Identification of the molecular interplay between dietary fatty acids and gut microbiota in Non Alcoholic Fatty Liver Disease
|Partner Organization||Partner Country|
|University of Bari||Italy|
1. Overall project description
Obesity promotes Non Alcoholic Fatty Liver Diseases (NAFLD) through mechanisms involving the gut microbiota. From 2 cohorts of obese women (FLORINASH FP7), in which a multilevel omics approach was used, we identified a microbiome architecture indicating increased dietary lipids processing. This was associated with hepatic insulin signaling perturbations, a transcriptomic profile indicative of NAFLD and triglyceride accumulation. From two other cohorts (MICIMAB & ROLIVER), transcriptomic analyses from intestinal biopsies and 16S sequencing of liver samples suggested: 1. impaired intestinal defense favoring translocation of bacteria towards the liver, inflammation and lipid deposition and 2. impaired intestinal and liver lipid metabolism. The interaction between dietary lipids and the gut microbiota in the etiology of NAFLD is unknown and could impair intestinal defense and lipid handling. Using original and complementary models of genetically modified mice and germ-free mice colonized with human microbiota, we will study the impact of different lipid-enriched diets on: 1. gut microbiota and its causal role in liver disease; 2. intestinal immune- and non-immune defense systems; 3. translocation of bacteria towards the liver responsible for inflammation; 4. lipid handling processes in the liver and the intestine (bile acids/FXR and fatty acids/PPAR); 5. gender by studying the role of the estrogen receptor (ER) in the gut microbiota/dietary lipids interplay.
Based on our first sets of experiments we have analyzed the host glucose and lipid metabolism including glucose- and insulin tolerance tests, MRI and gut transit time weight and plasma biochemistry. we also have performed liver and adipose tissue inflammation and metabolism: characterized using histology, biochemistry, immunohistochemistry,; and characterized the inflammation of tissue by qPCR. Briefly, Liver histology quantifying inflammation, steatosis and fibrosis, TG, Cholesterol esters and cholesterol and qPCR analysis of liver reporter genes for inflammation, lipogenesis have been run.
We have identified 2 fat-enriched diets out of 7 which control in the opposite way the liver lipid storage. The diets used are rich in saturated fatty acids (palm oil), saturated/mono-unsaturated fatty acids (lard), mono-unsaturated fatty acids (oleic sunflower oil), omega-6 poly-unsaturated fatty acids (linoleic sunflower oil), omega-3 poly-unsaturated fatty acids (fish oil), saturated long- and medium chain fatty acids (milk fat) or medium-length fatty acids (man-made formula), respectively (7 fat-modified diets +1 control diet).
Although the diets have similar amount of proteins, carbohydrate and energy content, the liver lipid are dramatically accumulating for one while not accumulating for the other. in addition, we have identified the associated gut microbiota which could be controling the differential impact of both diets. eventually, lipidomics revealed that the bile acids could be at play in the differential phenotypes. we have run the experiments in males and females.
We have analyzed the potential mechanisms associated with the differential liver lipid storage. The caecum has been analysed for SCFA, bile acids, and fatty acids. We have analysed diet and caecum content for soluble and insoluble fatty acid salts. We did not yet perform liver lipidomics but only TG/ChoE and Cholesterol in the liver and the gallbladder bile.
we have analyzed the role of the intestinal, and liver immune systems. the data are currently being analyzed.
we have collected liver, gut, and adipose tissue samples to analyze the corresponding microbiota and study the bacterial translocation process potentially at play on the control of liver lipid accumulation. the data should be obtained before summer.
In the WP3 we have initiated some experiments to demonstrate causality using PPARa/g, ERa; FXR ko mice fed the above selected diets. the results are on going. In vivo experiments with Alb-Cre is on-going while the in vivo part of the Villin-Cre is finished but downstream analyzes still remains to be performed. Metabolic in vivo characterization similar to what is outlined above (GTT, fating glucose, insulin, liver fat etc) is being performed.
