Gene expression profiling of white adipose tissue reveals paternal transmission of proneness to obesity

Abstract

Previously, we found that C57BL/6J (B6) mice are more prone to develop obesity than PWK mice. In addition, we analyzed reciprocal crosses between these mice and found that (PWK × B6) F1 mice, which have B6 fathers, are more likely to develop dietary obesity than (B6 × PWK) F1 mice, which have B6 mothers. These results suggested that diet-induced obesity is paternally transmitted. In this study, we performed transcriptome analysis of adipose tissues of B6, PWK, (PWK × B6) F1, and (B6 × PWK) F1 mice using next-generation sequencing. We found that paternal transmission of diet-induced obesity was correlated with genes involved in adipose tissue inflammation, metal ion transport, and cilia. Furthermore, we analyzed the imprinted genes expressed in white adipose tissue (WAT) and obesity. Expression of paternally expressed imprinted genes (PEGs) was negatively correlated with body weight, whereas expression of maternally expressed imprinted genes (MEGs) was positively correlated. In the obesity-prone B6 mice, expression of PEGs was down-regulated by a high-fat diet, suggesting that abnormally low expression of PEGs contributes to high-fat diet-induced obesity in B6 mice. In addition, using single-nucleotide polymorphisms that differ between B6 and PWK, we identified candidate imprinted genes in WAT.

Figure 1. Number of differentially expressed genes (>2.0-fold) in WAT of mice fed a HFD vs. mice fed a control diet.

(a) Generation of reciprocal crosses between B6 and PWK mice. (B6 × PWK) F1 is the offspring of a B6 mother and PWK father, and (PWK × B6) F1 is the offspring of a PWK mother and B6 father. (b) Number of genes shown by NGS analysis to be differentially expressed in WAT of HFD mice relative to WAT of mice fed a control diet. Red bars indicate the number of up-regulated genes (p < 0.05, >2-fold), and blue bars indicate the number of down-regulated genes (p < 0.05, >2-fold).

Figure 2. Number of genes with paternal or maternal-dependent expression changes in WAT.

The upper panel shows up-regulated genes, and the lower panel shows down-regulated genes. For example, a total of 271 genes were significantly up-regulated (p < 0.05, >2.0-fold) in both B6 and (PWK × B6) F1, but not in PWK or (B6 × PWK) F1; these were defined as B6 paternal allele-dependent up-regulated genes. By contrast, 226 genes were significantly up-regulated (p < 0.05, >2.0-fold) in both B6 and (B6 × PWK) F1, but not in PWK and (PWK × B6) F1; these were defined as B6 maternal allele-dependent up-regulated genes.

Figure 3. Correlation between body weight and expression of PEGs and MEGs.

(a) Correlation between body weight and expression of 27 PEGs and 32 MEGs in WAT, presented as box plots. (b) Examples of correlation between body weight and expression of PEGs or MEGs. Igf2, Plagl1, Peg3, and Gpr1 are PEGs, and Dhcr7, Gatm, Pon2, and Rian are MEGs. Expression were baselined to the median of each gene. (c) Differential expression of 27 PEGs and 32 MEGs between HFD and control diet B6 mice (HFD – Con). (d) Examples of differential expression of imprinted genes between HFD and control diet B6 mice. Expression of HFD and control diet B6 mice were presented. Expression were baselined to the median of each gene. Dbc, Slc38a, Dlk1, and Igf2 are PEGs, and Asb4, Tnfrsf26, Tnfrsf23, and Gatm are MEGs.

Figure 4. Screening for PEGs and MEGs in WAT.

(a) Strict selection criteria for identification of new PEGs. (b) Mild selection criteria for identification of new PEGs. (c) Strict selection criteria for identification of new MEGs. (d) Mild selection criteria for identification of new MEGs.

Figure 5. PWK allele expression at SNP loci.

(a) PWK allele expression at several SNP loci in known imprinted genes (Igf2, H13) and candidate imprinted genes (Bmp3, Ces1f). The map of each locus is provided beneath the graph. (b) Ratio of expression (B6 allele: PWK allele) of candidates. These results were confirmed by RT-PCR followed by digestion with restriction enzymes whose recognition sites contained SNPs between B6 and PWK.

Figure 6. Venn diagram of differentially expressed genes (>5.0-fold) in WAT of PWK fed a control diet or HFD, relative to WAT of B6 mice fed a control diet.

(a) Yellow circle indicates genes up-regulated in WAT of PWK fed a control diet relative to WAT of B6 fed a control diet (p < 0.05, >5.0-fold). Pink circle indicates genes up-regulated in WAT of PWK fed a HFD relative to WAT of B6 fed a control diet (p < 0.05, >5.0-fold). (b) Blue circle indicate genes down-regulated in WAT of PWK fed control diet relative to WAT of B6 with control diet (p < 0.05, >5.0-fold). Purple circle indicates genes down-regulated in WAT of PWK fed a HFD relative to WAT of B6 fed a control diet (p < 0.05, >5.0-fold). (c) Assessment of mouse brown and beige adipocyte markers in WAT of PWK and B6 mice. Numbers indicate fold change in expression in WAT of PWK relative to WAT of B6. (−) indicates no significant difference in expression between PWK and B6.