Involvement of heat shock protein 90 in the degradation of mutant insulin receptors by the proteasome

We previously reported three families with type A insulin-resistant syndrome who had mutations, either Asp1179 or Leu1193, in the kinase domain of the insulin receptor. The extreme insulin resistance of these patients was found to be caused by the decreased number of insulin receptors on the cell surface, due to the intracellular rapid degradation (Imamura, T., Takata, Y., Sasaoka, T., Takada, Y., Morioka, H., Haruta, T., Sawa, T., Iwanishi, M., Yang, G. H., Suzuki, Y., Hamada, J., and Kobayashi, M. (1994) J. Biol. Chem. 269, 31019-31027). In the present study, we first examined whether these mutations caused rapid degradation of unprocessed proreceptors, using the exon 13 deleted mutant insulin receptors (DeltaEx13-IR), which were accumulated in the endoplasmic reticulum as unprocessed proreceptors. The addition of Asp1179 or Leu1193 mutation to DeltaEx13-IR caused accelerated degradation of the unprocessed DeltaEx13-IR in the transfected COS-7 cells. Next, we tested whether these mutant receptors were degraded by the proteasome. Treatment with proteasome inhibitors Z-Leu-Leu-Nva-H (MG-115) or Z-Leu-Leu-Leu-H (MG-132) prevented the accelerated degradation of these mutant receptors, resulting in increased amounts of the mutant receptors in the COS-7 cells. Essentially the same results were obtained in the patient's transformed lymphocytes. Finally, we found that these mutant receptors bound to heat shock protein 90 (Hsp90). To determine whether Hsp90 played an important role in the accelerated receptor degradation, we examined the effect of anti-Hsp90 antibody on the mutant receptor degradation. The microinjection of anti-Hsp90 antibody into cells prevented the accelerated degradation of both Asp1179 and Leu1193 mutant insulin receptors. Taken together, these results suggest that Hsp90 is involved in dislocation of the mutant insulin receptors out of the endoplasmic reticulum into the cytosol, where the mutant receptors are degraded by the proteasome.     

In vivo properties of the conjugates of mitomycin C with estradiol benzoate and estradiol: pharmacokinetics and antitumor characteristics against P388 leukemia and sarcoma 180

Conjugates of mitomycin C (MMC) with estradiol benzoate and estradiol via glutaric acid, abbreviated to EB-glu-MMC and E-glu-MMC, respectively, as previously reported, were examined concerning their pharmacokinetic behaviors and antitumor effects against two kinds of general and popular tumors, P388 leukemia and Sarcoma 180. EB-glu-MMC and E-glu-MMC were dissolved in propylene glycol. Their solution was administered intraperitoneally to rats and mice in order to examine their plasma concentration-time profiles and antitumor characteristics, respectively. After the administration of EB-glu-MMC, EB-glu-MMC was detected slightly in blood only in the initial stage, while E-glu-MMC and MMC were observed there for a prolonged period. In the administration of E-glu-MMC, a similar phenomenon was observed but the drug retention effect seemed lower than that in EB-glu-MMC. In the antitumor test against P388 leukemia, E-glu-MMC exhibited a better effect than EB-glu-MMC; however, neither conjugate surpassed the effect of MMC. The toxic side effect was improved in each conjugate. As to the growth inhibition against Sarcoma 180, EB-glu-MMC and E-glu-MMC produced good effect and improved the toxic side effect. Especially, in the administration of EB-glu-MMC at the dose of 30 mg eq MMC/kg, a decrease in tumor volume was observed in the latter stage. EB-glu-MMC and E-glu-MMC were demonstrated to produce prolonged retention, to enter the systemic circulation to a fair extent, and to exhibit a good effect against the general solid tumor, Sarcoma 180, in vivo.     

Measurement and analysis of differential work hardening behavior of pure titanium sheet using spline function

Biaxial stress tests and in-plane tension/compression tests of pure titanium sheet (JIS #1) have been carried out in order to elucidate its anisotropic plastic deformation behavior under linear stress paths. Contours of plastic work and the directions of plastic strain rates at different levels of plastic work have been precisely measured in the stress space. The measured work contours bulged significantly toward the equibiaxial direction and showed strong asymmetry, and moreover, changed its shapes significantly with increasing plastic work (differential work hardening). Using the data of the measured work contours, the applicability of selected anisotropic yield functions, i.e., Hill’s quadratic, the Yld2000-2d and Cazacu’s yield functions, to the accurate prediction of the plastic deformation behavior of the pure titanium has been discussed. It was found that these yield functions were not able to reproduce the measured data. A new method for analyzing the differential work hardening behavior of the pure titanium sheet has been developed. This method uses the spline function of Bezier curves which approximates a work contour, inspired by the methodology proposed by Vegter and Boogaard (Int. J. Plasticity 22 (2006) 557-580). The procedure for determining the spline function is described in detail. The calculated results have been in good agreement with the differential work hardening behavior of the pure titanium sheet.     

