Add Gastrokine-1, an Anti-amyloidogenic Protein Secreted by the Stomach, Regulates Diet-induced Obesity

Gastrokine-1%2C an Anti-amyloidogenic Protein Secreted by the Stomach%2C Regulates Diet-induced Obesity.-.md

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+<br>All methods were carried out in accordance with relevant guidelines and regulations. All animal protocols were approved by the University of Notre Dame (Boone) or the University of Cincinnati (Seeley) Institutional Animal Care and Use Committees. All experiments were carried out in accordance with the ARRIVE (Animal Research: Reporting of In Vivo experiments) guidelines and in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals. − mice on a C57Bl/6 genetic background were generated by the trans-NIH Knockout-Out Mouse Project (KOMP) and bred and maintained in our facility on that background. Mice were provided food and  [MedicGLP Product](https://qrly.app/betsydobbi) water ad libitum and kept on a 12/12-h light/dark cycle. All cages contained at least one enrichment device in the form of a cardboard tube or "house". To test the effects of a thermoneutral environment mice were maintained in a room kept at 30 °C. For normal chow diet, mice were maintained on Teklad diet 2919 global 19% protein-extruded rodent diet (22% calories from fat).<br>
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+<br>For high-fat diet, mice were fed research diet D12492 (60% calories from fat). RNA was extracted from tissues using TRIZOL (Thermo Fisher) and cDNA was generated using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). GKN1 was amplified by 30 cycles of PCR using the primers listed in supplementary Table 1, and resolved by agarose gel electrophoresis. Proteins were extracted from tissues using RIPA buffer and 30 μg total protein was resolved by SDS-PAGE using NuPAGE 4-12% precast gels and transferred to PVDF membranes (Thermo Fisher). Membranes were blocked with 5% non-fat milk, immunoblotted with rabbit anti-GKN1 antibody (1:2000 in 5% non-fat milk) (Proteintech) overnight at 4 °C, and developed with anti-rabbit IgG-HRP antibody. Actin immunoblotting and Ponceau red staining of membranes were used as controls. To assess Gkn1 protein in the gut lumen, contents were flushed in PBS, mechanically dissociated and filtered through nylon mesh and filtrates were precipitated in cold acetone, resuspended in Laemmli buffer and assessed by Western blotting.<br>
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+<br>Formalin fixed paraffin embedded (FFPE) 5 μm sections were deparaffinized to water and blocked in 2% horse serum before overnight incubation with rabbit anti-GKN1 antibody (Proteintech) (1:250 in 2% horse serum). Sections were washed and developed with anti-rabbit antibody using the ImmPRESS-HRP polymer detection kit (Vector Labs). Standard protocols were followed for H&E staining of FFPE tissues. Magnetic resonance imaging was used to assess whole body composition of live subjects using the EchoMRI system (EchoMRI, Houston, TX). To assess the formation of tumors, mice were injected i.v. 200 μCi of (18F) fluorodeoxyglucose (Spectron MRC, South Bend, IN, USA) and scanned 30 min later with a trimodal ALBIRA PET/SPECT/CT image station (Carestream Health, Woodbridge, CT, USA) and analyzed as described16. T7532-120) according to the manufacturer’s specifications. Histological assessment of fat in liver was performed on FFPE tissues using H&E staining. In addition, to visualize fat accumulation, OCT-embedded frozen sections were stained with oil red O. Sections of 8 mm were dried, fixed in 4% paraformaldehyde, pretreated with 60% isopropanol, stained in 0.3% oil red O (w/v in 60% isopropanol), de-stained in 60% isopropanol, counterstained in hematoxylin and photographed in aqueous media.<br>
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+<br>For assessment of food caloric extraction, food intake was measured by weighing food daily and total stool was collected, dried and analyzed by bomb calorimetry (UAB small animal phenotyping core, Birmingham AL). Efficiency of calorie absorption was determined by the percent of calories consumed vs calorie content of stool. Fecal lipid content was assessed as previously described17. Briefly, diet was laced with 2.5% sucrose polybehenate and  [MedicGLP Official Website](https://www.100seinclub.com/bbs/board.php?bo_table=E04_1&wr_id=81848) animals allowed to feed for 2 days. Fecal samples were collected 24 h later. Fecal lipid content was assayed by gas chromatography of [fatty acid](https://wideinfo.org/?s=fatty%20acid) methyl esters. Dietary lipid absorption was estimated using a ratio of total fecal fatty acids to sucrose polybehenate. Blood glucose was measured with the Accucheck meter and strips (Roche, Indianapolis, IN) following an i.p. 1.5 g/kg dextrose in un-anesthetized mice previously fasted for 4 h. Fasting plasma insulin was measured by ELISA (Crystal Chem Inc., Downers Grove,  [MedicGLP](http://tangxj.cn:6012/francisboldt65) IL). For measurements of CCK, GIP, and ANGPLT4, tissue scrapings were collected from washed intestinal samples and processed for RNA isolation and assessment of gene expression by qPCR.<br>
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+<br>Ghrelin mRNA levels were assessed from mucosal scrapings of the stomach. Leptin and GLP-1 levels in serum were assessed by ELISA (EMD-Millipore, Burlington, MA). For immunolocalization of ghrelin and leptin in stomach tissue FFPE tissue sections were treated for antigen retrieval by boiling 10 min in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20 pH6.0) and stained with monoclonal anti-Gkn1 (R&D Systems), and polyclonal anti-ghrelin or anti-leptin antibodies (4C overnight; Thermo-Fisher), washed and stained with FITC or PE-conjugated secondary antibodies, mounted anti-fade media (prolong gold with DAPI) and photographed using fluorescence microscopy. Mice were individually housed in metabolic cages (TSE Systems, Chesterfield, MO) for indirect calorimetry. Mice were acclimated for 48 h prior to measurements and assessed for 48 h of oxygen consumption and carbon dioxide production and RER was calculated by the ratio of CO2 produced to O2 consumed. Gross morphological examination of secondary lymphoid organs and histology of tissues was performed to [identify signs](https://www.medcheck-up.com/?s=identify%20signs) of overt inflammation. Flow cytometry was used to analyze lymphocyte activation from spleens and the intestinal lamina propria, as described18. Sequencing of the 16S ribosomal RNA gene V4-V5 region was performed at Argonne National Laboratories (Argonne, IL) using the Illumina MiSeq instrument (San Diego, CA). Sequences were analyzed with the Quantitative Insights into Microbial Ecology (QIIME) toolkit version 1.9.119. Reads were quality filtered and demultiplexed using a PHRED score of 30. Quality filtering resulted in 544,839 high-quality sequences total. The quality filtered reads were clustered using the open reference OTU picking workflow against the April 2018 release of the SILVA database with a sequence identity of 97% and uclust used as the clustering algorithm. Chimeras were removed using ChimeraSlayer. Low confidence OTUs were removed. Samples with less that 5,000 reads were removed from the OTU table. Statistical analyses were performed with Prism software version 7 (Graphpad, LaJolla, CA). ANOVA was used for multiple comparisons with post-hoc Tukey’s test. Paired comparisons were performed using the Welch t test.<br>