Gut microbiota

ORIGINAL ARTICLE

Edward S Chambers,1 Alexander Viardot,1,10 Arianna Psichas,1 Douglas J Morrison,2
Kevin G Murphy,3 Sagen E K Zac-Varghese,3 Kenneth MacDougall,4 Tom Preston,2Catriona Tedford,4 Graham S Finlayson,5 John E Blundell,5 Jimmy D Bell,6
E Louise Thomas,6 Shahrul Mt-Isa,7 Deborah Ashby,7 Glen R Gibson,8 Soﬁa Kolida,8Waljit S Dhillo,3 Stephen R Bloom,3 Wayne Morley,9 Stuart Clegg,9 Gary Frost1▸ Additional material ispublished online only. To viewplease visit the journal online(http://dx.doi.org/10.1136/
gutjnl-2014-307913).
For numbered afﬁliations see
end of article.
Correspondence toProfessor Gary Frost, Nutrition
and Dietetic Research Group,
Section of Investigative
Medicine, Imperial College
London, 6th Floor
Commonwealth Building,Hammersmith Hospital,
London W12 0NN, UK;g.frost@imperial.ac.uk
ESC, AV, AP and DJM
contributed equally.
Received 24 June 2014
Revised 5 September 2014
Accepted 23 September 2014
Published Online First
10 December 2014

ABSTRACT
Objective The colonic microbiota ferment dietaryﬁbres, producing short chain fatty acids. Recent evidence
suggests that the short chain fatty acid propionate may
play an important role in appetite regulation. We
hypothesised that colonic delivery of propionate would
increase peptide YY (PYY) and glucagon like peptide-1(GLP-1) secretion in humans, and reduce energy intake
and weight gain in overweight adults.
Design To investigate whether propionate promotes
PYY and GLP-1 secretion, a primary cultured humancolonic cell model was developed. To deliver propionate
speciﬁcally to the colon, we developed a novel inulinpropionate ester. An acute randomised, controlled crossover study was used to assess the effects of this inulinpropionate ester on energy intake and plasma PYY and
GLP-1 concentrations. The long-term effects of inulinpropionate ester on weight gain were subsequently
assessed in a randomised, controlled 24-week study
involving 60 overweight adults.
Results Propionate signiﬁcantly stimulated the releaseof PYY and GLP-1 from human colonic cells. Acuteingestion of 10 g inulin-propionate ester signiﬁcantly
increased postprandial plasma PYY and GLP-1 andreduced energy intake. Over 24 weeks, 10 g/day inulinpropionate ester supplementation signiﬁcantly reduced
weight gain, intra-abdominal adipose tissue distribution,
intrahepatocellular lipid content and prevented thedeterioration in insulin sensitivity observed in the inulincontrol group.
Conclusions These data demonstrate for the ﬁrst timethat increasing colonic propionate prevents weight gainin overweight adult humans.
Trial registration number NCT00750438.

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INTRODUCTION

To cite: Chambers ES,
Viardot A, Psichas A, et al.
Gut 2015;64:1744–1754.

1744

Evidence published over the last 25 years demonstrates that hormonal and neuronal signals from the
GI tract play an important role in appetite regulation.1 There is increasing evidence that the gut
microbiota inﬂuences energy regulation and can bea major determinant in the development ofobesity.2 Recent investigations suggest that diet, thegut microbiota and fat storage can be linked

Signiﬁcance of this study
What is already known about this subject?

▸ Increased intake of dietary ﬁbre has been
associated with reduced appetite and weightloss.
▸ The short chain fatty acids (SCFAs) produced by
microbial fermentation of dietary ﬁbre in thecolon stimulate the release of the anorectic gut
hormones peptide YY (PYY) and glucagon like
peptide-1 (GLP-1) from rodent enteroendocrine
L cells via activation of the G protein coupledfree fatty acid receptor (FFAR) 2.
▸ Of the SCFAs produced by colonic fermentationof dietary ﬁbre, propionate has the highest
afﬁnity for FFAR 2.
▸ Mice receiving a faecal transplant from a donor
with a gut microbiota composition that
produces elevated levels of propionate in thecolon have reduced weight gain and adiposity.

What are the new ﬁndings?

▸ Propionate stimulates the release of PYY and
GLP-1 from primary cultured human colonic
cells.
▸ This ﬁrst-in-human study demonstrates thatdelivery of propionate directly to the colon,
acutely increases the release of PYY and GLP-1
and reduces energy intake.
▸ Long-term colonic propionate delivery prevents
body weight gain and reduces intra-abdominal
fat accretion in overweight adults.
▸ Long-term colonic propionate delivery
signiﬁcantly reduces intrahepatocellular lipid
content in adults that meet the diagnostic
criteria for non-alcoholic fatty liver disease.

How might it impact on clinical practice inthe foreseeable future?

▸ Optimising colonic propionate production
through selection of propiogenic dietary ﬁbres
may represent a novel route to prevent weightgain throughout life and improve publichealth.

Chambers ES, et al. Gut 2015;64:1744–1754. doi:10.1136/gutjnl-2014-307913

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Effects of targeted delivery of propionate to thehuman colon on appetite regulation, body weight
maintenance and adiposity in overweight adults

Gut microbiota

Chambers ES, et al. Gut 2015;64:1744–1754. doi:10.1136/gutjnl-2014-307913

METHODS
In vitro gut hormone secretion experiments
The effect of propionate on PYY and GLP-1 release from humancolonic crypts was determined using a modiﬁed version of anestablished method32 (see online supplementary material).

Inulin-propionate ester for colonic delivery of propionate inhumansWe developed a novel carrier molecule whereby propionate ischemically bound by an ester bond to inulin, a natural polymer
composed mainly of fructose. This inulin-propionate ester was
synthesised, as detailed in the online supplementary material.
The majority of propionate chemically bound to inulin should
only be released when the inulin polymer is fermented by thecolonic microbiota, thus providing targeted colonic delivery.
Isotope labelling studies were conducted to assess the stability ofthe molecule through the stomach and small intestine, and toprovide information about site and extent of propionate release,as described in the online supplementary information. In addition, the effects of inulin-propionate ester on fermentation proﬁles and gut microbial populations were studied using an invitro culture system (see online supplementary material).

Clinical studies
All subjects provided informed, written consent prior to theclinical trial which was approved by the Hammersmith andQueen Charlotte’s Research Ethics Committee (08/H0707/99).
All studies were carried out in accordance with the Declarationof Helsinki. All clinical trials where registered (Registration No:
NCT00750438).

Investigation of acute supplementation withinulin-propionate ester on appetite regulation
In ﬁrst-in-human studies, the acute effects of inulin-propionate
ester on appetite regulation, hormone release and energy intake
were studied in 20 volunteers. The primary outcome was energy
intake, and gut hormone release was a secondary outcome. The
effects on gastric emptying were examined in 14 volunteers in a
separate study. Detailed inclusion and exclusion criteria andmethodology for each acute study are described in the online
supplementary material.

Proof-of-principle investigation of the effect of long-term
supplementation with inulin-propionate ester on bodyweight maintenanceWe hypothesised that daily intake of inulin-propionate ester over
24 weeks would decrease weight gain in overweight adults. The
predeﬁned coprimary outcomes were changes in body weight
and food intake. A change in adipose tissue distribution was asecondary outcome. Men and women aged 40–65 years, with abody mass index (BMI) of 25–40 kg/m2 were recruited. Potential
participants were excluded if they met any of the following criteria: clinically signiﬁcant illness (including type 1 or type 2 diabetes), medication known to affect appetite and/or body weight,
a weight loss of 3 kg or greater in the preceding 2 months,
smoking, substance abuse, psychiatric illness and any abnormalities detected on physical examination, electrocardiography or
screening blood tests (measurement of complete blood count,electrolytes, fasting glucose, thyroid function and liver function).
Women were ineligible if they were pregnant or breast feeding.
From an initial 167 persons who responded to letters of invitation, 60 were randomly assigned to either the inulin-control or
inulin-propionate ester supplementation group.
1745

