Alterations in oviductal cilia morphology and reduced expression of axonemal dynein in diabetic NOD mice

In the present study, we examined the morphology of cilia and expression of the dynein intermediate chain 2 (DNAI2) in the oviduct of non-obese diabetic (NOD) mice. Results obtained with immunohistochemistry showed that DNAI2 expression was reduced in oviducts of diabetic NOD (dNOD) mice, as compared to that observed in the normoglycemic NOD (cNOD) group, especially in the acyclic dNOD mice. Oviductal cilia of dNOD mice appeared to be reduced in number. Results obtained with Western blot analysis revealed that the expression of DNAI2 protein was significantly less in oviducts of dNOD mice as compared to that of cNOD mice corroborating the results obtained with immunohistochemistry. Electron microscopic examination and quantitative imaging of thin sections of Epon-embedded oviducts of both dNOD and cNOD mice confirmed the reduction of the number of cilia in the oviduct of the dNOD group which also displayed aberrant axonemal ultrastructure, including disorganization of the axoneme and alteration of microtubule doublets into singlets as well as disruption of the plasma membrane in many cilia. Taken together, the present findings suggest that structural alterations of oviductal cilia in female diabetic NOD mice might be detrimental to the normal function of these particular cell structures in gamete transport.     

Modulation of mammalian circadian rhythms by tumor necrosis factor-α

Systemic low doses of the endotoxin lipopolysaccharide (LPS, 100?µg/kg) administered during the early night induce phase-delays of locomotor activity rhythms in mice. Our aim was to evaluate the role of tumor necrosis factor (Tnf)-alpha and its receptor 1/p55 (Tnfr1) in the modulation of LPS-induced circadian effects on the suprachiasmatic nucleus (SCN). We observed that Tnfr1-defective mice (Tnfr1 KO), although exhibiting similar circadian behavior and light response to that of control mice, did not show LPS-induced phase-delays of locomotor activity rhythms, nor LPS-induced cFos and Per2 expression in the SCN and Per1 expression in the paraventricular hypothalamic nucleus (PVN) as compared to wild-type (WT) mice. We also analyzed Tnfr1 expression in the SCN of WT mice, peaking during the early night, when LPS has a circadian effect. Peripheral inoculation of LPS induced an increase in cytokine/chemokine levels (Tnf, Il-6 and Ccl2) in the SCN and in the PVN. In conclusion, in this study, we show that LPS-induced circadian responses are mediated by Tnf. Our results also suggest that this cytokine stimulates the SCN after LPS peripheral inoculation; and the time-related effect of LPS (i.e. phase shifts elicited only at early night) might depend on the increased levels of Tnfr1 expression. We also confirmed that LPS modulates clock gene expression in the SCN and PVN in WT but not in Tnfr1 KO mice.

Highlights: We demonstrate a fundamental role for Tnf and its receptor in circadian modulation by immune stimuli at the level of the SCN biological clock.     

Endometrial receptivity defects and impaired implantation in diabetic NOD mice

Implantation failure is a major hurdle to a successful pregnancy. The high rate of postimplantation fetal loss in nonobese diabetic (NOD) mice is believed to be related to an abnormal decidual production of interferon (IFN)gamma. To address whether diabetes alters the natural events associated with successful implantation, certain morphological and molecular features of uterine receptivity in diabetic NOD (dNOD) mice were examined in normally mated pregnancy and in concanavalin A (ConA)-induced pseudopregnancy. As opposed to normoglycemic NOD (cNOD) mice, dNOD mice expressed retarded maturation of their uterine pinopodes and overexpressed MUC1 mucin at implantation sites (P < 0.001). Uterine production of leukemia inhibitory factor (LIF) and phosphorylation of uterine NFkappaBp65 and STAT3-Ty705 were found to be low (P < 0.01) during Day 4.5 postcoitum, whereas IFNgamma was aberrantly overexpressed. Loss of temporal regulation of progesterone receptor A (PR A) and PR B, together with aberrantly increased expression of the protein inhibitor of activated STAT-y (PIASy) (P < 0.01) and reduced recruitment (P < 0.01) of the latter to nuclear progesterone receptor sites were prominent features of decidualization failure occurring at peri-implantation in dNOD mice. In conclusion, the aberrant expression of endometrial IFNgamma in dNOD mice is associated with a nonreceptive endometrial milieu contributing to peri-implantation embryo loss in type 1 diabetes.     

