The ubiquitously expressed IQ motif-containing GTPase activating protein-1 (IQGAP1) is a scaffolding protein implicated in an array of cellular functions, in particular by binding to cytoskeletal elements and signaling proteins. A role of IQGAP1 in adipocytes has not been reported. We therefore investigated the cellular IQGAP1 interactome in primary human adipocytes. Immunoprecipitation and quantitative mass spectrometry identified caveolae and caveolae-associated proteins as the major IQGAP1 interactors alongside cytoskeletal proteins. We confirmed co-localization of IQGAP1 with the defining caveolar marker protein caveolin-1 by confocal microscopy and proximity ligation assay. Most interestingly, insulin enhanced the number of IQGAP1 interactions with caveolin-1 by five-fold. Moreover, we found a significantly reduced abundance of IQGAP1 in adipocytes from patients with type 2 diabetes compared with cells from nondiabetic control subjects. Both the abundance of IQGAP1 protein and mRNA were reduced, indicating a transcriptional defect in diabetes. Our findings suggest a novel role of IQGAP1 in insulin-regulated interaction between caveolae and cytoskeletal elements of the adipocyte, and that this is quelled in the diabetic state.

Introduction

Caveolae are microdomains that form invaginations of the plasma membrane in most cell types. Caveolae are especially abundant in adipocytes, with 1 million caveolae per cell and one-third of the plasma membrane constituting caveolae membrane [1]. The structural integrity of caveolae is dependent both on lipid composition, in particular cholesterol and sphingolipids [2,3], and on protein constituents, for example, caveolin-1, cavin-1, cavin-2, cavin-3, and EH domain-containing protein 2 (EHD2) [4]. In adipocytes, caveolae are active membrane regions supporting transfer of cholesterol and fatty acids in, and likely out of, the cell [5]. The insulin receptor is localized to caveolae [68] and is rapidly endocytosed in a caveolae-mediated process in response to insulin stimulation [9,10]. Triggered endocytosis, and its counterpart recycling exocytosis, appears to be integral functions of caveolae. Endocytosis/exocytosis requires restructuring of the cytoskeletal interactions [1113], and caveolae are probably dynamically linked to the plasma membrane-associated cytoskeletal network [14].

Several studies have suggested a link between caveolae and the cytoskeleton, in particular actin filaments [1517], but also microtubules [11,18] and intermediate filaments [19]. EHD2 has been identified as a caveolar protein in adipocytes [4], and the oligomerized caveolar protein EHD2 has been suggested to provide a link between caveolae and actin filaments [20]. Cavin-3 is another caveolar protein [4] that has been implicated in microtubular trafficking of internalized caveolae vesicles [18]. The IQ motif-containing GTPase activating protein-1 (IQGAP1, Uniprot accession number P46940) has been shown to stabilize microtubule dynamics in keratinocytes, which in turn may promote stable insertion of caveolae in the plasma membrane [11]. While intimate dynamic caveolar association with the cytoskeleton is recognized, the molecular underpinnings are far from clear.

IQGAP1 is a ubiquitously expressed multimodular scaffolding protein reported to interact with many different proteins in a variety of cell types [2130]. In particular, IQGAP1 has been reported to interact with microtubules and cytoskeletal proteins and to be involved in several different cellular functions [29,3134]. IQGAP1 has also been implied in diseases, such as cancer and diabetes [35,36], and may be involved in both production and secretion of insulin [37] as well as endocytosis and plasma membrane fusion of vesicles in pancreatic β-cells [38].

A role of IQGAP1 in adipocytes, a major cell type in energy homeostasis, has not been reported. In the present study, we have analyzed the interactome of IQGAP1 in human primary mature adipocytes and identified caveolae proteins as a set of primary interactors with IQGAP1, in addition to cytoskeletal proteins. Our findings suggest that IQGAP1 may possibly provide an insulin-regulated link between the cytoskeleton and caveolae.

Experimental procedures

Subjects

The study was approved by the Regional Ethics Board at Linköping University, Sweden, and the study has been carried out in accordance with the Declaration of Helsinki; all patients obtained written information and gave their informed approval before surgery. During elective surgery at the University Hospital in Linköping, Sweden, a small piece of subcutaneous abdominal fat tissue was excised from female subjects and immediately treated to isolate adipocytes.

Materials

Antibodies used in this work are as follows: anti-IQGAP1 (05-504, Millipore, Temecula, CA, USA), anti-β-tubulin (#T4026, Sigma-Aldrich, St. Louis, MO, USA), normal mouse IgG (sc-2025) and HRP-conjugated IgG secondary goat-anti-mouse antibody (sc-2005; Santa Cruz Biotechnical, Santa Cruz, CA, USA), and anti-caveolin (#3267, Cell Signaling Technology, Inc.). Chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise stated in the text.

