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Inhibition of Phosphatidylinositol 3-Kinase Enhances Mitogenic Actions of Insulin in Endothelial Cells*

Open AccessPublished:November 13, 2001DOI:https://doi.org/10.1074/jbc.M103728200
      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.
      NO
      nitric oxide
      PI 3-kinase
      phosphatidylinositol 3-kinase
      VCAM-1
      vascular cellular adhesion molecules-1
      eNOS
      endothelial nitric-oxide synthase
      HUVEC
      human umbilical vein endothelial cell
      IRS-1
      insulin receptor substrate-1
      MAP
      mitogen-activated protein kinase
      RPA
      RNase protection assay
      GGTase I
      geranylgeranyltransferase I
      FTase
      farnesyltransferase
      EBM
      endothelial basal media
      Insulin profoundly influences the function of the vascular endothelium (
      • Scherrer U.
      • Randin D.
      • Vollenweider P.
      • Vollenweider L.
      • Nicod P.
      ,
      • Shoemaker J.K.
      • Bonen A.
      ,
      • Abe H.
      • Yamada N.
      • Kamata K.
      • Kuwaki T.
      • Shimada M.
      • Osuga J.
      • Shionoiri F.
      • Yahagi N.
      • Kadowaki T.
      • Tanemoto H.
      • Ishibashi S.
      • Yazaki Y.
      • Makuuchi M.
      ,
      • Montagnani M.
      • Quon M.J.
      ,
      • Steinberg H.O.
      • Brechtel G.
      • Johnson A.
      • Fineberg N.
      • Baron A.D.
      ). In humans, physiological levels of insulin stimulate increased production of nitric oxide (NO)1 in the vasculature resulting in vasodilation and increased blood flow (
      • Scherrer U.
      • Randin D.
      • Vollenweider P.
      • Vollenweider L.
      • Nicod P.
      ,
      • Steinberg H.O.
      • Brechtel G.
      • Johnson A.
      • Fineberg N.
      • Baron A.D.
      ). Intriguingly, vasodilator actions of insulin are impaired in individuals who are also resistant to metabolic actions of insulin (
      • Steinberg H.O.
      • Chaker H.
      • Leaming R.
      • Johnson A.
      • Brechtel G.
      • Baron A.D.
      ). 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 vivostudies, 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 nitric-oxide synthase (eNOS) (
      • Zeng G.
      • Quon M.J.
      ,
      • Kuboki K.
      • Jiang Z.Y.
      • Takahara N.
      • Ha S.W.
      • Igarashi M.
      • Yamauchi T.
      • Feener E.P.
      • Herbert T.P.
      • Rhodes C.J.
      • King G.L.
      ,
      • Zeng G.
      • Nystrom F.H.
      • Ravichandran L.V.
      • Cong L.-N.
      • Kirby M.
      • Mostowski H.
      • Quon M.J.
      ). Activation of phosphatidylinositol 3-kinase (PI 3-kinase) is necessary to promote both increased expression and activity of eNOS in response to insulin (
      • Zeng G.
      • Quon M.J.
      ,
      • Kuboki K.
      • Jiang Z.Y.
      • Takahara N.
      • Ha S.W.
      • Igarashi M.
      • Yamauchi T.
      • Feener E.P.
      • Herbert T.P.
      • Rhodes C.J.
      • King G.L.
      ,
      • Zeng G.
      • Nystrom F.H.
      • Ravichandran L.V.
      • Cong L.-N.
      • Kirby M.
      • Mostowski H.
      • Quon M.J.
      ). Interestingly, PI 3-kinase is also a key signaling molecule mediating metabolic actions of insulin in adipose tissue and skeletal muscle (reviewed in Ref.
      • Shepherd P.R.
      • Withers D.J.
      • Siddle K.
      ). 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 (
      • Montagnani M.
      • Quon M.J.
      ).
      Recent studies (
      • Jiang Z.Y.
      • Lin Y.-W.
      • Clemont A.
      • Feener E.P.
      • Hein K.D.
      • Igarashi M.
      • Yamauchi T.
      • White M.F.
      • King G.L.
      ,
      • Cusi K.
      • Maezono K.
      • Osman A.
