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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.

Andersson M, Warolén M, Nilsson J, Selander M, Sterky C, Bergdahl K, Sörving C, James SR, Doverskog M - BMC Cell Biol. (2007)

Bottom Line: Therefore, we investigated the effect of adenovirus infection on insulin unresponsiveness by means of Akt activation in cultured cells.Our data further indicate that the use of recombinant baculovirus does not increase the phosphorylation of Akt in SHSY-5Y and C3A cells.In addition, our data may contribute to an understanding of the molecular mechanisms underlying baculovirus infection of human cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, R&D, Biovitrum AB, SE-11276 Stockholm, Sweden. monica.andersson@biovitrum.com <monica.andersson@biovitrum.com>

ABSTRACT

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.

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Insulin stimulation of SHSY-5Y and C3A cells infected with adenovirus or baculovirus vectors. Cultured SHSY-5Y (A and C) and C3A (B and D) cells were transduced with adenovirus (AdGFP) at MOI10 and 50 or baculovirus (BacGFP) at MOI1000 and 10000, respectively, and the responsiveness to insulin was determined. Non-infected (No virus) and infected cells were treated with 100 nM insulin for 10 (A and B) and 30 minutes (C and D), respectively. Cells were harvested after incubation for total cellular protein, and ELISA was performed with antibodies specific for Akt phosphorylated on serine 473 and total Akt, respectively.
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Figure 3: Insulin stimulation of SHSY-5Y and C3A cells infected with adenovirus or baculovirus vectors. Cultured SHSY-5Y (A and C) and C3A (B and D) cells were transduced with adenovirus (AdGFP) at MOI10 and 50 or baculovirus (BacGFP) at MOI1000 and 10000, respectively, and the responsiveness to insulin was determined. Non-infected (No virus) and infected cells were treated with 100 nM insulin for 10 (A and B) and 30 minutes (C and D), respectively. Cells were harvested after incubation for total cellular protein, and ELISA was performed with antibodies specific for Akt phosphorylated on serine 473 and total Akt, respectively.

Mentions: 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 100 nM insulin for 10 and 30 minutes, respectively and the phosphorylation of Akt was determined with phospho-specific ELISA (Figure 3; no virus). The response was most pronounced for SHSY-5Y cells with a transient 4.9-fold increase in Akt phosphorylation 10 minutes after addition of insulin which remained 3.1 – fold elevated after 30 minutes (Figure 3A and 3C, no virus). This data was again verified for SHSY-5Y cells with western blot using specific Ser473-phosho-Akt antibodies (Figure 2, lane 1 and 2). The C3A cells also responded to insulin albeit to a lower degree reaching a sustainable 1.6 – fold increase in Akt phosphorylation after 10 and 30 minutes incubation, respectively (Figure 3B and 3D, no virus). 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 (Table 1; Figure 2, lane 1 and 3), infection with AdGFP at MOI10 and 50 in the absence of insulin gave a dose dependent increase in Akt phosphorylation (Figure 3A and 3C). In addition, when the cells were incubated in the presence of 100 nM insulin for 10 minutes no additional phosphorylation of Akt was obtained (Figure 3A; Figure 2, lane 3 and 4). After 30 minutes of incubation with 100 nM insulin, Akt was further activated 1.6 and 1.7-fold over the activation already induced by adenovirus infection (Figure 3C). By contrast, SHSY-5Y cells infected with baculovirus showed no sign of basal Akt phosphorylation after 10 or 30 minutes (Figure 3A and 3C). Moreover, upon insulin stimulation of baculovirus infected cells, the response was analogous to that for non infected cells incubated with insulin. At 10 minutes incubation, a 5.7 and 5.4-fold increased phosphorylation was obtained at MOI1000 and 10000 (Figure 3A), and at 30 minutes incubation a 3.6 and 3.8-fold increase in Akt phosphorylation was obtained at MOI1000 and 10000, respectively (Figure 3C). Qualitatively similar data were obtained for C3A cells (Figure 3B and 3D). 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 1.6 to 2.1-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 HepG2 cells (data not shown). Interestingly, when we sought to examine the effects of virus infection on ERK1/2 activity in hepatoma cells, we found elevated basal ERK1/2 phosphorylation such that further increases could not be observed (data not shown). Thus our data show that cells transduced with baculovirus remain responsive to insulin stimulation as measured by phosphorylation of Akt.