Isolation of adipocytes and sample preparation

Adipocytes were isolated from adipose tissue samples by collagenase (type 1, Worthington, NJ, USA) digestion in modified Krebs–Ringer solution [53] and treated as described previously [54]. For analyses, adipocytes were lysed in 2 volumes of lysis buffer [25 mM Tris, 100 mM NaCl, 1 mM EGTA, 10 µM leupeptin, 1 µM pepstatin, 1 µM aprotinin, 50 µM phenylmethanesulfonylfluoride, 0.3% (w/v) CHAPS, pH 7.4] by passing three times through a 27-gauge needle. After lysis, all procedures were carried out on ice or at 4°C. Floating fat and pelleted debris were removed after centrifugation at 500×g for 10 min and then at 20 000×g for 30 min. Protein concentration in the remaining cell lysate was determined by Bradford (Bio-Rad, Richmond, CA) using bovine serum albumin in lysis buffer as reference. Adipocytes from two subjects were analyzed by in-gel digestion and from three subjects by on-beads digestion.

Immunoprecipitation

Samples were immunoprecipitated according to the manufacturer's instructions with the addition of a pre-clearing step. In short, 1 mg of protein in 1 ml of lysis buffer was precleared on 50 µl of protein G plus-agarose (sc-2002, Santa Cruz Biotechnology) before overnight incubation with 4 µg of anti-IQGAP1 antibody or with 4 µg of normal mouse IgG for control. Protein G plus-agarose was used to capture antibody–protein complexes, and unbound proteins were removed by washing with lysis buffer.

In-gel digestion

After immunoprecipitation, the beads were incubated for 15 min at 65°C in SDS–PAGE sample buffer and subjected to SDS–PAGE (Ready Gel Tris–HCl, 4–20%, Bio-Rad, Richmond, CA). Gels were silver-stained and each lane was cut into 29 bands, reduced with dithiothreitol, alkylated with iodoacetamide, and digested by trypsin (Thermo Scientific, Rockford, IL, USA) overnight at 37°C, as described previously [55]. Generated peptides were dried and dissolved in 0.1% (v/v) formic acid in water prior to mass spectrometry analyses.

On-beads digestion

After immunoprecipitation, beads with bound proteins were washed three times with 50 mM ammonium bicarbonate and digested with 0.1 μg trypsin in 100 µl for 3 h at 23°C with continuous shaking at 400 rpm. The supernatant was removed from the beads and reduced by the addition of dithiothreitol to a final concentration of 1 mM and the digestion was continued overnight at 37°C. Peptides were then alkylated with iodoacetamide (final concentration 6 mM) for 30 min at room temperature in darkness and reactions were terminated with the addition of 1 µl of concentrated triflouroacetic acid. Samples were desalted on Millipore C18 ZipTips (Millipore, Temecula, CA, USA), and peptides were eluted from the tips with 20% (v/v) acetonitrile and 1% (v/v) formic acid in water, dried, and dissolved in 0.1% (v/v) formic acid in water prior to LC–MS/MS analyses.

LC–MS/MS peptides were loaded on an EASY-nanoLCII system coupled online to an LTQ Orbitrap Velos Pro hybrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). Peptides from in-gel digestion were separated during 60 min by reverse-phase chromatography on a 20 × 0.1 mm C18 pre-column followed by a 100 × 0.075 mm C18 analytical column (particle size 5 µm, NanoSeparations, Nieuwkoop, Netherlands) at a flow rate of 300 nl/min. A gradient of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) was applied as follows: starting with 2% B; linear gradient 2–40% B in 0–40 min; 40–90% B in 40–60 min. On-beads-digested peptides were analyzed with the same experimental set-up as in-gel-digested peptides except for an LC gradient which was distributed as follows: linear gradient 2–25% B in 0–145 min and 25–100% B in 145–180 min. The top 20 most intense multiply charged ions were selected with an isolation window of 2.0 and fragmented in the linear ion trap by collision-induced dissociation with normalized collision energy of 30%. Dynamic exclusion of sequenced peptides for 60 s and charge state-filtering disqualifying singly charged peptides were activated and predictive AGC was enabled.

