1 Department of Biochemistry, Kyorin University School of Medicine, Mitaka,
Tokyo 181-8611, Japan
2 Department of Internal Medicine (III), Kyorin University School of Medicine,
Mitaka, Tokyo 181-8611, Japan
* Author for correspondence (e-mail: shinya{at}kyorin-u.ac.jp)
Accepted 18 September 2002
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Summary |
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Key words: Gene therapy, Type 2 diabetes, Insulin, Adipocyte, Adenovirus, Glucose transporter
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INTRODUCTION |
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Materials and Methods |
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Cell culture and adenovirus infection
3T3L1 adipocytes were differentiated by incubation in medium containing 10%
fetal bovine serum (FBS), 1.7 µM insulin, 0.5 mM 3-isobutyl-1-methylxantine
(IBMX) and 1 mM dexamethasone. Differentiated adipocytes were used for the
experiments 10 days after initiation of differentiation. For introducing the
recombinant adenovirus, differentiated adipocytes were infected by the
indicated adenovirus at multiples of infection (MOI) of 20 plaque forming
units (pfu)/cell. 2 days later experiments were performed.
Immunocytochemistry
For immunofluorescence studies of 3T3L1 adipocytes, 3T3L1 cells were
differentiated to adipocytes on glass chamber slides (eight wells: Lab-Tek
slides, Nunc) and infected with the indicated adenovirus. Two days later cells
were fixed with 2% paraformaldehyde, then immunostained with mouse monoclonal
anti-insulin antibody (Sigma) and/or rabbit polyclonal anti-GLUT4 antibody
and/or anti-GLUT1 antibody (a gift from K. Takata, Gunma University, Maebashi,
Japan), using appropriate second antibodies as described previously (Nagamatsu
et al., 2001b). Slides were examined using a Carl Zeiss LSM510 laser-scanning
confocal microscopy (Carl Zeiss, Co. Ltd., Jene, Germany) at the excitation
wavelength of 488 nm for GFP and 543 nm for rhodamine using a band pass filter
so as not to overlap the emitted light from GFP and rhodamine as described
previously (Ohara-Imaizumi et al.,
2002).
Time-lapse confocal microscopy
3T3L1 adipocytes were cultured and infected with Adex1CA pchi-GFP on the
glass chamber slide for imaging with confocal microscopy. Prior to imaging,
cells were incubated for 1 hour in Krebs-Ringer buffer (KRB) containing 110 mM
NaCl, 4.4 mM KCl, 1.45 mM KH2PO4, 1.2 mM
MgCl2, 2.3 mM calcium gluconate, 4.8 mM NaHCO3, 11 mM
glucose, 10 mM HEPES (pH 7.4) and 0.3% bovine serum albumine (BSA) without
insulin, then transferred to the thermostat-controlled (37°C) stage of
confocal microscopy. The cells were, then, stimulated by adding
10-7 M bovine insulin. A time-course of the change of GFP-labeled
vesicles was obtained from time-lapse images every 1 minute at the excitation
wavelength of 488 nm during stimulation. The data were analyzed by Metamorph
software (Nippon Roper Co. Ltd.).
Proinsulin release
After 3T3L1 adipocytes expressing human insulin gene were preincubated for
1hour in KRB buffer containing 11 mM glucose without insulin, cells were
washed several times, and they were challenged by a various concentration of
bovine insulin for 1 hour in a 500 µl of KRBG buffer. At the end of
incubation, supernatants were collected, and proinsulin in the medium was
measured by proinsulin ELISA assay kit using the human proinsulin standard
(DAKO, Denmark), which does not react with bovine insulin at all.
