Articles in PresS. Am J Physiol Cell Physiol (April 8, 2015). doi:10.1152/ajpcell.00343.2014

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Functional characterization of the human facilitative glucose transporter 12

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(GLUT12) by electrophysiological methods

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Short title: Functional characterization of hGLUT12

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Authors

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Jonai Pujol-Giménez1, Alejandra Pérez2, Alejandro Reyes3, Donald D.F. Loo3 and

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Maria Pilar Lostao1

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Affiliations

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1

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University of Navarra, Pamplona, Spain;

Department of Nutrition, Food Science and Physiology, School of Pharmacy, 2

Instituto de Bioquímica y Microbiología, 3

Department of

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Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile;

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Physiology, David Geffen School of Medicine at University of California, Los Angeles,

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California, USA.

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Corresponding author

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Maria Pilar Lostao

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E-mail: [email protected]

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Phone: 34-948425649

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Fax: 34-948425740

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ABSTRACT

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GLUT12 is a member of the facilitative family of glucose transporters. The goal of this

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study was to characterize the functional properties of GLUT12, expressed in Xenopus

21

laevis oocytes, using radiotracer and electrophysiological methods. Our results showed

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that GLUT12 is a facilitative sugar transporter with substrate selectivity: D-glucose≥α-

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methyl-D-glucopyranoside(α-MG)>2-deoxy-D-glucose(2-DOG)>D-fructose=D-

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galactose. α-MG is a characteristic substrate of the Na+/glucose (SGLT) family, and has

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not been shown to be a substrate of any of the GLUTs. In the absence of sugar, 22Na+

Copyright © 2015 by the American Physiological Society.

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was transported through GLUT12 at a higher rate (40%) than non-injected oocytes,

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indicating that there is a Na+ - leak through GLUT12. Genistein, an inhibitor of GLUT1,

28

also inhibited sugar uptake by GLUT12. Glucose uptake was increased by the PKA

29

activator 8-Br-CAMP, but not by the PKC activator PMA. In high K+ concentrations,

30

glucose uptake was blocked. Addition of glucose to the external solution induced an

31

inward current with a reversal potential of ~-15 mV, and was blocked by Cl- channel

32

blockers, indicating the current was carried by Cl- ions.

33

currents were unaffected by genistein. In high external K+ concentrations, sugar-

34

activated Cl- currents were also blocked, indicating that GLUT12 activity is voltage-

35

dependent. Furthermore, glucose induced current was increased by the PKA activator 8-

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Br-cAMP, but not by the PKC activator PMA. These new features of GLUT12 are very

37

different from those described for other GLUTs, indicating that GLUT12 must have a

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specific physiological role within glucose homeostasis, still to be discovered.

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Key words

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GLUT; GLUT12; Sugar transport; Two-electrode voltage clamp; Xenopus laevis

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oocytes

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The sugar-activated Cl-

3 43

INTRODUCTION

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Glucose uptake into the cells takes place through specific transporter proteins located

45

within the plasma membrane. These transporters belong to two families of integral

46

membrane proteins, the facilitative glucose transporter family GLUT/SLC2A (20, 27),

47

and the Na+/ glucose co-transporter family SGLT/SLC5A (49). The GLUTs transport

48

monosaccharides passively down the hexose concentration gradient. Until now, 14

49

different GLUT isoforms have been identified. The distinct GLUT isoforms are

50

distributed in a specific manner within the different tissues and cell types to satisfy their

51

metabolic needs. Each particular physiological environment determines the expression,

52

cellular location and regulation of the GLUT transporters.

53

GLUT12 was isolated from the mammary cancer cell line MCF-7 (34) and its

54

expression has been described in insulin-sensitive tissues such as muscle and adipose

55

tissue (34), where it seems to act as a secondary insulin-sensitive transporter (37, 38).

56

GLUT12 expression has also been found in some tumor tissues (11, 33, 39) and it has

57

been proposed as one of the key proteins in the energy supply to malignant cells through

58

glycolysis (50).

59

Previous functional studies have found that GLUT12 is a facilitative sugar transporter,

60

but surprisingly, protons influence sugar transport. In GLUT12-transfected MCDK

61

cells, uptake of 2-deoxy-D-glucose was increased by external acidic pH against its

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concentration gradient, implicating a role of protons in sugar transport (48). It was

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demonstrated that 2-deoxy-D-glucose (2-DOG) was transported by GLUT12, and that

64

the uptake of [3H]-2-DOG was inhibited by D-glucose>2-DOG> D-galactose>D-

65

fructose>L-glucose (31). Since these are competition studies, except for glucose (27,

66

29), it is not known if the sugars are substrates or inhibitors.

