Background: The clinical metabolic syndrome (Met Syn) is associated with systemic insulin resistance and with increased mass and dysfunction of subcutaneous (SAT) and visceral adipose tissue. Accumulation and dysfunction of epicardial adipose tissue (EAT) have been associated with cardiovascular risk in Met Syn. We previously characterized a porcine dietary model of Met Syn that demonstrates defective insulin signaling in cardiac and skeletal muscle and arterial wall, and impaired cardiac tolerance to ischemia. We used this model in this study to assess effects of MetSyn on insulin signaling in SAT and EAT. Methods: Yucatan micropigs (n=16) were fed a diet enriched in saturated fat (coconut oil) and high fructose corn syrup for 6 months to induce obesity and MetSyn. Yucatan micropigs or domestic farm pigs fed standard low-fat chow served as lean controls (n=23). In each pig, SAT and EAT were sampled in the absence of exogenous insulin stimulation and/or 10 min after a saturating dose of insulin (10 U/kg, ...

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.

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.

Serine (ser) phosphorylation of IRS-1 may be involved in a feedback inhibition of insulin signaling and, under certain circumstances, in the pathogenesis of insulin resistance (IR). On the other hand, variations in expression of p85α subunit of phosphatidylinositol (PI) 3-kinase could also modulate the strength of insulin signaling by altering the competition between the p85α monomer and the p85α-p110 heterodimer for the IRS-1 binding sites. In this study we examined the relationship between ser-phosphorylation of IRS-1 and expression of p85α in the development of IR in cultured 3T3 L1 adipocytes. IR was induced by the transfection of the constitutively active myristilated Akt (myr-Akt). Myr-Akt caused activation and phosphorylation of mTOR and p70S6 kinase. The latter promoted ser-phosphorylation of IRS-1 with a subsequent decline in its association with p85α and p110 as well as a decline in PI 3-kinase activity and insulin-stimulated 2-deoxyglucose (2-DOG) uptake-hence IR. A concomitant transfection of p85α siRNA reduced p85α expression by 70% and rescued insulin-stimulated 2DOG uptake from the inhibition induced by myr-Akt by diminishing the competition between p85α monomer and p85α-p110 heterodimer. In conclusion, IR induced by the Akt-mTOR-p70S6 kinase via ser-phosphorylation of IRS-1 can be ameliorated by a reduction in expression of p85α. Thus, interactions between ser-phosphorylation of IRS-1 and expression of p85α are important in the development of and the defense against IR.

Phosphatidylinositol 3-kinase (PI 3-k) plays a critical role in mediating metabolic signaling of insulin. PI 3-k consists of a regulatory p85 (with p85α being the major isoform) and a catalytic p110 subunits. The regulatory subunit p85 exists in excess to p110, creating a certain balance between the free p85 monomer and a p85-p110 heterodimer complex that possesses PI 3-k activity. Because p85 monomer competes with the heterodimer for the IRS-1 binding sites, increases in p85 expression displace p85-p110 heterodimer from IRS-1, resulting in a decreased PI 3-k activity. This can occur in type 2 diabetes, obesity, and pregnancy, when insulin resistance is induced by human placental growth hormone (GH). In this study we tested the direct effect of GH on p85a expression in 3T3-L1 cells and in fat cells obtained from wild-type (WT) mice and mice with heterozygous deletion of p85α+/-. Results Incubation of 3T3-L1 pre-adipocytes with increasing concentrations of rat recombinant GH (0, 500, and 2,500 ng/mL) for 24 hr increased p85α protein expression by approximately 250% (p < .001). In contrast, expression of p110 was not affected. In vivo insulin injection (10 U/kg BW) activated PI 3-k in the epididymal fat of WT and p85α+/- mice (p < .01). Administration of GH for 3 days (1 mg/kg s/c twice daily) blocked insulin-stimulated IRS-1-associated PI 3-k activity in the WT but not in p85α+/- mice. Conclusions GH induces insulin resistance by increasing expression of p85α and creating an imbalance between the subunits of PI 3-k. Increases in p85α compete with p85-p110 heterodimer at the IRS-1 binding sites, resulting in decreased PI 3-k activity.

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.

Restricted accessAbstractFirst published January 2006Effect of Growth Hormone on Expression of P85α and Phosphatidylinositol 3-Kinase Activity in 3T3-L1 Cells and Epididymal Fat of Wild-Type and P85α+/- MiceY. Vo, J.W. Leitner, […], L. Barbour, J. Friedman, and B. Draznin+2-2View all authors and affiliationsVolume 54, Issue 1_supplhttps://doi.org/10.1177/108155890605401S33

