Maintenance of our metabolic health is emerging as a major challenge of our time. The consequences of obesity include cardiovascular and metabolic disease (eg type 2 diabetes), as well as increased cancer, musculoskeletal (eg osteoarthritis), and infectious disease risk, notably corona virus mortality. Therapeutic approaches to body weight regulation have focused on appetite regulating pathways in the brain or nutrient absorption in the gut; with neither approach yielding adequate success. Therefore, new, effective and safe strategies are needed.

Our proposal investigates the function of relatively unknown enzyme, BUD23, in regulating body weight, body composition and energy metabolism. Genetic studies in humans have associated BUD23 and its partner TRMT112 to obesity and elevated circulating triglycerides, and our studies in mice suggest that the action of this factor may be particularly influential in adipose tissue and the liver, key sites regulating our response to food intake, fasting and obesity. BUD23 is an established and conserved regulator of ribosomal maturation, the component of the cell responsible for making proteins. Our work to date suggests that the impact of BUD23 on ribosome function is selective, and has a particularly strong influence over production and functioning of mitochondria (a critical site of energy metabolism within our cells). Indeed, we find that deletion of BUD23 specifically from adipose tissue in mice re-programmes the balance of energy storage and expenditure (use), and results in a significantly lower proportion of body fat. In this proposal, we will fully define the role of BUD23 in directing metabolism in key tissues to gain therapeutic insight and target processes, and delineate the fundamental mechanisms (from translation efficiency to mitochondrial function). This work is in line with MRC priorities in nutrition and obesity.

Specifically, we will investigate the phenotype of BUD23 in adipose and liver, and examine how BUD23 in these tissues regulates responses to high fat diet. We will explore links between BUD23 action and the cellular metabolic circadian clock, as our evidence to date reveals a striking loss of the typical change in energy substrate utilisation through circadian time when BUD23 is deleted in adipose. We will also investigate links between BUD23 and mitochondria dynamics and activity through proteomic and functional assessment in genetically targeted tissues. Finally, we will use ribosome profiling to identify the mechanism responsible for selective impacts on mRNA translation efficiency, which we think are encoded in mRNA sequences. We will integrate mRNA expression, mRNA translation, and steady-state protein abundance, using computational approaches to build an integrated model of how RNA biology affects energy metabolism, and how these processes may be therapeutically targeted.

Technical Abstract:
BUD23 as an important regulator of systemic metabolism, with selective deletion of BUD23 from adipose tissue driving a reprogramming of energy expenditure and storage with a resultant lean phenotype. We will now further characterise the AdipoCREBud23FL/FL mice, and extend these studies to include BAT-specific targeting (via Ucp1CRE) to delineate white and brown adipose contributions. This will be important in defining the broader impact of Bud23-dependent alteration in BAT activity and thermogenesis on systemic energy expenditure and adiposity. We also include a tamoxifen inducible hepatocyte-specific BUD23 null mouse line (via AlbcreERTBud23FL/FL). As the mechanism of action of BUD23 is through differential translation of mRNA species it is essential to take a global view of the tissue proteome. We did this for heart, and it was highly revealing, identifying a role in mitochondrial function. Here, we will characterise in detail mitochondrial number, structure and activity in each of our tissues of interest. We will also interrogate tissue metabolic activity using the Oroboros technology, which allows for high-resolution respirometry with monitoring of O2 consumption, ROS production (AmR), and mt-membrane potential in small tissue samples or isolated mitochondria. We will also use Seahorse technology to study intact cells in culture. We will investigate de-novo lipogenesis (DNL), and fatty acid content of adipose beds, and liver will be determined post-mortem.
In order to identify candidate mRNA targets, we shall apply ribosome profiling (ribo-seq) to generate quantitative, transcriptome-wide and high-resolution maps of the tissue translatomes across genotypes. Quantifying footprint vs. RNA abundances (RNA-seq data is always produced in parallel from the samples) allows calculating relative translation efficiencies transcriptome-wide.