Female Non-obese Diabetic Research Articles

Sex-dependent effects of bisphenol A on type 1 diabetes development in non-obese diabetic (NOD) mice.

Type 1 diabetes (T1D) is an autoimmune disease caused by immune-mediated pancreatic β-cell destruction. The endocrine disrupting chemical bisphenol A (BPA) has widespread human exposure and can modulate immune function and the gut microbiome (GMB), which may contribute to the increasing T1D incidence worldwide. It was hypothesized that BPA had sex-dependent effects on T1D by modulating immune homeostasis and GMB. Adult female and male non-obese diabetic (NOD) mice were orally administered BPA at environmentally relevant doses (30 or 300µg/kg). Antibiotic-treated adult NOD females were exposed to 0 or 30µg/kg BPA. BPA accelerated T1D development in females, but delayed males from T1D. Consistently, females had a shift towards pro-inflammation (e.g., increased macrophages and Bacteroidetes), while males had increases in anti-inflammatory immune factors and a decrease in both anti- and pro-inflammatory GMB. Although bacteria altered during sub-acute BPA exposure differed from bacteria altered from chronic BPA exposure in both sexes, the GMB profile was consistently pro-inflammatory in females, while males had a general decrease of both anti- and pro-inflammatory gut microbes. However, treatment of females with the antibiotic vancomycin failed to prevent BPA-induced glucose intolerance, suggesting changes in Gram-positive bacteria were not a primary mechanism. In conclusion, BPA exposure was found to have sex dimorphic effects on T1D with detrimental effects in females, and immunomodulation was identified as the primary mechanism.

Evaluation of different mucosal microbiota leads to gut microbiota-based prediction of type 1 diabetes in NOD mice

Type 1 diabetes (T1D) is a progressive autoimmune disease in which the insulin-producing beta cells are destroyed by auto-reactive T cells. Recent studies suggest that microbiota are closely associated with disease development. We studied gut, oral and vaginal microbiota longitudinally in non-obese diabetic (NOD) mice. We showed that the composition of microbiota is very different at the different mucosal sites and between young and adult mice. Gut microbiota are more diverse than oral or vaginal microbiota and the changes were more evident in the mice before and after onset of diabetes. Using alpha-diversity, Gram-positive/Gram-negative ratio as well as the relative abundance of Bacteroidetes and Erysipelotrichaceae in the gut microbiota, at 8 weeks of age, we formulated a predictive algorithm for T1D development in a cohort of 63 female NOD mice. Using this algorithm, we obtained 80% accuracy of prediction of diabetes onset, in two independent experiments, totaling 29 mice, with Area Under the Curve of 0.776 by ROC analysis. Interestingly, we did not find differences in peripheral blood mononuclear cells of the mice at 8 weeks of age, regardless of later diabetes development. Our results suggest that the algorithm could potentially be used in early prediction of future T1D development.

Exacerbation of Type 1 Diabetes in Perinatally Genistein Exposed Female Non-Obese Diabetic (NOD) Mouse Is Associated With Alterations of Gut Microbiota and Immune Homeostasis.

Despite various hypothesized benefits of dietary isoflavone genistein (GEN) from soy-based products, many questions surrounding GEN's immunotoxic effects, especially during perinatal exposure, have yet to be answered. The objective of the study was to determine if there existed a sex-specific effect of GEN on type 1 diabetes (T1D) following perinatal exposure. We exposed offspring of non-obese diabetic (NOD) mice to GEN per oral at a physiological dose (20 mg/kg body weight) from embryonic day 7 to postnatal day (PND) 21. In female offspring, perinatal GEN dosing significantly increased the incidence of T1D at early time points, and the exacerbation was associated with decreased serum levels of interleukin (IL)-10, IgG2a, and IgM. In male offspring dosed with GEN, a decrease in serum IgG1 was also observed. Flow cytometric analysis in females suggested an increased pro-inflammatory splenic CD5+CD24- and CD4-CD8+ cell counts, while both %T cells and %CD4+ T cells were significantly decreased in males, suggesting an anti-inflammatory effect. Gut microbiota (GMB) analysis indicated that fecal microbiota from PND 90 female offspring exhibited an increased level of Enterobacteriales (suggesting a pro-inflammatory response), while the similar changes were not found in PND 30 females. Moreover, RNA sequencing showed that intestinal α-defensin expression was down-regulated in GEN-treated females, supporting a pro-inflammatory response. However, perinatal GEN administration perturbed GMB toward an anti-inflammatory response in PND 90 males. Taken together, a strong sex-specific effect was found in the perinatal GEN exposure window, and the T1D exacerbation in NOD females was associated with GMB-related immunomodulatory mechanisms.

