Preadipocyte differentiation is extensively studied in 3T3L1 and 3T3F442A cells as surrogate models for deciphering the role of fat cell differentiation in human obesity. In this study, preadipocyte differentiation induced by Ad36 can be compared to conventional induction of differentiation by MDI. Overall, the adipogenic program induced by Ad36 was very similar to that induced by MDI with respect to expression of key adipogenic genes, followed by lipid accumulation, indicating the ability of Ad36 to simulate the MDI effect and induce adipogenesis. A noticeable difference was the relative down-regulation of C/EBPα by Ad36 in absence of MDI. C/EBPα suppresses cell cycle progression (23
), therefore, considering the increased cell proliferation by Ad36 in absence of MDI, its down-regulation by Ad36 would be expected. Down regulation of C/EBPα by Ad36 agrees well with our earlier report that showed Ad36 down regulated expression and secretion of leptin (13
), a gene under the transcriptional control of C/EBPα (24
). Down regulation of leptin may further promote adipogenesis in autocrine/paracrine way as previously discussed (13
While C/EBPα and PPARγ coordinately upregulate adipocyte differentiation, Ad36 induced differentiation appears to proceed without upregulation of C/EBPα but with upregulation of PPARγ2 and the downstream genes G3PDH and aP2. Effect of Ad36 was perhaps most remarkable for PPARγ2, compared to uninfected control, in presence or absence of MDI. Probably due to concomitantly increasing gene expression in MDI treated control group, statistical significance between the infected vs the uninfected groups was reached later (day 5) compared to that for the no-MDI group (day 3).
A virus may not infect every cell in a plate. Ad36 infects estimated 30% of 3T3-L1 cells at 3.8 MOI (unpublished observations). Thus, it should be noted that even within the Ad36 infected group, there are two cell populations, one infected with the virus and other uninfected. Presence of these two cell populations in Ad36 groups was evident from two peaks of gene expression Wnt10 and C/EBPβ, an early peak due to Ad36 infection and a late peak coinciding with normal gene expression of the uninfected cell population. Moreover, unlike human primary adipocyte progenitors, 3T3-L1 cells do not support Ad36 viral particle formation ((10
) and unpublished observations). Therefore, the direct effect of Ad36 is limited to only the cells it enters. This also implies that the effect of Ad36 on infected cells is so robust that the infection of only a fraction of cells significantly increases average response for the entire group.
Infection of cells by a viral agent is likely to induce non-specific changes in cell metabolism. We used Ad2 to control for such non-specific effect of Ad36 infection. As we observed before, Ad2 is not adipogenic (19
) and in fact may reduce adipocyte differentiation. Despite successful cellular entry, it did not modulate adipogenic program. However, caution should be used in interpreting the effects of a negative control virus. While it is reasonable in principle, in practice it is difficult to find an idea negative control virus. An ideal negative control virus for Ad36 would be the one that exactly mimics the effects of Ad36, but is non-adipogenic. A virus that enters a cell will induce its own set of modulation of cellular machinery, which may need another control. Hence, finding a perfect negative control for Ad36 is extremely difficult and perhaps, a proper control for “infection” is “no infection”. Nonetheless, it could be concluded that not all human adenoviruses posses the adipogenic effect like Ad36.
Subsequent to observing the modulation of cellular mRNA expression by Ad36, we wished to determine how the virus modulates cellular response. In a permissive cell, Ad36 attaches to a cell receptor, next it is internalized, which is followed by viral mRNA expression, DNA replication and virus assembly followed by the release of newly formed virus particles. A cellular change may be induced by attachment of a virus to a cell-receptor, or more actively, by viral mRNA, DNA or protein interaction with cellular counterparts. Broadly, Ad36 genes are grouped under early, and late genes, based on their initiation in virus replication cycle. Early genes are involved in inducing viral gene expressions, promoting cell cycle progression and blocking apoptosis, and viral DNA replication. The expression of late genes is involved in completing assembly of new virus particles. Despite the expression of early or late viral genes, 3T3-L1 cells do not support assembly of new Ad36 particles as indicated by a decrease in viral DNA(21
) or a lack of viral structural protein formation (unpublished observations).
