Gut Microbiome (2022), 3, e5, 1–14

doi:10.1017/gmb.2022.2

ARTICLE

Involvement of microbiota and short-chain fatty acids

on non-alcoholic steatohepatitis when induced by feeding
a hypercaloric diet rich in saturated fat and fructose
Iñaki Milton-Laskibar1,2 , Laura Judith Marcos-Zambrano3, Saioa Gómez-Zorita2,4,5,
Enrique Carrillo de Santa Pau3, Alfredo Fernández-Quintela2,4,5, Jose Alfredo Martínez2 and
María Puy Portillo2,4,5
1
Precision Nutrition and Cardiometabolic Health Program, IMDEA Food Institute (Madrid Institute for Advanced Studies),
Campus of International Excellence (CEI) UAMþCSIC, Spanish National Research Council, Madrid, Spain
2
CIBEROBN Physiopathology of Obesity and Nutrition, Institute of Health Carlos III (ISCIII), Madrid, Spain
3
Computational Biology Group, Precision Nutrition and Cancer Research Program, IMDEA Food Institute, Madrid, Spain
4
Nutrition and Obesity group, Department of Pharmacy and Food Science, Faculty of Pharmacy, Lucio Lascaray Research
Center, University of the Basque Country (UPV/EHU), Vitoria-Gasteiz, Spain
5
BIOARABA Health Research Institute, Vitoria-Gasteiz, Spain
Corresponding author. Email: [email protected]
I.M.-L. and L.J.M.-Z. contributed equally to this work.

(Received 16 June 2021; revised 08 March 2022; accepted 05 April 2022)

ABSTRACT
Consumption of high-energy-yielding diets, rich in fructose and lipids, is a factor contributing to the current
increase in non-alcoholic fatty liver disease prevalence. Gut microbiota composition and short-chain fatty
acids (SCFAs) production alterations derived from unhealthy diets are considered putative underlying
mechanisms. This study aimed to determine relationships between changes in gut microbiota composition
and SCFA levels by comparing rats featuring diet-induced steatohepatitis with control counterparts fed a
standard diet. A high-fat high-fructose (HFHF) feeding induced higher body, liver and mesenteric adipose
tissue weights, increased liver triglyceride content and serum transaminase, glucose, non-HDL-c and MCP-
1 levels. Greater liver malondialdehyde levels and glutathione peroxidase activity were also observed after
feeding the hypercaloric diet. Regarding gut microbiota composition, a lowered diversity and increased
abundances of bacteria from the Clostridium sensu stricto 1, Blautia, Eubacterium coprostanoligenes group,
Flavonifractor, and UBA1819 genera were found in rats featuring diet-induced steatohepatitis, as well as
higher isobutyric, valeric and isovaleric acids concentrations. These results suggest that hepatic alterations
produced by a hypercaloric HFHF diet may be related to changes in overall gut microbiota composition and
abundance of specific bacteria. The shift in SCFA levels produced by this unbalanced diet cannot be
discarded as potential mediators of the reported hepatic and metabolic alterations.

Keywords: Non-alcoholic steatohepatitis; rat; microbiota; high-fat high-fructose diet; short-chain fatty acids

Introduction
Obesity is a chronic metabolic disease featuring an excessive body fat accumulation that may impair
health status and life expectancy (WHO, 2020). The prevalence of obesity has worldwide exponentially
increased in the last decades, becoming one of the most prevalent chronic non-communicable diseases,
which is expected to affect more than 1-billion people by the year 2025 (World obesity, 2021). Obesity

© The Author(s), 2022. Published by Cambridge University Press on behalf of The Nutrition Society. This is an Open Access article, distributed
under the terms of the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted
re-use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 2 I. Milton-Laskibar et al.