Salivary-gland-protective regulatory T-cell dysfunction underlies female-specific sialadenitis in the non-obese diabetic mouse model of Sjögren syndrome.

Immune cell-mediated destruction of salivary glands is a hallmark feature of Sjögren syndrome. Similar to the female predominance in humans, female non-obese diabetic (NOD) mice develop spontaneous salivary gland autoimmunity. However, in both humans and mice it is unclear what factors contribute to the initial immune infiltration of the salivary glands. Here, we used an adoptive transfer model of Sjögren syndrome to determine if female mice harbor a sex-specific defect in salivary-gland-protective regulatory T (Treg) cells. Transfer of cervical lymph node (LN) cells from female NOD mice into sex-matched NOD-severe combined immunodeficient (SCID) recipients resulted in sialadenitis, regardless of the presence or absence of Treg cells. In contrast, transfer of cervical LN cells from male NOD mice into sex-matched NOD-SCID recipients only resulted in sialadenitis when Treg cells were depleted before transfer, suggesting that male NOD mice have functional salivary-gland-protective Treg cells. Notably, the host environment affected the ability of Treg cells to prevent sialadenitis with testosterone promoting salivary gland protection. Treg cells from male mice did not protect against sialadenitis in female recipients. Testosterone treatment of female recipients of bulk cervical LN cells decreased sialadenitis, and Treg cells from female mice were capable of protecting against development of sialadenitis in male recipients. Hence, our data demonstrate that female NOD mice develop sialadenitis through a defect in salivary-gland-protective Treg cells that can be reversed in the presence of testosterone.

Anti-IL-7 receptor-α treatment ameliorates newly established Sjögren's-like exocrinopathy in non-obese diabetic mice

The levels of interleukin (IL)-7 and its receptor are elevated in the salivary glands of patients with Sjögren's syndrome (SS). Our previous study indicates that IL-7 plays a critical pathogenic role in the development and onset of SS in a mouse model of this disease. The present study aims at determining whether IL-7 also plays a role in sustaining SS pathologies after the disease onset, by using the non-obese diabetic (NOD) model. Intraperitoneal administration of a blocking antibody against the IL-7 receptor α chain (IL-7Rα) to female NOD mice aged 10 weeks, which exhibited newly onset clinical SS, for the duration of 3 weeks significantly ameliorated characteristic SS pathologies including hyposalivation and leukocyte infiltration of the submandibular glands (SMGs). These changes were accompanied by a decrease in IFN-γ-producing CD4 T- and CD8 T cells, B cells, and lymphocyte chemoattractants CXCL9, −10, −11 and −13 in the SMGs. Anti-IL-7Rα treatment markedly diminished the amount of TNF-α in the SMGs and increased the level of claudin-1 and aquaporin 5, two molecules critical for normal salivary secretion. Furthermore, neutralization of IFN-γ and TNF-α, individually or in combination, considerably improved salivary secretion, reduced leukocyte infiltration and down-regulated CXCL9 and −13 expression in the SMGs. Collectively, the results indicate that endogenous IL-7R signals promote Th1 and Tc1 responses and IFN-γ- and TNF-α production to sustain the persistence of SS-like sialadenitis in NOD mice. These findings suggest that IL-7 and Th1 cytokines could serve as promising therapeutic targets for this prevalent autoimmune disease.

Beta‐cell‐specific loss of the inhibitory G protein, Gαz, prevents development of Type 1 Diabetes in NOD mice.

