Ghrelin uses Galphai2 and activates voltage-dependent K+ channels to attenuate glucose-induced Ca2+ signaling and insulin release in islet beta-cells: novel signal transduction of ghrelin

Ghrelin reportedly serves as a physiological regulator of insulin release. This study aimed to explore signaling mechanisms for insulinostatic ghrelin action in islet beta-cells, with special attention to heterotrimeric GTP-binding proteins and K(+) channels. Plasma insulin and growth hormone (GH) concentrations in rats were measured by enzyme-linked immunosorbent assay (ELISA). Islets were isolated from rats, ghrelin-knockout (Ghr-KO) mice, and wild-type mice by collagenase digestion, and insulin release was determined by ELISA. In rat single beta-cells, cytosolic Ca(2+) concentration ([Ca(2+)](i)) was measured by fura-2 microfluorometry, and membrane potentials and whole cell currents by patch-clamp technique. In rats, systemic ghrelin administration decreased plasma insulin concentrations, and this effect was blocked by treatment with pertussis toxin (PTX), whereas stimulation of GH release remained unaffected. In rat islets, ghrelin receptor antagonist increased and exogenous ghrelin suppressed glucose-induced insulin release in a PTX-sensitive manner. Glucose-induced insulin release from islets was greater in Ghr-KO than wild-type mice, and this enhanced secretion was blunted with PTX. Ghrelin PTX sensitively increased voltage-dependent K(+) (Kv) currents without affecting ATP-sensitive K(+) channels in rat beta-cells. In the presence of Kv channel blockers, ghrelin failed to suppress insulin release. Ghrelin attenuated glucose-induced action potentials and [Ca(2+)](i) increases in beta-cells. Suppressions of [Ca(2+)](i) increase and insulin release by ghrelin were blunted in beta-cells treated with PTX and with antisense oligonucleotide specific for G-protein Galpha(i2)-subunit. Ghrelin attenuates glucose-induced insulin release via PTX-sensitive Galpha(i2)-mediated activation of Kv channels and suppression of [Ca(2+)](i) in beta-cells, representing the unique signaling of ghrelin distinct from that for GH release.     

Inhibition by simvastatin, but not pravastatin, of glucose-induced cytosolic Ca2+ signalling and insulin secretion due to blockade of L-type Ca2+ channels in rat islet beta-cells

1. Hypercholesterolaemia often occurs in patients with type 2 diabetes, who therefore encounter administration of HMG-CoA reductase inhibitors. Alteration of pancreatic beta-cell function leading to an impaired insulin secretory response to glucose plays a crucial role in the pathogenesis of type 2 diabetes. Therefore, it is important to examine the effects of HMG-CoA reductase inhibitors on beta-cell function. 2. Cytosolic Ca2+ concentration ([Ca2+]i) plays a central role in the regulation of beta-cell function. The present study examined the effects of HMG-CoA reductase inhibitors on the glucose-induced [Ca2+]i signalling and insulin secretion in rat islet beta-cells. 3. Simvastatin, a lipophilic HMG-CoA reductase inhibitor, at 0.1-3 microg ml(-1) concentration-dependently inhibited the first phase increase and oscillation of [Ca2+]i induced by 8.3 mM glucose in single beta-cells. The less lipophilic inhibitor, simvastatin-acid, inhibited the first phase [Ca2+]i increase but was two orders of magnitude less potent. The hydrophilic inhibitor, pravastatin (100 microg ml(-1), was without effect on [Ca2+]i. 4. Simvastatin (0.3 microg ml(-1)), more potently than simvastatin-acid (30 microg ml(-1)), inhibited glucose-induced insulin secretion from islets, whereas pravastatin (100 microg ml(-1)) had no effect. 5. Whole-cell patch clamp recordings demonstrated a reversible inhibition of the beta-cell L-type Ca2+ channels by simvastatin, but not by pravastatin. Simvastatin also inhibited the [Ca2+]i increases by L-arginine and KCl, agents that act via opening of L-type Ca2+ channels. 6. In conclusion, lipophilic HMG-CoA reductase inhibitors can inhibit glucose-induced [Ca2+]i signalling and insulin secretion by blocking L-type Ca2+ channels in beta-cells, and their inhibitory potencies parallel their lipophilicities. Precaution should be paid to these findings when HMG-CoA reductase inhibitors are used clinically, particularly in patients with type 2 diabetes.     

