Unexpected functional consequences of xenogeneic transgene expression in β-cells of NOD mice

We describe unexpected alterations in the non-obese diabetic (NOD/Lt) mouse model of type 1 diabetes (T1D) following forced β-cell expression of non-mammalian genes ligated to an insulin promoter sequence. These include the jellyfish green fluorescent protein (GFP), useful for β-cell identification, and the bacteriophage P1 Cre recombinase, necessary for β cell–specific ablation of a gene using a Cre- loxP system. Homozygous expression of GFP, driven by the mouse insulin 1 gene promoter (MIP-GFP) in a single transgenic line of NOD mice, produced T1D in postnatal mice that was not associated with insulitis, but rather β cell–depleted islets. Hemizygous transgene expression suppressed spontaneous autoimmune T1D in females, and produced a male glucose intolerance syndrome associated with age-dependent declines in plasma insulin content. Among lines of transgenic NOD/Lt mice expressing Cre recombinase driven by the rat insulin 2 promoter (RIP-Cre), high, non-mosaic expression correlated with suppressed T1D development. These findings emphasize the need for careful characterization of genetically manipulated NOD mouse stocks to insure that model characteristics have not been compromised.     

Pancreatic deletion of insulin receptor substrate 2 reduces beta and alpha cell mass and impairs glucose homeostasis in mice

Aims/hypothesis Insulin signalling pathways regulate pancreatic beta cell function. Conditional gene targeting using the Cre/loxP system has demonstrated that mice lacking insulin receptor substrate 2 (IRS2) in the beta cell have reduced beta cell mass. However, these studies have been complicated by hypothalamic deletion when the RIPCre (B6.Cg-tg(Ins2-cre)25Mgn/J) transgenic mouse (expressing Cre recombinase under the control of the rat insulin II promoter) is used to delete floxed alleles in insulin-expressing cells. These features have led to marked insulin resistance making the beta cell-autonomous role of IRS2 difficult to determine. To establish the effect of deleting Irs2 only in the pancreas, we generated PIrs2KO mice in which Cre recombinase expression was driven by the promoter of the pancreatic and duodenal homeobox factor 1 (Pdx1, also known as Ipf1) gene. Materials and methods In vivo glucose homeostasis was examined in PIrs2KO mice using glucose tolerance and glucose-stimulated insulin secretion tests. Endocrine cell mass was determined by morphometric analysis. Islet function was examined in static cultures and by performing calcium imaging in Fluo3am-loaded beta cells. Islet gene expression was determined by RT-PCR. Results The PIrs2KO mice displayed glucose intolerance and impaired glucose-stimulated insulin secretion in vivo. Pancreatic insulin and glucagon content and beta and alpha cell mass were reduced. Glucose-stimulated insulin secretion and calcium mobilisation were attenuated in PIrs2KO islets. Expression of the Glut2 gene (also known as Slc2a2) was also reduced in PIrs2KO mice. Conclusions/interpretation These studies suggest that IRS2-dependent signalling in pancreatic islets is required not only for the maintenance of normal beta and alpha cell mass but is also involved in the regulation of insulin secretion. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Induction and therapy of autoimmune diabetes in the non-obese diabetic (NOD/Lt) mouse by a 65-kDa heat shock protein.

Insulin-dependent diabetes mellitus is caused by autoimmune destruction of the insulin-producing beta cells of the pancreas. The results described here indicate that a beta-cell target antigen in non-obese diabetic (NOD/Lt) mice is a molecule cross-reactive with the 65-kDa heat shock protein (hsp65) of Mycobacterium tuberculosis. The onset of beta-cell destruction is associated with the spontaneous development of anti-hsp65 T lymphocytes. Subsequently hsp65 cross-reactive antigen becomes detectable in the sera of the prediabetic mice and some weeks later anti-hsp65 antibodies, anti-insulin antibodies, and anti-idiotypic antibodies to insulin antibodies become detectable. The hsp65-cross-reactive antigen, the autoantibodies, and the T-cell reactivity then decline with the development of overt insulin-dependent diabetes. The importance of hsp65 in the pathogenesis of insulin-dependent diabetes was confirmed by the ability of clones of anti-hsp65 T cells to cause insulitis and hyperglycemia in young NOD/Lt mice. Moreover, hsp65 antigen could be used either to induce diabetes or to vaccinate against diabetes, depending on the form of its administration to prediabetic NOD/Lt mice. Other antigens such as the 70-kDa heat shock protein (hsp70) had no effect on the development of diabetes.     

