Adjusting the 17ß-Estradiol-to-Androgen Ratio Ameliorates Diabetic Nephropathy.

Diabetes is manifested predominantly in males in experimental models, and compelling evidence suggests that 17ß-estradiol (E2) supplementation improves hyperglycemia in humans. We previously generated a severely diabetic transgenic (Tg) mouse model by ß-cell­specific overexpression of inducible cAMP early repressor (ICER) and found that male but not female ICER-Tg mice exhibit sustained hyperglycemia and develop major clinical and pathologic features of human diabetic nephropathy (DN). Thus, we hypothesized that differences in circulating hormone levels have a key role in determining susceptibility to diabetes. Here, we examined whether DN in male ICER-Tg mice is rescued by adjusting the androgen-to-E2 ratio to approximate that in normoglycemic female ICER-Tg mice. We treated hyperglycemic male ICER-Tg mice with orchiectomy (ORX), E2 pellet implantation, or both. E2 pellet implantation at an early stage of DN with or without ORX caused a rapid drop in blood glucose and a dramatic increase in ß-cell number, and it markedly inhibited DN progression [namely, E2 reduced glomerulosclerosis, collagen 4 deposition and albuminuria, and prevented hyperfiltration]. Furthermore, E2 pellet implantation was more effective than ORX alone and induced a remarkable improvement, even when initiated at advanced-stage DN. In contrast, induction of normoglycemia by islet transplant in ICER-Tg mice eliminated albuminuria but was less effective than E2 + ORX in reducing glomerulosclerosis, collagen 4 deposition, and hyperfiltration. These findings indicate that E2 treatment is effective, even after establishment of DN, whereas glucose normalization alone does not improve sclerotic lesions. We propose that E2 intervention is a potential therapeutic option for DN.

Carbonic anhydrase II-positive pancreatic cells are progenitors for both endocrine and exocrine pancreas after birth.

The regenerative process in the pancreas is of particular interest because diabetes results from an inadequate number of insulin-producing beta cells and pancreatic cancer may arise from the uncontrolled growth of progenitor/stem cells. Continued and substantial growth of islet tissue occurs after birth in rodents and humans, with additional compensatory growth in response to increased demand. In rodents there is clear evidence of pancreatic regeneration after some types of injury, with proliferation of preexisting differentiated cell types accounting for some replacement. Additionally, neogenesis or the budding of new islet cells from pancreatic ducts has been reported, but the existence and identity of a progenitor cell have been debated. We hypothesized that the progenitor cells are duct epithelial cells that after replication undergo a regression to a less differentiated state and then can form new endocrine and exocrine pancreas. To directly test whether ductal cells serve as pancreatic progenitors after birth and give rise to new islets, we generated transgenic mice expressing human carbonic anhydrase II (CAII) promoter: Cre recombinase (Cre) or inducible CreER(TM) to cross with ROSA26 loxP-Stop-loxP LacZ reporter mice. We show that CAII-expressing cells within the pancreas act as progenitors that give rise to both new islets and acini normally after birth and after injury (ductal ligation). This identification of a differentiated pancreatic cell type as an in vivo progenitor of all differentiated pancreatic cell types has implications for a potential expandable source for new islets for replenishment therapy for diabetes.

Nuclear co-expression of p21 and p27 induced effective cell-cycle arrest in T24 cells treated with BCG.

A proposed mechanism underlying the effect of bacillus Calmette-Guérin (BCG) treatment for bladder cancer cells is as follows: BCG-induced crosslinking of cell-surface receptors results in the activation of signaling cascades, including cell-cycle regulators. However, the clinical significance of cell-cycle regulators such as p21 and p27 is controversial. Here we investigated the relationship between BCG exposure and p21 and p27. We used confocal laser microscopy to examine the expression levels of pKi67, p21 and p27 in T24 cells (derived from human urothelial carcinoma) exposed six times to BCG. We performed dual immunofluorescence staining methods for p21 and p27 and observed the localization of nuclear and cytoplasm expressions. We investigated the priority of p27 over p21 regarding nuclear expression by using p27 Stealth RNAi™ (p27-siRNA). With 2-h BCG exposure, the nuclear-expression level of p21 and p27 was highest, while pKi67 was lowest. The percentage of double nuclear-expression of p21 and p27 in BCG cells was significantly higher than that in control cells during the 1st to 6th exposure (P < 0.05), and the expression of pKi67 showed the opposite of this pattern. Approximately 10% of the nuclear p21 was independent of p27, whereas the cytoplasmic p21 was dependent on p27. Our results suggested that the nuclear co-expression of p21 and p27 caused effective cell-cycle arrest, and thus the evaluation of the nuclear co-expression of p21 and p27 might help determine the effectiveness of BCG treatment.

