Hisafumi Yasuda

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.

A possible association of Pro12Ala polymorphism in peroxisome proliferator–activated receptor γ2 gene with obesity in native Javanese in Indonesia

Peroxisome proliferators–activated receptor γ (PPAR γ) is a nuclear hormone receptor that serves as a master regulator of adipocytes‐specific genes contributing to adipocytes differentiation, susceptibility to obesity, and insulin sensitivity. The substitution of proline to alanine at codon 12 of the PPAR γ2 gene (Pro12Ala polymorphism) is most frequently identified and the associations with diabetes, obesity, and other clinical parameters have been reported and discussed in several ethnic groups. Among native Javanese ethnicity, however, there is no report about this polymorphism.

T lymphocyte response against pancreatic beta cell antigens in fulminant Type 1 diabetes

Aims/hypothesisFulminant Type 1 diabetes is a novel subtype of Type 1 diabetes that involves the abrupt onset of insulin-deficient hyperglycaemia. This subtype appears to be non-autoimmune because of the absence of diabetes-related autoantibodies in the serum, and of insulitis in pancreatic biopsy specimens. The pathogenesis of the disease is still unknown. In this study, we investigated whether T cell autoimmune responses are involved in fulminant Type 1 diabetes.MethodsCellular immune responses to beta cell autoantigens were studied by enzyme-linked immunospot (ELISPOT) assay in 13 fulminant Type 1 diabetic patients and 49 autoantibody-positive autoimmune Type 1 diabetic patients. Results were compared with those of 18 Type 2 diabetic patients, six secondary diabetic patients (diabetes due to chronic pancreatitis) and 35 healthy controls.ResultsNine of 13 (69.2%) GAD-reactive Th1 cells, and three of 12 (25%) insulin-B9-23-reactive Th1 cells were identified in fulminant Type 1 diabetic patients by ELISPOT, as in autoantibody-positive Type 1 diabetic patients. Four fulminant Type 1 diabetic patients possessed the highly diabetes-resistant allele DR2, three of whom had GAD-reactive Th1 cells in the periphery.Conclusions/interpretationPeripheral immune reaction was observed in 69.2% of fulminant Type 1 diabetic patients, indicating that autoreactive T cells might contribute, at least in part, to the development of fulminant Type 1 diabetes.

CD8 cytotoxic T-cell clone rapidly transfers autoimmune diabetes in very young NOD and MHC class I-compatible scid mice.

Summary A CD8 T-cell clone (YNK1.3) generated from acutely diabetic NOD mouse islets, showed proliferation and cytotoxicity when challenged with NOD and BALB/c islet cells and NOD-derived insulinoma cells. When 1–2 × 107 YNK1.3 cells were administered to 7–10-day-old NOD mice, the cells transferred overt diabetes very rapidly in each of the 16 recipients within 4 days of cell transfer. However, of 14 recipients receiving YNK 1.3 cells above 14 days of age none became diabetic. Fluorescent dye-labelled YNK1.3 cells extensively accumulated in the islets by 36 h after transfer in 7-day-old NOD recipients, while no significant insulitis was seen in 21-day-old recipients. Over half of NOD-scid recipients (5/9) rapidly became diabetic within 5 days after transfer of 1–2 × 107 YNK1.3 cells at 7 days of age, whereas only one of 12 recipients over 14 days of age became diabetic. Furthermore, YNK1.3 cells also transferred diabetes to H-2Kd-matched very young BALB/c-scid and CB17-scid mice, but not to C57BL/6-scid mice. Thus, optimally activated islet-specific CD8 T-cell clones are able to rapidly transfer diabetes to NOD and MHC class I compatible scid mice when a large enough number is administered at 7 days of age. Administration of monoclonal antibodies against adhesion molecules involved in the trafficking of lymphocytes from the circulation into the inflammatory tissues, could not prevent the cellular infiltration of YNK1.3 cells into the islets in 7-day-old NOD recipients. The results indicate that islet cells in the mouse around 7 days of age are generally susceptible to cytotoxic CD8 T cells, suggesting, therefore, that CD8 T cells may play an important role in the initiation of autoimmune diabetes in NOD mice. [Diabetologia (1997) 40: 1044–1052]

Regulatory CD8+ T cells induced by exposure to all-trans retinoic acid and TGF-β suppress autoimmune diabetes