Database search

Raw files were searched using Sequest in Proteome Discoverer (Thermo Fisher Scientific, San Jose, CS, USA; version 1.4.0.288) against a Uniprot Human 9606 (20130118) database with the following parameters: digestion enzyme trypsin; maximum number of missed cleavages 2; fragment ion mass tolerance 0.60 Da; parent ion mass tolerance 10.0 ppm; fixed modification carbamidomethylation of cysteine; variable modifications were oxidation of methionine, acetylation of lysine and the N-terminus, phosphorylation of serine, threonine, and tyrosine. Percolator was used for postprocessing and data were filtered at 1% false discovery rate. For in-gel digestion, a protein had to be identified in three out of four runs (two subjects, each with a technical replicate) to be considered a true identification. For on-beads digestion, a protein had to be identified both in technical repeats of a subject and in two out of three subjects. Samples immunoprecipitated with normal mouse IgG control antibody were used to identify unspecific interactions and, at least twice, the spectrum count in the immunoprecipitated sample, compared with the control sample, was required for identification as an immunoprecipitate-specific protein.

Bioinformatics analysis

To reveal described interactions between the identified proteins, we submitted the Uniprot accessions to STRING 9.1 [39] using a medium confidence score (minimum 0.400) and all prediction methods were activated. Pathway Studio, version 11.0 (Elsevier Inc.), was also used to identify interactions among the immunoprecipitated proteins and their involvement in different interaction pathways.

Confocal microscopy

The cells were fixed in 3% paraformaldehyde for 15 min and attached to poly-l-lysine-coated coverslips. Coverslips were then washed in glycine/phosphate buffer (50 mM glycine, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4). Cells were permeabilized in 0.1% saponin and blocked with 3% normal horse serum with 1% bovine serum albumin in the glycine/phosphate buffer for 1 h at 37°C in a humidity chamber. Incubation with primary antibody IQGAP1 (Millipore #05-504) was done overnight at 4°C. Following washes, cells were incubated with fluorescent secondary antibody (Alexa Fluor 488, Molecular Probes, Eugene, USA) for 2 h at room temperature. The nuclei were stained with DAPI (Vector Laboratories, Burlingame, USA). Confocal scanning microscopy was performed with an LSM 700 (Carl Zeiss, Jena, Germany).

Proximity ligation assay

In situ PLAs were performed using a Duolink kit (Olink Bioscience, Uppsala, Sweden) generally according to the manufacturer's instructions with some modifications. The cells were fixed in 4% paraformaldehyde for 10 min and attached to poly-l-lysine-coated 8-well chamber slides. The cells were then permeabilized with 0.01% Triton in Tris-buffered saline (TBS; 50 mM Tris, pH 7.6, 150 mM NaCl) and incubated with 100 mM glycine in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4) for 20 min. Permeabilized cells were incubated overnight at 4°C with primary antibodies diluted as follows: mouse IQGAP1 1:50 and rabbit caveolin 1:250. Cells were washed three times in TBS with 0.05% Tween-20 for 5 min each with gentle agitation. Secondary antibodies conjugated with oligonucleotides, PLA probe anti-mouse MINUS and PLA probe anti-rabbit PLUS, were added to the cells and incubated for 90 min at 37°C in a humidity chamber. Finally, after ligation and amplification steps, cells were counterstained with the DNA-binding dye Hoechst (Molecular Probes). Images were observed using an AxioCam MRm Rev.3 camera coupled to a Zeiss Axio Imager Z2 microscope (Carl Zeiss Microimaging GmbH, Jena, Germany) and analyzed with ZEN Pro imaging software. Negative controls were one primary antibody with both of the secondary antibodies, as well as no primary antibody with both of the secondary antibodies.

Immunoblotting

After separation by SDS–PAGE, proteins were transferred to PVDF membranes (Immobilon-P, Millipore, MA, USA). Membranes were blocked, incubated with anti-IQGAP1 or anti-β-tubulin antibody, visualized by chemiluminescence imaging (LAS 1000; Image Guage v.3.0, Fujifilm, Tokyo, Japan), and analyzed with MultiGauge V3.0 (Fujifilm). β-Tubulin was used for loading normalization between samples. The concentrations of primary and secondary antibodies were adjusted to ascertain a linear relationship between the amount of specific protein and the chemiluminescence signal. To allow comparison between different gels, two identical standard samples (mixture of aliquots from multiple subjects) were loaded on each gel, and all analyzed samples were normalized against the mean of the standard samples. Student's t-test is used for statistical analysis of the immunoblotting results.

