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Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California 92093
Address all correspondence and requests for reprints to: Jerrold M. Olefsky, Department of Medicine (0673), University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0673. E-mail: jolefsky{at}ucsd.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is known that hyperosmolarity has potent insulin-like properties on glucose metabolism, including activation of glucose transport in adipocytes and skeletal muscle (4, 5). Chen et al. (6) have shown that osmotic shock-stimulated GLUT4 translocation and glucose transport activity occur via a novel tyrosine kinase-dependent pathway. They have further shown that osmotic shock pretreatment of 3T3-L1 adipocytes induces resistance to insulin-stimulated GLUT4 translocation and glucose transport (7). Although insulin and osmotic shock use distinct signaling pathways to activate GLUT4 translocation and glucose transport, it is clear that the exposure of cells to osmotic shock attenuates the function of molecules required for insulin signal transduction. Specifically, osmotic shock pretreatment markedly inhibited insulin stimulation of Akt/PKB and p70S6 kinase activities; however, the precise mechanisms for these effects are unknown. Given the molecular cross-talk between the two pathways suggested by these data (7), we hypothesized that chronic insulin exposure would conversely induce cellular resistance to osmotic shock-induced glucose transport and GLUT4 translocation.
In the present work, we provide evidence that prolonged insulin treatment of 3T3-L1 adipocytes decreases both insulin- and osmotic shock-induced glucose transport and GLUT4 translocation. Furthermore, it inhibits Gab-1 tyrosine phosphorylation and Gab-1 associated PI 3-kinase activity, events that are necessary for stimulation of glucose transport (8).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-N-SH3 antibodies,
wortmannin, and recombinant protein A-agarose were purchased from
Upstate Biotechnology, Inc. (Lake Placid, NY). The GLUT4
antibody was obtained from Chemicon (Temecula, CA). Mouse monoclonal
antiphosphotyrosine (PY-20) was from Transduction Laboratories, Inc. (San Diego, CA). Fluorescein isothiocyanate (FITC)-,
tetramethyl-rodamine isothiocyanate (TRITC)-and aminomethylcoumarin
acetate-conjugated antimouse were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Horseradish
peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All radioisotopes were obtained from NEN Life Science Products (Boston, MA). Enhanced chemiluminescence reagent was obtained from
Pierce Chemical Co. (Rockford, IL).
Cell culture
3T3-L1 fibroblasts were maintained in DMEM, high glucose,
containing 10% calf serum. Postconfluency fibroblasts were
differentiated into adipocytes by changing the medium with DMEM, high
glucose, containing 10% FCS, 1 µg/ml insulin, 0.1 µg/ml
dexamethasone, and 112 µg/ml isobutylmethylxanthine. The medium was
removed after 2 days and replaced with DMEM, low glucose, containing
10% FCS, Glutamax, and 1% penicillin-streptomycin. Three to 7 days
after the addition of the differentiation mix, the cells were plated in
6- or 12-well dishes at densities of 8 x
105 and 4 x 105,
respectively. The medium was changed every second day until use, 10–12
days post differentiation. Approximately 90% of the cells exhibited
large lipid droplets indicative of adipocytes. Twenty-four hours before
the start of all experiments, cells were given fresh DMEM low glucose
media. In the case of the chronic insulin treatment, fully
differentiated cells were incubated with 10 nM insulin for
10 h in DMEM, low glucose media. After 10 h, cells were
rinsed and placed in a Krebs-Ringer phosphate buffer (KRP) containing
0.1% BSA for 30 min at 37 C before being stimulated. This protocol was
used in all experiments.
2-Deoxyglucose uptake in 3T3-L1 adipocytes
Chronically insulin-treated and control 3T3-L1 adipocytes were
treated as described above, followed by stimulation with 16.6
nM insulin or 600 mM sorbitol for 20 min at 37
C. Glucose transport was determined by the addition of 0.1
mM 2-deoxyglucose containing 0.2µCi of
2-[3H] deoxyglucose as described previously
(9). Nonspecific uptake was assessed using 0.1
mM L-glucose containing 0.2 µCi of
L-[3H]glucose. The reaction was
stopped after 10 min by aspiration, and extraneous glucose was removed
by four washes with ice-cold PBS. Cells were lysed in 1 N
NaOH, and glucose uptake was assessed by scintillation counting.
Samples were normalized for protein content by Bradford protein
assay.