Metabolic labeling and immunoprecipitation
Two days after 3T3L1 differentiated adipocytes were infected with
Adex1CA-pchi at an MOI of 20 pfu/cell, cells were pulse-labeled in methionine
(Met)-cysteine (Cys)-free RPMI 1640 medium containing 10% (vol/vol) dialyzed
FBS with 400 µCi of 35S-Met/Cys (New England Nuclear-Dupont,
Boston, MA) for 1 hour at 37°C. After washing out the labeling medium
several times, cells were chased for 1 hour. Labeled cells were disrupted by
sonication in a lysis buffer (0.1 M Tris-HCl, 0.05 M NaCl, 0.25% (w/v) BSA,
0.1% (v/v) Triton X-100, pH 7.5) containing a cocktail of protease inhibitors
(1 mM phenylmethylsulfonyl fluoride, 50 µg/ml trasylol, 10 µg/ml
leupeptin, and 5 µg/ml pepstatin A), and cell lysates were
immunoprecipitated with a guinea pig anti-insulin antibody (DAKO) by using
protein A sepharose beads (Pierce), as described previously
(Nagamatsu et al., 1999). The
immunoprecipitates were treated with acetic acid, then neutralized and
separated on a superdex peptide column (Amersham Pharmacia Biotech,
Buckinghamshire, UK) equipped with an HPLC (Beckman instruments, Inc.,
Fullerton, CA), and radioactivity of each fraction was measured as described
previously (Nagamatsu et al.,
1999
).
HPLC and insulin measurement
For detecting only mature insulin, we used the reverse phase HPLC, a model
obtained from Beckman (System Gold), connected to a TSK-GEL ODS-80TM
column (length 15.0 cm, internal diameter 4.6 mm) (TOSOH Co. Ltd., Kanagawa,
Japan). Ultraviolet absorption was monitored at 210 nm using a Power Chrom
(Eicom Co. Ltd., Kyoto, Japan) connected to a Macintosh computer. The two
buffers used were as follows: Buffer A, 0.05% trifluoroacetic acid (TFA) and
10% (v/v) acetonitrile. Buffer B, 0.05% TFA and 80% (v/v) acetonitrile. The
flow rate was 1 ml/minute, and 1 minute fractions were collected. The % of B
was increased linearly from 10% to 33% for the first 15 minutes, then from 33
to 38% for 15 minutes, finally from 38% to 80% for 30 minutes. The HPLC
fraction was neutralized and diluted by insulin assay buffer, and insulin was
measured by insulin ELISA kit using a human insulin standard (Molecular
Biology Laboratories, Co. Ltd., Nagoya, Japan). Recombinant human insulin and
bovine insulin (Sigma) as the standard were dissolved in 0.01 N acetic acid
containing 0.05% BSA.
Glucose uptake assay
Two days after 3T3L1 adipocytes were infected with Adex 1CA-pchi, cells
were placed in Hanks' solution (0.02 g/dl BSA, 136.9 mM NaCl, 5.6 mM KCl, 0.34
mM Na2HPO4-7H2O, 0.44 mM
KH2PO4, 1.27 mM CaCl2 and 4.20 mM
NaHCO3, 0.1 mM 2-deoxy-D-glucose, pH 7.4) for 1 hour prior to
glucose uptake experiments. The assay was essentially performed as described
previously (Nagamatsu and Steiner,
1992). Briefly, after 1 hour of preincubation, cells were washed
several times with Hanks' solution, and treated with different concentrations
of insulin for 30 minutes at 37°C, and then 0.5 µCi of
[14C]-2-deoxy-D-glucose (NEN Life Science Products Inc., Boston,
MA) was added to the medium containing 0.1 mM unlabeled 2-deoxy-D-glucose.
After 20 minutes of incubation at room temperature, cells were washed several
times with ice-cold phosphate buffered saline (PBS), solubilized by the
addition of 0.2 M NaOH, and the radioactivity was counted using a liquid
scintillation counter.
Statistical analysis
Results are means±s.e.m. from at least three different experiments
performed independently unless stated otherwise. Statistical analysis was
performed by ANOVA followed by Fisher's test and regression analysis using the
Statview software (Abacus Concepts, Inc., Berkeley, CA).
