BACKGROUND
the insulin receptor  is a heterotetrameric protein tyrosine kinase whose kinase activity is activated upon binding of insulin  <cit> .
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  <cit>  and differential properties, both in terms of common steps in their pathways  <cit>  as well as in differential activation  <cit>  make signalling diverse.
protein phosphorylation and dephosphorylation play a major role in intracellular signal transduction regulation  <cit>  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  <cit> .
in contrast, viral transduction utilises gene transfer by infection of host cells with a modified virus .
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  <cit> .
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 , and subsequently transcription and translation of the gene of interest .
it is well documented that such mechanisms involved in adenovirus infection and host inflammatory response modulate several host cell signalling pathways  <cit>  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 , and p38/map kinase pathways, respectively  <cit> .
downstream of p38/map kinase, mapkap kinase  <dig>  was shown to be activated upon adenovirus infection  <cit> .
interaction of adenovirus with αv integrins induces the activation of phosphatidylinositol 3-kinase  which triggers endocytosis  <cit> .
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  <cit> .
in addition, the phosphorylation of glycogen synthase kinase -3β and nuclear translocation of the p <dig> subunit of nf-κb, both downstream targets of the pi3-kinase/akt pathway, was demonstrated in adenovirus-infected corneal fibroblasts  <cit> .
furthermore, it was shown that adenovirus overrides cellular protein translation in a process involving the downstream activation of mtor  <cit> .
similarly, downstream of the p38/map kinase pathway, map kinase interacting kinase  <dig>   <cit>  was displaced from the eukaryotic initiation factor 4f  complex in hek <dig> cells, thereby blocking the phosphorylation of eif4e by mnk <dig> and the subsequent translation of cellular mrna in hek <dig> cells  <cit> .
over the past  <dig> years, the insect cell/baculovirus expression vector system, bevs  <cit> , has become an important tool for laboratory as well as for industrial scale expression of various heterologous proteins  <cit> .
bevs has also emerged as a versatile and powerful expression system for functional studies of proteins such as protein kinases  <cit>  and g protein-coupled receptors  <cit> , for protein-protein interactions  <cit> , cell based screening  <cit>  and for viral surface display  <cit> .
moreover, bevs has been shown to serve as an efficient gene-transfer vehicle in mammalian cells  for transient and stable expression of recombinant proteins  <cit> .
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 .
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  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  <cit> .
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 .
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 .
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.
RESULTS
infection of human neuroblastoma shsy-5y and liver c3a cells with recombinant adenovirus induces akt-ser <dig> phosphorylation
adenoviral gene delivery is commonly utilized for functional over-expression and characterization of a gene-of-interest in mammalian cells.
however, it is widely recognized that viral infection of human cell lines with homologous adenovirus vectors induces the activation of phosphatidylinositol 3-kinase , subsequently leading to the phosphorylation of protein kinase b/akt on serine  <dig>  <cit> .
akt is central for cellular responses mediated by insulin  and activation of akt is hence used as a common marker in insulin signalling.
therefore, while studying the impact of over-expression of various protein kinases on insulin signalling in experimental human cell lines, we investigated the influence of recombinant adenovirus on akt phosphorylation in shsy-5y and c3a cells.
cultured cells were first infected with recombinant adenovirus expressing green fluorescent protein  as a model protein, and phosphorylation of akt was studied with specific ser473-phosho-akt antibodies .
viral titration was performed at multiplicity of infection  between  <dig> and  <dig> and gfp expression was determined  <dig> hours post infection  by flow cytometry gated for intact adenovirus infected cells.
figure  <dig> displays side scatter against forward scatter for shsy-5y and c3a cells infected with adgfp at moi  <dig>   <dig>  and  <dig>  respectively.
the resulting transduction efficiencies were determined from expression histograms of the populations of intact cells  which are shown in the lower panels of figure  <dig> for each respective cell line, and was found to be  <dig> ,  <dig> , and  <dig>  % for shsy-5y cells, and  <dig> ,  <dig> , and  <dig>  % for c3a cells infected at moi  <dig>   <dig>  and  <dig>  respectively.
next, the influence on akt phosphorylation following adenovirus transduction was investigated.
as anticipated, the degree of phosphorylation of akt increased upon adenovirus infection and this increase was dose-dependent when both shsy-5y as well as c3a cells were subjected to adenovirus infection.
