Pre Biotic Os

Gut microbiota
Original research
Prebiotic diet changes neural correlates of food
Gut: first published as 10.1136/gutjnl-2023-330365 on 4 October 2023. Downloaded from http://gut.bmj.com/ on June 30, 2024 by guest. Protected by copyright.
decision-making in overweight adults: a randomised
controlled within-subject cross-over trial
Evelyn Medawar ‍ ‍,1,2,3 Frauke Beyer ‍ ‍,1,4 Ronja Thieleking ‍ ‍,1
Sven-Bastiaan Haange ‍ ‍,5 Ulrike Rolle-Kampczyk ‍ ‍,5 Madlen Reinicke ‍ ‍,6
Rima Chakaroun,7,8 Martin von Bergen,5 Michael Stumvoll ‍ ‍,8 Arno Villringer ‍ ‍,1,4
A Veronica Witte ‍ ‍1,4
► Additional supplemental ABSTRACT

material is published online Objective Animal studies suggest that prebiotic, WHAT IS ALREADY KNOWN ON THIS TOPIC
only. To view, please visit the ⇒ Targeting high-caloric food craving and
journal online (http://d x.doi.org/ plant-derived nutrients could improve homoeostatic
10.1136/gutjnl-2023-330365). and hedonic brain functions through improvements in unhealthy eating behaviour is crucial for
microbiome–gut–brain communication. However, little prevention and treatment of the worldwide
For numbered affiliations see obesity pandemic. The gut microbiome has
end of article. is known if these results are applicable to humans.
Therefore, we tested the effects of high-dosed prebiotic been implicated in feeding behaviour through
Correspondence to fibre on reward-related food decision-making in a modifying gut-brain crosstalk, for example,
Dr A Veronica Witte, Cognitive randomised controlled within-subject cross-over study short chain fatty acid production.
Neurology, University of and assayed potential microbial and metabolic markers.
Leipzig Medical Center, Leipzig, WHAT THIS STUDY ADDS
Design 59 overweight young adults (19 females,
Germany; ⇒ We here present causal evidence for effects of
v eronica.witte@medizin.uni- 18–42 years, body mass index 25–30 kg/m2) underwent
functional task MRI before and after 14 days of supplementary prebiotics on reward-related
leipzig.d e
supplementary intake of 30 g/day of inulin (prebiotics) food decision making in a group of 59 well-
Received 5 June 2023 and equicaloric placebo, respectively. Short chain fatty characterised overweight adults. Leveraging
Accepted 12 September 2023
acids (SCFA), gastrointestinal hormones, glucose/lipid advanced neuroimaging, next-generation
Published Online First sequencing and multiomics, our results suggest
4 October 2023 and inflammatory markers were assayed in fasting blood.
Gut microbiota and SCFA were measured in stool. functional microbial changes that underly these
Results Compared with placebo, participants showed effects.
decreased brain activation towards high-caloric wanted HOW THIS STUDY MIGHT AFFECT RESEARCH,
food stimuli in the ventral tegmental area and right PRACTICE OR POLICY
orbitofrontal cortex after prebiotics (preregistered, family
⇒ Our findings strengthen the hypothesis that
wise error-corrected p <0.05). While fasting blood levels
remained largely unchanged, 16S-rRNA sequencing dietary prebiotics cause a reduction of reward-
showed significant shifts in the microbiome towards related brain activation in response to high-
increased occurrence of, among others, SCFA-producing caloric food stimuli. A better understanding of
Bifidobacteriaceae, and changes in >60 predicted underlying microbiome–gut–brain mechanisms
functional signalling pathways after prebiotic intake. could help to develop novel strategies towards
Changes in brain activation correlated with changes fostering healthier eating behaviour in humans.
in Actinobacteria microbial abundance and associated
activity previously linked with SCFA production, such as
ABC transporter metabolism. including feeding5 and psychological functioning6
Conclusions In this proof-of-concept study, a prebiotic via the microbiota–gut–brain axis, however, direct
intervention attenuated reward-related brain activation experimental evidence is still limited.
during food decision-making, paralleled by shifts in gut Microbiota-derived metabolites of plant- based
microbiota. dietary fibre such as short-chain fatty acids (SCFA),
Trial registration number NCT03829189. can cross the blood–brain barrier7 to modulate
hypothalamic signalling.8 First experimental studies
showed that oral intake of the SCFA butyrate or
© Author(s) (or their
employer(s)) 2024. Re-use of the butyrate- producing bacteria Akkermansia
permitted under CC BY-NC. No INTRODUCTION spp lowered body weight (in humans9) and
commercial re-use. See rights Plant-based diets, recognised as a major effector of restored obesity-induced functional brain changes
and permissions. Published planetary health,1 are more beneficial for cardio- (in mice10). Moreover, 1 week of colonic SCFA
by BMJ.
vascular and brain health compared with conven- delivery modulated hypothalamic-pituitary-adrenal
To cite: Medawar E, Beyer F, tional Western diets.2 3 Plant-based food and related axis-
dependent stress- induced cortisol response
Thieleking R, et al. Gut prebiotic nutrients are less dense in calories and in a study including 66 healthy men,11 and intake
2024;73:298–310. have been claimed to modulate brain function4 of autologous faeces- derived microbiota from a
298 Medawar E, et al. Gut 2024;73:298–310. doi:10.1136/gutjnl-2023-330365
Gut microbiota
Figure 1 Study design. Within-subject cross-over dietary intervention design with two study arms and up to six measurement timepoints (upper
panel, T0: screening; BL1/2: baseline 1/2, FU1/2: follow-up 1/2, T6: additional follow-up). Participants were randomly assigned to receive first prebiotics
and second placebo (arm 1), or vice versa (arm 2), for 14 days each, separated by a 14-day wash-out period. Following the same timeline, at BL1,
FU1, BL2 and FU2, participants provided stool samples and underwent fasting blood draw (1), anthropometric measurements (2), received a standard
breakfast shake (3) and MRI assessments (4), followed by brief surveys (5), food remuneration (6) and further tests and questionnaires (7–8). Steps
(9–11) indicate data processing and statistical analysis. Screens give fMRI wanting task paradigm scheme and timing. BL, baseline; FU, follow-up;
fMRI, functional magnetic resonance imaging (MRI); LC-MS/MS, liquid chromatography–mass spectrometry; SPM, statistical parametric mapping, SwE,
sandwich estimator, WGNCA, weighted graph network correlational analysis. Created with BioRender.com.
dietary weight-loss period enhanced weight loss maintenance in food cues.20 In parallel, disinhibition and unhealthy food
humans.12 craving, sometimes controversially described as food addic-
Earlier trials in humans showed that supplementary intake of tion,21 have been linked with subtle structural differences in the
prebiotic fibre such as inulin-type fructans reduced subjective reward network22 23 and with differential brain activation in the
hunger and improved gut hormonal-driven appetite regulation ventromedial prefrontal cortex (vmPFC) in response to high-
through changes in postprandial glucagon-like peptide (GLP)-1, caloric food stimuli.24 Whether these effects can be mitigated by
neuropeptide y (PYY)13 (n=10) and ghrelin14 15 (both n<50). In prebiotic dietary targeting the gut–brain axis25 is yet unknown.
another randomised clinical trial (RCT) in >100 patients with We here aimed to test the hypothesis that a high-dosed prebi-
obesity, inulin compared with placebo induced greater weight otic fibre intervention can alter the gut microbiome and thereby
loss16 and exploratory results indicated mood improvements in neural activation patterns of food reward in a population at risk
a microbiota-based subgroup with elevated relative Coprococcus for weight gain and insulin resistance. To this end, we conducted
abundance at baseline.17 Own results from two cross-sectional an RCT in overall healthy adults in a randomised within-subject
analyses indicated that habitual overall dietary fibre intake links cross-over design and assessed food wanting using fMRI before
to specific microbiota genera including Parabacteriodes, which in and after 14 days of daily 30 g supplementary intake of inulin
turn explained variance in eating behaviour in adults with over- (prebiotic fibre) and equicaloric maltodextrin (placebo), respec-
weight and treatment success after bariatric surgery.18 tively. Suggested microbial and metabolic mediators of potential
However, neuroimaging evidence of how prebiotic diets and effects were measured using faeces and serum proxies collected
diet-related microbial changes affect the brain with regard to at all four timepoints. The study and analyses were preregistered
eating behaviour remains to be shown. At the brain level, food at ClinicalTrials.gov/NCT03829189 and osf.io/ynkxw.
decision-making is thought to rely on a complex interplay of
homoeostatic and hedonic signalling, orchestrated by a variety
of subcortical and cortical networks involving the brainstem METHODS
and hypothalamus, striatum and prefrontal cortex areas.19 The Study design
neurobiological underpinnings of (unhealthy) eating behaviour In this within-subject cross-over design, participants underwent
and their neuroimaging correlates, however, have not been screening and, if eligible, received both verum and placebo in a
fully understood. Functional MRI (fMRI) studies indicated that randomised order (two arms) for 14 days each, separated by a
presentation of highly palatable food cues leads to a stronger wash-out period of at least 2 weeks (figure 1). Verum (prebiotic
brain response in reward areas than equicaloric, non-palatable fibre) consisted of 30 g inulin (63 kcal, 26.7 g fibre, Orafti Beneo
Medawar E, et al. Gut 2024;73:298–310. doi:10.1136/gutjnl-2023-330365 299
Gut microbiota
Figure 2 Consolidated Standards of Reporting Trials (CONSORT) flow diagram. Participants underwent a randomised controlled dietary intervention
trial in a within-subject cross-over design. BMI, body mass index; fMRI, functional MRI.
Synergy1, BENEO, Mannheim, Germany) per day compared contraindications, aged 18–45 years, women: intake of oral
with calorie-matched placebo consisting of 16 g maltodextrin contraceptives. Exclusion criteria were: neurological or psychi-
