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In order to study a long-term effect along with adverse action of epalrestat, an aldose reductase inhibitor, a randomized, prospective study was conducted over the period of 3 years at 112 facilities. Six hundred and three diabetic patients with median motor conduction velocity (MCV)>40 m/s, HbA1c

The effect of the addition of different metals (Cu, Rh, Pd, Ir, and Pt) into supported Ni catalysts on the catalytic performance of methane decomposition into pure hydrogen and carbon was examined. The addition of Pd brought about considerable increases in the catalytic life and in the accumulated yields of hydrogen and carbon at the complete deactivation of the catalyst, while modification with the other metals decreased the yields compared to those for Ni/SiO2. For the Ni catalysts modified with Pd, the appropriate preparation conditions of the catalysts, the effect of catalytic supports, and the optimum reaction temperature on the methane decomposition were examined. The hydrogen yield attained a high value, 16,000 molH2/molPd+Ni, when Pd and Ni were of a mole ratio Pd/(Pd+Ni)=0.5 and a total loading Pd+Ni=37 wt% on carbon nanofiber support. This yield is the highest among those reported so far. Methane decomposition over Ni catalysts modified with Pd produced carbon nanofibers with unique structures, i.e., branched carbon nanofibers. XRD studies on the Ni catalysts modified with Pd indicated the formation of Pd–Ni alloys. The alloys are responsible for the increase in the catalytic life and the formation of carbon nanofibers with unique structures.

In this work we proposed and investigated a new technology using gasoline as a fuel for solid polymer electrolyte fuel cell through the decomposition of gasoline range alkanes into hydrogen and carbon. The method can supply a high purity hydrogen without CO and CO2. The decompositions of various alkanes (, and C8H18) have been examined over Ni/fumed silica (Cab–O–Sil) at a temperature of . Main products were hydrogen and carbon fibers with a low concentration by-product CH4. The number of carbons deposited per one Ni atom after complete deactivation of the catalyst was in the range of 750–1000 depending on the starting alkanes. The selectivity of H2 formation increased as the number of carbon in the molecular structure of alkanes increased. Among C6-alkanes, the H2 selectivity improved as 3-methylpentane (92%)

A number of diabetic patients with diabetic neuropathy were treated with epalrestat, an aldose reductase inhibitor, since this drug was launched into the market in Japan. More than 5000 patients with diabetic neuropathy who were treated with epalrestat for 3-12 months were treated to analyze the efficacy and the adverse reactions of the drug in this study. The improvement rates of subjective symptoms (i.e., spontaneous pain, numbness, coldness, and hypoesthesia) was 75% (slightly improved or better) and those of nerve function tests (i.e., motor nerve conduction velocity, sensory nerve-conduction velocity, and vibration threshold) 36%. Adverse drug reactions were encountered in 129 cases (2.5%) out of 5249 patients, none of which were severe ones. Although data are limited, they strongly suggest that epalrestat is a highly effective and safe agent for the treatment of diabetic neuropathy.

Lysophosphatidylcholine (LPC increased monocyte chemoattractant protein-1 (MCP-1) messenger RNA concentrations in human umbilical vein endothelial cells (HUVECs). A time-course study showed that the increase in MCP-1 mRNA levels peaked at 6 hours after treatment with LPC. The effect of LPC on the accumulation of MCP-I mRNA levels in HUVECs depended on LPC concentration, and the maximal effect was obtained at 50 micromol / L LPC, which induced a sixfold increase in MCP-1 mRNA levels. The amount of MCP-1 released from HUVECs measured using an enzyme-linked immunosorbent assay (ELISA) showed a 38% increase in the presence of 50 micromol/L LPC, but not in the presence of phosphatidylcholine or lysophosphatidylethanolamine. Coincubation with staurosporine, a potent inhibitor of protein kinase C (PKC) activity, attenuated the LPC-induced increase in MCP-1 mRNA levels by 53%. These results indicate that LPC can induce an increase in MCP-1 mRNA concentrations and stimulate the release of MCP-1 protein from HUVECs, and that the effect of LPC on the MCP-1 gene may be mediated through activation of the PKC pathway.

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.

To investigate the significance of fructose-induced protein modification, we examined both fructose- and glucose-induced protein oxidation and the formation of advanced glycation end products (AGE) in vitro. Albumin incubated in the presence of 100 mM fructose at 37 degrees C for 1 week showed 5.1-fold and 3.1-fold increases in the content of carbonyl, which is a marker for oxidized protein, when compared with either control incubated without sugar or with 100 mM glucose. Similarly, the same incubation with fructose increased the fluorescence intensity over 100-fold and 15-fold formation compared with that of no sugar and glucose controls, respectively. Both fructose-induced fluorescence and protein oxidation were almost completely suppressed in the presence of the iron chelator; deferoxamine (100 microM), the hydroxyl radical scavenger; MK-447 (1 mM), or aminoguanidine (200 mM), which is an inhibitor of AGE formation. In contrast, the fructose-induced formation of fluorescent albumin was potentiated in the presence of 100 microM FeCl2. This was completely inhibited in the presence of 60 microM or more deferoxamine. These results suggest that fructose promotes both AGE formation and protein oxidation possibly through the formation of hydroxyl radicals.