leads to the development of other pathological conditions and morbid manifestations, such as diabetes,
cardiovascular diseases, hypertension, certain types of cancer and non-alcoholic fatty liver disease
(NAFLD; Blüher, 2019; Silveira et al., 2021). Indeed, obesity is currently considered as one of the main
causes of early deaths in most developed countries (Blüher, 2019).
Sedentary lifestyle habits and/or excessive energy intake are usually the main contributors to the
development of this multifactorial disease. Moreover, endogenous factors such as genetic predispos-
ition (mainly single nucleotide polymorphisms) and altered gut microbiota composition are also
involved (Hwalla and Jaafar, 2021). In this scenario, the food industry has modified the composition
of a wide variety of foods and foodstuffs, exchanging calories coming from fat (the most energy-
yielding nutrient) for those coming from different sugars in order to reduce their energy density and
thus the energy intake of consumers. Unfortunately, this strategy not only has not been effective in
blunting the aforementioned obesity prevalence increase, but it has contributed to the increase in
added dietary fructose consumption, which has been related to NAFLD development (Softic et al.,
2016). Indeed, once fructose is absorbed in the intestine, the majority of this monosaccharide in the
portal vein enters the liver for subsequent metabolic utilization (Hannou et al., 2018). Within the
liver, fructose is known to impair hepatic lipid metabolism by enhancing de novo lipogenesis, as well
as reducing hepatic fatty acid oxidation, which may result in hepatic lipid accumulation (Hannou
et al., 2018). Moreover, it must be noted that fructose can also induce further dysfunction in the liver
(inflammation, oxidative stress and mitochondrial dysfunction), thus contributing to the onset and
progression of NAFLD from relatively benign conditions (hepatic steatosis) towards more harmful
ones (steatohepatitis, cirrhosis and hepatocellular carcinoma; Jegatheesan and De Bandt, 2017). Of
note, high-fructose intake is also known to induce hepatic insulin resistance due to enhanced fatty
acid synthesis and decreased oxidation (resulting in mitochondrial dysfunction), increased reactive
oxygen species (ROS) production and/or endoplasmic reticulum stress induction (Softic et al., 2019;
Wang et al., 2015).
Moreover, the relationship between fructose and NAFLD is not restricted to the effects exerted by
the sugar in the liver (Jegatheesan et al., 2016). Indeed, gut microbiota composition and permeability
alterations have been reported in trials conducted in rodents fed diets rich in fructose (Jegatheesan
et al., 2016). In this context, the “multiple hit” theory, which is currently used to describe NAFLD
development, considers gut microbiota alteration as one of the potential mechanisms underlying this
morbid liver condition (Buzzetti et al., 2016). In addition, the alterations induced by fructose in gut
microbiota composition can also impair the metabolite profile produced by intestinal bacteria
(Buzzetti et al., 2016). Among them, much attention has been paid to short-chain fatty acids
(SCFAs) due to their effects on energy metabolism or immune response, as well as their ability to
act as signalling molecules (Chakraborti, 2015). These lipid species are the product of indigestible
carbohydrate fermentation by intestinal bacteria (Aragonès et al., 2019), and when gut microbiota
dysbiosis occurs, their concentration may be impaired, affecting hepatic lipid metabolism (Alves-
Bezerra and Cohen, 2017).
In this scenario, the aim of this study was to determine the relationship between changes in gut
microbiota composition and SCFA levels induced by a high-fat, high-fructose (HFHF) feeding in rats.
Likewise, the role played by SCFAs in the development of steatohepatitis was also analysed.

Material and methods

Animals, diets and experimental design
This study was conducted using 20 six-week-old male Wistar rats (Envigo, Barcelona, Spain), and all
the experimental procedures were carried out in agreement with the Ethical Committee of the
University of the Basque Country (document reference CUEID CEBA/30/2010), according to the
European regulations (European Convention-Strasburg 1986, Directive 2003/65/EC and Recommen-
dation 2007/526/EC).

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Rats were housed in polycarbonate metabolic cages (Tecniplast Gazzada, Buguggiate, Italy) in an
air-conditioned room (22°C) with a 12-hour light/dark cycle. After a 6-day adaptation period, the
animals were randomly distributed into two groups of ten animals each: the control group, in which
animals were fed a standard diet (AIN-93G, OpenSource Diets, Denmark, D10012G) and the HFHF
group, in which animals were fed an HFHF diet (OpenSource Diets, Denmark, D09100301; Supple-
mentary Table S1). These experimental conditions were maintained for 8 weeks and animals had free
access to food and water throughout this time frame. Once the whole experimental period was
completed, animals were sacrificed after overnight fasting under anaesthesia (chloral hydrate) by
cardiac exsanguination.
Body weight and food intake were monitored daily. Faecal samples were collected and processed as
explained elsewhere (Milton-Laskibar et al., 2021). Serum was obtained by blood sample centrifugation
after clotting (1,000 g for 10 minutes, at 4ºC). Liver, as well as different white adipose tissue depots
(subcutaneous, epididymal, perirenal and mesenteric) were dissected, weighed and immediately frozen
in liquid nitrogen. Fresh faecal samples were collected at the end of the intervention period, prior to the
overnight fasting. To do so, the animals were taken one at a time and housed in a clean, single cage to
separately obtain faeces directly after defecation induced by a soft abdominal massage. All samples were
stored at –80ºC until analysis.

Determination of liver triacylglycerol content and blood markers

Total liver lipids were extracted following the method described by Folch et al. (1957), dissolved in
isopropanol and subsequently measured using a commercial spectrophotometric kit (SpinReact, Girona,
Spain). Commercially available spectrophotometric kits were also used to determine serum glucose
(Biosystems, Barcelona, Spain), alanine aminotransferase (ALT) and aspartate aminotransferase (AST)
levels. An enzyme-linked immunosorbent assay kit was used to measure serum monocyte chemoattract-
ant protein-1 (MCP-1; Abyntek, Derio, Spain) levels.