4 67

Initial studies from our laboratory on Xenopus laevis oocytes expressing GLUT12

68

indicated that glucose transport is enhanced in the presence of external Na+ and that

69

glucose induced an inward current (29). In the present work, we set out to characterize

70

the functional properties of GLUT12 using a combination of radiolabeled tracer and

71

electrophysiological methods. We measured the transport of radiolabeled glucose, 2-

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DOG, galactose, fructose and α-methyl-D-glucopyranoside (α-MG) directly. We

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determined the dependence of sugar transport on ions (Na+ and H+), and the effect of

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activators of PKA and PKC on sugar transport. Finally, we used the two-electrode

75

voltage clamp technique to identify and characterize the currents associated with the

76

activation of the transporter.

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MATERIALS AND METHODS

78

Expression of glucose transporters in Xenopus laevis oocytes

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Stage VI oocytes from X. laevis (Xenopus Express, France) were obtained as previously

80

described (15). They were microinjected with 50 ng of cRNA coding for human

81

GLUT12 or GLUT1 and maintained at 18 ºC in Barth´s medium (88 mM NaCl, 1 mM

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KCl, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, 2.4 mM NaHCO3 and 10

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mM HEPES-Tris, pH 7.4) supplemented with gentamycin (50 mg/L), ciprofloxacin (50

84

mg/L) and amikacin (50 mg/L). Experiments were performed at room temperature (20-

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23ºC) 2-5 days after cRNA microinjection. The experimental protocol to manipulate the

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animals was approved by the Animal Ethics Committee of the University of Navarra (nº

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008-11).

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Uptake assays

89

Sugars uptake was measured by the radiotracer method (24). Briefly, groups of 6-10

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oocytes were incubated for 15 min in 400 µl of Na+ buffer (100 mM NaCl, 2 mM KCl,

91

1 mM MgCl2, 1 mM CaCl2, and 10 mM HEPES-Tris, pH 7.5) containing the indicated

5 92

concentration of the respective unlabeled sugar and traces of the corresponding

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radiolabeled sugar. Once the incubation period has elapsed, the [14C] content of each

94

oocyte was determined by liquid scintillation counting and uptake was calculated as

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pmol/oocyte/15min. Uptake by non-injected oocytes was measured in all experiments.

96

Figures show representative experiments. Additionally, in some figures, uptake is also

97

expressed as percentage of the mean uptake of the indicated control condition. To this

98

end, the mean uptake by the non-injected oocytes was subtracted from the uptake value

99

of each GLUT12-expressing oocyte and results represent the mean of the indicated

100

number of oocytes obtained from at least 3 independent experiments.

101

The radiolabeled sugars used were: [14C] D-glucose (specific activity 55 mCi/mmol),

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[14C] D-galactose (specific activity 53 mCi/mmol), [14C] methyl-α-D-glucopyranoside

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(α-MG, specific activity 55 mCi/mmol), [14C] D-fructose (specific activity 300

104

mCi/mmol) and [14C] 2-deoxy-D-glucose (2-DOG, specific activity 58 mCi/mmol), all

105

purchased from American Radiolabeled (St Louis, MO, USA).

106

Sodium uptake was measured in Na+ buffer containing 22Na+ (specific activity 100-2000

107

Ci/g) purchased from Perkin Elmer. Uptake of

108

cpm/oocyte/15min, and results were normalized as above indicated.

109

Electrophysiology methods

110

The two-electrode voltage clamp method was used to control the membrane potential

111

and monitor the transporter activity as previously described (7, 28). Microelectrodes

112

were filled with 3M KCl solution (resistance 0.5-1.5 MΩ). Oocytes were maintained in

113

a chamber continuously perfused with Na+ buffer solution in the presence or absence of

114

substrate. Continuous current measurements were made at a holding potential (Vh) of -

115

50 mV, low-pass filtered at 0,05 Hz and digitized at 1 Hz using Axoscope V1.1.1.14

116

(Molecular Devices, Sunnyvale, CA, USA).

22

Na+ was calculated as

6 117

Substrate-induced currents, under voltage clamp conditions, were determined as the

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difference between the steady-state current measured in the presence and absence of

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sugars. In some experiments, the oocyte membrane potential was not clamped and

120

variations on the resting membrane potential were measured.

121

To determine the theoretical reversal potential for ions (Ex), the Nernst equation was

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used: Ex= (RT/zF) ln (([xout])/( [xin]))

123 124

Where R is the gas constant, T is the absolute temperatures, z is the electric charge of

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the ion, [x]in ionic intracellular concentration and [x]out ionic extracellular concentration.

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At room temperature (RT/F) is around 25.8 mV, so according to this datum, utilized

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Nernst equation was: Ex= 2.303 (25.8 mV/z) log (([xout])/([xin]))

128 129

Some figures show a representative experiment. In other figures, substrate-induced

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currents are expressed as percentage of the indicated control current obtained in the

131

same oocyte, and represented as the mean of the indicated number of oocytes. All the

132

experiments were performed with oocytes from at least 3 different frog donors.

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Effect of Na+, K+, and H+

134

The previously described Na+ buffer was modified in indicated experiments by

135

replacing 100 mM NaCl with 100 mM choline chloride (choline buffer) or 100 mM KCl

136

(K+ buffer). The pH was varied between 7.5 and 6 by using Tris-HCl.