Antigen-specific regulatory CD4(+) T cells have been described but there are few reports on regulatory CD8(+) T cells. We generated islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific regulatory CD8(+) T cells from 8.3-NOD transgenic mice. CD8(+) T cells from 8.3-NOD splenocytes were cultured with IGRP, splenic dendritic cells (SpDCs), TGF-beta, and all-trans retinoic acid (ATRA) for 5days. CD8(+) T cells cultured with either IGRP alone or IGRP and SpDCs in the absence of TGF-beta and ATRA had low Foxp3(+) expression (1.7+/-0.9% and 3.2+/-4.5%, respectively). In contrast, CD8(+) T cells induced by exposure to IGRP, SpDCs, TGF-beta, and ATRA showed the highest expression of Foxp3(+) in IGRP-reactive CD8(+) T cells (36.1+/-10.6%), which was approximately 40-fold increase compared with that before induction culture. CD25 expression on CD8(+) T cells cultured with IGRP, SpDCs, TGF-beta, and ATRA was only 7.42%, whereas CD103 expression was greater than 90%. These CD8(+) T cells suppressed the proliferation of diabetogenic CD8(+) T cells from 8.3-NOD splenocytes in vitro and completely prevented diabetes onset in NOD-scid mice in cotransfer experiments with diabetogenic splenocytes from NOD mice in vivo. Here we show that exposure to ATRA and TGF-beta induces CD8(+)Foxp3(+) T cells ex vivo, which suppress diabetogenic T cells in vitro and in vivo.

Development of fulminant Type 1 diabetes with thrombocytopenia after influenza vaccination: a case report

Diabet. Med. 29, 88–89 (2012)

NO-mediated cytotoxicity contributes to multiple low-dose streptozotocin-induced diabetes but not to NOD diabetes

Type 1 diabetes (T1D) is caused mostly by autoimmune destruction of pancreatic beta-cells, the precise mechanism of which remains unclear. Two major effector mechanisms have been proposed: direct cell-mediated and indirect cytokine-mediated cytotoxicity. Cytokine-mediated beta-cell destruction is presumed mainly caused by NO production. To evaluate the role of iNOS expression in T1D, this study used a novel iNOS inhibitor ONO-1714. ONO-1714 significantly reduced cytokine-mediated cytotoxicity and NO production in both MIN6N9a cells and C57BL/6 islets in the presence of IL-1beta, TNF-alpha, and IFN-gamma. To evaluate whether NO contributes to diabetes progression in vivo, ONO-1714 was administered to four different mouse models of autoimmune diabetes: multiple low-dose STZ (MLDS)-induced C57BL/6, CY-induced, adoptive transfer and spontaneous NOD diabetes. Exposure to STZ in vitro induced NO production in MIN6N9a cells and C57BL/6 islets, and in vivo injection of ONO-1714 to MLDS-treated mice significantly reduced hyperglycemia and interestingly, led to complete suppression of cellular infiltration of pancreatic islets. In contrast, when ONO-1714 was injected into spontaneous NOD mice and CY-induced and adoptive transfer models of NOD diabetes, overt diabetes could not be inhibited in these models. These findings suggest that NO-mediated cytotoxicity significantly contributes to MLDS-induced diabetes but not to NOD diabetes.

Elevated plasma nitrate in patients with crush syndrome caused by the Kobe earthquake

We investigated the nitric oxide profile in the plasma of ten healthy controls and 29 patients hurt by the Kobe Earthquake. The levels of nitrite (NO2-) and nitrate (NO3-) were measured simultaneously by high-performance liquid chromatography (HPLC) with UV detection using the Griess reaction after the reduction of nitrate to nitrite. Arginine consumed in food or diet-derived nitrite had no effect on the plasma nitrite and nitrate contents of the healthy volunteers. Plasma nitrate was elevated in the crush syndrome patients; whereas neither nitrite nor nitrate was increased in patients with normal renal function. This finding suggests increased nitric oxide synthesis, decreased excretion of nitric oxide in the crush syndrome or both.

Role of the mTOR complex 1 pathway in the in vivo maintenance of the intestinal mucosa by oral intake of amino acids

Aim:  Oral intake of nutrients is often compromised in elderly, multimorbid patients, but parenteral nutrition causes intestinal atrophy and impairs intestinal function. To uncover the molecular mechanisms by which amino acids are involved in intestinal atrophy and recovery, we studied whether the rapamycin‐sensitive mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) pathway is involved in this process.

Administration of a determinant of preproinsulin can induce regulatory T cells and suppress anti-islet autoimmunity in NOD mice.

Antigen-specific immunotherapy is expected to be an ideal strategy for treating type 1 diabetes (T1D). We investigated the therapeutic efficacy of a peptide in the leader sequence of preproinsulin, which was selected because of its binding affinity to the MHC I-A(g7) molecule. Preproinsulin-1 L7-24 peptide (L7-24) emulsified in Freunds incomplete adjuvant was administered subcutaneously to NOD mice. Administration of L7-24 increased the proportion of regulatory T cells in the spleen. Splenocytes of NOD mice immunized with this peptide secreted IL-4 and IL-10 in response to L7-24. This peptide also significantly prevented the development of diabetes and cured some newly diabetic NOD mice without recurrence. L7-24 peptide, which has a high affinity for pockets of I-A(g7), induced regulatory T cells and showed therapeutic effects. This peptide may provide a new approach for developing antigen-specific immunotherapy for autoimmune diabetes.