Articles in PresS. Am J Physiol Cell Physiol (April 8, 2015). doi:10.1152/ajpcell.00343.2014
1 1
Functional characterization of the human facilitative glucose transporter 12
2
(GLUT12) by electrophysiological methods
3
Short title: Functional characterization of hGLUT12
4
Authors
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Jonai Pujol-Giménez1, Alejandra Pérez2, Alejandro Reyes3, Donald D.F. Loo3 and
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Maria Pilar Lostao1
7
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;
11
Physiology, David Geffen School of Medicine at University of California, Los Angeles,
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California, USA.
13
Corresponding author
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Maria Pilar Lostao
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E-mail:
[email protected] 16
Phone: 34-948425649
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Fax: 34-948425740
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ABSTRACT
19
GLUT12 is a member of the facilitative family of glucose transporters. The goal of this
20
study was to characterize the functional properties of GLUT12, expressed in Xenopus
21
laevis oocytes, using radiotracer and electrophysiological methods. Our results showed
22
that GLUT12 is a facilitative sugar transporter with substrate selectivity: D-glucose≥α-
23
methyl-D-glucopyranoside(α-MG)>2-deoxy-D-glucose(2-DOG)>D-fructose=D-
24
galactose. α-MG is a characteristic substrate of the Na+/glucose (SGLT) family, and has
25
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.
2 26
was transported through GLUT12 at a higher rate (40%) than non-injected oocytes,
27
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-
36
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
38
specific physiological role within glucose homeostasis, still to be discovered.
39
Key words
40
GLUT; GLUT12; Sugar transport; Two-electrode voltage clamp; Xenopus laevis
41
oocytes
42
The sugar-activated Cl-
3 43
INTRODUCTION
44
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
62
concentration gradient, implicating a role of protons in sugar transport (48). It was
63
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-
72
DOG, galactose, fructose and α-methyl-D-glucopyranoside (α-MG) directly. We
73
determined the dependence of sugar transport on ions (Na+ and H+), and the effect of
74
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.
77
MATERIALS AND METHODS
78
Expression of glucose transporters in Xenopus laevis oocytes
79
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
82
KCl, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, 2.4 mM NaHCO3 and 10
83
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-
85
23ºC) 2-5 days after cRNA microinjection. The experimental protocol to manipulate the
86
animals was approved by the Animal Ethics Committee of the University of Navarra (nº
87
008-11).
88
Uptake assays
89
Sugars uptake was measured by the radiotracer method (24). Briefly, groups of 6-10
90
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
93
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
95
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),
102
[14C] D-galactose (specific activity 53 mCi/mmol), [14C] methyl-α-D-glucopyranoside
103
(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
118
difference between the steady-state current measured in the presence and absence of
119
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
122
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
125
the ion, [x]in ionic intracellular concentration and [x]out ionic extracellular concentration.
126
At room temperature (RT/F) is around 25.8 mV, so according to this datum, utilized
127
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
130
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.
133
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.
137
Inhibition by Cl- channel blockers and genistein
138
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
146
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.
151
Statistical Analysis
152
The results are expressed as mean values ± SE. Differences between groups were
153
analyzed by unpaired Student’s test or Mann-Whitney U-test, depending to the result of
154
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
168
substrate, is transported by GLUT12.
169
Effect of Na+ and H+
170
We have found in preliminary studies that GLUT12 glucose transport rate is increased
171
in the presence of Na+ (29). In MDCK cells over-expressing GLUT12, proton-coupled
172
active glucose transport has been reported (48). Furthermore, in some Na+-coupled
173
transporters, such as the SGLT family, protons (H+) can substitute for Na+ in driving
174
sugar transport (49). We therefore investigated the role of Na+ and H+ in sugar transport
175
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-
177
injected controls. The mean of 5 independent experiments (normalized to glucose
178
uptake at pH 7.5) is shown in Fig. 2B. At pH 7.5, glucose uptake in the absence of Na+
179
decreased by ~45 %, but acidification of the medium (pH 6) in the absence of Na+ did
180
not further modify uptake (~55 % inhibition), indicating that H+ were not involved in
181
glucose transport in the absence of Na+. Interestingly, in Na+ buffer, decrease of pH
182
diminished the uptake by ~60 %, indicating that protons, under certain conditions, also
183
interact with GLUT12. Higher glucose uptake in the presence of Na+ suggested that
184
GLUT12 activity could be coupled to the Na+ entry. To test this hypothesis, we studied
185
the uptake of
186
GLUT12-expressing oocytes was higher (40 %) than in non-injected oocytes. This
187
indicated that there was a Na+ leak (or Na+ uniport) mediated by GLUT12 (see also Fig.
188
5 below). When glucose was present in the external solution, Na+ uptake was ~30 %
189
higher than in the absence of glucose, although the difference between both conditions
190
was not statistically significant (Fig. 2D). As controls, and to rule out a possible
191
unspecific effect of Na+ in GLUT12-mediated glucose transport, we measured the
22
Na+. As shown in Fig. 2C, in the absence of glucose, Na+ uptake in
9 192
uptake of glucose in the presence and the absence of Na+ by GLUT1. Uptake of 20 mM
193
glucose, saturating concentration for GLUT1 (3), was the same in the presence and in
194
the absence of Na+ (data not shown), confirming that co-transport of Na+ and glucose
195
was a specific property of GLUT12.
196
Inhibition by genistein
197
Genistein is a competitive inhibitor of glucose transport by GLUT1, with a Ki of 12 μM
198
(41), and acts as a direct inhibitor of insulin-induced glucose uptake by GLUT4 in 3T3-
199
L1 adipocytes, with a Ki of 20 μM (5). We examined the effect of genistein on glucose
200
uptake by GLUT12. As shown in Fig. 3A, 100 μM genistein inhibited glucose uptake
201
almost completely.
202
Voltage dependence
203
We examined the dependence of GLUT12 on membrane potential by bathing the
204
oocytes in high K+ buffer (100 mM K+), where the membrane potential is depolarized to
205
a low value (eg. -4 mV, Fig. 10B). Fig. 3B shows that in K+ buffer, glucose uptake by
206
GLUT12 was almost completely inhibited, indicating glucose-uptake by GLUT12 is
207
voltage dependent. To rule out the possibility of an unspecific effect of K+, we
208
measured the uptake of glucose by GLUT1 in the presence of K+ buffer, and found that
209
it was not significantly modified (Fig. 3C).
210
Effect of PKA and PKC activators
211
It has been described that GLUT12 possesses internalization di-leucine motifs at the N-
212
and C-terminal ends of the protein, which retains this transporter into intracellular
213
compartments in the absence of stimulus (32, 34, 47). In agreement with this, GLUT12
214
has been described to localize in the plasma membrane and in the perinuclear region of
215
MCF-7 cells (34). On the other hand, it has been demonstrated in X. laevis oocytes, that
216
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
219
expression. Fig. 3D shows glucose uptake in control (untreated) oocytes and in oocytes
220
pre-incubated with 8-Br-cAMP (PKA activator) or PMA (PKC activator). Treatment
221
with 8-Br-cAMP (30 min) increased glucose-uptake by ~50 %; in contrast, treatment
222
with PMA (15 min) did not modify glucose uptake. These results indicated that
223
GLUT12 could be regulated by PKA.
224
II. Electrophysiological properties of GLUT12
225
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.
244
Dependence on Na+, H+ and Cl-.
245
Experiments were performed to determine the effect of Na+ on the currents evoked by
246
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.
283
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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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