Two distinct groups of obesity-discordant MZ pairs
The obesity-discordant pairs had a mean ∆weight of 17.4 kg (24%) and large differences in all adiposity measures (Table 1). However, for liver fat two very distinct subgroups emerged within the obesity-discordant pairs (Fig. 1). In half of these pairs, the obese co-twins had liver fat percentages as low as those of their leaner co-twins (∆liver fat 8%, Wilcoxon’s test between co-twins p = 0.21, group 1, Fig. 1, n = 8 pairs), whereas in the other half, the obese co-twins had strikingly increased liver fat content (∆liver fat 718%, p = 0.012, group 2, Fig. 1, n = 8 pairs).
Obesity-discordant pairs concordant and discordant for liver fat (Fig. 2a) did not differ for measures of overall fatness. Both groups had similar ∆weight (group 1, mean 17.4 kg, range 11.1–30.3 kg; group 2, mean 17.1 kg, range 9.6–25.8 kg) and ∆BMI (group 1, mean 5.9 kg/m2; range 3.8–10.0 kg/m2; group 2, mean 5.5 kg/m2, range 3.1–8.3 kg/m2). Neither did the groups differ for ∆SAT (Fig. 2b). However, ∆VAT was larger in group 2 than in group 1 pairs (Fig. 2c). Examples of twin pairs in each of the groups are presented in ESM Fig. 1.
Glucose and lipid metabolism and blood pressure
In group 1, there were no differences between the obese and lean co-twins in glucose and insulin curves during the OGTT (Fig. 3). The co-twins also had similar insulin resistance (HOMA-IR, Fig. 2d) and insulin sensitivity (Matsuda, Fig. 2e). In group 2, the obese co-twins had a significantly higher AUC for glucose (23%, p = 0.028) and insulin (78%, p = 0.028) during the OGTT (Fig. 3), as well as a 119% (p = 0.018) higher HOMA-IR and 55% (p = 0.028) lower Matsuda index than their lean co-twins (Fig. 2d, e). The obese co-twins in group 2 had significantly higher LDL- and lower HDL-cholesterol than their leaner co-twins (Table 1), while in group 1 the circulating lipids did not differ between co-twins. The blood pressure of the co-twins in both groups were similar, but a trend for increased blood pressure was observed in obese co-twins in group 2 (Table 1).
The obese co-twins in group 2 had significantly more liver fat (p = 0.0008) and VAT (p = 0.021), lower Matsuda index (Mann–Whitney U test p = 0.006) and higher HOMA-IR (p = 0.049) than the obese co-twins in group 1. Lean co-twins in group 2 did not differ from lean co-twins in group 1 in any of the metabolic values. Furthermore, co-twins from the weight-concordant group had similar metabolic measures (Table 1).
SAT cellularity and gene expression
Compared with the leaner co-twins, the obese co-twins had 11% more adipocytes in group 1 (p = 0.069) but 8% less in group 2 (p = 0.13) and, accordingly, ∆adipocyte cell number differed significantly between the groups (p = 0.037). The mean adipocyte cell size was increased in all obese co-twins (53%, p = 0.017 in group 1 and 69%, p = 0.018 in group 2) (Table 1).
Initially, a hypothesis-free GO enrichment analysis was done using the ‘Molecular function’ category to identify significant pathways. No differences were identified in the group of weight-concordant twins. Group 1 discordant twins differed only for ‘structural constituent of ribosome’ (GO:0003735) (FDR corrected p = 0.016). Next, a mean centroid of this pathway was constructed, revealing a decreased transcriptional activity in every obese co-twin (also in group 2, although not reaching statistical significance) (Fig. 2f). In contrast, the group 2 obese and lean co-twins differed significantly for mitochondrial pathways in the obese co-twin, namely ‘oxidoreductase activity’ (GO:0016491) (FDR corrected p = 0.045) and ‘Cofactor binding’ (GO:0048037) (p = 0.0096), the latter of which also included several mitochondrial genes. These pathway activities were lower in the obese co-twins and restricted to group 2 (Fig. 2g, h).
