Role for a Novel Signaling Intermediate, Phosphatidylinositol 5-Phosphate, in Insulin-Regulated F-Actin Stress Fiber Breakdown and GLUT4 Translocation

Article (PDF Available)inEndocrinology 145(11):4853-65 · December 2004with27 Reads
DOI: 10.1210/en.2004-0489 · Source: PubMed
Abstract
The cellular functions and regulation of phosphatidylinositol (PtdIns) 5-phosphate (5-P), the newest addition to the family of phosphoinositides (PIs), are still elusive. Here we have examined a plausible role of PtdIns 5-P as a signaling intermediate in acute insulin action. A wortmannin-insensitive transient increase of PtdIns 5-P mass levels that peaked at 10 min, and declined 20-30 min after insulin stimulation, was observed in both Chinese hamster ovary (CHO)-T cells stably expressing the insulin receptor and 3T3-L1 adipocytes. Similarly to insulin, found to induce a rapid disassembly of Texas-Red phalloidin-labeled actin stress fibers in CHO-T cells, microinjected PtdIns 5-P, but not other PIs, decreased the number and length of F-actin stress fibers in this cell type to a magnitude seen in response to insulin. Likewise, increases of PtdIns 5-P by ectopic expression of the PtdIns 5-P-producing enzyme PIKfyve yielded a similar effect. As with insulin, the PtdIns 5-P-induced loss of actin stress fibers was independent of PI 3-kinase activation. Furthermore, sequestration of functional PtdIns 5-P, either by ectopic expression of 3xPHD domains that bind selectively PtdIns 5-P or by microinjecting the GST-3xPHD fusion peptide, abrogated insulin-induced F-actin stress fiber disassembly in CHO-T cells. In 3T3-L1 adipocytes, microinjected PtdIns 5-P, but not other PIs, partially mimicked insulin's effect of translocating enhanced green fluorescent protein-GLUT4 to the cell surface. Conversely, insulin-induced myc-GLUT4 vesicle dynamics was arrested in the presence of coexpressed enhanced green fluorescent protein-3xPHD. Involvement of PIKfyve membrane recruitment, but not activation, and/or a decrease in PtdIns 4,5-bisphosphate levels are likely to be among the mechanisms underlying the insulin-induced PtdIns 5-P increase. Together, these results identify PtdIns 5-P as a novel key intermediate for insulin signaling in F-actin remodeling and GLUT4 translocation.

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Role for a Novel Signaling Intermediate,
Phosphatidylinositol 5-Phosphate, in
Insulin-Regulated F-Actin Stress Fiber
Breakdown and GLUT4 Translocation
DIEGO SBRISSA, OGNIAN C. IKONOMOV, JANA STRAKOVA, AND ASSIA SHISHEVA
Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201
The cellular functions and regulation of phosphatidylinositol
(PtdIns) 5-phosphate (5-P), the newest addition to the family
of phosphoinositides (PIs), are still elusive. Here we have ex-
amined a plausible role of PtdIns 5-P as a signaling interme-
diate in acute insulin action. A wortmannin-insensitive tran-
sient increase of PtdIns 5-P mass levels that peaked at 10 min,
and declined 20 –30 min after insulin stimulation, was ob-
served in both Chinese hamster ovary (CHO)-T cells stably
expressing the insulin receptor and 3T3-L1 adipocytes. Sim-
ilarly to insulin, found to induce a rapid disassembly of Texas-
Red phalloidin-labeled actin stress fibers in CHO-T cells, mi-
croinjected PtdIns 5-P, but not other PIs, decreased the
number and length of F-actin stress fibers in this cell type to
a magnitude seen in response to insulin. Likewise, increases
of PtdIns 5-P by ectopic expression of the PtdIns 5-P-produc-
ing enzyme PIKfyve yielded a similar effect. As with insulin,
the PtdIns 5-P-induced loss of actin stress fibers was inde-
pendent of PI 3-kinase activation. Furthermore, sequestra-
tion of functional PtdIns 5-P, either by ectopic expression of
3xPHD domains that bind selectively PtdIns 5-P or by micro-
injecting the GST-3xPHD fusion peptide, abrogated insulin-
induced F-actin stress fiber disassembly in CHO-T cells. In
3T3-L1 adipocytes, microinjected PtdIns 5-P, but not other
PIs, partially mimicked insulin’s effect of translocating en-
hanced green fluorescent protein-GLUT4 to the cell surface.
Conversely, insulin-induced myc-GLUT4 vesicle dynamics
was arrested in the presence of coexpressed enhanced green
fluorescent protein-3xPHD. Involvement of PIKfyve mem-
brane recruitment, but not activation, and/or a decrease in
PtdIns 4,5-bisphosphate levels are likely to be among the
mechanisms underlying the insulin-induced PtdIns 5-P
increase. Together, these results identify PtdIns 5-P as a novel
key intermediate for insulin signaling in F-actin remodeling
and GLUT4 translocation. (Endocrinology 145: 4853– 4865,
2004)
P
HOSPHORYLATED METABOLITES OF phosphatidyl-
inositol (PtdIns), collectively called phosphoinositides
(PIs), represent a minor fraction of the eukaryotic cell lipids
but play a major regulatory role in diverse cellular processes
such as signaling, membrane trafficking, cytoskeletal reor-
ganization, DNA synthesis, and cell cycle (for recent reviews,
see Refs. 1–8). In most cases, PIs serve as regulatory
membrane-localized signals to recruit/activate downstream
protein effectors that display PI-specific binding domains. A
growing number of PI-binding protein modules have been
recently identified, including the PH, the FYVE finger
(Fab1p, YOTB, Vac1p, and EEA1), the FERM (Four.1-Ezrin-
Radixin-Moesin), the Epsin N-Terminal Homology, the PX
(Phox Homology) and PHD finger (Plant HomeoDomain)
domains (for recent reviews, see Refs. 9 –13). Although their
specificity could be broad in some instances, the PI-binding
modules have been critical for elucidating the role of PIs in
cellular regulation and the mechanism of their function.
Of the seven PIs, the cellular roles of PtdIns 5-phosphate
(PtdIns 5-P) are the least well known. This is related to the
fact that, in mammalian cells, PtdIns 5-P represents only a
minor fraction of PIs and is poorly separated from the abun-
dant PtdIns 4-P upon HPLC resolution. Since its initial dis-
covery as a substrate for type II PI phosphate kinases (PIPKs)
(14), PtdIns 5-P has been the subject of several studies ad-
dressing its role and regulation of its metabolism in a cellular
context. This was made possible, in part, due to the imple-
mentation of an alternative approach for PtdIns 5-P intra-
cellular determination, the mass assay, resting upon the sub-
strate preference of type II PIPKs for PtdIns 5-P and the
effective detection of PtdIns 4,5-bisphosphate (-P
2
) by HPLC
(15). By mass assay, PtdIns 5-P levels were found to acutely
increase in response to thrombin stimulation of platelets (15).
Conversely, a robust decrease of PtdIns 5-P was seen upon
hypo-osmotic shock in mouse 3T3-L1 fibroblasts and adipo-
cytes, thus implicating PtdIns 5-P as a regulatory interme-
diate in the osmotic response pathway (16). Recently, a
marked increase of PtdIns 5-P levels was shown to accom-
pany Shigella flexneri bacteria invasion due to a potent inositol
4-phosphatase activity of the virulence factor IpgD that con-
verts PtdIns 4,5-P
2
to PtdIns 5-P and induces dramatic
changes of F-actin remodeling in mammalian cells (17). Pt-
Abbreviations: CHO, Chinese hamster ovary; EGFP, enhanced green
fluorescent protein; F-actin, filamentous actin; FITC, fluorescein isothio-
cyanate; GroPIns, glycerophosphorylinositol; GST, glutathione-S-trans-
ferase; IM, intracellular membrane; -P, phosphate; -P
2,
bisphosphate; -P
3,
trisphosphate; PI, phosphoinositide; PIPK, PI phosphate kinase; PM,
plasma membrane; PtdIns, phosphatidylinositol; PX, Phox Homology;
TLC, thin-layer chromatography.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
0013-7227/04/$15.00/0 Endocrinology 145(11):4853–4865
Printed in U.S.A. Copyright © 2004 by The Endocrine Society
doi: 10.1210/en.2004-0489
4853
dIns 5-P has also been reported to exist and function in the
nucleus (18). The PHD finger of the tumor suppressor ING2,
a motif common in many chromatin-regulatory proteins,
specifically interacts with PtdIns 5-P and was proposed to
function as a nuclear PtdIns 5-P receptor to regulate nuclear
responses to DNA damage (19). Clearly, although limited,
the available studies imply that PtdIns 5-P is a signaling
molecule in its own right and functions in the above or other
yet-to-be-identified cellular responses.
