EFFECT OF THE FTO LOCUS ON MITOCHONDRIAL THERMOGENESIS AND LIPID STORAGE

To identify the biologic processes affected by altered IRX3 and IRX5 expression in adipocytes, we used genomewide expression patterns in brown adipocyte–containing perirenal adipose tissue from a separate cohort of 10 nongenotyped, healthy kidney donors to identify genes with expression that was positively or negatively correlated with IRX3 and IRX5 expression. Genes that are associated with mitochondrial functions were found to have a negative correlation with IRX3 and IRX5, and genes with FXR and RXR lipid-metabolism functions were found to have a positive correlation, which suggests that IRX3 and IRX5 may play roles in energy dissipation and storage (Fig. 3A, and Table S1 in the Supplementary Appendix). IRX3 and IRX5 had consistently higher mean expression in white adipose tissue from nine participants, as well as negative correlation with PGC1A and UCP1 expression, as assessed with the use of interindividual expression patterns in perithyroid brown adipose tissue (Fig. S2B and S2C in the Supplementary Appendix); these findings indicated potential roles for IRX3 and IRX5 in the repression of thermogenesis.

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Figure 3

Regulation of Obesity-Associated Cellular Phenotypes in Human Adipocytes by IRX3 and IRX5

Panel A shows the mitochondrial and FXR and RXR activation genes with strongest positive (in red) or negative (in green) correlation with IRX3 and IRX5 in human perirenal adipose tissue from 10 participants. Panels B and C show box plots of the increased adipocyte diameter and decreased mitochondrial DNA content in isolated differentiated adipocytes from risk-allele carriers (16 and 8 participants, respectively) relative to nonrisk-allele carriers (26 and 8, respectively). The vertical line within each box represents the median, the left and right margins of each box indicate the interquartile range, and I bars indicate the range. Panel D shows box plots of the altered basal and isoproterenol-stimulated oxygen consumption rate (OCR) on small interfering RNA (siRNA) knockdown and doxycycline (DOX)–mediated overexpression of IRX3 and IRX5 in 8 risk-allele carriers and 10 nonrisk-allele carriers. The siRNA efficiency was 62% for IRX3 and 71% for IRX5. The horizontal line within each box represents the median, the top and bottom of each box indicate the interquartile range, and I bars indicate the range.

To examine the trans-eQTL genetic control of energy balance by the FTO obesity locus, we used primary preadipocytes from risk-allele carriers and nonrisk-allele carriers to evaluate the genes with mitochondrial and FXR and RXR functions that had expression patterns most closely correlated with those of IRX3 and IRX5, as well as several known markers of energy-balance regulation (Fig. S2D and S2E in the Supplementary Appendix). As compared with nonrisk-allele carriers, risk-allele carriers had lower expression of mitochondrial, browning, and respiration genes and higher expression of lipid-storage markers, which indicated a shift from energy dissipation to energy storage.

These differences in expression were also reflected in the cellular signatures of obesity. Risk-allele carriers had increased adipocyte size, reduced mitochondrial DNA content, and a loss of UCP1 response to β-adrenergic stimulus or cold exposure (Fig. 3B and 3C, and Fig. S2F in the Supplementary Appendix), as well as resistance to isoproterenol-mediated uncoupling, a decreased basal oxygen consumption rate, and a reduction in mitochondrial thermogenesis by a factor of 5 (Fig. S2G in the Supplementary Appendix); this indicated excessive accumulation of triglycerides, reduced mitochondrial oxidative capacity, reduced white adipocyte browning, and reduced thermogenesis.

ADIPOCYTE-AUTONOMOUS EFFECTS OF IRX3 AND IRX5 ON ENERGY BALANCE

We next quantified the effect that manipulation of IRX3 and IRX5 expression had on thermogenesis in primary preadipocytes that were isolated from both risk-allele carriers and nonrisk-allele carriers. In preadipocytes from risk-allele carriers, IRX3 and IRX5 knockdown restored oxygen consumption and thermogenesis response to nonrisk levels, increased thermogenesis by a factor of 7 (Fig. 3D), and restored UCP1 expression levels (Fig. S3A in the Supplementary Appendix). In preadipocytes from nonrisk-allele carriers, IRX3 and IRX5 overexpression reduced basal respiration and thermogenesis to risk-allele levels (with thermogenesis reduced by a factor of 8) (Fig. 3D) and decreased the expression of UCP1, other regulators of mitochondrial function and thermogenesis (PGC1A, PGC1B, and PRDM16), and the β-adrenergic receptor (ADRB3), which also regulates UCP1-independent thermogenesis programs (Fig. S3B and S3C in the Supplementary Appendix). These manipulations had no significant effect on preadipocytes from participants with the reciprocal genotypes, which indicated that IRX3 and IRX5 levels recapitulate the effect that the FTO genetic variant has on thermogenesis.