Subcellular fractionation of 3T3-L1 adipocytes and GLUT4
immunodetection
Cells from one 10-cm dish were incubated with 16.6
nM insulin or 600 mM sorbitol for 20 min at 37
C, and then washed three times with ice-cold PBS. Cells were scraped
into ice-cold HES buffer (255 mM sucrose, 20 mM
HEPES, 1 mM EDTA, pH 7.4) supplemented with protease
inhibitors (1 µg/ml leupeptin, 1 µg/ml pepstatin, and 200
µM PMSF). Cells were then homogenized using an LSC
homogenizer. Subcellular fractionation was carried out as described
previously (10).
Proteins from different fractions were were solubilized in Laemmli sample buffer and separated by SDS-PAGE with a 10% resolving gel. The proteins were then transferred to PVDF membrane and blotted overnight at 4 C with GLUT4 antibodies. After incubation with a secondary horseradish peroxidase-conjugated goat antirabbit antibody, the proteins were visualized by enhanced chemiluminescence, autoradiography, and densitometric quantitation.
Assessment of Gab-1 tyrosine phosporylation and association with
p85
Adipocytes plated in six-well dishes were stimulated at 37 C
with 16.6 nM insulin or 600 mM sorbitol for 20
min. Cells were lysed at 4 C in a lysis buffer containing 50
mM HEPES, 150 mM NaCl, 1% Triton X-100, 4
mM sodium orthovanadate, 20 mM sodium
pyrophosphate, 200 mM sodium fluoride, 0.5 M
EDTA, 80% glycerol, pH 7.4. Lysates were centrifuged at 14,000 x
g for 10 min at 4 C. Supernatants were incubated with Gab-1
antibody and recombinant protein A-agarose overnight at 4 C. Pellets
were washed three times in lysis buffer. Laemmli‘s buffer was added to
the pellets and boiled for 5 min. Samples were separated by SDS-PAGE on
7.5% polyacrylamide gels. Proteins were transferred to PVDF membrane
and blotted with PY20, p85, and Gab-1 antibodies according to the
manufacturer‘s instructions. Following incubation with horseradish
peroxidase-conjugated secondary antibodies, proteins were visualized by
enhanced chemiluminescence, autoradiography, and densitometric
quantitation.
Measurement of PI 3-kinase activity
3T3-L1 adipocytes were stimulated as described in the figure
legends and then lysed as described above. Supernatants were incubated
with Gab-1 antibody and recombinant protein A-agarose overnight at 4 C.
Bead pellets were washed three times with Buffer A (Tris-buffered
saline, pH 7.4, 1% Nonident P-40, and 100 µM
Na3VO4), three times with
Buffer B (100 mM Tris, pH 7.4, 500 mM
LiCl2, and 100 µM
Na3 VO4), and twice with
Buffer C (10 mM Tris, pH 7.4, 100 mM NaCl, 1
mM EDTA and 100 µM
Na3VO4). Pellets were
resuspended in Buffer C without the
Na3VO4. As described
previously (11), PI 3-kinase activity was assessed by the
phosphorylation of phosphatidylinositol in the presence of 20µCi of
[
-32P]ATP for 10 min. The reactions were
stopped with 20 µl of 8 N HCl and 160 µl of
CHCl3:methanol (1:1) and centrifuged. The lower
organic phase was removed and applied to potassium oxalate (1%)-
coated silica gel TLC plates. Following the separation of lipids by TLC
using the borate-buffered system (12),
phosphatidylinositol 3-phosphate was visualized by autoradiography.
National Institutes of Health Image scanning software was used for
quantitation.
Actin localization
As done previously (8, 9), 3T3-L1 adipocytes were
stimulated with 16.6 nM insulin or 600 mM
sorbitol for 10 min and fixed with 3.7% formaldehyde in PBS for 10 min
at room temperature. Cells were permeabilized in 0.2% Triton X-100 for
5 min, washed in PBS, and incubated at room temperature for 45 min with
TRITC-phalloidin (0.5 µg/ml) in PBS together with FITC-antimouse
antibodies (1:100) for 45 min. After staining, coverslips were washed
successively in PBS and deionized water for 15 min and mounted with
Gelvatol. Cytoskeletal changes were quantitated blindly, counting at
least 100 cells in random fields. Cells that showed actin staining at
the periphery were scored as positive for membrane ruffles. The
percentage of total counted cells displaying membrane ruffles is
represented as ruffling index. The cells were inspected and
photographed with a Carl Zeiss (New York, NY) Axiophot
fluorescence microscope.
Statistical analysis
Values are expressed as means ± SEM. Results
were analyzed by using the Student’s t test. A value of
P < 0.05 was considered significant.
