Biomedical subjects

Kenju Shimomura

Publications and source records attributed to Kenju Shimomura.

10 recordsLinked to original sources

PubMed2006

Functional analysis of six Kir6.2 (KCNJ11) mutations causing neonatal diabetes.

ATP-sensitive potassium (K(ATP)) channels, composed of pore-forming Kir6.2 and regulatory sulphonylurea receptor (SUR) subunits, play an essential role in insulin secretion from pancreatic beta cells. Binding of ATP to Kir6.2 inhibits, whereas interaction of Mg-nucleotides with SUR, activates the channel. Heterozygous activating mutations in Kir6.2 (KCNJ11) are a common cause of neonatal diabetes (ND). We assessed the functional effects of six novel Kir6.2 mutations associated with ND: H46Y, N48D, E227K, E229K, E292G, and V252A. K(ATP) channels were expressed in Xenopus oocytes and the heterozygous state was simulated by coexpression of wild-type and mutant Kir6.2 with SUR1 (the beta cell type of SUR). All mutations reduced the sensitivity of the K(ATP) channel to inhibition by MgATP, and enhanced whole-cell K(ATP) currents. Two mutations (E227K, E229K) also enhanced the intrinsic open probability of the channel, thereby indirectly reducing the channel ATP sensitivity. The other four mutations lie close to the predicted ATP-binding site and thus may affect ATP binding. In pancreatic beta cells, an increase in the K(ATP) current is expected to reduce insulin secretion and thereby cause diabetes. None of the mutations substantially affected the sensitivity of the channel to inhibition by the sulphonylurea tolbutamide, suggesting patients carrying these mutations may respond to these drugs.

ATP-Binding Cassette Transporters↗

PubMed2006

Nicotinamide nucleotide transhydrogenase: a key role in insulin secretion.

The C57BL/6J mouse displays glucose intolerance and reduced insulin secretion. QTL mapping identified Nicotinamide Nucleotide Transhydrogenase (Nnt), a nuclear-encoded mitochondrial protein thought to be involved in free radical detoxification, as a candidate gene. To investigate its functional role, we used siRNA to knock down Nnt in insulin-secreting MIN6 cells. This produced a dramatic reduction in insulin secretion and the rise in [Ca2+]i evoked by glucose, but not tolbutamide. We identified two ENU-induced point mutations in Nnt (N68K, G745D). Nnt mutant mice were glucose intolerant and secreted less insulin during a glucose tolerance test. Isolated islets showed impaired insulin secretion in response to glucose, but not to tolbutamide, and glucose failed to enhance ATP levels. Glucose utilization and production of reactive oxygen species were increased in Nnt beta cells. We hypothesize that Nnt mutations/deletion uncouple beta cell mitochondrial metabolism leading to less ATP production, enhanced KATP channel activity, and consequently impaired insulin secretion.

Adenosine Triphosphate↗

PubMed2006

Mutations at the same residue (R50) of Kir6.2 (KCNJ11) that cause neonatal diabetes produce different functional effects.

Heterozygous mutations in the human Kir6.2 gene (KCNJ11), the pore-forming subunit of the ATP-sensitive K(+) channel (K(ATP) channel), are a common cause of neonatal diabetes. We identified a novel KCNJ11 mutation, R50Q, that causes permanent neonatal diabetes (PNDM) without neurological problems. We investigated the functional effects this mutation and another at the same residue (R50P) that led to PNDM in association with developmental delay. Wild-type or mutant Kir6.2/SUR1 channels were examined by heterologous expression in Xenopus oocytes. Both mutations increased resting whole-cell currents through homomeric and heterozygous K(ATP) channels by reducing channel inhibition by ATP, an effect that was larger in the presence of Mg(2+). However the magnitude of the reduction in ATP sensitivity (and the increase in the whole-cell current) was substantially larger for the R50P mutation. This is consistent with the more severe phenotype. Single-R50P channel kinetics (in the absence of ATP) did not differ from wild type, indicating that the mutation primarily affects ATP binding and/or transduction. This supports the idea that R50 lies in the ATP-binding site of Kir6.2. The sulfonylurea tolbutamide blocked heterozygous R50Q (89%) and R50P (84%) channels only slightly less than wild-type channels (98%), suggesting that sulfonylurea therapy may be of benefit for patients with either mutation.

Adenosine Triphosphate↗

PubMed2005

3-D structural and functional characterization of the purified KATP channel complex Kir6.2-SUR1.

ATP-sensitive potassium (K(ATP)) channels conduct potassium ions across cell membranes and thereby couple cellular energy metabolism to membrane electrical activity. Here, we report the heterologous expression and purification of a functionally active K(ATP) channel complex composed of pore-forming Kir6.2 and regulatory SUR1 subunits, and determination of its structure at 18 A resolution by single-particle electron microscopy. The purified channel shows ATP-ase activity similar to that of ATP-binding cassette proteins related to SUR1, and supports Rb(+) fluxes when reconstituted into liposomes. It has a compact structure, with four SUR1 subunits embracing a central Kir6.2 tetramer in both transmembrane and cytosolic domains. A cleft between adjacent SUR1s provides a route by which ATP may access its binding site on Kir6.2. The nucleotide-binding domains of adjacent SUR1 appear to interact, and form a large docking platform for cytosolic proteins. The structure, in combination with molecular modelling, suggests how SUR1 interacts with Kir6.2.