Mitogen-activated protein kinase pathway controls autoimmune regulator (AIRE) gene expression in granulo-monocyte colony stimulating factor (GM-CSF)-stimulated myelomonocytic leukemia OTC-4 cells.

Autoimmune regulator (AIRE) gene is a responsible gene for the rare autosomal recessive autoimmune disease: autoimmune-polyendocrinopathy-candidiasis ectodermal dystrophy (APECED). Although it has been reported that AIRE is expressed in the thymic epithelial cells and monocyte-dendritic cell lineage, the regulatory mechanisms of AIRE gene expression have as yet been poorly understood. Here we show that the expression of AIRE gene was induced in granulo-monocyte colony stimulating factor (GM-CSF)-stimulated myelomonocytic leukemia OTC-4 cells. In GM-CSF-stimulated OTC-4 cells, stat5 was not phosphorylated, while mitogen-activated protein kinases (MAPKs), including MAPK kinase (MEK) 1/2 and p38 MAPK, were phosphorylated, indicating activation of MAPK pathway. In addition, the expression of AIRE gene was inhibited by specific p38 MAPK inhibitor (SB203580), whereas the expression was rather enhanced by the MEK1/2 inhibitor (U0126), suggesting that AIRE gene expression is regulated by mitogen-activated protein kinase pathway.

[B cells as key contributors in determining the level of immune responses -B-cell-targeted therapy in patients with autoimmune diseases].

The levels and types of immune responses are determined dependent on the extent of pathogen invasion, reactions to antigens mediated by macrophage-dendritic cells, T cells and antibodies. Recently, accumulating evidence suggests that B cells also play an important role in the regulation of immune responses. Here we have made a review to present a role of B cells in determining the level of immune responses and discussed about the clinical significance of B cell-targeted therapy in patients with autoimmune diseases. Type 1 diabetes is a T cell-mediated autoimmune disease characterized by the destruction of insulin-producing pancreatic beta cells. We and other groups have elucidated that B cells play a critical role in the development of insulitis and diabetes, as B-cell-deficient NOD mice are protected from developing type 1 diabetes. B cells are essential for the T cell receptor clonotype spreading of islet-infiltrating T cells, indicating that B cells may play a role in determining the level of immune responses by antigen presentation to antigen specific T cells. There are now numerous case reports and small series of clinical trials regarding rituximab therapy in autoimmune diseases, such as refractory autoimmune hemolytic anemia, IgM antibody-associated polyneuropathy, systemic lupus erythematosus and rheumatoid arthritis. Rituximab is a genetically engineered chimeric anti-CD 20 monoclonal antibody that is approved for the treatment of lymphoma. CD20 is a B-cell surface antigen that is expressed only on pre- B and mature B cells. Thus, rituximab causes a selective transient depletion of the CD20+ B -cell subpopulation. Rationale and strategy for targeting B cells in the treatment of autoimmune diseases consist of the inhibition of antigen-presentation and co-stimulation that induces T cell expansion and activation. Further careful mechanistic studies are required to develop therapies in patients with autoimmune diseases.

[A case of metabolic syndrome with coronary artery disease advanced over 13 years. Case report].

A 61-year-old man was diagnosed with obesity, diabetes mellitus, and hyperlipidemia associated with insulin resistance in 1988. His condition was complicated with asymptomatic coronary artery disease in 1992. His coronary artery disease gradually progressed during the subsequent 13 years of observation, and he underwent percutaneous coronary intervention four times and also received a coronary artery bypass graft. This is a case of metabolic syndrome with multiple risk factors for arteriosclerosis as visceral obesity, insulin resistance, diabetes, hypertension and hyperlipidemia, in which the recent rapid progression of coronary artery disease might be associated with the discontinuation of statin after coronary artery bypass graft, accompanied with hyper-LDL-choleterolemia. Patients with metabolic syndrome require most comprehensive and strict therapies against multiple risk factors.

