Characterization of protein factor(s) in rat bronchoalveolar lavage fluid that enhance insulin transport via transcytosis across primary rat alveolar epithelial cell monolayers

Abstract

The aim of this study was to characterize factor(s) in rat bronchoalveolar lavage fluid (BALF) that enhance(s) insulin transport across primary rat alveolar epithelial cell monolayers (RAECM) in primary culture. BALF was concentrated 7.5-fold using the Centricon device and the retentate was used to characterize the factor(s) involved in enhancing apical-to-basolateral transport of intact 125 I-insulin across various epithelial cell monolayers. These factor(s) enhanced transport of intact insulin across type II cell-like RAECM (3-fold increase) and type I cell-like RAECM (2-fold increase), but not across Caco-2 or MDCK cell monolayers. The insulin transport-enhancing factor(s) were temperature-and trypsin-sensitive. The mechanism of enhancement did not seem to involve paracellular transport or fluid-phase endocytosis, since fluxes of sodium fluorescein and FITC-dextran (70 kDa) were not affected by the factor(s) in the apical bathing fluid. BALF enhancement of intact 125 I-insulin transport was abolished at 4°C and in the presence of monensin, suggesting involvement of transcellular pathways. Sephacryl S-200 purification of BALF retentate, followed by LC-MS/MS, indicated that the high molecular weight (>100 kDa) fractions (which show some homology to alpha-1-inhibitor III, murinoglobulin gamma 2, and pregnancyzone protein) appear to facilitate transcellular transport of insulin across RAECM.

Conjugation with Cationic Cell-Penetrating Peptide Increases Pulmonary Absorption of Insulin

“…Lowering temperature from 37 to 4 °C caused ∼80% decrease in absorption of INS−cr9 across RAECM. By contrast, decreasing temperature to 4 °C results in approximately 37% and 40% inhibition of paracellular transport of INS and FITC−dextran across RAECM. A much greater decrease in absorption observed at 4 °C for INS−cr9 conjugates indicates the involvement of process(es) other than passive leak through paracellular pathways.…”

“…RAECM grown on 24 mm tissue culture-treated Transwells were pre-equilibrated with MDS (without NBS) for 1 h prior to transport studies. Radiolabeled INS or INS−CPP conjugates were dosed in the apical compartment and incubated at 37 °C for 2 h, after which media from the basolateral compartment were collected and subjected to the trichloroacetic acid (TCA) precipitation assay , to determine the fraction of intact INS or INS−CPP conjugate. Radioactivity associated with the supernatant and with the pellet was measured using a gamma counter.…”

Proper antiviral therapy for hepatitis B virus–associated acute-on-chronic liver failure

“…The half‐life of complexes is only a few minutes and they bind to receptors with high affinity (K d = 10 −8 − 10 −10 M). The affinity of receptors is similar for α 2 Ms from all mammalian sources (Chiabrando et al, 2002; Mann, 2007; Bahhady et al, 2008; Bowers et al, 2009) and is not affected by the nature of the complexed proteinase. As reported by Van Dijk et al (1992), activation of α 2 M by methylamine induces a charge distribution on the protein which results in the uptake of the complex by the scavenger receptor on endothelial cells.…”

“…The proteolytic cleavage could occur at any residue in this hexapeptide sequence. The additional proteolytic cleavage sites are also present at two other regions situated ∼15 and ∼27 residues away from the hexapeptide region (Bahhady et al, 2008). The structural transformation following cleavage of the bait region results in an increase in electrophoretic mobility (Shi et al, 2011), sedimentation coefficient (Habtemichael et al, 2011), decrease in the radius of gyration (Osterberg and Pap, 1983), and Stokes' radius (Akashi et al, 2008).…”

Insulin receptor binding motif tagged with IgG4 Fc (Yiminsu) works as an insulin sensitizer to activate Akt signaling in hepatocytes

“…Malabsorption might be overcome by the induction of receptor-mediated transcytosis (Bahhady et al, 2008). Transcytosis is the process by which cargo proteins internalized at one plasma membrane of a polarized cell are transported via vesicular carriers to the contralateral plasma membrane (Lee et al, 2007).…”