The exact mechanism of Ad-36-induced adiposity is unknown. The amount of viral DNA in the adipose tissue of the experimentally infected animal correlates with the mass of the adipose tissue [10
] and in vitro infection of rodent preadipocytes by Ad-36 enhanced lipid accumulation [19
]. These findings suggested that adiposity induced by Ad-36 in animal models involves a direct interaction of the virus with adipose tissue. Considering the strong potential of adipose tissue-resident stem cells to replicate and differentiate into adipocytes, we hypothesized that the observed in vivo adipogenic effect of the virus may involve recruitment of these cells. We tested the hypothesis in hASC, the human adipose tissue-resident multipotent stem cells [28
Adipose-derived stem/stromal cells (ASC) are derived from the stromal vascular fraction cell population; however, as we previously described [32
], they are the adherent population that has been expanded in culture and, based on their surface immunophenotype, represent <5% of the original stromal vascular fraction cells. Clonal analysis of the culture-expanded hASC shows that up to 52% of clonal cells were capable of differentiation into two or more lineages [34
]. Under appropriate stimuli, these cells commit to the adipocyte lineage and accumulate lipid [35
]. To establish human relevance, the cells were obtained from human subjects. Admittedly, intersubject variability is a limitation for human primary cells extracted from lipoaspirate [36
]. However, the protein expression of hASC is highly homogeneous in different donors, and expression of cell surface proteins is similar [20
]. More importantly, we focused on the adipogenic response with or without infection in the same individual instead of interindividual comparison. Samples from different age and BMI groups () exhibited similar behavior when exposed to Ad-36, minimizing the concerns about intersubject variability. Multiple subjects were used to ensure that the cellular response to Ad-36 was not an individual aberration. Also, the cells isolated from an individual are not adequate to conduct all experiments reported here without expanding by passaging several times. To avoid multiple passaging, cells from an individual were used for a limited number of experiments. Critical experiments were repeated in cells obtained from multiple individuals. It is worth noting that cells from all individuals studied showed a consistent response to Ad-36.
Demonstration of permissiveness of hASC to Ad-36 or Ad-2 infection is important to investigate the effect of these viruses on the cells. For instance, Ad-36 enhances differentiation of 3T3-L1 cells (a rodent preadipocyte cell line), but the infection is abortive [19
], whereas Ad-2 requires Coxsackie virus adenovirus receptor for cell entry, and unless overexpressed in 3T3-L1 cells [40
], its infectivity of the cell line is poor. A time-dependent increase in viral gene expression and DNA amount indicated successful entry and replication of Ad-36 and Ad-2 in hASC. It is noteworthy that despite a successful entry and replication in hASC, Ad-2 did not induce adipogenesis, which is consistent with earlier observations of the lack of its adipogenic ability in 3T3-L1 [19
] and in animals [21
]. This indicated that the adipogenic effect of an adenovirus is not due merely to the cell entry but depends on its subsequent specific interaction with host cell machinery. A complete lack of adipogenic effect of Ad-2 on hASC allowed us to focus only on the adipogenic effects of Ad-36 on commitment, differentiation, and lipid accumulation. Unlike A549 cells (human lung cancer cell line), which show characteristic cytopathic effect (CPE) and cell lysis in response to adenovirus infection, no CPE was observed in Ad-36-infected hASC even up to 2 weeks postinfection. Considering the affinity of adenoviruses for the respiratory tract, viral replication and lysis in lung cells are expected. Since increasing amounts of viral mRNA and DNA indicated successful viral replication in hASC, we believe the response of hASC to adenovirus infection may be peculiar to the cell type.
Similar to the approach of McBeath et al. [41
], we used osteogenic media to determine the potency of stimulus by Ad-36 to drive commitment of hASC to adipogenic lineage. Despite the presence of osteogenic media, which induced RunX2, a marker of osteogenic induction in uninfected control cells, Ad-36 induced commitment of hASC to adipocyte lineage. It should be noted that the Ad-36-induced adipogenesis may occur through the process of initiating commitment to the adipocyte lineage by previously undifferentiated ASC and/or by accelerating differentiation of cells that are already committed to the adipocyte lineage.
During the process of preadipocyte differentiation, expressions of C/EBP-β
are followed by those of C/EBP-α
and culminate in lipid accumulation [42
expression is critical for activation of PPAR-γ
and other downstream proadipogenic genes [43
expression, together with that of C/EBP-α
, leads to activation of several downstream genes, including glycerol 3-phosphate dehydrogenase and aP2 [42
], and consequent completion of the differentiation process. Robust upregulation of expression of these key indicators of adipogenic pathway convincingly demonstrated the induction of adipogenesis by Ad-36 in hASC. In addition to a direct effect on the cellular adipogenic program, Ad-36 may contribute to adipogenesis by modulating inflammatory cytokine response, which was not tested in this study.
LPL is a key enzyme for uptake of triglyceride-delivered fatty acids to adipose or muscular tissue [46
]. Increased adipocyte LPL activity increases lipid storage in adipocytes and clears free fatty acid from circulation [48
]. Activation of LPL, suggested by its extracellular translocation, shows transformation of Ad-36-infected hASC to cells with potentially functional capabilities as adipocytes.
Because of the potential significance of the findings, Ad-36-induced lipid accumulation was determined in multiple experiments and by using oil red O and BODIPY assays. The oil red O assay allowed quantification of lipid after extraction from cells, whereas BODIPY helped visualize and quantitate lipid in intact cells. Viral dose determined the amount of lipid accumulated and the number of infected cells. At a given viral dose, infection spread with time. Lipid accumulation per cell was also increased in a time-dependent manner for a given MOI. This increase may be due to accumulation of more lipid in an infected cells and/or recruitment of more cells due to the spreading infection. Collectively, this suggests a time- and virus dose-dependent increase in adiposity in vivo.
In light of the prevailing view that specific adipogenic inducers are required to commit hASC to the adipocyte lineage [20
], the ability of Ad-36 to induce spontaneous lipid accumulation merits attention. In the presence of adipogenic inducers, Ad-36 accelerated lipid accumulation, which eventually occurred in the uninfected hASC. Overall, Ad-36 appears to be a robust inducer of commitment, differentiation, and lipid accumulation in hASC.
Adipose tissue-resident stem cells are an important source for recruitment to adipogenic lineage and conversion to adipocytes. Excessive or impaired adipogenesis leading to obesity or lipodystrophy, respectively, can play a major role in destabilizing insulin secretion and action, glucose and lipid metabolism, energy balance, immune functions, and reproduction [51
]. This underscores the need to determine various intra- and extracellular regulators of the adipogenic process.
The experiment that screened human adipose tissue samples for natural Ad-36 infection was a blinded study conducted by coauthors in two locations. The presence of Ad-36 DNA in human adipose tissue was determined at one location, whereas the adipogenic potential of hASC obtained from the same adipose tissue samples was determined at the other location. Post hoc comparisons showed significantly greater differentiation potential of ASC from Ad-36 DNA+ subjects. This is the first report of a functional difference associated with natural Ad-36 infection in humans, consistent with our similar observations in hASC experimentally infected with Ad-36.
In summary, these findings support our hypothesis that natural infection with Ad-36 commits ASC to the adipocyte lineage and increases adiposity. The findings facilitate further research to screen other human adenoviruses for their potential adipogenic effects and to screen and identify subgroups of humans with adiposity of adenoviral origin. We believe that recognizing novel etiological factors of human obesity will eventually lead to specific and more effective treatment and/or prevention strategies. In addition, these findings may have implications in other areas of stem cell metabolism, particularly in identifying regulatory controls of adult stem cell commitment. The ability of Ad-36 to induce adipogenic programming in uncommitted cells may provide additional tools to elucidate some of the as yet unknown pathways and the basis to more effectively manipulate adult stem cell commitment.