Targeted oncogene activation by site-specific recombination in transgenic mice.

An efficient and accurate method for controlled in vivo transgene modulation by site-directed recombination is described. Seven transgenic mouse founder lines were produced carrying the murine lens-specific alpha A-crystallin promoter and the simian virus 40 large tumor-antigen gene sequence, separated by a 1.3-kilobase-pair Stop sequence that contains elements preventing expression of the large tumor-antigen gene and Cre recombinase recognition sites. Progeny from two of these lines were mated with transgenic mice expressing the Cre recombinase under control of either the murine alpha A-crystallin promoter or the human cytomegalovirus promoter. All double-transgenic offspring developed lens tumors. Subsequent analysis confirmed that tumor formation resulted from large tumor-antigen activation via site-specific, Cre-mediated deletion of Stop sequences.     

Conditional deletion of insulin receptor in thyrocytes does not affect thyroid structure and function

Thyroid-stimulating hormone (TSH) is the primary regulator of thyroid growth and function acting via cyclic AMP signaling cascades. In cultured thyrocytes, insulin and/or insulin-like growth factor-1 (IGF-1) are required for mediating thyrocyte proliferation in concert with TSH. To determine the role of insulin signaling in thyroid, growth in vivo, mice with thyrocyte-selective ablation of the insulin receptor (IR) were generated by crossing mice homozygous for a floxed IR allele with transgenic mice in which thyrocyte-specific expression of Cre recombinase was driven by the human thyroid peroxidase (TPO) gene promoter. Immunohistochemistry and Western blot analysis confirmed near complete loss of IR expression in the thyroid of thyrocyte IR knockout mice. These mice are viable and have no obvious thyroid dysfunction and macro- and microscopic thyroid morphology was normal. Thus, insulin signaling in thyrocytes does not play an essential role in the architecture and function of the thyroid in vivo.     

Rat insulin promoter 2-Cre recombinase mice bred onto a pure C57BL/6J background exhibit unaltered glucose tolerance

Beta-cell-specific gene targeting is a widely used tool when studying genes involved in beta-cell function. For this purpose, several conditional beta-cell knockouts have been generated using the rat insulin promoter 2-Cre recombinase (RIP2-Cre) mouse. However, it was recently observed that expression of Cre alone in beta-cells may affect whole body glucose homeostasis. Therefore, we investigated glucose homeostasis, insulin secretion, and beta-cell mass in our line of RIP2-Cre mice bred onto the C57BL/6J genetic background. We used 12- and 28-week-old female RIP2-Cre mice for analyses of insulin secretion in vitro, glucose homeostasis in vivo and beta-cell mass. Our mouse line has been backcrossed for 14 generations to yield a near 100% pure C57BL/6J background. We found that fasting plasma glucose and insulin levels were similar in both genotypes. An i.v. glucose tolerance test revealed no differences in glucose clearance and insulin secretion between 12-week-old RIP2-Cre and WT mice. Moreover, insulin secretion in vitro in islets isolated from 28-week-old RIP2-Cre mice and controls was similar. In addition, beta-cell mass was not different between the two genotypes at 28 weeks of age. In our experiments, we observed no differences in glucose tolerance, insulin secretion in vivo and in vitro, or in beta-cell mass between the genotypes. As our RIP2-Cre mice are on a near 100% pure genetic background (C57BL/6J), we suggest that the perturbations in glucose homeostasis previously reported in RIP2-Cre mouse lines can be accounted for by differences in genetic background.     