Insulin stimulates glucose uptake in fat and muscle by
inducing the translocation of an intracellular membrane (IM)
pool, containing GLUT4 glucose transporters (GLUT4 vesi-
cles), to the cell surface (for recent reviews, see Refs. 7, 20, and
21). This is a complex multistep signaling cascade, which is
still not completely understood. Initiated by the activated
insulin receptor, this cascade appears to require both PI 3-
kinase-dependent and -independent signals for optimal per-
formance (for recent reviews, see Refs. 2224). Over the last
several years, it became apparent that, like other vesicle
trafficking events, the filamentous (F)-actin cytoskeleton
plays a major role in insulin-regulated GLUT4 vesicle dy-
namics (2531). In fact, insulin has been shown to be a major
trigger of F-actin remodeling in a number of cell types that
may or may not express insulin-regulatable GLUT4. A rapid
decrease of F-actin stress fibers, followed by a transient in-
crease in membrane ruffling (lamellipodia), has been readily
observed in insulin-stimulated Chinese hamster ovary
(CHO)-T or HIRc-B cells, both expressing the insulin recep-
tor, and 3T3-L1 fibroblasts (29, 30, 3234). In differentiated
3T3-L1 adipocytes, which are large, round, lipid-laden cells
that do not contain typical stress fibers, increased membrane
ruffling and cortical F-actin formation have been seen in
response to insulin (28 30). Insulin-regulated F-actin cy-
toskeleton remodeling, whether linked or not with GLUT4
vesicle translocation, is likely mediated by two types of sig-
nals: one dependent on PI 3-kinase activity, whereas another
appears to be PI 3-kinase independent. Here we have tested
whether PtdIns 5-P is involved in the mechanisms used by
insulin to signal F-actin remodeling and GLUT4 vesicle
translocation. We found acutely elevated PtdIns 5-P mass
upon insulin stimulation in both CHO-T and 3T3-L1 adipo-
cytes. Insulin-induced disassembly of F-actin stress fibers in
CHO-T cells and cell surface redistribution of GLUT4 were
mimicked by microinjected PtdIns 5-P in a wortmannin-
independent manner and hindered by sequestering intracel-
lular PtdIns 5-P with PHD finger domains. Thus, PtdIns 5-P
emerges as a new key signaling intermediate in insulin action.
Materials and Methods
Cell cultures
Mouse 3T3-L1 fibroblasts were differentiated into insulin-sensitive
adipocytes as previously described (35). CHO-T cells, stably expressing
the human insulin receptor, were maintained in Hams F-12 medium,
containing 10% fetal bovine serum, 50 U/ml penicillin, and 50
g/ml
streptomycin sulfate as specified elsewhere (35).
Glutathione-S-transferase (GST)-PHD constructs and
protein production
To generate a GST-3xPHD domain, we used the clone pEGFP-C2
3xPHD of ING2, a kind gift by Dr. Or Gozani (19). The BglII-SalI frag-
ment of the latter construct was ligated in the compatible cohesive ends
of a BamH1-SalI digest of pGEX5X-3 in-frame with GST. Escherichia coli
strain XA-90 was used for transformation. Production and purification
of the GST fusion proteins on GSH-agarose beads (Sigma, St. Louis, MO)
were performed essentially as previously described (36). The concen-
tration and quality of the eluted purified proteins were determined
electrophoretically by the intensity of the Coomassie-stained protein
bands vs. BSA standard (Pierce, Rockford, IL).
Transient transfection
CHO-T cells were transfected with the pEGFP-3xPHD constructs
(kind gift by Or Gozani; Ref. 19) using LipofectAmine as a transfection
reagent. Twenty hours post transfection, the cells were serum-starved
for 4 h and, after microinjection and/or treatments indicated in the figure
legends, were processed for fluorescence microscopy analysis. In some
experiments, after transfection, CHO-T cells were serum-starved for 12 h
in media supplemented with 0.5% BSA before microinjection and stim-
ulation, yielding similar results. Differentiated 3T3-L1 adipocytes were
transfected ond7ofthedifferentiation program with the pEGFP-GLUT4
(a kind gift by Jeff Pessin) or pEGFP-3xPHD or cotransfected with
pcDNA 3.17xMyc-GLUT4 (a kind gift by Kostya Kandror) and pEGFP-
3xPHD cDNA constructs using the electroporation method as we de-
tailed previously (35). Experiments were performed 20 h post transfec-
tion, subsequent to serum starvation (4 h).
Cell treatment and PtdIns 5-P mass assay
Cells were serum-starved (4 h) in DMEM or Hams F-12 media and
then treated at 37 C with or without insulin (100 nm) for the indicated
time periods. In some experiments, cells were treated with wortmannin
(100 nm; 20 min) before insulin. PtdIns 5-P mass assay was performed
as we previously specified in detail (16). Briefly, PIs were isolated from
cell lipid extracts on neomycin-coated glass beads. Samples, supple-
mented with PtdIns as carrier, were subjected to PtdInsP-conversion
assay using bacterially produced and purified recombinant His-tagged
type II
PIPK (cDNA was a kind gift by Richard Anderson) in a buffer
consisting of 50 mm Tris-HCl (pH 7.4), 80 mm KCl, 10 mm magnesium
acetate, 2 mm EGTA, 0.01% sodium deoxycholate, and 5
m [
-
32
P]ATP
(5
Ci). In some cases, GST-3XPHD domain or GST peptide fragments
(each at 2
g) were added to the assay. The reaction, continued for 1 h
at 30 C, was stopped with 200
l1-n HCl and extracted with 160
l
chloroform:methanol (1:1; vol/vol). Lower layers were washed and then
spotted on an oxalate-treated and activated thin-layer chromatography
(TLC) plate (Whatman, PE SIL G, 250
m). Plates were developed in
65:35 (vol/vol) n-propanol-2 m acetic acid and exposed with Kodak
X-omat (Rochester, NY) autoradiography film. For quantitation, 210
pmol PtdIns 5-P standards were processed in parallel. Control samples
with no enzyme and/or no lipids were run in each experiment. Because
type II PIPK can also convert PtdIns 3-P to some degree, the identity of
the PtdIns 4,5-P
2
radioactive spots at both basal or insulin-stimulated
conditions was confirmed by HPLC-inositol head group analysis after
lipid extraction from the silica scrapings and deacylation.
Cell metabolic labeling with [
32
P]orthophosphate and
lipid extraction
Serum-starved (1 h) 3T3-L1 adipocytes or CHO-T cells were washed
in phosphate-free DMEM and then labeled for3hat37Cinphosphate-
free, serum-free DMEM supplemented with 0.5% BSA, 2 mm sodium
pyruvate, and 0.8 mCi/ml of [
32
P]orthophosphate as described previ
-
ously (37). Cells were stimulated at 37 C with or without insulin (100 nm)
for 10 min, and then washed with ice-cold PBS containing protease (1
mm phenylmethylsulfonylfluoride, 1 mm benzamidine, 5
g/ml leu-
peptin, 5
g/ml aprotinin, and 1
g/ml pepstatin) and phosphatase
inhibitors (50 mm NaF, 10 mm sodium pyrophosphate, 25 mm sodium
-glycerophosphate, and 2 mm sodium metavanadate) and scraped with
CH
3
OH/1 m HCl (1:1; vol/vol) in the presence of 5 mm EDTA and 5 mm
tetrabutylammonium hydrogen sulfate. Extracted radiolabeled lipids
were deacylated as previously specified (16, 37) and analyzed by HPLC
(see below).
4854 Endocrinology, November 2004, 145(11):4853 4865 Sbrissa et al. PtdIns 5-P Mediates Insulin Signaling
Microinjections, fluorescence, and
immunofluorescence microscopy
Microinjection in single CHO-T cells, seeded on glass coverslips in
35-mm dishes (Corning Inc., Corning, NY), was performed as described
previously (38, 39). Briefly, PIs [all in dipalmitoyl form from Echelon,
except for di-C
16
-PtdIns 3-P, which was from Matreya and di-C
16
-PtdIns
3,4,5-trisphosphate (-P
3
) , from Sigma] were sonicated and mixed with
goat IgG (2.8 mg/ml; Jackson Immune Research Laboratories, West
Grove, PA) to allow detection of injected cells. Purified recombinant
protein samples were concentrated in the injection buffer (100 mm KCl,
4mm sodium phosphate, pH 7.4) to 3 mg/ml protein and mixed with
goat IgG to visualize injected cells. Before microinjection of serum-
starved cells, the cell culture dish was filled with 5.0 ml prewarmed
starvation medium supplemented with 20 mm HEPES (pH 7.4) and
placed in a dish heater maintaining media temperature constant (36 C)
by a temperature controller (Warner Instruments, New York, NY). The
reagents were microinjected into the cytosol within a period of 20 min
(50 microinjected cells). Where indicated, cells were treated with in-
sulin (100 nm; 10 min) and/or pretreated with wortmannin (100 nm;20
min). After microinjection of the protein samples, cells were returned to
the CO
2
/O
2
incubator to recover for approximately 45 min and then
treated with insulin. Cells were fixed in 4% formaldehyde and double
stained with fluorescein isothiocyanate (FITC) antigoat IgG and rho-
damine-phalloidin (Molecular Probes; Eugene, OR) to visualize injected
cells and F-actin cytoskeleton, respectively. Coverslips were mounted on
slides using the Slow Fade Antifade Kit (Molecular Probes). Fluores-
cence analysis was performed in a Nikon Eclipse TE 200 inverted flu-
orescence microscope (Mager Scientific, Dexter, MI) using a 60 1.4 oil
immersion lens and a standard green fluorescence filter for GFP. Images
were captured with a SPOT RT Slider charge-coupled device camera
(Diagnostic Instruments, Sterling Heights, MI) mounted on the
microscope.
