Diabetic Female Mice Research Articles

Abstract 337: G Protein-coupled Estrogen Receptor Contributes To The Vascular Effects Of Aldosterone And Type Two Diabetes-associated Vascular Dysfunction

Aldosterone (Aldo) excess aggravates endothelial dysfunction in diabetes. Aldo exerts its effects via activation of both mineralocorticoid receptors (MR) and G protein-coupled estrogen receptors (GPER). Considering that GPER activation has beneficial effects in the vasculature, we hypothesized that GPER-mediated vascular effects of aldosterone are decreased/abrogated in diabetes. Second-order mesenteric arteries from control (B6BKS-Leprdb/+) and diabetic (db/db) female mice were incubated with 10 nM Aldo, in the presence of either vehicle (veh), the MR antagonist eplerenone (Eple, 10 μM) or the GPER antagonist G15 (1μM), and the effects on phenylephrine (Phe) vascular reactivity were determined. Aldo increased Phe maximal response (Emax, % of KCl contraction) in arteries from control (veh: 112.5±3.2 vs. Aldo: 129.1±2.8 p0.05). In control vessels, Eple did not alter Phe Emax either in the presence of Aldo or veh (p>0.05), whereas G15 abrogated Aldo-induced increase in Phe Emax (Aldo: 129.1±2.8 vs. G15+Aldo: 110.3±3.6 p<0.05). In db/db arteries, the MR and GPER antagonist decreased Phe Emax both in the presence of veh and Aldo (veh: 143.8±4.9 vs Aldo: 136.0±5.2 vs. Eple+veh: 105.4±4.8 vs. Eple+Aldo: 94.6±4.4 vs. G15+veh: 96.4±4.2 vs. G15+Aldo: 104.3±4.1 p<0.05). Arteries from diabetic mice exhibited increased ERK1/2 activation vs. control [arbitrary units (Au), 229.4±3 vs. 100.0±11.8, respectively, p<0.05]. Aldo increased ERK1/2 activation in control (Au, 99.8±0.4 vs. 272.2±58.0 p<0.05) and db/db mice (Au, 99.7±4.0 vs. 162.4±28.2 p<0.05). Eple and G15 reduced Aldo-induced ERK 1/2 activation in control arteries (p<0.05). In db/db arteries, G15 decreased ERK1/2 activation both in the presence of veh and Aldo (Au, Aldo: 162.4±28.2 vs. G15+veh: 81.1±9.1 vs. G15+aldo: 82.7±7.5 p<0.05). In summary, Aldo acutely increases contractile responses to PhE in mesenteric arteries from control animals by GPER-dependent mechanisms. In diabetic animals, both MR and GPER seem to contribute to Aldo effects in the vasculature. Contrary to our hypothesis, GPER contributes to the vascular effects of Aldo in diabetic animals as well as to diabetes-associated vascular dysfunction.

Autoimmunity as an Endogenous Source of Heterologous Alloreactivity.

In autoimmune Type 1 diabetic (T1D) recipients, the disease can recur in pancreatic islet transplants. However, because autoimmune T cells recognize autoantigens in the context of self MHC antigens, it is not clear how autoreactive (islet-specific) T cells may recognize MHC-unrelated islet allografts. In this study, determined the specificity of T cells infiltrating islet allografts in the non-obese diabetic (NOD) mouse model of T1D. We grafted 400-500 syngeneic (NOD, H-2g7) or allogeneic (C3H, H-2k) islets beneath the kidney capsule of spontaneously diabetic female NOD mice. Curiously, although the autoimmune repertoire is self (H-2g7) MHC-restricted, C3H allografts were more rapidly rejected in NOD mice (n = 6; MST 7days) than were syngeneic (NOD) islet grafts (n = 19; MST 11 days), p < .001. We tested the hypothesis that within the activated autoimmune T cell repertoire was a sub-population of T cell receptors (TCR) that cross-reacted with allogeneic MHC. To determine whether islet-autoreactive T cells infiltrate C3H islet allografts, we used 454 high-throughput TCR sequencing to compare the TCR repertoires expressed in the NOD recipient's residual endogenous pancreatic islets to those expressed by islet allograft-infiltrating T cells. A portion of TCRs were shared between the endogenous pancreatic islets and the C3H allograft. We next determined the specificity of TCRs used in high frequency in rejected C3H islet allografts. To this end, TCRs expressed in high frequency in the allograft (>1%) were cloned and tested for islet autoreactivity and/or alloreactivity following TCR cDNA transfection into IL-2 producing hybridoma cells. Of note, three TCRs studied that were the most frequent in the graft were also detected in the endogenous pancreas. As expected, all three TCRs were islet reactive. Surprisingly, all three of these TCR also were directly alloreactive to C3H spleen cells in the absence of either islet antigens or NOD APCs. Two of the three TCRs were MHC class I reactive (anti-H-2Dk), and the other was MHC class II-reactive (anti-I-Ak). These results suggest that dual auto/allo-reactive T cells are selectively expanded in islet allografts. As such, we propose that autoimmunity is an unusual source of endogenous heterologous alloreactivity that may target an allograft via simultaneous auto- and allo-reactive specificities.

