An edible fungi Pleurotus ostreatus inhibits adipogenesis via suppressing expression of PPAR γ and C/EBP α in 3T3-L1 cells: In vitro validation of gene knock out of RNAs in PPAR γ using CRISPR spcas9

Biomed Pharmacother. 2019 Aug;116:109030. doi: 10.1016/j.biopha.2019.109030. Epub 2019 May 29.

Abstract

Objective: Obesity is now well recognized as a disorder, one that is essentially preventable through changes in lifestyle. Obesity is also a main concern associated with expanded morbidity and mortality from many noncommunicable illnesses (NCDs). The study aimed to determine the antiobesity effect of Pleurotus ostreatus (PO) and its bioactive anthraquinone (AQ). The overall promoter genes CEBPα (CCAAT enhancer binding protein α) and PPARγ (Peroxisome proliferator activated receptor γ) in controlling the homeostasis of glucose was analysed using 3T3-L1 cell line. Finally, an insilico study was carried out using CRISPR software to identify the RNA's involved in adipogenesis especially of the control gene PPARγ.

Materials and methods: Preliminary screening of the edible fungi and their bio actives led to the marvellous discovery of side effect free agonists for treating obesity (adipogenesis). An edible fungi Pleurotus ostreatus (PO) were analysed in a screening platform with different series of tests for adipocyte differentiation, triglyceride analysis, lipolysis determination, glucose uptake assay, cytotoxicity assay and lipase activity followed by specific gene expression analysis. The gene knockout mechanism was also elucidated by CRISPR spcas 9 tool.

Results: The antiadipogenic (antiobesity) activity of DMSO extract of PO were found to stimulate the insulin dependent uptake of glucose. The extract also decreased the levels of triglycerides and glycerol accumulation in differentiated adipocyte cells. The binding FABP4 (Fatty acid binding protein) and transport protein FATP1 (Fatty acid transport protein) along with the fat breaking LPL (lipoprotein lipase) was found to be inhibited after the PO treatment at varying concentration (0-300 μg/ml). CRISPR spcas9 genome editing software was used as an insilico approach in validating the efficiency of mouse embryonic and human adipogenic cell line (3T3-L1). These tool analysed and found 4 RNAs gene knock out possibilities in PPARγ and their efficiency for further treating obesity.

Conclusion: These novel finding contribute to the confirmation that edible fungi PO and it's bioactive AQ is an adequate supplement for constraining the lipid and triglycerides in differentiated mature adipocytes by reversing the fat deposition. Thereby, forbidding the enzymes linked with fat absorption. Besides, the CRISPR tool identified gene knock out possibilities of control gene PPARγ, will pave a way in further research for treating obesity.

Keywords: 3T3-L1; Adipogenesis; Anthraquinone; CRISPR/Cas9; PPARγ; Pleurotus ostreatus; SREBP-1c.

MeSH terms

  • 3T3-L1 Cells
  • Adipogenesis*
  • Animals
  • Base Sequence
  • CCAAT-Enhancer-Binding Protein-alpha / metabolism*
  • CRISPR-Associated Protein 9 / metabolism*
  • CRISPR-Cas Systems / genetics*
  • Cell Differentiation
  • Gene Knockout Techniques*
  • Humans
  • Mice
  • PPAR gamma / metabolism*
  • Pleurotus / chemistry*
  • RNA / metabolism*

Substances

  • CCAAT-Enhancer-Binding Protein-alpha
  • PPAR gamma
  • RNA
  • CRISPR-Associated Protein 9