Koichi Yokono

Cognitive dysfunction associates with white matter hyperintensities and subcortical atrophy on magnetic resonance imaging of the elderly diabetes mellitus Japanese elderly diabetes intervention trial (J‐EDIT)

Type 2 diabetes is associated with cognitive dysfunction and increases the risk of dementia in the elderly. The aim of this study was to explore, by means of magnetic resonance (MR) imaging, possible relationships among clinical profiles of diabetes, cognitive function, white matter hyperintensities (WMHs) and subcortical brain atrophy.

Upregulation of the Mammalian Target of Rapamycin Complex 1 Pathway by Ras Homolog Enriched in Brain in Pancreatic β-Cells Leads to Increased β-Cell Mass and Prevention of Hyperglycemia

OBJECTIVE Components of insulin/IGF-1 receptor–mediated signaling pathways in pancreatic β-cells have been implicated in the development of diabetes, in part through the regulation of β-cell mass in vivo. Studies in vitro have shown that the protein Ras homolog enriched in brain (Rheb) plays a key role as a positive upstream regulator of the mammalian target of rapamycin complex 1 (mTORC1) pathway in integrating inputs from nutrients and growth factors for cell growth. Our objective was to investigate the role of the mTORC1 pathway in the regulation of β-cell mass in vivo. RESEARCH DESIGN AND METHODS We generated transgenic mice that overexpress Rheb in β-cells. We examined the activation of the mTORC1 pathway and its effects on β-cell mass, on glucose metabolism, and on protection against hyperglycemia. RESULTS Immunoblots of islet extracts revealed that the phosphorylation levels of ribosomal protein S6 and eukaryotic initiation factor 4E binding protein 1, downstream effectors for mTORC1, were upregulated in transgenic β-cells. Immunostaining of the pancreatic sections with anti–phospho-S6 antibody confirmed upregulation of the mTORC1 pathway in β-cells in vivo. The mice showed improved glucose tolerance with higher insulin secretion. This arose from increased β-cell mass accompanied by increased cell size. The mice also exhibited resistance to hyperglycemia induced by streptozotocin and obesity. CONCLUSIONS Activation of the mTORC1 pathway by Rheb led to increased β-cell mass in this mouse model without producing obvious unfavorable effects, giving a potential approach for the treatment of β-cell failure and diabetes.

Purification and Characterization of Insulin-Degrading Enzyme From Human Erythrocytes

An insulin-degrading enzyme (IDE) was purified from the cytosol of human erythrocytes via the use of ammonium sulfate precipitation and chromatography on columns composed of DEAE-Sephadex, pentylagarose, hydroxylapatite, chromatofocusing resins, and Ultrogel AcA-34. The final preparation was purified >50,000-fold and exhibited a single protein band of Mr = 110,000 on reduced sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Cross-linking of 125I-labeled insulin to the enzyme preparation labeled a protein of the same molecular weight, indicating that this band was in fact the enzyme. Intact insulin, insulin B chain, andglucagon inhibited this cross-linking half-maximally at concentrations of 0.1,1, and 1.5 μM, respectively. Under nondenaturing conditions, the enzyme had an Mr, = 300,000, suggesting that the enzyme may exist under physiological conditions as a dimer or trimer. The purified enzyme was inhibited by both sulfhydrylmodifying reagents and chelating agents, indicating that a free thiol and metal were both required for the activity of the enzyme. The purified enzyme was found to degrade physiological concentrations of intact insulin more rapidly than insulin B chain,although at high substrate concentrations (>1 μM) the enzyme degraded B chain to a greater extent. Additional characteristics of the enzyme were a pl of 5.2 and a pH optimum of 7.0. These properties of the red blood cell (RBC) enzyme were very similar to those reported for IDEs from other tissues. Moreover, a polyclonal antiserum to the IDE from skeletal muscle was found to recognize the RBC enzyme. These results indicate that the human erythrocyte enzyme is very similar tothe enzyme in other tissues and that these cells are a good source of material for purification of the human IDE.

Age-Associated Increase in Abdominal Obesity and Insulin Resistance, and Usefulness of AHA/NHLBI Definition of Metabolic Syndrome for Predicting Cardiovascular Disease in Japanese Elderly with Type 2 Diabetes Mellitus

