Changes in the signalling status of the small GTP-binding proteins Rac and Rho do not influence insulin-stimulated hexose transport

Post-receptor signalling molecules that convey the signal from the activated insulin receptor to the actual process of Glut4 translocation and hexose uptake are poorly understood. Various studies have suggested a requirement of the lipid kinase phosphatidylinositol-3 kinase (PI3-kinase) in this process. PI3kinase regulates the activation status of the small GTP-binding protein Rac which, in turn, is able to activate another G-protein Rho. Rac and Rho are known to regulate the structure of the membrane- and cytoplasmic actin-cytoskeleton. We have examined whether Rac and Rho transfer the signals generated by PI3kinase towards insulin-stimulated hexose uptake. For that purpose, we expressed in 3T3-L1 adipocytes the dominant-negative mutant of RacN17 using vaccinia virus-mediated gene transfer. The expression levels of the RacN17 protein were monitored by Western blotting. The abrogation of endogenous Rac signalling by expression of RacN17 was inferred from the observed loss of arachidonic acid release in response to insulin. Basal and insulin-stimulated hexose transport were not affected by expression of the RacN17 mutant. A possible contribution of Rho.GTP to stimulation of hexose uptake was examined by pre-incubation of adipocytes with lysophosphatidic acid (LPA). We observed a profound effect of LPA on the structure of the cytoskeleton and on the phosphorylation of Focal Adhesion Kinase (p125FAK), indicating that 3T3-L1 adipocytes respond to LPA and that Rho was activated by LPA. However, no effect was detected on the basal or on the insulin-stimulated hexose transport. We conclude that Rac and Rho are unlikely to be involved in insulin-stimulated hexose transport, suggesting a possible contribution of other signalling pathways, downstream of PI3kinase to this process.     

Agents that inhibit Rho, Rac, and Cdc42 do not block formation of actin pedestals in HeLa cells infected with enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) induces formation of actin pedestals in infected host cells. Agents that inhibit the activity of Rho, Rac, and Cdc42, including Clostridium difficile toxin B (ToxB), compactin, and dominant negative Rho, Rac, and Cdc42, did not inhibit formation of actin pedestals. In contrast, treatment of HeLa cells with ToxB inhibited EPEC invasion. Thus, Rho, Rac, and Cdc42 are not required for assembly of actin pedestals; however, they may be involved in EPEC uptake by HeLa cells.     

Adenovirus endocytosis requires actin cytoskeleton reorganization mediated by Rho family GTPases

Adenovirus (Ad) endocytosis via alphav integrins requires activation of the lipid kinase phosphatidylinositol-3-OH kinase (PI3K). Previous studies have linked PI3K activity to both the Ras and Rho signaling cascades, each of which has the capacity to alter the host cell actin cytoskeleton. Ad interaction with cells also stimulates reorganization of cortical actin filaments and the formation of membrane ruffles (lamellipodia). We demonstrate here that members of the Rho family of small GTP binding proteins, Rac and CDC42, act downstream of PI3K to promote Ad endocytosis. Ad internalization was significantly reduced in cells treated with Clostridium difficile toxin B and in cells expressing a dominant-negative Rac or CDC42 but not a H-Ras protein. Viral endocytosis was also inhibited by cytochalasin D as well as by expression of effector domain mutants of Rac or CDC42 that impair cytoskeletal function but not JNK/MAP kinase pathway activation. Thus, Ad endocytosis requires assembly of the actin cytoskeleton, an event initiated by activation of PI3K and, subsequently, Rac and CDC42.     