the degree of phosphorylation increased  <dig> and 5-fold for shsy-5y cells, and  <dig>  and  <dig> -fold for c3a cells, infected at moi  <dig> and  <dig>  respectively, as determined with an elisa assay measuring phosphoserine  <dig> in pkb.
these results were confirmed under various experimental conditions and are summarized in the upper part of table  <dig> for adgfp.
to further validate the results, the data obtained with the commercial elisa were verified with western blot using specific ser473-phosho-akt antibodies.
again, phosphorylation on serine  <dig> in infected but unstimulated cells was seen, thus confirming the activation of pkb by adenovirus infection measured by two different methods .
taken together, the results from these experiments show that when transduced with adenovirus up to moi  <dig>  shsy-5y and c3a cells respond with an increased phosphorylation of ser <dig> akt indicating that using adenovirus as a gene transfer vehicle in shsy-5y and c3a cells may influence the cells responsiveness to insulin treatment.
time notations refer to time of incubation at experimental conditions determined by the elisa method.
baculovirus mediated gene transduction and recombinant protein expression do not influence the level of phosphorylation of akt in human shsy-5y and c3a cells
the use of engineered baculovirus as a gene transfer vehicle in mammalian cells has emerged as a powerful tool for several applications.
one advantage of using this heterologous viral system is the virtual absence of endogenous viral gene expression and cytotoxicity in mammalian cells  <cit> .
in view of the results obtained above, we therefore investigated the use of baculovirus as a potential tool for gene delivery in human cell lines used to study insulin signalling.
high efficiency baculovirus transduction of various liver  <cit>  and neural cell lines  <cit>  has been established previously.
however, the use of the human neuroblastoma shsy-5y cells has to our knowledge not been reported and transduction efficiency and expression of reporter protein in shsy-5y cells was therefore first confirmed.
similar to that for adenovirus , a viral construct containing gfp under the control of a cytomegalovirus promoter  was first constructed .
recombinant virus was propagated in sf <dig> insect cells and prepared as described in the methods section.
adherent cultures of shsy-5y cells were infected with bacgfp and expression of reporter protein was confirmed by fluorescence microscopy  <dig> hours p.i.
.
for quantification and titration of transduction ratio, the cells were infected at moi's between  <dig> and  <dig> and for practical reasons the transduction efficiency  and expression level  were analysed  <dig> hours p.i.
using a chip-based flow cytometry application  <cit> .
without further optimisation of the infection and expression conditions, the transduction ratio increased in a dose dependent manner with increased moi to a maximum of  <dig> % .
however, when butyrate was added to the cells to enhance protein expression at a final concentration of  <dig> mm, the relative amount of gfp expressing-, and thus apparently transduced cells, was significantly increased from about  <dig> to  <dig> %.
in addition, the maximum relative mean expression level increased more than 4-fold in the shsy-5y cells when butyrate was present .
butyrate is a histone deacetylase  inhibitor, inducing a hyperacetylation of chromatin and enhancement of transcription.
the importance of the chromatin state of the baculovirus genome in the transduced cells for transgene expression and the role of hdac inhibitors has been well described  <cit>  and will not be further discussed.
besides the more thorough investigation of shsy-5y cells, chinese hamster overy cells  stably expressing the human insulin receptor  <cit> , human embryonic kidney cells , and rat fao- and human c3a hepatic-derived cell lines, were screened for the ability to be transduced by bacgfp .
this screen indicated that the cho.hir and the human hek <dig> cell lines were easily transduced by bacgfp as previously been reported by others  <cit> .
however, under our conditions butyrate was required to confirm efficient transduction in the cho.hir cell line.
whether this was conditional or caused by the heterologous ir was not further investigated.
the human hepatic-derived c3a cell line was also easily transduced by bacgfp and similarly to hek <dig> and cho.hir, butyrate enhanced the expression of gfp and thus the apparent transduction ratio.
by contrast, only minor gfp expression was detected in the rat hepatic-derived cell line fao  and was essentially unaffected by the addition of butyrate.
thus rat fao hepatoma cells appear to be relatively resistant to bacmam viral transduction.
