Volume 272, Number 33, Issue of August 15, 1997 pp. 20636-20640
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Upstream Stimulatory Factor-2 (USF2) Activity Is Required for Glucose Stimulation of L-Pyruvate Kinase Promoter Activity in Single Living Islet -Cells

(Received for publication, March 5, 1997, and in revised form, June 3, 1997)

Helen J. Kennedy , Benoit Viollet , Imran Rafiq , Axel Kahn and Guy A. Rutter

From the Department of Biochemistry, School of Medical Sciences, University Walk, University of Bristol, Bristol BS8 1TD, United Kingdom and the  Institut Cochin de Génétique Moléculaire, U. 129 INSERM, Université René Descartes, 24, rue du Faubourg Saint-Jacques, 75 014 Paris, France

Elevated glucose concentrations stimulate L-pyruvate kinase (L-PK) gene transcription in liver and islet -cells. A glucose response element 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 transacting factor(s) which binds to this site remains unclear. We have used photon counting digital imaging of firefly luciferase activity to monitor promoter activity continuously in single living islet  and derived INS-1 cells, and to analyze the molecular basis of the regulation by glucose. L-PK promoter activity, normalized to cytomegalovirus promoter activity using the distinct Renilla reniformis luciferase, was 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 - and INS-1 cells incubated at 30 mM glucose by 71-87%. Anti-USF2 antibodies had a much smaller effect on promoter activity in INS-1 cells cultured at 3 mM glucose, and on the activity of a modified promoter construct lacking an L4 box. These data support the view that glucose enhances L-PK gene transcription in -cells by modifying the transactivational capacity of USF2 bound to the upstream L4 box.

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