Glucose transport across lagomorph jejunum epithelium is modulated by AMP-activated protein kinase under hypoxia

The gastrointestinal epithelium possesses adaptation mechanisms to cope with huge variations in blood flow and subsequently oxygenation. Since sufficient energy supply is crucial under hypoxic conditions, glucose uptake especially must be regulated by these adaptation mechanisms. Therefore, we investigated glucose transport under hypoxic conditions. Jejunal epithelia of rabbits were incubated in Ussing chambers under short-circuit current conditions. Hypoxia was simulated by gassing with 1% O2 instead of 100% O2. The activity of sodium-coupled glucose transporter-1 (SGLT-1) was assessed by measuring the increase of short circuit current ( Isc) after the addition of 2 mM glucose to the mucosal buffer solution. We observed decreased activity of SGLT-1 after hypoxia compared with control conditions. To investigate underlying mechanisms, epithelia were exposed to agonists and antagonists of AMP-activated protein kinase (AMPK) before assessment of SGLT-1-mediated transport and the pAMPK/AMPK protein ratio. Preincubation with the antagonist restored SGLT-1 activity under hypoxic conditions to the level of control conditions, indicating an involvement of AMPK in the downregulation of SGLT-1 activity under hypoxia, which was confirmed in Western blot analysis of pAMPK/AMPK. Transepithelial flux studies using radioactively labeled glucose, ortho-methyl-glucose, fructose, and mannitol revealed no changes after hypoxic incubation. Therefore, we could exclude a decreased transepithelial glucose transport rate and increased paracellular conductance under hypoxia. In conclusion, our study hints at a decreased activity of SGLT-1 under hypoxic conditions in an AMPK-dependent manner. However, transepithelial transport of glucose is maintained. Therefore, we suggest other transport mechanisms, especially glucose transporter 1 and/or 2 to substitute SGLT-1 under hypoxia. NEW & NOTEWORTHY To our knowledge, this is the first approach to simulate hypoxia and study its effects in the jejunal epithelium using the Ussing chamber technique. We were able show that AMPK plays a role in the downregulation of SGLT-1 and that there seems to be an upregulation of other glucose transport mechanisms in the apical membrane of lagomorph jejunum epithelium under hypoxia, securing the epithelial energy supply and thus integrity.

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The Formation of an Insulin-responsive Vesicular Cargo Compartment Is an Early Event in 3T3-L1 Adipocyte Differentiation

Molecular Biology of the Cell ◽

10.1091/mbc.10.5.1581 ◽

1999 ◽

Vol 10 (5) ◽

pp. 1581-1594 ◽

Cited By ~ 51

Author(s):

Amr K. El-Jack ◽

Konstantin V. Kandror ◽

Paul F. Pilch

Keyword(s):

Glucose Transport ◽

Glucose Transporter ◽

Adipocyte Differentiation ◽

Receptor Trafficking ◽

Early Event ◽

Glucose Transporter 1 ◽

Glut4 Expression ◽

Velocity Gradients ◽

Dependent Inhibition ◽

Intracellular Glucose

Differentiating 3T3-L1 cells exhibit a dramatic increase in the rate of insulin-stimulated glucose transport during their conversion from proliferating fibroblasts to nonproliferating adipocytes. On day 3 of 3T3-L1 cell differentiation, basal glucose transport and cell surface transferrin binding are markedly diminished. This occurs concomitant with the formation of a distinct insulin-responsive vesicular pool of intracellular glucose transporter 1 (GLUT1) and transferrin receptors as assessed by sucrose velocity gradients. The intracellular distribution of the insulin-responsive aminopeptidase is first readily detectable on day 3, and its gradient profile and response to insulin at this time are identical to that of GLUT1. With further time of differentiation, GLUT4 is expressed and targeted to the same insulin-responsive vesicles as the other three proteins. Our data are consistent with the notion that a distinct insulin-sensitive vesicular cargo compartment forms early during fat call differentiation and its formation precedes GLUT4 expression. The development of this compartment may result from the differentiation-dependent inhibition of constitutive GLUT1 and transferrin receptor trafficking such that there is a large increase in, or the new formation of, a population of postendosomal, insulin-responsive vesicles.

