Differential targeting of facilitative glucose transporters in polarized epithelial cells

1996 ◽  
Vol 271 (2) ◽  
pp. C547-C554 ◽  
Author(s):  
W. S. Pascoe ◽  
K. Inukai ◽  
Y. Oka ◽  
J. W. Slot ◽  
D. E. James
Keyword(s):  
Epithelial Cells ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Mdck Cells ◽  
Intracellular Localization ◽  
Apical Plasma Membrane ◽  
Cellular Polarity ◽  
Glut 1 ◽  
Polarized Epithelial Cells ◽  
High Level

We have examined the intracellular localization of five facilitative glucose transporter proteins, one endogenous (GLUT-1) and four exogenous (GLUT-2, -3, -4, and -5), in polarized epithelial cells. GLUT-2, -3, -4, and -5 were stably transfected into Madin-Darby canine kidney (MDCK) cells, and peptide-specific antibodies were used to establish their distribution by immunofluorescence and immunoelectron-microscopic techniques. GLUT-1 and -2 were predominantly targeted to the basolateral domain of the cell, whereas GLUT-3 and -5 were targeted to the apical plasma membrane. The insulin-regulatable glucose transporter GLUT-4 was found in intracellular tubulovesicular structures beneath the surface of the cell. Vectorial 2-deoxy-D-glucose uptake measurements revealed that approximately 95% of glucose entry into wild-type MDCK cells occurs via the basolateral membranes. In GLUT-3-transfected cells, however, apical glucose uptake increased to approximately 55%; this was not observed in cells expressing the other GLUT isoforms. The discrete and differential intracellular localizations of the various GLUTs, in addition to the high level of sequence homology and predicted secondary structure similarity, render the GLUT family ideal for the study of intrinsic targeting motifs involved in the establishment and maintenance of cellular polarity.

Visualization of transcytosis in MDCK cells using laser scanning confocal microscopy

1994 ◽  
Vol 52 ◽  
pp. 220-221
Author(s):  
Greg Martin ◽  
Rohit Cariappa ◽  
Ann L. Hubbard
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Epithelial Cells ◽  
Laser Scanning ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Basolateral Membrane ◽  
Apical Plasma Membrane ◽  
Plasma Membrane Proteins ◽  
Polarized Epithelial Cells

The plasma membrane of polarized epithelial cells is composed of two structurally and functionally distinct domains -- the apical and basolateral -- that also differ in molecular composition. The routes followed by integral membrane proteins from their site of synthesis to their site of function varies between different kinds of epithelia. Madin-Darby canine kidney (MDCK) cells deliver plasma membrane proteins directly to the correct domain, while polarized hepatocytes deliver all newly synthesized plasma membrane proteins initially to the basolateral membrane, then retrieve and redirect the apical membrane proteins. We are studying the targeting signals and delivery routes of DPPIV, a single transmembrane protein whose destination is the apical domain in polarized epithelial cells.DPPIV transfected into MDCK cells is delivered to the basolateral plasma membrane after long (13hr) treatment with Brefeldin A (BFA). After BFA’s removal these molecules are retrieved from the basolateral membrane and transcytosed to the apical plasma membrane. This protocol provides a useful model for studies of the indirect route of protein sorting in polarized epithelial cells, since DPPIV at the basolateral surface can be labeled with specific antibody and then subsequently followed in living cells.

Differential glycolytic and glycogenogenic transduction pathways in male and female bovine embryos produced in vitro

2012 ◽  
Vol 24 (2) ◽  
pp. 344 ◽  
Author(s):  
M. Garcia-Herreros ◽  
I. M. Aparicio ◽  
D. Rath ◽  
T. Fair ◽  
P. Lonergan
Keyword(s):  
Glucose Metabolism ◽  
Protein Expression ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Glycogen Synthase ◽  
Bovine Embryos ◽  
High Concentration ◽  
Glucose Transporter 1 ◽  
Male And Female ◽  
Glut 1

