The impact of circadian rhythmicity in cold-induced thermogenesis in lean and obese subjects

Brown adipose tissue (BAT) recently emerged as a novel player in energy
expenditure (EE) in humans as it combusts fatty acids and glucose towards heat.
Human BAT can be activated by sympathetic stimulation resulting from cold
exposure or treatment with sympathomimetic drugs. Very interestingly,
short-term acclimation to mild cold was shown to reduce fat mass in obese
subjects and decrease peripheral insulin resistance of patients with T2DM by
43%. These findings have increased interest in the therapeutic potential of BAT
as a target to combat obesity and diabetes. Recently, in preclinical studies we
showed that BAT has a circadian rhythm. It is currently unknown whether this is
also the case in humans. We postulate that BAT activity should display a
circadian rhythm that adapts to changes in circadian behavior, and may
determine glucose/lipid levels throughout the day.

Primary objective:
To assess whether maximum cold-induced non-shivering thermogenesis (e.g.
thermogenesis as a consequence of BAT activity) differs between morning and
evening.

Secondary objectives:
a. To study whether cold-induced changes in plasma lipid and glucose
concentrations differ between morning and evening.
b. To investigate whether rhythmicity in cold-induced non-shivering
thermogenesis, glucose and lipid metabolism differ between lean, obese and
obese subjects with pre-diabetes.

The study design encompasses a single-arm randomized intervention study using
cold exposure. Prior to participation, subjects will undergo a screening to
exclude subjects with diabetes as well as other chronic diseases and
distinguish obese subjects that are glucose tolerant from those that are
prediabetic. After inclusion, the week prior to the study days, participants
will be asked to report their sleep-wake pattern physical activity and dietary
habits in two separate simple diaries as well as fill in a questionnaire to
determine their chronotype.

In the main study, subjects will be exposed to a cooling protocol twice within
a total timeframe of 72 h, once in the morning and once in the evening (for
details, see protocol page 13-14). We will include male and female lean, obese
glucose tolerant and obese impaired glucose tolerant men who will undergo
identical measurements in the morning and evening. An intravenous catheter will
be placed, a first blood sample will be drawn and wireless iButtons will be
attached to the skin. Thereafter, an infrared thermal image will be made.
Subjects will lie between two water-perfused mattresses and will then be
exposed to 32°C (= thermoneutrality) for 15 min. Resting energy expenditure
(REE) will be measured by ventilated hoods during thermoneutrality. Thereafter,
a venous blood sample will be obtained. Next, the individualized cooling
protocol starts. During the cooling protocol, energy expenditure will be
continuously measured by ventilated hood. From the start of the cooling on,
every 15 min, a blood sample will be taken. The personalized cooling protocol
stops when the subject starts shivering. Then, the temperature will be slightly
increased until shivering just stops and the stable cooling period starts. 60
min into the cooling procedure (approx. 60 min into stable cold exposure), REE
will be measured again via ventilated hoods to assess cold-induced
thermogenesis (CIT), which is an important outcome measurement of the study.
After this, the last venous blood sample will be drawn and an infrared thermal
image will be made.

As an intervention, a personalized cooling protocol will be used in order to
activate BAT and induce non-shivering thermogenesis. During the cooling
procedure, subjects will be exposed to mild cold (approx. 14°C) for 150 min.
Since the onset temperature of shivering shows a high interindividual
variation, we will use a personal cooling protocol to ensure maximum
non-shivering EE (and thus an equal maximum activation of BAT). The right
temperature will be determined via a subjective method, e.g. to ask the subject
if he or she experiences shivering. The time needed to achieve the right
temperature is approximately 30-45 minutes . Then, the stable cooling period of
120 min is started. During this time the subject will be asked every 15 minutes
whether he is experiencing shivering. If so, temperature will be increased with
2-3°C so that shivering just stops.

The risks of the current study are negligible.