Role of glucosamine in development of diabetic neuropathy independent of the aldose reductase pathway.

Long-term metabolic aberrations contribute to the development of diabetic neuropathy but the precise mechanism or mechanisms remains elusive. We have previously shown that aldose reductase-deficient mice exhibit delayed onset and progression of neuropathy following induction of diabetes, suggesting a role both for downstream metabolites of this enzyme and also for other unrelated pathways. In this study, we have utilized comprehensive metabolomics analyses to identify potential neurotoxic metabolites in nerve of diabetic mice and explored the mechanism of peripheral nerve injury. Aldose reductase knockout and control C57Bl/6J mice were made diabetic by injection of streptozotocin and followed for 8-16 weeks. Diabetic aldose reductase knockout mice exhibited delayed onset of nerve conduction slowing compared to diabetic wild-type mice. The sciatic nerves from aldose reductase knockout mice exposed to 12 weeks of diabetes were used for metabolomics analysis and compared with analyses of nerves from age-matched diabetic wild-type mice as well as non-diabetic aldose reductase knockout and wild-type mice. Neurotoxicity of candidate metabolites was evaluated using cultured Schwann cells and dorsal root ganglion neurons, and further confirmed in vivo. Metabolomics analysis identified elevated glucosamine levels in both diabetic aldose reductase knockout and diabetic wild mice. Exposure to glucosamine reduced survival of cultured Schwann cells and neurons accompanied by increased expression of cleaved caspase 3, CCAT-enhancer-binding homologous protein and mitochondrial hexokinase-I, along with ATP depletion. These changes were suppressed by siRNA to hexokinase-I or the ATP donor, inosine, but not by the antioxidant N-acetylcysteine or the endoplasmic reticulum-stress inhibitor 4-phenylbutyrate. The O-GlcNAcylation enhancer, O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino N-phenylcarbamate, did not augment glucosamine neurotoxicity. Single dose glucosamine injection into mice caused a reduction of sciatic nerve Na, K-ATPase activity, ATP content and augmented expression of hexokinase-I, which were suppressed by pretreatment with inosine but not with 4-phenylbutyrate. Mice implanted with a subcutaneous pump to infuse glucosamine for 12 weeks developed nerve conduction slowing and intraepidermal nerve fibre loss, recapitulating prominent indices of diabetic neuropathy. While acute glucosamine neurotoxicity is unlikely to contribute substantially to the slowly developing neuropathy phenotype in humans, sustained energy deprivation induced by glucosamine may well contribute to the pathogenesis of diabetic neuropathy. Our data thus identifies a novel pathway for diabetic neuropathy that may offer a potential new therapeutic target.

Impaired nerve fiber regeneration in axotomized peripheral nerves in streptozotocin-diabetic rats.

AIM/INTRODUCTION: Impaired nerve fiber regeneration is a salient feature of diabetic neuropathy. Its pathogenesis is still unclear. We attempted to characterize the structure of regenerated myelinated fibers after transection in streptozotocin-diabetic rats. MATERIALS AND METHODS: Streptozotocin-diabetic rats underwent transection of the sciatic nerve. Two and 4 weeks post-axotomy, regenerated myelinated fibers of the cut end and fibers at its proximal site were morphometrically examined. Non-diabetic control rats with axotomy were also examined for comparison. RESULTS: At 4 weeks post-axotomy, diabetic rats showed an increased myelinated fiber density and total fiber number with a trend toward reduced fiber size at the cut end compared with those in control rats. The average number of myelin lamellae relative to axonal size in regenerated fibers at the cut end was significantly reduced in diabetic rats compared with that in control rats. The proximal site showed a reduced size of fibers and axons in both diabetic and control rats to a similar extent compared with those in a non-axotomized state. At 2 weeks post-axotomy, these findings were less apparent. CONCLUSIONS: The nerves of diabetic rats when axotomized undergo impaired regeneration characterized by increased fiber density with hypomyelination.

Methylcobalamin effects on diabetic neuropathy and nerve protein kinase C in rats.

