Mice for this experiment were bred at the Max Planck Institute for Metabolism Research, Cologne, Germany. At the age of 7 weeks they were shipped to Dr. Kamal Rahmouni, University of IOWA, where the study was conducted as previously described (). hM3Dqor hM3Dqmice were anesthetized by i.p. injection of a 9.1 mg/kg ketamine and 9.1 mg/kg xylazine mixture and a tapered micro-renathane tubing (MRE-40 for mice) was inserted into the jugular vein for i.v. injection. For monitoring of blood pressure, an MRE-40 arterial catheter was inserted into the left carotid artery. The trachea was then cannulated with PE-50 tube allowing the mice to spontaneously breath oxygen-enriched room air. The level of anesthesia was sustained throughout the experimental protocol by a slow infusion of α-chloralose through the jugular vein (12 mg/kg bolus followed by a 6 mg/kg constant infusion). Body temperature was monitored with a thermometer inserted into the rectum and was maintained at 37.0 –37.5°C using a heating pad. To record liver sympathetic nerve activity (Liv-SNA), the celiac and liver branch of the ventral splanchnic nerve were identified and exposed at the level of liver artery using a dissecting microscope. The nerve was attached to a pair of 36-gauge stainless steel wire electrodes and quickly fixed with a silicon gel (Kwik-Sil; WPI) to prevent dehydration and for electrical insulation. After surgery, each animal was allowed to stabilize for 20–40 min. Electrical activity in each nerve was amplified 50,000 –100,000 times with a band path of 100–1000 kHz and monitored by an oscilloscope. The amplified and filtered nerve activity was converted to standard pulses by a window discriminator, which separated discharge from electrical background noise that was determined postmortem. Both the discharge rates and the neurogram were sampled with a Power-Lab analog-to-digital converter for recording and data analysis on a computer. Background noise, which was determined 30–60 min after the animal was sacrificed, was subtracted. Nerve activity was rectified and integrated with baseline nerve activity normalized to 100%. Baseline measurements of liver SNA was obtained during 5–10 min before iv injection of vehicle or CNO (3 mg/kg) and recorded for the following hour.

Experiments were performed 4 weeks after viral injections to allow for expression of the transgene. Animals were single housed for 1.5 weeks prior to the experiments. Five days before the first experimental day, animals were attached to fiber cable and kept connected throughout the experimental period. Photometry was performed using a custom-built fiber photometer (Model 925, Michael Dübbert, Electronic Laboratory, Institute for Zoology, University of Cologne), which was built following the principal specification described in () and as previously described (). For ensuring compatability throughout all studies, mice were fasted from ZT15.30 to ZT5.45. At ZT5.45 the laser was turned on. The recordings consisted of a baseline period of 15 min followed by exposure to either food, caged food or caged fake food at ZT6 and the calcium signal was recorded for 30 min. Each experimental day was separated by 8-10 days. The mice were allowed to sense the food as for the other experiments. Photometry data were subjected to minimal processing using InstantClue. Since the signal was recorded every second and remains 0 in between, we calculated the maximum value within in a rolling window of 4500 data points. Smoothing of data was performed by accessing the moving average (4500 data points). To correct for differences in the baseline, we calculated the mean of the first 10 min and divided the data by this value. This procedure was performed individually for each recording. The area under curve was calculated using trapezium rule between the smoothed signal data and a horizontal line at y = 0.

Fiber optic cables were firmly attached to the implanted fiber optic cannulae. ChR2 POMC and control ChR2 WT mice were acclimated to this procedure in their experimental cages for one week prior to optogenetic stimulation. On the day of the photostimulation experiment, food was removed and animals were attached to fiber optic cannulae at ZT0. At ZT6, blue laser (473 nm) light stimulation consisting of pulse trains (5 ms pulses of 20 Hz; 3 s on, 1 s off) was delivered. Following 30 min of photostimulation, mice were deeply anesthetized and liver samples were rapidly removed. Then, animals were perfused transcardially, their brains were removed and the locations of fiber tips were identified post hoc.

For all stereotaxic surgeries, animals were anesthetized with isoflurane and placed into a stereotaxic apparatus. For pain relief and postoperative care, mice were injected with buprenorphine (0.1 mg/kg) and meloxicam (5 mg per kg). Post-surgery, animals received tramadol in the drinking water (1 mg/mL), were inspected twice daily and body weight was monitored to ensure regain of pre-surgery weight.

C57BL/6N male mice, 5 in each cage, from Charles River, France, were fasted for 14 hr at ZT13 to ZT13.25 and injected from ZT4.50 to Z5.15 with prazosin (0.5 mg/kg) at ZT 5.20 to ZT5.45 and after 30 min injected with either saline or norepinephrine (2 mg/kg) as previously described (). The liver was removed 30 min following injection of saline or norepinephrine at ZT5.50 to ZT6.15.

