Longer-term effects of a novel nutritional combination on muscle insulin sensitivity and mitochondrial function, and vascular function in abdominally obese subjects

Type 2 diabetes mellitus (T2DM) is a progressive disease and early intervention
and prevention strategies are therefore very important. An important early
hallmark in the development of T2DM is insulin resistance. Since the majority
of postprandial glucose disposal occurs in skeletal muscle, improving muscle
insulin sensitivity will thus have a major impact on disease prevention.
Abdominally obese men and women have an increased risk to develop T2DM, and are
also characterized by an impaired vascular function. This may hamper proper
delivery of insulin, glucose and oxygen to muscles, thereby contributing to -
and possibly causing - muscle insulin resistance. Earlier it has been shown
that supplementation with L-arginine improves vascular function by improving
nitric oxide (NO) bioavailability. These NO-mediated beneficial effects on
vascular function may improve delivery of insulin, glucose and oxygen to the
muscle tissue, thereby improving muscle insulin sensitivity and mitochondrial
function. However, the doses needed of this amino acid cannot be provided by
regular diets or supplements, also due to the bitter taste of L-arginine.
Alternatively, smaller amounts of L-arginine with a specific combination of
other nutritional components (i.e. nitrate and nitrite), which are already part
of the regular diet and support alternative pathways to improve NO-mediated
vascular function, may also induce beneficial effects. We now hypothesize that
in abdominally obese adults with impaired fasting glucose concentrations
L-arginine combined with nitrate/nitrite increases muscle insulin sensitivity.

The primary objective is to investigate the longer-term effects of a novel
nutritional combination, containing L-arginine and nitrate/nitrite after eight
weeks on muscle insulin sensitivity (i.e. insulin-stimulated plasma glucose
rate of disappearance [Rd]) as compared to an iso-caloric placebo. Secondary
objectives are to examine effects on mitochondrial function of skeletal muscle
biopsies, vascular function (i.e. arterial stiffness, and vascular endothelial
and microvascular function), and characteristics of the muscle vasculature
(i.e. blood flow).

The trial will have a randomized, double-blind, cross-over design. The total
study duration will be at least twenty-four weeks, including an intervention
and control period of eight weeks, separated by a washout period of at least
eight weeks.

At the end of the intervention and the control period, study measurements will
be performed during two test days. After an overnight fast, a one-step
hyperinsulinemic-euglycemic clamp with glucose tracer combined with indirect
calorimetry (ventilated hood) will be performed to assess insulin sensitivity
(Rd). In addition, a muscle biopsy will be taken to assess mitochondrial
function. Vascular function and characteristics of muscle vasculature will be
assessed during another test day.

Subjects will be screened to determine eligibility during two visits of 15
minutes. During these screening visits, anthropometric measurements will be
performed and a fasting blood sample (5.5 mL + 2.0 mL = 7.5 mL) will be drawn.
During the trial on different occasions, tests will be performed and blood will
be sampled. During these tests, subjects have to stay at the university and are
not allowed to eat. Venipuncture can occasionally cause a local hematoma or
bruise to occur. Some subjects may report pain during venipuncture. Insertion
of the cannula can cause some discomfort and possible a hematoma or bruise.
Some study subjects may also report pain during the insertion of the cannula.
Indirect calorimetry might evoke claustrophobic reactions, but there are no
physical risks involved. Sampling skeletal muscle tissue biopsies is performed
under local anaesthesia and muscle pain can occur due to invasive method for
taking muscle tissue biopsy. In principle, all measurements are routine in our
metabolic research unit (MRUM) and are not expected to lead to physical side
effects. There are no directs benefits for the subjects. Subjects that not
fully adhere to the protocol will be excluded from the statistical analyses
because a per protocol analysis will be performed. Time investment is 19.5
hours (1170 minutes), excluding travel time, and blood sampling volume will be
447.5 mL during the whole study. Finally, the novel supplement and co-factors
are safe and there are no expected side effects related to the intervention
treatment.