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European Journal of Nutrition
ISSN 1436-6207
Eur J Nutr
DOI 10.1007/s00394-017-1524-x
Influence of gut microbiota on the
development and progression of
nonalcoholic steatohepatitis
Fabiana de Faria Ghetti, Daiane
Gonçalves Oliveira, Juliano Machado de
Oliveira, Lincoln Eduardo Villela Vieira
de Castro Ferreira, et al.
1 23
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Vol.:(0123456789)
1 3
Eur J Nutr
DOI 10.1007/s00394-017-1524-x
REVIEW
Influence ofgut microbiota onthedevelopment andprogression
ofnonalcoholic steatohepatitis
FabianadeFariaGhetti1,4 · DaianeGonçalvesOliveira1·
JulianoMachadodeOliveira1· LincolnEduardoVillelaVieiradeCastroFerreira1·
DionéiaEvangelistaCesar2· AnaPaulaBoroniMoreira3
Received: 31 January 2017 / Accepted: 6 August 2017
© Springer-Verlag GmbH Germany 2017
unaltered in NASH patients. There is an increase in the
Escherichia and Bacteroides genus. There is depletion of
certain taxa, such as Prevotella and Faecalibacterium.
Conclusion Although few studies have evaluated the com-
position of the gut microbiota in patients with NASH, it is
observed that these individuals have a distinct gut microbi-
ota, compared to the control groups, which explains, at least
in part, the genesis and progression of the disease through
multiple mechanisms. Modulation of the gut microbiota
through diet control offers new challenges for future studies.
Keywords Nonalcoholic fatty liver disease· Dysbiosis·
Steatohepatitis· Gut microbiota· Microbiome
Introduction
Nonalcoholic steatohepatitis (NASH) is the most severe his-
tological form of nonalcoholic fatty liver disease (NAFLD)
[1], characterized by the presence of hepatic steatosis and
inflammation, associated with ballooning degeneration, with
or without fibrosis [2]. Even as the prevalence of NASH in
the general population is approximately 2–5% [3, 4], around
70% of morbidly obese individuals are affected by this con-
dition [5]. Although in most cases, NASH carriers present
no symptoms, this condition may increase the risk of cirrho-
sis, liver failure, and hepatocellular carcinoma [6–9].
The exact cause of NASH is not yet clear, but studies have
suggested the role of the gut microbiota in the pathogenesis
of this disease [10–12]. In fact, it has been shown in animal
models that gut microbiota increase intrahepatic fat through
mechanisms associated with increased dietary energy extrac-
tion or change in lipogenesis and β-oxidation [10, 11]. Fur-
thermore, hepatocellular inflammation may be secondary
to increased intestinal permeability and translocation of
Abstract
Introduction Nonalcoholic steatohepatitis (NASH) is char-
acterized by the presence of steatosis, inflammation, and bal-
looning degeneration of hepatocytes, with or without fibro-
sis. The prevalence of NASH has increased with the obesity
epidemic, but its etiology is multifactorial. The current stud-
ies suggest the role of gut microbiota in the development
and progression of NASH. The aim is to review the studies
that investigate the relationship between gut microbiota and
NASH. These review also discusses the pathophysiological
mechanisms and the influence of diet on the gut–liver axis.
Result The available literature has proposed mechanisms
for an association between gut microbiota and NASH, such
as: modification energy homeostasis, lipopolysaccharides
(LPS)–endotoxemia, increased endogenous production of
ethanol, and alteration in the metabolism of bile acid and
choline. There is evidence to suggest that NASH patients
have a higher prevalence of bacterial overgrowth in the
small intestine and changes in the composition of the gut
microbiota. However, there is still a controversy regarding
the microbiome profile in this population. The abundance
of Bacteroidetes phylum may be increased, decreased, or
* Fabiana de Faria Ghetti
bia.ghetti@hotmail.com
1 Universitary Hospital andSchool ofMedicine, Federal
University ofJuiz de Fora, JuizdeFora, MinasGerais,
Brazil
2 Department ofBiology, Federal University ofJuiz de Fora,
JuizdeFora, MinasGerais, Brazil
3 Department ofNutrition, Federal University ofJuiz de Fora,
JuizdeFora, MinasGerais, Brazil
4 Unidade de Nutrição Clínica, Hospital Universitário, Rua
Catulo Breviglieri, s/n, Bairro Santa Catarina, JuizdeFora,
MinasGeraisCEP36036-330, Brazil
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microbial cell components to the circulation [12]. Finally,
gut microbiota can contribute to fibrogenesis through activa-
tion of hepatic stellate cells [13].
Although animal experiments have associated gut micro-
biota with the histological components of NAFLD, there
are few clinical studies with emphasis on the composition
and functionality of the microbiome in NASH. There is evi-
dence that NASH patients have a higher prevalence of small
intestinal bacterial overgrowth (SIBO) [14, 15] and the low-
est percentage of Bacteroidetes in their fecal content when
compared to healthy subjects [16]. On the other hand, other
studies have observed a higher abundance of Bacteroidetes
in the gut microbiota of patients with NASH, compared to
healthy controls [17, 18]. Thus, the results of the studies are
still controversial regarding the microbiome profile in this
population.
A detailed study of the composition of the gut microbiota
and its metabolic functions can determine which microor-
ganisms contribute to gut health maintenance and what
changes can lead to the development of pathologies [19].
Therefore, the aim of this review is to highlight the studies
that investigate the relationship between gut microbiota and
NASH. Pathophysiological mechanisms and the influence of
diet on the gut–liver axis are also discussed.
Development andprogression ofnonalcoholic
steatohepatitis
Traditionally, the pathogenesis of NASH is explained by
the hypothesis of “two hits” proposed by Day and James
[20], in 1998. According to the authors, insulin resistance
would be the first stimulus (the first “hit”) that determines
the accumulation of fat in hepatocytes, resulting in steatosis.
The steatosis itself increases the sensitivity of the liver to the
second “hit”. The second “hit” would be the oxidative stress,
which promotes liver injury, characterized by tissue lesions,
inflammation, and fibrosis [20].
According to the hypothesis of the “two hits”, insulin
resistance promotes hepatic lipogenesis and lipolysis in the
adipose tissue, increasing the amount of fatty acids released
to the liver in the initial event. On a smaller scale, the avail-
ability of fatty acids in the liver may result from the trans-
port mediated by lipoproteins after intestinal absorption of
dietary fats [21]. Upon entering the hepatocytes, the free
fatty acids are oxidized by mitochondria to generate energy
or are esterified in triacylglycerols (TG), incorporated into
very-low density lipoprotein (VLDL) particles, and exported
from the liver to the peripheral tissues [22]. When free fatty
acids in the hepatocytes exceed their metabolization and
export capacity, they can cause hepatic steatosis [22]. The
accumulation of fatty acids in the liver results in excessive
increase in the production of reactive oxygen species (ROS)
from the mitochondria [23]. In addition, peroxisomes and
microsomal oxidation pathways are activated and generate
more ROS, culminating in hepatic oxidative stress [24]. Oxi-
dative stress appears to be responsible for initiating necroin-
flammation. The consequence of oxidative stress is hepatic
lipid peroxidation in cell membranes and mitochondria, pro-
ducing malondialdehyde and hydroxynonenal, resulting in
mitochondrial dysfunction [25]. Malondialdehyde activates
the regulatory transcription factor and the expression of pro-
inflammatory cytokines and adhesion molecules (NF-kB),
stimulating production of the tumor necrosis factor alpha
(TNF-α), interleukin 8 (IL-8), and selectin E [26]. Hydrox-
ynonenal activates hepatic stellate cells, promoting colla-
gen deposition, and hence, the development of fibrosis [27].
