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• W3044996075 abstract "Nonalcoholic fatty liver disease (NAFLD) is the most common hepatic disorder related to type 2 diabetes (T2D). The disease can evolve toward nonalcoholic steatohepatitis (NASH), a state of hepatic inflammation and fibrosis. There is presently no drug that effectively improves and/or prevents NAFLD/NASH/fibrosis. GLP-1 receptor agonists (GLP-1Ra) are effective in treating T2D. As with the endogenous gut incretins, GLP-1Ra potentiate glucose-induced insulin secretion. In addition, GLP-1Ra limit food intake and weight gain, additional beneficial properties in the context of obesity/insulin-resistance. Nevertheless, these pleiotropic effects of GLP-1Ra complicate the elucidation of their direct action on the liver. In the present study, we used the classical methionine-choline deficient (MCD) dietary model to investigate the potential direct hepatic actions of the GLP-1Ra liraglutide. A 4-week infusion of liraglutide (570 µg/kg/day) did not impact body weight, fat accretion or glycemic control in MCD-diet fed mice, confirming the suitability of this model for avoiding confounding factors. Liraglutide treatment did not prevent lipid deposition in the liver of MCD-fed mice but limited the accumulation of C16 and C24-ceramide/sphingomyelin species. In addition, liraglutide treatment alleviated hepatic inflammation (in particular accumulation of M1 pro-inflammatory macrophages) and initiation of fibrosis. Liraglutide also influenced the composition of gut microbiota induced by the MCD-diet. This included recovery of a normal Bacteroides proportion and, among the Erysipelotrichaceae family, a shift between Allobaculum and Turicibacter genera. In conclusion, liraglutide prevents accumulation of C16 and C24-ceramides/sphingomyelins species, inflammation and initiation of fibrosis in MCD-diet-fed mice liver, suggesting beneficial hepatic actions independent of weight loss and global hepatic steatosis. Nonalcoholic fatty liver disease (NAFLD) is the most common hepatic disorder related to type 2 diabetes (T2D). The disease can evolve toward nonalcoholic steatohepatitis (NASH), a state of hepatic inflammation and fibrosis. There is presently no drug that effectively improves and/or prevents NAFLD/NASH/fibrosis. GLP-1 receptor agonists (GLP-1Ra) are effective in treating T2D. As with the endogenous gut incretins, GLP-1Ra potentiate glucose-induced insulin secretion. In addition, GLP-1Ra limit food intake and weight gain, additional beneficial properties in the context of obesity/insulin-resistance. Nevertheless, these pleiotropic effects of GLP-1Ra complicate the elucidation of their direct action on the liver. In the present study, we used the classical methionine-choline deficient (MCD) dietary model to investigate the potential direct hepatic actions of the GLP-1Ra liraglutide. A 4-week infusion of liraglutide (570 µg/kg/day) did not impact body weight, fat accretion or glycemic control in MCD-diet fed mice, confirming the suitability of this model for avoiding confounding factors. Liraglutide treatment did not prevent lipid deposition in the liver of MCD-fed mice but limited the accumulation of C16 and C24-ceramide/sphingomyelin species. In addition, liraglutide treatment alleviated hepatic inflammation (in particular accumulation of M1 pro-inflammatory macrophages) and initiation of fibrosis. Liraglutide also influenced the composition of gut microbiota induced by the MCD-diet. This included recovery of a normal Bacteroides proportion and, among the Erysipelotrichaceae family, a shift between Allobaculum and Turicibacter genera. In conclusion, liraglutide prevents accumulation of C16 and C24-ceramides/sphingomyelins species, inflammation and initiation of fibrosis in MCD-diet-fed mice liver, suggesting beneficial hepatic actions independent of weight loss and global hepatic steatosis. AT A GLANCE COMMENTARYEmmanuel S, et al.BackgroundNonalcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) are common hepatic disorders related to type 2 diabetes (T2D). Presently, no drug effectively improves and/or prevents NAFLD/NASH/liver fibrosis. GLP-1 receptor agonists (GLP-1Ra) are effective in treating T2D but their direct action on