Initial entry of IRAP into the insulin-responsive storage compartment occurs prior to basal or insulin-stimulated plasma membrane recycling
Gang Liu,1,*
June Chunqiu Hou,2,*
Robert T. Watson,2 and
Jeffrey E. Pessin2
1Department of Pediatric and Adolescent Medicine and Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota; and 2Department of Pharmacological Sciences, Stony Brook University, Stony Brook, New York
Submitted 22 April 2005
; accepted in final form 25 May 2005
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ABSTRACT
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To examine the acquisition of insulin sensitivity after the initial biosynthesis of the insulin-responsive aminopeptidase (IRAP), 3T3-L1 adipocytes were transfected with an enhanced green fluorescent protein-IRAP (EGFP-IRAP) fusion protein. In the absence of insulin, IRAP was rapidly localized (13 h) to secretory membranes and retained in these intracellular membrane compartments with little accumulation at the plasma membrane. However, insulin was unable to induce translocation to the plasma membrane until 69 h after biosynthesis. This was in marked contrast to another type II membrane protein (syntaxin 3) that rapidly defaulted to the plasma membrane 3 h after expression. In parallel with the time-dependent acquisition of insulin responsiveness, the newly synthesized IRAP protein converted from a brefeldin A-sensitive to a brefeldin A-insensitive state. The initial trafficking of IRAP to the insulin-responsive compartment was independent of plasma membrane endocytosis, as expression of a dominant-interfering dynamin mutant (Dyn/K44A) inhibited transferrin receptor endocytosis but had no effect on the insulin-stimulated translocation of the newly synthesized IRAP protein.
insulin-responsive aminopeptidase; trafficking; insulin; cargo selection; biosynthesis
IT IS WELL ESTABLISHED that striated muscle and adipose tissue contain specialized intracellular storage compartments that are enriched for the glucose transporter isoform GLUT4 and the insulin-responsive aminopeptidase (IRAP). In the basal state, these cargo proteins slowly recycle to and from the cell surface membrane. However, insulin stimulation increases the rate of exocytosis, resulting in a marked redistribution of these proteins to the cell surface by a process usually referred to as translocation (6, 12, 20, 34, 40). Despite intensive investigation, we have only a rudimentary understanding of the identity and nature of the insulin-responsive compartment(s). For example, subcellular fractionation has demonstrated that GLUT4 and IRAP primarily translocate from a post-trans-Golgi Network (TGN) structure(s) that is enriched for vesicle-associated membrane protein 2 but is cellugyrin negative (26, 28, 31, 39). However, whether these cargo proteins are stored in small vesicles similar to synaptic vesicles, concentrated in larger endosomal-like compartments, or retained through a dynamic equilibrium between these two states still remains unresolved.
Multiple studies have examined the trafficking itinerary of GLUT4 and IRAP that has been continuously expressed and is therefore in equilibrium with the plasma membrane and multiple intracellular compartments. In addition, it is generally assumed that these cargo proteins gain access to the insulin-responsive storage compartment(s) by first trafficking to the plasma membrane and subsequent endocytosis, followed by recycling and sorting through endosomes (18, 19, 33, 35, 40). Thus current analyses of mutant and chimeric proteins have not considered the pathway by which newly synthesized proteins are initially sorted from the TGN into the insulin-responsive storage compartment(s).
To address these issues, we have taken the approach of analyzing the initial entry of newly synthesized IRAP protein into the insulin-responsive storage compartment by assessing the time-dependent acquisition of insulin-stimulated translocation. Our data demonstrate that newly synthesized IRAP protein enters the insulin-responsive storage compartment(s) directly after exit from the Golgi apparatus without traversing the plasma membrane or undergoing internalization and subsequent endosomal sorting.
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EXPERIMENTAL PROCEDURES
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Materials.
