Objective: Alternative splicing (AS) plays a crucial role in diversifying the cellular protein
landscape. Previous studies identified distinct AS pattern in pancreatic islet cells
of diabetes-resistant B6-ob/ob compared to diabetes-prone New Zealand Obese (NZO)
mice. Our objective is to investigate specific splice events to understand their potential
impact on metabolic heath in mice predisposed to diabetes [1]
[2]
[3].
Methods: RNA-sequencing analysis was performed in pancreatic islets of male NZO and B6-ob/ob
mice. Candidate genes were characterized by AVV-mediated overexpression of specific
transcript variants in the MIN6 beta-cell line. Transfected cells were evaluated for
glucose-stimulated insulin secretion.
Results: RNA sequencing analysis of primary pancreatic islets revealed 894 significant alternative
splicing events (∆PSI>0.1) in 730 genes in NZO mice compared to B6-ob/ob mice. Different
selection criteria were applied to identify promising candidates, including a ΔPSI>0.3,
expression in human islets and protein functionality with the potential to affect
beta-cell function. Vps39 and Slc7a2 were selected for further investigation. Vps39
(vacuole protein sorting 39; subunit of HOPS complex) encodes a protein crucial for
the fusion of the late endosome and the autophagosome with the lysosome (van der Beek
et al., 2019). Since lysosomes are involved in insulin granule degradation in beta
cells (Müller et al., 2017) Vps39 might play an important role in this process. The
alternatively spliced form of Vps39, which skips exon 3 (∆PSI>0.7), was overexpressed
in MIN6 cells and tended to reduce glucose-stimulated insulin secretion in comparison
to the full-length protein. Slc7a2 (solute carrier family 7 member 2) encodes a cationic
amino acid transporter predominantly expressed in alpha and beta cells. The two relevant
isoforms (CAT2A and CAT2B) of this transporter, which differ in alternative exon 7
(mutually exclusive exons), are differentially expressed in the two mouse strains
and demonstrate markedly different substrate affinities (Closs et al., 1997). Both
mouse strains show robust expression of the low-affinity amino acid transporter CAT2A.
In contrast, CAT2B, characterized by its high substrate affinity, is significantly
higher expressed in B6-ob/ob mice. After arginine stimulation of primary islets from
B6-ob/ob and NZO mice, significant differences in insulin secretion were observed.
Notably, NZO islets exhibited markedly higher insulin secretion to arginine stimulation,
suggesting a pivotal role for amino acid transporters in islet cell function.
Conclusion: Our data suggest that of the splice product of Vps39 lacking exon 3 tends to result
in diminished glucose-stimulated insulin secretion. Further experiments have to be
performed to elucidate the effects of alternative splicing of Vps39 and Slc7a2 also
on morphological changes and their cellular localization.
