Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California 92093; and San Diego Veterans Affairs Medical Center, San Diego, California 92161
Address all correspondence and requests for reprints to: Jerrold M. Olefsky or Dorothy Sears Worrall, Department of Medicine (0673), University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0673. E-mail: jolefsky@ ucsd.edu or dsears{at}ucsd.edu
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ABSTRACT |
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BAPTA-AM pretreatment inhibited other insulin-induced phosphorylation events including phosphorylation of Akt, MAPK (ERK1 and 2) and p70 S6K. Phosphorylation of Akt on threonine-308 was more sensitive to Ca2+ depletion than phosphorylation of Akt on serine-473 at the same insulin dose (10 nM). In vitro 3'-phosphatidylinositol-dependent kinase 1 activity was unaffected by BAPTA-AM. Insulin-stimulated insulin-responsive glucose transporter isoform translocation and glucose uptake were both inhibited by calcium depletion. In summary, these data demonstrate a positive role for intracellular Ca2+ in distal insulin signaling events, including initiation/maintenance of Akt phosphorylation, insulin-responsive glucose transporter isoform translocation, and glucose transport. A negative role for Ca2+ is also indicated in proximal insulin signaling steps, in that, depletion of intracellular Ca2+ blocks IRS1 serine/threonine phosphorylation and enhances insulin-stimulated protein-protein interaction and PI3K activity.
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INTRODUCTION |
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The present studies were conducted to investigate the importance of intracellular Ca2+ in a variety of insulin actions including insulin-responsive glucose transporter isoform (GLUT4) translocation and glucose transport. Using the intracellular Ca2+ chelator 1,2- bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, sodium (BAPTA-AM), our results show that Ca2+ has both negative and positive effects on the transduction of insulin signals in 3T3-L1 adipocytes.
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RESULTS |
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Chelation of Ca2+ Enhances Insulin-Activated
Protein-Protein Interaction and PI3K Activity
Figure 1, A and B, shows that chelation of intracellular calcium
does not impair overall tyrosine phosphorylation of IRS1 and IRß.
Antiphosphotyrosine immunoprecipitation studies were conducted to
further assess tyrosine phosphorylation of these proteins with or
without BAPTA-AM. Figure 2A
shows that
the amount of IRS1 immunoprecipitated with PY20 antibody after 5 min of
submaximal insulin stimulation (1 nM) is enhanced by
pretreatment with BAPTA-AM. PY20 immunoprecipitation of IRß was also
slightly enhanced (Fig. 2B
). Additionally, an antibody to the p85
regulatory subunit of PI3K was able to pull down 60% (±10%,
SE) more IRS1 from the BAPTA-AM-pretreated,
insulin-stimulated lysates than the non-pretreated, insulin-stimulated
controls (Fig. 2C
). Immunoprecipitation studies using an antibody
against the p110
subunit of PI3K generated similar results with
respect to IRS1-PI3K interaction (data not shown).
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GLUT4 Translocation Requires Intracellular Calcium
Insulin stimulation of 3T3-L1 adipocytes leads to translocation of
GLUT4 from an intracellular low density microsomal compartment (LDM) to
the plasma membrane (PM). Cell fractionation and fluorescence
microscopy studies were employed to observe the effects of
intracellular calcium chelation on the location of GLUT4 in basal and
insulin-stimulated cells. Cells were fractionated into their
subcellular compartments as described in Materials and
Methods, and the levels of GLUT4 protein in the LDM and PM
fractions are shown in Fig. 7. Insulin
stimulation causes a decrease in GLUT4 levels in the LDM fraction (Fig. 7A
, lane 3, and 7B, left graph) and an increase in GLUT4
levels in the PM fraction (Fig. 7A
, lane 7, and 7B, right
graph). Pretreatment of cells with BAPTA-AM inhibits both the
GLUT4 decrease in the LDMs and the increase in the PMs.
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Subcellular Localization of Akt
We next analyzed the subcellular distribution of Akt,
hypothesizing that the BAPTA-AM-induced decrease in Akt phosphorylation
may be a consequence of Akt inaccessibility to PDK1 and 2 at the PM.
