[Diabetes-induced abnormality in protein metabolism].
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Animals↗
Biomedical subjects
R Kikkawa
Publications and source records attributed to R Kikkawa.
[Diabetic nephropathy--definition, stages, measurement of albuminuria, and therapy].
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Diabetic Nephropathies↗
SHPTP2 serves adapter protein linking between Janus kinase 2 and insulin receptor substrates.
To investigate the role of Janus kinase family (JAK1 and JAK2) in insulin signaling, we assessed their insulin-induced associations with other molecules in the cells overexpressing insulin receptors (HIRc and CHO-IR). After insulin stimulation, pp185 proteins (insulin receptor substrate, IRS) were co-immunoprecipitated with both kinases by alpha JAK1 and alpha JAK2 antibodies. However, JAK2 constitutively associated with pp95 protein (IR beta). Moreover, JAK2 also constitutively bound to a protein tyrosine phosphatase containing Src 2 regions (SHPTP2), but JAK1 did not. In HIRc cells expressing PTPase-negative mutant SHPTP2, no association of JAK2 with either pp185 or pp95 was detected. Thus, SHPTP2 might serve as an adapter protein linking between JAK2 and IRS. These results suggest that JAK1 and JAK2 behave differently and they may constitute a new regulatory component in insulin signaling.
Animals↗
[Treatment of diabetes mellitus and its complications. 3. Diabetic nephropathy].
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Diabetic Nephropathies↗
Expression of dominant negative mutant SHPTP2 attenuates phosphatidylinositol 3'-kinase activity via modulation of phosphorylation of insulin receptor substrate-1.
To clarify the role of protein-tyrosine phosphatase (PTPase) containing Src homology 2 regions (SHPTP2) in insulin signaling, either wild-type or mutant SHPTP2 (delta PTP; lacking full PTPase domain) was expressed in Rat 1 fibroblasts overexpressing human insulin receptors. In response to insulin, phosphorylation of insulin receptor substrate 1 (IRS-1), IRS-1-associated PTPase activities and phosphatidylinositol (PI) 3'-kinase activities were slightly enhanced in wild-type cells when compared with those in the parent cells transfected with hygromycin-resistant gene alone. In contrast, introduction of delta PTP inhibited insulin-induced association of IRS-1 with endogenous SHPTP2 and impaired both insulin-stimulated phosphorylation of IRS-1 and activation of PI 3'-kinase. Furthermore, decreased content of p85 subunit of PI 3'-kinase was also found in mutant cells. Consistently, the insulin-stimulated mitogen-activated protein kinase activities and DNA synthesis were also enhanced in wild-type cells, but impaired in mutant cells. Thus, the interaction of SHPTP2 with IRS-1 may be associated with modulation of phosphorylation levels of IRS-1, resulting in the changes of PI 3'-kinase and mitogen-activated protein kinase activity. Furthermore, an impaired insulin signaling in mutant cells may be partly reflected in a decreased content of p85 protein of PI 3'-kinase.
Animals↗
Atrial natriuretic peptide induces the expression of MKP-1, a mitogen-activated protein kinase phosphatase, in glomerular mesangial cells.
Atrial natriuretic peptide (ANP) has been shown to inhibit the proliferation of various types of cells including glomerular mesangial cells. The activation of mitogen-activated protein kinase (MAPK) is one of the main signal transduction systems leading to cell proliferation. MAPK is tightly regulated by the activating kinase, MEK, and specific phosphatase MKP-1. Constitutive expression of MKP-1 has been shown to inhibit cell proliferation by suppressing MAPK activity. In order to understand the mechanism of the anti-proliferative effect of ANP, we examined whether ANP could inhibit MAPK by inducing MKP-1 in cultured rat glomerular mesangial cells. ANP increased the expression of MKP-1 mRNA in a dose-dependent (10 nM maximum) and time-dependent, with a peak stimulation at 30 min, manner. Receptor for ANP is a transmembrane guanylyl cyclase. Activation of guanylyl cyclase of ANP receptor by ligand plays an essential role in ANP signal transduction. 8-Bromo-cGMP, a cell permeable analogue of cyclic GMP, and sodium nitroprusside, an activator of soluble guanylyl cyclase, could mimic the effects of ANP and were able to induce the expression of MKP-1 in a similar time course as ANP. The protein expression of MKP-1 was maximally stimulated by ANP at 120 min. Treatment of the cells with ANP for 120 min resulted in an inhibition of phorbol ester-induced activation of MAPK, while the activation of MEK was not affected by ANP. These results indicate that ANP might inhibit the proliferation of mesangial cells by inactivating MAPK through the induction of MKP-1.
