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 3

TET3 is required for leptin-induced repression of AGRP expression in cell lines.

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TET3 is required for leptin-induced repression of AGRP expression in cel...
(A) Schematic diagram of a post-fast refeeding study of Cas9+ mice injected with AAV or AAV-sgTet3. (B) Food intake of mice injected with AAV at the indicated time points following administration of leptin or saline. n = 6 mice per group. *P < 0.05 and **P < 0.01, by 2-tailed Student’s t test. (C) Food intake of mice injected with AAV-sgTet3 following administration of leptin or saline. n = 6 mice per group. Significance was determined by 2-tailed Student’s t test. (D) Mouse GT1-7 cells maintained in a high leptin concentration (Lept H, 1 × 10–8 M) were switched to a low leptin concentration (Lept L, 1 × 10–10 M), followed by RNA extraction and qPCR of Tet3 and Agrp mRNA 24 hours after the switch. n = 3 per group. *P < 0.05 and ***P < 0.001, by 2-tailed Student’s t test. (E) qPCR of Tet3 and Agrp mRNA from GT1-7 cells maintained in Lept L and then switched to Lept H for 24 hours. n = 3 per group. **P < 0.01 and ***P < 0.001, by 2-tailed Student’s t test. (F) 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). RNA was extracted 12 hours (for Tet3) or 36 hours (for Agrp) after the switch and analyzed by qPCR. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA with Tukey’s post test. (G) Representative immunoblots for TET3 and AGRP from GT1-7 cells treated as in F. GAPDH was used as a loading control. Proteins were isolated at the 36-hour time point. (H) Human SH-SY5Y neuroblastoma 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 24 hours after the switch and analyzed by qPCR. n = 3 per group. *P < 0.05 and **P < 0.01, by 1-way ANOVA with Tukey’s post test. (I) Representative immunoblots for TET3 and AGRP from SH-SY5Y cells treated as in H. Proteins were isolated at the 36-hour time point. All data represent the mean ± SEM.