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through a molecular mechanism involving short chain fatty
acids (SCFAs), the major products of dietary ﬁbre fermentationby the gut microbiome.3–5
A major public health challenge is the development of effective strategies that can prevent the increased prevalence inobesity and the reported average 0.3–0.8 kg/year weight gain in
adults.6–9 Such gradual long-term weight gain can be the resultof a small habitual positive energy balance of 50–100 kcal/day.10
Interventions that can be safely applied at the population level
to reverse this minor energy imbalance and prevent weight gain
throughout life would therefore have substantial beneﬁts topublic health. Increased intake of dietary ﬁbre has been associated with reduced appetite and weight loss.11–13 In particular,
evidence suggests that the fermentable component of dietaryﬁbre is critical in mediating these satiating effects.14 Feedingrodents a high level of fermentable dietary ﬁbre protects against
high-fat, diet-induced increases in body weight and fat
mass.15 16 There is also evidence that fermentable dietary ﬁbre
can suppress appetite and decrease body weight in humans.17–19
However, large amounts of dietary ﬁbre (>30 g/day) are
required for these beneﬁcial effects, and compliance with highﬁbre diets is poor, due to GI side effects, which may also
explain the inconsistent reports regarding their effects on appetite and body weight.20 Targeting the mechanisms by which fermentable dietary ﬁbre suppresses appetite may thus provide amore effective approach to weight control than the use of highﬁbre diets.
The SCFAs produced by microbial fermentation of dietary ﬁbrein the colon have been shown to stimulate the release of the anorectic gut hormones peptide YY (PYY) and glucagon like peptide-1(GLP-1) from rodent enteroendocrine L cells.21–23 These hormones are released from the gut and are involved in the short-term
signal of satiation and satiety to the appetite centres of the brain.24
Peripheral administration of PYY3–36 or GLP-1 enhances satiety
and reduces food intake in animals and man.25–27 Recent evidence
suggests that SCFAs stimulate GLP-1 release in rodents via stimulation of the G protein coupled free fatty acid receptor (FFAR) 2 oncolonic L cells.21 Of the SCFAs produced by colonic fermentationof dietary ﬁbre, propionate has the highest afﬁnity for
FFAR 2.28 29 Furthermore, propionate is an end product of bacterial metabolism, and thus, unlike acetate, does not undergo conversion to other SCFAs.30 Intriguingly, Roux-en-Y gastric bypass,which results in weight loss and reduced adiposity, promotes
increased levels of propionate in the colon.5 In keeping with theseﬁndings, a signiﬁcant negative correlation between adiposity andcaecal propionate concentrations has also been reported in germfree mice receiving faecal transplants from human twin donors discordant for obesity.4
Increasing colonic propionate is therefore an attractive target
for appetite modulation. However, orally administered SCFAs
are unpalatable and are rapidly absorbed in the small intestinewhere L cells are sparse. Furthermore, supplementing diets with
mixed high ﬁbre does not predictably or reliably increasecolonic production or circulating levels of propionate in all
human populations because of the variability in gut microbialactivity.31 To overcome the unpalatably high levels of fermentable dietary ﬁbre needed to signiﬁcantly increase colonic propionate levels, and the unpredictability in the production of theresulting SCFAs, we have developed a novel delivery system targeting the release of gram quantities of propionate in the proximal colon. We hypothesised that propionate would stimulateanorectic gut hormone release from the colon and that targeted
delivery of propionate to the colon would decrease appetite andprevent long-term weight gain in humans.

Gut microbiota
Study design

Body weight and composition
Body weight was measured in all subjects to the nearest 0.1 kg(Tanita BC-418MA) while subjects were wearing light clothing.
Body composition was assessed using MRI and MR spectroscopy(MRS), as previously described.33 MRI and MRS data could not
be collected in 20 subjects, due to metal implants (n=8), claustrophobia (n=9) or technical issues with the scanner (n=3).

Appetite regulation, gut hormone release and glucose
homoeostasis
A cannula was inserted into an antecubital vein and baseline blood
samples collected at −10 min and 0 min to assess plasma concentrations of glucose, insulin, PYY and GLP-1. Following the 0 min
sample, subjects were served a standardised breakfast (398 kcal;
71.2 g carbohydrate, 7.9 g fat, 10.3 g protein). At week 24, thebreakfast also contained 10 g of inulin-propionate ester or 10 ginulin-control depending on supplementation group. Postprandial
blood samples were taken at 15 min, 30 min, 60 min, 90 min,
120 min, 180 min, 240 min and 300 min and collected into
heparin-coated tubes containing 0.2 mL of aprotinin (Bayer, UK).
GLP-1-like and PYY-like immunoreactivity were measured using
established inhouse radioimmunoassay.34 35 Insulin and leptin
were measured by radioimmunoassay using commercially available
kits (Millipore, USA). Plasma glucose was measured using an
Abbott Architect ci8200 analyser (Abbott Diagnostics, USA). At
300 min subjects were offered a buffet lunch with food served inexcess, and asked to eat until they felt comfortably full. The
amount of food was quantiﬁed and energy intake calculated.
Subjective hunger, satiety and nausea were monitored with the useof 100 mm visual analogue scales (VAS).36 Subjects were asked tocomplete the VAS before each blood sample.

Risk factors for cardiovascular disease and diabetes
A fasting blood sample was collected and analysed for levels of
triglycerides, total cholesterol, low-density lipoprotein
1746

cholesterol, high-density lipoprotein cholesterol, glycosylated
haemoglobin (HbA1c), C reactive protein and liver function
tests (alanine transaminase, alkaline phosphatase, aspartatetransaminase). All analytes were measured by the Department ofChemical Pathology, Imperial College Healthcare National
Health Service Trust. Blood pressure and pulse were also measured after subjects had been in a supine position for at least
15 min.

Statistical analysis
The treatment group size for the acute energy intake study was
based on a power calculation, assuming a decrease of 15% inenergy intake with a SD of 20% (α=0.05, power=0.85), resulting in an estimated required sample size of 20 subjects. Data
from the acute supplementation study suggested a sample size of
50 individuals (25 in each group) was needed for the long-term
investigation. Sixty volunteers were therefore recruited to allow
for an estimated attrition rate of 15%. χ2 tests were performed
to compare percentages of subjects in each group who gained≥3% and ≥5% of their initial weight. For comparison of variables with a single measurement pre supplementation and post
supplementation, we calculated the change from baseline at
24 weeks and compared means within groups using paired
t-tests. The mean changes between groups for each of these variables were estimated using a multiple linear regression adjusted
for its baseline measurement, baseline weight and randomised
group. The linear model was also run on logit-transformed variables that were expressed as percentages to ensure their predictive values are within the 0–100% range.37 Variables measured
multiple (>2) times during the supplementation period (bodyweight, postprandial glucose, postprandial insulin response, PYY,
GLP-1, VAS, side effects assessment) were analysed using multilevel mixed effects models to account for the variability within
and between subjects. Area under the curve (AUC), unadjusted
for covariates, for postprandial glucose and postprandial insulin
response, was also calculated and compared between groups. In
the case of data missing at random, the regression techniques
described previously were applied after data were imputed using
the multiple imputation by chained equations technique toaccount for random biases of the unobserved covariates. Data
are presented as mean±SEM or 95% CI. p Values <0.05 wereconsidered statistically signiﬁcant.

RESULTS
Propionate stimulates PYY and GLP-1 release from human L
cells in vitro
Propionate signiﬁcantly stimulated PYY secretion from humancolonic cells, with concentrations of 200 mmol/L and
400 mmol/L inducing 1.8-fold and threefold rises above basal
secretion, respectively (p<0.05 and p<0.001; ﬁgure 1A).
Propionate also increased GLP-1 secretion, with 200 mmol/L
and 400 mmol/L inducing 1.6-fold and 2.4-fold increases in
GLP-1 release, respectively ( p<0.001; ﬁgure 1B).

Inulin-propionate ester delivers propionate to the colon
Propionate production from in vitro faecal fermentations was
signiﬁcantly higher from inulin-propionate ester compared withinulin, while no signiﬁcant differences in acetate or butyrate
production were observed (see online supplementary ﬁgure S1).
Following ingestion of 10 g inulin-propionate ester, breath H2started to increase at 180 min and peaked at 240 min post ingestion. A small gradual release of 13C was apparent between 0
min and 180 min post ingestion. More than 80% (82.9±2.3%)of the 13C recovered in breath over 24 h appeared coincident
Chambers ES, et al. Gut 2015;64:1744–1754. doi:10.1136/gutjnl-2014-307913

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The study was conducted using a randomised, double-blind,placebo-controlled, parallel design. Two-day study visits wererequired at baseline (week 0) and after 24 weeks of dietary supplementation. On the day prior to each study visit, subjects wereasked to consume a standard evening meal, to fast overnightfrom 22:00 and to avoid strenuous physical activity and alcohol.
All study visits commenced between 08:00 and 09:00 and wereconducted at the National Institute for Health Research (NIHR)/
Wellcome Trust Imperial Clinical Research Facility. After all baseline measurements had been taken, subjects were randomly
assigned to either the 10 g/day inulin-propionate ester group, or
the 10 g/day inulin-control group. Subjects were randomised as
described in the online supplementary material. The dietary supplement was supplied to subjects in ready-to-use sachets and they
were instructed to mix the contents into their normal diet once a
day during the 24-week supplementation period. All subjects
were instructed to maintain their usual dietary and physical activity habits during the supplementation period. Self-reported foodintake and physical activity were assessed at baseline and after
24 weeks of supplementation (see online supplementarymaterial). Regular communication between subjects and study
investigators encouraged good compliance. At week 8 and week
16 of the supplementation period, subjects attend follow-up
visits to monitor compliance and adverse events. At week 24,
measurements taken at baseline were repeated. Subjects returned
all their used and unused sachets to estimate compliance.

Gut microbiota

with and after breath H2 onset (ﬁgure 1C), suggesting deliveryof the majority of the tracer to the colon. It is estimated that10 g inulin-propionate ester delivered 2.36 g propionate to thecolon after accounting for small intestinal losses (0.49 g). The
isotopic data show that propionate released from the inulinpropionate ester appeared in the blood and was thus available
systemically. Where circulating plasma propionate was detectable, signiﬁcant increases in propionate 13C enrichment andtotal concentration were measured in peripheral blood 360 min
post ingestion compared with baseline (ﬁgure 1D, E). No
Chambers ES, et al. Gut 2015;64:1744–1754. doi:10.1136/gutjnl-2014-307913

signiﬁcant differences were observed in plasma acetate enrichment or total concentration (ﬁgure 1D, F). We estimate that theaddition of 10 g inulin-propionate ester to the diet would leadto a 2.5-fold increase in daily colonic propionate production(see online supplementary material).