*values are given with s.d.
for n =  <dig>
next, the influence of baculovirus infection and recombinant protein expression on the phosphorylation of akt was investigated.
in these studies shsy-5y and c3a cells were infected with bacgfp and total and phosphorylated akt were analysed using an akt specific elisa, as previously reported with adgfp.
baculovirus titres of  <dig> and  <dig> were chosen to achieve experimentally desired transduction ratios.
in contrast to cells infected with adenovirus at moi  <dig> and  <dig>  where pkb activation was observed , cells transduced with bacmam at moi  <dig> and  <dig> showed no apparent increase in pkb phosphorylation.
this was true for both shsy-5y and c3a cells .
taken together, the results from the experiments summarized in table  <dig> show that when transduced with adenovirus at moi  <dig> and  <dig>  shsy-5y and c3a cells respond with an increased phosphorylation of ser <dig> akt  and that this can be avoided by utilizing baculovirus transduction, at moi's as high as  <dig>
baculovirus mediated gene transduction and recombinant protein expression do not impair insulin mediated phosphorylation of akt in shsy-5y and c3a cells
since infection of shsy-5y and c3a cells with baculovirus apparently did not influence the phosphorylation of akt, the response to insulin following infection was investigated.
first the response to insulin of uninfected cells was determined.
shsy-5y and c3a cells were incubated with  <dig> nm insulin for  <dig> and  <dig> minutes, respectively and the phosphorylation of akt was determined with phospho-specific elisa .
the response was most pronounced for shsy-5y cells with a transient  <dig> -fold increase in akt phosphorylation  <dig> minutes after addition of insulin which remained  <dig>  – fold elevated after  <dig> minutes .
this data was again verified for shsy-5y cells with western blot using specific ser473-phosho-akt antibodies .
the c3a cells also responded to insulin albeit to a lower degree reaching a sustainable  <dig>  – fold increase in akt phosphorylation after  <dig> and  <dig> minutes incubation, respectively .
next, the effect of insulin stimulation of cells infected with adenovirus and baculovirus, respectively, were investigated.
as previously found for shsy-5y cells infected with adenovirus , infection with adgfp at moi <dig> and  <dig> in the absence of insulin gave a dose dependent increase in akt phosphorylation .
in addition, when the cells were incubated in the presence of  <dig> nm insulin for  <dig> minutes no additional phosphorylation of akt was obtained .
after  <dig> minutes of incubation with  <dig> nm insulin, akt was further activated  <dig>  and  <dig> -fold over the activation already induced by adenovirus infection .
by contrast, shsy-5y cells infected with baculovirus showed no sign of basal akt phosphorylation after  <dig> or  <dig> minutes .
moreover, upon insulin stimulation of baculovirus infected cells, the response was analogous to that for non infected cells incubated with insulin.
at  <dig> minutes incubation, a  <dig>  and  <dig> -fold increased phosphorylation was obtained at moi <dig> and  <dig> , and at  <dig> minutes incubation a  <dig>  and  <dig> -fold increase in akt phosphorylation was obtained at moi <dig> and  <dig>  respectively .
qualitatively similar data were obtained for c3a cells .
thus adenovirus infection increased basal akt phosphorylation and insulin was not able to increase this further.
by contrast, bacmam-transduced cells displayed no elevated basal akt phosphorylation and insulin was able to induce a  <dig>  to  <dig> -fold activation of the enzyme, as for control cells.
the apparently smaller akt response in c3a cells compared to shsy-5y cells may be related to the higher basal level of akt phosphorylation in unstimulated cells and is a feature of other hepatoma cells lines in culture including rat fao and human hepg <dig> cells .
interestingly, when we sought to examine the effects of virus infection on erk1/ <dig> activity in hepatoma cells, we found elevated basal erk1/ <dig> phosphorylation such that further increases could not be observed .
thus our data show that cells transduced with baculovirus remain responsive to insulin stimulation as measured by phosphorylation of akt.
discussion
in our earlier work, we reported that common lipid transfection reagents are able to induce a state of cellular insulin resistance which hampers significantly subsequent experiments designed to examine insulin signal transduction  <cit> .
we hypothesised that the activation of the insulin receptor which is causal to this state of insulin unresponsiveness is due to lipid phase separations in the plasma membrane.
in the current work, we have confirmed that the use of adenoviral transduction vectors in place of lipid transfection reagents leads to activation of components of the insulin signalling pi3k pathway, with subsequent induction of a state of insulin unresponsiveness.
