Colocalization of insulin receptor and insulin receptor substrate-1to caveolae in primary human adipocytes Cholesterol depletion blocks insulin signalling for metabolic and mitogenic con
Trang 1Colocalization of insulin receptor and insulin receptor substrate-1
to caveolae in primary human adipocytes
Cholesterol depletion blocks insulin signalling for metabolic and mitogenic control Margareta Karlsson1, Hans Thorn1, Anna Danielsson1, Karin G Stenkula1, Anita O¨st1, Johanna Gustavsson1, Fredrik H Nystrom2and Peter Stra˚lfors1
1 Department of Cell Biology and Diabetes Research Centre, and 2 Department of Medicine and Care and Diabetes Research Centre, University of Linko¨ping, Sweden
Caveolae are plasma membrane invaginations with several
functions, one of which appears to be to organize receptor
mediated signalling Here we report that in primary human
subcutaneous adipocytes the insulin receptor was localized
to caveolae by electron microscopy/immunogold detection
and by isolating caveolae from plasma membranes Part of
insulin receptor substrate 1 (IRS1), the immediate
down-stream signal mediator, was colocalized with the insulin
receptor in the plasma membrane and caveolae, as
demon-strated by immunofluorescence microscopy, immunogold
electron microscopy, and immunogold electron microscopy
of transfected recombinant HA-IRS1 In contrast, rat
epi-didymal adipocytes lacked IRS1 at the plasma membrane
Depletion of cholesterol from the cells using b-cyclodextrin
blocked insulin stimulation of glucose uptake, insulin
inhibition of perilipin phosphorylation in response to iso-proterenol, and insulin stimulation of protein kinase B and Map-kinases extracellular signal-related kinase (ERK)1/2 phosphorylation Insulin-stimulated phosphorylation of the insulin receptor and IRS1 was not affected, indicating that caveolae integrity is required downstream of IRS1 In conclusion we show that insulin receptor and IRS1 are both caveolar proteins and that caveolae are required for both metabolic and mitogenic control in human adipocytes Our results establish caveolae as foci of insulin action and stress the importance of examining human cells in addition to animal cells and cell lines
Keywords: extracellular signal-related kinase; ultrastructure; protein kinase B; b-cyclodextrin; glucose transport
Insulin exerts control over cell metabolism by binding to its
cell surface receptor, which has been characterized in great
detail [1–5] The occupied receptor is autophosphorylated
on tyrosine residues and can thereby tyrosine phosphorylate
other cellular proteins, to transduce the insulin signal into
the signal network of the cell Chief among these proteins
are the insulin receptor substrate (IRS) family of proteins
[1] When tyrosine is phosphorylated they can transmit
metabolic and mitogenic signals The further downstream
events involve the generation of second messengers and
phosphorylation of protein kinase B/Akt (PKB) Eventually
glucose transporter GLUT4 is translocated to the plasma
membrane for glucose uptake and other target proteins (e.g
perilipin [6]) are phosphorylated/dephosphorylated
Insu-lin’s ability for mitogenic signalling is transmitted via the
Map-kinases extracellular signal-related kinase (ERK)1/2
for phosphorylation control of transcription factors The
precise mechanisms for insulin’s cellular control are not yet known in detail, and especially not so in human cells and tissues
Caveolae are 25–150 nm invaginations of the plasma membrane and are found in most cell types They are particularly abundant in rat adipocytes and increase dramatically in number when 3T3-L1 fibroblasts are differentiated into fat cells and become insulin responsive [7–9] Cholesterol and sphingolipids together with the principal structural protein caveolin are required for caveo-lae to form Caveocaveo-lae are involved in numerous cellular processes such as receptor mediated uptake, receptor mediated signalling, and vesicular trafficking, reviewed in [10] A number of proteins involved in signal transduction have been found in caveolae [11–15] We have shown that in rat adipocytes and in 3T3-L1 adipocytes the insulin receptor
is localized to caveolae [16,17] The immediate downstream signal-mediator IRS1, on the other hand, has been reported not to be associated with the plasma membrane in rat adipocytes [18] or in other cell types [19–21] In rat adipocytes the disruption of caveolar integrity by cholesterol depletion using b-cyclodextrin, reversibly made the cells insulin resistant by inhibiting the insulin receptor from phosphorylating IRS1 On the other hand, insulin signalling for mitogenic control via Map-kinases ERK1 and 2 was not impaired in the rat cells [17,22] The importance of caveolae for insulin action is reinforced by the finding that, in response to insulin stimulation, the insulin regulated glucose transporter GLUT4 is translocated to caveolae for glucose
Correspondence to P Stralfors, Department of Cell Biology and
Diabetes Research Centre, University of Linko¨ping, SE58185
Linko¨ping, Sweden Fax: + 46 13 224314, Tel.: + 46 13 224315,
E-mail: peter.stralfors@ibk.liu.se
Abbreviations: ERK, extracellular signal-related kinase; GLUT4,
insulin-stimulated glucose transporter 4; IRS, insulin receptor
sub-strate; Map, mitogen activated protein; PKB, protein kinase B;
TEM, transmission electron microscopy.