ATP-Binding Cassette Transporters↗

PubMed2004

Fenofibrate, troglitazone, and 15-deoxy-Delta12,14-prostaglandin J2 close KATP channels and induce insulin secretion.

It is known that peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands stimulate acute-phase insulin secretion with a rapid Ca2+ influx into pancreatic beta-cells, but the precise mechanisms are not clear. The effects of PPAR-alpha ligands on pancreatic beta-cells also remain unclear. We investigated the effects of PPAR-alpha ligands (fenofibrate and fenofibric acid), a PPAR-gamma ligand (troglitazone), and an endogenous ligand of PPAR-gamma [15-deoxy-Delta12,14-prostaglandin J2 (15-deoxy-Delta12,14-PGJ2)] on KATP channel activity in clonal hamster insulinoma cell line, HIT-T15 cells. As assessed by whole-cell patch clamp, fenofibrate, fenofibric acid, troglitazone, and 15-deoxy-Delta12,14-PGJ2 reduced the KATP channel currents, and inhibition continued after washout of these agents. The concentration-response curves of fenofibrate, fenofibric acid, troglitazone, and 15-deoxy-Delta12,14-PGJ2 showed half-maximal inhibition of KATP channel currents (IC50) at 3.26, 94, 2.1, and 7.3 micromol/l, respectively. Fenofibrate (> or = 10(-6) mol/l), 15-deoxy-Delta12,14-PGJ2 (> or = 5 x 10(-5) mol/l), and troglitazone (> or = 10(-6) mol/l) inhibited [3H]glibenclamide binding, but fenofibric acid did not. In addition, fenofibrate (> or = 10(-6) mol/l), fenofibric acid (10(-4) mol/l), troglitazone (10(-4) mol/l), and 15-deoxy-Delta12,14-PGJ2 (> or = 10(-5) mol/l) increased insulin secretion from HIT-T15 when applied for 10 min. Our data suggest that PPAR-alpha and -gamma ligands interact directly with the beta-cell membrane and stimulate insulin secretion.

Animals↗

PubMed2003

Syntaxin 4 and Synip (syntaxin 4 interacting protein) regulate insulin secretion in the pancreatic beta HC-9 cell.

Although syntaxin 1 is generally thought to function as the primary target-N-ethylmaleimide-sensitive factor attachment protein receptor required for pancreatic beta cell insulin secretion, we have observed that overexpression of a dominant-interfering syntaxin 4 mutant (syntaxin 4/DeltaTM) attenuated glucose-stimulated insulin secretion in betaHC-9 cells. Furthermore, these cells express the selective syntaxin 4-binding protein Synip (syntaxin 4 interacting protein), and Synip was specifically co-immunoprecipitated with syntaxin 4 but not syntaxin 1. Overexpression of the full-length Synip protein (Synip/wild type) inhibited VAMP2 association with syntaxin 4 and decreased glucose-stimulated insulin secretion. This did not occur with a Synip mutant (Synip/ DeltaEF) that was incapable of binding syntaxin 4. Consistent with a functional role of syntaxin 4 in this process, expression of syntaxin 4/DeltaTM also inhibited glucose-stimulated insulin secretion. Furthermore, analysis of first and second phase insulin secretion demonstrated that syntaxin 4/DeltaTM mainly suppressed the second phase of insulin secretion. In contrast, overexpression of Synip resulted in an inhibition of both the first and second phase of glucose-stimulated insulin secretion. These data demonstrate that syntaxin 4 plays a functional role on insulin release and granule fusion in beta cells and that this process is regulated by the syntaxin 4-specific binding protein Synip.

Antigens, Surface↗

PubMed2003

Troglitazone inhibits isolated cell proliferation, and induces apoptosis in isolated rat mesangial cells.

BACKGROUND/AIMS: Troglitazone is one of thiazolidinedione derivatives as a high affinity ligand for peroxisome proliferator-activated receptor-gamma (PPAR-gamma). The in vivo studies demonstrated that troglitazone ameliorated microalbuminuria. There have been few reports about direct effect of thiazolidinedione derivatives on mesangial cell function. We determined the effect of troglitazone on isolated rat mesangial cell proliferation. METHODS: We determined PPAR-gamma mRNA expression in isolated rat mesangial cells. Chronic effects of 10(-6) to 10(-4) mol/l troglitazone on mesangial cell proliferation and mitogen-activated protein (MAP) kinase activity were also determined. The effects of troglitazone on apoptosis were investigated in rat mesangial cells. RESULTS: Rat PPAR-gamma mRNA was detected in isolated rat mesangial cells. Living cell number, assessed by colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) assay, was significantly decreased with 10(-4) mol/l troglitazone. The addition of 10(-6) to 10(-4) mol/l troglitazone dose-dependently inhibited 5-bromo-2'-deoxyuridine (BrdU) uptake into isolated rat mesangial cells. The addition of 10(-4) and 10(-5) mol/l troglitazone significantly reduced MAP kinase activity. Troglitazone at the concentrations of 10(-6) to 10(-4) mol/l dose-dependently increased DNA fragmentation rates, indicating that troglitazone may cause apoptosis in rat mesangial cells. Bax and Bcl-xL proteins were not changed, although Bcl-2 proteins increased with troglitazone. CONCLUSIONS: The present data demonstrated that troglitazone inhibits cell proliferation, and induces apoptosis in rat mesangial cells, raising a possibility that it directly affects renal function.

