FIGURE 1.

Glucose promotes turnover of tomosyn protein. A, primary human islets were infected with adenovirus containing tomosyn-2 or LacZ at 200 multiplicities of infection in 8 mm glucose for 48 h in standard supplemented RPMI 1640 growth medium. Following incubation, glucose-stimulated insulin secretion was performed as described under “Experimental Procedures.” Fractional insulin secretion in response to varying concentrations of glucose from tomosyn-2-infected cells was normalized to that of cells infected with LacZ control. Values are means ± S.E. of N ≥4. *, p ≤ 0.05 for the fold change in fractional insulin secretion from cells overexpressing tomosyn-2 versus LacZ. NS, not significant. B, INS1 (832/13) cells were cultured overnight in RPMI 1640 growth media containing 3 mm glucose. Following incubation, 50 μm cycloheximide was added to the cells. After 2 h, cells were either treated with 3 or 15 mm glucose, and samples were collected for protein measurement at various time points. The abundance of tomosyn protein was determined using anti-tomosyn antibody. The graph shows relative abundance of tomosyn at 3 and 15 mm glucose. Values are means ± S.E. of N ≥4. *, p ≤ 0.05 for the tomosyn protein abundance in cells treated with 15 mm glucose versus 3 mm. The protein abundance of tomosyn at time = 0 was set to one for both glucose concentrations and was normalized to a nonspecific band. C, INS1 (832/13) cells were cultured overnight in standard supplemental RPMI 1640 growth media containing 3 mm glucose. Following incubation, cells were treated with 15 mm glucose in the presence and absence of 50 μm MG132 for 4 h. Samples were collected for protein measurements. Protein abundance of tomosyn was determined, and data were normalized to β-actin. The graph shows relative abundance of tomosyn. Values are means ± S.E. of n = 4. *, p ≤ 0.05 for the change in tomosyn protein abundance over 4 h in cells treated with 15 mm glucose. NS, not significant.