Identification of persistent defects in insulin receptor structure and function capillary endothelial cells from diabetic rats.

Abstract

Insulin actions and receptors were studied in capillary endothelial cells cultured from diabetic BB rats and their nondiabetic colony mates. The endothelial cells from diabetic rats of 2 mo duration had persistent biological and biochemical defects in culture. Compared with normal rats, endothelial cells from diabetic rats grew 44% more slowly. Binding studies of insulin and insulin-like growth factor I (IGF-I) showed that cells from diabetic rats had 50% decrease of insulin receptor binding (nondiabetic: 4.6±0.7; diabetic: 2.6±0.4% per milligram protein, P < 0.01), which was caused by a 50% decrease in the number of binding sites per milligram protein, whereas IGF-I binding was not changed. Insulin stimulation of 2-deoxy-glucose uptake and a-aminoisobutyric acid uptake were also severely impaired with a 80-90% decrease in maximal stimulation, in parallel with a 62% decrease of insulin-stimulated autophosphorylation (P < 0.05). 125I-insulin cross-linking revealed an 140-kD a subunit of the insulin receptor similar to that in cells from nondiabetic rats, although bands at > 200 kD were also detected. The molecular weight of the insulin receptor,8 subunit (by SDS-PAGE) was smaller in cells from diabetic than from normal rats (88-90 vs. 95 kD). Neuraminadase treatment of the partially purified insulin receptors decreased the molecular weight of the insulin receptors from nondiabetic rats to a greater degree than its diabetic counterpart. In contrast, Northern blot analysis of insulin receptor mRNAs using human cDNA probes revealed two species of 9.4 and 7.2 kb with no difference in mRNA abundance between cells from diabetic and nondiabetic rats. We conclude that the exposure of capillary endothelial cells to a diabetic milieu in vivo can cause specific and persistent changes in the insulin receptor and insulin action.

Evidence for direct action of alloxan to induce insulin resistance at the cellular level

“…The failure to observe changes in interstitial insulin dynamics in diabetic dogs despite previous results [14], however, is also consistent with reports in our laboratory which indicate that in contrast to in vitro studies, insulin transport is not a receptor-mediated process in vivo [16]. Thus, defects in capillary endothelial insulin receptor function would not necessarily be reflected in altered interstitial insulin dynamics or lymph:arterial concentration gradients.…”

“…An additional function was proposed in which insulin receptors mediated the unidirectional movement of insulin across an endothelial cell monolayer in vitro [15]. These receptors showed pronounced abnormalities in several rodent models of insulin-deficient diabetes (BB rat, streptozotocin-injected rat), suggesting that insulin transport in diabetes is impaired [14]. No alteration in interstitial insulin kinetics, however, could be detected from our studies in the dog [16].…”

“…Most importantly, diabetic microangiopathy is a common complication of poorly controlled Type I diabetes and is characterized by thickening of the capillary basement membrane, impaired vasodilatory response to nitric oxide, and altered permeability to fluorescein dyes and macromolecules such as albumin which greatly exceed the size of insulin [13]. Furthermore, it has been reported that capillary endothelial cells of streptozotocin-diabetic rats exhibit defects in autophosphorylation and action of insulin receptors [14], which could cause altered TET, if insulin transport was receptor-mediated [15], although this has not been shown in vivo [16]. Thus, it is possible that alterations in TET could contribute to the insulin resistance of insulin deficient diabetes.…”

RETRACTED: Activation of protein kinase C by elevation of glucose concentration: proposal for a mechanism in the development of diabetic vascular complications.

“…Since an increase in DAG levels in the hearts of diabetic animals has recently been reported (41), we suggest that this activation ofPKC by glucose by means of de novo synthesis of DAG could be generalized to all vascular tissues involved in diabetic vascular complications. The elevation of PKC activities has been shown to alter many cellular functions that are common in diabetic vascular complications (1)(2)(3)(4)(5)(6)22), such as stimulating neovascularization (20), altering collagen synthesis (42), and affecting hormone and growth factor receptor recycling (12,43). In conclusion, we suggest that the alterations in the levels of metabolites encountered in pathophysiological state such as diabetes mellitus can affect PKC activities.…”

Preferential elevation of protein kinase C isoform beta II and diacylglycerol levels in the aorta and heart of diabetic rats: differential reversibility to glycemic control by islet cell transplantation.

“…The alteration of PKC by diabetes and glucose is interesting since PKC activity has been shown to modulate smooth muscle growth (12) and contraction (13,14), endothelial cell permeability (15), expression of signal transduction of hormones and growth factors (16,17), and cardiomyocyte contractility (18). All of these functions have been reported to be abnormal in diabetic animals or patients.…”