Comparison of the insulin and insulin-like growth factor 1 mitogenic intracellular signaling pathways

We compared the intracellular insulin-like growth factor-1 (IGF-1) and insulin signaling pathways in Rat1 fibroblasts expressing the equivalent number of insulin receptors and endogenous IGF-1 receptors. Insulin and IGF-1 stimulated tyrosine phosphorylation of IRS-1 and Shc in a similar dose- and time-dependent manner. The time course of Shc phosphorylation by both IGF-1 and insulin was slower than that of IRS-1. Both phosphorylated IRS-1 and Shc associated with Grb2.Sos complexes, leading to p21ras activation. To compare the functional importance of p21ras for IGF-1-and insulin-induced DNA synthesis, single cell microinjection studies were performed. BrdU incorporation into newly synthesized DNA was measured by immunofluorescence microscopy to assess the functional importance of p21ras. Both IGF-1 and insulin stimulated BrdU incorporation, but the effect of IGF-1 was greater. Microinjection of anti-p21ras antibody completely inhibited both IGF-1-and insulin-induced DNA synthesis, indicating the central role of p21ras in signaling by both hormones. Signal transduction from these receptors to Grb2.Sos complexes can occur through IRS-1 and/or Shc. To assess these two possible pathways, we performed Western blots for Grb2 in anti-Shc and anti-IRS-1 immunoprecipitates and found that 5-fold more Grb2 was associated with Shc than with IRS-1 after either IGF-1 or insulin stimulation. Microinjection of anti-Shc antibody inhibited IGF-1 and insulin stimulation of DNA synthesis by 78% and 74%, respectively. By microinjecting Shc subdomains of GST fusion proteins, we found that Shc N-terminus, but not the Shc SH2, was the functionally important domain through which Shc interacts with IGF-1 and insulin receptors. Insulin stimulation caused hyperphosphorylation and decreased electrophoretic mobility of Sos, and a similar effect was seen with IGF-1, although the time course was delayed compared with insulin. Finally, IGF-1 activated mitogen-activated proten kinase activity more effectively than insulin. These data indicate that Shc, rather than IRS-1, appears to be the predominant functional link to Grb2.Sos complexes from the IGF-1 receptor, as it is from the insulin receptor. Although IGF-1 and insulin stimulate cell cycle progression with similar coupling mechanisms from the receptor to Shc, to Grb2.Sos, to p21ras, the delayed IGF-1 induced mobility shift of Sos could lead to, at least in part, more efficient coupling to mitogen-activated protein kinase. These findings might explain the greater mitogenic activity of IGF-1 compared with insulin.     

Removal of endotoxin and cytokines by plasma exchange in patients with acute hepatic failure

OBJECTIVES: To compare the circulating concentrations of endotoxin and cytokines in patients with fulminant hepatitis and patients with the severe form of acute hepatitis, and to assess the effects of plasma exchange on the circulating concentrations of these inflammatory mediators in patients with acute hepatic failure. DESIGN: Prospective, consecutive entry study of patients meeting fulminant hepatitis criteria and the severe form of acute hepatitis criteria. SETTING: University hospital, intensive care unit. PATIENTS: Five patients with fulminant hepatitis, eight patients with the severe form of acute hepatitis, two patients with acute-on-chronic hepatic failure, and one patient with postoperative hepatic failure. INTERVENTIONS: Plasma endotoxin, serum tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6 were determined on admission in five patients with fulminant hepatitis and eight patients with the severe form of acute hepatitis. Circulating concentrations of the inflammatory mediators were measured before and after a single course of plasma exchange in eight patients with acute liver failure, including five patients with fulminant hepatitis, two patients with acute-on-chronic hepatic failure, and one patient with postoperative hepatic failure. MEASUREMENTS AND MAIN RESULTS: TNF-alpha and IL-6 in patients with fulminant hepatitis were significantly higher than in patients with the severe form of acute hepatitis, whereas endotoxin concentrations did not differ between patients with fulminant hepatitis or the severe form of acute hepatitis. IL-1beta was not detectable in patients with either fulminant hepatitis or the severe form of acute hepatitis. Plasma endotoxin concentrations decreased immediately after plasma exchange. Serum concentrations of TNF-alpha and IL-6 were significantly lower after plasma exchange than before plasma exchange. CONCLUSION: TNF-alpha and IL-6 may be important in the pathogenesis of the clinical symptoms that differentiate fulminant hepatitis from the severe form of acute hepatitis, and plasma exchange removes these inflammatory mediators from the circulation of patients with severe liver disease.     

