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. 2017 Nov 2;171(4):836-848.e13.
doi: 10.1016/j.cell.2017.09.015. Epub 2017 Oct 5.

Crosstalk Between KCNK3-Mediated Ion Current and Adrenergic Signaling Regulates Adipose Thermogenesis and Obesity

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Crosstalk Between KCNK3-Mediated Ion Current and Adrenergic Signaling Regulates Adipose Thermogenesis and Obesity

Yi Chen et al. Cell. .
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Abstract

Adrenergic stimulation promotes lipid mobilization and oxidation in brown and beige adipocytes, where the harnessed energy is dissipated as heat in a process known as adaptive thermogenesis. The signaling cascades and energy-dissipating pathways that facilitate thermogenesis have been extensively described, yet little is known about the counterbalancing negative regulatory mechanisms. Here, we identify a two-pore-domain potassium channel, KCNK3, as a built-in rheostat negatively regulating thermogenesis. Kcnk3 is transcriptionally wired into the thermogenic program by PRDM16, a master regulator of thermogenesis. KCNK3 antagonizes norepinephrine-induced membrane depolarization by promoting potassium efflux in brown adipocytes. This limits calcium influx through voltage-dependent calcium channels and dampens adrenergic signaling, thereby attenuating lipolysis and thermogenic respiration. Adipose-specific Kcnk3 knockout mice display increased energy expenditure and are resistant to hypothermia and obesity. These findings uncover a critical K+-Ca2+-adrenergic signaling axis that acts to dampen thermogenesis, maintain tissue homeostasis, and reveal an electrophysiological regulatory mechanism of adipocyte function.

Keywords: Adrenergic signaling; Brown fat; Calcium influx; Kcnk3; PKA signaling; Prdm16; Task-1; Thermogenesis; lipolysis.

