Obese Adipose Tissue Extracellular Vesicles Enhance Stemness of Colonic Stem Cells Dependent on Activation of Fatty Acid Oxidation

Abstract

Adipose tissue is the largest endocrine organ in the body, secreting hormones, cytokines, and extracellular vesicles (EVs). EVs are nanometer in size, can reach distant cells via the circulation, and carry molecular cargo that can cause phenotypic changes in the recipient cells. High fat diet (HFD)-induced obesity can enhance stemness of Lgr5+ crypt based columnar (CBC) stem cells, altering homeostasis of the crypt niche. However, modulation of Lgr5+ CBCs directly by EVs secreted from adipose tissue remains unknown. Here, we aim to characterize EV cargo from obese and lean adipose tissue and demonstrate the role of obese adipose-derived EVs in altering colonic CBC function. As a mouse model of obesity, C57BL6 (WT) mice were fed a purified HFD containing 45% kcal fat or a purified matched diet containing 10% kcal fat from 4-20 weeks of age. Visceral adipose tissue was cultured ex vivo and EVs were isolated by polyethylene glycol-based method or differential centrifugation. Additionally, human EVs were isolated from abdominal visceral fat from 5 obese patients (BMIs 40, 48, 60, 74, 96) undergoing bariatric surgery and 4 non-obese patients (BMIs 24.5, 24.7, 25, 25) undergoing foregut surgery. Mouse and human obese and non-obese adipose EVs were characterized by Nanosight analysis, transmission electron microscopy (TEM), and proteomics. Colon organoids (colonoids) were cultured from WT mice for 2 days and then treated with 10^7 or 10^8 obese or non-obese mouse EVs/ml every 2 days in exosome-free media for 8 days. To inhibit fatty acid oxidation (FAO), colonoids were treated with 25 mM Etomoxir (ETX) or vehicle concurrently with EVs. Using KEGG Pathway Database and Panther Protein Classification Software, 40% of EV proteins from mouse and human were associated with metabolism, including fatty acid (FA) metabolism, glycolysis, TCA cycle, and oxidative phosphorylation. The most abundantly expressed EV proteins associated with FA metabolism (FASN, FABP4, FABP5) and were specifically increased in obese adipose EVs from mouse and human; results were confirmed by western blotting. Colonoids treated with adipose tissue EVs derived from obese mice exhibited increased survival, growth, and persistence of stem/progenitor function, which were abolished by ETX. These results suggest that the contents of obese adipose EVs are poised to fuel FAO and to promote obesity-induced alterations in colonic CBCs.