Brefeldin A (BFA) was purchased from Sigma Chemical, and secondary Texas Red and FITC-conjugated donkey anti-rabbit IgG antibodies were obtained from Jackson ImmunoResearch Laboratories, respectively. Texas Red-conjugated human transferrin was purchased from Molecular Probes. The anti-HA antibody was from Santa Cruz Biotechnology. The transferrin receptor (TR) cDNA was obtained from the American Type Culture Collection. The EGFP-IRAP, EGFP-syntaxin 3 (Stx3), wild-type dynamin 1 (Dyn/WT), and Dyn/K44A cDNAs were all obtained as previously described (5, 41, 42, 47, 48).
Culture and transfection of 3T3-L1 adipocytes.
Murine 3T3-L1 adipocytes were cultured and electroporated as previously described (49). After electroporation, cells were allowed to recover for 124 h before insulin stimulation. Cells that were treated with insulin at the 3-h posttransfection time point were plated directly into serum-free medium. For all other time points, cells were first plated in complete medium and then switched to serum-free medium 23 h before insulin stimulation. Under these conditions, the transfection efficiency ranged from
15 to 25%.
Transferrin labeling.
After transfection with the TR cDNA, the adipocytes were cultured for 6 h, cooled to 4°C, and incubated with 50 µg/ml Texas Red-conjugated human transferrin for 1 h. The cells were then washed and warmed to 37°C for 30 min before fixation.
Confocal fluorescent microscopy and image analysis.
Cells were fixed for 15 min in 4% paraformaldehyde containing 0.2% Triton X-100, washed in PBS, and blocked with 5% donkey serum plus 1% BSA for 1 h. Primary and secondary antibodies were used at 1:100 dilutions (unless otherwise indicated) in blocking solution and samples were mounted on glass slides with Vectashield (Vector Laboratories). Cells were imaged using a Zeiss LSM 510 confocal fluorescent microscope. Quantification of plasma membrane translocation was determined by adjusting the laser power to within the linear range of emission and then counting the number of transfected cells displaying a continuous cell surface fluorescence as previously described (49). Images were then imported into Adobe Photoshop (Adobe Systems) for processing, and composite files were generated.
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RESULTS
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Intracellular localization and trafficking of EGFP-Stx3 and EGFP-IRAP after transfection.
To study the initial intracellular trafficking of newly synthesized IRAP, we examined the time-dependent localization of EGFP-IRAP after transfection (Fig. 1). We (14) have previously demonstrated that the EGFP-IRAP fusion displays an identical subcellular distribution and insulin-stimulated translocation as the endogenous IRAP protein. In the absence of insulin, EGFP-IRAP was localized in the perinuclear region of the cells with little, if any, detectable plasma membrane accumulation (Fig. 1A, ad). Insulin stimulation of cells expressing IRAP from 3 h had no significant difference in the distribution of the newly synthesized IRAP protein (Fig. 1A, e). However, at 6 h posttransfection, there was a small but readily observable insulin-stimulated cell surface localization of EGFP-IRAP (Fig. 1A, f). At 9 and 12 h after transfection, acute insulin stimulation resulted in a robust plasma membrane translocation of IRAP (Fig. 1A, gh). Quantification of selected time points is presented in Fig. 1B and demonstrates that acquisition of insulin-stimulated IRAP translocation requires between 6 and 9 h after expression of the EGFP-IRAP protein.

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Fig. 1. Newly synthesized insulin-responsive aminopeptidase (IRAP) does not accumulate at the plasma membrane. A: differentiated 3T3-L1 adipocytes were electroporated with 50 µg of enhanced green fluorescent protein-IRAP (EGFP-IRAP) cDNA. After transfection, cells were immediately incubated for various times and left either untreated (Basal, ad) or stimulated for 30 min with 100 nM insulin (Insulin, eh). Cells were then fixed and processed for confocal fluorescent microscopy. These images are a representative composite of individual cells that display typical distribution of EGFP-IRAP fluorescence at each time point. B: quantification of number of cells displaying EGFP-IRAP translocation was determined by counting of 50 representative cells per condition independently performed 4 times for a total of 200 cells.
