Interrogating the role of retriever sorting complex in adipose insulin-sensitivity and GLUT4 recycling

Abstract

GLUT4 is a mammalian facilitative glucose transporter expressed in adipose tissue and skeletal muscle. It is mobilised from intracellular Glucose Storage Vesicles (GSVs) to the plasma membrane in response to insulin and other stimuli. Trafficking events involving sorting GLUT4 and other proteins from a late endosomal compartment are essential for the formation of the GSVs and defects in these events may lead to insulin resistance.

Retromer and retriever are endosomal complexes that mediate the late-endosomal sorting of many cargoes involved in different physiological and pathophysiological processes. This study aimed to investigate whether retriever is involved in the endosomal sorting of GLUT4 in adipocytes. We generated CRISPR/Cas9 knockout cell lines for two essential components of retromer and retriever sorting complexes, VPS35 and VPS35L respectively, in 3T3-L1 embryonic mouse preadipocytes. In the VPS35L KO cells, we observed that the retriever knockout preadipocyte cell line failed to differentiate efficiently into adipocytes and readily detached from plasticware during differentiation. The insulin signalling pathway was not however altered, demonstrating that this part of the differentiation program was not affected by VPS35L knockout. Surface proteomics analysis identified potential cargoes recycled by retriever complex. Some are already known retriever cargos (LRP1, integrin α5, integrin α6) and some are new (PDGFRα and β; AOC3, DLK1). As surface levels of integrin α5, α6 and other adhesion molecules were affected we propose that the defect of adipogenesis in VPS35L KO preadipocytes might be mainly contributed to by a cell adhesion defect.

siRNA knockdown of VPS35L revealed increased surface GLUT4 upon insulin stimulation. We hypothesised that more GLUT4 and LRP1 were being diverted from a constitutive recycling pathway into the insulin sensitive GSV compartment upon VPS35L knockdown. Alternative methods, CRISPRi and inducible CRISPR, to further characterise the role of retriever in GLUT4 trafficking were unsuccessful.

The finding that perturbation of retriever function can improve glucose uptake in response to insulin may be of significance in the development of novel interventions for treating metabolic disorders such as type 2 diabetes.

Date of Award	13 Nov 2024
Original language	English
Awarding Institution	University of Bath
Supervisor	Francoise Koumanov (Supervisor) & Paul Whitley (Supervisor)