Secretion of intelectin-1 from malignant pleural mesothelioma into pleural effusion.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a rare but fatal tumour. Although most MPM patients show pleural effusion at even the early stage, it is hard to diagnose as MPM at the early stage because a sensitive and reliable diagnostic marker for MPM has not been found in plasma or pleural effusion. METHODS: In this study, we investigated whether intelectin-1 was specifically contained in MPM cells and the pleural effusion of MPM patient by immunohistochemistry, western blotting, and enzyme-linked immunosorbent assay. RESULTS: Malignant pleural mesothelioma cell lines, but not lung adenocarcinoma cell lines, secreted intelectin-1. In immunohistochemistry, epithelioid-type MPMs, but neither pleura-invading lung adenocarcinomas nor reactive mesothelial cells near the lung adenocarcinomas, were stained with anti-intelectin antibodies. Pleural effusion of MPM patients contained a higher concentration of intelectin-1 than that of lung cancer patients. CONCLUSION: These results suggest that detection of intelectin-1 may be useful for a differential diagnosis of epithelioid-type MPM in immunohistochemistry and that a high concentration of intelectin-1 in pleural effusion can be used as a new marker for clinical diagnosis of MPM.

Expression of c-kit protein in proliferative lesions of human breast: sexual difference and close association with phosphotyrosine status.

PURPOSE: The c-kit gene which codes transmembrane tyrosine kinase receptor protein plays an important role in several types of normal and/or neoplastic human tissues. We examined the expression patterns of c-kit protein in proliferative lesions of human breast tissues in both sexes. METHODS: The localization of c-kit protein was examined immunohistochemically in human breast, consisting of 366 normal tissue, 156 benign lesions (fibroadenoma, fibrocystic change, intraductal papilloma, benign phyllodes tumor, and gynecomastia), 13 borderline diseases (atypical ductal hyperplasia, atypical lobular hyperplasia, and borderline malignant phyllodes tumor), and 197 malignant lesions (non-invasive and/or invasive ductal carcinoma and malignant phyllodes tumor). RESULTS: In normal tissues and benign proliferative lesions, c-kit product was consistently detected on epithelial cell membranes and/or cytoplasms regardless of gender difference. In contrast, we failed to find c-kit product in female borderline epithelial lesions, including atypical lobular hyperplasia, or in female malignant lesions, except for two carcinomas. In situ hybridization analysis of c-kit mRNA in female tissues gave results comparable to those obtained by immunohistochemistry. On the other hand, c-kit product was consistently detected in male benign and malignant proliferative lesions. Apart from the female breast carcinomas which lacked c-kit, c-kit expression was almost always accompanied by positivity for phosphotyrosine in the breast tissues examined, suggesting possible phosphorylation of tyrosine residues of the c-kit receptor protein. CONCLUSIONS: Loss of c-kit product was related to malignant transformation in female breast, but not in the case of male breast. We suggest that the oncogenesis pathway of breast epithelium is different between males and females in terms of c-kit expression.

Recovery of function and mass of endogenous beta-cells in streptozotocin-induced diabetic rats treated with islet transplantation.

Islet transplantation corrects chronic hyperglycemia by augmentation of insulin supply from the graft tissue, but the role of endogenous beta-cells after transplantation is not clear. In the present study, we examined endogenous beta-cell function after glucose homeostasis had been reestablished by islet graft in streptozotocin (STZ)-induced diabetic rats. Fed plasma glucose levels in diabetic rats transplanted with a large number of islets (2500 islets) into the left kidney capsule soon became lower (139.8 +/- 8.2 mg/dl) and close to the level in controls (129.7 +/- 11.3 mg/dl), and IPGTT exhibited a pattern of plasma glucose response almost identical to control. The insulin and DNA contents, islet area, and the distribution of beta-cells that were markedly deteriorated in islets of STZ rats were significantly restored in transplanted rats. The insulin release in response to glucose or alpha-ketoisocaproate was less in STZ rats, while in islets of transplanted rats the secretion recovered to levels similar to controls. On the other hand, arginine-induced insulin release was conversely hyperresponsive in STZ rats, but in transplanted rats, the response was decreased similar to controls. Thus, as the plasma glucose level normalizes, residual beta-cells show a recovery of function that cannot be accounted for by the increase in mass alone.

