Dominant negative alpha-subunit of farnesyl- and geranylgeranyl-transferase I inhibits insulin-induced differentiation of 3T3-L1 pre-adipocytes.

OBJECTIVE: To investigate whether the expression of a dominant negative (DN) farnesyl- and geranygeranyl-transferase I (FTase/GGTase I) alpha-subunit in 3T3-L1 pre-adipocytes can inhibit insulin's ability to induce differentiation. DESIGN: 3T3-L1 pre-adipocytes were stably transfected with vector alone or vector expressing a mutated DN FTase/GGTase I alpha-subunit (S60A)(S62A) and incubated in serum-free medium in the absence and presence of insulin. MEASUREMENTS: Various assays were performed to determine the effect of DN FTase/GGTase I alpha-subunit expression in 3T3-L1 pre-adipocyte on insulin-induced DNA synthesis, cell count, phosphorylation of the FTase/GGTase I alpha-subunit, FTase and GGTase I activity, amounts of prenylated p21Ras and RhoA, phosphorylation of MAP kinase and Akt, and differentiation to mature fat cells. RESULTS: Expression of DN FTase/GGTase I alpha-subunit inhibited insulin's ability to increase DNA synthesis, cell count, FTase and GGTase I activity, amounts of prenylated p21Ras and RhoA, and magnitude of phosphorylation of MAP kinase. Expression of DN FTase/GGTase I alpha-subunit in 3T3-L1 pre-adipocytes was without effect on insulin-induced Akt phosphorylation. CONCLUSION: Expression of DN FTase/GGTase I alpha-subunit inhibits insulin-induced differentiation of 3T3-L1 pre-adipocytes to mature adipocytes, and thus could indicate potential therapeutic avenues to assuage the deleterious effects of obesity and type 2 diabetes.

Effect of insulin on cell cycle progression in MCF-7 breast cancer cells. Direct and potentiating influence.

We recently demonstrated that in MCF-7 breast cancer cells, insulin promoted the phosphorylation and activation of geranylgeranyltransferase I (GGTI-I), increased the amounts of geranylgeranylated Rho-A and potentiated the transactivating activity of lysophosphatidic acid (LPA) (Chappell, J., Golovchenko, I., Wall, K., Stjernholm, R., Leitner, J., Goalstone, M., and Draznin, B. (2000) J. Biol. Chem. 275, 31792-31797). In the present study, we explored the mechanism of this potentiating effect of insulin on LPA. Insulin (10 nm) potentiated the ability of LPA to stimulate cell cycle progression and DNA synthesis in MCF-7 cells. The potentiating effect of insulin appears to involve increases in the expression of cyclin E and decreases in the expression of the cyclin-dependent kinase inhibitor p27Kip1. All potentiating effects of insulin were inhibited in the presence of an inhibitor of GGTase I, GGTI-286 (3 microm) or by an expression of a dominant negative mutant of Rho-A. In contrast to its potentiating action, a direct mitogenic effect of insulin in MCF-7 cells involves activation of phosphatidylinositol 3-kinase and increased expression of cyclin D1. We conclude that the ability of insulin to increase the cellular amounts of geranylgeranylated Rho-A results in potentiation of the LPA effect on cyclin E expression and degradation of p27Kip1 and cell cycle progression in MCF-7 breast cancer cells.

Fetal hyperinsulinemia increases farnesylation of p21 Ras in fetal tissues.

Even though the role of fetal hyperinsulinemia in the pathogenesis of fetal macrosomia in patients with overt diabetes and gestational diabetes mellitus seems plausible, the molecular mechanisms of action of hyperinsulinemia remain largely enigmatic. Recent indications that hyperinsulinemia "primes" various tissues to the mitogenic influence of growth factors by increasing the pool of prenylated Ras proteins prompted us to investigate the effect of fetal hyperinsulinemia on the activitiy of farnesyltransferase (FTase) and the amounts of farnesylated p21 Ras in fetal tissues in the ovine experimental model. Induction of fetal hyperinsulinemia by direct infusion of insulin into the fetus and by either fetal or maternal infusions of glucose resulted in significant increases in the activity of FTase and the amounts of farnesylated p21 Ras in fetal liver, skeletal muscle, fat, and white blood cells. An additional infusion of somatostatin into hyperglycemic fetuses blocked fetal hyperinsulinemia and completely prevented these increases, specifying insulin as the causative factor. We conclude that the ability of fetal hyperinsulinemia to increase the size of the pool of farnesylated p21 Ras may prime fetal tissues to the action of other growth factors and thereby constitute one mechanism by which fetal hyperinsulinemia could induce macrosomia in diabetic pregnancies.