Previous studies have shown that developmental kinetic rates following IVF are lower in female than in male blastocysts and that this may be related to differences in glucose metabolism. In addition, an inhibition of phosphatidylinositol 3-kinase (PI3-K) inhibits glucose uptake in murine blastocysts. Therefore, the aim of this study was to identify and compare the expression of proteins involved in glucose metabolism (hexokinase-I, HK-I; phosphofructokinase-1, PFK-1; pyruvate kinase1/2, PK1/2; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; glucose transporter-1, GLUT-1; and glycogen synthase kinase-3, GSK-3) in male and female bovine blastocysts to determine whether PI3-K has a role in the regulation of the expression of these proteins. Hexokinase-I, PFK-1, PK1/2, GAPDH and GLUT-1 were present in bovine embryos. Protein expression of these proteins and GSK-3 was significantly higher in male compared with female blastocysts. Inhibition of PI3-K with LY294002 significantly decreased the expression of HK-I, PFK-1, GAPDH, GSK-3 A/B and GLUT-1. Results showed that the expression of glycolytic proteins HK-I, PFK-1, GAPDH and PK1/2, and the transporters GLUT-1 and GSK-3 is regulated by PI3-K in bovine blastocysts. Moreover, the differential protein expression observed between male and female blastocysts might explain the faster developmental kinetics seen in males, as the expression of main proteins involved in glycolysis and glycogenogenesis was significantly higher in male than female bovine embryos and also could explain the sensitivity of male embryos to a high concentration of glucose, as a positive correlation between GLUT-1 expression and glucose uptake in embryos has been demonstrated.

The GTPase Rac1 selectively regulates Salmonella invasion at the apical plasma membrane of polarized epithelial cells

2001 ◽  
Vol 114 (7) ◽  
pp. 1331-1341 ◽  
Author(s):  
A.K. Criss ◽  
D.M. Ahlgren ◽  
T.S. Jou ◽  
B.A. McCormick ◽  
J.E. Casanova
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Bacterial Pathogen ◽  
Small Gtpases ◽  
Apical Plasma Membrane ◽  
Membrane Domains ◽  
Intestinal Epithelial ◽  
Actin Structure

The bacterial pathogen Salmonella typhimurium colonizes its animal hosts by inducing its internalization into intestinal epithelial cells. This process requires reorganization of the actin cytoskeleton of the apical plasma membrane into elaborate membrane ruffles that engulf the bacteria. Members of the Ρ family of small GTPases are critical regulators of actin structure, and in nonpolarized cells, the GTPase Cdc42 has been shown to modulate Salmonella entry. Because the actin architecture of epithelial cells is organized differently from that of nonpolarized cells, we examined the role of two ‘Rgr; family GTPases, Cdc42 and Rac1, in invasion of polarized monolayers of MDCK cells by S. typhimurium. Surprisingly, we found that endogenous Rac1, but not Cdc42, was activated during bacterial entry at the apical pole, and that this activation required the bacterial effector protein SopE. Furthermore, expression of dominant inhibitory Rac1 but not Cdc42 significantly inhibited apical internalization of Salmonella, indicating that Rac1 activation is integral to the bacterial entry process. In contrast, during basolateral internalization, both Cdc42 and Rac1 were activated; however, neither GTPase was required for entry. These findings, which differ significantly from previous observations in nonpolarized cells, indicate that the host cell signaling pathways activated by bacterial pathogens may vary with cell type, and in epithelial tissues may further differ between plasma membrane domains.

scholarly journals Chlorpromazine Induces Basolateral Aquaporin-2 Accumulation via F-Actin Depolymerization and Blockade of Endocytosis in Renal Epithelial Cells

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 1057
Author(s):  
Richard Bouley ◽  
Naofumi Yui ◽  
Abby Terlouw ◽  
Pui W. Cheung ◽  
Dennis Brown
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Rhodamine Phalloidin ◽  
Renal Epithelial Cells ◽  
Aquaporin 2 ◽  
Basolateral Plasma Membrane

