Regulation of hepatic lipogenesis by the transcription complex Prep1-Pbx1
Cabaro, Serena (2011) Regulation of hepatic lipogenesis by the transcription complex Prep1-Pbx1. [Tesi di dottorato] (Inedito)
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Prep1 is an homeodomain transcription factor belonging to the TALE proteins, including also Pbx1, which plays an essential role in hematopoiesis, organogenesis and development. Prep1 forms transcriptionally active complexes with Pbx1 and regulates the activity of several genes. The Prep1 null mutation leads to embryonic death at a very early stage. Therefore, Prep1 hypomorphic (Prep1i/i) mice have been generated. Prep1 heterozygous (Prep1i/+) mice, which express only 55-57% of protein, have a complex metabolic phenotype with at least two relevant features. One is the presence of smaller but otherwise normally structured islets with reduced fasting and post-loading plasma insulin levels. The second is increased insulin sensitivity in skeletal muscle and in liver which is accompanied by protection from streptozotocin-induced diabetes. In muscle, decreased Prep1 levels are followed by an increase of the PGC1alpha/Glut4-mediated glucose uptake. In liver, better insulin sensitivity is due to a reduced SHP1 tyrosine phosphatase expression followed by an increase of insulin signaling. Also, triglyceride levels are significantly reduced in the liver of Prep1i/+ mice. However, the molecular mechanism by which Prep1 controls lipogenesis is unclear. In this study, we have focused our attention on the role of Prep1 on the regulation of the triglyceride synthesis. To study the lipogenesis in the liver of the Prep1 heterozygous mice, we have examined the expression of the lipogenetic enzyme FAS (Fatty Acid Synthase) by real-time RT-PCR analysis. Hepatic expression of FAS is significantly decreased. Western Blot analysis have shown increased phosphorylation of PKCzeta, LKB1, AMPK and ACC, which may control FAS expression and triglycerides production in Prep1i/+ mice liver. Protein and mRNA levels of the lipid phosphatase SHIP2, an inhibitor of PI3Kinase/PKCzeta signaling, are reduced by 40% in the liver of Prep1i/+ mice. Consistent with these data, HepG2 (Human Hepatocarcinoma cell line) cells overexpressing Prep1 display increased triglyceride levels and FAS expression, while PKCzeta, LKB1, AMPK and ACC phosphorylation is strongly reduced. Moreover, SHIP2 levels are increased by 50%. Interestingly, overexpression of Pbx1 cDNA in HepG2 cells mimicked Prep1-induced triglyceride synthesis. At the opposite, Prep1HR1 mutant, which is unable to bind Pbx1, fails to elicit these effects. ChIP and Re-ChIP experiments indicate that Prep1/Pbx1 complex can bind SHIP2 promoter region and regulate its expression. These data suggest that Prep1/Pbx1 dimer regulates hepatic triglycerides production by increasing SHIP2 levels and thereby inhibiting the PKCzeta/AMPK signaling.
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