Acute supplementation with inulin-propionate ester
increases PYY and GLP-1 secretion and reduces food intake
Acute supplementation with inulin-propionate ester signiﬁcantly reduced food intake from 1175 kcal (95% CI 957 to1747

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Figure 1 Propionate increases peptide YY (PYY) and glucagon like peptide-1 (GLP-1) release from primary human colonic cells andinulin-propionate ester supplementation delivers propionate to the colon in vivo. Cells isolated from human colonic tissue were incubated withincreasing concentrations of propionate. (A) PYY and (B) GLP-1 levels were measured in the supernatants and lysed cells by radioimmunoassay.
Percentage gut hormone release per well is expressed relative to the basal release measured (n=4–6). (C) The increase in breath H2 at 240 min
suggests that >80% of the labelled propionate entered the colon. (D) Plasma acetate and propionate 13C enrichment (δ13C per mil) at baseline and360 min. Plasma propionate was signiﬁcantly more enriched at 360 min whereas no difference was seen in acetate enrichment. Total plasma
propionate (E) and acetate (F) concentrations (mmol/L) at baseline and 360 min. Data are presented as mean±SEM, *p<0.05, ***p<0.001.

Gut microbiota1392) to 1013 kcal (95% CI 816 to 1210) (ﬁgure 2A, B;
p<0.01), a mean reduction of 13.8%. It takes an estimated
240 min for the inulin-propionate ester to enter the colon(ﬁgure 1C) and compared with inulin-control, inulinpropionate ester signiﬁcantly increased plasma PYY (ΔAUC240–
420 min 429 min×pmol/L (95% CI −543 to 1400) inulincontrol vs 3349 min×pmol/L (841 to 5857) inulin-propionate
ester, p<0.05) and GLP-1 levels (ΔAUC240–420 min 3495
min×pmol/L (95% CI −1567 to 8558) inulin-control vs 10
801 min×pmol/L (5897 to 15 704) inulin-propionate ester,1748

p<0.05) between 240 min and 420 min. Prior to 240 min
there was no signiﬁcant difference in the concentration of PYY
and GLP-1 between the inulin-control and the inulinpropionate ester (ﬁgure 2D). Glucose, insulin and leptin levels
and subjective ratings of appetite and nausea were not signiﬁcantly different following acute inulin-propionate ester andinulin-control supplementation (see online supplementaryﬁgures S2 and S3). Acute supplementation with inulinpropionate ester did not inﬂuence the rate of gastric emptying(see online supplementary information).
Chambers ES, et al. Gut 2015;64:1744–1754. doi:10.1136/gutjnl-2014-307913

Gut: first published as 10.1136/gutjnl-2014-307913 on 10 December 2014. Downloaded from http://gut.bmj.com/ on August 24, 2024 by guest. Protected by copyright.

Figure 2 Acute inulin-propionate ester supplementation increases plasma peptide YY (PYY) and glucagon like peptide-1 (GLP-1) levels and reduces
energy intake in humans. (A) The mean reduction in energy intake following inulin-control versus inulin-propionate ester. (B) A reduction in energy
intake occurred in 16 of the 20 volunteers. (C–F) Plasma gut hormone levels following acute supplementation of inulin-control versus
inulin-propionate ester. Arrows indicate standardised meals. Dotted lines signify the time point after which >80% inulin-propionate ester enters thecolon as determined by the enrichment of 13C in expired air and breath H2 methodology (ﬁgure 1C). Data are presented as mean±SEM, *p<0.05,**p<0.01. AUC, area under the curve.

Gut microbiota

Of the 60 volunteers randomised, data were analysed from the49 participants that completed the 24 week supplementation(table 1). Eleven participants (18%) did not complete the24 week supplementation and there were no signiﬁcant differences in attrition between the two groups (see online supplementary ﬁgure S4). Baseline and postsupplementation bodycomposition data was collected from 17 participants in theinulin-propionate ester group and 15 participants in the inulincontrol group. There was no difference between groups in compliance (95% (95% CI 92% to 98%) inulin-propionate ester vs
94% (95% CI 92% to 97%) inulin-control; p=0.864). Ratingsof nausea were not different between supplementation groups( p=0.736), though there were signiﬁcantly greater ratings ofﬂatulence ( p=0.004) in the inulin-control group during the supplementation period compared with the inulin-propionate ester
group (see online supplementary table S1).

Body weight and composition
Inulin-propionate ester supplementation resulted in beneﬁcialchanges in body weight and composition. There was a signiﬁcant difference in weight gain between groups. One of 25

participants gained ≥3% of their baseline body weight following
inulin-propionate ester supplementation (4%), as comparedwith 6 of 24 participants (25%) in the inulin-control group(ﬁgure 3A, p=0.036). Furthermore, none of the participants in
the inulin-propionate ester group had substantial weight gain(≥5% baseline weight) compared with 4 of 24 (17%) following
inulin-control supplementation (ﬁgure 3A; p=0.033). Although
the primary aim of the study was to prevent weight gain, it is ofinterest to note that weight loss after 24 weeks was greater inthe propionate ester group, though this effect was not signiﬁcantly different between groups (0.38 kg (95% CI −0.95 to1.72) inulin-control vs −1.02 kg (95% CI −2.10 to 0.04) propionate ester, p=0.099). Following the supplementation period,
the change in the distribution of intra-abdominal adipose tissue,
expressed as a percentage of total adipose tissue content, wassigniﬁcantly lower in the inulin-propionate ester group compared with inulin-control supplementation (table 2; p=0.027).
Furthermore, internal adipose tissue ( p=0.002) and the ratio ofinternal adipose tissue: subcutaneous adipose tissue was signiﬁcantly increased within the inulin-control group ( p=0.002), but
not in the inulin-propionate ester group. There was no signiﬁcant change in total adipose tissue content between groups.
Within the inulin-propionate ester group there was a trend for
reduced intrahepatocellular lipid (IHCL) content post supplementation ( p=0.061). However, subjects meeting the baseline
diagnostic criteria for non-alcoholic fatty liver disease (NAFLD)

Table 1 Baseline characteristics of subjects and changes in cardiovascular and diabetes risk factors following 24 weeks of inulin-control andinulin-propionate ester supplementation
Inulin-control (N=24)
Variable

Week 0Sex (% no. of subjects)Male
37.5 (9)Female62.5 (15)Race or ethnicity (% no. of subjects)
White75.0 (18)Black
8.3 (2)Asian
17.7 (4)Age (years)
53.4±1.5
Weight (kg)91.0±2.8
Glucose (mmol/L)
5.1±0.1
Insulin (mU/mL)
11.5±1.3
HOMA-IR
2.7±0.3
HbA1c (mmol/mol)
37.8±0.5
Triglycerides (mmol/L)
1.5±0.2
Cholesterol (mmol/L)
Total
5.3±0.2
Low-density lipoprotein
3.3±0.2
High-density lipoprotein
1.4±0.1
Liver function tests (IU/L)
Alanine transaminase
32.4±3.9
Alkaline phosphatase78.2±3.4
Aspartate transaminase29.8±1.6
Leptin (ng/mL)
31.9±4.0
C reactive protein (mg/L)
3.7±0.5
Blood pressure (mm Hg)
Systolic
137±2
Diastolic87±2
Pulse (bpm)70±2

Week 24

Inulin-propionate ester (N=25)
Value

Week 0Week 24

p Value

Inulin-propionate Ester−inulin-control
Difference (95% CI)

p Value87.5±3.0
5.0±0.1
8.8±0.8
2.0±0.237.8±0.7
1.4±0.2

0.062
0.898
0.702
0.644
0.297
0.176

−1.40 (−3.07 to 0.27)
0.05 (−0.21 to 0.32)−0.56 (−2.64 to 1.51)
0.14 (−0.63 to 0.34)
0.18 (−0.85 to 1.21)−0.29 (−0.68 to 0.11)

0.099
0.675
0.582
0.549
0.729
0.148

40.0 (10)60.0 (15)91.4±3.0
5.0±0.1
10.6±0.7
2.3±0.2
37.1±0.5
1.6±0.2

0.559
0.624
0.329
0.372
0.086
0.283

64.0 (16)16.0 (4)20.0 (5)55.3±1.488.5±2.9
5.0±0.1
9.0±1.0
2.0±0.2
38.2±0.7
1.5±0.2

5.0±0.2
3.1±0.2
1.3±0.1

0.014
0.132
<0.0015.5±0.2
3.5±0.2
1.4±0.1

5.1±0.2
3.2±0.2
1.3±0.1<0.001<0.001
0.009−0.10 (−0.39 to 0.19)−0.08 (−0.34 to 0.18)
0.02 (−0.06 to 0.10)

0.494
0.532
0.617

25.9±3.3
71.7±3.528.7±3.0
33.3±4.5
3.7±0.6

0.001<0.001
0.543
0.690
0.818

29.5±3.2
74.6±3.728.9±1.3
25.1±3.0
2.1±0.4

23.7±2.270.5±3.326.2±1.0
24.1±2.4
1.8±0.3

0.015<0.001
0.007
0.625
0.484

−0.28 (−9.30 to 8.75)
1.41 (−2.40 to 5.22)−1.43 (−7.41 to 4.55)−2.36 (−10.31 to 3.92)−0.27 (−1.39 to 0.84)

0.949
0.458
0.627
0.551
0.622

137±3
85±2
67±1

0.707
0.143
0.212

140±3
86±2
66±2

139±2
84±2
66±2

0.659
0.101
0.934

−0.83 (−4.98 to 3.81)−0.86 (−4.92 to 3.19)
0.63 (−3.36 to 4.61)

0.688
0.664
0.763

Mean±SEM.
HbA1C, glycosylated haemoglobin; HOMA-IR, homoeostasis model assessment of insulin resistance.