(Received 11 March 2004, revised 16 April 2004,
accepted 21 April 2004)
Trang 2uptake [9,23,24], and that some of the downstream
signalling for enhanced glucose uptake may take place in
caveolae [25]
The role of caveolae in insulin signalling has, however,
not been examined in human cells As animal cells in general
and immortalized cell lines in particular often deviate from
primary human cells, which are the cells most relevant to the
study of human disease, we set out to investigate this in
human adipocytes Here we report that in primary human
adipocytes the insulin receptor along with its immediate
downstream signal mediator IRS1 is localized in caveolae of
the plasma membrane Destruction of caveolae interrupts
insulin signal transduction downstream of IRS1 and makes
these cells insulin resistant to both metabolic and mitogenic
signalling
Materials and methods
Subjects
Samples of subcutaneous abdominal fat were obtained,
after the informed consent, from patients (male or female,
age 40–76 years) undergoing elective abdominal surgery at
the University Hospital of Linko¨ping Patients diagnosed
with diabetes were excluded The Local Ethics Committee
approved the study
Materials
Rabbit anti-insulin receptor b-chain and anti-caveolin
polyclonal and mouse anti-phosphotyrosine (PY20)
mono-clonal antibodies were from Transduction Laboratories
(Lexington, KY, USA) Rabbit
anti-phospho(Thr308)-PKB/Akt polyclonal antibodies were from Upstate Biotech
(Charlottesville, VI, USA) Rabbit polyclonal antibodies
against phospho-ERK1/2 and against phospho-p38
Map-kinase were from Cell Signalling Techn (Beverly, MA,
USA) Anti-insulin receptor b-chain monoclonal, anti-IRS1
rabbit polyclonal antibodies (sc-559, sc-560), and anti-HA
rabbit polyclonal antibodies were from Santa Cruz
Biotechnology (Santa Cruz, CA, USA) Colloidal gold
conjugated anti-(IgG) Ig was from Aurion (Wageningen,
the Netherlands) 2-Deoxy-D-[1-3H] glucose was from
Amersham Biotech (UK) Insulin, b-cyclodextrin, and other
chemicals were from Sigma-Aldrich (St Louis, MO, USA)
or as indicated in the text
Isolation and incubation of adipocytes
Human adipocytes were isolated by collagenase (type 1;
Worthington, NJ, USA) digestion as described [26] except
that the cells were allowed to floatate by gravity At a final
concentration of 100 lL packed cell volume per ml, cells
were incubated in Krebs–Ringer solution (0.12M NaCl,
4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM
KH2PO4) containing 20 mM Hepes, pH 7.40, 1% (w/v)
fatty acid-free bovine serum albumin, 100 nM
phenyliso-propyladenosine, 0.5 UÆmL)1 adenosine deaminase with
2 mM glucose, at 37C on a shaking water bath For
analysis of b-cyclodextrin effects on insulin control of
insulin receptor, IRS1, PKB, ERK1/2, and p38, cells were
incubated overnight before analysis Rat adipocytes were
isolated [26] from the epididymal adipose tissue of Sprague–Dawley rats (130–160 g, B and K Universal, Sollentuna, Sweden) The animals were treated in accor-dance with Swedish Animal Care regulations
Transfection of adipocytes with myc-tagged caveolin-1
or HA-tagged IRS1 Isolated adipocytes were transfected as follows: 200 lL cells (400 lL cell volume per ml) was mixed with 200 lL of buffer (137 mMNaCl, 2.7 mMKCl, 10 mMNa2HPO4and 1.8 mMKH2PO4, pH 7.5) containing 5 lg of empty vector pcis2, pcis2-myc-caveolin-1, or pcis2-HA-IRS1 (kindly supplied by M Quon, NIH, Bethesda, MD, USA) in an electroporation cuvette Cells were electroporated with 6 pulses at 600 V and 25 lF using Gene pulser II (Bio-Rad) Cells from 15 cuvettes were pooled and kept at 37C in 10% (v/v) CO2 After 1 h an equal volume of Dulbecco’s modified Eagle’s medium pH 7.5, containing 25 mM glu-cose, 50 unitsÆmL)1 penicillin, 50 lgÆmL)1 streptomycin,
200 nMphenylisopropyladenosine, 7% (w/v) bovine serum albumin, and 25 mM Hepes, was added After 18 h of incubation cells were collected and plasma membranes prepared for electron microscopy
Electron microscopy of plasma membranes Plasma membrane sheets were prepared for electron microscopy as described previously [27] After rinsing the adipocytes in ice-cold phosphate-buffer (10 mMNa2HPO4/ NaH2PO4, 150 mM NaCl, pH 7.5) they were attached
to nickel grids [28]: poly(L-lysine) and formvar coated grids were rehydrated by floating on ice-cold phosphate-buffer containing the cells Grids with captured adipocytes were flushed with ice-cold 150 mMKCl, 1.9 mMTris/HCl buffer, pH 7.6 Plasma membranes remaining on the grids were washed three times in 150 mM Hepes,
pH 7.5, and fixed in 0.1M sodium cacodylate buffer, containing 0.1M sucrose, 3% (w/v) paraformaldehyde, and 0.05% (v/v) glutaraldehyde, for 30 min at room temperature
Plasma membranes were blocked for 1 h in phosphate-buffer, containing 5% (w/v) bovine serum albumin (BSA-c, Aurion, the Netherlands), 0.1% (w/v) gelatine, and 5% normal goat serum (Aurion) at 37C This was followed by incubation with mouse anti-insulin receptor b-chain mono-clonal antibodies, or anti-IRS1 polymono-clonal antibodies, or rabbit anti-caveolin polyclonal antibodies for 1.5 h at
37C Grids were rinsed with phosphate-buffer, containing 0.1% (w/v) BSA-c before incubation with secondary antibodies Goat (rabbit IgG) Ig and/or goat anti-(mouse IgG) Ig, conjugated with 15 nm or 6 nm colloidal gold, was added to plasma membranes and incubated overnight at 4C