Hepatic oxidative stress markers

A commercial thiobarbituric acid reactive substances (TBARSs) assay kit (Cayman Chemical¸ Ann
Arbor, MI, USA) was used to analyse lipid peroxidation in rat liver lysates. The malondialdehyde (MDA)
and TBARS adduct resulting from their reaction in an acid medium was measured using an Infinite
200Pro plate reader (Tecan, Männedorf, Zürich, Switzerland). The obtained results were expressed as μg
MDA/mg of tissue.
The activity of catalase (CAT) was studied as described elsewhere (Gómez-Zorita et al., 2020)
following the method described by Aebi (1984), measuring the H2O2 disappearance spectrophotomet-
rically at a wavelength of 240 nm. Catalase activity was expressed as nmol/min/μg of protein. Glutathione
peroxidase (GPx) activity was measured spectrophotometrically in liver lysates using a commercial kit
(Biovision, Milpitas, CA, USA) and following the manufacturers’ instructions in an Infinite 200Pro plate
reader. Results were expressed as GPx U/mg of protein.

SCFA analysis
Faecal samples (about 30 mg of faeces) were directly weighted to a 1.5-mL LoBind Eppendorf tube and
mixed with 10 μL of internal standard mixture (BA-LAB, PA-LAB and AA-LAB) and 990 μL of a
methanol:water (50:50) mixture. Samples were vortexed for 5 minutes and centrifuged (5 minutes,
15,000 rpm at 4ºC). A volume of 80 μL of the supernatant was mixed with 10 μL BHA 0.1M and 10 μL
EDC 0.25M. Then, samples were vortexed and incubated at room temperature for 1 hour in darkness.
After incubation, the faecal extract was diluted 20-fold in 50 per cent aqueous MeOH. 200μL of diluted
sample was extracted by 600 μL of diethyl ether and 10 minutes of vigorous shaking. Then, samples were

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 4 I. Milton-Laskibar et al.

centrifuged (5 minutes, 15,000 rpm at 4ºC), and 40 μL of the upper organic layer was transferred and
evaporated to dryness using an StarlettePlus-E (SPE)-dryer. The residual was reconstituted in 200 μL of
50 per cent aqueous MeOH, briefly vortexed and centrifuged (5 minutes, 15,000 rpm at 4ºC) prior to 1-
μL injection on LC-MS/MS (Zeng and Cao, 2018).
The chromatographic separation was performed with a gradient, which was 0.1 per cent formic acid
in water with 10 mM of ammonium formate for mobile phase A and 0.1 per cent formic acid in
methanol:isopropanol (9:1 v/v) for mobile phase B. The column temperature was set at 45ºC, and the
injection volume was 1 μL. The source parameters were optimised operating in positive electrospray
ionisation to obtain the maximum response. The validation of the analytical methodology was carried
out by analysing a faecal sample pool by standard addition, using the internal standards mentioned
above. The quality parameters determined were linearity, limit of detection (MLD), limit of quanti-
fication (MQL) and both intraday and interday precision (repeatability and intermediate precision,
respectively).

Faecal DNA extraction and 16S rRNA gene amplification for microbiota composition analysis
DNA extraction was performed in fresh faecal samples collected at the end of the intervention period
using the QIAamp DNA stool MiniKit according to the manufacturer´s instructions (QIAGEN, Hilden,
Germany). The variable V3 and V4 regions of the bacterial 16S ribosomal RNA gene (16S rRNA) were
amplified from the faecal DNA and sequenced with the Illumina MiSeq platform (2  300). Briefly,
amplicon preparation was performed using the 16S Metagenomic Sequencing Library Preparation
Protocol (Illumina, San Diego, CA, USA), which includes overhang adapter sequences for compatibility
with Illumina index and sequencing adapters. Amplicon size was subsequently verified by electrophor-
esis (LabChip GX; PerkinElmer, Waltham, MA, USA). DNA libraries for 16S rRNA amplicons
sequencing were prepared with the Nextera XT DNA Library Preparation Kit (Nextera XT; Illumina)
according to the manufacturer’s instructions.
The 16S rRNA gene sequence data were processed using the Quantitative Insights Into Microbial
Ecology program (QIIME 2; Bolyen et al., 2019). Low-quality reads were filtered, and chimeric sequences
were removed afterwards. Clean reads were clustered as amplicon sequence variants (ASVs) using
DADA2 (Callahan et al., 2016) and annotated with the SILVA v.132 16S rRNA gene reference database
(Quast et al., 2013). The relative abundance of each ASV and alpha diversity (Shannon, Chao and
Simpson indexes) were calculated using the phyloseq R package (McMurdie and Holmes, 2013).
Weighted UniFrac distances were used to calculate Beta-diversity and then visualised with principal
coordinate analysis (PCoA). Statistical significance was determined by permutational multivariate
analysis of variance (PERMANOVA) with 999 random permutations using the function adonis from
the vegan R package (version 2.5.7; Dixon, 2003). We performed Linear Discriminant Analysis effect size
(LEfSe) to identify the bacterial taxa differentially enriched in different bacterial communities and
establish microbial biomarkers (Segata et al., 2011).