To examine the organism-level effects of the repression of Irx3 in adipose tissue, we used adipose Irx3 dominant-negative (aP2-Irx3DN) mice. These mice had pronounced antiobesity characteristics, including reduced body size, body weight, fat mass, white and brown fat depots, and adipocyte size (Fig. S4A through S4G in the Supplementary Appendix). These aP2-Irx-3DN mice also had resistance to weight gain on a high-fat diet, increased energy expenditure both at night and during the day, and increased oxygen consumption both at room temperature (22°C) and in thermoneutral conditions (30°C), but they did not have significant differences from control mice in food intake or locomotor activity (Fig. S4A and S4H through S4L in the Supplementary Appendix). At the molecular and cellular levels, these mice had increased mitochondrial activity and thermogenesis marker expression, reduced lipid-storage marker expression in both white and brown fat compartments, and markedly smaller adipocytes than did control mice (Fig. S4M, S4N, and S4O in the Supplementary Appendix).

We next evaluated the tissue-autonomous versus brain-mediated roles of Irx3 by comparing the aP2-Irx3DN mice with hypothalamus dominant-negative Ins2-Irx3DN mice.¹⁹ The aP2-Irx3DN mice had a reduction in fat-mass ratio that was 3 times as great as that in Ins2-Irx3DN mice (a reduction of 57% vs. 19%), despite the fact that transgene expression in the hypothalamus was 3 times lower than that in Ins2-Irx3DN mice (Fig. S4P and S4Q in the Supplementary Appendix), which indicated that Irx3 has a hypothalamus-independent regulatory role in whole-body energy regulation. The phenotypic effects of Irx3 repression in aP2-Irx3DN mice were also stronger than those in whole-body Irx3 knockout mice, which suggested potential dominant repressor effects in adipocytes or other tissues, and were independent of Fto gene expression, which did not change (Fig. S4P and S4R in the Supplementary Appendix).

Our findings indicate that both Irx3 and Irx5 have cell-autonomous roles: manipulation of Irx3 and Irx5 led to energy-balance differences in three mouse cellular models, including mouse embryonic fibroblast–derived adipocytes, white 3T3-L1 preadipocytes, and β-adrenergic–stimulated beige ME3 preadipocytes (Fig. S5 in the Supplementary Appendix). In each case, our results indicated that Irx3 and Irx5 induced adipocyte lipid accumulation and repressed thermogenesis in a cell-autonomous way.

DETERMINATION OF THE CAUSAL VARIANT AND DISRUPTION OF REPRESSION BY ARID5B

To predict the causal variant, the disruption of which is necessary and sufficient to cause IRX3 and IRX5 dysregulation in human preadipocytes, we used phylogenetic module complexity analysis (PMCA)²⁵ (Fig. 4A and 4B, and Fig. S6A and S6B in the Supplementary Appendix). The highest PMCA score was found for the rs1421085 T-to-C SNV, which is in perfect linkage disequilibrium with the most significant reported SNV, rs1558902, across multiple populations (1000 Genomes Phase 1 data), a finding that is consistent with a potentially causal role.

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Figure 4

Disruption of a Conserved ARID5B Repressor Motif by Causal SNV rs1421085 in Humans

Panel A shows disruption of an ARID5B repressor motif in the evolutionarily conserved motif module surrounding rs1421085. The sequences shown at the top of the panel indicate the frequencies of each nucleotide, with the size scaled to indicate the information content (measured as entropy) at each position. Panel B shows adapted phylogenetic module complexity analysis (PMCA)²⁵ scores in the FTO region for all 82 noncoding SNPs in LD (r²≥0.8) with tag SNV rs1558902, which was identified in a genomewide association study²⁶; rs1421085 had the maximal score. Chromatin state annotation is shown for Roadmap Epigenomics reference genome E025, which corresponds to adipose-derived mesenchymal stem cells; for information on the colors used to denote chromatin states, see Figure S1A in the Supplementary Appendix. Panel C shows increased endogenous expression of IRX3 and IRX5 on single-nucleotide T-to-C editing of rs1421085 in the nonrisk haplotype of a nonrisk-allele carrier, using CRISPR–Cas9 (five clonal expansions). CRISPR–Cas9 reediting from the engineered C risk allele back to a T nonrisk allele with the use of an alternative single guide RNA restores low endogenous IRX3 and IRX5 gene expression. Panel D shows reduced expression of IRX3 and IRX5 on C-to-T editing of the risk allele in adipocyte progenitors from a risk-allele carrier. Knockdown of ARID5B increases IRX3 and IRX5 levels, as compared with the nontargeting control (siNT), only in the rescued allele, but not in the risk allele.

