Consistent with other studies on the

Consistent with other studies on the metabolic effects of an OM3 enriched diet [26,104,105], irrespective of gender, the lowest glucose levels were seen in the Bob-Cat groups fed OM3 diet. Sexual dimorphism was observed in insulin levels. The highest average levels were in the HFD fed [Tg(CAT)±] males, while the HFD [Tg(CAT)±] females had the overall lowest levels of plasma insulin. The same results were reflected in the HOMA-IR. Furthermore, males in general had higher levels of insulin than females regardless of diet or genotype. This could be a direct effect of the increased visceral adipose tissue and liver weights in males compared to females [15]. Insulin levels are key in the metabolic function of the liver [106], so it was interesting that the males had an increase in liver weight, but not the females, as well as NC and HFD fed [Tg(CAT)±] mice groups compared to WT littermates. Despite increases in insulin, plasma ketone levels showed no significant differences in male HFD fed [Tg(CAT)±] mice compared to the WT mice fed HFD. In fact, ketone levels were >2 fold in the WT vs. [Tg(CAT)±] HFD group indicating insulin is repressing ketone body production in the [Tg(CAT) ±] mice [103,107]. Nevertheless, most importantly, Bob-Cat mice fed OM3 rich diet maintained glucose and insulin homeostasis throughout the duration of the 8 week study. Mechanistically, this result was expected to occur in part by the production of OM3-derived inflammatory resolution mediators [29], the higher ketones generated, “metabolic flexibility [88],” and potential alterations in “metabolic partitioning” [89] (indicated by the CLAMS fat oxidation analysis) in the Bob-Cat mice. However, in addition to these effects, the major contributor to the favorable metabolic profile of the Bob-Cat mice fed OM3 diet is through the activation of its receptor, GPR120 [26,108,109]. GPR120 (FFAR4) is a long-chain fatty Aliskiren receptor highly expressed in adipose tissue [26] and activated by OM3 fatty acids [22,26,92,110]. It plays beneficial roles in anti-inflammatory pathways in adipose tissue, food preference, glucose homeostasis, and insulin sensitivity, all of which are interrelated to regulate metabolic energy homeostasis in both physiological and pathophysiological conditions [111]. Though there are contradicting reports of GPR120 not required for these beneficial effects of OM3 fatty acids [112], the support for its role in OM3 mediated effects stems from genetic studies performed in humans. The human studies showed that mutations in GPR120 were associated with increased risk of obesity and IR [108]. GPR120 is also a novel risk factor for diet induced obesity (DIO) [26,113]. For these reasons, it was most compelling that within the OM3 fed Bob-Cat mice groups, which overexpresses antioxidant catalase within an obese parent background, we observed the highest levels of both mRNA and protein expression of GPR120 in the perigonadal adipose tissue. Based on our measurements of redox stress markers, the OM3 fed Bob-Cat mice also had an increased level of oxidized carbonyl groups within the adipose tissue. Together, this might suggest that redox regulation is playing a role in the upregulation of GPR120 expression and beneficial outcomes of the OM3 diet within the antioxidant-overexpressing mice of both genders. Redox regulation of GPR120 has not been previously shown in prior studies. Furthermore, nuclear factor erythroid-2-related factor 2 (Nrf2), is a redox sensitive transcription factor activated by long chain fatty acids (including EPA and DHA), phenolic antioxidants, and imbalances in redox stress [29,114]. Raising levels of Nrf2 by endogenous production of electrophilic products or pharmacological agents has been shown to prevent or act as therapies for type 2 diabetes, metabolic syndrome, obesity, and cardiovascular disease through activating anti-inflammatory pathways [30,[115], [116], [117], [118]] in addition to lowering body weight and fat mass [119]. Specifically, in the Bob-Cat mice groups, when fed a diet high in OM3 fatty acids, there were higher levels of adipose tissue Nrf2 mRNA expression. We speculate this to have occurred as a result of the synergistic effect of antioxidant overexpression and consumption of OM3 rich diet. Studies concentrated on the beneficial effects of polyunsaturated fatty acids (primarily EPA and DHA) have shown that their oxidized derivatives regulate the redox environment by covalently and reversibly reacting with nucleophilic residues on target proteins [29,120]. These reactions trigger the activation of cytoprotective pathways, including the Nrf2 antioxidant response [121]. Nrf2 activation subsequently causes an upregulation of phase II enzymes/antioxidants thus balancing oxidant: antioxidant ratios in addition to suppressing the NF-κB proinflammatory pathway [29]. Both antioxidant catalase and HO-1 are two of the key antioxidants upregulated in response to induction of Nrf2 [31,32,122]. In our model, the Bob-Cat mice group fed OM3 diet had the highest levels of catalase activity in addition to mRNA expression of HO-1, providing further evidence of Nrf2 induction and subsequent activation of the antioxidant and cytoprotective response.