Abstract
Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is vital for whole-body
glucose homeostasis, and loss of β-cell function can result in type 2 diabetes
(T2D), a major worldwide health burden. The main hallmark of β-cell dysfunction in
T2D is loss of GSIS, but the exact mechanisms behind this are still unknown. The main
aim of this thesis was to investigate how endogenous lipid metabolism contributes to
GSIS, which could help develop new therapeutic targets for T2D.
Using mass spectrometry (MS) to comprehensively profile lipids during GSIS revealed
that neutral lipid levels changed the most during insulin secretion. Further examination
revealed that saturated triacylglycerol (TG) decreased, and saturated diacylglycerol
(DG) increased. To examine the mechanism of action of saturated DG, the activity of
the lipid regulated protein kinase D (PKD) was investigated. Increased saturated DG
potentiated insulin secretion and increased PKD phosphorylation. Inhibition of PKD
activity blocked this potentiation of insulin secretion, indicating for the first time that
PKD signalling is important for GSIS.
Using tracer studies and subcellular fractionation, neutral lipid localisation was
investigated. Interestingly, TG pools appeared to show spatial changes with glucose
stimulation, and there was also evidence of lysosomal neutral lipid pools. The role of
lysosomal lipid metabolism was investigated by inhibiting lysosomal acid lipase (LAL)
with either siRNA or a small molecule inhibitor. Inhibition of LAL increased insulin
secretion due to accumulation of cytosolic neutral lipid. These increased lipids were
mobilised acutely to generate downstream signalling molecules to potentiate insulin
secretion.
Protein acylation is postulated to be involved in insulin secretion due to increased fatty
acyl-CoA levels after glucose stimulation. The role of protein acylation in GSIS was
also investigated. A specific MS technique was developed to identify acylated proteins,
and successfully identified a variety of acylated proteins with some evidence for their
differential regulation during GSIS.
In summary, this research has shown for the first time that: (1) saturated DG species
increase during GSIS (likely due to TG lipolysis) and modulate downstream signalling
cascades; (2) lysosomal lipid degradation is important in β-cells and; (3) protein
acylation can be measured by MS in β-cells.