Unusual resistance of ALR/Lt mouse beta cells to autoimmune destruction: role for beta cell-expressed resistance determinants

Genetic analysis of autoimmune insulin-dependent diabetes mellitus (IDDM) has focused on genes controlling immune functions, with little investigation of innate susceptibility determinants expressed at the level of target beta cells. The Alloxan (AL) Resistant (R) Leiter (Lt) mouse strain, closely related to the IDDM-prone nonobese diabetic (NOD)/Lt strain, demonstrates the importance of such determinants. ALR mice are unusual in their high constitutive expression of molecules associated with dissipation of free-radical stress systemically and at the beta-cell level. ALR islets were found to be remarkably resistant to two different combinations of beta-cytotoxic cytokines (IL-1beta, tumor necrosis factor alpha, and IFN-gamma) that destroyed islets from the related NOD and alloxan-susceptible strains. The close MHC relatedness between the NOD and ALR strains (H2-Kd and H2-Ag7 identical) allowed us to examine whether ALR islet cells could survive autoimmune destruction by NOD-derived Kd-restricted diabetogenic cytotoxic T lymphocyte clones (AI4 and the insulin-reactive G9C8 clones). Both clones killed islet cells from all Kd-expressing strains except ALR. ALR resistance to diabetogenic immune systems was determined in vivo by means of adoptive transfer of the G9C8 clone or by chimerizing lethally irradiated ALR or reciprocal (ALR x NOD)F1 recipients with NOD bone marrow. In all in vivo systems, ALR and F1 female recipients of NOD marrow remained IDDM free; in contrast, all of the NOD recipients became diabetic. In conclusion, the ALR mouse presents a unique opportunity to identify dominant IDDM resistance determinants expressed at the beta cell level.     

Illegitimate Cre-dependent chromosome rearrangements in transgenic mouse spermatids

The bacteriophage P1 Cre/loxP system has become a powerful tool for in vivo manipulation of the genomes of transgenic mice. Although in vitro studies have shown that Cre can catalyze recombination between cryptic "pseudo-loxP" sites in mammalian genomes, to date there have been no reports of loxP-site infidelity in transgenic animals. We produced lines of transgenic mice that use the mouse Protamine 1 (Prm1) gene promoter to express Cre recombinase in postmeiotic spermatids. All male founders and all Cre-bearing male descendents of female founders were sterile; females were unaffected. Sperm counts, sperm motility, and sperm morphology were normal, as was the mating behavior of the transgenic males and the production of two-celled embryos after mating. Mice that expressed similar levels of a derivative transgene that carries an inactive Cre exhibited normal male fertility. Analyses of embryos from matings between sterile Cre-expressing males and wild-type females indicated that Cre-catalyzed chromosome rearrangements in the spermatids that lead to abortive pregnancies with 100% penetrance. Similar Cre-mediated, but loxP-independent, genomic alterations may also occur in somatic tissues that express Cre, but, because of the greater difficulty of assessing deleterious effects of somatic mutations, these may go undetected. This study indicates that, following the use of the Cre/loxP site-specific recombination systems in vivo, it is prudent to eliminate or inactivate the Cre recombinase gene as rapidly as possible.     

