Table of Contents Author Guidelines Submit a Manuscript
International Journal of Endocrinology
Volume 2014, Article ID 435481, 5 pages
http://dx.doi.org/10.1155/2014/435481
Research Article

Prevention and Reversal of Diabetes by All-Trans Retinoid Acid and Exendin-4 in NOD Mice

1Division of Endocrinology and Metabolism, Department of Internal Medicine, Chang Gung University and Chang Gung Memorial Hospital, 5 Fu-Shin Street, Kweishan, Taoyuan 333, Taiwan
2Division of Pediatric Endocrinology, Department of Pediatrics, Chang Gung Memorial Hospital, 5 Fu-Shin Street, Kweishan, Taoyuan 333, Taiwan
3Biomedical Technology and Device Research Laboratories, Industrial Technology Research Institute of Taiwan, 195 Sec. 4, Chung Hsing Road, Chutung, Hsinchu 31040, Taiwan

Received 21 December 2013; Revised 1 May 2014; Accepted 15 May 2014; Published 3 June 2014

Academic Editor: Tien-Jyun Chang

Copyright © 2014 Jyuhn-Huarng Juang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

It has been shown that all-trans retinoid acid (ATRA) hinders the development of autoimmune diabetes by inducing immune tolerance status. Meanwhile, exendin-4 increases beta-cell function and mass. Thus, we hypothesized that ATRA and exendin-4 combination therapy would prevent and reverse autoimmune diabetes. NOD/scid mice were intravenously transferred with splenocytes isolated from 12-week-old female NOD mice. After adoptive transfer, mice were treated with vehicle, ATRA (0.5 mg/mouse intraperitoneally every other day), exendin-4 (3 μg/kg subcutaneously twice daily), or combination for 6 weeks. Compared with vehicle, ATRA () and ATRA plus exendin-4 () treatment delayed the onset of diabetes. The pancreatic insulin content in mice treated with ATRA () and exendin-4 () was significantly higher than that of control mice. All but one spontaneous diabetic NOD mouse treated with ATRA and/or exendin-4 remained persistent hyperglycemic. ATRA and/or exendin-4 treatment did not alter their blood glucose levels and survival. Our results indicate that, before the onset of autoimmune diabetes, ATRA and exendin-4 treatment alone preserves pancreatic beta cells; ATRA and ATRA plus exendin-4 treatment delays the onset of autoimmune diabetes. However, after the onset of autoimmune diabetes, ATRA and/or exendin-4 treatment is unable to reverse hyperglycemia or improve survival.

1. Introduction

Type 1 diabetes is characterized by the progressive loss of pancreatic beta cells caused by autoimmune attack [1]. Although beta-cell mass is markedly diminished in long-standing type 1 diabetics, residual beta cells can be detected and new beta-cell formation may occur in these patients several decades after the disease onset [2]. This observation has led to researches to induce remission of diabetes by targeting beta-cell autoimmunity and regeneration.

All-trans retinoid acid (ATRA) is a potent vitamin A derivative that has been clinically used for the treatment of acute promyelocytic leukemia [3] and skin disease [4]. Recently, Van et al. demonstrated ATRA treatment inhibited diabetes in NOD mice by inducing Treg cell-dependent immune tolerance [5]. Exendin-4 (exenatide) is a glucagon-like peptide-1 (GLP-1) receptor agonist resistant to dipeptidyl peptidase-IV-mediated inactivation [6] which exhibits sustainable GLP-1 effects including stimulation of glucose-dependent insulin secretion and biosynthesis and suppression of glucagon secretion, gastric emptying, and appetite [7]. In addition, exendin-4 stimulates beta-cell growth and differentiation [8], inhibits beta-cell apoptosis [9], and delays the onset of diabetes [10]. Thus, we hypothesized that the combination therapy of ATRA and exendin-4 would inhibit autoimmune destruction and enhance the growth and function of pancreatic beta cells, therefore, prevent and reverse autoimmune diabetes.

