Insulin Binding and Degradation by Human Erythrocytes at Physiological Temperature2

Abstract

When evaluated at 15 C, insulin binding to human erythrocytes is similar to that of human adipocytes fibroblasts, monocytes and placental membranes. At 37 C, however, both insulin binding and degradation by human erythrocytes have a unique character. At this temperature, by the end of the first 30 minutes, erythrocyte specific insulin binding is 3 to 4% of the total available insulin. This percentage of binding remains until the end of the first hour, then for the next four hours, increases linearly to 24%. Intact erythrocytes had negligible degradation of the free 125I-insulin but 56% of the 125I-insulin associated with the erythrocytes was degraded after five hours of incubation at 37 C. The degradation of the bound insulin was determined to be an intracellular property of erythrocytes. This degradation may be the mass action driving force responsible for the increased association of 125I-insulin observed after one hour of incubation. On the other hand, erythrocyte ghosts reached a steady state with 2% of the 125I-insulin bound after 1.5 hours of incubation at 37 C. More than 94% of the bound and free insulins were intact after 5 hours of incubation. These observations indicate, for the first time, that erythrocyte insulin degrading activity is localized inside the cells, not in their membranes, and that the human erythrocyte with its insulin receptors may be one of the important cell types in the metabolism of insulin.

The UK study of therapies of Type 2 (non-insulin-dependent) diabetes

“…In contrast with the second conclusion of Bihr et al [1], our published data [5][6][7][8] provided the following evidence of insulin internalization by human erythrocytes at 37~ (a) human erythrocyte cell membranes lack an insulin degrading enzyme [6]; (b) there is a very specific and potent insulin degrading enzyme in cell cytosol (7.8); (c) there is a time-dependent continuous increase in insulin association with the cells at 37 ~ [6]; and (d) about 56% of the cell-associated insulin is degraded at the end of 5 h of incubation at 37 ~ [6]. Moreover, according to biochemical studies performed to evaluate insulin receptor down-regulation (and thereby assess insulin internalization) in 1M-9 lymphocytes (nucleated cells), contradictory results were obtained by Baldwin et al [9], that is, insulin internalization was not observed.…”

“…Patients with Type 2 diabetes tend to have a low state of physical training [4] and, even in normal subjects, VO2 max correlates closely with carbohydrate disposal [5]. A programme of exercise added to a dietary regimen can carry additional metabolic and emotional benefits [6]. Two symposia have been devoted to the role of exercise in the management of diabetes [7,8], and the subject has been reviewed very recently [91.…”

Reduction of glucose and insulin concentrations during in vitro incubation of human whole blood at different temperatures

“…This was unexpected, as insulin is mostly degraded in liver and kidney, but to some extent also in blood cells and extracellularly [25]. Insulin concentrations in normal whole blood has in previous studies been stable at room temperature for 5 hours [26], at 37 ºC insulin have been reported to be bound to human erythrocytes followed by subsequent internalization and degradation [27,28]. Thus, insulin is degraded by viable erythrocytes at 37 ºC while the erythrocytes are unable to degrade insulin at room temperature, presumably due to reduced metabolic activity at this temperature.…”

“…Wilcoxon signed rank test revealed that endogenous insulin concentration after incubation at 37 ºC for 6 hours was significantly reduced from baseline 48 (36-94) to 23 (18)(19)(20)(21)(22)(23)(24)(25)(26)(27) pmol/L (p=0.03) (Figure 2). …”

Insulin causes insulin-receptor internalization in human erythrocyte ghosts

“…Specific insulin binding to the surface of ghosts was assayed by a method modified from that described by Gambhir et al (1981), in which filtration, rather than centrifugation, was used to remove free from bound insulin. Quadruplicate samples of erythrocyte ghosts (3 mg of protein/ml) in a total volume of 0.25 ml were incubated for 90 min at 15°C with 125I-insulin, with or without an excess of unlabelled insulin.…”