Regarding the role of PPARg: We used High Fat Diet (HFD) feeding as a model of obesity in C57BL/6 J male Wild-Type mice (WT), in whole-body Pparα- deficient mice (Pparα−/−) and in mice lacking Pparα only in hepatocytes (Pparαhep−/−). We provide evidence that Pparα deletion in hepatocytes promotes NAFLD and liver inflammation in mice fed a HFD. This enhanced NAFLD susceptibility occurs without development of glucose intolerance. Moreover, our data reveal that non-hepatocytic PPARα activity predominantly contributes to the metabolic response to HFD.Glucose and lipid homeostasis was analyzed, as well as liver histology, transcriptomics, lipidomics and 1H-NMR based metabolomics. No study of gut microbiota was performed in these mice. In addition, we used male and female WT and mice lacking Pparα only in hepatocytes (Pparαhep−/−) treated with several dietary models of steatosis (high-fat diet, choline-deficient high-fat diet and western-diet). Extensive hepatic phenotyping was performed (histology, transcriptomics, 1H-NMR based metabolomics, lipidomics). Upon all dietary challenges, we recapitulate the male-specific hepatic alterations (triglyceride accumulation, inflammation, fibrosis…) and female-specific protection, as observed in NAFLD pathology in humans. We show that hepatic PPARa pathway is involved in these sex-specific features and confirm the involvment of PPARa in the etiology of NAFLD in mens using a human cohort of male and female NAFLD patients.
Regarding the role of FXR: In order to evaluate the role of nuclear receptor FXR in diet-induced NAFLD and gut microbiota composition, we performed in vivo experiments in both genders. The male and female transgenic mice with selective constitutive intestinal FXR activation (VP16FXR) and their littermate controls (VP16) have been fed with 3 selected diets. In vivo experiment with Reference and Chow diets are ended, but further analyses have to be done. For glucose and lipid metabolism, GTT and ITT have been performed, as well as serum cholesterol, TG, ALT and AST have been measured. For these mice, gut microbiota and bile acid composition are being analysed. Liver histology quantifying inflammation, steatosis and fibrosis and qPCR analysis of liver reporter genes for inflammation, lipogenesis and fibrosis have not yet been performed.
Regarding the role of ERa: we have generarted mice deleted for ERa in the intestinal epithelial cells. the colony is just been raised and the data should be available in the next 6 months
The causality of gut microbiota through conventionalization of germ free mice had to be delayed. However, we have treated conventional mice with antibiotics prior to colonization. the results are being analyzed.
4.1 List of publications
|Authors||Title||Year, Issue, PP||Partners Number||Doi|
|Molinaro A, Koh A, Wu H, Schoeler M, Faggi I, Carreras A, Hallén A, Bäckhed F, Caesar R.||Hepatic expression of Lipopolysaccharide Binding Protein (Lbp) is induced by the gut microbiota through Myd88 and impairs glucose tolerance in mice independent of obesity||:100997. doi: 10.1016/j.molmet.2020.100997|
|Cariello M, Contursi A, Gadaleta RM, Piccinin E, De Santis S, Piglionica M, Spaziante AF, Sabbà C, Villani G, Moschetta A||Extra-Virgin Olive Oil from Apulian Cultivars and Intestinal Inflammation||10.3390/nu12041084|
|Gadaleta RM, Garcia-Irigoyen O, Cariello M, Scialpi N, Peres C, Vetrano S, Fiorino G, Danese S, Ko B, Luo J, Porru E, Roda A, Sabbà C, Moschetta A||Fibroblast Growth Factor 19 modulates intestinal microbiota and inflammation in presence of Farnesoid X Receptor||10.1016/j.ebiom.2020.102719|
|Piccinin E, Cariello M, De Santis S, Ducheix S, Sabbà C, Ntambi JM, Moschetta A||Role of Oleic Acid in the Gut-Liver Axis: From Diet to the Regulation of Its Synthesis via Stearoyl-CoA Desaturase 1 (SCD1).||10.3390/nu11102283|
|Piccinin E, Ducheix S, Peres C, Arconzo M, Vegliante MC, Ferretta A, Bellafante E, Villani G, Moschetta A||PGC-1beta induces susceptibility to acetaminophen-driven acute liver failure||10.1038/s41598-019-53015-6.|
|De Santis S, Cariello M, Piccinin E, Sabbà C, Moschetta A||Extra Virgin Olive Oil: Lesson from Nutrigenomics||10.3390/nu11092085|
|Olsson LM, Poitou C, Tremaroli V, Coupaye M, Aron-Wisnewsky J, Bäckhed F, Clément K, Caesar R.||Gut microbiota of obese subjects with Prader-Willi syndrome is linked to metabolic health||doi: 10.1136/gutjnl-2019-319322|
4.2 Presentation of the project
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4.3 List of submitted patents and other outputs
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