in this case, the mechanistic basis of the effects of adenovirus transduction are better established, with the potential involvement of integrin proteins at the cell surface leading to pi3k activation  <cit> .
alternatively, it was recently shown by o'shea and co-workers that adenovirus encodes two proteins that activate the mammalian target of rapamycin  and that one of these  mimics growth factor signalling by activating pi3k  <cit> .
in our search for transduction agents of recombinant dna that do not affect the cellular insulin signalling apparatus, we examined the effects of the bacmam baculovirus vector encoding the model protein gfp.
although known for a long time that baculoviruses can efficiently be taken up by mammalian cells  <cit> , the mode and kinetics of baculovirus infection of which are poorly understood.
recently, evidence for clathrin-mediated endocytosis has emerged as a major entry route in mammalian cells  <cit> .
our data show that this vector is effective as a transduction agent of mammalian cells in culture and that it has no apparent effect on components of the insulin signalling cascade leading to the activation of pkb.
as such, it does not induce a state of insulin unresponsiveness and may be a promising tool for investigations of recombinantly-expressed proteins on insulin signal transduction.
possible reasons for why bacmam transduction avoids cellular insulin resistance would include lack of interaction with surface integrin proteins and the fact that the heterologous virus does not hijack cellular protein translational machinery to the same degree as adenovirus.
it is however possible that that transient effects on pkb activation and other insulin signalling events upon baculovirus transduction may still be possible and may occur at time points that we have not mapped.
however, the major advantage of the baculovirus system is that cells remain responsive to insulin stimulation showing clear activation of pkb after transduction with the virus, which is not possible with adenovirus vectors.
interestingly, the effectiveness of bacmam as a transduction agent is greatly enhanced in the presence of the hdac inhibitor butyrate, which presumably leads to enhanced chromatin modification and facilitates dna transcription.
differential repression of baculovirus mediated transgene expression in different cell types is well known and the use of hdac inhibitors such as butyrate and trichostatin a are widely use to increase gene expression  <cit> .
considering that hdac <dig> is a negative regulator of insulin signalling via interactions with insulin receptor substrate proteins and that global inhibition of hdac activity enhances insulin signalling  <cit> , it is unlikely that bacmam transduction in the presence of hdac inhibitors will be appropriate for subsequent insulin signalling studies.
however, considering the relatively benign effects of bacmam exposure on cellular well-being, it may be possible to increase mois further should higher expression levels of the delivered gene of interest in the studied cell line be required.
in addition, individual cell lines demonstrate different susceptibilities to transduction by baculovirus  and therefore transduction conditions such as virus dosage, temperature, viral exposure time, and transduction media should be optimized for individual cell lines in order to increase the transduction efficiency  <cit> , finally, baculovirus vectors utilizing enhancer elements such as hr <dig>  <cit>  and episomal orip/ebna- <dig> elements  <cit>  in combination with mammalian promoters have been shown to increase and prolong transgene expression in mammalian cells.
CONCLUSIONS
the bacmam baculovirus vector is a promising reagent for the transduction of mammalian cells in culture allowing subsequent insulin signalling studies.
the virus does not induce a state of insulin unresponsiveness as do adenovirus vectors and lipid transfection reagents.
as such, bacmam is an agent of choice to examine the effects of recombinantly expressed proteins on insulin signal transduction.