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As a control for constitutive membrane trafficking, we also compared the time-dependent intracellular localization of syntaxin 3 (Stx3) (Fig. 2). Stx3 was chosen for comparison because this protein is a resident of the plasma membrane and has a similar topology as IRAP, being a type II single membrane-spanning protein (4, 24). Similarly to IRAP, 3 h after expression, the EGFP-Stx3 protein displayed a diffuse localization that was predominantly perinuclear (Fig. 2A, a). A small fraction of EGFP-Stx3 was localized to plasma membrane at 3 h but was quite pronounced by 6 h and remained persistent throughout the time course examined (Fig. 2A, bd). In contrast to EGFP-IRAP, the localization of pattern of EGFP-Stx3 was completely unaffected by insulin stimulation (Fig. 2A, eh). Quantification of these results is presented in Fig. 2B, and these data demonstrate that intracellular sequestration and subsequent insulin sensitivity are specific to the IRAP protein.

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Fig. 2. Newly synthesized syntaxin 3 (Stx3) rapidly accumulates at the plasma membrane. A: differentiated 3T3-L1 adipocytes were electroporated with 50 µg of EGFP-Stx3 cDNA. After transfection, cells were immediately incubated for various times and left either untreated (Basal, ad) or stimulated for 30 min with 100 nM insulin (Insulin, eh). Cells were then fixed and processed for confocal fluorescent microscopy. These images are a representative composite of individual cells that display typical distribution of EGFP-IRAP fluorescence at each time point. B: quantification of number of cells displaying EGFP-Stx3 at cell surface was determined by counting of 50 representative cells per condition independently performed 4 times for a total of 200 cells.
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Insulin-responsive IRAP translocation occurs concomitantly with a loss in BFA sensitivity.
BFA is a fungal metabolite that induces the collapse of Golgi membranes into the endoplasmic reticulum and prevents pre-TGN membrane trafficking (9, 11, 32). However, BFA does not inhibit the insulin-stimulated translocation of endogenous GLUT4, indicating that the insulin stimulates the exocytosis of GLUT4 from a post-TGN BFA-insensitive compartment (8, 29). Therefore, to determine the time-dependent exit of newly synthesized IRAP out of the TGN, we compared BFA sensitivity with the acquisition of insulin responsiveness (Fig. 3). Three hours after transfection, adipocytes were treated with BFA and incubated an additional 9 h for a total of 12 h after EGFP-IRAP expression. BFA treatment resulted in a dispersion of the EGFP-IRAP distribution with little compartment-specific localization (Fig. 3A, a). In addition, insulin was unable to induce any significant plasma membrane translocation (Fig. 3A, e). Cells expressing EGFP-IRAP for 6 h, followed by BFA treatment for 6 h, began to display a distinct perinuclear localization (Fig. 3A, b). At this time, insulin stimulation resulted in weak translocation of EGFP-IRAP and in only a small subfraction of the cell population (Fig. 3A, f). In contrast, cells expressing EGFP-IRAP for 9 h and then treated with BFA for an additional 3 h had a strong perinuclear distribution that underwent the typical extent of insulin-stimulated translocation (Fig. 3A, c and g). In Fig 3A, d and h, are the IRAP 12-h expression without BFA treatment that served as control. These data are quantified in Fig. 3B and demonstrate that, between 6 and 9 h after expression, newly synthesized IRAP exits a BFA-sensitive compartment(s) and concomitantly acquires insulin responsiveness.

