From the
Glucose exerts inverse effects upon the secretory function of
islet
Mammalian glucose homeostasis is controlled by glucose sensor
mechanisms in the endocrine pancreas which adjust release of insulin
and glucagon to variations in extracellular glucose concentration
(1) . Experiments on isolated rodent islets of Langerhans
indicate that glucose metabolism is necessary for
The present study confirms
(18) that the kinetics of
glucose uptake by unpurified islet cells is characterized by two
components, one with a high K
Isolated
Endocrine islet non-
The
absence of low K
Comparison of overall flux via glucose utilization and
3-OMG uptake is possible since both processes were studied under the
same experimental conditions and since the kinetic constants for
glucose uptake and 3-OMG uptake are similar
(35) . In pure rat
Data represent mean values ± S.E. of
( n) independent experiments. ND, not determined.
Apparent
V
We thank Erik Quartier for his excellent technical
assistance, the personnel of the Dept. of Endocrinology and Metabolism
for islet cell purification, and Dr. L. Kaufman for statistical advice.
We gratefully acknowledge Dr. M. Birnbaum, Harvard Medical School, for
providing the rat GLUT1 cDNA.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
- and
-cells, suppressing glucagon release and
increasing insulin release. This diverse action may result from
differences in glucose transport and metabolism between the two cell
types. The present study compares glucose transport in rat
- and
-cells.
-Cells transcribed GLUT2 and, to a lesser extent,
GLUT 1;
-cells contained GLUT1 but no GLUT2 mRNA. No other
GLUT-like sequences were found among cDNAs from
- or
-cells.
Both cell types expressed 43-kDa GLUT1 protein which was enhanced by
culture. The 62-kDa
-cell GLUT2 protein was converted to a 58-kDa
protein after trypsin treatment of the cells without detectable
consequences upon glucose transport kinetics. In
-cells, the rates
of glucose transport were 10-fold higher than in
-cells. In both
cell types, glucose uptake exceeded the rates of glucose utilization by
a factor of 10 or more. Glycolytic flux, measured as
D-[5
H]glucose utilization, was
comparable in
- and
-cells between 1 and 10 mmol/liter
substrate. In conclusion, differences in glucose transporter gene
expression between
- and
-cells can be correlated with
differences in glucose transport kinetics but not with different
glucose utilization rates.
-cells to sense
extracellular glucose
(2, 3, 4) . The glucose
transporter GLUT2 and glucokinase are candidate glucose sensor
molecules in
-cells
(5, 6, 7, 8) .
Both proteins are expressed in
-cells and hepatocytes, and their
low affinity for glucose results in catalytic rates that are linearly
dependent on the blood glucose concentration. GLUT2 gene expression is
down-regulated in islets of several diabetic animal models
(6, 9) , seems required for glucose responsiveness in
ATt-20 cells
(7) , but appears not responsible for heterogeneity
in glucose sensitivity of normal rat
-cells
(10) .
Glucokinase gene expression has been correlated with the rates of
glucose-induced insulin synthesis and release in intact islets
(11, 12, 13) as well as with the intercellular
differences in
-cell function
(10) . Virtually no
information is available on the glucose sensor in
-cells which
represent 10% or less of the cell mass. The ability to purify
-cells from rat pancreata creates the possibility of investigating
this cell type
(14) . Glucose inhibits glucagon release through
a direct effect on
-cells
(15) . Glucose transport in
-cells is slower than in
-cells and therefore may qualify as
a rate-limiting step for overall glucose metabolism
(16) . This
study compares purified rat
- and
-cell preparations for
their kinetics of glucose transport and their metabolic flux through
glycolysis. It investigates to which extent differences in glucose
handling are caused by differential expression of glucose transporter
genes.
Materials
D-[5-H]Glucose
(21 Ci/mmol) was purchased from Amersham (Bucks, United Kingdom);
L-[1-
H]glucose (20 Ci/mmol),
[
C]urea (56 mCi/mmol), and
3-[
H]OMG
(
)
(79 Ci/mmol)
from DuPont NEN.
