Hypomorphism of Fto and Rpgrip1l causes obesity in mice
George Stratigopoulos, … , Dieter Egli, Rudolph L. Leibel
George Stratigopoulos, … , Dieter Egli, Rudolph L. Leibel
Published April 11, 2016
Citation Information: J Clin Invest. 2016;126(5):1897-1910. https://doi.org/10.1172/JCI85526.
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Research Article Metabolism

Hypomorphism of Fto and Rpgrip1l causes obesity in mice

Abstract

Noncoding polymorphisms in the fat mass and obesity-associated (FTO) gene represent common alleles that are strongly associated with effects on food intake and adiposity in humans. Previous studies have suggested that the obesity-risk allele rs8050136 in the first intron of FTO alters a regulatory element recognized by the transcription factor CUX1, thereby leading to decreased expression of FTO and retinitis pigmentosa GTPase regulator-interacting protein-1 like (RPGRIP1L). Here, we evaluated the effects of rs8050136 and another potential CUX1 element in rs1421085 on expression of nearby genes in human induced pluripotent stem cell–derived (iPSC-derived) neurons. There were allele-dosage effects on FTO, RPGRIP1L, and AKT-interacting protein (AKTIP) expression, but expression of other vicinal genes, including IRX3, IRX5, and RBL2, which have been implicated in mediating functional effects, was not altered. In vivo manipulation of CUX1, Fto, and/or Rpgrip1l expression in mice affected adiposity in a manner that was consistent with CUX1 influence on adiposity via remote effects on Fto and Rpgrip1l expression. In support of a mechanism, mice hypomorphic for Rpgrip1l exhibited hyperphagic obesity, as the result of diminished leptin sensitivity in Leprb-expressing neurons. Together, the results of this study indicate that the effects of FTO-associated SNPs on energy homeostasis are due in part to the effects of these genetic variations on hypothalamic FTO, RPGRIP1L, and possibly other genes.

Authors

George Stratigopoulos, Lisa Cole Burnett, Richard Rausch, Richard Gill, David Barth Penn, Alicja A. Skowronski, Charles A. LeDuc, Anthony J. Lanzano, Pumin Zhang, Daniel R. Storm, Dieter Egli, Rudolph L. Leibel

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Figure 1

Functional consequences of rs1421085/rs8050136 on expression.

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Functional consequences of rs1421085/rs8050136 on expression. (A) RPGRIP...
(A) RPGRIP1L/FTO/AKTIP genomic location and implicated SNPs. (B) Nonradioactive gel-shift assay with double-stranded (DS) oligonucleotides carrying the obesity-risk C or -protective T allele at rs1421085 mixed with N2a cellular extracts overexpressing P200 or P110. P200 has higher affinity for the rs1421085 C allele, whereas P110 has higher affinity for the rs1421085 T allele. Each error bar represents 3 biological replicates. The experiment was repeated twice. (C) A luciferase assay was used to measure promoter activity. P200 overexpression (CMV-P200) repressed FTO minimal promoter (FTOp) activity in the presence of the putative enhancer sequence carrying the C obesity-risk allele [rs14(C)FTOp], whereas P200 overexpression failed to repress the FTO promoter in the presence of the putative enhancer carrying the T protective allele [rs14(T)FTO]. FTO promoter activity was enhanced upon P110 overexpression (CMV-P110) in the presence of the enhancer carrying the rs1421085 C allele and even more so in the presence of the enhancer carrying the rs1421085 T allele, to which P110 binds with higher affinity than the C allele in vitro. P110 overexpression also increased activity of the RPGRIP1L minimal promoter (RPGRIP1Lp) in the presence of the putative enhancer containing the rs1421085 T allele [rs14(T)RPGRIP1Lp] more so than in the presence of the putative enhancer carrying the rs1421085 C allele [rs14(C)RPGRIP1Lp]. P200 overexpression failed to repress RPGRIP1L promoter activity in the presence of the enhancer carrying the C or T allele. Error bars represent 4 biological replicates. (D) Allele dose-dependent expression of FTO, RPGRIP1L, and AKTIP in neurons heterozygous (n = 4) or homozygous for the obesity-risk (C/A) (n = 4) or -protective (T/C) (n = 6) alleles at rs1421085 and rs8050136. In box-and-whisker plots, horizontal bars indicate the medians, boxes indicate 25th to 75th percentiles, and whiskers indicate 10th and 90th percentiles. Error bars represent SEM. Statistical significance was determined by 2-tailed paired Student’s t test or ANOVA. P α values lower than 0.05 were considered significant.