This narrowed the focus to cellular attachment of Ad36, or to subsequent mRNA expression as mediators of cellular modulations. Sufficient time was allowed for cellular attachment and internalization of Ad36 before the CF treatment. Once the virus is internalized, CF interferes with its replication as evident by a reduction in viral mRNA expression of early viral genes E1a and E4orf1. This was accompanied with attenuation of adipogenic effects induced by Ad36. This suggests the process requires viral mRNA (more likely viral proteins) and that the cellular attachment of Ad36 is not adequate for inducing cellular modulations. These findings also agree with our recent data showing a lack of adipogenic effect of ultra-violet light (UV) inactivated Ad36(25
). UV irradiation induces DNA damage, but preserves surface proteins of a virus particle for cellular attachment, which was not adequate for inducing adipogenic effects of Ad36. Another approach to determine the effect of receptor engagement is to block internalization of the attached virus, which is particularly challenging because the exact receptor used by Ad36 is unknown.
Adipogenic inducers, such as M,D and I are required to experimentally stimulate differentiation of confluent preadipocyte cell lines. Lipid accumulation by Ad36 was 3-fold greater when MDI was added, compared to that without MDI (), which may be due to a synergistic effect of Ad36 and MDI or an additive effect of Ad36 through a novel pathway. Greater upregulation of adipogenic gene expression by Ad36 + MDI compared to MDI alone () supports a synergistic effect. Also, M,D and I can potentially induce adipogenesis in most cells in a plate, whereas, the effect of Ad36 may be limited to only the infected cells. This, coupled with lack of spread of virus infection in 3T3-L1 cells can limit the lipid accumulation attained by Ad36. Despite the limitations, even in the absence of MDI, Ad36 nearly doubled lipid accumulation compared to uninfected control. This was further assessed by observing Ad36 induced differentiation in selective presence of inducers. Percent increase in lipid accumulation in Ad36 group compared to respective uninfected control varied greatly between groups (MDI 27.5%, No-MDI 72%, MD 100%, MI 18% and DI 11%). Overall, those groups without insulin stimulation had much lower levels of lipid accumulation, suggesting the significance of insulin in preadipocyte differentiation and lipid accumulation. Interestingly, Ad36 was unable to increase lipid accumulation (DI group) in absence of M – which is required for increasing cAMP levels. The critical role of cAMP in Ad36 induced lipid accumulation is also supported by our previous findings showing marked increase in its levels by Ad36 in adipocyte progenitors(19
). Overall, it is well documented that adipogenic induction is required for differentiation of preadipocytes in vitro. Ability of Ad36 to induce adipogenic program without conventional inducers suggests the presence of exogenous modulators of the program. It is likely that Ad36 interacts with an unknown upstream target, which initiates an adipogenic program similar to that induced by MDI.
Ad36 appears to strongly promote cell proliferation. Due to increasing cell number in Ad36 infected group, lipid expression was normalized to cell count. The total lipid unadjusted for cell number was even greater for Ad36 group, compared to control. Thus, Ad36 increases accumulation of lipid in an infected cell and the cell number. This led us to determine the lipogenic ability of Ad36 in proliferating vs confluent cells. Reaching confluence before adipogenic induction is important for adequate differentiation of 3T3-L1 cells. Thus, the two potential variables, number of days post infection as well as post confluence, may influence lipid accumulation. Ad36 infection of confluent cells induced greater lipid accumulation vs that of subconfluent proliferating cells, determined after similar days post infection or confluence in presence or absence of MDI. Possibly, the lower lipogenic response of Ad36-infected subconfluent 3T3-L1 cells was due to fewer cells present for viral infection compared to those infected at confluence and because the infection does not spread in 3T3-L1 cells. Future experiments will determine if infection of proliferating human preadipocytes carry Ad36 in daughter cells, which may increase the adipogenic impact of the virus.
In summary, Ad36 is an inducer of adipogenic program in preadipocytes, and this single factor appears to qualitatively mirror the combined adipogenic effects of M, D, and I. This phenomenon is of particular relevance since Ad36 increases adiposity in experimentally infected animal models(5
) and preadipocytes of humans naturally infected with the virus show greater potential to differentiate and accumulate lipid(10
). Adipogenic potential of Ad36 has been recently re-confirmed in vivo by others(26
) and several other adipogenic adenoviruses have been reported (27
). Considering very high prevalence of adenovirus infection in population, it would be important to determine the adipogenic effect, if any, of certain adenovirus serotypes on preadipocyte differentiation. Importantly, the exact cellular target(s) of Ad36 that initiate the adipogenic response is(are) unclear. Identifying hitherto unknown regulators of cellular adipogenesis may provide additional tools to better understand and effectively manipulate preadipocyte differentiation.