Reduced Tyk2 gene expression in ß-cells due to natural mutation determines susceptibility to virus-induced diabetes.

Accumulating evidence suggests that viruses play an important role in the development of diabetes. Although the diabetogenic encephalomyocarditis strain D virus induces diabetes in restricted lines of inbred mice, the susceptibility genes to virus-induced diabetes have not been identified. We report here that novel Tyrosine kinase 2 (Tyk2) gene mutations are present in virus-induced diabetes-sensitive SJL and SWR mice. Mice carrying the mutant Tyk2 gene on the virus-resistant C57BL/6 background are highly sensitive to virus-induced diabetes. Tyk2 gene expression is strongly reduced in Tyk2-mutant mice, associated with low Tyk2 promoter activity, and leads to decreased expression of interferon-inducible genes, resulting in significantly compromised antiviral response. Tyk2-mutant pancreatic ß-cells are unresponsive even to high dose of Type I interferon. Reversal of virus-induced diabetes could be achieved by ß-cell-specific Tyk2 gene expression. Thus, reduced Tyk2 gene expression in pancreatic ß-cells due to natural mutation is responsible for susceptibility to virus-induced diabetes.

TYK2 Promoter Variant and Diabetes Mellitus in the Japanese.

BACKGROUND: Recently, natural mutation of Tyrosine kinase 2 (Tyk2) gene has been shown to determine susceptibility to murine virus-induced diabetes. In addition, a previous human genome-wide study suggested the type 1 diabetes (T1D) susceptibility region to be 19p13, where the human TYK2 gene is located (19p13.2). METHODS: Polymorphisms of TYK2 gene at the promoter region and exons were studied among 331 healthy controls, and 302 patients with T1D and 314 with type 2 diabetes (T2D) in the Japanese. FINDINGS: A TYK2 promoter haplotype with multiple genetic polymorphisms, which are in complete linkage disequilibrium, named TYK2 promoter variant, presenting decreased promoter activity, is associated with an increased risk of not only T1D (odds ratio (OR), 2.4; 95% confidence interval (CI), 1.2 to 4.6; P = 0.01), but also T2D (OR, 2.1; 95% CI, 1.1 to 4.1; P = 0.03). The risk is high in patients with T1D associated with flu-like syndrome at diabetes onset and also those without anti-glutamic acid decarboxylase autoantibody. INTERPRETATION: The TYK2 promoter variant is associated with an overall risk for diabetes, serving a good candidate as a virus-induced diabetes susceptibility gene in humans. FUNDING: Ministry of Education, Culture, Sports, Science and Technology and of Health, Labor and Welfare of Japan.

Acceptance of islet allografts in the liver of mice by blockade of an inducible costimulator.

BACKGROUND: An inducible costimulator (ICOS) has been found to be a novel costimulator for T-cell activation, although its precise role in transplant immunobiology remains unclear. This study determined whether ICOS plays an essential role in rejection of intrahepatic islet allografts in streptozotocin-induced diabetic mice. METHODS: Mononuclear cells in the liver of mice were isolated and examined by flow cytometry with respect to expression of ICOS in association with rejection, and the effects of in vivo treatment with an anti-ICOS antibody on survival of intrahepatic islet allografts were determined. RESULTSFlow cytometric analysis of mononuclear cells in the liver of normal untreated mice revealed that ICOS is expressed on CD4+CD3int natural killer T cells. The expression of ICOS was up-regulated on CD4+CD3bright T cells and expanded CD8 T cells in the liver in association with rejection. Posttransplant short-term administration of anti-ICOS antibody alone produced a significant prolongation of islet allograft survival. Administration of the antibody in conjunction with a subtherapeutic regimen of FK506 prevented rejection, leading to the acceptance of islet allografts. CONCLUSION: ICOS has an essential role in rejection of intrahepatic islet allografts and the blockade of ICOS interaction might be a novel approach for preventing islet allograft rejection.

