Preventive effect of Ninjin-to (Ren-Shen-Tang), a Kampo (Japanese traditional) formulation, on spontaneous autoimmune diabetes in non-obese diabetic (NOD) mice

We previously found that ingestion of an extract of Ninjin-to (NJT; Ren-Shen-Tang) suppressed the development of autoimmune diabetes in C57BL/KsJ mice induced by multiple low doses of streptozotocin. To verify this effects on spontaneous autoimmune diabetes, the effects of NJT on NOD mice were investigated in the present study. NJT, provided in drinking water (0.25%, 450 mg/kg/day) from 6 weeks of age, significantly prevented the incidence of spontaneous diabetes in female NOD mice at 30 weeks of age (2/10) compared with that of the controls (7/10), with no effects on body growth or food intake. Even in non-diabetic mice, the blood glucose levels of the NOD controls gradually increased with age, while such increase in NJT-treated mice was significantly suppressed by preventing any deficiency of glucose tolerance. NJT also significantly suppressed the progression of insulitis, which causes insulin deficiency and diabetes. It is well known that NOD mice develop insulitis and diabetes because of their Th1-dominant autoimmune response. IFN-gamma production from splenic T lymphocytes stimulated with anti-CD3 monoclonal antibodies was increased, whereas IL-4 production was decreased in NOD controls compared to age- and sex-matched normal ICR mice. NJT-treatment reduced these deviations of cytokine production in NOD mice. These data all suggest that NJT can prevent spontaneous insulitis and diabetes by the modification of deviated cytokine production in NOD mice.     

Lack of effect of vitamin D administration during pregnancy and early life on diabetes incidence in the non-obese diabetic mouse.

BACKGROUND AND AIMS: Several studies have suggested that vitamin D supplementation in early life may reduce the risk of developing type 1 diabetes in later life. The non-obese diabetic (NOD) mouse is a model of spontaneous type 1 diabetes currently used for testing hypothesis/compounds aimed at disease prevention. In this study, we tested the effect of vitamin D (16 IU by gavage) on diabetes incidence in NOD/Ba mice treated from conception with olive oil containing vitamin D via maternal dosing up to 10 weeks of age and followed up until 32 weeks of age. METHODS: Twelve breeding pairs were administered olive oil containing vitamin D during pregnancy, 15 days following the birth of the pups and for the next 10 weeks subsequently. The same breeding pairs were bred again after a clearance period of 15 days using a control solution to produce a control litter. This control group received a control solution for the same period of time. Diabetes incidence, degree of insulitis, and insulin content in the pancreas were investigated in the two groups. RESULTS: 12 vitamin D-treated NOD mice developed diabetes compared to 15 animals in the control group (Log rank test p = 0.899, NS). There were no significant differences between the groups in diabetes incidence, time of onset of the disease, degree of insulitis, or the insulin content in the pancreas. CONCLUSION: Vitamin D administered in utero and in the early stages of life at the dosage used does not change the incidence of diabetes or modify the disease process that leads to beta cell destruction in the NOD mouse.     

Activated protein C inhibits pancreatic islet inflammation, stimulates T regulatory cells, and prevents diabetes in non-obese diabetic (NOD) mice

Activated protein C (aPC) is a natural anticoagulant with strong cyto-protective and anti-inflammatory properties. aPC inhibits pancreatic inflammation and preserves functional islets after intraportal transplantation in mice. Whether aPC prevents the onset or development of type 1 diabetes (T1D) is unknown. In this study, when human recombinant aPC was delivered intraperitoneally, twice weekly for 10 weeks (from week 6 to 15) to non-obese diabetic (NOD) mice, a model for T1D, the incidence of diabetes was reduced from 70% (saline control) to 7.6% by 26 weeks of age. Islets of aPC-treated mice exhibited markedly increased expression of insulin, aPC/protein C, endothelial protein C receptor, and matrix metalloproteinase (MMP)-2 when examined by immunostaining. The insulitis score in aPC-treated mice was 50% less than that in control mice. T regulatory cells (Tregs) in the spleen, pancreatic islets, and pancreatic lymph nodes were increased 37, 53, and 59%, respectively, in NOD mice following aPC treatment. These Tregs had potent suppressor function and, after adoptive transfer, delayed diabetes onset in NOD.severe combined immunodeficiency mice. The culture of NOD mouse spleen cells with aPC reduced the secretion of inflammatory cytokines interleukin (IL)-1β and interferon-γ but increased IL-2 and transforming growth factor-β1, two cytokines required for Treg differentiation. In summary, our results indicate that aPC prevents T1D in the NOD mouse. The aPC mechanism of action is complex, involving induction of Treg differentiation, inhibition of inflammation, and possibly direct cyto-protective effects on β cells.     