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Inhibition by Cl- channel blockers and genistein

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Inhibition of glucose uptake and glucose-induced currents by the GLUTs inhibitor

139

genistein (Sigma-Aldrich) or the Cl- channel blockers niflumic acid (NFA, Sigma-

140

Aldrich),

141

Biotechnology) and diphenylanthranic acid (DPC, Santa Cruz Biotechnology), was

5-nitro-2-(3-phenylprpyl-amino)

benzoic

acid

(NPPB,

Santa

Cruz

7 142

measured by adding these compounds to the Na+ buffer solution in the presence of

143

glucose. The concentrations used were: 100-200 µM genistein, 100 µM NFA, 100 µM

144

NPPB, 1 mM DPC.

145

Effect of PKA and PKC activators

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To analyze the effect of protein kinases, oocytes were incubated with 0.1 mM of the

147

PKA activator 8-bromo adenosine 3´,5´-cyclic monophosphate (8-br-cAMP) (Sigma-

148

Aldrich) or 0.1 µM of the PKC activator Phorbol-12-myristate-13-acetate (PMA) (Santa

149

Cruz Biotechnology) for 30 and 15 min respectively (12, 17). Then, uptake of glucose

150

or glucose-induced currents were determined and compared with control conditions.

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Statistical Analysis

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The results are expressed as mean values ± SE. Differences between groups were

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analyzed by unpaired Student’s test or Mann-Whitney U-test, depending to the result of

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the test of normal distribution of the data; significance was set at p 2-DOG >fructose = galactose. These

166

results demonstrated for the first time that α-MG, a characterizing substrate for the

8 167

SGLT transporter family (49), which has never been described as a GLUT family

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substrate, is transported by GLUT12.

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Effect of Na+ and H+

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We have found in preliminary studies that GLUT12 glucose transport rate is increased

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in the presence of Na+ (29). In MDCK cells over-expressing GLUT12, proton-coupled

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active glucose transport has been reported (48). Furthermore, in some Na+-coupled

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transporters, such as the SGLT family, protons (H+) can substitute for Na+ in driving

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sugar transport (49). We therefore investigated the role of Na+ and H+ in sugar transport

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by GLUT12. Fig. 2A shows a representative experiment, in the presence of external Na+

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at pH 7.5, glucose uptake was 2-fold higher for GLUT12-expressing oocytes than non-

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injected controls. The mean of 5 independent experiments (normalized to glucose

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uptake at pH 7.5) is shown in Fig. 2B. At pH 7.5, glucose uptake in the absence of Na+

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decreased by ~45 %, but acidification of the medium (pH 6) in the absence of Na+ did

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not further modify uptake (~55 % inhibition), indicating that H+ were not involved in

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glucose transport in the absence of Na+. Interestingly, in Na+ buffer, decrease of pH

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diminished the uptake by ~60 %, indicating that protons, under certain conditions, also

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interact with GLUT12. Higher glucose uptake in the presence of Na+ suggested that

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GLUT12 activity could be coupled to the Na+ entry. To test this hypothesis, we studied

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the uptake of

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GLUT12-expressing oocytes was higher (40 %) than in non-injected oocytes. This

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indicated that there was a Na+ leak (or Na+ uniport) mediated by GLUT12 (see also Fig.

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5 below). When glucose was present in the external solution, Na+ uptake was ~30 %

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higher than in the absence of glucose, although the difference between both conditions

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was not statistically significant (Fig. 2D). As controls, and to rule out a possible

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unspecific effect of Na+ in GLUT12-mediated glucose transport, we measured the

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Na+. As shown in Fig. 2C, in the absence of glucose, Na+ uptake in

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uptake of glucose in the presence and the absence of Na+ by GLUT1. Uptake of 20 mM

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glucose, saturating concentration for GLUT1 (3), was the same in the presence and in

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the absence of Na+ (data not shown), confirming that co-transport of Na+ and glucose

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was a specific property of GLUT12.

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Inhibition by genistein

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Genistein is a competitive inhibitor of glucose transport by GLUT1, with a Ki of 12 μM

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(41), and acts as a direct inhibitor of insulin-induced glucose uptake by GLUT4 in 3T3-

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L1 adipocytes, with a Ki of 20 μM (5). We examined the effect of genistein on glucose

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uptake by GLUT12. As shown in Fig. 3A, 100 μM genistein inhibited glucose uptake

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almost completely.

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Voltage dependence

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We examined the dependence of GLUT12 on membrane potential by bathing the

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oocytes in high K+ buffer (100 mM K+), where the membrane potential is depolarized to

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a low value (eg. -4 mV, Fig. 10B). Fig. 3B shows that in K+ buffer, glucose uptake by

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GLUT12 was almost completely inhibited, indicating glucose-uptake by GLUT12 is

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voltage dependent. To rule out the possibility of an unspecific effect of K+, we

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measured the uptake of glucose by GLUT1 in the presence of K+ buffer, and found that

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it was not significantly modified (Fig. 3C).

