TET3 epigenetically controls feeding and stress response behaviors via AGRP neurons
Di Xie, … , Tamas L. Horvath, Yingqun Huang
Di Xie, … , Tamas L. Horvath, Yingqun Huang
Published October 3, 2022
Citation Information: J Clin Invest. 2022;132(19):e162365. https://doi.org/10.1172/JCI162365.
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Research Article Metabolism Neuroscience

TET3 epigenetically controls feeding and stress response behaviors via AGRP neurons

Abstract

The TET family of dioxygenases promote DNA demethylation by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). Hypothalamic agouti-related peptide–expressing (AGRP-expressing) neurons play an essential role in driving feeding, while also modulating nonfeeding behaviors. Besides AGRP, these neurons produce neuropeptide Y (NPY) and the neurotransmitter GABA, which act in concert to stimulate food intake and decrease energy expenditure. Notably, AGRP, NPY, and GABA can also elicit anxiolytic effects. Here, we report that in adult mouse AGRP neurons, CRISPR-mediated genetic ablation of Tet3, not previously known to be involved in central control of appetite and metabolism, induced hyperphagia, obesity, and diabetes, in addition to a reduction of stress-like behaviors. TET3 deficiency activated AGRP neurons, simultaneously upregulated the expression of Agrp, Npy, and the vesicular GABA transporter Slc32a1, and impeded leptin signaling. In particular, we uncovered a dynamic association of TET3 with the Agrp promoter in response to leptin signaling, which induced 5hmC modification that was associated with a chromatin-modifying complex leading to transcription inhibition, and this regulation occurred in both the mouse models and human cells. Our results unmasked TET3 as a critical central regulator of appetite and energy metabolism and revealed its unexpected dual role in the control of feeding and other complex behaviors through AGRP neurons.

Authors

Di Xie, Bernardo Stutz, Feng Li, Fan Chen, Haining Lv, Matija Sestan-Pesa, Jonatas Catarino, Jianlei Gu, Hongyu Zhao, Christopher E. Stoddard, Gordon G. Carmichael, Marya Shanabrough, Hugh S. Taylor, Zhong-Wu Liu, Xiao-Bing Gao, Tamas L. Horvath, Yingqun Huang

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Figure 5

TET3 negatively regulates the expression of NPY and VGAT.

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TET3 negatively regulates the expression of NPY and VGAT. (A) GT1-7 cell...
(A) GT1-7 cells transfected with NT siRNA were maintained in Lept L (NT siRNA/Lept L) or Lept H (NT siRNA/Lept H) or were transfected with Tet3 siRNA and maintained in Lept H (Tet3 siRNA/Lept H). RNAs were extracted at 12 hours (for Tet3) or 36 hours (for Npy and Slc32a1) following the switch and analyzed by qPCR. n = 3 per group in technical replicates. **P < 0.01 and ***P < 0.001, by 1-way ANOVA with Tukey’s post test. (B) Representative immunoblots for TET3, NPY, and VGAT from GT1-7 cells treated as in A. Proteins were isolated at the 36-hour time point. (C) SH-SY5Y cells transfected with NT siRNA were maintained in Lept L (NT siRNA/Lept L) or Lept H (NT siRNA/Lept H) or were transfected with TET3 siRNA and maintained in Lept H (TET3 siRNA/Lept H). RNA was extracted at 12 hours (for TET3) or 36 hours (for NPY and SLC32A1) following the switch and analyzed by qPCR. n = 3 per group in technical replicates. **P < 0.01 and ***P < 0.001, by 1-way ANOVA with Tukey’s post test. (D) Representative immunoblots for TET3, NPY, and VGAT from SH-SY5Y cells treated as in C. Proteins were isolated at the 36-hour time point. (E and F) Representative micrographs of NPY (green) and VGAT (green) in the ARCs of Cas9+ mice injected with AAV or AAV-sgTet3. AGRP neurons from the injected viruses are labeled red. Scale bars: 50 μm. All data represent the mean ± SEM.