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INTRODUCTION |
The insulin-regulated membrane aminopeptidase
(IRAP)1 was first identified
as a major protein in intracellular vesicles isolated from the low
density microsomes of fat and muscle cells that also harbor the
insulin-responsive glucose transporter isotype GLUT4 (1-3). IRAP was
earlier referred to as vp165 (vesicle protein of 165 kDa) (1) or gp160
(glycoprotein of 160 kDa) (2). IRAP
has been well characterized in muscle and fat cells with regard to its
subcellular localization and the regulation of its trafficking by
insulin (1, 2, 4-8). The results from these studies showed that IRAP
behaves almost identically to GLUT4. Under basal conditions, IRAP and
GLUT4 are efficiently sequestered in intracellular membrane
compartments. When cells are treated with insulin, IRAP and GLUT4
translocate to the plasma membrane. In adipocytes, this results in an
8- and 10-20-fold increase of IRAP and GLUT4, respectively, at the
cell surface (4, 9, 10). GLUT4 and IRAP are the only known molecules in
fat and muscle cells that exhibit such marked translocation in response to insulin.
The physiological role of GLUT4 and the significance of its
differential subcellular distribution under basal and
insulin-stimulated conditions are well established. The number of GLUT4
at the cell surface is the major determinant for the amount of glucose
transported into muscle and fat tissues (10, 11). Since skeletal muscle accounts for 80% of glucose disposal after feeding (12), and glucose
uptake is the rate-limiting step in glucose metabolism in skeletal
muscle (13), the regulation of the subcellular distribution of GLUT4 is
key to the maintenance of glucose homeostasis. The physiological
function of IRAP and the implication of its differential subcellular
distribution under basal and insulin-stimulated conditions, however,
are unknown.
The cloning of IRAP revealed that it is a member of the family of
zinc-dependent aminopeptidases (14). IRAP's closest
relatives in this family, the mammalian membrane aminopeptidases A and
N, were known to process peptide hormones in vitro (15, 16). Recent studies in mice using specific inhibitors for the two
aminopeptidases support their role in the processing of angiotensin II
and III, respectively, in vivo (17). We have previously
shown that IRAP removes the N-terminal amino acids from vasopressin,
Lys-bradykinin, and angiotensins III and IV efficiently (18). Insulin
itself is not a substrate. Angiotensins III and IV are also well
cleaved by aminopeptidase N (17, 19). However, vasopressin, with its N-terminal cystine, was known to be cleaved only by the human placental leucine aminopeptidase/oxytocinase (P-LAP) (19). The cloning
of P-LAP revealed that it is 87% identical to IRAP at the amino acid
level (20). In agreement with our results, P-LAP cleaves vasopressin
and angiotensin IV, as well as oxytocin (21). Whether the peptides
cleaved by IRAP and P-LAP in vitro are also in
vivo substrates has not been established.
The aminopeptidases homologous to IRAP are constitutively expressed at
the cell surface and so have continued access to their extracellular
substrates (22). IRAP, however, was expected to process its
extracellular substrates only after translocation to the cell surface.
In support of this, we have found that insulin, concomitant with the
translocation of IRAP to the plasma membrane, increases aminopeptidase
activity toward extracellular vasopressin in isolated adipocytes (18).
The cloning of IRAP and characterization of it as an aminopeptidase
that can process regulatory peptide hormones revealed thus a hitherto
unrecognized possible action of insulin, the potential to modify,
through IRAP, the action of other peptide hormones and thus broaden its
own spectrum of action. This mode of action may not be restricted to
insulin. It has been shown that endothelin-1 recruits IRAP to the
plasma membrane in adipocytes and cardiomyocytes (23, 24). Furthermore, IRAP may also play a role in processing peptide hormones in other tissues than fat and muscle. IRAP is well expressed in all major tissues with the exception of liver, where its expression levels are
low (14). Recently, it has been reported that stimulation of human
umbilical vascular endothelial cells with oxytocin leads to the
translocation of IRAP to the cell surface (25).
To address the question of the function of IRAP in a physiological
context, we have generated mice with a targeted disruption of the IRAP
gene (referred to as IRAP
/ mice). We first tested these mice for
defects in the regulation of glucose homeostasis, a major insulin
action that is dependent on the proper regulation of GLUT4.
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EXPERIMENTAL PROCEDURES |
Generation of IRAP/ Mice--
Genomic clones containing
fragments of the IRAP gene were isolated from a mouse embryonic stem
cell genomic P1 library (mouse strain 129) by PCR screening with two
primers derived from the rat IRAP cDNA (5'-GTCTTGGTGAGCATGAGATGG-3'
and 5'-CTAAGGTCCTGGCAGAGGGTA-3', corresponding to nucleotides 241-261
and 385-405, respectively (14)) by Genome Systems Inc. The genomic
clones were analyzed by restriction digestions, Southern blotting, and
sequencing, and a detailed exon-intron map of the IRAP gene was
obtained.2 Based on this
information, a targeting vector was constructed as follows (Fig.
1A). An XhoI (blunt-ended)-HindIII
genomic IRAP fragment corresponding to 3.8 kb of intron sequence with
its 3'-end 0.4 kb upstream of exon 2 was cloned into the
ClaI (blunt-ended)-HindIII restriction sites in
the vector pPol II long neo bpA 5' to the neomycin
resistance cassette. The neomycin resistance gene in this cassette is
oriented such that transcription proceeds in the opposite direction to
the introduced IRAP sequences. A 5.6-kb XhoI-ClaI
(blunt-ended) restriction fragment of IRAP genomic DNA with its 5'-end
0.5 kb downstream of exon 6 and its 3'-end 2 kb downstream of exon 8 was inserted into the XhoI and EcoRI
(blunt-ended) restriction sites in the vector downstream of the
neomycin resistance cassette and upstream of the thymidine kinase gene.
The thymidine kinase gene was derived from the pMC1TK vector. Correct
insertion of the fragments in the targeting construct was verified by
restriction analysis and sequencing of the junctions. The targeting
vector was linearized with SrfI and introduced into mouse
embryonic stem cells (from strain 129 SvEv) by electroporation.
Embryonic stem cell clones were selected for resistance to G418.
