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Elevated dietary ω-6 polyunsaturated fatty acids induce reversible peripheral nerve dysfunction that exacerbates comorbid pain conditions

Abstract

Chronic pain is the leading cause of disability worldwide1 and is commonly associated with comorbid disorders2. However, the role of diet in chronic pain is poorly understood. Of particular interest is the Western-style diet, enriched with ω-6 polyunsaturated fatty acids (PUFAs) that accumulate in membrane phospholipids and oxidise into pronociceptive oxylipins3,4. Here we report that mice administered an ω-6 PUFA-enriched diet develop persistent nociceptive hypersensitivities, spontaneously active and hyper-responsive glabrous afferent fibres and histologic markers of peripheral nerve damage reminiscent of a peripheral neuropathy. Linoleic and arachidonic acids accumulate in lumbar dorsal root ganglia, with increased liberation via elevated phospholipase (PLA)2 activity. Pharmacological and molecular inhibition of PLA2G7 or diet reversal with high levels of ω-3 PUFAs attenuate nociceptive behaviours, neurophysiologic abnormalities and afferent histopathology induced by high ω-6 intake. Additionally, ω-6 PUFA accumulation exacerbates allodynia observed in preclinical inflammatory and neuropathic pain models and is strongly correlated with multiple pain indices of clinical diabetic neuropathy. Collectively, these data reveal dietary enrichment with ω-6 PUFAs as a new aetiology of peripheral neuropathy and risk factor for chronic pain and implicate multiple therapeutic considerations for clinical pain management.

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Fig. 1: An ω-6 fatty acid–enriched diet induces a peripheral neuropathy–like phenotype in mice.
Fig. 2: The H6D increases membrane loading of ω-6 PUFAs and stimulates PLA2 activity in peripheral afferent neurons.
Fig. 3: An ω-3 fatty acid–enriched diet rescues the H6D-induced neuropathy-like phenotype.
Fig. 4: Diet-specific modulation of nociceptive behaviours associated with inflammatory and neuropathic pain.

Data availability

Data that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

The project described was supported in part by the National Center for Advancing Translational Sciences, National Institutes of Health (NIH), through grant UL1TR002645 (K.M.H.). Additional support from the NIH includes grants R01NS110948 (K.M.H.), T32DE14318 (P.M.L., A.R.F., K.M.H.), T32GM113896 (J.T.B.), F30AT009949 (J.T.B.), F32DK118841 (P.M.L.), F30DE028486 (A.R.F.) and a grant from the Ella and Williams Owen’s Foundation (K.M.H.). Clinical data were managed using REDCap software supported by UL1RR024982. Certain mass spectrometric analyses were carried out on equipment supported by the US Department of Agriculture, Agricultural Research Service, under agreement no. 58-3094-8-012. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the US Department of Agriculture. We thank X. Han and his laboratory for expertise and guidance on shotgun lipidomics. We thank M. Patil and P. Wu for technical assistance as well as A. Diogenes, N. Ruparel, A. Khan and A. Akopian for fruitful discussions.

Author information

Authors and Affiliations

Authors

Contributions

J.T.B., P.M.L., S.B.R. and K.M.H. conceived and designed the studies; J.T.B., P.M.L., M.R.B. and M.T. conducted behavioural experiments; A.R.F., P.M.L. and F.-M.C. conducted single-fibre electrophysiology; P.M.L. and J.T.B. performed histology; Q.L., F.-M.C. and P.M.L. performed BODIPY experiments; P.M.L., D.A.A. and M.T. performed total tissue lipid extractions; M.E.C., G.M.S. and S.B.H.B. conducted quantitative LC–MS/MS; P.M.L. and F.-M.C. performed western blots; E.E.L. and A.T. conducted neurological assessments on trial participants and collected skin punch biopsies; J.T.B., P.M.L. and K.M.H. performed data analysis for all experiments; P.M.L. and J.T.B. prepared figures, images and illustrations; P.M.L., J.T.B. and K.M.H. wrote the manuscript; all authors revised the manuscript.

