Biomedical subjects
Publications and source records attributed to R Kikkawa.
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An excessive production of extracellular matrix (ECM) proteins in glomerular mesangial cells is considered to be responsible for the development of mesangial expansion seen in diabetic nephropathy. Mechanical stretch due to glomerular hypertension has been proposed as one of the factors leading to an increase in the production of ECM proteins in mesangial cells, but the precise mechanism of stretch-induced overproduction of ECM proteins has not been elucidated. Herein, we provide the evidence that mitogen-activated protein kinase (MAPK) may play a key role in the overproduction of fibronectin (FN) in mesangial cells exposed to mechanical stretch. MAPK, also termed extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK), was activated by mechanical stretch in time- and intensity-dependent manners. Stretch-induced activation of ERK was inhibited by herbimycin A, a tyrosine kinase inhibitor, but not by GF109203X or calphostin C, the inhibitors of protein kinase C. Mechanical stretch also enhanced DNA-binding activity of AP-1, and this enhancement was inhibited by PD98059, an inhibitor of MAPK or ERK kinase (MEK). Furthermore, mechanical stretch stimulated the expression of FN mRNA followed by a significant increase in its protein accumulation. PD98059 could prevent stretch-induced increase in the expression of FN mRNA and protein. These results indicate that the activation of ERK may mediate the overproduction of ECM proteins in mesangial cells exposed to mechanical stretch, an in vitro model for glomerular hypertension seen in diabetes.
A newly identified 3'-untranslated region (UTR) polymorphism of the gene for skeletal muscle-specific glycogen-targeting subunit of protein phosphatase 1 (PPP1R3) was associated with insulin resistance and type 2 diabetes in Pima Indians (Xia J, Scherers W, Cohen PTW, Majer M, Xi T, Norman RA, Knowler WC, Bogardus C, Prochazka M: A common variant in PP1R3 associated with insulin resistance and type 2 diabetes. Diabetes 47:1519-1524, 1998). Thus, we investigated the frequency of polymorphism of the adenine- and thymine-rich element (ARE-1 and its variant ARE-2) in 426 Japanese type 2 diabetic and 380 nondiabetic subjects using a polymerase chain reaction (PCR)-restriction enzyme fragment length polymorphism (RFLP) method. The allele frequency of the ARE-2 variant in diabetic subjects was higher than that in nondiabetic subjects (0.34 vs. 0.29; P < 0.05), even though its frequency in Japanese subjects was lower (P < 0.001) than the reported value in Pima Indians (0.56). An aspartate polymorphism at codon 905 was 100% coupled to the ARE-2 allele, and its allele frequency was higher also in diabetic subjects. Although a serine substitution at codon 883 was partially linked with the ARE-2 allele, there was no difference between diabetic and nondiabetic subjects. These results indicate that the frequency of polymorphism of the PPP1R3 gene (ARE-2 and Asp905) is different between two ethnic groups and is increased in Japanese people with type 2 diabetes, suggesting that these variants may be a possible marker for searching for diabetogenic genes.
To characterize the morphology of the endoneurial microvasculature of degenerating nerves under hyperglycemia, the morphology of endoneurial microvessels in transected sciatic nerves was examined in normal and streptozotocin-induced diabetic rats. Three months after transection, the fascicular area and median vascular luminal area at the proximal level of the distal stump were significantly larger in diabetic than in control animals, whereas the number of vessels per fascicle was the same in the two groups. Arterioles in various stages of development were found in the centrifascicular region in some transected nerves. Serial sections revealed that these vessels originated from transperineural arterioles. The frequency and magnitude of vascular wall thickening were both greater in diabetic rats. These results suggest that the endoneurial microvasculature responds abnormally to nerve injury under hyperglycemia.
Apolipoprotein (apo)A-I alone or as a component of high density lipoprotein particles has antiatherogenic properties. The age-dependent decline in abundance of this protein may underlie the higher risk for developing occlusive coronary artery disease (CAD) in older individuals. Similar to humans, expression of rat apoA-I also declines with age. Results in rats showed that levels of serum apoA-I protein, hepatic mRNA, and transcription of the gene were decreased to 39%, 18%, and 38%, respectively, in 180-day-old animals compared to those of newborn rats. These findings suggest that a nuclear mechanism(s) may account for the decline in apoA-I expression. Accordingly, we examined hepatic nuclear binding activity to four specific cis-acting elements of the rat apoA-I promoter. There were age-dependent changes of binding activity to two proximal sites, B and C, but not to the more distal elements, IRCE and A. Decreased B-site binding activity correlated with lower mRNA levels encoding the activator, HNF-3beta. The age-dependent change in the pattern of binding to site C was due to a switch from the activator, HNF-4, to the repressor, ARP-1. In summary, the age-related decline in apoA-I expression may arise from a reduction in the activity of both cis-acting elements, B and C.
