Effect of dietary macronutrient composition on AMPK and SIRT1 expression and activity in human skeletal muscle.

Adenosine monophosphate-activated protein kinase (AMPK), silent mating type information regulation 2 homologue 1 (SIRT 1), and peroxisome proliferator-activated receptor γ co-activator α (PGC1α) constitute an energy sensing cellular network that controls mitochondrial biogenesis. Caloric restriction activates both AMPK and SIRT-1 to increase ATP production from fat oxidation. We characterized AMPK and SIRT 1 expression and activity in human skeletal muscle in response to dietary fat or carbohydrate intake on the background of either overfeeding or caloric restriction. AMPK phosphorylation and acetylation of PGC1α (as a measure of SIRT activity) were determined. Euglycemic-hyperinsulinemic clamp and muscle biopsies were performed in human subjects participating in 2 separate studies. In study 1, 21 lean healthy individuals were overfed for 5 days, while in study 2, 18 obese otherwise healthy individuals consumed a calorie-restricted diet for 5 days. Under both conditions - overfeeding and caloric restriction - high fat/low carbohydrate (HF/LC) diet significantly increased phosphorylation of AMPK and deacetylation of PGC1α in skeletal muscle without affecting total amounts of AMPK, PGC1α, or SIRT 1. In contrast, low fat/high carbohydrate (LF/HC) hypocaloric diet reduced phosphorylation of AMPK and deacetylation of PGC1α. Our data indicate that a relative deficiency in carbohydrate intake or, albeit less likely, a relative excess of fat intake even in the absence of caloric deprivation is sufficient to activate the AMPK-SIRT 1-PGC1α energy-sensing cellular network in human skeletal muscle.

Mitogenic action of insulin: friend, foe or 'frenemy'?

Either endogenous or exogenous hyperinsulinaemia in the setting of insulin resistance promotes phosphorylation and activation of farnesyltransferase, a ubiquitous enzyme that farnesylates Ras proteins. Increased availability of farnesylated Ras at the plasma membrane enhances mitogenic responsiveness of cells to various growth factors, thus contributing to progression of cancer and atherosclerosis. This effect is specific to insulin, but is not related to the type of insulin used. The stimulatory effect of hyperinsulinaemia on farnesyltransferase in the presence of insulin resistance represents one potential mechanism responsible for mitogenicity and atherogenicity of insulin.

In vivo knockdown of p85alpha with an antisense oligonucleotide improves insulin sensitivity in Lep(ob/ob) and diet-induced obese mice.

Phosphoinositide 3-kinase is a key signaling intermediate necessary for the metabolic actions of insulin. In this study, we assessed the effects of in vivo knockdown of the p85alpha subunit of phosphoinositide 3-kinase on insulin sensitivity, using an antisense oligonucleotide, in lean mice, diet-induced obese mice, and obese leptin-deficient Lep (ob/ob) mice. Mice were injected with either p85alpha-targeted antisense oligonucleotide or saline twice weekly for 4 weeks. Fasting levels of glycemia and insulinemia and insulin and glucose tolerance tests were used to determine insulin sensitivity. Western blot analysis and real-time polyacrylamide chain reaction were used to assess p85alpha protein and mRNA expression. IN VIVO administration of antisense oligonucleotide resulted in 50 and 60% knockdown of liver p85alpha protein and mRNA, respectively, in the lean, diet-induced obese and Lep (ob/ob) mice. This was associated with increased phosphoinositide 3-kinase activity and improved insulin sensitivity in diet-induced obese and Lep (ob/ob) mice. Thus, p85alpha could be an important therapeutic target to ameliorate insulin resistance.

Discordance between intramuscular triglyceride and insulin sensitivity in skeletal muscle of Zucker diabetic rats after treatment with fenofibrate and rosiglitazone.