the liver remains elusive.Translational SignificanceWe presently show that infusion of the GLP-1Ra liraglutide prevents accumulation of C16 and C24-ceramides/sphingomyelins species, inflammation and initiation of fibrosis in liver from mice on a methionine-choline deficient diet, suggesting beneficial hepatic actions independent of weight loss and global hepatic steatosis. GLP-1Ra may present added-value in hepatic complications of T2D. Emmanuel S, et al. Nonalcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) are common hepatic disorders related to type 2 diabetes (T2D). Presently, no drug effectively improves and/or prevents NAFLD/NASH/liver fibrosis. GLP-1 receptor agonists (GLP-1Ra) are effective in treating T2D but their direct action on the liver remains elusive. We presently show that infusion of the GLP-1Ra liraglutide prevents accumulation of C16 and C24-ceramides/sphingomyelins species, inflammation and initiation of fibrosis in liver from mice on a methionine-choline deficient diet, suggesting beneficial hepatic actions independent of weight loss and global hepatic steatosis. GLP-1Ra may present added-value in hepatic complications of T2D. Nonalcoholic fatty liver disease (NAFLD) is the most common hepatic disorder in industrialized countries.1Rinella ME Nonalcoholic fatty liver disease: a systematic review.JAMA. 2015; 313: 2263-2273Crossref PubMed Scopus (1348) Google Scholar,2Younossi Z Anstee QM Marietti M et al.Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention.Nat Rev Gastroenterol Hepatol. 2018; 15: 11-20Crossref PubMed Scopus (2033) Google Scholar Obesity and type 2 diabetes (T2D) are frequent causes of NAFLD.1Rinella ME Nonalcoholic fatty liver disease: a systematic review.JAMA. 2015; 313: 2263-2273Crossref PubMed Scopus (1348) Google Scholar,2Younossi Z Anstee QM Marietti M et al.Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention.Nat Rev Gastroenterol Hepatol. 2018; 15: 11-20Crossref PubMed Scopus (2033) Google Scholar Nevertheless, NAFLD can occur in lean patients due to various factors, including overconsumption of specific nutrients (such as fructose or cholesterol), accumulation of visceral fat, genetic predisposition, dyslipidemia, or lipodystrophy.1Rinella ME Nonalcoholic fatty liver disease: a systematic review.JAMA. 2015; 313: 2263-2273Crossref PubMed Scopus (1348) Google Scholar,2Younossi Z Anstee QM Marietti M et al.Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention.Nat Rev Gastroenterol Hepatol. 2018; 15: 11-20Crossref PubMed Scopus (2033) Google Scholar A significant proportion of patients with NAFLD develop a state of hepatic inflammation [nonalcoholic steatohepatitis, NASH], which can lead to fibrosis and cirrhosis, potentially resulting in hepatocellular carcinoma.3Michelotti GA Machado MV Diehl AM NAFLD, NASH and liver cancer.Nat Rev Gastroenterol Hepatol. 2013; 10: 656-665Crossref PubMed Scopus (643) Google Scholar Whilst lifestyle changes, including a modification in eating habits, weight loss or physical activity, have beneficial effects on liver steatosis, there is currently no efficient long-term treatment available to improve and/or avoid the development and progression of NAFLD, NASH, and fibrosis.1Rinella ME Nonalcoholic fatty liver disease: a systematic review.JAMA. 2015; 313: 2263-2273Crossref PubMed Scopus (1348) Google Scholar, 2Younossi Z Anstee QM Marietti M et al.Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention.Nat Rev Gastroenterol Hepatol. 2018; 15: 11-20Crossref PubMed Scopus (2033) Google Scholar, 3Michelotti GA Machado MV Diehl AM NAFLD, NASH and liver cancer.Nat Rev Gastroenterol Hepatol. 2013; 10: 656-665Crossref PubMed Scopus (643) Google Scholar Studying NAFLD is challenging in humans due to the invasive nature of liver biopsies and the slow progression rate of the disease. This situation has led to the development of many animal models,4Hebbard L George J. Animal models of nonalcoholic fatty liver disease.Nat Rev Gastroenterol Hepatol. 2011; 8: 35-44Crossref PubMed Scopus (330) Google Scholar of which the methionine and choline deficient (MCD) diet is most frequently used.4Hebbard L George J. Animal models of nonalcoholic fatty liver disease.Nat Rev Gastroenterol Hepatol. 