Synergistic action of advanced glycation end products and endogenous nitric oxide leads to neuronal apoptosis in vitro: a new insight into selective nitrergic neuropathy in diabetes.
OBJECTIVE
We have previously shown that in diabetes nitrergic neurones innervating the urogenital and gastrointestinal organs undergo a selective degenerative process. This comprises an initial insulin-reversible decrease in neuronal nitric oxide synthase (nNOS) in the axons, followed by apoptosis of the nitrergic neurones, a process that is not reversible by insulin. Since apoptosis was independent of serum glucose concentrations, and advanced glycation endproducts (AGEs) have been implicated in the pathogenesis of diabetic complications, we have now measured AGEs in the serum and penis, pyloric sphincter and pelvic ganglia of diabetic animals at different times after streptozotocin treatment. Furthermore, we have studied their effect in vitro on human neuroblastoma (SH-SY5Y) cells in the presence or absence of nNOS expression.


METHODS
Serum AGEs were measured using fluorometry and ELISA. Accumulation of AGEs in the tissues was evaluated with immunohistochemistry. The viability, apoptosis and oxidative stress in SH-SY5Y cells were measured upon exposure to AGEs or high concentrations of glucose.


RESULTS
AGEs increased gradually in the serum and tissues of streptozotocin-induced diabetic rats; this process was not affected by delayed insulin treatment. In SH-SY5Y cells, AGEs, but not high glucose concentrations, increased the reactive oxygen species and caspase-3-dependent apoptosis in a synergistic fashion with endogenous nitric oxide (NO). Apoptosis was prevented by treatment with a NOS inhibitor, a pan-caspase inhibitor, a soluble receptor of AGEs or an anti-oxidant, but not an inhibitor of soluble guanylate cyclase.


CONCLUSIONS
The synergistic actions of NO and AGEs account for the irreversible nitrergic degeneration in diabetes.
Cellek
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Wolfson Institute for Biomedical Research, University College London, London, UK. s.cellek@ucl.ac.uk
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Schmidt
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Moncada
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D000818:Animals; D017209:Apoptosis; D001786:Blood Glucose / Q000187:drug effects; D001835:Body Weight / Q000187:drug effects; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism / Q000502:physiology; D045744:Cell Line, Tumor; D002795:Choline O-Acetyltransferase / Q000378:metabolism; D003921:Diabetes Mellitus, Experimental / Q000150:complications / Q000378:metabolism / Q000503:physiopathology*; D003929:Diabetic Neuropathies / Q000209:etiology* / Q000503:physiopathology; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004943:Esophagogastric Junction / Q000737:chemistry; D005724:Ganglia / Q000737:chemistry; D015870:Gene Expression / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D017127:Glycation End Products, Advanced / Q000097:blood / Q000494:pharmacology / Q000502:physiology*; D007150:Immunohistochemistry; D007328:Insulin / Q000494:pharmacology; D008297:Male; D026602:Nitrergic Neurons / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D010413:Penis / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000378:metabolism; D012709:Serum Albumin / Q000737:chemistry; D014212:Tretinoin / Q000494:pharmacology
D001786:Blood Glucose; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D017127:Glycation End Products, Advanced; D007328:Insulin; D017382:Reactive Oxygen Species; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; D012709:Serum Albumin; D009569:Nitric Oxide; D014212:Tretinoin; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D002795:Choline O-Acetyltransferase; C505407:CASP3 protein, human; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D005947:Glucose