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.
19 - 21 Sep 2019
European Society for Paediatric Endocrinology