Chambers ES, et al. Gut 2015;64:1744–1754. doi:10.1136/gutjnl-2014-307913

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Long-term supplementation with inulin-propionate ester
prevents body weight gain, intra-abdominal adipose tissue
accretion and reduces intrahepatocellular lipid content inthose with non-alcoholic fatty liver disease

Gut microbiota(IHCL >5.5%)38 had a signiﬁcant reduction in IHCL content
following inulin-propionate ester supplementation (22.1% (95%
CI 7.7 to 36.6) to 15.9% (95% CI 5.2 to 26.5), p=0.038,
n=11; ﬁgure 3B). This effect was not observed in similar

subjects within the inulin-control group (19.1% (95% CI 2.0 to36.1) to 18.7% (95% CI 7.1 to 30.3), p=0.576, n=5; ﬁgure
3B). In vitro analysis suggested that the protective effects of theinulin-propionate ester on weight gain and adipose tissue

Table 2 Body fat depots at baseline and following 24 weeks of inulin-control and inulin-propionate ester supplementation
Inulin-control (N=15)

Inulin-propionate ester (N=17)

AT distribution (% total AT)

Week 0Week 24

p Value

Week 0Week 24

p Value

Inulin-propionate ester−inulin-control
Difference (95% CI)

p Value

Subcutaneous AT
Internal AT
Intra-abdominal AT
Abdominal
subcutaneous AT
1H-MRS
IHCL
Soleus IMCL
Tibialis IMCL

81.3±1.918.7±1.910.6±1.3
23.1±0.980.6±1.819.4±1.8
11.1±1.422.7±0.8

0.002
0.002
<0.0010.300

76.3±1.7
23.7±1.713.2±1.2
21.9±0.7

76.0±1.6
24.0±1.6
13.1±1.1
21.6±0.7

0.624
0.624
0.723
0.171

−0.59 (1.69 to 0.50)−0.23 (−1.09 to 0.64)−0.46 (−0.87 to −0.05)−0.23 (−0.88 to 0.42)

0.288
0.608
0.027
0.483

7.8±2.918.8±2.0
10.0±1.5

7.4±2.518.8±1.8
10.3±1.1

0.708
0.973
0.869

15.8±5.0
21.8±3.0
9.1±1.0

11.5±3.724.0±3.7
9.5±0.9

0.061
0.274
0.701

−0.83 (−5.04 to 3.38)
2.70 (−1.04 to 6.44)−0.42 (−2.83 to 1.99)

0.699
0.157
0.733

Mean±SEM.
IHCL was measured relative to liver water content and IMCL was measured relative to total muscle creatine signal.33
AT, adipose tissue; IHCL, intrahepatocellular lipid; IMCL, intramyocellular lipid; MRS, MR spectroscopy.

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Figure 3 The effect of 24 weeks inulin-control and inulin-propionate ester supplementation on weight gain, liver fat content and gut hormone
response. (A) The proportion of subjects who gained 3% or more and 5% or more of their baseline weight at 24 weeks. (B) Intrahepatocellular lipid(IHCL) content at baseline and following 24 weeks of inulin-control and inulin-propionate ester supplementation in subjects with non-alcoholic fatty
liver disease (NAFLD). Subjects were identiﬁed as having NAFLD on the basis of an IHCL content >5.5% at baseline.38 Postprandial plasma(C) peptide YY (PYY) and (D) GLP-1 release at baseline and following 24 weeks of inulin-control and inulin-propionate ester supplementation. Data
are presented as mean±SEM, *p<0.05.

Gut microbiota

distribution were not due to changes in gut bacterial populations
compared with inulin-control (see online supplementarymaterial; ﬁgure 4A–F).

Food intake and gut hormone release following long-term
supplementation with inulin-propionate ester
The change in food intake at an ad libitum meal following
24 weeks of supplementation was not statistically signiﬁcantbetween groups ( p=0.972), though long-term inulin-propionate
ester intake showed a trend towards a lower food intake by
8.7% from 836 kcal (95% CI 724 to 948) to 763 kcal (95% CI
654 to 872) ( p=0.100). Inulin-control supplementation
reduced food intake from 678 kcal (95% CI 535 to 820) to
Chambers ES, et al. Gut 2015;64:1744–1754. doi:10.1136/gutjnl-2014-307913

645 kcal (95% CI 514 to 776) (p=0.197), a mean reduction of
4.0%. Subjective ratings of appetite were signiﬁcantly reduced
within the inulin-propionate ester group following the supplementation period, while there were no differences in ratings ofnausea (see online supplementary ﬁgure S5). Interestingly, there
were no signiﬁcant differences in postprandial PYY (difference−1.48%; 95% CI −6.29% to 3.33%; ﬁgure 3C; p=0.546) or
GLP-1 secretion (difference 3.69%; 95% CI −4.48% to12.33%; ﬁgure 3D, p=0.361).

Glucose homoeostasis
Multilevel mixed effects models found no differences in postprandial glucose (p=0.350) or insulin response (p=0.924) between
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Figure 4 The effect of inulin-propionate ester on the gut microbiota. Bacterial concentrations expressed in Log10 cells/mL culture ﬂuid enumeratedusing ﬂuorescent in situ hybridisation (FISH) targeting (A) Biﬁdobacterium spp (Bif164), (B) Bacteroides/Prevotella (Bac303), (C) Atopobium cluster(Ato291), (D) Lactobacillus/Enterococcus (Lab158), (E) Clostridium histolyticum (Chis150) and (F) Eubacterium rectale/Clostridium coccoides (Erec482)
at 0 h, 10 h, 24 h, 34 h and 48 h anaerobic, pH controlled faecal batch culture fermentation with control (no substrate), inulin-control andinulin-propionate ester. Data are presented as mean±SEM (n=3), *<0.05, †<0.001, ‡<0.0001 with respect to the 0 h sample.

Gut microbiota

Risk factors for cardiovascular disease and diabetes
Inulin-propionate ester and inulin-control supplementation signiﬁcantly reduced circulating levels of total cholesterol, highdensity lipoprotein, alanine transaminase and alkaline phosphatase (table 1). Signiﬁcant reductions in low-density lipoprotein( p<0.001) and aspartate transaminase ( p=0.007) were only
observed within the propionate ester group.

DISCUSSIONThe GI tract is an important organ in the short-term control ofappetite.1 24 The production of SCFAs by microbial fermentation of dietary ﬁbre has been linked to positive physiologicaleffects, including improvements in body weight, adiposity andglucose metabolism.3 Our data demonstrate that the SCFA propionate stimulates the release of the anorectic gut hormones
PYY and GLP-1 from human colonic cells in vitro, supporting
observations made in animal models.21 To increase colonic propionate production in vivo, we designed and synthesised a novel
inulin-propionate ester, whereby propionate is conjugated by anester linkage to the carrier molecule inulin. Stable isotope methodology revealed that >80% of the propionate load from theinulin-propionate ester is released in the colon coincident withor after a rise in breath H2. This would suggest that only a relatively small amount of the esteriﬁed propionate is released andabsorbed in the small intestine. We have estimated that 10 ginulin-propionate ester ingestion leads to a 2.5-fold increase indaily colonic propionate production, a level very difﬁcult toachieve through feeding a mixed fermentable ﬁbre diet.20 We
also demonstrated that ingestion of the inulin-propionate ester
increases plasma propionate levels.
We subsequently, in the ﬁrst-in-human studies, demonstrated
that increased delivery of propionate to the colon acutely modulates gut hormone release and reduces food intake in healthy
subjects. The inulin-propionate ester did not suppress subjective
appetite responses, but signiﬁcantly reduced meal size, consistent with the action of a physiological satiation signal. We
observed a signiﬁcantly greater postprandial release of PYY and
GLP-1 when a mixed calorie breakfast contained 10 g inulinpropionate ester compared with 10 g inulin-control. It has beenpreviously shown that a sustained increase in circulating PYY
and GLP-1 can inﬂuence appetite-regulating circuits of the brain
and inhibit food intake.25 27 In this study, the rise in PYY and
1752