Lipid microinjection in differentiated, pEGFP-GLUT4 electroporated,
serum-starved 3T3-L1 adipocytes was performed in a similar manner
except for mixing sonicated lipids with Texas-Red dextran (70,000; Mo-
lecular Probes) for visualization of microinjected cells, as described
previously (38). Individual cells were monitored live within a 30-min
postmicroinjection period with a Nikon Eclipse TE 200 inverted fluo-
rescence microscope using a Hoffman Modulation contrast system with
a 40 objective and a standard green fluorescence filter for GFP. Cells
were subsequently stimulated with insulin (100 nm) for 20 min and
imaged live as described above. Images were captured with a SPOT RT
Slider charge-coupled device camera. Serum-starved 3T3-L1 adipocytes
cotransfected with pcDNA3.17xMyc-GLUT4 and either pEGFP-C2
3xPHD or pEGFP-C23xPHD3K cDNAs were stimulated with insulin
and fixed in 4% formaldehyde as described previously (35). Cells were
visualized by the enhanced green fluorescent protein (EGFP) fluores-
cence and a monoclonal anti-Myc antibody (ATCC, Manassas, VA; CRL
1729) that was subsequently detected with Texas-Red conjugated anti-
mouse IgG. Coverslips were mounted on slides, and the fluorescence
analysis was performed in a Nikon Eclipse TE 200 inverted fluorescence
microscope as described above.
Evaluation and quantitation of actin stress fibers and
GLUT4 translocation in single cells
Consistent with previously published criteria (32, 34), individual
CHO-T cells displaying parallel actin fibers that colocalized with the
nucleus were scored positive for F-actin stress fibers, whereas those
showing actin staining in the periphery were scored negative for F-actin
stress fibers but positive for membrane ruffles. Injected or transfected
individual 3T3-L1 adipocytes were scored positive for GLUT4 vesicle
translocation if appearance of a plasma membrane (PM) rim of GLUT4
fluorescence was documented. F-actin- or cell-surface-GLUT4-positive
cells were presented as percentage of the total number of microinjected
or transfected cells, given in the figure legends. Cells evaluated for
F-actin or GLUT4 PM redistribution were scored independently by two
observers.
3T3-L1 adipocyte subcellular fractionation and
GLUT4 translocation
After transfection (24 h) with indicated 3xPHD cDNA constructs,
3T3-L1 adipocytes were serum-starved (4 h), treated with insulin (100
nm, 20 min), and then subjected to subcellular fractionation exactly as
described in Ref. 35. Intracellular membrane (IM) and PM fractions were
subjected to SDS-PAGE and Western blotting with anti-GLUT4 poly-
clonal antibodies (a kind gift by Mike Czech) as described previously
(35).
HPLC analysis and data quantitation
Deacylated
32
P-labeled lipids were analyzed by HPLC on a Whatman
235-mm 4.60-mm column packed with 5-micron Partisphere SAX
(H
2
PO
4
) and eluted with a shallow ammonium phosphate gradient at
a flow rate of 1.0 ml/min as detailed elsewhere (16, 37). Coinjected
internal HPLC standards were prepared or purchased from sources
detailed previously (16, 37). Fractions were collected every 0.25 min, and
their radioactivity was analyzed simultaneously for
3
H- and
32
P-labeled
standard and products, respectively, with 2.0 ml of ScintiVerse liquid
scintillation cocktail on a liquid scintillation counter (Packard Instru-
ment Co., Inc.). The radioactivity of the TLC-scraped [
32
P]glycerophos
-
phorylinositol (GroPIns) 4,5-P
2
formed during PtdIns 5-P conversion
assay was analyzed with an on-line flow scintillation analyzer (Packard,
Meriden, CT; Radiomatic 525TR).
The radioactivity of the PI peaks was quantified by area integration
and is presented as a percentage of the radioactivity determined in
corresponding control samples or as a percentage of total PI radioac-
tivity, as indicated in figure legends. Because the separation of PtdIns
5-P from PtdIns 4-P was not complete, for quantitation of the counts
under the PtdIns 5-P peak riding on top of the PtdIns 4-P tail, the peak
was skimmed and presented as percent of the radioactivity determined
in control samples by the same approach.
PIKfyve lipid kinase activity
The in vitro activity was analyzed using anti-PIKfyve immunopre-
cipitates, derived from lysates of insulin-treated (100 nm, 10 min, 37 C)
or control cells, which were incubated with PtdIns and [
-
32
P]ATP for
15 min at 37 C. Radiolabeled products were extracted and analyzed by
TLC as previously described (38, 40).
Results
Insulin transiently increases PtdIns 5-P mass levels
To examine a plausible effect of insulin on the dynamics
of PtdIns 5-P intracellular production, we took advantage of
the mass assay that allows one to quantify PtdIns 5-P mass
levels in cells (16). We used two types of insulin-sensitive
cells: 3T3-L1 adipocytes, considered as prototypic insulin-
responsive cells; and CHO-T cells stably expressing the hu-
man insulin receptor. As illustrated in Fig. 1, both cell types
demonstrated marked increases of PtdIns 5-P mass levels
upon acute insulin treatment, as judged by the amounts of
PtdIns 4,5-P
2
synthesized in vitro from extracted PtdIns 5-P
and the action of type II PIPK. The effect reached a maximum
after 10 min of insulin stimulation, exceeding the basal PtdIns
5-P mass levels by 2.5-fold and 4.2-fold in 3T3-L1 adipocytes
and CHO-T cells, respectively (Fig. 1, C and D). The effect
was transient in both cell types and returned toward the basal
levels after 2030 min of insulin challenge (Fig. 1, AD).
Importantly, CHO-T cell pretreatment with wortmannin did
not abolish the insulin-induced PtdIns 5-P elevation (Fig. 1E).
Calculation of the PtdIns 5-P mass levels showed substan-
tially higher amounts (70-fold) of basal PtdIns 5-P in 3T3-L1
adipocytes compared with CHO-T cells, equal to 580 70
and 8.5 3 pmol/mg protein in 3T3-L1 adipocytes and
Sbrissa et al. PtdIns 5-P Mediates Insulin Signaling Endocrinology, November 2004, 145(11):4853 4865 4855
FIG. 1. Acute insulin induces robust increases of PtdIns 5-P mass levels in 3T3-L1 adipocytes and CHO-T cells in a wortmannin-independent
manner. Serum-starved 3T3-L1 adipocytes (A and C) or CHO-T cells (B, D, and E) were treated with or without insulin (100 nM)at37Cfor
the indicated time intervals (AD) or for 10 min subsequent to pretreatment with or without wortmannin (100 nM) for 20 min as indicated (E).
Cells were then washed and the lipids extracted as detailed in Materials and Methods. PIs were isolated on neomycin-coated glass beads and
subjected to in vitro conversion by type II PIP kinase for1hat30Cinthepresence of [
-
32
P]ATP.
32
P-labeled products were separated by TLC
and visualized by autoradiography. Shown are representative autoradiograms (A, B, and E) and quantitation from five (C) and four (D)
independent experiments for 3T3-L1 adipocytes and CHO-T cells, respectively, presented as a percentage of PtdIns 4,5-P
2
from the corresponding
nontreated cells for each individual time point (mean
SEM). PtdIns 5-P intracellular mass (see text) was calculated from PtdIns 5-P standards
run in parallel in each experiment (lane 7 in B, 10 pmol).
4856 Endocrinology, November 2004, 145(11):4853 4865 Sbrissa et al. PtdIns 5-P Mediates Insulin Signaling
CHO-T cells, respectively. Together, these results clearly
demonstrate a transient elevation of PtdIns 5-P mass asso-
ciated with acute insulin stimulation in insulin-sensitive cell
types. Importantly, this effect could be detected at very low
basal PtdIns 5-P mass (see below).