Amino Acid Sequence↗
Tolrestat improves nerve regeneration after crush injury in streptozocin-induced diabetic rats.
To delineate the ability of diabetic nerves to regenerate and to determine the effect of aldose reductase (AR) inhibitors (ARIs) on nerve regeneration in diabetic neuropathy, we evaluated nerve regeneration electrophysiologically and morphologically after sciatic nerve crush injury in three groups of male Sprague-Dawley rats: untreated diabetic (streptozocin [STZ]-induced, n = 16), tolrestat-treated diabetic (n = 16), and age-matched controls (n = 16). Compound muscle action potentials (CMAPs) appeared 4 weeks after crush injury in the control group and 5 weeks after injury in both diabetic groups. Motor nerve conduction velocity (MNCV) in the crushed nerves was decreased in both diabetic groups compared with the control group throughout the experiment. However, this decrease was significantly prevented at 24 weeks with tolrestat treatment. Morphologically, the density of myelinated nerve fibers (MNFs) and the number of MNFs per fascicle were significantly decreased in untreated diabetic rats, but tolrestat significantly prevented the former decrease at 5 weeks and the latter at 24 weeks. The mean diameter of large MNFs (>4 microm) was smaller in the untreated diabetic group than in the control group, but this decrease also was significantly prevented with tolrestat treatment. These results suggest that nerve regeneration is impaired in diabetic neuropathy and that tolrestat can prevent this impairment.
Animals↗
Differential inhibition of mesangial MAP kinase cascade by cyclic nucleotides.
The agents which increase intracellular cyclic AMP (cAMP) or cyclic GMP (cGMP) have been found to counteract the effects of the vasoconstrictive agents such as endothelin-1 (ET-1). To clarify the mechanism of this interaction, we evaluated the activities of mitogen-activated protein kinase (MAPK) cascade, one of the important signal transduction system of ET-1. Beraprost sodium, an analogue of PGI2, and adrenomedullin, a cAMP-raising agent, inhibited ET-1-induced activation of MAPK. Dibutyryl cAMP (Bt2-cAMP) and 8-bromo-cGMP (8-Br-cGMP), cell permeable analogues of cAMP and cGMP, were also able to inhibit the activation of MAPK and MAPK kinase (MAPKK) by ET-1 without interfering basal activities. In contrast, phorbol 12, 13-dibutylate (PDBu)-induced activation of MAPK and MAPKK was inhibited by Bt2-cAMP but not by 8-Br-cGMP. Interestingly, atrial natriuretic peptide (ANP) partially inhibited PDBu-induced activation of MAPK and MAPKK. These results indicate that cAMP and cGMP inhibit ET-1-induced activation of MAPK in cultured mesangial cells at different steps; the former might inhibit at a step downstream of PKC and the latter prior to PKC. The data also suggest that ANP might have cGMP-independent effect on MAPK.
Adrenomedullin↗
Insulin signaling and its regulation of system A amino acid uptake in cultured rat vascular smooth muscle cells.
Hyperinsulinemia has been recognized as an independent risk factor for atherosclerosis. However, its exact mechanisms are still unclear. In our previous work, we showed that 10 nmol/L insulin stimulated neither mitogen-activated protein kinase (MAP kinase) activity nor [3H]thymidine incorporation but did stimulated S6 kinase through the specific insulin receptors in cultured rat vascular smooth muscle cells (VSMCs). In this study, we observed that > or = 1 nmol/L insulin stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and activated IRS-1-dependent phosphatidylinositol 3'-kinase (PI 3'-kinase) and p70 S6 kinase (p70S6K) but not MAP kinase (extracellular signal-regulated kinase 2) and p90 S6 kinase (p90RSK). However, 10 nmol/L insulin-like growth factor I stimulated all these pathways. Finally, 10 nmol/L insulin stimulated alpha-amino-isobutyric acid (AIB) uptake, and wortmannin (100 nmol/L) completely inhibited insulin-stimulated AIB uptake, whereas rapamycin (20 nmol/L) had no such effect. Furthermore, cycloheximide (10 micrograms/mL) completely inhibited insulin-stimulated AIB uptake, but actinomycin D (5 micrograms/mL) failed to inhibit this. Thus, we reached the following conclusions: (1) Insulin (1 nmol/L) induced phosphorylation of IRS-1 and activated the PI 3'-kinase and p70S6K pathways in VSMCs, even though 10 nmol/L insulin did not significantly stimulate MAP kinase or p90RSK. (2) Stimulation of AIB uptake by insulin was regulated at the translational level via wortmannin-sensitive pathways but not p70S6K pathways.
