Baculovirus-mediated gene transfer and recombinant protein expression do not interfere with insulin dependent phosphorylation of PKB/Akt in human SHSY-5Y and C3A cells.

BACKGROUND: Recombinant adenovirus vectors and transfection agents comprising cationic lipids are widely used as gene delivery vehicles for functional expression in cultured cells. Consequently, these tools are utilized to investigate the effects of functional over-expression of proteins on insulin mediated events. However, we have previously reported that cationic lipid reagents cause a state of insulin unresponsiveness in cell cultures. In addition, we have found that cultured cells often do not respond to insulin stimulation following adenovirus treatment. Infection with adenovirus compromises vital functions of the host cell leading to the activation of protein kinases central to insulin signalling, such as protein kinase B/Akt. Therefore, we investigated the effect of adenovirus infection on insulin unresponsiveness by means of Akt activation in cultured cells. Moreover, we investigated the use of baculovirus as a heterologous viral gene delivery vehicle to circumvent these phenomena. Since the finding that baculovirus can efficiently transduce mammalian cells, the applications of this viral system in gene delivery has greatly expanded and one advantage is the virtual absence of cytotoxicity in mammalian cells. RESULTS: We show that infection of human neuroblastoma SHSY-5Y and liver C3A cells with recombinant adenovirus results in the activation of Akt in a dose dependent manner. In addition, this activation makes treated cells unresponsive to insulin stimulation as determined by an apparent lack of differential phosphorylation of Akt on serine-473. Our data further indicate that the use of recombinant baculovirus does not increase the phosphorylation of Akt in SHSY-5Y and C3A cells. Moreover, following infection with baculovirus, SHSY-5Y and C3A cells respond to insulin by means of phosphorylation of Akt on serine-473 in the same manner as uninfected cells. CONCLUSION: Widely-used adenovirus vectors for gene delivery cause a state of insulin unresponsiveness in human SHSY-5Y and C3A cells in culture due to the activation of central protein kinases of the insulin signalling pathway. This phenomenon can be avoided when studying insulin signalling by using recombinant baculovirus as a heterologous viral expression system. In addition, our data may contribute to an understanding of the molecular mechanisms underlying baculovirus infection of human cells.

Oxidation of protein tyrosine phosphatases as a pharmaceutical mechanism of action: a study using 4-hydroxy-3,3-dimethyl-2H-benzo[g]indole-2,5(3H)-dione.

Growth factor and insulin signal transduction comprise series of protein kinases and protein phosphatases whose combined activities serve to propagate the growth factor signal in a regulated fashion. It was shown previously that such signaling cascades generate hydrogen peroxide inside cells. Recent work has implied that one function of this might be to enhance the feed-forward signal through the reversible oxidation and inhibition of protein tyrosine phosphatases (PTPs). We identified compound 4-hydroxy-3,3-dimethyl-2H-benzo[g]indole-2,5(3H)-dione (BVT.948) as an agent that is able to inhibit PTP activity in vitro noncompetitively, a mechanism involving oxidation of the catalytic cysteine residue. We investigated the pharmaceutical utility of this compound by examining its effects in a series of in vitro cellular and in vivo assays. Results showed that BVT.948 was able to enhance insulin signaling in cells, although it did not increase tyrosine phosphorylation globally. Furthermore, the compound was active in vivo, enhancing insulin tolerance tests in ob/ob mice, therefore apparently enhancing insulin sensitivity. BVT.948 was able to inhibit several other PTPs tested and also was efficient at inhibiting several cytochrome P450 (P450) isoforms in vitro. The data suggest that inhibitors of PTPs that display noncompetitive kinetics must be viewed with caution because they may oxidize the enzyme irreversibly. Furthermore, although such compounds display interesting biological effects in vitro and in vivo, their general pharmaceutical utility may be limited due to undesired effects on P450 enzymes.

Insulin receptor activation and down-regulation by cationic lipid transfection reagents.

BACKGROUND: Transfection agents comprised of cationic lipid preparations are widely used to transfect cell lines in culture with specific recombinant complementary DNA molecules. We have found that cells in culture are often resistant to stimulation with insulin subsequent to treatment with transfection agents such as LipofectAMINE 2000 and FuGENE-6. This is seen with a variety of different readouts, including insulin receptor signalling, glucose uptake into muscle cells, phosphorylation of protein kinase B and reporter gene activity in a variety of different cell types RESULTS: We now show that this is due in part to the fact that cationic lipid agents activate the insulin receptor fully during typical transfection experiments, which is then down-regulated. In attempts to circumvent this problem, we investigated the effects of increasing concentrations of LipofectAMINE 2000 on insulin receptor phosphorylation in Chinese hamster ovary cells expressing the human insulin receptor. In addition, the efficiency of transfection that is supported by the same concentrations of transfection reagent was studied by using a green fluorescent protein construct. Our data indicate that considerably lower concentrations of LipofectAMINE 2000 can be used than are recommended by the manufacturers. This is without sacrificing transfection efficiency markedly and avoids the problem of reducing insulin receptor expression in the cells. CONCLUSION: Widely-used cationic lipid transfection reagents cause a state of insulin unresponsiveness in cells in culture due to fully activating and subsequently reducing the expression of the receptor in cells. This phenomenon can be avoided by reducing the concentration of reagent used in the transfection process.

Detection of insulin-regulated GLUT4-translocation by the insertion of a protein C epitope in L6 myoblasts.

Insulin stimulates glucose transport by translocation of the membrane glucose transporter GLUT4 from intracellular vesicles to the plasma membrane. GLUT4 is highly expressed in adipose tissue and skeletal muscle. We have constructed a cDNA containing the human GLUT4 inserted by a 12 amino acid protein C epitope in the first extracellular (exofacial) domain of the human GLUT4 (GLUT4-PC). Stable expression of GLUT4-PC in L6 myoblasts (L6-GLUT4-PC) was confirmed in immunofluorescence using monoclonal antibodies against protein C. The protein C staining yielded labeling in perinuclear vesicles strongly co-localizing with GLUT4 detected with antibodies directed against the endofacial part of GLUT4. The L6-GLUT4-PC cells were further characterized in a direct cell-based enzyme-linked immunosorbent assay by the use of beta-galactosidase. Cell surface binding of monoclonal protein C antibodies was detected with beta-galactosidase-conjugated secondary antibodies and chlorophenolred-beta-D-galactopyranoside (CPRG) as substrate in 2% paraformaldehyde fixed cells. In this assay, stimulation with insulin created a rapidly detectable recruitment of GLUT4-PC to the cell surface. This cell-based enzyme-linked immunosorbent GLUT4 assay was shown to be comparable with that of previously reported radioactive assays.