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Results |
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). Consistent with
previous studies (6, 7), osmotic shock, induced by
sorbitol treatment, resulted in a significant increase in
2-deoxyglucose uptake. Sorbitol treatment was not as effective as
insulin but led to a 4-fold increase in glucose transport,
approximately 60% the effect of insulin (Fig. 1B, open
bars). In contrast, as seen in Fig. 1B (solid bars)
after 10 h of chronic insulin treatment, osmotic shock and acute
insulin stimulation of glucose transport were markedly reduced. In
other words, the cells had become resistant to both osmotic shock and
insulin. We observed a tendency for the basal glucose transport to be
higher in resistant cells compared with control cells; however, this
difference did not reach statistical significance.
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). After insulin
stimulation of control cells, the amount of GLUT4 in the LDM fraction
decreased by 58%, and increased 7.8-fold in the PM fraction. Osmotic
shock led to a 43% decrease in LDM GLUT4 and 2.2-fold increase in the
PM GLUT4. In contrast, resistant cells show no significant effect of
acute insulin or osmotic shock stimulation on GLUT4 localization to
LDMs or PMs, compared with basal localization.
Osmotic shock-induced Gab-1 phosphorylation is decreased in cells
chronically treated with insulin
We have previously shown that osmotic shock leads to Gab-1
tyrosine phosphorylation with PI 3-kinase association and that Gab-1 is
a key component of the shock-induced glucose transport signaling
pathway (8). To determine whether Gab-1 phosphorylation
following osmotic shock stimulation is altered in chronically
insulin-treated cells, lysates from insulin- or sorbitol-stimulated
cells were immunoprecipitated with anti-Gab-1 antibody and
immunoblotted with antiphosphotyrosine antibody. In control,
nonresistant cells, an 8- to 10-fold increase in tyrosine
phosphorylated Gab-1 was observed upon sorbitol stimulation compared
with a 6-fold increase with insulin stimulation (Fig. 2, upper panel). In resistant
cells, the phosphorylation of Gab-1 was markedly decreased in response
to either osmotic shock or acute insulin stimulation. Changes in Gab-1
phosphorylation, were not due to change in the total amount of the
Gab-1 protein, as detected by immunoblotting (Fig. 2, lower
panel).
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, in control cells, sorbitol treatment
led to a 10-fold increase in the association of Gab-1 with the p85
subunit of PI 3-kinase in contrast to a 4-fold increase upon insulin
stimulation. In insulin resistant cells, this association was decreased
by 54% upon sorbitol stimulation, and by 64% (mean ±
SEM of three experiments) in response to an acute insulin
stimulation. The levels of Gab-1 protein in these immunoprecipitates
were similar (data not shown).
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, lane 4),
compared with an 7.8-fold increase upon sorbitol stimulation (Fig. 4B, lane 4). As expected, addition of 100 nM wortmannin to the
reaction mixture completely inhibited Gab-1-associated PI 3-kinase
activity stimulated by either osmotic shock or insulin (Fig. 4, A and
B, lane 5). In insulin-resistant cells, the Gab-1 associated PI
3-kinase activity was decreased by 61% upon insulin (Fig. 4A, lane 3),
and by 70% upon osmotic shock stimulation (Fig. 4B, lane 3).
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, open
bars). In contrast, in insulin-resistant cells, the appearance of
membrane ruffles was decreased by 42 ± 2% in sorbitol-stimulated
cells and by 67 ± 2% in insulin-stimulated cells (Fig. 5, solid bars).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In skeletal muscle and in adipocytes, insulin resistance may be caused by defects in insulin-stimulated glucose transport, which results from impairment in the translocation, fusion, or activation of GLUT4 glucose transporters (3, 17). Chronic insulin treatment of 3T3-L1 adipocytes produces a persistent defect in the cells’ ability to respond to subsequent acute insulin stimulation with an increase in GLUT4 translocation and glucose transport activity. In addition, chronic hyperinsulinemia can also affect the expression of GLUT4 protein. Previously published chronic insulin treatment conditions dramatically decreased the total GLUT4 amount in 3T3-L1 adipocytes (13) and adipocytes from obese and type 2 diabetic patients also exhibit reduced levels of GLUT4 protein (18, 19). In the current series of experiments, we wanted to produce a state of cellular insulin resistance without a loss of GLUT4 protein content, so that any defects would be attributable to signaling abnormalities. Therefore, we used a reduced insulin concentration of 10 nM and, importantly, a reduced insulin treatment time of 10 h, and found that these conditions caused cellular insulin resistance with no loss of total cellular GLUT4 protein content. With this approach, we studied the effects of 10 h insulin treatment on the subsequent ability of cells to respond to an osmotic shock stimulus and found that, independent of changes in GLUT4 content, the insulin-resistant cells were also resistant to the stimulatory effects of osmotic shock on glucose transport and GLUT4 translocation to the plasma membrane. Although insulin- and osmotic shock-induced GLUT4 translocation to the plasma membrane seems to be completely blocked in resistant cells, there remains a residual amount of glucose transport following either acute stimulus. Kozka et al. (13) have shown that chronic insulin treatment, in addition to causing GLUT4 down-regulation, causes an increase in cell surface GLUT1 that is correlated with an increase in basal glucose uptake. Thus, it is possible that the residual glucose uptake we observe in resistant, acutely stimulated cells is due to transport via the GLUT1 transporter, although we did not measure GLUT1 levels. Additionally, enhanced intrinsic activity of either transporter may be responsible for the residual glucose transport we observe in resistant, acutely stimulated cells (20).
