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Endocrinology, doi:10.1210/en.2003-1197
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Endocrinology Vol. 145, No. 4 1579-1586
Copyright © 2004 by The Endocrine Society

Annexin II Is a Thiazolidinedione-Responsive Gene Involved in Insulin-Induced Glucose Transporter Isoform 4 Translocation in 3T3-L1 Adipocytes

Jie Huang, Stanley H. Hsia, Takeshi Imamura, Isao Usui and Jerrold M. Olefsky

Department of Medicine (J.H., S.H.H., T.I., I.U., J.M.O.), Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California 92093; and San Diego Veterans Administration Medical Research Service and the Whittier Institute for Diabetes (J.M.O.), La Jolla, California 92037

Address all correspondence and requests for reprints to: Jerrold M. Olefsky, M.D., Department of Medicine (0673), University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0673. E-mail: jolefsky{at}ucsd.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The target genes of peroxisomal proliferator-activated receptor-{gamma} ligands that lead to insulin sensitization are not fully understood. In this study, we have found that the thiazolidinedione, troglitazone, increases expression of annexin II at both the mRNA and protein levels, raising the possibility that annexin II plays a role in insulin-stimulated glucose transporter isoform 4 (GLUT4) translocation and glucose transport. To assess this, we microinjected annexin II antibody or annexin II small interfering RNA into 3T3-L1 adipocytes and found that insulin-stimulated GLUT4 translocation was inhibited by 54 and 60%, respectively. Furthermore, microinjection of annexin II antibody inhibited constitutively active G{alpha}q (Q209L-G{alpha}q)-induced but not osmotic shock-induced GLUT4 translocation. When cells were cotransfected with wild-type annexin II, along with an enhanced green fluorescent protein-cmyc-GLUT4 construct, and the percentage of cells expressing cmyc-GLUT4 at the cell surface was measured by immunofluorescence microscopy, there was a marked increase in the ability of insulin to stimulate recruitment of cmyc-GLUT4 protein to the cell surface. In summary, our results show that annexin II is a newly described thiazolidinedione response gene involved in insulin-induced GLUT4 translocation in 3T3-L1 adipocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN STIMULATES GLUCOSE uptake into muscle and adipose tissue by increasing the translocation of glucose transporter isoform 4 (GLUT4), an insulin-responsive glucose transporter, from an intracellular localization to the cell surface (1, 2). Thiazolidinediones (TZDs) represent a class of insulin-sensitizing agents that have proved clinically useful for the treatment of type II diabetes and other insulin-resistant disorders (3). These agents work by binding to the peroxisomal proliferator-activated receptor-{gamma} (PPAR{gamma}) nuclear receptor, which then modulates expression levels of target genes (3). It has been shown that TZDs increase glucose uptake, GLUT4 translocation, and the expression levels of glucose transporters in adipocytes and myocytes (4, 5, 6). However, the molecular mechanisms by which TZDs sensitize cells to insulin action remain unclear. It would be of great interest to identify genes that are differentially regulated by TZDs and to elucidate the effects that the gene products may have to improve insulin sensitivity. Along these lines, we have used suppression subtractive hybridization (SSH) to identify genes that are differentially induced by troglitazone in 3T3-L1 adipocytes. SSH, a method based on suppression PCR, can selectively amplify differentially expressed cDNA fragments and simultaneously suppress amplification of nontarget sequences, therefore allowing enrichment for the differentially expressed cDNAs. It is a potent technique for identifying disease, tissue-specific, or other differentially expressed genes in response to experimental stimuli (7). Using this SSH approach, we have identified annexin II as a gene whose expression is up-regulated by TZDs in 3T3-L1 adipocytes.

Annexin II, a member of the annexin family, binds to acidic phospholipids, heparin, and F-actin in a calcium-dependent manner (8). Within the cell, annexin II exists as both a 36-kDa monomer and a 94-kDa heterotetramer, which is composed of two copies of annexin II and a dimer of p11, an S100-related protein (9, 10). Whereas annexin II monomer is cytosolic, the heterotetramer is localized to both the intracellular and extracellular surfaces of the plasma membrane, to the cortical cytoskeleton, early endosomes, clathrin-coated pits, and secretory vesicles (8). Although the physiological functions of this protein are still not clear, annexin II has been implicated in membrane-trafficking events such as exocytosis and endocytosis (8, 10, 11, 12). Cell surface annexin II has also been identified as a coreceptor for both plasminogen and tissue plasminogen activator (t-PA) on the endothelial cell surface, facilitating the production of plasmin (13, 14). In addition to its intracellular localization, annexin II is also found in the extracellular milieu in some tissues and cell culture media (15, 16). Extracellular annexin II may be important in several biological processes, such as plasminogen activation, cell adhesion, immunoglobulin transport, and viral infection (17).