To investigate the difference in signaling between insulin and insulin-like growth factor I (IGF-I), we studied the effects of these hormones on the phosphorylation state of Crk-associated substrate (Cas) in cells expressing human insulin receptor (HIRc). In the basal state, Cas was heavily tyrosine-phosphorylated, and insulin dephosphorylated Cas in a time- and dose-dependent manner. On the other hand, IGF-I phosphorylated rather than dephosphorylated Cas in HIRc cells. In HIRY/F2 cells expressing a mutant insulin receptor lacking a binding site of SHP-2, a protein-tyrosine phosphatase containing src homology 2 (SH2) regions, insulin accelerated phosphorylation of Cas, as did IGF-I. In HIRc cells expressing a mutant SHP-2 lacking a PTPase domain (DeltaPTP), which interfered with SHP-2 function, insulin failed to dephosphorylate Cas. In whole cell lysate obtained in the basal state, Cas bound to a glutathione-S transferase fusion protein containing SH2 domains of SHP-2 and dissociated from this GST protein in response to insulin. These results indicate that the opposite regulation of Cas phosphorylation by insulin and IGF-I may be mediated through different properties of their receptors, and that the interaction of the insulin receptor with SHP-2 may play an important role in determining the tyrosine-phosphorylation state of Cas.
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We investigated heparin-binding epidermal growth factor-like growth factor (HB-EGF) gene and protein expression in the central nervous system of prenatal and early postnatal rats. Assay by northern blot analysis showed that the HB-EGF mRNA was markedly expressed in the brain. In situ hybridization and immunohistochemical techniques showed that concordant expression of HB-EGF mRNA and protein was widely observed in the neurons and interfascicular oligodendrocytes, especially in the cerebellum, the hippocampus, the cerebral cortex, the subventricular area, and the brain stem nuclei. The intense expression of the HB-EGF mRNA was related anatomically and temporally to the proliferating neuroblasts in the external granular layer of the cerebellum and the subventricular layer of the cerebrum. These findings suggest that HB-EGF acts as a mitogen for the neuroblasts. Moreover, HB-EGF expression was observed in the post-mitogenic cells, such as in the cells of the molecular layer, the white matter, the IGL, or the Purkinje cells of the cerebellum. Since EGF receptors are abundantly expressed in the post-mitogenic period, the HB-EGF mRNA expression observed in the post-mitogenic period in our study suggests that HB-EGF also has a non-mitogenic function. These results suggest that HB-EGF significantly contributes to the development of the brain.
It is known that Wallerian degeneration (WD) is prerequisite for nerve regeneration, which is impaired in experimental diabetic rats. To elucidate the effect of hyperglycemia on WD, we studied the time course of WD after axotomy in streptozocin-diabetic (DM) and control rats. Sciatic nerves were removed at several time points after axotomy (days 0-24). Morphometric analysis indicated that WD was delayed in DM throughout experimental period. Quantitative immunohistochemical analysis showed that the early recruitment of macrophage did not differ between the two groups, although its late recruitment was significantly decreased in DM at 15 and 24 days post-axotomy, which suggested that the macrophage-associated process did not contribute to delayed WD in diabetes. Immunoblot analysis showed a delay in the degradation of neurofilaments (NFs) in DM during WD. Phosphorylated NFs detected by SMI31 were more recognized in DM, while the opposite was true for unphosphorylated NFs detected by SMI32. Since it is known that the sensitivity of NF to calpain-mediated proteolysis is modulated by its carboxyl-terminal phosphorylation state and phosphorylated NFs are resistant to calpains, we concluded that abnormal NF phosphorylation state in diabetes could be one of the mechanisms by which axonal degeneration was delayed.
To evaluate the role of nitric oxide synthase (nNOS) in the pathogenesis of diabetic neuropathy, we investigated nociception and nNOS expression in dorsal root ganglion (DRG) of rats with streptozocin-induced diabetes. Paw withdrawal threshold to noxious mechanical stimuli was decreased in both L-NAME-treated and diabetic rats. The number of NADPH-diaphorase positive neurons was significantly decreased in untreated diabetic compared with control rats. Decreased expression of nNOS protein was confirmed by immunoblotting. Insulin treatment completely prevented decreases in withdrawal threshold and nNOS expression. Cyclic GMP content paralleled nNOS expression in experimental animals. These results suggest that decreased nNOS-cGMP system in DRG may play a role in the pathogenesis of diabetic sensory neuropathy.
