Download Glucose transporter gene expression in early mouse

Development 113, 363-372 (1991)
Printed in Great Britain © The Company of Biologists Limited 1991
363
Glucose transporter gene expression in early mouse embryos
AILEEN HOGAN1, SUSAN HEYNER2, MAUREEN J. CHARRON3, NEAL G. COPELAND4,
DEBRA J. GILBERT4, NANCY A. JENKINS4, BERNARD THORENS5 and GILBERT A. SCHULTZ1*
1
Department of Medical Biochemistry, University of Calgary Health Sciences Center, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1
Canada
2
Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, 36'h and Hamilton Walk, Philadelphia, PA
19104-6080 USA
^Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY, 10461 USA
^Mammalian Genetics Laboratory, ABL-Basic Research Program, NCI/Frederick Cancer Research Land Development Center, P.O. Box B,
Building 539, Frederick, MD 21702 USA
5
Whitehead Institute for Biomedical Research, Cambridge, MA 02142 USA
* Author for correspondence
Summary
The glucose transporter (GLUT) isoforms responsible
for glucose uptake in early mouse embryos have been
identified. GLUT 1, the isoform present in nearly every
tissue examined including adult brain and erythrocytes,
is expressed throughout preimplantation development.
GLUT 2, which is normally present in adult liver,
kidney, intestine and pancreatic /} cells is expressed from
the 8-cell stage onward. GLUT 4, an insulin-recruitable
isoform, which is expressed in adult fat and muscle, is
not expressed at any stage of preimplantation development or in early postimplantation stage embryos.
Genetic mapping studies of glucose transporters in the
mouse show that Glut-1 is located on chromosome 4,
Glut-2 on chromosome 3, Glut-3 on chromosome 6, and
Glut-4 on chromosome 11.
Introduction
stimulation by insulin of glucose influx is caused by the
translocation of intracellular transporter molecules to
the plasma membrane (Cushman and Wardzala, 1980;
Suzuki and Kono, 1980; James et al. 1988). GLUT 5, the
most recently described isoform, is involved in glucose
uptake in the small intestine (Bell et al. 1990). GLUT 2,
which has a high Km for glucose (15-20 mivi) in the
concentration range that stimulates insulin release, is
involved in the glucose sensing mechanism of j3 cells as
well as in glucose uptake (Orci etal. 1989; Thorens etal.
1990; Unger, 1991).
The consensus GLUT molecule is a protein of about
500 amino acids with twelve membrane-spanning
domains. There is considerable sequence conservation
in these domains between the five members of the
GLUT family characterized so far. It is probable that
the transmembrane domains are necessary to form the
channel through which glucose is carried. The specific
mechanism of glucose transport and the molecules
involved during early development are of considerable
interest since the intracellular level of glucose has a
direct bearing on metabolic activity (Leese, 1990).
From in vitro studies, glucose does not seem to be
essential for embryonic growth until the time of
compaction and may even be inhibitory for early
Glucose can be transported across membranes by two
different mechanisms: via a sodium-coupled active
carrier system that is expressed in epithelial cells
(Esposito, 1984) or by a sodium-independent facilitative glucose transporter system (Wheeler and Hinkle,
1985). Five facilitative glucose transport proteins
(GLUT 1 to GLUT 5) have been described to date and
their cDNAs have been cloned (reviewed in Bell et al.
1990; Thorens etal. 1990; Kasanicki and Pilch, 1990). In
adult tissues, the GLUT 1 isoform that is expressed in
many tissues and transformed cells and the GLUT 3
isoform that is expressed primarily in human brain and
kidney appear to be responsible for constitutive glucose
uptake. The GLUT 2 isoform mediates glucose
transport in hepatocytes and insulin-producing fl cells.
The GLUT 4 isoform is the major transporter
responsible for the insulin-stimulated uptake that
occurs in muscle and adipose tissue although GLUT 1
expression is also modulated by insulin in muscle and
fat cells, but the effect is less significant because GLUT
4 is the primary and most abundant transporter
expressed in these cells (Walker et al. 1989; de Herreros
and Birnbaum, 1989; Kasanicki and Pilch, 1990). This
Key words: mouse embryos, glucose transporters,
polymerase chain reaction
364
A. Hogan and others
cleavage-stage embryos (Seshagiri and Bavister, 1989;
Chatot et al. 1989). However, from the late morula
stage onwards, glucose becomes increasingly important
to development and is the preferred energy substrate
(Biggers and Borland, 1976; Wordinger and Brinster,
1976).
Previous studies have shown that preimplantation
embryos in culture respond to exogenous insulin with
increased rates of DNA, RNA and protein synthesis
(Harvey and Kaye, 1988; Heyner etal. 1989a; Rappolee
et al. 1990) and that insulin receptor gene expression
can be detected from the 8-cell stage onwards (Rosenblum et al. 1986; Mattson et al. 1988; Rappolee et al.
1990). One aim of this study was to determine which
class of glucose transporter molecule was responsible
for uptake of glucose by the preimplantation embryo
and at which stage the corresponding genes were
expressed. Using the reverse transcription-polymerase
chain reaction technique (RT-PCR) as well as
immunohistochemistry, we have shown that the GLUT
1 isoform is expressed throughout preimplantation
development, that GLUT 2 expression is switched on at
the 8-cell stage, and that the insulin-recruitable GLUT
4 isoform is not expressed at any stage of preimplantation development.