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The Fast Lane of Hypoxic Adaptation: Glucose Transport Is Modulated via A HIF-Hydroxylase-AMPK-Axis in Jejunum Epithelium

International Journal of Molecular Sciences ◽

10.3390/ijms20204993 ◽

2019 ◽

Vol 20 (20) ◽

pp. 4993 ◽

Cited By ~ 3

Author(s):

Dengler ◽

Gäbel

Keyword(s):

Glucose Transport ◽

Glucose Transporter ◽

Ussing Chamber ◽

Transepithelial Transport ◽

Adaptation To Hypoxia ◽

Regulation Mechanism ◽

Short Term ◽

Glucose Transporter 1 ◽

Functional Changes ◽

Short Term Adaptation

The intestinal epithelium is able to adapt to varying blood flow and, thus, oxygen availability. Still, the adaptation fails under pathologic situations. A better understanding of the mechanisms underlying the epithelial adaptation to hypoxia could help to improve the therapeutic approach. We hypothesized that the short-term adaptation to hypoxia is mediated via AMP-activated protein kinase (AMPK) and that it is coupled to the long-term adaptation by a common regulation mechanism, the HIF-hydroxylase enzymes. Further, we hypothesized the transepithelial transport of glucose to be part of this short-term adaptation. We conducted Ussing chamber studies using isolated lagomorph jejunum epithelium and cell culture experiments with CaCo-2 cells. The epithelia and cells were incubated under 100% and 21% O2, respectively, with the panhydroxylase inhibitor dimethyloxalylglycine (DMOG) or under 1% O2. We showed an activation of AMPK under hypoxia and after incubation with DMOG by Western blot. This could be related to functional effects like an impairment of Na+-coupled glucose transport. Inhibitor studies revealed a recruitment of glucose transporter 1 under hypoxia, but not after incubation with DMOG. Summing up, we showed an influence of hydroxylase enzymes on AMPK activity and similarities between hypoxia and the effects of hydroxylase inhibition on functional changes.

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Reactive oxygen species downregulate glucose transport system in retinal endothelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.00140.2010 ◽

2011 ◽

Vol 300 (4) ◽

pp. C927-C936 ◽

Cited By ~ 29

Author(s):

Rosa Fernandes ◽

Ken-ichi Hosoya ◽

Paulo Pereira

Keyword(s):

Oxidative Stress ◽

Endothelial Cells ◽

Plasma Membrane ◽

Endothelial Cell ◽

Glucose Transport ◽

Glucose Transporter ◽

Cell Damage ◽

Proteasome Inhibition ◽

Glucose Transporter 1 ◽

Retinal Endothelial Cells

Retinal endothelial cells are believed to play an important role in the pathogenesis of diabetic retinopathy. In previous studies, we and others demonstrated that glucose transporter 1 (GLUT1) is downregulated in response to hyperglycemia. Increased oxidative stress is likely to be the event whereby hyperglycemia is transduced into endothelial cell damage. However, the effects of sustained oxidative stress on GLUT1 regulation are not clearly established. The objective of this study is to evaluate the effect of increased oxidative stress on glucose transport and on GLUT1 subcellular distribution in a retinal endothelial cell line and to elucidate the signaling pathways associated with such regulation. Conditionally immortalized rat retinal endothelial cells (TR-iBRB) were incubated with glucose oxidase, which increases the intracellular hydrogen peroxide levels, and GLUT1 regulation was investigated. The data showed that oxidative stress did not alter the total levels of GLUT1 protein, although the levels of mRNA were decreased, and there was a subcellular redistribution of GLUT1, decreasing its content at the plasma membrane. Consistently, the half-life of the protein at the plasma membrane markedly decreased under oxidative stress. The proteasome appears to be involved in GLUT1 regulation in response to oxidative stress, as revealed by an increase in stabilization of the protein present at the plasma membrane and normalization of glucose transport following proteasome inhibition. Indeed, levels of ubiquitinated GLUT1 increase as revealed by immunoprecipitation assays. Furthermore, data indicate that protein kinase B activation is involved in the stabilization of GLUT1 at the plasma membrane. Thus subcellular redistribution of GLUT1 under conditions of oxidative stress is likely to contribute to the disruption of glucose homeostasis in diabetes.

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Caffeine inhibits glucose transport by binding at the GLUT1 nucleotide-binding site

AJP Cell Physiology ◽

10.1152/ajpcell.00001.2015 ◽

2015 ◽

Vol 308 (10) ◽

pp. C827-C834 ◽

Cited By ~ 14

Author(s):

Jay M. Sage ◽

Anthony J. Cura ◽

Kenneth P. Lloyd ◽

Anthony Carruthers

Keyword(s):