TLR4 regulates cardiac lipid accumulation and diabetic heart disease in the nonobese diabetic mouse model of type 1 diabetes

Toll-like receptor (TLR)4 regulates inflammation and metabolism and has been linked to the pathogenesis of heart disease. TLR4 is upregulated in diabetic cardiomyocytes, and we examined the role of TLR4 in modulating cardiac fatty acid (FA) metabolism and the pathogenesis of diabetic heart disease in nonobese diabetic (NOD) mice. Both wild-type (WT) NOD and TLR4-deficient NOD animals had increased plasma triglyceride levels after the onset of diabetes. However, by comparison, TLR4-deficient NOD mouse hearts had lower triglyceride accumulation in the early stages of diabetes, which was associated with a reduction in myeloid differentiation primary response gene (88) (MyD88), phosphorylation of p38 MAPK (phospho-p38), lipoprotein lipase (LPL), and JNK levels but increased phospho-AMP-activated protein kinase (AMPK). Oleic acid treatment in H9C2 cardiomyocytes also led to cellular lipid accumulation, which was attenuated by TLR4 small interfering RNA. TLR4 deficiency in the cells decreased FA-induced augmentation of MyD88, phospho-p38, and LPL, suggesting that TLR4 may modulate FA-induced lipid metabolism in cardiomyocytes. In addition, although cardiac function was impaired in both diabetic WT NOD and TLR4-deficient NOD animals compared with control nondiabetic mice, this deficit was less in the diabetic TLR4-deficient NOD mice, which had greater ejection fraction, greater fractional shortening, and increased left ventricular developed pressure in the early stages after the development of diabetes compared with their diabetic WT NOD counterparts. Thus, we conclude that TLR4 plays a role in regulating lipid accumulation in cardiac muscle after the onset of type 1 diabetes, which may contribute to cardiac dysfunction.     

The quest for the sea urchin egg receptor for sperm

The incretin glucagon-like peptide-1 (GLP-1) and other GLP-1 receptor agonists have been shown to cause both antiapoptotic as well as regenerative effects on beta-cells in different animal models for diabetes. Our aim of this study was to test the hypothesis that spontaneously diabetic non obese diabetic (NOD) mice show an altered expression of GLP-1 compared to normoglycemic age-matched controls as a consequence of a diabetic state. To do this we used an ELISA prototype for mouse GLP-1 to measure plasma total GLP-1 from recently diabetic NOD mice as well as from age-matched normoglycemic NOD mice (controls). We also stained sections of pancreatic glands for GLP-1 from diabetic NOD mice and controls. We found increased levels of plasma total GLP-1 in diabetic NOD mice, when compared to control mice, both from non-fasted mice and from mice fasted for 2h. Furthermore, diabetic NOD mice displayed a higher GLP-1 response to an oral glucose tolerance test, compared to control mice. We also found that sections of pancreatic glands from diabetic NOD mice had an increased GLP-1 positive islet area in regard to relative islet area (i.e. total islet area / total pancreas area of the sections) compared to control mice. To our knowledge, this study is the first to show increased levels of GLP-1 in plasma in spontaneously diabetic NOD mice. We suggest that these results might represent a compensatory mechanism of the diabetic NOD mice to counteract beta-cell loss and hyperglycemia.     

Immunohistochemical study of monocyte chemoattractant protein-1 in the pancreas of NOD mice following cyclophosphamide administration and during spontaneous diabetes

