Obesity increases the risk for diabetes and cardiovascular disease. Genetic predisposition exacerbates environmental drivers of obesity such as energy dense diets and sedentary lifestyle. We have exploited divergently selected Fat (23% fat as bodyweight) and Lean (4% fat as bodyweight) lines of mice originating from a common base population to identify genes underlying divergent adiposity. A stratified approach using quantitative trait loci (QTL; heritable genetic intervals segregating with adiposity in Fat x Lean F2 populations), metabolic tissue transcriptomics and comparative cross-species bioinformatics identified candidate obesity and leanness genes in adipose tissue (Morton et al., 2011, 2016). Using a similar approach, we have identified novel muscle-expressed genes that segregate with adiposity. A specific phospholipase A2 isoform (we name here PlaX), positioned in the Found in obesity (Fob)-1 QTL, exhibited ~5-fold elevated mRNA levels in the skeletal muscle of Fat mice compared to Lean mice. PlaX activity has been previously linked to the regulation of intracellular membrane vesicle trafficking and generation of lipid signalling mediators (Cervera et al., 2021). Overexpression of PlaX in C2C12 myotubes impaired cellular energetics, glucose transport and increased levels of the active form of AMP-activated protein kinase (AMPK). This led us to hypothesise that skeletal muscle PlaX-overexpression may drive obesity by compromising myocyte energetics and nutrient utilisation. To test this hypothesis, we have generated global PlaX knockout transgenic mice. Our aim in this project was to characterise the skeletal muscle role of PlaX. To achieve this, we performed RNA-sequencing on samples taken from the extensor digitorum longus (EDL) muscle on the Illumina NextSeq 2000 platform. Differential gene expression analysis and functional enrichment analysis was carried out on samples from five wild type (WT) mice and five gene knock-out mice (KO), which lack a functional PlaX gene. Reactome pathway and Gene Ontology (GO) enrichment analyses were performed using the differentially expressed genes identified at the adjusted statistical threshold (FDR-adjusted (adj.) p<0.05). A threshold of adj.p<0.05 was used to define significant differentially expressed genes. 339 genes were differentially expressed between KO and WT (adj.p<0.05). As expected, RNA-sequencing revealed PlaX as the most significantly downregulated gene with a -1.82 fold change (adj.p=2.92e-5) in KO vs WT.  Among the most significantly differentially expressed genes were Per3, which was downregulated in KO vs WT (-1.5 fold change, adj.p=0.012), while Pdk4 was upregulated (2.2 fold change, adj.p=0.022). Both Per3 (Azevedo et al., 2021) and Pdk4 (Jeon et al., 2021)  are metabolism-linked genes that have been associated with obesity.

These differentially expressed genes indicate a metabolic response to PlaX loss-of-function in skeletal muscle and support the role of PlaX as a candidate obesity target. To further characterise the role of PlaX in skeletal muscle, we have overexpressed PlaX in L6 skeletal muscle cell line using lentiviral transduction and are currently performing RT-qPCR to measure expression of targeted genes from our RNA-sequencing data. In summary, our genetic strategy has identified a novel potential skeletal muscle driver of obesity that could be a tractable target for therapeutic development.