Statistical analysis
Descriptive results are presented as mean  SEM. Statistical analyses were performed using SPSS 24.0
(SPSS, Chicago, IL, USA). In the current analysis, all variables, with the exception of microbiome
variables, were normally distributed according to the Shapiro-Wilks test. Data were tested by an
independent Student’s t-test, being p < 0.05 values considered as statistically significant. In the case of
differences in the abundance of taxa, statistical analysis was performed with the Kruskal–Wallis test.
Significance was also set up at the p < 0.05 level.
Correlations between differentially enriched bacterial taxa and the studied SCFAs, and phenotypic
and inflammatory parameters were estimated by Spearman’s rank method, using the Microbiome

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package in R (available at https://microbiome.github.io/tutorials/; accessed 28 May 2021). The correl-

ation was assessed as Coefficient ≥ 0.2 and FDR ≤ 0.05.

Results
Body weight, liver weight, adipose tissue weights, liver triacylglycerol content and serum parameters
As shown in Figure 1A, the body weights of the animals fed the HFHF diet became significantly higher
than those observed in the control group since the second week of the study and remained until the end of
the experimental period (Week 8). Higher liver weights were also found in the animals of the HFHF
group in comparison to the animals in the control group (Figure 1B). Indeed, this same pattern was also
found regarding liver triacylglycerol content, where the values found in the HFHF group were signifi-
cantly higher than those in the control group (Table 1). As far as the weight of different adipose deposits
is concerned, significant differences were only found in the case of mesenteric adipose tissue. In this case,
the values observed in the HFHF group were significantly higher than those found in the control group
(Figure 1C). In turn, no significant changes were found between the two groups regarding visceral
adipose tissue nor total adipose tissue weights, although non-statistically significant trends were found in
both cases (p < 0.1) towards increased weights in the HFHF group.
With regard to serum parameters, the fasting serum glucose level observed in the animals from the
HFHF group was significantly higher in comparison to that found in the control group (Table 1). Serum
non-high-density lipoprotein cholesterol (non-HDL-c), which was calculated by subtracting serum
high-density lipoprotein cholesterol (HDL-c) levels from total serum cholesterol levels, was significantly
increased in the HFHF group compared to the control group. Finally, the analysis of serum MCP-1 levels

Figure 1. Body weight evolution (A), liver weights (B), weights of different adipose tissue depots (C) and serum transaminase levels
(D) of rats fed the experimental diets for 8 weeks. Values are means  SEM (n = 10). Statistical analyses were performed using unpaired
Student’s t-test. *The level of probability was set up at p < 0.05 as statistically significant, and # was used to represent values of p < 0.1.
ALT, alanine aminotransferase; AST, aspartate aminotransferase; AT, adipose tissue; C, control group; HFHF, high-fat high-fructose fed
animals; VAT, visceral adipose tissue.

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Table 1. Serum glucose, insulin, non-HDL-c and MCP-1 levels, TyG index, hepatic triglyceride content, MDA content, and
activities of CAT and GPx in the liver of rats fed on the experimental diets for 8 weeks.

Control HFHF

Serum

Glucose (mmol/dL) 5.41  0.31 6.55  0.08*

Non-HDL-c 51.5  3.5 108.4  9.0**

MCP-1 (pg/mL) 210.5  11.0 322.1  23.7**

Liver

Liver triglycerides (mg/g tissue) 17.1  2.1 53.4  9.5**

MDA (μM/mg tissue) 720.2  50.5 1,008.8  88.8*

CAT (nanomols/min/μg) 169.1  23.1 179.6  13.4

GPx (U/mg tissue) 1.26  0.16 5.99  1.70*

Notes: Values are means  SEM (n = 10). Statistical analyses were performed using unpaired Student’s t-test.
Abbreviations: CAT, catalase; GPx, glutathione peroxidase; HDL-c, high-density lipoprotein cholesterol; MCP-1, monocyte chemoattractant
protein-1; MDA, malondialdehyde; TyG, triglyceride-glucose index; NS, not significant.
*p < 0.05.
**p < 0.01.

revealed that this parameter was significantly increased in the HFHF group in comparison to the control
group (Table 1).

Hepatic oxidative stress markers

The hepatic oxidative stress analysis revealed that the amounts of MDA found in the liver samples of
animals in the HFHF group were significantly greater than those found in the animals in the control
group (Table 1). In addition, while no changes were observed between the two groups regarding CAT
activity, a higher GPx activation was observed in the livers of the animals in the HFHF group compared
to those in the control group (Table 1).