Our findings provide data on the mechanisms of this effect. Because shock does not lead to activation of the insulin receptor or IRS-1 (7), we focused our attention on other potential signaling molecules. We have previously observed that osmotic shock leads to extensive tyrosine phosphorylation of a new member of the IRS family called Gab-1, which then associates with the p85 subunit of PI 3-kinase with subsequent activation of the enzyme (8). Our earlier studies also showed that Gab-1 is a necessary signaling element of shock-induced GLUT4 translocation (8). The current results show that chronic-insulin exposure attenuates the tyrosine phosphorylation of Gab-1 induced by both osmotic shock and insulin. This attenuation presumably results from a chronic insulin-induced modification of Gab-1 protein or inhibition of a Gab-1 kinase. Additionally, we have found that a decrease in both insulin- and shock-stimulated Gab-1 phosphorylation led to a marked impairment in the ability of Gab-1 to associate with the p85 regulatory subunit of PI 3-kinase following acute insulin or osmotic shock stimulation. The chronic insulin-induced inhibition of Gab-1 associated PI 3-kinase activity correlates with the reduction in Gab-1 tyrosine phosphorylation and Gab-1-p85 association upon shock or insulin stimulation, indicating that the phosphotyrosines involved in PI 3-kinase association were inhibited to a similar extent as overall Gab-1 phosphorylation. Given our previous results showing the importance of Gab-1 in the shock signaling pathway (8), it is reasonable to conclude that defective Gab-1 phosphorylation is an important contributor to the osmotic shock resistance we have observed.
It is interesting to compare our current study with that of Chen et al. (7), who have shown that pretreatment of cells with hyperosmotic shock induces cellular insulin resistance. In their study, cells exposed to hyperosmotic conditions and subsequently acutely stimulated with insulin responded normally with respect to insulin receptor and IRS1 tyrosine phosphorylation, as well as PY20-associated PI 3-kinase activity; however, insulin-stimulated Akt/PKB phosphorylation was defective in these cells. The defect in Akt/PKB phosphorylation in their study was apparently due to the osmotic shock-induced activation of an Akt/PKB-directed phosphatase. Thus, the target of osmotic shock-induced insulin resistance is downstream of PI 3-kinase activation. In contrast, our study indicates that the target of insulin-induced osmotic shock resistance is upstream of PI 3-kinase, presumably at the level of Gab-1 tyrosine phosphorylation. Akt/PKB phosphorylation does not occur in response to osmotic shock (7),and we also find no Akt/PKB phosphorylation in insulin-resistant cells treated with acute osmotic shock (Janez, A., D. S. Worrall, and J. M. Olefsky, unpublished observation).
We have previously shown that insulin has a robust effect on stimulation of membrane ruffling in a PI 3-kinase-dependent manner (9, 14, 15, 16). We have also observed that sorbitol stimulation is capable of inducing membrane ruffling, but only up to 50% of the insulin effect ( Ref. 8 and this study). Data from our present study demonstrate that insulin resistance inhibits both insulin- and sorbitol-induced membrane ruffling. Thus, measuring two PI 3-kinase dependent effects, GLUT4 translocation and membrane ruffling, we found that both effects are decreased in chronically insulin-treated cells.
The studies described above clearly demonstrate that chronic insulin exposure leads to desensitization of the osmotic shock stimulated pathway and induces a state of cellular osmotic shock resistance for glucose transport and GLUT4 translocation. In addition, chronic insulin treatment decreases Gab-1 phosphorylation and Gab-1 associated PI 3-kinase activity, events that are necessary for full stimulation of osmotic shock-induced glucose transport. It is possible that these findings may underlie the effect of insulin resistance on other insulinomimetic agent-regulated pathways leading to GLUT4 translocation and glucose transport.
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Acknowledgments |
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Footnotes |
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2 Supported by NIH/NIDDK Individual NRSA Grant DK-09595.
Received April 14, 2000.
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References |
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