Non-Invasive,Simultaneous Quantification of Vascular Oxygenation and Glucose Uptake in Tissue

We report the development of non-invasive, fiber-based diffuse optical spectroscopy for simultaneously quantifying vascular oxygenation (SO₂) and glucose uptake in solid tumors in vivo. Glucose uptake was measured using a fluorescent glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG). Quantification of label-free SO₂ and 2-NBDG-fluorescence-based glucose uptake 60 minutes after administration of the tracer (2-NBDG₆₀) was performed using computational models of light-tissue interaction. This study was carried out on normal tissue and 4T1 and 4T07 murine mammary tumor xenografts in vivo. Injection of 2-NBDG did not cause a significant change in optical measurements of SO₂, demonstrating its suitability as a functional reporter of tumor glucose uptake. Correction of measured 2-NBDG-fluorescence for the effects of absorption and scattering significantly improved contrast between tumor and normal tissue. The 4T1 and 4T07 tumors showed significantly decreased SO₂, and 4T1 tumors demonstrated increased 2-NBDG₆₀ compared with normal tissue (60 minutes after the administration of 2-NBDG when perfusion-mediated effects have cleared). 2-NBDG-fluorescence was found to be highly sensitive to food deprivation-induced reduction in blood glucose levels, demonstrating that this endpoint is indeed sensitive to glycolytic demand. 2-NBDG₆₀ was also found to be linearly related to dose, underscoring the importance of calibrating for dose when comparing across animals or experiments. 4T1 tumors demonstrated an inverse relationship between 2-NBDG₆₀ and SO₂ that was consistent with the Pasteur effect, particularly when exposed to hypoxic gas breathing. Our results illustrate the potential of optical spectroscopy to provide valuable information about the metabolic status of tumors, with important implications for cancer prognosis.     

Delivery Rate Affects Uptake of a Fluorescent Glucose Analog in Murine Metastatic Breast Cancer

We demonstrate an optical strategy using intravital microscopy of dorsal skin flap window chamber models to image glucose uptake and vascular oxygenation in vivo. Glucose uptake was imaged using a fluorescent glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG). SO₂ was imaged using the differential absorption properties of oxygenated [HbO₂] and deoxygenated hemoglobin [dHb]. This study was carried out on two sibling murine mammary adenocarcinoma lines, 4T1 and 4T07. 2-NBDG uptake in the 4T1 tumors was lowest when rates of delivery and clearance were lowest, indicating perfusion-limited uptake in poorly oxygenated tumor regions. For increasing rates of delivery that were still lower than the glucose consumption rate (as measured in vitro), both 2-NBDG uptake and the clearance rate from the tumor increased. When the rate of delivery of 2-NBDG exceeded the glucose consumption rate, 2-NBDG uptake decreased with any further increase in rate of delivery, but the clearance rate continued to increase. This inflection point was not observed in the 4T07 tumors due to an absence of low delivery rates close to the glucose consumption rate. In the 4T07 tumors, 2-NBDG uptake increased with increasing rates of delivery at low rates of clearance. Our results demonstrate that 2-NBDG uptake in tumors is influenced by the rates of delivery and clearance of the tracer. The rates of delivery and clearance are, in turn, dependent on vascular oxygenation of the tumors. Knowledge of the kinetics of tracer uptake as well as vascular oxygenation is essential to make an informed assessment of glucose demand of a tumor.     

External Beam Radiotherapy for Head and Neck Cancers Is Associated with Increased Variability in Retinal Vascular Oxygenation

Background

Radiation retinopathy is a possible post-treatment complication of radiation therapy. The pathophysiologic mechanism is hypothesized to be microvascular in origin, but evidence is limited. In an effort to study retinal oxygenation in these patients, we herein evaluate the repeatability and variability of retinal oximetry measurements in subjects who had previously received radiation and make comparisons to a cohort of unirradiated subjects.

Methods

Using retinal oximetry, a non-invasive imaging modality, we performed in vivo measurements of arteriole (SaO₂) and venule SO₂ (SvO₂) in subjects (n = 9, 18 retinas) who had received incidental radiation to their retinas (≥ 45 Gy to one retina) and in healthy subjects (n = 20, 40 retinas). A total of 1367 SO₂ observations on 593 vessels in 29 persons were analyzed to assess three sources of variance in vessel SO₂: 1) variance in repeated measurements of the same vessel (“repeatability”), 2) variance in different vessels within the same subject (“within-subject variability”), and 3) variance between subjects (“between-subject variability”).