Thus, ROS, lipid peroxidation products, and cytokines are
involved in the second hit, which induces the progression of
simple steatosis to NASH.
At present, it is believed that the process of “two hits”
is insufficient to explain the pathogenesis of this heteroge-
neous disease, particularly in non-obese individuals [28].
Furthermore, simple hepatic steatosis, which is benign and
nonprogressive in a majority of patients, and NASH, may
reflect different pathogenesis [29]. In fact, the accumulation
of free fatty acids in the liver occurs mainly in the form of
TG [30]. There is evidence to indicate that TG by themselves
is not hepatotoxic, at least in mice (BKS.Cg-m/Leprdb/J)
with steatohepatitis [30]. Therefore, TG synthesis seems
to be an adaptive, beneficial response insituations, where
hepatocytes are exposed to potentially toxic TG metabolites
[30]. On the contrary, several other lipids, such as free fatty
acids, diacylglycerol, cholesterol, ceramide, and phospholip-
ids, also accumulate in the liver, and they are considered as
‘‘aggressive’’ lipids [31], that induce endoplasmic reticulum
(ER) stress, mitochondrial dysfunction, and oxidative stress,
resulting in hepatic inflammation and fibrogenesis [32, 33].
In this sense, a new theory was proposed by Tilg etal.
[31]: the hypothesis of “multiple parallel hits”. This hypoth-
esis proposes that several concurrent and not consecutive
stimuli (“multiple parallel hits”) induce oxidative stress,
which results in both hepatic steatosis and steatohepatitis.
Even as the two “hits” hypothesis suggests that steatosis
always precedes inflammation, the “multiple parallel hits”
hypothesis indicates that inflammation in NASH may pre-
cede steatosis in some cases [31]. In this new paradigm,
NASH results not only from oxidative stress, but also from
the interaction between different “hits”, including altered
lipid metabolism, mitochondrial dysfunction, ER stress,
genetic predisposition, and gut microbiota alterations [31].
It is increasingly recognized that the gut microbiota is impli-
cated in the pathogenesis and progression of NASH [34].
Thus, the gut microbiota has become a topic of interest in
recent investigations and a potential target of intervention
[35, 36].
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Gut microbiota
The human gut contains a large number of microorganisms,
mostly bacteria, collectively called gut microbiota [37]. The
latest estimate for the total number of bacteria cells in the
body is around 40 trillion (3.8×1013), the same order as the
number of human cells (3.0×1013), and their total mass is
about 0.2kg [38]. More than 1000 cultured gastrointestinal
species have been identified in the human microbiota [39].
Recent advances in molecular techniques, sequencing and
bioinformatic programs, have allowed the identification of
specific taxonomic groups, that is, phyla, classes, orders,
families, genera, and bacterial species [40]. Currently,
metagenomic techniques have been used to characterize the
composition, diversity, and potential physiological effects of
entire microbial communities, without cultivation and isola-
tion of the members of the community [41].
The Firmicutes and Bacteroidetes are the most prevalent
phyla in adults, followed by Actinobacteria, Proteobacteria,
Fusobacteria, Spirochaetae, and Verrucomicrobia [42]. The
stomach and small intestine are rich in Firmicutes (Lac-
tobacillaceae) and Proteobacteria (Enterobacteriaceae),
whereas the large intestine shows higher counts of Bacte-
roidetes (Bacteroidaceae, Prevotellaceae, and Rikenellaceae)
and Firmicutes (Lachnospiraceae and Ruminococcaceae)
[43]. The composition of adult microbiota remains relatively
stable, although the microbial diversity is acquired within
the first hours post birth, and is shaped over time as the diet
becomes more complex and the immune-system matures
[44]. A combination of multiple factors, including genetic
and environmental characteristics (type of delivery, antibi-
otic therapy, diet composition, lifestyle, social interactions,
and exposure to various xenobiotics) shapes the gut micro-
biota to make every individual microbially unique [45].
The gut microbiota can also be very dynamic and change
rapidly, for example, in response to dietary changes. One
study shows that an increase in caloric intake from 2400 to
3400kcal/day (with a similar nutrient profile that includes
24% protein, 16% fat, and 60% carbohydrates) over 3days
increases Firmicutes ratio and decreases the ratio of Bacte-
roidetes [10]. Diet is a major factor driving the composition
and metabolism of the gut microbiota [40] and the influence
of the diet on gut microbiota will be described in more detail
in the next sessions.
Nowadays, the gut microbiota is considered a metabolic
organ, which performs a wide range of functions including
an important role in the physiology of energy homeostasis
[46]. For example, some members of the Firmicutes phylum
are among the butyrate-producing bacteria that increase the
energy extraction from the diet [47]. In contrast, members
of the phylum Bacteroidetes participate in carbohydrate
metabolism and accomplish this by expressing enzymes
similar to glycosyl transferases, glycoside hydrolases, and
polysaccharide lyases [48]. In this context, the knowledge of
the composition and functions associated with the microbial
community is fundamental, as alterations in the composition
of the gut microbiota and/or its functions (called ‘dysbiosis’)
are associated with metabolic diseases, such as NASH [17,
18, 49]. Although human studies are scarce in the literature
(Table1), animal experiments support the link between gut
microbiota and the development of NASH.
Dysbiosis andnonalcoholic steatohepatitis
Experimental data
Animal experiments have demonstrated direct roles for gut
microbiota in the development and progression of nonalco-
holic steatohepatitis (NASH). Using germ-free C57BL/6J
mice, Bäckhead etal. [50] have previously demonstrated that
mice devoid of gut microbiota are resistant to diet-induced
obesity, steatosis, and insulin resistance. Subsequently,
Le Roy etal. [51], using the transplantation experiment,
have shown that differences in microbiota composition can
determine responses to a high-fat diet (HFD) in mice and
contribute to the development of steatosis, independent of
obesity. In this study, the conventional C57BL/6J mice, fed
with an HFD, have generally displayed liver steatosis, hyper-
glycemia, and systemic inflammation (called the ‘respond-
ers’), but some mice are nonresponders, normoglycemic,
and have a lower level of systemic inflammation, with the
same diet. Germ-free mice have been colonized with gut
microbiota from either the responders or the nonrespond-
ers and then fed the same HFD. Despite a similar weight
gain, responder–receiver mice have been found to develop a
higher level of liver steatosis, glycemia, and insulin resist-
ance than nonresponder–receivers. Pyrosequencing of the
16S ribosomal RNA genes has revealed that responder and
nonresponder mice have distinct gut microbiota includ-
ing differences at the phylum, genera, and species levels.
