Sciatic Nerve Of Diabetic Mice Research Articles

BMP5 ameliorates diabetic peripheral neuropathy by augmenting mitochondrial function and inhibiting apoptosis in Schwann cells

Diabetic peripheral neuropathy is a common and serious complication of diabetes. Bone morphogenetic protein 5 (BMP5) is a multifunctional protein involved in the nervous system. Nevertheless, its effect on diabetic peripheral neuropathy remained uncharacterized. In this study, diabetic neuropathy in mice was induced by a single dose of 150 mg/kg streptozotocin (STZ) via intraperitoneal injection. Lentivirus expressing BMP5 (LV-BMP5) administration improved pain sensitivity, nerve conduction velocities and morphological alterations of the sciatic nerve of diabetic mice. Elevated BMP5 by LV-BMP5 suppressed cell apoptosis in the sciatic nerve, as evidenced by declined TUNEL-positive cells and down-regulated cleaved caspase-3 and cleaved caspase-9 levels. BMP5 enhanced mitochondrial membrane potential and ATP level. BMP5 also increased the phosphorylation of Smad1/5/9. Besides, the role of BMP5 in high glucose (HG)-stimulated Schwann cells was determined. Results of in vitro studies were in line with the in vivo findings. These experimental data seem to imply that BMP5 prevents the development of diabetic neuropathy via the maintenance of Smad1/5/9-mediated mitochondrial function.

Lycorine improves peripheral nerve function by promoting Schwann cell autophagy via AMPK pathway activation and MMP9 downregulation in diabetic peripheral neuropathy

Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes mellitus and no effective therapy is approved. Here, lycorine, a natural alkaloid, was identified as a potential drug for DPN by the bioinformatics analysis of GEO datasets and Connectivity Map database. Lycorine administration improved peripheral nerve function and autophagy-associated proteins of diabetic mice. Again, in vitro high glucose-cultured rat Schwann cells (RSC96) showed enhanced autophagosome marker LC3-II with the treatment of lycorine. Additionally, beclin-1 and Atg3 were decreased in high glucose-stimulated RSC96 cells, which were reversed by lycorine treatment. Furthermore, DPN-associated differentially expressed genes (DEGs) from GEO datasets and lycorine-drug targets from PubChem and PharmMapper were visually analyzed and revealed that MMP9 was both DPN-associated DEGs and lycorine-drug target. Functional enrichment analysis of MMP9-relevant genes showed that cell energy metabolism was involved. Moreover, lycorine reduced high glucose-enhanced MMP9 expression in RSC96 cells. Overexpression of MMP9 attenuated lycorine-induced the expression of beclin-1, Atg3 and LC3-II in high glucose-cultured RSC96 cells. In addition, AMPK pathway activation was confirmed in lycorine-treated high glucose-cultured RSC96 cells. Then AMPK pathway inhibition attenuated lycorine-reduced MMP9 expression in high glucose-treated RSC96 cells. Molecular docking analysis revealed that lycorine bound the domain of AMPK containing Thr 172 site, which affected AMPK (Thr 172) phosphorylation. Finally, AMPK pathway activation and MMP9 downregulation were also revealed in the sciatic nerves of diabetic mice administrated with lycorine. Taken together, lycorine was advised to promote Schwann cell autophagy via AMPK pathway activation and MMP9 downregulation-induced LC3-II transformation in diabetic peripheral neuropathy.

TXNIP, a novel key factor to cause Schwann cell dysfunction in diabetic peripheral neuropathy, under the regulation of PI3K/Akt pathway inhibition-induced DNMT1 and DNMT3a overexpression

Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes mellitus (DM) and the dysfunction of Schwann cells plays an important role in the pathogenesis of DPN. Thioredoxin-interacting protein (TXNIP) is known as an inhibitor of thioredoxin and associated with oxidative stress and inflammation. However, whether TXNIP is involved in dysfunction of Schwann cells of DPN and the exact mechanism is still not known. In this study, we first reported that TXNIP expression was significantly increased in the sciatic nerves of diabetic mice, accompanied by abnormal electrophysiological indexes and myelin sheath structure. Similarly, in vitro cultured Schwann cells TXNIP was evidently enhanced by high glucose stimulation. Again, the function experiment found that knockdown of TXNIP in high glucose-treated RSC96 cells led to a 4.12 times increase of LC3-II/LC3-I ratio and a 25.94% decrease of cleaved caspase 3/total caspase 3 ratio. Then, DNA methyltransferase (DNMT) inhibitor 5-Aza has been reported to benefit Schwann cell in DPN, and here 5-Aza treatment reduced TXNIP protein expression, improved autophagy and inhibited apoptosis in high glucose-treated RSC96 cells and the sciatic nerves of diabetic mice. Furthermore, DNMT1 and DNMT3a upregulation were found to be involved in TXNIP overexpression in high glucose-stimulated RSC96 cells. Silencing of DNMT1 and DNMT3a effectively reversed high glucose-enhanced TXNIP. Moreover, high glucose-inhibited PI3K/Akt pathway led to DNMT1, DNMT3a, and TXNIP upregulation in RSC96 cells. Knockdown of DNMT1 and DNMT3a prevented PI3K/Akt pathway inhibition-caused TXNIP upregulation in RSC96 cells. Finally, in vivo knockout of TXNIP improved nerve conduction function, increased autophagosome and LC3 expression, and decreased cleaved Caspase 3 and Bax expression in diabetic mice. Taken together, PI3K/Akt pathway inhibition mediated high glucose-induced DNMT1 and DNMT3a overexpression, leading to cell autophagy inhibition and apoptosis via TXNIP protein upregulation in Schwann cells of DPN.

Trichostatin A increases BDNF protein expression by improving XBP-1s/ATF6/GRP78 axis in Schwann cells of diabetic peripheral neuropathy

Diabetic peripheral neuropathy (DPN) is the common complication of diabetes mellitus. Histone deacetylase (HDAC) inhibitor trichostatin A (TSA) is reported to ameliorate the peripheral nerves degeneration of DPN. However, the exact mechanism is still not well elucidated. Here, we first revealed that TSA promoted nerve conduction and brain derived neurotrophic factor (BDNF) expression in the sciatic nerves of diabetic mice. In line, TSA also reversed high glucose-reduced mature BDNF expression in vitro cultured rat Schwann cells (RSC96). Then unexpectedly, the downstream targets of TSA HDAC1 and HDAC5 were not involved in TSA-improved BDNF expression. Furthermore, unfolded protein response (UPR) chaperone GRP78 was revealed to be downregulated with high glucose stimulation in RSC96 cells, which was avoided with TSA treatment. Also, GRP78 upregulation mediated TSA-improved mature BDNF expression in high glucose-cultured RSC96 cells by binding with BDNF. As well, TSA treatment enhanced the binding of GRP78 with BDNF in RSC96 cells. Again, UPR-associated transcription factors XBP-1s and ATF6 were involved in TSA-increased GRP78 expression in high glucose-stimulated RSC96 cells. Finally, conditioned medium from high glucose-cultured RSC96 cells delayed neuron SH-SY5Y differentiation and that from TSA-treated high glucose-cultured RSC96 cells promoted SH-SY5Y cell differentiation. Taken together, our findings suggested that TSA increased BDNF expression to ameliorate DPN by improving XBP-1s/ATF6/GRP78 axis in Schwann cells.

IFN-γ/mTORC1 decreased Rab11 in Schwann cells of diabetic peripheral neuropathy, inhibiting cell proliferation via GLUT1 downregulation.

Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes mellitus. Rab11 is conserved gene-regulating vesicle traffic and reported to be involved in the pathogenesis of diabetes mellitus by affecting insulin sensitivity. We aimed to investigate the role of Rab11 in the pathogenesis of DPN. In this study, Rab11 expression decreased in the sciatic nerves of diabetic mice with impaired conduction function versus those of normal mice. In vitro experiment revealed interferon-γ (IFN-γ), not high glucose and interleukin 1β was the main factor to lead to Rab11 downregulation in RSC96 cells. Again, both Rab11 knockdown and IFN-γ treatment caused cell viability inhibition and the decrease in BrdU-positive cells. In contrast, overexpression of Rab11 reversed IFN-γ-reduced cell proliferation. Furthermore, mTORC1 not mTORC2 was proven to be suppressed by IFN-γ treatment in RSC96 cells, indicated in decreased phospho-p70S6K. Inhibition of the mTORC1 pathway resulted in Rab11 expression downregulation in RSC96 cells. Activation of the mTORC1 pathway effectively prevented IFN-γ-reduced Rab11 expression in RSC96 cells. Also, glucose transporter 1 (GLUT1) was found to be downregulated in RSC96 cells with Rab11 silence and overexpression of GLUT1 reversed Rab11 blocking-caused proliferation inhibition. Taken together, our findings suggest that IFN-γ decreases Rab11 expression via the inhibition of the mTORC1 signaling pathway, causing reduced cell proliferation in Schwann cells of DPN by GLUT1 downregulation.

The Akt/mTOR cascade mediates high glucose-induced reductions in BDNF via DNMT1 in Schwann cells in diabetic peripheral neuropathy

Brain-derived neurotropic factor (BDNF) deficiency in Schwann cells plays an important role in the pathogenesis of diabetic peripheral neuropathy (DPN). Little is known about the mechanism involved in BDNF downregulation in Schwann cells in DPN. In this study, we first confirmed downregulation of BDNF and neurotrophin 3 expression in the sciatic nerves of diabetic mice, which was accompanied by myelin sheath abnormalities. Moreover, in vitro, high glucose was revealed to cause downregulation of BDNF, but not neurotrophin 3, expression in RSC96 cells, which was accompanied by DNA hypermethylation of BDNF promoters I and II. DNMT1 was subsequently revealed to be enhanced at the mRNA and protein levels in high glucose-stimulated RSC96 cells, and inhibition of DNMT1 with 5-Aza treatment or shRNA vector transfection reversed high glucose-induced reductions in BDNF expression. Furthermore, the mTOR and upstream Akt pathways were indicated to mediate high glucose-induced DNMT1 and BDNF expression in RSC96 cells. Taken together, our results suggest that the Akt/mTOR cascade mediates high glucose-induced reductions in BDNF via DNMT1 in Schwann cells in DPN.

STAT3 phosphorylation mediates high glucose-impaired cell autophagy in an HDAC1-dependent and -independent manner in Schwann cells of diabetic peripheral neuropathy.

Schwann cells are the main supportive cells of the peripheral nerves. Schwann cells suffer inhibition of autophagy under hyperglycemia treatment in diabetic peripheral neuropathy (DPN). However, the exact mechanism is still not fully elucidated. We first observed the decrease of autophagy markers (LC3-II/LC3-I, P62) in the sciatic nerves of diabetic mice vs. normal mice, accompanied with the loss of myelinated nerve fibers and abnormal myelin sheath. In line with this, LC3-II/LC3-I and P62 were also significantly reduced in high glucose-treated rat Schwann cell 96 (RSC96) cells compared with normal glucose-treated cells. Furthermore, we found that trichostatin A [an inhibitor of histone deacetylase (HDAC)] evidently improved LC3-II/LC3-I in high glucose-treated RSC96 cells, without an effect on P62 expression. Again, HDAC1 and HDAC5 were revealed to be increased in RSC96 cells stimulated with high glucose. Inhibition of HDAC1 but not HDAC5 by small hairpin RNA vector enhanced LC3-II/LC3-I in high glucose-cultured RSC96 cells. In addition, LC3-II conversion regulators [autophagy-related protein (Atg)3, Atg5, and Atg7] were detected in high glucose-treated and HDAC1-knockdown RSC96 cells, and Atg3 was proven to be the key target of HDAC1. The presuppression of Atg3 offset the improvement of LC3-II/LC3-I resulting from HDAC1 inhibition in high glucose-treated RSC96 cells. The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway was activated in RSC96 cells treated with high glucose, which was indicated by increased STAT3 phosphorylation. Blocking STAT3 phosphorylation by chemical inhibitor AG490 induced HDAC1 down-regulation followed by increases in Atg3 and LC3-II/LC3-I. Interestingly, we also found that AG490 treatment enhanced P62 expression in high glucose-stimulated RSC96 cells. Taken together, our findings demonstrate that hyperglycemia inhibits LC3-II/LC3-I in an HDAC1-Atg3-dependent manner and decreases P62 expression in an HDAC-independent manner via the JAK-STAT3 signaling pathway in the Schwann cells of DPN.-Du, W., Wang, N., Li, F. Jia, K., An, J., Liu, Y., Wang, Y., Zhu, L., Zhao, S. Hao, J. STAT3 phosphorylation mediates high glucose-impaired cell autophagy in an HDAC1-dependent and -independent manner in Schwann cells of diabetic peripheral neuropathy.