Results

Retinal oximetry measurements were highly repeatable in both irradiated patients and unirradiated subjects. The within-subject variability of SvO₂ and SaO₂ measurements constituted the highest component of variance in both groups and was significantly higher in venules vs. arterioles (relative effect size 1.8, p<0.001) and in irradiated subjects vs. unirradiated subjects (relative effect size 1.6, p<0.001).

Conclusions

Retinal oximetry is a highly repeatable technology and can be reliably used to study vascular oxygenation in irradiated subjects. Different vessels within the same subject exhibit a high degree of variability, suggesting that pooled analyses of multiple vessels are most likely to be informative of regional retinal oxygenation. Finally, irradiated subjects exhibited significantly higher within-subject variability in SO₂ measurements, suggesting that radiation may cause regional alterations in retinal oxygen delivery and/or metabolism.     

Mesophyll Resistances to SO(2) Fluxes into Leaves

Uptake of label from solutions containing ³⁵SO₂, H³⁵SO₃⁻ and ³⁵SO₃²⁻ into mesophyll protoplasts, vacuoles, and chloroplasts isolated from young barley leaves was measured at different pH values. Uptake was fast at low pH, when the concentration of SO₂ was high, and low at high pH, when the concentration of SO₂ was low. When the resistance (R) of plasmalemma, tonoplast, and chloroplast envelope to the penetration of SO₂ was calculated from rates of uptake of label, comparable values were obtained for the different biomembranes at low pH values. R was close to 8000 seconds per meter and permeability coefficients were close to 1.25 × 10⁻⁴ meters per second. Under these conditions R may describe resistance to SO₂ diffusion across a lipid bilayer. At higher pH values, R decreased. As R was calculated on the assumption that SO₂ is the only penetrating molecular species, the data suggest that carrier-mediated anion transport contributes to the uptake of sulfur at physiological pH values thereby decreasing apparent R_SO2. The contribution of anion transport appeared to be smaller for transfer across the plasmalemma than for transfer across the tonoplast. It was large for transfer across the chloroplast envelope. The phosphate translocator of the chloroplast envelope catalyzed uptake of SO₃²⁻ into chloroplasts at neutral pH. Uptake was decreased in the presence of high levels of phosphate or sulfate and by pyridoxal phosphate. SO₂ transfer into cells leads to the intracellular liberation of one or two protons, depending on pH and oxidizing conditions. When the divalent sulfite anion is exchanged across the chloroplast envelope, bisulfite formation results in proton uptake in the chloroplast stroma, whereas SO₂ uptake into chloroplasts lowers the stroma pH.     

Examining glucose transport in single vascular smooth muscle cells with a fluorescent glucose analog

Changes in vascular smooth muscle glucose transport are thought to contribute to the pathogenesis of vascular disease in conditions such as diabetes, yet no single-cell assay for glucose uptake by VSM exists. Therefore, we examined the uptake of the fluorescent glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) in isolated pig vascular smooth muscle cells (VSMC) using digital imaging microscopy. Uptake of 2-NBDG by VSMC was inhibited by D-glucose but not by L-glucose, suggesting that 2-NBDG enters VSMC via glucose transporters. Uptake of 2-NBDG was linear in the presence of 10 mM D-glucose (n=6, R2=0.9408) but not in its absence (n=4, R2=0.9993), indicating that 2-NBDG is not metabolized and accumulates within the cells. 2-NBDG fluorescence in VSMC was often non-uniform and appeared to represent binding of 2-NBDG to some cytoplasmic component. The present study demonstrates that 2-NBDG is a useful tool for examining vascular smooth muscle glucose uptake at the single cell level.     

