Hisamitsu Ishihara

Islet beta-cell secretion determines glucagon release from neighbouring alpha-cells.

Homeostasis of blood glucose is maintained by hormone secretion from the pancreatic islets of Langerhans. Glucose stimulates insulin secretion from β-cells but suppresses the release of glucagon, a hormone that raises blood glucose, from α-cells. The mechanism by which nutrients stimulate insulin secretion has been studied extensively: ATP has been identified as the main messenger and the ATP-sensitive potassium channel as an essential transducer in this process. By contrast, much less is known about the mechanisms by which nutrients modulate glucagon secretion. Here we use conventional pancreas perfusion and a transcriptional targeting strategy to analyse cell-type-specific signal transduction and the relationship between islet α- and β-cells. We find that pyruvate, a glycolytic intermediate and principal substrate of mitochondria, stimulates glucagon secretion. Our analyses indicate that, although α-cells, like β-cells, possess the inherent capacity to respond to nutrients, secretion from α-cells is normally suppressed by the simultaneous activation of β-cells. Zinc released from β-cells may be implicated in this suppression. Our results define the fundamental mechanisms of differential responses to identical stimuli between cells in a microorgan.

Pancreatic beta cell line MIN6 exhibits characteristics of glucose metabolism and glucose-stimulated insulin secretion similar to those of normal islets

SummaryGlucose-stimulated insulin secretion, glucose transport, glucose phosphorylation and glucose utilization have been characterized in the insulinoma cell line MIN6, which is derived from a transgenic mouse expressing the large T-antigen of SV40 in pancreatic beta cells. Glucose-stimulated insulin secretion occurred progressively from 5 mmol/l glucose, reached the maximal level approximately seven-fold above the basal level at 25 mmol/l, and remained at this level up to 50 mmol/l. Glucose transport was very rapid with the half-maximal uptake of 3-O-methyl-d-glucose being reached within 15 s at 22 °C. Glucose phosphorylating activity in the cell homogenate was due mainly to glucokinase; the Vmax value of glucokinase activity was estimated to be 255±37 nmol·h−1·mg protein−1, constituting approximately 80% of total phosphorylating activity, whereas hexokinase activity constituted less than 20%. MIN6 cells exhibited mainly the high Km component of glucose utilization with a Vmax of 289±18 nmol·h−1·mg protein−1. Thus, glucose utilization quantitatively and qualitatively reflected glucose phosphorylation in MIN6 cells. In contrast, MIN7 cells, which exhibited only a small increase in insulin secretion in response to glucose, had 4.7-fold greater hexokinase activity than MIN6 cells with a comparable activity of glucokinase. These characteristics in MIN6 cells are very similar to those of isolated islets, indicating that this cell line is an appropriate model for studying the mechanism of glucose-stimulated insulin secretion in pancreatic beta cells.

A Critical Role for a Rho-Associated Kinase, p160ROCK, in Determining Axon Outgrowth in Mammalian CNS Neurons

We tested the contribution of the small GTPase Rho and its downstream target p160ROCK during the early stages of axon formation in cultured cerebellar granule neurons. p160ROCK inhibition, presumably by reducing the stability of the cortical actin network, triggered immediate outgrowth of membrane ruffles and filopodia, followed by the generation of initial growth cone-ike membrane domains from which axonal processes arose. Furthermore, a potentiation in both the size and the motility of growth cones was evident, though the overall axon elongation rate remained stable. Conversely, overexpression of dominant active forms of Rho or ROCK was suggested to prevent initiation of axon outgrowth. Taken together, our data indicate a novel role for the Rho/ROCK pathway as a gate critical for the initiation of axon outgrowth and the control of growth cone dynamics.

Type I Phosphatidylinositol-4-phosphate 5-Kinases CLONING OF THE THIRD ISOFORM AND DELETION/SUBSTITUTION ANALYSIS OF MEMBERS OF THIS NOVEL LIPID KINASE FAMILY

