A missense mutation in zinc finger homeobox-3 (ZFHX3) impedes growth and alters metabolism and hypothalamic gene expression in mice

Objective: A protein altering variant in the gene encoding zinc finger homeobox-3 (ZFHX3) has recently been associated with lower BMI in a human genome-wide association study. We investigated metabolic parameters in mice harbouring a missense mutation in Zfhx3 (Zfhx3Sci/+) and looked for altered expression of transcripts that are associated with energy balance in the hypothalamus to understand how ZFHX3 may influence metabolic effects. Methods: Body weight, length and composition were measured in 1 year old male and female Zfhx3Sci/+ and Zfhx3+/+ mice, and fat depot weights were measured along with fasted anabolic hormone concentrations. In a second cohort of male and female Zfhx3Sci/+ and Zfhx3+/+ mice weekly food intake was measured from the age of 9 - 20 weeks. A third cohort of female-only Zfhx3Sci/+ and Zfhx3+/+ mice underwent body composition analysis every two weeks from 6 - 14 weeks of age, and 24 h energy expenditure was assessed. At 16 weeks old, brains were collected in the light and dark phases for mRNA in situ hybridisation analysis of candidate genes identified in previous RNA-seq analysis of hypothalamic tissue of Zfhx3Sci/+ mice. Results: We found that 1 year old Zfhx3Sci/+ mice weighed less and had a shorter body length than wildtype littermates, and had reduced fat mass, smaller mesenteric fat depots, and lower circulating insulin, leptin and insulin-like growth factor-1 (IGF1) concentrations. Male and female Zfhx3Sci/+ mice ate less than wildtype controls from 10 weeks of age. In the final experiment, female Zfhx3Sci/+ mice weighed less and had lower lean mass, but fat mass didn't differ. Energy expenditure was lower in Zfhx3Sci/+ mice. We detected increased expression of somatostatin, and decreased expression of growth hormone-releasing hormone and growth hormone-receptor mRNAs in the arcuate nucleus (ARC), but these transcripts were unaltered in other hypothalamic nuclei. Similarly, ARC expression of orexigenic neuropeptide Y was decreased and ventricular ependymal layer expression of orphan G protein-coupled receptor Gpr50 was decreased. Conclusions: The Sci missense mutation conferred a protective metabolic phenotype in mice and implied a somatic growth effect. The promoters of mouse Sst and Gpr50 genes contain ZFHX3 binding AT-motifs, suggesting growth axis effects are driven by transcriptional activation of Sst in the ARC. It is likely that altered Gpr50 gene expression led to decreased energy expenditure and decreased Npy expression in the ARC decreased appetite. Together these results point to a pleiotropic effect of ZFHX3 in regulating energy balance, independent of its circadian effects. ### Competing Interest Statement The authors have declared no competing interest.