      • Pendergrass M.
      • Patti M.E.
      • Pratipanawatr T.
      • DeFronzo R.A.
      • Kahn C.R.
      • Mandarino L.J.
      ) 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 of the Ras/mitogen-activated protein kinase (MAP kinase)-dependent insulin signaling pathway. In fact, in many models of metabolic insulin resistance, insulin signaling via the IRS-1/PI 3-kinase pathway is impaired, whereas the MAP kinase pathway is unaffected (
      • Jiang Z.Y.
      • Lin Y.-W.
      • Clemont A.
      • Feener E.P.
      • Hein K.D.
      • Igarashi M.
      • Yamauchi T.
      • White M.F.
      • King G.L.
      ,
      • Cusi K.
      • Maezono K.
      • Osman A.
      • Pendergrass M.
      • Patti M.E.
      • Pratipanawatr T.
      • DeFronzo R.A.
      • Kahn C.R.
      • Mandarino L.J.
      ). Compensatory hyperinsulinemia resulting from insulin resistance may stimulate increased production of plasminogen activator inhibitor-1, endothelin, and various proliferative events in vascular smooth muscle cells via MAP kinase-dependent pathways (
      • Oliver F.J.
      • de la Rubia G.
      • Feener E.P.
      • Lee M.-E.
      • Loeken M.R.
      • Shiba T.
      • Quertermous T.
      • King G.L.
      ,
      • Hsueh W.A.
      • Law R.E.
      ,
      • Nordt T.K.
      • Bode C.
      ,
      • Hu R.M.
      • Levin E.R.
      • Pedram A.
      • Frank H.J.
      ,
      • Grenett H.E.
      • Benza R.I.
      • Li X.N.
      • Aikens M.L.
      • Grammer J.R.
      • Brown S.L.
      • Booyse F.M.
      ). We have demonstrated recently that the ability of insulin to increase the prenylation of Ras and Rho proteins is mediated via the Shc/MAP kinase pathway and is completely independent of PI 3-kinase activity (
      • Goalstone M.L.
      • Carel K.
      • Leitner J.W.
      • Draznin B.
      ,
      • Goalstone M.L.
      • Leitner J.W.
      • Berhanu P.
      • Sharma P.G.
      • Olefsky J.M.
      • Draznin B.
      ). Enhanced prenylation of these small molecular weight GTPases increases the mitogenic responsiveness of cells to a variety of growth factors.
      One might envision that hyperinsulinemia resulting from insulin resistance would drive intact Shc/MAP kinase-dependent pathways to increase prenylation of Ras and Rho leading to enhanced mitogenic responsiveness of endothelial cells. In the present study, we designed experiments to mimic the typical “insulin-resistant” state by blocking PI 3-kinase-dependent signaling and exposing cells to increased concentrations of insulin (“compensatory hyperinsulinemia”). The effects of insulin to stimulate prenylation of Ras and Rho proteins, expression of eNOS and adhesion molecules (VCAM-1 and E-selectin), and the interaction of leukocytes with the endothelium were examined in human umbilical vein endothelial cells (HUVEC). We found that inhibition of PI 3-kinase led to enhanced mitogenic actions of insulin in endothelial cells. Thus, we have identified an additional plausible mechanism for insulin resistance and hyperinsulinemia to contribute to vascular complications of diabetes.

      RESULTS

      We have shown previously that wortmannin completely inhibits the acute effect of insulin to stimulate production of NO in HUVEC (
      • Zeng G.
      • Quon M.J.
      ). In the present study we demonstrate that treatment of HUVEC with 50 nm wortmannin also fully inhibits the effect of insulin on the expression of eNOS in these cells (Fig.1). These experiments are consistent with previous results demonstrating that the effect of insulin to increase eNOS expression and activity in endothelial cells is mediated by the PI 3-kinase pathway (
      • Zeng G.
      • Quon M.J.
      ,
      • Kuboki K.
      • Jiang Z.Y.
      • Takahara N.
      • Ha S.W.
      • Igarashi M.
      • Yamauchi T.
      • Feener E.P.
      • Herbert T.P.
      • Rhodes C.J.
      • King G.L.
      ,
      • Zeng G.