Escherichia coli cytotoxic necrotizing factor 1 (CNF1), a toxin that activates the Rho GTPase

Cytotoxic necrotizing factor 1 (CNF1), a 110-kDa protein toxin from pathogenic Escherichia coli induces actin reorganization into stress fibers and retraction fibers in human epithelial cultured cells allowing them to spread. CNF1 is acting in the cytosol since microinjection of the toxin into HEp-2 cells mimics the effects of the externally applied CNF1. Incubation in vitro of CNF1 with recombinant small GTPases induces a modification of Rho (but not of Rac, Cdc42, Ras, or Rab6) as demonstrated by a discrete increase in the apparent molecular weight of the molecule. Preincubation of cells with CNF1 impairs the cytotoxic effects of Clostridium difficile toxin B, which inactivates Rho but not those of Clostridium sordellii LT toxin, which inhibits Ras and Rac. As shown for Rho-GTP, CNF1 activates, in a time- and dose-dependent manner, a cytoskeleton-associated phosphatidylinositol 4-phosphate 5-kinase. However, neither the phosphatidylinositol 4,5-bisphosphate (PIP2) nor the phosphatidylinositol 3,4-bisphosphate (PI 3,4-P2) or 3,4,5-trisphosphate (PIP3) cellular content were found increased in CNF1 treated HEp-2 cells. Cellular effects of CNF1 were not blocked by LY294002, a stable inhibitor of the phosphoinositide 3-kinase. Incubation of HEp-2 cells with CNF1 induces relocalization of myosin 2 in stress fibers but not in retraction fibers. Altogether, our data indicate that CNF1 is a toxin that selectively activates the Rho GTP-binding protein, thus inducing contractility and cell spreading.     

Regulation of the actin cytoskeleton by thrombin in human endothelial cells: role of Rho proteins in endothelial barrier function

Endothelial barrier function is regulated at the cellular level by cytoskeletal-dependent anchoring and retracting forces. In the present study we have examined the signal transduction pathways underlying agonist-stimulated reorganization of the actin cytoskeleton in human umbilical vein endothelial cells. Receptor activation by thrombin, or the thrombin receptor (proteinase-activated receptor 1) agonist peptide, leads to an early increase in stress fiber formation followed by cortical actin accumulation and cell rounding. Selective inhibition of thrombin-stimulated signaling systems, including Gi/o (pertussis toxin sensitive), p42/p44, and p38 MAP kinase cascades, Src family kinases, PI-3 kinase, or S6 kinase pathways had no effect on the thrombin response. In contrast, staurosporine and KT5926, an inhibitor of myosin light chain kinase, effectively blocked thrombin-induced cell rounding and retraction. The contribution of Rho to these effects was analyzed by using bacterial toxins that either activate or inhibit the GTPase. Escherichia coli cytotoxic necrotizing factor 1, an activator of Rho, induced the appearance of dense actin cables across cells without perturbing monolayer integrity. Accordingly, lysophosphatidic acid, an activator of Rho-dependent stress fiber formation in fibroblasts, led to reorganization of polymerized actin into stress fibers but failed to induce cell rounding. Inhibition of Rho with Clostridium botulinum exoenzyme C3 fused to the B fragment of diphtheria toxin caused loss of stress fibers with only partial attenuation of thrombin-induced cell rounding. The implication of Rac and Cdc42 was analyzed in transient transfection experiments using either constitutively active (V12) or dominant-interfering (N17) mutants. Expression of RacV12 mimicked the effect of thrombin on cell rounding, and RacN17 blocked the response to thrombin, whereas Cdc42 mutants were without effect. These observations suggest that Rho is involved in the maintenance of endothelial barrier function and Rac participates in cytoskeletal remodeling by thrombin in human umbilical vein endothelial cells.     

Distinct regulation of glucose transport by interleukin-3 and oncogenes in a murine bone marrow-derived cell line

