Gut-Resident Microbes Modulate Hepatic Metabolism and Susceptibility to Disease Open Access

Abstract

The microbes that reside within the intestine are powerful modifiers of host fitness and susceptibility to disease. In the past 20 years, advances in 16S sequencing have allowed for a more thorough investigation of the community architecture of the gut microbiome. Germ-free or antibiotic treated model organisms, coupled with metabolomics, proteomics, and metagenomics, have provided some insight into the functional consequences of perturbations within these communities. While significant progress has been made, the vast majority of these studies have scrutinized the effects of the microbiome within the context of host intestinal health. Recent research, however, has identified profound impacts of the microbiome on organs distal to the gut, with implications for cardiovascular, immunological, and even neurological health. However, the pace of phenotypic characterization has outstripped that of mechanistic understanding. In the studies that follow, we pair big data approaches (small molecule metabolomics and RNA sequencing) with validation in experimentally tractable systems (cell lines, transgenic mice, and Drosophila) to assess the effects and underlying molecular mechanisms of the gut microbiome on hepatic metabolism and susceptibility to disease. We identify a microbiome-derived small molecule, δ-valerobetaine, that is absorbed systemically and acts to lower carnitine levels and inhibit fatty acid oxidation. We characterize the effect of microbial colonization on hepatic signaling, and observe that the master regulator of the cellular antioxidant response Nrf2 is tonically activated by the presence of a microbiome. Furthermore, we narrow down the contributing microbial genera, and demonstrate that exogenous administration of Lactobacilli can augment this signaling in conventional mice with consequences for oxidative liver injury. Finally, we develop and validate a novel model of oxidative liver injury in Drosophila. These studies together contribute some mechanistic understanding to our burgeoning appreciation of the systemic effects of the microbiome.

Table of Contents

THE MICROBIOME-LIVER AXIS IN HEALTH AND DISEASE ................................................................... 1

1.1 INTRODUCTION ....................................................................................................................................... 2

1.2 STRUCTURE AND FUNCTION OF THE GUT MICROBIOME ............................................................................ 2

1.3 SYSTEMIC EFFECTS OF THE MICROBIOME ............................................................................................... 3

1.4 THE PORTAL CIRCULATION ..................................................................................................................... 4

1.5 DETOXIFICATION ACTIVITIES OF THE LIVER .............................................................................................. 5

1.6 LIVER DISEASE AND THE MICROBIOME .................................................................................................... 7

1.7 THE MICROBIOME AS A THERAPEUTIC MODALITY ..................................................................................... 8

1.8 PROBIOTICS AND LIVER DISEASE .......................................................................................................... 11 1.9 DROSOPHILA AS A MODEL FOR THE MICROBIOME-LIVER AXIS ................................................................ 12 1.10 CONCLUSION ..................................................................................................................................... 14 1.11 REFERENCES ..................................................................................................................................... 17

THE MICROBIOME-DERIVED METABOLITE Δ-VALEROBETAINE INHIBITS HEPATIC MITOCHONDRIAL FATTY ACID OXIDATION AND EXACERBATES HEPATIC STEATOSIS. .............. 30

2.1 INTRODUCTION ............................................................................................................................... 32

2.2 RESULTS .......................................................................................................................................... 34

2.2.1 Identification of the microbiome-derived mitochondrial metabolite δ-valerobetaine (VB) ......... 34

2.2.2 The gut microbiome produces VB.............................................................................................. 37

2.2.3 δ-valerobetaine (VB) decreases cellular carnitine in cultured HepG2 cells............................... 39

2.2.4 δ-valerobetaine (VB) decreases palmitate-dependent mitochondrial respiration in HepG2 cells ............................................................................................................................................................. 41

2.2.5 VB decreases formation of labeled acetyl-CoA production from labeled palmitic acid ............. 41

2.2.6 VB decreases carnitine and decreases mitochondrial fatty acid oxidation in conventional mice ............................................................................................................................................................. 44