GLP-1 was apparent between 240 min and 420 min following
oral administration of the inulin-propionate ester, and reachedlevels similar to those observed following a 1000 kcal meal.39
Such a rise did not occur following ingestion of the inulincontrol, suggesting that it is a speciﬁc effect of the inulinpropionate ester rather than the standardised 356 kcal lunch
provided in both trials. This would suggest that compared with
the inulin-propionate ester, a 10 g dose of inulin-control does
not raise colonic SCFA to a sufﬁcient concentration to stimulate
gut hormone release.40 Recent evidence suggests the colonicmicrobiota adapt rapidly to a change in substrate availability.41
Data from batch culture experiments demonstrated that inulincontrol and inulin-propionate ester stimulate changes to the gut
microbiota, although only inulin-control had a selective effecton Biﬁdobacterium. This would suggest that the observed shortterm effects on appetite regulation were independent of alterations to gut microbial composition.
Longitudinal studies demonstrate that adults gain weight gradually through middle age, with an average yearly weight gain of
0.3–0.8 kg.6–9 This accumulation of weight would result from asmall daily positive energy balance of around 50–100 kcal.10
The 14% (162 kcal) reduction in food intake observed following acute administration of 10 g/day inulin-propionate ester
would have a signiﬁcant impact on weight gain and health if sustained over the long term.42 We therefore investigated if longterm elevation of colonic propionate would prevent weight gain
by conducting a 24 week randomised controlled trial of inulinpropionate ester supplementation in overweight middle-aged
adults. We demonstrated lower weight gain in the inulinpropionate ester group, with signiﬁcantly fewer volunteers
gaining ≥3% or ≥5% body weight. This was coupled with areduced gain in intra-abdominal adipose tissue compared with
the inulin-control group and prevention of the deterioration of
postprandial glucose response. Furthermore, long-term elevations in colonic propionate production reduced IHCL content
in subjects meeting the diagnostic criteria for NAFLD. A reduction in IHCL is a reproducible ﬁnding in rodents fed a highlevel of fermentable dietary ﬁbre,16 43 however the mechanism
behind this is not well understood. Intra-abdominal adipose
tissue and NAFLD are regarded as major risk factors in thedevelopment of insulin resistance and type 2 diabetes.44
Interestingly, in this long-term study we were unable to detect
any change in PYY or GLP-1 release following ingestion of theinulin-propionate ester compared with inulin-control, in contrast
to our acute administration studies. This suggests that there may
be a desensitisation of the FFAR2/3 receptor response over time
and that the beneﬁcial effects of long-term inulin-propionate
ester supplementation may not be mediated by PYY and GLP-1.
However, subjective ratings of postprandial appetite were signiﬁcantly reduced within the inulin-propionate ester group and we
observed a trend towards a signiﬁcant decrease in food intake of
8.7% (73 kcal), suggesting that propionate may inﬂuence appetite and energy intake via mechanisms unrelated to PYY or GLP-1
release. It has been demonstrated that propionate can stimulate
leptin release through activation of FFAR2 on adipocytes,45
although we did not observe any changes in circulating leptin
concentrations following acute or long-term supplementation
with inulin-propionate ester. Recent reports suggest that propionate could also have a positive effect on energy balance and bodyweight independent of energy intake. An investigation observed
weight loss in germ-free mice transplanted with microbiota fromanimals which had undergone gastric bypass surgery. The
reduced body weight was associated with increased microbial
production of propionate, but no differences in energy intake
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supplementation groups. However, the change in postprandial
glucose AUC to the standardised breakfast at week 24 was signiﬁcantly different between groups (see online supplementary ﬁgure
S6; p=0.037). Glycaemic response signiﬁcantly deteriorated
following inulin-control supplementation (AUC0–300 min 1600
min×mmol/L (95% CI 1495 to 1705) to 1691 min×mmol/L(95% CI 1580 to 1802), p=0.010) but remained unchanged
following inulin-propionate ester supplementation (AUC0–300 min
1630 min×mmol/L (95% CI 1534 to 1727) to 1630 min×mmol/L(95% CI 1543 to 1717), p=0.993), in keeping with the differences in weight gain and body fat distribution observed within
the groups. Insulin AUC was not signiﬁcantly different following
inulin-propionate ester (AUC0–300 min 7685 min×mU/mL (95%
CI 5858 to 9511) to 7969 min×mU/mL (95% CI 5955 to 9982),
p=0.612) or inulin-control supplementation (AUC0–300 min
9285 min×mU/mL (95% CI 7454 to 11 115) to 8781 min×mU/
mL (95% CI 7265 to 10 297), p=0.464) and the change ininsulin AUC was not signiﬁcantly different between groups (seeonline supplementary ﬁgure S7; p=0.372), suggesting that thedifference in glycaemic response reﬂects a difference in insulin
sensitivity.

Gut microbiota

Chambers ES, et al. Gut 2015;64:1744–1754. doi:10.1136/gutjnl-2014-307913

data that have observed changes in the gut microbiome andassociated SCFA production proﬁles in weight loss. In humans,
the beneﬁcial actions of propionate appear to be mediated bydifferent mechanisms in the short term compared with the longterm, which warrants further study. Optimum delivery of propionate to the colon through selection of propiogenic componentsof the diet may represent a novel route to improve weight management at the population level.
Author afﬁliations
1
Nutrition and Dietetic Research Group, Section of Investigative Medicine, Imperial
College London, 6th Floor Commonwealth Building, Hammersmith Hospital, London,UK
2
Stable Isotope Biochemistry Laboratory, Scottish Universities Environmental ResearchCentre, University of Glasgow, Glasgow, UK
3
Section of Investigative Medicine, Imperial College London, Hammersmith Hospital,
London, UK
4
School of Science, University of the West of Scotland, Hamilton, UK
5Institute of Psychological Sciences, University of Leeds, Leeds, UK
6
Metabolic and Molecular Imaging Research Group, MRC Clinical Science Centre,
Imperial College London, Hammersmith Hospital, London, UK
7Imperial Clinical Trials Unit, School of Public Health, Imperial College London,
London, UK
8
Department of Food and Nutritional Sciences, University of Reading, Reading, UK
9Leatherhead Food Research, Randall’s Road Leatherhead, Surrey, UK
10
Diabetes and Metabolism Division, Garvan Institute of Medical Research,
Darlinghurst, NSW 2010, Australia
Acknowledgements The authors thank Robin Stewart, David Barn, Emma
Hamilton and Scott McLachlan for technical assistance in the synthesis of the
propionate ester and Sandra Small and Eleanor McKay for assistance in the
preparation and analysis of isotopically labelled samples. This study was supportedby the UK Biotechnology & Biological Sciences Research Council (BB/H004971/1).
All clinical trial were conducted at the NIHR/Wellcome Trust Imperial ClinicalResearch Facility. The Department at Imperial College is funded by grants from the
MRC, BBSRC, NIHR, an Integrative Mammalian Biology (IMB) Capacity BuildingAward, an FP7- HEALTH- 2009- 241592 EurOCHIP grant and funding from the
NIHR Imperial Biomedical Research Centre Funding Scheme.
Contributors All authors contributed to the design of the study. ESC, AV, AP and
SEKZ-V conducted the human studies. DJM, CT and TP developed and produced theinulin-propionate ester. All authors contributed to writing the manuscript.
Competing interests GSF is supported by a NIHR senior investigator award, AV is
supported by a NHMRC overseas based clinical research fellowship (535976) and a
FP7-People-2009-256365 reintegration grant. ESC, AP, SM-I and KMD aresupported by the BBSRC. WSD is supported by a NIHR Career development
Fellowship.
Ethics approval Hammersmith and Queen Charlotte’s Research Ethics Committee.
Provenance and peer review Not commissioned; externally peer reviewed.
Open Access This is an Open Access article distributed in accordance with theterms of the Creative Commons Attribution (CC BY 4.0) license, which permits
others to distribute, remix, adapt and build upon this work, for commercial use,
provided the original work is properly cited. See: http://creativecommons.org/licenses/
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acetate and butyrate are comparable. Previous studies haveshown that much larger doses of inulin-type fructans (>30 g/day) are required to modulate gut hormone release and appetite
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support a role speciﬁcally for colonic propionate in weight management and may provide a molecular explanation of recent

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Glucagon-Like Peptide-1 Secretion via the G-Protein-Coupled Receptor FFAR2.
Diabetes 2012;61:364–71.
Cherbut C, Ferrier L, Roze C, et al. Short-chain fatty acids modify colonic motility
through nerves and polypeptide YY release in the rat. Am J Physiol 1998;275(6 Pt
1):G1415–22.
Anini Y, Fu-Cheng X, Cuber JC, et al. Comparison of the postprandial release of
peptide YY and proglucagon-derived peptides in the rat. Pﬂugers Arch
1999;438:299–306.
Murphy KG, Bloom SR. Gut hormones and the regulation of energy homeostasis.
Nature 2006;444:854–9.
Batterham RL, Cohen MA, Ellis SM, et al. Inhibition of food intake in obese subjects
by peptide YY3–36. N Engl J Med 2003;349:941–8.
Turton MD, O’Shea D, Gunn I, et al. A role for glucagon-like peptide-1 in thecentral regulation of feeding. Nature 1996;379:69–72.
Batterham RL, Cowley MA, Small CJ, et al. Gut hormone PYY(3–36) physiologicallyinhibits food intake. Nature 2002;418:650–4.
Brown AJ, Goldsworthy SM, Barnes AA, et al. The Orphan G protein-coupled
receptors GPR41 and GPR43 are activated by propionate and other short chain
carboxylic acids. J Biol Chem 2003;278:11312–19.
Le Poul E, Loison C, Struyf S, et al. Functional characterization of human receptors
for short chain fatty acids and their role in polymorphonuclear cell activation. J Biol
Chem 2003;278:25481–9.

Boosting chemical by-product of dietary fibre fermentation in gut slims and trims
Propionate-friendly fibre intake may offer new weight management option, say researchers[Effects of targeted delivery of propionate to the human colon on appetite regulation, body weight
maintenance and adiposity in overweight adults Online First: 11 Dec 2014. doi 10.1136/gutjnl-2014307913]
Boosting levels of a naturally produced by-product of dietary fibre fermentation in the bowel can help
trim the waistline and stave off weight gain, reveals a small study published online in the journal Gut.
This approach may offer a new weight management option, suggest the researchers.
Animal studies have shown that the natural fermentation of dietary fibre by gut bacteria produces
short chain fatty acids, one of which is propionate.
These fatty acids stimulate the release of the gut hormones PYY and GLP-1, which in turn suppress
appetite. And propionate seems to be the most effective at stimulating PYY and GLP-1 release.
To find out if increasing levels of propionate in the bowel could reduce food intake and stave off
weight gain in people, the researchers developed a propionate supplement primed to target
propionate release in the bowel.
First, 20 volunteers were given either the propionate supplement or just inulin, a predominantly
fructose-containing plant fibre, and allowed to eat as much as they liked from a buffet. When given the
propionate supplement, participants ate 14% less, on average, and had higher levels of PYY and
GLP-1 in their blood.
Next, 60 overweight adults between the ages of 40 and 65 received either a daily 10 g dose of the
propionate supplement or 10 g of inulin alone over a period of 24 weeks. They were asked to follow
their normal dietary and exercise routines throughout.
Body weight was assessed at the beginning and end of the study period, as was the distribution of fat
around the body.
A fasting blood sample was also taken to check on risk factors for cardiovascular disease anddiabetes, including blood fats, liver enzymes, and markers of inflammation.
Forty nine of the original 60 participants completed the trial.
Among the 25 people taking the propionate supplement, just one put on more than 3% of theirbaseline weight, compared with six of the 24 treated with inulin alone.
Furthermore, the propionate supplement altered the distribution of body fat, significantly trimming
abdominal fat tissue compared with inulin alone, and lowering the total amount of fat in the liver.
Both the propionate supplement and inulin cut the risk factors for cardiovascular disease anddiabetes, although only the propionate supplement significantly reduced ‘bad’ low density cholesterol
and the enzyme aspartate transaminase, high levels of which are associated with tissue damage,
particularly of the heart and liver.
The evidence suggests that adults gain around half to 1 kilo in weight every year throughout middle
age by just consuming 50-100 extra calories a day. And the findings provide the first direct evidence
that raising propionate levels in the bowel can cut energy intake and stave off longer term weight gain,
say the researchers.

