Methods
Six obese, but otherwise healthy women (29-56 years; BMI 36.2-43.1 kg/m²) were studied before and after 8-16 days on a very low calorie diet (VLCD). Weight loss ranged from 2.4-7.2 kg. Six lean women (BMI 21.7-25.7 kg/m²) on an isocaloric diet served as controls. A subcutaneous adipose tissue biopsy was taken, under local anesthesia, from the abdominal area. The Ethical Committee at Huddinge Hospital approved the study protocol. Isolated adipocytes were prepared by collagenase treatment and incubated in vitro without or with insulin (3 or 1000 nM) for 10 minutes. Phosphatidylinositol (PI) 3-kinase activity was assessed by thin-layer chromatography. Tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 and protein expression of IRS-1/2, insulin receptor (IR), GLUT4 and p85 were assessed by SDS-PAGE and subsequent Western Blot analysis.

Results
Insulin-stimulated glucose transport in isolated adipocytes was reduced 20% in obese women (p<0.05). VLCD in obese women led to a further reduction in both basal and insulin-stimulated glucose transport and a 30% reduction (p<0.05) in GLUT4 protein expression. Fat cell volume was 2-fold greater in obese women and was not changed after VLCD. Insulin-stimulated (1000 nM) IRS-1 associated PI 3-kinase activity was reduced 65% in obese women (p<0.05), and was not affected by VLCD. IRS-1 protein expression was not altered by obesity or VLCD. IRS-2 associated PI 3-kinase activity was similar between lean and obese women. Protein expression of the insulin receptor and p85 subunit of PI 3-kinase was reduced in obese women, and not changed after VLCD.

Conclusions
Obesity in women is associated with impaired insulin signal transduction in adipocytes. Impaired insulin action on IRS-1, rather than IRS-2 appears to contribute to reduced glucose transport in isolated adipocytes from obese women. Short-term low calorie diet is not sufficient to improve impaired signal transduction or glucose transport in obese women.