Expression of AIRE gene in peripheral monocyte/dendritic cell lineage.

The responsible gene for autoimmune polyglandular syndrome type 1, known as autoimmune regulator (AIRE), was identified by positional cloning. The AIRE gene was reported to be expressed in the thymus medulla and lymph nodes. However, an expression of the AIRE gene in peripheral blood cells has not yet been reported. In the present study, we found that the AIRE gene was restrictively expressed in peripheral CD14-positive monocytes but not in CD4-positive T cells nor polymorphonuclear cells, as assessed by RT-PCR. Moreover, immunocytochemical study revealed the expression of the AIRE protein not only in CD14-positive monocytes but also in differentiated dendritic cells, cultured in RPMI1640 medium containing 800 U/ml GM-CSF, 1000 U/ml IL-4 and 100 U/ml TNF-alpha. Thus, it was concluded that the AIRE gene is restrictively expressed in the peripheral monocyte/dendritic cell lineage.

A delayed type hypersensitivity (DTH) skin reaction to hepatitis B surface antigen (HBsAg) and intradermal hepatitis B vaccination.

The significance of a delayed type hypersensitivity skin reaction to hepatitis B surface antigen (HBsAg) (HBs-DTH) in type B viral hepatitis (VHB) and in intradermal hepatitis B (HB) vaccination is reviewed. HBs-DTH could be developed by the intradermal injection of HB vaccine in anti-HBs positive people and also in persons immunized with HB vaccine. Thus, HBs-DTH could serve as a useful marker for the acquisition of an active Th1 type immunoreactivity to HBsAg. HBs-DTH was absent in patients with chronic VHB. In contrast, HBs-DTH developed early in the convalescent phase of the acute VHB, whereas the production of anti-HBs was significantly delayed, thus suggesting that HBs-DTH may be involved in the recovery mechanisms of acute VHB. Intradermal HB vaccination is useful not only in lowering the cost, but also in the rapid development of anti-HBs, reversing non-responsiveness, improving postexposure prophylaxis and in immunizing immunosuppressed people. A similar vaccination strategy should prove to be useful in prevention and control of not only other infectious diseases but also malignant neoplasms.

Birth and death of human ß-cells in pancreases from cadaver donors, autopsies, surgical specimens, and islets transplanted into mice.

There is great interest in the potential of the human endocrine pancreas for regeneration by ß-cell replication or neogenesis. Our aim was to explore this potential in adult human pancreases and in both islet and exocrine tissue transplanted into mice. The design was to examine pancreases obtained from cadaver donors, autopsies, and fresh surgical specimens and compare these findings with those obtained from islet and duct tissue grafted into the kidney. Islets and exocrine tissue were transplanted into normoglycemic ICR-SCID mice and studied 4 and 14 weeks later. ß-Cell replication, as assessed by double staining for insulin and Ki67, was 0.22 ± 0.03% at 4 weeks and 0.13 ± 0.03% at 14 weeks. In contrast, no evidence of ß-cell replication could be found in 11 cadaver donor and 10 autopsy pancreases. However, Ki67 staining of ß-cells in frozen sections obtained at surgery was comparable to that found in transplanted islets. Evidence for neogenesis in transplanted pancreatic exocrine tissue was supported by finding ß-cells within the duct epithelium and the presence of cells double stained for insulin and cytokeratin 19 (CK19). However, ß-cells within the ducts never constituted more than 1% of the CK19-positive cells. With confocal microscopy, 7 of 12 examined cells expressed both markers, consistent with a neogeneic process. Mice with grafts containing islet or exocrine tissue were treated with various combinations of exendin-4, gastrin, and epidermal growth factor; none increased ß-cell replication or stimulated neogenesis. In summary, human ß-cells replicate at a low level in islets transplanted into mice and in surgical pancreatic frozen sections, but rarely in cadaver donor or autopsy pancreases. The absence of ß-cell replication in many adult cadaver or autopsy pancreases could, in part, be an artifact of the postmortem state. Thus, it appears that adult human ß-cells maintain a low level of turnover through replication and neogenesis.