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.

To elucidate the role of hyperinsulinemia in the development of atherosclerosis, we evaluated insulin-specific signaling in cultured vascular smooth muscle cells (SMCs) and its desensitization by continuous exposure to insulin. The concentration of unlabeled insulin that inhibited specific [A14-125I]-insulin binding by 50% (IC50) was 0.33 +/- 0.02 nM, which was 100 times less than the IC50 of unlabeled IGF-I. For [125I]-IGF-I binding, the IC50 of unlabeled IGF-I was found to be 6.6 +/- 0.88 nM, which was 100 times less than the IC50 of unlabeled insulin. The binding capacities for insulin and IGF-I were found to be 1.28 +/- 0.86 and 1200 +/- 170 fmol/0.5 mg protein, respectively. Autophosphorylation of the beta-subunit of the insulin receptor was stimulated at above 0.17 nM (24 microU/ml) insulin. Insulin concentrations exceeding 1 nM significantly activated the S6 kinase in a dose-dependent manner. In contrast, 10 nM insulin did not activate MAP kinase nor [3H]thymidine incorporation into DNA, while both were activated by 38% and 44% with 1 microM insulin and by 52% and 67% with 10 nM IGF-I, respectively. By pre-exposing cells to 10 nM insulin for 12 h, the binding capacity for insulin decreased by 34% (P0.05), and activation of S6 kinase by insulin almost disappeared, while both IGF-I binding and the activation of S6 kinase by IGF-I were not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

The clinical efficacy of epalrestat (150 mg/day, 50 mg tid, po; A group), an aldose reductase inhibitor, was evaluated in 196 patients with diabetic neuropathy by a double-blind study using placebo (9 mg/day, 3 mg tid, po; P group) as a control for 12 weeks. The disappearance rates of upper limb spontaneous pain were 42.9% and 12.0% in the A and P groups, respectively, and those of lower limb spontaneous pain 48.6% and 22.6%, thus being significantly higher in the A group (p0.05, logrank-test). The motor nerve conduction velocity of the peroneal nerve significantly increased only in the A group (delta 1.6 +/- 0.6 m/sec, p0.01, paired t-test), and the extent of increase in that of the median nerve was significantly greater in the A group than in the P group (p0.05). Thresholds of vibratory sensation and autonomic nerve function were also significantly improved in the A group (p0.05). The data were reanalyzed by dividing patients into two groups according to their HbA1c values. The improvement ratings of subjective symptoms and of nerve function tests for cases with HbA1cor = 7.5% were both significantly different between the A and P groups, with the improvement rate being higher in the A group, and also higher as compared to the analysis for cases with HbA1c7.5%.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the dose-dependent effects of prostaglandin E1 (PGE1) analogue, OP1206.alpha CD (OP), on motor nerve conduction velocity (MNCV), nerve blood flow (NBF) and Na(+)-K(+)-ATPase (ATPase) activity in streptozocin-induced diabetic rats. At 10 micrograms/kg/day, OP ameliorated MNCV and NBF, but no ATPase activity, whereas at 30 micrograms/kg/day it increased MNCV and ATPase activity, but not NBF. These results suggested a possible direct metabolic effect of OP, at least at a certain dose, on ATPase activity independent of NBF. Since PGE1 exerts an effect on nerve cAMP content, we conducted an in vitro study to clarify the relationship of cAMP to the modulation of ATPase activity in diabetic nerves. We studied sciatic nerves isolated from 53 rats with streptozocin-induced diabetes that had exhibited hyperglycemia for 6 wk. OP increased the activity of ATPase and the accumulation of cAMP in a dose-dependent manner. Dibutyryl cAMP, a cAMP analogue, and aminophyline, which increases nerve cAMP content, enhanced ATPase activity in a dose-dependent manner. In addition, the increased activity of ATPase in diabetic nerves produced by OP was suppressed by a protein kinase inhibitor, H8. These results suggest that ATPase activity in diabetic nerves might be regulated or modified by cAMP and, possibly, by protein kinase A, a finding that is important for clarifying the pathogenesis of diabetic neuropathy and for developing new approaches to treatment.

To clarify the role of protein tyrosine phosphatase (PTPase) containing a pair of Src homology 2 (SH2) regions upon insulin signaling, we studied the interactions between the insulin receptor and SH-PTP2 coupled to glutathione-S-transferase. A full length SH-PTP2 was phosphorylated by insulin receptor kinase and associated with the insulin receptor in vitro. The N-terminal SH2 domain was more phosphorylated than the other SH2 domain of SH-PTP2. However, both SH2 domains of SH-PTP2 were necessary for association with insulin receptors. Phosphorylation of the SH2 domains of SH-PTP2 resulted in decreased PTPase activities toward the phosphorylated insulin receptor. These results indicate that the insulin receptor can negatively regulate SH-PTP2 activity by means of phosphorylating the SH2 domains.