The distribution of Akt and phosphoserine-473 Akt was analyzed using
protein- and phospho-specific antibodies that recognize all three Akt
isoforms (Fig. 9). The amount of Akt
protein in the basal and insulin-stimulated PM fractions was greater in
BAPTA-AM-treated cells compared with control cells. Insulin stimulation
did not cause an increase in Akt localization at the PM, compared
with basal levels, in control or BAPTA-pretreated cells. BAPTA-AM
strongly inhibited insulin-stimulated, serine-473 phosphorylation of
Akt in the cytosol and PM (Fig. 9A
, compare lanes 3 with lanes 4 in
pS-Akt blot). The ratio of phosphoserine-473 Akt signal relative to
total Akt protein signal in each fraction is shown in Fig. 9B
.
Interestingly, in control cells, the PM fraction contained 9.4 times
more serine-phosphorylated Akt protein than the cytosol fraction,
although the majority of total Akt protein was in the cytosol (Fig. 9B
).
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Discussion |
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Treatment of cells with BAPTA-AM to deplete intracellular calcium had no inhibitory effect on early steps of insulin action, such as insulin receptor autophosphorylation, IRS-1 tyrosine phosphorylation, or activation of PI3K. In fact, we found some evidence for increased activity of these initial events. For example, there was an increase in the amount of PI3K activity associated with IRS-1 in the insulin-stimulated state. Most likely, this was due to the ability of calcium depletion to inhibit IRS-1 serine/threonine phosphorylation and thus, enhance the efficiency of PI3K binding to phosphorylated IRS-1. In agreement with these results, it has been shown that serine/threonine phosphorylation of IRS1 is associated with insulin resistance (1519) and that serine phosphorylation causes a reduction in IRS1s ability to act as a docking site for PI3K (20, 21).
Akt phosphorylation is downstream of PI3K activity, and we found that in the BAPTA-AM-treated cells, there was a marked impairment in insulin-stimulated and heat shock-stimulated Akt phosphorylation. Clearly, in insulin-stimulated cells, this was not due to decreased PI3K activity because, if anything, PI3K activity was increased. Therefore, the data show that intracellular calcium depletion interferes with insulin-stimulated Akt activation at a step downstream of PI3K, assuming that in vitro PI3K activity reflects in vivo activity.
The level of insulin-induced serine-473 phosphorylation per Akt protein is inhibited in the cytosol and absent in the PM fractions from BAPTA-AM- pretreated cells. In contrast, generation of serine-473 phosphorylation per Akt protein in insulin-stimulated control cells was significantly greater in the PM fraction compared with the cytosol. This distinct subcellular distribution of phosphorylated Akt protein in control cells is not surprising given that activation of Akt by insulin requires its translocation to the PM and subsequent phosphorylation by PDK1 and the as yet uncharacterized PDK2 (22). These data suggest that Ca2+ is not required for Akt to fractionate with the PM and that the inhibition/lack of phospho-Akt signal in these fractions results from some Akt-localization-independent effect of Ca2+ chelation. Interestingly, insulin-mediated phosphorylation of Akt on threonine-308 and serine-473 exhibit different sensitivities to intracellular Ca2+ depletion that may be mediated by site-specific kinases and/or phosphatases. We have shown that the in vitro intrinsic activity of the threonine-308 kinase PDK1 is unaffected by BAPTA-AM pretreatment. Although the serine-473 candidate kinases are not known to be dependent on Ca2+ (2225), conflicting data exists regarding the role of Ca2+ and Akt serine-473 phosphorylation (26, 27). Ca2+ depletion may also activate Akt phosphatase(s), similar to the work of Draznin et al. (2) showing that elevated levels of intracellular Ca2+ inhibit phosphatases specific for glycogen synthase and GLUT4.