Liver-derived systemic factors drive ß cell hyperplasia in insulin-resistant states.

Integrative organ crosstalk regulates key aspects of energy homeostasis, and its dysregulation may underlie metabolic disorders such as obesity and diabetes. To test the hypothesis that crosstalk between the liver and pancreatic islets modulates ß cell growth in response to insulin resistance, we used the liver-specific insulin receptor knockout (LIRKO) mouse, a unique model that exhibits dramatic islet hyperplasia. Using complementary in vivo parabiosis and transplantation assays, as well as in vitro islet culture approaches, we demonstrate that humoral, nonneural, non-cell-autonomous factor(s) induces ß cell proliferation in LIRKO mice. Furthermore, we report that a hepatocyte-derived factor(s) stimulates mouse and human ß cell proliferation in ex vivo assays, independent of ambient glucose and insulin levels. These data implicate the liver as a critical source of ß cell growth factor(s) in insulin-resistant states.

Regulation of human autoimmune regulator (AIRE) gene translation by miR-220b.

Although mutations of autoimmune regulator (AIRE) gene are responsible for autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), presenting a wide spectrum of many characteristic and non-characteristic clinical features, some patients lack AIRE gene mutations. Therefore, something other than a mutation, such as dysregulation of AIRE gene, may be a causal factor for APECED or its related diseases. However, regulatory mechanisms for AIRE gene expression and/or translation have still remained elusive. We found that IL-2-stimulated CD4(+) T (IL-2T) cells showed a high expression of AIRE gene, but very low AIRE protein production, while Epstein-Barr virus-transformed B (EBV-B) cells express both AIRE gene and AIRE protein. By using microarray analysis, we could identify miR-220b as a possible regulatory mechanism for AIRE gene translation in IL-2T cells. Here we report that miR-220b significantly reduced the expression of AIRE protein in AIRE gene with 3'UTR region transfected 293T cells, whereas no alteration of AIRE protein production was observed in the open reading frame of AIRE gene alone transfected cells. In addition, anti-miR-220b reversed the inhibitory function of miR-220b for the expression of AIRE protein in AIRE gene with 3'UTR region transfected cells. Moreover, when AIRE gene transfected cells with mutated 3'UTR were transfected with miR-220b, no reduction of AIRE protein production was observed. Taken together, it was concluded that miR-220b inhibited the AIRE gene translation through the 3'UTR region of AIRE gene, indicating that miR-220b could serve as a regulator for human AIRE gene translation.

Subpopulations of GFP-marked mouse pancreatic ß-cells differ in size, granularity, and insulin secretion.

There is growing information about the heterogeneity of pancreatic ß-cells and how it relates to insulin secretion. This study used the approach of flow cytometry to sort and analyze ß-cells from transgenic mice expressing green fluorescent protein (GFP) under the control of the mouse insulin I gene promoter. Three populations of ß-cells with differing GFP brightness could be identified, which were classified as GFP-low, GFP-medium, and GFP-bright. The GFP-medium population comprised about 70% of the total. The GFP-low population had less insulin secretion as determined by the reverse hemolytic plaque assay and reduced insulin gene expression. Additionally, all three subpopulations of ß-cells were found in mice of varying ages (embryonic d 15.5 and postnatal wk 1-9). The three populations from the youngest had larger cells (forward scatter) and less granularity (side scatter) than those from the adults. This approach opens up new ways to advance knowledge about ß-cell heterogeneity.

Protective unfolded protein response in human pancreatic beta cells transplanted into mice.

BACKGROUND: There is great interest about the possible contribution of ER stress to the apoptosis of pancreatic beta cells in the diabetic state and with islet transplantation. METHODS AND FINDINGS: Expression of genes involved in ER stress were examined in beta cell enriched tissue obtained with laser capture microdissection (LCM) from frozen sections of pancreases obtained from non-diabetic subjects at surgery and from human islets transplanted into ICR-SCID mice for 4 wk. Because mice have higher glucose levels than humans, the transplanted beta cells were exposed to mild hyperglycemia and the abnormal environment of the transplant site. RNA was extracted from the LCM specimens, amplified and then subjected to microarray analysis. The transplanted beta cells showed an unfolded protein response (UPR). There was activation of many genes of the IRE-1 pathway that provide protection against the deleterious effects of ER stress, increased expression of ER chaperones and ERAD (ER-associated protein degradation) proteins. The other two arms of ER stress, PERK and ATF-6, had many down regulated genes. Downregulation of EIF2A could protect by inhibiting protein synthesis. Two genes known to contribute to apoptosis, CHOP and JNK, were downregulated. CONCLUSIONS: Human beta cells in a transplant site had UPR changes in gene expression that protect against the proapoptotic effects of unfolded proteins.