A new oral agent, pioglitazone, increases insulin sensitivity by activating receptor kinase in insulin-resistant rats. To clarify the mechanism, we studied in vitro effects of glucose and pioglitazone on the insulin receptor function using Rat 1 fibroblasts which expressed human insulin receptors. Insulin receptor kinase activity was impaired by incubating cells for 4 days in the presence of 27mM D-glucose. The glucose effect was time- and dose-dependent and also specific for D-glucose, since D-raffinose incubation had no effect. Pioglitazone treatment did not have any effect on intact receptor kinase. However, exposure of both 27mM D-glucose and 0.1 μ M pioglitazone to the cells completely prevented the glucose-induced impairment of insulin receptor kinase activity, suggesting that pioglitazone might reverse the processes which are critical for the glucose-induced desensitization of insulin receptor kinase.

Alteration of mesangial response to ANP and angiotensin II by glucose. To test the hypothesis that the function of glomerular mesangial cells is impaired in diabetes, we examined the responsiveness of mesangial cells cultured under high concentrations of glucose to atrial natriuretic peptide (ANP 1 ) and angiotensin II (Ang II). The ANP-induced accumulation of cGMP was enhanced in mesangial cells cultured under high glucose conditions, possibly due to the activation of particulate guanylate cyclase. Ang II action in mesangial cells was evaluated by measuring the ability of Ang II to inhibit ANP-induced cGMP accumulation through both activating phosphodiesterase (initial phase) and inhibiting guanylate cyclase (maintenance phase). The inhibition of both ANP-induced cellular cGMP accumulation and particulate guanylate cyclase activity by Ang II was significantly reduced in mesangial cells cultured under high concentrations of glucose. Moreover, in the cells exposed to high concentrations of glucose, both basal and Ang II-stimulated levels of inositol 1,4,5-trisphosphate (IP 3 ) were significantly reduced. These results indicate that, in high glucose conditions, the actions of ANP and Ang II are modulated differently, resulting in the impairment of contractile responsiveness of mesangial cells.

Impairments of the glutathione redox cycle in cultured endothelial cells under acidic pH conditions were measured. Glutathione-dependent H2O2-degrading activities decreased by 20% (P < .01) at pH 6 and by 51% (P < .01) at pH 4 compared with activities at pH 7.4 1 hour after a change with fresh medium. Intracellular reduced glutathione (GSH) content increased by 85% (P < .01) following the change with pH 7.4 medium. Such increases in GSH content were impaired after exposure to acidic medium. After exposure to 500 mumol/LH2O2, intracellular GSH content decreased by 61% compared with the level obtained in the absence of H2O2 at pH 7.4 (P < .01). Compared with the level at pH 7.4, the H2O2-induced decrease in intracellular GSH content was 32% lower (P < .01) at pH 6 and did not change at all at pH 4. After exposure to 500 mumol/L H2O2, the intracellular oxidized glutathione (GSSG) content increased by 160% at pH 7.4 (P < .01), 370% at pH 6 (P < .01), and 90% at pH 4 compared with treatment without H2O2, respectively. After exposure to 500 mumol/L H2O2, the release of GSSG from cells at pH 6 decreased by 38% compared with the value found at pH 7.4 (P < .05), and the release at pH 4 completely disappeared. Both glutathione peroxidase (GPO) and glutathione reductase activities decreased as a function of a decrease in pH from 7.4 to 4.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanism of anti-proliferative action of atrial natriuretic peptide (ANP) was examined in cultured mesangial cells. HS-142-1, a selective antagonist for ANP biological receptors, inhibited ANP-induced cellular cGMP accumulation and prevented ANP-induced inhibition of both serum-stimulated incorporation of 3H-thymidine and cell proliferation. Des-[Gln18, Ser19, Gly20, Leu21, Gly22]-ANP4-23-NH2 (C-ANP) failed to inhibit 3H-thymidine incorporation. These results indicate that anti-proliferative action of ANP is mediated by the biological receptors in cultured mesangial cells.

Dorsal column function and peripheral motor and sensory conduction velocities (MCV, SCV) were evaluated in experimental diabetic rats and compared with those in 2,5-hexanedione (2,5-HD) intoxicated rats. Hyperglycemia was induced by a single injection of streptozocin, and electrophysiological studies were performed 4 and 12 weeks after the injection. For 8 weeks 2,5-HD was administered daily by drinking water to make the 2,5-HD neuropathy rats. Age-matched rats were used as control. In diabetic rats, gracile surface potentials evoked by electrical stimulation of the lumbosacral trunk remained normal during the experimental period, whereas the N and P waves of the evoked potentials were suppressed and the duration of the N wave was prolonged in the 2,5-HD rats. In 4-week diabetic rats, the antidromic compound action potentials of the gracile tract recorded at the most proximal site of lumbosacral trunk were normal. In 12-week diabetic rats, the gracile tract conduction velocity (GTCV) was decreased, although the duration of these potentials was normal. By contrast, the GTCV was decreased and the duration was markedly prolonged in 2,5-HD rats. These findings might indicate that temporal dispersion of incoming volleys in the gracile tract is increased in 2,5-HD rats, but not in diabetic rats. These results suggest that diabetic myelopathy exists that but the magnitude and progression of this condition are quite different from those of 2,5-HD intoxication, typical dying-back-type neuropathy and that the dorsal column is less vulnerable than the peripheral nerve in diabetes mellitus.

