Leptin signaling in astrocytes regulates hypothalamic neuronal circuits and feeding (original) (raw)

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In the PDF version of the supplementary information initially published, Supplementary Figures 1a,b and 5a and the corresponding legends were missing. In the HTML version, the legends were present but the figure panels were missing. The errors have been corrected in the HTML and PDF versions of the supplementary information as of 10 June 2014.

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Acknowledgements

This work was supported by the US National Institutes of Health (DP1 DK098058, R01AG040236, P01NS062686 and R01 DK097566), the American Diabetes Association, the Helmholtz Society (ICEMED) and the Klarmann Family Foundation.

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Authors and Affiliations

  1. Program in Integrative Cell Signaling and Neurobiology of Metabolism, Section of Comparative Medicine, Yale University School of Medicine, New Haven, Connecticut, USA
    Jae Geun Kim, Shigetomo Suyama, Marco Koch, Sungho Jin, Zhong-Wu Liu, Marcelo R Zimmer, Jin Kwon Jeong, Klara Szigeti-Buck, Sabrina Diano, Marcelo O Dietrich & Tamas L Horvath
  2. Institute of Anatomy, University of Leipzig, Leipzig, Germany
    Marco Koch
  3. Department of Endocrinology, Hospital Infantil Universitario Niño Jesús, Instituto de Investigación La Princesa and Centro de Investigación Biomédica en Red de la Fisiopatología (CIBER) de Fisiopatología de Obesidad y Nutrición, Instituto de Salud Carlos III, Madrid, Spain
    Pilar Argente-Arizon, Jesús Argente & Julie Chowen
  4. Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, Connecticut, USA
    Jin Kwon Jeong, Sabrina Diano & Tamas L Horvath
  5. Institute for Diabetes and Obesity, Helmholtz Zentrum München & Technische Universität München, Germany
    Yuanqing Gao, Cristina Garcia-Caceres, Chun-Xia Yi & Matthias H Tschöp
  6. Child Study Center, Yale University School of Medicine, New Haven, Connecticut, USA
    Natalina Salmaso & Flora M Vaccarino
  7. Department of Neurobiology, Yale University School of Medicine, New Haven, Connecticut, USA
    Flora M Vaccarino, Sabrina Diano & Tamas L Horvath

Authors

  1. Jae Geun Kim
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  2. Shigetomo Suyama
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  3. Marco Koch
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  4. Sungho Jin
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  5. Pilar Argente-Arizon
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  6. Jesús Argente
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  7. Zhong-Wu Liu
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  8. Marcelo R Zimmer
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  9. Jin Kwon Jeong
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  10. Klara Szigeti-Buck
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  11. Yuanqing Gao
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  12. Cristina Garcia-Caceres
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  13. Chun-Xia Yi
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  14. Natalina Salmaso
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  15. Flora M Vaccarino
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  16. Julie Chowen
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  17. Sabrina Diano
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  18. Marcelo O Dietrich
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  19. Matthias H Tschöp
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  20. Tamas L Horvath
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Contributions

J.G.K., M.O.D. and T.L.H. designed the study. J.G.K., M.H.T. and T.L.H. interpreted the results. J.G.K. and S.J. performed the experiments and analyzed the data. M.K. and K.S.-B. contributed to Figures 1h,j and 2a–c. J.K.J. and S.D. contributed to Figure 1c. S.S. and Z.-W.L. contributed to Figure 2d–g and Supplementary Figure 6. M.R.Z., N.S. and F.M.V. contributed to Supplementary Figures 1 and 4a. P.A.-A., J.C. and J.A. contributed to Supplementary Figure 2b. Y.G., C.G.-C. and C.-X.Y. contributed to the generation of the animal model. J.G.K. and T.L.H. wrote the paper with input from the other authors.

Corresponding author

Correspondence toTamas L Horvath.

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Integrated supplementary information

Supplementary Figure 1 Presence of leptin receptors in hypothalamic astrocytes.

(a) Double fluorescence labeling of the astrocyte marker GFAP (red) and leptin receptors (leptin receptor-driven expression of EGFP, green) shows co-localization of GFAP-immunolabeling with EGFP-tagged leptin receptor-containing profiles (white arrows) in the arcuate nucleus (Arc). Scale bar = 100 μm. (b) The truncated leptin receptor (exon 17) allele was confirmed by in situ hybridization combined with immunohistochemistry. Red fluorescence indicates GFAP-positive astrocyte and green fluorescence indicates POMC neurons. White dots indicate mRNA signals of the leptin receptor containing exon 17. White arrows indicate cells expressing mRNA of leptin receptors. White arrowheads indicate leptin receptor-negative cells. Scale bar = 30 μm. (c) To further confirm astrocyte-specific expression of leptin receptors, transcript of leptin receptor b (LepRb) was amplified from RNA bound to the ribosomes selectively in astrocytes isolated from AldH-EGFP-L10a mice and in AgRP neurons isolated from AgRP-cre:_floxed Rpl22_HA mice. To check purification and contamination of the samples, we analyzed enrichment of AgRP (marker for AgRP purification), NeuN (Marker for neuronal cell contamination) and s100β (marker for astrocyte contamination) compared to that of input samples. Purple bar graphs represent enrichment of LepRb in the AgRP neurons or astrocytes of the hypothalamus compared to that of input controls.