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Effect of PKA and PKC activators

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It has been described that GLUT12 possesses internalization di-leucine motifs at the N-

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and C-terminal ends of the protein, which retains this transporter into intracellular

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compartments in the absence of stimulus (32, 34, 47). In agreement with this, GLUT12

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has been described to localize in the plasma membrane and in the perinuclear region of

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MCF-7 cells (34). On the other hand, it has been demonstrated in X. laevis oocytes, that

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the expression at the plasma membrane of several membrane transporters is increased

10 217

by PKA or PKC activation (17, 23). Therefore, we decided to study the effect of the

218

protein kinase activators 8-Br-cAMP and PMA in the regulation of GLUT12 functional

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expression. Fig. 3D shows glucose uptake in control (untreated) oocytes and in oocytes

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pre-incubated with 8-Br-cAMP (PKA activator) or PMA (PKC activator). Treatment

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with 8-Br-cAMP (30 min) increased glucose-uptake by ~50 %; in contrast, treatment

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with PMA (15 min) did not modify glucose uptake. These results indicated that

223

GLUT12 could be regulated by PKA.

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II. Electrophysiological properties of GLUT12

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Sugar-activated currents

226

To compare GLUT12 with the electrogenic SGLT family, which couples glucose and

227

Na+ transport, electrophysiological measurements were performed to detect currents

228

that might be associated with sugar transport through GLUT12 (29). Figure 4A shows a

229

continuous current record from a GLUT12-expressing oocyte bathed in Na+ buffer. The

230

oocyte membrane potential was clamped at -50 mV. Addition of glucose to the external

231

solution induced an inward current. When glucose was washed out from the external

232

solution in choline buffer, and subsequently in Na+ buffer, the holding current returned

233

to the original baseline. The amplitude of the inward current increased with increasing

234

glucose concentrations (1-100 mM) (~ 5, 10, 15, 25, 40 and 90 nA), but did not saturate

235

at the highest concentration tested. In contrast, GLUT12-mediated glucose uptake

236

saturated at a glucose concentration below 100 mM (data not shown). Glucose-induced

237

currents were not observed in non-injected oocytes (data not shown) (29), indicating

238

they were associated with GLUT12.

239

The sugar specificity of the currents induced by various sugars was studied. In contrast

240

to sugar uptake by GLUT12, where the rate of uptake depended on the sugar (Fig. 1B),

241

the currents induced by glucose, galactose, fructose, α-MG and 2-DOG (100 mM) were

11 242

of similar magnitude (80-110 nA) (Fig. 4B). These results suggested that the currents

243

induced by various sugars were uncoupled from their uptake by GLUT12.

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Dependence on Na+, H+ and Cl-.

245

Experiments were performed to determine the effect of Na+ on the currents evoked by

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glucose through GLUT12. We found there was a Na+ leak current mediated by

247

GLUT12, which is shown in the experiment of Fig. 5. A GLUT12-expressing oocyte

248

was held at -50 mV, and bathed initially in choline buffer. Replacing choline with Na+

249

in the external solution induced an inward current of 35 nA, and addition of glucose

250

(100 mM) to the Na+ buffer induced an inward current of 40 nA (Fig. 5A). In non-

251

injected control oocytes, the increase in current in switching from choline to Na+ buffers

252

was much smaller, and addition of glucose to Na+ buffer did not induce an inward

253

current (Fig. 5B). The current difference for non-injected oocytes was 20% of that of

254

the GLUT12-expressing oocytes (Fig. 5C). The current difference (switching from

255

choline to Na+ buffers) between GLUT12- and non-injected oocytes is the Na+ leak

256

through GLUT12. This is consistent with the increase in

257

compared to non-injected oocytes (Fig. 2C).

258

Fig. 6A shows the addition of 100 mM glucose to Na+ buffer induced a current of ~120

259

nA. In choline buffer, 100 mM glucose induced a current of ~80 nA. Similar to the

260

uptake experiments (Fig. 2A and B), the presence of H+ in choline buffer did not modify

261

the magnitude of the current (Fig. 6B), indicating that H+ were not involved in the

262

generation of the glucose-induced currents under this condition. Currents induced by

263

glucose in the presence of Na+ were 25 % higher than in its absence, demonstrating a

264

contribution of Na+ to the glucose-evoked currents. On the other hand, sugar-activated

265

currents observed in the absence of Na+ indicated that Na+ is not essential for induction

266

of the inward current by GLUT12.

22

Na+ uptake in GLUT12

12 267

To identify the current evoked by glucose through GLUT12, we recorded the changes in

268

membrane potential (Vm) in response to sugar. In a GLUT12-expressing oocyte bathed

269

in Na+ buffer, addition of 100 mM glucose to the bath solution depolarized Vm from -32

270

to -21 mV (Fig. 7A). After sugar exposure, the bath solution was replaced with choline

271

buffer, resulting in a hyperpolarization of Vm from -21 to -48 mV. When Na+ was

272

restored in the bath solution, Vm returned to -32 mV. To confirm the depolarization

273

induced by glucose was mediated by GLUT12, we repeated the experiment on control

274

non-injected oocytes from the same batch. There was no specific effect of glucose on

275

the Vm on these oocytes (Fig. 7B). The experiment also confirmed that there was no

276

endogenous expression of SGLT in the non-injected oocytes.