Responder mice harbour a significantly increased number of
sequences belonging to the Firmicutes phylum, Barnesiella,
Roseburia genera, Lachnospiraceae bacterium 609, and
Barnesiella intestinihominis species [51].
Barnesiella intestinihominis, that is part of the Porphy-
romonadaceae family, in particular, showed an increase in
inflammasome-deficient mice (C57Bl/6) associated with
the progression of NASH. It was revealed that the nucle-
otide-binding domain, leucine-rich repeat protein (NLRP)
6, NLRP3 inflammasomes, and the effector protein IL-18
negatively regulated the exacerbated hepatic steatosis and
inflammation via modulation of the gut microbiota. Antibi-
otic treatment with ciprofloxacin and metronidazole reduced
the severity of NASH in inflammasome-deficient mice
and abolished transmission of the phenotype to wild-type
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animals, showing that gut microbiota drove NASH progres-
sion in this model [52].
Progression of NASH was also strictly related to reduced
microbial diversity and an increased ratio of Firmicutes to
Bacteroidetes in model C57BL/6J mice [53]. The abundance
of Bacteroides spp., Bacteroides vulgatus, Desulfovibrio
spp., Atopobium spp., Clostridium cocleatum, and Clostrid‑
ium xylanolyticumin was increased in these animals and
positively correlated with the increased levels of lipopoly-
saccharides (LPS) [53], an endotoxin present on the cell sur-
face of Gram-negative bacteria, which induced inflammation
[54]. The authors also observed a reduction in the abundance
of gut barrier-protecting bacteria, such as the Lactobacillus
spp. [53]. Another study with rats also showed that during
the progression of NASH, the levels of LPS were highly
increased. In addition, an increase was found in Escherichia
coli and Enterococcus as well as a decrease was seen in
Lactobacillus, Bifidobacteria, and Bacteroide [36]. Based on
the connection between the intestine and liver, also termed
‘gut–liver axis’, the gut microbiota and their metabolic by-
products may influence liver pathology [36].
Dysbiosis could also promote liver fibrogenesis. Indeed,
C57BL/6 mice fed an HFD developed more severe liver
fibrosis than control mice that were fed a standard chow
diet, by changes in gut microbiota, activating an inflamma-
some cascade [55]. HFD-related increases in liver fibrosis
were associated with an increase in the percentage of Gram-
negative (mainly Proteobacteria) versus Gram-positive bac-
teria (mainly reduction in Erysipelotrichaceae and a com-
plete disappearance of Bifidobacteriaceae) and a reduced
ratio between Bacteroidetes and Firmicutes [55]. Bifido-
bacteriaceae (Firmicutes) was known to exert a protective
role during liver injury [56, 57], whereas Proteobacteria was
considered the main pathogen bacteria, expressing endotox-
ins [58]. Thus, the outcome suggested that dietary habits,
by increasing the percentage of intestinal Gram-negative
Table 1 Human studies that have evaluated the gut microbiota in nonalcoholic steatohepatitis
↑, increase; ↓, decrease; 16S rRNA, 16S ribosomal RNA sequencing; NASH, nonalcoholic steatohepatitis; qRT-PCR, quantitative real-time
polymerase chain reaction; SS, simple steatosis
References Study Sample Method Results and p value
Boursier etal. [49] Cross-sectional Adult NASH (n=35), obese no-
NASH (n=22)
16S rRNA ↑Bacteroides and ↓Prevotella
in NASH vs. obese no-NASH
(p=0.013 e p=0.053, respec-
tively)
Del Chierico etal. [69] Cross-sectional Children NASH (n=26), healthy
controls (n=54)
16S rRNA ↑Ruminococcus, Blautia, Dorea
↓Oscillospira in NASH vs. healthy
controls (p<0.05)
Zhu etal. [18] Cross-sectional Children NASH (n=22), obesity
(n=25) and healthy controls
(n=16)
16S rRNA ↑Bacteroidetes and ↓Firmicutes
in NASH vs. healthy controls
(p=0.009 e p=0.002, respec-
tively)
↑Proteobacteria, Enterobacteriaceae
and Escherichia in NASH vs.
healthy controls and obesity
(p=0.0007)
Wong etal. [17] Longitudinal (6months) Adult NASH (n=16) and healthy
control (n=22)
16S rRNA ↓Faecalibacterium and Anaero‑
sporobacter in NASH vs. healthy
controls (p<0.05)
↑Parabacteroides and Allisonella
in NASH vs. healthy control
(p<0.05)
↓Firmicutes in NASH vs. healthy
controls (p<0.05)
Mouzaki etal. [16] Cross-sectional Adult NASH (n=22), SS (n=11)
and healthy controls (n=17)
qRT-PCR ↓Bacteroidetes in NASH vs. SS and
healthy controls (p=0.006)
Shanab etal. [60] Cross-sectional Adult NASH (n=18) and healthy
controls (n=16)
Lactulose
breath
hydrogen
test
SIBO in NASH (77.78%) vs. SIBO
in healthy controls (31.25%)
(p<0.0001)
Wigg etal. [14] Cross-sectional Adult NASH (n=22) and healthy
controls (n=23)
Combined
xylose and
lactulose
breath test
SIBO in NASH (50%) vs. SIBO in
healthy controls (22%) (p=0.048)
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endotoxin producers, might accelerate liver fibrogenesis,
introducing dysbiosis as a cofactor that contributed to
chronic liver injury in NASH [55].
Human data
The first report, on humans, of the relationship between
gut microbiota and pathogenesis of NASH was published
by Drenick etal. [59]. In this study, patients undergoing
intestinal bypass developed parallel NASH and SIBO. After
being treated with antibiotics, the patients showed regres-
sion of hepatic steatosis, suggesting that microbiota were the
possible cause of NASH [59]. Subsequently, other studies
investigated gut microbiota in patients with NASH (Table1).
Wigg etal. [14] observed small intestinal bacterial over-
growth (SIBO) in 50% of the patients with NASH and in
22% of the control subjects (p=0.048). Shanab etal. [60]
also observed a higher prevalence of SIBO in the NASH
group, compared to the control group (77 vs. 31%).
SIBO may be characterized by an increase in the num-
ber of bacteria in the proximal small intestine (≥105 col-
ony-forming units/mL of intestinal content) or a change in
microbial composition, with a profile that is typical of the
microorganism species that colonize the large intestine [61].
Although the “gold standard” for the diagnosis of SIBO is
still thought to be jejunal aspiration and culture, this tech-
nique requires intestinal intubation that may not be well tol-
erated and may not detect the un-culturable species [62]. To
investigate the possible presence of SIBO, all the studies in
patients with NASH used the breath test (Table1), because
it provides the simplest noninvasive and widely available
diagnostic modality for suspected SIBO, by determination of
hydrogen and/or methane concentration produced by intes-
tinal bacterial metabolism in the exhaled air [63]. However,
there is a lack of consensus on the breath test interpretation
[64]. Studies seeking to validate breath testing have calcu-
lated sensitivities and specificities ranging from 31 to 77
and 44 to 100%, respectively [65, 66], leading to high false-
positive rates [67]. In addition, a variety of test methods
and diagnostic criteria are used in studies and they are not
standardized to define a positive test for SIBO. These factors
have led to a controversy regarding the diagnostic utility of
breath testing in SIBO [64].
