ESPE Abstracts (2019) 92 P1-30

Effects of Glypican-4 Protein on INS1E Cell Viability and Insulin Signalling

Joseph Buhl1, Antje Garten1,2, Sandy Richter1, Wieland Kiess1,3, Melanie Penke1

1Center for Pediatric Research Leipzig, University Hospital for Children and Adolescents, Leipzig University, Leipzig, Germany. 2Institute for Metabolism and Systems Research, University of Birmingham, Birmingham, United Kingdom. 3LIFE Leipzig Research Center for Civilization Diseases, Leipzig University, Leipzig, Germany

Background: Glypican-4 is a heparan sulphate proteoglycan. In addition to a membrane-bound glypican-4, a soluble form exists. Human and rodent adipose tissue were identified as source of circulating glypican-4. Glypican-4 serum levels are associated with obesity and insulin resistance, as in type 2 diabetes (T2D). Because of its positive effect on insulin sensitivity, glypican-4 might play a role in the development of obesity, insulin resistance, and T2D. We asked whether or not glypican-4 plays a role in β-cell function.

Methods: INS1E served as β-cell model. Glypican-4 mRNA and protein were detected by quantitative PCR and Western Blot, respectively. Rat adipose tissue served as control. INS1E cell viability was measured using WST1-Assay. Cells were incubated with recombinant rat glypican-4 over 6h, 24h, and 72h. To assess signalling, INS1E cells were incubated for 15 minutes with different stimulants (insulin, IGF1, glypican-4, and glypican-4 + insulin). Insulin signalling targets (phosphorylated and total InsR β-subunit, AKT, ERK) were detected by Western Blot and densitometrically analysed using ImageJ.

Results: Both glypican-4 mRNA and protein were detected in INS1E cells in comparable amounts to rat adipose tissue. Moreover, we found that rat glypican-4 mRNA expression is very high in the metabolic organs kidney, muscle, pancreas, and liver. WST1-Assays revealed that exogenous glypican-4 has no effect on INS1E cell viability in all tested conditions (low vs. high serum culture media, incubation times 6–72h, recombinant glypican-4 protein of different manufacturers, and altered starting cell numbers). Consistent with these results, we detected no effect of glypican-4 stimulation on INS1E insulin signalling. Whereas phosphorylation of insulin signalling pathway proteins InsR β-subunit, AKT and ERK increased with insulin and IGF1 as expected, treatment with recombinant glypican-4 protein did not enhance phosphorylation of these proteins. Furthermore, combined treatment of INS1E cells with insulin and glypican-4 did not lead to an increased phosphorylation of the insulin signalling pathway compared to sole insulin stimulation.

Discussion: Extensive testing of recombinant glypican-4 stimulation did not result in any effect on INS1E cell viability or insulin signalling pathway. We will test the effects of downregulating or overexpressing endogenous glypican-4 on INS1E cell function, and plan to measure glypican-4 in serum samples of lean and obese children to unravel a possible association between glypican-4 levels and parameters of obesity and insulin resistance.

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