In the mouse, at least two genetic loci for diabetes
mellitus and obesity have been mapped, db and ob
(Coleman, 1978). Both of these mutations give rise to
obese, insulin-resistant animals and are associated with
embryonic malformation and lowered fertility. It was of
considerable interest to map the glucose transporter
genes (Glut-l-Glut-4) in the mouse genome to determine linkage to the above markers of diabetes. The
results of these analyses are presented below.
stage embryos and cultured F9 cells exactly as described by
Telford et al. (1990).
Reverse transcription and polymerase chain reaction
RNA from embryos was reverse transcribed into cDNA as
described by Telford et al. (1990). In brief, purified RNA was
incubated at 43 °C for 60min with a mixture of 20 units of
AMV reverse transcriptase (Molecular Genetics Resources),
0.2ng oligo-dT (Pharmacia), 3min MgCl2, 50min Tris-HCl
pH8.3, 60mM KC1, 1/ig nuclease-free bovine serum albumin
(BSA), lmM each of dATP, dCTP, dGTP and dTTP
(Pharmacia), and 5 units of RNAguard (Pharmacia) in a
volume of lOfil. The reverse transcription reaction was then
repeated by addition of 12.5 units of fresh AMV reverse
transcriptase after a 95 °C, 3min denaturation step and flash
cooling to 4°C. The reaction mix was diluted with sterile
distilled water to 30^1 and 3^1 aliquots were used for each
PCR reaction. PCR (Saiki et al. 1988) was performed as
described previously (Rappolee et al. 1988; Telford et al.
1990). One tenth of the reverse transcription reaction (3^1)
was amplified with 1 unit Taq polymerase (Perkin ElmerCetus) in a final volume of 50 fi\ containing 10 mM Tris-HCl
pH8.3, 2.5 mM MgCl2, 50mM KC1, 5jug nuclease-free BSA,
1.5//M of each sequence-specific primer, and 0.6mM of each
dNTP. The mixture was overlaid with mineral oil to prevent
evaporation and then amplified by PCR for 40 cycles in a
DNA thermal cycler (Perkin Elmer-Cetus), where each cycle
included denaturation at 94°C for lmin, reannealing of
primers to target sequences at 55°C for 2min, and primer
extension at 72°C for 2min. The PCR products (20 ^tl) were
run on 2% agarose gels containing 0.5^gmF 1 ethidium
bromide and photographed.
Primer pairs
The Glut-1 primer pair was constructed from the sequence of
the rat Glut-1 cDNA (Birnbaum et al. 1986). The 5' primer is
identical to nucleotides 1263 to 1286 (24-mer) except for
nucleotide 1280 which was changed from a T to an A residue
in order to create a PstI site for cloning purposes. The 3'
primer is the reverse complement of nucleotides 1600 through
1623 (24-mer). The Glut-2 primers were derived from the
Materials and methods
corresponding mouse cDNA sequence (Asano et al. 1989).
The 5' Glut-2 primer is identical to nucleotides 1211 through
Embryo recovery
1231 (21-mer); its 3' partner is the reverse complement of
Random-bred CD1 mice (Charles River Breeding Laboranucleotides 1606 through 1628 (23-mer). The Glut-3 primer
tories) were used for the RT-PCR and immunofluorescence
pair was derived from the human Glut-3 cDNA sequence
studies. Procedures for superovulation and recovery of
(Kayano et al. 1988). The 5' primer is identical to nucleotides
oocytes and preimplantation embryos were as described by
1290 through 1319 (30-mer); the 3' primer is the reverse
Giebelhaus et al. (1983) and Rosenblum et al. (1986).
complement of nucleotides 1647 through 1670 (24-mer). The
Procedures for dissection of 7.5 to 9.5 day postimplantation
Glut-4 primer pair was derived from the sequence of the rat
stage embryos and culture of F9 embryonal carcinoma cells
cDNA (Charron et al. 1989). The 5' primer is the same as
were as described by Telford et al. (1990).
nucleotides 1104 to 1127 (24-mer) of the cDNA and its 3'
partner is the reverse complement of nucleotides 1324
through 1347 (24-mer).
RNA extraction
A primer pair for mouse /3-actin cDNA was included as an
RNA was extracted from pools of 100-300 preimplantation
internal control. Cytoplasmic /3-actin transcripts are expected
embryos in the presence of 10 ng E. coli ribosomal RNA
to be present in all cell types, and the particular primer pair
(Boehringer-Mannheim) as described by Hahnel et al. (1990).
