Shin-Ichiro Yamagishi

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 NH2-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.

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

Pioglitazone, one of thiazolidinediones, a peroxisome proliferator‐activated receptor (PPAR)‐γ 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‐γ 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‐γ. Diabetic rats showed suppressed protein kinase C (PKC) activity of endoneurial membrane fraction with decreased expression of PKC‐α. 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.

Enhanced in situ expression of aldose reductase in peripheral nerve and renal glomeruli in diabetic patients

Abstract.To explore the relationships between polyol pathway-related enzymes and pathologic features, we examined the immunohistochemical expression of aldose reductase (AR) and sorbitol dehydrogenase (SDH) in the peripheral nerve and kidney tissues collected postmortem from diabetic patients and compared it with those from non-diabetic patients. Tissue AR protein concentrations were also quantified. In non-diabetic patients, AR distributed in pericytes, smooth muscle cells of endo- and epi-neurial microvessels, Schwann cells in the sciatic nerve, and tubular cells of the renal medulla. By contrast, positive SDH reactions were observed in tubular cells of the renal cortex but were faint in the sciatic nerve. Diabetic patients frequently showed dense AR expressions in the sciatic nerve. In nephropathic diabetic patients, the glomerular mesangial area showed diffuse positive reactions for AR. The severity of structural changes in glomeruli correlated with the intensity of immunoreactive AR (r2=0.626, P<0.01). AR contents in the renal cortex and sciatic nerve from diabetic patients were 1.5- and 1.8-fold greater than those from non-diabetic patients, respectively (P<0.05 for both). These findings are the first to demonstrate enhanced AR expressions in peripheral nerve and renal glomeruli in diabetic patients and its relevance to the characteristic pathology.

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.

Galactosemic neuropathy in transgenic mice for human aldose reductase.

We studied the functional consequences of an enhanced polyol pathway activity, elicited with galactose feeding, on the peripheral nerve of transgenic mice expressing human aldose reductase. Nontransgenic littermate mice were used as controls. With a quantitative immunoassay, the expression level of human aldose reductase in the sciatic nerve was 791 ± 44 ng/mg protein (mean ± SE), about 25% of that in human sural nerve. When the transgenic mice were fed food containing 30% galactose, significant levels of galactitol accumulated in the sciatic nerve. Galactose feeding of nontransgenic littermate mice led to a 10-fold lower accumulation of galactitol. Galactose feeding for 16 weeks caused a significant and progressive decrease in motor nerve conduction velocity in transgenic mice to 80% of the level of galactose-fed littermate mice, which was not significantly different from that of galactose-free littermate mice. A morphometric analysis of sciatic nerve detected > 10% reduction of mean myelinated fiber size but no alterations of myelinated fiber density in galactose-fed transgenic mice compared with other groups. The functional and structural changes that develop in galactose-fed transgenic mice are similar to those previously reported in diabetic animals. The results of these studies suggest that transgenic mice expressing human aldose reductase may be a useful model not only for defining the role of the polyol pathway in diabetic neuropathy but also for identifying and characterizing effective inhibitors specific for human aldose reductase.

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 PKCα in endoneurium, whereas there was an increased expression of PKCβII 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.

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

Eur J Clin Invest 2011; 41 (4): 442–450

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

Abstract. Recent studies have shown that neurofibrillary tangles frequently coexist with α-synuclein (α-S)-positive fibrillary inclusions in the limbic system in Alzheimers disease. To elucidate whether α-, β- and γ-S immunoreactivity is present in Pick bodies (PBs), we examined immunohistochemically and immunoelectron microscopically the brains from three patients with Picks disease. Numerous PBs were distributed widely, and were occasionally immunoreactive for α-S and β-S, but not for γ-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 α-S or β-S (as evidenced by double-labeling immunohistochemistry), immunoelectron microscopy revealed that α-S and β-S immunoreactivity occurs in granular and vesicular structures, but not in filamentous structures. These findings suggest that α-S and β-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 α-S- and β-S-positive PBs in the dentate gyrus may represent the cellular response to PB formation in this particular system.

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)‐α 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. Copyright

Impaired nerve fiber regeneration in axotomized peripheral nerves in streptozotocin‐diabetic rats

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.