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Results |
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Secretion of proinsulin from 3T3L1 adipocytes
If (pro)insulin produced in adipocytes is to be included in GLUT4 vesicles,
then the peptide would be released in parallel with the exocytosis of the
GLUT4 vesicle and in response to exogenously administered insulin. It would
need to use the mechanism by which insulin receptor signaling causes movement
of the GLUT4 vesicle from the intracellular pool to the plasma membrane.
Therefore, we examined whether adipocyte (pro)insulin is released in response
to exogenously administered insulin. We measured the proinsulin release from
these cells by adding exogenous bovine insulin from 10-9 M to
10-7 M. Since we cannot measure the human insulin release from
these cells by insulin ELISA assay system, which crossreacts with bovine
insulin in the medium, we measured human proinsulin in the medium using a
human intact proinsulin ELISA assay system, which does not react with
10-7 M bovine insulin (data not shown). As shown in
Fig. 3, proinsulin release was
increased in response to exogenously administered insulin (no insulin,
5.1±1.8; 10-9 M insulin, 6.7±2.9; 10-8 M
insulin 22.4±6.2; 10-7 M insulin 29.5±7.2
pg/ml/105 cells).
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Processing of proinsulin and the content of proinsulin and mature
insulin in 3T3L1 adipocytes
We next examined whether the produced peptide derived from preproinsulin
mRNA in 3T3L1 adipocytes can be processed into mature insulin. For this
purpose, 3T3L1 adipocytes were labeled with 35S-Met/cys for 1 hour
and chased for 1 hour, then immunoprecipitated materials of cell lysates with
an anti-insulin antibody were analyzed by the peptide column.
Fig. 4A shows the
column-profile of the material, demonstrating that at least a proportion of
the proinsulin can be converted to mature insulin even in 3T3L1 adipocytes [%
radiolabeled insulin in the cell=18.3±4.3% (n=3)].
Proinsulin-converting enzymes, prohormone convertases PC2 and PC3, are known
to be expressed in most endocrine and neuroendocrine cells
(Halban and Irminger, 1994;
Neerman-Arbez et al., 1994
),
while furin is expressed ubiquitously
(Hatsuzawa et al., 1990
;
Hosaka et al., 1991
). Although
PC enzymes are not expressed in adipocytes, we actually detected the presence
of furin mRNA in adipocytes by RT-PCR analysis (data not shown). Human
proinsulin does not have the exact consensus sequence recognized by furin
(Yanagita et al., 1993
);
however, furin is still able to convert proinsulin to insulin
(Vollenweider et al., 1995
).
Therefore, it is conceivable that proinsulin in 3T3L1 adipocytes was processed
to insulin by furin, although its efficiency for proinsulin conversion must be
low.
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We then tried to determine the actual content of proinsulin and insulin in
3T3L1 adipocytes. 3T3L1 adipocytes were disrupted by sonication, and
proinsulin content was measured using a proinsulin ELISA assay. The proinsulin
content was 512±91 pg/105 cells (n=10). In order to
quantify the mature insulin content, we used the reverse-phase HPLC system. In
our system, recombinant human insulin was eluted at 21 minutes, as the % B was
linearly increased from 33% to 38% (Fig.
4B). The fraction sample of 3T3L1 adipocyte lysates by HPLC system
was measured by insulin ELISA assay, and
Fig. 4C shows the mature
insulin peak eluted from retention time 21 minutes, which was calculated to be
36±11.2 pg per 105 cells (n=10). Thus, the molar
ratio of proinsulin versus insulin in the cell was about 14:1. As these cells
secrete approximately 30 pg proinsulin into 1 ml culture medium per
105 cells in response to 10-7 M bovine
insulin-stimulation as shown in Fig.
3, it is assumed that roughly 23 pg insulin is simultaneously
released in response to exogenously administered insulin.
