The biological actions of insulin have been associated with activation of phosphatidylinositol 3-kinase (PI3-K), protein kinase C and mitogen-activated protein (MAP) kinases signalling pathways in mammalian cells (Bevan, 2001). Insulin (8 h) increases L-arginine transport through system y⁺/CATs (cationic amino acid transporters, Sobrevia et al. 1996) and acutely (minutes) activates endothelial NO synthase (eNOS) via insulin receptors (Zeng & Quon, 1996) in human umbilical vein endothelial cells (HUVECs). It has been reported that infusion of insulin induces a long-lasting (6-7 h) vasodilatation in humans (Utriainen et al. 1996), and incubation of bovine aortic endothelial cells with insulin led to increased level of eNOS protein and gene expression (Kuboki et al. 2000). We have investigated the effects of insulin on the activity and expression of L-arginine transporters and eNOS, and the involvement of PI3-K, PKC and p42/p44^mapk, in human fetal endothelium.

Endothelial cells were isolated (0.2 mg ml^-1 collagenase) from umbilical veins of normal pregnancies (Ethics Committee approval and informed patient consent were obtained) and cultured in medium 199 (M199), supplemented with 10 % fetal and 10 % newborn calf serum, 3.2 mM L-glutamine and 5 mM D-glucose. Transport of L-[³H]arginine (100 µM, 2 µCi ml^-1, 37°C, 1 min) was measured in the presence or absence of insulin (1 nM, 2 min-24 h), wortmannin (50 nM, 30 min, PI3-K inhibitor), PD-98059 (10 µM, 30 min, MAP kinase inhibitor), phorbol 12-myristate 13-acetate (PMA, 100 nM, 8 h, activator of PKC), calphostin C (100 nM, 30 min, inhibitor of PKC), or N ^G-nitro-L-arginine methylester (L-NAME, 100 µM, 30 min, eNOS inhibitor). Activity of eNOS was estimated by conversion of L-[³H]arginine into L-[³H]citrulline (L-[³H]arginine, 100 µM, 4 µCi ml^-1, 37°C, 30 min). Total protein level for eNOS was determined by Western blot. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to determine mRNA levels for human CAT-1, CAT-2B and eNOS.

Incubation of endothelial cells with 1 nM insulin for 2 min induced a significant increase (P < 0.05, Student’s unpaired t test) of L-arginine transport (6.1 ± 0.6 vs. 4.0 ± 0.3 pmol (µg protein)^-1 min^-1, means ± S.E.M., n = 8), associated with an elevated V_max (5.4 ± 0.8 vs. 21.6 ± 5.0 pmol (µg protein)^-1 min^-1) with no significant changes in the apparent K_m (176 ± 38 vs. 217 ± 22 µM). Insulin-induced increase of L-arginine transport was blocked (P < 0.05) by wortmannin (3.3 ± 0.6 pmol (µg protein)^-1 min^-1) and calphostin C (3.4 ± 0.9 pmol (µg protein)^-1 min^-1), but was unaltered by PD-98059 (6.4 ± 1.1 pmol (µg protein)^-1 min^-1, P > 0.05) or L-NAME (5.0 ± 0.8 pmol (µg protein)^-1 min^-1, P > 0.05). The mRNA levels for hCAT-2B are increased in cells incubated with insulin for 2 h (1.7-fold), 4 h (1.66-fold), 8 h (1.53-fold), 12 h (1.2-fold) and 24 h (1.3-fold). When cells were incubated for 1 or 24 h with insulin there was no significant change of hCAT-2B mRNA level compared with cells in the absence of insulin. hCAT-1 mRNA level remained unaltered between 1-12 h of incubation with insulin, but increased 1.5-fold after 24 h incubation. Parallel experiments show that L-arginine transport is significantly increased by incubation of cells with insulin for 8 h (2.9 ± 0.3 vs. 4.5 ± 0.6 pmol (µg protein)^-1 min^-1, means ± S.E.M., n = 3), an effect blocked by wortmannin (1.7 ± 0.7 pmol (µg protein)^-1 min^-1), calphostin C (2.3 ± 0.2 pmol (µg protein)^-1 min^-1), PD-98059 (2.4 ± 0.9 pmol (µg protein)^-1 min^-1) and L-NAME (2.5 ± 1.0 pmol (µg protein)^-1 min^-1). Incubation with insulin for 8 h also increased eNOS mRNA 1.4-fold, eNOS protein level (1.7-fold) and activity (control 4100 ± 120, insulin 8500 ± 350 d.p.m. (10⁶ cells)^-1 30 min^-1, P < 0.05).

These data suggest that regulation of the L-arginine/NO pathway by insulin is rapidly activated via a PI3-K signalling pathway involving protein kinase C in human umbilical vein endothelial cells. In addition, insulin-induced activity of the L-arginine/NO pathway could also result from a higher expression of hCAT-1, hCAT-2B and eNOS genes in this cell type.Zeng, G. & Quon, M.J. (1996). J. Clin. Invest. 98, 894-898.

This work was supported by FONDECYT (1000354 and 7000354) and DIUC-Iniciativa de Grupos de Investigaciùn de Avanzada (201034006-1.4, University of Concepciùn)-Chile, and The Wellcome Trust (UK). C.A.F. holds University of Concepciùn-MSc fellowship.

Bevan, P. (2001). J. Cell. Science 114, 1429-1430.

Kuboki, K., Jiang, Z.Y., Takahara, N., Ha, S.W., Igarashi, M., Yamauchi, T., Feener, E.P., Herbert, T.P., Rhodes, C.J. & King, J.L. (2000). Circulation 101, 676-681.

Sobrevia, L., Nadal, A., Yudilevich, D.L. & Mann, G.E. (1996). J. Physiol. 490, 775-781. abstract

Utriainen, T., Makimattila, S., Virkamaki, A., Bergholm, R. & Yki-Jarvinen, H. (1996). Diabetologia 39, 1477-1482.