Prolonged high‐glucose exposure decreased SREBP‐1/FASN/ACC in Schwann cells of diabetic mice via blocking PI3K/Akt pathway

Abnormal lipid metabolism and SREBP-1 downregulation are reported to be involved in the pathogenesis of diabetic peripheral neuropathy (DPN). In the current study, the relationship between PI3K/Akt signaling pathway and SREBP-1 expression was explored in Schwann cells of DPN. The phospho-Akt (Ser 473), phospho-Akt (Thr 308), and SREBP-1 expression were inhibited in the sciatic nerves of diabetic mice versus those of normal mice, accompanied with the atrophy of nerve fiber and the irregular myelin sheath. High concentration glucose suppressed phospho-Akt (Ser 473), phospho-Akt (Thr 308), and SREBP-1 expression in cultured Schwann cell (RSC96 cell) in vitro, and 25 mmol/L glucose was enough to lead to the maximum inhibitory effect. The time-course effect of high glucose showed that Akt phosphorylation gradually decreased with the extension of stimulation time. Somewhat differently, short-term high-glucose exposure enhanced SREBP-1 expression and prolonged high-glucose stimulation reduced the SREBP-1 expression in RSC96 cells. Similarly, prolonged high-glucose stimulation also downregulated FASN messenger RNA (mRNA), ACC mRNA, intracellular triglyceride, and cholesterol. LY294002 suppressed Akt activation followed by the decreased SREBP-1, FASN, ACC, triglyceride, and cholesterol. Contrarily, the PI3K/Akt signaling pathway agonist insulin pretreatment avoided prolonged high-glucose stimulation-blocked Akt activation and increased SREBP-1, FASN, and ACC expression in the levels of protein and mRNA in RSC96 cells. The knockdown of SREBP-1 by shRNA prevented insulin-induced enhanced FASN, ACC mRNA expression, triglyceride, and cholesterol in high-glucose-treated RSC96 cells. In conclusion, prolonged high-glucose exposure inhibits the SREBP-1/FASN/ACC expression in the Schwann cells of DPN via the blockage of the PI3K/Akt signaling pathway.

Hyperglycemia-induced Bcl-2/Bax-mediated apoptosis of Schwann cells via mTORC1/S6K1 inhibition in diabetic peripheral neuropathy

Schwann cell apoptosis is one of the characteristics of diabetic peripheral neuropathy (DPN). The mammalian target of rapamycin (mTOR) is a multifunctional signaling pathway that regulates cell apoptosis in various types of tissues and cells. To investigate whether the mTOR pathway is involved in cell apoptosis in the Schwann cells of DPN, diabetic mice and rat Schwann cells (RSC96) were chosen to detect phospho-mTOR (Ser 2448), phospho-S6K1 (Thr 389), phospho-4EBP1 (Thr 37/46), Bcl-2, Bax and cleaved caspase-3 by diverse pathological and biological techniques. The results showed that phospho-mTOR (Ser 2448) was decreased in the sciatic nerves of diabetic mice, concomitant with decreased Bcl-2, increased Bax, cleaved caspase-3 and cell apoptosis. In addition, high glucose treatment for 72 h caused a 35.95% decrease in the phospho-mTOR (Ser 2448)/mTOR ratio, a 65.50% decrease in the phospho-S6K1 (Thr 389)/S6K1 ratio, a 3.67-fold increase in the Bax/Bcl-2 ratio and a 1.47-fold increase in the cleaved caspase-3/caspase-3 ratio. Furthermore, mTORC1 inhibition, rather than mTORC2 inhibition, resulted in mitochondrial controlled apoptosis in RSC96 cells by silencing RAPTOR or RICTOR. Again, suppression of the mTORC1 pathway by a chemical inhibitor led to mitochondrial controlled apoptosis in cultured RSC96 cells in vitro. By contrast, activation of the mTORC1 pathway with MHY1485 prevented decreased phospho-S6K1 (Thr 389) levels caused by high glucose and cell apoptosis. Additionally, constitutive activation of S6K1 avoided high glucose-induced cell apoptosis in RSC96 cells. In summary, our findings suggest that activating mTORC1/S6K1 signaling in Schwann cells may be a promising strategy for the prevention and treatment of DPN.

