Factors involved in GLUT-1 glucose transporter gene transcription in cardiac muscle.

Glucose constitutes a major fuel for the heart, and high glucose uptake during fetal development is coincident with the highest level of expression of the glucose transporter GLUT-1 during life. We have previously reported that GLUT-1 is repressed perinatally in rat heart, and GLUT-4, which shows a low level of expression in the fetal stage, becomes the main glucose transporter in the adult. Here, we show that the perinatal expression of GLUT-1 and GLUT-4 glucose transporters in heart is controlled directly at the level of gene transcription. Transient transfection assays show that the -99/-33 fragment of the GLUT-1 gene is sufficient to drive transcriptional activity in rat neonatal cardiomyocytes. Electrophoretic mobility shift assays demonstrate that the transcription factor Sp1, a trans-activator of GLUT-1 promoter, binds to the -102/-82 region of GLUT-1 promoter during the fetal state but not during adulthood. Mutation of the Sp1 site in this region demonstrates that Sp1 is essential for maintaining a high transcriptional activity in cardiac myocytes. Sp1 is markedly down-regulated both in heart and in skeletal muscle during neonatal life, suggesting an active role for Sp1 in the regulation of GLUT-1 transcription. In all, these results indicate that the expression of GLUT-1 and GLUT-4 in heart during perinatal development is largely controlled at a transcriptional level by mechanisms that might be related to hyperplasia and that are independent from the signals that trigger cell hypertrophy in the developing heart. Furthermore, our results provide the first functional insight into the mechanisms regulating muscle GLUT-1 gene expression in a live animal.

Mechanism of action of pituitary adenylate cyclase-activating polypeptide on human glycoprotein hormone alpha-subunit transcription in alphaT3-1 gonadotropes.

Pituitary adenylate cyclase activating polypeptide (PACAP) has been shown to increase glycoprotein hormone alpha-subunit synthesis and release from pituitary cells. We have used alphaT3-1 clonal gonadotropes to investigate the intracellular mechanisms involved in PACAP regulation of alpha-subunit gene transcription; and using deletion, mutation, and heterologous constructs of the alpha-promoter linked to a luciferase reporter gene, we have defined DNA sequences responsive to PACAP. Stimulation of alphaT3-1 cells for 24 h with PACAP, GnRH, or vasoactive intestinal peptide (VIP) resulted in a time- and concentration-dependent increase in alpha-promoter transcription at 100 nM for GnRH (17.5-fold, P < 0.001), PACAP (12.7-fold, P < 0.01), and VIP (4.1-fold, P < 0.05). Incubation of alphaT3-1 cells in calcium-depleted medium suggested that the transcriptional response to PACAP was less dependent on changes in intracellular calcium concentration, in contrast to the results seen with GnRH or VIP, where alpha-subunit transcription was significantly reduced. Transfection of an alpha-promoter construct containing a mutant cAMP response element (CRE) suggested that the CRE region is involved in PACAP and VIP responsiveness, with stimulatory effects on the mutant construct by PACAP (11.1-fold) and VIP (7.6-fold) being significantly (P < 0.001) reduced, compared with their stimulatory effects (PACAP: 25.6-fold, VIP: 23.1-fold) on the native alpha-promoter. In the same experiment, the transcriptional response of the mutant CRE construct and the native CRE construct to GnRH was not significantly different. Both PACAP and VIP enhanced GnRH-stimulated alpha-subunit gene transcription, but this additive effect was lost when their combined effects on the mutant CRE were examined. Deletion analysis indicated that sequences between -244 and -195 bp were involved in mediating the response to PACAP, with a dramatic reduction in fold-stimulation by PACAP (2.0-fold) of the -195-bp construct, compared with the -244-bp construct (15.8-fold). Constructs containing only upstream alpha-promoter sequences from -517 bp to -98 bp, fused to the heterologous thymidine kinase promoter, exhibited a similar loss of responsiveness to PACAP below -298 bp. Thus, our studies show that, unlike GnRH, PACAP stimulation of alpha-subunit gene transcription in alphaT3-1 cells is less dependent on changes in intracellular calcium concentration; and full transcriptional activation of the alpha-subunit by PACAP requires an intact CRE. PACAP responsiveness involves sequences between -244 and -195 bp of the alpha-promoter. These sequences have been implicated also in GnRH-responsiveness and may thus provide a mechanism for coordinated regulation of the alpha-subunit gene by PACAP and GnRH in alphaT3-1 cells.