Embryos in 100 [A of buffer (0.2 M NaCl, 1 mm EDTA, 25 mM design was such that it spanned the first intron of the rodent fiactin gene (Nudel et al. 1983), which is 87bp in length. This
Tris-HCl pH7.4) were vortexed with an equal volume of
primer pair can, therefore, be used to detect both the
phenol and chloroform and phases were separated by
presence of /S-actin mRNA (cDNA) via a predicted 243 bp
centrifugation. The aqueous phase was re- extracted with
PCR fragment and contaminating genomic DNA, if present,
chloroform, recovered after centrifugation and precipitated in
as a 330bp fragment. The 5' primer (21-mer) is identical to
ethanol. RNA pellets were re-dissolved in 5 jA of sterile
nucleotides 182 to 202 in the cDNA sequence (Tokunaga et al.
distilled water and reverse transcribed. RNA from adult
1986). The 3' primer is the reverse complement of nucleotides
tissues was extracted by lysis in guanidinium isothiocyanate
403 through 424 (22-mer). The target sequence bracketed by
followed by ultracentrifugation through CsTFA (Berger and
the /3-actin primer pair is approximately 2000 bp from the 3'
Kimmel, 1987). RNA was purified from postimplantation
Glucose transporters in mouse embryos
end of the cDNA; therefore, detection of a ^-actin PCR
fragment indicates that reverse transcription produced /3-actin
cDNAs at least 2 kb in length.
All primers were obtained from the Regional DNA
Synthesis Laboratory, University of Calgary.
Antibodies
All antibodies were anti-peptide antibodies produced in
rabbits. Antibodies to the GLUT 1 transporter were directed
against the COOH-terminal 16 amino acids (Kahn etal. 1991);
antibodies to the murine GLUT 2 were also raised against
COOH-terminal amino acids (a.a. 512-523) using the
procedure outlined by Thorens et al. (1988). Two antibodies
to GLUT 4 were raised against the COOH-terminal 16 amino
acids (Baldini et al. 1991) and the NH2-terminal 13 amino
acids, respectively (Kahn et al. 1991). Control sera were either
preimmune sera (GLUT 1, 4) or reconstituted rabbit IgG
(GLUT 2). In addition, experiments were designed so that
several developmental stages were examined under identical
conditions, thus providing an internal control. The specificity
of the antisera to GLUT 1 and GLUT 4 was checked by
immunoblotting from target tissues, and from Xenopus
oocytes after microinjection with an mRNA containing the
appropriate transcript (Baldini et al. 1991). In the case of
GLUT 2, preabsorption of the antibody with the immunizing
peptide completely blocked the reactivity of the antiserum to
GLUT 2 as detected by western blotting of total liver lysate.
Immunofluorescent labelling
All manipulations were carried out in microdrops with the
exception of a few early experiments in which the embryos
were immobilized on a lawn of fibroblasts. Oocytes and
developmental stages were fixed in 3 % neutral buffered
formaldehyde for 30min. They were then permeabilized by
incubation in 0.1% Tween 20 for lOmin, and blocked by
incubating for 60min in 20% donkey serum in phosphatebuffered saline containing 2 % bovine serum albumin
(PBS-BSA). Oocytes and embryos were then washed three
times with PBS-BSA, and incubated in the primary antibody
at dilutions ranging from 1:200 to 1:1000 for 30min. The
developmental stages were then washed extensively with
PBS-BSA and incubated with the second antibody, rhodamine-conjugated swine anti-rabbit IgG (Accurate Chemical
and Scientific Corp.) at a concentration of 1:80 for 20min.
Following a final washing, the developmental stages were
mounted in drops of 30 % glycerol in PBS under a supported
coverslip, and evaluated with a Zeiss photomicroscope
equipped for epifluorescence. Developmental stages were
photographed using Kodak Tri X film using a standard
exposure time for all fluorescent records and were also
recorded under bright-field phase contrast.
Cloning and sequencing
The GLUT 1 PCR fragment was isolated from an agarose gel
using Geneclean (Bio 101), cut with Pstl, which cleaved
within the 5' primer, and Sau3A, which cleaved 31 bp from
the 3' end, then cloned into Ps/I-ZtomHI-cleaved pBluescript
KS+ (Stratagene). The gel-purified GLUT 2 PCR fragment
was blunt ended with Klenow fragment in the presence of
dNTPs, cut with Pstl which cleaved 77 bp from the 3' end, and
cloned into Ps/I-Z/irccII-cleaved pBluescript KS+. Sequencing was done by the dideoxy method as described by
Sanger et al. (1977) using the M13 universal primer for one
strand and the M13 reverse sequencing primer for the
opposite strand.
365
Interspecific backcross mapping
Interspecific backcross progeny were generated by mating
(C57BL/6JXM. spretus) F t females and C57BL/6J males as
previously described (Copeland and Jenkins, 1991). A total of
205 N2 progeny were obtained; a subset of these N2 mice were
used to map the Glut loci (see text for details). DNA
isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transfer and hybridization were
performed essentially as described (Jenkins et al. 1982). All
blots were prepared with Zetabind nylon membrane (AMFCuno); probes were labelled with [a-32P] dCTP using a nick
translation labelling kit (Boehringer Mannheim). The probes
and RFLPs for the Glut loci are as follows. The Glut-1 probe
(Birnbaum et al. 1986) detected a 2.4kb M. spretus-speafic
Hindlll fragment that was followed in these studies. The
Glut-2 locus was mapped by following the inheritance of a
9.0 kb Pstl fragment present in M. spretus DNA when probed
with a rat cDNA clone (Thorens etal. 1988). The Glut-3 probe
(Kayano et al. 1988) detected 7.8 and 2.7 kb Pstl M. spretusspecific fragments that cosegregated in this analysis. Taql
digestion of M. spretus DNA and hybridization with the Glut4 probe (Charron et al. 1989) revealed a 2.6 kb fragment that
segregated in backcross mice.
