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

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. 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. 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

A novel crosstalk between Alk7 and cGMP signaling differentially regulates brown adipocyte function

Objective: Obesity is an enormous burden for patients and health systems world-wide. Brown adipose tissue dissipates energy in response to cold and has been shown to be metabolically active in human adults. The type I transforming growth factor β (TGFβ) receptor Activin receptor-like kinase 7 (Alk7) is highly expressed in adipose tissues and is down-regulated in obese patients. Here, we studied the function of Alk7 in brown adipocytes. Methods: Using pharmacological and genetic tools, Alk7 signaling pathway and its effects were studied in murine brown adipocytes. Brown adipocyte differentiation and activation was analyzed. Results: Alk7 is highly upregulated during differentiation of brown adipocytes. Interestingly, Alk7 expression is increased by cGMP/protein kinase G (PKG) signaling, which enhances brown adipocyte differentiation. Activin AB effectively activates Alk7 and SMAD3 signaling. Activation of Alk7 in brown preadipocytes suppresses the master adipogenic transcription factor PPARγ and differentiation. Stimulation of Alk7 during late differentiation of brown adipocytes reduces lipid content and adipogenic marker expression but enhances UCP1 expression. Conclusions: We found a so far unknown crosstalk between cGMP and Alk7 signaling pathways. Tight regulation of Alk7 is required for efficient differentiation of brown adipocytes. Alk7 has differential effects on adipogenic differentiation and the development of the thermogenic program in brown adipocytes

Direct lentivirus injection for fast and efficient gene transfer into brown and beige adipose tissue

Brown adipose tissue is a special type of fat contributing to energy expenditure in human newborns and adults. Moreover, subcutaneous white adipose tissue has a high capacity to adapt an energy-consuming, brown-like/beige phenotype. Here, we developed an easy to handle and fast to accomplish method to efficiently transfer genes into brown and beige fat pads in vivo. Lentiviral vectors are directly injected into the target fat pad of anaesthetized mice through a small incision using a modified, small needle connected to a microsyringe, which is well suited for infiltration of adipose tissues. Expression of the target gene can be detected in brown/beige fat one week after injection. The method can be applied within minutes to efficiently deliver transgenes into subcutaneous adipose tissues. Thus, this protocol allows for studying genes of interest in a timely manner in murine brown/beige fat and could potentially lead to new gene therapies for obesity. Brown adipose tissue is a special type of fat contributing to energy expenditure in human newborns and adults. Moreover, subcutaneous white adipose tissue has a high capacity to adapt an energy-consuming, brown-like/beige phenotype. Here, we developed an easy to handle and fast to accomplish method to efficiently transfer genes into brown and beige fat pads in vivo. Lentiviral vectors are directly injected into the target fat pad of anaesthetized mice through a small incision using a modified, small needle connected to a microsyringe, which is well suited for infiltration of adipose tissues. Expression of the target gene can be detected in brown/beige fat one week after injection. The method can be applied within minutes to efficiently deliver transgenes into subcutaneous adipose tissues. Thus, this protocol allows for studying genes of interest in a timely manner in murine brown/beige fat and could potentially lead to new gene therapies for obesity

The Gq signalling pathway inhibits brown and beige adipose tissue.

Brown adipose tissue (BAT) dissipates nutritional energy as heat via the uncoupling protein-1 (UCP1) and BAT activity correlates with leanness in human adults. Here we profile G protein-coupled receptors (GPCRs) in brown adipocytes to identify druggable regulators of BAT. Twenty-one per cent of the GPCRs link to the Gq family, and inhibition of Gq signalling enhances differentiation of human and murine brown adipocytes. In contrast, activation of Gq signalling abrogates brown adipogenesis. We further identify the endothelin/Ednra pathway as an autocrine activator of Gq signalling in brown adipocytes. Expression of a constitutively active Gq protein in mice reduces UCP1 expression in BAT, whole-body energy expenditure and the number of brown-like/beige cells in white adipose tissue (WAT). Furthermore, expression of Gq in human WAT inversely correlates with UCP1 expression. Thus, our data indicate that Gq signalling regulates brown/beige adipocytes and inhibition of Gq signalling may be a novel therapeutic approach to combat obesity