Figures

Figure 1
Figure 1. Kcnk3 is a Prdm16 Target Enriched in Thermogenic Adipocytes
(A) Prdm16 ChIP-Seq peaks at the Kcnk3 locus. (B) Kcnk3 qPCR in the BAT and iWAT of control and adipose-specific prdm16 knockout mice (Prdm16 KO); n=3 mice per group. (C) Kcnk3 qPCR in in the BAT and iWAT of control and adipose-specific Prdm16 transgenic mice (aP2-Prdm16); n=4 mice per group. (D–E) Kcnk3 expression level (normalized to Adiponectin level) shown by translating ribosomal affinity purification from (D) Adiponectin-positive cells (Adiponectin-TRAP) and (E) Ucp1-positive cells (Ucp1-TRAP); n=3 mice per group; eWAT, epididymal white adipose tissue. (F) Kcnk3 expression levels in BAT and iWAT upon cold exposure for 4 hours and 2 days, respectively; n=3 mice per group. (G) Kcnk3 expression levels in BAT and iWAT under cold exposure for 24 hours followed by 24-hour acclimation to room temperature; n=3 mice per group. Data are represented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. two-tailed Student’s t-test. See also Figure S1.
Figure 2
Figure 2. Kcnk3 Mediates Outwards Potassium Currents in Brown Adipocytes
(A) Kcnk3 qPCR in different tissues of control and AdK3KO mice; n=4 mice per group. (B) Representative outward current traces in control and kcnk3-deficient (AdK3KO) brown adipocytes. (C) Outward currents in control (n=6) and kcnk3-deficient (n=8, from AdK3KO mice) brown adipocytes represented by peak amplitude (I)–voltage (V) relationship (maximal of first 500 ms) and steady-state amplitude (I)–voltage (V) relationship (2000–2500 ms). (D) Outward currents in brown adipocytes from control (n=4) and kcnk3-deficient (n=6, from global kcnk3 knockout mice) brown adipocytes represented by steady-state amplitude (I)–voltage (V) relationship (2000–2500 ms). (E) Representative outward current traces at pH 6.4, 7.4 and 8.4 in control and kcnk3-deficient (from AdK3KO mice) brown adipocytes differentiated in vitro. Hold potential is at +30 mV. (F) The Acid-sensitive current component, as a percentage of total outward currents, in control (n=10) and kcnk3-deficient (AdK3KO, n=5) brown adipocytes differentiated in vitro. Hold potential is at +30 mV. Data are represented as means ± SEM. *P < 0.05, ***P < 0.001, two-tailed Student’s t-test. See also Figure S1.
Figure 3
Figure 3. Kcnk3 Negatively Regulates Thermogenesis by Dampening NE-induced cAMP Production
(A) Representative oxygen consumption traces and quantification of respiration rates in in control and kcnk3-deficient brown adipocytes. Induced, NE rates minus Basal rates. (B) Lipolysis rates (amount of glycerol released per min) of brown adipocytes under different concentrations of NE stimulation. (C) Western blot of HSL phosphorylation in NE-stimulated control and kcnk3-deficient brown adipocytes. (D) Lipolysis rates of white adipocytes from the epididymal WAT. (E) Cyclic-AMP levels, (F) Lipolysis rates and (G) HSL phosphorylation levels of control and kcnk3-deficient brown adipocytes treated with NE and IBMX as indicated. Cells were pooled from 10 mice and 3 replicate measurements were taken per experiment. Data are represented as means ± SEM of 3 measurements. *P < 0.05, **P < 0.01, ***P < 0.001. two-tailed Student’s t-test. Each experiment was repeated at least 3 times. See also Figure S2 and S3.
Figure 4
Figure 4. Kcnk3 Dampens cAMP Production by Limiting NE-induced Ca2+ Entry Through VDCCs
(A) Representative traces of NE-induced membrane potential (Vm) changes in brown adipocytes. NE, 1 μM. (B) Vm of brown adipocytes before and after 1 μM NE stimulation. Basal Vm, 50 s average before NE; NE-peak Vm, maximum depolarization after NE; NE-average Vm,120 s average post peak depolarization; n=7 cells per group. (C) Fluo-4 signal in brown adipocytes before & after NE stimulation, scale bar 40 μm. (D) Averaged Fluo-4 intensity trace (normalized to intensity at 0 min) of control and kcnk3-deficient brown adipocytes in normal or Ca2+-free buffer before & after NE stimulation; n=30 cells per group. (E) cAMP production and (F) Lipolysis rates in control and kcnk3-deficient brown adipocytes in normal and Ca2+-free buffer. (G) Averaged Fluo-4 intensity trace (normalized to intensity at 0 min) of control and kcnk3-deficient brown adipocytes before & after NE stimulation treated with VDCC inhibitors (VDCCi): L651, 582 (1 μM, L-type VDCC inhibitor) and ω-Agatoxin TK (1 μM, P/Q-type VDCC inhibitor); n=21 cells per group. (H) cAMP production (cAMP accumulation in a 5-min window) in control and kcnk3-deficient brown adipocytes in normal Ca2+ buffer treated with L651, 582 (1 μM) and ω-Agatoxin TK (1 μM), as indicated. Data are represented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. (B) and (E)-(H), two-tailed Student’s t-test; (D) and (G), two-way ANOVA. In (E)–(H), Cells were pooled from 10 mice and 3 replicate measurements were taken per experiment. Each experiment was repeated at least 3 times. (E) and (H), cAMP production was measured in a 5-minitue time window. See also Figure S4.
Figure 5
Figure 5. Kcnk3 Negatively Regulates Adipose Thermogenesis in vivo
(A) BAT temperatures measured with probes implanted in the interscapular region of single-housed mice upon transition from 30 °C to 4 °C; n=8 mice per group. (B) Energy expenditure rates (VO2 and VCO2) of chow-fed mice under basal conditions; n=7 mice per group. (C) Energy expenditure rates (VO2 and VCO2) of chow-fed mice after CL316, 243-injection (1 mg/kg); n=7 mice per group. (D) Western blot of HSL phosphorylation in BAT from control and AdK3KO mice 20 min after CL316, 243-injection (10 mg/kg). n=4 mice per group. Data are represented as means ± SEM *P < 0.05, **P < 0.01, ***P < 0.001. (A), two-tailed Student’s t-test; (B) and (C), two-way ANOVA. Each experiment was repeated at least twice. See also Figure S5 and S6.
Figure 6
Figure 6. Ablating Kcnk3 from Adipose Tissue Protects Mice from Diet-induced Obesity
(A) Male and female mice body weight curves on HFD; n=7 mice per group. (B) Fat and lean mass of HFD-fed male mice; n=7 mice per group. (C) Energy expenditure rates (VO2 and VCO2) of HFD-fed mice under basal conditions; n=7 mice per group. (D) Energy expenditure rates (VO2 and VCO2) of HFD-fed mice after CL316, 243-injection (1 mg/kg); n=6 mice per group. (E) Blood glucose levels of HFD-fed male mice; Control, n=7; AdK3KO, n=6. (F) H&E staining of liver and BAT of HFD-fed control and AdK3KO mice following 14-weeks of HFD feeding. Scale bar, 200 μm. Data are represented as means ± SEM *P < 0.05, **P < 0.01. (B) and (E) two-tailed Student’s t-test; (A), (C) and (D), two-way ANOVA. Each experiment was repeated at least twice. See also Figure S7.
Figure 7
Figure 7. A Model Showing How Kcnk3-Mediated K+ Currents Regulate Thermogenesis in Brown Adipocytes
Kcnk3 mediates outward K+ flux, which antagonizes NE-induced membrane depolarization. This limits calcium influx through voltage-dependent calcium channels (VDCCs) and limits adrenergic signaling, thereby attenuating lipolysis and thermogenesis.

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