Mitochondrial dysfunction occurs in sensory neurons and may contribute to distal axonopathy in animal models of diabetic neuropathy. The adenosine monophosphate-activated protein kinase and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) signalling axis senses the metabolic demands of cells and regulates mitochondrial function. Studies in muscle, liver and cardiac tissues have shown that the activity of adenosine monophosphate-activated protein kinase and PGC-1α is decreased under hyperglycaemia. In this study, we tested the hypothesis that deficits in adenosine monophosphate-activated protein kinase/PGC-1α signalling in sensory neurons underlie impaired axonal plasticity, suboptimal mitochondrial function and development of neuropathy in rodent models of type 1 and type 2 diabetes. Phosphorylation and expression of adenosine monophosphate-activated protein kinase/PGC-1α and mitochondrial respiratory chain complex proteins were downregulated in dorsal root ganglia of both streptozotocin-diabetic rats and db/db mice. Adenoviral-mediated manipulation of endogenous adenosine monophosphate-activated protein kinase activity using mutant proteins modulated neurotrophin-directed neurite outgrowth in cultures of sensory neurons derived from adult rats. Addition of resveratrol to cultures of sensory neurons derived from rats after 3-5 months of streptozotocin-induced diabetes, significantly elevated adenosine monophosphate-activated protein kinase levels, enhanced neurite outgrowth and normalized mitochondrial inner membrane polarization in axons. The bioenergetics profile (maximal oxygen consumption rate, coupling efficiency, respiratory control ratio and spare respiratory capacity) was aberrant in cultured sensory neurons from streptozotocin-diabetic rats and was corrected by resveratrol treatment. Finally, resveratrol treatment for the last 2 months of a 5-month period of diabetes reversed thermal hypoalgesia and attenuated foot skin intraepidermal nerve fibre loss and reduced myelinated fibre mean axonal calibre in streptozotocin-diabetic rats. These data suggest that the development of distal axonopathy in diabetic neuropathy is linked to nutrient excess and mitochondrial dysfunction via defective signalling of the adenosine monophosphate-activated protein kinase/PGC-1α pathway.

The onset of diabetic neuropathy, a complication of diabetes mellitus, has been linked to poor glycemic control. We tested the hypothesis that the mitogen-activated protein kinases (MAPK) form transducers for the damaging effects of high glucose. In cultures of adult rat sensory neurons, high glucose activated JNK and p38 MAPK but did not result in cell damage. However, oxidative stress activated ERK and p38 MAPKs and resulted in cellular damage. In the dorsal root ganglia of streptozotocin-induced diabetic rats (a model of type I diabetes), ERK and p38 were activated at 8 wk duration, followed by activation of JNK at 12 wk duration. We report activation of JNK and increases in total levels of p38 and JNK in sural nerve of type I and II diabetic patients. These data implicate MAPKs in the etiology of diabetic neuropathy both via direct effects of glucose and via glucose-induced oxidative stress.