(63 kcal, 0 g fibre), each provided as two sachets per day. atric disease; intake of medication acting on the central nervous
Data acquisition took place between 2019 and 2022 with system; diabetes mellitus type 2; severe untreated internal disease
some breaks due to lockdown regulations during the SARS- including the gastrointestinal tract, lung, heart, vasculature, liver
CoV-2 pandemic. All participants were invited to baseline and and kidneys; eating disorder or unconventional eating habits;
follow-up visits for each condition, resulting in four study visits women: pregnancy, breastfeeding as well as daily consumption
with faeces and fasting blood sample collection, fMRI and ques- of >50 g alcohol, >10 cigarettes, or >6 cups of coffee. Out of
tionnaires. Briefly, after fasting blood draw and anthropometrics 106 initially recruited volunteers with screening assessment, 59
(~45 min), participants received a neutral drink covering 10% participants (19 women, 40 men) took part in the study, with
of their individual daily energy requirement. Right after, the 45 completing all 4 measurement visits (figure 2). For power
MRI assessment followed (~2 hours), which was then followed analysis and sample size rationale, see online supplemental
by further computer-based assessments (~1.5 hours) (see online file_general.
supplemental file_general for further details).
Registration and blinding
Participants Participants received a small reimbursement of €9–€10/hour
Volunteers of all gender were recruited via online and local for testing days and additionally €30 for study completion.
advertisements and the institute’s local database. Inclusion The study was registered at https://clinicaltrials.gov/ct2/show/
criteria were a body mass index of 25–30 kg/m2, no MRI NCT03829189 and https://osf.io/f6qz5 (14 January 2019)
Gut microbiota
prior to recruitment and data acquisition. Additionally, details Microbial analysis
on fMRI (pre)processing were uploaded before the start of data For 16S-rRNA gene profiling, DNA was extracted and V3–V4
analysis https://osf.io/ynkxw (11 May 2021). Participants and variable regions of the 16S-rRNA genes were amplified by PCR
staff members were blinded regarding the study intervention/ and a library was constructed, followed by paired-end 2×250 bp
placebo allocation. Sachets were labelled with either A or B Illumina sequencing. Raw sequencing data analysis was done on
through a random assignment performed by author AVW, who the inhouse Galaxy server using a pipeline implemented with the
was not involved in data collection, prior to the study. Alloca- DADA2 R-package processed data in fastq format.27 For further
tion to the A-B or B-A study arm was determined following a details, see online supplemental file_microbiome.
randomised order generated using the R software’s ‘sample()’
function by author RT. Authors EM and RT enrolled partici-
Statistical analysis
pants and assigned them to the intervention arm accordingly.
On a behavioural level, we hypothesised that participant’s
wanting ratings scored higher for food compared with art (H_
Patient and public involvement behav_1), and that wanting would change after prebiotic inter-
The authors acknowledge a missed opportunity of not following vention (H_behav_2), dependent on caloric density of the food
a tailored approach to involve patients or the public in the design item (H_behav_3). Linear mixed models were performed in R
of the study. We invited and collected comments and assessments (version>3.6) using lmer(), for a model-of-interest and a null
from all participants throughout the study to inform the design model for each effect of interest. Model residuals were tested
of upcoming research studies. for normal distribution using the R package performance() with
the command check_normality(x, effects=‘random’), see online
MRI supplemental file_behav for details.
MRI was performed on a 3T Siemens Prismafit scanner with a On a neural level, we hypothesised that food evaluation elicits
32-channel head coil. FMRI was done in an event-related design different regional brain activation compared with art evaluation
assessing wanting of food and art, respectively. Participants (H_neural_1), and that this differential brain response changes
were presented with four sets of images across four sessions after prebiotic intervention (H_neural_2). Inference tests were
(randomised order). Each stimulus was shown for 4000 ms with performed using a homoeostatic and reward-related region-of-
the question ‘How much do you want this now?’, followed by interest brain mask on first-level contrasts (designs A–C) and
a 4000 ms response period, followed by 500–4000 ms inter- second-level factors time (baseline, follow-up), group (prebiotics/
stimulus interval with a 500 ms jitter until the next stimulus was placebo), and time×group interactions, using the Sandwich Esti-
presented (figure 1). Wanting ratings were done on a 8-point mator (SwE V.2.2.2, https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/Swe,
Likert scale with 1 labelled as ‘not at all’ and 8 as ‘absolutely’. implemented in SPM V.12.7486 run in MATLAB V.>9.0) and
Participants were informed about receiving a reward right after R (V.>3.6). All main analyses were run in a homoeostatic regu-
the scanning session outside the scanner, for food and art, respec- lation and reward-related region-of-interest brain mask defined
tively, based on their highest ratings in that session. The reward by a combination of two meta-analyses of available previous
was given as a dish to eat right away and as a carton-based art independent studies at neurosynth. org using the keywords
print to take home with. ‘hypothalmus’ and ‘reward’, respectively, integrating functional
Preprocessing was done using fMRIPprep V.1.2.5.26 As prereg- brain responses of 922 and 98 studies, respectively (created in
istered, first-level contrasts of interest were global difference April 2021; figure 3 in online supplemental file_fMRI). Signif-
between food and art viewing, food compared with art wanting icant results were reported according to threshold-free cluster
slope, and wanting modulation (design A), food wanting by enhancement methods with alpha <0.05 and family wise error
caloric or fibre density (design B) and considering liking ratings (FWE) correction for multiple comparisons. For details, see
as modulator (design C). See online supplemental file_fMRI for fMRI preregistration and online supplemental file_fMRI.
further details. Further exploratory analyses were done with the aim to
generate hypotheses on potential mechanisms between changes
Additional behavioural assessments in the microbiome/metabolism and changes in brain activa-
Dietary habits, lifestyle factors including gastrointestinal quality tion. First, intervention effects versus placebo were explored
of life, sleep, physical activity, mental well-being and mood were in anthropometrics and blood and faeces markers according to
assessed at each timepoint. Additionally, we assessed potential mixed effects inference with (restricted) maximum likelihood
traits associated with food decision-making at baseline, that is, fitting and χ2 test for comparison. Microbiome composition and
on personality, eating behaviour, anxiety and well-being, as well predicted functional pathways based on Kyoto Encyclopaedia
as on art knowledge (see online supplemental file_behav for of Genes and Genomes (KEGG 28) were analysed using Stress
details). test on non-metric multidimensional scaling (NMDS) prior to
individual genera/pathway testing with linear mixed effects
modelling. Second, bivariate correlation analyses were done on
Blood and faeces markers
the difference (delta) post versus pre after prebiotic treatment,
To assess serum SCFA, gut hormones (ghrelin, GLP- 1,
in those outcomes that showed a significant group×time inter-
PYY), markers of glucose/lipid metabolism (glucose, insulin,
action effect only. Significance threshold for exploratory anal-
glycated haemoglobin A1c, high and low density lipopro-
yses was set at p<0.05, follow-up microbiome analyses were
tein, triglycerides), inflammatory markers (high sensitive C
corrected for multiple comparisons using false discovery rate.
reactive protein, interleukin-6, TNFalpha) and other markers
(trimethylamine-n-oxid and amino acids), blood was obtained
in fasting state (12.5±2.2 hours fasted) at the same time per Data and code availability
participant for each session. Stool samples were taken within Data are available at https://doi.org/10.17605/OSF.IO/FC4 and
1–2 days before the testing day to assess faecal SCFA and micro- code is available at https://gitlab.gwdg.de/gut_brain_study/food-
bial markers. wanting/task-fmri-behavior-analysis and https://gitlab.gwdg.
Gut microbiota
Figure 3 Behavioural (A, B) and neural response (C, D) to food and art stimuli in overweight adults during decision-making. Participants responded
to food with higher wanting scores compared with art (nobs=32 111, nsubj=59) (A), showing highest mean values for moderately high caloric stimuli,
and lowest mean values for art objects (nobs=32 111, nsubj=59) (B). Food compared with art valuation elicited stronger brain activation particularly
in subcortical areas of the reward network (nsubj=57) (C), while additional parametric modulation with wanting scores indicated a stronger brain
activation in ventromedial prefrontal cortex and orbitofrontal cortex when comparing food versus art (nsubj=57) (D). Statistics were done with linear
mixed effect modelling, up to 4 time points per participant×120 stimuli on wanting scores (main analysis) (A), (exploratory analysis) (B) and on
voxel-wise blood-oxygen-level-dependent signal using the sandwich estimator toolbox with threshold-free cluster enhancement (TFCE) family wise-
error correction (FWE) of multiple comparisons (C,D),(main analyses) (C,D). Colour bars depict parametric TFCE statistic (TFCE-t >50 for visualisation
purposes) with wild-boot strapped pFWE<0.05 marked in red outline.
de/gut_brain_study/food-wanting/fmri-analysis.29 Statistical Neurobehavioural correlates of reward-related decision-

