Background
The worldwide prevalence of obesity has markedly increased since 1980, with more than 609 million individuals classified as obese in 2015 [
1]. Obesity (defined as a body mass index (BMI) of 30 kg/m
2 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]. CVD (and indirectly, T2DM) is the leading cause of death and disability due to high BMI [
4]. The pathogenesis of obesity is not completely understood. A substantial number of behavioural and lifestyle factors influence susceptibility to obesity; however, the risk conferred by genetic and epigenetic traits is also significant with a heritability of 40–70% [
5]. Genome-wide association studies (GWAS) have identified a large number of loci robustly associated with obesity-related traits, such as BMI and body fat percentage (BF%), although the causal genes in these loci remain to be established and characterised.
One such locus identified in GWAS meta-analyses of BF% [
6,
7] is near the Sprouty RTK signalling antagonist 2 (
SPRY2) gene with no other genes within a 1 Mb window around the GWAS signal (Additional file
1: Figure S1). The lead variant (rs534870), 54 kb downstream of
SPRY2, exhibited a modest association with BMI, body weight and risk of obesity; importantly, its major allele was associated with a 0.14% decrease in BF% in individuals of European descent [
6]. In a larger follow-up study, the lead variant (rs693839, in high linkage equilibrium with rs534870,
R2 > 0.95) near
SPRY2 was similarly found to have a greater effect on BF% than BMI, suggesting a primary association with adiposity and body fat distribution rather than overall body weight. Additional experiments in
Drosophila supported
SPRY2 as the likely causal gene [
7]. Furthermore, several studies have implicated
SPRY2 as a potential candidate gene for T2DM. The rs1359790 variant [
8], situated 193 kb upstream to
SPRY2, was significantly associated with susceptibility to T2DM in Chinese [
9] and Japanese [
10] individuals. Sprouty proteins are negative regulators of receptor tyrosine kinase (RTK) signalling pathways [
11], which mediate a wide variety of key cellular processes, including critical roles in proliferation, communication and differentiation, as well as influences on motility, metabolism and survival [
12].
SPRY2 specifically modulates the Ras/mitogen activated protein (MAP) kinase pathway [
13,
14] and may function as a tumour suppressor gene, since its expression has been found to be repressed in a variety of cancers (reviewed in [
15]). Other examples of RTK families include vascular endothelial growth factors (VEGF), insulin-like growth factors (IGF), fibroblast growth-factors (FGF) and platelet-derived growth factors (PDGF).
In a recent study utilising whole-genome RNAi [
16],
SPRY2 was identified as a novel regulator of insulin transcription, and deletion of
SPRY2 in adult mouse β-cells led to mild hyperglycaemia and hypoinsulinaemia. However, based on the GWAS findings, there is reason to believe that
SPRY2 may also be involved in peripheral insulin resistance, metabolic syndrome or hepatosteatosis, rather than just insulin secretion. To our knowledge, no prior studies have explored the potential role of
SPRY2 in cells or tissues relevant to these conditions.
The liver is a central metabolic organ and plays a critical role in lipid metabolism and glucose homeostasis. Hence, we aimed to functionally characterise
SPRY2 in HepG2 cells, an in vitro model of human hepatocytes widely studied in the context of glucose and lipid metabolism and insulin resistance [
17‐
19]. We observed a marked increase in glucose uptake, along with an increase in lipid droplet accumulation in HepG2 cells after knockout of
SPRY2. Transcriptomic profiling revealed differentially expressed genes related to cholesterol biosynthesis, regulation of cell cycle and cellular signalling. These findings suggest a role for
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.
Discussion
To our knowledge, this is the first study to functionally characterise
SPRY2, a gene highlighted by GWAS for BF% [
6] and T2DM [
8], in human hepatocytes. Our results show increased glucose uptake and elevated lipid droplet accumulation to be the major functional consequences of
SPRY2 KO in these cells, as well as modifications to transcript levels of several genes and biological pathways relevant to the pathogenesis of metabolic diseases, such as obesity and T2DM.
The liver is a central metabolic organ and plays a critical role in lipid metabolism and glucose homeostasis. These processes can be dysregulated in obesity, leading to metabolic abnormalities that are associated with the development of NAFLD, insulin resistance and the pathogenesis of T2DM [
36]. HepG2 cells are widely used for the study of glucose and lipid metabolism, insulin signalling and mechanisms of insulin resistance in vitro [
17‐
19], and were therefore selected as a model of human hepatocytes in the present study.
The observed significant increase in glucose uptake following
SPRY2 KO in HepG2 cells suggests a possible role for
SPRY2 in glucose metabolism in hepatocytes. However, the mechanism by which this enhanced glucose uptake occurs is unclear; our RNA-Seq analysis revealed a nominal reduction in transcript levels of
SLC2A1 (GLUT1, the major glucose transporter in HepG2 cells [
37]) in
SPRY2 KO cells compared to mock.