In this study, we identified annexin II as one of the TZD response genes in 3T3-L1 adipocytes and demonstrated a novel role for annexin II in insulin-stimulated GLUT4 translocation. Inhibition of annexin II by microinjection of annexin II antibody or small interfering RNA (siRNA) into 3T3-L1 cells inhibited insulin-induced GLUT4 translocation. Microinjection of annexin II antibody also inhibited constitutively active G{alpha}q (Q209L-G{alpha}q)-induced GLUT4 translocation. Expression of wild-type annexin II led to an increase in insulin-induced GLUT4 translocation and glucose uptake. Taken together, our results show that annexin II is a TZD-responsive gene and a positive mediator in insulin-induced GLUT4 translocation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Murine annexin II cDNA was kindly provided by Dr. Tony Hunter (The Salk Institute, La Jolla, CA). Enhanced green fluorescent protein (EGFP)-cmyc-GLUT4 construct was kindly provided by Dr. Jeffrey E. Pessin (University of Iowa, Iowa City, IA). Rabbit polyclonal anti-GLUT4 and anti-GLUT1 antibodies were from Chemicon International (Temecula, CA). pcDNA3.1/Hygro expression vector was from Invitrogen (Carlsbad, CA). Monoclonal anti-annexin II antibody was from Transduction Laboratories (Lexington, KY), monoclonal anti-GLUT4 antibody was from Biogenesis (Kingston, NH), and monoclonal anti-ß-actin antibody was from Sigma Chemical Co. (St. Louis, MO). Monoclonal anti-cmyc antibody and horseradish peroxidase-linked antimouse, antirabbit antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Sheep IgG and rhodamine-, fluorescein isothiocyanate-conjugated antirabbit, antimouse, and antisheep IgG antibodies were obtained from Jackson Immmunoresearch Laboratories Inc. (West Grove, PA). Annexin II siRNA and a scrambled siRNA were purchased from Dharmacon Research, Inc. (Lafayette, CO). DMEM and fetal calf serum (FCS) were purchased from Life Technologies (Grand Island, NY). Troglitazone was from Sankyo Pharmaceutical Inc. (Tokyo, Japan). 2-[3H]Deoxyglucose and L-[3H]glucose were from ICN (Costa Mesa, CA). All other reagents were purchased from Sigma.

Cell treatment and transient transfection
3T3-L1 cells were cultured and differentiated as described previously (18). For preparation of total RNA for SSH and whole-cell lysates for immunoblotting experiments, d-9 3T3-L1 adipocytes were treated with 10 µg/ml troglitazone or vehicle control dimethylsulfoxide (DMSO) for 72 h. For 2-deoxyglucose (2-DOG) uptake and GLUT4 translocation assays, differentiated cells were treated with 10 µg/ml troglitazone or vehicle control (DMSO) for 72 h and serum starved for 4 h in DMEM containing 0.1% BSA before insulin stimulation.

The wild-type annexin II cDNA was subcloned into a mammalian expression vector, pcDNA3.1/Hygro. Differentiated 3T3-L1 adipocytes were transiently transfected with wild-type annexin II construct or cotransfected with EGFP-cmyc-GLUT4 and annexin II construct by electroporation as previously described (19). After electroporation, the cells were replated on collagen-coated tissue culture plates and incubated in DMEM containing 10% FCS at 37 C for 48 h before GLUT4 translocation and 2-DOG uptake experiments.

SSH
SSH between troglitazone-treated and untreated 3T3-L1 adipocyte RNA was performed using the PCR-Select cDNA Subtraction kit (Clontech, Palo Alto, CA) according to the manufacturer’s protocol. Briefly, total RNA was isolated using RNAzol B (Tel-Test, Friendswood, TX). mRNA was extracted using the Oligotex system (QIAGEN, Chatsworth, CA). Driver and tester cDNA was prepared using the PCR-Select cDNA Subtraction kit (Clontech). After two steps of subtractive hybridization, 27 primary PCR cycles and 12 secondary PCR cycles were performed. The PCR products were cloned into the pCR 2.1 vector using the T/A Cloning kit (Invitrogen).