Genomic DNA was isolated from resistant clones, digested with
XbaI, and screened for homologous recombination by Southern
blotting. The 1032-bp probe used for screening was derived from exon 9 and 0.5 kb of intron sequence upstream and 0.35 kb of intron sequence
downstream of exon 9 and was labeled with dUTP-digoxigenin by PCR
(primers 5'-CAAGCTCAGTCTGGAGTCTTAGTG-3' and
5'-GATTCACAGGGCTTCATAGAGAC-3'). The hybridized probe was
detected with an anti-digoxigenin antibody conjugated to alkaline
phosphatase and chemiluminescence according to the manufacturer's
instructions (Roche Molecular Bio-chemicals). Of the 384 clones analyzed, three showed the expected fragments of 12 and 8.5 kb
characteristic for the endogenous intact and the disrupted IRAP allele,
respectively (see Fig. 1B). The XbaI-digested genomic DNA of these three clones was also hybridized with a 783-bp dUTP-digoxigenin-labeled probe that was obtained by PCR with primers derived from the neomycin resistance gene (5'-GGGATCGGCCATTGAACAAG-3' and 5'-AAGGCGATAGAAGGCGATGC-3') to test for correct recombination between the 5' homologous IRAP region in the targeting vector and the
endogenous IRAP. All three clones showed the expected 8-kb
XbaI fragment (Fig. 1A and data not shown).
One of these clones was injected into blastocysts derived from Black
Swiss mice, and the blastocysts were implanted into pseudopregnant recipients. Male chimeric offspring were bred with wild type Black Swiss female mice (Taconic). In the agouti offspring, germ line transmission of the mutant IRAP allele was identified by Southern blotting of genomic DNA obtained from tail snips. The
dUTP-digoxigenin-labeled IRAP probe was used as described above and
marked in Fig. 1A. Six male and female mice heterozygous for
the mutant IRAP allele (IRAP+/) were bred to obtain the IRAP null
(IRAP/) together with wild type (IRAP+/+) and heterozygous
(IRAP+/) mice. The mice were genotyped by Southern blotting as
described above. Representative results from Southern blots are shown
in Fig. 1B. Nine male and female IRAP/ mice as well as
eight male and female IRAP+/+ mice were paired, and the offspring from
these breeding pairs were used for all of the experiments described
below unless stated otherwise. Offspring from different breeding pairs
for each genotype were used in each experiment to assure equal
representation of the different mixed genetic pools obtained through
the breeding of the outbred Black Swiss with the inbred 129 SvEv mice.
The mice were housed under a constant light (6 a.m. to 6 p.m.) and dark cycle and fed Teklad LM 485 mouse/rat diet (Harlan Teklad) with
free access to water.
Analysis of Blood Glucose and Plasma Insulin Concentrations and
Glucose and Insulin Tolerance Tests--
The determination of blood
glucose and plasma insulin concentrations as well as the oral glucose
tolerance test were carried out as described in Ref. 26 with the
exception that the blood samples for the determination of fed glucose
and insulin were taken between 8 and 9 a.m. The insulin tolerance
test was performed as described in Ref. 27.
Determination of Glucose Uptake in Isolated Muscles--
Mice
were anesthetized with sodium pentobarbital (Nembutal) (0.1 mg/g body
weight), and soleus and EDL were isolated. Glucose transport into the
isolated muscles was assayed by the uptake of 3H-labeled
3-O-methylglucose after the method described in Ref. 28 as
follows. The isolated muscles were incubated at 29 °C for 60 min in
1.5 ml of continuously gassed (95% O2/5% CO2)
Krebs-Henseleit bicarbonate buffer (116 mM NaCl, 4.6 mM KCl, 1.2 mM KH2PO4,
25.3 mM NaHCO3, 2.5 mM
CaCl2, 1.16 mM MgSO4, pH 7.2)
containing 0.1% (w/v) BSA, 8 mM glucose, and 32 mM mannitol. The muscles were transferred to fresh vials
containing 1.5 ml of Krebs-Henseleit bicarbonate buffer with 0.1% BSA,
40 mM mannitol, and either no or 13 nM insulin
(Humulin R; Lilly). After a 15-min incubation at 29 °C, the muscles
were placed into fresh vials containing 1.5 ml of Krebs-Henseleit
bicarbonate buffer with 0.1% (w/v) BSA, 8 mM
3-O-methylglucose (0.25 mCi of 3H-labeled
3-O-methylglucose/mmol (PerkinElmer Life Sciences)), 30 mM mannitol (10 µCi of [14C]mannitol/mmol
(PerkinElmer Life Sciences)), 2 mM pyruvate, and either 0 or 13 nM insulin. After 10 min of incubation at 29 °C, glucose uptake was terminated, and muscle pieces were processed as
described in Ref. 28. Glucose uptake was determined from the
intracellular 3H-labeled 3-O-methylglucose by
subtracting extracellular 3H-labeled
3-O-methylglucose from the total muscle-associated
3H-labeled 3-O-methylglucose. The extracellular
3H-labeled 3-O-methylglucose was estimated from
the content of the extracellular marker [14C]mannitol in
each muscle piece. Data are expressed as µmol of 3-O-methylglucose/g of muscle/10 min.
Determination of Glucose Uptake In Isolated
Adipocytes--
Adipocytes were isolated from the epididymal and
parametrial fat pads from male and female mice, and the uptake of
[U-14C]D-glucose (ICN) was measured as
described in Ref. 26 with the exception that glucose uptake was
terminated after 15 min of incubation. Aliquots of the adipocyte
preparation used for the glucose uptake assays were also subjected to
lipid extraction to determine lipid weight as described in Ref. 26.
Glucose uptake was normalized to the lipid content of each sample and
expressed as fmol of glucose/min/mg of lipid.
Analysis of Tissue Homogenates--
Mice were euthanized with
100% CO2. Tissues were isolated, rinsed in ice-cold
phosphate-buffered saline, blotted dry, immediately frozen in liquid
nitrogen, and stored at 80 °C until further processing. The frozen
tissues were weighed and homogenized in a buffer containing 25 mM Hepes, pH 7.4, 100 mM NaCl, 1 mM
Na3VO4, 20 mM sodium pyrophosphate,
3 mM N-ethylmaleimide, 1 µg/ml pepstatin, 10 µM EP475, 10 µg/ml aprotinin, and 0.2 mM
phenylmethylsulfonyl fluoride. Experimentally determined protein
content per mg of tissue was used as the basis to calculate the amount
of homogenization buffer to be added to each tissue such that the
protein concentrations of the final homogenates were between 10 and 20 mg/ml. Aliquots of the homogenates were mixed with 4× SDS sample
buffer (370 mM Tris, pH 6.8, 4 mM EDTA, 40%
(v/v) glycerol, 0.016% (w/v) bromphenol blue, 16% SDS, 80 mM dithiothreitol, 4 µg/ml pepstatin, 40 µM EP475, 4 mM phenylmethylsulfonyl fluoride, 40 µM leupeptin, and 40 µg/ml aprotinin).