The dramatic difference in the PtdIns 5-P intracellular
levels in 3T3-L1 adipocytes vs. CHO-T cells observed here by
mass assay is consistent with our previously published data,
reproduced herein (Refs. 16 and 37, and data not shown)
using HPLC inositol-head group analysis for determining
the basal
32
P-PtdIns 5-P accumulation in [
32
P]orthophos
-
phate-labeled cells. This analysis found radiolabeled PtdIns
5-P in quiescent 3T3-L1 adipocytes at quite substantial
amounts, comprising as much as 12% of PtdIns 4-P, in con-
trast to undetectable levels in CHO-T cells (16, 37). To de-
termine whether insulin-dependent increases of PtdIns 5-P
levels could be detected by similar analysis, deacylated lip-
ids, extracted from serum-starved
32
P-labeled 3T3-L1 adipo
-
cytes or CHO-T cells, stimulated or not, for 10 min with the
hormone, were resolved on HPLC columns by a shallow
ammonium phosphate gradient, shown previously to give a
better resolution of PtdIns 5-P from the descending edge of
the PtdIns 4-P peak (16, 40). Using a quantitation method
described in Materials and Methods, we detected only small
increases of [
32
P]PtdIns 5-P accumulation in response to in
-
sulin stimulation of 3T3-L1 adipocytes and no
32
P-PtdIns 5-P
accumulation in insulin-stimulated CHO-T cells (Fig. 2). This
is most likely due to the low basal levels of radiolabeled
PtdIns 5-P in the latter cell type that are below the sensitivity
of the HPLC-inositol head group detection, as we have dem-
onstrated previously (37). Comparative analysis of data from
both HPLC and mass assays reveals that, even at a 4-fold
increase,
32
P-PtdIns 5-P will represent only approximately
0.75% of the PtdIns 4-P peak in CHO-T cells and will most
likely remain buried within the trailing edge of the abundant
PtdIns 4-P peak long before the latter reaches base line.
Combined data from 3T3-L1 adipocytes and CHO-T cells
indicate that HPLC head group analyses are insufficient for
measuring basal or stimulated PtdIns 5-P in these and likely
in other cell types.
PtdIns 5-P, but not other PIs, mimics insulin in stress fiber
disassembly in CHO-T cells
The observed insulin-induced robust increase of PtdIns
5-P mass, demonstrated above, poses a question about the
nature of insulin-regulated cellular processes where PtdIns
5-P operates. One well-established acute insulin response in
CHO-T cells is the marked reduction of the number and
length of F-actin stress fibers that are seen in serum-starved
cells (30, 32, 33). Intriguingly, similarly to insulin-induced
changes in PtdIns 5-P mass (Fig. 1), the insulin-regulated
disassembly of F-actin stress fibers has been found to be
transient with a maximum at approximately 15 min (Fig. 3A)
and restoration of the actin stress fiber network after 30 min
of insulin presence (Ref. 32 and this study, not shown). Re-
markably, unlike the membrane ruffling that operates in a PI
3-kinase-dependent manner, insulin-induced loss of F-actin
stress fibers is shown to proceed by a PI 3-kinase-indepen-
dent (29, 30) or PtdIns 3,4,5-P
3
-independent mechanism (41,
42). Therefore, we analyzed the role of PtdIns 5-P in insulin-
regulated actin stress fiber disassembly in CHO-T cells. We
used several approaches to modulate intracellular PtdIns 5-P
levels. First, to increase PtdIns 5-P intracellular levels, the
lipid was microinjected in the cytoplasm of serum-starved
CHO-T cells. Cells were then fixed, and the organization of
F-actin was examined by rhodamine-conjugated phalloidin
and fluorescence microscopy. Remarkably, PtdIns 5-P, like
insulin, was found to induce stress fiber breakdown (Fig. 3B).
Quantitation from three independent experiments demon-
strated that the potency of microinjected PtdIns 5-P to induce
stress fiber breakdown is equal to that of insulin (Fig. 3C). To
test the specificity of this PtdIns 5-P effect we have examined
other PI derivatives under similar conditions. Intriguingly,
none of the other PIs tested here was found to display the
ability to induce stress fiber breakdown (Fig. 3C). In all cases
the F-actin stress fiber network displayed a similar appear-
ance to that in the control noninjected quiescent cells (Fig.
3B). Furthermore, CHO-T cell pretreatment with the PI 3-
kinase inhibitor wortmannin failed to significantly affect the
loss of stress fibers not only due to insulin (Ref. 30 and this
study, not shown) but also those due to microinjected PtdIns
5-P (Fig. 3B). These data are consistent with the notion that,
in CHO-T cells, PtdIns 5-P mediates insulins effect on stress
fiber depolymerization and that this proceeds independently
of PI 3-kinase activation. Under the experimental conditions
used here, we were unable to observe PtdIns 5-P-dependent
membrane ruffling in different optical planes of the cells,
indicating differential mechanisms underlying the stress fi-
ber breakdown and membrane ruffling.
We sought to confirm the observed effect of high PtdIns
5-P levels on the F-actin stress fiber loss by an alternative
FIG. 2. HPLC analysis reveals
32
P-PtdIns 5-P accumulation in re
-
sponse to acute insulin in 3T3-L1 adipocytes but not in CHO-T cells.
3T3-L1 adipocytes or CHO-T cells were serum/phosphate starved for
1 h and then labeled with [
32
P]orthophosphate for3hinphosphate/
serum-free DMEM as described in Materials and Methods. Cells were
then treated with insulin (100 n
M) for 10 min at 37 C or left untreated.
Cell lipids were extracted, deacylated, and coinjected with [
3
H]GroPIns
5-P, [
3
H]GroPIns 4-P, [
3
H]GroPIns 3-P, and [
3
H]GroPIns 4,5-P
2
as
internal HPLC standards. Fractions, collected every 0.25 min were
monitored for [
3
H] and [
32
P] radioactivity by liquid scintillation count
-
ing.
32
P-radioactivity was plotted and the counts within the elution
times corresponding to GroPIns 5-P determined and normalized to the
GroPIns 4,5-P
2
counts. Shown is a quantitation of HPLC elution
profiles with respect to insulin-stimulated [
32
P]PtdIns 5-P accumu
-
lation expressed as a percentage of basal [
32
P]PtdIns 5-P in 3T3-L1
adipocytes of two independent experiments with similar results. A
[
32
P]PtdIns 5-P peak was not detected in basal or insulin-stimulated
CHO-T cells in four independent cell labelings.
Sbrissa et al. PtdIns 5-P Mediates Insulin Signaling Endocrinology, November 2004, 145(11):4853 4865 4857
approach. We have recently demonstrated that the PIKfyve
enzyme produces intracellularly PtdIns 5-P, because a
HEK293 stable cell line expressing PIKfyve
WT
at a level 7-fold
higher than the endogenous protein displayed approxi-
mately 2-fold elevated PtdIns 5-P mass (16). To examine
whether increased PtdIns 5-P due to PIKfyve action is also
associated with actin stress fiber breakdown, we ectopically
expressed PIKfyve
WT
in CHO-T cells and assessed the F-actin
structures by rhodamine-phalloidin labeling of serum-
starved cells. We found that expression of EGFP-PIKfyve
WT
also induced loss of actin stress fibers (Fig. 4), whereas con-
trol EGFP expression did not (see Fig. 6A, a and b). Loss of
actin stress fibers was observed in approximately 45% of
EGFP-PIKfyve
WT
-transected CHO-T cells, representing ap
-
proximately 60% of insulins effect on stress fiber disassem-
bly in this cell type (see Fig. 3C). Clearly, the combined data
indicate that PtdIns 5-P, elevated either by direct lipid mi-
croinjection or enzymatically, mimics insulin action on stress
fiber breakdown.
Scavenging intracellular PtdIns 5-P inhibits insulin-
regulated actin stress fiber disassembly
If high PtdIns 5-P mimics insulin, then its elimination from
cells would be expected to oppose insulin effects. To test this
hypothesis, we explored recently identified PHD domain
modules, shown to preferentially bind PtdIns 5-P (19). The
PHD domain of ING2, a candidate tumor suppressor protein,
binds PtdIns 5-P with high affinity but could also interact
with PtdIns 4-P and PtdIns 3-P, although to a lesser extent
(19). In contrast, the PHD domain of ACF protein is exclusive
for PtdIns 5-P, but the binding affinity is lower than that of
ING2-PHD (19). Therefore, we tested the PHD domain fin-
gers from both proteins and used their three tandem repeat
forms to increase binding (19). We first confirmed, by the
PtdIns 5-P mass assay, the ability of recombinantly produced
GST-3xPHD domain fusion-peptide fragment to knock
down functional PtdIns 5-P. Results presented in Fig. 5, dem-
onstrating a dramatic inhibition of in vitro PtdIns 4,5-P
2
pro
-
duction from both basal or insulin-stimulated PtdIns 5-P
intracellular pools in the presence of GST-ING23xPHD pu-
rified peptide, but not GST alone, are consistent with the
notion that the 3xPHD peptide acts as a powerful PtdIns 5-P
scavenger.