277

Glucose-induced depolarization in GLUT12-expressing oocytes was variable, but never

278

reached values more positive than -15 mV. According to the X. laevis oocytes

279

intracellular ionic concentrations (45), and the extracellular concentration of ions in the

280

Na+ buffer, the theoretical reversal potentials calculated for Na+, K+ and Cl- were +57

281

mV, -100 mV and -16 mV respectively. Since the depolarization induced by glucose

282

was near -15 mV, we hypothesized the currents were carried by Cl- ions.

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Effect of chloride channel inhibitors

284

To confirm our hypothesis of a Cl- conductance through GLUT12, we tested some Cl-

285

channel inhibitors, widely described as blockers of Cl- channels in X. laevis oocytes

286

(45, 46), on glucose-induced currents through GLUT12. Fig. 8A shows that in a

287

GLUT12-expressing oocyte the addition of 100 mM glucose induced a current of ~80

288

nA, which was completely inhibited by the addition of 100 μM NFA (Fig. 8A and D).

289

Similar results were obtained with NPPB (100 μM) (Fig. 8B and D), while DPC (at 1

290

mM) did not significantly inhibit the glucose-induced currents (Fig. 8C and D). Thus

13 291

the pharmacology supports the view that the glucose-induced currents mediated by

292

GLUT12 were carried by Cl- ions.

293

To probe the link between the Cl- channel activated by glucose and sugar transport by

294

GLUT12, we investigated the effect of NFA on GLUT12 glucose uptake. Fig. 8E shows

295

that NFA had no effect on glucose uptake, but the compound completely blocked the Cl-

296

current induced by glucose (Fig. 8A).

297

Effect of genistein

298

We have shown that genistein is an inhibitor of GLUT12 sugar uptake (Fig. 3A). In

299

contrast, the current induced by 100 mM glucose (~60 nA) was unaffected by genistein

300

(Fig. 9).

301

Voltage dependence

302

Fig. 10A shows that, under voltage-clamp conditions, 100 mM glucose induced currents

303

of ~80 nA in a GLUT12-expressing oocyte in K+ buffer, and ~100 nA in Na+ buffer,

304

indicating that K+ did not affect the glucose-induced current. When Vm was not

305

clamped, in Na+ buffer 100 mM glucose depolarized Vm from -31 to -20 mV, but in K+

306

buffer, glucose did not affect Vm (Fig. 10B). In K+ buffer, Vm is depolarized to -4 mV,

307

beyond (more positive than) the reversal potential for Cl-, we anticipate that activation

308

of a Cl- current by glucose would result in an outward current or Cl- influx into the cell,

309

and hyperpolarize the membrane potential to the reversal potential for Cl- (~ -16 mV).

310

The absence of glucose-induced hyperpolarization (in K+ buffer) suggested the

311

GLUT12 glucose-induced Cl- currents were voltage-dependent, and appeared to be

312

inactivated at low membrane potential.

313

Mechanism of Cl- channel activation

314

As described above, the Cl- currents activated by different sugars depended more on the

315

concentration than on the nature of the sugar. We examined the effect of sorbitol and

14 316

mannitol, sugars that are not transported by GLUTs. Sorbitol (Fig. 11) and mannitol

317

(data not shown) induced currents through GLUT12 with similar magnitudes and time

318

course to those of glucose. The effects of sorbitol and mannitol were not observed in

319

non-injected oocytes. These results indicated that the GLUT-mediated currents might be

320

activated by an osmotic mechanism associated with the transporter.

321

Effect of PKA and PKC activators

322

We examined the effect of PKA and PKA activators on the glucose-induced currents.

323

Treatment with 8-Br-cAMP increased the glucose-induced currents by ~50 % (Fig. 12A

324

and C); however, treatment with PMA did not modify the currents (Fig. 12B and C).

325

These results are similar to the effect of PKA and PKC on glucose-uptake by GLUT12

326

(Fig. 3D).

327

DISCUSSION

328

Substrate selectivity.

329

From the direct uptake of radiolabeled sugars, we have determined the selectivity of

330

GLUT12: D-glucose ≥ α-MG> 2-DOG > D-fructose = D-galactose. This confirms the

331

previous finding (D-glucose ≥ 2DOG > D-galactose) based on competitive inhibition

332

studies of 2-DOG uptake

333

characteristic of the SGLT family, is also transported by GLUT12, and genistein, a

334

common inhibitor of sugar transport by the GLUTs, also inhibits GLUT12 glucose

335

transport.

