2012 Annual Report
1a.Objectives (from AD-416):
Inflammatory pathways are implicated in promoting insulin resistance, and recent research highlights the potential role of saturated fatty acids to more strongly promote so-called “sterile” pro-inflammatory pathways (i.e., NFkB activation) relative to polyunsaturated fatty acids (PUFAs). In fact, the latter--along with certain phytonutrients--have been shown to reduce the pro-inflammatory effects of saturated fatty acids. The effects of saturated FAs are thought to be in part through activation of cell-surface receptors such as the Toll-like receptors (TLRs) or intracellular proteins such as Nod. This has raised the questions: do circulating or local FAs activate TLRs/sterile inflammation and in turn drive diabetes, and might this be prevented or reversed through increasing dietary intakes of certain PUFAs or select phytonutrients? One conundrum facing the field has been the observation that saturated medium-chain FAs (i.e., C12:0, C14:0) are particularly potent activators of TLRs and NFkB-related pathways, yet are of negligible abundance in the diet or circulation; thus, one may ask whether these pathways are physiologically-relevant. A breakthrough in this regard has come from our recent discovery that in T2DM, there is an increased plasma accumulation of MCFA-carnitines, and these acylcarnitines activate NFkB in vitro more potently than MCFAs. Build-up of these chain-shortened metabolites derived from LCFA is believed to occur due to a relative mismatch of TCA cycle capacity relative to LCFA availability, and subsequent conversion by carnitine acyltransferases. In the current proposal, we will test if acylcarnitines activate NFkB through the TLR or NOD systems, well-known modulators of inflammatory responses. If true, one may contemplate that ultimately, dietary interventions that inhibit TLRs (i.e., PUFAs, phytonutrients) and/or increase TCA cycle capacity to minimize acylcarnitine generation (i.e., anaplerotic odd-chain FA-containing lipids) could be tested for anti-diabetic properties in human subjects.
1b.Approach (from AD-416):
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).
This is the final report for the INTERNATIONAL LIFE SCIENCES INSTITUTE project terminated in September 1, 2011.
Poor insulin sensitivity and frank type 2 diabetes typically occur in the setting of reduced or inefficient muscle long chain fatty acid (LCFA) catabolism in mitochondria. In the course of this 2 year project, substantial progress was made toward understanding how LCFA metabolism and inflammation intersect. The research team has published a significant paper outlining the finding that metabolite by-products of inefficient LCFA combustion (chain-shortened acylcarnitine moieties) are elevated in the plasma of type 2 diabetic African-American women. Most remarkably, follow-up studies revealed that these acylcarnitines act as triggers for pro-inflammatory pathways implicated in driving insulin resistance. Studies to date indicate that select acylcarnitines trigger activation of specific pattern recognition receptor pathways in immune cells, which in turn elicits pro-inflammatory outcomes such as cell cytokine release, activation of downstream proteins intracellularly, etc. This indicates that when fat oxidation is impaired or inefficient, as often seen in states such as obesity in which blood sugar control is poor, metabolites can accumulate that promote inflammation and may contribute to sub-optimal metabolic health.
This research contributed to objective 3 of the in-house parent project. Metabolites that are increased in the diabetic or pre-diabetic/insulin resistant state may be involved in exacerbating disease risk and poor metabolic health. The team has made significant progress toward characterizing the pro-inflammatory effects of acylcarnitine metabolites on a murine macrophage cell line, muscle cells, and a human colonic cell line. In summary, the investigators have found a significant acylcarnitine effect on inflammatory cytokine release and NFkB-luciferase expression. Several specific members of the pattern recognition receptor family that include the toll-like receptors (TLRs) have been excluded as targets of these effects, but knockdown of a downstream effect or molecule of these receptors did abolish the acylcarnitine-based inflammation, indicating that effects are specific to this family. Work in a C2C12 muscle cell culture system established that acylcarnitines evoke a pro-inflammatory response with respect to IL-6 cytokine secretion, and studies are underway to examine whether the response is accompanied by insulin resistance.