PIP2 and ATP cooperatively prevent cytosolic Ca2+-induced modification of ATP-sensitive K+ channels in rat pancreatic beta-cells

The factors that influence functional coupling between the sulfonylurea receptor (SUR1) and Kir6.2 subunits of ATP-sensitive K+ (K+(ATP)) channels were studied in rat pancreatic beta-cells using patch clamp and microfluorometric techniques. Tolbutamide at 10 micromol/l inhibited K+(ATP) channels in association with occurrence of action currents, but further exposure of beta-cells to the drug for 30 min or longer resulted in reappearance of K+(ATP) channel events. Half-maximal inhibition concentration (IC50) for tolbutamide was 1.5 microl/mol in 2.8 mmol/l glucose, and it was increased to 13.3 micromol/l when the cellular metabolism was inhibited by 0.5 mmol/l 2,4-dinitrophenol (DNP) for 5 min. Tolbutamide at 10 micromol/l induced an increase in cytosolic Ca2+ concentration ([Ca2+]i), and its amplitude was markedly reduced following exposure to 0.5 mmol/l DNP or long-term (30 min) exposure to 10 micromol/l tolbutamide. This tolbutamide insensitivity, as assessed by the [Ca2+]i response, was not observed when the external Ca2+ was omitted during the long-term exposure to tolbutamide. In cell-attached membrane patches, the tolbutamide insensitivity was also produced by treatment of cells with 150 micromol/l diazoxide and 25 mmol/l KCl in the presence, but not absence, of 2 mmol/l Ca2+ in the external solution. When the cytoplasmic face of inside-out membrane patches was treated with higher Ca2+ concentrations (2 micromol/l), both ADP-evoked activation and tolbutamide-induced inhibition of K+ ATP channels were attenuated with retaining ATP-induced inhibition, indicating the modification of K+(ATP) channels. The Ca2+-induced channel modification was prevented partially by phosphatidylinositol 4,5-bisphosphate (PIP2) and completely by ATP and PIP2 together, but not by ATP alone. Treatment of the channel with cytochalasin D, a disrupter of F-actin, evoked channel modification similar to that induced by Ca2+. The modification was prevented completely by phalloidin, a stabilizer of F-actin. In conclusion, long-term exposure to tolbutamide or metabolic inhibition causes modification of K+ ATP channels via mechanisms involving Ca2+-dependent reaction. The modification, which may reflect functional disconnection between SUR1 and Kir6.2, is prevented by ATP and PIP2, which may act cooperatively to stabilize membrane cytoskeletons (F-actin structures).     

Morphology of single axons of tectospinal neurons in the upper cervical spinal cord

