The GLP-1R agonist liraglutide limits hepatic lipotoxicity and inflammatory response in mice fed a methionine-choline deficient diet

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Reference

The GLP-1R agonist liraglutide limits hepatic lipotoxicity and inflammatory response in mice fed a methionine-choline deficient diet

SOMM, Emmanuel, et al.

Abstract

Nonalcoholic fatty liver disease (NAFLD) is the most common hepatic disorder related to type 2 diabetes (T2D). The disease can evolve towards 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 four-week infusion of liraglutide (570µg/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 [...]

SOMM, Emmanuel, et al . The GLP-1R agonist liraglutide limits hepatic lipotoxicity and

inflammatory response in mice fed a methionine-choline deficient diet. Translational Research , 2020

DOI : 10.1016/j.trsl.2020.07.008 PMID : 32711187

Available at:

http://archive-ouverte.unige.ch/unige:139520

Disclaimer: layout of this document may differ from the published version.

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The GLP-1R agonist liraglutide limits hepatic lipotoxicity and inflammatory response in mice fed a methionine-choline deficient diet

Q1 X XD1X XEMMANUEL SOMMD2X X¹,D3X XSOPHIE A. MONTANDOND4X X¹,D5X XURSULA LOIZIDES-MANGOLDD6X X,D7X XNADIA GAI¨AD8X X,

D9X X

VLADIMIR LAZAREVICD10X X,D11X XCLAUDIO DE VITOD12X X,D13X XELODIE PERROUDD14X X, D15X X

MARIE-LUCE BOCHATON-PIALLATD16X X,D17X XCHARNA DIBNERD18X X,D19X XJACQUES SCHRENZELD20X X, and D21X X

FRAN¸COIS R. JORNAYVAZD22X X

GENEVA, SWITZERLAND

Nonalcoholic fatty liver disease (NAFLD) is the most common hepatic disorder related to type 2 diabetes (T2D). The disease can evolve towardD23X Xnonalcoholic 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. AD24X X4-week infusion of liraglutide (570 mg/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 normalBacteroidespro- portion and, among the Erysipelotrichaceae family, a shift between Allobaculum andTuricibacter genera. In conclusion, liraglutide prevents accumulation of C16 and C24-ceramides/sphingomyelins species, inflammation and initiation of fibrosis

1These authors contributed equally to this work.

From the Service of Endocrinology, Diabetes, Nutrition and Patient Education, Department of Internal Medicine, Geneva University Hospitals/Uni- versity of Geneva, Geneva, Switzerland; Diabetes Center, Faculty of Medicine, University of Geneva, Geneva, Switzerland; Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland; Institute of Genetics and Genomics in Geneva (iGE3), University of Geneva, Geneva, Switzerland; Genomic Research Laboratory, Service of Infectious Diseases, Geneva University Hospitals, University of Geneva, Geneva, Switzerland; Division of Clinical Pathology, Geneva University Hospitals, Geneva, Switzerland; Department of Pathology and Immunol- ogy, Faculty of Medicine, University of Geneva, Geneva, Switzerland; Bacteriology Laboratory, Service of Laboratory Medicine, Geneva Univer- sity Hospitals, Geneva, Switzerland.

Submitted for Publication February 25, 2020; received submitted July 14, 2020; accepted for publication July 16, 2020.

Reprint requests: Emmanuel Somm, Diabetes Center, Faculty of Medicine, Hopitaux Universitaires de Geneve, Geneva, Switzerland.

Reprint requests: Francois R. Jornayvaz, Service of Endocrinology, Diabetes, Nutrition and Patient Education, Department of Internal Medicine,¸ Geneva University Hospitals/University of Geneva, Geneva, Switzerland

Q2 X X. e-mail:emmanuel.somm@unige.ch,Francois.Jornayvaz@hcuge.ch.

