Inositol 1,4,5-trisphosphate mass changes from fertilization through first cleavage in Xenopus laevis.

After fertilization in Xenopus laevis, inositol 1,4,5-trisphosphate (IP3) mass increased from 53 to 261 fmol/cell and returned to near basal by 10 min after insemination. IP3 was also elevated over control egg levels during first mitosis and first cleavage. Because IP3 levels and the fertilization calcium wave decline at about the same time and because calcium ionophore or pricking the egg increased IP3, the fertilization calcium wave may be due to calcium-induced IP3 production. In addition, the onset of sperm motility was associated with an increase, whereas the acrosomal reaction was accompanied by a decrease in IP3 mass. Combining our published data with this report, the first chronology of the levels of IP3 from the induction of meiosis (maturation) through fertilization and cleavage in one cellular system is summarized. These data suggest an in vivo dose response for IP3 and calcium release. A small (17 fmol/cell) IP3 change during the induction of meiosis may not be associated with a calcium change. Larger IP3 changes at cleavage (40 fmol/cell) and mitosis (125 fmol/cell) are associated with localized small calcium increases, whereas the largest IP3 change (208 fmol/cell) is associated with the large calcium increase at fertilization.

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.

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.

Protein kinase C initially inhibits the induction of meiotic cell division in Xenopus oocytes.

We have used one activator and two inhibitors of protein kinase C (PKC) to examine the role of this enzyme in the induction of meiotic cell division. At 1 U/ml, phosphatidylcholine-specific phospholipase C increases DAG, alters intracellular pH and inhibits the induction of meiosis by insulin or progesterone. However, when added about 1.6 h after progesterone, the enzyme speeds the induction of cell division. Microinjection of inhibitor peptide (19-36) of PKC has little effect on progesterone action but stimulates the induction of meiosis by insulin. When the inhibitor peptide is injected about 2h after insulin addition, the peptide inhibits. A second PKC inhibitor, staurosporine, decreases PKC-dependent intracellular pH and in vitro oocyte PKC activity. At similar concentrations, staurosporine stimulates insulin or progesterone action, but, when added after about 2 h, the drug inhibits induction by insulin. We conclude that PKC is initially inhibitory to the induction of meiotic cell division but then may become synergistic.

Insulin and progesterone increase 32PO4-labeling of phospholipids and inositol 1,4,5-trisphosphate mass in Xenopus oocytes.

After a 4-6 h induction period, insulin or progesterone induces Xenopus oocytes to enter prophase of meiosis. During the period of induction, both insulin and progesterone induced an increase in 32PO4 labeling of phosphatidylcholine and phosphatidylinositol. Through a mass assay, we found that insulin and progesterone increase inositol 1,4,5-trisphosphate (IP3) at about 15-30 s, 15 min and at about 2-3 h (0.5 GVBD50) after hormone addition. Since IP3 increases were small (from a basal of 66 to 104 nM), the results agree with prior conclusions that progesterone does not induce a large, cytosolic calcium elevation. Insulin is probably acting through the insulin-like growth factor-1 receptor as insulin concentrations greater than about 50 nM are required to increase IP3.

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.

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.

The antidiabetic drug metformin elevates receptor tyrosine kinase activity and inositol 1,4,5-trisphosphate mass in Xenopus oocytes.

Although metformin is an important antidiabetic, its mechanism of action is still unknown. To study its mechanism, we examined metformin stimulation of insulin action on the Xenopus oocyte. Similar to therapeutic concentrations, maximal stimulation of insulin-induced meiotic cell division was achieved at about 1-10 microg/ml (or 7.7-77 /microM) metformin. An equivalent concentration of metformin was required to elevate receptor tyrosine kinase activity (in whole cells or a membrane-cortex preparation) and, through this tyrosine kinase activation, inositol 1,4,5-trisphosphate (IP3) production. With whole cells, the preincubation time for metformin stimulation of insulin action (approximately 1 h) was equivalent to the time required for metformin to maximize tyrosine phosphorylation and raise IP3, levels. With the membrane-cortex preparation, metformin was active within minutes; thus, metformin may act at an intracellular site. Since metformin can increase IP3, mass, we prevented elevation of calcium by prior microinjection of a calcium chelator or heparin (a drug that inhibits IP3 binding to the IP3 receptor). Both the chelator and heparin blocked metformin stimulation of insulin action on whole cells. Since microinjection of IP3, also stimulates insulin action, metformin may stimulate insulin action by elevation of intracellular calcium in addition to activation of the receptor tyrosine kinase.

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.

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.

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 activity in 3T3-L1 adipocytes.

Activation of p21(ras) by GTP loading is a critical step in a cascade of intracellular insulin signaling. Farnesylation of p21(ras) protein is an obligatory event that facilitates Ras migration to the plasma membrane and subsequent activation. Farnesyltransferase (FTase) is a ubiquitous enzyme that catalyzes the lipid modification of p21(ras) by the addition of farnesyl to the C-terminal "CAAX" motif. In vitro and in vivo FTase activities were studied in 3T3-L1 adipocytes in response to insulin challenge. Insulin exerted a biphasic stimulatory effect on FTase activity measured in vitro with a 31% increase at 5 min and a 130% increase at 60 min. Insulin-stimulated farnesylation of p21(ras) pools in vivo correlated with FTase activity seen in vitro by displaying an increase in farnesylated p21(ras) from 40% of total cellular Ras in control cells to 63% by 5 min and 80% by 60 min (p < 0.05) in insulin-treated cells. Insulin challenge of 3T3-L1 adipocytes increased incorporation of tritiated mevalonic acid in p21(ras) in a dose-dependent manner and stimulated a 2-fold increase in phosphorylation of the alpha-subunit of FTase at 5 min and a 4-fold increase at 60 min.

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.

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.

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.

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.

Characterization of Xenopus laevis oocyte farnesyl transferase.

Farnesyl transferase (FTase) activity, characterized in extracts of Xenopus laevis oocytes using an in vitro filtration assay, was observed to be at least 80% cytosolic with optimal product formation at pH 7.0. Oocyte FTase displayed enzyme activity that was specific for the Ras-CVIM construct but not Ras-CAIL or Ras-SVLS. Labeling of Ras-CVIM using [3H]farnesyl pyrophosphate was inhibited by addition of unlabeled farnesyl pyrophosphate to the assay mixture but not by addition of either geranylgeranyl pyrophosphate or stearic acid. Polyclonal rabbit antibodies against recombinant human FTase subunits were tested for cross-reactivity with oocyte proteins and were used to determine the apparent molecular masses of oocyte FTase enzyme subunits. Anti-alpha subunit antibodies labeled a band of approximately 53 kDa. Anti-beta subunit antibodies bound both a high molecular mass band (140 kDa) and a lower molecular mass band (38 kDa) on immunoblots of cytosolic oocyte proteins. When oocyte FTase activity was resolved by gel filtration, the peak of enzyme activity correlated with immunoblot detection of the 38- and 53-kDa bands as well as a doublet of 86- and 95-kDa. This correlation suggests that Xenopus laevis oocyte FTase is a heterodimer under reducing conditions with subunits of 53 +/- 1 and 38 +/- 2 kDa, and that the holoenzyme migrates on SDS-polyacrylamide gels with an apparent molecular mass of 86-95 kDa.

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.

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.

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.