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Cited by 264Pain Mechanisms and TreatmentsPeripheral Neuropathies and DisordersAldose Reductase and Taurine

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Dyslipidemia-Induced Neuropathy in Mice

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Vincent, Andrea M., Hayes, John M., McLean, Lisa L., Vivekanandan-Giri, Anuradha, Pennathur, Subramaniam, Feldman, Eva L.·2009·Diabetes
DOI 10.2337/db09-0047PMC PMC2750230
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Abstract

OBJECTIVE: Neuropathy is a frequent and severe complication of diabetes. Multiple metabolic defects in type 2 diabetic patients result in oxidative injury of dorsal root ganglia (DRG) neurons. Our previous work focused on hyperglycemia clearly demonstrates induction of mitochondrial oxidative stress and acute injury in DRG neurons; however, this mechanism is not the only factor that produces neuropathy in vivo. Dyslipidemia also correlates with the development of neuropathy, even in pre-diabetic patients. This study was designed to explore the contribution of dyslipidemia in neuropathy. RESEARCH DESIGN AND METHODS: Mice (n = 10) were fed a control (10% kcal %fat) or high-fat (45% kcal %fat) diet to explore the impact of plasma lipids on the development of neuropathy. We also examined oxidized lipid–mediated injury in cultured DRG neurons from adult rat using oxidized LDLs (oxLDLs). RESULTS: Mice on a high-fat diet have increased oxLDLs and systemic and nerve oxidative stress. They develop nerve conduction velocity (NCV) and sensory deficits prior to impaired glucose tolerance. In vitro, oxLDLs lead to severe DRG neuron oxidative stress via interaction with the receptor lectin-like oxLDL receptor (LOX)-1 and subsequent NAD(P)H oxidase activity. Oxidative stress resulting from oxLDLs and high glucose is additive. CONCLUSIONS: Multiple metabolic defects in type 2 diabetes directly injure DRG neurons through different mechanisms that all result in oxidative stress. Dyslipidemia leads to high levels of oxLDLs that may injure DRG neurons via LOX-1 and contribute to the development of diabetic neuropathy.

