The persistent state of subclinical inflammation, which characterizes obesity, is thought to play an important role in the pathophysiological link between obesity and cardiometabolic disease risk. Central to this 'inflammatory hypothesis'is the role of adipose tissue macrophages (ATMs) in mediating the inflammatory response to chronic over-nutrition. Despite displaying reductions in adipose tissue (AT) inflammation and insulin resistance (IR) following high fat feeding, Toll-like Receptor 4 (TLR4) deficient mice exhibit a marked accumulation of ATMs. Preliminary data from our laboratory are consistent with the idea that TLR4 deficiency reduces AT inflammation and IR in diet-induced obesity by shifting ATM polarization towards an, anti- inflammatory, alternatively activated state. To date, no studies have explored the role of TLR4 signaling in ATM polarization. Thus, the Specific Aims of this project are: 1) to determine the influence of hematopoietic cell TLR4 signaling on AT inflammation and IR in diet-induced obesity;2) to determine the influence of parenchymal and hematopoietic cell TLR4 signaling on the in vivo polarization of ATMs in diet-induced obesity;3) to determine the influence of TLR4 signaling on macrophage substrate metabolism and lipid accumulation. To accomplish these aims we will utilize a bone marrow transplant (BMT) model, whereby C57BL/6 (WT) and TLR4-/- mice will receive either WT or TLR4-/- BM following lethal irradiation. Subsequently, recipient mice will be placed on a high fat diet (HFD) for 16 weeks. AT inflammation and IR will be assessed via AT expression of inflammatory markers and hyperinsulinemic-euglycemic clamps, respectively. A combination of real time rtPCR, flow cytometry, and immunofluorescent staining, and mixed chimeras will be used to determine the influence of parenchymal and hematopoietic cell TLR4 signaling on ATM polarization. Finally, we will utilize radio-labeled substrates to determine the influence of TLR4 signaling on macrophage substrate metabolism and neutral lipid synthesis at baseline and following Th1 and Th2 cytokine stimulation. Our hypotheses are: 1) hematopoietic cell TLR4 deficiency will reduce AT inflammation and IR following a HFD. Conversely, the restoration of hematopoietic cell TLR4 signaling in TLR4-/- mice will exacerbate AT inflammation and IR following a HFD;2) Hematopoietic cell TLR4 signaling will be the primary determinant of ATM polarization. That is, TLR4-/- ATMs will be primarily M2 polarized WT ATMs will primarily display an M1 phenotype regardless of the parenchymal cell genotype;3) TLR4 deficiency will reduce ATM lipid accumulation and promote a shift in substrate metabolism towards fatty acid oxidation.

Public Health Relevance

Overweight and obese individuals are at increased risk of developing cardiovascular disease and type 2 diabetes. Part of this increased risk is thought to result from the chronic low-grade inflammation associated with excess body fat. The goal of the current research is to determine how a specific inflammatory pathway (i.e., Toll-like Receptor 4) in cells of the immune system contributes to the development of type 2 diabetes in obesity.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32DK091040-02
Application #
8389811
Study Section
Special Emphasis Panel (ZDK1-GRB-9 (J1))
Program Officer
Castle, Arthur
Project Start
2011-07-01
Project End
2013-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
2
Fiscal Year
2012
Total Cost
$53,042
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Physiology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
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Orr, Jeb S; Kennedy, Arion J; Hasty, Alyssa H (2013) Isolation of adipose tissue immune cells. J Vis Exp :e50707
Kennedy, Arion; Gruen, Marnie L; Gutierrez, Dario A et al. (2012) Impact of macrophage inflammatory protein-1α deficiency on atherosclerotic lesion formation, hepatic steatosis, and adipose tissue expansion. PLoS One 7:e31508
Orr, Jeb S; Puglisi, Michael J; Ellacott, Kate L J et al. (2012) Toll-like receptor 4 deficiency promotes the alternative activation of adipose tissue macrophages. Diabetes 61:2718-27