dietary supplementation with high doses of regular vitamin d3 safely reduces diabetes incidence in nod mice when given early and long term

High doses of the active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) prevent diabetes in the non-obese diabetic (NOD) mouse but also elicit unwanted calcemic side-effects. Because immune cells themselves can convert vitamin D3 into 1,25(OH)2D3 locally, we hypothesized that dietary vitamin D3 can also prevent disease. Thus, we evaluated whether dietary administration of high doses of regular vitamin D3 (800 IU per day) during different periods of life (pregnancy and lactation, early-life (3-14 weeks of age), or lifelong (3-35 weeks of age)) safely prevents diabetes in NOD mice. We found that only lifelong treatment raised serum 25-hydroxyvitamin D3 from 173 nmol/L in controls to 290 nmol/L, without inducing signs of calcemic or bone toxicity, and significantly reduced diabetes development in both male and female NOD mice. This diabetes protection by vitamin D3 correlated with preserved pancreatic insulin content and improved insulitis scores. Moreover, vitamin D3 treatment decreased interferon-γ-positive CD8+ T-cells and increased CD4+(CD25+)FoxP3+ T-cells in pancreatic draining lymph nodes. In conclusion, this study shows for the first time that high doses of regular dietary vitamin D3 can safely prevent diabetes in NOD mice when administered lifelong, although caution is warranted with regards to administering equivalently high doses in humans

The active enhancer network operated by liganded RXR supports angiogenic activity in macrophages

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Increased β-Cell Mass by Islet Transplantation and PLAG1 Overexpression Causes Hyperinsulinemic Normoglycemia and Hepatic Insulin Resistance in Mice

OBJECTIVE-It is believed that an organism remains normoglycemic despite an increase in the beta-cell mass because of decreased insulin production by beta-cells on a per-cell basis However, some transgenic mouse models with beta-cell hyperplasia suggest that insulin production remains excessive and that normoglycemia is maintained by insulin resistance METHODS-Here, we investigated the effect of an increased beta-cell mass on glycemia and insulin resistance by grafting excess normal islets in normoglycemic mice, as well as using targeted PLAG1 expression in beta-cells, which leads to beta-cell expansion. RESULTS-In both models, fasting plasma insulin levels were increased, even though animals were normoglycemic. After an intraperitoneal glucose tolerance test, plasma insulin levels increased, which was associated with improved glucose clearing Under these conditions, normoglycemia is maintained by hepatic insulin resistance as demonstrated by hyperinsulinemic euglycemic clamp experiments. CONCLUSIONS-In conclusion, we demonstrate that when excess beta-cells are grafted, insulin production on a per beta-cell basis is not sufficiently decreased, leading to hyperinsulinemia and hepatic insulin resistance. This observation might be important for the design of stem cell-based islet replacement therapies. Diabetes 59:1957-1965, 2010Diabetes mellitus: pathophysiological changes and therap

Tumor hypoxia does not drive differentiation of tumor-associated macrophages but rather fine-tunes the M2-like macrophage population

Tumor-associated macrophages (TAM) are exposed to multiple microenvironmental cues in tumors, which collaborate to endow these cells with protumoral activities. Hypoxia, caused by an imbalance in oxygen supply and demand because of a poorly organized vasculature, is often a prominent feature in solid tumors. However, to what extent tumor hypoxia regulates the TAM phenotype in vivo is unknown. Here, we show that the myeloid infiltrate in mouse lung carcinoma tumors encompasses two morphologically distinct CD11b(hi)F4/80(hi)Ly6C(lo) TAM subsets, designated as MHC-II(lo) and MHC-II(hi) TAM, both of which were derived from tumor-infiltrating Ly6C(hi) monocytes. MHC-II(lo) TAM express higher levels of prototypical M2 markers and reside in more hypoxic regions. Consequently, MHC-II(lo) TAM contain higher mRNA levels for hypoxia-regulated genes than their MHC-II(hi) counterparts. To assess the in vivo role of hypoxia on these TAM features, cancer cells were inoculated in prolyl hydroxylase domain 2 (PHD2)-haplodeficient mice, resulting in better-oxygenated tumors. Interestingly, reduced tumor hypoxia did not alter the relative abundance of TAM subsets nor their M2 marker expression, but specifically lowered hypoxia-sensitive gene expression and angiogenic activity in the MHC-II(lo) TAM subset. The same observation in PHD2(+/+) → PHD2(+/-) bone marrow chimeras also suggests organization of a better-oxygenized microenvironment. Together, our results show that hypoxia is not a major driver of TAM subset differentiation, but rather specifically fine-tunes the phenotype of M2-like MHC-II(lo) TAM

