Steviol glycosides (SGs), such as stevioside and rebaudioside A, are natural, non-caloric sweet-tasting organic molecules, present in extracts of the scrub plant Stevia rebaudiana, which are widely ...used as sweeteners in consumer foods and beverages. TRPM5 is a Ca
-activated cation channel expressed in type II taste receptor cells and pancreatic β-cells. Here we show that stevioside, rebaudioside A and their aglycon steviol potentiate the activity of TRPM5. We find that SGs potentiate perception of bitter, sweet and umami taste, and enhance glucose-induced insulin secretion in a Trpm5-dependent manner. Daily consumption of stevioside prevents development of high-fat-diet-induced diabetic hyperglycaemia in wild-type mice, but not in Trpm5
mice. These results elucidate a molecular mechanism of action of SGs and identify TRPM5 as a potential target to prevent and treat type 2 diabetes.
Restoration of immune tolerance to disease-relevant antigens is an appealing approach to prevent or arrest an organ-specific autoimmune disease like type 1 diabetes (T1D). Numerous studies have ...identified insulin as a key antigen of interest to use in such strategies, but to date, the success of these interventions in humans has been inconsistent. The efficacy of antigen-specific immunotherapy may be enhanced by optimising the dose, timing, and route of administration, and perhaps by the inclusion of adjuvants like alum. The aim of our study was to evaluate the effect of an insulin peptide vaccine formulated with alum to prevent T1D development in female non-obese diabetic (NOD) mice when administered during late-stage pre-diabetes.
Starting at 10 weeks of age, female NOD mice received four weekly subcutaneous injections of an insulin B:8-24 (InsB:8-24) peptide with (Ins+alum) or without Imject
alum (Ins) as adjuvant. Diabetes incidence was assessed for up to 30 weeks of age. Insulin autoantibodies and C-peptide concentrations were measured in plasma and flow cytometric analysis was performed on pancreatic-draining lymph nodes (PLN) and pancreas using an InsB:12-20-reactive tetramer.
InsB:8-24 peptide formulated in alum reduced diabetes incidence (39%), compared to mice receiving the InsB:8-24 peptide without alum (71%, P < 0.05), mice receiving alum alone (76%, P < 0.01), or mice left untreated (70%, P < 0.01). This was accompanied by reduced insulitis severity, and preservation of C-peptide. Ins+alum was associated with reduced frequencies of pathogenic effector memory CD4
and CD8
T cells in the pancreas and increased frequencies of insulin-reactive FoxP3
Tregs in the PLN. Of interest, insulin-reactive Tregs were enriched amongst populations of Tregs expressing markers indicative of stable FoxP3 expression and enhanced suppressive function.
An InsB:8-24 peptide vaccine prevented the onset of T1D in late-stage pre-diabetic NOD mice, but only when formulated in alum. These findings support the use of alum as adjuvant to optimise the efficacy of antigen-specific immunotherapy in future trials.
Multipotent Adult Progenitor Cells (MAPCs) are one potential stem cell source to generate functional hepatocytes or β-cells. However, human MAPCs have less plasticity than pluripotent stem cells ...(PSCs), as their ability to generate endodermal cells is not robust. Here we studied the role of 14 transcription factors (TFs) in reprogramming MAPCs to induced endodermal progenitor cells (iENDO cells), defined as cells that can be long-term expanded and differentiated to both hepatocyte- and endocrine pancreatic-like cells. We demonstrated that 14 TF-iENDO cells can be expanded for at least 20 passages, differentiate spontaneously to hepatocyte-, endocrine pancreatic-, gut tube-like cells as well as endodermal tumor formation when grafted in immunodeficient mice. Furthermore, iENDO cells can be differentiated in vitro into hepatocyte- and endocrine pancreatic-like cells. However, the pluripotency TF OCT4, which is not silenced in iENDO cells, may contribute to the incomplete differentiation to mature cells in vitro and to endodermal tumor formation in vivo. Nevertheless, the studies presented here provide evidence that reprogramming of adult stem cells to an endodermal intermediate progenitor, which can be expanded and differentiate to multiple endodermal cell types, might be a valid alternative for the use of PSCs for creation of endodermal cell types.