The mechanisms that underlie the beta‐cell pathophysiology of Type 1 Diabetes (T1D) are not fully understood. Our lab has discovered a role for the unique inhibitory G‐protein, Gαz, in beta‐cell function, survival, and replication in both normal conditions and those of pathological stress. Gαz, when acting as a canonical inhibitory G‐protein, reduces production of cyclic adenosine monophosphate (cAMP) through inhibition of adenylate cyclase. cAMP acts as essential player in the amplification of glucose‐stimulated insulin secretion, while also mediating pathways that regulate beta‐cell death and replication. We've shown previously that removal of this inhibitory protein is sufficient to promote insulin secretion, beta‐cell growth and survival using chemically‐induced beta‐cell death and diabetes in C57BL/6J mice. Previous work in our lab has also shown that non‐obese diabetic (NOD) mice with global loss of Gαz are protected from developing T1D‐like hyperglycemia through a multi‐faceted maintenance of beta‐cell health. Whole body Gαznull animals exhibited a significant increase in beta‐cell function and replication and decreased beta‐cell death. Most interestingly, islets from Gαz‐null NOD animals exhibited a marked decrease in immune infiltration. In order to determine if Gαz in the beta‐cell was responsible for the protected phenotype, we generated a beta‐cell‐specific Gαz knockout by breeding Gz Flox/Flox mice with Rat Insulin Promoter (RIP) driven CreHerr mice, on the NOD background. After 10 generations of backcrossing, we began by determining if loss of beta cell Gαz was sufficient to protect NOD animals from developing T1D‐like hyperglycemia. Weekly random‐fed blood glucose measurements were taken beginning at weaning. In the initial cohort, we saw a 12% penetrance of hyperglycemia in male Cre‐positive NOD mice and a 23% penetrance of hyperglycemia in female Cre‐positive NOD mice by 30 weeks of age. In contrast, Gαzβ‐cell‐KO males and females were entirely protected from developing hyperglycemia during this period. Despite this stark contrast, fasting blood glucose was no different between genotypes, grouped by sex, at either 12 or 28 weeks of age. Glucose tolerance was also not different between Cre‐positive NOD controls and Gαzβ‐cell‐KO animals. Experiments are ongoing to further investigate other facets of beta‐cell health, including function, replication and survival. Additionally, it will be crucial to evaluate the presence and extent of insulitis in our Gαzβ‐cell‐KO animals. Thus far, our results show a critical role for beta‐cell Gαz in promoting T1D pathophysiology, although the precise mechanism remains to be determined.Support or Funding InformationNIH F31 DK109698 to RFThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Clostridium butyricum CGMCC0313.1 Protects against Autoimmune Diabetes by Modulating Intestinal Immune Homeostasis and Inducing Pancreatic Regulatory T Cells.

Recent evidence indicates that indigenous Clostridium species induce colonic regulatory T cells (Tregs), and gut lymphocytes are able to migrate to pancreatic islets in an inflammatory environment. Thus, we speculate that supplementation with the well-characterized probiotics Clostridium butyricum CGMCC0313.1 (CB0313.1) may induce pancreatic Tregs and consequently inhibit the diabetes incidence in non-obese diabetic (NOD) mice. CB0313.1 was administered daily to female NOD mice from 3 to 45 weeks of age. The control group received an equal volume of sterile water. Fasting glucose was measured twice a week. Pyrosequencing of the gut microbiota and flow cytometry of mesenteric lymph node (MLN), pancreatic lymph node (PLN), pancreatic and splenic immune cells were performed to investigate the effect of CB0313.1 treatment. Early oral administration of CB0313.1 mitigated insulitis, delayed the onset of diabetes, and improved energy metabolic dysfunction. Protection may involve increased Tregs, rebalanced Th1/Th2/Th17 cells and changes to a less proinflammatory immunological milieu in the gut, PLN, and pancreas. An increase of α4β7+ (the gut homing receptor) Tregs in the PLN suggests that the mechanism may involve increased migration of gut-primed Tregs to the pancreas. Furthermore, 16S rRNA gene sequencing revealed that CB0313.1 enhanced the Firmicutes/Bacteroidetes ratio, enriched Clostridium-subgroups and butyrate-producing bacteria subgroups. Our results provide the basis for future clinical investigations in preventing type 1 diabetes by oral CB0313.1 administration.

Anti-TCRβ mAb in Combination With Neurogenin3 Gene Therapy Reverses Established Overt Type 1 Diabetes in Female NOD Mice.

