Decreased glycolytic and tricarboxylic acid cycle intermediates coincide with peripheral nervous system oxidative stress in a murine model of type 2 diabetes

Abstract

Diabetic neuropathy (DN) is the most common complication of diabetes and is characterized by distal-to-proximal loss of peripheral nerve axons. The idea of tissue-specific pathological alterations in energy metabolism in diabetic complications-prone tissues is emerging. Altered nerve metabolism in type 1 diabetes models is observed; however, therapeutic strategies based on these models offer limited efficacy to type 2 diabetic patients with DN. Therefore, understanding how peripheral nerves metabolically adapt to the unique type 2 diabetic environment is critical to develop disease-modifying treatments. In the current study, we utilized targeted liquid chromatography-tandem mass spectrometry (LC/MS/MS) to characterize the glycolytic and tricarboxylic acid (TCA) cycle metabolomes in sural nerve, sciatic nerve, and dorsal root ganglia (DRG) from male type 2 diabetic mice (BKS.Cg-m+/+Lepr(db); db/db) and controls (db/+). We report depletion of glycolytic intermediates in diabetic sural nerve and sciatic nerve (glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate (sural nerve only), 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, and lactate), with no significant changes in DRG. Citrate and isocitrate TCA cycle intermediates were decreased in sural nerve, sciatic nerve, and DRG from diabetic mice. Utilizing LC/electrospray ionization/MS/MS and HPLC methods, we also observed increased protein and lipid oxidation (nitrotyrosine; hydroxyoctadecadienoic acids) in db/db tissue, with a proximal-to-distal increase in oxidative stress, with associated decreased aconitase enzyme activity. We propose a preliminary model, whereby the greater change in metabolomic profile, increase in oxidative stress, and decrease in TCA cycle enzyme activity may cause distal peripheral nerves to rely on truncated TCA cycle metabolism in the type 2 diabetes environment.

Figure 1. Increased oxidative stress in diabetic sciatic nerve and DRG

A. Fold changes in oxidative stress measures from whole sciatic nerve and DRG were calculated from the ratio of db/db to db/+. The nitrated protein content and HODEs were increased in whole sciatic nerve and DRG from diabetic mice compared with those from db/+ controls (* p<0.05; ** p<0.01; *** p<0.001 vs. db/+). The diabetic fold increase in oxidized lipids was greater in sciatic nerve than DRG (^ф p<0.05). Data expressed as mean fold-change ±SEM; 6 db/+ and db/db DRG; 12 db/+ and db/db sciatic nerve. There were no changes in oxidative stress measures between proximal and distal segments of db/+ control sciatic nerves (B, C). B. Oxidized lipids (HODEs) were elevated in proximal and distal db/db sciatic nerve, respectively, compared with those of db/+ mice. Diabetic sciatic nerve HODEs were greater distally than proximally. C. The nitrotyrosine-to-tyrosine ratio was elevated in proximal and distal db/db sciatic nerve, respectively, compared with those of db/+ mice. Data expressed as mean ±SEM; 6 db/+ and db/db sciatic nerve. * p<0.05; ** p<0.01. SCN, sciatic nerve; DRG, dorsal root ganglia; P, proximal; D, distal; NT, 3-nitrotyrosine. All data from male mice.

Figure 2. Decrease in aconitase enzyme activity in diabetic sciatic nerve and DRG

Aconitase activity was 1.6-fold greater in DRG than sciatic nerve in control (db/+) mice (^ϕϕ p<0.01), with this tissue difference maintained in the db/db mice (^ф p<0.05). Type 2 diabetes was associated with a decrease in aconitase activity in both tissues (** p<0.01 compared with db/+). Data expressed as mean ±SEM; 6 db/+ and db/db. SCN, sciatic nerve; DRG, dorsal root ganglia. All data from male mice.

Figure 3. Proposed relationship between diabetes, ROS, and the TCA cycle

ROS-mediated inhibition of aconitase may truncate the TCA cycle in diabetic sural nerve, sciatic nerve and DRG (bold arrows). This would result in the observed maintenance of succinate, fumarate, and malate levels in the face of decreased citrate and isocitrate. Reduced glycolytic intermediates feeding into the TCA cycle in sural nerve and sciatic nerve may contribute to the observed decreases in citrate and isocitrate and be interpreted as a glucose-deficient state. Activation of the α-KG-utilizing truncated TCA cycle pathway in the sural and sciatic nerves, would further contribute to ROS production, and to the greater oxidative stress observed in diabetic sciatic nerve than DRG. α-KGDH, alpha-ketoglutarate dehydrogenase; DRG, dorsal root ganglia; TCA, tricarboxylic acid; ROS, reactive oxygen species.