      • Nystrom F.H.
      • Ravichandran L.V.
      • Cong L.-N.
      • Kirby M.
      • Mostowski H.
      • Quon M.J.
      ). In contrast, under similar conditions, the ability of insulin to stimulate the phosphorylation of MAP kinase remained unaffected (Fig. 2), that is insulin (10 nm) promoted comparable phosphorylation of MAP kinase both in control and the wortmannin-treated cells.
      Figure thumbnail gr1
      Figure 1Effect of insulin and wortmannin on the expression of eNOS in HUVEC. Insulin-stimulated induction of eNOS expression is blocked by wortmannin (Wort) in HUVEC. Cells starved in EBM-B for 14 h were treated without or with insulin (100 nm, 20 h) in the absence or presence of wortmannin (50 nm). One group was treated with wortmannin alone. Cell lysates (125 μg of total protein) were subjected to immunoprecipitation with anti-eNOS antibody. The samples were then separated by 8% SDS-PAGE followed by immunoblotting with anti-eNOS antibody. A representative blot from experiments that were repeated independently three times is shown. The relative levels of expression were quantified by scanning densitometry of the immunoblots. Results are the mean ± S.E. of three independent experiments expressed in arbitrary density units.
      Figure thumbnail gr2
      Figure 2Effect of insulin and wortmannin on the phosphorylation of MAP kinase in HUVEC. HUVEC were serum-starved for 24 h and then treated without or with insulin (10 nm) in the absence or presence of wortmannin (WORT) (50 nm) for 24 h. Cell lysates were immunoprecipitated with p44/42 MAP kinase antibodies. Samples were subjected to SDS-PAGE, and levels of phosphorylated MAP kinase were determined by Western blotting using phospho-p44/42 MAP kinase antibodies.
      We then examined the effect of insulin on the activity of farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I) in wortmannin-treated cells. In control HUVEC, insulin (10 nm) stimulated both prenyltransferases at 1 h of incubation with a return toward basal activity at 24 h (Fig.3, A and B). In the wortmannin-treated cells, the effect of insulin on FTase was significantly increased at 24 h (Fig. 3A). A pattern of activation of GGTase I was somewhat different in the wortmannin-treated HUVEC. The effect of insulin was significantly greater both at 1 and 24 h of incubation (Fig. 3B). As expected, increased activity of the prenyltransferases was followed by the corresponding increases in the amounts of farnesylated p21Ras and geranylgeranylated Rho-A in wortmannin-treated cells exposed to either 10 or 100 nm insulin for 24 h (Fig.4).
      Figure thumbnail gr3
      Figure 3Effect of insulin and wortmannin on the activity of FTase (A) and GGTase I (B) in HUVEC. HUVEC were serum-starved for 24 h and then stimulated with insulin (10 nm) for 1 or 24 h, without (solid lines) or with (broken lines) pretreatment with wortmannin (100 nm, added 30 min before insulin). FTase and GGTase-I activities were determined as described previously (
      • Goalstone M.L.
      • Carel K.
      • Leitner J.W.
      • Draznin B.
      ,
      • Goalstone M.L.
      • Leitner J.W.
      • Berhanu P.
      • Sharma P.G.
      • Olefsky J.M.
      • Draznin B.
      ,
      • Goalstone M.L.
      • Natarajan P.
      • Standley P.R.
      • Walsh M.F.
      • Leitner J.W.
      • Carel K.
      • Scott S.
      • Nadler J.
      • Sowers J.R.
      • Draznin B.
      ,
      • Goalstone M.L.
      • Leitner J.W.
      • Wall K.
      • Dolgonos L.
      • Rother K.I.
      • Accili D.
      • Draznin B.
      ,
      • Golovchenko I.
      • Goalstone M.
      • Watson P.
      • Brownlee M.
      • Draznin B.
      ,
      • Chappell J.
      • Golovchenko I.
      • Wall K.
      • Stjernholm R.
      • Leitner J.
      • Goalstone M.
      • Draznin B.
      ). Results are expressed as mean ± S.E. (n = 4). *, p < 0.05versus insulin alone.
