Graduate Student Medical College of Wisconsin Milwaukee, Wisconsin, United States
Darcy Knaack (Medical College of Wisconsin)| Michael Thomas (Medical College of Wisconsin)| Mary Sorci-Thomas (Medical College of Wisconsin, Medical College of Wisconsin)| Daisy Sahoo (Medical College of Wisconsin, Medical College of Wisconsin, Medical College of Wisconsin)
Obesity continues to be an epidemic in the United States, placing individuals at a higher risk of developing other comorbidities such as type 2 diabetes and cardiovascular disease. Specialized cells called adipocytes accumulate excess triglycerides and undergo hypertrophic expansion as a protective mechanism to avoid lipotoxicity and organ failure. Since adipocytes have a reduced ability to synthesize their own cholesterol, they rely heavily on circulating lipoproteins for their cholesterol supply to support membrane expansion. Scavenger receptor BI (SR-BI) is believed to be important in the adipocyte cholesterol delivery process, as this integral membrane protein is the primary receptor for high-density lipoproteins (HDL) and regulates bi-directional cholesterol transport. Collaborative studies demonstrated that SR-BI-mediated HDL-cholesteryl ester (HDL-CE) uptake in the liver requires the extracellular matrix protein procollagen C-endopeptidase enhancer 2 (PCPE2). These published studies have prompted a novel hypothesis that PCPE2 facilitates SR-BI’s cholesterol transport functions to regulate adipocyte cholesterol homeostasis. To test our hypothesis, we have replicated an adipocyte model system where ear mesenchymal stem cells (EMSCs) isolated from the outer ears of mice can be differentiated into adipocytes and further used to study cholesterol transport in vitro. We validated our adipocyte model system by Oil Red O staining, mRNA expression of adipogenesis markers (PPARγ, LPL, FABP4, cEBP2α), and adiponectin secretion over the 9-day post-differentiation period in wild-type (WT), SR-BI knock-out (SR-BI-/-), and PCPE2-/- adipocytes. All three genotypes showed increased Oil Red O staining, increased mRNA expression of adipogenesis markers, and increased adiponectin secretion over the post-differentiation period. Tandem mass spectrometry analysis showed that SR-BI-/- and PCPE2-/- adipocytes had half the amount of cholesteryl esters compared to WT adipocytes, indicating that there may be disrupted cholesterol transport in the SR-BI-/- and PCPE2-/- adipocytes. Early pilot data indicate no major changes in cholesterol efflux from [3H]-cholesterol-labeled cells to HDL between WT, SR-BI-/-, and PCPE2-/- adipocytes. Experiments to assess HDL binding and HDL-CE uptake using double radiolabeled HDL particles ([125I]-HDL-[3H]-CHE) are currently ongoing. Interestingly, using an assay that assesses cholesterol distribution in membranes, we were able to show that loss of PCPE2 causes changes in cholesterol distribution within the membrane, suggesting that it may play a role in lipid raft formation. All together, we are confident that we have developed a unique in vitro adipocyte model system that can help us assess how PCPE2 may impact SR-BI-mediated cholesterol delivery.