Red wine polyphenols improve cardiometabolic risk in obese subjects by alleviating
inflammation and microvascular dysfunction

The global epidemic of obesity is bringing in its wake a catastrophic increase
in the prevalence of metabolic diseases. As a result, obesity-related diseases,
such as diabetes, hypertension, dyslipidaemia have surpassed tobacco use as a
cause of death(1;2). Obesity is a major cause of insulin resistance, which has
been implicated in the rising prevalence of the metabolic syndrome, a cluster
of risk factors which confers an increased risk for type 2 diabetes and
cardiovascular disease (CVD)(3). The mechanisms underlying this clustering are
incompletely understood. Obesity-associated microvascular dysfunction explains
part of this clustering and predisposes obese subjects to CVD(4;5).
Microvascular dysfunction, by affecting both flow resistance and perfusion, is
important not only in the development of obesity-related target-organ damage in
the heart and kidney, but also in the development of cardiovascular risk
factors such as hypertension and insulin resistance (6-9).

Beneficial effects of moderate use of alcoholic beverages (10), red wine in
particular (11), on
cardiovascular disease have been acknowledged for many years. In addition,
however, there are reported beneficial effects on components of the metabolic
syndrome (MS). Most impressive of these have been the reported effects of
moderate alcohol use on the incidence type 2 diabetes mellitus (DM).
Epidemiological studies suggest an impressive 40 to 60% crude reduction in DM
incidence.(12;13) This preventive effect is largely maintained (RR estimate:
~30%) after multivariable adjustment for possible confounders.(14)

The potential hazards of regular alcohol use are obvious. Thus, a key issue is
to identify the
favourable components of alcoholic beverages, red wine in particular. In this
context, several leads point to Red Wine Polyphenols (RWPs). Firstly, multiple
experimental studies have shown beneficial effects of mixed or separate RWPs
(i.e. without alcohol) on cardiometabolic parameters associated with obesity.
(15-20) In addition, studies using pure alcohol have failed to confirm that
alcohol itself is beneficial. In fact, alcohol may reduce insulin secretion and
thereby partially offset the favourable effects of red wine on glucose
homeostasis (21), eventually resulting in increased rates of DM as alcohol
intake increases.(22) Finally, observations of favourable effects, similar to
those of RWPs, of other polyphenol-rich food stuff, such as cocoa and green tea
(23) point towards favourable effects of
polyphenols rather than of the alcohol component of red wine.

Recently, a freely available RWP mix (Provinols*) was administered to obese
Zucker rats and
dramatically improved several parameters of glucose homeostasis.(17) Data in
humans, however, are remarkably scarce. A small study in DM patients (n=9)
suggested that red wine (not the RWP fraction in specific), improved
insulin-mediated glucose disposal by an impressive 43%.(24)

As for the specific polyphenol components of red wine, most research has
focused on resveratrol and quercetin. Resvertrol is quite specific to red wine
and has, at least in animal studies, beneficial effects on insulin sensitivity,
insulin secretion, and endothelial function.(18;19) Quercetin, another dominant
RWP compound, has also been implicated in RWPs favourable cardiometabolic
effects.(18) Ina recent study, quercetin attenuated atherosclerosis in ApoE*/*
gene*knockout mice by alleviating inflammation and improving NO
bioavailability. (25) Red wine polyphenol powder had beneficial effects on the
coronary microcirculation in patients with coronary artery disease (26).
Interestingly, another
study showed that RWPs improve endothelial NO-mediated relaxation using the
same PI3-kinase/Akt pathway as does insulin.(16) In addition, another study
suggested that the RWP resveratrol acts to reduce endothelin expression.(27)
Hence, a more favourable balance of insulin*s vascular effects by relative
amplification of insulin*s activation of nitric oxide is conceivable. In part,
these effects may be mediated by activation of Sirtuin 1 (SIRT-1) and
AMP-activated protein kinase (AMPK) by RWPs.(15)

Reference List

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Primary Objective: To study effects of RWPs on insulin sensitivity, glucose
tolerance, microvascular function (skin and muscle), insulin-mediated
microvascular responsiveness, and blood pressure.
Secondary objectives: To study effects of RWPs on markers of low-grade
inflammation, and couple the effects of RWPs to changes in the vascular insulin
signaling cascade.

randomized controlled trial (double blind)

mixed RWP 600mg/day (Provinols*, SEPPIC-France; corresponds to polyphenol
content of 6 glasses of red wine) or matching placebo for a total duration of 8
weeks

We expect that volunteers who have been using the RWP will have a health
benefit by volunteering for this study, however this health benefit is not
direct noticeable. The volunteers will be compensated for their time and
effort, as well as the invasive procedures, they will receive a total of ¤500
after completion of the investigations. Volunteers will also receive a
compensation for their transport-costs.