      Figure thumbnail gr4
      Figure 4Effect of insulin and wortmannin on the amounts of prenylated p21Ras and Rho-A. HUVEC were serum-starved for 24 h and then stimulated with insulin (10 and 100 nm) for 24 h (A and B) without or with pretreatment with wortmannin (Wort) (100 nm, added 30 min before insulin). Amounts of prenylated p21Ras (A) and Rho-A (B) were determined as described previously (
      • Goalstone M.L.
      • Carel K.
      • Leitner J.W.
      • Draznin B.
      ,
      • Goalstone M.L.
      • Leitner J.W.
      • Berhanu P.
      • Sharma P.G.
      • Olefsky J.M.
      • Draznin B.
      ,
      • Goalstone M.L.
      • Natarajan P.
      • Standley P.R.
      • Walsh M.F.
      • Leitner J.W.
      • Carel K.
      • Scott S.
      • Nadler J.
      • Sowers J.R.
      • Draznin B.
      ,
      • Goalstone M.L.
      • Leitner J.W.
      • Wall K.
      • Dolgonos L.
      • Rother K.I.
      • Accili D.
      • Draznin B.
      ,
      • Golovchenko I.
      • Goalstone M.
      • Watson P.
      • Brownlee M.
      • Draznin B.
      ,
      • Chappell J.
      • Golovchenko I.
      • Wall K.
      • Stjernholm R.
      • Leitner J.
      • Goalstone M.
      • Draznin B.
      ). Results are expressed as mean ± S.E. *, p < 0.05 versuscontrols; #, p < 0.05 versus insulin alone.
      We have shown previously that increased availability of farnesylated p21Ras augmented the mitogenic effectiveness of other growth factors, such as platelet-derived growth factor, epidermal growth factor, and insulin-like growth factor-1 in various cells (
      • Goalstone M.L.
      • Natarajan P.
      • Standley P.R.
      • Walsh M.F.
      • Leitner J.W.
      • Carel K.
      • Scott S.
      • Nadler J.
      • Sowers J.R.
      • Draznin B.
      ,
      • Goalstone M.L.
      • Leitner J.W.
      • Wall K.
      • Dolgonos L.
      • Rother K.I.
      • Accili D.
      • Draznin B.
      ). Similarly, cells with increased availability of geranylgeranylated Rho-A also displayed increased mitogenic responsiveness to agents working via the Rho pathway (
      • Golovchenko I.
      • Goalstone M.
      • Watson P.
      • Brownlee M.
      • Draznin B.
      ,
      • Chappell J.
      • Golovchenko I.
      • Wall K.
      • Stjernholm R.
      • Leitner J.
      • Goalstone M.
      • Draznin B.
      ). Therefore, we hypothesized that increased availability of farnesylated p21Ras and geranylgeranylated Rho-A in HUVEC might increase the responsiveness of these cells to other growth-promoting agents. To investigate this point, we examined the effect of insulin, in the presence of wortmannin, on the vascular endothelial growth factor (VEGF)-induced expression of the adhesion molecules, VCAM-1 and E-selectin. As expected, VEGF significantly increased the expression of VCAM-1 and E-selectin in control cells (Fig. 5). Insulin alone did not exert any significant effect on the expression of mRNA of these adhesion molecules. Nevertheless, in concert with previously published observations (
      • Aljada A.
      • Saadeh R.
      • Assian E.
      • Chanim H.
      • Dandona P.
      ,
      • Kim I.
      • Moon S.-O.
      • Kim S.H.
      • Kim H.J.
      • Koh Y.S.
      • Koh G.Y.
      ), it significantly attenuated the effect of VEGF (Fig. 5). However, in the presence of wortmannin, insulin lost its attenuating effect and potentiated the effect of VEGF on the expression of VCAM-1 and E-selectin (Fig. 5).
