Participants will be recruited in and near the vicinity of Maastricht by advertising on
local billboards, in local newspapers, public spaces, contacting previous participants
who gave consent to be contacted again, and on the internet. Those who express interest
will be invited for a screening. Upon arrival at the research facility, participant will
sign an informed consent form (see chapter 11.2 for complete procedure). Subsequently,
measurements of height, weight and blood pressure will be performed. The blood pressure
measurement will be performed 3 times to obtain an average blood pressure value (to avoid
measuring a peak or dip in the pressure). Furthermore, a 10-lead electrocardiogram (ECG)
will be recorded and the subjects will fill out a medical history (see document F1.1).
Following this, a two-hour oral glucose tolerance test (OGTT) will be performed to assess
glucose tolerance (applies only for potential pre-diabetic subjects). Fasting blood
sample (19 mL in total) from the T2DM patients will be obtain via venepuncture and no
OGTT will be performed. After inclusion in the study, participants will undergo two study
periods (period 1 and period 2) of eight days. During each period, participants will
visit the research facilities three times.
Sample size:
The primary aim of this study is to determine if acute cold-induced shivering will
decrease the area under the curve (AUC) for 24-hour glucose of individuals with a
different degree of metabolic impairment, including pre-diabetic participants and
patients with T2DM. Thus, the study will be powered accordingly to allow for detecting
the expected minimal effect in both groups. It is expected to see a clinically meaningful
decrease in AUC for 24-hour glucose of at least 15% in the pre-diabetic participants.
Based on literature, investigators anticipate a more pronounced (20%) effect in 24-hour
AUC for glucose in T2DM patients. To ensure that both groups are powered appropriately,
the sample size calculation of the T2DM patients will be followed and 12 participants
will be included in each group. When a drop-out rate of 20% is considered, investigators
anticipate that up to 15 participants will need to be included in each group to reach
this sample size. From experience with similar studies it is estimated that there will be
a screening failure of 50% and 70% for the T2DM and pre-diabetes groups, respectively.
Therefore, it is expected that researchers will need to screen 30 and 50 individuals,
respectively, from each study population.
Use of co-interventions: to minimise the variability in blood glucose induced by diet,
all participants will be asked to consume standardised meals during the two study
periods. Additionally, subjects will be asked to avoid strenuous physical activity (e.g.
walking long distances, stair climbing for >10 minutes or cycling for >30 min, hiking) or
structured exercise during the study periods to minimize exercise induced variability in
blood glucose. In addition, participants are asked to withstand from alcohol and
caffeine-containing drinks 24 hours before and after cold exposure. Participants will be
instructed to keep their normal sleeping pattern throughout the entire study. During
wash-out participants are advised to maintain their normal dietary habits and activity
pattern.
Measurements:
Participants will be equipped with a continuous glucose monitor (CGM) to continuously
measure blood glucose during each study period. Participants will be asked to wear a
FreeStyle Libre Pro iQ (Abbott) glucose monitor, which is a system able to measure
reliable blood glucose levels over a longer period; it is a standard technique in daily
diabetes practice. To determine physical activity levels throughout both study periods,
participants will be asked to wear an activPAL activity monitor (PAL Technologies Ltd).
Respiratory gas exchange will be measured using an open-air circuit respirometry with an
automated ventilated hood system (Omnical, Maastricht Instruments, Maastricht University,
Maastricht, The Netherlands). The O2 (oxygen) consumption and CO2 (carbondioxide)
production will be measured, from which energy expenditure and substrate oxidation can be
calculated. Core temperature will be measured during the two cold exposure interventions
with a telemetric pill (BodyCap, France) that will be swallowed in the evening before
each cold exposure day. During the two cold exposure days (visit 2 and 5) surface
electromyography (EMG) sensors (Trigno wireless system, Delsys Inc., USA) will be
attached to the surface of 6 muscles (upper trapezius fibres, latissimus dorsi,
pectoralis major, vastus lateralis, vastus medialis, rectus femoris) to determine
skeletal muscle electrical activity. Prior to the start of the cold exposure intervention
during visit 2 and 5 a catheter will be inserted in the antecubital vein of the
participant to repeatedly draw blood during the intervention. Blood samples will be
collected in the appropriate tubes and immediately centrifuged at high speed and plasma
will be immediately frozen in liquid nitrogen and stored at -80 ̊C for later analysis.
