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AIDA directly connects sympathetic innervation to adaptive thermogenesis by UCP1

Nature Cell Biology volume 23, pages 268–277 (2021)Cite this article

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Abstract

The sympathetic nervous system–catecholamine–uncoupling protein 1 (UCP1) axis plays an essential role in non-shivering adaptive thermogenesis. However, whether there exists a direct effector that physically connects catecholamine signalling to UCP1 in response to acute cold is unknown. Here we report that outer mitochondrial membrane-located AIDA is phosphorylated at S161 by the catecholamine-activated protein kinase A (PKA). Phosphorylated AIDA translocates to the intermembrane space, where it binds to and activates the uncoupling activity of UCP1 by promoting cysteine oxidation of UCP1. Adipocyte-specific depletion of AIDA abrogates UCP1-dependent thermogenesis, resulting in hypothermia during acute cold exposure. Re-expression of S161A-AIDA, unlike wild-type AIDA, fails to restore the acute cold response in Aida-knockout mice. The PKA–AIDA–UCP1 axis is highly conserved in mammals, including hibernators. Denervation of the sympathetic postganglionic fibres abolishes cold-induced AIDA-dependent thermogenesis. These findings uncover a direct mechanistic link between sympathetic input and UCP1-mediated adaptive thermogenesis.

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Fig. 1: AIDA is required for thermogenesis under cold stress.
Fig. 2: AIDA is required for sympathetic activation of BAT.
Fig. 3: Phosphorylation of AIDA by PKA promotes adaptive thermogenesis.
Fig. 4: AIDA-mediated adaptive thermogenesis is dependent on UCP1.
Fig. 5: AIDA interacts with UCP1.
Fig. 6: AIDA enhances the oxidative modification of UCP1.

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Data availability

GenBank database accession numbers: MT114182, MT114183, MT114184, MT114185, MT114186, MT114187, MT759803 and MT759804. All data that support the findings of this study are available on request from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank Y. Li, E. Gnaiger, T. Kuwaki, J. R. B. Lighton, E. T. Chouchani and D. Jiang for technical instruction; X. Li and X.-D. Jiang (Core Facility of Biomedical, Xiamen University) for raising the p-S161-AIDA antibody; the Xiamen University Laboratory Animal Center for the mouse in vitro fertilization service and all the other members of S.C.L. laboratory for their technical assistance. This work was supported by grants from the National Key Research and Development Project of China (grant no. 2016YFA0502001) and the National Natural Science Foundation of China (grant nos 31822027, 31871168, 31690101, 91854208 and 82088102), the Fundamental Research Funds for the Central Universities (grant nos 20720190084 and 20720200069), Project ‘111’ sponsored by the State Bureau of Foreign Experts and Ministry of Education of China (grant no. BP2018017), the Youth Innovation Fund of Xiamen (grant no. 3502Z20206028), the Natural Science Foundation of Fujian Province of China (grant no. 2017J01364) and XMU Training Program of Innovation and Entrepreneurship for Undergraduates (grant no. 2019×0666).

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  1. These authors contributed equally: Meng Shi, Xiao-Yu Huang.

Authors and Affiliations

  1. State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, China

    Meng Shi, Xiao-Yu Huang, Xin-Yi Ren, Xiao-Yan Wei, Yue Ma, Zhi-Zhong Lin, Dong-Tai Liu, Lintao Song, Tong-Jin Zhao, Guang Li, Luming Yao, Mingxia Zhu, Cixiong Zhang, Changchuan Xie, Yaying Wu, Shu-Yong Lin & Sheng-Cai Lin

  2. Department of Neurology, First Affiliated Hospital, Xiamen University, Xiamen, China

    Han-Ming Wu, Li-Ping Fan & Yi-Hong Zhan

  3. Department of Hepatic Surgery and Liver Transplantation Centre of the Third Affiliated Hospital, Guangdong Province Engineering Laboratory for Transplantation Medicine, Guangzhou, China

    Jingxing Ou

  4. Department of Digestive Diseases, School of Medicine, Xiamen University, Xiamen, China

    Shu-Yong Lin

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Contributions

Conceptualization: S.-C.L., S.-Y.L. and M.S. Methodology: M.S., X.-Y.H., Z.-Z.L., D.-T.L., L.S., L.Y., M.Z., C.Z., C.X., Y.W., L.-P.F., H.-M.W. and S.-Y.L. Formal analysis: S.-Y.L. and M.S. Investigation: M.S., X.-Y.H., X.-Y.R., Y.M., X.-Y.W. and S.-Y.L. Resources: T.-J.Z., G.L., J.O. and Y.-H.Z. Writing: S.-C.L., S.-Y.L. and M.S. Supervision: S.-C.L. and S.-Y.L. Project administration: S.-C.L. and S.-Y.L. Funding acquisition: S.-C.L., S.-Y.L., X.-Y.W. and Y.-H.Z.

