Exercise during mild hypoxia exposure to reverse impaired glucose metabolism in overweight and obese humans

The obesity epidemic calls for new therapeutic opportunities to prevent and
treat obesity and its comorbidities amongst which are insulin resistance and
cardiovascular diseases. Recent evidence suggests that tissue oxygenation plays
an important role in cardiometabolic health. Remarkably, individuals residing
at high altitude (hypobaric hypoxia) are less prone to develop type 2 diabetes
mellitus as compared to individuals living at sea-level (normobaric normoxia).
Furthermore, there is evidence to suggest that normobaric hypoxia exposure may
improve glucose homeostasis and insulin sensitivity in both rodents and humans.
The level of physical activity is an important determinant of insulin
sensitivity and glucose homeostasis. It is well established that performing
physical activity improves glucose uptake in the short term, and glycemic
control in the long term. Interestingly, recent studies have demonstrated that
an acute bout of exercise under hypoxic conditions (inhalation of air
containing less oxygen) may lead to a more pronounced improvement in plasma
glucose concentrations and/or insulin sensitivity as compared to normoxic
exercise. However, the effects of repeated hypoxic exercise bouts on glucose
profile throughout the day (i.e. 24h continuous glucose monitoring) remain
elusive.

In the present randomized, placebo-controlled, single-blind, cross-over study
study, we will investigate the effects of exercise under mild normobaric
hypoxic conditions (FiO2, 15%) for 4 consecutive days (2 x 30-min cycling
session at 50% WMAX) on postprandial substrate metabolism and 24h-glucose level
in overweight/obese subjects with impaired glucose tolerance. We hypothesize
that 4 consecutive days of exposure to mild hypoxia while performing moderate
intensity exercise improves glucose homeostasis in overweight and obese
individuals with impaired glucose homeostasis. The objective of the study is to
investigate the effect of moderate-intensity exercise under normobaric hypoxic
(FiO2 15%) as compared to normoxic (FiO2 21%) conditions for 4 consecutive days
(2 x 30min per day) on average 24h glucose levels in overweight and obese
individuals with impaired glucose tolerance.

In the present randomized, single-blind, placebo-controlled cross-over study,
subjects will be exposed to normobaric 1) mild hypoxia (FiO2 15%) and 2)
normoxia (FiO2 21%) during exercise (2 x 30min/day on a cycle ergometer) of the
same relative exercise intensity (equal to 50%WMAX under normoxic conditions)
for 4 consecutive days. Subjects will be randomly assigned to each condition
(computer-generated randomization plan; block size, n=4), separated by a
washout period (3-6 weeks). To accomplish this, subjects will exercise in an
oxygen chamber in which oxygen concentration of the ambient air and, as such,
FiO2 can be tightly controlled and monitored. Subjects will cycle two times a
day for 30 minutes at 50% WMAX, determined by an incremental workload test.
Since we will allow 5-10 min for subjects to get ready to start the 30-min
exercise session, and take into account a 5-min cooling down period before
leaving the hypoxic room again, subjects will be in the room for 45 min for
each session.
After initial screening, subjects are asked to visit the university for two
periods of 5 consecutive days each with a washout period of 3-6 weeks (for
details regarding the procedures, please see *Methods * *Study procedures*).
During the first 4 days (time investment: 4.5 hours/day), subjects will be
undergoing the exercise regimen, as described above.
- At day 1, on the first morning of each regimen, a glucose sensor (Enlite
Glucose Sensor MiniMed; Medtronic). The sensor will be inserted subcutaneously,
will be inserted subcutaneously, at 5 cm from the umbilicus, on the right side
of the abdomen, and will be connected to a continuous glucose monitor (iPro2
Professional CGM MiniMed; Medtronic, Northridge, CA, USA). The sensor will
remain inserted throughout the study (days 1-5). Furthermore, a physical
activity monitor (ActivPAL3 micro monitor) will be applied at the same moment,
to monitor physical activity of participants. At the end of day 5, the glucose
sensor, and the physical activity monitor will be removed.
- At days 1-5 (time investment: 4.5 hours), fasting blood samples will be
collected to determine plasma metabolites, inflammatory markers, and blood
pressure and body weight will be monitored.
- At day 5 (time investment: 8 hours), a mixed liquid meal challenge will be
performed to determine fasting and postprandial metabolite concentrations, and
substrate oxidation (using indirect calorimetry). Moreover, HOMA-IR will be
used to estimate insulin resistance, using fasting plasma glucose and insulin
values measured on the day after completion of the 4 day regimen. Furthermore,
a (fasting) skeletal muscle biopsy (m. vastus lateralis) will be collected in
the morning during this day.
After initial screening, the assessment of basal metabolic rate (BMR) and the
incremental workload test (to determine the maximal workload, WMAX), subjects
will have to invest approximately 52 hours.