For the preparation of adipocyte homogenates, adipocytes were isolated
from the epididymal and parametrial fat pads as described in Ref. 26 in
Krebs-Ringer-Hepes-BSA buffer (120 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM
MgSO4, 20 mM HEPES, pH 7.4, 1% BSA, 3 mM glucose, and 200 nM adenosine). To remove the BSA and thus avoid complications of interference in immunoblotting for GLUT4, the cells were washed twice in KRBH without BSA, and a 5%
fat cell suspension in KRBH was prepared. Aliquots of the suspension
were either processed for the determination of lipid weights as
described in Ref. 26 or were used to prepare adipocyte lysates as
follows. Cells were allowed to float, the infranatant was removed, and
1.2× SDS sample buffer was added to the cells. The protein
concentrations in all of the SDS samples were determined by a
precipitating Lowry assay with BSA as the standard (29).
Preparation of Subcellular Fractions from Isolated
Adipocytes--
Mice were euthanized with 100% CO2.
Adipocytes were isolated from the epididymal and parametrial fat pads
from male and female mice as described in Ref. 26 in KRBH-BSA buffer
(120 mM NaCl, 4 mM
KH2PO4, 1 mM CaCl2, 1 mM MgSO4, 10 mM NaHCO3,
30 mM HEPES, pH 7.4, 1% BSA, 3 mM glucose, and
200 nM adenosine). After the digestion with collagenase,
the cells were washed twice in KRBH-BSA. A 10% cell suspension was
prepared in KRBH-BSA and split into two equal aliquots. To one aliquot
insulin was added to 10 nM. All cells were incubated for
30-45 min at 37 °C with gentle shaking (40 rpm). The cells were
then washed once in homogenization buffer (255 mM sucrose,
20 mM Hepes, pH 7.4, with or without insulin) at room
temperature and subsequently homogenized with 10 up and down strokes in
a Teflon homogenizer rotating at 1250 rpm in the homogenization buffer
containing 1 µg/ml pepstatin, 10 µM EP475, 10 µg/ml
aprotinin, 10 µM leupeptin, and 1 mM
phenylmethylsulfonyl fluoride with or without insulin. Subcellular
fractions were obtained from the homogenates as described in Ref. 30.
The fractions were resuspended in homogenization buffer containing 1 µg/ml pepstatin, 10 µg/ml aprotinin, 10 µM leupeptin.
The protein concentrations in each fraction were determined by the
precipitating Lowry assay (29). Aliquots from each of the fractions
were used for the measurement of aminopeptidase activities as described
below. SDS samples containing equal amounts of protein for each
fraction from the IRAP/ and IRAP+/+ mice were prepared from
aliquots from the fractions and were analyzed by immunoblotting.
Immunoblotting of Tissue Homogenates and Subcellular
Fractions--
SDS samples of tissue homogenates containing equal
amounts of protein were immunoblotted for IRAP with an antibody raised against the N-terminal cytoplasmic domain of IRAP as described in Ref.
4. Equal amounts of protein of muscle tissue homogenates in SDS sample
buffer were separated on 16 × 16 × 0.15-cm 10%
SDS-polyacrylamide gels and transferred for 4 h or overnight to
Immobilon-P membranes (Millipore Corp.) at 100 mA. SDS samples of
adipocyte homogenates and subcellular fractions were separated on
9 × 13 × 0.1-cm 10% SDS-polyacrylamide gels and
transferred for 2 h to Immobilon-P membranes (Millipore) at 100 mA. Membranes were probed for GLUT4 with antibodies raised against the
C-terminal peptide of GLUT4 as described in Ref. 4. Immunoblots for
GLUT1 in heart and adipocytes were carried out with polyclonal
antibodies raised against the C-terminal peptide of GLUT1 (Research
Diagnostics Incorporation; catalog no. RDI-MGLUT1Cabr) following the
same protocol as described for GLUT4 in Ref. 4 except that the antibody was used at a concentration of 2.5 µg/ml. For the detection of GLUT1
in soleus and EDL, a polyclonal antibody raised against the entire
purified mouse GLUT1 (a gift from G. E. Lienhard) was used under
the same conditions as described for GLUT4 in Ref. 4 except that BSA
was used as the blocking reagent, and Tween 20 was added to the
antibody solution to a final concentration of 0.3%. Relative
expression levels were determined by analyzing different dilutions of
the samples and quantitating the signals by densitometry using
ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).
Aminopeptidase Assays--
Aliquots of plasma membrane and low
density microsomal fractions containing 5 µg of total protein in 100 µl of homogenization buffer were solubilized with the nonionic
detergent C12E9 (0.5%) by incubation for 10 min at room temperature. The aliquots were then added to 400 µl of
substrate in 120 mM NaCl, 4 mM
KH2PO4, 1 mM CaCl2, 1 mM MgSO4, 30 mM HEPES, pH 7.4, at
35 °C. The substrate used was L-lysine 
-naphthylamide
(Sigma), and it was present in the assay at a final concentration of
500 µM. The fluorescence of the liberated
-naphthylamine was recorded at excitation of 340 nm and emission of
410 nm over a 6-min incubation period at 35 °C (Hitachi Instruments
F-2500). The rates of cleavage were determined from the linear slope of
the graph.
Statistical Analysis--
For each experiment, unpaired
two-tailed t tests were used to compare the mean values for
the wild type and the IRAP+/ and IRAP/ mice. Differences were
taken to be not significant for p values greater than 0.05. Data are expressed as means ± S.E.
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RESULTS |
Growth and Development of IRAP/ Mice--
The IRAP gene was
disrupted in 129 SvEv embryonic stem cells by replacing exons 2-6 and
the intervening introns (in total 10 kb of IRAP genomic sequence) with
the neomycin resistance gene through homologous recombination with the
targeting vector (Fig. 1A).
Exons encoding domains that are crucial for the function of IRAP were
thus eliminated. Exon 2 encodes the cytoplasmic tail that confers to
IRAP its specific subcellular localization and regulation by insulin
(31) as well as the transmembrane domain. The zinc-binding site that is
encoded in exon 6 is essential for IRAP's catalytic activity (14, 32).
Mice heterozygous for the mutant allele were obtained on a mixed Black
Swiss/129SvEv genetic background. They were bred to yield IRAP/
mice together with IRAP+/+ and IRAP+/ mice. The offspring from six
heterozygous breeding pairs, a total of 280 mice (126 female and 154 male), were genotyped by Southern blotting (Fig. 1B).
IRAP+/+, IRAP+/, and IRAP/ were obtained at a ratio of 26:53:21
for the male and 22.2:52.4:25.4 for the female mice. The average litter
size was 6.4 pups. The ratios for the different genotypes reflect the expected Mendelian frequencies for the different genotypes. The lack of
IRAP thus does not affect embryonic viability.
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Fig. 1.