BACKGROUND: Methyl-base-attached cobalamin (Methycobalamin) (MC) has a special affinity for nerve tissues to promote myelination and transport of axonal cytoskeleton. It is not known, however, how MC influences on peripheral nerve in experimental diabetic neuropathy. MATERIALS AND METHODS: We studied the effects of MC on expressions and activities of protein kinase C (PKC) in peripheral nerve of streptozotocin-induced diabetic rats. Wistar rats, 8 weeks of age, were rendered diabetic by streptozotocin (40 mg kg(-1), iv) and followed for 16 weeks. A half of diabetic animals were treated with MC (10 mg kg(-1) per every other day, im) after the induction of diabetes. Normal Wistar rats were served as control. RESULTS: At the end, untreated diabetic animals developed significant delay of nerve conduction velocity (NCV), and MC treatment normalized the NCV. Nerve PKC activity was significantly suppressed in untreated diabetic rats, while the activity was normalized in treated animals. While PKCα located in Schwann cells, PKCßΙα and ßII distributed in axoplasm, vascular walls and macrophages. The decreased PKC activity in diabetic nerve was associated with reduced expression of membrane PKCα and increased membrane expression of PKCßII, and MC treatment corrected these changes. Diabetic nerve contained an increased number of macrophages and 8-hydroxydeoxyguanosine-positive cells in the endoneurium, the latter of which was significantly suppressed by MC treatment. Elevated nerve polyol levels in diabetic nerve were partially corrected by MC treatment. CONCLUSIONS: This study suggested that correction of impaired neural signalling of PKC and oxidative stress-induced damage may be a major attribute to the beneficial effects of MC on diabetic nerve.

Polyol pathway and diabetic nephropathy revisited: Early tubular cell changes and glomerulopathy in diabetic mice overexpressing human aldose reductase.

UNLABELLED: Aims/Introduction: The polyol pathway has long been involved in the pathogenesis of diabetic nephropathy. It remains still unclear, however, how the polyol pathway is implicated in this process. We explored the effects of the enhanced polyol pathway on renocortical tubular cells and glomeruli in experimentally-induced diabetes. MATERIALS AND METHODS: Transgenic mice (Tg) overexpressing human aldose reductase were made diabetic by streptozotocin and followed for 8 weeks. Renocortical pathology, expressions of tonicity-responsive enhancer binding protein (TonEBP) and carboxymethyllysine of advanced glycation end-products, were examined. Wild-type non-transgenic mice (Wt) were also made diabetic and served as controls. RESULTS: Diabetic Tg showed augmented expression of TonEBP in renocortical tubular cells with vacuolated degenerative changes. These structural changes were associated with pronounced deposition of carboxymethyllysine. There was a significant increase in kidney weight, glomerular size, and mesangial area in diabetic animals and there was a trend for more severe changes in these measures in diabetic transgenic mice compared with those in control diabetic mice. Treatment with aldose reductase inhibitor significantly prevented polyol accumulation, mesangial expansion and expressions of TonEBP and carboxymethyllysine in diabetic Tg, but its effects on the renal structure were equivocal in control diabetic Wt. CONCLUSIONS: Our findings suggest that tubuloglomerular change might contribute to early diabetic nephropathy under the influence of the enhanced polyol pathway. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2010.00071.x, 2010).

The role of the polyol pathway in acute kidney injury caused by hindlimb ischaemia in mice.

The polyol pathway, a collateral glycolytic process, previously considered to be active in high glucose milieu, has recently been proposed to play a crucial role in ischaemia/reperfusion tissue injury. In this study, we explored the role of the polyol pathway in acute kidney injury (AKI), a life-threatening condition, caused by hindlimb ischaemia, and determined if inhibition of the polyol pathway by aldose reductase (AR) inhibitor is beneficial for this serious disorder. Mice 8 weeks of age rendered hindlimb ischaemic for 3 h by the clipping of major supporting arteries revealed marked muscle necrosis with accumulation of sorbitol and fructose in ischaemic muscles. Serum concentrations of blood urea nitrogen (BUN), creatinine phosphokinase (CPK), creatinine, tumour necrosis factor (TNF)-alpha as well as interleukin (IL)-6 were all elevated in these mice. Treatment with AR inhibitor (ARI) effectively suppressed muscle necrosis and accompanying inflammatory reactions and prevented renal failure. Similar to ARI-treated mice, AR-deficient mice were protected from severe ischaemic limb injury and renal failure, showing only modest muscle necrosis and significant suppression of serum markers of renal failure and inflammation. Thus, these findings suggest that the polyol pathway is implicated in AKI caused by ischaemic limb injury and that AR may be a potential therapeutic target for this condition.