Purification and Culture of Cells from the Endocrine
Pancreas
Preparations of dispersed islet cells (20% -cells
and 65%
-cells), purified
-cells (>90% purity), isolated
endocrine non-
-cells (>60%
-cells and <20%
-cells), and purified
-cells (>80%
-cells and <5%
-cells) were isolated from adult male Wistar rats using previously
published methods
(14) . The purity of these preparations was
routinely determined by immunocytochemistry and radioimmunoassays for
insulin and glucagon. Cells were more than 90% viable as assessed by
neutral red uptake
(14) . Mean intracellular space was 622
± 48 and 270 ± 34 pl/10
cells for
-cells
( n = 5) and non-
-cells ( n = 7),
respectively. Dispersed islet cells,
-cells, non-
-cells, and
-cells were cultured for 16 h in Ham's F-10 medium, 1%
bovine serum albumin, 2 mmol/liter glutamine, 50 µmol/liter
isobutylmethylxanthine, and 6 mmol/liter glucose at 37 °C
(17) .
Glucose Uptake
Measurements
Zero- trans-3-OMG uptake was measured at 15
°C or 37 °C using 1 mmol/liter [C]urea
(25 mCi/mmol) as an intracellular space marker and 2 mmol/liter
L-[
H]glucose (200 mCi/mmol) to correct
for contamination with extracellular volume
(16, 18) .
Initial rates of 3-OMG uptake were measured at 15 s for islet cells and
-cells (1
10
cells per measurement) or at 1
min for non-
-cells (1.5
10
cells per
measurement). Uptake was stopped by the addition of 150 µl of
ice-cold Earle's-Hepes buffer containing 50 mmol/liter glucose
and 1 mmol/liter phloridzin and centrifuged through a 150-µl oil
phase (4:1 dibutyl/dinonylphthalate). The bottom phase of the tubes was
counted in liquid scintillation mixture. 3-OMG uptake was expressed per
liter cellular volume after correction for the extracellular space.
Apparent V
and K
of glucose uptake were determined by Eadie-Hofstee plots with the
best-fit lines drawn using the method of least squares.
Glucose Utilization in Purified
Glucose utilization was measured as the
conversion of
D-[5--Cells and
Non-
-cells
H]glucose into
tritiated water using batches of 5
10
cells and 2-h
incubations at 37 °C (see Ref. 19). Cellular metabolism was stopped
by the addition of 20 µl of 0.4 mol/liter citrate buffer, pH 4.9,
containing 5 mmol/liter KCN, 10 µmol/liter antimycin A, and 10
µmol/liter rotenone. Tritiated water production was measured via
liquid scintillation counting.
mRNA Analysis
Total RNA was isolated from 10cells by microadaptation of the guanidinium isothiocyanate/cesium
chloride method
(20) , separated according to its size on 1.5%
agarose gels containing formaldehyde and transferred by capillary blot
to nylon membranes (GeneScreen, Dupont NEN). Control RNAs were taken
from tissues (brain, liver, and muscle) of the same rats as were used
for
-cell preparation. The blots were UV-cross-linked and
hybridized with
P-labeled cDNA probes (Megaprime,
Amersham, Bucks, UK). Two stringent washings were done for 30 min in
0.2
SSC and 2.5% SDS at 65 °C. The intensity of the signals
was scanned on an Ultroscan XL densitometer (Pharmacia, Uppsala,
Sweden).
Analysis of Amplified cDNA
Total RNA extracted
from the control tissues and from 2.5 10
-cells or
-cells was reverse-transcribed in 50
mmol/liter Tris-Cl, pH 8.3, 40 mmol/liter KCl, 6 mmol/liter
MgCl
, 1 mmol/liter dithiothreitol with 1 mmol/liter dNTP,
200 nmol/liter poly(dT), 50 units of RNase inhibitor, and 10 units of
avian myeloblastosis virus reverse transcriptase for 1 h at 42 °C.
