Kaoru Aida

Enterovirus Infection, CXC Chemokine Ligand 10 (CXCL10), and CXCR3 Circuit: A Mechanism of Accelerated β-Cell Failure in Fulminant Type 1 Diabetes

OBJECTIVE Fulminant type 1 diabetes is characterized by the rapid onset of severe hyperglycemia and ketoacidosis, with subsequent poor prognosis of diabetes complications. Causative mechanisms for accelerated β-cell failure are unclear. RESEARCH DESIGN AND METHODS Subjects comprised three autopsied patients who died from diabetic ketoacidosis within 2–5 days after onset of fulminant type 1 diabetes. We examined islet cell status, including the presence of enterovirus and chemokine/cytokine/major histocompatibility complex (MHC) expressions in the pancreata using immunohistochemical analyses and RT-PCR. RESULTS Immunohistochemical analysis revealed the presence of enterovirus-capsid protein in all three affected pancreata. Extensive infiltration of CXCR3 receptor–bearing T-cells and macrophages into islets was observed. Dendritic cells were stained in and around the islets. Specifically, interferon-γ and CXC chemokine ligand 10 (CXCL10) were strongly coexpressed in all subtypes of islet cells, including β-cells and α-cells. No CXCL10 was expressed in exocrine pancreas. Serum levels of CXCL10 were increased. Expression of MHC class II and hyperexpression of MHC class I was observed in some islet cells. CONCLUSIONS These results strongly suggest the presence of a circuit for the destruction of β-cells in fulminant type 1 diabetes. Enterovirus infection of the pancreas initiates coexpression of interferon-γ and CXCL10 in β-cells. CXCL10 secreted from β-cells activates and attracts autoreactive T-cells and macrophages to the islets via CXCR3. These infiltrating autoreactive T-cells and macrophages release inflammatory cytokines including interferon-γ in the islets, not only damaging β-cells but also accelerating CXCL10 generation in residual β-cells and thus further activating cell-mediated autoimmunity until all β-cells have been destroyed.

Anti-platelet action of isoliquiritigenin, an aldose reductase inhibitor in licorice

The mechanism was studied by which isoliquiritigenin, a new aldose reductase inhibitor purified from licorice (Glycyrrhizae radix), inhibits platelet aggregation. This new agent significantly inhibited the phosphorylation of 40,000- and 20,000-dalton proteins, and inhibited the formation of 12 (S)-hydroxy-5,8,10-heptadecatrienoic acid, 12-hydroxyeicosatetraenoic acid and thromboxane B2. The inhibitory effect of isoliquiritigenin on platelet aggregation in vitro was comparable to that of aspirin. Our findings may indicate that isoliquiritigenin elicits an anti-platelet action by inhibiting not only cyclooxygenase but also lipoxygenase or peroxidase activity in platelets. Isoliquiritigenin also showed an anti-platelet action in vivo. Isoliquiritigenin appears to be the only aldose reductase inhibitor with a significant anti-platelet action. Since the hyperaggregability of platelets has been implicated in the pathogenesis of diabetic complications, isoliquiritigenin may offer a unique benefit as an aldose reductase inhibitor.

Z-4 allele upstream of the aldose reductase gene is associated with proliferative retinopathy in Japanese patients with NIDDM, and elevated luciferase gene transcription in vitro.

We determined by PCR the number of (A-C)n repeats in the 2. 1 kb upstream of the aldose reductase (AR2) gene in healthy control subjects and in patients with NIDDM in Japanese. Sixty-one patients were recruited based on the severity of retinopathy and subdivided into two groups with proliferative retinopathy and without retinopathy. Japanese exhibited 10 different alleles in this region. The most prevalent allele was designated Z ((A-C)24 repeats) allele. The Z-4 allele was significantly associated with patients with proliferative retinopathy, whereas the Z+2 allele was significantly associated with patients without retinopathy. Erythrocyte AR2 protein levels were significantly elevated in patients exhibiting the Z-4 allele compared to those exhibiting other alleles. When Z-4 allele was ligated in transfection experiments to luciferase vector containing the promoter region of the AR2 gene, the construct showed significantly higher transcription of the reporter gene compared to constructs without (A-C) repeat or with Z-2, Z or Z+2 alleles. Our results suggest that the Z-4 allele in the 2. 1 kb upstream of the AR2 gene may enhance gene transcription and may be a genetic risk factor, which determines the predisposition to retinopathy in Japanese patients with NIDDM.

