Postprandial insulin release and the impact on muscle perfusion

Aging is accompanied by a progressive loss of skeletal muscle mass and
strength, leading to the loss of functional capacity and an increased risk of
developing chronic metabolic disease.5 One of these metabolic diseases
interacting with muscle mass is Diabetes Mellitus type 2. Diabetes Mellitus
type 2 is characterized by high blood glucose in the context of insulin
resistance and relative insulin deficiency. It has become clear that amongst
its many actions, insulin is also a vasoactive hormone. Its effect to cause
endothelial-nitric oxidedependent vasodilation is physiologic and
dose-dependent. Recent data suggest that insulin*s metabolic and vascular
actions are closely linked. This also means that an increase in microvascular
perfusion following food intake is more resistant to postprandial insulin
release. This physiological process is brought into prominence with increasing
age, and even more in type 2 diabetics, and contributes to diminishing
glycaemic control.

To fulfil the increasing demand for real-time evaluation of micro vascular flow
in muscle tissue, new techniques have been evaluated. The conventional systemic
hemodynamic and oxygenation parameters are neither specific nor sensitive
enough to detect regional perfusion. A more complete evaluation of tissue
oxygenation can be achieved by adding noninvasive assessment of perfusion in
peripheral tissues to global parameters. Noninvasive monitoring of peripheral
perfusion could be a complementary approach that allows very early application
throughout the hospital and interventional research. Orthogonal polarization
spectral (OPS) is a non invasive technique that uses reflected light to produce
real-time images of the microcirculation. The technology has been incorporated
into a small hand-held video-microscope which can be used in both research and
clinical settings. OPS can assess tissue perfusion using the functional
capillary density (FCD), i.e., the length of perfused capillaries per
observation area (measured as cm/cm2). FCD is a very sensitive parameter for
determining the status of nutritive perfusion to the tissue. So far, one of the
most easily accessible sites in humans for peripheral perfusion monitoring is
the mouth. OPS produces excellent images of the sublingual microcirculation by
placing the probe under the tongue. Movement artifacts, semiquantitative
measure of perfusion, the presence of various secretions such as saliva and
blood, observer-related bias, and malfunction of the apparatus are some of the
limitations of the technique.

In the present study we will investigate the impact of postprandial insulin
release on microvascular recruitment in the oral cavity.

We hypothesize, that during an oral glucose tolerance test, the OPS arterial
flow measured sublingual will be comparable within the same subject, but
different between young, elderly and type 2 diabetic patients. Objective is to
quantify differences in micro vascular recruitment between healthy young,
elderly and elderly type 2 diabetes patients, in fasting conditions or
postpandrial, due to increasing insulin resistance. Therefore, we measure micro
vascular perfusion by sublingual OPS measurements in sublingual position in
young, elderly and age-matched Diabetes mellitus type 2 patients, before and
during an oral glucose tolerance test.

Primary outcome for this study will be micro vascular flow, expressed in
glycocalyx volume.

Secondary outcome will be: correlation between basal and postprandial micro
vascular blood flow and oral glucose tolerance. Additional information will be
obtained about ageing, DM2 and total and fiber specific muscle mass.

The study will be an interventional case control study. The experiments will be
conducted over a period of 6 months, in a clinical setting. A time schedule can
be found in the "onderzoeksprotocol" on page 8.

One week before the start of the study, all subjects will visit the laboratory
for an intake procedure. A fasting blood sample will be drawn from an
antecubital vein to determine basal blood glucose, insulin and HbA1c content.
Anthropometric measurements (body mass, height, waist circumference and leg
volume) will be determined and medical history and medication will be
discussed. All subjects will be subjected to a questionnaire regarding their
last year*s physical health. Before the start of the intervention, subjects
will receive instructions on diet and physical activity prior to the morning of
the study.

One week after inclusion, subjects arrive at the laboratory at 0800 h by car or
public transportation after an overnight fast. A catheter (Baxter BV, Utrecht,
the Netherlands) will be inserted into an antecubital vein for venous blood
sampling. After collection of a fasting blood sample (15 ml), a first OPS
measurement under the tongue will be performed. If possible, subjects will grip
the OPS-camera with both hands, in order to perform their own measurements,
assisted by B. Groen (see picture 1, page 9 "onderzoeksprotocol").