Growth factors and oncogenes promote glucose uptake, but the extent to which increased uptake is regulated at the level of glucose transporter function has not been clearly established. In this paper, we show that interleukin-3 (IL-3), a cytokine growth factor, and the transforming oncogenes ras and abl alter the activation state of glucose transporters by distinct mechanisms. Using bone marrow-derived IL-3-dependent 32Dc13 (32D clone 3) cells and 32D cells transformed with ras and abl oncogenes, we demonstrated that IL-3 enhanced [3H]-2-deoxyglucose (2-DOG) uptake in parental 32Dc13 cells by 40-50% at 0.2 mM 2-DOG, and this was associated with a 2.5-fold increase in transporter affinity for glucose (reduced Km). In comparison, ras and abl oncogenes enhanced 2-DOG uptake by 72-112%, associated with a 2-fold greater transporter affinity for glucose. The tyrosine kinase inhibitor genistein reversed the effects of both IL-3 and oncogenes on glucose uptake and reduced transporter affinity for glucose. Likewise, with exponentially growing 32D cells in the presence of IL-3, a protein kinase C inhibitor, staurosporine, and a phosphatidylinositol 3-kinase (PI-3) kinase inhibitor, wortmannin, inhibited 2-DOG uptake and decreased transporter affinity for glucose. In contrast, in oncogene-transformed cells, staurosporine inhibited 2-DOG uptake but failed to decrease transporter affinity for glucose, whereas wortmannin did not affect 2-DOG uptake. Inhibition of protein tyrosine phosphatases with vanadate enhanced 2-DOG uptake and transporter affinity for glucose in parental cells and in ras-transformed cells but had little effect on abl-transformed cells. Consistently, the serine/threonine phosphatase type 2A inhibitor okadaic acid enhanced 2-DOG uptake and transporter affinity for glucose in parental cells but had little effect on ras- or abl-transformed cells. These results demonstrate differences in the regulation of glucose transport in parental and oncogene-transformed 32D cells. Thus, IL-3 responses are dependent upon tyrosine, serine/threonine, and PI-3 kinases, whereas ras and abl effects on glucose transport depend upon tyrosine phosphorylation but are compromised in their dependence upon serine/threonine and PI-3 kinases.     

Comparative effects of GTPgammaS and insulin on the activation of Rho, phosphatidylinositol 3-kinase, and protein kinase N in rat adipocytes. Relationship to glucose transport

Electroporation of rat adipocytes with guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) elicited sizable insulin-like increases in glucose transport and GLUT4 translocation. Like insulin, GTPgammaS activated membrane phosphatidylinositol (PI) 3-kinase in rat adipocytes, but, unlike insulin, this activation was blocked by Clostridium botulinum C3 transferase, suggesting a requirement for the small G-protein, RhoA. Also suggesting that Rho may operate upstream of PI 3-kinase during GTPgammaS action, the stable overexpression of Rho in 3T3/L1 adipocytes provoked increases in membrane PI 3-kinase activity. As with insulin treatment, GTPgammaS stimulation of glucose transport in rat adipocytes was blocked by C3 transferase, wortmannin, LY294002, and RO 31-8220; accordingly, the activation of glucose transport by GTPgammaS, as well as insulin, appeared to require Rho, PI 3-kinase, and another downstream kinase, e.g. protein kinase C-zeta (PKC-zeta) and/or protein kinase N (PKN). Whereas insulin activated both PKN and PKC-zeta, GTPgammaS activated PKN but not PKC-zeta. In transfection studies in 3T3/L1 cells, stable expression of wild-type Rho and PKN activated glucose transport, and dominant-negative forms of Rho and PKN inhibited insulin-stimulated glucose transport. In transfection studies in rat adipocytes, transient expression of wild-type and constitutive Rho and wild-type PKN provoked increases in the translocation of hemagglutinin (HA)-tagged GLUT4 to the plasma membrane; in contrast, transient expression of dominant-negative forms of Rho and PKN inhibited the effects of both insulin and GTPgammaS on HA-GLUT4 translocation. Our findings suggest that (a) GTPgammaS and insulin activate Rho, PI 3-kinase, and PKN, albeit by different mechanisms; (b) each of these signaling substances appears to be required for, and may contribute to, increases in glucose transport; and (c) PKC-zeta may contribute to increases in glucose transport during insulin, but not GTPgammaS, action.     