Animals↗

PubMed2003

Relationship between hyperinsulinemia and pulse wave velocity in moderately hyperglycemic patients.

AIM: Arterial stiffness assessed by pulse wave velocity (PWV) reflects early stage arteriosclerosis. The influence of hyperinsulinemia on peripheral vascular disease (PVD) is still unknown. We determined the influences of hyperinsulinemia on PVD assessed by PWV in moderately hyperglycemic patients. METHODS: Thirty-six moderately hyperglycemic, outcoming patients were recruited in this study. All subjects were divided into two groups by fasting immunoreactive insulin (F-IRI) concentrations; group A; F-IRI> or =5 microU/ml, group B; F-IRI<5 microU/ml. Both hbPWV (from heart to brachial artery) and baPWV (brachial to artery to ankle) were evaluated by using Form PWV/ABI, in addition to ankle-brachial pressure index (ABPI). RESULTS: In group A, both hbPWV and baPWV showed significantly higher values than in group B. ABPIs were not different between two groups. Although age, FPG, plasma HbA1c, serum total-cholesterol, HDL-cholesterol concentrations, and systolic and diastolic blood pressure were at same levels in group A as group B, body mass index, HOMA-R, serum triglyceride concentrations were significantly higher in group A, indicating the existence of insulin resistance in group A. CONCLUSION: Hyperinsulinemia may be involved in the development of PVD in moderately hyperglycemic patients.

Adult↗

PubMed2002

Circulating concentrations of soluble leptin receptor: influence of menstrual cycle and diet therapy.

Concentrations of the soluble leptin receptor (sOB-R) may be related to leptin resistance in obesity. We measured sOB-R concentrations in serum in 103 non-diabetic Japanese men and women. All subjects were grouped according to body mass index (BMI; in kg/m(2)). Serum sOB-R concentrations did not differ significantly between normal-weight (18.5 < or = BMI < 25.0) men and women, but were significantly higher in underweight subjects (BMI < 18.5) than in normal-weight subjects. In contrast, overweight (25 < or = BMI < 30) and obese (30 < or = BMI < 35.0, 35.0 < or = BMI < 40, and BMI > or = 40) subjects had significantly lower sOB-R concentrations than did normal-weight subjects. Serum sOB-R concentrations were inversely correlated with BMI and serum immunoreactive leptin concentrations. Very low-energy diet therapy for 4 wk significantly lowered serum immunoreactive leptin concentrations but did not significantly affect serum sOB-R concentrations. Serum sOB-R concentrations did not change significantly during the menstrual cycle. Our results showed that serum sOB-R concentrations decrease with increasing BMI and that sex hormones likely do not affect serum sOB-R concentrations in non-pregnant women. The reduction in serum sOB-R concentrations in overweight and obese persons may reflect downregulation of hypothalamic leptin receptor production as a result of an increase in circulating leptin and might be an important factor in leptin resistance.

Adult↗

PubMed2002

Is leptin a key factor which develops obesity by ovariectomy?

Withdrawal of estrogen by ovariectomy increases adiposity, but decreases the circulating levels of the ob gene product, leptin, which inhibits food intake. The reduction of circulating leptin levels may thus play an important role in the induction of obesity by ovariectomy. To examine this hypothesis, body weight change by ovariectomy was investigated in leptin-deficient genetically obese (ob/ob) mice with leptin supplement. Prior to the operation, obese (ob/ob) female mice were treated with intraperitoneal administration of recombinant mouse leptin (1.0 microg/g body weight/day) for 8 days. Then, half of the leptin-treated mice and their lean littermates were bilaterally ovariectomized and their body weight changes were observed for 56 days. From 16 days after the operation, a significant increase in body weight by ovariectomy was observed only in lean mice without leptin treatment. From 44 days, a significant body weight gain by ovariectomy was observed in leptin-treated obese mice. Ovariectomy significantly increased retroperitoneal white adipose tissue weight in their lean littermates, but not in leptin-treated obese mice. It was suggested that the reduction of circulating leptin levels may play an important role in the increases of acute phase body weight gain by ovariectomy, but during static phase, the direct effects of estrogen withdrawal may appear independent of leptin-mediated effects.

Adipose Tissue↗