Volume 25, Issue 2, Pages 335-344 (February 2017) Gastric Bypass Surgery Recruits a Gut PPAR-α-Striatal D1R Pathway to Reduce Fat Appetite in Obese Rats  Mohammed K. Hankir, Florian Seyfried, Constantin A. Hintschich, Thi-Ai Diep, Karen Kleberg, Mathias Kranz, Winnie Deuther-Conrad, Luis A. Tellez, Michael Rullmann, Marianne Patt, Jens Teichert, Swen Hesse, Osama Sabri, Peter Brust, Harald S. Hansen, Ivan E. de Araujo, Ute Krügel, Wiebke K. Fenske  Cell Metabolism  Volume 25, Issue 2, Pages 335-344 (February 2017) DOI: 10.1016/j.cmet.2016.12.006 Copyright © 2017 Elsevier Inc. Terms and Conditions

Cell Metabolism 2017 25, 335-344DOI: (10.1016/j.cmet.2016.12.006) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 RYGB Surgery Induces Robust Body Weight Loss and Hypophagia and Reduces Preference for HF Food in DIO Rats (A) Weekly postoperative body weight measurements of high-fat (HF) DIO rats that had undergone Roux-en-Y gastric bypass surgeries with (RYGB-VAG) or without (RYGB) total sub-diaphragmatic truncal vagotomy compared to sham-operated control groups. Postoperatively, animals were maintained on a regular chow diet with the exception of Sham-HF rats, which were constantly maintained on an HF diet (mean ± SEM, n = 7–16/group; repeated-measures two-way ANOVA; #p < 0.05 for Sham-HF versus Sham-LF and Sham-VAG; ##p < 0.01, ###p < 0.001, and ####p < 0.0001 for RYGB, RYGB-VAG, and Sham-BWM versus Sham-LF, Sham-VAG, and Sham-HF). (B) Corresponding cumulative energy intake (mean ± SEM, n = 7–16/group; one-way ANOVA; ∗∗∗p < 0.001 and ∗∗∗∗p < 0.001). (C) Weekly preference for HF diet over regular chow for a separate cohort of RYGB-operated and sham-operated control rats given free access to both (mean ± SEM, n = 6/group; repeated-measures two-way ANOVA; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001). Cell Metabolism 2017 25, 335-344DOI: (10.1016/j.cmet.2016.12.006) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 RYGB Surgery Increases Intestinal OEA Synthesis in DIO Rats (A–E) Intestinal OEA concentrations determined by LC-MS of RYGB-operated and sham-operated control rats either food restricted overnight or refed with an HF meal for 60 min. Top panel shows a schematic of the gastrointestinal anatomy of “sham groups” (Sham-LF, Sham-VAG, Sham-HF, and Sham-BWM) and “RYGB groups” (RYGB and RYGB-VAG). (A) Duodenum/biliopancreatic limb, (B) proximal jejunum/Roux limb, and (C) proximal ileum/common channel. Concentrations of OEA in the common channel of RYGB and RYGB-VAG rats were compared to (D) the duodenum and (E) the proximal jejunum of sham-operated control animals (mean ± SEM, n = 3–7/group; repeated-measures two-way ANOVA; ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 for effects of refeeding; a = p < 0.05 and aa = p < 0.001 versus Sham-HF, b = p < 0.001 versus RYGB and RYGB-VAG, c = p < 0.05 versus RYGB and RYGB-VAG, and d = p < 0.0001 versus RYGB and RYGB-VAG for group effects in the refed state). (F) Corresponding HF intake after 60 min refeeding (mean ± SEM, n = 6/group; one-way ANOVA; e = p < 0.01 versus RYGB, ee = p < 0.001 versus RYGB, f = p < 0.05 versus RYGB-VAG, and ff = p < 0.001 versus RYGB-VAG). Cell Metabolism 2017 25, 335-344DOI: (10.1016/j.cmet.2016.12.006) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 RYGB Surgery Potentiates HF Feeding-Induced Dorsal Striatal Dopamine Release in DIO Rats (A) Dorsolateral striatal dopamine levels determined by HPLC of RYGB-operated and sham-operated control rats that were food restricted overnight and refed with an HF meal for 30 min (indicated by the gray area). Samples were collected by in vivo cerebral microdialysis at 10 min intervals at baseline, during the HF meal, and for 90 min afterward. Values for each group are expressed relative to baseline (mean ± SEM, n = 4–8/group). (B and C) Associated area under the curve (B) and corresponding HF intake (C) after 30 min refeeding (mean ± SEM, n = 4–8/group; one-way ANOVA; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001). Cell Metabolism 2017 25, 335-344DOI: (10.1016/j.cmet.2016.12.006) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 RYGB Surgery Increases