The insulin receptor (IR) is a heterotetrameric protein tyrosine kinase whose kinase activity is activated upon binding of insulin . Several aspects of cell metabolism and physiology are controlled and regulated by insulin, and through interactions with the IR at the cell surface, insulin causes the modulation of several interacting signal transduction pathways subsequently leading to physiological responses at the cellular level. The function of the transduction pathways is to coordinate the activation or inactivation of proteins in response to external stimuli. Moreover, pleiotropic  and differential properties, both in terms of common steps in their pathways  as well as in differential activation  make signalling diverse. Protein phosphorylation and dephosphorylation play a major role in intracellular signal transduction regulation  and the phosphorylation state of a target protein is dependent on the equilibrium between these activities. Disturbances in any element of such regulatory systems may lead to pathological conditions such as diabetes type II and cancer and are therefore the subject of extensive studies. In our research work we have performed various studies which investigate the effects of over-expression of a variety of different proteins on insulin-mediated events in cells in culture. The outcome of such studies places an emphasis on the ability to perform quantitative analyses with low back-ground noise.
Gene delivery into mammalian cells for protein over-expression and assessment of gene function involves viral as well as non-viral methods. Commonly, the non-viral methods comprise plasmid transfection with cationic lipids or polymers . In contrast, viral transduction utilises gene transfer by infection of host cells with a modified virus (reviewed in ). Advantages of using viral delivery systems are related to less cell membrane damage and a higher degree of transduction efficiency as compared to the non-viral methods, thus providing viruses as excellent tools for gene delivery to study cell biological processes.
Nevertheless, the use of viral delivery systems may suffer from lengthy construction time requiring transfection steps, cloning of producer cell lines to generate virus stocks, stock amplification and purification. In addition, homologous viral systems of animal or human origin, such as the commonly utilized adenovirus expression system, must be engineered to remove functions involved in the expression of viral genes and viral replication . In addition, the use of recombinant viruses for gene delivery comprises vital functions such as cell membrane adhesion and entry, cytoplasmic transport, replication of viral genome (DNA viruses), and subsequently transcription and translation of the gene of interest (adenovirus endocytosis reviewed in ). It is well documented that such mechanisms involved in adenovirus infection and host inflammatory response modulate several host cell signalling pathways  which therefore may interfere with functional mechanisms in study. For example, incoming adenoviruses have been shown to up-regulate two distinct cell signalling pathways leading to the activation of integrins and cAMP-dependent proteinkinase A (PKA), and p38/MAP kinase pathways, respectively . Downstream of p38/MAP kinase, MAPKAP kinase 2 (MK2) was shown to be activated upon adenovirus infection . Interaction of adenovirus with αv integrins induces the activation of phosphatidylinositol 3-kinase (PI3-kinase) which triggers endocytosis [12, 13]. Downstream of PI3-kinase, both protein kinase B/Akt and ERK/MAP kinase signalling pathways have been shown to be activated in a dose-dependent manner within minutes following adenovirus infection [14, 15]. In addition, the phosphorylation of glycogen synthase kinase (GSK)-3β and nuclear translocation of the p65 subunit of NF-κB, both downstream targets of the PI3-kinase/Akt pathway, was demonstrated in adenovirus-infected corneal fibroblasts . Furthermore, it was shown that adenovirus overrides cellular protein translation in a process involving the downstream activation of mTOR . Similarly, downstream of the p38/MAP kinase pathway, MAP kinase interacting kinase 1 (Mnk1) [18, 19] was displaced from the eukaryotic initiation factor 4F (eIF4F) complex in HEK293 cells, thereby blocking the phosphorylation of eIF4E by Mnk1 and the subsequent translation of cellular mRNA in HEK293 cells .
Over the past 20 years, the insect cell/baculovirus expression vector system, BEVS , has become an important tool for laboratory as well as for industrial scale expression of various heterologous proteins [22–24]. BEVS has also emerged as a versatile and powerful expression system for functional studies of proteins such as protein kinases  and G protein-coupled receptors , for protein-protein interactions , cell based screening  and for viral surface display . Moreover, BEVS has been shown to serve as an efficient gene-transfer vehicle in mammalian cells (termed BacMam) for transient and stable expression of recombinant proteins . Lately, there has been an increasing interest in the use of baculovirus for gene delivery in mammalian cells for protein production, functional expression, and gene therapy (reviewed in [31–33]. For example, the BacMam expression system was recently used to study estrogen receptor function in osteosarcoma cells , functional characterization of a KATP channel protein in CHO and HEK293 cells , and for RNA interference in human primary cells . It has also been developed for the application of nuclear- [37, 38] and G protein-coupled-receptor [39, 40] drug discovery. This interest has been enthused by the relative ease of construction and propagation of recombinant baculovirus vectors, restricted host range for viral replication and viral gene expression, combined with high transduction efficiency and no to low observable cytotoxicity in a wide range of mammalian cell types (reviewed in ). The use of specially engineered recombinant baculoviruses as efficient vehicles for gene transfer into mammalian cells is rapidly emerging as a powerful system for a variety of applications.
In this work we have investigated the use of engineered Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) as a gene delivery tool to study insulin mediated events in human neuroblastoma SHSY-5Y and hepatic C3A cells. Previously, investigating the effects of over-expression of proteins on insulin mediated events, we found that cells in culture often became resistant to stimulation with insulin subsequent to treatment with transfection agents comprised of cationic lipid preparations . This was shown to be caused by an induced state of insulin unresponsiveness due to insulin receptor activation and subsequent down-regulation. Similarly, we have found that cells in culture often become resistant to stimulation with insulin subsequent to treatment with adenovirus vectors (unpublished). In the present work we highlight the fact that adenovirus infection of human cells in culture involves the activation of PI3-kinase and subsequently the downstream activation of Akt. The PI3-kinase/Akt pathway is central to insulin mediated signalling and Akt is central for cellular responses mediated by insulin (reviewed in ). We show that adenovirus mediated gene transfer in human neuroblastoma SHSY-5Y and liver C3A cells cause a dose dependent phosphorylation of Akt and that this makes the cells unresponsive to insulin. Furthermore, we suggest that this phenomenon can be avoided by using recombinant AcMNPV baculovirus mediated gene transfer when studying insulin signalling in human neuroblastoma or liver cells.