Diabetes-associated hyperglycemia generates advanced glycation end products (AGEs) that contribute to vascular inflammation and atherosclerosis through signaling via the receptor for AGEs (RAGE). RAGE is a transmembrane receptor whose extracellular domain can be cleaved (ectodomain shedding), a process that reduces cell-surface RAGE signaling. Prior work showed that insulin can induce RAGE shedding via the metalloprotease ADAM10, but the mechanism for how insulin enhances ADAM10 activity and trafficking in endothelial cells remained unresolved. The authors aimed to determine whether insulin promotes ADAM10-mediated RAGE ectodomain shedding in human aortic endothelial cells (HAECs) and to delineate the roles of AKT isoforms and the small GTPase Rab14 in ADAM10 cell surface translocation.
Primary HAECs from a commercial source were used between passages 3 and 5. Cells were serum‑reduced prior to treatment and maintained in serum‑free, calcium‑containing medium for experiments. Treatments included insulin (dissolved in PBS) at concentrations reported across 0.1–100 nM, and AGE‑modified bovine serum albumin (AGE‑BSA) at 100 µg/mL to model AGE exposure that induces ICAM‑1 expression without compromising cell viability under the stated conditions.
The study used siRNA-mediated knockdown for ADAM10, Rab14, and individual AKT isoforms (AKT1, AKT2, AKT3). Pharmacologic inhibitors included MK‑2206 (pan‑AKT inhibitor, 1 µM) and GI254023X (ADAM10 inhibitor, 2 µM); both agents were chosen based on prior reports and did not alter gross cell morphology or total protein abundance in the experimental setting.
Authors assessed activation of AKT isoforms, ADAM10 localization, and RAGE ectodomain shedding using standard biochemical approaches described in the methods. Whole‑cell lysates and conditioned media were analyzed by Western blot. Co‑immunoprecipitation was used to probe interactions between Rab14 and ADAM10. Cell surface translocation of ADAM10 and Rab14 was examined by the approaches reported in the paper.
In HAECs, insulin treatment activated AKT1, AKT2, and AKT3. Concomitant with AKT activation, insulin promoted translocation of ADAM10 to the cell surface and increased RAGE ectodomain shedding into conditioned media. Inhibition of ADAM10 activity with GI254023X or knockdown of ADAM10 by siRNA prevented insulin‑induced RAGE shedding, demonstrating that the shedding response to insulin was mediated by ADAM10.
Pharmacologic blockade of AKT with MK‑2206 or siRNA knockdown of each AKT isoform inhibited insulin‑induced ADAM10 cell surface translocation and RAGE shedding, indicating that AKT activation is required for insulin’s effect on ADAM10 trafficking and function.
Co‑immunoprecipitation experiments showed an interaction between Rab14 and ADAM10. Insulin enhanced this interaction and promoted translocation of both Rab14 and ADAM10 to the cell surface. The authors note prior mechanistic links whereby phosphorylation of Rab GTPase‑regulatory proteins reduces their inhibition of Rab14, enabling Rab14 activation downstream of kinases such as AKT; this conceptual framework supports the observed insulin‑dependent recruitment of Rab14 in HAECs.
Functional consequences were examined using AGE‑BSA stimulation to induce ICAM‑1 expression. Insulin pretreatment attenuated AGE‑BSA‑induced ICAM‑1 upregulation. This protective effect required the ADAM10‑mediated RAGE shedding pathway: ADAM10 inhibition or knockdown abolished insulin‑induced RAGE shedding. Similarly, Rab14 knockdown blocked insulin‑induced ADAM10 surface translocation and RAGE ectodomain shedding and eliminated insulin’s ability to suppress AGE‑BSA‑induced ICAM‑1 expression. These findings position Rab14 as a necessary mediator linking AKT activation to ADAM10 trafficking and consequent RAGE shedding in HAECs.
The reported data indicate that in cultured human aortic endothelial cells, insulin activates all three AKT isoforms, which promotes Rab14‑dependent trafficking of ADAM10 to the plasma membrane. This trafficking increases ADAM10‑mediated RAGE ectodomain shedding and reduces downstream inflammatory signaling as measured by ICAM‑1 induction in response to AGE‑BSA. ADAM10 activity and Rab14 function were both required for insulin’s protective effect against AGE‑induced ICAM‑1 expression.
These results provide a mechanistic explanation for part of insulin’s anti‑atherogenic actions in endothelial cells by linking insulin receptor signaling through AKT to vesicular trafficking machinery that mobilizes ADAM10 to the cell surface to limit RAGE signaling. The paper reports these findings using primary HAECs and a combination of pharmacologic and genetic perturbations as described in the methods. Details such as quantitative fold changes, full time courses, and specific experimental replicates are those reported in the original article and supporting information.