 the insulin receptor 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. 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, and subsequently transcription and translation of the gene of interest. 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, and p38/map kinase pathways, respectively. downstream of p38/map kinase, mapkap kinase was shown to be activated upon adenovirus infection. interaction of adenovirus with αv integrins induces the activation of phosphatidylinositol 3-kinase which triggers endocytosis. 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. in addition, the phosphorylation of glycogen synthase kinase -3β and nuclear translocation of the p 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 was displaced from the eukaryotic initiation factor 4f complex in hek cells, thereby blocking the phosphorylation of eif4e by mnk and the subsequent translation of cellular mrna in hek cells. over the past 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. 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 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. 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 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. 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. 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.  infection of human neuroblastoma shsy-5y and liver c3a cells with recombinant adenovirus induces akt-ser phosphorylation adenoviral gene delivery is commonly utilized for functional over-expression and characterization of a gene-of-interest in mammalian cells. however, it is widely recognized that viral infection of human cell lines with homologous adenovirus vectors induces the activation of phosphatidylinositol 3-kinase, subsequently leading to the phosphorylation of protein kinase b/akt on serine. akt is central for cellular responses mediated by insulin and activation of akt is hence used as a common marker in insulin signalling. therefore, while studying the impact of over-expression of various protein kinases on insulin signalling in experimental human cell lines, we investigated the influence of recombinant adenovirus on akt phosphorylation in shsy-5y and c3a cells. cultured cells were first infected with recombinant adenovirus expressing green fluorescent protein as a model protein, and phosphorylation of akt was studied with specific ser473-phosho-akt antibodies. viral titration was performed at multiplicity of infection between and and gfp expression was determined hours post infection by flow cytometry gated for intact adenovirus infected cells. figure displays side scatter against forward scatter for shsy-5y and c3a cells infected with adgfp at moi and respectively. the resulting transduction efficiencies were determined from expression histograms of the populations of intact cells which are shown in the lower panels of figure for each respective cell line, and was found to be,, and % for shsy-5y cells, and,, and % for c3a cells infected at moi and respectively. next, the influence on akt phosphorylation following adenovirus transduction was investigated. as anticipated, the degree of phosphorylation of akt increased upon adenovirus infection and this increase was dose-dependent when both shsy-5y as well as c3a cells were subjected to adenovirus infection. the degree of phosphorylation increased and 5-fold for shsy-5y cells, and and -fold for c3a cells, infected at moi and respectively, as determined with an elisa assay measuring phosphoserine in pkb. these results were confirmed under various experimental conditions and are summarized in the upper part of table for adgfp. to further validate the results, the data obtained with the commercial elisa were verified with western blot using specific ser473-phosho-akt antibodies. again, phosphorylation on serine in infected but unstimulated cells was seen, thus confirming the activation of pkb by adenovirus infection measured by two different methods. taken together, the results from these experiments show that when transduced with adenovirus up to moi shsy-5y and c3a cells respond with an increased phosphorylation of ser akt indicating that using adenovirus as a gene transfer vehicle in shsy-5y and c3a cells may influence the cells responsiveness to insulin treatment. time notations refer to time of incubation at experimental conditions determined by the elisa method. baculovirus mediated gene transduction and recombinant protein expression do not influence the level of phosphorylation of akt in human shsy-5y and c3a cells the use of engineered baculovirus as a gene transfer vehicle in mammalian cells has emerged as a powerful tool for several applications. one advantage of using this heterologous viral system is the virtual absence of endogenous viral gene expression and cytotoxicity in mammalian cells. in view of the results obtained above, we therefore investigated the use of baculovirus as a potential tool for gene delivery in human cell lines used to study insulin signalling. high efficiency baculovirus transduction of various liver and neural cell lines has been established previously. however, the use of the human neuroblastoma shsy-5y cells has to our knowledge not been reported and transduction efficiency and expression of reporter protein in shsy-5y cells was therefore first confirmed. similar to that for adenovirus, a viral construct containing gfp under the control of a cytomegalovirus promoter was first constructed. recombinant virus was propagated in sf insect cells and prepared as described in the methods section. adherent cultures of shsy-5y cells were infected with bacgfp and expression of reporter protein was confirmed by fluorescence microscopy hours p.i.. for quantification and titration of transduction ratio, the cells were infected at moi's between and and for practical reasons the transduction efficiency and expression level were analysed hours p.i. using a chip-based flow cytometry application. without further optimisation of the infection and expression conditions, the transduction ratio increased in a