CRISPR-Cas9-mediated knockout of SPRY2 in human hepatocytes leads to increased glucose uptake and lipid droplet accumulation

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.

Differential and spatial expression meta-analysis of genes identified in genome-wide association studies of depression

“…SPRY2 was significantly associated with body fat percentage and type 2 diabetes mellitus in large genetic studies [67][68][69] . A knockout analysis of SPRY2 found a significant increase in glucose uptake and lipid droplet accumulation in an in vitro model of human hepatocyte cells 70 . This suggests that decreased expression of SPRY2 in human hepatocytes contributes to the pathogenesis of obesity and type 2 diabetes.…”

Differential and spatial expression meta-analysis of genes identified in genome-wide association studies of depression

“…SPRY2 was significantly associated with body fat percentage and type 2 diabetes mellitus in large genetic studies [64][65][66] . A knockout analysis of SPRY2 found a significant increase in glucose uptake and lipid droplet accumulation in an in vitro model of human hepatocyte cells 67 . This suggests that decreased expression of SPRY2 in human hepatocytes contributes to the pathogenesis of obesity and type 2 diabetes.…”

Harnessing the potential of CRISPR-based platforms to advance the field of hospital medicine

“…It is easy to foresee how gene-editing may be useful for polygenetic syndromes, including hypercholesterolemia, hypertension, and diabetes, among others [80,86,87]. For example, one group used CRISPR-Cas9 genome editing to target genes linked to insulin production, SPRY2, and the functional consequences of SPRY2 knockout and overexpression subsequently assessed using glucose uptake and lipid assays [88]. This work implicates a novel mechanism in the development of glucose intolerance and has profound implications for the treatment of diabetes, which is known to be an important risk factor for heart disease.…”