Binding Site ◽

Glucose Transport ◽

Glucose Transporter ◽

Cytochalasin B ◽

Sugar Transport ◽

Nucleotide Binding ◽

Nucleotide Binding Site ◽

Sugar Uptake ◽

Glucose Transporter 1 ◽

Intracellular Atp

Glucose transporter 1 (GLUT1) is the primary glucose transport protein of the cardiovascular system and astroglia. A recent study proposes that caffeine uncompetitive inhibition of GLUT1 results from interactions at an exofacial GLUT1 site. Intracellular ATP is also an uncompetitive GLUT1 inhibitor and shares structural similarities with caffeine, suggesting that caffeine acts at the previously characterized endofacial GLUT1 nucleotide-binding site. We tested this by confirming that caffeine uncompetitively inhibits GLUT1-mediated 3- O-methylglucose uptake in human erythrocytes [ Vmax and Km for transport are reduced fourfold; Ki(app) = 3.5 mM caffeine]. ATP and AMP antagonize caffeine inhibition of 3- O-methylglucose uptake in erythrocyte ghosts by increasing Ki(app) for caffeine inhibition of transport from 0.9 ± 0.3 mM in the absence of intracellular nucleotides to 2.6 ± 0.6 and 2.4 ± 0.5 mM in the presence of 5 mM intracellular ATP or AMP, respectively. Extracellular ATP has no effect on sugar uptake or its inhibition by caffeine. Caffeine and ATP displace the fluorescent ATP derivative, trinitrophenyl-ATP, from the GLUT1 nucleotide-binding site, but d-glucose and the transport inhibitor cytochalasin B do not. Caffeine, but not ATP, inhibits cytochalasin B binding to GLUT1. Like ATP, caffeine renders the GLUT1 carboxy-terminus less accessible to peptide-directed antibodies, but cytochalasin B and d-glucose do not. These results suggest that the caffeine-binding site bridges two nonoverlapping GLUT1 endofacial sites—the regulatory, nucleotide-binding site and the cytochalasin B-binding site. Caffeine binding to GLUT1 mimics the action of ATP but not cytochalasin B on sugar transport. Molecular docking studies support this hypothesis.

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Regulation of lactate production and glucose transport as well as of glucose transporter 1 and lactate dehydrogenase A mRNA levels by basic fibroblast growth factor in rat Sertoli cells

Journal of Endocrinology ◽

10.1677/joe.0.1730335 ◽

2002 ◽

Vol 173 (2) ◽

pp. 335-343 ◽

Cited By ~ 31

Author(s):

MF Riera ◽

SB Meroni ◽

HF Schteingart ◽

EH Pellizzari ◽

SB Cigorraga

Keyword(s):

Growth Factor ◽

Glucose Transport ◽

Glucose Transporter ◽

Lactate Production ◽

Mrna Levels ◽

Dependent Manner ◽

Short Term ◽

Glucose Transporter 1 ◽

Ldh Activity

By using cultured rat Sertoli cells as a model, both the action of basic fibroblast growth factor (bFGF) on lactate production and the site of this action were studied. bFGF stimulated Sertoli cell lactate production in a dose-dependent manner (basal: 7.3+/-0.5; 0.1 ng/ml bFGF: 7.5+/-0.5; 1 ng/ml bFGF: 7.5+/-0.6; 10 ng/ml bFGF: 10.3+/-1.0; 30 ng/ml bFGF: 15.2+/-1.5; 50 ng/ml bFGF: 15.4+/-1.6 microg/microg DNA). Two major sites for the action of this growth factor were identified. First, bFGF was shown to exert short- and long-term stimulatory effects on glucose transport (basal: 1170+/-102; 30 ng/ml bFGF for 120 min: 1718+/-152 and basal: 718+/-64; 30 ng/ml bFGF for 48 h: 1069+/-69 d.p.m./microg DNA respectively). Short-term bFGF stimulation of glucose transport was not inhibited by the protein synthesis inhibitor cycloheximide. These results indicate that short-term bFGF stimulation of glucose uptake does not involve an increase in the number of glucose transporters. On the other hand, stimulation with bFGF for periods of time longer than 12 h increased glucose transporter 1 (GLUT1) mRNA levels. These increased mRNA levels were probably ultimately responsible for the increments in glucose uptake that are observed in long-term treated cultures. Secondly, bFGF increased lactate dehydrogenase (LDH) activity (basal: 31.0+/-1.4; 30 ng/ml bFGF: 45.7+/- 2.4 mIU/microg DNA). The principal subunit component of those LDH isozymes that favors the transformation of pyruvate to lactate is subunit A. bFGF increased LDH A mRNA levels in a dose- and time-dependent manner. In summary, the results presented herein show that glucose transport, LDH activity and GLUT1 and LDH A mRNA levels are regulated by bFGF to achieve an increase in lactate production. These observed regulatory actions provide unequivocal evidence of the participation of bFGF in Sertoli cell lactate production which may be related to normal germ cell development.