A novel technique for temporally regulated cell type-specific Cre expression and recombination in the pituitary gonadotroph

Inducing tissue-specific genetic alterations under temporal control allows for the analysis of gene function in particular cell types at specified points in time. We have generated a system for tetracycline-controlled expression of Cre recombinase in mice using the unique CreTeR vector. The gonadotroph-specific bovine alpha-subunit (Balpha) promoter fragment was subcloned into the CreTeR vector, creating a technique for highly regulated expression of Cre recombinase exclusively in pituitary gonadotrophs. Control of Cre recombinase in the CreTeR vector was demonstrated in LbetaT2 pituitary cell lines, where Cre protein was detected in cells treated with doxycycline, but not in untreated cells. In transgenic mice, Cre was expressed in pituitary gonadotrophs of mice treated with doxycycline, but not in non-pituitary tissues or in transgenic mice not treated with doxycycline. We demonstrated Cre expression in the gonadotroph by immunostaining showing co-localization of Cre recombinase with the beta-subunit of LH (LH-beta). Furthermore, by crossing Balpha/CreTeR with R26R mice, we were able to demonstrate functional recombination within pituitary gonadotrophs, detected by lacZ expression. The Balpha/CreTeR mice described here can be used to study the function of virtually any gene in the gonadotroph; in particular, this will be useful in studying genes, which may have distinct roles in development and in the adult.     

Transplantation analysis of B cell destruction in (NOD x CBA)F1 mouse bone marrow chimeras

Summary F1 hybrids produced by outcross of non-obese diabetic (NOD) mice with diabetes resistant strains are also diabetes resistant. This resistance is abrogated if F1 hybrids are lethally irradiated and then haematopoietically reconstituted with NOD bone marrow. This model was employed to determine whether T lymphocyte recognition and elimination of pancreatic B cells in NOD mice is restricted by the MHC haplotype of the target B cell. Diabetes resistant (NOD/Lt x CBA/J)F1 hybrids were lethally irradiated and reconstituted with NOD/Lt bone marrow. Following haematopoietic reconstitution, donor matched NOD/Lt or CBA/J pancreatic islet and anterior pituitary grafts were grafted under a renal capsule to determine whether effector cells derived from NOD/Lt marrow progenitors would reject islet grafts in a MHC restricted fashion. The H-2^k haplotype expressed by CBA/J mice differs from all known loci of the unique H-2 haplotype of NOD; therefore, if NOD/Lt T lymphocytes eliminate pancreatic B cells in a MHC restricted fashion, NOD islet grafts would be eliminated in these chimeras while CBA islet grafts would be retained. Overt diabetes developed in 80% of the female and 40% of the male F1 hybrids following reconstitution with NOD/Lt marrow, while no hybrids reconstituted with CBA/J marrow became diabetic through a year of age. The retention of CBA/J skin and pituitary grafts in NOD/Lt marrow reconstituted F1 hybrids confirmed that the F1 thymic environment imparted tolerance to CBA/J alloantigens. Nonetheless, responses to a T cell dependent model antigen were restricted to the unique MHC haplotype of NOD. This was associated in the hyperglycaemic chimeras with rejection (8–21 days post-implantation) of both CBA/J and NOD/Lt islet grafts. This indicated generation of islet specific effectors that were not restricted by the MHC haplotype of the target B cell. Islet specific effectors were less active in those chimeras remaining normoglycaemic following NOD/Lt marrow reconstitution, since both NOD/Lt and CBA/J islet grafts remained intact 21 days post-implantation. Islet and pituitary grafts of each genotype remained free of leucocytic involvement in CBA/J marrow reconstituted F1 hybrids. The generation of B cell specific, but not genotype specific, effectors elicited from NOD/Lt marrow was age-dependent. Following priming with porcine insulin in vivo, T lymphocytes from 8-weekold NOD male mice failed to respond to porcine insulin in vitro, while primed T lymphocytes isolated from 16-weekold mice proliferated vigorously. This suggested that as pancreatic insulitis progressed in aging NOD/Lt mice, T lymphocytes capable of responding to B cells in a tissue specific, but not genotype specific fashion, were activated. Thus, we propose that immunological effectors derived from NOD/Lt, but not CBA/J marrow, are capable of eliminating islet grafts in non-MHC restricted fashion. However, this population could be activated as a consequence of B cell erosion initated in the pancreas by another class of effectors which may be restricted by the MHC haplotype of the target B cell.     

