Role of the central brain clock in the pathophysiology of insulin resistance

Diabetes mellitus (DM) has an increasing worldwide incidence with over 422
million affected individuals. Hyperglycemia in DM causes major morbidity and
mortality due to its macrovascular and microvascular complications. Insulin
resistance is a key pathophysiological process in the development of
hyperglycemia in most patients with diabetes mellitus. Lifestyle interventions
represent the initial treatment of insulin resistance, but they are frequently
ineffective in clinical practice, possibly because they currently do not
acknowledge the importance of meal timing and circadian synchrony.
Disruption of circadian synchrony leads to insulin resistance. Animal studies
and post-mortem human brain studies suggest that the master brain clock in the
hypothalamic suprachiasmatic nucleus (SCN) plays a role in the development of
insulin resistance, but it is unknown if in vivo SCN rhythms are disturbed in
patients with insulin resistance. In the present project we will therefore
determine the SCN activity rhythm using advanced functional brain imaging.
The circadian timing system regulates daily rhythms in food intake, energy
expenditure and glucose metabolism. The circadian timing system consists of the
master clock in the SCN, and peripheral clocks in other tissues. The SCN
regulates daily rhythms in energy expenditure and food intake. Peripheral
clocks in metabolic tissues such as liver, muscle, and adipose tissue, regulate
local insulin sensitivity and glucose metabolism. The SCN generates an
endogenous rhythm of approximately 24 hours (that is, a circadian rhythm) and
uses light input from the retina to synchronize this rhythm with the
environment. The core molecular timekeeping process in the central and
peripheral clocks is a transcriptional-translational feedback loop.
Circadian disruption is defined as disturbed synchrony between the daily
rhythms of darkness/light, fasting/eating, sleep/wake, and the central and
peripheral clock rhythms of the circadian timing system. Due to the ubiquitous
availability of food and artificial light, nowadays the synchrony between the
daily rhythms of food intake, physical activity, and the solar day is often
disrupted in our 24-hour society. Evidence suggests that circadian disruption
leads to insulin resistance, but most authors focus exclusively on the
disturbed peripheral clock rhythms. For example, several studies showed
evidence of disturbed peripheral clocks in insulin resistant patients.
We now hypothesize that also the central brain clock has an important role in
the development of human insulin resistance. In support, in rodents electrical
stimulation of the SCN causes hyperglycemia and SCN lesions induce insulin
resistance. Furthermore, our group showed in 2019 in postmortem brains of
patients with type 2 DM that the SCN contains reduced numbers of arginine
vasopressin (AVP) and vasoactive intestinal polypeptide (VIP) neurons, which
are the key timekeeping neurons in the SCN, compared to subjects with normal
insulin sensitivity. In line, patients with type 2 DM, as well as type 1 DM
have disturbed sleep-wake patterns. However, up to date no-one has investigated
whether the in vivo activity rhythm of the SCN is affected in patients with
insulin resistance. In the present project, we will use state-of-the-art
functional imaging of the SCN to determine the activity rhythm of the SCN in
humans with progressive stages of insulin resistance.

Primary Objective: to determine the activity rhythm of the SCN in humans with
progressive stages of insulin resistance
Secondary Objective(s): to determine the daily rhythm of the peripheral clock
in buccal cells in humans with progressive stages of insulin resistance

This is an observational cohort study with a duration of two weeks. For group 1
(obese people with normal insulin sensitivity) and group 2 (obese people with
insulin resistance) we will also assess fasting plasma glucose and insulin at
6-month and 12-month follow-up, to assess potential correlations within these
groups between SCN dysfunction and the development of insulin resistance or
type 2 diabetes.The study will be performed at Amsterdam UMC, location AMC,
with imaging performed at the Spinoza Institute for neuroimaging.

Participants will fill in a food diary, a sleep-wake diary for two weeks, and
participants will wear an actigraphy unit.
Subsequently, they will be admitted to our research unit for 24 hr. They will
be allowed to adhere to their regular sleep wake cycle except for a 60-min
awakening for SCN imaging.
An average Zeitgeber time (ZT) 0:00 will be calculated for each participant
based on the baseline MCTQ questionnaire and sleep-wake dairy. Zeitgeber time
0:00 represents the average wake-up time of the individual subject. At 4
time-points across the diurnal cycle (ZT 2:00, 08:00, 14:00, and 20:00), we
will image baseline SCN perfusion using fMRI.
In addition to the fMRI measurements, we will assess peripheral clock rhythms
by non-invasive collection of buccal cells at 4 timepoints across the diurnal
cycle followed by qPCR of the clock genes PER1, PER2, PER3, CRY, and ARNTL.
For group 3 (obese people with type 2 diabetes) we will assess glucose levels
using a finger stick blood test at the same 4 time points across the diurnal
cycle.

There is a small chance that the MRI scan shows an incidental finding. In that
case, Spinoza Neuroimaging center procedure is that the MRI scan is forwarded
to an Amsterdam UMC radiologist to check this finding. If the radiologist
confirms a finding for which medical treatment may be necessary, this
information will be passed on to the participant and the participant*s general
practitioner by the researcher. This information is also included in the
patient information and informed consent.
There is a chance that patients with insulin resistance at baseline will be
diagnosed with DM2 during follow-up. This information will be shared with the
participant and the participant*s general practitioner, as stated in the
informed consent form. The general practitioner will treat participants with
newly diagnosed type 2 diabetes or the general practitioner will refer these
participants to a specialist in internal medicine, if necessary.

Participant* sleep will be interrupted for one hour to obtain fMRI images at ZT
20:00. Therefore, for safety reasons, subjects will be instructed not to
participate in traffic and to have someone accompany them when leaving the
research unit after the 24-hour admission.

Patients with type 2 DM will be instructed to stop metformin treatment from
three days prior to imaging and to resume metformin treatment after imaging (4
days in total). This may cause a temporary elevation of plasma glucose but has
no long term risks. Patients with type 2 diabetes will be instructed to
measure their blood glucose once in the morning prior to the MRI sessions,
using a finger stick blood glucose test. We will determine the blood glucose to
determine the increase of fasting plasma glucose after temporarily stopping
metformin treatment, and to perform correlation analyses of increased glucose
on SCN activity.

Participants are not allowed to wear a face mask during fMRI imaging, as a face
mask may alter the BOLD signal. Scanning operators will wear a face mask.