The ability of 3xPHD peptides to arrest stress fiber break-
down induced by insulin was examined in CHO-T cells
ectopically expressing ING23xPHD or ACF-3xPHD con-
structs fused with EGFP. Remarkably, expression of both
EGFP-ING23xPHD and EGFP-ACF-3xPHD peptides, but
not EGFP alone, profoundly blocked the loss of F-actin stress
FIG. 3. Microinjected PtdIns 5-P, but not other PIs, mimics insulin in
stimulating stress fiber breakdown in CHO-T cells. A, Serum-starved
CHO-T cells expressing the insulin receptor were treated with insulin
(100 nM, 10 min, 37 C) or left untreated. Cells were washed, fixed in
formaldehyde, and permeabilized. F-actin was visualized with
rhodamine-phalloidin. Note the loss of actin stress fibers upon insulin
stimulation. B, Serum-starved CHO-T cells were comicroinjected
within a 20-min period with indicated PIs and goat IgG to visualize
injected cells. Cells in panels e and f were pretreated with wortmannin
(Wort) (100 nM) for 20 min at 37 C and then microinjected. Cells were
washed, fixed, permeabilized, and stained for F-actin and goat IgG
with rhodamine-phalloidin (a, c, and e), and FITC-conjugated donkey
antigoat IgG (b, d, and f), respectively. Individual cells, positive for
lipid microinjection were scored for the presence of parallel stress
fibers with the cell nucleus as described in Materials and Methods.
Shown are representative images from three independent experiments
with similar results. Arrows in a, c, and e depict microinjected cells,
visualized by FITC-conjugated donkey antigoat IgG (b, d, and f). Note
that only the PtdIns 5-P-microinjected cells display loss of actin stress
fibers, which is seen in both the absence (panel c, the cell pointed to
by an arrow) or presence of wortmannin pretreatment (panel e, the
two cells pointed to by arrows). C, Quantitation of positive cells for
actin stress fibers after microinjection of indicated PIs, presented as
a percentage of the total number of microinjected cells (50100 cells/
condition). Control (con) or insulin (ins)-treated cells were microin-
jected only with vehicle (goat IgG in injection buffer). Each bar rep-
resents the mean SEM from three independent experiments.
4858 Endocrinology, November 2004, 145(11):4853 4865 Sbrissa et al. PtdIns 5-P Mediates Insulin Signaling
fibers in response to insulin (Fig. 6, A and B). The peptides
had no significant effect on the stress fiber F-actin structures
in quiescent cells (Fig. 6, A and B). By contrast, expression of
a PtdIns 5-P-binding-deficient version of the EGFP-ING2
3xPHD domain, bearing substitutions in K
49
,K
51
, and K
56
(3xPHD3K; Ref. 19), had practically no effect on insulin-
induced actin stress fiber breakdown in CHO-T cells (Fig. 6,
A and B).
To confirm the inhibitory effect of PtdIns 5-P scavenging
by an alternative approach, we have inspected the organi-
zation of F-actin stress fibers in CHO-T cells that were mi-
croinjected with the GST-ING23xPHD peptide before stim-
ulation with insulin. As illustrated in Fig. 6C, whereas the
GST-3xPHD domain did not significantly change the actin-
stress fiber network in quiescent cells, it almost completely
prevented the stress fiber breakdown due to insulin. By con-
trast, control microinjection of GST alone failed to abort
insulin-induced loss of actin stress fibers (data not shown).
Effect of PtdIns 5-P and other PIs on GLUT4 translocation
in 3T3-L1 adipocytes
Until recently, PI 3-kinase activation and 3-polyphospho-
inositide production were considered to be necessary and
sufficient for insulin to stimulate GLUT4 vesicle transloca-
tion from intracellular storage sites to the PM and the sub-
sequent glucose uptake into fat and muscle cells. The obser-
vation that cell permeable derivatives of PtdIns 3,4,5-P
3
failed to mimic insulins effect on glucose transport (43) not
only suggested an additional PI 3-kinase-independent sig-
naling pathway in adipocytes (22, 23) but also has proven
critical the need for direct lipid-derived studies. Because
PtdIns 5-P not only constitutes a substantial subfraction of
total PtdInsP in resting 3T3-L1 adipocytes (16) but was also
found up-regulated in response to acute insulin stimulation
of this cell type (Fig. 1), we examined whether microinjected
PtdIns 5-P could affect GLUT4 vesicle dynamics. We fol-
lowed the dynamics of transiently expressed EGFP-GLUT4
shown previously to effectively reflect the behavior of en-
dogenous GLUT4 in these cells (35, 44, 45). In addition to
PtdIns 5-P, in this setting we have examined PtdIns 4-P,
PtdIns 3-P, PtdIns 3,5-P
2
, and PtdIns 3,4,5-P
3
to test the spec
-
ificity of the effect. In all cases, 24 h post transfection of
3T3-L1 adipocytes with pEGFP-GLUT4 construct, lipids
mixed with Texas-Red dextran were microinjected in the
cytoplasm of serum-starved cells. EGFP-GLUT4 vesicle ap-
pearance at the cell surface was monitored in single cells over
a time course of 30 min, while keeping the dish at 36 C by
a temperature controller. At the end of the observation pe-
riod, cells were stimulated with insulin for an additional 20
min to examine the responsiveness of the individual cells to
translocate EGFP-GLUT4 to the cell surface. This last step is
essential to avoid an artificial underestimation of the micro-
injected PIs effect due to two reasons. First, even if nonin-
jected, only half of the 3T3-L1 adipocytes display the ability
to translocate perinuclear EGFP-GLUT4 to the cell surface in
response to insulin, as observed previously in several stud-
ies, including our own (35). Second, unresponsiveness due to
cell damage associated with the PI microinjection is also
possible. Therefore, in the quantitative data, summarized in
Fig. 7B, we score the appearance of the characteristic PM rim
of the EGFP-GLUT4 fluorescence signals due to microin-
jected PI as well as the individual responsiveness of injected
cells to a subsequent insulin challenge. Importantly, under
the specified experimental conditions, the only one of the
here-examined PIs that displayed an ability to mimic insulin
in translocating EGFP-GLUT4 to the cell surface was PtdIns
5-P (Fig. 7, A and B). This effect was apparent in approxi-
mately 50% of the injected cells that responded positively to
a subsequent insulin challenge (Fig. 7B). Figure 7A (df)
FIG. 4. Expression of PIKfyve
WT
results in stress fiber breakdown in
CHO-T cells. Twenty-four hours post transfection with pEGFP-PIK-
fyve
WT
and subsequent to serum deprivation, CHO-T cells, stably
expressing the insulin receptor, were fixed in formaldehyde, perme-
abilized, and stained with rhodamine-phalloidin (A), as described in
Materials and Methods. PIKfyve
WT
-expressing cells, determined by
the EGFP fluorescence signals (B), were scored for the presence of
parallel actin stress fibers with the cell nucleus as described in Ma-
terials and Methods. Note the loss of actin stress fibers in the PIK-
fyve
WT
-expressing cells (arrowheads), seen in 45% of the transfected
cells (insulin-induced stress fiber breakdown occurred in 75% of the
nontransfected cells as illustrated in Fig. 3C).
FIG. 5. 3xPHD domain sequesters functional PtdIns 5-P. Serum-
starved 3T3-L1 adipocytes were treated with or without insulin (100
nM) for 10 min at 37 C. Cells were then washed and the lipids ex-
tracted. PIs were isolated on neomycin-coated glass beads, preincu-
bated with GST or GST-ING23xPHD peptides (2
g each) for 10 min,
and subjected to in vitro conversion by type II PIP kinase for1hat
30 C in the presence of [
-
32
P]ATP.
32
P-labeled products were sepa
-
rated by TLC and visualized by autoradiography. Shown is a repre-
sentative autoradiogram of three independent experiments with sim-
ilar results. Note the marked inhibition of PtdIns 4,5-P
2
synthesis in
the presence of the GST-ING23xPHD peptide (lanes 2 and 4).
Sbrissa et al. PtdIns 5-P Mediates Insulin Signaling Endocrinology, November 2004, 145(11):4853 4865 4859
illustrates one typical cell, in which the cell surface EGFP-
GLUT4 appearance is readily seen 30 min after PtdIns 5-P
microinjection. The effect is comparable with that observed
in a vehicle-microinjected cell after 20 min of insulin chal-
lenge (Fig. 7A, ac). By contrast, cells microinjected with
PtdIns 3,4,5-P
3
did not exhibit a noticeable effect in EGFP-
GLUT4 cell surface accumulation under similar conditions
(Figs. 7A, gi, and B). Given the GLUT4-PM rim is apparent
only 1520 min after PtdIns 5-P microinjection, together with
the lack of visible cell surface EGFP-GLUT4 before micro-
injection, and kinetic data for slow constitutive exocytosis of
GLUT4 in this cell type (230 min; Ref. 46), these results are
consistent with the notion that PtdIns 5-P acts by triggering
GLUT4 exocytosis rather than inhibiting GLUT4 internaliza-
tion. Clearly, these results demonstrate that direct adminis-
tration of PtdIns 5-P mimics, at least in part, the insulin effect
on cell-surface translocation of GLUT4 vesicles.
