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1 Supporting Information Shirazi et al /pnas SI Materials and Methods Brain Surgery: Rats. A lateral ventricle (LV) guide cannula was positioned and attached to the skull with dental acrylic and jeweler s screws and closed with an obturator, as described previously (1, 2). Briefly, a lateral ventricular guide cannula [26 gauge (Plastics One); coordinates: ±1.6 from the midline, 0.9 mm posterior to bregma, and 2.0 mm ventral to dura mater, with injector aimed 4.0 mm ventral to the dura) was implanted under ketamine/xylazine anesthesia. The cannula placement was verified before behavioral studies commenced with the angiotensin II (Tocris) drinking test (angiotensin II dose: 10 ng/1 μl, 2-μL injection bolus). Only the rats that passed the inclusion criteria were included in the study. Telemetric Transponder Surgery. For recording core body temperature and spontaneous physical activity, rats were implanted with telemetric transponders (G2 VitalView; Mini Mitter/Respironics) as described previously. Transponders were inserted into the abdominal cavity and secured to the abdominal muscles with sutures. The use of this telemetric system allows for continuous (every 5 min) measurements without any disturbance or stress to the animal. Interaction of Central GLP-1 Receptors Stimulation Effect on Food Intake, Body Weight, and Temperature with IL-6 and IL-1β. To determine whether signaling at the IL-1R or IL-6 (or the combination of the two) is mediating the anorexic, body weight-suppressing, and thermoregulatory effects of central glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) stimulation, rats received four counterbalanced injection conditions over 4 experimental days: (i) vehicle for IL-1Ra artificial cerebrospinal fluid (acsf) + vehicle for exendin-4 (EX4) (acsf); (ii) vehicle for IL-1Ra + EX4 0.2 μg/ 1 μl; (iii) IL-1Ra 1 μg/1 μl + EX4 0.2 μg/1 μl; and (iv) IL-1Ra + vehicle for EX4. For the determination of IL-6 mediation, rats received the following four counterbalanced conditions: (i) vehicle for IL-6ab (acsf) + vehicle for EX4 (acsf); (ii) vehicle for IL-6ab + EX4 0.2 μg/1 μl; (iii) IL-6ab 0.05 μg/1 μl + EX4 0.2 μg/1 μl; and (iv) IL-6ab+ vehicle for EX4. Body temperature and spontaneous activity were measured telemetrically every 5 min and plotted for a period of 4 h. Food intake was measured at 1, 2, 4, and 22 h after injection. Body weight was measured just before injection and 22 h postinjection. The light cycle period was chosen for the temperature measurement because previous studies indicated that the hypothermic effect of EX4 can be best captured during this period for the rat (3, 4). IL-1Ra dose was previously shown to attenuate IL-1β induced fever in the preoptic area of the hypothalamus (5).The same dose and route of administration of IL-6ab used here were previously shown to reduce leptin s and insulin s effects on food intake (6). To determine whether cooperative action of IL-1 and IL-6 is required, the following conditions were used: (i) vehicle for IL-1Ra + vehicle for IL-6ab (acsf) + vehicle for EX4 (acsf); (ii) vehicle for IL-1Ra + vehicle for IL-6ab + EX4 0.2 μg/ 1 μl; (iii) IL-1Ra with IL-6ab + EX4 0.2 μg/1 μl; and (iv)il-1ra+ IL-6ab + vehicle for EX4. RNA Isolation and mrna Expression. Hypothalamic and hindbrain gene expression levels after lateral ventricle injection of the GLP-1 analog, EX4, or vehicle (acsf) in two separate groups of rats as previously described (2). One group was restricted to 10 g ( 50% of average overnight intake) of chow overnight, and the second was allowed to eat ad libitum. The cytokines measured were the following: IL-1β, IL-6, TNFα, and TGF-β1. The neuropeptides measured included Pomc, neuropeptide Y (Npy), choleocystokinin (Cck), melanocortin 4 receptor (Mc4r), and leptin receptor (Lepr). Ninety minutes after EX4 injection the brains were rapidly removed, and the hypothalamus, the hindbrain (cut just caudal to the ventral tegmental area), and the hippocampus were dissected using a brain matrix, frozen in liquid nitrogen and stored at 80 C for later