Slot-blotting and clone identification
Probes for Northern slot blotting were synthesized by PCR using the nested PCR primers for the SSH-PCR. The PCR products were radiolabeled internally with [{alpha}-32P]dATP (3000 mCi/mmol, 10 mCi/ml) (NEN Life Science Products, Boston, MA) at 37 C for 30 min using a Strip-EZ DNA Kit (Ambion, Austin, TX). Total RNA (10 µg) was denatured at 65 C for 15 min in a formaldehyde-containing mixture before being loaded on nylon membranes (MSI, Westborough, MA). After UV cross-linking, membranes were prehybridized at 42 C for 1 h (50% deionized formamide, 5x sodium chloride-sodium phosphate-EDTA (SSPE), 5x Denhardt’s solution, 0.2% SDS, and 0.1 mg/ml freshly denatured sheared salmon sperm DNA) and then hybridized with radiolabeled probes at 42 C overnight in hybridization solution (50% deionized formamide, 5x SSPE, 5x Denhardt’s solution, 0.2% SDS, and 10% dextran sulfate) containing 100 µg salmon sperm DNA. After washing in 1x SSPE, 0.1% SDS four times, blots were exposed in a PhosphorImager, and images were analyzed using ImageQuant version 1.2 software (Molecular Dynamics, Sunnyvale, CA). For each set of slot blots made, hybridization with a ß-actin probe (Ambion) was also performed. Clones of interest were sequenced using the ABI 373 Sequencer System (Applied Biosystems, Foster City, CA) and the M13 forward and reverse primers (Life Technologies, Gaithersburg, MD). Sequence homologies of the inserts were then searched using the BLAST database (National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD).

Adenoviral infection and microinjection
GTPase-deficient (constitutively active) Q209L mutant G{alpha}q expression vector and recombinant adenovirus have been described elsewhere (20). 3T3-L1 adipocytes were transduced at a multiplicity of infection of 20 plaque-forming units per cell for 16 h with either a control recombinant adenovirus or the recombinant adenovirus expressing Q209L-G{alpha}q. Transduced cells were incubated for 60 h at 37 C in DMEM with 10% FCS before microinjection.

Microinjection was performed using a semiautomatic Eppendorf microinjection system. Antibodies and siRNAs for microinjection were dissolved in microinjection buffer containing 5 mM sodium phosphate (pH 7.2) and 100 mM KCl. The siRNAs were designed to target the following sequences: scrambled, 5'-CAGTAGATTGGCAATGACA-3', and annexin II, 5'-AAGGTACAAGAGCTACAGCCC-3'. To study insulin- and osmotic shock-induced GLUT4 translocation, serum-starved cells were microinjected with anti-annexin II antibody or sheep IgG, or annexin II siRNA or scrambled siRNA, followed by insulin or 600 mM sorbitol treatment for 20 min. Transduced cells were microinjected with anti-annexin II antibody or sheep IgG, followed by incubation in serum-free medium for 4 h. Cells were then fixed and processed for immunostaining as described below. For measuring the mRNA level of annexin II in siRNA-injected cells, approximately 200 cells were reseeded on a coverslip and each cell was microinjected with either scrambled siRNA or annexin II siRNA. At 24 h after microinjection, total RNA was extracted from all the cells on the coverslip, and RT-PCR was performed as described below.

Immunofluorescence microscopy
Immunostaining of endogenous GLUT4 and measurement of GLUT4 translocation were performed as described before (21). Cell surface GLUT4 was analyzed using immunofluorescence microscopy. In cotransfection experiments, cells were fixed with 3.7% formaldehyde. Without permeabilization, cells were blocked with 3% FCS in PBS for 10 min and incubated with anti-cmyc antibody at room temperature for 1 h, followed by incubation with rhodamine-conjugated secondary antibody at room temperature for 1 h. The transfected cells were recognized by green fluorescence from EGFP, and the percentage of cells with a positively stained fluorescence ring for cmyc among EGFP-positive cells was measured. The observer was blinded to the experimental condition of each coverslip.

