79 Serum Levels of

79 Serum Levels of Dioxin-Like Pollutants Are Positively Associated With the Cardiometabolic Disease Risk Biomarker TMAO in Leaner Individuals Michael Petriello, PhD 1 • Richard Charnigo • Manjula Sunkara • Sony Soman • Marian Pavuk 2 • Linda Birnbaum 3 • Andrew Morris, PhD 4 • Bernhard Hennig 5 1Cardiovascular Research Center, University of Kentucky • 2 CDC Agency for Toxic Substances and Disease Registry • 3 NCI at NIEHS • 4 Cardiovascular Medicine, University of Kentucky • 5 Superfund Research Center, University of Kentucky Postdoc Trimethylamine N-oxide (TMAO) is a diet and gut microbiota-derived metabolite that has been linked to cardiovascular disease risk in human studies and animal models. TMAO levels show wide inter and intra individual variability in humans that can likely be accounted for by multiple factors including diet, the gut microbiota, levels of the TMAO generating liver enzyme Flavin-containing monooxygenase 3 (FMO3) and kidney function. We recently found that dioxin-like (DL) environmental pollutants increased FMO3 expression to elevate circulating diet-derived TMAO in mice, suggesting that exposure to this class of pollutants might also contribute to inter-individual variability in circulating TMAO levels in humans. To begin to explore this possibility we examined the relationship between body burden of DL pollutants (reported by serum lipid concentrations) and serum TMAO levels (n=340) in the Anniston, AL cohort, which was highly exposed to polychlorinated biphenyls (PCBs). TMAO concentrations in archived serum samples from the Anniston Community Health Survey (ACHS-II) were measured, and associations of TMAO with 28 indices of pollutant body burden, including total dioxins toxic equivalent (TEQ), were quantified. Twenty-three (22 after adjustment for multiple comparisons) of the 28 indices were significantly positively associated with TMAO. Multivariate modeling revealed that total dioxins TEQ was significantly associated with TMAO among females (except at high BMIs) but not among males. Our results from this cross-sectional study indicate that exposure to DL pollutants may contribute to elevated serum TMAO levels. With the observation that dioxins were only associated with TMAO in leaner individuals, we have begun to mechanistically study the relationship between dioxin exposure, FMO3/TMAO, and cardiometabolic disease by characterizing a mouse model that develops atherosclerosis but not adipose tissue expansion. We examined the effects of PCB 126 on markers of systemic inflammation and atherosclerotic lesion size. Exposed mice exhibited significantly increased plasma cytokine levels and accelerated atherosclerotic lesion formation. More work needs to be completed to determine the role of TMAO and FMO3 in these processes (Supported in part by NIEHS/NIH grant P42ES007380). 95

80 Insulin signaling regulates apolipoprotein (Apo) AI secretion from hepatocytes Ailing Ji, PhD 1 • Xuebing Wang 1 • Lisa Bennett 2 • David Graf 2 • Gregory Graf, PhD 2 • Deneys van der Westhuyzen, PhD 1 1Cardiovascular Research Center, University of Kentucky • 2 Pharmaceutical Sciences, University of Kentucky Staff Insulin resistance is associated with increased risk for cholesterol gallstones as well as the development of diabetic dyslipidemia in which plasma levels of HDL cholesterol are reduced. HDL is the primary cholesterol carrier in the reverse cholesterol transport (RCT) pathway, the process by cholesterol is delivered from peripheral organs to the liver for elimination in the bile. Therefore, we hypothesized that insulin signaling is a regulator of hepatobiliary cholesterol clearance from the plasma and secretion into bile. To directly test the role of insulin signaling, we utilized mice harboring insulin receptor flanked by loxP sites (IRfl/fl) in combination with adenoassociated viral vectors containing no transgene (empty) or Cre recombinase to generate control and liver insulin receptor knock out mice (LIRKO), respectively. As with previous LIRKO strains (albumin-Cre, adenoviral-Cre), LIRKO mice showed markedly reduced insulin receptor mRNA and protein in liver, but not skeletal muscle or adipose tissue as well as impaired glucose tolerance when compared to controls. LIRKO mice had increased biliary cholesterol secretion as well as increased expression of the ABCG5 ABCG8 sterol transporter, the primary mediator of biliary cholesterol secretion. Levels of SR-BI, the primary HDL receptor were unchanged as were rates of HDL clearance from plasma and selective delivery of HDL cholesterol to the liver. None-the-less, plasma HDL cholesterol and ApoAI were reduced. Despite these reductions, ApoAI protein, but not mRNA, was increased in the liver of LIRKO mice. Immunofluorescence microscopy revealed that ApoAI accumulated in membrane-bound inclusions that stained positively for markers of early, late and recycling endosomes and of lysosomes. These structures persisted in primary hepatocyte cultures where rates of ApoAI secretion into the culture medium were reduced. To determine the intracellular itinerary of ApoAI, we developed a chimera of mCherry fluorescent protein and human ApoAI. mCherry ApoAI was capable of forming nascent HDL, associated with HDL in plasma, and partially restored HDL cholesterol in ApoAI-deficient mice. However, this mCherry-ApaAI failed to colocalize with a probe for acidic compartments in live cells, suggesting that the compartment is unique or that the accumulation of ApoAI neutralizes the endosomal/lysosomal vesicles in which it accumulates. Additional studies will be required to investigate the route by which ApoAI arrives and accumulates in cells with impaired insulin signaling. 96