Prevention of diabetes with a novel butyrate-enriched triglyceride

Obesity has become a serious global health and socioeconomic problem of the
twenty-first century due to the easy availability of energy-dens meals and
increasingly sedentary lifestyles. Currently, one-third of the world*s
population is overweight, with 10% of the population being obese. It is even
expected that by 2030, half of the world's population will be overweight or
obese. Obesity is also an economic burden, with costs doubling every decade,
reaching $860 to $956 by 2030. Moreover, obesity increases the risk to develop
metabolic disorders such as type 2 diabetes (T2D). Individuals with
overweight/obesity have an increased risk of at least sixfold to develop T2D
compared to normal-weight individuals. The primary cause of T2D is
obesity-driven insulin resistance (IR) in adipose tissue (AT), liver, and
skeletal muscle. This results in the first phase of pancreatic insulin over
secretion to overcome this resistance. As a result, the pancreas become
exhausted and the β-cells undergo apoptosis. It has been estimated that more
than 300 million people are predicted to have T2D as a consequence of obesity
by 2025.

The Western diet (WD) has been linked to the worldwide rise in obesity,
obesity-related T2D and IR. The typical WD is rich in
high-glycaemic/insulinemic carbohydrates, dairy products, and fat while being
low in fibres. Food survey data show that the daily intake of fibres for people
living in countries of the Western World is one-third below the recommend
level. Dietary fibres are the preferred energy source of the gut microbiota. In
individuals with metabolic disorders show indeed altered gut microbial profiles
have been reported, resulting in decreased functioning of the microbiota.
Hence, targeting the gut microbiota could be one potential therapeutical
strategy to combat the obesity and obesity related T2D pandemic since
accumulating research suggest that the microbiome may play a crucial role in
the aetiology of obesity, IR and T2D.

Over the past years, the gut microbiota has emerged as a critical regulator of
host energy and substrate metabolism. One of the essential roles of the gut
microbiota is the fermentation of indigestible carbohydrates, such as dietary
fibres, into short-chain fatty acids (SCFA). The most abundant SCFA in the gut
are acetate, butyrate and propionate. They are important regulators of both the
gut and host homeostasis. SCFA regulate many metabolic processes including
insulin sensitivity, energy balance, inflammation, glucose homeostasis en lips
metabolism by activating the G-protein coupled cell surface receptors (GPR)
expressed in the gut, AT, skeletal muscle and liver.

A body of animal studies as well as observational studies in humans
demonstrated that butyrate is one SCFA that has pronounced positive effects on
body weight control, inflammation, and insulin resistance. We have previously
demonstrated that increased concentrations of butyrate in the systemic
circulation after one-day dietary fibre supplementation are linked to
improvements in postprandial insulin sensitivity. Recently, we showed that a
novel butyrate/hexanoate-enriched triglyceride oil temporarily enhanced
systemic butyrate and hexanoate concentrations. While butyrate has attracted a
lot of scientific interest, the SCFA hexanoate has also been proposed to poses
anti-inflammatory effects. However, hexanoate remains a metabolite that has
been scarcely investigated, and there have been no reported human clinical
studies involved with hexanoate thus far. The increased postprandial
circulating butyrate showed no effect on metabolic markers in overweight/obese
males, but the intervention was short term. Therefore, the long-term effect of
increased systemic butyrate on insulin sensitivity and other markers of
metabolic health has never been studied before in humans.

We believe that a chronic increase of butyrate and hexanoate in the systemic
circulation improves adipose tissue fat storage capacity, oxidative metabolism
and inflammatory processes, resulting in decreased chronic low-grade
inflammation and ectopic lipid accumulation, thereby improving insulin
sensitivity in people with overweight/obesity and a high risk of developing
T2D. Combining butyrate and hexanoate could be a potential strategy to treat
obesity-related chronic low-grade inflammation and tissue-specific metabolic
dysfunctions because of the features of both SCFA.

Primary Objective:
The main goal of the study is to investigate the effects of a chronic increase
in the gut microbial metabolite butyrate and hexanoate in the systemic
circulation on peripheral insulin sensitivity in individuals with
overweight/obesity and a high risk to develop T2D.

