Abstract
BackgroundThe prevalence of obesity and its comorbidities, including type 2 diabetes mellitus (T2DM), is dramatically increasing throughout the world; however, the underlying aetiology is incompletely understood. Genome-wide association studies (GWAS) have identified hundreds of genec susceptibility loci for obesity and T2DM, although the causal genes and mechanisms are largely unknown. SPRY2 is a candidate gene identified in GWAS of body fat percentage and T2DM, and has recently been linked to insulin production in pancreatic β-cells. In the present study, we aimed to further understand SPRY2 via functional characterisation in HepG2 cells, an in vitro model of human hepatocytes widely used to investigate T2DM and insulin resistance.MethodsCRISPR-Cas9 genome editing was used to target SPRY2 in HepG2 cells, and the functional consequences of SPRY2 knockout (KO) and overexpression subsequently assessed using glucose uptake and lipid droplet assays, measurement of protein kinase phosphorylation and RNA sequencing.ResultsThe major functional consequence of SPRY2 KO was a significant increase in glucose uptake, along with elevated lipid droplet accumulation. These changes were attenuated, but not reversed, in cells overexpressing SPRY2. Phosphorylation of protein kinases across key signalling pathways (including Akt and mitogen activated protein kinases) was not altered after SPRY2 KO. Transcriptome profiling in SPRY2 KO and mock (control) cells revealed a number of differentially expressed genes related to cholesterol biosynthesis, cell cycle regulation and cellular signalling pathways. Phospholipase A2 group IIA (PLA2G2A) mRNA level was subsequently validated as significantly upregulated following SPRY2 KO, highlighting this as a potential mediator downstream of SPRY2.ConclusionThese findings suggest a role for SPRY2 in glucose and lipid metabolism in hepatocytes and contribute to clarifying the function of this gene in the context of metabolic diseases.
Highlights
The prevalence of obesity and its comorbidities, including type 2 diabetes mellitus (T2DM), is dramatically increasing throughout the world; the underlying aetiology is incompletely understood
Transcriptomic profiling revealed differentially expressed genes related to cholesterol biosynthesis, regulation of cell cycle and cellular signalling. These findings suggest a role for Sprouty RTK signalling antagonist 2 (SPRY2) in hepatocyte metabolism and provide further evidence that SPRY2 is the likely causal gene in a well-established locus associated with body fat distribution and T2DM
Confirmation of successful CRISPR-Cas9 genome editing For the SPRY2 KO studies, HepG2 cells were transduced with two distinct CRISPR-Cas9 Single guide RNAs (sgRNA)-lentiviral constructs to minimise the risk of off-target effects, which can compromise specificity [30]
Summary
The prevalence of obesity and its comorbidities, including type 2 diabetes mellitus (T2DM), is dramatically increasing throughout the world; the underlying aetiology is incompletely understood. Genome-wide association studies (GWAS) have identified hundreds of genec susceptibility loci for obesity and T2DM, the causal genes and mechanisms are largely unknown. Obesity (defined as a body mass index (BMI) of 30 kg/m2 or higher) is a risk factor for the development of serious chronic diseases, including type 2 diabetes mellitus (T2DM), cardiovascular disease (CVD), non-alcoholic fatty liver disease (NAFLD) and chronic kidney disease [2, 3]. Genomewide association studies (GWAS) have identified a large number of loci robustly associated with obesity-related traits, such as BMI and body fat percentage (BF%), the causal genes in these loci remain to be established and characterised
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