This document discusses the role of gut microbiota and dysbiosis in non-alcoholic fatty liver disease (NAFLD). It notes that dysbiosis, or an imbalance in gut bacteria, can lead to increased production of short-chain fatty acids which promote hepatic lipogenesis and gluconeogenesis. Dysbiosis also increases intestinal permeability and bacterial translocation, activating toll-like receptors and causing inflammation in the liver through cytokines and chemokines. Other effects include decreased levels of the fasting-induced adipose factor, increased lipoprotein lipase activity, and impaired choline and bile acid metabolism. Modulating the gut microbiota through prebiotics, probiotics, or fecal microbiota transplantation may be a potential

1. M O H A M E D N A G U I B , M D
A S S O C I A T E P R O F E S S O R O F I N T . M E D .
H E P A T O G A S T R O E N T E R O L O G I S T
C A I R O U N I V E R S I T Y
Dysbiosis:
The new face of NAFLD

2. Agenda ….
 Intro ..
 Hot Topic
 Microbiota and dysbiosis
 NAFLD and Obesity
 Microbiota composition in Obesity
 NAFLD and microbiota
 Could it be the new therapy?
 In a capsule …

3. Intro …
 Human gut consists of a large number of commensal
microorganisms, collectively known as “intestinal
microbiota”, which are essential for:
 Integrity of the mucosal barrier function
 Absorption of nutrients
 Energy homeostasis.
 Recent evidence suggests that enteric microbiota may
play a significant role in the development of obesity and
its complications.
 Obesity, insulin resistance and dyslipidemia, are co-
morbidities often associated to the presence of NAFLD

4. Intro …
 Nonalcoholic fatty liver disease (NAFLD) describes a
condition caused by a deposition of fat within the
liver cells in the absence of alcohol consumption.
 It is the commonest hepatic disease worldwide (16-
30% of the whole population  50-90% among
obese).
 Wide disease spectrum: from simple steatosis 
nonalcoholic steatohepatitis (NASH) 
Steatocirrhosis (37% of patients).

5. Hot topic …

6. Microbiota
 Microbiota: The microorganisms that typically inhabit a
bodily organ or part.
 The human intestine contains a very crowded and
heterogeneus microbial system, consisting at least 100 trillion
(1014) microbial cells weighing about 1.5 kg and composed of
more than 2000 species
 Gut microbiota should not be considered as an amalgam of
microbes, but as a real organ that interplays with human
beings.
 This complex community contains taxa from bacteria,
eukaryotes, viruses, and at least one archaeon, that interact
with one another and with the host, involving regulation of
local/systemic immunity, metabolic and trophic functions

7.  Gut microbiota has evolved with humans as a
mutualistic partner; however, changes in the
composition of the gut microbiota (dysbiosis) have
been found to be related with several clinical
conditions
 Dysbiosis: The condition that results when the
natural flora of the gut are thrown out of balance

8. Beneficial/
Commensal
Harmful/
Opportunistic
 Gut permeability, Endostoxaemia; Septicemia,
Systemic inflammation, Insulin resistance
Gut Dysbiosis
High-fat/High-sugar diets, over-nutrition, sedentary
lifestyle, antibiotic abuse

9. Could Microbiota Dysbiosis Be the Cause?
IBD/IBS/UC/CD
Diarrhea/Constipation/
Celiac disease/Gastroenteritis
Colorectal Cancer
Endocrine imbalance
Hypertension
NAFLD/NASH
Cardiovacular diseases
Metabolic
Syndrome
Endotoxaemia
septicaemia
Systemic
Inflammation
Diabetes/
Insulin resistance
Obesity
Asthma
Rheumatoid
arthritis

10. NAFLD and Obesity

11. NAFLD and Obesity
 Frequent disease, 16–30% of the general population,
depending on the assessment method.
 Its prevalence rises in parallel with the worldwide
epidemic of obesity and metabolic diseases, reaching
50–90% of obese, more than one-third present with
overt NASH.
 The diagnosis of NAFLD is made on histological
findings (goldstandard) to non-invasive methods like
scoring system, MRI, Fibromax®,…..etc

12. NAFLD and Obesity
 Pathogenesis of NAFLD, a ‘two-hit’
mechanism.
 The first hit consists in hepatocyte lipid
accumulation mainly due to obesity and
insulin resistance.
 Then a second hit, occurring in some
patients only, plays a role in the shift from
steatosis to NASH.