Magnolia dealbata Zucc and its active principles honokiol and magnolol stimulate glucose uptake in murine and human adipocytes using the insulin-signaling pathway

Some Magnolia (Magnoliaceae) species are used for the empirical treatment of diabetes mellitus, but the antidiabetic properties of Magnolia dealbata have not yet been experimentally validated. Here we report that an ethanolic extract of Magnolia dealbata seeds (MDE) and its active principles honokiol (HK) and magnolol (MG) induced the concentration-dependent 2-NBDG uptake in murine 3T3-F442A and human subcutaneous adipocytes. In insulin-sensitive adipocytes, MDE 50 μg/ml induced the 2-NBDG uptake by 30% respect to insulin, while HK and MG, 30 μM each, did it by 50% (murine) and 40% (human). The simultaneous application of HK and MG stimulated 2-NBDG uptake by 70% in hormone-sensitive cells, on which Magnolia preparations exerted synergic effects with insulin. In insulin-resistant adipocytes, MDE, HK and MG induced 2-NBDG uptake by 57%, 80% and 96% respect to Rosiglitazone (RGZ), whereas HK and MG simultaneously applied stimulated 2-NBDG uptake more efficiently than RGZ (120%) in both murine and human adipocytes. Inhibitors of the insulin-signaling pathway abolished the glucose uptake induced by Magnolia dealbata preparations, suggesting that their antidiabetic effects are mediated by this signaling pathway. In addition, MDE, HK and MG exerted only mild to moderate proadipogenic effects on 3T3-F442A and human preadipocytes, although the combined application of HK and MG markedly increased the lipid accumulation in both cell types. In summary, Magnolia dealbata and its active principles HK and MG stimulate glucose uptake in insulin-sensitive and insulin-resistant murine and human adipocytes using the insulin signaling pathway.     

Assessing Glucose Uptake through the Yeast Hexose Transporter 1 (Hxt1)

The transport of glucose across the plasma membrane is mediated by members of the glucose transporter family. In this study, we investigated glucose uptake through the yeast hexose transporter 1 (Hxt1) by measuring incorporation of 2-NBDG, a non-metabolizable, fluorescent glucose analog, into the yeast Saccharomyces cerevisiae. We find that 2-NBDG is not incorporated into the hxt null strain lacking all glucose transporter genes and that this defect is rescued by expression of wild type Hxt1, but not of Hxt1 with mutations at the putative glucose-binding residues, inferred from the alignment of yeast and human glucose transporter sequences. Similarly, the growth defect of the hxt null strain on glucose is fully complemented by expression of wild type Hxt1, but not of the mutant Hxt1 proteins. Thus, 2-NBDG, like glucose, is likely to be transported into the yeast cells through the glucose transport system. Hxt1 is internalized and targeted to the vacuole for degradation in response to glucose starvation. Among the mutant Hxt1 proteins, Hxt1^N370A and HXT1^W473A are resistant to such degradation. Hxt1^N370A, in particular, is able to neither uptake 2-NBDG nor restore the growth defect of the hxt null strain on glucose. These results demonstrate 2-NBDG as a fluorescent probe for glucose uptake in the yeast cells and identify N370 as a critical residue for the stability and function of Hxt1.     

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) (2.6×10⁴±1.4×10³ vs. 5.4×10³±1.3×10³ A.U., P = 0.008). Similarly, for 6-NBDG, the ligated LCs emitted 4.3-fold brighter fluorescent signals than the control RCs (1.6×10²±2.7×10¹ vs. 3.8×10¹±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.     

Enhancement of Glucose Uptake in Mouse Skeletal Muscle Cells and Adipocytes by P2Y6 Receptor Agonists

Glucose uptake by peripheral tissues such as skeletal muscles and adipocytes is important in the maintenance of glucose homeostasis. We previously demonstrated that P2Y₆ receptor (P2Y₆R) agonists protect pancreatic islet cells from apoptosis and stimulate glucose-dependent insulin release. Here, we investigated the effects of P2Y₆R activation on glucose uptake in insulin target tissues. An agonist of the P2Y₆R, P¹-(5′-uridine)-P³-(5′-N⁴-methoxycytidine)-triphosphate (MRS2957), significantly increased the uptake of [³H]2-deoxyglucose in mouse C2C12 myotubes and 3T3-L1 adipocytes, and this stimulation was significantly decreased by a selective P2Y₆R antagonist N,N″-1,4-butanediyl-bis[N′-(3-isothiocyanatophenyl)thiourea] (MRS2578). Pre-incubation with Compound C (an inhibitor of 5′-AMP-activated protein kinase, AMPK), or AMPK siRNA abolished the stimulatory effect of MRS2957 on glucose uptake. Also, MRS2957 (60 min incubation) increased recruitment of the facilitated glucose transporter-4 (GLUT4) to the cell membrane, which was blocked by MRS2578. Treatment of C2C12 myotubes with MRS2957 induced significant phosphorylation of AMPK, which increase GLUT4 expression through histone deacetylase (HDAC)5 signaling. Glucose uptake in primary mouse adipocytes from wild-type mice was stimulated upon P2Y₆R activation by either MRS2957 or native agonist UDP, and the P2Y₆R effect was antagonized by MRS2578. However, in adipocytes from P2Y₆R-knockout mice P2Y₆R agonists had no effect on glucose uptake, and there was no change in the glucose uptake by insulin. Our results indicate that the P2Y₆R promotes glucose metabolism in peripheral tissues, which may be mediated through AMPK signaling.     