      Figure thumbnail gr5
      Figure 5Effect of insulin, VEGF, and wortmannin on the expression of VCAM-1 and E-selectin mRNA in HUVEC. RNase protection assay of VCAM-1 and E-selectin mRNAs in insulin- and VEGF-stimulated HUVEC pretreated without or with wortmannin.A, HUVEC were incubated with insulin (100 nm) or VEGF-165 (20 ng/ml) for 4 h in the absence or presence of wortmannin (WT, 30 nm). Total RNA (10 μg) isolated from the cells was subjected to RPA as described under “Experimental Procedures.” B, densitometric analyses are presented as the relative ratio of VCAM-1 or E-selectin mRNA to cyclophilin mRNA. Results were similar in three independent experiments and expressed as the mean ± S.D. from three experiments. *, p < 0.05 versus control buffer; +, p < 0.05 versus control buffer plus insulin (100 nm); #, p < 0.05versus control buffer plus VEGF (20 ng/ml).
      We then addressed the functional implications of an increased expression of the adhesion molecules. Because the process of monocyte adhesion to endothelium begins with rolling interactions and progresses to a complete stop, we assessed these two phases of monocyte interaction with the insulin-treated endothelial cells in the presence of wortmannin (Fig. 6). The experiments were performed in a flow chamber under a low shear stress (0.3 dynes/cm2). We found that rolling interactions of monocytes with the endothelial cells in response to insulin were significantly increased by the blockade of PI 3-kinase (Fig. 6A). In addition, under these conditions, leukocytes were more likely to come to complete stops than in control experiments or in the presence of either insulin or wortmannin alone (Fig. 6B). Thus, the two essential early steps required for monocyte adhesion to endothelium (i.e. initial rolling and arrest of motion) were significantly increased when the cells were treated with insulin in the presence of wortmannin.
      Figure thumbnail gr6
      Figure 6Effect of insulin- and wortmannin-treated endothelial cells on monocyte adhesion and arrest.Insulin/wortmannin treatment of endothelial cells increases adhesion of Wehi 274.1 monocytes under flow. A, rolling cells/min. Wehi 274.1 monocytes were injected into a laminar flow chamber at 106 cells/ml at the shear stress of 0.3 dynes/cm2. Images were collected at 30 frames/s and analyzed to count the number of cells/min that formed rolling adhesions to localized endothelial cells. Endothelial cells were either untreated or treated with 100 nm wortmannin, 10 nminsulin, and a combination of both for 24 h. B, number of cell arrests/min. In the same experiments, recorded digital images were examined to determine the number of cells that arrested (adhered and stopped completely) per min on localized endothelial cells. Three independent experiments were performed. Results are expressed as mean ± S.E. *, p < 0.05 versuscontrol (A) and <0.03 versus all other treatments (B). #, p = 0.05versus other treatments (A).

      DISCUSSION

      Investigators exploring contributions of insulin resistance and the resulting compensatory hyperinsulinemia to the pathogenesis of vascular diseases that accompany the metabolic syndrome X must reconcile an apparent paradox. On the one hand, insulin resistance signifies impaired insulin action. On the other hand, hyperinsulinemia raises the possibility of increased insulin action in certain contexts. For example, in endothelial cells, diminished insulin action secondary to acquired or inherited insulin resistance readily explains the impaired ability of insulin to stimulate production of NO and normal vasodilation (
      • Montagnani M.
      • Quon M.J.
      ,
      • Steinberg H.O.
      • Brechtel G.
      • Johnson A.
      • Fineberg N.
      • Baron A.D.
      ,
      • Steinberg H.O.
      • Chaker H.
      • Leaming R.
      • Johnson A.
      • Brechtel G.
      • Baron A.D.
      ). At the same time, hyperinsulinemia may cause overproduction of endothelin-1 and plasminogen activator inhibitor-1 by endothelial cells that contribute to vascular disease (
      • Oliver F.J.
      • de la Rubia G.
      • Feener E.P.
      • Lee M.-E.
      • Loeken M.R.
      • Shiba T.
      • Quertermous T.
      • King G.L.
      ,
      • Hsueh W.A.
      • Law R.E.
      ,
      • Nordt T.K.
      • Bode C.
      ,
      • Hu R.M.
      • Levin E.R.
      • Pedram A.
      • Frank H.J.
      ,
      • Grenett H.E.
      • Benza R.I.
      • Li X.N.
      • Aikens M.L.
      • Grammer J.R.
      • Brown S.L.
      • Booyse F.M.