We evaluated the effects of chronic verapamil treatment on the cardiac alpha 1-adrenoceptor signalling system in streptozocin-induced diabetic rats. The decrease in maximum cell surface [3H]bunazosin binding (Bmax) in isolated cardiac myocytes from the diabetic group (-46%, P0.01) was completely reversed by a 4-week course of verapamil, while Bmax in the verapamil-treated control group was unchanged. Similarly, the reduction in ventricular inositol 1,4,5-trisphosphate (IP3) production after stimulation with 10 microM noradrenaline (NA) seen in diabetes (-30%, P0.01) was completely normalized by verapamil, while the response in the verapamil-treated control group was unaffected. These results indicate that verapamil can induce complete recovery of the impaired cardiac alpha 1-adrenoceptor signalling system in the diabetic heart without affecting glucose metabolism.

The effect of glucose concentration and insulin on glucose incorporation was studied in primary cultures of rat hepatocytes. The rate of glucose incorporation into hepatocytes was proportional to the medium glucose concentration from 100 to 800 mg/dl. At 800 mg/dl glucose the rate reached a plateau. Of the glucose taken up by hepatocytes, 16 and 18% was incorporated into glycogen and lipid, respectively, and 58% into the nucleotide fraction after incubation for 4 h. In the medium, lactate was the major product found. Insulin stimulates glucose incorporation by 20–112% into all the above pathways at glucose concentrations between 100 and 800 mg/dl. The insulin effect was noted as early as 2–4 h (early effect) and up to 24 h (delayed effect). This effect of insulin was observed to be dose dependent from 5 to 200 ng/ml insulin. While the delayed insulin effect was abolished by cycloheximide, the early effect of insulin was not affected. With respect to the key enzyme activities of glucose utilization, activation of glycogen synthase (increase of I-activity/total activity) and pyruvate kinase (activation at 0.2 mM phosphoenolpyruvate) was noted 4 h after insulin addition, and these effects were not abolished by cycloheximide. These two enzymes increased in total activity after 24 h. Both glucokinase and glucose-6-phosphate dehydrogenase activities increased by 30–35% and 65–93% at 4 and 24 h, respectively. The results indicate that hepatocytes directly utilize glucose in a dose-dependent manner with respect to glucose and insulin. A major early and delayed effect of insulin appeared due to the activation and induction of the key hepatic enzymes of glucose utilization, respectively.

Dual mechanism of angiotensin II inhibits ANP-induced mesangial cGMP accumulation. To evaluate an interaction between vasoconstrictive (Ang II) 1 and vasodilating (Ang) peptides, we examined the effect of Ang II on ANP-induced accumulation of cGMP in cultured glomerular mesangial cells. ANP rapidly increased intracellular cGMP levels, with a peak stimulation at one minute in the absence of IBMX and at ten minutes in the presence of IBMX. The ANP-induced cGMP accumulation was significantly inhibited when the cells were treated with Ang II simultaneously with ANP for one minute in the absence of IBMX. This inhibitory effect of Ang II was completely abolished by IBMX and significantly reduced in calcium-free media or by W7, but not affected by H7. Similar inhibitory effect was observed when cells were treated with A23187 but not with TPA for one minute. In the presence of IBMX, Ang II inhibited ANP-induced cGMP accumulation when cells were treated with Ang II for 15 minutes prior to the stimulation by ANP. This inhibition by Ang II was blocked by H7. ANP-induced increase in particulate guanylate cyclase activity was significantly reduced in the cells treated with Ang II or TPA. This reduction of enzyme activity was also prevented by H7. These results indicate that Ang II inhibits ANP-induced cGMP accumulation in cultured glomerular mesangial cells through at least two mechanisms; one is the activation of calcium-dependent, calmodulin-stimulated cyclic nucleotide phosphodiesterase in the initial phase, and the other is the inhibition of guanylate cyclase resulting from protein kinase C activation in the maintenance phase.

