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Hypothalamic Grb10 enhances leptin signalling and promotes weight loss

Nature Metabolism volume 5, pages 147–164 (2023)Cite this article

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Abstract

Leptin acts on hypothalamic neurons expressing agouti-related protein (AgRP) or pro-opiomelanocortin (POMC) to suppress appetite and increase energy expenditure, but the intracellular mechanisms that modulate central leptin signalling are not fully understood. Here we show that growth factor receptor-bound protein 10 (Grb10), an adaptor protein that binds to the insulin receptor and negatively regulates its signalling pathway, can interact with the leptin receptor and enhance leptin signalling. Ablation of Grb10 in AgRP neurons promotes weight gain, while overexpression of Grb10 in AgRP neurons reduces body weight in male and female mice. In parallel, deletion or overexpression of Grb10 in POMC neurons exacerbates or attenuates diet-induced obesity, respectively. Consistent with its role in leptin signalling, Grb10 in AgRP and POMC neurons enhances the anorexic and weight-reducing actions of leptin. Grb10 also exaggerates the inhibitory effects of leptin on AgRP neurons via ATP-sensitive potassium channel-mediated currents while facilitating the excitatory drive of leptin on POMC neurons through transient receptor potential channels. Our study identifies Grb10 as a potent leptin sensitizer that contributes to the maintenance of energy homeostasis by enhancing the response of AgRP and POMC neurons to leptin.

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Fig. 1: Grb10 regulates leptin signalling by interaction with LepRb.
Fig. 2: Grb10 in AgRP neurons contributes to maintenance of energy balance.
Fig. 3: Grb10 in AgRP neurons enhances leptin sensitivity.
Fig. 4: Grb10 potentiates the inhibitory effects of leptin on AgRP neurons through the KATP channel.
Fig. 5: Grb10 in POMC neurons ameliorates diet-induced obesity.
Fig. 6: Grb10 in POMC neurons enhances leptin sensitivity.
Fig. 7: Grb10 potentiates the excitatory effects of leptin on POMC neurons through TrpC currents.

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All data generated or analysed during this study are included in this published article (and its Supplementary information files). Source data are provided with this paper.

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Acknowledgements

Investigators involved in this work were supported by grants from USDA/CRIS (no. 51000-064-01 S to Y.X. and a fellowship award to K.M.C.), the National Nature Science Foundation of China (nos. 8173022 to F.L., 81870601 to J.B. and 31871180 to F.H.), the National Key R&D Program of China (nos. 2018YFC2000100 and 2019YFA0801903 to F.L., no. 2020YFA0803604 to F.H.), the American Heart Association (to Y.H. and L.T.) and the American Diabetes Association (no. 1-19-IBS-147 to J.B.). LepR-IRES-Cre mice were kindly provided by M. Myers at University Michigan.

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  1. These authors contributed equally: Hailan Liu, Yang He, Juli Bai.

Authors and Affiliations

  1. National Clinical Research Center for Metabolic Diseases, Metabolic Syndrome Research Center, Key Laboratory of Diabetes Immunology, Ministry of Education, Department of Metabolism and Endocrinology, The Second Xiangya Hospital of Central South University, Changsha, China

    Hailan Liu, Juli Bai, Feng Zhang, Hairong Luo, Fang Hu & Feng Liu

  2. USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, TX, USA

    Hailan Liu, Yang He, Yongjie Yang, Meng Yu, Hesong Liu, Longlong Tu, Nan Zhang, Na Yin, Junying Han, Zili Yan, Nikolas Anthony Scarcelli, Kristine Marie Conde, Mengjie Wang, Jonathan Carter Bean, Camille Hollan Sidell Potts, Chunmei Wang & Yong Xu

  3. Department of Cell Systems & Anatomy and Department of Pharmacology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA

    Juli Bai & Chuanhai Zhang

  4. Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX, USA

    Yong Xu

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  1. Hailan Liu
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Contributions

Hailan Liu performed stereotaxic injections, histology study, metabolic characterization and data analysis. Y.H. conducted all electrophysiological experiments and data analysis. J.B. and C.Z. performed cell culture, co-immunoprecipitation (Co-IP) and yeast two-hybrid experiments. F.Z., Y.Y., H. Luo, M.Y., Hesong Liu, L.T., N.Z., N.Y., J.H., Z.Y., N.A.S., K.M.C., M.W., J.C.B., C.H.S.P., C.W. and F.H. contributed to the generation of study mice, data analysis and discussion. Y.X. and F.L. conceptualized and designed this study, supervised the work and wrote the manuscript with inputs from other authors. Y.X. and F.L. had full access to all data in the study and take responsibility for their integrity and the accuracy of data analysis.