Background: Management of metabolic syndrome (MetS) seems to constitute an efficient strategy to attain successful ageing. Although the clinical entity of MetS in patients with diabetes mellitus has been discussed, there is very little information on MetS-type cardiometabolic risk factor clustering in diabetic elderly. Objective: To determine the relationship among age-associated changes in obesity, insulin resistance, and clustering of MetS-type risk factors, in association with vascular complications, in Japanese elderly with type 2 diabetes. Methods: A cross-sectional study was conducted of 812 diabetic elderly enrolled in the Japanese Elderly Diabetes Intervention Trial. Information on diabetes, blood examinations and complications was obtained. Abdominal obesity, insulin resistance and prevalence of MetS risk factor clustering, defined by three sets of criteria from the International Diabetes Federation (IDF), the Japanese Society of Internal Medicine (JSIM), and the American Heart Association and the National Heart, Lung, and Blood Institute (AHA/NHLBI), were analyzed. Results: Waist circumference and insulin resistance estimated by homeostasis model assessment insulin resistance (HOMA-IR) increased with age, followed by a partial decrease at age 80 and over. Prevalence of IDF-MetS and JSIM-MetS also increased with age at least until the age of 80, whereas the incidence of AHA/NHLBI-MetS did not show any apparent age changes. There was a significant crude linear association between waist circumference and HOMA-IR, which was highly elevated in IDF and AHA/NHLBI overlapping with MetS, and also elevated in AHA/NHLBI without abdominal obesity. Although IDF-MetS and JSIM-MetS, which specify abdominal obesity, did not always appear to be associated with cardiovascular diseases, AHA/NHLBI-MetS, comprising both abdominal obesity and non-abdominal obesity, independently correlated with coronary heart disease and stroke after adjustment for other risk factors of atherosclerotic diseases. Conclusion: There was an age-associated increase in the prevalence of abdominal obesity and insulin resistance in elderly diabetic Japanese subjects, with a clear relationship between waist circumference and insulin resistance. However, insulin resistance was elevated not only in cases with but also in those without abdominal obesity if accompanied by clustering of metabolic disorders. The AHA/NHLBI definition of MetS proved to be the most useful to predict cardiovascular disease in the diabetic elderly.

Expression of intercellular adhesion molecule 1 on pancreatic beta-cells accelerates beta-cell destruction by cytotoxic T-cells in murine autoimmune diabetes

Intercellular adhesion molecule 1 (ICAM-1) plays an important role in the pathogenesis of insulin-dependent diabetes mellitus (IDDM) by being involved in the extravasation of lymphocytes from the circulation into the inflamed pancreas. However, the mechanism of β-cell destruction by which expression of ICAM-1 on β-cells may facilitate adhesion of effector cells still remains to be elucidated. Several lines of evidence suggest that this adhesion molecule is involved in the destruction of pancreatic β-cells by killer lymphocytes in the NOD mouse, which shows an autoimmune diabetic syndrome similar to that of human IDDM. Immunohistochemical study under light microscopy demonstrated that all of the mononuclear cells infiltrating the islets strongly expressed ICAM-1 and leukocyte function-associated antigen 1 (LFA-1), a counterreceptor of ICAM-1, whereas ICAM-1 expression on islet cells was not apparent. However, immunohistochemical staining under electron microscopy revealed that islet β-cells adjacent to infiltrating lymphocytes were clearly stained by an anti-ICAM-1 monoclonal antibody (mAb). Flow cytometric analysis showed that the ICAM-1 expression on NOD islet cells and NOD-derived insulinoma cells (MIN6N8a) was inducible by interferon (IFN)-γ or tumor necrosis factor-α. These cytokines had an additive effect on the ICAM-1 induction. Susceptibility of MIN6N8a cells to lysis by a NOD islet-derived CD8+ cytotoxic T-cell clone was greatly enhanced by IFN-γ pretreatment, and this enhancement was abolished by anti-ICAM-1 and anti-LFA-1 mAbs. When both mAbs were administered into NOD mice with spontaneous or adoptively transferred diabetes, the development of diabetes was significantly prevented. These results suggest that cytokines secreted by isletinfiltrating mononuclear cells could induce the ICAM-1 expression on γ-cells, which accelerates the γ-cell destruction by cytotoxic T-cells. Therefore, immunointervention of the ICAM-l/LFA-1 pathway would be an excellent strategy to prevent human IDDM.

In Vivo X-Ray Angiography in the Mouse Brain Using Synchrotron Radiation

Background and Purpose— We, for the first time, performed in vivo x-ray angiography in the mouse brain using SPring-8, a third-generation synchrotron radiation facility. Methods— A thin PE-50 tube was placed in the unilateral external carotid artery in adult male C57BL/6J mice. While maintaining the blood flow in the internal carotid artery, 33 &mgr;L of contrast agent was injected and then selective angiography of the hemisphere was performed. Results— The average diameters of cerebral artery were as follows: 142.5±7.90 &mgr;m in middle cerebral artery, 138.3±9.35 &mgr;m in anterior cerebral artery, 120.5±5.53 &mgr;m in posterior cerebral artery, and 162.6±10.87 &mgr;m in internal carotid artery (n=5). To demonstrate the changes in diameter, we induced hypercapnia and detected the dilatation of the vessels between 121% and 124% of the original diameters (n=5). We also repeated angiography in the mice before and after intracarotid injection of vasodilatation drugs papaverine hydrochloride, ATP disodium, and fasudil hydrochloride hydrate and demonstrated the chronological changes in the diameters in each artery at 1, 5, 15, and 30 minutes after injection (n=1 for each drug). Conclusions— Using only a minimum volume of the contrast agent, synchrotron radiation enables us to study x-ray angiography in the mouse brain. The morphology of the vessels can be clearly observed under physiological conditions. The diameters and their changes can also be successfully studied in vivo.