Secondary Objective(s):
We will also look at the effect of a chronic increase of systemic butyrate on
other metabolic health markers including the following;
1. Hepatic and adipose tissue sensitivity
2. Energy and substrate metabolism (energy harvest, energy intake, energy
expenditure, fat and carbohydrate oxidation)
3. Circulating metabolites, hormones and inflammatory factors
4. Gut permeability
5. Composition and functionality of the microbiome
6. Body weight and composition
7. Adipose tissue gene/protein expression
8. Faecal and circulating SCFA

Finally, we will monitor dietary food intake and physical activity, assess
gastro-intestinal side-effects and evaluate general well-being and stress.

The proposed study will be a double-blind, randomized placebo-controlled
parallel trial, to evaluate the effect of an increased systemic butyrate
concentrations on peripheral insulin sensitivity and other markers of metabolic
health and gut functioning. Individuals aged 20-70 years with overweight or
obesity (BMI >= 28 kg/m2< 40 kg/m2) and impaired fasting glucose (defined as
fasting glucose 5.6-6.9 mmol/L), impaired glucose tolerance (two-hours plasma
glucose 7.8-11.1 mmol/L) or insulin resistance (HOMA-IR >2.2) 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+.

Recruitment and screening period
Volunteers will be recruited by means of posters (for example hung up in the
hospital and university), advertisements in local newspapers and in social
media, as well as from existing co-hort of subjects that have previously
approved by the investigator for future studies within our department.
Potentially interested participants will be contacted by the researcher via
e-mail or telephone, and will subsequently receive the written information
brochure via their e-mail. After this initial contact, the participants will
get one week to consider if they are willing to participate. In case the
volunteer wants to participate, an appointment for the screening will be made.
The screening is the first visit at the University. The participant will visit
the University after an overnight fast (>10h) and the screening will consist of
the following components;
- Checking an individual*s health, medical history and determining eligibility
and safety of participation
- Signing informed consent
- Measuring vital parameters and body measurements (body weight, length,
waist-to-hip ratio, systolic blood pressure and diastolic blood pressure)
- Taking blood samples to determine eligibility based on specific inclusion
criteria
o 20 mL for fasting glucose, insulin, Alanine Aminotransferase (ALAT),
creatinine, Hemoglobin A1c (HbA1c)
o Oral Glucose Tolerance Test (OGTT): 5 mL blood sampling for two hours after a
75 g glucose drink for plasma glucose and insulin

Intervention period
All eligible participants will be equally randomized over the two arms
(butyrate/hexanoate-enriched oil vs oil devoted of butyrate/hexanoate) with
stratification for age, BMI and sex. This randomized clinical trial with the
primary outcome peripheral insulin sensitivity has a placebo controlled, double
blind, parallel design which allows the evaluation of the role of a developed
oil containing butyrate/hexanoate-enriched triglyceride in human substrate and
energy metab-olism in 48 adult volunteers with overweight/obesity and impaired
glucose homeostasis.
After initial screening, participants will come to the university for the
clinical investigation day (CID) 1 and CID2, along with small follow-up
sessions at week 2 and 4.5 months, and a major follow-up visit at week 12 (3rd
month). In total, the participants will need to come to the university for at
least eight times (screening, CID1, CID2, week 2, week 12 and the 4.5-month
visit).

A CID will consist of the following measurements;
- Two-step hyperinsulinemic euglycemic clamp with parallel measurements of
indirect calorimetry
- Dual-energy x-ray absorptiometry (DEXA)
- Adipose tissue biopsy
- Multi-sugar permeability test
- Collecting stool samples
- Checking filed in questionnaires about dietary intake, physical activity,
general health status (Rand-36), stress (perceived stress scale; PSS) and
gastrointestinal symp-toms rate (GSRS) with the Bristol stool scale

Before each CID and for week 12, the food intake, physical activity and
Gastrointestinal Symptom Rating Scale (GSRS) with Bristol stool scale will be
filled in. The food intake is recorded by filling out a three-day food
questionnaire. Physical activity is measured via the Short questionnaire to
Assess Health-enhancing physical activity (SQUASH). The GSRS is used to screen
for whether the participant might experience gastrointestinal discomforts
during the intervention period such as bloating and the Bristol stool scale for
the type of faecal sample. Furthermore, their general health status will be
assessed using the Rand-36 questionnaire, and their stress level will be
evaluated through the PSS questionnaire. Also, the day before each CID and week
12 visit, the participants will collect a faecal sample for the analysis of
SCFA and microbiota.