Dominant negative farnesyltransferase alpha-subunit inhibits insulin mitogenic effects.

Farnesylation of p21Ras is required for translocation to the plasma membrane and subsequent activation by growth factors. Previously we demonstrated that insulin stimulates the phosphorylation of farnesyltransferase (FTase) and its activity, whereby the amount of farnesylated p21Ras anchored at the plasma membrane is increased. Herein we report that substitution of alanine for two serine residues (S60A)(S62A) of the alpha-subunit of FTase creates a dominant negative (DN) mutant. VSMC expressing the FTase alpha-subunit (S60A)(S62A) clone showed a 30% decreased basal FTase activity concurrent with a 15% decrease in the amount of farnesylated p21Ras compared to controls. Expression of alpha-subunit (S60A,S62A) blunted FTase phosphorylation and activity in the presence of hyperinsulinemia, and inhibited insulin-stimulated increases in farnesylated p21Ras. Insulin-stimulated VSMC expressing the FTase alpha-subunit (S60A,S62A) showed decreased (i) phosphorylation of FTase, (ii) FTase activity, (iii) amounts of farnesylated p21Ras, (iv) DNA synthesis, and (v) migration. Thus, down-regulation of FTase activity appears to mitigate the potentially detrimental mitogenic effects of hyperinsulinemia on VSMC.

Insulin-induced adipocyte differentiation. Activation of CREB rescues adipogenesis from the arrest caused by inhibition of prenylation.

Insulin is a potent adipogenic hormone that triggers an induction of a series of transcription factors governing differentiation of pre-adipocytes into mature adipocytes. However, the exact link between the insulin signaling cascade and the intrinsic cascade of adipogenesis remains incompletely understood. Herein we demonstrate that inhibition of prenylation of p21ras and Rho-A arrests insulin-stimulated adipogenesis. Inhibition of farnesylation of p21ras also blocked the ability of insulin to activate mitogen-activated protein (MAP) kinase and cyclic AMP response element-binding (CREB) protein. Expression of two structurally different inducible constitutively active CREB constructs rescued insulin-stimulated adipocyte differentiation from the inhibitory influence of prenylation inhibitors. Constitutively active CREB constructs induced expression of PPARgamma2, fatty acid synthase, GLUT-4, and leptin both in control and prenylation inhibitors-treated cells. It appears that insulin-stimulated prenylation of the Ras family GTPases assures normal phosphorylation and activation of CREB that, in turn, triggers the intrinsic cascade of adipogenesis.

Insulin signals to prenyltransferases via the Shc branch of intracellular signaling.

We assessed the roles of insulin receptor substrate-1 (IRS-1) and Shc in insulin action on farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I) using Chinese hamster ovary (CHO) cells that overexpress wild-type human insulin receptors (CHO-hIR-WT) or mutant insulin receptors lacking the NPEY domain (CHO-DeltaNPEY) or 3T3-L1 fibroblasts transfected with adenoviruses that express the PTB or SAIN domain of IRS-1 and Shc, the pleckstrin homology (PH) domain of IRS-1, or the Src homology 2 (SH2) domain of Shc. Insulin promoted phosphorylation of the alpha-subunit of FTase and GGTase I in CHO-hIR-WT cells, but was without effect in CHO-DeltaNPEY cells. Insulin increased FTase and GGTase I activities and the amounts of prenylated Ras and RhoA proteins in CHO-hIR-WT (but not CHO-DeltaNPEY) cells. Overexpression of the PTB or SAIN domain of IRS-1 (which blocked both IRS-1 and Shc signaling) prevented insulin-stimulated phosphorylation of the FTase and GGTase I alpha-subunit activation of FTase and GGTase I and subsequent increases in prenylated Ras and RhoA proteins. In contrast, overexpression of the IRS-1 PH domain, which impairs IRS-1 (but not Shc) signaling, did not alter insulin action on the prenyltransferases, but completely inhibited the insulin effect on the phosphorylation of IRS-1 and on the activation of phosphatidylinositol 3-kinase and Akt. Finally, overexpression of the Shc SH2 domain completely blocked the insulin effect on FTase and GGTase I activities without interfering with insulin signaling to MAPK. These data suggest that insulin signaling from its receptor to the prenyltransferases FTase and GGTase I is mediated by the Shc pathway, but not the IRS-1/phosphatidylinositol 3-kinase pathway. Shc-mediated insulin signaling to MAPK may be necessary (but not sufficient) for activation of prenyltransferase activity. An additional pathway involving the Shc SH2 domain may be necessary to mediate the insulin effect on FTase and GGTase I.