Inhibition of anion transport in corn root protoplasts

The effects of several amino-reactive disulfonic stilbene derivatives and N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate on Cl⁻, SO₄²⁻, and inorganic phosphate (Pi) uptake in protoplasts isolated from corn root tissue were studied. 4-Acetamido-4′-isothiocyano-2,2′-stilbenedisulfonic acid, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, 4,4′-diamino-2,2′-stilbenedisulfonic acid, and NAP-taurine inhibited Cl⁻ and SO₄²⁻ but not Pi and K⁺ uptake in corn root protoplasts; whereas mersalyl inhibited Pi but not Cl⁻ or SO₄²⁻ uptake. The rate of uptake of all anions decreased with increasing external pH. In addition, these reagents markedly inhibited plasmalemma ATPase activity isolated from corn root tissue. Excised root segments were less sensitive to Cl⁻ and SO₄²⁻ transport inhibitors.     

Investigating the role of potassium and urea to control fruit drop and to improve fruit quality of “Dhakki” date palm

Fruit drop is a key issue with date palm cultivars that can be addressed with a variety of methods and strategies. Foliar application of macronutrients can be more effective in inhibiting fruit drop and improving the quality of date fruits. The current study was carried out to investigate the possible role of potassium (K) and urea to reduce fruit drop and improve the fruit quality of “Dhakki” date palm. It was conducted in a complete randomised block design with seven treatments and three replications at Pakistan''s Agricultural Research Institute, Dera Ismail Khan. The treatments used were: (i) Control (distilled water spray); (ii) Potassium sulphate (K₂SO₄) at 1 %; (iii) K₂SO₄ at 1 % + Urea at 2 %; (iv) K₂SO₄ at 2 %; (v) K₂SO₄ at 2 % + Urea at 2 %; (vi) K₂SO₄ at 3 % and; (vii) K₂SO₄ at 3 % + Urea at 2 %. All the concentrations were sprayed at Kimri stage of fruit development during two consecutive growing seasons. Twenty-one date palms of equal size and age were chosen for the assessments to measure percent fruit drop and other physicochemical variables, including fruit length, fruit diameter, fruit weight, pulp percentage, yield/bundle, pH, total soluble solids (TSS), K content in fruit, and all sugars (percent) of harvested date fruit. The results revealed that bunch spray of K significantly affected all the parameters during both seasons. Application of K₂SO₄ alone and in combination with urea not only effectively reduced the fruit drop but also improved fruit quality in date where, K₂SO₄ applied at 2 % combined with urea was the best concentration in reducing fruit drop, enhancing other physicochemical attributes, and improving fruit quality of “Dhakki” date palm. This study may effectively contribute to reduce the fruit drop and enhance the fruit quality by using K and urea, enabling farmers to improve the date yield and increase economic growth.     

Vomifoliol 9-O-α-arabinofuranosyl (1→6)-β-d-glucopyranoside from the leaves of Diospyros Kaki stimulates the glucose uptake in HepG2 and 3T3-L1 cells

A novel α-glucosidase inhibitor, vomifoliol 9-O-α-arabinofuranosyl (1→6)-β-d-glucopyranoside, was isolated for the first time from leaves of Diospyros Kaki and its bioactivity analyzed. This inhibitor exhibited strong anti-α-glucosidase activity with an IC₅₀ value of 170.62 nM and stimulated a dose-dependent increase in the uptake of a fluorescent d-glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG), in HepG2 cells at a rate higher than that of insulin controls. It was also found to be associated with adipocyte differentiation and moderate increases in 2-NBDG uptake by 3T3-L1 cells. These findings suggest that vomifoliol 9-O-α-arabinofuranosyl (1→6)-β-d-glucopyranoside could augment peripheral glucose as an insulin-sensitizing agent against Type 2 diabetes mellitus.     

