Luciano Adezati

Increased Expression of Tissue Plasminogen Activator and Its Inhibitor and Reduced Fibrinolytic Potential of Human Endothelial Cells Cultured in Elevated Glucose

In diabetic patients, elevated plasma levels of t-PA and PAI-1 accompany impaired fibrinolysis. To identify mechanisms for these abnormalities, we examined whether vascular endothelial cells exposed to high glucose upregulate t-PA and PAI-1 production and whether ambient PA activity is decreased concomitantly. In 17 cultures of human umbilical vein endothelial cells grown to confluency in 30 mM glucose, the t-PA antigen released to the medium in 24 h was (median) 52 ng/106 cells (range 10–384) and the PAI-1 antigen was 872 ng/106 cells (range 217–2074)—both greater (P < 0.02) than the amounts released by paired control cultures grown in 5 mM glucose—29 ng/106 cells (range 7.5–216) and 461 ng/106 cells (range 230–3215), respectively. In the presence of high glucose, the steady-state levels of t-PA and PAI-1 mRNAs were increased correspondingly (median 142 and 183% of control, respectively, P < 0.05); high glucose per se and hypertonicity contributed to the upregulation in additive fashion. The PA activity of conditioned medium from cultures exposed to high glucose was 0.4 IU/ml (range 0.2–0.6), which was significantly lower (P < 0.02) than the PA activity of control medium (0.5 IU/ml, range 0.2–0.9). No difference was observed when comparing the PA activities of acidified conditioned media, expected to be depleted of inhibitors. Thus, high glucose coordinately upregulates endothelial t-PA and PAI-1 expression through effects exerted at the pretranslational level and enhanced by even mild degrees of hypertonicity. The decrease in ambient fibrinolytic potential may reflect an overwhelming effect of the increased availability of PAI-1. These findings propose a contributory mechanism for the fibrinolytic abnormalities of diabetes and the thrombotic tendency of the hyperglycemic hyperosmolar state.

Effect of two different glucose concentrations on insulin receptor mRNA levels in human hepatoma HepG2 cells

Glucose is known to affect mRNA levels of several genes. In order to investigate possible effects of glucose on insulin receptor mRNA levels, we cultured human hepatoma cells (HepG2) in two different culture media: DMEM containing 100 mg/dl glucose and DMEM containing 450 mg/dl glucose. Insulin receptor mRNA levels and insulin binding activity were reduced in HepG2 cultured at lower glucose concentrations. These data suggest that glucose affects insulin receptor gene expression.

Inhibition of insulin and epidermal growth factor (EGF) receptor autophosphorylation by a human polyclonal IgG

The immunoglobulin G of a polyclonal antiserum (pIgG) from a patient with insulin resistance and hypoglycemia was tested for its ability to inhibit insulin binding and to affect the autophosphorylation of partially-purified insulin receptors extracted from rat liver membranes. pIgG, when added 4 hr prior to insulin, inhibited subsequent insulin binding by 50% at 30 micrograms added protein; however, insulin previously bound to the receptor could not be displaced by a 4 hr subsequent exposure of up to 70 micrograms pIgG. pIgG, independent of its effect on insulin binding, inhibited both basal and insulin-stimulated autophosphorylation of the insulin receptor in a dose-dependent manner with a half maximal effect at 3.3 to 7 micrograms protein. Furthermore, pIgG also reduced basal autophosphorylation of the EGF receptor. The effect of pIgG to inhibit basal autophosphorylation of insulin and EGF receptors, together with its ability to reduce autophosphorylation of insulin receptors fully occupied by insulin, imply that the effect of pIgG on receptor autophosphorylation is largely independent of its effect on ligand binding. Moreover, these findings suggest that pIgG may inhibit autophosphorylation by acting on domains which are similar in the insulin and EGF receptors.

An instrument for real-time spectral estimation of heart rate variability signals

A Digital Signal Processor (DSP)-based instrument is proposed for estimating and displaying the Heart Rate Variability (HRV) spectrum in real-time. It consists of an intelligent module which is properly interfaced to an IBM PC and whose operations are independent from the computers other tasks. In this way, the simultaneous recording of the ECG sequence, needed for the more complete off-line analysis, can be performed by the same host. The employed hybrid spectral estimator (in which a classical FFT analysis follows the autoregressive extrapolation of data) appears to be the most apt for the present fixed point arithmetics implementation. The reliability of the instrument and its accuracy are checked both with suitable test signals and by comparison with the results obtained through off-line analysis of the same ECG tracks. The instrument is presently used for cardiovascular investigations, in particular for quickly picking patients with cardiac autonomic neuropathy (CAN) out of a population of diabetic subjects.

Regulation of insulin receptor-associated tyrosine kinase by a polyclonal IgG.