2011; 8: 35-44Crossref PubMed Scopus (330) Google Scholar The MCD diet is rich in sucrose and devoid of methionine and choline, 2 nutrients required for mitochondrial β-oxidation and very-low density lipoprotein synthesis in the liver.5Yao ZM Vance DE. Reduction in VLDL, but not HDL, in plasma of rats deficient in choline.Biochem Cell Biol. 1990; 68: 552-558Crossref PubMed Scopus (91) Google Scholar,6Weltman MD Farrell GC Liddle C Increased hepatocyte CYP2E1 expression in a rat nutritional model of hepatic steatosis with inflammation.Gastroenterology. 1996; 111: 1645-1653Abstract Full Text PDF PubMed Scopus (364) Google Scholar As a consequence, the MCD diet induces hepatic steatosis, inflammation, and fibrosis without weight gain.4Hebbard L George J. Animal models of nonalcoholic fatty liver disease.Nat Rev Gastroenterol Hepatol. 2011; 8: 35-44Crossref PubMed Scopus (330) Google Scholar In addition, MCD diet-fed mice present liver-specific, but not global, insulin-resistance.7Rinella ME Green RM. The methionine-choline deficient dietary model of steatohepatitis does not exhibit insulin resistance.J Hepatol. 2004; 40: 47-51Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar Introduced about 15 years ago, GLP-1Ra are considered effective drugs for the treatment of T2D, with the advantage of limiting hypoglycemia. As does endogenous GLP-1 secreted by the enteroendocrine L-cells in the intestine, GLP-1Ra primarily potentiate glucose-induced insulin secretion.8Drucker DJ. Mechanisms of action and therapeutic application of glucagon-like peptide-1.Cell Metab. 2018; 27: 740-756Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar In addition, GLP-1Ra protect pancreatic islets, reduce food intake, and stimulate energy expenditure,8Drucker DJ. Mechanisms of action and therapeutic application of glucagon-like peptide-1.Cell Metab. 2018; 27: 740-756Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar all additional beneficial outcomes in the context of obesity/insulin-resistance. Interestingly, in vitro studies suggest that GLP-1Ra act directly on hepatocytes to limit endoplasmic reticulum stress,9Sharma S Mells JE Fu PP Saxena NK Anania FA GLP-1 analogs reduce hepatocyte steatosis and improve survival by enhancing the unfolded protein response and promoting macroautophagy.PLoS One. 2011; 6: e25269Crossref PubMed Scopus (198) Google Scholar activate AMP-activated protein kinase,10Ben-Shlomo S Zvibel I Shnell M et al.Glucagon-like peptide-1 reduces hepatic lipogenesis via activation of AMP-activated protein kinase.J Hepatol. 2011; 54: 1214-1223Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 11Svegliati-Baroni G Saccomanno S Rychlicki C et al.Glucagon-like peptide-1 receptor activation stimulates hepatic lipid oxidation and restores hepatic signalling alteration induced by a high-fat diet in nonalcoholic steatohepatitis.Liver Int. 2011; 31: 1285-1297Crossref PubMed Scopus (297) Google Scholar, 12Wang YG Yang TL. Liraglutide reduces fatty degeneration in hepatic cells via the AMPK/SREBP1 pathway.Exp Ther Med. 2015; 10: 1777-1783Crossref PubMed Scopus (7) Google Scholar dampen lipogenesis10Ben-Shlomo S Zvibel I Shnell M et al.Glucagon-like peptide-1 reduces hepatic lipogenesis via activation of AMP-activated protein kinase.J Hepatol. 2011; 54: 1214-1223Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar or interact with the insulin signaling pathways.11Svegliati-Baroni G Saccomanno S Rychlicki C et al.Glucagon-like peptide-1 receptor activation stimulates hepatic lipid oxidation and restores hepatic signalling alteration induced by a high-fat diet in nonalcoholic steatohepatitis.Liver Int. 2011; 31: 1285-1297Crossref PubMed Scopus (297) Google Scholar, 12Wang YG Yang TL. Liraglutide reduces fatty degeneration in hepatic cells via the AMPK/SREBP1 pathway.Exp Ther Med. 2015; 10: 1777-1783Crossref PubMed Scopus (7) Google Scholar, 13Gupta NA Mells J Dunham RM et al.Glucagon-like peptide-1 receptor is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway.Hepatology. 2010; 51: 1584-1592Crossref PubMed Scopus (338) Google Scholar Nevertheless, the pleiotropic effects of GLP-1Ra on the pancreas and hypothalamus complicate the elucidation of their direct action on the liver in vivo. In fact, the anti-steatotic action of GLP-1Ra has been mainly observed in situations of weight loss and/or insulin sensitization in both animal models14Moreira GV Azevedo FF Ribeiro LM et al.Liraglutide modulates gut microbiota and reduces NAFLD in obese mice.J Nutr Biochem. 