dose dependent manner with increased moi to a maximum of %. however, when butyrate was added to the cells to enhance protein expression at a final concentration of mm, the relative amount of gfp expressing-, and thus apparently transduced cells, was significantly increased from about to %. in addition, the maximum relative mean expression level increased more than 4-fold in the shsy-5y cells when butyrate was present. butyrate is a histone deacetylase inhibitor, inducing a hyperacetylation of chromatin and enhancement of transcription. the importance of the chromatin state of the baculovirus genome in the transduced cells for transgene expression and the role of hdac inhibitors has been well described and will not be further discussed. besides the more thorough investigation of shsy-5y cells, chinese hamster overy cells stably expressing the human insulin receptor, human embryonic kidney cells, and rat fao- and human c3a hepatic-derived cell lines, were screened for the ability to be transduced by bacgfp. this screen indicated that the cho.hir and the human hek cell lines were easily transduced by bacgfp as previously been reported by others. however, under our conditions butyrate was required to confirm efficient transduction in the cho.hir cell line. whether this was conditional or caused by the heterologous ir was not further investigated. the human hepatic-derived c3a cell line was also easily transduced by bacgfp and similarly to hek and cho.hir, butyrate enhanced the expression of gfp and thus the apparent transduction ratio. by contrast, only minor gfp expression was detected in the rat hepatic-derived cell line fao and was essentially unaffected by the addition of butyrate. thus rat fao hepatoma cells appear to be relatively resistant to bacmam viral transduction. *values are given with s.d. for n = next, the influence of baculovirus infection and recombinant protein expression on the phosphorylation of akt was investigated. in these studies shsy-5y and c3a cells were infected with bacgfp and total and phosphorylated akt were analysed using an akt specific elisa, as previously reported with adgfp. baculovirus titres of and were chosen to achieve experimentally desired transduction ratios. in contrast to cells infected with adenovirus at moi and where pkb activation was observed, cells transduced with bacmam at moi and showed no apparent increase in pkb phosphorylation. this was true for both shsy-5y and c3a cells. taken together, the results from the experiments summarized in table show that when transduced with adenovirus at moi and shsy-5y and c3a cells respond with an increased phosphorylation of ser akt and that this can be avoided by utilizing baculovirus transduction, at moi's as high as baculovirus mediated gene transduction and recombinant protein expression do not impair insulin mediated phosphorylation of akt in shsy-5y and c3a cells since infection of shsy-5y and c3a cells with baculovirus apparently did not influence the phosphorylation of akt, the response to insulin following infection was investigated. first the response to insulin of uninfected cells was determined. shsy-5y and c3a cells were incubated with nm insulin for and minutes, respectively and the phosphorylation of akt was determined with phospho-specific elisa. the response was most pronounced for shsy-5y cells with a transient -fold increase in akt phosphorylation minutes after addition of insulin which remained – fold elevated after minutes. this data was again verified for shsy-5y cells with western blot using specific ser473-phosho-akt antibodies. the c3a cells also responded to insulin albeit to a lower degree reaching a sustainable – fold increase in akt phosphorylation after and minutes incubation, respectively. next, the effect of insulin stimulation of cells infected with adenovirus and baculovirus, respectively, were investigated. as previously found for shsy-5y cells infected with adenovirus, infection with adgfp at moi and in the absence of insulin gave a dose dependent increase in akt phosphorylation. in addition, when the cells were incubated in the presence of nm insulin for minutes no additional phosphorylation of akt was obtained. after minutes of incubation with nm insulin, akt was further activated and -fold over the activation already induced by adenovirus infection. by contrast, shsy-5y cells infected with baculovirus showed no sign of basal akt phosphorylation after or minutes. moreover, upon insulin stimulation of baculovirus infected cells, the response was analogous to that for non infected cells incubated with insulin. at minutes incubation, a and -fold increased phosphorylation was obtained at moi and, and at minutes incubation a and -fold increase in akt phosphorylation was obtained at moi and respectively. qualitatively similar data were obtained for c3a cells. thus adenovirus infection increased basal akt phosphorylation and insulin was not able to increase this further. by contrast, bacmam-transduced cells displayed no elevated basal akt phosphorylation and insulin was able to induce a to -fold activation of the enzyme, as for control cells. the apparently smaller akt response in c3a cells compared to shsy-5y cells may be related to the higher basal level of akt phosphorylation in unstimulated cells and is a feature of other hepatoma cells lines in culture including rat fao and human hepg cells. interestingly, when we sought to examine the effects of virus infection on erk1/ activity in hepatoma cells, we found elevated basal erk1/ phosphorylation such that further increases could not be observed. thus our data show that cells transduced with baculovirus remain responsive to insulin stimulation as measured by phosphorylation of akt. discussion in our earlier work, we reported that common lipid transfection reagents are able to induce a state of cellular insulin resistance which hampers significantly subsequent experiments designed to examine insulin signal transduction. we hypothesised that the activation of the insulin receptor which is causal to this state of insulin