A description of probes and RFLPs for the loci linked to the
Glut loci have been reported (Ceci et al. 1989; Mucenski et al.
1988; Siracusa et al. 1991; Cox et al. 1991; de Lapeyriere et al.
1990; Goodwin et al. 1991; and Buchberg et al. 1989) with the
exception of the Ret proto-oncogene. The probe for the Ret
locus (Takahashi and Cooper, 1987) detected 3.3 and 2.0kb
Taql M. spreto-specific fragments that cosegregated and
placed the locus on mouse chromosome 6. Recombination
distances were calculated as described (Green, 1981) using the
computer program SPRETUS MADNESS. Gene order was
determined by minimizing the number of recombination
events required to explain the allele distribution patterns.
Results
RNA was isolated from batches of oocytes and from
embryos at the 2-cell, 8-cell, morula and blastocyst
stages of development. The purified RNA was primed
with oligo-dT and reverse transcribed. One-tenth of the
product was used for amplification by PCR for each
primer pair specific for actin cDNA or for cDNAs for
the various glucose transporters. The assays were
repeated at least three times with different embryo
batches. Each cDNA preparation was tested for
genomic DNA contamination by PCR amplification
using primers that flank a target sequence of the actin
gene that contains an 87 bp intron. If genomic DNA
were present in the cDNA, a 330bp target sequence
would be amplified as well as the 243 bp fragment
representing the cDNA (Telford etal. 1990). No 330bp
PCR product for actin was detected in any of the
RT-PCR reactions with embryo RNA samples (see
Fig. 1A).
The primers designed to amplify target sequences of
the glucose transporter cDNAs were chosen from the 3'
region of the coding sequences. The amino acid
sequences of GLUT 1 and GLUT 4 share 65 % identity
and the amino acid sequences of GLUT 1 and 2 and
GLUT 2 and 4 share 55% and 54% identity,
respectively (Gould and Bell, 1990). Thus, the nucleic
366
A. Hogan and others
A
Fig. 1. Detection of transcripts in RNA from
preimplantation embryos by RT-PCR. RNA from 200
oocytes (Oc), 225 two-cell (2), 200 eight-cell (8), 238
morula (M), and 184 blastocyst (B) stage embryos was
reverse transcribed with oligo-dT, and one-tenth of the
reverse transcription product was amplified by 40 cycles of
PCR with oligonucleotides specific for /?-actin or GLUT
cDNAs as described in Materials and Methods. Part (20 fA)
of the PCR product was resolved on 2 % agarose gels
along with 2 fig of 1 kb ladder (Bethesda Research
Laboratories; lanes marked L). (A) RT-PCR using /3-actin
primers. (B) RT-PCR with GLUT 1 primers. The arrow
marks the position of the expected 361 bp PCR product for
GLUT 1. (C) RT-PCR using GLUT 2 primers. The arrow
marks the position of the expected 418 bp PCR product for
GLUT 2. (D) RT-PCR with GLUT 4 primers. The lane
marked LM contains the 244 bp PCR product of reverse
transcribed leg muscle RNA using GLUT 4 primers.
Abbreviations are as follows: L=lkb ladder; LI=liver;
LM=leg muscle; BR=brain.
1018
••—243
L
O c 2
8
M
B
1018-
— 361
L
O c2
8 M B
1018
<—418
L
O c2
8 M B
1018-
200-
L Oc
2
8
M B
Li
LM BR
acid sequences are sufficiently dissimilar to allow the
design of primers that do not cross-react during
amplification. The primers were tested on liver, muscle
and brain cDNA to ensure that the appropriate-sized
target sequence was amplified. The primers for this
study spanned the regions from the 3' end of the ninth
transmembrane domain to 60 bp from the end of the
coding sequence in the case of GLUT 1; from the 3' end
of the ninth transmembrane domain to the end of the
coding sequence for GLUT 2; and from the 3' end of
the ninth to the 5' end of the twelfth transmembrane
region for GLUT 4.
Examples of results of PCR amplification of cDNA
from preimplantation mouse embryos are shown in
Fig. 1. Each PCR assay contained reverse transcribed
RNA from roughly equivalent numbers of embryos
because total embryo mass does not change greatly
during the preimplantation period (Schultz, 1986).
Glut-1 mRNA was detected at all stages of development from egg through to blastocyst as is shown by a
band of the expected 361 bp target size (see arrow,
Fig. IB). To verify that this band was a PCR product of
Glut-1 cDNA, it was excised, cloned and sequenced. A
comparison of the cloned GLUT 1 sequence from
mouse embryos and the rat Glut-1 sequence (Birnbaum
et al. 1986) is shown (Fig. 2A). Over the 321 bp of
sequence presented, which includes the PstI and the
internal Sau3A restriction sites used for cloning, there
is 96% nucleotide sequence identity and 99% amino
acid sequence identity between the mouse and the rat
sequences.