MRI maps are available at https://identifiers.org/neurovault. making
collection:14111. Overall, wanting and liking ratings in the fMRI preference
task were higher for food than for art stimuli (H_behav_1;
RESULTS nobs=32 111, nsubj=59, b=1.03, t=7.78, 8, p<0.001,
A total of 59 well-c haracterised overweight/obese adults figure 3A,B, online supplemental file_behav-table 2). Food eval-
were included in main analyses (19 women, 40 men, mean uation activated large parts of the reward network, including
age 28 years±6.2 SD, body mass index (BMI) 27.3 kg/ ventral tegmental area (VTA), hypothalamus, nucleus accumbens
m 2±1.4 SD, socioeconomic status 14.2±3.2; table 1, online (NAc), basal ganglia and ventromedial thalamus, as well as ante-
supplemental file_general-t able1). rior insula, amygdala, cingulate, vmPFC and distinct parts of
the orbitofrontal cortex (OFC) (H_neural_1, n=57, design A,
pFWE<0.05; figure 3C). Similarly, higher wanting ratings for
Table 1 Baseline characteristics food compared with art elicited higher brain activation ubiqui-
tously across these brain areas, yet particularly in the vmPFC and
n=59
OFC (design A, pFWE<0.05; figure 3D).
Gender (n) Women 19
Men 40
Age (years) Mean (SD) 28.3 (6.55) Effect of prebiotics on food decision-making
Median (min, max) 28 (19.0, 45.0) At the behavioural level, individuals’ overall wanting scores were
BMI (kg/m2) Mean (SD) 27.3 (1.51) not different after the 2-week prebiotic intervention regarding
Median (min, max) 27.0 (25 30) food versus art and when accounting for calories or fibre,
SES index (score) Mean (SD) 14.5 (2.98) contrary to our hypothesis (H_behav_2+3; nobs=32 111/16,071,
Median (min, max) 14.4 (5.10, 19.2) nsubj=59, ball < ∣0.07∣, tall < ∣1.0∣; pall>0.32, online supple-
Habitual dietary fibre (g/day) Mean (SD) 16.3 (6.31) mental file_behav tables 7a, 10 and 11). Exploratory analysis
Median (min, max) 15.4 (1.54, 30.5) showed however that prebiotics compared with placebo led to
Blood HbA1c (%) Mean (SD) 5.31 (0.20) significantly lower overall wanting scores (online supplemental
Median (min, max) 5.30 (4.6, 5.8) file_behav table 7b). That is, when looking at stimulus subcat-
Missing 2 (3.4%) egory, participants reported decreases in wanting for very low
BMI, body mass index; HbA1c, glycated haemoglobin A1c; Max, maximum; Min, minimum; SD, standard and very high caloric content as well as for plants after prebiotics
deviation; SES, socioeconomic status.
(approximatively −0.3 points on the Likert scale, figure 4A;
Gut microbiota
Figure 4 Effects of prebiotic intervention on food decision-making. After the intervention, participants decreased wanting scores for food from
caloric quartiles 1 and 4 as well as animals (exploratory analysis, nsubj=59, (A). At the neural level, brain activation decreased in the ventral tegmental
area (VTA) and in two clusters in the orbitofrontal cortex (OFC) towards high-caloric, wanted food stimuli (main analysis, nsubj=57, (B, C). Statistics
according to linear mixed effects modelling, up to 4 time points per participant×120 stimuli on wanting scores and on voxel-wise blood-oxygen-
level-dependent signal using the sandwich estimator toolbox with threshold-free cluster enhancement (TFCE) family wise-error correction (FWE) of
multiple comparisons. Colour bars depict parametric TFCE statistic with wild-boot strapped pFWE<0.05 marked in red outline (upper right panel) and as
enlargement (lower right panel).
nobs=32 111, nsubj=59, group×time×subcategory: all p<0.01; (ball>0.09, tall>2.4, pall<0.013; examples figure 5C,D). BMI,
online supplemental file_fMRI-Results). waist-to-hip ratio, and blood pressure did not change signifi-
According to fMRI (H_neural_2), we did not observe changes cantly, which was also true for fasting ghrelin, GLP-1 and PYY,
in regional brain response after prebiotics in food compared glucose, insulin, amino acids, as well as inflammatory markers
with art viewing, food compared with art wanting slope, or (see online supplemental file_general tables 2–5).
wanting modulation (design A). However, brain activation In exploratory bivariate correlation analysis on change
towards wanted, high-caloric food (design B) decreased after scores after the prebiotic intervention, mean bold activa-
prebiotics compared with placebo in three clusters, in the VTA tion in the three outlined VTA and OFC clusters decreased
(pFWE-corr=0.042), in the right OFC (rOFC, pFWE-corr<0.05) in correlation with decreases in fasting PYY (Spearman’s
and in the right medial OFC (rmOFC, pFWE-corr<0.05) (n=57, r all>0.32, p all<0.05).
figure 4B,C, table 2). In addition, art liking compared with food
liking increased in a small cluster in the right NAc after prebiotics
compared with placebo (design C, table 2). See online supple- Changes in gut microbiota and parameters
mental file_fMRI-Results for secondary and sensitivity results. The prebiotic intervention led to increases in stool frequency
In addition, after prebiotics, participants reported less subjec- (b=1.2, t=2.1, p=0.04, figure 6A). Through 16S- rRNA
tive hunger during the fMRI task, compared with placebo analysis, we detected significantly decreased richness, even-
(exploratory analyses, ß=−0.39, p<0.001; figure 5A, online ness and alpha diversity after prebiotics compared with
supplemental file_behav tables 18 and 19). placebo (nobs=200, n subj=57, all p<0.001 figure 6B–Donline
While both intervention and placebo supplements contained, supplemental file_microbiome_table 1). Beta diversity on
the same amounts of calories and participants reported equally Amplicon Sequencing Variant was significantly different
high compliance in taking the daily supplements, we observed in after prebiotic intervention (NMDS, prebiotics: padj=0.001;
exploratory analysis decreases in body fat after placebo (time- figure 6E), and there were abundance changes in families
point×intervention, b=0.16, p=0.005; figure 5B). In addi- of Actinobacteria and Firmicutes (all p adj<0.02, figure 6F).
tion, lipid markers were significantly lower after placebo intake Zooming at the genera level, prebiotics induced significant
compared with prebiotics, as well as alanin- aminotransferase shifts in various abundances, including profound increases in
Table 2 Localisation of significant changes in brain activation to visual food and art stimuli during functional MRI, after prebiotic compared with
placebo intervention (main analysis)
TFCE P (FWE- TFCE cluster Peak X Y Z
Prebiotic compared with placebo corr) size Peak Z P(unc) (mm) (mm) (mm) Region
Parametric modulation, food wanting×kcal; decreases in activation 0.038 51 3.595 0.002 26 32 16 Right OFC
0.042 43 3.388 0.001 4 20 14 VTA
0.043 41 3.075 0.003 10 36 20 Right (medial) OFC
Art liking>food liking slope; increases in activation 0.039 3 3.827 0.001 8 16 6 Right NAc
NAc, nucleus accumbens; OFC, orbitofrontal cortex; TFCE, threshold-free cluster enhancement; unc, uncorrected; VTA, ventral tegmental area.