Transcriptional networks of progressive diabetic peripheral neuropathy in the db/db mouse model of type 2 diabetes: An inflammatory story

Diabetic peripheral neuropathy is the most common complication of diabetes and a source of considerable morbidity. Numerous molecular pathways are linked to neuropathic progression, but it is unclear whether these pathways are altered throughout the course of disease. Moreover, the methods by which these molecular pathways are analyzed can produce significantly different results; as such it is often unclear whether previously published pathways are viable targets for novel therapeutic approaches. In the current study we examine changes in gene expression patterns in the sciatic nerve (SCN) and dorsal root ganglia (DRG) of db/db diabetic mice at 8, 16, and 24 weeks of age using microarray analysis. Following the collection and verification of gene expression data, we utilized both self-organizing map (SOM) analysis and differentially expressed gene (DEG) analysis to detect pathways that were altered at all time points. Though there was some variability between SOM and DEG analyses, we consistently detected altered immune pathways in both the SCN and DRG over the course of disease. To support these results, we further used multiplex analysis to assess protein changes in the SCN of diabetic mice; we found that multiple immune molecules were upregulated at both early and later stages of disease. In particular, we found that matrix metalloproteinase-12 was highly upregulated in microarray and multiplex data sets suggesting it may play a role in disease progression.

Potential utility of aldose reductase-deficient Schwann cells IKARS1 for the study of axonal degeneration and regeneration.

Potential utility of aldose reductase-deficient Schwann cells IKARS1 for the study of axonal degeneration and regeneration.

The induction of the transcription factor Nrf2 enhances the antinociceptive effects of delta-opioid receptors in diabetic mice.

The involvement of heme oxygenase 1 (HO-1) in the modulation of the antinociceptive effects of opioids in type 1 diabetes has been demonstrated but the role played by the transcription factor Nrf2 in the regulation of painful neuropathy and in the effects and expression of δ-opioid receptors (DOR) in type 2 diabetes, has not been studied. In male BKS.Cg-m+/+Leprdb/J (db/db) mice, the anti-allodynic effects produced by a Nrf2 transcription factor activator, sulforaphane (SFN) administered alone and combined with two DOR agonists, [d-Pen(2),d-Pen(5)]-Enkephalin (DPDPE) and (+)-4-[(αR)-α-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N diethylbenzamide (SNC-80), were evaluated. The effects of SFN on glucose levels and body weight as well as on the proteins levels of Nrf2, HO-1, NAD(P)H: quinone oxidoreductase 1 (NQO1), MAPKs (JNK) and DOR in sciatic nerve from db/db mice were also assessed. This study showed that the administration of SFN dose dependently reversed mechanical allodynia, reduced hyperglycemia and body weight gain associated to type 2 diabetes and significantly increased the anti-allodynic effects of DPDPE and SNC-80 in db/db mice. This treatment normalized the down regulation of Nrf2 and NQO1 and enhanced the protein levels of HO-1 in db/db mice. Moreover, the administration of SFN also inhibited the JNK phosphorylation and DOR down-regulation in the sciatic nerve of diabetic mice. Our data indicated that SFN treatment is effective in reversing mechanical allodynia and enhancing DOR antinociceptive effects in db/db mice which effects might be mediated by activating Nrf2 signaling, reducing hyperglycemia, inhibiting JNK phosphorylation and avoiding DOR down-regulation in the sciatic nerve of these animals. These results propose SFN, alone and/or combined with DOR agonists, as interesting approaches for the treatment of painful diabetic neuropathy associated to type 2 diabetes in mice.

