Development and comparison of two 3T3-L1 adipocyte models of insulin resistance: increased glucose flux vs glucosamine treatment.

Insulin resistance can be induced in vivo by intravenous infusion of glucosamine or in cells by incubation with glucosamine. However, a publication (Hresko, R. C., et al. (1998) J. Biol. Chem. 273, 20658-20668) suggests a trivial explanation of glucosamine-induced insulin resistance whereby intracellular ATP pools are depleted presumably due to the phosphorylation of glucosamine to glucosamine 6-phosphate, a hexosamine pathway intermediate. The reduced ATP level impaired insulin receptor (IR) autophosphorylation and tyrosine kinase activity toward substrates. The present work describes the development and comparison of two methods for inducing insulin resistance, by treating 3T3-L1 adipocytes overnight using either 25 mM glucose/5 nM insulin or 2 mM glucosamine. Under these conditions basal glucose transport rates were comparable with controls. Insulin-stimulated 2-deoxyglucose uptake, however, was reduced by approximately 45% in response to both high glucose/insulin and glucosamine treatment, relative to control cells. The total relative amounts of the insulin-responsive glucose transporter, Glut4, remained constant under both treatment conditions. The relative phosphotyrosine (Tyr(P)) contents of the insulin receptor and its substrate 1 (IRS-1) were assessed in whole cell homogenates. With both methods to induce insulin resistance, IR/IRS-1 Tyr(P) levels were virtually indistinguishable from those in control cells. Insulin-stimulated phosphorylation of Akt on Ser(473) was not impaired in insulin-resistant cells. Furthermore, the relative Tyr(P) content of the PDGF receptor was comparable in high glucose/insulin- or glucosamine-treated 3T3-L1 adipocytes upon subsequent challenge with PDGF. Finally, the relative amounts of glutamine:fructose-6-phosphate amidotransferase and O-linked N-acetylglucosamine transferase, two important hexosamine pathway enzymes, were similar in both treatments when compared with controls. Thus, 3T3-L1 adipocytes can be used as a model system for studying insulin resistance induced by increased influx of glucose. Under appropriate experimental conditions, glucosamine treatment can mimic the effects of increased glucose flux without impairment of tyrosine phosphorylation-based signaling.

Isolation and characterization from porcine serum of a soluble sulfotransferase responsible for 6-O-sulfation of the galactose residue in 2'-fucosyllactose: implications in the synthesis of the ligand for L-selectin.

A soluble sulfotransferase from porcine serum which catalyzes the transfer of sulfate from adenosine 3'-phosphate 5'-phosphosulphate (PAPS) to 2'-fucosyllactose (2'-FL) was purified 36,333-fold using a combination of conventional and affinity chromatographic steps. The purified enzyme preparation after non-denaturing discontinuous-PAGE exhibited a molecular mass of about 80 kDa by reducing SDS-PAGE. However, when a partially purified enzyme preparation was subjected to gel filtration on Sephacryl S-300, the enzyme activity eluted in the void volume, which indicated that the native enzyme existed as an oligomer. The purified enzyme showed Km values of 9.15 microM for PAPS and 15.38 mM for 2'-FL at the optimum pH value of 7.4. The substrate specificity of the purified enzyme was evaluated with various sugars that are structurally similar to sialyl LewisX (sLeX). Results indicated that 3'-sialyllactose and lactose were efficient acceptors of sulfation, whereas 6'-sialyllactose and 6'-sialyllactosamine were poor substrates for this sulfotransferase. Further, the reaction product analysis revealed that the sulfate substitution, when using 2'-FL as the substrate, was at the C-6 position of the galactose residue. Coincidentally, a similar enzyme activity was also found in porcine lymphoid tissues such as, lymph nodes (peripheral and mesenteric) and spleen. Collectively, these findings suggest that this enzyme might be involved in the synthesis of the ligand for L-selectin.

SC-54684A: an orally active inhibitor of platelet aggregation.

