---
title: "Insulin promotes RAGE ectodomain shedding via AKT‑ and Rab14‑dependent ADAM10 trafficking in HAECs"
id: "plos-one-22-insulin-enhances-rage-ectodomain-shedding-by-inducing-rab14-dependent-adam10"
canonical_url: "https://medichelpline.com/clinical-feed/plos-one-22-insulin-enhances-rage-ectodomain-shedding-by-inducing-rab14-dependent-adam10"
content_type: "clinical_feed_article"
specialty: "Cardiology"
source_name: "PLOS ONE (Medicine)"
source_url: "https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0358445"
published_at: "2026-09-18T14:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# Insulin promotes RAGE ectodomain shedding via AKT‑ and Rab14‑dependent ADAM10 trafficking in HAECs
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/plos-one-22-insulin-enhances-rage-ectodomain-shedding-by-inducing-rab14-dependent-adam10
- **Specialty:** [Cardiology](https://medichelpline.com/clinical-feed/cardiology.md)
- **Primary Source:** PLOS ONE (Medicine)
- **Source URL:** [Original Journal Publication](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0358445)
- **Published At:** 2026-09-18T14:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- The study examined how **insulin** enhances ectodomain shedding of the receptor for advanced glycation end products (**RAGE**) in cultured human aortic endothelial cells (HAECs). Insulin was tested across 0.1–100 nM concentrations. - Insulin attenuated AGE-BSA–induced upregulation of intercellular adhesion molecule-1 (ICAM-1), indicating a protective, anti‑inflammatory effect against AGE stimulation. - Mechanistically, insulin activated all three **AKT** isoforms (AKT1, AKT2, AKT3) in HAECs and promoted translocation of **ADAM10** from intracellular compartments to the cell surface, increasing RAGE ectodomain shedding. - Pharmacologic inhibition of ADAM10 (GI254023X) or ADAM10 knockdown by siRNA abolished insulin-induced RAGE shedding, demonstrating ADAM10 dependence. - Inhibition of AKT with MK-2206 or knockdown of individual AKT isoforms blocked insulin-induced ADAM10 cell surface translocation and RAGE shedding, implicating AKT activation as necessary for the effect. - Co-immunoprecipitation revealed an interaction between **Rab14** and ADAM10; insulin enhanced this interaction and promoted translocation of both Rab14 and ADAM10 to the cell surface. - Rab14 knockdown prevented insulin-driven ADAM10 surface trafficking and RAGE shedding and eliminated insulin’s suppression of AGE-BSA–induced ICAM-1 expression, indicating a required role for Rab14 in the pathway. - The authors conclude that insulin promotes Rab14-mediated trafficking of ADAM10 to the plasma membrane through AKT activation, resulting in increased **RAGE** ectodomain shedding and reduced AGE-induced inflammatory signaling in HAECs.
## Clinical Analysis & Structured Key Points
Insulin enhances RAGE ectodomain shedding by inducing Rab14-dependent ADAM10 cell surface trafficking in human aortic endothelial cells | PLOS One Browse Subject Areas ? Click through the PLOS taxonomy to find articles in your field. For more information about PLOS Subject Areas, click here . Article Authors Metrics Comments Media Coverage Reader Comments Figures Figures Abstract Insulin attenuates the effects of advanced glycation end products (AGEs) by inducing a disintegrin and metalloprotease 10 (ADAM10)-mediated cleavage of the receptor for AGEs (RAGE); however, the molecular mechanism underlying this process remains incompletely understood. We investigated the mechanism by which insulin promotes ADAM10-mediated RAGE shedding in cultured human aortic endothelial cells (HAECs). AGE-modified bovine serum albumin (AGE-BSA) increased intercellular adhesion molecule-1 (ICAM-1) expression, whereas insulin pretreatment (0.1–100 nM) attenuated this effect. Mechanistically, insulin activated AKT1, AKT2, and AKT3, promoted ADAM10 translocation to the cell surface, and enhanced RAGE ectodomain shedding. In contrast, treatment with GI254023X (an ADAM10 inhibitor) or ADAM10 knockdown using siRNA abolished insulin-induced RAGE ectodomain shedding. Likewise, knockdown of AKT1, AKT2, or AKT3 using siRNA, as well as treatment with the pan-AKT inhibitor MK-2206, inhibited insulin-induced ADAM10 cell surface translocation and RAGE ectodomain shedding. Co-immunoprecipitation analysis further demonstrated an interaction between Rab14 and ADAM10. Insulin enhanced this interaction and promoted