Endocrine Abstracts

Type 2 diabetes is a metabolic disorder characterised by insulin resistance and is associated with diminished signalling through the insulin receptor. Protein tyrosine phosphatase 1B (PTP1B), encoded by the PTPN1 gene, has been shown to attenuate insulin’s actions by de-phosphorylating the activated insulin receptor and downstream signalling components such as IRS1. Several studies have highlighted PTP1B as a potential therapeutic target for type 2 diabetes and obesity; however, small molecule drug discovery efforts have proved to be challenging due to issues with achieving selectivity and bioavailability. Inhibition of PTP1B expression via the RNAi pathway represents an alternative therapeutic approach; however, due to their large size and charge these molecules cannot enter cells unaided. Here we aim to characterize a novel siRNA delivery method using a fusion protein containing the Drosophila melanogaster Antennapedia homeoprotein (AntpHD) transduction domain, which has the innate ability to cross cell membranes, and the double-stranded RNA binding domain (dsRBM1) from human protein kinase R, which binds dsRNA with high avidity. Fusion proteins consisting of either the AntpHD, or its third helix, Penetratin, fused to dsRBM1 will be tested as a means to deliver PTP1B siRNA into HepG2 cells with the aim of knocking down PTP1B and consequently potentiating insulin-mediated effects. Multiple constructs have been successfully cloned, expressed and purified using an E. coli expression system and characterized by SDS–PAGE and mass spectrometry. Indirect immunofluorescence studies using a myc-tagged construct have demonstrated the ability of the proteins to cross the cell membrane and localize within cytoplasmic vesicles after 2 and 24 h treatment, at 1 μM concentrations, while higher concentrations (10 μM), resulted in time-dependent peri-membrane aggregation with some intracellular localization in ≥50% of cells. Ongoing experiments are underway to assess the effectiveness of this method as an approach to knockdown PTP1B and potentiate insulin signalling.

Declaration of funding: Work was supported by Diabetes UK (grant number 11/0004331), the Leventis Foundation Scholars Association (2011-2013) and a fee waiver was provided by the University of Westminster, School of Life Sciences.

Leventis Foundation 2011–2013.