Insulin-producing β cells in patients with type 1 diabetes (T1D) are destroyed by T lymphocytes. We investigated whether targeting the T-cell receptor (TCR) with a monoclonal antibody (mAb) abrogates T-cell response against residual and newly formed islets in overtly diabetic nonobese diabetic (NOD) mice. NOD mice with blood glucose levels of 250 to 350 mg/dL or 350 to 450 mg/dL were considered as new-onset or established overt diabetes, respectively. These diabetic NOD mice were transiently treated with an anti-TCR β chain (TCRβ) mAb, H57-597, for 5 days. Two weeks later, some NOD mice with established overt diabetes further received hepatic gene therapy using the islet-lineage determining gene Neurogenin3 (Ngn3), in combination with the islet growth factor gene betacellulin (Btc). We found that anti-TCRβ mAb (50 µg/d) reversed >80% new-onset diabetes in NOD mice for >14 weeks by reducing the number of effector T cells in the pancreas. However, anti-TCRβ mAb therapy alone reversed only ∼20% established overt diabetes in these mice. Among those overtly diabetic NOD mice whose diabetes was resistant to anti-TCRβ mAb treatment, ∼60% no longer had diabetes when they also received Ngn3-Btc hepatic gene transfer 2 weeks after initial anti-TCRβ mAb treatment. This combination of Ngn3-Btc gene therapy and anti-TCRβ mAb treatment induced the sustained formation of periportal insulin-producing cells in the liver of overtly diabetic mice. Therefore, directly targeting TCRβ with a mAb potently reverses new-onset T1D in NOD mice and protects residual and newly formed gene therapy-induced hepatic neo-islets from T-cell‒mediated destruction in mice with established overt diabetes.

The role of bone marrow mesenchymal stromal cell derivatives in skin wound healing in diabetic mice.

Mesenchymal stromal cells (MSCs) have shown to be a promising tool in cell therapies to treat different conditions. Several pre-clinical and clinical studies have proved that the transplantation of MSCs improves wound healing. Here, we compare the beneficial effects of mouse bone marrow-derived allogeneic MSCs (allo-mBM-MSCs) and their acelullar derivatives (allo-acd-mMSCs) on skin wound healing in Non-Obese Diabetic (NOD) mice. One dose of allo-mBM-MSCs (1×106 cells) or one dose of allo-acd-mMSCs (1X) were intradermally injected around wounds in 8–10 week old female NOD mice. Wound healing was evaluated macroscopically (wound closure) every two days, and microscopically (reepithelialization, dermoepidermal junction, skin appendage regeneration, leukocyte infiltration, vascularization, granulation tissue formation, and density of collagen fibers in the dermis) after 16 days of MSC injection. In addition, we measured growth factors and specific proteins that were present in the allo-acd-mMSCs. Results showed significant differences in the wound healing kinetics of lesions that received allo-acd-mMSCs compared to lesions that received vehicle or allo-mBM-MSCs. In particular, mice treated with allo-acd-mMSCs reached significantly higher percentages of wound closure at day 4, 6 and 8, relative to the allo-mBM-MSCs and vehicle groups (p < 0.05), while wound closure percentages could not be statistically distinguished between the allo-mBM-MSCs and vehicle groups. Also, allo-acd-mMSCs had a greater influence in the skin would healing process. Specifically, they caused a less pronounced inflammatory severe response (p < 0.0001), more granulation tissue formation at an advanced stage (p < 0.0001), and higher density of collagen fibers (p < 0.05) compared to the other groups. Nevertheless, at day 16, both allo-mBM-MSCs and allo-acd-mMSCs revealed a higher effect on the recovery of the quality skin (continuous epidermis; regular dermoepidermal junction and skin appendages) relative to untreated lesions (p < 0.0001), but not between them. On the other hand, ELISA analyses indicated that the allo-acd-mMSCs contained growth factors and proteins relevant to wound healing such as IGF-1, KGF, HGF, VEGF, ANG-2, MMP-1, CoL-1 and PGE2. Compared to allo-acd-mMSCs, the administration of allo-mBM-MSCs is insufficient for wound healing in diabetic mice and delays the therapeutic effect, which maybe explained by the fact that trophic factors secreted by MSCs are critical for skin regeneration, and not the cells per se, suggesting that MSCs may require some time to secrete these factors after their administration.

