1. Volume 18, Issue 7, Pages 1587-1597 (February 2017)
Inhibition of P2Y6 Signaling in AgRP Neurons Reduces Food Intake and Improves Systemic Insulin Sensitivity in Obesity 
Sophie Marie Steculorum, Katharina Timper, Linda Engström Ruud, Nadine Evers, Lars Paeger, Stephan Bremser, Peter Kloppenburg, Jens Claus Brüning 
Cell Reports 
Volume 18, Issue 7, Pages (February 2017)
DOI: /j.celrep
Copyright © 2017 Max Planck Institute for Metabolism Research Terms and Conditions

2. Cell Reports 2017 18, 1587-1597DOI: (10.1016/j.celrep.2017.01.047)
Copyright © 2017 Max Planck Institute for Metabolism Research Terms and Conditions

3. Figure 1 UDP/P2Y6 Signaling Promotes Feeding in Obese Mice
(A) Representative microphotographs of GFP immunostaining (P2Y6-EGFP, green) and corresponding nuclear counterstaining (DAPI, blue) in the medio-basal hypothalamus containing the arcuate nucleus of the hypothalamus (ARH), the ventromedial nucleus of the hypothalamus (VMH), the dorsomedial nucleus of the hypothalamus (DMH), and the lateral hypothalamic area (LHA) of diet-induced obese P2Y6-EGFP mice.
(B) Representative microphotographs of ARH depicting co-immunostaining of P2Y6-EGFP (P2Y6-EGFP, green) and fluorescent Nissl staining (NeuroTrace, neurons, red).
(C and D) 2-hr food-intake measurement (depicted as food intake to body weight relative to control) after intracereborventricular (i.c.v.) administration of (C) UDP (100 μM) or vehicle (saline) (n = 18 versus 18) and (D) of the P2Y6-antagonist MRS 2578 (1 μM) or vehicle (DMSO 0,1%) (n = 14 versus 14).
Scale bars, 150 μm. 3V, third ventricle. Data are presented as mean ± SEM, ∗p ≤ 0.05 as determined by unpaired Student’s t test. See also Figure S1.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 Max Planck Institute for Metabolism Research Terms and Conditions

4. Figure 2
Mouse Models for the Study of Global or AgRP Neuron-Restricted P2Y6 Deficiency
(A and B) Real-time qPCR analysis of pyrimidinergic receptor P2Y, G protein coupled, 6 (P2ry6) mRNA expression in hypothalamus (Hyp), white adipose tissue (WAT), liver and skeletal muscle (SKM) of (A) whole-body P2Y6-deficient mice (P2Y6 Δ/Δ) and their littermates control (n = 7–10 versus 9) and of (B) P2Y6 ΔAgRP and their littermates control (n = 6–17 versus 6–18).
(C) Representative microphotographs of RNAscope in situ hybridization of P2ry6-mRNA (red) and AgRP-mRNA (green) and of corresponding nuclear counterstaining (DAPI, blue) in the arcuate nucleus (ARH) of top row, control mice; middle row, P2Y6 Δ/Δ mouse; bottom row, P2Y6 ΔAgRP mouse. Arrows depict double-labeled cells positive for P2ry6- and AgRP-mRNA and stars highlight cells that are P2ry6-mRNA positive but AgRP-mRNA negative.
(D) Left: representative traces of AgRP neurons firing frequency in response to the application of 3 μM UDP in control mouse (upper panel) and in P2Y6 ΔAgRP mouse (lower panel). Middle: quantification of action potential (AP) frequency (control, upper panel n = 21; P2Y6 ΔAgRP bottom panel, n = 20; responsive and non-responsive pooled). Light-gray and dark-gray circles mark, respectively, single recordings responding with a significant increase or decrease in AP frequency and open circles are non-responsive neurons. Right: overall responsiveness to 3 μM UDP of the recorded AgRP neuron population. All recordings have been conducted in synaptically isolated neurons. Scale bars, 50 μm.
Data are represented as presented as mean ± SEM and ∗p ≤ 0.05 and ∗∗∗p ≤ as determined by unpaired Student’s t (A and B) and as boxplots generated according to the “Tukey method” (mean: “+,” median: horizontal line) (D), ∗p ≤ 0.05 as determined by paired Student’s t test.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 Max Planck Institute for Metabolism Research Terms and Conditions

5. Figure 3
AgRP Neuron-Restricted P2Y6 Deficiency Limits High-Fat-Diet-Induced Adiposity and Hyperphagia
(A–H) Analysis of high-fat diet (HFD)-fed P2Y6 Δ/Δ (left panels) and P2Y6 ΔAgRP (right panels) and their respective control littermates. (A and B) Body weight curves from 3 to 18 weeks (A, n = 13 versus 12 and B, n = 11 versus 9) and naso-anal length at sacrifice (A, n = 12 versus 12 and B, n = 11 versus 9; 20 weeks). (C and D) Body-fat composition in percentages of lean and fat mass (C, n = 12 versus 12 and D, n = 11 versus 9; 20 weeks). (E and F) Quantification of adipocytes area in perigonadal white adipose tissue (E, n = 14 versus 17 and F, n = 13 versus 7; 20 weeks) and representative images of H&E staining (scale bar, 50 μm). (G and H) Cumulative 24-hr food intake (G, n = 13 versus 20 and H, n = 11 versus 17; 16 weeks). Data are represented as presented as mean ± SEM, p ≤ 0.05 and ∗∗∗p ≤ as determined by two-way ANOVA followed by Bonferroni post hoc test (A, B, G, and H) or two-tailed unpaired Student’s t test (C, D, E, and F).
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 Max Planck Institute for Metabolism Research Terms and Conditions

6. Figure 4
AgRP-Neuron-Restricted P2Y6 Deficiency Improves Obesity-Associated Insulin Resistance
(A and B) Glucose tolerance tests (GTT) performed in 12-week-old HFD-fed (A) P2Y6 Δ/Δ and control mice (n = 15 versus 12) and (B) P2Y6 ΔAgRP mice and control mice (n = 14 versus 16).
(C and D) Insulin tolerance tests (ITT) in 13-week-old HFD-fed (C) P2Y6 Δ/Δ and control mice (n = 16 versus 21) and (D) P2Y6 ΔAgRP mice and control mice (n = 16 versus 17). Area under the curves (AUCs) of each GTT and ITT are plotted next to the corresponding curves.
(E) Quantification and representative immunoblots of phosphorylated and total AKT as well as respective calnexin loading controls in skeletal muscle (SKM; n = 4 versus 4), brown adipose tissue (BAT; n = 5 versus 6), and liver (n = 5 versus 6) of 11-week-old mice fed a HFD. Data are represented as mean ± SEM. ∗p ≤ 0.05 and ∗∗p ≤ 0.01 as determined by two-way ANOVA followed by Bonferroni post hoc test for the curves or unpaired one-tailed Student’s t test.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2017 Max Planck Institute for Metabolism Research Terms and Conditions