Location: Obesity and Metabolism Research Unit
Project Number: 2032-51530-019-20
Start Date: Mar 01, 2009
End Date: Sep 01, 2011
Specific Aim 1. Determine if the Pro-Inflammatory, NFkB-Stimulating Activities of Medium-Chain Acylcarnitines Require Activation of Toll-like Receptors or Nod: Knockdown Studies. It is hypothesized that similar to the saturated fatty acids laurate (C12:0) and myristate (C14:0), laurylcarnitine and myristoylcarnitine will trigger NFkB-induced pro-inflammatory pathways through stimulation of cell surface TLR4 or TLR2, and/or via intracellular Nod. The studies will assess NFkB activation following manipulations that lead to loss-of-function for TLR4, TLR2, and Nod. Approach: The RAW264.7 murine monocyte cell line expresses TLR4, TLR2, and Nod, and is used routinely to test for NFkB activation using a transfection approach that enables luciferase detection driven by an NFkB-activated promoter construct. In addition, upon stimulation with pro-inflammatory agents such as C12:0 or LPS, these cells secrete cytokines such as IL-8 and TNFa that can be readily measured in the tissue culture medium using standard ELISA protocols routinely used by the laboratory. Thus, this model is ideal to test the effects of TLR4, TLR2, or Nod gene knockdown on NFkB activation in response to D,L-carnitine hydrochloride, D,L-decanoylcarnitine chloride (C10-carn), D,L-laurylcarnitine (C12-carn), D,L-myristoylcarnitine chloride (C14-carn). Specific Aim 2. Determine if the Pro-Inflammatory, NFkB-Stimulating Activities of Medium-Chain Acylcarnitines Require Activation of Toll-like Receptors or Nod: Reconsitution Studies. Approach: The studies will assess NFkB activation following manipulations that lead to gain-of-function for TLR4, TLR2, and Nod. Luciferase studies will be used in HEK293T cells to assess NFkB activation in response to acylcarnitines and LPS, using protocols similar to those outlined in Aim 1. Since this cell line does not express TLR2, TLR4, or Nod, activation of NFkB by LPS or saturated FAs can only be detected upon transfection of the cells with the target protein(s). We will compare NFkB activity in sham-transfected vs. TLR2-, TLR4- or Nod-transfected cells treated with LPS or acylcarnitines (dose and timeframe for the latter to be determined in pilot studies). Documents Truat Trust (International Life Sciences Institute ILSI 2009 Future Leader Award.) Log 37584. Formerly 5306-51530-016-20T (8/09).