Gut microbiota
Figure 5 Changes in secondary eating behaviour-related outcomes after prebiotic (red) compared with placebo condition (light blue). Hunger
ratings during fMRI significantly decreased after prebiotics (A), while gender-standardised body fat mass (FM-stand, B), serum lipid markers low-
density lipoprotein (LDL, C) and cholesterol (CHOL, D) significantly decreased after placebo (linear mixed effects modelling, all p<0.05, exploratory
analyses). FMRI, functional magnetic resonance imaging.
Bifidobacteria (table 3, online supplemental file_microbiome not provide compelling evidence that clusters of microbial taxa
table 2). related to neurobehavioural outcomes (exploratory analyses,
online supplemental file_microbiome).
Changes in microbiota genera link to changes in
neurobehavioural outcomes SCFA and microbial functional capacity prediction
We further explored whether the observed changes in micro- We could not detect changes in SCFA acetate, butyrate and
bial genera predicted intervention-induced changes in neurobe- propionate after intervention, neither in fasting serum nor in
haviour. According to these exploratory analyses, a less severe faecal concentrations (exploratory analyses, nobs≥122, nsubj≥40,
decrease in Subdoligranulum correlated with intervention- pall>0.39, table 4).
induced decreases in VTA brain activation towards wanted, Next, we explored changes induced by microbial shifts on the
high caloric food stimuli after prebiotic intervention (r=−0.38, metagenomic level according to KEGG28 analysis. Changes in
p=0.01). Note that bacterial abundance was measured in KEGG orthologue relative abundance were significantly different
percentage, thus a relative decrease in Subdoligranulum does after prebiotics (figure 7A, NMDS: prebiotics padj=0.001,
not necessarily display absolute decrease after prebiotics. Addi- placebo padj=0.99, posthoc- pairwise permutational multivar-
tionally, increases in Lactiplantibacillus (lactic acid producing iate analysis of variance (PERMANOVA): F=11.46, padj=0.002,
bacteria) were significantly related to increases in rmOFC activa- online supplemental file_microbiome table 3). The KEGG
tion (r=0.40, p=0.008), however abundance of this bacterium orthologues were annotated to 158 pathways out of which about
did not change in all participants. 44%, that is, 69 were significantly altered in relative abundance
Complementary weighted network analyses in a subgroup of after prebiotic intervention compared with placebo, including
available participant data from all four timepoints (n=35) did pathays related to carbohydrate, protein and fat metabolism,
Gut microbiota
Figure 6 Microbiota-related shifts after 2-week prebiotic intervention (exploratory analyses). Increases in stool frequency (A) and decreases in
(B) microbiota richness, (C) evenness, (D) Shannon index, (E) beta diversity changes compared by dissimilarity gradients according to group and
timepoint after prebiotics (pink) compared with placebo (blue), and (F) shifts in microbial family distribution. Asterisks in (A–D) indicating significant
ANOVA results for null-full model comparisons (p<0.05). ANOVA, analysis of variance; ASV, amplicon sequencing variant; NMDS, non-metric
multidimensional scaling.
plant degradation or cell repair (padj<0.05, online supplemental DISCUSSION