      ). An unresolved question is how pathological responses related to either insulin resistance or hyperinsulinemia can occur simultaneously in the same cell. One potential explanation for this important pathophysiological conundrum was suggested by the demonstration that insulin resistance along the IRS-1/PI 3-kinase pathway of insulin signaling does not necessarily coincide with resistance in other signaling pathways. In fact, the Ras-MAP kinase signaling pathway retains normal sensitivity to insulin in both humans and animals who are resistant to the metabolic actions of insulin (
      • Jiang Z.Y.
      • Lin Y.-W.
      • Clemont A.
      • Feener E.P.
      • Hein K.D.
      • Igarashi M.
      • Yamauchi T.
      • White M.F.
      • King G.L.
      ,
      • Cusi K.
      • Maezono K.
      • Osman A.
      • Pendergrass M.
      • Patti M.E.
      • Pratipanawatr T.
      • DeFronzo R.A.
      • Kahn C.R.
      • Mandarino L.J.
      ).
      We have demonstrated recently (
      • Goalstone M.L.
      • Wall K.
      • Leitner J.W.
      • Kurowski T.
      • Ruderman N.
      • Pan S.J.
      • Ivy J.L.
      • Moller D.E.
      • Draznin B.
      ,
      • Draznin B.
      • Miles P.
      • Kruszynska Y.
      • Olefsky J.
      • Friedman J.
      • Golovchenko I.
      • Stjernholm R.
      • Wall K.
      • Reitman M.
      • Accili D.
      • Cooksey R.
      • McClain D.
      • Goalstone M.
      ) that the effect of insulin to stimulate prenylation of Ras and Rho proteins is actually increased in tissues of insulin-resistant humans and animals. Insulin promotes the phosphorylation of the α-subunit of FTase and GGTase I and stimulates the activities of both prenyltransferases (
      • Goalstone M.L.
      • Carel K.
      • Leitner J.W.
      • Draznin B.
      ,
      • Goalstone M.L.
      • Leitner J.W.
      • Berhanu P.
      • Sharma P.G.
      • Olefsky J.M.
      • Draznin B.
      ,
      • Goalstone M.L.
      • Natarajan P.
      • Standley P.R.
      • Walsh M.F.
      • Leitner J.W.
      • Carel K.
      • Scott S.
      • Nadler J.
      • Sowers J.R.
      • Draznin B.
      ,
      • Goalstone M.L.
      • Leitner J.W.
      • Wall K.
      • Dolgonos L.
      • Rother K.I.
      • Accili D.
      • Draznin B.
      ,
      • Golovchenko I.
      • Goalstone M.
      • Watson P.
      • Brownlee M.
      • Draznin B.
      ,
      • Chappell J.
      • Golovchenko I.
      • Wall K.
      • Stjernholm R.
      • Leitner J.
      • Goalstone M.
      • Draznin B.
      ). Even though stimulatory effects of insulin on prenyltransferases and the amounts of prenylated Ras and Rho proteins are relatively small (15–30%, see Figs. 3 and 4), they are of high physiological significance, for they result in a 2-fold potentiation of action of other growth-promoting agents working via the Ras- and Rho-dependent pathways (
      • Goalstone M.L.
      • Leitner J.W.
      • Berhanu P.
      • Sharma P.G.
      • Olefsky J.M.
      • Draznin B.
      ,
      • Goalstone M.L.
      • Natarajan P.
      • Standley P.R.
      • Walsh M.F.
      • Leitner J.W.
      • Carel K.
      • Scott S.
      • Nadler J.
      • Sowers J.R.
      • Draznin B.
      ,
      • Goalstone M.L.
      • Leitner J.W.
      • Wall K.
      • Dolgonos L.
      • Rother K.I.
      • Accili D.
      • Draznin B.
      ,
      • Golovchenko I.
      • Goalstone M.
      • Watson P.
      • Brownlee M.
      • Draznin B.
      ,
      • Chappell J.
      • Golovchenko I.
      • Wall K.
      • Stjernholm R.
      • Leitner J.
      • Goalstone M.
      • Draznin B.
      ).
      Although insulin at high concentrations may act through the receptors for insulin-like growth factor-1 (
      • Sowers J.R.