RT-PCR
Total RNA was extracted from the cells microinjected with siRNAs using RNeasy Mini Kit (QIAGEN). One microliter of eluted RNA (<10 ng) was used for RT-PCR using OneStep RT-PCR Kit (QIAGEN). The following primers were used: annexin II-P1, 5'-CAGTGGTACCAATGTCTACTGTCCAC-3'; annexin II-P2, 5'-CAGTTCTAGATCAGTCATCCCCACC-3'; ß-actin-P1, 5'-GGTGAAGGCGACAGCAGTTGG-3'; and ß-actin-P2, 5'-AGGGTGAGGGACTTCCTGTAACC-3'. The RT reaction was carried out at 50 C for 30 min, followed by initial PCR activation at 95 C for 15 min. The PCR cycling for annexin II was performed as denaturation at 94 C for 30 sec, annealing at 56 C for 40 sec, and extension at 72 C for 1 min for 30 cycles. The final extension reaction was at 72 C for 10 min. RT-PCR products were analyzed by agarose gel electrophoresis.

Western blotting
Troglitazone or vehicle control (DMSO)-treated 3T3-L1 adipocytes were lysed in a solubilizing buffer containing 50 mM HEPES, 1 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 50 U/ml aprotinin, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 10 mM NaF (pH 7.4) for 20 min at 4 C. The whole-cell lysates were boiled in Laemmli sample buffer containing 100 mM dithiothreitol, and an equal amount of proteins was resolved by SDS-PAGE. Immunoblotting was conducted as described before (21).

2-DOG uptake
Differentiated 3T3-L1 adipocytes were serum starved for 4 h, followed by insulin treatment at 100 ng/ml for 30 min at 37 C. Glucose uptake was performed as described before (22).

Subcellular fractionation
After treatment with 10 µg/ml troglitazone or vehicle for 72 h, 3T3-L1 adipocytes were stimulated with or without insulin (100 ng/ml) for 20 min before washing in cold PBS and harvesting in ice-cold homogenization buffer (255 mM sucrose; 20 mM HEPES; 1 mM EDTA, pH 7.4; 200 µM phenylmethylsulfonyl fluoride; 1 µM leupeptin; and 1 µM pepstatin). Cells were then homogenized using a Teflon pestle homogenizer. Subcellular fractionation was performed as described previously (23). An equal amount of proteins from each fraction was resolved by SDS-PAGE and analyzed by Western blotting as described above.

Statistical analysis
Values are expressed as the mean ± SE. Results were analyzed using ANOVA. A value of P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Troglitazone increases glucose uptake and GLUT4 translocation in 3T3-L1 adipocytes
It has been shown that troglitazone increased glucose uptake and GLUT4 translocation in adipocytes and myocytes (4, 5). In this study, we first examined the effect of troglitazone on glucose uptake in 3T3-L1 adipocytes. As shown in Fig. 1AGo, cells treated with 10 µg/ml troglitazone for 72 h showed a 2.8-fold increase in basal 2-DOG uptake, as well as a 2.2-fold and 2.1-fold increase in insulin-stimulated 2-DOG uptake at 1 ng/ml and 10 ng/ml insulin concentration, respectively. We also examined the effect of troglitazone on GLUT4 translocation in these adipocytes using immunofluorescence microscopy. As shown in Fig. 1Go, B and C, cells treated with 10 µg/ml troglitazone for 24 h (Fig. 1BGo) or 72 h (Fig. 1CGo) showed little change in the basal cell-surface GLUT4 level but displayed a significant increase in insulin-stimulated GLUT4 translocation at 0.5 and 1 ng/ml insulin concentration by 150 and 175%, respectively, with 24-h troglitazone treatment and by 200 and 237%, respectively, with 72-h treatment. The concentrations of insulin causing 50% of the maximal response were approximately 70% lower in troglitazone-treated cells than those in control (DMSO-treated) cells, indicating an insulin-sensitizing effect of troglitazone on GLUT4 translocation. The GLUT4 translocation results are consistent with a study showing that troglitazone sensitizes cells to insulin action by increasing insulin-stimulated GLUT4 translocation in rat adipocytes (4).