determination of mrna expression. The extraction of the mrna, cdna synthesis, RT-PCR, and gene expression values were calculated as previously described (2). Individual brain samples were homogenized in Qiazol (Qiagen) using a TissueLyzer (Qiagen). Total RNA was extracted using RNeasy Lipid Tissue Mini Kit (Qiagen) with additional DNase treatment (Qiagen). RNA quality and quantity were assessed by spectrophotometric measurements (Nanodrop 1000, NanoDrop Technologies). For cdna synthesis, iscript cdna Synthesis kit (BioRad) was used. Real-time RT-PCR was performed using TaqMan probe, and primer sets for target genes were chosen from an on-line catalog (Applied Biosystems; reference numbers were as follows: Il1b- Rn _m1, Il6-Rn _m1, Tnf-Rn _g1, Tgfb1- Rn _m1). Gene expression values were calculated based on the ΔΔC t method (7), where the vehicle-injected group was designated as the calibrator. β-actin was used as the reference gene. Western Blot Analysis. Thirty minutes after EX4 injection the brains were rapidly removed, and the hypothalamus and a DVCenriched section of the hindbrain were dissected using a brain matrix, frozen in liquid nitrogen, and stored at 80 C. Wholetissue extracts for protein preparations and Western blot analyses were carried out as described (8). Protein concentrations were determined with the Bradford assay. Laemmli loading buffer (1 ) was added to the samples, which were boiled for 10 min and fractionated by gel electrophoresis on 4 12% (vol/vol) Bis Tris gels (Invitrogen). Nonspecific protein binding sites on the polyvinyldifluoride membranes (Amersham International) were blocked by incubating the membrane with 5% (wt/vol) nonfat milk in TBS Tween 20 buffer (10 mm Tris, 150 mm NaCl, and 0.1% Tween 20, ph 8.0) for 4 h. Blots generated with these extracts were probed with primary antibodies (Table S1) overnight at 4 C. The membranes were washed three times for 5 min each in TBS 0.05% Tween 20 and incubated with secondary antibodies (Table S1) for 2 h. Protein bands were visualized with SuperSignal West Dura Extended Duration Substrate (34076; Thermo Scientific, Pierce Biotechnology). To reprobe the blot with another antibody, the blot was rehydrated in methanol, rinsed in TBS, and incubated with stripping buffer (46430; Thermo Scientific) for 15 min. Immunoblotted signals were visualized with an LAS 1000 cooled charge-coupled device camera (Fuji Film). Individual bands were quantified directly from membranes by densitometry with Image Gauge software (Fuji Film). Proper loading was evaluated by staining the gels with Coomassie blue. All steps were carried out at room temperature unless otherwise stated. All targets chosen (Table S1, primary antibodies) were previously connected to IL-1β, IL-6, or both (9 11). Mouse Strains Used. All mice [IL-1 R1 KO, WT, and IL-6Ra fl/fl ] were purchased from The Jackson Laboratory. All mice used in the experiments were males. IL-1R KO Mice. The following IL-1R KO mice were used: strain name B6.129S7-Il1r1 tm1imx /J; stock no ; age at delivery 7 8 wk; and age at start of experiment 8 9 wk. 1of7

2 WT Mice. The following WT mice were used: strain name C57BL/6J; stock no: ; age at delivery 8 wk; and age at start of experiment 9 wk. IL-6Rα fl/fl Mice. The following IL-6Rα fl/fl mice were used: strain name B6;SJL-Il6ra tm1.1drew /J and stock no Brain IL-6R Knockdown. The IL-6Rα fl/fl mice were purchased from The Jackson Laboratory in October 2011 and were bred for five generations in the animal facility of the University of Gothenburg. At the time of the Tat-cre injection in the lateral ventricle, they were 5 mo (body weight: g). A Tat-Cre fusion protein, synthesized as described (12), was stereotaxically infused to the lateral ventricle (1.5 μl over 5 min, 2.1 mg/ml; anteroposterior, 0.3 mm bregma, 1.0 mm lateral, 2.5 mm dorsal ventral) according to a previously described protocol (13) to 5-mo-old male isoflurane anesthetized mice that were homozygously floxed for IL-6Rα (strain B6; SJL-Il6ra tm1.1drew ) (14) and purchased from The Jackson Laboratory. Control mice (also B6; SJL-Il6ra tm1.1drew ) were