After immunolabelling, the plasma membranes were rinsed and fixed in 2% (v/v) glutaraldehyde for 10 min followed by 1% (w/v) OsO4 for 30 min in 0.1M sodium cacodylate buffer, with 0.1M sucrose, pH 7.5, at room temperature Grids were rinsed with water, frozen, lyo-philized, and coated with 2 nm tungsten by magnetron sputtering directly in the freeze-dryer [29] Transmission electron microscopy was performed with Jeol EM1230
Trang 3TEM-SCAN (Tokyo, Japan) No labelling was observed
in the absence of primary antibody, nor was any
cross-reactivity detected between secondary and primary
antibodies
Immunofluorescence microscopy of plasma membrane
sheets
Plasma membrane sheets were prepared as described [17, 30]
and fixed in 3% (w/v) paraformaldehyde for 30 min at
room temperature After blocking in bovine serum albumin
and gelatine membranes were incubated with rabbit
anti-IRS1, caveolin, or insulin receptor antibodies Primary
antibodies were detected with fluorescent secondary
anti-bodies (Alexa fluor 488 or 594, from Molecular Probes,
USA) by fluorescence microscopy (Nikon, D-Eclipse CI
confocal microscope, Tokyo, Japan) No labelling was
observed in the absence of the primary antibody, nor was
any cross-reactivity detected between secondary and
pri-mary antibodies
Isolation of caveolae-enriched membrane fraction
A caveolae-enriched membrane fraction was isolated as
described [22] Adipocytes were homogenized in 10 mM
Tris/HCl, pH 7.4, 1 mM EDTA, 0.5 mM EGTA, 0.25M
sucrose, 25 mMNaF, 1 mM pyrophosphate with protease
inhibitors (10 lM leupeptin, 1 lM pepstatin, 1 lM
apro-tinin, 4 mM iodoacetate and 50 mM
phenylmethanesulfo-nyl fluoride) using a motor-driven Teflon/glass
homogenizer at room temperature Subsequent
proce-dures were carried out at 0–4C A plasma membrane
containing pellet, obtained by centrifugation at 16 000 g
for 20 min, was resuspended in 10 mMTris/HCl, pH 7.4,
1 mM EDTA and protease inhibitors Purified plasma
membranes were isolated by sucrose gradient
centrifuga-tion [31,32], referred to as the plasma membrane fraccentrifuga-tion
Aliquots of plasma membrane fraction were pelleted and
resuspended in 0.5M Na2CO3, pH 11, and protease
inhibitors [33], and sonicated with a probe-type sonifier
(MSE, Soniprep 150) 3· 20 s The homogenate was then
adjusted to 45% sucrose in 12 mM Mes, pH 6.5, 75 mM
NaCl, 0.25 M Na2CO3 and loaded under a 5–35%
discontinuous sucrose gradient in the same buffer and
centrifuged at 39 000 r.p.m for 16–20 h in a SW41 rotor
(Beckman Instruments, Fullerton, CA, USA) The
light-scattering band at the 5–35% sucrose interface, enriched
in caveolae, was collected and referred to as the caveolae
fraction A cytosolic fraction was obtained as the
supernatant from centrifuging the 16 000 g supernatant
at 150 000 g 75 min and discarding the pellet One third
of the plasma membrane from rat adipocytes constitutes
caveolae membrane [27], which thus is a minimum figure
for human adipocyte plasma membranes that have a
higher density of caveolae To examine the purity of our
caveolae preparation we isolated nuclear and
mitochond-rial membranes, which are the chief contaminants of
adipocyte plasma membrane [31], and identified seven
proteins by peptide sequence analysis using collision
induced dissociation and electrospray ionization mass
spectrometry The same analysis was performed in
parallel on the caveolae fraction and no nuclear/
mitochondrial contamination was detected (N Aboulaich,
J Vainonen, P Stra˚lfors, and A Vener, unpublished data)
SDS/PAGE and immunoblotting Proteins were separated by SDS/PAGE, transferred to a poly(vinylidene difluoride) blotting membrane (Immobi-lone-P, Millipore, Bedford, MA, USA), and incubated with indicated primary antibodies Bound antibodies were detected using Renaissence+ (New England Nuclear) with horseradish peroxidase-conjugated anti-IgG as secondary antibody Blots were evaluated by chemiluminescence imaging (Las1000, Fuji, Japan) By two-dimensional elec-trofocusing (pH 3–10) SDS/PAGE [34] analysis and immu-noblotting against phosphotyrosine and IRS1, we found that > 95% of the tyrosine phosphorylated 180 kDa band represents IRS1
Cholesterol and protein determination Cholesterol was quantitated spectrofluorometrically [35] by measuring the amount of H2O2 produced by cholesterol oxidase (Rhodococus erythropolis, from Boehringer Mann-heim, MannMann-heim, Germany) on a Fluoroscan spectrofluo-rometer (Labsystems, Finland) Protein was determined with the Micro BCA kit from Pierce Bovine serum albumin was used as standard
Determination of glucose transport Glucose transport was determined as uptake of
2-deoxy-D-[1-3H]glucose [36] after transfer of cells to medium without glucose Cells were treated with or without insulin for 15 min when 2-deoxy-D-[1-3H]glucose was added to a final concentration of 50 lM (10 lCiÆmL)1) and the cells were incubated for another 30 min; uptake was linear for at least 30 min Glucose uptake was stopped by centrifuging through dinonylphtalate and freezing in an aluminium block kept at)20 C Non-specific uptake was determined
in the presence of 25 lMcytochalasin B by terminating the incubation immediately after addition of the
2-deoxy-D-[1-3H]glucose Tubes were cut frozen through the oil-layer, the cell cakes were dissolved in 0.4 mL 1% (w/v) SDS, and radioactivity was measured in 3 mL of scintillant (QuickSafe A, Zinsser Analytic Ltd)