Corresponding author

Correspondence to Kenneth M. Hargreaves.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Metabolism thanks Jing Kang, Ru-Rong Ji and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Christoph Schmitt.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 H6D induces persistent nociceptive hypersensitivities in both male and female mice.

a-f, Time courses of changes in mechanical withdrawal threshold (a,c,e) and heat withdrawal latency (b,d,f) for male and female mice on the H6D and L6D. The top two plots (a,b) represent compiled male and female responses (L6D, n = 24; H6D, n = 29). a, ***P = 0.0003 week 4, ****P < 0.0001 weeks 8–24. b, ****P < 0.0001 weeks 4–24. The middle plots (c,d) are male-only responses (L6D, n = 12; H6D, n = 16). c, *P = 0.0401 week 4, ****P < 0.0001 weeks 8–24. d, ****P < 0.0001 weeks 4–24. The bottom plots (e,f) are female-only responses (L6D, n = 12; H6D, n = 13). e, **P = 0.0055 week 16, 0.0023 week 20, ***P = 0.0009 week 4, 0.0005 week 8, ****P < 0.0001 weeks 12,24. f, **P = 0.0022 week 4, ****P < 0.0001 weeks 8–24. Data are mean ± SEM. Error bars for some data points are within the size of the symbol. The statistical test used was two-way repeated-measures ANOVA with Sidak’s post-hoc test (a-f).

Source data

Extended Data Fig. 2 The H6D sensitizes afferent fibers to mechanical and heat stimuli.

a, Representative recording wavemarks from L6D and H6D mice during mechanical force application. The number of action potentials are denoted beneath each stimulation for each recording. b, Percentage of fibers exhibiting post-stimulus afterdischarge following mechanical force application. Values represent the number of fibers exhibiting afterdischarge over the total recorded fibers for each group (L6D, n = 34; H6D, n = 44). **P = 0.0096. c, The Peltier-based heat delivery system setup. d, Conduction velocities determined for recorded C (left) and AM (right) fibers from L6D and H6D mice (L6D-C, n = 19; H6D-C, n = 20; L6D-AM, n = 25; H6D-AM, n = 24). Dotted line represents cut-off value for C fiber classification. e,f, Glabrous IENF densities (e) and percentage of ATF3+ neurons in lumbar DRG (f) after 4 weeks on the L6D or H6D (L6D, n = 3; H6D, n = 3). g, Representative immunofluorescent staining of ATF3 expression in trigeminal ganglia (TG) from L6D and H6D mice co-localized with NeuN, scale bars: 50 μm (n = 2 mice/group). h,i, Immunofluorescent staining of Iba1 (h) and c-Fos (i) expression in the lumbar spinal cord of L6D and H6D mice. Positive control tissue was utilized from db/db mice. White arrowheads designate microglia (h), 10X magnification, scale bar: 50 μm (n = 2 mice/group). Red arrowheads designate c-Fos+ nuclei (i), 20X magnification, scale bar: 50 μm (n = 2 mice/group). All data are mean ± SEM. The statistical test used was a two-sided Fisher’s exact test (b).

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Extended Data Fig. 3 The H6D does not induce a diabetic phenotype.

a,b, Scatter plots of fasting blood glucose levels (a) and HbA1c levels (b) from mice on L6D and H6D for 8 weeks. Mice on normal chow (NC) and 16-week-old db/db mice served as negative and positive controls, respectively (NC, n = 5; L6D, n = 5; H6D, n = 5; db/db, n = 5). Dotted lines in each figure represent established cut-offs for type 2 diabetes. ****P < 0.0001 (db/db vs NC). c,d, Weekly monitoring of body weights (c) and food intake (d) for both male and female mice on either L6D or H6D. Data are mean ± SEM. Error bars for some data points are within the size of the symbol. Statistical test used was one-way ANOVA with Tukey’s post-hoc test (a,b).