Immune cells carry receptors for 1,25‐dihydroxyvitamin D3 1,25(OH)2D3; vitamin D receptor (VDR) and individuals with severe vitamin D deficiency have immune abnormalities. The aim of this study was ...to investigate the role of vitamin D in the immune system by studying VDR‐knockout (VDR‐KO) mice. VDR‐KO mice had the same metabolic phenotype as rachitic animals with severe hypocalcemia. Leukocytosis, lymphocyte subset composition in different immune organs, and splenocyte proliferation to several stimuli were normal, except for a lower response to anti‐CD3 stimulation (simulation index SI of 13 ± 4 vs. 24 ± 9 in wild‐type mice; p < 0.01). Macrophage chemotaxis was impaired (41 ± 19% vs. 60 ± 18% in wild‐type mice; p < 0.01) but phagocytosis and killing were normal. In vivo rejection of allogeneic (31 ± 12 days vs. 45 ± 26 days of survival in wild‐type mice, NS) or xenogeneic (10 ± 2 days vs. 16 ± 9 days of survival in wild‐type mice, NS) islet grafts was comparable with wild‐type mice. Surprisingly, VDR‐KO mice were protected from low‐dose streptozotocin‐induced diabetes mellitus (LDSDM; 5% vs. 65% in wild‐type mice; p < 0.001). Correcting hypocalcemia by use of lactose‐rich or polyunsaturated fat‐rich diets fully restored the immune abnormalities in vitro and the sensitivity to diabetes in vivo. On the other hand, treatment with 1,25(OH)2D3 protected wild‐type mice against diabetes but did not protect normocalcemic VDR‐KO mice. We conclude that immune defects observed in VDR‐KO mice are an indirect consequence of VDR disruption because they can be restored by calcium homeostasis normalization. This study proves that although 1,25(OH)2D3 is a pharmacologic and probably a physiological immunomodulator, its immune function is redundant. Moreover, we confirm the essential role of calcium in the immune system.
1,25-Dihydroxyvitamin D3 1,25(OH)2D3 has potent antiproliferative actions but calcemic effects obstruct its application in the treatment of hyperproliferative disorders. Therefore, analogs of ...1,25-(OH)2D3 are designed with a clear dissociation between both effects. Here the biological activity of the trans-decalin CD-ring analog CY10012 is discussed.
Proliferation/differentiation/transactivation assays as well as mouse models were used to determine the activity of CY10012 in vitro and in vivo.
CY10012, has ten-fold higher antiproliferative actions than 1,25(OH)2D3 but is also twice as calcemic. To determine the role of the Vitamin D Receptor (VDR) in mediating the calcemic actions of CY10012, the analog was daily administered to VDRwt and VDRko mice. This treatment caused drastic weight loss and death in VDRwt mice but not in VDRko mice.
Analog CY10012 has greater antiproliferative action but also two-fold higher calcemic effects which depended entirely on VDR-mediated signalling pathways.
Early graft failure, graft rejection, and autoimmune recurrence remain unresolved issues in islet xenotransplantation in type 1 diabetes. The first aim of this study was to examine the existence of ...early graft failure in spontaneously diabetic autoimmune NOD mice after rat islet transplantation under technically controlled circumstances. The second aim was to examine the mediators of this early xenograft dysfunction. First, we demonstrated a higher percentage of early xenograft failure (48%) in spontaneously diabetic NOD mice as compared with chemically diabetic old NOD (13%, P < 0.05) and C57Bl/6 (7%, P < 0.01) mice. In addition, in spontaneously diabetic NOD mice, xenogeneic islets displayed early graft failure more frequently than allogeneic (23%, P < or = 0.05) or isogeneic islets (7%, P < 0.01). No early graft failure was observed in allotransplantation or isotransplantation in chemically diabetic mice. Reverse transcriptase-polymerase chain reaction analysis of cytokine mRNA in islet xenografts 8 h after transplantation showed higher levels of interleukin (IL)-1 mRNA in autoimmune diabetic mice compared with chemically diabetic old NOD mice (1.40 +/- 0.32 vs. 0.90 +/- 0.14 IL-1 copies/beta-actin copies, P < 0.05). In contrast, mRNA levels of transforming growth factor (TGF)-beta were lower in spontaneously diabetic NOD mice than in chemically diabetic old NOD mice (0.67 +/- 0.16 vs. 1.36 +/- 0.50 TGF-beta copies/beta-actin copies, P < 0.05). No differences in tumor necrosis factor-alpha, IL-6, and inducible nitric oxide synthase were seen between autoimmune and nonautoimmune diabetic mice. T-cell cytokines (IL-2, IL-4, IL-10, and gamma-interferon) were absent in all mice until 48 h after transplantation. These data suggest that early islet xenograft failure is more common in spontaneously diabetic NOD mice and could be due to a nonspecific inflammatory reaction locally in the grafts.