Perilipin protein phosphorylation analysis For analysis of protein phosphorylation, adipocytes (50 lL packed cell volume per ml) were prelabelled with [32 P]phos-phate for 1 h [37] Total cell protein was prepared for SDS/ PAGE as described [26].32P-incorporation in perilipin was evaluated by radioimaging (Bas1000, Fuji)
Results
Localization of the insulin receptor in caveolae
of human adipocytes
We analyzed isolated adipocytes from human abdominal subcutaneous adipose tissue As seen by transmission
Trang 4electron microscopy, the inside of the plasma membrane of
a human fat cell was covered with large numbers of
caveolae These were seen as bulbs protruding from the
plasma membrane, either singularly (Fig 1A) or in clusters
of varying size (Figs 1B and 2) Caveolae clusters were
frequent in the plasma membrane of these cells, in contrast
to rat adipocytes [27] The caveolae structures were labelled
with antibodies against the caveolae specific protein caveolin
(6 nm gold particles) and against the insulin receptor
(15 nm gold particles) Labelling of the insulin receptor
was largely restricted to the caveolae structures: as single
caveolae (Fig 1A) or in clusters of caveolae (Fig 1B)
A close up view of a single caveola showed that only the
neck attaching the caveolar bulb to the plasma membrane
was significantly labelled by antibodies against caveolin
(Fig 2) The specific localization of caveolin in the necks of
caveolae has been described for rat adipocytes [27] and human fibroblasts [27] We verified this finding here by transfecting human adipocytes with myc-tagged caveolin-1 and examination with gold-conjugated antibodies against the myc tag Only the necks and not the bulbs of caveolae were labelled (not shown)
We also isolated a caveolae fraction, without using detergent [17], from purified human adipocyte plasma membranes and examined the presence of insulin receptor
by immunoblotting after gel electrophoresis The insulin receptor was present and enriched in this caveolae fraction, compared to the total plasma membrane fraction (Fig 3A) The chief structural constituents of caveolae, caveolin (Fig 3C), and cholesterol (0.59 ± 0.05 nmol cholesterol
Fig 1 Transmission electron microscopy of the inside of human
adi-pocyte plasma membrane Plasma membrane sheets were immunogold
labelled against the insulin receptor (15 nm gold particles, black
arrowheads) and caveolin (6 nm gold particles, white arrowheads) (A)
Membrane area with single caveolae, (B) a cluster of caveolae Images
contrast inverted.
Fig 2 Stereo image of a human adipocyte caveola by transmission electron microscopy Plasma membrane sheets were immunogold labelled against the insulin receptor (15 nm gold particle, black arrowheads) and caveolin (6 nm gold particles, white arrowheads) Image contrast inverted.
Fig 3 Insulin receptor in plasma membrane and isolated caveolae fractions Human adipocytes were incubated with or without 1 n M
insulin for 20 min, as indicated; plasma membrane (pm) and caveolae (cav) fractions were prepared Aliquots of 3 lg protein was subjected
to SDS/PAGE and immunoblotting with antibodies against (A) the insulin receptor b-subunit, (B) phospho-tyrosine and (C) caveolin.
Trang 5per lg protein (mean ± SE, n¼ 6) in the caveolae fraction
and 0.31 ± 0.02 in the plasma membrane fraction), were
also enriched The enrichment was compatible with
caveo-lae constituting between a third and half of the plasma
membrane; in human adipocytes the numerous caveolae
clusters increase the caveolae membrane more than in rat
adipocytes, which have a third of their plasma membrane
being caveolae [27]
Insulin receptor signalling in caveolae
We next examined the functional status of
caveolae-localized receptors After incubating cells with a
submax-imal concentration of insulin the insulin-induced increase in
receptor autophosphorylation was comparable in the
iso-lated caveolae fraction and in the total plasma membrane
fraction (Fig 3B)
We have previously shown that reduction of the amount
of cholesterol in the plasma membrane with b-cyclodextrin
destroys the structural integrity of caveolae [27] Depletion
of cholesterol made the human adipocytes insulin resistant:
insulin stimulation of glucose uptake was impaired by prior
cholesterol depletion, but without any effect on the basal
glucose uptake (Fig 4) The protein perilipin is believed to
be involved in the hormonal regulation of lipolysis [6]
Insulin’s ability to counteract catecholamine-stimulated phosphorylation of perilipin was also inhibited by the cholesterol depletion (Fig 5)
In line with these findings, insulin signalling to increased phosphorylation of protein kinase B (Fig 6A) was curtailed The insulin-stimulated phosphorylation of Map-kinases ERK1 and 2 (Fig 6B) was also attenuated by b-cyclodextrin In contrast, insulin-stimulated phosphory-lation (Fig 6C) of the insulin receptor was not affected by this level of prior cholesterol depletion, nor was the total tyrosine phosphorylation of IRS1 affected (Fig 6D) A more detailed examination of the effect of cholesterol depletion, at different concentrations of insulin, still failed to
Fig 4 Effect of cholesterol depletion on insulin control of glucose
up-take Isolated human adipocytes were incubated with (closed symbols)
or without (open symbols) 10 m M b-cyclodextrin for 50 min and were
then incubated with the indicated concentration of insulin for 15 min.