Evaluation of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose, a new fluorescent derivative of glucose, for viability assessment of yeast Candida albicans

A new fluorescent derivative of d-glucose, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG), which had been previously developed for the analysis of glucose uptake activity by living cells, was investigated to evaluate its applicability for assaying the viability of yeast Candida albicans. Lineweaver-Burk plots showed the uptake of 2-NBDG to be competitively inhibited by d-glucose and not by l-glucose, which suggested the involvement of the glucose transporting system of C. albicans in the uptake of 2-NBDG. A good correlation was obtained between the yeast viability, determined by the plate-count method, and the 2-NBDG uptake activity of yeast cells (correlation constant: r=0.97). This is expected to lead to the development of a new fluorescent probe for the determination of yeast cell viability.     

Light Response of CO(2) Assimilation, Dissipation of Excess Excitation Energy, and Zeaxanthin Content of Sun and Shade Leaves

Intact attached sun leaves of Helianthus annuus and shade leaves of Monstera deliciosa and Hedera helix were used to obtain light response curves of CO₂ uptake, the content of the carotenoid zeaxanthin (formed by violaxanthin de-epoxidation), as well as nonphotochemical quenching (q_NP), and the rate constant of radiationless energy dissipation (k_D). The latter two parameters were calculated from the decrease of chlorophyll a fluorescence at closed photosystem II traps in saturating pulses in the light. Among the three species, the light-saturated capacity of CO₂ uptake differed widely and light saturation of CO₂ uptake occurred at very different photon flux densities. Fluorescence quenching and zeaxanthin content exhibited features which were common to all three species: below light-saturation of CO₂ uptake nonphotochemical quenching occurred in the absence of zeaxanthin and was not accompanied by a decrease in the yield of instantaneous fluorescence. Nonphotochemical quenching, q_NP, increased up to values which ranged between 0.35 and 0.5 when based on a control value of the yield of variable fluorescence determined after 12 hours of darkness. As light saturation of CO₂ uptake was approached, q_NP showed a secondary increase and the zeaxanthin content of the leaves began to rise. This was also the point from which the yield of instantaneous fluorescence began to decrease. The increase in zeaxanthin was paralleled by an increase in the rate constant for radiationless energy dissipation k_D, which opens the possibility that zeaxanthin is related to the rapidly relaxing “high-energy-state quenching” in leaves.     

Vitamin D Up-regulates Glucose Transporter 4 (GLUT4) Translocation and Glucose Utilization Mediated by Cystathionine-γ-lyase (CSE) Activation and H2S Formation in 3T3L1 Adipocytes

A scientific explanation for the beneficial role of vitamin D supplementation in the lowering of glycemia in diabetes remains to be determined. This study examined the biochemical mechanism by which vitamin D supplementation regulates glucose metabolism in diabetes. 3T3L1 adipocytes were treated with high glucose (HG, 25 mm) in the presence or absence of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) (25, 50 nm), the active form of vitamin D. 1,25(OH)₂D₃ treatment caused significant up-regulation of GLUT4 total protein expression and its translocation to cell surface, and an increase in glucose uptake as well as glucose utilization in HG-treated cells. 1,25(OH)₂D₃ also caused cystathionine-γ-lyase (CSE) activation and H₂S formation in HG-treated adipocytes. The effect of 1,25(OH)₂D₃ on GLUT4 translocation, glucose utilization, and H₂S formation was prevented by propargylglycine, an inhibitor of CSE that catalyzes H₂S formation. Studies using antisense CSE also demonstrated the inhibition of GLUT4 translocation as well as glucose uptake and utilization in 1,25(OH)₂D₃-supplemented CSE-siRNA-transfected adipocytes compared with controls. 1,25(OH)₂D₃ treatment along with insulin enhanced GLUT4 translocation and glucose utilization compared with either insulin or 1,25(OH)₂D₃ alone in HG-treated adipocytes. 1,25(OH)₂D₃ supplementation also inhibited monocyte chemoattractant protein-1 and stimulated adiponectin secretion in HG-treated adipocytes, and this positive effect was prevented in propargylglycine-treated or CSE-knockdown adipocytes. This is the first report to demonstrate that 1,25(OH)₂D₃ up-regulates GLUT4 translocation and glucose utilization and decreases inflammatory markers, which is mediated by CSE activation and H₂S formation in adipocytes. This study provides evidence for a novel molecular mechanism by which 1,25(OH)₂D₃ can up-regulate the GLUT4 translocation essential for maintenance of glucose metabolism.     