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  		FIG. 1. Effects of troglitazone on glucose uptake, GLUT4 translocation, and GLUT1 protein expression in 3T3-L1 adipocytes. 3T3-L1 cells were treated with 10 µg/ml troglitazone (Tgz) ({diamondsuit}) or vehicle control DMSO ({blacksquare}) for 72 h (A, C, and D) or 24 h (B), followed by serum starvation for 3 h (A–C). A, The cells were then stimulated with insulin for 30 min at 0, 1, 10, and 100 ng/ml concentrations, followed by measurement of [3H]2-DOG uptake. B and C, The cells were stimulated with insulin for 20 min at 0, 0.1, 0.5, 1, 10, and 100 ng/ml concentrations. The cells were fixed, immunostained with anti-GLUT4 antibody, and scored using immunofluorescence microscopy. D, Total protein (10 µg) from whole-cell lysates was resolved by SDS-PAGE and immunoblotted with anti-GLUT1 antibody. The data represent the means ± SE of three independent experiments. *, P < 0.05; **, P < 0.01.

 
Figure 1AGo shows an increase in basal 2-DOG uptake after troglitazone treatment, whereas Fig. 1Go, B and C, show no change in basal cell-surface GLUT4. Because GLUT1 can contribute to basal glucose uptake, we measured the effects of troglitazone treatment on GLUT1 expression. As seen in Fig. 1DGo, troglitazone treatment led to an increase in GLUT1 expression, and because endogenous GLUT1 constitutively partitions largely to the plasma membrane compartment, this is likely an important contributor to the increased basal 2-DOG uptake in these cells. These results are also consistent with a previous, more detailed report showing that TZD treatment leads to increased GLUT1 expression and activity in 3T3-L1 adipocytes (6).

Annexin II is a TZD-responsive gene in 3T3-L1 adipocytes as shown by SSH
To study differential expression of genes induced by troglitazone, we used SSH to detect changes in mRNA expression in cells treated with the drug compared with vehicle control (DMSO). Over 200 cDNA plasmids were screened. Clones that showed a more than 2-fold increase of the signal intensity ratio between troglitazone-treated and control RNA samples were subsequently sequenced for identification. Sequence analysis of two of those clones revealed the coding sequence of the phospholipid- and actin-binding protein annexin II. SSH and slot blots showed a 5-fold increase in annexin II mRNA level in cells treated with troglitazone relative to control (Fig. 2AGo). We further examined annexin II protein expression in response to troglitazone treatment using Western blots. As shown in Fig. 2BGo, cells treated with 10 µg/ml troglitazone for 72 h displayed a 2-fold increase in the expression of annexin II protein compared with control cells. Our results indicate that in 3T3-L1 adipocytes, troglitazone increases the expression of annexin II at both mRNA and protein level, raising the possibility that annexin II might participate in TZD-induced insulin sensitization.



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  		FIG. 2. Effects of troglitazone on the expression of annexin II in 3T3-L1 adipocytes. Cells were treated with 10 µg/ml troglitazone (Tgz) or vehicle control (Ctrl) for 72 h. A, Total RNA was isolated, cDNAs were prepared, and SSH and slot blotting were conducted as described in Materials and Methods. Total RNA (10 µg) from each sample was denatured and loaded on nylon membranes and hybridized with [{alpha}-32P]dATP radiolabeled probes derived from SSH-PCR or a ß-actin probe. After stringent washing, blots were exposed in a PhosphorImager. Two clones were identified as annexin II by sequencing analysis. B, Whole-cell lysates were collected, and 10 µg of total proteins were resolved by SDS-PAGE and immunoblotted with anti-annexin II or anti-ß-actin antibody. A bar graph depicts the data representing the means ± SE of three independent experiments. *, P < 0.05.

 
Subcellular distribution of GLUT4 and annexin II
Both GLUT4 and annexin II exist in plasma membrane and subcellular membrane fractions. We examined the effects of troglitazone on the membrane distribution of GLUT4 and annexin II using subcellular fractionation. As shown in Fig. 3AGo, insulin stimulation caused translocation of GLUT4 molecules predominantly from low-density microsomal compartments (LDMs) to the plasma membranes in control cells, as expected. After troglitazone treatment for 72 h, there was a little decrease in GLUT4 level in the LDM in the basal state and a greater decrease in GLUT4 level in the high-density microsomal compartment (HDM) and LDM fractions upon insulin stimulation compared with control cells, suggesting that troglitazone may enhance mobilization of GLUT4 from LDMs and HDMs, contributing to increased GLUT4 translocation to the plasma membrane. Insulin stimulation did not affect the subcellular distribution of annexin II in either the control or troglitazone-treated cells (Fig. 3BGo). Troglitazone treatment caused an increase in annexin II protein content in LDM and cytoplasm fractions, with little change in HDM and plasma membrane fractions (Fig. 3BGo), leading to an overall increase in total annexin II protein in these fractions combined, which is consistent with our results showing that troglitazone treatment increased annexin II protein expression detected by Western blotting (Fig. 2BGo).