Change of incidence of antinuclear antibodies in serum of NOD mouse with aging

Extractable nuclear antigens (ENA) were prepared from liver of C57BL/6J mouse and analyzed by SDS PAGE Western-immunoblotting techniques. Some protein components of the ENA, with molecular weights of 94 K, 65 K, 32 K, and 26 K, reacted with antinuclear antibodies in the sera of NOD mice. Incidence of antinuclear antibodies in the sera of NOD mice with aging were measured by ELISA method using the ENA as antigen. The antinuclear antibodies were not detected in young NOD mice (10 weeks old). However, the incidence increased with aging and reached 100% in the female NOD mice of 40 weeks. In the male NOD mice, the incidence of antinuclear antibodies was delayed and low in comparison with that in female.     

Developing a new model for non-insulin dependent diabetes mellitus (NIDDM) by using the Philippine wild mouse, Mus musculus castaneus.

The Philippine wild-caught castaneus mouse (Mus musculus castaneus) and laboratory mouse (C57BL/6J: B6) were used to develop a new non-insulin dependent diabetes mellitus (NIDDM) model. Offspring from the cross between a wild male and B6 female were backcrossed to the sire. One male which exhibited highest fasting hyperglycemia (190 mg/dl) among eighty-seven backcross offspring was selected at 10 weeks of age, and crossed with a B6 female to comprise the fundamental stock (F0). Thereafter, full-sib mating was performed to develop a new inbred strain named CBD (Castaneus-B6 diabetic) mouse. Mice with relatively higher fasting hyperglycemia among F0 and F1 generations were selected for breeding. From the F2 generation, mice were defined as diabetic when blood glucose levels exceeded 200 mg/dl at 120 min in intraperitoneal glucose tolerance test (IPGTT) at 10 weeks of age, and have been selectively bred. The incidence of diabetic males from the F3-F6 generation fluctuated 45-75% at 10 weeks of age and 59-72% at 20 weeks of age. Diabetic males had about two-fold higher fasting glucose and insulin levels than B6 males. Glucose-stimulated insulin secretion was impaired in diabetic CBD mice compared to B6 males at 20 weeks. Moreover, diabetic mice had slight obesity compared to B6 mice. These facts indicated that diabetic features of CBD mice resemble NIDDM in humans. The CBD strain, characterized by high incidence and early onset of diabetes with mild obesity would be of value as a new NIDDM model. The method, utilizing wild castaneus mouse of different origin from laboratory mice, maybe useful in the development of other animal models.     

B lymphocyte depletion by CD20 monoclonal antibody prevents diabetes in nonobese diabetic mice despite isotype-specific differences in Fc gamma R effector functions

NOD mice deficient for B lymphocytes from birth fail to develop autoimmune or type 1 diabetes. To assess whether B cell depletion influences type 1 diabetes in mice with an intact immune system, NOD female mice representing early and late preclinical stages of disease were treated with mouse anti-mouse CD20 mAbs. Short-term CD20 mAb treatment in 5-wk-old NOD female mice reduced B cell numbers by approximately 95%, decreased subsequent insulitis, and prevented diabetes in >60% of littermates. In addition, CD20 mAb treatment of 15-wk-old NOD female mice significantly delayed, but did not prevent, diabetes onset. Protection from diabetes did not result from altered T cell numbers or subset distributions, or regulatory/suppressor T cell generation. Rather, impaired CD4+ and CD8+ T cell activation in the lymph nodes of B cell-depleted NOD mice may delay diabetes onset. B cell depletion was achieved despite reduced sensitivity of NOD mice to CD20 mAbs compared with C57BL/6 mice. Decreased B cell depletion resulted from deficient FcgammaRI binding of IgG2a/c CD20 mAbs and 60% reduced spleen monocyte numbers, which in combination reduced Ab-dependent cellular cytotoxicity. With high-dose CD20 mAb treatment (250 microg) in NOD mice, FcgammaRIII and FcgammaRIV compensated for inadequate FcgammaRI function and mediated B cell depletion. Thereby, NOD mice provide a model for human FcgammaR polymorphisms that reduce therapeutic mAb efficacy in vivo. Moreover, this study defines a new, clinically relevant approach whereby B cell depletion early in the course of disease development may prevent diabetes or delay progression of disease.     