Glut-2 mRNA was detectable after the 8-cell stage as
shown by the expected 418 bp band (see arrow,
Fig. 1C). The identity of the band was confirmed by
cloning and sequencing (Fig. 2B). The cloned 342 bp
component of the mouse Glut-2 PCR fragment shares
100 % nucleotide sequence identity with the published
mouse sequence (Asano et al. 1989) and 94 % nucleotide sequence identity and 96% amino acid sequence
identity with the corresponding rat cDNA sequence
(Thorens et al. 1988). Other amplified products were
Glucose transporters in mouse embryos
LeuLeuGluGlnLeuProTrpMetSerTyrLeuSerlleValAlallePlieGly
Primer - CTGCAGGAGCAGCTGCCTTGGATGTCCTATCTGAGCATCGTGGCCATCTTTGGC
CTGCTGGAGCAGCTGCCCTGGATGTCCTATCTGAGTATCGTGGCCATCTTTGGC
LeuLeuGluGlnLeuProTrpMetSerTyrLeuSerlleValAlallePheGly
PheValAlaPhePheGluValGlyProGlyProIleProTrpPhelleValAlaGluLeu
TTTGTGGCCTTCTTTGAAGTAGGCCCTGGTCCTATTCCATGGTTCATTGTGGCCGAGCTG
TTTGTGGCCTTCTTTGAAGTAGGCCCTGGTCCTATTCCATGGTTCATTGTGGCCGAGCTG
PheValAlaPhePheGluValGlyProGlyProIleProTrpPhelleValAlaGluLeu
PhoSarGlnGlvProArqProAlaAlalleAlavalAlaGlYPhegerAanTrpThrSer
TTCAGCCAGGGGCCCCGTCCTGCTGCTATTGCTGTGGCTGGCTTCTCCAACTGGACCTCA
TTCAGCCAGGGGCCCCGACCTGCTGCTGTTGCTGTGGCTGGCTTCTCTAACTGGACCTCA
PheSerGlnGlyProArgProAlaAlaValAlaValAlaGlyPheSerAsnTrpThrSer
AanPhelleValGlyHetcysPheGlnTyrValGluGlnLeuCysGlyProTyrValPhe
AACTTCATTGTGGGCATGTGCTTCCAGTATGTGGAGCAACTGTGCGGCCCCTACGTCTTC
AACTTCATCGTGGCCATGTGCTTCCAATATGTGGAGCAACTGTGTGGCCCCTACGTCTTC
AsnPhelleValGlyMetCysPheGlnTyrValGluGlnLeuCysGlyProTyrValPhe
IlallaPhaThrValLauLauValLauPhaPtiallaPhaThrTyrPheLysvalProGlu
ATCATCTTCACGGTGCTCCTCGTGCTCTTCTTCATCTTCACCTACTTCAAAGTCCCTGAG
ATCATCTTCACGGTGCTGCTGGTACTCTTCTTCATCTTCACCTACTTCAAAGTTCCTGAG
IlellePheThrValLeuLeuValLeuPhePhellePheThrTyrPheLysValProGlu
ThrLyaGlyArgThrPhaAspOluIla
ACCMAGGCCGAACCTTCGATGAGATC - 3' Primer
ACCAAAGGCCGGACCTTCGATGACATC
ThrLysGlyArgThrPheAspGluIle
primer -
ValGlyLauValLeuLauAapLysPhaAlaTrpMatSerTyrValSarMetThrAla
CGGTGGOACTTGTGCTGCTGGATAAATTCGCCTGGATGAGTTACGTGAGCATGACTGCC
CGCTGGGACTGGTGTTGCTGGATAAGTTCACCTGGATGAGTTATGTGAGCATGACGGCC
LeuGlyLeuValLeuLeuAspLysPheThrTrpMetSerTyrValSerMetThrAla
IlaPhaLauPhaValSarPhaPhaGluIlaGlyProGlyProIlaProTrpPheHetVal
ATCTTCCTCTTTGTCAGTTTCTTTGAGATTGGGCCAGGTCCAATCCCTTGGTTCATGGTT
ATCTTCCTCTTCGTCAGTTTCTTTGAGATTGGGCCAGGTCCAATCCCTTGGTTCATGGTT
I lePheLeuPheValSerPhePheGluI leGlyProGlyProI leProTrpPheMetVal
AlaGluPhaPhasarGlnGlyProArgProThrAlaLauAlaLeuAlaAlaPheSerAsn
GCTGAATTTTTCAGCCAAGGACCCCGTCCTACGGCTCTGGCACTCGCTGCCTTCAGCAAC
GCTGAATTTTTCAGCCAAGGACCCCGTCCCACGGCTCTGGCACTGGCTGCCTTTAGCAAC
AlaGluPhePheSerGlnGlyProArgProThrAlaLeuAlaLeuAlaAlaPheSerAsn
TrpvalCysAsnPheyallleAlaLeuCysPheGlnTyrlleAlaAspPheLeuGlyPro
TGGGTCTGCAATTTTGTCATCGCCCTCTGCTTCCAGTACATTGCGGACTTCCTTGGGCCT
TGGGTCTGCAATTTCATCATCGCCCTCTGCTTCCAGTACATTGCGGACTTCCTCGGGCCT
TrpValCysAsnPhellelleAlaLeuCysPheGlnTyrIleAlaAspPheLeuGlyPro
TyrValPhePheLeuPheAlaGlyvalvalLauValPheThrLeuPheThrPhePheLys
TACGTGTTCTTCCTCTTCGCTGGGGTGGTCCTGGTCTTCACCCTGTTTACATTCTTTAAA
TACGTGTTCTTCCTTTTTGCTGGGGTGGTCCTGGTCTTCACCCTGTTCACATTTTTTAAA
TyrVa 1 PhePheLeuPheAlaG ly Va 1 Va 1 LeuVa 1 PheThrLeuPheThrPhePheLys
valProGluThrLysGlyLysSerPhaG_luGluIleAlaAla
GTTCCAGAAACCAAAGGAAAGTCTTTTGAGGAAATCGCTGCAG
- 3' primer
GTTCCAGAAACCAAAGGAAAGTCTTTTGACGAAATTGCTGCAG
valProGluThrLysGlyLysSerPheAspGluIleAlaAla
Fig. 2. Sequences of the cloned Glut-1 and Glut-2 PCR
products. (A) The PCR target sequence of the mouse Glut1 cDNA is shown in the upper row in bold face. The
corresponding rat cDNA is shown below the mouse
sequence (Bimbaum et al. 1986). The Pstl and Sau3A
restriction sites used to clone the fragment are underlined.
(B) The sequence of the mouse Glut-2 PCR fragment is
presented in the upper row. The corresponding rat Glut-2
sequence is shown in the lower row (Thorens et al. 1988).
The Pstl site used to clone the fragment is underlined.
Dots between nucleotides indicate sequence identity.
Amino acids corresponding to the nucleotide sequence are