Adult mouse intrahepatic biliary epithelial cells induced in vitro to become insulin-producing cells.

Transdifferentiation of cells from a patient's own liver into pancreatic beta-cells could be useful for beta-cell replacement. We hypothesized that intrahepatic biliary epithelial cells (IHBECs) could become a new source of insulin-producing cells. IHBECs isolated from adult mice were expanded using our novel culture method termed, collagen-embedded floating culture method (CEFCM). With CEFCM, IHBECs formed three-dimensional ductal cysts and rapidly expanded their number by about 15-fold within 2 weeks. Over 90% of cells were positive for cytokeratin 7 and 19. At day 14, IHBECs were transfected with adenoviral (Ad)- pancreas duodenum homeobox 1 (Pdx-1), NeuroD or Pdx-1/VP16. After 7 additional days in serum- and insulin-free differentiation medium (DM), cell phenotypes were determined by RT-PCR, immunostaining and ELISA for insulin. In DM control IHBECs started to express some endocrine progenitor genes (Neurog3, NeuroD, Nkx6.1, and Pdx-1) but lacked insulin gene (Ins) mRNA. Transduced expression of PDX-1, NEUROD or PDX-1/VP16 led to expression of not only INS but also GLUT2 and prohormone convertase 1 and 2. About 3% of 4000 cells counted in PDX-1/VP16 transduced cultures stained strongly for C-peptide suggesting that a subpopulation may have the capacity for differentiation. Transduced cells released insulin (Ad-PDX-1 0.08+/-0.05, Ad-NEUROD 0.33+/-0.09, Ad-PDX-1/VP16 0.37+/-0.14 ng/1x10(5) cells after 48 h in culture). IHBECs can be markedly expanded, and then with molecular manipulation a subpopulation of these cells can differentiate towards a beta-cell phenotype. This approach may lead to a new source of beta-cells that can be used for transplantation in diabetes.

Adverse effects of obesity on ß-cell function in Japanese subjects with normal glucose tolerance.

SUMMARY: The purpose of the present study was to elucidate the role of obesity in both early- and late-phase insulin secretion during an oral glucose tolerance test (OGTT) performed with 75 g glucose in Japanese subjects. This was performed using indices of ß-cell function adjusted for insulin sensitivity. Of 155 subjects assessed, 68 had normal glucose tolerance (NGT) and 87 had impaired glucose tolerance (IGT). We used the homeostasis model assessment-insulin resistance (HOMA-IR) index as an indicator of insulin sensitivity. As indicators of ß-cell function, we used the HOMA-ß index, an insulinogenic index (ΔI30/ΔG30), and ΔAUC I/G(0-120), which were obtained in the OGTT. We then reevaluated the results after adjusting the ß-cell function for insulin sensitivity ([ΔI30/ΔG30]/HOMA-IR index and [ΔAUC I/G(0-120)]/HOMA-IR index). ß-Cell function was observed to reduce as the glucose tolerance deteriorated from NGT to IGT. However, when the effects of obesity were considered, the obese subjects with NGT already showed a decline in the (ΔAUC I/G(0-120))/HOMA-IR index value when compared with the nonobese subjects with NGT, despite the fact these subjects did not differ with regard to (ΔI30/ΔG30)/HOMA-IR index. As the glucose tolerance deteriorated to IGT, both (ΔI30/ΔG30)/HOMA-IR index and (ΔAUC I/G(0-120))/HOMA-IR index decreased to an identical extent in both subgroups. These data indicate that obesity causes a decrease in insulin secretion, especially during the late phase following a glucose load, even if the glucose tolerance remains normal.: