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. 2022 Sep 27:12:1016200.
doi: 10.3389/fcimb.2022.1016200. eCollection 2022.

Adipocyte commitment of 3T3-L1 cells is required to support human adenovirus 36 productive replication concurrent with altered lipid and glucose metabolism

Verónica Márquez  1 , Grisel Ballesteros  1 , Thomas Dobner  2 , Ramón A González  1
Affiliations

Adipocyte commitment of 3T3-L1 cells is required to support human adenovirus 36 productive replication concurrent with altered lipid and glucose metabolism

Verónica Márquez et al. Front Cell Infect Microbiol. .

Abstract

Human adenovirus 36 (HAdV-D36) can cause obesity in animal models, induces an adipogenic effect and increased adipocyte differentiation in cell culture. HAdV-D36 infection alters gene expression and the metabolism of the infected cells resulting in increased glucose internalization and triglyceride accumulation. Although HAdV-D36 prevalence correlates with obesity in humans, whether human preadipocytes may be targeted in vivo has not been determined and metabolic reprogramming of preadipocytes has not been explored in the context of the viral replication cycle. HAdV-D36 infection of the mouse fibroblasts, 3T3-L1 cells, which can differentiate into adipocytes, promotes proliferation and differentiation, but replication of the virus in these cells is abortive as indicated by short-lived transient expression of viral mRNA and a progressive loss of viral DNA. Therefore, we have evaluated whether a productive viral replication cycle can be established in the 3T3-L1 preadipocyte model under conditions that drive the cell differentiation process. For this purpose, viral mRNA levels and viral DNA replication were measured by RT-qPCR and qPCR, respectively, and viral progeny production was determined by plaque assay. The lipogenic effect of infection was evaluated with Oil Red O (ORO) staining, and expression of genes that control lipid and glucose metabolism was measured by RT-qPCR. In the context of a viral productive cycle, HAdV-D36 modulated the expression of the adipogenic genes, C/EBPα, C/EBPβ and PPARγ, as well as intracellular lipid accumulation, and the infection was accompanied by altered expression of glucolytic genes. The results show that only adipocyte-committed 3T3-L1 cells are permissive for the expression of early and late viral mRNAs, as well as viral DNA replication and progeny production, supporting productive HAdV-D36 viral replication, indicating that a greater effect on adipogenesis occurs in adipocytes that support productive viral replication.

Keywords: adipocyte commitment; human adenovirus 36; lipid and glucose metabolism; obesity; permissive replication.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Experiment workflow. 3T3-L1 cells were infected with HAdV-D36 or mock-infected at 2 or 12 days postconfluence (2 dpc or 12 dpc, respectively), in the absence or presence of differentiation factors (MM or MDI, respectively). The cells were harvested at the indicated time-points when measurements of viral and cellular mRNA, viral DNA, viral progeny production and intracellular lipids were performed as described in materials and methods.
Figure 2
Figure 2
3T3-L1 cells at 12 dpc show characteristics of differentiation committed preadipocytes. Differentiation of 3T3-L1 preadipocytes was induced by incubation postconfluence and addition of MDI. RNA was isolated from 3T3-L1 cells at 2 dpc (A) or 12 dpc (B), and expression levels of C/EBPβ, C/EBPα, and PPARγ were analyzed by RT-qPCR. Intracellular lipid accumulation was observed by ORO staining and white field microscopy (C). Scale bar = 100 μm. Cell numbers were measured using a Neubauer chamber (D). Changes in the percentage of cells with lipid accumulation were counted using ORO staining and white field microscopy (E), and the relative quantities of lipid accumulation were determined by elution of ORO staining, measured at 510 nm Optical Density (OD) (F). Primers were designed to hybridize at exon-exon junctions to measure mature mRNA. All values represent the mean of two independent experiments, measured in technical duplicates. Data are expressed as the mean and error bars represent standard deviations. Significant differences from each time-point relative to 3h (A, B) and between Mock- and HAdV-D36-infected cells at each time-point (D-F) are indicated by **p < 0,005. ***p < 0,0005. ****p < 0,00001. ns, not significant.
Figure 3
Figure 3
3T3-L1 cells at 12 dpc, but not at 2 dpc, are permissive for HAdV-D36 replication. 3T3-L1 cells, 2dpc or 12dpc, were infected with HAdV-D36 in the presence of MDI. Viral mRNA expression levels of E1A (A), and Hexon (B) were measured by RT-qPCR. DNA was isolated and analyzed by qPCR (C) and viral progeny production was determined by plaque assay (D). The expression levels of C/EBPβ, C/EBPα and PPARγ was measured by RT-qPCR (E, F). Intracellular lipid accumulation was observed by ORO staining and white field microscopy (G). Scale bar = 100 μm. Cell numbers were measured using a Neubauer chamber (H). Change in the percentage of cells with lipid accumulation in the presence of MDI were counted using ORO staining and white field microscopy (I), the relative quantities of lipid accumulation were determined by elution of ORO staining, measured at 510 nm Optical Density (OD) (J). Primers were designed to hybridize at exon-exon junctions to measure mature mRNA and to hybridize at sequences that correspond to intron-exon junctions to measure DNA. All values represent the mean of two independent experiments, measured in technical duplicates. Data are expressed as the mean and error bars represent standard deviations. Significant differences from each time-point relative to 3h (A–F) and between 2 and 12 dpc at each time-point (H-J) are indicated by **p < 0,005. ***p < 0,0005. ****p < 0,00001. ns, not significant.
Figure 4
Figure 4
HAdV-D36 infection increases adipogenesis in 3T3-L1 cells. 3T3-L1 cells at 12 dpc were infected with HAdV-D36 without adipocyte differentiation inducers (MM) and the expression levels of C/EBPβ, C/EBPα and PPARγ were analyzed by RT-qPCR (A, B). Intracellular lipid accumulation was observed by ORO staining and white field microscopy (C). Scale bar = 100 μm. Cell numbers were measured using a Neubauer chamber (D). Change in the percentage of cells with lipid accumulation in the absence of MDI were counted using ORO staining and white field microscopy (E), the relative quantities of lipid accumulation were determined by elution of ORO staining, measured at 510 nm Optical Density (OD) (F). Primers were designed to hybridize at exon-exon junctions to measure mature mRNA. All values represent the mean of two independent experiments, measured in technical duplicates. Data are expressed as the mean and error bars represent standard deviations. Significant differences from each time-point relative to 3h (A, B) and between Mock- and HAdV-D36-infected cells at each time-point (D-F) are indicated by *p < 0,05. **p < 0,005. ***p < 0,0005. ****p < 0,00001. ns, not significant.
Figure 5
Figure 5
The effect of HAdV-D36 on 3T3-L1 adipogenesis is enhanced by adipocyte differentiation inducers. The effect of infection on 12 dpc 3T3-L1 cells cultured in MDI was evaluated. 12 dpc 3T3-L1 cells were infected with HAdV-D36 in the presence of adipocyte differentiation inducers (MDI) and the expression levels of C/EBPβ, C/EBPα and PPARγ were analyzed by RT-qPCR (A, B). Intracellular lipid accumulation was observed by ORO staining and white field microscopy (C). Scale bar = 100 μm. Cell numbers were measured using a Neubauer chamber (D). Change in the percentage of cells with lipid accumulation in the presence of MDI were counted using ORO staining and white field microscopy (E) the relative quantities of lipid accumulation were determined by elution of ORO staining, measured at 510 nm Optical Density (OD) (F). Primers were designed to hybridize at exon-exon junctions to measure mRNA. All values represent the mean of two independent experiments, measured in technical duplicates. Data are expressed as the mean and error bars represent standard deviations. Significant differences from each time-point relative to 3h (A, B) and between Mock- and HAdV-D36-infected cells at each time-point (D-F) are indicated by *p < 0,05. **p < 0,005. ***p < 0,0005. ****p < 0,00001. ns, not significant.
Figure 6
Figure 6
Adipocyte commitment of 3T3-L1 cells is required to support productive replication of HAdV-D36. 3T3 cells treated with or without adipocyte differentiation inducers (MDI or MM) were infected 12dpc, and RNA and DNA were purified as described in materials and methods. Expression levels of E1A (A), IVa2 (B) and Hexon (C) were measured by RT-qPCR and DNA by qPCR (D). Viral progeny production was determined by plaque assay (E). Primers were designed to hybridize at exon-exon junctions to measure mature mRNA and at sequences that correspond to intron-exon junctions to measure DNA. All values represent the mean of two independent experiments, measured in technical duplicates. Data are expressed as the mean and error bars represent standard deviations. Significant differences from each time-point relative to 3h (A-D) and relative to 6 dpi (E) are indicated by *p < 0,05. **p < 0,005. ***p < 0,0005. ****p < 0,00001. ns, not significant.
Figure 7
Figure 7
HAdV-D36 infection of adipocyte committed cells promotes the expression of MYC- glycolytic target genes. To determine the effect of infection on MYC-target genes, 3T3-L1 cells at 12dpc were HAdV-D36-infected (B, D) or mock-infected (A, C), without adipocyte differentiation inducers (MM) (A, B) or with (MDI) (C, D). RNA was isolated at the indicated time-points and the expression levels of hexokinase (Hk), phosphofructokinase (PFk), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Lactate dehydrogenase (LDHA) were measured by RT-qPCR. All values represent the mean of two independent experiments, measured in technical duplicates. Data are expressed as the mean and error bars represent standard deviations. Significant differences from each time-point relative to 3h are indicated by *p < 0,05. **p < 0,005. ***p < 0,0005. ****p < 0,00001. ns, not significant.
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