2. Materials and Methods

2.1. Animals

NOD/scid (National Laboratory Animal Center and National Taiwan University, Taiwan) and NOD (National Defense Medical Center, Taiwan) mice were bred and housed in a specific pathogen-free environment in the animal facility at the Chang Gung Memorial Hospital. Blood was obtained from the snipped tail, and glucose was measured with a portable glucose meter (One Touch II, Lifescan Inc., Milpitas, CA, USA). Normoglycemia was defined as nonfasting blood glucose levels <200 mg/dL. All animal protocols were approved by the Ethics Committee of Chang Gung Memorial Hospital.

2.2. Adoptive Transfer

Splenocytes were isolated from 12-week-old female NOD mice. Nondepleted splenocytes (/mouse) were intravenously transferred into NOD/scid mice [5].

2.3. ATRA and Exendin-4 Treatment

NOD/scid mice with adoptive transfer and spontaneous diabetic NOD mice were treated with vehicle (as control), ATRA (Sigma, St. Louis, MO, 0.5 mg/mouse intraperitoneally every other day) [5], exendin-4 (Sigma, St. Louis, MO, 3 μg/kg subcutaneously twice daily) [11], or combination for 6 weeks.

2.4. Immunohistochemistry of the Pancreas

The pancreases of NOD/scid mice were removed before death, fixed in formalin solution, and processed for paraffin embedding and sectioning. Sections of the pancreases were stained for the endocrine beta cells with a guinea pig antiswine insulin antibody (Dako Co., Glostrup, Denmark) [11], Pdx1, and Ki67 (Abcam, California, CA). The degree of insulitis was evaluated by the mononuclear cell infiltration around or in the islet.

2.5. Insulin Content of the Pancreas

The pancreases of NOD/scid mice were removed before death and homogenized in acid ethanol. After homogenization, the samples were extracted overnight at 4°C. On the following day, they were centrifuged at 2,400 rpm for 30 min, and the supernatant was stored at −20°C. The pellet was again homogenized in acid ethanol and left 2 h at 4°C. Above procedure was repeated and insulin was extracted overnight. After centrifugation, the supernatant was added to the previous extraction sample and kept in −20°C freezer till assay. Insulin was measured by radioimmunoassay with INSI-PR kit (CIS US Inc., USA) [11].

2.6. Statistical Analysis

Results were expressed as mean and standard deviation. Kaplan-Meier survival analysis was used to compare cumulative diabetes incidence. ANOVA were employed for comparisons among multiple groups. A value of was considered significant.

3. Results

3.1. Prevention Study

After adoptive transfer, the mean diabetes-free time in NOD/scid mice treated with vehicle (), ATRA (), exendin-4 (), and ATRA plus exendin-4 () was 6.5, 8.0, 7.6, and 11.2 weeks, respectively. Compared with vehicle, ATRA () and ATRA plus exendin-4 () treatment delayed the onset of diabetes (Figure 1). Although mice with different treatments had varied degrees of insulitis and a variable number of beta cells per islet (Figure 2), the pancreatic insulin content in mice treated with ATRA ( ng/mL, , ) and exendin-4 ( ng/mL, , ) was significantly higher than that of control mice ( ng/mL, ) (Figure 3). Moreover, ATRA treated group had more Pdx-1- and Ki67-positive beta cells compared with control and Ex-4-treated animals (Figure 4).

435481.fig.001
Figure 1: Kaplan-Meier curve of the percentage of diabetes-free mice over time in vehicle (brown line), ATRA- (blue line), exendin-4- (EX-4, green line), and ATRA plus exendin-4- (orange line) treated groups. Adoptive-transfer was performed and treatment was started at day 0. Compared with vehicle, ATRA () and ATRA plus exendin-4 () treatment delayed the onset of diabetes.
435481.fig.002
Figure 2: Varied degrees of insulitis with abundant (upper panel) to scanty (lower panel) beta cells were observed in the pancreases of adoptive transferred NOD/scid mice treated with vehicle, ATRA, and exendin-4.
435481.fig.003
Figure 3: The pancreatic insulin content in adoptive transferred NOD/scid mice treated with vehicle, ATRA, and exendin-4.
435481.fig.004
Figure 4: Insulin, Pdx-1, and Ki67 staining for islets in the pancreases of adoptive transferred NOD/scid mice treated with vehicle, ATRA, and exendin-4 (Ex-4).
3.2. Reversal Study