The ras signaling pathway mimics insulin action on glucose transporter translocation

Recent observations suggest that insulin increases cellular levels of activated, GTP-bound Ras protein. We tested whether the acute actions of insulin on hexose uptake and glucose-transporter redistribution to the cell surface are mimicked by activated Ras. 3T3-L1 fibroblasts expressing an activated mutant (Lys-61) N-Ras protein exhibited a 3-fold increase in 2-deoxyglucose uptake rates compared with non-transfected cells. Insulin stimulated hexose uptake by approximately 2-fold in parental fibroblasts but did not stimulate hexose uptake in the N-Ras61K-expressing fibroblasts. Overexpression of N-Ras61K also mimicked the large effect of insulin on 2-deoxyglucose transport in 3T3-L1 adipocytes, and again the effects of the two agents were not additive. Total glucose transporter protein (GLUT) 1 was similar between parental and N-Ras61K-expressing 3T3-L1 fibroblasts or adipocytes, whereas total GLUT-4 protein was actually lower in the N-Ras61K-expressing compared with parental adipocytes. However, expression of N-Ras61K in 3T3-L1 adipocytes markedly elevated both GLUT-1 and GLUT-4 in plasma membranes relative to intracellular membranes, and insulin had no further effect. These modulations of glucose transporters by N-Ras61K expression are not due to upstream regulation of insulin receptors because receptor tyrosine phosphorylation and association of phosphatidylinositol 3-kinase with tyrosine-phosphorylated proteins were unaffected. These results show that activated Ras mimics the actions of insulin on membrane trafficking of glucose transporters, consistent with the concept that Ras proteins function as intermediates in this insulin signaling pathway.     

Localization of the glucosyltransferase activity of Clostridium difficile toxin B to the N-terminal part of the holotoxin

Clostridium difficile toxin B that is one of the largest cytotoxins (270 kDa) known acts on Rho subfamily proteins by monoglucosylation (Just, I., Selzer, J., Wilm, M., von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) Nature 375, 500-503). By deletion analysis we identified the enzyme and cytotoxic activity of the toxin to be located at the N terminus of the holotoxin. A 63-kDa fragment of toxin B covering the first 546 amino acid residues glucosylated Rho, Rac, and Cdc42, but not Ras, by using UDP-glucose as a cosubstrate. As known for the holotoxin, glucosylation by the toxin fragment was favored with the GDP-bound form of the low molecular mass GTPases. Microinjection of the toxin fragment into NIH-3T3 cells induced rounding up of cells and redistribution of the actin cytoskeleton. In contrast, a toxin fragment encompassing the first 516 amino acid residues was at least 1000-fold less active than toxin fragment 1-546 and cytotoxically inactive. The data give direct evidence for location of the enzyme activity of C. difficile toxin B at the N-terminal 546 amino acids residues and indicate a functionally and/or structurally important role of the region from amino acid residues 516 through 546 for enzyme and cytotoxic activities.     

Inhibition of receptor signaling to phospholipase D by Clostridium difficile toxin B. Role of Rho proteins

Rho proteins have been reported to activate phospholipase D (PLD) in in vitro preparations. To examine the role of Rho proteins in receptor signaling to PLD, we studied the effect of Clostridium difficile toxin B, which glucosylates Rho proteins, on the regulation of PLD activity in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor (mAChR). Toxin B treatment of HEK cells potently and efficiently blocked mAChR-stimulated PLD. In contrast, basal and phorbol ester-stimulated PLD activities were not or only slightly reduced. Cytochalasin B and Clostridium botulinum C2 toxin, mimicking the effect of toxin B on the actin cytoskeleton but without involving Rho proteins, had no effect on mAChR-stimulated PLD. Toxin B did not alter cell surface mAChR number and mAChR-stimulated binding of (guanosine 5'-O-(thio)triphosphate (GTP gamma S) to G proteins. In addition to mAChR-stimulated PLD, toxin B treatment also inhibited PLD activation by the direct G protein activators, AlF4- and GTP gamma S, studied in intact and permeabilized cells, respectively. Finally, C. botulinum C3 exoenzyme, which ADP-ribosylates Rho proteins, mimicked the inhibitory effect of toxin B on GTP gamma S-stimulated PLD activity. In conclusion, the data presented indicate that toxin B potently and selectively interferes with receptor coupling mechanisms to PLD, and furthermore suggest an essential role for Rho proteins in receptor signaling to PLD.     

