1. Gut Microbial triggers that influence the obesity associated phenotype
Samodha Fernando Department of Animal Science University of Nebraska-Lincoln

2. Gut Microbes
Kinross et al. Genome Medicine :14  

3. Influence of gut microbiome on obesity
“Two groups of beneficial bacteria are dominant in the human gut, the Bacteroidetes and the Firmicutes. Here we show that the relative proportion of Bacteroidetes is decreased in obese people by comparison with lean people, and that this proportion increases with weight loss on two types of low-calorie diet.” (Nature, 2006; 444, )

4. Influence of gut microbiome on obesity
Metabolic pathway-based clustering and analysis of the human gut microbiome of MZ twins
Microbiome of 154 individuals was studies
based on:
- 9,920 near full-length and 1,937,461 partial
bacterial 16S rRNA sequences,
gigabases shotgun data (454/Roche).
The human gut microbiome is shared among family
members, but each person’s gut microbial community
varies in the specific bacterial lineages present, with a
comparable degree of co-variation between adult
monozygotic and dizygotic twin pairs.
There is an extensive identifiable ‘core microbiome’
at the gene, rather than at the organismal lineage
level.
Obesity is associated with phylum-level changes in the microbiota, reduced bacterial diversity, and altered representation of bacterial genes and metabolic pathways.
There is a core microbiome at a functional level, deviations from this core are associated with different physiologic states (obese versus lean).
Turnbaugh et al, Nature 2009

5. Diet-Induced Obesity Is Linked to Marked but Reversible Alterations in the Mouse Distal Gut Microbiome
- 48 Mb of high quality sequence data
Conventionalized mice fed a low-fat, high polysaccharide (CHO) or high-fat/high-sugar (Western) diet.
The relative abundance of Firmicutes and Bacteroidetes divisions in the distal gut (cecal) microbiota.
Diet-induced obesity (DIO) is associated with a marked reduction in the overall diversity.
DIO is linked to a bloom of the Mollicutes class of bacteria within the Firmicutes division.
Microbiota transplantation experiments reveal that the DIO community has an increased capacity to promote host fat deposition.
Turnbaugh et al, Cell Host and Microbe 2008

6. Significance
Studies have helped understand the changes in microbial community structure, and the metabolic potential in obese and lean phenotypes.
Failed to illuminate the signals of the gut microbiome that ameliorate the obesity phenotype.
Large individual-to-individual variation in the human gut flora
Genotypic variations
Environmental exposure (current or historical)
Inability to control or monitor dietary and caloric intake
Consequently, previous studies have failed to identify microbial regulation of host genes that promote deposition of lipids in adipocytes.
In addition, how diversity relates to the collective function of the microbiome and its host remains obscure.  

7. Significance
These factors complicate efforts to understand how the human microbiome, its population structure and function, interacts and affects human pathophysiology 
Partly due to the lack of a good model system.
Cecum cannulated humanized pig model allowing direct sampling from the gut providing new opportunities to understand microbe-microbe interactions, host-microbe interactions and microbial triggers using meta-transcriptomics.

8. Specific Aims
Validate a cecum cannulated humanized pig model with capabilities to perform continuous real-time monitoring to evaluate gut microbial community dynamics and microbial gene expression.
First animal model that will allow continuous real-time monitoring of the human microbiome
Glimpse into the workings of the human microbiome
Identify signals of the gut microbiome in high fat, high carbohydrate diets that ameliorate the obesity phenotype, specifically, microbial genes that regulate adipogenesis.
New targets and strategies to control obesity
Identify the therapeutic potential of the human gut microbiome towards controlling obesity and type 2 diabetes

9. Innovation
First ever model that allows continuous, real-time sampling from the gut to perform functional studies
Opportunity to bypass the small intestines and directly add substrates and drugs to the large intestines to investigate the influence on the gut microbiota
Groundbreaking in its potential to revolutionize the way we study the human gut microbiome
A “core” microbial community is yet to be identified
A “core set of genes and gene families representing key metabolic functions has been discovered
microbial gene expression and its influence on host gene expression
Turnbaugh et al, Nature, 2007

10. Approach
To identify the influence of the gut microbiota in obesity and obesity related diseases, it is essential to understand the interaction between diet and the microbiome.
Microbial metabolism of dietary molecules (nutrients) in the gut drives the release of bioactive compounds (including lipid metabolites and short chain fatty acids), which interacts with host cellular targets to control energy metabolism
To assess the relevance of the gut microbiome to obesity, it is important to understand how the gut microbiome interacts with the host, with respect to metabolic response to the diet.
The microbial metabolites produced under different dietary conditions, influence interactions between different gut microbial communities (in situ effects).
These metabolites also have systemic effects on host tissues by acting as metabolic regulators.
Ex. Diet – Microbes – SCFA – pH – gut microbial ecosystem
SCFA – monocarboxylate transporters – metabolic substrates or regulators

