Dietary Omega-3 Polyunsaturated Fatty-Acid Supplementation Upregulates Protective Cellular Pathways in Patients with Type 2 Diabetes Exhibiting Improvement in Painful Diabetic Neuropathy

Abstract

Background: Omega-3 polyunsaturated fatty acids (PUFAs) have been proposed to improve chronic neuroinflammatory diseases in peripheral and central nervous systems. For instance, docosahexaenoic acid (DHA) protects nerve cells from noxious stimuli in vitro and in vivo. Recent reports link PUFA supplementation to improving painful diabetic neuropathy (pDN) symptoms, but cellular mechanisms responsible for this therapeutic effect are not well understood. The objective of this study is to identify distinct cellular pathways elicited by dietary omega-3 PUFA supplementation in patients with type 2 diabetes mellitus (T2DM) affected by pDN.

Methods: Forty volunteers diagnosed with type 2 diabetes were enrolled in the "En Balance-PLUS" diabetes education study. The volunteers participated in weekly lifestyle/nutrition education and daily supplementation with 1000 mg DHA and 200 mg eicosapentaenoic acid. The Short-Form McGill Pain Questionnaire validated clinical determination of baseline and post-intervention pain complaints. Laboratory and untargeted metabolomics analyses were conducted using blood plasma collected at baseline and after three months of participation in the dietary regimen. The metabolomics data were analyzed using random forest, hierarchical clustering, ingenuity pathway analysis, and metabolic pathway mapping.

Results: The data show that metabolites involved in oxidative stress and glutathione production shifted significantly to a more anti-inflammatory state post supplementation. Example of these metabolites include cystathionine (+90%), S-methylmethionine (+9%), glycine cysteine-glutathione disulfide (+157%) cysteinylglycine (+19%), glutamate (-11%), glycine (+11%), and arginine (+13.4%). In addition, the levels of phospholipids associated with improved membrane fluidity such as linoleoyl-docosahexaenoyl-glycerol (18:2/22:6) (+253%) were significantly increased. Ingenuity pathway analysis suggested several key bio functions associated with omega-3 PUFA supplementation such as formation of reactive oxygen species (p = 4.38 × 10^-4, z-score = -1.96), peroxidation of lipids (p = 2.24 × 10^-5, z-score = -1.944), Ca²⁺ transport (p = 1.55 × 10^-4, z-score = -1.969), excitation of neurons (p = 1.07 ×10^-4, z-score = -1.091), and concentration of glutathione (p = 3.06 × 10^-4, z-score = 1.974).

Conclusion: The reduction of pro-inflammatory and oxidative stress pathways following dietary omega-3 PUFA supplementation is consistent with the promising role of these fatty acids in reducing adverse symptoms associated with neuroinflammatory diseases and painful neuropathy.

Keywords: metabolism; metabolomics; omega-3; painful diabetic neuropathy; polyunsaturated fatty acids.

Scheme 1

Study Description. DHA, Docosahexaenoic acid; EPA, Eicosapentaenoic acid; T2DM, type 2 diabetes mellitus; SF-MPQ, Short-form McGill Pain Questionnaire.

Figure 1

Dendrogram from Hierarchical Cluster Analysis. Baseline and three-month samples from the same subject clustered together in multiple instances. Samples from the same time point had a moderate tendency toward adjacency.

Figure 2

RF classification of plasma samples collected at baseline and 3-months after omega-3 PUFA supplementation. (A) Classification of all participants’, regardless of pain status, metabolites were 93% accurate for samples. Top factors contributing to group separation shown in the biochemical importance plot. (B) Classification of all participants’, who reported pain symptoms, metabolites were 92% accurate for samples. (C) A one-way ANOVA revealed that there was a statistically significant difference in mean 1-linoleoyl-GPA (18:2) relative plasma levels (fold change) between at least two groups (F(3, 42) = (14.63), p ≤ 0.0001). Bonferroni’s post hoc analyses showed significant 1-linoleoyl-GPA (18:2) metabolite plasma level change when comparing No Pain BL to Mod-High Pain BL groups (p = 0.0143, 95% C.I. = (−1.158, −0.097)), No Pain BL to No Pain 3mo groups (p = 0.0220, 95% C.I. = (0.063, 1.100)), and Mod-High Pain BL to Mod-High Pain 3mo (p ≤ 0.0001, 95% C.I. = [0.578, 1.66]). * p ≤ 0.05 and **** p ≤ 0.0001.

Figure 3

Changes in cysteine, methionine, and glutathione metabolism overlaid on a metabolic map. The size of each colored bubble is proportional to the fold change (increase, red; decrease, blue). The baseline bubble in the legend corresponds to a fold change of 1. 5-MeTHF, 5-Methyltetrahydrofuran-2-ol; NADH, nicotinamide adenine dinucleotide (NAD) + hydrogen (H); NAD, nicotinamide adenine dinucleotide; Propionyl CoA, propionyl coenzyme A; TCA cycle, tricarboxylic acid cycle.

Figure 4

Glycerolipid and glucose metabolism changes overlaid on a metabolic map. The size of each colored bubble is proportional to the fold change (increase, red; decrease, blue). The baseline bubble in the legend corresponds to a fold change of 1. glucose-6-P, glucose-6-phosphate; fructose-6-P, Fructose-6-phosphate; fructose 1,6 bisP, fructose 1,6, bisphosphate; glyceraldehyde-3-P, glyceraldehyde-3-phosphate; DHAP, Dihydroxyacetone phosphate; acyl-CoA, acetyl coenzyme A; ATP, adenosine triphosphate; CTP, Cytidine triphosphate; CDP-choline, cytidine diphosphate-choline; CDP-ethanolamine, cytidine diphosphate-ethanolamine; CMP, cytidine monophosphate.

Figure 5

Changes in oxidation of fatty acids overlaid on a metabolic map. The size of each colored bubble is proportional to the fold change (increase, red; decrease, blue). The baseline bubble in the legend corresponds to a fold change of 1. C Acyl-Carnitines, Long Chain Acetyl-Carnitine; LCFA, Long Chain Fatty Acids; MCFA, Medium Chain Fatty Acids; EFA, Essential Fatty Acids; MUFA, Monounsaturated Fatty Acids; PUFA, Polyunsaturated Fatty Acids; LC acyl-CoA, Long Chain acetyl-coenzyme A; MC acyl-CoA, Medium Chain acetyl-coenzyme A; MC acyl-carnitine, Medium Chain acetyl-carnitine.