identified above and below each codon with differences in
the mouse sequence relative to the rat sequence
underlined.
367
observed in some of the PCR assays but the identification of these bands was not attempted.
In preliminary RT-PCR experiments, no expression
of GLUT 3 was detected in preimplantation embryos
but antibodies are not yet available to verify this result.
The nucleotide sequence of Glut-5 has not been
published so the design of primers for RT-PCR assays
is not yet possible.
No expression of GLUT 4 was detected at any stage
of preimplantation development. However, a strong
band of the expected target size (244 bp) was generated
when cDNA made from leg muscle RNA was used in
the PCR assay (Fig. ID). The identity of the Glut-4
PCR band generated from muscle cDNA was verified
by cleavage with Alul which cut the fragment at a site
predicted from the cDNA sequence (data not shown).
The pattern of GLUT expression in postimplantation
embryos at 7.5 to 9.5 days post coitum and in F9
embryonal carcinoma cells was also analysed. As was
the case during preimplantation development, GLUT 1
and GLUT 2 RT-PCR products were detected but no
expression of GLUT 4 was observed (see Fig. 3A for
8.5 day embryo RNA and Fig. 3B for F9 cell RNA). At
9.5 days of development, the mouse embryo has about
20 somites (muscle primordia) but these cells apparently do not express GLUT 4 characteristic of
differentiated muscle cells. A possible explanation may
be that GLUT 1 is sufficient for glucose uptake at this
stage of development since Glut-1 mRNA has been
detected in most tissues (Bell etal. 1990). Alternatively,
Glut-3 was originally cloned from fetal muscle (Kayano
et al. 1988) and could be mediating the transport of
glucose.
Indirect immunofluorescence studies carried out with
antibodies directed against mouse GLUT 1 transporter
peptides showed that developmental stages from the
oocyte through to the blastocyst reacted positively
(Fig. 4). Similar studies with antibodies directed against
the GLUT 2 transporter peptides showed positive
reactions in mouse embryos at the morula through
blastocyst stages (Fig. 5). These results show that the
protein products of Glut-1 and Glut-2 are present as
well as their mRNAs. No immunofluorescence was
detected when antibodies to GLUT 4 were reacted with
mouse embryos of similar developmental stages
(Fig. 5).
The mouse chromosomal locations of the four genes
encoding glucose transporter proteins (designated Glut1, Glut-2, Glut-3 and Glut-4) were determined by
interspecific backcross analysis using progeny derived
from matings of [(C57BL/6J x Mus spretus)¥lx
C57BL/6J] mice (see Materials and methods). This
interspecific backcross mapping panel has been typed
for over 675 loci that are well distributed among all the
autosomes as well as the X chromosome (Copeland and
Jenkins, 1991). C57BL/6J and M. spretus DNAs were
digested with several restriction endonucleases and
analyzed by Southern blot hybridization for informative
restriction fragment length polymorphisms (RFLPs)
using the GLUT probes. The strain distribution pattern
(SDP) of each RFLP in the interspecific backcross mice
368
A. Hogan and others
A
E
-418
-361
200
2
4
B
-418
-361
L
1
2
4
Fig. 3. Detection of Glut transcripts in RNA from
postimplantation embryos and from F9 embryonal
carcinoma cells by RT-PCR. (A) \y.g of RNA from
embryos at 8.5 days post coitum was reverse transcribed
using oligo-dT, and one-tenth of the reverse transcription
product was amplified by 40 cycles of PCR with
oligonucleotides specific for Glut-1 (1), Glut-2 (2), and
Glut-4 (4). The lkb ladder (L) is used as molecular weight
marker. (B) 1 fig of RNA from F9 embryonal carcinoma
cells was reverse transcribed as in the panel above and
one-tenth of the RT product was used for PCR with
primers for Glut-1 (1), Glut-2 (2) and Glut-4 (4).
was then determined and used to position the Glut loci
on the interspecific map (Fig. 6).