Hyperinsulinemia enhances transcriptional activity of nuclear factor-kappaB induced by angiotensin II, hyperglycemia, and advanced glycosylation end products in vascular smooth muscle cells.

Pathogenesis of macrovascular complications of diabetes may involve an activation of the transcription factor nuclear factor-kappaB (NF-kappaB) by hyperglycemia and advanced glycosylation end products (AGEs). Activation of NF-kappaB 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% (P:<0.05). GGTI-286, an inhibitor of GGTase I, blocked both effects of insulin. Increased availability of prenylated Rho-A significantly augmented the abilities of angiotensin II (Ang II), hyperglycemia, and AGEs to activate NF-kappaB, as measured by NF-kappaB response-element luciferase reporter activity. Preincubations of VSMCs with insulin for 24 hours doubled NF-kappaB transactivation by Ang II, hyperglycemia, and AGEs. This priming effect of insulin was completely inhibited by GGTI-286. We demonstrate for the first time, to our knowledge, that insulin potentiates NF-kappaB-dependent transcriptional activity induced by hyperglycemia, AGEs, and Ang II in VSMCs by increasing the activity of GGTase I and the availability of geranylgeranylated Rho-A.

Potentiation of Rho-A-mediated lysophosphatidic acid activity by hyperinsulinemia.

We have shown previously that insulin promotes phosphorylation and activation of farnesyltransferase and geranylgeranyltransferase (GGTase) II. We have now examined the effect of insulin on geranylgeranyltransferase I in MCF-7 breast cancer cells. Insulin increased GGTase I activity 3-fold and augmented the amounts of geranylgeranylated Rho-A by 18%. Both effects of the insulin were blocked by an inhibitor of GGTase I, GGTI-286. The insulin-induced increases in the amounts of geranylgeranylated Rho-A resulted in potentiation of the Rho-A-mediated effects of lysophosphatidic acid (LPA) on a serum response element-luciferase construct. Preincubation of cells with insulin augmented the LPA-stimulated serum response element-luciferase activation to 12-fold, compared with just 6-fold for LPA alone (p < 0.05). The potentiating effect of insulin was dose-dependent, inhibited by GGTI-286 and not mimicked by insulin-like growth factor-1. We conclude that insulin activates GGTase I, increases the amounts of geranylgeranylated Rho-A protein, and potentiates the Rho-A-dependent nuclear effects of LPA in MCF-7 breast cancer cells.

Effects of insulin on prenylation as a mechanism of potentially detrimental influence of hyperinsulinemia.

To investigate the cause and effect relationship between hyperinsulinemia and the increased amounts of farnesylated p21Ras, we performed hyperinsulinemic euglycemic clamps in normal weight volunteers as well as in normal mice and dogs. Insulin infusions significantly raised the amounts of farnesylated p21Ras in the white blood cells of humans, in liver samples of mice and dogs, and in aorta samples of mice. Obese hyperinsulinemic individuals and dogs (made hyperinsulinemic by surgical diversion of the pancreatic outflow from the portal vein into the vena cava) displayed increased amounts of farnesylated p21Ras before the hyperinsulinemic clamps. Infusions of insulin did not alter the already increased levels of farnesylated p21Ras in these experimental models. To further investigate the role of acquired insulin resistance in modulating insulin's effect on p21Ras prenylation, we induced insulin resistance in rats by glucosamine infusion. Insulin-resistant glucosamine-treated animals displayed significantly increased farnesylated p21Ras in response to insulin infusion compared to that in control saline-treated animals. Transgenic models of insulin resistance (heterozygous insulin receptor substrate-1 knockout mice, A-ZIP/F-1 fatless mice, and animals overexpressing glutamine:fructose-6-phosphate amidotransferase) contained increased amounts of farnesylated p21Ras. We conclude that hyperinsulinemia, either endogenous (a prominent feature of insulin resistance) or produced by infusions of insulin, increases the amounts of farnesylated p21Ras in humans, mice, and dogs. This aspect of insulin action may represent one facet of the molecular mechanism of the potentially detrimental influence of hyperinsulinemia.