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Correspondence to Feng Liu or Yong Xu.

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Nature Metabolism thanks Nicola Heller and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Ashley Castellanos-Jankiewicz, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Generation and validation of AgRP-Grb10-KD mice.

a. Representative immunofluorescence images and quantification of Grb10 in the ARH of AgRP-IRES-Cre/Rosa26-LSL-tdTomato mice (n = 3). b. Representative RNAscope images showing tdTomato, Agrp, Grb10 mRNAs and their co-localization in the ARH of LepR-IRES-Cre/Rosa26-LSL-tdTomato mice. c. Quantification for the percentage of tdTomato+Grb10+ AgRP neurons (n = 3). d. Schematic diagram of virus injection and representative validation of GFP expression in the ARH of 11 mice. e-f. Representative immunofluorescence images (e) and quantification (f) of Grb10 in the ARH of control and AgRP-Grb10-KD mice (n = 3 per group). Data are expressed as mean ± SEM. Two-tailed Student’s t test (f).

Source data

Extended Data Fig. 2 Disruption of Grb10 in AgRP neurons induces energy imbalance in male and female mice.

a-b. Body weight (a) and percentage of mass weight (b) in male control (n = 7) and AgRP-Grb10-KD (n = 5) mice. c-d. Area under curve (AUC) for GTT (c) and ITT (d) in male control (n = 7) and AgRP-Grb10-KD (n = 5) mice. e-g. Body weight (e), body composition (f) and food intake (g) in female control (n = 6) and AgRP-Grb10-KD (n = 5) mice. h. Temporal changes in energy expenditure and the regression of energy expenditure with body mass in female control (n = 6) and AgRP-Grb10-KD (n = 5) mice over a 24 hr period in the TSE PhenoMaster metabolic cages. i. Predicted energy expenditure with 45 g body mass for each individual mouse calculated by the regression lines in female control (n = 6) and AgRP-Grb10-KD (n = 5) mice. j-m. Serum leptin (j) and insulin (k) levels, GTT and its AUC (l) and ITT and its AUC (m) in female control (n = 6) and AgRP-Grb10-KD (n = 5) mice. n. Representative BAT, iWAT and gWAT histology in male control and AgRP-Grb10-KD mice (n = 4 per group), scale bar=50 µm. Data are expressed as mean ± SEM. Two-way ANOVA with Sidak’s or Tukey’s test (a, b, e-g, l, m) or regression-based ANCOVA (d) or two-tailed Student’s t test (c, d, i-k, l, m). *p < 0.05, **p < 0.01.

Source data

Extended Data Fig. 3 Generation and validation of AgRP-Grb10-OE mice.

a. Schematic diagram of virus injection into the ARH. b. Representative GFP expression in the ARH of AgRP-IRES-Cre/Rosa26-LSL-tdTomato mice. c. Quantification for the percentage of GFP cells that are tdTomato+ (n = 3). d. Grb10 immunofluorescence staining in the ARH with one side of the AgRP neurons infected with AAV-DIO-GFP and the other side infected with AAV-DIO-Grb10-GFP. e. Representative immunofluorescence images of GFP and Grb10 expression in the ARH. f. Quantification for the percentage of GFP+ AgRP neurons (n = 5). g-h. Body weight (g) and percentage of mass weight (h) in female control (n = 7) and AgRP-Grb10-OE (n = 8) mice. i-j. Area under curve (AUC) for GTT (i) and ITT (j) in female control (n = 7) and AgRP-Grb10-OE (n = 8) mice. Data are expressed as mean ± SEM. Two-way ANOVA with Sidak’s or Tukey’s test (g, h) or two-tailed Student’s t test (i, j). *p < 0.05.

