Defective processing of insulin-receptor precursor in cultured lymphocytes from a patient with extreme insulin resistance.

We have studied a patient with extreme insulin resistance, acanthosis nigricans, and decreased erythrocyte insulin binding. EBV-transformed lymphocytes from this patient exhibited markedly reduced binding of 125I-insulin. Radioiodination of cell surface receptors followed by immunoprecipitation with anti-receptor antibodies revealed the presence of increased amounts of a 210-kD protein but no detectable alpha or beta subunits. Continuous labeling with 2-[3H]mannose revealed the synthesis of a 190-kD precursor and a 210-kD protein. The 210-kD protein was phosphorylated in an insulin-dependent manner at high insulin concentrations. These results suggest that in this patient the biosynthesis of 190-kD receptor precursor, its terminal glycosylation, and intracellular transport to the cell surface proceed normally, while proteolytic maturation to alpha and beta subunits does not occur. We postulate that this defect either results from mutation(s) within the insulin-receptor gene, which render the precursor resistant to cleavage, or from a defect in the receptor processing enzyme.

Sustained contraction and loss of NO production in TGFbeta1-treated endothelial cells.

BACKGROUND AND PURPOSE: Transforming growth factor beta1 (TGFbeta1) is generated in atherosclerotic and injured vessel walls. We examined whether the endothelial-to-mesenchymal transdifferentiation induced by TGFbeta1 affects endothelial functions. EXPERIMENTAL APPROACH: Bovine aortic endothelial cells (BAECs) were treated with 3 ng ml(-1) TGFbeta1 for 7 days. Contraction of TGFbeta1-treated BAECs was assessed by collagen gel contraction assay. Protein expression and phosphorylation were assessed by Western blotting. Intracellular Ca2+ concentration and NO production were measured using fura2 and DAF-2, respectively. KEY RESULTS: TGFbeta1-treated BAECs showed dense actin fibers and expressed smooth muscle marker proteins; they also changed into smooth muscle-like, spindle-shaped cells in collagen gel cultures. ATP (10 microM) induced a gradual contraction of collagen gels containing TGFbeta1-treated BAECs but not of gels containing control BAECs. ATP-induced contraction of TGFbeta1-treated BAECs was not reversed by the removal of ATP but was partially suppressed by a high concentration of sodium nitroprusside (1 microM). TGFbeta1-treated BAECs showed sustained phosphorylation of myosin light chain in response to ATP and low levels of basal MYPT1 expression. ATP-induced Ca2+ transients as well as eNOS protein expression were not affected by TGFbeta1 in BAECs. However, ATP-induced NO production was significantly reduced in TGFbeta1-treated BAECs. Anti-TGFbeta1 antibody abolished all of these TGFbeta1-induced changes in BAECs. CONCLUSIONS AND IMPLICATIONS: Mesenchymal transdifferentiation induced by TGFbeta1 leads to sustained contraction and reduced NO production in endothelial cells. Such effects, therefore, would not be beneficial for vascular integrity.

Endoscopic band ligation therapy for upper gastrointestinal bleeding related to Mallory-Weiss syndrome.

BACKGROUND: No consensus exists as to the best endoscopic treatment for Mallory-Weiss syndrome. Endoscopic band ligation is a readily available and easily learned technique. This prospective study evaluated the efficacy and safety of endoscopic band ligation therapy for Mallory-Weiss syndrome. METHODS: From August 1998 to June 2005, a clinical trial assessed 37 patients with a diagnosis of Mallory-Weiss syndrome who had active bleeding, exposed vessels, or both. Their lesions were treated using endoscopic band ligation. RESULTS: Endoscopic band ligation was successful in 36 of 37 cases, with a follow-up period ranging from 1 to 24 months. The remaining patient had severe liver failure and disseminated intravascular coagulation. The patient bled again at 12 h and subsequently died. Except for this case, no recurrent bleeding, perforation, or other complications occurred. CONCLUSIONS: The study results suggest that endoscopic band ligation is an effective, safe, and easily learned procedure for treating upper gastrointestinal bleeding related to Mallory-Weiss syndrome.