The role of PtdIns 5-P in insulin-regulated GLUT4 trans-
location in 3T3-L1 adipocytes was further substantiated by
the ability of the 3xPHD domain of ING2 or ACF to inhibit
this effect (Fig. 8). As illustrated in Fig. 8, A and B, in the
presence of expressed EGFP-3xPHD domains, acute insulin
was unable to induce cell surface redistribution of ectopically
expressed Myc-GLUT4. Conversely, expression of a PtdIns
5-P-binding-deficient version of the EGFP-ING23xPHD do-
main, bearing substitutions in K
49
,K
51
, and K
56
(3xPHD3K;
Ref. 19), had practically no effect on insulin-induced cell
surface appearance of myc-GLUT4 (Fig. 8, A and B). Similar
FIG. 6. 3xPHD peptides inhibit insulin-regulated loss of F-actin stress fiber disassembly. A, CHO-T cells expressing the insulin receptor were
transfected with pEGFP alone, pEGFP-ING23xPHD, pEGFP-ING23xPHD3K, or pEGFP-ACF-3xPHD as indicated. Twenty hours post
transfections, serum-starved cells were stimulated with insulin (100 nM) for 10 min at 37 C or left untreated as indicated. Cells were washed,
fixed in formaldehyde, and stained with rhodamine-phalloidin to visualize F-actin as described in Materials and Methods. Arrowheads in a,
c, e, g, i, and k depict the transfected cells seen in b, d, f, h, j, and l by the GFP fluorescence signals. Note in g and k the transfected cells that
show stress fibers despite the insulin treatment. The second arrow in e points to a low-level expressing cell. B, Quantitation of transfected cells
positive for actin stress fibers presented as a percentage of the total number of cells transfected with each construct (counted 200 cells/
experiment/condition). Each bar represents the mean SEM from three experiments. C, Serum-starved CHO-T cells were microinjected with
GST-ING23xPHD peptide (3 mg/ml) mixed with goat IgG to visualize the injected cells. Approximately 45 min after microinjection, cells were
stimulated with insulin (100 nM) for 10 min at 37 C or left untreated as indicated. Cells were washed, fixed, and stained with rhodamine-
conjugated phalloidin and FITC-conjugated donkey antigoat IgG to visualize F-actin (a and c) and microinjected cells (b and d). Note in c the
three noninjected cells at the image top demonstrating the typical loss of actin stress fibers, in contrast to the six injected cells showing multiple
F-actin stress fibers despite the insulin treatment.
4860 Endocrinology, November 2004, 145(11):4853 4865 Sbrissa et al. PtdIns 5-P Mediates Insulin Signaling
results were obtained by assessing the endogenous GLUT4
distribution in subcellular fractions derived from 3T3-L1 adi-
pocytes transfected with the PHD domain reagents. Whereas
expression of EGFP-ING23xPHD3K (Fig. 8C) or EGFP alone
(not shown) did not affect insulin-induced GLUT4 translo-
cation to the PM fraction, expression of EGFP-ING23xPHD
was inhibitory to a considerable extent (40%). The trans-
fection efficiency of these constructs was at approximately
50%, implying that EGFP-ING23xPHD expression arrested
almost completely insulins effect on GLUT4 translocation.
Clearly, these results demonstrate that insulins effect on PM
translocation of GLUT4 is specifically influenced by PtdIns
5-P-binding reagents, consistent with the requirement for
PtdIns 5-P.
PtdIns 4,5-P
2
levels and PIKfyve enzymatic activity in
insulin-stimulated 3T3-L1 adipocytes and CHO-T cells
Mobilization of several pathways may underlie the insulin-
induced robust increase of PtdIns 5-P mass in 3T3-L1
adipocytes and CHO-T cells, demonstrated above. These
involve up-regulation of the pathways for 5-polyphospho-
inositide breakdown or PtdIns 5-P synthesis from PtdIns
and/or down-regulation of pathways responsible for PtdIns
5-P clearance. We first tested whether the PtdIns 4,5-P
2
break
-
down could account for the observed insulin-dependent
increase of PtdIns 5-P mass. Such a mechanism has been
recently reported to underlie the PtdIns 5-P rise in mamma-
lian cells due to S. flexneri invasion (17). We examined the
amounts of accumulated radiolabeled PtdIns 4,5-P
2
after a
10-min insulin stimulation of serum-starved
32
P-labeled
3T3-L1 adipocytes and CHO-T cells. As illustrated in Fig. 9,
the HPLC-head group analysis of extracted and deacylated
radiolabeled lipids failed to demonstrate significant insulin-
dependent changes in [
32
P]PtdIns 4,5-P
2
accumulation in
both cell types. This result is therefore consistent with a lack
of significant insulin-dependent increases of the PtdIns
4,5-P
2
breakdown and/or inhibition of the PtdIns 5-P clear
-
ance through the type II PIPK pathway. However, it should
be emphasized that, due to the markedly greater levels of
PtdIns 4,5-P
2
vs. PtdIns 5-P (Refs. 16 and 37 and this study),
even an insignificant decrease in PtdIns 4,5-P
2
could yield
substantial increases of PtdIns 5-P. Therefore, subtle, spa-
tially restricted variations in PtdIns 4,5-P
2
breakdown by a
yet-to-be-identified 4P-phosphatase and/or in PtdIns 4,5-P
2
synthesis by the type II PIPK pathway to explain the insulin-
induced rise in PtdIns 5-P, could not be ruled out, even in the
absence of significant changes in the overall
32
P-PtdIns 4,5-P
2
accumulation in response to insulin.
In a cellular context, PIKfyve is the enzyme responsible for
the synthesis of PtdIns 5-P from PtdIns (16). Possible insulin-
regulated PIKfyve activation in the time course of the ob-
served elevated PtdIns 5-P production in response to insulin
was tested in both 3T3-L1 adipocytes and CHO-T cells. In
agreement with our previous observation in insulin-stimu-
lated 3T3-L1 adipocytes, insulin action in CHO-T cells did
not result in significant changes of the in vitro synthesized
PtdIns 5-P by PIKfyve immunoprecipitates derived from cell
lysates of basal or stimulated cells (Ref. 47, and data not
shown). However, it should be emphasized that, although no
FIG. 7. PtdIns 5-P microinjection, but not other PIs, mimics insulin-
induced GLUT4 translocation in 3T3-L1 adipocytes. A, 3T3-L1 adi-
pocytes were electroporated with pEGFP-GLUT4 as described in Ma-
terials and Methods. On the next day, cells were serum-starved, and
the EGFP-GLUT4-expressing cells (a, d, and g) were injected with
Texas-Red (TxR) dextran in injection buffer alone (b) or mixed with
the indicated PIs (e and h). Twenty to 30 min post injection, individual
cells were monitored live for EGFP-GLUT4 translocation (d and g).
Cells were treated with insulin (100 nM) for an additional 20 min
(ac). Shown are representative images depicting the characteristic
GLUT4 PM rim by insulin in a vehicle-injected cell (a c) and the effect
of PtdIns 5-P microinjection (5-P, df) or PtdIns 3,4,5-P
3
microinjec
-
tion (3,4,5-P3, gi) on EGFP-GLUT4 localization. Note that PtdIns
5-P, but not PtdIns 3,4,5-P
3
produces a EGFP-GLUT4-plasma-mem
-
brane rim in the absence of insulin. B, Quantitation of cells positive
for EGFP-GLUT4 cell-surface fluorescence in response to microin-
jection of TxR alone or TxR mixed with indicated PIs, without (black
bars) or with (open bars) a subsequent insulin treatment, expressed
as a percentage of the total number of monitored microinjected cells
(24 cells for TxR dextran; 14 for PtdIns 3-P; 12 for PtdIns 4-P; 33 for
PtdIns 5-P; 14 for PtdIns 3,5-P
2
, and 22 for PtdIns 3,4,5-P
3
). Note that
only PtdIns 5-P was able to trigger the EGFP-GLUT4 redistribution
to the PM, and that only around 30% of the microinjected cells were
insulin responsive.
Sbrissa et al. PtdIns 5-P Mediates Insulin Signaling Endocrinology, November 2004, 145(11):4853 4865 4861
activation of in vitro PtdIns 5-P-producing activity was ob-
served in response to insulin, PIKfyves functional signifi-
cance to the insulin-induced elevation of PtdIns 5-P could be
confined to relocation, as we have documented previously in
3T3-L1 adipocytes (48).
