Effects of moderate intermittent hypoxia exposure on peripheral insulin sensitivity and substrate metabolism in humans

The obesity epidemic calls for novel strategies to prevent and treat obesity
and its comorbidities. Several studies have indicated that the amount of oxygen
to which tissues are exposed may substantially impact cardiometabolic health.
Interestingly, living at high altitude (hypobaric hypoxia) seems to be
associated with improved glucose homeostasis and a decreased prevalence of type
2 diabetes. Furthermore, normobaric hypoxia exposure has been shown to exert
beneficial effects on glucose homeostasis and insulin sensitivity in rodents
and humans. This may, at least in part, be explained by the effects of altered
adipose tissue and skeletal muscle oxygen tension. The present study aims to
investigate the effects of moderate intermittent hypoxia exposure on whole-body
and tissue-specific insulin sensitivity and substrate metabolism in humans, and
potential underlying mechanisms (adipose tissue and skeletal muscle oxygen
tension, oxidative capacity and inflammation) will be examined. This study may
open up a novel therapeutic avenue with direct clinical relevance for obese
insulin resistant subjects.

The current research proposal intends to study the therapeutic potential of
moderate intermittent hypoxia (IH) exposure in subjects at high risk of
developing type 2 diabetes, and aims to address the following key objectives:
1) To investigate the effects of moderate IH exposure for 7 consecutive days on
whole-body and tissue-specific insulin sensitivity, substrate metabolism,
adipose tissue and skeletal muscle mitochondrial capacity and PO2 in obese
insulin resistant men.
2) To unravel the mechanisms underlying the effects of moderate IH exposure on
insulin sensitivity, mitochondrial function, and glucose uptake using
differentiated human adipose tissue-derived mesenchymal stem cells (hMSC) and
skeletal muscle satellite cells.
3) To elucidate the effects of moderate IH exposure on adipose tissue and
systemic inflammation.

We will perform a randomized, placebo-controlled, cross-over study to
investigate the effects of moderate intermittent hypoxia exposure on whole-body
and tissue-specific insulin sensitivity and substrate metabolism in humans, and
examine potential underlying mechanisms (adipose tissue and skeletal muscle
oxygen tension, oxidative capacity and inflammation). Therefore, subjects will
be exposed to 1) moderate intermittent hypoxia (15% O2; equivalent to ~2400m
above sea level) and 2) normoxia (21% O2) for 7 consecutive days (3 cycles of
2h exposure/d in a normobaric room, with 1h of normoxia exposure between
hypoxic cycles) in a randomized fashion (computer-generated randomization plan;
block size, n=4), separated by a 4 week wash-out period.
After initial screening, subjects are asked to visit the university for two
periods of 7 consecutive days with a wash-out period of 4 weeks (Table 1, for
details please see *Methods* * *Study procedures*):
- During the 7 days (time investment: 8 hours/day), subjects will be exposed to
1) moderate intermittent hypoxia (15% O2; equivalent to ~2400m above sea level)
and 2) normoxia (21% O2) for 7 consecutive days (3 cycles of 2h exposure/d in a
normobaric room, with 1h of normoxia exposure between hypoxic cycles) in a
randomized fashion (computer-generated randomization plan; block size, n=4),
separated by a 4 week wash-out period. On days 1, 3, 6 and 7 of treatment,
blood samples will be collected to determine circulating metabolite
concentrations and systemic inflammatory markers, and a questionnaire to assess
hunger and satiety (VAS-scores) has to be completed at days 1, 3 and 7.
- At day 6 (time investment: 8 hours), we will adipose tissue and skeletal
muscle PO2 (two-channel optochemical measurement system), adipose tissue blood
flow (133Xe wash-out technique).
- At day 7 (time investment: 5 hours), a high-fat mixed-meal test will be
performed to determine fasting and postprandial metabolite concentrations,
substrate metabolism (indirect calorimetry) and systemic inflammatory markers
- At day 8 (time investment: 8 hours), peripheral and hepatic insulin
sensitivity will be determined (2-step hyperinsulinemic-euglycemic clamp) and
skeletal muscle (m. vastus lateralis) and adipose tissue biopsies will be
collected at baseline and during insulin-stimulated conditions (steady-state of
the clamp).
Thus, after initial screening and BMR measurement and DEXA scan, subjects will
have to invest approximately 106 hours for the total study.

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 12.1.
Each subject will undergo two exposure regimens, in a randomized fashion, with
a 4-week wash-out period in between:
- 7 consecutive days of intermittent exposure to 15% O2 (intervention
condition)
- 7 consecutive days of 21% O2 exposure (control condition)

- 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.
- During the visits at day 5 (twice because of the cross-over design), a
microdialysis probe will be placed in the abdominal subcutaneous adipose tissue
under sterile conditions 6-8 cm lateral from the umbilicus. One hour before
insertion of the probe, the skin will be anaesthetised by means of a cream
containing lidocaine (25 mg/g) and prolocaine (25 mg/g) (EMLA, Astra
Pharmaceutica, Zoetermeer, The Netherlands). The microdialysis catheter will be
inserted in skeletal muscle (gastrocnemius muscle) under local anesthesia.
Insertion of the microdialysis probes is as good as painless.
- Due to local anesthesia, the adipose tissue and skeletal muscle biopsies are
as good as painless. Subjects may experience some discomfort (pressure during
the introduction of the needle) during the muscle biopsy procedure.
Occasionally, desensitization 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
minimize the risk of hemotoma, the muscle biopsy place will be taped with an
elastic adhesive compression bandage, and the adipose tissue biopsy place will
be compressed for at least 5min after the biopsy has been taken. The place of
incision will leave a small scar, which will be minimized by sealing the
incision with sterile steristrips and a waterproof bandaid.
- The total radiation exposure (effective dose) during this research (including
133Xe and DEXA scan) is 0.09 mSv (4% of total background radiation in the
Netherlands per year).
- 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. This will be monitored using the Lake
Louise Acute Mountain Sickness (AMS) score questionnaire. To ensure that
moderate intermittent hypoxia exposure will not cause adverse effects, oxygen
saturation (Sp,o2, %) will be monitored continuously throughout the exposure
regimen by means of finger pulse oximetry, and blood pressure will be monitored
each day (automatic inflatable cuff; Omron Healthcare, Hamburg, Germany).