2018; 62: 143-154Crossref PubMed Scopus (67) Google Scholar, 15Mells JE Fu PP Sharma S et al.Glp-1 analog, liraglutide, ameliorates hepatic steatosis and cardiac hypertrophy in C57BL/6J mice fed a Western diet.Am J Physiol Gastrointest Liver Physiol. 2012; 302: G225-G235Crossref PubMed Scopus (165) Google Scholar, 16Zhu W Feng PP He K Li SW Gong JP Liraglutide protects non-alcoholic fatty liver disease via inhibiting NLRP3 inflammasome activation in a mouse model induced by high-fat diet.Biochem Biophys Res Commun. 2018; 505: 523-529Crossref PubMed Scopus (31) Google Scholar, 17Yamazaki S Satoh H Watanabe T Liraglutide enhances insulin sensitivity by activating AMP-activated protein kinase in male Wistar rats.Endocrinology. 2014; 155: 3288-3301Crossref PubMed Scopus (34) Google Scholar, 18Gao H Zeng Z Zhang H et al.The glucagon-like peptide-1 analogue liraglutide inhibits oxidative stress and inflammatory response in the liver of rats with diet-induced non-alcoholic fatty liver disease.Biol Pharm Bull. 2015; 38: 694-702Crossref PubMed Scopus (46) Google Scholar, 19Decara J Arrabal S Beiroa D et al.Antiobesity efficacy of GLP-1 receptor agonist liraglutide is associated with peripheral tissue-specific modulation of lipid metabolic regulators.Biofactors. 2016; 42: 600-611Crossref PubMed Scopus (24) Google Scholar, 20Ao N Yang J Wang X Du J Glucagon-like peptide-1 preserves non-alcoholic fatty liver disease through inhibition of the endoplasmic reticulum stress-associated pathway.Hepatol Res. 2016; 46: 343-353Crossref PubMed Scopus (39) Google Scholar and humans.21Armstrong MJ Gaunt P Aithal GP et al.Liraglutide safety and efficacy in patients with non-alcoholic steatohepatitis (LEAN): a multicentre, double-blind, randomised, placebo-controlled phase 2 study.Lancet. 2016; 387: 679-690Abstract Full Text Full Text PDF PubMed Scopus (951) Google Scholar,22Petit JM Cercueil JP Loffroy R et al.Effect of liraglutide therapy on liver fat content in patients with inadequately controlled type 2 diabetes: the lira-NAFLD study.J Clin Endocrinol Metab. 2017; 102: 407-415PubMed Google Scholar To investigate the liver-specific action of GLP-1Ra, we infused liraglutide to mice fed with the MCD diet, hypothesizing that liraglutide might improve NAFLD/NASH by reducing steatosis, inflammation and fibrosis independently of weight loss. All experimental protocols were performed in accordance with the Swiss animal welfare laws. Fourteen-week-old C57BL/6J male mice (Charles River Laboratories) were acclimatized for 1 week in standard conditions (22°C, 12h-light/12h-dark cycle, free access to water and food). Thereafter, mice were fed with a regular chow diet (RM3, SDS; Chow group) or a MCD diet (A02082002BR, Research Diets; MCD and MCD/Lira groups) for 7 weeks. During the last 4 weeks of the experiment, mice were either infused with isotonic saline solution (Chow and MCD groups) or with liraglutide (0.1 µl/h of Victoza, Novo Nordisk [6 mg liraglutide/ml]; MCD/Lira group) via micro-osmotic pumps (Model 1004D, Alzet). Food intake and body weight were monitored weekly during the experiment. A schematic representation of the study protocol is illustrated in Fig 1. At the end of the experiment, mice were fasted 4 hours, anesthetized with isoflurane and immediately sacrificed. The osmotic pumps were retrieved and emptying was checked to confirm liraglutide release. Blood samples were collected, and organs were dissected and weighed before fixation or cryopreservation in liquid nitrogen. Blood samples were collected in EDTA-coated tubes and plasma was stored at −80°C. Plasma levels of glucose, ALT, AST, total cholesterol, triglycerides and free fatty acids were assessed using a Cobas C111 robot and appropriate reagents (Roche Diagnostics). Plasma levels of insulin were measured using an ultrasensitive mouse insulin ELISA kit (Mercodia). Total RNA was isolated from 50 to 100 mg liver samples using TRI Reagent Solution (ThermoFisher). Five hundred ng of total RNA were reverse transcribed