Autoimmunity as an endogenous source of heterologous alloimmunity (TRAN3P.868)

Abstract Curiously, although autoimmunity is restricted to self MHC molecules, spontaneously diabetic NOD mice reject MHC-unrelated islet allografts more rapidly than syngeneic (NOD) grafts. We hypothesized that the autoimmune (islet-reactive) T cell repertoire harbors a subset of TCRs that also cross-react to allogeneic MHC molecules. To test this concept, we performed high throughput TCR sequencing of T cells infiltrating MHC-mismatched (C3H, H-2k) islet allografts on the day of rejection in diabetic female NOD mice. TCRs expressed in high frequency in the allograft (&amp;gt;1%) were cloned and tested for islet autoreactivity and/or alloreactivity following TCR cDNA transfection into IL-2 producing hybridoma cells. Of note, three TCRs studied that were the most frequent in the graft were also detected in the endogenous pancreas. All three transfectants expressing these TCRs were islet reactive. Importantly, all three of these TCR also were directly alloreactive to C3H spleen cells. Two of the three TCRs were MHC class I reactive (anti-H-2Dk), and the other was MHC class II-reactive (anti-I-Ak). These results suggest that dual auto/allo-reactive T cells are selectively expanded in islet allografts. As such, we propose that autoimmunity is an unusual source of endogenous heterologous alloreactivity that may accelerate allograft rejection by simultaneous auto- and allo-reactivity.

Rapid onset of cardiomyopathy in STZ-induced female diabetic mice involves the downregulation of pro-survival Pim-1

BackgroundDiabetic women are five times more likely to develop congestive heart failure compared with two fold for men. The underlying mechanism for this gender difference is not known. Here we investigate the molecular mechanisms responsible for this female disadvantage and attempt safeguarding cardiomyocytes viability and function through restoration of pro-survival Pim-1.Methods and ResultsDiabetes was induced by injection of streptozotocin in CD1 mice of both genders. Functional and dimensional parameters measurement using echocardiography revealed diastolic dysfunction in female diabetic mice within 8 weeks after STZ-induced diabetes. This was associated with significant downregulation of pro-survival Pim-1 and upregulation of pro-apoptotic Caspase-3, microRNA-1 and microRNA-208a. Male diabetic mice did not show any significant changes at this time point (P < 0.05 vs. female diabetic). Further, the onset of ventricular remodelling was quicker in female diabetic mice showing marked left ventricular dilation, reduced ejection fraction and poor contractility (P < 0.05 vs. male diabetic at 12 and 16 weeks of STZ-induced diabetes). Molecular analysis of samples from human diabetic hearts confirmed the results of pre-clinical studies, showing marked downregulation of Pim-1 in the female diabetic heart (P < 0.05 vs. male diabetic). Finally, in vitro restoration of Pim-1 reversed the female disadvantage in diabetic cardiomyocytes.ConclusionsWe provide novel insights into the molecular mechanisms behind the rapid onset of cardiomyopathy in female diabetics. These results suggest the requirement for the development of gender-specific treatments for diabetic cardiomyopathy.

