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. 2014 Oct 9;159(2):306-17.
doi: 10.1016/j.cell.2014.09.010.

O-GlcNAc transferase enables AgRP neurons to suppress browning of white fat

Hai-Bin Ruan  1 , Marcelo O Dietrich  2 , Zhong-Wu Liu  1 , Marcelo R Zimmer  3 , Min-Dian Li  4 , Jay Prakash Singh  1 , Kaisi Zhang  4 , Ruonan Yin  1 , Jing Wu  1 , Tamas L Horvath  5 , Xiaoyong Yang  6
Affiliations

O-GlcNAc transferase enables AgRP neurons to suppress browning of white fat

Hai-Bin Ruan et al. Cell. .

Abstract

Induction of beige cells causes the browning of white fat and improves energy metabolism. However, the central mechanism that controls adipose tissue browning and its physiological relevance are largely unknown. Here, we demonstrate that fasting and chemical-genetic activation of orexigenic AgRP neurons in the hypothalamus suppress the browning of white fat. O-linked β-N-acetylglucosamine (O-GlcNAc) modification of cytoplasmic and nuclear proteins regulates fundamental cellular processes. The levels of O-GlcNAc transferase (OGT) and O-GlcNAc modification are enriched in AgRP neurons and are elevated by fasting. Genetic ablation of OGT in AgRP neurons inhibits neuronal excitability through the voltage-dependent potassium channel, promotes white adipose tissue browning, and protects mice against diet-induced obesity and insulin resistance. These data reveal adipose tissue browning as a highly dynamic physiological process under central control, in which O-GlcNAc signaling in AgRP neurons is essential for suppressing thermogenesis to conserve energy in response to fasting.