Since diabetes now accounts for 35% of all new cases of end-stage renal disease in the United States, it is really important to prevent the onset of diabetic nephropathy. Activation of protein kinase C (PKC) is implicated to be one of the causal factors in the development of renal dysfunctions in diabetes. In this study, we have demonstrated that total diacylglycerol (DAG) contents and PKC activity in glomeruli were significantly increased in diabetic rats as compared to control rats, but intraperitoneal injection of d-alpha-tocopherol prevented these biochemical abnormalities in parallel with normalization of glomerular dysfunction such as increased glomerular filtration rate (GFR) in diabetic rats. Albuminuria in diabetic rats was also significantly increased as compared to control rats, whereas d-alpha-tocopherol treatment again ameriolated increased albuminuria in parallel with the inhibition of glomerular PKC activation by diabetes. Moreover, we have observed that the activity of DAG kinase, which metabolizes DAG to phosphatidic acid and acts as an attenuator for the DAG-PKC pathway, was enhanced by d-alpha-tocopherol treatment. These results suggest that the increase in the DAG-PKC pathway might play an important role for the development of glomerular dysfunctions in diabetes and d-alpha-tocopherol treatment could be helpful in diabetic nephropathy.
Otsuka Long-Evans Tokushima Fatty (OLETF) rats are reported to be obese Type II (non-insulin-dependent) diabetic rats with insulin resistance and impaired insulin secretion. To investigate the contribution of intestinal glucose absorption to postprandial hyperglycaemia, we determined the plasma xylose concentrations after an 0.8 g/kg oral xylose load which was used as a test of small intestinal glucose absorption in 6-week-old OLETF rats and weight-matched Long-Evans Tokushima Otsuka (LETO) rats. An oral glucose tolerance test showed that OLETF rats developed hyperglycaemia at 60 and 90 min after the glucose load, though the fasting plasma glucose concentration, insulin concentration and insulin-induced in vivo glucose utilization rate were similar. Consistently, in an oral D-xylose loading test, the peak concentration of plasma xylose in OLETF rats was increased by 58.7% compared with that of LETO rats (p < 0.005). The disappearance rate of plasma xylose concentrations after intravenous xylose loading did not differ between the two strains. Co-treatment with 0.4 g/kg phlorizin, a specific inhibitor of sodium-dependent glucose transporter 1 (SGLT1), abolished both plasma glucose and xylose concentrations after the loads. Morphological studies showed that both the small intestinal wet weight and surface area were 30% larger in the OLETF rats than in the LETO rats. Furthermore, the SGLT1 mRNA content of OLETF rats also increased compared with LETO rats. These results suggest that an increased SGLT1 expression concomitant with intestinal hypertrophy in OLETF rats is partly associated with postprandial hyperglycaemia before the onset of insulin resistance and hyperinsulinaemia.
In spite of the clarification in the temporal and spatial expression pattern of a cyclin-dependent kinase (Cdk) 5 and its neuron-specific activator, p35, in the CNS, it remains to be elucidated in the PNS. In addition, it is not known whether Cdk5 activity exists in the PNS. Therefore, we have examined their expression and activity in the PNS by immunoblot analysis, immunohistochemistry, and in vitro kinase assay. Immunoblot analysis indicated the expression of Cdk5 and p35 proteins in dorsal root ganglion (DRG) and sciatic nerve alike in the CNS. By immunohistochemistry, both proteins were shown to be present in the cell body and axon (sciatic nerve) of both DRG neurons and anterior horn cells. A co-immunoprecipitation study indicated the in vivo association between Cdk5 and p35 in both DRG and sciatic nerve. However, Cdk5 kinase activity was found only in DRG, but not in sciatic nerve. These results suggest that Cdk5 kinase activity exists and functions physiologically in the PNS and may be regulated by unknown mechanisms other than the availability of p35 as reported in developing brains.