infused with vehicle (saline). Tomato expression was decreased in cells surrounding the ventricles following treatment with Tat-cre according to a similar protocol in dttomato loxp/+ mice (Fig. S4). In Vitro Treatment of Neuro2A Cells. Neuro2A neuroblastoma cells (American Type Culture Collection no. CCL-131) that express the GLP-1R were maintained at 37 C in 5% CO 2 and cultured in DMEM with 1 mg/ml glucose (Biochrom AG), 10% (vol/vol) FBS (Biochrom AG), and 1% nonessential amino acids (Biochrom AG). Cells were treated with EX4 (0 or 10 nm) for 15 or 45 min, respectively. At the end of the incubation RNA was isolated for quantitative real-time PCR to detect IL-6, whereas a β-actin expression assay (15) was used as endogenous control. SI Results Additional Statistical Analysis Details for Fig. 3 in Main Text. IL-6 antibody (ab) attenuates food intake and weight loss responses to intracerebroventricular injection of EX4. Anorexic response to central EX4 after 4 h of food access was not altered by IL-6 blockade (Fig. 3A). Two-way ANOVA revealed no interaction but one-way ANOVA [F(3,45) = 42.06, P < ] showed a significant effect of treatment. Post hoc Tukey test revealed a significant effect of EX4 alone (P < 0.005) and of EX4/IL-6ab (P < 0.005) combination to reduce 4-h chow intake. In contrast, the 22-h (overnight) food intake was abolished by the IL-6ab treatment (Fig. 3B). The IL-6ab administration led to a complete reversal of the EX4-induced anorexia (one-way ANOVA [F (3,45) = 15.85, P < ; P < for EX4 alone and P = not significant (ns) for EX4/IL-6ab combination]. Comparison of the EX4 alone and EX4/IL-6ab groups indicated a significant difference (P < 0.05). Two-way ANOVA indicated a strong trend for an interaction [F(1,60) = 3.00, P = 0.08]. Similarly, the EX4- induced weight loss was significantly attenuated by IL-6ab (Fig. 3C). One-way ANOVA was F(3,45) = 17.65, P < ; P < for EX4 alone; and P < 0.01 for EX4/IL-6ab combination. Further comparison of the EX4 alone and EX4/IL-6ab groups indicated a significant difference between these two treatments (P < 0.05). Two-way ANOVA indicated a significant interaction [F(1,60) = 3.68, P = 0.05]. Also, IL-1Ra attenuates food intake and weight loss responses to i.c.v. injection of EX4. Anorexic response to central EX4 after 4 h of food access was not altered by IL-1R blockade (Fig. 3D). Two-way ANOVA revealed no interaction, and post hoc Tukey test after a one-way ANOVA [F (3,33) = 12.82, P < ] showed a significant reduction in food intake after both EX4 (P < 0.005) and EX4/ IL-1Ra (P < 0.05) combination. In contrast, the 22-h (overnight) food intake was abolished by the IL-1Ra treatment (Fig. 3E), and blockade of IL-1R led to a complete reversal of the EX4-induced anorexia [one-way ANOVA: F(3,33) = 10.26, P < ; P < for EX4 alone and P = ns for EX4/IL-1Ra combination compared with vehicle-treated rats]. Comparison of the EX4 alone and EX4/IL-1βRa groups indicated a significant difference (P < 0.05). Two-way ANOVA revealed a significant interaction of EX4 with the antagonist [F(1,44) = 3.73, P = 0.05]. Similarly, the EX4-induced weight loss was significantly attenuated by IL-1Ra (Fig. 3F). Blockade of the IL-1R also led to a complete reversal of the EX4-induced weight loss [one-way ANOVA: F(3,33) = 19.20, P < ; P < for EX4 alone and P = ns for EX4/ IL-1Ra combination compared with vehicle-treated rats]. Further comparison of the body weight changes after the EX4 alone and EX4/IL-1Ra groups indicated a significant difference (P < 0.005). Two-way ANOVA indicated a significant interaction [F (1,44) = 7.15, P < 0.05]. Data are expressed as mean ± SEM. n = 12 16/treatment condition. *P < 0.05, **P < 0.01, ***P < Additional Statistical Analysis Details for Fig. 4 in Main Text. Simultaneous IL-1R and IL-6 blockade synergistically attenuates food intake and weight loss responses to i.c.v. injection of EX4. Anorexic response to central EX4 after 1 h (Fig. 4A), 4 h (Fig. 4B), and 22 h (Fig. 4C) were significantly attenuated by the combination of IL-1Ra and IL-6ab treatment. Similarly, the EX4-induced weight loss was significantly