Integrin-mediated signals regulated by members of the rho family of GTPases

The organization of the actin cytoskeleton can be regulated by soluble factors that trigger signal transduction events involving the Rho family of GTPases. Since adhesive interactions are also capable of organizing the actin-based cytoskeleton, we examined the role of Cdc42-, Rac-, and Rho-dependent signaling pathways in regulating the cytoskeleton during integrin-mediated adhesion and cell spreading using dominant-inhibitory mutants of these GTPases. When Rat1 cells initially adhere to the extracellular matrix protein fibronectin, punctate focal complexes form at the cell periphery. Concomitant with focal complex formation, we observed some phosphorylation of the focal adhesion kinase (FAK) and Src, which occurred independently of Rho family GTPases. However, subsequent phosphorylation of FAK and paxillin occurs in a Rho-dependent manner. Moreover, we found Rho dependence of the assembly of large focal adhesions from which actin stress fibers radiate. Initial adhesion to fibronectin also stimulates membrane ruffling; we show that this ruffling is independent of Rho but is dependent on both Cdc42 and Rac. Furthermore, we observed that Cdc42 controls the integrin-dependent activation of extracellular signal-regulated kinase 2 and of Akt, a kinase whose activity has been demonstrated to be dependent on phosphatidylinositol (PI) 3-kinase. Since Rac-dependent membrane ruffling can be stimulated by PI 3-kinase, it appears that Cdc42, PI 3-kinase, and Rac lie on a distinct pathway that regulates adhesion-induced membrane ruffling. In contrast to the differential regulation of integrin-mediated signaling by Cdc42, Rac, and Rho, we observed that all three GTPases regulate cell spreading, an event that may indirectly control cellular architecture. Therefore, several separable signaling pathways regulated by different members of the Rho family of GTPases converge to control adhesion-dependent changes in the organization of the cytoskeleton, changes that regulate cell morphology and behavior.     

Requirement of atypical protein kinase clambda for insulin stimulation of glucose uptake but not for Akt activation in 3T3-L1 adipocytes

Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimulation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKCzeta and PKClambda) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKClambda in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a dominant negative mutant of PI 3-kinase). Overexpression of kinase-deficient mutants of PKClambda (lambdaKD or lambdaDeltaNKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKClambda, indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone- or hyperosmolarity-induced glucose uptake, were inhibited by lambdaKD or lambdaDeltaNKD in a dose-dependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKClambda was approximately 50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKClambda mutant that lacks the pseudosubstrate domain (lambdaDeltaPD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of lambdaDeltaPD in quiescent 3T3-L1 adipocytes resulted in the stimulation of glucose uptake and translocation of GLUT4 but not in the activation of Akt. Furthermore, overexpression of an Akt mutant in which the phosphorylation sites targeted by growth factors are replaced by alanine resulted in inhibition of insulin-induced activation of Akt but not of PKClambda. These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKClambda pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.     

Decreased basal, noninsulin-stimulated glucose uptake and metabolism by skeletal soleus muscle isolated from obese-hyperglycemic (ob/ob) mice