Corresponding authors

Correspondence to Shu-Yong Lin or Sheng-Cai Lin.

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Peer review information Nature Cell Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 AIDA is required for thermogenesis under cold stress.

a,c, Energy expenditure (EE) of Aida-GKO (a) and Aida-AKO (c) mice. Data were collected from 7 (a) or 8 (c) mice per group, expressed as mean ± s.e.m. of values normalized to the body weight0.75 (left), and average EE under basal and cold conditions, **P = 0.0044 (a), **P = 0.0084 (c) (two-way RM ANOVA with Sidak) (right). Basal EE was calculated as the average of 30–60 min before ambient temperature shift, EE under cold was calculated as the average of 100–130 min after temperature shift. b, Protein abundance of AIDA in different tissues of WT, Aida-GKO, Aidaf/f and Aida-AKO mice. Data are representative of two independent experiments with similar results. d, EE of Aidaf/f and Aida-AKO female mice on chow diet housed at room temperature (24 °C). Data were collected from 8 mice per group for 2 days, expressed as means of values normalized to the body weight0.75 (left), individual EE under dark or light phase (middle), and adjusted means ± s.e.m. based on a normalized mouse weight of 31.8610 g determined by ANCOVA (right). N.S., P = 0.134. Uncropped blots for b and numerical source data for a, c, d are provided in Source Data Extended Data Fig. 1.

Source data

Extended Data Fig. 2 AIDA mediates sympathetic activation of BAT.

a, EE of mice two weeks after BAT denervation. WT sham n = 14 mice, Aida-GKO sham n = 11 mice, WT denerved or Aida-GKO denerved n = 10 mice. Mean ± s.e.m. of values normalized to the body weight0.75 (left), individual values with mean ± s.e.m. (right). Basal, mean of 30–60 min before ambient temperature shift; cold, mean of 100–130 min after temperature shift. WT sham versus WT denerved, ***p < 0.0001; WT sham versus Aida-GKO sham, *P = 0.0303 (two-way RM ANOVA with Tukey). b, Cold tolerance test (CTT) of male mice pre-treated with 10 mg/kg propranolol. Mean ± s.e.m., n = 9 mice per group, Aidaf/f PBS versus Aida-AKO PBS, **P = 0.0059 (two-way RM ANOVA with Geisser-Greenhouse correction, followed by Tukey). c, Immunoblotting analysis of BAT. Relative p-TH to total TH and β3-Tubulin to β-Tubulin are shown as mean ± s.e.m., n = 5 mice (two-tailed unpaired Student’s t test). d,e, OCR (d) and EE (e) of mice treated with CL316243 (1 mg/kg). Data are from 7 mice per group, presented similar as in (a), *P = 0.0320 (d), *P = 0.0316 (e) (two-way RM ANOVA with Sidak). Basal, the average of the 40 min period before injection; CL stimulation, the average during 40 min after CL-injection. f, Aida-knockout specificity. Data are representative of two independent experiments with similar results. g, Body composition of 16-week-old female mice on chow diet. Data are mean ± s.e.m., n = 8 mice per group (two-way RM ANOVA with Sidak). Uncropped blots for c, f and numerical source data for a-e, g are provided in Source Data Extended Data Fig. 2.