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.

Andersson M, Warolén M, Nilsson J, Selander M, Sterky C, Bergdahl K, Sörving C, James SR, Doverskog M - BMC Cell Biol. (2007)

Insulin stimulation of SHSY-5Y and C3A cells infected with adenovirus or baculovirus vectors. Cultured SHSY-5Y (A and C) and C3A (B and D) cells were transduced with adenovirus (AdGFP) at MOI10 and 50 or baculovirus (BacGFP) at MOI1000 and 10000, respectively, and the responsiveness to insulin was determined. Non-infected (No virus) and infected cells were treated with 100 nM insulin for 10 (A and B) and 30 minutes (C and D), respectively. Cells were harvested after incubation for total cellular protein, and ELISA was performed with antibodies specific for Akt phosphorylated on serine 473 and total Akt, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1808450&req=5

Figure 3: Insulin stimulation of SHSY-5Y and C3A cells infected with adenovirus or baculovirus vectors. Cultured SHSY-5Y (A and C) and C3A (B and D) cells were transduced with adenovirus (AdGFP) at MOI10 and 50 or baculovirus (BacGFP) at MOI1000 and 10000, respectively, and the responsiveness to insulin was determined. Non-infected (No virus) and infected cells were treated with 100 nM insulin for 10 (A and B) and 30 minutes (C and D), respectively. Cells were harvested after incubation for total cellular protein, and ELISA was performed with antibodies specific for Akt phosphorylated on serine 473 and total Akt, respectively.
Mentions: 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 100 nM insulin for 10 and 30 minutes, respectively and the phosphorylation of Akt was determined with phospho-specific ELISA (Figure 3; no virus). The response was most pronounced for SHSY-5Y cells with a transient 4.9-fold increase in Akt phosphorylation 10 minutes after addition of insulin which remained 3.1 – fold elevated after 30 minutes (Figure 3A and 3C, no virus). This data was again verified for SHSY-5Y cells with western blot using specific Ser473-phosho-Akt antibodies (Figure 2, lane 1 and 2). The C3A cells also responded to insulin albeit to a lower degree reaching a sustainable 1.6 – fold increase in Akt phosphorylation after 10 and 30 minutes incubation, respectively (Figure 3B and 3D, no virus). 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 (Table 1; Figure 2, lane 1 and 3), infection with AdGFP at MOI10 and 50 in the absence of insulin gave a dose dependent increase in Akt phosphorylation (Figure 3A and 3C). In addition, when the cells were incubated in the presence of 100 nM insulin for 10 minutes no additional phosphorylation of Akt was obtained (Figure 3A; Figure 2, lane 3 and 4). After 30 minutes of incubation with 100 nM insulin, Akt was further activated 1.6 and 1.7-fold over the activation already induced by adenovirus infection (Figure 3C). By contrast, SHSY-5Y cells infected with baculovirus showed no sign of basal Akt phosphorylation after 10 or 30 minutes (Figure 3A and 3C). Moreover, upon insulin stimulation of baculovirus infected cells, the response was analogous to that for non infected cells incubated with insulin. At 10 minutes incubation, a 5.7 and 5.4-fold increased phosphorylation was obtained at MOI1000 and 10000 (Figure 3A), and at 30 minutes incubation a 3.6 and 3.8-fold increase in Akt phosphorylation was obtained at MOI1000 and 10000, respectively (Figure 3C). Qualitatively similar data were obtained for C3A cells (Figure 3B and 3D). 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 1.6 to 2.1-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 HepG2 cells (data not shown). Interestingly, when we sought to examine the effects of virus infection on ERK1/2 activity in hepatoma cells, we found elevated basal ERK1/2 phosphorylation such that further increases could not be observed (data not shown). Thus our data show that cells transduced with baculovirus remain responsive to insulin stimulation as measured by phosphorylation of Akt.

Bottom Line: Therefore, we investigated the effect of adenovirus infection on insulin unresponsiveness by means of Akt activation in cultured cells.Our data further indicate that the use of recombinant baculovirus does not increase the phosphorylation of Akt in SHSY-5Y and C3A cells.In addition, our data may contribute to an understanding of the molecular mechanisms underlying baculovirus infection of human cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, R&D, Biovitrum AB, SE-11276 Stockholm, Sweden. monica.andersson@biovitrum.com <monica.andersson@biovitrum.com>

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus