Bioactive peptides from human brown fat cells mediating metabolic control

Project Information

BALDER

Grant agreement ID: 101002725

DOI

10.3030/101002725

Project closed

EC signature date 8 April 2021

Start date 1 May 2021

End date 30 April 2026

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 2 000 000,00

EU contribution

€ 2 000 000,00

Coordinated by

KOBENHAVNS UNIVERSITET
Denmark

Periodic Reporting for period 3 - BALDER (Bioactive peptides from human brown fat cells mediating metabolic control)

Reporting period: 2024-05-01 to 2025-10-31

Summary of the context and overall objectives of the project

Obesity doubles the risk of dying from a cardiovascular-related event and affects about 1 in 3 people in the Western world. Despite recent progress in developing anti-obesity drugs, these medications present side-effects and are not equally efficient nor easily accessible for all. Increasing the spectrum of drugs for personalized treatment strategies is therefore warranted.

The central nervous system (CNS) plays a major role in obesity by regulating appetite based on feedback systems from peripheral organs, including adipose tissue. The neuronal connections between adipose tissue and the CNS include sympathetic motor neurons as well as sensory neurons in the adipose tissue and a bidirectional interconnection between white and brown adipose tissue. White adipose tissue also communicates its energetic state to the CNS via adipokines e.g. Leptin, while mutations in this gene result in severe obesity, which can be cured by supplying recombinant Leptin protein. Less is known about endocrine feedback mechanism arising from brown adipose tissue (BAT), whereas the concept of so-called batokines is increasingly recognized. BAT is a heat-producing, energy-consuming subtype of adipose tissue. Importantly, people with a retained ability to activate their BAT are less prone to develop diabetes and other cardiometabolic diseases even when living with obesity. Exploring the BAT-brain axis therefore holds great promise in identifying novel drug candidates for counteracting the development of cardiometabolic diseases, which would be a valuable health gain for society world-wide.

In conclusion, we investigate the endocrine BAT-brain axis to delineate BAT’s role in the neuroendocrine regulation of feeding behavior. Our overall hypothesis is that the energetic state of BAT is communicated to the CNS, via batokines, to mediate appetite control (Figure 1). The overall objective is to discover novel appetite-regulating circuits controlled by batokines via the crosstalk between BAT and CNS. We are interested in signaling molecules from BAT that act in an endocrine fashion, or act in a paracrine fashion on nerve ends, in turn affecting dorsla root ganglia and modulating the innervation of BAT. Among batokines candidate molecules, we primarily focus on peptides and small proteins. Peptides have previously proven to be powerful in mediating endocrine crosstalk and our strategy allows for the discovery of unknown molecules.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

We have performed in-depth analyses of human brown and white adipocytes resulting in the discovery of a new cellular subtype, the “SWAT cell” differentiating in parallel with adipocytes, from the same progenitor. This high-resolution data is key for interpreting our future work as SWAT cells are secreting the double number of factors compared to adipocytes and therefore have the potential to be highly involved in neuronal crosstalk. These findings are published in Nature Metabolism.

We have established a valuable collaboration with Dr Søren Nielsen at Rigshospitalet who has expertise within long read sequencing. This collaboration has accelerated our work on predicting microproteins from small open reading frames. In collaboration with the Nielsen group, we are establishing Ribo-seq. Integrating data from ribo-seq, long read sequencing and mass-spectrometry peptidomics is at the core of this project and we have already identified overlaps between mass-spectrometry and long read sequencing, which is used to predict bioactive microproteins.

We have used an in-house developed and published machine learning based prediction tool to shortlist our human brown adipocyte (including both adipogenic and SWAT cells) peptidomics data for different bioactivity classes. We found hundreds of peptides with a high prediction score for neuropeptides. To date, we have tested around 150 peptides in different cell systems using high content screening. We have optimized the methodology for RNAscope based detection of neuronal specificity and activity using automized quantification and are further establishing live cell imaging and microelectrode arrays for monitoring the effects of candidate molecules. Neuropeptides signal both centrally and peripherally and our in vitro models include hypothalamic neurons, sensory neurons, pancreatic alpha cells, hepatocytes and cardiomyocytes. We have a shortlist of bioactive peptides which we are currently investigating in depth and have initial in vivo data on one of these peptides.

I collaboration with Dr Thomas Frimurer at University of Copenhagen, we have developed an approach to use Alphafold to predict the receptors that our candidate peptides bind to and are currently validating these peptide-receptor pairs in vitro.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Through our high-resolution investigation of human brown and white adipocytes, we discovered a novel cell type, the SWAT cell, which is highly relevant in terms of studying adipocyte secretomes. Our findings are published in Nature Metabolism and was further supported by a back-to back publication from Silvia Corvera’s lab at UMASS. Corvera obtained supporting and complementary findings demonstrating that SWAT cells are held in a multipotent state by Wnt signaling. We have furthermore characterized inguinal adipose tissue in humans. This work has revealed novel insights into how thermogenic adipose tissue is suppressed in humans and couples back to our findings of SWAT cells and Wnt signaling.

Using a machine-learning based tool for predicting peptide bioactivity classes, and long-read sequencing and prediction of microproteins, we have identified hundreds of peptides with sequence similarities to neuropeptides and thus likely to signal in a neuronal context, supporting our initial hypothesis of an existing crosstalk.
These peptides are now being tested in our neuronal platform for high content screening and we have so far had great progress in identifying promising candidates in neurite outgrowth and acute neuronal activation or deactivation.

We have initial data on a small candidate protein being secreted from brown adipocytes and playing a key role in neuronal differentiation. We are currently designing in vivo and in vitro models to assess this crosstalk further.
By the end of the project, we expect to have identified several peptides forming a bidirectional BAT-neuro axis. From a larger perspective, this project has the potential of providing deeper insights into which mechanisms promote a healthy regulation of human metabolism.

Figure 1