A New Model of Primary Human Adipocytes Reveals Reduced Early Insulin Signalling in Type 2 Diabetes

Abstract

The aim of this study was to establish a diabetic model of primary human adipocytes for investigating potential defects in early insulin signalling. Specimens of human subcutaneous adipose tissue were obtained during orthopaedic surgical procedures. Preadipocytes were isolated and differentiated to adipocytes. Western blot analysis and immunoprecipitation were performed to determine protein content of IRS-1, IRS-2, p85, phosphorylation of IRS-1, IRS-2, Akt and MAPK as well as association between p85 and IRS-1/IRS-2. In addition to short-term insulin stimulation, the effect of hyperinsulinaemia was investigated by treating cells with insulin over a period of 36 hours. We found a significantly reduced basal expression of IRS-1 (54 +/- 15%) in adipocytes from type 2 diabetic subjects compared to controls with a further significant reduction in expression after long-term treatment (30 +/- 12%) compared to short-term treatment. IRS-2 expression also showed a significant reduction under hyperinsulinaemic conditions (20 +/- 2%) in diabetics vs. controls. Furthermore, long-term treatment with insulin in diabetic adipocytes led to a significant reduction in the phosphorylation of IRS-1(68 +/- 11%), IRS-2 (82 +/- 11%), Akt (42 +/- 2%), and MAPK (92 +/- 12%) and in the subsequent association between p85 to IRS-1 and IRS-2 (100 +/- 16% and 96 +/- 12%) in comparison to controls. Investigating glucose uptake diabetic adipocytes revealed a significant reduction of 90 +/- 2%. In this study, we were able to establish a new diabetic model of primary human adipocytes. A defect in early insulin signalling in type 2 diabetic patients under hyperinsulinaemic conditions was determined. These results might help to give further insights in early insulin action; additionally, this human model represents a useful target for the study of new therapeutic approaches.

Impact of Smoking on the Metabolic Action of Subcutaneous Regular Insulin in Type 2 Diabetic Patients

“…In addition, most population−based studies did not show any difference in daily insulin requirements between smoking and non−smoking patients with diabetes [23,24], which would have been expected with higher insulin re− sistance in smokers. Thus, smoking might not only lead to hyper− insulinemia through a deterioration of insulin resistance (poten− tially mediated by effects on insulin signaling [25] or leptin [26]), but also through other mechanisms (such as decreased insulin clearance, possibly through impaired action of atrial natriuretic peptide [27]).…”

Alterations of insulin signaling in type 2 diabetes: A review of the current evidence from humans

“…Studies regarding insulin resistance and type 2 diabetes in adipose tissue have also been performed, but to a lesser extent than in muscle tissue. The techniques used for the study of adipose tissue is based on isolated adipocytes from biopsy prepared by the collagenase method developed by Rodbell [51] in rats and modified for use in human adipocytes [23,[52][53][54][55][56][57][58][59][60][61][62]. Other techniques that have been used include whole tissue adipose explants [63].…”

“…Morbidly obese (56,57) Nonobese (52) Obese (23,52,55,58,60,62,64,69,125,126) Unchanged versus controls Impaired versus controls Unchanged/Impaired versus control…”

Adipose cell size changes are associated with a drastic actin remodeling

“…The enlarged adipocytes are, per se , less insulin responsive and exhibit impaired glucose uptake 1,5–7 . Short-term overfeeding in mice rapidly causes hypertrophy, and onset of both systemic and cellular insulin resistance in adipocytes 8 , and numerous studies have reported impaired insulin signaling at the level of insulin receptor substrate (IRS)-1 9,10 , and Akt 11 in adipocytes from obese and insulin resistant subjects 12,13 . Still, the exact mechanisms behind this impairment, or how this is associated with adipocyte expansion, have not yet been resolved.…”