Adenosine A2B receptor activation stimulates glucose uptake in the mouse forebrain

ATP consumption during intense neuronal activity leads to peaks of both extracellular adenosine levels and increased glucose uptake in the brain. Here, we investigated the hypothesis that the activation of the low-affinity adenosine receptor, the A_2B receptor (A_2BR), promotes glucose uptake in neurons and astrocytes, thereby linking brain activity with energy metabolism. To this end, we mapped the spatiotemporal accumulation of the fluorescent-labelled deoxyglucose, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), in superfused acute hippocampal slices of C57Bl/6j mice. Bath application of the A_2BR agonist BAY606583 (300 nM) triggered an immediate and stable (>10 min) increase of the velocity of 2-NBDG accumulation throughout hippocampal slices. This was abolished with the pretreatment with the selective A_2BR antagonist, MRS1754 (200 nM), and was also absent in A_2BR null-mutant mice. In mouse primary astrocytic or neuronal cultures, BAY606583 similarly increased ³H-deoxyglucose uptake in the following 20 min incubation period, which was again abolished by a pretreatment with MRS1754. Finally, incubation of hippocampal, frontocortical, or striatal slices of C57Bl/6j mice at 37 °C, with either MRS1754 (200 nM) or adenosine deaminase (3 U/mL) significantly reduced glucose uptake. Furthermore, A_2BR blockade diminished newly synthesized glycogen content and at least in the striatum, increased lactate release. In conclusion, we report here that A_2BR activation is associated with an instant and tonic increase of glucose transport into neurons and astrocytes in the mouse brain. These prompt further investigations to evaluate the clinical potential of this novel glucoregulator mechanism.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-015-9474-3) contains supplementary material, which is available to authorized users.     

Translational Diffusion of Macromolecule-sized Solutes in Cytoplasm and Nucleus

Fluorescence recovery after photobleaching (FRAP) was used to quantify the translational diffusion of microinjected FITC-dextrans and Ficolls in the cytoplasm and nucleus of MDCK epithelial cells and Swiss 3T3 fibroblasts. Absolute diffusion coefficients (D) were measured using a microsecond-resolution FRAP apparatus and solution standards. In aqueous media (viscosity 1 cP), D for the FITC-dextrans decreased from 75 to 8.4 × 10⁻⁷ cm²/s with increasing dextran size (4–2,000 kD). D in cytoplasm relative to that in water (D/D_o) was 0.26 ± 0.01 (MDCK) and 0.27 ± 0.01 (fibroblasts), and independent of FITC-dextran and Ficoll size (gyration radii [R_G] 40–300 Å). The fraction of mobile FITC-dextran molecules (f_mob), determined by the extent of fluorescence recovery after spot photobleaching, was >0.75 for R_G < 200 Å, but decreased to <0.5 for R_G > 300 Å. The independence of D/D_o on FITC-dextran and Ficoll size does not support the concept of solute “sieving” (size-dependent diffusion) in cytoplasm. Photobleaching measurements using different spot diameters (1.5–4 μm) gave similar D/D_o, indicating that microcompartments, if present, are of submicron size. Measurements of D/D_o and f_mob in concentrated dextran solutions, as well as in swollen and shrunken cells, suggested that the low f_mob for very large macromolecules might be related to restrictions imposed by immobile obstacles (such as microcompartments) or to anomalous diffusion (such as percolation). In nucleus, D/D_o was 0.25 ± 0.02 (MDCK) and 0.27 ± 0.03 (fibroblasts), and independent of solute size (R_G 40–300 Å). Our results indicate relatively free and rapid diffusion of macromolecule-sized solutes up to approximately 500 kD in cytoplasm and nucleus.     

