MyD88 determines the protective effects of fish oil and perilla oil against metabolic disorders and inflammation in adipose tissue from mice fed a high-fat diet

The study addresses whether plant-derived ω-3 PUFA (ALA-rich perilla oil) can mimic the metabolic and anti-inflammatory benefits of marine ω-3 PUFA (EPA/DHA-rich fish oil) in the context of diet-induced obesity, and through which arm(s) of TLR4 signaling these effects are mediated. Obesity-driven adipose inflammation contributes to dyslipidemia and insulin resistance. ω-3 PUFA benefit cardiovascular and metabolic health and may suppress TLR4 signaling, which activates MyD88-dependent and -independent pathways leading to pro-inflammatory cytokine production. However, comparative evidence between marine and plant ω-3 PUFA and the specific involvement of MyD88 in their protective effects is limited. This study evaluates FO and PO effects on glucolipid metabolism, inflammation, adipokines, and TLR4 pathway activation in adipose tissue, including confirmation using MyD88 knockout mice.

Prior work suggests both similarities and differences between marine and plant ω-3 PUFA: FO and PO each improved lipid profiles and inflammation in various rodent and human studies, though some trials found EPA/DHA more potent than ALA for triglyceride lowering or atherogenesis modulation. The ω-3/ω-6 ratio can influence metabolic outcomes independent of total ω-3 content. TLR4 and MyD88 expression are elevated in obesity and HF diet models, and ω-3 PUFA have been linked to suppression of TLR4/NF-κB signaling. These mixed findings underscore the need for direct comparisons and mechanistic evaluations focusing on TLR4/MyD88 pathways.

Design: Controlled animal experiment with two genotypes: wild-type C57BL/6J males (6 weeks) and MyD88−/− males on C57BL/6J background (8 weeks). Housing under standard conditions with ad libitum access to food and water. Randomization to four groups per genotype: NC diet (AIN-93M; 9.5% kcal fat), HF diet (D12451; 45.4% kcal fat), HF+fish oil (FO), HF+perilla oil (PO). Dosing: FO or PO at 0.5 g/kg body weight per day by oral gavage for 4 weeks, dose based on prior work. Sample sizes: n=10/group (C57BL/6J), n=6/group (MyD88−/−). No blinding reported. Fatty acid profiles of diets/oils provided in supplementary tables. Outcomes: Serum biochemistry (glucose, TG, TC, HDL-C, LDL-C) via DxC800 analyzer; serum insulin, MCP-1, IL-6, TNF-α, leptin, resistin via MILLIPLEX kits. Adipose histology: H&E staining of epididymal adipose; adipocyte counts per 25 mm². Molecular analyses in epididymal adipose: RT-qPCR for TLR4, MyD88, TRAF6, RIP1, IRF3, NF-κB p65 (normalized to GAPDH; 2^-ΔΔCt). Western blotting for corresponding proteins (TLR4, MyD88, TRAF6, IKKβ, RIP1, IRF3, NF-κB p65; GAPDH control); densitometry with ImageJ. Statistics: Mean±SD; one-way ANOVA with Tukey post hoc; P<0.05 deemed significant. Ethical approval by Southeast University IACUC.

- In C57BL/6J mice after 4 weeks HF feeding, HF vs NC significantly increased body weight, glucose, insulin, TG, TC, HDL-C, LDL-C, IL-6, TNF-α, leptin, and induced adipocyte hypertrophy (P<0.05). - FO and PO vs HF significantly decreased body weight, glucose, insulin, TG, TC, HDL-C, LDL-C, and IL-6, and reduced adipocyte size/increased adipocyte counts (P<0.05). HDL-C and LDL-C remained higher in PO vs FO; other variables showed no significant FO–PO differences. - At the molecular level in C57BL/6J adipose, HF upregulated TLR4, MyD88, TRAF6, IKKβ, RIP1, IRF3, and NF-κB p65 mRNA/protein. FO and PO significantly downregulated TLR4, MyD88, TRAF6, IKKβ, and NF-κB p65 (P<0.05), but not RIP1 or IRF3. Some differences between oils: PO showed higher MyD88 and IKKβ mRNA/protein and lower TRAF6 and NF-κB p65 protein vs FO in certain comparisons. - In MyD88−/− mice, HF elevated glucose, insulin, TC, HDL-C, leptin, resistin vs NC (P<0.05), with trends for TG, LDL-C, MCP-1, IL-6, TNF-α increases and reduced adipocyte counts that were not significant. FO and PO did not improve glucose, insulin, TC, HDL-C, leptin, or resistin vs HF (no significant differences), indicating loss of benefit without MyD88. - In MyD88−/− adipose, HF increased TLR4, RIP1, IRF3, and NF-κB p65 expression vs NC. FO and PO reduced TLR4 mRNA (PO < FO) but did not significantly affect RIP1, IRF3, or NF-κB p65, indicating no impact on the MyD88-independent arm. - Overall, beneficial metabolic and anti-inflammatory effects of FO and PO require MyD88 and are associated with suppression of the MyD88-dependent TLR4 signaling components (TRAF6, IKKβ, NF-κB p65).

The study demonstrates that both marine (EPA/DHA) and plant (ALA) ω-3 PUFA comparably ameliorate HF diet-induced metabolic abnormalities and adipose inflammation in wild-type mice, primarily via inhibition of the MyD88-dependent branch of TLR4 signaling, as evidenced by reductions in MyD88, TRAF6, IKKβ, and NF-κB p65 expression. The abolition of FO/PO benefits in MyD88−/− mice confirms MyD88’s necessity for these protective effects, while the lack of change in RIP1/IRF3/NF-κB p65 under MyD88 deficiency suggests limited engagement of the MyD88-independent pathway. These findings address the research question by establishing mechanistic parity between FO and PO in modulating TLR4/MyD88 signaling in adipose tissue, supporting plant ω-3 PUFA as a viable alternative to marine sources for mitigating obesity-related metabolic dysfunction. Notably, HDL-C responses highlight that changes in HDL quantity do not straightforwardly reflect cardiometabolic risk and suggest future profiling of HDL functionality/subfractions.

FO and PO confer similar protection against HF diet-induced dysmetabolism and adipose inflammation by suppressing TLR4 signaling in a MyD88-dependent manner. Plant-derived ω-3 PUFA (ALA-rich PO) can serve as an effective alternative to marine ω-3 PUFA (EPA/DHA-rich FO) for preventing obesity-associated metabolic disturbances. Future work should directly compare matched doses of purified EPA, DHA, and ALA; assess dose–response and time-course effects; and evaluate HDL subfractions and functionality to refine cardiometabolic risk assessment.

- The EPA+DHA content in FO and ALA content in PO were not dose-matched on a molar or bioactive-equivalent basis, complicating efficacy comparisons across ω-3 species. - Only a single dose and a single 4-week time point were tested, limiting insights into dose–response and temporal dynamics. - No blinding was implemented. - Outcomes were limited to epididymal adipose tissue; systemic and other tissue-specific mechanisms were not evaluated. - Lack of detailed HDL subfraction/functional assessments despite notable HDL-C changes.