1931-5244/$ - see front matter

Ó2020 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license. (http://creativecommons.org/licenses/by/

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in MCD-fed mice liver, suggesting beneficial hepatic actions independent of weight loss and global hepatic steatosis. (Translational Research 2020;&&:&&-&&)

Abbreviations: ALT = Alanine aminotransferase; AST = Aspartate aminotransferase; BAT = Brown adipose tissue; Cer = Ceramide; CerS = Ceramide synthase; DIO = Diet-induced obesity; FFA = Free fatty acid; GLP-1 = Glucagon like peptide-1; GLP-1Ra = GLP-1 receptor agonist;

HCC = Hepatocellular carcinoma; HOMA = Homeostasis model assessment; HSC = Hepatic stellate cell; LC = Long chain; LC-MS = Liquid chromatography mass spectrometry; Lira = Lira- glutide; MCD = Methionine-choline deficient; MUFA = Monounsaturated fatty acid; NAFLD = Nonalcoholic fatty liver disease; NASH = Nonalcoholic steatohepatitis; NGS = Next generation sequencing; PUFA = Polyunsaturated fatty acid; SFA = Saturated fatty acid; SM = Sphingomye- lin; T2D = Type 2 diabetes; TNF-a= Tumor necrosis factor alpha; VLC = Very long chain; WAT = White adipose tissue

Keywords:Glucagon like peptide-1 receptor agonist; Nonalcoholic fatty liver disease; Nonal- coholic steatohepatitis; Methionine-choline deficient diet; inflammation; Type 2 diabetes; Cer- amide; microbiota

Brief Commentary

Background: 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.

Translational significance: 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.

INTRODUCTION

Nonalcoholic fatty liver disease (NAFLD) is the most common hepatic disorder in industrialized coun- tries.^1,2Obesity and type 2 diabetes (T2D) are frequent causes of NAFLD.^1,2Nevertheless, NAFLD can occur in lean patients due to various factors, including over- consumption of specific nutrients (such as fructose or cholesterol), accumulation of visceral fat, genetic pre- disposition, dyslipidemia, or lipodystrophy.^1,2A 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 carcinomaD25X X.³ 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.^1-3

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,⁴of which the methionine and choline deficient (MCD) diet is most frequently used.⁴The MCD diet is rich in sucrose and devoid of methionine and choline,D26X X2 nutrients required for mitochondrial b-oxidation and very-low density lipoprotein synthesis in the liver.^5,6As a consequence, the MCD diet induces hepatic steatosis, inflammation, and fibrosis without weight gain.⁴ In addition, MCD diet-fed mice present liver-specific, but not global, insulin-resistance.⁷

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.⁸In addition, GLP-1Ra protect pancre- atic islets, reduce food intake, and stimulate energy expenditure,⁸ 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 reticulumD27X X stress,⁹ activate AMP-activated protein kinaseD28X X,^10-12dampen lipogenesis¹⁰ or interact with the insulin signaling pathways.^11-13Nev- ertheless, the pleiotropic effects of GLP-1Ra on the pan- creas 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 models^14-20and humans.^21,22

To investigate the liver-specific action of GLP-1Ra, we infused liraglutide to mice fed with the MCD diet,

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hypothesizing that liraglutide might improve NAFLD/

NASH by reducing steatosis, inflammation and fibrosis independently of weight loss.

MATERIAL AND METHODS

Animals, diets, treatments.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 D29X X1 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, SDSD30X X; Chow group) or aD31X XMCDD32X Xdiet (A02082002BR, Research DietsD33X X; MCD and MCD/Lira groups) for 7 weeks. During the

last D34X X4 weeks of the experiment, mice were either

infused with isotonic saline solution (Chow and MCD groups) or with liraglutide (0.1ml/hD35X Xof Victoza, Novo Nordisk [6 mg liraglutide/ml]D36X X; 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 inFig 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 analyses. Blood samples were collected in EDTA-coated tubes and plasma was stored at D37X X80˚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 ultrasensi- tive mouse insulin ELISA kit (Mercodia).

Gene expression.Total RNA was isolated from 50D38X Xto 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.