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Salicylketoximes That Target Glucose Transporter 1 Restrict Energy Supply to Lung Cancer Cells

ChemMedChem ◽

10.1002/cmdc.201500320 ◽

2015 ◽

Vol 10 (11) ◽

pp. 1892-1900 ◽

Cited By ~ 15

Author(s):

Carlotta Granchi ◽

Yanrong Qian ◽

Hyang Yeon Lee ◽

Ilaria Paterni ◽

Carolina Pasero ◽

...

Keyword(s):

Lung Cancer ◽

Cancer Cells ◽

Energy Supply ◽

Glucose Transporter ◽

Lung Cancer Cells ◽

Glucose Transporter 1 ◽

Target Glucose

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THE EFFECTS OF PORCINE CALCITONIN ON RENAL FUNCTION IN THE RABBIT

Journal of Endocrinology ◽

10.1677/joe.0.0500485 ◽

1971 ◽

Vol 50 (3) ◽

pp. 485-491 ◽

Cited By ~ 9

Author(s):

L.A. SALAKO ◽

A. J. SMITH ◽

R. N. SMITH

Keyword(s):

Filtration Rate ◽

Short Circuit ◽

Renal Plasma Flow ◽

Short Circuit Current ◽

Urinary Flow ◽

Transport Mechanisms ◽

Acid Clearance ◽

Tubular Transport ◽

Renal Tubular Transport ◽

Renal Tubular

SUMMARY Extracts of porcine thyroid containing calcitonin produced increases in urinary flow and urinary electrolyte content when infused or injected into anaesthetized rabbits. This response occurred more rapidly after intraaortic than after intravenous injection and was accompanied by an increase in glomerular filtration rate (inulin clearance) and renal plasma flow (paraaminohippuric acid clearance). Preparations of calcitonin failed to affect the short-circuit current in isolated frog skin. Although an effect of calcitonin on renal tubular transport mechanisms cannot be excluded it seems likely that one mechanism responsible for the diuretic effect of this compound in the rabbit is an increase in renal blood flow.

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Glucocorticoids Enhance Intestinal Glucose Uptake Via the Dimerized Glucocorticoid Receptor in Enterocytes

Endocrinology ◽

10.1210/en.2011-1747 ◽

2012 ◽

Vol 153 (4) ◽

pp. 1783-1794 ◽

Cited By ~ 25

Author(s):

Sybille D. Reichardt ◽

Michael Föller ◽

Rexhep Rexhepaj ◽

Ganesh Pathare ◽

Kerstin Minnich ◽

...

Keyword(s):

Small Intestine ◽

Glucose Uptake ◽

Molecular Mechanism ◽

Glucose Transport ◽

Transcriptional Control ◽

Glucose Transporter ◽

Gene Activation ◽

Mouse Strains ◽

Glucose Transporter 1

Glucocorticoid (GC) treatment of inflammatory disorders, such as inflammatory bowel disease, causes deranged metabolism, in part by enhanced intestinal resorption of glucose. However, the underlying molecular mechanism is poorly understood. Hence, we investigated transcriptional control of genes reported to be involved in glucose uptake in the small intestine after GC treatment and determined effects of GC on electrogenic glucose transport from transepithelial currents. GRvillinCre mice lacking the GC receptor (GR) in enterocytes served to identify the target cell of GC treatment and the requirement of the GR itself; GRdim mice impaired in dimerization and DNA binding of the GR were used to determine the underlying molecular mechanism. Our findings revealed that oral administration of dexamethasone to wild-type mice for 3 d increased mRNA expression of serum- and GC-inducible kinase 1, sodium-coupled glucose transporter 1, and Na+/H+ exchanger 3, as well as electrogenic glucose transport in the small intestine. In contrast, GRvillinCre mice did not respond to GC treatment, neither with regard to gene activation nor to glucose transport. GRdim mice were also refractory to GC, because dexamethasone treatment failed to increase both, gene expression and electrogenic glucose transport. In addition, the rise in blood glucose levels normally observed after GC administration was attenuated in both mutant mouse strains. We conclude that enhanced glucose transport in vivo primarily depends on gene regulation by the dimerized GR in enterocytes, and that this mechanism contributes to GC-induced hyperglycemia.