Discussion
The present studies identify PtdIns 5-P as a novel key
signaling intermediate in acute insulin action on F-actin
stress fiber disassembly and GLUT4 translocation in insulin-
sensitive CHO-T cells and 3T3-L1 adipocytes, which operates
by a PI 3-kinase-insensitive mechanism. This finding is based
on several lines of experimental evidence: first, acute insulin
markedly increased PtdIns 5-P levels in both CHO-T cells
and 3T3-L1 adipocytes, which proceeded in a wortmannin-
independent mechanism (Fig. 1); second, PtdIns 5-P, when
FIG. 8. 3xPHD peptide, but not PtdIns 5-P-binding-deficient
3xPHD3K mutant, inhibits insulin-regulated GLUT4 vesicle trans-
location in 3T3-L1 adipocytes. A, 3T3-L1 adipocytes were electropo-
rated to coexpress Myc-GLUT4 with either EGFP alone, EGFP-ING2-
3xPHD, or EGFP-ING2-3xPHD3K mutant as described in Materials
and Methods. On the next day, cells were serum-starved, then stim-
ulated with insulin (100 nM, 20 min) or left untreated, washed, fixed,
and processed for fluorescence microscopy as detailed in Materials
and Methods. Expressed GLUT4 was detected with anti-Myc mono-
clonal antibody and Texas-Red-conjugated secondary antibodies (a, d,
g, and j). The expression of GFP-based constructs was visualized by
the GFP fluorescence (b, e, h, and k). The images shown are repre-
sentative from three independent experiments in cells coexpressing
the plasmids. Bar,20
m. B, Quantitation of cells positive for Myc-
GLUT4-cell-surface fluorescence in basal or insulin-stimulated 3T3-L1
adipocytes doubly transfected with pcDNA3.1-Myc-GLUT4 and pEGFP
vector alone, pEGFP-ING2-3xPHD, or pEGFP-ING2-3xPHD3K,
FIG.9.
32
P-PtdIns 4,5-P
2
intracellular accumulation remains un
-
changed by acute insulin. 3T3-L1 adipocytes or CHO-T cells were
serum/phosphate starved for 1 h and then labeled with
[
32
P]orthophosphate for3hinphosphate/serum-free DMEM as de
-
scribed in Materials and Methods. Cells were then treated with in-
sulin (100 n
M) for 10 min at 37 C or left untreated. Cell lipids were
extracted, deacylated and coinjected with [
3
H]GroPIns 5-P,
[
3
H]GroPIns 4-P, [
3
H]GroPIns 3-P and [
3
H]GroPIns 4,5-P
2
as internal
HPLC standards. Fractions, collected every 0.25 min, were monitored
for [
3
H] and [
32
P] radioactivity by liquid scintillation counting.
32
P-
radioactivity was plotted and the counts within the elution times
corresponding to the [
32
P]GroPIns peaks determined by the above
[
3
H]-labeled standards were summed (total radioactivity). Shown is
a quantitation of HPLC elution profiles with respect to
32
P-PtdIns
4,5-P
2
, presented as a percentage (mean SEM) of total radioactivity
of three (for 3T3-L1 adipocytes) or two independent experiments
(CHO-T) with similar results.
presented as a percentage of the total number of coexpressing cells for
each condition. Results shown are from three independent experi-
ments, with observation of approximately 200 cells/condition/exper-
iment, mean SEM. C, 3T3-L1 adipocytes were electroporated to
express EGFP-ING23xPHD or EGFP-ING23xPHD3K as described
in Materials and Methods. On the next day, cells were serum-starved,
stimulated with insulin (100 nM, 20 min) or left untreated, and then
fractionated to obtain IM, PM, and cytosol. Indicated fractions were
resolved by SDS-PAGE, and insulin-induced GLUT4 translocation
was determined by immunoblotting [Western blot (WB)] with poly-
clonal anti-GLUT4 antibodies. Shown is a chemiluminescence detec-
tion of a representative blot of two independent experiments with
similar results. Note the inhibition (3040%) of insulins effect on
GLUT4 in both IM and PM under 3xPHD vs. 3xPHD3K expression.
4862 Endocrinology, November 2004, 145(11):4853 4865 Sbrissa et al. PtdIns 5-P Mediates Insulin Signaling
elevated in CHO-T cells by PtdIns 5-P cytoplasmic micro-
injection or by PIKfyve
WT
ectopic expression, mimicked in
-
sulins effect on F-actin stress fiber breakdown (Figs. 3 and
4); third, wortmannin pretreatment did not prevent the Pt-
dIns 5-P-induced loss of F-actin stress fibers in these cells
(Fig. 3); fourth, PtdIns 5-P was able to partially induce EGFP-
GLUT4 translocation onto the 3T3-L1 adipocyte cell surface
(Fig. 7); and fifth, both the insulin-induced loss of F-actin
stress fibers and insulin-induced GLUT4 cell surface redis-
tribution were largely abrogated in the presence of ectopi-
cally expressed or microinjected 3xPHD finger domain pep-
tides that sequester functional PtdIns 5-P (Figs. 5, 6, and 8).
Thus, PtdIns 5-P functions as a positive regulatory interme-
diate in insulin-regulated organization of F-actin and dy-
namics of GLUT4 vesicles.
A central role in actin filament remodeling stimulated by
growth factors, such as insulin or IGF-1, has been attributed
to the activated PI 3-kinase pathway and the subsequent
activation of the small GTP-binding proteins (34, 49). How-
ever, more recent data indicate that insulin action relays two
types of signals to modulate F-actin dynamics, one that is PI
3-kinase dependent and another that proceeds in a PI 3-
kinase-independent manner. Thus, pharmacological inhibi-
tion of PI 3-kinase did not affect insulin action on F-actin
stress fiber breakdown in CHO-T and 3T3-L1 fibroblasts,
whereas insulins effect on membrane ruffling was abrogated
(29, 30). Likewise, SHIP and GRP1, which hydrolyze or se-
quester the PI 3-kinase product PtdIns 3,4,5-P
3
, respectively,
did not inhibit insulins effect on stress fiber disassembly in
HIRcB cells, yet they both inhibited membrane ruffling (41,
42). Based on the data presented herein, we suggest that
PtdIns 5-P selectively mediates the PI 3-kinase-independent
insulin signaling to F-actin stress fiber breakdown but it is
not involved in membrane ruffling. The latter requires acti-
vation of the PI 3-kinase pathway, as suggested previously
(34, 50). The predicted role of PtdIns 5-P in the molecular
mechanisms of F-actin stress fiber disassembly is further
supported by recent studies demonstrating disappearance of
actin stress fibers, along with membrane bleb formation,
upon expression of the S. flexneri virulence factor IpgD in
mammalian cells (17). However, IpgD expression is associ-
ated with rather complex changes in the overall PI intracel-
lular metabolism, whereby, in addition to the rise of PtdIns
5-P and a massive PtdIns 4,5-P
2
hydrolysis, an activation of
the PI 3-kinase pathway occurs to increase higher 3-PIs such
as PtdIns 3,5-P
2
, PtdIns 3,4-P
2
, and PtdIns 3,4,5-P
3
(17), each
able to induce actin cytoskeleton remodeling on its own (8).
The data presented herein, based on a direct and selective
manipulation in PtdIns 5-P levels, provide compelling and
unequivocal evidence to implicate PtdIns 5-P in the regula-
tion of F-actin stress fiber dynamics. Moreover, loss of F-actin
stress fibers was not produced by any of the microinjected
3-PIs used here (Fig. 3, B and C), consistent with the notion
that activation of the PI 3-kinase pathway does not play a role
in this insulin effect. Rather, our data infer that an increase
in PtdIns 5-P mass is both required and sufficient to induce
the loss of actin stress fibers in response to insulin.
According to a currently held view, insulin-regulated
translocation of GLUT4 vesicles from intracellular storage
compartments to the 3T3-L1 adipocyte cell surface appears
to require not only a PI 3-kinase-dependent but also a PI
3-kinase-independent cascade (2224). Our observations for
activated GLUT4 vesicle exocytosis to the cell surface in
response to PtdIns 5-P microinjection in 3T3-L1 adipocytes
(Fig. 7), together with insulin-induced increases in PtdIns 5-P
in this cell type (Fig. 1), are consistent with the notion that the
PtdIns 5-P pathway may be necessary in the insulin-activated
PI 3-kinase-independent cascade. Studies in several labora-
tories have implicated the mobilization of actin filament re-
arrangement in insulin action on GLUT4 because various
pharmacological agents that stabilize or disrupt F-actin
structures are inhibitory (25, 26, 51). Recently it has been
demonstrated that GLUT4 vesicle translocation in response
to insulin action in 3T3-L1 adipocytes requires remodeling of
two distinct compartmentalized F-actin populations (31).
They both need activated TC10, a Rho family GTPase specific
for 3T3-L1 adipocytes, and proceed in a PI 3-kinase-inde-
pendent manner (31). Although we did not examine the
effect of PtdIns 5-P on the complex F-actin reorganization in
3T3-L1 adipocytes in this study, it is conceivable that insulin-
elevated PtdIns 5-P mass promotes an active F-actin break-
down as a first step to a complex F-actin remodeling for
optimal efficiency of GLUT4 vesicle delivery to the adipocyte
PM. Further work will be necessary to support a plausible
relationship between the elevated PtdIns 5-P and activated
TC10 pathway in response to insulin, as well as the relevance
of a PtdIns 5-P pathway in insulin action on glucose uptake.
