Volume 5, Issue 6, Pages 438-449 (June 2007) Insulin Action in AgRP-Expressing Neurons Is Required for Suppression of Hepatic Glucose Production  A. Christine Könner, Ruth Janoschek, Leona Plum, Sabine D. Jordan, Eva Rother, Xiaosong Ma, Chun Xu, Pablo Enriori, Brigitte Hampel, Gregory S. Barsh, C. Ronald Kahn, Michael A. Cowley, Frances M. Ashcroft, Jens C. Brüning  Cell Metabolism  Volume 5, Issue 6, Pages 438-449 (June 2007) DOI: 10.1016/j.cmet.2007.05.004 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Generation of POMC- and AgRP-Specific IR Knockout Mice (A) Cre-mediated recombination was visualized by immunohistochemistry for β-galactosidase (β-gal) in brains of double-heterozygous reporter mice (PomcCre-LacZ) at the age of approximately 10 weeks. Blue (DAPI), DNA; green, β-gal (POMC neurons). Scale bar = 100 μm. (B) Cre-mediated recombination was visualized by immunohistochemistry for β-galactosidase (β-gal) in brains of double-heterozygous reporter mice (AgRPCre-LacZ) at the age of approximately 10 weeks. Blue (DAPI), DNA; green, β-gal (AgRP neurons). Scale bar = 100 μm. (C) Western blot analysis of IR-β subunit, Akt, and α-tubulin (loading control) in whole brain, hypothalamus, liver, skeletal muscle, and pancreas in control, IRΔPOMC, and IRΔAgRP mice (n = 3 in each group). Cell Metabolism 2007 5, 438-449DOI: (10.1016/j.cmet.2007.05.004) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 PIP3 Formation in Hypothalamic Neurons of Control, IRΔPOMC, and IRΔAgRP Reporter Mice (A) Double immunohistochemistry of ARC neurons of control and IRΔPOMC reporter mice was performed in overnight-fasted animals, which were intravenously injected with either saline or insulin and sacrificed 10 min after stimulation. Arrows indicate one POMC and one non-POMC neuron in each panel. Blue (DAPI), DNA; red, β-gal (POMC neurons); green, PIP3. (B) Quantification of PIP3 levels in control reporter mice in the basal state (−) and after insulin stimulation (+). Values are means ± SEM of sections obtained from three unstimulated and four insulin-stimulated control mice. ∗∗p ≤ 0.01. (C) Quantification of PIP3 levels in IRΔPOMC reporter mice in the basal state (−) and after insulin stimulation (+). Values are means ± SEM of sections obtained from three unstimulated and three insulin-stimulated IRΔPOMC mice. (D) Double immunohistochemistry of ARC neurons of control and IRΔAgRP reporter mice was performed in overnight-fasted animals, which were intravenously injected with either saline or insulin and sacrificed 10 min after stimulation. Arrows indicate one AgRP and one non-AgRP neuron in each panel. Blue (DAPI), DNA; red, β-gal (AgRP neurons); green, PIP3. (E) Quantification of PIP3 levels in control reporter mice in the basal state (−) and after insulin stimulation (+). Values are means ± SEM of sections obtained from two unstimulated and two insulin-stimulated control mice. ∗p ≤ 0.05. (F) Quantification of PIP3 levels in IRΔAgRP reporter mice in the basal state (−) and after insulin stimulation (+). Values are means ± SEM of sections obtained from one unstimulated and four insulin-stimulated IRΔAgRP mice. (G) Examples of different magnitudes of PIP3 immunoreactivity as described in Experimental Procedures. Cell Metabolism 2007 5, 438-449DOI: (10.1016/j.cmet.2007.05.004) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Effects of Insulin and Tolbutamide on Electrical Activity of Control and IRΔAgRP-Z/EG Neurons (A) Representative recordings of identified AgRP neurons in ARC slices from AgRPCre-Z/EG (control) and IRΔAgRP-Z/EG mice before and after addition of 200 nM insulin. (B) Mean membrane potential of identified AgRP neurons in ARC slices from AgRPCre-Z/EG (n = 6) and IRΔAgRP-Z/EG (n = 6) mice before (−) and after (+) addition of 200 nM insulin. Displayed values are means ± SEM. ∗∗∗p ≤ 0.001. (C) Mean firing frequency of AgRPCre-Z/EG (n = 6) and IRΔAgRP-Z/EG (n = 6) neurons before (−) and after (+) addition of 200 nM insulin (control versus IRΔAgRP before addition of insulin, p = 0.11). (D) Representative recording of an identified