336

Until now, 14 different GLUT isoforms have been identified and divided into three

337

different classes, based on sequence homology. Class I contains GLUT1, GLUT2,

338

GLUT3, GLUT4 and GLUT14 (gene duplication of GLUT3); Class II comprises the

339

fructose transporter GLUT5, GLUT7, the urate transporter GLUT9 and GLUT11; and

340

class III includes GLUT6, GLUT8, GLUT10, GLUT12 and GLUT13 (3, 29). Fructose

(31). Furthermore, we show that α-MG, a substrate

15 341

is a common substrate for the Class II of GLUTs (3), and the transport of the sugar has

342

been related to the lack of the QLS motif in the helix 7 of the structure of GLUT

343

transporters. The lack of the QLS motif in GLUT12 is consistent with its ability to

344

transport fructose. However, unlike the Class II transporters, GLUT12 transports 2-

345

DOG and galactose, typical substrates of Class I (27). Overall, GLUT12 transports all

346

the hexoses that are substrates of the GLUT family (29); it also transports α-MG, which

347

until now had not been reported to be a GLUT substrate (8). Moreover, α-MG appears

348

to be as good substrate as glucose. Thus GLUT12 seems to be less restrictive among the

349

members of the GLUT family.

350

Effect of Na+ and H+

351

We have found that glucose uptake by GLUT12 is increased by ∼40% in the presence of

352

external Na+ compared to the absence, and there is a Na+-leak (uniport) through

353

GLUT12 in the absence of sugar. Na+ uptake by GLUT12 does not significantly

354

increase in the presence of glucose, and in the absence of external Na+, there is glucose

355

uptake (∼60 %). Taken all together, these results indicate the activity of GLUT12 is

356

dependent on Na+, but Na+ is not essential for GLUT12 function. Further experiments

357

are needed to determine if there is Na+-glucose coupling, or to establish the mechanism

358

of interaction between Na+ and GLUT12.

359

Glucose uptake was not modified by acidic pH, indicating that GLUT12 is independent

360

of H+ as well. In the presence of Na+, protons further decreases sugar uptake, indicating

361

that H+ could also interact with GLUT12. In relation to this, Wilson-O´Brien et al.

362

(2010) have demonstrated in MDCK cells that, in the presence of both Na+ and H+,

363

GLUT12 was able to transport glucose against its concentration gradient, coupling the

364

transport to the favorable proton gradient. However, under favorable glucose

16 365

concentration gradient, protons slightly inhibited glucose uptake

(48) as we here

366

demonstrate.

367

The use of Na+ or H+ gradients has been described for human transporters such as

368

SGLT1 (16), CNT3 (15), and the EAATs (21). For GLUT12, the inhibition of glucose

369

uptake observed in the presence of both Na+ and H+ could be the result of competition

370

of both ions for a common binding site, which would alter the conformation and

371

function of the transporter; nevertheless, further experiments are needed to address this

372

question. In this regard, the nucleoside transporter hCNT3 presents different substrate

373

affinity and maximal transports rate depending on whether Na+, H+ or both cations are

374

coupled to the substrate during the transport (35). Likewise, glutamate uptake through

375

EAAT4, which involves the transport of both Na+ and H+, is inhibited by low pH

376

through the decrease of the apparent affinity for Na+ (6).

377

Effect of K+

378

At high external K+ concentrations, the cell resting membrane potential is depolarized to

379

a low value. Voltage-dependence has been described for different Na+-driven co-

380

transporters (15, 44, 49), but has not been reported for facilitative transporters. The

381

blockade of glucose uptake in the absence of Na+ (in choline buffer) under high external

382

K+, suggests that the facilitative transport of glucose by GLUT12 is voltage dependent.

383

In Na+-coupled co-transporters such as SGLT1, depolarizing membrane voltages reduce

384

sugar transport due to the reduction in the electrochemical potential gradient for Na+

385

ions as well as the preference of the empty (Na+- and sugar- free) transporter for the

386

inward-facing conformation at positive membrane voltages (28).

387

It has been demonstrated that glutamate transport through the EAAT transporters is

388

driven by the co-transport of Na+ and counter-transport a K+ ion, which relocates the

389

transporter into the outward facing conformation

(4, 22). However, as high

17 390

extracellular K+ was not able to inhibit glucose-induced currents through GLUT12

391

under voltage-clamp conditions, it seems unlikely that K+ counter-transport is involved

392

in GLUT12 transport mechanism.

393

Regulation of GLUT12 by PKA and PKC

394

Activation of PKA but not PKC increases glucose uptake by GLUT12. The regulation

395

of GLUT12 by protein kinases could occur directly via phosphorylation of the

396

transporter, changing its transport kinetics, or indirectly, by regulating its insertion into

397

the plasma membrane (17, 23). Interestingly, in L6E9 rat muscle cells, the activation of

398

PKA by 8-br-cAMP increases expression of GLUT1, while decreases GLUT4

399

expression (42). By contrast, PMA up-regulates the translocation of GLUT4 to the

400

plasma membrane in isolated rat adipocytes, without any effect on GLUT1 (43).