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Reduced Chemosensitivity of FLT3/ITD Mutated Cells to Cytarabine and Quizartinib Under Hypoxic Conditions

Blood ◽

10.1182/blood.v128.22.1579.1579 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1579-1579

Author(s):

Heiko Konig ◽

Adriana L Rogozea ◽

Garrett H Kinnebrew ◽

Mircea Ivan

Keyword(s):

Drug Resistance ◽

Growth Inhibition ◽

Oxygen Tension ◽

Glucose Transporter ◽

Conflicts Of Interest ◽

Unmet Need ◽

Leukemic Cells ◽

Primary Cells ◽

Glucose Transporter 1 ◽

Hypoxic Conditions

Abstract Background: FLT3/ITD mutated AML is characterized by short remission duration and high relapse rates due to the survival of a small fraction of leukemic cells (LCs) that outlive initial therapy. There is compelling evidence that the hypoxic niche in the bone marrow (BM) provides a sanctuary where subpopulations of LCs evade cytotoxic therapy and acquire drug resistance. In order to define the mechanisms involved in this process, multiple studies have focused on the interactions between LCs and the BM microenvironment but less is known about to the role of hypoxia as a modulator of drug resistance. Hence, the effects of standard and investigational therapies under hypoxic conditions are largely unknown. Here, we investigated the cytotoxic response of FLT3/ITD-mutated cells to conventional and targeted therapy under normoxic (21% O2) and hypoxic (1% O2) conditions. Methods: Molm14 (M14) cells and primary cells from relapsed/refractory FLT3/ITD mutated AML patients were incubated in culture medium under normoxic and hypoxic conditions. Cytarabine (Cy) or Quizartinib (Quiz) were added as single agents at the indicated concentrations. After 48 hours proliferation and apoptosis assays were performed using MTT assays, annexin V/PI staining and FACS analysis (M14). Cells were also assessed for FLT3 protein and Hypoxia inducible factor (HIF) target gene expression by western blotting and PCR arrays (RT2 ProfilerTM PCR Array, Human Hypoxia Signaling Pathway Plus, Qiagen) (M14 and primary cells), respectively. Results: We found that M14 cells were significantly less susceptible to treatment under hypoxic conditions, both when exposed to Cy and targeted FLT3 inhibition with Quiz (Growth inhibition, 0.25nM Quiz: 8.5±4.2% (1% O2) vs. 22±2.5% (21% O2), p<.05; 49.6±2.5% (1% O2) vs. 58.1±2.3% (21% O2), n=8, p<.05; 0.25µM Cy: 3.7±2.5% (1% O2) vs. 14.4±1.5% (21% O2), p<.01; 1 µM Cy: 26.3±4.1% (1% O2) vs. 53.3±3.8% (21% O2), n=10-12, p<.001). In line with these findings, the apoptotic response of M14 cells to Cy treatment was significantly blunted under hypoxic conditions (Fold increase in apoptotic cells vs. untreated control, 1 µM: 5.7±0.8 fold [21% O2] vs. 2.7±0.5 fold [1% O2], n=4, p<.05). While not reaching the significance threshold, the trend was similar for the Quiz-treated cells. This effect was extended to primary, relapsed/refractory FLT3/ITD-mutated cells derived from patients (n=2) treated at our institution, where growth inhibition was consistently decreased in hypoxia at all doses of Cy and Quiz tested. Not unexpectedly, these primary cells required significantly higher doses compared to the established M14 cell line (1-10µM for Cy, and 50-200nM for Quiz). Importantly, Quiz appeared to be similarly effective in inactivating FLT3 activity under low and high oxygen tension. Therefore identifying tractable pro-survival genes induced by the low oxygen microenvironment should represent a viable strategy to increase the effectiveness of anti-leukemic agents. In order to identify such target candidates, we surveyed the effects of Quiz and Cy on a panel of 84 hypoxia-regulated genes using PCR arrays. Our preliminary studies revealed that while HIF pathway remains largely functional in the presence of Quiz or Cy, these agents severely blunt induction of several hypoxia genes (including Glucose transporter 1 [Glut1] and Carbonic Anhydrase 9 [CA9]) in both primary cells and M14 cells. Conclusions: 1.Hypoxia limits the cytotoxic effects of conventional and targeted therapeutic agents in primary and established FLT3/ITD mutated AML cells. 2. Quizartinib effectively abrogates FLT3 signaling under both normoxic and hypoxic conditions, indicating that other signaling pathways are critical for leukemic cell survival under reduced oxygen tension. 3. The induction of several potentially druggable HIF targets is disrupted by anti-AML agents, warranting the further investigation of combinational approaches between standard anti-AML agents and HIF targeted strategies. 4. Such therapeutic combinations are likely to be particularly effective undermicro-environmental stress conditions (e.g. severe hypoxia) thus addressing an unmet need in AML therapy. Disclosures No relevant conflicts of interest to declare.

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