적혈구를 이용한 인슐린 내재화율 ( Internalization Rate ) 의 측정에 관한 연구 = A Study on Internalization Rate of Insulin in Human Erythrocytes

다국어 초록 (Multilingual Abstract)

Plasma membrane insulin receptor complexes are internalized by endocytotic uptake. The internalized complexes are dissociated within these endosomal vesicles. After dissociation, the insulin and receptor physically segregate and progressively degraded to intermediates and small MW peptides. The rate of insulin internalization is incubation time-, temperature- and energy-de- pendent. This insulin internalization is directly related to the amount of insulin bound at the cell membrane and may play a role in the cellular resistance to insulin. Prior studies with various cells, including isolated hepatocytes, pancreatic acinar cells, adipocytes, cultured fibroblasts and monricytes, indicated that insulin is internalized into target cells. Trischitta et al has demonstrated that little ar no insulin internalized at 15and 24℃ in human erythrocytes. However, this study did not assess insulin internalization at the physiolagic temperature (37℃). Furthermore, the major metabolic pathways that persist in the erythrocyte are glycolytic and phosphogluconate pathway, which together meet the energy needs. Insulin enhance from fructose-6 phosphate to fructose-2-6-biphosphate by activation of fructose-2-6-biphosphatase for acceleration of glucose-phosphate entry into glycolytic pathway. Therefore, we have hypothesized that insulin internalization is important for human erythrocytes to survive. We studied the internalization of (125) I-Insulin in human erythrocytes by using an acid extraction technique. To remove cell surface-bound radioactivity from erythrocytes, we used an extraction wash procedure at optimal temperature (37℃) with a buffer at optimal pH(pH 6.0) to avoid cell damage and hemolysis. The rate of insulin internalization in human erythrocytes at 37℃ was progressively increased and maxi- mal after 3-4haur~s of incubation (78-94%) in control subjects. A plateau is then maintained up to 3 hours of incubation. Analysis by Sephadex G-50 column, HPLC with radioisotope detector of the degradation products of insulin after 3, 4hr of incubation at 37℃ indicated that total, nonextractable and extractable degradation products of insulin were progressively increased as the incubation time. Nearly 100% of labeled insulin was degraded and detected as nonextractable degradation products in 4 hrs. The rate of insulin internalization in human erythrocytes was inversely correlated with glucose concentration in media, Rapid internalization of insulin was also observed with a low glucose concentration (50mg/ dl). We have also studied the insulin internalization in human erythrocytes from patients with various insulin resistant conditions (type II DM, third trimester of pregrancy and hyperprolactinemia). The rate of insulin internalization is lower in insulin resistant conditions than in control subjects. In conclusion, (1) insulin rapidly internalizes into human erythrocytes at physiologic temperatures (37℃). 78-94% of the bound radioactivity is intracellular in the control subjects. (2) this process is incubation time-, temperature-and glucose concentration-dependent. (3) erythrocytes from insulin resistant conditions (type II DM, the third trimester of pregn8ncy and hyperprolactinemia) have a signifieantly decreased ability in internalize insulin Therefore, human erythrocytes are a suitable subject for assay of insulin internalization and decreased internalization may play a role in the cellular resistance to insulin action that occurs in these patients.