401

GLUT12, as GLUT1, has been proposed as one of the key proteins involved in the

402

glycolytic metabolism of cancer cells (50), which would fit with the fact that both are

403

up-regulated by PKA. Activation of PKC is also involved in the insulin-stimulated

404

translocation of GLUT4 to the plasma membrane (25). Comparison of the glucose

405

uptake levels in response to PKC or insulin in isolated rat adipocytes showed that, even

406

though PKC activation increased glucose transport, insulin-mediated glucose transport

407

was still higher (43), indicating the activity of another glucose transporter, probably

408

GLUT12, which would be activated by another pathway. As GLUT4 and GLUT12 are

409

differently regulated by PKA and PKC, it would make sense that each transporter could

410

counter balance the lack of the other, depending on the physiological situation, as it has

411

been proposed (29).

412

The glucose-activated current

413

The reversal potential of the glucose-activated inward current approaches the theoretical

414

Nernst potential for Cl-, indicating the current is predominantly carried by Cl- ions

18 415

exiting the cell. Blockade of the glucose-induced current by Cl- channel blockers NFA

416

and NPPB is consistent with this interpretation. However, Na+ may also contribute to

417

the glucose-activated current since the current is increased in the presence of Na+ (Fig.

418

6A). Several findings indicate the sugar-activated Cl- current and GLUT12 sugar

419

transport are uncoupled: 1) glucose-uptake saturated, but the glucose-induced Cl-

420

current did not saturate with increasing sugar concentrations (Fig. 4A); 2) sugar-uptake

421

showed a sugar specificity, while the currents induced by different sugars at the same

422

concentration were similar (Fig. 1B and 4B); 3) genistein inhibited sugar uptake but not

423

the sugar-induced current (Fig. 3 and 9); and 4) the chloride channel inhibitor NFA

424

completely block the glucose-induced current, in contrast, NFA did not block glucose-

425

uptake (Fig. 8D and E).

426

The magnitude of the sugar-activated current depended on the sugar concentration and

427

not on the nature of the sugar. Mannitol and sorbitol, which are not transported by

428

GLUT12, also activated the Cl- current, indicating the sugar-activated current might be

429

induced by osmotic pressure. Interestingly, GLUT1 has also shown to be influenced by

430

osmotic pressure, since their mRNA and protein levels, and plasma membrane location

431

are increased by high external mannitol concentrations (18). It has been found that on

432

X. laevis oocytes expressing the amiloride-sensitive Na+ sensitive channel (rENaC),

433

osmotic pressure activated Na+ currents through the protein (19).

434

Does GLUT12 possess dual transporter/channel activity? Alternatively, given that X.

435

laevis oocytes express several endogenous Cl- channels (36, 40), could the expression

436

of GLUT12 or perhaps our experimental conditions activate endogenous channels? Four

437

types of Cl- channels have been described in X. laevis oocytes, differing in their mode of

438

activation

439

Ca2+-activated channels. Our experiments were conducted at -50 mV, in the presence of

(36): hyperpolarization-activated, Ca2+-inactivated, volume-sensitive and

19 440

extracellular Ca2+ and using isosmotic solutions, discarding the activation of the first

441

three endogenous channels. Hypertonic solutions have been shown to activate

442

endogenous ionic conductance in X. laevis oocytes (1), but this activation was only

443

observed with solutions with an osmolarity higher than 480 mosmol l-1 (52). Therefore,

444

Ca2+-activated Cl- channels would remain as the main candidates to be activated by

445

GLUT12 expression in X. laevis oocytes, as they are blocked as well by NFA and

446

NPPB and its activity can be modulated by foreign proteins (45). However, even

447

though the sugar-induced currents associated with GLUT12 are inhibited by both NFA

448

and NPPB, they do not show the characteristic biphasic currents (channel activation

449

followed by inactivation) of the endogenous Ca2+-activated Cl- channels

450

Therefore, the sugar-activated Cl- current is likely due to the GLUT12 transporter.

451

Ligand-gated

452

demonstrated in X. laevis oocytes for the human amino acid transporters EAAT1-5 (2,

453

13, 44), ASCT1-2 (9, 51) and NaPi-1 (10), and the trout band 3 anion exchanger (14).

454

GLUT12 behavior resembles that of an hSGLT1 mutant in which neutralization of

455

Asp204 (D204C and D204N) results in a H+ channel activated by glucose that allows

456

proton independent glucose transport, which is increased in the presence of either Na+

457

or H+ (30).

458

Physiological relevance

459

In summary, GLUT12 is a versatile transporter: it transports a wide diversity of

460

hexoses, it can work as a Na+ or H+/glucose symporter (48), and shows electrogenic

461

properties (29). In addition, it can be translocated to the plasma membrane by a wide

462

variety of stimulus such as nutrients availability through the mTOR pathway (47),

463

growth factors and estradiol (26), insulin under the activation of PI3K pathway (37) and

464

by PKA. Therefore, it is clear that GLUT12 has unique functional features, indicating

Cl-

conductance

through

co-transporters

has

been

(46).

previously

20 465

that it must have a specific role in the whole-body glucose homeostasis, instead of being

466

an evolutionary GLUT4 ancestor maintained as a backup, as previously proposed (37).