For this experiment, 8-week-old male C57BL/6N mice were obtained from Charles River, France, and acclimatized to the facility for 8 days prior to the experiment. As mice were always sacrificed at ZT6 ± 30 min, mice were fasted accordingly. If mice were to be refed for either 30 min or 2 hr and sacrificed at ZT6, the former group was fasted at ZT13.5 and the latter ZT12. The mice were then refed at ZT5.5 and ZT4, respectively. In order to minimize the time of sacrificing the first to the last mouse in the same cage, the number of mice in each cage varied among experiments. For the induction of Fos mRNA in POMC neurons, mice were housed in pairs, as only two mice were perfused at the same time ( Figures 6 A and 6B). For studies, in which the liver was used for RNA sequencing ( Figure 1 A) or lipidomics ( Figure 2 A), mice were housed in groups of 3. For protein analysis and phosphoproteomic experiments, mice were housed in groups of 5 for the 30 min, 1 hr, 2 hr, and 4 hr time point and groups of 2 for the 5 min and 10 min exposure ( Figures 3 and 4 ). The estimated time from killing the first mouse to the next, including removing the liver and snap freezing in liquid nitrogen, was 45 s for all studies except for the lipidomics, which was closer to 1 min due to washing of the liver in ice cold PBS to remove blood. Overall, the approximate time to collect 5 livers was 4 min. We did not observe any correlation between liver Xbp1-splicing from the first to the fifth mouse indicating that the 4 min between the first and the fifth mouse in a group did not affect the results.

The circadian clock impacts on almost all metabolic pathways in the murine liver and ER-stress networks have a periodicity of 12 hr with lowest activity mid through the light cycle (Zeigtgeber, ZT6) (). ER volume (), norepinephrine levels (), ribosomal biogenesis (), all oscillate in a circadian manner. Therefore, to control for this effect, livers used for any analysis in this paper were removed halfway through the light cycle at ZT6 ± 30 min.

Cell experiments

Hepa1-6 cell Experimental procedure: 24 hours before the experiment, the cells were trypsinized, counted by using an automated cell counter (EVE automatic Cell counter NanoEnTek) and plated at a density of 2∗105 cells/well in 12-well plates (Becton Dickinson labware, France). Six hours before the stimulation, the medium DMEM (1x) + GlutaMAX™ -I Dulbeccos Modified Eagle Medium 4.5g/L D-glucose was replaced with serum free medium. Cells were stimulated with norepinephrine (10 μM dissolved in PBS (GIBCO)). The experiment was stopped by rapid removal of the cell medium and by directly submerging the plate in liquid nitrogen and kept at −80°C until processed for either RNA or protein preparation. For technical replicates the mean of three to six wells were used. At least three independent experiments were performed for each study.

Preparations of solutions for in vitro experiments Liu et al., 2016 Liu D.

Bordicchia M.

Zhang C.

Fang H.

Wei W.

Li J.L.

Guilherme A.

Guntur K.

Czech M.P.

Collins S. Activation of mTORC1 is essential for β-adrenergic stimulation of adipose browning. Jefferies et al., 1997 Jefferies H.B.

Fumagalli S.

Dennis P.B.

Reinhard C.

Pearson R.B.

Thomas G. Rapamycin suppresses 5'TOP mRNA translation through inhibition of p70s6k. Norepinephrine was dissolved in PBS to a final concentration of 1 mM and 10 μl of this was added to each well containing 1 mL of medium. Rapamycin was dissolved in pure DMSO at a concentration of 10 μM. This was further diluted in cell medium to a final concentration of 100 nM for inhibition of protein phosphorylation () and 20 nM for blocking transcription as described before (

Tissue, serum, and plasma processing Blood measurement. Blood glucose was measured by a hand-held glucometer ContourXT, with Contour Next strips (REF #84167879) (Bayer, Germany). Serum and plasma analysis. For the fasted, 30, 60 and 120 minutes study, leptin and insulin concentrations were determined in duplicates using 5 μl of serum according to the manufacturer’s ELISA instructions. To generate serum, whole blood was allowed to clot at RT for 30 minutes followed by a 30 minutes centrifugation at 3000 g and stored at −80°C until used. For the determination of insulin and norepinephrine concentrations in mice which had been fasted or exposed to caged food or refed for 5, 10 and 30 minutes, plasma was used. Plasma was prepared in the following way: 4 μl of 1 M sodium metabisulfite was added to EDTA-coated tubes, mixed with 400 - 500 μl blood and put on ice for 30 min before centrifugation for 30 minutes at 3000 g.

Processing of murine livers for the different analysis For all studies, the gall bladder was quickly removed before freezing the livers in liquid nitrogen. For lipidomics, the livers were briefly washed in ice cold PBS to remove blood. During the perfusion of the mice with 0.9% saline, a small piece of the liver was removed and quickly frozen in liquid nitrogen.