Aminoisobutyric Acids↗
Insulin resistance, hypertension and nephropathy.
Although insulin resistance has been involved in the pathogenesis of essential hypertension in non-diabetic patients, few studies were performed regarding to the association between insulin resistance, hypertension and nephropathy in diabetes mellitus. We observed the changes of blood pressure and proteinuria for 7 years in normotensive 28 patients with non-insulin-dependent diabetes mellitus (NIDDM), following measurement of insulin sensitivity. Patients were over 40 years old and not obese, and fasting plasma glucose levels were less than 140 mg/dl. Insulin sensitivity was determined using glucose-clamp method or glucose, insulin, and somatostatin infusion method. In 28 subjects, 12 subjects developed hypertension and 16 subjects were remained normotensive. Insulin induced glucose clearance was significantly decreased in subjects developed hypertension (30 +/- 12 ml/kg/10 min) than in subjects remained normotensive (50 +/- 19 ml/kg/10 min). Furthermore, we found significantly higher incidence of proteinuria in patients developed hypertensive (7 out of 12 patient) than in patients remained normotensive (one out of 16 patients; p < 0.05). These results suggest that insulin resistance is involved in the etiology of hypertension in NIDDM patients, and that this derangement has an important role for the progression of diabetic nephropathy.
Blood Pressure↗
Glycation, oxidative stress, and scavenger activity: glucose metabolism and radical scavenger dysfunction in endothelial cells.
It has been reported that oxidative stress is increased in vivo in the diabetic state. Increased oxidative stress is caused not only by accelerated production of oxygen-free radicals but also by decreased scavenging of those molecules. Endothelial cells are extremely sensitive to oxidative stress, resulting in impairments of various endothelial cell function. In this report, we studied the association of intracellular glucose metabolism and oxygen radical scavenging function via the glutathione redox (GR) cycle in cells exposed to high-glucose conditions using cultured human umbilical vein endothelial cells. Glutathione-dependent H2O2 degradation in cells exposed to 33 mmol/l glucose (HG) for 5-7 days was reduced by 48% vs. 5.5 mmol/l glucose (NG). This impairment under the oxidative stress was D-glucose-specific and concentration-dependent and was also associated with a 42% decrease in intracellular NADPH content. Exposure of cells to 200 micromol/l H2O2 stimulated the GR cycle and the pentose phosphate pathway (PPP) at the same time. In the HG condition, activation of PPP was reduced by 50%, which was consistent with a decrease in NADPH content. Inhibition of glycolysis by H2O2 was less marked in HG cells versus NG cells. Activation of polyol pathway in HG cells is not responsible for the decrease in intracellular NADPH content. These results indicate that activation of the PPP and NADPH supply to the GR cycle is impaired in HG cells exposed to H2O2, which may result in increased oxidative stress to endothelial cells.
Cells, Cultured↗
[Tyrosine phosphorylation of focal adhesion kinase (p125FAK) and paxillin in glomeruli from diabetic rats].
Focal adhesion kinase (p125FAK) is a novel non-receptor cytosolic tyrosine kinase which is activated through the phosphorylation of its tyrosine residue by ligands that bind to integrins and ligands that activate protein kinase C (PKC). In diabetic glomeruli, extracellular matrix proteins such as fibronectin, laminin and type IV collagen, which bind to integrins, were found to be increased in the mesangial area. Furthermore, PKC was shown to be activated in diabetic glomeruli. These changes might be able to cause the activation of p125FAK in diabetic glomeruli. To test this hypothesis, we examined tyrosine phosphorylation of p125FAK and paxillin, a proposed substrate of p125FAK, in glomeruli isolated from streptozotocin (STZ)-induced diabetic rats. Tyrosine phosphorylation of p125FAK or paxillin was evaluated by immunoblot analysis using anti-phosphotyrosine antibody after immunoprecipitation with anti-p125FAK or anti-paxillin antibody. Three and seven weeks after STZ injection, tyrosine phosphorylation of both p125FAK and paxillin was increased in diabetic glomeruli. The increase in tyrosine phosphorylation of p125FAK and paxillin was not observed in glomeruli from diabetic rats treated with insulin. To investigate the mechanism of increase in tyrosine phosphorylation of p125FAK, we examined tyrosine phosphorylation of p125FAK in mesangial cells plated on a fibronectin-coated dish or cultured under conditions of high glucose concentration (conditions under which PKC can be activated). Attachment of the cells to fibronectin induced tyrosine phosphorylation of p125FAK, while a high glucose concentration did not modulate tyrosine phosphorylation of p125FAK. In conclusion, tyrosine phosphorylation of p125FAK and paxillin was increased in diabetic glomeruli and these alternations may have been caused by changes in extracellular matrix proteins in diabetes.