The negative control contained no RNA. First strand cDNA was dialyzed
on a 0.025-mm filter and amplified via polymerase chain reaction
(21) in 20 mmol/liter Tris-Cl, pH 8.3, 25 mmol/liter KCl, 2
mmol/liter MgCl
, 200 mmol/liter dNTP, 50 pmol of 5`- and
3`-primer mixes, and 2.5 units of Taq polymerase for 40 cycles
in a Techne PHC1 thermocycler (Techne Ltd., Duxford, Cambridge, UK)
with blanks in each assay. PCR products were controlled for their
length on a 1.5% agarose gel. Gene-specific primers, recognizing
sequences encoding the first extracellular and the third intracellular
loop
(22) , were: GLUT1-5` (codon 48-54),
5`AACCACCGCTATGGAGAG; GLUT1-3` (codon 222-228),
5`GCACACTCTTGGCCCGGT; GLUT2-5` (codon 78-84),
5`GCCTGGGAAGAAGAGACT; GLUT2-3` (codon 252-258),
5`AGCTTTTCTTTGCCCTGA; GLUT4-5` (codon 64-70),
5`GGTCCTGGGGGACCGGAC; GLUT4-3` (codon 238-244),
5`GACTCTTTCGGGCAGGCC, yielding amplified fragments of 540 base pairs.
Mixed oligonucleotide primed amplification of cDNA
(23) was
performed using two degenerate primer sets, 5`-primer set:
5`GG(A/T)GCC(C/T)TGGG(A/C)AC(A/C/T)CT(C/G/T)(C/A)ACCA (codon
154-161 of GLUT1) and 3`-primer set:
3`GTCGTC(G/A)A(C/G)AG(C/A/T)CC(G/T)TAGTTAC (codon 282-289 of
GLUT1), yielding an amplified fragment of 406 base pairs. Polymerase
chain reaction products were diluted (1/3, 1/10, and 1/20) and
dot-blotted to nylon membranes that were hybridized with
5`-radiolabeled oligonucleotides complementary to isoform-specific
sequences encoding parts of the third intracellular loop:
GLUT1-3`CTTGGCCCGGTTCTCACACG (codon 221-228),
GLUT4-3`CCCCGGACGGGCTTTCTCAG (codon 237-244), and
GLUT2-3`TCAGTCCCGTTTCTTTTCGA (codon 251-258). After three
washing steps (twice in 3
SSC, 5% SDS at 50 °C for 30 min
and once in 1
SSC, 1% SDS, at 50 °C for 30 min), the
hybridized membranes were exposed for autoradiography. To facilitate
subcloning, EcoRI and XbaI restriction sites were
included in all the 5`- and 3`-primers, respectively. Polymerase chain
reaction-derived fragments were eluted from agarose gel and cloned in
XbaI and EcoRI cut pBluescript SK
(Stratagene). Clones were sequenced via the dideoxy method using
modified T7 DNA polymerase (Pharmacia, Uppsala, Sweden).
Protein Analysis
Protein blotting and analysis was
performed as described before
(5) with some minor adaptations.
Cell and tissue samples were homogenized by sonication in 5% SDS, 5%
-mercaptoethanol, 80 mmol/liter Tris-Cl (pH 6.8), 5 mmol/liter
EDTA, and 10% glycerol in the presence of 1 mmol/liter
phenylmethylsulfonyl fluoride. Homogenates were separated on a 10%
SDS-polyacrylamide gel and electroblotted to nitrocellulose membranes.
Membranes were incubated with anti-GLUT1 (1/1000) or anti-GLUT2
(1/4000) for 60 min at room temperature. The second antibody
(anti-rabbit peroxidase) was incubated at room temperature for 50 min.
Peroxidase activity was detected via chemiluminescence (ECL, Amersham,
Bucks, UK). The intensity of the signals was quantified via laser
densitometry.
3-O-Methylglucose Uptake Kinetics in Purified
Initial rates of
zero- trans-3- O-methylglucose (3-OMG) uptake in islet
cells, purified islet -Cells and Non-
-cells
-cells, and non-
-cells were measured at
15 °C to slow down the process sufficiently for accurate
calculations (Fig. 1). In islet cells, uptake approached
saturation at 40 mmol/liter substrate (Fig. 1 a).
Transformation of the data into Eadie-Hofstee plots (Fig. 1 b)
and calculation of the least squares fit for either a one- or a
two-component model revealed that the latter was more adequate ( p < 0.05). A predominant kinetic component
( K
= 16 mmol/liter;
V
= 11 mmol/min/liter) was distinguished
from a second component with apparent K
= 3 mmol/liter and V
= 3
mmol/min/liter. Uptake of 3-OMG in purified
-cells was not
saturated at 40 mmol/liter substrate (Fig. 1 c). In contrast to
dispersed islet cells, Eadie-Hofstee transformation of data from pure
-cells showed only one component of transport with
V
= 14 mmol/min/liter and
K
= 18 mmol/liter
(Fig. 1 d). Transport in islet non-
-cells was at
least one order of magnitude slower than that measured in
-cells
and dose dependently increased between 1 and 15 mmol/liter 3-OMG
(Fig. 1 e). Kinetic analysis of 3-OMG transport in
endocrine non-
-cells revealed one component with
K
= 8.5 mmol/liter and
V
= 0.8 mmol/min/liter
(Fig. 1 f).