“The present results support a role specifically for colonic propionate in weight management and may
provide a molecular explanation of recent data that have observed changes in the gut [range ofbacteria] and associated [short chain fatty acid production] profiles in weight loss,” they conclude.
Contacts:
Professor Gary Frost, Nutrition and Dietetic Research Group, Section of Investigative Medicine,
Imperial College London, London UK.
Email: g.frost@imperial.ac.uk
Or
Dr Douglas Morrison, Stable Isotope Biochemistry Laboratory, Scottish Universities Environmental
Research Centre, University of Glasgow, Glasgow, UK.
Email: Douglas.Morrison@glasgow.ac.uk

SUPPLEMENTARY MATERIALCONTENTSPAGE
SUPPLEMENTARY METHODS
Isolation of human colonic cells

2

In vitro gut hormone secretion experiments

2

Production of inulin-propionate ester

3

In vitro fermentation profiles of inulin-propionate ester and inulin-control

4

Acute intervention with inulin-propionate ester study subjects

5

Colonic delivery investigation

5

Gut hormone and energy intake investigation

6

Gastric emptying investigation

6

Long-term supplementation with inulin-propionate ester

7

Fluorescent in situ hybridisation to assess gut microbial changes in response7

to inulin propionate ester and control
Statistical analysis

9

SUPPLEMENTARY RESULTS

10

SUPPLEMENTARY FIGURES12SUPPLEMENTARY TABLES

20

REFERENCES

22

1

SUPPLEMENTARY METHODS

Isolation of human colonic cells
Colonic biopsies were obtained from patients undergoing diagnostic colonoscopy at Hammersmith
Hospital. Biopsies were usually obtained from the right side of the colon. All subjects provided
informed, written consent prior to the study, approval for which was granted by the Hammersmith
and Queen Charlotte’s Research Ethics Committee (Registration No: 2000/5795).
The colonic tissue was prepared as described previously 1 2. Briefly, the tissue was digested with
0.4mg/ml collagenase XI (Sigma, UK) in Dulbecco’s Modified Eagle Medium (DMEM) at 37oC.
The resulting cell suspensions were centrifuged for 5 minutes at 500 × g and the pellets resuspended

in

DMEM

(supplemented

with

10%

foetal

calf

serum

(FCS)

and

1%

penicillin/streptomycin). The digestion process was repeated three times and the combined cellsuspensions plated onto 24-well, 1% Matrigel-coated plates and incubated at 37oC in an atmosphereof 5% CO2.

In vitro gut hormone secretion experiments
Secretion experiments were carried out 18-24 hours after the colonic cells were plated. Cells were
washed twice with secretion buffer (4.5mM KCl, 138mM NaCl, 4.2mM NaHCO3, 1.2mM
NaH2PO4, 2.6mM CaCl2, 1.2mM MgCl2 and 10mM HEPES, adjusted to pH 7.4 with NaOH)
containing 0.1% bovine serum albumin (BSA) 1 and incubated with sodium propionate (Sigma,UK) for 2 hours at 37oC in an atmosphere of 5% CO2. Concentrations of 200 and 400 mmol/L wereused as estimates suggest that the concentration of SCFA in the human colon is approximately 150
mmol/L and modelling data from in vitro systems demonstrate that these levels will more thandouble with a high fermentable carbohydrate diet 3 4. In addition, these concentrations are in linewith those previously shown to stimulate PYY release from the rodent colon (500 mmol/L) 5.
Following incubation, cell supernatants were centrifuged and the plates treated with lysis buffer andfreeze-thawed. Percentage gut hormone release was calculated for each well and normalised tobasal secretion 1. Cell health was confirmed using a CytoScanTM lactate dehydrogenase assay (GBiosciences, USA). PYY and GLP-1 data was obtained from separate cell cultures.

2

Production of inulin-propionate ester
The inulin-propionate ester was synthesized by reacting inulin with propionic anhydride (0.8 L/Kg
inulin) in water whilst maintaining pH between 8 - 8·5 and the reaction temperature < 20°C. Excess
unreacted propionate was removed by filtration twice through activated carbon columns whilst the
mixture was maintained at pH 2. Finally, the product was recovered as a fine amorphous crystalline
product through spray drying. A 13C stable isotope labelled variant of the propionate ester was
synthesised in an identical fashion to produce 13C-propionate ester with (1-13C1)-propionate boundto inulin.

To determine the amount of free propionate in the final product, 100 mg of ester product wasdissolved in 2 ml water containing 10 mM butyric acid as the internal standard (IS). To quantify
free propionate, 200 µl of the solution was treated with 100 µl of concentrated orthophosphoric acidfollowed immediately by ether extraction (1 ml). To quantify total propionate (free + bound),another 200 µl of the solution was treated with 100 µl of concentrated orthophosphoric acid andheated at 80°C for 1 hour before being extracted with 1 ml ether. Propionate and butyrate in theether extracts were quantified by gas chromatography with flame ionisation detection 6. Propionate
yield was calculated relative to the IS and the amount of free propionate calculated by the ratio(free/total) × 100 (%). The degree of esterification (de) was also calculated using this analysis byusing the yield of bound propionate (total – free) per gram of ester to compute moles of propionate
yielded per mole of propionate ester.

An inulin-propionate ester was produced with a degree of esterification of 0.74 ± 0.02, meaning on
average that 24.6 ± 0.67% (0.74 out of maximum of 3 per monosaccharide unit) of all hydroxyl
groups were replaced by an ester group. The level of free propionate was 2.57 ± 0.26 % of the total
propionate available from the molecule, demonstrating that more than 97% of propionate waschemically bound to the inulin polymer. Less than 1% of the bound propionate is released when theinulin-propionate ester is solubilised in acid (pH 1-2), a similar low pH to the environment of thestomach. Heating to >80ºC is required in order to release the propionate from the ester in thispreparation.

3

In vitro fermentation profiles of inulin-propionate ester and inulin-control
Faecal samples were collected from three healthy volunteers (who had no history of gastrointestinal
complaints and were antibiotic free for >6 months prior to faecal collection) and preparedseparately in triplicate for each substrate. Faecal fermentation systems consisted of the fermentation
medium (2.25g tryptone in 450 ml of distilled water), 112.5 µl of micromineral solution (13.2 g of
CaCl2, 10.0 g of MnCl2, 1.0 g CoCl2, FeCl3 made up to 100ml with distilled water), 225 ml ofmacromineral solution (2.85 g Na2HPO4, 3.1 g KH2PO4, 0.3 g MgSO4 made up to 500 ml with
distilled water), 225 ml of buffer solution (2 g NH4HCO3, 17.5 g NaHCO3 made up to 500 ml with
distilled water) and 1 ml of 0.1% (w/v) resazurin solution (a redox indicator). This medium wasadjusted to pH 7 using 6M HCl, after which it was sterilized by boiling for 5 min. Reducing
solution prepared on the day of fermentation (312.5 mg cysteine hydrochloride, 2 ml 1M NaOH,
312.5 mg sodium sulfide, and 47.5 ml distilled water) was added at 0.5 ml per 10 ml of medium
after the solution was cooled to 37°C under oxygen-free nitrogen (OFN) until anaerobic conditions
were achieved as indicated by a colour change from pale indigo to colourless. A 32% faecal slurry
was prepared for each subject in 66.6 mM phosphate buffer (pH=7), homogenized in a household
blender for 2 min and strained through a nylon stocking. The final in vitro system containing 0.5 mlof the slurry was added to 4.2 ml of the pre-reduced fermentation medium in 10 ml autoclaved
fermentation bottles. Each batch culture consisted of 4.9 ml of the above slurry mixture to which
100 mg of inulin-propionate ester or inulin-control was added. At 0 and 24 h, 800 µL of theaqueous volume was removed from each vial. For SCFA extraction, 100 µL of internal standard(IS; 2-ethylbutyrate, 73.8 mM) and 25 μL concentrated orthophosphoric acid was added to 225 μL
of sample and thoroughly mixed and extracted with 3 x 1 mL of ether. The ether aliquots werepooled and a sub-sample transferred to a clean vial for analysis. Samples were analysed by GC-FID(Trace GC, ThermoFisher, UK) using a ZB-WAX column (15 m × 0.53 mm × 0.25 um;
Phenomenex, Cheshire UK). The GC operating parameters were nitrogen carrier gas (1.89 ml/min)
and GC oven parameters starting at an initial temp of 80°C, ramp temp (10°C /min) to 210°C withsplitless injection. The concentration of acetate, propionate and butyrate calculated using the area
ratio to the IS and the calibrated response factor of each SCFA to the IS as determined by agravimetrically prepared external standard. The concentration (production) of SCFAs and molar
ratios were calculated.