We previously showed that in polarized Madin–Darby canine kidney (MDCK) cells, aquaporin-2 (AQP2) is continuously targeted to the basolateral plasma membrane from which it is rapidly retrieved by clathrin-mediated endocytosis. It then undertakes microtubule-dependent transcytosis toward the apical plasma membrane. In this study, we found that treatment with chlorpromazine (CPZ, an inhibitor of clathrin-mediated endocytosis) results in AQP2 accumulation in the basolateral, but not the apical plasma membrane of epithelial cells. In MDCK cells, both AQP2 and clathrin were concentrated in the basolateral plasma membrane after CPZ treatment (100 µM for 15 min), and endocytosis was reduced. Then, using rhodamine phalloidin staining, we found that basolateral, but not apical, F-actin was selectively reduced by CPZ treatment. After incubation of rat kidney slices in situ with CPZ (200 µM for 15 min), basolateral AQP2 and clathrin were increased in principal cells, which simultaneously showed a significant decrease of basolateral compared to apical F-actin staining. These results indicate that clathrin-dependent transcytosis of AQP2 is an essential part of its trafficking pathway in renal epithelial cells and that this process can be inhibited by selectively depolymerizing the basolateral actin pool using CPZ.

scholarly journals Microtubule perturbation inhibits intracellular transport of an apical membrane glycoprotein in a substrate-dependent manner in polarized Madin-Darby canine kidney epithelial cells.

1990 ◽  
Vol 1 (12) ◽  
pp. 921-936 ◽  
Author(s):  
M J van Zeijl ◽  
K S Matlin
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Intracellular Transport ◽  
Mdck Cells ◽  
Sodium Dodecyl ◽  
Apical Plasma Membrane ◽  
Dependent Manner ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

The effects of microtubule perturbation on the transport of two different viral glycoproteins were examined in infected Madin-Darby canine kidney (MDCK) cells grown on both permeable and solid substrata. Quantitative biochemical analysis showed that the microtubule-depolymerizing drug nocodazole inhibited arrival of influenza hemagglutinin on the apical plasma membrane in MDCK cells grown on both substrata. In contrast, the microtubule-stabilizing drug taxol inhibited apical appearance of hemagglutinin only when MDCK cells were grown on permeable substrata. On the basis of hemagglutinin mobility on sodium dodecyl sulfate gels and its sensitivity to endo H, it was evident that nocodazole and taxol arrested hemagglutinin at different intracellular sites. Neither drug caused a significant increase in the amount of hemagglutinin detected on the basolateral plasma membrane domain. In addition, neither drug had any noticeable effect on the transport of the vesicular stomatitis virus (VSV)-G protein to the basolateral surface. These results shed light on previous conflicting reports using this model system and support the hypothesis that microtubules play a role in the delivery of membrane glycoproteins to the apical, but not the basolateral, domain of epithelial cells.

scholarly journals Effects of Chronic Undernutrition on Glucose Uptake and Glucose Transporter Proteins in Rat Heart

Endocrinology ◽  
2002 ◽  
Vol 143 (11) ◽  
pp. 4295-4303 ◽  
Author(s):  
M. Lucia Gavete ◽  
Maria Agote ◽  
M. Angeles Martin ◽  
Carmen Alvarez ◽  
Fernando Escriva
Keyword(s):  
Glucose Uptake ◽  
Glucose Transporter ◽  
Surface Membrane ◽  
Subcellular Fractionation ◽  
High Energy ◽  
Regulatory Subunits ◽  
Glut 1 ◽  
Glut 4 ◽  
Energy Demands ◽  
Glucose Transporter Proteins

Abstract The high energy demands of myocardium are met through the metabolism of lipids and glucose. Importantly, enhanced glucose utilization rates are crucial adaptations of the cardiac cell to some pathological conditions, such as hypertrophy and ischemia, but the effects of undernutrition on heart glucose metabolism are unknown. Our previous studies have shown that undernutrition increases insulin-induced glucose uptake by skeletal muscle. Consequently, we considered the possibility of a similar adaptation in the heart. With this aim, undernourished rats both in the basal state and after euglycemic hyperinsulinemic clamps were used to determine the following parameters in myocardium: glucose uptake, glucose transporter (GLUT) content, and some key components of the insulin signaling cascade. Heart membranes were prepared by subcellular fractionation in sucrose gradients. Although GLUT-4, GLUT-1, and GLUT-3 proteins and GLUT-4/1 mRNAs were reduced by undernutrition, basal and insulin-stimulated 2-deoxyglucose uptake were significantly enhanced. Phosphoinositol 3-kinase activity remained greater than control values in both conditions. The abundance of p85α and p85β regulatory subunits of phosphoinositol 3-kinase was increased as was phospho-Akt during hyperinsulinemia. These changes seem to improve the insulin stimulus of GLUT-1 translocation, as its content was increased at the surface membrane. Such adaptations associated with undernutrition must be crucial to improvement of cardiac glucose uptake.

Effect of diet on insulin- and contraction-mediated glucose transport and uptake in rat muscle

1995 ◽  
Vol 269 (3) ◽  
pp. R544-R551 ◽  
Author(s):  
X. Han ◽  
T. Ploug ◽  
H. Galbo
Keyword(s):  
Glucose Uptake ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Fiber Types ◽  
Transport Capacity ◽  
Glut 1 ◽  
Glut 4 ◽  
Red Muscle ◽  
Muscle Glucose Transport ◽  
Stimulation Of

A diet rich in fat diminishes insulin-mediated glucose uptake in muscle. This study explored whether contraction-mediated glucose uptake is also affected. Rats were fed a diet rich in fat (FAT, 73% of energy) or carbohydrate (CHO, 66%) for 5 wk. Hindquarters were perfused, and either glucose uptake or glucose transport capacity (uptake of 3-O-[14C]-methyl-D-glucose (40 mM)) was measured. Amounts of glucose transporter isoform GLUT-1 and GLUT-4 glucose-transporting proteins were determined by Western blot. Glucose uptake was lower (P < 0.05) in hindlegs from FAT than from CHO rats at submaximum and maximum insulin [4 +/- 0.4 vs. 5 +/- 0.3 (SE) mumol.min-1.leg-1 at 150 microU/ml insulin] as well as during prolonged stimulation of the sciatic nerve (4.4 +/- 0.4 vs. 5.6 +/- 0.6 mumol.min-1.leg-1). Maximum glucose transport elicited by insulin (soleus: 1.7 +/- 0.2 vs. 2.6 +/- 0.2 mumol.g-1.5 min-1, P < 0.05) or contractions (soleus: 1.8 +/- 0.2 vs. 2.6 +/- 0.3, P < 0.05) in red muscle was decreased in parallel in FAT compared with CHO rats. GLUT-4 content was decreased by 13-29% (P < 0.05) in the various fiber types, whereas GLUT-1 content was identical in FAT compared with CHO rats. It is concluded that a FAT diet reduces both insulin and contraction stimulation of glucose uptake in muscle and that these effects are associated with diminished skeletal muscle glucose transport capacities and GLUT-4 contents.

scholarly journals Sphingomyelinase has an insulin-like effect on glucose transporter translocation in adipocytes

1998 ◽  
Vol 274 (5) ◽  
pp. R1446-R1453 ◽  
Author(s):  
T. S. David ◽  
P. A. Ortiz ◽  
T. R. Smith ◽  
J. Turinsky
Keyword(s):  
Plasma Membrane ◽  
Insulin Receptor ◽  
Glucose Uptake ◽  
Signaling Pathway ◽  
Insulin Signaling ◽  
Glucose Transporter ◽  
Insulin Signaling Pathway ◽  
Glut 1 ◽  
Glut 4 ◽  
Glut 4 Translocation

Rat epididymal adipocytes were incubated with 0, 0.1, and 1 mU sphingomyelinase/ml for 30 or 60 min, and glucose uptake and GLUT-1 and GLUT-4 translocation were assessed. Adipocytes exposed to 1 mU sphingomyelinase/ml exhibited a 173% increase in glucose uptake. Sphingomyelinase had no effect on the abundance of GLUT-1 in the plasma membrane of adipocytes. In contrast, 1 mU sphingomyelinase/ml increased plasma membrane content of GLUT-4 by 120% and produced a simultaneous decrease in GLUT-4 abundance in the low-density microsomal fraction. Sphingomyelinase had no effect on tyrosine phosphorylation of either the insulin receptor β-subunit or the insulin receptor substrate-1, a signaling molecule in the insulin signaling pathway. It is concluded that the incubation of adipocytes with sphingomyelinase results in insulin-like translocation of GLUT-4 to the plasma membrane and that this translocation does not occur via the activation of the initial components of the insulin signaling pathway.

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