Histology, morphometry, pathological score.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 D39X X80˚C. Paraffin-embedded sections were stained with hematoxylin-eosin (H&E), Sirius red, Periodic Acid Schiff’sD40X Xsolutions and Ki67 antibody using classical procedures. Frozen sections were stained with Oil red O solution using classical procedures. Pictures were acquired using a VS120 micro- scope (Olympus). Steatosis and fibrosis quantifications were achieved with Oil red O and Sirius red D41X Xlabeled 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.²³

Lipidomic analysis. Lipid extraction and lipidomic analyses were performed as previously described.^24,25

Chow diet Chow diet + NaCl MCD diet MCD diet + NaCl MCD diet MCD diet + Liraglutide

Osmotic pumps

3 weeks 4 weeks

Diet-induced liver defects

Treatment Chow

MCD MCD/Lira C57BL/6 mice

Fig 1. Schematic representation of the study protocol. Fourteen-week-old C57BL/6J male mice were fed with either a regular chow diet or a methionine-choline deficient (MCD) diet for 3 weeks to initiate liver defects mim- icking human NASH. During the four last weeks of the experiment, mice were infused either with saline solution (Chow and MCD groups) or with liraglutide (6 mg/ml, 0.1ml/h) (MCD/Lira group) via micro-osmotic pumps.

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Lipids were isolated from 20 mg of liver tissue. After extraction, the organic lipid containing phase was dried in a vacuum concentrator (CentriVap, Labconco). Lip- ids were then dissolved in chloroform/methanol and divided into D42X X3 aliquots. One aliquot was treated by alkaline hydrolysis to enrich for sphingolipids²⁶ and the otherD43X X2 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 Spec- trometer (Thermo Fisher Scientific) equipped with a robotic nanoflow ion source (Nanomate HD, Advion Biosciences), using multiple reaction monitoringD44X X. Each individual ion dissociation pathway was optimized with regard to collision energy. Lipid concentrations were calculated relative to the relevant internal stand- ards 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.

Microbiota analysis. 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 bacte- rial 16S rRNA genes (Escherichia coli positions 341 805) was amplified using 1 ng of DNA extracted from colon content samples or 5mL of the DNA extract obtained from negative controls in a 25D45X XmL volume of ZymoBIOMICS PCR PreMix (Zymo Research) containing each of 0.4 mM 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 (1mL) on a 2100 Bioanalyzer (Agi- lent 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. Trim- momatic v.0.32²⁷ 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.0.9.11²⁸ using the following settings: maximum assembly length ( m) 470; minimum assembly length

( D46X Xn) 390; minimum overlap ( D47X Xv) 20; minimum read

size after trimming ( D48X Xt) 240; P value ( D49X XP) 0.0001;

maximal proportion of uncalled bases ( D50X Xu) 0; and quality score threshold in trimming ( D51X Xq) 33. The merged sequences were subjected to the UPARSE pipeline (USEARCH v.8.1.1861)²⁹ for clustering into opera- tional taxonomic units (OTUs) at 97% identity. Taxo- nomic assignment of the representative OTUs was performed using MOTHUR v.1.39.5³⁰ classify.seqs command (with method = wang, D52X Xcutoff = 80) and the EzBioCloud 16S rRNA gene sequence database³¹ downloaded on 4 January 2018. We removed OTUs that (D53X X1) had an average relative abundance higher in the negative controls than in colon content samples or

(D54X X2) had<90% identity to reference prokaryotic EzBio-

Cloud 16S sequences as revealed by USEARCH (-usearch_global -id 0.9 -query_cov 0.99).³²Normali- zation 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/enaD55X X; study number: PRJEB36797).

Statistical analysesX X. Statistical analyses of the data Q3

usingD56X X1-way ANOVA was performed using the Graph- Pad 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. AD57X XPvalue<0.05 was considered statistically significant.

RESULTS

Liraglutide does not change energy homeostasis or carbohydrate metabolism in mice on an MCD dietD58X X.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.³³Here, 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

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solution (Fig 2A). Similarly, body weight (Fig 2B), epididymal white adipose tissue mass (Fig 2C), interscap- ular 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 assessmentD59X Xindex, 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, D60X XP= 0.22 for MCD/Lira vsD61X XMCD 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.