Genomic variation in pancreatic ion channel genes in Japanese type 2 diabetic patients.

BACKGROUND: Many genetic diseases are caused by mutations in ion channel genes. Because type 2 diabetes is characterized by pancreatic beta-cell insensitivity to glucose, the genes responsible for glucose metabolism and calcium signaling in pancreatic beta-cells are candidate type 2 diabetes susceptibility genes. METHODS: We have examined genomic variations in two ion channel genes relevant to the molecular pathology of diabetes mellitus, the Kir6.2 subunit of the ATP-sensitive potassium channel gene and alpha(1D) subunit of the voltage-dependent calcium channel (VDCC) gene among Japanese type 2 diabetic patients. RESULTS: There are two alleles in the Kir6.2 gene: EI, glutamic acid at codon 23 and isoleucine at codon 337 and KV, lysine at codon 23 and valine at codon 337. The allelic frequencies of these polymorphisms are similar in type 2 diabetic patients and normal subjects. We also detected trinucleotide repeat polymorphisms in the amino terminus and the carboxyl terminal region of the alpha(1D) gene. Expansion of the ATG trinucleotide repeat from seven to eight was detected only in type 2 diabetic patients, but the frequency was low and was similar in type 2 diabetic patients and normal subjects. CONCLUSIONS: Although variations of the Kir6.2 and alpha(1D) genes are not associated with the development of common type 2 diabetes, further studies may determine the role of these genomic variations, especially those in the alpha(1D) VDCC gene, in the pathogenesis of certain subsets of type 2 diabetes, or as a co-factor in the polygenic disorder generally.

Distinct effect of diazoxide on insulin secretion stimulated by protein kinase A and protein kinase C in rat pancreatic islets.

Protein kinase activation is known to stimulate glucose-induced insulin secretion in the presence of diazoxide. Diazoxide opens the ATP-sensitive K(+) channel and inhibits FAD-linked glycerophosphate dehydrogenase activity in a concentration-dependent manner. In the present study, we examined the effect of lower (100 microM) and higher (250 microM) concentrations of diazoxide on insulin release by protein kinase A (PKA) and protein kinase C (PKC) activation. Forced depolarization by a high potassium concentration, augmented the intracellular Ca(2+) concentration ([Ca(2+)](i)) similarly in the presence of both concentrations of diazoxide. Under this condition, 250 microM diazoxide inhibited insulin release enhanced by PKA activation but not that by PKC. Under a basal concentration of [Ca(2+)](i), PKC activation elicited glucose-induced insulin secretion at 100 and 250 microM diazoxide, while PKA activation did so only at 100 microM. These augmentations were completely inhibited by mannoheptulose, a glucokinase inhibitor. Glyceraldehyde, in place of glucose, enhanced insulin secretion by PKC activation under both concentrations of diazoxide. On the other hand, it did not affect PKA-stimulated insulin release under either conditions, but in the case of 100 microM, glucose augmented the insulin secretion in the presence of glyceraldehyde and db-cAMP concentration-dependently. These data suggest that insulin release stimulated by PKA and PKC activation under diazoxide is dependent on glucose metabolism, and that a signal derived from proximal steps in glycolysis may be necessary for the secretion by PKA activation.

Heat shock restores insulin secretion after injury by nitric oxide by maintaining glucokinase activity in rat islets.

Heat shock protein (hsp), including hsp70, has been reported to restore the glucose-induced insulin release suppressed by nitric oxide (NO). However, the mechanism underlying this recovery remains unclear. In the present study, we examine the effects, in rat islets, of heat shock on insulin secretion inhibited by a small amount of NO and also on glucose metabolism, the crucial factor in insulin release. Exposure to a higher dose (15 U/ml) of interleukin-1beta (IL-1beta) abolished the insulin release by stimulation of glucose or KCl in both control and heat shocked islets. In rat islets exposed to a lower dose (1.5 U/ml) of IL-1beta, insulin secretion in response to glucose, but not to glyceraldehydes (GA), ketoisocaproate (KIC), or KCl, was selectively impaired, concomitantly with lower ATP concentrations in the presence of 16.7 mM glucose, while such suppression of insulin secretion and ATP content was not observed in heat shock-treated islets. NO production in islets exposed to 1.5 U/ml IL-1beta was significantly, but only partly, decreased by heat shock treatment. The glucose utilization rate measurement using [5-3H]-glucose and [2-3H]-glucose and the glucokinase activity in vitro were reduced in islets treated with 1.5 U/ml IL-1beta. In heat shock-treated islets, glucose utilization and glucokinase activity were not affected by 1.5 U/ml IL-1beta. These data suggest that heat shock restores glucose-induced insulin release inhibited by NO by maintaining glucokinase activity and the glucose utilization rate in islets in addition to reducing endogenous NO production.

