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The present study aimed to analyze the mechanism of action of a hydrolyzed gamma-conglutin fraction (Cgh) extracted from Andean L. mutabilis legume on glucose metabolism. At 5 mg/mL, Cgh from L. mutabilis inhibited dipeptidyl peptidase IV activity (DPP-IV) (100%), and increased (p < 0.05) 6.5-fold the glucose uptake in a dual-layered enterocyte/adipocyte culture system compared to untreated cells, through an enhancement of glucose transporter type-4 translocation. In this model, insulin-induced glucose uptake was potentiated by addition of L. mutabilis hydrolysates. Also, Cgh L. mutabilis hydrolysates decreased (p < 0.05) by 50% gluconeogenesis in a dual-layered enterocyte/hepatocyte system and reduced phosphoenolpyruvate carboxykinase expression. These data showed that L. mutabilis hydrolysates can affect glucose metabolism inhibiting DPP-IV enzymatic activity, improving insulin receptor …