13. NAFLD and Obesity
 Pathogenesis of NAFLD, a ‘two-hit’
mechanism.
 The first hit consists in hepatocyte lipid
accumulation mainly due to obesity and
insulin resistance.
 Then a second hit, occurring in some
patients only, plays a role in the shift from
steatosis to NASH.

14. NAFLD and Obesity
 Several factors have been incriminated in the
“second hit” of NAFLD such as:
 Oxidative stress
 Systemic inflammatory mediators
 Chronic intermittent hypoxia.
 Adipose tissue inflammatory tone (increased macrophage
accumulation in visceral adipose tissue and increased
inflammatory gene expression).

16. NAFLD and Obesity
 More recently, an alternative theory favours the
determinant effect of free fatty acid lipotoxicity in
liver injury.
 Hepatic steatosis is known to develop in the context
of an imbalance of triglycerides afflux in the liver,
which is dramatically increased during obesity and
insulin resistance:
 Increased supply (coming from diet, de novo lipogenesis and
adipose tissue)
 Decreased degradation (via impaired b-oxidation).

18. NAFLD and Obesity
 Focus on Fructose consumption, which is
dramatically increased in the western diet
 Its metabolism induces both de novo hepatic
lipogenesis and reactive oxygen species (ROS)
production  ‘two hits’ of NAFLD.

19. Microbiota
composition in
Obesity

21. Microbiota composition in Obesity
 There are many animal and human studies
investigating the relationship between intestinal
microbiota and obesity or body weight changes.
 Bacterial gene sequences from the distal intestinal
microbiota of genetically obese ob/ob mice, lean ob/+
and wild-type siblings, the investigators found that
genetically obese mice had a 50% reduction in
abundance of Bacteriodetes and a proportional
increase in Firmicutes phyla compared to lean
sibling mice although they were fed with the same
polysaccharide-rich diet.

22. MECHANISMS LINKING THE INTESTINAL
MICROBIOTA AND OBESITY
 Intestinal microbiota and energy harvest
from the diet (caloric salvage).
 Fiaf (fasting-induced adipocyte factor).
 Increased intestinal permeability and
inflammation (LPS-induced chronic
inflammation).
 Releasing of gut hormones (gut-derived
peptide secretion).

23. 1. Intestinal microbiota and energy harvest from the
diet (caloric salvage).
 Metagenomic analyses of the microbiota performed in obese
mice and humans revealed an increased capacity for the
degradation (fermentation) of carbohydrates.
 This microbial fermentation increases the amount of short-
chain fatty acids (SCFAs), such as acetate, propionate,
butyrate, and L-lactate (gluconeogenesis).
 SCFAs are also physiological ligands of G-protein coupled
receptors GPR43 and 41 (also called free fatty acid receptor 2
and 3, respectively), which are expressed in several cell types
(immune cells, endocrine cells, and adipocytes) of host tissues.
 Monosaccharides (microbial fermentation)  portal vein
activate the hepatic carbohydrate response element binding
protein (ChREBP) (lipogenesis).
-Turnbaugh PJ, et al. Nature 2006
-Delzenne NM, Cani PD. Annu Rev Nutr 2011;
- Bjursell M, et al., Am J Physiol Endocrinol Metab 2011

24. 2. Fiaf (fasting-induced adipocyte factor)
 Many studies showed that colonizing germ-free mice
with gut microbiota had led to a decrease in the
intestinal expression of angiopoietin-like factor Ⅳ
[ANGPTL4, also called fasting-induced adipose
factor (Fiaf)], thereby blunting the inhibition of
lipoprotein lipase in the adipose tissue.
--Bäckhed F, et al. Proc Natl Acad Sci USA 2004
-Mandard S, et al., J Biol Chem 2004
-Harris K, et al. J Obes 2012

25. 3. Increased intestinal permeability and
inflammation
 Obesity, diabetes and insulin resistance are associated with a
low grade systemic inflammation.
 Intestinal microorganisms have highly conserved microbial
molecules, called “pathogen- associated molecular patterns”
(PAMPs) and endogeneous products called “damage-
associated molecular patterns” (DAMPs), which are
recognized by pattern recognition receptors  membranous
toll-like receptors (TLRs) and intracellular NOD-like receptors
(NLRs).
 Stimulation of TLRs results in the activation of several
different intracellular signaling cascades including stress-
activated and mitogen activated protein (MAP) kinases, Jun N-
terminal kinases, p38 MAP kinase, interferon regulatory factor
3 and nuclear factor kappa B (NF-κB) pathways.
- Ge H, et al.,. Endocrinology 2008
- Jumpertz R, et al., Am J Clin Nutr 2011

26. 4. Releasing of gut hormones
 Intestinal microbial system regulates entero-
endocrine cells and promotes the release of several
gut hormones e.g: Peptide YY (PYY). (enteroendocrine cell-
derived hormone normally inhibits gut motility).
 Dietary fructooligosaccharides increased the
abundance of Bifidobacterium in the distal intestine,
which led to increased colonic fermentation and
glucagon-like peptide 1 (GLP-1) levels, decreased
serum orexigenic peptide ghrelin and decreased food
intake, fat mass, and hepatic steatosis in rats.
-Samuel BS, et al., Proc Natl Acad Sci USA 2008

29. NAFLD and
microbiota

30. NAFLD and microbiota
 Studies suggest that intestinal microbiota may stimulate
liver steatosis though several mechanisms:
(1) Induction of obesity by harvesting energy from
otherwise indigestible dietary polysaccharides.
(2) Regulation of gut permeability and stimulation of low
grade inflammation.
(3) Modulation of dietary choline metabolism
(4) Regulation of bile acid metabolism
(5) Stimulation of endogenous ethanol production by
enteric bacteria.

31. 1. Induction of obesity by harvesting energy from
otherwise indigestible dietary polysaccharides.
Short-chain fatty acids (SCFAs)
G-protein coupled receptors GPR43
and 41
Hepatic carbohydrate response
element binding protein (ChREBP).

32. 2. Small intestinal bacterial overgrowth, increased
intestinal permeability and low-grade inflammation
 In human studies NAFLD has been associated with
increased LPS plasma levels, through mechanisms
involving:
 Increased intestinal permeability (Leaky-gut syndrome)
 Small intestinal bacterial overgrowth (SIBO)
 Tight junction alteration (Leaky-gut syndrome)
 Bacterial translocation (into portal circulation)
- Ge H, et al.,. Endocrinology 2008
- Jumpertz R, et al., Am J Clin Nutr 2011

33.  This results in TLR-4 signaling  CD14 activation 
inflammation.
 Also activation of lipid peroxidation and production
of ROS  serve as 2nd hit.
 Inflammasomes, major contributors of inflammation,
are cytoplasmic multiprotein complexes, which
include nucleotide-binding domain (NLRPSs).
 NLRPSs are sensors of the bacterial PAMPs and
DAMPs  manipulate cleavage of pro-inflammatory
cytokines.

34. 3. Altered choline metabolism
 Choline is a water-soluble essential nutrient.
 It is an important phospholipid component of the cell
membrane and is the precursor molecule for the
neurotransmitter acetylcholine.
 Choline has important roles in fat metabolism in the liver
and a very-low-lipoprotein assembly, and also it promotes
lipid transport from the liver.
 Gut microbiota secrete enzymes that cleave the dietary
choline to its toxic metabolites (dimethylamine and
trimethylamine).
 Liver uptakes these toxic methylamines and converts
them to trimethylamine-N-oxide which induce
inflammation in the liver.
Vance DE. Curr Opin Lipidol 2008

35.  The role of dietary choline in NAFLD can be
explained by the bioavailibility of free choline to for
lipoproteins in the liver (especially very- low-
density-lipoprotein-VLDL), which allows the export
of free fatty acids from this organ
 If the gut microbiota converts excessive amounts of
dietary choline into trimethylamine, this leads to
reduced choline bioavailibility and consequent fatty
liver disease

36. 4. Altered bile acid metabolism
 Bile acids modulate lipid absorption and cholesterol
homeostasis.
 The nuclear bile acid receptor, called farnesoid X receptor
(FXR), is strongly expressed at bile acid excretion (liver)
and absorption (intestine) regions.
 Bile acids also act as signaling molecules and activate FXR
and The G-protein coupled receptor TGR5.
 FXR plays a key role in the control of hepatic de novo
lipogenesis, VLDL triglyceride export and plasma
triglyceride turnover.
 Gut microbiota can modulate bile acid metabolism.
Wang Z, et al.. Nature 2011

