Is microvascular dysfunction an early phenomenon in the development of skeletal muscle insulin resistance? A dietary intervention study in healthy men

Excessive caloric intake causes overweight, obesity, insulin resistance and
ultimately type 2 diabetes and cardiovascular diseases (CVD). In rodents with
obesity-associated insulin resistance induced by high-fat diet, insulin
resistance develops in the vasculature well before these responses are detected
in skeletal muscle, liver or adipose tissue. Insulin signaling in vascular
endothelium, therefore, seems more susceptible than other tissues to the
deleterious effects of nutrient overload. Furthermore, insulin signaling in
vascular endothelium has been proposed to regulate its own delivery to skeletal
muscle, by increasing blood flow and recruiting perfused capillaries and thus
may be an important therapeutic target to improve insulin resistance in
skeletal muscle. In humans, the early adaptation of the microvasculature during
the initial phase of overfeeding and weight gain has not been studied yet.
Therefore, it remains unclear whether insulin resistance in endothelial cells
precede insulin resistance in skeletal muscle and if improving endothelial
insulin signaling/microvascular function may thus serve as a
preventive/therapeutic strategy for ameliorating peripheral insulin resistance.

Primary objective:
To assess the effects of hypercaloric overfeeding in humans on vascular and
metabolic insulin sensitivity and moreover, to establish whether insulin
resistance develops in the vasculature before it occurs in peripheral tissue
during hypercaloric overfeeding.

Secondary objectives:
To assess whether the effects on hepatic, skeletal muscle tissue, adipose
tissue and microvascular insulin sensitivity induced by caloric overfeeding are
reversible during a subsequent hypocaloric diet.
To determine if hypercaloric overfeeding in humans influences insulin-mediated
recruitment in skeletal muscle and adipose tissue differently.
To determine if direct pharmacological stimulation of capillary recruitment by
a stable prostacyclin (PGI2) analogue can overcome diet-induced resistance to
glucose disposal in skeletal muscle?
To determine if hypercaloric overfeeding in humans influences adipocyte size,
cell types and paracrine effects of PVAT surrounding skeletal muscle
arterioles.
To determine if hypercaloric overfeeding in humans influences insulin-signaling
in subcutaneous adipose fat tissue.
To determine if hypercaloric overfeeding affects gen expression in endotheial
cells.

This is an expiremental dietary intervention study: 30 days hypercaloric diet
and a subsequent hypocaloric diet.
Measurements will take place at baseline, 7 days after the start of the
hypercaloric diet, at the end of the hypercaloric diet and at the end of the
hypocaloric diet. These measurements will include a 2-step
hyperinsulinaemic-euglymic clamp, contrast enhanced ultrasounds (CEUS) to
determine metabolic and vascular insulin sensitivity. Furthermore, we will take
1 muscle and adipose tissue biopsy per person, either at the end of the
hypercaloric or hypocaloric phase.

The intervention consists of a hypercaloric (60% caloric excess) diet, during a
period of approximately 30 days, followed by a hypocaloric phase (1.0 x resting
energy expenditure), until subjects return to their baseline weight. The
surplus of engery will be provided asunhealthy foods, such as sugared bevarages
and patato chips.

After inclusion, participants will be randomized to either study group (n=24)
or control group (n=10). The control group will undergo all measurmenets
without any dietary interventions. The study group participants will enter a
diet intervention during a total of 60 days, starting with a high caloric diet,
followed by a subsequent low caloric diet. The hypocaloric diet will be
continued until participants return to baseline body weight. We expect that all
effects caused by the hypercaloric diet are reversible. During the whole diet
intervention participants will visit the medical centre a total of five times.
The first visit is a screening day, which will include anamnesis, physical
examination and obtaining blood samples for analysis. The other four visits
will consist of a two step hyperinsulinaemic-euglymic clamp and two
contrast-enhanced ultrasounds. During the end of the hypercaloric diet, half
of the participants will receive an iloprost infusion during the clamp.
Furthermore, to compare muscle microvessels between the hypercaloric and
hypocaloric phase we will take a muscle biopsy. To minimize the discomfort we
will randomly divide all subjects into two groups and take only one biopsy per
subject.

Risks associated with hyperinsulinaemic-euglymic clamps are hypo- or
hyperglycemia, which we will prevent by adjusting pump levels. Iloprost
infusion is known to have some side effects, but non are severe at a low dose.
Muscle biopsies require a local anaesthesia, which can give some discomfort to
the participants. Also, after the biopsy participants can expect some muscle
ache. We expect no severe or serious events during our study.

There is no health benefit for the participants. As a compensation for their
time and effort, as well as the burden/discomfort of the invasive procedures,
subjects will receive 1000 euros after completion of the investigation.