Phospholipid binding by certain phospholipid-binding domains, including the Akt PH (pleckstrin homology) domain, can be affected by relatively low levels of Ca2+ (28, 29) and by phosphorylation levels of phosphatidylinositol. Chelation of intracellular calcium did not inhibit the localization of Akt at the PM in our study but may have perturbed the PH domain-mediated localization of PDK1 and/or the hypothetical PDK2. This would also explain the BAPTA-AM sensitivity of insulin-induced p70 S6K phosphorylation, the activation of which is dependent upon PDK1 (30). Alternatively, phosphatidylinositol phosphatase (e.g. SHIP, SH2-containing inositol polyphosphate 5-phosphatase) activity may be affected by intracellular Ca2+ depletion, resulting in PH domain-mediated effects on Akt phosphorylation. The mode of Ca2+s site-specific effects on Akt phosphorylation are unclear; however, we demonstrate that the minimum level of Ca2+ required for Akt threonine-308 phosphorylation is higher than that required for serine-473 phosphorylation.
We, in the present study, and others have shown that intracellular calcium is required for glucose uptake stimulated by insulin and other stimuli (2, 31, 32). We also find that BAPTA-AM preincubation completely blocked insulin-stimulated GLUT4 translocation as assayed using subcellular fractionation and fluorescence imaging. The role of Akt in mediating these effects is unclear from our studies. Whether Akt is upstream of insulin-activated glucose transport is controversial, BAPTA-AMs effect on glucose transport and Akt phosphorylation may be correlative rather than causal. The requirement of Ca2+ in synaptic vesicle fusion and in vesicle fusion/exocytosis in nonneuronal cells is well documented. Specific levels of Ca2+ regulate interactions between vesicle associated proteins (synaptotagmins and syntaxins) required for vesicle fusion in an isoform-specific manner (33). Studies of vesicle fusion in nonneuronal cell types suggest that exocytosis is mediated by small, localized changes in Ca2+ flux that occur at the site of fusion (34, 35). Such small changes would not be detectable by traditional means of measuring intracellular Ca2+ levels (e.g. the fluorescent Ca2+ indicator Fura-2) but are inhibitable with BAPTA-AM (34). Ca2+/calmodulin has been shown to be required for vacuolar fusion (36) and also for insulin-stimulated GLUT4 translocation (3) and glucose transport (Refs. 37 and 38, and our unpublished observations). Thus, calmodulin and some vesicle-associated proteins (SNAREs, soluble N-ethyl maleimide sensitive factor attachment protein receptor) are Ca2+-regulated proteins required for vesicle fusion, possibly including fusion of GLUT4 vesicles with the PM after insulin-mediated translocation.
There are many studies in the literature regarding the involvement of Ca2+ in insulin signaling, and although large changes in intracellular levels of Ca2+ do not occur in response to insulin (5), it is clear that an optimal Ca2+ concentration within cells is essential for insulin mediated events (2). It has been suggested that small changes in Ca2+ concentration or Ca2+ fluxes may be mechanistically important for insulin signaling (1, 2). Also, elevated levels of intracellular calcium are associated with in vitro and in vivo insulin-resistant states (711, 39), and it is possible that high intracellular levels of Ca2+ would prevent an insulin target cell from sensing acute insulin-induced small changes in Ca2+ flux. This may provide one reason why elevated intracellular Ca2+ levels are associated with insulin resistant states.
In summary, we have found that Ca2+ depletion results in enhanced proximal insulin action including PI3K activation and IRS1/PI3K association. In contrast, distal insulin signaling events are inhibited by Ca2+ depletion. We find that Ca2+ is required for insulin-induced Akt phosphorylation and that this Ca2+-dependent activation of Akt is downstream of PI3K. Lastly, we have demonstrated that Ca2+ depletion blocks insulin-induced GLUT4 translocation and glucose transport. Thus, intracellular Ca2+ is an important component of insulin action at multiple steps in the insulin signaling cascade.
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MATERIALS AND METHODS |
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Cell Culture
3T3-L1 fibroblasts were maintained in DMEM-high glucose (25
mM glucose, 1.8 mM CaCl2)
medium containing 10% calf serum. Postconfluent fibroblasts were
differentiated into adipocytes by adding 1 µg/ml insulin, 0.1 µg/ml
dexamethasone, and 112 µg/ml isobutylmethylxanthine to the medium.