In type 1 diabetes mellitus (T1DM), the processes which control the recruitment of immune cells into pancreatic islets are poorly defined. Complex interactions involving adhesion molecules, chemokines and chemokine receptors may facilitate this process. The chemokine, monocyte chemoattractant protein-1 (MCP-1), previously shown to be important in leukocyte trafficking in other disease systems, may be a key participant in the early influx of blood-borne immune cells into islets during T1DM. In the non-obese diabetic (NOD) mouse, the expression of MCP-1 protein has not been demonstrated. We employed dual-label immunohistochemistry to examine the intra-islet expression, distribution and cellular source of MCP-1 in the NOD mouse following cyclophosphamide administration. NOD mice were treated with cyclophosphamide at day 72–73 and MCP-1 expression studied at days 0, 4, 7, 11 and 14 after treatment and comparisons were made between age-matched NOD mice treated with diluent and non-diabetes-prone CD-1 mice. Pancreatic expression of MCP-1 was also examined in NOD mice at various stages of spontaneous diabetes. In the cyclophosphamide group at day 0, MCP-1 immunolabelling was present in selective peri-islet macrophages but declined at day 4. It increased slightly at day 7 but was more marked from day 11, irrespective of diabetes development. The pattern of MCP-1 expression in macrophages was different over time in both the cyclophosphamide and control groups. In the cyclophosphamide group, there was a change over time with an increase at day 11. In the control group, there was little evidence of change over time. There was no significant difference in the mean percentage of MCP-1 positive macrophages between the cyclophosphamide-treated diabetic and non-diabetic mice. During spontaneous diabetes in the NOD mouse, only a few peri-islet MCP-1 cells appeared at day 45. These became more numerous from day 65 but were absent at diabetes onset. We speculate that a proportion of early islet-infiltrating macrophages which express MCP-1 may attract additional lymphocytes and macrophages into the early inflamed islets and intensify the process of insulitis.     

The Endogenous Melatonin (MT) Signal Facilitates Reentrainment of the Circadian System to Light-Induced Phase Advances by Acting Upon MT2 Receptors

The indolamine melatonin is an important rhythmic endocrine signal in the circadian system. Exogenous melatonin can entrain circadian rhythms in physiology and behavior, but the role of endogenous melatonin and the two membrane-bound melatonin receptor types, MT1 and MT2, in reentrainment of daily rhythms to light-induced phase shifts is not understood. The present study analyzed locomotor activity rhythms and clock protein levels in the suprachiasmatic nuclei (SCN) of melatonin-deficient (C57BL/6J) and melatonin-proficient (C3H/HeN) mice, as well as in melatonin-proficient (C3H/HeN) mice with targeted deletion of the MT1, MT2, or both receptors, to determine effects associated with phase delays or phase advances of the light/dark (LD) cycle. In all mouse strains and genotypes, reentrainment of locomotor activity rhythms was significantly faster after a 6-h phase delay than a 6-h phase advance. Reentrainment after the phase advance was, however, significantly slower than in melatonin-deficient animals and in mice lacking functional MT2 receptors than melatonin-proficient animals with intact MT2 receptors. To investigate whether these behavioral differences coincide with differences in reentrainment of clock protein levels in the SCN, mPER1, mCRY1 immunoreactions were compared between control mice kept under the original LD cycle and killed at zeitgeber time 04 (ZT04) or at ZT10, respectively, and experimental mice subjected to a 6-h phase advance of the LD cycle and sacrificed at ZT10 on the third day after phase advance. This ZT corresponds to ZT04 of the original LD cycle. Under the original LD cycle, the numbers of mPER1- and mCRY1-immunoreactive cell nuclei were low at ZT04 and high at ZT10 in the SCN of all mouse strains and genotypes investigated. Notably, mouse strains with intact melatonin signaling and functional MT2 receptors showed a significant increase in the number of mPER1- and mCRY1-immunoreactive cell nuclei at the new ZT10 as compared to the former ZT04. These data suggest the endogenous melatonin signal facilitates reentrainment of the circadian system to phase advances on the level of the SCN molecular clockwork by acting upon MT2 receptors. (Author correspondence: M.Pfeffer@em.uni-frankfurt.de)     

Circadian locomotor analysis of male mice lacking the gene for neuronal nitric oxide synthase (nNOS-/-)