CS-045, (+/-)-5-[4-(6-hydroxy-2,5,7,8-tetramkethylchroman-2- ylmethoxy)benzyl]-2,4-thiazolidinedione, lowers plasma glucose in several animal models of non-insulin dependent diabetes mellitus (NIDDM) presumably by increasing insulin sensitivity. Little adverse effect was found in a phase 1 study on healthy male subjects. In order to test its efficacy in lowering plasma glucose in NIDDM in man, a pilot multi-center clinical trial of CS-045 was carried out in 146 patients with NIDDM whose glycemic control was inadequate (FPG greater than 140 mg/dl) on diet and/or other oral hypoglycemic agents. CS-045 was given orally in a daily dose of 200 mg or 400 mg for 12 weeks in addition to the previous treatment. The mean fasting plasma glucose (FPG) and fructosamine began to decrease within 2 weeks and the mean HbA1c within 8 weeks. After 12 weeks, the FPG fell from 192 +/- 41 to 155 +/- 45 mg/dl (P less than 0.01), fructosamine from 3.7 +/- 0.6 to 3.3 +/- 0.6 (P less than 0.01), and HbA1c from 8.9 +/- 1.5 to 8.1 +/- 1.5% (P less than 0.01). The drug was effective in 39% of patients in that FPG fell by more than 20% of the initial value. This rate of efficacy was the same when CS-045 was given alone or together with other oral hypoglycemic agents. The drug was more effective in a dosage of 400 mg than with 200 mg (the rate of efficacy 46% vs 25%) and more effective in obese patients than in lean patients (46% vs 25%).(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein (apo) B subspecies were separated by high-performance liquid chromatography (HPLC) to analyze Apo B-48 contents in very-low-density lipoproteins (VLDL) more easily. Apo B-100 and B-48 were eluted through two connected column of Shim-pack Diol-300 at a retention time of 24 min and 32.3 min, respectively. The molecular masses estimated by this method were approximately 600 kDa in apo B-100, 220 kDa in apo B-48. % apo B-48 in total apo B (% B-48) determined by measuring peak area of HPLC-separated apo B in a healthy subject was 76% in chylomicrons, 13% in fasting VLDL, and 20% in 2 h-postprandial VLDL. No peak of apo B-48 was detected in LDL. Recovery of Apo B determined by re-chromatography of HPLC-separated sample was 91 +/- 4.0% and 95 +/- 3.6% in Apo B-100 and apo B-48, respectively. 125I-labeled apo B in VLDL were also analyzed by both HPLC and SDS-PAGE. Percent radioactivity of apo B-48 fraction in the total apo B determined by the HPLC (17.3%) was similar to the value obtained through the measurement of radioactivity separated by the SDS-PAGE (17.6%). Coefficient of variation in % B-48 determined by the peak area was 2.5%. Percent B-48 determined by the HPLC method was significantly correlated with % B-48 by SDS-PAGE, but 6-8 times higher, which might be accounted for in part by the reported difference of chromogenicity between apo B-100 and apo B-48. % B-48 in VLDL separated from fasting plasma were 17.1 +/- 5.6% in 14 healthy subjects, and positively related to VLDL triglyceride and cholesterol concentrations. Apo B isoprotein analysis by the HPLC method is reliable and easy to perform for studying apo B metabolism in triglyceride-rich lipoproteins in physiological as well as pathological conditions.

Although activation of polyol pathway has been proposed as one of the etiologic factors of diabetic complications, precise mechanism of the effect of polyol accumulation is still unclear. In order to test the hypothesis that there is an association of polyol pathway with myo-inositol metabolism, we measured myo-inositol content in cultured rat glomerular mesangial cells. By exposing the cells to high concentrations of glucose, intracellular myo-inositol content was reduced from 12.39 +/- 0.64 nmol/mg protein at 0 mmol/L glucose to 6.54 +/- 0.38 nmol/mg protein at 27.5 mmol/L glucose and 4.88 +/- 0.43 nmol/mg protein at 55 mmol/L glucose. This decrease of myo-inositol content was partially prevented by co-incubation with aldose reductase inhibitor, sorbinil. To examine further the mechanism of myo-inositol depletion, myo-inositol uptake by mesangial cells was studied. Major myo-inositol uptake process was sodium-dependent, saturable, and ouabain sensitive with Vmax of 171 pmol/mg protein/20 min and Km of 33 mumol/L. Sodium-dependent myo-inositol uptake was significantly inhibited by glucose in a dose-dependent manner only when glucose was present during uptake experiment, and kinetic analysis revealed the inhibition was competitive. Aldose reductase inhibition failed to prevent inhibitory effect of glucose on myo-inositol uptake. These data suggest that myo-inositol content of glomerular mesangial cells, which is reduced by high concentrations of glucose, is maintained by two processes: a glucose-sensitive but sorbitol-insensitive process, sodium-dependent myo-inositol uptake; and a sorbitol (aldose reductase) sensitive process, myo-Inositol depletion under high glucose condition may induce dysfunction of mesangial cells seen in diabetes.

In order to clarify the mechanism of mesangial cell dysfunction in diabetes, we examined the activities of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK), important kinases in various cellular functions, and also evaluated the isoenzymes of PKC in mesangial cells cultured under high glucose conditions. Exposure of cells to high concentrations (27.8 mM) of glucose for 5 days resulted in a significant elevation of PKC activities in the membrane fraction. MAPK was also activated in cells cultured under high glucose conditions. Of the PKC isoenzymes, the levels of PKC alpha and zeta were significantly increased in the membrane fraction after 5 days of exposure to high concentrations of glucose. These results indicate that the translocation of PKC alpha and zeta and the activation of MAPK under high glucose conditions might be underlying mechanisms of the functional disturbance of mesangial cells in diabetes.

Previous studies by other investigators have shown that sodium depletion causes down-regulation of angiotensin II receptors in renal glomeruli, which is ascribed to elevation of circulating angiotensin II levels. The present study was designed to determine whether elevation of circulating angiotensin II levels alone can down-regulate its own receptors without changes in the sodium balance. Rats were infused with angiotensin II at 50 ng/min intraperitoneally for 24 h or 7 d, then glomeruli were isolated by a mechanical sieving technique and used in a radioreceptor assay for angiotensin II. Angiotensin II infusion for 24 h and for 7 d both significantly suppressed plasma renin activity, and elevated plasma angiotensin II level (3-fold) but did not affect the plasma sodium and potassium or differences between dietary intake and urinary excretion of these electrolytes. By Scatchard analyses significant down-regulation of angiotensin II receptor number was demonstrated in renal glomeruli derived from rats infused with angiotensin II for either 24 h (32% decrease from the control value) or 7 d (37% decrease). No significant changes in receptor affinity were observed after 24 h of angiotensin II infusion, although the infusion for 7 d slightly but significantly decreased K d (to 2.57 ± 0.08 nM from 3.17 ± 0.19 , P ). From these results, we conclude that circulating angiotensin II level itself can regulate the number of its own receptors in renal glomeruli even in the absence of changes in the sodium balance.