Targeted disruption of the IRAP gene.
A, maps of the mouse IRAP gene locus, the targeting vector,
and the mutant IRAP gene locus after homologous recombination. Exons
are shown as boxes and are numbered. The position
of the probe used for genotyping by Southern blotting and the
XbaI fragments recognized by the probe for the endogenous
(12-kb) and the mutated (8.5-kb) IRAP are marked. Maps are drawn to
scale. B, genotyping of wild type (+/+), IRAP-heterozygous
(+/), and IRAP null (/) mice by Southern blot analysis.
XbaI-digested genomic DNA was hybridized with the probe
derived from a sequence downstream of the 3' homologous recombination
region. The location of the probe is shown in A. The
XbaI fragments derived from the wild type and IRAP
recombinant alleles (indicated in A) were 12 and 8.5 kb,
respectively, as judged by comparison with size markers and are marked
with arrows on the right of the blot.
C, immunoblots of tissue homogenates from wild type (+/+)
and IRAP null (/) mice. SDS samples containing 100 µg of total
protein from each tissue were separated on a 6% SDS-polyacrylamide gel
and immunoblotted for IRAP as described in Ref. 4. Lanes
1 and 4, skeletal muscle; lanes
2 and 5, diaphragm; lanes 3 and 6, heart; lanes 7 and
9, brown adipose tissue; lanes 8 and
10, white adipose tissue from female wild type and IRAP/
mice, respectively. The position of IRAP is indicated with an
arrow on the left of the blot. The signal
above the position of IRAP at slightly higher molecular
weight in skeletal muscle and diaphragm is due to nonspecific reaction
of the antibody with a protein not related to IRAP.
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The absence of the IRAP protein was confirmed by immunoblotting tissue
homogenates derived from IRAP/ mice with an antibody against IRAP.
Immunoblots for muscle and fat tissues are shown in Fig. 1C.
No IRAP was detected in these or in any other of the tissues where IRAP
is normally expressed. An additional protein of slightly higher
molecular weight than IRAP is detected by the antibody in skeletal
muscle and diaphragm of both IRAP+/+ and IRAP/ mice (Fig.
1C). In contrast to IRAP itself, this protein is not
immunoprecipitated with the antibody used (14), and we conclude that it
is a protein that is not related to IRAP.
Subcellular fractions that were obtained from isolated adipocytes were
tested for aminopeptidase activity toward L-lysine -naphthylamide. L-Lysine -naphthylamide is a good
synthetic substrate for IRAP (14) and is not cleaved or is poorly
cleaved by the membrane aminopeptidases A and N (33). The
aminopeptidase activities toward L-lysine -naphthylamide
we measured in the plasma membrane and in the low density microsomal
membranes derived from the IRAP/ cells were 37 ± 3 and
14 ± 1%, respectively, of the activities we determined in the
corresponding fractions from wild type cells (Fig.
2). We have previously determined that the aminopeptidase activities toward the L-lysine
-naphthylamide that cannot be immunoprecipitated with the antibody
against IRAP from the plasma and low density microsomal fractions
prepared from isolated rat adipocytes were 30 ± 4 and 7 ± 4% of the total activities (n = 4 individual
preparations and immunoprecipitations), respectively
(18).3 The remaining
aminopeptidase activities in the fractions after depletion of IRAP are
thus similar to the activities remaining in each of the corresponding
fractions from IRAP/ cells. This suggests that there is no increase
in an alternative aminopeptidase that compensates for the lack of IRAP
in the IRAP/ cells. The remaining activity is most likely due to
aminopeptidase N, an enzyme that is abundant in the plasma membrane
(22).
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Fig. 2.
Aminopeptidase activities in subcellular
fractions from isolated adipocytes. Aminopeptidase activities
toward L-lysine--naphthylamide were measured in plasma
membrane (PM) and low density microsomal fractions
(LDM) as described under "Experimental Procedures." The
values obtained for each individual fraction for the IRAP/ cells
(/) were compared with the respective fractions obtained from
IRAP+/+ cells (+/+) in the preparation carried out simultaneously and
expressed as a percentage. Three separate subcellular fractionations
with 20 mice for each genotype and with basal and insulin-stimulated
cells were carried out. Two preparations were done for male mice, and
one was done for female mice. The relative values for the respective
male and female IRAP/ fractions and the respective fractions from
basal and insulin-stimulated cells were the same, and the values were
thus pooled (n = 6). The data in the graph
represent the means ± S.E.
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Growth of the offspring from the heterozygous breeding pairs was
assessed by measuring the body weights of male IRAP+/+, IRAP+/, and
IRAP/ mice at 30-day intervals from 30 to 150 days of age (Fig.
3). We found that the weights were not
significantly different between the three genotypes at any time point.
We also could not detect any differences in weights between the three
genotypes in the same mice at 1 year of age (Table
I and data not shown). From these
results, we conclude that in the absence of IRAP, postnatal growth and
weight gain during adult life is normal.
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Fig. 3.
Growth curves. Body weights for male
IRAP+/+ (rectangles) (n = 7-9),
IRAP+/ (n = 10) (reversed
triangles), and IRAP/ (triangles)
(n = 6-7) mice at 30-day intervals from 30 to 150 days
of age were measured. The means ± S.E. of the weights for each
genotype for each age were plotted. Littermates from the original
heterozygous breeding pairs were used.
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Table I
Blood glucose and plasma insulin levels for wild type (+/+) and IRAP
null (/) mice in the fed and fasted state
Measurements were made for 6-month-old male and female mice and for
12-month-old male mice. Values are displayed as mean ± S.E. Data
are from eight mice for each genotype and sex for the 6-month-old mice
and from six male mice for each genotype for the 12-month-old mice
(except insulin-fed, n=5 for each genotype). For the
measurements of blood glucose and insulin on 1-year-old mice, IRAP+/+
and IRAP/ offspring from the original heterozygous breeding pairs
were used. Blood samples were obtained from the cut tail vein between 8 and 9 a.m. for the determination of fed values and at 9-10 a.m.
after a 16-h overnight fast for the fasted values. Glucose values were
determined with the Precision-G blood glucose testing system
(MediSense). Insulin values were determined in plasma using an insulin
radioiummune assay (Linco Research Inc.).
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Eight pairs of male and female wild type and nine pairs of male and
female IRAP/ mice were bred. The ages of the mice used for breeding
were matched for the two genotypes. The IRAP+/+ pairs yielded an
average of 4.5 ± 0.3 litters with 6.5 ± 0.4 pups/litter. The average interval between the litters was 27.6 ± 2 days.