Nitric oxide (NO) is an endogenous gas that functions as a neurotransmitter. Because NO is very labile with a half-life of less than 5 sec, most functional studies of NO have manipulated its synthetic enzyme, NO synthase (NOS). Three isoforms of NOS have been identified: (1) in the endothelial lining of blood vessels (eNOS), (2) an inducible form found in macrophages (iNOS), and (3) in neurons (nNOS). Most pharmacological studies to date have blocked all three isoforms of NOS. Previous studies using such agents have revealed that NO might be necessary for photic entrainment of circadian rhythms; general NOS inhibitors attenuate phase shifts of free-running behavior, light-induced c-fos expression in the suprachiasmatic nucleus (SCN), and phase shifts of neural firing activity in SCN maintained in vitro. To assess the specific role of nNOS in mediating entrainment of circadian rhythms, mice with targeted deletion of the gene encoding the neuronal isoform of NOS (nNOS-/-) were used. Wild-type (WT) and nNOS-/- mice initially were entrained to a 14:10 light:dark (LD) cycle. After 3 weeks, the LD cycle was either phase advanced or phase delayed. After an additional 3 weeks, animals were held in either constant dim light or constant dark. WT and nNOS-/- animals did not differ in their ability to entrain to the LD cycle, phase shift locomotor activity, or free run in constant conditions. Animals held in constant dark were killed after light exposure during either the subjective day or subjective night to assess c-fos induction in the SCN. Light exposure during the subjective night increased c-fos expression in the SCN of both WT and nNOS-/- mice relative to animals killed after light exposure during the subjective day. Taken together, these findings suggest that NO from neurons might not be necessary for photic entrainment.     

Cyclin-dependent kinase 4 hyperactivity promotes autoreactivity in the immune system but protects pancreatic cell mass from autoimmune destruction in the nonobese diabetic mouse model

Cyclin-dependent kinase 4 (Cdk4) plays a central role in perinatal pancreatic beta cell replication, thus becoming a potential target for therapeutics in autoimmune diabetes. Its hyperactive form, Cdk4R24C, causes beta cell hyperplasia without promoting hypoglycemia in a nonautoimmune-prone mouse strain. In this study, we explore whether beta cell hyperproliferation induced by the Cdk4R24C mutation balances the autoimmune attack against beta cells inherent to the NOD genetic background. To this end, we backcrossed the Cdk4R24C knockin mice, which have the Cdk4 gene replaced by the Cdk4R24C mutated form, onto the NOD genetic background. In this study, we show that NOD/Cdk4R24C knockin mice exhibit exacerbated diabetes and insulitis, and that this exacerbated diabetic phenotype is solely due to the hyperactivity of the NOD/Cdk4R24C immune repertoire. Thus, NOD/Cdk4R24C splenocytes confer exacerbated diabetes when adoptively transferred into NOD/SCID recipients, compared with NOD/wild-type (WT) donor splenocytes. Accordingly, NOD/Cdk4R24C splenocytes show increased basal proliferation and higher activation markers expression compared with NOD/WT splenocytes. However, to eliminate the effect of the Cdk4R24C mutation specifically in the lymphocyte compartment, we introduced this mutation into NOD/SCID mice. NOD/SCID/Cdk4R24C knockin mice develop beta cell hyperplasia spontaneously. Furthermore, NOD/SCID/Cdk4R24C knockin females that have been adoptively transferred with NOD/WT splenocytes are more resistant to autoimmunity than NOD/SCID WT female. Thus, the Cdk4R24C mutation opens two avenues in the NOD model: when expressed specifically in beta cells, it provides a new potential strategy for beta cell regeneration in autoimmune diabetes, but its expression in the immune repertoire exacerbates autoimmunity.     

Increased expression of GPI-specific phospholipase D in mouse models of type 1 diabetes

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is a high-density lipoprotein-associated protein. However, the tissue source(s) for circulating GPI-PLD and whether serum levels are regulated are unknown. Because the diabetic state alters lipoprotein metabolism, and liver and pancreatic islets are possible sources of GPI-PLD, we hypothesized that GPI-PLD levels would be altered in diabetes. GPI-PLD serum activity and liver mRNA were examined in two mouse models of type 1 diabetes, a nonobese diabetic (NOD) mouse model and low-dose streptozotocin-induced diabetes in CD-1 mice. With the onset of hyperglycemia (2- to 5-fold increase over nondiabetic levels), GPI-PLD serum activity and liver mRNA increased 2- to 4-fold in both models. Conversely, islet expression of GPI-PLD was absent as determined by immunofluorescence. Insulin may regulate GPI-PLD expression, because insulin treatment of diabetic NOD mice corrected the hyperglycemia along with reducing serum GPI-PLD activity and liver mRNA. Our data demonstrate that serum GPI-PLD levels are altered in the diabetic state and are consistent with liver as a contributor to circulating GPI-PLD.     