Diabetes acceleration or prevention by a coxsackievirus B4 infection: critical requirements for both interleukin-4 and gamma interferon

Type 1 diabetes acceleration in nonobese diabetic (NOD) mice through coxsackievirus B4 (CVB4) infection requires a preexisting critical mass of autoreactive T cells in pancreatic islets, and in the absence of this insulitic threshold, CVB4 infection leads to long-term disease protection. To understand this acceleration and protection process, we challenged 8- and 12-week-old NOD mice containing a disruption in interleukin-4 (IL-4) or gamma interferon (IFN-gamma) genes (NOD IL-4-/- and NOD IFN-gamma-/-, respectively) with a diabetogenic, pancreatropic Edwards strain of CVB4. The elimination of IL-4 did not alter the rate of insulitis or diabetes development in NOD mice, while the elimination of IFN-gamma delayed these events several weeks. CVB4 infection in 8-week-old mice only significantly accelerated the onset of diabetes in a subset of standard, but not IL-4- or IFN-gamma-deficient, NOD mice. Long-term diabetes protection was established in standard NOD mice as well as in the NOD IFN-gamma-/- mice that did not rapidly develop disease following CVB4 infection at 8 weeks of age. When mice were infected at 12 weeks of age, the onset of diabetes was accelerated in NOD IL-4-/- mice, while neither acceleration nor long-term protection was elicited in NOD IFN-gamma-/- mice. No differences were observed in the kinetics of CVB4 clearance in pancreases from NOD, NOD IL-4-/-, and NOD IFN-gamma-/- mice. Collectively, these results suggest that at the insulitis threshold at which CVB4 infection can first accelerate the onset of diabetes in NOD mice, IL-4 as well as IFN-gamma contributes to this pathogenic process. The protective mechanism against diabetes elicited in NOD mice infected with CVB4 prior to the development of a critical threshold level of insulitis requires neither IL-4 nor IFN-gamma.     

Localization of Idd11 using NOD congenic mouse strains: elimination of Slc9a1 as a candidate gene

 Type 1 diabetes is a multigenic autoimmune disease, the genetic basis for which is perhaps best characterized in the nonobese diabetic (NOD) mouse model. We previously located a NOD diabetes susceptibility locus, designated Idd11, on mouse Chromosome (Chr) 4 by analyzing diabetic backcross mice produced after crossing NOD/Lt with the nondiabetic resistant strain C57BL/6 (B6) strain. In order to confirm Idd11 and further refine its location, three NOD congenic mouse strains with different B6 derived intervals within Chr 4 were generated. Two of the congenic strains had a significant decrease in the cumulative incidence of diabetes compared with NOD/Lt control mice. The third NOD congenic strain, containing a B6 interval surrounding the Slc9a1 locus, was not protected against diabetes. These results define a new distal boundary for Idd11 and eliminate the Slc9a1 gene as a candidate. The Idd11 locus has now been definitively mapped to a 13cM interval on mouse Chr 4. Received: 15 May 1999 / Revised: 25 September 1999     