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Fig. 3. Time-dependent acquisition of insulin-stimulated IRAP translocation occurs in parallel with conversion from a Brefeldin A (BFA)-sensitive to a BFA-insensitive step. A: differentiated adipocytes were electroporated with 50 µg of EGFP-IRAP cDNA. At 3 (a and e), 6 (b and f), 9 (c and g), and 12 (d and h) h posttransfection, cells were incubated with 5 µg/ml of BFA for an additional 9, 6, 3, and 0 h. After total expression period of 12 h, cells were left either untreated (Basal, ad) or stimulated for 30 min with 100 nM insulin (Insulin, eh). Cells were then fixed and processed for confocal microscopy. Images are representative composite of individual cells that display typical distribution of EGFP-IRAP fluorescence at each time point. B: quantification of number cells displaying EGFP-IRAP translocation was determined by counting of 50 representative cells per condition independently performed three times for a total of 150 cells.
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Inhibition of dynamin-dependent endocytosis does not impair acquisition of insulin-stimulated EGFP-IRAP translocation.
In the case of regulated membrane protein trafficking, current models assume that, following biosynthesis, the newly synthesized protein defaults to the plasma membrane through a constitutive exocytotic pathway. The cell surface protein is then internalized and recycled through the endosomal system and subsequently sorted into insulin-responsive storage compartments (18, 19, 33, 35, 40). However, recent studies examining the initial endomembrane trafficking of GLUT4 suggest a direct sorting to the insulin-responsive GLUT4 storage compartment (47). To test this hypothesis experimentally for the compartmentalization of IRAP, we took advantage of a dominant-interfering dynamin mutant (Dyn/K44A) that has been successfully used to inhibit GLUT4 endocytosis (1, 7, 23, 45). Because dynamin is a soluble protein, BFA treatment immediately after transfection will not affect dynamin expression but will trap integral membrane proteins in the endoplasmic reticulum. After BFA washout, normal secretory membrane trafficking ensues, allowing the subsequent analysis of membrane transport events.
Initially, to confirm that Dyn/K44A is an effective inhibitor of endocytosis, we cotransfected adipocytes with the TR and either Dyn/WT or the Dyn/K44A mutant. The cells were immediately incubated with BFA for 3 h to allow dynamin expression, followed by extensive washing and incubation for an additional 6 h in the absence of BFA. The cell surface TR was then labeled with Texas Red-transferrin at 4°C and examined for its internalization after warming to 37°C for 30 min (Fig. 4). As expected, both Dyn/WT and Dyn/K44A were primarily localized to the plasma membrane and were also found to distribute diffusely throughout the cytoplasm (Fig. 4, eh). At 4°C, TR displayed a characteristic cell surface localization without any significant intracellular labeling in both Dyn/WT-and Dyn/K44A-expressing cells (Fig. 4, a and c). Incubation of the Dyn/WT-expressing cells for 30 min at 37°C resulted in the endocytosis and accumulation of the Texas Red-transferrin-labeled TR in numerous intracellular compartments (Fig. 4b). In contrast, cell expression of Dyn/K44A has little, if any internalized Texas Red-transferrin-labeled transferrin receptor following warming to 37°C for 30 min (Fig. 4d).

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Fig. 4. Expression of a dominant-interfering dynamin mutant (Dyn/K44A) inhibits transferrin receptor (TR) endocytosis. Differentiated 3T3-L1 adipocytes were electroporated with 50 µg of human transferrin cDNA plus 200 µg of the dynamin wild type (Dyn/WT) cDNA (a, b, e, f, i, and j) or the dominant-interfering Dyn/K44A cDNA (c, d, g, h, k, and l). Cells were cooled 6 h later to 4°C and incubated with 50 µg/ml Texas Red-labeled transferrin for 60 min. One-half of the cells were washed and warmed to 37°C (b, d, f, h, j, and l), and the others remained at 4°C for 30 min (a, c, e, g, i, and k). Cells were fixed and subjected to confocal fluorescent microscopy for Texas Red-transferrin (ad) and dynamin expression (eh). Merge images are presented in i-l. These are representative images from 3 independent experiments. and , Perinuclear localized TR after endocytosis.