Receptor-binding kinetics and degradation of tyrosine A-14 and A-19 125I-labelled insulin was studied using cultured human lymphocytes. Receptor-binding ability of A-14 insulin was 1.5-times as high as that of A-19 insulin. Dissociation from receptors on lymphocytes showed no difference between these two labelled insulins. In association studies percent bound of A-14 insulin was 1.5-times as high as that of A-19 insulin at any time after incubation. These results suggested that lower binding affinity of A-19 insulin was due to decreased association rate, but not due to increased dissociation rate. Degradation of A-14 insulin by incubation media of lymphocytes was also 1.5-times as high as that of A-19 insulin.

Shen et al.1 and Reaven et al.2 have recently reported increased insulin resistance in adult-onset type diabetes based on the steady state plasma glucose (SSPG) method. In this method, epinephrine and propranolol were used to suppress endogenous insulin secretion. Epinephrine and propranolol themselves elevate SSPG levels.3 Also, plasma levels of growth hormone (HGH) and glucagon may not be suppressed under these conditions, since paradoxical rises or less suppressibility of HGH4 and glucagon5 following glucose loads have been reported in diabetic subjects. We have therefore used somatostatin, which has been shown to have no direct effect on glucose and lipid metabolism,6 to suppress endogenous secretion of insulin, glucagon, and HGH. Using the newly revised method, we have assessed insulin sensitivity in diabetics treated with diet alone, sulfonylureas, or insulin, and in obese nondiabetic subjects with hyperinsulinism.

Summary The metabolism of a mutant insulin, [Leu B25 ]-insulin, was studied in vitro and in vivo. Porcine or mutant insulin (4 μg/kg body weight) was injected i.v. into streptozotocin-induced diabetic rats and their plasma glucose and insulin levels were determined. The half-lives of porcine and mutant insulin were 3 min and 18 min, respectively. The ability of the mutant insulin to lower the blood glucose levels was 38% of that of normal when the glucose levels at the nadir were compared. Receptor-mediated degradation of the mutant insulin assessed by chromatography of the degraded materials in the media after incubation with cells was less compared with that of porcine insulin (4% vs. 24%). The media containing the insulin degrading enzyme (IDE) of IM-9 cells and rat livers degraded porcine and mutant insulin to the same extent. These results suggest that the decreased clearance of insulin is due to the decreased receptor binding and the decreased receptor-mediated degradation, but is not due to the decreased degradation by IDE.

The contractile function of renal glomerulus was studied in vitro using isolated glomeruli from streptozotocin-diabetic rats. Glomerular contraction was assessed by the reduction of extracellular [3H]inulin space of glomerulus, mostly composing of intracapillary space, produced by angiotensin II. The inulin space was dose-dependently reduced after angiotensin II addition in both diabetic and control rats but the degree of reduction significantly smaller in the former. The radioreceptor assay revealed rather increased angiotensin II receptors in diabetic glomeruli. Since the contractile response of glomerulus to angiotensin II is mediated via mesangial cell contraction, these results suggest the presence of mesangial cell dysfunction in diabetes.

Insulin receptors and IGF-I receptors in cultured fibroblasts were investigated in a patient with extreme insulin resistance due to unprocessed insulin receptors. Insulin binding to cultured fibroblast monolayers and partially purified insulin receptors was extremely decreased to 27% and 18% of control value, respectively. Affinity cross-linking study revealed that molecular weight of the insulin receptor was 210 kDa and that it could not be dissociated to alpha- and beta-subunit with dithiothreitol treatment. Because IGF-I binding to the fibroblasts from the patient was normal and alpha-subunit of IGF-I receptor was 135 KDa, the defect was specific to the insulin receptor. Autophosphorylation of the 210 kDa unprocessed insulin proreceptor was stimulated by insulin in a dose-dependent manner. In the fibroblasts from the patient, insulin-stimulated alpha-aminoisobutyric acid uptake was fivefold shifted to the right in the dose-response curve (ED50 20 ng/mL for the patient v 3.5 ng/mL for the control subjects), but the maximally stimulated uptake was normal. With 0.025% trypsin treatment, insulin binding and alpha-aminoisobutyric acid uptake were normalized. These results suggested that (1) abnormal processing of insulin proreceptor also occurred in the cultured fibroblasts, (2) the postreceptor steps of insulin action were totally intact, and (3) IGF-I receptors were normally processed in this patient.

To elucidate the role of calmodulin in insulin action, we examined the effect of the calmodulin antagonists, W-7 and W-5, on glucose transport in isolated rat adipocytes. W-7 inhibited insulin-stimulated 2-deoxyglucose uptake by 18% at 100 microM, but it did not affect basal uptake levels. W-5, a less potent analogue of W-7, however, had no significant effect at the same concentration, indicating that the effect was specific to calmodulin. Similar results were observed in a 3-O-methylglucose uptake study. Kinetic analysis of 2-deoxyglucose uptake revealed that W-7 affected the insulin-induced increase in Vmax but not Km. These results suggest that calmodulin modifies insulin action in the glucose transport system.

In order to evaluate the value of diabetic Japanese monkeys (Macaca fuscatus) as an animal model for studying the pathogenesis of diabetic neuropathy, morphological examinations were performed on myelinated nerve fibers and endoneurial microvessels at three levels of the lower limb nerve in eight streptozocin (STZ)-diabetic monkeys with the duration of diabetes up to 36 months and in four roughly age-matched control monkeys using a computer-assisted image analyzer. Nerve fiber loss was not found, although a tendency for nerve fiber atrophy was found in diabetic monkeys. Endoneurial microvessels did not show either endothelial or pericyte proliferation or basement membrane thickening. The results suggest that chronically STZ-diabetic Japanese monkeys with the duration of diabetes up to 36 months might be useful for studying diabetic axonopathy, but do not closely mimic the nerve pathology found in human diabetic neuropathy.

We studied the effect of the duration of diabetic state on insulin action in skeletal muscle by measuring insulin binding, 2-deoxyglucose uptake, and intracellular glucose metabolism in isolated soleus muscles from streptozotocin-induced diabetic rats. Insulin binding to soleus muscles from diabetic rats was increased over that from controls. Glucose transport activity was determined by measuring the 2-deoxyglucose uptake at the concentration of 1 mmol/L at 25 degrees C. In the rats with diabetes of one week duration, insulin-stimulated 2-deoxyglucose uptake was not impaired, whereas basal 2-deoxyglucose uptake was decreased. However, the diabetic rats with two weeks duration revealed a 35.6% decrease in the insulin-stimulated 2-deoxyglucose uptake. Furthermore, four week duration of diabetic state led to a 60% decrease both in basal and insulin-stimulated 2-deoxyglucose uptake. Total glucose utilization was estimated as the total amount of glucose incorporated into muscle and lactate released into the medium following incubation at 37 degrees C, with 5 mmol/L glucose. The diabetic rats with one week duration did not demonstrate any changes in total glucose utilization both in basal and insulin-stimulated state. However more than two weeks duration of diabetes led to a 30% to 35% decrease both in basal and insulin-stimulated total glucose utilization, similar to the findings in the 2-deoxyglucose uptake study. We concluded that prolonged insulinopenia led to decreased glucose transport and intracellular glucose metabolism and resulted in insulin resistance in skeletal muscles.

A highly sensitive and simplified method for the differential determination of serum ketone bodies has been developed. Serum was deproteinized with perchloric acid, and acetoacetate contained in the supernate was reacted with newly synthesized p-nitrobenzene diazonium fluoroborate at 37 degrees C for 10 min. The formed hydrazo compound was converted by alkali to the more stable azo compound which has a peak absorbance at 645 nm. For the determination of 3-hydroxybutyrate, this was enzymatically converted to acetoacetate using 3-hydroxybutyrate dehydrogenase, LDH, NAD and pyruvate. Using 0.2 ml serum, acetoacetate and 3-hydroxybutyrate could be quantitated in 30 min. The described method is five times more sensitive than the enzymatic photometric method and can detect individual ketone bodies at concentrations as low as 20 mumol/l. Differential determination of serum levels of ketone bodies is clinically useful for the diagnosis of type 1 diabetes and in monitoring diabetic control.

[SerB24]-insulin, the second structurally abnormal mutant insulin, and [SerB25]-insulin were semisynthesized and were studied for receptor binding and biological activity. Receptor binding and biological activity determined by its ability to increase 2-deoxy-glucose uptake in rat adipocytes were 0.7-3% of native insulin for [SerB24]-insulin and 3-8% for [SerB25]-insulin. Negative cooperative effect of these analogues was also markedly decreased. Immunoreactivity of [SerB24]-insulin was decreased whereas that of [SerB25]-insulin was normal. Markedly decreased receptor binding of [SerB24]-insulin appeared to be due to substitution of hydrophobic amino acid, Phe, with a polar amino acid, Ser, at B24.

We studied the effect of chronic exposure to insulin on insulin receptors of cultured lymphocytes (RPMI-1788 line). The cells treated with insulin (2 micrograms/ml) at 37 degrees C for 12 h, showed a 36.5% decrease in the number of binding sites. Solubilized extract from the cells treated with insulin showed a 35.9% decrease of binding capacity, suggesting that insulin exposure induced the loss of total (cell surface and intracellular) receptors. Insulin-induced loss of receptors was blocked by chloroquine, suggesting that receptor loss is mediated by a chloroquine sensitive pathway. Bacitracin, which inhibited the insulin degradation, had no effect on insulin-induced receptor loss in this cell line. We also found that vitamin K5, one of the insulin mimickers, induced a 31.5% loss of insulin receptors. Therefore, the post-receptor process appeared to mediate down regulation. In cultured lymphocytes, insulin exposure caused a significant loss of total receptors, suggesting that insulin-induced receptor loss may be due to receptor degradation. Insulin-induced receptor loss is mediated by a chloroquine sensitive pathway, and is related to the post-binding process stimulated by vitamin K5.