Source data

Extended Data Fig. 4 Grb10 enhances leptin sensitivity in AgRP neurons.

a-b. Representative images (a) and quantification (b) for leptin-induced pSTAT3 in the VMH of control (n = 5) and AgRP-Grb10-KD (n = 5) mice treated with saline or leptin. c-d. Representative images (c) and quantification (d) for leptin-induced pSTAT3 in the VMH of control (n = 5) and AgRP-Grb10-OE (n = 5) mice treated with saline or leptin. Scale bar = 100 µm. Data are expressed as mean ± SEM. Two-tailed Student’s t test (b, d).

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Extended Data Fig. 5 Grb10 has no effect on baseline activity of AgRP neurons.

a. Representative traces of action potentials in untreated control AgRP neurons and those with Grb10 being deleted or overexpressed. b-c. Quantification of resting membrane potential (b) and firing frequency (c) in the three groups (n = 4 per group). Data are expressed as mean ± SEM and were collected from both male and female mice. One-way ANOVA with Tukey’s test (b, c).

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Extended Data Fig. 6 JAK2 or PI3K inhibition blocks the effects of leptin on AgRP neurons.

a. Representative traces of leptin-induced hyperpolarization in control or Grb10 overexpressed AgRP neurons in the presence of AG490. b-c. Quantification of resting membrane potential (b) and firing rate (c) in the two groups. Hyperpolarization is defined as > 2 mV decrease in resting membrane potential. d. Representative traces of KATP conductance in control or Grb10 overexpressed AgRP neurons in the presence of AG490 (n = 3 per group). e. Quantification of KATP conductance in these groups (n = 3 per group). f. Representative traces of leptin-induced hyperpolarization in Grb10 overexpressed AgRP neurons in the presence or absence of ETP45658. g-h. Quantification of resting membrane potential (g) and firing rate (h) in the two groups. i. Representative traces of KATP conductance in Grb10 overexpressed AgRP neurons (n = 4 per group). KATP currents from Grb10 overexpressed AgRP neurons were recorded in the presence or absence of ETP35658. j. Quantification of KATP conductance in the two groups (n = 4 per group). Data are expressed as mean ± SEM and were collected from both male and female mice. Two-way ANOVA with Sidak’s or Tukey’s test (b, c, g, h) or two-tailed Student’s t test (e, j).

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Extended Data Fig. 7 Ablation of Grb10 in POMC neurons has no effect on body weight and food intake under chow-fed condition.

a. Representative RNAscope images showing tdTomato, Pomc, Grb10 mRNAs in the ARH of LepR-IRES-Cre/Rosa26-LSL-tdTomato mice (n = 3) and quantification for the co-localization of Grb10 and pomc and tdTomato. b-c. Representative immunofluorescence images (b) and quantification analysis (c) of Grb10 in the ARH of POMC-CreER/Rosa26-LSL-tdTomato mice (n = 3) and POMC-Grb10-KD (n = 4) mice upon tamoxifen injection. d-f. Body weight gain (d), cumulative food intake (e) and body composition (f) of chow-fed male control (n = 10) and POMC-Grb10-KD (n = 7) mice at 4 weeks after tamoxifen injection. Data are expressed as mean ± SEM. Two-way ANOVA with Sidak’s or Tukey’s test (d-f) or two -tailed Student’s t test (c).

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Extended Data Fig. 8 Grb10 deletion in POMC neurons accelerates the development of obesity in HFD-fed male and female mice.

