SCFA production and their metabolic effects after inulin ingestion

The gut microbiota play an important role in metabolic diseases. One of the
major roles of the gut microbiota is to ferment undigested food particles, for
example resistant starch and carbohydrates, into SCFAs. The most abundant SCFAs
are acetate, propionate and butyrate. SCFAs can have beneficial effects on body
weight and insulin sensitivity, as is shown in several animal and human
studies. Underlying mechanisms how SCFAs can influence metabolism still need to
be unravelled.

An example of a fermentable carbohydrate is the prebiotic inulin, which occurs
naturally in chicory-based products such as endive and radicchio. Prebiotics
escape digestion and are fermented by bacteria in the gut. Inulin has been
proven to produce SCFA in in vitro models in a ratio of 65:20:15
(acetate:propionate:butyrate). Most fermentation of inulin occurs in the
proximal part of the colon, and our own research proves that increasing SCFA in
the proximal part of the colon can have a beneficial effect on human substrate
and energy metabolism, as can be seen by a significant lowering of plasma
glucose and insulin 60 minutes after SCFA infusion in the colon.

Several studies examined the role of inulin on substrate and energy metabolism.
Most studies focus on a chronic effect of 9-14 gram inulin supplementation for
4 to 8 weeks. A significant lowering of triglycerides was observed in several
human studies. Lowering of insulin, lowering of fasting glucose, significant
lowering of LDL-C and total cholesterol, and a significant decrease of body
weight, BMI, and intake of energy and total fat was observed. A study in beagle
dogs confirms that total energy expenditure measured in these dogs is
significantly higher after an inulin-containing diet for 21 days. No direct
measurements of energy expenditure after inulin ingestion in humans have been
conducted so far. Inulin can also significantly increase the bifidobacteria,
which are considered beneficial for human health due to their ability to
increase butyrate formation.

Acute effects (< 8 hour) of 24 gram inulin include increasing plasma GLP-1 and
decreasing plasma ghrelin in healthy human subjects. Also serum acetate,
propionate and butyrate were significantly higher after inulin intake from 4-6
hours, with acetate peaking at 4.5h, propionate at 5h and butyrate at 5.5h. A
study conducted in healthy and hyperinsulinemic volunteers showed a lower
fasting and post-prandial serum SCFA concentrations in healthy volunteers after
24 gram inulin intake, however no difference in SCFA formation after inulin
intake was found between the two groups. They also did not find any significant
increase in plasma SCFAs 4h after inulin ingestion. A longer study period of
6-8h is needed to see the differences in SCFA production.

We hypothesize in the current study that inulin is fermented into different
SCFAs, which are taken up by the colon, enter the blood stream and have
beneficial signalling and/or direct metabolic effects.

Based on our hypothesis that orally ingested inulin is fermented into different
SCFAs, and these SCFAs can have beneficial metabolic effects, we aim to address
the following primary objective: To investigate the human metabolic effects of
SCFA derived from inulin.

This is a double-blind randomized placebo-controlled study.
After initial screening, the subjects come to the university for 3 testdays.
Each testday they will be provided with a high-fat mixed meal, and measurements
will follow. Different products will be diluted into the high-fat mixed meal
(see intervention: inulin, or maltodextrin). Each testday measurements via a
ventilated hood will take place, next to blood sampling and breath sampling at
different timepoints. At two of the three testdays, also muscle and fat
biopsies will take place, one in the morning before intake of the mixed-meal
and one at the end of the testday, totalling 4 fat biopsies and 4 muscle
biopsies.

In total there will be three test days with a wash-out period of one week in
between. The interventions will be diluted into a high-fat milkshake.
The three test days consist of:
1. 13C inulin (500mg) + 23,5gram inulin
2. 24 gram inulin (as background for 13C inulin measurements)
3. 24 gram maltodextrin (placebo)

All subjects will be screened before participation and thereby receive
information about their health status. In the future there can be general
health benefits for the public, but the volunteers will have no personal
benefits by participating in the study. The general interest of this study is
that there have never been investigations with 13C labelled inulin combined
with an oral inulin load in human beings. This is the first study where 13C
label incorporation in venous plasma will be measured, and linked to metabolic
responses after an inulin load.
There are different burdens volunteers can experience during the study. Burdens
that volunteers can experience are the time spent with the study (subjects will
have to invest approximately 27 hours in the study, divided among 3 days and a
screening visit) and the dietary and healthy regimen they have to follow. Also
the collection of fecal samples can be experienced as a burden for the
volunteers, because they have to handle them their selves.
During the study, blood will be collected via a venous catheter. Venepunctures
can occasionally cause a local hematoma or bruise to occur. Some participants
report pain during venepuncture. The total amount of blood sampled is 13x10mL
per test day, totalling 390mL during the whole test period. The adipose tissue
biopsy might cause local hematoma as well. Some participants report pain which
is experienced as muscle pain after the muscle biopsy. More often the muscle
feels stiff for a couple of days after the biopsy. To minimize the risk for a
hematoma, the biopsy place will be compressed for approximately 5 minutes after
biopsy. The place of incision will leave a small scar (3 mm for adipose tissue
biopsy and 8 mm for skeletal muscle biopsy). To promote good wound healing, the
incision will be sealed with sterile steristrips and a waterproof band-aid. The
muscle biopsy will, in addition, be sealed with a compression bandage. For the
indirect calorimetry, healthy volunteers have to lay on a bed for a couple of
hours in a row, and this can be tiring. We try to reduce the invasiveness of
the ventilated hood by using blocks of 25-50 minutes where the hood stays on.
In between the blocks, the volunteers still need to lie on the bed, however the
hood is off and they can get some fresh air. All other measurements, such as
the breath samples and VAS-scoring cause no discomfort.
There have been few reports of side-effects after inulin ingestion. The effects
reported were cramps and flatulence, however after chronic inulin intake for 2
months at an amount of 14 gram per day. Even dosages up to 40 gram of inulin
per day caused no nausea or headache in obstipated patients. Bowel frequency
was however increased, and four out of 25 patients complained about moderate
abdominal pain at the high dosage of 40 gram for 7 days. At a dosage of 20 gram
inulin per day, no side-effects were reported at all. No side-effects after a
single oral dose of 24 gram inulin were reported in two different studies. The
use of 13C labelled inulin is not considered as harmful. Inulin is a natural
product and 1-2% of the C-atoms in natural occurring inulin are 13C-atoms.
These 13C-atoms are stable and do not degrade into harmful products. The
departments of Surgery and Human Biology have experience with the use of stable
isotopes, even within the same TIFN project (METC 11-03-72).