Exposure to 15% O2 is comparable to an altitude of ~2800 m. Adverse Events
(e.g. Acute mountain sickness symptoms) may occur above ~2500 m, although most
people do not experience symptoms at this altitude. Importantly, the exposure
to normobaric moderate hypoxia (15% O2) will be under strict control, as
described in detail in section 13.1. Performing moderate intensity exercise at
50% WMAX under the mild hypoxic conditions that will be applied in the present
study is expected to decrease SpO2 to about 88-90% O2. To ensure that mild
hypoxia during moderate-intensity exercise will not cause adverse effects
(headache, nausea, etc.) we will continuously monitor SpO2 by finger pulse
oximetry during exercise. Although not expected, we will ask subjects to stop
exercising in case SpO2% drops below 80% O2 to assure safety

Each subject will undergo two exposure regimens whilst performing exercise, in
a randomised fashion with a 3-6 weeks wash-out period in between:
- 4 consecutive days of mild hypoxic exposure while performing exercise
(50%WMAX) for 2 x 30 minutes each day.
- 4 consecutive days of normoxic exposure while performing exercise (50%WMAX)
for 2 x 30 minutes each day.

During both regimens, day 5 will consist of a mixed meal challenge, which will
be executed under normoxic conditions.

One of the burdens that participants might experience may include the time
investment of participating in the study. Including screening, measurement of
basal metabolic rate (BMR) and maximal aerobic capacity/workload, subjects need
to invest approximately 57 hours to complete the study. The following risks may
be associated with participating in the study:
- For accurate measurements with the 24h glucose monitoring method, four
calibrations per day are essential. Calibrations will be performed by finger
prick using a capillary blood glucose meter. Participants could experience
local sensitivity in the fingers.
- During several visits blood samples will be collected via a catheter.
Occasionally, a local hematoma or bruise may occur. Some participants report
pain during insertion of a catheter.
- Due to local anaesthesia, the skeletal muscle biopsy are as good as painless.
Subjects may experience some discomfort (pressure during the introduction of
the needle) during the muscle biopsy procedure. Occasionally, desensitisation
or increased sensitivity of the skin at the site of the muscle biopsy may
occur, which may last for several weeks/months. Furthermore, the biopsy
procedures may cause a local hematoma or bruise. To minimise the risk of
hematoma, the muscle biopsy place will be taped with an elastic adhesive
compression bandage. The place of incision will leave a small scar (5 mm),
which will be minimised by sealing the incision with sterile steristrips and a
waterproof bandaid.
- Exposure to 15% O2 (~2800 m) rather than lower pO2 will be applied to prevent
or at least minimize Adverse Events. Acute mountain sickness symptoms (e.g.
headache, nausea) may occur above ~2500 m, although most people do not
experience symptoms at this altitude. To ensure that mild hypoxia exposure will
not cause adverse effects, oxygen saturation (SpO2, %) will be monitored
continuously throughout the exercise sessions by means of finger pulse
oximetry, and blood pressure will be monitored each day (automatic inflatable
cuff; Omron Healthcare, Hamburg, Germany).
- Performing moderate intensity exercise at 50%Wmax under normoxic conditions
lowers systemic oxygen saturation as compared to resting conditions. Under
hypoxic conditions (FiO2 15%), SpO2 will further decrease. Performing moderate
intensity exercise at 50%Wmax under the mild hypoxic conditions that will be
applied in the present study is expected to decrease SpO2 to about 90% O2. To
ensure that mild hypoxia exposure during moderate-intensity exercise will not
cause adverse effects (headache, nausea, etc.) we will continuously monitor
SpO2 by finger pulse oximetry during exercise performance. Although not
expected, we will ask subjects to stop exercising in case SpO2 drops below 80%
to assure safety, based on guidelines developed by American Thoracic
Society/American College of Chest Physicians on cardiopulmonary exercise
testing.
- No risks are known regarding the oral glucose tolerance test (OGTT),
mixed-meal challenge and indirect calorimetry. These methods are routinely
applied in clinical trials, and SOPs are available at our research facility
(MRUM).
- Dietary products to provide subjects standardized breakfast, lunch and dinner
are freely available for costumers. Therefore, these food products will not
cause risks for the participants.