Mitsuhiro Nakazaki

Ischemic preconditioning in the hippocampus of a knockout mouse lacking SUR1-based KATP channels

Background and Purpose— ATP-sensitive K+ (KATP) channels have been implicated in the mechanism of neuronal ischemic preconditioning. To evaluate the role of neuronal/&bgr;–cell-type KATP channels, SUR1 null (Sur1KO) mice lacking (KIR6.x/SUR1)4 KATP channels were subjected to a preconditioning protocol with the use of double carotid occlusion. Methods— Wild-type C57BL/6 and Sur1KO mice were subjected to a double carotid block for 40 minutes with or without a 20-minute preconditioning block. After a 10-day reperfusion period, damage was assessed histologically in the hippocampal CA1, CA2, and CA3 areas and in the dentate gyrus. The neuroprotective effects of intracerebroventricular injections of diazoxide, which selectively affects mitochondria versus opening SUR1-type KATP channels, and 5-hydroxydecanoate, a selective blocker of mitoKATP channels, were evaluated with the same protocol. Results— Neurons in the CA1 region of both Sur1KO and wild-type animals subjected to a 20-minute ischemic insult were protected equally from neuronal damage produced by a subsequent 40-minute ischemic period. Pretreatment with diazoxide protected both Sur1KO and wild-type neurons, while 5-hydroxydecanoate augmented neurodegeneration in both strains of animals when administered before a 20-minute bout of ischemia. Conclusions— SUR1-based KATP channels are not obligatory for neuronal preconditioning or augmentation of neurodegeneration by 5-hydroxydecanoate.

Involvement of ATP-Sensitive K+ Channels in Free Radical–Mediated Inhibition of Insulin Secretion in Rat Pancreatic β-Cells

To explore the mechanisms of inhibition of insulin secretion in pancreatic β-cells by oxygen free radicals, we studied the effects of H2O2 on membrane currents using the patch-clamp technique. Exposure of β-cells to H2O2 (≥30 (μmol/1) increased the activity of ATP-sensitive potassium (K+ATP) channels without changing the single channel conductance in cell-attached membrane patches. Action currents observed during superfusion of 11.1 mmol/1 glucose were suppressed. In inside-out membrane patches, the activity of K+ATP channels was not influenced by H2O2. In conventional whole-cell clamp experiments using a pipette solution containing 3 mmol/1 ATP, H2O2 did not influence the membrane currents. However, H2O2 did activate the K+ATP channel current in perforated whole-cell clamp configurations. The increased K+ATP channel current was reversed by subsequent exposure to 11.1 mmol/12-ketoisocaproic acid. In cell-attached membrane patches, the K+ATP channel current evoked by exposure to 30 μmol/l H2O2 was inhibited by exposure to 11.1 mmol/l glyceraldehyde, whereas the channel was again activated by exposure to 0.3 mmol/l H2O2. Subsequent superfusion of 11.1 mmol/l 2-ketoisocaproic acid inhibited the channel; this effect was counteracted by exposure to 10 mmol/l H2O2. Transient inhibition of K+ATP channels with provocation of action potentials was observed after washout of 100 μmol/l H2O2 during superfusion of 2.8 or 11.1 mmol/l glucose. We conclude that H2O2 has no direct effect on the K+ATP channels but that it indirectly activates the channels when it is exposed to β-cells under conditions in which the cellular metabolism is physiologically regulated.

Inhibition of the ATP‐sensitive potassium channel by class I antiarrhythmic agent, cibenzoline, in rat pancreatic β‐cells