SCFA analysis
Among all the studied SCFAs, significant differences were found in isobutyric, isovaleric and valeric
acids, whose faecal concentrations were significantly greater in the HFHF group compared to the control
group (Table 2).

Dietary induced shifts in microbiota composition

To understand the underlying mechanisms by which HFHF diet contributed to hepatic damage, the effects
of dietary strategies on the gut microbiota composition were explored. As measured by β-diversity, there
was a significant difference in overall microbial composition between the two experimental groups
(p < 0.001, PERMANOVA). Figure 2 shows PCoA using the weighted UniFrac distance matrix. The
microbiota of HFHF group was clearly separated from that of the control group.
A significant decrease in alpha diversity was observed in the HFHF group, as showed by the Chao1
index (C = 73.2 and HFHF = 57.2; p < 0.01; Figure 3A). In addition to the overall microbial composition,
gut microbiota was evaluated at different levels to establish differences in the abundance of microbial
taxa according to both dietary groups, and to select those bacteria which can represent potential
microbial biomarkers of each condition by performing a LEfSe analysis (Figure 3B,C). In the case of
the animals in the control group, the most abundant bacteria were from the Class Clostridia, order

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Table 2. Concentration of SCFAs in faecal samples of rats fed on the experimental diets for 8 weeks.

Control HFHF

Acetic acid C2:0 (nmol/g) 14,234  2,094 16,924  2,034

Propionic acid C3:0 (nmol/g) 2,915  456 5,337  1,334

Isobutyric acid C4:0 (nmol/g) 449  74 852  125*

Butyric acid C4:0 (nmol/g) 2,126  384 2,006  330

Isovaleric acid C5:0 (nmol/g) 340  65 870  139**

Valeric acid C5:0 (nmol/g) 515  56 1,539  297*

Hexanoic acid C6:0 (nmol/g) 18  4 12 1

Notes: Values are means  SEM (n = 10). Statistical analyses were performed using unpaired Student’s t-test.
*p < 0.05.
**p < 0.01.
Abbreviation: NS, not significant.

Figure 2. Principal coordinate analysis (PCoA) weighted UniFrac plot. Components PCoA1 and PCoA2 are shown (p < 0.001,
PERMANOVA). All samples are connected to the centroid (shown as a point). C, control group, represented in red circles; HFHF,
high-fat high-fructose fed animals, represented in blue triangles.

Clostridiales, family Ruminococcaceae (particularly the Ruminiclostridium 9, Ruminococacceae UCG

005 and Ruminococacceae UCG 014 genera), as well as bacteria from Class Clostridia (Lachnospiraceae
UCG 004 and Coprococcus 3 genera) and phylum Tenericutes (Anaeroplasma and Mollicutes RF39
genera) were observed. Another genus of less-described bacteria (ie. Muribaculaceae uncultured and
Lachnospiraceae UCG 004) was also characteristic of this group.
As far as the rats fed the HFHF diet (HFHF group) are concerned, bacteria from the phylum
Firmicutes, class Bacilli, (particularly the Lactococcus genus), as well as bacteria from the class
Clostridia, particularly the genera Clostridium sensu stricto 1, Blautia, Eubacterium coprostanoligenes
group, Flavonifractor and the uncharacterized genus UBA1819 genera were the most abundant ones.

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 8 I. Milton-Laskibar et al.

A B

Figure 3. Linear discriminant analysis (LDA) integrated with effect size (LEfSe). Cladogram representing the differentially abundant
taxonomic groups (LDA score > 4, p < 0.001) (A), microbial diversity according to Chao1, Shannon and Simpson indexes (B) and
histogram representing the 20 most abundant genera (C) in rats fed on the experimental diets for 8 weeks. C, control group,
represented in red; HFHF, high-fat high-fructose fed animals, represented in green.

Interestingly, these changes found in the HFHF group occurred despite at class (Clostridia) and order
(Clostridiales) levels, the relative abundances in the control group were greater (Figure 3C). In addition,
bacteria from the class Erysipelotrichia (particularly genus Fecalitalea) were also overrepresented in the
animals from the HFHF group.
A correlation analysis looking for an association between the observed microbial biomarkers and the
changes reported in phenotypic and biochemical parameters, SCFA abundances and markers of hepatic
oxidative stress in the liver was conducted. A negative correlation was found between bacteria enriched
in the control group fed with a standard diet (Ruminococcaceae UCG 005, Ruminococcaceae UCG
014 and Ruminiclostridium 9) and liver weight, transaminase (ALT) and non-HDL-c levels. On the other
hand, bacteria found in HFHF group (Clostridium sensu stricto 1, Clostridiaceae 1) was correlated
positively with liver weight, non-HDL-c and GPx (Table 3 and Supplementary Figure S1)

Discussion
The worldwide increase in NAFLD prevalence has converted this morbid liver condition in a global
health problem, becoming the most common hepatic alteration not only in adults, but also in children

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Table 3. Significant correlations between the relative abundance (%) of microbial biomarkers selected after LEfSe analysis
A and several phenotypic and metabolic parameters in rats fed on the experimental diets for 8 weeks.