37. 5. Stimulation of endogenous ethanol
production by enteric bacteria
 Intestinal microbiota produces a number of potentially
hepatotoxic compounds such as ethanol, phenols,
ammonia and they are transported to liver by portal
system.
 These toxins stimulate hepatic Kupffer cells for
production of nitric oxide and cytokines such as TNF-α.
 Acetaldehyde and acetate are two major metabolites of
ethanol:
 Acetate is a substrate for fatty acid synthesis (via inhibition of the
Krebs cycle).
 Actaldehyde and its metabolites may lead to the formation of ROS.
Wagnerberger S, et al., . Dig Dis Sci 2012

38. NAFLD and microbiota

39. Summary of Effects of gut microbiota on the development of
nonalcoholic fatty liver disease and nonalcoholic
steatohepatitis through the gut-liver axis.
 Altered intestinal bacterial composition (dysbiosis) results
in degradation of carbohydrates in the intestinal lumen and
produces short-chain fatty acids (SCFAs), which are
substrates for hepatic lipogenesis and gluconeogenesis.
 The interaction of SCFAs with G-protein coupled receptors
(GPRs) releases the peptide YY (PYY), which modulates gut
motility and nutrient absorption.
 SCFAs stimulate hepatic carbohydrate response element
binding protein (ChREBP) and increase lipogenesis.

40. Summary of Effects of gut microbiota on the development of
nonalcoholic fatty liver disease and nonalcoholic
steatohepatitis through the gut-liver axis.
 Bacterial translocation to the portal circulation causes
interaction of bacterial endotoxins (lipopolysaccharides,
LPS) with hepatic toll-like receptors (TLR4 and TLR9) and
results in the release of cytokines and chemokines.
 Decreased Fiaf (fasting-induced adipose factor) levels
enhance lipoprotein lipase (LPL) activity and cause fat
accumulation.
 Choline deficiency causes liver steatosis via decreased
secretion of very- low-density-lipoprotein-(VLDL) from the
liver.

41. Therapeutic
Influences

42. 1. Antibiotics
 No concise supporting evidence at the current time.
 May alter activation of TLRs and therefore limit the
extent of inflammation and increased intestinal
permeability.
 Decreased intestinal permeability would allow for
increased expression of tight junction proteins and
improve the mucosal integrity.
 Potential role of Rifaximin in NAFLD under clinical
investigations.
Kalambokis G, et al.,
Hepatology. 2012

43. 2. Pre-Biotics
 Originally defined as:
“nondigestible food ingredients
that beneficially affect the host
by selectively stimulating the
growth and/or activity of one
or a limited number of
bacteria in the colon
 Now more loosely defined as:
“selectively fermented
ingredients that allow specific
changes, both in the
composition and/or activity in
the gastrointestinal
microflora, that confer
benefits.

44. Potential Roles of Prebiotics
 May decrease luminal pH to impede the growth of pathogens.
 Attenuate the levels of lipogenesis
 May alter de novo lipogenesis through modifications in the level of gene
expressiono
 May alter the by-products of microbial fermentation (short chain fatty-acids)
 Increase level of Bifidobacterium, which is associated with
decreased endotoxemia and levels of proinflammatory cytokines.
 Increased production of glucagon-like-peptide-2 in prebiotic treated
mice is associated with decreased intestinal permeability and
decreased LPS.
 Associated with decreased levels of cholesterol and triglycerides in
human subjects
 Potential alteration of appetite and caloric intake mediated by
changes in the levels of ghrelin and Peptide YY.
Parnell J a,et al.Liver Int. 2012

45. 3. Pro-Biotics
 Probiotics are live
microoganisms
that, when
administered in
adequate
quantities, confer a
health benefit to
the host

46. Potential Roles of Pro-biotics
 Decreased intestinal pH inhibition of some
pathogenic gram negative bacteria.
 May compete with and displace bacteria from
epithelial surface receptors in the gut.
 Increase mucin secretion through upregulation of
(mucine-producing genes) MUC2 and MUC3 genes.
 Potential reductions in LDL, cholesterol, and
triglycerides.
 Histologic evidence for the improvement in
inflammation and steatosis in some studies
Sanders ME. Clin Infect Dis. 2008

48. In a capsule …..
 Human gut contains 1014 micoorganisms called
“Microbiota”. They are considered a new organ and
becoming the new hit in medicine.
 Dysbiosis: results when the natural flora of the gut are
thrown out of balance. It is claimed to be the cause of
many diseases.
 Dysbiosis is linked to obesity by several mechanisms.
 Dysbiosis is linked to NAFLD, independet of its role in
causing obesity.
 Re-adjustement of such a balance by means of
Antibiotics, Pre- and/or Pro-biotics may play a very
important therapeutic role in the near future.