Down-regulation of a Mr 90,000 heat shock cognate protein during granulocytic differentiation in HL-60 human leukemia cells.

Modulation of the synthesis of heat shock proteins in HL-60 human promyelocytic leukemia cells during differentiation was studied by metabolic labeling with [35S]methionine and Northern blot analysis. HL-60 cells were found to synthesize constitutively a high level of a Mr 90,000 protein (heat shock cognate protein, hsc90), which was very closely related to Mr 90,000 heat shock protein, hsp90, as far as molecular weight, isoelectric point, peptide maps, immunoreactivity, and encoded mRNA were concerned. Differentiation induction by dimethyl sulfoxide markedly decreased the level of the hsc90 expression, but preserved the ability to preferentially express hsp90 in response to heat stress. These results suggest distinct regulatory mechanisms of the synthesis between hsc90 and hsp90 although they are indistinguishable by conventional protein or mRNA analysis, and indicate that hsc90 has some role in cell growth and differentiation.

Induction of two estrogen-responsive proteins by antiestrogens in R27, a tamoxifen-resistant clone of MCF7 cells.

In an attempt to approach the mechanism by which estrogen and antiestrogen regulate the growth of estrogen receptor-positive breast cancer, we have studied the effects of the antiestrogens tamoxifen (TAM) and 4-hydroxytamoxifen (OH-TAM) on the induction of two estrogen-specific proteins in a variant of MCF7 cells termed R27, cloned for its ability to grow in the presence of TAM. We found that, in the R27 variant, antiestrogens as well as estradiol were able to increase specifically the production of a Mr 52,000 protein which was released into the culture medium. This protein was shown to be identical to the Mr 52,000 glyco-protein induced by estrogen and released by MCF7 cells on the basis of its specific inducibility by physiological concentrations of 17 beta-estradiol and of its resolution in two-dimensional gel analysis. Dose-response analysis showed that, in the R27 variant, TAM and OH-TAM acquired the ability to induce the Mr 52,000 protein at concentrations compatible with their relative affinities for the estrogen receptor, while these antiestrogens were inefficient in the wild MCF7 cells. Whereas the relative increase of the Mr 52,000 protein was similar with TAM, OH-TAM, and 17 beta-estradiol, the general production of Mr 52,000 and of total labeled proteins was less with the antiestrogens than with 17 beta-estradiol. Moreover, OH-TAM displayed a biphasic dose-response curve with inhibitory effects at concentrations above 10 nM, suggesting an additional mechanism. Neither TAM nor OH-TAM had any antiestrogenic effect when added in the presence of 17 beta-estradiol. In the R27 variant, both estradiol and antiestrogens induced the progesterone receptor sites; however, the extent of the stimulation was lower with antiestrogens than with estradiol. This study shows that, in addition to the classical antiestrogen-resistant breast cancer cells, in which the estrogen receptor is absent or inactive, there is another class of antiestrogen-resistant cells in which the drug becomes a full estrogen agonist as evidenced by the induction of the Mr 52,000 estrogen-specific protein, which is not induced in the wild-type cells.

Hepatitis C and hepatitis B in the etiology of hepatocellular carcinoma in the Japanese population.

We conducted case-control studies of hepatocellular carcinoma (HCC) and liver cirrhosis (LC) in relation to hepatitis C virus (HCV) and hepatitis B virus infection, involving 91 patients with HCC, 75 patients with LC who had no evidence of HCC, and 410 control subjects from the Japanese population. Serum antibody to HCV (anti-HCV) was detected by both enzyme-linked immunosorbent assay and recombinant immunoblot assay in 51, 51, and 3% of HCC, LC, and controls, respectively, whereas the corresponding prevalence of serum hepatitis B surface antigen (HBsAg) was 21, 11, and 2%, respectively. The relative risks (and 95% confidence intervals) for the presence of serum anti-HCV were estimated as 52.3 (23.9-114.3) for HCC and 64.4 (27.4-151.4) for LC. These values exceeded the relative risk of HCC (15.3) and that of LC (6.1) for positive serum HBsAg. Among male patients with HCC or LC, anti-HCV rates were very high in blood recipients (about 70%), heavy drinkers (46-62%), and those who had no identifiable risk factors (65-75%), indicating possible transmission of HCV via routes other than transfusion. No significant difference in anti-HCV status was observed between the HCC and LC groups. It was notable that anti-HCV was much less prevalent among HBsAg-positive patients with HCC or LC than among HBsAg-negative ones. There was a slight to moderate increase in HCC or LC risk among blood recipients and heavy drinkers after adjustment for anti-HCV status. These results indicate that, in Japan, the possible role of HCV infection in the etiology of HCC and LC is extremely large and seems to be more important than chronic hepatitis B virus infection.