Determination of gastric emptying in nonobese diabetic mice

Animal studies on diabetic gastroparesis are limited by inability to follow gastric emptying changes in the same mouse. The study aim was to validate a nonlethal gastric emptying method in nonobese diabetic (NOD) LtJ mice, a model of type 1 diabetes, and study sequential changes with age and early diabetic status. The reliability and responsiveness of a [(13)C]octanoic acid breath test in NOD LtJ mice was tested, and the test was used to measure solid gastric emptying in NOD LtJ mice and nonobese diabetes resistant (NOR) LtJ mice. The (13)C breath test produced results similar to postmortem recovery of a meal. Bethanechol accelerated gastric emptying [control: 92 +/- 9 min; bethanechol: 53 +/- 3 min, mean half emptying time (T(1/2)) +/- SE], and atropine slowed gastric emptying (control: 92 +/- 9 min; atropine: 184 +/- 31 min, mean T(1/2) +/- SE). Normal gastric emptying (T(1/2)) in nondiabetic NOD LtJ mice (8-12 wk) was 91 +/- 2 min. Aging had differing effects on gastric emptying in NOD LtJ and NOR LtJ mice. Onset of diabetes was accompanied by accelerated gastric emptying during weeks 1-2 of diabetes. Gastric emptying returned to normal by weeks 3-5 with no delay. The [(13)C]octanoic acid breath test accurately measures gastric emptying in NOD LtJ mice, is useful to study the time course of changes in gastric emptying in diabetic NOD LtJ mice, and is able to detect acceleration in gastric emptying early in diabetes. Opposing changes in gastric emptying between NOD LtJ and NOR LtJ mice suggest that NOR LtJ mice are not good controls for the study of gastric emptying in NOD LtJ mice.     

链脲佐菌素诱导C57小鼠1型糖尿病模型的研究

目的:通过小剂量多次腹腔注射链脲佐菌素(STZ)诱导建立与人类1型糖尿病相似的C57小鼠糖尿病模型,研究建模剂量和成模率。方法:将32只C57小鼠随机分为正常对照组(A)和实验组(B)。实验组(B)可分为低、中、高剂量组(50 mg/kg、70mg/kg、90 mg/kg)(n=8)。两组都喂普通饲料1周后,B组连续5天腹腔注射不同剂量STZ,测定注射前、注射后1周、2周、3周、4周、5周的空腹血糖和体重,观察小鼠饮食、饮水和排尿情况。STZ注射第3周进行口服糖耐量实验(OGTT)。结果:给药前A、B组体重和血糖无显著差异,给药1周后,B组饮水量和进食量明显增加,体重减轻。C57小鼠用药2周后,中剂量组达到建模标准,成模率75%。各剂量组均出现了糖耐量异常。结论:诱导建立C57小鼠1型糖尿病模型方法是连续5日腹腔注注射STZ,适宜剂量为70 mg/kg。     

Granzyme B is dispensable in the development of diabetes in non-obese diabetic mice

Pancreatic beta cell destruction in type 1 diabetes is mediated by cytotoxic CD8(+) T lymphoctyes (CTL). Granzyme B is an effector molecule used by CTL to kill target cells. We previously showed that granzyme B-deficient allogeneic CTL inefficiently killed pancreatic islets in vitro. We generated granzyme B-deficient non-obese diabetic (NOD) mice to test whether granzyme B is an important effector molecule in spontaneous type 1 diabetes. Granzyme B-deficient islet antigen-specific CD8(+) T cells had impaired homing into islets of young mice. Insulitis was reduced in granzyme B-deficient mice at 70 days of age (insulitis score 0.043±0.019 in granzyme B-deficient versus 0.139±0.034 in wild-type NOD mice p<0.05), but was similar to wild-type at 100 and 150 days of age. We observed a reduced frequency of CD3(+)CD8(+) T cells in the islets and peripheral lymphoid tissues of granzyme B-deficient mice (p<0.005 and p<0.0001 respectively), but there was no difference in cell proportions in the thymus. Antigen-specific CTL developed normally in granzyme B-deficient mice, and were able to kill NOD islet target cells as efficiently as wild-type CTL in vitro. The incidence of spontaneous diabetes in granzyme B-deficient mice was the same as wild-type NOD mice. We observed a delayed onset of diabetes in granzyme B-deficient CD8-dependent NOD8.3 mice (median onset 102.5 days in granzyme B-deficient versus 57.50 days in wild-type NOD8.3 mice), which may be due to the delayed onset of insulitis or inefficient priming at an earlier age in this accelerated model of diabetes. Our data indicate that granzyme B is dispensable for beta cell destruction in type 1 diabetes, but is required for efficient early activation of CTL.     

Suppression of overt diabetes in NOD mice by anti-thymocyte serum or anti-Thy 1, 2 antibody

Effects of anti-thymocyte serum (ATS) and anti-Thy 1, 2 monoclonal antibody on the spontaneously occurring diabetes in NOD mice were examined. Spontaneous diabetes in female mice was markedly suppressed by intravenous injection of rabbit anti-mouse thymocyte serum diluted to 1:4 on three consecutive days during the time period from 70 to 100 days after birth; the cumulative incidence of overt diabetes upto 195 days of age was greatly reduced and the onset of diabetes was delayed. Similar effect was observed with anti-Thy 1, 2 antibody treatment. These findings suggest that T lymphocytes play a role in the production of spontaneous diabetes in this mouse strain.     

