Positional Cloning of a Type 2 Diabetes Quantitative Trait Locus; Tomosyn-2, a Negative Regulator of Insulin Secretion
Figure 8
Overexpression of tomosyn-2 inhibits insulin secretion in INS1 (832/13) cells and binds syntaxin-1A and -4.
A) INS1 (832/13) cells were plated in a 96-well plate (105 cells/well). After overnight incubation, the cells were cultured in RPMI supplemental media and transfected with a bicistronic mammalian expression plasmid containing either GFP or b-tomosyn-2 and GFP. After 36 h of incubation, the insulin secretion was measured. Fractional insulin secretion in response to 3 mM 8-bromo cAMP at 7 mM glucose from b-tomosyn-2 transfected cells was normalized to that of cells transfected with GFP. Values are means ± S.E. of N≥4. * p≤0.05 for the fractional insulin secretion from the cells overexpressing tomosyn-2 vs. GFP expressing cells. B) HEK 293T cells were transfected with mock, tomosyn-1-myc, or tomosyn-2-myc isoforms (b, s, m, and xb) mammalian expression plasmids. After 24 h, the cells were harvested and whole cell lysates (WCL) were prepared. GST-pull-down experiments were performed as described in Methods. Ponceau S staining shows input of the GST fusion proteins. The overexpression of the exogenous protein in HEK 293T cells was determined by subjecting the WCL to western blot using anti-myc antibody. C) The graphs show the binding of each tomosyn-2 isoform relative to the amount of bound tomosyn-1. Values are means ± S.E. of N = 4.