Changes in phosphoinositide turnover, Ca2+ mobilization, and protein phosphorylation in platelets from NIDDM patients.

Enhanced platelet functions have been demonstrated in patients with non-insulin-dependent diabetes mellitus (NIDDM). This study evaluated abnormalities in platelet signal transduction in diabetic patients, including turnover of phosphoinositides, mobilization of intracellular Ca2+, and phosphorylation of 20,000- and 47,000-Mr proteins (P20 and P47). Washed platelets were obtained from 6 patients with NIDDM whose platelet aggregation rates were abnormally elevated (DM-A group), 11 NIDDM patients with normal platelet aggregation rates (DM-B group), and 8 age-matched healthy control subjects. The mass and specific radioactivity of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol (PI), and phosphatidic acid (PA) in 32P-labeled platelets were not different among the three groups. Hydrolysis of PIP2, PIP, and PI; accumulation of PA; and phosphorylation of P20 in platelets stimulated by 0.05 U/ml thrombin were significantly increased in the DM-A group compared with the control or DM-B group. There was no difference in P47 phosphorylation among the three groups. On the contrary, P20 and P47 phosphorylation induced by 50 nM of 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, was significantly decreased in the DM-A group. Additionally, the intracellular free Ca2+ concentration [( Ca2+]i) was measured with the fluorescent Ca2+ indicator fura 2. Although the basal [Ca2+]i value was similar in the three groups, the rise in [Ca2+]i induced by 0.05 U/ml thrombin in the presence and the absence of extracellular Ca2+ was significantly higher in the DM-A group than the other groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Relative hypersecretion of amylin to insulin from rat pancreas after neonatal STZ treatment.

With isolated perfused pancreases from normal and diabetic model rats, we studied alterations of the secretion of islet amyloid polypeptide, or amylin, which has been recently identified as a major component of amyloid deposits in the pancreatic islets of patients with non-insulin-dependent diabetes mellitus. Neonatal (n) Wistar-King albino rats given streptozocin (STZ) on the 2nd (n2STZ) or 5th (n5STZ) neonatal day exhibited moderate and marked elevations, respectively, of plasma glucose and HbA1 as adults compared with control rats given the vehicle. The release of amylin from the perfused pancreases in response to glucose and arginine paralleled that of insulin in all three groups. However, the molar ratio of secreted amylin to insulin in response to 16.7 mM glucose by n5STZ pancreases (6.55 +/- 0.71%) was significantly greater than that for either n2STZ (1.71 +/- 0.24%, P less than 0.05) or the control (0.60 +/- 0.03%, P less than 0.05) pancreases. The secreted amylin-insulin ratio of n2STZ pancreases also was significantly greater than that of the controls (P less than 0.05). The increased amylin-insulin molar ratios of both n2STZ and n5STZ pancreases also occurred during infusions of 33.3 mM glucose and 10 mM arginine. These findings suggest that amylin secretion may be preserved in diabetic rats with reduced beta-cell mass and that hyperglycemia may increase amylin production independently of that of insulin, which may be significant in the pathogenesis of non-insulin-dependent diabetes mellitus.

Release of amylin from perfused rat pancreas in response to glucose, arginine, beta-hydroxybutyrate, and gliclazide.