RNA isolation from murine liver The same part of the outer part of the left lobe was always taken for analysis. The piece of liver (approx. 10 mg) was snap-frozen in liquid nitrogen in 1.5 mL tube containing beads (1.4mm Zirconium oxide beads, Cat-No. KT03961-1-103.BK, Bertin Technoloiges, France). One mL ice-cold Qiazol (QIAGEN) was added to a small piece of liver (approx. 10 mg) on ice, a maximum of 12 samples was processed at one time. The samples were homogenized at RT using the fast prep machine (MP Biomedicals, Ohio, USA) for 40 s speed 6 m/s, allowed to stand at RT for 5 min, then 300 μL of chloroform was added. The tubes were vigorously shaken, spun for 15 min at 13000 rpm at RT. 300 μl of supernatant was mixed with 400 μL of 70% EtOH. From this step on the RNeasy Mini kit (QIAGEN) was used according to the manufacturers instruction. No DNase treatment was used for regular cDNA synthesis, qPCR and Xbp1-splicing assay, but only used for RNA sequencing. RNA was eluted with 50 μL RNase-free water and kept on ice. The concentration was measured by Nanodrop ND-100 (Peqlab Biotechnology, France), and diluted to a final concentration of 1200-1500 ng/ μl. This RNA was used for RNA sequencing and cDNA synthesis. Expression of spliced Xbp1 using cDNA synthesized from this part correlated well with the expression using cDNA generated from RNA from a piece of liver from the inner part of the right lobe (data not shown).

RNA isolation from Hepa1-6 cells For RNA isolations 12 well plates were allowed to thaw from −80°C. Then 300 μL of the RTL buffer (RNeasy Mini Kit (QIAGEN)) with 1 μL per 1 mL β-mercaptoethanol was added to each well. The solution was transferred to 300 μL of 70% EtOH. From this step on, the procedure was done according to the manual provided by the manufacturer of the RNeasy Mini kit (QIAGEN).

cDNA synthesis From total liver mRNA, 2 μg were reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosytems, Cat # 4368814). 2 μL buffer, 4.2 μL of MilliQ-purified H 2 O, 2 μL random hexamer primers, 1 μL RT enzym and 0.8 μL dNTP were added to 2 μg of total liver RNA in 10 μL H 2 O. Reverse transcription was performed at 25°C for 10 min, at 37°C for 60 min, and at 85°C for 5 min. 2 μg cDNA/RNA were diluted to a total of 200 μL with MilliQ-purified H 2 O. RNA from Hepa1-6 was reverse-transcribed as described above, except that cDNA was synthesized from only 1 μg of total Hepa1-6 RNA and diluted to 100 μl.

qPCR Yang et al., 2015 Yang L.

Calay E.S.

Fan J.

Arduini A.

Kunz R.C.

Gygi S.P.

Yalcin A.

Fu S.

Hotamisligil G.S. METABOLISM. S-Nitrosylation links obesity-associated inflammation to endoplasmic reticulum dysfunction. Shao et al., 2014 Shao M.

Shan B.

Liu Y.

Deng Y.

Yan C.

Wu Y.

Mao T.

Qiu Y.

Zhou Y.

Jiang S.

et al. Hepatic IRE1α regulates fasting-induced metabolic adaptive programs through the XBP1s-PPARα axis signalling. qPCR was performed using the QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems) for amplification. Quantification was done using the delta Ct method and normalized to the control group in the given experiment. Hypoxanthine guanine phosphoribosyl transferase (Hprt) was used to normalize within each sample. See Table S4 for list of primers, TaqMan probes and SYBRGreen primer pairs. The SYBRGreen primers were synthesized by Eurogentec (Germany) and TaqMan probes were obtained from Applied Biosystems. The primers for Dnajc3 (p58ipk) and Herpud1 have previously been described () and the Xbp1(s) and Xbp1(u) primers, which share a common antisense primer, have been described before ().

Xbp1-splicing assay 2 μl of cDNA (concentration 10 μg/μL), same as for qPCR, were used for amplification of unspliced and spliced Xbp1 in a total volume of 25 μL containing 1 × PCR buffer, MgCl (2.5 mM) mXbp11.3S (0.4 μM), mXbp1.12AS (0.4 μM) dNTPs (2.5 mM) Taq (5 U/mL) and MilliQ-purified H 2 O. Mxbp 1.3S (5′ A AAC AGA GTA GCA GCG CAG ACT GC 3′) and mxbp 1.12AS (5′ TC CTT CTG GGT AGA CCT CTG GGA A 3′). PCR protocol: 94°C 4 min, (94°C 10 s, 68°C 30 s 72°C 30 s) for 35 cycles 72°C 10 min. Expected products: 473 bp unspliced and 450 bp spliced. PCR products were separated on a 2% agarose gel with ethidium bromide for 2.5 hr at 150 V.

Protein isolation from murine liver Frozen livers were broken into smaller pieces in liquid nitrogen and 60 - 80 mg were transferred to 1.5 mL-tubes containing beads as for total RNA isolation. Livers were homogenized for 40 s at RT in 1 mL of the following buffer: Tris-HCL 25 mM, Na 3 VO 4 100 mM, NaF 100mM, Na 4 P 2 O 4 50 mM, EGTA 10 mM, EDTA 10 mM, NP-40 1%, PMSF 1mM pH 7.4 and cOmplete mini (1 tablet/10 mL) were freshly added. The samples were left on ice for 5 min, visually inspected for complete homogenization. If samples were not completely homogenized then the procedure was repeated. Samples were then centrifuged for 30 min at 17.000 g, the supernatant was taken for determination of protein concentration using the BCA assay (Pierce) relative to a BSA standard. Protein was mixed with Laemmli buffer (Bio-Rad) (1:4) containing β-Mercaptoethanol (1:10) and H 2 O was added to a final concentration of 2 μg protein per μL.