Analysis of mRNA expression

RNA was prepared from isolated adipocytes by Trizol (Life Technologies, CA, USA) extraction and solid-phase extraction using RNeasy MinElute Cleanup columns (Qiagen, Germantown, MD, USA). Reagents for real-time PCR analysis of IQGAP1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; iQ™ SYBR® Green Supermix) were from Bio-Rad (Hercules, CA, USA). GAPDH was used as an internal reference to normalize expression levels between samples. First-strand cDNA was synthesized from 1.5 µg of adipocyte total RNA using the iScript Advanced cDNA Synthesis Kit (Bio-Rad Laboratories) for qRT-PCR according to the manufacturer's instructions (Bio-Rad Laboratories). All real-time PCR measurements were performed in triplicates (CFX96 Real-Time PCR Detection System, Bio-Rad Laboratories) with default cycle parameters. Primers were IQGAP1 forward: 5′-CACTGGCTAAGACGGAAGTGTC-3′ and reverse: 5′-TCCTGGCTGGAACCGGAT-3′ and for GAPDH forward: 5′-GTCAGTGGTGGACCTGACCT-3′ and reverse: 5′-CACCACCCTGTTGCTGTAGC-3′.

Results

Identification of IQGAP1-associated proteins

To identify proteins associated with IQGAP1 in human adipocytes, we lysed isolated mature primary human adipocytes in a nonprotein-denaturing CHAPS-containing buffer to maintain the integrity of protein complexes. IQGAP1-interacting proteins were immunoprecipitated with antibodies against IQGAP1 and analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). To minimize methodological bias in the identification of the IQGAP1 interactome, we digested immunoprecipitated proteins with trypsin using both in-gel digestion and on-beads digestion prior to analysis by mass spectrometry. Careful elimination of unspecifically immunoprecipited proteins and of potentially false positive identifications, as described in Experimental Procedures, rendered a list of 32 highly confident IQGAP1-associated proteins (Table 1). Out of these 32 proteins, 16 were confidently identified both after in-gel and on-beads digestion, 12 only after in-gel digestion, and 4 only after on-beads digestion. No IQGAP1 peptides were detected by immunoblotting or by mass spectrometry in controls immunoprecipitated without IQGAP1 antibody. We also analyzed the whole cell lysate and no IQGAP2- or IQGAP3-specific peptides were detected by mass spectrometry after in-gel digestion, indicating that IQGAP1, identified by 30 unique peptides (not shown), is the dominating isoform in human adipocytes.

Table 1
IQGAP1 interactome identified in primary human adipocytes

Adipocytes from five nondiabetic subjects [mean age 64 years (age 45–87); mean BMI 29 kg/m2 (range 23–32)] were analyzed, with two replicates each.