Antimicrobial peptide and mucin expression differ between the diabetic NOD mouse and non‐diabetic NOR mouse (650.16)

Type 1 Diabetes (T1D) is defined as the selective immune destruction of insulin producing β‐cells within the islet. Alterations in the intestinal microbiota, increased intestinal permeability, and an aberrant immune system are thought to play key roles in the development of T1D. Recent studies have shown that mice that develop T1D have a different microbiota compared to mice that do not develop T1D. However why mice that are on the same diet in the same facilities have different microbiota is unknown. We hypothesize that altered antimicrobial peptide (AMP) and mucin production early in life determines ones microbiota, protecting some, but not others from T1D. Non‐obese diabetic (NOD) female mice were compared to age and sex matched Non‐obese diabetic resistant (NOR) mice. AMP and mucin gene expression was measured in the ileum and colon of the two strains and protein expression was examined by immunohistochemistry. At 2 weeks of age there is a significant increase in the AMP, defcr‐4, in the ileum of NOD mice compared to NOR mice. In addition a decrease in the mucins, Muc 1, 2, and 3 was measured in the colons of NOD mice compared to NOR mice. This correlates with alterations that we have measured in Bacteroidetes and Firmicutes between the two strains of mice. We postulate that these differences in AMP and mucin expression between the two strains leads to alterations in microbiota, which leads to differences in disease outcome.

Plasminogen Activator Inhibitor-1 Is Involved in Impaired Bone Repair Associated with Diabetes in Female Mice

Previous studies suggest that fracture healing is impaired in diabetes; however, the underlying mechanism remains unclear. Here, we investigated the roles of plasminogen activator inhibitor-1 (PAI-1) in the impaired bone repair process by using streptozotocin (STZ)-induced diabetic female wild-type (PAI-1 +/+) and PAI-1-deficient (PAI-1 −/−) mice. Bone repair and the number of alkaline phosphatase (ALP)-positive cells at the site of a femoral bone damage were comparable in PAI-1 +/+ and PAI-1 −/− mice without STZ treatment. Although the bone repair process was delayed by STZ treatment in PAI-1 +/+ mice, this delayed bone repair was blunted in PAI-1 −/− mice. The reduction in the number of ALP-positive cells at the site of bone damage induced by STZ treatment was attenuated in PAI-1 −/− mice compared to PAI-1 +/+ mice. On the other hand, PAI-1 deficiency increased the levels of ALP and type I collagen mRNA in female mice with or without STZ treatment, and the levels of Osterix and osteocalcin mRNA, suppressed by diabetic state in PAI-1 +/+ mice, were partially protected in PAI-1 −/− mice. PAI-1 deficiency did not affect formation of the cartilage matrix and the levels of types II and X collagen and aggrecan mRNA suppressed by STZ treatment, although PAI-1 deficiency increased the expression of chondrogenic markers in mice without STZ treatment. The present study indicates that PAI-1 is involved in the impaired bone repair process induced by the diabetic state in part through a decrease in the number of ALP-positive cells.

Fecal metabonomic study of a polysaccharide, MDG-1 from Ophiopogon japonicus on diabetic mice based on gas chromatography/time-of-flight mass spectrometry (GC TOF/MS)

Type 2 Diabetes Mellitus (T2DM) is a chronic metabolic disorder with systemic complications and has been a worldwide epidemic. Ophiopogon japonicus is a traditional Chinese medicine used to treat diabetes for thousands of years. From our previous work, we know that MDG-1, a water-soluble β-D-fructan polysaccharide from O. japonicas could treat T2DM experimentally. However, MDG-1 is poorly absorbed and its mechanism of action is still unknown. Therefore, a GC TOF/MS-based metabonomic approach in combination with multivariate statistical analysis was performed to investigate the mechanism of MDG-1 in a spontaneous diabetic model. Female diabetic KKay mice (21 weeks old) were randomly divided into a diabetic group (n = 6, gavaged with distilled water) and a MDG-1-Diabetic group (n = 7, gavaged with MDG-1, 300 mg kg(-1)) and female C57BL/6 mice (21 weeks old) were set as controls (n = 6, gavaged with distilled water). After 8-weeks of treatment, feces samples were collected for GC-TOF/MS analysis. Consequently, 12 potential biomarkers were identified, including monosugars (D-tagatose, D-lyxose, D-erythrose, xylo-hexos-5-ulose, 2-deoxy-galactose), butanedioic acid, amino acids (phenylalanine, L-lysine, L-methionine, L-aspartic acid) and purine derivatives (7H-purine, 2'-deoxyinosine). We assume the monosugars and butanedioic acid were the fermentation products of MDG-1 by intestinal microbes and MDG-1 actions against diabetes might be accomplished through the absorbable monosugars and butanedioic acid via suppressing intestinal glucose absorption, enhancing liver glycogenesis, inhibiting glycogenolysis and promoting GLP-1 secretion. Besides, MDG-1 might alleviate diabetes and diabetic nephropathy by reducing 7H-purine and 2'-deoxyinosine. Further omics-driven studies including genomics, proteomics and metabonomics were considered to be carried out to provide direct evidence of gut microbiome contribution to MDG-1 actions.