Protein isolation from Hepa1-6 cells Cells were lysed by first thawing the cells on ice and then adding 150 μL cell lysis buffer (Tris-HCl 25 mM, NaCl 25 mM, Na 3 VO 4 10 mM, NaF 10 mM Na 4 P 2 O 7 10 mM and EGTA 1 mM with freshly added NP-40 1% and cOmplete Mini 1 tablet /10 mL pH 7.4) to each well. After pipetting up and down, cell lysates were transferred to a cold 1.5 mL-tube and centrifuged for 15 min at 17.000 g. The supernatant was mixed with Laemmli buffer (Bio-Rad) (1:4) and β-Mercaptoethanol (1:10). 10 μL were separated by SDS-PAGE using the same system and antibodies as for the liver (see below).

Western blotting Proteins were separated using precasted gels from BioRad 4%–15%, 10% or 7,5% with either 26 or 18 wells. The voltage was set to 200 V and kept constant. Transfer of protein to PVDF membranes was done using the Trans-Blot Turbo system (Bio-Rad) with either the 7 or 10 min protocol. Tris/glycine running buffer (1x TGS buffer, Bio-Rad) was used as running buffer. After the transfer, the membranes were blocked in (1:20) western blotting reagent (Roche 11829200) for 1–2 hr and incubated over night in primary antibody. The next day, membranes were washed three times for 5–7 min in TBS-T pH 7.4 and incubated for 1–2 hr in secondary antibody, and then washed 4 times for 10 min in TBS-T pH 7.4. Proteins were visualized using the Fusion Solo Vilber Lourmat system. Band intensities were quantified using ImageJ (National Institutes of Health, Bethesda, United States). As a loading control, all membranes were probed with either beta actin or calnexin. The intensity of phospho protein bands was devided by the intensity of beta actin or calnexin. No stripping was done. For individual primary and secondary antibodies, lot numbers, company, and dilutions see Table S5. For the amount of protein loaded and blotting conditions, please see Table S5 for details. All primary and secondary antibodies were diluted in TBS-T pH 7.4, 5% 1:20 western blotting reagent. Membranes: Trans-Blot (R)Turbo Bio-Rad Midi Format, 0.2 μm PVDF, single application. Gels 7.5%, 26 wells, 15 μL, 1.0 mm Criterion TGX Precast gel. Gels 10%, 26 wells, 15 μL, 1.0 mm Criterion TGX Precast gel. Gels 4 - 15%, 26 wells, 15 μL, 1.0 mm Criterion TGX Precast gel.

Ultrathin sections and electron microscopy Tissues were post-fixed in 2% glutaraldehyde (Electron Microscopy Sciences) in 0.12 M phosphate buffer and treated with 1% osmium tetroxide (Electron Microscopy Sciences). After dehydration using ethanol and propylene oxide, tissues were embedded in Epon (Sigma). For electron microscopy, 70 nm ultrathin sections were cut from Epon-blocks and stained with uranyl acetate (Plano GMBH) and lead nitrate (Sigma). Images were acquired using a transmission electron microscope (JEM-2100 Plus) equipped with Gatan ONE View camera. ER surface area was measured on individual electron micrographs from three mice per treatment condition with 8 images from each mouse and presented as a mean per picture. All quantifications were done in a blinded fashion using ImageJ (National Institutes of Health, Bethesda, United States).

Perfusion for RNA scope and immunohistochemistry, confocal imaging, and quantification Tissue processing. Animals were perfused transcardially with 0.9% saline followed by ice cold 4% paraformaldehyde (PFA; pH 7.4). The brains were removed and post-fixed in 4% PFA at RT for 18 hr, followed by incubation with 25% sucrose in 0.1 M phosphate buffered saline (PBS, pH 7.4) at 4°C for 24 hr. The brains were cut at 14 μm for RNAscope or 30 μm for immunohistochemistry on a freezing microtome and collected in bins containing sterile anti-freeze solution (30% ethylene glycol and 20% glycerol in PBS) and subsequently stored at −20°C until further processing.