BACKGROUND: Intravenous therapy has been shown to be beneficial in the prevention of acute platelet-associated thrombotic events. However, orally active agents would be advantageous for chronic therapy. Fibrinogen receptor antagonists block the fibrinogen/platelet interaction and thus inhibit a step required for thrombus formation. To date, no orally active fibrinogen binding inhibitors have been characterized. SC-54684A, now in clinical trial, is the orally active prodrug of a potent and specific fibrinogen binding antagonist. METHODS AND RESULTS: We measured inhibition of 125I-fibrinogen binding to activated platelets and inhibition of aggregation in platelet-rich plasma to selected agonists and showed IC50s of 1.0 x 10(-8) and 3 to 7 x 10(-8) mol/L, respectively. Specificity of the active moiety was determined by studying its effect on the binding of (1) neutrophils to interleukin (IL)-1 beta-stimulated endothelial cells, (2) endothelial cells to fibronectin, and (3) vitronectin to isolated vitronectin and fibrinogen receptors. No effect was observed on the binding neutrophils to IL-stimulated endothelial cells or endothelial cell binding to fibronectin. There was a fivefold separation between binding to isolated receptors of vitronectin and fibrinogen. Collagen-induced aggregation was inhibited by 80%, and bleeding time was increased approximately 2.5-fold when the active moiety was infused to steady state at 0.2 micrograms/kg per minute in dogs. When the ester prodrug was given orally and the active moiety was given intravenously, the oral systemic activity was approximately 20%. Pharmacokinetic analysis after intravenous infusion of the prodrug or active moiety showed that the prodrug was rapidly converted to the active moiety; the active moiety had a t1/2 of 6.5 hours. When the prodrug was administered both orally and intravenously, the systemic availability of the active moiety was 62%. CONCLUSIONS: SC-54684A, an orally active antiplatelet drug now in clinical trial, is shown to be a potent, specific fibrinogen binding inhibitor that blocks platelet aggregation to a wide variety of known stimuli and has good bioavailability in animals.

Studies on selectin-carbohydrate interactions.

Recruitment of neutrophils to sites of inflammation is now believed to occur through an initial rolling interaction at the luminal surface of activated endothelium and is mediated by a class of mammalian lectins referred to as the selectins. Selectins recognize carbohydrate determinants on co-receptors. It is generally believed that many selectin molecules must bind to many carbohydrate receptor molecules i.e. multivalent binding, to enable sufficient binding strength to elicit the rolling response between the neutrophil and the endothelial cell. One of the approaches to the generation of more potent molecular antagonists of the selectin-mediated cell-cell interaction is to mimic the multivalent interaction in a single compound. Recent experiments utilising conjugated forms of sialyl Lewisx-BSA have explored this feasibility (Welply et al., 1994). In that study, monovalent sLex (sialic acid alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc), the minimum binding determinant for E-selectin, as well as monovalent sialyllactosamine (sialic acid alpha 2-3Gal beta 1-4GlcNAc), a non-binding structure, and the corresponding multivalent BSA-conjugated forms were tested for their ability to inhibit binding of HL-60 cells to immobilised E-selectin. As expected, only sLex and sLex-BSA were found to do so. sLex16-BSA (16 mol tetrasaccharide/mol BSA) showed a dose-dependent inhibition of HL-60 binding with a measured IC50 of 1 microM; demonstrating close to a three-order of magnitude enhancement of inhibitory activity compared to free sLex. This result indicated that multivalent forms of sLex are capable of binding to E-selectin with higher affinity than do monovalent glycans. In another study, fluorescent forms of monovalent sLex were synthesized and used to measure a true thermodynamic dissociation constant for the monovalent sLex:E-selectin interaction of 120 +/- 31 microM (Jacob et.al., 1995).

Multivalent sialyl-LeX: potent inhibitors of E-selectin-mediated cell adhesion; reagent for staining activated endothelial cells.

Free, monovalent, SLeX (Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)-GlcNAc), SLn (Neu5Ac alpha 2-3Gal beta 1-4GlcNAc) and corresponding BSA-conjugated forms--displaying different ratios of SLeX and SLn to protein--were tested for their ability to inhibit binding of HL-60 cells to immobilized E-selectin. Free SLeX and conjugated SLeX-BSA inhibited cell binding in a dose-dependent manner. SLn and SLn-BSA did not inhibit binding. SLeX16BSA (16 mol tetrasaccharide/mol BSA) and monovalent SLeX inhibited cell binding with measured inhibitory concentrations (IC50S) of 1 microM and 1 mM, respectively, demonstrating a three-order-of-magnitude enhancement of inhibitory activity with the multivalent form of SLeX. A SLex7BSA conjugate was 10-fold less potent than those with 11 or 16 mol SLeX/mol BSA. An assay which measured neutrophil rolling on interleukin (IL)-1 beta-activated human umbilical vein endothelial cells (HUVECs) showed 50% reduction in the number of rolling neutrophils in the presence of 1 microM SLeX16BSA, whereas the level of free, monovalent SLeX oligosaccharide required to produce the same effect was approximately 0.3 mM. SLeX-BSA was found to be an excellent reagent for staining endothelial cells expressing E-selectin. Biotinylated SLeX-BSA in conjunction with Texas red avidin-stained lipopolysaccharide (LPS)-activated HUVECs, and co-incubation of activated cells with anti-E-selectin, specifically blocked staining. The distribution of E-selectin, as determined by binding of SLeX-BSA, was virtually identical with that obtained by binding of anti-E-selectin antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

