(72a) Fat Cell-Derived Nanocarriers Transfer Inflammatory Cargo to Distant Organs

Problem: Obesity is a global health challenge, with over half the population expected to be overweight or obese by 2035. Obesity leads to various complications, ranging from mechanical to metabolic complications. Recently, weight-loss therapeutics demonstrated the pivotal role of decreased weight in alleviating various obesity-associated comorbidities, including type II diabetes, fatty liver disease, and cardiovascular disease, among many others actively undergoing clinical trials. Therefore, understanding the physiological differences that manifest with increased fat tissue is of utmost importance. A key cell type in fat tissue is the adipocyte, also referred to as a fat cell. Although the primary function of the adipocyte is to serve as a metabolic cell that stores excess lipids, adipocytes have recently been implicated in inflammatory processes. Adipocytes can perform innate immune functions by secreting adipokines that recruit inflammatory cells, while also participating in antigen presentation and influencing the adaptive immune system. However, the method by which the adipocyte influences distant organs remains a mystery. Therefore, we propose that cell-derived nanocarriers, known as extracellular vesicles (EVs) that contain bioactive cargo, are responsible for trafficking inflammatory signals to distant organs.

Methods: To investigate the role of adipocyte-derived extracellular vesicles (AdEVs) on interorgan signaling, we employed a multifaceted approach that combined engineering and pre-clinical research. Adipocytes were harvested from patients with and without obesity undergoing bariatric surgery who voluntarily participated under informed consent, as well as from lean and obese mice. AdEVs were purified using tangential flow filtration (TFF) and characterized using electron microscopy, tunable resistive pulse sensing (TRPS), and single-EV phenotyping. Single-EV experiments were conducted using a biosensor that utilizes surface plasmon resonances to amplify fluorescence signals at the coverslip surface when coupled with total internal reflective fluorescence microscopy. Furthermore, co-culture experiments were performed to investigate the cellular uptake of AdEVs stained with a lipophilic dye utilizing confocal microscopy. On the other hand, AdEVs were introduced into a mouse model that mimics complex human obesity-associated diseases, including insulin resistance, metabolic dysfunction-associated fatty liver disease (MAFLD), and atherosclerosis, the underlying major cause of cardiovascular disease. After injecting AdEVs, various metabolic tests were performed to assess the extent of insulin resistance, MAFLD, and atherosclerosis. Lastly, microRNA (miRNA) sequencing was performed to identify novel cargoes responsible for inducing inflammatory signals at distant sites.

Results: AdEVs were successfully purified using TFF, resulting in low concentrations of unwanted proteins while maintaining vesicular morphology and sterility. TRPS demonstrated that AdEVs from patients with and without obesity fell within the small EV range (<200 nm) and exhibited similar sizes. Single-EV phenotyping revealed the presence of EV-specific biomarkers on the surface of AdEVs, with no significant differences observed between patients with and without obesity when corrected for AdEV concentration. Furthermore, adipocyte-specific biomarkers were enriched on the surface of AdEVs. The presence of adipocyte-specific biomarkers on AdEVs enabled the tracking of AdEVs in complex biofluids, such as plasma, revealing an increased production of circulating AdEVs in patients with obesity compared to lean patients. Interestingly, AdEVs from obese mice, but not AdEVs from lean mice, accelerated atherosclerosis in the recipient mice while worsening insulin resistance and promoting the deposition of fatty reserves in the liver. AdEVs were engulfed by the local immune milieu, indicating interorgan signaling. Confocal microscopy revealed the colocalization of AdEVs with subcellular compartments in macrophages, a significant inflammatory cell type associated with obesity-associated diseases, indicating the specific uptake of AdEVs. Furthermore, macrophages produced pro-inflammatory responses when co-cultured with AdEVs from patients with obesity but not from lean patients, implying a differential cargo in the AdEVs. Sequencing of AdEVs confirmed the presence of various differentially expressed miRNAs, with bioinformatic analyses identifying the Metabolic Pathway as the most significant pathway, corroborating our in vivo data.

Implications: Our results provide insights into the mechanisms that occur when individuals consume a high-fat diet and the signaling mechanisms involved in obesity, particularly those affecting obesity-associated comorbidities, including atherosclerosis, MAFLD, and insulin resistance. Our robust approach, which investigates physiological pathology, cellular mechanisms, subcellular compartments, and molecular differences, integrates pre-clinical and engineering methods to provide a comprehensive perspective of adipocyte interorgan signaling. Lastly, the molecular differences between AdEVs from patients with and without obesity encourage potential therapeutic targets, which can attenuate obesity-associated comorbidities.