Analysis of farnesyl transferase activity during hormone-induced maturation of Xenopus laevis oocytes.

Preincubation of Xenopus laevis oocytes with insulin or insulin-like growth factor 1 (IGF-1) resulted in inhibition of farnesyl transferase (FTase) activity measured both in vivo (after microinjection of tritiated farnesyl pyrophosphate and Ras-CVIM into oocytes) and in extracts using a filtration assay. FTase activity measured in oocyte extracts was inhibited 55% after a 20 min treatment of oocytes with 1 microM insulin or 10 nM IGF-1. The apparent IC(50) for inhibition of oocyte FTase by IGF-1 is 0.3 nM. The observed decrease in FTase activity was apparently not due to translocation of enzyme from cytosol to membrane, since activities measured both in soluble extracts and resuspended crude pellets displayed comparable levels of inhibition following hormone treatment. Using a hexapeptide (TKCVIM) as substrate, FTase activity was also inhibited 65% when oocytes were pretreated with 10 nM IGF-1. Two FTase inhibitors [(alpha-hydroxyfarnesyl) phosphonic acid (HFPA) and chaetomellic acid A (CA)] effectively inhibited Xenopus oocyte FTase by 80-90% when added to assay mixtures (IC(50) values of 338 +/- 96 nM HFPA and 232 +/- 80 nM CA) or after incubation of oocytes in drug before preparation of soluble extracts for assay (IC(50) values of 7 +/- 6 nM HFPA and 328 +/- 128 nM CA). The farnesyl transferase inhibitors were observed to slow the time course of oocyte maturation but did not block the IGF-1-induced maturation response. J. Exp. Zool. 286:193-203, 2000.

Increased amounts of farnesylated p21Ras in tissues of hyperinsulinaemic animals.

We have recently demonstrated that insulin activates farnesyltransferase (FTase) and thereby increases the amounts of cellular farnesylated p21Ras in 3T3-L1 fibroblasts, adipocytes and vascular smooth muscle cells. We postulated that hyperinsulinaemia might considerably increase the the cellular pool of farnesylated p21Ras available for activation by other growth factors. To examine the role of in vivo hyperinsulinaemia in regulating farnesylated p21Ras, we measured the amounts of farnesylated p21Ras in tissues of hyperinsulinaemic animals. Liver, aorta, and skeletal muscle of ob/ob mice, and mice made obese and hyperinsulinaemic by injection of gold-thioglucose contained greater amounts of farnesylated p21Ras than tissues of their lean normoinsulinaemic counterparts. Similarly, farnesylated p21Ras was increased (67 vs. 35 % in control animals, p<0.01) in the livers of hyperinsulinaemic Zucker rats (fa/fa). Reduction of hyperinsulinaemia by exercise training (2 h/day for 7-8 weeks) resulted in decreases in the amounts of farnesylated p21Ras in these animals. Increased farnesylated p21Ras in hyperinsulinaemic animals reflected increasing increments in the activity of FTase in ob/ob mice (2-fold increase) and fa/fa Zucker rats (3.5-fold increase), while the total amounts of Ras proteins remained unchanged. In contrast to insulin-resistant hyperinsulinaemic animals, denervated insulin-resistant rat soleus muscle (in the presence of normoinsulinaemia) showed normal amounts of farnesylated p21Ras. In summary, these data confirm increased amounts of farnesylated p21Ras in tissues of hyperinsulinaemic animals.

Insulin promotes phosphorylation and activation of geranylgeranyltransferase II. Studies with geranylgeranylation of rab-3 and rab-4.

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, alpha-hydroxyfarnesylphosphonic acid. Concomitantly, insulin stimulated the phosphorylation of the GGTase II alpha-subunit without any effect on the GGTase II beta-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 alpha-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.

Effect of insulin on farnesyltransferase gene transcription and mRNA stability.