Sodium-Glucose Transporter-2 (SGLT2; SLC5A2) Enhances Cellular Uptake of Aminoglycosides

Aminoglycoside antibiotics, like gentamicin, continue to be clinically essential worldwide to treat life-threatening bacterial infections. Yet, the ototoxic and nephrotoxic side-effects of these drugs remain serious complications. A major site of gentamicin uptake and toxicity resides within kidney proximal tubules that also heavily express electrogenic sodium-glucose transporter-2 (SGLT2; SLC5A2) in vivo. We hypothesized that SGLT2 traffics gentamicin, and promotes cellular toxicity. We confirmed in vitro expression of SGLT2 in proximal tubule-derived KPT2 cells, and absence in distal tubule-derived KDT3 cells. D-glucose competitively decreased the uptake of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), a fluorescent analog of glucose, and fluorescently-tagged gentamicin (GTTR) by KPT2 cells. Phlorizin, an SGLT2 antagonist, strongly inhibited uptake of 2-NBDG and GTTR by KPT2 cells in a dose- and time-dependent manner. GTTR uptake was elevated in KDT3 cells transfected with SGLT2 (compared to controls); and this enhanced uptake was attenuated by phlorizin. Knock-down of SGLT2 expression by siRNA reduced gentamicin-induced cytotoxicity. In vivo, SGLT2 was robustly expressed in kidney proximal tubule cells of heterozygous, but not null, mice. Phlorizin decreased GTTR uptake by kidney proximal tubule cells in Sglt2^+/− mice, but not in Sglt2^−/− mice. However, serum GTTR levels were elevated in Sglt2^−/− mice compared to Sglt2^+/− mice, and in phlorizin-treated Sglt2^+/− mice compared to vehicle-treated Sglt2^+/− mice. Loss of SGLT2 function by antagonism or by gene deletion did not affect gentamicin cochlear loading or auditory function. Phlorizin did not protect wild-type mice from kanamycin-induced ototoxicity. We conclude that SGLT2 can traffic gentamicin and contribute to gentamicin-induced cytotoxicity.     

Does Marriage Make Us Healthier? Inter-Country Comparative Evidence from China,Japan, and Korea

Objectives

This study focuses on East Asian countries and investigates the difference in the marriage premium on the health-marriage protection effect (MPE) between younger and older generations and the intra-couple education concordance effect (ECE) on the health of married individuals. This study used inter-country comparative data from China, Japan, and Korea.

Methods

This study focused on individuals (n = 7,938) in China, Japan, and Korea who were sampled from the 2010 East Asian Social Survey. To investigate MPE and ECE, four health indicators were utilized: a physical and mental components summary (PCS and MCS), self-rated health status (D_self), and happiness level (D_happy). Ordinary least squares regression was conducted by country- and gender-specific subsamples.

Results

We found that the MPE on PCS, MCS, and D_self was more significant for the older generation than for the younger generation in both China and Japan, whereas the results were inconclusive in Korea. With regard to the ECE on happiness (D_happy), for both men and women, couples tend to be happier when both the husband and the wife are well educated (“higher balanced marriage”) compared to couples with a lower level of educational achievement (“lower balanced marriage”). Significant benefits from a “higher balanced marriage” on MCS and D_self were observed for women only. In contrast, no statistically significant differences in health status were observed between “higher balanced marriage” couples and couples with different levels of educational achievements (“upward marriage” or “downward marriage”).

Conclusions

This study found that (1) the MPE was more significant for the older generation, and (2) the health gap, particularly the happiness gap, between higher- and lower-balanced married couples was significant. The inter-country comparative findings are useful to explain how the role of marriage (and therefore of family) on health has been diluted due to the progress of industrialization and modernization.     

Some observations on the biosynthesis of the plant sulpholipid by Euglena gracilis

1. dl-Cysteine decreases the uptake of ³⁵SO₄²⁻ by Euglena gracilis but does not decrease the relative incorporation of the isotope into sulpholipid; cysteic acid, on the other hand, does not affect the uptake of ³⁵SO₄²⁻ but does dilute out its incorporation into the sulpholipid. 2. Both l-[³⁵S]cysteic acid and dl-+meso-[3-¹⁴C]cysteic acid appear almost exclusively in 6-sulphoquinovose. 3. Molybdate inhibits the incorporation of ³⁵SO₄²⁻ into sulpholipid but not its uptake into the cells; this suggests that adenosine 3′-phosphate 5′-sulphatophosphate may be concerned with the biosynthesis of sulpholipid, and it was shown to be formed by chloroplast fragments. 4. An outline scheme for sulpholipid biosynthesis based on these observations is discussed.