Kenju Shimomura

PPAR gamma 2 Prevents Lipotoxicity by Controlling Adipose Tissue Expandability and Peripheral Lipid Metabolism

Peroxisome proliferator activated receptor gamma 2 (PPARg2) is the nutritionally regulated isoform of PPARg. Ablation of PPARg2 in the ob/ob background, PPARg2−/− Lepob/Lepob (POKO mouse), resulted in decreased fat mass, severe insulin resistance, β-cell failure, and dyslipidaemia. Our results indicate that the PPARg2 isoform plays an important role, mediating adipose tissue expansion in response to positive energy balance. Lipidomic analyses suggest that PPARg2 plays an important antilipotoxic role when induced ectopically in liver and muscle by facilitating deposition of fat as relatively harmless triacylglycerol species and thus preventing accumulation of reactive lipid species. Our data also indicate that PPARg2 may be required for the β-cell hypertrophic adaptive response to insulin resistance. In summary, the PPARg2 isoform prevents lipotoxicity by (a) promoting adipose tissue expansion, (b) increasing the lipid-buffering capacity of peripheral organs, and (c) facilitating the adaptive proliferative response of β-cells to insulin resistance.

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

ATP‐sensitive potassium (KATP) 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 KATP channel complex composed of pore‐forming Kir6.2 and regulatory SUR1 subunits, and determination of its structure at 18 Å 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.

Permanent neonatal diabetes caused by dominant, recessive, or compound heterozygous SUR1 mutations with opposite functional effects.

Heterozygous activating mutations in the KCNJ11 gene encoding the pore-forming Kir6.2 subunit of the pancreatic beta cell K(ATP) channel are the most common cause of permanent neonatal diabetes (PNDM). Patients with PNDM due to a heterozygous activating mutation in the ABCC8 gene encoding the SUR1 regulatory subunit of the K(ATP) channel have recently been reported. We studied a cohort of 59 patients with permanent diabetes who received a diagnosis before 6 mo of age and who did not have a KCNJ11 mutation. ABCC8 gene mutations were identified in 16 of 59 patients and included 8 patients with heterozygous de novo mutations. A recessive mode of inheritance was observed in eight patients with homozygous, mosaic, or compound heterozygous mutations. Functional studies of selected mutations showed a reduced response to ATP consistent with an activating mutation that results in reduced insulin secretion. A novel mutational mechanism was observed in which a heterozygous activating mutation resulted in PNDM only when a second, loss-of-function mutation was also present.

A novel mutation causing DEND syndrome A treatable channelopathy of pancreas and brain

Objectives: Activating mutations in the human KCNJ11 gene, encoding the pore-forming subunit (Kir6.2) of the ATP-sensitive potassium (KATP) channel, are one cause of neonatal diabetes mellitus. In a few patients, KCNJ11 mutations cause a triad of developmental delay, epilepsy, and neonatal diabetes (DEND syndrome). The aim of this study was to determine the clinical effects, functional cause, and sensitivity to sulfonylurea treatment of a novel KCNJ11 mutation producing DEND syndrome. Methods: We screened the DNA of a 3-year-old patient with neonatal diabetes, severe developmental delay, and therapy-resistant epilepsy for mutations in KCNJ11. We carried out electrophysiologic analysis of wild-type and mutant KATP channels heterologously expressed in Xenopus oocytes. Results: We identified a novel Kir6.2 mutation (I167L) causing DEND syndrome. Functional analysis showed both homomeric and heterozygous mutant channels were less inhibited by MgATP leading to an increase in whole-cell KATP currents. This effect was due to an increase in the intrinsic open probability. Heterozygous channels were strongly inhibited by the sulfonylurea tolbutamide. Treatment of the patient with the sulfonylurea glibenclamide not only enabled insulin therapy to be stopped, but also resulted in improvement in epilepsy and psychomotor abilities. Conclusions: We report a case of developmental delay, epilepsy, and neonatal diabetes (DEND) syndrome that shows neurologic improvement with sulfonylurea therapy. Early recognition of patients with DEND syndrome may have considerable therapeutic benefit for the patient.

Nicotinamide nucleotide transhydrogenase: a link between insulin secretion, glucose metabolism and oxidative stress

This paper reviews recent studies on the role of Nnt (nicotinamide nucleotide transhydrogenase) in insulin secretion and detoxification of ROS (reactive oxygen species). Glucose-stimulated insulin release from pancreatic beta-cells is mediated by increased metabolism. This elevates intracellular [ATP], thereby closing KATP channels (ATP-sensitive potassium channels) and producing membrane depolarization, activation of voltage-gated Ca2+ channels, Ca2+ influx and, consequently, insulin secretion. The C57BL/6J mouse displays glucose intolerance and reduced insulin secretion, which results from a naturally occurring deletion in the Nnt gene. Transgenic expression of the wild-type Nnt gene in C57BL/6J mice rescues the phenotype. Knockdown of Nnt in the insulin-secreting cell line MIN6 with small interfering RNA dramatically reduced Ca2+ influx and insulin secretion. Similarly, mice carrying ENU (N-ethyl-N-nitrosourea)-induced loss-of-function mutations in Nnt were glucose intolerant and secreted less insulin during a glucose tolerance test. Islets isolated from these mice showed impaired insulin secretion in response to glucose, but not to the KATP channel blocker tolbutamide. This is explained by the fact that glucose failed to elevate ATP in Nnt mutant islets. Nnt is a nuclear-encoded mitochondrial protein involved in detoxification of ROS. beta-Cells isolated from Nnt mutant mice showed increased ROS production on glucose stimulation. We hypothesize that Nnt mutations enhance glucose-dependent ROS production and thereby impair beta-cell mitochondrial metabolism, possibly via activation of uncoupling proteins. This reduces ATP production and lowers KATP channel activity. Consequently, glucose-dependent electrical activity and insulin secretion are impaired.

