Effects of Enzyme Inhibitors and Insulin Concentration on Transepithelial Transport of Insulin in Rats

Abstract

The objective of this study was to determine whether transepithelial transport of insulin can be improved by enzyme inhibitors and whether insulin concentration affects its ileal absorption. Ussing chambers and radioimmunoassay were used to study insulin transport across the rat ileum, and circular dichroic spectra were used to determine whether insulin aggregated at high concentrations. Inhibitors that inhibit insulin-degrading enzyme, including N-ethylmaleimide, 1,10-phenanthroline and p-chloromercuribenzoate, dramatically improved insulin transport across the ileum. At 100 nm, the ileal permeability of immunoreactive insulin was 10(-6) cm s-1 in the presence of inhibitors, and was negligible when inhibitors were not used. Ammonium chloride, a lysosomotropic agent that increases intralysosomal pH, and aprotinin, a proteasome inhibitor, did not increase transport of insulin to a detectable extent. Insulin permeability decreased as its concentration increased from 100 nm to 83.3 microM, and at 83.3 microM insulin aggregated. It is concluded that insulin transport is improved by enzyme inhibitors, but is impaired by insulin aggregation at high concentrations.

Transport of Insulin in Modified Valia-Chien Chambers and Caco-2 Cell Monolayers

“…The P app of phenol red across the duodenum, jejunum, and ileum were the 4.04 × 10 −6 , 4.54 × 10 −6 , 5.20 × 10 −6 cm/s, respectively. These data were in accordance with those obtained from the Ussing chambers (Bai & Chang, 1996), but lower than those using the everted gut sac technique (Schilling & Mitra, 1990).…”

“…When the intestinal mucosal cells were destroyed, and aqueous pores in the mucosa would open up, hydrophilic molecules could more easily pass through the enlarged intercellular junctions. Such methods as the constant of transepithelial electrical resistance, accumulation of [ 3 H]-D-glucose, appearance of lactate dehydrogenase and permeability of phenol red were used to determine whether the membranes remained intact throughout the experiments (Bai & Chang, 1996). Table 1 summarizes the permeability experiment of phenol red using the Valia-Chien chambers.…”

Novel Mucosal Insulin Delivery Systems Based on Fusogenic Liposomes

“…This implies that insulin, which was directly introduced into the cytosol by the FL membrane fusion, was strongly degraded by the cytosolic IDE. PCMB, a sulfhydryl-modifying agent, has been used to inhibit the degradation of insulin by IDE in various tissue cytoplasms (15,16). In the present study, no absorptionenhancing effect was observed following insulin solution coadministration of PCMB and the simple liposomes loaded with both insulin and PCMB together.…”

“…Regarding the permeation of insulin into the intestinal epithelium, Bai and Chang (15,16) reported that insulin was highly degraded by IDE in the intestinal cytoplasm, and it potentially limits the transepithelial transport of insulin. In this study, we found that the coencapsulation of PCMB as the IDE inhibitor further increased FL's enhancement effect of insulin colonic absorption (Fig.…”

“…We then examined the absorption of peptide drugs from various intestinal mucosae with the aid of FL, using insulin as a model peptide drug. One of the limiting factors for improving insulin absorption from the intestinal epithelium may be the degradation of insulin in the cytosol by insulin-degrading enzyme (IDE) (15,16). IDE is the neutral thiol metalloproteinase involved in the intracellular metabolism of insulin in hepatocytes, adipocytes, kidney, muscle cells, intestinal tissue, and other cells (15Y17).…”