the translocation of both Rab14 and ADAM10 to the cell surface. Conversely, Rab14 knockdown blocked insulin-induced ADAM10 cell surface translocation and RAGE ectodomain shedding, thereby abolishing the protective effect of insulin against AGE-BSA-induced ICAM-1 expression. Collectively, these findings demonstrate that insulin promotes Rab14-mediated trafficking of ADAM10 to the cell surface through AKT activation in HAECs, resulting in enhanced RAGE ectodomain shedding. Citation: Baek CH, Kim H, Moon SY, Lee EK, Yang WS (2026) Insulin enhances RAGE ectodomain shedding by inducing Rab14-dependent ADAM10 cell surface trafficking in human aortic endothelial cells. PLoS One 21(9): e0358445. https://doi.org/10.1371/journal.pone.0358445 Editor: Yung-Hsiang Chen, China Medical University, TAIWAN Received: May 6, 2026; Accepted: September 1, 2026; Published: September 18, 2026 Copyright: © 2026 Baek et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This study was supported by a grant (2024IL0025) from the Asan Institute for Life Sciences, Asan Medical Center, Seoul, Korea. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. 1. Introduction Diabetes mellitus is a major risk factor for atherosclerosis [ 1 ]. Advanced glycation end products (AGEs), which are generated under hyperglycemic conditions, contribute to the development of diabetic atherosclerosis by promoting inflammatory responses in the arterial wall [ 2 ]. The biological effects of AGEs are mediated primarily through the receptor for AGEs (RAGE), a cell surface receptor composed of an extracellular domain, a transmembrane region, and a short cytoplasmic tail [ 3 ]. Insulin lowers blood glucose levels by promoting glucose uptake in skeletal muscle cells and adipocytes [ 4 ]; however, it also exhibits anti-atherogenic properties. For example, insulin has been shown to reduce aortic atherosclerotic lesions in apolipoprotein E knockout mice [ 5 ] and to slow the progression of carotid intima-media thickness in patients with type 1 diabetes [ 6 ]. The anti-atherogenic effects of insulin may be partly attributed to its ability to counteract AGE-mediated responses. Insulin induces ectodomain shedding of RAGE through a disintegrin and metalloprotease 10 (ADAM10) [ 7 ], thereby reducing RAGE-mediated inflammatory responses to AGEs. However, the mechanism by which insulin enhances ADAM10 activity remains controversial. In macrophages, insulin has been reported to increase ADAM10 activity by upregulating ADAM10 mRNA and protein expression [ 7 ]. In contrast, insulin enhances ADAM10 activity in COS-7 cells without altering ADAM10 mRNA or protein levels [ 8 ]. ADAM10 is synthesized as pro-ADAM10 in the endoplasmic reticulum (ER). In the Golgi apparatus, furin or other proprotein convertases remove the prodomain of pro-ADAM10, generating the mature, active form of ADAM10 [ 9 ]. Under basal conditions, ADAM10 is predominantly localized in the Golgi apparatus, particularly the trans-Golgi network [ 10 , 11 ]. To function as a sheddase, ADAM10 must translocate to the cell surface [ 12 ]. In human aortic endothelial cells (HAECs), we previously demonstrated that extracellular calcium influx- or 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR)-induced AMP-activated protein kinase (AMPK) activation, as well as SC79 (a cell-permeable AKT activator)-induced AKT (protein kinase B) activation, promotes ADAM10 translocation to the cell surface [ 13 – 15 ]. Rab14, a small GTPase, plays a critical role in ADAM10 cell surface translocation induced by AMPK and AKT activation [ 14 , 15 ]. TBC1D1 and TBC1D4 (AS160) function as Rab GTPase-activating proteins that stimulate GTP hydrolysis on Rab14, thereby maintaining Rab14 in its inactive GDP-bound state [ 16 ]. However, phosphorylation of TBC1D1 and TBC1D4 by activated AMPK or AKT diminishes their inhibitory effects on Rab14, allowing Rab14 activation [ 16 ]. AKT is a serine/threonine protein kinase comprising three isoforms, AKT1, AKT2, and AKT3, which are encoded by distinct genes [ 17 ]. In our previous study, SC79 activated all three AKT isoforms in HAECs, promoting ADAM10 cell surface translocation and enhancing its shedding activity. Insulin is also known to activate all three AKT isoforms [ 18 ]. Therefore, insulin may enhance ADAM10 shedding activity by promoting Rab14-dependent ADAM10 cell surface translocation through AKT activation. However, this hypothesis has not yet been experimentally tested. This study aimed to elucidate how insulin promotes ADAM10 shedding activity in cultured HAECs. We first examined whether insulin induces ADAM10-mediated RAGE ectodomain shedding. We then investigated whether insulin promotes ADAM10 cell surface translocation and whether AKT is required for this process. Finally, we examined the role of Rab14 in insulin-induced ADAM10 cell surface translocation. 