using the PrimeScript RT reagent kit (Takara) and gene expression levels were assessed by quantitative PCR using the Power SYBR Green master mix (Thermofisher) and a Light-Cycler 480 (Roche Diagnostics). Normalization was done using the 40S ribosomal protein S29 (RPS29) gene. Liver and the distal part of the intestine (ileum) were either fixed overnight in 10% formalin before dehydration and embedded in paraffin or immediately embedded in OCT medium (Cell path LTD) and frozen on dry ice before storage at −80°C. Paraffin-embedded sections were stained with hematoxylin-eosin (H&E), Sirius red, Periodic Acid Schiff's solutions and Ki67 antibody using classical procedures. Frozen sections were stained with Oil red O solution using classical procedures. Pictures were acquired using a VS120 microscope (Olympus). Steatosis and fibrosis quantifications were achieved with Oil red O and Sirius red labeled slides using the area measurement tool of the ImageJ software (NIH). For fibrosis quantification, vessels, exhibiting a massive confounding collagen staining, were excluded from the analysis which represents only collagen fibers emerging in the liver parenchyma. Two independent liver lobes per animal were used for these quantifications. Ileal villi and crypt sizes were measured on H&E stained sections with the strait segment tool of ImageJ software. The pathological score, including steatosis and activity (ballooning and lobular inflammation) was evaluated by a trained pathologist (CDV) blinded to the diets as previously described.23Bedossa P Poitou C Veyrie N et al.Histopathological algorithm and scoring system for evaluation of liver lesions in morbidly obese patients.Hepatology. 2012; 56: 1751-1759Crossref PubMed Scopus (469) Google Scholar Lipid extraction and lipidomic analyses were performed as previously described.24Loizides-Mangold U Perrin L Vandereycken B et al.Lipidomics reveals diurnal lipid oscillations in human skeletal muscle persisting in cellular myotubes cultured in vitro.Proc Natl Acad Sci U S A. 2017; 114: E8565-E8E74Crossref PubMed Scopus (58) Google Scholar,25Matyash V Liebisch G Kurzchalia TV Shevchenko A Schwudke D Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics.J Lipid Res. 2008; 49: 1137-1146Crossref PubMed Scopus (1239) Google Scholar Lipids were isolated from 20 mg of liver tissue. After extraction, the organic lipid containing phase was dried in a vacuum concentrator (CentriVap, Labconco). Lipids were then dissolved in chloroform/methanol and divided into 3 aliquots. One aliquot was treated by alkaline hydrolysis to enrich for sphingolipids26Clarke NG Dawson RM Alkaline O leads to N-transacylation. A new method for the quantitative deacylation of phospholipids.Biochem J. 1981; 195: 301-306Crossref PubMed Scopus (183) Google Scholar and the other 2 aliquots were used for glycerophospholipids and phosphorus assay, respectively. Mass spectrometry analysis for the identification and quantification of phospho- and sphingolipid species was performed on a TSQ Vantage Triple Stage Quadrupole Mass Spectrometer (Thermo Fisher Scientific) equipped with a robotic nanoflow ion source (Nanomate HD, Advion Biosciences), using multiple reaction monitoring. Each individual ion dissociation pathway was optimized with regard to collision energy. Lipid concentrations were calculated relative to the relevant internal standards and normalized to the phosphate content of each lipid extract to account for variability in the amount of starting material and to correct for sample loss during the extraction procedure. DNA extraction. DNA was extracted from about 30 mg colon content using Quick-DNA Fecal/Soil Microbe Miniprep Kit (Zymo). Purified DNA was quantified using the Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific) and stored at –20°C. Two negative controls were performed by extracting DNA using the same extraction procedure but omitting the addition of colon content. Amplicon Sequencing. The V3–4 region of the bacterial 16S rRNA genes (Escherichia coli positions 341–805) was amplified using 1 ng of DNA extracted from colon content samples or 5 µL of the DNA extract obtained from negative controls in a 25 µL volume of ZymoBIOMICS PCR PreMix (Zymo Research) containing each of 