Animals
F344 and WKY/NCr1 rats were obtained from Charles River Laboratories Japan, Inc. (Kanagawa, Japan). Rats were maintained in a 12 h light/dark cycle (light on at 08:00) at room temperature (23 ± 2°C) with food and water provided ad libitum. Animal experiments have been approved by the Animal Care and Use Committee of Okayama University (permission number: # OKU-2017523, OKU-2017592, OKU-2020016, OKU-2020144, OKU-2020536, OKU-2020840 ) and the Shigei Medical Research Institute (authorization number: #17007, #20005), and were performed in accordance with the Guidelines for Animal Experimentation at Okayama University and the Shigei Medical Research Institute. During study and care, the housing and use of animals was strictly carried out in accordance with appropriate guidelines and regulations. Every effort has been made to minimize animal suffering and to reduce the number of animals used in experiments. Reporting of animal data in this study followed the recommendations outlined in the ARRIVE guidelines.
Generation of NMU KO rats
NMU KO rats were generated by the rGONAD method as previously described31.32. Pre-oestrus female F344 rats were mated with male rats overnight, then the mating plugs were visually inspected the next morning and used for electroporation experiments. Electroporation experiments were carried out on anesthetized female rats (intraperitoneal injection of an anesthetic agent: mixture of Midazolam, Vetorphale and Domitor). Single-stranded DNA containing three stop codons (5ʹ-TAGCTAGCTAGAATTCCCGG-3ʹ), Cas9 guide RNA against the first exon of nmu (5ʹ-CGAGCAGCTAATCGCCGCCCAGG-3ʹ) (Fig. 1A) and Cas9 protein were injected into the oviductal lumen of pregnant female rats (E0.75) with the Alt-RTM CRISPR-Cas9 system (Integrated DNA Technologies, Coralville, IA) and subsequent electroporation by NEPA21 (Neppa Gene. Co., Ltd. Chiba, Japan). The guide RNA was designed using CHOPCHOP (http://chopchop.cbu.uib.no/). Genetic alterations were verified by PCR and then DNA sequencing. Genomic DNA was isolated from a piece of ear. Sequencing was performed using PCR products. PCR products were purified and directly sequenced and analyzed on an Applied Biosystems 3500 DNA Sequencer (Thermo Fisher Scientific) using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific). The selected founder generation rat was crossed with a wild-type rat to generate the F1 generation nmu+ /- rats. WT and NMU KO rats were generated by crossing nmu+ /- rats. Genotyping was performed by PCR with genomic DNA isolated from an ear tip. PCR amplification was performed using EmeraldAmp MAX PCR Master Mix (Takara Bio Inc., Shiga, Japan) according to manufacturer’s instructions. Oligonucleotide-specific primers for the WT and KO alleles were Nmu- Fw (5′-GATTTAAAAGTTGGTGCGCG-3′) and nmu -Rv (5′-GACAGGAGAGGGAGATGCAGTT-3′). Amplicon sizes were confirmed by 2% agarose gel electrophoresis (product sizes: WT allele, 222 bp; KO allele, 242 bp) (Fig. 1D).
Body weight measurement
The rats were kept at a rate of two animals per cage after weaning. Body weight was measured weekly from 4 to 24 weeks during the normal diet (MF, Oriental Yeast Co., Ltd, Shiga, Japan) and from 4 to 15 weeks during the high fat diet (D12451, Research diet , Inc., NJ, USA).
Food and water consumption
Rats were kept in an individual cage from 8 weeks of age with food and water provided ad libitum. The amounts of food and water ingested were measured weekly from 9 to 24 weeks to calculate average daily amounts. To analyze circadian patterns of food and water intake, we assessed the light and dark phases of food intake for 4 continuous days and calculated the average amounts.
Home cage locomotor activity and body temperature
Locomotor activity and body temperature were measured using the E-mitter telemetry system (Starr Life Sciences Corp., PA, USA). Rats were implanted subcutaneously with G2E transmitters under isoflurane anesthesia. Rats were kept individually in plastic home cages [276 × 445 × 204 (H) mm; CLEA Japan, Inc., Tokyo, Japan] with food and water provided ad libitum. Data was collected for 4 continuous days from 5 days postoperatively to calculate average activity volumes and average body temperature.
Preparation of an anti-NMU monoclonal antibody
We produced a rat monoclonal antibody against rNMU as previously described.44. Briefly, a synthetic peptide of 19 amino acid residues with the sequence YKVNEYQGPVAPSGGFFLF-Cys, which corresponds to part of the C-terminal side of mature rat NMU, was conjugated to limpet hemocyanin and an emulsion of The antigen was injected into WKY/NCrl rats anesthetized with sevoflurane. Treated rats were euthanized 17 days after injection and lymphocytes were fused with SP2/0-Ag14 myeloma cells. After cell fusion, culture supernatants were screened to confirm positive clones by solid-phase enzyme immunoassay (ELISA).