Lotus (Nelumbo nucifera Gaertn) plumule polysaccharide ameliorates pancreatic islets loss and serum lipid profiles in non-obese diabetic mice

To unravel possible protective effects of a newly isolated lotus plumule polysaccharide (LPPS) on type 1 diabetes (T1D), this study isolated LPPS and administered it to non-obese diabetic (NOD) female mice for 15 weeks. Oral glucose tolerance, serum ketone body, glucose, insulin, and lipid levels, as well as pancreatic islet cell numbers and the insulin secretion ability of the experimental mice were determined. The results showed that LPPS administration in vivo significantly (P<0.05) increased pancreatic islet cell numbers and slightly enhanced the basal insulin secretion ability compared to the control group. LPPS administration improved serum lipid profiles in the diabetic mice via relatively increasing serum high density lipoprotein-cholesterol, but decreasing low density lipoprotein-cholesterol and total cholesterol levels. The present study suggests that LPPS supplementation may ameliorate T1D progress and its complications through protecting pancreatic islets and modulating serum lipid profiles.

A Bispecific Protein Capable of Engaging CTLA-4 and MHCII Protects Non-Obese Diabetic Mice from Autoimmune Diabetes

Crosslinking ligand-engaged cytotoxic T lymphocyte antigen-4 (CTLA-4) to the T cell receptor (TCR) with a bispecific fusion protein (BsB) comprised of a mutant mouse CD80 and lymphocyte activation antigen-3 (LAG-3) has been shown to attenuate TCR signaling and to direct T-cell differentiation toward Foxp3+ regulatory T cells (Tregs) in an allogenic mixed lymphocyte reaction (MLR). Here, we show that antigen-specific Tregs can also be induced in an antigen-specific setting in vitro. Treatment of non-obese diabetic (NOD) female mice between 9–12 weeks of age with a short course of BsB elicited a transient increase of Tregs in the blood and moderately delayed the onset of autoimmune type 1 diabetes (T1D). However, a longer course of treatment (10 weeks) of 4–13 weeks-old female NOD animals with BsB significantly delayed the onset of disease or protected animals from developing diabetes, with only 13% of treated animals developing diabetes by 35 weeks of age compared to 80% of the animals in the control group. Histopathological analysis of the pancreata of the BsB-treated mice that remained non-diabetic revealed the preservation of insulin-producing β-cells despite the presence of different degrees of insulitis. Thus, a bifunctional protein capable of engaging CTLA-4 and MHCII and indirectly co-ligating CTLA-4 to the TCR protected NOD mice from developing T1D.

Administration of IL-1 Trap prolongs survival of transplanted pancreatic islets to type 1 diabetic NOD mice

We previously reported that IL-1 Trap (a hybrid molecule consisting of the extracellular domain of IL-1 receptor accessory protein and IL-1 receptor type 1 arranged inline and fused to the Fc-portion of IgG1) can protect rat pancreatic islets in vitro against noxious effects induced by IL-1β. In this study we tested the effect of administration of a murine IL-1 Trap on the recurrence of disease (ROD) model in non-obese diabetic (NOD) mice. Spontaneously diabetic female NOD mice received implantation of a curative number (600) of syngeneic pancreatic islets beneath their left kidney capsule from young healthy NOD mouse donors. Once a day, the mice were injected subcutaneously with IL-1 Trap (30mg/kg bodyweight), or an equimolar dose Fc-control protein (8.4mg/kg bodyweight) or saline. The treatments were maintained until ROD (i.e. a blood glucose value ⩾11.1mM for 2 consecutive days) or until 5days after transplantation. 3 out of 11 mice treated with IL-1 Trap showed a significantly increased graft survival compared to all other mice, and analysis of relative cytokine mRNA levels in isolated spleen cells showed elevated IL-4 mRNA levels, but no differences in FoxP3 or iNOS staining of grafts, from mice treated with IL-1 Trap, at both endpoints, compared to both control groups. Administration of IL-1 Trap counteracts islet cell destruction in the NOD mouse model of type 1 diabetes. In part this could be due to a shift towards Th2 cytokine production seen in IL-1 Trap treated animals.