Reversal of hyperglycemia by insulin-secreting rat bone marrow- and blastocyst-derived hypoblast stem cell-like cells

β-cell replacement may efficiently cure type 1 diabetic (T1D) patients whose insulin-secreting β-cells have been selectively destroyed by autoantigen-reactive T cells. To generate insulin-secreting cells we used two cell sources: rat multipotent adult progenitor cells (rMAPC) and the highly similar rat extra-embryonic endoderm precursor (rXEN-P) cells isolated under rMAPC conditions from blastocysts (rHypoSC). rMAPC/rHypoSC were sequentially committed to definitive endoderm, pancreatic endoderm, and β-cell like cells. On day 21, 20% of rMAPC/rHypoSC progeny expressed Pdx1 and C-peptide. rMAPCr/HypoSC progeny secreted C-peptide under the stimulus of insulin agonist carbachol, and was inhibited by the L-type voltage-dependent calcium channel blocker nifedipine. When rMAPC or rHypoSC differentiated d21 progeny were grafted under the kidney capsule of streptozotocin-induced diabetic nude mice, hyperglycemia reversed after 4 weeks in 6/10 rMAPC- and 5/10 rHypoSC-transplanted mice. Hyperglycemia recurred within 24 hours of graft removal and the histological analysis of the retrieved grafts revealed presence of Pdx1-, Nkx6.1- and C-peptide-positive cells. The ability of both rMAPC and HypoSC to differentiate to functional β-cell like cells may serve to gain insight into signals that govern β-cell differentiation and aid in developing culture systems to commit other (pluripotent) stem cells to clinically useful β-cells for cell therapy of T1D

The role of interferon regulatory factor-1 in cytokine-induced mRNA expression and cell death in murine pancreatic beta-cells.

Combinations of cytokines, including interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma), induce nitric oxide (NO) production and cell death in pancreatic islet cells. We have previously shown that these events are preceded by increased expression of the transcription factor interferon regulatory factor-1 (IRF-1). We utilized an IRF-1 knockout mouse (IRF-1-/-) to investigate the role of IRF-1 in cytokine-induced islet- and beta-cell gene expression and cell death. For this purpose, pancreatic islets or FACS-purified beta-cells were isolated from wild type (wt) or IRF-1-/- mice. These cells were exposed for different time points to IL-1beta (50 U/mI), IFN-gamma (1,000 U/ml) and/or TNF-alpha (1,000 U/ml) before being harvested for determination of viability (by nuclear dyes) and mRNA expression (by RT-PCR with specific primers). Following a 24 hours exposure to IL-1beta or IL-1beta + IFN-gamma, pancreatic islets isolated from IRF-1-/- mice presented a 30-50% reduction in medium nitrite accumulation and inducible NO-synthase (iNOS) expression. Interestingly, both wt and IRF-1-/- purified beta-cells failed to produce NO in response to IL-1beta alone, but presented a similar increase in nitrite accumulation and iNOS expression following exposure to IL-1beta + IFN-gamma. The basal expression of MHC class I mRNA was lower in IRF-1-/- islet cells (30% reduction), but there was a similar 2-4 fold-increase in MHC expression in islet cells from both strains following cytokine exposure. IL-1beta induced serine protease inhibitor-3 (SPI-3; a putative cellular "defense" protein) mRNA expression in both wt and IRF-1-/- islets or beta-cells. IFN-gamma decreased the IL-1beta-induced SPI-3 expression in wt islets or beta-cells, but induced a 5-fold increase in the expression of this mRNA in IRF-1-/- islets cells, suggesting that IRF-1 mediates an inhibitory effect of IFN-gamma on SPI-3 expression. Treatment of whole islets for 3 days with IL-1beta + IFN-gamma induced significantly more islet cell death in wt than in IRF-1-/- mice (respectively 85 +/- 3% versus 31 +/- 4% dead cells). On the other hand, prolonged exposure (3-9 days) of FACS-purified beta-cells to the same cytokines, or a combination of 3 cytokines, led to a similar increase in cell death in both IRF-1-/- and wt islets. In conclusion, IRF-1 contributes to cytokine-induced islet iNOS expression and cell death. These effects are absent in purified beta-cells, suggesting that IRF-1 may mediate its effects on whole islets via activation of non-endocrine cells (e.g. macrophages and ductal cells) present in these preparations.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Dual role of interferon-gamma signalling pathway in sensitivity of pancreatic beta cells to immune destruction.