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Extended Data Fig. 4 The H6D alters lipid composition in DRG, but not spinal cord.

a,b, Heatmaps of lipid species from lumbar DRG (a) and spinal cord (b) from male (♂) and female (♀) mice on either the H6D or L6D. Lipid classes are designated to the left of each heatmap. Scale bar represents z-score transformations for each lipid species. c,d, Scatter plots of LA- and AA-esterified lipids (c) as well as ω-3 content (d, EPA, DHA levels) in DRG sub-profiled by lipid class for male and female mice on either diet (n = 3 mice/group/sex). c, Acyl carnitine (LA): *P = 0.0346, ***P = 0.0004. Acyl carnitine (AA): *P = 0.0417 (♂), *P = 0.0214 (♀). Ethanolamine plasmalogens (LA): ***P = 0.0008, ****P < 0.0001. Ethanolamine plasmalogens (AA): **P = 0.0027, ***P = 0.0007. Fatty acyl chains (LA): **P = 0.0069. Lyso-phosphatidylcholine (LA): ***P = 0.0002, ****P < 0.0001. Lyso-phosphatidylcholine (AA): *P = 0.0161, *P = 0.0215. Lyso-phosphatidylethanolamine (LA): ****P < 0.0001. Phosphatidic acid (LA): **P = 0.005, *P = 0.0166. Phosphatidic acid (AA): ****P < 0.0001, ***P = 0.0001. Phosphatidylethanolamine (LA): ***P = 0.0006, ***P = 0.0002. Phosphatidylethanolamine (AA): **P = 0.0015, ***P = 0.0007. Phosphatidylglycerol (AA): **P = 0.0033. Phosphatidylinositol (AA): **P = 0.003, ***P = 0.001. Phosphatidylserine (LA): ***P = 0.0001, **P = 0.0072. d, Lyso-phosphatidylcholine: *P = 0.0316. Phosphatidylglycerol: **P = 0.0062. Data are mean ± SEM. Statistical tests used were two-way ANOVA with Tukey’s post-hoc test (c) and one-way ANOVA with Tukey’s post-hoc test (d).

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Extended Data Fig. 5 PLA2g7 expression predominates in neuronal subpopulations of the lumbar DRG.

a, Heatmap indicating PLA2 isozyme expression across established neuronal subpopulations of the mouse lumbar DRG. Single-cell RNA-seq data were reproduced with permission36. b,c, qPCR data showing PLA2 isozyme expression (b) and change in cycle threshold values relative to 18 S rRNA (c) in lumbar DRG from H6D and L6D mice (b: n = 3/group. c: L6D, n = 6; H6D, n = 6). d, Representative immunofluorescent staining of PLA2g7 expression in mouse lumbar DRG and co-localization with neuronal subtype-specific markers. No primary controls are included for each marker as designated. The magenta arrows highlight two small diameter neurons, one with high PLA2g7 expression and one with low to moderate expression, that are negative for NFH. White arrows designate cell bodies with PLA2g7+ staining and their co-localization with the respective subtype marker. White arrowheads highlight axons projecting through the ganglia that exhibit virtually no PLA2g7 expression compared to cell bodies. Scale bars: 50 μm (n = 2 mice). e, Representative immunofluorescent staining of lumbar DRG from naïve control mice that received either scrambled or PLA2g7-directed siRNA intrathecally for the purpose of PLA2g7 antibody validation. White arrows highlight PLA2g7 + staining of neuronal cell bodies, whereas the white arrowheads designate cells exhibiting loss of PLA2g7 immunoreactivity. Scale bars: 50 μm, (n = 2 mice/group). f,g, Circulating plasma PLA2g7 levels (f) and plasma LA accumulation (g) from L6D and H6D mice after 8 weeks (L6D, n = 4; H6D, n = 5). **P < 0.0022. Data are mean ± SEM. Error bars for some data points are within the size of the symbol. Statistical test used was unpaired two-tailed Student’s t test (g).

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Extended Data Fig. 6 Pharmacological inhibition and genetic knockdown of PLA2g7 in DRG neurons reduces PLA2 activity and attenuates nociceptive hypersensitivities.