RNAscope ®. All reagents were purchased from Advanced Cell Diagnostics (ACD, Hayward, CA) if not stated otherwise. The Fos probe (Cat No. 316921-C3) contained 20 oligo pairs and targeted region 407-1427 (Acc. No. ® Multiplex Fluorescent Assay (Cat No. 320851; for Pomc+Fos), which in the end labeled the POMC probe with Atto 550 and the Fos probe with Atto 647, or, for the simultaneous detection of Pomc+AgRP+Fos, the tyramide-based RNAscope® Multiplex Fluorescent v2 Assay (Cat. No. 323110) that rendered probes labeled with OpaI520 (1:750), Cy3 (1:3000) and Cy5 (1:3000) respectively. Sections were counterstained with DAPI and coverslipped with ProLong Gold Antifade Mountant (Cat. No. P36931; ThermoFisher) and stored in the dark at 4°C until imaging. Fluorescent in situ hybridization for the simultaneous detection of Fos, Pomc and Agrp mRNA was performed using RNAscope. All reagents were purchased from Advanced Cell Diagnostics (ACD, Hayward, CA) if not stated otherwise. The Fos probe (Cat No. 316921-C3) contained 20 oligo pairs and targeted region 407-1427 (Acc. No. NM_010234.2 ) of the Fos transcript, and the POMC probe (Cat No. 314081-C2) contained 10 oligo pairs and targeted region 19-995, (Acc. No: NM_008895.3 ) of the Pomc transcript. The AgRP probe (Cat No. 400711-C2) constituted 16 oligo pairs and targeted region 11-764 of the AgRP transcript (Acc. No. NM_001271806.1 ). Three-plex negative and three-plex positive control probes recognizing bacterial dihydrodipicolinate reductase, DapB (Cat No. 320871) and PolR2A, cyclophilin and Ubiquitin (Cat No. 320881), were processed in parallel with the target probes to ensure tissue RNA integrity and optimal assay performance. All incubation steps were performed at 40°C using the ACD HybEz hybridization system (Cat No. 321462). On the day before the assay, every 12th section throughout the ARH was mounted on SuperFrost Plus Gold slides (Cat No. FT4981GLPLUS; ThermoFisher), dried at RT, briefly rinsed in autoclaved MilliQ-purified water, air-dried and baked at 60°C overnight. From each animal, one section from the same region of the brain was also mounted for use with the negative control probe to enable subsequent calculation of background. On the day of the assay, slides were first incubated for 7 min in ACD, submerged in Target Retrieval (Cat No. 322000) at a temperature of 98.5 - 99.5°C for 8 min, followed by two brief rinses in autoclaved MilliQ-purified water. The slides were quickly dehydrated in 100% ethanol and allowed to air dry for 5 min. A hydrophobic barrier was then created around the sections using an ImmEdge hydrophobic barrier pen (Cat No. 310018). The sections were incubated with Protease III (Cat No. 322340) for 40 min for the detection of Pomc and Fos, or Protease Plus (Cat. No. 322330) for 25 min for the detection of Pomc, AgRP and Fos. The subsequent steps, i.e., hybridization of the probes and the amplification and detection steps, were performed according to the manufacturer’s protocol for RNAscopeMultiplex Fluorescent Assay (Cat No. 320851; for Pomc+Fos), which in the end labeled the POMC probe with Atto 550 and the Fos probe with Atto 647, or, for the simultaneous detection of Pomc+AgRP+Fos, the tyramide-based RNAscopeMultiplex Fluorescent v2 Assay (Cat. No. 323110) that rendered probes labeled with OpaI520 (1:750), Cy3 (1:3000) and Cy5 (1:3000) respectively. Sections were counterstained with DAPI and coverslipped with ProLong Gold Antifade Mountant (Cat. No. P36931; ThermoFisher) and stored in the dark at 4°C until imaging.

Immunofluorescence Following the RNAscope procedure for AgRP and POMC (see above, with the modification that incubation with Protease III was only 10 minutes to avoid degradation of the GFP epitope), slides were briefly washed in PBS and then blocked in 3% goat serum/1 x PBS (Triton-X) for 1 hr at RT. After a brief wash in 1 x PBS the slides were incubated over night in chicken anti-GFP antibody (Abcam no. 13970, 1:500). The following morning, sections were washed intensively for 30 min in 1 x PBS and incubated with a goat anti-chicken antibody (1:500) for 1 hr at RT. After washing for 30 min, sections were counterstained with DAPI and coverslipped with ProLong Gold Antifade Mountant (Cat No. P36931; ThermoFisher) and stored in the dark at 4°C until imaging. Staining of POMC and GFP: Brain sections were washed in PBS with Tween-20, (pH 7.4 (PBST)) and blocked in 3% normal donkey serum in PBST for 1 hr at RT. Brain sections were then incubated overnight at RT in blocking solution containing primary antiserum (rabbit anti-POMC precursor, Phoenix Pharmaceuticals H-029-30, 1:1.000; chicken anti-GFP, Life Technologies A10262, 1:1.000). The next morning, sections were extensively washed in PBS for 30 min followed by incubation in Alexa-fluorophore secondary antibody (Molecular Probes, R37119, A-11039, both 1:1,000) for 1 hr at RT. After several washes in PBS, sections were mounted on slides after being counterstained with DAPI.

Imaging and quantification Images were captured using a confocal Leica TCS SP-8-X microscope, equipped with a 40x/1.30 oil objective. Tile scans and Z stacks (optical section of 1.0 μm) of the ARC were captured unilaterally from rostral to caudal, rendering approx. 5 sections per animal. Laser intensities for the two probe channels were kept constant throughout the entire material. Images were imported into Fijii (NIH) where maximum intensity projections were made. The DAPI channel was enhanced regarding brightness and contrast, but the probe channels were left unmodified. The images were then imported and fused into the Halo software (Indica Labs) for quantification of double-labeled neurons. The software relies on the DAPI stain for cellular identification and automatically calculates the cell intensity for each cell and probe (an integrated number containing both the fluorescent intensity and the area covered by the probe within the designated cell). The threshold for probe recognition was calculated as the mean cell intensity present in the negative control sections + 3xSD. All labeling above this value was considered to be true signal.

Transcriptomics Dobin et al., 2013 Dobin A.

Davis C.A.

Schlesinger F.

Drenkow J.

Zaleski C.

Jha S.

Batut P.

Chaisson M.