4

Acute intervention with inulin-propionate ester
Nine healthy subjects (8 males and 1 female) were recruited for the colonic delivery investigation.
The mean (± SEM) age, weight and body mass index (BMI) were 32 ± 4 years, 75.0 ± 4.0 kg and25.0 ± 1.3 kg/m2, respectively. Twenty healthy subjects (15 males and 5 females) were recruited forthe acute food intake investigation. The mean (± SEM) age, weight and BMI were 31 ± 2 years,
75.0 ± 3.0 kg and 25.4 ± 0.8 kg/m2, respectively. Fourteen healthy subjects (8 males and 6 females)
were recruited for the gastric emptying investigation. The mean (± SEM) age, weight and BMI were
32 ± 4 years, 69.4 ± 3.5 kg and 24.0 ± 0.9 kg/m2, respectively. The inclusion criteria for theseinvestigations were a BMI of 20 to 35 kg/m2 and 21 to 65 years of age. The exclusion criteria weresmoking, substance abuse, pregnancy, use of medications (except for oral contraceptives), a change
in body weight >3 kg in the previous 2 months, medical or psychiatric illness, and any
abnormalities detected on physical examination, electrocardiography, or screening blood tests(measurement of complete blood count, electrolytes, fasting glucose, thyroid function and liverfunction).

Colonic delivery investigation
This investigation compared the appearance of 13C in breath CO2 with the appearance of breath H2,
a methodology previously used to investigate gut transit times 7. Subjects arrived at Hammersmith
Hospital at 08:30 and were served a standardized breakfast (533 kcal; 78 g carbohydrate (CHO), 17
g fat, 18 g protein) containing 10g of propionate ester and 100mg 13C labelled inulin propionate
ester (containing ~30 mg of bound (1-13C1)-propionate). Breath H2 was collected and measured inreal-time using a handheld H2 monitor (Bedfont Scientific Ltd, Kent UK). Breath CO2 was collected
serially over 24 h (excluding the sleep period) by exhaling alveolar breath through a straw into
Exetainers (Labco, Buckinghamshire, UK). 13CO2 enrichment was determined by isotope ratio mass
spectrometry (IRMS). Breath H2 was expressed as parts per million (ppm) and 13CO2 as ppm xs,
defined as ppm 13C enrichment above baseline samples collected before isotope ingestion.
Cumulative 13CO2 excretion was also calculated. Plasma was collected at -15, 0 and 360 min, andurine over 24 h for analysis of 13C propionate enrichment by GC-combustion-IRMS (GC-C-IRMS)
6. Acetate 13C enrichment was also measured in the same analysis as a control for dietary influenceon SCFA 13C enrichment and for evidence of inter-conversion between propionate and acetate.
Although our experimental methodology was not designed to explore gluconeogenesis, 13C
enrichment was measured in plasma glucose and plasma alanine by LC-IRMS 8 and GC-C-IRMS 9,
respectively. Enrichment in plasma 13C glucose would occur through gluconeogenesis frompropionate directly but alanine would indicate the enrichment in the triose pool, with which it
5

comes rapidly into isotopic equilibrium 10. Data were expressed as δ13C (per mil or ‰) which
represents the change in the measured ratio in parts per thousand from the internationally accepted
standard carbon Vienna Pee Dee Belemnite (VPDB). Isotopic enrichment was expressed as δ13C(‰). SCFA concentrations (µmol/L) were measured relative to an internal standard (3-methyl
valerate).

Energy intake and gut hormone investigation
The study was performed in a randomized, double-blind, crossover manner, with each subject
studied on two occasions > 7 days apart. Subjects refrained from alcohol and strenuous exercise forthe 24 hours prior to each study day and consumed an identical meal between 19:00 and 20:00 theevening before. Subjects then fasted overnight and arrived at Hammersmith Hospital at 08:30 oneach study day. A cannula was inserted into a forearm vein and baseline blood samples werecollected at -10 and 0 min. Following the 0 min sample, subjects were served a standardized
breakfast (398 kcal; 71 g CHO, 8 g fat, 10 g protein) containing either 10 g of inulin-propionate
ester, or 10 g inulin control. At 180 min a standardized lunch (356 kcal; 34 g CHO, 12 g fat, 28 g
protein) was provided, and at 420 min subjects were offered a buffet dinner with food served inexcess and asked to eat until they felt comfortably full. The amount of food was quantified andenergy intake calculated. Postprandial blood samples were taken at 15, 30, 60, 90, 120, 180, 240,
300, 360 and 420 min and collected into heparin-coated tubes containing 0.2 ml of aprotonin(Bayer, UK). Plasma was separated immediately by centrifugation at 4°C and then stored at -70°C
until analyzed. Subjective hunger, satiety, and nausea were monitored with the use of 100 mm
visual analog scales (VAS) 11. Subjects were asked to complete the VAS before each blood sample.

Gastric emptying investigation
The study was performed in a randomized, double-blind, crossover manner, with each subject
studied on two occasions > 7 days apart. Subjects refrained from alcohol and strenuous exercise forthe 24 hours prior to each study day and asked to consume a standard meal between 19:00 and
20:00 the evening before. On the study days, subjects attended having fasted overnight. Following
baseline blood samples, subjects were given 10 g inulin-propionate ester or 10 g inulin control in astandardized breakfast (641 kcal; 113 g CHO, 15 g fat, 16 g protein). At 300 min volunteers wereserved a standard lunch (354 kcal; 47 g CHO, 12 g fat, 12 g protein) together with 100 mg 13Coctanoic acid 12. Breath CO2 was collected serially for 480 min after the standard lunch by exhaling
alveolar breath through a straw into Exetainers (Labco, Buckinghamshire, UK). 13CO2 enrichment
6was determined by isotope ratio mass spectrometry (IRMS). The time to 50% AUC excretion of 13C
in breath (T1/2) was calculated as a proxy for gastric emptying rate.

Long-term supplementation with inulin-propionate ester
Randomisation
Four strata were defined according to sex (male, female) and BMI (<30.0 kg/m2, ≥30.0 kg/m2) andrandomisation sequences for each stratum were generated using the random number generator using
Stata (version 11.0). The two supplementation groups were labelled ‘A’ and ‘B’ by DJM who held
the key to the allocations for the duration of the trial. Participants were randomised to either ‘A’ or‘B’ by the trial statistician (SMI) according to the randomisation sequence and assigned arandomisation number. Throughout the trial, none of the participants or investigators involved in
the trial had access to complete information on the randomisation allocations.

Self-reported assessment of energy intake and physical activity
Participants were asked to record energy intake by 3-day food diary at baseline and during the last
week of the 24 week supplementation period. Food diaries were analysed using Dietplan6(Forestfield Software, West Sussex, UK). Physical activity was measured at baseline and at week
24 using the short self-administered format of the International Physical Activity Questionnaire(IPAQ)13. IPAQ measures the frequency and duration of any walking and other moderate-tovigorous intensity physical activity undertaken for more than 10-continuous minutes across allcontexts (e.g. work, home and leisure) over a 7-day period. IPAQ calculates metabolic equivalents(MET-h/week), derived by assigning standardised MET values for walking, moderate-intensity andvigorous-intensity of 3.3, 4.0 and 8.0, respectively.

Fluorescent in situ hybridisation to assess gut microbial changes in response to inulinpropionate ester and control
The effects of inulin-propionate ester on gut microbial populations were studied using an in vitro
culture system. Faecal samples were obtained from three healthy human volunteers (two males; age
30 – 50 years; BMI 25-31 kg/m2). Volunteers were excluded if they suffered from any
gastrointestinal disorder (e.g. ulcerative colitis, Crohn’s disease, irritable bowel syndrome, peptic
7

ulcers and cancer) and/or had taken antibiotics in the six months preceding sample donation.
Samples were kept under anaerobic conditions (10% H2, 10% CO2 and 80% N2) and used within amaximum of 30 min after voiding. Faecal samples were diluted 1/10 w/w in anaerobic phosphatebuffered saline (0.1 mol/l phosphate buffer solution, pH 7.4) and homogenised in a stomacher(Stomacher 400, Seward, West Sussex, UK) for 2 min at normal speed.

Batch culture fermentations
Sterile anaerobic batch culture fermenters (150ml working volume) were set up in parallel andaseptically filled with 135ml pre-reduced, sterile basal culture medium (peptone water 2 g/l(Oxoid), yeast extract 2 g/l (Oxoid, Basingstoke, UK), NaCl 0.1 g/l, K2HPO4 0.04 g/l, KH2PO4 0.04
g/l, MgSO4.7H2O 0.01 g/l, CaCl2.6H2O 0.01, NaHCO3 2 g/l, Tween 80 2 ml (BDH, Poole, UK),haemin 0.05 g/l, vitamin K1 10 l, cysteine.HCl 0.5 g/l, bile salts 0.5 g/l). The fermenters were
gassed overnight with O2 free N2 at a rate of 15mL/min. Inulin-propionate ester and inulin control
substrates (1/10 w/v) were added to their respective fermenters just prior to the addition of the
faecal slurry. A substrate-free vessel was set up for each volunteer as negative controls. Cultures
were continuously stirred and kept at 37°C by means of a circulating water bath. Culture pH was
kept between 9.7 and 6.9 using automated pH controllers (Fermac 260, Electrolab, Tewkesbury,UK). Each vessel was inoculated with 15 ml fresh fecal slurry (1/10 w/w). Batch fermentations
were ran for 48 h and 6 ml samples were obtained from each vessel at 0, 10, 24, 34 and 48 h formicrobial enumeration by fluorescent in situ hybridisation (FISH) analysis. Three replicate
fermentations were set up, each inoculated with one of three different human fecal samples.

