Abstract
Hepatic gluconeogenesis is tightly balanced by opposing stimulatory (glucagon) and inhibitory (insulin) signaling pathways. Hepatocyte growth factor (HGF) is a pleiotropic growth factor that mediates diverse biological processes. In this study, we investigated the effect of HGF and its family member, macrophage-stimulating factor (MSP), on hepatic gluconeogenesis in primary hepatocytes. HGF and MSP significantly repressed expression of the key hepatic gluconeogenic enzyme genes, phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (Glc-6-Pase) and reduced glucose production. HGF and MSP activated small heterodimer partner (SHP) gene promoter and induced SHP mRNA and protein levels, and the effect of HGF and MSP on SHP gene expression was demonstrated to be mediated via activation of the AMP-activated protein kinase (AMPK) signaling pathway. We demonstrated that upstream stimulatory factor-1 (USF-1) specifically mediated HGF effect on SHP gene expression, and inhibition of USF-1 by dominant negative USF-1 significantly abrogated HGF-mediated activation of the SHP promoter. Elucidation of the mechanism showed that USF-1 bound to E-box-1 in the SHP promoter, and HGF increased USF-1 DNA binding on the SHP promoter via AMPK and DNA-dependent protein kinase-mediated pathways. Adenoviral overexpression of USF-1 significantly repressed PEPCK and Glc-6-Pase gene expression and reduced glucose production. Knockdown of endogenous SHP expression significantly reversed this effect. Finally, knockdown of SHP or inhibition of AMPK signaling reversed the ability of HGF to suppress hepatocyte nuclear factor 4alpha-mediated up-regulation of PEPCK and Glc-6-Pase gene expression along with the HGF- and MSP-mediated suppression of gluconeogenesis. Overall, our results suggest a novel signaling pathway through HGF/AMPK/USF-1/SHP to inhibit hepatic gluconeogenesis.
Highlights
Glucose homeostasis is tightly regulated by a hormonal network in which glucagon, glucocorticoids, and insulin are the main agents [1, 2]
Using Primary Human Hepatocyte (PHH), we demonstrated that Hepatocyte growth factor (HGF) activated the AMPK signaling pathway rapidly, and AMPK inhibitor compound C abolished HGF-mediated induction of small heterodimer partner (SHP) mRNA expression (Fig. 3D)
Using transient transfection assay with pepck and Glc-6-Pase gene promoters, we demonstrated that HGF and Macrophage-stimulating protein (MSP) repressed cAMP/Dex-mediated increase in promoter transactivation, which was reversed upon blocking the AMPK signaling pathway or by using siSHP oligonucleotides (Fig. 7C)
Summary
Reagents and Plasmids—Recombinant HGF and MSP were from R & D Systems; wortmannin, H89, U0126, 8-bromocAMP, and dexamethasone were from Sigma; SB203580, SP600125, and compound C were from Calbiochem; and insulin (Norvolin R) was from Green Cross (Korea). Maintenance of cell lines and transient transfections were performed as described previously [22]. Cells were transfected with indicated reporter plasmids together with expression vectors encoding various transcription factors or treated with various chemicals. Semiquantitative and qPCR analysis in primary rat hepatocytes and PHHs were performed using primers for PEPCK, Glc-6-Pase, SHP, and -actin as described previously [5, 22]. Western Blot Analysis—Cell lysate preparation and Western blot analysis in primary rat hepatocytes and PHHs, using rabbit monoclonal AMPK␣, rabbit monoclonal phospho-AMPK␣ (Thr-172), rabbit polyclonal ACC, rabbit polyclonal phosphoACC (Ser-79), rabbit polyclonal USF-1 (C-20), rabbit polyclonal SHP (H-160), and -tubulin antibodies (Santa Cruz Biotechnology) were described previously [5, 22]. HepG2 cells were transfected with reporter plasmids, and treatments were performed as indicated.
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