1. The Alterations on Gut Microbiota and its Effects on Obesity
Nikki Huynh
Bioinformatics: Sequence Analysis
20 November 2017

2. Objectives
Focus on how gut microbiota can be modulated by dietary intervention + prebiotics (using high generation sequencing)
First paper: Dietary intervention and identifying metabolic characteristics of LGC (low gene richness) and HGC (high gene richness)
Second Paper: How Ganoderma Lucidum alters the gut microbiota to prevent obesity

3. Dietary Intervention impact on gut microbial gene richness
Aure´lie Cotillard1,2*, Sean P. Kennedy3*, Ling Chun Kong1,2,4*, Edi Prifti1,2,3*, Nicolas Pons3*, Emmanuelle Le Chatelier3,
Mathieu Almeida3, Benoit Quinquis3, Florence Levenez3,5, Nathalie Galleron3, Sophie Gougis4, Salwa Rizkalla1,2,4,
Jean-Michel Batto3,5, Pierre Renault5, ANR MicroObes consortium{, Joel Dore´3,5, Jean-Daniel Zucker1,2,6, Karine Cle´ment1,2,4
& Stanislav Dusko Ehrlich3

4. Introduction
Complex gene-environment interactions are important in the development of obesity
Collective genome of microorganisms in our body, microbiome, may have major role in the pathogenesis of obesity by environmental factors
Composition of gut microbiota and changes in dietary composition
Study on dietary intervention on gut microbial gene richness

5. Methods Clinical Investigation 38 obese and 11 overweight subjects
No antibiotics/drugs
(Weight-induced Loss) First 6-weeks: Energy-restricted high protein diet (1200 kcal per..
(Weight Stabilization) Second 6-weeks: 20% Increase in total energy intake, above their resting energy metabolic rate
0,6,12 weeks: blood and fecal samples
Adipose tissue samples
7-day dietary record and interview with dietician

6. Metagenomic Sequencing
ABI SOLiD sequencing technology on fecal sample
Retained an average of 14.2 million ± 8.1 million uniqued mapped reads/individuals
Abundance of each gene: # of reads that are uniquely mapped to a gene/nucleotide length
Frequencies: Abundance of each gene/total # of uniquely mapped reads for a given sample

7. Gut microbial composition of LGC and HGC subjects

8. Differences between LGC and HGC subjects in bioclinical variables.

9. Gene richness of LGC and HGC groups during the intervention

10. Analysis Gut Microbial Composition at Baseline
At a threshold of 480,000 genes…
LGC harboring 379,436 genes
HGC harboring 561,499 genes
Phenotypes at Baseline
Individuals of LGC present phenotypes that expose them to an increased risk of obesity-associated co-morbidities
LGC seemed to consume less fruits/veggies and less fishery products than HGC
Bacterial Species
Identified 6,230 genes different (P < )
Vast majority of these genes (3,966; 89%) were found in only 18 clusters
Relative abundance of the 18 clusters was computed as a mean frequency of the 25 tracer genes for each cluster
All more abundant among HGC individuals

11. Conclusion
Gene richness increased significantly and remained the same after stabilization phase in LGC group
Gene richness had no significant change in HGC
Low gene richness associated with increased adipose tissue (6 weeks) and increased systemic inflammation (12 weeks)
Inflammation refractory to dietary intervention
Gene richness may therefore help to predict the efficacy of dietary intervention on inflammatory variables in overweight/obese individuals

12. Ganoderma lucidum reduces obesity in mice by modulating the composition of the gut microbiota
Chih-Jung Chang1,2,3,4,5,*, Chuan-Sheng Lin1,2,3,5,*, Chia- Chen Lu6, Jan Martel1, Yun-Fei Ko7,8, David M. Ojcius1,9,
Shun-Fu Tseng5, Tsung-Ru Wu2,3, Yi-Yuan Margaret Chen4, John D. Young1,7,8,10 & Hsin-Chih Lai1,2,3,5