AAV-mediated pancreatic overexpression of Igf1 counteracts progression to autoimmune diabetes in mice

ObjectiveType 1 diabetes is characterized by autoimmune destruction of β-cells leading to severe insulin deficiency. Although many improvements have been made in recent years, exogenous insulin therapy is still imperfect; new therapeutic approaches, focusing on preserving/expanding β-cell mass and/or blocking the autoimmune process that destroys islets, should be developed. The main objective of this work was to test in non-obese diabetic (NOD) mice, which spontaneously develop autoimmune diabetes, the effects of local expression of Insulin-like growth factor 1 (IGF1), a potent mitogenic and pro-survival factor for β-cells with immunomodulatory properties. MethodsTransgenic NOD mice overexpressing IGF1 specifically in β-cells (NOD-IGF1) were generated and phenotyped. In addition, miRT-containing, IGF1-encoding adeno-associated viruses (AAV) of serotype 8 (AAV8-IGF1-dmiRT) were produced and administered to 4- or 11-week-old non-transgenic NOD females through intraductal delivery. Several histological, immunological, and metabolic parameters were measured to monitor disease over a period of 28–30 weeks. ResultsIn transgenic mice, local IGF1 expression led to long-term suppression of diabetes onset and robust protection of β-cell mass from the autoimmune insult. AAV-mediated pancreatic-specific overexpression of IGF1 in adult animals also dramatically reduced diabetes incidence, both when vectors were delivered before pathology onset or once insulitis was established. Transgenic NOD-IGF1 and AAV8-IGF1-dmiRT-treated NOD animals had much less islet infiltration than controls, preserved β-cell mass, and normal insulinemia. Transgenic and AAV-treated islets showed less expression of antigen-presenting molecules, inflammatory cytokines, and chemokines important for tissue-specific homing of effector T cells, suggesting IGF1 modulated islet autoimmunity in NOD mice. ConclusionsLocal expression of Igf1 by AAV-mediated gene transfer counteracts progression to diabetes in NOD mice. This study suggests a therapeutic strategy for autoimmune diabetes in humans.

Genistein prevention of hyperglycemia and improvement of glucose tolerance in adult non-obese diabetic mice are associated with alterations of gut microbiome and immune homeostasis

Although studies have linked soy phytoestrogen 4,7,4-trihydroxyisoflavone genistein (GEN) to reduced type 1 diabetes (T1D) risk, the mechanism of dietary GEN on T1D remains unknown. In our studies, adult non-obese diabetic (NOD) mouse model was employed to investigate the effects of GEN exposure on blood glucose level (BGL), glucose tolerance, gut microbiome, and immune responses. Adult male and female NOD mice were fed with either soy-based or casein-based diet, and received GEN at 20mg/kg body weight by gavage daily. The BGL and immune responses (represented by serum antibodies, cytokines and chemokines, and histopathology) were monitored, while the fecal gut microbiome was sequenced for 16S ribosomal RNA to reveal any alterations in gut microbial communities. A significantly reduced BGL was found in NOD males fed with soy-based diet on day 98 after initial dosing, and an improved glucose tolerance was observed on both diets. In addition, an anti-inflammatory response (suggested by reduced IgG2b and cytokine/chemokine levels, and alterations in the microbial taxonomy) was accompanied by an altered β-diversity in gut microbial species. Among the NOD females exposed to GEN, a later onset of T1D was observed. However, the profiles of gut microbiome, antibodies and cytokines/chemokines were all indicative of pro-inflammation. This study demonstrated an association among GEN exposure, gut microbiome alteration, and immune homeostasis in NOD males. Although the mechanisms underlying the protective effects of GEN in NOD mice need to be explored further, the current study suggested a GEN-induced sex-specific effect in inflammatory status and gut microbiome.