a-b. Body weight (a) and percentage of mass weight (b) in HFD-fed male control (n = 9) and POMC-Grb10-KD (n = 6) mice. c-d. Area under curve (AUC) for GTT (c) and ITT (d) in HFD-fed male control (n = 9) and POMC-Grb10-KD (n = 6) mice. e-i. Body weight (e), weight gain (f), body composition (g-h) and cumulative food intake (i) in HFD-fed female control (n = 7) and POMC-Grb10-KD (n = 9) mice. j. Temporal changes in energy expenditure and the regression of energy expenditure with body mass in HFD-fed female control (n = 7) and POMC-Grb10-KD (n = 7) mice over a 24 hr period in the TSE PhenoMaster metabolic cages. k. Predicted energy expenditure with 40 g body mass for each individual mouse calculated by the regression lines in HFD-fed female control (n = 7) and POMC-Grb10-KD (n = 7) mice. l-m. Serum leptin (l) and insulin (m) levels in HFD-fed female control (n = 6) and POMC-Grb10-KD mice (n = 6). n-o. GTT and its AUC (n) and ITT and its AUC (o) in HFD-fed female control (n = 7) and POMC-Grb10-KD (n = 9) mice. p. Representative BAT, iWAT and gWAT histology in HFD-fed female control and POMC-Grb10-KD mice (n = 4 per group), scale bar=50 µm. Data are expressed as mean ± SEM. Two-way ANOVA with Sidak’s or Tukey’s test (a, b, e-i, n, o) or two sided regression-based ANCOVA (j) or two-tailed Student’s t test (c, d, k-o). *p < 0.05, **p < 0.01, ***p < 0.001.

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Extended Data Fig. 9 Overexpression of Grb10 in POMC neurons has no effect on body weight and food intake under chow-fed condition.

a. Schematic diagram of virus injection into the ARH. b. Beta-endorphin immunofluorescence staining in the ARH with POMC neurons infected with AAV-DIO-Grb10-GFP. c. Quantification for the percentage of beta-endorphin+ GFP cells (n = 3). d. Representative Grb10 immunofluorescence staining in the ARH with one side of the POMC neurons infected with AAV-DIO-GFP and the other side infected with AAV-DIO-Grb10-GFP (n = 4). e. Representative immunofluorescence images of GFP and Grb10 expression in the ARH. f. Quantification for the percentage of GFP+ POMC neurons (n = 5). g-i. Body weight (g), cumulative food intake (h) and body composition (i) of chow-fed male control (n = 6) and POMC-Grb10-OE (n = 7) mice. Data are expressed as mean ± SEM. Two-way ANOVA with Sidak’s or Tukey’s test (g-i).

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Extended Data Fig. 10 Grb10 overexpression in POMC neurons ameliorates the development of obesity in HFD-fed male and female mice.

a-b. Body weight (a) and percentage of mass weight (b) in HFD-fed male control (n = 6) and POMC-Grb10-OE (n = 7) mice. c-d. Area under curve (AUC) for GTT (c) and ITT (d) in HFD-fed male control (n = 6) and POMC-Grb10-OE (n = 7) mice. e-i. Body weight (e), weight gain (f), body composition (g-h) and cumulative food intake (i) in HFD-fed female control (n = 8) and POMC-Grb10-OE (n = 6) mice. j. Temporal changes in energy expenditure and the regression of energy expenditure with body mass in HFD-fed female control (n = 8) and POMC-Grb10-OE (n = 6) mice over a 24 hr period in the TSE PhenoMaster metabolic cages. k. Predicted energy expenditure with 40 g body mass for each individual mouse calculated by the regression lines in HFD-fed female control (n = 8) and POMC-Grb10-OE (n = 6) mice. l-m. Serum leptin (l) and insulin (m) levels in HFD-fed female control (n = 6) and POMC-Grb10-OE (n = 6) mice. n-o. GTT and its AUC(n) and ITT and its AUC (o) in HFD-fed female control (n = 8) and POMC-Grb10-OE (n = 6) mice. p. Representative BAT, iWAT and gWAT histology in HFD-fed female control and POMC-Grb10-OE mice (n = 4 per group), scale bar=50 µm. Data are expressed as mean ± SEM. Two-way ANOVA with Sidak’s or Tukey’s test (a, b, e-i, n, o) or two sided regression-based ANCOVA (j) or two-tailed Student’s t test (c, d, k-o). *p < 0.05, **p < 0.01.

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Liu, H., He, Y., Bai, J. et al. Hypothalamic Grb10 enhances leptin signalling and promotes weight loss. Nat Metab 5, 147–164 (2023). https://doi.org/10.1038/s42255-022-00701-x

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