Sympathetic regulation of hepatic autophagy by CREB/CRTC-2

Abstract

Autophagy is one of the main processes of degradation of intracellular components, such as lipids (lipophagy), and essential for the maintenance of cellular homeostasis. In the liver, dysregulation of the autophagic process leads to the accumulation of hepatic triglycerides and the development of steatosis. It is known that the cAMP responsive element-binding protein (CREB) and its co-activator type 2 (CRTC-2) regulate the autophagy gene program. Recent studies from our laboratory have shown that noradrenergic innervation in the liver of mice is able to regulate the gene program for gluconeogenesis through the recruitment of CREB/CRTC-2. However, practically nothing is known about the neural regulation of hepatic autophagy and the signaling pathways involved. Therefore, the main objective of this project is to investigate the role of catecholamines (norepinephrine and adrenaline) in the control of liver autophagy and the participation of CREB/CRTC-2 in this response. For this, necessary hepatocytes from mice (C57 / Bl6) will be used to investigate the direct effect of catecholamines, in the presence or not of glucagon, no autophagic flow and expression of autophagic genes and proteins, as well as proteins of the signaling pathways of the cAMP and Ca2+. The importance of CREB/CRTC-2 will be assessed in hepatocytes transfected with adenovirus expressing the CREB negative dominant protein (ACREB) and CRTC-2 iRNA. In in vivo conditions, the autophagic flow will be evaluated in the liver of sympathectomized or adrenodemedicated mice exposed to specific models of noradrenergic sympathetic activation (cold) or of the adrenal (containment), respectively, as well as in liver of steatotic animals treated with phenylephrine (±1 adrenergic agonist) or clenbuterol (²2 adrenergic agonist). The transgenic mice LC3-GFP and CRE-luc will also be used to count LC3 punctas (autophagy indicator) and CREB transcriptional activity, respectively. An expression of the genes will be analyzed by RT-PCR and the protein content by western blot and immunofluorescence. These findings will be important for the sympathetic knowledge of the regulation of the hepatic autophagic process in stressful situations.