Morphology of single axons of tectospinal (TS) neurons was investigated by intraaxonal injection of horseradish peroxidase (HRP) at the upper cervical spinal cord of the cat. TS axons were electrophysiologically identified by their direct responses to stimulation of the contralateral superior colliculus (SC). None of these axons responded to thoracic stimulation at Th2. Three-dimensional reconstructions of the axonal trajectories were made from 20 well-stained TS axons at C1-C3. Cell bodies of these axons were located in the intermediate or deep layers of the caudal two-thirds of the SC. Usually, TS axons had multiple axon collaterals, and up to seven collaterals were given off per stem axon [2.7 ± 1.6 (mean ± S.D.); n = 20]. Collaterals had simple structures and ramified a few times mainly in the transverse plane. The number of terminals for each collateral was small. These collaterals terminated in the lateral parts of laminae V–IX, mainly in laminae VI, VII, and VIII. There were usually gaps free from terminal arborizations between adjacent collaterals, because the rostrocaudal spread of each collateral (mean = 700 μm) was narrower than the intercollateral interval (mean = 2,500 μm). Seven of the 19 TS axons had terminals in the lateral parts of laminae V–VIII, with little projection to lamina IX, and the other 12 axons had terminals in lamina IX besides the projection to the lateral parts of laminae V–VIII. Axon terminals in lamina IX did not appear to make contacts with the somata or proximal dendrites of retrogradely labeled motoneurons, but contacts were found with the somata of counterstained interneurons in the lateral parts of laminae V–VIII. Three spinal interneurons (two in lamina VIII and one in lamina V at C1) that received monosynaptic excitation from the SC were stained, and their axonal trajectories were reconstructed. They had multiple axon collaterals at C1-C2 and mainly projected to laminae VIII and IX, with smaller projections to lamina VII. Many axon terminals of the interneurons were found in multiple neck motor nuclei, where some of them made contacts with retrogradely labeled motoneurons. The present finding provides evidence that the direct TS projection to the spinal cord may influence activities of multiple neck muscles, mainly via spinal interneurons, and may play an important role in control of head movement in parallel with the tectoreticulospinal system. © 1996 Wiley-Liss, Inc.     

Enhanced urinary adiponectin excretion in IgA-nephropathy patients with proteinuria

Adiponectin is secreted specifically by adipose tissue. It was reported that the serum adiponectin level was markedly increased in patients with end-stage renal disease and was positively associated with abnormal renal function in type 2 diabetes. Recently, we found that urinary adiponectin level was significantly increased in type 2 diabetic patients with overt diabetic nephropathy, but not in those without nephropathy. The aim of the present study was to evaluate whether the urinary adiponectin level is increased not only in diabetic patients with macroalbuminuria but also in IgA-nephropathy patients with macroalbuminuria. We measured urinary adiponectin levels in 24 healthy control subjects, 12 IgA-nephropathy patients, and 19 type 2 diabetic nephropathy patients, and they were, in medians, 2.24 microg/g creatinine (ranges of 0.85 to approximately 3.70), 59.2 microg/g creatinine (4.95 to approximately 186), and 33.1 microg/g creatinine (4.69 to approximately 114), respectively. In the two patient groups, urinary adiponectin levels were significantly higher than in control subjects (P<0.01). Moreover, positive correlations between urinary adiponectin levels and albumin-to-creatinine ratios were observed in IgA-nephropathy (R2=0.53, P<0.01) and diabetic nephropathy patients (R2=0.61, P<0.01), but not in control subjects. Serum adiponectin levels were unchanged in these three groups. These findings suggested that the increase of urinary adiponectin levels partly results from enhanced filtration of circulating adiponectin through the changes of glomerular permselectivity and intraglomerular hydruric pressure. However, clinical implication of urinary adiponectin excretion in healthy control remains to be elucidated.     

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.     

Oral melanoma: a multicenter study of 69 patients from Japan

Clinical Oral Investigations - The purpose of this multicenter retrospective study was to investigate the demographic characteristics and treatment outcomes of patients with mucosal malignant...     

Dosimetric correction for a six degrees carbon fiber couch

We investigated experimentally and clinically the influence of a six degree (6D) carbon fiber couch on conventional radiation therapy. We used 4, 6 and 10 MV X-rays and compared dose distributions based on correction methods, i.e. monitor unit (MU) addition, including computed tomography (CT) couch, and the couch modeling. Additionally, we evaluated the clinical value of dosimetric correction for the 6D couch in 30 patients treated with multi-field irradiation. In the phantom study, the maximum difference of isocenter doses attributable to the 6D couch was 5.1%; the difference was reduced with increasing X-ray energy. Although the isocenter dose based on each correction method was precise within ±1%, MU addition underestimated the surface dose. In the clinical study, the maximum difference of isocenter doses attributable to the 6D couch was 2.7%. The correction methods for the 6D couch provide for highly precise treatment planning. However, the clinical indication of complicated correction methods should be considered for each institution or each patient, because the influence of the 6D couch was reduced with multi-field irradiation.