11. SCFA as metabolic regulators
SCFA produced by the gut microbes has different metabolic features
Acetate – fatty acid or cholesterol precursor
Butyrate – energy substrates for colonocytes
Propionate – Gluconeogenic substrate in the liver
Ligands for G-protein coupled receptors (GPR 43 & 41 and fatty acid receptor 1 & 2) – adipose tissue expansion
Can play a role in regulating host metabolism.
Ex. Conjugated linoleic acids (CLAs), bile acids, gases such as methane and hydrogen sulfide, and polysaccharides such as, lipopolysaccharide (LPS), and peptidoglycan can bind to specific receptors in the host and change gene expression and metabolic activity of the host.
LPS is found in higher levels in the serum of obese individuals, can create metabolic endotoxemia that stimulates insulin resistance, obesity and systemic inflammation.
CLAs are beneficial.

12. SCFA as metabolic regulators
Influence of the gut microbiome on adipogenesis. Different dietary molecules can result in changes in the gut microbial flora. The fermentation of carbohydrates and other nutrients by the gut microbiota on a high fat/high carbohydrate diet can result in an increase SCFA concentration and can result in increased absorption. The SCFA absorbed can promote fat storage via activation of GPR43 and 41 receptors. The presence of gut microbiota can suppress the intestinal synthesis of FIAF (fasting-induced adipose factor), and influence the activity of lipoprotein lipase (LPL) and the fat storage in the adipose tissue. In addition, hepatic and muscle fatty acid oxidation can be influenced/altered by the gut microbiota via the 5’ adenosinmonophosphate-activated protein kinase (AMPK)-dependent mechanism. Finally, low grade inflammation and insulin resistance observed in obesity can be triggered by alteration of the gut barrier (namely, by a decrease in tight junction proteins (ZO-I and Occludin) by activating the endocannabinoid (eCB) system tone, leading to higher plasma lipopolysaccharide levels (LPS). These events contribute to fat deposition mainly in high fat diets.

13. Gut Microbial Stimulation
Gut microbial stimuli and transcriptional control of adipogenesis
Gut Microbial Stimulation
The proposed influence of the gut microbial stimuli on transcriptional control of adipogenesis (modified from Rosen et al. Genes and Dev., 2000) The transcription proteins are expressed in a network in which microbial gene expression or metabolites (SCFAs) can activate, C/EBPβ and C/EBPδ, followed by PPARγ, directly PPARγ, or C/EBPα. Microbial stimuli can also activate ADD1/SREBP1 followed by PPARγ activation. All of these factors contribute to the expression of genes that lead to the obesity phenotype.

14. Central hypothesis
In the obesity phenotype, signals of the gut microbiome influences host gene expression resulting in increased differentiation of preadipocytes to adipocytes, increasing the susceptibility to type 2 diabetes.
To test the above hypothesis…
A new cannulated humanized pig model will be established and validated.
The influence of high starch and high fat diets on microbial gene expression and the influence of microbial gene expression on host gene expression, specifically on the transcriptional cascade involving the nuclear receptor PPAR, members of the C/EBPs family and basic helix- loop-helix family (ADD1/SREBP1c) that regulates adipogenesis, will be investigated.

15. Specific Aim #1 – Experimental Design
Week 3
Week 4
Week 5
Week 6

16. Specific Aim #1 – Experimental Design
Daily sampling
Week 8
Week 9
Week 10
Week 11
Weekly body weight
Back-fat thickness
Intake
Weekly Biopsy from sub-cutaneous fat and gastrointestinal wall to monitor host responses.

17. Analysis – Gut microbial community
DNA and RNA extraction from donor and recipient fecal and recipient cecal microbiota
Amplification and sequencing of the V1-V3 region of microbial 16S rRNA gene (using 454-pyrosequencing)
At CAGE under the supervision of Dr. Andy Benson (secondary mentor)
Microbial community analysis
Quality filter reads (primer seq. barcode, length, quality)
Daisy chopper – unequal sampling
Taxonomy based analysis
“Classifier”
NCBI-NR

18. Analysis – Gut microbial community
Operational Taxonomic Unit (OUT) based analysis
MOTHUR
Align with SILVA
Pre-cluster – pseudo linkage clustering
UCHIME – chimera
Cluster at 97% to generate OTUs
AMOVA, ACE, Chao1, and rarefaction curves
Weighted UniFrac – clearcut and FastUnifrac
Ordination plots – PCoA and NMDS (non-metric multidimensional scaling)