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Having established that Dyn/K44A is an effective inhibitor of plasma membrane endocytosis, we next cotransfected Dyn/WT and Dyn/K44A with EGFP-IRAP and then examined the time-dependent localization and acquisition of insulin responsiveness of the newly synthesized IRAP protein (Fig. 5). In the presence of Dyn/WT, the distribution of EGFP-IRAP remained primarily in the perinuclear region, with only a small extent of plasma membrane accumulation in the unstimulated state (Fig. 5A). Expression of Dyn/WT had no significant effect on the acquisition of insulin-responsive IRAP translocation that began 6 h after transfection and increased to maximal levels between 9 and 12 h. In contrast, in the absence of insulin, the cells expressing Dyn/K44A displayed a slow, time-dependent accumulation of EGFP-IRAP at the cell surface that was detectable between 9 and 12 h after transfection (Fig. 5B). These data are consistent with previous studies demonstrating that Dyn/K44A inhibits GLUT4 endocytosis but that the accumulation is relatively slow in unstimulated cells (23, 47). However, the ability of insulin to stimulate the translocation of newly synthesized EGFP-IRAP, even in the presence of Dyn/K44A, was readily apparent at 912 h and was essentially identical to that observed in the presence of Dyn/WT (Fig. 5B). Together, these data demonstrate that plasma membrane endocytosis is not necessary for newly synthesized IRAP protein to acquire insulin responsiveness.
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DISCUSSION
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Insulin stimulation of adipocytes results in a marked redistribution of the insulin-responsive glucose transporter (GLUT4) from intracellular storage sites to the plasma membrane (6, 12, 20, 34, 40, 46). Although fibroblasts have a limited capacity to display insulin-stimulated GLUT4 translocation, the formation of the unique adipocyte retention compartment occurs early in the differentiation process before the expression of GLUT4 (13, 16). In contrast to GLUT4, IRAP is ubiquitously expressed but becomes sequestered into the same insulin-responsive GLUT4 intracellular retention compartments during adipogenesis (37).
At present, the mechanism that accounts for the selective sequestration of both GLUT4 and IRAP has remained enigmatic. For example, previous immunofluorescent, subcellular fractionation, vesicle immunoadsorption, and electron microscopy studies have indicated a substantial codistribution of IRAP with markers of the general recycling endosome pathway (2, 14, 15, 22, 30, 36, 38). Mutation analysis of the IRAP cytoplasmic domain has suggested the presence of several targeting motifs, but the potential retention receptor(s) or intracellular membrane targets remain unknown. More importantly, IRAP continuously cycles to and from the plasma membrane through multiple intracellular membrane compartments (21, 27, 41). This inherent property of IRAP makes conclusions on the basis of steady-state expression studies highly problematic.
To address this issue, we hypothesized that establishing the trafficking of IRAP after initial biosynthesis under non-steady-state conditions would provide a system independent of endocytosis and recycling. Our data demonstrated that, after the initial biosynthesis, IRAP was rapidly retained within the perinuclear region without any significant accumulation at the plasma membrane. This is in contrast to other constitutive trafficking proteins (Stx3) that rapidly defaulted to the plasma membrane at 3 h after protein expression. Furthermore, the acquisition of insulin-stimulated IRAP translocation required 69 h of expression. This temporal delay was not due to the inhibition of insulin signaling, because the endogenous GLUT4 protein undergoes normal translocation in cells examined 3 h posttransfection (47). In addition, the transfection protocol had no effect on the insulin stimulation of phosphatidylinositol 3-kinase or protein kinase B activation (data not shown). Thus our results are consistent with a model wherein newly synthesized IRAP undergoes a time-dependent sorting process that is necessary for the acquisition of insulin responsiveness.