Pathogenic role of endogenous TNF-α in the development of Sjögren's-like sialadenitis and secretory dysfunction in non-obese diabetic mice

Patients with Sjögren's syndrome (SS), an autoimmune disease primarily affecting exocrine glands, exhibit enhanced TNF-α expression in the saliva and salivary glands. However, the precise in vivo role of TNF-α during the initiation and development of SS is not clearly defined. The present study is undertaken to determine the function of endogenously produced TNF-α in the pathogenesis of SS in non-obese diabetic (NOD) mice, a model of this human disease. Administration of a neutralizing anti-TNF-α antibody to female NOD mice during the stage prior to disease onset significantly improved salivary secretion, indicating a remission of clinical symptoms of SS. TNF-α blockade also decreased the number of leukocyte foci and reduced the number of T cells and B cells in the submandibular glands (SMG). Moreover, TNF-α blockade reduced T-bet protein levels in the SMG, suggesting a decrease in T helper 1 and T cytotoxic 1 cells. These cellular changes induced by TNF-α neutralization were associated with a reduction in T- and B-cell chemoattractants CXCL9 and CXC13. In addition, TNF-α blockade markedly increased the expression level of tight junction protein claudin-1 and water channel protein aquaporin-5, two key factors required for normal salivary secretion, in the SMG. Collectively, these findings indicate that endogenous TNF-α has a pathogenic role in the development of SS in the NOD model of this disease.

Experimental cannabidiol treatment reducesearly pancreatic inflammation intype 1 diabetes.

Destruction of the insulin-producing beta cells in type 1 diabetes (T1D) is induced by invasion of immune cells causing pancreatic inflammation. Cannabidiol (CBD), a phytocannabinoid, derived from the plant, Cannabis sativa, was shown to lower the incidence of diabetes in non-obese diabetic (NOD) mice, an animal model of spontaneous T1D development. The goal of this study was to investigate the impact of experimental CBD treatment on early pancreatic inflammation in T1D by intravital microscopy (IVM) in NOD mice. Seven-week-old female NOD mice were prophylactically administered daily 5 mg/kg CBD or control vehicle i.p. five times weekly for ten weeks. Animals underwent IVM following confirmation of T1D diagnosis by blood glucose testing. Leukocyte activation and functional capillary density (FCD) were quantified via IVM. CBD-treated NOD mice developed T1D later and showed significantly reduced leukocyte activation and increased FCD in the pancreatic microcirculation. Experimental CBD treatment reduced markers of inflammation in the microcirculation of the pancreas studied by intravital microscopy.

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.

Peroxisome Proliferator-activated Receptor-γ Activation Augments the β-Cell Unfolded Protein Response and Rescues Early Glycemic Deterioration and β Cell Death in Non-obese Diabetic Mice

Type 1 diabetes is an autoimmune disorder that is characterized by a failure of the unfolded protein response in islet β cells with subsequent endoplasmic reticulum stress and cellular death. Thiazolidinediones are insulin sensitizers that activate the nuclear receptor PPAR-γ and have been shown to partially ameliorate autoimmune type 1 diabetes in humans and non-obese diabetic (NOD) mice. We hypothesized that thiazolidinediones reduce β cell stress and death independently of insulin sensitivity. To test this hypothesis, female NOD mice were administered pioglitazone during the pre-diabetic phase and assessed for insulin sensitivity and β cell function relative to controls. Pioglitazone-treated mice showed identical weight gain, body fat distribution, and insulin sensitivity compared with controls. However, treated mice showed significantly improved glucose tolerance with enhanced serum insulin levels, reduced β cell death, and increased β cell mass. The effect of pioglitazone was independent of actions on T cells, as pancreatic lymph node T cell populations were unaltered and T cell proliferation was unaffected by pioglitazone. Isolated islets of treated mice showed a more robust unfolded protein response, with increases in Bip and ATF4 and reductions in spliced Xbp1 mRNA. The effect of pioglitazone appears to be a direct action on β cells, as islets from mice treated with pioglitazone showed reductions in PPAR-γ (Ser-273) phosphorylation. Our results demonstrate that PPAR-γ activation directly improves β cell function and survival in NOD mice by enhancing the unfolded protein response and suggest that blockade of PPAR-γ (Ser-273) phosphorylation may prevent type 1 diabetes.

Inducing Specific Immune Tolerance to Prevent Type 1 Diabetes in NOD Mice.