Many microbial studies have focused on the fecal micro-
biota. It is important to highlight the major drawback of the
use of stool analyses. It is the fact that a fecal sample does
not reflect the microbiota composition from the small intes-
tine, because it represents mainly fecal samples from the end
of the colon [68]. Therefore, studies with NASH patients
show changes in the composition of the fecal microbiota, but
there is controversy regarding the profile of resident bacte-
ria in the gut. For example, Mouzaki etal. [16] show a low
percentage of Bacteroidetes (Bacteroidetes to total bacteria
counts) and no differences in Firmicutes, in the stool sam-
ples of NASH patients. Instead, two studies [17, 18] have
observed an increase in Bacteroidetes and decrease in Fir-
micutes in NASH patients, compared to the healthy controls.
There is recent evidence that shows that the abundance of
Bacteroidetes and Firmicutes is similar between NASH and
no-NASH patients [49].
Other changes in the gut microbiota are related to NASH
(Table1). Recent evidence shows that the percentage of Bac‑
teroides genus, one of the most important genera within the
Bacteroidetes phylum, is significantly increased in NASH,
whereas the percentage of the Prevotella genus is decreased
[49]. In addition, pediatric NASH patients have a lower fecal
abundance of Faecalibacterium and Anaerosporobacter, but
higher abundance of Parabacteroides and Allisonella. A sig-
nificant difference is observed at the phylum, family, and
genera level in the fecal samples of children with NASH.
Proteobacteria/Enterobacteriaceae/Escherichia are higher in
NASH compared to healthy controls and obese patients [18].
Another study with pediatric patients shows a decrease of
Oscillospira and increases of Ruminococcus, Blautia, and
Dorea in NASH compared to the controls [69].
The variability of methods (qPCR vs. pyrosequencing),
exclusion of all taxa with an abundance below 1% and pro-
file of subjects (adults vs. children) may explain, in part, why
there is still no consensus in the literature about which bac-
terial groups are increased or reduced in the gut of NASH
patients, compared to no-NASH patients. In these studies, all
NASH patients have a high body mass index (BMI) (>29kg/
m2), and the BMI of NASH patients is significantly higher
than that of healthy controls in two studies [16, 18]. As obe-
sity itself is linked to gut microbiota composition changes
[47, 70], BMI can be a major confounder [28]. Thus far, no
study has directly assessed the gut microbiota composition
in non-obese patients with NASH, but recent evidence has
shown that non-obese patients with nonalcoholic fatty liver
disease (NAFLD) have 20% more Bacteroidetes phylum and
24% fewer Firmicutes phylum, compared to healthy con-
trols [28]. Future studies should include non-obese NASH
patients in their analyses, to exclude the impact of obesity.
Although few studies have evaluated the composition of
the gut microbiota in NASH patients, it was observed that
these individuals have a distinct gut microbiota, compared to
the healthy control groups, which explains, at least in part,
the genesis and progression of the disease through multiple
mechanisms.
Mechanistic pathways inthedevelopment
andprogression ofNASH
The mechanisms involved in the relationship between gut
microbiota and NASH are not yet fully known, but the
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proposed mechanisms in the literature are described below
(Fig.1). The proposed mechanisms may potentiate each
other through shared molecular pathways of fat accumula-
tion, activation of inflammation, and fibrogenesis in the liver
[71].
Modification ofenergy homeostasis
Energy harvest fromthediet
The gut microbiota has the ability to extract energy from
food via glycoside hydrolases and polysaccharide lyases,
which are not encoded by the human genome (Fig.1). Such
enzymes in the colon metabolize undigested polysaccharides
into monosaccharides and short chain fatty acids (SCFA)
[50]. The monosaccharides produced by microbial fer-
mentation are absorbed and transferred to the liver through
portal circulation, where they activate factors like the car-
bohydrate-responsive element-binding protein (ChREBP),
which increases the transcription of proteins involved in
hepatic lipogenesis [72]. The SCFA (acetate, propionate,
and butyrate) can be used for lipid or gluconeogenesis [73].
Thus, bacterial SCFA provide an additional source of energy
for the body, promoting fatty liver accumulation [74].
The first investigation in this line of evidence has been
conducted by Bäckhed etal. [11]. Colonization of C57BL/6
mice, germ-free (raised in absence of microorganisms), with
cecal content from mice that were colonized with a normal
microbiota at birth (termed ‘conventionally raised’) resulted
in 60% of increased total body fat and consequently hepatic
TG accumulation, without any increase in food consumption
or energy expenditure [11]. Subsequently, Turnbaugh etal.
[47] showed that the C57BL/6J obese mice had a higher con-
centration of SCFA and fewer calories in their stool, suggest-
ing that in these animals, the microbiota contribute to the
extraction of additional calories from their diet. These ani-
mals showed higher levels of Firmicutes than Bacteroidetes
compared to their lean counterparts. The changes observed
in obese mice microbiota could increase energy delivery to
the liver and reduce fecal energy loss [47].
Although the experimental data indicate that the gut
microbiota influence the energy balance, it remains uncer-
tain as to what extent gut microbiota are an important regu-
lator of nutrient absorption in humans. One clinical study
showed that the total amount of SCFA and propionate were
higher in the obese group than in the lean group [73]. How-
ever, another study found no difference in energy excre-
tion in the stools and no difference in bacterial abundance
between the obese and lean groups [10].
There is evidence that SCFA produced in the colon con-
tribute to approximately 5–10% of the energy requirements
[75]. It may be possible that the additional calories provided
to the host by the microbiota, due to the fermentation of
undigested dietary molecules, are not sufficient to induce
significant changes in weight [76]. One of the arguments to
support this hypothesis is that consumption of a high-fiber
diet could increase SCFA production, which usually helps
to reduce weight and adipose tissue [77, 78]. Studies with
Fig. 1 Mechanisms proposed
in the relationship between gut
microbiota and nonalcoholic
steatohepatitis. LPL lipoprotein
lipase, LPS lipopolysaccharide,
NASH nonalcoholic steato-
hepatitis, ROS reactive oxygen
species, TMAO trimethylamine-
N-oxide, TLR toll-like recep-
tor, VLDL very-low density
lipoprotein
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prebiotics also have indicated that a higher intestinal pro-
duction of SCFA is associated with an increase in satiety
and a consequent reduction in dietary intake. These effects
are in part related to the increase of glucagon-like peptides
(GLP-1 and GLP-2) and peptide YY (PYY), which lead to
hypothalamic effects related to the reward mechanism [79,
80]. Thus, more mechanistic studies are required to under-
stand the role of each of the SCFA on NASH.