Given the association of the rs534870 variant near
SPRY2 with BF% [
6], as well as its postulated specific effect on adiposity [
7], we hypothesised that
SPRY2 KO could affect lipid droplet accumulation in HepG2 cells. Indeed, there were significantly more lipid droplet in the
SPRY2 KO cells compared to mock; this may be a result of de novo fatty acid synthesis due to increased glucose uptake [
38]. Our RNA-Seq data showed that
SPRY2 KO led to an increase in transcript level of enzymes involved in lipogenesis, including acetyl-CoA carboxylase alpha (
ACACA), the rate-limiting step in long-chain fatty acid synthesis [
39], as well as fatty acid synthase (
FASN). Furthermore,
SPRY2 KO may enhance the glycolytic pathway that provides pyruvate for fatty acid synthesis: our RNA-Seq results showed decreased transcript levels of
GCKR, which negatively regulates hepatic glucokinase (GCK), a glycolytic enzyme that phosphorylates glucose to produce glucose-6-phosphate [
40], as well as a significant increase in transcript level of
SREBF1, a transcription factor that mediates insulin-stimulated upregulation of GCK [
41]. Therefore,
SPRY2 KO may affect glucose uptake and lipid droplet accumulation in part by modulating activation and expression of insulin-sensitive GCK enzymes, although these observations require validation in future studies.
A clear phenotype resulting from
SPRY2 OE in HepG2 cells did not emerge in the present study, with similar levels of glucose uptake and lipid droplet accumulation observed in
SPRY2 OE and mock cells. Gain- and loss-of-function studies of a target gene are generally expected to produce opposite phenotypes, however, this is not always observed [
42,
43], particularly with regard to tumour suppressor genes [
44], which may apply in the case of
SPRY2. Ideally, the
SPRY2 OE experiments would be repeated to further characterise the resultant phenotypes in HepG2 cells.
Further investigation into cellular signalling pathways via phospho-kinase profiling did not reveal any significant changes to protein kinase phosphorylation levels between HepG2 mock and
SPRY2 KO cells, despite the array encompassing a number of protein kinases relevant to glucose metabolism and insulin signalling. Transcriptome profiling via RNA-Seq identified a total of 421 DE genes between mock and
SPRY2 KO HepG2 cells (178 upregulated and 243 downregulated genes). The most significant DE gene,
PLA2G2A, was validated by RT-qPCR as significantly upregulated at the mRNA level following
SPRY2 KO.
PLA2G2A is highly expressed in liver and elevated circulating levels of its encoded protein, secretory phospholipase A2 group IIA (sPLA
2), are a risk factor for atherogenesis [
31]. Furthermore, sPLA
2 influences hepatic cholesterol uptake [
32] and improves insulin sensitivity in mice [
33]. Dipeptidyl peptidase 4 (
DPP4) mRNA level was validated as significantly decreased in the
SPRY2 KO2 cells. Elevated DPP4 expression has been linked to insulin resistance in obesity [
45] and NAFLD [
35], and DPP4 inhibitors are currently in clinical use as anti-diabetic drugs. Further studies will be required to explore the potential link between
SPRY2 and these genes in the context of obesity and T2DM, and in particular, the potential role of
SPRY2 in the pathogenesis of liver diseases, such as NAFLD.
Pathway analysis revealed many DE upregulated genes arising from
SPRY2 KO to be involved with DNA replication and cell cycle regulation; this is consistent with the established role for
SPRY2 in inhibiting cell proliferation and acting as a tumour suppressor in certain types of cancer [
14,
46]. Among the highly significant terms several were related to the metabolic processes that regulate cholesterol biosynthesis and fatty acid metabolism, which correlates with our experimental findings suggesting that
SPRY2 regulates metabolic processes, such as glucose uptake and lipid accumulation.
Collectively, the increase in glucose uptake and lipid droplet accumulation, as well as the modulation of transcript levels in
SPRY2 KO cells suggest that
SPRY2 may be involved in metabolic homeostasis in hepatocytes, although whether this contributes to the pathogenesis of obesity, T2DM or NAFLD remains to be determined using further in vitro experiments. However, it would be interesting to speculate that the changes in transcript levels of multiple genes associated with fatty acid synthesis and alterations to cholesterol synthesis pathways observed in the
SPRY2 KO cells could constitute a potential mechanism leading to increased lipid droplet accumulation in hepatocytes, and potentially, a contribution by
SPRY2 to conditions such as hepatic steatosis and NAFLD. Furthermore, the present study is unable to disentangle if the numerous DE cell cycle regulation genes identified in the RNA-Seq experiments are relevant to obesity and T2DM or may also be indicative of neoplastic changes, given that the loss of
SPRY2 is associated with hepatocarcinogenesis [
47‐
49] and that HepG2 cells themselves are a hepatoma cell line.
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