Affinity of T cells for chromogranin A remain constant in type I diabetic NOD mice. (P4119)

Abstract In type 1 diabetes numerous autoantigens have been identified in the non-obese mouse model including insulin B 9-23, chromogranin A (ChgA29-42), and glutamic acid decarboxylase 65 (GAD65) to list a few, but the kinetics of antigen specificity during the progression from insulitis to overt diabetes is not known. A T cell clone, BDC2.5, isolated from a diabetic NOD was recently identified as being reactive to ChgA. The two-dimensional (2D) micropipette adhesion frequency assay was able to determine that BDC2.5 TCR transgenic cells specifically recognize ChgA29-42, albeit with 100-fold lower affinity compared to the highly stimulatory mimotope. As this TCR was originally derived from a diabetic NOD, we wanted to see if this highly sensitive assay could detect ChgA29-42 specific cells in the pancreatic lymph nodes (PLN) of diabetic NODs. ChgA29-42 specific CD4 T cells were detected in the PLN at a high frequency and their 2D effective affinity was not statistically different from the BDC2.5 affinity for ChgA29-42. Tracking both the frequency and affinity of antigen specific cells isolated from normoglycemic to overtly diabetic female NOD mice, we have determined that the frequency of antigen specific cells present in the PLN but not the overall affinity range changes for ChgA29-42 between these time points. Thus the micropipette adhesion frequency assay allows one to track the frequency and affinity of antigen specific T cells during the progression to autoimmune disease.

Stimulation of Autophagy Improves Endoplasmic Reticulum Stress–Induced Diabetes

Accumulation of misfolded proinsulin in the β-cell leads to dysfunction induced by endoplasmic reticulum (ER) stress, with diabetes as a consequence. Autophagy helps cellular adaptation to stress via clearance of misfolded proteins and damaged organelles. We studied the effects of proinsulin misfolding on autophagy and the impact of stimulating autophagy on diabetes progression in Akita mice, which carry a mutation in proinsulin, leading to its severe misfolding. Treatment of female diabetic Akita mice with rapamycin improved diabetes, increased pancreatic insulin content, and prevented β-cell apoptosis. In vitro, autophagic flux was increased in Akita β-cells. Treatment with rapamycin further stimulated autophagy, evidenced by increased autophagosome formation and enhancement of autophagosome–lysosome fusion. This was associated with attenuation of cellular stress and apoptosis. The mammalian target of rapamycin (mTOR) kinase inhibitor Torin1 mimicked the rapamycin effects on autophagy and stress, indicating that the beneficial effects of rapamycin are indeed mediated via inhibition of mTOR. Finally, inhibition of autophagy exacerbated stress and abolished the anti-ER stress effects of rapamycin. In conclusion, rapamycin reduces ER stress induced by accumulation of misfolded proinsulin, thereby improving diabetes and preventing β-cell apoptosis. The beneficial effects of rapamycin in this context strictly depend on autophagy; therefore, stimulating autophagy may become a therapeutic approach for diabetes.