Each locus segregated independently. The Glut-1
locus was assigned to mouse chromosome 4, 1.7 cM
proximal of Lmyc. This placement of Glut-1 is
consistent with the recent results of Bahary et al. (1990).
The Glut-2 locus was placed on mouse chromosome 3.
This gene did not recombine with Evi-1 in 150 mice
typed in common for both probes. This suggests that
Glut-2 and Evi-1 reside within 2.0cM (upper 95%
confidence level). Glut-3 was placed on mouse chromosome 6, 2.0cM distal to Ret and 0.7 cM proximal of Ly-
Fig. 4. Immunofluorescent localization of GLUT 1
transporter protein. The left-hand panel shows a
developmental series of preimplantation mouse embryos
under bright-field phase-contrast optics and the right-hand
panel shows the image under epifluorescence. Stages from
oocyte (la,b), two-cell (2a,b), eight-cell (3a,b), and
blastocyst (4a,b) showed positive reactivity, while a blastocyst
treated under the same conditions with preimmune serum
was negative (5a,b). The bar represents 40,urn.
Glucose transporters in mouse embryos
369
Fig. 5. Immunofluorescent localization of GLUT 2
transporter protein. The left-hand panel shows a
developmental series of preimplantation mouse embryos
under bright-field phase-contrast optics and the right-hand
panel shows the image under epifluorescence. Embryos at
the two-cell (la,b) and eight-cell (2a,b) stages were
negative, while morula and blastocyst stages showed
positive reactions (3a,b; 4a,b). All stages were negative
when incubated with antisera directed against GLUT 4;
5a,b shows a representative result of incubating blastocyst
stage embryos. The bar represents 40 fim.
Discussion
4. Finally, Glut-4 was placed in the middle region of
mouse chromosome 11, tightly linked to Trp53-1. The
fact that no recombination between the two genes in
135 mice typed in common was observed suggests that
they reside within 2.2cM of each other (upper 95%
confidence level).
The facilitative glucose transporters are encoded by a
small multi-gene family and various members have
distinct tissue distributions (Bell et al. 1990; Thorens et
al. 1990). This study focuses on the three most
extensively characterized isoforms: GLUT 1, GLUT 2,
and GLUT 4. Human Glut-3 cDNA has been isolated
from a fetal skeletal muscle library and is expressed in
various adult tissues, primarily brain and kidney
(Kayano et al. 1988). The murine Glut-3 cDNA has now
been cloned and the sequence (as yet unpublished) is
significantly divergent from the human Glut-3 cDNA
(G. Bell, personal communication). This may explain,
in part, our inability to detect this mRNA using
oligonucleotides based on the human cDNA sequence.
When the murine Glut-3 sequence becomes available,
we will be able to address this question accurately.
Preimplantation mouse embryos require pyruvate
and lactate as energy sources until the 8-cell stage and
then switch to a glucose-based metabolism (Gardner
and Leese, 1986). Glucose is a precursor of a number of
macromolecular cell constituents: complex sugars for
mucopolysaccharides, ribose moieties for nucleic acid
synthesis and glycerol phosphate for phospholipids.
Using the selective inhibitors phlorizin and phloretin, it
was determined that glucose uptake in preimplantation
embryos is mediated by sodium-independent facilitative glucose transporters (Gardner and Leese, 1988) so
it is important to examine the functional role of GLUT
proteins in early development. GLUT 1 is expressed
throughout preimplantation development and appears
to be responsible for the low levels of glucose uptake
observed from the 1-cell stage onwards by Gardner and
Leese (1988). Since the zygotic genome in the mouse is
not activated until the 2-cell stage, the Glut-1 PCR band
from oocyte RNA is presumably due to the presence of
maternal mRNA. At the 8-cell stage of development,
GLUT 2 begins to be expressed and stays switched on
through the morula and blastocyst stages. This may be
responsible, along with an increase in GLUT 1, for the
rise in glucose uptake that begins at the 8-cell stage
(Gardner and Leese, 1988). Since insulin receptor gene
expression can be detected in embryos from the 8-cell
stage onwards (Heyner et al. 1989a; $>chx\\\z et al. 1990),
it seemed possible that the insulin-recruitable GLUT 4
isoform might also be involved in glucose transport
after the 8-cell stage. However, no expression of GLUT
4 was detected with either RT-PCR methods or
370
A. Hogan and others
1
}
0/122 ( 2.4 ) -
'Ifa
9pl3-p22
Ip31-p32
13/119, 10.9 +/-2.9
- - 11-7
14/191, 7.3 +/- 1.9
2/119. 1.7+/- 1.2 \
5/122,4.1 +/- 1.8
--
Lmyc
Ip31.3-p35
Ip32
Lck
Ip32-p35
Glut-l
. Evi-1
-Glut-2
0/150(2.0)-
2/178, 1.1 +/-0.8
3/147.2.0+/- 1.2
1/145. 0.7 +/- 0.7 ZJI
Raf-1
Ret
Glul-3
Ly-4
Ftf-6
3p25
10qll.2
12pl3.3
12pl2-pter
12pl3
3q25-q27
3q26.I-q26.3
8/136, 5.9 +/- 2.0
- " Fgfb
1
3/188, 1.6+/- 0.9
8ql2-ql3
Myhs
4q25
17pll-pter
7/144,4.9+/- 1.8
0/135 ( 2.2 ) •
• Trp53-]
17pl3.1
'Glut-4
17pl3
4/135, 3.0+/- 1.5
--
antibodies specific to GLUT 4 at any stage up to the
blastocyst.