AIM: Intramyocellular triglyceride (IMTG) correlates with insulin resistance, but there is no clear causal relationship. Insulin resistance and associated hyperinsulinaemia may increase IMTG, via the insulin-regulated transcription factor, sterol regulatory element-binding protein 1 (SREBP-1). PPAR agonists may also affect IMTG via changes in insulin sensitivity, SREBP-1 or other factors. METHODS: We examined skeletal muscle IMTG and SREBP-1 expression, and metabolic parameters in Zucker diabetic fatty rats (ZDF) after 25 weeks of PPAR-gamma or PPAR-alpha administration. RESULTS: Compared with Zucker lean rats (ZL), untreated ZDF had significantly higher weights, serum glucose, insulin, free fatty acids, total cholesterol and triglycerides. IMTG and SREBP-1c messenger RNA (mRNA) were also higher in untreated ZDF; both were decreased by fenofibrate (FF). Rosiglitazone (Rosi), despite marked improvement in glycaemia, hyperinsulinaemia and hyperlipidaemia, failed to affect SREBP-1 expression, and increased body weight and IMTG. Rosi/FF combination caused less weight gain and no IMTG increase, despite metabolic effects similar to Rosi alone. CONCLUSIONS: IMTG and SREBP-1c mRNA are high in the ZDF. FF and Rosi both improved insulin sensitivity but had opposite effects on IMTG. Thus, there was a clear discordance between insulin sensitivity and IMTG with PPAR agonists, indicating that IMTG and insulin sensitivity do not share a simple relationship.

Nutritional upregulation of p85alpha expression is an early molecular manifestation of insulin resistance.

AIMS/HYPOTHESIS: We sought to define early molecular alterations associated with nutritionally induced insulin resistance in humans. METHODS: Insulin sensitivity was assessed using a hyperinsulinaemic-euglycaemic clamp in eight healthy women while on an isocaloric diet and after 3 days of overfeeding (50% above eucaloric diet). Expression of phosphatidylinositol (PI) 3-kinase subunits p85alpha and p110 was assessed and measurements were made of IRS-1-associated PI 3-kinase activity, tyrosine and serine phosphorylation of IRS-1, and serine and threonine phosphorylation of p70S6 kinase. Measurements were made in skeletal muscle biopsies obtained before and after overfeeding. RESULTS: Three days of overfeeding resulted in a reduction of insulin sensitivity accompanied by: (1) increased expression of skeletal muscle p85alpha; (2) an alteration in the ratio of p85alpha to p110; (3) a decrease in the amount of IRS-1-associated p110; and (4) a decrease in PI 3-kinase activity. Increases in expression of p85alpha and in the p85alpha:p110 ratio demonstrated a highly significant inverse correlation with insulin sensitivity, and changes in PI 3-kinase activity correlated with changes in insulin sensitivity. Tyrosine and serine phosphorylation of IRS-1 and serine and threonine phosphorylation of p70S6 kinase were unaffected by 3 days of overfeeding. CONCLUSIONS/INTERPRETATION: We identified a novel mechanism of nutritionally induced insulin resistance in healthy women of normal weight. We conclude that increased expression of p85alpha may be one of the earliest molecular alterations in the mechanism of the insulin resistance associated with overfeeding.

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.

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.

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.

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.

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.

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. 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.

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, cation metabolism and insulin resistance.

There is considerable evidence that insulin and insulin-like growth factors regulate a number of important physiological functions in a variety of tissues, some not considered to be classically insulin sensitive. Impaired biological responses to insulin and related insulin-like growth factors are referred to as insulin resistance. Persons with insulin resistance often display clinical abnormalities other than impaired glucose tolerance, including central obesity, hypertension, dyslipidemia, microalbuminuria, and abnormal coagulation and fibrinolytic systems. The mechanisms leading to development of insulin resistance are not fully understood. However, in addition to abnormalities of phosphorylation processes, it appears that alterations in cellular cation metabolism contribute to diminished cellular actions of insulin (i.e., glucose transport and hemodynamic actions). This review focuses on known cellular cation abnormalities and associated insulin resistance and cardiovascular disease.

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.