Amylin is a 37-amino acid peptide isolated from the islet amyloid of patients with non-insulin-dependent diabetes mellitus. The isolated perfused normal rat pancreas was used to evaluate the effects of glucose and insulin secretagogues, such as arginine, beta-hydroxybutyrate, and gliclazide, on amylin secretion. Glucose and the other stimulants tested elicited a significant release of amylin from the rat pancreas in a biphasic pattern, similar to that of insulin. Dose-response studies of the glucose-induced release of amylin and insulin revealed that they possessed a similar dependency on glucose. However, the release of amylin induced by high concentrations of glucose was partially dissociated from that of insulin; that is, the amylin-insulin molar ratios induced by 22.2 and 33.3 mM glucose (1.11 +/- 0.05 and 1.05 +/- 0.04%, respectively) were significantly higher than those induced by 16.7 mM glucose (0.90 +/- 0.04%, P less than 0.01 vs. 22.2 mM glucose, P less than 0.05 vs. 33.3 mM glucose). Additionally, when the basal concentration of glucose in the perfusate was increased from 5.6 to 11.1 mM, the response of amylin was unchanged. These data suggest that amylin may be an islet hormone whose abundant response to high concentrations of glucose might contribute to the oversecretion of amylin in the hyperglycemia that accompanies diabetes mellitus.

High glucose reduces specific binding for D-alpha-tocopherol in cultured aortic endothelial cells.

We investigated the effects of glucose on specific D-alpha-tocopherol binding to cultured bovine aortic endothelial cells. Our results confirmed that cultured bovine aortic endothelial cells have specific binding sites for D-alpha-tocopherol. These binding sites exhibited time- and temperature-dependent saturation. The specific binding affinity of D-alpha-tocopherol was significantly lower in endothelial cells cultured in high concentrations of glucose (16.8 or 22.4 mM) for > 7 days compared with cells cultured in a physiological glucose concentration (5.6 mM). No significant reduction occurred in D-alpha-tocopherol binding when 11.1 mM mannitol was added to cells cultured in 5.6 mM glucose. The addition of an aldose reductase inhibitor (ICI-128436, Statil) did not significantly affect the high-glucose-induced reduction of D-alpha-tocopherol binding, although it reduced sorbitol levels in the cells compared with those from cells cultured in high concentrations of glucose. Moreover, significantly higher amounts of lipid peroxides were produced in aortic endothelial cells cultured in high concentrations of glucose (16.8 or 22.4 mM) for > 3 days compared with cells cultured in a physiological concentration of glucose. These results indicate that high concentrations of glucose reduce D-alpha-tocopherol binding through mechanisms independent of putative osmotic effects of sorbitol accumulation in the cells. Possible mechanisms include glycation of protein or oxidative damage of cells and/or redox and metabolic imbalances associated with increased flux of glucose via the sorbitol pathway. A glucose-mediated reduction in D-alpha-tocopherol binding could diminish the beneficial effects of D-alpha-tocopherol to vascular endothelial cells and thereby may increase the vascular toxicity of hyperglycemia in diabetes mellitus.

Normalization of diacylglycerol-protein kinase C activation by vitamin E in aorta of diabetic rats and cultured rat smooth muscle cells exposed to elevated glucose levels.

Hyperglycemia and diabetes have been shown to increase diacylglycerol (DAG) level and activate protein kinase C (PKC) activity in the vascular tissues, possibly altering vascular function. We have characterized the effects of D-alpha-tocopherol (vitamin E) on PKC activities and DAG levels in rat aortic smooth muscle cells (ASMCs) cultured with elevated glucose levels as well as in the vascular tissues obtained from control and diabetic rats. In ASMCs, the specific PKC activity from the membraneous fraction and total DAG level were increased by 31 +/- 4% (P < 0.05) and 50 +/- 7% (P < 0.05), respectively, when the glucose levels were changed from 5.5 to 22 mmol/l. The addition of D-alpha-tocopherol and another lipophilic antioxidant, probucol, prevented the glucose-stimulated increases in DAG level and PKC activity. By immunoblotting studies, D-alpha-tocopherol treatment was able to reduce the enhancement of PKC beta II isoform in the membraneous fraction isolated from ASMCs. Comparing streptozotocin-induced diabetic rats with their nondiabetic controls, both membraneous-specific PKC activities and total cellular DAG levels were increased in aorta by 162% (P < 0.05) and 60% (P < 0.05), respectively. Intraperitoneal injection of D-alpha-tocopherol (40 mg/kg) every other day prevented the increases in membraneous-specific PKC activities and total DAG levels in parallel with a significant increase of D-alpha-tocopherol contents in the aorta and plasma. These findings have demonstrated that D-alpha-tocopherol can prevent the activation of PKC activities in the vascular cells and tissues induced by hyperglycemia by lowering DAG levels, possibly via its antioxidant effect.