13. Obesity Closely linked with chronic, low-grade inflammation:
Insulin resistance
Type-2 diabetes
Fatty liver disease
Cardiovascular disease
Gut dysbiosis and metabolic endotoxemia
Gut microbiota of obese humans: decreased levels of Bacteroides + increased levels of Firmicutes
-Chronic inflammation + obesity-related disorders: gut dysbiosis and metabolic endotoxemia
-Metabolic endotoxemia: increased levels of LPS in the blood
-Recent study indicates that changes in the composition of the gut microbiota are associated with the development of obesity

14. Gut Microbiota
Trillions of bacteria = nutrient acquisition and energy regulations
Associated with the development of obesity + its metabolic disorders
Increased ratio of Firmicutes/Bacteroidetes + other Obesity
Obesity-induced gut dysbiosis impairs intestinal integrity
Release of the endotoxin lipopolysaccharide (LPS) blood stream  inflammation and insulin resistance
- Gut dysbiosis: Leads to release of the endotoxin lipopolysaccharide (LPS) from intestinal Gram-Neg. bacteria into blood stream, leading to inflammation and insulin resistance
due to stimulation of Toll-like receptor 4 (TLR4)-mediated inflammation2

15. Ganoderma Lucidum
Chinese Medicinal mushrooms used to promote health and longevity
High molecular weight of polysaccharides (>300kDA)
Triterpenes and polysaccharides inhibit adipocyte differentiation + produce hypoglycaemia
Induce antidiabetic and antioxidant activities
Effect on gut microbiota, inflammation, and obesity: unknown
High molecular weight may the active components of WEGL

16. WEGL decreases pro-inflammatory cytokine expression in the liver and adipose tissues of HFD-fed mice
-Relative expression of TNF-a (a), IL-1b (b), IL-6 (c), IL-10 (d) and PAI-1 (e) in hepatic and adipose tissues was assessed using qRT–PCR and in comparison
with the Chow group
-Graph bars with different letters on top represent statistically significant results (Po0.05), bars with the same letter correspond to results that show no statistically significant differences.

17. WEGL reduces serum LPS and TLR4-related signaling pathways in HFD mice
-Effects of WEGL treatment on serum endotoxin
TLR4 protein protein production
JNK phosphorylation
DE) lkB-alpha production were examined in the liver and epididymal adipose tissues of chow- and HFD-fed mice using limulus amebocyte lysate assay kit (immunoblots)

18. WEGL alters microbiota composition in HFD-fed mice
Microbiota compoisition in faeces of chow-fed mice treated with/without 8% WEGL and HFD mice treated with 2,4,8 % WEGL wre anaylsed using next generation sequencing
Plots were generated
Multivariate analysis of variance from PCoA matrix scores
Bacterial taxonomic profiling in the phylum level of intestinal bacteria from different mouse groups

19. d) Heatmap showing the abundance of 91 OTUs significantly altered by WEGL in HFD-fed mice based on RDA
e)Represented bacterial taxa information (species, genus, family and phylum) of 91 OTUs

20. Analysis of gut microbiota following faecal transplantation
Faecal transplantation from Chow-, HFD-, and Chow/HFD + 8% WEGL fed mice was performed and relevant microbiota anaylsis was done
Plots shown were generated
Multivariate analysis of variance from PCoA matrix scores
Bacterial taxonomic analysis of intestinal bacterium from each mouse groups

21. D) Heatmap showing the abundance of 155 OTUs significantly altered by Chow, Chow + 8% WEGL and HFD + 8% transplanted mice based on RDA analysis
Represented bacterial taxa information
White circles and black diamonds indicate the OTUs that increased or decreased compared with HFD recipient mice

22. Results
Anti-obesity effects of WEGL transferrable through faecal transplantation
Major bioactive ingredients: High molecular weight polysaccharides
Alters and produce specific gut microbiota
Altering the Firmicutes: Bacteroidetes ratio
Decreases TLR4 signaling
Reduces endotoxemia
Regulate expression of lipogenic genes
Affect pro-inflammatory signaling pathways (TLR2)
-Decreased several inflammation bacterial species
-TLR2: controls adiposity and insulin resistance
-Reduces expression of genes involved in fatty acid synthesis

23. References
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