2. Materials and methods 2.1. HAEC culture and treatments Primary HAECs, obtained from Lonza Walkersville (Walkersville, MD, USA), were cultured in endothelial growth medium-2 (Lonza Walkersville) for 3–5 passages. Cells from each culture dish were cryopreserved separately in individual cryovials. For experiments, cells were thawed, seeded onto tissue culture plates, and cultured for 24 h. The cells were then incubated for 16 h in Medium 199 supplemented with Hank’s salts (Thermo Fisher Scientific) and 2% fetal bovine serum to reduce variations in basal cellular conditions. Before treatment, the culture medium was replaced with serum-free Medium 199 supplemented with Hank’s salts (Ca² ⁺ 1.26 mM) to minimize the effects of undefined serum components and potential background interference. The following reagents were used for cell treatments: insulin (MedChemExpress, HY-P0035), AGE-modified bovine serum albumin (AGE-BSA; Cayman Chemical), MK-2206 (MedChemExpress, HY-10358), GI254023X (Sigma-Aldrich), and dimethyl sulfoxide (DMSO; Sigma-Aldrich). Insulin was dissolved in phosphate-buffered saline (PBS), whereas MK-2206 and GI254023X were dissolved in DMSO. AGE-BSA was used at a concentration of 100 µg/mL, as this concentration did not significantly affect HAEC viability and effectively induced ICAM-1 expression, as demonstrated in our previous study [ 13 ]. To inhibit AKT and ADAM10 activity, HAECs were treated with 1 µM MK-2206 and 2 µM GI254023X, respectively. These concentrations were selected based on previously reported conditions [ 19 , 20 ], and neither treatment altered cell morphology or total protein abundance under the experimental conditions. 2.2. Small interfering RNA (siRNA) transfection The following siRNAs were used: ADAM10-siRNA (Ambion®), Rab14-siRNA (Ambion®), control siRNA (Ambion®), AKT1-siRNA (Santa Cruz Biotechnology, sc-29195), AKT2-siRNA (Santa Cruz Biotechnology, sc-29197), AKT3-siRNA (Santa Cruz Biotechnology, sc-38911), and control siRNA (Santa Cruz Biotechnology, sc-37007). HAECs were seeded and cultured in EBM-2 endothelial growth basal medium for 24 h. siRNA–Lipofectamine complexes were prepared by mixing siRNAs with Lipofectamine (Life Technologies/Thermo Fisher Scientific) diluted in Opti-MEM medium (Life Technologies/Thermo Fisher Scientific) and incubating the mixture at room temperature for 15 min. The culture medium was then replaced with serum-free Medium 199 supplemented with Hank’s salts, and cells were transfected with the siRNA–Lipofectamine complexes for 6 h. After transfection, cells were maintained in EBM-2 endothelial growth basal medium for an additional 18 h before subsequent experiments. 2.3. Western blot analysis Western blotting was performed using whole-cell lysates and conditioned media as indicated. Whole-cell lysates were prepared as follows: cells were lysed with cold RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.25% sodium deoxycholate, 1% NP-40, and protease and phosphatase inhibitors) and incubated on ice for 10 min. Lysates were collected, transferred to microcentrifuge tubes, and centrifuged at 10,000 × g for 5 min at 4°C. The resulting supernatants were collected as whole-cell lysates. Protein concentrations were determined using the Bradford method. Equal amounts of protein from whole-cell lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-P membranes (EMD Millipore, Bedford, MA, USA). Membranes were incubated with primary antibodies, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. The primary antibodies used were as follows: anti-RAGE (Santa Cruz Biotechnology, sc-80652; mouse monoclonal antibody recognizing a truncated extracellular domain of RAGE), anti-RAGE (JF0975; NBP2–67095, R&D Systems; rabbit monoclonal antibody recognizing amino acids 350–390 corresponding to the C-terminal region of human RAGE), anti-ADAM10 (Santa Cruz Biotechnology, sc-28358), anti-Rab14 (Santa Cruz Biotechnology, sc-271401), anti-ICAM-1 (Santa Cruz Biotechnology, sc-7891), anti-actin (Santa Cruz