Spearman correlation (r) values for microbial groups founds after LEfSe analysis

Microorganism Liver markers Correlation (r) p.adj (FDR)

Ruminococcaceae UCG-014 Non-HDL-c 0.802 0.0151

Clostridium sensu stricto 1 Liver weight 0.765 0.0241

Clostridium sensu stricto 1 Non-HDL-c 0.755 0.0278

Clostridiaceae 1 Liver weight 0.778 0.0347

Clostridiaceae 1 Non-HDL-c 0.768 0.0347

Ruminococcaceae UCG-014 Liver weight 0.729 0.0387

Ruminococcaceae UCG-014 ALT 0.716 0.0387

Ruminococcaceae UCG-005 Liver weight 0.725 0.0388

Ruminiclostridium 9 Liver weight 0.722 0.0388

Clostridium sensu stricto 1 GPx 0.742 0.0425

Abbreviations: ALT, alanine aminotransferase; GPx, glutathione peroxidase; HDL-c, high-density lipoprotein cholesterol.

(DiStefano and Shaibi, 2021; Masarone et al., 2014). NAFLD encompasses a wide spectrum of liver
alterations, from a relatively benign steatosis to more harmful situations such as cirrhosis or hepatocel-
lular carcinoma (Engin, 2017). Besides excessive lipid accumulation in the liver, events such as oxidative
stress, inflammation and fibrosis are involved in the progression of hepatic damage (Brunt et al., 2015).
High-fat intake, a common feature of “westernised diets,” is considered among the triggering factors of
NAFLD development. In this regard, excessive dietary fat intake (specially saturated fats) can alter
hepatic lipid metabolism, impairing the balance between liver lipid “input” (plasma fatty acid uptake and
de novo lipogenesis) and “output” (mitochondrial fatty acid oxidation and very-low-density lipoprotein
release), thus resulting in an excessive liver lipid accumulation (Lian et al., 2020). Additionally, much
attention has also been paid to added fructose intake as another factor leading to NAFLD development.
The consumption of this sugar has increased in the last decades concomitantly with the expansion of
NAFLD (DiStefano and Shaibi, 2021). Indeed, excessive fructose consumption may not only increase
hepatic lipid accumulation, but also produce liver inflammation and fibrosis, leading to the development
of non-alcoholic steatohepatitis (Jegatheesan and De Bandt, 2017).
In accordance with these facts, in the present study, rats fed with the HFHF diet showed increased
liver weight and triglyceride content, which were accompanied by an increase in the levels of serum
transaminases (ALT and AST), commonly used as markers of liver function impairment (Sattar et al.,
2014). Moreover, alterations in glycaemic control (higher fasting glucose) and dyslipidaemia (higher
non-HDL-c levels) were also found in these same animals. These observations are in agreement with the
available scientific literature, where fructose derived alterations in glucose and lipid metabolisms have
been described (Softic et al., 2020; Stanhope et al., 2015).
In addition, the enhanced hepatic MDA content found in the rats fed the HFHF diet suggests that a
greater ROS production occurred in these animals, which in turn resulted in higher lipid peroxidation.
Moreover, the enhanced GPx activity found in the same rats points towards a higher antioxidant
response, most likely as an attempt to revert the oxidative damage produced by the HFHF diet.
Additionally, the higher circulating MCP-1 levels found in the HFHF group could be expected since
the involvement of this cytokine in liver damage progression has been previously reported (Glass et al.,
2018; Kirovski et al., 2011).

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 10 I. Milton-Laskibar et al.

Traditionally, the so-called “two hit” theory has been used to describe the events resulting in NAFLD.
According to this theory, the “first hit” is originated by insulin resistance mediated excessive hepatic lipid
accumulation due to enhanced de novo lipogenesis and altered fatty acid transport, whereas the “second
hit” accounts for hepatic oxidative stress, inflammation and mitochondrial dysfunction. All these events
would lead to NAFLD development, as well as progression to nonalcoholic steatohepatitis (NASH)
(Engin, 2017). However, this theory has been considered as too simplistic, and thus the “multiple hit”
theory has been proposed, which, besides the aforementioned mechanisms, also considers the alterations
in gut microbiota composition as one of the contributing factors (Buzzetti et al., 2016).
In the present study, the HFHF diet not only led to a decreased microbial α-diversity as revealed by
lowered values in the Chao1 index, but it also affected the abundances of specific gut bacteria. Similar
results were previously reported in Wistar rats fed a diet rich in fat and sucrose (45 and 17 per cent of the
total energy as fat and sucrose, respectively) for 6 weeks (Etxeberria et al., 2015). Indeed, bacteria from
Ruminococcaceae family, known to be inversely correlated with NAFLD in humans (Astbury et al.,
2020), were abundant in the control group and significantly diminished in the animals fed the HFHF
diet. In addition, the abundance of several bacteria genera that have been related to liver alterations, such
as Clostridium sensu stricto 1 or Blautia, were increased in the HFHF group. Interestingly, these changes
occurred despite at class (Clostridia) and order (Clostridiales) levels the abundances in these bacteria
were greater in the control group. These findings suggest that the negative effects elicited by the HFHF
feeding affects the overall gut microbiota diversity, and therefore the abundance of specific bacteria. The
results obtained in this study regarding gut microbiota composition are in good accordance with those
reported by other authors using rodent models fed with unbalanced experimental diets (Chen et al., 2019;
Daniel et al., 2014; Duparc et al., 2017; Leal-Díaz et al., 2016). One of the limitations of this study is that
the 16S rRNA analysis of gut microbiota composition was only carried out at one timepoint, using faecal
samples collected at the end of the study. The main reason for not collecting faecal samples at previous
time points (at the beginning of the study) is that after the adaptation period, the animals were randomly
distributed in the experimental groups assuming that there were no differences between both experi-
mental groups at baseline. Other limitation is the lack of resolution at species level of amplicon
sequencing allowing characterisation of microbial changes only to genus level, and the representation
of less characterised genera such as UBA1819.
Besides gut microbiota composition, the different metabolic products that are produced by intestinal
bacteria have also gain interest as mediators of microbioma-host crosstalk (Bashiardes et al., 2016).
Among them, SCFAs have received much attention since these metabolites are known to be involved in
the maintenance of body weight and energy homeostasis or glucose and lipid metabolism, as well as
being related to NAFLD development (Bashiardes et al., 2016). In this regard, SCFAs may protect against
NAFLD, improving gut barrier integrity, reducing visceral adipose tissue accumulation and derived
excessive fatty acid entrance into the liver, as well as by directly exerting anti-inflammatory effects once
having reached the liver (Dai et al., 2020).
Acetate, propionate and butyrate are the three major volatile SCFAs produced by gut bacteria and are
known to participate in a variety of processes (Deleu et al., 2021). In the case of the liver, studies
conducted in rodents and humans have described hepato-protective effects for these SCFAs. Several
mechanisms of action have been described to date regarding the effects of SCFAs in liver protection
(Dangana et al., 2020) including the modulation of visceral adipose tissue fat accumulation and lipid
metabolism. Moreover, the improvement produced by these SCFAs in gut barrier function is also
potentially responsible for their hepato-protective effects. Additionally, the activation of nod-like
receptor family pyrin domain containing 3 inflammasome and resulting release of interleukin 18 pro-
duced by acetate, butyrate and propionate has also been described to improve gut barrier integrity (Macia
et al., 2015). Despite the well characterised hepato-protective effects of acetate, propionate and butyrate,
no differences on their levels were found between the control and HFHF groups. As far as the enhanced
faecal level of isobutyric acid found in the HFHF group of our study, it could be considered as expected
since increased faecal level of this bacterial product has been found in patients with NAFLD (Da Silva
et al., 2018; Jumpertz et al., 2011). In addition, a greater level of isobutyric acid has been reported in faecal

