Positional Cloning of a Type 2 Diabetes Quantitative Trait Locus; Tomosyn-2, a Negative Regulator of Insulin Secretion

Abstract

We previously mapped a type 2 diabetes (T2D) locus on chromosome 16 (Chr 16) in an F2 intercross from the BTBR T (+) tf (BTBR) Lep^ob/ob and C57BL/6 (B6) Lep^ob/ob mouse strains. Introgression of BTBR Chr 16 into B6 mice resulted in a consomic mouse with reduced fasting plasma insulin and elevated glucose levels. We derived a panel of sub-congenic mice and narrowed the diabetes susceptibility locus to a 1.6 Mb region. Introgression of this 1.6 Mb fragment of the BTBR Chr 16 into lean B6 mice (B6.16^BT36–38) replicated the phenotypes of the consomic mice. Pancreatic islets from the B6.16^BT36–38 mice were defective in the second phase of the insulin secretion, suggesting that the 1.6 Mb region encodes a regulator of insulin secretion. Within this region, syntaxin-binding protein 5-like (Stxbp5l) or tomosyn-2 was the only gene with an expression difference and a non-synonymous coding single nucleotide polymorphism (SNP) between the B6 and BTBR alleles. Overexpression of the b-tomosyn-2 isoform in the pancreatic β-cell line, INS1 (832/13), resulted in an inhibition of insulin secretion in response to 3 mM 8-bromo cAMP at 7 mM glucose. In vitro binding experiments showed that tomosyn-2 binds recombinant syntaxin-1A and syntaxin-4, key proteins that are involved in insulin secretion via formation of the SNARE complex. The B6 form of tomosyn-2 is more susceptible to proteasomal degradation than the BTBR form, establishing a functional role for the coding SNP in tomosyn-2. We conclude that tomosyn-2 is the major gene responsible for the T2D Chr 16 quantitative trait locus (QTL) we mapped in our mouse cross. Our findings suggest that tomosyn-2 is a key negative regulator of insulin secretion. Humans carry many genetic variants that confer small effects on metabolic traits relevant to type 2 diabetes. These effects are amplified by environmental stressors like obesity. We used morbid obesity as a sensitizer to identify genes that contribute to the diabetes susceptibility of the BTBR mouse strain. Using mapping and breeding strategies, we were able to narrow a genetic region to one containing just 13 genes. One of these genes, tomosyn-2, emerged as a prime candidate. Our functional studies showed that tomosyn-2 is an inhibitor of insulin secretion, and it binds to the proteins involved in the fusion of insulin containing granules with the plasma membrane. We found a coding mutation and demonstrated that this mutation affects the stability of the protein product. Our work with Tomosyn-2 provides new insights into the regulation of insulin secretion and emphasizes that negative regulation is critical for avoiding insulin-induced hypoglycemia.