Proinsulin is the first autoantigen in type 1 diabetes (T1D). We reasoned that coupling hematopoietic stem cells (HSCs) transplantation with ex vivo transduction of syngeneic HSCs with lentiviral vectors to express proinsulin II could prevent T1D in nonobese diabetic (NOD) mice. Hematopoietic stem cells were isolated from 6- to 8-week-old NOD female mice and transduced in vitro with lentiviral vectors encoding proinsulin II. Preconditioned 3- to 4-week-old female NOD mice were transplanted with transduced or nontransduced HSCs and compared with age-matched unmanipulated control. The insulitis, T1D development, and immune reconstitution were assessed. The mean (SD) insulitis score was significantly reduced (1.156 [0.575] vs 2.156 [0.892] or 3.043 [0.728], P = 0.009 or <0.001), and diabetes was nearly completely prevented (1/13 vs 5/12 or 4/9, P = 0.031 or 0.013) in recipients of transduced HSCs expressing proinsulin II as compared with recipients of nontransduced HSCs or unmanipulated control. Sialitis, reconstitution of peripheral blood leukocytes, and in vitro recall responses to ovalbumin were not different between 3 groups of mice. Syngeneic transplantation of HSCs transduced ex vivo with lentiviral vectors to encode proinsulin II is a novel strategy to prevent T1D.

Sexual dimorphism in myocardial acylcarnitine and triglyceride metabolism.

BackgroundCardiovascular disease is the leading cause of death among diabetic patients. Importantly, recent data highlight the apparent sexual dimorphism in the pathogenesis of cardiovascular disease in diabetics with respect to both frequency- and age-related risk factors. The disposition to cardiovascular disease among diabetic patients has been attributed, at least in part, to excess lipid supply to the heart culminating in lipotoxicity of the heart and downstream derangements. A confounding factor in obese animal models of diabetes is that increased peripheral lipid availability to the heart can induce cardio-metabolic remodeling independent of the underlying pathophysiology of diabetes, thus masking the diabetic phenotype. To that end, we hypothesized that the use of non-obese diabetic (NOD) animal models will reveal metabolic signatures of diabetes in a sex-specific manner.MethodsTo test this hypothesis, male and female NOD Goto-Kakizaki (GK) rats were used to assess the expression profile of 84 genes involved in lipid metabolism. In parallel, targeted lipidomics analysis was performed to characterize sex differences in homeostasis of non-esterified fatty acids (NEFA), acylcarnitines (AC), and triglycerides (TG).ResultsOur analysis revealed significant sex differences in the expression of a broad range of genes involved in transport, activation, and utilization of lipids. Furthermore, NOD male rats exhibited enhanced oxidative metabolism and accumulation of TG, whereas female NOD rats exhibited reduced TG content coupled with accumulation of AC species. Multi-dimensional statistical analysis identified saturated AC16:0, AC18:0, and AC20:0 as dominant metabolites in mediating sex differences in AC metabolism. Confocal microscopy of rat cardiomyocytes exposed to AC14:0, AC16:0, and AC18:0 confirmed induction of ROS with AC18:0 being more potent followed by AC14:0.ConclusionOverall, we demonstrate sex differences in myocardial AC and TG metabolism with implications for therapy and diagnosis of diabetic cardiovascular disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13293-016-0077-7) contains supplementary material, which is available to authorized users.

The pathogenic effect of TNF-α on development of Sjögren’s syndrome in non-obese diabetic mice

Abstract Tumor necrosis factor-alpha (TNF-α), is a proinflammatory cytokine involved in the pathogenesis of various autoimmune diseases including rheumatoid arthristis, inflammatory bowel disease and psoriasis. Previous studies showed that patients with Sjögren’s syndrome(SS), a prevalent systemic autoimmune disease primarily affecting tear and saliva secretion, exhibit enhanced expression of TNF-α in saliva and salivary gland. However, the exact role of TNF-α in SS disease development is not yet fully determined. Our current study employed non-obese diabetic (NOD) mice, a model of SS-like diseases, to investigate the function of endogenous TNF-α in pathogenesis of SS. We observed that administration of a neutralizing anti-TNF-α antibody significantly improved salivary secretion in female NOD mice, indicating a remission of clinical symptom of SS. TNF-α neutralization also decreased the number of leukocytic foci and reduced the number of total and activated CD4 T cells and B cells in the submandibular gland of NOD mice. Moreover, neutralization of TNF-α reduced the number of CXCR3-expressing CD4 T cells and T-bet protein level in the submandibular gland, suggesting a decrease in the infiltrating T helper 1 and T cytotoxic 1 cells. In addition, the decrease in CXCR3+ CD4 T cells was associated with reduced expression of CXCR3 ligand CXCL9. Overall, the results presented here indicate that endogenous TNF-α plays a pathogenic role in the initiation and development of SS disease and neutralization of TNF-α may be greatly beneficial for the prevention and treatment of this prevalent autoimmune disease.