Insulin-like Growth Factor 1 mRNA Expression in the Uterus of Streptozotocin-treated Diabetic Mice

Reproductive functions decline with the onset of diabetes in female mice. Diabetic mice have smaller uteri with an underdeveloped endometrium, suggesting diminished estrogen-induced growth. We aimed to clarify the changes in the estrous cycle and in insulin-like growth factor 1 (IGF1) expression in the uteri of streptozotocin (STZ)-treated diabetic mice, because IGF1 is one of the main growth factors involved in estrogen-induced uterine growth. ICR female mice were intraperitoneally administered STZ (10 mg/100 g BW), and blood glucose levels were determined. Mice with blood glucose levels > 200 mg/dl were classified as diabetic mice. The onset of diabetes was associated with acyclic estrous cycles. Diabetes was also induced with STZ in ovariectomized mice. Uterine Igf1 mRNA levels were reduced in ovariectomized STZ-treated diabetic mice. Estrogen is known to stimulate Igf1 mRNA expression in the uterus, but estrogen action was abolished in the uteri of STZ-treated diabetic mice. mRNA expressions of estrogen receptor α (ERα) and steroid hormone receptor coactivators (SRC-1/Ncoa1, SRC-2/Ncoa2, SRC-3/Ncoa3 and CBP/p300/Crebbp) were reduced in the uteri of ovariectomized STZ-treated diabetic mice. The present study demonstrates that diabetes induces a decline in female reproductive functions in mice. Igf1 expression in ovariectomized diabetic female mice was decreased, and decreased responsiveness to estrogen in the uteri of diabetic mice is probably associated with a reduction in ERα and steroid receptor coactivator mRNA expression.

Suppressed G‐protein‐coupled receptor kinase 2 activity protects female diabetic‐mouse aorta against endothelial dysfunction

Pre-menopausal women have less cardiovascular disease and lower cardiovascular morbidity and mortality than men the same age. Previously, we noted in mice that G-protein-coupled receptor kinase 2 (GRK2) negatively regulates the Akt/eNOS pathway in male diabetic aortas and that endothelial function via the Akt/eNOS pathway is less affected in female diabetic aortas. The cellular mechanisms underlying these sex differences remain unclear. We aimed to investigate the ways in which GRK2 might modulate vascular functions in male and female diabetic mice (DM). Vascular functions were examined in aortic rings. GRK2, β-arrestin 2 and Akt/eNOS-signalling-pathway protein levels and activities were assayed by Western blotting. Phenylephrine-induced contraction was greater, while both clonidine-induced and insulin-induced relaxations were weaker (vs. male controls), in aortas from male type 2 DM, suggesting impairments of the Akt/eNOS pathway and α-adrenoceptor function. GRK2-inhibitor reversed only the impairment in Akt/eNOS-pathway-mediated relaxation in male DM. Increases in GRK2 activity, GRK2 expression in the membrane, plasma Ang II and systolic blood pressure were seen in male DM (vs. male controls) but not in female DM; these increases were attenuated by GRK2-inhibitor treatment. Repeatedly obtaining clonidine concentration-response curves led to reduced relaxation in male and in female DM aortas, indicating similar desensitization between female DM and male DM. This effect was reversed by GRK2-inhibitor in both sexes. GRK2 plays a key role in modulating the aortic vasodilator effect of clonidine by selectively affecting the Akt/eNOS pathway. This action of GRK2 is more powerful in male than in female DM.

Protective effect of endogenous estrogen on streptozotocin-induced diabetes mellitus in female mice

Objective To investigate the effects of endogenous estrogen on blood glucose level,serum insulin level,and plasma total antioxidant capacity in streptozotocin-induced diabetic female mice,and to explore possible protective effects of estrogen on pancreatic islet cells.Methods Female mice were randomly according to body weight divided into four groups:( 1 ) Sham( Sham operation and vehicle administration) ; ( 2 ) Ovx( ovariectomy and vebicle administration ) ; ( 3 ) Sham + STZ ( Sham operation and STZ administration ) ; and ( 4 ) Ovx + STZ ( ovariectomy and STZ administration).The diabetic mice were induced by intraperitoneal injection of streptozotocin (STZ,50 mg/kg ).Blood glucose levels were measured once a week.Results The blood glucose level and malondialdehyde of Ovx group were higher than that in Sham group,while total anti-oxidant capacity ( T-AOC ) was lower than those in Sham group.the blood glucose level and MDA of Ovx+ STZ group were higher than those in Sham +STZ group,while T-AOC and serum insulin level were lower than those in Sham + STZ group.Conclusions Endogenous estrogen may have some protective effects on pancreatic islet function from streptozotocin-induced diabetes mellitus in female mice. Key words: Estrogen; Diabetes mellitus, experimental; Antioxidant capacity; Insulin