The enzymology underlying the observed robust insulin-
dependent PtdIns 5-P increase was also addressed in this
study, but due to the complex PI intracellular metabolism,
the data still do not allow a definitive conclusion. We have
previously shown that the synthetic arm in PtdIns 5-P pro-
duction, at least in part, is due to PIKfyve, because ectopic
expression of PIKfyve
WT
or the dominant-negative kinase-
deficient PIKfyve
K1831E
mutant increases or decreases, re
-
spectively, the intracellular PtdIns 5-P levels in HEK293 sta-
ble cell lines (16). Consistent with these data, we have found
here that PIKfyve
WT
expression in CHO-T cells partially re
-
produced the PtdIns 5-P effect in actin stress fiber breakdown
(Fig. 4). However, the in vitro PIKfyve lipid kinase activity
appears to be insensitive to insulin-directed activation in
both 3T3-L1 adipocytes and CHO-T cells (Ref. 47, and this
study). Because acute insulin action in 3T3-L1 adipocytes
causes a recruitment of cytosolic PIKfyve to membranes (48)
where the PtdIns substrate resides, it is conceivable that
elevation in PtdIns 5-P mass in response to insulin would be
seen even in the absence of detectable increases in PIKfyve
intrinsic activity. The role of the PIKfyve pathway in acute
insulin action on glucose metabolism is further substantiated
by data demonstrating inhibition of insulin-regulated
GLUT4 vesicle translocation in 3T3-L1 adipocytes expressing
dominant-negative kinase-deficient PIKfyve
K1831E
mutant
(35). Thus, these data and considerations are consistent with
the concept that the robust increase in PtdIns 5-P mass in
response to insulin is due, at least in part, to PIKfyve.
Another source for a PtdIns 5-P mass increase is an aug-
mented breakdown of PtdIns 4,5-P
2
. Such a pathway has
been recently seen upon Shigella flexneri invasion of epithelial
cells (17) and found to coincide with an active F-actin re-
modeling. Although a mechanism of PtdIns 4,5-P
2
hydrolysis
Sbrissa et al. PtdIns 5-P Mediates Insulin Signaling Endocrinology, November 2004, 145(11):4853 4865 4863
is plausible to operate in response to insulin, HPLC analyses
of the PtdIns 4,5-P
2
head group showed no significant de
-
creases in PtdIns 4,5-P
2
levels upon insulin stimulation of
metabolically labeled 3T3-L1 adipocytes and CHO-T cells
(Fig. 9). However, it is worth emphasizing that even subtle
diminution of total PtdIns 4,5-P
2
would produce a substantial
PtdIns 5-P increase due to the high levels of intracellular
PtdIns 4,5-P
2
vs. PtdIns 5-P in these cell types (16, 37). More
-
over, expected are spatially restricted changes, which may
not result in an overall alteration in
32
P-PtdIns 4,5-P
2
accu
-
mulation. Furthermore, though currently unknown, the
pathway of PtdIns 5-P hydrolysis could also represent a
potential insulin-sensitive target to yield a PtdIns 5-P in-
crease. Recent studies in Dictyostelium, identifying a new
PtdIns 5-P-specific phospholipidinositol phosphatase PLIP
(52), indicate that this assumption may be correct. Finally, the
increase in PtdIns 5-P could be associated with insulin-de-
pendent negative regulation of type II PIPK activities that
consume PtdIns 5-P, converting it to PtdIns 4,5-P
2
. This hy
-
pothesis is supported by new data, published while this
manuscript was in its final stages of preparation, demon-
strating decreased insulin signaling to PI 3-kinase and Akt
upon expression of type II PIPK in CHO cells (53). Clearly,
whereas the enzymology of the rapid increase/attenuation
of PtdIns 5-P is rather complex and may involve up- or
down-regulation of more than one pathway, the data pre-
sented herein identify, for the first time, an insulin-regulated
PtdIns 5-P pathway that signals to F-actin depolymerization
and GLUT4 dynamics. PtdIns 5-P target proteins relevant to
actin remodeling in the context of insulin action remain to be
identified.
Acknowledgments
We thank Linda McCraw for the excellent secretarial assistance. We
thank Drs. Richard Anderson, Or Gozani, Junying Yuan, Jeff Pessin,
Kostya Kandror, and Mike Czech for the kind gifts of His-Type II
PIPK,
EGFP-ING2-3xPHD, EGFP-ING2-3xPHD3K, EGFP-ACS-3xPHD, EGFP-
GLUT4 or Myc-GLUT4 cDNAs, and GLUT4 antibodies.
Received April 16, 2004. Accepted July 20, 2004.
Address all correspondence and requests for reprints to: Assia Shi-
sheva, Department of Physiology, Wayne State University School of
Medicine, 540 East Canfield, Detroit, Michigan 48201. E-mail: ashishev@
med.wayne.edu.
This work was supported by the National Institutes of Health
(DK58058) and American Diabetes Association research grants (to A.S.).
Part of this work was presented at the 63rd Scientific Session of Amer-
ican Diabetes Association, New Orleans, 2003.
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Sbrissa et al. PtdIns 5-P Mediates Insulin Signaling Endocrinology, November 2004, 145(11):4853 4865 4865
    • "Recent discoveries showed that nonphosphoglycans-as IP7 and PI5P – also participate in modulating glucose metabolism. IP7 is required for efficient exocytosis of insulin containing secretory granules from pancreatic β cells [84], while PI5P has been demonstrated mimicking insulin effect in facilitating GLUT4 translocation to cell surface, this way enhancing glucose uptake [85]. "
    [Show abstract] [Hide abstract] ABSTRACT: Introduction: Inositol and its derivatives comprise a huge field of biology. Myo-inositol is not only a prominent component of membrane-incorporated phosphatidylinositol, but participates in its free form, with its isomers or its phosphate derivatives, to a multitude of cellular processes, including ion channel permeability, metabolic homeostasis, mRNA export and translation, cytoskeleton remodeling, stress response. Areas covered: Bioavailability, safety, uptake and metabolism of inositol is discussed emphasizing the complexity of interconnected pathways leading to phosphoinositides, inositol phosphates and more complex molecules, like glycosyl-phosphatidylinositols. Expert opinion: Besides being a structural element, myo-inositol exerts unexpected functions, mostly unknown. However, several reports indicate that inositol plays a key role during phenotypic transitions and developmental phases. Furthermore, dysfunctions in the regulation of inositol metabolism have been implicated in several chronic diseases. Clinical trials using inositol in pharmacological doses provide amazing results in the management of gynecological diseases, respiratory stress syndrome, Alzheimer's disease, metabolic syndrome, and cancer, for which conventional treatments are disappointing. However, despite the widespread studies carried out to identify inositol-based effects, no comprehensive understanding of inositol-based mechanisms has been achieved. An integrated metabolomics-genomic study to identify the cellular fate of therapeutically administered myo-inositol and its genomic/enzymatic targets is urgently warranted.
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    • "Physiological roles for PI5P associated with the cytoskeleton have also been reported (reviewed in detail in (Bulley et al., 2015) ). For example, overexpression of the PI5P- generating enzyme PIKfyve or microinjection of PI5P mimics insulin's effects on Factin stress fibres (Sbrissa et al., 2004), and studies from Wesche's lab (Haugsten et al., 2013, Oppelt et al., 2014, Oppelt et al., 2013) imply that PI5P is a signalling intermediate in FGF1-stimulated cell migration. FGF1 induces PI5P production and promotes cell migration in BJ fibroblasts (Oppelt et al., 2013), effects that are attenuated by knockdown of the PI5P-generating enzymes PIKfyve and MTMR3. "
    [Show abstract] [Hide abstract] ABSTRACT: The phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are an important family of enzymes, whose physiological roles are being teased out by a variety of means. Phosphatidylinositol-5-phosphate 4-kinase γ (PI5P4Kγ) is especially intriguing as its in vitro activity is very low. Here we review what is known about this enzyme and discuss some recent advances towards an understanding of its physiology. Additionally, the effects of the ATP-competitive inhibitor I-OMe Tyrphostin AG-538 on all three mammalian PI5P4Ks was explored, including two PI5P4Kγ mutants with altered ATP- or PI5P-binding sites. The results suggest a strategy for targeting non-ATP binding sites on inositol lipid kinases.
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    • "It has been shown that the engagement of the T receptor (TCR) in T cells induces an increase in PtdIns5P, which in turns recruits DOK (Downstream of Tyrosine Kinases) family proteins to promote their phosphorylation and to activate a negative feedback loop [34]. Stimulation of adipocytes with insulin also induces a peak production of PtdIns5P resulting in the loss of actin stress fibers and the acceleration of the translocation of the glucose transporter GLUT4 to the plasma membrane [35]. Recently, we have shown that PtdIns5P was coordinating membrane dynamics and actin cytoskeleton reorganization. "
    [Show abstract] [Hide abstract] ABSTRACT: By interacting specifically with proteins, phosphoinositides organize the spatiotemporal formation of protein complexes involved in the control of intracellular signaling, vesicular trafficking and cytoskeleton dynamics. A set of specific kinases and phosphatases ensures the production, degradation and inter-conversion of phosphoinositides to achieve a high level of precision in the regulation of cellular dynamics coordinated by these lipids. The direct involvement of these enzymes in cancer, genetic or infectious diseases, and the recent arrival of inhibitors targeting specific phosphoinositide kinases in clinic, emphasize the importance of these lipids and their metabolism in the biomedical field.
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