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

ATP-sensitive potassium (KATP) 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. KATP 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 KATP channel to inhibition by MgATP, and enhanced whole-cell KATP 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 KATP 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.

The role of CHOP messenger RNA expression in the link between oxidative stress and apoptosis

Low expression of antioxidant enzymes makes pancreatic beta-cells susceptible to cell damage by oxidative stress. Pancreatic beta-cell loss caused by endoplasmic reticulum stress is associated with the onset of diabetes mellitus. The present studies were undertaken to investigate a possible involvement of proapoptotic gene CHOP in pancreatic beta-cells damage by oxidative stress. The induction of CHOP messenger RNA and apoptosis were investigated in betaHC-9 cells after the oxidative stress by hydrogen peroxide and ribose. Latter was examined after the suppression of CHOP by small interfering RNA. For in vivo study, the pancreatic beta-cells were examined in CHOP-knockout (KO) mice after multiple low-dose streptozotocin (MLDS) administration. In betaHC-9 cells, both hydrogen peroxide and ribose obviously increased apoptotic cells, accompanied with enhanced CHOP messenger RNA expression. However, the number of apoptotic cells by those stimulations was significantly reduced by the addition of small interfering RNA against CHOP. In vivo study also showed that CHOP-KO mice were less susceptible to diabetes after MLDS administration. Although the oxidative stress marker level was similar to that of MLDS-treated wild type, the pancreatic beta-cell area was maintained in CHOP-KO mice. The present studies showed that CHOP should be important in pancreatic beta-cell injury by oxidative stress and indicate that CHOP may play a role in the development of pancreatic beta-cell damage on the onset of diabetes mellitus.

Adjacent mutations in the gating loop of Kir6.2 produce neonatal diabetes and hyperinsulinism.

KATP channels regulate insulin secretion from pancreatic β‐cells. Loss‐ and gain‐of‐function mutations in the genes encoding the Kir6.2 and SUR1 subunits of this channel cause hyperinsulinism of infancy and neonatal diabetes, respectively. We report two novel mutations in the gating loop of Kir6.2 which cause neonatal diabetes with developmental delay (T293N) and hyperinsulinism (T294M). These mutations increase (T293N) or decrease (T294M) whole‐cell KATP currents, accounting for the different clinical phenotypes. The T293N mutation increases the intrinsic channel open probability (Po(0)), thereby indirectly decreasing channel inhibition by ATP and increasing whole‐cell currents. T294M channels exhibit a dramatically reduced Po(0) in the homozygous but not in the pseudo‐heterozygous state. Unlike wild‐type channels, hetT294M channels were activated by MgADP in the absence but not in the presence of MgATP; however, they are activated by MgGDP in both the absence and presence of MgGTP. These mutations demonstrate the importance of the gating loop of Kir channels in regulating Po(0) and further suggest that Mg‐nucleotide interaction with SUR1 may reduce ATP inhibition at Kir6.2.

Chapter 25 Insulin Secretion from β-Cells is Affected by Deletion of Nicotinamide Nucleotide Transhydrogenase

Nicotinamide nucleotide transhydrogenase (NNT) is an inner mitochondrial membrane transmembrane protein involved in regenerating NADPH, coupled with proton translocation across the inner membrane. We have shown that a defect in Nnt function in the mouse, and specifically within the beta-cell, leads to a reduction in insulin secretion. This chapter describes methods for examining Nnt function in the mouse. This includes generating in vivo models with point mutations and expression of Nnt by transgenesis, and making in vitro models, by silencing of gene expression. In addition, techniques are described to measure insulin secretion, calcium and hydrogen peroxide concentrations, membrane potential, and NNT activity. These approaches and techniques can also be applied to other genes of interest.

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Endolysosomal Two-pore Channels Modulate Membrane Excitability and Stimulus-Secretion Coupling in Mouse Pancreatic β Cells

Background: TPCs are regulated by NAADP and other factors. Results: NAADP-induced Ca2+ release from acidic stores evokes depolarizing currents in pancreatic β cells. Inhibition of NAADP signaling or TPC knock out attenuates Ca2+ signaling and insulin secretion. Conclusion: NAADP-evoked Ca2+ release enhances β cell excitability and insulin secretion in response to glucose or sulfonylureas. Significance: NAADP signaling pathways offer novel therapeutic targets for diabetes treatment. Pancreatic β cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca2+ action potentials due to the activation of voltage-dependent Ca2+ channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca2+ release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic β cells. NAADP-regulated Ca2+ release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca2+ from the endolysosomal system, resulting in localized Ca2+ signals. We show here that NAADP-mediated Ca2+ release from endolysosomal Ca2+ stores activates inward membrane currents and depolarizes the β cell to the threshold for VDCC activation and thereby contributes to glucose-evoked depolarization of the membrane potential during stimulus-response coupling. Selective pharmacological inhibition of NAADP-evoked Ca2+ release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca2+ signals, and insulin secretion. Our findings implicate NAADP-evoked Ca2+ release from acidic Ca2+ storage organelles in stimulus-secretion coupling in β cells.