We studied insulin binding to cultured differentiating muscle cell line L6. Insulin binding to the cells reached a plateau after incubation with 125I-insulin for 4 h at 22 degrees C, and was at an optimum at pH 7.8. Preincubation with 10 microM of hydrocortisone for 36 h at 37 degrees C resulted in significantly increased insulin binding (1.73 +/- 0.12 ng/mg protein for treated cells vs. 1.13 +/- 0.025 ng/mg protein for control cells, mean +/- SD, P less than 0.001). Preincubation with 1 microM of hydrocortisone or 1 microM of dexamethasone also led to increased binding. The number of insulin-binding sites per cell increased 2.5-fold in glucocorticoid-treated cells (9.7 X 10(3) sites/cell for treated vs. 3.8 X 10(3) sites/cell for control cells). Preincubation with trifluoperazine (5 microM), a calmodulin inhibitor, did not affect insulin binding to the cells. These results indicate that glucocorticoid might have some important role in regulating the number of insulin receptors in L6 muscle cells.

[D-Phe B24 ]- and [D-Phe B25 ]-human insulin were semisynthesized from porcine insulin by enzyme assisted coupling method. Receptor binding ability of [D-Phe B24 ]- and [D-Phe B25 ]-insulin was 180% and 4%, respectively, of that of human insulin. Increased affinity of [D-Phe B24 ]-insulin was ascribed to markedly decreased dissociation rate in binding to human cultured lymphocytes. Negative cooperative effect of [D-Phe B24 ]insulin was also increased to twice of that of human insulin. Biological activity of these analogues was assessed by 2-deoxy-glucose uptake studies in isolated adipocytes and the ability of [D-Phe B24 ]- and [D-Phe B25 ]-insulin was 140% and 4%, respectively, of that of human insulin. These findings suggest that B25 L-Phe is more crucial for receptor binding and that [D-Phe B24 ]-insulin is the first semisynthetic insulin to show increased affinity for insulin receptors.

The long-term in vitro effects of insulin on insulin binding and glucose transport were studied using rat adipocytes in a time-dependent manner. Isolated fat cells were incubated with insulin (100 ng/ml) for 4, 8 and 24 h in a TCM 199 medium, at 37 degrees C, and then insulin binding (37 degrees C, 60 min) and 3-O-methylglucose transport (37 degrees C, 2 s) were determined. Decreased insulin binding was demonstrated in the cells incubated with insulin for 8 h, and Scatchard analysis revealed that receptor number was decreased to 61.7% of that of control cells. Thus, insulin-induced down regulation of receptors was evident after 8 h incubation with insulin. On the other hand, 8 h incubation with insulin resulted in markedly increased basal (i.e., in the absence of insulin) glucose transport up to 246% of control values. In the cells incubated with insulin for 24 h, maximally insulin-stimulated glucose transport was significantly increased up to 248% of control value. Thus, these results suggested that insulin-induced down regulation of receptors appeared to be coupled with increased cell-surface glucose transporters, and that there was a time-lag between down regulation of insulin receptors and increase of available glucose transporters in the plasma membrane.

Two sisters presented with severe insulin resistance and markedly decreased insulin binding to erythrocytes, cultured fibroblasts and transformed lymphocytes. The dose-response curve of insulin-stimulated amino acid uptake in the fibroblasts was shifted to the right. The molecular weight of the insulin receptor on the transformed lymphocytes from the patients was 210,000 and could not be dissociated to α- and β-subunits by dithiothreitol treatment. However, the proreceptor was cleaved by trypsin and this led to the production of α-subunit with normal insulin binding. We performed cDNA sequence analysis of the cleavage site of the insulin proreceptor from the patients. The polymerase chain reaction was used to obtain a large amount of cDNA coding for the region including the interconnecting site. A thermostable DNA polymerase, Taq polymerase, successfully produced enough cDNA for the region to be sequenced. The results showed an AG G (Arg) to AG T (Ser) point mutation, resulting in the change of the interconnecting sequence of the two subunits from -Arg-Lys-Arg- Arg - to -Arg-Lys-Arg- Ser -. These results suggest that the tertiary structure change of the cleavage site leads to production of unprocessed insulin proreceptors.

Conversion of C 14 into fatty acids of liver and carcass from acetate-2-C 14 , and into blood glucose and liver glycogen from DL-alanine-1-C 14 were studied during and following a dynamic phase of obesity caused in rats by feeding of high caloric diet. (1) There were found to be no significant differences among normal rats and rats in dynamic or static (postdynamic) phases in cumulative expired C 14 O 2 after injection of acetate-2-C 14 . (2) Conversion of C 14 from acetate-2-C 14 into fatty acids of the liver and carcass was increased in the dynamic phase and decreased in the static phase. (3) Incorporation of C 14 from DL-alanine-I-C 14 into blood glucose and liver glycogen was found to be increased in the dynamic and static phases of high-caloric fed rats.