Blood will be analyzed for: glucose, insulin, cholesterol, triglycerides, free fatty
acids and other relevant metabolic health markers. Subjects will be provided with
standardised meals for both study periods (same period as the CGM measurements are
performed). The total daily caloric intake will be determined by multiplying the basal
metabolic rate (BMR) of the subject by a PAL value, where the BMR will be estimates using
the Harris-Benedict formula. Since our participants are sedentary by study design, a PAL
value of 1.5 for participants aged <50 years and 1.4 for subject aged 50-75 will be used,
as done in a previous study within the same research group (NL78281.068.21). The caloric
distribution throughout the day and the macronutrient composition of the meals will
maximally reflect the habitual diet of the Dutch population. As such, the breakfast,
lunch and dinner will account for ~20-25%, ~20-25% and ~40-45% of the total caloric
intake, respectively, and snacks will account for up to 10%.
Statistical analysis:
For all study parameters data will be reported as minimum, maximum, mean ± SD and/or ±
SEM, or as median and range in case of a highly skewed distribution. All primary,
secondary, and explorative study parameters are numerical values or an average of
numerical values of a defined time period. Statistical analyses will be performed using
the statistical computer program SPSS version 26.0. P-values of ≤ 0.05 (two-sided
testing) will be considered statistically significant. Missing values will not be
replaced. If a missing value occurs for a specific parameter, the subject in question
will not contribute to the analysis of that parameter and therefore no intention-to-treat
procedure will be followed but a per protocol analysis. Only participants who fully
complied with the study protocol and fulfilled the entire study will be included in the
analysis of the outcome parameters. Paired sample t-test analysis will be conducted to
investigate the effects of shivering on the primary outcome (24-hour AUC for glucose
measured before vs. after acute shivering) in the two study groups. The secondary outcome
(24-hour AUC for glucose in response to acute mild vs. moderate shivering) will be
investigated using a linear mixed model with treatment (shivering intensity) and time as
fixed factors.
Safety reporting:
In accordance to section 10, subsection 4, of the WMO, the sponsor will suspend the study
if there is sufficient ground that continuation of the study will jeopardise subject
health or safety. The sponsor will notify the accredited METC (Medical Ethics Commitee)
without undue delay of a temporary halt including the reason for such an action. The
study will be suspended pending a further positive decision by the accredited METC. The
investigator will take care that all subjects are kept informed.
Adverse events are defined as any undesirable experience occurring to a subject during
the study, whether or not considered related to the cold exposure intervention/trial
procedure. All adverse events reported spontaneously by the subject or observed by the
investigator or his staff will be recorded.
The investigator will report all (Serious Adverse Events) SAEs to the sponsor without
undue delay after obtaining knowledge of the events.
The sponsor will report the SAEs through the web portal ToetsingOnline to the accredited
METC that approved the protocol, within 7 days of first knowledge for SAEs that result in
death or are life threatening followed by a period of maximum of 8 days to complete the
initial preliminary report. All other SAEs will be reported within a period of maximum 15
days after the sponsor has first knowledge of the serious adverse events.
Data monitoring:
The study will be monitored by the Clinical Trails Centre Maastricht (CTCM). The study is
classified as a clinical trial with negligible. In accordance with this risk
classification, in total, three monitor visits will be performed: initial, interim and
close out visit. During these visits source data verification (including electronic case
report forms, medical records, etc.) and overall quality assurance will be performed by
the accredited monitor.
The sponsor/investigator will submit a summary of the progress of the trial to the
accredited METC once a year. Information will be provided on the date of inclusion of the
first subject, numbers of subjects included and numbers of subjects that have completed
the trial, serious adverse events/ serious adverse reactions, other problems, and
amendments.
The investigator/sponsor will notify the accredited METC of the end of the study within a
period of 8 weeks. The end of the study is defined as the last patient's last visit. The
sponsor will notify the METC immediately of a temporary halt of the study, including the
reason of such an action.
Handling and storage of data and documents:
At the start of the study, participants will be assigned a study code (SHVR + number of
inclusion) that will not change during the study. The code will consist of the study
acronym and a number in sequential order of screening; i.e. SHVR001, SHVR002, SHVR003,
etc. This code will be followed with the period, P1 or P2. This code is linked with the
name, address, date of birth, and telephone number of the participant in a password
protected file. For all purposes, this code will be used for participant identification.
Only the responsible principal investigator (Joris Hoeks) and the researcher conducting
this study (Dzhansel Hashim) can access this file. The privacy of the participants who
take part in the study will be protected. This means that the study code will not contain
the participants' initials or birth date as explained above. In case data, documents or
samples will be analyzed outside the scope of this proposed study, the participants will
be asked for permission but only if the participant provided permission to be approached
for follow-up research on the informed consent. Data files containing personal
information of participants are stored separately and password protected. All human
material, research data and documents will be stored for 15 years.
Ethical consideration:
This study will be conducted according to the principles of the Declaration of Helsinki
(64th WMA General Assembly, Fortaleza, Brazil, October 2013) and in accordance with the
Medical Research Involving Human Subjects Act (WMO) and other guidelines, regulations and
Acts.