Tropomyosin prevents depolymerization of actin filaments from the pointed end.

Regulation of the pointed, or slow-growing, end of actin filaments is essential to the regulation of filament length. The purpose of this study is to investigate the role of skeletal muscle tropomyosin (TM) in regulating pointed end assembly and disassembly in vitro. The effects of TM upon assembly and disassembly of actin monomers from the pointed filament end were measured using pyrenyl-actin fluorescence assays in which the barbed ends were capped by villin. Tropomyosin did not affect pointed end elongation; however, filament disassembly from the pointed end stopped in the presence of TM under conditions where control filaments disassembled within minutes. The degree of protection against depolymerization was dependent upon free TM concentration and upon filament length. When filaments were diluted to a subcritical actin concentration in TM, up to 95% of the filamentous actin remained after 24 h and did not depolymerize further. Longer actin filaments (150 monomers average length) were more effectively protected from depolymerization than short filaments (50 monomers average length). Although filaments stopped depolymerizing in the presence of TM, they were not capped as shown by elongation assays. This study demonstrates that a protein, such as TM, which binds to the side of the actin filament can prevent dissociation of monomers from the end without capping the end to elongation. In skeletal muscle, tropomyosin could prevent thin filament disassembly from the pointed end and constitute a mechanism for regulating filament length.

Cytochalasin B induces cellular DNA fragmentation.

Cellular DNA fragmentation can be induced in many biological instances without plasma membrane damage. The fungal metabolite, cytochalasin B, is capable of modifying numerous cellular functions related to DNA synthesis. In this work it is demonstrated that cytochalasin B is capable of inducing DNA fragmentation in a number of cells lines. This DNA fragmentation occurs before plasma membrane lysis and over a period of hours. Cytochalasin E and villin, agents that act on the microfilaments, also induce DNA fragmentation. Phorbol dibutyrate, a diacylglyceral analog, is able to inhibit cytochalasin B-induced DNA fragmentation in a dose-dependent fashion. These findings support the interpretation that cytochalasin B is inducing DNA fragmentation via its effect on the actin filaments.

Tropomyosin stabilizes the pointed end of actin filaments by slowing depolymerization.

Tropomyosin is postulated to confer stability to actin filaments in nonmuscle cells. We have found that a nonmuscle tropomyosin isolated from the intestinal epithelium can directly stabilize actin filaments by slowing depolymerization from the pointed, or slow-growing, filament end. Kinetics of elongation and depolymerization from the pointed end were measured in fluorescence assays using pyrenylactin filaments capped at the barbed end by villin. The initial pointed end depolymerization rate in the presence of tropomyosin averaged 56% of the control rate. Elongation from the pointed filament end in the presence of tropomyosin occurred at a lower free G-actin concentration, although the on rate constant, kappa p+, was not greatly affected. Furthermore, in the presence of tropomyosin, the free G-actin concentration was lower at steady state. Therefore, nonmuscle tropomyosin stabilizes the pointed filament end by lowering the off rate constant, kappa p-.

Tropomyosin distinguishes between the two actin-binding sites of villin and affects actin-binding properties of other brush border proteins.

