The concept of "selective insulin resistance" has emerged as a unifying hypothesis in attempts to reconcile the influence of insulin resistance with that of hyperinsulinemia in the pathogenesis of macrovascular complications of diabetes. To explore this hypothesis in endothelial cells, we designed a set of experiments to mimic the "typical metabolic insulin resistance" by blocking the phosphatidylinositol 3-kinase pathway and exposing the cells to increasing concentrations of insulin ("compensatory hyperinsulinemia"). Inhibition of phosphatidylinositol 3-kinase with wortmannin blocked the ability of insulin to stimulate increased expression of endothelial nitric-oxide synthase, did not affect insulin-induced activation of MAP kinase, and increased the effects of insulin on prenylation of Ras and Rho proteins. At the same time, this experimental paradigm resulted in increased expression of vascular cellular adhesion molecules-1 and E-selectin, as well as increased rolling interactions of monocytes with endothelial cells. We conclude that inhibition of the metabolic branch of insulin signaling leads to an enhanced mitogenic action of insulin in endothelial cells.Insulin profoundly influences the function of the vascular endothelium (1-5). In humans, physiological levels of insulin stimulate increased production of nitric oxide (NO) 1 in the vasculature resulting in vasodilation and increased blood flow (1, 5). Intriguingly, vasodilator actions of insulin are impaired in individuals who are also resistant to metabolic actions of insulin (6). Although associations between vascular disease and insulin-resistant states such as diabetes, obesity, and hypertension have been firmly established, the mechanisms linking endothelial dysfunction and accelerated atherosclerosis with insulin resistance (typically defined as decreased sensitivity or responsiveness to metabolic actions of insulin) have not been fully elucidated. With in vivo studies, it is particularly challenging to differentiate potentially distinct influences of insulin resistance per se from effects of compensatory hyperinsulinemia. In vitro studies in vascular endothelial cells demonstrate that insulin may stimulate production of NO by increasing both the expression and the activity of endothelial nitricoxide synthase (eNOS) (7-9). Activation of phosphatidylinositol 3-kinase (PI 3-kinase) is necessary to promote both increased expression and activity of eNOS in response to insulin (7-9). Interestingly, PI 3-kinase is also a key signaling molecule mediating metabolic actions of insulin in adipose tissue and skeletal muscle (reviewed in Ref. 10). Thus, abnormalities in PI 3-kinase-dependent pathway that are shared among different tissues may provide one molecular explanation for the frequent associations of vascular disease and insulin-resistant states (4).Recent studies (11, 12) in both humans and animals demonstrate that regulation of the insulin receptor substrate-1 (IRS-1)/PI 3-kinase-dependent branch of insulin signaling may be distinct from regulation ...

Abstract-Pathogenesis of macrovascular complications of diabetes may involve an activation of the transcription factor nuclear factor-B (NF-B) by hyperglycemia and advanced glycosylation end products (AGEs). Activation of NF-B is believed to be dependent on activation of the Rho family of GTPases. Although the precise mechanism of the Rho-mediated action is not completely understood, posttranslational modification of the Rho proteins by geranylgeranylation is required for their subsequent activation. We observed that in cultured vascular smooth muscle cells (VSMCs), insulin stimulated the activity of geranylgeranyltransferase (GGTase) I and increased the amounts of geranylgeranylated Rho-A from 47% to 60% (PϽ0.05). GGTI-286, an inhibitor of GGTase I, blocked both effects of insulin.

Rab proteins play a crucial role in the trafficking of intracellular vesicles. Rab proteins are GTPases that cycle between an inactive GDP-bound form and an active GTP-bound conformation. A prerequisite to Rab activation by GTP loading is its post-translational modification by the addition of geranylgeranyl moieties to highly conserved C-terminal cysteine residues. We examined the effect of insulin on the activity of geranylgeranyltransferase II (GGTase II) in 3T3-L1 fibroblasts and adipocytes. In fibroblasts, insulin increased the enzymatic activity of GGTase II 2.5-fold after 1 h of incubation, an effect that is blocked by perillyl alcohol, an inhibitor of prenyltransferases, but not by the geranylgeranyltransferase I inhibitor, GGTI-298, or the farnesyltransferase inhibitor, ␣-hydroxyfarnesylphosphonic acid. Concomitantly, insulin stimulated the phosphorylation of the GGTase II ␣-subunit without any effect on the GGTase II ␤-subunit. At the same time, insulin also increased the amounts of geranylgeranylated Rab-3 in 3T3-L1 fibroblasts from 44 ؎ 1.2% in control cells to 63 ؎ 3.8 and 64 ؎ 6.1% after 1 and 24 h of incubation, respectively. In adipocytes, insulin increased the amounts of geranylgeranylated Rab-4 from 38 ؎ 0.6% in control cells to 56 ؎ 1.7 and 60 ؎ 2.6% after 1 and 24 h of incubation, respectively. In both fibroblasts and adipocytes, the presence of perillyl alcohol blocked the ability of insulin to increase geranylgeranylation of Rab-4, whereas GGTI-298 and ␣-hydroxyfarnesylphosphonic acid were without effect, indicating that insulin activates GGTase II. In summary, insulin promotes phosphorylation and activation of GGTase II in both 3T3 L1 fibroblasts and adipocytes and increases the amounts of geranylgeranylated Rab-3 and Rab-4 proteins.The family of Rab proteins is believed to play a crucial role in the intracellular trafficking of vesicles along the endocytic and exocytic pathways (1, 2). To date, over 30 members of this family have been identified (3, 4). Although the exact role and mechanism of action of Rab proteins remain to be elucidated, it appears that Rab proteins are involved in the process of vesicular transport and precise targeting of membranous vesicles to their docking and/or fusion sites (5, 6). Rab proteins are GTPases that cycle between an inactive GDP-bound form, cystosolic Rab, and an active GTP-bound conformation, membraneassociated Rab, under the influence of Rab-associated guanine nucleotide exchange factors and guanosine triphosphatase-activating proteins (1-7). Although Rab protein activity is based on GTP loading, a prerequisite to Rab activation is its posttranslational modification (4 -6, 8, 9).Post-translational modification of small molecular mass GTP-binding proteins is accomplished by the isoprenylation of conserved cysteine residues found on their C termini (10, 11). Ras and Rho proteins are prenylated on a single cysteine residue of the CAAX box (where C is cysteine, A is the aliphatic residue, and X is methionine, serine, or glutamine for Ras and leucine for ...