Liraglutide changes hepatic lipid composition qualitatively but not quantitativelyD62X X.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, hypo- cholesterolemia (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 (iD63X Xe,D64X Xmacro- steatosisD65X X;Fig 3G, I). Liraglutide infusion did not quantitatively affect hepatosteatosis in MCD diet-fed mice (Fig 3GD66X X J), in accordance with unchanged gene expression of central regulators of lipid metabolism (Fig 3K). Nevertheless, lipid species differ in terms of lipotoxicity.^34,35To evaluate potential changes in lipid composition, we analyzed the hepatic content of over 900 lipid species by targeted mass spectrometry. Hier- archical 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. Differ- ent biochemical pathways (de novo ceramide biosynthesis vsD67X Xdegradation of sphingomyelin into ceramide) converge towardD68X Xthe generation of ceramide (Fig 4D69X XD).

Gene expression of key enzymes involved in ceramide production was increased in MCD diet-fed mice 0

2 4 6

Dailyfoodintake(g)

Chow MCD MCD/Lira

Before treatment

* * * *

After treatment

0.0 0.2 0.4 0.6

eWATmass(g) ^Chow

MCD MCD/Lira

* *

0 10 20 30

Finalbodyweight(g)

* *

0.00 0.02 0.04 0.06 0.08 0.10

BATmass(g)

*

0 5 10 15

Glucose(mmol/L) Chow

MCD MCD/Lira

* *

0.0 0.2 0.4 0.6

Insulin(ng/ml) ^ChowMCD

MCD/Lira

*

A B C

D E F

Fig 2. Liraglutide infusion does not modify energy homeostasis or carbohydrate metabolism in mice fed a MCD diet. (A) Food intake before and after saline/liraglutide infusion (g per day). (B) Body weight at the end of the experiment (after 7 weeks of chow/MCD-diet consumption and 4 weeks of saline/liraglutide infusion) (g). (C) Epididymal white adipose tissue (eWAT) mass (g). (D) Brown adipose tissue (BAT) mass (g). (E) Circulating glucose levels (mmol/L). (F) Circulating insulin levels (ng/mL). Results are expressed as the mean§SEM.

n = 9 12 male mice per group. One-way ANOVA: *Pvalue for MCD or MCD/Lira vs Chow<0.05,^#Pvalue for MCD/Lira vs MCD<0.05.

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compared to mice fed with a chow diet (Fig 4D70X XE). Fur- thermore, gene expression of Sptlc2, cerS4, and cerS6 was downregulated by liraglutide, suggesting a broad effect on sphingolipid metabolism.

Beyond their structural function, the degree of fatty acid saturation of lipid species also impacts the hepatic status.^36-38To analyze the degree of fatty acid saturation/desaturation, we measured the levels of phospholipids harboring (D71X X1) only 1 double bond in both fatty acyl chains combined (considered as monounsaturated fatty acids, MUFA); (D72X X2) between 2 6 double bonds in theirD73X X2 fatty acyl chains (considered as polyunsaturated fatty acids, PUFA); and (D74X X3) no double bonds in theirD75X X2 fatty acyl chains (considered as saturated fatty acids, SFA).

Hepatic PUFA lipids showed a trend to decrease in MCD/Lira mice compared to chow diet-fed mice while MUFA lipids were similarly decreased in the MCD and MCD/Lira groups compared to the Chow group (Fig 4F). Saturated fatty acid containing lipids were specifi- cally decreased in the MCD/Lira group compared to the

Chow and MCD groups (Fig 4F). Among qualitative changes in lipid composition, the length of the fatty acid chain is another important feature for cell membrane permeability/fluidity.³⁵ Phospholipids with long chains (LC), harboring a combined carbon number of 28 34 in both fatty acyl chains, were reduced in both the MCD and MCD/Lira groups (Fig 4G). In contrast, phospholipids with very long chains (VLC), harboring 38D76X X 44 carbons, remained unchanged in the Chow, MCD, and MCD/Lira groups (Fig 4G).

