Concurrent Inhibition of AKT and GLUT1 Reveals Endpoint-Specific Effects on Glucose Metabolism and Cell Death in HepG2 Hepatocyte-Derived Cells

Background: Glucose handling in hepatocyte-derived cells integrates insulin-responsive AKT signaling with GLUT1-mediated glucose transport, jointly regulating substrate trafficking, glycogen synthesis, and cell survival. MK-2206, a potent allosteric AKT inhibitor, and BAY-876, a highly selective GLUT1 inhibitor, represent targeted approaches to disrupt these processes. However, the consequences of simultaneous inhibition of both nodes in hepatocyte-derived cells remain incompletely understood. Objective: This study aims to evaluate the impact of simultaneous AKT inhibition and GLUT1 blockade on target engagement, cell viability, proliferation, cell death, glucose uptake, and glycogen storage in HepG2 cells. Methods: HepG2 cells were treated with MK-2206, BAY-876, or their combination under basal hyperglycemic and acute insulin-challenged conditions. Multi-model synergy, AKT/GLUT1 pharmacological effects, cell viability, proliferation, cell death, glucose uptake, and glycogen storage were assessed, alongside exploratory molecular docking analyses of both inhibitors against their respective targets. Results: Dual AKT and GLUT1 inhibition produced combination-specific reductions in cell viability and showed a pattern of concurrent antiproliferative effects and increased Annexin V/PI positivity without detectable caspase-3 activation under insulin-challenged conditions. Furthermore, the dual inhibition of AKT signaling and GLUT1-mediated glucose uptake produced combination-specific reductions in basal glycogen content, while glucose uptake reductions showed AKT-dominant patterns without significant combination superiority over MK-2206 alone under either metabolic state. Exploratory molecular docking of MK-2206 and BAY-876 against AKT1 and SLC2A1/GLUT1, respectively, generated computationally predicted binding poses consistent with the established binding modes of these inhibitors. Conclusions: Dual AKT/GLUT1 targeting produces endpoint-specific patterns of pharmacological interaction on insulin-responsive metabolism, proliferation, and cell death in HepG2 hepatocyte-derived cells, distinguishing combination-specific effects from AKT-driven effects. These findings support an endpoint-specific pharmacological framework in which AKT signaling and BAY-876-mediated GLUT1 pharmacological effects jointly influence glucose handling and cell fate in a hepatocyte-derived model.