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The contribution of mucous/glycocalyx layers, as a diffusional or enzymatic barrier, to the absorption of insulin was investigated in situ and in vitro studies using rats. To remove the mucous/glycocalyx layers, ileal segments were exposed to a hyaluronidase solution in situ. The removal of the layers was confirmed by transmission electron microscopy, and the safety of the hyaluronidase pretreatment was established based on light microscopy, a constant mucosal membrane electrical resistance and the absence of lactate dehydrogenase leakage. In the in situ loop absorption experiment, hyaluronidase pretreatment significantly increased the plasma insulin level accompanied by an obvious hypoglycemic response. In the in vitro transport experiment, the apparent permeability coefficient of insulin was significantly increased by the hyaluronidase pretreatment, whereas that of 4.4 kDa fluorescein isothiocyanate-labeled dextran and of antipyrine, respective markers for passive para- and transcellular permeation, was unaffected. In the insulin degradation experiment in vitro, a significant amount of insulin was degraded in the compartment removed by hyaluronidase pretreatment. Thus, the mucous/glycocalyx layers functioned in insulin absorption as an enzymatic barrier and insignificantly affected diffusive absorption. In addition, co-administration of aprotinin, a protease inhibitor, further increased insulin absorption from ileum pretreated with hyaluronidase, implying the existence of another enzymatic barrier that influences insulin mucosal absorption.