2-Deoxyglucose uptake was then determined Because the maximal
response to insulin was variable between individuals (131, 53, and
58 nmol 2-deoxyglucoseÆmin)1ÆmL)1packed cells), glucose uptake was
normalized to the maximal uptake for each experiment and expressed
as percent of max Mean ± SE, n ¼ 3 separate experiments of three
determinations each on cells from three subjects.
Fig 5 Effect of cholesterol depletion on insulin control of perilipin phosphorylation Isolated human adipocytes were incubated with or without 10 m M b-cyclodextrin (bCD) as indicated for 50 min and then they were incubated with 20 n M isoproterenol (iso) or 20 n M isopro-terenol and 1 n M insulin (iso + ins), or with vehicle (contr) for 15 min Shown is the32P-phosphorylation of perilipin after SDS/PAGE of whole-cell lysates The indicated doublet represents perilipin; the phosphorylation is typically shifted from the lower molecular mass band to an enhanced phosphorylation of the higher molecular mass band in response to cyclic AMP elevation [6].
Fig 6 Effect of cholesterol depletion on insulin signal mediators Human adipocytes were treated with or without 7 m M b-cyclodextrin for 50 min, and then incubated with or without 10 n M insulin for
20 min, as indicated Whole-cell lysate aliquots were subjected to SDS/ PAGE and immunoblotting with antibodies against (A) phospho-PKB; (B) phospho-ERK1/2; (C) phospho-tyrosine (insulin receptor b-subunit); (D) phospho-tyrosine (IRS1).
Trang 6reveal any significant effects on insulin receptor (Fig 7A) or
IRS1 (Fig 7B) phosphorylation at any concentration of the
hormone However, as we determined total
tyrosine-phos-phorylation of IRS1, we cannot rule out effects on specific
phosphorylation sites on the protein
Localization of IRS1 in the plasma membrane and
caveolae of human adipocytes
As IRS1 phosphorylation by the insulin receptor was not
dependent on plasma membrane cholesterol/caveolae
integ-rity, as has been found to be the case in rat adipocytes [22],
we examined if IRS1 was localized in the plasma membrane
and caveolae in human adipocytes Immunofluorescence
microscopy of plasma membrane sheets, from cells not
exposed to insulin, using anti-IRS1 Igs revealed that IRS1
was localized in the plasma membrane (Fig 8B) Antibodies against different epitopes (N- and C-terminal) of the protein similarly detected IRS1 in the plasma membrane In contrast, under identical conditions, IRS1 was repeatedly not detected by immunofluorescence microscopy in the plasma membrane of rat epididymal adipocytes (Fig 8E) The punctuate pattern of IRS1 binding in the plasma membrane (Fig 8B) suggested that part of IRS1 may be associated with caveolae and hence the insulin receptor To examine the possibility of caveolar localization of IRS1 we analyzed plasma membrane sheets for colocalization with caveolin (Fig 8A–C) or the insulin receptor (Fig 9A–C) The merged immunofluorescence images for IRS1 and caveolin or IRS1 and the insulin receptor indicates colocalization (as demonstrated by the yellow colour) of IRS1 with both caveolin and the insulin receptor
To further examine a caveolar localization of IRS1 we next examined plasma membrane sheets by electron micro-scopy after immunogold labelling with antibody against IRS1 (Fig 10A,B) IRS1 was detected in the caveolae structures as well as outside: 63% of IRS1 was found in caveolae (293 of 567 identified gold particles, from 10 different cells, were found in or directly associated with caveolae structures, which constituted 30% of the plasma membrane sheets examined) Similar results were obtained with antibodies against the N- and against the C-terminal part of IRS1 IRS1 remained bound to the plasma membrane after depletion of cholesterol with b-cyclodextrin (not shown)
To dispel any uncertainty of antibody cross-reactivity and detection of other plasma membrane and caveolar proteins,
we transfected human adipocytes with the human IRS1 with an HA-tag (hemagglutinin) and used immunogold-antibodies against the HA-tag to examine IRS1 localization
in the human adipocyte plasma membrane Figure 10C shows that the HA-tag of HA-IRS1 was detected by electron microscopy in the plasma membrane and in caveolae, similarly to the findings with antibody against the wild-type IRS1
Discussion
Our findings demonstrate that caveolae are central to insulin action in human adipocytes, similarly to the previously described situation in rat and 3T3-L1 cytes [15,17,22–24]: the insulin receptor in human adipo-cytes is located in caveolae, is autophosphorylated in caveolae, and is dependent on caveolae for its cellular control Intriguingly, in human adipocytes the immediate downstream signal mediator IRS1 was, under basal noninsulin-stimulated conditions, colocalized with the insulin receptor in caveolae In type 2 diabetes the tissues respond poorly to insulin, exhibiting insulin resistance that can be overcome by increasing concentrations of circula-ting insulin In the majority of cases it is still not known what defect in the insulin target cell causes insulin resistance Our finding that insulin’s cellular control is dependent on caveolae/cholesterol in human adipocytes indicates that caveolae has to be taken into account when trying to understand normal insulin signal transduction as well as the dysfunction that causes insulin resistance in different human conditions
Fig 7 Effect of cholesterol depletion on dose–response effect of insulin
on tyrosine phosphorylation of insulin receptor and IRS1 Human
adi-pocytes were treated with (closed symbols) or without (open symbols)
7 m M b-cyclodextrin for 50 min, and incubated with indicated
con-centration of insulin for 20 min Whole-cell lysate aliquots were
sub-jected to SDS/PAGE and immunoblotting with antibodies against (A)
phospho-tyrosine (insulin receptor b-subunit) or (B) phospho-tyrosine
(IRS1) It was verified that in each experiment insulin-stimulated
phosphorylation of PKB was blocked Insulin-stimulated
phosphory-lation was obtained by setting the value with no insulin to 0% and at
100 n M insulin to 100% effect Dose–response curves were fitted to
experimental data using the sigmoid dose–response algorithm in
GraphPad Prism 3 software Mean ± SE, n ¼ 5 subjects.
