PAI-1 is increased by 3- to 4-fold in blood from patients with type 2 diabetes and obese subjects with insulin resistance, as shown by studies in Schneider's laboratory. The increased concentration of PAI-1 was associated with increased functional activity of PAI-1. This increased expression of PAI-1 is associated with a diminished fibrinolytic response. Patients were exposed to a physiological stress (venous occlusion) and the change in tPA activity was measured to determine fibrinolytic response. A decreased fibrinolytic response was found in the obese (1.8 IU/ml) and diabetic (1.5 IU/ml; p<0.05 vs lean) subjects compared to the lean subjects (2.7 IU/ml).

Analysis of atherectomy specimens from diabetic and non-diabetic subjects showed increased immunoreactivity of PAI-1 in samples from diabetics, regardless of the type of diabetic treatment (Fig. 2). This increase in PAI-1 was associated with a decrease in uPA. These results in association with their observations made in PAI-1 deficient mice led them to suggest that the overexpression of PAI-1 combined with the decreased fibrinolytic activity in the vessel wall may potentiate the genesis of unstable plaques.

The infusion of glucose and triglycerides into healthy subjects increased concentrations of insulin and PAI-1 in blood. By contrast, the infusion of insulin and glucose with the use of euglycemic clamp techniques led to a decrease in the concentration of free fatty acid, while the concentration of triglycerides remained low throughout the infusion. As expected, insulin levels rose and glucose was maintained normally but the concentration of PAI-1 was similar to that seen in subjects in whom normal saline was infused. These experiments strongly support that an interaction between lipids, particularly triglycerides and free fatty acids, glucose, and insulin modulates expression of PAI-1 in diabetic subjects.

In vitro studies with a human hepatoma cell line (HepG2 cells) support this observation. In the HepG2 cells, exposure to insulin alone and free fatty acids alone led to a modest (2-fold) increase in PAI-1. Combining insulin and free fatty acids resulted in a synergistic increase in the expression of PAI-1. Exposure to insulin and free fatty acid concentrations similar to those seen in diabetic subjects led to a 3- to 4-fold increase in the accumulation of PAI-1 in conditioned media

The molecular mechanisms by which insulin alters expression of PAI-1 was characterized in HepG2 cells. Nuclear run-on assays demonstrated that insulin does not increase the rate of transcription of PAI-1. The degradation of PAI-1 mRNA was determined after transcription was inhibited with actinomycin D. Insulin prolongs of the half-life of PAI-1 mRNA. Thus, insulin-mediated increased expression of PAI-1 in HepG2 cells is post-transcriptional.

Free fatty acid studies

A chimeric gene construct was created in which approximately 1300 base pairs of the 5'-flanking DNA were inserted upstream to a luciferase reporter. Free fatty acids increased the luciferase activity nearly 2-fold when HepG2 cells were transfected with the construct (Fig. 3). A promoter-deletion analysis showed that the fatty acid responsive element was located upstream of -610.

Deletion of a 72-base pair segment from the full-length construct of the 5'-flanking PAI-1 DNA led to abolition of the response to free fatty acids. Further, when this 72-base pair segment was placed upstream of a minimal promoter the response to free fatty acids returned. Thus, the 72-base pair segment contains a fatty acid responsive region.

Gel shift mobility assays and Dnase 1 footprinting identified a specific sequence that is repeated four times in the 72-base pair segment (Fig. 4). Substantial structural homology between the repeated sequence and an SP-1 binding site exists. However, super shift assays with an anti-SP-1 antibody did not demonstrate a 'supershift'. Thus, further studies are underway to identify the SP-1 like transcription factor that mediates the effect of PAI-1.

Diabetes is clearly associated with an increased concentration of PAI-1 in the blood and in the arterial wall. Increased PAI-1 may potentiate atherosclerosis by promoting thrombosis (limiting fibrinolysis) and by impairing vessel wall proteolytic capacity that alters the composition of the plaque. Potential mechanisms responsible for increased expression of PAI-1 in diabetic subjects include hyperinsulinemia, hyperlipidemia, and hyperglycemia. Insulin-mediated effects in HepG2 cells are secondary to stabilization of PAI-1 mRNA. Free fatty acids increase transcription of PAI-1 mRNA. The combination is synergistic.