Studies of SNAP-25 in regulated membrane fusion : metabolic consequences and tuning of intracellular Ca(2+) dynamics in beta cells

Increased release of insulin is usually regarded as a symptom of metabolic syndrome, contributing to insulin resistance in peripheral organs thus affecting glucose and insulin homeostasis. The existing animal models to address the metabolic syndrome are currently not optimal. In neuronal, neuroendocrine and endocrine cells, stimulus-dependent membrane fusion occurs via the SNARE complex, formed by the membrane-associated proteins SNAP-25 and syntaxin and the vesicle-associated protein VAMP-2, and requires intracellular Ca2+ elevations. The SNAP-25 protein exists as two splicing variants, SNAP-25a and SNAP-25b, differing in only 9 out of 206 amino acids. Both isoforms can mediate membrane fusion but their specific functions still remain unknown. In this thesis, we have investigated if an apparently small modification in the exocytotic machinery could act as triggering factor for development of metabolic syndrome. We used a genetically modified mouse expressing normal levels of SNAP-25, but with only the SNAP-25a isoform available.

In Paper I, by monitoring a number of metabolic parameters during 7 weeks on control or Western (high fat/high sucrose) diet, we found that SNAP-25b-deficiency leads to metabolic syndrome, characterised by hyperinsulinemia, obesity, hyperglycaemia, liver steatosis and adipocyte hypertrophy. These conditions were even more pronounced when the mutation was combined with Western diet. The metabolic phenotype caused by SNAP-25b-deficiency was accompanied by increased insulin secretion from the islets of Langerhans partially involving beta cell hyperplasia in a sex dependent manner.

In Paper II we addressed these issues closer and focused on islet physiology by monitoring intracellular Ca2+ dynamics in beta cells upon glucose stimulation. SNAP-25b-deficiency impaired the collective control of Ca2+ oscillations in beta cells with early initiation and delayed termination of activity as well as decreased synchronicity. Derangements of intracellular Ca2+ oscillatory patterns can be related to the increased insulin secretion found in SNAP-25b-deficient mice. In Paper III we observed that the SNAP-25 isoforms mediate different interactions with proteins important for the strict control of exocytosis both in neurons and beta cells, such as Munc18-1 and the Gβγ subunits of the heterotrimeric G proteins.

In summary, we have shown that even a small modification in the machinery regulating membrane fusion, such as replacing SNAP-25b with SNAP-25a, acts as a triggering factor for the development of metabolic syndrome in mice. This condition was associated with loss of preciseness of Ca2+ oscillations in beta cells and increased insulin secretion. Thus, we propose the SNAP-25b-deficient mouse as a new model of metabolic syndrome and prediabetes.

List of scientific papers

I. Valladolid-Acebes I, Daraio T, Brismar K, Harkany T, Ögren SO, Hökfelt TG, Bark C. Replacing SNAP-25b with SNAP-25a expression results in metabolic disease. Proc Natl Acad Sci U S A. 2015; 112(31): E4326-4335.
https://doi.org/10.1073/pnas.1511951112

II. Daraio T, Križančić Bombek L, Gosak M, Valladolid-Acebes I, Skelin Klemen M, Refai E, Berggren PO, Brismar K, Slak Rupnik M, Bark C. SNAP-25b-deficiency increases insulin secretion and changes spatiotemporal profile of Ca2+ oscillations in β cell networks. Sci Rep. 2017;7(1):7744.
https://doi.org/10.1038/s41598-017-08082-y

III. Daraio T, Valladolid-Acebes I, Brismar K, Bark C. SNAP-25a and SNAP-25b mediate different interactions with Munc18-1 and Gβγ subunits. [Manuscript]