Acute glucose deprivation leads to apoptosis in a cell model of acute diabetic neuropathy.
OBJECTIVE
Our aims were to better understand the mechanisms underlying peripheral neuropathy with diabetes mellitus and to test the hypothesis that acute lowering of glucose levels induces apoptosis in hypoxic neurons.


METHODS
We used rat dissociated dorsal root ganglion (DRG) neurons incubated in a medium high in glucose concentration (700 mg%) and room air (PO2 150 torr). After 5 days, DRG neurons were placed in hypoxic conditions (PO2 7.6 torr) with a normal-glucose (100 mg%) or high-glucose (700 mg%) medium containing 3 or 100 ng/mL of nerve growth factor. Acute lowering of glucose levels under hypoxic conditions led to apoptosis of DRG neurons. Apoptosis was demonstrated by bis-benzimide staining for nuclear fragmentation, electron microscopy, DNA laddering, and TUNEL staining. Caspase 3 immunocytochemistry and inhibition of neuronal death by the caspase inhibitor z-VAD-fmk (100 microM) confirmed that death was apoptotic.


RESULTS
Hypoxia-induced death was decreased when DRG neurons were maintained in high-glucose medium, suggesting that high levels of substrate protected against hypoxia. Apoptosis was completely prevented by increasing the concentration of nerve growth factor from 3 to 100 ng/mL and was partially prevented by the addition of the antioxidant alpha-lipoic acid (500 microM).


CONCLUSIONS
This model provides a novel means for studying the pathogenesis and treatment of early stages of diabetic neuropathy.
Honma
Hiroyuki
H
Molecular Neuroscience PhD Program, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.
Podratz
Jewel L
JL
Windebank
Anthony J
AJ
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology*; D001549:Benzamides; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002452:Cell Count; D015687:Cell Hypoxia / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival; D002478:Cells, Cultured; D053938:DNA Fragmentation; D003929:Diabetic Neuropathies / Q000378:metabolism / Q000473:pathology*; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian; D005727:Ganglia, Spinal / Q000473:pathology* / Q000648:ultrastructure; D005947:Glucose / Q000172:deficiency / Q000378:metabolism*; D007094:Imides / Q000493:pharmacokinetics; D007150:Immunohistochemistry / Q000379:methods; D020287:In Situ Nick-End Labeling / Q000379:methods; D008854:Microscopy, Electron / Q000295:instrumentation / Q000379:methods; D020932:Nerve Growth Factor / Q000502:physiology; D011092:Polyethylene Glycols / Q000493:pharmacokinetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D008063:Thioctic Acid / Q000494:pharmacology; D013997:Time Factors
D000975:Antioxidants; D001549:Benzamides; D007094:Imides; C109983:bis-benzimide-polyethylene glycol; D011092:Polyethylene Glycols; D008063:Thioctic Acid; D020932:Nerve Growth Factor; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D005947:Glucose