Bacterial enumeration
Fluorescent in situ hybridization targeting Bifidobacterium spp. (Bif164), Bacteroides/ Prevotella(Bac303), Lactobacillus/ Enterococcus (Erec482), Clostridium histolyticum (Chis150), Atopobium
cluster (Ato291) and Eubacterium rectale/ Clostridium coccoides (Erec482), was used as describedby Sarbini et al (2011). Cells were visualized by fluorescent microscopy (Eclipse 400, Nikon,
Surrey, UK) using the Fluor 100 lens. For each sample, 15 different fields of view wereenumerated.

8

Statistical analysis
One-way ANOVA was performed to determine differences in in vitro secreted gut hormone levels.
Differences in energy intake and area under the curve (AUC) for plasma hormone levels and gastric
emptying between trials were assessed using Paired Student’s t test. Two-way (trial×time) repeatedmeasures ANOVA was performed to determine differences in plasma hormone levels, VAS, breathhydrogen, and bacterial group populations. Significant effects were followed up by Tukey’s post
hoc comparisons. Data are presented as means ± SEM or ± 95% Confidence Interval [CI]. P values< 0.05 were considered statistically significant.

9

SUPPLEMENTARY RESULTS

Colonic delivery investigation
13

C enrichment in plasma glucose and plasma alanine

Enrichments in plasma δ13C glucose and δ13C alanine were 1.5 ± 1.7 per mil (p=0.27) and 0.9 ± 0.8
per mil (p=0.11) at 360 min compared with baseline, respectively.

Estimation of daily colonic short chain fatty acid production from inulin-propionate ester
Data from sudden death victims suggests that the average propionate pool size in the proximal large
intestine is 4.5 mmol and 10 g of inulin-propionate ester releases an additional 15.0 mmol and 36.2
mmol from inulin fermentation and bound propionate respectively 3. Using stoichiometric equations
for hexose fermentation by intestinal bacteria 14, we calculate that a daily dietary intake of 15 g nonstarch polysaccharide yields a daily production of 94.1, 34.5 and 28.2 mmol for acetate, propionate
and butyrate, respectively. Addition of 10 g inulin-control leads to a 2.0-, 1.6- and 1.4-fold change
for daily acetate, propionate and butyrate production, respectively, whereas addition of 10 g inulinpropionate ester leads to a 1.7-, 2.5- and 1.3-fold change for daily acetate, propionate and butyrate
production respectively.

Gastric emptying investigation
Rates of gastric emptying were not significantly different between acute propionate ester andcontrol treatments (Siegel T1/2 185 min [95% CI, 168 to 204] inulin-propionate ester vs. 180 min[95% CI, 163 to 198] control, P=0.506).

Gut microbial changes to inulin-propionate ester and inulin-control
Inulin control significantly increased Bif164 labelled cells at all time points compared to baseline.
Bif164 levels with control were significantly higher compared to the propionate ester cultures
throughout fermentation. Both propionate ester and inulin control significantly increased Bac303
labelled cells at all sampling points compared to baseline levels. Ato291 labelled cells increased10significantly with both test substrates between 10 and 34 h compared with baseline concentrations.
No other significant changes were seen in any of the groups targeted or total bacteria levels. It
appears that propionate ester was fermentable by Bacteroides and Atopobium but not by
Bifidobacterium spp.

11

SUPPLEMENTARY FIGURES

Suppl. Figure 1. A. Short chain fatty acid production and B. molar ratios of inulin-control andinulin-propionate ester in fecal fermentations. Mean ± SEM, *P<0.05 (n=3).

12

Suppl. Figure 2. The effect of acute inulin-propionate ester supplementation on postprandial
A. glucose, B. insulin or C. leptin response. Arrows indicate timings of standardized meals. 10 ginulin-control or 10 g inulin-propionate ester were provided with breakfast at 0 min. Dotted lines
signify the time point after which >80% inulin-propionate ester enters the colon as determined by
the enrichment of 13C in expired air and breath H2 methodology (Figure 1C). Data are presented asmeans ± SEM (n=20).

13

Suppl. Figure 3. An acute increase in colonic propionate content does not affect postprandial
ratings of appetite or nausea. A. Hunger, B. Desire To Eat, C. Fullness, and D. Nausea. Ratings
were made using 100 mm visual analogue scales (VAS), with extreme statements anchored at eachend of the rating scale (e.g. 0 mm Not at all hungry, 100 mm Extremely hungry). Arrows indicate
standardized meals. 10 g inulin-control or 10 g inulin-propionate ester were provided with breakfast
at 0 min. Data are presented as means ± SEM (n=20).

14

Suppl. Figure 4. Flow chart showing recruitment and retention in the 24 week
supplementation study.

15

Suppl. Figure 5. Postprandial ratings of appetite and nausea at baseline and following 24 weeks supplementation with inulin-control andinulin-propionate ester. A. and B. Hunger, C. and D. Desire To Eat, E. and F. Fullness, and G. and H. Nausea. Ratings were made using 100 mm
visual analogue scales (VAS), with extreme statements anchored at each end of the rating scale (e.g. 0 mm Not at all hungry, 100 mm Extremely
hungry). *P<0.05, Mean ± SEM (inulin-control, n = 24; inulin-propionate ester, n = 25).

16

Suppl. Figure 6. Effect of 24 weeks supplementation with inulin-control and inulin-propionate
ester on glucose response. A. Postprandial glucose response at baseline and following 24 weeks ofsupplementation with inulin-control and inulin-propionate ester. B. Glucose AUC0-300min at week 0
and week 24. C. Change from baseline (week 0) glucose AUC0-300min at week 24. *P<0.05. Mean ±SEM (inulin-control, n = 24; inulin-propionate ester, n = 25).

17

Suppl. Figure 7. Effect of 24 weeks supplementation with inulin-control and inulin-propionate
ester on insulin response. A. Postprandial insulin response at baseline and following 24 weeks ofsupplementation with inulin-control and inulin-propionate ester. B. Insulin AUC0-300min at week 0
and week 24. C. Change from baseline (week 0) insulin AUC0-300min at week 24. Mean ± SEM(inulin-control, n = 24; inulin-propionate ester, n = 25).

18

Suppl. Figure 8. Effect of 24 weeks supplementation with inulin-control and inulin-propionate
ester on breath hydrogen. A. Fasting and postprandial breath hydrogen at baseline and following
24 weeks of supplementation with inulin-control and inulin-propionate ester and B. Breathhydrogen AUC0-300min at week 0 and week 24. *P<0.05. Mean ± SEM (Propionate Ester, n = 9;Control, n = 8).

19

SUPPLEMENTARY TABLES

Suppl. Table 1. Gastrointestinal adverse events reported during the 24 week supplementation
period.
Inulin-Control

Inulin-Propionate Ester(N = 24)(N = 25)

Inulin-Propionate
Ester- Inulin-Control

Side EffectMean ± SD

Mean ± SD

Difference (95% CI)

P Value

Stomach Discomfort

31.0 ± 26.6

10.5 ± 13.0

-11.1 (-29.0, 6.7)

0.221

Nausea7.5 ± 10.6

4.6 ± 5.8

1.5 (-7.4, 10.4)

0.736

Bloating

34.0 ± 27.4

19.7 ± 22.7

-6.8 (-24.4, 10.8)

0.448

Flatulence

56.0 ± 28.4

25.2 ± 22.6

-24.9 (-41.9, -8.0)

0.004

Heartburn17.8 ± 19.5

9.9 ± 14.4

-1.9 (-18.0, 14.2)

0.820

Belching

15.1 ± 14.3

10.0 ± 14.0

-0.0 (-11.8, 11.8)

1.000

Ratings were made at weeks 8, 16 and 24 using 100 mm visual analogue scales (VAS). Subjects were asked torate the occurrence of each side effect with extreme statements anchored at each end of the rating scale (0 mm
Never, 100 mm All the time).

20

Suppl. Table 2. Self-reported food intake and physical activity of subjects at baseline andfollowing 24 weeks of inulin-control and inulin-propionate ester supplementation. Means ± SEM.
Inulin-Control

Inulin-Propionate Ester

Week 0Week 24

Week 0Week 24

Energy(kcal)

2215 ± 247

2508 ± 317

1957 ± 249

1753 ± 359

Energy(kJ)9306 ± 1034

10544 ± 1316

8213 ± 1043

7385 ± 1522

Protein(g)

95 ± 1595 ± 1175 ± 970 ± 11Fat(g)

84 ± 1294 ± 1882 ± 1373 ± 12

Carbohydrate(g)

256 ± 19294 ± 43237 ± 29

213 ± 68

Fibre
(g)

20 ± 421 ± 517 ± 212 ± 5

Total physical activity(MET-h/week)

33.3 ± 8.0

31.3 ± 6.8

25.7 ± 4.3

26.8 ± 4.0

Vigorous-intensity(MET-h/week)

3.5 ± 3.3

4.9 ± 3.4

4.3 ± 1.8

4.4 ± 1.9

Moderate-intensity(MET-h/week)

5.9 ± 2.5

4.9 ± 2.5

3.6 ± 1.1

4.1 ± 0.9

Walking(MET-h/week)

23.9 ± 5.0

21.5 ± 3.8

17.8 ± 3.7

18.3 ± 3.6

Energy intake was recorded with 3-day food diaries. Physical activity was measured using the short selfadministered format of the International Physical Activity Questionnaire (IPAQ) 13. IPAQ calculates metabolic
equivalents (MET-h/week), derived by assigning standardised MET values for walking, moderate-intensity andvigorous-intensity of 3.3, 4.0 and 8.0, respectively. No significant differences were found in any measure either
within- or between-groups (P>0.05).

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