Correction of protein kinase C activity and macrophage migration in peripheral nerve by pioglitazone, peroxisome proliferator activated-gamma-ligand, in insulin-deficient diabetic rats.

Pioglitazone, one of thiazolidinediones, a peroxisome proliferator-activated receptor (PPAR)-gamma ligand, is known to have beneficial effects on macrovascular complications in diabetes, but the effect on diabetic neuropathy is not well addressed. We demonstrated the expression of PPAR-gamma in Schwann cells and vascular walls in peripheral nerve and then evaluated the effect of pioglitazone treatment for 12 weeks (10 mg/kg/day, orally) on neuropathy in streptozotocin-diabetic rats. At end, pioglitazone treatment improved nerve conduction delay in diabetic rats without affecting the expression of PPAR-gamma. Diabetic rats showed suppressed protein kinase C (PKC) activity of endoneurial membrane fraction with decreased expression of PKC-alpha. These alterations were normalized in the treated group. Enhanced expression of phosphorylated extracellular signal-regulated kinase detected in diabetic rats was inhibited by the treatment. Increased numbers of macrophages positive for ED-1 and 8-hydroxydeoxyguanosine-positive Schwann cells in diabetic rats were also corrected by the treatment. Pioglitazone lowered blood lipid levels of diabetic rats, but blood glucose and nerve sorbitol levels were not affected by the treatment. In conclusion, our study showed that pioglitazone was beneficial for experimental diabetic neuropathy via correction of impaired PKC pathway and proinflammatory process, independent of polyol pathway.

Pathology and pathogenetic mechanisms of diabetic neuropathy: correlation with clinical signs and symptoms.

Drastic increase in diabetic patients poses serious problems in the care of neuropathy so that there needs to explore the pathogenesis and to establish the effective treatment. Recent clinical and basic studies revealed characteristic pathophysiology of diabetic neuropathy and some clue to the direction of the treatment. The pathology of diabetic neuropathy is characterized by progressive nerve fiber loss that gives rise to positive and negative clinical signs and symptoms such as pain, paresthesia and loss of sensation. The nerve fiber loss takes the form of pan-modal pattern with proximo-distal gradient. Endoneurial microangiopathic change is also a constant feature of peripheral nerve pathology and negatively correlates with nerve fiber density. The vascular change and distal nerve fiber loss of small caliber, in particular, at the site of epidermis, commence even in subjects with impaired glucose tolerance and precede loss of nerve fibers in the nerve trunk of lower extremities. Pathogenetic mechanisms underlying the progressive nerve fiber loss seem to be multifactorial, including polyol pathway, glycation, reactive oxygen species, and altered protein kinase C activity. Clinical trials based on this background confirmed that fundamental treatment is in fact beneficial for the prevention and halting of this intractable disorder.

Aldose reductase-deficient mice are protected from delayed motor nerve conduction velocity, increased c-Jun NH2-terminal kinase activation, depletion of reduced glutathione, increased superoxide accumulation, and DNA damage.