There are two possible trafficking routes that could account for this time-dependent appearance of insulin responsiveness. After biogenesis and secretory membrane trafficking (Golgi/TGN), IRAP could default to the plasma membrane and then undergo internalization and endosomal sorting to the insulin-responsive compartment(s). Alternatively, after constitutive transport, a slow, time-dependent sorting step could occur in the TGN or post-TGN compartment that is required for IRAP vesicle transport to the insulin-responsive retention compartment.
To address these possibilities, we first examined the sensitivity of the newly synthesized IRAP protein to BFA treatment. Previous studies have demonstrated that the insulin-responsive compartment is insensitive to BFA, indicating that the responsive compartment is post-TGN (3, 8, 29). Our data demonstrated that IRAP was BFA sensitive up to 6 h after expression, but by 9 h was BFA insensitive. Because constitutive plasma membrane-trafficking proteins such as Stx3 rapidly localize to the plasma membrane, these data are consistent with IRAP undergoing a slow TGN/post-TGN sorting step directly to the insulin-responsive compartment.
More specifically, we utilized a dominant-interfering dynamin mutant to block plasma membrane receptor endocytosis (10, 17, 43, 44, 50). Several laboratories have demonstrated that the Dyn/K44A mutant is also an effective inhibitor of GLUT4 endocytosis in adipocytes (1, 23, 45). We confirmed this observation, as Dyn/K44A prevented TR endocytosis. Despite inhibition of plasma membrane endocytosis, the time-dependent acquisition of insulin-stimulated IRAP translocation was not significantly different. These data provide compelling evidence that the initial sorting of newly synthesized IRAP occurs independently of plasma membrane trafficking and therefore must result exclusively from intracellular trafficking events.
Recently, we observed that the newly synthesized GLUT4 protein also undergoes a slow, intracellular sorting process before the acquisition of insulin responsiveness (47). In addition, analysis of the time-dependent trafficking of GLUT4/GLUT1 loss-of-function and gain-of-function chimeras demonstrated the requirement of both the cytoplasmic amino terminus and large intracellular loop between transmembrane domains 6 and 7 to recapitulate these initial trafficking events (25). In this regard, it was reported that amino acids 5684 of the cytoplasmic IRAP domain were sufficient to display insulin-stimulated translocation, endocytosis, and recycling (21). Although the initial time-dependent acquisition of insulin responsiveness was not determined, the IRAP minimal domain does contain a related dileucine motif similar to one in the large intracellular loop of GLUT4. However, there are no apparent sequence similarities between the amino-terminal domain of GLUT4 and the minimal functional 5684 IRAP domain. Thus future studies will be needed to determine the precise sequence requirements for the initial sorting and acquisition of insulin responsiveness.
In sum, our data are most consistent with the following model. After translation and integration into the endoplasmic reticulum, proteins are transported to the Golgi and arrive at the TGN. Constitutive trafficking proteins destined for the plasma membrane (Stx3) take a default pathway, similar to constitutive secretion, and are directly transported to the plasma membrane. In contrast, proteins destined for the insulin-responsive storage compartment (IRAP, GLUT4) are sorted from constitutive trafficking proteins (GLUT1, vesicular stomatitis virus-G protein, Stx3). After sorting and entry of GLUT4 and IRAP into the insulin-responsive compartment, there is a slow rate of exit in the basal state. However, in the presence of insulin, the rate of exit from this compartment is markedly enhanced. Future studies will be necessary to identify the specific motifs and protein interactions responsible for these individual trafficking/sorting decisions.
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GRANTS
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This study was supported by research grants (DK-55811 and DK-33823) from the National Institute of Diabetes and Digestive and Kidney Diseases and PF-03-133-01-TBE from the American Cancer Society.
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ACKNOWLEDGMENTS
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We thank Bintou Diouf and Jeffery Smith for the care and maintenance of the 3T3-L1 adipocytes.
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FOOTNOTES
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Address for reprint requests and other correspondence: J. E. Pessin, Dept. of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794-8651 (e-mail: pessin{at}pharm.stonybrook.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* These authors contributed equally to this study. 
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