Supplementary Figure 2 Strategy of transgenic mice with selective impairment of leptin receptor signaling in astrocytes.

(a) Schematic drawing of floxed leptin receptor construct, which was used to generate a mouse line allowing cell and time-specific knockout of leptin receptors in astrocytes. (b) RT-PCR shows a deletion of exon 17 of the leptin receptor in primary astrocytes from GFAP-LepR–/– mice. Representative images were selected from at least 3 time repeated experiments.

Supplementary Figure 3 Verification of astrocyte-specific Cre-recombination.

(a) Schematic drawing of human GFAP-driven Cre promoter construct under control of estrogen receptors. (b) GFAP-CreERT2 mice were crossed with tdTomato-loxP reporter mice to confirm successful Cre-mediated recombination in GFAP-positive cells. (c) Ai14 tdTomato mice express red fluorescence in GFAP-positive cells following cre-mediated recombination. tdTomato fluorescences were not co-localized with signals of Iba-1, a biomarker for microglia or NeuN, a biomarker for neuron. White arrows indicate double-labeled cells. Representative images were selected from at least 3 time repeated experiments. Scale bar = 50 μm.

Supplementary Figure 4 Effect of astrocyte-specific deletion of leptin receptors on morphology of astrocytes in the hippocampal CA3.

(a) To confirm expression of leptin receptor in hippocampal astrocytes, we performed ribosome profiling to isolated RNA bound to the ribosomes selectively from hippocampal astrocytes and analyzed enrichment of LepRb. Purple bar graph represents enrichment of LepRb in astrocytes of the hippocampus compared to that of input control. (b) Representative image of GFAP-immunolabeling in the hippocampal CA3 of GFAP-LepR+/+ (+/+) or GFAP-LepR–/– (–/–) mice. (c) The number of GFAP-positive cells (n=5 slices for GFAP-LepR+/+; n=4 slices for GFAP-LepR–/–, p=0.7150, t(7)=0.3803) and (d) length (n=8 cells for GFAP-LepR+/+; n=8 cells for GFAP-LepR–/–, p>0.9999, t(14)=0.0) and (e) number (n=8 cells for GFAP-LepR+/+; n=8 cells for GFAP-LepR–/–, p=0.456, t(14)=0.7667) of astrocyte primary projections did not differ between GFAP-LepR+/+ and GFAP-LepR–/–mice. Results are means ± the s.e.m. P values for unpaired comparisons were analyzed by two-tailed Student's _t_-test.

Supplementary Figure 5 Leptin receptor signaling in astrocytes affects contact formation between astrocytes and melanocortin cells.

(a) Representative electron micrograph showing astrocyte coverage (green pseudo-color and blue arrows) onto POMC-labeled cells. POMC cells of GFAP-LepR–/– mice had less coverage of their perikaryal membranes by astrocytic processes compared to controls. Scale bar = 1 μm. Coronal sections labeled with GFP in AgRP or POMC cells were incubated with GFAP antibody. (b, d) Representative pictures of double-labeled AgRP or POMC-GFP and GFAP-positive cells in the Arc of GFAP-LepR+/+ or GFAP-LepR–/–mice. Percentage of (c) POMC (n=7 slices for GFAP-LepR+/+; n=6 slices for GFAP-LepR–/–, p<0.0001, t(11)=8.226) or (e) AgRP cells (n=8 slices for GFAP-LepR+/+; n=8 slices for GFAP-LepR–/–, p=0.0004, t(14)=4.672) contacted with GFAP-fiber signals was reduced in GFAP-LepR–/– mice. White arrows indicate cells interacted with astrocyte fibers. ***, p<0.001 versus GFAP-LepR+/+ mice. Results are means ± the s.e.m. P values for unpaired comparisons were analyzed by two-tailed Student's _t_-test. Scale bar = 100 μm.

Supplementary Figure 6 The probability distribution and average peak amplitude of miniature postsynaptic currents on AgRP or POMC neurons.