A novel glucokinase regulator in pancreatic beta cells: precursor of propionyl-CoA carboxylase beta subunit interacts with glucokinase and augments its activity.

A glucokinase regulatory protein has been reported to exist in the liver, which suppresses enzyme activity in a complex with fructose 6-phosphate, whereas no corresponding protein has been found in pancreatic beta cells. To search for such a protein in pancreatic beta cells, we screened for a cDNA library of the HIT-T15 cell line with the cDNA of glucokinase from rat islet by the yeast two hybrid system. We detected a cDNA encoding the precursor of propionyl-CoA carboxylase beta subunit (pbetaPCCase), and glutathione S-transferase pull-down assay illustrated that pbetaPCCase interacted with recombinant rat islet glucokinase and with glucokinase in rat liver and islet extracts. Functional analysis indicated that pbetaPCCase decreased the K(m) value of recombinant islet glucokinase for glucose by 18% and increased V(max) value by 23%. We concluded that pbetaPCCase might be a novel activator of glucokinase in pancreatic beta cells.

Augmentation of basal insulin release from rat islets by preexposure to a high concentration of glucose.

We have found that preexposure to an elevated concentration of glucose reversibly induces an enhancement of basal insulin release from rat pancreatic islets dependent on glucose metabolism. This basal insulin release augmented by priming was not suppressed by reduction of the intracellular ATP or Ca(2+) concentration, because even in the absence of ATP at low Ca(2+), the augmentation was not abolished from primed electrically permeabilized islets. Moreover, it was not inhibited by an alpha-adrenergic antagonist, clonidine. A threshold level of GTP is required to induce these effects, because together with adenine, mycophenolic acid, a cytosolic GTP synthesis inhibitor, completely abolished the enhancement of basal insulin release due to the glucose-induced priming without affecting the glucose-induced increment in ATP content and ATP-to-ADP ratio. In addition, a GDP analog significantly suppressed the enhanced insulin release due to priming from permeabilized islets in the absence of ATP at low Ca(2+), suggesting that the GTP-sensitive site may play a role in the augmentation of basal insulin release due to the glucose-induced priming effect.

The insulinotropic mechanism of the novel hypoglycaemic agent JTT-608: direct enhancement of Ca(2+) efficacy and increase of Ca(2+) influx by phosphodiesterase inhibition.

We examined the effects of the novel hypoglycaemic agent JTT-608 [trans-4-(4-methylcyclohexyl)-4-oxobutyric acid] on insulin secretion using rat pancreatic islets, and analysed the mechanism of its effect. JTT-608 augmented 8.3 mM glucose-induced insulin secretion dose-dependently, and there was a stimulatory effect of 100 microM JTT-608 at both moderate and high concentrations (8.3, 11. 1 and 16.7 mM) of glucose, but not at low concentrations (3.3 and 5. 5 mM). In perifusion experiments, both phases of insulin release were enhanced, and the effect was eliminated 10 min after withdrawal of the agent. In the presence of 200 microM diazoxide and a depolarizing concentration (30 mM) of K(+), there was an augmentation of insulin secretion by 100 microM JTT-608, not only under high levels of glucose but also under low levels, and the effects were abolished by 10 microM nitrendipine. JTT-608 augmented insulin secretion from electrically permeabilized islets in the presence of stimulatory concentrations (0.3 and 1.0 microM) of Ca(2+), and the intracellular Ca(2+) concentration ([Ca(2+)](i)) response under 16.7 mM glucose, 200 microM diazoxide, and 30 mM K(+) was also increased. The cyclic AMP content in the islets was increased by 100 microM JTT-608, and an additive effect to 1 microM forskolin was observed, but not to 50 microM 3-isobutyl-1-methylxanthine (IBMX). JTT-608 inhibited phosphodiesterase (PDE) activity dose-dependently. We conclude that JTT-608 augments insulin secretion by enhancing Ca(2+) efficacy and by increasing Ca(2+) influx. This appears to be a result of the increased intracellular cyclic AMP concentration due to PDE inhibition.