Mitochondrial dysfunction has been proposed as a mediator of neurodegeneration in diabetes complications. The aim of this study was to determine whether deficits in insulin-dependent neurotrophic support contributed to depolarization of the mitochondrial membrane in sensory neurons of streptozocin (STZ)-induced diabetic rats. Whole cell fluorescent video imaging using rhodamine 123 (R123) was used to monitor mitochondrial inner membrane potential (⌬ m ). Treatment of cultured dorsal root ganglia (DRG) sensory neurons from normal adult rats for up to 1 day with 50 mmol/l glucose had no effect; however, 1.0 nmol/l insulin increased ⌬ m by 100% (P < 0.05). To determine the role of insulin in vivo, STZ-induced diabetic animals were treated with background insulin and the ⌬ m of DRG sensory neurons was analyzed. Insulin therapy in STZ-induced diabetic rats had no effect on raised glycated hemoglobin or sciatic nerve polyol levels, confirming that hyperglycemia was unaffected. However, insulin treatment significantly normalized diabetes-induced deficits in sensory and motor nerve conduction velocity (P < 0.05). In acutely isolated DRG sensory neurons from insulin-treated STZ animals, the diabetes-related depolarization of the ⌬ m was corrected (P < 0.05). The results demonstrate that loss of insulin-dependent neurotrophic support may contribute to mitochondrial membrane depolarization in sensory neurons in diabetic neuropathy. Diabetes 52: 2129 -2136, 2003 M itochondrial dysfunction has been proposed as a central mediator of neurodegeneration in the central and peripheral nervous systems (1) and has been discussed as a critical modulator of diabetes complications in neurons (2) and endothelial cells (3). In the peripheral nerves of humans with diabetes, there is mitochondrial ballooning and disruption of internal cristae, although this is localized to Schwann cells and is rarely observed in axons (4). Similar structural abnormalities in mitochondria have been described in Schwann cells of galactose-fed rats (4) and in dorsal root ganglion (DRG) neurons of long-term streptozocin (STZ)-induced diabetic rats (2). Furthermore, acutely isolated adult sensory neurons from STZ-induced diabetic rats exhibit depolarization of the mitochondrial inner membrane (5). One current hypothesis is that high glucose concentrations induce elevated levels of oxidative phosphorylation, resulting in damaging amounts of reactive oxygen species (ROS)-the latter then mediate degenerative changes in mitochondrial structure and cell function (3).A variety of hyperglycemia-induced secondary metabolic defects have been identified as possible causal factors in the etiology of the symmetrical sensory polyneuropathy observed in diabetes. These include polyol pathway flux (6), protein glycosylation (7), oxidative stress (8), and impaired neurotrophic support (9). Insulin and IGF-I and -II are trophic factors for embryonic (10) and adult sensory neurons (11) and modulate axon outgrowth and expression of cytoskeletal proteins (12,13). Trigeminal sens...

OBJECTIVEImpairments in mitochondrial function have been proposed to play a role in the etiology of diabetic sensory neuropathy. We tested the hypothesis that mitochondrial dysfunction in axons of sensory neurons in type 1 diabetes is due to abnormal activity of the respiratory chain and an altered mitochondrial proteome.RESEARCH DESIGN AND METHODSProteomic analysis using stable isotope labeling with amino acids in cell culture (SILAC) determined expression of proteins in mitochondria from dorsal root ganglia (DRG) of control, 22-week-old streptozotocin (STZ)-diabetic rats, and diabetic rats treated with insulin. Rates of oxygen consumption and complex activities in mitochondria from DRG were measured. Fluorescence imaging of axons of cultured sensory neurons determined the effect of diabetes on mitochondrial polarization status, oxidative stress, and mitochondrial matrix-specific reactive oxygen species (ROS).RESULTSProteins associated with mitochondrial dysfunction, oxidative phosphorylation, ubiquinone biosynthesis, and the citric acid cycle were downregulated in diabetic samples. For example, cytochrome c oxidase subunit IV (COX IV; a complex IV protein) and NADH dehydrogenase Fe-S protein 3 (NDUFS3; a complex I protein) were reduced by 29 and 36% (P < 0.05), respectively, in diabetes and confirmed previous Western blot studies. Respiration and mitochondrial complex activity was significantly decreased by 15 to 32% compared with control. The axons of diabetic neurons exhibited oxidative stress and depolarized mitochondria, an aberrant adaption to oligomycin-induced mitochondrial membrane hyperpolarization, but reduced levels of intramitochondrial superoxide compared with control.CONCLUSIONSAbnormal mitochondrial function correlated with a downregulation of mitochondrial proteins, with components of the respiratory chain targeted in lumbar DRG in diabetes. The reduced activity of the respiratory chain was associated with diminished superoxide generation within the mitochondrial matrix and did not contribute to oxidative stress in axons of diabetic neurons. Alternative pathways involving polyol pathway activity appear to contribute to raised ROS in axons of diabetic neurons under high glucose concentration.