Recently, we have shown that hyperinsulinemia increases the activity of farnesyltransferase (FTase) in vitro (1) and in hyperinsulinemic animals (2), stimulates the phosphorylation of the FTase alpha-subunit (3), increases the amounts of cellular farnesylated p21Ras (4), and potentiates the nuclear effects of other peptide growth factors, such as EGF, IGF-1 and PDGF (5). To further investigate the mechanism by which insulin stimulates FTase activity we tested the effect of insulin on the rate of FTase transcription, the rate of FTase mRNA degradation, and the amounts of FTase protein. Insulin increased the amounts of FTase alpha- and beta-subunit mRNA in 3T3-L1 fibroblasts 2.5-fold to 4-fold after 6 h and 24 h incubation, respectively, but did not increase the rate of FTase transcription over a 24 h period. Insulin did, however, increase the stability of both alpha- and beta-subunit mRNA. The half-life for both FTase alpha- and beta-subunit mRNA was approximately 3 h and 6h in the absence and in the presence of insulin, respectively. Although insulin stabilized the alpha- and beta-subunit mRNA of FTase, there was no increase in amounts of protein of either subunit. These data suggest that although insulin increases the stability of the FTase mRNA, it stimulates FTase enzymatic activity only at the post-translational level.

Effect of insulin on farnesyltransferase. Specificity of insulin action and potentiation of nuclear effects of insulin-like growth factor-1, epidermal growth factor, and platelet-derived growth factor.

We have previously demonstrated that insulin activates farnesyltransferase (FTase) and augments the amounts of farnesylated p21 (Goalstone, M. L., and Draznin, B. (1996) J. Biol. Chem. 271, 27585-27589). We postulated that this aspect of insulin action might explain the "priming effect" of insulin on the cellular response to other growth factors. In the present study, we show the specificity of the effect of insulin on FTase. Insulin, but not insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), or platelet-derived growth factor (PDGF), stimulated the phosphorylation of the alpha-subunit of FTase and the amounts of farnesylated p21. Even though all four growth factors utilized the Ras pathway to stimulate DNA synthesis, only insulin used this pathway to influence FTase. Insulin failed to stimulate FTase in cells expressing the chimeric insulin/IGF-1 receptor and in cells derived from the insulin receptor knock-out animals. Insulin potentiated the effects of IGF-1, EGF, and PDGF on DNA synthesis in cells expressing the wild type insulin receptor, but this potentiation was inhibited in the presence of the FTase inhibitor, alpha-hydroxyfarnesylphosphonic acid. We conclude that the effect of insulin on FTase is specific, requires the presence of an intact insulin receptor, and serves as a conduit for the "priming" influence of insulin on the nuclear effects of other growth factors.

Insulin potentiates platelet-derived growth factor action in vascular smooth muscle cells.

Correlative studies have indicated that hyperinsulinemia is present in many individuals with atherosclerosis. Insulin resistance has also been linked to cardiovascular disease. It has proved to be difficult to decipher whether hyperinsulinemia or insulin resistance plays the most important role in the pathogenesis of atherosclerosis and coronary artery disease. In this study, we demonstrate that insulin increases the amount of farnesylated p21Ras in vascular smooth muscle cells (VSMC), thereby augmenting the pool of cellular Ras available for activation by platelet-derived growth factor (PDGF). In VSMC incubated with insulin for 24 h, PDGF's influence on GTP-loading of Ras was significantly increased. Furthermore, in cells preincubated with insulin, PDGF increased thymidine incorporation by 96% as compared with a 44% increase in control cells (a 2-fold increment). Similarly, preincubation of VSMC with insulin increased the ability of PDGF to stimulate gene expression of vascular endothelial growth factor 5- to 8-fold. The potentiating influence of insulin on PDGF action was abrogated in the presence of a farnesyltransferase inhibitor. Thus, the detrimental influence of hyperinsulinemia on the arterial wall may be related to the ability of insulin to augment farnesyltransferase activity and provide greater amounts of farnesylated p21Ras for stimulation by various growth promoting agents.

What does insulin do to Ras?

