Using a complex carbohydrate mixture added to a high-protein DIet to STeer fermentation to improve metabolic, gut and brain heALth

The worldwide prevalence of obesity has nearly tripled in the past 40 years,
totalling up to over 650 million adults in 2016. Obesity is related to the
onset of metabolic disorders, such as metabolic syndrome, type 2 diabetes
mellitus (T2DM), non-alcoholic fatty liver disease (NAFLD) and cardiovascular
disease (CVD). These diseases are associated with a high morbidity and
mortality, making T2DM the ninth leading cause of death worldwide. Apart from
causing a high burden on a patients wellbeing, this also leads to a global
health and socio-economic burden.

The human diet plays an important role in (metabolic) health and can be a major
component in the aetiology of obesity, T2DM and NAFLD. The western diet, being
energy and relatively low in fibres, is seen as a major contributor to the
development of these diseases. However, diet composition may affect food
digestion and fermentation, which may have either a beneficial or detrimental
effect on human metabolism and health. The gut microbiome, consisting of
trillions of microbes and being responsible for the fermentation of
indigestible food components, is being more and more thoroughly understood, and
is recognized for its important role in gut health, mucosal immunity and
integrity, and in metabolic and psychological health of its host.

The microbiome is able to ferment indigestible food components, such as
prebiotics, dietary fibres and resistant starches. Fermentation of these
carbohydrates, called saccharolytic fermentation, occurs mainly in the proximal
colon and produces short-chain fatty acids (SCFA), of which acetate, propionate
and butyrate are the most common. These SCFA, which are the main fuel for
colonocytes, are absorbed quickly, leading to an almost absolute absence of
SCFA in the transverse colon. In absence of indigestible carbohydrates, the
microbiome shifts to proteolytic fermentation in the transverse and distal
colon, producing numerous substrates, such as indoles, tryptophane, ammonia and
hydrogen sulphide.

In general, SCFA have a beneficial effect on human health. They improve gut
health by improving epithelial function, intestinal barrier function and
mucosal quality, and reducing gut permeability. SCFA also have a positive
impact on metabolic health by reducing body weight, increasing energy
expenditure, satiety, insulin sensitivity and adipose tissue lipid buffering
capacity, and preventing low-grade inflammation and ectopic lipid accumulation.
Furthermore, evidence shows that an increase in circulating SCFA reduces
inflammation in the gut, pancreas and throughout the body, including the brain,
by reducing cytotoxic T cell activity and increasing anti-inflammatory
regulatory T-cells. Lastly, SCFA are found to reduce stress in rodents, as a
result of SCFA-induced reduced release of corticotropin-releasing hormone and
downregulation of mineralocorticoid receptor expression.

Proteolytic fermentation, more prevalent in the transverse and distal colon,
produces substrates, such as branched-chain fatty acids, ammonia and phenols,
that can have detrimental health effects. On the other hand, there are also
indications that substrates of proteolytic digestion may have positive health
effects. Proteolytic digestion, and especially digestion of tryptophan rich
proteins, in the small intestine is associated with reduced inflammation and
improved gut barrier function and increased satiety and weight
loss/maintenance. Overall, the balance between saccharolytic and proteolytic
fermentation products in different parts of the gut may be an important
determinant of metabolic health.

Our recent studies have shown that delivering SCFA, acetate in particular,
specifically to the distal colon yields beneficial health effects, such as
increased fat oxidation, reduced lipolysis, increased satiety hormone
concentrations and reduced inflammatory cytokines (TNF-α). Apart from this,
other studies have shown that delivering SCFA directly to the distal colon
improves human metabolism and increases levels of satiety hormones and energy
expenditure, which may be partly due to SCFA bypassing the liver and increasing
peripheral SCFA concentrations. These effects were not present when acetate was
administered to the proximal colon. We therefore hypothesize that an increased
delivery of indigestible carbohydrates throughout the entire colon may have the
most pronounced health effects by increasing distal saccharolytic fermentation
and SCFA production, and thereby inhibiting proteolytic fermentation.

Increasing the SCFA concentration in the distal colon can be achieved by using
complex carbohydrates, such as resistant starches or dietary fibres. These
complex, indigestible carbohydrates escape fermentation in the small intestine,
since the composition of the small intestinal microbiome differs from the
colonic microbiome, lacking specific bacteria that are capable of fermenting
these carbohydrates.

Many previous studies regarding manipulation of intestinal digestion and
fermentation are performed on rodents, while evidence in humans is lacking. The
few available human trials mainly describe acute interventions and present
inconsistent data. Furthermore, the beneficial effects of SCFA on glucose
homeostasis and insulin sensitivity are mainly found in lean individuals, and
not in obese/(pre)diabetic adults.
The few human trials on long term effects of introduction of SCFA in the colon
are mostly conducted in healthy adults, and results were not consistent. This
may be related to interindividual variation in response. We hypothesize that
this is partly due to a different composition of initial microbiome, as well as
differences in metabolic phenotypes (i.e. being insulin resistant or having
prediabetes).