1 Cibenzoline, a class I antiarrhythmic agent, was investigated for its effect on the ATP‐sensitive K+ channel of pancreatic β‐cells by the patch clamp technique. 2 In perforated patch clamp experiments, cibenzoline depolarized the membrane of single β‐cells and thereafter, caused firing of action potentials in the presence of 2.8 mm glucose. 3 Cibenzoline inhibited the activity of the ATP‐sensitive K+ channel in cell‐attached recordings in the presence of 2.8 mm glucose and evoked repetitive fluctuations of the baseline current, apparently reflecting the action potentials of the β‐cell. 4 In whole‐cell clamp experiments, time‐independent outward current was induced by depleting cytoplasmic ATP with 0.1 mm ATP and 0.1 mm ADP in the solution contained in the pipette. The outward current was inhibited by cibenzoline in a dose‐dependent manner in the concentration range of 1 μm to 100 μm and half maximum inhibition occurred at 1.5 μm. 5 Cibenzoline blocked substantially the ATP‐sensitive K+ channel current when applied at the inner side of the membrane in isolated inside‐out membrane patches. 6 It is concluded that cibenzoline blocks the ATP‐sensitive K+ channel of pancreatic β‐cells and, thereby, stimulates insulin secretion at sub‐stimulatory levels of glucose.

Repetitive mitochondrial Ca2+ signals synchronize with cytosolic Ca2+ oscillations in the pancreatic beta-cell line, MIN6

Summary We examined the relationship between cytosolic Ca2+ concentration ([Ca2+]c) and mitochondrial matrix Ca2+ concentration ([Ca2+]m) in the pancreatic beta-cell line, MIN6. [Ca2+]c was monitored in a single or a group (30 cells) of fura-2-loaded MIN6 cells, and [Ca2+]m was measured in a group (1 × 106 cells) of MIN6 cells stably transfected with aequorin targeted at the mitochondria. Exogenous ATP (0.25 mmol/l) produced a single transient increase in [Ca2+]c whereas 22 mmol/l KCl produced a sustained plateau increase. ATP and KCl evoked transient increases in [Ca2+]m but with distinct time courses of [Ca2+]m decline: the [Ca2+]m increase induced by ATP decreased more rapidly than that induced by KCl. Nitrendipine (3 μmol/l), a blocker of L-type Ca2+ channels, inhibited both [Ca2+]c and [Ca2+]m signals in response to KCl and tolbutamide, but not those to ATP. Peak levels of [Ca2+]m increase (around 2 μmol/l) exceeded those of [Ca2+]c increase (around 500 nmol/l). A rise in glucose concentration from 3 to 30 mmol/l induced oscillations of [Ca2+]c that overlay the sustained increases in [Ca2+]c in single cells. An oscillatory increase in [Ca2+]m was similarly observed in response to glucose. Addition of 10 mmol/l 2-ketoisocaproic acid at 20 mmol/l glucose further increased the plateau level of [Ca2+]c and the frequency of [Ca2+]c oscillations, which were correlated with a further increase in [Ca2+]m. In response to pulsatile exposure to KCl, [Ca2+]c and [Ca2+]m increased synchronously. These data suggest that an oscillatory increase in [Ca2+]m in beta cells, the signal which is thought to be necessary for continuous stimulation of mitochondrial metabolism, is produced synchronously with the [Ca2+]c oscillations. [Diabetologia (1998) 41: 279–286]

Cyclic AMP/cAMP‐GEF pathway amplifies insulin exocytosis induced by Ca2+ and ATP in rat islet β‐cells

Cyclic AMP (cAMP) plays a pivotal role in insulin secretion induced by incretins. The effects of the second messenger extend to many sites and there has been much controversy on the mechanisms. The aim of this study was to examine how cAMP amplified insulin exocytosis.

Effect of Mitochondrial and/or Cytosolic Glycerol 3-Phosphate Dehydrogenase Overexpression on Glucose-Stimulated Insulin Secretion From MIN6 and HIT Cells