Protective role of adenovirus vector-mediated interleukin-10 gene therapy on endogenous islet β-cells in recent-onset type 1 diabetes in NOD mice.

The aim of the present study was to provide an animal experimental basis for the protective effect of the adenoviral vector-mediated interleukin-10 (Ad-mIL-10) gene on islet β-cells during the early stages of type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. A total of 24 female NOD mice at the onset of diabetes were allocated at random into three groups (n=8 per group): Group 1, intraperitoneally injected with 0.1 ml Ad-mIL-10; group 2, intraperitoneally injected with 0.1 ml adenovirus vector; and group 3, was a diabetic control. In addition to groups 1, 2 and 3, 8 age- and gender-matched NOD mice were intraperitoneally injected with 0.1 ml PBS and assigned to group 4 as a normal control. All mice were examined weekly for body weight, urine glucose and blood glucose values prior to onset of diabetes, and at 1, 2 and 3 weeks after that, and all mice were sacrificed 3 weeks after injection. Serum levels of interleukin (IL)-10, interferon (IFN)-γ, IL-4, insulin and C-peptide were evaluated, and in addition the degree of insulitis and the local expression of IL-10 gene in the pancreas were detected. The apoptosis rate of pancreatic β-cells was determined using a TUNEL assay. Compared with groups 2 and 3, IL-10 levels in the serum and pancreas were elevated in group 1. Serum IFN-γ levels were decreased while serum IL-4 levels and IFN-γ/IL-4 ratio were significantly increased in group 1 (P<0.01). C-peptide and insulin levels were higher in group 1 compared with groups 2 and 3, (P<0.01). Furthermore, compared with groups 2 and 3, the degree of insulitis, islet β-cell apoptosis rate and blood glucose values did not change significantly (P>0.05). The administration of the Ad-mIL-10 gene induced limited immune regulatory and protective effects on islet β-cell function in NOD mice with early T1D, while no significant reduction in insulitis, islet β-cell apoptosis rate and blood glucose was observed.

Vaccination with a co-expression DNA plasmid containing GAD65 fragment gene and IL-10 gene induces regulatory CD4(+) T cells that prevent experimental autoimmune diabetes.

The non-obese diabetic (NOD) mouse is a commonly used animal model for studying type 1 diabetes (T1D). The aims of our study were to explore the diabetes-preventive effect in NOD mice and the potential mechanisms of an optimized co-expression DNA vaccine containing GAD65 fragment gene with the IL-10 gene (SGAD65190-315 /IL-10). Female NOD mice at the age of 3-4weeks old were randomly divided into two groups and received intra-muscular injection of either blank pBudCE4.l vector (n = 34) or pBudCE4.l carrying the SGAD65190-315 /IL-10 (n = 32). The incidence of diabetes was monitored up to 30weeks of age. The severity of insulitis, apoptosis rate of β cells and relevant mechanisms were examined. Administration with SGAD65190-315 /IL-10 blocked the onset of autoimmune diabetes in NOD mice, significantly suppressed islet inflammation, inhibited the apoptosis of islet β cells, induced immune tolerance to autoantigen GAD65 and proinsulin and shifted the Th1/Th2 balance towards Th2. More importantly, the frequencies of CD4(+) CD25(+) Foxp3(+) regulatory T cells (Tregs) in the spleen and pancreatic lymph nodes in vaccine-immunized mice were significantly increased, and these Tregs were GAD65-reactive. In addition, Treg depletion by anti-CD25 mAb administration abolished the protective effects of SGAD65190-315 /IL-10 on diabetes and insulitis. Moreover, depletion of CD4(+) CD25(+) T cells using magnetic-activated cell sorting impaired the protective effect of SGAD65190-315 /IL-10 vaccination on adoptive transfer of diabetes. Our data suggested that SGAD65190-315 /IL-10 DNA vaccine had protective effects on T1D by upregulating autoantigen-reactive Tregs. Our findings may provide a novel preventive therapy for T1D. Copyright © 2016 John Wiley & Sons, Ltd.