Volume 17, Issue 10, Pages 2512-2521 (December 2016) Insulin-Dependent Activation of MCH Neurons Impairs Locomotor Activity and Insulin Sensitivity in Obesity  A. Christine Hausen, Johan Ruud, Hong Jiang, Simon Hess, Hristo Varbanov, Peter Kloppenburg, Jens C. Brüning  Cell Reports  Volume 17, Issue 10, Pages 2512-2521 (December 2016) DOI: 10.1016/j.celrep.2016.11.030 Copyright © 2016 The Author(s) Terms and Conditions

Cell Reports 2016 17, 2512-2521DOI: (10.1016/j.celrep.2016.11.030) Copyright © 2016 The Author(s) Terms and Conditions

Figure 1 Insulin Increases Excitability of MCH Neurons (A) Cre-mediated recombination was visualized by immunohistochemistry for EGFP on brain sections of double-heterozygous reporter mice (MCH-GFP). Cre-mediated recombination removes the loxP-flanked neomycin resistance gene; thus, GFP is transcribed only in MCH-expressing cells. GFP-positive cells are indicated in brown. 3V, third ventricle. Scale bar, 50 μm. (B) Higher magnification of GFP-positive cells in the lateral hypothalamus shown in (A). (C) Electrophysiological identification of an MCH neuron by a series of hyperpolarizing current injections (0 pA to −100 pA in 20-pA steps) revealing a prominent outward rectification (arrowhead). (D) Immunohistochemical identification of a recorded MCH neuron expressing GFP (green, arrowhead) that was injected with biocytin (red) in the lateral hypothalamus of an MCH-GFP mouse. Scale bars, 20 μm. (E) Left: representative recording from a spontaneously active MCH-GFP neuron that fired action potentials. Right: representative current-clamp recording from an MCH-GFP neuron that was silent. Inset: proportion of silent and spontaneously (spont.) active MCH-GFP neurons. (F) Representative voltage responses to increasing depolarizing current injections (from 20 to 80 pA in 20-pA steps) elicited from a holding potential of −60 mV before and during the application of insulin (200 nM) in the presence of synaptic blockers. (G) Top: mean spike frequency during the current pulse as a function of injected current of insulin-excited cells. The asterisks represent significant differences in spike frequency before and during insulin application. Data are represented as means ± SEM; ∗p ≤ 0.05; ∗∗p ≤ 0.01, as determined by paired Student’s t test. AP, action potential. Bottom: percentage of MCH neurons that responded to insulin with an increase, a decrease, or no change in evoked spike frequency (in the presence of synaptic blockers). See also Figure S1. Cell Reports 2016 17, 2512-2521DOI: (10.1016/j.celrep.2016.11.030) Copyright © 2016 The Author(s) Terms and Conditions

Figure 2 Increased Locomotor Activity and Improved Insulin Sensitivity in Obese IRΔMCH Mice (A) Left: quantification of PIP3 levels in control reporter mice (MCH-LacZ) in the basal state (-) and after insulin stimulation (+). Values are means ± SEM of sections obtained from two unstimulated (saline-treated) and three insulin-stimulated control mice. Right: representative double immunohistochemistry of lateral hypothalamic neurons of an insulin-stimulated control reporter mouse. (B) Left: quantification of PIP3 levels in IRΔMCH LacZ reporter mice in the basal state (-) and after insulin stimulation (+). Values are means ± SEM of sections obtained from five unstimulated (saline-treated) and five insulin-stimulated IRΔMCH mice. Right: representative double immunohistochemistry of lateral hypothalamic neurons of an insulin-stimulated IRΔMCH reporter mouse. Blue (DAPI) indicates DNA; red indicates LacZ (MCH cells); green indicates PIP3. (C) Average body weight of male control (♢, n = 19) and IRΔMCH (♦, n = 21) mice on a high-fat diet (HFD). (D) Epididymal fat pad weight of male control (n = 15) and IRΔMCH (n = 18) mice at the age of 20 weeks on a HFD. (E) Brown adipose tissue (BAT) weight of male control (n = 15) and IRΔMCH (n = 18) mice on a HFD at the age of 20 weeks. (F) Average body fat content of male control (n = 15) and IRΔMCH (n = 18) mice on a HFD at the age of 20 weeks measured by nuclear magnetic resonance. (G) Serum leptin concentrations measured by ELISA of male control (n = 6) and IRΔMCH (n = 10) mice on a HFD at the age of 19 weeks. (H) Daily food intake of male control (n = 18) and IRΔMCH (n = 23) mice on a HFD at the age of 18 weeks. (I) Mean oxygen consumption (VO2) corrected for lean body mass of male control (n = 18) and IRΔMCH (n = 23) mice on a HFD measured by indirect calorimetry at the age of 18 weeks. (J) Basal locomotor activity of male control (n = 18) and IRΔMCH (n = 23) mice on a HFD at the age of 18 weeks. (K) Serum insulin concentrations of male control (n = 6) and IRΔMCH (n = 10) mice on a HFD at the age of 19 weeks. (L) Glucose tolerance test of overnight-fasted male control (n = 18) and IRΔMCH (n = 14) mice on a HFD at the age of 12 weeks. (M) Insulin tolerance test of male control (n = 28) and IRΔMCH (n = 28) mice on a HFD at the age of 13 weeks. (N) Pyruvate tolerance test of male control (n = 14) and IRΔMCH (n = 17) mice on a HFD at the age of 15 weeks. (O) Upper panel: immunoblot analysis (left) and quantification (right) of insulin-stimulated Akt phosphorylation (S473) in livers of control and IRΔMCH mice. An immunoblot was performed for phosphorylated Akt, Akt, and calnexin as a loading control. Lower panel: immunoblot analysis (left) and quantification (right) of insulin-stimulated Akt phosphorylation in skeletal muscles of control and IRΔMCH mice. Data are represented as means ± SEM. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001, as determined by unpaired Student’s t test for the evaluation of PIP3 formation in LacZ-positive neurons (A), locomotor activity (J), and quantification of insulin-stimulated Akt phosphorylation (O); and by two-way ANOVA followed by Holm-Sidak’s post hoc test for the insulin tolerance test (ITT) and pyruvate tolerance test (PTT) (M and N). See also Figure S2. Cell Reports 2016 17, 2512-2521DOI: (10.1016/j.celrep.2016.11.030) Copyright © 2016 The Author(s) Terms and Conditions