RIG-I– and MDA5-Initiated Innate Immunity Linked With Adaptive Immunity Accelerates β-Cell Death in Fulminant Type 1 Diabetes

OBJECTIVE The contribution of innate immunity responsible for aggressive β-cell destruction in human fulminant type 1 diabetes is unclear. RESEARCH DESIGN AND METHODS Islet cell expression of Toll-like receptors (TLRs), cytoplasmic retinoic acid–inducible gene I (RIG-I)-like receptors, downstream innate immune markers, adaptive immune mediators, and apoptotic markers was studied in three autopsied pancreata obtained 2 to 5 days after onset of fulminant type 1 diabetes. RESULTS RIG-I was strongly expressed in β-cells in all three pancreata infected with enterovirus. Melanoma differentiation–associated gene-5 was hyperexpressed in islet cells, including β- and α-cells. TLR3 and TLR4 were expressed in mononuclear cells that infiltrated islets. Interferon (IFN)-α and IFN-β were strongly expressed in islet cells. Major histocompatibility complex (MHC)-class I, IFN-γ, interleukin-18, and CXC motif ligand 10 were expressed and colocalized in affected islets. CD11c+ MHC-class II+ dendritic cells and macrophage subsets infiltrated most islets and showed remarkable features of phagocytosis of islet cell debris. CD4+ forkhead box P3+ regulatory T cells were not observed in and around the affected islets. Mononuclear cells expressed the Fas ligand and infiltrated most Fas-expressing islets. Retinoic acid–receptor responder 3 and activated caspases 8, 9, and 3 were preferentially expressed in β-cells. Serum levels of IFN-γ were markedly increased in patients with fulminant type 1 diabetes. CONCLUSIONS These findings demonstrate the presence of specific innate immune responses to enterovirus infection connected with enhanced adoptive immune pathways responsible for aggressive β-cell toxicity in fulminant type 1 diabetes.

Clinical efficacy of a stable prostacyclin analog, iloprost, in diabetic neuropathy

Iloprost, a stable prostacyclin analog, was evaluated clinically for its ability to ameliorate the symptoms of peripheral neuropathy associated with diabetes. In an open, nonrandomized trial, 13 diabetic patients with neuropathy but without proliferative retinopathy received an intravenous infusion of Iloprost at a dose of 10 micrograms, at a rate of 0.1 micrograms/kg/h, twice daily for two weeks. The administration of Iloprost relieved the majority of such subjective symptoms as pain, numbness or sensation of cold and to a lesser extent, such autonomic symptoms as dizziness. In contrast, there was little evidence of objective improvement, e.g., in motor nerve conduction velocity. Iloprost treatment significantly inhibited the platelet aggregation rate stimulated by collagen in vitro. In the one patient tested, thermography revealed an increase in skin temperature by more than 2 degrees C. Side effects associated with Iloprost included headache (3 patients) or aggravation of pain in the extremities (2 patients) and could be ameliorated by slowing the infusion rate or by discontinuing the drug (one patient). Iloprost appears to be safe and effective for relieving the symptoms of diabetic neuropathy. Our results provide the rationale for a double-blind, clinical trial in larger populations of diabetics with peripheral neuropathy.

New Mitochondrial DNA Homoplasmic Mutations Associated With Japanese Patients With Type 2 Diabetes

Epidemiological studies have shown that patients with type 2 diabetes are more likely to have affected mothers than fathers and that the disease is often transmitted in a mode of maternal inheritance (1,2). Because transmission of mitochondria is exclusively maternal (3), mitochondrial DNA (mtDNA) mutations have been implicated in the maternal inheritance of diabetes (4). However, mtDNA mutation at 3243 has been reported in only 1-1.5% of patients with type 2 diabetes (5,6). Therefore we postulated that other mtDNA mutations may be associated with type 2 diabetes. Recently, using polymerase chain reaction (PCR)-restriction fragment (RF)-single-strand conformation polymorphism (SSCP) analysis (7), we confirmed 56 mtDNA mutations in Japanese subjects (8). In this study, we investigated the prevalence of 5 mtDNA mutations, including 3 mutations that we reported (8) in Japanese subjects. The study was performed in accordance with the principles of the Declaration of Helsinki. Informed consent was obtained from all subjects, and the study design was approved by the Ethical Committee of Yamanashi Medical University. Peripheral blood was obtained from 253 unrelated patients (mean age: 55.7 ± 1.8 years) with type 2 diabetes in the order of their visits to outpatient Clinic of Yamanashi Medical University Hospital. We also recruited 345 healthy control subjects (mean age: 43.6 ± 1.6 years) with no family history of diabetes and normal oral glucose tolerance or HbAlc, who visited the Isawa Curehouse or Koseiren Health Center for medical checkups. Genomic DNA was obtained from peripheral leukocytes using a DNAQuick kit (Dainippon Pharmaceutical, Osaka, Japan). mtDNA mutations were detected either by PCR-SSCP or PCR-restriction fragment length polymorphism (RFLP) analyses. Two primer pairs were used: primer pair A, for-

Ligand-bound Thyroid Hormone Receptor Contributes to Reprogramming of Pancreatic Acinar Cells into Insulin-producing Cells