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 11

samples of subjects with hypercholesterolemia in the study reported by Granado-Serrano et al. (2019),
where a positive correlation between faecal isobutyric acid level and serum low-density lipoprotein
cholesterol level was observed. Moreover, in that study, a higher faecal isovaleric acid concentration was
also found in patients with hypercholesterolemia, which fits well with the results of our study, where the
rats fed the HFHF diet showed the same effect (Granado-Serrano et al., 2019). However, in our case, no
correlation was found between the level of this SCFA and non-HDL-c-levels. As valeric acid is
concerned, the studies addressing the effects of this SCFA in liver steatosis are scarce.
In order to better understand the potential associations between the studied microbial measurements
and the phenotypical, biochemical and SCFA levels, correlation studies were carried out. According to
the results obtained, bacteria from the Ruminococcaceae family could be considered as markers of low
liver weight and/or serum transaminase and non-HDL-c levels, since negative correlations were found in
the control group between these bacteria and the aforementioned markers. These results could be
explained because these bacteria have been related to gastrointestinal health in humans due to their effect
in the maintenance of intestinal structure and functions, such as permeability, nutrient uptake and
immunocompetence (Rajilić‐Stojanović and de Vos, 2014; Tang et al., 2018). As far as the positive
correlations found in the HFHF group between Clostridium sensu stricto 1 and Clostridiaceae 1 with
parameters such as non-HDL-c and GPx, these results are in line with data reported in studies in
humans, in which the abundance of these bacteria was positively correlated with HDL-c molecule
diameter (Vojinovic et al., 2019). However, other studies have also reported negative correlations of these
bacteria with markers such as liver stiffness measurement and controlled attenuation parameter in
humans (Lanthier et al., 2021). Indeed, reductions in abundance have been related to the development of
liver fibrosis in patients with severe steatosis (Lanthier et al., 2021). Therefore, despite Clostridium sensu
stricto 1 and Clostridiaceae 1 are indicators of less healthy microbiota (Yang et al., 2019), further studies
are warranted in order to better understand this apparent discrepancy. With regard to SCFAs, despite
significant differences in their levels were found between the control and HFHF groups, no correlations
were found with gut microbiota composition. These outcomes may result unexpected, especially for rats
in the control group whose faeces revealed a greater presence of butyrate-producing bacteria such as
Ruminococcaceae, but without an actual change of the levels of butyrate. Similarly, increased acetate
levels could also been expected in the animals fed the HFHF diet since a relationship between this SCFA
and metabolic syndrome has been proposed through a microbiota-brain axis (Perry et al., 2016). In this
scenario, it could be hypothesised that the observed changes in gut microbiota composition were not big
enough to shift the production of certain SCFA. In this regard, a longer experimental period could have
resulted in a clearer relationship between shifts in gut microbiota composition and SCFA levels.
In conclusion, the current study demonstrates that the alterations induced by a high-energy diet rich
in saturated fat and fructose in the liver, as well as the impairment of several metabolic markers involved
in glycaemic control and lipid homeostasis, may be related, at least in part, to changes in overall gut
microbiota composition and the abundance of specific bacteria derived by this dietary pattern since
several mechanisms of action have been characterised (increased gut permeability, enhanced bacterial
translocation and production/release of pro-inflammatory mediators or inflammation of adipose tissue
and liver) linking the two events (Deleu et al., 2021). Nevertheless, further determinations are warranted
to better elucidate/confirm this hypothesis. As far as SCFA is concerned, the differences found between
the experimental groups were not extensive despite the significant changes induced by the HFHF diet in
gut microbiota richness and diversity. It seems that under these experimental conditions, the role played
by SCFAs in diet-induced steatohepatitis development seems limited and may not be associated with gut
microbiota shifts.

Acknowledgement. The SCFA analyses were performed with the equipment of the Centre for Omic Sciences (COS), Joint
Unit of the Universitat Rovira i Virgili and Eurecat.

Supplementary Materials. To view supplementary material for this article, please visit http://doi.org/10.1017/gmb.2022.2.

Disclosure statement. The authors declare no conflict of interest.

https://doi.org/10.1017/gmb.2022.2 Published online by Cambridge University Press

 12 I. Milton-Laskibar et al.

Author contributions. Conceptualisation: M.P.P., J.A.M. and I.M.-L.; Data curation: I.M.-L., L.J.M.-Z., S.G.-Z., E.C.S.P. and
A.F.-Q.; Formal analysis: I.M.-L., L.J.M.-Z. and E.C.S.P.; Funding acquisition: M.P.P.; Investigation: M.P.P., J.A.M., I.M.-L.,
L.J.M.-Z., S.G.-Z. and A.F.-Q.; Methodology: I.M.-L., S.G.-Z. and M.P.P.; Writing – original draft: M.P.P., J.A.M. and I.M.-L;
Writing – review & editing: I.M.-L., L.J.M.-Z., S.G.-Z., E.C.S.P., A.F.-Q., M.P.P. and J.A.M.

Funding. I.M.-L. acknowledges financial support from the Juan de la Cierva Programme-Training Grants of the Spanish State
Research Agency of the Spanish Ministerio de Ciencia e Innovación y Ministerio de Universidades (FJC2019-038925-I). L.J.M.-
Z. has a Juan de la Cierva Grant (IJC2019-042188-I) from the Spanish State Research Agency of the Spanish Ministerio de
Ciencia e Innovación y Ministerio de Universidades. This research was funded by the Ministerio de Economía y Competiti-
vidad-Fondo Europeo de Desarrollo Regional (grant number AGL-2015-65719-R MINECO/FEDER, UE), Instituto de Salud
Carlos III CIBERobn (grant number CB12/03/30007) and University of the Basque Country (grant number GIU 18/173).

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Cite this article: Milton-Laskibar I., Marcos-Zambrano L.J., Gómez-Zorita S., Carrillo de Santa Pau E., Fernández-Quintela A.,
Martínez J.A., and Portillo M.P. 2022. Involvement of microbiota and short-chain fatty acids on non-alcoholic steatohepatitis
when induced by feeding a hypercaloric diet rich in saturated fat and fructose. Gut Microbiome, 3, e5, 1–14. https://doi.org/
10.1017/gmb.2022.2

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