The endogenous melatonin (MT) signal facilitates reentrainment of the circadian system to light-induced phase advances by acting upon MT2 receptors

The indolamine melatonin is an important rhythmic endocrine signal in the circadian system. Exogenous melatonin can entrain circadian rhythms in physiology and behavior, but the role of endogenous melatonin and the two membrane-bound melatonin receptor types, MT1 and MT2, in reentrainment of daily rhythms to light-induced phase shifts is not understood. The present study analyzed locomotor activity rhythms and clock protein levels in the suprachiasmatic nuclei (SCN) of melatonin-deficient (C57BL/6J) and melatonin-proficient (C3H/HeN) mice, as well as in melatonin-proficient (C3H/HeN) mice with targeted deletion of the MT1, MT2, or both receptors, to determine effects associated with phase delays or phase advances of the light/dark (LD) cycle. In all mouse strains and genotypes, reentrainment of locomotor activity rhythms was significantly faster after a 6-h phase delay than a 6-h phase advance. Reentrainment after the phase advance was, however, significantly slower than in melatonin-deficient animals and in mice lacking functional MT2 receptors than melatonin-proficient animals with intact MT2 receptors. To investigate whether these behavioral differences coincide with differences in reentrainment of clock protein levels in the SCN, mPER1, mCRY1 immunoreactions were compared between control mice kept under the original LD cycle and killed at zeitgeber time 04 (ZT04) or at ZT10, respectively, and experimental mice subjected to a 6-h phase advance of the LD cycle and sacrificed at ZT10 on the third day after phase advance. This ZT corresponds to ZT04 of the original LD cycle. Under the original LD cycle, the numbers of mPER1- and mCRY1-immunoreactive cell nuclei were low at ZT04 and high at ZT10 in the SCN of all mouse strains and genotypes investigated. Notably, mouse strains with intact melatonin signaling and functional MT2 receptors showed a significant increase in the number of mPER1- and mCRY1-immunoreactive cell nuclei at the new ZT10 as compared to the former ZT04. These data suggest the endogenous melatonin signal facilitates reentrainment of the circadian system to phase advances on the level of the SCN molecular clockwork by acting upon MT2 receptors.     

Circadian organization in male mice lacking the gene for endothelial nitric oxide synthase (eNOS-/-)

Circadian (approximately 24 h) rhythms in physiology and behavior are generated by the bilateral suprachiasmatic nucleus (SCN) of the anterior hypothalamus. For these endogenous rhythms to be synchronized with the external environment, light information must be transmitted to pacemaker cells within the SCN. This transmission of light information is accomplished via a direct retino-hypothalamic tract (RHT). Nitric oxide (NO), an endogenous gas that functions as a neurotransmitter, has been implicated as a messenger necessary for photic entrainment. Three isoforms of the enzyme that form NO, NO synthase, have been identified (a) in neurons (nNOS), (b) in the endothelial lining of blood vessels (eNOS), and (c) as an inducible form in macrophages (iNOS). The present study was undertaken to determine the specific role of eNOS in circadian organization and photic entrainment. Wild-type (WT) and eNOS-/- mice were initially entrained to a 14:10 light:dark (LD) cycle. After 3 weeks, the LD cycle was phase advanced. After an additional 3 weeks, animals were held in constant darkness (DD). eNOS-/- animals did not exhibit a deficit in the ability to entrain to the LD cycle, phase-shift locomotor activity, or free-run in constant conditions. Animals held in DD were killed after light exposure during either the subjective day or the subjective night to assess c-fos induction in the SCN. Light exposure during the subjective night increased c-fos protein expression in the SCN of both WT and eNOS-/- mice relative to animals killed after light exposure during the subjective day. Taken together, these findings suggest that endothelial isoform of NOS may not be necessary for photic entrainment in mice.     