Type I phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinases (PIP5K) catalyze the synthesis of phosphatidylinositol 4,5-bisphosphate, an essential lipid molecule in various cellular processes. Here, we report the cloning of the third member (PIP5Kγ) and the characterization of members of the type I PIP5K family. Type I PIP5Kγ has two alternative splicing forms, migrating at 87 and 90 kDa on SDS-polyacrylamide gel electrophoresis. The amino acid sequence of the central portion of this isoform shows approximately 80% identity with those of the α and β isoforms. Northern blot analysis revealed that the γ isoform is highly expressed in the brain, lung, and kidneys. Among three isoforms, the β isoform has the greatest V max value for the PtdIns(4)P kinase activity and the γ isoform is most markedly stimulated by phosphatidic acid. By analyzing deletion mutants of the three isoforms, the minimal kinase core sequence of these isoforms were determined as an approximately 380-amino acid region. In addition, carboxyl-terminal regions of the β and γ isoforms were found to confer the greatest V max value and the highest phosphatidic acid sensitivity, respectively. It was also discovered that lysine 138 in the putative ATP binding motif of the α isoform is essential for the PtdIns(4)P kinase activity. As was the case with the α isoform reported previously (Shibasaki, Y., Ishihara, H., Kizuki, N., Asano, T., Oka, Y., Yazaki, Y. (1997) J. Biol. Chem.272, 7578–7581), overexpression of either the β or the γ isoform induced an increase in short actin fibers and a decrease in actin stress fibers in COS7 cells. Surprisingly, a kinase-deficient substitution mutant also induced an abnormal actin polymerization, suggesting a role of PIP5Ks via structural interactions with other molecules.

Type Iα phosphatidylinositol-4-phosphate 5-kinase mediates Rac-dependent actin assembly

Action polymerization is essential for a variety of cellular processes including movement, cell division and shape change. The induction of actin polymerization requires the generation of free actin filament barbed ends, which results from the severing or uncapping of pre-existing actin filaments [1] [2], or de novo nucleation, initiated by the Arp2/3 complex [3] [4] [5] [6] [7]. Although little is known about the signaling pathways that regulate actin assembly, small GTPases of the Rho family appear to be necessary [8] [9] [10] [11]. In thrombin-stimulated platelets, the Rho family GTPase Rac1 induces actin polymerization by stimulating the uncapping of actin filament barbed ends [2]. The mechanism by which Rac regulates uncapping is unclear, however. We previously demonstrated that Rac interacts with a type I phosphatidylinositol-4-phosphate 5-kinase (PIP 5-kinase) in a GTP-independent manner [12] [13]. Because PIP 5-kinases synthesize phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), a lipid that dissociates capping proteins from the barbed ends of actin filaments [14] [15] [16], they are good candidates for mediating the effects of Rac on actin assembly. Here, we have identified the Rac-associated PIP 5-kinase as the PIP 5-kinase isoforms alpha and beta. When added to permeabilized platelets, PIP 5-kinase alpha induced actin filament uncapping and assembly. In contrast, a kinase-inactive PIP 5-kinase alpha mutant failed to induce actin assembly and blocked assembly stimulated by thrombin or Rac. Furthermore, thrombin- or Rac-induced actin polymerization was inhibited by a point mutation in the carboxyl terminus of Rac that disrupts PIP 5-kinase binding. These results demonstrate that PIP 5-kinase alpha is a critical mediator of thrombin- and Rac-dependent actin assembly.

Wolfram syndrome 1 gene negatively regulates ER stress signaling in rodent and human cells

Wolfram syndrome is an autosomal-recessive disorder characterized by insulin-dependent diabetes mellitus, caused by nonautoimmune loss of beta cells, and neurological dysfunctions. We have previously shown that mutations in the Wolfram syndrome 1 (WFS1) gene cause Wolfram syndrome and that WFS1 has a protective function against ER stress. However, it remained to be determined how WFS1 mitigates ER stress. Here we have shown in rodent and human cell lines that WFS1 negatively regulates a key transcription factor involved in ER stress signaling, activating transcription factor 6alpha (ATF6alpha), through the ubiquitin-proteasome pathway. WFS1 suppressed expression of ATF6alpha target genes and repressed ATF6alpha-mediated activation of the ER stress response element (ERSE) promoter. Moreover, WFS1 stabilized the E3 ubiquitin ligase HRD1, brought ATF6alpha to the proteasome, and enhanced its ubiquitination and proteasome-mediated degradation, leading to suppression of ER stress signaling. Consistent with these data, beta cells from WFS1-deficient mice and lymphocytes from patients with Wolfram syndrome exhibited dysregulated ER stress signaling through upregulation of ATF6alpha and downregulation of HRD1. These results reveal a role for WFS1 in the negative regulation of ER stress signaling and in the pathogenesis of diseases involving chronic, unresolvable ER stress, such as pancreatic beta cell death in diabetes.