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Figures

Figure 1
Figure 1. Fasting and AgRP neurons suppress WAT browning
(A–C) Expression of thermogenic genes in different fat depots from ad libitum fed or 24 h-fasted mice (n = 5). Same amount of RNA was used for reverse transcription followed by real time PCR. Gene expression was first normalized to 36b4 and then relative mRNA amount per depot was calculated based on total RNA levels (Figure S1A). Total levels of thermogenic genes per depot in BAT (A), rWAT (B) and iWAT (C) are shown. (D–H) Mice were either fed ad libitum or fasted overnight at room temperature (RT) or 4 °C. (D) Expression of Ucp1 transcript in different fat depots (n = 5). (E) Immunoblotting showing protein levels of Ucp1, TH, and Uchl1. Densitometric analyses are shown at the bottom. (F–H) NE levels in (F) BAT, (G) iWAT, and (H) rWAT. Data are shown as mean ± SEM. *, p < 0.01; **, p < 0.01 by unpaired student’s t-test. See also Figure S1.
Figure 2
Figure 2. Acute activation of AgRP neurons suppresses the thermogenic program in WAT
(A) 10 mg/kg body weight of capsaicin was injected to Trpv1−/−;AgRP-Cre−;R26Trpv1 (control) or Trpv1−/−;AgRP-Cre+;R26Trpv1 transgenic mice. Food was removed during all the experiments. (B) Thermogenic gene expression in adipose tissues, 1 h after capsaicin injection (n = 4). (C) Changes in energy expenditure of mice after capsaicin injection (n = 4). (D) Body temperature of mice during cold challenge immediately after capsaicin injection (n = 9–11). (E, F) Ucp1 expression in BAT (E) and rWAT (F) of capsaicin-injected mice at RT or 4 °C for 2h (n = 4–6). (G) Serum levels of NE of mice after 2h of capsaicin injection (n = 8). (H) Levels of NE in iWAT and rWAT of mice after 2h of capsaicin injection (n = 8). (I, J) Mice were injected with saline or 1 mg/kg body weight of CL-316, 243 at the same time with capsaicin. (I) Change in core body temperature (n = 6–8). (J) Levels of Ucp1 transcript in rWAT (n = 4–6). Data are shown as mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001 by unpaired student’s t-tests.
Figure 3
Figure 3. OGT is required for AgRP neuron activity
(A) Ribosome-associated mRNAs were isolated from the acuate nucleus of AgRP-Cre+; RPL22HA mice, and real-time PCR was performed to determine the enrichment of Ogt transcripts in HA immunoprecipitation compared to the input (n = 4). Agrp and Pomc transcripts were used as controls. (B) Immunostaining of OGT in the hypothalamus of fed and overnight-fasted Npy-hrGFP mice. (C) Immunostaining of O-GlcNAc in the hypothalamus of fed and overnight-fasted Npy-hrGFP mice. (D) Immunostaining of O-GlcNAc in the hypothalamus of Npy-hrGFP mice injected with saline or 120 mmol/kg body weight of ghrelin for 1 h. (E) AgRP-Cre−;Ogtflox (CT) and AgRP-Cre+;Ogtflox (KO) mice were crossbred onto Npy-hrGFP background for the whole-cell current-clamp recordings. (F) Basal membrane potential of AgRP neurons (n = 19). (G) Representative tracing of action potentials of AgRP neurons. (H) Firing rate of AgRP neurons (n = 19). Data are shown as mean ± SEM. *, p < 0.05 by unpaired student’s t-test. 3V, 3rd ventricle. Bar = 50 µm. See also Figure S2.
Figure 4
Figure 4. O-GlcNAcylation modulates the voltage-dependent Kcnq3 channel
(A) 10 mV- stepwise whole-cell voltage-clamp (-50 mV to 40 mV) was performed to record K+ current in Npy-hrGFP-positive cells (n = 6–8). (B) Current-voltage curve of K+ currents at 300 ms in panel A. (C) Immunoprecipitation showing interaction between OGT and Kcnq3 in fed and fasted hypothalamus. (D) Myc-tagged wildtype and T655A Kcnq3 were expressed in HEK 293 cells. O-GlcNAc levels were determined by Myc immunoprecipitation followed by western blotting. 6-Ac-CAS, a specific inhibitor of O-GlcNAcase to increase O-GlcNAc levels. (E) HEK 293 cells were transfected with wildtype or T655A Kcnq, K+ currents were recorded by whole-cell voltage-clamp (-60 to 80 mV), and then current density was calculated. (F) Current density - voltage curve of wildtype and T655A Kcnq3 at 300 ms in E. Data are shown as mean ± SEM. *, p < 0.05 by unpaired student’s t-test
Figure 5
Figure 5. Loss of Ogt in AgRP neurons promotes browning and improves glucose metabolism in mice fed on normal chow
(A, B) Expression of thermogenic genes in rWAT (A) and BAT (B) of 6-month-old female mice (n = 4). (C) Oxygen consumption rate of BAT and rWAT in the presence of oligomycin, an ATPase inhibitor (n = 8). (D) Expression of Ucp1 in rWAT from mice treated with 3 days of saline or SR59230A (n = 4–6). (E–F) Expression of thermogenic genes in BAT (E) and rWAT (F) from fed and 24 h-fasted AgRP-OGT KO female mice (n = 3–4). Total amounts of mRNA were calculated based on relative mRNA levels and total amounts of RNA isolated. (G, H) Loss of energy expenditure (G) and body weight (H) in CT and KO female mice after fasting for 24 h (n = 6–15). (I) Expression of gluconeogenic genes in liver of 6-month-old female mice (n = 3–4). (J) Pyruvate tolerance test in 5-month-old female mice (n = 4–7). Insert, area under curve (AUC). (K) Glucose tolerance test in 5-month-old female mice (n = 8–12). Insert, AUC. Data are shown as mean ± SEM. *, p < 0.05 by unpaired student’s t-test. See also Figure S3.
Figure 6
Figure 6. Browning phenotypes in AgRP-Ogt mice on HFD
(A) Thermogenic gene expression in different fat depots from 10-month-old female HFD mice (n = 5–7). (B) Weight of fat depots in 10-month-old female HFD mice (n = 5–6). (C) H&E staining of adipose tissues from 10-month-old female HFD mice. (D) Immunoblotting of UCP1 and tyrosine hydroxylase in rWAT of 10-month-old female HFD mice. (E) Norepinephrine levels in fat depots (n = 12). (F) Energy expenditure in 6-month-old female HFD mice determined by metabolic cage study followed by regressing to body weight using ANCOVA analysis (n = 11). Data are shown as mean ± SEM. *, p < 0.05; **, p < 0.01 by unpaired student’s t-test.
Figure 7
Figure 7. Loss of Ogt in AgRP neurons protects mice from diet-induced obesity and insulin resistance
(A) Growth curve of female mice fed with HFD (n =12–13). (B) Fat mass of 5-month-old female HFD mice (n =12–13). (C) Daily intake of HFD in 2-month-old female mice (n = 6). (D–F) Fasting blood glucose (D), fasting serum insulin (E), and HOMA-IR (F) in 6-month-old female HFD mice (n = 6). (G) Glucose tolerance test in 5-month-old female HFD mice. Area under curve (AUC) is shown to the right (n = 17–19). (H) Insulin tolerance test in 5-month-old female HFD mice. Area under curve (AUC) is shown to the right (n = 12–13). Data are shown as mean ± SEM. *, p < 0.05 by unpaired student’s t-test. See also Figure S4.
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