Source data

Extended Data Fig. 3 AIDA is not required for long-term cold adaptation.

a, Body temperatures of WT and Aida-GKO mice after a stepwise cold-adaptation (24 °C 1 week, 18 °C 1 week, 4 °C 4 weeks). Data are mean ± s.e.m., n = 7 mice per group, N.S., P = 0.7166 (two-tailed unpaired Student’s t test). b, Body weight curve of single-housed WT and Aida-GKO mice during stepwise cold-adaptation. Data are mean ± s.e.m., n = 6 mice per group. The linear regression slopes of the growth curves between WT and Aida-GKO mice are not significant (P = 0.9047). The body weights between the two groups of mice are significantly different (***P = 0.0003, two-way RM ANOVA with Geisser-Greenhouse correction). c-g, Blood glucose (c), serum non-esterified fatty acids (NEFA) (d), triacylglycerol (TG) (e), insulin (f) and leptin (g) levels of cold-adapted WT and Aida-GKO mice. Data are individual values with mean ± s.e.m., n = 8 mice per group (c), WT mice n = 7, Aida-GKO mice n = 8 (d-g) (two-tailed unpaired Mann-Whitney test). Numerical source data for a-g are provided in Source Data Extended Data Fig. 3.

Source data

Extended Data Fig. 4 Phosphorylation of AIDA by PKA.

a,b, AIDA phosphorylation after stimulation with epinephrine (a) or dopamine (b). Brown adipocytes were treated with 1 μM epinephrine or 1 μM dopamine. Cell extracts were subjected to electrophoresis using phos-tag gels (phos.) or normal gels. The relative levels of phosphorylated AIDA to total AIDA are standardized among three independent experiments and shown as mean ± s.e.m. in the right (ordinary one-way ANOVA with Dunnett). c, Mass spectrometry analysis of AIDA phosphorylation site. HEK293T cells expressing HA-AIDA were treated with 10 μM forskolin. The mobility-impeded band of AIDA resolved by phos-tag gel (phos.) was subjected to mass spectrometry analysis. d, PKA phosphorylates AIDA. HEK293T cells were transfected with WT-PRKACA (catalytic subunit of PKA) or a kinase-dead mutant of PKA (KD-PRKACA) together with HA-AIDA. The total cell lysates (TCL) were subjected to immunoprecipitation (IP) against HA. e, AIDA phosphorylation is eliminated by calf-intestinal alkaline phosphatase (CIAP). Lysates of HEK293T cells expressing Myc-PRKACA and HA-AIDA were subjected to IP against HA. Immunoprecipitated AIDA was treated with 2 U/μl CIAP for 1 h at 37 °C. f, Inhibition of PKA attenuates forskolin-induced AIDA phosphorylation. HEK293T cells transfected with HA-tagged AIDA were pre-treated with inhibitors against AMPK (Dorsomorphin, 1 μM), Aurora B (Barasertib, 1 μM), AKT (Akt inhibitor VIII, 2 μM) or PKA (H89, 30 μM) prior to stimulation with forskolin (10 μM) for 60 min. The relative intensities of p-S161-AIDA to HA-AIDA are standardized among three independent experiments and shown as mean ± s.e.m. in the right (ordinary one-way ANOVA with Dunnett). g, CL316243 treatment increases endogenous AIDA-S161 phosphorylation in WT BAT. Mice were i.p. injected with 1 mg/kg CL. 1 h post injection, BATs were homogenized and subjected to IP against endogenous AIDA. Data in d, e, g are representative of two independent experiments with similar results. Uncropped blots for a, b, d-g and numerical source data for a, b, f are provided in Source Data Extended Data Fig. 4.

Source data

Extended Data Fig. 5 Phosphorylation of AIDA promotes adaptive thermogenesis.

a, Protein abundance of AIDA in different tissues of mice with adeno-associated virus (AAV)-mediated BAT-specific expression of AIDA. WT and Aida-GKO mice were injected with Ucp1 mini-promoter and enhancer-driven AAV carrying WT-, S161A-, S161D-AIDA or GFP as a control. Blots in the left are representative samples of two independent experiments with similar results. The relative protein levels of AIDA to β-tubulin in BAT (GFP n = 18, WT n =18, S161A n = 17, S161D n = 16 mice) are quantified and shown as mean ± s.e.m. in the right (Kruskal-Wallis test with Dunn’s multiple comparisons test). b, EE of Aida-GKO mice after re-expression of WT-, S161A-, S161D-AIDA or GFP as a control to BAT under cold exposure. Aida-GKO GFP or Aida-GKO S161A-AIDA mice n = 9, Aida-GKO WT-AIDA or Aida-GKO S161D-AIDA mice n = 8. Data are mean ± s.e.m. normalized to the body weight0.75 (upper), or average EE under basal and cold conditions (lower). Basal EE of each mice was calculated as the average of 30–60 min before ambient temperature shift, EE under cold was calculated as the average of 100–130 min after temperature shift. Aida-GKO GFP versus Aida-GKO WT-AIDA, **P = 0.0028; Aida-GKO WT-AIDA versus Aida-GKO S161A-AIDA, ***P = 0.0003; Aida-GKO WT-AIDA versus Aida-GKO S161D-AIDA, **P = 0.0076; N.S., not significant (two-way RM ANOVA with Tukey). Uncropped blots for a and numerical source data for a, b are provided in Source Data Extended Data Fig. 5.