Neuronal Pathway from the Liver Modulates Energy Expenditure and Systemic Insulin Sensitivity

Coordinated control of energy metabolism and glucose homeostasis requires communication between organs and tissues. We identified a neuronal pathway that participates in the cross talk between the liver and adipose tissue. By studying a mouse model, we showed that adenovirus-mediated expression of peroxisome proliferator–activated receptor (PPAR)–g2 in the liver induces acute hepatic steatosis while markedly decreasing peripheral adiposity. These changes were accompanied by increased energy expenditure and improved systemic insulin sensitivity. Hepatic vagotomy and selective afferent blockage of the hepatic vagus revealed that the effects on peripheral tissues involve the afferent vagal nerve. Furthermore, an antidiabetic thiazolidinedione, a PPARg agonist, enhanced this pathway. This neuronal pathway from the liver may function to protect against metabolic perturbation induced by excessive energy storage.

Regulation of Pancreatic β Cell Mass by Neuronal Signals from the Liver

Metabolic regulation in mammals requires communication between multiple organs and tissues. The rise in the incidence of obesity and associated metabolic disorders, including type 2 diabetes, has renewed interest in interorgan communication. We used mouse models to explore the mechanism whereby obesity enhances pancreatic β cell mass, pathophysiological compensation for insulin resistance. We found that hepatic activation of extracellular regulated kinase (ERK) signaling induced pancreatic β cell proliferation through a neuronal-mediated relay of metabolic signals. This metabolic relay from the liver to the pancreas is involved in obesity-induced islet expansion. In mouse models of insulin-deficient diabetes, liver-selective activation of ERK signaling increased β cell mass and normalized serum glucose levels. Thus, interorgan metabolic relay systems may serve as valuable targets in regenerative treatments for diabetes.

Overexpression of Catalytic Subunit p110α of Phosphatidylinositol 3-Kinase Increases Glucose Transport Activity with Translocation of Glucose Transporters in 3T3-L1 Adipocytes

To elucidate the mechanisms of phosphatidylinositol (PI) 3-kinase involvement in insulin-stimulated glucose transport activity, the epitope-tagged p110α subunit of PI 3-kinase was overexpressed in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Overexpression of p110α was confirmed by immunoblot using anti-tagged epitope antibody. p110α overexpression induced a 2.5-fold increase in PI 3-kinase activity associated with its regulatory subunits in the basal state, an increase exceeding that of the maximally insulin-stimulated control cells, while PI 3-kinase activity associated with phosphotyrosyl protein was only modestly elevated. Overexpression of p110α induced an approximately 14-fold increase in the basal glucose transport rate, which was also greater than that observed in the stimulated control. No apparent difference was observed in the cellular expression level of either GLUT1 or GLUT4 proteins between control and p110α-overexpressing 3T3-L1 adipocytes. Subcellular fractionation revealed translocation of glucose transporters from intracellular to plasma membranes in basal p110α-overexpressing cells. The translocation of GLUT4 protein to the plasma membrane was further confirmed using a membrane sheet assay. These findings indicate that an increment in PI 3-kinase activity induced by overexpression of p110α of PI 3-kinase stimulates glucose transport activity with translocation of glucose transporters, i.e., mimics the effect of insulin.

Overexpression of monocarboxylate transporter and lactate dehydrogenase alters insulin secretory responses to pyruvate and lactate in beta cells

Previous investigations revealed low activities of lactate dehydrogenase (LDH) and plasma membrane monocarboxylate transporters (MCT) in the pancreatic beta cell. In this study the significance of these characteristics was explored by overexpressing type A LDH (LDH-A) and/or type 1 MCT (MCT-1) in the clonal INS-1 beta cells and isolated rat islets. Inducible overexpression of LDH-A resulted in an 87-fold increase in LDH activity in INS-1 cells. Adenovirus-mediated overexpression of MCT-1 increased lactate transport activity 3.7-fold in INS-1 cells. Although overexpression of LDH-A, and/or MCT-1 did not affect glucose-stimulated insulin secretion, LDH-A overexpression resulted in stimulation of insulin secretion even at a low lactate concentration with a concomitant increase in its oxidation in INS-1 cells regardless of MCT-1 co-overexpression. Adenovirus-mediated overexpression of MCT-1 caused an increase in pyruvate oxidation and conferred pyruvate-stimulated insulin release to isolated rat islets. Although lactate did not stimulate insulin secretion from control or MCT-1-overexpressing islets, co-overexpression of LDH-A and MCT-1 evoked lactate-stimulated insulin secretion with a concomitant increase in lactate oxidation in rat islets. These results suggest that low expression of MCT and LDH is requisite to the specificity of glucose in insulin secretion, protecting the organism from undesired hypoglycemic actions of pyruvate and lactate during exercise and other catabolic states.