To this end, the aim of the present project is to determine the long-term
effects (12 weeks) of an optimal balance between saccharolytic and proteolytic
fermentation products on gut health, human metabolism and psychological state
in humans with prediabetes and overweight. The present study might give rise to
new possible preventative and therapeutic measures to reduce obesity and its
related metabolic disorders, including insulin resistance, metabolic syndrome,
type 2 diabetes mellitus and non-alcoholic fatty liver disease. Additionally,
we expect that improving microbial composition and functionality may also
improve brain health by supporting cognitive functioning and appetite
regulation, altering reactions to food cues and reducing experienced stress.

Primary Objective:
The main goal of this study is to provide evidence that steering
saccharolytic/proteolytic fermentation by a specific indigestible fibre mixture
supplemented against the background of a high-protein diet optimizes beneficial
and minimizes detrimental substrate production. Most importantly, we will be
looking into changes in peripheral insulin sensitivity.

Secondary Objectives:
Apart from determining possible effects on insulin sensitivity, we will also
investigate the following parameters and analyse whether steering fermentation
can influence the following secondary outcomes:
1. Hepatic and adipose tissue insulin sensitivity
2. Energy and substrate metabolism (energy harvest, energy intake, energy
expenditure, fat and carbohydrate oxidation).
3. Circulating metabolites, inflammatory factors and blood lipid spectrum
4. Gut permeability
5. Brain health (neurocognitive functioning and food reward-related brain
activity)
6. Adipose tissue and skeletal muscle metabolism
7. Composition and functionality of microbiome
8. Gastro-intestinal side-effects of provided dietary supplement against the
background of a high-protein diet

Additionally, we will explore possible correlations in the gathered data,
including anthropometric measurements, physical activity, dietary intake,
glucose sensitivity, composition of gut microbiota, etc.

The proposed study will be a double-blind, randomized placebo-controlled trial,
to evaluate the effect of the dietary supplement on gut, metabolic, immune and
brain health by optimizing saccharolytic and proteolytic fermentation.
Individuals aged 30-70 years with overweight or obesity (BMI >= 28 kg/m2 < 40
kg/m2) and prediabetes (defined as fasting glucose 5.6-6.9 mmol/l or HbA1c
42-47 mmol/mol or HOMA-IR >1.85) will be included in the study.
All visits regarding this study will take place at the facilities of the
department of human biology or the Metabolism Research Unit Maastricht of
Maastricht University, or at the clinical facilities of Maastricht University
Medical Center+.

After the application of an individual via (e-)mail or phone, the researcher
will contact the potential participant by email or phone to provide additional
information regarding the study, answer questions and determine possible
eligibility, after which a week of consideration will be given. After a week,
the potential participant will be contacted again and, if they are still
interested in participating, a screening visit will be planned.

The first visit to our facilities will consist of the following components:
- Providing additional information, answering questions and checking if
all information is understood correctly, ensuring participants are still
interested in participating
- Signing informed consent
- Checking an individual*s wellbeing, medical history and determining
eligibility and safety of participation.
- Measuring vital parameters and body measurements (body weight, length,
waist-to-hip ratio, systolic and diastolic blood pressure)
- Taking blood samples to determine eligibility based on specific inclusion
criteria (20ml to determine glucose, insulin, ALAT, creatinine, HbA1c)
The participant will visit our facilities after a >10h overnight fasted state.

All eligible participants will be equally randomized over the two arms (dietary
supplement vs placebo) with minimisation for age, sex and BMI. The entire trial
period after screening and randomization will take 13 weeks, which will be
explained in more detail below.

Intervention period
This randomized clinical trial with the primary outcome tissue-specific insulin
sensitivity has a placebo-controlled, double blind, randomized parallel design,
which allows evaluation of the role of a fibre mixture against the background
of a high protein diet on host metabolism in male and female adult volunteers
with overweight/obesity and prediabetes.

The approach is as follows:

1. Preceding this human intervention trial, a pilot study already has been
performed to screen for the most optimal fibre mixture increasing distal
colonic saccharolytic fermentation (and thus SCFA production) and inhibiting
proteolytic fermentation. Faeces have been collected in 13 human volunteers
with insulin resistance and overweight. The pooled faecal microbiota has been
inoculated in TIM-2 to analyse SCFA and BCFA production after addition of
different fibres or fiber combinations in a 24-h protocol against the
background of a high-plant based protein mixture (which was comparable to the
protein ratio as proposed in the human trial). The fibre mixture that resulted
in the highest microbial SCFA production in the part of TIM-2 that represented
the distal colon in vitro will be supplemented in the in vivo study. The hereby
defined fibre mixture is composed out of potato and pectin fibres
2. After randomization, the 12 week impact of the fiber supplement on
tissue-specific insulin sensitivity will be determined in a randomized, double
blind, parallel designed study.