Protein name Gene name Uniprot accession number Theoretical Mw (kDa) Coverage (%) No. of peptides 
Alcohol dehydrogenase 1B ADH1B P00325 39.9 19 
Calmodulin CALM1/2 P62158 16.8 32 
Caveolin-1 CAV1 Q03135 20.4 24 
Dihydrolipoyllysine residue acetyltransferase component of pyruvate dehydrogenase complex (DLAT) DLAT P10515 69 26 13 
EH domain-containing protein 2 EHD2 Q9NZN4 61.2 15 
Fatty acid synthase FASN P49327 273.4 11 
Filaggrin FLG P20930 431.1 
Filaggrin-2 FLG2 Q5D862 248 
Glycogen phosphorylase PYGL P06737 97.1 16 11 
Hormone-sensitive lipase LIPE Q05469 116.6 18 14 
Inverted formin-2 INF2 Q27J81 135.6 
Myosin light polypeptide 6 MYL6 P60660 16.9 30 
Myosin regulatory light chain 12A MYL12A P19105 19.8 24 
Unconventional myosin-Ic MYO1C O00159 121.7 36 31 
Myosin-9 MYH9 P35579 226.6 43 64 
Myosin-10 MYH10 P35580 229 39 61 
Myosin-14* MYH14 Q7Z406 228.6 39 60 
Perilipin-1 PLIN1 O60240 56 16 
Perilipin-4 PLIN4 Q96Q06 134.4 40 42 
Polymerase I and transcript release factor (Cavin 1) PTRF Q6NZI2 43.50 34 13 
Protein kinase Cδ-binding protein (Cavin 3) PRKCDBP Q969G5 27.70 28 
Pyruvate dehydrogenase E1 component subunit β (PDHB) PDHB P11177 39.20 11 
Redox-regulatory protein FAM213A FAM213A Q9BRX8 25.7 29 
Serum deprivation response protein (Cavin 2) SDPR O95810 47.1 16 
Spectrin α chain, nonerythrocytic 1 SPTAN1 Q13813 284.5 38 73 
Spectrin β chain, nonerythrocytic 1 SPTBN1 Q01082 274.6 33 60 
Tensin-1 TNS1 Q9HBL0 185.7 
Trifunctional enzyme subunit α HADHA P40939 83 36 21 
Trifunctional enzyme subunit β HADHB P55084 49 20 
Tubulin α TUBA Q9BQE3 49.9 13 
Tubulin β§ TUBB Q5JP53 49.9 
Vimentin VIM P08670 53.6 64 51 
Protein name Gene name Uniprot accession number Theoretical Mw (kDa) Coverage (%) No. of peptides 
Alcohol dehydrogenase 1B ADH1B P00325 39.9 19 
Calmodulin CALM1/2 P62158 16.8 32 
Caveolin-1 CAV1 Q03135 20.4 24 
Dihydrolipoyllysine residue acetyltransferase component of pyruvate dehydrogenase complex (DLAT) DLAT P10515 69 26 13 
EH domain-containing protein 2 EHD2 Q9NZN4 61.2 15 
Fatty acid synthase FASN P49327 273.4 11 
Filaggrin FLG P20930 431.1 
Filaggrin-2 FLG2 Q5D862 248 
Glycogen phosphorylase PYGL P06737 97.1 16 11 
Hormone-sensitive lipase LIPE Q05469 116.6 18 14 
Inverted formin-2 INF2 Q27J81 135.6 
Myosin light polypeptide 6 MYL6 P60660 16.9 30 
Myosin regulatory light chain 12A MYL12A P19105 19.8 24 
Unconventional myosin-Ic MYO1C O00159 121.7 36 31 
Myosin-9 MYH9 P35579 226.6 43 64 
Myosin-10 MYH10 P35580 229 39 61 
Myosin-14* MYH14 Q7Z406 228.6 39 60 
Perilipin-1 PLIN1 O60240 56 16 
Perilipin-4 PLIN4 Q96Q06 134.4 40 42 
Polymerase I and transcript release factor (Cavin 1) PTRF Q6NZI2 43.50 34 13 
Protein kinase Cδ-binding protein (Cavin 3) PRKCDBP Q969G5 27.70 28 
Pyruvate dehydrogenase E1 component subunit β (PDHB) PDHB P11177 39.20 11 
Redox-regulatory protein FAM213A FAM213A Q9BRX8 25.7 29 
Serum deprivation response protein (Cavin 2) SDPR O95810 47.1 16 
Spectrin α chain, nonerythrocytic 1 SPTAN1 Q13813 284.5 38 73 
Spectrin β chain, nonerythrocytic 1 SPTBN1 Q01082 274.6 33 60 
Tensin-1 TNS1 Q9HBL0 185.7 
Trifunctional enzyme subunit α HADHA P40939 83 36 21 
Trifunctional enzyme subunit β HADHB P55084 49 20 
Tubulin α TUBA Q9BQE3 49.9 13 
Tubulin β§ TUBB Q5JP53 49.9 
Vimentin VIM P08670 53.6 64 51 
*

Evidence of MYH14 isoform 6 in one subject.

Peptides also correspond to accession number P11277.

Peptides also correspond to accession numbers Q71U36 and P68363.

§

Evidence of TUBB 4B chain in one subject and TUBB 2A chain in one subject.

Potential functional interactions between the identified proteins were analyzed with STRING [39] and Pathway Studio (Experimental Procedures). Both analyses revealed two major clusters of proteins that interact with IQGAP1 in adipocytes: cytoskeletal proteins and caveolae proteins (Figure 1). The group of cytoskeletal proteins comprised myosins, tubulins, spectrins, fillagrins, vimentin, calmodulin, formin, and tensin. We also identified four proteins involved in lipid metabolism [hormone-sensitive lipase (HSL), perilipin-1 (PLIN1), perilipin-4, and fatty acid synthase] and mitochondrial functions (Figure 1). The group of mitochondrial interactors was represented by trifunctional enzyme (subunits α and β) and by two components of the multimeric pyruvate dehydrogenase complex: pyruvate dehydrogenase E1 component subunit β and dihydrolipoyllysine residue acetyltransferase component.

IQGAP1 interactome in primary human adipocytes.

Figure 1.
IQGAP1 interactome in primary human adipocytes.

Most of the proteins that were coimmunoprecipitated with IQGAP1 can be clustered into four different groups: cytoskeletal proteins, proteins associated with caveolae, proteins involved in TAG metabolism, and mitochondrial proteins. The abbreviations used: TAG, triacylglycerol; PDHB, pyruvate dehydrogenase E1 component subunit β; HSL, hormone-sensitive lipase; PLIN1, perilipin-1; PLIN4, perilipin-4; FAM213A, redox-regulatory protein; TFE, trifunctional enzyme subunit α and β.

Figure 1.
IQGAP1 interactome in primary human adipocytes.

