Long-term omega-3 fatty acid supplementation ameliorates cochlear homocysteine metabolism and progressive hearing loss in mice

Póster presentado a la Conferencia de la International Academy on Nutrition and Aging (IANA), celebrada en Barcelona (España) del 18 al 19 de junio de 2015.[Background]: Ageing is associated with a variety of chronic illnesses, including cancer and cardiovascular disease and also with progressive sensory impairment. In fact, about 30% of the human population over 65 is affected by age-related hearing loss (ARHL). Several studies have shown an association between deficiencies in essential nutrients and hearing loss, including: i) an inverse association between high plasma levels of omega-3 (w3) polyunsaturated fatty acids (PUFAs) and AHRL; and ii) a correlation between low serum folate, and the resulting elevated plasma homocysteine (tHcy) levels, and hearing loss. High levels of tHcy have been long considered and independent risk factor for cardiovascular disease, but recently increases in this parameter were also proposed a risk factor of human hearing loss. These associations between hearing loss and nutritional deficits have suggested the preventive potential of nutrition intervention in this field, and especially in ARHL. Essential nutrients such as PUFAs are well-known for their beneficial effects, among others on cognitive development and maintenance, inflammation and oxidative stress. The general basis for this protection seems to rely on the relationship between vascular disease and hearing loss. Precisely, microvascular disease seems to cause the atrophy of the stria vascularis, which is the structure within the cochlea that maintains its metabolic and ionic homeostasis. Altogether the accumulated data suggest that a reduction in the blood supply of essential nutrients into the cochlea, and the subsequent metabolic alterations, could be among the main triggers of hearing loss progression and that supplementation may preclude these effects. [Methods]: Eight-week old C57BL/6J female mice (N=28) were divided into two experimental groups that were fed up to eight months either control (N= l 9) or w3-supplemented diets (N=9). For comparative purposes, the control group was further divided into 4- (C-4M, N=4) and 10-months old (C-IOM, N=l 5) subgroups. Control and supplemented diets differed in the content of soybean oi, that was partially substituted by fish oil Eupoly-3 EPA(1.6g/kg) in the w3-diet. Hearing was evaluated by auditory brainstem response (ABR) analysis to broadband click and 8, 16, 20, 28, and 40 kHz pure tone frequencies. The recording of distortion product otoacoustic emissions (DPOAE) was performed after stimulation with f l and f2 primary tones, with a ratio f2/f l = 1.2. Primary tones for 8, 1 O, 14, 18 and 22 kHz frequencies were tested. RT -qPCR and Western blotting were used to determine cochlear levels of the methionine cycle enzymes, cytokines and stress biomarkers. Total plasma Hcy (tHcy) was determined by HPLC analysis and serum folate levels were analyzed by a microbiological method using Lactobacillus casei.[Results]: No differences in ABR thresholds, wave amplitudes and latencies were detected at 7 months of age between groups. Nonetheless, a significant decrease in the DPOAE 2fl-f2 amplitude at 8 kHz measured at intensities of 20 dB SPL above threshold was detected in the control group compared to C-4M mice. Hearing loss was evident at 10 months of age, where significant increases in the ABR thresholds at all frequencies were detected in control versus w3 mice. Ageing in the control group was associated with imbalanced cochlear cytokine expression toward increased pro-inflammatory cytokines as determined by RT- qPCR. 111b and 116 expression levels decreased in w3 compared to C-IOM cochleae. Moreover, decreased expression of lgfl and lgfl r was lower in w3 than C-IOM cochleae when compared to C-4M samples. Western blotting analysis of cochlear stress signaling pathways revealed higher P-JNK levels in the C-10M than in w3 cochleae. Additionally, Cat and Gss cochlear expression was reduced in C-10M compared to C-4M cochleae, a change partially prevented in the w3 group. During aging a reduction in the expression of Hcy metabolism genes was observed in C-10M cochleae, only alterations in Bhmt and Cbs being significantly prevented by w3 feeding. Western blotting showed that w3 supplementation precluded the CBS protein increase detected in C-10M cochleae, but induced BHMT protein levels. Furthermore, anti-Hcy western blotting of cochlear extracts showed no differences in global protein homocysteinylation between groups. Finally, similar tHcy and serum folate levels were measured in CIOM and w3-10M groups. [Conclusion]: Long-term w3-supplementation partially prevents progression of hearing loss in C57BL/6J mice. This effect is mainly exerted through maintenance of JGF-1 signaling and the balance between pro- and anti-inflammatory cytokines. The increased transsulfuration/remethylation ratio for Hcy elimination observed in C-lOM cochleae is changed by the w3-diet towards an increased BHMT remethylation, indicating an attempt to preserve the flux through the methionine cycle. Altogether, the results obtained suggest a long-term protective role of w3-supplementation on cochlear metabolism and progression of hearing loss.RMY and NY are fellows of the JAE-CSIC predoctoral program and FP7-AFHELO, respectively. This work was supported by grants SAF2011-24391, BFU2009-08977, FP7-AFHELO, FP7-TARGEAR and Puleva-LACTALIS. SAF2014-53979-R and BFU2009-08977.Peer Reviewe