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FIG. 1. Mild neuropathy after 12 weeks of high-fat diet. Following 12 weeks of high-fat or control diet, weight (A), blood glucose (B), hindpaw withdrawal latency (C), and sciatic NCV (D) were assessed. n = 10/group. *P < 0.05 vs. control diet.
FIG. 1. Mild neuropathy after 12 weeks of high-fat diet. Following 12 weeks of high-fat or control diet, weight (A), blood glucose (B), hindpaw withdrawal latency (C), and sciatic NCV (D) were assessed. n = 10/group. *P < 0.05 vs. control diet.CC BY-NC-ND · PubMed Central
FIG. 2. Neuropathy and impaired glucose tolerance after 34 weeks on high-fat diet. Complete phenotyping after 34 weeks on control (Ctrl) or high-fat (HF) diet was performed. The figure displays the glucose tolerance test (A), weight (B), plasma insulin (C), GHb (D), sural NCV (E), sciatic NCV (F), hindpaw withdrawal latency (G), and IENFD (H). *P < 0.05 vs. the control diet group. In all panels, n = 10. A: ♦, control; □, high fat. I: Representative IENFD images from one control-fed and one high-fat–fed sample. Bar = 50 μm; d = dermis, e = epidermis. White dots indicate nerve fibers counted. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 2. Neuropathy and impaired glucose tolerance after 34 weeks on high-fat diet. Complete phenotyping after 34 weeks on control (Ctrl) or high-fat (HF) diet was performed. The figure displays the glucose tolerance test (A), weight (B), plasma insulin (C), GHb (D), sural NCV (E), sciatic NCV (F), hindpaw withdrawal latency (G), and IENFD (H). *P < 0.05 vs. the control diet group. In all panels, n = 10. A: ♦, control; □, high fat. I: Representative IENFD images from one control-fed and one high-fat–fed sample. Bar = 50 μm; d = dermis, e = epidermis. White dots indicate nerve fibers counted. (A high-quality digital representation of this figure is available in the online issue.)CC BY-NC-ND · PubMed Central
FIG. 3. Plasma lipid profiles in the plasma at 34 weeks. Pooled plasma samples (two pools per group, each pool analyzed three times) were subjected to fractionation by FPLC; then cholesterol (A) and triglycerides (B) were measured in each fraction. The graph shows the means ± SE for n = 2 pools/group. ■ regular diet; ▴, high-fat diet.
FIG. 3. Plasma lipid profiles in the plasma at 34 weeks. Pooled plasma samples (two pools per group, each pool analyzed three times) were subjected to fractionation by FPLC; then cholesterol (A) and triglycerides (B) were measured in each fraction. The graph shows the means ± SE for n = 2 pools/group. ■ regular diet; ▴, high-fat diet.CC BY-NC-ND · PubMed Central
FIG. 4. LOX-1 expression on DRG neurons. DRG neurons from adult rat were assessed for LOX-1 by Western blotting (A and B) and immunocytochemistry (C) after 3 days in culture. A: A total of 40 mg protein from untreated control (C) cultures in lane 1 display the glycosylated 48-kDa isoform of LOX-1 that is predicted to be at the cell surface. The lower–molecular weight precursor (40 kDa) and nonglycosylated (32 kDa) isoform are also present. Exposure to high glucose (G) (20 mmol/l added, total 25.7 mmol/l glucose) or 1 μg/ml oxLDL for 3 h did not alter LOX-1 expression. A total of 5, 10, or 30 mg/ml oxLDL for 3 h increased the level of the 48-kDa isoform, and the two higher doses appear to decrease the lower–molecular weight forms. +, a positive control peptide based on the 40 kDa predicted precursor form of LOX-1. B: Densitometry was performed on three replicates of the representative blot shown in A. Mean pixel densities for the 48-kDa band are normalized against actin. The graph shows the means ± SE. *P < 0.05 vs. the untreated control. C: A representative image of untreated DRG neurons is shown. White arrows indicate neurons labeled for LOX-1. Bar = 50 μm. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 4. LOX-1 expression on DRG neurons. DRG neurons from adult rat were assessed for LOX-1 by Western blotting (A and B) and immunocytochemistry (C) after 3 days in culture. A: A total of 40 mg protein from untreated control (C) cultures in lane 1 display the glycosylated 48-kDa isoform of LOX-1 that is predicted to be at the cell surface. The lower–molecular weight precursor (40 kDa) and nonglycosylated (32 kDa) isoform are also present. Exposure to high glucose (G) (20 mmol/l added, total 25.7 mmol/l glucose) or 1 μg/ml oxLDL for 3 h did not alter LOX-1 expression. A total of 5, 10, or 30 mg/ml oxLDL for 3 h increased the level of the 48-kDa isoform, and the two higher doses appear to decrease the lower–molecular weight forms. +, a positive control peptide based on the 40 kDa predicted precursor form of LOX-1. B: Densitometry was performed on three replicates of the representative blot shown in A. Mean pixel densities for the 48-kDa band are normalized against actin. The graph shows the means ± SE. *P < 0.05 vs. the untreated control. C: A representative image of untreated DRG neurons is shown. White arrows indicate neurons labeled for LOX-1. Bar = 50 μm. (A high-quality digital representation of this figure is available in the online issue.)CC BY-NC-ND · PubMed Central