The glycerol phosphate shuttle consists of FAD-linked mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) and its cytosolic NAD-linked isoform (cGPDH). Impaired mGPDH activity has recently been suggested to be one of the primary causes of insulin secretory defects in β-cells. We found that mGPDH and cGPDH activities in MIN6 cells are comparable to those of isolated islets and higher than those in HIT cells by eightfold and threefold, respectively. Therefore, we selected the MIN6 cell line as a β-cell model with normally regulated insulin secretion and normal shuttle enzyme activities and the HIT cell line as a β-cell model with impaired insulin secretion and lower activities of these enzymes. The role of these dehydrogenases in glucose-stimulated insulin secretion was addressed by examining the effects of overexpression of mGPDH and/or cGPDH via recombinant adenoviruses in these cells. Infection with recombinant adenovirus with a cDNA encoding the Escherichia coli β-galactosidase gene resulted in expression of its gene in 90% of MIN6 and HIT cells. Infection with a recombinant adenovirus with mGPDH cDNA (AdexlCAmGPDH) caused 2.1- fold and 5.7-fold increases in dehydrogenase activity as compared with those of control MIN6 and HIT cells, respectively. Infection with a recombinant adenovirus with cGPDil cDNA (AdexlCAcGPDH) caused a more than 50-fold increase in activity in both cell lines. Glycerol phosphate shuttle flux, as estimated by [2-3H]glycerol conversion to [3H]H2O, was increased to 120–130% by infection with AdexlCAmGPDH, but not with AdexlCAcGPDH infection, in both MIN6 and HIT cells. No further increase in flux through the glycerol phosphate shuttle was detected when the cells were infected with AdexlCAmGPDH together with AdexlCAcGPDH. Furthermore, neither [U-14C]glucose oxidation nor the insulin secretory response to glucose was affected in either cell line. Thus, mGPDH abundance in MIN6 and HIT cells is not directly related to their insulin secretory capacity in response to glucose, and reduced expression of mGPDH is not the primary cause of abnormal insulin secretory responses in HIT cells. The present data indicate that the emerging hypothesis pointing to mGPDH deficiency as a possible cause of NIDDM needs to be carefully evaluated.

Genome-wide linkage analysis of type 2 diabetes mellitus reconfirms the susceptibility locus on 11p13–p12 in Japanese

AbstractType 2 diabetes mellitus is a heterogeneous disorder, and the development of type 2 diabetes mellitus is associated with both insulin secretion defect and insulin resistance. The primary metabolic defect leading to type 2 diabetes mellitus has been thought to be varied among populations, especially in Japanese and Caucasians. Here, we have done the genome-wide scan for type 2 diabetes mellitus using 102 affected Japanese sib-pairs to identify the genetic factors predisposing to type 2 diabetes mellitus. Nonparametric linkage analysis showed one suggestive evidence for linkage to 11p13-p12 [D11S905: two-point maximum LOD score (MLS) of 2.89 and multipoint MLS of 2.32] and one nominally significant evidence for linkage to 6q15-q16 (D6S462: two-point MLS of 2.02). Interestingly, the 11p13-p12 region was reported to be a susceptibility locus for Japanese type 2 diabetes mellitus with suggestive evidence of linkage, and D11S905 was within 5 cM to D11S935 with the highest MLS in the previous linkage analysis reported. The only overlapped susceptibility region with suggestive evidence of linkage for Japanese type 2 diabetes mellitus was D11S935-D11S905 among the three reports including this study. These results taken together suggest that a susceptibility gene for type 2 diabetes mellitus in Japanese will reside in 11p13-p12.

Characterization of the effect of serum bilirubin concentrations on coronary endothelial function via measurement of high-sensitivity C-reactive protein and high-density lipoprotein cholesterol