Immunohistochemical study of prostacyclin-stimulating factor (PSF) in the diabetic and atherosclerotic human coronary artery.

Prostacyclin (PGI2) synthesis by vascular endothelial cells (ECs) decreases in diabetic subjects, possibly leading to the development of diabetic angiopathy, such as that seen in atherosclerosis. We recently found a novel bioactive peptide, prostacyclin-stimulating factor (PSF), which stimulates PGI2 synthesis by cultured aortic ECs. Our previous studies demonstrated that PSF is dominantly expressed by arterial smooth muscle cells (SMCs). In the present study, we found PSF to exist in the SMCs of human coronary arteries by means of immunohistochemical methods. Human coronary arteries obtained from autopsies were divided into four subgroups, with or without NIDDM and/or myocardial infarction. Immunostaining for PSF was performed by the avidin-biotin peroxidase complex method using a purified anti-PSF antibody, and the immunostaining for PSF was assessed semiquantitatively. PSF staining was markedly reduced in coronary arterial SMCs from patients with NIDDM and/or myocardial infarction. In addition, the effect of a high glucose culture on PSF mRNA expression and PSF production in bovine aortic SMCs was examined by immunocytochemical staining and both Western and Northern blot analyses. The immunostaining and immunoblot band for PSF also significantly decreased when bovine aortic SMCs were cultured with high concentrations of glucose. Furthermore, as compared with the SMCs cultured with a physiological glucose concentration, the density ratio of PSF mRNA to 28S rRNA expression significantly decreased when the SMCs were cultured with high concentrations of glucose. These results strongly suggest that the decreased PSF production may thus results in a decreased production of PGI2 in the coronary artery, thus leading to the development of both diabetic macroangiopathy and atherosclerosis.

Vitamin E normalizes diacylglycerol-protein kinase C activation induced by hyperglycemia in rat vascular tissues.

Hyperglycemia, a major cause of vascular complications in diabetes, has been shown to activate the diacylglycerol (DAG)-protein kinase C (PKC) pathway in vascular tissues. We have found that D-alpha-tocopherol (vitamin E) treatment reversed the adverse effects of hyperglycemia both in vitro and in vivo. In aortic smooth muscle cells (ASMCs), the PKC specific activity from the membranous fraction and total DAG were increased by 31 +/- 4% (P < 0.05) and 50 +/- 7% (P < 0.05), respectively, when the glucose levels were changed from 5.5 to 22 mmol/l. D-alpha-tocopherol prevented the glucose-stimulated increases in DAG level and PKC activity as well as the amount of PKC beta II isoform in ASMCs cultured with elevated glucose levels. Comparing streptozotocin-induced diabetic rats after 2 weeks of disease to controls, specific membranous PKC activities and total DAG levels were increased in aorta (162%, P < 0.05; 60%, P < 0.05). Intraperitoneal injection of D-alpha-tocopherol (40 mg/kg) every other day resulted in a significant decrease of the elevated membranous PKC specific activity and total DAG levels in parallel with a significant increase of D-alpha-tocopherol content in the aorta. These findings suggested that D-alpha-tocopherol can prevent the activation of PKC in the vascular cells and aorta induced by hyperglycemia by normalizing the elevated levels of DAG.

High glucose induces alteration of gap junction permeability and phosphorylation of connexin-43 in cultured aortic smooth muscle cells.