All but one spontaneous diabetic NOD mouse remained persistent hyperglycemic. The lowest blood glucose level in control () and mice treated with ATRA (), exendin-4 (), and ATRA plus exendin-4 () was , , , and  mg/dL, respectively (, Figure 5(a)). Meanwhile, ATRA and/or exendin-4 treatment did not alter their survival time [, , , and days in control () and mice treated with ATRA (), exendin-4 (), and ATRA plus exendin-4 (), respectively; , Figure 5(b)]. One mouse achieved normoglycemia 10 days after ATRA treatment and then had occasional hyperglycemia between 88 and 366 days (Figure 6).

fig5
Figure 5: The lowest blood glucose levels (a) and survival time (b) in spontaneous diabetic NOD mice treated with and without ATRA and exendin-4 (Ex-4).
435481.fig.006
Figure 6: One spontaneous diabetic NOD mouse achieved normoglycemia 10 days after ATRA treatment but then had occasional hyperglycemia between 88 and 366 days.

4. Discussion

In the present study, we tested if ATRA and exendin-4 treatment could prevent and reverse autoimmune diabetes. In the prevention experiment, due to limited space in our animal facility, NOD/scid mice were adoptive transferred with splenocytes isolated from 12-week-old female instead of new-onset diabetic NOD mice presented by Van et al. [5]. Even though 100% of our control NOD/scid mice developed diabetes with the mean onset of 6.5 weeks after adoptive transfer [12], in contrast, those treated with ATRA and ATRA plus exendin-4 developed diabetes with the mean onset of 8.0 and 11.2 weeks, respectively. Clearly, ATRA and ATRA plus exendin-4, but not exendin-4, treatment delayed the onset of autoimmune diabetes. This finding indicates that before the onset of diabetes, the inhibition of autoimmunity (exerted by ATRA) is more important than the promotion of beta-cell regeneration (exerted by exendin-4). Since there were varied degrees of insulitis and a variable number of beta cells per islet in the pancreas of mice before death with different treatment, we compared their pancreatic insulin content, an indicator of pancreatic beta-cell mass. Higher pancreatic insulin content was observed in mice treated with ATRA and exendin-4 than in control mice after adoptive transfer. It supports the beneficial effect of ATRA and exendin-4 treatment on beta-cell preservation before the onset of autoimmune diabetes. Our finding of more Pdx-1- and Ki67-positive beta cells in ATRA-treated group compared with control and Ex-4-treated animals indicating that ATRA has replicating and differentiating effects on pancreatic beta cells.

In the reversal experiment, we used spontaneous diabetic NOD mice instead of NOD/scid mice with adoptive transfer. In this slowly progressive diabetic model [13], residual beta cells in the pancreas could be rescued. Unfortunately, nearly all diabetic NOD mice remained persistent hyperglycemic regardless of ATRA and/or exendin-4 treatment. Meanwhile, ATRA and/or exendin-4 treatment did not improve their survival time. Therefore, after the onset of autoimmune diabetes, ATRA cannot halt autoimmune beta-cell destruction. There was only one mouse that achieved normoglycemia after ATRA treatment. It needs to be further studied.

5. Conclusions

Before the onset of autoimmune diabetes, ATRA and exendin-4 treatment alone preserves pancreatic beta cells; ATRA and ATRA plus exendin-4 treatment delays the onset of autoimmune diabetes. After the onset of autoimmune diabetes, ATRA and/or exendin-4 treatment is unable to reverse hyperglycemia or improve survival.

Conflict of Interests

The authors declared no conflict of interests.

Authors’ Contribution

Jyuhn-Huarng Juang and Yang-Hau Van contributed equally to this paper.

Acknowledgments

This work was supported by Grants from the Chang Gung Memorial Hospital (nos. CMRPG391701-2 and CMRPG3B1551-2), Taiwan, and the National Science Council (no. 99-2314-B-182A-010-MY3) of Taiwan.