PDE5 inhibitors promote recovery of peripheral neuropathy in diabetic mice.

PDE5 inhibitors promote recovery of peripheral neuropathy in diabetic mice.

Pepino polyphenolic extract improved oxidative, inflammatory and glycative stress in the sciatic nerves of diabetic mice.

The effect of pepino polyphenolic extract (PPE) on diabetic neuropathy was examined. Using HPLC/ESI-MS-MS analysis, PPE was demonstrated to contain coumaroyl and caffeoyl derivatives among polyphenols. PPE at 0.5 or 1% was supplied to diabetic mice for 12 weeks. The PPE intake at two doses significantly improved glycaemic control. These treatments reserved the glutathione (GSH) level, and decreased the thiobarbituric acid reactive substances (TBARS) level, reactive oxygen species (ROS), interleukin (IL)-6, tumour necrosis factor (TNF)-alpha, fructose, and glycation intermediates and precursors of advanced glycation end products (AGEs), such as methylglyoxal (MG) and N-(carboxymethyl)lysine (CML), in the sciatic nerves of diabetic mice. In a histological study of sciatic nerves, PPE had the effects in improving the nerves of diabetic mice, showing disorganization of the fascicle with numerous small myelinated fibers. The PPE intake at two doses retained the activity, and the protein and mRNA levels of glutathione peroxidase (GPX), and decreased protein expressions of aldose reductase (AR) and the receptor for the advanced glycation end product (RAGE) in sciatic nerves. These findings support that pepino polyphenolic extract could attenuate oxidative, inflammatory and glycative stress in diabetic peripheral nerves.

Insulin treatment, but not protein kinase C inhibition, restores glutathione peroxidase activity in sciatic nerve of diabetic mice

Experimental diabetes promotes changes in biochemical activities of peripheral nervous tissue. Glutathione peroxidase (GSHPx) activity decreases in sciatic nerve of diabetic mice very early after onset of experimental diabetes. Bffective glycemic control with so0 mU/g of insulin on days 4,5 and 6 after ahoxan treatment, restores the early lost GSHPx activity in peripheral nerve of diabetic mice to control values. GSHh( activity in diabetic mouse peripheral nerve is not modified by the constant delivery (with a constant delivery device consisting of miniosmotic pumps implanted in the back of the animals) of calphostin C, a protein kinase C (PKC) inhibitor. In our hands, this same experimental setun was able to restore Na.K-ATPase activity of peripheral nerve (Diabetes 4?Z:257-262, 1993).-Therefore the decrease of GSHPx activity seems to be independent on a PKC mediated mechanism. On the other hand, this early GSHPx activity decrease in peripheral nerve of diabetic mice is closely related to hyperglycemia, and a tight glycemic control is lather effective in restoring the control levels of this enzymatic activity.

Parameters of conduction via afferent nerve fibers in mice with streptozotocin-induced and genetically determined diabetes

The parameters of conduction via afferent nerve fibers were studied in mice with streptozotocin-induced and genetically determineddiabetes mellitus (9- to 12-week-old animals; streptozotocin was injected into 5-week-old mice). Recording of spinal cord dorsal surface potentials evoked by stimulation of the sciatic nerve showed that within the studied time interval the mice of the two diabetic groups were characterized by a moderate decrease (by 7.9% and 5.8%, on average) in the conduction velocity for afferent volleys (measured according to the delay of the peak of positivity of a volley) and by a considerable increase in the duration of the positive phase of these volleys (by 36% and 33%, respectively, as compared with the values in intact animals). Therefore, the population of relatively “slow” group A afferent fibers becomes noticeably larger in the sciatic nerve of diabetic mice even at early stages of the pathology, but at the same time a considerable amount of the fastest-conducting (about 45–60 m/sec) fibers is still preserved. The changes in mice with diabetes of different etiology were very similar, in spite of different hyperglycemia levels in these groups. Possible factors determining diabetes-induced modifications of the conduction velocity via the nerve fibers are discussed.