file_microbiome table 4). In this proof-of-concept study, we tested the effects of a prebiotic
More specifically, exploratory analyses indicated that increases intervention on food decision-making in a randomised within-
in relative abundance of Bifidobacteria correlated significantly subject cross-over design including 59 well-characterised, over-
with increases in metabolic pathways related to taurine, seleno weight adults. In preregistered analyses, we found that 14 days
compounds, nicotinate and amino acids, and with decreases of high-dose dietary prebiotics, compared with placebo, led to
related to porphyrin metabolism, steroid degradation, (unsatu- decreases in bold-related brain activation towards high caloric,
rated) fatty acid biosynthesis, and DNA repair functions (exem- wanted food in the VTA and right OFC measured using 3T
plary figure 7B,C; Spearman’s rall>0.32, pall<0.05). In addition, fMRI. In parallel, prebiotics led to significant shifts in relative
increases in Lactobacillus and decreases in Gordonibacter abundance of the gut microbiota, including increases in SCFA-
correlated with increases in pyruvate metabolism pathway, a producers such as Bifidobacteria and Collinsella. Exploratory
precursor of SCFA (note that not all participants changed in analyses indicated intervention- induced changes in relative
Lactobacillus and Gordonibacter abundance, though). Further abundance and predicted metabolic pathways correlated with
exploratory analyses indicated that decreases in VTA brain acti- changes in VTA brain activation. While fasting gut hormones,
vation after prebiotic intervention correlated with intervention- inflammatory markers and SCFA in blood and faeces remained
induced significant decreases in pathways involved in flavonoid unchanged, we observed that prebiotics- induced decreases in
and stilbenoid biosynthesis, two-component signal transduction, brain activation in reward areas related to decreases in fasting
biofilm formation, amino sugar and nucleotide sugar metabo- PYY.
lism, citrate cycle (rall>0.37, pall<0.05), and with significant
increases in ATP-binding cassette transporters (ABC, r=−0.39,
p<0.05, exemplary figure 7D,E). Decreases in rOFC activation Changes in functional brain activation
after prebiotics correlated with significant decreases in aromatic Only few studies with moderate sample size have addressed
hydrocarbon degradation (r=0.32, p<0.05). Decreases in whether manipulating the microbiome can alter brain func-
rmOFC activation after prebiotics correlated with significant tions. A parallel trial in 34 females indicated that 4 weeks of
increases in oxidative phosphorylation (r=−0.31, p<0.05). For fermented milk consumption (including Bifidobacteria) induced
details, see online_supplemental_file_microbiome figure 2a and resting-state functional connectivity changes in the midbrain.30
2b. Another randomised trial reported that 4 weeks of probiotic
Gut microbiota
Table 3 Significant shifts in microbiota relative abundances on the genera level after prebiotic intervention, according to 16S-rRNA sequencing
and linear mixed effects modelling after FDR-correction for multiple comparisons
Interaction effect time (follow-up)×intervention (prebiotic) ANOVA null model comparison
Increased abundance b t P value padj
Anaerostipes 0.73 3.01 0.003 0.017
Bifidobacterium 9.82 10.42 <0.001 <0.001
Collinsella 2.66 4.96 <0.001 <0.001
Holdemanella 0.37 3.13 0.002 0.011
Lachnospiraceae FCS020 group 0.21 3.31 0.001 0.006
Lacticaseibacillus 0.10 2.05 <0.001 0.002
Lactiplantibacillus 0.03 2.82 <0.001 <0.001
Lactobacillus* 2.08 2.65 0.008 0.045
Ligilactobacillus 0.28 2.67 0.008 0.045
Limosilactobacillus 0.28 5.10 <0.001 <0.001
Decreased abundance
Desulfovibrio 0.20 3.41 0.001 0.006
Eggerthella 0.33 3.46 0.001 0.006
Eubacterium brachy group 0.11 3.18 0.002 0.011
Eubacterium eligens group 0.21 2.76 0.006 0.033
Roseburia 1.10 3.86 <0.001 0.001
Ruminococcus gauvreauii group 0.69 3.86 <0.001 0.001
Shuttleworthia 0.08 2.78 0.006 0.033
Subdoligranulum 1.30 2.82 0.005 0.028
Linear mixed effects modelling outcome compared to null model and model of interest as follows (ANOVA model comparison with p<0.05): with the Formula: bacterial_genus_
of_interest−time point×intervention+time point+intervention+(1+(intervention+time point)|subject). All models run on nobs =204 in nsubj=58 and listed in alphabetical order of
genera of interest.
*Statistics refer to models without random slopes due to non-convergence.
ANOVA, analysis of variance; FDR, false-discovery rate.
supplementary powder containing Bifidobacteria and Lacto- increase in body fat after prebiotics which was not statistically
bacillae resulted in changes in microbial genera abundance significant when comparing pre versus post, but in the interac-
that correlated with improvements of emotional attention and tion model, that is, when taking into account a marginal decrease
memory, paralleled by differences in related brain activation.31 after placebo (discussed below). This anthropometric data might
Our findings now present prebiotics-induced changes in brain speak against a significant translation of the observed changes in
activation with potential implications for food craving and brain activation to healthier eating behaviour, however two weeks
decision-making: While the neuronal processes underlying may be too short to generate robust trends in body composition
human eating behaviour are far from fully understood,32 neuro- and studies incorporating longer durations are needed.
imaging studies indicate neural responses within VTA and OFC
to underly dopamine-related reward anticipation and subjective
value attribution of food, respectively, linking stronger BOLD- Microbiota-related mechanisms
related activation to higher reward values and decision-making.33 The gut microbiome has only recently been shown to be rele-
Indeed, midbrain and medial OFC activation during fMRI in vant for host nutritional foraging in rats, for example, through
response to milkshake taste predicted the amount of milkshake changing circulating amino acids and bacterial tryptophan.38
intake after the scan.34 Another faecal transplantation rat study indicated that micro-
Consistently, drivers of reward considering food (such as biota from obese donors resulted in changes in food prefer-
caloric content) modulate subjective value particularly in the ence and expression of dopaminergic markers in the striatum.39
OFC,35 and the right OFC has been specifically implicated in In humans, a single-group study in 26 females suggested that
food-related motivation.36 Notably, decreases in brain activation increased consumption of vegetables rich in inulin-type fruc-
towards high caloric food cues such as ice-cream in the OFC tans over two weeks increased Bifidobacteria and decreased the
has for example been shown using fMRI when participants desire to eat sweet, salty, and fatty food.40 In the current study,
were instructed to consider health aspects or long-term conse- we similarly observed changes in multiple bacterial genera abun-
quences of consumption, compared with ‘naive’ viewing.37 The dances after prebiotics compared with placebo, mainly increases
intervention-induced decreases in VTA and rOFC in the current in Actinobacteria phylum (eg, Bifidobacteria) and Firmicutes
study might thus indicate a diminished anticipation of reward, phylum (eg, Lactobacillus). This suggests a marked increase in
and a smaller subjective value attribution to high-caloric wanted fiber-degrading, SCFA producing bacteria that are present in the
foods after prebiotic treatment, potentially translating in a subtle gut, which is in line with previous human trials.16 41–44
reduction of the desire for high-caloric food. At the behavioural Functional capacity prediction analyses further yielded a
level, we could not confirm a general reduction in food wanting multitude of different pathways that were selectively changed
ratings, yet exploratory analysis indicated less wanting of very after prebiotic intervention, among them pathways involved in
high and very low caloric food, as well as certain art objects, SCFA production capable to modify systemic SCFA signalling.
and less hunger after prebiotics. We also observed a marginal For example, one of the most strongly upregulated pathways
Gut microbiota
related to the ABC transporters (ko02010). It has been shown
that Lactobacillus use dietary fibre (e.g., inulin) via ABC trans-
P value
porters to produce acetate,45 which can be further degraded to
0.60
0.84
butyrate.46 Multiple of the upregulated microbiota genera after
prebiotics have been classified in previous studies to produce
Change
−12.7
SCFA, eg, Anaerostipes, Bifidobacterium and Holdemanella.47
−7.0
20.0
(%)
0.0
Moreover, pointing to a dose–effect relationship, a less severe
decrease in relative Subdoligranulum abundance (also SCFA
mean±SD
6.2±3.2
6.6±3.4
0.6±0.6
0.4±0.4
producers), as well as the increases in prebiotics-induced upregu-
Post,
lation of ABC transporters, correlated with significant decreases