Trang 7We found that in human adipocytes IRS1 is associated
with the plasma membrane and the caveolae, and hence in
close proximity to the insulin receptor This finding was
demonstrated by immunofluorescence microscopy and
immunogold electron microscopy, using antibodies against
the N- or C-terminal part of the protein, thus reducing the
possibility of antibodies crossreacting with and detecting an
unrelated plasma membrane/caveolae protein Moreover,
transfected cells expressed HA-IRS1 in the caveolae and the
plasma membrane as analyzed with antibodies against the
HA-tag Plasma membrane sheets on grids were extensively
washed before incubation with the antibodies, thus reducing
the possibility of unspecific binding of IRS1 to the plasma
membrane Also, after the same treatment no IRS1 was
associated with the plasma membrane in rat epididymal
adipocytes In rat adipocytes (herein and [18]) and other cells [19–21] IRS1 has been shown not to be associated with the plasma membrane, but has been found in the cytosol or bound to intracellular membranes and the cytoskeleton, pointing to the importance of exercising great caution when extrapolating to the situation in human beings from findings
in animal cells and cell lines commonly used
A consequence of IRS1 localization in the plasma membrane and caveolae appears to be that depletion of cholesterol and destruction of caveolae does not block insulin interaction with and stimulation of IRS1 tyrosine phosphorylation as happens in rat adipocytes [22], but instead blocks the further downstream phosphorylation of PKB and the target effects on glucose uptake and perilipin phosphorylation It can be hypothesized that the occupied
Fig 8 IRS1 colocalization with caveolin in the plasma membrane Plasma membrane sheets attached to glass cover-slips were incubated with antibodies against caveolin and against the C-terminal part of IRS1 (A–C) Human adipocyte examined by immunofluorescence microscopy using red fluorescent antibodies against caveolin (A) and green fluorescent antibodies against IRS1 (B); merged image (C) (D–F) Rat adipocyte examined
by immunofluorescence microscopy using green fluorescent antibodies against caveolin (D) and red fluorescent antibodies against IRS1 (E); merged image (F).
Fig 9 IRS1 colocalization with the insulin receptor in the plasma membrane Plasma membrane sheets attached to glass coverslips were incubated with antibodies against the insulin receptor and IRS1, and examined by immunofluorescence microscopy using green fluorescent antibodies for the insulin receptor (A) and red fluorescent antibodies for IRS1 (B); merged image (C).
Trang 8insulin receptor and, in human adipocytes,
tyrosine-phos-phorylated IRS1 have to be internalized via caveolae in
order to interact with and activate downstream signal
mediators Our finding then poses the question of IRS2
localization in these cells Differential localization of IRS1
and IRS2 could explain why IRS1 is a preferred mediator of
insulin signalling under normal conditions Receptor
inter-nalization by way of caveolae was recently reported for the
kinin B(2) receptor [38] Intriguingly, it has been suggested that a larger cyclic AMP response to stimulation of the b1-compared to the b2-adrenergic receptor in rat cardio-myocytes depends on colocalization of the b1-receptor, but not the b2-receptor, with the downstream mediator adenylyl cyclase in caveolae [39] It will be important to examine the mechanism for IRS1 binding to caveolae in the human fat cells and whether this binding is subject to control, by, e.g insulin Human IRS1 does not contain the consensus sequence for binding to caveolin, as has been described for a number of signalling proteins including the insulin receptor [40]
A further property that distinguishes primary human from rat adipocytes was that insulin stimulation to increased phosphorylation of Map-kinases ERK1/2 was blocked by cholesterol depletion/caveolae destruction in the human cells This is contrary to the situation in rat adipocytes, where only metabolic and not mitogenic control by insulin was dependent on intact caveolae [22] Apparently this difference reflects different properties of insulin receptor signalling in rat and human tissues This again stresses the importance of examining human cells and tissues b-Cyclodextrin is widely used to control cellular levels of cholesterol through its ability to mildly extract cholesterol from the plasma membrane of intact cells without itself incorporating into the membrane The effects of b-cyclodextrin treatment were critically dependent on its concentration The effective concentration had to be experimentally tried out for each batch of the compound, which varied between manufacturers and between different lots from the same manufacturer
In conclusion, we report that in human adipocytes caveolae contain the insulin receptor and its immediate downstream signal mediator IRS1, and that cholesterol depletion and caveolae destruction make these cells insulin resistant, downstream of IRS1, for both metabolic and mitogenic control
Acknowledgements
We thank Kurt Borch, Preben Kjolhede, and colleagues at the departments of Surgery and Obstetrics at the University Hospital in Linko¨ping for providing us with adipose tissue Financial support was obtained from Lions Foundation, Swedish Society for Medical Research, Swedish National Board for Laboratory Animals, O¨stergo¨t-land County Council’s Medical Research Funds, Swedish Foundation for Strategic Research (National Network for Cardiovascular Research), Swedish Diabetes Association and the Swedish Research Council.