Gap junction is thought to have a crucial role in maintaining tissue homeostasis. We examined the effect of a high glucose level on gap junctional intercellular communication (GJIC) activity in cultured vascular smooth muscle cells (VSMCs) using the fluorescent dye transfer method. After a 48-h incubation with 22 mmol/l glucose (high glucose level), GJIC activity of VSMCs was significantly reduced compared with incubation with 5.5 mmol/l glucose (normal glucose level) (P < 0.05). Treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate (TPA; 5 x 10(-8) mol/l), a protein kinase C (PKC) activator, for 1 h also reduced GJIC activity (P < 0.01). In addition, treatment of the cells with calphostin C, a specific PKC inhibitor, for 3 h completely restored the GJIC activity inhibited by the high glucose level. Western blot analysis showed that connexin 43 (Cx43), which is the major functional protein of gap junction, is present in multiphosphorylated forms: a nonphosphorylated form (P0) and phosphorylated forms (P1, P2, and P3). Incubation of VSMCs with a high glucose level significantly increased the density ratio of P3/P0 compared with a normal glucose level (P < 0.05). Similarly, treatment of the cells with TPA significantly increased the P3/P0 ratio compared with controls (P < 0.01). In addition, the increase in the P3/P0 density ratio induced by a high glucose level was restored to the control level by both staurosporine and calphostin C. These results suggest that the high glucose level induced the inhibition of GJIC activity in cultured VSMCs through excessive phosphorylation of Cx43, mediated by PKC activation. This may contribute to the development of the macroangiopathy associated with diabetes.

Decreased VIP content in peripheral nerve from streptozocin-induced diabetic rats.

After induction of diabetes with streptozocin (STZ-D) in rats, we measured vasoactive intestinal polypeptide (VIP) content in sciatic nerve and spinal cord obtained from nondiabetic, untreated STZ-D, and insulin-treated STZ-D rats. Eight weeks after the onset of diabetes, caudal nerve conduction velocity (NCV) in the untreated STZ-D rats (n = 13) was slower than in the controls (n = 11; mean +/- SE 30.9 +/- 0.6 vs. 41.4 +/- 1.8 m/s, P less than 0.001). The decrease in NCV was less marked in the insulin-treated STZ-D rats (n = 11; 36.3 +/- 0.9 m/s, P less than 0.05 vs. control). VIP content in sciatic nerve decreased in the untreated STZ-D rats (1.33 +/- 0.23 ng/g wet wt) compared with the other groups (control, 3.10 +/- 0.44, P less than 0.01; insulin-treated STZ-D, 2.44 +/- 0.55, P less than 0.05). However, in spinal cord, VIP content was not significantly different among the three groups. The VIP levels in sciatic nerve showed a positive correlation with NCV (r = 0.430, P less than 0.01). In addition, an inverse correlation between VIP levels and blood glucose levels was observed (r = -0.5624, P less than 0.001). NCV was also inversely correlated with blood glucose levels (r = -0.7662, P less than 0.001). Together with a previous morphological study, these findings suggest a possible causal relationship between reduced VIP content and diabetic neuropathy.

Prostacyclin-stimulating factor, novel protein, and diabetic angiopathy.

We recently purified and cloned a new protein that stimulates the synthesis of prostacyclin (PGI2) by the vascular endothelial cells (ECs). We have termed this protein "PGI2-stimulating factor" (PSF). The present study evaluated the expression of PSF mRNA in tissues of Wistar rats, including the kidneys of rats with streptozotocin-induced diabetes, and in cultured cells. Furthermore, we evaluated the presence of PSF in human sera and the immunohistochemical localization of PSF in tissues of patients obtained at autopsy. The latter included a coronary atherosclerotic lesion of a patient who died of acute myocardial infarction. PSF was observed by Northern blot analysis to be expressed in all rat tissues examined (brain, lung, liver, kidney, skeletal muscle, and fat tissue) and was expressed in cultured vascular ECs, smooth muscle cells (SMCs), and fibroblast cells (FCs). A decreased expression of PSF was observed in the kidneys of diabetic rats versus those of normal rats. The presence of PSF in human serum was confirmed by Western blot analysis. In humans, PSF was mainly localized in vascular ECs and SMCs of arterial media and in SMCs of bronchi. Reduced staining for PSF was found in an atherosclerotic versus a normal coronary artery of humans. PSF may be involved in the production of PGI2 in the vessel wall and may participate in the maintenance of vascular homeostasis. PSF abnormalities may be involved in the development of such vascular lesions as atherosclerosis and diabetic angiopathy.