Degradation of Insulin by Isolated Mouse Pancreatic Acini: Evidence for Cell Surface Protease Activity

In the present study, we have used isolated mouse pancreatic acini to investigate the relationship between 125I-insulin binding and its degradation in order to probe the nature and cellular localization of the degradative process. In these cells, the proteolysis of 125I-insulin was dependent on time and cell concentration, and was saturated by unlabeled insulin with a Km of 290 nM. Since this value was much higher than the Kd for insulin binding to its receptor (1.1 nM), the data indicated that 125I-insulin degradation by acini occurred primarily via nonreceptor mechanisms. Several lines of evidence suggested that insulin was being degraded by the neutral thiol protease, insulin degrading enzyme (IDE). First, insulin degradation was inhibited by thiolreacting agents such as N-ethylmaleimide and p-chloromercuribenzoate. Second, the Km for degradation in acini was similar to the reported Km for IDE in other tissues. Third, the enzyme activity had a relative mol wt of approximately 130,000 by gel filtration, a value similar to that reported for purified IDE. Fourth, the degrading activity was removed with a specific antibody to IDE. Other lines of evidence suggested that enzymes located on the cell surface played a role in insulin degradation by acini. First, the nonpenetrating sulfhydryl reacting agent 5,5′dithiobis-2-nitrobenzoic acid blocked 125I-insulin degradation. Second, a specific antibody to IDE identified the presence of the enzyme on the cell surface. Third, chloroquine, leupeptin and antipain, agents that inhibit lysosomal function, did not influence 125I-insulin degradation. Fourth, highly purified pancreatic plasma membranes degraded 125I-insulin.

Carbamylation of Insulin and Its Biological Activity

A nonspecific binding reaction between cyanic acid formed from urea and protein or peptide is called carbamylation. In the present study, insulin, a peptide hormone, was subjected to carbamylation and the activity of carbamylated insulin was determined. Both immunological and biological activities of insulin changed on carbamylation. The decrease in biological activity in respect to glucose oxidation of fat cells or receptor-binding capacity of rat hepatocytes was greater than that in immunological activity.

Long-term multiple risk factor interventions in Japanese elderly diabetic patients: the Japanese Elderly Diabetes Intervention Trial--study design, baseline characteristics and effects of intervention.

Aim:  To evaluate long‐term, multiple risk factor intervention on physical, psychological and mental prognosis, and development of complications and cardiovascular disease in elderly type 2 diabetes patients.

Prevention of Cyclophosphamide-Induced and Spontaneous Diabetes in NOD/Shi/Kbe Mice by Anti-MHC Class I Kd Monoclonal Antibody

The immune mechanisms directly responsible for β-cell destruction in insulin-dependent diabetes are undefined. We studied the role of MHC class I–restricted T lymphocytes in the development of diabetes in cyclophosphamide (CY)-treated male and untreated female NOD mice (H-2Kd,Db). After administration of CY to 10-wk-old male NOD/Shi/Kbe mice, 37 of 64 (58%) phosphate-buffered saline–injected control mice and 13 of 22 (59%) anti-Kb and 12 of 27 (44%) anti-Db monoclonal antibody (MoAb)-injected mice became diabetic by 14 wk of age, whereas only 3 of 38 (8%) anti-Kd and 2 of 13 (15%) anti-Lyt-2 MoAb-injected mice did. In untreated female NOD/Shi/Kbe mice, 30 of 46 (65%) mice developed spontaneous diabetes by 30 wk of age, whereas none of 9 anti-Kd MoAb-injected mice became diabetic. Immunohistochemical studies showed that islet-infiltrating cells in CY-treated control mice were composed mainly of both L3T4+ and Lyt-2+ T lymphocytes, whereas many L3T4+ and very few Lyt-2+ lymphocytes infiltrated within the islets in anti-Kd MoAb-injected mice. Administration of anti-Lyt-2 MoAb induced the absence of Lyt-2+ T lymphocytes in the islet and spleen. However, anti-Kd MoAb did not change the number of spleen cells or the T-lymphocyte subset and response to concanavalin A. These results suggest that MHC class I Kd-restricted Lyt-2+ T lymphocytes play an important role as direct effector cells in destruction of β-cells in NOD/Shi/Kbe mice.