Activation ofG protein‑coupled receptors
The SCFA act on the G protein-coupled receptors, such as
Gpr41 and Gpr43, expressed in intestine and adipose tissues
[81]. GPR41 and GPR43 have been renamed free fatty acid
receptors FFA3 and FFA2, respectively [82], based on their
responsiveness to SCFA. There is a power order of SCFA in
activating human FFA2 and FFA3 receptors, where FFA2 is
activated more potently by acetate=propionate>butyrate,
whereas for FFA3, it is propionate=butyrate>acetate [83].
The FFA3 activation stimulates enteroendocrine cells
to increase production of the PYY, a hormone that reduces
intestinal motility and provides greater absorption of nutri-
ents, particularly of SCFA [84]. The FFA2 activation con-
tributes to inhibition of lipolysis and adipocyte differen-
tiation, leading to increased adipose tissue [85]. FFA2 is
also present on intestinal neutrophils and might, therefore,
contribute to NASH pathogenesis by increasing intestinal
inflammation and permeability [86, 87].
Effects ofadenosine 5′‑monophosphate protein kinase
andfasting‑induced adipose factor
Experimental studies suggest that the presence of microbiota
inhibits the enzyme adenosine 5′-monophosphate protein
kinase (AMPK) pathway and suppresses intestinal expres-
sion of the protein fasting-induced adipose factor (FIAF)
[11, 50]. AMPK is a key enzyme that controls the cellular
energy status, which in turn activates the key enzymes of
mitochondrial fatty acid oxidation, including acetyl-CoA
carboxylase (ACC) and carnitine-palmitoyltransferase I
(CTP1). When inhibited, the AMPK suppresses muscle
oxidation of fatty acids, favoring adiposity [50]. FIAF is
a circulating lipoprotein lipase (Lpl) inhibitor produced by
the intestine, liver, and adipose tissue [88]. Inhibition of
FIAF increases the activity of the lipoprotein lipase, lead-
ing to fat accumulation in the adipose tissue and increases
hepatic uptake of free fatty acids [11]. Inhibition of FIAF
further decreases expression of the peroxisome prolifera-
tor-activated receptor gamma coactivator 1 alpha (PGC1-α)
and enzymes involved in mitochondrial fatty acid oxidation
[50]. Together, these effects may increase insulin resistance,
resulting in obesity and hepatic steatosis (Fig.1) [11, 50].
Lipopolysaccharide–endotoxemia
It has been proposed that changes in the gut microbiota
favor an increase in circulatory lipopolysaccharides (LPS),
particularly when the diets are rich in fat and energy [89,
90]. Increased circulatory LPS may contribute to meta-
bolic endotoxemia (low-grade inflammation) [91], which
plays a pivotal role in the development and progression of
NASH (Fig.1) [90]. LPS is recognized by pattern recogni-
tion receptors. These receptors include membranous toll-
like receptors (TLRs) (especially TLR-4) and intracellular
NOD-like receptors (NLRP3 and NLRP6 inflammasomes).
Stimulation of TLR-4 results in the activation of several dif-
ferent intracellular signaling cascades, inducing the synthe-
sis of a variety of inflammatory cytokines (especially TNF-
α), which induce inflammation, oxidative stress, and insulin
resistance [92]. Kupffer cells, which express the highest lev-
els of TLR-4 liver, are cells that respond to LPS to produce
cytokines and ROS [93]. The interaction between LPS and
TLR-4 also activates receptors on stellate cells, resulting
in hepatic fibrogenesis [15]. Increase in the levels of LPS
also leads to liver injury through a mechanism mediated by
the inflammasome, which includes NLRPs, a group of cyto-
plasmatic and multiprotein complexes [94]. NLRPs manip-
ulate the cleavage of proinflammatory interleukins (ILs),
such as pro-IL-1β and pro-IL-18 [94]. Henao-Mejia etal.
[52] experimentally revealed that the NLRP6 and NLRP3
inflammasome alterations or IL-18 deficiency cause intesti-
nal microbial changes by enhancing portal influx of TLR-4
and TLR-9 ligands, which in turn increase hepatic TNF-α
production in C57Bl/6 mice. Apart from LPS, TNF-α, ILs,
and plasminogen activator inhibitor-1 (PAI-1) may represent
a good marker of NASH. The increased portal endotoxemia
could induce the expression of PAI-1, a fibrinolysis inhibi-
tor [95]. Elevated PAI-1 has been correlated with enhanced
LPS-induced liver damage and induction of liver inflamma-
tory response [96, 97].
Several studies conducted on animals and humans support
the link between LPS–endotoxemia and the development of
NASH [54, 98–100]. An animal study demonstrated that
chronic infusion of LPS caused endotoxemia, hepatic stea-
tosis, and changed gut microbiota in HFD C57bl6/J mice
[54]. Genetically obese mice exhibit increased sensitivity
to endotoxin hepatotoxicity, quickly developing steatohepa-
titis after exposure to low doses of LPS [99]. In addition,
intraperitoneal administration of LPS-augmented hepatic
inflammation, apoptosis, and reactive substances in the
methionine choline-deficient nutritional model of NASH/
C57/BL6 mice [100]. In human studies, increased levels of
endotoxin were found in NASH patients, as compared to
healthy individuals [101, 102]. Similarly, it was reported that
morbidly obese subjects have increased levels of LPS and
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LPS-binding proteins (LBP), which correlate with a major
liver expression of TNF-α and the presence of NASH [98].
Possible mechanisms for endotoxemia in patients with
NASH include SIBO and disruption of the intestinal mucosa
barrier integrity, which may lead to an increased intestinal
permeability and excessive absorption of LPS, resulting
in low-grade inflammation and hepatic fibrosis [15, 103].
This association is supported by a growing body of experi-
mental and human data. Genetically obese C57BL/6J mice
display enhanced intestinal permeability, leading to portal
endotoxemia. Moreover, murine hepatic stellate cells iso-
lated from the livers of the animals were more sensitive to
LPS, developing a stronger inflammatory and fibrogenic
phenotype [103]. In human studies, Miele etal. [15] have
shown that patients with NAFLD have increased intestinal
permeability, and this abnormality is related to the increased
prevalence of SIBO and disrupt tight junctions compared
to healthy adults. Another study has associated SIBO with
the expression of TLR-4 and IL-8 in NASH patients [60].
Wigg etal. [14] have found increased prevalence of SIBO
and elevated TNF-α levels in patients with NASH, but have
found no difference in the intestinal permeability or serum
endotoxin levels. Despite the negative result of this study,
the authors have suggested that endotoxin may still be an
important factor in the pathogenesis of NASH. Some pos-
sible explanations for the paradox in this study are under-
estimated endotoxin levels, due to a retrospective collection
of endotoxins. Endotoxins bound to plasma proteins are not
measured and systemic levels may not reflect portal endo-
toxins [14]. In addition, NASH patients may have significant
susceptibility to gut leakiness, and gut leakiness may still be
an important pathogenic factor in patients with NASH and
‘normal’ intestinal permeability [104].
Increased endogenous ethanol production
The hypothesis that endogenous ethanol contributes to the
pathogenesis of NASH dates back from the Cope etal. study
[105]. The authors have reported elevated alcohol concentra-
tion in the breath of obese mice and have demonstrated that
breath alcohol concentration can be reduced by gut micro-
bial intervention with antibiotics [105]. Human studies have
reported increased endogenous ethanol in NASH. Nair etal.