High glucose level and free fatty acid stimulate reactive oxygen species production through protein kinase C--dependent activation of NAD(P)H oxidase in cultured vascular cells.

Recent studies have revealed that vascular cells can produce reactive oxygen species (ROS) through NAD(P)H oxidase, which may be involved in vascular injury. However, the pathological role of vascular NAD(P)H oxidase in diabetes or in the insulin-resistant state remains unknown. In this study, we examined the effect of high glucose level and free fatty acid (FFA) (palmitate) on ROS production in cultured aortic smooth muscle cells (SMCs) and endothelial cells (ECs) using electron spin resonance spectroscopy. Exposure of cultured SMCs or ECs to a high glucose level (400 mg/dl) for 72 h significantly increased the free radical production compared with low glucose level exposure (100 mg/dl). Treatment of the cells for 3 h with phorbol myristic acid (PMA), a protein kinase C (PKC) activator, also increased free radical production. This increase was restored to the control value by diphenylene iodonium, a NAD(P)H oxidase inhibitor, suggesting ROS production through PKC-dependent activation of NAD(P)H oxidase. The increase in free radical production by high glucose level exposure was completely restored by both diphenylene iodonium and GF109203X, a PKC-specific inhibitor. Exposure to palmitate (200 micromol/l) also increased free radical production, which was concomitant with increases in diacylglycerol level and PKC activity. Again, this increase was restored to the control value by both diphenylene iodonium and GF109203X. The present results suggest that both high glucose level and palmitate may stimulate ROS production through PKC-dependent activation of NAD(P)H oxidase in both vascular SMCs and ECs. This finding may be involved in the excessive acceleration of atherosclerosis in patients with diabetes and insulin resistance syndrome.

Risk factor analyses for macrovascular complication in nonobese NIDDM patients. Multiclinical Study for Diabetic Macroangiopathy (MSDM).

To examine the characteristic features of risk factors for macroangiopathy (MA) in nonobese Japanese NIDDM patients, 899 NIDDM patients with and without MA were registered from 40 facilities. Of these, 386 subjects were identified as having any form of MA (total MA); these included 211 with ischemic heart disease (IHD), 163 with cerebrovascular disease (CVD), and 77 with peripheral vascular disease (PVD). Univariate analyses revealed the following common risk factors for total MA, IHD, CVD, and PVD: age, hypertension, systolic blood pressure (sBP) or diastolic blood pressure (dBP), duration of diabetes, diabetic microangiopathy (retinopathy, nephropathy, and neuropathy), low HDL cholesterol level, and higher LDL cholesterol/HDL cholesterol ratio. Additional significant risk factors for specific conditions were also identified, respectively, as male sex for total MA, IHD, and PVD, smoking for IHD and PVD, and high fasting plasma glucose level for total MA and CVD. With stepwise multivariate logistic regression analysis, older age, duration of diabetes, smoking, and low LDL cholesterol/HDL cholesterol ratio were identified as significant and independent risk factors for total MA, IHD, CVD, and PVD. Other risk factors identified were high dBP for IHD, CVD, and PVD, high sBP for total MA, and low BMI for PVD. These results clearly demonstrated that duration of diabetes, smoking, hypertension, and dyslipidemia are major risk factors for MA in NIDDM patients. Since the mean BMI was similar for both groups (approximately 23 kg/m2) and there were no significant differences in immunoreactive insulin levels before and after 75-g oral glucose challenge testing, obesity and hyperinsulinism at the time of the analyses were not considered to play an important role for the pathogenesis of MA in Japanese NIDDM patients. By using the chi 2 test, cutoff points were determined for six of the most commonly measured risk factors. The cutoff point was the level beyond which a significantly higher prevalence of MA occurred. The cutoff points (rounded slightly upward in some cases) for fasting plasma glucose, sBP, dBP, serum total cholesterol level, serum triglyceride level, and BMI were 140 mg/dl, 140 mmHg, 80 mmHg, 180 mg/dl, 120 mg/dl, and 23 kg/m2, respectively. When these cutoff points were used as control criteria, the prevalence of MA was significantly lower in subjects whose risk factor measurements remained under the proposed control criteria for four or more of the six variables. In conclusion, in nonobese NIDDM patients, age, hypertension, and dyslipidemia were found to be risk factors for MA. Duration of diabetes was also demonstrated as an independent risk factor, indicating the close association of deranged glucose metabolism with the pathogenesis of MA in NIDDM patients. It seems to be crucial to control these risk factors for the prevention of MA in NIDDM patients.

