Enhancement Effect of Poly(amino acid)s on Insulin Uptake in Alveolar Epithelial Cells

Abstract

In this study, we elucidated the effect of poly(amino acid)s such as poly-L-ornithine (PLO) on FITC-insulin uptake in cultured alveolar type II epithelial cells, RLE-6TN. FITC-insulin uptake by RLE-6TN cells as well as its cell surface binding was markedly increased by PLO without cytotoxicity. The uptake of FITC-insulin in the presence of PLO was shown to be mediated by endocytosis, but in contrast to the uptake in the absence of PLO, the contribution of macropinocytosis emerged. Colocalization of FITC-insulin and LysoTracker Red was observed by confocal laser scanning microscopy both in the absence and presence of PLO, indicating that FITC-insulin was partly targeted to lysosomes in the cells and degraded. The half-life of the intracellular degradation of FITC-insulin was, however, prolonged by the presence of PLO. PLO also stimulated the uptake of other FITC-labeled compounds. Among them, the enhancement effects of PLO on FITC-albumin and FITC-insulin uptake were prominent. The effect of PLO on insulin absorption was also examined in in-vivo pulmonary administration in rats, and co-administration of PLO enhanced the hypoglycemic action of insulin. These findings suggest that co-administration of poly(amino acid)s such as PLO is a useful strategy for enhancing insulin uptake by alveolar epithelial cells and subsequent absorption from the lung.

Receptor-mediated endocytosis of macromolecules and strategy to enhance their transport in alveolar epithelial cells

“…The effect of PLO on insulin absorption was also examined in in-vivo pulmonary administration in rats. Co-administration of PLO enhanced the pharmacological effect of insulin (hypoglycemic action), most likely due to the enhanced pulmonary absorption of intact insulin [104]. These findings suggest that co-administration of poly(amino acid)s is a useful strategy for enhancing insulin uptake by alveolar epithelial cells and subsequent absorption from the lung, and finally for enhancing its pharmacological effect.…”

“…Thus, cationic poly(amino acid)s such as PLO is likely to be effective in stimulating insulin uptake in alveolar epithelial cells. In fact, insulin uptake by RLE-6TN cells was markedly increased by PLO without apparent cytotoxicity [104]. As albumin uptake, PLO showed the bell-shaped concentration-dependent stimulation effect on insulin uptake, and excess amount of PLO resulted in the decreased stimulation effect in RLE-6TN cells ( Figure 5).…”

Enhancement Effect of Poly-L-ornithine on the Nasal Absorption of Water-Soluble Macromolecules in Rats

“…The obtained pharmacokinetic parameters are summarized in Table 1. PLOs with different molecular weights improved the nasal absorption of FD-4 at all concentrations and significantly increased the C max and the AUC compared to the control solution (FD-4 alone), except for 0.025% (w/v) PLO (5) and PLO (20). Furthermore, PLO increased the plasma FD-4 levels and tended to prolong the T max in a concentration-dependent manner, indicating that the nasal absorption-enhancing effect was increased and prolonged with increasing PLO concentration.…”

“…Reagents Poly-L-ornithine hydrobromide (MW, ca. 5.8, 20 and 78 kDa for PLO (5), PLO (20) and PLO (80), respectively) was purchased from Alamanda Polymers, Inc. (Huntsville, AL, U.S.A.). Fluorescein isothiocyanate-dextran (FD-4, MW 4.0 kDa) and poly-L-arginine hydrochloride (PLA, MW 44.3 kDa for PLA (45)) were obtained from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.).…”

“…Intranasal (i.n.) doses of FD-4 (33 mg/kg, 165 mg/mL in saline) containing different concentrations (0.025, 0.05, 0.1, 0.5 and 1.0% (w/v)) of PLO (5), PLO (20) and PLO (80) were administered at a distance of 8 mm from the entrance of the left nostril via a polyethylene tube attached to a microsyringe. For comparison, FD-4 solutions with/without 0.5% (w/v) PLA (45) were also administered at the same dose as that in the groups co-administered with PLO.…”