Liraglutide limits inflammation and initiation of fibrosis in mice on an MCD-dietD77X X.To further explore the pathology of hepatic inflammatory infiltrates in the MCD/Lira and the MCD groups of mice, we extended the analysis to expression of immune cell markers by means of quantitative real-time PCR. In the liver, macrophages are considered key players, even initiators, of the inflammatory process and are classified according to their tissue of origin or their activation state.^39,40 Molecular markers of both liver-resident Kupffer cells Chow

MCD

MCD/

Lira

0 200 400 600

ALT(U/L)

Ctrl MCD-NaCl MCD-Lira

* *

H&E Oil Red O

50 um

0 200 400 600 800

AST(U/L) *

*

0.0 0.5 1.0 1.5 2.0 2.5

Cholesterol(mmol/L)

* *

0.0 0.5 1.0 1.5

Triglycerides(mmol/L)

* *

0.0 0.5 1.0 1.5 2.0

FFA(mmol/L) *

*

0 10 20 30

%areaOilRedO

* *

0 20 40 60 80 100

%hepatocytes

Microsteatosis Macrosteatosis

MCD MCD/

Lira

0 2 4 6

Pathologicalscore

Steatosis Ballooning Inflammation

MCD MCD/

Lira

A B C D E F

G

K

H I J

0.0 0.5 1.0 1.5 2.0

Geneexpression(A.U.)

* *

PPARa 0.0 0.5 1.0 1.5 2.0

* *

Fasn 0.0 0.5 1.0 1.5 2.0

*

Cpt1a 0 2 4 6 8 * *

Cd36 0.0 0.5 1.0 1.5 2.0

Atgl 0.0 0.5 1.0 1.5 2.0

Dgat2 0

2 4 6

Livermass(%B.W.)

C h o w M C D M C D / L ir a

Fig 3. Liraglutide infusion does not quantitatively change hepatic lipid accumulation in MCD diet-fed mice. (A) Relative liver mass (expressed as percentage of body weight). (B) Circulating alanine aminotransferase (ALT) levels (U/L). (C) Circulating aspartate aminotransferase (AST) levels (U/L). (D) Circulating free fatty acid (FFA) levels (mmol/L). (E) Circulating total cholesterol levels (mmol/L). (F) Circulating triglyceride levels (mmol/L). (G) Haematoxylin and eosin (H&E) and Oil-red O staining of liver sections. Scale bars 50mm, black arrows: macrosteatosis, grey arrow: microsteatosis, white arrows: immune cell infiltration. (H) Quantification of lipids expressed as percent of area of liver sections stained with Oil red O. (I) Evaluation of the percent of hepa- tocyte containing micro- and macrosteatosis. (J) Pathological score with steatosis and activity (lobular inflammation + ballooning) evaluated by a pathologist blinded to the diets and treatments. (K) Hepatic gene expression of lipids enzymes and transporters (A.U). Results are expressed as the mean§SEM. n = 9 10 male mice per group (panel A-J), n = 7 9 male mice per group (panel K). One-way ANOVA: *Pvalue for MCD or MCD/Lira vs Chow<0.05,^#Pvalue for MCD/Lira vs MCD<0.05.

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(F4/80, Tlr4, Tlr9) and monocyte-derived macrophages (Ly6c, Ccr2) were all overexpressed in the MCD group and normalized in the MCD/Lira group (Fig 5A). Simi- larly, markers of classically activated (pro-inflammatory) M1 macrophages (Cd14, Tnfa) were up-regulated in the MCD group and significantly reduced in the MCD/Lira group (Fig 5A). In contrast, no difference was observed between the MCD and the MCD/Lira group regarding the expression of alternatively activated (anti-inflammatory) M2 macrophage markers (Arg1, Cd206, Retnla, Cd163D78X X; Fig 5D79X XA). Markers of myeloid dendritic cells (Cd11c, MHCII, Cd86D80X X; Fig 5, B) and activated hepatic stellate cells (Acta1 and Vim, involved in the deleterious epithelial-mesenchymal transition, Fig 5D81X XC) were significantly reduced by liraglutide treatment. In accordance, the expression of sev- eral pro-fibrogenic genes was lower in the MCD/Lira group compared to the MCD group (Fig 5D82X XD), including Timp1, Serpine1, Mmp13. Even more strikingly,

Tgfb1 and genes encoding collagen proteins showed the same basal levels of expression in the MCD/Lira as in the Chow group (Fig 5D83X XD). Our experimental set-up (7 weeks of MCD-diet consumption) allowed initiating but not observing a massive fibrosis. Nevertheless, histological staining of hepatic sections with Sirius red showed that the administration of liraglutide reduced the number and the size of collagen fibers in livers of MCD diet-fed mice (Fig 5D84X XE). Quantitatively, MCD diet-fed mice exhibited a D85X X7-fold increase in the Sirius red positive area of liver parenchyma compared to mice fed a chow diet (Fig 5D86X XF). Per comparison, MCD diet-fed mice treated with liraglutide exhibited a significantly attenuatedD87X X4-fold increase in Sirius red positive area compared to chow diet-fed mice (Fig 5D88X XF).