AgRP neuron in an ARC slice from an AgRPCre-Z/EG (control) mouse in the absence of drug, after addition of 200 nM insulin, and after addition of 200 μm tolbutamide. Cell Metabolism 2007 5, 438-449DOI: (10.1016/j.cmet.2007.05.004) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Unaltered Energy Homeostasis in IRΔPOMC and IRΔAgRP Mice (A) Average body weights of male control (♢) and IRΔPOMC (♦) mice on normal diet (n = 26–31). (B) Average body weights of male control (♢) and IRΔAgRP (♦) mice on normal diet (n = 18–25). (C) Serum leptin levels of male control (n = 10) and IRΔPOMC (n = 10) mice on normal diet (ND) and high-fat diet (HFD) at 15 weeks of age. Displayed values are means ± SEM. (D) Serum leptin levels of male control (n = 11–15) and IRΔAgRP (n = 8–15) mice on normal diet (ND) and high-fat diet (HFD) at 15 weeks of age. (E) Daily food intake of male control (n = 15) and IRΔPOMC (n = 20) mice on normal diet at 11–13 weeks of age. (F) Daily food intake of male control (n = 11) and IRΔAgRP (n = 11) mice on normal diet at 11–13 weeks of age. (G) Neuropeptide expression in male control (n = 8) and IRΔPOMC (n = 12) mice at 12 weeks of age. (H) Neuropeptide expression in male control (n = 8) and IRΔAgRP (n = 8) mice at 12 weeks of age. Cell Metabolism 2007 5, 438-449DOI: (10.1016/j.cmet.2007.05.004) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Regulation of Glucose Homeostasis in IRΔPOMC and IRΔAgRP Mice (A) Blood glucose levels during the clamp period in control (□, n = 13), IRΔPOMC (▪, n = 9), and IRΔAgRP (▴, n = 11) mice at 15 weeks of age. Displayed values are means ± SEM. (B) Glucose infusion rates during the clamp period in control (□, n = 13), IRΔPOMC (▪, n = 9), and IRΔAgRP (▴, n = 11) mice at 15 weeks of age. (C) Hepatic glucose production (HGP) measured under basal and steady-state conditions during the clamp in control (n = 13), IRΔPOMC (n = 9), and IRΔAgRP (n = 11) mice. ∗p ≤ 0.05; ∗∗p ≤ 0.01. (D) Percentage suppression of HGP by insulin as measured under steady-state conditions during the clamp in control (n = 13), IRΔPOMC (n = 9), and IRΔAgRP (n = 11) mice. (E) Tissue-specific insulin-stimulated glucose uptake rates in control (n = 13), IRΔPOMC (n = 9), and IRΔAgRP (n = 11) mice. Cell Metabolism 2007 5, 438-449DOI: (10.1016/j.cmet.2007.05.004) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 Insulin Action in AgRP Neurons Regulates Hepatic IL-6 and G6Pase Expression (A) IL-6 mRNA expression in liver at the end of euglycemic-hyperinsulinemic clamps in control (n = 13), IRΔPOMC (n = 9), and IRΔAgRP (n = 11) mice. Displayed values are means ± SEM. ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001. (B) Top panel shows western blot analysis of the glucose-6-phosphatase α subunit and β-actin (loading control) in liver at the end of euglycemic-hyperinsulinemic clamps in control (n = 4), IRΔPOMC (n = 5), and IRΔAgRP (n = 4) mice. Bottom panel shows densitometric analysis of glucose-6-phosphatase α subunit expression in IRΔPOMC and IRΔAgRP mice as compared to controls. Cell Metabolism 2007 5, 438-449DOI: (10.1016/j.cmet.2007.05.004) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 Model of HGP Regulation via Insulin Action in AgRP Neurons Cell-autonomous action of insulin on AgRP neurons results in activation of the PI3 kinase (p85, regulatory subunit; p110, catalytic subunit) and generation of PIP3. PIP3 activates KATP channels, resulting in hyperpolarization and a decreased neuronal firing rate with subsequent reduced release of AgRP and other transmitters of AgRP neurons. This regulates innervation of liver, leading to increased IL-6 expression. IL-6 acting on liver parenchymal cells results in Stat3 phosphorylation via activation of Jak2, thereby leading to decreased expression of G6Pase. G6P, glucose-6-phosphate; gp130, glycoprotein 130; IL-6R, interleukin-6 receptor α; Jak, Janus kinase 2; PIP2, phosphatidylinositol 4,5-biphosphate; Pten, phosphatase and tensin homolog. Cell Metabolism 2007 5, 438-449DOI: (10.1016/j.cmet.2007.05.004) Copyright © 2007 Elsevier Inc. Terms and Conditions