The Trafficking and Processing of Insulin and Insulin Receptors in Cultured Rat Hepatocytes*

Abstract

The processing and trafficking of insulin in cultured rat hepatocytes were studied. A time course of binding of radiolabeled insulin to hepatocytes at 37 C revealed a rapid rise in cell-associated radioactivity that reached a steady state by 20 min. Using an acid medium to extract insulin bound to surface receptors, the time courses of receptor binding and internalization of the ligand were characterized. The earliest event in insulin processing was the binding of insulin to surface receptors, reaching steady state by 20 min with a t1/2 of 4 min. The internalization rate of ligand was initially slower than the binding rate, with a t1/2 of 6 min. Similar internalization rates of the insulin receptor were found by measuring the trypsin sensitivity of hepatocyte insulin receptors covalently occupied with a photo-affinity-labeled derivative of insulin [( 125I]B2 (2-nitro-4-azido-phenylacetyl)Des-PheB1-insulin). At steady state, the internalized ligand and receptor comprised approximately 40-45% of the cell-associated radioactivity. The time course of intracellular degradation was assessed by trichloroacetic acid (TCA) precipitability and Sephadex G-50 gel chromatography of solubilized cells containing only internalized radioactivity. Intracellular TCA-soluble and low mol wt degradation products first appeared by 5 min and were released from the cell 3 min later. Chloroquine (100 microM) completely inhibited the formation of intracellular low mol wt degradation products as well as their appearance in the medium. The release of intracellular radioactivity was assessed by first removing surface-bound insulin with acid extraction. Eighty percent of the intracellular radioactivity was released in 45 min with a t1/2 of 8 min. The released radioactivity was assessed by TCA precipitability and gel chromatography. These results demonstrate that after 20 min, 43% of the released intracellular radioactivity is intact insulin. The percentage of intact insulin released increases in a dose-dependent fashion as the amount of insulin bound and internalized increases. In conclusion, the earliest event in insulin processing is binding to surface receptors. After a short delay, insulin and its receptor are internalized and trafficked into either a chloroquine-sensitive degradative pathway or a chloroquine-insensitive retroendocytotic pathway. The amount of insulin that traverses the nondegradative retroendocytotic pathway is proportional to the amount of insulin bound and internalized by the cell.

Insulin transport across capillaries is rate limiting for insulin action in dogs.

“…Intracellular insulin degradation will change free insulin concentration in lymph, which in turn may influence diffusion across the capillary wall. Lymph insulin levels may also be altered by retroendocytosis (54,55), a process by which internalized intact hormone is recycled to the plasma membrane and extruded into the interstitial space; however the quantitative importance of this reverse process in vivo is unclear. Lastly, one must be cautious of the use of inulin as a reference molecule for transcapillary insulin diffusion.…”

Pulsatile Insulin Secretion Dictates Systemic Insulin Delivery by Regulating Hepatic Insulin Extraction In Humans

“…Thus, maximal insulin binding by hepatocytes coincides with the insulin concentration range present in the insulin concentration wavefront within a pulse presented to the liver in the portal vein, ϳ1,000 -4,000 pmol/l. In contrast, hepatocyte insulin binding is much less avid at insulin concentrations typically present in the trough of an insulin pulse train, which represents a basal nonpulsatile component of release (32)(33)(34). High binding in insulin pulses would be expected to result in endocytosis of a high proportion of insulin delivered to the hepatic sinusoids.…”

“…Written informed consent was obtained from all study participants. Five healthy subjects (two men and three women) mean age 32 years (range [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39], with a mean BMI of 24.9 kg/m 2 (21.2-27.1) participated. All subjects were nondiabetic based on fasting plasma glucose concentration (mean fasting plasma glucose 5.4 mmol/l [5.1-5.8]) and HbA 1c values (mean 4.8% [4.5-5.0]).…”

Epsin binds to the EH domain of POB1 and regulates receptor-mediated endocytosis

“…Screening with a multivalent nonapeptide phage display library has revealed that ten of thirteen EH domains from (Tang and Cai, 1996;Di Fiore et al, 1997;Haner et al, 1997;Tang et al, 1997;Wendland and Emr, 1998). Similar to many cell surface receptors, the insulin receptor is internalized into intracellular vesicular compartments following ligand binding and tyrosine kinase activation (Levy and Olefsky, 1987;Backer et al, 1989). It has been shown that insulin receptor kinase tyrosine autophosphorylation in conjugation with the dileucine motifs located in juxtamembrane domain of the bsubunit is essential for the insulin receptor internalization (Backer et al, 1992;Hamer et al, 1997) and that the insulin receptor internalization occurs through a dynamin-and clathrin-mediated endocytic pathway (Ceresa et al, 1998).…”