An insulinotropic effect of vitamin D analog with increasing intracellular Ca2+ concentration in pancreatic beta-cells through nongenomic signal transduction.

The effect of 1alpha,25-dihydroxylumisterol3 (1alpha,25(OH)2lumisterol3) on insulin release from rat pancreatic beta-cells was measured to investigate the nongenomic action of vitamin D via the putative membrane vitamin D receptor (mVDR). 1Alpha,25(OH)2lumisterol3, a specific agonist of mVDR, dose-dependently augmented 16.7 mM glucose-induced insulin release from rat pancreatic islets and increased the intracellular Ca2+ concentration ([Ca2+]i), though not increasing Ca2+ efficacy in the exocytotic system. These effects were completely abolished by an antagonist of mVDR, 1beta,25-dihydroxyvitamin D3 (1beta,25(OH)2D3), or by a blocker of voltage-dependent Ca2+ channels, nitrendipine. Moreover, both [Ca2+]i elevation, caused by membrane depolarization, and sufficient intracellular glucose metabolism are required for the expression of these effects. 1Alpha,25(OH)2lumisterol3, therefore, has a rapid insulinotropic effect, through nongenomic signal transduction via mVDR, that would be dependent on the augmentation of Ca2+ influx through voltage-dependent Ca2+ channels on the plasma membrane, being also linked to metabolic signals derived from glucose in pancreatic beta-cells. However, further investigations will be needed to discuss physiologically the meaning of insulinotropic effects of vitamin D through mVDR.

Regulation of intracellular ATP concentration under conditions of reduced ATP consumption in pancreatic islets.

ATP is the most important factor in glucose-induced insulin secretion in pancreatic beta-cells, but examination of intracellular differences in ATP concentration is difficult because ATP production and consumption occur simultaneously. In the present study, we measured the ATP concentration under the condition of a reduced ATP requirement by omitting extracellular Ca(2+) and inhibiting Na-K ATPase. The ATP concentration in islets incubated with 16.7 mM glucose in the absence of Ca(2+) for 30 min was increased by about 1. 9-fold more than in the presence of Ca(2+). The increment was extracellular Ca(2+)-dependent, and was completely abolished by the metabolic inhibitors dinitrophenol and iodoacetic acid. The Ca channel blockers including nitrendipine and Ni(2+) did not affect the ATP concentration in islets incubated with 16.7 mM glucose in the presence of Ca(2+). However, when thapsigargin and suramin, inhibitors of Ca-ATPase at the endoplasmic reticulum, were added to Ca channel blockers in the presence of ambient Ca(2+), the intraislet ATP content was increased, similarly to that under Ca-free conditions. But thapsigargin did not further augment the ATP concentration in the islet with 16.7 mM glucose in the absence of Ca(2+). On the other hand, the suppression of Na-K ATPase by ouabain rather reduced the ATP concentration augmented by omission of extracellular Ca(2+). In addition, vanadate, a blocker of Ca-ATPase at the plasma membrane, failed to increase the ATP concentration in the islets. These data suggest that the increment of ATP concentration in the absence of Ca(2+) is attributable to the reduced ATP requirement due to stopping of the Ca-ATPase activity at the endoplasmic reticulum, and that the intracellular ATP concentration is differently regulated by Na-K ATPase at plasma membrane and by Ca-ATPase at endoplasmic reticulum.

Endogenous nitric oxide inhibits glucose-induced insulin secretion by suppression of phosphofructokinase activity in pancreatic islets.