Efficient in vivo manipulation of mouse genomic sequences at the zygote stage.

We describe a transgenic mouse line carrying the cre transgene under the control of the adenovirus EIIa promoter that targets expression of the Cre recombinase to the early mouse embryo. To assess the ability of this recombinase to excise loxP-flanked DNA sequences at early stages of development, we bred EIIa-cre transgenic mice to two different mouse lines carrying loxP-flanked target sequences: (i) a strain with a single gene-targeted neomycin resistance gene flanked by 1oxP sites and (ii) a transgenic line carrying multiple transgene copies with internal loxP sites. Mating either of these loxP-carrying mouse lines to EIIa-cre mice resulted in first generation progeny in which the loxP-flanked sequences had been efficiently deleted from all tissues tested, including the germ cells. Interbreeding of these first generation progeny resulted in efficient germ-line transmission of the deletion to subsequent generations. These results demonstrate a method by which loxP-flanked DNA sequences can be efficiently deleted in the early mouse embryo. Potential applications of this approach are discussed, including reduction of multicopy transgene loci to produce single-copy transgenic lines and introduction of a variety of subtle mutations into the line.     

Changing patterns of cell surface mono (ADP-ribosyl) transferase antigen ART2.2 on resting versus cytopathically-activated T cells in NOD/Lt mice

Aims/hypothesis. ART2.2 is a mouse T-cell surface ectoenzyme [mono (ADP-ribosyl) transferase] shed upon strong activation. We analysed temporal changes in ART2.2 expression in unmanipulated and cyclophosphamide-treated NOD/Lt mice compared with diabetes-resistant control strains. We used NAD, the ART2.2 substrate, to test whether ART-mediated ADP-ribosylation could retard diabetogenic activation of islet-reactive T cells in vitro. Methods. ART2.2 and CD38, another NAD-utilizing enzyme, were measured by flow cytometry. ADP-ribosylation from ethano-NAD was followed by flow cytometry using a reagent specific for etheno-ADP ribose. Results. Although mature NOD CD4 + and C D8 + T cells expressed ART2.2, this expression was delayed in young NOD mice when compared with control strains. This ontological delay at 3 weeks of age correlated with an early burst of CD25 expression unique to NOD splenic T cells. This pattern was reproduced in cyclophosphamide-accelerated diabetes in young NOD/Lt males, wherein a retarded repopulation of ART2.2 T cells in spleen and islets correlated with development of heavy insulitis and diabetes. NAD inhibited anti-CD3 induced activation of splenic T cells in vitro and also retarded killing of beta-cell targets by NOD islet-reactive CD8 effectors in vitro at concentrations equal to or greater than 1 μmol/l. Evidence suggested that CD38 on B lymphocytes competes with ART2.2 for substrate needed by B lymphocytes for ADP ribosylation. Conclusions. ART2.2 on T cells may not simply mark the resting state, but could also contribute to it via ADP-ribosylation. [Diabetologia (2001) 44: 848–858] Received: 27 November 2000 and in revised form: 1 March 2001     

Endoplasmic reticulum stress caused by overexpression of islet-specific glucose-6-phosphatase catalytic subunit-related protein in pancreatic Beta-cells

The high rate of protein synthesis in beta-cells renders them susceptible to endoplasmic reticulum (ER) stress, a condition that can be aggravated by additional imbalances in ER homeostasis and could potentially contribute to the pathogenesis of type-1 and type-2 diabetes. Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is an ER-resident protein that is specifically expressed in pancreatic beta-cells and is a major target of diabetogenic CD8(+) T cell responses in non-obese diabetic (NOD) mice. We produced transgenic mice expressing human IGRP (hIGRP) under the control of rat insulin promoter (RIP) to study epitopes in hIGRP capable of driving diabetogenic human leukocyte antigen (HLA)-restricted CD8(+) T-cell responses in hIGRP/HLA transgenic NOD mice. Surprisingly, we found that 3 out of 14 lines expressing RIP-hIGRP in a non-T1D-prone genetic background developed a form of early-onset diabetes that was dissociated from autoimmune inflammation of pancreatic islets. We show that diabetes in these 3 lines resulted from increased rates of beta-cell death because of ER stress. We hypothesize that IGRP compounds the viability of beta-cells undergoing ER stress by generating unfolded proteins in the ER lumen, and that IGRP's location in the ER accounts, in part, for its exquisite immunogenicity in T1D-prone genetic backgrounds.     