The active form of vitamin D, 1,25(OH)2D3, can prevent various forms of experimentally induced autoimmune disorders. The aim of this study was to confirm these findings in NOD mice that spontaneously ...develop an autoimmune type of diabetes mellitus. Therefore, the effect of a long-term 1,25(OH)2D3 treatment on the incidence of insulitis, the histological lesion preceding diabetes, was studied. Forty-three NOD mice were treated with 1,25(OH)2D3 (5 micrograms/kg) i.p. every other day from age 21 days on, when no insulitis was present yet. At day 100, 16 control mice receiving the treatment vehicle (arachis oil) had an incidence of insulitis of 75%, whereas only 41% of the 1,25(OH)2D3-treated animals developed insulitis (P < 0.025). Calcemia, determined 24 h after the last 1,25(OH)2D3 injection was 2.5 +/- 0.1 mM, which was higher than in control animals (2.3 +/- 0.1 mM), but was well tolerated. Cellular immunity, as assessed with the mixed lymphocyte reaction performed at day 100, was not impaired significantly. This study demonstrates that long-term treatment with high doses of 1,25(OH)2D3 is able to decrease the incidence of insulitis in spontaneous autoimmune diabetes without major side effects.
High doses of the active form of vitamin D3, 1,25-dihydroxyvitamin D3 1,25(OH)2D3, prevent diabetes in the 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/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.
High doses of the active form of vitamin D3, 1,25-dihydroxyvitamin D3 1,25(OH)2D3, prevent diabetes in the 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/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.
Type 1 diabetes is characterized by the presence of an autoimmune memory, responsible for the destruction of even syngeneic islet grafts. This recurrence of autoimmunity is partly responsible for the ...need of extensive immunosuppression in pancreas and islet transplantation in type 1 diabetic patients. The aim of the study was to evaluate the capacity of a 20-epi-analog of vitamin D3, KH1060, both alone and in combination with cyclosporine (CsA) to prevent diabetes recurrence in syngeneic islet grafts in nonobese diabetic (NOD) mice.
Spontaneously diabetic NOD mice grafted with syngeneic islets (n=500) under the kidney capsule were treated with KH1060, CsA, or a combination of both drugs from the day before transplantation until recurrence or 60 days after transplantation.
Vehicle-treated mice showed a recurrence of diabetes in 100% of cases (n=17) within 4 weeks. Treatment with high doses of CsA (15 mg/kg/day) or KH1060 (1 microg/kg/2 days) significantly prolonged islet survival (60 days and 50 days, respectively, versus 9.5 days in controls; P<0.001 and P<0.0001). Mice treated with subtherapeutical doses of both drugs combined (KH1060 0.5 microg/kg/2 days + CsA 7.5 mg/kg/day) had significant prolongation of graft survival (48 days; P<0.001) and more importantly, four of five mice that were still normoglycemic 60 days after transplantation showed no recurrence after discontinuation of all treatment. Histology of the grafts of control and combination-treated mice demonstrated that graft infiltration and islet destruction were less severe in grafts of combination-treated mice. Cytokine mRNA analysis in the grafts 6 days after transplantation revealed a clear suppression of interleukin-12 and T helper 1 cytokines and higher levels of interleukin-4 in combination-treated mice.
KH1060, an analog of 1,25(OH)2D3, delays autoimmune disease recurrence after syngeneic islet transplantation in NOD mice, both alone and especially in combination with CsA, possibly restoring tolerance to beta cells in 30% of cases.
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