Mechanism Underlying Defective Interferon Gamma-Induced IDO Expression in Non-obese Diabetic Mouse Fibroblasts

Indoleamine 2,3-dioxygenase (IDO) can locally suppress T cell-mediated immune responses. It has been shown that defective self-tolerance in early prediabetic female non-obese diabetic (NOD) mice can be attributed to the impaired interferon-gamma (IFN-γ)- induced IDO expression in dendritic cells of these animals. As IFN-γ can induce IDO in both dendritic cells and fibroblasts, we asked the question of whether there exists a similar defect in IFN-γ-induced IDO expression in NOD mice dermal fibroblasts. To this end, we examined the effect of IFN-γ on expression of IDO and its enzymatic activity in NOD dermal fibroblasts. The results showed that fibroblasts from either prediabetic (8 wks of age) female or male, and diabetic female or male (12 and 24 wks of age respectively) NOD mice failed to express IDO in response to IFN-γ treatment. To find underlying mechanisms, we scrutinized the IFN- γ signaling pathway and investigated expression of other IFN-γ-modulated factors including major histocompatibility complex class I (MHC-I) and type I collagen (COL-I). The findings revealed a defect of signal transducer and activator of transcription 1 (STAT1) phosphorylation in NOD cells relative to that of controls. Furthermore, we found an increase in MHC-I and suppression of COL-I expression in fibroblasts from both NOD and control mice following IFN-γ treatment; indicating that the impaired response to IFN-γ in NOD fibroblasts is specific to IDO gene. Finally, we showed that an IFN-γ-independent IDO expression pathway i.e. lipopolysaccharide (LPS)-mediated-c-Jun kinase is operative in NOD mice fibroblast. In conclusion, the findings of this study for the first time indicate that IFN-γ fails to induce IDO expression in NOD dermal fibroblasts; this may partially be due to defective STAT1 phosphorylation in IFN-γ-induced-IDO signaling pathway.

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Sir:FigureWe appreciate Dr. Wagner's interest in our article and her comments regarding sex differences between the TallyHo/JnJ mice. In our recent article, “The TallyHo Polygenic Mouse Model of Diabetes: Implications in Wound Healing,”1 we used only male TallyHo mice. Sex dimorphism for the diabetic phenotype is commonly observed in murine models of type 2 diabetes, and the TallyHo strain is no exception. In 2006, Kim et al. published an extensive phenotypic analysis of the TallyHo strain, demonstrating that male mice developed diabetes whereas female mice did not.2 It has been postulated that the protection of diabetes in female mice is at least partially attributable to estrogen-dependent increases in insulin receptors in the hepatocyte membranes and low hepatic estrogen sulfotransferase activity, which prevent insulin resistance and virilization of liver metabolism, respectively.3,4 Although female TallyHo mice do not express an overt diabetic phenotype, they are more obese, with mild hyperinsulinemia, mild hypertriglyceridemia, and pancreatic islet cell hypertrophy when compared with nondiabetic control mice. As a result, they do represent an obese, nondiabetic murine model. Although there is a paucity of literature specifically defining blood glucose levels required for the diagnosis of diabetes in mice, there is substantial evidence regarding the development of the cellular derangements characteristic of diabetes and the correlative blood glucose levels at which they occur.2 The progression of type 2 diabetes is characterized by impaired glucose tolerance, insulin resistance, and finally failure of insulin secretion. Simplistically, these events could be characterized by hyperglycemia, hyperinsulinemia, and pancreatic islet cell hypertrophy and/or atrophy. The male TallyHo mouse begins this progression at 4 weeks of age and, by the eighth week of life, exhibits significantly impaired glucose tolerance, insulin resistance, and islet cell hypertrophy, consistent with diabetes. The blood glucose levels at 8 weeks are above the 200-mg/dl range and continue to rise, peaking in the 300- to 400-mg/dl range by 14 weeks of life.1 Donald W. Buck, II, M.D. Thomas A. Mustoe, M.D. Division of Plastic and Reconstructive Surgery, Northwestern University, Feinberg School of Medicine, Chicago, Ill.