At CID1, the baseline measurements are taken. After CID1, the participants
receive the intervention products and are instructed to take the dietary
supplement on the following day for 24 weeks (six months).
1. Oil containing butyrate/hexanoate-enriched triglycerides (dosage
approximately 2g of butyrate) twice a day
2. Placebo oil without the butyrate/hexanoate-enriched triglycerides twice a day

The intervention period will be 24 weeks (168 days) with a minimum of 159 days
and a maxi-mum of 178 days intervention due to practical reasons. The oil will
be incorporated in the participant*s breakfast and dinner. The type of
treatment will be blinded for both the volunteers and researchers. After the
24-week intervention, CID2 is performed in week 24 in which the same
measurements occur as CID1. This way, we can detect long-term effects of the
butyrate/hexanoate-enriched triglyceride oil on peripheral insulin sensitivity
and other important health markers. This study is designed to examine the
butyrate effect independent of any diet or exercise intervention. For this
reason, participants will be encouraged to maintain their regular lifestyle, to
eat till they feel satiated, and not to consciously try to gain or lose weight
throughout the study. Finally, after completion of the trial, all data will be
gathered and analyzed. The participants will receive a financial compensation
for their participation as well as reimbursements for their travel expenses.

This study has two different groups receiving either the investigational
product or placebo:
- Investigational product: butyrate/hexanoate-enriched triglyceride oil
(approximately 12.5 mL per day for 24 weeks)
- Placebo: similar oil in volume and amount but without
butyrate/hexanoate-enriched triglyceride (approximately 12.5 mL per day for 24
weeks)

The type of intervention will be blinded for both the volunteers and the
researchers and provided in randomized order.

In general, obesity and its related diseases bring a high burden to a patient*s
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 and gut 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 participating in the study, the
participants will receive information about their health and may profit from
general health benefits if they are randomized in the intervention group.

By testing the pre-established, possibly beneficial dietary component, it will
help us understand particular metabolic pathways, examine the relationship
between food and the gut, and determine how this affects the regulation of
metabolic health. 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, carbohydrate and fat
metabolism and emotional wellbeing.

The butyrate-hexanoate enriched oil will not pose any risk to general health in
participants, since the components are natural and present in foods such as
milk, cheese and yoghurt. However, they might cause mild gastro-intestinal
discomfort. In an acute setting, we showed that the butyrate-hexanoate enriched
oil was well-tolerated and did not increase gastrointestinal complaints or
discomforts which was determined by GSRS (METC NL75253.068.20). Furthermore,
adhering to a diet for 24 consecutive weeks requires motivation, compliance,
time and 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 participant*s health,
but come with possible side-effects or complications. All questionnaires,
collecting and providing of faecal and urine sample, DEXA scan, indirect
calorimetry and gut permeability test will not harm the participant, nor pose a
threat to their health. For all the 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 (~31 hours in total, see table 1). These tests will not pose a
health risk for participants. However, the mental burden posed by these may be
relevant. The filling in of the questionnaires and the intake record of the
supplements can be viewed as time consuming. Moreover, the collection of faecal
samples can be experienced as a burden as they have to handle them themselves
and have to store them at home. Also, the 24-week intake of the oil might be
seen as a burden for the subjects. Individual results will only be shared with
the participant at their own request.
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 invasive tests (i.e., blood samples, tissue biopsies,
hyperinsulinemic-euglycemic clamp) can pose a burden to participants. This is
particularly true for the hyperinsulinemic-euglycemic clamp, as it involves
drawing blood samples and inserting intravenous cannulas on multiple occasions,
for which a sharp needle will need to penetrate skin tissue. This procedure may
hurt a participant or cause bleeding and hematoma, although the risk can be
minimized with good clinical practice. Furthermore, the clamp is a long test
day (approximately 9 hours) during which 205 mL of blood per CID will be
sampled. The remaining will be 25 mL for screening and 10 mL during the week 12
visit resulting in a total of 445 mL blood withdrawn over the course of the
entire study. Additionally, the clamp brings a small risk of hypo- or
hyperglycemia. However, from our own extensive expertise, these conditions do
not occur very often and can be reserved immediately. A medical doctor is
always available during the clamp. To summarize, while the measurements of the
study, particularly the clamp procedure, imposes a burden for the participants,
the method is well-performed in our department mitigating the associated risks.
Finally, the tissue biopsy is also an invasive test and might cause pain,
hematoma, bruising or bleeding to a participant. Pressure will be applied to
the insertion site after the adipose tissue biopsy to reduce risk of hematoma.
The insertion site will leave a small scar (~ 3 mm).

Standard operating procedures (SOPs) for each measurement are available on the
UM Human Biology Department*s server.