New paradigm of GPCR signalling at intracellular sites in metabolic diseases

Aims: To investigate the involvement of GPCR signalling at intracellular sites in the regulation of adipocyte metabolism and its potential exploitation for the pharmacological therapy of metabolic diseases.

Background: Complex metabolic diseases are a major cause of morbidity and mortality, mainly due to cardiovascular complications. Adipose tissue functions as energy store and is a major regulator of energy expenditure, food intake and insulin sensitivity. Thus, a better knowledge of the mechanisms implicated in adipocyte regulation will be crucial to develop innovative therapies for metabolic diseases. G-proteincoupled receptors (GPCRs) play fundamental roles in the control of adipocyte metabolism1. Recent studies by our and other groups have revealed that GPCRs signal not only at the cell surface, as previously thought, but also at intracellular sites, such as early endosomes or the Golgi/trans-Golgi network2-5. This new concept is revolutionizing our understanding of GPCR biology and might provide new strategies to treat diabetes, obesity and other complex diseases.

Hypothesis: the central hypothesis of this project is that GPCR signalling at intracellular sites plays an important role in the regulation of adipocyte metabolism and that its pharmacological modulation might be used to elicit specific therapeutic effects.

Experimental methods: Experiments will be performed in the murine 3T3-L1 adipocyte cell line as well as in primary mouse adipocytes (WAT/BAT) differentiated in vitro7-9. Advanced microscopy methods that are well established in our labs, such as fluorescence resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS) and single-molecule microscopy, will be used to investigate the kinetics and subcellular location of GPCR signaling10-12. In particular, we will use FRET sensors tethered to different subcellular compartments (plasma membrane, cytosol, Golgi/TGN, mitochondria, nucleus)5. In addition, we will use primary adipocytes isolated from transgenic mice expressing of a cAMP FRET sensor2. Receptor internalization will be inhibited pharmacologically (dynasore, Dyngo-4a) or with a dominant-negative dynamin mutant (K44A). Fluorescent markers will be employed to identify the subcellular compartments where GPCR signalling is occurring5. As a complementary approach, we will use cell fractionation and standard assays to measure ligand binding, G-protein activation or enzymatic activity (e.g. adenylyl cyclase) in subcellular fractions (plasma embrane, cytosol, mitochondria, etc.). Energy and metabolic pathway usage (e.g. lipogenesis, lipolysis) will be assessed using a range of analytical methods including high-resolution mitochondrial respiration (Oroboros), seahorse technology, targeted lipidomics and metabolic tracing.

Research plan: We will initially focus on the long-chain free fatty acid receptor GPR120 and the hydroxycarboxylic acid receptor 2 (HCA2), which have antilipolytic effects and are emerging pharmacological targets for metabolic diseases1. First, we will investigate the kinetics and subcellular location of the Gi/o and Gq responses induced by receptor activation in 3T3-L1 cells and mouse primary adipocytes. Subsequently, we will evaluate the effect of inhibiting receptor internalization on the kinetics and spatial dynamics of these events. In parallel, we will perform trafficking experiments in living cells, combined with superresolution microscopy (dSTORM) in fixed cells to study the subcellular localization
and trafficking of receptors, G-proteins and effectors in adipose cells. In addition, we will evaluate the contribution of cell surface vs. intracellular receptor signalling on adipocyte metabolic parameters, including lipolysis and oxygen consumption. Finally, we will compare a panel of new synthetic receptor agonists in their ability to induce signalling at intracellular sites as well as to modulate adipocyte metabolism.