References
1 Yenush, L & White, M.F (1997) The IRS-signalling system during insulin and cytokine action Bioessays 19, 491–500.
2 Olefsky, J.M (1990) The insulin receptor a multifunctional pro-tein Diabetes 39, 1009–1016.
3 White, M.F & Kahn, C.R (1994) The insulin signaling system.
J Biol Chem 269, 1–4.
4 Cohen, P (1999) Identification of a protein kinase cascade of major importance in insulin signal transduction Philos Trans Royal Soc 354, 485–495.
5 Nystro¨m, F.H & Quon, M.J (1999) Insulin signalling: metabolic pathways and mechanisms for specificity Cell Sign 11, 563–574.
Fig 10 IRS1 localization in the plasma membrane and caveolae by
electron microscopy Plasma membrane sheets attached to grids were
incubated with antibodies against IRS1 N-terminus (A,B) Human
adipocytes were transfected with the recombinant gene for HA-IRS1.
Plasma membrane sheets attached to grids were then incubated with
antibodies against the HA-tag (C) After incubation with colloidal
gold-derivatized secondary antibodies (15 nm gold particles,
arrow-heads) membranes were examined by transmission electron
micros-copy Images contrast inverted.
Trang 96 Clifford, G.M., Londos, C., Kraemer, F.B., Vernon, R.G &
Yeaman, S.J (2000) Translocation of hormone-sensitive lipase
and perilipin upon lipolytic stimulation of rat adipocytes J Biol.
Chem 275, 5011–5015.
7 Fan, J.Y., Carpentier, J.L., Obberghen, E.V., Grunfeld, C.,
Gorden, P & Orci, L (1983) Morphological changes of the
3T3-L1 fibroblast plasma membrane upon differentiation to the
adi-pocyte form J Cell Sci 61, 219–230.
8 Kandror, K.V., Stephens, J.M & Pilch, P.F (1995) Expression
and compartmentalization of caveolin in adipose cells: coordinate
regulation with and strucural segregation from GLUT4 J Cell
Biol 129, 999–1006.
9 Scherer, P.E., Lisanti, M.P., Baldini, G., Sargiacomo, M., Corley
Mastick, C & Lodish, H.F (1994) Induction of caveolin during
adipogenesis and association of GLUT4 with caveolin-rich
vesi-cles J Cell Biol 127, 1233–1243.
10 Razani, B., Woodman, S.E & Lisanti, M.P (2002) Caveolae:
from cell biology to animal physiology Pharmacol Rev 54,
431–467.
11 Smart, E.J., Graf, G.A., McNiven, M.A., Sessa, W.C., Engelman,
J.A., Scherer, P.E., Okamoto, T & Lisanti, M.P (1999) Caveolins,
liquid-ordered domains and signal transduction Mol Cell Biol.
19, 7289–7304.
12 Anderson, R.G.W (1998) The caveolae membrane system Annu.
Rev Biochem 67, 199–225.
13 Kurzchalia, T.V & Parton, R.G (1999) Membrane
microdo-mains and cavolae Curr Opin Cell Biol 11, 424–431.
14 Simons, K & Ikonen, E (1997) Functional rafts in cell
mem-branes Nature 387, 569–572.
15 Cohen, A.W., Combs, T.P., Scherer, P.E & Lisanti, M.P (2003)
Role of caveolin and caveolae in insulin signaling and diabetes.
Am J Physiol 285, E1151–E1160.
16 Stra˚lfors, P (1997) Insulin second messengers Bioessays 19,
327–335.
17 Gustavsson, J., Parpal, S., Karlsson, M., Ramsing, C., Thorn, H.,
Borg, M., Lindroth, M., Peterson, K.H., Magnusson, K.E &
Stra˚lfors, P (1999) Localisation of the insulin receptor in caveolae
of adipocyte plasma membrane FASEB J 13, 1961–1971.
18 Anai, M., Ono, H., Funaki, M., Fukushima, Y., I nukai, K.,
Ogihara, T., Sakoda, H., Onishi, Y., Yazaki, Y., Kikuchi, M.,
Oka, Y & Asano, T (1998) Different subcellular distribution and
regulation of expression of insulin receptor substrate (IRS) -3 from
those of IRS-1 and IRS-2 J Biol Chem 273, 29686–29692.
19 I noue, G., Cheatham, B., Emkey, R & Kahn, C.R (1998)
Dynamics of insulin signaling in 3T3-L1 adipocytes Differential
compartmentalization and trafficking of insulin receptor substrate
(IRS)-1 and IRS-2 J Biol Chem 273, 11548–11555.