Most of the proteins that were coimmunoprecipitated with IQGAP1 can be clustered into four different groups: cytoskeletal proteins, proteins associated with caveolae, proteins involved in TAG metabolism, and mitochondrial proteins. The abbreviations used: TAG, triacylglycerol; PDHB, pyruvate dehydrogenase E1 component subunit β; HSL, hormone-sensitive lipase; PLIN1, perilipin-1; PLIN4, perilipin-4; FAM213A, redox-regulatory protein; TFE, trifunctional enzyme subunit α and β.

IQGAP1 associates with caveolae in primary human adipocytes

The most striking IQGAP1 partners in human adipocytes were caveolae-associated proteins (Table 1 and Figure 1). Caveolin-1, cavin-1, cavin-2, cavin-3, and EHD2 are proteins required for the integrity of caveolae structures [4,40]. Moreover, myosin-Ic (Table 1 and Figure 1) has previously been identified as a caveolar protein [4], and the triacylglycerol (TAG)-metabolizing HSL and PLIN1 (Table 1 and Figure 1) are known to be associated with caveolae in adipocytes [4143].

By confocal microscopy, we found that IQGAP1 was evenly distributed along the plasma membrane of the human adipocyte, including the expanded region harboring the nucleus (Figure 2), in line with the distribution of caveolae in the plasma membrane [1]. However, the extreme architecture of the adipocyte, with a thin film of cytosol between the central lipid droplet and the plasma membrane, precludes a distinction between a pure caveolar and cytosolic localization of IQGAP1 by this technique.

IQGAP1 distribution in primary human adipocytes.

Figure 2.
IQGAP1 distribution in primary human adipocytes.

(A) IQGAP1 was visualized with a fluorescent antibody (green) using confocal microscopy. The nucleus was stained with DAPI (blue). The experiment was repeated with adipocytes from four subjects. (B) Close-up of plasma membrane region.

Figure 2.
IQGAP1 distribution in primary human adipocytes.

(A) IQGAP1 was visualized with a fluorescent antibody (green) using confocal microscopy. The nucleus was stained with DAPI (blue). The experiment was repeated with adipocytes from four subjects. (B) Close-up of plasma membrane region.

Next, to confirm the colocalization of IQGAP1 with caveolin-1, the defining marker protein for caveolae, we used in situ proximity ligation assay (PLA). PLA is an antibody-based method to visualize and quantify specific protein interactions in the cell. In this method, two different proteins of interest are recognized by their respective specific primary antibody and then with a corresponding pair of secondary antibodies conjugated to complementary oligonucleotides. In close proximity (<40 nm), the oligonucleotides hybridize and are ligated and amplified. A fluorescent signal from each pair of PLA probes can then be detected and quantified by counting the number of fluorescent spots in microscopic images of the cells [44]. Our results indicate a clear colocalization between caveolin-1 and IQGAP1, with a large number of fluorescent interaction spots in adipocytes incubated with antibodies against both proteins (Figure 3C), but not in control samples (Figure 3A,B).

IQGAP1 and caveolin-1 colocalization.

Figure 3.
IQGAP1 and caveolin-1 colocalization.

Specific colocalization of IQGAP1 and caveolin-1 was demonstrated by PLA. Each red spot represents a single IQGAP1–caveolin-1 complex (interaction). (A) Control, under basal conditions, with antibodies against IQGAP1 only. (B) Control with antibodies against caveolin-1 only. (C) Colocalization of IQGAP1 and caveolin-1 under basal conditions. (D) Colocalization of IQGAP1 and caveolin-1 in the presence of insulin. Adipocytes were incubated with 10 nM insulin for 30 min, when the interaction of IQGAP1 with caveolin-1 was analyzed. The nucleus was stained with DAPI (blue). (E) Adipocytes were incubated without (basal) or with 10 nM insulin for 30 min, as indicated, when the interaction of IQGAP1 with caveolin-1 was quantified by PLA analysis. The number of PLA interaction spots was counted in five independent experiments (3–8 pictures each) from three different patients. Mean values of control cells (incubated without primary antibodies against IQGAP1 and caveolin-1) were subtracted from those of each experiment and noninsulin-treated cells were set to 1. Data were presented as mean ± SE (error bar), one-sample t-test.

Figure 3.
IQGAP1 and caveolin-1 colocalization.