AIMS/HYPOTHESIS: Disruption of the interferon-gamma (IFN-gamma) signalling pathway at the level of interferon regulatory factor-1 (IRF-1) protects islets against cytokine-induced nitric oxide production and cell death in vitro. The aim of this study was to investigate the effects of a global disruption of IFN-gamma signalling, or a selective disruption of IRF-1, on beta-cell sensitivity to in vivo immune destruction. METHODS: In a first set of experiments, IFN-gamma receptor knockout mice (IFN-gammaR-/-) and interferon regulatory factor-1 knockout mice (IRF-1-/-) were rendered diabetic by injections of 50 mg streptozotocin i. p. on 5 consecutive days (MLDSTZ). RESULTS: Whereas no difference in sensitivity to MLDSTZ-induced diabetes could be observed between IFN-gammaR-/- mice and their 129/Sv/Ev controls (50% vs 55%, NS), there was an increased incidence of diabetes in IRF-1-/- mice (100% vs 67% in C57B1/6 mice, p < 0.05). A similar increased sensitivity to immune destruction of IRF-1-/- islets was observed when these islets were used as allografts. Islet graft survival rate of IFN-gammaR-/- and 129/Sv/Ev islets, when transplanted in alloxan-diabetic BALB/c recipients, was comparable (12.0 +/- 1.9 days vs 12.9 +/- 2.3 days, NS). Allograft rejection, however, of IRF-1-/- islets by BALB/c recipients occurred more rapidly than following transplantation to their C57B1/6 controls (9.1 +/- 2.0 days vs 13.1 +/- 1.5 days, p < 0.003). CONCLUSIONS/INTERPRETATION: These data indicate that IFN-gamma signal transduction at the beta-cell level is not essential for immune beta-cell destruction in vivo. Moreover, disruption of the IRF-1 gene in pancreatic islets increases susceptibility to beta-cell killing, suggesting that IRF-1 might be necessary for the expression of putative beta-cell "defence and/or repair" genes.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Interferon regulatory factor-1 is a key transcription factor in murine beta cells under immune attack.

AIMS/HYPOTHESIS: IFN-gamma, together with other inflammatory cytokines such as IL-1beta and TNF-alpha, contributes to beta cell death in type 1 diabetes. We analysed the role of the transcription factor interferon regulatory factor (IRF)-1, a downstream target of IFN-gamma/signal transducer and activator of transcription (STAT)-1, in immune-mediated beta cell destruction. METHODS: Islets from mice lacking Irf-1 (Irf-1 (-/-)) and control C57BL/6 mice were transplanted in overtly diabetic NOD mice. Viability and functionality of islets were evaluated in vitro. Chemokine expression by Irf-1 (-/-) islets and INS-1E cells transfected with Irf-1 short interfering RNA (siRNA) was measured by real-time PCR as well as in functional assays in vitro. RESULTS: IRF-1 deletion in islets was associated with higher prevalence of primary non-function (63% vs 25%, p <or= 0.05) and shorter functioning graft survival (6.0 +/- 2.6 vs 10.4 +/- 4.8 days, p <or= 0.05) in contrast to similar skin graft survival. Although Irf-1 (-/-) islets were resistant to cytokine-induced cell death, insulin secretion by them was lower than that of control C57BL/6 islets under medium and cytokine conditions. IL-1 receptor antagonist partly restored the cytokine-induced secretory defect in vitro and completely prevented primary non-function in vivo. Cytokine-exposed Irf-1 (-/-) islets and INS-1E cells transfected with Irf-1 siRNA showed increased expression of Mcp-1 (also known as Ccl2), Ip-10 (also known as Cxcl10), Mip-3alpha (also known as Ccl20) and Inos (also known as Nos2) mRNA and elevated production of monocyte chemoattractant protein-1 (MCP-1) and nitrite compared with controls. In vivo, Irf-1 (-/-) islets displayed a higher potential to attract immune cells, reflected by more aggressive immune infiltration in the grafted islets. CONCLUSIONS/INTERPRETATION: These data indicate a key regulatory role for IRF-1 in insulin and chemokine secretion by pancreatic islets under inflammatory attack.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Deletion of STAT-1 pancreatic islets protects against streptozotocin-induced diabetes and early graft failure but not against late rejection.