The effect of a polyclonal anti-insulin receptor antibody (pIgG) on the insulin receptor tyrosine kinase (IRTK) activity toward poly-(Glu-Tyr) was examined using wheat germ agglutinin agarose-purified insulin receptors from rat liver membranes. The main effect of pIgG was a reduction of Vmax (from 60.8 to 31.8 pmol/min/mg), without changes of Km, when IRTK was activated by insulin. In contrast, when IRTK was activated by ATP preincubation, pIgG was unable to affect the reaction, suggesting that IRTK possesses at least two regulatory mechanisms, one of which can be affected by pIgG.

Antipeptide antibodies toward the extracellular domain of insulin receptor beta-subunit

In order to investigate structure and function of beta-subunit extracellular portion, four polyclonal antibodies (AP1, AP2, AP3 and AP4) toward peptides comprised in this region were generated. None of them recognizes native human and rat insulin receptor both in vitro and in whole cells. Two antibodies, AP1 and AP2, immunoprecipitate isolated (DTT-reduced) human beta-subunits and bind to human IM-9 cell after alpha-subunit tryptic cleavage. Only AP1 recognizes rat beta-subunit both in vitro and in trypsin treated rat FAD cells. These findings suggest that: (i) the extracellular portion of the insulin receptor beta-subunit is partially covered by the alpha-subunit in human and rat native insulin receptors; (ii) human and rat beta-subunit extracellular domains are different, at least in the amino acid sequence corresponding to residues 785-796 of the human insulin receptor.

Insulin Receptor Regulation in Human Mature Red Cells in vitro

We have studied the ability of mature red cells to regulate the number and affinity of their insulin receptors, in vitro. Our data show that mature red cells are not able to change either the number and the affinity of their insulin receptors, after preincubation with high concentrations of insulin alone or insulin and glucose. We conclude that mature red cells possess an insulin receptor system not completely similar to that of major target cells such as hepatocytes and adipocytes, and therefore we suggest some criticism in evaluating these cells in clinical studies, regarding the insulin receptor status.

I.V. Glucose tolerance test: Correlation between FFA, glucose and IRI in normal, obese and diabetic subjects

SummaryInsulin response and FFA behavior have been evaluated during an IVGTT in 63 subjects of whom 18 were normal, 31 were obese (with varying degrees of carbohydrate tolerance) and 14 were mild non insulin-dependent diabetics. The extreme reduction of insulin secretion in the early phase (Δ 0–15 min) and the less severe impairment of the late phase (Δ 15–60 min) have been confirmed; obese subjects showed on the average an active insulin response to venous loading; this was more marked and more consistent in the late phase. Compared to controls, FFA concentration both in basal conditions and during IVGTT was progressively higher in obese and diabetic patients. When analyzing the interplay between IRI, KG and FFA in the course of IVGTT, it was observed that: (1) a close correlation exists between KG and early insulin response (r=0.72); (2) a correlation between Δ IRI 0–15 min and percentage decrease of FFA at 45 min is found only in normal subjects; (3) a negative highly significant correlation is found between Kg and mean FFA plasma level 0–60 min. This last correlation is evidence of the important role played by FFA in carbohydrate tolerance. The conflicting results reported by others have been discussed.

Insulin-like growth factor I (IGF I) receptor autophosphorylation and kinase activity. Effect of a human polyclonal antibody (pIgG)

IGF I receptor is a tyrosine kinase capable of phosphorylating the receptor itself and other substrates. A high degree of homology does exist in tyrosine kinase domain among receptors for several polypeptide growth factor receptors and this enzymic activity has been indicated as a possible mediator of biological action. Nevertheless growth factor receptors possess peculiar specificities both in their functions and tissue distribution. A human polyclonal IgG (pIgG), previously characterized as anti insulin receptor antibody, able to inhibit insulin receptor kinase activity, was used to further investigate subunit homologies and differences in antigenicity and functional regulation between IGF I and insulin receptors, IGF I receptor tyrosine kinase was stimulated by a IGF I analog (aIGF I), produced by DNA recombinant technology, pIgG was able to inhibit IGF I receptor kinase activity, thus revealing antigenic homologies between the kinase domains of insulin and IGF I receptors. However the more pronounced inhibition of IGF I receptor-compared with insulin receptor kinase activity by pIgG suggests the existence of different regulatory mechanisms.

Exploration of the early insulin response by two small successive loads of I.V. glucose in normal and obese subjects.

SummaryTwo 5 g glucose loads at 1-h interval were given to healthy controls and obese subjects with slightly altered or normal OGTT in order to explore the capacity of restoration of the ‘rapid insulin response’ to i.v. glucose. In the normal subjects, the two successive loads gave rise to identical responses as far as maximum increase (Δmax), average increase at 2–5 min (Δ2–5 min), area of increase 0–15 min (Δ0–15 min) for both glucose and IRI, were concerned. Obese subjects could be divided on the basis of their insulin response to the first load into normal responders (group I) and high-responders (group II). In group I obese subjects, the responses to the second load were identical to those to the first. In group II obese subjects Δmax, Δ2–5 min and Δ0–15 min of the insulin response to the second load were reduced as compared to the first.