Biotechnology, sc-47778), anti-AKT1 (ABclonal, A11016), anti-AKT2 (Cell Signaling Technology, #3063), anti-AKT3 (ABclonal, A12909), anti-p-AKT1 (Ser473) (ABclonal, AP0140), anti-p-AKT2 (Ser474) (ABclonal, AP0005), and anti-p-AKT3 (Ser472) (Abnova Corporation, PAB8141). After washing, membranes were incubated with Luminata Forte Western HRP Substrate (EMD Millipore), and chemiluminescent signals were detected by exposure to X-ray film. Films were scanned, and band intensities were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). For Western blot analysis of conditioned media, equal volumes of samples were concentrated using Amicon® Ultra centrifugal filters (Ultracel®-10K, EMD Millipore) before loading onto SDS-PAGE gels. Following electrophoretic separation, proteins were transferred to Immobilon-P membranes and incubated with an antibody against the truncated extracellular domain of RAGE (Santa Cruz Biotechnology, sc-80652). 2.4. Cell surface ADAM10 immunofluorescence staining Cells were cultured in 60-mm dishes containing coverslips placed on the bottom of each dish. Following experimental treatments, the coverslips were removed, and cells were fixed with 4% paraformaldehyde for 10 min. To detect cell surface ADAM10, immunofluorescence staining was performed without membrane permeabilization. Cells were blocked with 1% bovine serum albumin in PBS for 60 min to prevent nonspecific antibody binding. The cells were then incubated overnight at 4°C with an antibody against the extracellular domain of ADAM10 (ABclonal, A10438; rabbit antibody recognizing amino acids 214–500 of ADAM10). After washing with PBS, cells were incubated with an Alexa Fluor 488-conjugated secondary antibody. Nuclei were subsequently stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Immunofluorescence images were acquired using a Zeiss LSM710 laser-scanning confocal microscope (Carl Zeiss, Oberkochen, Germany). Fluorescence intensities were quantified in cells completely contained within the field of view, excluding cells partially captured at the image borders. The fluorescence intensity of each cell was measured using ImageJ software (National Institutes of Health) and calculated using the following formula: Corrected cell fluorescence = integrated density − (cell area × mean background fluorescence). Cells remaining in the culture dishes outside the coverslips were collected and used to determine the levels of ADAM10, AKT1, AKT2, AKT3, or Rab14 by Western blot analysis. 2.5. Co-immunoprecipitation Co-immunoprecipitation was performed to analyze the interaction between Rab14 and ADAM10. Briefly, cells were seeded in 100-mm culture dishes and incubated with or without insulin for 20 min. Cells were then lysed in IP lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, and protease inhibitors), and whole-cell lysates were prepared. For preclearing, 500 µg of lysate was incubated with 20 µL of protein A agarose (Santa Cruz Biotechnology, sc-2001) and 1 µg of rabbit control IgG (Cell Signaling Technology, #2729) for 30 min. After centrifugation, the supernatants were collected and incubated overnight with 4 µg of rabbit control IgG or rabbit anti-ADAM10 monoclonal antibody (JM32–11; Thermo Fisher Scientific, MA5–32616), followed by incubation with 20 µL of protein A agarose beads for 4 h. The protein A agarose beads were collected, washed, and incubated with 5 × SDS loading buffer to elute the immunoprecipitated proteins. Western blot analysis was performed on whole-cell lysates (input) and immunoprecipitated samples using mouse monoclonal antibodies against ADAM10 (Santa Cruz Biotechnology, sc-28358) and Rab14 (Santa Cruz Biotechnology, sc-271401). 2.6. Isolation of cell surface and intracellular proteins The Pierce™ Cell Surface Protein Biotinylation and Isolation Kit (Thermo Fisher Scientific) was used to evaluate the cell surface localization of Rab14 and ADAM10 following insulin treatment. Briefly, cells were seeded and cultured in 100-mm culture dishes. After incubation with or without insulin for 20 min, cells were treated with the membrane-impermeable sulfo-NHS-SS-biotin reagent for 10 min at room temperature to biotinylate cell surface proteins. Whole-cell lysates were then prepared and incubated with NeutrAvidin agarose beads to capture biotinylated proteins. After centrifugation, the supernatants were collected as the intracellular protein fraction. Equal amounts of intracellular proteins were concentrated using Amicon® Ultra centrifugal filters (Ultracel®-10K, EMD Millipore). The NeutrAvidin agarose bead-bound biotinylated surface proteins were washed and incubated with dithiothreitol-containing elution buffer to release cell surface proteins. The levels of Rab14 and ADAM10 in whole-cell lysates, intracellular fractions, and cell surface fractions were determined by Western blot analysis. 