attenuated by this combination treatment (Fig. 4D). Two-way ANOVA revealed a significant interaction: F(1,40) = 5.37, P < 0.05 for 1-h intake, F(1,40) = 3.78, P < 0.05 for 4-h intake, and F(1,40) = 6.77, P < 0.05 for 22-h intake. Also one-way ANOVA [F(3,30) = 83.94, P < , F (3,30) = 82.05, P < and F(3,30) = 33.77, P < for 1-, 4-, and 22-h intake, respectively] showed a significant effect of treatment. Post hoc tests revealed a significant effect of EX4 alone (P < for all: 1, 4, and 22 h) and the EX4/(IL-1Ra+IL-6ab) combination (P < for both 1 and 4 h and P < 0.05 for 22 h) to reduce chow intake. Importantly, however, the comparison of the EX4 alone and the EX4/(IL-1Ra+IL-6ab) groups indicated a significant difference (P < 0.05 for 1-h intake, P < 0.01 for 4-h intake, and P < for 22-h intake). The combined interleukin blockade was successful at attenuating the EX4-induced weight loss [one-way ANOVA: F(3,30) = 36.18, P < ; P < for EX4 alone; and P < 0.05 for EX4/(IL-1βRant+IL-6ab) combination). The comparison of the EX4 alone and EX4/(IL-1βRa+ IL-6ab) groups indicated a significant difference (P < 0.005), and two-way ANOVA indicated a significant interaction [F(1,40) = 16.86, P < ]. Data are expressed as mean ± SEM. n = 11/ treatment condition. *P < 0.05, **P < 0.01, ***P < Skibicka KP, Hansson C, Alvarez-Crespo M, Friberg PA, Dickson SL (2011) Ghrelin directly targets the ventral tegmental area to increase food motivation. Neuroscience 180: Skibicka KP, Hansson C, Egecioglu E, Dickson SL (2012) Role of ghrelin in food reward: Impact of ghrelin on sucrose self-administration and mesolimbic dopamine and acetylcholine receptor gene expression. Addict Biol 17(1): Skibicka KP, Alhadeff AL, Grill HJ (2009) Hindbrain cocaine- and amphetamineregulated transcript induces hypothermia mediated by GLP-1 receptors. J Neurosci 29(21): Hayes MR, Skibicka KP, Grill HJ (2008) Caudal brainstem processing is sufficient for behavioral, sympathetic, and parasympathetic responses driven by peripheral and hindbrain glucagon-like-peptide-1 receptor stimulation. Endocrinology 149(8): Fabricio AS, et al. (2006) Interleukin-1 mediates endothelin-1-induced fever and prostaglandin production in the preoptic area of rats. Am J Physiol Regul Integr Comp Physiol 290(6):R1515 R Flores MB, et al. (2006) Exercise improves insulin and leptin sensitivity in hypothalamus of Wistar rats. Diabetes 55(9): Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25(4): Shao R, et al. (2012) Coordinate regulation of heterogeneous nuclear ribonucleoprotein dynamics by steroid hormones in the human fallopian tube and endometrium in vivo and in vitro. Am J Physiol Endocrinol Metab 302(10):E1269 E Wang J, Campbell IL (2002) Cytokine signaling in the brain: Putting a SOCS in it? J Neurosci Res 67(4): of7

3 10. Linossi EM, Babon JJ, Hilton DJ, Nicholson SE (2013) Suppression of cytokine signaling: The SOCS perspective. Cytokine Growth Factor Rev 24(3): Venieratos PD, Drossopoulou GI, Kapodistria KD, Tsilibary EC, Kitsiou PV (2010) High glucose induces suppression of insulin signalling and apoptosis via upregulation of endogenous IL-1beta and suppressor of cytokine signalling-1 in mouse pancreatic beta cells. Cell Signal 22(5): Peitz M, Pfannkuche K, Rajewsky K, Edenhofer F (2002) Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: A tool for efficient genetic engineering of mammalian genomes. Proc Natl Acad Sci USA 99(7): Langlet F, et al. (2013) Tanycytic VEGF-A boosts blood-hypothalamus barrier plasticity and access of metabolic signals to the arcuate nucleus in response to fasting. Cell Metab 17(4): McFarland-Mancini MM, et al. (2010) Differences in wound healing in mice with deficiency of IL-6 versus IL-6 receptor. J Immunol 184(12): Hesse D, et al. (2010) Altered GLUT4 trafficking in adipocytes in the absence of the GTPase Arfrp1. Biochem Biophys Res Commun 394(4): Fig. S1. EX4 did not change the hippocampal IL-1 expression. The histograms represent the mrna levels of cytokines in ad-libitum fed and food-restricted rats following i.c.v. EX4 treatment. Data are normalized to β-actin and expressed as relative quantity compared with vehicle treatment. n = 9 11/treatment condition. Data are expressed as mean ± SEM. 3of7