2.2.7 VB is associated with the severity of hepatic steatosis in adolescents and with visceral adiposity in adults ................................................................................................................................ 48

2.3 DISCUSSION..................................................................................................................................... 50

2.4 METHODS ......................................................................................................................................... 53

2.5 REFERENCES .................................................................................................................................. 62

A DROSOPHILA MODEL OF ACETAMINOPHEN TOXICITY .................................................................. 71

3.1 INTRODUCTION ............................................................................................................................... 73

3.2 RESULTS .......................................................................................................................................... 75

3.2.1 Acetaminophen Accumulates in Drosophila. ............................................................................. 75

3.2.2 Acetaminophen exposure results in dose-dependent mortality in WT Drosophila. ................... 77

3.2.3 APAP Administration Increases ROS in the Drosophila Fat Body and Depletes Systemic Glutathione. ......................................................................................................................................... 78

3.2.4 Fat body-specific depletion of Cyp18a1 attenuates APAP toxicity. ........................................... 80

3.2.5 Genetic and pharmacologic modifiers of APAP toxicity in Drosophila. ..................................... 81

3.2.6 The microbiome protects against acetaminophen toxicity in Drosophila................................... 83

3.2.7 Effects of age on APAP-induced mortality ................................................................................. 84

3.3 DISCUSSION..................................................................................................................................... 86

3.4 METHODS ......................................................................................................................................... 88

3.5 REFERENCES .................................................................................................................................. 91

GUT-RESIDENT LACTOBACILLI ACTIVATE HEPATIC NRF2 AND PROTECT AGAINST OXIDATIVE LIVER INJURY ............................................................................................................................................ 95

4.1 INTRODUCTION ............................................................................................................................... 97

4.2 RESULTS ........................................................................................................................................ 100

4.2.1 Conventionalization of germ-free mice alters the hepatic metabolome................................... 100

xi

4.2.2 Conventionalization of germ-free mice induces the hepatic Nrf2 antioxidant response pathway ........................................................................................................................................................... 102

4.2.3 Symbiotic Lactobacilli sp. activate the Nrf2 pathway in the Drosophila melanogaster fat body ........................................................................................................................................................... 104

4.2.4 Oral administration of Lactobacillus rhamnosus GG activates Nrf2 signaling in the murine liver ........................................................................................................................................................... 106

4.2.5 Lactobacillus rhamnosus GG protects the Drosophila fat body against toxicity in a Nrf2-dependent manner ............................................................................................................................ 108

4.2.6 Lactobacillus rhamnosus GG administration attenuates acetaminophen hepatotoxicity in conventional mice.............................................................................................................................. 111

4.2.7 Hepatic Nrf2 mediates Lactobacillus rhamnosus GG protection against acetaminophen hepatotoxicity .................................................................................................................................... 114

4.2.8 Hepatic Nrf2 mediates Lactobacillus rhamnosus GG protection against acute alcohol induced injury .................................................................................................................................................. 116

4.2.9 Lactobacillus rhamnosus GG alters the small molecule milieu of portal serum. ..................... 118

4.2.10 Lactobacillus rhamnosus GG mediated generation of 5-methoxyindoleacetic acid activates hepatic Nrf2 ....................................................................................................................................... 119

4.2.11 Lactobacilli produce 5-MIAA in vitro....................................................................................... 122

4.3 DISCUSSION................................................................................................................................... 124

4.4 METHODS ....................................................................................................................................... 128

4.5 REFERENCES ................................................................................................................................ 135

DISCUSSION AND FUTURE DIRECTIONS ............................................................................................. 141

5.1 DISCUSSION................................................................................................................................... 142

5.2 FUTURE DIRECTIONS ................................................................................................................... 144

5.3 REFERENCES ................................................................................................................................ 148

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Cell
Keyword	Liver Microbiome Metabolism
Committee Chair / Thesis Advisor	Andrew Neish, Emory University
Committee Members	Paul Dawson, Emory University Michael Koval, Emory University Ken Moberg, Emory University Eric Ortlund, Emory University

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