Liraglutide impacts the gut-liver axisD89X X.As an enterokine, GLP-1 production is regulated by the gut microbiota.^41,42In turn, GLP-1R agonism can modify intesti- nal bacteria populations.^14,43 In this context, we

MCD MCD/

Lira Cer

C16 SM C24

SM C16

Cer C24

A B

Ceramide species

Sphingomyelin species

0 500 1000 1500 2000 2500

*_#

* C16

0 20 40 60 80

fmol/nmolphosphate

*

* C14

0 1 2 3 4

*

# C24

0.0 0.5 1.0 1.5

pmol/nmolphosphate C22

0 100 200 300 400 500

* * C20

0 100 200 300 400 500

* * C18

0 200 400 600 800

*

* C16

0 50 100 150 200

fmol/nmolphosphate C14

0 200 400 600 **

C18

0 100 200 300 400 **

C20

0.0 0.5 1.0 1.5 2.0

pmol/nmolphosphate C22

0 2 4 6

*_#

*

C

C24

F G

D

0.0 0.5 1.0 1.5 2.0CerS5

* *

0 1 2 3 4 DegS2

*

0.0 0.5 1.0 1.5 2.0Smpd1

* * 0 1 2 3 CerS4

*

#

*

E

0.0 0.5 1.0 1.5 CerS2

0.0 0.5 1.0 1.5DegS1

0.0 0.5 1.0 1.5Smpd2 0.0

0.5 1.0 1.5Sptlc1

0 1 2 3 4 5Sptlc2

*^#

*

0 1 2 3 4 CerS6

*#

*

p=0.09

0 2 4 6

SFA

#

*

pmol/nmolphosphate

0 10 20 30 40

MUFA

**

0 50 100 150

LC

**

pmol/nmolphosphate

0 100 200 300 400

VLC

Ceramide enzymes

Fatty acid saturation Fatty acid chain length

0 200 400 600

PUFA Palmitoyl-CoA

Serine Acyl-CoA

Dihydroceramide Ceramide Sphingosine

Sphingomyelin Sptlc CerS

DegS Smpd

Acyl-CoA CerS

DE NOVO PATHWAY

SALVAGE PATHWAY

SPHINGOMYELIN PATHWAY

Fig 4. Liraglutide infusion qualitatively changes hepatic lipid composition in MCD diet-fed mice. (A) Hierar- chical 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) Concentration of hepatic ceramides normalized to the phosphate content. (C) Concentration of hepatic sphingomyelins normalized to the phosphate content. (D) Schematic representation of biochemical pathways leading to ceramide production. (E) Hepatic gene expression of enzymes involved in the ceramide biosynthesis. (F) Total amount of hepatic phospholipids according to their degree of saturation (normalized to the phosphate content). (G) Total amount of hepatic phospholipids according to their chain length (normalized to the phosphate content). Results are expressed as the mean§SEM. n = 5 6 male mice per group. Cer, ceramide, SM, sphingomyelin, SFA, saturated fatty acids, MUFA, monounsaturated fatty acids, PUFA, polyunsaturated fatty acids. Diacyl phospholipids were sorted into those that have no double bonds in their 2 fatty acyl chains combined (SFA), one double bond (MUFA) or 2 6 double bonds. LC: long chains (total sum of 28 34 carbons in both fatty acyl chains), VLC:

very long chains (total sum of 36 44 carbons in both fatty acyl chains). One-way ANOVA: *Pvalue for MCD or MCD/Lira vs Chow<0.05,^#Pvalue for MCD/Lira vs MCD<0.05.

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