Macrophage Function in Processing of Excess Dietary Fat
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Amal Bajonaid DMSc Thesis.pdf (2.200Mb)
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Bajonaid, Amal
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Bajonaid, Amal. 2023. Macrophage Function in Processing of Excess Dietary Fat. Doctoral dissertation, Harvard University School of Dental Medicine.Abstract
Background: Chronic low-grade metabolic inflammation is one of the hallmarks of metabolic diseases. The risk of periodontitis is approximately threefold higher in diabetic individuals. To examine this relationship, we used a strain of transgenic mice overexpressing the receptor for Resolvin E1 (ERV1tg) that have a reduced inflammatory phenotype depicted by higher resolving (M2) to inflammatory macrophage (M1) ratio and are resistant to periodontitis and compared them to wild type (WT). Resolving macrophages secrete anti-inflammatory cytokines and lipid mediators and contribute to resolution of inflammation. Our previous work showed that when wild type and ERV1tg mice are fed a high-fat diet (HFD), ERV1tg mice have higher resolving to inflammatory macrophage ratio, WT males become obese, but both genders of the ERV1tg are protected from diabetes and periodontitis. In addition, WT female mice were protected from morbid weight gain, impaired fasting glucose and glucose intolerance compared to the male mice, and ERV1tg female mice had higher energy expenditure and lower lean/fat mass ratio compared to ERV1tg male mice.Objective: investigate the role of resolving macrophages in processing of excess dietary fat in ERV1 tg mice and determine the difference that leads to sexual dimorphism in energy utilization by metabolic organs.
Materials and Methods: 6-week-old ERV1tg and WT male and female mice were fed HFD for 16 weeks, 5 mice per group. Differences in expression of uncoupling protein 1 (UCP-1), peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1⍺), nuclear respiratory factor 1 (NRF-1), sirtuin1 (SIRT1) and estrogen-related receptor alpha (ERRα) in visceral adipose tissue (VAT), liver and brown adipose tissue (BAT) were assessed using real-time
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quantitative PCR and western blot. mRNA expression was normalized to peptidylprolyl isomerase A (PPIA) as a reference gene. Protein expression was normalized to Actin-B. to investigate the impact of RvE1 on cholesterol uptake by macrophages and efflux to HDL, we used RAW264.7 murine macrophage cell line and performed in vitro cholesterol uptake and efflux assay and investigated the impact of varying concentrations of RvE1 on cholesterol uptake and efflux to HDL. Statistical analysis: One-way ANOVA with ⍺=0.05.
Results: Expression of PGC1-α mRNA in VAT was significantly higher in ERV1 tg female mice (4.4-fold ± 0.66) compared to ERV1 tg male mice (0.65-fold ± 0.12, p .0001). Relative NRF- 1 mRNA expression in VAT was significantly higher in ERV1 tg female mice (1.6-fold ± 0.12) compared to ERV1 tg male mice (0.65 ± 0.13, p 0.0001). ERV1 tg female mice showed significantly higher expression of Sirt-1 mRNA in VAT (3.1-fold ± 0.26) compared to ERV1 tg male mice (1.7-fold ± 0.29, p 0.0001). NRF-1 mRNA expression in the liver was significantly higher in ERV1 tg female mice (2.5-fold ± 0.21) compared to ERV1 tg male mice (2.0-fold ± 0.59, p = 0.0002). Relative Sirt-1 mRNA expression in BAT was significantly higher in ERV1 tg female mice (2.2-fold ± 0.12) compared to WT female (0.5-fold ± 0.08, p 0.0001), and ERV1 tg male mice (1.3-fold ± 0.29) compared to WT male mice (0.47-fold ± 0.09, p 0.0001). In vitro cholesterol uptake was significantly higher in M2 macrophages (190.6 ± 14.2 RFU) compared to M1 (109.6 ± 13.3 RFU, p 0.0001). M1 and M2 macrophages treated with RvE1 showed significantly higher uptake of cholesterol compared to vehicle. In vitro cholesterol efflux to HDL was significantly higher in M2 macrophages (4.8% ± 0.9) compared to M1 (2.06% ± 0.33, p 0.0001) and M0 (1.9% ± 0.45, p 0.0001). M1 and M2 macrophages treated with RvE1 showed significantly higher efflux of cholesterol to HDL compared to vehicle.
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Conclusions: RvE1, the ligand for ERV1, is known to directly regulate inflammation and prevent low-grade inflammation in type 2 diabetes. This study provides direct evidence for molecular targets of RvE-mediated regulation of obesity that display sexual dimorphism suggesting that female ERV1 tg mice have higher metabolic flexibility through increased mitochondrial function to adapt to excess energy. Future studies will investigate the molecular basis for differential responses to RvE1 based on gender focusing on the intracellular signaling pathways of RvE1.
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