      ), we have demonstrated that the effect of insulin on the prenyltransferases is not mimicked by other growth factors, requires the presence of an intact insulin receptor, and is mediated via the Shc-MAP kinase pathway (
      • Goalstone M.L.
      • Leitner J.W.
      • Berhanu P.
      • Sharma P.G.
      • Olefsky J.M.
      • Draznin B.
      ,
      • Goalstone M.L.
      • Leitner J.W.
      • Wall K.
      • Dolgonos L.
      • Rother K.I.
      • Accili D.
      • Draznin B.
      ). Analogous to congenital adrenal hyperplasia, where the influence of ACTH is directed along an unaffected branch of steroidogenesis, we postulate that insulin signaling via the MAP kinase pathway is unaffected (and its signaling to the prenyltransferases is actually increased) in the presence of insulin resistance along the PI 3-kinase pathway. Thus, metabolic insulin resistance caused by impaired PI 3-kinase pathways may result in compensatory hyperinsulinemia that increases the activity of the prenyltransferases through unopposed Shc-MAP kinase pathways (Fig. 7).
      Figure thumbnail gr7
      Figure 7Model of insulin resistance in endothelium and its potential role in the pathogenesis of atherosclerosis. In the presence of insulin resistance, where the PI 3-kinase-dependent (metabolic) branch of insulin signaling is blocked, the mitogenic branch is normal and insulin signaling to the prenyltransferases is augmented. Insulin signaling to prenyltransferases flows via activation of Shc and MAP kinase (
      • Goalstone M.L.
      • Leitner J.W.
      • Berhanu P.
      • Sharma P.G.
      • Olefsky J.M.
      • Draznin B.
      ). Greater availability of prenylated Ras and Rho proteins and decreased production of NO in endothelial cells potentiate the effects of other growth factors leading to increased expression of adhesion molecules.f-p21 Ras, farnesylated p21Ras;ggRho-A, geranylgeranylated Rho-A.
      Our current experiments strongly support our hypothesis and demonstrate an increased responsiveness of HUVEC to VEGF in the presence of an experimental paradigm mimicking the state of metabolic insulin resistance and compensatory hyperinsulinemia. In vascular endothelial cells, blockade of the PI 3-kinase signaling pathway with wortmannin increased the ability of insulin to augment the activities of FTase and GGTase I as well as the amounts of prenylated p21Rasand Rho-A. Concomitantly, the effects of VEGF on the production of VCAM-1 and E-selectin in HUVEC were increased, and the endothelial cells attracted significantly greater numbers of mononuclear cells (Fig. 6). Because insulin-stimulated production of NO is a PI 3-kinase-mediated event (
      • Zeng G.
      • Quon M.J.
      ,
      • Zeng G.
      • Nystrom F.H.
      • Ravichandran L.V.
      • Cong L.-N.
      • Kirby M.
      • Mostowski H.
      • Quon M.J.
      ), our results are also consistent with a recent study showing that the effect of insulin to inhibit expression of ICAM-1 in endothelial cells may be dependent on production of NO (
      • Aljada A.
      • Saadeh R.
      • Assian E.
      • Chanim H.
      • Dandona P.
      ).
      Increased expression of cellular adhesion molecules, including ICAM-1, VCAM-1, and E-selectin, is believed to be among the earliest steps in the process of atherogenesis (
      • Ross R.
      ). These adhesion molecules are not only expressed in the endothelial cells but are also released into the circulation via either shedding or an alternative pathway (
      • Gearing A.J.
      • Neuman W.
      ). Increased levels of circulating adhesion molecules are correlated with future coronary events (
      • Shyer K.G.
      • Chang H.
      • Lin C.C.
      • Kuan P.
      ,
      • Wallen N.H.
      • Held C.
      • Rehnquist N.
      • Hjemdahl P.
      ,
      • Ridker P.M.
      • Hennekens C.H.
      • Roitman-Johnson B.
      • Stampfer M.J.
      • Allen J.
      ). Similarly, increased levels of circulating adhesion molecules are consistently found in patients with type 2 diabetes and other conditions associated with insulin resistance (reviewed in Refs.
      • Chen N.-G.
      • Holmes M.
      • Reaven G.M.
      and
      • Kado S.
      • Nagata N.