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  		FIG. 3. Effects of troglitazone on the subcellular distribution of GLUT4 and annexin II in 3T3-L1 adipocytes. Cells were treated with 10 µg/ml troglitazone (Tgz) or vehicle control (Ctrl) for 72 h, followed by stimulation with or without insulin (100 ng/ml) for 20 min. Cells were then harvested for subcellular fractionation as described in Materials and Methods. Protein (10 µg) from each fraction was resolved by SDS-PAGE and immunoblotted with anti-GLUT4 antibody (A) or anti-annexin II antibody (AII-Ab) (B).

 
Annexin II is involved in insulin-induced GLUT4 translocation in 3T3-L1 adipocytes
To assess the possible role of annexin II in GLUT4 translocation, we microinjected monoclonal anti-annexin II antibody or control sheep IgG into 3T3-L1 adipocytes and measured GLUT4 translocation to the cell surface using immunofluorescence microscopy. As shown in Fig. 4AGo, microinjection of annexin II antibody did not affect the basal cell-surface GLUT4 level, but significantly inhibited insulin-stimulated GLUT4 translocation by 80 and 54% with 1 and 10 ng/ml insulin, respectively. To confirm this effect using a different approach, we also microinjected annexin II siRNA, or a scrambled siRNA as a control, into 3T3-L1 adipocytes and found that insulin-stimulated GLUT4 translocation was inhibited by 60% (Fig. 4BGo). To demonstrate the efficiency of the injected siRNA, we determined the effect of annexin II siRNA on annexin II mRNA levels in these cells using RT-PCR. Approximately 200 cells were reseeded on a coverslip, and each one of them was microinjected with either control siRNA or annexin II siRNA. At 24 h after microinjection, total RNA was extracted from all the cells on the coverslip, and RT-PCR was performed using annexin II primers. As shown in Fig. 4CGo, the annexin II RT-PCR product was decreased dramatically in cells microinjected with annexin II siRNA, compared with cells injected with control siRNA. As a control, ß-actin primers were also used, and there was no decrease in ß-actin RT-PCR product in the annexin II siRNA-injected sample (Fig. 4CGo). The RT-PCR results demonstrated that the injected annexin II siRNA was able to effectively knock down endogenous annexin II mRNA levels in 3T3-L1 cells. Taken together, the results indicate that annexin II plays a novel role in insulin-stimulated GLUT4 translocation.



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  		FIG. 4. Annexin II is involved in insulin-induced GLUT4 translocation in 3T3-L1 adipocytes. A, Serum-starved 3T3-L1 adipocytes were microinjected with control IgG or anti-annexin II antibody (AII-Ab), followed by stimulation with or without insulin for 20 min. B, At 24 h after microinjection with a scrambled siRNA (control) or annexin II siRNA (AII), cells were serum starved, followed by stimulation with or without insulin for 20 min. GLUT4 translocation was analyzed using immunofluorescence microscopy. The data represent the mean ± SE of three independent experiments. C, Approximately 200 cells were reseeded on a coverslip, and each cell was microinjected with either a scrambled siRNA (control) or annexin II siRNA (AII). At 24 h after microinjection, total RNA was extracted from all the cells on the coverslip, and RT-PCR was performed using annexin II primers and ß-actin primers. RT-PCR products were analyzed by agarose gel electrophoresis. These experiments were repeated twice.

 
We have previously shown that G{alpha}q/11 is a component of the insulin-stimulated GLUT4 translocation pathway upstream of phosphatidylinositol 3-kinase and that constitutively active G{alpha}q (Q209L) can induce GLUT4 translocation (22). To further assess the role of annexin II in insulin-stimulated GLUT4 translocation, we microinjected annexin II antibody into 3T3-L1 adipocytes transduced with adenovirus expressing Q209L and measured GLUT4 translocation. As shown in Fig. 5AGo, similar to the results reported previously (22), Q209L expression significantly increased the cell-surface GLUT4 level to approximately 70% of the maximal insulin effect. Microinjection of annexin II antibody completely inhibited Q209L-induced GLUT4 translocation. In addition to insulin, osmotic shock can also stimulate glucose transport and GLUT4 translocation (24, 25), and as shown in Fig. 5BGo, microinjection of annexin II antibody did not affect GLUT4 translocation induced by 600 mM sorbitol treatment.