FIG. 5. High glucose and oxLDL induce oxidative stress in adult DRG neurons. After 3 days in culture, oxidative stress assays were performed in cultured DRG neurons. A: DRG neurons were exposed to increasing concentrations of oxLDL in the presence of basal (5.7 mmol/l) or high (25.7 mmol/l) glucose for 1 h, then loaded with MitoSOX for 15 min. n = 9, *P < 0.01 vs. control (basal glucose, 0 μg/ml oxLDL). ■, basal glucose; ▩, high glucose. B: DRG neurons were exposed to increasing concentrations of oxLDL alone or in the presence of the mitochondrial uncoupler FCCP (2.5 μmol/l) or the antioxidant α-lipoic acid (LA) (100 μmol/l) for 1 h, then loaded with DHE for 15 min. n = 9; *P < 0.01, +P < 0.05 vs. control. C: TRAP was assessed in untreated DRG neurons and those exposed to 30 μg/ml oxLDL for 3 h, n = 3, *P < 0.01. D: Nitrotyrosine fluorescence was assessed 24 h following exposure to high glucose or increasing concentrations of oxLDL in the presence or absence of LOX-1–neutralizing antibody (100 mg/ml). ■, none; □, anti–LOX-1. n = 9, *P < 0.01 vs. untreated control; †P < 0.01 vs. no antibody.
FIG. 5. High glucose and oxLDL induce oxidative stress in adult DRG neurons. After 3 days in culture, oxidative stress assays were performed in cultured DRG neurons. A: DRG neurons were exposed to increasing concentrations of oxLDL in the presence of basal (5.7 mmol/l) or high (25.7 mmol/l) glucose for 1 h, then loaded with MitoSOX for 15 min. n = 9, *P < 0.01 vs. control (basal glucose, 0 μg/ml oxLDL). ■, basal glucose; ▩, high glucose. B: DRG neurons were exposed to increasing concentrations of oxLDL alone or in the presence of the mitochondrial uncoupler FCCP (2.5 μmol/l) or the antioxidant α-lipoic acid (LA) (100 μmol/l) for 1 h, then loaded with DHE for 15 min. n = 9; *P < 0.01, +P < 0.05 vs. control. C: TRAP was assessed in untreated DRG neurons and those exposed to 30 μg/ml oxLDL for 3 h, n = 3, *P < 0.01. D: Nitrotyrosine fluorescence was assessed 24 h following exposure to high glucose or increasing concentrations of oxLDL in the presence or absence of LOX-1–neutralizing antibody (100 mg/ml). ■, none; □, anti–LOX-1. n = 9, *P < 0.01 vs. untreated control; †P < 0.01 vs. no antibody.CC BY-NC-ND · PubMed Central
FIG. 6. High glucose and oxLDL cause cell death in DRG neurons. Adult DRG neurons were exposed to high glucose (25.7 mmol/l) or increasing concentrations of oxLDL, and then cell death was quantitated by caspase 3 activation after 5 h (A) or TUNEL after 24 h (B). DRG neurons were additionally pretreated with LOX-1–neutralizing antibody (□, 100 mg/ml), apocyanin (▨, 1 μmol/l), or α-lipoic acid (▩, 100 μmol/l). n = 9, *P < 0.01 vs. untreated control, +P < 0.01 vs. no pretreatment (■, None).
FIG. 6. High glucose and oxLDL cause cell death in DRG neurons. Adult DRG neurons were exposed to high glucose (25.7 mmol/l) or increasing concentrations of oxLDL, and then cell death was quantitated by caspase 3 activation after 5 h (A) or TUNEL after 24 h (B). DRG neurons were additionally pretreated with LOX-1–neutralizing antibody (□, 100 mg/ml), apocyanin (▨, 1 μmol/l), or α-lipoic acid (▩, 100 μmol/l). n = 9, *P < 0.01 vs. untreated control, +P < 0.01 vs. no pretreatment (■, None).CC BY-NC-ND · PubMed Central
FIG. 7. High glucose and oxLDL activate DRG neuron NAD(P)H oxidase. A: Adult DRG neurons were exposed to 30 μg/ml oxLDL and then immunolabeled for NAD(P)H oxidase subunits p47 or gp91. B: Adult DRG neurons were exposed to high glucose (25.7 mmol/l) or oxLDL (30 μg/ml) for 1 or 3 h, then lysed for biochemical assays of NAD(P)H oxidase. *P < 0.01 vs. untreated control, +P < 0.05 vs. untreated control. ■, control; ▩, glucose; □, oxLDL. C: Adult DRG neurons were exposed to high glucose (G) (25.7 mmol/l) or oxLDL (30 μg/ml) for 3 h then lysed for Western blotting. The representative image shows the immunoblot for p47 and the loading control actin, and the bar graph contains the means ± SE of three repeats of the illustrated blot. *P < 0.01 vs. untreated control (C). (A high-quality digital representation of this figure is available in the online issue.)
FIG. 7. High glucose and oxLDL activate DRG neuron NAD(P)H oxidase. A: Adult DRG neurons were exposed to 30 μg/ml oxLDL and then immunolabeled for NAD(P)H oxidase subunits p47 or gp91. B: Adult DRG neurons were exposed to high glucose (25.7 mmol/l) or oxLDL (30 μg/ml) for 1 or 3 h, then lysed for biochemical assays of NAD(P)H oxidase. *P < 0.01 vs. untreated control, +P < 0.05 vs. untreated control. ■, control; ▩, glucose; □, oxLDL. C: Adult DRG neurons were exposed to high glucose (G) (25.7 mmol/l) or oxLDL (30 μg/ml) for 3 h then lysed for Western blotting. The representative image shows the immunoblot for p47 and the loading control actin, and the bar graph contains the means ± SE of three repeats of the illustrated blot. *P < 0.01 vs. untreated control (C). (A high-quality digital representation of this figure is available in the online issue.)CC BY-NC-ND · PubMed Central

