Confocal microscopy study of the different patterns of 2-NBDG uptake in rabbit enterocytes in the apical and basal zone

Román, Yolanda; Alfonso, Amparo; Louzao, M. Carmen; Vieytes, Mercedes R.; Botana, Luís M.

doi:10.1007/s004240100677

Cited by 21 publications

(14 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The fluorescent dye 2-NBDG was a good tool to study glucose transport. We found that the dye is incorporated into freshly isolated rabbit enterocytes through glucose transporters as it was previously demonstrated in other cellular models (5)(6)(7)(9)(10)(11). The 2-NBDG was actively transported and accumulated in cells to a steady-state level that depended upon its extracellular concentration, the glucose transport capacity of the cells, and the intracellular degradation Figure 6.…”

Section: Discussionsupporting

confidence: 71%

Glucose Uptake in Enterocytes: A Test for Molecular Targets of Okadaic Acid

Louzao¹,

Vieytes²,

Fontal³

et al. 2003

Journal of Receptors and Signal Transduction

View full text Add to dashboard Cite

The main diarrheic shellfish poisoning (DSP) toxin is okadaic acid (OA). Although OA is a protein phosphatase 1 and 2A inhibitor less is known about the involvement of the toxin in diarrhea. The initial statement was that OA, by altering the phosphorylation state of proteins, might modify glucose uptake and consequently ionic and water reabsorption across the small intestine. This report presents studies of glucose transport in isolated rabbit enterocytes by using a fluorescent derivative of D-glucose. The dye allowed examining the relation between the toxic effect of OA and cellular mechanisms involved in glucose transport. The central findings are: (i) OA potentiates decrease on glucose uptake due to protein kinase A (PKA) inhibitors such as H89; and (ii) the increase of sugar uptake induced by the protein kinase C (PKC) inhibitor chelerythrine is enhanced by OA. Importance of this work is justified by the need to determine molecular targets of diarrheic toxins in intestinal cells.

show abstract

Section: Discussionsupporting

confidence: 71%

Glucose Uptake in Enterocytes: A Test for Molecular Targets of Okadaic Acid

Louzao¹,

Vieytes²,

Fontal³

et al. 2003

Journal of Receptors and Signal Transduction

View full text Add to dashboard Cite

show abstract

“…We observed the cellular uptake of 2-NBDG in the proximal tubule was similar from the apical and basolateral side of the cell, suggesting uptake by both SGLT and the glucose transporter (GLUT) as observed in studies in isolated cells (2,7,23,33).…”

Section: D)supporting

confidence: 69%

Novel application of complementary imaging techniques to examine in vivo glucose metabolism in the kidney

Hato

Friedman

Mang

et al. 2016

American Journal of Physiology-Renal Physiology

View full text Add to dashboard Cite

-The metabolic status of the kidney is a determinant of injury susceptibility and a measure of progression for many disease processes; however, noninvasive modalities to assess kidney metabolism are lacking. In this study, we employed positron emission tomography (PET) and intravital multiphoton microscopy (MPM) to assess cortical and proximal tubule glucose tracer uptake, respectively, following experimental perturbations of kidney metabolism. Applying dynamic image acquisition PET with 2-18 fluoro-2-deoxyglucose ( 18 F-FDG) and tracer kinetic modeling, we found that an intracellular compartment in the cortex of the kidney could be distinguished from the blood and urine compartments in animals. Given emerging literature that the tumor suppressor protein p53 is an important regulator of cellular metabolism, we demonstrated that PET imaging was able to discern a threefold increase in cortical 18 F-FDG uptake following the pharmacological inhibition of p53 in animals. Intravital MPM with the fluorescent glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) provided increased resolution and corroborated these findings at the level of the proximal tubule. Extending our observation of p53 inhibition on proximal tubule glucose tracer uptake, we demonstrated by intravital MPM that pharmacological inhibition of p53 diminishes mitochondrial potential difference. We provide additional evidence that inhibition of p53 alters key metabolic enzymes regulating glycolysis and increases intermediates of glycolysis. In summary, we provide evidence that PET is a valuable tool for examining kidney metabolism in preclinical and clinical studies, intravital MPM is a powerful adjunct to PET in preclinical studies of metabolism, and p53 inhibition alters basal kidney metabolism. positron emission tomography; multiphoton microscopy; kidney; p53 IT IS WIDELY APPRECIATED THAT kidney tubule metabolism is a critical determinant of both kidney function under physiological conditions and dysfunction in a variety of disease processes such as acute kidney injury (AKI) (31). More specifically, there is a growing awareness that understanding proximal tubule metabolism is a key component toward elucidating the overall susceptibility of the kidney to disease and injury. The application of drugs targeting glucose uptake in the proximal tubule for the treatment of diabetes mellitus has recently underscored this issue. Historical interpretation of studies from heterogeneous ex vivo preparations suggests that glucose utilization for glycolysis by the proximal tubule is minimal (17, 32). However, recent studies suggest that proximal tubule glycolytic flux plays an important role under various stressors and in diverse pathophysiological conditions (14,15,24,34). Nonetheless, a detailed examination and understanding of kidney glucose metabolism in vivo is lacking in large extent to the absence of tools to examine this process in vivo.Positron emission tomography (PET) has revolutionized the study and clinical detection of n...

show abstract

“…8 Consistently, glucose uptake was higher in HEPTECs from diabetes patients than in controls. Radiolabeled substrate, 14 C-labeled a-methyl-d-glucopyranoside ([ 14 C]AMG), is commonly used to measure SGLTmediated glucose uptake in primary cultures of human or animal PT cells and in the PT cell line LLC-PK 1 . [8][9][10] Similarly, we have used [ 14 C]AMG in our toxicological studies to show the inhibitory effect of cadmium on SGLT activity.…”

Section: Introductionmentioning

confidence: 99%

“…11,12 A fluorescent derivative of glucose, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), is available. 13 Microscopy can be performed to examine 2-NBDG uptake in living cells, 14 and the intracellular concentration of transported 2-NBDG can be measured with fluorescence spectroscopy. 15 In enterocytes, 2-NBDG uptake could be blocked with phlorizin, an inhibitor of SGLTs, 14,16 whereas cytochalasin B, a GLUT inhibitor, cells, 18,19 suggesting that 2-NBDG is transported by SGLTs and GLUTs.…”

Section: Introductionmentioning

confidence: 99%

A Fluorescence Method for Measurement of Glucose Transport in Kidney Cells

Blodgett

Kothinti

Kamyshko

et al. 2011

Diabetes Technology & Therapeutics

View full text Add to dashboard Cite

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 a-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 378C in buffer containing 50-200 mM 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

show abstract

Confocal microscopy study of the different patterns of 2-NBDG uptake in rabbit enterocytes in the apical and basal zone

Cited by 21 publications

References 22 publications

Glucose Uptake in Enterocytes: A Test for Molecular Targets of Okadaic Acid

Glucose Uptake in Enterocytes: A Test for Molecular Targets of Okadaic Acid

Novel application of complementary imaging techniques to examine in vivo glucose metabolism in the kidney

A Fluorescence Method for Measurement of Glucose Transport in Kidney Cells

Contact Info

Product

Resources

About