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F Sentinelli, E Filippi, M G Cavallo, S Romeo, M Fanelli, and M G Baroni

The insulin receptor substrate-1 (IRS-1) plays a central role in insulin sensitivity, and association studies have shown that the IRS-1 G972R variant is a risk factor for insulin resistance. However, how this mutation may lead to impaired insulin sensitivity is still to be determined. Our study aimed to evaluate, after transfection of the IRS-1 G972R variant in 3T3L1 adipocytes, the effect of this mutation on insulin signaling and on cell differentiation. The 3T3L1 cells were transfected with pcDNA3 expression vector containing either the human wild-type IRS-1 or the G972R variant. After induction of differentiation, the 3T3L1 transfected with wild-type IRS-1 differentiated in 6–8 days, while the cells transfected with G972R variant did not differentiate. To determine whether the defect in IRS-1 was responsible for this, we analyzed the expression of several genes involved in the insulin signaling pathway. Results showed that PPARγ expression was significantly reduced in cells transfected with the mutated IRS-1, together with a significant decrease in binding of phosphatidylinositol-3 kinase (PI 3-kinase) to IRS-1 G972R and in PI 3-kinase activity. In addition, we observed that the interaction between the insulin receptor (IR) and the IRS-1 G972R protein was increased and that the autophosphorylation of the IR was significantly inhibited in 3T3L1-G972R cells compared with 3T3L1-WT. Treatment of the 3T3L1-G972R cells with pioglitazone (PIO), a PPARγ agonist, restored differentiation with higher level of PPARγ expression and restoration of PI 3-kinase binding to IRS-1 G972R and PI 3-kinase activity. IR autophosphorylation was also increased. Withdrawal of PIO in fully differentiated 3T3L1-G972R cells determined the reappearance of the insulin signaling defect. Finally, we observed higher levels of IRS-2 expression, suggesting that IRS-2 may play a more important role in adipocyte insulin signaling. In conclusion, IRS-1 G972R variant impairs insulin signaling, and treatment with PPARγ agonist restores the normal phenotype of 3T3L1 cells.

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M G Cavallo, F Dotta, L Monetini, S Dionisi, M Previti, L Valente, A Toto, U Di Mario, and P Pozzilli

Abstract

In the present study we have evaluated the expression of different beta-cell markers, islet molecules and autoantigens relevant in diabetes autoimmunity by a human insulinoma cell line (CM) in order to define its similarities with native beta cells and to discover whether it could be considered as a model for studies on immunological aspects of Type 1 diabetes.

First, the positivity of the CM cell line for known markers of neuroendocrine derivation was determined by means of immunocytochemical analysis using different anti-islet monoclonal antibodies including A2B5 and 3G5 reacting with islet gangliosides, and HISL19 binding to an islet glycoprotein. Secondly, the expression and characteristics of glutamic acid decarboxylase (GAD) and of GM2-1 ganglioside, both known to be islet autoantigens in diabetes autoimmunity and expressed by human native beta cells, were investigated in the CM cell line. The pattern of ganglioside expression in comparison to that of native beta cells was also evaluated. Thirdly, the binding of diabetic sera to CM cells reacting with islet cytoplasmic antigens (ICA) was studied by immunohistochemistry. The results of this study showed that beta cell markers identified by anti-islet monoclonal antibodies A2B5, 3G5 and HISL-19 are expressed by CM cells; similarly, islet molecules such as GAD and GM2-1 ganglioside are present and possess similar characteristics to those found in native beta cells; the pattern of expression of other gangliosides by CM cells is also identical to human pancreatic islets; beta cell autoantigen(s) reacting with antibodies present in islet cell antibodies (ICA) positive diabetic sera identified by ICA binding are also detectable in this insulinoma cell line.

We conclude that CM cells show close similarities to native beta cells with respect to the expression of neuroendocrine markers, relevant beta cell autoantigens in Type 1 diabetes (GAD, GM2-1, ICA antigen), and other gangliosides. Therefore, this insulinoma cell line may be considered as an ideal model for studies aimed at investigating autoimmune phenomena occurring in Type 1 diabetes.

Journal of Endocrinology (1996) 150, 113–120