Insulin resistance of diaphragms of ob/ob mice has been repeatedly demonstrated previously both in vitro and in vivo. In the present study, transport and metabolism of glucose with and without insulin stimulation were compared in a skeletal muscle more likely than diaphragm or heart to be representative of the overall striated muscle mass, i.e. isolated soleus muscle. Compared with soleus muscle from lean controls, unstimulated lactate release in the presence of exogenous glucose was depressed from 16.2 to 12.3 nmol/60 min per mg wet wt in soleus from ob/ob mutants; glycolysis was decreased from 6.6 to 3.7 and [14C]glucose oxidation to 14CO2 from 0.90 to 0.33 nmol glucose/60 min per mg wet wt. Uptake of 2-deoxyglucose (2-DOG), both with and without insulin, was very much less for soleus from ob/ob than from lean mice, at 2-DOG concentrations ranging from 0.1 to 10 mM, and in mice of 6-15 wk. When 2-DOG concentration was 1 mM, its basal uptake was 0.53 nmol/30 min per mg wet wt for soleus of ob/ob as against 0.96 for soleus of lean mice. The absolute increment due to 1 mU/ml insulin was 0.49 in muscle of ob/ob as against 1.21 in that of lean mice. When the resistance to insulin action was decreased by pretreatment in vivo by either streptozotocin injection or fasting, the decreased basal 2-DOG uptake of subsequently isolated soleus muscle was not improved. Inhibition of endogenous oxidation of fatty acids by 2-bromostearate, while greatly increasing 14CO2 production from [14C]glucose, did not affect basal [5-3H]glucose metabolism or 2-DOG uptake. It is suggested that transport and/or phosphorylation of glucose under basal, unstimulated conditions are depressed in soleus muscle of ob/ob mice, whether or not resistance to insulin and hyperinsulinemia are also present. Although the origin of the decreased basal glucose uptake remains unknown it might be related to a similar decrease in basal glucose uptake by ventromedial hypothalamic cells, an event presumably resulting in a tendency to hyperphagia. Decreased basal glucose uptake by soleus muscle of ob/ob mice might explain the hyperglycemia, and hence partly the hyperinsulinemia and excessive fat deposition of those animals.     

Structural domains that contribute to substrate specificity in facilitated glucose transporters are distinct from those involved in kinetic function: studies with GLUT-1/GLUT-2 chimeras

GLUT-2 differs from other members of the facilitated glucose transporter family because it transports a wider range of substrates and exhibits a higher Km for transport of glucose analogs such as 2-deoxyglucose (2-DOG). In order to investigate the structural determinants of the unique substrate specificity and kinetic function of GLUT-2, recombinant adenoviruses were used to express native, mutant, and chimeric glucose transporters in the kidney cell line CV-1, yielding the following key observations. (1) A chimera consisting of GLUT-1 with the C-terminal tail of GLUT-2 had a Km for 2-DOG of 9.9 +/- 1.5 that was intermediate between that of native GLUT-1 (3.7 +/- 0.4) and native GLUT-2 (26.3 +/- 3.3). In contrast to the effect of the GLUT-2 C terminus on Km for 2-DOG, this substitution did not confer enhanced uptake of three alternative substrates (fructose, arabinose, or streptozotocin) which are transported efficiently by native GLUT-2 but not by GLUT-1. (2) A chimera consisting of GLUT-2 with the N-terminal 87 amino acids of GLUT-1 exhibited no change in Km for 2-DOG relative to native GLUT-2 but exhibited a significant reduction in capacity for transport of the three alternative substrates. (3) Mutation of asparagine 62 in GLUT-2 to glutamine produced a transporter lacking its N-linked oligosaccharide that exhibited a 2.5-fold increase in Km for 2-DOG but equally efficient transport of the three alternative substrates relative to native GLUT-2. These data provide insight into structural domains that affect substrate specificity in facilitated glucose transporters and demonstrate that they are distinct from elements involved in glucose transport kinetics.     

Activation of Rho GTPases by Escherichia coli cytotoxic necrotizing factor 1 increases intestinal permeability in Caco-2 cells

The cytotoxic necrotizing factor 1 (CNF1) activates Rho GTPases by deamidation of glutamine-63 and thereby induces redistribution of the actin cytoskeleton and formation of stress fibers. Here, we have studied the effects of CNF1 on the transepithelial resistance of Caco-2 cells, a human intestinal epithelial cell line, in comparison with the Rho-inactivating toxin B of Clostridium difficile. Whereas toxin B decreased the transepithelial resistance of Caco-2 cells by about 80% after 4 h, CNF1 reduced it by about 40%. Significant changes of the transepithelial resistance induced by CNF1 were detected after 3 h of incubation. Half-maximal effects were observed with 10 and 41 ng of CNF1 and toxin B per ml, respectively. Flux measurement revealed no CNF1-induced increase of fluorescein isothiocyanate-dextran permeation within the first 4 h of incubation and a 2.9-fold increase after 24 h of incubation. In contrast, toxin B induced a 28-fold increase of permeation after 24 h. As detected by rhodamine-phalloidin staining, CNF1 increased polymerization of F actin at focal contacts of adjacent cells and induced formation of stress fibers. The data indicate that not only depolymerization but also polymerization of actin and subsequent reorganization of the actin cytoskeleton alter the barrier function of intestinal tight junctions.     