The Ras pathway lies in the center of signalling cascades of numerous growth-promoting factors. The Ras pathway appears to connect signalling events that begin at the plasma membrane with nuclear events. Insulin is one of the major stimulants of the Ras signalling pathway. The influence of insulin on this pathway consists of five important events: (1) p21Ras activation is promoted by insulin stimulation of the guanine nucleotide exchange factor, Sos, resulting in increased GTP-loading of p21Ras; (2) p21Ras deactivation involves the hyperphosphorylation of Sos; (3) insulin increases farnesyltransferase (FTase) activity that farnesylates p21Ras; (4) increased amounts of farnesylated p21Ras translocate to the plasma membrane where they can be activated by other growth-promoting agents; and (5) cellular responses to other growth factors are potentiated by insulin-stimulated pre-loading of the plasma membrane with farnesylated p21Ras.

Insulin stimulates the phosphorylation and activity of farnesyltransferase via the Ras-mitogen-activated protein kinase pathway.

Farnesylation of p21Ras by farnesyltransferase (FTase) is obligatory for anchoring p21Ras to the plasma membrane, where it can be activated by growth factors. Insulin significantly stimulates the phosphorylation of the alpha-subunit of FTase (4-fold) and the enzymatic activity of FTase in 3T3-L1 fibroblasts and adipocytes. FTase activity was assessed by the amount of [3H] mevalonate (a precursor of farnesyl) incorporated into p21Ras in vivo and by quantitating the amount of farnesylated p21Ras before and after insulin administration. Insulin-stimulated phosphorylation of the alpha-subunit of FTase in 3T3-L1 fibroblasts and adipocytes was blocked by the mitogen-activated protein/extracellular-signal regulated kinase-kinase inhibitor, PD98059, but not by wortmannin or bisindolylmaleimide. Additionally, PD98059 blocked insulin-stimulated [3H]mevalonic incorporation and farnesylation of unprocessed p21Ras in both cell lines. Furthermore, expression of the dominant negative mutant of p21Ras precluded insulin-stimulated phosphorylation of the FTase alpha-subunit and activation of its enzymatic activity. In contrast, 3T3-L1 fibroblasts, expressing the constitutively active Raf-1, exhibited enhanced phosphorylation of the FTase alpha-subunit. It seems that insulin's effect on the phosphorylation and activation of FTase in both fibroblasts and adipocytes is mediated via the Ras pathway, resulting in a positive feedback augmentation of the cellular pool of farnesylated p21Ras.

GTP loading of farnesylated p21Ras by insulin at the plasma membrane.

Insulin promotes the phosphorylation and activation of farnesyltransferase (FTase) in a time- and a dose-dependent manner. Increased FTase activity results in a larger pool of farnesylated p21Ras and allows for enhanced GTP loading. Insulin significantly increases the pool of farnesylated p21Ras from 20-25% in quiescent 3T3-L1 fibroblasts to approximately 70%, most of which is targeted to the plasma membrane. Furthermore, insulin promotes GTP loading of plasma membrane and not cytosolic p21Ras. The half-life of plasma membrane-associated farnesylated p21Ras is approximately 6 hours, and is identical in control and insulin-treated cells. We have also observed a direct correlation between the amounts of farnesylated p21Ras at the plasma membrane and the magnitude of insulin-induced GTP loading of p21Ras.

Insulin signaling.

The following article is another in a series of papers presented at the Annual Meeting of the Western Association of Physicians. The WAP meets in Carmel, usually in the first week of February, along with sister organization the Western Society for Clinical Investigation and the Western Federation for Clinical Research. These meetings are designed to offer members and guest physicians broad updates in multiple specialties of medicine and basic science. This eclectic approach provides broad cross-fertilization of ideas, often leading to scientific collaboration. Readers of The Western Journal of Medicine are encouraged to register and attend the meetings. The scheduled program for future meetings will be published in the December issue of the journal.

Hyperinsulinemia potentiates activation of p21Ras by growth factors.

Incubation of 3T3-L1 fibroblasts with insulin (10 nM or 100 nM) for 24 or 48 hours resulted in a significant increase in the amount of farnesylated p21Ras with a concomitant increase in the amount of GTP-loaded p21Ras. Cells preincubated with 100 nM insulin for 24 or 48 hours exhibited further 5-8 fold increases in p21Ras.GTP loading in response to an acute (10 minute) challenge with either insulin, EGF, or IGF-1. Effects of hyperinsulinemia were completely abolished by the presence of 1 microM alpha-hydroxyfarnesylphosphonic acid, a potent inhibitor of farnesyltransferase. These novel observations indicate that hyperinsulinemia increases the cellular pool of farnesylated p21Ras and thereby potentiates activation of p21Ras by growth factors.