4 Fig. S2. Activity is expressed here on a line graph as a group average for each treatment (A, C, ande). In the IL-6 blockade experiment a small but significant elevation in activity was observed when the data were expressed as 2.5-h postinjection sums [one-way ANOVA F(3,45) = 4.15, P < 0.05; two-way ANOVA: F(1,60) = 7.06, P < 0.05]. This hyperactivity seemed to be blocked by the IL-6ab (B). None of the treatments changed the activity expressed here on a line graph as a group average for each treatment (C and E) or histogram of 2.5-h postinjection sum (D and F) in the IL-1 or combination blockade experiments. Data are expressed as mean ± SEM. n = 16 (A and B), n = 12 (C and D), and n = 11 (E and F)/treatment condition. *P < of7

5 Fig. S3. Core temperature after central EX4 alone and in combination with IL-6ab or IL-1βRa. Fast onset and long-lasting hypothermia was observed after i.c.v. injection of EX4 and IL-6 or IL-1β blockade did not change this response (A and C). Body temperature differed significantly across the four treatments [F(3, 45) = P < (Fig. 4B), F(3, 33) = 24.41, P < ]. Post hoc Tukey comparisons of the four groups indicated that both EX4 (P < 0.005) and EX4/IL-6ab (P < 0.005) produced a significant hypothermia (B). Similarly, Tukey post hoc comparisons of the four groups indicated that both the EX4 (P < 0.005) and EX4/IL-1βRa (P < 0.01) produced a significant hypothermia (D). Meanwhile, comparison of the EX4 alone and EX4/IL-6ab or EX4 alone and EX4/ IL-1βRa groups did not indicate any significant differences. Furthermore, a two-way ANOVA did not indicate any significant interaction. In contrast to single interleukin blockade, the hypothermia was attenuated by the concurrent IL1R/IL-6 blockade (E and F). Body temperature differed significantly across the four treatments [F(3, 30) = 19.46, P < ] (E and F). Post hoc comparisons of the four groups indicated that both EX4 (P < 0.005) and EX4/(IL-1βRa+IL-6ab) (P < 0.05) produced hypothermia. Comparison of the EX4 alone and EX4/(IL-1a+IL-6ab) groups indicated a significant difference between the two treatments (P < 0.05). Furthermore, a two-way ANOVA indicated a trend for a significant interaction [F(1,40) = 3.69, P = 0.06]. The arrows indicate the injection timing of the antagonist or respective vehicle (first arrow) and the EX4 or respective vehicle (second arrow). The histograms (B, D, and F) represent 2.5-h postinjection averages of core temperature. Data are expressed as mean ± SEM. n = 16 (A and B), n = 12 (C and D), and n = 11 (E and F)/treatment condition. *P < 0.05, **P < 0.01, ***P < of7

6 Fig. S4. Representative images showing tomato expression (red) in cells in dttomatoloxp/+ mice in which the Tat-cre recombinant protein has been infused to the right lateral ventricle to indicate the CNS distribution of cre-mediated knockdown. A general view of the third ventricle (3V) at the level of the praventricular nucleus of the hypothalamus (PVH) (A) and a zoom on the ependymal layer bordering the PVH (B). Hoechst (white) DNA staining and tomato (red) as a marker of tat-cre induced recombination are shown in A and B. RCH, retrochiasmatic area. 6of7

7 Table S1. Antibodies used for Western blot experiments Name Name MW (kda) Dilution (method) Source Primary antibodies Stat3 (catalog #8768) Rabbit monoclonal antibody (IgG) 86 1:1,000 (WB) Cell Signaling Technology Phospho-Stat3 (catalog #9145) Rabbit monoclonal antibody (IgG) 79/86 1:1,000 (WB) Cell Signaling Technology SOCS1 (sc-9021) Rabbit polyclonal antibody (IgG) 24 1:500 (WB) Santa Cruz Biotechnology SOCS2 (sc-9022) Rabbit polyclonal antibody (IgG) 33 1:500 (WB) Santa Cruz Biotechnology β-actin (AC-74) Mouse monoclonal antibody (IgG) 42 1:1,000 (WB) Sigma-Aldrich Secondary antibodies Anti-rabbit IgG HRP-linked antibody (catalog #7074) 1:10,000 (WB) Cell Signaling Technology Anti-mouse IgG HRP-linked antibody (catalog #7076) 1:10,000 (WB) Cell Signaling Technology MW, molecular weight; WB, Western blot analysis. 7of7