in prebiotics-induced VTA brain activation in the current study.
Propionate (μmol/g)
This may suggest a potential mechanistic route of higher SCFA

41
42
24
28
mean±SD n
production leading to lessened reward anticipation, however,

7.1±2.4
7.1±3.1
0.5±0.5
0.4±0.4
these considerations need to be taken with caution due to the
Pre,
lack of direct evidence.

In contrast to our a priori hypothesis, we did not observe
42
42
28
27
changes in faecal or fasting blood levels of acetate, butyrate

value n
or propionate, suggesting that, in principle, changes in brain

0.78
0.88
activity may have been driven by other indirect factors. Similar

P
to our trial, previous small scale studies could not show increases
Change
in faecal SCFA after inulin, for example, in healthy young adults

22.6
29.2
mean±SD (%)
2.5
0.0
(subgroup, n=49).48 Others observed SCFA increases, for

example, in type 2 diabetes mellitus (n=25,49), or even decreases
8.1±2.4
7.4±3.2
3.8±3.0
3.1±3.2
in faecal SCFA (n=30).42 These conflicting results might be

Post,
explained by unknown complexity of local and systemic micro-

bial effects, and/or by pre-existing differences such as micro-
41
42
32
28
n
biota patterns at baseline (note higher relative Firmicutes in

Acetate (μmol/g)
mean±SD
our overweight/obese group compared with obesity studies), or

7.9±2.7
7.4±2.4
3.1±3.2
2.4±2.3
differences in stool frequency, weight and fluidity (note signifi-

Pre,
cant changes in Bristol stool scale after prebiotics in the current

study). The latter opens the possibility that changes in, for
42
42
30
32
value n
example, gut motility (specifically anticipatory contractions on

0.70
0.59
seeing food stimuli in the scanner) may underlie the observed

P
changes in brain responses.

Change
Body fat and lipid markers slightly improved after placebo

−20.0
−8.2
−4.3
(%)
0.0
condition and worsened after prebiotics in the current study.

While we did not observe changes in lifestyle habits according
mean±SD
16.8±8.0
15.5±9.4
to questionnaires, beneficial effects of for example increased

0.5±0.4
0.4±0.3
Post,
energy expenditure in the placebo phase cannot be ruled out.

P, according to ANOVA null-full model p value, total=sum of butyrate, acetate and proprionate.
Also, inulin, particularly at high doses, might challenge liver

cholesterol metabolism, as postulated in mice under certain
41
42
34
37
n
Butyrate (μmol/g)
conditions.50 A recent human study further reported spikes in

mean±SD
18.3±8.7
16.2±6.9
liver enzymes, cytokines and cholesterol in some participants

0.5±0.3
0.5±0.4
after 30 g/day inulin,51 underlining the possibility that the dosage

Pre,
of inulin in the current study might have exceeded optimal levels.

42
42
36
34
For serum SCFA, others did find short-term increases in SCFA

value n
Concentrations of faeces and serum SCFA levels
after inulin,52 and the postprandial increase in SCFA correlated

0.39
0.65
with decreases in serum ghrelin.53 Prebiotics and SCFA also stim-

P
ulate the expression of PYY and GLP-1 in the gut,54 that may
Change
ANOVA, analysis of variance; SCFA, short chain fatty acid.
contribute to changes in central reward-related food responses.

−6.8
−2.9
−5.2
−3.6
(%)
In humans, PYY injections induced changes in BOLD-related

fMRI signalling in the hypothalamus, VTA and OFC.55 56 We
mean±SD
23.2±12.2
24.6±8.5
5.5±1.5
5.3±1.5
found that decreases in fasting PYY correlated with decreases in

Post,
brain activation in the VTA and OFC clusters after intervention,

pointing to a similar mechanisms. However, a (postprandial)
40
42
27
25
n
increase in serum SCFA or gut hormones due to prebiotics in

mean±SD
our sample might have been masked after overnight fasting.

26.4±10.9
Total (μmol/g)
23.9±8.8
5.8±1.3
5.5±1.4
Pre,
Limitations
Prebiotics 42
42
Prebiotics 27
29
n
Our study should be discussed in light of several limitations.