OBJECTIVE: Exposure of beta-cells to inflammatory cytokines leads to apoptotic cell death through the activation of gene networks under the control of specific transcription factors, such as interferon-gamma-induced signal transducer and activator of transcription (STAT)-1. We previously demonstrated that beta-cells lacking STAT-1 are resistant to cytokine-induced cell death in vitro. The aim of this study was to investigate the effect of STAT-1 elimination on immune-mediated beta-cell destruction in vivo. RESEARCH DESIGN AND METHODS: Multiple low-dose streptozotocin (STZ) was given to C57BL/6 mice after syngeneic STAT-1(-/-) or wild-type islet transplantation. STAT-1(-/-) and wild-type islets were also transplanted in alloxan-diabetic BALB/c and spontaneously diabetic nonobese diabetic (NOD) mice. Additionally, mice were treated with interleukin (IL)-1 blockade (IL-1 receptor antagonist [IL-1ra]) and low-dose T-cell suppression (cyclosporine A [CsA]). RESULTS: When exposed to multiple low-dose STZ in an immune-competent host, STAT-1(-/-) islets were more resistant to destruction than wild-type islets (28 vs. 100% diabetes incidence, P < or = 0.05). STAT-1 deletion also protected allogeneic islet grafts against primary nonfunction in autoimmune NOD mice (0 vs. 17% using wild-type islets). However, no difference in survival time was observed. Additionally, treating recipients with IL-1ra and CsA prolonged graft survival in chemically diabetic BALB/c mice, whereas no difference was seen between STAT-1(-/-) and C57BL/6 grafts. CONCLUSIONS: These data indicate that STAT-1 is a key player in immune-mediated early beta-cell dysfunction and death. When considering the many effector mechanisms contributing to beta-cell death following islet transplantation, multiple combined interventions will be needed for prolongation of beta-cell survival in the autoimmune context of type 1 diabetes.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Deletion of STAT-1 pancreatic islets protects against streptozotocin-induced diabetes and early graft failure but not against late rejection

OBJECTIVE: Exposure of beta-cells to inflammatory cytokines leads to apoptotic cell death through the activation of gene networks under the control of specific transcription factors, such as interferon-gamma-induced signal transducer and activator of transcription (STAT)-1. We previously demonstrated that beta-cells lacking STAT-1 are resistant to cytokine-induced cell death in vitro. The aim of this study was to investigate the effect of STAT-1 elimination on immune-mediated beta-cell destruction in vivo. RESEARCH DESIGN AND METHODS: Multiple low-dose streptozotocin (STZ) was given to C57BL/6 mice after syngeneic STAT-1(-/-) or wild-type islet transplantation. STAT-1(-/-) and wild-type islets were also transplanted in alloxan-diabetic BALB/c and spontaneously diabetic nonobese diabetic (NOD) mice. Additionally, mice were treated with interleukin (IL)-1 blockade (IL-1 receptor antagonist [IL-1ra]) and low-dose T-cell suppression (cyclosporine A [CsA]). RESULTS: When exposed to multiple low-dose STZ in an immune-competent host, STAT-1(-/-) islets were more resistant to destruction than wild-type islets (28 vs. 100% diabetes incidence, P < or = 0.05). STAT-1 deletion also protected allogeneic islet grafts against primary nonfunction in autoimmune NOD mice (0 vs. 17% using wild-type islets). However, no difference in survival time was observed. Additionally, treating recipients with IL-1ra and CsA prolonged graft survival in chemically diabetic BALB/c mice, whereas no difference was seen between STAT-1(-/-) and C57BL/6 grafts. CONCLUSIONS: These data indicate that STAT-1 is a key player in immune-mediated early beta-cell dysfunction and death. When considering the many effector mechanisms contributing to beta-cell death following islet transplantation, multiple combined interventions will be needed for prolongation of beta-cell survival in the autoimmune context of type 1 diabetes.status: publishe