To evaluate whether rs1421085 plays a causal role in enhancer activity, we introduced the C allele into the nonrisk haplotype in our luciferase reporter assay. The T-to-C single-nucleotide alteration increased enhancer activity levels for 10-kb and 1-kb segments centered on the variant, in both orientations and both upstream and downstream of the transcription start, which indicated a gain of enhancer activity in association with the rs1421085 risk allele (Fig. S6C and S6D in the Supplementary Appendix).

To evaluate the effect of the variant on regulator binding, we used electrophoretic mobility-shift assays (EMSAs) of adipocyte nuclear extract with probes for the risk allele and the nonrisk allele of rs1421085. We found binding for the nonrisk allele, T, which lacked enhancer activity, but no binding for the risk allele, C; this indicated that the increased enhancer activity associated with the risk allele is probably due to a loss of repressor binding rather than to a gain of activator binding (Fig. S6E in the Supplementary Appendix).

We examined disrupted motifs and regulator expression to identify potential upstream regulators. The T-to-C substitution disrupted conserved motifs for NKX6-3, LHX6, and the ARID family of regulators (Fig. 4A). Among them, ARID5B had the highest expression in adipose tissue and adipocytes and was bound specifically to the nonrisk allele in EMSA competition experiments (Fig. S6E and S6F in the Supplementary Appendix). ARID5B is known to play both repressive and activating roles and was previously implicated in adipogenesis and lipid metabolism in mice.^27,28. Among nonrisk-allele carriers, expression of ARID5B was negatively correlated with expression of IRX3 and IRX5, a finding consistent with ARID5B having a repressive role. No correlation was found in risk-allele carriers, which indicates a loss of ARID5B regulation (Fig. S6G in the Supplementary Appendix).

To evaluate the causal role of ARID5B, we next examined the effects of its knockdown and overexpression on IRX3 and IRX5. ARID5B knockdown increased IRX3 and IRX5 expression in primary preadipocytes from nonrisk-allele carriers to risk-allele levels, which indicates a loss of repression, but it had no effect on preadipocytes from risk-allele carriers, which indicates epistasis with the obesity-risk haplotype (Fig. S6H in the Supplementary Appendix). Consistent with this finding, in SGBS enhancer assays, ARID5B knockdown increased the activity of preadipocytes with the nonrisk allele to risk-allele levels, which indicates a loss of repression, but had no effect on risk-allele constructs, indicating epistasis with the rs1421085 risk allele (Fig. S6I in the Supplementary Appendix). ARID5B overexpression further reduced IRX3 and IRX5 levels in nonrisk-allele carriers, which indicated that repression was strengthened, but had no significant effect on risk-allele carriers, a finding consistent with impaired ARID5B repression in association with the risk haplotype (Fig. S6J in the Supplementary Appendix).

We also evaluated the cellular effects of ARID5B-directed perturbations in primary preadipocytes from risk-allele carriers and nonrisk-allele carriers. In preadipocytes from nonrisk-allele carriers, ARID5B knockdown reduced basal oxygen consumption and lipolysis (Fig. S6K and S6L in the Supplementary Appendix) and shifted expression patterns from mitochondrial to lipid markers (Fig. S2E in the Supplementary Appendix), which indicated that ARID5B plays causal roles in energy-balance regulation. In contrast, ARID5B knockdown had no effect on preadipocytes from risk-allele carriers, a finding consistent with a loss of ARID5B control.

These results suggest that the FTO obesity variant acts through disruption of ARID5B binding in the risk haplotype, leading to a loss of repression, a gain of enhancer activity, and increases in IRX3 and IRX5 expression (Fig. S6M in the Supplementary Appendix).

Supported by grants from the Clinical Cooperation Group “Nutrigenomics and Type 2 Diabetes” of the Helmholtz Zentrum München, German Research Center for Environmental Health, and Technische Universität München, Freising-Weihenstephan (to Dr. Claussnitzer); fellowships from the Heart and Stroke Foundation of Canada and the Hospital for Sick Children (to Dr. Kim); grants from the Canadian Institute of Health Research (to Drs. Hui and Drucker); and grants from Else Kröner-Fresenius Foundation, Virtual Institute “Molecular Basis of Glucose Regulation and Type 2 Diabetes” received from the Helmholtz Zentrum München, Western Norway Regional Health Authority, the Swedish federal government under the LUA/ALF agreement, and the National Institutes of Health (R01HG004037, R01GM113708, R01HG008155, and RC1HG005334).