[106] have demonstrated that obese women with NASH have
higher breath ethanol concentrations than healthy controls
detected by gas chromatography. Another study has shown
that pediatric NASH patients have higher plasma concentra-
tions of ethanol when compared to healthy or obese indi-
viduals [18]. In addition, an increased expression of etha-
nol-metabolizing enzymes, alcohol dehydrogenase, catalase,
and aldehyde dehydrogenase is seen in NASH liver [107].
In summary, these outcomes suggest that the microbiota of
patients with NASH produce more ethanol, which induces
the expression of ethanol-metabolizing enzymes in the liver
[107].
The normal microbiota in the human large intestine is
capable of producing and metabolizing ethanol [108]. It has
been shown that under anaerobic conditions, the bacterial
metabolism of pyruvate, produced during the breakdown of
carbohydrates, generates acetaldehyde, which can then be
further reduced to form ethanol [109]. This metabolic fate of
carbohydrates is favored when there is intestinal overgrowth
of bacteria or yeast [110] or if carbohydrates, particularly
sugar (e.g., glucose, sucrose, and fructose), are consumed
excessively [111].
Despite the lack of consistent NASH-related gut micro-
biota changes, the possible overgrowth of ethanol-producing
bacteria may underlie an increase in the circulation of etha-
nol levels in NASH. Zhu etal. [18] have shown a higher E.
coli rate in the NASH group, compared to groups without
NASH. E. coli is a member of the Family of Enterobacte-
riaceae, which typically aerobically degrade carbohydrates
by mixed acid fermentation [112]. Ethanol is one of the
common-end products of this pathway [112]. Furthermore,
it is possible to suggest that the intestinal overgrowth of
other bacteria-producing ethanol or yeast (e.g., Lactobacil‑
lus fermentum, Weissella confusa, and Saccharomyces cer‑
evisiae) explain the higher plasma concentration of ethanol
in some NASH patients [113], mainly in those with rich-in-
carbohydrate diets [114]. L. fermentum and W. confusa are
both heterolactic organisms [115, 116]. Ethanol is one of the
dominating metabolites of heterolactic intestinal microbes
by mixed acid fermentation [115]. Finally, S. cerevisiae,
typically for yeasts, metabolizes hexoses via ethanol fer-
mentation, yielding just ethanol and carbon dioxide [117].
The intestinally derived ethanol may contribute to the
pathogenesis of NASH (Fig.1), because gut-derived ethanol
can induce hepatic steatosis [118]. In addition, increases in
the production of ethanol by gut microbiota may injure the
intestinal barrier and promote increasing permeability and
endotoxemia [105, 119, 120]. Consequently, tissues, includ-
ing the liver, that are exposed to this blood flow are stimu-
lated to produce cytokines, such as TNF-α [105] and ROS,
causing liver injury [119].
Altered bile acid metabolism
Primary bile acid (BA) species (cholic and chenode-
oxycholic acids) are synthesized and conjugated with
glycine or taurine in the liver, stored in the gallbladder,
and released into the duodenum until ingestion of a fat
meal (Fig.2) [121]. In the intestine, BA are metabolized
by bacteria to more hydrophobic BA species, through
7α-dehydroxylation and/or deconjugation of hydrophilic
groups, resulting in secondary BA species (deoxycholic
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Eur J Nutr
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and lithocholic acid) [122]. Over 95% of BA are reab-
sorbed in the distal ileum and then recycled via the portal
vein into the liver [123].
In addition to promoting the absorption of fat, cholesterol,
and fat-soluble vitamins in the intestinal tract, BA also act
as signaling molecules that modulate a variety of physio-
logical processes [124]. Regulatory actions of BA are medi-
ated through specific BA-activated receptors, including the
farnesoid X receptor (FXR), and members of the G protein-
coupled receptor, mainly the TGR5 [125]. FXR, which is
highly expressed in hepatocytes and enterocytes, is activated
by free and conjugated primary BA [124]. FXR induces the
expression of a short heterodimer partner (SHP), which
inhibits CYP7A1 activation, the first enzyme of BA syn-
thesis [121]. In the small intestine, FXR induces the fibro-
blast growth factor (human FGF19 and mouse FGF15), an
intestinal hormone, to repress hepatic BA synthesis through
FGF receptor 4 (FGFR4) expressed in the liver [125]. Acti-
vation of the FXR pathways not only regulates the synthesis
and enterohepatic cycle of BA, but also acts on the control
of hepatic de novo lipogenesis in the liver, exportation of
TG by VLDL, and gluconeogenesis [126]. TGR5 is widely
distributed and expressed in various tissues, including the
intestines, enteroendocrine cells, and liver [121]. Activation
of TGR5 by secondary BA induces intestinal glucagon-like
peptide-1 (GLP-1) release from the intestinal enteroendo-
crine L cells and GLP-1-associated improvements in glucose
tolerance and liver function [127]. Therefore, BA plays a
crucial role in lipid and glucose homeostasis [126].
By altering BA metabolism and its regulated signaling
pathways, gut microbiota could contribute to the patho-
genesis of NASH (Figs.1, 2) [128]. Although the precise
mechanism is unknown, altered BA concentrations have
been reported in patients with NASH [34, 129, 130]. Patients
with NASH have higher fasting and postprandial total serum
BA concentrations, including secondary BA, which tend to
be a more hydrophobic and cytotoxic species [129]. In a
similar study [130], total and secondary BA were increased
in the liver tissues of NASH patients. This increase in BA
concentration could be the consequence of a higher rate
of BA synthesis or possibly be an adaptive response to
the accumulation of TG in the liver [131]. A healthy liver
is very efficient in removing BAs from the enterohepatic
cycle. When the liver function is compromised, more BA
appears in the circulation, because the liver is not adequately
removing them [131]. Higher levels of serum 7α-hydroxy-
4-cholesten-3-one (C4), a BA synthesis intermediate and a
reliable marker of de novo BA synthesis, were also observed
in NASH patients. C4 may represent the hepatic response
to the increased fecal BA losses [35]. Indeed, higher fecal
BA levels have been demonstrated in patients with NASH.
In this study, higher levels of unconjugated primary BA in
the stool correlated with dysbiosis [35].