The exaggerated flux through polyol pathway during diabetes is thought to be a major cause of lesions in the peripheral nerves. Here, we used aldose reductase (AR)-deficient (AR(-/-)) and AR inhibitor (ARI)-treated mice to further understand the in vivo role of polyol pathway in the pathogenesis of diabetic neuropathy. Under normal conditions, there were no obvious differences in the innervation patterns between wild-type AR (AR(+/+)) and AR(-/-) mice. Under short-term diabetic conditions, AR(-/-) mice were protected from the reduction of motor and sensory nerve conduction velocities observed in diabetic AR(+/+) mice. Sorbitol levels in the sciatic nerves of diabetic AR(+/+) mice were increased significantly, whereas sorbitol levels in the diabetic AR(-/-) mice were significantly lower than those in diabetic AR(+/+) mice. In addition, signs of oxidative stress, such as increased activation of c-Jun NH(2)-terminal kinase (JNK), depletion of reduced glutathione, increase of superoxide formation, and DNA damage, observed in the sciatic nerves of diabetic AR(+/+) mice were not observed in the diabetic AR(-/-) mice, indicating that the diabetic AR(-/-) mice were protected from oxidative stress in the sciatic nerve. The diabetic AR(-/-) mice also excreted less 8-hydroxy-2'-deoxyguanosine in urine than diabetic AR(+/+) mice. The structural abnormalities observed in the sural nerve of diabetic AR(+/+) mice were less severe in the diabetic AR(-/-) mice, although it was only mildly protected by AR deficiency under short-term diabetic conditions. Signs of oxidative stress and functional and structural abnormalities were also inhibited by the ARI fidarestat in diabetic AR(+/+) nerves, similar to those in diabetic AR(-/-) mice. Taken together, increased polyol pathway flux through AR is a major contributing factor in the early signs of diabetic neuropathy, possibly through depletion of glutathione, increased superoxide accumulation, increased JNK activation, and DNA damage.

Immunohistochemical expressions of cytokeratins, mucin core proteins, p53, and neuroendocrine cell markers in epithelial neoplasm of appendix.

Epithelial neoplasms of appendix are infrequent, and their pathological features are not fully characterized. We collected 33 cases of appendiceal tumors and examined immunohistochemically the expression of cytokeratins (CK, CK7, and CK20), mucin core protein (MUC1, MUC2, MUC5AC, and MUC6), E-cadherin, chromogranin A, and p53 protein. Gene analysis of TP53 was also conducted on exons 5 to 8. Clinically, mucinous tumors were predominant in females. Immunohistochemically, all the tumors expressed CK20, whereas CK7 was positive in one third of the cases. Similarly, MUC2 was expressed in all the tumors, whereas MUC1 and MUC5AC were detected in about a half of the cases. Although chromogranin A-positive cells are generally sparse in normal appendix, they were more common in mucinous tumors than in nonmucinous tumors. Contrary to the previous data reported (Mod Pathol 2002;15:599-605), mucinous carcinoma exhibited a higher frequency of p53-positive cells (mean 29%) compared with mucinous adenoma (2.8%) (P < .001), whereas nonmucinous tumors showed high levels of p53-positive cells to similar extent (51%-67%) in both adenoma and carcinoma. The high expression of p53 protein coincided with the presence of mutations in multiple sites of TP53 gene in mucinous tumors. This is the first report that characterized the immunophenotypic profile of appendiceal epithelial neoplasms with an emphasis of a higher frequency of p53 positivity in mucinous carcinoma cases compared with mucinous adenoma in the appendix.

[Perspective for the treatment of diabetic neuropathy: translation from molecular studies to bedside].

Drastic increase in a population of diabetic patients urges to establish effective treatment or management of peripheral neuropathy, the most common complication of diabetes. Recent studies emphasize the occurrence of peripheral neuropathy in patients with impaired glucose tolerance, which condition is now shown to augment polyol pathway as well as non-enzymatic protein glycation in the peripheral nerve, exerted by postprandial hyperglycemia. Such metabolic cascades in turn result in tissue-specific alterations of cellular signaling, represented by decreased protein kinase C (PKC) activity and Na, K-ATPase activity in the peripheral nerve and contrariwise increased PKC activity in microvessels. The decrease in nerve PKC activity was demonstrated to associate with reduced membrane alpha-isoform expression and the increase in vessel PKC activity was due to membrane beta-isoform expression, respectively. Dual mechanisms of nerve and vascular alterations in addition to multiple metabolic factors may operate in the development of diabetic neuropathy in a complicated manner. Consequently, inhibition of hyperglycemia as well as specific intervention of single molecules related to altered cellular signaling is the essential approach for the primary prevention of diabetic neuropathy.

