the role of subcutaneous and visceral fat tissue in the mechanism of obesity induced insulin resistance

The increasing number of patients with obesity becomes globally one of the
major health problems. Obesity is recognized as one of the major risk factors
in the development of diabetes, coronary heart disease and subfertility. This
is explained by the obesity related hyperinsulinaemia, hyperandrogenism and
dyslipidaemia. Not only the total amount of body fat , but especially the body
fat distribution contributes to these fenomena. It is recognized that metabolic
activity of visceral fat tissue is different from subcutaneous fat tissue. Both
types of fat tissue secrete numerous peptides (adipokines) which affect
metabolic processes such as regulation of food-intake, and insuline
sensitivity. This makes fat tissue an important target organ for the
identification of the mechanism of obesity related insulin resistance the
precursor of type II diabetes. The bowel influences adipokine production by
peptide hormones such as GIP and GLP-1 of which is demonstrated that they
influence insulin sensitivity, possibly by changing adipokine levels in serum.
Also, short chain fatty acids formed by fermitation in the colon, can influence
adipokine secretion. An important hypothesis for the development of insulin
resistance states that obesity leads to a chronical inflammation of the fat
tissue which leads to oxidative stress and changes in the production of
adipokines, resulting in insulin resistance in peripheral tissues such as
muscle and liver. Not only adipokines but also the recently discovered
exosomes could play a role in the induction of insulin resistance. Exosomes are
membrane vesicles that are produced, amongst others, by adipocytes. It has been
demonstrated that in immune cells exosomes can hold proteines and RNA that
influence the gene expression by other cell types. The possibility thus exists
that next to adipokines, exosomes produced by fat tissue, in this manner
influence insulin resistance of the peripheral tissues. These mechanisms will
be studied in this research project.

The main goal of the study is the identification of the mechanism of obesity
related insulin resistance. We will study the relation between the different
cell types in visceral and subcutaneous fat tissue and its influence on
adipokine proflie. Especially the interactions between macrophages and
adipocytes will be studied, including the role of exosomes.
Furthermore, we will study the interactions between bowel factors such as the
bowel hormones GIP and GLP-1 and the short chain fatty acids and subcuteanous
and visceral fat tissue.
To compare the effects of the factors mentioned on the different types of fat
tissue, a quantitative proteomic analysis will be developed using stabile
isotope labelled amino acids that- incorporated in proteins- enable us to
quantify differences in protein expression using mass spectometry.
To validate the proteins found by this proteomic analyis in vivo, we will
correlate these to body fat distribution measured by waist hip ratio and BMI
and serum levels of these factors.

Explorative study in the basal mechanisms of insulin resistance using fat
biopsies from subcutaneous and visceral fat tissue of patients undergoing
laparotomy for gynaecological disorders.

Material and methodes
On the day of operation during general anesthesia a serum sample will be taken
by venous punction (20 CC). Serum is frozen and stored at -80 degree celcius. A
fat biopsy (2-5 gram) will be taken from subcuteanous fat ( at the incision
line of the operation) and visceral fat (omentum).
Briefly, adipose tissue explants are transported from the operating room to the
laboratory in transport buffer (PBS, 5.5 mM glucose, 50 µg/ml gentamicin) at
room temperature. Adipose tissue pieces will be cultured at 37 ºC and 5% CO2
following the different culture set-ups (the procedure by Fried and Moustaid-
Moussa). The procedure is optimized for proteomic analysis using fat tissue
by our own research group. The final secretome sample and the media collected
after culture will be stored frozen and analysed afterwards. Analysis of the
culture media will be performed using LC-MS/MS. This method has beeen described
by Alvarez-Llamas. The proteins and the media will be concentrated,
fractionated, igested and than analysed by mass spectometry showing the
protein distribution of the culture medium. The fat tissue itself will be used
for the analysis of adipokine gene expression using RT-PCR and DNA arrays. For
serum measurements of adipokines commercially available multiplex ELISA kits
will be used.

The biopsy of subcutaneous and visceral fat tissue do not provide additional
risk to the patients that will be operated for gynaecological disorder. The
biopsies will be taken from the incision (subcutaneous) and the omentum.