Gingeras T.R. STAR: ultrafast universal RNA-seq aligner. Love et al., 2014 Love M.I.

Huber W.

Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. 2 ± 1), a maximum Benjamini-Hochberg corrected p value of 0.05, and a minimum combined mean of 5 reads were deemed to be significantly differentially expressed After total RNA isolation (see above), 2 μg of total RNA was sent to Cologne Center for Genomics. The quality of the RNA integrity was assessed by Agilent 2200 TapeStation System and all samples had an RNA integrity number above 7. PolyA mRNA libraries were prepared using TruSeq® RNA sample preparation Kit v2 (Illumina). Libraries were sequenced for 50 million reads 50 bp single-end on a Illumina HiSeq 2000 sequencer with a paired-end (101x7x101 cycles) protocol. After sequencing, raw reads were assessed for quality, adaptor content and duplication rates with FastQC ( http://www.bioinformatics.babraham.ac.uk/projects/fastqc ). Reaper version 13 - 100 was employed to trim reads after a quality drop below a mean of Q20 in a window of 10 nucleotides. Only reads between 30 and 150 nucleotides were cleared for further analysis. Trimmed and filtered reads were aligned versus the Ensemble mouse genome version mm10 (GRCm38) using STAR 2.4.0a with the parameter –outFilterMismatchNoverLmax 0.1 to increase the maximum ratio of mismatches to mapped length to 10% (). The number of reads aligning to genes was counted with featureCounts 1.4.5-p1 tool from the Subread package. Only reads mapping at least partially inside exons were admitted and aggregated per gene. Reads overlapping multiple genes or aligning to multiple regions were excluded. Differentially expressed genes were identified using DESeq2 version 1.62 (). The Ensemble annotation was enriched with UniProt data (release 06.06.2014) based on Ensemble gene identifiers (Activities at the Universal Protein Resource (UniProt)). Only genes with a minimum fold change of ± 2 (Log± 1), a maximum Benjamini-Hochberg corrected p value of 0.05, and a minimum combined mean of 5 reads were deemed to be significantly differentially expressed

Phosphoproteomics Nolte et al., 2014 Nolte H.

Konzer A.

Ruhs A.

Jungblut B.

Braun T.

Krüger M. Global protein expression profiling of zebrafish organs based on in vivo incorporation of stable isotopes. Protein lysis and digestion. Five biological replicates of each experiment (refed, caged food and fasted) were performed. Livers were crushed in liquid nitrogen using a mortar. Six Lys6 labeled SILAC livers were used to generate an internal standard that was the same for all experiments. Liver powder was then dissolved in RIPA buffer (150 mM NaCl, 1% NP-40, 0.2% Na-Desoxycholat, 0.1% SDS in 10 mM Tris HCl pH 7.5) containing protease and phosphatase inhibitors (Biotol) using 1 mL buffer per 10 mg powder. Samples were sonicated on ice and centrifuged at 14.000 g at 4°C. Supernatant was then used for acetone precipitation overnight at −20°C, the pellet was washed with 90% acetone, dried and dissolved in 6 M Urea, 2 M Urea in 10 mM HEPES. Then, protein concentration was determined and 5 mg of protein were mixed with the heavy SILAC standard at a one to one ratio. Proteins were reduced by 10 mM dithiothreitol and alkylated using 55 mM Iodacetamide as described before (). Lys-C endopeptidase was added in an enzyme to protein ratio of 1 to 100 and incubated for 2 hr at RT. Urea was diluted to 2 M by adding 50 mM Ammonium bicarbonate and additional Lys-C was used in the same ratio. Digestion was stopped by adding 5% Acetonitrile and 0.2% TFA in a one to one ratio. th fraction was pooled and adjusted to binding conditions of TiO 2 beads 80% acetonitrile and 7% TFA in a 5 mL tube (final volume 2-3 mL). Prior to phosphopeptide enrichment TiO 2 beads were washed in 40% ACN and 10% ammonium hydroxide, followed by 2 washing steps of 70% acetonitrile and 80% acetonitrile/ 5% TFA. Then TiO 2 beads (5 μm Titansphere, GL Sciences) were dissolved in binding buffer (80% ACN, 7% TFA by 10 μL/mg of beads) as described previously ( Krishnan et al., 2015 Krishnan R.K.

Nolte H.

Sun T.

Kaur H.

Sreenivasan K.

Looso M.

Offermanns S.

Krüger M.

