Alk7 signaling and direct lentivirus injection as novel approaches to investigate brown fat development and function

Abstract

The obesity pandemic is increasing worldwide and is a major threat to human health. Comorbidities associated with obesity are type 2 diabetes, cardiovascular disorders, non-alcoholic fatty liver disease and even some types of cancer. Presently, we lack reliable and easy applicable medication without major side-effects to treat obesity. Recents studies have unvailed the existance of energy-consuming brown adipose tissue (BAT) in human adults. This tissue has therefore become the focus of research to develop novel anti-obesity therapies. <br /> The presented thesis depicts two novel approaches to investigate the development and function of brown adipocytes. The first approach identifies the type I TGFß receptor Activin receptor-like kinase 7 (Alk7) as a novel cyclic guanosin monophosphate (cGMP)-regulated target in brown adipocytes. cGMP is an important second messenger in adipocytes that activates PKGI to induce adipogenic and thermogenic differentiation. Interestingly, exogenous cGMP treatment of brown adipocytes increases Alk7 expression. Activin AB is a potent ligand activating Alk7 downstream-signaling in brown adipocytes, which is mediated by phosphorylation of SMAD3. Activation of Alk7 during terminal differentiation of brown adipocytes differentially regulates adipogenic and thermogenic protein expression. It induces downregulation of several adipogenic markers and upregulation of the major thermogenic marker UCP1. Importantly, this effect is augmented in cGMP-treated brown adipocytes. Alk7 could therefore serve as potential endogenous brake of the cGMP signaling pathway, avoiding overstimulation of the adipogenic program by cGMP and at the same time enhancing UCP1 expression to facilitate energy expenditure. <br /> To be able to analyze effects of the Alk7/cGMP signaling pathway as well as of other signaling pathways in BAT in vivo, the second part of this work describes the development and validation of an easy-to-handle and fast-to-accomplish method for direct lentiviral injections into subcutaneous adipose tissues. Lentiviral vectors are directly injected into the target fat pad of anesthetized mice through a small incision using a microsyringe connected to a modified, small needle, which is well suited for infiltration of adipose tissues. Expression of the target gene can be detected in the respective adipose tissue as early as one week after injection and is stable over months due to the use of lentiviral vectors, which integrate stably into the host genome. Delivery of transgenes into the fat pads of one mouse is carried out within minutes. The method therefore allows for studying genes of interest in murine brown/beige fat in a timely manner. Consequently, it could be of substantial interest for any researcher investigating signaling pathways in adipose tissues, potentially leading to new gene therapies for the treatment of obesity.