The differentiation medium was removed after 3 d and replaced with
DMEM-low glucose (5 mM glucose, 1.8 mM
CaCl2) medium containing 10% FCS, Glutamax, and
1% penicillin-streptomycin. Seven days after the addition of the
differentiation mix, the cells were plated in culture dishes for each
given experiment. The medium was changed every third day until use,
1016 d post differentiation. Approximately 90% of the cells
exhibited large lipid droplets indicative of adipocytes. Before each
experiment, cells were serum-starved for 24 h in DMEM-low
glucose containing 0.1% BSA. This study protocol was used in all our
experiments. During experiments, DMSO concentration in the
media/incubation buffer was equal in control samples to that used as
vehicle for inhibitors in all experiments and never exceeded 0.1%.
BAPTA-AM was diluted in media or buffer and cells were pretreated for
1030 min before initiating stimulation with insulin, etc. The drug
was present during stimulation only unless otherwise noted.
Lysates, Immunoprecipitations, and Immunoblottting
Cells were rinsed two times with ice-cold PBS and lysed at 4 C
in 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, 2 mM phenylmethylsulfonyl fluoride, 1
mM EDTA, 10% glycerol, pH 7.4. Lysates were vortexed well
and placed on ice for 10 min. Lysates were then centrifuged at
14,000 x g for 10 min at 4 C. Supernatants were
separated from the resulting fat layer and protein concentrations were
determined by Bradford assay. Lysates for immunoprecipitation or
affinity chromatography were incubated with 24 µg of the indicated
antibody or GST-fusion with protein A-sepharose or glutathione-agarose,
respectively, at 4 C for 214 h with gentle agitation. Pellets were
washed three times in lysis buffer. Laemmlis buffer was added to the
pellets and boiled for 5 min. Samples were separated by SDS-PAGE and
transferred to polyvinylidene difluoride membrane. Immunoblotting with
various antibodies was conducted as per company instructions.
Immunoblots were incubated with the appropriate horseradish
peroxidase-conjugated secondary antibody and subsequently analyzed by
enhanced chemiluminescence and autoradiography. Autoradiographs were
quantitated using densitometric scanning and NIH Image software.
2-Deoxyglucose Uptake
After pretreatment as indicated in figure legends, 3T3-L1
adipocytes were stimulated with 16.7 nM insulin, 600
mM sorbitol, or 3 mM
H2O2 for 20 min at 37 C.
Glucose transport was determined by the addition of 0.1 mM
2-deoxyglucose containing 0.2 µCi of 2-[3H]
deoxyglucose as described previously (40) in HEPES/salts
buffer with 0.1% BSA (10 mM HEPES, 2.5 mM
NaH2PO4, 130 mM
NaCl, 4.7 mM KCl, 1.24 mM
MgSO4, 2.47 mM
CaCl2, pH 7.4). 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 three 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
After pretreatment as indicated, cells from one 10-cm dish per
condition were incubated with insulin as described and washed three
times with ice-cold PBS. Cells were scraped into ice-cold HES buffer
(255 mM sucrose, 20 mM HEPES, 1 mM
EDTA, 4 mM
Na3VO4, 200 mM
sodium fluoride, 20 mM sodium pyrophosphate, pH 7.4)
supplemented with protease inhibitors ("Complete" protease cocktail
tablet). Cells were then homogenized using an Potter-Ejlerham
homogenizer. Subcellular fractionation was carried out as described
previously (41).
In Vitro PI3K and PDK1 Activity Assays
3T3-L1 adipocytes were pretreated and stimulated as indicated,
then lysed and immunoprecipitated as described above with the following
modifications. After incubation with the antibody, bead pellets were
washed three times with Buffer A (Tris-buffered saline, 1% NP-40, and
100 µM
Na3VO4, pH 7.4), three
times with Buffer B (100 mM Tris, 500 mM
LiCl2, and 100 µM
Na3VO4, pH 7.4), and twice
with Buffer C (10 mM Tris, 100 mM NaCl, 1
mM EDTA, and 100 µM
Na3VO4, pH 7.4). Pellets
were resuspended in Buffer C without
Na3VO4. As described
previously (42), PI3K activity was assessed by the
phosphorylation of phosphatidylinositol in the presence of 20 µCi of
[-32P]ATP for 20 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 1%-CDTA-coated silica gel TLC
plates. After the separation of lipids by TLC using the borate-buffered
system (43), phosphatidylinositol 3-phosphate was
visualized by autoradiography. Autoradiographs were scanned and
quantitated as described above.