Swiercz J.M. Quantitative analysis of the TNF-α-induced phosphoproteome reveals AEG-1/MTDH/LYRIC as an IKKβ substrate. 2 beads were added to each fraction and incubated on a rotating wheel for 30 min at RT. Beads were collected by centrifugation (1000 g, 30 s) and supernatant was transferred to a fresh 5 mL tube containing further 2.5 mg of TiO 2 beads. Beads were washed with 2 × 2 mL 80% ACN and 7% TFA and loaded onto C8-StageTips. Beads were washed further three times on-tip using 30% ACN, 1% TFA, followed by 60% ACN, 1% TFA and 80% ACN, 1% TFA. Beads were dried with a syringe and phosphopeptides were eluted with 5% ammonium hydroxide and twice with 10% ammonium hydroxide, 40% ACN. Eluate was collected fraction-wise in a 96 well plate and concentrated in a speed-vac for 2.5 hr for complete dryness and resuspended in 10 μL 2.5% ACN and 5% formic acid. High pH fractionation and phosphopeptide enrichment. Peptides were desalted by C18 Cartridges (Waters GmbH) including several washing steps of bound peptides by 0.1% TFA and three-step elution by 40% and twice 60% Acetonitrile (ACN) containing 0.1% TFA (600 μL each). Samples were dried in a speed-vac and dissolved in 10 mM Ammonium hydroxide to a final volume of 1.6 mL. High pH fractionation was done on an Ultimate 3000 HPLC (Thermo Scientific). Complete samples were loaded via 4-step injection onto a Waters X-bridge column (Waters XBridge BEH130 C18 3.5 μm 4.6 × 250 mm column). The column temperature was set to 35°C. For peptide separation a binary buffer system was used: Buffer A) 10 mM ammonium hydroxide and B) 90% ACN, 10 mM ammonium hydroxide. Due to the relative high sample volume and smaller sample-loop volume (400 μL) each sample was injected four times within a high pH run. Peptides were loaded onto the column using 5% B at 1 mL/min between each injection step for 1 min and after the fourth injection this was held for further 2 min. In a linear shape the buffer B content was increased from 10% to 25%, ramped to 40% in 5 min, further increased to 95% B within 5 min and held for 3 min. Followed by a re-equilibration step of 5 min after ramping the gradient back to loading conditions (5% B). Fractions were collected for a total volume of 1000 μL and prior collection 200 μL of 80% ACN and 5% TFA was added to each vial to immediately shift the pH to acidic conditions. Fractions were concentrated in a speed-vac for at least 12 h to almost complete dryness and pooled as follows. Every 13fraction was pooled and adjusted to binding conditions of TiObeads 80% acetonitrile and 7% TFA in a 5 mL tube (final volume 2-3 mL). Prior to phosphopeptide enrichment TiObeads were washed in 40% ACN and 10% ammonium hydroxide, followed by 2 washing steps of 70% acetonitrile and 80% acetonitrile/ 5% TFA. Then TiObeads (5 μm Titansphere, GL Sciences) were dissolved in binding buffer (80% ACN, 7% TFA by 10 μL/mg of beads) as described previously (). Then, 2.5 mg TiObeads were added to each fraction and incubated on a rotating wheel for 30 min at RT. Beads were collected by centrifugation (1000 g, 30 s) and supernatant was transferred to a fresh 5 mL tube containing further 2.5 mg of TiObeads. Beads were washed with 2 × 2 mL 80% ACN and 7% TFA and loaded onto C8-StageTips. Beads were washed further three times on-tip using 30% ACN, 1% TFA, followed by 60% ACN, 1% TFA and 80% ACN, 1% TFA. Beads were dried with a syringe and phosphopeptides were eluted with 5% ammonium hydroxide and twice with 10% ammonium hydroxide, 40% ACN. Eluate was collected fraction-wise in a 96 well plate and concentrated in a speed-vac for 2.5 hr for complete dryness and resuspended in 10 μL 2.5% ACN and 5% formic acid. Kelstrup et al., 2012 Kelstrup C.D.

Young C.

Lavallee R.

Nielsen M.L.

Olsen J.V. Optimized fast and sensitive acquisition methods for shotgun proteomics on a quadrupole orbitrap mass spectrometer. Liquid chromatography and mass spectrometry. LC-MS/MS instrumentation consisted out of an Easy nLC 1000 (Thermo Fisher) coupled via a nono-spray ionization source to a QExactive Plus mass spectrometer. Peptides were separated on a 50 cm column (I.D. = 75 μm, packed with 1.7 μm C18 Beads, Dr. Maisch) using a two-buffer system: A) 0.1% formic acid and B) 0.1% formic acid in acetonitrile. Content of buffer B was increased over 90-120 min gradient time from 5% to 23% within 70% of the total time, followed by an increase to 55% and 85% and a re-equilibration step to 5% before the next sample was loaded onto the column. MS1 level spectra were acquired at a resolution of 70,000 at 200 m/z, using 3e6 as an AGC target within a maximum injection time of 20 ms. The instrument operated in a top 10 mode and sequentially isolated the top 10 most intense peaks using an isolation window of 1.9 m/z for HCD fragmentation at normalized collision energy of 27. The resolution was set to 35,000 at 200 m/z. We used an AGC target of 5e5 and a maximum injection time of 110–120 ms maximizing parallelization and high quality MS2 spectra for phosphosite localization and identification ().

LC-MS/MS data analysis Cox and Mann, 2008 Cox J.

Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Cox et al., 2011 Cox J.

Neuhauser N.

Michalski A.

Scheltema R.A.

Olsen J.V.

Mann M. Andromeda: a peptide search engine integrated into the MaxQuant environment. 2 SILAC ratios were normalized by median substraction. In order to identify significantly different phosphorylation sites a t test was applied. The set of similar regulated phosphorylation sites were identified by the fold change compared to fasted (1.5 fold in the same direction e.g., up or downregulated compared to fasted). Protein annotations such as Gene Ontology terms, Pfam and KEGG pathways are based on Uniport while substrate motif information were annotated via Perseus ( Tyanova et al., 2016 Tyanova S.