Bilirubin can prevent oxidation of low-density lipoprotein (LDL) and may protect against atherosclerosis and coronary heart disease (CHD). The goal of this study was to characterize the relationship between bilirubin and CHD through measurements of bilirubin concentration, coronary endothelial function, and markers of oxidative stress, inflammation, and lipid/glucose metabolism. The study population consisted of 141 patients without CHD who underwent Doppler flow study. Vascular reactivity was examined by intracoronary administration of papaverine, acetylcholine (ACh) and nitroglycerin using a Doppler guide wire. Serum bilirubin, high-sensitivity C-reactive protein (hsCRP), malondialdehyde-modified LDL, LDL cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), fasting plasma glucose (FPG), and immunoreactive insulin were also measured. Homeostasis model assessment insulin resistance index and estimated glomerular filtration rate (eGFR) were calculated. Univariate analysis revealed that both percent change in coronary blood flow (CBF) and coronary artery diameter induced by ACh correlated positively with log-transformed bilirubin (r = 0.22, P < 0.05; r = 0.20, P < 0.05, respectively). Percent change in CBF in response to ACh correlated positively with eGFR (r = 0.24, P < 0.05) and correlated inversely with age, LDL-C, and log-transformed FPG (r = −0.24, P < 0.05; r = −0.17, P < 0.05, r = −0.22, P < 0.05, respectively). Multivariate analysis revealed that log-transformed bilirubin was the only independent predictor of percent change in CBF in response to ACh. Multivariate analysis revealed that log-transformed hsCRP and HDL-C were independent predictors of log-transformed bilirubin. These results suggest that a high level of bilirubin is associated with favorable coronary endothelial function, which may be mediated via the effect of bilirubin on inflammation and HDL-C.

Successful treatment of Good syndrome with cytomegalovirus duodenoenteritis using a combination of ganciclovir and immunoglobulin with high anti-cytomegalovirus antibody titer

We describe the case of a 64-year-old woman with Good syndrome who presented with watery diarrhea and abdominal distention caused by cytomegalovirus (CMV) duodenoenteritis. Thymoma and hypogammaglobulinemia were first identified when the patient was 58 years old. She had repeatedly complained of symptoms even after thymectomy. Abdominal radiography revealed multiple air-fluid levels, and computed tomography revealed ascites and dilation of the small intestine. Immunofluorescent staining of specimens obtained by duodenal mucosal biopsy revealed intracellular inclusion bodies of CMV, although serum CMV pp65 antigenemia assays yielded negative results. CMV infection of the small intestine caused mucosal edema resulting in malabsorption. The patient was treated using ganciclovir and an immunoglobulin preparation with a high titer of antibodies against CMV (CMV-Ig), and subsequently made a rapid recovery from abdominal symptoms. When patients with Good syndrome complain of abdominal symptoms, particularly chronic diarrhea, a diagnosis of CMV gastroenteritis should not be excluded, even if negative results are obtained for CMV pp65 antigenemia assays. Combination therapy of ganciclovir and CMV-Ig seems useful for patients with CMV gastroenteritis.

Association of upregulated activity of KATP channels with impaired insulin secretion in UCP1-expressing insulinoma cells

Insulin‐secreting MIN6 cells overexpressing uncoupling protein‐1 (UCP1) were studied regarding insulin secretion in response to various secretagogues. Overexpression of UCP1 prevented an increase of cytosolic ATP levels induced by glucose. In contrast, glucose utilization was not affected, nor was glycerol phosphate flux. The UCP1‐expressing cells showed an inability to increase cytosolic Ca2+ concentration ([Ca2+]i) in response to glucose or α ketoisocaproate and this resulted in less insulin secretion, whereas initial reduction in [Ca2+]i occurring upon either nutrient addition was not affected. Moreover, the effectiveness of tolbutamide on [Ca2+]i increase was reduced and the dose‐response relations for insulin secretion induced by the agent was shifted toward the right in the UCP1‐expressing cells. The resting membrane potential of the UCP1‐expressing cells was significantly hyperpolarized by 6.2 mV compared with control cells. In the perforated and conventional whole‐cell patch‐clamp configurations, the conductance density of ATP‐sensitive K+ (KATP) channels of the UCP1‐expressing cells was 6‐fold and 1.7‐fold greater than that of the control cells, respectively. The sensitivity of KATP channels for tolbutamide was not different between two groups, indicating that in intact cells more than 6‐fold higher concentrations of tolbutamide were required to reduce the KATP channel currents of UCP1‐expressing cells to the same levels as of the control cells. The current density of the voltage‐dependent Ca2+ channels was not influenced. In conclusion, UCP1‐expressing cells showed a refractoriness to respond to tolbutamide as well as nutrients. An upregulated activity of KATP channels was associated with unresponsiveness to the agent in the cells with impaired mitochondrial function.