Biomedical subjects

M L Goalstone

Publications and source records attributed to M L Goalstone.

At least 19 recordsLinked to original sources

PubMed2003

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.

3T3 Cells↗

PubMed2001

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.

Breast Neoplasms↗

PubMed2001

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.

Alkyl and Aryl Transferases↗

PubMed2001

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.

Adipocytes↗

PubMed2001

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.

3T3 Cells↗

PubMed2001

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.

Adipose Tissue↗

PubMed2000

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.

Alkyl and Aryl Transferases↗

PubMed2000

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.

Alkyl and Aryl Transferases↗

PubMed1999

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.

3T3 Cells↗

PubMed1999

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.

3T3 Cells↗

PubMed1999

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.

3T3 Cells↗

PubMed1998

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.

3T3 Cells↗

PubMed1998

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.

Animals↗

PubMed1998

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.

Animals↗

PubMed1997

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.

Blood Glucose↗

PubMed1996

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.

3T3 Cells↗

PubMed1996

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.

Alkyl and Aryl Transferases↗

PubMed1996

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.

Animals↗