Mechanism of insulin action--signal transductions after binding to insulin receptor

Insulin binds to the alpha subunit of the insulin receptor which activates the tyrosine kinase in the beta subunit and tyrosine-phosphorylates the insulin receptor substrates-1 (IRS-1). Insulin promotes the formation of a complex between tyrosine-phosphorylated IRS-1 and several proteins including phosphoinositide(PI) 3-kinase, a heterodimer consisting of regulatory 85-kDa (p85) and catalytic 110-kDa (p110) subunits, GRB2 and Syp via the Src homology region 2 (SH2) domains. Recently, it was suggested that GRB2-Sos complex binding to IRS-1 was linked to Ras activation and that PI 3-kinase binding to IRS-1 was linked to activation of glucose transport. Since the mechanism of insulin-stimulated glucose uptake is mainly due to translocation of glucose transporters from an intracellular vesicle pool to the plasma membrane, PI 3-kinase activity may be involved in vesicle transport in mammalian cells.     

A common motif of eukaryotic glycosyltransferases is essential for the enzyme activity of large clostridial cytotoxins

A fragment of the N-terminal 546 amino acid residues of Clostridium sordellii lethal toxin possesses full enzyme activity and glucosylates Rho and Ras GTPases in vitro. Here we identified several amino acid residues in C. sordellii lethal toxin that are essential for the enzyme activity of the active toxin fragment. Exchange of aspartic acid at position 286 or 288 with alanine or asparagine decreased glucosyltransferase activity by about 5000-fold and completely blocked glucohydrolase activity. No enzyme activity was detected with the double mutant D286A/D288A. Whereas the wild-type fragment of C. sordellii lethal toxin was labeled by azido-UDP-glucose after UV irradiation, mutation of the DXD motif prevented radiolabeling. At high concentrations (10 mM) of manganese ions, the transferase activities of the D286A and D288A mutants but not that of wild-type fragment were increased by about 20-fold. The exchange of Asp270 and Arg273 reduced glucosyltransferase activity by about 200-fold and blocked glucohydrolase activity. The data indicate that the DXD motif, which is highly conserved in all large clostridial cytotoxins and also in a large number of glycosyltransferases, is functionally essential for the enzyme activity of the toxins and may participate in coordination of the divalent cation and/or in the binding of UDP-glucose.     

Distribution of prepro-nociceptin/orphanin FQ mRNA and its receptor mRNA in developing and adult mouse central nervous systems