a, Optimization of DRG protein concentration used with the PLA2 BODIPY activity assay. H6D DRG homogenates demonstrate increased activity at multiple concentrations compared to L6D (BSA, n = 4; L6D-300, n = 6; L6D-150, n = 7; L6D-75, n = 7; H6D-300, n = 5; H6D-150, n = 7; H6D-75, n = 7 DRG replicates/group). **P = 0.0055 (300), 0.0023 (150). b, qPCR data showing annexin isozyme expression in L6D and H6D DRG (L6D, n = 6; H6D, n = 5). c, Immunoblots of PLA2g7 and GAPDH protein expression in membrane and cytosolic fractions from homogenized DRG (L6D, n = 4 mice; H6D, n = 3 mice). Molecular weight markers (kDa) are adjacent to each target. d, Concentration-response curves for darapladib-mediated inhibition of PLA2 activity for DRG (n = 3/group). e, Darapladib half-maximal inhibitory concentrations (IC50) as determined by nonlinear regression (n = 3/group). f, Total LA and AA levels determined from glabrous hindpaw skin punches (L6D, n = 6; H6D, n = 6). **P = 0.0098 (LA), 0.0071 (AA). g, Dose-response timecourses for i.pl. darapladib on heat- and mechanical-evoked nociception (L6D-VEH, n = 6; L6D-3, n = 4; L6D-30, n = 9; H6D-VEH, n = 6; H6D-3, n = 5; H6D-30, n = 9). h,i, PLA2g7 qPCR data for lumbar DRG (h) and spinal cord (i) following intrathecal siRNA treatment (q.d. x 3d) (h: L6D-scr, n = 7; H6D-scr, n = 6; L6D-PLA2g7, n = 8; H6D-PLA2g7, n = 6. i: n = 3/group). h, *P = 0.0429 (L6D-PLA2g7), 0.0371 (H6D-PLA2g7). i, *P = 0.0333. j, Immunofluorescent images of glabrous IENFs from siRNA-treated mice, scale bar: 50 μm (n = 2 mice/group). k,l, Mechanical force-response curves (k) and EF50 values (l) for 16-week db/db mice injected i.pl. with either vehicle or darapladib (db/db-veh, n = 5; db/db-DARA, n = 6). l, **P = 0.0020. m,n, Mechanical force-response curves (m) and EF50 values (n) for a different cohort of 16-week db/db mice following i.t. siRNA injections (db/db-veh, n = 5; db/db-darapladib, n = 5). n, **P = 0.0066. All data are mean ± SEM. Error bars for some data points are within the size of the symbol. Statistical tests used were one-way ANOVA with Sidak’s post-hoc test (a), Tukey’s post-hoc test (d), or Dunnett’s post-hoc test (h,i), two-way ANOVA with Tukey’s post-hoc test (c), and unpaired two-tailed Student’s t test (f,l,n).

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Extended Data Fig. 7 H3D reverses H6D-induced changes in afferent fibers.

a, Discharge frequencies of spontaneously-active fibers (H6D, n = 30; H3D, n = 41). b, Discharge frequencies of AM fibers (H6D, n = 13; H3D, n = 21). *P = 0.0353 (75), 0.0335 (100), **P = 0.0047 (150). c,d, Representative immunofluorescence staining of glabrous hindpaw skin IENFs (c) and ATF3 expression in lumbar DRG neurons (d) in H6D and H3D mice. Scale bars: 25 μm (c; n = 4 mice/group), 50 μm (d; n = 3 mice/group). The representative images supplement Figs. 3h and 3i, respectively. Data are mean ± SEM. Error bars for some data points are within the size of the symbol. Statistical test used was two-way ANOVA with Sidak’s post-hoc test (b).

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Extended Data Fig. 8 Increased LA content in skin of diabetic subjects with painful neuropathy.

a, Chromatogram snapshots of the endogenous LA peak (labeled A) overlaid with the LA-d4 internal control peak (labeled B) for skin biopsy extracts from diabetic and control subjects. Integrated AUC values (a.u.) for each peak are beneath each chromatogram. b-d, Correlation analyses between subject skin LA levels and their respective LANSS scores (b), NPSI scores (c), and hallux vibration detection thresholds (d) (control, n = 12; diabetic, n = 16). Linear regression identified the best-fit line (solid line) with 95% confidence intervals (dotted lines). Inset boxes contain Spearman coefficients (rs) and corresponding P-values. Statistical test used was two-tailed Spearman correlation.

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Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1. Diet breakdowns. Supplementary Table 2. Clinical data.

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Boyd, J.T., LoCoco, P.M., Furr, A.R. et al. Elevated dietary ω-6 polyunsaturated fatty acids induce reversible peripheral nerve dysfunction that exacerbates comorbid pain conditions. Nat Metab 3, 762–773 (2021). https://doi.org/10.1038/s42255-021-00410-x

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