467

ACKNOWLEDGMENTS

468

We thank A. Redín for technical assistance and Dr. E. Gorraitz for her important

469

contributions to the preliminary experiments.

470

GRANTS

471

This work has been supported by “Fundación Marcelino Botín” and PIUNA (University

472

of Navarra). J. Pujol-Giménez was a recipient of a fellowship from “Asociación de

473

Amigos” (University of Navarra).

474

DISCLOUSURES

475

All authors disclose any actual or potential conflict of interest including any financial,

476

personal or other relationships with other people or organizations that could

477

inappropriately influence this work.

478

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479

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614

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616

Xenopus oocytes and their relationship to single mechanically gated channels. J Physiol 523 Pt

617

1: 83-99, 2000.

618

Legends to figures

619

Figure 1 - Substrate selectivity of GLUT12 - A) A representative experiment showing

620

the uptake by GLUT12-expressing oocytes of 20 mM radiolabeled glucose, α-MG,

621

galactose, fructose and 2-DOG in Na+ buffer. Data represent the mean of 5-10

622

measurements and error bars indicate standard errors (SE). Sugar uptake by non-

623

injected oocytes (NI) is also shown. *p< 0.05 vs. NI. B) Mean GLUT12 sugar uptake-

624

rate was pooled from 3 independent experiments (13-19 oocytes). Values were

625

corrected for basal uptake in non-injected oocytes and normalized to the mean glucose

26 626

uptake (568 + 137 to 1610 + 774 pmol/oocyte/15min) and are represented as the

627

mean+SE. Not significant (NS); *p< 0.05; ***p< 0.001 vs. Glucose.

628

Figure 2 - Effect of Na+ and H+ on glucose uptake - A) A representative experiment

629

showing the uptake of 5 mM glucose by GLUT12-expressing and non-injected oocytes

630

(NI) at the indicated pH in Na+ and choline buffers. Data represent the mean (± SE) of

631

5-10 measurements. *p< 0.05 vs. NI. B) Mean GLUT12 glucose uptake-rate was

632

obtained from 5 independent experiments (22-34 oocytes), normalized to the mean

633

glucose uptake in Na+ at pH 7.5 (71 ± 9 to 340 ± 70 pmol/oocyte/15 min) and

634

represented as the mean + SE. ***p< 0.001 vs. Na+ pH 7.5. C) Uptake of 100 mM Na+

635

by GLUT12-expressing oocytes was determined in the absence and presence of 5 mM

636

glucose. Data represent the mean (± SE) of 5-10 measurements. Sugar uptake by non-

637

injected oocytes (NI) is also shown. *p< 0.05 vs. NI. D) Mean GLUT12 Na+ uptake-rate

638

was obtained from 3 independent experiments (11-13 oocytes). Values were corrected

639

for basal non-mediated uptake in non-injected oocytes, normalized to the mean Na+

640

uptake in the presence of glucose (399 + 153 to 911 + 239 cpm/oocyte/15min) and

641

represented as the mean + SE. Not significant (NS) vs. 22Na++ glucose.

642 643

Figure 3 - Effect of genistein, K+ and PKA and PKC activators on glucose uptake

644

A) Inhibition of GLUT12-mediated glucose transport by genistein. Uptake of 5 mM

645

glucose by GLUT12-expressingand non-injected oocytes (NI) was determined in Na+

646

buffer in the absence and presence of 100 μM genistein. Data represent the mean (± SE)

647

of 5-10 measurements. *p< 0.05; Not significant (NS) vs. NI. B) Effect of K+ on glucose

648

transport. Uptake of 5 mM glucose by GLUT12-expressing oocytes was determined in

649

choline and K+ buffers. Data represent the mean (± SE) of 5-10 measurements. *p
10 experiments.

29 700

Figure 8 - Effect of chloride channel inhibitors on GLUT12-mediated glucose

701

induced-currents and transport - A) A GLUT12-expressing oocyte was held at -50

702

mV and perfused with Na+ buffer (black line). The addition of 100 mM glucose in Na+

703

buffer (dotted black line) induced a current of 60 nA, which was totally inhibited by 100

704

μM NFA (dashed black line). The oocyte was washed out with choline buffer (grey

705

line). Similar experiments were performed using 100 μM NPPB (B) or 1mM DPC (C).

706

Traces are representative of 4 (A), 4 (B) and 3 (C) experiments D) Results are the mean

707

± SE (3-4 oocytes) of glucose-induced current in the presence of chloride channel

708

inhibitors normalized to mean glucose-induced currents before their perfusions (25-90

709

nA). Not significant (NS); *p< 0.05; **p

Functional characterization of the human facilitative glucose transporter 12 (GLUT12) by electrophysiological methods.

GLUT12 is a member of the facilitative family of glucose transporters. The goal of this study was to characterize the functional properties of GLUT12,...
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