UPSTREAM STIMULATORY FACTOR-II (USF2) ACTIVITY IS REQUIRED FOR GLUCOSE STIMULATION OF L-PYRUVATE KINASE PROMOTER ACTIVITY IN SINGLE LIVING ISLET BETA-CELLS

Elevated glucose concentrations stimulate L-pyruvate kinase (L-PK) gen e transcription in liver and islet beta-cells, A glucose response elem ent termed the L4 box (two noncanonical E-boxes located -165 and -154 base pairs upstream of the transcriptional start point) has previously been defined within the proximal promoter region of the gene, However , the identity of the trans acting factor(s) which binds to this site remains unclear, We have used photon counting digital imaging of firef ly luciferase activity to monitor promoter activity continuously in si ngle living islet beta and derived INS-1 cells, and to analyze the mol ecular basis of the regulation by glucose, L-PK promoter activity, nor malized to cytomegalovirus promoter activity using the distinct Renill a reniformis luciferase, was greater than or equal to 6-fold higher in cells cultured at 16 mM glucose or above compared with cells cultured at 3 mM glucose, Microinjection of antibodies against the ubiquitous transcription factor USF2 inhibited L-PK promoter activity in beta- an d INS-1 cells incubated at 30 mm glucose by 71-87%. Anti-USF2 antibodi es had a much smaller effect on promoter activity in INS-1 cells cultu red at 3 mM glucose, and on the activity of a modified promoter constr uct lacking an L4 box, These data support the view that glucose enhanc es L-PK gene transcription in beta-cells by modifying the transactivat ional capacity of USF2 bound to the upstream L4 box.