References

  1. G. S. Eisenbarth, “Type I diabetes mellitus. A chronic autoimmune disease,” The New England Journal of Medicine, vol. 314, no. 21, pp. 1360–1368, 1986. View at Google Scholar · View at Scopus
  2. J. J. Meier, A. Bhushan, A. E. Butler, R. A. Rizza, and P. C. Butler, “Sustained beta cell apoptosis in patients with long-standing type 1 diabetes: indirect evidence for islet regeneration?” Diabetologia, vol. 48, no. 11, pp. 2221–2228, 2005. View at Publisher · View at Google Scholar · View at Scopus
  3. S. Castaigne, C. Chomienne, M. T. Daniel et al., “All-trans retinoic acid as a differentiation therapy for acute promyelocytic leukemia. I. Clinical results,” Blood, vol. 76, no. 9, pp. 1704–1709, 1990. View at Google Scholar · View at Scopus
  4. J. J. Leyden, “Therapy for acne vulgaris,” The New England Journal of Medicine, vol. 336, no. 16, pp. 1156–1162, 1997. View at Publisher · View at Google Scholar · View at Scopus
  5. Y. H. Van, W.-H. Lee, S. Ortiz, M.-H. Lee, H.-J. Qin, and C.-P. Liu, “All-trans retinoic acid inhibits type 1 diabetes by T regulatory (Treg)- dependent suppression of interferon-γ-producing T-cells without affecting Th17 cells,” Diabetes, vol. 58, no. 1, pp. 146–155, 2009. View at Publisher · View at Google Scholar · View at Scopus
  6. R. Goke, H.-C. Fehmann, T. Linn et al., “Exendin-4 is a high potency agonist and truncated exendin-(9-39)-amide an antagonist at the glucagon-like peptide 1-(7-36)-amide receptor of insulin- secreting β-cells,” The Journal of Biological Chemistry, vol. 268, no. 26, pp. 19650–19655, 1993. View at Google Scholar · View at Scopus
  7. D. J. Drucker, “The role of gut hormones in glucose homeostasis,” The Journal of Clinical Investigation, vol. 117, no. 1, pp. 24–32, 2007. View at Publisher · View at Google Scholar · View at Scopus
  8. J. M. Egan, A. Bulotta, H. Hui, and R. Perfetti, “GLP-1 receptor agonists are growth and differentiation factors for pancreatic islet beta cells,” Diabetes/Metabolism Research and Reviews, vol. 19, no. 2, pp. 115–123, 2003. View at Publisher · View at Google Scholar · View at Scopus
  9. I. A. Urusova, L. Farilla, H. Hui, E. D'Amico, and R. Perfetti, “GLP-1 inhibition of pancreatic islet cell apoptosis,” Trends in Endocrinology and Metabolism, vol. 15, no. 1, pp. 27–33, 2004. View at Publisher · View at Google Scholar · View at Scopus
  10. I. Hadjiyanni, L. L. Baggio, P. Poussier, and D. J. Drucker, “Exendin-4 modulates diabetes onset in nonobese diabetic mice,” Endocrinology, vol. 149, no. 3, pp. 1338–1349, 2008. View at Publisher · View at Google Scholar · View at Scopus
  11. J.-H. Juang, C.-H. Kuo, C.-H. Wu, and C. Juang, “Exendin-4 treatment expands graft β-cell mass in diabetic mice transplanted with a marginal number of fresh islets,” Cell Transplantation, vol. 17, no. 6, pp. 641–647, 2008. View at Publisher · View at Google Scholar · View at Scopus
  12. S. W. Christianson, L. D. Shultz, and E. H. Leiter, “Adoptive transfer of diabetes into immunodeficient NOD-scid/scid mice: relative contributions of CD4+ and CD8+ T-cells from diabetic versus prediabetic NOD.NON-Thy-1a donors,” Diabetes, vol. 42, no. 1, pp. 44–55, 1993. View at Google Scholar · View at Scopus
  13. M. S. Anderson and J. A. Bluestone, “The NOD mouse: a model of immune dysregulation,” Annual Review of Immunology, vol. 23, pp. 447–485, 2005. View at Publisher · View at Google Scholar · View at Scopus