Daily injection of insulin attenuated impairment of liver circadian clock oscillation in the streptozotocin-treated diabetic mouse

Recent studies have shown a dampened amplitude of clock gene rhythm in the heart and liver of streptozotocin (STZ)-treated rats and mice, however it is unknown whether impairment is due to dysfunction of the suprachiasmatic nucleus (SCN) or not. Rhythmic expression of mPER2 was dampened in the STZ-treated mouse liver but not SCN and cerebral cortex. Injection of insulin could normalize an impairment of mPer2 and mPER2 expression rhythm in the liver, when it was injected at nighttime, but not at daytime. In the present study, we demonstrated that insulin-dependent diabetes impaired oscillation of the peripheral clock gene and its product. Insulin injection can recover dampened oscillation of the peripheral clock depending on its injection time.     

Behavioral responses of Vipr2-/- mice to light

Vasoactive intestinal polypeptide and its receptor, VPAC2, play important roles in the functioning of the dominant circadian pacemaker, located in the hypothalamic suprachiasmatic nuclei (SCN). Mice lacking VPAC2 receptors (Vipr2-/-) show altered circadian rhythms and impaired synchronization to environmental lighting cues. However, light can increase phosphoprotein and immediate early gene expression in the Vipr2-/- SCN demonstrating that the circadian clock is readily responsive to light in these mice. It is not clear whether these neurochemical responses to light can be transduced to behavioral changes as seen in wild-type (WT) animals. In this study we investigated the diurnal and circadian wheel-running profile of WT (C57BL/6J) and Vipr2-/- mice under a 12-h light:12-h complete darkness (LD) lighting schedule and in constant darkness (DD) and used 1-h light pulses to shift the activity of mice in DD. Unlike WT mice, Vipr2-/- mice show grossly altered locomotor patterns making the analysis of behavioral responses to light problematic. However, analyses of both the onset and the offset of locomotor activity reveal that in a subset of these mice, light can reset the offset of behavioral rhythms during the subjective night. This suggests that the SCN clock of Vipr2-/- mice and the rhythms it generates are responsive to photic stimulation and that these responses can be integrated to whole animal behavioral changes.     

Dynamics of pathogenic and suppressor T cells in autoimmune diabetes development

In the nonobese diabetic (NOD) mouse, pathogenic and suppressor CD4(+) T cells can be distinguished by the constitutive expression of CD25. In this study, we demonstrate that the progression of autoimmune diabetes in NOD mice reflects modifications in both T cell subsets. CD4(+)CD25(+) suppressor T cells from 8-, but not 16-wk-old NOD mice delayed the onset of diabetes transferred by 16-wk-old CD25-depleted spleen cells. These results were paralleled by the inhibition of alloantigen-induced proliferation of CD4(+)CD25(-) cells, indicating an age-dependent decrease in suppressive activity. In addition, CD4(+)CD25(-) pathogenic T cells became progressively less sensitive to immunoregulation by CD4(+)CD25(+) T cells during diabetes development. CD4(+)CD25(-) T cells showed a higher proliferation and produced more IFN-gamma, but less IL-4 and IL-10, whereas CD4(+)CD25(+) T suppressor cells produced significantly lower levels of IL-10 in 16- compared with 8-wk-old NOD mice. Consistent with these findings, a higher frequency of Th1 cells was observed in the pancreas of 16-wk-old compared with 8-wk-old NOD mice. An increased percentage of CD4(+)CD25(-) T cells expressing CD54 was present in 16-wk-old and in diabetic NOD, but not in BALB/c mice. Costimulation via CD54 increased the proliferation of CD4(+)CD25(-) T cells from 16-, but not 8-wk-old NOD mice, and blocking CD54 prevented their proliferation, consistent with the role of CD54 in diabetes development. Thus, the pathogenesis of autoimmune diabetes in NOD mice is correlated with both an enhanced pathogenicity of CD4(+)CD25(-) T cells and a decreased suppressive activity of CD4(+)CD25(+) T cells.     