After 30 minutes, a bolus of 75 g glucose (dissolved in 250 ml water) will be
ingested (t=0 min). After the ingestion of the bolus, venous blood (8 ml) will
be sampled every 30 min until t=120 min. The type 2 diabetes patients
participating in this study will cease the use of anti-diabetic medication
during the last 2 days prior to the OGTT. Plasma glucose concentrations are
measured to classify impaired glucose tolerance and type 2 diabetes according
to ADA criteria of 2006. In addition, plasma glucose, and insulin
concentrations will be used to assess insulin sensitivity using the Oral
Glucose Insulin Sensitivity (OGIS) Model.

At time point t = 15, t=30, t=45, t=60 t=75 t=90 t=105 and t=120, micro
vascular flow will be measured with the use of OPS in sublingual position.
After completion of the OGTT and OPS measurements, a small amount of muscle
tissue will be obtained from the M. Vastus Lateralis. Muscle samples will be
cut into 5 µm thick cross sections and will be stained for immunohistochemical
determination of fiber type distribution and vascularisation. Finally a CT-scan
of the right upper leg will be made. Radiation exposere is extimated to be 0.03
mSe. Imaging will last approximately 5 minutes. After completion of the test,
all subjects will be observed during a short period of time (30 minutes).
During this period, a lunch will be served consisting of bread, marmalade,
currant loaf and coffee or tea, before heading home.

All subjects will perform an oral glucose tolerance test (OGTT). After an
overnight fast, subjects arrive at the laboratory at 0800 h by car or public
transportation after an overnight fast. A catheter (Baxter BV, Utrecht, the
Netherlands) will be inserted into an antecubital vein for venous blood
sampling. After collection of a fasting blood sample (15 ml), a bolus of 75 g
glucose (dissolved in 250 ml water) will be ingested (t=0 min). After the
ingestion of the bolus, venous blood (8 ml) will be sampled every 30 min until
t=120 min. The type 2 diabetes patients participating in this study will cease
the use of anti-diabetic medication during the last 2 days hours prior to the
OGTT. Plasma glucose concentrations are measured to classify impaired glucose
tolerance and type 2 diabetes according to ADA criteria of 2006. In addition,
plasma glucose, HbA1c and insulin concentrations are used to assess insulin
sensitivity using the Oral Glucose Insulin Sensitivity (OGIS) Model.

At time point t=0, t=15, t=30, t=45, t=60, t=75, t=90, t=105 and t=120, micro
vascular flow will be measured with the use of OPS in sublingual position.
After completion of the OGTT, a muscle biopsy will be obtained from the middle
region of the vastus lateralis muscle (15 cm above the patella) and
approximately 2 cm away from the fascia by the percutaneous needle biopsy
technique described by Bergström. The muscle biopsy will be carefully freed
from any visible fat and blood, and rapidly frozen in liquid nitrogen cooled
isopentane and embedded in Tissue-Tek (Sakura Finetek Europe B.V., Zoeterwoude,
Netherlands), and stored at -80 ºC for subsequent histochemical analysis. A
single slice CT-scan will be made. After the biopsy and CT-scan, testing is
completed. Subjects will receive a lunch before heading home.

The OGTT will be performed twice: once with glucose monohydrate, and once with
plain water. Testing will be done in a randomized order. After the last OGTT, a
small amount of muscle tissue is obtained in a muscle biopsy.

The risks involved in participating in this experiment are minimal. Insertion
of the catheters in a vein is comparable to a normal blood drawn and the only
risk is of a small local haematoma. This is the same for the muscle biopsy. The
incision made for obtaining the muscle biopsy (performed by an experienced
physician) will heal completely. Withholding oral blood glucose lowering
medication for a period of two days does not result in a serious disturbance of
blood glucose homeostasis and blood glucose concentrations will be normalized
within 1-2 days after testing. After the test, subjects can resume their normal
medication routine.