The physiological role of nitric oxide (NO) on the mechanism of insulin secretion is unknown, but some studies suggest that NO affects glucose metabolism in pancreatic beta-cells. We have aimed at clarifying the physiological role of endogenous NO and its target in the glucose metabolism of beta-cells. The expression of brain-type NO synthase (bNOS) was detected in pancreatic islets by Western blotting. Under the condition of elevated intracellular Ca2+ concentration induced in the beta-cells by high glucose and forced depolarization by 40 mM K+, the generation of NO from the islets was enhanced. This increase was suppressed by the NOS blockers, N-iminoethyl-l-ornithine (L-NIO), and exposure to Ca2+-free extracellular solution. In addition, the NOS blockers L-NIO and 7-nitro indazole (7-NI) enhanced glucose-induced but not glyceraldehyde- or KIC-induced insulin secretion. In an in vitro enzyme study, the NO donor sodium nitroprusside (SNP) suppressed phosphofructokinase activity and activated glucokinase and glucose-6-phosphate isomerase activity, but SNP significantly inhibited the combined activity of the enzymes. This suggests that endogenous NO has an inhibitory role on insulin release induced by glucose and that its underlying mechanism is the suppression of phosphofructokinase activity in glycolysis.

Ouabain suppresses ATP elevation in response to fuel secretagogues in pancreatic islets.

The alterations of the ATP concentration in response to fuel secretagogues such as glucose, glyceraldehyde, and ketoisocaproate (KIC) were investigated in a single islet. The intraislet ATP concentration was transiently elevated and then decreased to a level slightly higher than basal. To asses the ATP content under conditions of reduced ATP consumption, the Na-K pump blocker ouabain was used. The elevation of ATP concentration was found unexpectedly to be suppressed under ouabain in the islet, even when incubated with any of the secretagogues. High glucose did not elevate the intracellular creatine phosphate during incubation with ouabain. Since the suppression rate for the intraislet ATP elevation was considerably smaller with KIC than with glyceraldehyde and glucose, we conclude that ouabain inhibits ATP production, at least in part, in the glycolytic pathway through a feedback mechanism.

Necessity of endogenous GTP derived from glucose-6-phosphate for insulin secretion augmented by glucose under protein kinase A activation.

To investigate the possible involvement of some intracellular metabolic signaling other than the ATP derived from glucose metabolism under protein kinase A (PKA) activation, we measured the insulin secretory capacity stimulated by glucose and other fuel secretagogues using diazoxide-treated pancreatic islets. Under these conditions, we found a signal from a site proximal to glyceraldehyde-3-phosphate (GA-3-P) in the glycolysis to be necessary for glucose-induced insulin secretion. By using several different glycolytic enzyme inhibitors, we found that this proximal signal is derived from glucose-6-phosphate (G-6-P), and that metabolic signaling distal to GA-3-P also is necessary. Mycophenolic acid completely inhibited the augmented glucose-induced insulin secretion, which guanosine could reverse, indicating that the proximal signaling is coupling with endogenous GTP production. In this novel system of metabolic signaling, endogenous GTP derived from G-6-P in the glycolysis elicits the augmentation of glucose-induced insulin secretion under PKA activation in diazoxide-treated pancreatic islets.

Metabolic inhibition impairs ATP-sensitive K+ channel block by sulfonylurea in pancreatic beta-cells.

The effect of metabolic inhibition on the blocking of beta-cell ATP-sensitive K+ channels (KATP channels) by glibenclamide was investigated using a patch-clamp technique. Inhibition of KATP channels by glibenclamide was attenuated in the cell-attached mode under metabolic inhibition induced by 2,4-dinitrophenol. Under a low concentration (0.1 microM) of ATP applied in the inside-out mode, KATP channel activity was not fully abolished, even when a high dose of glibenclamide was applied, in contrast to the dose-dependent and complete KATP channel inhibition under 10 microM ATP. On the other hand, cibenzoline, a class Ia antiarrhythmic agent, inhibits KATP channel activity in a dose-dependent manner and completely blocks it, even under metabolic inhibition. In sulfonylurea receptor (SUR1)- and inward rectifier K+ channel (Kir6.2)-expressed proteins, cibenzoline binds directly to Kir6.2, unlike glibenclamide. Thus, KATP channel inhibition by glibenclamide is impaired under the condition of decreased intracellular ATP in pancreatic beta-cells, probably because of a defect in signal transmission between SUR1 and Kir6.2 downstream of the site of sulfonylurea binding to SUR1.

Insulin secretion and its modulation by antiarrhythmic and sulfonylurea drugs.

