Fig. 5.

The effects of glucose, IBMX and DETAnonoate on insulin mRNA levels (A) and insulin (B) and total protein (C) biosynthesis in INS-1 cells. (A) INS-1 cells were cultured for 3 h in RPMI 1640 medium with 1.67 mM glucose (LG), 16.7 mM glucose (HG), 11.1 mM glucose (C), 11.1 mM glucose and 1 mM IBMX (IBMX) or 11.1 mM glucose and 1 mM DETAnonoate (DNO) and were analyzed using real time PCR. Insulin mRNA was normalized against GAPDH and expressed as percent of control. Bars are means ± SD for 9 observations. * denotes p < 0.05 using Student’s t-test when comparing HG to LG and IBMX and DNO to C. (B) INS-1 cells were labeled with ³H-leucine for three h in a KRBH buffer containing BSA and 1.67 mM glucose (LG), 16.7 mM glucose (HG), 11.1 mM glucose (C), 11.1 mM glucose and 1 mM IBMX (IBMX) or 11.1 mM glucose and 1 mM DETAnonoate (DNO). Insulin was immunoprecipitated with guinea pig anti-insulin antibodies and protein A sepharose, and total proteins were precipitated with TCA. Incorporated radioactivity was quantified by liquid scintillation counting. Results are means ± SEM for 3 observations. * denotes p < 0.05 using Student’s t-test when comparing HG to LG and IBMX and DNO to C.