Endocrine Abstracts

Background/aim: Patients with germline mutations in the tumor suppressor PTEN frequently develop single or multiple lipomas. PTEN antagonizes the phosphatidylinositide3-kinase/AKT/mechanistic target of rapamycin (PI3K/AKT/mTOR) pathway, which promotes cell proliferation and is involved in adipocyte differentiation. The aim of this study was to investigate the mechanisms leading to aberrant adipose tissue growth using PTEN knock-down cell models.

Methods: Primary cells of the stromal vascular fraction (SVF) from human fat biopsies were transfected with siRNA against PTEN after 40 or more days in culture and compared to scramble siRNA transfected cells. Additionally the PTEN gene was mutated in SVF cells using the CRISPR/Cas9 system.

Results: PTEN was transiently down regulated in SVF cells via siRNA to 65±5% resulting in an elevated AKT phosphorylation (7.7±5.1 fold) compared to control cells. Lipid accumulation was 1.4±0.2 fold higher in differentiated PTEN knock-down cells compared to controls as measured by Oil-Red O staining. PPAR gamma expression increased 1.7±0.1 fold. Cell count was increased 2.3±0.3 fold in PTEN knock-down cells after 7 days. An elevated AKT phosphorylation as well as increased lipid accumulation (2.5±0.3 fold, measured via Nile Red staining) and cell count (1.7±0.2 fold) were also observed for PTEN CRISPR cells.

Conclusion: Primary human preadipocytes lose their ability to differentiate into adipocytes after several weeks in culture. Their differentiation capacity could be partly recovered with reduction in PTEN levels. An enhanced proliferation of these cells corresponds with the enhanced activation of AKT.