In vitro PDK1 kinase assays were conducted as per
manufacturers instructions using immunoprecipitated PDK1 from cells
pretreated 30 min with DMSO or 100 µM BAPTA-AM
before stimulation with 10 nM insulin for 5 min.
One protocol exception was that rabbit antisheep antibody and protein
A-agarose were used to precipitate the primary sheep anti-PDK1
antibody. Briefly, PDK1 was immunoprecipitated from cell lysates
[Lysis buffer A: 50 mM Tris-HCl, pH 7.5, 1
mM EDTA, 1 mM EGTA, 0.5
mM
Na3VO4, 0.1% (vol/vol)
2-mercaptoethanol, 1% (vol/vol) Triton X-100, 50
mM sodium fluoride, 5 mM
sodium pyrophosphate, 10 mM sodium
ß-lycerophosphate, 0.1 mM
pheynylmethylsulfonyl fluoride, 1 µM
microcystin, 1 mg/ml of aprotinin, pepstatin, leupeptin] overnight at
4 C. Activity of PDK1 is measured by the PDK1-dependent activation of
SGK (serum- and glucocorticoid-induced protein kinase) in a
two-step reaction. In step 1, immunoprecipitated PDK1 was
incubated with purified, inactivated SGK protein in PDK1 assay
dilution buffer (supplied in kit) for 30 min at 30 C with shaking. In
step 2, exogenous Akt/PKB specific substrate peptide was added with
[-32P] ATP for an additional 10 min. Labeled
substrate peptide was spotted onto P81 paper and washed with 0.75%
phosphoric acid. Incorporation of radioisotope was measured by
scintillation counting. Results are the average of three independent
experiments. Data from negative controls (including control IgG IPs and
kinase reactions without exogenous substrate) were all less than 1% of
the maximal signal in each experiment.
Staining of GLUT4 Translocation
3T3-L1 adipocytes on coverslips were pretreated and stimulated
as indicated, then rinsed two times with ice-cold PBS. Cells were fixed
in 3.7% formaldehyde in PBS for 10 min. Coverslips were rinsed two
times with PBS, and all further incubations were shielded from light.
Cells were stained with TRITC-concanavalin A for 30 min then washed two
times with PBS. Cells were permeabilized in 0.1% Triton X-100, 2% FCS
in PBS for 10 min. Coverslips were incubated with GLUT4 antibody
overnight at 4 C then washed in 2% FCS in PBS for 10 min.
Coverslips were then incubated with FITC-conjugated antirabbit
secondary antibody for 60 min then washed with 2% FCS in PBS for 10
min. Nuclei were stained with 50 ng/ml DAPI
(4',6-diamidino-2-phenylindole, dihydrochloride) in PBS for 5 min then
washed with 2% FCS in PBS for 10 min. Coverslips were rinsed in water
before mounting on Gelvatol on microscope slides.
Imaging of GLUT4 Translocation
Images were captured using a DeltaVision deconvolution
microscope system (Applied Precision, Inc., Issaquah, WA). The system
includes a Photometrics charge-coupled device mounted on a
Nikon microscope. In general, 75 optical sections spaced
by 0.2 µm were taken of cells mounted and stained on coverslips.
Pixel intensities were kept in the linear response range of the digital
camera. The lens used for these images was 40x (NA1.3); the resulting
pixel size is 120 nm2. Data sets were
deconvoluted and analyzed using SoftWorx software (Applied Precision,
Inc.) on a Silicon Graphics (Mountain View, CA) Octane
workstation. Images used for quantitation were volume views made from 5
consecutive sections of the 75 sections that include the entire volume
of the cells. We used the 5 sections beginning at section no. 17,
counting away from the cell bottom/coverslip for all measurements (Fig. 8A). These five-section, volume views were used for analysis because
they best represented the cell edges and the staining distribution
within the PM of each cell.