Within the same time period, the IRAP/ pairs produced an average of 4.9 ± 0.4 litters with 5.9 ± 0.4 pups/litter. The average
interval between the litters was 28.6 ± 2.3 days. The values
obtained for the IRAP/ and IRAP+/+ breeding pairs were not
significantly different. Thus, reproduction is not impaired in the
IRAP/ mice. We also found that the survival of the pups from the
IRAP/ mice was the same as for the wild type mice, indicating that
the IRAP/ mothers nursed their pups well.
Glucose Homeostasis in IRAP/ Mice--
To determine whether
glucose homeostasis was affected by the lack of IRAP, blood glucose and
plasma insulin levels were measured in fasted and randomly fed male and
female mice at 6 months of age (Table I). The values that were obtained
for the IRAP/ mice for both sexes were not significantly different
from the IRAP+/+ mice. To determine whether disturbances in glucose
homeostasis would become apparent with advanced age, fasted and fed
blood glucose and plasma insulin levels were also determined in
1-year-old male mice (Table I). As in the 6-month-old mice, no
differences were found between the IRAP/ and the wild type mice.
Next, glucose and insulin tolerance tests were performed on male and
female IRAP/ and wild type mice (Fig.
4). In the oral glucose tolerance test,
the blood glucose values for the male IRAP/ mice were slightly
higher than the ones for the control mice at each time point after the
administration of glucose (Fig. 4A). However, the values
were not statistically significantly different from the values obtained
for the wild type mice. The blood glucose values for the female mice
were identical for IRAP+/+ and IRAP/ mice (Fig. 4B). In
the insulin tolerance test, the blood glucose levels in the male
IRAP/ mice did not decline to the same degree as in the IRAP+/+
mice (Fig. 4C). Minimal glucose values obtained for
IRAP/ and IRAP+/+ mice were 76.4 ± 5 and 53 ± 3.4% of
basal levels before the injection of insulin, respectively. In the
female IRAP/ mice, the blood glucose levels at each time point were higher than in the IRAP+/+ mice, but the values were not significantly different (Fig. 4D). The results from these tests show that
the IRAP/ mice maintain normal glucose homeostasis under randomly fed conditions and handle an oral glucose challenge similar to wild
type mice. The effectiveness of a bolus of insulin to lower blood
glucose is impaired in the male but not in the female IRAP/ mice.
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Fig. 4.
Oral glucose and insulin tolerance
tests. A and B, oral glucose tolerance tests
with fasted 10-month-old male (A) and 6-month-old female
(B) wild type (rectangles) and IRAP/ mice
(triangles) (n = 8 for each genotype and
sex). 1 mg of D-glucose/g of body weight was given with a
feeding needle to mice fasted for 16 h, and glucose concentrations
were determined in blood drops obtained from the cut tail vein at the
indicated times. C and D, insulin tolerance tests
with randomly fed 8-10-month-old male (C)
(n = 15 for each genotype) and female (D)
(n = 16 for each genotype) wild type
(rectangles) and IRAP/ mice (triangles).
Insulin (Humulin R; 0.75 units/kg of body weight) was given by
intraperitoneal injection at 2 p.m. to randomly fed mice, and
blood glucose measurements were taken at the indicated times as
described under oral glucose tolerance tests. Data
points and error bars in the graphs
represent means ± S.E. Only the values in the insulin tolerance
test for the male (C) were statistically significantly
different between the IRAP/ and wild type mice (p < 0.05). The average body weights for the IRAP/ and wild type mice
used in the tests were not statistically significantly different.
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Glucose Uptake into Isolated Muscles and Adipocytes--
To
determine whether the impaired response to the glucose-lowering effect
of insulin in the IRAP/ mice could be due to a decrease in glucose
uptake into the tissues responsible for the major fraction of
insulin-mediated glucose disposal, we measured glucose transport into
isolated muscles and adipocytes. 3-O-Methylglucose uptake
into soleus and EDL was measured in the absence and presence of a
maximally stimulating dose of insulin (13 nM) as described in Ref. 28. Glucose uptakes in soleus were the same for male wild type
and IRAP/ mice under basal (0.53 ± 0.07 and 0.52 ± 0.03 µmol/g of muscle/10 min) as well as under insulin-stimulated conditions (1.22 ± 0.08 and 1.13 ± 0.13 µmol/g of
muscle/10 min) (Fig. 5A). For
the soleus isolated from female wild type and IRAP/ mice, glucose
uptake was not different under basal conditions (0.59 ± 0.07 and
0.56 ± 0.06 µmol/g of muscle/10 min). The insulin-stimulated glucose uptake, however, was decreased by 23% in the IRAP/ when compared with the wild type mice (1.25 ± 0.09 compared with
1.62 ± 0.07 µmol/g of muscle/10 min) (Fig. 5A). The
insulin-stimulated increases in glucose uptake over basal level were
2.3- and 2.2-fold for the male IRAP+/+ and IRAP/ mice,
respectively, and 2.7- and 2.2-fold for the female IRAP+/+ and
IRAP/ mice, respectively. In the EDL isolated from IRAP/ mice,
glucose uptake was decreased by 30% for both male and female when
compared with wild type muscles under basal (for male 0.40 ± 0.02 compared with 0.61 ± 0.07 µmol/g of muscle/10 min; for female
0.39 ± 0.05 compared with 0.56 ± 0.09 µmol/g of muscle/10
min) and insulin-stimulated conditions (for male 0.58 ± 0.04 compared with 0.85 ± 0.06 µmol/g of muscle/10 min; for female
0.68 ± 0.05 compared with 1.05 ± 0.08 µmol/g of muscle/10
min) (Fig. 5B). The stimulation of glucose uptake by insulin
was 1.4-fold for the male IRAP+/+ and IRAP/ mice and 1.9- and
1.8-fold for the female IRAP+/+ and IRAP/ mice, respectively.
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Fig. 5.
Glucose uptake in skeletal muscles and
adipocytes. A and B,
3-O-methylglucose uptake into isolated soleus (A)
and EDL (B) from 7-8-month-old male and female IRAP/
(/) and wild type mice (+/+) in the absence (open
bars) or presence of 13 nM insulin
(black bars). C, glucose uptake into
isolated adipocytes from 12-month-old male and female IRAP/ (/)
and wild type mice (+/+) in the absence (open
bars) or presence of 10 nM (male) and 3 nM (female) insulin (black bars).
Data in each graph represent the means ± S.E. from 3-4
experiments each carried out in triplicates (A and
B) and quadruplicates (C). An asterisk
above the bars for the IRAP/ mice indicates
that the value was statistically significantly different from the
respective value obtained for the IRAP+/+ mice (p < 0.05, except for glucose uptake into male adipocytes in the absence of
insulin, where p = 0.06). Glucose uptake assays into
adipocytes were also performed in 6-month-old male mice, and glucose
uptake was expressed per cell. The relative changes in glucose uptake
between the wild type and the IRAP/ mice were the same as for the
assays shown in the figure.