20 Clark, S.F., Martin, S., Carozzi, A.J., Hill, M.M & James, D.E.
(1998) Intracellular localization of phosphatidylinositide 3-kinase
and insulin receptor substrate-1 in adipocytes: Potential
involve-ment of a membrane skeleton J Cell Biol 140, 1211–1225.
21 Kriaucinas, K.M., Meyers, M.G & Kahn, C.R (2000) Cellular
compartmentalization in insulin action: altered signaling by a
lipid-modified IRS-1 Mol Cell Biol 20, 6849–6859.
22 Parpal, S., Karlsson, M., Thorn, H & Stra˚lfors, P (2001)
Cho-lesterol depletion disrupts caveolae and insulin receptor signaling
for metabolic control via IRS-1, but not for MAP-kinase control.
J Biol Chem 276, 9670–9678.
23 Gustavsson, J., Parpal, S & Stra˚lfors, P (1996) Insulin-stimulated
glucose uptake involves the transition of glucose transporters to a
caveolae-rich fraction within the plasma membrane: implications
for type II diabetes Mol Med 2, 367–372.
24 Karlsson, M., Thorn, H., Parpal, S., Stra˚lfors, P & Gustavsson, J.
(2002) Insulin induces translocation of glucose transporter
GLUT4 to plasma membrane caveolae in adipocytes FASEB J.
16, 249–251.
25 Baumann, C.A., Ribon, V., Kanzaki, M., Thurmond, D.C., Mora, S., Shigematsu, S., Bickel, P.E., Pessin, J.E & Saltiel, A.R (2000) CAP defines a second signalling pathway required for insulin-stimulated glucose transport Nature 407, 202–207.
26 Stra˚lfors, P & Honnor, R.C (1989) Insulin-induced dephospho-rylation of hormone-sensitive lipase Correlation with lipolysis and cAMP-dependent protein kinase activity Eur J Biochem 182, 379–385.
27 Thorn, H., Stenkula, K.G., Karlsson, M., O¨rtegren, U., Nystrom, F.H., Gustavsson, J & Stra˚lfors, P (2003) Cell surface orifices of caveolae and localization of caveolin to the necks of caveolae in adipocytes Mol Biol Cell 14, 3967–3976.
28 Voldstedlund, M., Tranum-Jensen, J & Vinten, J (1993) Quan-tification of Na+/K+-ATPase and glucose transporter isoforms
in rat adipocyte plasma membrane by immunogold labeling.
J Membr Biol 136, 63–73.
29 Lindroth, M., Fredriksson, B.-A & Bell, P.B (1991) Cryosput-tering – a combined freeze-drying and sputCryosput-tering method for high-resolution electron microscopy J Microsc 161, 229–239.
30 Robinson, L.J., Pang, S., Harris, D.S., Heuser, J & James, D.E (1992) Translocation of the glucose transporter (GLUT4) to the cell surface in permeabilized 3T3-L1 adipocytes: effects of ATP, insulin, and GTPcS and localization of GLUT4 to clathrin lattices J Cell Biol 117, 1181–1196.
31 McKeel, D.W & Jarett, L (1970) Preparation and characteriza-tion of a plasma membrane fraccharacteriza-tion from isolated fat cells J Cell Biol 44, 417–432.
32 Oka, Y & Czech, M.P (1984) Photoaffinity labeling of insulin-sensitive hexose transporters in intact rat adipocytes J Biol Chem 259, 8125–8133.
33 Song, K.S., Li, S., Okamoto, T., Quilliam, L.A., Sargiacomo, M.
& Lisanti, M.P (1996) Co–purification and direct interaction of ras with caveolin, an integral membrane protein of caveolae microdomains Detergent-free purification of caveolae mem-branes J Biol Chem 271, 9690–9697.
34 Rabilloud, T., Blisnick, T., Heller, M., Luche, S., Aebersold, R., Lunardi, J & Braun-Breton, C (1999) Analysis of membrane proteins by two-dimensional electrophoresis: Comparison of the proteins extracted from normal or Plasmodium falciparum-infected erythrocyte ghosts Electrophoresis 20, 3603–3610.
35 Heider, J.G & Boyett, R.L (1978) The picomole determination
of free and total cholesterol in cells in culture J Lipid Res 19, 515–518.
36 Frost, S.C., Kohanski, R.A & Lane, M.D (1987) Effect of phenylarsine oxide on insulin-dependent protein phosphorylation and glucose transport in 3T3-L1 adipocytes J Biol Chem 262, 9872–9876.
37 Alemany, S., Mato, J.M & Stra˚lfors, P (1987) Phospho-dephospho-control by insulin is mimicked by a phospho-oligo-saccharide in adipocytes Nature 330, 77–79.
38 Marceau, F., Sabourin, T., Houle, S., Fortin, J.P., Petitclerc, E., Molinaro, G & Adam, A (2002) Kinin receptors: functional as-pects Int Immunopharmacol 2, 1729–1739.
39 Ostrom, R.S., Gregorian, C., Drenan, R.M., Xiang, Y., Regan, J.W & Insel, P.A (2001) Receptor number and caveolar co-localization determined receptor coupling efficiency to adenylyl cyclase J Biol Chem 276, 42063–42069.
40 Couet, J., Li, S., Okamoto, T., Ikezu, T & Lisanti, M.P (1997) Identification of peptide and protein ligands for the caveolin-scaffolding domain Implications for the interaction of caveolin with caveolae-associated proteins J Biol Chem 272, 6525–6533.