Specificity for the newly produced antibody was confirmed by Western blot analysis to recognize the synthetic rat NMU peptide (Cat. No. 350285, Abbiotec, Escondido, CA). Synthetic rat NMU peptide (3 ng) was subjected to 16% SDS-PAGE and transferred to PVDF blotting membranes. The membranes were blocked with 5% skimmed milk powder in Tris-buffered saline solution containing 0.1% Tween-20 for 1 h. The immunoblot was performed with the anti-NMU antibody, followed by a secondary antibody conjugated with horseradish peroxidase (1:5000 dilution). Immune complexes were visualized by enhanced chemiluminescence (Thermo Fisher Scientific) using an ImageQuant 800 (Cytiva). The single bands corresponded to the expected sizes (Supplementary Figure S1). Immunoreactivities disappeared completely with a synthetic antibody absorbed by a peptide.
Immunohistochemistry
Immunohistochemical detection of NMU was performed by the avidin-biotin complex (ABC) method. Male WT and NMU KO rats were sacrificed by decapitation under isoflurane anesthesia and the brains were rapidly removed. Brain tissues fixed with 4% paraformaldehyde were dehydrated in an ascending series of ethanol, immersed in xylene, embedded in paraffin (Palaplast, Sakura Finetek USA, Inc., CA, USA) and sectioned at a thickness of 10 µm. Sections were deparaffinized, incubated for 2 h with 10 mM sodium citrate (pH 6.0) for antigen retrieval, treated with 0.5% sodium metaperiodate to block endogenous peroxidases for 15 min , then incubated with TNBS (1% normal horse serum and 0.4% Triton X-100 in PBS) for 1 h. After washing with PBS, the sections were incubated overnight in a humid chamber with the anti-NMU antibody diluted 1:10 in TNBS. The ABC method was performed with a staining kit (VECTASTAIN Elite ABC Kit Peroxidase; VECTOR Laboratories, CA, USA). Reactions were visualized with 0.02% 3ʹ3-diaminobenzidine tetrachloride in 0.006% H2O2 in 50 mM Tris-HCl, pH 7.6. Stained sections were visualized under an optical microscope (BX60; Olympus, Tokyo, Japan) and photographed with a digital camera (DP70; Olympus).
Hematoxylin and eosin staining
Male WT and NMU KO rats were sacrificed by decapitation under isoflurane anesthesia. Liver and adipose tissues were fixed in 4% paraformaldehyde, dehydrated in an ascending series of ethanol, immersed in xylene, embedded in paraffin, and sectioned at a thickness of 10 μm. Sections were deparaffinized, washed with DW, and stained with hematoxylin and eosin (3 min for each stain). The stained sections were washed in running tap water, dehydrated with a series of alcohols and with xylene, then mounted for observation under a light microscope and imaging with a digital camera. For adipose tissue analysis, three regions were randomly selected from each section and photographed, and the number of cells was counted to calculate the average cell number.
On the spot hybridization (ISH)
Male F344 rats were sacrificed by decapitation under isoflurane anesthesia and the brains were rapidly removed. ISH was performed on 8 μm thick frozen frontal sections as previously described.30.45. Rat antisense and digoxigenin-labeled sense nmu cRNA probes (GenBank accession number NM_022239; positions 231-625) and rat Nmur2cRNA probes (GenBank accession number NM_022275; positions 473 to 1270) were synthesized using a labeling kit (Roche Diagnostics GmbH, Mannheim, Germany) with SP6 or T7 RNA polymerase.
Laser microdissection (LMD) and cDNA sample preparation
Male F344 rats were sacrificed by decapitation under isoflurane anesthesia and the brains were rapidly removed. Samples of the rat suprachiasmatic nucleus (SCN), PVN, dorsomedial hypothalamic nucleus (DMH), VMH, arcuate nucleus (ARC), PT, and third ventricle (3V) ependymal cell layer were taken from serial frontal sections of rat brains (30 µm thick) using an LMD system (LMD 7000; Leica Microsystems, Wetzlar, Germany) as previously described30.46. Briefly, sections were fixed with ice-cold acetone for 2 min, rehydrated with an ethanol series, and stained with 0.1% toluidine blue. Representative micrographic images of the dissections are shown in Supplementary Figure S3. Total RNA from samples was extracted using an RNeasy Micro Kit (QIAGEN GmbH, Hilden, Germany). cDNA was synthesized from total RNA using Prime Script RT Master Mix (Takara Bio).
Quantitative RT-PCR (qPCR)
RT-qPCR was performed using the LightCycler 96 system (Roche Diagnostics) with SYBR Premix Ex Taq (Takara Bio). Primers used for RT-qPCR are shown in Supplementary Table S1. Rat Rpl19expression was evaluated as an internal control. Amplicon size and specificity were confirmed by melting curve analysis and 2% agarose gel electrophoresis.
Blood tests
Plasma glucose (GLU), total cholesterol (T-CHO) and triglycerides (TG) were analyzed using a Hitachi 7180 automatic analyzer (Hitachi High-Technologies Corporation, Japan) with Wako commercial kits ( FUJIFILM Wako Shibayagi Corp., Gunma, Japan). The analysis was carried out by Oriental Yeast Co., Ltd. Insulin levels were measured using a Rebis Insulin-rat T ELISA kit (FUJIFILM Wako Shibayagi). Leptin levels were measured using a leptin ELISA kit (Morinaga Institute of Biological Science, Yokohama, Japan).
statistical analyzes
Data are presented as means ± SEM. Comparisons between the two groups were made using Student’s t test. All statistical analyzes were performed using GraphPad Prism 6 software (GraphPad Software, La Jolla, CA). P
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