First, 14 days of intervention can be considered too short to
Table 4
Placebo
Placebo
Faeces
Serum
induce long-lasting effects on neuronal processes involved in

eating behaviour. Also, secondary analyses did not replicate the
Gut microbiota
Figure 7 Predicted functional shifts and their correlations with changes in microbiota genera and in reward-related brain activation after prebiotic
intervention (exploratory analyses). (A) Dissimilarity of functional composition of microbiome preprebiotic to versus postprebiotic intervention based
on NMDS stress test (p=0.001) and principal component analysis of relative abundance of predicted KEGG orthologues statistics, calculated by
PERMANOVA (padj=0.002). (B) Change scores of Bifidobacterium abundance and arginine biosynthesis (ko00220), (C) Bifidobacterium abundance and
cysteine and methionine metbolism (ko00270), (D) flavonoid biosynthesis (ko00941) and changes in reward-related brain response, (E) stilbenoid,
gingerol biosynthesis (ko00945) and reward-related brain response. (B–E), all r>0.32, all p<0.05 according to Spearman’s correlation, line gives
regression fit with 95% CI. KEGG, Kyoto Encyclopaedia of Genes and Genomes; NMDS, non-metric multidimensional scaling; PERMANOVA,
permutational multivariate analysis of variance; VTA, ventral tegmental area.
exact same activation clusters at the whole brain level or when SCFA production, gut motility or PYY and reduced reward-
further constraining fMRI analyses to very small peak areas of related brain activation during food- decision making. While
the reward network. By following recommendations to fully the current data does not allow us to conclude that the prebi-
preregister the applied brain mask and statistical thresholding otic treatment-induced changes in brain responses were benefi-
in addition to further preprocessing steps, we, however, aimed cial for behavioural control, neural response in reward-related
to ensure confidence in the robustness of the observed effects. areas during fMRI have previously shown to predict behaviour
Exploratory analyses need to be interpreted with caution due to change,57 underlining implications for the treatment of unhealthy
their non-confirmative nature. In addition, KEGG analyses need eating behaviours or overnutrition using microbiome-changing
to be considered indirect only and microbiome samples were not
interventions. Future studies are needed to explore whether such
time-locked to MRI sessions. Due to the within-subject cross-
treatments could open avenues for less invasive approaches to
over design, however, interindividual differences at baseline
obesity.
determining microbiota responses could be kept to a minimum.
Also, participants belonged to a Western, Educated, Indus-
Author affiliations
trialised, Rich and Democratic society and we did not recruit 1
Department of Neurology, Max Planck Institute for Human Cognitive and Brain
representative shares of female and diverse gender, limiting Sciences, Leipzig, Germany
generalisability of results difficult. 2
Berlin School of Mind and Brain, Humboldt-Universität zu Berlin, Berlin, Germany
3
Charité Universitätsmedizin Berlin, Berlin, Germany
4
CONCLUSIONS Cognitive Neurology, University of Leipzig Medical Center, Leipzig, Germany
5
Department of Molecular Systems Biology, Helmholtz-Centre for Environmental
According to preregistered RCT analysis of advanced 3T-fMRI,
Research - UFZ, Leipzig, Germany
this proof- of-
concept study suggests that a high- dosed 6
Institute for Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics,
microbiome- changing prebiotic intervention decreases brain University of Leipzig Medical Center, Leipzig, Germany
responses to high- caloric food cues during decision- making 7
Department of Molecular and Clinical Medicine, University of Gothenburg,
within 2 weeks in overweight adults. Based on 16S- rRNA Goteborg, Sweden
8
combined with functional pathway prediction and metabo- Medical Department III Endocrinology Nephrology Rheumatology, University of
lomics, exploratory findings offer the possibility of a mecha- Leipzig Medical Center, Leipzig, Germany
nistic link between prebiotic dietary intake, related changes in Twitter Evelyn Medawar @EvelynMedawar and A Veronica Witte @witte1veronica