Heterogeneity of anterograde fast-pathway and retrograde slow-pathway conduction patterns in patients with the fast-slow form of atrioventricular nodal reentrant tachycardia: electrophysiologic and electrocardiographic considerations.

OBJECTIVES: This study sought to define the electrophysiologic and electrocardiographic characteristics of fast-slow atrioventricular nodal reentrant tachycardia (AVNRT). BACKGROUND: In fast-slow AVNRT the retrograde slow pathway (SP) is located in the posterior septum, whereas the anterograde fast pathway (FP) is located in the anterior septum; however, exceptions may occur. METHODS: Twelve patients with fast-slow AVNRT were studied. To determine the location of the retrograde SP, atrial activation during AVNRT was examined while recording the electrograms from the low septal right atrium (LSRA) on the His bundle electrogram and the orifice of the coronary sinus (CS). Further, to investigate the location of the anterograde FP, single extrastimuli were delivered during AVNRT both from the high right atrium and the CS. RESULTS: The CS activation during AVNRT preceded the LSRA in six patients (posterior type); LSRA activation preceded the CS in three patients (anterior type), and in the remaining three both sites were activated simultaneously (middle type). In the anterior type, CS stimulation preexcited the His and the ventricle without capturing the LSRA electrogram (atrial dissociation between the CS and the LSRA), suggesting that the anterograde FP was located posterior to the retrograde SP. In the posterior and middle types, high right atrial stimulation demonstrated atrial dissociation, suggesting that the anterograde FP was located anterior to the SP. In the posterior and middle types, retrograde P waves in the inferior leads were deeply negative, whereas they were shallow in the anterior type. CONCLUSIONS: Fast-slow AVNRT was able to be categorized into posterior, middle and anterior types according to the site of the retrograde SP. The anterior type AVNRT, where an anteriorly located SP is used in the retrograde direction and a posteriorly located FP in the anterograde direction, appears to represent an anatomical reversal of the posterior type which uses a posterior SP for retrograde and an anterior FP for anterograde conduction. Anterior type AVNRT should be considered in the differential diagnosis of long RP (RP > PR intervals) tachycardias with shallow negative P waves in the inferior leads.

Phosphotyrosine phosphatase activity prevents the detection of P210bcr/abl protein in mature cells in chronic myelogenous leukemia even by an immunoblotting technique.

P210bcr/abl protein with tyrosine protein kinase has been implicated in the proliferation and differentiation of chronic myelogenous leukemia cells. Using an immunoblotting technique with antiphosphotyrosine (anti-P-Tyr) antibodies, we examined whether P210bcr/abl protein was expressed in chronic phase cells in patients with chronic myelogenous leukemia (CML). We could detect P210bcr/abl protein in blast cells regardless of myeloid or lymphoid lineage but not in chronic phase cells from patients. However, in a patient with both blast cells and chronic phase cells, we could identify the protein only after the enrichment of the blast crisis cells by Percoll gradient centrifugation. When K562 cells were mixed with mature granuloid cells, the P210bcr/abl in K562 cells detected by immunoblotting was decreased. Using phosphotyrosyl proteins in K562 cells as substrates, high phosphotyrosyl (P-Tyr) phosphatase activity was observed, not only in the lysate of chronic phase cells from CML patients but also in the lysate of neutrophils from normal subjects. These findings suggest the possibility that high P-Tyr phosphatase activity prevents the detection of P210bcr/abl in CML cells in the chronic phase. The activity may be characteristic of mature cells and may regulate cellular events through dephosphorylation of P210bcr/abl.