19. Metagenomic analysis of the Gut microbial community
Metagenome sequencing will be done using 454-pyrosequencing
Metabolic potential and as a scaffold for metatranscriptome analysis
Assembled into contigs via velvet and SOAPdenovo (short oligonucleotide assembly package)
Gene prediction via MATAGENEMark – Bacterial
Fungal gene prediction via Dr. Etsuko Moriyama’s (primary mentor) fungal genome database and CAZy database
MuMer – compare metagenome reads to reference bacterial and fungal genomes
KEGG, COG and NCBI-nr using BLASTX
Blasted to each other
MEGAN

20. Metatranscriptomic analysis of the Gut microbial community
Total RNA
Poly A mRNA
RNA (18S,16S, 23S, 28S, 5S) +
Bacterial mRNA
Bacterial total RNA
(16S, 23S, 5S, mRNA)
Bacterial mRNA & 5S RNA
Identify responses of the
fungi, and protozoa
in the gut
Metatranscriptome analysis
Metatranscriptome sequencing will be done using Illumina
Metagenome as a scaffold - BFAST
Similar to metagenome analysis

21. Host responses Transcriptomic analysis of biopsy samples
Total RNA – poly A Isolation
High throughput Illumina sequencing
Align to genome (NCBI build 3.1, based on Sscrofa 10) for annotation
Quantitative real-time PCR (qPCR) analysis
Quantify gene expression of transcription factors C/EBP, C/EBP, C/EBP, PPAR, and C/EBP, GPR43 and 41
SCFA analysis
Statistical analysis – Dr. Steve Kachman (secondary mentor)

22. Expected results and interpretations
Differences in microbial species composition in lean and obese individuals and recipient animals
Differences in SCFA composition with high lactic acid levels under high carbohydrate dietary condition
Differential gene expression is expected, with more carbohydrate utilization genes under high carbohydrate diet
Host gene expression in the gut wall and sub cutaneous tissue are also expected to change – genes regulating adipogenesis is expected to be upregulated in obese recipient pigs

23. Potential problems
Cecum cannulations – None expected (Dr. Doug Hostetler)
How sampling will effect anaerobic environment?
Quick sampling, limited exposure to oxygen, oxygen scavenged by facultatives
Major portion is facultative
Use of cannula in rumen microbiology studies

24. Specific Aim #2 – Experimental Design
High
Low
Low
High
High
Low
NO TRANSPLANTATION

25. Analysis
Microbial community analysis, metagenomics and meta transcriptomic analysis performed as described in Aim #1
To identify differentially expressed genes that influence obesity
Critical to understand origin of observed changes
Independently estimate transcript abundance and genome abundance
Expression level of a gene;
o(gene,genome) = e(gene,genome) * a(genome)
(observable gene expression o(gene,genome) is a product of two factors: the gene expression level within the host genome, e(gene,genome); and the abundance of that genome in the environment, a(genome))
Host transcriptomics, realtime PCR analysis and SCFA profiles will be monitored as before.

26. Expected results and interpretations
Pinpoint specific genes and pathways that are influenced by microbial gene expression
Dissect the influence of diet and microbiome
Obese microbiota on high fat/ high carb diet will gain more than lean microbiota on High fat/ high carb diet
Down-regulation of host adipogenic genes under above conditions
Obese microbiota on low fat/ low carb diet will gain more than lean microbiota on low fat/ low carb diet
Up-regulation of host adipogenic genes under above conditions
Obese pigs that receive lean microbiota and the low fat/low carb diet is expected to reduce fat deposition and down-regulate adipogenic genes
Lean pigs that receive obese microbiota and the high fat/high carb diet is expected to increase fat deposition and up-regulate adipogenic genes

27. Summary and future directions
Potential problems
Removing a majority of the cecum microbiota during transplantation
Stirpump will be used to rapidly remove cecum contents
Summary and future directions
Powerful tool to gain insight into the workings of the human microbiome and identify microbial genes and populations that influence obesity
Provide a glimpse into the metabolic and molecular interactions that occur between diet, host and its gut microflora.
Expand the model to study other metabolic diorders ( metabolic syndrome, Inflammatory bowel disease etc.)

28. Use of core facilities Computational and data sharing core
Computational analysis
Storage
Platform for collaboration and data sharing
Core for applied Genomics and Ecology (CAGE)
Fully equipped for molecular biology and genome-based studies
All 454-pyrosequencing will be performed in this facility
CAGE Director Dr. Andy Benson (secondary mentor)
Data analysis pipelines for quality filtering and to perform taxonomy and phylogeny based analysis.