(a, b) Cummulative probability distribution and average peak amplitude (inserts) of mIPSC or mEPSC onto AgRP neurons (Fig. 6a: n=9 cells for GFAP-LepR+/+; n=9 cells for GFAP-LepR–/–, p=0.7715, t(798)=0.2906; Fig. 6b: n=9 cells for GFAP-LepR+/+; n=9 cells for GFAP-LepR–/–, p=0.2788, t(798)=1.084). No-differences were observed between control and GFAP-LepR–/– mice. (c) GFAP-LepR–/– mice showed a significantly increase in probability distribution and average peak amplitude (inserts) of mIPSC onto POMC neurons (n=9 cells for GFAP-LepR+/+; n=9 cells for GFAP-LepR–/–, p<0.0001, t(896)=7.604). (d) GFAP-LepR–/– mice revealed an increase in probability distribution of mEPSC amplitude onto POMC neurons (n=23 cells for GFAP-LepR+/+; n=25 cells for GFAP-LepR–/–, p<0.0001, t(3998)=5.255). **, p<0.01; ***, p<0.001 versus GFAP-LepR+/+. Results are means ± the s.e.m. P values for unpaired comparisons were analyzed by two-tailed Student's _t_-test.

Supplementary Figure 7 Metabolic phenotypes of GFAP-LepR+/+ and GFAP-LepR–/–mice.

Five-week-old male mice were administrated tamoxifen (Tx) or vehicle (Veh) and then analyzed metabolic phenotype at three-month-old age. GFAP-LepR–/–mice did not show significant changes of (a) body weight (n=6 mice for GFAP-LepR+/+-veh; n=8 mice for GFAP-LepR+/+-tx; n=9 mice for GFAP-LepR–/–-tx, p=0.3720, t(12)=0.9274 for GFAP-LepR+/+-veh versus GFAP-LepR+/+-tx; p=0.08, t(15)=1.874 for GFAP-LepR+/+-tx versus GFAP-LepR–/–-tx), (b) fat mass (n=6 mice for GFAP-LepR+/+-veh; n=6 mice for GFAP-LepR+/+-tx; n=5 mice for GFAP-LepR–/–-tx, p=0.588, t(10)=0.5630 for GFAP-LepR+/+-veh versus GFAP-LepR+/+-tx; p=0.1974, t(9)=1.392 for GFAP-LepR+/+-tx versus GFAP-LepR–/–-tx) (c) lean mass (n=6 mice for GFAP-LepR+/+-veh; n=6 mice for GFAP-LepR+/+-tx; n=5 mice for GFAP-LepR–/–-tx, p=0.405, t(10)=0.8686 for GFAP-LepR+/+-veh versus GFAP-LepR+/+-tx; p=0.1410, t(9)=1.614 for GFAP-LepR+/+-tx versus GFAP-LepR–/–-tx) (d) food intake (n=5 mice for GFAP-LepR+/+-veh; n=7 mice for GFAP-LepR+/+-tx; n=7 mice for GFAP-LepR–/–-tx, p=0.8471, t(10)=0.1979 for GFAP-LepR+/+-veh versus GFAP-LepR+/+-tx; p=0.6162, t(12)=0.05146 for GFAP-LepR+/+-tx versus GFAP-LepR–/–-tx) (e, f) energy expenditure (n=8 mice for GFAP-LepR+/+; n=8 mice for GFAP-LepR–/–, p=0.8378, t(14)=0.2086 for light period; p=0.8641, t(14)=0.1743 for dark period; p=0.9966, t(14)=0.004295 for total) and (g, h) physical activity (n=8 mice for GFAP-LepR+/+; n=8 mice for GFAP-LepR–/–, p=0.1184, t(14)=1.663 for light period; p=0.2703, t(14)=1.148 for dark period; p=0.1692, t(14)=1.149 for total) when compared to littermate control mice. Results are means ± the s.e.m. P values for unpaired comparisons were analyzed by two-tailed Student's _t_-test.

Supplementary Figure 8 Representative images of Fos-positive POMC or AgRP neurons in the Arc of GFAP-LepR+/+ and GFAP-LepR–/– mice.

(a) Representative images show double labeled POMC-GFP and Fos cells in the Arc of GFAP-LepR+/+ and GFAP-LepR–/– mice.Scale bar = 100 μm. (b) Representative images show double labeled AgRP-GFP and Fos cells in the Arc of GFAP-LepR+/+ and GFAP-LepR–/– mice. Scale bar = 100 μm.

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Kim, J., Suyama, S., Koch, M. et al. Leptin signaling in astrocytes regulates hypothalamic neuronal circuits and feeding.Nat Neurosci 17, 908–910 (2014). https://doi.org/10.1038/nn.3725

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