Introduction:
HS
is a complex polysaccharide which is synthesised directly onto HSPG core
proteins. High intracellular levels of HS are essential for islet beta cell
survival (1). T2D-prone db/db mice are characterised by obesity, insulin
resistance, endoplasmic reticulum (ER) stress, defective protein maturation and
ultimately beta cell failure.
Objective: This study investigated
whether ER stress constitutes the molecular basis for HS loss in islet beta
cells of T2D-prone db/db mice.
Methods: The intra-islet expression
of HSPG core proteins (collagen type XVIII (Col18), syndecan-1 (sdc1) and CD44)
and HS was examined by immunohistochemistry and Alcian blue histochemistry,
respectively, in pancreases of db/db female mice and control db/+ mice at 4, 5,
6, 9 and 12 weeks of age (n=3/time-point). The %HS+ve and %HSPG+ve islet tissue
was determined by morphometry. ER stress was induced in MIN6 beta cells using thapsigargin
(50nM) and confirmed by real-time RT-PCR analysis of Atf3, CHOP and BiP gene expression.
Intracellular staining of HSPG core proteins and HS in MIN6 cells and apoptosis
by 7AAD uptake was examined by flow cytometry.
Results: Intra-islet HS in db/db
islets declined to 56% and 44% of control db/+ islets at 4 and 12 (p<0.001)
weeks, respectively. Intra-islet levels of col18, CD44 and sdc1 declined to 67%,
29%(p<0.001) and 41%(p<0.05) of db/+ controls at 4 weeks, respectively,
and were significantly reduced to 12%, 25% and 9% of controls by 12 weeks
(p<0.001) at which time 67% of db/db mice were hyperglycaemic and 100% were
obese. ER-stressed MIN6 cells showed decreased intracellular sdc1 (p<0.01)
and CD44 (p<0.05) core proteins to 24%-42% of controls and a significant 1.4-fold
increase in apoptosis (p<0.05).
Conclusions: Unlike T1D where beta
cell HS is degraded by heparanase, our findings suggest that ER stress in T2D down-regulates
HSPG core protein levels, leading to diminished HS synthesis and beta cell
failure.