Thrombosis and llaemostasis - @ F. K. SchattauerVerlagsgesellschaft mbH (Stuttgart) 60 (3) 491-494 (1988) lnsulin Stimulates the Synthesis of Plasminogen Activator Inhibitor 1 by the Human Hepatocellular Cell Line Hep G2 M. C. Alessil, l. Juhan-Vague1, T Kooistra2, P. J. Declerck3, and D. Collen3 From the Laboratory of Hematologyr, CHU Timone, Marseille, France, the Gaubius lnstitute2, TNO, Leiden, The Netherlands, and the Center for Thrombosis and Vascular Research3, Leuven, Belgium Key words Insulin - PAI-1 - Hepatocyte - Endothelial cell Summary Secretion of plasminogen activator inhibitor L (PAI-l) by cultures of human umbilical vein endothelial cells and human hepatocellular cell line Hep G2 was evaluated after insulin stimulation. The secretion of PAI-1 antigen and activity was measured in the conditioned medium and the cellular extracts after incubation of confluent cultures with l"/" serum medium for 24 hours. Insulin induced a dose dependent increase of the PAI-1 secretion by Hep G2 cell line. At 10-a 1v1 a two fold increase of PAI-1 antigen and activity were observed whereas o,2 antiplasrnin and fibrinogen were not significantly modified. No effect of insulin was observed on PAI-1 antigen and PAI activity produc- tion by human endothelial cells whereas endotoxin resulted in a two fold increase in PAI-1 secretion. In recent clinical studies we have demonstrated that the level of plasma insulin correlated with that of PAI-I. Thus we hypothesize that hepatocytes represent a physiological source of plasma PAI-1 which is modulated by plasma insulin level. lntroduction Increased plasma levels of plasminogen activator inhibitor (PAI) activity occur in various prethrombotic states including coronary artery disease (1), venous thrombosis (2, 3), and the post operative state (4). Elevated plasma PAI activity levels have a predictive value for the recurrence of myocardial infarction (5) which suggests that PAI is of pathophysiological importance. PAI-1 is synthesized by endothelial cells and by human hepatocytes in culture (6,7), and occurs in the alpha-granules of blood platelets; PAI-1 seems to be the main physiological inhibitor of t-PA in plasma (8). Several mechanisms which remain poorly understood appear to be involved in the regulation of PAI-1 secretion (8); endotoxin (9) and interleukin 1 (10) stimu- late its synthesis in endothelial cells, corticosteroids increase hepatic synthesis (11), whereas the PAI-1 level in blood correlates with the triglyceride level (3, 5, L2). In recent studies (12, 13) we have demonstrated that the level of plasma insulin correlates with that of PAI-I. In the present study we have therefore compared the effect of insulin on PAI-1 secretion by human endothelial cell and human hepatocyte (Hep G2 line) cultures. It appears that insulin stimulates PAI-1 synthesis by the hepatoma cell line, but not by endothelial cells. Insulin stimulation of heipatic synthesis may explain the increased plasma PAI-1 concentration in hyperinsulinemic subjects (12,13). Correspondence to: Prof. I. Juhan-Vague, Laboratory of Hematology, CHU Timone, 13385 Marseille C6dex 5, France Materials and Methods Material Human insulin was purchased from and proinsulin was a kind gift from Novo industries (Paris). Cycloheximide, Actinomycin D and gelatin were obtained from Sigma Chemical Co (St Louis, MO). Lipopolysaccharide 01.1L : 84 from Escherichia coli was from Osi Laboratories (Paris). Plasminogen, the chromogenic substrate D-Va1-Leu-Lys-p-nitroanilide (52251) and CNBr-digested fibrinogen fragments were from Kabi Vitrum (Amsterdam). The protein assay kit was from BIORAD (Richmond, CA). Fibrinogen, and antiserum against fibrinogen were from Stago (Paris), orthophenylene diamine was from Fluka (Darmstadt, W Ger- many), and medium 199, Dulbecco's modified eagle medium and fetal calf serum were from Flow Laboratories (Irvine, UK). Human serum was obtained from a pool of healthy subjects. Purified PAI-I, t-PA, a.2 antiplasmin and monoclonal antibodies against PAI-1 and o,2 antiplasmin were prepared as described elsewhere ('!,4,15). The human hepatoma cell line Hep G2 was provided by Dr B. B. Knowles, Wistar Institute of Anatomy and Biology, Philadelphia, PA. All other reagents were of laboratory grade quality. Cell Culture Human endothelial cells were prepared from fresh umbilical cord veins by the method of Jaffe et al. (16). Cells were used after one passage and plated in tissue culture flasks (25 cm2) coated with gelatin. Cetls were grown to confluency at 37" C with atmospheric air and 5"/" COrin medium 199 supplemented with Hepes L5 mM, NaHCO3 L5 mM, glutamine}ffiM, penicillin 100 IU/ml, streptomycin 100 pglml, l0% heat inactivated human serum and 10% heat inactivated fetal calf sbrum. The cells were identified as endothelial cells by their typical cobblestone pattern, by immunofluorescence staining for von Willebrand factor and by electronic microscopy. The hepatoma cell line Hep GZ was grown in DMEM supplemented with 10% heat inactivated fetal calf serum and 5"/" heat inactivated human serum. Cell cultures were grown in 9.6 cmz dishes until confluency. The culture medium was then replaced by 2 ml culture medium containingl"/" serum. Endotoxin, insulin or proinsulin were dissolved in the same medium and added in the cell cultures. After incubation, the conditioned medium was harvested and centrifuged for 5 min at 3,500 x g. The samples were either immediately assayed or kept frozen at -20" C until use. Inhibition studies with Cycloheximide (2.5 pM) and Actinomycin D (2 pM) were performed by addition of inhibitors together with insulin to the cell cultures and incubation for 20 hours. Cell extracts were prepared by incubation of cell monolayers with L ml 0.5% Tliton X100 for L hour at room temperature. The concentrations of PAI-1 and o2 antiplasmin were measured with specific ELISA's (L4, 15). PAI activity was determined by the method of Verheijen et al. (17); the results were expressed in units equivalent to the amount of IU of t-PA neutralized. The activity of t-PA was determined by comparison with the international reference preparation (8315L7). Fib- rinogen was quantified by an ELISA assay purchased from Stago Laboratories. Protein concentration was determined by the method of Bradford (18). 49L This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.