Mechanism of Insulin Uptake in Rat Alveolar Type II and Type I-Like Epithelial Cells

Abstract

The alveolar epithelium is comprised of type II and type I cells, which are two morphologically and functionally different epithelial cells. Alveolar type I cells, which are squamous and cover more than 90% of the alveolar surface area, play a major role in gas exchange. 1,2) On the other hand, alveolar type II cells are cuboidal and outnumber type I cells, although type II cells occupy less than 10% of the surface area even when their apical microvilli are taken into consideration.1,2) In addition, type II cells have multiple functions such as surfactant production and secretion, and serve as type I cell progenitors.2)The majority of protein and peptide therapeutic drugs were developed as injection formulations, and have a variety of problems, especially for patients, in terms of safety, pain, and needle phobia. 3,4) Recently, the lung has attracted a great deal of interest as an alternative administration route for protein and peptide drugs. 3,5) The inhalation pulmonary delivery system, which may enable the systemic absorption of protein and peptide drugs, has been extensively studied.6) In particular, clinical research on inhaled insulin has confirmed the efficacy and safety of inhaled insulin in the treatment of diabetes mellitus. 7) In addition, the bioavailability of inhaled insulin via the lung is greater compared to other non-invasive routes such as the gut and nose. 8)Previous studies concerning protein transport across alveolar epithelia have suggested that the transport system most likely to be involved is endocytosis. 9) We also have studied the transport of albumin in alveolar epithelial cells, and found that albumin is internalized into type II and type I cells via a clathrin-mediated endocytic pathway with greater uptake activity in type II cells than in type I cells.10) These findings indicate that despite the much smaller surface area of type II cells compared to type I cells, type II cells most likely play an important role in the endocytic transport of proteins in alveolar epithelia.In the case of small proteins (peptides) such as insulin, both paracellular and transcellular routes may be involved in alveolar epithelial transport.1) Bur et al. 11) reported that in human primary cultured alveolar type I-like epithelial cells, the apparent permeability coefficient of insulin was comparable to that reported for dextran, indicating that a specific, transcellular transport process is not involved. On the other hand, the alveolar epithelial junction was reported to be tighter than other epithelia, 1) suggesting that the contribution of a paracellular route for insulin in alveolar epithelial transport may not be so high. Furthermore, it has been reported that megalin serves as an endocytic receptor for insulin uptake in rat renal proximal tubule cells, 12) and that megalin is also expressed in alveolar epithelial cells.13) Thus, it is possible that an endocytic pathway is involved in insulin transport in alveolar epithelia. However, little information is available concerning the handling of insu...

Albumin and mammalian cell culture: implications for biotechnology applications

“…In the RLE-6TN alveolar type II epithelial cell line, albumin transported by a non-caveolae transport mechanism, predominantly a clathrin-dependent process, was tracked to lysosomes and the rate of degradation measured (Yumoto et al 2006). In the latter study the apparent uptake of fluorescently labelled albumin was approximately 1,100 ng/mg cell protein/60 min, in general agreement with their other measurements in primary type II and type I rat alveolar epithelial cells of between 800 and 6,000 ng/mg cell protein/60 min; the latter is equivalent to 0.2–0.7 pg/cell/60 min (calculated from: Ikehata et al 2008; Ikehata et al 2009). Moreover, fluorimetric analysis after SDS–PAGE indicated that during this continuous uptake process approximately 16% of the albumin was degraded after 60 min.…”

Mechanism underlying insulin uptake in alveolar epithelial cell line RLE-6TN

“…These results indicate that energy-dependent, saturable mechanism, most likely receptor-mediated endocytosis, is involved in FITC-insulin uptake by RLE-6TN cells. These characteristics are essentially the same as those observed for primary cultured alveolar type II epithelial cells (Ikehata et al, 2009), though the apparent affinity seems to be somewhat lower in RLE-6TN cells.…”

“…Uptake experiments were performed as described previously (Ikehata et al, 2009;Tagawa et al, 2008;Takano et al, 2002). Briefly, RLE-6TN cells grown on 35-mm culture dishes were used.…”

“…Rat alveolar type II epithelial (AT II) cells were isolated from Sprague-Dawley male rats as described previously (Ikehata et al, 2008(Ikehata et al, , 2009. Total RNA was extracted from RLE-6TN cells and AT II cells using an RNeasy Mini kit (Qiagen, Valencia, CA, USA), and RT-PCR was performed as described previously (Takano et al, 2009).…”

“…Assuming that the number of type II cells is about 1.5-fold of that of type I cells, the contribution of type II cells to total albumin uptake was estimated to be more than 75% (Ikehata et al, 2008). Similarly, the contribution of type II cells to total insulin uptake was estimated to be more than 50% (Ikehata et al, 2009). Thus, though the surface area occupied by type II cells is quite small, type II cells may play an important role in the uptake of protein and peptide drugs in the lung.…”