Before the start of the intervention, participants will visit our facilities
for the first clinical investigation days (CID 1 and 2). These days consist of
the following test: (see Chapter 8.3 Study procedures for detailed description
of the tests)
- Providing stool samples by participant
- Taking skeletal muscle and adipose tissue biopsies
- Performing two-step hyperinsulinemic-euglycemic clamp
- Performing indirect calorimetry during the clamp
- Performing gut permeability tests by intake of specific sugars dissolved in
water and analysis of urine samples
- Conducting Functional Magnetic Resonance Imaging to study food-reward-related
brain activity
- Conducting a DEXA scan to determine body composition
- Filling in questionnaires and performing neuropsychological tests
- Food diaries will be filled in prior to CID 1.
- Distributing and explaining of assigned intervention (fiber supplement or
placebo)

After CID 1, participants will be provided with a standardized meal. This meal
needs to be consumed at home on the evening before CID 2 ad libitum.
Afterwards, they should remain fasted until the hyperinsulinemic-euglycemic
clamp on CID 2. CID 2 will take place at least 2 days and preferably within 2
weeks after CID 1.

After the 12 weeks of intervention, two additional CIDs (CID 3+4) will take
place, which are similar to CID 1+2, respectively.

After completing the screening and CID 1+2, the intervention will consist of
either a dietary supplement or a placebo which will have to be taken once daily
during 12 consecutive weeks. Furthermore, all participants will receive a
background diet high in protein 25EN% protein mixture, 30EN% fat, and 45EN%
carbohydrates. Protein content will consists for 45-50% out of plant-based
protein and for 50-55% out of animal-based proteins. The protein-rich products
will be consumed during breakfast and dinner. The fibre mixtures and control
product can be easily consumed with a high-protein diet. The exact diet
composition will be discussed individually to establish a diet suited to the
participants* needs and taste. The energy requirements for this diet will be
estimated using indirect calorimetry based on resting energy expenditure and an
activity factor.
Both the participants and investigators will be blinded to the treatment. The
study is designed to study the dietary fibre effects against the background of
a high protein diet independent of any diet or exercise intervention.

In week 2, 6 and 9, participants will return for a short interview or will be
contacted via telephone to assess their wellbeing and compliance, answer arisen
questions and fill in questionnaires. Additionally, a stool sample will be
provided in week 2 and a stool and blood sample will be provided in week 6.
Questionnaires will mainly be filled in before and after the intervention, with
the addition of SQUASH and three-day food diary in week 6, and BSS and GSRS in
week 2 and 6.

To screen their food intake and physical activity (SQUASH), participants will
fill in a three-day food diary before each CID 1+3 and on week 6.
Gastrointestinal Symptom Rating Scale (GSRS) questionnaire will be completed
the day before the CID1+3 and on weeks 2 and 6. Therefore, we can screen,
whether the participant might experience gastrointestinal discomforts during
the intervention period such as bloating and the outcomes are also relevant for
our faecal microbiota analysis.

After completion of the trial, all data will be gathered and analyzed.
Participants will receive a financial compensation for their efforts, as well
as reimbursements of their travel expenses.
The total amount of time participants spend at our facilities will be around 40
hours (Screening: 0.5h; CID 1+2: 2 x 9.5h; Interview week 2, 6, 9: 3 x 0.5h;
CID 3+4: 2 x 9.5h).

As mentioned before, CID 3 will be planned 12 weeks after the start of the
intervention. The dietary supplement/placebo and the protein-rich diet will
have to be continued until CID 3, which means that in some cases the
intervention period will be not exactly 12 weeks, but a few days longer or
shorter, depending on the availability of the participant. Logically, the
investigators aim for CID 3 to be planned exactly 12 weeks after the start of
the intervent

All participants will receive a high-protein diet during the entire trial (12
consecutive weeks in total, starting the day directly after CID 2 until the day
prior to CID3). This diet will consist of 25 energy percentage (E%) protein,
30E% fat and 45E% carbohydrates. Proteins will be 45-50% plant-based and 50-55%
animal-based, to ensure a resemblance to the current western diet consisting of
mainly animal-based proteins, while simultaneously acting on the currently
shifting trend in society towards more plant-based diets.