Specific colocalization of IQGAP1 and caveolin-1 was demonstrated by PLA. Each red spot represents a single IQGAP1–caveolin-1 complex (interaction). (A) Control, under basal conditions, with antibodies against IQGAP1 only. (B) Control with antibodies against caveolin-1 only. (C) Colocalization of IQGAP1 and caveolin-1 under basal conditions. (D) Colocalization of IQGAP1 and caveolin-1 in the presence of insulin. Adipocytes were incubated with 10 nM insulin for 30 min, when the interaction of IQGAP1 with caveolin-1 was analyzed. The nucleus was stained with DAPI (blue). (E) Adipocytes were incubated without (basal) or with 10 nM insulin for 30 min, as indicated, when the interaction of IQGAP1 with caveolin-1 was quantified by PLA analysis. The number of PLA interaction spots was counted in five independent experiments (3–8 pictures each) from three different patients. Mean values of control cells (incubated without primary antibodies against IQGAP1 and caveolin-1) were subtracted from those of each experiment and noninsulin-treated cells were set to 1. Data were presented as mean ± SE (error bar), one-sample t-test.

Insulin control of IQGAP1 colocalization with caveolae

Insulin is known to bind to its receptor and to signal in caveolae [8]. Moreover, insulin induces the phosphorylation of caveolin-1 in caveolae in human adipocytes [9], which may control the interaction with IQGAP1 [45]. We therefore examined the effect of insulin stimulation on the colocalization of caveolin-1 and IQGAP1. PLA demonstrated an increased number of interactions between IQGAP1 and caveolin-1 after treatment with 10 nM insulin (this concentration produces maximal effects throughout the insulin signaling network [46]) (Figure 3D). Quantitation of the number of caveolin-1/IQGAP1 interaction spots demonstrated a five-fold increased abundance of colocalized caveolin-1 and IQGAP1 in response to insulin stimulation of the adipocytes (Figure 3E). No change in the abundance of IQGAP1 protein was observed after insulin stimulation by SDS–PAGE and immunoblotting (not shown), indicating a redistribution and increased interaction of IQGAP1 with caveolae.

IQGAP1 in adipocytes from nondiabetic and type 2 diabetic subjects

We compared the total abundance of IQGAP1 in adipocytes obtained from nondiabetic subjects and from patients with type 2 diabetes. The abundance of IQGAP1 protein was 40% lower in adipocytes from diabetic subjects compared with that from nondiabetic controls (Figure 4A,B). Likewise, the abundance of mRNA transcripts for IQGAP1 was 33% lower in the diabetic adipocytes, as determined by real-time PCR (Figure 4C).

Abundance of IQGAP1 protein and mRNA transcript in primary human adipocytes normally and in type 2 diabetes.

Figure 4.
Abundance of IQGAP1 protein and mRNA transcript in primary human adipocytes normally and in type 2 diabetes.

(A) Quantification of total IQGAP1 protein by SDS–PAGE and immunoblotting of adipocytes from patients diagnosed with type 2 diabetes (n = 8 subjects) [mean age 55 years (range 28–81); mean BMI 39 kg/m2 (range 28–49)], compared with adipocytes from nondiabetic subjects (n = 10 subjects) [mean age 60 years (range 33–91); mean BMI 24 kg/m2 (range 19–27)]. Data were presented in arbitrary units as mean ± SE (error bars), Student's t-test. A representative blot was used for quantification of total IQGAP1 protein in adipocytes obtained from nondiabetic and type 2 diabetic subjects. β-Tubulin was used for loading normalization between samples. (B) Real-time PCR quantification of IQGAP1 mRNA in primary human adipocytes from patients diagnosed with type 2 diabetes (n = 4 subjects) [mean age 68 years (range 45–79); mean BMI 35 kg/m2 (range 29–43)], compared with adipocytes from nondiabetic subjects (n = 4 subjects) [mean age 62 years (range 57–69); mean BMI 23 kg/m2 (range 22–24)]. Mean values of normal (nondiabetic) adipocytes were subtracted from the individual values of T2D cells, and normal cells values were set to 1. Data were presented as mean ± SE (error bar), one-sample t-test.

Figure 4.
Abundance of IQGAP1 protein and mRNA transcript in primary human adipocytes normally and in type 2 diabetes.

(A) Quantification of total IQGAP1 protein by SDS–PAGE and immunoblotting of adipocytes from patients diagnosed with type 2 diabetes (n = 8 subjects) [mean age 55 years (range 28–81); mean BMI 39 kg/m2 (range 28–49)], compared with adipocytes from nondiabetic subjects (n = 10 subjects) [mean age 60 years (range 33–91); mean BMI 24 kg/m2 (range 19–27)]. Data were presented in arbitrary units as mean ± SE (error bars), Student's t-test. A representative blot was used for quantification of total IQGAP1 protein in adipocytes obtained from nondiabetic and type 2 diabetic subjects. β-Tubulin was used for loading normalization between samples. (B) Real-time PCR quantification of IQGAP1 mRNA in primary human adipocytes from patients diagnosed with type 2 diabetes (n = 4 subjects) [mean age 68 years (range 45–79); mean BMI 35 kg/m2 (range 29–43)], compared with adipocytes from nondiabetic subjects (n = 4 subjects) [mean age 62 years (range 57–69); mean BMI 23 kg/m2 (range 22–24)]. Mean values of normal (nondiabetic) adipocytes were subtracted from the individual values of T2D cells, and normal cells values were set to 1. Data were presented as mean ± SE (error bar), one-sample t-test.