Effects of polyol pathway hyperactivity on protein kinase C activity, nociceptive peptide expression, and neuronal structure in dorsal root ganglia in diabetic mice.

We explored the specific impact of polyol pathway hyperactivity on dorsal root ganglia (DRG) using transgenic mice that overexpress human aldose reductase because DRG changes are crucial for the development of diabetic sensory neuropathy. Littermate mice served as controls. Half of the animals were made diabetic by streptozotocin injection and followed for 12 weeks. After diabetes onset, diabetic transgenic mice showed a significant elevation of pain sensation threshold after transient decrease and marked slowing of motor and sensory nerve conduction at the end of the study, while these changes were modest in diabetic littermate mice. Protein kinase C (PKC) activities were markedly reduced in diabetic transgenic mice, and the changes were associated with reduced expression of membrane PKC-alpha isoform that was translocated to cytosol. Membrane PKC-betaII isoform expression was contrariwise increased. Calcitonin gene-related peptide-and substance P-positive neurons were reduced in diabetic transgenic mice and less severely so in diabetic littermate mice. Morphometric analysis disclosed neuronal atrophy only in diabetic transgenic mice. Treatment with an aldose reductase inhibitor (fidarestat 4 mg x kg(-1) x day(-1), orally) corrected all of the changes detected in diabetic transgenic mice. These findings underscore the pathogenic role of aldose reductase in diabetic sensory neuropathy through the altered cellular signaling and peptide expressions in DRG neurons.

Differential influence of increased polyol pathway on protein kinase C expressions between endoneurial and epineurial tissues in diabetic mice.

To explore the relationship between polyol pathway and protein kinase C (PKC), we examined PKC activities and expressions of PKC isoforms separately in endoneurial and vessel-rich epineurial tissues in diabetic mice transgenic for human aldose reductase (Tg). Tg and littermate control mice (Lm) were made diabetic by streptozotocin at 8 weeks of age and treated orally with aldose reductase inhibitor (ARI) (fidarestat 3-5 mg/kg/day) or placebo for 12 weeks. At the end, compared with non-diabetic state, sorbitol contents were increased 6.4-fold in endoneurium and 5.1-fold in epineurium in diabetic Tg, whereas the increase was detected only in endoneurium in diabetic Lm. Endoneurial PKC activity was significantly reduced in diabetic Tg. By contrast, epineurial PKC activity was increased in both diabetic Lm and diabetic Tg and there was no significant difference between the two groups. These changes were all corrected by ARI treatment. Consistent with the changes of PKC activities, diabetic Tg showed decreased expression of PKC alpha in endoneurium, whereas there was an increased expression of PKC beta II in epineurium in both diabetic Tg and diabetic Lm. These findings suggest the presence of dichotomous metabolic pathway between neural and vascular tissues in the polyol-PKC-related pathogenesis of diabetic neuropathy.

Pick's disease: alpha- and beta-synuclein-immunoreactive Pick bodies in the dentate gyrus.

Recent studies have shown that neurofibrillary tangles frequently coexist with alpha-synuclein (alpha-S)-positive fibrillary inclusions in the limbic system in Alzheimer's disease. To elucidate whether alpha-, beta- and gamma-S immunoreactivity is present in Pick bodies (PBs), we examined immunohistochemically and immunoelectron microscopically the brains from three patients with Pick's disease. Numerous PBs were distributed widely, and were occasionally immunoreactive for alpha-S and beta-S, but not for gamma-S in all three cases. However, these immunoreactive PBs were almost all restricted to the dentate gyrus. Despite the co-localization of phosphorylated tau and alpha-S or beta-S (as evidenced by double-labeling immunohistochemistry), immunoelectron microscopy revealed that alpha-S and beta-S immunoreactivity occurs in granular and vesicular structures, but not in filamentous structures. These findings suggest that alpha-S and beta-S are up-regulated in the neuronal perikarya but they are not incorporated into the constituent filaments of PBs, and that the preferential distribution of alpha-S- and beta-S-positive PBs in the dentate gyrus may represent the cellular response to PB formation in this particular system.