Significant role for Fas in the pathogenesis of autoimmune diabetes

Programmed cell death represents an important pathogenic mechanism in various autoimmune diseases. Type I diabetes mellitus (IDDM) is a T cell-dependent autoimmune disease resulting in selective destruction of the beta cells of the islets of Langerhans. beta cell apoptosis has been associated with IDDM onset in both animal models and newly diagnosed diabetic patients. Several apoptotic pathways have been implicated in beta cell destruction, including Fas, perforin, and TNF-alpha. Evidence for Fas-mediated lysis of beta cells in the pathogenesis of IDDM in nonobese diabetic (NOD) mice includes: 1) Fas-deficient NOD mice bearing the lpr mutation (NOD-lpr/lpr) fail to develop IDDM; 2) transgenic expression of Fas ligand (FasL) on beta cells in NOD mice may result in accelerated IDDM; and 3) irradiated NOD-lpr/lpr mice are resistant to adoptive transfer of diabetes by cells from NOD mice. However, the interpretation of these results is complicated by the abnormal immune phenotype of NOD-lpr/lpr mice. Here we present novel evidence for the role of Fas/FasL interactions in the progression of NOD diabetes using two newly derived mouse strains. We show that NOD mice heterozygous for the FasL mutation gld, which have reduced functional FasL expression on T cells but no lymphadenopathy, fail to develop IDDM. Further, we show that NOD-lpr/lpr mice bearing the scid mutation (NOD-lpr/lpr-scid/scid), which eliminates the enhanced FasL-mediated lytic activity induced by Fas deficiency, still have delayed onset and reduced incidence of IDDM after adoptive transfer of diabetogenic NOD spleen cells. These results provide evidence that Fas/FasL-mediated programmed cell death plays a significant role in the pathogenesis of autoimmune diabetes.     

Islet Remodeling in Female Mice with Spontaneous Autoimmune and Streptozotocin-Induced Diabetes

Islet alpha- and delta-cells are spared autoimmune destruction directed at beta-cells in type 1 diabetes resulting in an apparent increase of non-beta endocrine cells in the islet core. We determined how islet remodeling in autoimmune diabetes compares to streptozotocin (STZ)-induced diabetes. Islet cell mass, proliferation, and immune cell infiltration in pancreas sections from diabetic NOD mice and mice with STZ-induced diabetes was assessed using quantitative image analysis. Serial sections were stained for various beta-cell markers and Ngn3, typically restricted to embryonic tissue, was only upregulated in diabetic NOD mouse islets. Serum levels of insulin, glucagon and GLP-1 were measured to compare hormone levels with respect to disease state. Total pancreatic alpha-cell mass did not change as autoimmune diabetes developed in NOD mice despite the proportion of islet area comprised of alpha- and delta-cells increased. By contrast, alpha- and delta-cell mass was increased in mice with STZ-induced diabetes. Serum levels of glucagon reflected these changes in alpha-cell mass: glucagon levels remained constant in NOD mice over time but increased significantly in STZ-induced diabetes. Increased serum GLP-1 levels were found in both models of diabetes, likely due to alpha-cell expression of prohormone convertase 1/3. Alpha- or delta-cell mass in STZ-diabetic mice did not normalize by replacement of insulin via osmotic mini-pumps or islet transplantation. Hence, the inflammatory milieu in NOD mouse islets may restrict alpha-cell expansion highlighting important differences between these two diabetes models and raising the possibility that increased alpha-cell mass might contribute to the hyperglycemia observed in the STZ model.     

Vitamin D and 1,25-dihydroxyvitamin D3 as modulators in the immune system

Treatment from weaning until old age with 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)) prevents diabetes in NOD mice. It is mainly through its actions on dendritic cells (DCs), that 1,25(OH)(2)D(3) changes the function of potentially autoreactive T lymphocytes. In contrast, early life treatment (from 3 to 70 days of age) of NOD mice with vitamin D or 1,25(OH)(2)D(3) did not influence final diabetes incidence at 200 days of age. Also in spontaneous diabetic BB rats, diabetes could not be prevented by early life treatment (from 3 to 50 days of age) with vitamin D (1000 IU per day) or 1,25(OH)(2)D(3) (0.2 microg/kg per day or 1 microg/kg per 2 days). However, when NOD mice were made vitamin D deficient in early life (until 100 days of age), diabetes onset occurred earlier and final incidence was increased. These data further support a role for vitamin D and its metabolites in the pathogenesis of type 1 diabetes in NOD mice.