Skeletal muscle-specific Cre recombinase expression,controlled by the human α-skeletal actin promoter,improves glucose tolerance in mice fed a high-fat diet

Aims/hypothesis

Cre-loxP systems are frequently used in mouse genetics as research tools for studying tissue-specific functions of numerous genes/proteins. However, the expression of Cre recombinase in a tissue-specific manner often produces undesirable changes in mouse biology that can confound data interpretation when using these tools to generate tissue-specific gene knockout mice. Our objective was to characterise the actions of Cre recombinase in skeletal muscle, and we anticipated that skeletal muscle-specific Cre recombinase expression driven by the human α-skeletal actin (HSA) promoter would influence glucose homeostasis.

Methods

Eight-week-old HSA-Cre expressing mice and their wild-type littermates were fed a low- or high-fat diet for 12 weeks. Glucose homeostasis (glucose/insulin tolerance testing) and whole-body energy metabolism (indirect calorimetry) were assessed. We also measured circulating insulin levels and the muscle expression of key regulators of energy metabolism.

Results

Whereas tamoxifen-treated HSA-Cre mice fed a low-fat diet exhibited no alterations in glucose homeostasis, we observed marked improvements in glucose tolerance in tamoxifen-treated, but not corn-oil-treated, HSA-Cre mice fed a high-fat diet vs their wild-type littermates. Moreover, Cre dissociation from heat shock protein 90 and translocation to the nucleus was only seen following tamoxifen treatment. These improvements in glucose tolerance were not due to improvements in insulin sensitivity/signalling or enhanced energy metabolism, but appeared to stem from increases in circulating insulin.

Conclusions/interpretation

The intrinsic glycaemia phenotype in the HSA-Cre mouse necessitates the use of HSA-Cre controls, treated with tamoxifen, when using Cre-loxP models to investigate skeletal muscle-specific gene/protein function and glucose homeostasis.

     

Calcium insufficiency accelerates type 1 diabetes in vitamin D receptor-deficient nonobese diabetic (NOD) mice

Vitamin D exerts important regulatory effects on the endocrine and immune systems. Autoimmune type 1 diabetes (T1D) development in the inbred NOD mouse strain can be accelerated by vitamin D insufficiency or suppressed by chronic treatment with high levels of 1α,25-dihydroxyvitamin D(3). Consequently, a report that T1D development was unaffected in NOD mice genetically lacking the vitamin D receptor (VDR) was unexpected. To further assess this result, the mutant stock was imported to The Jackson Laboratory, backcrossed once to NOD/ShiLtJ, and progeny rederived through embryo transfer. VDR-deficient NOD mice of both sexes showed significant acceleration of T1D. This acceleration was not associated with alterations in immune cells targeting pancreatic β-cells. Rather, the capacity of β-cells to produce and/or secrete insulin was severely impaired by the hypocalcaemia developing in VDR-deficient NOD mice fed a standard rodent chow diet. Feeding a high-lactose calcium rescue diet that circumvents a VDR requirement for calcium absorption from the intestine normalized serum calcium levels, restored β-cell insulin secretion, corrected glucose intolerance, and eliminated accelerated T1D in VDR-deficient NOD mice. These findings suggest that calcium and/or vitamin D supplementation may improve disease outcomes in some T1D-prone individuals that are calcium deficient.