2.7. Statistical analysis Data are presented as mean ± SEM. The Shapiro–Wilk test was used to assess data normality, and no significant deviations from a normal distribution were observed. Comparisons between two independent groups were performed using Student’s t -test. Differences among three or more groups were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons test. Data involving two independent variables (siRNA knockdown and cell treatment) were analyzed using two-way ANOVA, followed by Šidák-adjusted multiple comparisons. Statistical analyses were performed using GraphPad Prism version 11.0.2 for Windows (GraphPad Software, Boston, MA, USA). A p value < 0.05 was considered statistically significant. 3. Results 3.1. Insulin pretreatment inhibits AGE-BSA-induced ICAM-1 expression In HAECs, AGE-BSA increased ICAM-1 expression; however, insulin pretreatment attenuated AGE-BSA-induced ICAM-1 expression in a concentration-dependent manner at concentrations ranging from 0.1 to 100 nM ( Fig 1 ). Our previous study demonstrated that AGE-BSA induces ICAM-1 expression through RAGE-mediated signaling, as RAGE knockdown by siRNA attenuated this response [ 13 ]. Download: PNG larger image TIFF original image Fig 1. Insulin pretreatment inhibits AGE-BSA-induced ICAM-1 expression. HAECs were pretreated with the indicated concentrations of insulin for 30 min, followed by incubation with AGE-BSA (100 μg/mL) for 24 h. Cell lysates were analyzed by Western blot using antibodies against ICAM-1 and actin. (n = 3 independent experiments, * p < 0.05 vs. control; # p < 0.05 vs. AGE-BSA). https://doi.org/10.1371/journal.pone.0358445.g001 3.2. Insulin promotes RAGE ectodomain shedding Ectodomain cleavage of RAGE by metalloproteases occurs near the cell surface, generating two fragments: an N-terminal ectodomain fragment and a C-terminal membrane-associated fragment. The N-terminal fragment is released into the culture medium, whereas the C-terminal fragment remains within the cells. HAECs were treated with 100 nM insulin for 5, 10, 30, or 60 min or with the indicated concentrations of insulin (0.1, 1, 10, or 100 nM) for 30 min. Cell lysates and conditioned media were analyzed by Western blotting. The shed N-terminal fragment was detected using a monoclonal antibody against the RAGE extracellular domain, whereas the C-terminal fragment was detected using a monoclonal antibody recognizing human RAGE amino acids 350–390. Insulin treatment reduced full-length RAGE levels in cell lysates while increasing the levels of the shed RAGE ectodomain fragment in conditioned media in a time- and concentration-dependent manner ( Fig 2A and 2B ). In addition, insulin induced a time- and concentration-dependent increase in the intracellular C-terminal fragment of RAGE, accompanied by a reduction in full-length RAGE ( Fig 2C and 2D ). Together, these findings demonstrate that insulin induces RAGE ectodomain shedding. Download: PNG larger image TIFF original image Fig 2. Insulin promotes RAGE ectodomain shedding. (A, B) HAECs were treated with 100 nM insulin for 5, 10, 30, or 60 min (n = 3 independent experiments) or with the indicated concentrations of insulin (0.1, 1, 10, or 100 nM) for 30 min (n = 3 independent experiments). Cell lysates and culture supernatants were analyzed by Western blot using antibodies against the RAGE extracellular domain and actin.(C, D) HAECs were treated with 100 nM insulin for 5, 10, 30, or 60 min (n = 4 independent experiments) or with the indicated concentrations of insulin (0.1, 1, 10, or 100 nM) for 30 min (n = 4 independent experiments). Cell lysates were analyzed by Western blot using antibodies against the RAGE C-terminal domain and actin. ( * p < 0.05 vs. control). https://doi.org/10.1371/journal.pone.0358445.g002 3.3. AKT or ADAM10 inhibition abolishes insulin-induced RAGE ectodomain shedding In the following experiments, 100 nM insulin was used to investigate the mechanism underlying insulin-induced RA
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