Figure 1:
3-OMG uptake in rat islet cells ( a and b), purified -cells ( c and d),
and non-
-cells ( e and f). Initial uptake
velocities were measured at 15 °C using zero- trans uptake
conditions. Data represent mean values from five independent
experiments for islet cells and purified
-cells and from seven
independent experiments for non-
-cells. One- or two-component
regression analysis of the Eadie-Hofstee plots ( b, d,
and f) of the data in a, c, and e was performed to calculate the K and V
values.
Comparison of 3-OMG Transport and Glucose
Utilization
Initial velocities of 3-OMG transport were compared
with overall rates of glycolysis in pure - and non-
-cells
(). Data were expressed per liter of intracellular space to
compensate for the differences in cell size between both cell
populations. In contrast to
-cells, 3-OMG uptake in
non-
-cells was slow enough to be measured at 37 °C, so that
direct comparison with glucose utilization was feasible. Rates of
glucose utilization increased proportionally to the extracellular
substrate concentration between 1 and 10 mmol/liter substrate and were
virtually identical in
- and non-
-cells (). On
the contrary, 3-OMG uptake was approximately 10 times faster in
-cells than in non-
-cells. Despite the fact that transport
was measured at a lower temperature than utilization, rates of 3-OMG
uptake in pure
-cells exceeded the corresponding rates of glucose
utilization at least by one order of magnitude (). In
non-
-cells, the increase from 15 °C to 37 °C accelerated
3-OMG transport 3- to 6-fold, resulting in a mean calculated
Q
value of approximately 2. Glucose transport in
non-
-cells was between 8 and 9 times more rapid than overall
glucose utilization, both at 5 and 10 mmol/liter substrate
().
Glucose Transporter Gene Transcription in Islet Cells and
in Purified
GLUT1 cDNA hybridized
with a 2.8-kb transcript in both -Cells and
-Cells
- and
-cells (Fig. 2).
The ratio of GLUT1 mRNA over
-actin mRNA was higher in
-cells
than in
-cells. Hybridization of
-cell RNA with a GLUT2 cDNA
probe resulted in detection of two transcripts, one of 2.8 kb that was
relatively abundant and also found in liver, and one of 3.9 kb that was
found in
-cells but not in liver. No GLUT2 expression was detected
in
-cells. GLUT4 was not transcribed in
-cells or in
-cells (data not shown).
Figure 2:
RNA analysis of purified -cells and
-cells. 5 µg of total RNA was Northern-blotted as described
under ``Experimental Procedures'' and successively hybridized
with cDNA probes specific for GLUT1, GLUT2, and
-actin.
Autoradiographic exposure times required to generate the signals that
are shown were 12 days for GLUT1, 3 days for GLUT2, and 12 h for
-actin. The blot shown here is representative of three independent
experiments.
Isoform-specific oligonucleotide
primers were used to amplify reverse-transcribed mRNA from - and
-cells. Amplification of control cDNAs from brain, liver, and
muscle resulted in DNA fragments of expected length (data not shown).
While GLUT1 sequences were detected in all cDNA mixtures, GLUT2 and
GLUT4 sequences were present only in liver and muscle cDNAs,
respectively (data not shown). The partial sequences obtained from
purified islet cells were identical with previously published
sequences:
-cells contained GLUT1 and GLUT2 sequences and
-cells contained GLUT1 and no GLUT2 sequences (data not shown). To
investigate whether new members of the GLUT family were present, we
performed mixed oligonucleotide primed amplification of cDNA
(23) with degenerate primer sets recognizing highly conserved
sequences of the GLUT family
(22) . Amplified cDNA was
concentrated in a single ethidium bromide-stained band with the
expected fragment length (data not shown). Dot blots of the polymerase
chain reaction products were hybridized with oligonucleotide probes
specific for GLUT1, GLUT2, and GLUT4 sequences (15% overall identity).
