GM3 and insulin sensitivity

Background
Diabetes is affecting 5% of the Western population, and is becoming more and
more a problem in all age categories. Although type II diabetes was at first
mainly a problem of the elderly, the incidence in children and adolescents is
rapidly increasing. This increment runs in parallel with the increment in the
prevalence of obesity.
Obesity induces hepatic and peripheral insulin resistance. The underlying
mechanism has not been elucidated completely. However it has been convincingly
demonstrated that elevated free fatty acids (FFA) are one of the major players
in the induction of obesity-induced insulin resistance. The increase in free
fatty acids originates from ongoing lipolysis under hyperinsulinemic and basal
conditions. Peripheral glucose disposal under hyperinsulinemic conditions
occurs mainly in skeletal muscle (1). Therefore many studies have focused on
this tissue to get more insight in the mechanisms behind the decreased glucose
uptake as seen in obesity-induced peripheral insulin resistance.
Insulin binding to the insulin receptor stimulates a complex insulin signaling
cascade, eventually resulting in translocation of the glucose transporter 4
(glut4) to the plasma membrane. Many ex vivo, in vitro and in vivo experiments
in cell lines, animals and humans have identified possible explanations for the
reduced glucose uptake in skeletal muscle. The following mechanisms have been
proposed:
1. increased reactive oxygen species (ROS) production (2;3)
2. a pro-inflammatory state of adipose tissue resulting in dysfunctional
adipocytes and ongoing production of local and systemic cytokines.
Inflammatory cytokines released from adipocytes or macrophages infiltrating
adipose tissue may antagonize insulin action. (4)
3. mitochondrial dysfunction induced by increased intracellular levels of
peroxidized fatty acids
4. Ectopic uptake of fat. Accumulation of intramyocellular lipid and its
metabolites, so called lipid toxicity, leads to insulin resistance. (5)
In this study we want to focus on this latter possible mechanism for insulin
resistance.
Once fatty acids enter the cell, they are activated to acyl-CoAs and follow two
predominant routes, i.e. incorporation into complex lipids (triglycerides,
diacylglycerol, phospholipids, cholesteryl esters) or intra-mitochondrial
beta-oxidation. Increased availability of the long chain fatty acids palmitate
and stearate may result in increased formation of the intracellular glycolipid
ceramide. Ceramide has been shown to interfere negatively with the insulin
signaling pathway(6). Higher levels of muscle ceramide have been reported in
obese subjects in some but not all human studies(7). Ceramide is a precursor
for gangliosides, which may also be involved in the induction of saturated
fatty acid-induced insulin resistance(8). Gangliosides reside within the plasma
membrane and are able to modulate insulin signaling at the level of the insulin
receptor (9-13). The most abundant ganglioside is GM3 (14) Indeed GM3 synthase
knockout mice have enhanced insulin sensitivity and are protected from high fat
diet-induced insulin resistance(15). Recently, we and others have shown in rats
that pharmacologically reducing GM2 and GM3 levels results in amelioration of
high fat diet induced insulin resistance (16;17). Whether this is also true for
the human situation is not known. (18)
Whether levels of muscle and adipose tissue gangliosides are elevated in obese
insulin resistant subjects remains to be answered. To explore whether
gangliosides are indeed elevated in obese subjects compared to matched healthy
controls and whether this is correlated to peripheral insulin resistance, we
propose a study with obese and lean subjects in whom, we will measure muscle
and adipocyte residing gangliosides and glucose metabolism using stable
isotopes.

We hypothesize that membrane-residing gangliosides are elevated in obese
insulin resistant subjects and correlate to peripheral insulin resistance.
Furthermore, we hypothesize that the perturbation of the insulin signaling
cascade by elevated muscle gangliosides is caused by a reduced phosphorylation
of the insulin receptor.

We will perform a hyperinsulinemic euglycemic clamp using stable isotopes, in
10 obese insulin resistant subjects and in 10 healthy lean subjects, matched
for age and sex. At the beginning and end of the clamp a muscle biopsy from the
vastus lateralis and a fat biopsy from the abdominal subcutaneous adipose
tissue will be performed.

-Stable isotopes behave as the natural substrate and have no side effects

-A possible side effect of the administration of insulin is hypoglycaemia.
Since we will measure the plasma glucose every 5 minutes and the scope of the
protocol is to maintain the blood glucose stable at 5 mmol/l, hypoglycaemia is
not expected

-The total volume of blood sample for the entire protocol is 150 ml. This
amount is not considered to be of negative influence to the subject*s health.

-During the muscle and fat biopsy, subjects should not experience any
discomfort, since the biopsy area will be adequately anaesthetized. The day
after the biopsy subjects may experience a soar feeling at the biopsy location,
which will resolve in a few days after the biopsy. Safety measurements will be
taken to reduce the risk for a haematoma (pressure bandage). In our experience,
muscle biopsy is tolerated well and leaves minimal scarring.