Background: One goal of diabetic regenerative medicine is to convert mature pancreatic acinar cells into insulin-producing cells. Results: Ligand-bound thyroid hormone receptor α (TRα), which interacts with p85α, induces phosphatidylinositol 3-kinase (PI3K) signaling and insulin expression. Conclusion: PI3K signaling must be activated for TRα-induced reprogramming of pancreatic acinar cells. Significance: TRα is critical for postnatal expansion of the β-cell mass. One goal of diabetic regenerative medicine is to instructively convert mature pancreatic exocrine cells into insulin-producing cells. We recently reported that ligand-bound thyroid hormone receptor α (TRα) plays a critical role in expansion of the β-cell mass during postnatal development. Here, we used an adenovirus vector that expresses TRα driven by the amylase 2 promoter (AdAmy2TRα) to induce the reprogramming of pancreatic acinar cells into insulin-producing cells. Treatment with l-3,5,3-triiodothyronine increases the association of TRα with the p85α subunit of phosphatidylinositol 3-kinase (PI3K), leading to the phosphorylation and activation of Akt and the expression of Pdx1, Ngn3, and MafA in purified acinar cells. Analyses performed with the lectin-associated cell lineage tracing system and the Cre/loxP-based direct cell lineage tracing system indicate that newly synthesized insulin-producing cells originate from elastase-expressing pancreatic acinar cells. Insulin-containing secretory granules were identified in these cells by electron microscopy. The inhibition of p85α expression by siRNA or the inhibition of PI3K by LY294002 prevents the expression of Pdx1, Ngn3, and MafA and the reprogramming to insulin-producing cells. In immunodeficient mice with streptozotocin-induced hyperglycemia, treatment with AdAmy2TRα leads to the reprogramming of pancreatic acinar cells to insulin-producing cells in vivo. Our findings suggest that ligand-bound TRα plays a critical role in β-cell regeneration during postnatal development via activation of PI3K signaling.

Clinical Significance of Erythrocyte Sorbitol—Blood Glucose Ratios in Type II Diabetes Mellitus

The polyol pathway has been implicated in the pathogenesis of diabetic complications. To determine the activity of the polyol pathway, the ratio of erythrocyte sorbitol to blood glucose, which reflects aldose reductase activity, was evaluated in 329 patients with type II (non-insulin-dependent) diabetes mellitus and in 100 nondiabetic age-matched control subjects. Although erythrocyte sorbitol levels were markedly elevated, sorbitol-glucose ratios were significantly lower in diabetic patients than in nondiabetic subjects. Sorbitol-glucose ratios in diabetic patients decreased progressively as blood glucose and hemoglobin A1c (HbA1c) levels increased. Sorbitol-glucose ratios were also studied during a 75-g oral glucose tolerance test. Ratios were again lower in diabetic patients than those in nondiabetic subjects and significantly decreased 120 min after glucose loading. The ratio in diabetic patients also fell with increasing age of the patients. In diabetic patients with neuropathy, retinopathy, or nephropathy, however, sorbitol-glucose ratios were significantly higher than in those without these complications; ratios increased further as complications became more severe. Our findings suggest that the affinity of aldose reductase for glucose in patients with diabetic complications may be increased and that the polyol pathway is implicated in the pathogenesis of diabetic complications.

A type 2 diabetic patient with liver dysfunction due to human insulin

A 45-year-old man had been complaining of thirst and polydypsia for the last 3 months and was diagnosed as having type 2 diabetes mellitus because his fasting blood glucose showed 221 mg/dl with positive urinary ketone. He was hospitalized to a private hospital and Penfil 30R was started. However, serum gamma-GTP and aminotransferases began to elevate after insulin treatment and exceeded 1000 IU/l. Insulin was discontinued and serum gamma-GTP and aminotransferases returned close to the normal range. Since his glycemic control became poor again, Penfil 30R was restarted and serum gamma-GTP and aminotransferases elevated again. Therefore, insulin was discontinued and the patient was referred to the Third Department of Internal Medicine, Yamanashi Medical University Hospital because of liver dysfunction. His plasma glucose decreased by diet therapy, and improved further by the administration of glibenclamide. After obtaining informed consent, Humalin R was challenged. Seven days after insulin injection, serum aminotransferases began to elevate again. Lymphocyte stimulation test was negative against three preparations (Penfil R, Penfil N and Humalin R). The present case suggests that human insulin itself can cause liver dysfunction and we need to pay more attention to liver function tests when we start insulin treatment.

Immunopathological and genetic features in slowly progressive insulin-dependent diabetes mellitus and latent autoimmune diabetes in adults.

Abstract:  In 1982 we proposed the presence of a subtype of type 1 diabetes [slowly progressive insulin‐dependent diabetes mellitus (SPIDDM)], which was characterized by persistently positive islet cell antibody, late age of onset, noninsulin‐dependent diabetes, and slowly progressive beta cell failure. Since then many studies demonstrated that this subtype of type 1 diabetes is prevalent in many ethnic groups and was later called the latent autoimmune diabetes in adults (LADA). Recent epidemiological studies reported that about 10% of patients with apparent type 2 diabetes have at least one autoantibodies against islet‐specific antigen with high potential to progress to insulin‐dependent state. Between SPIDDM and LADA some differences are reported in terms of some genetic predispositions including HLA class II and class I genes, vitamin D receptor gene, and CTLA4 genes. Common features in SPIDDM and LADA including preserved beta cells at the onset of diabetes and weak T cell response to residual beta cells suggest that these subtypes of type 1 diabetes are suitable candidates for prevention treatment for further progression of beta cell failure.