Discussion

The findings here demonstrate a novel role of IQGAP1 as an insulin-regulated caveolae interactor. Furthermore, in addition to caveolae, various cytoskeletal proteins that IQGAP1 interacts with suggest that IQGAP1 may act as an insulin-regulated link between caveolae and the cytoskeleton. This is interesting as the insulin receptor is exclusively localized in caveolae in human adipocytes, and caveolae are critically involved in the endocytosis of the receptor [7,9], which most probably requires cytoskeletal restructuring. The insulin-regulated translocation of GLUT4 in intracellular vesicles to the plasma membrane involves caveolae [68] and also requires cytoskeletal traffic and restructuring, as reviewed in ref. [14]. Moreover, fatty acid uptake and synthesis of TAG in the caveolae membrane [5,47] require cytoskeletal restructuring, especially in connection with its fusion with the central lipid droplet of the adipocyte.

Of the cytoskeletal proteins, F-actin is a well-studied IQGAP1-interacting protein [11,2224,33,35]. Although we detected more actin in the IQGAP1 immunoprecipitates than in the controls after both in-gel and on-beads digestion, we have not included actin in the list of IQGAP1-interacting proteins in human adipocytes (Table 1), because too much actin was present in the controls. Several proteins have been reported to interact with IQGAP1 [22] that were not detected in our experiments. This discrepancy can be explained not only by different cell types examined, but also by different experimental conditions that will affect the stringency of the coimmunoprecipitation.

It should be noted that caveolae are relatively detergent-resistant structures of the plasma membrane; therefore, our findings do not necessarily imply that IQGAP1 directly interacts with each one of the coimmunoprecipitated caveolar proteins. Moreover, caveolae are highly enriched in cholesterol [2,48], and a recent study demonstrates that, in prostate cancer cells, IQGAP1 localizes to cholesterol-rich membrane ruffles and this localization is regulated by caveolin-1 [45]. It has also been demonstrated that IQGAP1 binds phosphoinositides, which are enriched in caveolae [49,50], through its C-terminal aPI domain [51]. Direct binding partner(s) of caveolae to IQGAP1 remain to be identified, as well as the IQGAP1 domain(s) involved.

Our finding that the abundance of IQGAP1 is substantially reduced in adipocytes from patients with type 2 diabetes is most interesting in light of insulin control of the interaction of IQGAP1 with caveolae and the critical functions of caveolae in insulin signaling [8] and in adipocyte metabolism [47]. The reduced abundance of both the IQGAP1 protein and the corresponding mRNA indicates that the transcriptional control of IQGAP1 may be affected in the diabetic state. The abundance of IQGAP1 mRNA has also been reported to be reduced in the insulin-producing β-cells from patients with type 2 diabetes [52]. It will be most interesting to investigate how the reduced abundance of IQGAP1 affects the adipocytes and to what extent that contributes to the diabetic state in these cells.

Abbreviations

EHD2, EH domain-containing protein 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HSL, hormone-sensitive lipase; IQGAP1, IQ motif-containing GTPase activating protein-1; LC–MS/MS, liquid chromatography–tandem mass spectrometry; PDHB, pyruvate dehydrogenase E1 component subunit β; PLA, proximity ligation assay; PLIN1, perilipin-1; TAG, triacylglycerol; TBS, Tris-buffered saline.

Author Contribution

M.V.T. and P.S. designed the study and supervised the project. Å.J. performed mass spectrometry experiments. M.R.R. and Å.J. performed confocal microscopy. M.R.R., Å.J., and C.J. performed PLA preparations and immunoblotting. C.J. and Å.J. made cell preparations and immunoprecipitation experiments. M.R.R. performed mRNA experiments. All authors analyzed the results and wrote the manuscript.

Funding

The present study was supported by University of Linköping, a 3-year program at the Swedish Diabetes Fund, and a 5-year program at the Swedish Research Council.

Acknowledgments

The in situ PLA was performed by the PLA Proteomics facility, which is supported by Swedish Science for Life Laboratory.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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