      ). Furthermore, in healthy volunteers, Chenet al. (
      • Chen N.-G.
      • Holmes M.
      • Reaven G.M.
      ) have found a significant correlation between the degree of insulin resistance and circulating concentrations of E-selectin, ICAM-I, and VCAM-1, that is the most insulin-resistant individuals displayed the highest concentrations of all three soluble adhesion molecules (
      • Chen N.-G.
      • Holmes M.
      • Reaven G.M.
      ). Our findings that insulin (with or without VEGF) can potentiate the expression of these adhesion molecules when the metabolic (PI 3-kinase-dependent) branch of insulin signaling is inhibited may provide a molecular explanation for the increased cellular expression and circulating concentrations of adhesion molecules observed in insulin-resistant states.
      The incidence of atherosclerosis is much higher in patients with type 2 diabetes than in the general population. However, much remains to be learned about the relationship between the pathophysiology of these diseases. It is well known that monocyte adhesion to endothelium is an essential early event in the development of the atherosclerotic plaque (
      • Prescott S.M.
      • McIntyre T.M.
      • Zimmerman G.A.
      ,
      • Glass C.K.
      • Witztum J.L.
      ). Nevertheless, little is known about the effects of hyperinsulinemia in the presence of insulin resistance on the adhesion process itself. We have addressed this topic using a functional assay to test adhesion of the monocyte cell line Wehi 274.1 to cultured endothelium under stress. Our data clearly demonstrate an increase in both rolling adhesion and monocyte arrest when cells were treated with insulin in the presence of wortmannin. These effects were especially pronounced at low shear stresses (commonly associated with areas that are prone to atherosclerotic plaque formation). Thus, concomitant insulin resistance and hyperinsulinemia may be an important contributor to increased adhesivity of arterial endothelial cells and other early events in the development of atherosclerosis.
      Insulin activated both FTase and GGTase I in HUVEC (Fig. 3). Further studies are needed to determine the precise aspects of VEGF action that are potentiated by the greater availability of farnesylated p21Ras as compared with the greater availability of geranylgeranylated Rho-A. VEGF action is initiated by its interaction with several transmembrane tyrosine kinase receptors, including flt-1 and flk-1 (
      • de Vries C.
      • Escobedo J.A.
      • Ueno H.
      • Houck K.
      • Ferrara N.
      • Williams L.T.
      ,
      • Waltenberger J.
      • Claesson-Welsh L.
      • Siegbahn A.
      • Shibuya M.
      • Heldin C.H.
      ). Both PI 3-kinase and MAP kinase have been shown to participate in VEGF signaling cascades (
      • Guo D.
      • Jia Q.
      • Song H.-Y.
      • Warren R.S.
      • Donner D.B.
      ,
      • Pedram A.
      • Razandi M.
      • Levin E.R.
      ). Recent studies (
      • Kim I.
      • Moon S.-O.
      • Kim S.H.
      • Kim H.J.
      • Koh Y.S.
      • Koh G.Y.
      ) indicate that effects of VEGF on expression of adhesion molecules involve nuclear factor κB (NFκB). Interestingly, activation of NFκB is dependent upon the availability of Rho-A (
      • Montaner S.
      • Perona R.
      • Saniger L.
      • Lacal J.C.
      ). In fact, our own studies have demonstrated that the ability of insulin to potentiate transactivation of NFκB by angiotensin II, hyperglycemia, and advance glycosylation end products is Rho-A-dependent (
      • Golovchenko I.
      • Goalstone M.
      • Watson P.
      • Brownlee M.
      • Draznin B.
      ). Thus, activation of prenyltransferases by insulin is likely to augment the vascular actions of VEGF as well.
      In summary, we show that in an in vitro model of metabolic insulin resistance with hyperinsulinemia, insulin is unable to stimulate eNOS expression but is able to increase production of adhesion molecules in the same endothelial cells (possibly via enhanced prenylation). We postulate that insulin resistance in vivosimultaneously results in decreased NO production and compensatory hyperinsulinemia that augments prenylation of Ras and Rho proteins. This may contribute to the increased responsiveness of endothelial cells to atherogenic actions of VEGF and other growth factors.

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