Dysbiosis could substantially alter BA homeostasis
[35], especially in the colon, where some bacteria, includ-
ing Bacteroides, Clostridium, and Escherichia, are able to
deconjugate and/or dehydroxylate BA, which may lead to an
increase in the circulation of unconjugated secondary BA
Fig. 2 Influence of gut micro-
biota in the development of
nonalcoholic steatohepatitis by
altering bile acid metabolism
and its regulated signaling path-
ways. BA bile acid, CYP7A1 the
first enzyme of bile acid syn-
thesis, FGF15 fibroblast growth
factor 15, FGFR4 FGF receptor
4, FXR farsenoide X receptor;
GLP‑1 glucagon-like peptide-1,
LPS lipopolysaccharide, NASH
nonalcoholic steatohepatitis,
ROS reactive oxygen species,
TNF‑α tumor necrosis factor
alpha, TRG‑5 G protein-coupled
receptor, TLR‑4 Toll-like recep-
tor 4, SHP short heterodimer
partner, VLDL very-low density
lipoprotein
Author's personal copy
Eur J Nutr
1 3
species [129]. At high levels, BA are able to activate inflam-
matory and oxidative stress, resulting in apoptosis or necro-
sis, and eventually fibrosis and cirrhosis [132]. In contrast,
the relative abundance of Clostridium leptum (C. leptum to
total bacteria counts) is decreased in patients with NASH
compared to the controls and correlates with higher cholic
and chenodeoxycholic acids in the stool [35]. Higher levels
of unconjugated primary BA in the stool are positively cor-
related with steatosis, ballooning, and fibrosis. These find-
ings may represent the hepatotoxic impact of hydrophobic
BA. BA can also contribute to the development of NASH
through its effects on intestinal permeability [35]. BA has
bactericidal activity and reduces the intestinal permeability
to endotoxin [133]. However, increased deconjugation of
BA reduces the bactericidal properties of the bile, causing
growth of bacteria that promotes more deconjugation of BA,
and ultimately, translocation and endotoxemia in homeo-
stasis conditions [134]. The interplay between BA and gut
microbiota in human NASH needs to be investigated further.
Altered choline metabolism
Choline is an essential nutrient and phospholipid component
of cell membranes required for the formation of VLDL and
exportation of liver lipids [135]. The metabolism of dietary
choline by microbiota reduces the bioavailability of choline
free to the secretion of VLDL, favoring the accumulation
of fat in the liver (Fig.1) [135, 136]. It is also known that
enzymes produced by the gut microbiota catalyze the con-
version of dietary choline into toxic methylamines, such as
trimethylamine (TMA) [1]. TMA is subsequently oxidized in
the liver, forming trimethylamine-N-oxide (TMAO), which
induces liver inflammation. Moreover, TMAO may affect
glucose and lipid metabolism, promoting the development
of fatty liver [136]. TMAO increases fasting insulin levels
and homeostasis model assessment-estimated insulin resist-
ance (HOMA-IR) and also exacerbates the impaired glucose
tolerance in HFD mice (C57BL/6). These effects are associ-
ated with the expression of genes related to the insulin signal
pathway, glycogen synthesis, gluconeogenesis, and glucose
transport in the liver [137]. The effects of TMAO on lipid
metabolism involve the overexpression of flavin contain-
ing monooxygenase (FMO3) in the human hepatoma cell
line, resulting in increased lipogenesis. These effects may
be mediated through the peroxisome proliferator-activated
receptor alpha (PPARα) and Kruppel-like factor 15 (KLF15)
pathways [138].
A few studies have examined the association of choline
and its metabolites with fatty liver disease. A choline-defi-
cient diet greatly exacerbates a fatty liver induced by HFD
consumption in C57Bl/6 mice [139]. Moreover, Pfp/Rag2
mice, submitted to a choline-deficient diet, develop a fatty
liver featuring fibrosis and elevation of the proinflammatory
markers serum amyloid A (SAA) and TNFα. Hepatic TG is
significantly increased as well as alanine aminotransferase,
demonstrating inflammation-linked hepatocyte damage
[140]. Human studies have shown that the consumption of a
low-choline diet promotes accumulation of TG in the liver
and worsens fibrosis [141]. In a recent study, Chen etal.
[136] have shown adverse associations between the circulat-
ing TMAO level and the presence and severity of NAFLD in
Chinese adults, but no significant choline–NAFLD associa-
tion has been observed. The composition of the gut micro-
biota can change with a low-choline diet. Healthy women
during choline depletion show rate variations of Erysipel‑
otrichi (Firmicutes) and Gammaproteobacteria (Proteobac-
teria) in their fecal contents, which are directly associated
with changes in the liver fat [142]. The Gammaproteobac‑
teria genera in particular, identified in their study, includ-
ing Klebsiella spp., Enterobacter spp., and Escherichia spp.,
are gram-negative bacteria with LPS-containing membranes
[142]. Therefore, the increase of circulating LPS can be one
of the possible mechanisms involved in NASH development
in choline-deficient patients.
On the basis of all these mechanistic pathways, it is
possible to suggest that modulation of the gut microbiota,
through strategies that can include diet, probiotics, antibiot-
ics, or fecal microbiota transplantation, is a possibility for
the treatment of NASH [143]. However, diet appears to be
the simplest, most physiological, and most effective method
to improve intestinal health [144].
Diet andgut microbiota
The diet is among the most easily controlled factors that
can potentially manipulate the gut microbiota [145]. As dis-
cussed above, a high-energy diet, HFD, high-carbohydrate
diet (mainly high-fructose diet), and decreased choline
intake can alter the gut microbiota, which has been shown
to be associated with NASH. Correcting dietary habits is
typically part of the standard recommendations for NASH
treatment [146]. However, we have not found studies in the
literature that have investigated the effects of dietary habit
modifications on the gut microbiota of NASH patients, with-
out the use of probiotics and/or prebiotics. There is some
evidence in animal models and no-NASH subjects that sug-
gests that the amount of dietary calories and the balance
between the three dietary macronutrients (fats, carbohy-
drates, and proteins) have the potential to improve the gut
microbiota [147].
The impact of calorie restriction on gut microbiota has
been demonstrated in C57BL/6J mice [148]. Calorie restric-
tion enriches phylotypes correlated with probiotic effects,
such as Lactobacillus and Bifidobacterium, and reduces
phylotypes correlated with inflammation and obesity, such
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as Streptococcaceae and Desulfovibrionaceae. These calo-
rie restriction-induced changes in the gut microbiota are
concomitant with significantly reduced serum levels of the
LPS-binding protein, suggesting that animals under calorie
restriction can establish a structurally balanced architecture
of the gut microbiota that may exert a health benefit to the
host via reduction of antigen load from the gut. In obese sub-
jects, the reduction in food energy content decreases the phy-
lum Firmicutes and increases Bacteroidetes [10, 149, 150].
Moreover, these calorie-restricted diets increase microbial
gene richness in subjects with obesity [151] and normalize
the circulating LPS levels [152].
The effects of dietary macronutrients on the human
microbiota are still poorly understood. A number of studies
focus on the impact of a “Western” diet (high in animal fat
and protein and low in fiber), compared to a “non-Western”
diet (low in animal fat and protein and high in fiber) [43,
153, 154]. Western and non-Western human diets are con-
sistently associated with distinct gut microbial communities
[43, 153, 154]. Amato etal. [154] observed elevated micro-
bial richness and a relatively higher abundance of Prevotella
in non-Western humans and a relatively elevated abundance
of Bacteroides in Western humans. These results are con-
cordant with a study of the human gut microbiota that asso-
ciates diets high in protein and animal fat with high levels
of Bacteroides and diets high in plant carbohydrates with
high levels of Prevotella [155, 156]. Similarly, all published
studies of Western and non-Western humans to date report
higher microbial richness and a higher abundance of Prevo‑
tella in non-Western populations [43, 153, 154]. One study
also shows a higher abundance of Xylanibacter [156]. The
genera Prevotella and Xylanibacter are known to contain
a set of bacterial genes for cellulose and xylan hydrolysis,
completely lacking in the non-Western population. In addi-
tion, Enterobacteriaceae (Shigella and Escherichia) were
significantly under-represented in non-Western populations
compared to Western populations [156]. Moreover, a clini-
cal trial has shown that healthy subjects with a prudent-style
diet (20% of fat) for 1month reduced plasma LPS levels by
38%, whereas a Western-style diet induced a 71% increase
in plasma levels of endotoxin [157].