The full‐length estrogen receptor ERα66 mediates ERK activation and downregulation of ERα36 and ERβ expression in kidneys of female diabetic mice

The kidney expresses multiple estrogen receptors, including the full‐length ERα66, its ERα36 splice variant, and ERβ. We previously reported that female ERα66KO mice (which lack a functional ERα66, but still express ERα36) were protected from diabetes‐induced glomerular enlargement, an early marker of renal complications. We hypothesized that diabetes triggers ER‐dependent pro‐hypertrophic signaling events, in concert with altered ER protein levels. Kidneys were harvested 2 wks after inducing type 1 diabetes (streptozotocin injection) in female wild‐type (WT) and ERα66KO mice, with insulin provided to temper the degree of hyperglycemia; sham mice received vehicle treatments. Western blot analysis revealed that phosphorylated‐to‐total ERK1/2 was increased in kidneys from diabetic WT mice, averaging 170±34 % of sham WT (P&lt;0.05); however, this parameter was unaltered by diabetes in ERα66KO mice (97±13 % of sham ERα66KO). Renal ERα36 and ERβ protein levels were reduced in diabetic WT mice, averaging 42±7 and 43±5 % of sham WT values, respectively (both P&lt; 0.05). In contrast, diabetes did not significantly decrease renal ERα36 and ERβ levels in ERα66KO mice. In summary, the renal ERα66‐dependent events triggered by diabetes include downregulation of both ERα36 and ERβ, as well as ERK activation. These processes may contribute to the glomerular enlargement that arises early in diabetes.

Live Imaging Reveals the Link Between Decreased Glucose Uptake in Ovarian Cumulus Cells and Impaired Oocyte Quality in Female Diabetic Mice

Maternal diabetes has been demonstrated to adversely affect preimplantation embryo development and pregnancy outcomes. Emerging data suggest that these effects are associated with compromised oocyte quality. However, direct evidence of a pathway by which maternal diabetes exerts its effects on the oocyte is still lacking. Cumulus cells are metabolically coupled to oocytes, and bidirectional communication between them is essential for the development and functions of both compartments. The primary focus of this work was to evaluate the connection between glucose uptake in cumulus cells and oocyte quality in diabetic mice. This experiment has been difficult, because cumulus cells need to be separated from oocytes and labeled with isotope in the process of measuring glucose uptake. Here, we report a method for live imaging glucose transport in single cumulus-oocyte complexes using a fluorescent glucose analog (6-(N-(7-nitrobenz-2-oxa-1,3-diazol- 4-yl)amino)-6-deoxyglucose). By tracking the ATP content and spindle/chromosome status in individual oocytes surrounded by cumulus cells with differing glucose uptake activity, we reveal that compromised oocyte quality in diabetic mice is linked to decreased glucose uptake in cumulus cells.

Rapamycin selectively alters serum chemistry in diabetic mice

The study was undertaken to explore the effect of rapamycin, an anti-inflammatory agent, on the metabolic profile of type 2 diabetic mice. Seven-month-old diabetic db/db mice and their lean littermate non-diabetic controls (db/m) were randomized to receive control chow or chow mixed with rapamycin (2.24 mg/kg/day) (each group n =20, males and females) for 4 months and sacrificed. Serum samples were analyzed for the measurement of glucose, creatinine, blood urea nitrogen (BUN), alkaline phosphatase (ALP), alanine aminotransferase (ALT), total cholesterol, total triglyceride, and total protein, using the automated dry chemistry analysis. Rapamycin elevated serum glucose in female diabetic mice. Serum creatinine tended to be higher in diabetic mice but was not affected by rapamycin; there was no difference in BUN levels among the groups. Serum ALP was elevated in diabetic mice and rapamycin lowered it only in female diabetic mice; serum ALT levels were increased in female diabetic mice, unaffected by rapamycin. Serum total protein was elevated in diabetic mice of both genders but was not affected by rapamycin. Diabetic mice from both genders had elevated serum cholesterol and triglycerides; rapamycin did not affect serum cholesterol but decreased serum total triglycerides in male diabetic mice. We conclude that rapamycin elicits complex metabolic responses in aging diabetic mice, worsening hyperglycemia in females but improving ALP in female diabetic and total triglycerides in male diabetic mice, respectively. The metabolic effects of rapamycin should be considered while performing studies with rapamycin in mice.