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.     

The enlarged population of marginal zone/CD1d(high) B lymphocytes in nonobese diabetic mice maps to diabetes susceptibility region Idd11

The NOD mouse is an important experimental model for human type 1 diabetes. T cells are central to NOD pathogenesis, and their function in the autoimmune process of diabetes has been well studied. In contrast, although recognized as important players in disease induction, the role of B cells is not clearly understood. In this study we characterize different subpopulations of B cells and demonstrate that marginal zone (MZ) B cells are expanded 2- to 3-fold in NOD mice compared with nondiabetic C57BL/6 (B6) mice. The NOD MZ B cells displayed a normal surface marker profile and localized to the MZ region in the NOD spleen. Moreover, the MZ B cell population developed early during the ontogeny of NOD mice. By 3 wk of age, around the time when autoreactive T cells are first activated, a significant MZ B cell population of adult phenotype was found in NOD, but not B6, mice. Using an F2(B6 x NOD) cross in a genome-wide scan, we map the control of this trait to a region on chromosome 4 (logarithm of odds score, 4.4) which includes the Idd11 and Idd9 diabetes susceptibility loci, supporting the hypothesis that this B cell trait is related to the development of diabetes in the NOD mouse.     

Rotavirus infection accelerates type 1 diabetes in mice with established insulitis

Infection modulates type 1 diabetes, a common autoimmune disease characterized by the destruction of insulin-producing islet beta cells in the pancreas. Childhood rotavirus infections have been associated with exacerbations in islet autoimmunity. Nonobese diabetic (NOD) mice develop lymphocytic islet infiltration (insulitis) and then clinical diabetes, whereas NOD8.3 TCR mice, transgenic for a T-cell receptor (TCR) specific for an important islet autoantigen, show more rapid diabetes onset. Oral infection of infant NOD mice with the monkey rotavirus strain RRV delays diabetes development. Here, the effect of RRV infection on diabetes development once insulitis is established was determined. NOD and NOD8.3 TCR mice were inoculated with RRV aged > or = 12 and 5 weeks, respectively. Diabetes onset was significantly accelerated in both models (P < 0.024), although RRV infection was asymptomatic and confined to the intestine. The degree of diabetes acceleration was related to the serum antibody titer to RRV. RRV-infected NOD mice showed a possible trend toward increased insulitis development. Infected males showed increased CD8(+) T-cell proportions in islets. Levels of beta-cell major histocompatibility complex class I expression and islet tumor necrosis factor alpha mRNA were elevated in at least one model. NOD mouse exposure to mouse rotavirus in a natural experiment also accelerated diabetes. Thus, rotavirus infection after beta-cell autoimmunity is established affects insulitis and exacerbates diabetes. A possible mechanism involves increased exposure of beta cells to immune recognition and activation of autoreactive T cells by proinflammatory cytokines. The timing of infection relative to mouse age and degree of insulitis determines whether diabetes onset is delayed, unaltered, or accelerated.     

Mechanism of action of a streptococcal preparation (OK-432) in prevention of autoimmune diabetes in NOD mice. Suppression of generation of effector cells for pancreatic B cell destruction

We have previously reported that treatment with a streptococcal preparation (OK-432), one of the biologic response modifiers, inhibits insulitis and development of autoimmune diabetes in nonobese diabetic (NOD) mice and Bio-Breeding rats used as animal models of insulin-dependent diabetes mellitus (IDDM). We studied the mechanism by which OK-432 inhibited development of IDDM in NOD mice. In female NOD mice diabetes spontaneously developed from 10 to 15 wk of age and the cumulative incidence of diabetes amounted to 74.7% at 30 wk of age. NOD mice, however, never developed diabetes over the observation period of up to 45 wk of age when they were i.p. injected with 0.1 mg of OK-432 weekly from 4 to 15 wk of age. OK-432 treatment in younger age had a stronger inhibitory effect on the development of diabetes. Diabetes was transferred to young, irradiated mice by spleen cells of nontreated, adult mice, but barely transferred by those of OK-432-treated mice. Furthermore, these spleen cells of OK-432-treated mice did not suppress transfer of diabetes by diabetic mice spleen cells. Treatment with cyclophosphamide promoted development of diabetes in nontreated NOD mice due to removal of suppressor cells. However, cyclophosphamide did not show the promotive effect in OK-432-treated mice. Taken together, these results indicate that OK-432 treatment prevented development of diabetes mainly by suppression in generation of the effector cells for pancreatic B cell destruction. The same OK-432 treatment did not suppress the immune response to exogenous AG such as xenogeneic SRBC and allogeneic tumor cells. The study suggests that BRM in the natural environment such as streptococci may suppress induction and progression of autoimmunity and be useful for the immunotherapy of human IDDM.     