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  		FIG. 5. Effects of anti-annexin II antibody microinjection on Q209L-G{alpha}q- and osmotic shock-induced GLUT4 translocation. A, After 60 h of adenovirus infection with either control adenovirus or adenovirus expressing Q209L-G{alpha}q mutant (Q209L) (multiplicity of infection = 20), 3T3-L1 cells were microinjected with control IgG or anti-annexin II antibody (AII-Ab). B, Serum-starved cells were microinjected with control IgG or anti-annexin II antibody (AII-Ab), followed by treatment with or without 600 mM sorbitol for 20 min. GLUT4 translocation was analyzed using immunofluorescence microscopy. The data represent the means ± SE of three independent experiments.

 
To further explore the role of annexin II in GLUT4 translocation, we cotransfected 3T3-L1 adipocytes with wild-type annexin II and an EGFP-cmyc-GLUT4 construct by electroporation and measured the percentage of cells with a positively stained fluorescence ring for cmyc among EGFP-positive cells by immunofluorescence microscopy. As shown in Fig. 6AGo, cells transfected with wild-type annexin II showed no significant change in basal surface cmyc-GLUT4 levels but displayed a significant increase in insulin-stimulated EGFP-cmyc-GLUT4 translocation at 0.5, 1, and 10 ng/ml insulin concentrations compared with cells cotransfected with control vector (P < 0.01). We also measured 2-DOG uptake in cells transfected with the wild-type annexin II construct. As shown in Fig. 6BGo, with 100 ng/ml insulin treatment, cells transfected with wild-type annexin II showed significantly increased 2-DOG uptake, consistent with the GLUT4 translocation results in Fig. 6AGo.



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  		FIG. 6. Effects of expression of annexin II on GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes. A, 3T3-L1 cells were cotransfected with EGFP-cmyc-GLUT4 and either wild-type annexin II ({diamondsuit}) or control vector ({blacksquare}) by electroporation, and 48 h after electroporation, cells were serum starved and stimulated with insulin for 20 min at 0, 0.1, 0.5, 1, and 10 ng/ml concentrations. Cells were then fixed, immunostained with anti-cmyc antibody, and analyzed using immunofluorescence microscopy. B, Cells were transfected with wild-type annexin II (WT) or vector control by electroporation, and 48 h after electroporation, cells were serum starved and stimulated with or without insulin (1 and 100 ng/ml) for 30 min, followed by measurement of [3H]2-DOG uptake. The data are presented as fold increase above basal, and all results are normalized to the basal 2-DOG uptake in control cells. The results represent the means ± SE of three independent experiments. *, P < 0.05; **, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
TZDs are antidiabetic insulin-sensitizing agents that improve insulin action in a variety of human and animal states of insulin resistance and diabetes (3). Although it is known that TZDs function as ligands for PPAR{gamma}, the molecular mechanisms of the insulin-sensitizing effects are not well understood. It is thought that these agents induce key glucoregulatory genes that enhance insulin action, and in this study, we used SSH to identify annexin II as one of the up-regulated genes in response to troglitazone in differentiated 3T3-L1 adipocytes. We also demonstrated a novel role for annexin II in the process of insulin-induced GLUT4 translocation and glucose transport.

Annexin II is a phospholipid-, Ca2+-, and F-actin-binding protein, and our data show that its mRNA and protein levels are up-regulated by TZD treatment. The heterotetramer form of annexin II is localized to plasma membranes, early endosomes, clathrin-coated pits, cortical cytoskeleton, and secretory vesicles and has been implicated in exocytotic and endocytotic processes (8). Indeed, recent data have demonstrated that annexin II can be an important component of the machinery controlling endosomal membrane dynamics, consistent with a role for annexin II in the endocytic pathway (26). This raises the possibility that annexin II may be involved in GLUT4 vesicle trafficking in insulin-responsive cells. To test this idea, we first examined the subcellular distribution of annexin II and GLUT4 in 3T3-L1 adipocytes by subcellular fractionation and found that annexin II, as well as GLUT4, was present in LDM, HDM, and plasma membrane fractions. Insulin stimulation caused GLUT4 translocation but had little effect on annexin II distribution. This result suggests that annexin II molecules in LDMs and HDMs do not traffic along with GLUT4 vesicles in response to insulin. Similar results were reported by Raynal et al. (27) who showed that annexin II was not redistributed after insulin treatment in rat adipocytes. Our subcellular fractionation data also showed that, in the presence of insulin, troglitazone pretreatment resulted in a greater insulin-induced reduction in GLUT4 content of LDMs and, even more dramatically, in the HDMs, both changes contributing to the increased GLUT4 translocation to the plasma membrane in the drug-treated cells. This interesting finding suggests that TZDs may sensitize cells to insulin by allowing mobilization of GLUT4 vesicles from different pools, including a pool that is normally insulin insensitive.