unresponsiveness is due to lipid phase separations in the plasma membrane. in the current work, we have confirmed that the use of adenoviral transduction vectors in place of lipid transfection reagents leads to activation of components of the insulin signalling pi3k pathway, with subsequent induction of a state of insulin unresponsiveness. in this case, the mechanistic basis of the effects of adenovirus transduction are better established, with the potential involvement of integrin proteins at the cell surface leading to pi3k activation. alternatively, it was recently shown by o'shea and co-workers that adenovirus encodes two proteins that activate the mammalian target of rapamycin and that one of these mimics growth factor signalling by activating pi3k. in our search for transduction agents of recombinant dna that do not affect the cellular insulin signalling apparatus, we examined the effects of the bacmam baculovirus vector encoding the model protein gfp. although known for a long time that baculoviruses can efficiently be taken up by mammalian cells, the mode and kinetics of baculovirus infection of which are poorly understood. recently, evidence for clathrin-mediated endocytosis has emerged as a major entry route in mammalian cells. our data show that this vector is effective as a transduction agent of mammalian cells in culture and that it has no apparent effect on components of the insulin signalling cascade leading to the activation of pkb. as such, it does not induce a state of insulin unresponsiveness and may be a promising tool for investigations of recombinantly-expressed proteins on insulin signal transduction. possible reasons for why bacmam transduction avoids cellular insulin resistance would include lack of interaction with surface integrin proteins and the fact that the heterologous virus does not hijack cellular protein translational machinery to the same degree as adenovirus. it is however possible that that transient effects on pkb activation and other insulin signalling events upon baculovirus transduction may still be possible and may occur at time points that we have not mapped. however, the major advantage of the baculovirus system is that cells remain responsive to insulin stimulation showing clear activation of pkb after transduction with the virus, which is not possible with adenovirus vectors. interestingly, the effectiveness of bacmam as a transduction agent is greatly enhanced in the presence of the hdac inhibitor butyrate, which presumably leads to enhanced chromatin modification and facilitates dna transcription. differential repression of baculovirus mediated transgene expression in different cell types is well known and the use of hdac inhibitors such as butyrate and trichostatin a are widely use to increase gene expression. considering that hdac is a negative regulator of insulin signalling via interactions with insulin receptor substrate proteins and that global inhibition of hdac activity enhances insulin signalling, it is unlikely that bacmam transduction in the presence of hdac inhibitors will be appropriate for subsequent insulin signalling studies. however, considering the relatively benign effects of bacmam exposure on cellular well-being, it may be possible to increase mois further should higher expression levels of the delivered gene of interest in the studied cell line be required. in addition, individual cell lines demonstrate different susceptibilities to transduction by baculovirus and therefore transduction conditions such as virus dosage, temperature, viral exposure time, and transduction media should be optimized for individual cell lines in order to increase the transduction efficiency, finally, baculovirus vectors utilizing enhancer elements such as hr and episomal orip/ebna- elements in combination with mammalian promoters have been shown to increase and prolong transgene expression in mammalian cells.  the bacmam baculovirus vector is a promising reagent for the transduction of mammalian cells in culture allowing subsequent insulin signalling studies. the virus does not induce a state of insulin unresponsiveness as do adenovirus vectors and lipid transfection reagents. as such, bacmam is an agent of choice to examine the effects of recombinantly expressed proteins on insulin signal transduction. 
recombinant adenovirus vectors and transfection agents comprising cationic lipids are widely used as gene delivery vehicles for functional expression 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. 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. in addition, we have found that cultured cells often do not respond to insulin stimulation following adenovirus treatment. in addition, this activation makes treated cells unresponsive to insulin stimulation as determined by an apparent lack of differential phosphorylation of akt on serine- our data further indicate that the use of recombinant baculovirus does not increase the phosphorylation of akt in shsy-5y and c3a cells. 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. however, we have previously reported that cationic lipid reagents cause a state of insulin unresponsiveness in cell cultures. therefore, we investigated the effect of adenovirus infection on insulin unresponsiveness by means of akt activation in cultured cells. moreover, following infection with baculovirus, shsy-5y and c3a cells respond to insulin by means of phosphorylation of akt on serine- in the same manner as uninfected cells. consequently, these tools are utilized to investigate the effects of functional over-expression of proteins on insulin mediated events. in addition, our data may contribute to an understanding of the molecular mechanisms underlying baculovirus infection of human cells. 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.
 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.  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- 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- in the same manner as uninfected cells.  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. 