Cellular and molecular basis for omega 3 polyunsaturated fatty acid regulation of brown adipose tissue

Obesity is a significant health challenge affecting more than 35% of the American population and is linked to higher risks for metabolic diseases namely cardiovascular diseases, and type 2 diabetes. Obesity is defined as excess accumulation of white adipose (fat) tissue, leading to dysregulation of energy balance. Obesity is a complex disease which results from the current obesogenic environment with underlying genetic susceptibility and other factors. Given the complexity of obesity, multiple prevention and treatment strategies are needed. These include caloric restriction, physical activity with lifestyle modifications, pharmacological treatments and/or surgery. Different adipose tissue depots exist in the human body, and include white adipose tissue (WAT) and brown adipose tissue (BAT). WAT is well established as a major culprit in obesity-associated metabolic diseases due to its endocrine and inflammatory functions. Indeed, WAT cells produce and secrete hormones and inflammatory substances known to raise blood pressure, insulin resistance and cause other metabolic dysfunctions. On the other hand, BAT plays a fundamental role in regulating energy homeostasis by increasing thermogenesis and energy expenditure. The amount of BAT is reduced with aging and obesity. BAT has been less studied especially in humans, but has gained significant attention in recent years as a potential novel target to prevent and/or treat obesity.
Long chain omega 3 polyunsaturated fatty acids from fish oil have several beneficial health effects such as anti-inflammatory, cardiovascular protective role, and in some cases weight-reducing properties. Previous studies from our laboratory have reported that one of these fatty acids, namely eicosapentaenoic acid (EPA), reduced high fat (HF) diet-induced obesity, inflammation, and insulin resistance in mice, independent of caloric intake. However, contribution of BAT to these beneficial metabolic effects of EPA was not determined. We hypothesized that EPA exerted some of these effects in part by activating BAT thermogenic program. Experimentally, we induced dietary obesity in B6 mice by feeding them high fat diet without (HF) or with EPA (HF-EPA) for 11 weeks then harvested BAT and WAT for gene, protein and metabolic analyses. To further determine direct effects of EPA in brown fat cells, we conducted in vitro studies using HIB 1B clonal brown adipocytes treated with or without EPA. Our results showed that BAT from EPA-supplemented mice expressed significantly higher levels of thermogenic genes such as PR domain containing 16 (Prdm16) and Peroxisome Proliferator-activated Receptor Gamma Coactivator -1 alpha (Pgc1α) and higher uncoupling protein 1 (UCP1) content compared to HF-fed mice. In contrast, WAT (both subcutaneous and visceral) had undetectable levels of these markers with no significant responses to EPA. Similarly, HIB 1B cells treated with EPA expressed significantly higher mRNA expression of the above thermogenic markers. Moreover, EPA treatment significantly increased maximal oxidative metabolism as well as peak glycolytic metabolism in HIB 1B cells. These results suggested that EPA may act in part by targeting BAT metabolism to prevent obesity. To gain further insight into molecular mechanisms mediating EPA effects, we performed RNA sequencing (RNA-Seq) and microRNA (miRNA) profiling (using Illumina Hi-Seq and Mi-Seq), two powerful genomic tools to identify novel gene and miRNA regulators of BAT metabolism, which were differentially modulated in HF vs. HF-EPA-fed mice. Analyses of our sequencing data revealed 479 genes that were differentially expressed (2-fold change, p<0.05) in BAT from HF compared to HF-EPA fed mice. These genes were further mapped to specific cellular pathways and functions using Ingenuity Pathway Analysis software (IPA®). Genes with negative correlation with thermogenesis such as hypoxia‐inducible factor 1 α inhibitor (Hif1an), and notch homolog 1, translocation-associated (Notch1) were downregulated by EPA. Moreover, pathways related to thermogenesis and energy expenditure such as peroxisome proliferator-activated receptor- (PPARα), retinoid X receptor (RXR), phosphatase and tensin homolog (PTEN) were upregulated by EPA. Furthermore, pathways associated with obesity and inflammation such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-β), Signal Transducer and Activator of Transcription 3 (STAT3), and Tumor necrosis factor receptor 1 (TNFR1) were downregulated by EPA. Global miRNA profiling identified ten miRNAs that were upregulated, and six miRNAs were downregulated by EPA, which included key regulatory miRNAs involved in BAT thermogenesis such as miR-455-3p and miR-30a-5p. While these two miRNAs have previously been associated with thermogenesis, this is the first report of their regulation by EPA. We also identified miR-129 that we identified for the first time as expressed in BAT, and involved in regulation of thermogenic genes in response to EPA. Overall, results from this study showed that EPA can be used to successfully target obesity and associated metabolic disorders. This is the first report to use a nutritional intervention (EPA) to characterize mechanisms underlying increased thermogenic program by identifying key target pathways, genes, and miRNAs mediating regulation of BAT by EPA. These findings provide evidence that dietary approaches such as fish oil may be effective in protecting against obesity and metabolic disorders. More studies are warranted to investigate additional mechanisms of EPA in obesity and BAT metabolism in mice. Importantly, future clinical studies to translate these findings to human subjects are warranted.

This dissertation won 1st Place in the Texas Tech University Outstanding Thesis and Dissertation Award, Biological Life Sciences, 2017.

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