This method was validated on amplified mRNA from brain
(GLUT1-positive), liver (GLUT2-positive), and muscle (GLUT4-positive;
Fig. 3
). From brain mRNA, a fragment was amplified that
reproducibly hybridized with the GLUT4-specific oligoprobe. In purified
-cells, GLUT1-cDNA was detected reproducibly, but the predominant
glucose transporter was GLUT2. Amplified cDNA from
-cells was only
positive for GLUT1 (Fig. 3). When the amplified DNA fragments were
cloned and sequenced, both GLUT1 and GLUT2 sequences were found in
-cells (1 GLUT1 clone for 100 GLUT2 clones), while the obtained
-cell cDNA fragments contained GLUT1 sequences (10% of all
clones), or sequences that were not homologous to any protein present
in the PIR (Protein Identification Resource, Washington D. C.) or
Swiss-prot data banks (remaining clones; data not shown).
Figure 3:
Dot-blot analysis of cDNA from -cells
and
-cells after amplification via mixed oligonucleotide primed
amplification of cDNA (23), starting from first strand cDNAs from 2.5
10
- or
-cells and from 1 µg of
control tissue RNA. The negative control contained no RNA. Per sample,
5 µl of amplified cDNA and a 3-, 10-, and 20-fold dilution was
blotted. Compared to
-cells and controls, 5 times more amplified
cDNA from
-cells was applied. Primers for amplification and probes
for hybridization are described under ``Experimental
Procedures.''
Glucose Transporter Protein Expression in Purified
Translation products of the
described mRNAs were studied via Western blotting (Fig. 4).
Positive control tissues showed the expected 40-kDa band for brain
GLUT1 and 62-kDa band for liver GLUT2. Isolated -Cells and
-Cells
-cells were weakly
positive for GLUT1 and strongly positive for GLUT2 immunoreactivity
(Fig. 4). The GLUT1 protein appeared larger in
-cells
(approximately 43 kDa) than in brain. GLUT2 immunoreactivity was
distributed over an intense band of approximately 58 kDa and a much
weaker
62-kDa band (Fig. 4). In
-cell preparations with
3-fold more cells, only weak GLUT1 and GLUT2 signals were detected. The
ratio of GLUT1 over GLUT2 abundance in
-cells was 60-fold higher
than in
-cells. Therefore, the GLUT2 signal in
-cells
probably originates from contaminating
-cells, while the GLUT1
signal can be considered as
-cell-specific. In
-cells
cultured for 16 h in serum-free medium complemented with 6 mmol/liter
glucose, GLUT1 protein was 4-fold more abundant than in freshly
isolated
-cells (Fig. 4). A comparable induction of GLUT1
expression by culture in 6 mmol/liter glucose was observed in
-cells.
Figure 4:
Analysis of -cell- and
-cell
proteins for presence of GLUT1 and GLUT2. Western blots from 25 µg
of protein from the control tissues or protein from 7
10
-cells or 2.5
10
-cells were
performed as described under ``Experimental Procedures.''
Blotting efficiency was established by Ponceau staining of total
protein. Exposure times were 30 min for GLUT1 and 5 min for GLUT2. The
blot shown here is representative for four independent experiments.
16h G6 = 16 h of culture in serum-free medium
supplemented with 6 mmol/liter glucose.
GLUT2 Protein Heterogeneity and Glucose Uptake
We
then investigated whether the molecular heterogeneity of GLUT2 protein
was correlated to the rate of glucose uptake. The ratio of the
densitometric intensities of the 62-kDa versus the 58-kDa
band was 10-fold lower in freshly isolated
-cells (0.09 ±
0.05) than in 16-h cultured
-cells (0.90 ± 0.10; p < 0.005; n = 3) (Fig. 5, lanes 1 and 2). This was not influenced by adding the trypsin
inhibitor antipain (3 mg/ml) to the culture medium (Fig. 5,
lane 3). Incubation of cultured
-cells with trypsin (3
mg/ml, 10 min, 37 °C) resulted in a rapid disappearance of the
62-kDa band (Fig. 5, lane 4); this effect did not occur
when antipain was present during trypsin exposure (Fig. 5,
lane 5). Freshly isolated islet cells exhibited only the
58-kDa GLUT2 protein (Fig. 5, lane 6), but after 16 h of
culture only 62-kDa GLUT2 immunoreactivity was found (Fig. 5,
lane 7). As for isolated
-cells, short trypsin treatment
of cultured islet cells shifted the 62-kDa GLUT2 isoform to the 58-kDa
isoform (Fig. 5, lane 8). The untreated and
trypsin-treated cultured islet cell preparations, with, respectively,
62-kDa and 58-kDa GLUT2 protein, were compared for their glucose uptake
kinetics, using the two-component model. Both preparations displayed
low and high K
uptake with similar
kinetic characteristics ().