Based on these findings, additional pathways of interest were studied to explain biological differences between groups 1 and 2. Three mitochondrial pathways were selected to represent different aspects of energy handling. All of them were downregulated in the SAT of group 2, but not group 1 obese co-twins: oxidative phosphorylation pathway (p = 0.48 in group 1, p = 0.028 in group 2) (Fig. 2i); BCAA catabolism pathway (p = 0.48 in group 1, p = 0.018 in group 2) (Fig. 2j) and fatty acid β oxidation pathway (p = 1.0 in group1, p = 0.018 in group 2) (Fig. 2k). Along with decreased catabolism, or clearance of BCAAs, circulating levels of BCAAs were increased in the obese co-twins compared with their lean counterparts (p = 0.016), but in within-group analyses the difference was statistically significant only in group 1 twins (Table 1).
We also tested two pathways relating to SAT enlargement (i.e. triacylglycerol synthesis and adipocyte cell differentiation). The triacylglycerol synthesis pathways did not differ between the obese and lean co-twins’ SAT in either of the groups (Fig. 2m). In contrast, the adipocyte differentiation pathway was significantly downregulated in group 2 obese co-twins (p = 0.004), whereas the pathway expressions in group 1 co-twins were similar (p = 0.33) (Fig. 2l). Further, the major transcript responsible for SAT lipolysis, LIPE, suggested that lipolysis was downregulated in group 2 but not in group 1 obese co-twins (ESM Fig. 2a).
Finally, significantly elevated activity of the chronic inflammatory response pathway (CIRP) was observed in the obese co-twins of group 2 (p = 0.028) but not group 1 (p = 0.31) (Fig. 2n). The CIRP pathway was also significantly more active in the obese co-twins from group 2 when compared with the equally obese co-twins from group 1 (p = 0.037) (Fig. 2n). Circulating hsCRP levels also differed between the co-twins only in group 2 (Fig. 2o).
Adipokines in serum and in SAT
In both groups 1 and 2, the obese co-twins had significantly higher SAT expression and plasma levels of leptin than the non-obese co-twins (Table 1 and ESM Fig. 2b). Comparisons of group 1 and 2 were performed on sex-adjusted values, because both circulating (p < 0.001) and SAT leptin mRNA levels (p = 0.0095) were higher in women than in men. Despite similar BMI differences, plasma leptin concentrations showed larger intra-pair differences in group 2 than in group 1 (p = 0.014) suggesting that circulating leptin levels were disproportionally increased in the obese twins with adipose tissue inflammation and fatty livers. The obese co-twins had lower expression levels of adiponectin in adipose tissue only in group 2 (p = 0.018 vs p = 0.58 in group 1) (ESM Fig. 2c). In plasma, ∆adiponectin was similar in both groups, but within-pair differences were statistically significant only in group 1 (Table 1).
Age, sex, lifestyle factors and onset of obesity
Groups 1 and 2 were similar with regards to age, sex, smoking habits, alcohol intake, total physical activity and mean daily energy intake (Table 1). In group 2, sports activity was lower and alcohol intake tended to be higher in the obese than in lean co-twins. The onset of obesity discordance between the two groups was similar (18.8 and 20.0 years in groups 1 and 2, respectively).
Independent associations of adipose tissue function and liver fat
∆Liver fat correlated with ∆VAT (r = 0.40, p = 0.038) but not with ∆SAT (r = 0.17, p = 0.39). Of the metabolic measures studied, ∆liver fat correlated with ∆CIRP pathway (r = 0.44, p = 0.026) and ∆plasma leptin (r = 0.51, p = 0.011). In a multivariate model including both ∆VAT and ∆SAT and confounding factors (twin-pair sex, age and ∆sports activity), ∆VAT was the only variable independently explaining ∆liver fat (β = 4,800 ± 1,600 cm3, p = 0.007, adjusted R
2 in the whole model 31%, p = 0.023). However, when we included the two metabolic measures of SAT that were in univariate analyses associated with ∆liver fat (∆CIRP pathway expression and ∆plasma leptin), the results changed completely. The independent contributors to ∆liver fat in the subsequent model were ∆CIRP (β = 2.4 ± 1.0, p = 0.025), ∆leptin (β = 0.00017 ± 0.000056 pg/ml, p = 0.008), age (β = 0.29 ± 0.12 years, p = 0.030) and reduced ∆SAT (β = −1,600 ± 600 cm3, p = 0.017). The whole model explained 48% of variation in ∆liver fat (p = 0.019).