DataInspector application was used to quantitatively analyze the
five-section volume views. The images shown in Fig. 8B are quantitative
volume views define the voxel intensity of each wavelength in a unique
false-color scale (very low voxel intensity as violet, very
strong as red). Importantly, these quantitations are
computer-derived using the original digital fluorescent image data and
are not dependent upon the brightness of the cell images projected on
the computer screen or prints. Voxel intensity values can be displayed
in a color scale form (Fig. 8B
) or can be displayed in numerical form
(not shown, but graphically represented in Fig. 8C
). Levels of
FITC-labeled (green), anti-GLUT4 antibody at the PM were
measured relative to staining of TRITC-concanavalin A (red)
used as a plasma membrane control marker. Levels of TRITC-concanavalin
A at the PM relative to TRITC-concanavalin A in the cytoplasm did
not change between matched control and BAPTA-AM-pretreated samples.
Thus, changes in the GLUT4/concanavalin A ratio reflect changes in
GLUT4 levels only. Each measurement was determined using the summed
voxel intensity within a circular cursor with a diameter of 480 nm.
Values graphed in Fig. 8C
are the average of five PM measurements for
each of five cells ± SEM.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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{ref} 1. Clausen T, Elbrink J, Martin BR 1974 Insulin controlling calcium distribution in muscle and fat cells. Acta Endocrinol 77:137143
2. Draznin B, Sussman K, Kao M, Lewis D, Sherman N 1987 The existence of an optimal range of cytosolic free calcium for insulin-stimulated glucose transport in rat adipocytes. J Biol Chem 262:1438514388
3. Yang C, Watson RT, Elmendorf JS, Sacks DB, Pessin JE 2000 Calmodulin antagonists inhibit insulin-stimulated GLUT4 (glucose transporter 4) translocation by preventing the formation of phosphatidylinositol 3,4,5-trisphosphate in 3T3-L1 adipocytes. Mol Endocrinol 14:317326
4. Klip A 1984 Is intracellular Ca2+ involved in insulin stimulation of sugar transport? Fact and prejudice. Can J Biochem Cell Biol 62:12281236
5. Kelly KL, Deeney JT, Corkey BE 1989 Cytosolic free calcium in adipocytes. Distinct mechanisms of regulation and effects on insulin action. J Biol Chem 264:1275412757
6. Sorensen SS, Christensen F, Clausen T 1980 The relationship between the transport of glucose and cations across cell membranes in isolated tissues. X. Effect of glucose transport stimuli on the efflux of isotopically labeled calcium and 3-O-methylglucose from soleus muscles and epididymal fat pads of the rat. Biochem Biophys Acta 602:433445
7. Zemel MB 1998 Nutritional and endocrine modulation of intracellular calcium: implications in obesity, insulin resistance and hypertension. Mol Cell Biochem 188:129136
8. Levy J, Gavin JR, Sowers JR 1994 Diabetes mellitus: A disease of abnormal cellular calcium metabolism? Am J Med 96:260273
9. Draznin B 1993 Cytosolic calcium and insulin resistance. Am J Kid Dis 21:3238
Abbreviations: BAPTA-AM, 1,2-bis(o-Aminophenoxy)ethane- N,N,N',N'-tetraacetic acid, sodium; CDTA, trans-1,2-diaminocyclo-hexane-N,N,N',N'-tetra-acetic acid; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate; GLUT4, insulin- responsive glucose transporter isoform; IR, insulin receptor; IRS, insulin receptor substrate; LDM, low density microsomal compartment; PDK1 or PDK2, 3'-phosphatidylinositol-dependent kinase 1 or 2; PH, pleckstrin homology; PM, plasma membrane; TRITC, tetramethyl-rodamine isothiocyanate.
Received for publication January 16, 2001. Accepted for publication October 19, 2001.