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Glucose uptake was measured into adipocytes isolated from the
epididymal and parametrial fat depots of male and female mice, respectively, in the absence and presence of maximally stimulating insulin concentrations (10 and 3 nM for male and female,
respectively) as described in Ref. 26 (Fig. 5C). The basal
glucose uptake for the adipocytes from male IRAP/ mice was
decreased by 30% when compared with wild type cells (17.5 ± 3.3 compared with 27.3 ± 2.8 fmol/min/mg of lipid). For the female
IRAP/ adipocytes, basal glucose uptake was decreased by 45%
versus wild type cells (46.3 ± 11.2 versus
84.9 ± 8.4 fmol/min/mg of lipid). The insulin-stimulated glucose
uptake into isolated adipocytes from male and female IRAP/ mice was
decreased by 52 and 57%, respectively, when compared with the glucose
uptake by wild type cells (for male 46.9 ± 8.7 compared with
103.9 ± 10.3 fmol/min/mg; for female 206.5 ± 42.9 compared
with 479.2 ± 35.6 fmol/min/mg). The -fold increases in glucose
uptake induced by insulin over basal were 3.8 and 2.9 for male
IRAP/ and IRAP+/+ mice, respectively, and 6 and 4.8 for female
IRAP/ and IRAP+/+ mice, respectively. The -fold increases were not
statistically significantly different between the two genotypes. The
insulin dose that elicited a half-maximal response (ED50)
was determined from four independent experiments in which glucose
uptake was measured at 0, 0.1, 1, and 10 nM insulin for the
epididymal adipocytes and at 0, 0.03, 0.3, and 3 nM for
parametrial adipocytes. The ED50 values were 0.11, 0.19, 0.05, and 0.06 for adipocytes from wild type and IRAP/ male and
wild type and IRAP/ female mice, respectively. The epididymal and
parametrial fat pads from which the adipocytes for the glucose uptake
assays were isolated weighed 0.44 ± 0.09 and 0.48 ± 0.1 g for male IRAP+/+ and IRAP/ mice, respectively, and
0.35 ± 0.05 and 0.44 ± 0.08 g for the female IRAP+/+
and IRAP/ mice, respectively. These weights were not statistically
significantly different. We conclude that basal as well as
insulin-stimulated glucose uptake into isolated EDL and adipocytes is
impaired in the IRAP/ mice. Glucose uptake into isolated soleus is
normal for male IRAP/ mice under basal and insulin-stimulated
conditions. It is normal under basal conditions but impaired after
insulin stimulation for female IRAP/ mice. Since the insulin dose
required for half-maximal stimulation of glucose uptake in isolated
adipocytes is the same for wild type and IRAP/ mice, we infer that
insulin sensitivity is normal in the IRAP/ mice.
Determination of Glucose Transporter Amounts in Muscle and Fat
Tissues--
To determine whether a decrease in the expression of
GLUT4 was responsible for the decreases in glucose uptake observed in EDL and adipocytes, immunoblots for GLUT4 were performed and analyzed by densitometry (Fig. 6). GLUT4 was
barely detectable in EDL from male and female IRAP/ and was
determined to be 15 ± 2 and 20 ± 2%, respectively, of wild
type levels (Fig. 6B). In isolated adipocytes, GLUT4 was
decreased to 53 ± 2 and 57 ± 5% for male and female
IRAP/ mice, respectively, when compared with wild type cells (Fig.
6C). Although glucose uptake was normal in soleus (male) or
slightly impaired (female), GLUT4 expression was decreased to 43 ± 3% in male and to 35 ± 7% in female IRAP/ mice when compared with wild type soleus (Fig. 6A). GLUT4 protein
levels were also assessed in heart. In hearts from male and female
IRAP/ mice GLUT4 was decreased to 15 ± 2 and 35 ± 6%
(Fig. 6D), respectively, when compared with wild type male
and female hearts. The decreases in GLUT4 are expressed relative to the
same amount of total protein analyzed for each tissue for the different
genotypes. Since the amounts of total protein obtained per mg of tissue
were the same for the IRAP/ mice and the wild type mice, the
relative decreases in GLUT4 reflect absolute decreases in GLUT4 amounts
in the cells. To correlate the relative decreases in GLUT4 to glucose
uptake in isolated adipocytes, we also determined the amount of total protein per mg of lipid. We found that the numbers were similar for
adipocytes isolated from IRAP/ and IRAP+/+ mice. The relative decreases of GLUT4 per the same amount of protein therefore are equivalent to the relative decreases in glucose uptake expressed per mg
of lipid. The decrease in GLUT4 expression could thus explain the
decrease in glucose uptake in IRAP/ adipocytes. However, in soleus
and EDL from IRAP/ mice, the relative level of glucose uptake was
higher than what could be expected from the relative decrease in
GLUT4.
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Fig. 6.
GLUT4 expression in muscle and adipose
tissues. A and B, quantitation of GLUT4 in
skeletal muscle homogenates, soleus (A), and EDL
(B). Homogenates containing 150, 75, 37.5, and 18.8 µg of
total protein for IRAP+/+ samples and 150 and 75 µg for IRAP/
samples were subjected to SDS-polyacrylamide gel electrophoresis and
immunoblotted with affinity-purified antibodies raised against the
C-terminal peptide of GLUT4 (4). The signals were quantitated by
densitometry, and the values obtained for the IRAP/ samples were
expressed as the percentage of the values for those IRAP+/+ samples for
which the signals in the respective dilutions were linear and in the
range of the values for the IRAP/ samples. The data in the graphs
represent the means ± S.E. from five male wild type (+/+) and
five male IRAP/ (/) mice (8 months old). The insets
show representative results for two IRAP+/+ and two IRAP/ samples
(75 µg of total protein loaded in each lane). Immunoblots performed
on muscle homogenates derived from female mice (n = 3 for each genotype) yielded similar results. C, quantitation
of GLUT4 in white adipocyte homogenates. Adipocytes were isolated from
five male 12-month-old IRAP+/+ and IRAP/ mice as described under
"Experimental Procedures." From the resulting suspensions of the
cells pooled for each genotype, SDS samples were prepared, and aliquots
containing 15 µg (2×), 7.5 µg (1×), and 3.75 µg (1/2×)
µg of total protein were immunoblotted with antibodies against GLUT4
(shown in the inset). Signals were quantitated as described
for A and B. The graph shows the
means ± S.E. from three different adipocyte preparations.