Source data

Extended Data Fig. 6 AIDA-mediated adaptive thermogenesis is dependent on UCP1.

a,b, EE under cold (n = 9 mice per group, except for Aida-AKO mice n = 11) (a) or i.p. CL316243 (1 mg/kg) treatment (n = 11 mice) (b). Mean ± s.e.m. of values normalized to the body weight0.75 (left) and individual values with mean ± s.e.m. (right). Basal, average of 30–60 min before temperature shift; cold, average of 100–130 min after temperature shift. Aidaf/f versus Aida-AKO, **P = 0.0026; Aidaf/f versus Aidaf/f Ucp1-KD, ***P < 0.0001 (a). Basal and CL-stimulated values were the average of 40 min before and after CL-injection, respectively. Aidaf/f versus Aida-AKO, *P = 0.0345; Aidaf/f versus Aidaf/f Ucp1-KD, **P = 0.0022 (b) (two-way RM ANOVA with Tukey). c,d, Ucp1 mRNA in BAT of mice housed at room temperature (c) or after a stepwise cold-adaptation (d). Data are mean ± s.e.m., n = 7 WT mice, n = 8 Aida-GKO mice (c), n = 8 mice per group (d) (two-tailed unpaired Student’s t test). e,f, UCP1 in BAT of mice after a stepwise cold-adaptation (e), or treated with CL (1 mg/kg) daily for 5 days (f). Relative UCP1 levels to β-Actin are quantified and shown as mean ± s.e.m. in the right (e, n = 5 mice per group, two-tailed unpaired Student’s t test; f, n = 3 mice, ordinary two-way ANOVA, Tukey). g, ERAD-associated proteins in BAT. n = 5 mice. Relative protein levels to HSP90 are quantified and shown as mean ± s.e.m. below (two-tailed unpaired Student’s t test with Welch’s correction). Uncropped blots for e-g and numerical source data for a-g are provided in Source Data Extended Data Fig. 6.

Source data

Extended Data Fig. 7 AIDA interacts with UCP1.

a, Co-IP of endogenous AIDA and UCP1 using BAT from WT mice treated with PBS or CL316243 (1 mg/kg) for 1 h. b, Immunofluorescence analysis of localization of ectopically expressed AIDA-HA and endogenous UCP1 in primary brown adipocytes. Red, AIDA; green, UCP1. Scale bar, 10 μm. c, Immunoblotting analysis of the fractionation patterns of AIDA and UCP1 in BAT from WT mice injected with PBS or CL316243 (1 mg/kg) for 1 h. Relative intensities of AIDA, UCP1, SDHA, CANX and β-Tubulin are quantified (lower). d, Validation of expression of APEX2-AIDA in BAT from cold-stimulated mice. e,f, Determination of UCP1-binding domain in AIDA (e) and AIDA-binding domain in UCP1 (f). FL, full length; CD, C-terminal deletion; ND, N-terminal deletion; MD, middle deletion. Experiment performed once. g, Alignment of AIDA S161 (in mouse) residue and the flanking amino acid residues in different species. h, Alteration of Pro163 to Ser of Branchiostoma floridae (bf) AIDA renders it a substrate for PKA. HEK293T cells were transfected with WT-bfAIDA or P163S-bfAIDA together with mPRKACA or bfPRKACA. Cells were lysed and subjected to IP against HA. i, PKA phosphorylates AIDA of marmot (mh), hedgehog (ee) and hamster (ma). HEK293T cells were transfected with HA-AIDA. Following 1 h treatment of forskolin (10 μM), the cells were lysed and subjected to IP against HA. The relative intensities of p-S161-AIDA to total AIDA are standardized among three independent experiments and shown as mean ± s.e.m. in the right (ordinary two-way ANOVA with Sidak). j-l, Interaction between AIDA and UCP1 of marmot (j), hedgehog (k) and hamster (l) after adrenergic stimulation. HEK293T cells were transfected with AIDA and UCP1. After 1 h forskolin (10 μM) treatment, the cells were lysed and subjected to IP against FLAG. Data in a-f, h, j-l are representative of two independent experiments with similar results. Uncropped blots for a, c-f, h-l and numerical source data for c, i are provided in Source Data Extended Data Fig. 7.

