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Coregulation of Glucagon-Like Peptide-1 Synthesis with Proglucagon and Prohormone Convertase 1 Gene Expression in Enteroendocrine GLUTag Cells¹

Section on Cellular Neurobiology Laboratory of Developmental Neurobiology (S.D.), National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland; Departments of Physiology (A.I., P.L.B.) and Medicine (P.L.B.), University of Toronto, Toronto, Ontario, M5S 1A8, Canada; and Laboratory for Molecular Oncology (E.J.), Center for Human Genetics, University of Leuven and Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium

Address all correspondence and requests for reprints to: Dr. P. L. Brubaker, Room 3366, Medical Sciences Building, University of Toronto, Toronto, Ontario, M5S 1A8, Canada. E-mail: p.brubaker{at}utoronto.ca

The insulinotropic hormone glucagon-like peptide-1 (GLP-1) is synthesized in the intestinal L cell by prohormone convertase 1 (PC1)-mediated posttranslational processing of proglucagon. Previous studies have demonstrated that proglucagon gene transcription in the L cell is stimulated by the protein kinase A (PKA) pathway through a cAMP response element (CRE). Because the PC1 gene contains two functional CREs, the present studies were conducted to investigate whether the PC1 and proglucagon genes are coregulated by PKA, and to elucidate the temporal relationship(s) of PC1 and proglucagon gene expression with production of GLP-1, in the intestinal cell. The GLUTag enteroendocrine cell line, which is known to express the proglucagon gene and to synthesize and secrete GLP-1, was used as a model. Proglucagon and PC1 messenger RNA transcript levels were both increased after 12 h (but not 24 h) of treatment of GLUTag cells with forskolin/isobutylmethylxanthine (IBMX), by 2.7 ± 0.3- and 2.4 ± 0.3-fold, respectively, compared with controls (P < 0.01–0.001). Activation of PKA resulted in a 2.1 ± 0.1-fold increase in PC1 reporter construct expression (P < 0.001) at 12 h, which was dependent on the presence of the CRE, and a 13- to 24-fold increment in PC1 protein levels (P < 0.01) at 12 and 24 h. Similarly, forskolin/IBMX increased secretion of GLP-1, by 1.8 ± 0.2- and 2.2 ± 0.6-fold at 12 and 24 h, respectively (P < 0.05–0.01). Although the cell content of GLP-1 was diminished after 12 h of treatment (P < 0.001), GLP-1 levels increased back to control values after 24 h of forskolin/IBMX treatment (P < 0.01 vs. 12-h levels). Thus, PKA-induced secretion of GLP-1 from the L cell is followed by restoration of the cellular peptide levels through a PKA-mediated, CRE-dependent up-regulation of proglucagon and PC1 gene expression.

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