High glucose via NOX-dependent ROS generation and AKT activity promotes adipose-derived stem cell de-differentiation

Abstract

NADPH-oxidase (NOX)-dependent reactive oxygen species (ROS) production is involved in self-renewal of stem and progenitor cells. Herein, we investigated whether high glucose (25 mM/L) (HG)-dependent NOX-mediated ROS generation is involved in self-renewal of visceral adipose tissue-derived stem cells (ASCs) as well. To this end ASCs cultured in HG or normal glucose (5 mM/L) used as control, were evaluated for their stem cell identity. We demonstrated that freshly isolated ASCs are pluripotent as they differentiate into adipocytes in-vitro and form neovessels in-vivo. However, only HG-cultured ASCs expressed octamer-binding transcription factor 4 (Oct4) and Nanog and formed spheroids. The assembly of p47phox and p67phox subunits is crucial for NOX-enzymatic activity. By knock-down of p47phox the role of NOX-generated ROS in driving ASC de-differentiation has been provided. siRNA technology was also applied to demonstrate the role of Akt activity in mediating HG-induced Oct-4 and Nanog expression as well as spheroid formation. Additionally, by knock-down of Oct4 we provided further evidence that Oct4 is essential for HG-mediated stem cell identity. Soluble factors released by ASCs are key elements in their mechanism of action. We found that NOX and Akt activity are required for cytokine production by "spheroids". Finally, as HG-cultured ASCs, diabetic patient-derived ASCs expressed higher levels of Oct-4 and Nanog than ASCs derived from healthy subjects and engaged ROS and Akt activity to turn on their secretion program. Thereby, our data indicate that HG via NOX-dependent Akt activity induces ASC de-differentiation, and suggest that HG pre-conditioning might be exploited for ASC ex-vivo expansion.