Animals↗
[Studies on type IV collagen production in cultured mesangial cells].
Mesangial expansion is one of the histological abnormalities observed among the various glomerular diseases. It is due largely to the accumulation of extracellular matrix (ECM) proteins, such as type IV collagen, laminin and fibronectin. In order to examine the abilities of mesangial cells to produce type IV collagen, we used enzyme-linked immunosorbent assay to measure type IV collagen secreted from cultured rat mesangial cells. After exposing confluent cells to endothelin-1 (ET-1, 0.01 microM), insulin-like growth factor-1 (100 ng/ml) angiotensin-II (1 microM) and transforming growth factor-beta (1 ng/ml) for 24 hours, type IV collagen production increased 1.5 approximately 2.5 times. ET-1 stimulated type IV collagen production in a dose-dependent fashion, and this effect was blocked by the protein kinase C inhibitor, staurosporine. Atrial natriuretic peptide (ANP) inhibited the increase in type IV collagen production caused by ET-1 in a dose-dependent fashion. Addition of 8-bromo-cGMP similarly suppressed the stimulation of type IV collagen production caused by ET-1. These findings indicate that ET-1 stimulates type IV collagen synthesis in cultured rat mesangial cells by a mechanism probably involving activation of PKC, and that ANP inhibits ET-1-induced production of type IV collagen through a cGMP-dependent process.
Animals↗
[Relationship between the stage of diabetic nephropathy and serum lipoprotein (a) concentrations--influence of hypoproteinemia].
UNLABELLED: Lipoprotein (a) [Lp(a)] has been reported to be an independent risk factor for coronary heart disease. Elevated levels of Lp(a) in diabetic subjects have also been reported, especially in subjects with nephropathy. However, the mechanism and the clinical implication of Lp(a) elevation in diabetics remain obscure. In the present study, to verify the change in Lp(a) concentration with the progression of nephropathy, serum Lp(a) levels were measured in 546 NIDDM patients, 33 hemodialysis NIDDM patients, and 145 non-diabetic controls. RESULTS: 1) The serum Lp(a) levels in diabetics were significantly higher than those in the non-diabetic controls. 2) Serum Lp(a) concentrations in microalbuminuric subjects were significantly higher than those in normoalbuminuric subjects, and were further increased in macroalbuminuric subjects. 3) In macroalbuminuric subjects, serum Lp(a) concentrations were inversely correlated with serum protein concentrations, but not with serum creatinine. 4) Once hemodialysis was started, the decrement of both serum Lp (a) and LDL-C concentrations were associated with the increment of serum protein concentrations. These data indicate that elevation of serum Lp (a) concentration in overt nephropathy could be, at least in part, due to hypoproteinemia.
Adult↗
Thiazolidine derivatives ameliorate high glucose-induced insulin resistance via the normalization of protein-tyrosine phosphatase activities.
The mechanisms for the insulin resistance induced by hyperglycemia were investigated by studying the effect of high glucose concentration (HG) and its modulation by thiazolidine derivatives, on insulin signaling using Rat 1 fibroblasts expressing human insulin receptors (HIRc). Incubating HIRc cells in 27 mM D-glucose for 4 days impaired the insulin-stimulated phosphorylation of pp185 and receptor beta-subunits. Both protein kinase C activities and phorbol dibutyrate binding to intact cells were unchanged; however, cytosolic protein-tyrosine phosphatase (PTPase) activity increased within 1 h prior to the impairment of insulin receptor kinase in HG cells (Maegawa, H., Tachikawa-Ide, R., Ugi, S., Iwanishi, M., Egawa, K., Kikkawa, R., Shigeta, Y., and Kashiwagi, A. (1993) Biochem. Biophys. Res. Commun. 197, 1078-1082). Increased PTPase activity was consistent with a 2-fold increase in the amount of PTP1B, and anti-PTP1B antibody inhibited this increment of cytosolic PTPase activity in HG cells. Co-incubating cells with pioglitazone prevented these abnormalities in cytosolic PTPase, the PTP1B content and the impaired phosphorylation of pp185 and receptor beta subunits in HG cells. Finally, HG cells had impaired insulin-stimulated alpha-amino-isobutyric acid uptake, which was ameliorated by exposure to thiazolidine derivatives. In conclusion, exposing cells to high glucose levels desensitizes insulin receptor function, and thiazolidine derivatives can reverse the process via the normalization of cytosolic PTPase, but not of protein kinase C.