Time-lapse images of insulin-induced (pro)insulin release from 3T3L1
adipocytes
In order to directly observe the (pro)insulin release from 3T3L1
adipocytes, we monitored the change in number of GFP-labeled vesicles
containing (pro)insulin using time-lapse confocal imaging. 3T3L1 adipocytes
infected with Adex1CA pchi-GFP were stimulated by the addition 10-7
M insulin. The number of GFP-labeled vesicles in the confocal image of 1
minute post-insulin stimulation was decreased to approximately 64% of that
counted in pre-stimulation. (Prestimulation; 100%; 1 minute post stimulation,
64±18%; 2 minutes post stimulation; 47±23%; 3 minutes post
stimulation, 68±11%; 6 minutes post-stimulation, 71±25%)
(Fig. 5B). The decline of the
number of GFP-labeled vesicles by insulin stimulation was not due to the
photobleaching, because the number of vesicles without insulin stimulation
showed no change during the same time course [0 minutes, 100%; 1 minutes,
84±19%; 2 minutes, 94±11% (n=3 cells)]. Thus, the data
indicate that (pro)insulin is exocytosed quickly, at least within 1 minute,
from 3T3L1 adipocytes expressing the human insulin gene.
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Glucose uptake by 3T3L1 adipocytes
Finally, we tested our hypothesis that insulin released from 3T3L1
adipocytes amplifies the insulin action through the endogenous insulin
receptor in an autocrine manner by measuring insulin-stimulated
14C-2-deoxy-D-glucose uptake by 3T3L1 adipocytes expressing the
human insulin gene. As shown in Fig.
6, these adipocytes led to a progressive increase in glucose
uptake in a dose-dependent manner of exogenously administered insulin compared
with that by control cells. Glucose uptake by 3T3L1 adipocytes expressing the
insulin gene was increased to approximately two-fold that of control cells
under 10-8 M insulin (10-8 M insulin; 21,900±1530
versus 12,100±1020 cpm/dish, P<0.001), and the
dosedependent curve was shifted to the left (no statistical difference under
10-7 M insulin); however, only a little difference in glucose
uptake was observed under the absence and presence of exogenous
10-9 M insulin between insulin expressing and control cells. Thus,
it appears that insulin released from 3T3L1 adipocytes augmented the
insulin-stimulated glucose uptake by exogenously administered insulin in an
autocrine manner.
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Discussion |
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In the present study, we first presented evidence that (pro)insulin
expressed in 3T3-L1 adipocytes is targeted to GLUT4 vesicles. The signal
peptide of preproinsulin seems to be important for its delivery to GLUT4
vesicle in 3T3L1 adipocytes, because the addition of GFP to the N-terminus of
preproinsulin disturbed the sorting of the peptide to the GLUT4 vesicle (data
not shown). Although the signal peptide of preproinsulin plays a crucial role
in sorting the peptide from rough endoplasmic reticulum (RER) to the insulin
secretory granules in pancreatic ß cells
(Rhodes, 2000), there is no
report showing the role of preproinsulin signal peptide in adipocytes. Our
data indicated that the signal peptide of preproinsulin has a functional role
in delivering the peptide to GLUT4 vesicle in adipocytes. Since GLUT4 vesicle
is exocytosed by insulin (Oatey et al.,
1997
; Czech and Corvera,
1999
), insulin action on adipocytes may be amplified by the
insulin released in parallel with GLUT4 vesicle exocytosis from adipocytes in
an autocrine manner (Fig. 1).
This autocrine release is probably able to improve the insulin resistance in
diabetic adipocytes via a local exposure to progressively increasing
concentrations of insulin. Indeed, we have reported that blood glucose levels
in diabetic KKAy mice and Zucker fa/fa rats, which have
insulin resistance in insulin target tissues
(Chang et al., 1986
;
King et al., 1992
), were
decreased by introducing human insulin gene into adipose tissues
(Nagamatsu et al., 2001a
).