Figure 3 Inactivation of the Insulin Receptor in MCH-Expressing Cells Improves Peripheral Glucose Metabolism in Obese Mice (A) Blood glucose levels during hyperinsulinemic-euglycemic clamp analysis in HFD-fed control (n = 10) and IRΔMCH (n = 9) mice. (B) Left: glucose infusion rates (GIRs) during hyperinsulinemic-euglycemic clamps in HFD-fed control (n = 10) and IRΔMCH (n = 9) mice. Right: analysis of areas under the curve (AUCs) for GIR. (C) Hepatic glucose production (HGP) of HFD-fed control (n = 10) and IRΔMCH (n = 9) mice before (basal) and during (steady state) hyperinsulinemic-euglycemic clamp analysis. (D) Tissue-specific insulin-stimulated [1-14C]-deoxy-D-glucose uptake in brain, white adipose tissue (WAT), and skeletal muscle (SM) of HFD-fed control (n = 10) and IRΔMCH (n = 9) mice under steady-state conditions in hyperinsulinemic-euglycemic clamp analysis. (E) Left: quantification of lateral hypothalamic PIP3 levels in overnight-fasted unstimulated (saline-treated) control and IRΔMCH reporter mice fed a HFD. Values are means ± SEM of sections obtained from four MCH-LacZ control and three IRΔMCH LacZ mice. Right: representative double immunohistochemistry of MCH neurons of unstimulated (saline-treated) control and IRΔMCH reporter mice fed a HFD. Blue (DAPI) indicates DNA; red indicates LacZ (MCH cells); green indicates PIP3. (F) Percentage of MCH-expressing cells with high levels of PIP3 of HFD-fed control MCH-LacZ (black and white stripes) and IRΔMCH (black) LacZ reporter mice in relation to high PIP3 levels of unstimulated control MCH-LacZ reporter mice on a normal chow diet (white). Data are represented as means ± SEM; ∗p ≤ 0.05, as determined by two-way ANOVA followed by Holm-Sidak’s post hoc test for glucose infusion rates (B); by unpaired Student’s t test for hepatic glucose production (C) and evaluation of PIP3 in LacZ-positive cells (E); and by one-way ANOVA followed by Tukey’s post hoc test for comparing high levels of PIP3 in MCH-expressing cells (F). Cell Reports 2016 17, 2512-2521DOI: (10.1016/j.celrep.2016.11.030) Copyright © 2016 The Author(s) Terms and Conditions

Figure 4 Acute Chemogenetic Activation of MCH Neurons Decreases Locomotor Activity (A) Representative rate histogram (top) and the respective original recording (bottom) of an MCH-expressing neuron in a hypothalamic slice of an hM3DGqMCH;tdTomato mouse before, during, and after application of 3 μM clozapine-N-oxide (CNO). (B) Quantification of change in membrane-potential (left) and action-potential firing frequency (right) of identified MCH neurons in hypothalamic slices of hM3DGqMCH;tdTomato mice after application of 3 μM CNO. (C) Food intake of hMD3GqWT (n = 7) and hM3DGqMCH-Cre (n = 8) mice after CNO administration (3 mg/kg [body weight; BW]) during the early phase of the light cycle. (D) Left: locomotor activity of hMD3GqWT (n = 7) and hM3DGqMCH-Cre (n = 8) mice in their home cage environment after CNO administration (3 mg/kg BW). Right: analysis of areas under the curve (AUCs) for locomotor activity. (E) Glucose tolerance test of overnight-fasted hMD3GqWT (n = 14) and hM3DGqMCH-Cre (n = 10) mice after CNO administration (3 mg/kg BW). (F) Insulin tolerance test of randomly fed hMD3GqWT (n = 11) and hM3DGqMCH-Cre (n = 7) mice after CNO administration (3 mg/kg BW). Data are represented as means ± SEM. In (B), whiskers indicate the minimum and maximum values, center lines indicate the median, and plus signs indicate the means. ∗p ≤ 0.05; ∗∗p ≤ 0.01; and ∗∗∗p ≤ 0.001, as determined by paired two-tailed Student’s t test for the effect of CNO versus control solution on membrane-potential and action-potential firing frequency (B); by two-way ANOVA followed by Holm-Sidak’s post hoc test for locomotor activity (D); and by unpaired Student’s t test for the AUC analysis of locomotor activity (D). Cell Reports 2016 17, 2512-2521DOI: (10.1016/j.celrep.2016.11.030) Copyright © 2016 The Author(s) Terms and Conditions