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Metadata

Authors
Vincent, A. M., Hayes, J. M., McLean, L. L., Vivekanandan-Giri, A., Pennathur, S., & Feldman, E. L.
Affiliations
  • University of Michigan (US)
  • Department of Medicine, University of Michigan, Ann Arbor, Michigan.
Corresponding author
Vincent, Andrea M.
ORCID
Venue
Diabetes
Publisher
American Diabetes Association
Published
2009-07-10
Volume
58
Issue
10
Pages
2376-2385
ISSN
  • 0012-1797 (ISSN-L)
  • 1939-327X
DOI
10.2337/db09-0047
PMC
PMC2750230
OpenAlex
W2155399617
Cited by
264
References
51
Citations by year
2026: 11 · 2025: 12 · 2024: 20 · 2023: 15 · 2022: 15 · 2021: 23 · 2020: 19 · 2019: 14 · 2018: 11 · 2017: 12 · 2016: 9 · 2015: 9 · 2014: 31 · 2013: 14 · 2012: 21
FWCI
10.86
Type
journal article
Language
en
Open access
Licence
Locations
  • Diabetes (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
Funders
  • American Diabetes Association
  • University of Washington
  • National Institutes of Health
  • National Institute of Diabetes and Digestive and Kidney Diseases (U01DK076160, R24DK082841)
Topics
  • Health Sciences › Medicine › Physiology (medical) › Pain Mechanisms and Treatments
  • Health Sciences › Medicine › Clinical Neurology › Peripheral Neuropathies and Disorders
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Aldose Reductase and Taurine
Keywords
dyslipidemia, diabetic neuropathy, dorsal root ganglion neurons, oxidized LDL, LOX-1, NADPH oxidase, oxidative stress, high-fat diet, nerve conduction velocity, sensory impairment, type 2 diabetes, hyperglycemia, plasma lipids
MeSH terms
Animals, Blood Glucose, Diabetes Mellitus, Type 2, Diabetic Neuropathies, Dietary Fats, Female, Ganglia, Spinal, Glucose Tolerance Test, Hindlimb, Insulin, Lipids, Lipoproteins, LDL, Mice, Inbred C57BL, Nerve Fibers, Neural Conduction, Neurons, Rats, Sprague-Dawley, Reactive Oxygen Species, Dyslipidemias, Scavenger Receptors, Class E, Mice, Rats, DNA Fragmentation

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