Striatal D1R Density in DIO Rats Postoperatively Maintained on a Choice Diet of LF and HF Food Left panel: representative brain PET images of RYGB-operated and sham-operated control rats that received the dopamine 1 receptor (D1R) selective radioligand [11C] SCH-23390. Animals were previously maintained on a choice diet of LF and HF food for 14 weeks postoperatively up until the time of scanning. Right panel: the mean standardized uptake value (SUV) of [11C] SCH-23390 in striatum normalized to that of the mean SUV in cerebellum and plotted against time (data are presented as linear regression curves with 95% confidence intervals denoted by the dotted lines; n = 5/group; p values for slopes of linear regression curves were obtained from one-way ANOVA). Cell Metabolism 2017 25, 335-344DOI: (10.1016/j.cmet.2016.12.006) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Reduced HF Appetite after RYGB Surgery Requires Intact PPAR-α, Vagus Nerve, and Dorsal Striatal D1R Signaling High-concentration fat emulsion (5% Intralipid) intakes and preferences of RYGB-operated and sham-operated control rats during oral two-bottle preference tests in response to interfering with gut-brain signaling. Schematic diagrams on the left demonstrate where stimulators/blockers were administered prior to experiments. (A and B) Intake (A) and preference (B) of 5% Intralipid following intra-intestinal infusion of the PPAR-α antagonist GW-6471 (4 mg/kg in DMSO/0.9% saline), the PPAR-α agonist WY-14643 (1 mg/kg in DMSO/0.9% saline), or vehicle (DMSO/0.9% saline) (mean ± SEM, n = 5–10/group; one-way ANOVA; ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001). (C and D) Intake (C) and preference (D) of 5% Intralipid following complete sub-diaphragmatic truncal vagotomy (mean ± SEM, n = 5–10/group; one-way ANOVA; ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, #p < 0.05 versus Sham-LF, and ##p < 0.05 versus Sham-LF). (E and F) Intake (E) and preference (F) of 5% Intralipid following intra-dorsolateral striatal infusion of the selective D1R antagonist SCH-23390 (0.6 μg/0.3 μL in ACSF) or ACSF as vehicle (mean ± SEM, n = 4–11/group; two-tailed t test; ∗p < 0.05 and ∗∗∗p < 0.001). Cell Metabolism 2017 25, 335-344DOI: (10.1016/j.cmet.2016.12.006) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 A Gut-Striatal Dopamine Circuit that Drives Reduced HF Appetite after RYGB Due to the intestinal reconfiguration caused by RYGB, ingested fat (1) is diverted away from the proximal small intestine (biliopancreatic limb/duodenum) toward the distal small intestine (Roux limb/proximal jejunum) (2). As ingested fat traverses through the gut, it eventually meets with bile in the common channel (distal jejunum/proximal ileum) (3). Here, fatty acids (FAs), liberated from the catalytic action of pancreatic lipases, cross the apical membrane of enterocytes (4) via CD36-mediated transport (Schwartz et al., 2008). Inside the enterocyte, FAs are converted into OEA through a series of enzymatic reactions. From this stage, the precise signaling pathway has not been defined, but two distinct possibilities exist: (5) within the enterocyte the lipid messenger OEA and/or FAs activate the nuclear receptor PPAR-α, resulting in rapid non-genomic membrane depolarization (Melis et al., 2008) and the release of an unidentified excitatory paracrine (across the basolateral membrane) onto vagal afferents, and/or (6) OEA is released by the enterocyte and enters vagal afferents, where it activates PPAR-α (Liu et al., 2014), also resulting in membrane depolarization. This signal is propagated via the vagus nerve to hindbrain neurons in the nucleus tractus solitarius (NTS) (7), which send indirect projections to midbrain dopaminergic substantia nigra pars compacta neurons (SNpc) (8). The increased function of nigrostriatal dopaminergic neurons culminates in the release of dopamine, which binds to and activates dopamine 1 receptors (D1Rs) predominantly expressed in direct pathway medium spiny neurons (9), thereby reducing fat appetite (10). Cell Metabolism 2017 25, 335-344DOI: (10.1016/j.cmet.2016.12.006) Copyright © 2017 Elsevier Inc. Terms and Conditions