Gut microbiota
Acknowledgements We thank all the individuals who took part in the study. 3 Chen X, Maguire B, Brodaty H, et al. Dietary patterns and cognitive health in older
For participant support, we thank Maria Dreyer, Ramona Menger, Bettina Johst adults: a systematic review. J Alzheimers Dis 2019;67:583–619.
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specifically Nicole Pampus, Sylvie Neubert, Mandy Jochemko, Anke Kummer and microbiota-gut-brain-a xis. Exp Biol Med (Maywood) 2021;246:796–811.
Domenica Klank, and Torsten Schlumm. For fMRI analysis support we thank Hannah 5 Yu KB, Hsiao EY. Roles for the gut microbiota in regulating neuronal feeding circuits. J
Sophie Heinrichs. For lab support we thank Laura Hesse, Charlotte Wiegank, Lina Clin Invest 2021;131:e143772.
Eisenberg, Emmy Töws and Anna-Luise Wehle. For all other data collection, we 6 Dalile B, Van Oudenhove L, Vervliet B, et al. The role of short-chain fatty
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thank Lorenz Lemcke and Anna Bujanow for medical assistance. For SCFA analysis, effects of propionate upon the blood-brain barrier. Microbiome 2018;6:55.
we thank Beatrice Engelmann and for blood analysis we thank Nicole Krebs and 8 Anastasovska J, Arora T, Sanchez Canon GJ, et al. Fermentable carbohydrate alters
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Contributors Study conceptualisation and design: EM, MvB, MS, AV and VW.
muciniphila in overweight and obese human volunteers: a proof-of-concept
Code for tasks: RT. Data collection: EM and RT. Behavioural and fMRI analysis: EM,
exploratory study. Nat Med 2019;25:1096–103.
FB and VW. Code review: RT, FB. Serum analysis: MR. Microbiome analysis: S-BH.
10 Arnoldussen IAC, Wiesmann M, Pelgrim CE, et al. Butyrate restores HFD-induced
Metabolomics analysis: URK. Network analysis: S-BH, EM, VW and RC. Manuscript
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draft and guarantor: EM and VW. Revision: FB, RT, RC, MvB, MS, EM and VW. All
(Lond) 2017;41:935–44.
authors agreed on the content of the material.
11 Dalile B, Vervliet B, Bergonzelli G, et al. Colon-delivered short-chain fatty acids
Funding This work was supported by grants of the German Research Foundation attenuate the cortisol response to psychosocial stress in healthy men: a randomized,
(DFG), contract grant number 209933838 CRC1052-03 A1 to VW and MS, and by placebo-c ontrolled trial. Neuropsychopharmacology 2020;45:2257–66.
the Berlin School of Mind and Brain (stipend for EM) and the German Foundation 12 Rinott E, Youngster I, Yaskolka Meir A, et al. Effects of diet-modulated
for Environment (stipend for EM). The inulin supplement was sponsored by the autologous fecal microbiota transplantation on weight regain. Gastroenterology
manufacturer BENEO, Mannheim, Germany. 2021;160:158–73.
13 Cani PD, Lecourt E, Dewulf EM, et al. Gut Microbiota fermentation of prebiotics
Competing interests None declared.
increases satietogenic and incretin gut peptide production with consequences
Patient and public involvement Patients and/or the public were not involved in for appetite sensation and glucose response after a meal. Am J Clin Nutr
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control in children with overweight and obesity: a randomized controlled trial. Am J
Ethics approval The ethics board of the Medical Faculty of the University of Clin Nutr 2017;105:790–9.
Leipzig, Germany, raised no concerns regarding the study protocol (228/18-ek) and 15 Parnell JA, Reimer RA. Weight loss during oligofructose supplementation is associated
all participants provided written informed consent. with decreased ghrelin and increased peptide YY in overweight and obese adults. Am
Provenance and peer review Not commissioned; externally peer reviewed. J Clin Nutr 2009;89:1751–9.
16 Hiel S, Gianfrancesco MA, Rodriguez J, et al. Link between gut microbiota and health
Data availability statement Data are available in a public, open access outcomes in inulin -treated obese patients: lessons from the Food4Gut multicenter
repository. Data and code availability. Data are available at 10.17605/OSF.IO/FC2G4 randomized placebo-controlled trial. Clin Nutr 2020;39:3618–28.
and code is available at https://gitlab.gwdg.de/gut_brain_study/food-wanting/task- 17 Leyrolle Q, Cserjesi R, D G H Mulders M, et al. Prebiotic effect on mood in obese
fmri-behavior-analysis and https://gitlab.gwdg.de/gut_brain_study/food-wanting/ patients is determined by the initial gut microbiota composition: a randomized,
fmri-analysis. Statistical MRI maps are available at https://identifiers.org/neurovault. controlled trial. Brain Behav Immun 2021;94:289–98.
collection:14111. 18 Medawar E, Haange S-B, Rolle-Kampczyk U, et al. Gut Microbiota link dietary fiber
Supplemental material This content has been supplied by the author(s). It intake and short-chain fatty acid metabolism with eating behaviour. Nutrition
has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have [Preprint] 2020.
been peer-reviewed. Any opinions or recommendations discussed are solely those 19 Berthoud H-R, Münzberg H, Morrison CD. Blaming the brain for obesity: integration of
of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and hedonic and homeostatic mechanisms. Gastroenterology 2017;152:1728–38.
responsibility arising from any reliance placed on the content. Where the content 20 DiFeliceantonio AG, Coppin G, Rigoux L, et al. Supra-additive effects of combining fat
includes any translated material, BMJ does not warrant the accuracy and reliability and carbohydrate on food reward. Cell Metab 2018;28:33–44.
of the translations (including but not limited to local regulations, clinical guidelines, 21 Fletcher PC, Kenny PJ. Food addiction: a valid concept. Neuropsychopharmacology
terminology, drug names and drug dosages), and is not responsible for any error 2018;43:2506–13.
and/or omissions arising from translation and adaptation or otherwise. 22 Beyer F, García-García I, Heinrich M, et al. Neuroanatomical correlates of food
addiction symptoms and body mass index in the general population. Hum Brain Mapp
Open access This is an open access article distributed in accordance with the 2019;40:2747–58.
Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which 23 Peng-Li D, Sørensen TA, Li Y, et al. Systematically lower structural brain connectivity in
permits others to distribute, remix, adapt, build upon this work non-commercially, individuals with elevated food addiction symptoms. Appetite 2020;155.
and license their derivative works on different terms, provided the original work is 24 Schulte EM, Yokum S, Jahn A, et al. Food cue reactivity in food addiction: a functional
properly cited, appropriate credit is given, any changes made indicated, and the use magnetic resonance imaging study. Physiol Behav 2019;208:112574.
is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/. 25 Gupta A, Osadchiy V, Mayer EA. Brain-gut-Microbiome interactions in obesity and
food addiction. Nat Rev Gastroenterol Hepatol 2020;17:655–72.
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Evelyn Medawar http://orcid.org/0000-0001-5011-8275 for functional MRI. Nat Methods 2019;16:111–6.
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Ronja Thieleking http://orcid.org/0000-0001-8934-3672 from illumina amplicon data. Nat Methods 2016;13:581–3.
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Ulrike Rolle-K ampczyk http://orcid.org/0000-0002-7728-6284 Biosystems 1998;47:119–28.
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Gut microbiota

Prebiotic diet changes neural correlates of food

► Additional supplemental ABSTRACT

de/​gut_brain_study/food-wanting/fmri-analysis.29 Statistical Neurobehavioural correlates of reward-related decision-

Medawar E, et al. Gut 2024;73:298–310. doi:10.1136/gutjnl-2023-330365 303

plant degradation or cell repair (padj<0.05, online supplemental DISCUSSION

lation of ABC transporters, correlated with significant decreases

This may suggest a potential mechanistic route of higher SCFA

production leading to lessened reward anticipation, however,

lack of direct evidence.

changes in faecal or fasting blood levels of acetate, butyrate

or propionate, suggesting that, in principle, changes in brain

activity may have been driven by other indirect factors. Similar

in faecal SCFA after inulin, for example, in healthy young adults

(subgroup, n=49).48 Others observed SCFA increases, for

in faecal SCFA (n=30).42 These conflicting results might be

explained by unknown complexity of local and systemic micro-

biota patterns at baseline (note higher relative Firmicutes in

our overweight/obese group compared with obesity studies), or

differences in stool frequency, weight and fluidity (note signifi-

cant changes in Bristol stool scale after prebiotics in the current

example, gut motility (specifically anticipatory contractions on

seeing food stimuli in the scanner) may underlie the observed

changes in brain responses.

Body fat and lipid markers slightly improved after placebo

condition and worsened after prebiotics in the current study.

to questionnaires, beneficial effects of for example increased

energy expenditure in the placebo phase cannot be ruled out.

Also, inulin, particularly at high doses, might challenge liver

conditions.50 A recent human study further reported spikes in

liver enzymes, cytokines and cholesterol in some participants

after 30 g/day inulin,51 underlining the possibility that the dosage

of inulin in the current study might have exceeded optimal levels.

For serum SCFA, others did find short-­term increases in SCFA

after inulin,52 and the postprandial increase in SCFA correlated

with decreases in serum ghrelin.53 Prebiotics and SCFA also stim-

ANOVA, analysis of variance; SCFA, short chain fatty acid.

contribute to changes in central reward-­related food responses.

In humans, PYY injections induced changes in BOLD-­related

found that decreases in fasting PYY correlated with decreases in

brain activation in the VTA and OFC clusters after intervention,

increase in serum SCFA or gut hormones due to prebiotics in

our sample might have been masked after overnight fasting.

Our study should be discussed in light of several limitations.

induce long-­lasting effects on neuronal processes involved in

308 Medawar E, et al. Gut 2024;73:298–310. doi:10.1136/gutjnl-2023-330365

Medawar E, et al. Gut 2024;73:298–310. doi:10.1136/gutjnl-2023-330365 309

310 Medawar E, et al. Gut 2024;73:298–310. doi:10.1136/gutjnl-2023-330365

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de/gut_brain_study/food-wanting/fmri-analysis.29 Statistical Neurobehavioural correlates of reward-related decision-

For serum SCFA, others did find short-term increases in SCFA

contribute to changes in central reward-related food responses.

In humans, PYY injections induced changes in BOLD-related

induce long-lasting effects on neuronal processes involved in