The obese volunteers in this study are needed as they are supposed to be
insulin resistant and have mild microvascular dysfunction.

Mixed RWP 600mg/day (Provinols*, SEPPIC-France; corresponds to polyphenol
content of 6 glasses of red wine, freely available) are composed of polyphenols
of Carbernet-Sauvignon red wine and selected for its antioxidant content. It is
an additive-free food product, obtained through simple physical extraction. No
side effects have been reported yet. However a small risk (e.g. allergic
reaction) is always present.

Risks of the hyperinsulemic-euglycemic clamp consist of nausea, headache
afterwards and allergic reactions; rare: (self-limiting) flushing, urticaria
and nausea, very rare: (<1:10.000) anaphylactic shock to one of the components
of the insulin solution (saline 0.9%, Glucose 20%, insulin (NovoRapid®, Novo
nordisk), Furthermore there is an obvious risk of hypo- (and hyper-) glycaemia,
which will be counteracted by drawing regular blood samples and adjusting the
glucose infusion rate.
Otherwise the risks are the same as those of a normal peripheral line, with
failure of placement and repetitive attempts, a hematoma or phlebitis (1/100)
being the most common. In the literature, clamping is a frequently used method,
however, no adverse events are described as such. In this research unit, there
is ample experience with clamping, no adverse events other than those mentioned
before are known, phlebitis has occurred only once as the most serious adverse
event in >200 (estimated) clamps performed over the previous years. Total blood
sampling volume during the clamp ~40 ml.
The drawing of blood samples has a risk of a hematoma, slight pain at the
insertion site during, and after the insertion, as well as the risk of failure
and thus repetitive attempts.
Drawing blood samples from a venous catheter has the risk of quick thrombus
formation. Patency is checked regularly and forming thrombi are removed as soon
as possible (the 3 way connector set-up makes this possible without the risk of
injecting thrombi intravascular).

Risks associated with the meal test only consist of the above described risk
during blood sampling. Total blood sampling volume during the meal test ~200 ml.

During CEUS we will make use of contrast (SonoVue ® microbubbles). The most
common side effects with SonoVue (seen in between 1 and 10% of subjects
undergoing studies with SonoVue) are headache, facial flushing, nausea,
dizziness, moderate hypotension, injection site pain, injection site reactions,
including bruising, burning, and paraesthesia at the injection site.
In a postmarketing study, serious adverse events occured in 0,009% of patients
(2/23188). The serious adverse events consisted of dyspnoea, bronchospasm,
slight hypotension and bradycardia in one patient who recovered in 30 minutes,
the other serious adverse event consisted of clouding of consciousness,
dorsolumbar pain, severe hypotension and a cutaneous rash, which lasted for 30
minutes. No fatalities occurred

Our own department has several years of experience with CEUS and SonoVue ® for
research purposes. The department of anaesthesiology has several years of
experience with MCE

During Capillary video microscopy and iontophoresis of acetylcholine and SNP
along with laser Doppler measurements can cause in a minority of patients- a
slight tingling feeling in the finger where iontophoresis is performed, because
of the electric current used.

Risks associated with the quadriceps muscle biopsy include myalgia, cutaneous
infection and bleeding at the biopsy site, to prevent the latter, adequate
pressure will be applied as well as steri-strips and compressive bandages.
Infections will be prevented by using sterile instruments and covers, as well
as disinfecting the skin using chlorhexidine.

As the above mentioned side effects are well known, care will be taken
throughout the protocol to prevent them from occurring (for example, adequate
application of pressure at the cannulation sites after removal of catheters or
frequent sampling of plasma glucose levels after initiation of insulin infusion
(and adequate co-infusion of glucose) to prevent hypoglycaemia).

Volunteers will stay at the clinical research unit for at least 30 minutes
after completion of all procedures in order to monitor any delayed adverse
events. Volunteers are studied in the immediate vicinity of a clinical unit,
where resuscitation facilities are present and can be at the bedside within 1
minute.