A novel prototype of auxiliary edge resonant bridge leg link snubber‐assisted soft‐switching sine‐wave PWM inverter

This paper proposes a new circuit topology of the three‐phase soft‐switching PWM inverter and PFC converter using IGBT power modules, which has the improved active auxiliary switch and edge resonant bridge leg‐commutation‐link soft‐switching snubber circuit with pulse current regenerative feedback loop as compared with the typical auxiliary resonant pole snubber discussed previously. This three‐phase soft‐switching PWM double converter is more suitable and acceptable for a large‐capacity uninterruptible power supply, PFC converter, utility‐interactive bidirectional converter, and so forth. In this paper, the soft‐switching operation and optimum circuit design of the novel type active auxiliary edge resonant bridge leg commutation link snubber treated here are described for high‐power applications. Both the main active power switches and the auxiliary active power switches achieve soft switching under the principles of ZVS or ZCS in this three‐phase inverter switching. This three‐phase soft‐switching commutation scheme can effectively minimize the switching surge‐related electromagnetic noise and the switching power losses of the power semiconductor devices; IGBTs and modules used here. This three‐phase inverter and rectifier coupled double converter system does not need any sensing circuit and its peripheral logic control circuits to detect the voltage or the current and does not require any unwanted chemical electrolytic capacitor to make the neutral point of the DC power supply voltage source. The performances of this power conditioner are proved on the basis of the experimental and simulation results. Because the power semiconductor switches (IGBT module packages) have a trade‐off relation in the switching fall time and tail current interval characteristics as well as the conductive saturation voltage characteristics, this three‐phase soft‐switching PWM double converter can improve actual efficiency in the output power ranges with a trench gate controlled MOS power semiconductor device which is much improved regarding low saturation voltage. The effectiveness of this is verified from a practical point of view. © 2006 Wiley Periodicals, Inc. Electr Eng Jpn, 155(4): 64–76, 2006; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/eej.20207     

Fatty acid induced insulin resistance in rat-1 fibroblasts overexpressing human insulin receptors: impaired insulin-stimulated mitogen-activated protein kinase activity

Saturated fatty acids cause insulin resistance but the underlying molecular mechanism is still unknown. We examined the effect of saturated nonesterified fatty acids on insulin binding and action in transfected Rat-1 fibroblasts, which over-expressed human insulin receptors. Incubation with 1.0 mmol/l palmitate for 1-4 h did not affect insulin binding, insulin receptor autophosphorylation, insulin-stimulated tyrosine kinase activity toward poly(Glu4:Tyr1), pp185 and Shc phosphorylation and PI3-kinase activity in these cells. However, the dose response curve of insulin-stimulated glucose transport was right-shifted. Palmitate inhibited the maximally insulin-stimulated mitogen activated protein (MAP) kinase activity toward synthetic peptide to 7% that of control. The palmitate treatment influenced neither cytosolic protein kinase A activity nor cAMP levels. These results suggested that 1) palmitate did not inhibit the early steps of insulin action from insulin binding to pp185 or Shc phosphorylation but inhibited insulin-stimulated MAP kinase, and that 2) palmitate decreased insulin sensitivity as manifested by inhibited insulin-stimulated glucose uptake. In conclusion, the mechanism of saturated non-esterified fatty acid induced insulin resistance in glucose uptake may reside at post PI3-kinase or Shc steps, including the level of MAP kinase activation.     

The dominant negative effect of a kinase-defective insulin receptor on insulin-like growth factor-I-stimulated signaling in Rat-1 fibroblasts

To study the interaction between insulin receptor (IR) and insulin-like growth factor-I (IGF-I) receptor (IGF-IR) tyrosine kinases, we examined IGF-I action in Rat-1 cells expressing a naturally occurring tyrosine kinase-deficient mutant IR (Asp 1048 IR). IGF-I normally stimulated receptor autophosphorylation, IRS-I phosphorylation, and glycogen synthesis in cells expressing Asp 1048 IR. However, the Asp 1048 IR inhibited IGF-I-stimulated thymidine uptake by 45% to 52% and amino acid uptake (aminoisobutyric acid [AIB]) by 58% in Asp 1048 IR cells. Furthermore, IGF-I-stimulated tyrosine kinase activity toward synthetic polymers, Shc phosphorylation, and mitogen-activated protein (MAP) kinase activity was inhibited. The inhibition of mitogenesis and AIB uptake was restored with the amelioration of the impaired tyrosine kinase activity and Shc phosphorylation by the introduction of abundant wild-type IGF-IR in Asp 1048 IR cells. These results suggest that the Asp 1048 IR causes a dominant negative effect on IGF-IR in transmitting signals to Shc and MAP kinase activation, which leads to decreased IGF-I-stimulated DNA synthesis, and that the kinase-defective insulin receptor does not affect IGF-I-stimulated IRS-I phosphorylation, which leads to the normal IGF-I-stimulated glycogen synthesis.