We will employ two well-characterized and widely used in vitro cell culture systems to address each of the objectives proposed. Namely, 3T3-L1 preadipocytes and RAW264.7 macrophages (available in both PI’s lab). Initial cell culture experiments will test the primary hypothesis that treatment of cranberry extract (CBE) promotes fatty acid oxidation and mitochondrial thermogenic biogenesis in 3T3-L1 adipocytes and anti-inflammatory effects in RAW264.7 macrophages. Our previous experience in these cell culture systems has shown that a doses of proanthocyanidin (PAC) contents (5% and 15% PAC) in CBE extract are sufficient to provide a clear dose-response effect on gene expression (iNos, Cox-2, Tnfα, Mcp-1 and Il-6). The details of 3T3-L1 adipocytes and macrophage cell culture and basic treatment protocols are described in the proposal. For the second objective to define potential mechanisms underlying the cross-talk between adipocytes and macrophages and the resultant adipocyte inflammatory and metabolic functions, the conditioned medium from macrophages activated with LPS or LPS plus CBE will be added to 3T3-L1 adipocytes. The contribution of the anti-inflammatory effect of CBE to fat cell inflammation and function will be assessed by qRT-PCR analysis for mRNA expression and western blot analysis for protein expression. Specifically, we will measure genes involved in lipolysis (ATGL, pHSL and perilipin), fatty acid oxidation (CPT-1, pAMPK, and pACC) and their upstream regulators such as a transcription factor, PPARβ/δ and NF-kB. Glucose uptake experiment will be conducted to correlate the molecular changes to biological effects of CBE in both basal and insulin-stimulated state. To determine the molecular mechanisms by which CBE promotes overall thermogenic program and improves inflammation and metabolic functions in adipocytes in the objective three, we will utilize pluripotent mesenchymal stem cells, C3H10T1/2 cell line (10T1/2 cells), a widely used for adipocyte differentiation and browning of white adipocytes. 10T1/2 cells will be cultured and maintained in DMEM media and differentiated and treated as described in the proposal in details. These experiments will test the hypothesis that CBE treatment increases browning of white adipocytes and fat oxidation by upregulating the expression of thermogenic biogenesis as evidenced by marker genes including AMPK, PRDM16, PGC-1a, CPT-1, and UCP-1. All of the procedures and molecular techniques to complete the proposed experiments are general scientific methods and are routinely performed in my and the collaborator’s laboratory. Thus, we do not expect any methods or particular needs that are significant departures from usual approaches.
Y.C.
Kim