Immunoblots on two separate preparations from female mice for each
genotype yielded similar results. D, quantitation of GLUT4
in heart. Homogenates containing 50, 25, 12.5, and 6.25 µg of total
protein for IRAP+/+ samples and 50 and 25 µg for IRAP/ samples
were immunoblotted for GLUT4, and the signals were quantitated as
described for A and B. The graphs show
the means ± S.E. from six 6-8-month-old male mice for each
genotype. The inset shows a representative immunoblot for
two IRAP+/+ and IRAP/ male mice with 50 µg of total protein
loaded in each lane. Immunoblots were also performed for female mice.
The relative expression of GLUT4 in the female IRAP/ hearts was
35 ± 6% of that in female IRAP+/+ hearts (n = 3 for each genotype) (data not shown).
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In fat and muscle cells, the glucose transporter isoform GLUT1 is also
expressed. It is much less abundant than GLUT4 and comprises only about
10% of the total glucose transporters in adipocytes and skeletal
muscle (34, 35). To determine whether changes in the expression of
GLUT1 could compensate for the decreases in GLUT4, immunoblots for
GLUT1 were performed on soleus and EDL, adipocyte, and heart
homogenates (Fig. 7). No increase in the expression of GLUT1 was detected in any of these tissues when compared
with the respective tissue homogenates from wild type mice. The
relative expression levels in the IRAP/ tissues were 93 ± 9 for soleus (n = 3), 95 ± 18% for EDL
(n = 3), 105% for adipocytes (pooled from five mice),
and 105 ± 30% for heart (n = 3). This finding
rules out the possibility that GLUT1 compensates for the decrease in
GLUT4.
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Fig. 7.
GLUT1 expression in muscle and adipose
tissues. 100 µg of total protein of soleus (A) and
EDL (B), 40 µg of adipocyte (C), and 135 µg
of heart (D) homogenates were subjected to immunoblotting
with GLUT1 antibodies as described under "Experimental Procedures."
Representative results are shown for one male wild type (+/+) and one
male IRAP/ (/) sample. The ages of the mice were 8 months
(soleus and EDL), 12 months (adipocytes), and 6 months (heart).
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Subcellular Distribution of GLUT4 in Adipocytes--
To evaluate
whether the absence of IRAP led to changes in the subcellular
distribution of GLUT4, subcellular fractions were prepared from basal
and insulin-stimulated adipocytes from male and female 6-month-old
IRAP/ and IRAP+/+ mice. Plasma membranes, low density microsomal
membranes, and homogenates were immunoblotted for GLUT4. Fig.
8 shows representative immunoblots of
different dilutions of plasma and low density microsomal membranes
isolated from male IRAP/ and IRAP+/+ adipocytes. We found that
GLUT4 was decreased to 48 ± 7 and 49 ± 2%, respectively,
in the plasma membrane fraction from basal and insulin-stimulated
IRAP/ adipocytes when compared with the respective fractions from
wild type cells. In the low density microsomes, the decreases were
similar to 46 ± 6 and 50 ± 2%, respectively, in the
fractions from basal and insulin-stimulated cells when compared with
the respective fractions from wild type cells. The relative decreases
in GLUT4 in each of the fractions from the IRAP/ mice were the same
as in the homogenates prepared from the same cells. The relative level
in these was 53 ± 4%. The -fold increase of GLUT4 in the plasma
membrane in response to insulin was 2 and 1.6 for IRAP/ and IRAP+/+
mice. From these results, we conclude that in the absence of IRAP the relative subcellular distribution of GLUT4 is unchanged at steady state. The glucose uptake assays for which the results are shown in
Fig. 5C were performed with adipocytes isolated from
1-year-old mice. However, we obtained similar results for glucose
uptake measurements in adipocytes from male IRAP/ mice at 6 months of age. Thus, the relative decrease in glucose uptake in adipocytes correlates exactly with the relative decrease of GLUT4 in the plasma
membrane fraction.
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Fig. 8.
Subcellular distribution of GLUT4 in basal
and insulin-stimulated adipocytes. Adipocytes were isolated from
twenty 6-month-old male IRAP/ (/) and wild type (+/+) mice and
incubated with (10 nM) (lanes 6-10)
or without insulin (lanes 1-5). Plasma
(PM) and low density microsomal membrane (LDM)
fractions were prepared from the adipocytes by differential
centrifugation. SDS samples from each of the fractions were
immunoblotted for GLUT4. Lanes 1-3 and
8-10 show fractions derived from wild type adipocytes, and
lanes 4-7 show fractions derived from IRAP/
cells. Total protein analyzed were for plasma membrane (10 µg (1×),
5 µg (1/2×), and 2.5 µg (1/4×)) and for low density
microsomal membranes (5 µg (1×), 2.5 µg (1/2×), and 1.25 µg (1/4×)). The subcellular fractionation and subsequent
immunoblotting of the fractions for GLUT4 was repeated for another set
of male mice as well as for female mice. Similar results to the ones
shown were obtained. Immunoblots for GLUT4 were also carried out on the
homogenates from which the plasma and low density microsomes were
derived. The results were identical to the ones for the adipocyte cell
lysates shown in Fig. 6C.
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Tissue Weights--
The weights of adipose tissue and skeletal
muscles were not significantly different between the wild type and the
IRAP/ mice. However, the weights of the hearts isolated from male
IRAP/ mice were on average 20% larger than the hearts obtained
from the IRAP+/+ mice (203.7 ± 13.4 mg versus
169.9 ± 8.6 mg, p = 0.048, n = 7-8, 11-month-old mice). The body weights were identical with 42.3 ± 3.1 g for the IRAP+/+ and 42.5 ± 2.4 g for
the IRAP/ mice. The ratios of heart weights over body weights were
0.0041 ± 0.0002 and 0.0048 ± 0.0004 (p = 0.0697).