Temu T.

Sinitcyn P.

Carlson A.

Hein M.Y.

Geiger T.

Mann M.

Cox J. The Perseus computational platform for comprehensive analysis of (prote)omics data. Cox and Mann, 2012 Cox J.

Mann M. 1D and 2D annotation enrichment: a statistical method integrating quantitative proteomics with complementary high-throughput data. Vizcaíno et al., 2016 Vizcaíno J.A.

Csordas A.

del-Toro N.

Dianes J.A.

Griss J.

Lavidas I.

Mayer G.

Perez-Riverol Y.

Reisinger F.

Ternent T.

et al. 2016 update of the PRIDE database and its related tools. All raw data were processed using MaxQuant 1.5.3.8 and the implemented Andromeda search engine (). Acquired MS/MS spectra were used for identification of modified and unmodified peptides using the Uniprot reference proteome of Mus musculus (2016). A Lys-C/P was set as the used protease and a maximum of 2 missed cleavages was tolerated. Heavy SILAC channel was defined by Lys6 as the label. Mass tolerances were kept as by default. The FDR was estimated by the implemented decoy algorithm and calculated to 0.01 at the protein, site and peptide-spectrum-match level by the revert method. Carbamidomethylation at cysteine residues was set as a fixed modification while phosphorylation at Serine, Threonine and Tyrosine as well as Methionineoxidation and protein N-term acetylation were defined as variable modifications. A minimal ratio count of 1 was required for quantification and peptide length of 7 amino acids was defined. The re-quantify and match-between runs algorithms were enabled using default settings. LogSILAC ratios were normalized by median substraction. In order to identify significantly different phosphorylation sites a t test was applied. The set of similar regulated phosphorylation sites were identified by the fold change compared to fasted (1.5 fold in the same direction e.g., up or downregulated compared to fasted). Protein annotations such as Gene Ontology terms, Pfam and KEGG pathways are based on Uniport while substrate motif information were annotated via Perseus (). Perseus was also used to analyze data by Principal Component Analysis (for all omics-levels). To identify enriched gene ontologies we performed a fisher exact test and used a specific target group (for example the group of phosphorylation sites that were regulated similarly between refed and caged food compared to fasted against all identified phosphorylation sites. We aimed to compare phosphorylation and mRNA adaptions in response to refed and caged food. To achieve this, we conducted a 2D enrichment (). We mapped mRNA expression data based on Uniprot IDs to the phosphorylation data and performed a 2D enrichment for refed compared to fasted and caged food compared to fasted on these two omics levels separately. Finally, we overlapped the two lists of systematically up/downregulated annotations. Phosphoproteomics data are available via ProteomeXchange with identifier PXD005681 ().

Lipids The whole liver was quickly removed without the gallbladder and washed in ice cold PBS on ice and then snap frozen in liquid nitrogen and stored at – 80°C until processed further. Livers were then transferred to a ceramic mortar containing liquid nitrogen also submerged in liquid nitrogen. Using the mortar, the livers were crushed to fine powder and 50-80 mg was weighed off and transferred to a plastic tube containing ceramic beads. Extraction was done by adding 1.5 mL methanol/H 2 0 (1:1) containing 2 mM deuterium labeled acetate, 40 μM norvaline and 2 μL of a deuterium-labeled lipid standard (Aventis). The samples were homogenized using the fast prep machine (MP Biomedicals, Ohio, USA) 4 × 40 s speed 6 m/s. After inspection of the sample for total tissue lysis, the samples were centrifuged 10 min 16.000 g at 4°C and the supernatant was transferred to a new 1.5 mL-tube, and dried using a SpeedVac (aqueous extract) used for GC-MS. 1.5 mL of ice cold dichlororomethane/methanol mixture (3:1) was added to the pellet and the samples were homogenized again using the fast prep machine 4 × 40 s speed 6 m/s. After centrifugation at 16.000 g for 10 min, the supernatant was collected and dried in a vacuum concentrator. This is the organic extract for GC-MS, which was sent to MS-Omics ApS (Copenhagen, Denmark) for lipidomics. Pluskal et al., 2010 Pluskal T.

Castillo S.

Villar-Briones A.

Oresic M. MZmine 2: modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data. Sample analysis was carried out by MS-Omics as follows: The samples reconstituted in an isopropanol/methanol/water (2:1:1) mixture. The analysis was carried out using a UPLC system (UPLC Acpuity, Waters) coupled with a time of flight mass spectrometer (Xevo G2 Tof, Waters). An electrospray ionization interface was used as ionization source. Analysis was performed in negative and positive ionization mode. The UPLC was performed using a slightly modified version of the Waters application note described by Isaac et al. (Giorgis Isaac, Stephen McDonald, and Giuseppe Astarita, Lipid separation using UPLC with charged surface hybrid technology, Waters application notes, 2011). Data processing was performed in MZmine 2 (T. Pluskal, S. Castillo, A. Villar-Briones, M. Orešič, MZmine 2: Modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data ()). Identification of compounds was performed using a both accurate mass (with an accepted deviation of 0.01 Da) and retention time in relation to chain length and double bonds.