IL-10-deficiency unmasks unique immune system defects and reveals differential regulation of organ-specific autoimmunity in non-obese diabetic mice

Interleukin (IL)-10 is a potent anti-inflammatory cytokine and ablation of IL-10 exacerbates Th1-type autoimmune diseases. Even though type 1 diabetes (T1D) in NOD mice is believed to be Th1-mediated, the incidence and severity of T1D is unaltered in IL-10-deficient NOD mice raised under pathogen-free conditions. We describe for the first time, the outcome of IL-10 deficiency on islet and other organ-specific autoimmunity in NOD mice raised in a conventional facility. IL-10-deficient NOD mice under such conditions were protected from spontaneous as well as cyclophosphamide-induced diabetes, but were susceptible to diabetes induced by adoptive transfer of splenocytes from spontaneously diabetic NOD mice. Whereas the incidence of rectal prolapse was very high in this NOD.IL-10(-/-) mouse colony, IL-10-deficient C57Bl/6 mice raised under similar conditions seldom developed rectal prolapse. While injection of complete Freund's adjuvant (CFA) significantly reduced insulitis, it did not ameliorate colitis in IL-10-deficient NOD mice indicating differential regulation of organ-specific autoimmunity by CFA. Phenotypic characterization of IL-10(-/-) mice revealed a significant increase in splenic macrophage numbers in NOD but not on the B6 background. This was accompanied by a heightened systemic inflammatory cytokine response and mortality following in vivo challenge with a toll-like receptor 9 agonist, CpG-containing DNA.     

Acceleration of autoimmune diabetes in Rheb-congenic NOD mice with β-cell-specific mTORC1 activation

The protein Ras homolog enriched in brain (Rheb) is a Ras-like small GTPase that activates the mechanistic target of rapamycin complex 1 (mTORC1), which promotes cell growth. We previously generated transgenic C57BL/6 mice overexpressing Rheb in β-cells (B6^Rheb), which exhibited increased β-cell size and improved glucose tolerance with higher insulin secretion than wild type C57BL/6 mice. The mice also showed resistance to obesity-induced hyperglycemia, a model of type 2 diabetes, and to multiple low-dose-streptozotocin (MLDS)-induced hyperglycemia, a model of type 1 diabetes (T1D). To investigate whether the effects of mTORC1 activation by Rheb in B6^Rheb mice would also be evident in NOD mice, a spontaneous autoimmune T1D model, we created two NOD mouse lines overexpressing Rheb in their β-cells (NOD^Rheb; R3 and R20). We verified Rheb overexpression in β-cells, the relative activation of mTORC1 and β-cell enlargement. By 35 weeks of age, diabetes incidence was significantly greater in the R3 line and tended to be greater in the R20 line than in NOD mice. Histological analysis demonstrated that insulitis was significantly accelerated in 12-week-old R3 NOD^Rheb mice compared with NOD mice. Furthermore, serum insulin autoantibody (IAA) expression was significantly higher than that of NOD mice. We also examined whether complete Freund’s adjuvant (CFA) treatment alone or with glucagon-like peptide-1 (GLP-1) analog would reverse the hyperglycemia of NOD^Rheb mice; unexpectedly, almost none achieved normoglycemia. In summary, diabetes progression was significantly accelerated rather than prevented in NOD^Rheb mice. Our results suggest that the β-cell enlargement might merely enhance the autoimmunity of pathogenic T-cells against islets, leading to acceleration of autoimmune diabetes. We conclude that not only enlargement but also regeneration of β-cells in addition to the prevention of β-cell destruction will be required for the ideal therapy of autoimmune T1D.