0.4 µM forward primer 5’-barcode-CCTACGGGNGGCWGCAG-3’ and reverse primer 5’-barcode-GACTACHVGGGTATCTAATCC-3’ tagged with 8-nt barcodes at their 5’ends (Fasteris, Plan-les-Ouates, Switzerland). The PCRs were carried out with an initial denaturation at 95°C for 3 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 51°C for 30 seconds, and extension at 72°C for 60 seconds, and a final extension at 72°C for 10 minutes. Duplicate PCRs of each sample were combined and run (1 µL) on a 2100 Bioanalyzer (Agilent Technologies) for quality analysis and quantification. The sequencing library was constructed using the MetaFast protocol (Fasteris, Plan-les-Ouates, Switzerland). The sequencing was carried out for 2 × 300 cycles on an Illumina MiSeq instrument using MiSeq Reagent Kit v3. Bioinformatics analysis. Libraries were demultiplexed using the Fasteris internal software. No mismatches were allowed in a barcode and a maximum of 2 mismatches were allowed per 16S primer sequences. Trimmomatic v.0.3227Bolger AM Lohse M Usadel B Trimmomatic: a flexible trimmer for Illumina sequence data.Bioinformatics. 2014; 30: 2114-2120Crossref PubMed Scopus (24610) Google Scholar was used to remove Illumina adapters and to trim reads, by cutting them once the average quality within the 4-base window fell below a quality score of 15. Reads with a final length <60 bases were discarded. Forward and reverse reads were further quality-filtered and paired-end-joined with PEAR v.0.9.1128Zhang J Kobert K Flouri T Stamatakis A PEAR: a fast and accurate Illumina Paired-End reAd mergeR.Bioinformatics. 2014; 30: 614-620Crossref PubMed Scopus (2321) Google Scholar using the following settings: maximum assembly length (−m) 470; minimum assembly length (−n) 390; minimum overlap (−v) 20; minimum read size after trimming (−t) 240; P value (−P) 0.0001; maximal proportion of uncalled bases (−u) 0; and quality score threshold in trimming (−q) 33. The merged sequences were subjected to the UPARSE pipeline (USEARCH v.8.1.1861)29Edgar RC.UPARSE highly accurate OTU sequences from microbial amplicon reads.Nature Methods. 2013; 10: 996-998Crossref PubMed Scopus (8498) Google Scholar for clustering into operational taxonomic units (OTUs) at 97% identity. Taxonomic assignment of the representative OTUs was performed using MOTHUR v.1.39.530Schloss PD Westcott SL Ryabin T et al.Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities.Appl Environ Microbiol. 2009; 75: 7537-7541Crossref PubMed Scopus (13794) Google Scholar classify.seqs command (with method = wang, −cutoff = 80) and the EzBioCloud 16S rRNA gene sequence database31Yoon S-H Ha S-M Kwon S et al.Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies.Int J Syst Evol Microbiol. 2017; 67: 1613-1617Crossref PubMed Scopus (3852) Google Scholar downloaded on 4 January 2018. We removed OTUs that (1) had an average relative abundance higher in the negative controls than in colon content samples or (2) had <90% identity to reference prokaryotic EzBioCloud 16S sequences as revealed by USEARCH (-usearch_global -id 0.9 -query_cov 0.99).32Edgar RC. Search and clustering orders of magnitude faster than BLAST.Bioinformatics. 2010; 26: 2460-2461Crossref PubMed Scopus (13244) Google Scholar Normalization of OTU counts for sequencing depth (110,000) was performed using the “rrarefy” command from the R vegan package. Sequencing data were submitted to the European Nucleotide Archive (ENA; www.ebi.ac.uk/ena; study number: PRJEB36797). Statistical analyses. Statistical analyses of the data using 1-way ANOVA was performed using the GraphPad Prism version 7.02 software. Graphs represent mean + standard error of the mean (SEM). Clustered heat map was generated in R using the heatmap.2 function of the “gplots” package. A Pvalue < 0.05 was considered statistically significant. First, we investigated the metabolic action of liraglutide in the nutritional context of the MCD-diet. Previous studies have shown that GLP-1Ra, like liraglutide, reduce food intake in rodents.33Kanoski SE Hayes MR Skibicka KP GLP-1 and weight loss: unraveling the diverse neural circuitry.Am J Physiol Regul Integr Comp Physiol. 