Furthermore, according to randomization, one group will receive the dietary
supplement and the remaining participants will receive a placebo.

The fibre supplement, as determined using the TIM-2 model, will consist of a
combination of potato and pectin fibre. It can be consumed with each meal.

The participants not receiving the fibre supplement will be provided with a
placebo in an isocaloric manner, which will be maltodextrin. Maltodextrin
Glucidex IT 12 (Roquette Freres, Lestrem, France) is a fully digestible
carbohydrate and will be used as isocaloric compensation to the investigated
fibre additive. Furthermore, maltodextrin will be comparable to the fibre in
terms of taste, mouthfeel and appearance.

In general, obesity and its related diseases bring a high burden to a patients
wellbeing and quality of life. Furthermore, these diseases are one of the
leading causes of death worldwide, have a high socio-economic impact on
society, and thus increase health care costs. By evaluating possible strategies
to improve metabolic, gut and brain health, we want to attribute to a healthier
lifestyle, a decrease in obesity and obesity-related diseases and in the
socioeconomic impact of said diseases.

By testing the pre-established, possibly beneficial dietary component, we will
gain insight in specific metabolic pathways, analyse interactions between food,
gut and brain, and find potential ways to steer the human digestive system to
provide beneficial substrates and reduce detrimental by-products. Along with
the purpose of finding fundamental evidence to support further preventative or
therapeutic interventions and studies, participants might benefit individually
from the intervention in terms of small short-term improvements in body weight,
body composition and carbohydrate and fat metabolism.

Nor the investigated dietary additive, placebo or high-protein diet will pose
any risk to general health in participants, since these are natural, commonly
used dietary components and possible allergies or intolerances are established
and ruled out during the screening visit. However, they might cause mild
gastro-intestinal discomfort. Furthermore, adhering to a diet for 12
consecutive weeks, consisting of relatively high-amounts of proteins and the
investigated fibre-combination, requires motivation, compliance, time and
willingness to change their normal lifestyle behaviour to a temporary lifestyle
compliant with the prescribed intervention. This will subsequently pose a
burden to participants, which is necessary to be able to evaluate the effects
on primary and secondary outcome parameters as stated in chapter 8.

All other conducted tests will not pose a threat to the participants* health,
but come with possible side-effects or complications. The physical examination,
all questionnaires, collecting and providing of faecal and urine sample,
neuropsychological evaluations, MRI and DEXA scan, the gut permeability test
and indirect calorimetry will not harm the participants, nor pose a threat to
their health. For all these tests, a participant will have to be cooperative
and motivated to actively and correctly fulfil all tests and interventions, and
will have to be willing to spend a vast amount of time at our facilities (40
hours in total). These tests will not pose a health risk for participants.
However, the mental burden posed by these tests may be relevant. Especially the
neurocognitive tests may have an impact on mental/psychological wellbeing, in
particular when confronted with less optimal test results.However, the mental
burden posed by these tests is relevant. Especially the neurocognitive tests
can have a significant impact on mental/psychological wellbeing in terms of
exhaustion and the possibility of confronting results if these tests are
performed inadequately.
The induced radiation during a DEXA scan (<20 µSv) is far less than a Dutch
citizen is exposed to on a yearly basis (2.5 mSv). This will not cause any
additional health effects.
The MRI is performed in a narrow space and is known to be noisy. Some
participants might experience claustrophobia, which will be examined during the
screening visit, and during the scan, participants will be monitored closely.
The invasive tests (i.e. blood samples, tissue biopsies,
hyperinsulinemic-euglycemic clamp) might cause more of a burden to a
participant. Blood samples and intravenous cannulas will be drawn or inserted
on multiple occasions, for which a sharp needle will need to penetrate skin
tissue. This might hurt a participant or cause bleeding and hematoma, but is in
general of minor relevance/importance/danger.
Tissue biopsies (adipose tissue and skeletal muscle) are the most invasive
tests and might cause pain, hematoma, bruising or bleeding to a participant,
mainly during and after the skeletal muscle biopsy. Pressure will be applied to
the insertion site after the muscle biopsy to reduce the risk of hematoma and
will be properly bandaged. The insertion site of both tissue biopsies will
leave a small scar (~3mm for scAT and ~8mm for SMT).
During the hyperinsulinaemic-euglycemic clamp there is a small risk of hypo- or
hyperglycemia. However, from our own extensive experience, these conditions do
not occur very often and can be reversed immediately.
All CIDs will be performed at the Metabolic Research Unit Maastricht (MRUM) at
Maastricht University. All tests will be performed following general safety and
health guidelines and Standard Operating Procedures (SOPs). Strict guidelines
in case of emergency are in place. Furthermore, a medical doctor will always be
either physically present or reachable via telephone during all tests.