To explore a role for annexin II in GLUT4 translocation, we microinjected annexin II antibody or annexin II siRNA into 3T3-L1 adipocytes and found that insulin-induced GLUT4 translocation was significantly inhibited. These results strongly indicate an involvement of annexin II in insulin-induced GLUT4 translocation. We have shown that G{alpha}q/11 plays a key role in insulin-induced glucose transport in 3T3-L1 adipocytes and that G{alpha}q can act upstream of phosphatidylinositol 3-kinase in this pathway (22, 28). To further assess the role of annexin II in insulin-induced GLUT4 translocation, we microinjected annexin II antibody into 3T3-L1 adipocytes transduced with Q209L-G{alpha}q-expressing adenovirus and found that Q209L-G{alpha}q-induced GLUT4 translocation was significantly inhibited. This result suggests that annexin II may act downstream of G{alpha}q in insulin signaling to GLUT4 translocation. It is known that hyperosmolarity has potent stimulatory effects on glucose metabolism, including activation of glucose transport (24, 25). Our microinjection data showed that osmotic shock-induced GLUT4 translocation was not affected by annexin II antibody injection, suggesting that annexin II may not play a role in this process.

To further confirm the role of annexin II in insulin-induced GLUT4 translocation and glucose transport, we also performed transfection experiments. Our transfection data showed that cells transfected with wild-type annexin II displayed an increase in both insulin-stimulated GLUT4 translocation and glucose uptake. These results clearly support the idea that annexin II is a positive mediator involved in insulin-induced GLUT4 translocation and glucose transport.

In conclusion, our results show that annexin II is a newly identified TZD response gene in 3T3-L1 adipocytes. Because one of the major pharmacological effects of TZDs is to induce insulin sensitization, and because annexin II is known to function in vesicular trafficking, it seemed reasonable to propose that annexin II might be involved in insulin-stimulated GLUT4 translocation. Our data argue strongly that this is the case, because microinjection of annexin II antibody or siRNA inhibited insulin- and Q209L-G{alpha}q-stimulated GLUT4 translocation, and overexpression of wild-type annexin II protein enhanced GLUT4 translocation and glucose uptake. However, it is not known from the current results whether annexin II acts at a GLUT4 vesicle trafficking event or in the insulin signaling pathway leading to GLUT4 translocation. Additional studies will be needed to determine the function of annexin II in these processes. Taken together, our results suggest a novel function for annexin II in the process of insulin-induced GLUT4 translocation and raise the possibility that annexin II may be an important target protein for TZD-induced insulin sensitization.


   Acknowledgments
 
We thank Dr. Tony Hunter for kindly providing the annexin II cDNA, Dr. Jeffrey E. Pessin for kindly providing the EGFP-cmyc-GLUT4 construct, and Ms. Elizabeth Hansen for editorial assistance.


   Footnotes
 
Present address for S.H.H.: Clinical Trials Unit, Charles R. Drew University of Medicine and Science, 1731 East 120th Street, Los Angeles, California 90059.

This work was supported by a grant from the NIH (DK33651 to J.M.O.) and the American Diabetes Association Mentor-Based Postdoctoral Fellowship Award (J.M.O.).

Abbreviations: DMSO, Dimethylsulfoxide; 2-DOG, 2-deoxyglucose; EGFP, enhanced green fluorescent protein; FCS, fetal calf serum; GLUT4, glucose transporter isoform 4; HDM, high-density microsomal compartment; LDM, low-density microsomal compartment; PPAR{gamma}, peroxisomal proliferator-activated receptor-{gamma}; siRNA, small interfering RNA; SSH, suppression subtractive hybridization; SSPE, sodium chloride-sodium phosphate-EDTA; TZD, thiazolidinedione.

Received September 10, 2003.

Accepted for publication January 6, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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