The composition of gut microbiota could also be influ-
enced by the quality of dietary lipids. One experiment
evaluated the effect of a fat-type diet, varying in polyun-
saturated-to-saturated fatty acid ratios in the gut microbiota
composition and hepatic TG accumulation [158]. C57Bl/6J
mice were fed purified HFDs (45E% fat) containing palm
oil (saturated lipids), olive oil (monounsaturated lipids), or
safflower oil (polyunsaturated lipids) for 8weeks. According
to the authors, HFD containing palm oil induced a higher
liver TG content, reduced microbial diversity, and increased
the Firmicutes:Bacteroidetes ratio, whereas HFDs contain-
ing olive oil or safflower did not change the gut microbiota
[157]. Similarly, Caesar etal. [159] fed mice isocaloric
diets that differed only in fat composition (either lard or fish
oil, which are rich in saturated and polyunsaturated lipids,
respectively) to assess how the dietary fat sources affected
the microbiota. This study showed that the genera Bacte‑
roides, Turicibacter, and Bilophila had increased in lard-fed
mice, while Actinobacteria (Bifidobacterium and Adlercreut‑
zia), lactic acid bacteria (Lactobacillus and Streptococcus),
Verrucomicrobia (Akkermansia muciniphila), Alphaproteo‑
bacteria, and Deltaproteobacteria had increased in fish-oil-
fed mice. Moreover, TLR-4 was activated by serum from
mice fed with lard, suggesting that a lard diet promotes an
increase in the influx of microbial factors into the systemic
circulation [159].
Regarding carbohydrate intake, there is evidence that
low-carbohydrate diets can impair the gut microbiota, since
they are also restricted in source foods, such as fruits, veg-
etables, and grains, which are rich in fiber (non-digestible
carbohydrates) [160, 161]. In general, the consumption of
fiber-rich diets is associated with greater richness and diver-
sity of the gut microbiota, being positively associated with
the presence of Bacteroidetes and Actinobacteria and with
reduction in the Firmicutes:Bacteroides ratio [150]. Fiber-
rich diets also promote a higher concentration of SCFA in
the fecal contents, especially of butyrate, which has a benefi-
cial effect on inflammation [162]. Thus, the replacement of
fructose, and other simple carbohydrates, with non-digesti-
ble carbohydrates can avoid the consequences of dysbiosis
in the gut–liver axis, especially in NASH.
Finally, the relationship between protein intake and gut
microbiota, specifically in NASH patients, is lacking. The
excess protein has been linked with potentially damaging
effects on the gut microbiota and health. Hoodia etal. [163]
have verified that a high-protein diet reducts Faecalibac‑
terium prausnitzii and increases colon permeability and
secretion of cytokines. Other evidence has reported high
levels of Clostridium spp. and Bacteroides spp., with con-
current reductions in Bifidobacterium spp., Roseburia spp.,
and Eubacterium spp., in subjects who consumed a high-
protein diet [161, 164, 165]. Reductions in Bifidobacterium
spp., Roseburia spp., and Eubacterium may increase the risk
of NASH, as these bacterial species are usually associated
with butyrate production and control of endotoxemia [166].
It is important to mention that, generally, the highest pro-
portion of dietary protein is accompanied by a reduction in
the amount of carbohydrates. Therefore, it is possible that
the impact of the consumption of high-protein diets on the
gut microbiota is related not only to the production of toxic
substances derived from protein fermentation, but also to the
reduction of dietary carbohydrate consumption, especially of
non-digestible carbohydrates [162]. Besides that, the source
of protein varies between studies. For example, populations
that consume meat-rich diets have higher fecal Bacteroides,
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Bifidobacterium, Peptococcus, and anaerobic Lactobacillus
species [167]. A vegetarian diet is associated with higher
rates of Bacteroides thetaiotaomicron, Clostridium clostridi‑
oforme, and F. prausnitzii compared to an omnivorous diet
[168].
Although the studies use different methods and varying
manipulations of diets, which make comparability and gen-
eralization of the outcomes difficult, it seems clear that the
various diets are important environmental factors that regu-
late and modify the gut microbiota.
Conclusions
The development and progression of NASH is a complex
and multifactorial process, which cannot be completely
explained by the “two hits” hypothesis. It is increasingly
recognized that the gut microbiota is implicated in the
pathogenesis and progression of NASH. There are evi-
dences suggesting that NASH patients have a higher prev-
alence of bacterial overgrowth of the small intestine and
changes in the composition of gut microbiota, but there
is controversy regarding the profile of resident bacteria in
the gut. An abundance of the Bacteroidetes phylum may
be increased, decreased, or unaltered in NASH patients.
There is an increase in the Enterobacteriaceae family (phy-
lum Proteobacteria), especially Escherichia. Moreover, the
Bacteroides genus (phylum Bacteroidetes) is also increased.
There is depletion of certain taxa, such as Prevotella, Fecali‑
bacterium, Anaerosporobacter, Oscillospira, Ruminococcus,
Blautia, and Dorea. Although few studies have evaluated the
gut microbiota in NASH patients, it was observed that these
subjects have a distinct gut microbiota compared to the con-
trol groups, which explains, at least in part, the genesis and
progression of the disease through multiple mechanisms.
Changes in the gut microbiota have consequences on
energy homeostasis, resulting in hepatic steatosis. Dysbiosis
is also responsible for increased intestinal permeability and
metabolic endotoxemia, which correlate with inflammation
and liver fibrosis. In addition, it is observed that the metabo-
lism of other related pathways is affected by the gut micro-
biome in NASH, such as choline and bile acid metabolism
and the endogenous production of ethanol. The role of LPS
and bile acids in NASH pathogenesis has been discussed
in several studies and they appear to be the most relevant
factors in humans.
It is essential to identify strategies to modulate the gut
microbiota and probably minimize the development and pro-
gression of NASH. Modulation of gut microbiota by diet
control offers new challenges for future studies.
Acknowledgements The authors gratefully acknowledge the com-
bined support of the Brazilian government organizations (Comissão de
Aperfeiçoamento de Pessoal do Nível Superior-CAPES and Fundação
de Amparo à Pesquisa do Estado de Minas Gerais-FAPEMIG).
Author contributions The author contributions were as follows:
FFG and DGO designed the concept of the study, and all authors were
involved in the literature search and review. FFG and DGO wrote the
manuscript. APBM, JMO, DEC and LEVVCF were involved with
editing the manuscript, and all authors read and approved the final
manuscript.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict
of interest.
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