impairing the structure of the canalicular membrane. METHODS: ATP8B1/Atp8b1 was knocked down in human and rat hepatocytes and Caco2 cells using adenoviral and oligonucleotide small interfering RNAs. RESULTS: ATP8B1 messenger RNA and protein expression was greatly reduced in human and rat cells. In contrast, FXR expression and several FXR-dependent membrane transporters (bile salt export pump [BSEP], multidrug resistance-associated protein [MRP] 2) were unchanged at messenger RNA or protein levels in ATP8B1-deficient cells, whereas Mrp3 and Mrp4 were up-regulated in rat hepatocytes. FXR activity remained intact in these cells, as evidenced by 6alpha-ethyl chenodeoxycholic acid-mediated induction of small heterodimer partner, BSEP, and multidrug-resistant protein (MDR) 3/Mdr2. Fluorescent substrate excretion assays indicate that Bsep function was significantly reduced in Atp8b1-deficient rat hepatocytes, although Bsep remained localized to the canalicular membrane. Exposure to the hydrophobic bile acid CDCA resulted in focal areas of canalicular membrane disruption by electron microscopy and luminal accumulation of NBD-phosphatidylserine, consistent with the function of Atp8b1 as an aminophospholipid flippase. CONCLUSIONS: ATP8B1 deficiency predisposes to cholestasis by favoring bile acid-induced injury in the canalicular membrane but does not directly affect FXR expression, which may occur in PFIC1 as a secondary phenomenon associated with cholestasis.

Evaluation and clinically relevant applications of a fluorescent imaging analog to fluorodeoxyglucose positron emission tomography.

A fluorescent analog to 2-deoxy-2 [(18)F] fluoro-D-glucose position emission tomography (FDG-PET) would allow for the introduction of metabolic imaging into intraoperative and minimally invasive settings. We present through in vitro and in vivo experimentation an evaluation of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), a fluorescently labeled glucose molecule, as a molecular beacon of glucose utilization. The competitive inhibition of 2-NBDG uptake by excess free glucose is directly compared against FDG uptake inhibition in cultured cells. 2-NBDG uptake in the brain of a mouse experiencing a generalized seizure is measured, as well as in subcutaneously implanted tumors in mice during fed and fasting states. Localization of 2-NBDG into malignant tissues is studied by laser scanning microscopy. The clinical relevance of 2-NBDG imaging is examined by performing fluorescence colonoscopy, and by correlating preoperative FDG-PET with intraoperative fluorescence imaging. 2-NBDG exhibits a similar uptake inhibition to FDG by excess glucose in the growth media. Uptake is significantly increased in the brain of an animal experiencing seizures versus control, and in subcutaneous tumors after the animals are kept nil per os (NPO) for 24 h versus ad libitum feeding. The clinical utility of 2-NBDG is confirmed by the demonstration of very high target-to-background ratios in minimally invasive and intraoperative imaging of malignant lesions. We present an optical analog of FDG-PET to extend the applicability of metabolic imaging to minimally invasive and intraoperative settings.