A number of studies on the anabolic and proliferative
effects of insulin on the early mouse embryo have been
reported (Harvey and Kaye, 1988, 1990; Heyner et al.
1989b; Gardner and Kaye, 1991) but the effects of
insulin on glucose uptake by preimplantation mammalian embryos is not entirely clear. Since insulin
causes an increase in glucose uptake in adult cells such
as adipocytes (Charron et al. 1989) and since expression
of the insulin receptor gene has been detected in
preimplantation embryos (Schultz et al. 1990), an
association between insulin and glucose uptake during
early development was suspected. Insulin at concentrations ranging from 0.0017 to 170 nM has been
reported to stimulate radiolabelled glucose uptake in
vitro in preimplantation mouse embryos (Gardner,
1990). In addition, blastocysts collected from streptozotocin-induced diabetic mice (Beebe and Kaye, 1991)
and rats (Brison and Leese, 1990) show impaired
development. The uptake of glucose in rabbit blastocysts is also mediated by sodium-independent glucose
transporters but in this species, glucose transport
appears to be unaffected by the addition of insulin at
concentrations of 100 nM (Robinson et al. 1990). Our
data show that the glucose transporter isoforms GLUT
1 and GLUT 2 are the likely mediators of glucose
uptake in the early mouse embryo and are in agreement
with observations obtained using western blot analysis
Evi-2
17ql 1.2
Fig. 6. Linkage maps showing
the chromosomal locations of
the four Glut loci in mouse.
The loci were mapped by
interspecific backcross analysis.
The number of recombinant
N2 animals over the total
number of N2 animals typed
plus the recombination
distance in centimorgans
(±one standard error) is
shown for each pair of loci on
the left of the chromosome
maps. Where no recombinants
were found between loci, the
upper 95 % confidence level of
the recombination distance is
given in parentheses. The
positions of loci in human
chromosomes are shown to the
right of the chromosome maps.
and high resolution immunocytochemistry (Rao et al.
1990). Further studies are needed to establish the
conditions under which transport of glucose in preimplantation embryos by GLUT 1 and GLUT 2 might
be influenced by insulin and to determine whether the
mitogenic and other physiological effects of insulin are
distinct from its role in sugar metabolism.
In this report, we also present the genetic mapping of
Glut-l-Glut-4 in the mouse genome. Glut-l is located
on chromosome 4, Glut-2 on chromosome 3, Glut-3 on
chromosome 6 and Glut-4 on chromosome 11. Alignment of the interspecific map with the composite map
compiled by M. T. Davisson, T. H. Roderick, A. L.
Hillyard, and D. P. Doolittle and provided by GBASE
(a computerized data base maintained at The Jackson
Laboratory, Bar Harbor, ME) has failed to identify any
known mouse mutation that maps in the vicinity of a
Glut locus that has a phenotype consistent with a defect
in one of these genes. Finally, many regions of
homology have been identified between mouse and
human chromosomes and it is often possible to predict
where a gene will map in one species based on its
position in the other. From the mouse mapping studies,
we would suggest that Glut-l is located on human lp,
Glut-2 on human 3q, Glut-3 on human 12p, and Glut-4
on human 17p. These expectations have been confirmed from human mapping studies and the precise
location of the GLUT loci in human chromosomes is
summarized in Fig. 6. These studies also demonstrate
Glucose transporters in mouse embryos
that the GLUT loci are dispersed in the human genome
as well.
The authors are grateful to Dr A. Hahnel for her help with
the embryo collection and RNA extraction. We also thank G.
Bell and G. Cooper for probes and D. Swing, B. Cho, and M.
Cybulski for excellent technical assistance. This work was
supported by NIH grant HD 23511 to S. H. and G. A. S., by
grant MT-4854 from the MRC (Canada) to A. H. and G. A.
S., by NIH grant DK-08101 to M. J. C , and by the National
Cancer Institute, DHHS, under contract NO1-CO-74101 with
ABL to N. G. C , D. J. G. and N. A. J.
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(Accepted 11 June 1991)