In previous studies we have shown that the insulin-responding glucose transporter isoform of 3T3-L1 adipocytes, GluT4, is almost completely located on microvilli. Furthermore, insulin caused the integration of these microvilli into the plasma membrane, suggesting that insulin-induced stimulation of glucose uptake may be due to the destruction of the cytoskeletal diffusion barrier formed by the actin filament bundle of the microvillar shaft regions [Lange et al. (1990) FEBS Lett. 261, 459-463; Lange et al. (1990) FEBS Lett. 276, 39-41]. Similar shape changes in microvilli were observed when the transport rates of adipocytes were modulated by glucose feeding or starvation. Here we demonstrate that the action of insulin on the surface morphology of hepatocytes is identical to that on 3T3L1 adipocytes; small and narrow microvilli on the surface of unstimulated hepatocytes were rapidly shortened and dilated on top of large domed surface areas. The aspect and mechanism of this effect are closely related to "membrane ruffling" induced by insulin and other growth factors. Pretreatment of hepatocytes with the PI 3-kinase inhibitor wortmannin (100 nM), which completely prevents transport stimulation by insulin in adipocytes and other cell types, also inhibited insulin-induced shape changes in microvilli on the hepatocyte surface. In contrast, vasopressin-induced microvillar shape changes in hepatocytes [Lange et al. (1997) Exp. Cell Res. 234, 486-497] were insensitive to wortmannin pretreatment. These findings indicate that PI 3-kinase products are necessary for stimulation of submembrane microfilament dynamics and that cytoskeletal reorganization is critically involved in insulin stimulation of transport processes. The mechanism of the insulin-induced cytoskeletal reorganization can be explained on the basis of the recent finding of Lu et al. [Biochemistry 35(1996) 14027-14034] that PI 3-kinase products exhibit much higher affinity for the profilin-actin complex than the primary products, PIP and PIP2. Thus, activated PI 3-kinase may direct a flux of profilin-actin complexes to the membrane locations of activated insulin receptors, where, due to the release of actin monomers after binding of profilactin to PI(3,4)P2 and PI(3,4,5)P3, massive actin polymerization is initiated. As a consequence, PI 3-kinase activation initiates a vectorial reorganization of the cellular actin system to membrane sites neighboring activated insulin receptors, giving rise to local membrane stress as visualized by extensive surface deformations and shortening of microvilli. In addition, extensive high-affinity binding of F-actin-barbed endcapping proteins enhances the cytoplasmic concentration of rapidly polymerizing filament ends. Consequently, the actin monomer concentration is lowered and the (cytoplasmic) pointed ends of the microvillar shaft bundle depolymerize and become shorter. The observations presented strengthen the previously postulated diffusion-barrier concept of glucose- and ion-uptake regulation and provide a mechanistic basis for explaining the action of insulin and other growth factors on transport processes across the plasma membrane.     

Tyrosine phosphatase inhibitors, vanadate and pervanadate, stimulate glucose transport and GLUT translocation in muscle cells by a mechanism independent of phosphatidylinositol 3-kinase and protein kinase C

Vanadate and pervanadate (pV) are protein tyrosine phosphatase (PTP) inhibitors that mimic insulin to stimulate glucose transport. To determine whether phosphatidylinositol (PI) 3-kinase is required for vanadate and pV, as it is for insulin, cultured L6 myotubes were treated with vanadate and pV. The two compounds stimulated glucose transport to levels similar to those stimulated by insulin; however, while PI 3-kinase activity and the increase in the lipid products PI 3,4-bisphosphate and PI 3,4,5-trisphosphate were inhibited by wortmannin after stimulation by all three agents--insulin, vanadate, and pV--wortmannin blocked glucose transport stimulated by insulin but not vanadate or pV. Vanadate and pV stimulated the translocation of GLUTs from an intracellular compartment to the plasma membrane; this stimulation was not blocked by wortmannin, but insulin-induced GLUT translocation was inhibited. Similar results were obtained in cultured H9c2 cardiac muscle cells in which wortmannin did not inhibit glucose transport or the vanadate-induced translocation of GLUT4 in c-myc-GLUT4 transfected cells. The ser/thr kinase PKB (Akt/PKB/RAC-PK) is activated by insulin, lies downstream of PI 3-kinase, and has been implicated in signaling of glucose transport. Insulin and pV stimulated PKB activity, and both were inhibited by wortmannin. In contrast, vanadate, at concentrations that maximally stimulated glucose transport, did not significantly increase PKB activity. To determine the potential role of protein kinase C (PKC), L6 cells were incubated chronically with phorbol myristate acetate (PMA) or acutely with the PKC inhibitors calphostin C and bisindolylmaleimide. There was no inhibition of glucose transport stimulation by insulin, vanadate, or pV, and a combination of wortmannin and PKC inhibitors also failed to block the effect of vanadate and pV. In contrast, disassembly of the actin network with cytochalasin D blocked the stimulation of glucose transport by all three agents. In conclusion, vanadate and pV are able to stimulate glucose transport and GLUT translocation by a mechanism independent of PI 3-kinase and PKC. Similar to that by insulin, glucose transport stimulation by vanadate and pV requires the presence of an intact actin network.