Sustained recovery of Na(+)-K(+)-ATPase activity in sciatic nerve of diabetic mice by administration of H7 or calphostin C, inhibitors of PKC.

We have previously shown that intraperitoneal injection of H-7, an inhibitor of PKC, restores completely the activity of Na(+)-K(+)-ATPase in sciatic nerve of diabetic mice; however, the effect was transient, with a half-life of approximately 1 h under the conditions used. This work assessed whether calphostin C, a new more potent and specific inhibitor of PKC, is also able to restore the activity of Na(+)-K(+)-ATPase in sciatic nerve of ALX-induced diabetic mice and also assessed if continuous administration of H-7 or calphostin C can afford sustained recovery of the ATPase. Small amounts of calphostin C (i.e., 2 micrograms/kg) restore entirely the activity of the enzyme. Larger doses (e.g., 30 micrograms/kg) can be administered with equal results. The ED50 was approximately 0.5 micrograms/kg. This indicates that calphostin C is approximately 20,000 times more potent than H-7 in restoring the ATPase activity in diabetic mice. A single intraperitoneal injection of 1 or 10 micrograms/kg of calphostin C maintains the enzyme for 4 and 8 h, respectively. Administration of H-7 by continuous delivery from micro-osmotic pumps implanted in the back of the mice maintains the Na(+)-K(+)-ATPase for 24 h, although the activity decreases thereafter. This is the result of instability of H-7 in solution. Continous administration of calphostin C maintains the activity of the ATPase at nearly normal values for at least 2 wk. The results support the hypothesis that, in sciatic nerve tissue of diabetic animals, the activity of PKC is increased, leading to higher phosphorylation of Na(+)-K(+)-ATPase, which results in the decreased activity observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Sustained recovery of Na(+)-K(+)-ATPase activity in sciatic nerve of diabetic mice by administration of H7 or calphostin C, inhibitors of PKC

Sustained recovery of Na(+)-K(+)-ATPase activity in sciatic nerve of diabetic mice by administration of H7 or calphostin C, inhibitors of PKC

Inhibition of protein kinase C restores Na+,K(+)-ATPase activity in sciatic nerve of diabetic mice.

We have tested if inhibition of protein kinase C is able to prevent and/or to restore the decrease of Na+,K(+)-ATPase activity in the sciatic nerve of alloxan-induced diabetic mice. Mice were made diabetic by subcutaneous injection of 200 mg of alloxan/kg of body weight. The activity of Na+,K(+)-ATPase decreased rapidly (43% after 3 days) and slightly thereafter (58% at 11 days). We show that intraperitoneal injection of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), an inhibitor of protein kinase C, prevents completely the loss of Na+,K(+)-ATPase activity produced by alloxan. Also, H7 injected into diabetic mice, 4-9 days after the injection of alloxan, restores the activity of the enzyme. The amount of activity recovered depends on the dose of H7 administered; complete recovery was reached with injection of 15 mg of H7/kg of body weight. The effect of H7 is transient, with a half-life of approximately 1 h.

Basal lamina thickness and permeability to horseradish peroxidase of intraneural capillaries in diabetic mice

The fine structural morphology, basal lamina thickness, and permeability to intravenously injected horseradish peroxidase (HRP) of intraneural capillaries have been examined in 10-month-old diabetic mice ( C57BL KsJ , db db ). The afflicted mice of this strain have previously been shown to develop peripheral neuropathy. In this study we observed that a very small percentage of endothelial cells in sciatic nerves of diabetic mice had abnormal accumulations of membrane whorls in their cytoplasm. Although quantitative measurements of intraneural capillary basal lamina thickness showed that there was no statistically significant increase in basal lamina thickness, occasional sciatic nerve capillaries in diabetic mice did have thickened basal laminae. Cytochemical studies showed that there was no apparent leakage of HRP from capillaries in the sciatic nerve, trigeminal nerve, and cerebral cortex from diabetic mice or their unafflicted littermates. These results suggest that microangiopathy, as assessed by these measures, is not prominent in diabetic mice and therefore, probably does not play a major role in the development of peripheral neuropathy in these mice.