Figure 5:
The effect of -cell culture on GLUT2
expression. 10
-cells were homogenized immediately
after isolation ( lane 1), after 16 h of culture ( lane
2), after 16 h of culture in the presence of antipain ( lane
3), after 16 h of culture followed by 10-min treatment with
trypsin (3 mg/ml, 37 °C) ( lane 4), and after 16 h of
culture followed by addition of trypsin and antipain ( lane 5).
Islet cells were used immediately after isolation ( lane 6) or
after 16 h of culture ( lane 7), and after 16 h of culture
followed by a 10-min incubation with trypsin ( lane 8). Ponceau
staining of total protein was comparable in all lanes (not shown).
Exposure time was 5 min. The blot shown here is representative for
three independent experiments.
and high
V
, the other with a low
K
and low V
.
Analysis of glucose transport in purified
- and non-
-cells
indicates that the high K
component is a
feature of the
-cells. The low K
component is, however, not recovered in one of the two purified
preparations, which suggests its association with islet cells, which
are not selected during the purification procedure. Alternatively, low
K
glucose uptake does occur in islet
-cells, but the absence of non-
-cells alters the intrinsic
activity of the expressed glucose transporters, for instance by
altering their phosphorylation/dephosphorylation state. The preparation
of endocrine non-
-cells displayed glucose transport with low
capacity and intermediate affinity compared to the low
V
/low K
transport
observed in islet cells. Our data do not exclude that high affinity
glucose transporters are expressed in non-
-cells and that their
intrinsic activity and affinity for glucose are dependent on
-cell
factors.
-cells transcribe the GLUT1 gene and express
the protein. The GLUT1 protein was more abundant after overnight
culture at 6 mmol/liter glucose, which is compatible with previous data
on isolated islets
(24, 25) . Accumulation of GLUT1
through decreased protein degradation at 6 mmol/liter glucose has been
proposed as part of the glucose sensing mechanism in fibroblasts
(26) , but its biological relevance for
-cells remains
elusive. It is for instance conceivable that GLUT1 is a stress protein
(27) which becomes more abundant during culture at low glucose
levels. Since the
-cell GLUT1 protein has not yet been detected
in situ, it is possible that its expression was induced by the
isolation procedure and maintained in cell culture. Whether this
phenomenon influences glucose uptake has not been investigated. At both
the mRNA and protein level, GLUT2 was the major glucose transporter in
isolated
-cells. The GLUT2(-like) mRNA occurred as a 2.8-kb and a
3.9-kb transcript. The cDNA corresponding to the 2.8-kb mRNA has been
cloned
(5) , but the sequence and significance of the larger
transcript is not yet known. GLUT2 protein also displayed molecular
heterogeneity, immunoblots reproducibly revealing proteins of 58 and
62 kDa. The 62-kDa protein can be rapidly converted to the 58-kDa
form by proteolytic cleavage, indicating that the enzymatic procedures
of islet isolation and cell preparation may have caused the appearance
of the lower molecular weight form. Proteolytic cleavage occurs in the
first extracellular loop of GLUT2
(28) . By combining functional
and molecular measurements, we have now shown that shifts between the
62- and 58-kDa forms do not affect the glucose transport kinetics of
-cells. Our data support the view that residues in the
carboxyl-terminal region of GLUT2 determine its kinetic properties for
glucose uptake
(29) . They also indicate that the two components
of glucose uptake in dispersed islet cells cannot be attributed to
heterogeneity in GLUT2 protein. Recovery of the large GLUT2 isoform was
more complete after culture of unpurified islet cell preparations than
of pure
-cells; it was also enhanced by culture at 20 mmol/liter
glucose (data not shown). The presence of endocrine non-
-cells may
add to the regulation of GLUT2 gene expression by the
-cells. The
glucose effect is probably caused by increased de novo synthesis, since glucose is known to increase GLUT2 gene
expression
(30) .
