Figure 5

Glucose and cAMP regulate transcriptional activation by AP-1 through induction of AP-1 component expression. A) Schematic representation of pAP-1-luc reporter. B) Min6 cells were transfected with pAP-1-luc (or control vector) and maintained at low glucose before stimulation with glucose (10 mM) and cpt-cAMP (0.2 mM) for 6 hours. C) Cells were transfected with AP-1 reporter (or control vector) and indicated quantity (in μg) of expression vector for c-fos and c-jun. D) pAP-1 reporter vector was co-transfected with either A-FOS (a c-FOS dominant negative form), empty vector (control) or A-C/EBP as additional control (dominant negative form of C/EBP, a transcription factor structurally related to c-FOS). Stimulations were performed as under B. E,F,G) Min6 cells cultured at low glucose were stimulated with high glucose (10 mM) and GLP-1 (10 nM) for indicated period of time. Nuclear extracts were analyzed by western (E). Specific binding of c-FOS and JUND to AP-1 sequence was measured in nuclear extracts with an ELISA-like assay (F,G). E, F) representative of two repeated experiments. ^#, p < 0.05 vs c-Jun alone (n = 3); *, p < 0.01 (n = 4), by Student T-test. Error bars: s.d.; ND: not determined.