Dopamine Receptors in Thermogenic Adipose Tissue - Research Stay at the Univeristy of Vienna
Francesca-Maria Raffaelli, University of Lübeck
This research stay is dedicated to help evaluate particular aspects regarding the doctoral project "Crosstalk of dopamine and thyroid hormone signalling in the control of thermogenic adipose tissue".
Previous observations and in vitro findings strongly suggest a role of dopamine in activating brown adipose tissue (BAT) thermogenesis. Therefore, we have designed a translational approach to investigate the role of dopamine, as well as D1- and D2- like agonists on murine and human BAT ex vivo and in vivo.
With our collaboration partners in Vienna, we will analyse in detail the presence of D1- and D2- like dopamine receptors (DRD1 and DRD2) in murine BAT.
The collaboration partner provides essential expertise, biological material and technical support for this project's progress. We are aiming to identify dopamine receptors by employing fractionated protein isolation, Weston blot analysis, Immunoprecipitation and LC-MS analysis. All analyses are controlled for by using DRD1 and DRD2 knock-out samples, provided by the collaboration partner.
Furthermore, we are aiming to identify whether DRD1 and DRD2 receptors - if present in BAT - are in a glycosylated or non-glycosylated state.
This collaboration will ultimately result in a joint publication of the accomplished results.
My primary research aim in Vienna was to elucidate whether or not dopamine receptors (DRDs) are present in adipose tissues. The collaboration partner provides essential expertise, biological material (knockout tissue) and technical support for this project's progress. We were aiming to identify dopamine receptors by employing fractionated protein isolation, Western blot and LCMS analyses. The experiment was controlled for by using DRD1 and DRD2 knockout samples, provided by the collaboration partner.
During this stay I had the opportunity to learn and apply an optimized protocol for membrane protein isolation and sample preparation for Western blotting. Furthermore, I was able to prepare membrane and cytosolic sample fractions for LCMS by performing protein solubilisation, filter aided proteolytic digestion and solid phase extraction, followed by hands on practice at the LCMS system and analysis software.
Additionally, multiple peptide sub-fractions of each tissue of interest were prepared by high pH reversed-phase peptide fractionation in order to aid answering our question regarding low abundant receptor proteins. All samples are currently queued to be run in the near future.
The results will not only clarify whether dopamine D1- and D2-like receptors are present on murine brown adipocytes, but also whether the receptors are in a glycosylated (active) state or not. Consequently, these analyses together with ex- and in vivo studies conducted at the home University of Lübeck, will reveal whether effects of dopamine on BAT thermogenesis are direct or indirect.
Grant awarded: €2,090