Aminoisobutyric Acids↗
Antibodies against GM1 ganglioside affect K+ and Na+ currents in isolated rat myelinated nerve fibers.
High titers of anti-GM1 ganglioside antibodies (anti-GM1 antibodies) may be implicated in lower motor neuron disease. We studied the pathogenic role of anti-GM1 antibody using the petroleum jelly-gap voltage clamp technique on isolated single myelinated rat nerve fibers. Anti-GM1 antisera were obtained from rabbits immunized with GM1 ganglioside. Extracellularly applied anti-GM1 antisera without complement activity increased both the rate of rise and the amplitude of the K+ current elicited by step depolarization, with little effect on Na+ current. In the presence of active complement, however, anti-GM1 antibodies decreased the Na+ current, and caused a progressive increase of nonspecific leakage current. Neither complement alone nor complement-supplemented antisera from which anti-GM1 antibodies were depleted by affinity chromatography had any effect on ionic current. These observations indicate that anti-GM1 antibodies themselves can uncover K+ channels in the paranodal region, while anti-GM1 antibodies bound to the nodal membrane in the presence of complement may form antibody-complement complexes that block Na+ channels and disrupt the membrane at the node of Ranvier.
Action Potentials↗
Prevention of glomerular hyperfiltration in rats with streptozotocin-induced diabetes by an atrial natriuretic peptide receptor antagonist.
The contribution of atrial natriuretic peptide (ANP) to the development of glomerular hyperfiltration in diabetes was investigated by examining the effects of HS-142-1, a non-peptide antagonist of biological receptors for ANP, on glomerular filtration rate (GFR) and renal plasma flow (RPF) in rats with streptozotocin-induced diabetes. Three to four weeks after streptozotocin injection, the plasma concentration of ANP, urinary cyclic GMP excretion rate, GFR, and RPF were significantly higher in diabetic rats than in control rats. The increase in GFR and RPF in diabetic rats was significantly reduced, in a dose-dependent manner, by a single intravenous injection of HS-142-1; the maximal effect was apparent at a dose of 10 mg per kg of body weight. Continuous subcutaneous administration of HS-142-1 with an osmotic minipump for 3 to 4 weeks, beginning 2 days after streptozotocin injection, prevented the increases in urinary cyclic GMP excretion rate, GFR, and RPF observed in untreated diabetic rats. These results highlight the importance of ANP in the development of diabetic glomerular hyperfiltration and indicate that this condition can be prevented by continuous inhibition of the action of ANP.
Animals↗
Increase of voltage-sensitive calcium channels and calcium accumulation in skeletal muscles of streptozocin-induced diabetic rats.
The number of voltage-sensitive calcium channels (VOCC) in triceps surae muscle membrane fractions isolated from control and streptozocin (STZ)-induced diabetic rats was determined using [3H]PN200-110, a dihydropyridine derivative, as a ligand. Furthermore, quantitative analysis of calcium in soleus muscle fibers was performed by the calcium oxalate-pyroantimonate method and x-ray microanalysis. The maximum binding (Bmax) of [3H]PN200-110 in skeletal muscle membrane isolated from 10-week diabetic rats (1,091 +/- 77 fmol/mg protein) was increased significantly by 91% as compared with the control value (572 +/- 32 fmol/mg protein), without a significant change in Kd. The increase in Bmax of [3H]PN200-110 was dependent on the duration of diabetes, and was not found until 6 weeks after STZ injection. Insulin treatment for 8 weeks after induction of diabetes normalized Bmax to the control level (583 +/- 53 fmol/mg protein). Precipitates of calcium antimonate, identified by x-ray microanalysis, were observed much more frequently in specimens from 10-week diabetic rats versus controls. The increase in the incidence of precipitates was not observed in 3-week diabetic rats and was suppressed by 8 weeks' insulin treatment. These results indicate that the number of VOCC in chronically diabetic rats was increased in the sarcolemmal membrane of skeletal muscle and that calcium was accumulated inside skeletal muscle fibers.
Analysis of Variance↗