Cardiovascular drugs such as antiarrhythmic agents with Vaughan Williams class Ia action have been found to induce a sporadic hypoglycemia. Recent investigation has revealed that these drugs induce insulin secretion from pancreatic beta-cells by inhibiting ATP-sensitive K+ (KATP) channels in a manner similar to sulfonylurea drugs. The mechanism underlying block of KATP channels by antiarrhythmic drugs was different, however, from that of sulfonylureas: firstly, because binding of radioactive glibenclamide could not be inhibited by unlabelled antiarrhythmic agents, and vice versa; secondly, because the two compounds differ in the kinetics and sidedness of drug action-antiarrhythmic drugs act on the channel from the inner surface of the cell membrane, whereas glibenclamide binds through the intramembrane pathway; finally, it was shown that functional KATP channels in beta-cells are composed of two distinct molecules-a sulfonylurea receptor (SUR) and a channel pore-forming subunit, an inwardly-rectifying K channel with two transmembrane regions (Kir6.2). Antiarrhythmic drugs reversibly inhibit the K+ conductance displayed by the Kir6.1 (a putative KATP channel clone)-transfected NIH3T3 cells. Therefore they appear to interact directly with the pore-forming subunit, thereby inhibiting KATP channel currents and exerting an insulinotrophic effect.

Alterations in basal and glucose-stimulated voltage-dependent Ca2+ channel activities in pancreatic beta cells of non-insulin-dependent diabetes mellitus GK rats.

In genetically occurring non-insulin-dependent diabetes mellitus (NIDDM) model rats (GK rats), the activities of L- and T-type Ca2+ channels in pancreatic beta cells are found to be augmented, by measuring the Ba2+ currents via these channels using whole-cell patch-clamp technique, while the patterns of the current-voltage curves are indistinguishable. The hyper-responsiveness of insulin secretion to nonglucose depolarizing stimuli observed in NIDDM beta cells could be the result, therefore, of increased voltage-dependent Ca2+ channel activity. Perforated patch-clamp recordings reveal that the augmentation of L-type Ca2+ channel activity by glucose is markedly less pronounced in GK beta cells than in control beta cells, while glucose-induced augmentation of T-type Ca2+ channel activity is observed neither in the control nor in the GK beta cells. This lack of glucose-induced augmentation of L-type Ca2+ channel activity in GK beta cells might be causatively related to the selective impairment of glucose-induced insulin secretion in NIDDM beta cells, in conjunction with an insufficient plasma membrane depolarization due to impaired closure of the ATP-sensitive K+ channels caused by the disturbed intracellular glucose metabolism in NIDDM beta cells.

Block of pancreatic ATP-sensitive K+ channels and insulinotrophic action by the antiarrhythmic agent, cibenzoline.

1. We investigated the effect of cibenzoline (a class Ia antiarrhythmic drug) on basal insulin secretory activity of rat pancreatic islets and ATP-sensitive K+ channels (KATP) in single pancreatic beta cells of the same species, using radioimmunoassay and patch clamp techniques. 2. Micromolar cibenzoline had a dose-dependent insulinotrophic action with an EC50 of 94.2 +/- 46.4 microM. The compound inhibited the activity of the KATP channel recorded from a single beta-cell in a concentration-dependent manner. The IC50 was 0.4 microM in the inside-out mode and 5.2 microM in the cell-attached mode, at pH 7.4. 3. In the cell-attached mode, alkalinization of extracellular solution increased the inhibitory action of cibenzoline and the IC50 was reduced from 26.8 microM at pH 6.2 to 0.9 microM at pH 8.4. On the other hand, the action of cibenzoline in the excised inside-out mode was acute in onset with a small IC50, indicating that the drug attains its binding site from the cytoplasmic side of the cell membrane. 4. In the inside-out mode, micromolar ADP reactivated the cibenzoline-blocked KATP channels in a manner similar to that by which ADP restored ATP-dependent block of the channel. 5. The binding of [3H]-glibenclamide to pancreatic islets was inhibited by glibenclamide but not by cibenzoline. In contrast, the [3H]-cibenzoline binding was displaced by unlabelled cibenzoline but not by glibenclamide. It is concluded that cibenzoline blocks pancreatic KATP channels via a binding site distinct from the sulphonylurea receptor.