Atrial natriuretic peptide (ANP) has been shown to counteract various actions of endothelin-1 (ET-1) in mesangial cells. We have reported that both extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) are activated by ET-1 and ET-1-induced activation of ERK is inhibited by ANP. To further clarify the action of ANP, we examined the effect of ANP on ET-1-induced activation of JNK. ANP inhibited ET-1-induced activation of JNK in a dose-dependent manner. This inhibitory effect of ANP was reversed by HS-142-1, an antagonist for biological receptors of ANP, while C-ANP, an analog specific to clearance receptors of ANP, failed to inhibit ET-1-induced activation of JNK. 8-Bromo-cGMP and sodium nitroprusside were also able to inhibit ET-1-induced activation of JNK, suggesting cGMP-dependent action of ANP. In contrast, ANP failed to inhibit interleukin-1 beta (IL-1 beta)-induced activation of JNK. Since an increase in intracellular calcium ([Ca2+]i) was shown to be necessary for ET-1-induced activation of JNK in mesangial cells, we measured [Ca2+]i using fura-2. ANP attenuated the ET-1-induced increase in [Ca2+]i in concentrations enough to inhibit ET-1-induced activation of JNK. Finally, ANP was able to inhibit ET-1-, but not IL-1 beta-induced increase in DNA-binding activity of AP-1 by gel shift assay. These results indicate that ANP is able to inhibit ET-1-induced activation of AP-1 by inhibiting both ERK and JNK, suggesting that ANP might be able to counteract the expression of AP-1-dependent genes induced by ET-1.
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We have reported that high glucose conditions (27 mM for 4 days) induces activation of protein tyrosine phosphatases (PTPases) which are associated with impaired insulin signaling in Rat 1 fibroblasts expressing human insulin receptors [Maegawa, H. et al. (1995) J. Biol. Chem. 270, 7724-7730]. In this study, we found increased mRNA-levels of a non-receptor type PTPase, protein tyrosine phosphatase 1B (PTP1B), and receptor type PTPases, leukocyte common antigen-related phosphatase (LAR), and LAR-related phosphatase (LRP), under high glucose conditions. In accordance with these results, LAR content was significantly increased, whereas LRP content was not increased. Cytosolic PTP1B content was increased, but membrane-associated PTP1B content showed no detectable change. Pioglitazone, a thiazolidinedione, normalized increased cytosolic PTPase activity through reduction of cytosolic PTP1B content, but it had no effect on mRNA levels of these PTPases. Under the high glucose condition, we also found that epidermal growth factor (EGF)-stimulated signaling, including tyrosine-phosphorylation of EGF receptor and phosphatidylinositol 3'-kinase activities, was attenuated. Nevertheless, pioglitazone failed to restore the attenuated EGF-signaling. These results indicate that the high glucose conditions cause dysfunction of EGF receptor. However, the increased cytosolic PTP1B content is not involved in the abnormal regulation of EGF-signaling, in contrast to insulin-signaling.
A 75-year-old woman was admitted because of pain and numbness in the extremities and trunk. She subsequently suffered from lymphadenopathy and spiky fever. The immunohistochemical analysis of the biopsied lymph nodes and sural nerve and electrophysiological examination supported a diagnosis of angioimmunoblastic lymphadenopathy-like T cell lymphoma with polyneuropathy. The infiltrating lymphoma cells of the sural nerve and lymph node shared the same phenotype (CD45RO, CD3, CD30 positive). An increased expression of HLA-DR antigen was observed in endothelial and Schwann cells. Chemotherapy with CHOP-Bleomycin markedly relieved her pain. These findings suggest that a direct lymphocytic infiltration in the nerve may be associated with neuropathy in this case.
Gene regulation in the cardiovascular tissues of diabetic subjects has been reported to be altered. To examine abnormal activities in transcription factors as a possible cause of this altered gene regulation, we studied the activity of two redox-sensitive transcription factors--nuclear factor-kappaB (NF-kappaB) and activating protein-1 (AP-1)--and the change in the mRNA content of heme oxygenase-1, which is regulated by these transcription factors in the cardiac tissues of rats with streptozotocin-induced diabetes. Increased activity of NF-kappaB and AP-1 but not nuclear transcription-activating factor, as determined by an electrophoretic mobility shift assay, was found in the hearts of 4-week diabetic rats. Glycemic control by a subcutaneous injection of insulin prevented these diabetes-induced changes in transcription factor activity. In accordance with these changes, the mRNA content of heme oxygenase-1 was increased fourfold in 4-week diabetic rats and threefold in 24-week diabetic rats as compared with control rats (P < 0.01 and P < 0.05, respectively). Insulin treatment also consistently prevented changes in the mRNA content of heme oxygenase-1. The oral administration of an antioxidant, probucol, to these diabetic rats partially prevented the elevation of the activity of both NF-kappaB and AP-1, and normalized the mRNA content of heme oxygenase-1 without producing any change in the plasma glucose concentration. These results suggest that elevated oxidative stress is involved in the activation of the transcription factors NF-kappaB and AP-1 in the cardiac tissues of diabetic rats, and that these abnormal activities of transcription factors could be associated with the altered gene regulation observed in the cardiovascular tissues of diabetic rats.