Insulin resistance is a cardinal feature of normal pregnancy and excess growth hormone (GH) states, but its underlying mechanism remains enigmatic. We previously found a significant increase in the p85 regulatory subunit of phosphatidylinositol kinase ( PI3-kinase) and striking decrease in IRS-1-associated PI 3-kinase activity in the skeletal muscle of transgenic animals overexpressing human placental growth hormone. Herein, using transgenic mice bearing deletions in p85 alpha, p85 beta, or insulin-like growth factor-1, we provide novel evidence suggesting that overexpression of p85 alpha is a primary mechanism for skeletal muscle insulin resistance in response to GH. We found that the excess in total p85 was entirely accounted for by an increase in the free p85 alpha-specific isoform. In mice with a liver-specific deletion in insulin-like growth factor-1, excess GH caused insulin resistance and an increase in skeletal muscle p85 alpha, which was completely reversible using a GH-releasing hormone antagonist. To understand the role of p85 alpha in GH-induced insulin resistance, we used mice bearing deletions of the genes coding for p85 alpha or p85 beta, respectively (p85 alpha(+/-) and p85 beta(-/-)). Wild type and p85 beta(-/-) mice developed in vivo insulin resistance and demonstrated overexpression of p85 alpha and reduced insulin-stimulated PI 3-kinase activity in skeletal muscle in response to GH. In contrast, p85 alpha(+/-) mice retained global insulin sensitivity and PI 3-kinase activity associated with reduced p85 alpha expression. These findings demonstrated the importance of increased p85 alpha in mediating skeletal muscle insulin resistance in response to GH and suggested a potential role for reducing p85 alpha as a therapeutic strategy for enhancing insulin sensitivity in skeletal muscle.

Sterol regulatory element-binding proteins (SREBPs) are transcription factors that regulate enzymes required for cholesterol and fatty acid synthesis. Expression of SREBP-1 is enhanced by insulin; however, the actual insulin-signaling cascades employed are yet unclear. We determined the roles of the phosphatidylinositol (PI) 3-kinase and mitogen-activated protein (MAP) kinase-dependent pathways in the effect of mediating insulin on SREBP-1 in L-6 skeletal muscle cells and 3T3 L1 adipocytes, using wortmannin or LY294002 to inhibit the PI 3-kinase pathway, and PD98059 to inhibit the MAP kinase-dependent pathway. In myocytes, insulin increased SREBP-1 protein in a dose-dependent manner. 1 and 10 nm insulin significantly increased expression of total cellular SREBP-1 protein at 24 and 48 h, nuclear SREBP-1 protein at 24 h, and SREBP-1a mRNA at 24 h. Although wortmannin and LY294002 had no effect on this aspect of insulin action, PD98059 completely blocked each of these responses. Transfection of a dominant negative mutant of Ras similarly blocked the insulin effect on SREBP-1. In contrast, in adipocytes, the insulin effect on SREBP-1 was mediated via the PI 3-kinase and not the MAP kinase pathway. In conclusion, although insulin increases skeletal muscle SREBP-1 expression in a dose-dependent fashion via the MAP kinase-dependent signaling pathway, insulin action on adipocyte SREBP-1 is mediated via the PI 3-kinase signaling pathway. In the state of insulin resistance, characterized by selective inhibition of the PI 3-kinase pathway, the usual stimulation of lipogenesis by insulin in adipocytes may be inhibited, whereas intramyocellular lipogenesis via the MAP kinase pathway of insulin may continue unabated.

OBJECTIVE: To investigate whether the expression of a dominant negative (DN) farnesyl- and geranygeranyl-transferase I (FTase/GGTase I) α-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 α-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 α-subunit expression in 3T3-L1 pre-adipocyte on insulin-induced DNA synthesis, cell count, phosphorylation of the FTase/GGTase I α-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 α-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 α-subunit in 3T3-L1 pre-adipocytes was without effect on insulin-induced Akt phosphorylation. CONCLUSION: Expression of DN FTase/GGTase I α-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.

Insulin is a mild mitogen and has been shown to potentiate mitogenic influence of other growth factors. Because hyperinsulinemia and/or overexpression of insulin receptors have been linked to development, progression, and outcome of breast cancer, we attempted to evaluate the mechanism of these associations. We have compared the expression of insulin receptors and the magnitude of insulin signaling in breast tumors and adjacent normal mammary tissue samples obtained from 20 patients. We observed that insulin binding more than doubled in the tumors as compared with the normal tissue (P < .01 by paired t test). Insulin signaling to Shc, judged by the magnitude of its phosphorylation, was also significantly enhanced in the tumors. In contrast, the phosphorylation of the insulin-receptor substrate-1 (IRS-1), Akt, and mitogen-activated protein (MAP) kinase were identical in the tumorous and normal mammary tissues. Finally, tumors displayed significantly increased amounts of farnesylated p21 Ras and geranylgeranylated Rho-A (P < .01), consistent with Shc-dependent activation of farnesyl (FTase) and geranylgeranyl transferases (GGTase) in the tumor tissue. We conclude that the mechanism of the mitogenic influence of insulin in breast cancer may include increased expression of insulin receptors, preferential hyperphosphorylation of Shc, and increased amounts of prenylated p21 Ras and Rho-A in tumor tissue as compared with adjacent normal mammary tissue.

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.

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-ΔNPEY) 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 α-subunit of FTase and GGTase I in CHO-hIR-WT cells, but was without effect in CHO-ΔNPEY cells. Insulin increased FTase and GGTase I activities and the amounts of prenylated Ras and RhoA proteins in CHO-hIR-WT (but not CHO-ΔNPEY) 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 α-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.

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 p21(ras) and Rho-A arrests insulin-stimulated adipogenesis. Inhibition of farnesylation of p21(ras) 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 PPAR gamma2, 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.

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 p27(Kip1). All potentiating effects of insulin were inhibited in the presence of an inhibitor of GGTase I, GGTI-286 (3 pm) 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 D-1. 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 p27(Kip1) and cell cycle progression in MCF-7 breast cancer cells.

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.

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-S and Rab-4 proteins.