As (pro)insulin is stored in GLUT4 vesicles, as shown in
Fig. 2A, it is conceivable
that, in adipocytes, exogenously administered insulin stimulates adipocyte
(pro)insulin release via the regulated vesicular transport system. Therefore,
we have examined the dose dependency of insulin-induced proinsulin release
from 3T3L1 adipocytes. Although it is the best way to directly measure human
insulin in the medium, we could not measure mature insulin released from
adipocytes into the medium by the standard immunoreactive insulin assay
system, because it detects not only human insulin but also bovine insulin, a
large amount of which was exogenously administered to the medium. Therefore,
we measured human proinsulin released in the medium, using the intact human
proinsulin assay system. It is of interest that proinsulin release was
increased in a dose-dependent manner with the increase in exogenously
administered insulin, suggesting the successful construction of the
insulin-regulated (pro)insulin release system in adipocytes. Furthermore,
confocal images have clearly shown that the total number of the vesicle
containing insulin-GFP was decreased by the addition of 10-7 M
insulin, confirming the insulin-induced release of (pro)insulin from 3T3L1
adipocytes Thus, we thought that adipocyte (pro)insulin secreted in parallel
with GLUT4 vesicle exocytosis might amplify the local insulin action. Indeed,
glucose uptake by these cells was almost increased to a maximum response by
10-8 M of exogenously administered insulin, showing that
insulin-dose-dependent curve for the glucose uptake was shifted to the left
(Fig. 6). Of course, it cannot
be ruled out that preproinsulin expressed in adipocytes may be sorted to
vesicles other than GLUT4 vesicles. Indeed, it is reported that adipsin, a
serine protease, and adipocyte complement related protein of 30 kDa (ACRP30)
are secreted by insulin stimulation
(Robinson et al., 1992;
Scherer et al., 1995
;
Yang and Mueckler, 1999
;
Bogan and Lodish, 1999
). As
Bogan and Lodish (Bogan and Lodish,
1999
) showed, insulin stimulates exocytosis of a regulated
secretory compartment containing ACRP30 in 3T3L1 adipocytes, and some of the
preproinsulin expressed in 3T3L1 adipocyte may be delivered to these vesicles.
However, it is emphasized that, importantly, in any case, insulin stimulates
proinsulin exocytosis in a regulated manner in our system.
The adipocyte insulin but not proinsulin must be released to enhance the
insulin-stimulated glucose uptake by these cells, because mature insulin has a
potent biological activity (Blundell et
al., 1978; Renscheidt et al.,
1984
). We have attempted to directly measure the mature insulin in
the medium using HPLC system; however, it was extremely difficult because it
is only present in small quantities. Therefore, we estimated the actual amount
of adipocyte insulin in the medium by calculation. The amount of human
proinsulin released by the addition of 10-8 M bovine insulin was
approximately 22 pg/ml, as shown in the results, so that the actual insulin
amount in the medium is assumed to be 1
2 pg/ml (approximately 0.2 pM), on
the basis of the data of proinsulin/insulin molar ratio (about 14:1) in the
cell, which was derived from HPLC results. On the other hand, this
concentration of adipose insulin in the medium, less than at least 1 pM, is
not enough to evoke the insulin action on the insulin target tissues in
general (Ellis et al., 1986
).
If so, how could insulin released from these cells enhance the insulin action
of exogenous administered insulin in an autocrine manner? We assume that the
local concentration of adipose insulin around the insulin receptor may be
extremely high, just when the vesicles containing insulin are fused to the
plasma membrane, which may be able to increase exogenous insulin action. Of
course, many mechanisms other than autocrine loop proposed cannot be ruled
out; overexpression or release of insulin in 3T3L1 adipocytes may affect the
release of leptin, or resistin, which may affect the adipocyte metabolism
(Unger et al., 1999
;
Steppan et al., 2001
).
In conclusion, we have succeeded in constructing the regulated insulin secretory pathway within adipocytes. This is a first report of a successful construction of an insulin regulated insulin secretory pathway in non-ß cells, and this method could provide potential clues for efficient gene therapy for type 2 diabetes, although many studies are required for the practical application to humans.
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Acknowledgments |
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