The intestinal epithelial cell brush border exhibits distinct localizations of the actin-binding protein components of its cytoskeleton. The protein interactions that dictate this subcellular organization are as yet unknown. We report here that tropomyosin, which is found in the rootlet but not in the microvillus core, can bind to and saturate the actin of isolated cores, and can cause the dissociation of up to 30% of the villin and fimbrin from the cores but does not affect actin binding by 110-kD calmodulin. Low speed sedimentation assays and ultrastructural analysis show that the tropomyosin-containing cores remain bundled, and that 110-kD calmodulin remains attached to the core filaments. The effects of tropomyosin on the binding and bundling activities of villin were subsequently determined by sedimentation assays. Villin binds to F-actin with an apparent Ka of 7 X 10(5) M-1 at approximate physiological ionic strength, which is an order of magnitude lower than that of intestinal epithelial cell tropomyosin. Binding of villin to F-actin presaturated with tropomyosin is inhibited relative to that to pure F-actin, although full saturation can be obtained by increasing the villin concentration. Villin also inhibits the binding of tropomyosin to F-actin, although not to the same extent. However, tropomyosin strongly inhibits bundling of F-actin by villin, and bundling is not recovered even at a saturating villin concentration. Since villin has two actin-binding sites, both of which are required for bundling, the fact that tropomyosin inhibits bundling of F-actin under conditions where actin is fully saturated with villin strongly suggests that tropomyosin's and one of villin's F-actin-binding sites overlap. These results indicate that villin and tropomyosin could compete for actin filaments in the intestinal epithelial cell, and that tropomyosin may play a major role in the regulation of microfilament structure in these and other cells.

Low Mr tropomyosin isoforms from chicken brain and intestinal epithelium have distinct actin-binding properties.

Tropomyosin isoforms of the low Mr class were isolated from chicken intestinal epithelium and brain, and their physical and functional properties were characterized. Tropomyosin from each tissue contains four distinct polypeptides, all of about 32,000 daltons. In two-dimensional gels, brain tropomyosin contains two major and two minor polypeptides; the major epithelium isoforms coelectrophorese with the two minor brain isoforms. Conversely, only small amounts of the major brain isoforms are detected in the epithelium. Actin-binding properties of brain tropomyosin isoforms are distinct from those of the intestinal epithelium. At 2.5 mM MgCl2 and physiological ionic strength, the intestinal epithelial tropomyosin binds to filamentous actin with an apparent Ka of 8 X 10(6) M-1 whereas brain tropomyosin has an apparent Ka of 8 X 10(5) M-1. Tropomyosin from either tissue binds actin cooperatively with a Hill coefficient of 2.3 for intestinal epithelial cell and 1.95 for brain tropomyosin. Isoforms from both tissues exhibit reduced head-to-tail polymerizability as compared to muscle tropomyosin. The actin-binding properties of intestinal epithelial cell tropomyosin are therefore similar to those of the muscle tropomyosins even though the isoforms have lower molecular weight, a paracrystal structure, and reduced head-to-tail polymerizability typical of the other nonmuscle tropomyosins. These results indicate that a heterogeneity of functional properties may be expressed among the low Mr tropomyosin isoforms.

Purification and characterization of GDP-D-mannose 4,6-dehydratase from porcine thyroid.

The enzyme GDP-D-mannose 4,6-dehydratase has been purified 1500-fold from porcine thyroid tissue. The enzyme exhibits a molecular mass of 251000 Da as determined by sedimentation techniques. Its subunit size was determined as 41500 Da by dodecyl sulfate gel electrophoresis. The enzyme has a Km of 3.3 microM with respect to GDP-D-mannose and appears specific with respect to this substrate. The enzyme appears to be inhibited by guanine nucleotides and by guanine nucleotide sugars. It is particularly susceptible to inhibition by GDP-L-fucose. It is suggested that this compound may have a physiological function as an end-product feedback inhibitor.

An assay for GDP-D-mannose-4,6-dehydratase.

The mechanism of GDP-D-mannose-4,6-dehydratase action with respect to loss of the C5 hydrogen has been established using GDP-D-[5-3H]-mannose as a substrate. This observation has been incorporated into a rapid assay for the enzyme based on the equilibration of 3H with the aqueous medium.

Phosphorylation controls brush border motility by regulating myosin structure and association with the cytoskeleton.

The intestinal epithelial cell brush border (BB) is a useful model for nonmuscle cell motility. We studied regulation of BB motility by analyzing myosin phosphorylation and its association with the cytoskeleton. Our results demonstrate that myosin associates with the cytoskeleton only when it is dephosphorylated. Myosin light chain kinase substrates release myosin, phosphorylated and in the form of filaments, from the cytoskeleton. Although ITP and GTP serve as myosin ATPase substrates, they do not cause BB contraction, myosin release, or phosphorylation. Brush border contraction occurs with ATP or with a mixture of ITP and ATP gamma S. Therefore, phosphorylation regulates myosin association with the cytoskeleton, myosin is not bound at the actin-myosin binding site, and when phosphorylated, myosin forms filaments for movement.