A role for EFR3A during insulin stimulated dispersal of GLUT4 at the plasma membrane

Laidlaw, Kamilla Margrethe Ebbesen (2018) A role for EFR3A during insulin stimulated dispersal of GLUT4 at the plasma membrane. PhD thesis, University of Glasgow.

Abstract

The regulation of blood glucose levels post-prandial relies on insulin-stimulated glucose uptake within adipocytes and striated muscle. The insulin effect in these tissues is a result of the translocation of the facilitative glucose transporter type IV (GLUT4) from intracellular storage vesicles to the plasma membrane. GLUT4 translocation results in the majority of glucose uptake in the body post-prandial. A major contribution to developing Type-2 diabetes is insulin-resistance, which is a result of ineffective insulin signalling, and glucose uptake.
GLUT4 is found in insulin sensitive tissue, such as adipose and striated muscle, it is sequestered and stored intracellularly in specialised GLUT4 storage vesicles (GSV) in the absence of insulin. GSVs are a dynamic vesicle which translocate in response to insulin stimulation to the plasma membrane. In the absence of insulin stimulation GSVs which arrive at the PM show reduced fusion to the membrane and are quickly re-endocytosed. Upon insulin stimulation there is an increase in GSVs translocation to the plasma membrane and upon arrival fusion with retention at the plasma membrane is increased.
At the plasma membrane GLUT4 arrives within in a cluster. Within this cluster GLUT4 is dynamic but corralled within in the cluster. In the absence of insulin GLUT4 is quickly re-endocytosed in a “kiss-and-run” type event. In the insulin-stimulated cell GLUT4 arrives in clusters which have been observed to disperse from the original site of fusion. In response to insulin stimulation GLUT4 mobility at the plasma membrane is increased with increased dispersal. This indicates that insulin stimulation has an effect on the behaviour of GLUT4 at the plasma membrane. This dispersal is hypothesised to increase GLUT4 dwell time at the plasma membrane increasing the effect of insulin signalling for a greater period of time. The dispersal of GLUT4 at the plasma membrane is an insulin mediated response which has no known molecular mechanism.
A genetic screen conducted in S. cerevisiae indicate a role for a mutant allele, fgy1-1, of the protein Efr3. This protein has two homologous mammalian orthologues EFR3A and EFR3B. The mammalian EFR3 is a palymitoylated protein responsible for membrane localisation and as a result the activity of the phosphoinositide kinase, PI4K type IIIα. PI4KIIIα activity is required for generation of phosphoinositide 4-phosphate (PI4P) at the plasma membrane inner leaflet.
The phospho-identity of the membrane phosphoinositide has been shown to affect a variety of cellular functions. Mobility of plasma membrane inserted proteins is dictated by the composition of the membrane and cytoskeletal network below the membrane. Single molecule tracking of GLUT4 at the plasma membrane shows increased mobility in the insulin-stimulated cell. Increased mobility and increased dispersal of GLUT4 in response to insulin stimulation has no known molecular mechanism which made EFR3 a promising candidate for further investigation.
This thesis aims to investigate EFR3A and of PI4P during insulin stimulated GLUT4 dispersal.
The results from these experiments showed that EFR3A is the expressed homolog within adipocyte cell types used in this investigation. Results indicating that in mice with impaired glucose tolerance EFR3A and PI4KIIIα protein levels are increased in insulin responsive striated muscle samples. This indicates a compensatory effect as insulin resistance occurs in these animals. Results showed that increased expression of EFR3A led to increasing the plasma membrane GLUT4 in the absence of insulin stimulation signifying GLUT4 is enriched in the plasma membrane instead of intracellularly within GSVs when EFR3A is over expressed. Inhibition of EFR3A plasma membrane localisation through expression of a cytosolic mutant leads to inhibition of the insulin stimulated increase of GLUT4 at the plasma membrane. This indicates that EFR3A at the plasma membrane is important for insulin stimulated GLUT4 increase. In concurrence with these data inhibition of EFR3A and PI4KIIIα through siRNA depletion resulted in inhibited insulin stimulated glucose uptake. Increased glucose uptake is the end result of insulin stimulation, and inhibition of EFR3A machinery inhibits this activity.
Taken together these findings indicate that EFR3A has a positive effect on plasma membrane GLUT4 and insulin stimulated glucose uptake. A role for the generation of PI4P at the plasma membrane is proposed to be behind this positive effect, which future work could aim to elucidate.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: GLUT4, Insulin, Diabetes, EFR3, Phosphatidylinositol 4-phosphate, phosphatidylinositol 4-kinase IIIα.
• Subjects: Q Science > QH Natural history > QH345 Biochemistry
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Supervisor's Name: Gould, Professor Gwyn W. and Bryant, Professor Nia J.
• Date of Award: 2018
• Depositing User: Ms Kamilla Margrethe Ebbesen Laidlaw
• Unique ID: glathesis:2018-38963
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 20 Dec 2018 16:08
• Last Modified: 24 Jan 2019 13:42
• URI: https://theses.gla.ac.uk/id/eprint/38963