2016; 310: R885-R895Crossref PubMed Scopus (126) Google Scholar Here, we confirmed that mice fed an MCD-diet eat approximately 30% less than mice fed a chow diet (Fig 2A). Nevertheless, liraglutide did not potentiate the anorexic effect of the MCD-diet. In fact, MCD diet-fed mice infused with liraglutide ate the same amount of food as those infused with saline solution (Fig 2A). Similarly, body weight (Fig 2B), epididymal white adipose tissue mass (Fig 2C), interscapular brown adipose tissue mass (Fig 2D), glycemia (Fig 2E), and insulinemia (Fig 2F) were all significantly decreased by the MCD-diet consumption but unaffected by liraglutide administration. The homeostasis model assessment index, considered as a good marker of whole body insulin sensitivity, was greatly improved in the MCD-diet group but unchanged by liraglutide (Chow: 28.9 ± 1.8, MCD: 2.5 ± 0.5, MCD/Lira: 4.3 ± 1.4, P= 0.22 for MCD/Lira vs MCD groups). Taken together, these results showed that liraglutide infusion did not impact energy homeostasis or basal glycemic control in mice on the MCD-diet, allowing us to investigate directly the hepatic action of liraglutide independently of potentially confounding metabolic effects. Relative liver weight was not statistically different between Chow, MCD and MCD/Lira groups (Fig 3A). Circulating levels of alanine aminotransferase (Fig 3B), aspartate aminotransferase (Fig 3C) and free fatty acids (Fig 3D), increased significantly by the MCD-diet, were not further modified by liraglutide treatment. Similarly, hypocholesterolemia (Fig 3E) and hypotriglyceridemia (Fig 3F) were comparable in MCD diet-fed mice infused with saline solution or liraglutide. As expected, histological hepatic sections stained with H&E and Oil-red O revealed that livers of MCD diet-fed mice displayed significant steatosis and lobular inflammation, while their hepatocytes presented only mild ballooning (Fig 3G, J). Lipids, representing more than 20% of the liver area in MCD diet-fed mice (Fig 3G, H), mostly accumulated as single droplets (ie, macrosteatosis; Fig 3G, I). Liraglutide infusion did not quantitatively affect hepatosteatosis in MCD diet-fed mice (Fig 3G–J), in accordance with unchanged gene expression of central regulators of lipid metabolism (Fig 3K). Nevertheless, lipid species differ in terms of lipotoxicity.34Liangpunsakul S Chalasani N. Lipid mediators of liver injury in nonalcoholic fatty liver disease.Am J Physiol Gastrointest Liver Physiol. 2019; 316: G75-G81Crossref PubMed Scopus (24) Google Scholar,35Musso G Cassader M Paschetta E Gambino R Bioactive lipid species and metabolic pathways in progression and resolution of nonalcoholic steatohepatitis.Gastroenterology. 2018; 155 (e8): 282-302Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar To evaluate potential changes in lipid composition, we analyzed the hepatic content of over 900 lipid species by targeted mass spectrometry. Hierarchical clustering analysis based on fold-change compared to the Chow group (Fig 4A) highlighted lipid species significantly changed between the MCD and the MCD/Lira groups. Interestingly, the marked increases in C16 and C24-ceramide (Fig 4B) and sphingomyelins (Fig 4C) levels observed in the liver of MCD diet-fed mice were partly alleviated in the liver of MCD diet-fed mice treated with liraglutide. Different biochemical pathways (de novo ceramide biosynthesis vs degradation of sphingomyelin into ceramide) converge toward the generation of ceramide (Fig 4D). Gene expression of key enzymes involved in ceramide production was increased in MCD diet-fed mice compared to mice fed with a chow diet (Fig 4E). Furthermore, gene expression of Sptlc2, cerS4, and cerS6 was downregulated by liraglutide, suggesting a broad effect on sphingolipid metabolism.Fig 4Liraglutide infusion qualitatively changes hepatic lipid composition in MCD diet-fed mice. (A) Hierarchical clustering of 797 lipid hepatic species presented as LOG2 of fold-change compared to the Chow diet group. Most relevant lipid species changed by liraglutide infusion are listed on the left. (B) Con" @default.
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• W3044996075 title "The GLP-1R agonist liraglutide limits hepatic lipotoxicity and inflammatory response in mice fed a methionine-choline deficient diet" @default.
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