-cells did not
exhibit specific GLUT2 expression, but they contained both GLUT1 mRNA
and protein albeit at low abundance. Preparations with more than 80%
-cells
(14) also exhibited these GLUT1 signals, suggesting
their association with
-cells. Contamination with non-
-cells
is still present, but we consider it unlikely that this explains the
GLUT1 signals for the following reasons: ( a) preparations
highly enriched in
-cells were negative or very weakly positive
for GLUT2, excluding a major contamination with
-cells;
( b) cell culture which leads to selective loss of
contaminating exocrine cells did not result in loss of GLUT1 expression
in
-cell preparations; and ( c) amplification of cDNA from
-cells resulted in GLUT-nucleotide sequences that were identical
with GLUT1. So far, GLUT1 protein has not been detected in
-cells
by immunocytochemical staining
(24) but this may be related to
the sensitivity limit of this technique. Since we sequenced only 15% of
the GLUT1 sequence, the glucose transporter in
-cells may be a
GLUT1-related protein, with a specific amino- or carboxyl-terminal
region, but cross-reacting on Western blots with an antibody directed
against the carboxyl terminus of GLUT1. It has been proposed that
insulin plays a permissive role for glucose-induced inhibition of
glucagon secretion
(31) . We therefore screened
-cell RNA
for the presence of sequences homologous to the insulin-responsive
glucose transporter GLUT4. No GLUT4 sequences were found, which is
compatible with the previous observation that insulin does not
stimulate glucose transport rates in
-cells
(16) .
3-OMG transport in
GLUT1-positive
-cells may be explained by cryptic expression of
the GLUT1 gene, but this seems unlikely, since GLUT1 protein has been
localized in the plasma membrane of cultured islet
-cells
(24, 25) . Alternatively, rates of glucose uptake via
GLUT1 in
-cells may be too low to detect under conditions of
simultaneously elevated rates of high
K
/high V
transport
via GLUT2. Rat pancreatic
-cells display metabolic and functional
heterogeneity in terms of glucose responsiveness
(32) . The
possibility that
-cell subpopulations with different sensitivity
for glucose differ in glucose transporter gene expression has been
excluded
(10) . Indeed, virtually all insulin-positive islet
cells also contain membrane-bound GLUT2 immunoreactivity in islet
sections
(24) . GLUT1 and GLUT2 may be expressed on the same
-cells at a different subcellular localization, for instance GLUT2
at the afferent pole (glucose sensor) and GLUT1 at the basal pole
(33) . Increased GLUT2 immunoreactivity in the microvillous
compartment of
-cells supports this concept
(34) .
Subcellular immunodetection of GLUT1 and GLUT2 in
-cells by
electron microscopy will be required to further examine this
possibility.
-cells, glucose uptake is such a rapid process that its rate
largely exceeds that of glycolysis even when measured at low
temperatures. Abundant expression of GLUT2 renders the plasma membrane
almost freely permeable to glucose, permitting the cytoplasmic glucose
sensor to measure minute-to-minute variations in the extracellular
glucose concentration. In
-cells, absence of GLUT2 and low
expression of GLUT1 result in glucose uptake rates which are 10-fold
lower than in
-cells, but they are still 10-fold higher than
overall metabolic flux, strongly suggesting that transport in this cell
type is not rate-limiting for overall glucose metabolism. It has been
proposed that some glucose transporter isoforms may influence the
process of glucose sensing via nonmetabolic properties, such as the
structural organization of sensor proteins close to or in the plasma
membrane
(7) . Alternatively, enzymatic steps distal to glucose
transport may be more directly responsible for glucose sensing in rat
- and
-cells.
Table:
Glucose uptake and glucose utilization in -
and non-
-cells
Table:
Effect of trypsin on
3-O-methylglucose transport kinetics of islet cells
and K
were
determined by Eadie-Hofstee plot of the mean values from five
independent experiments with the best-fit lines drawn using the method
of least squares.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.