PMID- 31664995 OWN - NLM STAT- MEDLINE DCOM- 20200403 LR - 20200403 IS - 1472-6823 (Electronic) IS - 1472-6823 (Linking) VI - 19 IP - 1 DP - 2019 Oct 29 TI - CRISPR-Cas9-mediated knockout of SPRY2 in human hepatocytes leads to increased glucose uptake and lipid droplet accumulation. PG - 115 LID - 10.1186/s12902-019-0442-8 [doi] LID - 115 AB - BACKGROUND: The 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 beta-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. METHODS: CRISPR-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. RESULTS: The 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. CONCLUSION: These 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. FAU - Cook, Naomi L AU - Cook NL AD - Molecular Epidemiology and Science for Life Laboratory, Department of Medical Sciences, Uppsala University, Uppsala, Sweden. FAU - Pjanic, Milos AU - Pjanic M AD - Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA, USA. FAU - Emmerich, Andrew G AU - Emmerich AG AD - Molecular Systems Biology, Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden. FAU - Rao, Abhiram S AU - Rao AS AD - Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA, USA. AD - Department of Bioengineering, Stanford University, Stanford, CA, USA. FAU - Hetty, Susanne AU - Hetty S AD - Molecular Epidemiology and Science for Life Laboratory, Department of Medical Sciences, Uppsala University, Uppsala, Sweden. FAU - Knowles, Joshua W AU - Knowles JW AD - Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA, USA. AD - Stanford Cardiovascular Institute, Stanford University, Stanford, CA, USA. AD - Stanford Diabetes Research Center, Stanford University, Stanford, CA, USA. FAU - Quertermous, Thomas AU - Quertermous T AD - Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA, USA. AD - Stanford Cardiovascular Institute, Stanford University, Stanford, CA, USA. AD - Stanford Diabetes Research Center, Stanford University, Stanford, CA, USA. FAU - Castillejo-Lopez, Casimiro AU - Castillejo-Lopez C AD - Molecular Epidemiology and Science for Life Laboratory, Department of Medical Sciences, Uppsala University, Uppsala, Sweden. AD - Department of Immunology, Genetics and Pathology and Science for Life Laboratory, Uppsala University, Uppsala, Sweden. FAU - Ingelsson, Erik AU - Ingelsson E AUID- ORCID: 0000-0003-2256-6972 AD - Molecular Epidemiology and Science for Life Laboratory, Department of Medical Sciences, Uppsala University, Uppsala, Sweden. eriking@stanford.edu. AD - Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA, USA. eriking@stanford.edu. AD - Stanford Cardiovascular Institute, Stanford University, Stanford, CA, USA. eriking@stanford.edu. AD - Stanford Diabetes Research Center, Stanford University, Stanford, CA, USA. eriking@stanford.edu. LA - eng GR - 20140422/Hjart-Lungfonden/ GR - R01 DK120565/DK/NIDDK NIH HHS/United States GR - R01 DK106236-01A1/NH/NIH HHS/United States GR - 2013.0126/Knut och Alice Wallenbergs Stiftelse/ GR - R01DK107437/NH/NIH HHS/United States GR - R01 DK106236/DK/NIDDK NIH HHS/United States GR - P30 DK116074/DK/NIDDK NIH HHS/United States GR - 2015-02907/Vetenskapsradet/ PT - Journal Article DEP - 20191029 PL - England TA - BMC Endocr Disord JT - BMC endocrine disorders JID - 101088676 RN - 0 (Intracellular Signaling Peptides and Proteins) RN - 0 (Membrane Proteins) RN - 0 (SPRY2 protein, human) RN - IY9XDZ35W2 (Glucose) SB - IM MH - *CRISPR-Cas Systems MH - Gene Expression Profiling MH - Glucose/*metabolism MH - Hep G2 Cells MH - Hepatocytes/cytology/*metabolism MH - Humans MH - Intracellular Signaling Peptides and Proteins/*antagonists & inhibitors/genetics/*metabolism MH - Lipid Droplets/*metabolism MH - *Lipogenesis MH - Membrane Proteins/*antagonists & inhibitors/genetics/*metabolism MH - Phosphorylation MH - Signal Transduction PMC - PMC6820957 OTO - NOTNLM OT - CRISPR-Cas9 OT - Glucose metabolism OT - Hepatocytes OT - Lipid metabolism OT - Obesity OT - SPRY2 OT - Type 2 diabetes mellitus COIS- The authors declare that they have no competing interests. EDAT- 2019/10/31 06:00 MHDA- 2020/04/04 06:00 PMCR- 2019/10/29 CRDT- 2019/10/31 06:00 PHST- 2019/08/01 00:00 [received] PHST- 2019/10/10 00:00 [accepted] PHST- 2019/10/31 06:00 [entrez] PHST- 2019/10/31 06:00 [pubmed] PHST- 2020/04/04 06:00 [medline] PHST- 2019/10/29 00:00 [pmc-release] AID - 10.1186/s12902-019-0442-8 [pii] AID - 442 [pii] AID - 10.1186/s12902-019-0442-8 [doi] PST - epublish SO - BMC Endocr Disord. 2019 Oct 29;19(1):115. doi: 10.1186/s12902-019-0442-8.