Source data

Extended Data Fig. 8 ROS production and the phosphorylation of AIDA are separate events downstream of adrenergic stimulation.

a, EE of mice before and after CL316243 injection pre-treated with NAC. Mice were i.p. injected with NAC (500 mg/kg) 10 min before injection of CL316243 (1 mg/kg). n = 8 mice. Data are shown as mean ± s.e.m. of values normalized to the body weight0.75 (left), and individual normalized values before and after CL-injection (right). N.S., P = 0.9391 (two-way RM ANOVA with Sidak). b,c, Effect of NAC (10 mM) pre-treatment on AIDA phosphorylation (b) and the interaction between AIDA and UCP1 (c) after forskolin (10 μM) stimulation in HEK293T cells. The relative intensities of p-S161-AIDA to total AIDA (b) and immunoprecipitated AIDA to UCP1 (c) are standardized among three independent experiments and shown as mean ± s.e.m. in the right (ordinary two-way ANOVA with Tukey). Uncropped blots for b, c and numerical source data for a-c are provided in Source Data Extended Data Fig. 8.

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Extended Data Fig. 9 Activation of UCP1 by fatty acids is independent of AIDA.

a, Fold change of NEFA released from BAT (left) or inguinal WAT (iWAT; right) of mice in response to NE (10 μM). Data are mean ± s.e.m., n = 6 BATs or iWATs per group (ordinary two-way ANOVA with Sidak). b, Immunoblotting analysis of lipases in mice-derived brown adipocytes (left) and white adipocytes (right) treated with forskolin (10 μM). Adipocytes are derived from n = 2 mice. c, CTT of mice pre-treated with ATGL inhibitor. Mice were i.p. injected with Atglistatin (50 μM/kg) at 10 min before cold exposure. Atglistatin was dissolved in 25% HCl. Atglistatin-HCl complex was obtained via evaporating excess HCl and dissolved in PBS containing 0.25% castor oil. Data are mean ± s.e.m., n = 8 mice per group, *P = 0.0193 (two-way RM ANOVA with Sidak). d, OCR of brown adipocytes from Aidaf/f and Aida-AKO mice. Brown adipocytes were pre-treated with DMSO or Atglistatin (40 μM) 1 h before test. OCR at each cycle is calculated as the percentage of initial OCR, shown as mean ± s.e.m., Aidaf/f brown adipocytes are from n = 7 mice, Aida-AKO brown adipocytes are from n = 8 mice. Graph on the right indicates the quantification of changes of ISO-induced mitochondrial uncoupling respiration rates over basal mitochondrial uncoupling respiration rates, data are mean ± s.e.m., ***P < 0.0001 (ordinary two-way ANOVA with Tukey). e, A schematic model showing that adrenergic signalling leads to phosphorylation of AIDA by PKA, phosphorylated AIDA in turn interacts with and promotes oxidation of UCP1, promoting adaptive thermogenesis by UCP1. Several elements of this image were adopted and modified from Servier Medical Art by Servier (https://smart.servier.com/) licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/). Uncropped blots for b and numerical source data for a, c, d are provided in Source Data Extended Data Fig. 9.

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Supplementary information

Reporting Summary

Peer Review Information

Supplementary Tables

Supplementary Table 1. Genotyping primers. Supplementary Table 2. Primers used for qRT–PCR. Supplementary Table 3. Antibody list.

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Shi, M., Huang, XY., Ren, XY. et al. AIDA directly connects sympathetic innervation to adaptive thermogenesis by UCP1. Nat Cell Biol 23, 268–277 (2021). https://doi.org/10.1038/s41556-021-00642-9

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