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DISCUSSION |
One of the most important results obtained in this initial
characterization of the IRAP/ mice is the finding of the
substantial reduction in GLUT4 protein in all of the tissues where
GLUT4 is predominantly expressed (36): in skeletal muscle, heart, and adipose tissue. The decreases for GLUT4 were similar in the tissues from male and female IRAP/ mice. These findings suggest that the
presence of IRAP is required for the maintenance of normal GLUT4 levels
independent of the tissue type and sex. Preliminary data that were
obtained for skeletal muscle and heart showed that the defect was
present as early as postnatal day 18 (data not shown), thus indicating
that it is also independent of the age of the mice. Reduced expression
of GLUT4 has been found in various insulin-resistant states in humans
and animals (36). In most of these, the decreased expression of GLUT4
is limited to fat. Only in a few animal models was the defect observed
in fat and muscle tissues: the Zucker diabetic fatty rat, the diabetic
(KK/Ay) mouse, the viable yellow (Avy/a) mouse,
and the obese SHR/N-cp rat (37-39). For these models evidence was
provided that the decrease in the expression of GLUT4 was a consequence
of hyperglycemia. IRAP/ mice do not have elevated blood glucose
levels, and thus hyperglycemia cannot be the cause for the decrease in
GLUT4 in the IRAP/ mice. Down-regulation of GLUT4 in skeletal
muscle, heart, and fat tissues has also been observed in
insulin-deficient states, such as fasting and streptozotocin-induced diabetes (40). The decrease in GLUT4 levels in the
streptozotocin-induced diabetic animals is due to insulin deficiency
per se. Only insulin therapy restores the expression of
GLUT4; the normalization of glycemia alone is not sufficient (41). The
molecular mechanisms by which insulin regulates GLUT4 levels in
vivo are not known. In the IRAP/ mice, insulin levels are
normal (Table I), and our data from the insulin dose response for
glucose uptake in adipocytes suggest that insulin sensitivity for the
IRAP/ cells is comparable with wild type cells. It therefore seems
unlikely that the decrease in GLUT4 levels in the IRAP/ mice is due
to an absolute or relative insulin deficiency.
Common to insulin-deficient animals and the IRAP/ mice, however, is
the lack of IRAP function at the cell surface. It would be expected
that in the former insulin-triggered translocation of IRAP to the cell
surface is severely diminished, and in the latter IRAP is lacking
entirely. We have previously shown that insulin increases the
processing of extracellular substrates concomitant with the
translocation of IRAP to the cell surface (18). Thus, when IRAP is
absent from the cell surface extracellular peptide hormones that are
substrates for IRAP may not be processed efficiently. We thus speculate
that the altered action of a peptide hormone that is a substrate for
IRAP or is under the control of a substrate for IRAP leads to the
down-regulation of GLUT4. We presently do not know what the in
vivo substrate for IRAP is, but we have previously shown that IRAP
cleaves the N-terminal amino acid from vasopressin, Lys-bradykinin,
angiotensin III, and angiotensin IV in vitro (18). The
removal of the N-terminal amino acid inactivates vasopressin (42) and
converts angiotensin III and angiotensin IV into products with
different receptor specificity (43). There is evidence that
angiotensins may be involved in the control of GLUT4 levels. An
angiotensin type-1 receptor antagonist prevents the decrease in GLUT4
levels in the hearts of streptozotocin-diabetic rats (44). Both
angiotensin II and angiotensin III bind to the angiotensin-1 receptor
and exert similar effects through the interaction (45). It is possible
that in the IRAP/ mice the half-life of angiotensin III is
increased and its prolonged action may lead to the down-regulation of GLUT4.
Another mechanism that could be responsible for the decrease in GLUT4
is that the absence of IRAP leads to a trafficking defect and
mistargeting of GLUT4 and subsequently to increased degradation of
GLUT4. This defect would most likely be due to the physical absence of
IRAP and would be independent of its catalytic activity. The specific
intracellular compartment where IRAP and GLUT4 predominantly reside has
not been established. One model is that they are in specialized
secretory vesicles that fuse with the cell surface when cells are
stimulated with insulin (46). Another model is that they are in the
endosomal recycling compartment from which they are sorted into
vesicles going to the plasma membrane upon stimulation with insulin
(46). The sorting mechanism that has to occur in either of the models
is unknown. Also, the molecular mechanisms responsible for the
sequestration of IRAP and GLUT4 in the intracellular compartment and
the insulin-triggered translocation to the cell surface are unknown.
The cytoplasmic domain of IRAP contains several potential trafficking
motifs (two dileucine motifs and two clusters of acidic amino acids)
similar to those found in the C-terminal cytoplasmic tail of GLUT4
(14). For IRAP, we have been able to show that the cytoplasmic tail
carries all of the information for its specific intracellular
localization and insulin-regulated trafficking (31). Extensive studies
with the three major cytoplasmic domains in GLUT4 (the N terminus, the
cytoplasmic loop between membrane-spanning domains 6 and 7, and the C
terminus) have identified roles for each of these in GLUT4 trafficking
(47). Injection of either a GST fusion protein with the cytoplasmic
tail of IRAP or a peptide derived from the C-terminal end of GLUT4
triggers the redistribution of both IRAP and GLUT4 to the cell surface
(48, 49). These findings suggest that the same mechanism may be
responsible for their sequestration within the cells under basal
conditions. However, the protein(s) interacting with the cytoplasmic
domains of IRAP and/or GLUT4 have not been identified. A strong
physical interaction between IRAP and GLUT4 has been ruled out; no IRAP
is found in immunoprecipitates derived from detergent cell lysates
using an antibody against GLUT4 and vice versa (4). Our analysis of
subcellular fractions obtained from isolated adipocytes from IRAP/
and IRAP+/+ mice does not support a role for IRAP in the sequestration
and insulin-stimulated trafficking of GLUT4. Under steady state
conditions in basal and insulin-treated cells, the relative
distribution of GLUT4 between the plasma membrane and the low density
microsomes is the same in the IRAP/ and IRAP+/+ adipocytes. The
subcellular fractionation method we used in this study does not
differentiate between specific compartments within the low density
microsomes. However, we find that in the IRAP/ adipocytes, GLUT4 is
targeted to an intracellular compartment from which it can be recruited
to the cell surface by insulin to the same extent as in wild type
cells. The finding of the normal relative subcellular distribution of
GLUT4 in the IRAP/ adipocytes contrasts with the finding for the
subcellular distribution for IRAP described in GLUT4 / adipocytes.
In the latter, IRAP is redistributed to the plasma membrane under basal conditions, and there is no further increase in IRAP in this fraction in response to insulin (50).
Yet a third explanation for the decrease in GLUT4 in the absence of
IRAP is that GLUT4 and IRAP may be routed through a specific common
degradation pathway. At present, we do not know what the mechanisms are
that guide the degradation of either GLUT4 or IRAP. We have shown that
IRAP and GLUT4 are approximately equally abundant in adipocytes (1),
and it is therefore conceivable that in the absence of IRAP GLUT4 would
be targeted for degradation more efficiently because of a lack of
competition. In this instance, it would be expected that in the absence
of GLUT4, IRAP levels would be decreased. A recent study reports that
IRAP is indeed decreased by 40 and 60% in skeletal muscle and heart,
respectively, in GLUT4/ mice (50).
Transgenic mouse models with decreased or lack of expression of GLUT4
in muscle and/or adipose tissues have confirmed the importance of GLUT4
in the maintenance of glucose homeostasis (51-54). The mice with GLUT4
l
§,
TOP
ABSTRACT
INTRODUCTION
